CN114457054A - 一种cGAS mRNA的制备及作为免疫激活剂的应用 - Google Patents
一种cGAS mRNA的制备及作为免疫激活剂的应用 Download PDFInfo
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Abstract
本申请公开了一种用于体外表达的cGAS mRNA,其表达载体、制备方法以及用途。
Description
技术领域
本发明涉及生物工程领域,具体涉及cGAS mRNA的制备方法及其作为免疫激动剂的应用。
背景技术
近年来,基于体外转录(IVT,in vitro transcription)的信使RNA(mRNA)的治疗正显示出巨大的潜力。它的原理是将体外制备的mRNA包裹成药物递送到体内组织被细胞内吞,外源mRNA到达细胞后被核糖体识别并根据其编码信息瞬时表达出蛋白质,mRNA药物在理论上有诸多优势:相对于蛋白药物,mRNA表达出的功能蛋白使用患者自身的蛋白翻译后修饰系统,解决了一些蛋白的不可成药性问题。相比于DNA类药物需要入核,mRNA不需要入核,不会整合到基因组避免了插入突变;然而mRNA一直受到体外制备、稳定性和递送问题的困扰。直到近几年IVT技术配合化学和酶学加帽法、修饰核苷酸的引入、HPLC纯化技术使得mRNA IVT能有效制备。同时脂质体和脂质纳米颗粒用于mRNA的包裹和递送,可以提高mRNA的稳定性和递送效率。因此mRNA在开发新的疗法方面的潜力已得到越来越多认可[1]。目前有超过25个mRNA药物包括mRNA疫苗,抗肿瘤药物和蛋白替代的临床研究正在展开,在抗击COVID-19斗争中SARS-CoV-2mRNA疫苗的开发速度非常快,世界卫生组织已经批准紧急使用两种SARS-CoV-2mRNA疫苗,mRNA疫苗表现出非常高的保护效率[2-4]。
环鸟苷酸-腺苷酸合成酶(cGAS)-干扰素基因刺激因子(STING)通路,是将DNA感知与诱导强大的先天免疫防御程序联系在一起的关键机制。在这一途径中,cGAS与双链DNA(dsDNA)的结合激活了其催化活性,并导致其产生环鸟甘酸-腺苷酸(cyclic GMP-AMP,cGAMP),这是一种第二信使分子,也是STING的强效激动剂,与cGAMP结合使STING多聚化,构象发生变化,募集TANK-结合激酶1(TBK1)蛋白,磷酸化并激活干扰素调节因子3(IRF3),IRF3入核诱导I型干扰素(IFN)产生,进而激活天然免疫。该通路的激活剂是抗病毒和抗癌干预的潜在靶点。多种cGAMP类似物瘤内给药在黑色素瘤、乳腺癌和结直肠癌的小鼠模型中介导了强大的治疗效果,同时建立了长期免疫记忆[5]。在临床前肿瘤模型中成功与多种免疫治疗药物联合使用,2019年报道的小分子BNBC是cGAS-cGAMP-STING途径的激动剂,不仅可以诱导针对多种病毒的先天抗病毒免疫力,而且还可以刺激适应性免疫应答的激活[6]。这些发现为启动cGAS-STING通路激活剂的临床开发提供了充分的依据。cGAS自然识别需要递送穿过细胞膜的dsDNA分子,因此目前正在致力于开发STING(而不是cGAS)激动剂,而天然STING激动剂是小的环状二核苷酸(CDN),可以很容易地被小分子模仿。局限的是,越来越多的证据表明,以高剂量局部使用STING激动剂可能失效,甚至过度的STING活化介导T细胞毒性效应的淋巴细胞凋亡,对抗癌效果不利[7]。
发明内容
本发明旨在利用mRNA药物的优势,直接通过递送cGAS mRNA识别內源的dsDNA,刺激干扰素的产生,以cGAS-STING通路为抗病毒和抗癌干预的靶点。具体而言,通过以下技术方案解决了本领域的技术问题。
1.一种mRNA,其包含编码cGAS或其活性片段的编码区,其中所述mRNA还包含以下的一种或多种:
5’-帽结构,优选为m7GPPP、m7G-PPPNm或m7G-PPPNmNm;
3’-聚腺苷酸,其包含约25至约400个腺苷酸的序列;
5’-UTR,所述5’-UTR的序列优选如SEQ ID NO:2所示;
