CN114617977A - 一种以ACE2靶向递送shRNA的ACE2siRNA药物 - Google Patents
一种以ACE2靶向递送shRNA的ACE2siRNA药物 Download PDFInfo
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- CN114617977A CN114617977A CN202210116885.6A CN202210116885A CN114617977A CN 114617977 A CN114617977 A CN 114617977A CN 202210116885 A CN202210116885 A CN 202210116885A CN 114617977 A CN114617977 A CN 114617977A
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Abstract
本发明涉及一种以ACE2靶向递送shRNA的ACE2siRNA药物，从冠状病毒保守基因或微卫星中筛选靶标siRNA，由两条互补的正反义siRNA链合成带loop环的小发夹shRNA，并合成以受体结合域RBD为配体的ACE2或穿胞肽ACE2，然后将ACE2分别连接到shRNA的正反义链上，合成由shRNA区和ACE2区构成的ACE2siRNA，其中双价ACE2起中和RBD和靶向递送shRNA的作用，以ACE2桥接的“shRNA‑ACE2‑RBD‑病毒”复合物使shRNA随着病毒的感染进入靶细胞，不但可避免将shRNA非特异性递送给未感染细胞的副作用，而且还能产生抗变异毒株和以ACE2中和病毒的作用，另外以两分子ACE2和1分子shRNA合成的ACE2siRNA及shRNA的免疫佐剂作用可使ACE2具有更强的抗原性，刺激机体产生的抗‑ACE2又具有抗病毒作用。
Description
技术领域
本发明涉及一种以ACE2靶向递送shRNA的ACE2siRNA药物，属于传染病防治的生物制药领域。
背景技术
新型冠状病毒的主要结构包括单股正链核酸(ssRNA)、刺突蛋白(S)、膜蛋白(M)、包膜蛋白(E)和核壳蛋白(N)，其中S蛋白的N端由结构域(S1-NTD)和受体结合域(S1-RBD)组成，新型冠状病毒通过其受体结合域S1-RBD与宿主细胞受体ACE2结合引起感染。
ACE2为I型跨膜糖蛋白，由805个氨基酸组成，包括跨膜区、胞内羧基端和胞外氨基端，冠状病毒通过其S1-RBD与ACE2的细胞外催化结构域互动结合，导致细胞内吞、膜融合，使病毒进入表达ACE2或含有ACE2受体的细胞。
RNA干扰(RNAi)作为一种高效的序列特异性基因沉默技术，正在给疾病的治疗带来难以想象的应用前景，已有多种siRNA药物被FDA审批上市。小干扰RNA(siRNA)大约21～23bp，以参与RNA干扰(RNAi)的方式调节基因表达,特异性降解与之互补的靶信使RNA(mRNA)，但无论是细胞水平还是活体内，使用siRNA进行基因干扰均要克服很多困难：1)膜通透性：siRNA带有大量的负电荷，分子量大(～13KD)，自身很难穿过细胞膜，运输siRNA主要靠化学修饰和一些运输载体；2)抗核酸酶降解：siRNA由大量的核糖核酸分子构成，很容易被外界的RNA酶降解，如果在设计siRNA及选择运输载体时不对碱基进行特定的化学修饰或采取载体保护方法，则在siRNA进入作用位点前即被RNA酶降解；3)靶向递送及载体：siRNA的作用位点主要在靶细胞浆内，所以需将siRNA特异性递送给靶细胞浆，如果不能有效地选用合适的靶向递送载体并及时使siRNA从内涵体释放到胞浆，通常可激活细胞免疫反应，导致干扰素等细胞因子的释放，所以，如何有效地将siRNA运输并释放到靶细胞浆是影响RNAi效果的瓶颈问题，有些siRNA能导致序列或浓度依赖性非特异性基因沉默即脱靶，因此，在设计siRNA时，应同时考虑靶向递送、基因抑制效果以及尽可能选择低脱靶效应的序列。
在COVID-19的防治中，如果能以合适的靶向递送载体将nCoVsiRNA稳定地、特异地依次递送给靶器官、靶组织、靶细胞、定位于靶细胞、穿越靶细胞膜、释放到靶细胞浆，并对多种变异毒株广谱有效，就应能更好地开展COVID-19的靶向基因治疗。
所以本发明要设计和合成以ACE2中和RBD及靶向递送shRNA的ACE2siRNA。
发明内容
本发明的目的是要提供一种以ACE2靶向递送shRNA的ACE2siRNA药物及其合成方法和用途，使双价ACE2能与RBD结合而起中和病毒和靶向递送shRNA作用，并使被ACE2靶向递送的shRNA间接结合RBD而形成“shRNA-ACE2-RBD-病毒”的复合物，进而使shRNA被复合物靶向递送，随着病毒的感染进入靶细胞，起广谱而靶向的抗变异毒株作用。
本发明的目的通过以下技术方案实施：
筛选共有基因：从数据库记载的各种致病性冠状病毒及其变异毒株中筛选不随病毒变异而改变的共有基因，包括保守基因、超保守基因和/或保守微卫星。
筛选siRNA：从共有基因中预选多对以该共有基因为干扰靶标的siRNA，使siRNA包含有保守基因、超保守基因和/或保守微卫星。
合成siRNA：合成2条互补的21-25nt的寡核苷酸siRNA及起间隔作用的碱基序列。
合成shRNA：将已合成的2条互补寡核苷酸多肽siRNA和起间隔作用的碱基序列进一步合成由中间碱基序列间隔成loop环的小发夹shRNA双链。
优选siRNA：将合成的shRNA构建干扰载体，检测其mRNA表达、蛋白表达和干扰效果，经siRNA设计、合成、筛选、迭代设计和验证，优选具有高沉默效率的siRNA。
合成优选的siRNA和shRNA：采用优选的siRNA序列按上述合成siRNA、shRNA，包括为增加稳定性和避免脱靶进行的化学修饰。
合成ACE2多肽或蛋白：合成的ACE2包括但不限于全长ACE2、第741-763位氨基酸的跨膜ACE2、第764-805位氨基酸的胞内ACE2、第1-740位氨基酸的胞外ACE2以及经氨基酸序列密码子优化的ACE2多肽或蛋白。
ACE2siRNA的合成：以二硫键、磷酸二酯键、二硫代磷酸脂键、硫醚键、肟键、酰胺键或马来酰亚胺-巯基键等偶联方法将已合成的shRNA和ACE2连接、合成为化合物；或根据shRNA的核苷酸序列和ACE2的氨基酸序列从氨基酸水平直接合成ACE2-shRNA-ACE2。
ACE2siRNA的提纯：以高效液相色谱、反向高效液相色谱或离子交换色谱进行提纯。
ACE2siRNA的脂质体修饰：通过带负电荷的shRNA吸附带正电荷的脂质体制备脂质体修饰化合物；通过ACE2氨基的巯基化使巯基与脂质体的马来酰胺形成马来酰亚胺-巯基键制备PEG内化的脂质体修饰化合物；通过ACE2氨基与脂质体形成氨甲酸酯键制备脂质体修饰化合物；通过ACE2或ACE2片段连接脂质体修饰的siRNA制备脂质体修饰化合物。
ACE2siRNA的验证：在体外细胞水平检测ACE2siRNA对2种或以上不同变异毒株的抗病毒效果，观察是否具有以保守基因为靶标的广谱抗变异毒株作用；检测ACE2siRNA在动物体内是否具有靶向递送shRNA的作用以及是否能刺激宿主产生ACE2-Ab。
本发明的有益效果在于：
首次设计抗冠状病毒变异靶标siRNA/shRNA，首次以ACE2作为靶向递送载体，首次以shRNA和ACE2合成具有靶向递送、中和病毒和产生ACE2-Ab作用的ACE2siRNA。
本发明基于保守基因为不同毒株的共有基因从不随病毒变异的病毒保守基因、超保守基因和/或保守微卫星中筛选siRNA，并以这种siRNA合成广谱抗变异毒株靶标shRNA。
进而，基于配体RBD和受体ACE2的关系设计以ACE2靶向递送shRNA的ACE2siRNA。
进而，以shRNA和ACE2合成ACE2siRNA，使之由短发夹shRNA区和双链ACE2区构成，其中ACE2起递送shRNA和结合RBD的作用，以ACE2桥接形成的“shRNA-ACE2-RBD-病毒”使shRNA随病毒感染进入靶细胞，这种以ACE2和病毒共同递送shRNA的新方法，不但可避免将shRNA非特异性递送给未感染细胞的副作用，而且还产生特异而广谱抗变异毒株的作用。
进而，原本siRNA/shRNA带负电荷、脂溶性，通透性和稳定性差，将siRNA/shRNA和ACE2合成ACE2siRNA后，则优化了siRNA/shRNA的通透性和稳定性，易被递送。
进而，以2个ACE2分子和1个shRNA分子合成的ACE2siRNA，因ACE2以双价结合RBD，所以实质上是以双价ACE2中和病毒，从而阻断病毒感染靶细胞。
进而，因ACE2siRNA中含有2个ACE2分子和1个shRNA分子，以及shRNA除了抗病毒外还能产生免疫佐剂的作用(shRNA为寡核苷酸，而寡核苷酸也是主要佐剂之一)，所以ACE2siRNA比单分子ACE2的抗原性更强，能刺激宿主产生更高效价的ACE2-Ab。因ACE2-Ab能与病毒竞争靶细胞的ACE2受体，所以ACE2siRNA(ACE2-Ab)具有阻断病毒感染的作用。
进而，脂质体修饰除了能使shRNA在体内缓慢释放、延长药效和内吞入浆等作用外，还能作为免疫佐剂增强ACE2siRNA(ACE2)的免疫效果。
进而，体外细胞实验表明合成的ACE2siRNA对2种不同变异毒株同时有效，说明具有以保守基因为靶标的抗变异毒株作用；体内动物实验表明ACE2siRNA在动物体内具有靶向递送的RNAi作用，刺激产生的ACE2-Ab可通过封闭ACE2受体而抑制病毒感染。
