WO2022000374A1 - 一种covid-19病毒抑制剂 - Google Patents
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Abstract
一种COVID-19病毒抑制剂，其至少包含SEQ ID NO.1～SEQ ID NO.12所示的miRNA。作为优选的方案，上述miRNA以经IL-21激活的NK细胞分泌的外泌体为载体。
Description
本发明涉及一种COVID-19病毒抑制剂。
MicroRNA(miRNA)是真核生物中存在的一类20个核苷酸长度左右的内源性非编码RNA，可以与mRNA结合从而调控基因的表达。目前,在人类基因组中已知的miRNA有2000种,miRNA几乎在所有生物途径中都发挥着重要作用，其表达谱的变化与许多人类疾病相关。miRNA通过抑制翻译和诱导mRNA降解来抑制基因的表达,在病毒的复制或抑制过程中也是如此。
病毒是一种个体微小，结构简单，只含一种核酸(DNA或RNA)，必须在活细胞内寄生并以复制方式增殖的非细胞型生物。病毒依赖宿主细胞而存活，其感染复制周期大致包括：吸附、穿入、脱壳、生物合成(病毒核酸复制；病毒蛋白质合成)、装配与释放。根据病毒核酸类型的不同，动物病毒可以分为三大类：DNA病毒、DNA和RNA逆转录病毒、RNA病毒。其中，DNA病毒包括腺病毒、疱疹病毒、痘病毒、肝炎病毒等，DNA和RNA逆转录病毒包括肉瘤病毒和艾滋病毒等，RNA病毒包括SARS冠状病毒、埃博拉病毒、流感病毒等。
目前针对病毒性传染病的防治主要以抗病毒抑制剂及特异性疫苗的研制与应用为主。然而，大多数病毒性疾病至今尚无特效抑制剂，而能够应用疫苗进行防治的病毒性疾病也很少。SARS-CoV-2作为全新发现的单股正链RNA病毒，依赖宿主细胞进行新病毒的合成。虽然目前COVID-19病毒基因组全测序已经完成，但是COVID-19病毒在宿主细胞内的复制和调控过程仍有待研究。冠状病毒的正链RNA进入宿主细胞后，可以直接作为mRNA链指导蛋白质的合成；也可以通过依赖RNA的RNA聚合酶(RDRP)作用，生成负链，再以负链为模板，在RDRP作用下，生成正链，达到复制的目的。同时，生成的正链也可作为mRNA指导蛋白质的合成。目前抗RNA病毒都是基于病毒复制过程依赖宿主细胞的RNA聚合酶等机理而研发特异性的RNA聚合酶抑制剂(小分子化合物，瑞德西韦)，而此类抑制剂的研发周期长、失败率高，使得开发针对逆转录酶的特异性抑制剂难度很大，此外，COVID-19病毒的RNA可以直接指导蛋白质合成，RNA聚合酶抑制剂对此根本无效。
发明内容
本发明的目的在于提供一种COVID-19病毒抑制剂，包含SEQ ID NO.1～SEQ ID NO.12所示的特异性miRNA。SEQ ID NO.1～SEQ ID NO.12所示的特异性miRNA与COVID-19病毒S蛋白的部分区域完全匹配，其亲和力强，因此具有有效的抗病毒能力。
本发明的目的在于提供一种COVID-19病毒抑制剂，包含SEQ ID NO.1～SEQ ID NO.12所示的特异性miRNA。该SEQ ID NO.1～SEQ ID NO.12所示的特异性miRNA与aNK细胞外泌体内包含的某些miRNA具有极高的相似性，因此，安全可控。
具体的，上述特异性miRNA在使用时，可导入到生物安全的载体中，这种载体包括但不限于外泌体(例如，aNK细胞分泌的外泌体)、人工合成的高分子聚合物等。在某些优选的实施例中，上述SEQ ID NO.1～SEQ ID NO.12所述的特异性miRNA以经IL-21激活的NK细胞(以下简称aNK细胞)分泌的外泌体为载体。外泌体是一类由细胞分泌的携带胞质组分的纳米级别的膜性小泡，机体各种各样的细胞都能分泌这类膜性小泡，其广泛分布于唾液、血浆、乳汁等体液中。外泌体中含有蛋白质、mRNA和miRNA等多种生物活性物质，通过膜融合的方式将内容物如miRNA和蛋白质传递给其他细胞，作为细胞之间相互交流的桥梁。采用aNK外泌体作为上述特异性miRNA的载体，可以进一步提高抑制剂的抗病毒能力，且安全可控。
