CN108611355B

CN108611355B - Gene medicine for X chromosome linked myotubule myopathy

Info

Publication number: CN108611355B
Application number: CN201810417620.3A
Authority: CN
Inventors: 田文洪; 马思思; 董小岩; 吴小兵
Original assignee: Beijing Jinlan Gene Technology Co ltd
Current assignee: Beijing Jinlan Gene Technology Co ltd
Priority date: 2018-05-04
Filing date: 2018-05-04
Publication date: 2021-08-31
Anticipated expiration: 2038-05-04
Also published as: CN108611355A

Abstract

The invention provides a recombinant adeno-associated virus mediated X chromosome linked myotubule myopathy treatment drug. The recombinant adeno-associated virus vector carries a myotubulin gene expression frame containing human miR-142-3p and miR-122 target sequences. In vivo experiments show that the recombinant adeno-associated virus vector can be efficiently introduced into a body to continuously and stably express myotubulin, prolong the life cycle of an animal model and recover the growth and development of the animal model. The results suggest that the recombinant adeno-associated virus vector is hopefully developed into a novel X chromosome-linked myotubule myopathy treatment drug.

Description

Gene medicine for X chromosome linked myotubule myopathy

Technical Field

The invention relates to the technical field of biology, in particular to an X chromosome linked myotubule myopathy gene drug with a recombinant adeno-associated virus vector carrying an expression frame of an artificially designed myotubulin gene (mtm 1).

Background

X-linked myotubule myopathy (XLMTM) is a congenital muscular dystrophy, mainly due to the failure of myofibrils to form myotubules, resulting in poor myotubule formation, thus disrupting muscle tissue development and staying in fetal muscle tissue, and muscle cells are found in patient muscle sections, but the nucleus is not scattered around the muscle fibers as mature muscle tissue, but is located in the center of the cell. XLMTM disease is caused by mutations in the X chromosome-linked myotubulin (myotubulin, mtm 1) gene. The mtm1 gene encodes aA phosphatase capable of modulating PIP and PIP in a cell₂The ratio of (a) and (b) plays an important role in the cell signaling process. Myotubulin (hereinafter referred to as "MTM 1 protein") is widely expressed in cells, but shows a function only in muscle cells. mutation of the mtm1 gene resulted in XLMTM disease with an incidence of 1/50000 newborn male infants (Amburgey K, et al neurology, 2017; 89(13): 1355-. Currently, XLMTM disease has no effective therapeutic drugs and methods, and enzyme replacement therapy and gene therapy are possible therapeutic strategies.

In 2013, Lawlor MW et al evaluated the effect of MTM1 protein, a fusion antibody binding to the Fv fragment, on an XLMTM mouse model (Lawlor MW, et al Human Molecular genetics. 2013; 22: 1525-1538.). The result shows that the protein can effectively improve the muscle strength and reverse the pathological change characteristics in a model mouse, and shows a certain therapeutic effect. Although the binding of the antibody to the Fv fragment increases the targeting of the MTM1 protein in vivo, the MTM1 protein acts in cells, and the protein is required to penetrate the cell membrane into cytoplasm, so that the efficiency is low and the effect is greatly influenced. As a protein drug, continuous administration is required, the compliance of patients is low, and the protein drug is not an ideal therapeutic drug candidate. In the aspect of gene and drug research, research results of Buj-Bello A and the like in 2008 show that an XLMTM mouse model carrying an mtm1 gene expression cassette AAV vector can effectively recover the muscle function near an injection site by intramuscular injection (Buj-Bello A, et al Human Molecular genetics. 2008; 17: 2132-. Subsequently, Childers MK and the like construct a mouse MTM1 gene expression frame and a dog MTM1 gene expression frame which are regulated and expressed by muscle-specific promoters, package the frames into AAV8 virus, and inject the virus into XLMTM model mice and model dogs to express and generate MTM1 protein, restore the normal function of the model and prolong the survival time (Childers MK, et al, Sci Transl Med 2014;6:220ra 10.). This study demonstrates the feasibility of systemic administration for the treatment of XLMTM. Mack DL and the like further prove that AAV 8-mediated systemic gene therapy can effectively correct systemic mtm1 gene mutation in an XLMTM dog model in 2017 (Mack DL, et al. Mol ther. 2017; 25(4): 839-854), and a scientific basis is laid for the clinical trial of the XLMTM gene drug based on AAV8 vectors. The patent (WO 2014/167253A 1) reports a gene therapy method of XLMTM, and the gene therapy method adopts AAV vectors carrying an mtm1 gene expression unit regulated by a muscle-specific promoter and is administrated by an intravenous injection system, wherein the serotypes of the AAV vectors are AAV8 and AAV 9. US patent (US 9895426) reports XLMTM gene therapy approaches similar to those of patent (WO 2014/167253 a 1). The main difference is that US patent (US 9895426) uses two animal models, XLMTM model mouse and XLMTMT model dog, to verify the effectiveness of the gene drug. Based on the above findings, various XLMTM gene drugs entered the clinical study stage (https:// clinicaltralals. gov /), with a number of relevant clinical trial items of 2.

Based on the full research of work of others, we designed a gene therapy drug rAAV-MCK-hMTM1-122T-142T for XLMTM. Firstly, the human mtm1 gene coding sequence (hMTM 1) is optimized and synthesized, the influence of mRNA secondary structure, rare codon and cryptic splice site on the transcription and translation of the mtm1 gene coding sequence is eliminated through sequence optimization, and the gene expression level is improved. Secondly, the transcription of hMTM1 is regulated by adopting a Mouse Creatine Kinase (MCK) promoter, and the MCK promoter has the characteristics of good muscle tissue specificity, high transcription level, short sequence and the like (Shield MA, et al. Mol Cell biol. 1996; 16(9): 5058-5068.), and is beneficial to the efficient expression of hMTM1 specificity in muscle tissue. In addition, the miR-142-3p target sequence is added in the 3' UTR (untranslated region) region of the Gene, so that the expression of the hMTM1 Gene in immune-related cells (such as antigen presenting cells) is maximally inhibited, and the generation probability of immune response to MTM1 protein is remarkably reduced (Boisgerault F, et al. hum Gene ther. 2013; 24 (4):393 and 405.). By utilizing the characteristic of specific high expression of miR-122 in normal liver (joint C. RNA biol. 2012; 9(2): 137-142.), a miR-122 target sequence is designed in the 3' UTR, so that the expression of hMTM1 gene in liver cells is reduced, and the safety of the hMTM1 gene is enhanced. A safe and non-pathogenic recombinant AAV vector (Dismuke DJ, et al Curr Gene ther, 2013; 13(6): 434-. Further improving the success possibility of drug development.

Glandular associationViruses (AAV) are known for their discovery in adenovirus preparations (atcheson RW,et al. Science. 1965; 149: 754-756.Hoggan MD, et alproc Natl Sci USA 1966; 55: 1467-. AAV is a member of the family parvoviridae (subvirus), and comprises multiple serotypes, the genome of which is single-stranded DNA (Rose JA,et alproc Natl Acad Sci USA 1969; 64: 863-. AAV is a dependent virus, requiring other viruses such as adenovirus, herpes simplex virus, and human papilloma virus (Geoffroy MC,et alcurr Gene ther 2005, (5 (3): 265-271), or an auxiliary factor provides an auxiliary function to copy. In the absence of helper virus, AAV infects cells and its genome integrates into the cell chromosome to become latent (Chiorini JA,et alcurr Top Microbiol Immunol. 1996; 218:25-33.) without production of progeny virus.

The first AAV virus isolated was serotype 2 AAV (AAV 2) (atcheson RW,et alscience 1965, 149: 754-. The AAV2 genome is about 4.7kb long, with Inverted Terminal Repeats (ITRs) of length 145bp at both ends of the genome, in a palindromic-hairpin structure (Lusby E,et alj Virol, 1980; 34: 402-409). There are two large Open Reading Frames (ORFs) in the genome, encoding the rep and cap genes, respectively. The full-length genome of AAV2 has been cloned into an e.coli plasmid (Samulski RJ,et al. Proc Natl Acad Sci USA. 1982; 79: 2077-2081. Laughlin CA, et al. Gene. 1983; 23: 65-73.)。

ITRs are cis-acting elements of the AAV vector genome that play important roles in integration, rescue, replication, and genome packaging of AAV viruses (Xiao X,et alj Virol, 1997, (71) (2) 941-948). The ITR sequences contain a Rep protein binding site (RBS) and a terminal melting site, trs (terminal resolution site), which are recognized by Rep protein binding and nicked at trs (Linden RM,et alproc Natl Acad Sci USA 1996; 93(15): 7966-. The ITR sequences can also form a unique 'T' letter type secondary structure and play an important role in the life cycle of AAV viruses(Ashktorab H, et al. J Virol. 1989; 63(7): 3034-3039.)。

The remainder of the AAV2 genome can be divided into 2 functional regions, the rep and cap gene regions (Srivastava a,et alj Virol, 1983, 45(2), 555-. The Rep gene region encodes four Rep proteins, Rep78, Rep68, Rep52 and Rep 40. Rep proteins play an important role in replication, integration, rescue and packaging of AAV viruses. Wherein Rep78 and Rep68 specifically bind to terminal melting sites trs (terminal resolution site) and the GAGY repeat motif in ITRs (Huser D,et alPLoS Patholog.2010, 6(7) e1000985. the replication process of AAV genome from single strand to double strand is initiated. The trs and GAGC repeat motifs in the ITRs are central to replication of the AAV genome, and therefore although the ITR sequences are not identical in all serotypes of AAV virus, both hairpin structures are formed and Rep binding sites are present. The AAV2 genome map has p19 promoter at position 19, and expresses Rep52 and Rep40, respectively. Rep52 and Rep40 have no function of binding to DNA, but have ATP-dependent DNA helicase activity. The cap gene encodes the capsid proteins VP1, VP2, and VP3 of AAV virus. Of these, VP3 has the lowest molecular weight but the highest number, and the ratio of VP1, VP2, and VP3 in mature AAV particles is approximately 1:1: 10. VP1 is essential for the formation of infectious AAV; VP2 assists VP3 in entering the nucleus; VP3 is the major protein that makes up AAV particles.

