CN108611355A - The genomic medicine of X-linkage myotublar myopathy - Google Patents

Abstract

The present invention provides recombined gland relative virus mediated X-linkage myotublar myopathy medicine.Recombined glandulae correlation viral vectors carry the flesh tubulin gene expression frame for including people miR 122 target sequences of 142 3p and miR.Experiment in vivo shows that the recombined glandulae correlation viral vectors can be imported efficiently in vivo, sustainedly and stably expresses flesh tubulin, extends the life cycle of animal model, restores its growth and development.As a result it prompts, which is hopeful to be developed into new X-linkage myotublar myopathy medicine.

Description

The genomic medicine of X-linkage myotublar myopathy

Technical field

The present invention relates to biotechnologies, and in particular to recombined glandulae correlation viral vectors carry artificial design flesh tubulin Gene（mtm1）The X-linkage myotublar myopathy genomic medicine of expression cassette.

Background technology

The chain myotublar myopathies of X (X-linked myotubular myopathy, XLMTM) are a kind of Congenital muscle mistakes Obstacle occurs when foster disease, mainly muscle fibril row are at myotubule, causes myotubule to be formed bad, thus musculature is made to develop Interrupt and rest on the musculature of fetal period, sufferer muscle section can find muscle cell, but nucleus not as at Ripe musculature is dispersed in around meat fiber, but positioned at cell center.XLMTM diseases are micro- by the flesh of X-linkage Guan Su（Myotubularin, mtm1）Gene mutation causes.A kind of phosphatase of mtm1 gene codes, can adjust PIP in cell And PIP₂Ratio, play a significant role during cell signalling.Flesh tubulin（Lower abbreviation " MTM1 albumen "）Thin Wide expression in born of the same parents, but function is only shown in muscle cell.Mtm1 gene mutations lead to XLMTM diseases, incidence 1/ 50000 newborn boy babies（Amburgey K, et al. Neurology. 2017; 89(13):1355-1364.）.At present For XLMTM diseases without effective medicine and method, enzyme replacement therapy and gene therapy are possible therapeutic strategies.

2013, Lawlor MW etc. had rated the MTM1 albumen of fusion antibody combination Fv segments in XLMTM mouse models Function and effect（Lawlor MW, et al. Human Molecular Genetics. 2013; 22: 1525-1538.）.Knot Fruit finds that the albumen can effectively improve the muscle strength in model mice and reverse lesion characteristics, shows that certain treatment is made With.Although antibody combination Fv segments increase the targeting of MTM1 albumen in vivo, since MTM1 albumen rises in the cell Effect, needs albumen penetration cell film to enter in cytoplasm, efficiency is relatively low, larger to influential effect.As protein drug, need It is administered continuously, patient dependence is relatively low, is not that a kind of ideal medicine is candidate.In terms of genomic medicine research, 2008 The display of the results of study such as Buj-Bello A carries mtm1 gene expression frame AAV carrier intramuscular injection XLMTM mouse models can be effective Restore the muscle function near injection site in ground（Buj-Bello A, et al. Human Molecular Genetics. 2008; 17: 2132-2143.）, the feasibility of gene therapy XLMTM is demonstrated from principle.Then, Childers MK etc. The mouse mtm1 gene expression frames and dog mtm1 gene expression frames for constructing muscle specific promoter regulating and expressing, are packaged into AAV8 viruses, in XLMTM model mices and model dog body, expression generates MTM1 albumen for intravenous injection, Restoration model it is normal Function extends life cycle（Childers MK, et al. Sci Transl Med. 2014;6:220ra10.）.The research is said The feasibility being administered systemically for treating XLMTM is illustrated.Mack DL in 2017 etc. are further confirmed in XLMTM dog models The gene therapy that is administered systemically that AAV8 is mediated can effectively correct the mtm1 gene mutations of whole body（Mack DL, et al. Mol Ther. 2017; 25(4): 839-854.）, move towards clinical test for the XLMTM genomic medicines based on AAV8 carriers and establish Scientific basic.Patent（WO2014/167253 A1）The gene therapy method of XLMTM is reported, this method is taken using AAV carriers Mtm1 gene expression units with muscle specific promoter regulation and control, intravenous system administration, AAV carrier serotypes are AAV8 And AAV9.United States Patent (USP)（US9895426）Report same patent（WO2014/167253 A1）The similar gene therapy sides XLMTM Method.Primary difference is that United States Patent (USP)（US9895426）Use two kinds of XLMTM model mices and XLMTMT model dogs etc. dynamic Object model verifies the validity of genomic medicine.Based on the studies above as a result, a variety of XLMTM genomic medicines enter clinical research rank Section（https://clinicaltrials.gov/）, relevant clinical pilot project number is 2.

On the basis of fully investigating others' work, we devise a kind of gene therapy medicament rAAV- for XLMTM MCK-hMTM1-122T-142T.First, our optimum synthesis people mtm1 gene coded sequences（hMTM1）, excellent by sequence Change, eliminates mRNA secondary structures, rare codon and cryptic splice site to mtm1 gene coded sequence transcription and translations It influences, improves gene expression dose.Secondly, using mouse creatine kinase（MCK）The transcription of promoter regulation hMTM1, MCK are opened Mover has the characteristics that musculature specificity is good, transcriptional level is high, sequence is short and small（Shield MA, et al. Mol Cell Biol. 1996; 16(9): 5058-5068.）, contribute to hMTM1 specificity high efficient expression in musculature.In addition, 3 ' UTR of gene（untranslated region）MiR-142-3p target sequences are added in area, inhibit hMTM1 bases to the maximum extent Because in immunity-associated cell（Such as antigen presenting cell）Middle expression significantly reduces the generation probability for the reaction of MTM1 protein immunizations （Boisgerault F, et al. Hum Gene Ther. 2013; 24 (4):393-405.）.It is special using normal liver Property height expression miR-122 the characteristics of（Jopling C. RNA Biol. 2012; 9(2): 137-142.）, set in 3 ' UTR MiR-122 target sequences are counted, hMTM1 genes is reduced and is expressed in liver cell, enhance its safety.Selection is safe, non-pathogenic heavy Group AAV carriers（Dismuke DJ, et al. Curr Gene Ther. 2013; 13(6): 434-452.）Carry hMTM1 Gene expression frame.Further improve the successful possibility of drug development.

Adeno-associated virus（Adeno-associated virus, AAV）It gains the name because being found in adenoviral preparation （Atchison RW,et al. Science. 1965; 149: 754-756.Hoggan MD, et al. Proc Natl Sci USA. 1966; 55: 1467-1474.）.AAV is Parvoviridae (Parvovirus) member, including a variety of serum Type, genome be single stranded DNA (Rose JA,et al. Proc Natl Acad Sci USA. 1969; 64: 863- 869.), wherein the Genome Size of AAV2 is 4682 nucleotide.AAV is dependovirus, needs other viruses such as adenopathy Poison, herpes simplex virus and human papilloma virus (Geoffroy MC,et al. Curr Gene Ther. 2005; 5(3): 265-271.) or cofactor provides miscellaneous function ability reproducible.In the presence of no helper virus, after AAV infection cells its Genome would be integrated into cell chromosome become latence (Chiorini JA,et al. Curr Top Microbiol Immunol. 1996; 218:25-33.), without generating progeny virus.