在某些实施例中，aNK外泌体是指经IL-21激活NK后所获得的外泌体，但不限于此。
通过大量实验证明，不同于现有的NK细胞和RNA聚合酶抑制抗病毒机制，上述SEQ ID NO.1～SEQ ID NO.12所示的特异性miRNA及其载体aNK外泌体，均能特异性靶向COVID-19病毒S蛋白mRNA，促进COVID-19病毒S蛋白mRNA在宿主细胞内被剪切和降解，阻断COVID-19病毒S蛋白的合成，抑制COVID-19病毒侵袭能力，快速和高效的降低病人体内的病毒拷贝数。
本发明的有益效果在于：
(1)从抑制侵袭蛋白的机制出发，特异性好，速度快，效果显著；
(2)不同于蛋白酶抑制剂和逆转录酶抑制剂等抗病毒制剂治疗病毒感染患者，本发明不具有副作用，安全可控；
(3)可通过与常规治疗方案联合使用增强治疗效果；
(4)miRNA可以人工合成、外泌体可低温储存，成本低，可以实现大规模的生产。
图1透射电镜观察aNK外泌体的形态图(A)和aNK外泌体对HEK-293细胞的细胞毒性测试图(B)；
图2 Werstern blot检测aNK外泌体的蛋白表达情况图；
图3流式细胞仪检测aNK细胞和aNK外泌体的表面蛋白表达情况图；
图4 miRNA靶向病毒基因组的生物信息学结果；
图5具有COVID-19病毒基因组靶向能力的miRNA的Energy和Score分布图；
图6初始NK细胞(1M/ml)、aNK细胞(1M/ml)，以及不同浓度的aNK外泌体抑制HEK-293细胞中COVID-19病毒spike的mRNA转录和蛋白表达；
图7在不同载体中的人工碱基替换miRNA抑制COVID-19病毒spike蛋白的表达。
下面结合实施例对本发明作进一步说明。
实施例1:aNK外泌体的获取及其结构特征
1.1收集IL-21培养18天的NK细胞培养液以获得激活后的NK细胞aNK，流式细胞仪检测aNK细胞的纯度达到95％以上，并且培养液无细菌和支原体污染。
1.2aNK细胞培养液通过台式低速离心机400g离心5min去除细胞沉淀，收集上清4度保存待用。
1.3采用中空纤维切向过滤系统(Spectrum Laboratories KrosFlo Research II TFF System)对培养液中的外泌体进行纯化。首先，利用0.45μm mPES中空纤维过滤柱(P-S02-E45U-10-N)去除细胞培养液中的细胞碎片；滤出液进一步通过截留分子量在300-kDa的mPES中空纤维过滤柱(S02-E300-05-N)进行浓缩，获得外泌体粗制品；为了进一步缩小体积并且去除残余的培养基和盐离子，用3倍体积的PBS对外泌体粗制品进行稀释，并采用截留分子量在300-kDa的mPES中空纤维过滤柱(D02-E300-05-N)进行浓缩，得到纯度很高的外泌体。
1.4将获取的外泌体用去离子水重悬，取少量外泌体置于有碳涂层的铜网，去除多余的水分，并用2％乙酸铀酰进行染色，自然干燥后利用透射电镜观察外泌体的形态和大小。如图1A所示，TEM结果显示分离的aNK外泌体具有典型 的外泌体结构，呈现内部半透明、椭圆、大小不一的封闭膜结构。
1.5用BCA蛋白定量试剂盒对外泌体进行蛋白定量，并用裂解液释放外泌体中的蛋白，采用Werstern blot方法对外泌体Marker蛋白CD63、ALIX，aNK外泌体特有的NKp30、NKp44、NKp46、NKG2D和perforin进行检测。如图2所示，与HEK-293细胞相比，aNK外泌体含有NK细胞特有的细胞毒受体NKp30、NKp44、NKp46、NKG2D和穿孔素。
1.6将获取的外泌体，用thermo fisher scientific的4微米latex beads与外泌体结合，然后用流式抗体孵育。采用流式细胞学方法对aNK外泌体表面蛋白NKp30、NKp44、NKp46和NKG2D进行检测。如图3所示，与aNK细胞相同，aNK外泌体含有NK细胞特有的细胞毒受体NKp30、NKp44、NKp46和NKG2D。
为了确认aNK细胞所分泌的外泌体是否具有细胞毒性，我们将aNK细胞外泌体作用于HEK-293细胞。将实施例1获得的aNK外泌体按照不同比例加入HEK-293细胞，通过CCK-8试剂盒检测细胞的活力来评估aNK外泌体对HEK-293细胞的细胞毒性，测试结果如图1B所示。与阴性对照(不加入任何制剂)相比，200μg/ml以下的aNK外泌体对细胞没有任何的细胞毒性。
