CN116875620A - 一种用于递送mRNA的生物制剂及其制备方法 - Google Patents
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Abstract
本发明提供了一种用于递送mRNA的生物制剂及其制备方法,所述的生物制剂为工程细胞所分泌、内部装载特定mRNA的微囊泡,主要涉及基因工程技术领域。本发明主要将装配蛋白连接到微囊泡膜蛋白的C端,将与装配蛋白结合的引导结合序列(一段特殊的RNA茎环结构)插入编码目的mRNA的3’‑非翻译区(3’‑UTR),来设计两种融合基因结构,并以此构建重组慢病毒载体和稳转细胞系,从而生产微囊泡。本发明所构微囊泡,生物相容性好,制备方法简单,可在重组细胞微囊泡生成时,通过装配蛋白和引导结合序列间的相互作用将特定mRNA装载到微囊泡内部。本发明所构微囊泡可将特定mRNA递送到细胞内,可以直接应用于体内。
Description
技术领域
本发明涉及基因工程技术领域,是一种用于递送mRNA的生物制剂及其制备方法。
背景技术
mRNA递送(mRNA delivery)是指将特定的mRNA分子输送到细胞内的过程,通常是为了引导细胞产生特定的蛋白质。这种技术可以用于基因治疗、癌症治疗和疫苗开发等领域。目前,常见的mRNA递送方法包括脂质体转染、电转染、纳米颗粒递送等。脂质体转染是将mRNA包裹在脂质体中,通过脂质体与细胞膜融合实现mRNA的递送。该方法简单易行,适用于多种细胞类型和组织,且对mRNA的保护效果较好。但是,脂质体转染的效率较低,且存在一定的毒性。电转染是通过电场作用将mRNA导入细胞内。该方法操作简单,适用于不易被脂质体转染的细胞类型,如神经元和成骨细胞等。但是,电转染的效率也较低,且对细胞有一定的毒性和损伤。纳米颗粒递送是将mRNA包裹在纳米颗粒中,通过颗粒与细胞膜的相互作用将mRNA递送到细胞内。该方法适用于多种细胞类型和组织,且具有较高的递送效率和稳定性。但是,纳米颗粒递送的制备过程较为复杂,且存在一定的毒性和免疫原性。病毒载体递送是将mRNA包裹在一些常见的病毒载体中,如腺病毒、腺相关病毒等,通过病毒与细胞的相互作用实现mRNA的递送。该方法具有较高的递送效率和稳定性,且可以实现精确的基因编辑和调控。但是,病毒载体递送存在一定的安全隐患和免疫原性,特别不适宜体内递送,且制备过程较为复杂。
微囊泡是细胞分泌的活性囊泡,其直径大小在100-1000nm,内含大量生物分子,包括核酸、蛋白质和脂质等,在细胞间的通信中发挥重要作用。微囊泡因其固有的稳定性、出色的生物相容性、低免疫原性和良好的组织穿透能力等优点,已成为体内外mRNA向细胞递送的理想载体。然而主要通过电穿孔将外源mRNA装载到微囊泡内部的方式,严重制约了微囊泡递送mRNA进一步的应用。
发明内容
有鉴于此,本发明的目的在于提供一种可以在体内外应用的递送mRNA的生物制剂,所述递送mRNA的生物制剂为改造过后膜内装载表达特定mRNA的微囊泡。
为了实现以上发明目的,本发明提供以下技术方案:
第一方面,提供一种膜内装载特定mRNA微囊泡的融合基因,包括第一融合基因和第二融合基因,所述第一融合基因包括微囊泡富含的膜蛋白的跨膜区基因和装配蛋白基因;第二融合基因包括目的mRNA的序列和装配蛋白的引导结合序列。
微囊泡膜蛋白包括Lamp2b,CD63,CD9,Alix,Tsg101,Flotillin,CD81、CD82、CD151、tetraspanin等。
优选地,Lamp2b的跨膜区基因为SEQ ID NO.1,CD63的跨膜区基因为SEQ ID NO.2。
装配蛋白可以和一段特定RNA茎环结构结合,如噬菌体包膜蛋白MS2,核糖体结合蛋白L7Ae,转录反激活子蛋白Tat等。本发明利用其与装配蛋白与其引导结合序列结合将目的mRNA导入到微囊泡中。
优选地,噬菌体包膜蛋白MS2序列为SEQ ID NO.3;核糖体结合蛋白L7Ae序列为SEQID NO.4,转录反激活子蛋白Tat序列为SEQ ID NO.5。
