Gene transfer expression cassettes for the treatment of muscular dystrophy

Abstract

The present invention relates to a recombinant hybrid protein, a nucleic acid molecule encoding the recombinant hybrid protein, a gene transfer expression cassette containing the nucleic acid molecule, and a gene delivery system containing the gene transfer expression cassette, which can be used to treat muscular dystrophy. The nucleic acid molecule, gene transfer expression cassette, and gene delivery system of the present invention can stably express a therapeutic protein in muscle tissue, and achieve good therapeutic effects on muscular dystrophy.

Description

[Technical Field]

[0001] The present disclosure relates to recombinant hybrid proteins, nucleic acid molecules encoding said recombinant hybrid proteins, transgenic expression cassettes comprising said nucleic acid molecules, and gene delivery systems comprising said transgenic expression cassettes that can be used to treat muscular dystrophy. [Background technology]

[0002] Muscular dystrophy (MD) is a broad range of degenerative muscle diseases that primarily manifest as muscle dysfunction in various areas, such as progressive muscle atrophy and weakness, to varying degrees. The most common and rapidly progressing of these is Duchenne muscular dystrophy (DMD). DMD is characterized by severe X-linked dystrophy. DMD is a multi-stranded recessive progressive neuromuscular disease. Patients with DMD suffer from a deficiency of dystrophin. DMD causes progressive degeneration of skeletal, cardiac, and respiratory muscles. Statistics show that DMD affects 100% of boys worldwide. DMD affects 1 in 3,600 people. Patients with DMD gradually lose the ability to walk independently between the ages of 12 and 15. The patient then experiences breathing difficulties, respiratory infections, and swallowing problems, which eventually progress to cardiomyopathy and death. There is also muscular dystrophy (BMD), in which the amount of dystrophin protein in muscle biopsy samples is or characterized by a decline in quality.

[0003] The dystrophin gene is approximately 2.5 Mb in size and is the largest known human gene. It is expressed primarily in skeletal and cardiac muscles. The gene is located on the X chromosome at Xp21, and contains 79 DMD is caused by mutations in the dystrophin gene. The most common mutations in DMD are large deletions of one or more exons (60-70%), duplications (5-10%), and single-nucleotide mutations (including small deletions or insertions, single-nucleotide changes, and splice site alterations). The basic function of dystrophin protein is to stabilize muscle fibers by binding to F-actin through its N-terminal domain and to β-dystroglycan through its C-terminal domain, thereby bridging and anchoring the proteins during contraction. Loss of dystrophin protein expression leads to severe muscle atrophy, respiratory failure, and cardiac failure. This is due to impaired dystrophin function, which inhibits the formation of the dystrophin-associated glycoprotein complex (DGC), leading to membrane instability, increased susceptibility to injury, and fibronecrosis (Findlay AR, Wein N, Kaminoh Y, et al., Clinical phenotypes as predictors of the outcome of skipping around DMD exon 45[J]. Ann Neurol, 2015, 77(4):668-74). Therefore, increasing dystrophin protein expression is important for the treatment of muscular dystrophies.

[0004] Currently, there are no effective drugs for the treatment of muscular dystrophy. Glucocorticoid therapy is the main conservative treatment, but it can only delay the progression of the disease by one to two years. Therefore, there is an urgent need to develop effective drugs for the treatment of muscular dystrophy.

[0005] Currently, gene delivery via adeno-associated virus (AAV) vectors is not available for the treatment of rare, single-episode diseases. AAV has low pathogenicity and can live for a long time in various organs and tissues. These characteristics have been clearly demonstrated in AAV in the field of gene therapy. AAV offers certain advantages and is suitable for the delivery of therapeutic genes. However, the gene size that can be packaged into an AAV vector is limited to a maximum of 4.7 kb, making it difficult to carry the large dystrophin protein gene. Researchers have been looking for ways to reduce the size of the large dystrophin protein gene and incorporate it into an AAV expression vector. Therefore, it is desirable to obtain a truncated dystrophin protein gene that can stably express a functional, small dystrophin protein with therapeutic activity.

[0006] Dystrophin-associated protein (Utrophin), a homologous protein of dystrophin, is also expressed ubiquitously in muscle and has the same functions as dystrophin. (Miura P, Jasmin B J. Utrophin upregulation for treating Duchenne or Becker muscular dystrophy: How close are we? [J]. Trends Mol Med, 2006, 12(3):122-9; Fairclough RJ, Wood MJ, Davies K E. Therapy for Duchenne muscular dystrophy: renewed optimism from genetic approaches [J]. Nat Rev Genet, 2013,14(6):373-8).Studies have shown that upregulation of utrophin expression also plays an important role in the treatment of muscular dystrophies and may be used as a therapeutic target for muscular dystrophies (Guiraud S, Chen H, Burns DT et al., Advances in genetic therapeutic strategies for Duchenne muscular dystrophy[J]. Exp Physiol, 2015, 100(12): 1458-67; Moorwood C, Khurana TS, Duchenne muscular dystrophy medicament discovery - the application of utrophin promoter activation screening[J]. ExpertOpin Medicament Discov, 2013, 8(5): 569-81; Ricoti V, Spinty S, Roper H, et al., Safety, Tolerability, and Pharmacokinetics of SMT C1100, an a2-Arylbenzoxazole Utrophin Modulator, following Single- and Multiple-Dose Administration to Pediatric Patients with Duchenne Muscular Dystrophy[J]. PLoSOne, 2016, 11(4): e0152840). Furthermore, studies have shown that upregulating the expression of utrophin, a dystrophin-associated protein, using small molecules or microRNAs can alleviate the symptoms of muscular dystrophy. Summary of the Invention

[0007] In order to solve the above technical problems, in a first aspect, the present disclosure provides a composition including: A recombinant hybrid protein is provided, which contains: the N-terminal domain of full-length human dystrophin-related protein (utrophin), hinge H1, spectrin-like repeat R1, and spectrin-like repeat R2. repeat R2, spectrin-like repeat R3 and the first half of hinge H2; and spectrin-like repeat R23, spectrin-like repeat R24, hinge H4 and the CR domain of full-length human dystrophin.

[0008] In one embodiment, the recombinant hybrid protein comprises the amino acid sequence shown in SEQ ID NO: 4. In a preferred embodiment, the recombinant hybrid protein consists of the amino acid sequence shown in SEQ ID NO:4.

[0009] The recombinant hybrid protein of the present disclosure can effectively improve muscle function and alleviate the symptoms of muscular dystrophy, and has good therapeutic effects on muscular dystrophy.

[0010] In a second aspect, the present disclosure provides a nucleic acid molecule encoding a recombinant hybrid protein according to the first aspect.

[0011] In one embodiment, the nucleic acid molecule is at least 50% identical to the nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:10, preferably at least 50% identical to the nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:10. The nucleotide sequence of the target gene is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identical to the nucleotide sequence of the target gene.

[0012] In one embodiment, the nucleic acid molecule comprises the nucleotide sequence as set forth in SEQ ID NO:8 or SEQ ID NO:10. In a preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence as set forth in SEQ ID NO:8 or SEQ ID NO:10. consists of the nucleotide sequence as shown in SEQ ID NO:10.

[0013] In a third aspect, the present disclosure provides a transgenic expression cassette comprising a promoter, a nucleic acid molecule according to the second aspect, and a mini-polyA.

