CN117065052A - 递送自复制rna分子的方法 - Google Patents
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Abstract
本发明提出了递送自复制RNA分子的方法和组合物,所述组合物包括核酸分子和递送载体,所述核酸分子包括自复制RNA分子,所述递送载体携带所述核酸分子,所述递送载体包括蛋白质、脂质体和外泌体的至少之一。利用本发明的组合物有助于实现核酸类药物尤其是自复制RNA分子的体内递送。
Description
技术领域
本发明涉及生物领域。具体地,本发明涉及递送自复制RNA分子的方法。
背景技术
近年来基于mRNA药物或mRNA疫苗的开发成为人们关注的热点之一。然而,传统mRNA不是很稳定,在细胞内几天内就会降解,导致不可持续的蛋白质表达水平。如果长期用于疾病治疗,可能需要患者注射大量的mRNA,这可能会增加mRNA治疗的毒副作用。
自复制RNA分子可以在细胞质内进行自我复制,因此可以在很低的剂量下达到不低于传统mRNA的蛋白表达水平,并且可以在一定时间内长效表达蛋白质,从而展现出比mRNA更有前景的技术优势。
然而,针对RNA分子,目前缺乏有效的递送手段,这极大地限制了自复制RNA分子技术的应用。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。
在本发明的一个方面,本发明提出了一种组合物。根据本发明的实施例,所述组合物包括:核酸分子,所述核酸分子包括自复制RNA分子;递送载体,所述递送载体携带所述核酸分子。
为了实现自复制RNA分子进入细胞且稳定存在于细胞内,需要递送载体将其递送至细胞内,有助于实现细胞内RNA自复制,维持长效的高蛋白水平,更好地发挥作用效果。根据本发明的实施例,利用本发明的组合物有助于实现核酸类药物尤其是自复制RNA分子的体内递送。
在本发明的另一方面,本发明提出了一种药物组合物。根据本发明的实施例,所述药物组合物包括:前面所述的组合物。
在本发明的又一方面,本发明提出了一种递送自复制RNA分子的方法。根据本发明的实施例,所述方法包括:将自复制RNA分子配制为前面所述的组合物;和任选的,为待处理对象提供所述组合物。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解,其中:
图1显示了根据本发明一个实施例的自复制RNA分子(reRNATM)的结构示意图;
图2显示了根据本发明一个实施例的含有自复制RNA分子的蛋白质-RNA复合物的结构示意图;
图3显示了根据本发明一个实施例的含自复制RNA分子的LNP递送系统示意图;
图4显示了根据本发明一个实施例的含自复制RNA分子的蛋白质递送系统结构示意图;
图5显示了根据本发明一个实施例的reRNATM-GFP入胞效率检测的分析示意图;
图6显示了根据本发明一个实施例的以LCMV-GP作为递送蛋白递送reRNATM-GFP至vero细胞的GFP荧光图;
图7显示了根据本发明一个实施例的以NDV-F/NDV-HN作为递送蛋白递送reRNATM-GFP至vero细胞的GFP荧光图;
图8显示了根据本发明一个实施例的以LNP作为递送载体递送reRNATM-GFP至vero细胞的GFP荧光图。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的,仅用于解释本发明,而不能理解为对本发明的限制。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
在本文中所使用的术语“自复制RNA分子”也可以称为“自我扩增RNA”,与普通mRNA相比,自复制RNA分子的重要的区别在于它可以使用自己的RNA序列作为模板进行自我复制。根据本申请的实施例,自复制RNA分子还可以在细胞质进行翻译和复制工作,不进入细胞核,可以避免与基因组发生整合所带来的潜在风险。通常mRNA编码需要表达的蛋白质,细胞内的核糖体用来完成翻译和蛋白质生产。根据本申请的实施例,自复制RNA分子会携带一个能够表达RNA聚合酶(RNA依赖RNA聚合酶)的序列,在该RNA分子在细胞质中通过翻译产生RNA聚合酶后,它可以用该自复制RNA分子作为模板来产生更多的自复制RNA分子。
组合物
在本发明的一个方面,本发明提出了一种组合物。根据本发明的实施例,所述组合物包括:核酸分子,所述核酸分子包括自复制RNA分子;递送载体,所述递送载体携带所述核酸分子。
为了实现自复制RNA分子进入细胞且稳定存在于细胞内,将其承载在递送载体上,递送载体可以携带自复制RNA分子进入细胞内,有助于实现细胞内RNA自复制,维持长效的高蛋白水平,更好地发挥作用效果。根据本发明的实施例,利用本发明的组合物有助于实现核酸类药物尤其是自复制RNA分子的体内递送。
发明人在研究过程中发现,由于自复制RNA分子将至少编码RNA聚合酶的序列与表达靶蛋白的序列连接起来,因此整个mRNA分子的分子量比传统mRNA的分子量大得多,分子量过大可能导致递送效率、翻译效率以及复制效率显著降低。为了对这些效率进行改善,发明人进行了深入研究,希望能够寻找到最短的核酸片段,可以正常发挥自我复制和翻译的功能。
根据本发明的实施例,所述自复制RNA分子包括:第一RNA序列,所述第一RNA序列编码N蛋白或其功能片段;第二RNA序列,所述第二RNA序列编码P蛋白或其功能片段;第三RNA序列,所述第三RNA序列编码L蛋白或其功能片段;和靶分子编码区,所述靶分子编码区编码至少一个靶分子。