目的mRNA为需要递送进细胞的信使RNA,可以在受体细胞内翻译成蛋白或者多肽。
装配蛋白的引导结合序列为一类特殊序列,能与装配蛋白结合将目的mRNA导入到微囊泡中。
优选地,与MS2结合的序列为MS2bs,其核苷酸序列如SEQ ID NO.6所示;与L7Ae结合的序列为C/D box,其核苷酸序列如SEQ ID NO.7所示;与Tat结合的序列为TAR-RNA,其核苷酸序列如SEQ ID NO.8所示。
第二方面,提供一种重组载体,包含由第一融合基因与第二融合基因的核苷酸序列,所述重组载体的框架为真核细胞表达载体。
在一些实施例中,所述真核细胞表达载体为pLVX-Puro载体;
通过XhoI和EcoRI双酶切和连接,将第一融合基因和第二融合基因的核苷酸序列分别或同时插入pLVX-Puro载体上,进行并联表达或串联表达。
在串联表达时,第一融合基因与第二融合基因间通过IRES链接,IRES核苷酸序列如SEQ ID NO.9所示。
第三方面,提供一种用于分泌膜内装载特定mRNA微囊泡的稳转细胞系,利用第二方面所述的重组载体构建稳转细胞系。
在一些实施例中,一种用于分泌膜内装载特定mRNA微囊泡的稳转细胞系,包括利用上述重组载体转化的293T细胞。
第四方面,提供一种膜内装载特定mRNA微囊泡的制备方法,其特征在于,包括以下步骤:将所述稳转细胞系接种于无血清的完全培养基中培养24-72h,上清液中含有所述微囊泡。
本发明还提供了一种制备膜内装载特定mRNA微囊泡的方法,包括以下步骤:将上述稳转细胞系接种于无血清完全培养基中培养2天,上清液中含有所述膜外表达膜内装载特定mRNA微囊泡。将得到的上清液依次250-450g离心12-16min,2500-4500g离心16-22min,8000-10000g离心18-35min,10000-15000g离心90-120min,所得沉淀为所述微囊泡。
本发明还提供了利用上述方法制备得到的膜内装载特定mRNA微囊泡。
本发明提供了一种可用于体内外的生物制剂,所述生物制剂的有效成分为膜内装载特定mRNA微囊泡。以及,一种药物组合物,所述药物组合物包括上述的融合基因、上述的核酸分子,上述的重组载体或重组病毒,以及药学上接受的载体。
本发明还提供了上述膜内装载特定mRNA微囊泡在体内外的应用。具体涉及上述的融合基因、上述的核酸分子,上述的重组载体或重组病毒、生物制剂在制备治疗药物中的应用。
有益效果:本发明通过装配蛋白,可以特异性识别结合特定的RNA茎环结构这一特性,将装配连接到微囊泡上膜蛋白的C端,并将装配蛋白的引导结合序列插入编码目的mRNA融合蛋白序列mRNA的3’-非翻译区(3’-UTR)。由于在微囊泡上膜蛋白表达较高,目的mRNA可以通过3’-UTR的引导结合序列和微囊泡上膜蛋白C端的装配蛋白间的相互结合作用主动封装到微囊泡中。由此获得的微囊泡,可以将装载的目的mRNA递送到受体细胞内,诱导其产生目的mRNA表达的蛋白或多肽。本发明所述微囊泡可以在微囊泡生成时将目的mRNA装载到微囊泡内,有效避免了电转导方式所带来的对微囊泡结构的破坏,同时因微囊泡良好的生物相容性,可以直接应用于体内mRNA药物的递送,意义重大。
附图说明
图1本发明实施例中微囊泡CARM-MVS的制备和表征示意图;
图2本发明实施例中CARM-MVS装载CAR mRNA检测结果示意图;
图3本发明实施例中CARM-MVS体外制备CAR-T流式分析;
图4本发明实施例中CARM-MVS体外制备CAR-T Westernblot检测;
图5本发明实施例中B7H3 CAR-T-MVS细胞毒性实验分析;
图6本发明实施例中B7H3 CAR-T-MVS细胞毒性细胞因子分泌检测;
图7本发明实施例中CARM-MVS体内制备CAR-T流式分析;
图8本发明实施例中利用微囊泡(CARM-MVS)制备CAR-T示意图。
具体实施方式