[0014] In one embodiment, the promoter is selected from the following: a CB promoter, a CAG promoter, or a promoter of a muscle-specific gene, including a muscle creatine kinase (MCK) promoter, a human creatine kinase (hCK) promoter, a shortened human creatine kinase (shCK) promoter, a skeletal α-actin promoter, a cardiac α-actin promoter, a myosin heavy chain (MyHC) promoter, a myosin light chain 2 (MLC2) promoter, a myosin light chain 3F promoter, a desmin gene promoter, and a promoter of a myogenic regulatory factor family (MyoG, Myf5, Mrf4, and Myogenin). In a preferred embodiment, the promoter is an hCK promoter or an shCK promoter. In a more preferred embodiment, the promoter is an shCK promoter. Higher levels of protein expression can be obtained by using shCK compared to hCK.

[0015] In one embodiment, the promoter has the nucleotide sequence as set forth in SEQ ID NO:9. It has columns.

[0016] In one embodiment, the transgenic expression cassette further comprises two ITRs flanked at either end. Each of these ITRs may be a normal ITR or a truncated ITR. For example, a 145 bp regular ITR or a 100 bp shortened ITR. In this case, both ITRs are normal ITRs of 145 bp.

[0017] In one embodiment, the transgenic expression cassette further comprises an origin of replication, a polyadenylation signal, an internal ribosome entry site (IRES), and / or a 2A signal such as P2A, T2A, and F2A.

[0018] In one embodiment, the transgenic expression cassette consists of a nucleotide sequence as set forth in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

[0019] In a fourth aspect, the present disclosure provides a gene transfer expression cassette according to the third aspect and an AAV cassette. A gene delivery system is provided, comprising a psylase protein.

[0020] In one embodiment, the AAV capsid protein is a natural AAV capsid protein or an artificial AAV capsid protein. In a preferred embodiment, the AAV is selected from the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, AAVrh10, AAVrh39, AAVrh43, AAV32.33, AAV3B, AAVv66, AAVXL32, and AAV.PHP.B.

[0021] The nucleic acid molecules, gene transfer expression cassettes and gene delivery systems disclosed herein can stably express the recombinant hybrid proteins in muscle tissue, thereby improving muscle function and achieving good therapeutic effects on muscular dystrophy.

[0022] In a fifth aspect, the present disclosure provides a method for preparing a pharmaceutical composition for treating muscular dystrophy. The present invention provides the use of a transgenic expression cassette according to the third aspect or a gene delivery system according to the fourth aspect in

[0023] In one embodiment, muscular dystrophies include Duchenne muscular dystrophy, Becker muscular dystrophy, and other muscle degenerative diseases.

[0024] In a preferred embodiment, the muscular dystrophy is Duchenne muscular dystrophy.

[0025] In a sixth aspect, the present disclosure provides a pharmaceutical composition comprising one of: a recombinant hybrid protein according to the first aspect, a nucleic acid molecule according to the second aspect, a transgenic expression cassette according to the third aspect, and a gene delivery system according to the fourth aspect; and excipient(s).

[0026] In one embodiment, the pharmaceutical composition is used to treat muscular dystrophies, including Duchenne muscular dystrophy, Becker muscular dystrophy, and other muscle degenerative diseases. In a preferred embodiment, the muscular dystrophy is Duchenne muscular dystrophy.

[0027] In a seventh aspect, the present disclosure provides a method of treating muscular dystrophy, comprising administering to a subject a therapeutically effective amount of a compound selected from the group consisting of steroids, steroid hormone ... and steroid hormones. Administering to a subject in need thereof an amount of a pharmaceutical composition according to the sixth aspect. Provide the law.

[0028] In one embodiment, the pharmaceutical composition is administered via a systemic or local route, such as intravenous, intramuscular, subcutaneous, oral, topical, intraperitoneal, or local administration.

[0029] In a preferred embodiment, the pharmaceutical composition is administered by a systemic route, for example, intravenous administration.

[0030] In a preferred embodiment, the pharmaceutical composition is administered via a topical route into a muscle, for example, the pharmaceutical composition is injected into the biceps brachii or gastrocnemius muscle. [Brief explanation of the drawings]

