USE OF DRUG WHICH REMOVES IgG / IgM ANTIBODIES OR B CELLS

Abstract

A use of a drug which removes IgG / IgM antibodies or B cells. IgG antibodies or B cells are removed via the drug, antibody aggregation is reduced, and muscle fibers can gradually change from a state of chronic injury to a state of promoting healing. By means of construction of an animal model, the drug which removes IgG antibodies or B cells is found to effectively reduce muscle injury, inflammation, and fibrosis levels in Duchenne muscular dystrophy mice, improve muscle strength, and to have no obvious side effects. Therefore, targeting B cells and antibody aggregation via the drug which removes antibodies or B cells can be used as a new method for treating incurable DMD.

Description

Application of a drug for clearing IgG / IgM antibodies or B cells Technical Field

[0001] The present invention relates to the field of treatment of genetic diseases caused by dystrophin mutations, and for the first time proposes that drugs that eliminate antibodies or B cells can effectively treat or alleviate Duchenne muscular dystrophy without obvious side effects. Background Art

[0002] Dystrophin mutations are a genetic disorder characterized by an X-linked recessive muscular dystrophy, including Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Therefore, patients are generally male, with an incidence of approximately 1 / 3,500-1 / 5,000 in boys. Females are often carriers, and the disease is rare (1 / 45,000-1 / 100,000), with milder symptoms. Duchenne muscular dystrophy is caused by mutations in the DMD gene, resulting in a loss of the encoded protein, dystrophin. Symptoms begin at approximately 2-3 years of age, and with increasing age, muscle damage and necrosis gradually worsen, manifesting as progressive muscle fiber degradation, significantly elevated creatine kinase (CK) levels, and proximal lower limb weakness. Generally, patients are unable to walk independently at around 10 years old and need to rely on crutches and wheelchairs. Most patients will develop spinal curvature, respiratory muscle weakness, dilated cardiomyopathy, etc., and eventually die of cardiopulmonary failure around the age of 30-40.

[0003] Currently, exon skipping strategies and gene replacement therapy are considered the most promising treatments for Duchenne muscular dystrophy, but they still have significant limitations. For example, corticosteroid therapy is the only available treatment for all types of Duchenne muscular dystrophy, but its efficacy is limited and its side effects are significant. There is currently a lack of effective clinical evidence for stop codon readthrough therapy. The efficiency of inducing exon skipping in Duchenne muscular dystrophy patients using anti-oligosense nucleotides is still relatively low and can only be used for patients with mutations in hotspot regions. There are currently no clinically successful cases of inducing exon skipping in Duchenne muscular dystrophy patients using gene editing (one clinical treatment case has been reported, but the patient has since died). Gene replacement therapy can currently successfully induce the production of artificially designed truncated Dystrophin proteins in clinical practice, but whether the resulting truncated Dystrophin proteins are functional remains to be determined. Furthermore, it may induce autologous humoral or cellular immune responses. Therefore, optimizing existing treatment options, further understanding the pathogenesis of Duchenne muscular dystrophy, and developing new, effective and safe treatment strategies for Duchenne muscular dystrophy are crucial for the treatment of Duchenne muscular dystrophy.

[0004] B cells, also known as B lymphocytes, develop from hematopoietic stem cells in the bone marrow. B cells play a vital role in the immune system. Abnormalities in B cell morphology and function can lead to a variety of diseases, including B cell tumors, autoimmune diseases, and multiple sclerosis. Upon antigen stimulation, B cells differentiate into plasma cells, which then produce and secrete antibodies. Antibodies primarily contribute to humoral immunity by neutralizing toxins and preventing pathogen invasion, activating complement to produce membrane attack complexes that lyse and destroy cells, and promoting opsonophagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies play a crucial role in a wide range of clinical diagnostic, preventive, and therapeutic procedures. B cell depletion therapy can effectively and transiently deplete B cells, reducing B cell antibody production and thereby ameliorating disease progression caused by abnormal B cell morphology and function. It is currently widely used in clinical treatment. However, the efficacy of antibody- or B cell-depleting drugs in the treatment of Duchenne muscular dystrophy has not yet been reported.

[0005] Summary of the Invention

[0006] This invention aims to develop new, effective, and safe treatment strategies for Duchenne muscular dystrophy by further understanding its pathogenesis. The present invention discovered that ectopic accumulation of antibodies on myofibers plays a crucial role in the pathogenesis of Duchenne muscular dystrophy. By reducing antibody accumulation through antibody-clearing or B-cell-depleting drugs, myofibers can gradually transition from a chronically damaged state to one that promotes healing. This phenomenon, along with the drug's therapeutic efficacy, has been effectively validated in a DMD mouse model.

