CN111876421A - 靶向KrasG12D突变转录本的gRNA序列、载体及其应用 - Google Patents
靶向KrasG12D突变转录本的gRNA序列、载体及其应用 Download PDFInfo
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Abstract
一种靶向KrasG12D突变转录本的gRNA序列，其基因序列由gRNA‑DR和间隔序列组成，所述的gRNA‑DR的基因序列如SEQ ID NO.7所示，所述的间隔序列如SEQ ID NO.1～6中的任意一种所示。本发明还提供了一种腺病毒载体，其携带靶向KrasG12D突变转录本的gRNA序列。还提供了一种腺病毒载体，其携带靶向KrasG12D突变转录本的gRNA序列和编码CasRx蛋白的基因序列。本发明还提供了上述的腺病毒载体在制备以KrasG12D作为靶点的治疗癌症的药物中的用途。本发明比之前的shRNA、siRNA的靶向性和特异性均提高。比CRISPR‑Cas9技术具有更低的脱靶效应。
Description
技术领域
本发明属于医学生物领域，涉及一种载体，具体来说是一种靶向KrasG12D突变转录本的gRNA序列、携带CasRx系统的腺相关病毒载体的构建方法和应用。
背景技术
基因改变(例如突变，扩增，重排等)通常导致癌基因的活化，从而驱动癌症的发展。在癌基因中，KRAS突变可能是人类癌症中最普遍的,其编码一种名为Kras的小GTP酶。Kras突变发生在大约90％的胰腺癌，30％到40％的结肠癌，15％到20％的肺癌以及其他的癌症类型中。Kras突变异常激活下游信号通路，从而有助于促进和维持癌症恶性肿瘤。与其他成功的抗肿瘤靶向抑制剂不同，尽管经过数十年的努力，临床上仍缺乏针对突变Kras药物的开发。目前，开发KrasG12C特异性抑制剂的进展虽然带来了希望，但尚无有效的针对KrasG12D的抑制剂。需要指出的是，一般情况下，KrasG12D比KrasG12C发生的频率更高。
胰腺癌是恶性程度非常高的肿瘤，其5年生存率低于10％。手术和辅助行化疗是目前最主要的治疗手段。同时，胰腺癌患者对靶向治疗或免疫治疗不获益。因此，开发新的治疗手段对与提高胰腺癌患者的预后非常重要。在胰腺癌中，Kras的突变常常发生，其突变频率为90％左右。
Kras突变是一种功能激活型突变，是驱使胰腺癌恶性化的一个重要因素。Kras突变中又以第12位氨基酸G突变位D为主(KrasG12D)，其次是KrasG12V和KrasG12C。因此，KrasG12D是胰腺癌中潜在的一个重要的治疗靶点。
目前临床上，开发针对KrasG12C的抑制剂在肺癌中的取得了重要进展，但是以KrasG12D作为靶点的治疗仍然缺乏。目前靶向KrasG12D的策略是基因编辑或者敲低。比如，基于CRISPR-Cas9，或者siRNA都被报道可以靶向突变KrasG12D的并且在体内体外抑制KrasG12D肿瘤包括胰腺癌的恶行进展。然而，这些现存的手段都面临一个严重的问题，即脱靶效应。脱靶效应可能造成严重的副作用，因此大大限制了其在临床中的应用。因此，开发新的靶向突变KrasG12D的方法对与胰腺癌的临床治疗具有极其重要的意义。
目前，具有转录组编辑能力的CRISPR-Cas13家族的蛋白显示出很好的应用潜力。但是，CasRx是否能被用来靶向突变的Kras转录本仍然不清楚。
发明内容
本发明的目的在于提供一种靶向KrasG12D突变转录本的gRNA序列、携带CasRx系统的腺相关病毒载体的构建方法和应用，所述的这种靶向KrasG12D突变转录本的gRNA序列、携带CasRx系统的腺相关病毒载体的构建方法和应用要解决现有技术中的药物对于以KrasG12D作为靶点的治疗效果不佳的技术问题。
本发明提供了一种靶向KrasG12D突变转录本的gRNA序列，其基因序列由gRNA-DR和间隔序列组成，所述的gRNA-DR的基因序列如SEQ ID NO.7所示，所述的间隔序列如SEQ IDNO.1～6中的任意一种所示。
本发明还提供了一种腺病毒载体，其携带靶向KrasG12D突变转录本的gRNA序列。
本发明还提供了一种腺病毒载体，其携带靶向KrasG12D突变转录本的gRNA序列和编码CasRx蛋白的基因序列,所述的编码CasRx蛋白的基因序列如SEQ ID NO.8所示。
本发明还提供了上述的腺病毒载体在制备以KrasG12D作为靶点的治疗癌症的药物中的用途。
CasRx蛋白已报告在哺乳动物细胞中具有高效的靶向能力且极低的脱靶效应。CasRx蛋白通过与gRNA一起而发挥基因编辑的功能。gRNA主要由DR和spacer两个部分组成，DR序列能够与CasRx蛋白结合，而spacer是一个具有22nt的序列，其与目的RNA序列互补配对。具有转录组编辑能力的CasRx蛋白在gRNA的引导下与目的RNA结合，对目的RNA进行切割，从而达到特异降解目的RNA到目的。
