CN114717230A - 成纤维细胞生长因子mRNA的无细胞和无载体体外RNA转录方法和核酸分子 - Google Patents
成纤维细胞生长因子mRNA的无细胞和无载体体外RNA转录方法和核酸分子 Download PDFInfo
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Abstract
本发明涉及治疗性mRNA的无细胞和无载体体外RNA转录方法和核酸分子。更具体地,本发明提供了一种核酸分子,其从5’端到3’端包含5’‑帽结构、人β‑珠蛋白5’非翻译区(5’‑UTR)、至少一个编码区、人α‑珠蛋白3’非翻译区(3’‑UTR)和3’‑聚腺苷酸尾,所述至少一个编码区可操作地连接到所述5’‑UTR和所述3’‑UTR,且所述编码区编码成纤维细胞生长因子(FGF),例如碱性成纤维细胞生长因子(bFGF),尤其是人bFGF,以及一种体外转录方法。本发明的体外转录方法和核酸分子不仅能够获得每小时可高达约2.3mg/mL甚至更高的mRNA的最大产量,而且所产生的mRNA显示出增强的基因表达稳定性和翻译效率。
Description
技术领域
本公开一般地涉及分子生物学领域,更具体地涉及成纤维细胞生长因子mRNA的无细胞和无载体体外RNA转录方法和核酸分子。
背景技术
信使RNA(mRNA)是一种相对较新的治疗性分子,其具有广泛的临床应用潜力,包括癌症治疗、疫苗和再生疗法。与基于重组蛋白的药物相比,基于mRNA的药物的优势包括:1.生产具有成本效益;2.更长的治疗效果;3.快速合成和纯化;4.无内毒素和传染原;5.翻译后修饰是有利的。mRNA也是基因治疗的一种非常有益的替代方法,因为它并未基因整合到基因组中,不需要进入细胞核,并且表达是可直接控制的。总体而言,基于mRNA的疗法是安全且具有成本效益的。
尽管基于mRNA的疗法是有希望的,但是mRNA的稳定性以及将mRNA递送至细胞的能力是不良的。
碱性成纤维细胞生长因子(basic fibroblast growth factor,简称bFGF,也称为FGF2)是成纤维细胞生长因子家族的成员,在神经退行性疾病、心脏病和难以愈合的伤口类病变中具有多种治疗用途。此外,bFGF通过诱导成纤维细胞和干细胞的增殖在组织发育中发挥重要作用,它还在干细胞的大规模生产方面起到重要作用。然而,目前bFGF蛋白的生产成本高,产量低,阻碍了其在医药行业的商业应用。例如,在干细胞培养条件下bFGF蛋白不稳定,易降解,而例行更换含有商购bFGF的新鲜培养基会极大提高研发成本。
因此,在本领域中对于改进的体外RNA转录方法以及更加稳定的mRNA例如碱性成纤维细胞生长因子mRNA仍然存在需求。
发明内容
在一个方面中,本公开提供了一种RNA核酸分子,其从5’端到3’端包含或由下列组成:5’-帽结构、人β-珠蛋白5’非翻译区(5’-UTR)、至少一个编码区、人α-珠蛋白3’非翻译区(3’-UTR)和3’-聚腺苷酸尾,所述至少一个编码区可操作地连接到所述5’-UTR和所述3’-UTR。
在一个实施方案中,所述编码区编码至少一种目的多肽或蛋白质,所述目的多肽或蛋白质任选地选自生长因子、抗原性多肽或蛋白质、变应原性多肽或蛋白质、治疗性多肽或蛋白质,或前述种类的片段、变体或衍生物。在一个实施方案中,所述编码区编码成纤维细胞生长因子(FGF),例如碱性成纤维细胞生长因子(bFGF),尤其是人bFGF。
在一个实施方案中,所述编码区还编码选自下列的至少一种:信号肽、肽标签或蛋白质标签、定位信号或定位序列、和肽接头。
在一个实施方案中,所述5’-UTR包含或组成为根据SEQ ID NO:1的RNA序列,或与根据SEQ ID NO:1的RNA序列具有至少80%、90%、95%、96%、97%、98%、或99%的序列同一性的RNA序列。
在一个实施方案中,所述3’-UTR包含或组成为根据SEQ ID NO:2的RNA序列,或与根据SEQ ID NO:2的RNA序列具有至少80%、90%、95%、96%、97%、98%、或99%的序列同一性的RNA序列。
在一个实施方案中,所述5’-帽结构是5’-抗反向帽类似物(5’-ARCA),优选地所述5’-ARCA是7m G(3’-O-Me)pppG。
在一个实施方案中,所述3’-聚腺苷酸尾包含10个至200个、20个至100个、40个至80个、或50个至70个腺嘌呤核苷酸。
在另一个方面中,本公开提供了一种DNA核酸分子,其从5’端到3’端包含或由下列组成:启动子、人β-珠蛋白5’-非翻译区(5’-UTR)、至少一个编码区、人α-珠蛋白3’-非翻译区(3’-UTR)和转录终止子,所述至少一个编码区可操作地连接到所述5’-UTR和所述3’-UTR。
在一个实施方案中,所述编码区编码至少一种目的多肽或蛋白质,所述目的多肽或蛋白质任选地选自肽生长因子、抗原性多肽或蛋白质、变应原性多肽或蛋白质、治疗性多肽或蛋白质,或前述种类的片段、变体或衍生物。在一个实施方案中,所述编码区编码成纤维细胞生长因子(FGF),例如碱性成纤维细胞生长因子(bFGF),尤其是人bFGF。
在一个实施方案中,所述编码区还编码选自下列的至少一种:信号肽、肽标签或蛋白质标签、定位信号或定位序列、和肽接头。
在一个实施方案中,所述启动子选自T3、T7、Sny5或SP6启动子。
在一个实施方案中,所述启动子选自是T3启动子,并且所述转录终止子选自T7转录终止子。
在一个实施方案中,所述5’-UTR包含或组成为根据SEQ ID NO:3的DNA序列,或与根据SEQ ID NO:3的DNA序列具有至少80%、90%、95%、96%、97%、98%、或99%的序列同一性的DNA序列。
在一个实施方案中,所述3’-UTR包含或组成为根据SEQ ID NO:4的DNA序列,或与根据SEQ ID NO:4的DNA序列具有至少80%、90%、95%、96%、97%、98%、或99%的序列同一性的DNA序列。
在一个实施方案中,所述DNA核酸分子进一步包含位于所述启动子上游的第一限制性酶切位点和位于所述转录终止子下游的第二限制性酶切位点。
在一个实施方案中,所述DNA核酸分子进一步包含位于所述启动子和所述5’-UTR之间的第三限制性酶切位点和位于所述3’-UTR和所述转录终止子之间的第四限制性酶切位点。
在又一个方面中,本公开提供了一种体外转录方法,其包括步骤:
(a)提供根据本公开所述的DNA核酸分子作为转录模板;
(b)任选地,扩增所述DNA核酸分子;
(c)将所述DNA核酸分子在5’-抗反向帽类似物(5’-ARCA)、优选7m G(3’-O-Me)pppG存在的情况下进行体外转录,以获得包含5’-ARCA封端的mRNA的反应混合物;
(d)任选地,通过添加DNA酶去除所述包含5’-ARCA封端的mRNA的反应混合物中的转录模板;和
(e)向所述包含5’-ARCA封端的mRNA的反应混合物添加聚腺苷酸聚合酶反应混合物进行3’-聚腺苷酸尾添加,以获得5’-ARCA封端的并具有3’-聚腺苷酸尾的mRNA。
在又一个方面中,本公开提供了一种组合物,其包含根据本公开的的RNA核酸分子和/或根据本公开的体外转录方法获得的RNA核酸分子,以及药学上可接受的载剂和/或赋形剂。
在一个实施方案中,所述组合物是药物组合物或疫苗或试剂盒。
在又一个方面中,本公开提供了本公开所述的RNA核酸分子和/或根据本公开的体外转录方法获得的RNA核酸分子在制备用于治疗或预防选自免疫疾病、遗传病、癌症、感染性疾病、炎性疾病、变态反应的疾病或病症和/或用于基因治疗和/或免疫调节的药物中的用途。
在一个实施方案中,本公开提供了本公开所述的编码成纤维细胞生长因子(FGF)例如碱性成纤维细胞生长因子(bFGF)、尤其是人bFGF的RNA核酸分子在化妆用途例如润肤、消除皱纹、消除或预防色斑、或抗皮肤衰老中的用途或在制备修复创伤、再生肌肤、防止脱发、促进组织发育或刺激骨形成或用于治疗或预防神经退行性疾病、心脏病或糖尿病性神经病变的药物中的用途。
附图说明
附图仅为了更清楚地说明本发明,而不是以任何方式限制本发明的公开范围和保护范围。
图1示出了根据本公开的实施方案的核酸分子的结构:DNA核酸构建体(图1A);RNA核酸分子(图1B)和优选的5’-ARCA(图1C)。
图2示出了EGF和bFGF mRNA的体外转录。
图3示出了GFP mRNA转染的293T细胞的荧光信号。
图4示出了通过qPCR对293T细胞中的细胞内EGF和bFGF mRNA进行定量的结果。
图5示出了EGF和FGF蛋白在293T细胞中的表达。通过凝胶电泳确认EGF和bFGFmRNA的分子量(图5A);在将EGF和bFGF mRNA转染到293T细胞中之后,在指定的时间点通过蛋白质印迹分析全细胞裂解物和细胞培养基,其中转染的293T细胞显示出细胞外hbFGF(图5B)、细胞内hbFGF(图5C)、细胞外hEGF(图5D)和细胞内hEGF(图5E)显著增加。
具体实施方式
为了促进对本发明原理的理解的目的,现参考在附图中举例说明的实施方案,并且将具体描述所述实施方案。然而,将理解的是,这些描述并不旨在对本发明的范围进行任何限制。
以下提供对一种无细胞和无载体的体外转录方法和用于这些方法的构建体的详细描述。这些方法和构建体满足了本领域中存在的至少一项需求。
本文中所用章节标题仅用于组织目的,不应理解为以任何方式限制所述主题。
除非另有明确定义,否则本文中所用术语应根据其在本领域中的通常含义来理解。除非文中另有规定或指示,否则没有数量词修饰的名词表示一个/种或更多个/种。
标准技术和流程通常根据本领域的常规方法和多种一般参考文献来进行(通常可参见,Sambrook et al.Molecular Cloning:A Laboratory Manual,2nd ed.(1989)ColdSpring Harbor Laboratory Press,Cold Spring Harbor,N.Y.)。
除非另有说明,否则本文中所用的术语“约”是指指定值的+/-10%,更优选+/-5%,例如+/-4%、+/-3%、+/-2%或+/-1%。
需要说明的是,在不冲突的情况下,本公开中的实施方案及实施例中的特征可以相互组合。
本公开提供了一种用于产生mRNA例如mRNA治疗剂的新型体外转录RNA平台。该平台可提供具有成本效益的mRNA生产,具有高的安全性和有效性,并且易于施用到细胞中。本公开提供的mRNA包含5’-抗反向帽类似物(5’-ARCA)、人β珠蛋白5’-非翻译区序列、信号序列和靶基因或目标基因编码序列、人α珠蛋白3'非翻译序列和3’-聚腺苷酸尾。在该平台上产生的mRNA显示出增强的基因表达稳定性和翻译效率。并且,该新型平台在孵育60分钟内显示出可高达约2.3mg/mL甚至更高的mRNA的最大产量。本公开至少部分基于令人惊讶的发现:本公开所选择的元件的组合不仅能够获得每小时可高达约2.3mg/mL甚至更高的mRNA的最大产量,而且所产生的mRNA显示出增强的基因表达稳定性和翻译效率。
在一些实施方案中,本公开的方法尤其适用于体外转录成纤维细胞生长因子(FGF)例如碱性成纤维细胞生长因子(bFGF)、尤其是人bFGF。
如本文所使用,术语“核酸”或“核酸分子”是指任何DNA或RNA分子,并且与多核苷酸可互换使用。在此提及编码特定蛋白质和/或肽的核酸或核酸序列时,所述核酸或核酸序列分别优选还包含调节序列,从而允许在合适的宿主例如人类中表达,即,转录和/或翻译编码特定蛋白质或肽的核酸序列。
RNA核酸分子
在一个方面,本公开提供了一种RNA核酸分子,其从5’端到3’端包含5’-帽结构、人β-珠蛋白5’非翻译区(5’-UTR)、至少一个编码区、人α-珠蛋白3’非翻译区(3’-UTR)和3’-聚腺苷酸尾,所述至少一个编码区可操作地连接到所述5’-UTR和所述3’-UTR。在一些实施方案中,所述编码区编码成纤维细胞生长因子(FGF)例如碱性成纤维细胞生长因子(bFGF)、尤其是人bFGF。