参见图1和图2,发明人通过对各种RNA病毒的RNA在细胞内的翻译及自我扩增机制进行了深入研究,发现通过采用编码来自弹状病毒的N蛋白,P蛋白及L蛋白的RNA分子作为核心区域,可以实现RNA在动物细胞内的自我复制和翻译,并且该核心区域作为强大的“引擎”,可以提供高效转录扩增和启动大分子蛋白的“动能”,能够进一步搭载“货物区”来复制或者翻译靶分子,这些靶分子几乎涵盖了目前市场上所有的蛋白质药物。根据本发明的实施例,“货物区”可设计不同的蛋白编码盒使机体在细胞内生产多种肽、酶、抗体、通道蛋白以及受体蛋白等,从而达到不同的预防或治疗目的,覆盖肿瘤管线、疫苗管线、罕见病管线及前瞻性通用型产品管线。本发明中将提出的新型自复制RNA分子命名为reRNATM。
在本文中所使用的术语“功能片段”是指蛋白质的全长序列的一部分,但仍能够发挥与RNA分子自我复制相关的功能,例如可以是全长序列的截断型的,也可以是蛋白质全长序列的氨基酸序列发生替换、突变或者删除等改变后的蛋白质。根据本申请的实施例,对于N蛋白的功能片段,可以结合RNA分子,保护RNA不受核酸酶的影响,对于P蛋白的功能片段,能够结合N蛋白,在模板上定位L聚合酶,同时也能够作为RNA聚合酶转录和复制复合体的基本组成部分,进一步L蛋白的功能片段能够发挥RNA聚合酶的功能,与RNA的转录及复制有关。
根据本发明的实施例,所述N蛋白、所述P蛋白、所述L蛋白的至少之一分别独立地来自弹状病毒科病毒。
所述N蛋白、所述P蛋白、所述L蛋白,可以来自水疱性口炎病毒印第安纳株,N蛋白包括但不限于Uniprot ID为:P03521、P11212、Q77E03、Q8B0H4、B7UCZ2的序列;P蛋白包括但不限于Uniprot ID为:P04880、Q8B0H8、P04879、P03520、B7UCZ3的序列;L蛋白包括但不限于Uniprot ID为:Q8B0H0、Q98776、Q8B0I0、Q8B0H5、P03523的序列。
所述N蛋白、所述P蛋白、所述L蛋白,可以来自水疱性口炎病毒新泽西株,N蛋白包括但不限于Uniprot ID为:P04881、Q89034、S5TKS4、Q89036、Q89037的序列;P蛋白包括但不限于Uniprot ID为:P04877、Q89057、Q89052、Q89050、Q89049的序列;L蛋白包括但不限于Uniprot ID为:P16379、P16379、I7DDL0、Q8B545、S5TC82的序列。
所述N蛋白、所述P蛋白、所述L蛋白,还可以来自其他水泡性病毒属(如钱迪普拉水疱病毒、马拉巴水泡病毒)、狂犬病毒属。
根据本发明的实施例,所述N蛋白具有SEQ ID NO:1所示氨基酸序列或与其具有至少80%同源性的氨基酸序列,所述P蛋白具有SEQ ID NO:2所示氨基酸序列或与其具有至少80%同源性的氨基酸序列,所述L蛋白具有SEQ ID NO:3所示氨基酸序列或与其具有至少80%同源性的氨基酸序列。
MSVTVKRIIDNTVVVPKLPANEDPVEYPADYFRKSKEIPLYINTTKSLSDLRGYVYQGLKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIGIFDLVSLKALDGVLPDGVSDASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQCKMINEQFEPLVPEGRDIFDVWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCAALATFGHLCKITGMSTEDVTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSPYSSVKNPAFHFWGQLTALLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFCVGDNKYTPDDSTGGLTTNAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGLREKTIGKYAKSEFDK(SEQ ID NO:1)
MDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEEHTKPSYFQAADDSDTESEPEIEDNQGLYAPDPEAEQVEGFIQGPLDDYADEEVDVVFTSDWKQPELESDEHGKTLRLTSPEGLSGEQKSQWLSTIKAVVQSAKYWNLAECTFEASGEGVIMKERQITPDVYKVTPVMNTHPSQSEAVSDVWSLSKTSMTFQPKKASLQPLTISLDELFSSRGEFISVGGDGRMSHKEAILLGLRYKKLYNQARVKYSL(SEQ ID NO:2)
MEVHDFETDEFNDFNEDDYATREFLNPDERMTYLNHADYNLNSPLISDDIDNLIRKFNSLPIPSMWDSKNWDGVLEMLTSCQANPIPTSQMHKWMGSWLMSDNHDASQGYSFLHEVDKEAEITFDVVETFIRGWGNKPIEYIKKERWTDSFKILAYLCQKFLDLHKLTLILNAVSEVELLNLARTFKGKVRRSSHGTNICRIRVPSLGPTFISEGWAYFKKLDILMDRNFLLMVKDVIIGRMQTVLSMVCRIDNLFSEQDIFSLLNIYRIGDKIVERQGNFSYDLIKMVEPICNLKLMKLARESRPLVPQFPHFENHIKTSVDEGAKIDRGIRFLHDQIMSVKTVDLTLVIYGSFRHWGHPFIDYYTGLEKLHSQVTMKKDIDVSYAKALASDLARIVLFQQFNDHKKWFVNGDLLPHDHPFKSHVKENTWPTAAQVQDFGDKWHELPLIKCFEIPDLLDPSIIYSDKSHSMNRSEVLKHVRMNPNTPIPSKKVLQTMLDTKATNWKEFLKEIDEKGLDDDDLIIGLKGKERELKLAGRFFSLMSWKLREYFVITEYLIKTHFVPMFKGLTMADDLTAVIKKMLDSSSGQGLKSYEAICIANHIDYEKWNNHQRKLSNGPVFRVMGQFLGYPSLIERTHEFFEKSLIYYNGRPDLMRVHNNTLINSTSQRVCWQGQEGGLEGLRQKGWSILNLLVIQREAKIRNTAVKVLAQGDNQVICTQYKTKKSRNVVELQGALNQMVSNNEKIMTAIKIGTGKLGLLINDDETMQSADYLNYGKIPIFRGVIRGLETKRWSRVTCVTNDQIPTCANIMSSVSTNALTVAHFAENPINAMIQYNYFGTFARLLLMMHDPALRQSLYEVQDKIPGLHSSTFKYAMLYLDPSIGGVSGMSLSRFLIRAFPDPVTESLSFWRFIHVHARSEHLKEMSAVFGNPEIAKFRITHIDKLVEDPTSLNIAMGMSPANLLKTEVKKCLIESRQTIRNQVIKDATIYLYHEEDRLRSFLWSINPLFPRFLSEFKSGTFLGVADGLISLFQNSRTIRNSFKKKYHRELDDLIVRSEVSSLTHLGKLHLRRGSCKMWTCSATHADTLRYKSWGRTVIGTTVPHPLEMLGPQHRKETPCAPCNTSGFNYVSVHCPDGIHDVFSSRGPLPAYLGSKTSESTSILQPWERESKVPLIKRATRLRDAISWFVEPDSKLAMTILSNIHSLTGEEWTKRQHGFKRTGSALHRFSTSRMSHGGFASQSTAALTRLMATTDTMRDLGDQNFDFLFQATLLYAQITTTVARDGWITSCTDHYHIACKSCLRPIEEITLDSSMDYTPPDVSHVLKTWRNGEGSWGQEIKQIYPLEGNWKNLAPAEQSYQVGRCIGFLYGDLAYRKSTHAEDSSLFPLSIQGRIRGRGFLKGLLDGLMRASCCQVIHRRSLAHLKRPANAVYGGLIYLIDKLSVSPPFLSLTRSGPIRDELETIPHKIPTSYPTSNRDMGVIVRNYFKYQCRLIEKGKYRSHYSQLWLFSDVLSIDFIGPFSISTTLLQILYKPFLSGKDKNELRELANLSSLLRSGEGWEDIHVKFFTKDILLCPEEIRHACKFGIAKDNNKDMSYPPWGRESRGTITTIPVYYTTTPYPKMLEMPPRIQNPLLSGIRLGQLPTGAHYKIRSILHGMGIHYRDFLSCGDGSGGMTAALLRENVHSRGIFNSLLELSGSVMRGASPEPPSALETLGGDKSRCVNGETCWEYPSDLCDPRTWDYFLRLKAGLGLQIDLIVMDMEVRDSSTSLKIETNVRNYVHRILDEQGVLIYKTYGTYICESEKNAVTILGPMFKTVDLVQTEFSSSQTSEVYMVCKGLKKLIDEPNPDWSSINESWKNLYAFQSSEQEFARAKKVSTYFTLTGIPSQFIPDPFVNIETMLQIFGVPTGVSHAAALKSSDRPADLLTISLFYMAIISYYNINHIRVGPIPPNPPSDGIAQNVGIAITGISFWLSLMEKDIPLYQQCLAVIQQSFPIRWEAVSVKGGYKQKWSTRGDGLPKDTRISDSLAPIGNWIRSLELVRNQVRLNPFNEILFNQLCRTVDNHLKWSNLRRNTGMIEWINRRISKEDRSILMLKSDLHEENSWRD(SEQ ID NO:3)
根据本申请的实施例,所述第一RNA序列具有如SEQ ID NO:4所示的核苷酸序列,所述第二RNA序列具有如SEQ ID NO:5所示的核苷酸序列,所述第三RNA序列具有如SEQ IDNO:6所示的核苷酸序列。
AUGUCCGUGACCGUGAAGAGGAUCAUCGACAAUACCGUGGUGGUGCCCAAGCUGCCCGCCAAUGAGGACCCUGUGGAGUACCCUGCCGAUUACUUUAGGAAGAGCAAGGAGAUCCCUCUGUACAUCAAUACAACCAAGAGCCUGAGCGAUCUGAGAGGCUACGUGUACCAGGGCCUGAAGUCCGGCAACGUGAGCAUCAUCCACGUGAACAGCUACCUGUACGGCGCCCUGAAGGAUAUCAGAGGCAAGCUGGAUAAGGAUUGGUCCUCCUUCGGCAUCAACAUCGGCAAGGCCGGCGACACCAUCGGCAUCUUUGAUCUGGUGUCCCUGAAGGCCCUGGACGGCGUGCUGCCCGACGGAGUUAGCGACGCCAGCAGGACAUCCGCCGACGACAAGUGGCUGCCUCUGUACCUGCUGGGCCUGUACAGAGUGGGCAGAACACAGAUGCCCGAGUACAGGAAGAAGCUGAUGGACGGCCUGACCAAUCAGUGCAAGAUGAUCAAUGAGCAGUUCGAGCCCCUGGUGCCCGAGGGCAGAGAUAUCUUUGACGUGUGGGGCAAUGAUAGCAACUACACCAAGAUCGUGGCCGCCGUGGACAUGUUUUUCCACAUGUUCAAGAAGCACGAGUGUGCCAGCUUUAGGUACGGCACAAUCGUGUCCAGGUUCAAGGACUGCGCCGCCCUGGCCACAUUUGGCCACCUGUGCAAGAUCACCGGCAUGUCCACAGAGGAUGUGACCACAUGGAUCUUGAACAGGGAGGUGGCCGAUGAGAUGGUGCAGAUGAUGCUGCCUGGCCAGGAGAUCGAUAAGGCCGACUCCUACAUGCCUUACCUGAUCGAUUUCGGCCUGAGCUCCAAGUCCCCUUACAGCUCCGUGAAGAACCCCGCCUUCCACUUUUGGGGCCAGCUGACAGCCCUGCUGCUGAGAUCCACCAGAGCCAGAAAUGCCAGGCAGCCUGACGAUAUCGAGUACACCAGCCUGACAACAGCCGGCCUGCUGUACGCCUACGCCGUGGGAAGCUCCGCCGAUCUGGCCCAGCAGUUCUGUGUGGGCGAUAAUAAGUACACCCCUGAUGAUUCCACAGGCGGCCUGACCACAAACGCCCCCCCUCAGGGCAGAGAUGUGGUGGAGUGGCUGGGCUGGUUCGAGGACCAGAAUAGGAAGCCUACACCCGACAUGAUGCAGUACGCCAAGAGAGCCGUGAUGUCCCUGCAGGGCCUGAGAGAGAAGACCAUCGGCAAGUACGCCAAGAGCGAGUUUGACAAGUAA(SEQ ID NO:4)
