WO2023124741A1 - 用于治疗肌营养不良症的转基因表达盒 - Google Patents
用于治疗肌营养不良症的转基因表达盒 Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
- C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
Definitions
- the present disclosure relates to a recombinant hybrid protein for treating muscular dystrophy, a nucleic acid molecule encoding the recombinant hybrid protein, a transgene expression cassette comprising the nucleic acid molecule, and a gene delivery system comprising the transgene expression cassette.
- Muscular dystrophy is a wide range of muscle degenerative diseases, mainly manifested as muscle function defects in different parts, such as progressive muscle atrophy and weakness in different degrees.
- DMD Duchenne muscular dystrophy
- Duchenne muscular dystrophy is the most common and progresses most rapidly.
- Duchenne muscular dystrophy is a severe X-linked recessively inherited neuromuscular disorder. Due to the lack of dystrophin (Dystrophin), DMD patients will gradually degenerate skeletal muscle, heart and respiratory muscles. According to statistics, about 1 in every 3600 boys in the world suffers from this disease.
- X-linked recessive muscular dystrophies also include Becker muscular dystrophy (Becker muscular dystrophy, BMD), which is characterized by a decrease in the quantity or quality of dystrophin protein in muscle biopsy samples.
- BMD Becker muscular dystrophy
- the dystrophin gene is the largest known human gene, approximately 2.5 Mb, and is mainly expressed in skeletal and cardiac muscle. The gene is located at the Xp21 position of the X chromosome and contains 79 exons. DMD is caused by mutations in the dystrophin gene. The most common types of mutations in DMD include large deletions (60-70%) of one or more exons, duplications (5-10%), single-nucleotide variations (including small deletions or insertions, single-base base changes and cut site changes). The basic function of dystrophin is to bind to F-actin through the N-terminal domain and to bind to ⁇ -dystroglycan through the C-terminal domain during contraction, acting as a bridging and anchoring protein.
- dystrophin function to stabilize muscle fibers. Loss of dystrophin expression results in severe muscle atrophy, respiratory and heart failure due to loss of dystrophin function, which disrupts the formation of its associated glycoprotein complex (DGC), leading to sarcolemma instability and critical Increased sensitivity to injury, and fibrous necrosis (Findlay A R, 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 the expression of dystrophin is critical for the treatment of muscular dystrophy.
- DGC glycoprotein complex
- Glucocorticoid therapy is the main conservative treatment, but it can only delay disease progression for one or two years. Therefore, there is an urgent need to obtain effective drugs for the treatment of muscular dystrophy.
- adeno-associated virus adeno-associated virus, AAV
- AAV adeno-associated virus
- AAV adeno-associated virus
- Dystrophin-associated protein (Utrophin), as a homologous protein of Dystrophin, is also ubiquitously expressed in muscle, and it has a similar function and structure to 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 R J,Wood M J,Davies K E.Therapy for Duchenne muscular dystrophy:renewed optimism from genetic approaches[J] . Nat Rev Genet, 2013, 14(6):373-8).
- the present disclosure provides a recombinant hybrid protein, which includes: the N-terminal domain of the full-length human dystrophin-related protein (Utrophin), hinge H1, membrane contractile protein-like repeat sequence R1, contractilein-like repeat R2, contractin-like repeat R3, and the first half of hinge H2; and, the contractilein-like repeat R23, contractin-like Repeat sequence R24, hinge H4 and CR domain.
- Utrophin human dystrophin-related protein
- hinge H1 membrane contractile protein-like repeat sequence R1, contractilein-like repeat R2, contractin-like repeat R3, and the first half of hinge H2
- the contractilein-like repeat R23, contractin-like Repeat sequence R24, hinge H4 and CR domain the contractilein-like Repeat sequence R24, hinge H4 and CR domain.
- the above-mentioned recombinant hybrid protein comprises the amino acid sequence shown in SEQ ID NO: 4.
- the amino acid sequence of the above-mentioned recombinant hybrid protein is shown in SEQ ID NO: 4.
- the recombinant hybrid protein of the present disclosure can effectively improve muscle function, alleviate the symptoms of muscular dystrophy, and has a good therapeutic effect on muscular dystrophy.
- the present disclosure provides a nucleic acid molecule encoding the recombinant hybrid protein according to the first aspect.
- the nucleotide sequence of the above-mentioned nucleic acid molecule has at least 50% identity with the nucleotide sequence shown in SEQ ID NO: 8 or SEQ ID NO: 10, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% identity.
- the above-mentioned nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 8 or SEQ ID NO: 10.
- the nucleotide sequence of the aforementioned nucleic acid molecule is shown in SEQ ID NO: 8 or SEQ ID NO: 10.
- the present disclosure provides a transgene expression cassette, which includes: a promoter, the nucleic acid molecule according to the second aspect, and mini polyA.
- the promoter is selected from: CB promoter, CAG promoter, muscle creatine kinase MCK promoter, human creatine kinase hCK promoter, truncated human creatine kinase CK (Shortened CK, referred to as shCK) Promoter, skeletal muscle ⁇ -actin promoter, cardiac ⁇ -actin promoter, myosin heavy chain (MyHC) promoter, myosin light chain 2 (MLC2) promoter, myosin light chain 3F promoter, The desmin (desmin) gene promoter and the myogenic regulatory factor family (MyoG, Myf5, Mrf4 and Myogenin) and other muscle-specific gene promoters.
- the promoter is hCK promoter or shCK promoter.
- the promoter is the shCK promoter. Higher protein expression levels were obtained using shCK compared to hCK.
- nucleotide sequence of the promoter is shown in SEQ ID NO: 9.
- the transgene expression cassette further includes regulatory elements, such as two ITRs located at both ends, and the ITRs are normal ITRs or shortened ITRs, such as 145bp normal ITR or 100bp shortened ITR. In a preferred embodiment, the two ITRs are normal ITRs of 145 bp.
- the transgenic expression cassette further includes an origin of replication, a polyadenylation signal, an internal ribosome entry site (IRES), and/or a 2A signal, eg, P2A, T2A, and F2A.
- an origin of replication e.g., a polyadenylation signal, an internal ribosome entry site (IRES), and/or a 2A signal, eg, P2A, T2A, and F2A.
- nucleotide sequence of the transgene expression cassette is shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
- the present disclosure provides a gene delivery system, which includes: the transgene expression cassette according to the third aspect and AAV capsid protein.
- the above-mentioned AAV capsid protein is a natural AAV capsid protein or an artificially modified AAV capsid protein.
- the above-mentioned AAV is selected from: 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.
- the nucleic acid molecule, transgene expression cassette and gene delivery system of the present disclosure can stably express the above-mentioned recombinant hybrid protein in muscle tissue, thereby improving muscle function and achieving a good therapeutic effect on muscular dystrophy.
- the present disclosure provides the application of the transgene expression cassette according to the third aspect or the gene delivery system according to the fourth aspect in the preparation of a medicament for treating muscular dystrophy.
- muscular dystrophy includes Duchenne muscular dystrophy, Becker muscular dystrophy, and other muscle degenerative diseases.