[0007] The technical solutions adopted in the present invention are as follows:

[0008] A drug that clears IgG / IgM antibodies or B cells is used to prepare a drug for treating or delaying the onset of genetic diseases caused by dystrophin mutations. Specifically, a drug that clears IgG / IgM antibodies or B cells refers to a drug that clears IgG antibodies, a drug that clears IgM antibodies, a drug that clears B cells, or a drug that simultaneously clears two or more of these: IgG antibodies, IgM antibodies, and B cells.

[0009] The IgG / IgM antibody or B cell clearing drug reduces the accumulation of IgG / IgM antibodies in the patient's muscles, reduces the infiltration of M1 macrophages, and increases the ratio of M2 macrophages through a strategy of gradually converting the muscle fibers from a chronic damaged state to a healing-promoting state.

[0010] The genetic diseases caused by dystrophin mutations include various mutation types of Duchenne muscular dystrophy and Becker muscular dystrophy.

[0011] The drug for clearing IgG / IgM antibodies or B cells is an injection.

[0012] The IgG / IgM antibody or B cell clearing drug is anti-CD20, anti-CD19, anti-CD19 CART, anti-FcRn, FcγRI inhibitor or BTK inhibitor, etc.

[0013] The effective effects of the present invention are as follows: the drug of the present invention utilizes an antibody or B cell-clearing agent to reduce the accumulation of antibodies on muscle fibers in DMD animal models, reduce the infiltration of M1 macrophages in the muscles of DMD animal models, and increase the ratio of M2 macrophages, thereby achieving the effect of treating or delaying the onset of Duchenne muscular dystrophy. The drug has a significant effect in treating or delaying the onset of Duchenne muscular dystrophy. The present invention provides a more optimal strategy for the clinical treatment of various mutation types of Duchenne muscular dystrophy and has good clinical prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 shows the results of immunoglobulin aggregation on the sarcolemma of the Duchenne muscular dystrophy mouse model. E4* Immunofluorescence staining of IgG and IgM on the tibialis anterior muscle membrane of Dmd and Mdx mice, where Laminin is a marker of the muscle cell membrane (scale bar: 20 μm); B is an 8-week-old Dmd E4* Immunofluorescence staining of IgG and IgM on the tibialis anterior muscle membrane of Mdx and Dmd mice (scale bar: 100 μm); C is a B cell-deficient Dmd E4* Mouse (Dmd E4* ΔB) IgG staining on the tibialis anterior muscle fascia after intraperitoneal injection of normal mouse-derived IgG (15 mg / kg, twice a week for 6 weeks) (Scale bar: 50 μm).

[0015] Figure 2 shows the accumulation of immunoglobulins on the sarcolemma of patients with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). The samples included muscle samples from seven DMD patients (including one from a 3-year-old DMD patient, three from 4-year-old DMD patients, two from 6-year-old DMD patients, and one from an 11-year-old DMD patient), three from BMD patients (aged 10, 21, and 23, with the 21-year-old BMD patient showing more severe symptoms), and four relatively normal controls with milder symptoms from other muscle diseases (one with spinal muscular atrophy, approximately 20 years old; one with ataxia, 23 years old; one with limb-girdle muscular dystrophy type 4, 34 years old; and one with myotonic dystrophy, 30 years old).

[0016] Figure 3 shows 8-week-old Dmd cells with B cell deficiency. E4* Mouse (Dmd E4* ΔB) Compared with 8-week-old Dmd E4* The results of improved muscle function in mice are shown in Figure 1. A shows the detection of Dmd by Western blotting. E4* ΔB shows the expression of immunoglobulins in skeletal muscle of mice; B is 8-week-old Dmd E4* ΔB mice and Dmd E4* Comparison curve of the remaining muscle tension of mice; C is Dmd at 8 weeks of age E4* ΔB mice and Dmd E4* Comparative bar graph of creatine kinase levels in mouse serum, where creatine kinase originates from myocardial and skeletal muscles, and creatine kinase levels reflect the damage to myocardial and skeletal muscles; D is Dmd observed by micro-CT at 8 weeks of age E4* ΔB mice and Dmd E4* Photo of the dorsal bone bending of mice; E is the Dmd counted by micro-CT at 8 weeks of age E4* ΔB mice and Dmd E4* Comparative bar graph of the degree of back bone curvature (hunchback coefficient) of mice; F is the Dmd observed and counted by hematoxylin-eosin staining at 8 weeks of age E4* ΔB mice and Dmd E4* Histogram of the ratio of centronuclear myocytes in the tibialis anterior and rectus femoris muscles of mice.