The AAV viruses being separated to earliest are 2 type AAV of serotype（AAV2）(Atchison RW,et al. Science. 1965; 149: 754-756.).AAV2 genomes are about 4.7kb, and genome both ends are " the opposing end weight of length 145bp Complex sequences "（ inverted terminal repeat, ITR）, in the palindrome-hairpin structure (Lusby E,et al. J Virol. 1980; 34: 402-409.).There are two great opening reading frames in genome（ORF）, it is separately encoded rep and cap bases Cause.The full-length genome of AAV2 be cloned into escherichia coli plasmid (Samulski RJ,et al. Proc Natl Acad Sci USA. 1982; 79: 2077-2081. Laughlin CA, et al. Gene. 1983; 23: 65-73.)。

ITR is the cis-acting elements of AAV vector gene groups, in the integration of AAV viruses, rescue, duplication and genome packet Play a significant role in dress (Xiao X,et al. J Virol. 1997; 71(2): 941-948. ).It is wrapped in ITR sequences Protein binding site containing Rep（Rep binding site, RBS）With end unwinding site trs（terminal resolution site）, can be identified by Rep protein bindings and at trs generate notch (Linden RM,et al. Proc Natl Acad Sci USA. 1996; 93(15): 7966-7972.).ITR sequences can also form unique " T " alpha type secondary structure, Play a significant role in the life cycle of AAV viruses (Ashktorab H,et al. J Virol. 1989; 63(7): 3034-3039.)。

AAV2 genomes rest part can be divided into 2 functional areas, the gene regions rep and the gene regions cap (Srivastava A,et al. J Virol. 1983; 45(2): 555-564.).The gene regions rep encode Rep78, Rep68, Rep52 and Rep40 Four kinds of Rep albumen.Rep albumen all plays an important roll the duplication of AAV viruses, integration, rescue and packaging.Wherein Rep78 With the end unwinding site trs in Rep68 and ITR（terminal resolution site）Motif is repeated with GAGY （repeat motif）Specific binding (H ü ser D,et al. PLoS Pathog. 2010; 6(7):E1000985.), Start AAV genomes from the single-stranded reproduction process to double-strand.It is in AAV genome duplications that trs and GAGC, which repeats motif, in ITR The heart thus while ITR sequences are all not quite similar in the AAV viruses of various serotypes, but can form hairpin structure and deposit In Rep binding sites.There are p19 promoters at AAV2 Genome Atlas position 19, expresses Rep52 and Rep40 respectively.Rep52 The DNA helicase activity for being not bound with the function of DNA with Rep40, and thering is ATP to rely on.The capsid of cap gene codes AAV viruses Albumen VP1, VP2 and VP3.Wherein, VP3 molecular weight is minimum, but quantity is most, VP1, VP2, VP3 in ripe AAV particles Ratio substantially 1:1:10.VP1 is formed with necessary to infective AAV；VP2 assists VP3 to enter nucleus；VP3 is group At the major protein of AAV particles.

With the understanding to AAV vial life periods and its relevant molecule biological mechanism, AAV viruses have been transformed into one The efficient foreign gene transfer tool of kind, i.e. AAV carriers.Only include the ITR of AAV viruses in improved AAV vector genes group Sequence and the exogenous gene expression frame for carrying transhipment, the Rep and Cap protein that virus packaging needs are carried by the way that exogenous plasmid is trans- For reducing rep and cap genes and being packaged into the harm that AAV carriers may be brought.In addition, AAV viruses itself, which do not have, causes a disease Property, so that AAV carriers is become generally acknowledged one of safest viral vectors.Delete the D sequences in the side ITR sequences of AAV viruses And trs（terminal resolution site）Sequence can also make the recombination AAV viral vectors being packaged to be carry gene Self complementation of group, forms double-strand, significantly improves the inside and outside transduction efficiency of AAV carriers（Wang Z,et al. Gene Ther. 2003;10(26):2105-2111. McCarty DM, et al. Gene Ther. 2003;10(26):2112-2118.）. The virus being packaged to be becomes scAAV（self-complementary AAV）Virus, i.e., so-called double-strand AAV viruses.It is different In the unmutated ssAAV of bilateral ITR（single-stranded AAV）, i.e., traditional AAV viruses.The packaging of scAAV viruses Capacity smaller, the only half of ssAAV bale capacities, about 2.2kb-2.5kb, but transduction efficiency higher after infection cell.AAV Virus serotype is numerous, and different serotype has different tissue infection preferendums, therefore application AAV carriers can be by external source base Because be transported to specific organ and tissue (Wu Z,et al. Mol Ther. 2006; 14(3): 316-327.).Certain blood Clear type AAV carriers can also pass through blood-brain barrier, and foreign gene is caused in cerebral neuron, be carried for the cerebripetal gene transfer of target Supplied may (Samaranch L,et al. Hum Gene Ther. 2012; 23(4): 382-389.).In addition, AAV is carried The stable in physicochemical property of body, to soda acid and high temperature embody stronger tolerance (Gruntman AM,et al. Hum Gene Ther Methods. 2015; 26(2):71-76.), it is easy to develop the biological products of high stability.

The AAV carriers also packaging system with relative maturity, is convenient for large-scale production.Common AAV is carried both at home and abroad at present Body packaging system is mainly auxiliary virus system, herpes simplex virus including three plasmid co-transfection systems, adenovirus（Herpes Simplex virus type 1, HSV1）Packaging system for helper virus and the packaging system based on baculoviral.Its In, three plasmid transfection packaging systems are safe because being not necessarily to helper virus, are the AAV vector packaging systems being most widely used, It is also the production system of current mainstream in the world.It shows slightly unfortunately, the missing of efficiently extensive transfection method limits three matter Application of the grain transfection system in AAV carriers are prepared on a large scale.Yuan etc. establishes extensive as the AAV of helper virus using adenovirus Packaging system（Yuan Z,et al. Hum Gene Ther, 2011,22(5):613-624.）, the system production is efficient, But trace of the adenovirus in last AAV finished products exists in packaging system, affects the safety of AAV finished products.HSV1 is as auxiliary It is another kind of widely used AAV vector packaging systems to help the packaging system of virus.Wu Zhijian and Conway etc. is almost same When propose AAV2 carrier package strategies using HSV1 as helper virus in the world（Wu Zhijian, Wu little Bing etc..Science Bulletin, 1999,44（5）：506-509.Conway JE,et al. Gene Ther, 1999,6:986-993.）.Subsequent Wustner Etc. the AAV5 carrier package strategies proposed using HSV1 as helper virus（Wustner JT,et al. Mol Ther, 2002, 6(4):510-518.）.On this basis, Booth etc. carries the rep/cap genes and AAV of AAV using two HSV1 respectively Opposing end sequence（Inverted terminal repeat, ITR）/ exogenous gene expression frame, so latter two recombination HSV1 is sick Malicious co-infection produces cell, and packaging generates AAV viruses（Booth MJ,et al. Gene Ther,2004,11:829- 837.）.Thomas etc. further establishes the suspension cell system of double HSV1 viruses AAV productions（Thomas DL,et al. Gene Ther,2009,20:861-870.）, make it possible more massive AAV virus productions.In addition, the utilizations such as Urabe Three baculovirals carry the structure of AAV, non-structural and ITR/ exogenous gene expression frames respectively, construct the rod-shaped of AAV carriers Viral Packaging System.In view of the unstability of baculoviral foreign gene-carrying, then reduce required bar in production system The number of shape virus, gradually three baculovirals of the needs since most to need two or baculovirals（Chen H. Mol Ther.2008;16(5):924-930. Galibert L,et al.J Invertebr Pathol, 2011,107 Suppl: S80-93.）And a baculoviral adds one plant of inducible cell line strategy（Mietzsch M,et al. Hum Gene Ther. 2014;25:212-222. Mietzsch M, et al. Hum Gene Ther. 2015;26(10):688- 697.）.Each packaging system all differs from one another, and can make suitable selection as needed.

Due to These characteristics, AAV carriers are increasingly becoming the gene that one kind being widely used in gene therapy, especially hereditary disease The foreign gene transfer tool for the treatment of.By in November, 2017, the clinical gene therapy for the opportunity AAV carriers ratified in the world tries Proved recipe case has 204（http://www.abedia.com/wiley/vectors.php）.What is more important is carried based on AAV The lipoprotein lipase gene medicine Glybera of body was ratified to list in 2012 by European Bureau of Drugs Supervision, became the Western countries First gene therapy medicament of approval（Ylä-Herttuala S.Mol Ther. 2012; 20(10): 1831- 1832.）；On December 19th, 2017, U.S. FDA ratified congenital amaurosis Before-daybreak diseases（RPE65 gene mutations cause）Gene therapy medicament Luxturna is listed, and becomes the gene therapy medicament of the rare disease in first, the U.S.（https://www.fda.gov/ newsevents/newsroom/pressannouncements/ucm589467.htm）.Hemophilia B（Kay MA, et al.Nat Genet. 2000; 24(3): 257-261.）AAV vector gene therapies drug obtain good clinical test effect Fruit, it is contemplated that in the near future can list marketing, benefit many patients.