非翻译区(UTR)
如本文所使用,术语“非翻译区(UTR)”是指位于本文所述核酸分子编码区上游(5’)和/或下游(3’)的“非翻译区”,从而通常位于编码区的侧翼。因此,术语“UTR”通常包括5’-非翻译区(“5’-UTR”)和3’-非翻译区(“3’-UTR”)。UTR通常可以包含或组成为不翻译成蛋白质的核酸序列。UTR可包含一种或多种调节元件。
如本文所使用,术语“调节元件”是指具有基因调节活性的核酸序列,其能够影响可操作地(以顺式或反式)连接的可转录核酸序列的转录或翻译。该术语可包括启动子、增强子、内部核糖体进入位点(IRES)、内含子、前导序列、转录终止信号例如多聚腺苷酸化信号、以及其他表达控制元件。
如本文所使用,术语“可操作地连接”指元件的排列,其中以该术语描述的元件被装配以执行其一般功能。举例而言,与核酸序列可操作地连接的指定启动子在恰当的酶存在时能影响该序列的表达。启动子无需与该序列毗邻,只要其能指导该序列表达即可。因此,例如,间隔的非翻译但被转录的序列可出现在启动子序列和核酸序列间,并且启动子序列仍然被认为与编码序列“可操作地连接”。
在一些实施方案中,UTR“可操作地连接”,即以功能关系位于编码区上游和下游,优选以允许它们控制(即调控或调节,优选增强)编码序列表达的方式位于编码区上游和下游。
本申请的发明人出乎意料地发现5’-和3’-非翻译区(UTR)的组合,优选地结合本公开的5’-帽结构和聚腺苷酸尾,一起协同作用从而协同地增强可操作连接的核酸序列例如成纤维细胞生长因子(FGF)例如碱性成纤维细胞生长因子(bFGF)、尤其是人bFGF的表达。具有本发明的UTR组合的核酸分子有利地使得能够实现为基因治疗或免疫治疗目的而递送的大量多肽或蛋白质的快速和瞬时表达。因此,本文提供的核酸分子对于体内的各种治疗应用特别有用,包括例如基因治疗、癌症免疫治疗或针对感染因子的疫苗接种。
UTR组合(优选地结合本公开的5’-帽结构和聚腺苷酸尾)的协同作用测试是本领域技术人员的常规操作,例如可以在mRNA转染后通过荧光素酶表达进行协同作用测试,以证明存在协同作用,即不只是累加作用。
如本文所使用,术语“5’-UTR”是指核酸分子的一部分,其位于开放阅读框的5’(即“上游”),并且不翻译成蛋白质。在本公开上下文中,5’-UTR以转录起始位点开始,并在开放阅读框的起始密码子之前的一个核苷酸终止。
如本文所使用,术语“3’-UTR”是指核酸分子的一部分,其位于开放阅读框的3’(即“下游”),并且不翻译成蛋白质。在本公开的上下文中,3’-UTR对应于位于蛋白质编码序列的终止密码子(优选紧邻蛋白质编码序列的终止密码子)3’与多聚腺苷酸序列之间的序列。
在一个实施方案中,本公开的UTR组合是人β-珠蛋白5’非翻译区(5’-UTR)和人α-珠蛋白3’非翻译区(3’-UTR)。在一个实施方案中,本公开的UTR组合包括(a)人β-珠蛋白5’非翻译区或其同源物、变体或片段和(b)人α-珠蛋白3’非翻译区或其同源物、变体或片段。在一些实施方案中,同源物或变体与UTR仅在所述UTR的调节元件之外存在核苷酸差异,例如一个或多个核苷酸差异,诸如1-20、2-15、3-10、4-9、5-8、6-7个核苷酸差异。在一些实施方案中,UTR的片段包含所述UTR的全部调节元件。
在一些实施方案中,人β-珠蛋白5’-UTR包含或组成为根据SEQ ID NO:1的RNA序列,或与根据SEQ ID NO:1的RNA序列具有至少80%、90%、95%、96%、97%、98%、或99%的序列同一性的RNA序列。在一些实施方案中,所述与根据SEQ ID NO:1的RNA序列具有所述同一性的RNA序列与SEQ ID NO:1的RNA序列仅在UTR中的调节元件之外存在核苷酸差异,例如一个或多个核苷酸差异,诸如1-20、2-15、3-10、4-9、5-8、6-7个核苷酸差异。
SEQ ID NO:1(人β-珠蛋白5’-UTR的RNA序列)
5’-ACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACC-3’
在一些实施方案中,人α-珠蛋白3’-UTR包含或组成为根据SEQ ID NO:2的RNA序列,或与根据SEQ ID NO:2的RNA序列具有至少80%、90%、95%、96%、97%、98%、或99%的序列同一性的RNA序列。在一些实施方案中,所述与根据SEQ ID NO:2的RNA序列具有所述同一性的RNA序列与SEQ ID NO:2的RNA序列仅在UTR中的调节元件之外存在核苷酸差异,例如一个或多个核苷酸差异,诸如1-20、2-15、3-10、4-9、5-8、6-7个核苷酸差异。
SEQ ID NO:2(人α-珠蛋白3’-UTR的RNA序列)
5’-GCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUG-3’
5’-帽结构
根据本发明的特别优选的实施方案,本公开的RNA核酸分子通过添加“5’-帽结构”来修饰,这样可以使所述RNA核酸分子进一步稳定化。
“5’-帽结构”可以由修饰的核苷酸、特别是由鸟嘌呤核苷酸的衍生物形成。优选地,5’-帽通过5’-5’-三磷酸键连接至5’末端。在一些实施方案中,5’帽可被甲基化,例如m7GpppN,其中N是带有5’-帽的核酸的5’末端核苷酸,通常是mRNA的5’-端。m7GpppN是5’-帽结构(帽0结构),其天然存在于由聚合酶II转录的mRNA中。
本领域已知的5’-帽结构的其他实例包括甘油基、反向脱氧无碱基残基(部分)、4’,5’亚甲基核苷酸、1-(β-D-赤型呋喃核糖基)核苷酸、4’-硫代核苷酸、碳环核苷酸、1,5-脱水己糖醇核苷酸、L-核苷酸、α-核苷酸、修饰的碱基核苷酸、苏型戊呋喃核糖核苷酸、无环3’,4’-断核苷酸、无环3,4-二羟基丁基核苷酸、无环3,5-二羟基戊基核苷酸、3’-3’-反向核苷酸部分、3’-3’反向无碱基部分、3’-2’反向核苷酸部分、3’-2’反向无碱基部分、1,4-丁二醇磷酸酯、3’-氨基磷酸酯、磷酸己酯、氨基己基磷酸酯、3’-磷酸酯、3’-硫代磷酸酯、二硫代磷酸酯或桥连或非桥连的甲基膦酸酯部分。
5’-帽结构可以是帽0、帽1(m7G的相邻核苷酸的核糖被甲基化)、帽2(m7G下游的第2个核苷酸的核糖被额外甲基化)、帽3(m7G下游的第3个核苷酸的核糖被额外甲基化)、帽4(m7G下游的第4个核苷酸的核糖被甲基化)。
在一些实施方案中,可用于本公开的特别优选的5’-帽结构是ARCA(抗反向帽类似物)。在一个实施方案中,ARCA是7m G(3’-O-Me)pppG。该ARCA通过将7mGpppG中的7mG残基的3’-OH基团由OCH3(“OMe”)取代而获得。本领域己知几种类型的ARCA类似物(参见例如美国专利号7,074,596)。然而,本申请令人惊讶地发现,相比于其它ARCA类似物,采用7m G(3’-O-Me)pppG,不需要相对于pppG的大幅摩尔过量的ARCA以确保大部分mRNA转录物分子具有5’-帽结构。在一些实施方案中,7m G(3’-O-Me)pppG与pppG(GTP)的摩尔比不大于4:1,例如3:1、2:1或1:1。
本申请还发现,采用ARCA尤其是7m G(3’-O-Me)pppG,其与本公开描述的UTR组合协同地增强可操作连接的核酸序列的表达。具有本发明的ARCA尤其是7m G(3’-O-Me)pppG以及UTR组合的核酸分子有利地使得能够实现为基因治疗或免疫治疗目的而递送的大量多肽或蛋白质的快速和瞬时表达。
3’-聚腺苷酸尾
根据另外的优选实施方案,本发明的RNA核酸分子可以包含聚腺苷酸序列。
如本文所使用,术语“聚腺苷酸序列”也称为“聚腺苷酸尾”或“3’-聚腺苷酸尾”,其指腺苷核苷酸的序列,例如最多约400个腺苷核苷酸,例如约20个至约400个,优选约50个至约400个,更优选约50个至约300个,甚至更优选约50个至约250个,最优选约60个至约250个腺苷核苷酸的序列。如本文所用,“聚腺苷酸序列”还可包含约10个至200个腺苷核苷酸,优选约10个至100个腺苷核苷酸,更优选约40个至80个腺苷核苷酸或甚至更优选约50个至70个腺苷核苷酸。聚腺苷酸序列通常位于RNA,特别是mRNA的3’末端。
RNA核酸分子中的聚腺苷酸序列可以优选地由DNA模板通过RNA体外转录获得。或者,也可以通过化学合成的常规方法在体外获得聚腺苷酸序列,而不必从DNA模板转录而来。
此外,可以使用可商购获得的聚腺苷酸化试剂盒和本领域已知的相应方案,通过RNA核酸分子的酶促聚腺苷酸化来产生聚腺苷酸序列或聚腺苷酸尾。通常将聚腺苷酸化理解为将聚腺苷酸序列添加至RNA核酸分子,例如成熟前mRNA。聚腺苷酸化可以通过所谓的聚腺苷酸化信号来诱导。该信号优选位于待聚腺苷酸化的核酸(RNA)序列的3’末端的核苷酸片段内。聚腺苷酸化信号通常包含由腺嘌呤和尿嘧啶/胸腺嘧啶核苷酸组成的六聚体,优选六聚体序列AAUAAA。
本申请的发明人进一步发现,在本公开的RNA核酸分子的3’末端添加聚腺苷酸尾进一步增加了RNA核酸分子的稳定性。该聚腺苷酸尾与本公开描述的ARCA尤其是7mG(3’-O-Me)pppG以及本公开描述的UTR组合协同地增强可操作连接的核酸序列的稳定性及其表达产量。
编码区
根据本发明的核酸分子包含至少一个编码区或编码序列,所述编码区或编码序列可操作地连接至(通常在两侧连接)本文限定的至少一种3’-UTR元件和至少一种5’-UTR元件。术语“编码序列”或“cds”和“编码区”在本文可互换使用,是指编码目的(基因)产物的核酸的区段或部分。基因产物是基因表达的产物,包括多肽和核酸,通常,本公开的核酸分子的至少一个编码区可以编码至少一种多肽或蛋白质,称为“目的多肽或蛋白质”。编码区通常由在其5’端以起始密码子(例如AUG)为边界和在其3’端以终止密码子(例如UAG、UAA或UGA)为边界的外显子构成。在本公开的核酸分子中,编码区以如本文所限定的至少一种5’-UTR元件和至少一种3’-UTR元件为边界。
目的多肽或蛋白质通常包括可以由具有至少一个编码区的核酸序列编码的任何多肽或蛋白质,并且可以在适当条件下表达以产生功能性多肽或蛋白质产物。在本文中,术语“功能性”是指“能够发挥期望的生物学功能”和/或“表现出期望的生物学特性”。目的多肽或蛋白质可以具有多种功能,并且包括例如生长因子、抗体、酶、信号传导蛋白、受体、受体配体、肽激素、转运蛋白、结构蛋白、神经递质、血清蛋白、载体、药物、免疫调节剂、癌基因、抑癌剂、毒素、肿瘤抗原等。在一些实施方案中,目的多肽或蛋白质可以是治疗性、抗原性和变应原性多肽或蛋白质。
细胞生长因子
在一些实施方案中,本公开的核酸分子的至少一个编码区可以编码至少一种生长因子。如本文所使用,术语“生长因子”是指一类通过与特异的、高亲和的细胞膜受体结合,调节细胞生长与其他细胞功能等多效应的多肽类物质。在一些实施方案中个,生长因子可选自血小板类生长因子(血小板来源生长因子,PDGF);骨肉瘤来源生长因子(ODGF)、表皮生长因子类(表皮生长因子EGF,转化生长因子TGFα和TGFβ)、成纤维细胞生长因子(αFGF、βFGF)、类胰岛素生长因子(IGF-Ⅰ、IGF-Ⅱ)、神经生长因子(NGF)、白细胞介素类生长因子(IL-1、IL-1、IL-3等)、红细胞生长素(EPO)、集落刺激因子(CSF)等。
在一个优选的实施方案中,生长因子是成纤维细胞生长因子(FGF)例如碱性成纤维细胞生长因子(bFGF)、尤其是人bFGF。在一些实施方案中,成纤维细胞生长因子是天然存在的成纤维细胞生长因子或其功能性变体或片段。
治疗性多肽或蛋白质
在一些实施方案中,本公开的核酸分子的至少一个编码区可以编码至少一种“治疗性多肽或蛋白质”。术语“治疗性多肽或蛋白质”是指能够介导期望的诊断、预防或治疗效果,优选导致疾病的检测、预防、改善和/或治愈的多肽或蛋白质。
