CN111057157A - Fusion protein mDp116 for resisting amyotrophy - Google Patents

Abstract

The present disclosure relates to an anti-muscular atrophy fusion protein mDp116, a nucleic acid encoding the fusion protein, a vector comprising the nucleic acid, a cell, use of the fusion protein, the nucleic acid, the vector and the cell for preparing a medicament for treating muscular atrophy, and a pharmaceutical composition comprising the nucleic acid, the vector or the cell. The invention adopts bioinformatics and proteomics model to reconstruct Dp116, which keeps the interaction between helices and the inherent stable protein structure; the internal structure of the anti-amyotrophic protein is changed, so that the binding force between the anti-amyotrophic protein and a second-class histocompatibility complex (MHCII) on the surface of a T cell is reduced, the T cell cannot stimulate the corresponding B cell to further divide and differentiate to generate an antibody corresponding to the foreign protein so as to achieve the aim of inhibiting immune reaction, and the stability of the anti-amyotrophic protein is greatly improved by modifying the immunogenicity of the anti-amyotrophic protein; the designed Dp116 gene therapy can not only improve the function of muscles and treat Duchenne muscular dystrophy, but also improve the cognitive function of patients with DMD.

Description

Fusion protein mDp116 for resisting amyotrophy

Technical Field

The disclosure relates to the technical field of fusion proteins, in particular to an anti-muscular atrophy fusion protein mDp 116.

Background

Duchenne Muscular Dystrophy (DMD) is one of the most common fatal childhood genetic diseases, and DMD disease results from variation of the largest gene (2.3 megabases, 79 exons) in humans [1,2], with most mutations being sporadic multiple exon frameshift deletions (DMD dystrophin-related protein) deletions; or exon deletions occur in-frame (in frame deletion) resulting in short pieces of dystrophin [3,4 ]. Dystrophin forms an important mechanical link between cytoskeletal actin and transmembrane Dystrophin Glycoprotein Complex (DGC), these proteins collectively protect the sarcolemma from stresses generated during muscle contraction [5,6 ]. In the absence of dystrophin, DGC is unstable, the sarcolemma becomes very fragile, leading to myofibronecrosis, inflammatory cell infiltration, myofiber regeneration and eventually replacement of myofibers by connective tissue with progressive myosclerosis and loss of contractility, and muscle gradually degenerates and disappears with age, including respiratory muscles, often leading to a decline in respiratory function due to the degeneration of respiratory muscles, leading to pulmonary complications or respiratory failure leading to death with a mean life of about 20-30 years [7,8 ]. Although the use of glucocorticoids, ACE inhibitors and mechanical ventilation support combined therapy can temporarily slow the rate of progression, the final clinical course is not obstructive and the final stages of the disease pay a significant cost in terms of care etc. [9 ].

In recent years, due to the pathophysiological complexity and the wide range of DMD, research into DMD molecules and cellular targets has been increasingly driven. These include interference with NFkappaB signaling [10,11], exon skipping and stop codon suppression [12], and gene editing the DMD gene [13-15], but these approaches have limited relief from the rate of pathophysiological development or are directed to a limited number of patients with specific mutations. Only if the primary cause is resolved, it is possible to "end Du's". Gene therapy refers to the introduction of exogenous normal genes into target cells to correct or compensate for diseases caused by gene defects and abnormalities, thereby achieving the therapeutic goal. That is, the exogenous gene is inserted into the proper recipient cell of a patient by gene transfer technology, so that the product produced by the exogenous gene can treat a certain disease. The possibility that gene therapy for DMD offers a durable curative treatment, as early as 20 years ago, Clemens [16] et al introduced a full-length dystrophin gene (12kb) using adenovirus as a vector and intramuscular injection, but this approach has been abandoned due to the strong immune response and limited dystrophin distribution produced by the complex vector capsid. Later, various vectors for deriving recombinant adeno-associated virus (rAAVs) were developed, and rAAVs was widely used in one of gene research and gene therapy vectors [17-20] due to its diverse species, low immunogen, wide host cell range, strong diffusion capacity, long in vivo gene expression time, etc., scientists used derived adenovirus to generate "mini dystrophin" using vectors in sequence in mice [21-24] and dogs [25,26] experimental results showed different degrees of improvement in pathology, physiology and muscle function of DMD, however, phase I clinical trials using this technology found evidence of cell-mediated immune response targeted in injected muscle of study subjects with DMD gene deletion, which resulted in the disappearance of mini dystrophin in a short time [27], and similarly, in preclinical studies using the same human transgene for systemic gene transfer, dystrophin deficient dogs (GRMD) cause clinically severe generalized myositis, and the immune response to mini-dystrophin exacerbates the progression of DMD disease [25 ]. Studies of other DMD dog models and non-human primates revealed limitations in induced peripheral tolerance to vector-encoded antigens, especially in DMD muscle, mini-dystrophin expression contributes to improving DMD exacerbation progression, but the immune system of the self is considered "heterosis", and thus, the challenge to solving the immune response of mini-dystrophin is a matter of primary design consideration in future trials of gene therapy DMD [28 ].

