CN117070566A - CD38基因敲除的Raji-Luc细胞系构建方法及其细胞系 - Google Patents
CD38基因敲除的Raji-Luc细胞系构建方法及其细胞系 Download PDFInfo
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Abstract
本发明提供一种CD38基因敲除的Raji‑Luc细胞系构建方法,所述方法包括以下步骤:设计靶向CD38的多个sgRNA序列,用多个sgRNA序列构建多个PB‑CRISPR‑CD38sgRNA质粒,用多个PB‑CRISPR‑CD38sgRNA质粒分别电转染Raji‑Luc细胞,用流式细胞仪检测电转染后Raji‑Luc细胞的CD38敲除效率和利用细胞培养确定细胞生长状态,筛选出最优的sgRNA序列,用最优的sgRNA序列构建的PB‑CRISPR‑CD38sgRNA质粒批量电转染Raji‑Luc细胞,和筛选稳定敲除的单克隆Raji‑Luc细胞株。本发明所构建的CD38基因敲除的Raji‑Luc细胞可以模拟肿瘤复发后抗原低表达甚至不表达的情况,荧光素酶基因的表达让后续研究人员可以在体内环境中对肿瘤进行长期观察和检测。
Description
技术领域
本发明涉及细胞技术领域,特别涉及CD38基因敲除的Raji-Luc细胞系构建方法及其细胞系。
背景技术
淋巴瘤是最常见的血液肿瘤之一,主要分为非霍奇金淋巴瘤(non-hodgkinlymphoma,NHL)和霍奇金淋巴瘤(hodgkin lymphoma,HL)两类。其中,非霍奇金淋巴瘤占所有淋巴瘤的90%,B细胞非霍奇金淋巴瘤的发病率是T细胞淋巴瘤的三倍。其发病率随年龄段呈上升趋势,年龄超过60岁的长者患病的可能性较大,致死率较高。
CD38,也称为环状ADP核糖水解酶,CD38是一种Ⅱ型跨膜糖蛋白,由45kD的单链组成,具有受体和酶双重功能,与其配体CD31结合后可与其他细胞表面受体相互作用,参与细胞与细胞间的黏附,激活细胞内信号传导途径,进而促进细胞增殖或凋亡。在CD38阳性的血液恶性肿瘤中,CD38可以调节肿瘤细胞在通过内皮细胞壁时的迁移活动,并且可以促进肿瘤生长增殖。最重要的CD38在多种血液恶性肿瘤上均匀的表达,且表达量高于正常造血细胞,这使CD38成为一个具有吸引力的治疗靶点。
嵌合抗原受体基因修饰T(CAR-T)细胞疗法是将T细胞进行改造,使其表达一个包括抗原识别域、共刺激域和T细胞激活域的融合蛋白,经过改造的CAR-T细胞可以特异性识别并消除表达靶抗原的肿瘤细胞,这项免疫疗法被誉为未来肿瘤治疗的希望。经美国食品药品监督管理局(FDA)批准上市的CAR-T细胞产品有六种,其中四款的靶点为CD19,CD19靶点研发的巨大成功刺激了其他靶点的研究,但是并不是所有接受治疗的患者。然而,约60%治疗后复发患者的癌细胞会出现CD19表达减少或完全丧失的现象。有临床研究指出,一名B细胞急性淋巴细胞白血病(B-cell Acute Lymphoblastic Leukemia,B-ALL)患者在接受抗CD19 CAR-T细胞注入19天后病情得到了完全缓解,但在治疗后第261天出现病情复发并最终死亡。研究人员发现,该患者复发后的白血病细胞均异常表达抗CD19 CAR,无法检测到CD19的表达。因此,尽管CD19 CAR-T细胞在治疗B细胞恶性肿瘤方面效果显著,但治疗后经常会出现肿瘤复发的现象,而肿瘤复发的主要原因与抗原逃逸有关,可以推论CD38 CAR-T推及临床后也会和CD19 CAR-T一样出现肿瘤复发的现象。
