Adeno-associated viral vectors expressing microdystrophin genes and uses thereof

Abstract

The application discloses an adeno-associated virus vector expressing a micro anti-dystrophin gene and application thereof. A human micro anti-dystrophin has an amino acid sequence shown as SEQ ID NO. 1. A gene encoding the human micro anti-dystrophin has a sequence shown as SEQ ID NO. 3. On this basis, a muscle tissue-specific promoter is used to efficiently produce the micro anti-dystrophin in C2C12 muscle cells. Finally, an AAV9 virus vector and a recombinant adeno-associated virus expressing the micro anti-dystrophin are constructed, the AAV9 virus vector and the recombinant adeno-associated virus can efficiently transduce muscle cells in a DMD disease model, and the micro anti-dystrophin can be efficiently expressed to realize the goal of DMD treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of gene drugs, and relates to an adeno-associated virus vector expressing a micro-anti-dystrophin gene and application thereof. BACKGROUND

[0002] Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disease, and patients show symptoms from infancy, and neuromuscular disorders are manifested as progressive muscle weakness, atrophy, wasting, fat infiltration and fibrosis, thereby causing patients to gradually lose the ability to walk independently, respiratory failure, cardiomyopathy and premature death (PMID: 33602943). The cause of DMD is mutation of the dystrophin gene, thereby causing deletion of dystrophin protein expression.

[0003] The dystrophin gene is composed of 79 exons, and encodes a dystrophin protein with a molecular weight of 427 kDa (PMID: 7719347). The dystrophin protein is composed of four domains: an N-terminal domain; a rod domain; a cysteine-rich domain and a C-terminal domain (PMID: 3607877). Dystrophin is a cytoskeletal protein, which, together with sarcoglycans, merosin, neuronal nitric oxide synthase (nNOS) and the like, forms a dystrophin-associated protein complex, participates in cell signal transduction, and plays a crucial role in the stability of the muscle fiber membrane (PMID: 26140716). Deletion of dystrophin can enhance the vulnerability of the muscle membrane, and then induce muscle fiber splitting, ischemia, free radical-mediated oxidative damage and cytoplasmic calcium overload during contraction, ultimately leading to mitochondrial dysfunction and muscle fiber death (PMID: 3173492; PMID: 18345011; PMID: 11087833). In addition, due to the impaired muscle fiber regeneration ability of DMD patients, muscle tissue gradually undergoes fibrosis (PMID: 26246170). The gene mutations that cause DMD are mainly frameshift mutations and nonsense mutations, thereby causing complete deletion of dystrophin protein expression (PMID: 21399986).

[0004] With the development and advancement of medical technology, an increasing number of emerging treatment technologies and methods for DMD are gradually entering the public eye, such as stem cell therapy, exon skipping therapy, gene editing, and gene replacement therapy. In February 2021, Sarepta Therapeutics' Amondys 45 (trade name casimersen) was approved by the U.S. Food and Drug Administration (FDA) for use in DMD patients with exon 45 skipping gene mutations. This drug is a phosphodiesteramide morpholine oligomer that utilizes exon skipping therapy. This involves altering the splicing process of the mRNA expressing anti-dystrophin, skipping the gene mutation that prematurely interrupts protein synthesis. While the resulting anti-dystrophin is smaller than the normal protein, it retains most of its binding domains to other proteins, thus still performing some of the protein's normal functions, achieving the goal of treating DMD patients with exon 45 skipping gene mutations.

