CN118005749A - 腺相关病毒突变体及其应用 - Google Patents
腺相关病毒突变体及其应用 Download PDFInfo
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- CN118005749A CN118005749A CN202410201063.7A CN202410201063A CN118005749A CN 118005749 A CN118005749 A CN 118005749A CN 202410201063 A CN202410201063 A CN 202410201063A CN 118005749 A CN118005749 A CN 118005749A
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- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于生物技术领域,公开了一种腺相关病毒突变体及其应用。本发明提供了一种腺相关病毒衣壳蛋白突变体,其氨基酸序列为如SEQ ID No.12所示。本发明基于RGD基序进行多肽组合库设计的策略,配合定向筛选的方法,通过单次筛选过程就能发掘出有效的AAV变体,筛选精度高。获得了具有肌肉特异性的AAV突变体,在肌肉组织中效果良好,并且肝嗜性低,特异性良好。本发明为肌肉疾病的基因治疗提供更多有用、可选的载体工具,具有巨大的临床价值和商业应用场景。
Description
本发明专利申请是基于2023年07月21日提交的发明名称为“腺相关病毒突变体及其应用”的中国专利申请号202310898798.5的分案申请。
技术领域
本发明涉及生物医药技术领域,具体涉及一种具有肌肉靶向性的腺相关病毒突变体及其应用。
背景技术
腺相关病毒(Adeno-associated virus,AAV)是一类包裹着线性单链DNA基因组的无包膜小病毒,属于微小病毒科(Parvoviridae)依赖病毒属(Dependovirus),需要辅助病毒(通常为腺病毒)参与复制。AAV基因组为单链DNA片段,包含于非包膜病毒衣壳中,可分成三个功能区域:两个开放阅读框(Rep基因、Cap基因)和末端反向重复序列(ITR)。重组腺相关病毒载体(rAAV)源于非致病的野生型腺相关病毒,由于其具有宿主范围广、非致病性、低免疫原性、长期稳定表达外源基因、良好的扩散性能和物理性质稳定等优点,作为基因转移载体在基因治疗和疫苗研究中得到广泛应用。在医学研究中rAAV被用于多种疾病的基因治疗的研究(包括体内、体外实验),如基因功能研究、构建疾病模型、制备基因敲除鼠等方面。
近年来,基因治疗已成为治疗肌肉类疾病的一种新型方法。以杜氏肌营养不良(DMD)为例,它是一种罕见的致命性神经肌肉遗传病,DMD是由编码肌营养不良蛋白的基因中的改变或突变引起的。DMD的症状通常出现在婴幼儿身上,患者会经历发育迟缓,如行走、爬楼梯或从坐姿站立困难。SRP-9001(AAVrh74.MHCK7.micro-Dystrophin)是Sarepta/罗氏的基因治疗候选产品,利用MHCK7启动子与AAVrh74载体结合,以递送微型抗肌营养不良蛋白转基因,该基因能够产生较小但起作用的肌营养不良蛋白(称为微肌营养不良蛋白),是DMD患者缺乏的功能性蛋白。2023年6月22日,Sarepta Therapeutics宣布其AAV基因疗法Delandistrogene moxeparvovec(SRP-9001)获得FDA批准上市,用于治疗杜氏肌营养不良(DMD),商品名为Elevidys。药物疗效良好,患者肌肉功能得到显著改善。然而,AAV治疗也存在着缺点。例如,靶向性、特异性差,使剂量过高会引起免疫系统的反应,导致副作用出现。此外,高剂量也意味着更高的生产难度以及更高的成本费用。因此开发具有靶向性更高的药物从而降低药物剂量,或者使药物具有更好的特异性从而规避不良反应,是进行AAV血清型改造的主要目的。
综合上述,虽然AAV是目前应用广泛且最安全的基因治疗载体之一,但体内更低剂量的肌肉靶向等方面仍需进一步改进。因此,开发出具有更好治疗效果、降低治疗剂量、减少副作用和使用成本的血清型类型,以满足患者的需求,并推动基于AAV的基因治疗方法朝向规模化、社会化应用。这对于提升基因治疗的效益,服务于广大患者具有重要意义。
发明内容
本发明的目的在于克服现有技术的不足之处而提供一种腺相关病毒突变体及其应用,该腺相关病毒突变体具有肌肉靶向性,特异性高、安全性好。
为实现上述目的,本发明采取的技术方案如下:
第一方面,本发明提供了一种腺相关病毒衣壳蛋白突变体,所述腺相关病毒衣壳蛋白突变体插入有异源肽;所述异源肽的氨基酸序列为如SEQ ID No.1~4中任一所示的序列。
本发明的腺相关病毒衣壳蛋白突变体对不同肌肉组织(股四头肌、肱二头肌和腹肌等)有良好的靶向性,与对照组AAV9相比,肌肉靶向性最多提高了约10倍,并且肝脏嗜性也低于对照组2~3倍,特异性好。
作为本发明所述的腺相关病毒衣壳蛋白突变体的优选实施方式,所述异源肽的插入位点位于腺相关病毒衣壳蛋白氨基酸583和591之间。