[0031] [Figure 1A] FIG. 1A shows the structures (subdomains) of the full-length human dystrophin protein, the full-length human dystrophin-associated protein, and the recombinant hybrid protein encoded by the M6 ​​construct constructed by the inventors. [Figure 1B] FIG. 1B is a schematic diagram of one embodiment of the hCK-opti-M6, hCK-M6, hCK-B84, shCK-opti-M6, and shCK-M6 expression cassettes. [Figure 2A] Figure 2A shows immunofluorescence analysis of therapeutic proteins in the hearts of mice injected with AAV9-hCK-opti-M6, AAV9-hCK-M6, AAV9-shCK-opti-M6, and AAV9-shCK-M6 6 weeks after injection. Frozen sections of hearts from WT, Mdx, and treated mice were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 2B] Figure 2B shows immunofluorescence analysis of therapeutic proteins in the gastrocnemius muscles of mice injected with AAV9-hCK-opti-M6, AAV9-hCK-M6, AAV9-shCK-opti-M6, and AAV9-shCK-M6 6 weeks after injection. Frozen sections of gastrocnemius muscles from WT, Mdx, and treated mice were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 2C] Figure 2C shows immunofluorescence analysis of therapeutic proteins in the biceps muscles of mice injected with AAV9-hCK-opti-M6, AAV9-hCK-M6, AAV9-shCK-opti-M6, and AAV9-shCK-M6 6 weeks after injection. Frozen sections of biceps muscles from WT, Mdx, and treated mice were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 2D]Figure 2D shows immunofluorescence analysis of therapeutic proteins in the diaphragms of mice injected with AAV9-hCK-opti-M6, AAV9-hCK-M6, AAV9-shCK-opti-M6, and AAV9-shCK-M6 6 weeks after injection. Frozen sections of diaphragms from WT, Mdx, and treated mice were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 3] Figure 3 shows a comparison of the expression of the opti-M6 sequence and the existing B84 sequence in mice. Frozen sections of the heart, gastrocnemius muscle, and diaphragm from WT mice, Mdx mice, and mice treated with hCK-opti-M6 and hCK-B84 were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 4A] Figure 4A shows immunofluorescence analysis of therapeutic proteins in the hearts of mice injected with AAV9-shCK-opti-M6 and AAV9-hCK-B84 4 weeks after injection. Frozen sections of hearts from WT mice, Mdx mice, and mice from the two treatment groups were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 4B] Figure 4B shows immunofluorescence analysis of therapeutic proteins in the gastrocnemius muscle of mice injected with AAV9-shCK-opti-M6 and AAV9-hCK-B84 4 weeks after injection. Frozen sections of gastrocnemius muscle from WT mice, Mdx mice, and mice from the two treatment groups were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 4C] Figure 4C shows immunofluorescence analysis of therapeutic proteins in the quadriceps muscles of mice injected with AAV9-shCK-opti-M6 and AAV9-hCK-B84 4 weeks after injection. Frozen sections of quadriceps muscles from WT mice, Mdx mice, and mice from the two treatment groups were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 4D]Figure 4D shows immunofluorescence analysis of therapeutic proteins in the biceps muscles of mice injected with AAV9-shCK-opti-M6 and AAV9-hCK-B84 4 weeks after injection. Frozen sections of biceps muscles from WT mice, Mdx mice, and mice from the two treatment groups were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 4E] Figure 4E shows immunofluorescence analysis of therapeutic proteins in the tibialis anterior muscle of mice injected with AAV9-shCK-opti-M6 and AAV9-hCK-B84 4 weeks after injection. Frozen sections of tibialis anterior muscle from WT mice, Mdx mice, and mice from the two treatment groups were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 4F] Figure 4F shows immunofluorescence analysis of therapeutic proteins in the diaphragms of mice injected with AAV9-shCK-opti-M6 and AAV9-hCK-B84 4 weeks after injection. Frozen sections of diaphragms from WT mice, Mdx mice, and mice from the two treatment groups were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 4G] Figure 4G shows immunofluorescence analysis of therapeutic proteins in the intercostal muscles of mice injected with AAV9-shCK-opti-M6 and AAV9-hCK-B84 4 weeks after injection. Frozen sections of intercostal muscles from WT mice, Mdx mice, and mice from the two treatment groups were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 4H] Figure 4H shows the expression levels of the shCK-opti-M6 and hCK-B84 expression cassettes. Western blot analysis of (a) heart, (b) gastrocnemius muscle, and (c) diaphragm from WT mice, Mdx mice, and mice treated with AAV9-shCK-opti-M6 and AAV9-hCK-B84 for 4 weeks. GAPDH was used as an internal reference. [Figure 5]Figure 5 shows serum creatine kinase (CK) levels in male and female mice. (A) Serum CK levels in male WT mice, Mdx mice, and mice treated with AAV9-shCK-opti-M6 and AAV9-hCK-B84 for 8–9 weeks. (B) Serum CK levels in WT mice, Mdx mice, and female mice treated with AAV9-shCK-opti-M6 and AAV9-hCK-B84 for 8–9 weeks. n=4, *p<0.05, **p<0.01, ***p<0.001, t-test. [Figure 6] Figure 6 shows the improvement of muscle function and behavior in mice. (A) Rotarod performance test and (B) grip strength test in WT mice, Mdx mice, and mice treated with AAV9-shCK-opti-M6 and AAV9-hCK-B84 for 8 weeks. n=4, *p<0.05, **p<0.01, t-test. [Figure 7A] Figure 7A shows long-term expression of AAV9-shCK-opti-M6 in the mouse heart. Immunofluorescence analysis of the therapeutic protein was performed on the hearts of mice at different time points after injection (4, 6, 9, and 12 weeks after injection). Immunofluorescence staining was performed using antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 7B] Figure 7B shows long-term expression of AAV9-shCK-opti-M6 in the gastrocnemius muscle of mice. Immunofluorescence analysis of the therapeutic protein was performed in the gastrocnemius muscle of mice at different time points after injection (4, 6, 9, and 12 weeks after injection). Immunofluorescence staining with antibodies was performed, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 7C] Figure 7C shows long-term expression of AAV9-shCK-opti-M6 in the quadriceps muscle of mice. Immunofluorescence analysis of the therapeutic protein was performed in the quadriceps muscle of mice at different time points after injection (4, 6, 9, and 12 weeks after injection). Immunofluorescence staining was performed using antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 7D]Figure 7D shows long-term expression of AAV9-shCK-opti-M6 in the biceps muscle of mice. Immunofluorescence analysis of the therapeutic protein was performed in the biceps muscle of mice at different time points after injection (4, 6, 9, and 12 weeks after injection). Immunofluorescence staining with antibodies was performed, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 7E] Figure 7E shows long-term expression of AAV9-shCK-opti-M6 in the tibialis anterior muscle of mice. Immunofluorescence analysis of the therapeutic protein was performed in the tibialis anterior muscle of mice at different time points after injection (4, 6, 9, and 12 weeks after injection). Immunofluorescence staining with antibodies was performed, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 7F] Figure 7F shows long-term expression of AAV9-shCK-opti-M6 in the mouse diaphragm. Immunofluorescence analysis of the therapeutic protein was performed on the diaphragm of mice at different time points after injection (4, 6, 9, and 12 weeks after injection). Immunofluorescence staining was performed using antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 7G] Figure 7G shows long-term expression of AAV9-shCK-opti-M6 in the intercostal muscles of mice. Immunofluorescence analysis of the therapeutic protein was performed in the intercostal muscles of mice at different time points after injection (4, 6, 9, and 12 weeks after injection). Immunofluorescence staining was performed using antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 7H] Figure 7H shows long-term expression of AAV9-shCK-opti-M6 in mouse tongues. Immunofluorescence analysis of the therapeutic protein was performed on mouse tongues at different time points after injection (4, 6, 9, and 12 weeks after injection). Immunofluorescence staining with antibodies was performed, and cell nuclei were counterstained with DAPI. Scale bar: 200 μm. [Figure 7I]Figure 7I shows the long-term expression levels of the shCK-opti-M6 expression cassette in mice in vivo. Western blot analysis of (a) heart, (b) gastrocnemius, (c) quadriceps, (d) biceps, (e) tibialis anterior, (f) diaphragm, (g) intercostal muscle, and (h) tongue from mice treated with AAV9-shCK-opti-M6 for 4, 6, 9, and 12 weeks. GAPDH was used as an internal reference. [Figure 8] FIG. 8 shows the amino acid sequence of the recombinant hybrid protein (ie, the protein product of the M6 ​​and opti-M6 constructs) (SEQ ID NO: 4). [Figure 9-1] FIG. 9-1 shows the nucleotide sequence of the codon-optimized M6 (opti-M6) construct (SEQ ID NO: 8). [Figure 9-2] Figure 9-2 shows the nucleotide sequence of the codon-optimized M6 (opti-M6) construct (SEQ ID NO: 8). [Figure 10] FIG. 10 shows the nucleotide sequence of the shCK promoter (SEQ ID NO: 9). [Figure 11-1] FIG. 11-1 shows the nucleotide sequence of the M6 ​​construct without codon optimization (SEQ ID NO: 10). [Figure 11-2] FIG. 11-2 shows the nucleotide sequence of the M6 ​​construct without codon optimization (SEQ ID NO: 10). DETAILED DESCRIPTION OF THE INVENTION

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0033] Unless otherwise noted, nucleic acid or polynucleotide sequences are presented herein in single-stranded form, from left to right, in a 5' to 3' orientation. Nucleotides and amino acids presented herein are presented in the form recommended by the IUPACIUB Commission on Biochemical Nomenclature, or (for amino acids) by one-letter or three-letter code.

[0034] Unless otherwise specified, "polynucleotide" is synonymous with "nucleic acid" and refers to a polymer of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, hybrid sequences or analogs thereof. Polynucleotides can contain modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs.

[0035] As used herein, the terms "comprising," "having," "including," "containing," and the like are used in conjunction with open-ended phrases (including "including but not limited to").

[0036] As used herein, the terms "patient" and "subject" are used interchangeably and in their conventional sense to refer to an organism suffering from or capable of suffering from a condition that can be prevented or treated by administration of a pharmaceutical composition of the present disclosure, and include humans and non-human animals (e.g., rodents or other mammals).

[0037] In one embodiment, the subject is a non-human animal (e.g., chimpanzees and other ape and monkey species; livestock such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; poultry such as chickens, turkeys, and other geese, ducks, geese, and other wild and game birds). In one embodiment, the subject is a mammal. In one embodiment, the subject is a human.

[0038] As used herein, the terms "treat," "treating," and " "Treatment" (and grammatical variations thereof) includes: (1) inhibiting a condition, disease, or disorder, i.e., preventing, alleviating, or delaying the progression of a disease or its recurrence, or the manifestation of at least one clinical or subclinical symptom thereof; or (2) alleviating the disease, i.e., alleviating a state, disease, or condition, or at least one of them. At the same time, it results in the resolution of one clinical or subclinical symptom.

[0039] As used herein, the term "therapeutically effective amount" refers to a dose that produces a therapeutic effect when administered. For example, a therapeutically effective amount of a pharmaceutical agent suitable for treating muscular dystrophy is an amount sufficient to prevent or ameliorate one or more symptoms associated with muscular dystrophy. .

[0040] As used herein, the terms "improve," "improving," and "improvement" (and grammatical variations thereof) refer to the improvement of symptoms associated with a disease. and may refer to an improvement in at least one parameter that measures or quantifies a symptom. .