优选地,根据本公开的核酸分子可以包含至少一个编码区域,该编码区域编码替代缺失、缺陷或突变的蛋白质的治疗性蛋白质;对治疗遗传性或获得性疾病、感染性疾病或肿瘤例如癌症或肿瘤疾病有益的治疗性蛋白质;辅助性或免疫刺激性治疗蛋白;治疗性抗体或抗体片段、变体或衍生物;肽激素;基因编辑剂;免疫检查点抑制剂;T细胞受体、或T细胞受体片段、变体或衍生物;和/或酶。
抗原性多肽或蛋白质
本公开的核酸分子的至少一个编码区可以编码至少一种“抗原性多肽或蛋白质”。术语“抗原性多肽或蛋白质”或简称“抗原”通常指在适当条件下能够与免疫系统的组分(例如抗体或免疫细胞通过其抗原受体,例如B细胞受体(BCR)或T细胞受体(TCR))相互作用/被其识别,并优选能够引发免疫应答的任何多肽或蛋白质“抗原性肽或蛋白质”优选通过其“表位”或“抗原决定簇”与免疫系统的组分相互作用而被其识别。
特定抗原性多肽或蛋白质的选择通常取决于要治疗或预防的疾病。一般而言,核酸分子可以编码任何与疾病(例如,癌症、感染性疾病)相关的抗原性多肽或蛋白质,所述疾病可以通过诱导针对所述抗原感染性疾病的免疫应答来治疗。
优选地,根据本发明的人工核酸分子可包含至少一个编码区,其编码肿瘤抗原、病原性抗原、自身抗原、同种抗原或变应原性抗原。特别优选的是肿瘤抗原NY-ESO-1、5T4、MAGE-C1、MAGE-C2、Muc-1、PSA、PSMA、PSCA、STEAP和PAP。
变应原性多肽或蛋白质
本公开的核酸分子的至少一个编码区可以编码至少一种“变应原性多肽或蛋白质”。术语“变应原性多肽或蛋白质”或“变应原”是指当暴露于对象时,能够诱导变态反应,即以改变的身体反应性(如超敏反应)为特征的病理性免疫反应的多肽或蛋白质。通常,“变应原”与“特应性”有关,即涉及免疫珠蛋白E(IgE)的不利免疫反应。因此,术语“变应原”通常是指与特应性有关并诱导IgE抗体的物质(此处为多肽或蛋白质)。在一些实施方案中,变应原可以包括昆虫衍生的变应原、哺乳动物变应原、软体动物衍生的变应原、植物变应原和真菌变应原等。
其他结构域、标签、接头、序列或元件
优选地,除了编码至少一种目的多肽或蛋白质之外,本公开的核酸分子的至少一个编码区还可以编码其他多肽结构域、标签、接头、序列或元件。将编码所述其他结构域、标签、接头、序列或元件的核酸序列在框内可操作地连接至编码目的多肽或蛋白质的区域,使得编码序列的表达优选产生目的多肽或蛋白质与其他结构域、标签、接头、序列或元件偶联的融合产物。
本公开的核酸分子的至少一个编码区还可以编码下列的至少一个:信号肽、肽或蛋白质标签、定位信号或序列、肽接头等元件。
术语“信号肽”(有时也称为分泌信号肽或信号序列)是指通常存在于将经由分泌途径而分泌的蛋白质末端的典型短肽(通常长度可以为16个至30个氨基酸)。当目的多肽或蛋白质是成纤维细胞生长因子时,信号肽序列是允许成纤维细胞生长因子(FGF)例如碱性成纤维细胞生长因子(bFGF)、尤其是人bFGF在翻译后分泌的信号肽。
优选地,可以将信号肽引入目的多肽或蛋白质中以促进多肽或蛋白质的分泌。特别是在编码抗原性多肽或蛋白质的核酸与信号肽融合的情况下,适当的分泌可有助于触发针对所述抗原的免疫应答。信号肽也可以有效地与本文公开的任何其他多肽或蛋白质组合。当与目的多肽或蛋白质组合编码时,这种信号肽可以位于目的多肽或蛋白质的N端、C端和/或内部,优选位于N端。在核酸水平上,通常将这种信号肽的编码序列置于框内(即,在同一阅读框中)。
可用于本公开的示例性信号肽包括但不限于经典或非经典MHC分子的信号序列(例如MHC I分子和MHC II分子的信号序列,例如MHC I类分子HLA-A*0201的信号序列),细胞因子或免疫珠蛋白的信号序列,免疫球蛋白或抗体恒定链的信号序列,Lamp1、Tapasin、Erp57、Calretikulin、钙连蛋白、PLAT、EPO、或白蛋白的信号序列,以及其他膜相关蛋白的信号序列或与内质网(ER)或内体-溶酶体区室相关的蛋白的信号序列。
术语“肽或蛋白质标签”是引入目的多肽或蛋白质中以赋予所需的生物学功能或特性的短氨基酸序列。通常,“肽标签”可用于检测、纯化、分离或添加某些所需的生物学特性或功能。
“肽接头”或“间隔子”是连接本文所公开的目的多肽或蛋白质的结构域、部分或部件,例如多结构域蛋白质或融合蛋白的结构域、部分或部件的短氨基酸序列。多肽或蛋白质、或其结构域、部分或部件优选是功能性的,即实现特定的生物学功能。
DNA核酸分子
本公开的RNA核酸分子可以优选地由DNA模板通过RNA体外转录获得。或者,也可以通过化学合成的常规方法在体外获得聚腺苷酸序列,而不必从DNA模板转录而来。
在一个实施方案中,本公开的RNA核酸分子可以优选地由DNA模板通过RNA体外转录获得。
因此,在一个方面,本公开提供了一种DNA核酸分子,其从5’端到3’端包含启动子、人β-珠蛋白5’-非翻译区(5’-UTR)、至少一个编码区、人α-珠蛋白3’-非翻译区(3’-UTR)和转录终止子,所述至少一个编码区可操作地连接到所述5’-UTR和所述3’-UTR。在一些实施方案中,所述编码区编码成纤维细胞生长因子(FGF)例如碱性成纤维细胞生长因子(bFGF)、尤其是人bFGF。
在一个实施方案中,本公开的DNA核酸分子包括人β-珠蛋白5’非翻译区(5’-UTR)和人α-珠蛋白3’非翻译区(3’-UTR)的UTR组合。在一个实施方案中,本公开的UTR组合包括(a)人β-珠蛋白5’非翻译区或其同源物、变体或片段和(b)人α-珠蛋白3’非翻译区或其同源物、变体或片段。在一些实施方案中,同源物或变体与UTR仅在所述UTR的调节元件之外存在核苷酸差异,例如一个或多个核苷酸差异,诸如1-20、2-15、3-10、4-9、5-8、6-7个核苷酸差异。在一些实施方案中,UTR的片段包含所述UTR的全部调节元件。
在一些实施方案中,人β-珠蛋白5’-UTR包含或组成为根据SEQ ID NO:3的RNA序列,或与根据SEQ ID NO:3的DNA序列具有至少80%、90%、95%、96%、97%、98%、或99%的序列同一性的DNA序列。在一些实施方案中,所述与根据SEQ ID NO:3的DNA序列具有所述同一性的DNA序列与SEQ ID NO:3的DNA序列仅在UTR中的调节元件之外存在核苷酸差异,例如一个或多个核苷酸差异,诸如1-20、2-15、3-10、4-9、5-8、6-7个核苷酸差异。
SEQ ID NO:3(人β-珠蛋白5’-UTR的DNA序列)
5’-ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC-3’
在一些实施方案中,人α-珠蛋白3’-UTR包含或组成为根据SEQ ID NO:4的DNA序列,或与根据SEQ ID NO:4的DNA序列具有至少80%、90%、95%、96%、97%、98%、或99%的序列同一性的DNA序列。在一些实施方案中,所述与根据SEQ ID NO:4的DNA序列具有所述同一性的RNA序列与SEQ ID NO:4的DNA序列仅在UTR中的调节元件之外存在核苷酸差异,例如一个或多个核苷酸差异,诸如1-20、2-15、3-10、4-9、5-8、6-7个核苷酸差异。
SEQ ID NO:4(人α-珠蛋白3’-UTR的DNA序列)
5’-GCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCTGGTCTTTG-3’
在一些实施方案中,根据所采用的RNA聚合酶来选择相应的启动子和转录终止子或终止元件。可用于本公开的RNA聚合酶包括但不限于T3、T7、Sny5或SP6 RNA聚合酶。因此,可用于本公开的启动子和转录终止子可选自T3、T7、Sny5或SP6 RNA聚合酶的启动子和转录终止子或终止元件。示例性的启动子选自T3、T7、Sny5或SP6启动子,优选T7启动子。示例性的转录终止子或终止元件可以是T7转录终止子。
在一些实施方案中,用于转录本公开的RNA分子的DNA模板可包括通过PCR方法或对化学合成的寡核苷酸进行退火所得到的线性模板、经克隆构建的质粒、以及基于RNA前体进行第一链及第二链合成(例如aRNA扩增)得到的cDNA模板。
用作转录模板的质粒载体需要通过限制性内切酶消化来线性化。因为转录反应会延续至DNA模板的末端,线性化可以确保获得确定长度及序列的RNA转录本。
在一些实施方案中,本公开的DNA核酸分子进一步包含位于启动子上游的第一限制性酶切位点和位于转录终止子下游的第二限制性酶切位点。
在一些实施方案中,第一限制性酶切位点和第二限制性酶切位点允许DNA核酸分子插入载体例如质粒。如本文所使用,术语“限制性内切酶”或“限制性核酸内切酶”是指可以识别并附着特定的脱氧核苷酸序列,并对每条链中特定部位的两个脱氧核糖核苷酸之间的磷酸二酯键进行切割的一类酶。切割方法是将糖类分子与磷酸之间的键切断,进而于两条DNA链上各产生一个切口,且不破坏核苷酸与碱基。切割形式有两种,分别是可产生具有突出单股DNA的黏状末端,以及末端平整无凸起的平滑末端。由于断开的DNA片段可由DNA连接酶连接,因此染色体或DNA上不同的限制片段,得以经由剪接作用而结合在一起。在一些实施方案中,第一限制性酶切位点和第二限制性酶切位点可以相同或不同,第一限制性酶切位点和第二限制性酶切位点都是EcoRI。可用于本公开的限制性内切酶可包括但不限于:EcoRI、PstI、XbaI、BamHI、HindIII、TaqI、NotI、HinfI、Sau3A、PovII、SmaI、HaeIII、AluI、SalI、Dra等。使用标准分子生物学方法进行目标多核苷酸的插入,例如,如Sambrook etal.(Sambrook et al.Molecular Cloning:A Laboratory Manual,Cold Spring HarbourLaboratory Press,1989)和/或Ausubel et al.(Current Protocols in MolecularBiology,Greene Pub.Associates and Wiley-Interscience(1988)所描述。连接核酸的方法对本领域技术人员是显而易见的,并描述于例如Sambrook et al.Molecular Cloning:ALaboratory Manual,Cold Spring Harbour Laboratory Press,1989和/或Ausubel etal.(编者),Current Protocols in Molecular Biology,Greene Pub.Associates以及Wiley-Interscience(1988)中。在一个实例中,利用连接酶(例如T4DNA连接酶)连接核酸。
在一些实施方案中,本公开的DNA核酸分子进一步包含位于启动子和5’-UTR之间的第三限制性酶切位点和位于3’-UTR和转录终止子之间的第四限制性酶切位点。在一些实施方案中,第三限制性酶切位点和第四限制性酶切位点允许通用克隆。在一些实施方案中,第三限制性酶切位点和第四限制性酶切位点独立地选自EcoRI、PstI、XbaI、BamHI、HindIII、TaqI、NotI、HinfI、Sau3A、PovII、SmaI、HaeIII、AluI、SalI和Dra。在一些实施方案中,第三限制性酶切位点和第四限制性酶切位点是不同的,例如分别是SalI和NotI。
体外转录方法