附图说明
图1是本发明制备ACE2siRNA的技术线路图。
图2是本发明ACE2siRNA的结构示意图
图3是本发明ACE2siRNA及其应用示意图。
图4是本发明的ACE2靶向递送脂质体修饰shRNA示意图。
图5是本发明的ACE2靶向递送脂质体修饰siRNA示意图。
在图1中，经保守基因筛选、以保守基因为靶标的广谱抗变异毒株靶标siRNA筛选、shRNA合成、ACE2合成，最终合成ACE2siRNA或ACE2shRNA。
在图2中，1为loop环，2为由两条互补的正反义链形成的shRNA，3为两条ACE2多肽(蛋白)，两条ACE2多肽分别与shRNA的正反义链相连接。shRNA被ACE2保护并被ACE2靶向递送至病毒受体结合域RBD或ACE2受体通道，然后随病毒RBD通过ACE2通道而特异性进入靶细胞浆，降解病毒靶基因。
在图3中，1是loop环，由间隔小发夹shRNA正反义链的碱基序列间隔形成；2是shRNA，由两条正反义链siRNA互补结合形成，该siRNA以冠状病毒保守基因为干扰靶标，具有广谱抗变异毒株的靶向基因治疗作用；3是与shRNA的正反义链相连接的ACE2；4是冠状病毒；5是冠状病毒S蛋白受体结合域RBD；6是细胞7表达的ACE2受体；8是不表达ACE2的细胞。如图2所示，由loop环1、shRNA 2和ACE2 3构成ACE2siRNA；冠状病毒4通过其受体结合域RBD5与受体ACE2 6特异性结合而感染细胞7，但冠状病毒4不会感染不表达ACE2的细胞8；ACE2siRNA中的ACE2 3与细胞7表达的ACE2 6一样，也能与冠状病毒4的RBD 5结合，所以ACE2 3能与ACE2 6竞争结合RBD 5，使ACE2 3具有抑制病毒4感染细胞7的作用；ACE2siRNA在后期又起疫苗的作用，因为ACE2 3能刺激宿主产生抗-ACE2,抗-ACE2能封闭ACE2 6，从而产生抑制病毒4感染细胞7的作用；更重要的是，通过ACE2 3的桥接，形成了“shRNA 2-ACE23-RBD 5-病毒4”的复合物，使shRNA 2随病毒4进入细胞7，靶向干扰病毒4在细胞7中复制，避免因shRNA 2非特异性进入未感染病毒4的细胞8而产生的毒副作用。
在图4中，1和2分别为被脂质体4包裹的loop环和shRNA，其中shRNA由两条正反义链siRNA互补结合形成，该siRNA以冠状病毒保守基因为干扰靶标；3为ACE2，裸露于脂质体4外，起到靶向递送作用；脂质体4起保护shRNA和引起细胞内吞作用；5为PEG，PEG可使siRNA缓慢释放和长效循环；如图3构成ACE2-shRNA/Lip的复合物。
在图5中，1为被脂质体包裹的siRNA，2为脂质体层，3为PEG层，4为ACE2。其中siRNA起RNAi作用，脂质体起保护siRNA和引起细胞内吞作用，PEG使siRNA缓慢释放和长效循环，ACE2在最外层，起到靶向递送siRNA的作用，如图4构成ACE2-siRNA/Lip的复合物。
具体实施方式
下面结合图1，2、3、4和5，对本发明的具体实施方法作详细的描述，但这些范例性的描述并不对本发明的权利要求所限定的保护范围构成任何限制。
一、设计以超保守基因、保守基因或保守微卫星为靶标的siRNA
1、超保守基因、保守基因及保守微卫星的设计
如技术线路图1所示，从Genbank数据库(http://www.NCBI.nlm.nih.gov/genome/)中下载β冠状病毒属(特别是新型冠状病毒及其变异毒株)全基因组(cDNA)序列，在全基因组序列中搜寻最长的公共子序列，获得超保守基因或保守基因；利用Clustal W软件对Genbank数据库下载的全基因组进行序列比对，检测不同序列之间的相似度，筛选保守微卫星序列；利用MEGA6.0分子进化遗传分析软件，用邻接法(Neighbour-Jioning，N-J)对下载的冠状病毒氨基酸序列构建氨基酸种系分子进化树，对氨基酸序列的分子变异特征进行分析，推断保守基因序列。
共找到以下3段最长及次长的超级保守子序列(没有插入或删除的完全相同的子序列)，其长度为22～30bp,与小RNA长度相当，但在高等生物特别是人类中不包含这3段子序列。
具体序列如下：
SEQ ID NO.1(Subsequence 1)＝ttaatacgacctctctgttggattttgaca(30bp)；
SEQ ID NO.2(Subsequence 2)＝ggttcgcaacttcacaca gagt(22bp)；
SEQ ID NO.3(Subsequence 3)＝caggcgtttgttggttgattaa(22bp)。
共找到以下3段最长及次长的保守子序列，它们的长度为22～30bp,与小RNA长度相当，但在高等生物特别是人类中不包含这3段保守子序列：
SEQ ID NO.4(Subsequence 1)＝gttttacgacaacgatgttggtttaggaca(30bp)；
SEQ ID NO.5(Subsequence 2)＝ggttcggttgttatatacgata(22bp)；
SEQ ID NO.6(Subsequence 3)＝ggttcagagagtctcctattta(22bp)。
共找到以下5个由核苷酸重复多次的保守微卫星位点，微卫星分别为CTCTCT、AGAGAG、AAAAAAA、TATATA、CACACA。
2、siRNA的常规设计及其常见变异株间的比对
根据上述从Genbank数据库(http://www.NCBI.nlm.nih.gov/genome/)下载的β冠状病毒属(特别是新型冠状病毒及其变异毒株)的完整基因组(cDNA)序列，利用Ambion公司的shRNA在线设计软件(http://www.ambion.com/techlib/misc/siRNAtools.html)获得长度约为19nt的多个siRNA备选序列，根据RNA结合的Tm值及特异性比对结果，优选siRNA。例如，据此本申请从最早出现的WUHAN株(NC_045512.2)、最常见的DELTA变异株、最近出现的OMICRON变异株的E、M、N、ORF1ab及S基因中分别优选到表1-5所示的候选siRNA，表中“钭黑体”序列为三种毒株的共有siRNA序列(如表1中的SEQ ID NO.9～11)，即尽管最早出现的WUHAN株变异为DELTA株和最近的OMICRON株，但是各毒株中仍保持不变且理论上具有靶向干扰作用的保守序列(siRNA)，如果靶向干扰这些siRNA序列，即可广谱杀灭相应的变异毒株。
表1新型冠状病毒WUHAN株、DELTA株、OMICRON株E基因的siRNA候选序列
表2新型冠状病毒WUHAN株、DELTA株、OMICRON株M基因的siRNA候选序列
表3新型冠状病毒WUHAN株、DELTA株、OMICRON株N基因的siRNA候选序列
表4新型冠状病毒WUHAN株、DELTA株、OMICRON株ORF1ab基因的siRNA候选序列
表5新型冠状病毒WUHAN株、DELTA株、OMICRON株S基因的siRNA候选序列
3、筛选以超保守基因、保守基因或保守微卫星为靶标的siRNA
利用Clustal W软件或其他软件，将上述设计的超保守基因、保守基因及保守微卫星与常规筛选的siRNA进行基因序列比对，检测不同序列之间的相似度，设计既为超保守基因、保守基因或保守微卫星，又为RNAi靶位点的多对siRNA(设计以超保守基因、保守基因或保守微卫星为靶标的siRNA)。
(1)以超保守基因和保守微卫星为靶标的siRNA(S1/S2)：
SEQ ID NO.1(Subsequence 1)＝ttaatacgacctctctgttggattttgaca(30bp)；
SEQ ID NO.2(Subsequence 2)＝ggttcgcaacttcacaca gagt(22bp)；
(2)以保守基因和保守微卫星为靶标的siRNA(S3/S4)：
SEQ ID NO.5(Subsequence 3)＝ggttcggttgttatatacgata(22bp)；
SEQ ID NO.6(Subsequence 4)＝ggttcagagagtctcctattta(22bp)。
通过上述设计，获得以超保守基因、保守基因或保守微卫星为干扰靶标的在理论上抗冠状病毒变异毒株的siRNA，命名为siRNA1/2/3/4。
二、验证以超保守基因、保守基因或保守微卫星为靶标的siRNA功能
1、合成siRNA/shRNA
根据pSilencer4.1.CMV.neo干扰载体的多克隆酶切位点设计能表达发夹结构的shRNA模板，每个模板由两条大部分互补的55bp的单链DNA构成，退火互补后能形成带有BamH I和Hind III酶切位点粘性末端的DNA双链，用于与线性化的pSilencer4.1.CMV.neo的连接。然后按设计的siRNA及其shRNA模板，委托公司合成，所合成的siRNA/shRNA序列如下：
SEQ ID NO.1(Subsequence 1)＝ttaatacgacctctctgttggattttgaca(30bp)；
SEQ ID NO.2(Subsequence 2)＝ggttcgcaacttcacaca gagt(22bp)；