3’-UTR,所述3’-UTR的序列优选来源于提供稳定的mRNA的基因的3’UTR,更优选如SEQ ID NO:5所示;
修饰的核苷酸,所述修饰的核苷酸优选自5-甲基-CTP、假-UTP、N1-甲基-假UTP和5-甲氧基-UTP中的一种或多种。
2.项目1所述的mRNA,其中所述cGAS为如SEQ ID NO:3所示的小鼠cGAS或如SEQ IDNO:4所示的人cGAS。
3.编码项目1-2任一项所述的mRNA的多核苷酸。
4.一种包含项目3所述的多核苷酸的表达载体,优选地,所述表达载体还包含启动子序列,优选T7启动子序列,优选地,所述表达载体为质粒。
5.项目1-2任一项所述的mRNA在制备免疫调节药物和/或免疫靶向药物中的用途。
6.项目5所述的用途,其中所述免疫调节药物和/或免疫靶向药物为抗癌或抗病毒药物。
7.项目1-2任一项所述的mRNA在制备免疫佐剂中的用途。
8.一种制备项目1-2任一项的mRNA的方法,其包括将项目4的表达载体引入宿主细胞,在培养基中培养所述宿主细胞,从宿主细胞中分离表达载体,将所述表达载体线性化后进行体外转录,从而得到所述mRNA。
9.项目8所述的方法,其中通过在含有经修饰的核苷酸的体外无细胞转录系统中进行体外转录,得到含有经修饰的核苷酸的mRNA,优选地,所述经修饰的核苷酸是N1-甲基-假UTP。
10.项目8-9任一项所述的方法,其中通过牛痘病毒加帽酶系统对所述转录的mRNA进行加帽反应,使得所述mRNA具有5’帽结构。
本发明的目的在于提供一种体外转录cGAS mRNA,得到含有cGAS mRNA序列的方法。本发明还提供相应的体外转录质粒。
本发明还提供了一种体外转录的cGAS mRNA,该体外转录mRNA是将cGAS的基因片段插入pCR2-Kan质粒中,作为含有体外转录元件的cGAS mRNA序列的体外转录模板。具体是将cGAS基因序列插入一般的商用表达质粒中。其中人cGAS(h-cGAS)基因的NCBI登录号为NC_000006.12(本文中SEQ ID NO:4),蛋白的NCBI登录号为NP_612450.2。小鼠cGAS(m-cGAS)基因的NCBI登录号为NC_000075.7(本文中SEQ ID NO:3),蛋白的NCBI登录号为NP_775562.2。人、小鼠cGAS与NCBI中NP_612450.2蛋白序列相比,体外转录表达的cGAS蛋白分子质量约55kDa,可为进一步的生物学研究创造条件。
本发明所述的“cGAS基因序列”也包括对NC_000006.12或NC_000075.7中一个或多个密码子被编码相同氨基酸的简并密码子所取代后产生的序列。由于密码子的简并性,所以与NC_000006.12或NC_000075.7核苷酸序列同源性低至约70%的简并序列也能编码出相同的氨基酸序列。该术语还包括能在中度严紧条件下,更佳地在高度严紧条件下,与NC_000006.12或NC_000075.7的核苷酸序列杂交的核苷酸序列。该术语还包括与NC_000006.12或NC_000075.7的核苷酸序列的同源性为至少70%,较佳的至少80%,更佳的至少90%的核苷酸序列。
本发明还提供了cGAS mRNA在细胞中作为免疫激动剂的使用方法。用脂质体包裹的cGAS mRNA转染细胞可以增强细胞中mRNA的表达。通过cGAS-STING-IFN通路活化检测免疫激动剂的效果,使用如下两种方法进行检测:1.利用过表达293T-STING-mNeongreen报告系统,在蛋白水平检测STING多聚化;2.利用THP1荧光素酶Lucia报告系统,在转录水平检验IFN的分泌,具体步骤可见实施例3。
本发明还提供所述的cGAS mRNA在联合mRNA类疫苗方面的应用,即利用cGAS mRNA作为疫苗组分。在该应用中体外转录的RNA可以与抗原RNA一同包装,或再与市场一般的常用佐剂一起联合使用,发挥类似佐剂的效果增强疫苗免疫效果。
本发明还提供所述的cGAS mRNA在构建其他核酸类疫苗方面的应用。提供了一种疫苗配方,cGAS的基因片段作为唯一核酸片段或片段之一,构建核酸类疫苗。该应用中,可将cGAS基因片段由人或鼠基因组中扩增得到,或再与市场一般的常用佐剂一起联合使用,增强疫苗免疫效果。
