CN110337493A - 用于治疗肌强直性营养不良的组合物和方法 - Google Patents
用于治疗肌强直性营养不良的组合物和方法 Download PDFInfo
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- CN110337493A CN110337493A CN201780067115.4A CN201780067115A CN110337493A CN 110337493 A CN110337493 A CN 110337493A CN 201780067115 A CN201780067115 A CN 201780067115A CN 110337493 A CN110337493 A CN 110337493A
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Abstract
本发明涉及用于治疗肌强直性营养不良的组合物和方法。
Description
技术领域
本发明涉及用于治疗肌强直性营养不良的组合物和方法。
背景技术
核苷酸重复序列扩张区(nucleotide repeat expansion),尤其是三核苷酸重复序列扩张区,涉及超过二十余种神经和发育障碍。已经提出治疗这些疾病的一种方法是使用高度特异性的核酸酶将重复序列缩短至非病理性长度(参见Richard GF的综述,TrendsGenet.2015年4月;31(4):177-186)。
高度特异性的核酸酶例如兆核酸酶(meganuclease)、ZFN、TALEN和CRISPR-Cas9核酸酶已用于这样的策略中。然而,本领域技术人员认为后者不适合用于切除三核苷酸重复序列扩张区(参见上文引用的Richard)。总之,TALEN被认为是更有前途的缩短三核苷酸重复序列的工具。
针对这种强烈的偏见,本发明人在此表明可以实施CRISPR-Cas9***以从DMPK基因中的基因组DNA切除核苷酸重复序列扩张区,从而提供用于治疗肌强直性营养不良的强大且意想不到的工具。更特别地,本发明人发现了如何使用源自于金黄色葡萄球菌(Staphylococcus aureus)的Cas9来改善DMPK基因内核苷酸重复序列扩张区的切除效率。
发明内容
本发明人已经表明,针对上文展现的强烈偏见,CRISPR-Cas9***可以有效地用于切除核苷酸重复序列扩张区。本发明涉及使用源自于金黄色葡萄球菌(S.aureus)并具有适当的单个向导RNA(single guide RNA)(sgRNA)的CRISPR-Cas9***来切除DMPK基因内核苷酸重复序列扩张区的改进工具。
在一个方面,本文公开了可用于从DMPK基因的3'-UTR特异性切除核苷酸重复序列扩张区、尤其是三核苷酸重复序列扩张区的单个向导RNA(sgRNA)分子。本文公开的sgRNA分子能够通过碱基配对与所靶向的核苷酸扩张区的5'或3'的基因组DNA靶序列(前间区序列)的互补序列结合,并且能够将Cas9内切核酸酶募集至sgRNA和基因组DNA之间的杂交位点或附近。更确切地说,本文用于切除三核苷酸重复序列扩张区的Cas9内切核酸酶源自于金黄色葡萄球菌(SaCas9)。本发明的sgRNA分子包含适合于在所述互补位点附近诱导SaCas9介导的双链断裂的所有序列元件。特别地,本申请公开了适合于实现DMPK基因的3'-非翻译区(3'-UTR)中存在的核苷酸重复序列扩张区的切除的sgRNA对,其中所述sgRNA对包含第一sgRNA和第二sgRNA,所述第一sgRNA与位于所述核苷酸重复序列扩张区的5'的靶基因组DNA序列互补,并且所述第二sgRNA与位于所述核苷酸重复序列扩张区的3'的靶基因组DNA序列互补。所述第一sgRNA分子能够在Cas9内切核酸酶的存在下在DMPK基因的3'-UTR内诱导所述核苷酸重复序列扩张区的5'的双链断裂。所述第二sgRNA分子能够在Cas9内切核酸酶的存在下在DMPK基因的3'-UTR内诱导所述核苷酸重复序列扩张区的3'的双链断裂。在本发明的上下文中,所述Cas9内切核酸酶源自于金黄色葡萄球菌(SaCas9),或所述Cas9内切核酸酶是SaCas9的功能性变体。
所述第二sgRNA分子包含15-40个核苷酸的向导序列,所述向导序列包含SEQ IDNO:12中所示的核苷酸序列。在一个特定实施方式中,所述第二sgRNA的向导序列由选自SEQID NO:5、SEQ ID NO:6和SEQ ID NO:21的核苷酸序列组成。
在一个特定实施方式中,所述第一sgRNA分子包含长度为15-40个核苷酸的向导序列,所述向导序列包含SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11中所示的核苷酸序列。
本发明的另一个方面涉及一种sgRNA,其包含能够通过碱基配对与靶基因组DNA序列的互补序列结合的序列,所述靶基因组DNA序列位于DMPK基因的3'-UTR内的核苷酸重复序列扩张区的5'或3';
其中所述sgRNA分子能够在源自于金黄色葡萄球菌的Cas9内切核酸酶(SaCas9)或作为SaCas9的功能性变体的Cas9内切核酸酶存在下在所述3'-UTR内诱导所述核苷酸重复序列扩张区的5'或3'的双链断裂;
其中所述sgRNA包含15-40个核苷酸的向导序列,所述向导序列包含选自SEQ IDNO:8-12的序列。
本文公开的另一个方面是所述CRISPR-Cas9***在切除靶细胞基因组DNA内DMPK基因的3'UTR内的核苷酸重复序列扩张区中的用途。
根据进一步的方面,本文公开了一种从细胞的基因组DNA中DMPK基因的3'-UTR切除核苷酸重复序列扩张区的方法,所述方法实施了所述CRISPR-Cas9***。所述方法可以包括向细胞中引入如上所述的sgRNA分子对以及源自于金黄色葡萄球菌的Cas9内切核酸酶或源自于金黄色葡萄球菌的Cas9内切核酸酶的功能性变体的编码基因。
在另一个方面,本文公开了一种用于治疗1型肌强直性营养不良的方法,其中至少使用如上所述的sgRNA分子对和源自于金黄色葡萄球菌的Cas9内切核酸酶或源自于金黄色葡萄球菌的Cas9内切核酸酶的功能性变体从DMPK基因切除核苷酸重复序列扩张区。
在一个特定实施方式中,被切除的核苷酸重复序列扩张区是位于DMPK基因的3'-UTR内的双、三、四、五或六核苷酸重复序列扩张区,优选三核苷酸重复序列扩张区。
在一个特定实施方式中,所述核苷酸重复序列扩张区可包含20个或更多的重复序列,例如20至10000个重复序列,特别是50至5000个重复序列。
更具体地,本发明的用途和方法可包括向细胞例如有需要的对象的细胞中引入:
(i)第一sgRNA分子;
(ii)长度为15-40个核苷酸的第二sgRNA分子,其包含SEQ IDNO:12中所示的序列;和
(iii)源自于金黄色葡萄球菌的CRISPR/Cas9内切核酸酶、或源自于金黄色葡萄球菌的CRISPR/Cas9内切核酸酶的功能性变体;
其中所述第一和第二sgRNA分别与位于DMPK基因的3'-UTR内的核苷酸重复序列扩张区的5'和3'的序列互补,从而适合于通过在DMPK基因的所述3'-UTR内诱导所述核苷酸重复序列扩张区的5'的双链断裂、以及在DMPK基因的所述3'-UTR内诱导所述核苷酸重复序列扩张区的3'的双链断裂来切除所述核苷酸重复序列扩张区。
本发明提供了一种用于治疗1型肌强直性营养不良(DM1)的治疗策略。
所述sgRNA分子被设计成通过碱基配对与DMPK基因的3'-UTR中的基因组DNA靶序列(或称为靶序列)的互补序列结合。该靶序列称为前间区序列(protospacer),并位于称为PAM(前间区序列邻近基序(Protospacer adjacent motif))的核苷酸基序旁边,PAM被所实施的源自于金黄色葡萄球菌的Cas9内切核酸酶(SaCas9)特异性识别。换句话说,所述sgRNA分子包含与所述前间区序列相对应的向导RNA序列,以便与所述前间区序列的互补序列结合。
在一些实施方式中,所述sgRNA分子包含向导RNA序列和支架序列,其中所述向导RNA序列具有15至40个核苷酸,特别是17至30个核苷酸,特别是20至25个核苷酸,例如20、21、22、23、24或25个核苷酸。在一个特定实施方式中,所述向导RNA序列具有21至24个核苷酸。在一个特定实施方式中,所述sgRNA分子包含由21或24个核苷酸组成的向导RNA序列。
另一个方面涉及编码如本文提供的sgRNA或sgRNA分子对的载体,所述载体优选是质粒或病毒载体,例如rAAV载体或慢病毒载体,特别是rAAV载体。
在一些实施方式中,所述SaCas9内切核酸酶和/或所述sgRNA分子由一种或几种载体例如一种或几种质粒或病毒载体表达。例如,所述SaCas9内切核酸酶可以由第一载体表达,而所述第一和第二sgRNA分子可以由单一的第二载体表达,或者一个由第二载体表达而另一个由第三载体表达。在另一个实施方式中,实施所述CRISPR-Cas9***所必需的所有元件都被包含在单一载体中。在另一个实施方式中,SaCas9内切核酸酶和sgRNA分子在体外预组装为核糖核蛋白复合物(RNP),然后通过转染方法递送给细胞。在进一步的实施方式中,纯化的重组SaCas9内切核酸酶蛋白和sgRNA分子在体外合成并分开地递送给细胞。
在另一个实施方式中,如上所述的sgRNA分子对与另一种sgRNA分子对组合使用。在这个实施方式中,组合使用的sgRNA分子对可以由一种或几种载体例如一种或几种质粒或病毒载体表达。
另一个方面涉及靶细胞,其用如本文所述的载体转染或转导。
在进一步的方面,本文公开了一种试剂盒,其包含SaCas9内切核酸酶、或SaCas9内切核酸酶的功能性变体,以及如上所述的第一和第二sgRNA分子。在另一个方面,本文公开了一种试剂盒,其包含编码SaCas9内切核酸酶的载体和编码如上所述的第一和/或第二sgRNA分子的载体,或表达所述SaCas9内切核酸酶和一种或两种sgRNA分子的单一载体。如上所述,试剂盒中的载体可以是质粒载体或病毒载体。在进一步的方面,本文公开了一种试剂盒,其包含SaCas9内切核酸酶和sgRNA分子的核糖核蛋白复合物。在另一个方面,本文公开了一种试剂盒,其包含在体外分开地合成的重组SaCas9内切核酸酶蛋白和sgRNA分子。另外,本发明的试剂盒可以包括任何其它试剂(例如缓冲剂和/或一种或多种转染试剂)或可用于实施本文公开的方法和用途的装置。
本发明的其它方面和实施方式将在以下详细描述中显而易见。
附图说明
图1.SaCas9和Sa sgRNA表达盒。addgene质粒61591及其衍生质粒MLS43(含有较小的启动子EFS而不是原始CMV)和MLS47(含有用于表达第二sgRNA的第二盒)中来自金黄色葡萄球菌的Cas9(SaCas9)的表达盒。来自金黄色葡萄球菌的Cas9的序列是具有以下GenBankID:CCK74173.1的序列,Addgene质粒61591。
图2.选择的sgRNA前间区序列,相应的基因组靶标和PAM,以及切割效率。与sgRNA的向导序列对应的sgRNA前间区序列靶向从基因DMPK的终止密码子到polyA信号在CTG重复序列的上游或下游的基因组区域。示出了SaCas9特异性的相应基因组靶序列和PAM(前间区序列邻近基序)。测试了所有sgRNA在它们的基因组靶标处切割DNA的能力。通过在线程序TIDE分析的切割效率的结果显示在表的最后一列中。
图3.从DMPK基因的终止密码子(此处任意指示为核苷酸1)到polyA在DMPK 3'-UTR的CTG重复序列周围的基因组区域。指示了在DMPK 3'-UTR内测试的所有sgRNA的位置。SaCas9特异性的相应PAM(前间区序列邻近基序)由长方形围绕。CTG重复序列、以及DMPK终止密码子和polyA信号加下划线。