- the muscular dystrophy is Duchenne muscular dystrophy.
- the present disclosure provides a medicine, which comprises: the recombinant hybrid protein according to the first aspect, the nucleic acid molecule according to the second aspect, the transgene expression cassette according to the third aspect and the One of the gene delivery systems described in the four aspects, and an excipient.
- the above-mentioned drug is used for treating muscular dystrophy, which includes Duchenne muscular dystrophy, Becker muscular dystrophy, and other muscle degenerative diseases.
- muscular dystrophy which includes Duchenne muscular dystrophy, Becker muscular dystrophy, and other muscle degenerative diseases.
- the muscular dystrophy is Duchenne muscular dystrophy.
- the present disclosure provides a method of treating muscular dystrophy, comprising administering a therapeutically effective amount of the drug according to the sixth aspect to a subject in need thereof.
- the drug is administered by systemic or local routes, such as intravenous, intramuscular, subcutaneous, oral, topical, intraperitoneal, and intralesional.
- systemic or local routes such as intravenous, intramuscular, subcutaneous, oral, topical, intraperitoneal, and intralesional.
- the drug is administered systemically, eg intravenously.
- the drug is administered to a muscle by a local route, for example injection into the biceps brachii or gastrocnemius.
- Figure 1A shows the structures (subdomains) of full-length human dystrophin, full-length human dystrophin-related protein, and recombinant hybrid protein encoded by the M6 construct constructed by the present inventors.
- Figure IB shows a schematic diagram of hCK-opti-M6, hCK-M6, hCK-B84, shCK-opti-M6 and shCK-M6 expression cassettes according to one embodiment.
- Figure 2A is an immunofluorescence analysis of therapeutic proteins in the hearts of mice injected with AAV9-hCK-opti-M6, AAV9-hCK-M6, AAV9-shCK-opti-M6, AAV9-shCK-M6 6 weeks after injection.
- the frozen sections of the hearts of WT mice, Mdx mice and mice in the treatment group were immunofluorescence stained with antibodies, and nuclei were counterstained with DAPI, scale bar: 200 ⁇ m.
- Figure 2B is an immunofluorescent analysis of therapeutic proteins in the gastrocnemius muscle of mice injected with AAV9-hCK-opti-M6, AAV9-hCK-M6, AAV9-shCK-opti-M6, and AAV9-shCK-M6 for 6 weeks.
- the frozen sections of the gastrocnemius muscles of WT mice, Mdx mice and mice in the treatment group were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 ⁇ m.
- Figure 2C is an immunofluorescence analysis of therapeutic proteins in the biceps brachii muscle of mice injected with AAV9-hCK-opti-M6, AAV9-hCK-M6, AAV9-shCK-opti-M6, AAV9-shCK-M6 6 weeks later.
- Frozen sections of biceps brachii of WT mice, Mdx mice and mice in the treatment group were immunofluorescence stained with antibodies, and cell nuclei were counterstained with DAPI.
- Scale bar 200 ⁇ m.
- Figure 2D is an immunofluorescence analysis of therapeutic proteins in the diaphragm 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 the diaphragm of WT mice, Mdx mice and mice in the treatment group were immunofluorescence stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 ⁇ m.
- Figure 3 shows the comparison of opti-M6 sequence and existing B84 sequence expressed in mice. Frozen sections of the heart, gastrocnemius muscle and diaphragm of WT mice, Mdx mice, hCK-opti-M6 and hCK-B84 treated mice were immunofluorescence stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 ⁇ m.
- Figure 4A is an immunofluorescence analysis of therapeutic proteins in the hearts of mice injected with AAV9-shCK-opti-M6 and AAV9-hCK-B84 4 weeks later. Immunofluorescence staining was performed on the frozen sections of hearts of WT mice, Mdx mice and mice in two treatment groups, and nuclei were counterstained with DAPI. Scale bar: 200 ⁇ m.
- Figure 4B is an immunofluorescence analysis of therapeutic proteins in the gastrocnemius muscle of mice injected with AAV9-shCK-opti-M6 and AAV9-hCK-B84 for 4 weeks. Frozen sections of gastrocnemius muscles of WT mice, Mdx mice, and two treatment groups were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 ⁇ m.
- Figure 4C is an immunofluorescence analysis of therapeutic proteins in the quadriceps muscles of mice injected with AAV9-shCK-opti-M6 and AAV9-hCK-B84 for 4 weeks. Frozen sections of quadriceps muscles of WT mice, Mdx mice, and mice in two treatment groups were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI, scale bar: 200 ⁇ m.
- Figure 4D is an immunofluorescence analysis of therapeutic proteins in the biceps brachii muscle of mice injected with AAV9-shCK-opti-M6 and AAV9-hCK-B84 for 4 weeks. Frozen sections of biceps brachii of WT mice, Mdx mice, and mice in two treatment groups were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 ⁇ m.
- Figure 4E is an immunofluorescence analysis of therapeutic proteins in the tibialis anterior muscle of mice injected with AAV9-shCK-opti-M6 and AAV9-hCK-B84 for 4 weeks.
- the frozen sections of the tibialis anterior muscle of WT mice, Mdx mice and mice in two treatment groups were immunofluorescence stained with antibodies, and cell nuclei were counterstained with DAPI.
- Scale bar 200 ⁇ m.
- Figure 4F is an immunofluorescent analysis of therapeutic proteins in the diaphragm of mice injected with AAV9-shCK-opti-M6 and AAV9-hCK-B84 for 4 weeks. Frozen sections of the diaphragm of WT mice, Mdx mice, and mice in two treatment groups were immunofluorescently stained with antibodies, and cell nuclei were counterstained with DAPI. Scale bar: 200 ⁇ m.
- Figure 4G is an immunofluorescence analysis of therapeutic proteins in the intercostal muscles of mice injected with AAV9-shCK-opti-M6 and AAV9-hCK-B84 4 weeks later. Frozen sections of intercostal muscles of WT mice, Mdx mice and mice in two treatment groups were immunofluorescence stained with antibodies, and cell nuclei were counterstained with DAPI, scale bar: 200 ⁇ m.
- Figure 4H shows the expression levels of the shCK-opti-M6 expression cassette and the hCK-B84 expression cassette.
- Western blot analysis of (a) heart, (b) gastrocnemius muscle and (c) diaphragm of WT mice, Mdx mice, AAV9-shCK-opti-M6 and AAV9-hCK-B84 treated for 4 weeks. GAPDH was used as an internal reference.
- Figure 5 shows serum CK levels in male and female mice.
- A is the serum CK level of male WT mice, Mdx mice, AAV9-shCK-opti-M6 and AAV9-hCK-B84 after 8-9 weeks of treatment
- B is the female WT mice, Mdx mice, AAV9 - Serum CK levels after 8-9 weeks of treatment with shCK-opti-M6 and AAV9-hCK-B84.
- n 4, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, t-test.
- Figure 6 shows the improvement of muscle function and behavior in mice.
- A Rotarod test and
- B grip test of WT mice, Mdx mice, AAV9-shCK-opti-M6 and AAV9-hCK-B84 after 8 weeks of treatment.