With the understanding of the life cycle of AAV and its related molecular biological mechanism, AAV is transformed into one efficient foreign gene transferring tool, AAV vector. The modified AAV vector genome only contains the ITR sequence of AAV virus and an exogenous gene expression frame carrying transport, Rep and Cap proteins required by virus packaging are provided in trans through exogenous plasmids, and possible harm caused by packaging Rep and Cap genes into AAV vectors is reduced. Moreover, the AAV virus itself is not pathogenic, making the AAV vector one of the most recognized safe viral vectors. Deletion of the D sequence and the trs (tertiary resolution site) sequence in the ITR sequence on one side of the AAV enables self-complementation of the genome carried by the packaged recombinant AAV vector to form double chains, thus remarkably improving the in vitro and in vivo transduction efficiency of the AAV vector (Wang Z,et al. Gene Ther. 2003;10(26):2105-2111. McCarty DM, et algene ther 2003, 10(26) 2112-2118). The resulting packaged virus becomes a scAAV (self-complementary AAV) virus, a so-called double-stranded AAV virus. Unlike ssAAV (single-stranded AAV), a classical AAV virus, in which neither ITR is mutated at both sides. The packaging capacity of scAAV virus is smaller, only half of the packaging capacity of ssAAV, about 2.2kb-2.5kb, but transduction efficiency is higher after infecting cells. AAV viruses are numerous in serotype, different serotypes having different tissue infection tropism, and thus the use of AAV vectors enables the transport of foreign genes to specific organs and tissues (Wu Z,et almol ther 2006, 14(3) 316-. Some serotype AAV vectors can also cross the blood brain barrier, leading foreign genes into brain neurons, providing the possibility for gene transduction targeting the brain (Samaranch L,et alhum Gene ther, 2012, 23(4) 382. 389.). In addition, AAV vectors have stable physicochemical properties, and exhibit strong tolerance to acids and bases and high temperatures (Gruntman AM,et alhum Gene their methods 2015, 26(2): 71-76), it is easy to develop biological products with higher stability.

AAV vectors also have relatively mature packaging systems, facilitating large-scale production. At present, the AAV vector packaging system commonly used at home and abroad mainly comprises a three-plasmid cotransfection system, a packaging system taking adenovirus as a helper virus, a packaging system taking Herpes simplex virus type 1 (HSV 1) as a helper virus and a packaging system based on baculovirus. Among them, the three plasmid transfection packaging system is the most widely used AAV vector packaging system because of no need of auxiliary virus and high safety, and is also the mainstream production system in the world at present. The lack of efficient large-scale transfection methods has somewhat limited the use of three-plasmid transfection systems for large-scale production of AAV vectors. Yuan et al established an AAV large-scale packaging system with adenovirus as the helper virus (Yuan Z,et alhum Gene Ther, 2011,22(5): 613-. HSV1 as a packaging system for helper virus is another AAV vector package with wide applicationAnd (5) assembling the system. Almost simultaneously, Wushijia and Conway et al internationally proposed the packaging strategy of AAV2 vector with HSV1 as helper virus (Wushijia, Wu soldier et al scientific bulletin, 1999, 44 (5): 506-509. Conway JE,et algene Ther, 1999,6: 986-. Subsequently, Wustner et al proposed an AAV5 vector packaging strategy with HSV1 as a helper virus (Wustner JT,et almol Ther, 2002,6(4): 510-. On the basis, Booth et al utilize two HSV1 to respectively carry the rep/cap gene of AAV and Inverted terminal sequence (ITR)/exogenous gene expression cassette of AAV, then two recombinant HSV1 viruses are co-infected with production cell, packaged to produce AAV virus (Booth MJ,et algene Ther,2004,11: 829-. Thomas et al further established the suspension cell system for AAV production of bis HSV1 virus (Thomas DL,et algene Ther,2009,20: 861-870), enabling larger scale AAV virus production. In addition, Urabe and the like construct a baculovirus packaging system of AAV vectors by using three baculoviruses to respectively carry AAV structural, non-structural and ITR/exogenous gene expression cassettes. Considering the instability of baculovirus carrying foreign genes, the number of baculovirus required in the production system is subsequently reduced, gradually from the first requiring three baculovirus to the second requiring two or one baculovirus (Chen H. Mol ther.2008;16(5):924-,et alj Invertebr Pathol, 2011,107 Suppl: S80-93.) and a baculovirus plus one strain inducing cell line strategy (Mietzsch M,et al. Hum Gene Ther. 2014;25:212-222. Mietzsch M, et alhum Gene ther 2015, 26(10) 688 697. Each packaging system has various characteristics, and can be selected as required.

Due to the above characteristics, AAV vectors are becoming an exogenous gene transfer tool widely used in gene therapy, particularly in gene therapy of genetic diseases. By 11 months 2017, there are 204 approved gene therapy clinical trials of AAV vectors worldwide (http:// www.abedia.com/willey/vectors. More importantly, the AAV vector-based lipoprotein lipase gene therapy drug Glybera has been approved by European drug administration to be on the market in 2012, and becomes the first approved drug in the Western worldGene therapy medicine (YL ä -Herttuala S.Mol Ther2012, 20(10) 1831 and 1832); the American FDA approved congenital black disease (caused by RPE65 gene mutation) gene therapy medicine Luxturna is marketed in 19.12.2017, and becomes the gene therapy medicine for the first rare diseases in the United states (https:// www.fda.gov/news events/news group/presentation uncementes/ucm 589467. htm). Hemophilia B (Kay MA, et al.Nat GenetThe AAV vector gene therapy medicaments of 2000, 24(3), 257 and 261) all have good clinical test effects, are expected to be sold in the near future and benefit a large number of patients.

In the invention, the AAV vector is selected to carry the hMTM1 gene expression cassette, and is mainly based on the following characteristics of the AAV vector. For one, AAV vectors retain only the two ITR sequences required for packaging in wild-type virus, and do not contain the protein-encoding genes in the wild-type virus genome (salenik M,et almicrobiol spectra. 2015; 3(4), which is low in immunogenicity. Secondly, AAV achieves sustained stable expression of the gene-carrying reading frame, usually in the form of non-integrated extrachromosomal genetic material (Chen ZY,et almol ther 2001, 3(3) 403-. Third, AAV vectors have high transduction efficiency both by intravenous and intramuscular injection (Wang Z,et al. Nat Biotechnol. 2005;23:321-328. Bish LT, et al. Hum Gene Ther. 2008;19:1359-1368.Zincarelli C, et al. Mol Ther. 2008;16:1073-1080.Prasad KM, et al. Gene Ther. 2011;18:43-52.Rebuffat A, et alhum Gene ther.2010, 21(4), 463-477) to ensure that the expression cassette of hMTM1 Gene can efficiently express MTM1 protein in vivo.

