CN114561381A - 免疫mRNA及其制备方法和应用 - Google Patents
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Abstract
本发明公开了免疫mRNA及其制备方法和应用，属于生物医药技术领域。所述免疫mRNA的制备方法，具体是在采用体外转录合成mRNA，其特征是，在体外转录合成mRNA时，掺入5～15mol％的5‑甲基胞苷和5～15mol％的2‑硫尿苷用于化学修饰体外转录合成。采用本发明所述方法制得的免疫mRNA递送后的稳定性及蛋白合成效率得到显著上升，可提高其在递送后抗口腔上皮细胞侵染的能力，且在递送口腔上皮细胞后能减弱与模式识别受体(TLR3、TLR7、TLR8及RIG‑I)的结合，降低mRNA递送的免疫反应。
Description
技术领域
本发明涉及免疫mRNA及其制备方法和应用，属于生物医药技术领域。
背景技术
mRNA是以DNA的一条链作为模板转录而来、携带有遗传信息、可以指导蛋白质合成的单链核糖核酸，从1961年发现mRNA至2019年，市面上尚无mRNA药物获批应用于临床。然而，近十年来，mRNA递送系统和核酸化学的不断发展推进了mRNA疗法的临床前开发，加速了mRNA发展成为一种新型核酸药物的进程。2021年获紧急授权使用的两款mRNA疫苗在新冠疫情爆发后短短一年时间左右便推向临床，且保护效力超过90％，体现了mRNA疫苗的优越性。
牙周炎与心血管疾病、糖尿病、妊娠不良、风湿性疾病、心理疾病等全身性疾病密切相关。2018年第四次“全国口腔健康流行病学调查报告”数据显示，我国80～97％的成年人存在不同程度的牙周问题。迄今，牙周炎已被认为是仅次于肿瘤和心血管疾病，危害人类健康的第三大疾病。牙周炎的抗生素治疗会带来病原菌抗性患者过敏及非特异性作用等问题，因此发展抗生素治疗的替代方法是亟待解决的问题。自2020年初新型冠状病毒肺炎疫情爆发以来，mRNA疫苗的应用使mRNA递送成为研究的热点。在应用过程中，mRNA递送的不稳定及诱发的免疫反应等问题一直是该领域研究的热点。
现有技术中体外转录合成天然免疫蛋白mRNA(如S100A8/A9、抗菌肽CAMP)递送口腔上皮细胞，能够表达出相应的蛋白，提高其抗李斯特菌或沙门氏菌侵染的能力，但存在以下不足：(1)mRNA递送的效率低；(2)递送后与细胞病原模式识别受体结合，诱发较强的免疫反应。
发明内容
本发明要解决的技术问题是提供递送效率高，且可降递送的免疫反应的免疫mRNA及其制备方法和应用。
为解决上述技术问题，本发明采用以下技术方案：
化学修饰合成免疫mRNA的方法，采用体外转录合成mRNA，具体是在体外转录合成mRNA时，掺入5～15mol％的5-甲基胞苷和5～15mol％的2-硫尿苷用于化学修饰体外转录合成。
本领域公知，在体外转录合成mRNA时需以核苷三磷酸混合物NTPs(包括腺苷三磷酸ATP，尿苷三磷酸UTPutp，胞苷三磷酸CTPctp和鸟苷三磷酸GTP)为原料，本发明所述方法中，所述的掺入5～15mol％的5-甲基胞苷和5～15mol％的2-硫尿苷是指，核苷三磷酸混合物中使用的尿苷由5～15mol％的2-硫尿苷和95～85mol％的尿苷三磷酸组成，使用的胞苷则是由5～15mol％的5-甲基胞苷和95～85mol％的胞苷三磷酸组成。申请人通过试验发现，通过掺入5～15mol％的5-甲基胞苷和5～15mol％的2-硫尿苷用于化学修饰体外转录合成能够显著提高免疫mRNA的稳定性及蛋白合成效率，并显著增强口腔上皮细胞抗牙龈卟啉单胞菌(ATCC 33277)侵染的能力；而且该化学修饰免疫mRNA递送口腔上皮细胞后也能显著减弱与模式识别受体(TLR3、TLR7、TLR8及RIG-I)的结合，降低mRNA递送的免疫反应。
