WO2022047876A1 - 杜氏肌营养不良症相关的外显子剪接增强子、sgRNA、基因编辑工具及应用 - Google Patents

杜氏肌营养不良症相关的外显子剪接增强子、sgRNA、基因编辑工具及应用 Download PDF

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WO2022047876A1
WO2022047876A1 PCT/CN2020/119361 CN2020119361W WO2022047876A1 WO 2022047876 A1 WO2022047876 A1 WO 2022047876A1 CN 2020119361 W CN2020119361 W CN 2020119361W WO 2022047876 A1 WO2022047876 A1 WO 2022047876A1
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sgrna
seq
gene
nucleotide sequence
exon51
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常兴
李甲
邱晗
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西湖大学
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Priority to CA3191505A priority patent/CA3191505A1/en
Priority to US18/043,874 priority patent/US20230287419A1/en
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Definitions

  • the invention belongs to the field of gene therapy, and relates to a Duchenne muscular dystrophy-related exon splicing enhancer, sgRNA, and a gene editing tool in mammals (experimental animal models and human patients) targeting gene mutation type hereditary diseases. Mutational transformation therapy. In particular, it relates to the gene editing therapy of Duchenne muscular dystrophy DMD in mouse models and human patients.
  • rare diseases are uncommon diseases in a certain area, with the number of patients accounting for 0.065% to 0.1% of the total population.
  • the pathogenesis of these diseases is often difficult to find, and there is a lack of targeted treatment drugs, which brings great harm to the health of patients and brings a great burden to their families and society.
  • the pathogenesis of rare diseases is often due to genetic mutations, resulting in complex and multiple clinical disease manifestations. Due to the limitations of diagnostic methods, patients are generally classified as a single disease after showing clinical symptoms in the early course of the disease, and only after long-term treatment does not improve, will they be further judged as difficult and undiagnosed diseases. Therefore, related research on difficult and undiagnosed diseases/rare diseases needs to be carried out urgently, including but not limited to: exploration of pathogenic mechanisms, optimization of diagnostic methods, tracking of disease progression, screening of drug targets, and combination of gene editing technology for targeted The development of genetic drugs, etc.; at the same time, the discovery and improvement of animal models of special rare diseases can also improve our comprehensive understanding of rare diseases and the innovation of targeted drugs.
  • the present invention takes muscular dystrophy (Muscular Dystrophy), a rare disease that has been clinically discovered earlier but lacks effective treatment methods for a long time, as the breakthrough point, takes Duchenne Muscular Dystrophy (DMD) as the research object, and combines new
  • Muscular Dystrophy DMD
  • the mouse model was discovered, the gene therapy for the disease was developed and optimized, and the gene therapy was applied to the human genome sequence.
  • Duchenne Muscular Dystrophy is an X-chromosome genetic disorder that can be detected in about 1 in 4,000 newborn males.
  • DMD Duchenne Muscular Dystrophy
  • myocardial tissue damage and dysfunction are the most deadly threats.
  • the clinical treatments that can be given are limited to the relief of symptoms: for example, the use of angiotensin inhibitors to relieve the discomfort caused by the deterioration of myocardial function, such drugs Including Pandinopril, and a variety of Lol beta-blockers.
  • interventional therapy also helps to relieve the symptoms of DMD patients, including cardiac circulatory assistance system and respiratory assistance system.
  • Sarepta Therapeutics is a biotechnology company focused on developing precision gene therapy for rare diseases.
  • the Golodirsen developed by it was approved by the U.S. FDA on December 12, 2019, for the treatment of DMD patients diagnosed with exon 53 skipping gene mutations. It is estimated that about 8% of people with DMD carry this mutation.
  • Golodirsen is an antisense oligonucleotide that works by targeting the dystrophin sequence. Therefore, drugs designed for other mutation sites are still a huge gap at present. At present, on a global scale, including DMD-targeted drugs that have entered clinical trials, the competition is fierce, but the demand is still huge.
  • the purpose of the present invention is to provide a Duchenne muscular dystrophy-related exon splicing enhancer, sgRNA, and gene editing tools for genetically mutated rare diseases, which can be used as medicines for mammals (disease animal models and human patients) ) in vivo gene editing therapy.
  • the present invention provides a Duchenne muscular dystrophy-related exon splicing enhancer, the exon splicing enhancer is an exon splicing enhancer element targeting human DMD gene Exon51, and its nuclear
  • the nucleotide sequence includes:
  • the DMD gene Exon51 can be induced to skip reading, thereby realizing gene editing therapy in mammals.
  • CRISPR nuclease which can disrupt the structure of ESE by introducing deletion inserts (Insertions and deletions, Indels) through DNA double-strand breaks; antisense oligonucleotide ASO, by targeting the corresponding element of pre-mRNA in cells position, preventing its retention in the final protein amino acid sequence.
  • the present invention also provides a Duchenne muscular dystrophy-related single-stranded guide RNA (single-strand guide RNA, sgRNA) that can target a specific genome, and the sequence of the sgRNA includes:
  • the nucleotide sequence of the sgRNA for the Dmd-E4 mouse mutation site is shown in SEQ ID No.4.
  • sgRNA-1 of human DMD gene Exon51 its nucleotide sequence is shown in SEQ ID No.8.
  • sgRNA-2 of human DMD gene Exon51 its nucleotide sequence is shown in SEQ ID No.9.
  • sgRNA-3 of human DMD gene Exon51 its nucleotide sequence is shown in SEQ ID No.10.
  • sgRNA-4 of human DMD gene Exon51 its nucleotide sequence is shown in SEQ ID No.11.
  • sgRNA-5 of human DMD gene Exon51 its nucleotide sequence is shown in SEQ ID No.12.
  • sgRNA-6 of human DMD gene Exon51 its nucleotide sequence is shown in SEQ ID No.13.
  • sgRNA-7 of human DMD gene Exon51 its nucleotide sequence is shown in SEQ ID No.14.
  • sgRNA-8 of human DMD gene Exon51 its nucleotide sequence is shown in SEQ ID No.15.
  • sgRNA-9 of human DMD gene Exon51 its nucleotide sequence is shown in SEQ ID No.16.
  • sgRNA-10 of human DMD gene Exon51 its nucleotide sequence is shown in SEQ ID No.17.
  • sgRNA-11 of human DMD gene Exon51 its nucleotide sequence is shown in SEQ ID No.18.
  • sgRNA-12 of human DMD gene Exon51 its nucleotide sequence is shown in SEQ ID No.19.
  • sgRNA-13 of human DMD gene Exon51 its nucleotide sequence is shown in SEQ ID No.20.
  • the sgRNA combined with the gene editing tool can be used in the preparation of a drug for treating Duchenne muscular dystrophy.