In order to reduce the probability of the organism generating immune response to MTM1 protein, the time for continuous and stable expression of hMTM1 gene is prolonged. We chose the MCK promoter for muscle-specific high-efficiency expression, and let the introduced hMTM1 gene be specifically and efficiently expressed in muscle. Furthermore, we utilized the characteristics of normal liver-specific high expression of miR-122 (Joint C. RNA biol. 2012; 9(2): 137-. Inhibiting gene expression by miRNABased on the principle (Kim VN. Nat Rev Mol Cell biol.2005;6(5): 376-385.), the miR-122 molecule in the liver Cell can inhibit the expression of MTM1 protein and obviously reduce the expression of MTM1 protein in the liver. The targeted expression of the MTM1 protein is realized through the regulation and control of a muscle specific promoter and a miR-122 target sequence. Meanwhile, in order to reduce immune response possibly brought by over-expression of MTM1 protein, a miR-142-3p target sequence regulation strategy is designed. Since miR-142-3p is highly expressed in hematopoietic stem cell line-derived cells (Chen CZ,et alscience 2004, 303(5654): 83-86.), immune cells differentiate equally from hematopoietic stem Cell lines, thus using the principle of miRNA-inhibitory Gene expression (Kim VN. Nat Rev Mol Cell biol.2005;6(5): 376-.

miRNAs (microRNAs) are single-stranded non-coding RNAs of 18 to 25 nucleotides (nt) in length that are widely found in humans and animals (Bartel DP. Cell. 2004; 116: 281-297.Kim VN. Nat Rev Mol Cell biol.2005; 6: 376-385.). miRNA was first found in caenorhabditis elegans (c. elegans) in 1993 (Lee RC,et al. Cell. 1993; 75: 843-854. Wightman B, et alcell. 1993; 75: 855 · 862.). The lin-4 gene in elegans is capable of down-regulating the expression of the lin-14 gene, but the encoded product of the lin-4 gene is not a protein, but is a small RNA molecule, indicating that the small RNA molecule itself encodes for the ability to regulate the expression of the gene. Subsequently, a number of similar small RNA molecules were sequentially found in different species and cells (Lagos-Quintana M,et al. Science. 2001; 294: 853-858. Lau NC, et al. Science. 2001; 294: 858-862. Lee RC, et alscience 2001, 294: 862-864), mirnas began to become a collective term for this class of small RNAs. mirnas regulate the expression of approximately 60% of genes in humans (Lewis BP,et al. Cell. 2005;120: 15-20. Friedman RC, et algenome res. 2009; 19: 92-105), plays an important role in a variety of physiological and pathological processes (carteon M,et al. Cell Cycle. 2007; 6: 2127-2132. Ambros V. Cell. 2003; 113:673-676. Schichel R, et al. Oncogene. 2008; 27: 5959-5974.)。

miRNA genes are typically located in exons, introns, and intergenic regions of the genome (Olena AF,et al. J Cell Physiol. 2010; 222: 540-545. Kim VN, et altrends Genet 2006; 22: 165-173.). In cells, miRNA is produced as follows (Winter J,et alnat Cell biol. 2009;11: 228-. Firstly, in a cell nucleus, miRNA genes are initiated to be transcribed by RNA polymerase II or III to generate an initial product pri-microRNA; the pri-microRNA self-folding partial sequence forms a stem-loop structure. Subsequently, the processing complex consisting of ribonuclease III Drosha and DGCR8 molecules acts on the pri-microRNA, cutting off the excess sequence, leaving around 60nt of stem-loop structure, the precursor miRNA molecule pre-microRNA (Lee Y,et al. Nature. 2003;425: 415-419.Denli AM, et al. Nature. 2004;432: 231-235. Gregory RI, et al. Nature. 2004; 432:235-240. Han J, et al. Genes Dev. 2004; 18: 3016-3027. Landthaler M, et alcurr biol. 2004;14: 2162-. The pre-microRNA then passes from the nucleus into the cytoplasm with the aid of the transporter Exportin-5 (Lund E,et al. Science. 2003; 303: 95-98. Yi R, et al. Genes Dev. 2003;17: 3011-3016. Bohnsack MT, et alRNA, 2004; 10: 185-191.) whose stem-loop structure is processed by Dicer enzyme to remove the loop portion and become a double-stranded RNA molecule (Jiang F,et al. Genes Dev. 2005;, 19: 1674-1679. Saito K, et alPLoS biol.2005; 3: e 235.). Finally, the double-stranded RNA molecule is bound by a protein factor such as AGO2, one strand of which is degraded, and the other strand of which forms an RNA-induced silencing complex (RISC) with the protein factor. RISC recognizes target sequences in mRNA, reduces mRNA expression levels by degrading mRNA molecules, promoting 3' end de-adenylation of mRNA molecules, and inhibiting translation, regulates gene expression at post-transcriptional levels (Storz G,et al. Curr Opin Microbiol.2004; 7: 140-144. Fabian MR, et al. Annu Rev Biochem. 2010; 79: 351-379. Valencia-Sanchez MA, et algenes Dev 2006; 20: 515-. Thus using highly expressed intracellular miRNAs, extracorporeallyThe 3' UTR (untranslated region) of the source gene is inserted into the target sequence of the miRNA, so that the expression of the foreign gene in the introduced cells can be effectively inhibited.

According to the design thought, rAAV-MCK-hMTM1-122T-142T virus is prepared, and rAAV-MCK-hMTM1 control virus without miRNA target sequence and rAAV-MCK-EGFP control virus carrying green fluorescent protein coding frame are designed and prepared. These viruses were each injected at equal doses into an XLMTM mouse model to evaluate the effectiveness of designing rAAV-MCK-hMTM 1-122T-142T. The result shows that compared with the rAAV-MCK-EGFP control virus, the rAAV-MCK-hMTM1-122T-142T and the rAAV-MCK-hMTM1 can express MTM1 protein in an XLMTM mouse model, restore the physiological function of the muscle of the XLMTM mouse model and prolong the survival period of the model mouse. Compared with rAAV-MCK-hMTM1 virus, the rAAV-MCK-hMTM1-122T-142T virus has lower expression level in the liver of a model mouse, obviously reduces the toxic effect possibly brought by over-expression of liver hMTM1, and more importantly, the stable and continuous expression time of hMTM1 is longer, thereby providing a new treatment option for XLMM patients.

Disclosure of Invention

In view of the above, the present invention provides a novel XLMTM gene therapeutic drug based on AAV vectors. The drug is characterized in that an AAV vector carries an hMTM1 gene expression cassette. The sequence is optimized to obtain a human mtm1 gene coding sequence hMTM1, a mouse muscle specific promoter MCK is adopted to regulate the expression of the hMTM1 sequence, and a miR-142-3p target sequence is added in a 3' UTR (untranslated region) region of a gene expression frame. Based on the design, the drug is expected to be capable of efficiently expressing and generating MTM1 protein in muscle tissues of XLMTM model mice after intravenous injection, recovering the physiological functions of the muscles of the model mice and prolonging the life cycle.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a gene therapy medicine for treating X-linked myotubule myopathy (XLMTM), which is characterized in that the medicine is based on a recombinant AAV vector, and the AAV vector is utilized to efficiently introduce a medicine effect element into a body through intravenous injection, so that the MTM1 protein which can generate a therapeutic effect by the efficient expression of the medicine effect element is realized. In order to realize the high-efficiency expression of MTM1, the selected AAV vector serotypes mainly comprise AAV8 and AAV9 according to the transduction characteristics of different AAV serotypes.

The XLMTM gene therapeutic drug provided by the invention is characterized in that the hMTM1 sequence is obtained by optimizing the coding region sequence of the original human MTM1 protein gene by eliminating rare codons, RNA secondary structure and the like, and the expression of the hMTM1 sequence is regulated by a mouse creatine kinase promoter (MCK promoter), so that the specific and high-efficiency expression of the human MTM1 protein in muscle is ensured; 2 completely complementary human miR-122 and miR-142-3p target sequences are respectively inserted into the 3' UTR of a gene expression frame, based on the principle that miRNA inhibits gene expression, miR-122 is specifically and highly expressed in liver cells, the expression of MTM1 protein in liver is remarkably reduced, the expression level of miR-142-3p in human hematopoietic stem cells is high, the expression of human MTM1 in human hematopoietic stem cell source cells including antigen presenting cells is inhibited, the probability that an organism generates immune response aiming at the human MTM1 protein is reduced, and the continuous expression of the human MTM1 protein is facilitated.

The XLMTM gene therapeutic drug provided by the invention is characterized in that after the drug is injected into an XLMTM model mouse body through intravenous injection, human MTM1 protein can be efficiently, stably and continuously expressed in the mouse body, the MTM1 protein generated by expression can recover the physiological function of the muscle of the model mouse, and the life cycle of the model mouse is prolonged, so that the effect of treating XLMTM is achieved.

The XLMTM gene therapeutic drug provided by the invention is further characterized in that the XLMTM gene therapeutic drug can be continuously expressed in an XLMTM model mouse for a long time through once intravenous injection administration to generate human MTM1 protein, thereby recovering the physiological function of the muscle of the model mouse and prolonging the life cycle.

The important original experimental materials used in the present invention are as follows:

pHelper plasmid, derived from AAV Helper Free System (Agilent Technologies, USA), was purchased from Agilent Technologies, Inc. and stored. The plasmid contains three plasmids to co-transfect HEK293 cells to prepare adenovirus-derived helper function genes E2A, E4, VA RNA and the like required by recombinant AAV.

pAAV-RC plasmid, derived from AAV Helper Free System (Agilent Technologies, USA), was purchased from Agilent Technologies, Inc. and stored. The pAAV-RC plasmid contains the Rep and cap genes of AAV2 intact, and provides the 4 Rep proteins (Rep 78, Rep68, Rep52 and Rep 40) and AAV2 coat proteins necessary for packaging in the preparation of recombinant AAV2 virus by three-plasmid co-transfection packaging.