[0005] Besides exon skipping and gene editing, gene replacement therapy is another promising gene therapy. This involves introducing exogenous genes into target cells via a suitable vector to replace or compensate for abnormal genes, thus achieving therapeutic goals. Currently, the most advanced gene therapy is SRP-9001 (also known as AAVrh74.MHCK7.micro-Dystrophin). This therapy combines the MHCK7 promoter with the AAVrh74 vector to provide expression of a miniature anti-dystrophin protein to restore muscle function in DMD patients. The miniature anti-dystrophin protein contains an N-terminal domain (NT), a helical repeat region (R(1,2,3,24)), a hinge region (H(1,2,4)), and a cysteine-rich functional region (CR), arranged in the pattern NT-H1-R1-R2-R3-H2-R24-H4-CR. Currently, SRP-9001 is undergoing clinical trials, but the latest results from the interim trial failed to meet the primary functional endpoint assessing improvement. Pfizer's fordadistrogene movaparvovec (PF-06939926) is an investigational intravenous gene therapy. It encapsulates a "mini" dystrophin transgene controlled by a human muscle-specific promoter within an adeno-associated virus 9 (AAV9) vector. The "mini" dystrophin's structure includes an N-terminal domain (NT), helical repeats (R(1,2,22,23,24)), a hinge region (H(1,3,4)), and a cysteine-rich region (CR), arranged in the pattern NT-H1-R1-R2-H3-R22-R23-R24-H4-CR. One patient died in the bedridden cohort of a phase Ib clinical trial. This news has raised new concerns in the industry regarding the safety of gene therapy for DMD.

[0006] Dystrophic myopathy (DMD) is inherited in an X-linked recessive manner. Its causative gene is the dystrophin gene, which is the largest gene in the human body, far exceeding the optimal packaging capacity of a single AAV (<4.7kb). Most current gene therapy strategies for DMD involve packaging a truncated dystrophin gene while retaining key structural domains, expressing a truncated but biologically functional dystrophin, thus addressing the problem of the large size of the complete DMD gene making packaging impossible. The most crucial aspect of DMD gene therapy strategies is the design of the truncated protein structure; the retention or removal of different structural domains significantly affects protein function. Studies have designed various truncated proteins with different combinations of structural domains based on the results of dystrophin, and different combinations of domains have shown different in vivo therapeutic effects (e.g., the proportion of dystrophin-positive myofibrils, the expression effect of truncated proteins, and the recruitment capacity of nNOS). Proteins resulting from the retention and combination of different structural domains exhibit different functional characteristics and expression levels. Neither SRP-9001 nor PF-06939926 contains the nNOS binding regions R16 and R17. Summary of the Invention

[0007] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a human miniature anti-muscular dystrophy protein μDystrophin and its encoding gene.

[0008] Another object of the present invention is to provide an adeno-associated virus vector and recombinant adeno-associated virus expressing the miniature anti-dystrophy protein gene.

[0009] Another object of the present invention is to provide the application of the said gene, adeno-associated virus vector, and recombinant adeno-associated virus.

[0010] The objective of this invention can be achieved through the following technical solutions:

[0011] The amino acid sequence of human micro-dystrophin (μDystrophin) is shown in SEQ ID NO.1.

[0012] The gene encoding the human miniature anti-muscular dystrophy protein μDystrophin described in this invention.

[0013] As a preferred embodiment of the present invention, the gene nucleotide sequence is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0014] A human μDysco protein expression cassette containing the gene encoding the human miniature anti-muscular dystrophy protein μDystrophin described in this invention.

[0015] As a preferred embodiment of the present invention, the human μDysco protein expression cassette is composed of an enhancer-promoter-target gene sequence-polyA signal or a promoter-target gene sequence-polyA signal. The promoter is selected from the muscle-specific Spc512 promoter (as shown in SEQ ID NO.4), the muscle-specific MCK promoter (as shown in SEQ ID NO.5), and the muscle-specific CK8 promoter (as shown in SEQ ID NO.7). The target gene is selected from the gene described in the present invention, preferably a codon-optimized gene encoding the human miniature anti-dystrophy protein μDystrophin described in the present invention.

[0016] As a preferred embodiment of the present invention, the enhancer is selected from the MCK enhancer sequence as shown in SEQ ID NO.6, and the polyA signal sequence is selected from rBG polyA, as shown in SEQ ID NO.8.

[0017] In a preferred embodiment of the present invention, the enhancer, promoter, target gene sequence and polyA are linked by a bond or nucleotide linkage sequence.