伯基特淋巴瘤(burkitt lymphoma,BL)就是一种侵袭性的B细胞淋巴瘤,属于非霍奇金淋巴瘤。1963年,Pulvertaft RJV在一位11岁男孩的左上颌骨的Burkitt淋巴瘤中分离得到Raji细胞,建立了第一个人类造血系统的连续传代细胞。Raji细胞的CD19和CD38呈双阳性表达,常被用做靶细胞进行非霍奇金淋巴瘤的相关研究。本发明通过CRISPR cas9技术,构建了Raji-Luc CD38 KO细胞,为后续探索如何解决CAR-T细胞疗法由于抗原免疫逃逸而产生的肿瘤复发问题奠定了基础。
发明内容
为了解决上述技术问题,本发明提供一种CD19基因敲除的Raji-Luc细胞系构建方法,所述方法包括以下步骤:
步骤1:设计靶向CD38的多个sgRNA序列;
步骤2:用步骤1的多个sgRNA序列构建多个PB-CRISPR-CD38 sgRNA质粒;
步骤3:用步骤2的多个PB-CRISPR-CD38 sgRNA质粒分别电转染Raji-Luc细胞;
步骤4:用流式细胞仪检测步骤3得到的电转染后Raji-Luc细胞的CD38敲除效率和利用细胞培养确定细胞生长状态,筛选出最优的sgRNA序列;
步骤5:利用最优的sgRNA序列构建的PB-CRISPR-CD38 sgRNA质粒批量电转染Raji-Luc细胞;
步骤6:筛选稳定敲除的单克隆Raji-Luc细胞株。
在一种实施方式中,所述最优的sgRNA序列是SEQ IDNo.1:GCTAGGTCCGAAACATTCCAC。
在一种实施方式中,在步骤6中,取电转后的Raji-Luc CD38细胞,用染色缓冲液重悬细胞,并向其中加入流式抗体避光染色;染色结束后用流式细胞仪检测细胞的转染效率,进行流式分选,将分选后的细胞极限稀释法筛选稳定敲除的单克隆Raji-Luc细胞株。
在一种实施方式中,提供一种利用上述方法构建获得的CD38基因敲除的Raji-Luc细胞系。
在一种实施方式中,提供一种特异性靶向CD38基因的sgRNA,所述sgRNA的靶向序列为SEQ ID No.1:GGCGGGACATGTTCACCCTGG。
在一种实施方式中,提供一种基于CRISPR特异性靶向CD38基因的sgRNA,所述sgRNA的靶向序列为SEQ ID No.1:GCTAGGTCCGAAACATTCCAC。
在一种实施方式中,提供上述sgRNA在敲除CD38基因或在制备CD38基因敲除细胞系中的应用。
在一种实施方式中,提供一种用于敲除sgRNA基因的试剂盒,其特征在于,所述试剂盒包括上述的sgRNA,或靶向敲除sgRNA基因的打靶载体;所述靶向敲除sgRNA基因的打靶载体包括上述的sgRNA和CRISPR蛋白基因的编码序列。
在一种实施方式中,提供一种基因敲除载体,含有上述的sgRNA序列。
近年来,抗CD19 CAR-T细胞在治疗复发或难治性急性B淋巴细胞白血病和非霍奇金淋巴瘤方面取得了快速而持久的显著疗效。然而,在CAR-T细胞临床治疗的过程中,部分患者出现了最初产生了高应答率,但在后续治疗中治疗失败的现象。2022年,6名患者接受了CARIBOU公司临床研究中第一个具有PD-1敲除的同种异体CAR-T细胞疗法CB-010的治疗(NCT04637763),其中有3名患者在治疗后6个月内出现复发现象。经研究表明,治疗失败的主要原因是由于肿瘤细胞部分或完全丧失靶抗原表达,即出现了抗原逃逸的现象。这种现象的出现,迫使我们去探究如何使CAR-T细胞在肿瘤细胞抗原丢失后仍然对其具有杀伤作用或是寻找更加合适的替代靶点。
本发明通过设计构建PB-CRISPR-CD38 sgRNA质粒,采用电转染的方法构建了CD38敲除的Raji-Luc细胞。通过流式分选筛选得到稳定敲除的单克隆细胞株。并采用荧光素酶化学发光法验证了Raji-Luc CD38 KO两组单克隆细胞与Raji-Luc细胞的荧光素酶表达无显著差异,且不能激活CD38 CAR-T细胞对其进行杀伤。本发明所构建的Raji-Luc CD38 KO细胞可以模拟肿瘤复发后抗原低表达甚至不表达的情况,荧光素酶基因的表达让后续研究人员可以在体内环境中对肿瘤进行长期观察和检测。对后续改善CAR-T细胞治疗过程中的抗原逃逸现象以及治疗霍奇金淋巴瘤提供了细胞模型,为进一步提升CAR-T的治疗能力,提高淋巴瘤患者治愈率和降低肿瘤复发率提供了帮助。