In the present invention, we select AAV carriers to carry hMTM1 gene expression frames, are mainly based upon the following of AAV carriers Feature.First, AAV carriers only retain two ITR sequences for packing needs in wild-type virus, wild-type virus gene is not contained Protein coding gene in group（Salgenik M,et al. Microbiol Spectr. 2015; 3(4).）, immunogenicity It is low.Second, AAV realizes the continual and steady expression for carrying gene frame usually in the form of unconformable extrachromosomal genetic element （Chen ZY,et al. Mol Ther. 2001; 3(3): 403-410.）, avoid being inducted into gene random integration and bringing Safety issue.Third, AAV carriers all have higher transduction efficiency by intravenous injection, 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 al. Hum Gene Ther.2010;21(4):463- 477.）, ensure that hMTM1 gene expression frames can efficiently express MTM1 albumen in vivo.

The probability reacted for MTM1 protein immunizations is generated in order to reduce body, extends hMTM1 genes and continually and steadily expresses Time.We select the MCK promoters of muscle specific high efficient expression, make the hMTM1 genes of importing special high in muscle Effect expression.Further, we utilize the characteristics of normal liver specificity overexpression miR-122（Jopling C. RNA Biol. 2012; 9(2): 137-142.）, miR-122 target sequences are cloned into the 3 ' areas UTR of hMTM1 gene expression frames.Borrow miRNA The principle of inhibition of gene expression（Kim VN. Nat Rev Mol Cell Biol.2005;6(5):376-385.）, in liver cell MiR-122 molecules can inhibit the expression of MTM1 albumen, significantly reduce expression of the MTM1 albumen in liver.Pass through muscle specific Property promoter and miR-122 target sequences regulation and control, realize MTM1 albumen targeting expression.Simultaneously table is crossed in order to reduce MTM1 albumen Up to the immune response that may be brought, we devise miR-142-3p target sequence regulating strategies.Since miR-142-3p is in hematopoiesis High expression in stem cell line derived cell（Chen CZ,et al. Science.2004; 303(5654): 83-86.）, exempt from Epidemic disease cell, which breaks up, derives from candidate stem cell system, therefore utilizes the principle of miRNA inhibition of gene expression（Kim VN. Nat Rev Mol Cell Biol.2005;6(5):376-385.）, the gene expression for carrying miR-142-3p target sequences can be immune Obviously inhibited in cell, to reduce probability of the body generation for gene expression product immune response（Boisgerault F, et al. Hum Gene Ther. 2013; 24 (4):393-405.）.

miRNAs（microRNAs）Be the length that is widely present in human and animal's body it is 18 to 25 nucleotide （Nucleotide, nt）Single-stranded non-coding RNA (Bartel DP. Cell. 2004; 116: 281-297. Kim VN. Nat Rev Mol Cell Biol. 2005; 6: 376-385.).MiRNA is first in caenorhabditis elegan within 1993 （C.elegans）It is middle find (Lee RC,et al. Cell. 1993; 75: 843-854. Wightman B, et al. Cell. 1993; 75: 855-862.).Lin-4 genes can lower the expression of lin-14 genes, but lin- in C.elegans The coded product of 4 genes not instead of protein, a kind of small RNA molecular show that itself coding small RNA molecular can adjust gene Expression.Then, small RNA molecular as multiple types in different species and cell in succession find (Lagos-Quintana M,et al. Science. 2001; 294: 853-858. Lau NC, et al. Science. 2001; 294: 858- 862. Lee RC, et al. Science. 2001;294:862-864.), miRNA starts the system as such tiny RNA Claim.MiRNA adjust about 60% gene of the mankind expression (Lewis BP,et al. Cell. 2005;120: 15-20. Friedman RC, et al. Genome Res. 2009; 19:92-105.), it is played in a variety of physiology and pathologic process Important function (Careton 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.)。

MiR-96 gene be usually located in the exon, introne and intergenic region of genome (Olena AF,et al. J Cell Physiol. 2010; 222: 540-545. Kim VN, et al. Trends Genet. 2006; 22: 165- 173.).In the cell, the generation process of miRNA it is for example following (Winter J,et al. Nat Cell Biol. 2009;11: 228-234.).First in nucleus, miR-96 gene starts transcription by rna plymerase ii or III and generates initial product pri- microRNA；Self folded portion sequence of pri-microRNA forms loop-stem structure.Then, by rnase iii Drosha Pri-microRNA is acted on the processing complex of DGCR8 molecular compositions, extra sequence is cut, leaves the stem ring knot of 60nt or so Structure, i.e. 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 al. Curr Biol. 2004;14: 2162-2167.).Then under the assistance of transport protein Exportin-5, pre-microRNA Enter in cytoplasm from nucleus (Lund E,et al. Science. 2003; 303: 95-98. Yi R, et al. Genes Dev. 2003;17: 3011-3016. Bohnsack MT, et al. RNA. 2004; 10:185-191.), Remove annular section in its loop-stem structure through the processing of Dicer enzymes, become double stranded rna molecule (Jiang F,et al. Genes Dev. 2005;, 19: 1674-1679. Saito K, et al. PLoS Biol. 2005; 3: e235.)。 Finally, double stranded rna molecule is combined by protein factors such as AGO2, wherein a chain is degraded, another chain and protein factor shape Silencing complex is induced at RNA（RNA induced silencing complex, RISC）.RISC identifies target sequence in mRNA, By mRNA molecules of degrading, promote 3 ' end of mRNA molecules that polyadenylation and inhibition translation is gone to reduce the expression of mRNA, is turning After record Level tune gene expression (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 al. Genes Dev. 2006; 20: 515-524.).Therefore using the miRNA of intracellular high expression, in foreign gene 3 ' UTR (untranslated region) be inserted into the target sequence of the miRNA, foreign gene can be effectively inhibited and imported Expression in cell.

According to the above mentality of designing, rAAV-MCK-hMTM1-122T-142T viruses are prepared in we, and design preparation Obtain the rAAV-MCK-hMTM1 comparison virus of no miRNA target sequences and the rAAV- of Carrying Green Fluorescent Protein encoder block MCK-EGFP comparison virus.Dosage are waited to be injected in XLMTM mouse model bodies respectively these viruses, evaluation design rAAV- The validity of MCK-hMTM1-122T-142T.The results show that comparing rAAV-MCK-EGFP comparison virus, rAAV-MCK- HMTM1-122T-142T and rAAV-MCK-hMTM1 can express MTM1 albumen in XLMTM mouse model bodies, restore The physiological function of XLMTM mouse model muscle, the life cycle of lengthening model mouse.And compared to rAAV-MCK-hMTM1 diseases Poison, rAAV-MCK-hMTM1-122T-142T viruses expression in model mice liver is lower, significantly reduces liver HMTM1 is overexpressed the toxic effect that may be brought, and the what is more important hMTM1 stabilization continuous expression times are longer, suffer from for XLMTM Person provides new therapeutic choice.

Invention content

In view of this, the present invention provides a kind of novel XLMTM gene therapy medicaments based on AAV carriers.The drug by AAV carriers carry hMTM1 gene expression frames.Sequence optimisation obtains people mtm1 gene coded sequence hMTM1, using mouse muscle spy The expression of Specific Promoters MCK regulation and control hMTM1 sequences, in 3 ' UTR of gene expression frame（untranslated region）Qu Jia Enter miR-142-3p target sequences.Based on above-mentioned design, it is contemplated that the drug is by can be in XLMTM model mice fleshes after intravenous injection High efficient expression generates MTM1 albumen in meat tissue, and the muscle physiological function of Restoration model mouse extends life cycle.