- n 4, *p ⁇ 0.05, **p ⁇ 0.01, t-test.
- Figure 7A is the long-term expression of AAV9-shCK-opti-M6 in mouse heart. Immunofluorescence analysis of therapeutic proteins in mouse hearts at different time points after injection (4 weeks, 6 weeks, 9 weeks, and 12 weeks), immunofluorescent staining with antibodies and counterstaining of nuclei with DAPI, scale bar: 200 ⁇ m.
- Figure 7B is the long-term expression of AAV9-shCK-opti-M6 in mouse gastrocnemius muscle. Immunofluorescence analysis of therapeutic proteins in mouse gastrocnemius muscle at different time points after injection (4 weeks, 6 weeks, 9 weeks and 12 weeks), immunofluorescent staining with antibodies and counterstaining of nuclei with DAPI, scale bar: 200 ⁇ m.
- Figure 7C is the long-term expression of AAV9-shCK-opti-M6 in mouse quadriceps muscle. Immunofluorescence analysis of therapeutic proteins in mouse quadriceps muscles at different time points after injection (4 weeks, 6 weeks, 9 weeks and 12 weeks), immunofluorescence staining with antibodies, counterstaining nuclei with DAPI, scale bar: 200 ⁇ m .
- Figure 7D is the long-term expression of AAV9-shCK-opti-M6 in mouse biceps. Immunofluorescence analysis of therapeutic proteins in biceps brachii of mice at different time points after injection (4 weeks, 6 weeks, 9 weeks and 12 weeks), immunofluorescent staining with antibodies, nuclei counterstained with DAPI, scale bar: 200 ⁇ m .
- Figure 7E is the long-term expression of AAV9-shCK-opti-M6 in mouse tibialis anterior muscle. Immunofluorescence analysis of therapeutic proteins in the tibialis anterior muscle of mice at different time points (4 weeks, 6 weeks, 9 weeks and 12 weeks) after injection, immunofluorescent staining with antibodies and counterstaining of nuclei with DAPI, scale bar: 200 ⁇ m.
- Figure 7F is the long-term expression of AAV9-shCK-opti-M6 in the mouse diaphragm. Immunofluorescence analysis of therapeutic proteins in the diaphragm of mice at different time points (4 weeks, 6 weeks, 9 weeks and 12 weeks) after injection, immunofluorescent staining with antibodies, counterstaining nuclei with DAPI, scale bar: 200 ⁇ m.
- Figure 7G is the long-term expression of AAV9-shCK-opti-M6 in mouse intercostal muscles. Immunofluorescence analysis of therapeutic proteins in the intercostal muscles of mice at different time points after injection (4 weeks, 6 weeks, 9 weeks and 12 weeks), immunofluorescent staining with antibodies and counterstaining of nuclei with DAPI, scale bar: 200 ⁇ m.
- Figure 7H is the long-term expression of AAV9-shCK-opti-M6 in mouse tongue. Immunofluorescence analysis of therapeutic proteins in mouse tongues at different time points after injection (4 weeks, 6 weeks, 9 weeks and 12 weeks), immunofluorescent staining with antibodies and counterstaining of nuclei with DAPI, scale bar: 200 ⁇ m.
- Figure 7I shows the long-term expression levels of the shCK-opti-M6 expression cassette in mice.
- GAPDH was used as an internal reference.
- Figure 8 shows the amino acid sequence (SEQ ID NO: 4) of the recombinant hybrid protein (i.e. M6 and opti-M6 construct protein products).
- Figure 9 shows the nucleotide sequence (SEQ ID NO: 8) of the codon-optimized M6 (opti-M6) construct.
- Figure 10 shows the nucleotide sequence (SEQ ID NO: 9) of shCK promoter.
- Figure 11 shows the nucleotide sequence (SEQ ID NO: 10) of the M6 construct without codon optimization.
- nucleic acid or polynucleotide sequences listed herein are in single-stranded form, oriented 5' to 3', left to right. Nucleotides and amino acids are presented herein using the format recommended by the IUPACIUB Biochemical Nomenclature Commission, using either the single-letter code or the three-letter code for the amino acids.
- polynucleotide is synonymous with “nucleic acid” and refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, mixed sequences thereof, or the like.
- a polynucleotide may include modified nucleotides, such as methylated or restricted nucleotides and nucleotide analogs.
- the terms "patient” and “subject” are used interchangeably and in their conventional sense to refer to an organism suffering from or susceptible to a condition that can be prevented or treated by administering the medicaments of the present disclosure, and Humans and non-human animals (eg, rodents or other mammals) are included.
- the subject is a non-human animal (e.g., chimpanzees and other ape and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; experimental animals including rodents) animals such as mice, rats and guinea pigs; birds including domestic, wild and game birds such as chickens, turkeys and other chickens, ducks, geese, etc.).
- the subject is a mammal. In one embodiment, the subject is a human.
- treating includes: (1) inhibiting the condition, disease or disorder, i.e., arresting, reducing or delaying the development of the disease or its recurrence or the development of at least one clinical or subclinical symptom thereof; or (2) Ameliorating a disease, ie, causing regression of at least one of a condition, disease or disorder, or clinical or subclinical symptoms thereof.
- a therapeutically effective amount refers to a dose that produces the therapeutic effect for which it is administered.
- a therapeutically effective amount of a drug useful in the treatment of muscular dystrophy may be an amount capable of preventing or ameliorating one or more symptoms associated with the muscular dystrophy.
- the term “amelioration” refers to an amelioration of a symptom associated with a disease, and may refer to an amelioration of at least one parameter that measures or quantifies the symptom.
- the term "preventing" a condition, disease or disorder includes preventing, delaying or reducing the incidence and/or likelihood of the occurrence of at least one clinical or subclinical symptom of a developing condition, disease or disorder in a subject,
- the subject may suffer from or be susceptible to the condition, disease or disorder but has not experienced or exhibited clinical or subclinical symptoms of the condition, disease or disorder.
- topical administration or “local route” refers to administration with a local effect.
- transduction refers to the process of delivering exogenous nucleic acid into a host cell, followed by transcription and translation of the polynucleotide product, which involves the transfer of exogenous A source polynucleotide is introduced into a host cell.
- gene delivery refers to the introduction of exogenous polynucleotides into cells for gene delivery, including targeting, binding, uptake, transport, replicon integration and expression.
- gene expression refers to the process of gene transcription, translation and post-translational modification to produce the gene's RNA or protein product.
- infection refers to the process by which a virus or virus particle comprising a polynucleotide component delivers a polynucleotide into a cell and produces its RNA and protein products, and may also refer to the process of virus replication in a host cell .
- targeting means that the virus preferentially enters some cells or tissues, and then further expresses the viral genome or the sequence carried by the recombinant transgene in the cells.
- vector refers to one or a series of macromolecules that encapsulate a polynucleotide, which facilitates the delivery of the polynucleotide to target cells in vitro or in vivo.
- Types of vectors include, but are not limited to, plasmids, viral vectors, liposomes, and other gene delivery vehicles.