The pAAV-R2C8 plasmid was constructed and stored by this company. The pAAV-RC plasmid in AAV Helper Free System (Agilent Technologies, USA) is used as basic skeleton, and the sequence from 2013 to 4220 in pAAV-RC plasmid is replaced by the coat protein coding sequence Cap8 (sequence from 2121 to 4337 in the genome) in AAV8 genome (GenBank ID: AF 513852), so as to obtain pAAV-R2C8 plasmid. The simple construction process is that pAAV-R2C8 plasmid sequence information is obtained according to the thought, a sequence between HindIII and PmeI restriction sites in the pAAV-R2C8 plasmid is artificially synthesized, and a standard molecular cloning method is adopted to replace the sequence between the HindIII and the PmeI restriction sites in the pAAV-RC plasmid by the synthetic sequence to obtain the pAAV-R2C8 plasmid. The pAAV-R2C8 plasmid contains the cap gene of AAV8 and the Rep gene of AAV2 in a complete form, and 4 Rep proteins (Rep 78, Rep68, Rep52 and Rep 40) and AAV8 coat proteins which are necessary for packaging are provided in the preparation of recombinant AAV8 virus by three-plasmid co-transfection packaging.

The pAAV-R2C9 plasmid was constructed and stored by this company. The pAAV-RC plasmid in AAV Helper Free System (Agilent Technologies, USA) is used as basic skeleton, and the sequences 2013 to 4220 in pAAV-RC plasmid are replaced by AAV9 coat protein coding sequence (GenBank ID: AY 530579), so that pAAV-R2C9 plasmid is obtained. The simple construction process is that pAAV-R2C9 plasmid sequence information is obtained according to the thought, a sequence between HindIII and PmeI restriction sites in the pAAV-R2C9 plasmid is artificially synthesized, and a standard molecular cloning method is adopted to replace the sequence between the HindIII and PmeI restriction sites of the pAAV-RC plasmid by the synthetic sequence to obtain the pAAV-R2C9 plasmid. The pAAV-R2C9 plasmid contains the cap gene of AAV9 and the Rep gene of AAV2 in a complete form, and 4 Rep proteins (Rep 78, Rep68, Rep52 and Rep 40) and AAV9 coat proteins which are necessary for packaging are provided in the preparation of recombinant AAV9 virus by three-plasmid co-transfection packaging.

Small X-linked myotubular myopathyMouse model species mice were prepared, purchased from The Jackson Laboratory, USA, under The trade name B6.Cg-Mtm1 ^tm1Itland/J, accession number 018153. The species is female in sex, the X chromosome-linked MTM1 (myocyte myophathy gene 1) gene exon 4 mutation heterozygote, and the mutated MTM1 gene cannot be expressed to produce the normal MTM1 protein (Pierson CR, et al. Hum Mol Gene t. 2012;21(4): 811-25.). After the mouse is hybridized with a male wild type C57BL/6J mouse, about half of the newborn male mouse mtm1 gene is mutated, and similar X-linked myotubule myopathy occurs to a human.

Control mice, C57BL/6J mice, purchased from Beijing Huafukang Biotech GmbH, used as wild-type controls for animal experiments.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a schematic diagram of the pAAV2neo vector structure. The AAV vector pAAV2neo (Dong X, et al, PLoS ONE. 2010; 5(10): e 13479.) with both ITRs on both sides being 145bp wild-type ITRs was stored by this company. ITR, inverted terminal repeat, length 145 bp. CMV promoter, human cytomegalovirus early promoter. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame. XhoI, KpnI, EcoRI, SalI, BglII, BamHI and ApaI are all restriction sites.

FIG. 2 is a schematic diagram of the structure of pAAV2-MCK-EGFP vector. ITR, inverted terminal repeat, length 145 bp. MCK, mouse creatine kinase promoter. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 3 is a schematic diagram of the structure of pAAV2-MCK-EGFP vector. ITR, inverted terminal repeat, length 145 bp. MCK, mouse creatine kinase promoter. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame. EGFP, enhanced green fluorescent protein coding region sequence.

FIG. 4 shows a schematic diagram of the vector structure of pAAV2-MCK-hMTM 1. ITR, inverted terminal repeat, length 145 bp. MCK, mouse creatine kinase promoter. hMTM1, optimized synthetic human mtm1 gene coding region sequence. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame. XhoI, KpnI and EcoRI are all restriction sites.

FIG. 5 shows a schematic structure of pAAV2-MCK-hMTM1-122T-142T vector. ITR, inverted terminal repeat, length 145 bp. MCK, mouse creatine kinase promoter. hMTM1, optimized synthetic human mtm1 gene coding region sequence. 2 × miR-122T, 2 copies of the fully complementary human miR-122 target sequence. 2 XmiR-142-3 pT, 2 copies of the fully complementary human miR-142-3p target sequence. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame. XhoI, KpnI, EcoRI and BglII are all restriction sites.

FIG. 6 injection of recombinant AAV carrying hMTM1 gene expression cassette to prolong survival of XLMTM model mice. 6 different recombinant AAV viruses (AAV 8-MCK-EGFP, AAV8-MCK-hMTM1, AAV8-MCK-hMTM1-122T-142T, AAV9-MCK-EGFP, AAV9-MCK-hMTM1, AAV9-MCK-hMTM 1-122T-142T) at 3 × 10¹³XLMTM model mice were injected caudally at a dose of vg/kg, with AAV8-MCK-EGFP and AAV9-MCK-EGFP as control viruses, and 5 mice of XLMTM model were injected for each virus, with XLMTM mice aged 3 weeks at the time of injection. After virus injection, the survival of the mice was recorded. WT, C57BL/6J wild-type control mice; KO-AAV8-MCK-EGFP, XLMTM model mouse injected with AAV8-MCK-EGFP virus; KO-AAV8-MCK-hMTM1, XLMTM model mouse injected with AAV8-MCK-hMTM1 virus; KO-AAV8-MCK-hMTM1-122T-142T, XLMM model mouse injected with AAV8-MCK-hMTM1-122T-142T virus; KO-AAV9-MCK-EGFP, XLMTM model mouse injected with AAV9-MCK-EGFP virus; KO-AAV9-MCK-hMTM1, AAV injectionXLMTM model mouse of 9-MCK-hMTM1 virus; KO-AAV9-MCK-hMTM1-122T-142T, XLMM model mouse injected with AAV9-MCK-hMTM1-122T-142T virus.

FIG. 7 weight change in XLMTM model mice injected with recombinant AAV virus carrying hMTM1 gene expression cassette. 6 different recombinant AAV viruses (AAV 8-MCK-EGFP, AAV8-MCK-hMTM1, AAV8-MCK-hMTM1-122T-142T, AAV9-MCK-EGFP, AAV9-MCK-hMTM1, AAV9-MCK-hMTM 1-122T-142T) at 3 × 10¹³XLMTM model mice were injected caudally at a dose of vg/kg, with AAV8-MCK-EGFP and AAV9-MCK-EGFP as control viruses, and 5 mice of XLMTM model were injected for each virus, with XLMTM mice aged 3 weeks at the time of injection. After injection of the virus, the body weight of the mice was recorded at various time points. WT, C57BL/6J wild-type control mice; KO-AAV8-MCK-EGFP, XLMTM model mouse injected with AAV8-MCK-EGFP virus; KO-AAV8-MCK-hMTM1, XLMTM model mouse injected with AAV8-MCK-hMTM1 virus; KO-AAV8-MCK-hMTM1-122T-142T, XLMM model mouse injected with AAV8-MCK-hMTM1-122T-142T virus; KO-AAV9-MCK-EGFP, XLMTM model mouse injected with AAV9-MCK-EGFP virus; KO-AAV9-MCK-hMTM1, XLMTM model mouse injected with AAV9-MCK-hMTM1 virus; KO-AAV9-MCK-hMTM1-122T-142T, XLMM model mouse injected with AAV9-MCK-hMTM1-122T-142T virus.

FIG. 8 results of the distance traveled in 90 minutes after 5 weeks of virus injection in mice. 6 different recombinant AAV viruses (AAV 8-MCK-EGFP, AAV8-MCK-hMTM1, AAV8-MCK-hMTM1-122T-142T, AAV9-MCK-EGFP, AAV9-MCK-hMTM1, AAV9-MCK-hMTM 1-122T-142T) at 3 × 10¹³XLMTM model mice were injected caudally at a dose of vg/kg, with AAV8-MCK-EGFP and AAV9-MCK-EGFP as control viruses, and 5 mice of XLMTM model were injected for each virus, with XLMTM mice aged 3 weeks at the time of injection. The distance traveled by the mice in 90 minutes was recorded 2 weeks after virus injection. WT, C57BL/6J wild-type control mice; KO-AAV8-MCK-EGFP, XLMTM model mouse injected with AAV8-MCK-EGFP virus; KO-AAV8-MCK-hMTM1, XLMTM model mouse injected with AAV8-MCK-hMTM1 virus; KO-AAV8-MCK-hMTM1-122T-142T, XLMM model mouse injected with AAV8-MCK-hMTM1-122T-142T virus; KO-AAV9-MCK-EGFP, XLMTM model injected with AAV9-MCK-EGFP virusA mouse; KO-AAV9-MCK-hMTM1, XLMTM model mouse injected with AAV9-MCK-hMTM1 virus; KO-AAV9-MCK-hMTM1-122T-142T, XLMM model mouse injected with AAV9-MCK-hMTM1-122T-142T virus.

