CN115029346A - 用于靶向敲降Htra2转录本的sgRNA、CRISPR/CasRx系统及应用 - Google Patents
用于靶向敲降Htra2转录本的sgRNA、CRISPR/CasRx系统及应用 Download PDFInfo
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Abstract
本发明公开了一种用于靶向敲降Htra2转录本的CRISPR/CasRx系统及应用,该系统包括特异性靶向Htra2 mRNA的sgRNA以及CasRx,并通过腺相关病毒载体递送,形成有效敲降Htra2转录本的RNA编辑系统。本发明通过实验评价所述的CRISPR/CasRx系统对新霉素诱导的小鼠听力损失的影响,结果发现该RNA编辑系统能特异性敲降小鼠中的Htra2转录本,且新霉素造模小鼠注射治疗系统后,可观察到听力功能的恢复,同时还观察到内外毛细胞存活率的提高、纤毛形态变规则,直观地验证了本发明提供的AAV‑CasRx治疗系统对氨基糖苷类药物导致的耳毒性损伤的改善作用,可用于制备防治获得性感音神经性聋的药物或保健品。
Description
技术领域
本发明涉及分子生物学领域、医药领域,具体涉及一种用于靶向敲降Htra2转录本的sgRNA、CRISPR/CasRx系统及应用。
背景技术
衰老、噪声、感染及耳毒性药物是引起人类后天获得性感音神经性聋的主要原因,其中,氨基糖苷类药物、顺铂药物等所致的耳毒性损伤在临床治疗中时有发生,如何避免和降低其诱导的内耳毒性一直是亟待解决的问题。然而,目前临床上对后天获得性感音神经性聋的治疗手段十分有限。
CRISPR-Cas9是现目前最强大的基因编辑工具,广泛应用于多种疾病的基因治疗,通过基因编辑的方法对获得性感音神经性耳聋进行干预已有研究报道,然而CRISPR-Cas9系统存在潜在的脱靶风险,Cas9蛋白可能会靶向切割与sgRNA序列相近的DNA序列,且CRISPR-Cas9编辑对基因组DNA的变化是不可逆的,其介导的永久敲除可能存在未知的副作用。此外,虽然RNA干扰(RNAi)可高效切割和抑制靶标转录本,但其会引起特异性的脱靶效应,这样的脱靶效应很难预测,也限制了该技术的实用性,特别是在治疗和临床应用方面。
CRISPR-Cas13是一种新型RNA编辑系统,其中,Cas13蛋白是一种2类VI型的CRISPR-Cas系统效应蛋白,具有RNA介导的RNA酶切活性,是目前第二大类CRISPR-Cas系统发现的唯一能够降解RNA的蛋白,比传统的RNA干预工具具有更高的特异性。Cas13蛋白家族已经鉴定出4个成员,包括Cas13a、Cas13b、Cas13c和Cas13d。有报道称CasRx(RfxCas13d)比其它Cas13s具有更高的活性和特异性。作为目前最小的Cas13酶,CasRx可以很容易地被包装进入AAVs载体,这使得CRISPR-CasRx系统在体内递送变得很方便。CasRx已被应用于肝脏和眼病小鼠模型的治疗工具,与其他基因编辑系统相比,RNA编辑系统可以在不改变基因组的情况下提供更安全的基因沉默方法,适合于后天获得性疾病。
然而,利用CRISPR-CasRx系统恢复体内功能的研究较少,能否利用CRISPR-CasRx系统来提供一种防治后天获得性感音神经性聋的方法,并提高现有基因编辑系统的特异性和效率,成为亟待解决的技术问题。
发明内容
本发明针对现有技术缺乏防治后天获得性感音神经性聋的问题,提供了一种用于靶向敲降Htra2转录本的sgRNA及RNA编辑系统,用以防治由氨基糖苷类药物、噪声等原因导致的耳毒性损伤,可用于制备防治获得性感音神经性聋的药物或保健品。
