CN113337544B - 表达car和微小rna的逆转录病毒载体及其应用 - Google Patents
表达car和微小rna的逆转录病毒载体及其应用 Download PDFInfo
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Abstract
本发明提供一种表达CAR和微小RNA的逆转录病毒载体，该病毒载体中微小RNA与CAR共表达，所述微小RNA优选是miR155。本发明将miR155与anti‑CD19CAR共表达于CAR‑T细胞，实现miR155对CAR‑T细胞功能的同步调节，构建联合治疗方法。并对miR155anti‑CD19CAR‑T细胞进行体内、外功能验证，证明miR155anti‑CD19CAR‑T细胞在体内、外均具有较好的细胞活性、增殖能力与抗肿瘤能力。
Description
技术领域
本发明涉及肿瘤治疗技术领域，特别涉及表达CAR和微小RNA的逆转录病毒载体及其应用。
背景技术
微小RNA155(microRNA155，miR155)是微小RNA(microRNAs，miRNAs)家族成员之一，在进化过程中高度保守，在转录后水平通过不完全碱基配对识别和结合相应mRNA并将其降解，从而抑制靶mRNA翻译，调节其基因表达，参与调控体内多种生理和病理过程。miR155是最早发现的与炎症反应功能相关的miRNAs之一。
miR155位于人21号染色体短臂21位点，由长链非编码RNA转录本B细胞整合簇(B-cell Integration Cluster，BIC)编码，其表达水平受BIC转录及成熟miRNA加工过程的影响。miR155的产生与成熟过程与绝大多数miRNAs相同：首先，由细胞核内RNA聚合酶II转录为加帽和加多聚腺苷酸尾的初级转录本，然后，经Drosha核糖核酸酶III剪切，产生含茎环结构的前体miRNA，再转运至细胞质，经Dicer核糖核酸酶剪切，产生成熟的miRNA和反义miRNA产物。成熟miRNA参与形成RNA诱导的沉默复合物(RNA-induced silencing complex，RISC)并发挥作用，识别靶mRNA，导致靶mRNA的翻译抑制。
miR155在机体免疫调节中发挥重要作用，参与固有免疫应答，促进巨噬细胞促炎表型的获得，影响树突状细胞迁移、表型和抗原提呈活性，并影响B细胞和T细胞功能，在维持淋巴细胞稳态和正常免疫反应中起着重要作用。miR155在T细胞的激活、增殖、分化发育和凋亡等各方面均发挥一定的调控作用。
miR155能直接靶向抑制CTLA-4从而降低T细胞的CTLA-4表达水平并促进其活化与增殖，直接靶向抑制FOXO3来抑制T细胞凋亡，或直接靶向SHIP-1间接影响FOXO3的活性从而提高活化的T细胞的存活率。
miR155参与调控T细胞的分化与功能，通过靶向抑制幼稚T细胞分化成不同亚群过程中所必需的细胞因子、信号通路及其特异性转录因子，调节基因的表达，从而影响T细胞的分化与功能。多种转录因子参与其中，例如SOCS1、SHIP1、S1PR1、PU.1、c-Maf、STAT、RoR-γt、Fosl2等。
miR155参与调控CD4+T细胞的分化，包括调节Th1/Th2细胞平衡、Th17/Treg细胞平衡、Tfh细胞分化。有研究发现miR155过表达能促进幼稚CD4+T细胞分化为Th1细胞，而miR155缺失能促进Th2细胞的分化。miR155主要通过调节IFN-γ信号而促进Th1细胞分化，同时抑制Th2细胞分化，对T细胞发挥抗肿瘤功能具有重要意义。Th1细胞主要分泌IFN-γ、TNF-α、IL-2等细胞因子，其中IFN-γ和IL-2能促进细胞毒性T淋巴细胞的增殖和分化，增强抗肿瘤免疫应答，TNF-α能诱导靶细胞凋亡，这些与T细胞抗肿瘤功能及T细胞增殖能力密切相关。
miR155在调节CD8+T细胞功能中发挥重要作用，CD8+T细胞即细胞毒性T细胞，主要通过特异性识别内源性抗原肽-MHC 1类分子复合物，释放穿孔素、颗粒溶素、颗粒酶等物质而杀伤靶细胞；表达FasL，与靶细胞表面的Fas结合，通过Fas-FasL途径诱导靶细胞凋亡；分泌TNF-α，与靶细胞表面的TNF受体(tumour-necrosis factor receptor，TNFR)结合，通过TNF-TNFR途径诱导靶细胞凋亡。miR155可通过靶向AP-1家族的转录因子Fosl2而调节衰竭的CD8+T细胞，促进其扩增及维持长期持久性。