更为具体的化学修饰合成免疫mRNA的方法，包括：将S100A8蛋白和S100A9蛋白或者是CAMP基因开放阅读框分别插入质粒pGEM4Z-2bgUTR-64A，获得重组质粒pGEM4Z-S100A8-2bgUTR-64A和pGEM4Z-S100A9-2bg UTR-64A或者是pGEM4Z-CAMP-2bgUTR-64A，采用载体特异性引物对对所得重组质粒进行扩增，得到PCR产物；以纯化后的PCR片段作为模板，体外转录合成mRNA，在体外转录合成时，掺入5～15mol％的5-甲基胞苷和5～15mol％的2-硫尿苷用于化学修饰，采用抗反向帽类似物加帽，纯化，即得到相应的免疫mRNA；其中，所述的载体特异性引物对分别如SEQ ID NO：1和SEQ ID NO：2所示。
上述方法中，采用现有常规方法构建质粒pGEM4Z-2bgUTR-64A，具体可按以下方法进行构建：通过基因工程方法在pGEM-4Z载体多克隆位点间插入2个β-珠蛋白非翻译区片段，并在2个β-珠蛋白非翻译区片段后Sp6端插入含有64个多聚腺苷酸的Poly(A)片段，构建载体pGEM4Z-2bgUTR-64A并测序验证。
上述方法中，当需要获得A8免疫mRNA、A9免疫mRNA或A8/A9免疫mRNA时，通过基因工程方法将S100A8蛋白和/或S100A9蛋白的基因开放阅读框分别插入质粒pGEM4Z-2bgUTR-64A以获得重组质粒pGEM4Z-S100A8-2bgUTR-64A和/或pGEM4Z-S100A9-2bgUTR-64A；在同时使用S100A8和S100A9时，所述S100A8和S100A9的摩尔比通常为1：1。而需要获得C AMP免疫mRNA，通过基因工程方法将CAMP基因开放阅读框分别插入质粒pGEM4Z-2bgUTR-64A以获得重组质粒pGEM4Z-CAMP-2bgUTR-64A。获得的重组质粒需要经测序验证(具体可采用常规方法)后再进行下一步。
上述方法中，扩增所得的PCR产物经过纯化后即为纯化后的PCR片段，具体的纯化操作与现有技术相同，如先进行常规纯化再采用苯酚/氯仿法抽提纯化。
上述方法中，所述的抗反向帽类似物可以是ARCA(anti-reverse cap analogue)，也可以是现有其它常规的商业化帽子。在完成加帽操作后的纯化操作与现有技术相同，优选是采用MEGAclear试剂盒进行纯化。
本发明所述方法中，在体外转录合成mRNA时，优选是掺入10mol％的5-甲基胞苷和10mol％的2-硫尿苷。
本发明所述方法中，未详细提及的操作与现有技术相同。
本发明还包括以上述方法制备得到的免疫mRNA。
本发明还包括以上述免疫mRNA递送口腔上皮细胞的方法，具体是将免疫mRNA采用TransIT-mRNA脂质体或其它现有常规的脂质体裹纯化后递送口腔上皮细胞。
本发明进一步包括免疫mRNA在制备增强口腔上皮细胞抗病原菌侵染能力的制剂中的应用，所述的抗病原菌具体为牙龈卟啉单胞菌(ATCC 33277)。
与现有技术相比，本发明的特点在于：
1.采用本发明所述方法制得的免疫mRNA递送后的稳定性及蛋白合成效率得到显著上升(通过Western blot法检测细胞内表达的蛋白量显著上升)；
2.采用本发明所述方法制得的免疫mRNA可提高其在递送后抗口腔上皮细胞(具体为牙龈卟啉单胞菌(ATCC 33277))侵染的能力(通过抗生素保护法检测细胞内侵染的病原菌数量显著降低)；
3.采用本发明所述方法制得的免疫mRNA递送口腔上皮细胞后能减弱与模式识别受体(TLR3、TLR7、TLR8及RIG-I)的结合，降低mRNA递送的免疫反应(在mRNA递送后，通过RNA免疫共沉淀，再以RT-qPCR技术检测靶mRNA的量明显降低)。
附图说明
图1为采用化学修饰的免疫mRNA递送口腔上皮细胞后蛋白的表达。
图2为采用化学修饰的免疫mRNA递送口腔上皮细胞后对牙龈卟啉单胞菌(ATCC33277)侵染能力的影响。
图3为采用化学修饰的免疫mRNA递送口腔上皮细胞后对模式识别受体结合的影响。
图4为对S100A8、S100A9和CAMP开放阅读框的核苷酸序列进行化学修饰的核苷酸位置示意图，其中粗体字母表示进行化学修饰的核苷酸位置。
具体实施方式