[0017] Figure 4 shows a 12-month-old Dmd with B cell deficiency. E4* Mouse (Dmd E4* ΔB) Compared with 12-month-old Dmd E4* The results of improvement of myocardial and skeletal muscle function in mice. A is Dmd E4* ΔB mice and Dmd E4* Comparison of mouse survival results; B is Dmd E4* ΔB mice and Dmd E4* Comparative bar graph of creatine kinase content in mouse serum; C is Dmd E4* ΔB mice and Dmd E4* Comparison curve of the remaining muscle tension of mice; D is the Dmd statistically calculated by Masson staining E4* ΔB mice and Dmd E4* Histogram of fibrosis ratios of mouse myocardium, tibialis anterior muscle, rectus femoris muscle, and diaphragm muscle; E is Dmd calculated by micro-CT E4* ΔB mice and Dmd E4* Comparative bar graph of the degree of bone curvature of the mouse back; F is the Dmd calculated by the ultrasound imaging systemE4* ΔB mice and Dmd E4* Comparative bar graph of left ventricular ejection fraction and left ventricular short-axis contraction rate in mice.

[0018] Figure 5 shows the improvement in muscle function in 12-month-old B cell-deficient Mdx mice (MdxΔB) compared to 12-month-old Mdx mice. A is a graph comparing the residual muscle tensile force of MdxΔB mice and Mdx mice; B is a bar graph comparing serum creatine kinase levels in MdxΔB and Mdx mice; C is a bar graph showing the ratio of centronuclear myocytes in the tibialis anterior and rectus femoris muscles of 12-month-old MdxΔB and Mdx mice as observed by hematoxylin-eosin staining; D is a bar graph showing the ratio of fibrosis in the myocardium, tibialis anterior, rectus femoris, and diaphragm muscles of 12-month-old MdxΔB and Mdx mice as observed by Masson staining.

[0019] Figure 6 shows the results of the ectopic aggregation of antibodies on myofibers caused by the expression of FcγRI in the DMD mouse model. E4* , Dmd E4* The skeletal muscle lysates of ΔB and WTΔB mice were incubated to enrich the proteins that may bind to immunoglobulins in the mouse muscles and analyzed by mass spectrometry. B is an 8-week-old Dmd E4* Immunofluorescence staining of FcγRI on the muscular membrane of the tibialis anterior muscle of Mdx and Mdx mice, where Laminin is a marker of the muscular membrane (scale bar: 100 μm); C is the detection of FcγRI on the muscular membrane of 8-week-old Dmd mice using RNA probe hybridization technology. E4* The expression results of FcγRI in the myocytes of the tibialis anterior muscle of mice, where Laminin is a marker of the myocyte membrane, DAPI marks the cell nucleus, and the signal in the cytoplasm pointed by the arrow is FcγRI mRNA labeled with RNA probe (scale bar: 20μm); D is 8-week-old Dmd E4* and Fc commonγchain knockout Dmd E4* (Fcer1g - / - Dmd E4* ) Immunofluorescence staining results of FcγRI on the fascia of the tibialis anterior muscle of mice (Scale bar: 100 μm).

[0020] Figure 7 shows Fcer1g at 6-7 weeks old - / - Dmd E4* Compared with 6-7 week old Dmd mice E4* The results of the improvement of mouse muscle function are shown in Figure 1. A shows the Fcer1g expression in 6-7 weeks old mice observed and counted by hematoxylin-eosin staining. - / - Dmd E4*mice and 6-7-week-old Dmd E4* The ratio of myocytes containing central nuclei in the tibialis anterior and rectus femoris muscles of mice; B is Fcer1g at 6-7 weeks of age - / - Dmd E4* mice and 6-7-week-old Dmd E4* Comparative bar graph of creatine kinase levels in mouse serum; C is Fcer1g at 6-7 weeks of age - / - Dmd E4* mice and 6-7-week-old Dmd E4* Comparison bar graph of residual muscle pulling force in mice.