[0018] As a preferred embodiment of the present invention, the nucleotide sequence of the Spc512-μDysco protein expression cassette is as shown in SEQ ID NO.9, the nucleotide sequence of the MCK-μDysco protein expression cassette is as shown in SEQ ID NO.11, and the nucleotide sequence of the CK8-μDysco protein expression cassette is as shown in SEQ ID NO.13.

[0019] A vector characterized by containing the gene described in this invention, or the human μDysco protein expression cassette described in this invention.

[0020] As a preferred embodiment of the present invention, the vector is selected from any of the following recombinant adeno-associated virus vector serotypes: AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV-LK03 or AAVAnc80d, preferably AAV2, AAV5, AAV8 or AAV9.

[0021] A recombinant adeno-associated virus that specifically expresses micro-dystrophin protein was prepared by co-transfecting HEK293 cells with AAV REP and CAP protein expression plasmids, helper plasmids, and the vector described in this invention.

[0022] As a preferred embodiment of the present invention, the AAV REP protein and CAP protein expression plasmids are selected from pAAV2 / 9; the helper plasmids are selected from pAdΔF6.

[0023] As a preferred embodiment of the present invention, the viral genome sequence of AAV9 expressing the μDysco protein is shown in SEQ ID NO. 10, SEQ ID NO. 12 or SEQ ID NO. 14.

[0024] The present invention relates to the use of the gene encoding the human miniature anti-muscular dystrophy protein μDystrophin, the human μDysco protein expression cassette, the vector, and the recombinant adeno-associated virus in the preparation of a drug for treating Duchenne muscular dystrophy.

[0025] As a preferred embodiment of the present invention, the gene encoding the human micro-dystrophin protein μDystrophin, the human μDysco protein expression cassette, the vector, and the recombinant adeno-associated virus described herein are used in the preparation of a drug for restoring the expression of Dystrophin and nNOS in the muscles of DMD mice, reducing the degree of muscle fibrosis in DMD mice, restoring the morphology of muscle tissue in DMD mice, and restoring the muscle strength in DMD mice.

[0026] Beneficial effects:

[0027] This invention first analyzed the structure of anti-dystrophy protein (ADD) and designed a miniaturized DDD protein. This protein contains the key structural domains necessary for DDD function and solves the problem that the large size of DDD cannot be completely packaged into AAV. Subsequently, we optimized the codons of the miniaturized DDD gene, improving its expression level. Based on this, we used a muscle tissue-specific promoter to efficiently produce the miniaturized DDD in C2C12 muscle cells. Finally, we constructed an AAV9 viral vector for efficient tissue-specific expression of the miniaturized DDD for gene therapy research in DMD. In a DMD model, the AAV9 vector containing the newly designed miniaturized DDD expression cassette (1×10⁻⁶) is used. 12Gene therapy efficacy was assessed after GC (150 μl / mouse) was injected into the circulatory system of diseased mice via tail vein. Eight weeks post-administration, immunofluorescence results showed expression of anti-dystrophin and neuronal nitric oxide synthase (nNOS) in the muscle tissue of all treatment groups, while no positive signal was observed in the untreated DMD mice. Masson staining results showed that the degree of muscle fibrosis and collagen volume fraction were improved in the treatment groups compared to the untreated groups. Pathological morphological examination of the muscle tissue of each group revealed varying degrees of muscle morphology restoration in the treatment groups, with a significant reduction in the area of ​​inflammatory infiltration. Muscle strength testing showed that the grip strength of the treatment group mice was higher than that of the untreated group, and the rate of muscle strength decline was slower. The results show that by designing a novel and optimized miniature anti-muscular dystrophy protein coding sequence and designing a miniature anti-muscular dystrophy protein AAV9 expression cassette, muscle cells can be efficiently transduced in a DMD disease model, and functional truncated anti-muscular dystrophy protein can be efficiently expressed, thus achieving the goal of treating Duchenne muscular dystrophy. Attached Figure Description

[0028] Figure 1 Schematic diagram of μDystrophin protein structure

[0029] Figure 2 Codon optimization validation expression vector plasmid map

[0030] Figure 3 Validation of μDystrophin expression vector in vitro expression levels

[0031] A represents a comparison of μDystrophin expression levels across different vector groups using Western blotting.