SEQ ID NO.5(Subsequence 3)＝ggttcggttgttatatacgata(22bp)；
SEQ ID NO.6(Subsequence 4)＝ggttcagagagtctcctattta(22bp)。
2、shRNA表达载体的构建
将上述合成的shRNA分别与线性化的干扰载体pSilencer4.1.CMV.neo进行连接和鉴定，构建shRNA表达质粒，转化DH5a，获得shRNA表达载体。
3、shRNA表达(干扰)载体的效果鉴定
根据合成的siRNA/shRNA序列，构建荧光标签载体，并分别与shRNA表达质粒共转染293T细胞，进行鉴定，或以PCR扩增shRNA。常规方法如下：
shRNA引物设计：参照网上在线引物设计软件，设计上、下游引物，在上游引物5'端添加起始密码，为将扩增产物克隆到pEGFP-N1中，引物的5'端添加用于与载体发生同源重组的同源臂。
shRNA基因扩增：按上海生工试剂盒所提供的基因扩增反应体系及反应条件进行基因扩增、产物回收和纯化，获得扩增产物。
pEGFP-N1的线性化：复苏含有pEGFP-N1质粒的DH5a菌种，按试剂盒提取质粒，测定浓度后进行酶切，0.8％琼脂糖凝胶电泳鉴定并回收线性化的载体。
将扩增的shRNA与荧光标签载体(pEGFP-N1)进行连接：使用金斯瑞公司的同源重组试剂盒进行连接，连接结束后可保存在-20℃备用或马上进行转化。
shRNA干扰载体的效果鉴定：分别将干扰载体(pSilencer-shRNA1/2/3/4)和荧光标签载体(pEGFP-shRNA1/2/3/4)共转染293T细胞，干扰载体与标签载体的质量比为1：2，同时设立对照，转染后48h观察细胞内GFP蛋白的融合表达，根据荧光强度评价干扰效果：
流式细胞检测：为定量分析不同干扰载体的干扰效果，用流式细胞术检测，分析荧光蛋白表达细胞在总细胞数中的比例。
Westernbolt分析：①细胞收集与裂解：以RIPA裂解细胞。②SDS-PAGE蛋白电泳：制备SDS-PAGE胶，将样品加入等体积的2xSDS缓冲液，沸水煮5min，冰浴2min，12000xg，10min。③Western blot检测：经转膜、封闭、一抗结合、洗涤、二抗结合和显色，观察结果。
RT-PCR检测mRNA：采用相对荧光定量RT-PCR法检测转染细胞中基因的相对表达量，根据标准曲线，由CT值换算目的基因以及B-actin内参基因拷贝数，以B-actin内参基因校正病毒基因mRNA相对表达量(目的基因拷贝数/B-actin拷贝数)，定量评价干扰效果。
4、获得高沉默效率的siRNA/shRNA
经上述设计、合成、筛选、迭代设计、合成和验证，获得以超保守基因、保守基因或保守微卫星为靶标的siRNA/shRNA：
SEQ ID NO.1(Subsequence 1(shRNA1))＝ttaatacgacctctctgttggattttgaca；
SEQ ID NO.2(Subsequence 2(shRNA2))＝ggttcgcaacttcacaca gagt；
SEQ ID NO.5(Subsequence 3(shRNA3))＝ggttcggttgttatatacgata。
三、合成以保守基因为靶标的shRNA
根据上述筛选的以超保守基因、保守基因或保守微卫星为靶标的shRNA1/2/3，委托生物公司，每个shRNA合成2条互补的19-25nt的寡核苷酸多肽siRNA，以及合成起间隔作用的9nt的碱基序列，然后将所合成的siRNA和碱基序列连接成由中间碱基序列间隔成loop环的小发夹shRNA双链，所合成的shRNA双链的每条单链均可以分别连接RBD多肽或蛋白。
例如，分别将SEQ ID NO.1(shRNA1)、SEQ ID NO.2(shRNA2)和SEQ ID NO.5(shRNA3)合成5'-ttaatacgacctctctgttggattttgacattcaagagatgtcaaatccaacagagaggtcgtattaa-3'(SEQ ID NO.42)、5'-ggttcgcaacttcacacagagtttcaagagaactctgtgtgaagttgcgaacc-3'(SEQ ID NO.43)和5'-ggtt cggt tgtta tatac gata ttcaagaga tatc gtatataaca accg aacc-3'(SEQ ID NO.44)，其中“TTCAAGAGA”为loop环，其左、右侧分别为互补的正反义链，SEQ ID NO.42～44可分别合成shRNA1、shRNA2和shRNA3。同理从表1-5中进一步优选高沉默效率的siRNA，合成shRNA，进而在其3'和/或5'连接RBD蛋白或其多肽。
四、靶向递送载体ACE2的设计和合成
1、ACE2的氨基酸序列
从GeneBank数据库(http://www.ncbi.nlm.gov/genbank)中查找人ACE2基因序列信息，ACE2由805个氨基酸组成，其中第1-740位氨基酸序列位于细胞膜外，第741-763位氨基酸位于跨膜区，第764-805位氨基酸位于细胞内。在构成ACE2蛋白的20种氨基酸中，亮氨酸占9.4％，半胱氨酸和组氨酸分别占1.0％和2.0％，以及带负电荷氨基酸残基(天冬氨酸+谷氨酸)和带正电荷氨基酸残基(精氨酸+赖氨酸)等，SARS-CoV及SARS-CoV 2通过病毒受体结合区与ACE2的细胞外催化结构域互动结合，这种互动能够导致细胞内吞、膜融合，从而使得SARS-CoV进入高表达ACE2或含有ACE2受体的细胞内。
2、ACE2的受体作用
ACE2为脂溶性的I型跨膜糖蛋白，有氨基末端催化结构域和羧基末端结构域，其中N端在细胞膜外，C端在细胞膜内，分为N末端信号肽区、羧基多肽酶活化区和跨膜区。当冠状病毒Spike蛋白接触ACE2催化结构域的亚结构域I的顶端(不影响亚结构域II和肽酶活性)时，ACE2的外结构域被裂解，跨膜结构域被内在化，使病毒-宿主细胞进一步融合，所以认为跨膜区与病毒-受体复合物从细胞膜转运到细胞质有关。
3、本申请ACE2的设计
从ACE2的氨基酸序列及ACE2的受体作用可知，ACE2表达细胞的细胞壁和细胞膜均存在ACE2受体通道，全长ACE2、跨膜区ACE2、胞内ACE2及胞外ACE2均属于穿膜多肽，具有靶向递送siRNA的功能，胞外ACE2的N端具有结合冠状病毒RBD的中和病毒功能。可设计并合成全长ACE2(SEQ ID NO.45)、第1-740位氨基酸的胞外ACE2(SEQ ID NO.46)、第741-763位氨基酸的跨膜ACE2(SEQ ID NO.47)、第764-805位氨基酸的胞内ACE2(SEQ ID NO.48)，以及优化氨基酸序列的ACE2多肽作为siRNA的靶向递送载体。
4本申请ACE2的合成
两个氨基酸脱水缩合形成肽键，多个氨基酸残基以肽键相连接形成多肽。可委托公司采用多肽合成仪自动化合成多肽，基本方法是，按合成多肽的序列逐个加入氨基酸，使肽链从C端到N端残基逐步延长，要求每一个氨基酸残基以一端保护和另一端活化的形式缩合，并且在每一轮肽链延长循环后除去氨基上的临时性保护基团，直至目标多肽的全部氨基酸序列缩合完毕。目前常用的固相合成多肽的反应原理是在密闭的防爆玻璃反应器中使氨基酸按照已知的序列从C端-羧基端向N端-氨基端的顺序不断添加所需氨基酸，进行合成反应，最终得到多肽。其步骤包括：①去保护：用碱性溶剂去除氨基的保护基团；②激活和交联：活化下一个氨基酸的羧基，使活化的单体羧基与游离的氨基交联，形成肽键，反复循环这两步反应，直到多肽合成完成。
五、以ACE2和shRNA合成ACE2siRNA
1、ACE2siRNA的设计
以胞外ACE2和shRNA合成为例，根据SEQ ID NO.1～2、SEQ ID NO.5、SEQ ID NO.7～39、SEQ ID NO.42～44及ACE2序列(SEQ ID NO.45～48)，将上述合成的siRNA/shRNA的正反义链的一端与loop环(5'-TTCAAGAGA-3’)相连，另一端分别与胞外ACE2(C端)相连，连接成“胞外ACE2-siRNA正义链-loop环-siRNA反义链-胞外ACE2”的结构，其中两条互补的正反义链会形成双链，但两条ACE2多肽不会形成双链，如图2所示，是带有两条ACE2多肽、两条siRNA正反义链和loop环的发夹状连接物shRNA。因为病毒RBD通过C端结合ACE2的N端感染细胞，以及ACE2多肽与siRNA的结合可增加siRNA的通透性、稳定性及干扰效果，所以如图3所示，本设计中的ACE2不但能中和病毒、阻止病毒感染，而且能将siRNA/shRNA靶向递送给病毒受体结合域RBD，形成“shRNA-ACE2-RBD-病毒”的复合物，进而使shRNA被病毒递送，并使shRNA随着病毒的感染进入靶细胞，发挥靶向干扰作用。
2、胞外ACE2-shRNA-胞外ACE2(缩写为2ACE2-shRNA)的合成