作为本发明所述的腺相关病毒衣壳蛋白突变体的优选实施方式,其氨基酸序列为如SEQ ID No.9~12中任一所示的序列。
第二方面,本发明提供了一种编码腺相关病毒衣壳蛋白突变体的核酸,其核苷酸序列包含如SEQ ID No.5~8所示的异源肽核苷酸序列。
作为本发明所述的编码腺相关病毒衣壳蛋白突变体的核酸的优选实施方式,其核苷酸序列为如SEQ ID No.13~SEQ ID No.16中任一所示的序列。
第三方面,本发明提供了一种重组腺相关病毒,包括所述的腺相关病毒衣壳蛋白突变体。
本发明的腺相关病毒衣壳蛋白突变体构建得到的重组腺相关病毒载体特异性更高,安全性更好,应用范围广
作为本发明所述的重组腺相关病毒的优选实施方式,还包括异源目的基因。
作为本发明所述的重组腺相关病毒的优选实施方式,所述异源目的基因编码干扰RNA、适配体、内切核酸酶、指导RNA中任一种基因产物。
第四方面,本发明将所述的腺相关病毒衣壳蛋白突变体、所述的重组腺相关病毒在制备用于将基因产物递送至受试者细胞或组织中的药物或制剂中应用。
作为本发明所述的应用的优选实施方式,所述细胞为肌肉细胞;所述组织为肌肉组织。
第五方面,本发明将所述的腺相关病毒衣壳蛋白突变体、所述的重组腺相关病毒在制备用于预防和/或治疗肌肉类疾病的药物递送工具中应用。
第六方面,本发明将所述的腺相关病毒衣壳蛋白突变体、所述的重组腺相关病毒在制备肌肉类疾病治疗药物中应用。
作为本发明所述的应用的优选实施方式,所述肌肉类疾病包括但不限于杜氏肌营养不良(DMD)、Becker型肌营养不良症(BMD)、X连锁肌小管性肌病(XLMTM)、肢带型肌营养不良(LGMD)、强直性肌营养不良症、面肩肱型肌营养不良症中的任意一种。
与现有技术相比,本发明的有益效果为:
本发明采用基于特定基序的方法构建小型AAV病毒库,通过单次筛选过程就能发掘出有效的AAV变体,筛选精度高,无需进行多轮的重复筛选及验证。本发明获得了4个具有肌肉特异性的AAV9突变体,从mRNA和蛋白表达水平验证了其在股四头肌,肱二头肌,腹肌等肌肉组织中效果良好(与对照组AAV9相比,肌肉靶向性最多提高了约10倍),并且肝嗜性低(低于对照组2~3倍),特异性良好。为肌肉疾病的基因治疗提供更多有用、可选的载体工具,造福于广大患者,具有巨大的临床价值和商业应用场景。
附图说明
图1为不同血清型对C57小鼠肌肉(股四头肌)靶向性分析(3周);图中,A为股四头肌的mRNA相对表达水平,B为股四头肌的蛋白表达水平;
图2为不同血清型对C57小鼠肌肉(肱二头肌)靶向性分析(3周);图中,A为肱二头肌的mRNA相对表达水平,B为肱二头肌的蛋白表达水平;
图3为不同血清型对C57小鼠肌肉(腹肌)靶向性分析(3周);图中,A为腹肌的mRNA相对表达水平,B为腹肌的蛋白表达水平;
图4为不同血清型对C57小鼠肝脏嗜性分析(3周);图中,A为肝脏的mRNA相对表达水平,B为肝脏的蛋白表达水平。
具体实施方式
为更好地说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。本领域技术人员应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
实施例中所用的试验方法如无特殊说明,均为常规方法;所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:新型突变体的筛选
(1)AAV9衣壳蛋白突变体库骨架质粒的构建
AAV9库骨架载体包含MHCK启动子、Intron、突变的AAV9 CAP序列[AAV9CAP序列的T582后序列被去除,T582核酸序列ACA突变为ACT,从而与polyA前段序列构成酶切位点BsrGI(TGTACA),用于后续骨架酶切线性化]和polyA。将上述序列通过基因合成的方式合成,并插入到AAV载体质粒的ITR之间,形成AAV9库骨架载体。
(2)突变Rep-CAP载体的构建
通过在AAV9中CAP序列的VP1、VP2、VP3蛋白的N端引入终止密码子的方式,使Rep-CAP载体正常表达Rep蛋白、AAP蛋白,而不能表达CAP的VP1、VP2、VP3蛋白,从而避免亲本AAV9中CAP序列的污染。将上述序列通过基因合成的方式合成,并插入替换AAV9 Rep-CAP载体的CAP序列。
(3)包含RGD基序的随机多肽载体库的构建
引物设计:AAV9 CAP的N583和A591序列间作为插入和修饰序列,原始序列为N583-HQSAQAQ-A591,在其中不同位点插入替换包含RGD的随机四肽序列形成以下18种组合:N583-HQSAQ(RGD+随机四肽)AQ-A591,N583-HQSAQ(RGD+随机四肽)Q-A591,N583-HQSAQ(RGD+随机四肽)-A591,N583-HQSA(RGD+随机四肽)AQ-A591,N583-HQSA(RGD+随机四肽)Q-A591,N583-HQSA(RGD+随机四肽)-A591,N583-HQS(RGD+随机四肽)AQ-A591,N583-HQS(RGD+随机四肽)Q-A591,N583-HQS(RGD+随机四肽)-A591,N583-HQ(RGD+随机四肽)AQ-A591,N583-HQ(RGD+随机四肽)Q-A591,N583-HQ(RGD+随机四肽)-A591,N583-H(RGD+随机四肽)AQ-A591,N583-H(RGD+随机四肽)Q-A591,N583-H(RGD+随机四肽)-A591,N583-(RGD+随机四肽)AQ-A591,N583-(RGD+随机四肽)Q-A591,N583-(RGD+随机四肽)-A591。