miR155可通过表观遗传重编程，沉默终端分化的驱动因素，下调Akt抑制剂Ship1促进多梳阻遏复合物2(Polycomb Repressor Complex2，PRC2)相关因子Phf19的表达，间接增强PRC2的活性，以抑制T细胞衰老和功能耗竭，增强CD8+T细胞的抗肿瘤功能。miR155可直接抑制Akt和Stat5信号转导的多个负调节因子的表达，通过直接靶向抑制Akt的负性调节因子Ship-1，以及直接靶向抑制Stat5的负性调节因子：Socs1和Ptpn2，从而增强CD8+T细胞的增殖和持续抗肿瘤效应功能。且miR155直接靶向SHIP-1，还能够调控效应记忆CD8+T细胞形成过程中起关键作用的转录因子T-bet，促进CD8+T细胞分化成效应记忆T细胞及促进CD8+T细胞增殖。
总之，miR155参与调控T细胞的激活、增殖、分化发育和凋亡等各方面，能促进T细胞活化与增殖，抑制T细胞凋亡，调节Th1细胞分化，促进细胞毒性CD8+T淋巴细胞的分化，并增强其抗肿瘤活性和增殖能力。因此，miR155在抗肿瘤免疫治疗中具有较好的前景，对T细胞抗肿瘤功能的增强具有重要意义，需要进一步探索其发挥功能的机制，以利于更好地将其应用于疾病的治疗。
靶向CD19抗原分子的CAR-T细胞疗法虽然已经取得了显著的临床效果，但如何增强效应细胞功能和提高T细胞增殖能力是CAR-T细胞免疫疗法目前面临的最具挑战的问题之一。
发明内容
为了解决上述技术问题，本发明提供将miR155整合到表达CAR(嵌合抗原受体)的逆转录病毒载体中，同时进行基因表达的调节，采用无功能的RNA作为对照。
在一种实施方式中，提供一种表达CAR和微小RNA的逆转录病毒载体，该病毒载体中微小RNA与CAR共表达，所述微小RNA优选是miR155。
在一种实施方式中，将U6启动子、miR155和EF1α启动子整合到所述表达CAR的逆转录病毒载体中，由U6启动子驱动miR155的表达，EF1α启动子驱动抗CAR的表达，所述抗CAR优选是抗CD19 CAR和/或CD38 CAR。
在一种实施方式中，所述逆转录病毒载体中包括依次串联的U6启动子、miR155、EF1α启动子、上游的信号肽和用于检测的myc标签；CD19 CAR抗原结合区域；CD8铰链-跨膜结构域；CD28或4-1BB协同激活结构域和CD3ζ胞内信号传导结构域。
在一种实施方式中，所述CD19 CAR抗原结合区域是抗CD19 CAR的ScFv氨基酸序列SEQ ID NO:1，DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS。
在一种实施方式中，提供一种靶向嵌合抗原受体T细胞，其包括由上述逆转录病毒载体表达的靶向嵌合抗原受体。
在一种实施方式中，提供一种治疗肿瘤的药物，其含有上述的嵌合抗原受体T细胞。
在一种实施方式中，所述肿瘤是多发性骨髓瘤。
在一种实施方式中，提供上述逆转录病毒载体的应用，将编码所述嵌合抗原受体的基因片段插入所述载体中，包装成病毒载体颗粒，感染人T细胞，制备得到嵌合抗原受体T细胞，用于表面CD19阳性和/或CD38的肿瘤治疗。
本发明采用基因工程方法构建MFG-miR155-anti-CD19 CAR逆转录病毒载体质粒，将U6启动子、miR155和EF1α启动子整合到表达CAR的逆转录病毒载体中，由U6启动子驱动miR155的表达，EF1α启动子驱动anti-CD19 CAR的表达。进行逆转录病毒载体包装，收获高滴度的逆转录病毒载体。将包装完成的miR155 anti-CD19 CAR逆转录病毒载体转导人原代T细胞，流式细胞术检测anti-CD19 CAR表达效率达50％以上，qPCR检测miR155表达水平明显增高。这说明成功构建miR155 anti-CD19 CAR-T细胞，miR155可以正常表达，实现miR155对CAR-T细胞功能的同步调节，且同时表达miR155没有影响anti-CD19 CAR的表达。
miR155增强anti-CD19 CAR-T细胞体外抗肿瘤功能。通过流式细胞术凋亡检测、萤光素酶检测、RTCA检测证明miR155能增强anti-CD19 CAR-T细胞体外杀伤肿瘤细胞功能；通过压力测试实验证明miR155能促进anti-CD19 CAR-T细胞在反复抗原刺激下的长期抗肿瘤功能；通过ELISA检测细胞因子，证明miR155能促进anti-CD19 CAR-T细胞杀伤肿瘤细胞时IFN-γ、TNF-α、IL-2的释放水平，从而促进anti-CD19 CAR-T细胞抗肿瘤功能。