本发明相对于现有技术具有的有益效果如下:
1.本发明所合成的mRNA可以由体外转录进行加帽反应获得,细胞内存在许多RNA酶,它们可从5’端攻击游离的RNA,使mRNA极易降解,当mRNA的5’端加上m7G-PPPNm帽子后,可阻止RNA酶的切割,延长mRNA的半衰期,提高翻译效率,同时真核生物mRNA必需通过5’帽结合蛋白才能接触核糖体从而正确起始翻译。
2.本方法使小鼠cGAS mRNA过表达的C57Bl-6J鼠诱导分化的骨髓来源巨噬细胞(BMDC)中表面的共刺激分子CD86表达增加。
3.本方法体外转录的cGAS mRNA可以在mRNA类疫苗中作为免疫激活剂,作为佐剂与疫苗免疫源RNA一同组装,而不需要引入其他成分。
4.本方法体外转录的cGAS mRNA可以在人源、鼠源的细胞中表达,并且具有生物学活性。联合HT-DNA刺激通路活化使STING多聚,即使在无外源的HT-DNA刺激时,也可以被内源的双链DNA激活,并且活化下游信号通路诱导干扰素产生。短期过表达cGAS mRNA免疫激活程度更高、更长效,相比使用信号通路的激动剂(如cGAMP)作为第二信直接活化STING通路。
附图说明
为了更清楚地说明本发明实施例,下面将对实施例涉及的附图进行简单地介绍。
图1为实施例1设计的模板DNA序列的结构图。
图2显示了实施例2中IVT mRNA转染293T细胞后,Western Blot检测蛋白表达的结果图。
图3为实施例3转染cGAS mRNA的293T-STING-mNeongreen荧光图。
图4A为实施例3中THP1荧光素酶活性光子计数。
图4B为实施例3中THP1荧光素酶显色图。
图5A为实施例4中BMDC成熟标记CD86的荧光强度统计图。
图5B为实施例5中DC2.4吞噬抗原的比例。
具体实施方式
下面结合实施例对本发明进行详细的说明,但本发明的实施方式不限于此,并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。对于本领域技术人员来讲,在不付出创造性劳动的前提下,获得其他的类似的实施例均落入本发明的保护范围。
实施例1 mRNA制备
本发明以人cGAS、小鼠cGAS为例。每种序列将被克隆到mRNA合成载体上:
1.构建体外转录载体:以pCR2-Kan(中国科学技术大学方芳教授实验室惠赠)为质粒载体,选择Cfr42I(SacII)和Bsu15I(ClaI)为目的基因两侧的酶切位点,插入目的基因后序列依次为T7启动子序列(SEQ ID NO:1)、5’UTR序列(SEQ ID NO:2)、cGAS序列、3’UTR序列(SEQ ID NO:5)(质粒酶切连接操作步骤均为本领域常规)。
2.进行质粒模板制备:扩增载体后,通过PCR使载体线性化。正向引物的核苷酸序列如SEQ ID NO:6所示,反向引物的核苷酸序列如SEQ ID NO:7所示。得到DNA模板序列不同元件的信息如表1所示,具体的核苷酸序列小鼠cGAS如SEQ ID NO:3,人cGAS如SEQ ID NO:4所示。
表1.DNA模板序列不同元件的信息
3.核酸纯化:酚/氯仿抽提纯化PCR片段。
以步骤2得到的线性目的基因片段,进一步酚/氯仿抽提纯化,并将其用作体外转录(IVT)的模板,具体为:
(1)加入160μl无RNA酶的水将产物稀释至180μl;
(2)加入20μl 3M的醋酸钠(pH5.2)到稀释后的产物中,用移液器充分混匀;
(3)加入200μl的酚/氯仿混合液(1:1)进行抽提,室温10000rpm离心5min,将上层溶液(水相)转移至新的无RNA酶的EP管中;
(4)加入与水相等体积的氯仿抽提2次,收集上层水相;
(5)加入2倍体积的无水乙醇并混匀,-20℃孵育至少30min,15000rpm离心15min;
(6)弃上清并加入500μl预冷的70%乙醇洗涤RNA沉淀,15000rpm离心,弃上清。重复此步骤一次;
(7)开盖干燥2min,加入20-50μl无RNA酶的水或其他缓冲液溶解RNA沉淀;