本发明和已有技术相比，其技术进步是显著的。本发明比之前的shRNA、siRNA的靶向性和特异性均提高。比CRISPR-Cas9技术具有更低的脱靶效应。因此，其应用的安全性更高。
附图说明
图1:显示了基于AAV2载体的CasRx-gRNA的构建，EF-1a后面跟着CasRx蛋白，hU6启动子后面跟gRNA序列。
图2：显示了gRNA分别携带6条靶向突变KrasG12D转录本的设计及效果验证。(a)gRNA由DR和spacer区域组成。spacer长度为22bp，6条设计的均靶向突变的Kras转录本。(b)RT-qPCR验证KRAS转录本水平的含量。结果表明6条spacer均可以在PANC-1细胞中靶向并敲低突变KrasG12D转录本。
图3：显示了CasRx-gRNA腺病毒感染的细胞中导致蛋白水平kras的降低及细胞质膜定位的减少。(a)Western-blot验证Kras蛋白水平。(b)灰度定量Western-blot的Kras的结果。(c)IF验证kras蛋白的质膜定位情况。
图4：显示了CasRx-gRNA腺病毒kras敲低，明显抑制kras突变的胰腺癌细胞增殖。(a-b)CCK-8验证细胞增殖。H6c7作为Kras野生型细胞。(c-d)克隆形成实验验证细胞增殖。(e-f)细胞对Gemcitabin敏感性实验。(g-h)小鼠皮下成瘤实验验证。
图5：显示了AAV8携带靶向突变Kras转录本的casRx-gRNA系统在胰腺癌原位模型中有效延长小鼠的生存。(a)图示工作原理。CasRx蛋白和靶向突变Kras转录本的gRNA同时克隆刀AAV2点质粒中，利用AAV8体系在293T细胞中包装出AAV8病毒。此病毒通过腹腔注射到胰腺原位癌模型的老鼠中。(b)小鼠生存时间的统计。(c)小鼠原位胰腺癌图示。(d)肿瘤体积的统计结果。(e)小鼠体重的测量统计结果。
具体实施方式
实施例1靶向KrasG12D转录本的gRNA的spacer序列的设计
靶向KrasG12D转录本的gRNA的一系列spacer序列按以下几个条件进行设计：
1)Spacer序列长度为22bp；
2)其22bp的序列与Kras的mRNA序列互补配对；(Kras的mRNA见NCBI ReferenceSequence:NM_004985.5)
3)Spacer序列必须涵盖Kras的G12D的单个核苷酸突变位点A>G；
4)Spacer的3’端末的一个碱基及/或者后的一个碱基为c。
按以上4个条件，设计的spacer序列如下：
1)cctacgccatcagctccaacta；如SEQ ID NO.1所示。
2)ccatcagctccaactaccacaa；如SEQ ID NO.2所示。
3)cgccatcagctccaactaccac；如SEQ ID NO.3所示。
4)cttgcctacgccatcagctcca；如SEQ ID NO.4所示。
5)ctcttgcctacgccatcagctc；如SEQ ID NO.5所示。
6)cactcttgcctacgccatcagc。如SEQ ID NO.6所示。
实施例2含有靶向KrasG12D转录本的gRNA的腺病毒的构建及鉴定
如图1所示，含有AmpR promoter,AmpR,ori,AAV2 ITR,EF-1αcore promoter,SV40-NLS,RfxCas13d,SV40-NLS,HA,hU6 promoter,gRNA-DR,gRNA(DR+spacer)，hGHpolyAsignal,AAV2 ITR,f1 ori的腺相关病毒载体骨架pAAV-EF1a-Cas13d-HA-U6-sgRNA，这种腺相关病毒载体骨架可以搭载不同的治疗性gRNA并广泛的用于基因治疗领域。
构建成功的载体为:pAAV-EF1a-Cas13d-HA-U6-sgRNA。
其中，以上的spacer序列：(1.cctacgccatcagctccaacta；2.ccatcagctccaactaccacaa；3.cgccatcagctccaactaccac；4.Cttgcctacgccatcagctcca；5.ctcttgcctacgccatcagctc；6.cactcttgcctacgccatcagc)通过BbsI的位点插入。
BbsI(NEB)酶切AAV2腺相关病毒载体骨架载体：BbsI 1ul,10×NEB buffer 2ul,骨架载体1ug,水定容到20ul。37℃1h。1％琼脂糖凝胶电泳15min，150V并回收线性的骨架载体。
以上的spacer序列通过合成正向和反向的DNAoligo(携带BbsI相应的互补序列)在体外变性退火后变成双链DNA后，通过T4连接酶连到骨架载体上。
连接体系：T4连接酶(NEB)1ul,10×buffer,dsDNA oligo(10uM),载体(1uM),水定容到20ul，16℃过夜。将连接产物转化到大肠杆菌Stable3菌株中进行转化，挑单克隆，小提质粒后，通过sanger测序验证正确的质粒。