可委托公司，按照设计(图2)，根据SEQ ID NO.42～48，采用多肽与寡核苷酸的常规合成方法，将上述合成的多肽(ACE2)和寡核苷酸(shRNA/siRNA)以羧腙键、肟键、酰胺键、硫醚键、二硫键、磷酰键、腙键、酰脲键、磷酸二酯键、二硫代磷酸脂键、马来酰亚胺-巯基键等形式偶联成缀合物，包括多肽和寡核苷酸的正义链(5'末端、3'末端)或反义链(3'末端)以较牢固的共价键、较松散的离子键、疏水键或以带间隔臂的羧腙键进行非共价或共价交联，合成多肽-寡核苷酸偶联物(POCs)。目前最常用的合成POCs法是共价交联-液相片段合成法，已广泛应用于合成各种POCs，其主要步骤是：分别在固相基质上分别合成多肽和寡核苷酸，然后同时将两个合成物从固相基质上剥离，所剥离的多肽和寡核苷酸在溶液中通过反应活性基团进行偶联。合成POCs主要包括：①马来酰亚胺-巯基偶联：在多肽或寡核苷酸上修饰马来酰亚胺，在另一单体上修饰巯基，然后将两个单体加入同一溶液中即可反应得到POCs；②二硫键或硫醚键偶联：用巯基对寡核苷酸的5'或3'位进行修饰，然后与C端用溴乙酰基进行修饰的多肽在pH7.0的缓冲溶液中进行反应；二硫键偶联可以通过两个巯基直接氧化，或先通过联吡啶二硫醚等活化剂将巯基活化再与另一含有巯基的寡聚物偶联，常用二硫键合成siRNA与多肽的偶联物；③肟键偶联：醛基与氨基相互反应产生肟，该反应条件温和、反应效率高，并且可以直接生成双链DNA与特定多肽的偶联产物，同时，还可以通过双功能化的寡核苷酸与多肽或糖类通过肟键在核酸的5’与3’端同时连接上两条多肽，该方法不需要各种保护过程并能一步完成，用于合成了“肽-寡核苷酸-肽”产物，具体方法为在寡核苷酸的5’和3’均引入醛基，再与羟基胺修饰的多肽反应，得到“肽-寡核苷酸-肽”，且所得产率较高，这种双官能团化的寡核苷酸与多肽的一步反应不需要任何保护策略和交联剂，在微酸的条件下就能得到较高的产率；④酰胺键偶联：直接利用含有活化羧酸或硫酯的寡聚物与另一修饰有氨基的聚合物反应得到产物；⑤腙键偶联：需要先将肼基引入到多肽上，然后加入pH值为3～5之间的柠檬酸缓冲溶液，再与修饰有乙酰醛基的寡核苷酸进行反应，才可得到以腙键连接的POCs，获得靶向目标基因的化合物。
3、胞外ACE2-shRNA-胞外ACE2的提纯
色谱方法一直是纯化和分析多肽与寡核苷酸偶联物的最常用的方法之一。根据偶联物的复杂程度需选择不同的色谱方法进行分离，主要方法有高效液相色谱(HPLC)、反向高效液相色谱(RP-HPLC)，离子交换色谱(IEC，通常为阴离子交换色谱)，或者将其中的两个或几个进行串联使用，具体按操作说明。将“胞外ACE2-shRNA-胞外ACE2”缩写为2ACE2-shRNA。
4、以ACE2靶向递送shRNA的其他化合物的合成
同理可将shRNA(SEQ ID NO.7～39)与胞外ACE2、跨膜ACE2、胞内ACE2或密码子优化的ACE2多肽连接成化合物，包括但不限于“跨膜ACE2-shRNA-跨膜ACE2”、“胞内ACE2-shRNA-胞内ACE2”；或将shRNA/siRNA插入到ACE2多肽中间，合成“跨膜ACE2-shRNA-胞外ACE2”、“胞内ACE2-shRNA-胞外ACE2”；也可同理设计以ACE2或其优化的多肽为靶向递送载体的化合物，包括但不限于“ACE2-siRNA、胞外ACE2-siRNA、跨膜ACE2-siRNA、胞内ACE2-siRNA”。
六、化合物的脂质体修饰
脂质体(Liposomes，简称Lip)修饰是为了运输和稳定shRNA，包括以带负电荷的shRNA吸附带正电荷的脂质体、以ACE2多肽中的氨基的巯基化使巯基与脂质体形成巯基-马来酰亚胺键或以ACE2氨基与脂质体形成氨甲酸酯键等方法(图4-5)。
实施例1：以脂质体DOTAP/Chol制备脂质体修饰化合物
(1)ACE2-shRNA-ACE2的合成
合成siRNA、shRNA，进而以shRNA和ACE2合成ACE2-shRNA-ACE2。
(2)脂质溶液的配制
DOTAP(MW＝698.5)：10mg/ml，精确称取DOTAP[N-(2,3-二油酰氧基-1-丙基)三甲基甲磺酸铵：N-1-(2,3-di-oleoyloxy)propyl)-N,N,N-trimeth ylammoniumethylsulfate]粉末100mg，加入10ml容量瓶中，加氯仿溶液至刻度线。
Chol(MW＝386)：5mg/ml，精确称取Chol[胆固醇：cholesterol]粉末50mg，加入10ml容量瓶中，加氯仿溶液至刻度线。
m-PEG2000-DSPE(MW＝2787)：10mg/ml，称取m-PEG2000-DSPE(甲氧基化聚乙二醇二硬脂酰磷脂酰乙醇胺：Methoxy-polyethylene glycol-distearoyl phosphatidyi-ethanolamine)粉末10mg，加入lml DEPC水，涡旋后超声1min，使之彻底溶解。
Mal-PEG2000-DSPE(MW＝2941.6)：10mg/ml，精确称取Mal-PEG2000-DSPE(Maleimide derivatized polyethylene glycol-distearoyl phosphatidyl-ethanolamine：马来酰亚胺化聚乙二醇二硬脂酰磷脂酰乙醇胺)粉末10mg，加1ml DEPC水，涡旋后超声1min，溶解。
(3)脂质体DOTAP/Chol的制备(薄膜水化法)
采用薄膜水化法(Lipid-film method)制备脂质体DOTAP/Chol，脂质浓度为10mM，主要步骤如下：按照制备1ml脂质体DOTAP/Chol的量，分别取DOTAP、Chol两种脂质的氯仿溶液，以DOTAP：Chol＝1：1(M：M)的比例加至500ml三角烧瓶，加3～4ml氯仿溶液；37℃真空旋转蒸发45～60min，使成为均匀的脂质薄膜，用高纯氮气吹尽痕量氯仿；加入1ml DEPC水振摇，将脂质薄膜从瓶壁洗下，得脂质混悬液；待充分水合后，超声lmin，依次通过400nm、200nm、80nm、50nm聚碳酸酯膜挤出，各10～20次，即得脂质体DOTAP/Chol。
(4)ACE2和2ACE2-shRNA的巯基化
2-IT(Traut'S reagent)是蛋白巯基化的常用试剂，可在ACE2蛋白N氨基处进行巯基化，步骤如下：取2ACE2-shRNA和2-IT(Traut'S reagent，2-iminothiolane-HCl)，混合均匀(2-IT与2ACE2-shRNA的摩尔比为200：1)，室温条件下反应2h；通过透析法除去多余的2-IT，每次以足量透析液(1×PBS，5mM EDTA，pH＝7.4)，4℃保存，隔夜透析，磁力搅拌，6～8h换透析液；分别采用BCA法和Ellman法测定蛋白浓度和巯基化程度。
(5)以脂质体DOTAP/Chol制备脂质体修饰化合物
(A)利用带负电何的siRNA/shRNA吸附带正电荷的脂质体进行制备
①取120μl的DOTAP/Chol脂质体(10mM)，加入20μ1的2ACE2-shRNA(约2μg/μ1，40μg)，加DEPC水11μl，室温静置10min，获得脂质体修饰复合物2ACE2-shRNA/ch1。
②取siRNA 90μ1(24μg，20mM)或shRNA 90μ1(24μg，10mM)，加DEPC水57.6μ1，室温静置10min,加入600μl的DOTAP/Chol脂质体(50mM)，获siRNA/ch1和shRNA/ch1。
③将①制备的脂质体修饰复合物2ACE2-shRNA/ch1与②制备的脂质体修饰复合物siRNA/ch或shRNA/ch等量混合，获得相应的混合型脂质体修饰复合物。
(B)以ACE2中的巯基与PEG中的马来酰亚胺的交联反应进行制备
为了增长脂质体的循环时间和靶向特异性，对(A)制备的各种脂质体修饰复合物做进一步的PEG化和ACE2修饰，获得以RBD为配体的PEG化脂质体修饰复合物。
取9.53μl(设对照6.36μl和12.7μl)的10mg/ml的MAL-DSPE-PEG，分别将其插入上述(A)制备的脂质体修饰复合物2ACE2-shRNA/ch1、siRNA/ch1或shRNA/ch1中(分别将两者混合)，50℃水浴孵育10min，室温静置10min，然后加入约200μg经上述巯基化的ACE2或2ACE2-shRNA/ch1，使ACE2中巯基化氨基上的巯基与MAL-DSPE-PEG中的马来酰亚胺发生交联反应，分别获得ACE2修饰并经PEG化(PEG化的PEG浓度分别为5mo1％PEG、7.5mo1％PEG和10mo1％PEG)的复合物，即该复合物为先以siRNA、shRNA和/或2ACE2-shRNA静电吸附脂质体DOTAP/Chol，再以MAL-DSPE-PEG包裹在外层，最后以MAL-DSPE-PEG连接ACE2或2ACE2-shRNA，制备脂质体(ch)修饰复合物，包括ACE2-siRNA/ch1、ACE2-shRNA/ch1、ACE2-(2ACE2-shRNA/ch1)、(2ACE2-shRNA)-siRNA/ch1、(2ACE2-shRNA)-shRNA/ch1、(2ACE2-shRNA)-(2ACE2-shRNA/ch1)，分别缩写为ACE2-siRNA/ch1、ACE2-shRNA/ch1、3ACE2-shRNA/ch1、2ACE2-shRNA/siRNA/ch1、2ACE2-2shRNA/ch1、4ACE2-2shRNA/ch1。如图3所示，其中ACE2起靶向递送siRNA/shRNA的作用，脂质体和PEG起保护siRNA/shRNA、缓慢释放siRNA/shRNA、向胞内转染siRNA/shRNA或免疫佐剂的作用。
实施例2：以脂质体liposomal制备脂质体修饰化合物
