CN117440834A - 核糖核酸的体内递送用组合物及其制备方法 - Google Patents
核糖核酸的体内递送用组合物及其制备方法 Download PDFInfo
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Abstract
本发明涉及包含基于阳离子脂质的脂质体的信使核糖核酸(mRNA)递送用组合物,本发明的信使核糖核酸递送用组合物的保管稳定性优秀,在生物体内(in vivo)表现出高的细胞内递送率和表达率,可以提高癌症治疗用信使核糖核酸疫苗或者病毒感染预防用信使核糖核酸疫苗等的稳定性及效率。
Description
【技术领域】
本发明涉及信使核糖核酸(mRNA)体内递送用组合物,具体地,涉及包含基于阳离子脂质的脂质体的信使核糖核酸体内递送用组合物及其制备方法。
本申请要求于2021年3月8日提交且申请号为第10-2021-0029929号的韩国专利申请的优先权,其全部内容通过引用结合在本申请中。
【背景技术】
基因治疗法及基因疫苗已在医药领域中得到证明,通常,该技术不仅适用于遗传疾病,而且还可以将自身免疫性疾病、感染性疾病、癌症或肿瘤相关疾病、炎症疾病等作为治疗对象。
据报告,在将编码目标基因的脱氧核糖核酸(DNA)及核糖核酸(RNA)直接向动物注入时,它们在存活有目标基因的动物中表达,通过该表达使免疫成为可能,由此,基因疫苗的开发也随之开启(Wolff JA et al.Science,247∶1465-8,1990)。
基因疫苗接种可以对细菌表面的特征结构要素、病毒粒子、肿瘤抗原等选择性的抗原引起所希望的免疫反应。大体上,疫苗接种为现代医学的中枢成果之一。然而,有效的疫苗现在只能在有限的疾病中使用。因此,无法通过疫苗接种预防的感染症每年都给数百万人带来影响。
在基因治疗或基因疫苗接种中,脱氧核糖核酸和核糖核酸可以用做用于基因给药的核酸分子,已知与核糖核酸相比,脱氧核糖核酸相对稳定且易于操作。
然而,在脱氧核糖核酸的情况下,若向患者的基因组内给药的脱氧核糖核酸-切片插入不希望的位置而引起基因损伤,则会发生潜在的危险。追加地,还会产生所不希望的抗脱氧核糖核酸抗体,而且还有问题就是,通过脱氧核糖核酸施与及其以后的转录/翻译所表达的肽或蛋白质的表达水平受限。调节脱氧核糖核酸转录的特定转录因子的存在与否给施与的脱氧核糖核酸的表达水平带来主要影响,若没有特定转录因子,则无法通过脱氧核糖核酸转录产生充分量的核糖核酸,从而使翻译后生成的肽或蛋白质的水平也受到限制。
相反,在将核糖核酸用作用于基因给药的工具的情况下,核糖核酸无需转录,无需像脱氧核糖核酸那样进入细胞核,而是可以直接在细胞质内直接合成蛋白质,因此没有进入细胞染色体引起所不希望的基因损伤的顾虑。并且,与脱氧核糖核酸相比,因半衰期短而不诱导长期基因变形(Sayour EJ et al.,J Immunother Cancer 2015;3∶13,2015)。通常的核糖核酸疫苗递送到细胞内后只短期激活来表达目标蛋白质,在几天内因酶反应而受到破坏,只留下对目标抗原(蛋白质)的特异性免疫反应。
并且,在将核糖核酸用作用于基因给药的工具的情况下,无需通过核膜,而只需通过细胞膜就起作用,因此,即使使用比脱氧核糖核酸少的量,也可以表达与脱氧核糖核酸相同量的蛋白质。并且,核糖核酸本身具有免疫增强原性,即使施与的量比脱氧核糖核酸少,也可以获得同样的免疫效果。
为了基因疫苗接种,使用核糖核酸替代脱氧核糖核酸,可以防止所不希望的基因组融合及抗脱氧核糖核酸抗体的生成或使之最小化。但是,核糖核酸却是非常易于被偏重的核糖核酸酶(RNAse)分解的相当不稳定的分子种类。