(8)-80℃保存待用;
(9)得到高质量的纯化DNA片段;利用纳米光度计(IMPLEN GMBH#T50947)对DNA进行定性和定量分析。
4.体外转录:合成修饰的mRNA,根据体外转录试剂盒T7 High Yield RNATranscription kit(Novoprotein#E131)、N1-甲基假-UTP(Jena Bioscience#NU-890L)。在含有RNA转录酶、缓冲液、核苷三磷酸等条件的体外无细胞系统中,以DNA为模板转录得到mRNA。
IVT操作如下:
(1)冰上融化表2中的各组分,室温下混合,反应体系置于37℃PCR仪中孵育3h。
表2.体外转录体系(20μl)
(2)先混匀其他组分,最后加入T7 RNA聚合酶混合物,轻轻混匀。
(3)转录完成后每1μg模板DNA加入1μl DNA酶Ⅰ,37℃孵育15min,以去除DNA模板。
5.酚/氯仿抽提纯化体外转录mRNA产物:
具体方法同实施例1.3酚/氯仿核酸纯化方法,mRNA片段也使用纳米光度计定性、定量分析。
6.5’端加帽反应:采用牛痘病毒加帽酶系统(Novoprotein#M072)对转录的RNA进行加帽反应,使RNA在甲基转移酶催化下,5’端携带cap1(m7G-PPPNm)帽子结构,具体过程如下:
(1)用无RNA酶的水将RNA稀释至16μl;
(2)将所得的RNA溶液置于65℃加热5min,结束后冰上放置5min;
(3)依次加入表3中的各组分;
(4)37℃反应60min。最终获得的序列的结构图如图1所示。
表3.加帽反应系统中的组分、
实施例2 cGAS mRNA转染293T表达验证
(1)转染前一天将293T细胞(美国典型培养物保藏中心ATCC)(人肾上皮细胞系)铺至12孔细胞培养板,添加10%热灭活胎牛血清(VivalCell#
2115106)和1%抗生素(biosharp#BL505A)的DMEM培养基培养(BI#06-1055-57-1ACS),观察细胞状态,使细胞密度生长铺板大约为60%-70%后,方可转染脂质体到293T;
(2)使用Endofectin MAX(GeneCopoeia#EF003)以质量体积比1:2(核酸:Endofectin MAX,1g核酸+2L Endofectin MAX)的比例将实施例1中所得cGAS的mRNA分别转染500ng到293T细胞中;
(3)25μl预先置于室温的无血清的培养液(invitrogen#31985070)分别稀释cGASmRNA、EndoFectin Max转染试剂。混匀,室温放置5min;
(4)将cGAS mRNA稀释液加入到Endofectin MAX稀释液中,充分混匀,室温静置15分钟;
(5)吸去细胞培养上清,用PBS洗1次,加入1ml无血清的培养液,加入上述转染mRNA混合液;
(6)加入Endofectin MAX脂质体后轻微震荡混匀,于37℃、5%CO2的饱和湿度培养箱中培养;所有操作无RNA酶;
(7)24h后,收集细胞制备裂解蛋白,蛋白裂解液用抗cGAS的抗体(BOSTER#A31676-1)做western blot检测细胞中蛋白表达情况,微管蛋白(文渊阁)作为阳性对照,使用显色试剂盒(biosharp#BL520B)进行化学发光显色,使用成像仪(勤翔#ChemiScope 6200)上层化学发光自动曝光模式采集图片,所得结果如图2所示。图2中,不转染的293T细胞本身几乎没有cGAS蛋白。人、小鼠cGAS mRNA可以在细胞内表达蛋白,由图2对比可知,体外转录的cGAS mRNA可以在人源细胞中成功表达。
实施例3 cGAS mRNA功能验证
1.cGAS mRNA转染293T-STING-mNeongreen细胞
293T-STING-mNeongreen细胞:来源于人肾上皮细胞293T(美国典型培养物保藏中心ATCC),是研究STING激活和的首选模型,293T本身不表达cCAS和STING,稳定转染过表达带有绿色荧光mNeongreen的STING。整个细胞体带有绿色荧光,STING蛋白活化时在高尔基体组装成多聚体复合物,即出现亮度更高的绿色荧光的点聚现象。