- polynucleotides to be delivered are sometimes referred to as "expression cassettes" or “transgene cassettes,” which may include, but are not limited to, certain proteins or synthetic polypeptides (which can enhance, inhibit, impair, protect, trigger, or prevent certain biological and physiological functions), coding sequences of interest in vaccine development (e.g., polynucleotides expressing proteins, polypeptides or peptides suitable for eliciting an immune response in mammals), coding sequences of RNAi materials (e.g., shRNA, siRNA, antisense oligonucleotides) or alternative biomarkers.
- expression cassettes or “transgene cassettes”
- coding sequences of interest in vaccine development e.g., polynucleotides expressing proteins, polypeptides or peptides suitable for eliciting an immune response in mammals
- RNAi materials e.g., shRNA, siRNA, antisense oligonucleotides
- expression cassette refers to a polynucleotide fragment encoding a specific protein, polypeptide or RNAi element, which can be cloned into a plasmid vector.
- a "cassette” can also be packaged into an AAV particle and used as the viral genome to deliver the transgene product into target cells.
- the "cassette” may also include other regulatory elements, such as specific promoters/enhancers, polyA, regulatory introns, etc., to enhance or attenuate the expression of the transgene product.
- the transgene cassette contains a number of regulatory elements to enable packaging of the transgene into the virus, such as a normal ITR of 145 bp, a shortened ITR of approximately 100 bp in length, in addition to the sequence encoding the protein product.
- the transgenic cassette further comprises polynucleotide elements for controlling the expression of the protein product, such as an origin of replication, a polyadenylation signal, an internal ribosome entry site (IRES), or a 2A signal (e.g., P2A, T2A, F2A), promoters and enhancers, for example, CMV promoters with vertebrate ⁇ -actin, ⁇ -globin or ⁇ -globin regulatory elements or other hybrid CMV promoters (referred to as CB and CAG promoters) , muscle creatine kinase MCK promoter, human creatine kinase hCK promoter, truncated creatine kinase shCK promoter, skeletal muscle ⁇ -actin promoter, cardiac ⁇ -actin promoter, myosin heavy chain (MyHC) Promoter, myosin light chain 2 (MLC2) promoter, myosin light chain 3F promoter, desmin gene promoter and myogenic regulatory factor family
- Promoters and enhancers can be activated by chemicals or hormones (such as doxycycline or tamoxifen) to ensure gene expression at specific time points.
- promoters and enhancers may be natural or artificial or chimeric sequences, ie prokaryotic or eukaryotic sequences.
- the inducible regulatory element for gene expression may be a tissue or organ-specific promoter or enhancer, including but not limited to: promoters specific to various types of muscle cells, such as , muscle creatine kinase MCK promoter, human creatine kinase hCK promoter, truncated creatine kinase shCK promoter, skeletal muscle ⁇ -actin promoter, cardiac ⁇ -actin promoter, myosin heavy chain (MyHC) Promoter, myosin light chain 2 (MLC2) promoter, myosin light chain 3F promoter, desmin (Desmin) gene promoter and myogenic regulatory factor family (MyoG, Myf5, Mrf4 and Myogenin) and other muscle-specific promoters of sex genes; and specific promoters of the osteoblast lineage (eg, the osteocalcin promoter).
- promoters specific to various types of muscle cells such as , muscle creatine kinase MCK promoter, human creatine kinase h
- ITR inverted terminal repeat
- AAV inverted terminal repeat
- AAV types 1-12 avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV.
- AAV terminal repeats need not have native terminal repeats, so long as the terminal repeats are available for viral replication, packaging and integration.
- trans-element refers to a transgene cassette packaged in AAV particles and expressed in target cells to produce a therapeutic protein product.
- full-length human dystrophin includes four structural domains: an N-terminal domain, a central rod domain, a CR domain, and a C-terminal (CT) domain, wherein the central rod domain includes 24 Contractin-like repeats (R1-R24) and 4 hinges (H1-H4).
- the full-length human dystrophin-associated protein also includes four domains: N-terminal domain, central rod domain, CR domain and C-terminal (CT) domain, wherein the central rod domain includes 22 membrane contractile proteins Like repeats (R1-R22) and 4 hinges (H1-H4).
- CT C-terminal domain
- R1-R22 Like repeats
- H1-H4 Like repeats
- the M6 constructs of the present disclosure comprise two forms: a non-codon-optimized M6 construct and a codon-optimized M6 (opti-M6) construct.
- cogniated refers to a polynucleotide sequence modified from its native form. Such modifications result in a difference of one or more base pairs, with or without a change in the corresponding amino acid sequence, which may enhance or inhibit gene expression and/or cellular response to the modified polynucleotide sequence.
- the AAV capsid protein can be any AAV serotype capsid protein, including native AAV capsid proteins (e.g., native AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV).
- native AAV capsid proteins e.g., native AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV.
- AAV capsid protein e.g., engineered AAV capsid proteins of types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV.
- engineered AAV capsid proteins eg, engineered capsid proteins of types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV.
- the present disclosure provides a therapeutic tool with the effect of improving muscle function, which can be used to treat various diseases with related pathological mechanisms, including but not limited to: Duchenne muscular dystrophy, Becker muscular dystrophy , and other muscle degenerative diseases.
- the clinical manifestations of a muscle degenerative disease include muscle atrophy and/or reduced or lost exercise capacity.
- the protein product of the therapeutic tool includes a protein that improves muscle function, such as, but not limited to, Dystrophin (full-length or truncated), Utrophin (full-length or truncated), ) and hybrid combinations of the two.
- the therapeutic tool is a protein such as, but not limited to, Dystrophin (full length or truncated), Utrophin (full length or truncated), and recombinant hybrids of the two.
- the transgenic expression cassette of the present disclosure includes: hCK promoter sequence (SEQ ID NO: 1), mini polyadenylation (polyA) sequence (SEQ ID NO: 2) and codon-optimized M6 (opti -M6) construct (SEQ ID NO:8), thus forming the hCK-opti-M6 expression cassette (SEQ ID NO:3).
- the hCK-opti-M6 expression cassette is flanked by 145 bp normal ITRs that allow packaging of the expression cassette into AAV virions as single-chain AAV vectors.
- the transgenic expression cassette of the present disclosure includes: hCK promoter sequence (SEQ ID NO: 1), mini polyadenylation (polyA) sequence (SEQ ID NO: 2) and M6 without codon optimization construct (SEQ ID NO: 10), thus forming the hCK-M6 expression cassette (SEQ ID NO: 5).
- the hCK-M6 expression cassette is flanked by 145 bp normal ITRs that allow packaging of the expression cassette into AAV virions as single-chain AAV vectors.
- the transgenic expression cassette of the present disclosure includes: shCK promoter sequence (SEQ ID NO: 9), mini polyadenylation (polyA) sequence (SEQ ID NO: 2) and codon-optimized M6 (opti -M6) construct (SEQ ID NO:8), thus forming the shCK-opti-M6 expression cassette (SEQ ID NO:6).