[0041] As used herein, "prevent" or "prevention" of a condition, disease, or disorder. The terms "preventing," "prevention," and "prevention" (and grammatical variations thereof) include preventing the development of at least one clinical or subclinical symptom of a condition, disease, or disorder in a subject who is suffering from or susceptible to the condition, disease, or disorder, but who has not yet experienced or exhibited a clinical or subclinical symptom of the condition, disease, or disorder. "Preventing, delaying, or reducing the incidence and / or likelihood of"

[0042] As used herein, the term "topical administration" or "topical route" refers to administration with a local effect.

[0043] As used herein, the terms "transduce," "transducing," "transduction," "transfect," "transfection," "transform," "transforming," and "transformation" (and grammatical variations thereof) refer to the process of delivering exogenous nucleic acid into a host cell followed by transcription and translation of the polynucleotide product. Such processes include the use of recombinant viruses to introduce exogenous polynucleotides into host cells.

[0044] As used herein, the term "gene delivery" (and grammatical variations thereof) refers to the introduction of an exogenous polynucleotide into a cell for gene transfer, and includes targeting, binding, uptake, transport, and replicon integration and expression.

[0045] As used herein, "gene expression" or "express" or "expressing" The terms "expressing" and "expression" (and grammatical variations thereof) refer to the processes of transcription, translation, and post-translational modification of a gene, resulting in the production of RNA or transcripts of that gene. Produces protein products.

[0046] As used herein, the terms "infect," "infecting," "infection" (and grammatical variations thereof) refer to the process by which a virus or viral particle containing a polynucleotide component delivers the polynucleotide into a cell and induces its RNA and protein production. It can also refer to the process of viral replication in a host cell.

[0047] As used herein, the term "targeting" (and grammatical variations thereof) means that the virus preferentially invades certain cells or tissues and then expresses sequences carried by the viral genome or recombinant transgene in those cells.

[0048] As used herein, the term "vector" refers to a vector that encapsulates a polynucleotide and A vector refers to a polymer or set of polymers that facilitate the delivery of a polynucleotide to a target cell, either in vitro or in vivo. Categories of vectors include, but are not limited to, plasmids, viral vectors, liposomes, and other gene delivery vectors. The polynucleotides delivered may also be referred to as "expression cassettes" or "gene transfer cassettes," but may include, but are not limited to, coding sequences for specific proteins or synthetic polypeptides that may enhance, inhibit, attenuate, protect against, induce, or prevent specific biological and physiological functions; coding sequences of interest in vaccine development (e.g., polynucleotides that express proteins, polypeptides, or peptides suitable for eliciting an immune response in mammals); coding sequences for RNAi materials (e.g., shRNA, siRNA, antisense oligonucleotides); or any biomarker.

[0049] As used herein, the terms "expression cassette," "transgene cassette," and "transgene expression cassette" are used interchangeably and refer to a polynucleotide fragment encoding a particular protein, polypeptide, or RNAi element that can be cloned into a plasmid vector.

[0050] In one embodiment, the "cassette" is packaged into an AAV particle and the gene is delivered to a target cell. It can also be used as a viral genome to deliver the transgene. The "cassette" can also contain other regulatory elements, such as specific promoters / enhancers, polyA, regulatory introns, etc., to enhance or reduce the expression of the transgene.

[0051] In one embodiment, in addition to the sequence encoding the protein product, the gene transfer cassette further comprises a number of regulatory elements to allow packaging of the transgene into the virus, such as 145 bp of regular ITRs, truncated ITRs of approximately 100 bp in length, etc. In some embodiments, the gene transfer cassette further comprises polynucleotide elements for controlling the expression of protein products, such as an origin of replication, a polyadenylation signal, an internal ribosome entry site (IRES), or a 2A signal (e.g., P2A, T2A, F2A), a promoter and enhancer, for example, the CMV promoter or other hybrid CMV promoters with vertebrate β-actin, β-globin, or β-globin regulatory elements (referred to as the CB and CAG promoters), or promoters of muscle-specific genes, including the muscle creatine kinase (MCK) promoter, human creatine kinase (hCK) promoter, truncated creatine kinase (shCK) promoter, skeletal α-actin promoter, cardiac α-actin promoter, myosin heavy chain (MyHC) promoter, myosin light chain 2 (MLC2) promoter, myosin light chain 3F promoter, desmin gene promoter, and promoters of the myogenic regulatory factor family (MyoG, Myf5, Mrf4, and Myogenin). Promoters and enhancers can be activated by chemicals or hormones (such as doxycycline or tamoxifen) to ensure gene expression at specific times. Furthermore, promoters and enhancers can be natural, artificial, or chimeric sequences, i.e., prokaryotic or eukaryotic.

[0052] In some preferred embodiments, the inducible regulatory element for gene expression is a tissue- or organ-specific promoter or enhancer, and may be, but is not limited to, promoters specific to various types of muscle cells, such as the muscle creatine kinase (MCK) promoter, human creatine kinase (hCK) promoter, truncated human creatine kinase (shCK) promoter, skeletal α-actin promoter, cardiac α-actin promoter, myosin heavy chain (MyHC) promoter, myosin light chain 2 (MLC2) promoter, myosin light chain 3F promoter, promoters of muscle-specific genes including the desmin gene promoter, and promoters of the myogenic regulatory factor family (MyoG, Myf5, Mrf4, Myogenin); and osteoblast lineage-specific promoters (such as the osteocalcin promoter).

[0053] As used herein, the term "inverted terminal repeat (ITR)" refers to a repeat that forms a hairpin structure. ITRs, as used herein, include any AAV viral terminal repeat or synthetic sequence that functions as a cis element that mediates viral replication, packaging, and integration. These include, but are not limited to, terminal repeats of types 1 to 12 (avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV terminal repeats). It is not necessary to have the native terminal repeat sequence, as long as the repeat is functional for viral replication, packaging and integration.

[0054] As used herein, the term "cis element" refers to a cis-like molecule that is packaged into an AAV particle and that It refers to a gene transfer cassette that is expressed in target cells to produce a protein product that has a therapeutic effect.

[0055] As shown in Figure 1A, full-length human dystrophin has four structural domains: an N-terminal domain, a central rod domain containing 24 spectrin-like repeats (R1-R24) and four hinges (H1-H4), a CR domain, and a C-terminal (CT) domain. Proteins also have four domains: an N-terminal domain, a central rod domain containing 22 spectrin-like repeats (R1-R22) and four hinges (H1-H4), a CR domain, and a C-terminal (CT) domain. These specific amino acid sequences are known in the art and can be found in the literature or in public databases such as the UniProt protein database.

[0056] The M6 ​​constructs of this disclosure come in two forms: non-codon optimized M6 constructs and codon optimized M6 constructs. This is the optimized M6 (opti-M6) construct.

[0057] As used herein, "codon optimization" or "codon optimal The term "codon-optimized" refers to a polynucleotide that has been modified from its native form. Such modifications may be made without or with the corresponding change in amino acid sequence. The modified polynucleotide sequence may result in a difference of one or more base pairs, which may enhance or inhibit gene expression and / or cellular response to the modified polynucleotide sequence.

[0058] In one embodiment, the AAV capsid protein can be an AAV capsid protein of any serotype, including native AAV capsid proteins (e.g., capsid proteins of AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV), and artificial AAV capsid proteins (e.g., artificial capsid proteins of AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV). The genomic sequences, ITR sequences, Rep and Cap proteins of various AAV serotypes are known in the art. These sequences can be found in the literature or public databases such as GenBank and / or WO 2021050970 A1; US ​​2019 / 036676 A1; Choudhury SR et al., InVivo Selection Yields AAV-B1 Capsid for Central Nervous System and Muscle GeneTherapy. Mol Ther., 2016, 24(7): 1247-57; Hsu HL. et al., Structural characterization of a novel human adeno-associated viral capsid with neurotropic properties. Nat Commun 11, 3279 (2020).