In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：

The chain myotublar myopathies of X are treated the present invention provides a kind of（XLMTM）Gene therapy medicament, which is characterized in that the medicine Object is based on recombination AAV carriers, and efficiently drug effect element can be imported in vivo by intravenous injection using AAV carriers, realized Drug effect element high efficient expression generates the MTM1 albumen of therapeutic effect.In order to realize the high efficient expression of MTM1, according to different blood The transduction feature of clear type AAV, the AAV carrier serotypes of selection are mainly AAV8 and AAV9.

XLMTM gene therapy medicaments provided by the invention, which is characterized in that the coding of original people MTM1 protein gene Region sequence has carried out the optimizations such as elimination rare codon, RNA secondary structures and has obtained hMTM1 sequences, is opened using mouse creatine kinase Mover（MCK promoters）The expression for regulating and controlling hMTM1 sequences, ensure that people's MTM1 albumen specific high-efficiency expression in muscle；In base Because being inserted into people's miR-122 and miR-142-3p target sequence of 2 complete complementaries in 3 ' UTR of expression cassette respectively, pressed down based on miRNA The principle of gene expression processed, miR-122 specificity overexpressions in liver cell significantly reduce table of the MTM1 albumen in liver It reaches, miR-142-3p expressions in human hematopoietic stem cell are high, and people MTM1 is dry thin in the artificial blood including antigen presenting cell Expression is suppressed in born of the same parents' derived cell, can be reduced the probability for the immune response that body is generated for people's MTM1 albumen, be contributed to The continuous expression of people's MTM1 albumen.

XLMTM gene therapy medicaments provided by the invention, which is characterized in that be injected to the drug intravenous administration After in XLMTM model mices body, it can constantly express people's MTM1 albumen by efficient stable in Mice Body, express the MTM1 eggs of generation It is capable of the muscle physiological function of Restoration model mouse, the life cycle of lengthening model mouse, to play the effect for the treatment of XLMTM in vain Fruit.

XLMTM gene therapy medicaments provided by the invention, it is further characterized in that, intravenous administration single administration can be grown Phase, constantly the expression in XLMTM model mice bodies generated people's MTM1 albumen, and the muscle physiological function of Restoration model mouse extends Life cycle.

The important Initial experiments material that the present invention uses is as follows：

PHelper plasmids derive from AAV Helper Free System（Agilent Technologies, the U.S.）, by this Company is purchased from AgilentTechnologies companies and preserves.The plasmid includes that three plasmid co-transfection HEK293 cells prepare weight Required adenovirus source helper function genes E2A, E4 and VA RNA of group AAV viruses etc..

PAAV-RC plasmids derive from AAV Helper Free System（Agilent Technologies, the U.S.）, It purchased from AgilentTechnologies companies and is preserved by our company.PAAV-RC plasmids include the rep and cap of complete AAV2 Gene provides 4 kinds of Rep albumen necessary to packaging in three plasmid co-transfections pack Prepare restructuring AAV2 viruses（Rep78、 Rep68, Rep52 and Rep40）With AAV2 coat protein.

PAAV-R2C8 plasmids are built by our company and are preserved.With AAV Helper Free System（Agilent Technologies, the U.S.）In pAAV-RC plasmids be basic framework, with AAV8 genomes（GenBank ID：AF513852） Middle coat protein coding sequence Cap8（2121st to 4337 bit sequence in genome）Replace pAAV-RC plasmids in the 2013rd to 4220 bit sequences are to get pAAV-R2C8 plasmids.Brief building process is to obtain pAAV-R2C8 plasmid sequences according to aforementioned thinking Column information, HindIII is to sequence between PmeI restriction enzyme sites in artificial synthesized pAAV-R2C8 plasmids, using the molecule gram of standard Grand method obtains pAAV-R2C8 with sequence between HindIII and PmeI restriction enzyme sites in composition sequence replacement pAAV-RC plasmids Plasmid.PAAV-R2C8 plasmids include the rep genes of the cap genes and AAV2 of complete AAV8, pack and make in three plasmid co-transfections 4 kinds of Rep albumen necessary to packaging are provided in standby recombination AAV8 viruses（Rep78, Rep68, Rep52 and Rep40）Outside AAV8 Glutelin.

PAAV-R2C9 plasmids are built by our company and are preserved.With AAV Helper Free System（Agilent Technologies, the U.S.）In pAAV-RC plasmids be basic framework, with AAV9 coat protein coding sequences（GenBank ID：AY530579）The 2013rd to 4220 bit sequence is to get pAAV-R2C9 plasmids in replacement pAAV-RC plasmids.Brief structure Process is, pAAV-R2C9 plasmid sequence information is obtained according to aforementioned thinking, and HindIII is extremely in artificial synthesized pAAV-R2C9 plasmids Sequence between PmeI restriction enzyme sites replaces pAAV-RC plasmids HindIII using the molecular cloning method of standard with composition sequence Sequence obtains pAAV-R2C9 plasmids between PmeI restriction enzyme sites.PAAV-R2C9 plasmids include the cap genes of complete AAV9 With the rep genes of AAV2,4 kinds of Rep eggs necessary to packaging are provided in three plasmid co-transfections pack Prepare restructuring AAV9 viruses In vain（Rep78, Rep68, Rep52 and Rep40）With AAV9 coat protein.

The chain myotublar myopathy mouse models of X prepare kind of a mouse, are purchased from U.S. The Jackson Laboratory, trade name B6.Cg-Mtm1 ^tm1Itl / J, conservation number 018153.This kind of mouse gender is female, X-linkage mtm1（myotubular myopathy gene 1）4 heterozygous mutation of gene extron, the mtm1 genes of mutation, which cannot express, generates normal MTM1 eggs In vain（Pierson CR, et al. Hum Mol Genet. 2012;21(4):811-25. ）.The same Male wild-type of this kind of mouse After C57BL/6J mouse hybrids, approximately half of neonatal male mouse mtm1 gene mutations occur small with the chain fleshes of the similar X of people Pipe myopathy.

Control mice, C57BL/6J mouse are purchased from Beijing HFK Bio-Technology Co., Ltd., are used as zoopery Wild type control.

Description of the drawings

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technology description to be briefly described.

Fig. 1 pAAV2neo carrier structure schematic diagrames.The both sides ITR that our company preserves is the AAV of 145bp wild types ITR Carrier pAAV2neo（Dong X, et al. PLoS ONE. 2010; 5(10):e13479.）.ITR, inverted Terminal repeat, length are the inverted terminal repeat of 145bp.CMV promoter, human cytomegalovirus early stage open Mover.BGH polyA, the polynucleotide tailing signal of bovine growth hormone.Amp, ampicillin resistance gene frame.Neo, Neomycin resistance gene frame.XhoI, KpnI, EcoRI, SalI, BglII, BamHI and ApaI are restriction enzyme site.

Fig. 2 pAAV2-MCK-EGFP carrier structure schematic diagrames.ITR, inverted terminal repeat, length are The inverted terminal repeat of 145bp.MCK, mouse creatine kinase promoter.BGH polyA, the polymerized nucleoside of bovine growth hormone Sour tailing signal.Amp, ampicillin resistance gene frame.Neo, neomycin resistance gene frame.

Fig. 3 pAAV2-MCK-EGFP carrier structure schematic diagrames.ITR, inverted terminal repeat, length are The inverted terminal repeat of 145bp.MCK, mouse creatine kinase promoter.BGH polyA, the polymerized nucleoside of bovine growth hormone Sour tailing signal.Amp, ampicillin resistance gene frame.Neo, neomycin resistance gene frame.EGFP, enhanced green Fluorescence protein coding region sequence.

Fig. 4 pAAV2-MCK-hMTM1 carrier structure schematic diagrames.ITR, inverted terminal repeat, length are The inverted terminal repeat of 145bp.MCK, mouse creatine kinase promoter.HMTM1, people's mtm1 gene codes of optimum synthesis Region sequence.BGH polyA, the polynucleotide tailing signal of bovine growth hormone.Amp, ampicillin resistance gene frame. Neo, neomycin resistance gene frame.XhoI, KpnI and EcoRI are restriction enzyme site.