为了更好的解释本发明的技术方案，下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1
1)通过基因工程方法在pGEM-4Z载体多克隆位点间插入2个β-珠蛋白非翻译区片段，并在2个β-珠蛋白非翻译区片段后Sp6端插入含有64个多聚腺苷酸的Poly(A)片段，构建载体pGEM4Z-2bgUTR-64A并测序验证；
2)通过基因工程方法将S100A8和S100A9)基因开放阅读框分别插入质粒pGEM4Z-2bgUTR-64A，获得重组质粒pGEM4Z-S100A8-2bgUTR-64A和pGEM4Z-S100A9-2bgUTR-64A(经测序验证)，采用载体特异性引物对(5’-CCTAAGCTTGCCACCATGAAGACCCAAAGGGATGGCC-3’(SEQ ID NO：1)/5’-ATTTGCGGCCGCCTAGGACTCTGTCCTGGGTACAAGATTCC-3’(SEQ ID NO：2))扩增，PCR产物常规纯化后，采用苯酚/氯仿法抽提纯化，得到纯化后的A8/A9 PCR片段；其中，S100A8和S100A9的开放阅读框的核苷酸序列分别为
和
所示，对它们进行化学修饰的核苷酸位置如图4(A)中的粗体字母所示；
3)通过基因工程方法将CAMP基因开放阅读框分别插入质粒pGEM4Z-2bgUTR-64A，获得重组质粒pGEM4Z-CAMP-2bgUTR-64A(经测序验证)，采用载体特异性引物对(5’-CCTAAGCTTGCCACCATGAAGACCCAAAGGGATGGCC-3’(SEQ ID NO：12)/5’-ATTTGCGGCCGCCTAGGACTCTGTCCTGGGTACAAGATTCC-3’(SEQ ID NO：2))扩增，PCR产物常规纯化后，采用苯酚/氯仿法抽提纯化，得到纯化后的CAMP PCR片段；其中，CAMP的开放阅读框的核苷酸序列为
所示，对该序列进行化学修饰的核苷酸位置如图4(B)中的粗体字母所示；
4)以步骤2)或步骤3)得到的纯化后的PCR片段作为模板，以核苷三磷酸混合物为原料，加入无RNA酶的水，T7 RNA聚合酶，抗反向帽类似物(anti-reverse cap analogue，ARCA)，RNA酶抑制剂以及掺入10mol％的5-甲基胞苷和10mol％的2-硫尿苷化学修饰。37℃水浴锅中体外转录合成2.5h，1.25h及2.5h时取出短暂离心后再放回水浴。水浴结束后需加入DNA酶I处理15min消化，再以MEGAclear试剂盒纯化并定量，得到A8/A9免疫mRNA或CAMP免疫mRNA。
细胞培养使用常规MEM培养基和10％的胎牛血清(FBS)，细胞生长密度达到60～80％时即可进行mRNA递送。取一干净的1.5mL EP管，加入250μL不含血清的MEM培养基，之后向内加入2.5μg所得免疫mRNA轻轻混合均匀(1：100)，再加入5μL mRNA Boost Reagent轻轻混合均匀，最后加入5μL TransIT-mRNA Reagent轻轻混合均匀，室温静置2～5min形成mRNA·Boost Reagent·TransIT-mRNA Reagent复合物，再均匀地滴加于口腔上皮细胞上。递送24h后，收集细胞，对蛋白的表达、对病原菌的侵染能力、与细胞病原模式识别受体结合后诱发的免疫反应进行检测：
递送24h后，收集细胞，采用western bLot法检测蛋白的表达，结果如图1所示；S100A8及S100A9 mRNA摩尔比为1:1。其中，1.100mol％假尿苷；2.25mol％的5-甲基胞苷、25mol％的2-硫尿苷；3.10mol％的5-甲基胞苷、10mol％的2-硫尿苷；4.100mol％的5-甲基胞苷及100mol％的假尿苷；5.20mol％的5-甲基胞苷，10mol％的2-硫尿苷及10％的假尿苷；6.未化学修饰；7.空转(对照)。误差线为3～6次实验结果的标准差。*P<0.05。