其中(RGD+随机四肽)本身也有5种RGD位置组合:RGDXXXX,XRGDXXX,XXRGDXX,XXXRGDX,XXXXRGD。因此总共构成90种序列引物,这些引物作为上游引物与下游引物组成引物对,在AAV9 CAP载体为模板的情况下,扩增出目的片段库,片段库两端具有同源臂,可与酶切后的AAV9库骨架质粒同源重组形成载体库。
具体操作步骤为:以包含AAV9 CAP的载体为模板,利用上述引物PCR扩增获得包含随机序列的片段。将片段进行凝胶电泳和胶回收,得到纯化的核酸片段;将核酸片段通过Gibson同源重组连接的方式连接入AAV9库骨架载体(经过BsrG I酶切和胶回收纯化),连接后的载体通过PCR产物纯化试剂盒进行纯化后,用Plasmid-Safe DNase酶消化,以去除没有连接上的片段;最后再通过PCR产物纯化试剂盒进行纯化后,即得构建好的包含RGD基序的AAV9随机多肽载体库。
(4)AAV9突变体病毒库的构建
将突变的Rep-Cap质粒、AAV9突变体质粒库、pHelper质粒共转于HEK-293T细胞中,采用碘克沙醇梯度超高速离心纯化腺相关病毒,测量病毒滴度在1012GC/mL~1013GC/mL为合适滴度,得AAV9突变体病毒库,放置-80℃备用。
(5)筛选AAV9突变体
(5.1)动物注射及解剖
动物实验使用6~8周龄的C57雄性小鼠,按不同剂量进行分组,每组按每只小鼠分别注射2E10GC、3E9GC病毒库,按照实验组配制相关病毒,在注射21天后进行动物解剖及器官取材(各部位肌肉、心脏、肝脏等),样本取材后立即进行液氮速冻,并用于后续RNA提取实验。
(5.2)总RNA提取和RT-PCR
样品的研磨:提前10min预冷研磨仪并设置好研磨参数。将保存于-80℃冰箱动物组织样品取出,取约50~100mg组织,于无菌培养皿内剪成黄豆粒大小后,转移至1.5mLRNase-free EP管内。按照每50~100mg组织:1mL TransZol Up的比例加入适量的TransZolUp,然后加入干净无菌的3mm研磨钢珠两颗,缠上封口膜。将样品置于24孔研磨适配器内并配平,拧紧螺旋,按下关盖按钮。启动研磨程序,待仪器运行结束后取出样品,观察样品研磨粒度,如无大块组织残留,即可进行后续提取操作。研磨后的样品在4℃,12,000×g转速离心2min,吸取上清转移至新的做好相应标记的1.5mL RNase-free EP管内。
样品总RNA的提取:具体参照TransZol Up Plus RNA Kit(北京全式金,货号:ER501)说明书。每使用1mL TranZol up,加0.2mL RNA Extraction Agent,剧烈震荡5min;12,000×g,4℃离心10min。此时样品分为三层,转移无色的水相于新的1.5mL RNase-freeEP管中,加入等体积的无水乙醇(此时可能会出现沉淀),轻轻颠倒混匀;将得到的溶液和沉淀一起加入离心柱中,12,000×g室温离心30s,弃滤液;加500μL CB9,12,000×g室温离心30s,弃滤液;重复上步骤一次;加入500μLWB9,12,000×g室温离心30s,弃滤液;重复上步骤一次;12,000×g室温离心2min,彻底去除残留的乙醇;将离心柱放入1.5mL RNase-free EP管中,加入30~50μL(视组织大小而定)RNase-free Water在离心柱的中央,室温静置1min;12,000×g室温离心1min,洗脱RNA;
样品核酸浓度测定:使用微量核酸定量仪检测器检测RNA浓度,记录浓度、OD260/280、OD260/230,把RNA保存于-80℃。
RT-PCR:提取RNA样品使用PrimeScriptTM IV 1st strand cDNA Synthesis Mix(Takara,6215A)进行第一链cDNA的合成。随后使用NEB Q5进行2轮PCR扩增(第一轮使用外侧引物扩增;第二轮使用胶回收的第一轮产物作为模板,用NGS引物进行扩增),胶回收对应条带大小的PCR产物送公司进行NGS测序。
NGS测序、数据分析及候选载体的挑选:测序后进行测序数据分析,选择出现频率排行靠前且在多个样本中多次出现的序列作为候选,进行后续AAV突变体的构建和验证工作。
实施例2:构建AAV衣壳蛋白突变体并生产病毒
(1)突变体血清型载体的构建和质粒提取
将AAV9 Rep-CAP质粒用Smi I和BshT I双酶切,凝胶电泳并切下5000bp左右的片段条带进行凝胶回收,得到酶切的骨架片段。
根据突变体1的Cap序列,设计以下引物,具体步骤为:以AAV9的Rep-CAP质粒为模板使用Cap-f+YJ221-R引物进行扩增并胶回收得到目的产物YJ221-1,以AAV9的Rep-CAP质粒为模板使用YJ221-F+cap-r引物进行扩增并胶回收得到目的产物YJ221-2,通过以下步骤和比例混合骨架片段、YJ221-1、YJ221-2,即可重组构建突变体1的Rep-CAP质粒;