Fig. 5 pAAV2-MCK-hMTM1-122T-142T carrier structure schematic diagrames.ITR, inverted terminal Repeat, length are the inverted terminal repeat of 145bp.MCK, mouse creatine kinase promoter.HMTM1, optimum synthesis People's mtm1 coding sequences.2 × miR-122T, people's miR-122 target sequences of 2 copy complete complementaries.2×miR-142- 3pT, people's miR-142-3p target sequences of 2 copy complete complementaries.BGH polyA, the polynucleotide tailing of bovine growth hormone Signal.Amp, ampicillin resistance gene frame.Neo, neomycin resistance gene frame.XhoI, KpnI, EcoRI and BglII It is restriction enzyme site.

Fig. 6 injections carry hMTM1 gene expression frames recombination AAV viruses and extend XLMTM model mice life cycles.6 kinds are not Same recombination AAV viruses（AAV8-MCK-EGFP、AAV8-MCK-hMTM1、AAV8-MCK-hMTM1-122T-142T、AAV9- MCK-EGFP、AAV9-MCK-hMTM1、AAV9-MCK-hMTM1-122T-142T）With 3 × 10¹³vg/kg（Viral genome, vg）Dosage through tail vein injection XLMTM model mices, wherein AAV8-MCK-EGFP and AAV9-MCK-EGFP are control disease Poison, 5 XLMTM model mices of each virus injection, XLMTM Mouse Ages are 3 weeks when injection.After injecting virus, mouse is recorded Life cycle.WT, C57BL/6J wildness control mice；KO-AAV8-MCK-EGFP, injection of AAV 8-MCK-EGFP viruses XLMTM model mices；KO-AAV8-MCK-hMTM1, the XLMTM model mices of injection of AAV 8-MCK-hMTM1 viruses；KO- AAV8-MCK-hMTM1-122T-142T, the XLMTM model mices of injection of AAV 8-MCK-hMTM1-122T-142T viruses；KO- AAV9-MCK-EGFP, the XLMTM model mices of injection of AAV 9-MCK-EGFP viruses；KO-AAV9-MCK-hMTM1, injection The XLMTM model mices of AAV9-MCK-hMTM1 viruses；KO-AAV9-MCK-hMTM1-122T-142T, injection of AAV 9-MCK- The XLMTM model mices of hMTM1-122T-142T viruses.

Fig. 7 injections carry the changes of weight situation of the XLMTM model mices of hMTM1 gene expression frames recombination AAV viruses. 6 The different recombination AAV viruses of kind（AAV8-MCK-EGFP、AAV8-MCK-hMTM1、AAV8-MCK-hMTM1-122T-142T、 AAV9-MCK-EGFP、AAV9-MCK-hMTM1、AAV9-MCK-hMTM1-122T-142T）With 3 × 10¹³vg/kg（viral Genome, vg）Dosage through tail vein injection XLMTM model mices, wherein AAV8-MCK-EGFP and AAV9-MCK-EGFP are Comparison virus, 5 XLMTM model mices of each virus injection, XLMTM Mouse Ages are 3 weeks when injection.After injecting virus, no The weight of mouse is recorded with time point determining.WT, C57BL/6J wildness control mice；KO-AAV8-MCK-EGFP, injection The XLMTM model mices of AAV8-MCK-EGFP viruses；KO-AAV8-MCK-hMTM1, injection of AAV 8-MCK-hMTM1 viruses XLMTM model mices；KO-AAV8-MCK-hMTM1-122T-142T, injection of AAV 8-MCK-hMTM1-122T-142T viruses XLMTM model mices；KO-AAV9-MCK-EGFP, the XLMTM model mices of injection of AAV 9-MCK-EGFP viruses；KO-AAV9- MCK-hMTM1, the XLMTM model mices of injection of AAV 9-MCK-hMTM1 viruses；KO-AAV9-MCK-hMTM1-122T-142T, The XLMTM model mices of injection of AAV 9-MCK-hMTM1-122T-142T viruses.

Displacement distance test result in mouse 90 minutes after Fig. 8 injecting virus 5 weeks.6 kinds of different recombination AAV viruses （AAV8-MCK-EGFP、AAV8-MCK-hMTM1、AAV8-MCK-hMTM1-122T-142T、AAV9-MCK-EGFP、AAV9- MCK-hMTM1、AAV9-MCK-hMTM1-122T-142T）With 3 × 10¹³vg/kg（Viral genome, vg）Dosage it is quiet through tail Arteries and veins injects XLMTM model mices, and wherein AAV8-MCK-EGFP and AAV9-MCK-EGFP are comparison virus, each virus injection 5 XLMTM model mices, XLMTM Mouse Ages are 3 weeks when injection.After injecting virus 2 weeks, moved in record mouse 90 minutes away from From.WT, C57BL/6J wildness control mice；KO-AAV8-MCK-EGFP, the XLMTM moulds of injection of AAV 8-MCK-EGFP viruses Type mouse；KO-AAV8-MCK-hMTM1, the XLMTM model mices of injection of AAV 8-MCK-hMTM1 viruses；KO-AAV8-MCK- HMTM1-122T-142T, the XLMTM model mices of injection of AAV 8-MCK-hMTM1-122T-142T viruses；KO-AAV9-MCK- EGFP, the XLMTM model mices of injection of AAV 9-MCK-EGFP viruses；KO-AAV9-MCK-hMTM1, injection of AAV 9-MCK- The XLMTM model mices of hMTM1 viruses；KO-AAV9-MCK-hMTM1-122T-142T, injection of AAV 9-MCK-hMTM1-122T- The XLMTM model mices of 142T viruses.

Displacement distance test result in mouse 90 minutes after Fig. 9 injecting virus 3 months.6 kinds of different recombination AAV viruses （AAV8-MCK-EGFP、AAV8-MCK-hMTM1、AAV8-MCK-hMTM1-122T-142T、AAV9-MCK-EGFP、AAV9- MCK-hMTM1、AAV9-MCK-hMTM1-122T-142T）With 3 × 10¹³vg/kg（Viral genome, vg）Dosage it is quiet through tail Arteries and veins injects XLMTM model mices, and wherein AAV8-MCK-EGFP and AAV9-MCK-EGFP are comparison virus, each virus injection 5 XLMTM model mices, XLMTM Mouse Ages are 3 weeks when injection.After injecting virus 3 months, record mouse is moved in 90 minutes Distance.WT, C57BL/6J wildness control mice；KO-AAV8-MCK-EGFP, the XLMTM of injection of AAV 8-MCK-EGFP viruses Model mice；KO-AAV8-MCK-hMTM1, the XLMTM model mices of injection of AAV 8-MCK-hMTM1 viruses；KO-AAV8-MCK- HMTM1-122T-142T, the XLMTM model mices of injection of AAV 8-MCK-hMTM1-122T-142T viruses；KO-AAV9-MCK- EGFP, the XLMTM model mices of injection of AAV 9-MCK-EGFP viruses；KO-AAV9-MCK-hMTM1, injection of AAV 9-MCK- The XLMTM model mices of hMTM1 viruses；KO-AAV9-MCK-hMTM1-122T-142T, injection of AAV 9-MCK-hMTM1-122T- The XLMTM model mices of 142T viruses.ND due to injecting virus dead mouse, therefore is not measured.