  • the present invention also provides a Duchenne muscular dystrophy-related gene editing tool, comprising a fusion protein of cytosine deaminase and a Cas9 mutant, the sgRNA of claim 2 and a vector.
  • the vectors are commonly used biological plasmids, such as AAV vector plasmids, pCDNA3.1 plasmids, and the like.
  • cytosine deaminase can be AID, apobec etc., preferably, cytosine deaminase is AID, and the amino acid sequence and nucleic acid sequence of the fusion protein of AID and Cas9 mutant are respectively as SEQ ID NO.1 and SEQ ID NO.2 is shown.
  • the gene editing tool is packaged by adeno-associated virus vector AAV.
  • Adeno-associated virus AAV can deliver the nucleic acid sequence expressing AID-Cas9 fusion protein and sgRNA to target cells, so that it can express proteins with DNA editing function and sgRNA molecules with guiding function in cells, wherein sgRNA can guide AID-
  • the Cas9 fusion protein is integrated into a specific genomic locus in target cells to induce and transform pathogenic mutations and inactivate them for the purpose of treating diseases.
  • the promoter of the adeno-associated virus vector AAV is Syn100 promoter or a promoter designed based on ck8a, mhck7 and the like.
  • nucleotide sequence of the adeno-associated virus vector AAV is shown in SEQ ID NO.3.
  • the present invention also provides the application of the above-mentioned gene editing tool in preparing a medicine for treating Duchenne muscular dystrophy.
  • the invention takes the DMD mouse model and the pathogenic mutation carried by human DMD patients as examples, through designing and constructing a gene editing tool, and using adeno-associated virus AAV to realize the treatment of the DMD mouse model in vivo; of pathogenic mutations, and designed a gene editing scheme to achieve the transformation of pathogenic mutations at the cellular level.
  • the invention provides an innovative treatment method for gene mutation type hereditary rare diseases, and is expected to achieve a breakthrough therapeutic effect on many hereditary rare diseases.
  • Figure 1 shows the composition of functional elements including gene editing tools, wherein A is a separate package of viruses, and B is a combined package of viruses;
  • Figure 2 is a flow chart of the treatment for the new DMD mouse disease model Dmd-E4, wherein A is the preventive treatment for newborn mice, and B is the reparative treatment for adult mice;
  • Figure 3 shows the partial sequencing results of the AAV plasmid
  • Figure A is the sequencing comparison result of the Syn100 promoter
  • Figure B is the sequencing comparison result of the fusion protein of AID and Cas9 mutant
  • Figure C is the sequencing comparison result of the U6 promoter.
  • Figure 4 AAV treatment successfully repaired the disease phenotype caused by defective Dystrophin expression in Dmd-E4 mice, in which: Panel A, reverse transcription PCR of RNA in the hearts of treated Dmd-E4 mice, The primers were designed in Exon3 and Exon5 to detect the occurrence of skipping in Exon4 carrying the mutation, Dmd is the gene encoding the dystrophin protein in mice, and Gapdh is the internal reference for PCR; Figure B, using the quantitative method of capillary electrophoresis, it was determined that Exon4 skipped reading.
  • FIG. 5 AAV treatment successfully restored muscle function and prolonged survival in Dmd-E4 mice.
  • Panel A the serum creatine kinase content of treated Dmd-E4 mice was measured, WT and untreated Dmd-E4 mouse samples were used as controls;
  • Panel B using HE staining and Masson staining methods, Assess the degree of myocardial inflammatory cell infiltration and fibrosis in Dmd-E4 mice after treatment;
  • Figure C according to the results of Masson staining, quantitative statistics on the recovery of myocardial fibrosis in Dmd-E4 mice after treatment;
  • Figure D using the micro-CT method to detect the degree of spinal curvature in Dmd-E4 mice, with WT mice and untreated mice as controls;
  • E the quantitative statistics of spinal curvature in D;
  • F Using the tension device to detect the degradation of the maximum muscle tension of the whole body in the treated Dmd-E4 mice during the cyclic force;
  • G graph the survival time statistics of W
  • Figure 6 Gene editing tools can successfully induce corresponding modifications of the DMD gene in human cells.
  • Picture A two sgRNAs were successfully screened in K562 cell line, which can induce Exon51 to skip reading. The picture shows the results of reverse transcription PCR after RNA extraction from edited K562 cells, showing that the combination of two sgRNAs can induce efficient induction K562 cells lacking Exon50 successfully skipped Exon51;
  • Panel B Exon51 inducing DMD gene skipped in normal human iPS and DMDExon50-deficient cells;
  • Panel C using immunofluorescence detection to determine the presence of Dystrophin protein in edited iPS cells The expression was restored in Figure D; the expression of Dystrophin protein was restored in the edited iPS cells by the method of western blot;
  • Figure E the quantitative statistics of the restored expression of the protein in Figure D.
  • the invention takes DMD mouse models and pathogenic mutations carried by human DMD patients as examples, and realizes the transformation of pathogenic mutations by designing and constructing gene editing tools.
  • the present invention will be further described below in conjunction with specific embodiments and accompanying drawings:
  • Example 1 AAV virus carrying gene editing tools
  • the gene editing tool designed according to the present invention is shown in Figure 1. Taking AID as an example, we clone the corresponding sequence into the AAV plasmid, including the following steps:
  • the pAAV2 backbone vector (purchased from addgene, but not limited to) was double-digested based on the restriction sites of XhoI and NotI.
  • the amino acid sequences of AID and Cas9 fusion proteins in the gene editing tool were designed.
  • the amino acid sequences and nucleic acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.
  • the double-stranded DNA fragment was directly synthesized, and the elements such as the Syn100 promoter and tailing signal were connected to the AAV backbone vector to obtain the AAV vector plasmid expressing the AID-Cas9 mutant fusion protein, the sequence of which is SEQ ID NO.3 shown.
  • the sequences of U6 promoter, H1 promoter and 7SK promoter can be linked with sgRNAs that recognize the exon splicing site of pathogenic mutation, and the Syn100 promoter and tailing can be used at the same time.
  • the U6 promoter was linked with the sgRNA targeting the exon splicing site of the pathogenic mutation to construct an AAV plasmid vector with a 4.9kbp insert sequence. Part of the results of the relevant plasmid cloning is shown in Figure 3 below.
  • a novel DMD mouse disease model Dmd-E4 with abnormal cardiac function was selected, which can be purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., but is not limited thereto.
  • Dmd-E4 showed cardiac hypertrophy, fibrosis and other phenotypes in the heart at 6-8 weeks, and showed severe degeneration of cardiac function at about 8 months. This process closely mimics the cardiac pathology of DMD patients.
  • the expression of Dystrophin protein can be retained to the maximum extent and its biological function can be restored.