[0032] B represents the statistical analysis of grayscale values ​​from Western blot (WB) detection of μDystrophin protein expression levels in vitro. Image J was used to analyze and statistically analyze the relative grayscale values ​​of image (A).

[0033] Figure 4 Gene therapy candidate vector expression validation

[0034] AC is a plasmid map of gene therapy candidate vectors.

[0035] D represents a comparison of μDystrophin expression levels in different vector groups using Western blotting.

[0036] E represents the statistical analysis of grayscale values ​​from Western blotting (WB) detection of μDystrophin protein expression levels in vitro. Image J was used to analyze and statistically analyze the relative grayscale values ​​of image (D).

[0037] Figure 5 Schematic diagram of recombinant adeno-associated virus structure

[0038] Figure 6 Dystrophin immunofluorescence assay

[0039] scale bar = 200μm

[0040] Figure 7 nNOS immunofluorescence assay

[0041] scale bar = 200μm

[0042] Figure 8 Muscle fibrosis detection

[0043] A shows the Masson staining results of muscle tissue in each group, scale bar = 200 μm.

[0044] B is the collagen volume fraction count analyzed and statistically determined from image J in graph (A).

[0045] Figure 9 Muscle tissue morphology detection

[0046] scale bar = 100μm

[0047] Figure 10 grip test results

[0048] A is a statistical graph of the continuous gripping force of the forelimbs of mice in each group.

[0049] B is a statistical chart of the continuous gripping force of the hind limbs of mice in each group.

[0050] C is a statistical chart of the average gripping force of the forelimbs of mice in each group.

[0051] D is a statistical chart of the average gripping force of the hind limbs of mice in each group.

[0052] Untreated: n=10, WT: n=15, Spc512: n=15, MCK: n=15, CK8: n=15; ns indicates no significant difference, *p<0.05, **p<0.01. Detailed Implementation

[0053] Example 1: Design of human miniature anti-muscular dystrophy protein

[0054] Dystrophin is a transmembrane cytoskeletal protein located in the sarcolemma. Its N-terminal (NT) binds to intracellular actin (F-actin). The rod-shaped region contains 24 helical repeat regions (R) and 4 hinge regions (H). Near the C-terminus is a cysteine-rich (CR) functional region linked to the β-dystrophin-glycoprotein complex (β-DGC), responsible for binding calmodulin during contraction. The C-terminus (CT) is linked to dystrobrevin and syntrophin, jointly contributing to maintaining muscle cell membrane stability. This invention's self-designed human micro-dystrophin (μDystrophin) includes the NT, H1, R1, R16, R17, R24, H4, CR, and CT domains of the Dystrophin protein. Figure 1 This is a truncated anti-dystrophy protein that retains some biological functions. It contains the most important structural domains necessary for the anti-dystrophy protein to function, and solves the problem that the large size of the anti-dystrophy protein cannot be completely packaged into AAV.

[0055] Example 2: Optimization of the target gene sequence of miniaturized anti-muscular dystrophy protein and construction of expression vector

[0056] The wild-type miniature anti-muscular dystrophy protein gene (μDyswt, SEQ ID NO.2) and the codon-optimized miniature anti-muscular dystrophy protein gene (μDysco, SEQ ID NO.3) were synthesized from the whole genome, and the whole plasmid pAAV.CB7.μDyswt.bGH was synthesized. Figure 2 A, SEQ ID NO.15) and pAAV.CB7.μDysco.bGH ( Figure 2 B, SEQ ID NO.16) plasmid expression vector.

[0057] Example 3: Validation of in vitro expression level of μDystrophin expression vector

[0058] pAAV.CB7.μDyswt.bGH and pAAV.CB7.μDysco.bGH from Example 2, along with the EGFP-expressing plasmid, were transfected into HEK293 cells cultured in 6-well plates using Lipo3000 transfection reagent at the same plasmid amount (1 μg). After 72 h, protein was extracted, and the expression level of μDystrophin was compared using Western blot (WB). The results showed that codon optimization significantly improved the protein expression level. Figure 3 A) By comparing the gray values ​​of protein bands, codon optimization showed that the expression level of μDystrophin protein increased by 1.2 times. Figure 3 B).