Although most of DMD does not express dystrophin in vivo, its homologous protein, Utrophin [29-31] dystrophin, is structurally about 50% identical to dystrophin, and is normally highly expressed only in the muscle of young children, with the expression of dystrophin gradually decreasing with age and only in small amounts in some parts of adults, such as the muscle nerve junctions, etc. Tinsley et al first demonstrated in mdx mouse animal models that proteins associated with muscular dystrophy can compensate for the function of dystrophin, significantly delaying the progression of the course of muscular dystrophy. The specific means of up-regulation are various: directly introducing intact or tailored anti-muscular atrophy-related proteins; introducing the tailored anti-muscular atrophy related protein into the viral vector; activating transcription of an anti-muscular atrophy-related protein; stabilize its mRNA after transcription, increase protein translation, and the like. Some of the small molecules screened are ready for clinical trials. The small molecule drug VOX C1100 used by VASTox corporation in the United kingdom for up-regulation of proteins related to muscular atrophy is certified in orphan medicine by European drug administration in 2007, and is ready to enter clinical trials in 2008, but is not really implemented. BMN195 of BioMarin corporation, usa, in 2010 began a phase I clinical trial in healthy volunteers, but the trial results showed that oral absorption of the drug was not ideal and the drug concentration required for treatment could not be achieved, thereby terminating further trials. Although the results of initial clinical trials are not ideal, the up-regulation of the dystrophia related protein is still an important way to treat DMD/BMD, and the selection of a proper drug becomes a key. This therapeutic strategy has the advantage of avoiding an immune response to new proteins using existing proteins in the body, as compared to mini-dystrophin gene therapy, but has the disadvantage that the up-regulated mini-dystrophin-related protein is expressed in too low an amount insufficient to recruit the transmembrane Dystrophin Glycoprotein Complex (DGC), thus greatly reducing the effect. Scientific research proves that the mini-dystrophin can replace the function of dystrophin to restore the muscle function of patients with Duchenne muscular dystrophy [32-34], and Song et al, 2019 and 10 months, use AVV9 to carry short-chain mini-dystrophin gene, and the expression of the mini-dystrophin successfully improves the muscle function and greatly reduces CK on DMD mouse and dog models, while the mini-dystrophin causes strong immune response.

In summary, most of the mini-dystrophin/dystrophin proteins designed in the existing documents are simply helix-spliced, and interaction between the helices is lost, so that the protein structure is changed, and the therapeutic effect is not ideal. In addition, the gene therapy is used to artificially introduce the sheared short dystrophin to better treat the muscular disease, but a great problem is that the immunogenicity of the dystrophin cannot be removed. Thus greatly reducing the potential for future applications of dystrophin. Therefore, there is still a need for dystrophin proteins with preserved protein structure and reduced immunogenicity.

Reference documents:

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disclosure of Invention

In order to solve the above problems in the prior art, the present invention provides a dystrophin fusion protein mDp116 using bioinformatics and proteomics models to reconstruct Dp116, wherein the fusion protein comprises the amino acid sequence shown as SEQ ID No. 1.

Preferably, the fusion protein further comprises the amino acid sequences shown as SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4.

Preferably, the amino acid sequence of the fusion protein comprises SEQ ID NO. 2, SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 4 in sequence from the N-terminus to the C-terminus.

Preferably, the fusion protein comprises the amino acid sequence shown in SEQ ID NO. 5.

In another aspect, the invention also provides a nucleic acid molecule encoding the fusion protein. The nucleic acid molecule may be DNA or RNA. The nucleotide sequence of the nucleic acid molecule can be obtained by the conventional bioinformatics operation of the amino acid sequence of the fusion protein, and can be further subjected to codon optimization according to the type of a host cell.

In another aspect, the invention also provides a nucleic acid vector comprising the nucleic acid molecule.

Preferably, the nucleic acid vector is an adenoviral vector.

In another aspect, the invention also provides a cell comprising said nucleic acid molecule and/or expressing said fusion protein. The cell may be a prokaryotic cell, such as E.coli, or a eukaryotic cell, such as yeast, Hela, HEK 293T. The cell may be a cell for amplifying the nucleic acid molecule or a cell for producing the fusion protein.

In another aspect, the invention also provides the use of the fusion protein, the nucleic acid molecule, the vector and the cell for preparing a medicament for treating duchenne muscular dystrophy and/or improving cognitive function of a patient.

In another aspect, the invention also provides a pharmaceutical composition comprising the fusion protein, and/or the nucleic acid molecule, and/or the nucleic acid vector, and/or the cell, and a pharmaceutically acceptable carrier or excipient.

The recombinant short-chain dystrophin is completely different from the previous design based on mDp116 dystrophin subtype existing in a human body and combined with the optimized design of a functional region of the dystrophin, so that the stability is greatly improved on the structure and the function, the immune response in the human body is avoided to a great extent, the protein is self-protein, the damage of muscle cells can be obviously protected, the function of the muscle is improved, the effect of treating DMD is achieved, and the safe and effective treatment scheme is provided. Compared with the prior art, the invention mainly achieves the following beneficial effects:

1) the Dp116 is reconstructed by adopting a bioinformatics and proteomics model, so that the interaction between the helices and the inherent stable protein structure are maintained;

2) the inherent structure of the anti-dystrophin is changed through the inherent Dp116 coding sequence in vivo, so that the binding force of the anti-dystrophin and a second-class histocompatibility complex (MHCII) on the surface of a T cell is reduced, the T cell cannot stimulate the corresponding B cell to further divide and differentiate to generate an antibody corresponding to a foreign protein so as to achieve the aim of inhibiting immune response, and the stability of the anti-dystrophin is greatly improved through modifying the immunogenicity of the anti-dystrophin.

3) The designed Dp116 gene therapy can not only improve muscle function, but also improve cognitive function of DMD patients. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.

FIG. 1 is a diagram of the structure of the full-length dystrophin protein and the recombinant fusion protein mDp 116.

FIG. 2 is a schematic diagram showing the combination of 30 types of the amino acid sequences of dystrophin proteins H1 and H3.

Figure 3 is a flexible ordering of different combinations of dystrophin proteins H1 and H3.

FIG. 4 is a graph showing the results of the immunogenicity calculations.

FIG. 5 shows the results of the immunogenicity analysis of various combinations of dystrophin proteins H1 and H3.