[0021] Figure 8 shows the Dmd of Anti-CD20 administered at 1 week of age E4* Compared with the control group, mice with Dmd E4* The results of improving mouse skeletal muscle function. A is Dmd E4* Diagram of drug administration time and myocardial and skeletal muscle function test time in mice; B is Dmd E4* The changes in the proportion of B cells in the blood of mice after administration; C is Dmd E4* Detection of IgG and IgM levels in the muscles of mice after 4 weeks of administration; D stands for Dmd E4* Mice in the drug-treated group and Dmd E4* Comparison of creatine kinase levels in the serum of mice in the control group; E is Dmd E4* Mice in the drug-treated group and Dmd E4* Comparison of the residual muscle tension of mice in the control group; F is the Dmd calculated by micro-CT E4* Mice in the drug-treated group and Dmd E4* Comparative bar graph of the degree of back bone curvature of mice in the control group; G stands for Dmd E4* Mice in the drug-treated group and Dmd E4* Comparison of the survival status of mice in the control group.

[0022] Figure 9 shows the Dmd of Anti-CD20 administered at 1 week of age E4* Compared with the control group, mice with Dmd E4* The results of pathological changes in myocardial and skeletal muscles of mice. A is Dmd E4* Mice in the drug-treated group and Dmd E4* The histogram of the fibrosis ratio of the rectus femoris and diaphragm muscles of the control group mice; B is the Dmd calculated by the ultrasound imaging system E4* Mice in the drug-treated group and Dmd E4* Comparative bar graph of left ventricular ejection fraction and left ventricular short axis contraction rate in control mice; C is the flow cytometry staining of immune cells isolated from mouse muscles to calculate Dmd E4*Mice in the drug-treated group and Dmd E4* Representative diagram of the changes in the ratio of M1 / M2 macrophages in the muscles of control mice; D stands for Dmd E4* Mice in the drug-treated group and Dmd E4* Statistical graph of the M1 / M2 macrophage ratio and the number of infiltrating immune cells in the muscles of control mice.

[0023] Figure 10 shows the improvement in muscle function in Mdx mice treated with Anti-CD20 starting at 1 week of age, compared to Mdx mice in the control group. A shows the dosing schedule for 1-week-old Mdx mice; B shows a comparison of serum creatine kinase levels in 1-week-old Mdx-treated mice and the Mdx control group; and C shows a comparison of residual muscle tension in 1-week-old Mdx-treated mice and the Mdx control group.

[0024] Figure 11 shows the Dmd of Anti-CD20 administered at 8 weeks of age E4* Compared with the control group, mice with Dmd E4* The results of the improvement of mouse muscle function. A is 8-week-old Dmd E4* Diagram of drug administration time for mice; B is 8-week-old Dmd E4* Figure 3: Changes in the proportion of B cells in the blood of mice after administration; C is 8-week-old Dmd E4* Mice in the drug-treated group and Dmd E4* Comparison of creatine kinase levels in the serum of mice in the control group; D is Dmd at 8 weeks of age E4* Mice in the drug-treated group and Dmd E4* Comparison of the remaining muscle tension of mice in the control group. DETAILED DESCRIPTION

[0025] The present invention proposes the use of an antibody or B cell-clearing drug in the preparation of a drug for treating or alleviating Duchenne muscular dystrophy (DMD). In this application, the antibody or B cell-clearing drug can effectively reduce the aggregation level of antibodies on muscle fibers of a DMD mouse model, reduce the infiltration of M1 macrophages in the muscle of the DMD mouse model, and increase the ratio of M2 macrophages in the muscle, thereby achieving the effect of treating or delaying Duchenne muscular dystrophy.

[0026] The present invention is further described below with reference to the accompanying drawings and examples. In the following examples, C57BL / 10-Mdx mice are a mouse model with dystrophin deficiency (caused by a point mutation in exon 23 of the Dmd gene) purchased from the Jackson Laboratory; WTΔB mice, i.e., μMT mice, are a mouse model with B cell deficiency (homozygous Ighmtm1Cgn targeted mutation) purchased from the Jackson Laboratory; C57BL / 10-Dmd E4* The mouse is a dystrophin-deficient mouse model (caused by a 4-base deletion in exon 4 of the Dmd gene) and was obtained from the National Model Mouse Resource Center (Nanjing, China); Fcer1g - / - The mice are Fc common γ chain (encoded by Fcer1g) knockout mice, which were donated by Shanghai Jiao Tong University and purchased from Jackson Laboratory; wild-type C57BL / 6 mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. C57BL / 10-Mdx mice and C57BL / 10-Dmd E4* Mice were backcrossed to the C57BL / 6 background for at least ten generations. E4* ΔB was respectively composed of Mdx mice, Dmd E4* After two generations of hybridization between mice and μMT mice, B cell-deficient DMD mice were obtained. - / - Dmd E4* By Dmd E4* Mouse and Fcer1g - / - After two generations of crossbreeding, mice lacking both the Fc common γ chain and dystrophin were obtained.