[0059] Example 4: Design, construction, and in vitro expression level validation of gene therapy candidate vectors

[0060] To further evaluate the expression of μDystrophin protein in vivo, the efficacy and safety of gene therapy, we synthesized a full-plasmid viral packaging cis-plasmid vector pAAV.Spc512.μDysco.rBG( Figure 4 A, SEQ ID NO.17), pAAV.MCK.μDysco.rBG( Figure 4 B, SEQ ID NO.18) and pAAV.CK8.μDysco.rBG ( Figure 4 C, SEQ ID NO. 19) three vectors. The three plasmid vectors mentioned above, as well as the pAAV.CB7.μDysco.bGH vector and the EGFP-expressing plasmid from Example 2, were transfected into C2C12 muscle cells cultured in 6-well plates using the same amount of plasmid (1 μg) via Lipo3000 transfection reagent. Proteins were extracted after 72 h, and the expression levels of μDystrophin in each vector were compared using Western blot. By comparing the gray values ​​of the protein bands, the pAAV.Spc512.μDysco.rBG vector showed the highest μDystrophin protein expression level, comparable to that of pAAV.CB7.μDysco.bGH using a broad-spectrum promoter; pAAV.CK8.μDysco.rBG was next; and pAAV.MCK.μDysco.rBG had the lowest expression level. Figure 4 (D and E), but all three muscle tissue-specific promoters can achieve expression of μDystrophin protein in muscle cells.

[0061] Example 5: Preparation and purification of AAV virus

[0062] Following the method reported by Martin Lock et al. for packaging and purifying recombinant AAV virus, PEI was used to co-transfect HEK293 cells with AAV Rep and Cap protein expression plasmids (pAAV2 / 9), helper plasmids (pAdΔF6), and AAV packaging cis plasmids (pAAV.Spc512.μDysco.rBG, SEQ ID NO.17; pAAV.MCK.μDysco.rBG, SEQ ID NO.18; pAAV.CK8.μDysco.rBG, SEQ ID NO.19) to prepare the virus AAV9.Spc512.μDysco.rBG. Figure 5 A,SEQ IDNO.10),AAV9.MCK.μDysco( Figure 5 B, SEQ ID NO.12) and AAV9.CK8.μDysco virus ( Figure 5 C (SEQ ID NO. 14), after 48 h of transfection, cells and culture supernatant were harvested. AAV virus was purified by ultracentrifugation with iodixanol. Viral titer was determined by digital quantitative PCR (ddPCR), and the AAV9.Spc512.μDysco virus titer was 4.44 × 10⁻⁶. 15 GC / mL, AAV9.MCK.μDysco viral titer is 2.35 × 10⁻⁶. 15 GC / mL, AAV9.CK8.μDysco viral titer is 5.51×10⁻⁶. 15 GC / mL.

[0063] Example 6: AAV9μDysco gene therapy restores Dystrophin expression in the muscles of mice with DMD.

[0064] Using the AAV9.Spc512.μDysco, AAV9.MCK.μDysco, and AAV9.CK8.μDysco viruses prepared in Example 5, DMD-affected mice (4 weeks old, 15 mice per virus) were injected via tail vein, with a viral injection dose of 1×10⁻⁶. 12GC / mouse. Eight weeks after viral injection, we collected muscle tissue from each group of mice (n=5 per group). Dystrophin expression in various muscle tissues (heart, triceps, tibialis anterior, gluteal, and gastrocnemius) of the treated, untreated, and wild-type mice was detected by frozen section and immunofluorescence. Results showed that eight weeks after viral administration, no Dystrophin protein signal was detected in any muscle tissue of the untreated group. Weak Dystrophin protein signal was detected in the tibialis anterior and gluteal muscles of the AAV9.Spc512.μDysco group. Strong signal was detected in the heart, gastrocnemius, and gluteal muscles of the AAV9.MCK.μDysco group. Strong signal expression was detected in all muscle tissues of the AAV9.CK8.μDysco group and wild-type mice. Figure 6 This indicates that after AAV9μDysco gene therapy, the expression of Dystrophin protein on the sarcolemma was restored, with the AAV9.CK8.μDysco group showing the highest expression level and the best treatment effect.