Figure 10 difference muscle groups agonistic muscle tubulin expressions.6 kinds of different recombination AAV viruses（AAV8-MCK- EGFP、AAV8-MCK-hMTM1、AAV8-MCK-hMTM1-122T-142T、AAV9-MCK-EGFP、AAV9-MCK-hMTM1、 AAV9-MCK-hMTM1-122T-142T）With 3 × 10¹³vg/kg（Viral genome, vg）Dosage through tail vein injection XLMTM model mices, wherein AAV8-MCK-EGFP and AAV9-MCK-EGFP are comparison virus, 5 XLMTM of each virus injection Model mice, XLMTM Mouse Ages are 3 weeks when injection.After injecting virus 3 months, separation musculus extensor digitorum longus pedis, musculus quadriceps and diaphragm flesh Deng representative musculature.Extract histocyte total protein, SDS-PAGE（Sodium dodecyl sulphate-polyacrylamide gel electricity Swimming）Afterwards, pvdf membrane, protein immunoblot are transferred to（western blot）Detection, it is micro- to be calculated flesh using GAPDH as internal reference The expression of Guan Su.WT, C57BL/6J wildness control mice；KO-AAV8-MCK-EGFP, injection of AAV 8-MCK-EGFP diseases The XLMTM model mices of poison；KO-AAV8-MCK-hMTM1, the XLMTM model mices of injection of AAV 8-MCK-hMTM1 viruses；KO- AAV8-MCK-hMTM1-122T-142T, the XLMTM model mices of injection of AAV 8-MCK-hMTM1-122T-142T viruses；KO- AAV9-MCK-EGFP, the XLMTM model mices of injection of AAV 9-MCK-EGFP viruses；KO-AAV9-MCK-hMTM1, injection The XLMTM model mices of AAV9-MCK-hMTM1 viruses；KO-AAV9-MCK-hMTM1-122T-142T, injection of AAV 9-MCK- The XLMTM model mices of hMTM1-122T-142T viruses.

Figure 11 different tissues hMTM1 gene expression dose testing results.6 kinds of different recombination AAV viruses（AAV8- MCK-EGFP、AAV8-MCK-hMTM1、AAV8-MCK-hMTM1-122T-142T、AAV9-MCK-EGFP、AAV9-MCK- hMTM1、AAV9-MCK-hMTM1-122T-142T）With 3 × 10¹³vg/kg（Viral genome, vg）Dosage noted through tail vein XLMTM model mices are penetrated, wherein AAV8-MCK-EGFP and AAV9-MCK-EGFP are comparison virus, each virus injection 5 XLMTM model mices, XLMTM Mouse Ages are 3 weeks when injection.After injecting virus 3 months, isolating cardiac, liver, skeletal muscle, The tissues such as lung, spleen and kidney.Total tissue RNA is extracted, hMTM1 RNA copy numbers and GAPDH RNA are copied in quantitative PCR detection total serum IgE Shellfish number calculates the ratio of hMTM1 RNA copy numbers and GAPDH RNA copy numbers, the expression for indicating hMTM1 genes. WT, C57BL/6J wildness control mice；The XLMTM models of KO-AAV8-MCK-EGFP, injection of AAV 8-MCK-EGFP viruses are small Mouse；KO-AAV8-MCK-hMTM1, the XLMTM model mices of injection of AAV 8-MCK-hMTM1 viruses；KO-AAV8-MCK-hMTM1- 122T-142T, the XLMTM model mices of injection of AAV 8-MCK-hMTM1-122T-142T viruses；KO-AAV9-MCK-EGFP, note Penetrate the XLMTM model mices of AAV9-MCK-EGFP viruses；KO-AAV9-MCK-hMTM1, injection of AAV 9-MCK-hMTM1 viruses XLMTM model mices；KO-AAV9-MCK-hMTM1-122T-142T, injection of AAV 9-MCK-hMTM1-122T-142T viruses XLMTM model mices.

Specific implementation mode

The invention discloses a kind of gene therapy medicaments of the chain myotublar myopathies of X, including drug is designed, prepared in a small amount And functional verification, those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular It is that all similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this Invention.The method of the present invention and application are described by preferred embodiment, and related personnel can obviously not depart from this Method described herein and application are modified or are suitably changed and combined in invention content, spirit and scope, realizing and Using the technology of the present invention.Wherein, unless otherwise specified, the various reaction reagents involved in embodiment can pass through commercial channel It is commercially available.

With reference to embodiment, the present invention is further explained：

1 plamid vector construction of embodiment

In order to build pAAV2-MCK-EGFP, pAAV2-MCK-hMTM1 and pAAV2- for obtaining packaging recombination AAV viruses and needing MCK-hMTM1-122T-142T plasmids, the pAAV2neo that we are preserved with company first（Fig. 1）Based on, bibliography （Shield MA, et al. Mol Cell Biol. 1996; 16(9): 5058-5068.）With MCK promoters (SEQ ID No.1 the CMV promoter in pAAV2neo carriers) is replaced, pAAV2-MCK is obtained.Next, enhanced green fluorescence will be contained The DNA sequence dna of albumen coded sequence（SEQ ID No.2）, artificial optimization design synthesis hMTM1 sequences (SEQ ID No.3) With hMTM1-122T-142T sequences（SEQ ID NO.4）（Add 2 people miR- successively after the terminator codon of hMTM1 sequences The target sequence of the target sequence of 122 complete complementaries and 2 people's miR-142-3p complete complementaries）It is cloned into pAAV2-MCK carriers respectively KpnI and EcoRI and KpnI and BglII restriction enzyme sites between, obtain pAAV2-MCK-EGFP, pAAV2-MCK-hMTM1 and PAAV2-MCK-hMTM1-122T-142T carriers.

（1）PAAV2-MCK vector constructions

Bibliography（Shield MA, et al. Mol Cell Biol. 1996; 16(9): 5058-5068.）, Search obtains mouse creatine kinase in GeneBank databases（MCK）The sequence number of promoter（M21390.1）, analysis obtains MCK promoter sequences hold addition XhoI restriction enzyme sites " 5 '-CTCGAG-3 ' ", in promoter sequence in promoter sequence 5 ' 3 ' end addition KpnI restriction enzyme sites " 5 '-GGTACC-3 ' " of row, obtain MCK sequences, sequence information such as SEQ ID Shown in NO.1.Nanjing Jin Sirui Bioisystech Co., Ltd is sent to synthesize MCK sequences, composition sequence is cloned into pUC57-1.8K loads Body（Nanjing Genscript Biotechnology Co., Ltd.）, obtain pUC57-1.8K-MCK.The double digested generations of KpnI and XhoI The MCK sequence fragments of 0.6kb and the carrier segments of 1.8kb, recycling MCK sequence fragments are spare.It is replaced with MCK sequence fragments CMV promoter sequence in pAAV2neo carriers between XhoI and KpnI restriction enzyme sites, digestion sequencing identification obtain pAAV2-MCK Carrier（See attached drawing 2）.

（2）Comparison virus package carrier pAAV2-MCK-EGFP structures

With pCMV-C-EGFP（Green skies Bioisystech Co., Ltd, China）For template, design primer EGFP-F/EGFP-R, PCR amplification EGFP gene coding region sequence contains KpnI and EcoRI restriction enzyme sites in EGFP-F and EGFP-R primers respectively.Expand Increasing obtains EGFP coding region sequence segments, spare with the double digested rear recycling of EcoRI and KpnI.Distinguished with EcoRI and KpnI Double digested pAAV2-MCK carriers recycle the pAAV2-MCK carrier segments of linearisation（About 6.7kb）.Segment is recycled by two It is ligated and transformed into E.coli JM109 competent cells（Precious biology, Dalian）, obtain expressing containing EGFP gene after screening, identification The AAV plasmid vectors pAAV2-MCK-EGFP of frame（See attached drawing 3）.

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

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

（3）PAAV2-MCK-hMTM1 vector constructions

Search obtains people's MTM1 histone amino sequence references in NCBI albumen databases（GenBank ID：NP_000243）, root According to principles such as people's codon preferences, Nanjing Genscript Biotechnology Co., Ltd. synthesizes to obtain hMTM1 sequences（SEQ ID No.3）.Synthesis hMTM1 sequences are cloned into pUC57 simple carriers（Nanjing Genscript Biotechnology Co., Ltd.）, obtain pUC57-hMTM1.KpnI and EcoRI distinguishes double digested pUC57- hMTM1 carriers and pAAV2-MCK carriers, recycling The pAAV2-MCK carrier segments of hMTM1 segments and linearisation, two segments are ligated and transformed into E.coli JM109 competent cells （Precious biology, Dalian）, screening, identification obtain pAAV2-MCK-hMTM1 carriers（See attached drawing 4）.