Figure 9 results of the distance traveled test within 90 minutes after 3 months of virus injection in mice. 6 different recombinant AAV viruses (AAV 8-MCK-EGFP, AAV8-MCK-hMTM1, AAV8-MCK-hMTM1-122T-142T, AAV9-MCK-EGFP, AAV9-MCK-hMTM1, AAV9-MCK-hMTM 1-122T-142T) at 3 × 10¹³XLMTM model mice were injected caudally at a dose of vg/kg, with AAV8-MCK-EGFP and AAV9-MCK-EGFP as control viruses, and 5 mice of XLMTM model were injected for each virus, with XLMTM mice aged 3 weeks at the time of injection. 3 months after virus injection, the distance traveled by the mice in 90 minutes was recorded. WT, C57BL/6J wild-type control mice; KO-AAV8-MCK-EGFP, XLMTM model mouse injected with AAV8-MCK-EGFP virus; KO-AAV8-MCK-hMTM1, XLMTM model mouse injected with AAV8-MCK-hMTM1 virus; KO-AAV8-MCK-hMTM1-122T-142T, XLMM model mouse injected with AAV8-MCK-hMTM1-122T-142T virus; KO-AAV9-MCK-EGFP, XLMTM model mouse injected with AAV9-MCK-EGFP virus; KO-AAV9-MCK-hMTM1, XLMTM model mouse injected with AAV9-MCK-hMTM1 virus; KO-AAV9-MCK-hMTM1-122T-142T, XLMM model mouse injected with AAV9-MCK-hMTM1-122T-142T virus. ND, injected virus mice death, so do not determine.

FIG. 10 different muscle tissue myotubulin expression levels. 6 different recombinant AAV viruses (AAV 8-MCK-EGFP, AAV8-MCK-hMTM1, AAV8-MCK-hMTM1-122T-142T, AAV9-MCK-EGFP, AAV9-MCK-hMTM1, AAV9-MCK-hMTM 1-122T-142T) at 3 × 10¹³XLMTM model mice were injected caudally at a dose of vg/kg, with AAV8-MCK-EGFP and AAV9-MCK-EGFP as control viruses, and 5 mice of XLMTM model were injected for each virus, with XLMTM mice aged 3 weeks at the time of injection. Representative muscle tissues such as extensor digitorum longus, quadriceps and diaphragm muscles were isolated 3 months after virus injection. Extracting total protein of tissue cells, transferring the total protein to a PVDF membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), detecting by western blot, and calculating by taking GAPDH as an internal reference to obtain the expression level of the myotubulin. The WT is able to perform a task,c57BL/6J wild-type control mice; KO-AAV8-MCK-EGFP, XLMTM model mouse injected with AAV8-MCK-EGFP virus; KO-AAV8-MCK-hMTM1, XLMTM model mouse injected with AAV8-MCK-hMTM1 virus; KO-AAV8-MCK-hMTM1-122T-142T, XLMM model mouse injected with AAV8-MCK-hMTM1-122T-142T virus; KO-AAV9-MCK-EGFP, XLMTM model mouse injected with AAV9-MCK-EGFP virus; KO-AAV9-MCK-hMTM1, XLMTM model mouse injected with AAV9-MCK-hMTM1 virus; KO-AAV9-MCK-hMTM1-122T-142T, XLMM model mouse injected with AAV9-MCK-hMTM1-122T-142T virus.

FIG. 11 shows the results of detecting the expression level of hMTM1 gene in different tissues. 6 different recombinant AAV viruses (AAV 8-MCK-EGFP, AAV8-MCK-hMTM1, AAV8-MCK-hMTM1-122T-142T, AAV9-MCK-EGFP, AAV9-MCK-hMTM1, AAV9-MCK-hMTM 1-122T-142T) at 3 × 10¹³XLMTM model mice were injected caudally at a dose of vg/kg, with AAV8-MCK-EGFP and AAV9-MCK-EGFP as control viruses, and 5 mice of XLMTM model were injected for each virus, with XLMTM mice aged 3 weeks at the time of injection. After 3 months of virus injection, tissues of heart, liver, skeletal muscle, lung, spleen and kidney were isolated. Extracting total RNA of the tissues, detecting the copy number of hMTM1 RNA and the copy number of GAPDH RNA in the total RNA by quantitative PCR, and calculating the ratio of the copy number of hMTM1 RNA and the copy number of GAPDH RNA to express the expression level of hMTM1 gene. WT, C57BL/6J wild-type control mice; KO-AAV8-MCK-EGFP, XLMTM model mouse injected with AAV8-MCK-EGFP virus; KO-AAV8-MCK-hMTM1, XLMTM model mouse injected with AAV8-MCK-hMTM1 virus; KO-AAV8-MCK-hMTM1-122T-142T, XLMM model mouse injected with AAV8-MCK-hMTM1-122T-142T virus; KO-AAV9-MCK-EGFP, XLMTM model mouse injected with AAV9-MCK-EGFP virus; KO-AAV9-MCK-hMTM1, XLMTM model mouse injected with AAV9-MCK-hMTM1 virus; KO-AAV9-MCK-hMTM1-122T-142T, XLMM model mouse injected with AAV9-MCK-hMTM1-122T-142T virus.

Detailed Description

The invention discloses a gene therapy medicine for X-linked myotubule myopathy, which comprises the design, the mini-preparation and the functional verification of the medicine. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention. In which, unless otherwise specified, the various reagents mentioned in the examples are commercially available.

The invention is further illustrated by the following examples:

example 1 plasmid vector construction

In order to construct the pAAV2-MCK-EGFP, pAAV2-MCK-hMTM1 and pAAV2-MCK-hMTM1-122T-142T plasmids required for obtaining packaged recombinant AAV, we first replaced the CMV promoter in the pAAV2neo vector with the MCK promoter (SEQ ID No.1) based on pAAV2neo (FIG. 1) stored in the company in the reference (Shield MA, et al. Mol Cell biol. 1996; 16(9): 5058 and 5068.) to obtain pAAV 2-MCK. Next, a DNA sequence (SEQ ID No. 2) containing an enhanced green fluorescent protein coding sequence, an artificially optimized and designed hMTM1 sequence (SEQ ID No.3) and an hMTM1-122T-142T sequence (SEQ ID No. 4) (after a stop codon of an hMTM1 sequence, 2 target sequences completely complementary to human miR-122 and 2 target sequences completely complementary to human miR-142-3p are sequentially added) are respectively cloned between KpnI and EcoRI and KpnI and BglII enzyme cutting sites of the pAAV2-MCK vector to obtain pAAV2-MCK-EGFP, pAAV2-MCK-hMTM1 and pAAV2-MCK-hMTM1-122T-142T vectors.

(1) pAAV2-MCK vector construction

In reference (Shield MA, et al. Mol Cell biol. 1996; 16(9): 5058-5068.), the sequence number of the Mouse Creatine Kinase (MCK) promoter was found by searching GeneBank database (M21390.1), the MCK promoter sequence was analyzed, XhoI restriction site "5 '-CTCGAG-3'" was added to the 5 'end of the promoter sequence, and KpnI restriction site "5' -GGTACC-3 '" was added to the 3' end of the promoter sequence, to obtain the MCK sequence, and the sequence information was shown in SEQ ID No. 1. The MCK sequence was synthesized by Nanjing Kingsry Biotechnology Co., Ltd, and the synthesized sequence was cloned into pUC57-1.8K vector (Nanjing Kingsry Biotechnology Co., Ltd.) to obtain pUC 57-1.8K-MCK. The KpnI and XhoI double digestion yielded a 0.6kb MCK sequence fragment and a 1.8kb vector fragment, and the MCK sequence fragment was recovered for use. The CMV promoter sequence between XhoI and KpnI enzyme cutting sites in the pAAV2neo vector is replaced by the MCK sequence fragment, and the pAAV2-MCK vector is obtained through enzyme cutting sequencing identification (shown in the attached figure 2).

(2) Construction of control Virus packaging vector pAAV2-MCK-EGFP

The method comprises the steps of designing a primer EGFP-F/EGFP-R by taking pCMV-C-EGFP (Biyuntian biotechnology limited company, China) as a template, amplifying an EGFP gene coding region sequence by PCR, wherein KpnI and EcoRI enzyme cutting sites are contained in the EGFP-F primer and the EGFP-R primer respectively. And amplifying to obtain an EGFP coding region sequence fragment, digesting by EcoRI and KpnI double enzyme, and recovering for later use. The pAAV2-MCK vector was digested with EcoRI and KpnI, and the linearized pAAV2-MCK vector fragment (about 6.7 kb) was recovered. The two recovered fragments are connected to transform E.coli JM109 competent cells (Baozhi, Dalian), and AAV plasmid vector pAAV2-MCK-EGFP (see figure 3) containing EGFP gene expression frame is obtained after screening and identification.