Detailed Description

Reference will now be made in detail to the exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Rather, they are merely illustrative of some aspects of the invention, which are detailed in the appended claims, and should not be construed to limit the scope of the invention.

Design scheme of one, mDp116 mini-sized anti-muscular atrophy protein

Dystrophin proteins can be structurally divided into four regions. One end of the protein is combined with F-actin through an N-terminal area, and the other end is combined with a muscle membrane through the interaction of a cysteine-rich area, a C-terminal area and DGC, so that the protein plays a role in mechanically stabilizing cell membranes in muscle tissues. Dystrophin has now been found to exist in at least 8 homologous proteins, which are widely distributed in the nervous system and muscle tissues, and expressed in other tissues as Dp427, Dp140, Dp116, etc. Although the functions of these homologous proteins are currently poorly known, the existence of these dystrophin isoforms provides a theoretical basis for our design of functional, stable mini-dystrophin proteins. The mDp140/116 mini-dystrophin protein of the present invention comprises all protein sequences of Dp140, Dp116, and its other end connects N terminal region and H1 region (FIG. 1, wherein ABD corresponds to SEQ ID NO:2, SP22/24 fragment corresponds to SEQ ID NO:3, H4 and CH fragment corresponds to SEQ ID NO:4), in order to further maintain the flexibility of the protein of Dp140, Dp116, and maintain the stability of the structure, the flexibility prediction is performed on H1/H3 fragment, each side of H3 away from the cell membrane is reduced by one amino acid, H1 is increased by one amino acid near the muscle filament end, the following 30 connection modes (FIG. 2) are designed, and the flexibility thereof is predicted on-line by using pep-fold3, wherein in 30 different combinations, design 1 has the best flexibility, but the number of amino acids related to N terminal is too small, designs 10,11 and 15 provide better flexibility (FIG. 3), in order to further ensure the safety of the recombinant protein, the IEDB is used for analyzing the immunogenicity, wherein the design 10 (shown as SEQ ID NO: 1) has very low immunogenicity (FIGS. 4 and 5), and in conclusion, the novel recombinant protein (namely the anti-muscular atrophy fusion protein mDp116 provided by the invention) containing the design 10 has obvious advantages in structural stability, function, immune response and the like compared with other aspects, and the amino acid sequence of the recombinant protein mDp116 is shown as SEQ ID NO: 5.

Process and method for preparing high titer rAAV-CMV-mDp116

1. Artificially synthesized gene expression cassette mDp116

Since AAV is typically capable of packaging genes within 5Kb in length, functional truncations were used for DMD repair genes. In order to achieve better gene expression activity, a gene expression cassette of codon-optimized (3492bp, 1164aa) mDp116 is synthesized by a synthetic method, a high-efficiency CMV promoter is used as the promoter, a reduced CW3L sequence (Choi et al. molecular Brain 2014,7:17) with WPRE and SV40PA functions is adopted as an RNA stabilizing element, and the gene expression cassette is placed between Not1 enzyme cutting sites of a pBluescript SK (+) vector to obtain a pBlue-CMV-mDp116-CW3L plasmid vector. The actual packaged gene fragment length of the rAAV-CMV-mDp116-CW3L is about 5028 bp.

2. Construction of shuttle vector plasmid pFD-Rep-Cap9- (ITR-CMV-mDp116-CW3L)

Next, the OptimUTRO gene expression cassette in pBlue-CMV-OptimUTRO-CW3L plasmid carrying rAAV-CMV-OptimUTRO genome is replaced into shuttle vector plasmid pFD-Rep-Cap9- (ITR-GOI) by Not1 enzyme cutting site to obtain pFD-Rep-Cap9- (ITR-CMV-mDp116-CW3L), the shuttle plasmid carries capsid protein gene Cap9 of type 9 AAV, so the packaging is the type 9 rAAV.

3. Large-scale preparation of rAAV-CMV-mDp116-CW3L by using novel OneBac system

In order to prepare a large amount of rAAV with high titer and high quality, a novel high-efficiency OneBac system (Wu et al. mol. the method Clin Dev.2018 Jul 4; 10:38-47.) for preparing rAAV by infecting Sf9 cells with baculovirus is adopted, recombinant Baculovirus (BEV) is prepared according to the operation method of the Bac-to-Bac system, and a recombinant shuttle plasmid pFD-Rep-Cap9- (ITR-CMV-OptimtUTRO-CW 3L) is transformed into escherichia coli DH10Bac containing a recombinant baculovirus genome to carry out Tn7 enzyme-mediated homologous recombination, so as to obtain recombinant bacmid DNA containing the rAAV-CMV-OptimtRO-CW 3L genome. Then, Bacmid DNA was transfected into insect Sf9 cells, the culture was continued until a significant cytopathic effect (CPE) caused by BEV infection was produced, and cell culture supernatant was collected, which contained a large amount of BEV/Rep-Cap9- (ITR-CMV-mDp116-CW 3L). Then, Sf9 cells cultured in suspension are infected by the BEV virus inoculation, and after 72h infection, Sf9 cell sediment is collected for subsequent purification of rAAV.