gRNA-DR的序列为5’-aacccctaccaactggtcggggtttgaaac-3’(如SEQ ID NO.7所示。gRNA-DR和spacer共同组成了gRNA。该序列能够与CasRx蛋白相互作用，帮助CasRx蛋白与gRNA序列结合。
实施例3构建携带CasRx-gRNA的AAV8(滴度大约1*10^13GC/ml)。
将pAAV-EF1a-Cas13d-HA-U6-sgRNA质粒进行腺相关病毒(AAV)包装，并进行滴度检测。
具体方法如下：
通过使用三种AAV质粒(pAAV8-rep/cap-Y-F突变体,pAAV-EF1a-Cas13d-HA-U6-sgRNA/pAAV2-GFP和pHelper(pAAV2-GFP,pAAV8-rep/cap-Y-F突变体,pHelper三种质粒均为市售产品)瞬时转染293T细胞，产生了重组AAV(rAAV)载体。用聚乙烯亚胺以80％汇合度瞬时转染293T细胞。转染后72小时收集细胞，裂解，并用25单位/mL的苯甲酸酶核酸酶处理。随后，通过基于碘克沙醇的梯度密度离心，然后通过柱色谱法纯化重组AAV。然后使用Amicon Ultra 10K离心过滤器(Millipore)在磷酸盐缓冲液中将重组AAV载体浓缩至0.5ml的最终体积。通过实时PCR分析定量含有病毒DNA的AAV载体滴度。
将对照质粒DNA(pAAV-EF1a-Cas13d-HA-U6-sgRNA)的10倍稀释系列用于生成标准曲线以确定AAV载体基因组滴度(GC/ml)。结果表明病毒滴度均大于1E+13GC/ml后。将AAV等分并保存在-80℃直至使用。
实施例4
对PANC-1细胞顺转CasRx的质粒，同时依次顺转一系列的gRNA，其体系如下：lipofectamine3000 7ul,P3000 6ul,opti-MEM 500ul,质粒1ug。室温10min后加入PANC-1上清中，48小时后，利用RT-qPCR验证每个gRNA的是否对突变的KrasG12D转录本有敲低作用。结果表明分别携带以上6个spacer序列的gRNA均在PANC-1细胞中表现出敲低KrasG12D转录本的能力(图2)。
实施例5
对PANC-1细胞加入相应的腺病毒48小时后获得稳定表达CasRx和gRNA的细胞,对细胞进行western-blot检测。结果发现Kras在蛋白水平明显的下降(图3)。
同时，通过CCK-8细胞活力测定(第一天96孔板谱1000个细胞，然后每天CCK-8试剂检测细胞活力)和克隆形成实验(6孔板铺200个细胞，2周后取出上清，用结晶紫对细胞进行染色)，表明PANC-1细胞的增殖能力明显下降。最后，CasRx和gRNA的共表达导致PANC-1对化疗药物Gemcitabine的敏感性提高10倍左右(图4)。
实施例6
将稳转CasRx系统的PANC-1细胞移植到老鼠皮下后(1×106细胞重选于100ul的生理盐水，通过注射器注射进入裸鼠BALB/C皮下)，持续观察，结果发现CasRx和gRNA的共表达导致皮下肿瘤生长严重受阻。且，其对Gemcitabine的敏感性大大提高(图4)。
实施例7
构建As-PC-1的原位胰腺癌模型：大约对As-PC-11×106细胞重选于50ul的生理盐水，从小鼠的右上腹部切开大约1cm左右的口子，用镊子夹出小鼠胰腺，用注射器将As-PC-1细胞注射进入胰腺的尾部。放回胰腺并缝合伤口。15天后，向构建的老鼠原位胰腺癌模型注射大约1*10^11浓度的AAV8CasRx-gRNA。持续观察，发现AAV8CasRx-gRNA显提高了老鼠的生存率。同时，AAV8CasRx-gRNA并没有对老鼠的肝造成可见的损伤，老鼠的体重也没有受到影响(图5)。
序列表
<110>蒋望
<120> 靶向KrasG12D突变转录本的gRNA序列、载体及其应用
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<170>SIPOSequenceListing 1.0
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<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ccatcagctccaactaccac aa 22
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cgccatcagctccaactacc ac 22
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<213> 人工序列(Artificial Sequence)
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cactcttgcctacgccatcagc 22
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<212> DNA