当pH＞8时，ACE2的氨基与pNP-PEG-DPPE(PEG-PE)反应形成稳定的氨甲酸酯键偶联物，并定向定量地插入脂质体的外层膜，制成脂质体修饰化合物。本实施例以ACE2片段和siRNA制备以ACE2靶向递送siRNA(ACE2-siRNA)的脂质体修饰化合物。
(1)ACE2的合成
采用上述ACE2合成方法合成ACE2或其片段。
(2)pNP-PEG-DPPE的合成
取20mg/mL DPPE(二棕榈酰乙醇胺)氯仿溶液10ml，置50mL圆底烧瓶中，滴加三乙胺(TEA)0.65mL，取约4.0g浓度为200mg/mL的(pNP)2-PEG3400(聚乙二醇3400二对硝基苯碳酸酯)氯仿溶液加入上述混合液中，吹入氮气，密封，避光，于室温下磁力搅拌过夜，减压蒸干溶剂，真空除尽残留氯仿，加入0.01mol/L HCl水溶液100mL，超声处理形成透明的胶束溶液。0.01mol/L HCI水溶液为洗脱液，经CL-4B Sepharose进行分离，除去未反应的(pNP)2-PEG3400和释放的pNP，收集含pNP-PEG-DPPE胶束的洗脱液，冷冻干燥，用TLC、HPLC、MS和NMR对pNP-PEG-DPPE进行定性和定量。
(3)ACE2-PEG-DPPE的合成：取pNP-PEG-DPPE 100mg溶于10mL氯仿中，置50mL烧瓶中，于旋转蒸发仪上减压除去氯仿，形成脂膜，真空除尽残留氯仿，将25mg ACE2溶于0.01mol/LHCl4mL中，加入内壁涂布脂膜的烧瓶中，于室温孵育30min，轻摇使脂膜分散。于混悬液中加10mum/L(pH 9.0)Tris缓冲液20mL，混匀，氮气保护，于4℃孵育过夜。将样品置于分子质量为5kD的透析袋中，在10mmol/L(pH 7.4)Tris缓冲液中透析约4h，再用去离子水于4℃透析24h，取出袋内溶液，冷冻干燥，置-20℃冰箱内保存。
(4)ACE2-siRNA/liposomal的合成：将ePC(蛋黄磷脂)、Ch(胆固醇)、PEG2000-DSPE(二硬脂酰乙醇胺聚乙二醇2000)和DOTAP(二油酰三甲胺丙烷)的氯仿溶液按摩尔比(60:34:3.0:3.0)混合，如需标记脂膜，于上述混合液中加入占总脂质量摩尔比为0.1％Rho-PE，减压除尽氯仿，形成脂膜。将一定量的siRNA溶于经DEPC处理的超纯水中，siRNA用量应完全中和DOTAP所带的正电荷。于50℃水浴中用含siRNA的水溶液将上述磷脂膜水化30min，形成包裹siRNA的脂质体。用手动挤出装置(Avanti Polar Lipids)，将初步形成的脂质体分别过0.4μm和0.1μm的聚碳酸酯核孔膜(Whatman)10次，制备粒径均一的脂质体。取适量ACE2-PEG-DPPE溶于甲醇中，置烧瓶，氮气吹干成膜，加入制备的脂质体悬液，于37℃水浴温浴2h，使ACE2-PEG-DPPE定向地插入到脂质体的外层膜上，并使ACE2占总脂质的摩尔比为0.5％～1.O％(可适当调整)，用动态激光散射、冰冻蚀刻电子显微镜和核酸电泳检验，制成如图3-4所示的脂质体(ch/lip)复合物ACE2-shRNA/siRNA/lip。
所述ACE2siRNA由ACE2或其多肽与shRNA/siRNA合成，及其进一步的脂质体修饰。
七、化合物(ACE2siRNA)的验证
1、体外验证广谱抗病毒效果
(1)病毒液的准备
在Vero E6细胞生长至30％汇合度的DMEM培养液(10％FBS)中加入病毒株，36℃、5％CO2培养箱培养5～7d，至出现细胞病变效应(CPE)时，分离病毒，用培养液配成103～105TCID50/ml病毒液备用。据此分别准备新冠病毒的两种变异毒株B.1.617.1和B.1.617.2的病毒液，用于验证ACE2siRNA是否对2种或以上含有相同保守基因的变异病毒同时有效，以证明本发明的shRNA/siRNA是否具有以保守基因为靶标的广谱抗病毒效果。
(2)化合物(ACE2siRNA)和病毒共培养
分别设立实验组和对照组，试验化合物抗B.1.617.1和B.1.617.2的效果。每组接种8孔板，每孔2×105个Vero-E6细胞、2mL DMEM培养液(10％FBS)，置36℃、5％CO2培养箱中培养至30％汇合度时更换培养液，同时加入试验化合物、B.1.617.1和B.1.617.2株病毒液。
其中实验组包括:2ACE2-shRNA组(0.1nmol 2ACE2-shRNA+0.6ml病毒液)、2ACE2-shRNA/ch1组(0.1nmol 2ACE2-shRNA/ch1+0.6ml病毒液)、4ACE2-2shRNA/ch1组(0.1nmol4ACE2-2shRNA/ch1+0.6ml病毒液)、ACE2-siRNA/lip组(0.1nmol ACE2-siRNA/lip+0.6ml病毒液)；对照组包括：naked shRNA组(0.1nmol naked shRNA+0.6ml病毒液)、naked siRNA组(0.1nmol naked siRNA+0.6ml病毒液)、ACE2对照组(0.1nmol ACE2+0.6ml病毒液)、阳性对照组(0.6ml病毒液)、阴性对照组(0.6ml DMEM培养液)(表1-6)。
然后于培养1小时、24小时和72小时后每组各取上清液，以1:4、1:12、1:36、1:108、1:324、1:972、1:2916、1:8748倍稀释，进行RT-PCR检测。
(3)实时荧光RT-PCR检测各组病毒RNA
病毒核酸提取和核酸(ORF1ab/N)检测按照试剂盒说明书操作。
(4)病毒RNA检测结果
①B.1.617.1株检测结果
表1所示，各组细胞培养1h后，阴性对照组病毒RNA检测结果为阴性，阳性对照组RNA检测结果的滴度为1:36，其他各组RNA检测结果滴度均为1:12。
表2所示，各组细胞培养24h后，阴性对照组病毒RNA检测结果仍为阴性，各对照组病毒RNA检测结果为1:324～1:2916，而实验组的病毒RNA检测结果均为1:108，明显低于阳性对照组和ACE2对照组(p＜0.01)。
表3所示，各组细胞培养72h后，阴性对照组病毒RNA检测结果仍为阴性，各对照组RNA检测结果滴度＞1:2916，对照组的RNA检测结果滴度＞1:2916～1:8748，而实验组的RNA检测结果滴度为1:324～1:972，明显低于对照组(p＜0.01)。
表1-3说明，实验组具有明显的抗B.1.617.1株作用，说明与ACE2连接的shRNA或siRNA能被递送至靶细胞内进行RNA干扰，而未与ACE2连接的shRNA或siRNA不能进入靶细胞内，在胞外不能发挥RNA干扰的作用。
表1化合物与B.1.617.1株共培养1小时培养液中病毒RNA RT-PCR检测结果(+/-)
表2化合物与B.1.617.1株共培养24小时培养液中病毒RNA RT-PCR检测结果(+/-)
表3化合物与B.1.617.1株共培养72小时培养液中病毒RNA RT-PCR检测结果(+/-)
②B.1.617.2株检测结果
表4所示，各组细胞培养1h后，阴性对照组病毒RNA检测结果为阴性，阳性对照组和ACE2对照组RNA检测结果均为1:36，其他各组RNA检测结果均为1:12。
表5所示，各组细胞培养24h后，阴性对照组病毒RNA检测结果仍为阴性，各对照组RNA检测结果滴度为1:324～1:972，而4组实验组的RNA检测结果滴度为1:36～1:108，明显低于对照组(p＜0.01)。
表6所示，各组细胞培养72h后，阴性对照组病毒RNA检测结果仍为阴性，各对照组RNA检测结果为1:2916～1:8748，而在4组实验组的RNA检测结果中有2组为1:324、另2组为1:972，与对照组相比仍有明显的差异(p＜0.01)。
表4-6说明，实验组具有明显的抗B.1.617.2株作用，说明与ACE2连接的shRNA或siRNA能被递送至靶细胞内进行RNA干扰，而未与RBD连接的shRNA或siRNA不能进入靶细胞内，从而不能发挥RNA干扰的作用。
表1-6表明，实验组(ACE2siRNA)同时具有抗B.1.617.1和B.1.617.2的作用，说明实验组以保守基因为干扰靶标的ACE2siRNA具有广谱抗变异毒株的效果。
表4化合物与B.1.617.2株共培养1小时培养液中病毒RNA RT-PCR检测结果(+/-)
表5化合物与B.1.617.2株共培养24小时培养液中病毒RNA RT-PCR检测结果(+/-)
表6化合物与B.1.617.2株共培养72小时培养液中病毒RNA RT-PCR检测结果(+/-)
2、体内验证ACE2的靶向递送功能
(1)动物分组和接种
动物分组：选择6-8周龄、40克左右的SPF级雌性BALB/c小鼠，随机分为2ACE2-shRNA组(接种2ACE2-shRNA+B.1.617.2)、2ACE2-shRNA/ch1组(接种2ACE2-shRNA/ch1+B.1.617.2)、4ACE2-2shRNA/ch1组(接种4ACE2-2shRNA/ch1+B.1.617.2)，ACE2-siRNA/lip组(接种ACE2-siRNA/lip+B.1.617.2)，shRNA组(接种shRNA+B.1.617.2)阳性对照组(接种B.1.617.2+生理盐水)和阴性对照组(仅接种生理盐水)，每组20只。
动物接种：经鼻腔喷雾接种40μl滴度为105/mlTCID50的B.1.617.2株病毒液，阴性对照组经鼻腔喷雾接种40μl生理盐水。以腹腔注射5％水合氯醛溶液麻醉，分别将0.1nmol的2ACE2-shRNA、2ACE2-shRNA/ch1、4ACE2-2shRNA/ch1、ACE2-siRNA/lip和shRNA缓慢注入小鼠气管，在感染后第7天每组处死10只小鼠，进行病毒检测；另外每组10只用于观察抗体。