根据突变体2的Cap序列,设计以下引物,具体步骤为:以AAV9的Rep-CAP质粒为模板使用Cap-f+YJ222-R引物进行扩增并胶回收得到目的产物YJ222-1,以AAV9的Rep-CAP质粒为模板使用YJ222-F+cap-r引物进行扩增并胶回收得到目的产物YJ222-2,通过以下步骤和比例混合骨架片段、YJ222-1、YJ222-2,即可重组构建突变体2的Rep-CAP质粒;
根据突变体3的Cap序列,设计以下引物,具体步骤为:以AAV9的Rep-CAP质粒为模板使用Cap-f+YJ223-R引物进行扩增并胶回收得到目的产物YJ223-1,以AAV9的Rep-CAP质粒为模板使用YJ223-F+cap-r引物进行扩增并胶回收得到目的产物YJ223-2,通过以下步骤和比例混合骨架片段、YJ223-1、YJ223-2,即可重组构建突变体3的Rep-CAP质粒;
根据突变体4的Cap序列,设计以下引物,具体步骤为:以AAV9的Rep-CAP质粒为模板使用Cap-f+YJ224-R引物进行扩增并胶回收得到目的产物YJ224-1,以AAV9的Rep-CAP质粒为模板使用YJ224-F+cap-r引物进行扩增并胶回收得到目的产物YJ224-2,通过以下步骤和比例混合骨架片段、YJ224-1、YJ224-2,即可重组构建突变体4的Rep-CAP质粒;
根据突变体5的Cap序列,设计以下引物,具体步骤为:以AAV9的Rep-CAP质粒为模板使用Cap-f+YJ225-R引物进行扩增并胶回收得到目的产物YJ225-1,以AAV9的Rep-CAP质粒为模板使用YJ225-F+cap-r引物进行扩增并胶回收得到目的产物YJ225-2,通过以下步骤和比例混合骨架片段、YJ225-1、YJ225-2,即可重组构建突变体5的Rep-CAP质粒;
根据突变体6的Cap序列,设计以下引物,具体步骤为:以AAV9的Rep-CAP质粒为模板使用Cap-f+YJ226-R引物进行扩增并胶回收得到目的产物YJ226-1,以AAV9的Rep-CAP质粒为模板使用YJ226-F+cap-r引物进行扩增并胶回收得到目的产物YJ226-2,通过以下步骤和比例混合骨架片段、YJ226-1、YJ226-2,即可重组构建突变体6的Rep-CAP质粒;
根据突变体7的Cap序列,设计以下引物,具体步骤为:以AAV9的Rep-CAP质粒为模板使用Cap-f+YJ227-R引物进行扩增并胶回收得到目的产物YJ227-1,以AAV9的Rep-CAP质粒为模板使用YJ227-F+cap-r引物进行扩增并胶回收得到目的产物YJ227-2,通过以下步骤和比例混合骨架片段、YJ227-1、YJ227-2,即可重组构建突变体7的Rep-CAP质粒;
根据突变体8的Cap序列,设计以下引物,具体步骤为:以AAV9的Rep-CAP质粒为模板使用Cap-f+YJ228-R引物进行扩增并胶回收得到目的产物YJ228-1,以AAV9的Rep-CAP质粒为模板使用YJ228-F+cap-r引物进行扩增并胶回收得到目的产物YJ228-2,通过以下步骤和比例混合骨架片段、YJ228-1、YJ228-2,即可重组构建突变体8的Rep-CAP质粒;
上述AAV衣壳蛋白突变体的Rep-CAP载体构建中涉及的引物如表1所示:
表1引物序列
取1个干净的200μL PCR管做好标记并放在冰盒上,将上述酶切骨架、各个目的片段,按骨架:片段摩尔比为1:3配制反应液,PCR仪中50℃反应30min进行重组连接。取50μL感受态细胞在冰上解冻,10μL的连接产物与DH5α感受态细胞混合,冰上放置20~30分钟;42℃热激45秒;快速置于冰上冰浴2分钟,加入400μL复苏SOC培养基(不含抗生素),37℃、200rpm培养1h;均匀涂于Amp抗性平板(50μg/mL),37℃培养14个小时。挑选单克隆菌,在4mL液体LB培养基(Amp+抗性)中扩大培养,37℃培养14小时。
菌液经过12000rpm离心1分钟,倒掉上清培养基;加入250μL的buffer P1/RNaseA混合液,高速涡旋重悬细菌;加入250μL的buffer P2,上下颠倒8~10次;加入350μL的buffer P3,立即颠倒混匀8~10次让溶液彻底中和;13000rpm离心10分钟,取上清过柱;12000离心1分钟,倒掉废液,加入500μL的PW1,12000离心1分钟,倒掉废液;加入600μL的PW2,12000离心1分钟,倒掉上清;加入600μL的PW2,12000离心1分钟,倒掉上清;12000rpm空转2分钟;加入55℃预热洗脱液30~50μL,静置2分钟,12000rpm离心1分钟。使用微量核酸定量仪进行浓度检测。
获得质粒经过浓度检测,酶切鉴定的阳性质粒取10μL送测序,阳性质粒保存在-20℃。测序结果表明,获得质粒能够编码变异型衣壳蛋白VP1。最后根据后期测试需要的病毒量提取相关的Helper质粒,各组Rep-Cap质粒(AAV9野生型及AAV9突变体1~8)质粒以及GOI质粒(ssAAV.CAG.Fluc-2a-eGFP.WPRE.SV40pA)。