在一个方面,本公开提供了一种体外转录方法,其包括步骤:(a)提供根据本公开所述的DNA核酸分子作为转录模板;(b)任选地,扩增所述DNA核酸分子;(c)将所述DNA核酸分子在5’-抗反向帽类似物(5’-ARCA)、优选7m G(3’-O-Me)pppG存在的情况下进行体外转录,以获得包含5’-ARCA封端的mRNA的反应混合物;(d)任选地,通过添加DNA酶去除所述包含5’-ARCA封端的mRNA的反应混合物中的转录模板;和(e)向所述包含5’-ARCA封端的mRNA的反应混合物添加聚腺苷酸聚合酶反应混合物进行3’-聚腺苷酸尾添加,以获得5’-ARCA封端的并具有3’-聚腺苷酸尾的mRNA。在一些实施方案中,所述核酸分子的编码区编码成纤维细胞生长因子(FGF)例如碱性成纤维细胞生长因子(bFGF)、尤其是人bFGF。
术语RNA“体外转录”涉及从无细胞系统(体外)中的DNA模板合成RNA的方法。DNA,优选线性DNA(例如线性化质粒DNA、线性化dbDNA)用作生成RNA转录物的模板。用于RNA体外转录的DNA模板可以通过克隆核酸,特别是对应于待体外转录的相应RNA的cDNA,并将其引入用于RNA体外转录的适当载体,例如进入质粒DNA来获得。
还可以通过本公开的DNA核酸分子为模板,通过PCR扩增,来制备用于体外转录的模板。PCR产物在纯化后,可通过标准的体外转录方法进行RNA合成。将所获得的PCR产物与包含5’-抗反向帽类似物(5’-ARCA)的体外RNA合成预混合液混合并孵育(例如在37℃下孵育30分钟),以生成5’-ARCA封端的RNA。在一些实施方案中,体外RNA合成预混合液包含:适用于体外转录的缓冲液(例如Tris-HCl,pH 7.9)、帽类似物(例如5’-抗反向帽类似物)、核糖核苷三磷酸(ATP、UTP、CTP和GTP)、核糖核酸酶抑制剂和RNA聚合酶(例如T7 RNA聚合酶)。在一些实施方案中,体外RNA合成预混合液还可包含MgCl2、抗氧化剂和多胺(例如亚精胺)的至少一种或全部。
为了获得适合在基于RNA的治疗中使用的高质量RNA,可以从最终RNA产物中高效且可靠地去除DNA模板,以确保基于RNA的治疗剂的功效和安全性。从RNA体外转录反应中的DNA模板去除可以例如通过酶(例如DNase I)消化DNA以及纯化RNA来实现。
通过额外的聚腺苷酸聚合酶反应混合物进行3’-聚腺苷酸拖尾。在一些实施方案中,聚腺苷酸聚合酶反应混合物包含缓冲液(例如Tris-HCl,pH 8.1)和聚腺苷酸聚合酶以及盐(例如NaCl和MgCl2的一种或两种)并孵育(例如在37℃下孵育30分钟),以获得5’-ARCA封端的并具有3’-聚腺苷酸尾的mRNA。
组合物和疫苗
在另一方面,本公开提供了一种组合物,其包含本公开的RNA核酸分子,以及至少一种药学上可接受的载剂和/或赋形剂。在一些优选的实施方案中,该组合物作为药物组合物提供。根据另外的优选实施方案,可以将组合物作为疫苗提供。“疫苗”通常被理解为提供至少一种抗原,优选抗原性肽或蛋白质的预防或治疗材料。“提供至少一种抗原”是指例如疫苗包含抗原或疫苗包含例如编码抗原的分子。因此,本公开的疫苗可包含至少一种RNA核酸分子,其编码至少一种如本文所定义的抗原性多肽或蛋白质,该抗原性多肽或蛋白质可以例如衍生自肿瘤抗原、细菌抗原、病毒抗原、真菌抗原或原生动物抗原、自身抗原、变应原或同种异体抗原,并且优选地当其被表达并呈递至免疫系统时诱导针对相应抗原的免疫应答。
本公开的组合物或疫苗优选包含本文所述的至少一种RNA核酸分子。本公开的组合物或疫苗中的每个RNA核酸分子可以编码至少一种或至少两种(相同或不同)的多种目的多肽或蛋白质。RNA核酸分子可以以“复合”或“游离”形式或其混合物的形式提供在组合物或疫苗中。组合物或疫苗还可以包含至少一种另外的活性剂,其用于治疗用RNA核酸分子或包含其的组合物或疫苗进行治疗的疾病或病症。
优选地,根据本发明的组合物或疫苗包含至少一种药学上可接受的载剂和/或赋形剂。术语“药学上可接受的”是指与一种或多于一种活性剂相容并且不干扰和/或不显著降低其药物作用的化合物或试剂。药学上可接受的载剂和赋形剂优选具有足够高的纯度和足够低的毒性,以使其适于施用于待治疗的对象。
药学上可接受的赋形剂可以发挥不同的功能作用,并且包括但不限于稀释剂、填充剂、膨胀剂、载剂、崩解剂、黏合剂、润滑剂、助流剂、包衣、溶剂和助溶剂、缓冲剂、防腐剂、佐剂、抗氧化剂、润湿剂、消泡剂、增稠剂、甜味剂、调味剂和保湿剂。
对于液体形式的组合物,有用的药学上可接受的载剂和赋形剂包括溶剂、稀释剂或载剂例如(无热原)水、(等渗)盐溶液例如磷酸盐或柠檬酸盐缓冲盐水、固定油、植物油例如花生油、棉籽油、芝麻油、橄榄油、玉米油、乙醇、多元醇(例如甘油、丙二醇、聚乙二醇等);卵磷脂;表面活性剂;防腐剂例如苯甲醇、对羟基苯甲酸酯、氯丁醇、苯酚、抗坏血酸、硫柳汞等;渗剂例如糖、多元醇例如甘露醇、山梨糖醇或氯化钠;单硬脂酸铝或明胶;抗氧化剂例如抗坏血酸或亚硫酸氢钠;螯合剂例如乙二胺四乙酸(EDTA);缓冲液例如乙酸盐、柠檬酸盐或磷酸盐;以及用于调节张力的试剂例如氯化钠或葡萄糖。可用酸或碱例如盐酸或氢氧化钠调节pH。相对于特定参考介质,缓冲液可以是高渗的、等渗的或低渗的,即,相对于特定参考介质,缓冲液可以具有更高的、相同的或更低的盐含量,其中优选地可以使用上述盐的这种浓度,其不会由于渗透或其他浓度效应而导致细胞损伤。参考介质是例如以“体内”方法产生的液体,例如血液、淋巴液、胞浆液体、或其他体液、或例如可以在“体外”方法中用作参考介质的液体,例如常用的缓冲液或液体。这种常用的缓冲液或液体是技术人员已知的。
对于(半)固体形式的组合物,有用的药学上可接受的载剂和赋形剂包括黏合剂,例如微晶纤维素,黄蓍胶或明胶;淀粉或乳糖;糖,例如乳糖、葡萄糖和蔗糖;淀粉,例如玉米淀粉或马铃薯淀粉;纤维素及其衍生物,例如羧甲基纤维素钠、乙基纤维素、乙酸纤维素;崩解剂,例如藻酸;润滑剂,例如硬脂酸镁;助流剂,例如硬脂酸、硬脂酸镁;硫酸钙、胶体二氧化硅等;甜味剂,例如蔗糖或糖精;和/或调味剂,例如薄荷、水杨酸甲酯或橙味剂。
合适的药学上可接受的载剂和赋形剂通常可以基于组合物的所需配方来选择。
通过注射,特别是静脉注射施用的液体组合物在制造和储存条件下应该是无菌和稳定的。这种组合物通常配制成胃肠外可接受的水溶液,该水溶液不含热原、具有合适的酸碱度、等渗并保持活性成分的稳定性。用于根据本发明的液体组合物的特别有用的药学上可接受的载剂和赋形剂包括水,通常是无热原的水;等渗盐水或缓冲溶液,例如磷酸盐、柠檬酸盐等缓冲溶液。特别是对于本公开的组合物的注射,可以使用水或优选缓冲液,更优选水性缓冲液,其包含钠盐,优选至少50mM的钠盐;钙盐,优选至少0.01mM的钙盐;和任选的钾盐,优选至少3mM的钾盐。
根据优选的实施方案,钠盐、钙盐和任选的钾盐可以以它们的卤化物的形式存在,例如氯化物、碘化物或溴化物,以它们的氢氧化物、碳酸盐、碳酸氢盐或硫酸盐等形式存在。此外,缓冲液中可以包含上述阳离子的有机阴离子。
根据优选的实施方案,如上文所定义的适合于注射目的的缓冲液可包含选自氯化钠(NaCl)、氯化钙(CaCl2)和任选的氯化钾(KCl)的盐,其中除氯离子外,还可存在其他阴离子。CaCl2也可以用另一种盐例如KCl代替。通常,注射缓冲液中的盐的浓度为至少50mM氯化钠(NaCl)、至少3mM氯化钾(KCl)和至少0.01mM氯化钙(CaCl2)。
于局部施用的组合物可使用本文别处所述的合适的液体和/或(半)固体赋形剂或载剂配制成乳剂、软膏剂、凝胶剂、糊剂或散剂。经口施用的组合物可以使用本文其他地方所述的合适的液体和/或(半)固体赋形剂或载剂配制成片剂、胶囊剂、液体剂、散剂或持续释放形式。
根据一些优选的实施方案,本公开的组合物或疫苗通过肠胃外施用,特别是通过皮内或肌内注射、经口、鼻内、肺部、吸入、局部、直肠、颊部、阴道或通过植入的贮库施用,并且以液体或冻干制剂的形式提供以用于本文其他地方所讨论的肠胃外施用。肠胃外制剂通常储存在小瓶、静脉输液袋、安瓿、药筒或预装注射器中,并且可以以注射剂、吸入剂或气雾剂的形式施用,优选以注射剂的形式施用。
根据优选的实施方案,本公开的组合物或疫苗可以包含与脂质复合的本公开的RNA核酸分子,其例如可以是脂质纳米颗粒、脂质体、脂质复合体或乳液的形式。
根据其他优选的实施方案,本公开的组合物或疫苗以冻干形式提供。优选地,冻干的组合物或疫苗在施用前在合适的缓冲液中重构,该缓冲液有利地基于水性载剂,例如乳酸林格溶液、磷酸盐缓冲液,优选乳酸林格溶液。
根据优选的实施方案,本公开的组合物或疫苗还可以包含至少一种佐剂。广义上的“佐剂”或“辅助组分”通常是可改变例如增强其他活性剂例如治疗剂或疫苗的作用的药理学和/或免疫学试剂。在本文中,“佐剂”可以理解为任何适合支持本公开的组合物的施用和递送的化合物。具体而言,佐剂可优选增强添加其的组合物或疫苗的免疫刺激特性。此外,此类佐剂可以但不限于,引发或增强先天免疫系统的免疫应答,即非特异性免疫应答。
试剂盒
在另一方面,本公开涉及包含本公开的RNA核酸分子和/或组合物或疫苗的试剂盒或试剂套装。
在本公开的试剂盒或试剂套装中,至少一种冻干或液体形式的RNA核酸分子,任选地与一种或多种药学上可接受的载剂和/或赋形剂一起。
任选地,本公开的试剂盒或试剂套装还可包含其他试剂,例如抗微生物剂、RNA酶抑制剂、增溶剂等。
试剂套装可以是两个或多于两个部分的套装,并且通常在合适的容器中包含其组分。例如,每个容器可以是小瓶、瓶子、挤压瓶、广口瓶、密封的小袋等形式,或任何其他合适的形式,条件是该容器被配置为防止组分的过早混合。可以分别提供每个不同的组分,或者可以与一些不同的组分一起提供(即,在同一容器中)。试剂套装还可以包含有关其成分的任何施用和剂量信息的技术说明。
医疗用途和治疗
本公开的RNA核酸分子或组合物或疫苗或试剂盒可以用于人类,也可以用于兽医目的,优选用于人类医学目的。
根据另一方面,本发明因此涉及用作药物的本公开的RNA核酸分子、组合物、疫苗、或试剂盒。
本公开的RNA核酸分子、组合物或疫苗或试剂盒可用于治疗遗传病、癌症、自身免疫疾病、炎性疾病和感染性疾病或其他疾病或状况。
根据另一方面,本发明因此涉及本公开的RNA核酸分子、组合物或疫苗或试剂盒,其用于治疗遗传疾病、癌症、自身免疫疾病、炎性疾病和感染性疾病或其他疾病或状况。
“基因疗法”优选地涉及调节对象中的基因表达,以达到治疗效果。为此,基因疗法通常包括将核酸引入细胞。基因疗法可能涉及宿主细胞的体内或体外转化。
术语“治疗”疾病包括预防疾病(即,导致临床症状不发展);抑制疾病(即,阻止或抑制临床症状的发展);和/或缓解疾病(即,导致临床症状的消退)。应当理解的是,由于一个或多于一个最终诱发事件可能是未知的或潜在的,因此并非总是可能在“预防”和“抑制”疾病或病症之间进行区分。因此,术语“预防”将被理解为构成涵盖“预防”和“抑制”两者的“治疗”的类型。因此,术语“治疗”包括“预防”。
如本文所用,术语“对象”、“患者”或“个体”通常包括人和非人动物,并且优选地包括哺乳动物(例如非人灵长类动物,包括狨猴、绢毛猴、蜘蛛猴、猫头鹰猴、长尾黑颌猴、松鼠猴、以及狒狒、猕猴、黑猩猩、猩猩、大猩猩;牛;马;绵羊;猪;鸡;猫;狗;小鼠;大鼠;兔;豚鼠等),包括嵌合和转基因动物和疾病模型。在本文中,术语“对象”优选是指非人灵长类动物或人,最优选人。
因此,本公开还提供了通过向有需要的对象施用药学上有效量的RNA核酸分子、组合物或疫苗或试剂盒来治疗本文公开的疾病的方法,其包括向需要的患者/对象施用药学上有效量的所述RNA核酸分子、组合物或疫苗或试剂盒。
可以例如全身或局部施用本公开的RNA核酸分子或组合物或疫苗或试剂盒。全身施用途径通常包括例如透皮、经口、肠胃外途径,包括皮下、静脉内、肌内、动脉内、皮内和腹膜内注射和/或鼻内施用途径。局部施用途径一般包括例如外用途径,但也包括皮内、透皮、皮下或肌内注射或病灶内、瘤内、颅内、肺内、心内、和舌下注射。