尽管过去数年已经取得了许多进展,但本发明所属技术领域中仍存在需要提供不至因抗原的早期分解或细胞中的信使核糖核酸的无效释放引起的信使核糖核酸的无效翻译使给药遭到严重破坏的方法的难题。进而,为了减少潜在的风险以及使疫苗能够在第三代中挑起大梁,切实需要减少信使核糖核酸疫苗的剂量。虽然疫苗等基于核酸的治疗剂具有突出的功能性,但为了实现这些功能,仍需在细胞或有机体内向适当的部位更为有效地递送。
然而,在治疗及预防中使用核酸面临两个问题。第一,游离核糖核酸在血浆中易于被核酸酶分解。第二,在相关翻译工具尚在的细胞内,游离核糖核酸接近该区域的能力有限。因此,尝试使用中性脂质、胆固醇、聚乙二醇(PEG)、聚乙二醇化脂质及核酸等其他脂质结构要素和从阳离子脂质形成的脂质纳米粒子在血浆中阻止核糖核酸的分解并促进细胞吸收寡核苷酸。
在利用脂质体(Liposome)或脂质纳米粒子(lipid nanoparticle)等基于脂质的运载体的情况下,信使核糖核酸通常使用吸附(adsorption)在外部或封装(encapsulation)在内部的方法。尤其,已知在将信使核糖核酸吸附在外部的情况下,通常是以不是单一的脂质体的脂质体与核酸的凝集物的形式存在,吸附力随着脂质的组合、核酸的状态及核酸与脂质体的比例的不同而不同,还给稳定性带来影响,因此需要使之最佳化。
并且,关于信使核糖核酸的表达能力,通常,即使运载体在生物体外(in vitro)的表达得到验证,在生物体内表达(in vivo expression)的结果也可能不同。在生物体内(Invivo),根据脂质的结构及运载体的物理化学特性,大多存在因血浆蛋白(plasma protein)的凝集引起的半衰期(half-life)及生物分布(bio-distribution)减少、随脂质结构的细胞摄取(cell uptake)方式不同、由细胞外基质(ECM,extracellular matrix)引起的屏障(barrier)等向减少细胞内递送效率的因素。因此,与体外递送不同,在体内递送中的运载体最优化是必需的。
【发明内容】
【技术问题】
本发明人致力于研究能够通过向体内稳定递送信使核糖核酸以提高细胞内表达效率来诱导稳定的蛋白质表达的运载体改良技术。结果,通过特定工序制备基于阳离子脂质体的“脂质体+信使核糖核酸复合物”并查明通过相关工序制备的“脂质体+信使核糖核酸复合物”在可以生物体内表现出高的细胞内递送率和表达率,从而完成本发明。
因此,本发明的目的在于,提供具有高的体内递送率和表达率的包含基于阳离子脂质的脂质体的信使核糖核酸递送用组合物。
本发明的再一目的在于,提供包含上述信使核糖核酸递送用组合物作为有效成分的用于预防或治疗选自由癌症、肿瘤、自身免疫性疾病、遗传疾病、炎症疾病、病毒感染及细菌感染组成的组中的疾病的药物组合物。
本发明的还一目的在于,提供包含上述信使核糖核酸递送用组合物作为有效成分的疫苗。
本发明的另一目的在于,提供包含上述信使核糖核酸递送用组合物作为有效成分的功能性化妆品组合物。
本发明的又一目的在于,提供信使核糖核酸递送用组合物的制备方法。
【技术方案】
本发明人致力于研究能够通过向体内稳定递送信使核糖核酸以提高细胞内表达效率来诱导稳定的蛋白质表达的运载体改良技术。结果,通过特定工序制备基于阳离子脂质体的“脂质体+信使核糖核酸复合物”并查明通过相关工序制备的“脂质体+信使核糖核酸复合物”可以在生物体内表现出高的细胞内递送率和表达率。
本发明涉及包含基于阳离子脂质的脂质体的信使核糖核酸递送用组合物、包含上述信使核糖核酸递送用组合物作为有效成分的用于预防或治疗选自由癌症、病毒感染及细菌感染组成的组中的疾病的药物组合物、包含上述信使核糖核酸递送用组合物作为有效成分的疫苗、包含上述信使核糖核酸递送用组合物作为有效成分的功能性化妆品组合物及上述信使核糖核酸递送用组合物的制备方法。
若无其他定义,则本说明书中使用的所有技术及科学术语都具有与本发明所属技术领域的普通技术人员通常理解的相同的含义。通常,本说明书中使用的命名法是本发明所属技术领域中众所周知并通常使用的。