附图说明
为了更清楚地说明本申请实施例中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本申请中记载的一些实施例,对于本领域普通技术人员来说,在不付出创造性劳动的前提下,还可以根据这些附图获得其它的附图。
图1是本发明的PB-CRISPR-CD38 sgRNA构造示意图;
图2是本发明的不同sgRNA电转染结果图;
图3是本发明的不同sgRNA电转染细胞存活率和细胞增值倍数结果图,其中3b是细胞存活率图,3c是细胞增值倍数图;
图4是流式检测Raji-Luc CD38 KO单克隆细胞表面蛋白表达结果图;
图5是Raji-Luc CD38 KO细胞系荧光素酶检测结果图;
图6是Raji-Luc CD38 KO19和22单克隆测序结果图;
图7是流式检测CD19 CAR-T和CD38 CAR-T的转导效率结果图,图7a是CD19CAR和CD38 CAR结构示意图,图7b是CD19 CAR-T和CD38 CAR-T的转导效率结果图;
图8是CAR-T细胞对Raji-Luc及Raji-Luc CD38 KO细胞的杀伤效率检测结果图,其中图8a是Raji-Luc细胞结果图,8b是Raji-Luc CD38 KO细胞结果图。
具体实施方式
为了使本领域技术领域人员更好地理解本申请中的技术方案,下面将结合实施例对本发明作进一步说明,显然,所描述的实施例仅仅是本申请一部分实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例,都应当属于本申请保护的范围。
一.实验材料
Raji-Luc细胞购自北京维通达生物技术有限公司、电转所用的原始质粒pCAG-PBase、PB-CRISPR-cas9、PB-CRISPR-sgrna均为商品化质粒。
RPMI 1640培养基、AIM-V培养基、PBS溶液、青霉素-链霉素溶液、限制性内切酶、T4连接酶、琼脂、酵母提取物、胰蛋白胨、氯化钠、氨苄青霉素、DH5α感受态细胞、50×TAE溶液、DNA marker DNA ladder、10×DNA Loading Buffer和琼脂糖均购自北京兰博利德商贸有限公司。胶回收试剂盒,质粒小提试剂盒、质粒大提试剂盒和DNA纯化试剂盒均购自南京诺唯赞生物科技股份有限公司有限公司。FBS胎牛血清购自北京百诺威生物科技有限公司公司。7-AAD抗体、小鼠抗人MYC抗体、APC小鼠抗人CD19抗体、FITC小鼠抗人CD38抗体购自深圳市达科为生物技术股份有限公司。
二.实验方法
1.构建PB-CRISPR-CD38 sgRNA质粒
sgRNA(small guide RNA)是向导RNA(guide RNA,gRNA),在RNA编辑的过程中引导尿苷残基插入或缺失到动质体(kinetoplastid)中,属于一种小型非编码RNA,可与pre-mRNA配对。在IDT网站设计并验证off-target以及on-traget,选择合适的sgRNA序列由擎科生物进行引物合成。本发明从数量众多的sgRNA序列中综合考虑各种因素,选择了4条的sgRNA序列进行合成。将合成的引物片段退火形成所需的目的片段并进行PCR扩增,将扩增产物进行纯化回收与线性化载体CRISPR-sgRNA-vector在16℃恒温条件下采用T4连接12h,转化至感受态大肠杆菌后扩大培养并提取质粒并进行浓缩,见图1。
表靶向CD38的sgRNA序列
2.Raji-Luc细胞的培养
复苏Raji-Luc细胞,于37℃、5%CO2细胞培养箱中无菌培养,每48h用含10%FBS的RPMI培养基传代一次,使接种密度为5×105个/ml,培养至对数生长期用于后续实验。