图4.在HeLa细胞(A)和DM1人细胞(iPS来源的MPC)(B)中DMPK CTG重复序列的删除。DMPK 3’-UTR区域是从所指示的细胞系中提取的gDNA中PCR扩增的,并在1.5%琼脂糖凝胶中分离PCR产物。用含有所指示的sgRNA对的质粒MLS43的衍生物转染细胞。将下游sgRNA12B和12与上游sgRNA 1、4、7和8B中的每一个一起进行测试。来自仅用SaCas9表达质粒或GFP表达质粒转染的细胞的gDNA用作对照(ctrl)。经删除的PCR片段的预期大小在琼脂糖凝胶图片下方的图块中指示。
图5.对带有DMPK CTG重复序列删除的基因组区域的测序检查。从琼脂糖凝胶中提取对应于含有CTG重复序列删除的那些(图4中由箭头指示)的PCR产物,纯化并通过标准测序进行测序。未经删除的基因组区域(WT)与经删除的区域(Δ后接的是sgRNA对的相应编号)之间的比对显示了Sa Cas9的准确位置(即前间区序列的核苷酸N3和N4之间)。
图6.用慢病毒载体CRISPR-Cas9处理的DM1细胞中DMPK CTG重复序列的删除和聚集点消失。A)分别处于CMV和U6启动子表达下的来自金黄色葡萄球菌(Sa)的慢病毒载体Cas9和sgRNA的双***的示意图。UP和DW:靶向CTG重复序列上游和下游的基因组区域的sgRNA。B)通过基因组PCR检测DM1永生化成肌细胞中的CTG重复序列切除。将来自带有2600个CTG重复序列的患者的细胞用MOI递增的慢病毒载体Cas9和sgRNA(4-12和8B-12对,如各琼脂糖凝胶图像的左侧所示)转导。编辑和未编辑的PCR扩增子分别由黑色和白色箭头指示。CTG重复序列删除百分比的估算在对应的PCR条带下方指示(#%DEL)。C)在用慢病毒载体CRISPR-Cas9处理后已经失去细胞核聚集点的DM1细胞的定量。
图7.通过AAV-CRISPR在杂合DMSXL小鼠中体内删除DMPK CTG重复序列扩张区。A)分别处于SPc5-12和U6启动子表达下的SaCas9和sgRNA的AAV。eGFP-K=与Kash肽连接的增强型GFP蛋白以处理进入核膜的蛋白。B)和C)显示了经受AAV载体Cas9和sgRNA的肌内注射的小鼠中的CTG重复序列切除的基因组PCR。将数量相等的AAV Cas9和sgRNA病毒粒子共注射到左侧胫骨前肌中(+),并测试了两个剂量:~1*10^11(B)和2*10^11(C)总Vg。(-):阴性对照,用PBS注射右侧胫骨前肌。黑色箭头:具有CTG删除的PCR产物(794bp)。星号:小于预期大小(>4200bp)的未经删除的PCR产物。
具体实施方式
发明人在本文中表明,源自于金黄色葡萄球菌的CRISPR-Cas9***可以有效地用于从DMPK基因切除核苷酸重复序列,从而提供用于治疗1型肌强直性营养不良(DM1)的有力工具。
因此,在第一个方面,本文公开了
(i)第一sgRNA分子,其能够通过碱基配对与基因组DNA中的靶序列(前间区序列)的互补序列结合,所述靶序列位于定位在DMPK基因的3'UTR内的核苷酸重复序列的5'。
(ii)第二sgRNA分子,其能够通过碱基配对与基因组DNA中的靶序列(前间区序列)的互补序列结合,所述靶序列位于定位在DMPK基因的3'UTR内的核苷酸重复序列的3'。
(iii)sgRNA分子对,所述sgRNA分子各自能够通过碱基配对与基因组DNA中的靶序列的互补序列结合,所述靶序列分别位于定位在DMPK基因的3'UTR内的核苷酸重复序列的5'和3’。
CRISPR-Cas9***
II型成簇规律性间隔短回文重复序列(Clustered Regularly InterspacedShort Palindromic Repeats)(CRISPR)***是一种RNA引导的内切核酸酶技术,最近已表现为一种有前途的基因组编辑工具。该***有两个不同的组分:(1)向导RNA和(2)内切核酸酶,在这种情况下是CRISPR相关(Cas)核酸酶,Cas9。向导RNA是细菌CRISPRRNA(crRNA)和反式激活crRNA(tracrRNA)的组合,其被工程化为单个嵌合向导RNA(sgRNA)转录物(Jinek等人,Science 2012 Aug 7;337(6096):816-21)。sgRNA将crRNA的靶向特异性与tracrRNA的支架特性组合在单个转录物中。当sgRNA和Cas9在细胞中表达时,可以修饰或永久性破坏基因组靶序列。
所述sgRNA/Cas9复合物通过sgRNA向导序列与基因组DNA中靶序列(前间区序列)的互补序列之间的碱基配对而被募集到靶序列。为了成功结合Cas9,所述基因组靶序列也必须含有紧跟在所述靶序列之后的正确的前间区序列邻近基序(PAM)序列。所述sgRNA/Cas9复合物的结合将Cas9定位于所述基因组靶序列,使得所述Cas9内切核酸酶可以切割DNA的两条链,导致双链断裂(DSB)。Cas9将在PAM序列上游的第3和第4个核苷酸之间切割。根据在本发明中实施的***,然后可以通过非同源末端连接(NHEJ)修复途径来修复所述DSB。
本发明涉及这种功能强大的***的实施,所述***在本文中以创新的方式得到改进,用于有效地切除已报道与1型肌强直性营养不良(DM1)相关的重复序列。
Cas9内切核酸酶
II型CRISPR-Cas***的DNA靶向机制涉及向导RNA,所述向导RNA指导Cas9内切核酸酶以序列特异性方式切割所靶向的DNA,这取决于所靶向的DNA上前间区序列邻近基序(PAM)的存在。
PAM序列取决于Cas9内切核酸酶所源于的细菌物种而异。
在本发明的上下文中,Cas9内切核酸酶源自于金黄色葡萄球菌(SaCas9)。因此,DNA的切割取决于SaCas9特异性的PAM的存在。SaCas9的共有PAM序列是NNGRRT,其中R=A或G(互补链中为AYYCNN,其中Y=T或C)(Ran FA等人,Nature 2015;KleinstiverBP等人,NatBiotech 2015)。
本发明中使用的Cas9内切核酸酶也可以是SaCas9内切核酸酶功能性变体。
“功能性变体”是指变体Cas9内切核酸酶,其具有与亲本SaCas9内切核酸酶不同的序列,能够通过识别与亲本SaCas9相同的前间区序列邻近基序(PAM)并与sgRNA的相同恒定部分匹配来诱导DNA中的定点双链断裂。所述变体可以来源于亲本SaCas9内切核酸酶,例如具有GenBank ID CCK74173.1或由Addgene质粒61591编码的SaCas9(具有如SEQ ID NO:47中所示的氨基酸序列)。例如,功能性变体SaCas9内切核酸酶与已知的SaCas9内切核酸酶相比可包含一个或多个氨基酸***、缺失或取代,并且可以与已知的SaCas9内切核酸酶具有至少50%、60%、70%、80%、90%、95%、96%、97%、98%或99%同一性。
向导RNA
本文公开了单个向导RNA(或sgRNA),其被具体设计成使用源自于金黄色葡萄球菌的Cas9内切核酸酶或源自于金黄色葡萄球菌的Cas9内切核酸酶的变体从DMPK切除三核苷酸重复序列扩张区。
如上所述,所述sgRNA是所述CRISPR-Cas9***中为所述***提供基因组DNA靶向特异性的部分。被靶向的基因组DNA序列包含15至40个核苷酸,特别是20至30个核苷酸,特别是20至25个核苷酸,例如20、21、22、23、24、25个核苷酸,其后面是如上所述的适当的前间区序列邻近基序(PAM)。在一个特定实施方式中,所述sgRNA分子包含向导RNA序列,其与在DMPK基因的3'-UTR内的PAM之前的15至40个核苷酸、特别是20至30个核苷酸、特别是20至25个核苷酸、例如20、21、22、23、24、25个核苷酸、更具体是21或24个核苷酸的基因组序列的互补序列互补。
在一个特定实施方式中,所述向导RNA序列与DMPK的所述基因组序列相同或具有至少80%同一性,优选至少85%、90%、95%、96%、97%、98%、或至少99%同一性并且能够与SaCas9特异性的PAM之前的15至40个核苷酸、特别是20至30个核苷酸、特别是20至25个核苷酸、例如20、21、22、23、24、25个核苷酸、更具体是21或24个核苷酸的所述基因组序列的互补序列杂交。如本领域技术人员公知的,sgRNA不含PAM基序,因此不与PAM的互补序列结合。靶序列可以位于DMPK基因的3'-UTR内的所述基因组DNA的任一条链上。因此,根据本发明,整个靶序列和PAM均在DMPK基因的3'-UTR中。
在本发明的上下文中,“3'-UTR”被定义为从DMPK基因的终止密码子到DMPK基因的多腺苷酸化的基因组区域。
生物信息学工具可用于鉴定包含适当的PAM的靶基因组DNA序列,例如由以下网络工具提供的那些:CRISPR Design(http://crispr.mit.edu),E-CRISP(http://www.e-crisp.org/E-CRISP/designcrispr.html),CasFinder(http://arep.med.harvard.edu/CasFinder/),和CRISPOR(http://tefor.net/crispor/crispor.cgi)。本领域技术人员也可以参考Doench等人,Nat Biotechnol.2014年12月;32(12):1262-7或Prykhozhij等人,PLoS One.2015年3月5日;10(3):e0119372,并且可以在以下网站http://www.cnb.csic.es/~montoliu/CRISPR/上找到关于CRISPR-Cas9***以及鉴定包含适当PAM的靶基因组DNA的其它信息和资源。或者,可以通过在序列比对工具例如BLAST或FASTA算法中使用PAM序列作为查询,在目标基因内鉴定这样的PAM序列。
然而,在本申请中,发明人表明在靶向DMPK基因中所述核苷酸重复序列的下游区域的那些sgRNA中,仅有数量有限的sgRNA能够提供这种重复序列的有效切除。
众所周知,sgRNA是crRNA和tracrRNA的融合体,其提供靶向特异性(这是由向导序列与靶基因组DNA序列的互补序列的碱基配对所赋予的)和对Cas9内切核酸酶的支架/结合能力两者。换句话说,sgRNA分子包括向导序列(对应于crRNA的与前间区序列的互补序列结合的特异性部分)和sgRNA恒定部分(包含crRNA的非特异性部分、接头环和tracrRNA)。在两者都源自于金黄色葡萄球菌的意义上,所述sgRNA恒定部分和所选择的Cas9内切核酸酶匹配。
在一个特定实施方式中,所述sgRNA的恒定序列是如SEQ ID NO:7所示的Sa sgRNA恒定部分(81个核苷酸的序列,源自于Addgene质粒61591)。
SEQ ID NO:7:
GUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUUU
在另一个实施方式中,所述sgRNA的恒定序列是SEQ ID NO:7中所示的Sa sgRNA恒定部分的功能性变体,其与SEQ ID NO:7中所示的序列具有至少60%、70%、80%、85%、90%、95%、96%、97%、98%或99%的同一性并且能够提供对于SaCas9内切核酸酶或对于SaCas9内切核酸酶的功能性变体的支架/结合能力。