[0027] In addition, unless otherwise specified, all percentages in this application are by mass.

[0028] Example 1: In vivo experiments in mice confirmed that the progression of Duchenne muscular dystrophy in DMD mice with B cell and antibody knockout was significantly improved

[0029] Obtain DMD mice at 2 and 8 weeks of age E4*The tibialis anterior muscle membrane of DMD and Mdx mice was immunostained for IgG and IgM, respectively, and it was found that compared with WT mice (as shown in Figures 1A and 1B), there was obvious aggregation of immunoglobulins on the surface of the muscle cell membrane of 2-week-old and 8-week-old DMD mice. Similarly, the sarcolemma of patients with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) was obtained for immunofluorescence staining of IgG and IgM, and the same phenomenon was observed in the muscle sample sections of DMD and BMD clinical patients, that is, compared with the control group patients (as shown in Figure 2A), there was obvious aggregation of immunoglobulins on the surface of the muscle cell membrane of DMD and BMD clinical patients. In addition, by giving 1-week-old Dmd E4* ΔB mice (DMD mice with B cell deficiency) were injected intraperitoneally with WT mouse IgG (15 mg / kg, twice a week for 6 weeks) and it was found that WT mouse IgG could bind to Dmd E4* The surface of the myocyte membrane of ΔB mice (Figure 1C). These results indicate that non-anti-self antibodies can bind to the surface of the myocyte membrane of Duchenne muscular dystrophy mice.

[0030] Detection of Dmd by Western blotting E4* Is there any residual immunoglobulin in the ΔB muscle? Intraperitoneally inject 2.5% Avertin 300-500μl, and use a syringe to take 10ml of 1×PBS for cardiac perfusion. Take an appropriate amount of skeletal muscle tissue, mince it with surgical scissors, add RIPA lysis buffer (150mM NaCl, 1% NP-40, 2mM EDTA, 50mM Tris pH 7.4, 0.1% SDS), and homogenize the tissue. Let it stand at 4℃ for 30min, and shake it once every 5 minutes. Centrifuge at 4℃, 14000rpm, and centrifuge for 30min. Take the supernatant, add a certain volume of 6×SDS loading dye, mix well, and denature at 95℃ for 10min. Then, let it stand at 4℃ and perform SDS-PAGE gel electrophoresis. Transfer to the membrane, block with skim milk, and develop with ECL chemiluminescence solution. Protein blotting results showed that compared with WT mice, Dmd E4* There is a significant enrichment of immunoglobulins (including IgG and IgM) in muscles. E4* No immunoglobulins were detected in ΔB muscles (Fig. 3A ).

[0031] The tensile strength of the forelimb muscles of mice was measured by a mouse muscle tensile tester, and the maximum force reading was recorded. The interval between each test was 10 seconds and repeated 10 times. The residual tensile test results showed that the 8-week-old Dmd E4* ΔB, Dmd at 12 months E4* ΔB and MdxΔB, compared with the Dmd of the control group at 8 weeks of age E4* , 12 months old DmdE4* The muscle function of Mdx mice was significantly improved (Figures 3B, 4C, and 5A).

[0032] The content of creatine kinase in mouse serum was quantified using the CREATINE KINASE-SL ASSAY (326-10, Sekisui) kit. First, the standard was diluted in series to draw a standard curve. At the same time, the standard and the sample to be tested were added to the prepared creatine kinase detection reaction solution (CK-SL Buffer Reagent (R1) and CK-SL Substrate Reagent (R2) were mixed at a ratio of 4:1). The reaction was allowed to proceed at room temperature for 5-10 minutes. The absorbance of the standard and the sample to be tested was detected at 340nm using an enzyme-labeled instrument. The standard curve was drawn and the creatine kinase content of the sample to be tested was calculated. The results of the creatine kinase content determination experiment showed that 8-week-old Dmd E4* ΔB, Dmd at 12 months E4* ΔB and MdxΔB, compared with the Dmd of the control group at 8 weeks of age E4* , 12 months old Dmd E4* The creatine kinase levels in the serum of Mdx and Mdx were significantly decreased (Figures 3C, 4B, and 5B).