为解决上述问题,本发明首先提供了一种用于靶向敲降Htra2转录本的sgRNA,所述sgRNA的序列选自SEQ ID NO:1-5所示序列中的任意一条。
优选地,所述sgRNA的序列如SEQ ID NO:1所示。
本发明另一方面还提供了一种用于靶向敲降Htra2转录本的CRISPR/CasRx系统,所述的CRISPR/CasRx系统包含前面任一所述的sgRNA和CasRx蛋白。
优选地,所述的CRISPR/CasRx系统通过腺相关病毒载体递送。
进一步地,所述的腺相关病毒为AAV-PHP.eB病毒。
本发明另一方面还提供了一种用于靶向敲降Htra2转录本的试剂盒,所述试剂盒包含:前面任一项所述的CRISPR/CasRx系统。
本发明另一方面还提供了前面任一项所述的CRISPR/CasRx系统在制备防治获得性感音神经性聋的药物或保健品中的应用。
优选地,所述的获得性感音神经性聋是由氨基糖甙类药物或铂类药物或噪声所致的耳毒性聋。
本发明另一方面还提供了一种防治获得性感音神经性聋的药物组合物,所述的药物组合物包括:前面任一项所述的CRISPR/CasRx系统,以及药学上和生理学上可接受的载体。
相对于现有技术,本发明的有益效果是:
(1)本发明提供了一种用于靶向敲降Htra2转录本的CRISPR/CasRx系统,且通过一系列实验评价其对新霉素诱导的小鼠听力损失的影响,结果发现该系统能在mRNA水平特异性敲降小鼠的Htra2基因,且新霉素造模小鼠注射治疗系统后,可观察到听力功能的恢复,同时还观察到内外毛细胞存活率的提高、纤毛形态变规则,直观地验证了本发明AAV-CasRx治疗系统对氨基糖苷类药物导致的耳毒性损伤的改善作用,可用于制备防治获得性感音神经性聋的药物或保健品。
(2)本发明还经实验证明了AAV-CasRx系统对靶基因的转录本具有高编辑效率和特异性,且对非靶向基因的脱靶效率很低,表明本发明提供的AAV-CasRx系统内耳注射的安全性,这些结果为进一步开展获得性非遗传性聋的临床治疗研究提供了基础和支撑。
附图说明
图1为特异性靶向Htra2的gRNA的筛选;其中:
A为设计靶向Htra2转录本的sgRNA示意图;
B为筛选靶向Htra2转录本的高效特异性sgRNAs的流程图。
图2为CasRx、sgRNA3和Htra2表达质粒转染HEK293细胞后的检测结果;其中:
A为EGFP荧光强度的流式分析结果;
B为不同gRNAs靶向的Htra2在mRNA水平的表达分析结果。
图3为编码CasRx和sgRNA3的AAV载体示意图。
图4为使用AAV-CasRx系统进行体内RNA敲降研究的实验流程图。
图5为sgRNA3在体内编辑RNA的脱靶效率分析。
图6为使用AAV-CasRx系统对新霉素诱导损伤小鼠听力功能的影响;其中:
A为4周龄时注射组和未注射组小鼠内耳的ABR阈值;
B为6周龄时注射组和未注射组小鼠内耳的ABR阈值;
C为4周龄时注射组和未注射组小鼠内耳的DPOAE阈值;
D为6周龄时注射组和未注射组小鼠内耳的DPOAE阈值。
图7为使用AAV-CasRx系统对毛细胞存活率的影响;其中:
A为4周龄时注射组和未注射组小鼠基底膜中Htra2、Caspase3和Caspas9 mRNA的相对表达水平对比;
B为4周龄时注射组和未注射组小鼠基底膜中Htra2蛋白表达水平对比;
C为注射4周后采集的100μm耳蜗切片的共聚焦图像;
D为每100μm耳蜗的顶圈、中圈和底圈毛细胞的数量。
图8为使用AAV-CasRx系统对小鼠毛束形态的影响。