(2)突变体血清型病毒的包装和纯化
将得到各组(AAV9野生型和AAV9突变体1~8)的Rep-Cap质粒,表达萤火虫荧光素酶(Fluc)和绿色荧光蛋白(EGFP)的GOI质粒,pHelper质粒以合适的量共转于HEK-293T细胞中,采用碘克沙醇梯度超高速离心纯化AAV病毒,测量病毒滴度在1E+12GC/mL~1E+13GC/mL为合适滴度,放置-80℃备用。
实施例3:突变体血清型各指标的对比测试
(1)动物注射及解剖
动物实验使用6~8周龄的C57雄性小鼠,按照设计好的实验组和对照组配制相关病毒,每组按每只小鼠注射2E11GC病毒,在注射3周后进行动物解剖及各器官取材,样本取材后立即进行液氮速冻,并分别用于后续RNA提取和WB检测等实验。
(2)目的基因mRNA表达水平检测
(2.1)总RNA提取及反转录:
样品的研磨:提前10min预冷研磨仪并设置好研磨参数。将保存于-80℃冰箱动物组织样品取出,取约50~100mg组织,于无菌培养皿内剪成黄豆粒大小后,转移至1.5mLRNase-free EP管内。按照每50~100mg组织:1mL TransZol Up的比例加入适量的TransZolUp,然后加入干净无菌的3mm研磨钢珠两颗,缠上封口膜。将样品置于24孔研磨适配器内并配平,拧紧螺旋,按下关盖按钮。启动研磨程序,待仪器运行结束后取出样品,观察样品研磨粒度,如无大块组织残留,即可进行后续提取操作。研磨后的样品在4℃,12,000×g转速离心2min,吸取上清转移至新的做好相应标记的1.5mL RNase-free EP管内。
样品总RNA的提取:具体参照TransZol Up Plus RNA Kit(北京全式金,货号:ER501)说明书。每使用1mL TransZol up,加0.2mL RNA Extraction Agent,剧烈震荡5min;12,000×g,4℃离心10min。此时样品分为三层,转移无色的水相于新的1.5mL RNase-freeEP管中,加入等体积的无水乙醇(此时可能会出现沉淀),轻轻颠倒混匀;将得到的溶液和沉淀一起加入离心柱中,12,000×g室温离心30s,弃滤液;加500μL CB9,12,000×g室温离心30s,弃滤液;重复上步骤一次;加入500μLWB9,12,000×g室温离心30s,弃滤液;重复上步骤一次;12,000×g室温离心2min,彻底去除残留的乙醇;将离心柱放入1.5mL RNase-free EP管中,加入30~50μL(视组织大小而定)RNase-free Water在离心柱的中央,室温静置1min;12,000×g室温离心1min,洗脱RNA;
样品核酸浓度测定:使用微量核酸定量仪检测器检测RNA浓度,记录浓度、OD260/280、OD260/230,把RNA保存于-80℃。
反转录:每组RNA样品使用All-in-One First-Strand cDNASynthesis SuperMix for qPCR(One-Step gDNA Removal)(北京全式金,货号:AE341-03),具体步骤参考说明书。
(2.2)定量PCR(qPCR)实验:
取每组cDNA作为模板,按照2×SYBR Green qPCR Master Mix(Bimake,货号:B21203)说明书进行qPCR体系配置,见表2~4:
表2qPCR体系
试剂 | 使用量 |
2×SYBR Green qPCR Master Mix | 10μL |
cDNA模板 | 1.5μL |
上游引物(10μM) | 1μL |
下游引物(10μM) | 1μL |
ROX Reference Dye | 0.4μL |
去离子水 | Up to 20μL |
表3引物序列
引物名称 | 引物序列(5’->3’) |
Fluc2-qPCR-F1 | AACCAGCGCCATTCTGATCA |
Fluc2-qPCR-R1 | TCGGGGTTGTTAACGTAGCC |
GAPDH-F2 | CAGGAGAGTGTTTCCTCGTCC |
GAPDH-R2 | TTCCCATTCTCGGCCTTGAC |
表4 qPCR程序设置
(2.3)数据分析
根据每组的Ct值,按公式2-ΔΔct计算相对表达量。
(3)WB检测目的蛋白的表达水平
样品预处理:
把组织剪切成细小的碎片,称量并且记录重量后,放置1.5mL或者2mL离心管中,标记管子,在-80℃中冷冻待用,预冷冷冻研磨仪;溶解RIPA(碧云天,P0013B)裂解液(在使用前数分钟内加入PMSF,使PMSF终浓度为1mM);
按照每20mg组织加入150~250μL裂解液的比例加入上述完全裂解液,然后加入两颗已灭菌的氧化锆研磨珠,直接在裂解液中研磨样品(脑、脊髓等组织样品:温度-20℃,频率70Hz,时间每震荡50s停顿10s,循环3~4次;肌肉、肝脏等样品:温度-20℃,频率70Hz,时间每震荡50s停顿10s,5~7次)。样品研磨完后,将样品在冷冻离心机中,4℃,12,000×g离心5~10min,后取上清转移至新的灭菌EP管中,-20℃或-80℃保存;
蛋白浓度测定:
按照改良型BCA法蛋白质浓度测定试剂盒(生工,货号C503051)中的方法测定蛋白浓度后,根据所需用量取适量的蛋白匀浆样品,与相应量的5×SDS-PAGE蛋白上样缓冲液混合,沸水浴10min,冷却后低速离心片刻,等待上样。
WB(Western Blot)检测:
a.SDS-PAGE电泳:根据蛋白浓度及表达水平确定适当的上样量,小于20μL/孔,组织匀浆蛋白上样量约为20~50μg,电泳具体操作流程如下:将预制胶上的梳子拔出,将凝胶安装到电泳槽,内、外槽均加入电泳缓冲液,其中内槽加入新配制的缓冲液,并检漏,若无漏则可在外槽加入电泳缓冲液;取适量处理好的蛋白样品上样,用预染的标准蛋白作为参照,在天能电泳装置上进行100V恒压电泳,电泳时间为100min,直到溴酚蓝到达胶底部。关闭电源,小心卸下预制胶板,取下凝胶,置于转膜缓冲液中等待后续操作;
b.转膜:据胶面积剪取6张滤纸和1张PVDF膜。PVDF膜在甲醇中浸泡5-10sec,然后转移至转膜缓冲液浸泡5min,滤纸也在转膜缓冲液中预湿润;安装转移装置:负极(黑板)-海绵-3层润湿的滤纸-凝胶-PVDF膜-3层润湿的滤纸-海绵-正极(透明板)。赶走每层气泡以避免影响转移效果,夹紧支架,放入电转槽内;使用100V恒压冰浴转膜100min;根据预染蛋白质分子量标准条带是否完全转至PVDF膜来判断转膜成功与否;将转好的PVDF膜浸泡在PBST溶液中室温洗涤5min,按照需求裁剪PVDF膜,裁膜过程中注意勿让PVDF膜变干;
c.封闭及抗体孵育:用封闭液(5%脱脂奶粉)孵育PVDF膜,室温2h或4℃过夜;将封闭好的PVDF膜转入一抗杂交液(Luciferase Rabbit Polyclonal antibody(Proteintech,27986-1-AP)按1:2000;GADPH Rabbit Polyclonal antibody(Proteintech,10494-1-AP)按1:2000;Rabbit GFP tag Polyclonal antibody(Proteintech,50430-2-AP)按1:2000,分别加入至4ml QuickBlockTM Western一抗稀释液(碧云天,P0256)中,即配为一抗杂交液),室温孵育1h或4℃孵育过夜,然后用PBST洗膜,3×5min;将洗涤后的PVDF膜转入二抗杂交液(HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L)(Proteintech,SA00001-2)按1:5000加入至4mL QuickBlockTM Western二抗稀释液(碧云天,P0258)中,即配为二抗杂交液),室温孵育1h,PBST洗膜,3×5min;
d.显色:将ECL化学发光试剂盒的A液与B液等体积混合,震荡混匀后,将发光液滴在PVDF膜上以使PVDF膜上全都覆盖有发光液,调整不同的曝光时间使蛋白条带清晰,仪器拍照。
经上述对不同的筛选突变体与亲本AAV9对照相比,如图1、2和3所示,突变体1、2、4、7对肌肉的靶向能力均有不同程度的提升。其中突变体1和2在股四头肌的mRNA水平较AAV9高8.34倍和10.35倍,突变体4和7则略低,分别为6.52倍和5.55倍。股四头肌的蛋白表达水平,则以突变体1、2和7有较为显著的提升。肱二头肌的蛋白表达结果与股四头肌基本相似,也是突变体1、2和7最为显著。而且其mRNA水平最高的为突变体2和7,为AAV9对照的2.73倍和2.26倍。在腹肌的mRNA表达水平,也是突变体1、2、4和7显著增加,分别达到AAV9对照的2.64倍,3.19倍,2.29倍和3.94倍,蛋白水平基本一致。综合以上3种肌肉组织中的表现,可以确定突变体1、2、4和7对肌肉的靶向性良好,从中我们也发现,虽然候选突变体均含有RGD基序,但是围绕RGD基序附近氨基酸种类及对AAV衣壳蛋白结构的影响,从而使靶向性有所差异。因此对已有确定基序的靶向序列,邻近氨基酸的优化也是一个必要的过程。
为更好的评估突变体在体内的特异性,分析了突变体对肝脏的嗜性。结果如图4所示,大部分突变体不管是mRNA水平还是蛋白质水平,均要低于AAV9对照组,如突变体1、2、4和7的mRNA水平分别为对照组的0.32倍,0.55倍,0.4倍和0.51倍。说明本发明的突变体不单在肌肉的靶向性提高了,而且肝嗜性降低了,特异性的显著提高对于后续的疗效和减少剂量、毒副作用等,都有非常重要的作用。
本发明筛选出的AAV衣壳蛋白突变体1、2、4、7,其VP1的氨基酸序列依次如SEQ IDNo.9~SEQ ID No.12所示、核苷酸序列依次如SEQ ID No.13~SEQ ID No.16所示;VP1中靶向肽的氨基酸序列依次如SEQ ID No.1~SEQ ID No.4所示、核苷酸序列依次如SEQ IDNo.5~SEQ ID No.8所示。具体如下:
SEQ ID No.1(靶向肽1氨基酸序列):QMHRGDA;
SEQ ID No.2(靶向肽2氨基酸序列):RRGDLVG;
SEQ ID No.3(靶向肽4氨基酸序列):SVRGDAS;
SEQ ID No.4(靶向肽7氨基酸序列):DLRSRGD;
SEQ ID No.5(靶向肽1核酸序列(5’->3’)):CAGATGCATAGAGGAGACGCT;