分子生物学试剂盒和工具,例如适当的质粒,可用于容易地产生在DMPK的被靶向的基因组DNA序列和SaCas9内切核酸酶这两个方面具有期望特异性的sgRNA。例如,许多质粒和工具可得自Addgene。在一个特定实施方式中,所述sgRNA或sgRNA对在U6启动子控制下由质粒表达。在一个特定实施方式中,本发明的sgRNA对的两个sgRNA由在同一载体中(例如在同一质粒中,或在同一重组病毒基因组例如AAV基因组或慢病毒基因组中)含有所述两个sgRNA支架的单个表达盒表达,每个支架在启动子、特别是U6启动子的控制下。在一个特定实施方式中,所述两个sgRNA支架以反向位置(或尾对尾取向)或以串联提供,特别是以串联(例如头对尾取向)提供。在另一个实施方式中,SaCas9内切核酸酶编码基因可操作地连接于启动子,例如诱导型或组成型启动子,特别是遍在性或组织特异性启动子,特别是肌肉特异性启动子。遍在性启动子包括例如EFS、CMV、SFFV或CAG启动子。肌肉特异性启动子包括但不限于本领域公知的肌肉肌酸激酶(MCK)启动子、desmin启动子或合成的C5.12启动子。另外,用于表达SaCas9内切核酸酶的启动子可以是诱导型启动子,例如四环素、他莫昔芬或蜕皮素诱导型启动子。
所述第一和第二sgRNA分子各自与分别在待切除的DMPK的核苷酸重复序列扩张区的5'和3'的区域互补。所述sgRNA分子因此被设计成特异性结合具有PAM的核苷酸重复序列扩张区的上游和下游区域,其中整个靶序列和PAM在DMPK基因的3'UTR内。
所述被靶向序列(同源区域+PAM)与被切除区域的距离可能不是关键的,但为了使基因结构的不稳定性最小化,所述被靶向序列可以被选择成距离所述核苷酸重复序列扩张区的最接近末端在小于500、400、300、200、100、90、80、70、60、50、40、30、20或小于10个核苷酸之内。例如,考虑到所述sgRNA被设计成引导诱发所述核苷酸重复序列扩张区的5'的DSB,被靶向序列的PAM的最3'核苷酸距离待切除的核苷酸重复序列扩张区的第一(考虑5'至3'方向)核苷酸的最5'核苷酸在小于500、400、300、200、100、90、80、70、60、50、40、30、20或小于10个核苷酸之内。
所述sgRNA分子被设计成使用SaCas9切除位于DMPK基因的3'非翻译区内的三核苷酸重复序列扩张区。在该实施方式的特定变体中,本发明涉及包含15-40个核苷酸的向导序列的sgRNA分子,所述向导序列包含选自以下的序列:
AGUCGAAGACAGUUC(SEQ ID NO:8)
ACAACCGCUCCGAGC(SEQ ID NO:9)
GGCGAACGGGGCUCG(SEQ ID NO:10)
AGGGUCCUUGUAGCC(SEQ ID NO:11)
GAACCAACGAUAGGU(SEQ ID NO:12)。
在一个更特定的实施方式中,本发明涉及包含选自以下的向导序列的sgRNA分子:
GCCCCGGAGUCGAAGACAGUUC(SEQ ID NO:1)
CAGUUCACAACCGCUCCGAGC(SEQ ID NO:2)
GCGGCCGGCGAACGGGGCUCG(SEQ ID NO:3)
GGCUCGAAGGGUCCUUGUAGCC(SEQ ID NO:4)
CUUUGCGAACCAACGAUAGGU(SEQ ID NO:5)
GCACUUUGCGAACCAACGAUAGGU(SEQ ID NO:6)
在一个更特定的实施方式中,本发明涉及包含选自以下的向导序列的sgRNA分子
GCCCCGGAGUCGAAGACAGUUC(SEQ ID NO:1)
GCAGUUCACAACCGCUCCGAGC(SEQ ID NO:20)
GCGGCCGGCGAACGGGGCUCG(SEQ ID NO:3)
GGCUCGAAGGGUCCUUGUAGCC(SEQ ID NO:4)
GCUUUGCGAACCAACGAUAGGU(SEQ ID NO:21)
GCACUUUGCGAACCAACGAUAGGU(SEQ ID NO:6)。
在如上所示的SEQ ID NO:20和21中,因为需要G来开始U6启动子的转录,所以与SEQ ID NO:2和5相比分别引入了加下划线的G碱基。然而,本领域技术人员将会理解,其它启动子在紧接在所述向导编码序列之前的这个位置可能不需要G,或者如本领域公知的,可能需要一种或多种其它核苷酸碱基。
本发明还涉及如上定义的载体,其包含编码sgRNA分子的序列,所述sgRNA分子包含选自SEQ ID NO:1至6、SEQ ID NO:20和SEQ ID NO:21的向导序列。在进一步的特定实施方式中,编码sgRNA分子的序列还包含编码如SEQ ID NO:7中所示的sgRNA恒定部分序列、或如上定义的其功能性变体的序列。更特别地,编码整个sgRNA分子的序列选自SEQ ID NO:13至18。
在一个特定实施方式中,所述sgRNA分子对是包含以下的sgRNA对:
-第一sgRNA分子,用于诱导位于DMPK基因的3'UTR内的三核苷酸重复序列扩张区的上游(或5')的双链断裂(DSB),其中所述上游DSB在所述3'-UTR内诱导;
-第二sgRNA分子,用于诱导DMPK的三核苷酸重复序列扩张区的下游(或3')的DSB,其中所述下游DSB在所述3'-UTR内诱导,其中所述第二sgRNA分子包含向导序列,所述向导序列的长度为15-40个核苷酸、特别是20至30个核苷酸、特别是20至25个核苷酸,例如由20、21、22、23、24或25个核苷酸、特别是21或24个核苷酸组成,并包含SEQ ID NO:12中所示的序列。
在一个更特定的实施方式中,所述第一sgRNA分子包含向导序列,所述向导序列的长度为15-40个核苷酸、特别是20至30个核苷酸、特别是20至25个核苷酸、例如由20、21、22、23、24或25个核苷酸、特别是21或24个核苷酸组成,并包含选自SEQ ID NO:8至SEQ ID NO:11的序列。在一个优选实施方式中,所述第一sgRNA的向导序列由选自SEQ ID NO:1至SEQID NO:4和SEQ ID NO:20的核苷酸序列组成。
在另一个优选实施方式中,所述第二sgRNA的向导序列由选自SEQ ID NO:5、SEQID NO:6和SEQ ID NO:21的核苷酸序列组成。
在另一个实施方式中,本发明的sgRNA分子对包含选自以下的向导序列对:
-SEQ ID NO:1和SEQ ID NO:5(或SEQ ID NO:21);
-SEQ ID NO:2和SEQ ID NO:5(或SEQ ID NO:21);
-SEQ ID NO:20和SEQ ID NO:5(或SEQ ID NO:21);
-SEQ ID NO:3和SEQ ID NO:5(或SEQ ID NO:21);
-SEQ ID NO:4和SEQ ID NO:5(或SEQ ID NO:21);
-SEQ ID NO:1和SEQ ID NO:6;
-SEQ ID NO:2和SEQ ID NO:6;
-SEQ ID NO:20和SEQ ID NO:6;
-SEQ ID NO:3和SEQ ID NO:6;和
-SEQ ID NO:4和SEQ ID NO:6。
如上所述,本发明的sgRNA可以从表达盒表达。所述sgRNA的表达可以特别地由启动子例如U6启动子控制。因此,本发明还包括sgRNA的表达盒,其包含置于启动子例如SEQID NO:19中所示的U6启动子控制下的sgRNA编码序列。
在一个特定实施方式中,所述表达盒包含以下序列用于从U6启动子表达sgRNA:
-以5'至3'取向,SEQ ID NO:19、SEQ ID NO:48和SEQ ID NO:54的组合;
-以5'至3'取向,SEQ ID NO:19、SEQ ID NO:49和SEQ ID NO:54的组合;
-以5'至3'取向,SEQ ID NO:19、SEQ ID NO:50和SEQ ID NO:54的组合;
-以5'至3'取向,SEQ ID NO:19、SEQ ID NO:51和SEQ ID NO:54的组合;
-以5'至3'取向,SEQ ID NO:19、SEQ ID NO:52和SEQ ID NO:54的组合;或
-以5'至3'取向,SEQ ID NO:19、SEQ ID NO:53和SEQ ID NO:54的组合。
本发明的方法和用途
本发明设想了用SaCas9内切核酸酶和sgRNA分子触及DMPK的靶基因组DNA序列的各种方式。在一些实施方式中,将所述SaCas9内切核酸酶以多肽形式引入细胞内。在一个变体中,所述SaCas9内切核酸酶与细胞穿透肽缀合或融合,所述细胞穿透肽是促进分子摄入到细胞中的肽。所述sgRNA分子也可以作为分离的寡核苷酸直接施用于细胞或使用转染试剂例如脂质衍生物、脂质体、磷酸钙、纳米粒子、显微注射或电穿孔施用于细胞。SaCas9内切核酸酶和sgRNA分子也可以在体外预组装为核糖核蛋白复合物,然后直接或使用转染试剂递送给细胞。
在另一个实施方式中,本发明设想了将所述SaCas9内切核酸酶和/或sgRNA分子以表达所述内切核酸酶和/或sgRNA分子的载体的形式引入靶细胞。因此,本发明还涉及编码本发明的sgRNA分子或sgRNA分子对的载体。在细胞中引入和表达基因的方法是本领域已知的。可以通过物理、化学或生物手段将表达载体转移到宿主细胞中。可以使用已知的物理方法例如磷酸钙沉淀、脂质转染、粒子轰击、显微注射、电穿孔将表达载体引入细胞中。用于将多核苷酸引入宿主细胞的化学手段包括胶体分散体系,例如大分子复合物、纳米囊、微球、珠子、以及脂质衍生物和脂质体。在其它实施方式中,通过生物手段、特别是通过病毒载体引入所述SaCas9内切核酸酶和/或sgRNA分子。可用于本发明的实践中的代表性病毒载体包括但不限于,源自于腺病毒、逆转录病毒、特别是慢病毒、痘病毒、单纯疱疹病毒I和腺相关病毒(AAV)的载体。适当的病毒载体的选择当然将取决于靶细胞和病毒嗜性。
在一个实施方式中,所述SaCas9内切核酸酶和sgRNA分子被提供在不同载体(例如两种载体,一种含有编码SaCas9内切核酸酶的基因,第二种编码两种sgRNA分子;或三种载体,一种编码SaCas9内切核酸酶,以及每种sgRNA分子各一种载体)内。在另一个实施方式中,从单一表达载体表达所述CRISPR-Cas9***的所有元件,包括从DMPK切除三核苷酸重复序列扩张区所需的SaCas9内切核酸酶和两种sgRNA分子。
在一个特定实施方式中,将本发明的SaCas9内切核酸酶和sgRNA分子与其它DM1处理组合,同时或顺序地使用。特别地,本发明的SaCas9内切核酸酶和sgRNA分子可以与另一种sgRNA分子对和另一种或相同的Cas9内切核酸酶组合,同时或顺序地使用,以切除所述重复序列扩张区。
在一个特定实施方式中,所述另一种sgRNA分子对选自EP16306427(其通过引用整体并入)中公开的对。
在一个特定实施方式中,所述另一种sgRNA分子对包含含有15-40个核苷酸的向导序列的sgRNA分子,所述向导序列包含EP16306427的SEQ ID NO:11中所示的核苷酸序列。
在另一个特定实施方式中,所述另一种sgRNA分子对包含第一sgRNA分子,其包含长度为15-40个核苷酸的向导序列,所述向导序列包含EP16306427的SEQ ID NO:7、EP16306427的SEQ ID NO:8、EP16306427的SEQ ID NO:9或EP16306427的SEQ ID NO:10中所示的核苷酸序列。
在另一个特定实施方式中,所述另一种sgRNA分子对包含第一sgRNA分子,其中所述第一sgRNA的向导序列由选自EP16306427的SEQ ID NO:1-4和EP16306427的SEQ ID NO:18的核苷酸序列组成。
在另一个特定实施方式中,所述另一种sgRNA分子对包含第二sgRNA分子,其中所述第二sgRNA分子的向导序列由EP16306427的SEQ ID NO:5中所示的核苷酸序列组成。
在一个方面,本发明还涉及包含本发明的sgRNA分子或本发明的sgRNA对的靶细胞,或者用本发明的载体转染或转导的靶细胞。任选地,所述靶细胞还表达SaCas9内切核酸酶,例如从与表达本发明的sgRNA分子或sgRNA对的载体相同的载体表达SaCas9内切核酸酶。例如,重组细胞可选自iPS来源的间充质祖细胞(MPC)或hESC来源的MPC。