实施例2:aNK外泌体miRNA测序和分析
根据实施例1获得的aNK外泌体，委托南京世和基因生物技术有限公司对aNK外泌体中miRNA进行测序。采用NEBNext Multiplex Small RNA Library Prep Set for Illumina(NEB,USA)进行建库，并对待测序样本添加测序接头。采用Illumina X-ten PE150平台对样本进行测序，每个样本至少300M reads。为了减少假阳性，信噪比大于100的独立序列纳入miRDeep log-odds score的计算，并设置miRDeep log-odds score的范围为-10～10.为了发掘aNK外泌体中全新的miRNA，以miRDeep score＝0作为cut-off值，所有超过cut-off值的miRNA作为全新的miRNA。测序结果显示，aNK细胞外泌体中存在大量的miRNA表达(43.92％)，说明miRNA在外泌体调控过程中具有重要作用。通过测序中发现了1162个已知的miRNA和全新序列的miRNA有148个。
从NCBI数据库下载COVID-19病毒COVID-19(MN908947.3)的基因组序列。如图4A所示，将aNK外泌体测序获得的miRNA序列和病毒基因组序列分别输入miRanda v3.3a软件进行比对，设置Gap Open Penalty:-9.0；Gap Extend Penalty:-4.0；Score Threshold:140；Energy Threshold:1 kcal/mol；Scaling Parameter:4.0。通过miRanda软件和Lunix服务器的大数据计算，我们获得了针对COVID-19病毒基因组具有靶向能力和结合位点的miRNA种类和数量。通过比对所有的 miRNA，我们发现其中有1079个miRNA对COVID-19病毒的基因组具有靶向能力。我们对COVID-19病毒基因组上能够匹配的miRNA的Energy和Score进行了分析，如图4B所示。我们还深度分析了新冠状病毒12个区域能够匹配的miRNA的Energy和Score，如图5A和B所示，表明激活后的NK细胞外泌体包含有靶向COVID-19病毒的几个核心结构mRNA的miRNA，包括E蛋白、N蛋白、M蛋白、ORF1ab等核心区域。我们特定的选择了S蛋白作为攻击目标，因为人体本身没有S蛋白，攻击COVID-19病毒S蛋白不会造成副作用。
实施例3:初始NK细胞、aNK细胞、aNK外泌体、aNK外泌体中的miRNA和碱基替换后的miRNA(M)抑制COVID-19病毒S(Spike)蛋白表达
COVID-19病毒S蛋白存在于病毒表面，是COVID-19病毒进入人体细胞的核心蛋白。病毒失去了S蛋白将不再能够进一步感染人体细胞，因此S蛋白是多种抑制剂设计的靶标。为了确定初始NK细胞、aNK细胞、aNK细胞所分泌的外泌体是否具有抗COVID-19病毒能力，我们首先以COVID-19病毒S蛋白过表达系统为模型，观察初始NK细胞、aNK细胞、aNK外泌体抑制病毒的S蛋白复制的效果。我们将COVID-19病毒S蛋白质粒表达于HEK-293细胞作为检测对象。将初始NK细胞(1M/ml)、按照实施例1激活得到的aNK细胞(1M/ml)、以及实施例1获得的aNK外泌体按照不同比例加入表达COVID-19病毒S蛋白的HEK-293细胞液中，通过ELISA检测细胞中COVID-19病毒spike蛋白的OD值来评估初始NK细胞、aNK细胞、aNK外泌体对COVID-19病毒的抑制能力，测试结果如图6A所示。与阴性对照(不加入任何制剂)相比，高浓度的初始NK细胞几乎不具有抗病毒效果，高浓度的aNK细胞具有一定的抗病毒效果；而低浓度下的aNK外泌体也比高浓度的aNK细胞表现出更好的抗病毒效果，而且随着外泌体浓度的增加，COVID-19病毒S蛋白的表达呈现浓度依赖性抑制。进一步，通过荧光定量PCR检测25ug/ml的aNK外泌体作用下，HEK-293细胞中COVID-19病毒S蛋白mRNA表达水平，我们发现，低浓度aNK外泌体的处理即可降低COVID-19病毒S蛋白的mRNA表达水平(图6B)。