根据优选的实施方案,RNA核酸分子、组合物或疫苗或试剂盒通过肠胃外途径,优选通过皮内、皮下或肌内途径施用。优选地,RNA核酸分子组合物或疫苗或试剂盒可通过注射例如皮下、肌内、或皮内注射施用,其可以是无针注射和/或针头注射。因此,在优选的实施方案中,根据本发明的医疗用途和/或治疗方法包括通过皮下、肌内或皮内注射,优选通过肌内或皮内注射,更优选通过皮内注射施用所述RNA核酸分子、组合物或疫苗或试剂盒。这种注射可以通过使用常规的针头注射或(无针)射流注射来进行,优选地通过使用(无针)射流注射来进行。
本公开的RNA核酸分子、(药物)组合物或疫苗或试剂盒可以每天几次、每天、每隔一天、每周或每月施用于有需要的对象;并且可以依次或同时施用。
如果施用不同的RNA核酸分子、或组合物或疫苗或包含几种组分例如不同的RNA核酸分子和任选单独本文所述的其他活性剂的试剂盒,则每种组分可以同时施用(通过相同或不同的施用途径同时施用)或分开施用(在不同时间通过相同或不同的施用途径施用)。
本公开的RNA核酸分子、组合物或疫苗或试剂盒可以优选以治疗有效量施用。如本文所用,“治疗有效量”是指足以在所寻求的组织、系统、动物或人类中引发期望的生物或药物反应的活性剂的量。治疗有效量优选地足以诱导要治疗的疾病的阳性改变,即用于减轻待治疗疾病的症状、减少疾病进展或预防待预防疾病的症状。然而,与此同时,“治疗有效量”优选小到足以避免严重的副作用,也就是说允许优势和风险之间的合理关系,即安全和治疗有效量。
“治疗有效量”还随待治疗的特定病症以及待治疗患者的年龄、身体状况、体重、性别和饮食、病症的严重程度、治疗持续时间、伴随治疗的性质、所用的特定药学上可接受的载剂或赋形剂、治疗方案和类似因素而变化。
RNA核酸分子的“治疗有效量”还可以根据RNA核酸分子的类型来选择,例如单顺反子、双顺反子或甚至多顺反子RNA,因为在于单顺反子RNA的量相等的情况下,双顺反子或甚至多顺反子RNA可以导致目的编码多肽或蛋白质的显著更高的表达。
本公开的RNA核酸分子、组合物或疫苗或试剂盒的治疗功效和毒性可以通过细胞培养物或实验动物中的标准药物程序来确定,例如,用于确定LD50(对50%的群体致死的剂量)和ED50(对50%的群体治疗有效的剂量)。毒性和治疗效果之间的剂量比是治疗指数,可以表示为LD50/ED50比。通常优选表现出大的治疗指数的RNA核酸分子、组合物或试剂盒。从细胞培养测定法和动物研究中获得的数据可用于配制用于人类的剂量范围。这类化合物的剂量优选在包括几乎没有或没有毒性的ED50的循环浓度的范围内。
例如,本文所述的RNA核酸分子、组合物或疫苗或试剂盒的治疗有效剂量可以为每剂量单位约0.001mg至10mg,优选每剂量单位约0.01mg至5mg,更优选每剂量单位约0.1mg至2mg或每剂量单位约0.01nmol至1mmol,特别是每剂量单位1nmol至1mmol,优选每剂量单位1μmol至1mmol。在一些实施方案中,本公开的RNA核酸分子、组合物或疫苗或试剂盒的治疗有效剂量可以为约0.01g/kg至10g/kg,优选约0.05mg/kg至5g/kg,更优选约0.1mg/kg至2.5g/kg(每kg体重)。
遗传病
在优选的实施方案中,RNA核酸分子、(药物)组合物或疫苗或试剂盒用于治疗或预防遗传病。
如本文所用,术语“遗传病”包括由基因组异常(即偏离野生型、健康和无症状状态)引起的、以其为特征的或与之相关的任何疾病、障碍或病症。此类异常可包括染色体拷贝数的变化(例如,非整倍体)或其部分变化(例如,缺失、重复、扩增);或染色体结构发生变化(例如,易位、点突变)。基因组异常可能是遗传性的(隐性或显性的)或非遗传性的。基因组异常可存在于生物体的某些细胞中或该生物体的所有细胞中,并且包括常染色体异常、X连锁异常、Y连锁异常和线粒体异常。
癌症
在优选的实施方案中,RNA核酸分子、组合物或疫苗或试剂盒用于治疗或预防癌症。
如本文所用,术语“癌症”指赘生物,其特征在于细胞不受控制且通常迅速增殖,从而趋于侵入周围组织并转移至远处身体部位。该术语包括良性肿瘤和恶性肿瘤。癌症中的恶性肿瘤通常以间变、侵袭和转移为特征;而良性肿瘤通常不具备这些性质。该术语包括以肿瘤生长的赘生物,以及血液和淋巴系统癌症。
在一些实施方案中,根据本公开的RNA核酸分子、组合物或疫苗或试剂盒可以用作药物,特别是用于治疗肿瘤或癌症疾病。在这种情况下,治疗优选涉及瘤内施用,特别是通过瘤内注射施用。因此,根据本公开的RNA核酸分子、组合物或疫苗或试剂盒可以用于制备用于治疗肿瘤或癌症疾病的药物,所述药物特别适合于瘤内应用(施用)以治疗肿瘤或癌症疾病。
优选地,本文提及的肿瘤和癌症疾病选自优选包括以下的肿瘤或癌症疾病:例如急性淋巴细胞白血病、急性髓细胞白血病、肾上腺皮质癌、AIDS相关癌症、AIDS相关淋巴瘤、肛门癌、阑尾癌、星形细胞瘤、基底细胞癌、胆管癌、膀胱癌、骨癌、骨肉瘤/恶性纤维组织细胞瘤、脑干胶质瘤、脑瘤、小脑星形细胞瘤、脑星形细胞瘤/恶性胶质瘤、室管膜瘤、髓母细胞瘤、幕上原始神经外胚层肿瘤、视觉通路和下丘脑神经胶质瘤、乳腺癌、支气管腺瘤/类癌、伯基特淋巴瘤、儿童类癌肿瘤、胃肠道类癌肿瘤、未知原发性、原发性中枢神经系统淋巴瘤、儿童小脑星形细胞瘤、儿童脑星形细胞瘤/恶性胶质瘤、宫颈癌、儿童癌症、慢性淋巴细胞白血病、慢性粒细胞白血病、慢性骨髓增生性疾病、结肠癌、皮肤T细胞淋巴瘤、结缔组织增生性小圆细胞肿瘤、子宫内膜癌、室管膜瘤、食道癌、尤因家族肿瘤中的尤因肉瘤、儿童颅外生殖细胞瘤、性腺外生殖细胞瘤、肝外胆管癌、眼内黑色素瘤、视网膜母细胞瘤、胆囊癌、胃癌、胃肠道类癌肿瘤、胃肠道间质瘤、颅外、性腺外或卵巢生殖细胞瘤、妊娠滋养细胞瘤、脑干胶质瘤、儿童脑星形细胞瘤、儿童视觉途径和下丘脑胶质瘤、胃类癌、毛细胞白血病、头颈癌、心脏癌、肝细胞癌、霍奇金淋巴瘤、咽下癌、儿童下丘脑和视觉通路神经胶质瘤、眼内黑色素瘤、胰岛细胞癌、卡波西肉瘤、肾癌、喉癌、白血病、急性淋巴细胞白血病、急性髓细胞白血病、慢性淋巴细胞白血病、慢性粒细胞白血病、多毛细胞白血病、唇和口腔癌、脂肪肉瘤、肝癌、非小细胞肺癌、小细胞肺癌、淋巴瘤、AIDS相关淋巴瘤、伯基特淋巴瘤、皮肤T细胞淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、原发性中枢神经系统淋巴瘤、巨珠蛋白血症、骨/骨肉瘤的恶性纤维组织细胞瘤、儿童髓母细胞瘤、黑色素瘤、眼内(眼)黑色素瘤、默克尔细胞癌、成人恶性间皮瘤、儿童间皮瘤、伴隐匿性原发性的转移性鳞状颈癌、口腔癌、儿童多发性内分泌肿瘤综合征、多发性骨髓瘤/血浆细胞瘤、蕈样肉芽肿病、骨髓增生异常综合征、骨髓增生异常/骨髓增生性疾病、慢性粒细胞白血病、成人急性髓细胞白血病、儿童急性髓细胞白血病、多发性骨髓瘤、慢性骨髓增生异常、鼻腔和鼻旁窦癌、鼻咽癌、神经母细胞瘤、口腔癌、口咽癌、骨肉瘤/骨恶性纤维组织细胞瘤、卵巢癌、卵巢上皮癌、卵巢生殖细胞肿瘤、卵巢低度恶性潜能肿瘤、胰腺癌、胰岛细胞胰腺癌、鼻旁窦和鼻腔癌、甲状旁腺癌、阴茎癌、咽喉癌、嗜铬细胞瘤、松果星形细胞瘤、松果生殖细胞瘤、儿童松果体细胞瘤和幕上原始神经外胚层肿瘤、垂体腺瘤、血浆细胞瘤/多发性骨髓瘤、胸膜肺母细胞瘤、原发性中枢神经系统淋巴瘤、前列腺癌、直肠癌、肾细胞癌、肾盂和输尿管癌、视网膜母细胞瘤、儿童横纹肌肉瘤、唾液腺癌、尤因家族肉瘤、卡波西肉瘤、软组织肉瘤、子宫肉瘤、Sézary综合征、皮肤癌(非黑色素瘤)、皮肤癌(黑色素瘤)、默克尔细胞皮肤癌、小肠癌、鳞状细胞癌、伴隐匿性原发性的转移性鳞状颈癌、儿童幕上原始神经外胚层肿瘤、睾丸癌、咽喉癌、儿童胸腺瘤、胸腺瘤和胸腺癌、甲状腺癌、儿童甲状腺癌、肾盂和输尿管移行细胞癌、妊娠滋养细胞肿瘤、尿道癌、子宫内膜子宫癌、子宫肉瘤、阴道癌、儿童视觉通路和下丘脑胶质瘤、外阴癌、巨珠蛋白血症和儿童威尔姆氏瘤(肾癌)。
感染性疾病
在优选的实施方案中,RNA核酸分子、组合物或疫苗或试剂盒用于治疗或预防感染性疾病。
术语“感染”或“感染性疾病”涉及通常不存在于体内的微生物例如细菌、病毒和寄生虫的入侵和繁殖。感染可能不会引起任何症状并且是亚临床的,或者可能引起症状并且在临床上是明显的。感染可能保持局部性,或者可能通过血液或淋巴系统传播,以成为全身性感染。在这种情况下,感染性疾病优选包括病毒、细菌、真菌或原生动物感染性疾病。
自身免疫疾病
在优选的实施方案中,RNA核酸分子、组合物或疫苗或试剂盒用于治疗或预防自身免疫疾病。
术语“自身免疫疾病”是指对象中的任何疾病、病症或病状,其特征在于由对象对其自身的细胞、组织和/或器官的免疫反应引起的细胞、组织和/或器官损伤。通常,“自身免疫疾病”是由与自身抗原(即健康人体细胞表达的抗原)具有反应性的抗体引起或加剧的。
自身免疫疾病可以分为全身性症状,包括但不限于系统性红斑狼疮(SLE)、干燥综合征、硬皮病、类风湿关节炎和多发性肌炎;或局部综合征,其可以是内分泌性的(I型糖尿病、桥本甲状腺炎、艾迪生病等)、皮肤病学的(寻常性天疱疮)、血液病学的(自身免疫性溶血性贫血)、神经系统的(多发性硬化症)或几乎可以涉及任何限定的身体组织。在本文中,自身免疫疾病可以选自I型自身免疫疾病或II型自身免疫疾病或III型自身免疫疾病或IV型自身免疫疾病,例如多发性硬化症(MS)、类风湿关节炎、糖尿病、I型糖尿病(1型糖尿病)、慢性多发性关节炎、突眼性甲状腺肿、慢性肝炎的自身免疫形式、溃疡性结肠炎、I型过敏性疾病、II型过敏性疾病、III型过敏性疾病、IV型过敏性疾病、纤维肌痛、脱发、别赫捷列夫症、克罗恩病、重症肌无力、神经性皮炎、风湿性多肌痛、进行性全身性硬化症(PSS)、赖特综合征、风湿性关节炎、银屑病、血管炎和II型糖尿病。
炎性疾病
在优选的实施方案中,RNA核酸分子、组合物或疫苗或试剂盒用于治疗或预防炎性疾病。
术语“炎性疾病”是指对象中的以炎症,优选为以慢性炎症为特征、由其引起、或伴随其的任何疾病、病症或病状。自身免疫疾病可能与炎症有关,也可能与炎症无关。而且,炎症可能是或不是由自身免疫疾病引起的。因此,某些疾病可以被表征为自身免疫疾病和炎性疾病两者。
在本文中,示例性炎性疾病包括但不限于类风湿关节炎、克罗恩病、糖尿病性视网膜病、银屑病、子宫内膜异位症、阿尔茨海默氏病、强直性脊柱炎、关节炎(例如骨关节炎、类风湿关节炎(RA)、银屑病性关节炎)、哮喘、动脉粥样硬化、结肠炎、皮炎、憩室炎、纤维肌痛、肝炎、肠易激综合症(IBS)、系统性红斑狼疮(SLE)、肾炎、帕金森病和溃疡性结肠炎。
变态反应
在优选的实施方案中,RNA核酸分子、组合物或疫苗或试剂盒用于治疗或预防变态反应。
术语“变态反应”或“过敏性超敏反应”是指通常在遗传上易感的个体(特应性)中,由免疫机制针对变应原引发的超敏反应引起或以其为特征的任何疾病、病症或病状。变态反应可以是抗体介导的或细胞介导的。在大多数患者中,通常引起过敏反应的抗体属于IgE同种型(IgE介导的变态反应,I型变态反应)。在非IgE介导的变态反应中,抗体可能属于IgG同种型。可以根据引起过敏反应的抗原来源对变态反应进行分类。在本文中,变态反应可以选自(a)食物过敏、(b)药物过敏、(c)屋尘过敏、(d)昆虫毒液或叮咬过敏、和(e)花粉过敏。或者,可以根据变态反应的主要症状对变态反应进行分类。在本文中,变态反应可以选自(a)哮喘、(b)鼻炎、(c)结膜炎、(d)鼻结膜炎(rhinoconjuctivitis)、(e)皮炎、(f)荨麻疹和(g)过敏反应。
在一些实施方案中,本公开的核酸分子编码成纤维细胞生长因子(FGF)例如碱性成纤维细胞生长因子(bFGF)、尤其是人bFGF。成纤维细胞生长因子是一类由约150-200氨基酸组成的多肽,以两种密切相关的形式存在,即碱性成纤维细胞生长因子(bFGF)和酸性成纤维细胞生长因子(aFGF)。已知现在有24种FGF成员。各种细胞合成bFGF,调节特定种类细胞的增殖和分化。此外,bFGF在生物体内和体外表现出有效的血管生成作用,刺激平滑肌细胞的生长,促进伤口愈合和组织再生。bFGF在神经退行性疾病、心脏病和难以愈合的伤口类病变中也具有多种治疗用途,并且还可通过诱导成纤维细胞和干细胞的增殖在组织发育中发挥重要作用。另外,bFGF可促进表皮细胞代谢和增殖,改善皮肤的生理状态,促进表皮组织的修复和代谢,从而可使皮肤细嫩年轻,淡化色斑,平复皱纹,减轻日晒对皮肤的损伤,还具有显著抗衰老作用。