本发明的***用于在DMPK基因的3'非翻译区内切除核苷酸重复序列扩张区,特别是三核苷酸重复序列。在一个特定实施方式中,所述核苷酸重复序列扩张区(例如三核苷酸重复序列扩张区)包含所述核苷酸基序的20至10000个重复序列,更特别是50至5000个重复序列。例如,待切除的核苷酸重复序列扩张区(例如三核苷酸重复序列扩张区)可包含所述核苷酸基序的任何数量的重复序列,例如至少20、30、40、50、60、70、80、90、100、200、300、400、500、600、700、800、900、1000个或至少超过2000个重复序列。更具体地,重复序列的数量是病理性的重复序列数量,这意味着所述核苷酸重复序列(例如三核苷酸重复序列)与疾病状态相关或可能相关。在一个特定实施方式中,所述重复序列是DMPK基因的3'非翻译区内的CTG重复序列,并且从20个以上重复序列或从50个以上重复序列起是病理性的。在一个特定实施方式中,所述核苷酸重复序列扩张区包含20至10000个重复序列,更特别是50至5000个重复序列。特别地,所述核苷酸重复序列扩张区包含1000至3000个重复序列,更特别是1200至2600个重复序列。
用于本文中时,术语“治疗”和“医治”是指向对象施用有效量的组合物,使得所述对象具有至少一种疾病症状的减轻或疾病的改善,例如,有益或期望的临床结果。出于本发明的目的,有益或期望的临床结果包括但不限于,减轻一种或多种症状、削弱疾病程度、稳定(即,不加重)疾病状态、延迟或减缓疾病进展、缓解或缓和疾病状态、以及消退(无论是部分还是全部),无论是可检测的还是不可检测的。治疗可以指与如果不接受治疗时的预期生存相比延长生存。或者,如果疾病的进展降低或停止,则治疗是“有效的”。“医治”也可以意指与如果不接受治疗时的预期生存相比延长生存。需要治疗的那些包括已经诊断患有与多核苷酸序列表达相关的病症的那些,以及由于遗传易感性或其它因素可能发展这样的病症的那些。用于本文中时,术语“治疗”和“医治”还指预防疾病或病症,这意味着延迟或防止这样的疾病或病症的发作。
本发明提供了核苷酸重复序列扩张区病症的治疗,所述病症是与DMPK基因的3'-非翻译区内的三核苷酸(例如CTG)重复序列扩张区相关的DM1。
本发明还涉及如上所述的sgRNA分子对,其用作药物。
本发明还涉及如上所述的sgRNA分子对,其用于治疗DM1的方法中。
本发明还涉及如上所述的sgRNA分子对在制造用于治疗DM1的药物中的用途。
本发明还涉及一种治疗DM1的方法,所述方法包括向有需要的对象施用有效量的如上所述的sgRNA分子对。
可以配制和施用本发明的sgRNA分子、sgRNA分子对、重组SaCas9内切核酸酶蛋白、载体(编码一种或多种sgRNA分子和/或SaCas9内切核酸酶)和细胞,以通过产生所述sgRNA分子、sgRNA分子对、载体和细胞与其在有需要的对象中的作用位点接触的任何手段来治疗肌强直性营养不良。
本发明还提供了药物组合物,其包含本发明的sgRNA或sgRNA对、或本发明的重组SaCas9内切核酸酶蛋白或载体(单独或与SaCas9内切核酸酶编码序列一起,编码本发明的sgRNA、或sgRNA对)、或本发明的细胞。这样的组合物包含治疗有效量的治疗剂(本发明的sgRNA、载体或细胞)和可药用载体。在一个具体实施方式中,术语“可药用”意指由联邦或州政府的管理机构批准或在美国或欧洲药典或其它公认的药典中列出用于动物和人类。术语“载体”是指治疗剂与之一起施用的稀释剂、佐剂、赋形剂或介质。这样的药物载体可以是无菌液体,例如水和油,包括石油、动物、植物合成来源的那些,例如花生油、大豆油、矿物油、芝麻油等。当所述药物组合物静脉内施用时,水是优选的载体。盐水溶液和右旋糖水溶液和甘油溶液也可用作液体载体,特别是用于可注射的溶液。合适的药物赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙二醇、水、乙醇等。
如果需要,所述组合物也可含有少量的润湿或乳化剂、或pH缓冲剂。这些组合物可采取溶液、悬液、乳液、片剂、丸剂、胶囊、粉末、缓释制剂等形式。口服制剂可以包含标准载体,例如药物级甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。合适的药物载体的实例描述于E.W.M Martin的“雷明顿药物科学(Remington's PharmaceuticalSciences)”中。这样的组合物将含有治疗有效量的所述治疗剂,优选为纯化的形式,以及适量的载体,以便提供恰当施用于对象的形式。
所述药物组合物适于对哺乳动物、特别是人类的任何类型的施用,并按照例行程序配制。所述组合物通过使用合适的常规药物载体、稀释剂和/或赋形剂来配制。所述组合物的施用可以经由任何常见途径,只要经由该途径可利用靶组织即可。这包括例如口服、鼻、皮内、皮下、肌内、腹膜内或静脉内施用。优选地,将所述组合物配制成适于静脉内施用于人类的药物组合物。通常,用于静脉内施用的组合物是在无菌等渗水性缓冲液中的溶液。必要时,所述组合物也可以包含增溶剂和局部麻醉剂例如利诺卡因以缓解注射部位的疼痛。
在治疗核苷酸重复序列扩张区中有效的本发明治疗剂的量可以通过标准临床技术来确定。此外,可任选地采用体内和/或体外测定来帮助预测最佳剂量范围。制剂中采用的精确剂量也将取决于施用途径、疾病严重度,并应根据从业者的判断和每个患者的情况来决定。向有需要的对象施用的sgRNA、载体或细胞的剂量将基于若干因素而变化,所述因素包括但不限于施用途径、对象年龄、或获得所需治疗效果必需的表达水平。本领域技术人员可以基于该领域中的知识,基于这些因素和其它因素而容易地确定所需的剂量范围。
实施例
下面提供了将SEQ ID NO与下面实验部分和图中使用的sgRNA编号匹配的表格。
sgRNA编号 | 1 | 4 | 7 | 8B | 12 | 12B |
SEQ ID NO | 1 | 20 | 3 | 4 | 21 | 6 |
材料和方法
质粒构建
编码金黄色葡萄球菌Cas9的质粒源自于质粒pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA(MLS42)[Ran等人,2015]。EFS启动子是用引物F-XhoI-MreI-EFS(MLS63)和R-XmaI-NruI-EFS(MLS64)PCR扩增的,并被克隆到无启动子的pX601-AAV-::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA的XhoI/AgeI位点,以获得pAAV-EFS::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA(MLS43)。
将第二个sgRNA盒(U6::BbsI-sgRNA)串联克隆到质粒MLS43中第一个sgRNA盒上游的Acc65I位点,以获得构建体pAAV-EFS::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BbsI-sgRNA;U6::BsaI-sgRNA(MLS47)。使用现有盒U6::BsaI-sgRNA的相同序列但将sgRNA前间区序列克隆位点更换成BbsI,通过合成来合成(GeneCust)***物U6::BbsI-sgRNA。
具有n ID编号的Sa sgRNA前间区序列,已经合成为正向和反向的寡核苷酸对(表2)并体外退火,然后将它们克隆到限制性位点BbsI(MLS47衍生质粒pAAV-EFS::NLS-SaCas9-NLS-3xHA-bGHpA;U6::n-DMPK-sgRNA;U6::BsaI-sgRNA)和BsaI(质粒pAAV-EFS::NLS-SaCas9-NLS-3xHA-bGHpA;U6::n-sgRNA;U6::n-sgRNA_DMPK)。
通过使用pCCL质粒[pCC-hPGK.GFP(MLS87);来自MarioAmendola博士的慷慨赠与]的骨架来构建慢病毒载体。将***物U6::4-sgRNA;U6::12-sgRNA_DMPK和U6::8B-sgRNA;U6::12-sgRNA_DMPK(源自于酶消化的质粒MLS83和MLS85)克隆到质粒MLS87的XhoI/EcoRV位点,以获得pCCL-U6::4-sgRNA;U6::12-sgRNA_DMPK-hPGK.GFP和pCCL-U6::8B-sgRNA;U6::12-sgRNA_DMPK-hPGK.GFP(MLS99和MLS101)。将源自于质粒MLS42的CMV启动子克隆到无启动子的pCCL-GFP(没有hPGK启动子的MLS87)的XhoI/AgeI位点而获得pCCL-CMV-GFP(MLS107)。慢病毒载体pCCL-CMV-SaCas9(MLS110)的构建是通过将SaCas9PCR***物[引物F-AgeI-SaCas9(MLS142)和R-SalI-SaCas9(MLS143);质粒MLS42作为模板]克隆到pCCL-CMV(没有GFP的MLS107)的SalI/AgeI位点来完成的。
用于SaCas9和sgRNA对8B-12的腺相关病毒(AAV)载体已通过使用具有已测序的ITR的pAAV质粒[Genethon质粒库]构建。用引物F-PmeI-SaCas9(MLS146)和R-NotI-SaCas9_3xHE(MLS147)并使用质粒MLS42作为模板来PCR扩增SaCas9。将凝胶纯化的***物SaCas9克隆到AAV质粒pC512-Int-smSVpolyA(MLS1)的PmeI/NotI位点中,从而获得pAAV-SPc5-12-SaCas9(MLS118)。pAAV-Des-eGFP-KASH-U6::8B-12-sgRNA_DMPK(MLS122)是通过将PCR***物U6::8B-12-sgRNA_DMPK[引物F-MCS-前-U6SasgRNA(MLS163)和R-PmlI-EndSasgRNA-up(MLS166);质粒MLS85作为模板]克隆到pAAV-Des-EGFP-KASH(MLS23/MLS27)的AflII/MssI位点中而获得的。
表1.质粒列表
表2.引物列表
sgRNA的设计
手工以及通过程序CasBLASTR(http://www.casblastr.org/)和CRISPOR(http://tefor.net/crispor)筛选DMPK 3’-UTR内所有可能的SaCas9靶标。PAM序列NNGRRT用于筛选,其中R=A或G(非编码链中为AYYCNN,其中Y=T或C)[Ran等人,Nature 2015;Kleinstiver等人,Nat Biotech 2015]。
对于每个sgRNA前间区序列,通过程序CasOFFinder(http://www.rgenome.net/cas-offinder/),基于人类基因组“智人(GRCh38/hg38)-人类(2014年4月2日更新)(Homosapiens(GRCh38/hg38)-Human(02April 2014Updated))”并设置截止错配数≤4来计算潜在脱靶数。潜在脱靶也已通过CRISPOR(http://tefor.net/crispor)基于人类基因组“智人-人类-UCSC 2013年12月(GRCh38/hg38)(Homo sapiens-Human-UCSC Dec.2013(GRCh38/hg38))”进行了检查。
sgRNA前间区序列的选择通过将相应的潜在脱靶数和它们在DMPK 3’-UTR区域内的靶位置考虑在内来进行。每个Sa sgRNA的长度从21到24不等。每当所述前间区序列不以G开始时,就将该核苷酸添加到所述序列的5'以优化U6驱动的转录。
细胞培养