其中,WT表示野生型小鼠,Neo表示未注射AAV-CasRx系统的新霉素造模小鼠,Neo+AAV-CasRx-g3表示注射AAV-CasRx系统的新霉素造模小鼠。
具体实施方式
以下结合附图和实施例对本发明的技术方案做进一步的说明。应当指出,以下实施例仅仅用于对本发明进行解释和说明,并不用于限定本发明。本领域技术人员根据上述发明内容所做出的一些非本质的改进和调整,仍属于本发明的保护范围。
前期研究证实,氨基糖苷类药物引起耳毒性的主要原因是氧化应激反应促进了细胞凋亡,进而导致毛细胞的损伤和听力缺失,这也是顺铂药物及噪声导致耳毒性聋的主要原因。丝氨酸蛋白酶Htra2在维持线粒体稳态和调控细胞凋亡中发挥重要作用,且在药物诱导耳毒性小鼠的内耳组织中高表达,降低Htra2的表达将有效干预氨基糖苷类药物、顺铂药物等原因诱导的耳毒性损伤。此外,腺相关病毒(AAV)已被应用于多项临床实验,并已经获得批准应用于临床,对人体细胞生长、形态或分化无明显病理影响,具有安全性高、免疫原性弱、宿主范围广、物理性质稳定、表达时间长及稳定等优点;且多项研究表明AAV是目前感音神经性耳聋基因治疗的首选载体。由此,本申请发明人试图构建AAV-CasRx系统来敲降Htra2转录本,以调节细胞凋亡,最终达到防治获得性感音神经性聋的目的。
本发明首先提供了一种用于靶向敲降Htra2转录本的sgRNA,所述sgRNA的序列选自SEQ ID NO:1-5所示序列中的任意一条。
优选地,所述sgRNA的序列如SEQ ID NO:1所示。
本发明另一方面还提供了一种用于靶向敲降Htra2转录本的CRISPR/CasRx系统,所述的CRISPR/CasRx系统包含前面任一所述的sgRNA和CasRx蛋白。
优选地,所述的CRISPR/CasRx系统通过腺相关病毒载体递送。
进一步地,所述的腺相关病毒为AAV-PHP.eB病毒。
本发明另一方面还提供了一种用于靶向敲降Htra2转录本的试剂盒,所述试剂盒包含:前面任一项所述的CRISPR/CasRx系统。
本发明另一方面还提供了前面任一项所述的CRISPR/CasRx系统在制备防治获得性感音神经性聋的药物或保健品中的应用。
优选地,所述的获得性感音神经性聋是由氨基糖甙类药物或铂类药物或噪声所致的耳毒性聋。
本发明另一方面还提供了一种防治获得性感音神经性聋的药物组合物,所述的药物组合物包括:前面任一项所述的CRISPR/CasRx系统,以及药学上和生理学上可接受的载体。
合适的药学上可接受的载体是本领域普通技术人员所熟知的。在Remington’sPharmaceutical Sciences中可找到关于药学上可接受的载体的充分说明。在组合物中药学上可接受的载体可含有液体,如水、磷酸盐缓冲液、ringer溶液、生理盐水、平衡盐溶液、甘油或山梨醇等。另外,这些载体中还可能存在辅助性的物质,如润滑剂、助流剂、润湿剂或乳化剂、pH缓冲物质和稳定剂,如白蛋白等。
下面对本发明的实验过程及实验结果进行详细说明,从而证明本发明提供的AAV-CasRx系统对氨基糖苷类药物诱导的耳毒性损伤的防治作用,能够用于制备防治获得性感音神经性聋的药物或保健品。
本发明实施例中无特别说明外,所用试剂及耗材均为市售商品。
本实施例ICR小鼠来源于上海杰思捷实验动物有限公司,实验动物的研究得到复旦大学实验动物伦理委员会和上海市医学实验动物管理委员会的批准。所有动物均在复旦大学实验动物科学系饲养,所有实验程序均按照动物研究的政策和伦理进行。
(一)设计、筛选和构建AAV-CasRx系统及其体外RNA编辑实验
1、sgRNA的设计