EGFP-F: 5’-ataggtaccgccaccatggtgagcaag-3’ (SEQ ID NO.5)

EGFP-R: 5’-gcggaattcttacttgtacagctcgtc-3’ (SEQ ID No.6)

(3) pAAV2-MCK-hMTM1 vector construction

The NCBI protein database is searched to obtain the amino sequence reference of human MTM1 protein (GenBank ID: NP-000243), and the hMTM1 sequence (SEQ ID No.3) is synthesized by Nanjing Kingsler Biotechnology GmbH according to the principle of human codon preference and the like. The synthetic hMTM1 sequence was cloned into pUC57 simple vector (Nanjing Kingsry Biotech Co., Ltd.) to obtain pUC57-hMTM 1. KpnI and EcoRI respectively digest pUC57-hMTM1 vector and pAAV2-MCK vector by double digestion, hMTM1 fragment and linearized pAAV2-MCK vector fragment are recovered, E.coli JM109 competent cell (Baozbio, Dalian) is transformed after the two fragments are connected, and pAAV2-MCK-hMTM1 vector (shown in figure 4) is obtained by screening and identifying.

(4) pAAV2-MCK-hMTM1-122T-142T vector construction

And sequentially splicing the completely complementary target sequences of the 2 serially repeated human miR-122 and the completely complementary target sequences of the 2 serially repeated human miR-142-3p with the optimized hMTM1 gene sequence to obtain an hMTM1-122T-142T sequence (SEQ ID No. 4). The hMTM1-122T-142T sequence was synthesized by Nanjing Kingsler Biotech Ltd. The synthesized hMTM1-122T-142T sequence was cloned into pUC57 simple vector (Nanjing Kingsry Biotech Co., Ltd.) to obtain pUC57-hMTM1-122T-142T vector. KpnI and BglII are subjected to double digestion to digest pUC57-hMTM1-122T-142T vector and pAAV2-MCK vector respectively, hMTM1-122T-142T fragment and linearized pAAV2-MCK vector fragment are recovered, E.coli JM109 competent cells (Takara Shuzo, Dalian) are transformed after the two fragments are connected, and pAAV2-MCK-hMTM1-122T-142T vector (shown in figure 5) is obtained through screening and identification.

Example 2 recombinant AAV Virus preparation and assay

Reference is made to the literature (Xiao X,et alj Virol, 1998, (72 (3): 2224-2232.) the AAV is obtained by packing the recombinant AAV with three plasmid packing systems and separating, purifying and packing with cesium chloride density gradient centrifugation. Briefly, AAV vector plasmids (pAAV 2-MCK-EGFP, pAAV2-MCK-hMTM1 or pAAV2-MCK-hMTM 1-122T-142T), helper plasmids (pHelper), and Rep and Cap protein expression plasmids (pAAV-R2C 8 or pAAV-R2C 9) of AAV are mixed uniformly according to a molar ratio of 1:1:1, HEK293 cells are transfected by a calcium phosphate method, the cells and culture supernatant are harvested after 48h transfection, and the recombinant AAV viruses are separated and purified by a cesium chloride density gradient centrifugation method. Packaging and purifying to obtain 6 recombinant viruses such as AAV8-MCK-EGFP, AAV8-MCK-hMTM1, AAV8-MCK-hMTM1-122T-142T, AAV9-MCK-EGFP, AAV9-MCK-hMTM1, AAV9-MCK-hMTM1-122T-142T and the like.

And determining the genome titer of the prepared AAV by a quantitative PCR method. The specific process is as follows:

designing primers and probes for quantitative PCR detection aiming at MCK promoter sequences:

MCK-Q-F：5’- ACACCATGGAGGAGAAGCTC-3’ (SEQ ID NO.7)

MCK-Q-R：5’- GCCGGGAACATGGAACAGTA-3’ (SEQ ID NO.8)

MCK-Q-P：5’- CCCTGGTGGAGCCCGTGCCT-3’ (SEQ ID NO.9)

MCK-Q-F and MCK-Q-R are used as primers, and MCK-Q-P is used as a probe. The 5 'end of the probe is marked by FAM fluorescent protein, and the 3' end is connected with BlackBerry query. Primers and probes were synthesized by thermolfisher Scientific. Specifically amplifying a segment with the length of 95bp in an MCK promoter by taking MCK-Q-F and MCK-Q-R as primers, adopting a TaqMan Probe combination method, taking pAAV2-MCK-EGFP plasmid with the concentration of 1 mu g/mu l and a sample diluted by 10 times of gradient as a standard, applying Premix Ex Taq (Probe qPCR) reagent (Takara, Dalian, China) and detecting the genome titer of the virus by using a fluorescent quantitative PCR instrument (model: ABI 7500 fast, ABI). The procedures are described in Premix Ex Taq (Probe qPCR) reagent Specification. Methods for the treatment of viruses are described in the literature (Aurnhammer C, et al Hum Gene their methods, 2012; 23(1): 18-28.).

Example 3 animal experiments

X-linked Myocostalis mouse model preparation of breeder mice, purchased from The Jackson Laboratory, USA under The trade name B6.Cg-Mtm1 ^tm1Itland/J, accession number 018153. The species is female in sex, the X chromosome-linked MTM1 (myocyte myophathy gene 1) gene exon 4 mutation heterozygote, and the mutated MTM1 gene cannot be expressed to produce the normal MTM1 protein (Pierson CR, et al. Hum Mol Gene t. 2012;21(4): 811-25.). The mtm1 gene mutation detection was performed on neonatal male mice after crossing the breeding mice with male wild-type C57BL/6J mice (Buj-Bello A, et al. Mtm1 gene mutant male mice were selected for subsequent experiments.

Dividing 30 3-week-old mtm1 gene mutation male mice into 6 groups at random, dividing 5 mice in each group into 6 groups, injecting AAV8-MCK-EGFP, AAV8-MCK-hMTM1, AAV8-MCK-hMTM1-122T-142T, AAV9-MCK-EGFP, AAV9-MCK-hMTM1 and AAV9-MCK-hMTM1-122T-142T viruses by vein, wherein the injection dosage of each mouse is 3 × 10¹³vg/kg, wherein AAV8-MCK-EGFP and AAV9-MCK-EGFP are control viruses. Wild type C57BL/6J without virus injection was used as a positive control. Recording the survival time of the mice after injecting the virus, and measuring the body weight of the mice at different time pointsTesting the movement locus distance of the mouse within 90 minutes after 2 weeks and 3 months of virus injection, and testing the muscle microtubule expression level of different types of muscle tissues and the muscle microtubule gene expression condition of different organs of the mouse after 3 months of virus injection.

Survival of mice was recorded after virus injection and the results are shown in figure 6. From the results in FIG. 6, it can be seen that mice injected with EGFP reporter viruses (AAV 8-MCK-EGFP or AAV 9-MCK-EGFP) group XLMM model had short survival time, not longer than 68 days. The survival of 4 groups of mice injected with AAV virus group carrying hMTM1 gene expression (AAV 8-MCK-hMTM1, AAV 8-MCK-hMTMT 1-122T-142T, AAV9-MCK-hMTM1 and AAV 9-MCK-hMTMT 1-122T-142T) XLMM model is prolonged. Moreover, the life cycle prolonging time of the mouse injected with the AAV8-MCK-hTMT1-122T-142T or AAV9-MCK-hTMT1-122T-142T virus is obviously longer than that of the mouse injected with the AAV8-MCK-hMTM1 or AAV9-MCK-hMTM1 virus, which shows that the effect of the drug structure design containing the miRNA target sequence in the mouse model is better. More importantly, the survival period of mice injected with AAV8-MCK-hTMT1-122T-142T or AAV9-MCK-hTMT1-122T-142T virus is not obviously different from that of wild C57BL/6J mice, further explaining that the drug design has good effectiveness.

Body weights of mice were measured at different times after virus injection. At each weight measurement time point, only the weight of the live mice was measured, and if the mice died at the time of measurement, there was no weight measurement result. The results are shown in FIG. 7. From the results of FIG. 7, it can be seen that the body weight of mice injected with EGFP reporter viruses (AAV 8-MCK-EGFP or AAV 9-MCK-EGFP) group XLMM model increased slowly or hardly until death. In contrast, the body weight of mice in 4 groups injected with AAV virus group carrying hMTM1 gene expression (AAV 8-MCK-hMTM1, AAV 8-MCK-hMTMT 1-122T-142T, AAV9-MCK-hMTM1 and AAV9-MCK-hMT 1-122T-142T) XLMM model increased with time, and the body weight increasing trend of the mice is not obviously different compared with that of wild type C57BL/6J mice. The result shows that the growth and development process of XLMTM model mice can be effectively recovered by injecting AAV carrying hMTM1 gene expression cassette.