3.筛选最优sgRNA序列
将Raji-Luc细胞分为4组,每组1×106个,命名为sgRNA1、sgRNA2、sgRNA3和sgRNA4,300g离心5分钟,弃去上清,加入无血清RPMI培养基重悬,每组加入pCAG-PBase转座酶,PB-CRISPR-cas9及PB-CD38 sgRNA质粒各4μg,总计12μg,混匀后转移至电转杯中。采用伯乐电转仪,选择K562模式进行电转染。电转染48小时后,取适量电转后的4组细胞和未电转的原始Raji-Luc细胞,400g离心5min,去除上清,用适量PBS洗涤细胞,离心,去除上清,分别加入染色缓冲液重悬细胞,APC anti human CD38流式抗体避光染色1h。染色结束后加入PBS洗涤细胞,离心后用PBS重悬,用流式细胞仪检测细胞的CD38敲除效率。不同sgRNA序列电转染结果如图2所示,细胞存活率和增殖情况如图图3b和c所示。
4.电转染PB-CRISPR-CD38 sgRNA1质粒制备Raji-Luc CD38 KO细胞并筛选稳定敲除的单克隆细胞株
取1×107个Raji-Luc细胞,300g离心5分钟,弃去上清,加入含有sgRNA1电转质粒的无血清RPMI培养基重悬,转移至电转杯进行电击操作。电击结束将细胞置于培养箱稳定30min后缓慢滴加双倍血清并放置于37℃、5%CO2细胞培养箱中无菌培养。电转染48小时后,取适量电转后的Raji-Luc CD38 KO细胞和未电转的原始Raji-Luc细胞,400g离心5min,去除上清,用适量PBS洗涤细胞,离心,去除上清,分别加入2%FBS-PBS染色缓冲液重悬细胞,并向其中加入PE anti human CD38,APC anti human CD19和7-AAD流式抗体避光染色1h。染色结束后加入PBS洗涤细胞,离心后用PBS重悬,用流式细胞仪检测细胞的转染效率,将7-AAD-CD38-CD19+的分群设为目标细胞,进行流式分选,将分选后的细胞极限稀释至2.5个/ml,在96孔板中每孔加入200μl筛选单克隆细胞。将96孔板中的单克隆细胞株进行扩大培养,再次取适量单克隆细胞用抗体PE anti human CD38,APC anti human CD19染色,以未转染的Raji-Luc细胞作为阳性对照,对比检测Raji-Luc CD38 KO细胞表面CD19和CD38的表达情况,以确定筛选得到的的细胞是否成功敲除表面CD38抗原。流式细胞术检测电转染效率并筛选稳定敲除的单克隆细胞株。
5.细胞系表面荧光素酶表达的检测
分别取原始的Raji-Luc细胞、Raji-Luc CD38 KO细胞19号单克隆、Raji-LucCD38KO细胞22号单克隆各1×105个,依次梯度稀释为1×104,1×103,1×102,10个/100μL接种于96孔白板中,每孔分别加入100μL Bright-LumiTM萤火虫荧光素酶检测试剂,反应5min后,选择化学发光模式,检测各组各孔细胞的相对发光光度(relative light unit,RLU)。
6.Raji-Luc CD38 KO细胞基因组测序
分别取原始的Raji-Luc细胞、Raji-Luc CD38 KO细胞19号、22号单克隆提取基因组,进行PCR扩增并将产物进行测序,将测序结果与原基因进行比对。
7.制备CD19 CAR-T和CD38 CAR-T细胞
健康志愿者采静脉血10ml,提取PBMC细胞,加入IL-2和OK-T3活化T细胞后培养48h,分为CD19 CAR-T组和CD38 CAR-T组,分别用实验室前期制备的逆转录病毒进行转导,CAR的结构为经典的二代CAR结构并带有检测的MYC标签,获得CD19CAR-T和CD38 CAR-T细胞。转导48h后,分别取适量CD19 CAR-T和CD38 CAR-T细胞,用PE-MYC抗体染色后,流式细胞仪检测转导效率。转导完成后的CAR-T细胞置于37℃、5%CO2细胞培养箱中无菌培养,每48h用含有100U/mL IL-2的AIM-Ⅴ完全培养基传代一次,接种密度为1×106个/ml。