在Genebank中找到Htra2基因的序列,针对Htra2基因的不同外显子(Exon)设计了23条sgRNA,sgRNA设计图(图1的A)显示了敲降效率较高(后续实验证明)的5条sgRNA的靶向位置。这5条sgRNA的DNA序列分别为:
sgRNA3:CCCACATTCAATCGTGCCCAGAGATCCGGG(SEQ ID NO:1);
sgRNA10:GCAACAACAAACTCCCCTTGCCGGACATCA(SEQ ID NO:2);
sgRNA11:GGCTTCCCATGGCAACAACAAACTCCCCTT(SEQ ID NO:3);
sgRNA14:CCAGAATTTCCAAAATCAATAGCTGCATCG(SEQ ID NO:4);
sgRNA21:GCCAAGATCACATCACCAGGCCGCAGACCA(SEQ ID NO:5)。
2、质粒的构建
如图1的B所示:本实施例分别构建了三种类型的质粒,分别为:表达CasRx的质粒,表示为CasRx-mCherry质粒;表达sgRNAs的质粒,表示为sgRNA质粒(sgRNA质粒共23个,分别表达前文设计的23条sgRNA);表达Htra2的荧光报告载体,表示为EGFP-Htra2质粒。
3、体外筛选编辑效率最高的sgRNA
为了筛选最佳sgRNA,一方面,在体外通过lipo3000转染系统将CasRx-mCherry质粒和sgRNA质粒导入到HEI-OC1细胞中表达,用以检测内源性Htra2的敲降效率;另一方面,在体外通过lipo3000转染系统将CasRx-mCherry质粒、sgRNA质粒和EGFP-Htra2质粒导入到HEK293T细胞中表达,用以检测外源性Htra2的敲降效率。检测过程和结果如下:
(1)流式细胞术(FACS)
EGFP与Htra2共表达,EGFP的荧光强弱反映了Htra2的表达强弱,间接反映了CasRx系统对Htra2的敲降效率。于是,本实施例在质粒转染培养48h后通过FACS技术测定各组HEK293T细胞的EGFP荧光强度,作为RNA敲降效率的指标。结果如图2的A所示:本发明设计的23条sgRNA均在不同程度成功切割靶基因的转录本,其中效率较高的sgRNA有5条,分别为sgRNA3、sgRNA10、sgRNA11、sgRNA14和sgRNA21,其中效率最高的是sgRNA3。
(2)qRT-PCR定量分析
进一步对靶向切割效率较高的5个sgRNA靶向的Htra2 mRNA表达水平进行分析。通过流式分选收集带mCherry和EGFP荧光的细胞并提取RNA,通过qRT-PCR检测Htra2 mRNA的表达。结果如图2的B所示:sgRNA3组Htra2 mRNA的表达水平最低,与荧光强度测定结果(图2的A)一致。
以上结果表明,sgRNA3是最佳的策略,故使用sgRNA3用于后续实验。需要说明的是,本发明对所述sgRNA的来源没有特殊限制,采用本领域所熟知的sgRNA的来源即可,例如委托基因合成公司合成。
4、AAV-CasRx系统的构建
如图3所示,基于前述研究成果,本实施例将sgRNA3和CasRx蛋白包装入同一个Php.eB-AAV病毒中,成功构建AAV-CasRx治疗系统,用于后续研究。
(二)使用AAV-CasRx系统进行体内RNA编辑实验
1、实验过程
图4中的A图表示为使用AAV-CasRx系统进行体内RNA编辑的实验流程:包装AAV-CasRx病毒治疗系统。C57新生小鼠出生后1天(P1),冰麻实验动物,在显微镜辅助下,暴露术野;缓慢通过内耳注入适量AAV-CasRx病毒治疗系统;注射结束后,缝合皮肤切口并复温,恢复后送回培养。小鼠在P11至P17连续7天皮下注射新霉素Neomycin造模。