HeLa细胞在具有高葡萄糖和GlutaMAX(Invitrogen)并补充有10%胎牛血清(FBS,Invitrogen)的Dulbecco改良Eagle培养基(DMEM)中培养。DM1细胞(iPS来源的MPC)在补充有20%FBS、1%MEM非必需氨基酸溶液(Thermo Fisher Scientific)和1%GlutaMAXTMSupplement(Thermo Fisher Scientific)的KnockOut DMEM(Thermo Fisher Scientific)中生长。
永生化WT和DM1成肌细胞在补充有15%FBS的骨骼肌细胞生长培养基(Promocell)中培养,或在与199培养基(1:4比率;Life Technologies)混合并补充有20%FBS、25μg/ml胎球蛋白、0.5ng/ml bFGF、5ng/ml EGF和0.2μg/ml***(Sigma-Aldrich)的DMEM中培养。
在汇合的细胞中通过用分化培养基(补充有5μg/ml胰岛素的DMEM)替换生长培养基来诱导成肌细胞的分化。
使用37℃标准温度和5%CO2来生长和维持培养中的细胞。
转染实验
在转染前一天将细胞接种在6或12孔板中,并在70-90%的汇合度下转染。使用转染试剂FuGENE HD(3:1比率的FuGENE-DNA;Promega)和lipofectamine 3000(ThermoFisher Scientific)分别转染HeLa细胞和DM1PS来源的MPC细胞。转染后2-3天通过离心收获细胞,并将细胞沉淀团保持在-80℃直至提取基因组DNA。
慢病毒载体和转导实验
如以前所述(Cantore等人,2015),通过磷酸钙沉淀进行293T细胞的瞬时四质粒转染来产生慢病毒载体。通过对感染的HCT116细胞的基因组DNA进行定量PCR(qPCR)来确定载体滴度[载体基因组/ml(vg/ml)](分别由Genethon Vector Core和Quality ControlServices进行病毒产生和滴定)。
将DM1成肌细胞在转导前一天接种于12孔板中,并在70%的汇合度下进行感染。在转导前除去生长培养基并代之以最小体积(400μl/皿)的转导培养基[骨骼肌基础培养基(Promocell)或DMEM,补充有10%FBS和4μg/ml聚凝胺]。将病毒直接添加到转导培养基中并将细胞温育5-6小时,然后添加完全生长培养基。在转导后第1天,将细胞转移到6孔板中并保持培养两代,然后1)收集并冷冻它们以供gDNA提取,2)将它们固定以供FISH/免疫荧光分析。
基因组DNA提取和基因组PCR
用GeneJet基因组DNA纯化试剂盒(Thermo Scientific)或用QIAmp DNA Microand Mini试剂盒(QIAGEN)根据制造商的说明书从HeLa细胞和DM1iPS来源的MPC细胞中提取基因组DNA。通过MagNA Pure96***与MagNA Pure LC总核酸分离试剂盒(Roche),从永生化DM1成肌细胞以及从小鼠肌肉中提取gDNA。
使用高保真Taq DNA聚合酶(Invitrogen)来扩增DMPK3’-UTR。按照制造商方案制备补充有10%DMSO的PCR主混合物。使用150ng的gDNA作为模板以及在Cas9预期切割位点的上游和下游退火的引物(表2)进行PCR。PCR条件如下:95℃下2分钟,35x[95℃下30秒,52℃下30秒,72℃下30秒];72℃下10分钟。使用更多的循环(38)和更长的延伸时间(5分钟)以扩增DMSXL小鼠肌肉中的长CTG重复序列扩张区。PCR产物通过在含有GelRed DNA染色的1.5-2%琼脂糖凝胶中电泳进行分离。在UV曝光下拍摄凝胶图像并调节亮度和对比度。
通过凝胶提取( Gel和PCR Clean-up,Macheray Nageland)纯化PCR产物,并通过Sanger DNA测序(Beckman Coulter Genomics)进行测序。
DM1成肌细胞的荧光原位杂交和免疫荧光
如Taneja KL所述[Taneja KL,1998]并加以一些修改[Denis Furlinglaboratory,Institut de Miologie,巴黎]进行FISH实验。简而言之,将培养在腔室载玻片(Corning)中的细胞在磷酸盐缓冲盐水(PBS)中洗涤并在4%多聚甲醛(PFA)中固定。固定后,将细胞在PBS中洗涤并储存在4℃下70%乙醇中。将细胞在PBS中水合并在杂交缓冲液(40%甲酰胺,2x盐水-柠檬酸钠(SSC),0,2%BSA)中与探针Cy3标记的2’OMe(CAG)7(Sigma-Aldrich)一起温育。杂交后,将显微镜载玻片洗涤数次,然后在PBS/0.25%TritonX-100中使细胞膜透化。SaCas9通过针对位于所述蛋白的C末端的HA标签表位的抗体来检测。纯化的小鼠单克隆抗HA标签(Covance)在5%BSA中1/400稀释下用作第一抗体,并在室温下温育1小时30分钟。山羊抗小鼠633第二抗体(Thermo Scientific)在5%BSA中1/1000稀释下使用,并在室温下温育1小时。使用具有DAPI(Southern Biotech)的安装溶液来组装显微镜载玻片与盖玻片。用共聚焦显微镜(Leica DMi8)获取显微镜图像,用Leica ApplicationSuite X软件分析,并用Adobe Photoshop或用ImageJ软件处理。
动物和AAV载体注射
携带从DM1患者克隆的45kb人类基因组DNA的DMSXL小鼠(90%C57BL/6背景)用于体内研究[Huguet等人,2012]。通过如Gomes-Pereira和合作者[Gomes-Pereira等人,2007]所述的PCR测定转基因状态。小鼠的笼养和处理根据法国动物保护委员会(French Councilon animal care)“实验动物护理和使用指南(Guide for the care and Use LaboratoryAnimals)”:EEC86/609理事会指令-2001-131号法令制定的准则进行。
RAAV载体由Genethon Vector Core和Quality Control Service如以前所述(Ronzitti等人,2016)来产生和滴定。
对被***/甲苯噻嗪混合物麻醉的6周龄DMSXL小鼠进行肌内注射。将AAV病毒注射到左侧TA中并测试两种不同的剂量:1*10^11和2*10^11总Vg/TA;将PBS注射到右侧TA中作为对照。注射后4周,收集TA肌肉并在液氮中冷冻。
结果
Sa Cas9和sgRNA表达盒
在该研究中研究的Cas9是来自金黄色葡萄球菌的Cas9(SaCas9)。因为SaCas9的尺寸小并可以装入腺相关病毒(AAV)载体中,该内切核酸酶特别令人感兴趣。所有含有SaCas9和Sa sgRNA支架的质粒均源自于质粒pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA(Feng Zhang Lab,Addegene编号#61591,图1)。为了在同一载体中包含SaCas9和两个sgRNA盒,首先将原始CMV启动子用较小的EFS替换(图1,MLS43)。Sa Cas9的表达及其核定位通过Western印迹和利用针对HA表位的Ab的免疫荧光来验证(数据未显示)。接下来,添加所述sgRNA的第二盒,与已存在的那个相同,但具有所述sgRNA前间区序列的不同克隆位点(图1,MLS47)。
SgRNA前间区序列的序列(对应于sgRNA的向导序列)列于图2中,它们靶向从基因DMPK的终止密码子到polyA信号在CTG重复序列的上游或下游的基因组区域。图3中指示了在DMPK 3'-UTR内测试的所有sgRNA的位置。
测试sgRNA在它们的基因组靶标处切割DNA的能力。简而言之,用带有Sa Cas9和只一个sgRNA(克隆在BbsI位点的前间区序列)的质粒转染HeLa细胞,并在转染后2-3天收集。将从那些转染细胞中提取的基因组DNA用作模板以PCR扩增每个sgRNA的基因组靶标周围的DMPK 3'-UTR区域。PCR产物被送去测序,转染和未转染细胞的色谱图文件用于通过在线程序TIDE(http://tide-calculator.nki.nl/)分析切割效率。
TIDE分析的结果显示在图2的表的最后一列中。
该表的最后一列显示了在所测试的sgRNA前间区序列之中切割效率不等。特别是,测试的所有上游前间区序列(1,4,7,8B)都非常有效,通过TIDE得到的切割百分比在42和47.4%之间。
关于下游前间区序列,本发明人意外地发现它们中的一些能更有效地切割CTG重复序列下游的区域。特别是,六个下游前间区序列(10,15B,22,17A,17B,和19)的切割百分比弱,在1.1到7.9%范围内,而两个下游前间区序列(12和12B)对于切割DNA特别有效,切割百分比分别为32.5%和28.8%。
这些结果表明,在DNA切割方面,所有sgRNA完全没有表现出相同的效率,并且出乎意料地,下游sgRNA 12和12B能够特别有效地切割CTG重复序列扩张区下游的DNA。
人DMPK基因中CTG重复序列的Sa Cas9介导删除
然后,我们使用SaCas9与适当sgRNA的组合在病理性CTG扩张区存在下的人DMPK基因座中测试了所述CRISPR-Cas9***的功效。为了删除CTG重复序列扩张区,我们制造了在同一质粒中带有Sa Cas9和两个sgRNA的构建体,两个sgRNA中的一个靶向CTG重复序列上游的区域并且另一个靶向CTG重复序列下游的区域(分别克隆到质粒MLS47的BbsI和BsaI位点,参见图1)。
所述sgRNA对基于它们的单一切割效率来选择(TIDE分析,图2)。更确切地说,将上游sgRNA 1、4、7和8B各自与相比下游sgRNA 10、15B、17A、17B、19和22而言提供最高切割百分比的下游sgRNA 12或12B一起进行测试。
带有Sa Cas9和所指示的sgRNA对的质粒用于转染HeLa细胞或DM1细胞(iPS来源的MPC,来自I-Stem,Evry)。如材料和方法中所述对DMPK 3’-UTR进行PCR扩增,并将PCR产物在1.5%琼脂糖凝胶中分离(图4)。从琼脂糖凝胶中提取相对于全长产物和含有CTG重复序列删除的产物的条带,并通过测序验证。未经删除的野生型DMPK3'-UTR区域和经删除的该区域之间的退火显示在图5中,并突出显示了每个测试的sgRNA对的切割位点(即前间区序列的核苷酸N3和N4之间)。
总之,这些结果表明所述SaCas9-sgRNA***适合于从人DMPK基因的3'-UTR区域有效切除CTG重复序列。
它还表明,下游sgRNA的选择是实现从DMPK基因的3'-UTR区域有效切除CTG重复序列的重要参数。
总之,本发明人鉴定了当与源自于金黄色葡萄球菌的Cas9内切核酸酶一起使用时导致有效的单一切割效率的特异性sgRNA前间区序列。此外,他们证明,利用SaCas9内切核酸酶与适当的sgRNA的组合的CRISPR-Cas9***适合且有效地从DMPK 3’UTR切除CTG重复序列。
DM1患者细胞系中CTG重复序列的CRISPR-Cas9介导删除
为了测试CRISPR-Cas9在删除长CTG重复序列扩张区中的能力,我们采用来自带有2600个CTG重复序列的患者的永生化DM1细胞(Arandel等人,2017)。我们构建了用于SaCas9和sgRNA的慢病毒载体,因为已知这些病毒高效转导成肌细胞。图6A中描绘了慢病毒构建体的图示:SaCas9在CMV启动子的控制下,靶向CTG重复序列上游和下游区域(UP和DW)的两个sgRNA的对是串联的并且均在U6启动子的控制下。