Purification and titer determination of rAAV-CMV-mDp116-CW3L

Sf9 cells collected after infection were resuspended in lysis buffer at a density of 2X 107cells/mL (50mM Trisand 2mM MgCl2[ pH 7.5 ]). The cells were freeze-thawed repeatedly 3 times in liquid nitrogen and 37 ℃ water bath, and 50U/mL nuclease (benzonase, Sigma) and 150mM NaCl were added to the lysate and treated at 37 ℃ for 1 hour. Then centrifuged at 2,500 Xg for 15min and the supernatant was further purified by density gradient centrifugation with iodixanol (iodioxanol) (Strobel et al. hum. Gene Ther. methods.2015; 26: 147-. The method mainly comprises the following steps: a15%, 25%, 40%, and 58% concentration gradient of 60% iodoxanol (OptiPrep; Sigma) in buffered PBS-MK Buffer (1 XPBS, 1mM MgCl2,2.5mM KCl) was added sequentially to a 39mL Quick-Seal tubes (Beckman Coulter) ultracentrifuge tube, followed by addition of the lysate supernatant containing the treated rAAV, and centrifuged at 18 ℃ for 2 hours using a 70Ti rotor of a Backman ultracentrifuge at 63,000 rpm. After centrifugation, the solution of 40% gradient phase is absorbed, dialyzed by PBS buffer solution, and then centrifugally ultrafiltered and concentrated by an Amicon Ultra-15(MWCO,100 kDa; Merck Millipore) ultrafiltration tube to obtain purified rAAV, and the rAAV is frozen and stored at minus 80 ℃. The titer of the purified rAAV virus was determined by fluorescent quantitative PCR using iQ SYBRGreen Supermix kit (Bio-Rad) and CMV promoter-specific quantitative primers CMV-F and CMV-R selected from the quantitative primers based on a 10-fold dilution of a plasmid containing a CMV sequence, and the titer of the virus was expressed by Vector Genome (VG)/ml (Grieger et al. Nat. Protoc.2006; 1: 1412. 1428).

Functional assessment of high titer rAAV-CMV-mDp116

1. Vector processing and tissue storage

We randomly blinded newborn MDX mice (day 7) and wild type (C57 mice, Jackson laboratories) and labeled with an Aramis Micro tatoo kit (Ketchum Manufacturing Inc, Canada) followed by intraperitoneal injection (Hamilton, Model 1710SN syring, Lot No.81008) at a 2.5X1012 rAAV-CMV-mDp116 dose. Based on this, C57BL/10SNJ andMDX pups were injected with either 50-250 μ LPBS as a negative control or PBS-diluted rAAV-CMV-mDp116 using a 32-gauge insulin syringe. Each mouse was weighed prior to injection. After dosing, all mice returned to the litter and were weaned off. At approximately 8 weeks of age, MDX and C57BL/10SNJ mice were euthanized according to institutional policy with CO 2. The heart, tibialis anterior, gastrocnemius, quadriceps femoris, triceps, abdomen, diaphragm, temporalis and liver were removed for the next study; according to studies, AAV9 showed less than 100-fold non-target gene expression in mice, and thus the other fraction was stored but not utilized. The designated tissue samples were placed in OCT (tissue-TEK) containing an embedding mold (Richard alan scientific) and snap frozen in liquid nitrogen cooled isopentane. Additional designated biological tissue samples were placed in tissue containers and flash frozen in liquid nitrogen. All samples were stored at-80 ℃. Frozen sections 5-7 μm thick were processed at-25 ℃ in a cryostat (Microm)^TMHM550, Thermo Scientific, usa) was cut and mounted on slides (Superfrost Plus, Fisher Scientific, usa), and all samples were stored in a "sample database of 6-week injections".

2. Dual N-terminal and C-terminal staining of dystrophia-related proteins

All sections of muscle specimens were immunostained for Utrophin using n-terminal polyclonal and c-terminal monoclonal antibodies (which bind to Utrophin without binding to native protein). First, incubation was carried out in 1% Triton X-100(Roche diagnostics GmbH, Mannheim, Germany) for 20 minutes, followed by dilution in 0.01M PBS (Roche diagnostics GmbH, Mannheim, Germany) and rinsing of the sample in PBS three times for 5 minutes (3X 5 minutes). Sections were incubated in 5% normal donkey serum for 15min and then with N-terminal anti-dystrophin-related antibody (N-19, SC-7460, goat polyclonal IgG, Santa Cruz, CA, USA, dilution 1:50) for 60 min at 37 ℃. After a second cycle of 3 × 5min PBS wash, incubate with 5% normal donkey serum for 15min at room temperature. The sections prepared were incubated in donkey serum containing goat polyclonal secondary antibody (sc-2024, Santa Cruz, Calif., USA, dilution 1:300) at 37 ℃ for 30 minutes. After a third pbs wash of 3 × 5 minutes, sections were first incubated with 10% normal goat serum (invitrogen, Scotland, UK) for 15 minutes, then with c-terminal utrophin antibody (mancho7, mouse monoclonal protein igG2a, santa cruz, ca, usa, diluted 1:25 at 37 ℃) for 60 minutes. After washing for 3 × 5 minutes in PBS and incubation with 10% normal goat serum, sections were incubated for 30 minutes in goat serum containing anti-mouse IgG2A phosphor 594(A-21140, Life Technologies, USA, dilution 1:300) at 37 ℃. The plates were washed again for 3X5 minutes in PBS and mounted on Vectashield blocking agent (H-1000) (vector laboratories, Calif., USA) or blocking agent with DAPI (H-1500) (veterinary laboratories). Photographs were taken using a Leica DM6000B microscope (Leica, Germany).