The distance of the mouse movement track is measured within 90 minutes after injection of the virus 2 weeks and 3 months after injection of the virus, the test method being described in the article (Childers MK, et al Sci Transl Med 2014;6:220ra 10.). The results are shown in FIG. 8 (2 weeks after virus injection) and FIG. 9 (3 months after virus injection). From the results of fig. 8, it can be seen that the difference between the group 6 mice injected with XLMTM virus model and the group wild C57BL/6J mice is significant, and no significant difference is observed between the groups injected with XLMTM virus, which may be related to the short injection time of the virus and the inefficient expression of the hMTM1 gene carried by the virus. From the results of fig. 9, except that XLMTM model mice injected with EGFP reporter gene viruses (AAV 8-MCK-EGFP or AAV 9-MCK-EGFP) all died and no detection data was obtained, the movement locus distances of 4 XLMTM model mice injected with hMTM1 gene expression AAV viruses (AAV 8-MCK-hMTM1, AAV8-MCK-hTMT1-122T-142T, AAV9-MCK-hMTM1 and AAV9-MCK-hTMT 1-122T-142T) were not significantly different from those of wild type C57 BL/xlj mice, which indicates that the 4 viruses can be effectively expressed and generate MTM1 protein when introduced into MTM model mice and recover the movement ability of the model mice.

After 3 months of virus injection, 1 mouse was sacrificed each group, and XLMTM model mice injected with EGFP reporter virus (AAV 8-MCK-EGFP or AAV 9-MCK-EGFP) all died and were not tested as "ND" (FIG. 10). Protein immunoblotting was used to detect expression of MTM1 protein in different muscle tissues, as described in detail in the article (Childers MK, et al. Sci Transl Med. 2014;6:220ra 10.). The difference from the reference is that the human MTM1 protein primary antibody used in the present invention is purchased from the pharmaceutical Mingkude (China, cat # AP6809 b-400), and the antibody is a rabbit polyclonal antibody recognizing the C-terminal amino acid sequence of MTM1 protein. The remaining reagents are in accordance with the reference. The detection results are shown in fig. 10. As can be seen from the results in FIG. 10, in three different muscle tissues, 4 groups of mice injected with AAV virus group carrying hMTM1 gene expression (AAV 8-MCK-hMTM1, AAV 8-MCK-hMTMT 1-122T-142T, AAV9-MCK-hMTM1 and AAV 9-MCK-hMTMT 1-122T-142T) XLMM model showed no significant difference in MTM1 protein expression level, and all were not lower than wild type C57BL/6J mice. The result shows that the injection of AAV virus carrying hMTM1 gene expression cassette can effectively express and produce MTM1 protein in mouse muscle of XLMTM model.

Selecting the killed mice, separating organs such as heart, liver, skeletal muscle, spleen, lung and kidney, extracting total RNA of various organs, determining the copy number of hMTM1 RNA and the copy number of mouse GAPDH RNA in the total RNA by quantitative PCR, wherein the copy number is expressed by Ct value, calculating the difference value of the Ct value of hMTM1 RNA and the Ct value of mouse GAPDH RNA, and the power of the difference value of the Ct value used for 2 represents the relative expression level of hMTM1 RNA.

Quantitative PCR method was used to determine Ct values of hMTM1 RNA and mouse GAPDH RNA in total RNA. The specific process is as follows:

primers and probes for quantitative PCR detection were designed for hMTM1 RNA sequence:

hMTM1-Q-F：5’- GCAGATCAGCAAGCTGACAA-3’ (SEQ ID NO.10)

hMTM1-Q-R：5’- CGTCATCGGACTCGTATCCT-3’ (SEQ ID NO.11)

hMTM1-Q-P：5’- CGCAAGGCCAAGCGTGAACGCA-3’ (SEQ ID NO.12)。

primers and probes for quantitative PCR detection were designed for the mouse GAPDH RNA sequence:

GAPDH-Q-F：5’-AACGGATTTGGCCGTATTGG-3’ (SEQ ID NO.13)

GAPDH-Q-R：5’-AATCTCCACTTTGCCACTGC-3’ (SEQ ID NO.14)

GAPDH-Q-P：5’-CGCCTGGTCACCAGGGCTGC-3’ (SEQ ID NO.15)

hMTM1-Q-F/hMTM1-Q-R and GAPDH-Q-F/GAPDH-Q-R are primers, and hMTM1-Q-P and GAPDH-Q-P are probes. The 5 'end of the probe is marked by FAM fluorescent protein, and the 3' end is connected with BlackBerry query. Primers and probes were synthesized by thermolfisher Scientific. Specifically amplifying a 92bp fragment in the hMTM1 sequence by using hMTM1-Q-F and hMTM1-Q-R as primers, specifically amplifying a 66bp fragment in the GAPDH sequence by using GAPDH-Q-F and GAPDH-Q-R as primers, measuring the amplification Ct values (representing copy numbers) of hMTM1 RNA and GAPDH RNA in a detection sample by using a One-Step reaction TaqMan probe combination method, applying One Step primer PCR RT-PCR Kit (Perfect Real Time) reagent (Takara, Dalian, China) and detecting by using a fluorescence quantitative PCR instrument (model: ABI 7500 fast, ABI). The procedure is described in the One Step PrimerScript RT-PCR Kit (Perfect Real Time) reagent instruction.

The results of the quantitative PCR assay are shown in FIG. 11. From the results shown in FIG. 11, it was found that in mice injected with AAV8-MCK-hMTM1, AAV8-MCK-hMTM1-122T-142T, AAV9-MCK-hMTM1 and AAV9-MCK-hMTM1-122T-142T virus groups, hMTM1 expression level in muscle tissues such as cardiac muscle and skeletal muscle was high, and hMTM1 expression level in organs such as spleen, kidney and lung was low, indicating that transduction properties of AAV8 and AAV9 viruses and tissue specificity of MCK promoter were effective in expressing hMTM1 gene specifically in muscle tissues. Furthermore, when the AAV vector is injected in an intravenous administration system, the transduction efficiency of the AAV vector to the liver is high, the expression of hMTM1 can be detected in the liver of mice with AAV8-MCK-hMTM1 and AAV9-MCK-hMTM1 virus groups, and although the expression level of hMTM1 is not high due to the regulation of MCK muscle-specific promoters, the safety risk is not small. The experimental result shows that the target sequence of miR-122 highly expressed in liver added into 3' UTR of hMTM1 expression frame can effectively inhibit the expression of hMTM1 gene (figure 11), remarkably enhances the muscle specificity expression of hMTM1 gene, and increases the safety in the use process. The results in fig. 11 show that the expression of hMTM1 gene was not detected in wild-type mice because the DNA sequence of hMTM1 was codon optimized and was different from the endogenous MTM1 RNA sequence of mice, and therefore the probes and primers for quantitative PCR detection of hMTM1 gene could not recognize and bind to the mouse MTM1 RNA sequence, and there was no detection signal, which also indicates that the quantitative PCR probes and primers used in the present invention can specifically recognize and bind to hMTM1 RNA sequence, and the reliability of the detection results is high. In the assay, mice injected with AAV8-MCK-EGFP or AAV9-MCK-EGFP died, and no assay results are shown in FIG. 11.

In conclusion, the results show that AAV8-MCK-hMTM1, AAV8-MCK-hMTM1-122T-142T, AAV9-MCK-hMTM1 and AAV9-MCK-hMTM1-122T-142T recombinant viruses designed by the invention can be introduced into XLMTM model mice through intravenous administration, and can express and generate human MTM1 protein in the muscle of the model mice, thereby prolonging the survival period, recovering development (weight gain) and physiological functions of the model mice. And AAV8-MCK-hMTM1-122T-142T and AAV9-MCK-hMTM1-122T-142T viruses show better tissue specificity, the survival time of model mice is prolonged, and the development potential is larger.

Sequence listing

<110> Acanthopanax beijing and institute of molecular medicine, Inc

<120> gene medicine for X chromosome linked myotubule myopathy

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 572

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ctcgagccac tatgggtcta ggctgcccat gtaaggaggc aaggcctggg gacacccgag 60

atgcctggtt ataattaacc cagacatgtg gctgctcccc cccccccaac acctgctgcc 120

tgagcctcac ccccaccccg gtgcctgggt cttaggctct gtacaccatg gaggagaagc 180

tcgctctaaa aataaccctg tccctggtgg agcccgtgcc tgggactccc aaagtattac 240

tgttccatgt tcccggcgaa gggccagctg tcccccgcca gctagactca gcacttagtt 300

taggaaccag tgagcaagtc agcccttggg gcagcccata caaggccatg gggctgggca 360

agctgcacgc ctgggtccgg ggtgggcacg gtgcccgggc aacgagctga aagctcatct 420

gctctcaggg gcccctccct ggggacagcc cctcctggct agtcacaccc tgtaggctcc 480

tctatataac ccaggggcac aggggctgcc cccgggtcac caccacctcc acagcacaga 540

cagacactca ggagccagcc agccagggta cc 572

<210> 2

<211> 758

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggtaccgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 60

gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 120

gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 180

ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc 240

gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 300

cgcaccatct tcttcaaggg cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 360

ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 420

atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 480

aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc 540

gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 600

cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc 660

gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag 720

ctgtacaagt aaagtggccg cgactctaga gggaattc 758

<210> 3

<211> 1833

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggtaccgcca ccatggcctc cgcctctacc agcaagtaca actcccactc tctggagaat 60