用MOI(感染复数)递增的Cas9和sgRNA慢病毒载体转导DM1细胞,并通过基因组PCR测试CTG删除(图6B)。如材料和方法部分中所述并使用引物对F1-DMPK-3UTR和R1-DMPK-3UTR进行PCR。来自未处理细胞(-/-)或仅用50MOI的sgRNA慢病毒载体(-/50)转导的细胞的gDNA用作阴性对照。较低分子量的条带(4-12对为587bp,8B-12对为698bp)代表有CTG重复序列的基因组删除的PCR产物,没有区分扩张和未扩张的等位基因。较高分子量的条带代表具有未扩张的CTG重复序列(870bp)的未经删除的基因组区域的PCR产物。因为长度巨大,我们无法PCR扩增所述扩张的未经删除的CTG重复序列(2600个重复序列对应于长于8600bp的PCR产物)。我们的结果显示,接种的病毒粒子的量与PCR条带的强度之间存在相关性:在载体的MOI递增时,我们可以观察到相对于CTG删除的条带的强度增加和相对于未经删除的PCR产物的条带的强度降低。这些数据显示Cas9和所选择的sgRNA对4-12和8B-12在DM1成肌细胞中被慢病毒载体有效递送并且它们可体外导致CTG重复序列的删除。
接下来,我们对于了解CTG删除是否会影响细胞核聚集点的存在有兴趣。因此,我们选择了用这两种载体在高MOI(25和50)下转导的DM1细胞,并且我们进行了FISH分析。未处理或仅用一种载体处理的DM1细胞用作对照。在通过共聚焦显微镜获取图像之后,我们手动计数聚集点消失的细胞的数量,并将该数量以整个群体的百分比报告(图6C)。要注意,并非所有细胞都被这两种慢病毒载体感染,因此如果对双阳性细胞Cas9-sgRNA进行归一化,图6C中报道的百分比将更高。如同对PCR删除所观察到的,聚集点的消失也与接种的病毒粒子的量相关,并且在最高MOI(MOI 50)下,没有聚集点的细胞数量更高。
我们的数据显示,CRISPR-Cas9介导的CTG重复序列切除决定了细胞核中聚集点的消失。
AAV Cas9和sgRNA诱导DMSXL小鼠中体内删除CTG重复序列扩张区
为了验证我们的sgRNA对在体内诱导CTG重复序列删除的能力,我们选择了DMSXL小鼠作为DM1疾病的动物模型(Huguet等人,2012)。DMSXL小鼠带有一个拷贝的有约1200个CTG重复序列的人DMPK基因,并再现了人类病理学的许多特征。为了将CRISPR-Cas9***递送到DMSXL小鼠的肌肉组织中,我们构建了用于SaCas9和sgRNA的腺相关病毒(AAV)载体。已知AAV有效感染肌肉,但它们的包装容量有限,约4.7Kb(Warrington等人,2006;Buj-Bello等人,2008)。出于这个原因,我们设计了两种AAV,一种用于Cas9,另一种用于两个串联的sgRNA。SaCas9在SPc5-12的控制下,SPc5-12是一种合成的小启动子,其在肌肉中驱动转基因的良好表达(Li等人,1999)。与sgRNA对8B-12及其U6启动子相关的序列从相应的慢病毒构建体(图6)进行PCR扩增,并克隆到AAV质粒中Desmin启动子驱动的eGFP-K表达盒的多腺苷酸化序列下游,其中K是用于核膜定位的Kash肽(图7A)。
用于Cas9和sgRNA 8B-12的两种AAV已被共注射到6周龄杂合DMSXL小鼠的左侧胫骨前肌(TA)中。四周后,对小鼠实施安乐死并收集肌肉。为了检测CTG重复序列删除,用从TA提取的基因组DNA以及引物F1-DMPK-3UTR和R2-DMPK-3UTR进行PCR(参见材料和方法)。来自仅用PBS注射的右侧TA的gDNA用作阴性对照(-)。基因组PCR的结果呈现在图7中并且它们相对于用两种不同剂量1*10^11(B)和2*10^11(C)总Vg注射的小鼠。所有注射了AAVCas9-sgRNA(+)的TA显示出对应于具有CTG重复序列删除的扩增子的预期大小(794bp)的PCR条带。此外,一些未处理和处理的TA(-和+)显示出一些小于未经删除的PCR产物(>4200bp)的预期大小的细条带。它们很可能代表含有CTG重复序列扩张区的未经删除的基因组区域的未完成扩增。
我们的结果证明,Cas9与sgRNA 8B-12对一起,能够体内删除DMSXL小鼠模型中人DMPK基因的3'-UTR处的长CTG重复序列扩张区。
序列表
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<212> DNA
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<220>
<223> DMPK 3'-UTR从终止密码子到polyA
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tgaaccctag aactgtcttc gactccgggg ccccgttgga agactgagtg cccggggcac 60
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ccagctccag tcctgtgatc cgggcccgcc ccctagcggc cggggaggga ggggccgggt 180
ccgcggccgg cgaacggggc tcgaagggtc cttgtagccg ggaatgctgc tgctgctgct 240
gctgctgctg ctgctgctgc tgctgctgct gctgctgctg ctgctggggg gatcacagac 300
catttctttc tttcggccag gctgaggccc tgacgtggat gggcaaactg caggcctggg 360
aaggcagcaa gccgggccgt ccgtgttcca tcctccacgc acccccacct atcgttggtt 420
cgcaaagtgc aaagctttct tgtgcatgac gccctgctct ggggagcgtc tggcgcgatc 480
tctgcctgct tactcgggaa atttgctttt gccaaacccg ctttttcggg gatcccgcgc 540
ccccctcctc acttgcgctg ctctcggagc cccagccggc tccgcccgct tcggcggttt 600
ggatatttat tgacctcgtc ctccgactcg ctgacaggct acaggacccc caacaacccc 660
aatccacgtt ttggatgcac tgagaccccg acattcctcg gtatttattg tctgtcccca 720
cctaggaccc ccacccccga ccctcgcgaa taaaa 755
<210> 47
<211> 1114
<212> PRT
<213> 人工序列
<220>
<223> 来自质粒Addgene61591的SaCas9
<400> 47
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser Val
20 25 30
Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly
35 40 45
Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg
50 55 60
Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile
65 70 75 80
Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His
85 90 95
Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu
100 105 110
Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu
115 120 125
Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr
130 135 140
Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala
145 150 155 160
Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys
165 170 175
Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr
180 185 190
Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln
195 200 205
Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg
210 215 220
Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys
225 230 235 240
Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe
245 250 255
Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr
260 265 270
Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn
275 280 285
Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe
290 295 300
Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu
305 310 315 320
Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys
325 330 335
Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr
340 345 350
Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala
355 360 365
Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu
370 375 380
Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser
385 390 395 400
Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile
405 410 415
Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala
420 425 430
Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln
435 440 445
Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro
450 455 460
Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile
465 470 475 480
Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg
485 490 495
Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys
500 505 510
Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr
515 520 525
Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp
530 535 540
Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu
545 550 555 560
Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro
565 570 575
Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys
580 585 590
Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu
595 600 605
Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile
610 615 620
Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu
625 630 635 640
Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp
645 650 655
Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu
660 665 670
Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys
675 680 685
Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp
690 695 700
Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp
705 710 715 720
Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys
725 730 735
Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys
740 745 750
Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu
755 760 765
Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp
770 775 780
Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile
785 790 795 800
Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu
805 810 815
Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu
820 825 830
Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His
835 840 845
Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly
850 855 860
Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr
865 870 875 880
Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile
885 890 895
Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp
900 905 910
Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr
915 920 925
Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val
930 935 940
Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser
945 950 955 960
Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala
965 970 975
Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly
980 985 990
Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile
995 1000 1005
Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn
1010 1015 1020
Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser
1025 1030 1035
Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1040 1045 1050
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys
1055 1060 1065
Gly Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys
1070 1075 1080
Lys Lys Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Tyr Pro
1085 1090 1095
Tyr Asp Val Pro Asp Tyr Ala Tyr Pro Tyr Asp Val Pro Asp Tyr
1100 1105 1110
Ala
<210> 48
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 编码序列向导序列sgRNA 1
<400> 48
gccccggagt cgaagacagt tc 22
<210> 49
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 编码序列向导序列sgRNA 4
<400> 49
gcagttcaca accgctccga gc 22
<210> 50
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 编码序列向导序列sgRNA 7
<400> 50
gcggccggcg aacggggctc g 21
<210> 51
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 编码序列向导序列sgRNA 8B
<400> 51
ggctcgaagg gtccttgtag cc 22
<210> 52
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 编码序列向导序列sgRNA 12
<400> 52
gctttgcgaa ccaacgatag gt 22
<210> 53
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 编码序列向导序列sgRNA 12B
<400> 53
gcactttgcg aaccaacgat aggt 24
<210> 54
<211> 81
<212> DNA
<213> 人工序列
<220>
<223> sgRNA恒定部分(如在质粒中)
<400> 54
gttttagtac tctggaaaca gaatctacta aaacaaggca aaatgccgtg tttatctcgt 60
caacttgttg gcgagatttt t 81
<210> 55
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 55
cgctcgagcg ccggcgtgag gctccggtgc ccgtcagtgg 40
<210> 56
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 56
cgcccgggtc gcgatcacga cacctgtgtt ctggcggcaa acc 43
<210> 57
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 57
<400> 57
gcgaccggtg ccaccatggc cccaaagaag 30
<210> 58
<211> 44
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 58
cgcgtcgacc ttaagcgtaa tctggaacat cgtatgggta agcg 44
<210> 59
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 59
gcggtttaaa cgccaccatg gccccaaaga ag 32
<210> 60
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 60
ccgcggccgc gcgagctcta ggaattctta agcgtaatc 39
<210> 61
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 61
ggaggtacct taagcaattg gacatagtcg tttaaacc 38
<210> 62
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 62
cctcacgtgt cctgcggccg caaaaatctc g 31
<210> 63
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 63
caccgcggcc ggcgaacggg gctcg 25
<210> 64
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 64
aaaccgagcc ccgttcgccg gccgc 25
<210> 65
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 65
caccggctcg aagggtcctt gtagcc 26
<210> 66
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 66
aaacggctac aaggaccctt cgagcc 26
<210> 67
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 67
caccgcggcc aggctgaggc cctgac 26
<210> 68
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 68
aaacgtcagg gcctcagcct ggccgc 26
<210> 69
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 69