3. Dual immunofluorescent staining for gamma-transmembrane/laminin, MuRF-1/laminin and MyHC-embryo/laminin, MYH16

The staining procedure for the proteins was the same as described previously. Rabbit anti-gamma-transmembrane protein (NBP1-59744, Novus Biologicals, Littleton, Co.) and MURF1(NBP1-31207, Novus Biologicals, Littleton, Co.) polyclonal antibodies were diluted 1:50 in PBS with Bovine Serum Albumin (BSA). MyHC-embryonic monoclonal antibody (f1.652) (development studios, Hybridoma Bank, Iowa, USA) was used at a dilution of 1:50 to 1:100 in pbs. The identification of myofibers was carried out using chicken laminin polyclonal antibodies (ab14055-50, Abcam, Cambridge, MA, USA) obtained by dilution at 1:500-1:1000 and anti-chicken IgY (TR) antibodies (ab7116, Abcam, Cambridge, MA, USA, dilution 1:300) as the secondary antibody. MYH16 rabbit polyclonal antibody peptide sequences were generated using human and canine MYH16 "2 loop" region sequences. The sensitive and fully specific binding was verified by the dominant myosin subtype expressed in the temporal muscle of dogs. Peptide sequence: LLALLFKEEEAPAGS

4. Hematoxylin-eosin staining (H & E)

The slices with a thickness of 7m were air-dried at room temperature for 15 minutes. The cells were then stained with Harris' hematoxylin for 2.5 minutes, rinsed in distilled water, soaked in 0.1% acetic acid for 15 seconds, then rinsed repeatedly in tap water for 4 minutes, and counterstained with 1% eosin for 1 minute. In the last step, dehydration was carried out three times in ethanol for 2 minutes each. Representative, non-overlapping sections were photographed and recorded under high power vision (HPF).

5. Immunoblot analysis

10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 20-40. mu.g of whole cell or whole muscle lysate loaded per well. The proteins were transferred to a polyvinylidene fluoride membrane (PVDF membrane). The primary antibody was a goat polyclonal antibody, mu UTrophin was detected by N-terminal localization, diluted 1:500 fold (N-19, SC-760, St. Cruis, USA), and the secondary antibody was an donkey anti-goat antibody conjugated to horseradish peroxidase (Sigma Aldrich), diluted 1: 5000. Protein detection and quantification was performed using the Odyssey Infrared imaging System (LI-COR). The gamma-transmembrane protein was detected with a mouse monoclonal antibody (vector laboratories VP-G803) and a donkey anti-mouse conjugated HRP secondary antibody ((Santa Cruz Biotechnology).

6. In situ end-marker analysis

Sections were initially fixed in 10% formalin phosphate buffer (Fisher Scientific, USA) for 20 min. The DNA fragments were then nicked end-labeled in situ using the TACS 2TdT fluorescein apoptosis detection kit (Trevigen, Gaithersburg, Md., USA) as described by the manufacturer.

7. Serum Creatine Kinase (CK) assay

Serum was collected via inframandibular venipuncture using a 5mm Animal scalpel (golden Animal Lancet, Braintree Scientific, Inc, Braintree, MA). Mu.l of the sample was collected in a heparinized blood collection tube (Terumo, Cat.: TMLH). Mice were monitored for signs of potential distress within 30 minutes after blood draw. Clinical pathology laboratory measurement.

8. In vitro evaluation of muscle contraction Performance

An Aurora Mouse 1200A system is adopted, dynamic muscle control v.5.3 software is utilized, and physiological indexes such as isometric contractility, isometric strength and relaxation tension, force reduction after ECCs and the like are measured on the muscle freshly separated from 2-month-old mdx mice. All these mice were tested for in vivo grip strength 24 hours prior to euthanasia and ex vivo testing. EDL muscle was kept in Ringer solution (100mM NaCl,4.7mM KCl,3.4mM CaCl2,1.2mM KH2PO4,1.2mM MgSO4,25mM HEPES and 5.5mM D-glucose) with continuous oxygenation at 24 ℃. The tic stimulation protocol used was a single stimulation with a duration of 0.2 milliseconds. To measure the diastolic force maximum, the same stimulation was repeated at a frequency of 120 hertz for 500 milliseconds. The interval between two tonic contractions was 5 minutes to ensure muscle recovery. Muscle length is adjusted to achieve maximal twitch response. This length is measured between the outermost visible ends of the tendon junctions and is recorded as the optimal length (L0). After testing the equidistant properties of the EDL, a series of six eccentric contractions (ECCS-once every five minutes) were applied to the cycle, repeated 500 milliseconds of isometric contraction, then the muscles were stretched 10% L0 for the last 200 milliseconds, while maximum tonic stimulation was given. The absolute force reported by each ECC corresponds to the peak force of the equidistant stages of ECC.

Physiological test in mdx mice

To reduce bias to ensure consistency and robustness of the blind experiments, we used IACUC approved protocol. Within 24 hours, the spontaneous wheel distance was measured. Baseline vertical movement was measured for 5 minutes and then rested for 3 minutes in the original cage. The force is measured using an axial force sensor. When measuring the downhill driving distance, the treadmill gradient is +15 degrees, and the speed is increased from 10m/min (10min), 11m/min (1min) to 12m/min (6 min). The test is terminated when three consecutive shocks or 17 minutes of testing are completed.

Sequence listing

<110> Beijing Huizui-Hezhongzhou Biotech Co., Ltd

<120> an dystrophin fusion protein mDp116

<141>2020-01-02

<160>5

<170>SIPOSequenceListing 1.0

<210>1

<211>57

<212>PRT

<213> Intelligent (Homo sapiens)

<400>1

Met Leu Pro Arg Pro Pro Lys Val Thr Lys Gln Pro Asp Leu Ala Pro

1 5 10 15

Gly Leu Thr Thr Ile Gly Ala Ser Pro Thr Gln Thr Val Thr Leu Val

20 25 30

Thr Gln Pro Val Val Thr Lys Glu Thr Ala Ile Ser Lys Leu Glu Met

35 40 45

Pro Ser Ser Leu Met Leu Glu Val Pro

50 55

<210>2

<211>251

<212>PRT

<213> Intelligent (Homo sapiens)