miR155增强anti-CD19 CAR-T细胞体外增殖能力。通过细胞计数计算生长倍数、监测生长曲线及CFSE增殖检测实验证明miR155增强anti-CD19 CAR-T细胞的增殖能力。流式细胞术检测miR155 anti-CD19 CAR-T细胞TCM含量增多，亦证明miR155能促进anti-CD19CAR-T细胞记忆表型分化，这与T细胞长期持续性相关。
miR155增强anti-CD19 CAR-T细胞体内抗肿瘤功能。使用免疫缺陷NOD-PrkdcscidIl2rgnull NPG小鼠构建Raji-Luc细胞肿瘤动物模型，miR155 anti-CD19 CAR-T细胞治疗后，通过小动物活体成像、体重监测、外周血T细胞流式细胞术检测等方法，证明miR155anti-CD19 CAR-T细胞在体内显示出良好的抗肿瘤功能，使肿瘤完全清除，并明显延长肿瘤模型小鼠的生存期。虽然miR155 anti-CD19 CAR-T细胞与对照组RNAU6 anti-CAR-T细胞在小动物活体成像生物发光信号强度和存活率上没有明显差异，但在第二次注射CAR-T细胞7天后对小鼠血清IFN-γ的ELISA检测实验，发现miR155共表达anti-CD19CAR-T细胞治疗组小鼠血清IFN-γ水平显著增加，miR155增强了CAR-T细胞体内效应功能。在第52天研究终止时，流式细胞术检测发现miR155 anti-CD19 CAR-T细胞治疗组小鼠外周血T细胞含量明显高于对照组，miR155促进了CAR-T细胞在体内的增殖能力。因此，miR155过表达增强anti-CD19 CAR-T细胞体内抗肿瘤活性及增殖能力。
本发明中miR155 anti-CD19 CAR-T细胞中TCM细胞含量增多，这与T细胞长期持续性相关。
miR155 anti-CD19 CAR-T细胞对肿瘤细胞的杀伤能力增强，在肿瘤细胞刺激下，释放细胞因子IFN-γ、TNF-α、IL-2水平也升高，且记忆细胞表型增多。
总之，本发明将miR155与anti-CD19 CAR共表达于CAR-T细胞，实现miR155对CAR-T细胞功能的同步调节，构建联合治疗方法。并对miR155 anti-CD19 CAR-T细胞进行体内、外功能验证，证明miR155 anti-CD19 CAR-T细胞在体内、外均具有较好的细胞活性、增殖能力与抗肿瘤能力。
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miR155联合anti-CD19 CAR表达质粒结构示意图，其中图1A是未改造的抗CD19CAR表达载体示意图，图1B是改造的对照U6抗CD19CAR表达载体示意图，图1C是U6-miR155-抗CD19CAR表达载体示意图。
图2是逆转录病毒载体表达质粒转染Phoenix-ECO细胞效率检测结果图，其中图2A是pMFG-U6-RNA-EF1α-anti-CD19 CAR质粒结果图，图2B是pMFG-U6-miR155-EF1α-anti-CD19 CAR质粒结果图；
图3是miR155 anti-CD19 CAR-T细胞体外杀伤Raji细胞效率检测(n＝3)结果图，与RNAU6 anti-CD19 CAR-T细胞比较，*P<0.05；
图4体外重复抗原刺激后CAR-T细胞杀伤的压力测试检测(n＝3)结果图，其中4A是第一个共培养周期后细胞杀伤效率检测结果图，4B是第二个共培养周期后细胞杀伤效率检测结果图；4C是第三个共培养周期后细胞杀伤效率检测结果图；与RNAU6 anti-CD19CAR-T细胞比较，**P<0.01；
图5是miR155 anti-CD19 CAR-T细胞杀伤Raji-Luc细胞效率检测(n＝3)结果图，与RNAU6 anti-CD19 CAR-T细胞比较，**P<0.01；
图6是miR155 anti-CD19 CAR-T细胞杀伤SW620细胞效率检测(n＝3)结果图，6A是SW620细胞指数变化图，6B是实验终止时间截点的SW620细胞指数图，与RNAU6anti-CD19CAR-T细胞比较，***P<0.001；
图7是anti-CD19 CAR-T细胞IFN-γ、TNF-α、IL-2释放水平检测(n＝3)结果图，其中7A：IFN-γ；7B：TNF-α；7C：IL-2释放水平；与RNAU6 anti-CD19 CAR-T细胞比较，*P<0.05，**P<0.01，***P<0.001；