在本发明中,关于利用核酸的体内基因递送,欲开发通过诱导对于病毒或细菌表面的特征结构要素、病毒粒子及肿瘤抗原等选择性抗原诱导所希望的免疫反应的治疗用蛋白质的表达来预防或治疗疾病的方法,从而寻找能够提高信使核糖核酸的细胞内表达效率来显出稳定的效果的方法。
于是,利用基于阳离子脂质的脂质体制备信使核糖核酸递送用组合物,确认到上述信使核糖核酸递送用组合物在生物体内表现出高的细胞内递送率和表达率。
在本发明的利用基于阳离子脂质的脂质体的信使核糖核酸递送用组合物中,信使核糖核酸能够以与基于阳离子脂质的脂质体形成复合物的形态存在,据此,利用基于阳离子脂质的脂质体的信使核糖核酸递送用组合物与“脂质体+信使核糖核酸的复合物”以相同含义来使用。
以下,更为详细地说明本发明。
根据本发明的一实施方式,本发明提供包含基于阳离子脂质的脂质体的信使核糖核酸递送用组合物。
在本发明中,“阳离子脂质”包括不受pH变化的影响而持续具有阳离子性的脂质或通过pH变化来转换为阳离子性的离子性脂质。
上述阳离子脂质可以为1,2-二油酰基-3-三甲基丙烷铵(DOTAP)、二甲基双十八烷基溴化铵(DDA)、3β-[N-(N',N'-二甲氨基乙烷)氨基甲酰胆固醇(3β-[N-(N',N'-dimethylaminoethane)carbamoyl cholesterol,DC-Chol)、1,2-二油酰氧基-3-二甲基丙烷铵(DODAP)、1,2-二-O-十八烯基-3-三甲基丙烷铵(1,2-di-O-octadecenyl-3-trimethylammonium propane,DOTMA)、1,2-二肉豆蔻酰基-sn-甘油-3-乙基磷酸胆碱(1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine,14:1Etyle PC)、1-棕榈酰-2-油酰-sn-甘油-3-乙基磷酸胆碱(1-palmitoyl-2-oleoyl-snglycero-3-ethylphosphocholine,16:0-18:1Ethyl PC)、1,2-二油酰-sn-甘油-3-乙基磷酸胆碱(1,2-dioleoyl-sn-glycero-3-ethylphosphocholine,18:1Ethyl PC)、1,2-二硬脂酰-sn-甘油-3-乙基磷酸胆碱(1,2-distearoyl-sn-glycero-3-ethylphosphocholin,18:0Ethyl PC)、1,2-二棕榈酰-sn-甘油-3-乙基磷酸胆碱(1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine,16:0Ethyl PC)、1,2-二肉豆蔻酰-sn-甘油-3-乙基磷酸胆碱(1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine,14:0Ethyl PC)、1,2-二月桂酰-sn-甘油-3-乙基磷酸胆碱(1,2-dilauroyl-sn-glycero-3-ethylphosphocholin,12:0EthylPC)、N1-[2-((1S)-1-[(3-氨基丙基)氨基]-4-[二(3-氨基丙基)氨基]丁基甲酰氨基)乙基]-3,4-二[油氧基]-苯甲酰胺(N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide,MVL5)、1,2-二肉豆蔻酰基-3-二甲基丙烷铵(1,2-dimyristoyl-3-dimethylammonium-propane,14:0DAP)、1,2-二棕榈酰基-3-二甲基丙烷铵(1,2-dipalmitoyl-3-dimethylammonium-propane,16:0DAP)、1,2-二硬脂酰基-3-二甲基丙烷铵(