递送24h后，牙龈卟啉单胞菌(ATCC 33277)侵染，采用抗生素保护法结合平板计数法检测细胞内存活病原菌的数量，分析细胞抗侵染性病原菌能力的大小，结果如图2所示。其中，1.100mol％假尿苷；2.25mol％的5-甲基胞苷、25mol％的2-硫尿苷；3.10mol％的5-甲基胞苷、10mol％的2-硫尿苷；4.100mol％的5-甲基胞苷及100mol％的假尿苷；5.20mol％的5-甲基胞苷，10mol％的2-硫尿苷及10mol％的假尿苷；6.未化学修饰；7.空转(对照)。误差线为3～6次实验结果的标准差。*P<0.05。
递送24h后，收集细胞，抽提RNA，选用TLR3、TLR7、TLR8或RIG-I抗体RNA免疫共沉淀，再通过RT-qPCR技术检测mRNA的含量，结果如图3所示。其中，1.100mol％假尿苷；2.25mol％的5-甲基胞苷、25mol％的2-硫尿苷；3.10mol％的5-甲基胞苷、10mol％的2-硫尿苷；4.100mol％的5-甲基胞苷及100mol％的假尿苷；5.20mol％的5-甲基胞苷，10mol％的2-硫尿苷及10mol％的假尿苷；6.未化学修饰；7.空转(对照)。误差线为3～6次实验结果的标准差。*P<0.05，**P<0.01。
序列表
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Claims (8)
1.化学修饰合成免疫mRNA的方法，采用体外转录合成mRNA，其特征是，在体外转录合成mRNA时，掺入5～15mol％的5-甲基胞苷和5～15mol％的2-硫尿苷用于化学修饰体外转录合成。
2.根据权利要求1所述的方法，其特征是，将S100A8和S100A9或者是CAMP基因开放阅读框分别插入质粒pGEM4Z-2bgUTR-64A，获得重组质粒pGEM4Z-S100A8-2bgUTR-64A和pGEM4Z-S100A9-2bgUTR-64A或者是pGEM4Z-CAMP-2bgUTR-64A，采用载体特异性引物对对所得重组质粒进行扩增，得到PCR产物；以纯化后的PCR片段作为模板，体外转录合成mRNA，在体外转录合成时，掺入5～15mol％的5-甲基胞苷和5～15mol％的2-硫尿苷用于化学修饰，采用抗反向帽类似物加帽，纯化，即得到相应的免疫mRNA；其中，所述的载体特异性引物对分别如SEQ IDNO：1和SEQ ID NO：2所示。
3.根据权利要求1或2所述的方法，其特征是，在体外转录合成mR NA时，掺入10mol％的5-甲基胞苷和10mol％的2-硫尿苷。
4.根据权利要求1或2所述的方法，其特征是，所述的抗反向帽类似物为ARCA。
5.根据权利要求1～4中任一项所述方法制备得到的免疫mRNA。
6.采用权利要求5所述免疫mRNA递送口腔上皮细胞的方法，其特征是，将免疫mRNA采用TransIT-mRNA脂质体包裹纯化后递送口腔上皮细胞。
7.权利要求5所述的免疫mRNA在制备增强口腔上皮细胞抗病原菌侵染能力的制剂中的应用。
8.根据权利要求7所述的应用，其特征是，所述的抗病原菌为牙龈卟啉单胞菌。
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| CN102947450A (zh) * | 2009-12-07 | 2013-02-27 | 宾夕法尼亚州大学信托人 | 用于重编程细胞的包含纯化的经修饰的rna的rna制剂 |
| CN105473163A (zh) * | 2013-01-11 | 2016-04-06 | 马克·C·赫茨伯格 | 涉及mRNA转染的治疗组合物和方法 |
| WO2020168466A1 (en) * | 2019-02-19 | 2020-08-27 | Stemirna Therapeutics Co., Ltd. | Modified nucleoside and synthetic methods thereof |
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