caccgctttg cgaaccaacg ataggt 26
<210> 70
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 70
aaacacctat cgttggttcg caaagc 26
<210> 71
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 71
caccgcactt tgcgaaccaa cgataggt 28
<210> 72
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 72
aaacacctat cgttggttcg caaagtgc 28
<210> 73
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 73
caccgggggg cgcgggatcc ccgaaaaa 28
<210> 74
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 74
aaactttttc ggggatcccg cgcccccc 28
<210> 75
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 75
caccggctcc gcccgcttcg gcggt 25
<210> 76
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 76
aaacaccgcc gaagcgggcg gagcc 25
<210> 77
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 77
caccgccggc tccgcccgct tcggcggt 28
<210> 78
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 78
aaacaccgcc gaagcgggcg gagccggc 28
<210> 79
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 79
caccgaaaac gtggattggg gttgtt 26
<210> 80
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 80
aaacaacaac cccaatccac gttttc 26
<210> 81
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 81
caccggggtc tcagtgcatc caaaac 26
<210> 82
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 82
aaacgttttg gatgcactga gacccc 26
<210> 83
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 83
gttcgccgtt gttctgtctc g 21
<210> 84
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 84
tccagagctt tgggcagatg g 21
<210> 85
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 85
ccgggtaccg ttcgccgttg ttctgtctcg 30
<210> 86
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 86
ccgctctaga tccagagctt tgggcagatg g 31
<210> 87
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 87
gtcccaggag ccaatcagag g 21
<210> 88
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 88
ctagctcctc ccagaccttc g 21
Claims (15)
1.一种sgRNA分子对,其中所述对包含能够通过碱基配对与靶基因组DNA序列的互补序列结合的第一sgRNA分子和第二sgRNA分子,所述第一sgRNA分子和第二sgRNA分子分别位于定位在DMPK基因的3'-非翻译区(3'-UTR)内的核苷酸重复序列扩张区的5'和3',
其中所述第一sgRNA分子能够在Cas9内切核酸酶的存在下在所述3'-UTR内诱导所述核苷酸重复序列扩张区的5'的双链断裂;
其中所述第二sgRNA分子能够在Cas9内切核酸酶的存在下在所述3'-UTR内诱导所述核苷酸重复序列扩张区的3'的双链断裂;
其中所述Cas9内切核酸酶源自于金黄色葡萄球菌(Staphylococcus aureus)(SaCas9),或其中所述Cas9内切核酸酶是SaCas9的功能性变体;
其中所述第二sgRNA分子包含15-40个核苷酸的向导序列,所述向导序列包含SEQ IDNO:12中所示的核苷酸序列。
2.权利要求1的sgRNA对,其中所述第一sgRNA包含长度为15-40个核苷酸的向导序列,所述向导序列包含SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11中所示的核苷酸序列。
3.权利要求1或2的sgRNA对,其中所述第一sgRNA的向导序列由选自SEQ ID NO:1-4和SEQ ID NO:20的核苷酸序列组成。
4.权利要求1-3任一项的sgRNA对,其中所述第二sgRNA的向导序列由选自SEQ ID NO:5、SEQ ID NO:6和SEQ ID NO:21的核苷酸序列组成。
5.一种sgRNA,其包含能够通过碱基配对与靶基因组DNA序列的互补序列结合的序列,所述靶基因组DNA序列位于DMPK基因的3'-UTR内的核苷酸重复序列扩张区的5'或3';
其中所述sgRNA分子能够在源自于金黄色葡萄球菌的Cas9内切核酸酶(SaCas9)或作为SaCas9的功能性变体的Cas9内切核酸酶存在下在所述3'-UTR内诱导所述核苷酸重复序列扩张区的5'或3'的双链断裂;
其中所述sgRNA包含15-40个核苷酸的向导序列,所述向导序列包含选自SEQ ID NO:8-12的序列。
6.权利要求5的sgRNA,其中所述sgRNA包含选自SEQ ID NO:1-6、SEQ ID NO:20和SEQID NO:21的向导序列。
7.一种编码权利要求1至6任一项的sgRNA或sgRNA分子对的载体,所述载体优选是质粒或病毒载体,例如rAAV载体或慢病毒载体。
8.一种靶细胞,其用权利要求7的载体转染或转导。
9.一种用于产生sgRNA或sgRNA对的方法,所述方法包括在允许产生所述sgRNA或sgRNA对的条件下培养权利要求8的靶细胞,并从所述培养步骤中回收所述sgRNA或所述sgRNA分子对。
10.一种用于切除位于细胞中DMPK基因的非编码区内的核苷酸重复序列的体外方法,所述方法包括在所述细胞中引入权利要求1-4任一项的sgRNA分子对或权利要求7的载体,以及源自于金黄色葡萄球菌(S.aureus)的CRISPR/Cas9内切核酸酶。
11.权利要求1-4任一项的sgRNA对或权利要求7的载体,其与源自于金黄色葡萄球菌的Cas9内切核酸酶组合用作药物。
12.权利要求1-4任一项的sgRNA对或权利要求7的载体,其与源自于金黄色葡萄球菌的Cas9内切核酸酶组合用于治疗1型肌强直性营养不良的方法中。
13.权利要求12的供使用的sgRNA对,其中所述核苷酸重复序列扩张区是位于DMPK基因的3'-UTR内的双、三、四、五或六核苷酸重复序列扩张区,特别是三核苷酸重复序列扩张区。
14.权利要求13的供使用的sgRNA对,其中所述核苷酸重复序列扩张区包含20个或更多的重复序列,例如20至10000个重复序列,更特别是50至5000个重复序列。
15.一种药物组合物,其包含权利要求1至6任一项的sgRNA或sgRNA分子对或权利要求7的载体,或源自于金黄色葡萄球菌的CRISPR/Cas9内切核酸酶,或权利要求8的细胞。
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CN111471712A (zh) * | 2020-04-22 | 2020-07-31 | 江苏同科医药科技有限公司 | CRISPR/Cas9双靶点靶向敲除目的基因载体构建方法 |
WO2021023307A1 (zh) * | 2019-08-08 | 2021-02-11 | 复旦大学 | CRISPR/Cas9基因编辑***及其应用 |
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Publication number | Priority date | Publication date | Assignee | Title |
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RU2725737C2 (ru) | 2015-05-29 | 2020-07-03 | Регенерон Фармасьютикалс, Инк. | Животные, отличные от человека, с нарушением в локусе c9orf72 |
WO2018002812A1 (en) | 2016-06-29 | 2018-01-04 | Crispr Therapeutics Ag | Materials and methods for treatment of myotonic dystrophy type 1 (dm1) and other related disorders |
KR102294755B1 (ko) | 2016-09-30 | 2021-08-31 | 리제너론 파마슈티칼스 인코포레이티드 | C9orf72 유전자좌에서 헥사뉴클레오티드 반복 확장을 갖는 비인간 동물 |
EP3532615A1 (en) | 2016-10-28 | 2019-09-04 | Genethon | Compositions and methods for the treatment of myotonic dystrophy |
KR102443358B1 (ko) | 2017-12-06 | 2022-09-14 | 어비디티 바이오사이언시스 인크. | 근위축증 및 근긴장성 이영양증을 치료하는 조성물 및 방법 |
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CN110337493B (zh) | 2024-03-12 |
JP7211940B2 (ja) | 2023-01-24 |
JP2019532662A (ja) | 2019-11-14 |
WO2018078131A1 (en) | 2018-05-03 |
CA3039733A1 (en) | 2018-05-03 |
US20190256868A1 (en) | 2019-08-22 |
EP3532614A1 (en) | 2019-09-04 |
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