  • Example 1 the method of Example 1 was used to construct a gene editing tool, wherein the designed sgRNA sequence for the Dmd-E4 mouse mutation site was shown in SEQ ID NO. 4, and the obtained expression was for the Dmd-E4 mouse mutation site
  • the AAV vector plasmid of the sgRNA the sequence of which is shown in SEQ ID NO.5.
  • the corresponding sequence comprising the AID-Cas9 fusion protein and the sgRNA against Dmd-E4 mice in the same AAV vector plasmid is shown in SEQ ID NO.6.
  • the virus with the serotype AAV9 was selected to synthesize and purify, and the Dmd-E4 mice were treated according to the preventive treatment of newborn mice and the repair treatment of adult mice, as shown in Figure 2.
  • Dmd-E4 mice were mated with KO male and female mice. After the female mice became pregnant, the male and female mice were separated into cages, and the pregnant female mice were observed every two days for production. After the birth of the newborn Dmd-E4 mice , observe the gender, select 3-5 male mice as the experimental group, and another 3-5 male mice as the negative control group;
  • adeno-associated virus AAV (titer 10 13 vg/mL) carrying gene editing tools was administered by intraperitoneal injection or facial vein injection, and control mice were simultaneously given an equal volume of sterile PBS, Afterwards, they were reared normally with the female mice;
  • mice When the mice grew to about 2 months, in addition to the experimental group and the control group, 3-5 WT male mice of the same age were taken, and the following treatments were performed at the same time: After the mice were anesthetized, the function of the tibialis anterior muscle was first performed. Test, echocardiography, etc., then collect cardiac arteriovenous blood to kill mice, centrifuge to separate serum and store at -80°C, and collect cardiac muscle, skeletal muscle, tibialis anterior muscle, back muscle, liver, brain, kidney at the same time and other tissues, and extract its protein, RNA, genomic DNA, and retain enough tissue for immunofluorescence staining, hematoxylin and eosin staining, etc.
  • FIG. 4D-F is the immunization of the protein in the mouse heart Blot detection, in which, Figure 4D is the band map, Figure 3E is the quantitative statistics of the bands in the 3D figure, Figure 4F Dystrophin protein expression, the results show that treated Dmd-E4 significantly restored Dystrophin protein expression.
  • small animal cardiac ultrasonography was used to investigate whether the changes of cardiac related physiological structures in Dmd-E4 mice were repaired after AAV treatment. The results are shown in Figure 4G, the results show that the heart-related physiological structures of Dmd-E4 mice were basically repaired after treatment.
  • Figure 5A is the measurement result of creatine kinase content in the serum of mice. It can be seen from the figure that compared with WT and untreated Dmd-E4 mouse samples, the creatine kinase content of Dmd-E4 mice after treatment Decreased significantly.
  • HE staining and Masson staining were used to evaluate the degree of myocardial inflammatory cell infiltration and fibrosis in the treated Dmd-E4 mice. At the same time, according to the results of Masson staining, the myocardial fibrosis in the treated Dmd-E4 mice was quantitatively counted.
  • Figure 5H shows the molecular biological evidence of gene editing in Dmd-E4 mouse cardiomyocytes.
  • the pre-mRNA of the corresponding cells was subjected to reverse transcription PCR, followed by high-throughput sequencing, and it was found that the expected sgRNA was near the target position.
  • the mutation of Dmd-E4 mice is the molecular basis and basis for the treatment of cardiac disease phenotype in Dmd-E4 mice.
  • adeno-associated virus AAV (titer 10 13 vg/mL) carrying gene editing tools was administered by tail vein injection or skeletal muscle in situ injection, and control mice were simultaneously given an equal volume of sterile PBS;
  • mice were treated for about 2 months, in addition to the experimental group and the control group, 3-5 WT male mice of the same age were taken, and the following treatments were performed simultaneously: Functional tests, echocardiography, etc., then the cardiac arteriovenous blood was collected to kill the mice, and the serum was centrifuged and stored at -80°C.
  • Example 3 Gene editing of the DMD model of human induced pluripotent stem cell iPSC successfully restored the expression of Dystrophin protein
  • the present invention has successfully implemented the gene editing therapy of human cells.
  • iPSCs induced pluripotent stem cells
  • CRISPR-cas9 method to specifically delete the exon 50 of the Dystrophin-encoding gene DMD, Exon50, so that the dystrophin protein encoding
  • the sequence produced a frameshift mutation, thereby constructing a mutation type that mimics a DMD patient, making it a good DMD disease model cell.
  • sgRNA is sgRNA-12 as shown in SEQ ID No.19 and sgRNA-13 as shown in SEQ ID No.20, and sgRNA-12 mainly targets the outer surface as shown in SEQ ID No.21 and SEQ ID No.22.
  • Exon splicing enhancers. sgRNA-13 mainly targets the exon splicing enhancer as shown in SEQ ID NO.24.
  • the above two sgRNA-12s were screened in the human K562 cell line and could induce the skip reading of Exon51.
  • Figure 6A the results of reverse transcription PCR after RNA extraction from the edited K562 cells showed that both sgRNAs could Induced mutation, combined use can efficiently induce K562 cells lacking Exon50 to successfully skip to Exon51.
  • the open reading frame of Dystrophin protein in iPS cells lacking Exon50 can be restored, thereby rebuilding the expression of Dystrophin protein.
  • the specific implementation is as follows:
  • mTeSR1 medium was replaced with RPMI/B27-insulin medium containing 6uM CHIR99021, and cultured for 2 days;
  • the medium was replaced with RPMI/B27-insulin medium containing 5 ⁇ M IWR1, and cultured for 2 days;
  • IWR1 stimulation was removed, the medium was replaced with RPMI/B27-insulin medium, and cultured for 2 days;
  • the cell culture medium was changed to RPMI/B27 medium, and then the medium was used for culturing, and the medium was changed every two days to obtain human pluripotent stem cells differentiated into cardiomyocytes.
  • iPS cells differentiated into cardiomyocytes were digested with Accutase and plated in 6-well plates, and 4 ⁇ 10 5 cells were plated in each well;
  • plasmid expressing AID and Cas9 mutant fusion protein such as Lenti-V2-AIDx-nSaCas9(KKH)-Ugi plasmid
  • 500 ng of UGI expressing plasmid such as pCDNA3.1-Ugi
  • 1.5 ⁇ g Mix the sgRNA plasmid in 150 ⁇ l opti-MEM, add 2.5 ⁇ l PLUS TM reagent, and mix gently;
  • the remaining sgRNA-1 to sgRNA-11 shown in SEQ ID No.7-SEQ ID No.18 all target the corresponding exons shown in SEQ ID No.21-SEQ ID No.24 of the present invention
  • the splicing enhancer when constructed as a gene editing tool, efficiently induces skipping Exon51, thereby enabling the treatment of human DMD.