8.荧光素酶化学发光法验证CAR-T对Raji-Luc和Raji-Luc CD38 KO细胞的杀伤作用
取对数生长期的Raji-Luc和Raji-Luc CD38 KO细胞,以4×104个/50μL每孔的密度接种于96孔白板中。各分为三个实验组:靶细胞+Pan-T细胞组、靶细胞+CD19 CAR-T细胞组、靶细胞+CD38 CAR-T细胞组,每组分别设置效靶比为1∶1、1:2、1:4、1:8,各三个复孔。取适量CAR-T和Pan-T细胞,处理至合适密度,在上述96孔板中每孔加入50μL CAR-T细胞或Pan-T细胞,置于37℃、5%CO2细胞培养箱中共培养。培养8h后,分别在各组每孔加入100μLBright-LumiTM萤火虫荧光素酶检测试剂,反应5min后,选择化学发光模式,检测各组各孔细胞的相对发光光度(relative light unit,RLU),计算杀伤效率,计算公式:杀伤效率=1-(实验孔-空白孔)/(最大裂解孔-空白空)×100%。
9.统计学方法
实验数据采用GraphPad Prism 8统计软件进行分析。计量资料以表示,组间比较采用t检验。P<0.05差异有统计学意义,*P<0.05,**P<0.01。
三.实验结果
1.构建PB-CRISPR-CD38 sgRNA质粒
测序结果显示,所构建的4个PB-CRISPR-CD38 sgRNA质粒序列与预期序列一致,成功构建PB-CRISPR-CD38 sgRNA1、PB-CRISPR-CD38 sgRNA2、PB-CRISPR-CD38 sgRNA3和PB-CRISPR-CD38 sgRNA4质粒。
2.不同的sgRNA序列转染效果
电转染48小时后,sgRNA1、sgRNA2、sgRNA3和sgRNA4四组细胞CD38的表达率都比原始Raji-Luc要低,分别为43.6%、37.7%、68.5%和68.8%,见图2,由此可见sgRNA1和sgRNA2的序列靶向性较强。在后续培养中,sgRNA1组的细胞状态恢复最好,增殖速度也较快,培养至第8天时sgRNA2组的细胞已经基本死亡,sgRNA3组虽然存活率超过百分之七十,增殖倍数最高,但是敲除效率低于sgRNA1组,sgRNA4组存活率和增殖倍数均不高。sgRNA1组的增殖倍数为20.26,电转后存活率持续升高,第8天达到了87%,见图3,分别是图3b和3c。由此可见sgRNA1组细胞电转染过后细胞状态最佳,由此选择sgRNA1作为后续构建Raji-LucCD38 KO细胞的质粒序列。
3.获得稳定表达的单克隆细胞
经流式细胞术分选后和有限稀释法获得19、22、31和59号单克隆Raji-Luc CD38KO细胞,结果显示(见图4),分选后所得两组细胞电转导效率均达到99%以上(即CD38阴性,CD19阳性细胞),因此成功制备Raji-Luc CD38 KO细胞。
4.Raji-Luc CD19 KO细胞与原始Raji-Luc细胞的荧光素酶表达无显著差异
经酶标仪检测结果所示(见图5),19号单克隆Raji-Luc CD38 KO细胞和22号单克隆Raji-Luc CD38 KO细胞与原始Raji-Luc细胞相比荧光素酶表达无显著差异,而31和59号表达荧光素酶较弱,因此选择19号和22号继续进行后续实验。
5.Raji-Luc CD19 KO细胞在对应基因位点发生突变
测序结果显示,Raji-Luc CD38 KO 19号单克隆在CD38基因处发生了大片段的碱基丢失,而22号单克隆则为突变了一个碱基,见图6。
6.制备CD19 CAR-T和CD38 CAR-T细胞