图8是CFSE染色法检测CAR-T细胞增殖(n＝3)结果图，其中8A是第0h细胞CFSE信号；8B是第0h细胞CFSE信号MFI；C是第24h细胞CFSE信号；D是第24h细胞CFSE信号MFI；与RNAU6 anti-CD19 CAR-T细胞比较，****P<0.0001；
图9是miR155 anti-CD19 CAR-T细胞增殖计数结果图，其中9A是细胞增殖曲线；9B是细胞存活率曲线；
图10是TCM细胞检测(n＝3)结果，其中10A是CAR-T细胞中TCM细胞群检测；10B是CAR-T细胞中TCM细胞群检测散点图；
图11是NPG小鼠肿瘤生物发光成像面积检测与信号强度检测(n＝6)结果图，其中10A是肿瘤面积检测结果图，10B是肿瘤生物发光整体信号强度检测结果图；
图12是NPG小鼠生存曲线(n＝6)；
图13是NPG小鼠体重变化曲线(n＝6)；
图14血清IFN-γ释放水平检测(n＝6)结果图，与RNAU6 anti-CD19 CAR-T细胞治疗组比较，*P<0.05。
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。
实施例一本发明的CAR结构与质粒图谱
为了使miR155和CAR能够更好地表达，我们将U6启动子和EF1α启动子分别整合到CAR表达载体中，代替长末端重复序列(LTR)来驱动表达，由U6启动子驱动miR155的表达，EF1α启动子驱动anti-CD19 CAR的表达，参见图1，EF1a启动子相比于CMV启动子可在免疫细胞中更持续表达从而延长CAR在T细胞中的表达。图1A是未改造的抗CD19CAR表达载体示意图，图1B是改造的对照U6抗CD19CAR表达载体示意图，图1C是U6-miR155-抗CD19CAR表达载体示意图。图1中MMLV(truncated)是MMLV编码的逆转录病毒辅助DNA。图1中gag基因编码病毒的核心蛋白，SP引导肽，myc蛋白标签和CD19 CAR ScFv是CD19 CAR单链可变区。逆病毒载体由LTR启动表达，可表达不超过6kb的外源片段，在CAR序列前加入U6-shRNA-EF1a，为了使目的片段更好的表达，U6启动子启动shRNA的表达，EF1a启动子启动CD19-CAR的表达。
本发明中靶向CD19的嵌合抗原受体采用鼠源FMC63 CD19单克隆抗体作为单链可变区序列，CD8作为铰链和跨膜区，CD28作为共刺激结构域，CD3ζ作为细胞内激活结构域，且在CAR序列之前，加入一个信号肽和myc标签用来检测转导效率。将U6启动子和EF1α启动子分别整合到CAR表达载体中，代替长末端重复序列(LTR)来驱动抗CD19 CAR的表达，由U6启动子驱动miR155的表达，EF1α启动子驱动anti-CD19 CAR的表达。
抗CD19 CAR的ScFv氨基酸序列：
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS((SEQ ID NO:1))
miR155的碱基序列：
CTGTTAATGCTAATCGTGATAGGGGTTCTTGCCTCCAACTGACTCCTACATATTAGCATTAACAG(SEQID NO:2)
实施例二MFG-U6-miR155-EF1α-anti-CD19 CAR质粒的构建
一.分子克隆中转化与筛选实验
使用DH5α感受态细胞(购自北京天根生化有限公司)筛选阳性克隆。
二.U6-RNA序列引物设计及PCR扩增
构建pMFG-U6-RNA-EF1α-anti-CD19 CAR(MFG为质粒载体)表达质粒作为对照组表达质粒。以人全基因组DNA为模板，设计特异的引物，分别PCR扩增U6启动子序列与RNA序列，然后重叠延伸PCR扩增U6-RNA序列。PCR扩增引物序列见表1，在生工生物工程(上海)股份有限公司进行引物合成。用无核酸酶水将引物稀释至工作浓度10μM以待使用。
采用基因工程方法将U6启动子与无功能RNA克隆入pMD18-T载体，再将EF1α启动子引入U6-RNA序列下游，将U6-RNA-EF1α片段引入pMFG-anti-CD19 CAR载体构建pMFG-U6-RNA-EF1α-anti-CD19 CAR表达质粒，由U6启动子驱动RNA的表达，EF1α启动子驱动anti-CD19 CAR的表达。
表1引物序列
三.miR155引物设计及获取
miRBase数据库(http://www.mirbase.org/cgi-bin/mirna_entry.pl？acc＝MI0000681)检索人miR155碱基序列：5’-CUGUUAAUGCUAAUCGUGAUAGGGGUUUUUGCCUCCAACUGACUCCUACAUAUUAGCAUUAACAG-3’。见表2。采用上下游引物退火的方法获得miR155片段(AgeI/EcoR I)。