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Abstract

一种杜氏肌营养不良症相关的外显子剪接增强子、sgRNA、基因编辑工具及应用。相关的外显子剪接增强子、sgRNA和基因编辑工具可在制备治疗杜氏肌营养不良症的药物中应用。其中，基于胞嘧啶脱氨酶AID的突变体与Cas9突变体设计的基因编辑工具，以腺相关病毒AAV为载体，可对哺乳动物的基因组进行定点改造。通过优化编辑工具的编码核酸序列以及元件组成结构，可以高效实现哺乳动物遗传物质DNA的定点靶向改造，通过对携带疾病突变的核酸序列进行靶向遗传操作，使其致病突变无法保留至成熟的蛋白质氨基酸序列中或使致病突变无法发挥其功能，从而达到治疗多种基因突变类遗传性罕见病的目的，具有高效，安全，稳定的优点。

Description

杜氏肌营养不良症相关的外显子剪接增强子、sgRNA、基因编辑工具及应用 技术领域
本发明属于基因治疗领域，涉及一种杜氏肌营养不良症相关的外显子剪接增强子、sgRNA、基因编辑工具在哺乳动物(实验动物模型以及人类病人)体内针对基因突变型遗传性疾病致病突变的改造治疗。尤其涉及杜氏肌营养不良症DMD在小鼠模型以及人类病人的基因编辑治疗。
背景技术
根据世界卫生组织WHO的定义，罕见病为在一定区域内，患病人数占总人口数0.065％～0.1％的非常见疾病。这类疾病往往发病机制难寻，缺少针对性治疗药物，对病人身体健康带来极大的危害，对其家庭以及社会均带来了极大的负担。由于我国极高的人口基数，罕见病发病的绝对数量不容忽视，近年来也引起了科研人员与临床专家的重视和关注。2018年5月，我国国家卫生健康委员会、科技部、工业和信息化部、国家药品监督管理局、国家中医药管理局等五部门联合发布了《第一批罕见病》目录，其中列出了121种疾病，这也标志着罕见病在我国得到了进一步的重视和关注。
罕见病的发病机制往往是由于基因突变，造成复杂的多种临床疾病表现。限于诊断手段的局限性，病人在病程早期表现出临床症状后被笼统归为单一性疾病，经过长期治疗并没有得到改善后，才会被进一步判断为疑难未诊断疾病。因此，对于疑难未诊断疾病/罕见病的相关研究亟待开展，包括但不限于：致病机制的探究、诊断手段的优化、发病进程的追踪、药物靶点的筛选以及结合基因编辑技术对针对性基因药物的开发等；同时对特殊罕见病动物模型的发现和改良，也可以提高我们对罕见疾病的全方位理解和针对性药物的创新。本发明以肌营养不良症(Muscular Dystrophy)这一临床上发现较早却长期缺失有效治疗手段的罕见病为切入点，以杜氏肌营养不良症(Duchenne Muscular Dystrophy,DMD)为研究对象，结合新发现的小鼠模型，对该疾病的基因治疗手段进行了开发和优化，并将基因治疗手段应用于人类基因组序列中。
杜氏肌营养不良症DMD是一种X染色体遗传性疾病，大约每4000个新生男性中可以检测出一个患儿,其致病原因在于肌营养不良蛋白Dystrophin因基因突变而造成的表达缺失。对于DMD病人而言，心肌的组织损伤与功能异常是最致命的威胁。在很长的一段时间内，DMD并没有十分有效的治疗手段，临床能够给予的治疗仅限于对症状的缓解：比如利用血管紧张素抑制剂来缓解心肌功能退化所带来的不适，这类药物包括配哚普利、以及多种洛尔类beta受体阻断剂。同时，随着医疗手段的提高，介入治疗也帮助缓解DMD病人的症状，这其中包括心脏循环辅助系统和呼吸辅助系统等。但是，这些治疗并无法本质性地提高DMD病人的生活质量，延长DMD病人的寿命，心脏功能的进行性退化仍旧是DMD病人致死的最主要原因。随着分子生物学的不断进展，结合临床的数据分析，人们发现有一类同样在Dystrophin蛋白的编码基因中存在突变的病人，并没有表现出与DMD病人一样的严重病理进程，这类病人被称为贝氏肌营养不良症BMD患者，他们所携带的Dystrophin基因突变不会造成完整蛋白open reading frame的破坏，因此可以产生具有一定生物学功能的Dystrophin蛋白，不会表现出严重的心肌功能障碍以及其他肌肉功能的缺陷。相对于DMD病人的严重病理进程，BMD病人的寿命不会得到明显的影响，可以几乎恢复如正常人一般的日常生活。这类BMD病人的出现给了科研人员一个启发，是否可以在不影响蛋白读码框的前提下，诱导DMD病人中携带突变的外显子发生跳读，从而产生一种接近全长的Dystrophin蛋白，用于治疗DMD病人。这种想法在近些年已经被付诸实践，现在已有多种携带突变的Exon可以被利用这种方案进行治疗，其中几类已经被批准开启临床实验。截止到2019年底，在全球范围内仅有有限的几种针对DMD的特效药物被批准上市。其中，Sarepta Therapeutics是一家专注于开发精准 基因疗法治疗罕见性疾病的生物技术公司。其研发的Golodirsen经美国FDA加速批准于2019年12月12日上市，用于治疗确诊为发生53外显子跳跃基因突变的DMD患者。据估计，约8％的DMD患者携带此突变。Golodirsen的本质是一种反义寡核苷酸，通过靶向肌营养不良蛋白的序列而发挥作用。因此，对其他突变位点设计的药物，在目前来说仍是一个极大的空白。目前来说，在全球范围内，包含已经进入临床试验的DMD针对性药物在内，竞争较为激烈，但需求依然巨大。目前针对DMD的药物据统计有5款上市药物，6款药物处在临床III期试验阶段，19款药物处在临床II期阶段，还有5款药物刚刚进入临床I期试验阶段。需要指出的是，在人类DMD病人中，这些药物只适用于携带某种特定突变的一类病人，对于其他DMD病人来说，仍然缺少足够的针对性治疗药物。由Sarepta Therapeutics研发的Eteplirsen是一种反义二胺吗啉代寡核苷酸(PMO)治疗剂，是该公司首款治疗DMD的上市(2016年)药物。然而这些目前已经上市的药物往往存在治疗效率低，需要持续给药，价格极其昂贵的缺点。而基因编辑治疗方案则是可以直接对基因突变型遗传病的致病突变进行靶向改造，一旦经过编辑，即可从根本上达到治愈疾病的效果，具有极大的优势。
不仅是DMD这一类疾病，目前在全球范围内，利用基因编辑工具针对罕见性遗传病治疗的应用极其罕见。
发明内容
本发明的目的在于针对基因突变型遗传性罕见病，提供一种杜氏肌营养不良症相关的外显子剪接增强子、sgRNA、基因编辑工具，作为药物用以哺乳动物(疾病动物模型以及人类病人)体内基因编辑治疗。