下面结合附图对本发明实施例进行详细阐述,以使本发明的技术方案和优点更易于被相关领域技术人员理解,从而对本发明的保护范围做出更为清楚明确的界定。
实施例1微囊泡CAR-MVS的获取及表征
1.融合基因SEQ ID NO.10:Lamp2b-MS2;SEQ ID NO.11:B7H3-CAR-MS2bs序列委托南京擎科生物合成。
2.通过XhoI和EcoRI双酶切和连接,将SEQ ID NO.10和SEQ ID NO.11分别插入到慢病毒载体pLVX-Puro上,构建重组慢病毒载体。
3.串联表达时通过同源重组方式,将SEQ ID NO.10和SEQ ID NO.11之间用IRES(SEQ ID NO.9)连接,插入到慢病毒载体pLVX-Puro。
3.利用转染试剂lipo2000(购自Thermo)将重组慢病毒载体、包装质粒pGag/Pol、pRev和pVSV-G(购自Addgene),按照一定比例转染293细胞,包装收集慢病毒。
4.将获取的慢病毒感染293细胞,经嘌呤霉素筛选获得稳定表达融合基因SEQ IDNO.10和SEQ ID NO.11的稳转细胞系。
5.利用获得的稳转细胞系,放于37℃含5% CO2的培养箱中培养,培养基为无血清完全培养基,48h收集上清;
6.上清液350g离心13min去除大的细胞碎片,3500g离心10min去除细胞碎片,8000g离心20min去除小的细胞碎片,100000g离心120min,所得沉淀即为微囊泡(CARM-MVS)。
7.微囊泡在20ml培养基中超速离心分离,利用磷酸钨染色,用TEM-2100记录图像(见图1)表征。
实例2CARM-MVS内装载CAR mRNA效率分析
我们通过荧光定量PCR对微囊泡CARM-MVS内CAR mRNA的装载效率进行分析,将各组微囊泡收集后分别提取RNA,反转录后得到cDNA,之后分别设计引物进行荧光定量PCR。结果显示(见图2),CARM-MSV内CAR mRNA的量均高于其他组,表明通过MS2-MS2bs的作用实现了CAR mRNA在微囊泡内的大量加载。
实例3微囊泡CARM-MVS体外制备CAR-T检测
将CARM-MVS添加到健康人激活后的T细胞中,2天后通过流式抗体APC anti-mouseF(ab)2antibody检测B7H3 CAR蛋白的在T细胞上表达情况,结果显示(见图3)CARM-MVS组的平均荧光强度(MFI)值为3468,明显高于对照组。从转导效率来看,CARM-MVS组T细胞CAR阳性率约为11.23±3.5%。随后,提取各处理组T细胞总蛋白,经凝胶电泳分离,转膜,利用anti-human CD3ζ抗体westernblot进一步检测B7H3 CAR蛋白在T细胞内的表达情况,结果显示(见图4)除了在每组细胞中检测到到内源性CD3ζ(15KD)条带外,在CARM-MVS组和阳性对照组CAR-T-LV(利用慢病毒构建CAR-T细胞)中明显观察到B7H3 CAR蛋白(64KD)条带,但在对照组中很难检测到。以上结果证实在激活T细胞中加入微囊泡CARM-MVS成功构建出了靶向B7H3的mRNA CAR-T细胞。
实例4微囊泡制备CAR-T(CAR-MVS)体外杀伤功能检测
利用荧光素酶标记的靶细胞,采用荧光素酶法检测微囊泡制备的靶向B7H3 CAR-T(B7H3 CAR-T-MVS)在体外对靶细胞特异性的杀伤功能。
检测公式如下:
对B7-H3 CAR-T-MVS或B7-H3 CAR-T-LV在效靶比(E:T)为10:1,5:1,1:1下的杀伤效力进行检测比较。结果显示(见图5),和未处理过的T细胞相比,,B7-H3 CAR-T-MVS和B7-H3 CAR-T-LV细胞均可以高效杀伤B7-H3高表达的SGC细胞(B7-H3-SGC),而且随着效靶比的提升,对靶细胞B7-H3-SGC的杀伤比率也不断升高,而两种CAR-T细胞在和B7-H3阴性的SGC细胞共培养时,则没有观察到明显的杀伤作用。