[0033] Mice aged 8 weeks or 12 months were intraperitoneally injected with 300μl-500μl of 2.5% Avertin. After the mice were anesthetized, they were fixed with tape and placed in a Micro CT machine for photography. Subsequently, the analysis software of Bruker SkyScan 1276Micro CT was used to perform 3D reconstruction of the multiple photos taken to obtain a complete skeletal image of the mouse. Among them, the calculation of the kyphosis coefficient refers to drawing a line (A1) between the caudal edge of the last cervical vertebra (C7) and the caudal edge of the sixth lumbar vertebra (L6). Another line (A2) was drawn perpendicular to the dorsal edge of the vertebra at the point of maximum curvature. The kyphosis coefficient was calculated as A1 / A2. The results of Micro CT showed that Dmd at 8 weeks of age E4* ΔB, Dmd at 12 months E4* ΔB, compared with the Dmd of the control group at 8 weeks of age E4* , 12 months old Dmd E4* The curvature of the back bones was significantly improved (Figures 3D, 3E, and 4E).

[0034] Muscle tissue was collected from 8-week-old or 12-month-old mice, fixed in 10% neutral formalin, dehydrated, embedded in paraffin, and then sectioned. Paraffin sections were then stained with hematoxylin-eosin and Masson's staining to assess the presence of newly formed myocytes (centronuclear myocytes) in the muscle tissue (Figures 3F, 5C) and the degree of fibrosis (Figures 4D, 5D). Hematoxylin-eosin staining revealed that the ratio of centronuclear myocytes in the tibialis anterior and rectus femoris muscles of 8-week-old B cell-knockout DMD mice was significantly decreased compared to DMD mice, approaching that of the WT group (Figures 3F, 5C). Masson's staining revealed that the ratio of fibrosis in the myocardium, tibialis anterior, rectus femoris, and diaphragm muscles of 12-month-old B cell-knockout DMD mice was significantly decreased compared to DMD mice (Figures 4D, 5D).

[0035] After removing the hair from the chest of 12-month-old mice with depilatory cream, the mice were placed in an anesthesia induction box for anesthesia. The anesthetized mice were fixed in a supine position on the echocardiography operating table, and the mouth and nose of the mice were stuffed with an anesthesia mask. The anesthetic flow rate was 0.8L / min, and the concentration was about 1%-1.5% to maintain the anesthesia of the mice. The electrocardiogram of the mice was collected using the Vevo 3100 small animal ultrasound imaging platform, and the B-model and M-model data were saved. Using the LV Trace tool, in the M-model mode, the tracing was performed along the endocardial and endocardial tracks of the anterior wall of the left ventricle, and three cardiac cycles were recorded continuously. Then, the posterior wall of the left ventricle was traced in the same way for three consecutive cardiac cycles. After that, LV Trace will automatically generate the left ventricular ejection fraction and the left ventricular short axis shortening rate (as shown in Figure 4F). The results of cardiac ultrasound showed that the Dmd knockout of B cells in 12-month-old mice E4* Compared with Dmd E4* In mice, left ventricular ejection fraction and left ventricular fractional shortening were significantly improved, closer to those of the WT group.

[0036] In summary, the present invention achieves the purpose of delaying the disease progression in different DMD mouse models by knocking out B cells and antibodies in DMD mice.

[0037] Example 2: FcγRI knockout can effectively reduce the accumulation of IgG on the muscle cell membrane surface in Duchenne muscular dystrophy mice

[0038] By combining Protein A / G beads with Dmd E4* Mouse muscle lysate incubation (negative control was Dmd E4* ΔB and WTΔB mice), incubated at 4°C for 3 h to enrich Dmd E4*The IgG and IgG complexes in the mouse muscle lysate were removed from the supernatant and washed with wash buffer 5 times, 5 minutes each time. Then, loading buffer was added and the samples were boiled at 95℃ for 10 minutes and stored at -80℃. The results of mass spectrometry analysis showed that compared with the negative control group, the Dmd E4* In the group, Protein A / G beads bound a large amount of immunoglobulins and complement proteins in muscle. In addition, FcγRI and Trim21 were expressed in Dmd E4* The results of immunofluorescence experiments showed that compared with WT mice, Mdx and Dmd E4* The expression of FcγRI can be detected on the surface of mouse muscle cell membrane (as shown in Figure 6B). The results of RNAscope experiments showed that FcγRI mRNA probe signals can be detected in the muscle cells of DMD mice, indicating that FcγRI transcripts exist in the muscle cells of DMD mice (as shown in Figure 6C). In addition, by constructing DMD with Fc commonγchain knockout E4* (Fcer1g - / - Dmd E4* ) mice, and the muscle tissue was taken for immunofluorescence experiments. The results showed that: Fcer1g - / - Dmd E4* FcγRI expression was undetectable on the surface of mouse skeletal muscle cells, and IgG binding was significantly reduced (Figure 6D). These results indicate that FcγRI is expressed in the muscle cells of DMD mice, and knocking out FcγRI can effectively reduce the accumulation of IgG on the muscle cell membrane surface of Duchenne muscular dystrophy mice.