后续进行一系列的检测和观察。
2、检测
(1)听力脑干反应(Auditory brainstem response,ABR):在小鼠4周龄和6周龄时,选取合适测听频率(4、8、16、24和32kHz)测试ABR反应,比较注射耳和未注射耳的听力情况。
(2)畸变产物耳声发射(DPOAE)测试:在小鼠4周龄和6周龄时,选取合适测听频率(4、8、16、24和32kHz)测试耳蜗外毛细胞主动发射的声能,评估注射AAV-CasRx治疗系统对外毛细胞功能的影响。
(3)免疫荧光染色计数毛细胞数量:部分内耳注射AAV-CasRx治疗系统的小鼠在4周龄测试ABR后颈椎脱臼处死,取双侧耳蜗,解剖感觉上皮区,通过免疫荧光染色并在激光共聚焦显微镜下拍照(Imaging),并计数毛细胞数量,比较注射耳和未注射耳的耳蜗毛细胞存活情况。其中,本实验使用Myosin7a染色(红色)。
(4)电镜法检测毛细胞纤毛形态:收获6周龄小鼠基底膜,制片后通过电子显微镜进行成像,观察经AAV-CasRx系统治疗后毛细胞纤毛形态的异同。
(5)编辑效率及脱靶检测:取注射AAV-CasRx治疗系统4周后的新霉素损伤模型鼠的双侧耳蜗,提取组织RNA,进行RNAseq深度测序,分析得到AAV-CasRx系统在体内的编辑效率及脱靶效率,同时以未注射AAV-CasRx系统的新霉素损伤耳作为对照。
3、实验结果
(1)AAV-CasRx系统体内RNA敲降的脱靶分析
对AAV-CasRx注射后4周收集的耳蜗进行RNA测序。通过小鼠全转录组比对sgRNA30bp序列,筛选出最可能脱靶的10个基因,并分析注射组(Neo+AAV-CasRx-g3组)与未注射组(Neo组)的表达差异。
结果如图5所示,Top10脱靶位点的表达在注射组与未注射组之间无显著差异,表明本发明提供的AAV-CasRx系统进行RNA敲降时没有脱靶效应,说明该系统内耳注射的安全性。
(2)注射AAV-CasRx系统能防治进行性听力损失
为了研究AAV-CasRx系统对新霉素诱导损伤小鼠听力功能的影响,本实施例记录了ABR,评估注射组小鼠和未注射组小鼠4kHz至32kHz频率范围内的听力功能。结果表明,在注射后4周,与未注射组相比,注射AAV-CasRx系统治疗的小鼠耳蜗在4kHz、8kHz和16kHz的ABR阈值有明显降低,平均阈值下降5~17dB,最好的阈值改善达到40dB(图6的A和B)。
此外,还测量了DPOAE来评估外毛细胞的功能。在注射后4周和6周,与未注射组相比,注射AAV-CasRx系统治疗的小鼠耳朵在8kHz和16kHz具有更低的DPOAE阈值(图6的C和D)。
以上结果表明,本发明AAV-CasRx治疗系统能改善听力功能,有效防治进行性听力损失。
(3)注射AAV-CasRx系统能提高毛细胞存活率
为了评估注射本发明AAV-CasRx系统对毛细胞存活率的影响,我们在小鼠4周龄时取其内耳基底膜,提取RNA并逆转录,通过qRT-PCR分别检测注射组(Neo组)和未注射组(Neo+AAV-CasRx-g3组)小鼠的Htra2、Caspase3和Caspas9的表达水平。结果显示,相较于未注射组小鼠,注射组小鼠的Htra2、Caspase3和Caspas9表达水平均显著降低(图7的A),有效地抑制了毛细胞的凋亡;同时WB结果同样显示相较于未注射组小鼠,注射组小鼠的Htra2蛋白表达水平均显著降低(图7的B)。