通过ELISA检测效靶比为5:1时,各培养体系中炎症因子IL-2和IFN-γ的分泌情况。结果显示(见图6),炎症因子分泌情况与细胞杀伤实验结果相吻合。。与未处理组T细胞相比,B7-H3 CAR-T-MVS和B7-H3 CAR-T-LV在和靶细胞B7-H3-SGC共培养后,培养体系中的炎症因子IL-2和IFN-γ的分泌量均显著升高,而在SGC细胞组里没有检测到此现象的发生。这些结果表明,利用微囊泡构建的CAR-T细胞,可以和正常通过慢病毒构建的CAR-T细胞一样,在体外对靶细胞发挥特异性的杀伤功能。
虽然本发明方法制备的CAR-T细胞在杀伤靶细胞功能与常规方法制备方法相差不大,但是,本发明由于只利用mRNA在T细胞内表达CAR分子链,因此所制备的CAR-T细胞(B7-H3 CAR-T-MVS)避免了常规方法制备CAR-T细胞(B7-H3 CAR-T-LV)将CAR分子链随机插入到T细胞基因组所带来的安全隐患,特别适用于体内直接制备CAR-T。
实例5微囊泡CARM-MVS体内制备CAR-T检测
相比较于慢病毒颗粒,微囊泡因其良好的稳定性、出色的生物相容性等优点,使得CARM-MVS有望进一步应用于体内CAR-T的制备。
将CARM-MVS及对照各组分别注射到小鼠淋巴结内,24h后,从各组小鼠淋巴结中提取T细胞,通过流式抗体APC anti-mouse F(ab)2antibody检测B7H3 CAR蛋白的在各组T细胞上表达情况,结果显示(见图7)CARM-MVS组的平均荧光强度和转导效率均明显高于其他对照组,表明构建的微囊泡CARM-MVS可以用于体内CAR-T的制备。这些结果说明采用本发明方法利用装配蛋白所制备的CARM-MVS比不用装配蛋白所制备的CAR-MV包含更多CAR mRNA,因此在体内制备CAR-T细胞时显示了更高的制备效率。
实例6利用Lamp2b跨膜区与装配蛋白L7Ae及其引导结合序列C/D box制备CARM-MVS内装载CAR mRNAB7H3-CAR的实验及效果验证;
1.融合基因SEQ ID NO.12:Lamp2b-L7Ae;SEQ ID NO.13:B7H3-CAR-C/Dbox序列委托南京擎科生物合成。
2.实验操作和检验步骤同实例1-4。
3.结果如表1所示:
表1为在不同效靶比(E:T)情况下,B7-H3 CAR-T-MVS细胞对靶细胞的杀伤效率(%)
结果显示,采取本发明通过装配蛋白引导mRNA方法制备CARM-MSV内CAR mRNA的量显著高于不用装配蛋白组,由此制备的B7-H3 CAR-T-MVS可以高效杀伤B7-H3高表达的靶细胞。
实例7利用CD63跨膜区与装配蛋白Tat及其引导结合序列TAR-RNA制备CARM-MVS内装载CAR mRNAB7H3-CAR的实验及效果验证;
1.融合基因SEQ ID NO.14:CD63-Tat;SEQ ID NO.15:B7H3-CAR-TARRNA序列委托南京擎科生物合成。
2.实验操作和检验步骤同实例1-4。
3.结果如表2所示:
表2为在不同效靶比(E:T)情况下,B7-H3 CAR-T-MVS细胞对靶细胞的杀伤效率(%)
结果显示,采取本发明通过装配蛋白引导mRNA方法制备CARM-MSV内CAR mRNA的量显著高于不用装配蛋白组,由此制备的B7-H3 CAR-T-MVS可以高效杀伤B7-H3高表达的靶细胞。
综上所述,本发明通过装配蛋白,可以特异性识别结合特定的RNA茎环结构这一特性,将装配连接到微囊泡上膜蛋白的C端,并将装配蛋白的引导结合序列插入编码目的mRNA融合蛋白序列mRNA的3’-非翻译区(3’-UTR)。由于在微囊泡上膜蛋白表达较高,目的mRNA可以通过3’-UTR的引导结合序列和微囊泡上膜蛋白C端的装配蛋白间的相互结合作用主动封装到微囊泡中。由此获得的微囊泡,可以将装载的目的mRNA递送到受体细胞内,诱导其产生目的mRNA表达的蛋白或多肽。本发明所述微囊泡可以在微囊泡生成时将目的mRNA装载到微囊泡内,有效避免了电转导方式所带来的对微囊泡结构的破坏,同时因微囊泡良好的生物相容性,可以直接应用于体内mRNA药物的递送,意义重大。