<213> 人工序列(Artificial Sequence)
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aacccctaccaactggtcggggtttgaaac 30
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<212> DNA
<213> Homo sapiens
<400> 8
atcgaaaaaaaaaagtccttcgccaagggcatgggcgtgaagtccacactcgtgtccggc 60
tccaaagtgtacatgacaaccttcgccgaaggcagcgacgccaggctggaaaagatcgtg 120
gagggcgacagcatcaggagcgtgaatgagggcgaggccttcagcgctgaaatggccgat 180
aaaaacgccggctataagatcggcaacgccaaattcagccatcctaagggctacgccgtg 240
gtggctaacaaccctctgtatacaggacccgtccagcaggatatgctcggcctgaaggaa 300
actctggaaaagaggtacttcggcgagagcgctgatggcaatgacaatatttgtatccag 360
gtgatccataacatcctggacattgaaaaaatcctcgccgaatacattaccaacgccgcc 420
tacgccgtcaacaatatctccggcctggataaggacattattggattcggcaagttctcc 480
acagtgtatacctacgacgaattcaaagaccccgagcaccatagggccgctttcaacaat 540
aacgataagctcatcaacgccatcaaggcccagtatgacgagttcgacaacttcctcgat 600
aaccccagactcggctatttcggccaggcctttttcagcaaggagggcagaaattacatc 660
atcaattacggcaacgaatgctatgacattctggccctcctgagcggactgaggcactgg 720
gtggtccataacaacgaagaagagtccaggatctccaggacctggctctacaacctcgat 780
aagaacctcgacaacgaatacatctccaccctcaactacctctacgacaggatcaccaat 840
gagctgaccaactccttctccaagaactccgccgccaacgtgaactatattgccgaaact 900
ctgggaatcaaccctgccgaattcgccgaacaatatttcagattcagcattatgaaagag 960
cagaaaaacctcggattcaatatcaccaagctcagggaagtgatgctggacaggaaggat 1020
atgtccgagatcaggaaaaatcataaggtgttcgactccatcaggaccaaggtctacacc 1080
atgatggactttgtgatttataggtattacatcgaagaggatgccaaggtggctgccgcc 1140
aataagtccctccccgataatgagaagtccctgagcgagaaggatatctttgtgattaac 1200
ctgaggggctccttcaacgacgaccagaaggatgccctctactacgatgaagctaataga 1260
atttggagaaagctcgaaaatatcatgcacaacatcaaggaatttaggggaaacaagaca 1320
agagagtataagaagaaggacgcccctagactgcccagaatcctgcccgctggccgtgat 1380
gtttccgccttcagcaaactcatgtatgccctgaccatgttcctggatggcaaggagatc 1440
aacgacctcctgaccaccctgattaataaattcgataacatccagagcttcctgaaggtg 1500
atgcctctcatcggagtcaacgctaagttcgtggaggaatacgcctttttcaaagactcc 1560
gccaagatcgccgatgagctgaggctgatcaagtccttcgctagaatgggagaacctatt 1620
gccgatgccaggagggccatgtatatcgacgccatccgtattttaggaaccaacctgtcc 1680
tatgatgagctcaaggccctcgccgacaccttttccctggacgagaacggaaacaagctc 1740