[0059] In one embodiment, the present disclosure provides a therapeutic tool for improving muscle function, which can be used to treat a variety of diseases with associated pathological mechanisms, including, but not limited to, Duchenne muscular dystrophy, Baker muscular dystrophy, and other muscle degenerative diseases.

[0060] In one embodiment, clinical symptoms of the muscle degenerative disease include muscle atrophy and / or a decrease or loss of exercise capacity.

[0061] In one embodiment, the protein product of the therapeutic tool (e.g., transgenic expression cassette) includes, but is not limited to, proteins for improving muscle function, such as dystrophin (full-length or truncated), utrophin (full-length or truncated), and hybrid combinations of both.

[0062] In one embodiment, the therapeutic tool is a protein such as, but not limited to, dystrophin (full length or truncated), utrophin (full length or truncated), and recombinant hybrid proteins of both.

[0063] In one embodiment, the transgenic expression cassette of the present disclosure is an hCK-opti-M6 expression cassette. The hCK promoter sequence (SEQ ID NO:1), minipolynucleotides (SEQ ID NO:3), and Denylation (polyA) sequence (SEQ ID NO:2), and the codon-optimized M6 (opti-M6) construct (SEQ The hCK-opti-M6 expression cassette is flanked by 145 bp of conventional ITRs. , which allows the expression cassette to be packaged into AAV particles as a single-stranded AAV vector.

[0064] In one embodiment, the transgenic expression cassette of the present disclosure comprises an hCK promoter sequence (SEQ ID NO:1), a mini-polyadenylation sequence, and a nucleotide sequence (SEQ ID NO:2) to form the hCK-M6 expression cassette (SEQ ID NO:5). The M6 ​​construct contains a codon-optimized (polyA) sequence (SEQ ID NO:2), and a non-codon-optimized M6 construct (SEQ ID NO:10). The hCK-M6 expression cassette is flanked by 145 bp of normal ITRs, which allow the expression cassette to be packaged into AAV particles as a single-stranded AAV vector.

[0065] In one embodiment, the transgenic expression cassette of the present disclosure comprises an shCK promoter sequence (SEQ ID NO:9), a mini-polyadenylation (polyA) sequence (SEQ ID NO:2), and a codon-optimized M6 (opti-M6) construct (SEQ ID NO:3) to form the shCK-opti-M6 expression cassette (SEQ ID NO:6). The shCK-opti-M6 expression cassette is flanked by 145 bp of conventional ITRs, which allow the expression cassette to be packaged into AAV particles as a single-stranded AAV vector.

[0066] In one embodiment, the transgenic expression cassette of the present disclosure is a shCK-M6 expression cassette (SEQ ID NO: 1). The shCK promoter sequence (SEQ ID NO: 9), minipolyadenylation sequence (SEQ ID NO: 7), and The shCK-M6 expression cassette contains a 145-bp ITR sequence, which allows the expression cassette to be packaged into AAV particles as a single-stranded AAV vector.

[0067] In some embodiments, AAV particles of dystrophin and dystrophin-associated proteins are expressed in HEK293 cells using three plasmids (plasmid 1: cis-element plasmid; Plasmid 2: AAV Rep / Cap plasmid; Plasmid 3: helper plasmid) produced by transfection.

[0068] In one embodiment, to produce therapeutic AAV particles, a three-plasmid transfection of HEK293 cells is performed as follows: Plasmid 1: a cis-element plasmid carrying ITRs (e.g., hCK-opti-M6, hCK-M6, shCK-opti-M6, and shCK-M6 expression cassettes); Plasmid 2: a plasmid carrying the coding sequence for a capsid protein (e.g., AAV9 capsid protein). Plasmid 3: a helper plasmid carrying adenoviral components capable of promoting replication, assembly, and packaging of AAV virions. In one embodiment, AAV particles produced by HEK293 cells are purified by cesium chloride (CsCl) density gradient centrifugation (e.g., as described in Example 2 of this disclosure).

[0069] Those skilled in the art will appreciate the techniques for producing recombinant and synthetic polypeptides or proteins, designing nucleic acid sequences, producing transformed cells, constructing recombinant AAV variants, modifying capsid proteins, and packaging vectors expressing AAV Rep and / or Cap sequences, and transiently growing packaging cells. For either transient or stable transfection, known standard methods can be used. These techniques are known to those skilled in the art. See, for example, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (Cold Spring Harbor, NY, 1989).

[0070] In some embodiments, the gene delivery system of the present disclosure is used in adjunctive cell transplantation therapy. AAV particles can be used to transduce various cell types in vitro and produce protein products. Stable cell lines expressing the α-glucanase inhibitor can be produced and introduced into the body for therapeutic purposes in a variety of cell types, including, but not limited to, endothelial cells, myoblasts, fibroblasts, astrocytes, Müller cells, oligodendrocytes, microglia, rods and cones, neurons, hematopoietic stem cells, monocytes, granulocytes, lymphocytes, osteoclasts, and macrophages.

[0071] In one embodiment, the cells used for transplantation are autologous to the subject and can be cultured in vitro. The principles and techniques for introducing or transplanting cells into a subject are known to those skilled in the art.

[0072] In one embodiment, AAV particles are harvested from the culture medium and lysates of HEK293 cells. The preparation methods include affinity chromatography, ion exchange chromatography, and chloride ion chromatography. These include cesium and iodixanol gradient ultracentrifugation. Related chemicals or reagents include, but are not limited to, chemicals or reagents used in cell culture (e.g., vertebrate serum such as bovine, horse, goat, or chicken serum, components of cell culture media such as glutamine, glucose, sucrose, sodium pyruvate, or phenol red, or antibiotics such as penicillin, kanamycin, streptomycin, or tetracycline), chemicals or reagents used in cell lysis, polynucleotide precipitation, or ultracentrifugation (e.g., Triton X-100, NP-40, sodium deoxycholate, sodium lauryl sulfate, domiphen bromide, dodecyl dodecyl phosphate), or the like). Sodium salicylate, sodium chloride, magnesium chloride, calcium chloride, barium chloride, nitrate, potassium chloride, ammonium chloride, ammonium persulfate, ammonium sulfate, PEG-20, PEG-40, PEG-400, PEG-2000, PEG-6000, PEG-8000, PEG-20000, Tris-HCl, Tris-acetate, manganese chloride, phosphate, bicarbonate, cesium chloride, methanol, ethanol alcohol, glycerin, iodixanol, isopropyl alcohol, butanol, benzonase, DNase I, RNase; affinity column materials (e.g., AAVX affinity resin, heparan sulfate proteoglycan and mucin resins, and other materials associated with AAV-specific antibodies); ion exchange chromatography materials and Acids, bases, and organic substances in the wash buffers (e.g., hydrochloric acid, sulfuric acid, acetic acid, formic acid, nitric acid, urea, acetone, chloroform, acetonitrile, trifluoroacetic acid, sodium hydroxide, potassium hydroxide, barium hydroxide, ammonium hydroxide, Tris base or other organic amines, poloxamer 188, Tween 20, Tween 40, Tween 80, guanidine hydrochloride).

[0073] In one embodiment, the exogenous polynucleoside delivered to the target cell by the AAV vector The tide encodes a therapeutic native protein, which may or may not be codon optimized.

[0074] In one embodiment, the exogenous polynucleoside delivered to the target cell by the AAV vector The tide encodes a synthetic polypeptide.