表2 miR155引物序列
七.pMFG-U6-miR155-EF1α-anti-CD19 CAR质粒表达验证
用转染试剂FuGene HD将表达质粒转染HEK-293T细胞，放置于37℃CO2培养箱中孵育24h。孵育24h后各组取1×106个细胞用于流式细胞术检测转染效率，取2×106个细胞用于细胞总RNA提取，RT-qPCR检测miR155的表达水平。
八.miR155表达水平检测
将质粒转染或病毒载体转导细胞进行总RNA提取，用特异的Taqman MicroRNA反转录试剂盒进行反转录，反转录反应结束后，进行RT-qPCR反应。数据处理用2-ΔΔct方法计算miR155表达的倍数变化。
九.pMFG-U6-RNA-EF1α-anti-CD19 CAR表达质粒构建
在pMFG-anti-CD19 CAR表达质粒基础上，构建pMFG-U6-RNA-EF1α-anti-CD19CAR表达质粒。首先PCR扩增U6-RNA序列，5’端加入限制性内切酶位点Sal I，3’端加入限制性内切酶位点Pac I、Nhe I、Sal I，克隆入pMD18-T载体，获得pMD18-T-U6-RNA。Pac I及Nhe I双酶切pPB-EF1α-EGFP-SV40-Puro载体获得EF1α片段并连接入pMD18-T-U6-RNA载体，获得pMD18-T-U6-RNA-EF1α质粒。然后使用Sal I酶切pMD18-T-U6-RNA-EF1α质粒获得U6-RNA-EF1α片段，采用同尾酶Xho I连接入pMFG-anti-CD19 CAR质粒，获得pMFG-U6-RNA-EF1α-anti-CD19 CAR表达质粒。
实施例二miR155 anti-CD19 CAR-T细胞的构建
一.miR155 anti-CD19 CAR-T细胞构建
1.人PBMC分离与T细胞活化
采集健康志愿者外周血，用Ficoll密度梯度离心法分离获得PBMC。将PBMC重悬于含有10％FBS的AIMV培养基中，加入CD3单克隆抗体(OKT3)、白细胞介素-2(IL-2)，激活T细胞。
2.CAR-T细胞构建
提取的PBMC细胞体外培养48h后进行逆病毒转导，构建RNAU6 anti-CD19 CAR-T细胞与miR155 anti-CD19 CAR-T细胞，RNAU6 anti-CD19 CAR-T细胞用于对照组实验，转导48h后，采用流式细胞术检测anti-CD19 CAR表达水平。用PE-c-Myc抗体标记CAR分子，使用流式细胞术分析测定CAR转导阳性率。
3.CAR-T细胞病毒载体基因组整合拷贝数检测
qPCR检测CAR-T细胞中病毒载体在基因组的整合拷贝数。首先，提取CAR-T细胞全基因组DNA、未转导的PanT细胞全基因组DNA(作为阴性对照)，以及anti-CD19CAR-T细胞全基因组DNA(作为阳性对照)。以人甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphatedehydrogenase，GAPDH)为内参，jw403与jw404为内参基因扩增引物，jw003与jw004为逆转录病毒载体目的基因扩增引物，参见表3，qPCR检测逆转录病毒载体整合拷贝数。
表3引物序列
4.miR155 anti-CD19 CAR亲嗜性逆转录病毒载体制备
pMFG-U6-miR155-EF1α-anti-CD19 CAR与pMFG-U6-RNA-EF1α-anti-CD19 CAR表达质粒分别转染Phoenix-ECO细胞，制备亲嗜性逆转录病毒载体。转染48h后，收获细胞上清，并流式细胞术检测Phoenix-ECO细胞Myc标签表达水平，结果显示：pMFG-U6-miR155-EF1α-anti-CD19 CAR与pMFG-U6-RNA-EF1α-anti-CD19 CAR能够在Phoenix-ECO细胞中高水平表达，见图2。
5.miR155 anti-CD19 CAR双嗜性逆转录病毒载体制备及载体生产细胞系构建
亲嗜性逆转录病毒载体分别转导PG13细胞，建立稳定生产miR155 anti-CD19 CAR双嗜性逆转录病毒载体的PG13细胞系。流式细胞术检测亲嗜性逆转录病毒载体转导PG13细胞的转导效率，结果显示：PG13细胞转导效率约70％，提示稳定生产miR155anti-CD19 CAR双嗜性逆转录病毒载体的PG13细胞系构建成功。