gagagcatca agcgcacctc ccgggatggc gtgaacagag acctgacaga ggccgtgcct 120

aggctgccag gagagaccct gatcacagat aaggaagtga tctacatctg cccattcaac 180

ggccccatca agggccgggt gtatatcacc aattaccgcc tgtatctgcg gtccctggag 240

acagatagct ccctgatcct ggacgtgcca ctgggcgtga tctctagaat cgagaagatg 300

ggaggagcca cctctagggg agagaacagc tacggcctgg atatcacctg taaggacatg 360

agaaatctga ggtttgccct gaagcaggag ggccactccc ggagagatat gttcgagatc 420

ctgaccagat atgcctttcc tctggcccac agcctgccac tgttcgcctt tctgaacgag 480

gagaagttca atgtggacgg ctggacagtg tacaaccctg tggaggagta taggcgccag 540

ggactgccaa accaccactg gcggatcacc tttatcaata agtgctacga gctgtgcgac 600

acatatcccg ccctgctggt ggtgccttac agagccagcg acgatgacct gcggagagtg 660

gccaccttca ggtcccgcaa ccggatccca gtgctgtctt ggatccaccc cgagaataag 720

acagtgatcg tgcgctgcag ccagcctctg gtgggcatgt ccggcaagcg gaacaaggat 780

gacgagaagt acctggatgt gatcagagag accaataagc agatcagcaa gctgacaatc 840

tatgacgcaa ggccaagcgt gaacgcagtg gcaaataagg caaccggagg aggatacgag 900

tccgatgacg cctatcacaa cgccgagctg ttctttctgg atatccacaa tatccacgtg 960

atgcgcgaga gcctgaagaa ggtgaaggac atcgtgtacc ccaacgtgga ggagagccac 1020

tggctgtcta gcctggagtc cacccactgg ctggagcaca tcaagctggt gctgacaggc 1080

gccatccagg tggccgataa ggtgtcctct ggcaagagct ccgtgctggt gcactgctcc 1140

gatggatggg acaggaccgc acagctgaca tctctggcca tgctgatgct ggactctttc 1200

tatagaagca tcgagggctt tgagatcctg gtgcagaagg agtggatctc tttcggccac 1260

aagtttgcca gcaggatcgg ccacggcgat aagaatcaca ccgatgccga ccgctctcca 1320

atcttcctgc agtttatcga ctgcgtgtgg cagatgagca agcagttccc caccgccttc 1380

gagtttaacg agcagtttct gatcatcatc ctggaccacc tgtacagctg caggttcggc 1440

acattcctgt ttaattgtga gtccgccaga gagaggcaga aggtgaccga gcgcacagtg 1500

agcctgtggt ccctgatcaa ctccaataag gagaagttca agaacccctt ttacacaaag 1560

gagatcaatc gcgtgctgta tcctgtggcc agcatgcggc acctggagct gtgggtgaac 1620

tactatatca gatggaatcc caggatcaag cagcagcagc caaaccccgt ggagcagcgg 1680

tacatggagc tgctggccct gcgcgatgag tatatcaagc ggctggagga gctgcagctg 1740

gccaattccg ccaagctgtc tgacccccct acctccccct ctagcccttc tcagatgatg 1800

cctcacgtgc agacacactt ttgataagaa ttc 1833

<210> 4

<211> 1942

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggtaccgcca ccatggcctc cgcctctacc agcaagtaca actcccactc tctggagaat 60

gagagcatca agcgcacctc ccgggatggc gtgaacagag acctgacaga ggccgtgcct 120

aggctgccag gagagaccct gatcacagat aaggaagtga tctacatctg cccattcaac 180

ggccccatca agggccgggt gtatatcacc aattaccgcc tgtatctgcg gtccctggag 240

acagatagct ccctgatcct ggacgtgcca ctgggcgtga tctctagaat cgagaagatg 300

ggaggagcca cctctagggg agagaacagc tacggcctgg atatcacctg taaggacatg 360

agaaatctga ggtttgccct gaagcaggag ggccactccc ggagagatat gttcgagatc 420

ctgaccagat atgcctttcc tctggcccac agcctgccac tgttcgcctt tctgaacgag 480

gagaagttca atgtggacgg ctggacagtg tacaaccctg tggaggagta taggcgccag 540

ggactgccaa accaccactg gcggatcacc tttatcaata agtgctacga gctgtgcgac 600

acatatcccg ccctgctggt ggtgccttac agagccagcg acgatgacct gcggagagtg 660

gccaccttca ggtcccgcaa ccggatccca gtgctgtctt ggatccaccc cgagaataag 720

acagtgatcg tgcgctgcag ccagcctctg gtgggcatgt ccggcaagcg gaacaaggat 780

gacgagaagt acctggatgt gatcagagag accaataagc agatcagcaa gctgacaatc 840

tatgacgcaa ggccaagcgt gaacgcagtg gcaaataagg caaccggagg aggatacgag 900

tccgatgacg cctatcacaa cgccgagctg ttctttctgg atatccacaa tatccacgtg 960

atgcgcgaga gcctgaagaa ggtgaaggac atcgtgtacc ccaacgtgga ggagagccac 1020

tggctgtcta gcctggagtc cacccactgg ctggagcaca tcaagctggt gctgacaggc 1080

gccatccagg tggccgataa ggtgtcctct ggcaagagct ccgtgctggt gcactgctcc 1140

gatggatggg acaggaccgc acagctgaca tctctggcca tgctgatgct ggactctttc 1200

tatagaagca tcgagggctt tgagatcctg gtgcagaagg agtggatctc tttcggccac 1260

aagtttgcca gcaggatcgg ccacggcgat aagaatcaca ccgatgccga ccgctctcca 1320

atcttcctgc agtttatcga ctgcgtgtgg cagatgagca agcagttccc caccgccttc 1380

gagtttaacg agcagtttct gatcatcatc ctggaccacc tgtacagctg caggttcggc 1440

acattcctgt ttaattgtga gtccgccaga gagaggcaga aggtgaccga gcgcacagtg 1500

agcctgtggt ccctgatcaa ctccaataag gagaagttca agaacccctt ttacacaaag 1560

gagatcaatc gcgtgctgta tcctgtggcc agcatgcggc acctggagct gtgggtgaac 1620

tactatatca gatggaatcc caggatcaag cagcagcagc caaaccccgt ggagcagcgg 1680

tacatggagc tgctggccct gcgcgatgag tatatcaagc ggctggagga gctgcagctg 1740

gccaattccg ccaagctgtc tgacccccct acctccccct ctagcccttc tcagatgatg 1800

cctcacgtgc agacacactt ttgataagaa ttccaaacac cattgtcaca ctccagatcc 1860

aaacaccatt gtcacactcc aacgcgctcc ataaagtagg aaacactaca gtcatccata 1920

aagtaggaaa cactacagat ct 1942

<210> 5

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ataggtaccg ccaccatggt gagcaag 27

<210> 6

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gcggaattct tacttgtaca gctcgtc 27

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

acaccatgga ggagaagctc 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gccgggaaca tggaacagta 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ccctggtgga gcccgtgcct 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gcagatcagc aagctgacaa 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cgtcatcgga ctcgtatcct 20

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cgcaaggcca agcgtgaacg ca 22

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

aacggatttg gccgtattgg 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aatctccact ttgccactgc 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cgcctggtca ccagggctgc 20

Claims

1. A human myotubulin gene expression cassette characterized in that,

(i) comprises a muscle-specific promoter sequence, said muscle-specific promoter being a creatine kinase (MCK) promoter;

(II) contains an optimized human myotubulin coding sequence, and the sequence information is shown in SEQ ID NO. 3;

(III) carrying human miRNA target sequences, which are completely complementary target sequences of human miR-122 and miR-142-3p, wherein the number of each miRNA target sequence is not less than 2, and the target sequences are connected in series through a spacer sequence.

2. The human myotubulin gene expression cassette of claim 1, wherein the muscle specific promoter is Mouse Creatine Kinase (MCK) promoter and the sequence information is shown in SEQ ID No. 1.

3. The human myotubulin gene expression cassette of claim 1, wherein the sequences of (ii) and (iii) are SEQ ID No. 4.

4. A recombinant adeno-associated viral vector comprising the human myotubulin gene expression cassette of any one of claims 1 to 3.

5. The recombinant adeno-associated viral vector according to claim 4, wherein the recombinant adeno-associated viral vector serotypes include AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10.

6. The recombinant adeno-associated viral vector according to claim 5, wherein the recombinant adeno-associated viral vector serotype is AAV8 or AAV 9.

7. A gene medicament comprising the human myotubulin gene expression cassette of any one of claims 1 to 3 or the recombinant adeno-associated virus vector of any one of claims 4 to 6.

8. The gene medicine of claim 7, wherein the administration is systemic.

9. The gene medicine of claim 8, wherein the administration is intravenous injection.

10. The gene medicine of claim 7, wherein the one-time administration can continuously express to produce human myotubulin, and the adverse symptoms caused by the mutation of the myotubulin gene can be effectively relieved or cured.