[0075] In one embodiment, the transgenic expression cassette or gene delivery system of the present disclosure is formulated into a pharmaceutical composition (e.g., an injectable solution, tablet, capsule, powder, eye drop) for administration to a human or other mammal. The pharmaceutical composition may also contain other components, such as pharmaceutical excipients, aqueous or organic solvents (such as water, glycerol, ethanol, methanol, isopropyl alcohol, chloroform, phenol, or polyethylene glycol), salts (such as sodium chloride, potassium chloride, phosphates, acetates, bicarbonates, Tris-hydrochlorides and Tris-acetates), agents for retarding dissolution (such as paraffin), surfactants, antibacterial agents, liposomes, lipoplexes, immunosuppressants (e.g., cortisone, prednisone, cyclosporine), nonsteroidal anti-inflammatory drugs (NSAIDs, e.g., aspirin, ibuprofen, acetaminophen), microspheres, rigid matrices, semi-solid carriers, nanospheres, or nanoparticles. Additionally, pharmaceutical compositions can be delivered in single or multiple doses by inhalation, systemic or local (e.g., intravenous, subcutaneous, intraocular, subretinal, suprachoroidal, parenteral, intramuscular, intracerebroventricular, oral, intraperitoneal, and intrathecal) administration.

[0076] In one embodiment, the present disclosure provides a pharmaceutical composition comprising a recombinant hybrid protein, nucleic acid molecule, transgenic expression cassette, or gene delivery system of the present disclosure; and excipient(s). The pharmaceutical composition of the present disclosure can be used to transduce cells in vitro or to transduce mammals (such as rodents, primates, and humans) in vivo, and can treat various diseases associated with muscle defects, such as muscular dystrophies, including Duchenne muscular dystrophy, Becker muscular dystrophy, and other muscle degenerative diseases. In one embodiment, the muscular dystrophy is Duchenne muscular dystrophy.

[0077] In one embodiment, treatment of muscular dystrophy refers to an improvement in the degree of muscle damage, creatine kinase levels, and NASS (North American Spine Society) muscle function scores in patients receiving treatment.

[0078] The present disclosure will be described in more detail below with reference to the accompanying figures and examples. The following examples are only used to explain the present disclosure and are not intended to limit the scope of the present disclosure. Experimental methods that do not specify specific conditions in the examples were carried out according to conventional conditions known in the art or conditions recommended by manufacturers. [Example]

[0079] Example 1: Design and construction of M6 and opti-M6 constructs and AAV vectors The B84 (mini-dystrophin) construct (SEQ ID NO: 11) was obtained by PCR cloning using human dystrophin cDNA as a template. The mini-human dystrophin protein is described, for example, in CN109641944A (designated Hopti-Dys3978 and Dys3978) or in Wang B, Li J, Xiao X. Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model [J]. Proc Natl Acad Sci USA, 2000, 97(25):13714-9.

[0080] The inventors also demonstrated, through extensive experimental studies and rational design, the highly truncated M6 construct (SEQ ID NO: 1) based on full-length human dystrophin and full-length human dystrophin-associated protein. The M6 ​​construct was codon-optimized to obtain the opti-M6 construct (SEQ ID NO: 8). As shown in Figure 1A, the recombinant hybrids encoded by the M6 ​​construct or the opti-M6 construct were The lid protein is a ligated protein that binds to the N-terminal region of the full-length human dystrophin-associated protein (utrophin). the terminal domain, hinge H1, spectrin-like repeat R1, spectrin-like repeat R2, spectrin-like repeat R3 and the first half of hinge H2; and the spectrin-like repeat R23, spectrin-like repeat R24, hinge H4 and CR domain of full-length human dystrophin.

[0081] Furthermore, to enhance the expression of target proteins, the present inventors further constructed a truncated human creatine kinase CK (shCK) promoter based on the human creatine kinase hCK promoter (SEQ ID NO: 1).

[0082] Next, the above M6 and opti-M6 genes were subcloned into AAV vector plasmids using the hCK promoter and shCK promoter, respectively, to generate AAV-hCK-M6-polyA, AAV-hCK-opti-M6-polyA, AAV-shCK-M6-polyA, and AAV-shCK-opti-M6-polyA vectors. Similarly, the B84 gene was cloned into an AAV vector plasmid containing the hCK promoter and minipolyA signal sequence to generate the AAV-hCK-B84 vector.

[0083] Example 2: Expression of M6 and opti-M6 constructs in Mdx mice To study the expression of M6 and opti-M6 constructs in mice, four treatment groups were established in this example: hCK-opti-M6, hCK-M6, shCK-opti-M6, and shCK-M6.

[0084] AAV-hCK-M6-polyA, AAV-hCK-opti-M6-polyA, AAV-shCK-M6-polyA, and The AAV-shCK-opti-M6-polyA vector was packaged into AAV9 viral particles. The three-plasmid cotransfection method (e.g., Xiao X, Li J, Samulski R J. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus [J]. J Virol, 1998, 72(3):2224-32) was used to transfect the four new systems described above. The transfection was performed using three plasmids: the element plasmid vector, the AAV9 capsid plasmid, and the adenovirus helper plasmid. HEK293 cells were transfected for 40 to 80 h. After transfection, the cells were lysed to remove the polynucleotides. AAV viral vectors were purified twice by CsCl density gradient ultracentrifugation according to the protocol described in [J], 1996. The viral titer was determined by qPCR and was approximately 2 × 10 13 ~1×10 14 The purity of the capsid protein was determined by SDS-PAGE. The presence of endotoxin was determined by gel electrophoresis and the results were satisfactory.

[0085] Then, add 3 x 10 AAV particles to 13 vg / kg was injected into Mdx mice via the tail vein. WT mice Age-matched littermates of Mdx mice were used as controls. Six weeks after injection, the mice were sacrificed. Heart, gastrocnemius, biceps, and diaphragm tissues were collected for frozen sectioning and immunofluorescence staining. Rabbit polyclonal antibodies used for immunofluorescence staining were purchased from Abcam (Cambridge, MA). This antibody recognizes the amino acid fragment between positions 2800 and 3000 of human dystrophin (spectrin-like repeats R23 and R24). Therefore, both the B84 and M6 constructs were able to recognize the amino acid fragment. The protein expressed by the antibody can be recognized by this antibody.

[0086] Immunofluorescent staining of the heart, gastrocnemius, biceps brachii, and diaphragm tissues revealed the Mdx mammary gland. This can reflect improvements in muscle pathology related to cardiac function, lower limb motor function, upper limb motor function, and respiratory function in mice. The pathological phenotype in Mdx mice began at approximately 3 weeks of age and was accompanied by myofiber degeneration and regeneration. The characteristic and primary pathological manifestation of this process was the central location of nuclei in myofibers.

[0087] As a result, in the hearts of Mdx mice 6 weeks after administration, the protein levels in the four treatment groups were The expression levels were significantly higher than those in the untreated Mdx control group. The expression levels of the hCK-opti-M6 and shCK-opti-M6 expression cassettes were significantly higher than those of the hCK-M6 and shCK-M6 expression cassettes (Figure 2A). Expression of the hybrid therapeutic protein was also observed in the gastrocnemius muscle of treated mice, and expression of opti-M6 was also observed. The expression levels were significantly higher than those of the unoptimized M6 (Figure 2B). In the biceps brachii muscle, the protein expression levels of each of the four treatment groups were significantly higher than those of the untreated Mdx control group. shCK-opti-M6 was significantly better than the other three treatment groups and was significantly higher than that of the WT mouse. The expression level of NF-κB1 was close to that of NF-κB1, and centrifugation of nuclei was observed in the skeletal muscle of Mdx mice. The expression in the diaphragm was similar to that in the biceps brachii muscle, and shCK-opti-M6 was expressed broadly in the visual field, superior to the other three expression cassettes (Figure 2D).