- shCK-opti-M6 expression cassette is flanked by normal ITRs of 145 bp, which allow packaging of the expression cassette into AAV virions as single-chain AAV vectors.
- the transgenic expression cassette of the present disclosure includes: shCK promoter sequence (SEQ ID NO: 9), mini polyadenylation (polyA) sequence (SEQ ID NO: 2) and M6 without codon optimization construct (SEQ ID NO: 10), thus forming the shCK-M6 expression cassette (SEQ ID NO: 7).
- shCK-M6 expression cassette is flanked by 145 bp normal ITRs, which allow packaging of the expression cassette into AAV virions as single-chain AAV vectors.
- dystrophin and related protein AAV particles are produced by three-plasmid (plasmid 1: cis-element plasmid; plasmid 2: AAV Rep/Cap plasmid; plasmid 3: helper plasmid) transfection of HEK293 cells.
- plasmid 1 cis-element plasmid
- plasmid 2 AAV Rep/Cap plasmid
- plasmid 3 helper plasmid
- Plasmid 1 cis-element plasmid with ITR (e.g., hCK-opti-M6, hCK-M6, shCK -opti-M6 and shCK-M6 expression cassettes); Plasmid 2: AAV Rep/Cap plasmid with coding sequence for capsid protein (e.g., AAV9 capsid protein); Plasmid 3: helper plasmid with adenoviral components, which can promote Replication, assembly and packaging of AAV virions.
- AAV particles produced by HEK293 cells are purified by cesium chloride (CsCl) density gradient centrifugation (eg, the method described in Example 2 of the present disclosure).
- CsCl cesium chloride
- the gene delivery systems of the present disclosure are used to aid in cell transplantation therapy.
- AAV particles with transgenes can be used to transduce various types of cells in vitro to generate stable cell lines expressing protein products, which can then be introduced in vivo for therapeutic purposes.
- Types of cells include, but are 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.
- the cells used for transplantation are autologous cells of the subject, which allow for in vitro culture.
- the principles and techniques of introducing or transplanting cells into a subject are known to those skilled in the art.
- AAV particles are harvested from the culture medium and lysates of HEK293 cells. Purification methods such as affinity chromatography, ion exchange chromatography, cesium chloride and iodixanol gradient ultracentrifugation.
- Chemicals or reagents related to AAV production and purification include, but are not limited to: Chemicals or reagents used in cell culture (e.g., components of cell culture media including bovine, equine, goat, chicken or other vertebrate serum, glutamine Amides, glucose, sucrose, sodium pyruvate, phenol red; antibiotics such as penicillin, kanamycin, streptomycin, tetracycline); chemicals or reagents for cell lysis, polynucleotide precipitation, or ultracentrifugation (such as Triton X-100, NP-40, sodium deoxycholate, sodium lauryl sulfate, domiphene bromide, sodium lauryl salicylate, sodium chloride, magnesium chloride, calcium chloride, barium chloride, nitric acid Salt, potassium chloride, ammonium chloride, ammonium persulfate, ammonium sulfate, PEG-20, PEG-40, PEG-400, PEG-2000
- the exogenous polynucleotide delivered by the AAV vector to the target cell encodes a native protein for therapeutic use, either codon-optimized or non-codon-optimized.
- the exogenous polynucleotide delivered by the AAV vector to the target cell encodes a synthetic polypeptide.
- the transgene expression cassette or gene delivery system of the present disclosure is made into pharmaceutical preparations (eg, injections, tablets, capsules, powders, eye drops) and administered to humans or other mammals.
- the pharmaceutical preparation also contains other ingredients such as pharmaceutical excipients, water-soluble or organic solvents (such as water, glycerol, ethanol, methanol, isopropanol, chloroform, phenol or polyethylene glycol), salts (such as sodium chloride, chloride Potassium, Phosphate, Acetate, Bicarbonate, Tris-HCl and Tris-Acetate), Delaying Dissolving Agents (e.g.
- Paraffin Paraffin
- Surfactants e.g, cortisone, prednisone, cyclosporine
- Immuno Inhibitors eg, cortisone, prednisone, cyclosporine
- nonsteroidal anti-inflammatory drugs e.g, aspirin, ibuprofen, acetaminophen
- microspheres rigid matrices, semisolid carriers, Nanospheres or nanoparticles.
- compositions can be administered by inhalation, systemic or topical (e.g., intravenous, subcutaneous, intraocular, intravitreal, subretinal, suprachoroidal, parenteral, intramuscular, intracerebroventricular, oral, intraperitoneal, and intrathecal)
- systemic or topical e.g., intravenous, subcutaneous, intraocular, intravitreal, subretinal, suprachoroidal, parenteral, intramuscular, intracerebroventricular, oral, intraperitoneal, and intrathecal
- the drug is delivered in single or multiple doses.
- the present disclosure provides a medicament comprising the recombinant hybrid protein, nucleic acid molecule, transgene expression cassette or gene delivery system of the present disclosure; and excipients.
- the drugs of the present disclosure can be used to transduce cells in vitro or mammals (such as rodents, primates and humans) in vivo to treat various diseases associated with defective muscle function, such as muscular dystrophy, including: Duchenne muscular dystrophy, Becker muscular dystrophy, and other muscle degenerative diseases.
- the muscular dystrophy is Duchenne muscular dystrophy.
- treating muscular dystrophy refers to improving the degree of muscle damage, creatine kinase level and NASS (The North American Spine Society) muscle function score of the treated patient.
- NASS The North American Spine Society
- Example 1 Design and construction of M6 and opti-M6 constructs and AAV vectors
- the cDNA of human dystrophin was used as a template to obtain the B84 (Mini-dystrophin) construct (SEQ ID NO: 11) by the PCR cloning method.
- the B84 construct and the small human dystrophin protein it encodes can be found for example in CN 109641944A (corresponding to Hopti-Dys3978 and Dys3978 respectively) or 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 U S A,2000,97(25):13714-9.
- the inventors also constructed a highly truncated M6 construct (SEQ ID NO: 10) based on full-length human dystrophin and full-length human dystrophin-related protein, and in this Codon optimization was performed on the basis of the M6 construct to obtain the opti-M6 construct (SEQ ID NO: 8).
- the recombinant hybrid protein encoded by the M6 construct or the opti-M6 construct contains: the N-terminal domain of the full-length human dystrophin-related protein, hinge 1 (H1), contractile protein-like repeat sequence 1 (R1), contractilin-like repeat 2 (R2), contractilin-like repeat 3 (R3), and the first half of hinge 2 (H2); and, the contractilin-like of full-length human dystrophin Repeat 23 (R23), contractrin-like repeat 24 (R24), hinge 4 (H4), and CR domains.
- the inventors also constructed a truncated human creatine kinase CK (shCK) promoter based on the human creatine kinase hCK promoter (SEQ ID NO: 1).
- the above-mentioned M6 and opti-M6 genes were subcloned into AAV vector plasmids, and hCK promoter and shCK promoter were used respectively to generate vectors AAV-hCK-M6-polyA, AAV-hCK-opti-M6-polyA, AAV- shCK-M6-polyA and AAV-shCK-opti-M6-polyA.