6.逆转录病毒载体滴度检测
将PG13逆转录病毒载体生产细胞系培养至细胞汇合度约95％时，收获细胞上清，连续收集4天。提取病毒RNA，RT-qPCR检测病毒载体滴度，制作标准曲线，计算病毒载体滴度。
7.miR155 anti-CD19 CAR-T细胞构建及转导效率检测
从健康志愿者捐献的外周血中分离PBMC，T细胞体外培养、活化。分别用miR155anti-CD19 CAR逆转录病毒载体、RNAU6 anti-CD19 CAR逆转录病毒载体转导T细胞，构建miR155 anti-CD19 CAR-T细胞与RNAU6 anti-CD19 CAR-T细胞，流式细胞术检测转导效率，结果显示：miR155 anti-CD19 CAR与RNAU6 anti-CD19 CAR逆转录病毒载体能够成功转导人原代T细胞，转导效率均达到50％以上。提示同时表达miR155没有影响anti-CD19CAR的表达。
8.miR155 anti-CD19 CAR-T细胞整合拷贝数检测
合适的逆转录病毒载体整合拷贝数可能与降低逆转录病毒载体整合突变的几率相关，因此qPCR检测miR155 anti-CD19 CAR-T细胞病毒载体在基因组的整合拷贝数。结果显示：所有的CAR-T细胞逆转录病毒载体整合拷贝数均小于3，anti-CD19 CAR拷贝数为2.26±0.12，RNAU6 anti-CD19 CAR拷贝数为1.81±0.03，miR155 anti-CD19 CAR拷贝数为2.17±0.21。
9.miR155表达水平检测
miR155 anti-CD19 CAR逆转录病毒载体转导人原代T细胞，成功构建miR155anti-CD19 CAR-T细胞后，采用FITC标记的CD19重组抗原分子作为流式抗体来检测anti-CD19 CAR的表达，结果显示：anti-CD19 CAR高效表达。提取细胞总RNA，用特异的TaqmanMicroRNA反转录试剂盒进行反转录，qPCR探针法检测miR155表达水平。结果发现miR155anti-CD19 CAR-T细胞的miR155表达水平明显增高(1.83±0.04:1)。这说明将miR155整合到表达CAR的逆转录病毒载体中、构建miR155 anti-CD19 CAR-T细胞后，能够同时进行基因表达的调节，且不影响CAR的表达。
实施例四.miR155增强anti-CD19 CAR-T细胞抗肿瘤功能的体外研究
一.羧基荧光素二醋酸盐琥珀酰亚胺酯(carboxyfluorescein diacetatesuccinimidyl ester，CFSE)增殖检测实验
从实验组和对照组细胞中取出相同数量的CAR-T细胞，进行CFSE染色，染色结束时，检测CAR-T细胞CFSE信号强度。每组取适量细胞，用APC标记的抗人CD3抗体染色30min后，流式细胞术检测CFSE信号强度，获得CD3-APC阳性细胞中FITC阳性细胞比例及FITC信号平均荧光强度(Mean fluorescence intensity，MFI)。
检测在肿瘤细胞抗原刺激的情况下CAR-T细胞增殖情况：每组取100μL CFSE染色均匀的细胞接种于96孔板，并各加入100μL密度为8×105个/mL的Raji细胞，使效应细胞和靶细胞数量比为1:2，在肿瘤细胞的刺激下培养24h，流式细胞术检测细胞增殖情况。
二.中央记忆T(central memory T，TCM)细胞检测
流式细胞术检测CAR-T细胞中TCM含量，TCM表达CD45RO和CD62L分子阳性。因此采用APC标记的抗人CD3抗体、PE/Cyanine7标记的抗人CD45RO抗体和PE标记的抗人CD62L抗体对CAR-T细胞进行流式细胞染色，分析CD3阳性细胞群中CD45RO和CD62L双阳性TCM细胞群比例。
三.miR155增强anti-CD19 CAR-T细胞体外杀伤肿瘤细胞功能检测
1.miR155 anti-CD19 CAR-T细胞体外杀伤Raji细胞效率检测
将miR155 anti-CD19 CAR-T细胞与Raji细胞按数量比1:16，1:8，1:4，1:2，1:1共培养12h，流式细胞术检测miR155 anti-CD19 CAR-T细胞杀伤Raji细胞的杀伤效率。结果显示：miR155 anti-CD19 CAR-T细胞较RNAU6 anti-CD19 CAR-T细胞杀伤效率明显增强，参见图3和下表。
2.miR155 anti-CD19 CAR-T细胞体外杀伤Raji细胞压力测试检测