[0088] These results demonstrate that the M6 ​​and opti-M6 constructs can broadly express hybrid therapeutic proteins in four representative muscle tissues, including the heart and gastrocnemius, and exert therapeutic effects. The optimized opti-M6 construct achieved higher expression levels than the M6 ​​construct. Furthermore, higher protein expression levels were achieved using shCK compared with hCK. In summary, the shCK-opti-M6 expression cassette exhibited the highest in vivo activity.

[0089] Example 3: Expression of Opti-M6 and B84 constructs in Mdx mice In this example, we compared the expression levels of the opti-M6 and B84 constructs in a mouse model. The virus was then packaged into AAV9-hCK-opti-M6 and AAV9-hCK-B84 viral particles. Mdx mice were injected with 3 × 10 viral particles. 13The treatment was carried out by tail vein injection at a dose of 0.05 mg / kg. WT mice and age-matched littermates of Mdx mice served as controls. Six weeks after treatment, the mice were dissected and tissues, including the heart, gastrocnemius muscle, and diaphragm, were collected. The expression of the therapeutic protein in each tissue was detected by tissue immunofluorescence.

[0090] The results showed that the expression levels of therapeutic proteins in the heart, gastrocnemius muscle, and diaphragm of mice treated with AAV9-hCK-opti-M6 and AAV9-hCK-B84 were significantly higher than those of untreated mdx controls. (Figure 3).

[0091] As shown in Figure 3, in the heart, the AAV9-hCK-opti-M6 treatment group showed similar results to the AAV9-hCK-B84 treatment group. The AAV9-hCK-opti-M6 treatment group showed significantly stronger fluorescence than the AAV9-hCK-B84 treatment group in the gastrocnemius muscle, and the AAV9-hCK-opti-M6 treatment group showed significantly stronger fluorescence and a significantly higher number of positive cells than the AAV9-hCK-B84 treatment group in the diaphragm.

[0092] In summary, the opti-M6 construct exhibited superior therapeutic efficacy compared to the B84 construct.

[0093] Example 4: Expression of shCK-opti-M6 and hCK-B84 vectors in Mdx mice In this example, the present inventors compared the expression ability of the shCK-opti-M6 vector and the hCK-B84 vector in a mouse model.

[0094] The AAV-shCK-opti-M6 and AAV-hCK-B84 vectors were packaged separately into AAV9-shCK-opti-M6 and AAV9-hCK-B84 viral particles as described in Example 2. Mdx mice were infected with 3 × 10 viral particles. 13 The mice were injected via the tail vein at a dose of 0.05 mg / kg. WT mice and age-matched littermates of Mdx mice served as controls. After 4 weeks of treatment, the mice were dissected and the heart, gastrocnemius, quadriceps, and diaphragm were examined. These tissues were then collected and protein expression in each tissue was measured using immunofluorescence and Western blotting.

[0095] As shown in Figure 4A, in the hearts of mice treated with AAV9-shCK-opti-M6 and AAV9-hCK-B84 The expression level of the therapeutic protein in the AAV9-shCK-opti-M6 cells was significantly higher than that in the untreated Mdx control group, and AAV9-shCK-opti-M6 cells showed stronger fluorescence.

[0096] As shown in Figures 4B–4E, after treatment with AAV9-shCK-opti-M6 and AAV9-hCK-B84, limb muscles Widespread expression of the therapeutic protein was also observed in tissues (gastrocnemius, tibialis anterior, biceps brachii, and quadriceps femoris). The expression level in limb muscle tissues of the shCK-opti-M6 treatment group was significantly higher than that of the hCK-B84 treatment group. The expression levels were significantly higher than those in the shCK-opti-M6-treated group. In mice treated with shCK-opti-M6, we observed peripheral nucleation of muscle fibers in areas where the protein was widely expressed, indicating that the pathological phenotype of central nucleation was corrected to some extent, preventing muscle degeneration and regeneration, and that treatment with the vector of the present invention can ameliorate the pathological phenotype of muscle.

[0097] As shown in Figures 4F–4H, in the diaphragm and intercostal muscle tissues related to respiratory function, widespread expression of the therapeutic protein was observed in both the shCK-opti-M6 and hCK-B84 treatment groups, and the fluorescence intensity of both groups was significantly higher. No significant differences in strength were observed.

[0098] In vivo protein expression of shCK-opti-M6 and hCK-B84 was compared with that of human dystrophin. The R23 and R24 fragments of the torin-like repeats were simultaneously detected using an antibody that recognizes them. Western blot analysis revealed that the expression level of shCK-opti-M6 in the mouse heart was significantly higher than that of hCK-B84. The expression levels of shCK-opti-M6 in the gastrocnemius muscle and diaphragm were higher than those of hCK-B84. It was close to the bell (Fig. 4G).

[0099] Taking these results together, shCK-opti-M6 showed significantly higher expression than hCK-B84. It turns out to be profitable.

[0100] Example 5: Creatinine in Mdx mice treated with shCK-opti-M6 and hCK-B84 vectors kinase levels In this example, to verify the therapeutic effects of shCK-opti-M6 and hCK-B84 vectors in Mdx mice, the inventors measured serum creatine nitrate, an important indicator of muscle damage in Mdx mice. Kinase (CK) levels were detected.

[0101] In this example, two treatment arms were established: AAV9-shCK-opti-M6 and AAV9-hCK-B84. The mouse is 3 x 10 13 The mice were injected into the tail vein at a dose of 0.05 mg / kg. Mdx mice of the same age as the WT mice were used as controls. Each group consisted of 8 mice: 4 males and 4 females. After 8-9 weeks of treatment, serum was measured by retro-orbital blood collection. were collected and the CK levels in the serum of the mice were detected.

[0102] As shown in Figure 5, serum CK levels in untreated Mdx mice were significantly higher than those in 4-month-old WT mice. Serum CK levels were significantly higher in Mdx mice treated with AAV than in those treated with shCK-opti-M6 (males, **p<0.01; females, ***p<0.001), whereas serum CK levels were significantly reduced in Mdx mice treated with AAV and in those treated with shCK-opti-M6 (males, **p<0.05; females, ***p<0.001).

[0103] The creatine kinase levels in Mdx mice treated with the shCK-opti-M6 vector were lower than those in Mdx mice treated with the hCK-B84 vector, and the shCK-opti-M6 vector It can be seen that the tar has a better therapeutic effect.

[0104] Example 6: shCK-opti-M6 and hCK-B84 vectors on motor performance and muscle strength in Mdx mice Effect In this example, to evaluate the effects of shCK-opti-M6 and hCK-B84 vectors on motor performance and muscle strength in Mdx mice, mice were subjected to a rotarod test (motor exercise) and a grip strength test (muscle strength).

[0105] The rotarod test was performed three times in WT mice, untreated Mdx mice, and Mdx mice treated with shCK-opti-M6 and hCK-B84 for 8 weeks (n = 4). The results are shown in Figure 6A. There was a significant difference in the duration of the rotarod between Mdx and WT mice (*p<0.05). Compared with untreated Mdx mice, the motor performance of Mdx mice treated with the shCK-opti-M6 vector was significantly improved. Improved (*p<0.05), and there was no statistical difference between Mdx mice treated with hCK-B84 vector In particular, Mdx mice treated with the shCK-opti-M6 vector exhibited motor duration similar to that of WT mice. showed.

[0106] On the other hand, the results of the mouse grip strength test showed that the limbs of Mdx mice treated with the shCK-opti-M6 vector showed Grip strength was significantly improved compared to untreated Mdx mice (**p<0.01), reaching the level of WT mice. This indicates that the temperature approaches 100°C (Figure 6B).

[0107] These results suggest that shCK-opti-M6 vector significantly improves myocardial function in mice compared with hCK-B84 vector. It has been shown to be highly effective in improving cognitive and behavioral performance.