- the B84 gene was cloned into an AAV vector plasmid containing the hCK promoter and mini polyA signal sequence, resulting in the vector AAV-hCK-B84.
- mice In order to study the expression of M6 and opti-M6 constructs in mice, four treatment groups were set up in this example: hCK-opti-M6, hCK-M6, shCK-opti-M6 and shCK-M6.
- the vectors AAV-hCK-M6-polyA, AAV-hCK-opti-M6-polyA, AAV-shCK-M6-polyA and AAV-shCK-opti-M6-polyA constructed in Example 1 were packaged into AAV9 virus particles.
- AAV virus is produced according to the three-plasmid co-transfection method (such as 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), transfection of the above four novel vector cis-element plasmids, AAV9 capsid plasmid and adenovirus helper plasmid by three plasmids.
- HEK293 cells were lysed 40-80 hours after transfection and polynucleotides were removed.
- AAV viral vectors were subsequently purified twice by CsCl density gradient ultracentrifugation using the protocol described by Snyder R, Xiao S, Samulski R J. Production of recombinant adeno-associated viral vectors [J], 1996.
- the virus titer determined by qPCR method is about 2 ⁇ 10 13 -1 ⁇ 10 14 vg/ml.
- the purity of capsid protein was detected by SDS-PAGE. Endotoxin was detected by gel method, and the result was qualified.
- mice 3 ⁇ 10 13 vg/kg of the above-mentioned AAV particles were injected into the Mdx mice through the tail vein. Same-age WT mice and littermate Mdx mice were used as controls. Six weeks after injection, mice were dissected. Heart, gastrocnemius, biceps, and diaphragm tissues were collected for cryosection and immunofluorescent staining.
- the rabbit-derived polyclonal antibody used for immunofluorescence staining was purchased from Abcam (Catalog No. ab251754), which can recognize the 2800-3000 amino acid fragment of human dystrophin (contractin-like repeat sequence R23 and R24). Thus, proteins expressed by both the B84 and M6 constructs were recognized by this antibody.
- Immunofluorescent staining of heart, gastrocnemius, biceps brachii, and diaphragm tissue can reflect the improvement of muscle pathology related to cardiac function, lower limb motor function, upper limb motor function and respiratory function in Mdx mice, respectively.
- the pathological phenotype of Mdx mice begins at about 3 weeks of age, accompanied by the degeneration and regeneration of muscle fibers.
- the nucleus is located in the center of the muscle fibers, which is the characteristic performance of this process and the main pathological manifestation.
- the M6 and opti-M6 constructs can widely express the hybrid therapeutic protein in four representative muscle tissues including heart and gastrocnemius muscle, and produce therapeutic effects.
- the optimized opti-M6 construct showed higher expression levels compared to the M6 construct. Also, higher protein expression levels were obtained with shCK compared to hCK.
- the shCK-opti-M6 expression cassette has the best in vivo activity.
- the inventors compared the expression levels of the opti-M6 construct and the B84 construct in model mice.
- Opti-M6 and B84 were respectively constructed into the original hCK promoter vector and packaged into viruses to obtain AAV9-hCK-opti-M6 and AAV9-hCK-B84 virus particles.
- Mdx mice were treated by tail vein injection of virus particles at a dose of 3 ⁇ 10 13 vg/kg. Same-age WT mice and littermate Mdx mice were used as controls. After 6 weeks of treatment, mice were dissected, and tissues such as heart, gastrocnemius muscle and diaphragm were collected. The expression of therapeutic proteins in different tissues was detected by tissue immunofluorescence.
- the AAV9-hCK-opti-M6 treatment group achieved similar fluorescence intensity to the AAV9-hCK-B84 treatment group, and the AAV9-hCK-opti-M6 treatment group had more positive cells;
- the fluorescence intensity of the AAV9-hCK-opti-M6 treatment group was significantly stronger than that of the AAV9-hCK-B84 treatment group; in the diaphragm, compared with the AAV9-hCK-B84 treatment group, the AAV9-hCK-opti- The fluorescence in the M6 treatment group was stronger, and the number of positive cells was significantly more.
- the opti-M6 construct produced a better therapeutic effect than the B84 construct.
- the inventors compared the expression capabilities of the shCK-opti-M6 vector and the hCK-B84 vector in model mice.
- AAV-shCK-opti-M6 and AAV-hCK-B84 vectors were packaged into AAV9-shCK-opti-M6 and AAV9-hCK-B84 virions, respectively, as described in Example 2.
- Mdx mice were treated by injecting virus particles via the tail vein at a dose of 3 ⁇ 10 13 vg/kg. Same-age WT mice and littermate Mdx mice were used as controls. After 4 weeks of treatment, the mice were dissected, and tissues such as the heart, gastrocnemius, quadriceps, and diaphragm were collected. Then, protein expression in different tissues was detected by immunofluorescence and western blotting.
- AAV9-shCK-opti-M6 and AAV9-hCK-B84 treatment were also observed Expression of a wide range of therapeutic proteins.
- the expression level in limb muscle tissue of shCK-opti-M6 treatment group was significantly higher than that of hCK-B84 treatment group.
- the inventors observed peripheral nucleation of myofibers in regions where the protein was widely expressed, which to some extent corrected the pathological phenotype of central nucleation and prevented muscle degeneration and regeneration, indicating that The vector can improve the muscle pathological phenotype after treatment.
- shCK-opti-M6 and hCK-B84 in vivo were detected simultaneously with antibodies that can recognize the contractile protein-like repeat sequence R23 and R24 fragments of human dystrophin.
- Western blot results showed that the expression of shCK-opti-M6 in mouse heart was higher than that of hCK-B84, and the expression level of shCK-opti-M6 in gastrocnemius muscle and diaphragm was close to that of hCK-B84 (Fig. 4G).
- Example 5 Creatine kinase levels in Mdx mice after treatment with shCK-opti-M6 carrier and hCK-B84 carrier
- the inventors detected the level of serum creatine kinase (Creatine Kinase, CK), which is the expression of muscle damage in Mdx mice. important indicators.
- two treatment groups were set up, namely AAV9-shCK-opti-M6 and AAV9-hCK-B84.
- Mdx mice were treated by tail vein injection at a dose of 3 ⁇ 10 13 vg/kg. Same-age WT mice and littermate Mdx mice were used as controls. 8 in each group, 4 males and 4 males. After 8-9 weeks of treatment, blood was collected from the orbit, serum was collected, and the level of CK in mouse serum was detected.
- the serum CK level of untreated Mdx mice was significantly higher than that of WT mice at 4 months of age (male, **p ⁇ 0.01; female, ***p ⁇ 0.001), while in Significantly decreased in AAV-treated Mdx mice, among which the serum CK value in the shCK-opti-M6 treatment group was significantly decreased (male, *p ⁇ 0.05; female, ***p ⁇ 0.001).
- Example 6 Effects of shCK-opti-M6 and hCK-B84 vectors on exercise capacity and muscle strength of Mdx mice
- mice were subjected to the rotarod test (locomotion) and the grip test (strength).