[0039] Further research results showed that when muscle tissues of mice aged 6-7 weeks were collected and stained with hematoxylin-eosin, newly generated myocytes (central nuclear myocytes) in the muscle tissues of mice were observed (as shown in Figure 7A), Fcer1g - / - Dmd E4* Compared with Dmd E4* In mice, the ratio of centronuclear myocytes was significantly decreased. - / - Dmd E4* Compared with Dmd E4* The creatine kinase content in the mouse serum was significantly decreased (as shown in Figure 7B), and the muscle function was significantly improved (as shown in Figure 7C).

[0040] In summary, the present invention can effectively reduce the binding of IgG on the surface of DMD mouse muscle cells by knocking out FcγRI in DMD mice, thereby delaying the disease progression of the DMD mouse model.

[0041] Example 3: Depletion of B cells in young mice by anti-CD20 antibody can effectively alleviate the disease progression of DMD in the long term

[0042] One-week-old DMD mice were intraperitoneally injected with anti-CD20 antibodies (2 mg / kg), and one-week-old DMD mice were intraperitoneally injected with IgG (2 mg / kg) as a control. The drugs were administered once a week for 8 consecutive weeks (Figures 8A and 10A). After 4 weeks of administration, blood was collected from the mice through the submandibular vein. 50 μl of peripheral blood was collected in an EP tube containing EDTA solution for the detection of B cells in the peripheral blood, and the rest was used to collect serum. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add 200 μl of red blood cell lysis buffer, pipet and mix, and let it stand for 5 minutes. Subsequently, 1 ml of PBS was added to stop the red blood cell lysis, centrifuge at 1500 rpm for 5 minutes, and collect the cell pellet. 50 μl of live / dead fix violet kit (thermo, L34964) was dissolved in PBS at a volume ratio of 1:1000 and incubated at 4°C in the dark for 30 minutes; 50 μl of anti-CD16 / CD32 (2.4G2, BD Pharmingen) was dissolved in Staining buffer (PBS + 1% FBS + 1mM EDTA) at a volume ratio of 1:400 and blocked at 4°C for 10 minutes; 50 μl of Staining buffer containing the corresponding surface antibodies anti-mouse CD45-APC / Cyanine7 (30-F11, Biolegend, 103115) and anti-mouse CD19-FITC (1D3 / CD19, Biolegend, 152403) (1:200 volume ratio) was added and incubated at 4°C in the dark for 30 minutes. The cells were washed twice with Staining buffer and 200 μl of staining buffer was added. After resuspending in buffer, the cells were tested on the machine. CD45 and CD19 staining showed that during the period of intraperitoneal injection of Anti-CD20 antibody to DMD mice, the ratio of B cells to immune cells was almost 0, which gradually recovered after drug withdrawal. The B cell ratio returned to normal levels 2-4 weeks after drug withdrawal (Figure 8B). At the same time, the results of Western blot detection showed that 1-week-old DMD E4* Four weeks after intraperitoneal injection of Anti-CD20 antibody, the mice showed a significantly higher expression of CD20 than those in Dmd E4* The immunoglobulin content in mouse muscles was significantly decreased (Figure 8C).

[0043] In addition, the experimental results of the two DMD mouse models showed that the serum creatine kinase content in the Anti-CD20 group continued to decrease compared with the Ctrl IgG group (as shown in Figures 8D and 10B), and remained at a low level for a long time after drug withdrawal. Muscle function also showed significant improvement (as shown in Figures 8E and 10C). Micro CT results showed that 8 months after drug withdrawal, the curvature of the back bones of the Anti-CD20 group mice was still significantly improved compared with the Ctrl IgG group mice (as shown in Figure 8F). Compared with the Ctrl IgG group DMD E4* Mouse, Anti-CD20 group Dmd E4* The survival time of mice was also significantly prolonged (Figure 8G). Further Masson staining results showed that compared with the Ctrl IgG group, Dmd E4* Mouse, Anti-CD20 group Dmd E4* The fibrosis rate in the rectus femoris and diaphragm muscles of mice was significantly reduced (Figure 9A). The results of cardiac ultrasound showed that 8 months after drug withdrawal, the left ventricular ejection fraction and left ventricular fractional shortening of mice in the Anti-CD20 group were significantly improved compared with those in the Ctrl IgG group, and were closer to those of the WT group (Figure 9B).