将CAR-T细胞与Raji细胞按数量比1:1共培养48h后，流式细胞术检测共培养细胞的CD19分子表达，结果发现miR155 anti-CD19 CAR-T细胞组与RNAU6 anti-CD19CAR-T细胞组均无CD19分子表达阳性的细胞。重复3个周期的共培养，每次共培养周期结束后，进行CAR-T细胞杀伤Raji细胞的流式凋亡检测实验，共检测3次。
三次结果均显示miR155 anti-CD19 CAR-T细胞与RNAU6 anti-CD19 CAR-T细胞杀伤Raji细胞的杀伤效率较无抗原刺激情况下的杀伤效率均有所增强，且miR155 anti-CD19CAR-T细胞较RNAU6 anti-CD19 CAR-T细胞杀伤Raji细胞的杀伤效率有更明显的增强，且三次结果的增强效果依次增强，这说明miR155对anti-CD19 CAR-T细胞长期抗肿瘤功能有益，结果见图4A、4B、4C。
具体参见下表。
3.miR155 anti-CD19 CAR-T细胞体外杀伤Raji-Luc细胞效率检测
将miR155 anti-CD19 CAR-T细胞与稳定表达萤光素酶的Raji-Luc细胞共培养12h后，萤光素酶检测CAR-T细胞杀伤Raji-Luc细胞效率。结果显示miR155 anti-CD19 CAR-T细胞较RNAU6 anti-CD19 CAR-T细胞杀伤Raji-Luc细胞效率明显增强，如图5，具体参见下表。
4.miR155 anti-CD19 CAR-T细胞杀伤SW620细胞效率检测
通过实时细胞无标记分析技术(RTCA)检测CAR-T细胞对肿瘤细胞SW620增殖的影响。检测结束后，数据分析发现，miR155 anti-CD19 CAR-T细胞组靶细胞细胞指数明显低于对照组RNAU6 anti-CD19 CAR-T细胞组，并有统计学差异，见图6，这说明miR155anti-CD19CAR-T细胞杀伤SW620细胞效率明显增强，具体参见下表。
5.miR155 anti-CD19 CAR-T细胞分泌细胞因子水平检测
miR155 anti-CD19 CAR-T细胞与靶细胞共培养(效应细胞:靶细胞＝1:1)12h后，ELISA检测细胞培养上清中IFN-γ、TNF-α、IL-2分泌水平。结果可见：miR155 anti-CD19CAR-T细胞组细胞上清中IFN-γ、TNF-α、IL-2释放水平明显增高，见图7。
6.miR155增强anti-CD19 CAR-T细胞体外增殖能力检测
6.1 CFSE增殖检测实验
对CFSE染色后的细胞进行流式细胞术分析，检测CD3-APC阳性细胞中FITC阳性细胞比例及FITC信号MFI。结果显示：CFSE染色第0h，组间CFSE染色较为均匀，FITC信号MFI无明显差别，如图8A与B。
将CFSE染色均匀的CAR-T细胞分别在有靶细胞抗原刺激情况下(效靶比为1:2)培养24h，流式细胞术检测细胞增殖情况。结果发现，miR155 anti-CD19 CAR-T细胞较RNAU6anti-CD19 CAR-T细胞，FITC信号MFI明显降低，即CFSE信号强度明显降低，miR155 anti-CD19 CAR-T细胞增殖速度明显较快。见图8C与D和下表。
6.2细胞计数法增殖倍数检测
每48h进行细胞计数，并将细胞传代，使其浓度为1×106个/mL，根据细胞增殖记录，制作miR155 anti-CD19 CAR-T细胞增殖曲线与存活率曲线。可见随着细胞培养时间延长，在第14天之后，miR155 anti-CD19 CAR-T细胞增殖能力与存活率较RNAU6 anti-CD19CAR-T细胞有所增强，见图9和下表。
6.3 CD4+T细胞与CD8+T细胞比例检测
CD4+CAR-T细胞和CD8+CAR-T细胞的比例影响CAR-T细胞疗效[42]，确定本研究CAR-T细胞混合液中CD4+T细胞与CD8+T细胞的比例具有一定的意义。结果发现：当只有效应细胞存在时，RNAU6 anti-CD19 CAR-T细胞以及miR155 anti-CD19 CAR-T细胞中CD4+T细胞与CD8+T细胞的比例无明显差别，miR155并没有对CD4+T细胞与CD8+T细胞的比例变化产生明显的影响；当效应细胞与靶细胞(Raji细胞)共培养12h后，RNAU6 anti-CD19 CAR-T细胞与miR155anti-CD19 CAR-T细胞中CD8+T细胞比例均明显增高，但组间无显著性差异。
6.4TCM细胞检测