<400>2

Met Leu Trp Trp Glu Glu Val Glu Asp Cys Glu Arg Glu Asp Val Gln

1 5 10 15

Lys Lys Thr Phe Thr Lys Trp Val Asn Ala Gln Phe Ser Lys Phe Gly

20 25 30

Lys Gln His Ile Glu Asn Leu Phe Ser Asp Leu Gln Asp Gly Arg Arg

35 40 45

Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Gln Lys Leu Pro Lys Glu

50 55 60

Lys Gly Ser Thr Arg Val His Ala Leu Asn Asn Val Asn Lys Ala Leu

65 70 75 80

Arg Val Leu Gln Asn Asn Asn Val Asp Leu Val Asn Ile Gly Ser Thr

85 90 95

Asp Ile Val Asp Gly Asn His Lys Leu Thr Leu Gly Leu Ile Trp Asn

100 105 110

Ile Ile Leu His Trp Gln Val Lys Asn Val Met Lys Asn Ile Met Ala

115 120 125

Gly Leu Gln Gln Thr Asn Ser Glu Lys Ile Leu Leu Ser Trp Val Arg

130 135 140

Gln Ser Thr Arg Asn Tyr Pro Gln Val Asn Val Ile Asn Phe Thr Thr

145 150 155 160

Ser Trp Ser Asp Gly Leu Ala Leu Asn Ala Leu Ile His Ser His Arg

165 170 175

Pro Asp Leu Phe Asp Trp Asn Ser Val Val Cys Gln Gln Ser Ala Thr

180 185 190

Gln Arg Leu Glu His Ala Phe Asn Ile Ala Arg Tyr Gln Leu Gly Ile

195 200 205

Glu Lys Leu Leu Asp Pro Glu Asp Val Asp Thr Thr Tyr Pro Asp Lys

210 215 220

Lys Ser Ile Leu Met Tyr Ile Thr Ser Leu Phe Gln Val Leu Pro Gln

225 230 235 240

Gln Val Ser Ile Glu Ala Ile Gln Glu Val Glu

245 250

<210>3

<211>354

<212>PRT

<213> Intelligent (Homo sapiens)

<400>3

Thr His Arg Leu Leu Gln Gln Phe Pro Leu Asp Leu Glu Lys Phe Leu

1 5 10 15

Ala Trp Leu Thr Glu Ala Glu Thr Thr Ala Asn Val Leu Gln Asp Ala

20 25 30

Thr Arg Lys Glu Arg Leu Leu Glu Asp Ser Lys Gly Val Lys Glu Leu

35 40 45

Met Lys Gln Trp Gln Asp Leu Gln Gly Glu Ile Glu Ala His Thr Asp

50 55 60

Val Tyr His Asn Leu Asp Glu Asn Ser Gln Lys Ile Leu Arg Ser Leu

65 70 75 80

Glu Gly Ser Asp Asp Ala Val Leu Leu Gln Arg Arg Leu Asp Asn Met

85 90 95

Asn Phe Lys Trp Ser Glu Leu Arg Lys Lys Ser Leu Asn Ile Arg Ser

100 105 110

His Leu Glu Ala Ser Ser Asp Gln Trp Lys Arg Leu His Leu Ser Leu

115 120 125

Gln Glu Leu Leu Val Trp Leu Gln Leu Lys Asp Asp Glu Leu Ser Arg

130 135 140

Gln Ala Pro Ile Gly Gly Asp Phe Pro Ala Val Gln Lys Gln Asn Asp

145 150 155 160

Val His Arg Ala Phe Lys Arg Glu Leu Lys Thr Lys Glu Pro Val Ile

165 170 175

Met Ser Thr Leu Glu Thr Val Arg Ile Phe Leu Thr Glu Gln Pro Leu

180 185 190

Glu Gly Leu Glu Lys Leu Tyr Gln Glu Pro Arg Glu Leu Pro Pro Glu

195 200 205

Glu Arg Ala Gln Asn Val Thr Arg Leu Leu Arg Lys Gln Ala Glu Glu

210 215 220

Val Asn Thr Glu Trp Glu Lys Leu Asn Leu His Ser Ala Asp Trp Gln

225 230 235 240

Arg Lys Ile Asp Glu Thr Leu Glu Arg Leu Gln Glu Leu Gln Glu Ala

245 250 255

Thr Asp Glu Leu Asp Leu Lys Leu Arg Gln Ala Glu Val Ile Lys Gly

260 265 270

Ser Trp Gln Pro Val Gly Asp Leu Leu Ile Asp Ser Leu Gln Asp His

275 280 285

Leu Glu Lys Val Lys Ala Leu Arg Gly Glu Ile Ala Pro Leu Lys Glu

290 295 300

Asn Val Ser His Val Asn Asp Leu Ala Arg Gln Leu Thr Thr Leu Gly

305 310 315 320

Ile Gln Leu Ser Pro Tyr Asn Leu Ser Thr Leu Glu Asp Leu Asn Thr

325 330 335

Arg Trp Lys Leu Leu Gln Val Ala Val Glu Asp Arg Val Arg Gln Leu

340 345 350

His Glu

<210>4

<211>314

<212>PRT

<213> Intelligent (Homo sapiens)