AUGGAUAACCUGACAAAGGUGAGAGAGUACCUGAAGAGCUACAGCAGACUGGACCAGGCCGUGGGCGAGAUCGACGAGAUCGAGGCCCAGAGAGCCGAGAAGUCCAAUUACGAGCUGUUCCAGGAGGAUGGCGUGGAGGAGCACACAAAGCCUAGCUACUUUCAGGCCGCCGAUGAUAGCGACACAGAGAGCGAGCCCGAGAUCGAGGAUAAUCAGGGCCUGUACGCCCCCGACCCUGAGGCUGAGCAGGUGGAGGGCUUCAUCCAGGGCCCUCUGGAUGACUACGCCGACGAGGAGGUGGACGUGGUGUUUACAAGCGAUUGGAAGCAGCCCGAGCUGGAGAGCGAUGAGCACGGCAAGACACUGAGGCUGACCAGCCCUGAGGGCCUGUCCGGCGAGCAGAAGAGCCAGUGGCUGUCCACAAUCAAGGCCGUGGUGCAGUCCGCCAAGUACUGGAAUCUGGCCGAGUGCACAUUUGAGGCCAGCGGCGAGGGCGUGAUCAUGAAGGAGAGACAGAUCACACCCGAUGUGUACAAGGUGACCCCUGUGAUGAACACACACCCCUCCCAGUCCGAGGCCGUGUCCGACGUGUGGUCCCUGAGCAAGACAAGCAUGACAUUCCAGCCCAAGAAGGCCUCCCUGCAGCCUCUGACCAUCUCCCUGGACGAGCUGUUCAGCAGCAGAGGCGAGUUUAUCAGCGUGGGCGGCGACGGCAGAAUGAGCCACAAGGAGGCCAUCCUGCUGGGCCUGAGAUACAAGAAGCUGUACAAUCAGGCCAGAGUGAAGUACUCCCUGUAA(SEQ ID NO:5)
AUGGAGGUGCACGAUUUUGAGACAGAUGAGUUCAACGAUUUUAACGAGGAUGAUUACGCCACCAGAGAGUUCCUGAACCCCGACGAGAGGAUGACAUACCUGAAUCACGCCGACUACAAUCUGAACUCCCCUCUGAUCAGCGACGAUAUCGAUAAUCUGAUCAGGAAGUUCAACUCCCUGCCUAUCCCCUCCAUGUGGGACUCCAAGAACUGGGAUGGCGUGCUGGAGAUGCUGACCUCCUGCCAGGCCAACCCCAUCCCUACCUCCCAGAUGCACAAGUGGAUGGGCUCCUGGCUGAUGUCCGAUAACCACGACGCCUCCCAGGGCUACUCCUUUCUGCACGAGGUGGAUAAGGAGGCCGAGAUCACCUUUGAUGUGGUGGAGACAUUCAUUAGAGGCUGGGGCAACAAGCCUAUCGAGUACAUCAAGAAGGAGAGAUGGACCGACUCCUUUAAGAUCCUGGCCUACCUGUGUCAGAAGUUCCUGGAUCUGCACAAGCUGACACUGAUCCUGAACGCCGUGUCCGAGGUGGAGCUGCUGAAUCUGGCCAGGACCUUCAAGGGCAAGGUGAGGAGGAGCUCCCACGGCACCAAUAUCUGUAGGAUCAGAGUGCCCAGCCUGGGCCCCACAUUCAUCAGCGAGGGCUGGGCCUACUUUAAGAAGCUGGACAUCCUGAUGGAUAGAAACUUCCUGCUGAUGGUGAAGGAUGUGAUCAUCGGCAGGAUGCAGACCGUGCUGAGCAUGGUGUGCAGGAUCGACAAUCUGUUCAGCGAGCAGGACAUCUUCUCCCUGCUGAAUAUCUACAGGAUCGGCGAUAAGAUCGUGGAGAGGCAGGGCAACUUCAGCUACGAUCUGAUCAAGAUGGUGGAGCCCAUCUGUAAUCUGAAGCUGAUGAAGCUGGCCAGAGAGAGCAGACCUCUGGUGCCCCAGUUUCCUCACUUCGAGAAUCACAUCAAGACAAGCGUGGAUGAGGGCGCCAAGAUCGAUAGAGGCAUCAGAUUUCUGCACGAUCAGAUCAUGUCCGUGAAAACUGUUGAUCUGACACUGGUCAUCUAUGGCUCCUUCAGACACUGGGGCCACCCCUUUAUCGAUUACUACACCGGCCUGGAGAAGCUGCACAGCCAGGUGACCAUGAAGAAGGACAUCGACGUGUCCUACGCCAAGGCCCUGGCCUCCGAUCUGGCCAGGAUCGUGCUGUUUCAGCAGUUCAACGACCACAAGAAGUGGUUUGUGAAUGGCGAUCUGCUGCCUCACGACCACCCCUUUAAGUCCCACGUGAAGGAGAAUACAUGGCCCACAGCCGCCCAGGUGCAGGACUUCGGCGAUAAGUGGCACGAGCUGCCCCUGAUCAAGUGUUUCGAGAUCCCUGAUCUGCUGGACCCCAGCAUCAUCUACUCCGAUAAGUCCCACAGCAUGAACAGAAGCGAGGUGCUGAAGCACGUGAGAAUGAACCCCAAUACCCCUAUCCCUUCCAAGAAGGUGCUGCAGACCAUGCUGGACACCAAGGCCACCAACUGGAAGGAGUUUCUGAAGGAGAUCGACGAGAAGGGCCUGGAUGAUGACGACCUGAUCAUCGGCCUGAAGGGCAAGGAGAGAGAGCUGAAGCUGGCCGGCAGAUUUUUCUCCCUGAUGUCCUGGAAGCUGAGAGAGUACUUCGUGAUCACCGAGUACCUGAUCAAGACCCACUUCGUGCCCAUGUUCAAGGGCCUGACCAUGGCCGAUGACCUGACCGCCGUGAUCAAGAAGAUGCUGGAUAGCUCCAGCGGCCAGGGCCUGAAGUCCUACGAGGCCAUCUGCAUCGCCAAUCACAUCGACUACGAGAAGUGGAAUAACCACCAGAGGAAGCUGAGCAACGGCCCCGUGUUCAGAGUGAUGGGCCAGUUCCUGGGCUACCCCUCCCUGAUCGAGAGGACACACGAGUUCUUCGAGAAGAGCCUGAUCUACUACAAUGGCAGACCUGACCUGAUGAGGGUGCACAAUAAUACCCUGAUCAAUUCCACAUCCCAGAGGGUGUGCUGGCAGGGCCAGGAGGGCGGACUGGAGGGACUGAGGCAGAAGGGCUGGAGCAUCCUGAACCUGCUGGUCAUUCAGAGAGAGGCCAAGAUCAGAAACACCGCCGUGAAGGUGCUGGCCCAGGGCGACAAUCAGGUCAUUUGCACACAGUACAAGACAAAGAAGAGCAGGAAUGUGGUGGAGCUGCAGGGCGCCCUGAAUCAGAUGGUGAGCAAUAACGAGAAGAUCAUGACAGCCAUCAAGAUCGGCACCGGCAAGCUGGGCCUGCUGAUCAACGAUGAUGAGACAAUGCAGUCCGCCGAUUACCUGAAUUACGGCAAGAUCCCCAUCUUCAGAGGCGUGAUCAGGGGCCUGGAGACAAAGAGGUGGAGCAGAGUGACAUGUGUGACCAACGAUCAGAUCCCUACAUGCGCCAACAUCAUGUCCUCCGUGUCCACAAAUGCCCUGACCGUGGCCCACUUUGCCGAGAAUCCUAUCAAUGCCAUGAUCCAGUACAAUUACUUCGGCACCUUUGCCAGGCUGCUGCUGAUGAUGCACGAUCCCGCCCUGAGGCAGAGCCUGUACGAGGUGCAGGACAAGAUCCCUGGCCUGCACAGCUCCACAUUCAAAUACGCCAUGCUGUACCUGGACCCUUCCAUCGGCGGCGUGAGCGGCAUGUCCCUGUCCAGAUUCCUGAUCAGAGCCUUCCCCGAUCCUGUGACCGAGAGCCUGAGCUUUUGGAGGUUUAUCCACGUGCACGCCAGAUCCGAGCACCUGAAGGAGAUGUCCGCCGUGUUCGGCAACCCUGAGAUCGCCAAGUUUAGGAUCACCCACAUCGACAAGCUGGUGGAGGACCCUACAUCCCUGAACAUCGCCAUGGGCAUGAGCCCCGCCAAUCUGCUGAAAACUGAAGUUAAGAAGUGCCUGAUCGAGUCCAGACAGACAAUCAGAAACCAGGUCAUUAAGGAUGCCACAAUCUACCUGUACCACGAGGAGGAUAGACUGAGGUCCUUCCUGUGGAGCAUCAAUCCCCUGUUUCCCAGAUUCCUGUCCGAGUUCAAGAGCGGCACCUUCCUGGGCGUGGCCGAUGGCCUGAUCUCCCUGUUCCAGAAUAGCAGAACAAUCAGAAAUUCCUUCAAGAAGAAGUACCACAGAGAGCUGGAUGACCUGAUCGUGAGAUCCGAGGUGUCCAGCCUGACCCACCUGGGCAAGCUGCACCUGAGAAGGGGCUCCUGCAAGAUGUGGACCUGCAGCGCCACCCACGCCGACACACUGAGAUACAAGUCCUGGGGCAGAACAGUGAUCGGCACCACCGUGCCUCACCCCCUGGAGAUGCUCGGCCCUCAGCACAGGAAGGAGACACCUUGUGCCCCUUGCAAUACAAGCGGCUUUAAUUACGUGUCCGUGCACUGUCCCGAUGGCAUCCACGAUGUGUUCUCCAGCAGAGGCCCUCUGCCUGCCUACCUGGGCUCCAAGACCUCCGAGUCCACAAGCAUCCUGCAGCCUUGGGAGAGAGAGUCCAAGGUGCCUCUGAUCAAGAGGGCCACCAGACUGAGAGAUGCCAUCAGCUGGUUCGUGGAGCCUGACUCCAAGCUGGCCAUGACCAUCCUGUCCAACAUCCACAGCCUGACCGGCGAGGAGUGGACCAAGAGGCAGCACGGCUUUAAGAGGACCGGCUCCGCCCUGCACAGAUUUUCCACCUCCAGAAUGUCCCACGGCGGCUUUGCCAGCCAGAGCACCGCCGCUCUGACCAGACUGAUGGCCACCACAGACACAAUGAGAGAUCUGGGCGAUCAGAAUUUCGAUUUCCUGUUCCAGGCCACACUGCUGUACGCCCAGAUCACAACCACCGUGGCCAGGGACGGCUGGAUCACCUCCUGUACCGACCACUACCACAUCGCCUGCAAGAGCUGCCUGAGGCCCAUCGAGGAGAUCACACUGGACAGCAGCAUGGAUUACACACCUCCUGAUGUGAGCCACGUGCUGAAAACUUGGCGUAACGGCGAGGGCAGCUGGGGCCAGGAGAUCAAGCAGAUCUACCCUCUGGAGGGCAACUGGAAGAAUCUGGCCCCUGCCGAGCAGUCCUACCAGGUGGGCAGGUGUAUCGGCUUCCUGUACGGCGACCUGGCCUACAGGAAGAGCACACACGCCGAGGACUCCUCCCUGUUCCCCCUGAGCAUCCAGGGCAGAAUCAGGGGCAGGGGCUUCCUGAAGGGCCUGCUGGACGGCCUGAUGAGGGCCAGCUGUUGUCAGGUCAUUCACAGGAGGUCCCUGGCCCACCUGAAGAGGCCCGCCAACGCCGUGUACGGCGGCCUGAUCUACCUGAUCGACAAGCUCUCCGUGAGCCCCCCUUUUCUGUCCCUGACAAGGUCCGGCCCCAUCAGAGAUGAGCUGGAGACAAUCCCUCACAAGAUCCCUACCAGCUACCCCACCAGCAAUAGGGACAUGGGCGUGAUCGUGAGAAACUACUUUAAGUACCAGUGCAGGCUGAUCGAGAAGGGCAAGUACAGAUCCCACUACUCCCAGCUGUGGCUGUUCAGCGAUGUGCUGUCCAUCGAUUUCAUCGGCCCCUUUAGCAUCAGCACCACACUGCUGCAGAUCCUGUACAAGCCUUUCCUGAGCGGCAAGGACAAGAAUGAGCUGAGGGAGCUGGCCAAUCUGUCCUCCCUGCUGAGGUCCGGCGAGGGCUGGGAGGACAUCCACGUGAAGUUUUUCACAAAGGAUAUCCUGCUGUGCCCUGAGGAGAUCAGACACGCCUGUAAGUUUGGCAUCGCCAAGGACAACAACAAGGAUAUGAGCUACCCCCCUUGGGGCAGGGAGAGCAGAGGCACAAUCACCACCAUCCCCGUGUACUACACAACAACCCCCUACCCUAAGAUGCUGGAGAUGCCUCCUAGAAUCCAGAAUCCUCUGCUGUCCGGCAUCAGACUGGGCCAGCUGCCCACAGGCGCCCACUACAAGAUCAGAUCCAUCCUGCACGGCAUGGGCAUCCACUACAGAGACUUUCUGUCCUGUGGCGAUGGCUCCGGCGGCAUGACCGCCGCUCUCCUGAGGGAGAAUGUGCACAGCAGAGGCAUCUUUAAUAGCCUGCUGGAGCUGUCCGGCAGCGUGAUGAGAGGCGCCUCCCCCGAGCCCCCUUCCGCUCUGGAAACACUGGGCGGCGAUAAGAGCAGGUGUGUGAAUGGCGAGACAUGCUGGGAGUACCCCAGCGAUCUGUGUGAUCCCAGAACAUGGGACUACUUUCUGAGGCUGAAGGCCGGCCUGGGCCUGCAGAUCGAUCUGAUCGUGAUGGAUAUGGAGGUGAGAGACAGCAGCACCAGCCUGAAGAUCGAGACAAAUGUGAGAAACUAUGUGCACAGGAUCUUGGAUGAGCAGGGCGUGCUGAUCUACAAGACCUACGGCACAUACAUCUGCGAGAGCGAGAAGAAUGCCGUGACCAUCCUGGGCCCUAUGUUUAAGACAGUGGACCUGGUGCAGACCGAGUUUAGCUCCAGCCAGACAUCCGAGGUGUACAUGGUGUGUAAGGGCCUGAAGAAGCUGAUCGAUGAGCCCAAUCCUGAUUGGAGCUCCAUCAAUGAGUCCUGGAAGAAUCUCUACGCCUUUCAGAGCAGCGAGCAGGAGUUCGCCAGGGCCAAGAAGGUGAGCACAUACUUCACCCUGACCGGCAUCCCCUCCCAGUUCAUCCCUGAUCCCUUCGUGAACAUCGAGACAAUGCUGCAGAUCUUUGGCGUGCCUACCGGCGUGAGCCACGCCGCUGCUCUGAAGAGCUCCGACAGGCCCGCCGAUCUGCUGACAAUCUCCCUGUUUUACAUGGCCAUCAUCUCCUACUACAACAUCAACCACAUCAGAGUGGGCCCCAUCCCCCCUAACCCUCCUAGCGACGGCAUCGCCCAGAAUGUGGGCAUCGCCAUCACCGGCAUCUCCUUUUGGCUGAGCCUGAUGGAGAAGGACAUCCCCCUGUACCAGCAGUGUCUGGCCGUGAUCCAGCAGAGCUUUCCCAUCAGGUGGGAGGCCGUGUCCGUGAAGGGCGGCUACAAGCAGAAGUGGUCCACAAGGGGCGAUGGCCUGCCUAAGGAUACAAGGAUCAGCGACAGCCUGGCCCCCAUCGGCAACUGGAUCAGGUCCCUGGAGCUGGUGAGGAAUCAGGUGAGACUGAACCCUUUCAACGAGAUCCUGUUUAACCAGCUGUGCAGAACAGUGGACAACCACCUGAAGUGGUCCAAUCUGAGAAGAAAUACAGGCAUGAUCGAGUGGAUCAACAGAAGGAUCUCCAAGGAGGACAGGAGCAUCCUGAUGCUGAAGAGCGACCUGCACGAGGAGAACUCCUGGAGAGACUAA(SEQ ID NO:6)
根据本发明的实施例,所述递送载体包括蛋白质、脂质体和外泌体的至少之一。