[0065] Example 7: AAV9μDysco gene therapy restores nNOS expression in the muscles of mice with DMD.

[0066] Eight weeks after viral injection, we collected muscle tissue from mice in each group (n=5 per group) and detected the expression of nNOS in various muscle tissues (heart, tibialis anterior, and gastrocnemius) of the treated, untreated, and wild-type mice using frozen sections and immunofluorescence. The results showed that eight weeks after viral administration, no nNOS protein signal was detected in any muscle tissue of the untreated group, while strong signals were detected in the tibialis anterior and gastrocnemius muscles of all three treated groups and wild-type mice. In heart tissue, weak nNOS signals were detected in the AAV9.Spc512.μDysco and AAV9.MCK.μDysco groups, while strong signals were detected only in the AAV9.CK8.μDysco and wild-type groups. Figure 7 The above results indicate that AAV9μDysco gene therapy can restore the expression of nNOS protein, with the AAV9.CK8.μDysco group showing the best treatment effect among the three treatment groups.

[0067] Example 8: AAV9μDysco gene therapy reduces muscle fibrosis in DMD mice

[0068] Eight weeks after viral injection, we collected muscle tissue from each group of mice (n=5 per group). We then used frozen sections and Masson staining to examine the fibrosis and interstitial calcification in muscle tissues (heart, tibialis anterior, and gastrocnemius) of the treated, untreated, and wild-type mice to assess muscle health. In Masson staining, small-molecule dyes penetrate muscle fibers, turning them red, while larger-molecule aniline dyes can only penetrate loosely structured collagen fibers, turning them blue. Furthermore, areas of calcification were stained dark red. Figure 8 (As indicated by arrow A). Statistical analysis of the collagen volume fraction results for each group showed that in the untreated group, the collagen volume fractions in the tibialis anterior muscle, gastrocnemius muscle, and myocardium were 16%, 25%, and 26%, respectively, indicating significant muscle fiber damage and fibrosis. After AAV9μDysco gene therapy, the collagen volume fraction in the gastrocnemius muscle of the AAV9.Spc512.μDysco group was 5%, and the collagen volume fraction in the myocardium was 8%, showing a certain degree of improvement in fibrosis compared to the untreated group. In the AAV9.MCK.μDysco group and the AAV9.CK8.μDysco group, the collagen volume fraction in each muscle tissue decreased to below 4%, indicating that the expression of μDystrophin protein protected the muscle membrane after gene therapy, greatly improving the muscle fiber's resistance to mechanical damage.

[0069] Example 9: AAV9μDysco gene therapy restores muscle tissue morphology in DMD mice

[0070] Eight weeks after viral injection, we collected muscle tissue from each group of mice (n=5 per group). We then examined the morphology of various muscle tissues (heart, triceps, tibialis anterior, gluteal muscles, and gastrocnemius) in the treated, untreated, and wild-type mice using frozen sections and Hematologic & E staining. H&E staining results showed that the untreated group exhibited significant inflammatory cell infiltration in all muscle tissues. Figure 9 (As indicated by the arrows) In mice treated with AAV9μDysco gene therapy for 8 weeks, muscle morphology in all three treatment groups showed varying degrees of recovery in different muscle tissues, and the area of ​​inflammatory infiltration in the muscle tissues was significantly reduced. The AAV9.Spc512.μDysco group still showed relatively obvious inflammatory infiltration in the triceps and gluteal muscles. The AAV9.CK8.μDysco group showed the best recovery level among the three treatment groups, with its muscle morphology recovering to a level comparable to the wild-type control group. Figure 9 ).