因此,本公开提供的编码成纤维细胞生长因子(FGF)例如碱性成纤维细胞生长因子(bFGF)的核酸分子可用于化妆用途例如润肤、消除皱纹、消除或预防色斑、或抗皮肤衰老。本公开提供的编码成纤维细胞生长因子(FGF)例如碱性成纤维细胞生长因子(bFGF)的核酸分子还可以用于修复创伤、防止脱发(例如促进毛发生长或毛囊再生或预防或治疗毛发脱落)或促进组织发育或用于治疗或预防神经退行性疾病或心脏病。
在一些实施方案中,本公开提供的编码成纤维细胞生长因子(FGF)例如碱性成纤维细胞生长因子(bFGF)的核酸分子还可用于刺激骨形成(Wang JS.Basic fibroblastgrowth factor for stimulation of bone formation in osteoinductive orconductive implants.Acta Orthop Scand Suppl.1996Apr;269:1-33.doi:10.3109/17453679609155229.PMID:8629452.)以及治疗糖尿病性神经病变(NAKAE,M.et al.,Diabetic Neuropathy Is Improved by bFGF Treatment.Diabetes,2004,53)。
联合疗法
本公开的RNA核酸分子、组合物或疫苗或试剂盒也可以用于联合疗法。可用于治疗或预防本文限定的疾病和病症的任何其他疗法可与本文公开的用途和方法组合。
例如,接受本公开的RNA核酸分子、组合物或疫苗或试剂盒的对象可以是接受化学治疗(例如一线或二线化学治疗)、放射治疗、化学放射治疗(化学治疗和放射治疗的组合)、酪氨酸激酶抑制剂(例如EGFR酪氨酸激酶抑制剂)、抗体治疗和/或抑制性和/或刺激性检查点分子(例如CTLA4抑制剂)的患有癌症或相关病症的患者,或在接受上述一种或多于一种治疗后已达到部分缓解或疾病稳定的患者。或者,接受本公开的RNA核酸分子、组合物或疫苗或试剂盒的对象可以是接受抗生素、抗真菌剂或抗病毒治疗的优选如本文所定义的患有感染性疾病的患者。
本公开的RNA核酸分子、组合物或疫苗或试剂盒的施用可以在施用另一种治疗剂或使患者接受对治疗特定疾病或病症有用的另一种治疗之前、同时和/或随后进行。
本公开以编码人碱性成纤维细胞生长因子(hbFGF)、人表皮生长因子(hEGF)和绿色荧光蛋白(GFP)的mRNA为示例,介绍了一种生产mRNA的新型无细胞体外转录RNA平台或系统。在该系统中具有以下优势的一个或多个:产生的mRNA具有很高的稳定性;可以很容易地施用到细胞中;具有更好的翻译效率;具有快速的生产时间(不长于1小时);产量可达2mg/ml/h,甚至高达约2.3mg/ml/h;易于管理;和生产成本低。该系统特别适用于成纤维细胞生长因子优选碱性成纤维细胞生长因子mRNA的体外转录。
实施例
本文中通过以下实施例对本公开进行描述,其仅旨在举例说明,对本公开的范围并无限制。
材料和方法:
用于RNA体外转录的DNA寡核苷酸的设计
如图1A所示,从T7启动子之前的EcoRI开始设计DNA寡核苷酸构建体。将目标基因的信号序列和编码序列(hEGF、hbFGF、GFP)分别添加到人β珠蛋白5'非翻译序列和人α珠蛋白3'非翻译序列之间。为了终止转录,在构建体的5'末端添加了T7终止子。为了通用克隆,在间隔区序列中添加了SalI和NotI。DNA寡核苷酸由ThermoFisher Scientific合成。该DNA寡核苷酸的序列分别如SEQ ID NO:5-7所示。
SEQ ID NO:5(hEGF)
5’-GGGAGAGTCGACAAATAAGAGAGAAAAGAAGAGTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGCTGCTCACTCTTATCATTCTGTTGCCAGTAGTTTCAAAAAATAGTGACTCTGAATGTCCCCTGTCCCACGATGGGTACTGCCTCCATGATGGTGTGTGCATGTATATTGAAGCATTGGACAAGTATGCATGCAACTGTGTTGTTGGCTACATCGGGGAGCGATGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGCTGAGCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCTGGTCTTTGGCGGCCGCCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATGAATTCGC-3’
SEQ ID NO:6(hbFGF)
5’-GGGAGAGTCGACAAATAAGAGAGAAAAGAAGAGTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGCAGCCGGGAGCATCACCACGCTGCCCGCCTTGCCCGAGGATGGCGGCAGCGGCGCCTTCCCGCCCGGCCACTTCAAGGACCCCAAGCGGCTGTACTGCAAAAACGGGGGCTTCTTCCTGCGCATCCACCCCGACGGCCGAGTTGACGGGGTCCGGGAGAAGAGCGACCCTCACATCAAGCTACAACTTCAAGCAGAAGAGAGAGGAGTTGTGTCTATCAAAGGAGTGTGTGCTAACCGTTACCTGGCTATGAAGGAAGATGGAAGATTACTGGCTTCTAAATGTGTTACGGATGAGTGTTTCTTTTTTGAACGATTGGAATCTAATAACTACAATACTTACCGGTCAAGGAAATACACCAGTTGGTATGTGGCACTGAAACGAACTGGGCAGTATAAACTTGGATCCAAAACAGGACCTGGGCAGAAAGCTATACTTTTTCTTCCAATGTCTGCTAAGAGCTGAGCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCTGGTCTTTGGCGGCCGCCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATGAATTCGC-3’
SEQ ID NO:7(GFP)
5’-GGGAGAGTCGACAAATAAGAGAGAAAAGAAGAGTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCTGGTCTTTGGCGGCCGCCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATGAATTCGC-3’
RNA的体外转录
通过以寡核苷酸P1(5’-AATTCATCCGGATATAGTTC-3’(SEQ ID NO:8))和P2(5’-TGTACATAATACGACTCACTAT-3’(SEQ ID NO:9))为引物,以上文设计的DNA寡核苷酸为模板,进行PCR延伸,制备用于体外转录的PCR模板。PCR产物通过New England Biolabs(美国马萨诸塞州伊普斯维奇)的PCR&DNA Cleanup试剂盒进行纯化,然后进行标准mRNA合成,该mRNA合成按照New England Biolabs的描述进行操作。将1μg纯化的PCR产物与体外mRNA合成预混液混合[1.5mM ATP,1.25mM UTP,1.25mM CTP,1mM GTP,4mM 7m G(3’-O-Me)pppG(ARCA),1U/μl RNase抑制剂,0.4U/μl T7 RNA聚合酶,40mM Tris-HCl(pH 7.9),6mMMgCl2、2mM亚精胺]并在37℃下孵育30分钟,以生成5’-ARCA封端的mRNA。然后将0.2U/μlDNase I添加到反应混合物中,并在37℃下孵育15分钟以去除PCR模板。通过额外的Poly(A)聚合酶反应混合物[50mM Tris-HCl(pH8.1),250mM NaCl,10mM MgCl2、0.05U/μl Poly(A)聚合酶]进行Poly(A)拖尾,并在37℃下孵育30分钟。最终的mRNA产物通过New EnglandBiolabs(美国马萨诸塞州伊普斯维奇)的RNA Cleanup试剂盒纯化,并保存用于分析。所获得的mRNA的结构如图1B所示,mRNA的序列分别如SEQ ID NO:10-12所示。
SEQ ID NO:10(hEGF)
5’-GGGAGAGUCGACAAAUAAGAGAGAAAAGAAGAGUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCAUGCUGCUCACUCUUAUCAUUCUGUUGCCAGUAGUUUCAAAAAAUAGUGACUCUGAAUGUCCCCUGUCCCACGAUGGGUACUGCCUCCAUGAUGGUGUGUGCAUGUAUAUUGAAGCAUUGGACAAGUAUGCAUGCAACUGUGUUGUUGGCUACAUCGGGGAGCGAUGUCAGUACCGAGACCUGAAGUGGUGGGAACUGCGCUGAGCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGGCGGCCGC-3’
SEQ ID NO:11(hbFGF)
5’-GGGAGAGUCGACAAAUAAGAGAGAAAAGAAGAGUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCAUGGCAGCCGGGAGCAUCACCACGCUGCCCGCCUUGCCCGAGGAUGGCGGCAGCGGCGCCUUCCCGCCCGGCCACUUCAAGGACCCCAAGCGGCUGUACUGCAAAAACGGGGGCUUCUUCCUGCGCAUCCACCCCGACGGCCGAGUUGACGGGGUCCGGGAGAAGAGCGACCCUCACAUCAAGCUACAACUUCAAGCAGAAGAGAGAGGAGUUGUGUCUAUCAAAGGAGUGUGUGCUAACCGUUACCUGGCUAUGAAGGAAGAUGGAAGAUUACUGGCUUCUAAAUGUGUUACGGAUGAGUGUUUCUUUUUUGAACGAUUGGAAUCUAAUAACUACAAUACUUACCGGUCAAGGAAAUACACCAGUUGGUAUGUGGCACUGAAACGAACUGGGCAGUAUAAACUUGGAUCCAAAACAGGACCUGGGCAGAAAGCUAUACUUUUUCUUCCAAUGUCUGCUAAGAGCUGAGCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGGCGGCCGC-3’
SEQ ID NO:12(GFP)
5’-GGGAGAGUCGACAAAUAAGAGAGAAAAGAAGAGUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCAUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAAGCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGGCGGCCGC-3’
mRNA转染