(2)以细胞半数感染量(TCID50)的百分率检测病毒
将感染后第7天处死小鼠肺组织制备10％匀浆，各取100pl离心后上清，以10倍递次稀释，接种于VeroE6单层生长的96孔板，每孔30μl，每个稀释度接种4个孔，轻摇匀浆，放37℃吸附lh，以Hank氏液清洗，加培养液，放37℃C02培养箱培养，观察细胞病变效应(CPE)，计算各组VeroE6半数感染量(TCID50)的百分率，百分率越大病毒含量越多(表7-13)。
表7 2ACE2-shRNA组小鼠肺组织匀浆致VeroE6半数感染量的百分率
表8 2ACE2-shRNA/ch1组小鼠肺组织匀浆致VeroE6半数感染量的百分率
表9 4ACE2-2shRNA/ch1组小鼠肺组织匀浆致VeroE6半数感染量的百分率
表10 ACE2-siRNA/lip组小鼠肺组织匀浆致VeroE6半数感染量的百分率
表11 shRNA组小鼠肺组织匀浆致VeroE6半数感染量的百分率
表12阳性对照组小鼠肺组织匀浆致VeroE6半数感染量的百分率
表13阴性对照组小鼠肺组织匀浆致VeroE6半数感染量的百分率
(3)ACE2靶向递送的效果
从表7-13可知，各组小鼠肺匀浆致VeroE6半数感染量的百分率(101孔)分别为：2ACE2-shRNA组为35.0％、2ACE2-shRNA/ch1组为27.5％、4ACE2-2shRNA/ch1组为32.5％、ACE2-siRNA/lip组为30.0％、shRNA组为95.0％、阳性对照组为95.0％和阴性对照组为5.0％。因为RNAi主要发生在细胞浆内，shRNA组中的shRNA不易通过细胞膜，所以几乎不起RNAi作用，其结果与阳性对照一致；而2ACE2-shRNA组、2ACE2-shRNA/ch1组、4ACE2-2shRNA/ch1组和ACE2-siRNA/lip组中的shRNA因被ACE2靶向递送至靶细胞浆，所以其RNAi效果较好，其VeroE6半数感染量的百分率与阳性对照组相比，均具有显著性差异(p＜0.05)。
3、ACE2-Ab的检测及功能验证
(1)样本与检测方法
采集上述另外每组10只小鼠的第2、4、6周静脉血，分离血清，并混合每组相同周次的小鼠血清,保存于-20℃备用。以双抗原夹心法测定ACE2-Ab，具体按照试剂盒操作。
(2)检测结果
由表14可知，各实验组(含有ACE2)小鼠的肺组织匀浆ACE2-Ab显著高于对照组(无ACE2)小鼠的肺组织匀浆ACE2-Ab(p＜0.05)，说明实验组的ACE2能刺激小鼠产生ACE2-Ab，而且产生的ACE2-Ab含量与ACE2分子组成和脂质体修饰有关。
表14各实验组(携带ACE2)小鼠的肺组织匀浆ACE2-Ab检测结果(ng/L)
(3)ACE2-Ab的功能验证
以2ACE2-shRNA组、2ACE2-shRNA/ch1组、4ACE2-2shRNA/ch1组和ACE2-siRNA/lip组为试验组(均含ACE2-Ab)，shRNA组为对照组(含病毒但不含ACE2-Ab)。混合经ACE2-Ab检测后的每组第2、4、6周血清，倍比稀释各组混合血清，然后各取30μl接种于VeroE6单层生长的96孔板，各试验组同时接种shRNA组的未稀释混合血清30μl，阴性对照组仅接种生理盐水30μl。轻摇混匀，置37℃吸附lh，以Hank氏液清洗，加培养液，置37℃C02培养箱培养，1周内观察细胞病变效应(CPE)，以“+”表示细胞正常生长，见表15。
表15因ACE2-Ab中和VeroE6细胞表面ACE2所致的抗病毒感染结果(+/CPE)
由表15可知，阴性对照组因未接种病毒，所以各孔VeroE6均未出现CPE；阳性对照组在无ACE2-Ab中和作用的前提下接种了病毒(shRNA组未稀释混合血清)，所以各孔VeroE6均产生CPE；shRNA组因无ACE2-Ab的中和作用，所以仅在含病毒的自身血清稀释1:128倍后才不使VeroE6出现CPE；而各试验组虽然和阳性对照组一样也接种了病毒，但因具有ACE2-Ab的中和作用，所以在血清即ACE2-Ab含量稀释1:64或以上时才使VeroE6出现CPE。说明ACE2-Ab能中和VeroE6细胞表面的病毒受体ACE2，从而产生有抗病毒作用。
综上，本发明的shRNA起抗变异毒株的作用；ACE2起靶向递送shRNA、双价结合RBD、抑制病毒通过RBD感染、保护shRNA及刺激宿主产生能封闭ACE2的ACE2-Ab的作用；而且多分子ACE2及佐剂作用的shRNA和脂质体均能促进ACE2-Ab的产生和作用。
序列表
<110> 杭州痴创生物科技有限公司
<120> 一种以ACE2靶向递送shRNA的ACE2siRNA药物
<141> 2021-12-31
<150> 2021113298926
<151> 2021-11-11
<160> 48
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Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala
1 5 10 15
Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe
20 25 30
Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp
35 40 45
Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn
50 55 60
Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala
65 70 75 80
Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln
85 90 95
Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys
100 105 110
Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser
115 120 125
Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu
130 135 140
Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu
145 150 155 160
Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu
165 170 175
Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg
180 185 190
Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu
195 200 205
Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu
210 215 220
Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu
225 230 235 240
His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile
245 250 255
Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly
260 265 270
Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys
275 280 285
Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala
290 295 300
Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu
305 310 315 320
Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro
325 330 335
Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly
340 345 350
Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp
355 360 365
Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala
370 375 380
Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe
385 390 395 400
His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys
405 410 415