蛋白质可以作为递送载体携带核酸分子进入细胞,以实现遗传物质的细胞内递送。
脂质体作为纳米级药物递送载体被广泛地应用于药物递送中,可以自组装成病毒大小的颗粒,并能够将mRNA从内质体释放到细胞质。同时,可以起到促进细胞摄取、提高核内体逃逸、保护核酸分子不被TLRs识别、避免先天免疫系统的过度激活等作用。如图3所示,LNP递送系统主要是由LNP电势及核酸电势决定包裹核酸的量,由此一个LNP递送系统可以包裹一个到多个核酸分子。
外泌体是从活细胞释放到细胞外微环境的50-150nm大小的囊泡,由于外泌体体积小和其本身就是细胞产物,通过外泌体递送药物可以避免巨噬细胞的吞噬作用或降解,还可以在体内长时间循环,保持效果。其中,外泌体能够穿过血脑屏障以将核酸药物递送到中枢神经系统是外泌体递送药物的一个显著优势。
根据本发明的实施例,所述蛋白质包括非人源蛋白和人源蛋白。本发明对于人源蛋白的类型不作严格限定,只要是能够作为递送载体将核酸分子递送到细胞内即可,包括但不限于SNARE蛋白家族。根据本发明的实施例,所述非人源蛋白包括病毒衣壳蛋白。相比于人源蛋白,病毒衣壳蛋白的递送效果更佳,由于病毒衣壳蛋白不具有病毒遗传物质,易感染进入细胞,靶向细胞特异性强,可以实现蛋白及遗传物质的细胞内递送。并且,病毒衣壳蛋白容易获得,提高了制备递送载体的效率,降低成本。如图4所示,病毒蛋白递送载体是源自病毒自身的特性,所以一个蛋白质递送载体只能包裹一个核酸分子。
除了弹状病毒科的外壳蛋白因其相似的结构可以完成reRNATM的递送外,其他病毒的外壳蛋白也可以用于reRNATM的递送。根据本发明的实施例,所述病毒包括痘类病毒、狂犬病病毒、黄病毒、麻疹病毒、冠状病毒、水疱性口炎病毒、新城疫病毒和淋巴细胞脉络丛脑膜炎病毒的至少之一。上述病毒均具有衣壳蛋白,可以用作递送载体。
根据本发明的实施例,所述蛋白质选自水疱性口炎病毒受体、淋巴细胞性脉络丛脑膜炎病毒外壳蛋白和/或新城疫病毒外壳蛋白。具体地,水疱性口炎病毒受体VSV-G具有如SEQ ID NO:7的氨基酸序列。淋巴细胞性脉络丛脑膜炎病毒外壳蛋白LCMV-GP的具体序列信息参考UniProtKB/Swiss-Prot:P09991;新城疫病毒外壳蛋白分别为NDV-F和NDV-HN,具体序列信息参考UniProtKB/Swiss-Prot:Q9DLD4和UniProtKB/Swiss-Prot:Q91UL0。发明人经过大量实验研究发现,上述三类蛋白质的自复制RNA递送效率高。
根据本发明的实施例,所述脂质体上含有靶向元件。由此,可以将携带的核酸分子靶向递送至特定组织或器官。
根据本发明的实施例,所述脂质体包括带永久正电荷的阳离子脂质体(如DOTAP、DOTMA)、辅助脂质体(如DSPC、DOPE)、结构脂质体、(如胆固醇、胆固醇脂)、长循环脂质体(如DMG-PEG2000)和可电离的阳离子脂质体(如DLin-MC3-DMA、DLin-KC2-DMA、DLin-DMA、DODMA、DODAP)的至少之一。
根据本发明的实施例,所述脂质体选自DOTAP、DSPC、胆固醇和DMG-PEG2000。发明人经过大量实验研究发现,上述脂质体的自复制RNA递送效率高。
药物组合物
在本发明的另一方面,本发明提出了一种药物组合物。根据本发明的实施例,所述药物组合物包括:前面所述组合物。如前所述,组合物中递送载体可以携带包含自复制RNA分子的核酸分子进入细胞内,自复制RNA分子可以在细胞质内进行自我复制,其搭载的靶分子编码区可以表达小分子、多种肽、酶、抗体、通道蛋白以及受体蛋白等,从而达到不同的预防或治疗目的。
根据本发明的实施例,药物组合物进一步包括药学上可接受的辅料。本发明对于辅料的种类不做严格限定,可以根据情况灵活选择。对于注射制剂,药物上可接受的辅料可以包括缓冲剂、增溶剂等,药物组合物可以被制备成例如一次剂量的剂型的安瓿或例如多剂量容器的单元型剂型。药物组合物还可以被制备成溶液、悬浮液、冻干剂、水剂、针剂和长效制剂。
需要说明的是,前面针对组合物所描述的特征和优点,同样适用于该药物组合物,在此不再赘述。
递送自复制RNA分子的方法
在本发明的又一方面,本发明提出了一种递送自复制RNA分子的方法,其为非诊断和治疗目的。根据本发明的实施例,所述方法包括:将自复制RNA分子配制为前面所述的组合物;和任选的,为待处理对象提供所述组合物。由此,递送载体可以有效地递送自复制RNA分子进入细胞内。
需要说明的是,前面针对组合物所描述的特征和优点,同样适用于该递送自复制RNA分子的方法,在此不再赘述。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1
水疱性口炎病毒(简称VSV病毒)的受体是低密度脂蛋白(LDLR),具有广泛的组织分布,发明人筛选出的特定的VSV-G(SEQ ID NO:7)具有较高的亲和力,以此VSV-G蛋白作为递送蛋白,具有很高的细胞递送效率。
具体实验如下:
1、将经培养的HEK 293T细胞用转染试剂Lipo8000TM进行reRNATM质粒(质粒上携带有reRNATM序列,该序列包含mRNA序列、SEQ ID NO:4-6序列、GFP序列)及辅助质粒(辅助质粒包含分别包括SEQ ID NO:4-6序列的单独质粒、VSV-G质粒、T7 RNA聚合酶质粒)的转染,转染完毕后,培养细胞,培养箱中培养48小时,收取上清进行纯化,得到reRNATM-GFP种子。
2、将经培养的HEK 293细胞用转染试剂Lipo8000TM进行辅助质粒(辅助质粒上携带编码VSV-G的核酸分子)的转染,转染完毕,加入reRNATM-GFP种子,培养箱中培养48小时,收取上清进行纯化,得到VSV-G包裹的reRNATM-GFP。
在6孔板上铺2×106个293细胞,使用含1%FBS的培养基培养细胞,分别将VSV-G包裹的reRNATM-GFP以1.4pg、14pg和140pg的添加量加入到2ml细胞液中,16小时后对培养的细胞进行流式检测。