第一方面，本发明提供了一种杜氏肌营养不良症相关的外显子剪接增强子，所述外显子剪接增强子为靶向人类DMD基因Exon51的外显子剪接增强子元件，其核苷酸序列包括：
1)如SEQ ID N0.21所示的序列及其反向互补序列。
2)如SEQ ID N0.22所示的序列及其反向互补序列。
3)如SEQ ID N0.23所示的序列及其反向互补序列。
4)如SEQ ID N0.24所示的序列及其反向互补序列。
通过改变或者阻断上述外显子剪接增强子(Exon Splicing Enhancer，ESE)等元件，可以诱导跳读DMD基因Exon51，从而实现对哺乳动物体内基因编辑治疗。例如：CRISPR nuclease,可以通过DNA的双链断裂引入的缺失插入片段(Insertions and deletions,Indels)破坏ESE的结构；反义寡聚核苷酸ASO，通过在细胞中靶向pre-mRNA的对应元件位置，阻止其保留至最终的蛋白质氨基酸序列中。
第二方面，本发明还提供了一种杜氏肌营养不良症相关的可以靶向特定基因组的单链向导RNA(single-strand guide RNA，sgRNA)，所述sgRNA的序列包括：
针对Dmd-E4小鼠突变位点的sgRNA，其核苷酸序列如SEQ ID N0.4所示。
针对人类DMD基因Exon50的sgRNA，其核苷酸序列如SEQ ID N0.7所示。
针对人类DMD基因Exon51的sgRNA-1，其核苷酸序列如SEQ ID N0.8所示。
针对人类DMD基因Exon51的sgRNA-2，其核苷酸序列如SEQ ID N0.9所示。
针对人类DMD基因Exon51的sgRNA-3，其核苷酸序列如SEQ ID N0.10所示。
针对人类DMD基因Exon51的sgRNA-4，其核苷酸序列如SEQ ID N0.11所示。
针对人类DMD基因Exon51的sgRNA-5，其核苷酸序列如SEQ ID N0.12所示。
针对人类DMD基因Exon51的sgRNA-6，其核苷酸序列如SEQ ID N0.13所示。
针对人类DMD基因Exon51的sgRNA-7，其核苷酸序列如SEQ ID N0.14所示。
针对人类DMD基因Exon51的sgRNA-8，其核苷酸序列如SEQ ID N0.15所示。
针对人类DMD基因Exon51的sgRNA-9，其核苷酸序列如SEQ ID N0.16所示。
针对人类DMD基因Exon51的sgRNA-10，其核苷酸序列如SEQ ID N0.17所示。
针对人类DMD基因Exon51的sgRNA-11，其核苷酸序列如SEQ ID N0.18所示。
针对人类DMD基因Exon51的sgRNA-12，其核苷酸序列如SEQ ID N0.19所示。
针对人类DMD基因Exon51的sgRNA-13，其核苷酸序列如SEQ ID N0.20所示。
所述sgRNA与基因编辑工具结合，可在制备治疗杜氏肌营养不良症的药物中应用。
第三方面，本发明还提供了一种杜氏肌营养不良症相关的基因编辑工具，包括胞嘧啶脱氨酶和Cas9突变体的融合蛋白、权利要求2所述sgRNA和载体。所述载体为常用的生物质粒，例如AAV载体质粒、pCDNA3.1质粒等。
进一步地，胞嘧啶脱氨酶可以为AID、apobec等，作为优选，胞嘧啶脱氨酶为AID，AID和Cas9突变体的融合蛋白的氨基酸序列以及核酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
进一步地，所述基因编辑工具由腺相关病毒载体AAV包装。腺相关病毒AAV可以将表达AID-Cas9融合蛋白以及sgRNA的核酸序列递送至靶细胞中，使其在细胞中表达出具有DNA编辑功能的蛋白质和具有引导功能的sgRNA分子，其中sgRNA可以引导AID-Cas9融合蛋白至靶细胞中的特定基因组位点，对致病突变进行诱导改造，使其失活以达到治疗疾病的目的。
进一步地，所述腺相关病毒载体AAV的启动子为Syn100启动子或基于ck8a、mhck7等设计的启动子。
进一步地，腺相关病毒载体AAV的核苷酸序列如SEQ ID NO.3所示。
本发明还提供了上述基因编辑工具在制备治疗杜氏肌营养不良症的药物中的应用。
本发明的有益效果是：
本发明以DMD小鼠模型和人类DMD病人携带的致病突变为例，通过设计构建基因编辑工具，利用腺相关病毒AAV在体内水平实现了对DMD小鼠模型的治疗；同时也针对人类DMD病人的致病突变，设计了基因编辑方案，在细胞水平实现了对致病突变的改造。本发明提供了对基因突变型遗传性罕见病的创新型治疗手段，有望对诸多遗传性罕见病实现突破性的治疗效果。
附图说明
图1为包含基因编辑工具的功能元件组成，其中，A为分开包毒、B为合并包毒；
图2为针对新型DMD小鼠疾病模型Dmd-E4治疗流程图，其中，A为新生小鼠预防性治疗、B为成年小鼠修复性治疗；
图3为AAV质粒的部分测序结果；A图为Syn100启动子的测序比对结果；B图为AID与Cas9突变体融合蛋白的测序比对结果；C图是U6启动子的测序比对结果。
图4.AAV治疗成功修复了Dmd-E4小鼠因Dystrophin表达缺陷所造成的疾病表型结果示意图，其中：A图，对经过治疗的Dmd-E4小鼠心脏中的RNA进行反转录PCR，引物设计在Exon3和Exon5中，检测携带突变的Exon4发生了跳读，Dmd为小鼠编码Dystrophin蛋白的基因，Gapdh为PCR的内参；B图，利用毛细管电泳定量的方法，确定Exon4发生跳读的条带与没有发生跳读(即包含)的条带所包含核酸量的比例；C图，对Exon4发生跳读的条带进行Sanger测序，确定Exon4发生了完整的跳读，Exon3和Exon5拼接到了一起；D图，对经过治疗的Dmd-E4小鼠心脏中的蛋白质进行免疫印迹检测，以WT小鼠以及没有经过治疗的小鼠样本为阳性与阴性对照，VCL为大分子量的内参；E图，D图中条带的定量统计；F图，免疫荧光染色的方法检测经过治疗的Dmd-E4小鼠心脏中Dystrophin蛋白表达的情况，包括两个治疗后样本；G图，利用小动物心脏超声检测的方法考察Dmd-E4小鼠的心脏相关生理结构的改变在经过AAV治疗后是否得到了修复；H图，为F图的定量，量化了Dystrophin阳性表达细胞的占比。P-value：*p<0.05,**p<0.01,***p<0.001.