[0108] Example 7: Long-term expression of shCK-opti-M6 vector in Mdx mice In this example, we demonstrate the long-term expression of the shCK-opti-M6 vector in Mdx mice. Four groups of Mdx mice were treated with 3 × 10 13 The vector was injected into the tail vein at a dose of 1000 mg / kg. Muscle samples were collected at different time points (4, 6, 9, and 12 weeks) and analyzed by immunofluorescence and Western blot. The expression was detected by immunoblotting. Mdx mice of the same age and litter size as WT mice were used as controls.

[0109] As a result, after 4 weeks of treatment with the shCK-opti-M6 vector, protein expression was observed in the heart. Expression peaked 6–9 weeks after injection, and the number of positive cells and fluorescence intensity significantly increased (Figure 7A). Six weeks after shCK-opti-M6 vector treatment, widespread protein expression was observed in the gastrocnemius muscle of mdx mice (Figure 7B). Four weeks after shCK-opti-M6 vector treatment, weak fluorescence intensity was observed in the quadriceps muscle, which peaked 6–9 weeks after injection and remained stable for up to 12 weeks after injection (Figure 7C). The biceps and quadriceps muscles showed similar immunofluorescence staining results (Figure 7D). In the tibialis anterior muscle of mdx mice, high levels of protein expression were already observed 4 weeks after injection, which further increased 6–9 weeks after injection and remained stable for up to 12 weeks after injection (Figure 7E). The diaphragm and tibialis anterior muscle showed similar immunofluorescence staining results (Figure 7F). Immunofluorescence staining of the intercostal muscles showed that high levels of protein expression were detected from 4 to 9 weeks after shCK-opti-M6 vector treatment, with a slight decrease in expression at 12 weeks after injection (Figure 7G). The tongue and intercostal muscles also showed similar immunofluorescence results (Figure 7H).

[0110] To detect protein expression, the spectrin-like repeats R23 and R24 of human dystrophin were used. The results of Western blot analysis using antibodies that recognize the R24 and R25 fragments are shown in Figure 7I. In the liver, protein expression was detected from 4 weeks after injection, with the highest expression level at 6-9 weeks. In the gastrocnemius muscle, high expression levels were observed 6 weeks after injection, and persisted for up to 12 weeks after injection. In the quadriceps muscle, protein expression peaked at 9 weeks after injection. In the biceps brachii muscle, the protein expression level was highest at 6 weeks after injection, and a relatively high level of expression was still detected at 12 weeks after injection. In the tibialis anterior muscle, a constant level of protein expression was detected 4 weeks after injection, and a high level of protein expression was maintained 6 to 9 weeks after injection. In the diaphragm, protein expression was significantly higher than that in the control group. Western blot analysis of the intercostal muscles showed similar results to those of the tongue: different levels of protein expression were detected between 4 and 9 weeks after injection, and the expression level decreased at 12 weeks after injection.

[0111] The above immunofluorescence and Western blot results show that shCK-opti-M6 inhibits the mitochondrial function in Mdx model mice. This indicates that long-term stable expression can be achieved.

[0112] Although the present disclosure has been illustrated and described with reference to the accompanying figures and preferred embodiments of the present disclosure, those skilled in the art should understand that the above is a further detailed description of the present disclosure in connection with specific embodiments, and that the present invention should not be construed as being limited to the embodiments described herein. Those skilled in the art may make various changes in both form and details, including making some simple inferences or substitutions, without departing from the spirit and scope of the present disclosure.

Claims

1. A recombinant hybrid protein comprising: the N-terminal domain, hinge H1, spectrin-like repeat R1, spectrin-like repeat R2, spectrin-like repeat R3, and the first half of hinge H2 of full-length human utrophin; and A recombinant hybrid protein comprising the spectrin-like repeat R23, spectrin-like repeat R24, hinge H4, and CR domain of full-length human dystrophin, wherein the recombinant hybrid protein comprises the amino acid sequence shown in SEQ ID NO:

4.

2. 2. The recombinant hybrid protein of claim 1, wherein the recombinant hybrid protein consists of the amino acid sequence shown in SEQ ID NO:

4.

3. A nucleic acid molecule encoding the recombinant hybrid protein of claim 1.

4. 4. The nucleic acid molecule of claim 3, wherein the nucleic acid molecule has a nucleotide sequence that is at least 90%, 95%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO:

10.

5. The nucleic acid molecule of claim 4, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO:

10.

6. The nucleic acid molecule of claim 4, wherein the nucleic acid molecule consists of the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO:

10.

7. 10. A transgenic expression cassette comprising a promoter, the nucleic acid molecule of claim 3, and a mini-polyA.

8. 8. The gene transfer expression cassette of claim 7, wherein the promoter is a promoter of a muscle-specific gene.

9. 8. The gene expression cassette of claim 7, wherein the promoter is selected from the following: CB promoter, CAG promoter, muscle creatine kinase (MCK) promoter, human creatine kinase (hCK) promoter, truncated human creatine kinase (shCK) promoter, skeletal α-actin promoter, cardiac α-actin promoter, myosin heavy chain (MyHC) promoter, myosin light chain 2 (MLC2) promoter, myosin light chain 3F promoter, desmin gene promoter, and promoters of the myogenic regulatory factor family (MyoG, Myf5, Mrf4, Myogenin).

10. 8. The gene transfer expression cassette of claim 7, wherein the promoter has the nucleotide sequence shown in SEQ ID NO:

9.

11. 8. The gene transfer expression cassette of claim 7, further comprising two regulatory elements located at both ends thereof.

12. 12. The gene transfer expression cassette of claim 11, wherein the two regulatory elements are each either a normal ITR or a truncated ITR.

13. 12. The gene transfer expression cassette of claim 11, wherein the two regulatory elements are each a 145 bp conventional ITR.

14. 8. The gene transfer expression cassette of claim 7, further comprising an origin of replication, a polyadenylation signal, an internal ribosome entry site (IRES), and / or a 2A signal.

15. 15. The gene transfer expression cassette of claim 14, wherein the 2A signal is P2A, T2A or F2A.

16. 8. The gene transfer expression cassette of claim 7, wherein the gene transfer expression cassette consists of the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:

7.

17. A gene delivery system comprising the gene transfer expression cassette of claim 7 and an AAV capsid protein.

18. 18. The gene delivery system of claim 17, wherein the AAV capsid protein is a natural AAV capsid protein or an engineered AAV capsid protein.

19. The gene delivery system of claim 18, wherein the AAV is selected from the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, AAVrh10, AAVrh39, AAVrh43, AAV32.33, AAV3B, AAVv66, AAVXL32, and AAV.PHP.B.

20. Use of the gene transfer expression cassette of any one of claims 7 to 16 or the gene delivery system of any one of claims 17 to 19 in the preparation of a pharmaceutical composition for treating muscular dystrophy.

21. 21. The use of claim 20, wherein the muscular dystrophies include Duchenne muscular dystrophy, Becker muscular dystrophy, and other muscle degenerative diseases.

22. 21. The use according to claim 20, wherein the muscular dystrophy is Duchenne muscular dystrophy.

23. 20. A pharmaceutical composition comprising any one selected from the group consisting of the recombinant hybrid protein of claim 1 or 2, the nucleic acid molecule of any one of claims 3 to 6, the gene transfer expression cassette of any one of claims 7 to 16, and the gene delivery system of any one of claims 17 to 19; and an excipient.

24. 24. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition is administered via a systemic or local route.

25. 25. The pharmaceutical composition of claim 24, wherein the pharmaceutical composition is administered intravenously, intramuscularly, subcutaneously, orally, topically, intraperitoneally, or intrarectally.

26. 25. The pharmaceutical composition of claim 24, wherein the pharmaceutical composition is administered by biceps brachii or gastrocnemius injection.

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