- the inventors studied the long-term expression of the shCK-opti-M6 vector in Mdx mice.
- Four groups of Mdx mice were injected through the tail vein at a dose of 3 ⁇ 10 13 vg/kg. Muscle samples were collected at different time points (4 weeks, 6 weeks, 9 weeks and 12 weeks) after vector injection for immunofluorescence and western blot detection. Same-age WT mice and littermate Mdx mice were used as controls.
- the protein expression was detected with an antibody that can recognize the membrane contractile protein-like repeat sequence R23 and R24 fragments of human dystrophin, and the results of Western blot analysis are shown in Figure 7I.
- protein expression can be detected as early as 4 weeks after injection, with the highest expression level at 6-9 weeks, and persists until 12 weeks after injection.
- the expression was higher at 6 weeks after injection, and the expression reached the peak at 12 weeks after injection.
- the protein expression reached a peak at 9 weeks after injection, and remained at a high level at 12 weeks after injection.
- the expression level of the protein was the highest at 6 weeks after injection, and a higher level of expression could still be detected at 12 weeks after injection.
- a certain level of protein expression can be detected 4 weeks after injection, and the expression has been maintained at a high level for 6-9 weeks after injection.
- protein expression was still detectable 12 weeks after injection.
- the results of Western blot analysis of the intercostal muscles were similar to those of the tongue, with different levels of protein expression detected at 4-9 weeks after injection, and the expression level decreased at 12 weeks after injection.
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Claims (18)
- 重组杂合蛋白,其包括:全长人Utrophin的N端结构域、铰链H1、膜收缩蛋白样重复序列R1、膜收缩蛋白样重复序列R2、膜收缩蛋白样重复序列R3和铰链H2的前半部分,以及全长人Dystrophin的膜收缩蛋白样重复序列R23、膜收缩蛋白样重复序列R24、铰链H4和CR结构域。
- 根据权利要求1所述的重组杂合蛋白,其中,所述重组杂合蛋白包含SEQ ID NO:4所示的氨基酸序列;优选地,所述重组杂合蛋白的氨基酸序列如SEQ ID NO:4所示。
- 编码权利要求1或2所述的重组杂合蛋白的核酸分子。
- 根据权利要求3所述的核酸分子,其核苷酸序列与SEQ ID NO:8或SEQ ID NO:10所示的核苷酸序列具有至少50%的同一性,优选具有至少50%、60%、70%、80%、85%、90%、95%、99%或100%的同一性。
- 根据权利要求4所述的核酸分子,其中,所述核酸分子包含SEQ ID NO:8或SEQ ID NO:10所示的核苷酸序列,优选地,所述核酸分子的核苷酸序列如SEQ ID NO:8或SEQ ID NO:10所示。
- 转基因表达盒,其包括:启动子、权利要求3至5中任一项所述的核酸分子、mini polyA。
- 根据权利要求6所述的转基因表达盒,其中,所述启动子选自:CB启动子、CAG启动子、肌肉肌酸激酶MCK启动子、人肌酸激酶hCK启动子、截短的人肌酸激酶CK(shCK)启动子、骨骼肌α-actin启动子、心肌α-actin启动子、肌球蛋白重链(MyHC)启动子、肌球蛋白轻链2(MLC2)启动子、肌球蛋白轻链3F启动子、结蛋白(desmin)基因启动子和生肌调控因子家族(MyoG、Myf5、Mrf4和Myogenin)等肌肉特异性基因的启动子;优选地,所述启动子为hCK启动子或shCK启动子;更优选地,所述启动子为shCK启动子。
- 根据权利要求6或7所述的转基因表达盒,其中,所述启动子的核苷酸序列如SEQ ID NO:9所示。
- 根据权利要求6至8中任一项所述的转基因表达盒,其中,所述转基因表达盒还包括调控元件,例如位于两端的两个ITR,所述ITR为正常ITR或缩短ITR;优选地,所述两个ITR均为145bp的正常ITR。
- 根据权利要求6至9中任一项所述的转基因表达盒,其中,所述转基因表达盒还包括复制起点、聚腺苷酸化信号、内部核糖体进入位点(IRES)和/或2A信号,例如P2A、T2A和F2A。
- 根据权利要求6至10中任一项所述的转基因表达盒,其中,所述转基因表达盒的核苷酸序列如SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:6或SEQ ID NO:7所示。
- 基因递送***,其包括:权利要求6至11中任一项所述的转基因表达盒和AAV衣壳蛋白。
- 根据权利要求12所述的基因递送***,其中,所述AAV衣壳蛋白为天然AAV衣壳蛋白或人工改造的AAV衣壳蛋白;优选地,所述AAV选自: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和AAV.PHP.B。
- 权利要求6至11中任一项所述的转基因表达盒或权利要求12或13所述的基因递送***在制备用于治疗肌营养不良症的药物中的应用。
- 根据权利要求14所述的应用,其中,所述肌营养不良症包括:杜氏型肌营养不良症、贝克型肌营养不良症,以及其他肌肉退行性疾病;优选地,所述肌营养不良症为杜氏型肌营养不良症。
- 药物,其包含:权利要求1或2所述的重组杂合蛋白、权利要求3至5中任一项所述的核酸分子、权利要求6至11中任一项所述的转基因表达盒和权利要求12或13所述的基因递送***中的一种,以及赋形剂。
- 一种治疗肌营养不良症的方法,包括向有需要的受试者施用治疗有效量的权利要求16所述的药物。
- 根据权利要求17所述的方法,其中,所述药物通过全身途径或局部途径施用,例如静脉内施用、肌内施用、皮下施用、经口施用、局部接触、腹膜内施用和病灶内施用;优选地,所述药物通过全身途径施用,例如静脉内施用;还优选地,所述药物通过局部途径施用于肌肉,例如注射至肱二头肌或腓肠肌。
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Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1439051A (zh) * | 2000-04-28 | 2003-08-27 | 肖啸 | 编码抗肌萎缩蛋白小基因的dna序列及其使用方法 |
US20170218037A1 (en) * | 2014-07-26 | 2017-08-03 | Consiglio Nazionale Delle Ricerche | Compositions and methods for treatment of muscular dystrophy |
CN107250364A (zh) * | 2015-01-16 | 2017-10-13 | 华盛顿大学 | 新的微小肌养蛋白及相关使用方法 |
US20190036676A1 (en) | 2016-01-27 | 2019-01-31 | Ntt Docomo, Inc. | User terminal, radio base station, and radio communication method |
CN109641944A (zh) | 2016-06-21 | 2019-04-16 | 班布疗法公司 | 优化的小型抗肌萎缩蛋白基因和表达盒和它们的用途 |
CN110997923A (zh) * | 2017-03-17 | 2020-04-10 | 全国儿童医院研究所 | 腺相关病毒载体递送肌肉特异性微肌营养不良蛋白以治疗肌营养不良症 |
CN111057157A (zh) * | 2020-01-02 | 2020-04-24 | 北京辉润合众生物科技有限公司 | 一种抗肌萎缩融合蛋白mDp116 |
CN111138549A (zh) * | 2020-01-02 | 2020-05-12 | 北京辉润合众生物科技有限公司 | 一种抗肌萎缩融合蛋白mUp113 |
WO2021050970A1 (en) | 2019-09-13 | 2021-03-18 | Rutgers, The State University Of New Jersey | Aav-compatible laminin-linker polymerization proteins |
RU2767335C1 (ru) * | 2021-03-02 | 2022-03-17 | Общество с ограниченной ответственностью «Марлин Биотех» | Химерные белки на основе утрофина и дистрофина человека и их применение для лечения миодистрофии Дюшенна |