用脂质纳米颗粒完成mRNA的转染。简而言之,将DC-胆固醇(3β-[N-(N',N'-二甲基氨基乙烷)-氨基甲酰基]胆固醇盐酸盐)和DOPE(二油酰基磷脂酰乙醇胺)以25mg/mL溶解在氯仿中。将40μL DC-胆固醇与80μL DOPE混合,并在真空浓缩器中干燥15分钟以蒸发氯仿。当将脂质重悬于1mL无核酸酶的水中并在35kHz的超声浴中乳化1小时,然后在小型挤出机中挤出。
将10μL脂质纳米颗粒稀释于100μL无血清DMEM培养基中,然后与1μg所获得的mRNA混合并在室温下孵育15分钟。然后将mRNA-脂质纳米颗粒复合物添加到293T细胞中,以温育指定的时间。
蛋白质检测
转染后24小时,在配备有Andor EM-CCD相机的尼康Eclipse Ti倒置显微镜下,在Plan Apo 20x/0.75DIC镜头下观察到了被GFP mRNA转染的293T细胞。在相应的滤镜设置下拍摄明场和GFP图像,如图3所示。用hEGF和hbFGF mRNA转染的293T细胞温育24小时。转染后0、2、4、6、8、24小时收集细胞和培养基。在裂解缓冲液(PBS+1%Triton和完全蛋白酶抑制剂)中裂解细胞。通过Western印迹分析细胞裂解物样品的hbFGF和hEGF表达。结果示于图5中。
实时PCR
裂解转染的293T细胞,并用RNAzol试剂(分子研究中心)提取其总RNA。用Qubit定量RNA的产量。使用Oligodt引物,用GoScript逆转录酶(Promega)对100ug RNA进行逆转录。通过预先设计的实时PCR引物/探针对cDNA样品进行分析,以QuantStudio 3和SYBR GreenI预混液检测hEGF和hbFGF。所有样品均一式三份运行。平均基因表达在3个独立实验中计算。如图2所示,用Qubit定量在不同时间点收集的EGF和bFGF mRNA的体外转录产量。当体外转录进行一个小时时,最大产量达到2250μg/mL。
结果:
mRNA的工程化和体外转录
DNA寡核苷酸由T7启动子、人β珠蛋白5’-非翻译序列、目的基因的信号序列和编码序列(hEGF、hbFGF和GFP)、人α珠蛋白3’-非翻译序列和T7终止子组成。该寡核苷酸充当hEGF、hbFGF和GFP的稳定的体外转录骨架。通过T7聚合酶转录mRNA,并在5’和3’末端分别添加抗反向帽类似物和polyA尾巴,以增强mRNA的稳定性和翻译效率。体外转录期间的各个时间点被用于分析。当反应时间为1小时时,每毫升mRNA的最大产量为约2.3mg。
图4示出了通过qPCR对293T细胞中的细胞内EGF和bFGF mRNA进行定量。将0.5μg用脂质纳米颗粒包裹的EGF和bFGF mRNA转染到293T细胞中。用qPCR定量转染后不同时间点收集的细胞内的EGF和bFGF mRNA。从图4可见,在标准293T细胞培养条件下,EGF和bFGF mRNA的浓度在8小时内保持稳定,并最终在转染后至24小时才有所下降。
表达GFP的mRNA转染293T细胞
293T细胞用包封有0.5ug表达GFP的mRNA的脂质纳米颗粒转染,并孵育24小时。在荧光显微镜下,超过85%的转染细胞在GFP滤光片下显示出荧光信号,如图3所示。通过脂质纳米颗粒包封的编码GFP的mRNA已成功转染到293T细胞中。GFP mRNA转染的293T细胞的荧光信号随时间增加。
qPCR检测hEGF和hbFGF mRNA
用分别包封有0.5ug hEGF mRNA和0.5ug hbFGF mRNA的脂质纳米颗粒转染293T细胞。采用了对hEGF和hbFGF具有高度特异性的RNA探针来定量mRNA水平。hEGF和hbFGF mRNA的水平在整个培养的前8小时都保持稳定,而在第24小时仅略有下降,这证明mRNA非常稳定,如图4所示。
蛋白质印迹法检测hEGF和hbFGF
在转染后0、2、4、6、8、24小时收集用hEGF和hbFGF mRNA转染的293T细胞。制备细胞裂解物和细胞培养基,并通过针对hEGF和hbFGF抗体的蛋白质印迹进行分析。如图5所示,在转染后第4小时后,细胞裂解物中的hEGF和hbFGF的表达均显著增加;另一方面,在转染后第6小时后,细胞培养基中的hEGF和hbFGF水平被观察到并显著增加。
虽然在本文已经非常详细地描述了RNA体外转录方法和构建体的各种实施方案,但这些实施方案仅仅作为本文描述的公开内容的非限制性实例而提供。因此,本领域技术人员将理解,可以对本公开描述的方案进行各种变化和改变,而不偏离本发明的精神。实际上,本公开内容不旨在是穷尽性的或旨在限制本发明的范围。
进一步,在代表性实施方案的描述中,本公开内容已经以特定步骤顺序给出本发明的方法和/或过程。然而,该方法或过程不应限于所描述的特定步骤顺序。其它步骤顺序是可能的。因此,本文公开的特定步骤顺序不应解释为对本发明的限制。此外,针对方法和/或过程的公开内容不应限于以所描述的顺序进行它们的步骤。这样的顺序可以变化并且仍在本发明的范围内。
序列表
<110> 梦芊科技知识产权有限公司
<120> 成纤维细胞生长因子mRNA的无细胞和无载体体外RNA转录方法和核酸分子
<130> GWHWW204171DI
<141> 2021-01-05
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50
<212> RNA
<213> 人(Homo sapiens)
<400> 1
acauuugcuu cugacacaac uguguucacu agcaaccuca aacagacacc 50
<210> 2
<211> 88
<212> RNA
<213> 人(Homo sapiens)
<400> 2
gcuggagccu cgguagccgu uccuccugcc cgcugggccu cccaacgggc ccuccucccc 60
uccuugcacc ggcccuuccu ggucuuug 88
<210> 3
<211> 50
<212> DNA
<213> 人(Homo sapiens)
<400> 3
acatttgctt ctgacacaac tgtgttcact agcaacctca aacagacacc 50
<210> 4
<211> 88
<212> DNA
<213> 人(Homo sapiens)
<400> 4
gctggagcct cggtagccgt tcctcctgcc cgctgggcct cccaacgggc cctcctcccc 60
tccttgcacc ggcccttcct ggtctttg 88
<210> 5
<211> 521
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gggagagtcg acaaataaga gagaaaagaa gagtacattt gcttctgaca caactgtgtt 60
cactagcaac ctcaaacaga caccatgctg ctcactctta tcattctgtt gccagtagtt 120
tcaaaaaata gtgactctga atgtcccctg tcccacgatg ggtactgcct ccatgatggt 180
gtgtgcatgt atattgaagc attggacaag tatgcatgca actgtgttgt tggctacatc 240
ggggagcgat gtcagtaccg agacctgaag tggtgggaac tgcgctgagc tggagcctcg 300
gtagccgttc ctcctgcccg ctgggcctcc caacgggccc tcctcccctc cttgcaccgg 360
cccttcctgg tctttggcgg ccgcctgcta acaaagcccg aaaggaagct gagttggctg 420
ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg 480
gttttttgct gaaaggagga actatatccg gatgaattcg c 521
<210> 6
<211> 785
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gggagagtcg acaaataaga gagaaaagaa gagtacattt gcttctgaca caactgtgtt 60
cactagcaac ctcaaacaga caccatggca gccgggagca tcaccacgct gcccgccttg 120
cccgaggatg gcggcagcgg cgccttcccg cccggccact tcaaggaccc caagcggctg 180
tactgcaaaa acgggggctt cttcctgcgc atccaccccg acggccgagt tgacggggtc 240
cgggagaaga gcgaccctca catcaagcta caacttcaag cagaagagag aggagttgtg 300
tctatcaaag gagtgtgtgc taaccgttac ctggctatga aggaagatgg aagattactg 360
gcttctaaat gtgttacgga tgagtgtttc ttttttgaac gattggaatc taataactac 420
aatacttacc ggtcaaggaa atacaccagt tggtatgtgg cactgaaacg aactgggcag 480
tataaacttg gatccaaaac aggacctggg cagaaagcta tactttttct tccaatgtct 540
gctaagagct gagctggagc ctcggtagcc gttcctcctg cccgctgggc ctcccaacgg 600
gccctcctcc cctccttgca ccggcccttc ctggtctttg gcggccgcct gctaacaaag 660
cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg 720
gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatgaa 780
ttcgc 785
<210> 7
<211> 1037
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gggagagtcg acaaataaga gagaaaagaa gagtacattt gcttctgaca caactgtgtt 60
cactagcaac ctcaaacaga caccatggtg agcaagggcg aggagctgtt caccggggtg 120
gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc 180
gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc 240
aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc 300
agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 360
tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag 420
gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 480
gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat 540
atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc 600
gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc 660
cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc 720
aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc 780
ggcatggacg agctgtacaa gtaagctgga gcctcggtag ccgttcctcc tgcccgctgg 840
gcctcccaac gggccctcct cccctccttg caccggccct tcctggtctt tggcggccgc 900
ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag 960
cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta 1020
tatccggatg aattcgc 1037
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aattcatccg gatatagttc 20
<210> 9
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tgtacataat acgactcact at 22
<210> 10
<211> 384
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 10
gggagagucg acaaauaaga gagaaaagaa gaguacauuu gcuucugaca caacuguguu 60
cacuagcaac cucaaacaga caccaugcug cucacucuua ucauucuguu gccaguaguu 120
ucaaaaaaua gugacucuga auguccccug ucccacgaug gguacugccu ccaugauggu 180
gugugcaugu auauugaagc auuggacaag uaugcaugca acuguguugu uggcuacauc 240
ggggagcgau gucaguaccg agaccugaag uggugggaac ugcgcugagc uggagccucg 300
guagccguuc cuccugcccg cugggccucc caacgggccc uccuccccuc cuugcaccgg 360
cccuuccugg ucuuuggcgg ccgc 384
<210> 11
<211> 648
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 11
gggagagucg acaaauaaga gagaaaagaa gaguacauuu gcuucugaca caacuguguu 60
cacuagcaac cucaaacaga caccauggca gccgggagca ucaccacgcu gcccgccuug 120
cccgaggaug gcggcagcgg cgccuucccg cccggccacu ucaaggaccc caagcggcug 180
uacugcaaaa acgggggcuu cuuccugcgc auccaccccg acggccgagu ugacgggguc 240
cgggagaaga gcgacccuca caucaagcua caacuucaag cagaagagag aggaguugug 300
ucuaucaaag gagugugugc uaaccguuac cuggcuauga aggaagaugg aagauuacug 360
gcuucuaaau guguuacgga ugaguguuuc uuuuuugaac gauuggaauc uaauaacuac 420
aauacuuacc ggucaaggaa auacaccagu ugguaugugg cacugaaacg aacugggcag 480
uauaaacuug gauccaaaac aggaccuggg cagaaagcua uacuuuuucu uccaaugucu 540
gcuaagagcu gagcuggagc cucgguagcc guuccuccug cccgcugggc cucccaacgg 600
gcccuccucc ccuccuugca ccggcccuuc cuggucuuug gcggccgc 648
<210> 12
<211> 900
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 12
gggagagucg acaaauaaga gagaaaagaa gaguacauuu gcuucugaca caacuguguu 60
cacuagcaac cucaaacaga caccauggug agcaagggcg aggagcuguu caccggggug 120
gugcccaucc uggucgagcu ggacggcgac guaaacggcc acaaguucag cguguccggc 180
gagggcgagg gcgaugccac cuacggcaag cugacccuga aguucaucug caccaccggc 240
aagcugcccg ugcccuggcc cacccucgug accacccuga ccuacggcgu gcagugcuuc 300
agccgcuacc ccgaccacau gaagcagcac gacuucuuca aguccgccau gcccgaaggc 360
uacguccagg agcgcaccau cuucuucaag gacgacggca acuacaagac ccgcgccgag 420
gugaaguucg agggcgacac ccuggugaac cgcaucgagc ugaagggcau cgacuucaag 480
gaggacggca acauccuggg gcacaagcug gaguacaacu acaacagcca caacgucuau 540
aucauggccg acaagcagaa gaacggcauc aaggugaacu ucaagauccg ccacaacauc 600
gaggacggca gcgugcagcu cgccgaccac uaccagcaga acacccccau cggcgacggc 660
cccgugcugc ugcccgacaa ccacuaccug agcacccagu ccgcccugag caaagacccc 720
aacgagaagc gcgaucacau gguccugcug gaguucguga ccgccgccgg gaucacucuc 780
ggcauggacg agcuguacaa guaagcugga gccucgguag ccguuccucc ugcccgcugg 840
gccucccaac gggcccuccu ccccuccuug caccggcccu uccuggucuu uggcggccgc 900
Claims (12)
1.一种RNA核酸分子,其从5’端到3’端包含5’-帽结构、人β-珠蛋白5’非翻译区(5’-UTR)、至少一个编码区、人α-珠蛋白3’非翻译区(3’-UTR)和3’-聚腺苷酸尾,所述至少一个编码区可操作地连接到所述5’-UTR和所述3’-UTR,所述编码区编码成纤维细胞生长因子(FGF)例如碱性成纤维细胞生长因子(bFGF)。
2.根据权利要求1所述的RNA核酸分子,其中所述编码区还编码选自下列的至少一种:信号肽、肽标签或蛋白质标签、定位信号或定位序列、和肽接头。
3.根据权利要求1或2所述的RNA核酸分子,其中所述5’-UTR包含或组成为根据SEQ IDNO:1的RNA序列,或与根据SEQ ID NO:1的RNA序列具有至少80%、90%、95%、96%、97%、98%、或99%的序列同一性的RNA序列;和/或其中所述3’-UTR包含或组成为根据SEQ IDNO:2的RNA序列,或与根据SEQ ID NO:2的RNA序列具有至少80%、90%、95%、96%、97%、98%、或99%的序列同一性的RNA序列。
4.根据权利要求1-3任一项所述的RNA核酸分子,其中所述5’-帽结构是5’-抗反向帽类似物(5’-ARCA),优选地所述5’-ARCA是7m G(3’-O-Me)pppG。
5.根据权利要求1-4任一项所述的RNA核酸分子,其中所述3’-聚腺苷酸尾包含10个至200个、20个至100个、40个至80个、或50个至70个腺嘌呤核苷酸。
6.一种DNA核酸分子,其从5’端到3’端包含启动子、人β-珠蛋白5’-非翻译区(5’-UTR)、至少一个编码区、人α-珠蛋白3’-非翻译区(3’-UTR)和转录终止子,所述至少一个编码区可操作地连接到所述5’-UTR和所述3’-UTR,所述编码区编码成纤维细胞生长因子(FGF),例如碱性成纤维细胞生长因子(bFGF)。
7.根据权利要求6所述的DNA核酸分子,其中所述编码区还编码选自下列的至少一种:信号肽、肽标签或蛋白质标签、定位信号或定位序列、和肽接头。
8.根据权利要求6或7所述的DNA核酸分子,其中所述启动子选自T3、T7、Sny5或SP6启动子,和/或所述启动子选自是T3启动子,并且所述转录终止子选自T7转录终止子。
9.根据权利要求6-8任一项所述的DNA核酸分子,其中所述5’-UTR包含或组成为根据SEQ ID NO:3的DNA序列,或与根据SEQ ID NO:3的DNA序列具有至少80%、90%、95%、96%、97%、98%、或99%的序列同一性的DNA序列;和/或其中所述3’-UTR包含或组成为根据SEQID NO:4的DNA序列,或与根据SEQ ID NO:4的DNA序列具有至少80%、90%、95%、96%、97%、98%、或99%的序列同一性的DNA序列。
10.一种体外转录方法,其包括步骤:
(a)提供根据权利要求6-9任一项所述的DNA核酸分子作为转录模板;
(b)任选地,扩增所述DNA核酸分子;
(c)将所述DNA核酸分子在5’-抗反向帽类似物(5’-ARCA)、优选7m G(3’-O-Me)pppG存在的情况下进行体外转录,以获得包含5’-ARCA封端的mRNA的反应混合物;
(d)任选地,通过添加DNA酶去除所述包含5’-ARCA封端的mRNA的反应混合物中的转录模板;和
(e)向所述包含5’-ARCA封端的mRNA的反应混合物添加聚腺苷酸聚合酶反应混合物进行3’-聚腺苷酸尾添加,以获得5’-ARCA封端的并具有3’-聚腺苷酸尾的mRNA。
11.一种组合物,其包含根据权利要求1-5任一项所述的RNA核酸分子和/或根据权利要求10所述的方法获得的RNA核酸分子,以及药学上可接受的载剂和/或赋形剂;其中优选地,所述组合物是药物组合物或试剂盒。
12.根据权利要求1-5任一项所述的RNA核酸分子和/或根据权利要求10所述的方法获得的RNA核酸分子在化妆用途例如润肤、消除皱纹、消除或预防色斑、或抗皮肤衰老中的用途或在制备用于修复创伤、再生肌肤、防止脱发、促进组织发育或刺激骨形成或用于治疗或预防神经退行性疾病、心脏病或糖尿病性神经病变的药物中的用途。
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