另外,我们还在小鼠4周龄对毛细胞进行了计数。结果表明,与未注射组(Neo组)相比,在注射组(Neo+AAV-CasRx-g3组)小鼠耳蜗的顶圈(Apex)和中圈(Mid)观察到更多的内毛细胞和外毛细胞(图7的C);注射组小鼠耳蜗中圈及底圈(Base)每100μm感觉上皮区的毛细胞计数结果分别为42.1±6.4及11.6±2.6个,而未注射组小鼠耳蜗的计数结果分别为26.8±8.5及9.0±1.4个(图7的D)。
以上结果表明,本发明AAV-CasRx系统能显著提高新霉素诱导损伤小鼠毛细胞的存活率。
(4)注射AAV-CasRx系统能改善毛束形态
毛细胞上的静纤毛束负责声音检测,静纤毛的形态缺陷会导致耳聋。因此,在本发明中,采用电子显微镜扫6周龄的治疗组和未治疗组小鼠基底膜,以评估毛束形态,同时扫描野生型小鼠基底膜作对照。结果如图8所示,野生型小鼠基底膜外毛细胞整齐有序地排列成3层;Neo组毛细胞大量丢失,且纤毛排列杂乱无序;Neo+AAV-CasRx-g3治疗组毛细胞的排列明显规则有序,且细胞的丢失显著减少。
以上结果表明,本发明AAV-CasRx系统能显著修复新霉素诱导损伤的纤毛束缺失和形变。
综上所述,本发明提供了一种用于靶向敲降Htra2转录本的CRISPR/CasRx系统,且通过新霉素造模小鼠,评价其对新霉素诱导的小鼠听力损失的影响,结果发现该系统能特异性在mRNA水平敲降小鼠中的Htra2基因,且新霉素造模小鼠注射治疗系统后,可观察到听觉功能恢复,同时还观察到内外毛细胞存活率的提高、纤毛形态变规则,直观地验证了本发明AAV-CasRx治疗系统对氨基糖苷类药物导致的耳毒性损伤的改善作用,可制备防治获得性感音神经性聋的药物或保健品。
尽管本发明的内容已经通过上述优选实施例作了详细介绍,但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后,对于本发明的多种修改和替代都将是显而易见的。因此,本发明的保护范围应由所附的权利要求来限定。
Claims (9)
1.一种用于靶向敲降Htra2转录本的sgRNA,其特征在于,所述sgRNA的序列选自SEQ IDNO:1至5所示序列中的任意一条。
2.如权利要求1所述的用于靶向敲降Htra2转录本的sgRNA,其特征在于,所述sgRNA的序列如SEQ ID NO:1所示。
3.一种用于靶向敲降Htra2转录本的CRISPR/CasRx系统,其特征在于,所述的CRISPR/CasRx系统包含权利要求1或2所述的sgRNA和CasRx蛋白。
4.如权利要求3所述的用于靶向敲降Htra2转录本的CRISPR/CasRx系统,其特征在于,所述的CRISPR/CasRx系统通过腺相关病毒载体递送。
5.如权利要求4所述的用于靶向敲降Htra2转录本的CRISPR/CasRx系统,其特征在于,所述的腺相关病毒为AAV-PHP.eB病毒。
6.一种用于靶向敲降Htra2转录本的试剂盒,其特征在于,所述试剂盒包含:权利要求3-5中任一项所述的CRISPR/CasRx系统。
7.权利要求3-5中任一项所述的CRISPR/CasRx系统在制备防治获得性感音神经性聋的药物或保健品中的应用。
8.如权利要求7所述的应用,其特征在于,所述的获得性感音神经性聋是由氨基糖甙类药物或铂类药物或噪声所致的耳毒性聋。
9.一种防治获得性感音神经性聋的药物组合物,其特征在于,所述的药物组合物包括:权利要求3-5中任一项所述的CRISPR/CasRx系统,以及药学上和生理学上可接受的载体。
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