His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn
420 425 430
Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly
435 440 445
Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe
450 455 460
Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met
465 470 475 480
Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr
485 490 495
Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe
500 505 510
Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala
515 520 525
Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile
530 535 540
Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu
545 550 555 560
Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala
565 570 575
Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe
580 585 590
Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr
595 600 605
Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu
610 615 620
Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met
625 630 635 640
Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu
645 650 655
Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val
660 665 670
Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro
675 680 685
Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile
690 695 700
Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn
705 710 715 720
Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln
725 730 735
Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val
740 745 750
Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg
755 760 765
Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile
770 775 780
Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp
785 790 795 800
Val Gln Thr Ser Phe
805
<210> 46
<211> 740
<212> PRT
<213> Unknown
<400> 46
Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala
1 5 10 15
Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe
20 25 30
Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp
35 40 45
Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn
50 55 60
Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala
65 70 75 80
Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln
85 90 95
Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys
100 105 110
Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser
115 120 125
Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu
130 135 140
Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu
145 150 155 160
Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu
165 170 175
Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg
180 185 190
Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu
195 200 205
Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu
210 215 220
Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu
225 230 235 240
His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile
245 250 255
Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly
260 265 270
Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys
275 280 285
Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala
290 295 300
Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu
305 310 315 320
Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro
325 330 335
Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly
340 345 350
Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp
355 360 365
Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala
370 375 380
Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe
385 390 395 400
His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys
405 410 415
His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn
420 425 430
Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly
435 440 445
Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe
450 455 460
Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met
465 470 475 480
Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr
485 490 495
Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe
500 505 510
Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala
515 520 525
Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile
530 535 540
Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu
545 550 555 560
Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala
565 570 575
Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe
580 585 590
Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr
595 600 605
Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu
610 615 620
Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met
625 630 635 640
Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu
645 650 655
Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val
660 665 670
Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro
675 680 685
Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile
690 695 700
Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn
705 710 715 720
Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln
725 730 735
Pro Pro Val Ser
740
<210> 47
<211> 23
<212> PRT
<213> Unknown
<400> 47
Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val Ile Val Val Gly
1 5 10 15
Ile Val Ile Leu Ile Phe Thr
20
<210> 48
<211> 42
<212> PRT
<213> Unknown
<400> 48
Gly Ile Arg Asp Arg Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn
1 5 10 15
Pro Tyr Ala Ser Ile Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe
20 25 30
Gln Asn Thr Asp Asp Val Gln Thr Ser Phe
35 40
Claims (10)
1.一种以ACE2靶向递送shRNA的ACE2siRNA药物，其特征在于，合成以ACE2中和冠状病毒和靶向递送抗变异靶标shRNA的ACE2siRNA，包括分别合成以冠状病毒保守基因为靶标的shRNA和以冠状病毒受体结合域RBD为配体的ACE2，然后将合成的ACE2分别连接到合成的shRNA的正反义链上，构成由ACE2和shRNA组成的ACE2siRNA，包括进一步的脂质体修饰，使被ACE2靶向递送的shRNA特异性间接结合RBD，形成shRNA-ACE2-RBD-病毒的复合物，进而使shRNA被复合物靶向递送而随着病毒的感染进入靶细胞，起抗变异靶标的RNA干扰作用。
2.根据权利要求1所述的一种以ACE2靶向递送shRNA的ACE2siRNA药物，其特征在于，所述以保守基因为靶标是指用于合成shRNA的siRNA选自数据库记载的各种致病性冠状病毒及其变异毒株的共有基因，使合成的shRNA靶向干扰所述共有基因，从而起广谱抗变异毒株的作用；所述共有基因包括但不限于超保守基因、保守基因和/或由保守微卫星拼接的基因。
3.根据权利要求1、2所述的一种以ACE2靶向递送shRNA的ACE2siRNA药物，其特征在于，所述合成shRNA包括但不限于首先合成2条互补的约21-25nt的siRNA寡核苷酸多肽以及合成起间隔作用的loop环碱基序列，然后将所合成的siRNA和loop环连接成由loop环间隔成的小发夹shRNA双链，继之在shRNA双链的每条单链上各连接ACE2多肽。
4.根据权利要求1、2所述的一种以ACE2靶向递送shRNA的ACE2siRNA药物，其特征在于，所述以保守基因为靶标是指靶向所述保守基因的siRNA序列包括但不限于SEQ ID NO.1～41；所述以超保守基因、保守基因或保守微卫星为靶标的siRNA序列包括但不限于SEQ IDNO.1、SEQ ID NO.2和SEQ ID NO.5；所述保守基因包括但不限于WUHAN株、DELTA变异株和/或OMICRON变异株共有的基因序列，其siRNA序列包括但不限于SEQ ID NO.9～11、SEQ IDNO.16～20、SEQ ID NO.23、SEQ ID NO.26～27、SEQ ID NO.31～33、SEQ ID NO.35或SEQ IDNO.37～39。
5.根据权利要求1、3所述的一种以ACE2靶向递送shRNA的ACE2siRNA药物，其特征在于，所述ACE2siRNA包括将所述ACE2与所述shRNA和/或siRNA连接成化合物，或将所述shRNA和/或siRNA插入到所述ACE2多肽中间，连接成化合物，包括进而以脂质体修饰化合物。
6.根据权利要求1、5所述的一种以ACE2靶向递送shRNA的ACE2siRNA药物，其特征在于，所述ACE2包括全长ACE2、胞外ACE2、跨膜ACE2、胞内ACE2或密码子优化的ACE2多肽。
7.根据权利要求1、6所述的一种以ACE2靶向递送shRNA的ACE2siRNA药物，其特征在于，所述ACE2包括但不限于第1-740位氨基酸的胞外ACE2、第741-763位氨基酸的跨膜ACE2、第764-805位氨基酸的胞内ACE2以及经氨基酸序列优化的ACE2多肽。
8.根据权利要求1、5所述的一种以ACE2靶向递送shRNA的ACE2siRNA药物，其特征在于，所述连接或合成包括但不限于将ACE2与shRNA的反义链3'末端、正义链5'末端或3'末端进行连接，并包括但不限于以带间隔臂的羧腙键、二硫键、磷酸二酯键、二硫代磷酸脂键、硫醚键、肟键、酰胺键、马来酰亚胺-巯基键的化学偶联或共价偶联进行连接。
9.根据权利要求1所述的一种以ACE2靶向递送shRNA的ACE2siRNA药物，其特征在于，所述ACE2siRNA包括在shRNA的正反义链上分别连接ACE2或RBD，或分别连接其多肽。
10.根据权利要求1、5所述的一种以ACE2靶向递送shRNA的ACE2siRNA药物，其特征在于，所述脂质体修饰ACE2siRNA包括但不限于制备ACE2-shRNA/ch1-ACE2、siRNA/ch1和shRNA/ch1、ACE2-siRNA/ch1、ACE2-shRNA/ch1、3ACE2-shRNA/ch1、2ACE2-shRNA/siRNA/ch1、2ACE2-2shRNA/ch1、4ACE2-2shRNA/ch1和ACE2-siRNA/lip。
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| US12144898B2 (en) | 2020-04-09 | 2024-11-19 | Finncure Oy | Virus-like particles for preventing the spreading and lowering the infection rate of viruses |
| US12194157B2 (en) | 2020-04-09 | 2025-01-14 | Finncure Oy | Carrier for targeted delivery to a host |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US12144898B2 (en) | 2020-04-09 | 2024-11-19 | Finncure Oy | Virus-like particles for preventing the spreading and lowering the infection rate of viruses |
| US12194157B2 (en) | 2020-04-09 | 2025-01-14 | Finncure Oy | Carrier for targeted delivery to a host |
| US12311061B2 (en) | 2020-04-09 | 2025-05-27 | Finncure Oy | Methods of fabricating carriers for targeted delivery to a host |
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