结果如图5所示,reRNATM-GFP添加量为1.4pg时,16小时细胞的阳性率可达96.5%;reRNATM-GFP添加量为14pg和140pg时,16小时细胞的阳性率可达98.2%。培养时间延长,可以一直维持这种表达,可见VSV-G蛋白具有很高的递送效率。
MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:7)
实施例2
淋巴细胞性脉络丛脑膜炎病毒(简称LCMV病毒)的外壳蛋白为LCMV-GP,其受体为α-肌营养不良蛋白,是一种普遍存在的蛋白质,也是reRNATM递送蛋白的优质备选。制备LCMV-GP作为递送蛋白的reRNATM-GFP产品,也可完成reRNATM的递送,具体实验如下:
1、LCMV-GP蛋白包裹reRNATM-GFP的步骤参考实施例1。
2、在6孔板上铺2×106个vero细胞,使用含1%FBS的培养基培养细胞,加入以LCMV-GP蛋白为递送蛋白的reRNATM-GFP(同实施例1),观察vreo形态及GFP荧光。
结果如图6所示,可以看出,reRNATM-GFP被有效地递送至vero细胞中。
实施例3
新城疫病毒(简称NDV病毒)也是一种有囊膜的病毒,其外壳蛋白有两个,分别为NDV-F和NDV-HN,两个蛋白共同完成入胞及溶酶体逃逸,实现RNA的递送,将两个蛋白同时作为递送蛋白,也可完成reRNATM-GFP的递送,具体实验如下:
1、LCMV-GP蛋白包裹reRNATM-GFP的步骤参考实施例1。
2、在6孔板上铺2×106个vero细胞,使用含1%FBS的培养基培养细胞,加入以NDV-F蛋白和NDV-HN蛋白为递送蛋白的reRNATM-GFP(同实施例1),观察vero形态及GFP荧光。
如图7所示,以新城疫病毒F蛋白和HN蛋白为递送蛋白,可以实现reRNATM-GFP的有效递送。
实施例4
裸露的reRNATM-GFP不能进入胞内,以LNP作为递送载体可以有效地实现递送reRNATM。具体实验如下:
1、将LNP溶液(DOTAP:DSPC:胆固醇:DMG-PEG2000=44:16:37.5:2.5v/v)与reRNATM-GFP种子(制备方法参考实施例1)通过微流控芯片进行混合。混合完成后混入PBS缓冲液中稀释3倍,浓缩纯化,过滤除菌,得到LNP包裹的reRNATM-GFP。
2、在6孔板上铺2×106个vero细胞,使用含1%FBS的培养基培养细胞,加入LNP包裹的reRNATM-GFP,观察vero形态及GFP荧光。
表1为LNP包裹前后reRNATM-GFP粒径及电势的变化,从结果可以看出LNP包裹之后的电势在+20mV左右,粒径在150nm左右,比较利于RNA的递送。
表1 LNP包裹前后reRNATM-GFP电势及粒径的变化
样品名称 | 电势(mV) | 粒径(nm) | PdI |
reRNATM-GFP | -22.1±7.3 | 367.4±8.3 | 0.373 |
LNP+reRNATM-GFP | 21.1±3.7 | 131.8±2.1 | 0.193 |
包裹后的reRNATM-GFP可以完成在vero细胞内的RNA递送,图8为LNP包裹reRNATM-GFP递送入vero细胞24小时之后的GFP表达情况,reRNATM-GFP量为18ng,vero细胞数量为2×105个。可以看出,脂质体可以实现reRNATM-GFP的有效递送。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (10)
1.一种组合物,其特征在于,包括:
核酸分子,所述核酸分子包括自复制RNA分子;
递送载体,所述递送载体携带所述核酸分子。
2.根据权利要求1所述的组合物,其特征在于,所述自复制RNA分子包括:
第一RNA序列,所述第一RNA序列编码N蛋白或其功能片段;
第二RNA序列,所述第二RNA序列编码P蛋白或其功能片段;
第三RNA序列,所述第三RNA序列编码L蛋白或其功能片段;和
靶分子编码区,所述靶分子编码区编码至少一个靶分子。
3.根据权利要求2所述的组合物,其特征在于,所述N蛋白、所述P蛋白、所述L蛋白的至少之一分别独立地来自弹状病毒科病毒。
4.根据权利要求2所述的组合物,其特征在于,所述N蛋白具有SEQ ID NO:1所示氨基酸序列或与其具有至少80%同源性的氨基酸序列,所述P蛋白具有SEQ ID NO:2所示氨基酸序列或与其具有至少80%同源性的氨基酸序列,所述L蛋白具有SEQ ID NO:3所示氨基酸序列或与其具有至少80%同源性的氨基酸序列。
5.根据权利要求1所述的组合物,其特征在于,所述递送载体包括蛋白质、脂质体和外泌体的至少之一。
6.根据权利要求5所述的组合物,其特征在于,所述蛋白质包括非人源蛋白和人源蛋白;
所述非人源蛋白包括病毒衣壳蛋白;
所述人源蛋白包括SNARE蛋白家族;
任选地,所述病毒包括痘类病毒、狂犬病病毒、黄病毒、麻疹病毒、冠状病毒、水疱性口炎病毒、新城疫病毒和淋巴细胞脉络丛脑膜炎病毒的至少之一。
7.根据权利要求5所述的组合物,其特征在于,所述蛋白质选自水疱性口炎病毒受体、淋巴细胞性脉络丛脑膜炎病毒外壳蛋白和/或新城疫病毒外壳蛋白。
8.根据权利要求5所述的组合物,其特征在于,所述脂质体包括带永久正电荷的阳离子脂质体、辅助脂质体、结构脂质体、长循环脂质体和可电离的阳离子脂质体的至少之一;
优选地,所述脂质体选自DOTAP、DSPC、胆固醇和DMG-PEG2000。
9.一种药物组合物,其特征在于,包括:权利要求1~8任一项所述的组合物。
10.一种递送自复制RNA分子的方法,其特征在于,包括:
将自复制RNA分子配制为权利要求1~8任一项所述的组合物;和
任选的,为待处理对象提供所述组合物。
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