以上所述仅是本发明的优选实施例,并非因此限制本发明的专利范围,对于相关技术领域的人员来说,在不脱离本发明原理的前提下,对前述记载的技术方案进行相关若干改进和润饰,均包括在本发明的专利保护范围内。
Claims (13)
1.一种膜内装载特定mRNA微囊泡的融合基因,其特征在于,包括第一融合基因和第二融合基因,所述第一融合基因包括微囊泡富含的膜蛋白的跨膜区基因和装配蛋白基因;第二融合基因包括目的mRNA的序列和装配蛋白的引导结合序列。
2.根据权利要求1所述的融合基因,其特征在于,所述微囊泡富含的膜蛋白的跨膜区包括分子Lamp2b、CD63、CD9、Alix、Tsg101、Flotillin、CD81、CD82、CD151、Tetraspanin的跨膜区;
优选地,所述微囊泡富含的膜蛋白的跨膜区为Lamp2b的跨膜区或与其具有90-99%同一性的氨基酸序列,包含的核苷酸序列如SEQ ID NO.1所示;其中所述CD63的跨膜区包含的核苷酸序列如SEQ ID NO.2所示。
3.根据权利要求1所述的融合基因,其特征在于,所述装配蛋白能和一段特定的RNA茎环序列结合,所述装配蛋白选自:
噬菌体衣壳蛋白MS2,其基因核苷酸序列为SEQ ID NO.3所示;
核糖体结合蛋白L7Ae,其基因序列核苷酸序列为SEQ ID NO.4所示;
转录反激活子蛋白Tat,其基因序列核苷酸序列为SEQ ID NO.5所示。
4.根据权利要求1所述的融合基因,其特征在于,所述装配蛋白的引导结合序列用于与装配蛋白结合将目的mRNA导入到微囊泡中;
当装配蛋白为噬菌体衣壳蛋白MS2,所述装配蛋白的引导结合序列为MS2bs,其核苷酸序列如SEQ ID NO.6所示;
当装配蛋白为核糖体结合蛋白L7Ae,所述装配蛋白的引导结合序列为C/D box,其核苷酸序列如SEQ ID NO.7所示;
当装配蛋白为转录反激活子蛋白Tat,所述装配蛋白的引导结合序列为TAR-RNA,其核苷酸序列如SEQ ID NO.8所示。
5.一种核酸分子,所述核酸分子编码权利要求1-4任一项中所述的融合基因。
6.一种载体或包含其的重组病毒,其特征在于,所述载体包含权利要求5中所述的核酸分子。
7.根据权利要求6所述载体或包含的其重组病毒的构建方法,其特征在于,所述方法包括:将所述的融合基因合成后导入质粒载体中,通过含有同源臂的PCR引物,扩增融合基因编码序列,并通过同源重组的方法将融合基因编码序列插入病毒载体即得。
8.一种重组细胞,所述重组细胞表达权利要求1-4任一项中所述的融合基因、权利要求5所述的核酸分子或权利要求6所述的载体或包含其的重组病毒,所述重组细胞包括经修饰的293T细胞。
9.一种膜内装载特定mRNA微囊泡的制备方法,其特征在于,包括以下步骤:将权利要求8所述重组细胞接种于培养基中培养24-72h,上清液中含有所述微囊泡。
10.根据权利要求9所述的方法,其特征在于,还包括:在培养后,将得到的上清液依次250-450g离心12-16min,2500-4500g离心16-22min,8000-10000g离心18-35min,10000-15000g离心90-120min,所得沉淀为所述微囊泡。
11.一种生物制剂,其特征在于,所述制剂以权利要求9或10所述方法制备得到的膜内装载特定mRNA微囊泡为有效成分。
12.一种药物组合物,其特征在于,所述药物组合物包括权利要求1-4所述的融合基因、权利要求5所述的核酸分子,权利要求6所述的重组载体或重组病毒,以及药学上接受的载体。
13.一种应用,所述应用包括以下任一项:权利要求1-4中任意一项所述的融合基因、权利要求5所述的核酸分子、权利要求6中重组载体或重组病毒、权利要求11所述的生物制剂、权利要求12所述的药物组合物在制备治疗药物中的应用。
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