图5.AAV治疗成功恢复了Dmd-E4小鼠的肌肉功能并延长了其生存期。A图，对经过治疗的Dmd-E4小鼠的血清中肌酸激酶含量进行了测定，WT与未经治疗的Dmd-E4小鼠样品为对照；B图，利用HE染色与Masson染色的方法，评估经过治疗后的Dmd-E4小鼠心肌炎症细胞浸润与纤维化的程度；C图，根据Masson染色的结果，定量统计经过治疗后的Dmd-E4小鼠心肌纤维化程度的恢复情况；D图，利用micro-CT的方法，检测Dmd-E4小鼠脊柱弯曲 的程度，以WT小鼠以及没有经过治疗的小鼠样本为对照；E图，D图中脊柱弯曲程度的定量统计；F图，利用拉力装置检测经过治疗的Dmd-E4小鼠全身肌肉最大拉力在循环发力过程中的降级幅度；G图，WT小鼠以及经过AAV治疗与未治疗的Dmd-E4小鼠的生存期统计；H图，Dmd-E4小鼠心肌细胞中基因编辑的分子生物学证据，将对应细胞的pre-mRNA进行逆转录PCR，后进行高通量测序，发现在sgRNA靶向的位置附近，产生了预期的突变，是对Dmd-E4小鼠的心脏疾病表型进行治疗的分子基础与依据。*p<0.05,**p<0.01,***p<0.001。
图6.基因编辑工具在人类细胞中可以成功诱导DMD基因的对应改造。A图，在K562细胞系中成功筛选出两个sgRNA，可以诱导Exon51发生跳读，如图为编辑后的K562细胞抽提RNA后进行逆转录PCR的结果，显示两个sgRNA联用可以高效诱导缺失Exon50的K562细胞成功跳读Exon51；B图，在正常人类iPS与缺失DMDExon50的细胞中诱导DMD基因的Exon51发生跳读；C图，利用免疫荧光检测的方法确定Dystrophin蛋白在经过编辑的iPS细胞中回复了表达；D图，利用western blot的方法确定Dystrophin蛋白在经过编辑的iPS细胞中回复了表达；E图，D图中蛋白回复表达的定量统计。
具体实施方式
本发明以DMD小鼠模型和人类DMD病人携带的致病突变为例，通过设计构建基因编辑工具，实现了对致病突变的改造。下面结合具体实施例及附图对本发明作进一步说明：
实施例1携带基因编辑工具的AAV病毒
根据本发明设计的基因编辑工具如图1所示，以AID为例，我们将对应的序列克隆至AAV质粒中，包括如下步骤：
首先，以XhoI和NotI的酶切位点为基础对pAAV2骨架载体(购自addgene，但不限于此)进行双酶切。同时设计基因编辑工具中AID和Cas9融合蛋白的氨基酸序列，氨基酸序列以及核酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。经过密码子优化后，直接合成双链DNA片段，与Syn100启动子以及加尾信号等元件连接至AAV骨架载体中获得表达AID-Cas9突变体融合蛋白的AAV载体质粒，其序列SEQ ID NO.3所示。此外，利用引物合成以及PCR的方法，可以将U6启动子、H1启动子以及7SK启动子的序列与识别致病突变外显子剪切位点的sgRNA连接起来，同时用Syn100启动子与加尾信号来表达绿色荧光蛋白以及相关元件，以增加蛋白表达标签以及辅助提升基因编辑效率；此外，还可以采用合并包毒的AAV质粒构建基因编辑工具，在AID和Cas9融合蛋白表达元件的基础上，连接U6启动子与靶向致病突变外显子剪切位点的sgRNA，构建成为4.9kbp插入序列的AAV质粒载体。相关质粒克隆的部分结果如下图3所示。
在构建完成的AAV载体质粒后，根据既往文献[Grieger,J.,Choi,V.&Samulski,R.Production and characterization of adeno-associated viral vectors.Nat Protoc 1,1412–1428(2006).]，包装并纯化得到滴度为1 x 10 13v.g./mL的血清型为AAV9的AAV病毒。分别包毒的按比例混合使用，合并包毒的可直接用于体内治疗；
实施例2利用携带基因编辑工具的AAV对DMD模型小鼠进行体内治疗
本实施例选择了一种具有心脏功能异常的新型DMD小鼠疾病模型Dmd-E4，该模型可购自江苏集萃药康生物科技有限公司，但不限于此。Dmd-E4在6-8周心脏即出现了心肌肥厚，纤维化等表型，而在8个月左右则表现出心脏功能的严重退化。这一过程很好地模拟了DMD病人的心脏病理进程。针对这一模型，我们应用胞嘧啶脱氨酶与Cas9设计基因编辑工具，以携带致病突变的外显子为目标，在其5’剪接位点附近进行诱导突变，使其发生跳读，在不影响蛋白开放阅读框的基础上，最大限度地保留Dystrophin蛋白的表达，恢复其生物学功能。
具体地，采用实施例1的方法构建基因编辑工具，其中设计的针对Dmd-E4小鼠突变位点的sgRNA序列如SEQ ID NO.4所示，得到的表达针对Dmd-E4小鼠突变位点的sgRNA的AAV载体质粒，其序列如SEQ ID NO.5所示。包含AID-Cas9融合蛋白与针对Dmd-E4小鼠的sgRNA于同一AAV载体质粒的对应序列如SEQ ID NO.6所示。
选择血清型为AAV9的病毒合成与纯化，并按照新生小鼠的预防性治疗与成年小鼠的修 复性治疗两种方案进行对Dmd-E4小鼠的治疗，如图2所示。
(A)新生小鼠的基因治疗
入组：将Dmd-E4小鼠纯和KO的雌雄小鼠交配，待雌鼠怀孕后，雌雄小鼠分笼，每隔两日观察怀孕雌鼠是否生产，待新生Dmd-E4小鼠出生后，观察性别，选取3-5只雄鼠作为实验组，另外3-5只雄鼠为阴性对照组；
给药：将50-75μL的携带基因编辑工具的腺相关病毒AAV(滴度10 13v.g./mL)通过腹腔注射或者面静脉注射的方式给药，对照小鼠同时给予等体积的无菌PBS，随后与母鼠一起正常饲养；
采样和检测：待小鼠成长至2个月左右时，除实验组与对照组外，取3-5只同龄WT雄鼠，同时进行如下处理：麻醉小鼠后，首先进行胫骨前肌的功能测试、超声心动图的检测等，随后采集心脏动静脉血以处死小鼠，离心分离血清后存于-80℃，同时收集心肌、骨骼肌、胫骨前肌、背部肌肉、肝脏、脑部、肾脏等组织，并提取其蛋白质、RNA、基因组DNA，以及留存足够组织进行免疫荧光染色、苏木精伊红染色等。