<400>4

Ala His Arg Asp Phe Gly Pro Ala Ser Gln His Phe Leu Ser Thr Ser

1 5 10 15

Val Gln Gly Pro Trp Glu Arg Ala Ile Ser Pro Asn Lys Val Pro Tyr

20 25 30

Tyr Ile Asn His Glu Thr Gln Thr Thr Cys Trp Asp His Pro Lys Met

35 40 45

Thr Glu Leu Tyr Gln Ser Leu Ala Asp Leu Asn Asn Val Arg Phe Ser

50 55 60

Ala Tyr Arg Thr Ala Met Lys Leu Arg Arg Leu Gln Lys Ala Leu Cys

65 70 75 80

Leu Asp Leu Leu Ser Leu Ser Ala Ala Cys Asp Ala Leu Asp Gln His

85 90 95

Asn Leu Lys Gln Asn Asp Gln Pro Met Asp Ile Leu Gln Ile Ile Asn

100 105 110

Cys Leu Thr Thr Ile Tyr Asp Arg Leu Glu Gln Glu His Asn Asn Leu

115 120 125

Val Asn Val Pro Leu Cys Val Asp Met Cys Leu Asn Trp Leu Leu Asn

130 135 140

Val Tyr Asp Thr Gly Arg Thr Gly Arg Ile Arg Val Leu Ser Phe Lys

145 150 155 160

Thr Gly Ile Ile Ser Leu Cys Lys Ala His Leu Glu Asp Lys Tyr Arg

165 170 175

Tyr Leu Phe Lys Gln Val Ala Ser Ser Thr Gly Phe Cys Asp Gln Arg

180 185 190

Arg Leu Gly Leu Leu Leu His Asp Ser Ile Gln Ile Pro Arg Gln Leu

195 200 205

Gly Glu Val Ala Ser Phe Gly Gly Ser Asn Ile Glu Pro Ser Val Arg

210 215 220

Ser Cys Phe Gln Phe Ala Asn Asn Lys Pro Glu Ile Glu Ala Ala Leu

225 230 235 240

Phe Leu Asp Trp Met Arg Leu Glu Pro Gln Ser Met Val Trp Leu Pro

245 250 255

Val Leu His Arg Val Ala Ala Ala Glu Thr Ala Lys His Gln Ala Lys

260 265 270

Cys Asn Ile Cys Lys Glu Cys Pro Ile Ile Gly Phe Arg Tyr Arg Ser

275 280 285

Leu Lys His Phe Asn Tyr Asp Ile Cys Gln Ser Cys Phe Phe Ser Gly

290 295 300

Arg Val Ala Lys Gly His Lys Met His Tyr

305 310

<210>5

<211>976

<212>PRT

<213> Intelligent (Homo sapiens)