将实施例2筛选得到的miRNA输入到miRanda v3.3a软件进行比对，设置Gap Open Penalty:-9.0；Gap Extend Penalty:-4.0；Score Threshold:140；Energy Threshold:1 kcal/mol；Scaling Parameter:4.0。通过miRanda软件和Lunix服务器的大数据计算，我们获得了miRNA针对COVID-19病毒S蛋白mRNA的结合能力(energy)评估，根据结合能力(energy)进行排序，优选出12个结合能力(energy)最强的miRNA：hsa-miR-6734-5p、hsa-miR-4793-3p-2、hsa-miR-12159、 hsa-miR-125a-3p、hsa-miR-30c-1-3p、hsa-miR-12238-1、hsa-miR-365a-5p、hsa-miR-221-3p、hsa-miR-1244、hsa-miR-4723-5p、hsa-miR-12238-2、hsa-miR-4793-3p-1(依次如SEQ ID NO.13～24所示)。
将上述12个结合能力(energy)最强的miRNA进行碱基替换，以使其与COVID-19病毒S蛋白的部分区域完全匹配，如下表1中(M)所示。
表1：12个miRNA及碱基替换后的miRNA(M)的结合能
| Name | Sequence | Score | Energy1 |
| miR-6734-5p | UUGAGGGGAGAAUGAGGUGGAGA | 154 | -29.27 |
| miR-6734-5p(M) | CUGAGGCAAGGACAUAAGAUGAUAG | 183 | -42.6 |
| miR-4793-3p-2 | UCUGCACUGUGAGUUGGCUGGCU | 143 | -28.69 |
| miR-4793-3p-2(M) | ACUACACCAUGAGGUGCUGACU | 200 | -44.87 |
| miR-12159 | UCUGGGAGUGGGGCUGUGGGUGG | 141 | -27.16 |
| miR-12159(M) | UCUGAGAGAGGGUCAAGUGCACAG | 210 | -51.73 |
| miR-125a-3p | ACAGGUGAGGUUCUUGGGAGCC | 140 | -27.13 |
| miR-125a-3p(M) | ACAAAUGAGGUCUCUAGCAGCA | 200 | -41.65 |
| miR-30c-1-3p | CUGGGAGAGGGUUGUUUACUCC | 150 | -27.03 |
| miR-30c-1-3p(M) | CUGAGAGAGGGUCAAGUGCACAGU | 210 | -52.2 |
| miR-12238-1 | UCCCAGGGUCGGCUGUGGUAGCCCU | 151 | -26.98 |
| miR-12238-1(M) | GCUGACUGAGGGAAGGACAUAAGAU | 215 | -50.86 |
| miR-365a-5p | AGGGACUUUUGGGGGCAGAUGUG | 158 | -26.66 |
| miR-365a-5p(M) | AGGGACUUCUGUGCAGUUAAC | 195 | -39.94 |
| miR-221-3p | AGCUACAUUGUCUGCUGGGUUUC | 146 | -26.49 |
| miR-221-3p(M) | AGCUACACUACGUGCCCGCCGAG | 205 | -53.08 |
| miR-1244 | AAGUAGUUGGUUUGUAUGAGAUGGUU | 154 | -26.42 |
| miR-1244(M) | GAGUAAGUUGAUCUGCAUGAAUAGCA | 220 | -46.53 |