如图4A所示，对经过治疗的Dmd-E4小鼠心脏中的RNA进行反转录PCR，引物设计在Exon3和Exon5中，检测携带突变的Exon4发生了跳读；同时，利用毛细管电泳定量的方法，确定Exon4发生跳读的条带与没有发生跳读(即包含)的条带所包含核酸量的比例；结果如图4B所示。进一步地，对Exon4发生跳读的条带进行Sanger测序，如图4C所示，Exon4发生了完整的跳读，Exon3和Exon5拼接到了一起；图3D-F是对小鼠心脏中的蛋白质进行免疫印迹检测，其中，图4D是条带图，图3E是3D图中条带的定量统计，图4F Dystrophin蛋白表达的情况，结果表明经过治疗的Dmd-E4显著恢复了Dystrophin蛋白表达。另外，利用小动物心脏超声检测的方法考察Dmd-E4小鼠的心脏相关生理结构的改变在经过AAV治疗后是否得到了修复。结果图4G所示，结果表明治疗后Dmd-E4小鼠的心脏相关生理结构基本得到修复。
进一步地，对Dmd-E4小鼠的肌肉功能及其生存期是否恢复及延长进行了验证。图5A是小鼠的血清中肌酸激酶含量测定结果，从图中可以看出，与WT和未经治疗的Dmd-E4小鼠样品相比，治疗后的Dmd-E4小鼠肌酸激酶含量显著下降。利用HE染色与Masson染色的方法，评估经过治疗后的Dmd-E4小鼠心肌炎症细胞浸润与纤维化的程度，同时根据Masson染色的结果，定量统计经过治疗后的Dmd-E4小鼠心肌纤维化程度的恢复情况，结果如图5B-5C所示，治疗后的Dmd-E4小鼠心肌纤维化程度明显得到了改善。另外，还利用micro-CT的方法，检测Dmd-E4小鼠脊柱弯曲的程度(图5D-5E)，利用拉力装置检测经过治疗的Dmd-E4小鼠全身肌肉最大拉力在循环发力过程中的降级幅度(图5F)，结果均表明Dmd-E4小鼠在经过治疗后，脊柱的弯曲程度得到了缓解，同时小鼠的全身肌肉拉力得到了增强，并且极大地延长了Dmd-E4小鼠的生存期(图5G)。图5H为Dmd-E4小鼠心肌细胞中基因编辑的分子生物学证据，将对应细胞的pre-mRNA进行逆转录PCR，后进行高通量测序，发现在sgRNA靶向的位置附近，产生了预期的突变，是对Dmd-E4小鼠的心脏疾病表型进行治疗的分子基础与依据。
上述结果表明，本发明的基因编辑工具能够有效治疗和预防新生Dmd-E4小鼠。
(B)成年小鼠的基因治疗
入组：取3-5只4-6周龄的纯和KO Dmd-E4雄鼠作为实验组给予基因治疗，取3-5只4-6周龄的纯和KO Dmd-E4雄鼠作为对照组给予等量PBS处理；
给药：将约50μL的携带基因编辑工具的腺相关病毒AAV(滴度10 13v.g./mL)通过尾静脉注射或骨骼肌原位注射的方式给药，对照小鼠同时给予等体积的无菌PBS；
采样和检测：待小鼠经过治疗后2个月左右时，除实验组与对照组外，取3-5只同龄WT雄鼠，同时进行如下处理：麻醉小鼠后，首先进行胫骨前肌的功能测试、超声心动图的检测等，随后采集心脏动静脉血以处死小鼠，离心分离血清后存于-80℃，同时收集心肌、骨骼肌、胫骨前肌、背部肌肉、肝脏、脑部、肾脏等组织，并提取其蛋白质、RNA、基因组DNA，以 及留存足够组织进行免疫荧光染色、苏木精伊红染色等。
结果表明：通过AAV作为基因编辑工具的运载介质，可以实现对突变外显子的高效基因修复。在经过治疗的Dmd-E4小鼠中，心肌及多个肌肉组织中均可以观察到致病外显子发生了跳读，并因此恢复了Dystrophin蛋白的表达，同时心肌损伤的表型也得到了明显的修复，使成年Dmd-E4小鼠得到了治疗。
实施例3对人类诱导多能干细胞iPSC的DMD模型进行基因编辑成功恢复Dystrophin蛋白的表达
本发明同时对人类细胞的基因编辑治疗进行了成功的实施。首先我们从正常人外周血单核细胞中构建了诱导多能干细胞(iPSC)，随后利用CRISPR-cas9的方法特异性地删除了Dystrophin编码基因DMD的第50号外显子Exon50，使得Dystrophin蛋白的编码序列产生了移码突变，从而构建了模拟DMD病人的突变类型，成为一个很好的DMD疾病模型细胞。针对此细胞，我们设计了AID和Cas9融合蛋白与对应的sgRNA的序列，用于靶向DMD基因的第51号外显子Exon51的一系列潜在调控外显子剪接的元件，本实施例中采用的sgRNA为如SEQ ID No.19所示的sgRNA-12和如SEQ ID No.20所示的sgRNA-13，sgRNA-12主要靶向如SEQ ID N0.21、SEQ ID N0.22所示的外显子剪接增强子。sgRNA-13主要靶向如SEQ ID N0.24所示的外显子剪接增强子。上述两个sgRNA-12在人类K562细胞系中筛选获得，可以诱导Exon51的跳读，如图6A所示，编辑后的K562细胞抽提RNA后进行逆转录PCR的结果，显示两个sgRNA均能诱导突变，联用可以高效诱导缺失Exon50的K562细胞成功跳读Exon51。同时通过诱导Exon51的跳读，可以使缺失Exon50的iPS细胞中Dystrophin蛋白的开放阅读框恢复，进而重建Dystrophin蛋白的表达。具体实施方案如下：
3.1诱导iPS细胞向心肌细胞分化
(1)用Accutase消化培养在基质胶上的人iPS细胞，37℃消化6分钟，用DMEM培养基终止反应，收集细胞，1500rpm离心3分钟，并在显微镜下计数；
(2)将iPS细胞铺在预先用基质胶包被过的12孔板中，调整细胞密度为10000-20000细胞/cm2，iPS细胞用mTeSR1培养基培养，并加入10μM ROCK抑制剂(Y-27632)，培养4天，每天更换新鲜培养基，更换培养基时不需要添加ROCK抑制剂；
(3)细胞培养4天后，将mTeSR1培养基更换为含有6uM CHIR99021的RPMI/B27-insulin培养基，培养2天；
(4)撤去CHIR99021刺激，将培养基更换为RPMI/B27-insulin培养基，培养1天；
(5)将培养基更换为含有5μM IWR1的RPMI/B27-insulin培养基，培养2天；
(6)撤去IWR1刺激，将培养基更换为RPMI/B27-insulin培养基，培养2天；
将细胞培养基更换为RPMI/B27培养基，之后一直用此培养基培养，每两天换一次培养基，获得向心肌细胞分化的人多能干细胞。
3.2在向心肌细胞诱导分化的人多能干细胞中进行基因编辑工具的转染
(1)在转染前一天，用Accutase消化向心肌细胞分化的iPS细胞，铺于6孔板中，每孔中铺4×10 5细胞；
(2)约24h后等到向心肌细胞分化的iPS细胞密度达到60％左右时，将细胞培养基换成无抗培养基；