[0044] In order to further reveal the reasons why Anti-CD20 antibodies produce sustained therapeutic effects, the present invention used Percoll (GE Healthcare) density gradient centrifugation to separate the Dmd of Anti-CD20 group and Ctrl IgG group 10 weeks after drug withdrawal. E4* Comparison of immune cells in mouse muscle tissue was performed as follows: Mice were euthanized, and muscle tissue from the upper and lower limbs was removed and placed in pre-chilled PBS buffer. The muscles were minced with surgical scissors and digested in tissue digestion buffer (containing 1640 medium, 1% FBS, 10mM HEPES, 1mM MgCl2, 1mM CaCl2, 0.1mg / ml DNase I, and 1mg / ml Collagenase II) at 37°C for 30 min. The cells were ground using a 100μm pore size cell sieve, centrifuged at 1500rpm for 10min at 4°C, resuspended in 5mL of 40vol% Percoll solution, and transferred to a 70vol% Percoll solution for density gradient centrifugation at 600×g at 22°C for 30min, with an acceleration of 2 and a deceleration of 0. After centrifugation, cells in the middle white cloud layer were collected, washed twice with pre-chilled PBS, counted, stained with antibodies, and analyzed by flow cytometry. The results of flow cytometry staining showed that Anti-CD20 antibodies can continuously reduce the number of infiltrating immune cells in the muscles of DMD mouse models, mainly reducing the infiltration of M1 macrophages, while increasing the ratio of M2 macrophages in the muscles, thereby producing a sustained therapeutic effect (as shown in Figures 9C and 9D).

[0045] In summary, the present invention reduces the level of immunoglobulins in the muscles of DMD mice, reduces the infiltration of M1 macrophages, and increases the ratio of M2 macrophages in the muscles by intraperitoneal injection of Anti-CD20 antibodies into one-week-old DMD mice, thereby producing a sustained therapeutic effect, improving the disease characteristics of DMD mice, and thus achieving the effect of delaying the disease progression of the DMD mouse model.

[0046] Example 4: Depletion of B cells in adult mice using anti-CD20 antibodies can effectively alleviate the disease progression of DMD

[0047] 8-week-old Dmd E4* Mice were intraperitoneally injected with Anti-CD20 antibody (2 mg / kg), and 8-week-old Dmd E4* Mice were intraperitoneally injected with IgG (2 mg / kg) as a control, once a week for 8 consecutive weeks (as shown in Figure 11A). Peripheral blood was collected from the submandibular vein of mice, and flow cytometry results showed that the ratio of B cells to immune cells in the peripheral blood was almost 0, which was the same as the case of intraperitoneal injection of Anti-CD20 antibodies in young mice. It gradually recovered after drug withdrawal, and the B cell ratio returned to normal levels 2-4 weeks after drug withdrawal (as shown in Figure 11B). In addition, further experimental results showed that 8-week-old Dmd E4* After about 6-8 weeks of intraperitoneal injection of Anti-CD20 antibodies, the serum creatine kinase level in mice continued to decrease compared to the Ctrl IgG group (as shown in Figure 11C), and remained at a low level for a long time after drug withdrawal. In terms of muscle function, the sustained muscle tension in the Anti-CD20 group was significantly improved compared to the Ctrl IgG group (as shown in Figure 11D). These results indicate that eliminating B cells with Anti-CD20 antibodies can effectively alleviate the progression of DMD in adult mice.

Claims

1. An application of a drug for removing IgG / IgM antibodies or B cells, characterized in that: Used to prepare drugs to treat or delay genetic diseases caused by dystrophin mutations.

2. The use according to claim 1, characterized in that: The IgG / IgM antibody or B cell clearing drug reduces the accumulation of IgG / IgM antibodies in the patient's muscles, reduces the infiltration of M1 macrophages, and increases the ratio of M2 macrophages through the strategy of clearing antibodies or B cells, thereby gradually transforming the muscle fibers from a chronic injury state to a healing-promoting state.

3. The use according to claim 1, characterized in that: The genetic diseases caused by the dystrophin mutation include various mutation types of Duchenne muscular dystrophy and Becker muscular dystrophy.

4. The use according to claim 1, characterized in that: The drug for clearing IgG / IgM antibodies or B cells is an injection.

5. The use according to claim 1, characterized in that: The IgG / IgM clearing antibody or B cell drug is anti-CD20, anti-CD19, anti-CD19 CART, anti-FcRn, FcγRI inhibitor or BTK inhibitor.

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