（4）PAAV2-MCK-hMTM1-122T-142T vector constructions

By the complete of the people miR-142-3p of the target sequence of the complete complementary of the people miR-122 of 2 tandem sequence repeats and 2 tandem sequence repeats Complete complementary target sequence optimizes together obtained hMTM1 gene orders and splices successively, obtains hMTM1-122T-142T sequences（SEQ ID No.4）.HMTM1-122T-142T sequences are synthesized by Nanjing Genscript Biotechnology Co., Ltd..By the hMTM1- of synthesis 122T-142T sequences are cloned into pUC57 simple carriers（Nanjing Genscript Biotechnology Co., Ltd.）, obtain pUC57- HMTM1-122T-142T carriers.KpnI and BglII distinguish double digested pUC57-hMTM1-122T-142T carriers and PAAV2-MCK carriers recycle the pAAV2-MCK carrier segments of hMTM1-122T-142T segments and linearisation, after the connection of two segments Transformed E .coli JM109 competent cells（Precious biology, Dalian）, screening, identification obtain pAAV2-MCK-hMTM1-122T- 142T carriers（See attached drawing 5）.

Embodiment 2 recombinates AAV viruses and prepares and examine and determine

Reference literature（Xiao X,et al. J Virol. 1998;72(3):2224-2232.）, system is packed using three plasmids System packaging recombination AAV viruses, are isolated and purified using cesium chloride density gradient centrifugation and are packaged to be AAV viruses.Briefly, AAV Vector plasmid（PAAV2-MCK-EGFP, pAAV2-MCK-hMTM1 or pAAV2-MCK-hMTM1-122T-142T）, helper plasmid （pHelper）With the Rep and Cap protein expression plasmid of AAV（PAAV-R2C8 or pAAV-R2C9）According to 1:1:1 molar ratio is mixed After even, using calcium phosphate procedure transfected HEK 293, after transfecting 48h, cell and culture supernatant are harvested, using cesium chloride density Gradient centrifugation isolates and purifies recombination AAV viruses.Packaging purifying obtains AAV8-MCK-EGFP, AAV8-MCK-hMTM1, AAV8- MCK-hMTM1-122T-142T, AAV9-MCK-EGFP, AAV9-MCK-hMTM1, AAV9-MCK-hMTM1-122T-142T etc. 6 Kind recombinant virus.

The genome titer that AAV viruses are prepared is measured using quantifying PCR method.Detailed process is as follows：

Quantitative PCR detection primer and probe is designed for 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 is primer, and MCK-Q-P is probe.The end of probe 5 ' FAM fluorescent protein labelings, 3 ' end connections BlackBerry quencher.Primer and probe is synthesized by Thermofisher Scientific.With MCK-Q-F and MCK-Q- R be primer specificity to expand length in MCK promoters be 95bp segments, using TaqMan probe combined techniques, with 1 μ g/ μ l's The sample of pAAV2-MCK-EGFP plasmids and its 10 times of gradient dilutions is standard items, using Premix Ex Taq (Probe QPCR) reagent（Takara, Dalian, China）, use fluorescence quantitative PCR instrument（Model：ABI 7500 fast, ABI）Detection virus Genome titer.Operating process is referring to Premix Ex Taq (Probe qPCR) reagent specification.The processing method ginseng of virus See document（Aurnhammer C, et al. Hum Gene Ther Methods. 2012; 23(1): 18-28.）.

3 zoopery of embodiment

The chain myotublar myopathy mouse models of X prepare kind of a mouse, are purchased from U.S. The Jackson Laboratory, trade name B6.Cg-Mtm1 ^tm1Itl / J, conservation number 018153.This kind of mouse gender is female, X-linkage mtm1（myotubular myopathy gene 1）4 heterozygous mutation of gene extron, the mtm1 genes of mutation, which cannot express, generates normal MTM1 eggs In vain（Pierson CR, et al. Hum Mol Genet. 2012;21(4):811-25. ）.The kind same Male wild-type of mouse After C57BL/6J mouse hybrids, mtm1 detection in Gene Mutation is carried out to neonatal male mouse, method is referring to document（Buj-Bello A, et al. PNAS. 2002; 99(23):15060-15065.）.Select mtm1 gene mutation male mices for follow-up real It tests.

30 3 week old mtm1 gene mutation male mices are randomly divided into 6 groups, every group of 5 mouse distinguish intravenous administration AAV8-MCK-EGFP、AAV8-MCK-hMTM1、AAV8-MCK-hMTM1-122T-142T、AAV9-MCK-EGFP、AAV9-MCK- HMTM1 and AAV9-MCK-hMTM1-122T-142T viruses, every mouse injection dosage are 3 × 10¹³Vg/kg, wherein AAV8- MCK-EGFP and AAV9-MCK-EGFP is comparison virus.Using the wild type C57BL/6J of not injecting virus as positive control.Note The life cycle of record mouse is penetrated after virus, different time points measure mouse weight, injecting virus 2 weeks and 90 points of progress after 3 months The test of mouse movement trajectory distance, injecting virus mouse different type muscle groups agonistic muscle tubulin expression after 3 months in clock And Different Organs muscle tubulin expression conditions.

The life cycle of mouse is recorded after injecting virus, as a result as shown in Fig. 6.From the result of Fig. 6 it is found that injection EGFP reports Accuse gene viruses（AAV8-MCK-EGFP or AAV9-MCK-EGFP）The life cycle of group XLMTM model mices is shorter, is no more than 68 days.4 groups of injections carry hMTM1 gene expression AAV virus groups（AAV8-MCK-hMTM1、AAV8-MCK-hTMT1-122T- 142T, AAV9-MCK-hMTM1 and AAV9-MCK-hTMT1-122T-142T）The life cycle of XLMTM model mices all extends.And And the life span extension of injection of AAV 8-MCK-hTMT1-122T-142T or AAV9-MCK-hTMT1-122T-142T virus mouse Time is significantly more than injection of AAV 8-MCK-hMTM1 or AAV9-MCK-hMTM1 virus mouse, illustrates containing miRNA target sequences Function and effect of the medicines structure design in mouse model body are more preferable.What is more important, injection of AAV 8-MCK-hTMT1- The life cycle of 122T-142T or AAV9-MCK-hTMT1-122T-142T virus mouse has no bright with wildness C57BL/6J mouse Significant difference is different, further illustrates that this kind of drug design validity is good.

Different time measures the weight of mouse after injecting virus.At each body weight determination time point, measurement lives small The weight of mouse, if dead mouse when measuring, does not have body weight determination result.As a result as shown in Fig. 7.It can from the result of Fig. 7 Know, injection EGFP reporter gene viruses（AAV8-MCK-EGFP or AAV9-MCK-EGFP）The weight of group XLMTM model mices increases Length is slow or hardly increases, until dead.On the contrary, 4 groups of injections carry hMTM1 gene expression AAV virus groups（AAV8-MCK- HMTM1, AAV8-MCK-hTMT1-122T-142T, AAV9-MCK-hMTM1 and AAV9-MCK-hTMT1-122T-142T）XLMTM The weight of model mice increases as time went on, and not compared to the weight gain trend of wild type C57BL/6J mouse See notable difference.The result shows that injection, which carries hMTM1 gene expression frame AAV viruses, can effectively restore the life of XLMTM model mices Long growth course.

It injects disease 2 weeks and carries out mouse movement trajectory distance in 90 minutes after 3 months after injecting virus and test, test method Referring to article（Childers MK, et al. Sci Transl Med. 2014;6:220ra10.）.As a result such as Fig. 8（Injection Virus is after 2 weeks）And Fig. 9（After injecting virus 3 months）It is shown.From the result of Fig. 8 it is found that 6 groups of injecting virus XLMTM model mices Group is apparent with equal difference between wild type C57BL/6J mouse groups, and injecting virus group between have no notable difference, Ke Nengyu The virus injection time is not grown, and it is related that virus carries the expression not yet in effect of hMTM1 genes.From the result of Fig. 9 it is found that in addition to injecting EGFP Reporter gene virus（AAV8-MCK-EGFP or AAV9-MCK-EGFP）Group XLMTM model mices are all dead, do not obtain testing number According to outer, 4 groups of injections carrying hMTM1 gene expression AAV virus groups（AAV8-MCK-hMTM1、AAV8-MCK-hTMT1-122T- 142T, AAV9-MCK-hMTM1 and AAV9-MCK-hTMT1-122T-142T）The movement locus distance of XLMTM model mices is homogeneous Than having no notable difference in wild type C57BL/6J mouse groups, illustrating can be effective in 4 kinds of virus importing XLMTM model mice bodies Ground expression generates MTM1 albumen, the locomitivity of Restoration model mouse.