如图7所示,经流式细胞仪检测,转导48小时后,CD19 CAR-T组和CD38 CAR-T组(见图7a)均出现明显细胞分群,转导效率分别为72.4%和72.9%,表明CD19 CAR和CD38 CAR分子在T淋巴细胞表面成功表达,成功制备CD19 CAR-T和CD38 CAR-T细胞(见图7b)。
7.Raji-Luc CD19 KO细胞不能激活对应的CAR-T细胞进行杀伤
分别以Raji-Luc细胞和构建所得Raji-Luc CD38 KO细胞为靶细胞,实验室此前构建成功的CD19 CAR-T和CD38 CAR-T细胞为效应细胞,荧光素酶测定杀伤效率实验结果如图8显示,当靶细胞为Raji-Luc细胞时,CD19 CAR-T与CD38 CAR-T细胞杀伤能力相当,且明显强于Pan-T细胞(图8a);当靶细胞为Raji-Luc CD38 KO细胞时,CD38CAR-T细胞与Pan-T细胞的杀伤能力相当,CD19 CAR-T细胞的杀伤能力明显强于Pan-T细胞和CD38 CAR-T细胞(图8b)。该结果表明敲除CD38的Raji-Luc细胞株不能激活对应的CAR-T细胞进行杀伤。
应该理解到披露的本发明不仅仅限于描述的特定的方法、方案和物质,因为这些均可变化。还应理解这里所用的术语仅仅是为了描述特定的实施方式方案的目的,而不是意欲限制本发明的范围,本发明的范围仅受限于所附的权利要求。
本领域的技术人员还将认识到,或者能够确认使用不超过常规实验,在本文中所述的本发明的具体的实施方案的许多等价物。这些等价物也包含在所附的权利要求中。
Claims (9)
1.CD38基因敲除的Raji-Luc细胞系构建方法,其特征在于,所述方法包括以下步骤:
步骤1:设计靶向CD38的多个sgRNA序列;
步骤2:用步骤1的多个sgRNA序列构建多个PB-CRISPR-CD38 sgRNA质粒;
步骤3:用步骤2的多个PB-CRISPR-CD38 sgRNA质粒电转染Raji-Luc细胞;
步骤4:用流式细胞仪检测步骤3得到的电转染后Raji-Luc细胞的CD38敲除效率和利用细胞培养确定细胞生长状态,筛选出最优的sgRNA序列;
步骤5:利用最优的sgRNA序列构建的PB-CRISPR-CD38 sgRNA质粒批量电转染Raji-Luc细胞;
步骤6:筛选稳定敲除的单克隆Raji-Luc细胞株。
2.根据权利要求1所述的方法,其特征在于,所述最优的sgRNA序列是SEQ IDNo.1:GGCGGGACATGTTCACCCTGG。
3.根据权利要求1所述的方法,其特征在于,在步骤6中,取电转后的Raji-Luc CD38细胞,用染色缓冲液重悬细胞,并向其中加入流式抗体避光染色;染色结束后用流式细胞仪检测细胞的转染效率,进行流式分选,将分选后的细胞极限稀释法筛选稳定敲除的单克隆Raji-Luc细胞株。
4.如权利要求1-3任一所述方法构建获得的CD38基因敲除的Raji-Luc细胞系。
5.一种特异性靶向CD38基因的sgRNA,其特征在于,所述sgRNA的靶向序列为SEQ IDNo.1:GGCGGGACATGTTCACCCTGG。
6.基于CRISPR特异性靶向CD38基因的sgRNA,其特征在于,所述sgRNA的靶向序列为SEQID No.1:GGCGGGACATGTTCACCCTGG。
7.如权利要求5所述的sgRNA在敲除CD38基因或在制备CD38基因敲除细胞系中的应用。
8.一种用于敲除sgRNA基因的试剂盒,其特征在于,所述试剂盒包括权利要求5所述的sgRNA,或靶向敲除sgRNA基因的打靶载体;所述靶向敲除sgRNA基因的打靶载体包括权利要求5所述的sgRNA和CRISPR蛋白基因的编码序列。
9.一种基因敲除载体,含有权利要求5所述的sgRNA序列。
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