aagaaaggcaagcacggcatgagaaatttcattattaataacgtgatcagcaataaaagg 1800
ttccactacctgatcagatacggtgatcctgcccacctccatgagatcgccaaaaacgag 1860
gccgtggtgaagttcgtgctcggcaggatcgctgacatccagaaaaaacagggccagaac 1920
ggcaagaaccagatcgacaggtactacgaaacttgtatcggaaaggataagggcaagagc 1980
gtgagcgaaaaggtggacgctctcacaaagatcatcaccggaatgaactacgaccaattc 2040
gacaagaaaaggagcgtcattgaggacaccggcagggaaaacgccgagagggagaagttt 2100
aaaaagatcatcagcctgtacctcaccgtgatctaccacatcctcaagaatattgtcaat 2160
atcaacgccaggtacgtcatcggattccattgcgtcgagcgtgatgctcaactgtacaag 2220
gagaaaggctacgacatcaatctcaagaaactggaagagaagggattcagctccgtcacc 2280
aagctctgcgctggcattgatgaaactgcccccgataagagaaaggacgtggaaaaggag 2340
atggctgaaagagccaaggagagcattgacagcctcgagagcgccaaccccaagctgtat 2400
gccaattacatcaaatacagcgacgagaagaaagccgaggagttcaccaggcagattaac 2460
agggagaaggccaaaaccgccctgaacgcctacctgaggaacaccaagtggaatgtgatc 2520
atcagggaggacctcctgagaattgacaacaagacatgtaccctgttcagaaacaaggcc 2580
gtccacctggaagtggccaggtatgtccacgcctatatcaacgacattgccgaggtcaat 2640
tcctacttccaactgtaccattacatcatgcagagaattatcatgaatgagaggtacgag 2700
aaaagcagcggaaaggtgtccgagtacttcgacgctgtgaatgacgagaagaagtacaac 2760
gataggctcctgaaactgctgtgtgtgcctttcggctactgtatccccaggtttaagaac 2820
ctgagcatcgaggccctgttcgataggaacgaggccgccaagttcgacaaggagaaaaag 2880
aaggtgtccggcaattcc 2898
Claims (4)
1.一种靶向KrasG12D突变转录本的gRNA序列，其特征在于，其基因序列由gRNA-DR和间隔序列组成，所述的gRNA-DR的基因序列如SEQ ID NO.7所示，所述的间隔序列如SEQ ID NO.1～6中的任意一种所示。
2.一种腺病毒载体，其特征在于，其携带权利要求1所述的靶向KrasG12D突变转录本的gRNA序列。
3.一种腺病毒载体，其特征在于，其携带权利要求1所述的靶向KrasG12D突变转录本的gRNA序列和编码CasRx蛋白的基因序列,所述的编码CasRx蛋白的基因序列如SEQ ID NO.8所示。
4.权利要求2或者3所述的腺病毒载体在制备以KrasG12D作为靶点的治疗癌症的药物中的用途。
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| CN115029346A (zh) * | 2022-04-22 | 2022-09-09 | 复旦大学附属眼耳鼻喉科医院 | 用于靶向敲降Htra2转录本的sgRNA、CRISPR/CasRx系统及应用 |
| CN115029346B (zh) * | 2022-04-22 | 2024-01-02 | 复旦大学附属眼耳鼻喉科医院 | 用于靶向敲降Htra2转录本的sgRNA、CRISPR/CasRx系统及应用 |
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| CN117701532B (zh) * | 2024-02-06 | 2024-04-16 | 深锐(天津)生物医学有限公司 | 用于KRAS-G12D基因编辑的gRNA及包含其的分子系统和应用 |
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