流式细胞术检测miR155 anti-CD19 CAR-T细胞中TCM细胞含量变化，即CD3阳性细胞群中，CD45RO和CD62L双阳性细胞群比例变化，结果发现：miR155 anti-CD19 CAR-T细胞TCM含量明显增多。见图10。
实施例四miR155增强anti-CD19 CAR-T细胞抗肿瘤功能的体内研究
NPG小鼠尾静脉注射Raji-Luc细胞4天后，小动物活体成像可见肿瘤信号，平均光量子数为(1.53±0.34)×104photons per sec，说明肿瘤动物模型构建成功。
1.miR155 anti-CD19 CAR-T细胞体内抗肿瘤活性检测
小动物活体成像监测NPG小鼠肿瘤生物发光成像信号强度变化。可见模型组和未转导CAR的PanT组小鼠肿瘤信号逐渐增强并伴随小鼠陆续死亡，而RNAU6 anti-CD19CAR-T细胞或miR155 anti-CD19 CAR-T细胞治疗后，肿瘤信号消失，并持续到第52天实验结束时未检测到肿瘤复发，肿瘤面积大小和整体信号强度变化见图11。
2.小鼠生存曲线
第0天尾静脉注射Raji-Luc细胞，持续监测小鼠生存情况，记录小鼠生存曲线，可见CAR-T细胞治疗组小鼠生存时间均明显延长，见图12。
3.小鼠体重监测
监测小鼠体重变化，可见模型组和PanT组小鼠在肿瘤细胞注射后第20天左右，体重开始急剧下降，并随后死亡。而RNAU6 anti-CD19 CAR-T细胞及miR155 anti-CD19CAR-T细胞治疗组小鼠体重变化较平稳，没有降低的趋势，见图13。
4.miR155 anti-CD19 CAR-T细胞体内增殖水平检测
在CAR-T细胞治疗组小鼠外周血中能持续检测到CD3+T细胞至实验终止的第52天。在肿瘤细胞Raji-Luc注射入NPG小鼠后第52天，即第一次注射CAR-T细胞后第48天或第二次注射CAR-T细胞后第41天，使用BV785标记的抗人CD3抗体流式细胞术检测T细胞含量。结果发现：anti-CD19 CAR-T细胞治疗组小鼠外周血中CD3+T细胞在外周血有核细胞比例为(1.29±0.99)％；RNAU6 anti-CD19 CAR-T细胞治疗组为(1.82±0.94)％；miR155 anti-CD19 CAR-T细胞治疗组为(7.12±2.15)％；根据标曲计算出细胞数，即：每100μL外周血中anti-CD19 CAR-T细胞5187.04±8329.14个；RNAU6 anti-CD19 CAR-T细胞7181.21±7951.34个；miR155 anti-CD19 CAR-T细胞51340.34±18192.49个，明显高于对照组RNAU6anti-CD19 CAR-T细胞治疗组。因此，miR155过表达对T细胞体内长期增殖能力有促进功能。
5.miR155 anti-CD19 CAR-T细胞体内释放IFN-γ水平检测
ELISA检测第二次注射CAR-T细胞7天后，NPG小鼠血清中IFN-γ的释放水平。结果可见miR155 anti-CD19 CAR-T细胞治疗组小鼠血清中IFN-γ释放水平明显增高，见图14和下表。
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。
序列表
<110> 北京中医药大学
<120> 表达CAR和微小RNA的逆转录病毒载体及其应用
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Claims (2)
1.一种表达CAR和微小RNA的逆转录病毒载体在制备治疗肿瘤药物中的应用，其特征在于，该病毒载体中微小RNA与CAR共表达，所述微小RNA是miR155；
将U6启动子、miR155和EF1α启动子整合到所述表达CAR的逆转录病毒载体中，由U6启动子驱动miR155的表达，EF1α启动子驱动抗CAR的表达，所述抗CAR是抗CD19 CAR；和
所述逆转录病毒载体中包括依次串联的U6启动子、miR155、EF1α启动子、上游的信号肽和用于检测的myc标签；CD19 CAR抗原结合区域；CD8铰链-跨膜结构域；CD28或4-1BB协同激活结构域和CD3ζ胞内信号传导结构域；
所述CD19 CAR抗原结合区域是抗CD19 CAR的ScFv氨基酸序列SEQ ID NO:1，DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS。
2.根据权利要求1所述的应用，所述肿瘤是多发性骨髓瘤。
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