[0071] Example 10: AAV9μDysco gene therapy restores muscle strength in DMD mice

[0072] Eight weeks after viral injection, we performed three repeated grip strength tests on mice in each group to evaluate muscle strength and grip strength decay. Statistical analysis of the grip strength test results showed that the three consecutive intervals of grip strength measurements reflected the changes in muscle strength decay in the mice. Figure 10 In mice treated with AAV9.Spc512.μDysco (A and B), the forelimb strength of mice in the AAV9.Spc512.μDysco group was improved compared to the untreated group, but the difference was not statistically significant. The strength improvement in the AAV9.MCK.μDysco group was statistically significant compared to the untreated group (p<0.05). The AAV9.CK8.μDysco group showed the best recovery level in forelimb strength, with a significant difference compared to the untreated group (p<0.01). Regarding hindlimb strength, all three treatment groups showed significant improvements in muscle strength, with statistically significant differences compared to the untreated group (p<0.05). Furthermore, we analyzed the overall grip strength of the mice after treatment by statistically analyzing the average grip strength. The statistical results show that… Figure 10 In both C and D groups, the AAV9.MCK.μDysco and AAV9.CK8.μDysco groups showed statistically significant differences compared to the untreated group (p<0.05). These results indicate that AAV9μDysco gene therapy can restore muscle strength in DMD mice, with the AAV9.CK8.μDysco group showing the best recovery after treatment.

Claims

1. A human mini-dystrophin protein, μDystrophin, characterized in that The amino acid sequence is shown as SEQ ID NO.

1.

2. A human μDysco protein expression cassette comprising a gene encoding the human mini-antidystrophin μDystrophin according to claim 1.

3. The human μDysco protein expression cassette of claim 2, wherein consisting of an enhancer-promoter-gene of interest sequence-polyA signal or consisting of a promoter-gene of interest sequence-polyA signal, wherein the promoter is selected from the group consisting of a muscle-specific Spc512 promoter having a sequence as shown in SEQ ID NO. 4, a muscle-specific MCK promoter having a sequence as shown in SEQ ID NO. 5, and a muscle-specific CK8 promoter having a sequence as shown in SEQ ID NO. 7; and the gene of interest is a gene encoding the human mini-antidystrophin μDystrophin according to claim 1.

4. The human μDysco protein expression cassette of claim 3, wherein The enhancer is selected from the group consisting of a MCK enhancer sequence having a sequence as shown in SEQ ID NO. 6, and the polyA signal sequence is selected from the group consisting of a rBG polyA having a sequence as shown in SEQ ID NO.

8.

5. The human μ Dysco protein expression cassette of claim 2, wherein The nucleotide sequence of the human μDysco protein expression cassette is selected from the group consisting of SEQ ID NO. 9, SEQ ID NO. 11, and SEQ ID NO.

13.

6. A vector, characterized in that The vector is an AAV9 recombinant adeno-associated virus vector serotype comprising the human μDysco protein expression cassette according to any one of claims 2-5.

7. A recombinant adeno-associated virus that expresses a micro-dystrophin protein tissue-specifically, characterized in that The AAV REP protein and CAP protein expression plasmid, the helper plasmid, and the vector according to claim 6 are co-transfected into HEK293 cells to package and prepare.

8. The recombinant adeno-associated virus of claim 7, wherein The AAV REP protein and CAP protein expression plasmid is selected from the group consisting of pAAV9, and the helper plasmid is selected from the group consisting of pAdΔF6.

9. The recombinant adeno-associated virus of claim 7, characterized in that The AAV9 μDysco protein expression viral genome sequence is shown as SEQ ID NO. 10, SEQ ID NO. 12, or SEQ ID NO.

14.

10. Use of the gene encoding the human mini-antidystrophin μDystrophin according to claim 1, the human μDysco protein expression cassette according to any one of claims 2-5, the vector according to claim 6, or the recombinant adeno-associated virus according to any one of claims 7-9 in the preparation of a medicament for treating Duchenne muscular dystrophy.

Images