<400>5

Met Leu Trp Trp Glu Glu Val Glu Asp Cys Glu Arg Glu Asp Val Gln

1 5 10 15

Lys Lys Thr Phe Thr Lys Trp Val Asn Ala Gln Phe Ser Lys Phe Gly

20 25 30

Lys Gln His Ile Glu Asn Leu Phe Ser Asp Leu Gln Asp Gly Arg Arg

35 40 45

Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Gln Lys Leu Pro Lys Glu

50 55 60

Lys Gly Ser Thr Arg Val His Ala Leu Asn Asn Val Asn Lys Ala Leu

65 70 75 80

Arg Val Leu Gln Asn Asn Asn Val Asp Leu Val Asn Ile Gly Ser Thr

85 90 95

Asp Ile Val Asp Gly Asn His Lys Leu Thr Leu Gly Leu Ile Trp Asn

100 105 110

Ile Ile Leu His Trp Gln Val Lys Asn Val Met Lys Asn Ile Met Ala

115 120 125

Gly Leu Gln Gln Thr Asn Ser Glu Lys Ile Leu Leu Ser Trp Val Arg

130 135 140

Gln Ser Thr Arg Asn Tyr Pro Gln Val Asn Val Ile Asn Phe ThrThr

145 150 155 160

Ser Trp Ser Asp Gly Leu Ala Leu Asn Ala Leu Ile His Ser His Arg

165 170 175

Pro Asp Leu Phe Asp Trp Asn Ser Val Val Cys Gln Gln Ser Ala Thr

180 185 190

Gln Arg Leu Glu His Ala Phe Asn Ile Ala Arg Tyr Gln Leu Gly Ile

195 200 205

Glu Lys Leu Leu Asp Pro Glu Asp Val Asp Thr Thr Tyr Pro Asp Lys

210 215 220

Lys Ser Ile Leu Met Tyr Ile Thr Ser Leu Phe Gln Val Leu Pro Gln

225 230 235 240

Gln Val Ser Ile Glu Ala Ile Gln Glu Val Glu Met Leu Pro Arg Pro

245 250 255

Pro Lys Val Thr Lys Gln Pro Asp Leu Ala Pro Gly Leu Thr Thr Ile

260 265 270

Gly Ala Ser Pro Thr Gln Thr Val Thr Leu Val Thr Gln Pro Val Val

275 280 285

Thr Lys Glu Thr Ala Ile Ser Lys Leu Glu Met Pro Ser Ser Leu Met

290 295 300

Leu Glu Val Pro Thr His Arg Leu Leu Gln Gln Phe Pro Leu Asp Leu

305 310 315 320

Glu Lys Phe Leu Ala Trp Leu Thr Glu Ala Glu Thr Thr Ala Asn Val

325 330 335

Leu Gln Asp Ala Thr Arg Lys Glu Arg Leu Leu Glu Asp Ser Lys Gly

340 345 350

Val Lys Glu Leu Met Lys Gln Trp Gln Asp Leu Gln Gly Glu Ile Glu

355 360 365

Ala His Thr Asp Val Tyr His Asn Leu Asp Glu Asn Ser Gln Lys Ile

370 375 380

Leu Arg Ser Leu Glu Gly Ser Asp Asp Ala Val Leu Leu Gln Arg Arg

385 390 395 400

Leu Asp Asn Met Asn Phe Lys Trp Ser Glu Leu Arg Lys Lys Ser Leu

405 410 415

Asn Ile Arg Ser His Leu Glu Ala Ser Ser Asp Gln Trp Lys Arg Leu

420 425 430

His Leu Ser Leu Gln Glu Leu Leu Val Trp Leu Gln Leu Lys Asp Asp

435 440 445

Glu Leu Ser Arg Gln Ala Pro Ile Gly Gly Asp Phe Pro Ala Val Gln

450 455 460

Lys Gln Asn Asp Val His Arg Ala Phe Lys Arg Glu Leu Lys Thr Lys

465 470 475 480

Glu Pro Val Ile Met Ser Thr Leu Glu Thr Val Arg Ile Phe Leu Thr

485 490 495

Glu Gln Pro Leu Glu Gly Leu Glu Lys Leu Tyr Gln Glu Pro Arg Glu

500 505 510

Leu Pro Pro Glu Glu Arg Ala Gln Asn Val Thr Arg Leu Leu Arg Lys

515 520 525

Gln Ala Glu Glu Val Asn Thr Glu Trp Glu Lys Leu Asn Leu His Ser

530 535 540

Ala Asp Trp Gln Arg Lys Ile Asp Glu Thr Leu Glu Arg Leu Gln Glu

545 550 555 560

Leu Gln Glu Ala Thr Asp Glu Leu Asp Leu Lys Leu Arg Gln Ala Glu

565 570 575

Val Ile Lys Gly Ser Trp Gln Pro Val Gly Asp Leu Leu Ile Asp Ser

580 585 590

Leu Gln Asp His Leu Glu Lys Val Lys Ala Leu Arg Gly Glu Ile Ala

595 600 605

Pro Leu Lys Glu Asn Val Ser His Val Asn Asp Leu Ala Arg Gln Leu

610 615 620

Thr Thr Leu Gly Ile Gln Leu Ser Pro Tyr Asn Leu Ser Thr Leu Glu

625 630 635 640

Asp Leu Asn Thr Arg Trp Lys Leu Leu Gln Val Ala Val Glu Asp Arg

645 650 655

Val Arg Gln Leu His Glu Ala His Arg Asp Phe Gly Pro Ala Ser Gln

660 665 670

His Phe Leu Ser Thr Ser Val Gln Gly Pro Trp Glu Arg Ala Ile Ser

675 680 685

Pro Asn Lys Val Pro Tyr Tyr Ile Asn His Glu Thr Gln Thr Thr Cys

690 695 700

Trp Asp His Pro Lys Met Thr Glu Leu Tyr Gln Ser Leu Ala Asp Leu

705 710 715 720

Asn Asn Val Arg Phe Ser Ala Tyr Arg Thr Ala Met Lys Leu Arg Arg

725 730 735

Leu Gln Lys Ala Leu Cys Leu Asp Leu Leu Ser Leu Ser Ala Ala Cys

740 745 750

Asp Ala Leu Asp Gln His Asn Leu Lys Gln Asn Asp Gln Pro Met Asp

755 760 765

Ile Leu Gln Ile Ile Asn Cys Leu Thr Thr Ile Tyr Asp Arg Leu Glu

770 775 780

Gln Glu His Asn Asn Leu Val Asn Val Pro Leu Cys Val Asp Met Cys

785 790 795 800

Leu Asn Trp Leu Leu Asn Val Tyr Asp Thr Gly Arg Thr Gly Arg Ile

805 810 815

Arg Val Leu Ser Phe Lys Thr Gly Ile Ile Ser Leu Cys Lys Ala His

820 825 830

Leu Glu Asp Lys Tyr Arg Tyr Leu Phe Lys Gln Val Ala Ser Ser Thr

835 840 845

Gly Phe Cys Asp Gln Arg Arg Leu Gly Leu Leu Leu His Asp Ser Ile

850 855 860

Gln Ile Pro Arg Gln Leu Gly Glu Val Ala Ser Phe Gly Gly Ser Asn

865 870 875 880

Ile Glu Pro Ser Val Arg Ser Cys Phe Gln Phe Ala Asn Asn Lys Pro

885 890 895

Glu Ile Glu Ala Ala Leu Phe Leu Asp Trp Met Arg Leu Glu Pro Gln

900 905 910

Ser Met Val Trp Leu Pro Val Leu His Arg Val Ala Ala Ala Glu Thr

915 920 925

Ala Lys His Gln Ala Lys Cys Asn Ile Cys Lys Glu Cys Pro Ile Ile

930 935 940

Gly Phe Arg Tyr Arg Ser Leu Lys His Phe Asn Tyr Asp Ile Cys Gln

945 950 955 960

Ser Cys Phe Phe Ser Gly Arg Val Ala Lys Gly His Lys Met His Tyr

965 970 975

Claims (10)

1. A fusion protein, characterized in that: the fusion protein comprises an amino acid sequence shown as SEQ ID NO. 1.

2. The fusion protein of claim 1, wherein: the fusion protein further comprises amino acid sequences shown as SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4.

3. The fusion protein of claim 1, wherein: the amino acid sequence of the fusion protein sequentially comprises SEQ ID NO 2, SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 4 from the N end to the C end.

4. The fusion protein of claim 1, wherein: the fusion protein comprises an amino acid sequence shown as SEQ ID NO. 5.

5. A nucleic acid molecule encoding the fusion protein of any one of claims 1-4.

6. A nucleic acid vector comprising the nucleic acid molecule of claim 5.

7. The nucleic acid vector of claim 6, wherein: the nucleic acid vector is an adenovirus vector.

8. A cell comprising the nucleic acid molecule of claim 5 and/or expressing the fusion protein of any one of claims 1-4.

9. Use of the fusion protein of any one of claims 1-4, the nucleic acid molecule of claim 5, the nucleic acid vector of claim 6, the cell of claim 8 for the manufacture of a medicament for treating duchenne muscular dystrophy and/or for improving cognitive function in a patient.

10. A pharmaceutical composition comprising the fusion protein of any one of claims 1-4, and/or the nucleic acid molecule of claim 5, and/or the nucleic acid vector of claim 6, and/or the cell of claim 8, and a pharmaceutically acceptable carrier or adjuvant.

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