CN114316070A (zh) * | 2021-12-29 | 2022-04-12 | 上海勉亦生物科技有限公司 | 用于治疗肌营养不良症的转基因表达盒 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB201720224D0 (en) * | 2017-12-05 | 2018-01-17 | Royal Holloway & Bedford New College | Treatment of muscular dystrophies |
-
2021
- 2021-12-29 CN CN202111642563.7A patent/CN114316070B/zh active Active
-
2022
- 2022-12-01 WO PCT/CN2022/135805 patent/WO2023124741A1/zh active Application Filing
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Patent Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1439051A (zh) * | 2000-04-28 | 2003-08-27 | 肖啸 | 编码抗肌萎缩蛋白小基因的dna序列及其使用方法 |
US20170218037A1 (en) * | 2014-07-26 | 2017-08-03 | Consiglio Nazionale Delle Ricerche | Compositions and methods for treatment of muscular dystrophy |
CN107250364A (zh) * | 2015-01-16 | 2017-10-13 | 华盛顿大学 | 新的微小肌养蛋白及相关使用方法 |
US20190036676A1 (en) | 2016-01-27 | 2019-01-31 | Ntt Docomo, Inc. | User terminal, radio base station, and radio communication method |
CN109641944A (zh) | 2016-06-21 | 2019-04-16 | 班布疗法公司 | 优化的小型抗肌萎缩蛋白基因和表达盒和它们的用途 |
CN110997923A (zh) * | 2017-03-17 | 2020-04-10 | 全国儿童医院研究所 | 腺相关病毒载体递送肌肉特异性微肌营养不良蛋白以治疗肌营养不良症 |
WO2021050970A1 (en) | 2019-09-13 | 2021-03-18 | Rutgers, The State University Of New Jersey | Aav-compatible laminin-linker polymerization proteins |
CN111057157A (zh) * | 2020-01-02 | 2020-04-24 | 北京辉润合众生物科技有限公司 | 一种抗肌萎缩融合蛋白mDp116 |
CN111138549A (zh) * | 2020-01-02 | 2020-05-12 | 北京辉润合众生物科技有限公司 | 一种抗肌萎缩融合蛋白mUp113 |
RU2767335C1 (ru) * | 2021-03-02 | 2022-03-17 | Общество с ограниченной ответственностью «Марлин Биотех» | Химерные белки на основе утрофина и дистрофина человека и их применение для лечения миодистрофии Дюшенна |
CN114316070A (zh) * | 2021-12-29 | 2022-04-12 | 上海勉亦生物科技有限公司 | 用于治疗肌营养不良症的转基因表达盒 |
Non-Patent Citations (15)
Title |
---|
"MOLECULAR CLONING: A LABORATORY MANUAL", 1989 |
BANKS GLEN B., CHAMBERLAIN JEFFREY S., ODOM GUY L.: "Microutrophin expression in dystrophic mice displays myofiber type differences in therapeutic effects", PLOS GENETICS, vol. 16, no. 11, pages e1009179, XP093076116, DOI: 10.1371/journal.pgen.1009179 * |
CHOUDHURY SR ET AL.: "In Vivo Selection Yields AAV B 1 Capsid for Central Nervous System and Muscle Gene Therapy", MOL THER., vol. 24, no. 7, 2016, pages 1247 - 57, XP055445527, DOI: 10.1038/mt.2016.84 |
DUAN, DONGSHENG: "Systemic AAV Micro-Dystrophin Gene Therapy for Duchenne Muscular Dystrophy", MOLECULAR THERAPY, vol. 26, no. 10, 31 October 2018 (2018-10-31), XP055925242, DOI: 10.1016/j.ymthe.2018.07.011 * |
FAIRCLOUGH R J, WOOD M JDAVIES K E: "Therapy for Duchenne muscular dystrophy: renewed optimism from genetic approaches [J", NAT REV GENET, vol. 14, no. 6, 2013, pages 373 - 8 |
FINDLAY A RWEIN NKAMINOH Y ET AL.: "Clinical phenotypes as predictors of the outcome of skipping around DMD exon 45[J", ANN NEUROL, vol. 77, no. 4, 2015, pages 668 - 74, XP071641498, DOI: 10.1002/ana.24365 |
GUIRAUD SCHEN HBURNS D T ET AL.: "Advances in genetic therapeutic strategies for Duchenne muscular dystrophy[J", EXP PHYSIOL, vol. 100, no. 12, 2015, pages 1458 - 67, XP071896481, DOI: 10.1113/EP085308 |
HSU HL ET AL.: "Structural characterization of a novel human adeno-associated virus capsid with neurotropic properties", NAT COMMUN, vol. 11, 2020, pages 3279, XP055822287, DOI: 10.1038/s41467-020-17047-1 |
LIN, A.Y. ET AL.: "Impacts of Dystrophin and Utrophin Domains on Actin Structural Dynamics: Implications for Therapeutic Design.", JOURNAL OF MOLECULAR BIOLOGY., vol. 420, 11 April 2012 (2012-04-11), XP028514779, DOI: 10.1016/j.jmb.2012.04.005 * |
MIURA PJASMIN B J: "Utrophin upregulation for treating Duchenne or Becker muscular dystrophy: how close are we?[J", TRENDS MOL MED, vol. 12, no. 3, 2006, pages 122 - 9, XP028058695, DOI: 10.1016/j.molmed.2006.01.002 |
MOORWOOD CKHURANA T S: "Duchenne muscular dystrophy medicament discovery - the application of utrophin promoter activation screening[J", EXPERT OPIN MEDICAMENT DISCOV, vol. 8, no. 5, 2013, pages 569 - 81 |
RICOTI VSPINTY SROPER H ET AL.: "Safety, Tolerability, and Pharmacokinetics of SMT C1100, a 2-Arylbenzoxazole Utrophin Modulator, following Single- and Multiple-Dose Administration to Pediatric Patients with Duchenne Muscular Dystrophy[J", PLOS ONE, vol. 11, no. 4, 2016, pages e0152840 |
SNYDER RXIAO SSAMULSKI R J, PRODUCTION OF RECOMBINANT ADENO-ASSOCIATED VIRAL VECTORS[J, 1996 |
WANG BLI JXIAO X: "Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model[J", PROC NATL ACAD SCI U S A, vol. 97, no. 25, 2000, pages 13714 - 9, XP002949073, DOI: 10.1073/pnas.240335297 |
XIAO XLI JSAMULSKI R J: "Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus[J", J VIROL, vol. 72, no. 3, 1998, pages 2224 - 32 |
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