SEQ ID No.6(靶向肽2核酸序列(5’->3’)):CGTAGAGGAGACTTGGTTGGG;
SEQ ID No.7(靶向肽4核酸序列(5’->3’)):TCGGTGAGAGGAGACGCGTCG;
SEQ ID No.8(靶向肽7核酸序列(5’->3’)):GATCTTCGTAGTAGAGGAGAC;
SEQ ID No.9(突变体1VP1氨基酸序列):
MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQMHRGDAAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL*
SEQ ID No.10(突变体2VP1氨基酸序列):
MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSARRGDLVGAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL*
SEQ ID No.11(突变体4VP1氨基酸序列):
MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSSVRGDASAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL*
SEQ ID No.12(突变体7VP1氨基酸序列):
MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQDLRSRGDAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL*
SEQ ID No.13(突变体1VP1核酸序列(5’->3’)):
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACTAACCACCAGATGCATAGAGGAGACGCTGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA;
SEQ ID No.14(突变体2VP1核酸序列(5’->3’)):
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACTAACCACCAGAGTGCCCGTAGAGGAGACTTGGTTGGGGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA;
SEQ ID No.15(突变体4VP1核酸序列(5’->3’)):
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACTAACCACCAGAGTTCGGTGAGAGGAGACGCGTCGGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA;
SEQ ID No.16(突变体7VP1核酸序列(5’->3’)):
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCG
CGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGAC
AACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACA
AGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACC
AGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCA
GGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCC
AAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAA
AGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATC
GGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTC
CCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAA
TGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTA
GTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAG
CACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGC
ACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTT
TGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACA
ACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGT
TACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTC
ACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGC
CGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGC
CAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAAC
GGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTC
ACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCA
AAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCA
GCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGT
CTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGG
CTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGG
AGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAG
ACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACC
CGGTAGCAACGGAGTCCTATGGACAAGTGGCCACTAACCACCAGGATCTTCGTAGTAGAGG
AGACGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGAC
AGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTC
ACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAA
AACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCAT
CACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAAC
AGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTG
AATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA。
综合以上结果,本发明采用基于特定基序的方法构建小型AAV病毒库,通过单次筛选过程就能发掘出有效的AAV变体,筛选精度高,无需进行多轮的重复筛选及验证。基于RGD基序进行多肽组合库设计的策略,配合定向筛选的方法,获得了4个具有肌肉特异性的AAV9突变体,从mRNA和蛋白表达水平验证了其在股四头肌,肱二头肌,腹肌等肌肉组织中效果良好(与对照组AAV9相比,肌肉靶向性最多提高了约10倍),并且肝嗜性低(低于对照组2~3倍),特异性良好。为肌肉疾病的基因治疗提供更多有用、可选的载体工具,造福于广大患者,具有巨大的临床价值和商业应用场景。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (10)
1.一种腺相关病毒衣壳蛋白突变体,其特征在于,其氨基酸序列为如SEQ IDNo.12所示。
2.一种编码腺相关病毒衣壳蛋白突变体的核酸,其特征在于,其核苷酸序列为如SEQID No.16所示。
3.一种重组腺相关病毒,其特征在于,包括权利要求1所述的腺相关病毒衣壳蛋白突变体。
4.根据权利要求3所述的重组腺相关病毒,其特征在于,还包括异源目的基因。
5.根据权利要求4所述的重组腺相关病毒,其特征在于,所述异源目的基因编码干扰RNA、适配体、内切核酸酶、指导RNA中任一种基因产物。
6.权利要求1所述的腺相关病毒衣壳蛋白突变体、权利要求3~5任一项所述的重组腺相关病毒在制备用于将基因产物递送至受试者细胞或组织中的药物或制剂中的应用。
7.根据权利要求6所述的应用,其特征在于,所述细胞为肌肉细胞;所述组织为肌肉组织。
8.权利要求1所述的腺相关病毒衣壳蛋白突变体、权利要求3~5任一项所述的重组腺相关病毒在制备用于预防和/或治疗肌肉类疾病的药物递送工具中的应用。
9.权利要求1所述的腺相关病毒衣壳蛋白突变体、权利要求3~5任一项所述的重组腺相关病毒在制备肌肉类疾病治疗药物中的应用。
10.根据权利要求8或9所述的应用,其特征在于,所述肌肉类疾病包括但不限于杜氏肌营养不良、Becker型肌营养不良症、X连锁肌小管性肌病、肢带型肌营养不良、强直性肌营养不良症、面肩肱型肌营养不良症中的任意一种。
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