After injecting virus 3 months, every group is respectively put to death 1 mouse, injection EGFP reporter gene viruses（AAV8-MCK-EGFP Or AAV9-MCK-EGFP）Group XLMTM model mices all deads, therefore this item detection is not done, label is ND " in figure（Figure 10）.MTM1 protein expression situations in different type musculature are detected using detected by Western blot, referring specifically to article （Childers MK, et al. Sci Transl Med. 2014;6:220ra10.）.Difference with bibliography exists The people's MTM1 albumen primary antibodies used in the present invention are purchased from the bright Kant of medicine（China, article No.：AP6809b-400）, antibody is that rabbit is more It is anti-, identify the C-terminal amino acid sequence of MTM1 albumen.Remaining reagent is consistent with bibliography.Testing result is as shown in Figure 10.From For the result of Figure 10 it is found that in three kinds of different types of musculature, 4 groups of injections carry hMTM1 gene expression AAV virus groups （AAV8-MCK-hMTM1, AAV8-MCK-hTMT1-122T-142T, AAV9-MCK-hMTM1 and AAV9-MCK-hTMT1-122T- 142T）The MTM1 protein expression levels of XLMTM model mices have no notable difference, and it is small to be all not less than wild type C57BL/6J Mouse.The result shows that injection carries hMTM1 gene expression frame AAV viruses can effectively express production in XLMTM model mice muscle Raw MTM1 albumen.

Above-mentioned execution mouse, the organs such as isolating cardiac, liver, skeletal muscle, spleen, lung and kidney is selected to extract various organs Total serum IgE, quantitative PCR measure hMTM1 RNA copy numbers and mouse GAPDH RNA copy numbers in total serum IgE, copy number Ct value tables Show, calculate the difference of the Ct values of hMTM1 RNA and the Ct values of mouse GAPDH RNA, the power table for 2 Ct value difference values shows The relative expression levels of hMTM1 RNA.

Quantifying PCR method measures the Ct values of hMTM1 RNA and mouse GAPDH RNA in total serum IgE.Detailed process is as follows：

Quantitative PCR detection primer and probe is designed for hMTM1 RNA sequences：

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).

Quantitative PCR detection primer and probe is designed for mouse GAPDH RNA sequences：

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 is primer, and hMTM1-Q-P and GAPDH-Q-P are to visit Needle.The end of probe 5 ' FAM fluorescent protein labelings, 3 ' end connection BlackBerry quencher.Primer and probe by Thermofisher Scientific synthesis.HMTM1 sequences as primer specificity are expanded using hMTM1-Q-F and hMTM1-Q-R Middle length is 92bp segments, and length in GAPDH sequences as primer specificity is expanded using GAPDH-Q-F and GAPDH-Q-R is 66bp segments measure the expansion of hMTM1 RNA and the GAPDH RNA in detection sample using single step reaction TaqMan probe combined techniques Increase Ct values（Indicate copy number）, using One Step PrimerScript RT-PCR Kit（Perfect Real Time）Examination Agent（Takara, Dalian, China）, use fluorescence quantitative PCR instrument（Model：ABI 7500 fast, ABI）Detection.Operating process is joined See One Step PrimerScript RT-PCR Kit（Perfect Real Time）Reagent specification.

Quantitative PCR detection result is as shown in Fig. 11.From the result of Figure 11 it is found that for injection of AAV 8-MCK-hMTM1, AAV8-MCK-hMTM1-122T-142T, AAV9-MCK-hMTM1 and AAV9-MCK-hMTM1-122T-142T virus group mouse, HMTM1 expressions are high in the musculatures such as cardiac muscle, skeletal muscle, and hMTM1 expressions in the organs such as spleen, kidney and lung Low, the tissue specificity of the transduction properties and MCK promoters that illustrate AAV8 and AAV9 viruses itself can effectively make hMTM1 bases Because specificity is expressed in musculature.Further, when intravenously administrable systemic injection, AAV carriers are efficient to liver transduction, The table of hMTM1 is can detect in the present invention in the liver of injection of AAV 8-MCK-hMTM1 and AAV9-MCK-hMTM1 virus group mouse It reaches, although due to the regulation and control of MCK muscle specific promoters, the expression of hMTM1 is not high, but still may bring no small peace Full property risk.Ours in 3 ' UTR of hMTM1 expression cassettes the experimental results showed that be added the target of the miR-122 of high expression in liver Sequence can effectively inhibit the expression of hMTM1 genes（Figure 11）, the muscle specific expression of hMTM1 genes is significantly enhanced, is increased The safety during use is added.The result of Figure 11 is shown in the expression that hMTM1 genes are not detected in wild-type mice, this It is because after the optimization of hMTM1 codons, DNA sequence dna has been different from the MTM1 RNA sequences of endogenous mouse, therefore is directed to The quantitative PCR detection probe and primer None- identified of hMTM1 genes simultaneously combine mouse MTM1 RNA sequences, do not detect letter Number, also illustrate that the quantitative PCR probe used in the present invention and primer can specifically identify and combine hMTM1 RNA sequences, inspection It is high to survey result reliability.In test experience, injection of AAV 8-MCK-EGFP or AAV9-MCK-EGFP mouse dead, therefore Figure 11 In there is no testing result to show.

In short, from the above it is found that AAV8-MCK-hMTM1, AAV8-MCK-hMTM1-122T- that the present invention designs 142T, AAV9-MCK-hMTM1 and AAV9-MCK-hMTM1-122T-142T recombinant virus import XLMTM models through intravenously administrable In Mice Body, people's MTM1 albumen can be generated in model mice myogenic expression, the life cycle of lengthening model mouse restores development （Weight gain）And physiological function.And AAV8-MCK-hMTM1-122T-142T and AAV9-MCK-hMTM1-122T-142T diseases Poison shows that better tissue specificity, the life span extension time of model mice are longer, the potentiality to be exploited with bigger.

Sequence table

<110>Beijing FivePlus Molecular Medicine Institute

<120>The genomic medicine of X-linkage myotublar 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 (9)

1. a kind of people's flesh tubulin gene expression frame, which is characterized in that

（Ⅰ）Including muscle specific promoter sequence；

（Ⅱ）People's flesh tubulin coded sequence containing optimization；

（Ⅲ）Carrier's miRNA target sequences.

2. muscle specific promoter as described in claim 1, specially mouse creatine kinase（MCK）Promoter, sequence letter Breath is shown in SEQ ID NO.1.

3. the people's flesh tubulin coded sequence optimized as described in claim 1, sequence information are shown in SEQ ID NO.3.

4. people miRNA target sequences as described in claim 1, the target of the specific complete complementary for people miR-122, miR-142-3p The number of sequence, each miRNA target sequence is no less than 2, and target sequence is connected in series with by intervening sequence, one of which target sequence SEQ ID NO.4 are shown in design.

5. a kind of recombined glandulae correlation viral vectors, it is characterised in that carry gene expression frame as described in claim 1 or right It is required that 2,3,4 require the combination of sequence.

6. recombined glandulae correlation viral vectors as claimed in claim 5, which is characterized in that recombined glandulae correlation viral vectors serotype Including AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, wherein preferred serotype is AAV8 and AAV9.

7. a kind of genomic medicine, which is characterized in that wanted including gene expression frame described in claim 1 or claim 2,3,4 The combination for seeking sequence and the recombined glandulae correlation viral vectors described in claim 5,6.

8. the genomic medicine described in claim 7, which is characterized in that administering mode is to be administered systemically, including but not limited to vein Drug administration by injection.

9. the genomic medicine described in claim 7, which is characterized in that the sustainable expression of single administration generates people's flesh tubulin, has The ill symptoms brought by flesh tubulin gene mutation are alleviated or cured to effect.