具体实施如下:
(1)转染前一天将293T-STING-mNeongreen细胞按转染密度转移到48孔细胞培养板;
(2)转染不同核酸(HT-DNA 100ng/孔或cGAS mRNA100ng/孔)。转染方案及脂质体比例依照实施例2方法;
(3)用cGAMP(1μg/mL)(Abexbio#B8362)刺激做观察STING点聚阳性对照;
(4)4、12h使用倒置荧光显微镜(明美#MF52-N)观察STING的绿色荧光点。
其中NT表示未加任何处理常规培养的细胞,反映细胞的密度和生长状态,pCR2表示转染无关的mRNA。4h、12h表示对应处理到采集图片时的时间间隔。由图3对比可知:
(1)转染cGAS同时转染HT-DNA(sigma#SLBW8354),4h可以看到STING的点聚,比cGAMP组稍弱。免疫激活效果:cGAMP>小鼠cGAS>人cGAS;
(2)转染无关的mRNA+HT-DNA无点聚,单独刺激HT-DNA也无点聚。说明cGAS mRNA才是有效的激活剂;
(3)使用cGAS mRNA 12h激活STING效果小鼠cGAS>人cGAS>cGAMP,仅转染cGAS没有外源HT-DNA刺激也出现STING活化,说明内源的HT-DNA可以在较长的时间之后慢慢激活cGAS。
2.cGAS mRNA转染THP1-IFN-lucia细胞
THP1-IFN-lucia细胞:来源于人单核细胞(THP1)(美国典型培养物保藏中心ATCC),是研究IFN激活和信号转导的模型。
THP1-IFN-lucia细胞IFN完整开放阅读框,被内源性启动子控制下的Lucia(荧光素酶)报告基因取代,在IFN启动子的控制下表达荧光素。
因此,THP1-IFN-lucia细胞可以通过测定Lucia荧光素酶的活性来监测IFN诱导分泌。使用荧光素酶检测底物腔肠素(启维#CZ10),通过荧光素酶促显色反应程度,可以很直观地评估细胞的干扰素表达水平。具体实施如下:
(1)将THP1-IFN-Lucia分到96孔细胞培养板中0.5-1×106个/ml,100μl培养基体系(0.5-1×105个/孔);
(2)m-cGAS-mRNA 0.1μg/孔或h-cGAS-mRNA 0.2μg/孔转染THP1-lucia,12h后转染HT-DNA 0.1μg/孔刺激,另设一组平行对照不加HT-DNA刺激,转染依照实施例2方法;
(3)刺激12h后,将腔肠素(1mg/ml溶于乙醇1000X)用PBS稀释10倍后,每孔加入1μl(1μg/ml终浓度);
(4)通过酶标仪(SuPerMax#3100)光子计数,统计可知荧光素酶Lucia的酶活性。或者将细胞培养板去掉盖子,用勤翔成像仪化学发光成像模式曝光1min,显色反应成黑白图。
其中阴性对照为同等条件下的转染试剂对照,不加核酸。
由图4荧光素酶显色对比可知:
(1)由图4A可知转染鼠源cGAS可以诱导干扰素产生,人源的激活免疫效果微弱,HT-DNA能进一步增强m-cGAS诱导干扰素。证明小鼠cGAS mRNA是有生物学活性的,可被HT-DNA激活,并且强烈诱导干扰素;
(2)同样使用上述方法增加转染用量,由图4B可知通过增加人cGAS mRNA用量可以增强干扰素的诱导;
(3)人、小鼠的cGAS mRNA都是有效的STING激动剂。
实施例4 cGAS mRNA转染BMDC细胞
1.cGAS mRNA诱导BMDC细胞(C57Bl-6J)鼠诱导分化的骨髓来源巨噬细胞)成熟。
(1)小鼠(6-10周龄)安乐死,在超净台内取小鼠后腿,放入无菌冰PBS中浸泡;
(2)在超净台内中取出所有股骨和胫骨,并将骨周围的肌肉组织尽量去除干净,将骨放入无菌RPMI 1640(美仑#MA0552)清洗两次;
(3)将骨放入已灭菌研钵中,轻轻按压至骨头完全变为白色,用200目尼龙网滤去小碎片和肌肉组织,收集骨髓悬液;
(4)1500rpm,4℃,离心5min,弃去上清;
(5)加入2ml红细胞裂解液(biosharp#BL503A)重悬细胞沉淀,室温裂解3min,期间震荡混匀;