| miR-4723-5p | UGGGGGAGCCAUGAGAUAAGAGCA | 141 | -26.02 |
| miR-4723-5p(M) | GAGGGAAGGACAUAAGAUGAUAGCC | 215 | -50.3 |
| miR-12238-2 | UCCCAGGGUCGGCUGUGGUAGCCCU | 143 | -24.86 |
| miR-12238-2(M) | ACUUGCUGUGGAAGAAAGUGAGUC | 210 | -45.75 |
| miR-4793-3p-1 | UCUGCACUGUGAGUUGGCUGGCU | 149 | -24.47 |
| miR-4793-3p-1(M) | GCUACACUACGUGCCCGCCGAGG | 205 | -55.13 |
将上述碱基替换后的miRNA(M)(SEQ ID NO.1～SEQ ID NO.12)进行结合能力(energy)测试，从表中可以看出，其结合能力(energy)显著增强。
上述碱基替换后的miRNA(M)(SEQ ID NO.1～SEQ ID NO.12)可以完全通过人工设计合成。
将hsa-miR-6734-5p、hsa-miR-4793-3p-2、hsa-miR-12159、hsa-miR-125a-3p、hsa-miR-30c-1-3p、hsa-miR-12238-1、hsa-miR-365a-5p、hsa-miR-221-3p、hsa-miR-1244、hsa-miR-4723-5p、hsa-miR-12238-2、hsa-miR-4793-3p-1及碱基替换后的miRNA(M)分别以终浓度5nM与转染载体Genmute、单核细胞所分 泌的外泌体、激活后的NK细胞外泌体(按照实施例1步骤1.2获得)分别进行共孵育，待孵育结束后以载体浓度12.5μg/ml加入HEK-293细胞中。通过ELISA检测细胞中COVID-19病毒spike蛋白的OD值来评估miRNA对COVID-19病毒的抑制能力。测试结果如图7A(转染载体Genmute)、7B(单核细胞分泌的外泌体)、7C(激活后的NK细胞分泌的外泌体)所示。图中C表示未经碱基替换的miRNA，M表示碱基替换后的miRNA。
通过与阴性对照Blank组对比，可以证明，碱基替换前后的miRNA均能抑制COVID-19病毒spike蛋白的表达，实现COVID-19病毒的抑制，尤其是碱基替换后的hsa-miR-6734-5p(M)、hsa-miR-4793-3p-2(M)、hsa-miR-12159、hsa-miR-125a-3p(M)、hsa-miR-30c-1-3p(M)、hsa-miR-12238-1(M)、hsa-miR-365a-5p(M)、hsa-miR-221-3p(M)、hsa-miR-1244(M)、hsa-miR-4723-5p(M)、hsa-miR-12238-2(M)、hsa-miR-4793-3p-1(M)抑制spike蛋白表达的效果最为明显。
单纯的单核细胞所分泌的外泌体和转染载体Genmute均无法抑制COVID-19病毒spike蛋白的表达，而人为的在单核细胞外泌体中加载碱基替换后的miRNA则具有抑制COVID-19病毒spike蛋白表达的能力；此外，利用激活后的aNK细胞分泌外泌体作为上述miRNA的载体，我们发现其具有显著的抑制COVID-19病毒spike蛋白表达的能力。
作为本领域的公知常识，上述负载有特异性miRNA的载体在形成抑制剂时，一般是将其以200μg/ml以下的浓度溶于PBS、生理盐水等类体液中，以进行注射治疗；或溶于PBS、生理盐水、去离子水等溶剂中，以作为喷剂使用。
Claims (2)
- 一种COVID-19病毒抑制剂，其特征在于，至少包含SEQ ID NO.1～SEQ ID NO.12所示的miRNA。
- 根据权利要求1所述的COVID-19病毒抑制剂，其特征在于，所述miRNA以经IL-21激活的NK细胞分泌的外泌体为载体。
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