(3)将2.5μg表达AID与Cas9突变体融合蛋白的质粒(例如Lenti-V2-AIDx-nSaCas9(KKH)-Ugi质粒)与500ng表达UGI的质粒(例如pCDNA3.1-Ugi)质粒以及1.5μg sgRNA质粒混匀在150μl opti-MEM中，并加入2.5μl PLUS TM试剂，轻轻混匀；
(4)将12μl Lipofectamine LTX与150μl opti-MEM培养基混匀，并将其加入到步骤(3)的质粒中，轻轻混匀，室温孵育15min，将反应产物加入到(2)的向心肌细胞分化的iPS细胞中；
(5)转染48h后，在转染的细胞中加入2μg/ml puromycin，筛选3天后撤药，转染后7天收取细胞进行分析。
3.3对经过编辑的iPSC进行相关指标的检测
(1)抽提编辑前后iPSC的基因组DNA，检测相应的Exon51是否发生了突变；
(2)抽提编辑前后iPSC的RNA，进行逆转录PCR，检测Exon51在RNA水平上是否发生了跳读，结果如图6B所示，在正常人类iPS与缺失DMD Exon50的细胞中诱导DMD基因的Exon51发生跳读；
(3)对编辑前后iPSC在蛋白水平上考察Dystrophin蛋白的表达，实验方法包括Western Blot，免疫荧光染色等。图6C是利用免疫荧光检测的方法确定Dystrophin蛋白在经过编辑的iPS细胞中恢复了表达的结果；图6D是利用western blot的方法确定Dystrophin蛋白在经过编辑的iPS细胞中恢复了表达；图6E是D图中蛋白回复表达的定量统计；
上述结果表明：在K562细胞系中成功构建出可以诱导人类DMD基因Exon51等发生跳读的基因编辑方式，鉴定出一系列潜在调控外显子跳读的序列元件，同时进一步利用这种基因编辑方案，可以将DMD疾病模型细胞iPSC成功进行治疗性改造，恢复Dystrophin蛋白的表达。
另外，其余SEQ ID No.7-SEQ ID No.18所示的sgRNA-1～sgRNA-11均是靶向本发明对应的如SEQ ID No.21-SEQ ID No.24所示的外显子剪接增强子，当采用其构建成基因编辑工具时，高效诱导跳读Exon51，从而实现治疗人类DMD。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其他不同形式的变化或变动。这里无需也无法把所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明的保护范围。

Claims (9)

  1. 一种杜氏肌营养不良症相关的外显子剪接增强子，其特征在于，所述外显子剪接增强子为靶向人类DMD基因Exon51的外显子剪接增强子元件，其核苷酸序列包括：
    1)如SEQ ID N0.21所示的序列及其反向互补序列；
    2)如SEQ ID N0.22所示的序列及其反向互补序列；
    3)如SEQ ID N0.23所示的序列及其反向互补序列；
    4)如SEQ ID N0.24所示的序列及其反向互补序列。
  2. 一种杜氏肌营养不良症相关的sgRNA，其特征在于，所述sgRNA的序列包括：
    针对Dmd-E4小鼠突变位点的sgRNA，其核苷酸序列如SEQ ID N0.4所示；
    针对人类DMD基因Exon50的sgRNA，其核苷酸序列如SEQ ID N0.7所示；
    针对人类DMD基因Exon51的sgRNA-1，其核苷酸序列如SEQ ID N0.8所示；
    针对人类DMD基因Exon51的sgRNA-2，其核苷酸序列如SEQ ID N0.9所示；
    针对人类DMD基因Exon51的sgRNA-3，其核苷酸序列如SEQ ID N0.10所示；
    针对人类DMD基因Exon51的sgRNA-4，其核苷酸序列如SEQ ID N0.11所示；
    针对人类DMD基因Exon51的sgRNA-5，其核苷酸序列如SEQ ID N0.12所示；
    针对人类DMD基因Exon51的sgRNA-6，其核苷酸序列如SEQ ID N0.13所示；
    针对人类DMD基因Exon51的sgRNA-7，其核苷酸序列如SEQ ID N0.14所示；
    针对人类DMD基因Exon51的sgRNA-8，其核苷酸序列如SEQ ID N0.15所示；
    针对人类DMD基因Exon51的sgRNA-9，其核苷酸序列如SEQ ID N0.16所示；
    针对人类DMD基因Exon51的sgRNA-10，其核苷酸序列如SEQ ID N0.17所示；
    针对人类DMD基因Exon51的sgRNA-11，其核苷酸序列如SEQ ID N0.18所示；
    针对人类DMD基因Exon51的sgRNA-12，其核苷酸序列如SEQ ID N0.19所示；
    针对人类DMD基因Exon51的sgRNA-13，其核苷酸序列如SEQ ID N0.20所示。
  3. 一种权利要求2所述sgRNA在制备治疗杜氏肌营养不良症的药物中的应用。
  4. 一种杜氏肌营养不良症相关的基因编辑工具，其特征在于，包括胞嘧啶脱氨酶和Cas9突变体的融合蛋白、权利要求2所述sgRNA和载体。
  5. 根据权利要求4所述的基因编辑工具，其特征在于，所述胞嘧啶脱氨酶为AID，AID和Cas9突变体的融合蛋白的氨基酸序列以及核酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
  6. 根据权利要求4所述的基因编辑工具，其特征在于，所述基因编辑工具由腺相关病毒 载体AAV包装。
  7. 根据权利要求6所述的基因编辑工具，其特征在于，所述腺相关病毒载体AAV的启动子为Syn100启动子或基于ck8a、mhck7设计的启动子。
  8. 根据权利要求6所述的基因编辑工具，其特征在于，腺相关病毒载体AAV的核苷酸序列如SEQ ID NO.3所示。
  9. 一种权利要求4-8任一项所述基因编辑工具在制备治疗杜氏肌营养不良症的药物中的应用。
PCT/CN2020/119361 2020-09-02 2020-09-30 杜氏肌营养不良症相关的外显子剪接增强子、sgRNA、基因编辑工具及应用 WO2022047876A1 (zh)

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