(6)加10ml无菌PBS终止裂解,1500rpm,4℃,离心5min,弃去上清;
(7)用1ml RPMI 1640完全培养液重悬细胞沉淀,过200目尼龙网g过滤去除杂质,血球计数板细胞计数
(8)取适量细胞悬液与含小鼠粒细胞-巨噬细胞集落刺激因子GM-CSF(20ng/ml)(同立海源#GMP-TL655-0050)的RPMI 1640完全培养基混合;如大约6×106个BM(骨髓细胞)铺至10cm细胞培养皿(Non-Treated)中,每皿10ml培养基,此为培养第0天;
(9)培养第3天时,收集5ml旧培养液,1500rpm,4℃,离心5min,弃去上清,用5ml含体外转录小鼠GM-CSF(20ng/ml)的RPMI 1640完全培养基重悬细胞沉淀,再将细胞悬液放回原皿(可全换液,据细胞状态决定);
(10)培养第6天时,用移液器轻轻吹打收集悬浮细胞,1500rpm,4℃离心5min,弃去上清用10ml含体外转录小鼠GM-CSF(20ng/ml)的RPMI 1640完全培养基重悬细胞沉淀,再将细胞悬液至10cm细胞培养皿(Treated);
(11)培养第9天时,用移液器轻轻吹打收集悬浮细胞,1500rpm,4℃离心5min,弃去上清,用10ml含体外转录小鼠GM-CSF(10ng/ml)和RPMI 1640完全培养基重悬细胞沉淀铺至12孔细胞培养板(Treated);
(12)同时LPS(1μg/ml)(sigma#L6529)、poly(I:C)(1μg/ml)(sigma#p1530)、cGAMP(10μM)、小鼠cGAS mRNA(500ng/ml)或仅转染试剂转染诱导BMDC成熟。转染方案参照实施例2,于37℃、5%CO2的饱和湿度培养箱中培养24h。
2.成熟BMDC流式细胞仪分析
上述BMDC细胞培养24h,通过流式细胞仪(Beckman#Cytoflex)检测成熟BMDC的比例。
(1)收集细胞(通常1×106/孔),离心(400g 5min,4℃),用1×PBS洗一遍,离心(400g 5min,4℃);
(2)提前配置实验中需要的工作液,分为内含Fc受体阻断剂α-CD16/32(1.5μg/ml)(Biolegend#101302)的封闭液和荧光抗体混合液Cocktail(表4);
(3)用60μl CD16/32(1.5μg/ml)(Biolegend#101302)的抗体稀释液重悬细胞封闭,4℃放置15-30min或室温标记10min;用1×PBS洗一遍,离心(400g 5min,4℃);
(4)加入60μl含有特异性表面标记荧光抗体混合液Cocktail,4℃避光放置15-30min,离心(400g 5min,4℃);
(5)表4.FACS分析的抗体概述
(6)补140μl 1×PBS洗一遍,离心(400g 5min,4℃);
(7)200μl 1×PBS重悬细胞;
(8)细胞悬液经200目尼龙网过滤去除杂质,转至1.5ml Ep管中,上机检测;
(9)收集后,使用FlowJo X版本10.0.7R2处理,补偿使用单抗体染色细胞对照进行数字化调整。BMDC细胞使用以下门控策略定义FSC-A/SSC-ACells/FSC-H/FSC-A/SSC-H/SSC-ASinglets/LiveDead-/CD11c+。统计DC细胞中CD86的信号强度。CD86是抗原提呈细胞表面分子,提供T细胞生存和活化所需的共刺激信号。是评价佐剂效果的指标。
(10)通过这种分析,图5A所示与仅用培养基对照(平均值:1903)相比、cGAMP上调CD86的强度更高(平均值:4113),要强于一般佐剂poly(I:C)(平均值:2753)。与仅转染试剂Endo对照(平均值:2547)相比,小鼠cGAS mRNA超过经典的方案LPS(平均值:5414)诱导CD86上调水平(平均值:6723)。说明小鼠cGAS RNA大幅上调了CD86表面的共刺激分子。
实施例5 m-cGAS mRNA诱导DC2.4对OVA-FITC的吞噬
(1)鼠源树突状细胞DC2.4细胞(北京索莱宝科技有限公司)培养至密度为5×105个/ml,接种在24孔细胞培养板中,DMEM培养基培养细胞使其数量达到培养板面积的60%左右;
(2)对细胞使用不同激动剂:cGAMP(10μM)、m-cGAS mRNA(500ng/ml)和等量的转染试剂,刺激24h。
(3)培养基中加入20ng/ml的OVA-FITC蛋白(Solarbio#SF069),再孵育4h。
(4)PBS清洗细胞三次,消化收集细胞,染色标记DC2.4,流式染色方案参考实施例4。
(5)使用CytExpert版本2.4.0.28统计分析流式数据。同样补偿使用单抗体染色细胞对照进行数字化调整。活细胞使用以下门控策略定义:FSC-A/SSC-A Cells/FSC-H/FSC-A/SSC-H/SSC-A Singlets/LiveDead-,最后统计活细胞中FITC阳性的比例,即为吞噬抗原的DC2.4细胞比例。
(6)如图5B所示,与仅用培养基阴性对照相比,转染小鼠cGAS mRNA的DC2.4细胞对OVA抗原的摄取显著增强,约有2倍的增长。同时我们观察到仅使用转染试剂Endo-MAX摄取抗原能力相比于培养基阴性对照略有下降,这可能是由于试剂对细胞有一定毒性,影响了细胞状态,后续需要优化mRNA体外递送体系,提高小鼠cGAS mRNA转染效率的同时降低细胞毒性。
综上可知,cGAS mRNA可以活化STING,并刺激干扰素的产生,是有效的STING激动剂。cGAS mRNA可以促进树突状细胞成熟,促进抗原吞噬,是一种具有广阔应用前景的疫苗联合递送佐剂。
以上所述的具体实施例,对本发明的目的、技术方案和有益效果进行了进一步详细说明,应理解的是,以上所述仅为本发明的具体实施例而已,并不用于限制本发明,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
参考文献
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Claims (10)
1.一种mRNA,其包含编码cGAS或其活性片段的编码区,其中所述mRNA还包含以下的一种或多种:
5’-帽结构,优选为m7GPPP、m7G-PPPNm或m7G-PPPNmNm;
3’-聚腺苷酸,其包含约25至400个腺苷酸的序列;
5’-UTR,所述5’-UTR的序列优选如SEQ ID NO:2所示;
3’-UTR,所述3’-UTR的序列优选来源于提供稳定的mRNA的基因的3’UTR,更优选如SEQID NO:5所示;
修饰的核苷酸,所述修饰的核苷酸优选自5-甲基-CTP、假-UTP、N1-甲基-假UTP和5-甲氧基-UTP中的一种或多种。
2.权利要求1所述的mRNA,其中所述cGAS为如SEQ ID NO:3所示的小鼠cGAS或如SEQ IDNO:4所示的人cGAS。
3.编码权利要求1-2任一项所述的mRNA的多核苷酸。
4.一种包含权利要求3所述的多核苷酸的表达载体,优选地,所述表达载体还包含启动子序列,优选T7启动子序列,优选地,所述表达载体为质粒。
5.权利要求1-2任一项所述的mRNA在制备免疫调节药物和/或免疫靶向药物中的用途。
6.权利要求5所述的用途,其中所述免疫调节药物和/或免疫靶向药物为抗癌或抗病毒药物。
7.权利要求1-2任一项所述的mRNA在制备免疫佐剂中的用途。
8.一种制备权利要求1-2任一项的mRNA的方法,其包括将权利要求4的表达载体引入宿主细胞,在培养基中培养所述宿主细胞,从宿主细胞中分离表达载体,将所述表达载体线性化后进行体外转录,从而得到所述mRNA。
9.权利要求8所述的方法,其中通过在含有经修饰的核苷酸的体外无细胞转录系统中进行体外转录,得到含有经修饰的核苷酸的mRNA,优选地,所述经修饰的核苷酸是N1-甲基-假UTP。
10.权利要求8-9任一项所述的方法,其中通过牛痘病毒加帽酶系统对所述转录的mRNA进行加帽反应,使得所述mRNA具有5’-帽结构。
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