1,2-distearoyl-3-dimethylammonium-propane,18:0DAP)、N-(4-羧基苄基)-N,N二甲基-2,3-双(油酰氧基)丙-1-铵(N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium,DOBAQ)、1,2-硬脂酰基-3-三甲基丙烷铵(1,2-stearoyl-3-trimethylammoniumpropane,18:0TAP)、1,2-二棕榈酰基-3-三甲基丙烷铵(1,2-dipalmitoyl-3-trimethylammonium-propane,16:0TA)、1,2-二肉豆蔻酰基-3-三甲基丙烷铵(1,2-dimyristoyl-3-trimethylammonium-propane,14:0TAP)和/或N4-胆固醇精胺(N4-Cholesteryl-Spermine,GL67),优选地,可以为1,2-二油酰基-3-三甲基丙烷铵或C12-200,但不限定于此。
具体地,上述“DOTAP(1,2-二油酰基-3-三甲基丙烷铵(1,2-Dioleoyl-3-trimethylammonium propane))”为具有下述化学式1的结构的阳离子性乳化剂,用作纤维软化剂,近来,也用作形成脂质体的核酸运载体。
【化学式1】
本发明的包含基于阳离子脂质的脂质体的信使核糖核酸递送用组合物还可以包含中性脂质。
在本发明中,“中性脂质”包括不受pH变化的影响而持续具有中的脂质或通过pH变化来转换为中性的离子性脂质。
上述中性脂质可以为1,2-二油酰-sn-甘油-3-磷酸乙醇胺(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine,DOPE)、1,2-二肉豆蔻酰-sn-甘油-3-磷酸胆碱(1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine,DMPC)、1,2-二油酰-sn-甘油-3-磷酸胆碱(1,2-dioleoyl-sn-glycero-3-phosphocholine,DOPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(1,2-dipalmitoyl-sn-glycero-3-phosphocholine,DPPC)、1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(1,2-distearoyl-sn-glycero-3-phosphocholine,DSPC)、1,2-二亚油酰-sn-甘油-3-磷酸胆碱(1,2-dilinoleoyl-sn-glycero-3-phosphocholine,DLPC)、磷酯酰丝氨酸(PS)、磷酸乙醇胺(PE)、磷脂酰甘油(PG)、磷酸(PA)和/或磷脂酰胆碱(PC),优选地,可以为1,2-二油酰-sn-甘油-3-磷酸乙醇胺,但不限定于此。
具体地,上述“DOPE(1,2-二油酰-sn-甘油-3-磷酸乙醇胺(1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine))”具有下述化学式2的结构,用作阳离子脂质体用辅助脂质。
【化学式2】
在本发明中,上述阳离子脂质与中性脂质的重量比可以为1:9至9.5:0.5、2:8至9:1、3:7至8:2或4:6至7:3,但不限定于此。
若脱离上述重量比例,则会显著降低信使核糖核酸递送效率。
并且,本发明的包含基于阳离子脂质的脂质体的信使核糖核酸递送用组合物还可以包含胆固醇。
在本发明中,上述阳离子脂质与胆固醇的重量比可以为6:1至1:3、4:1至1:2.5、3:1至1:2或2:1至1:1.5,但不限定于此。
并且,在本发明的脂质体中包含阳离子脂质、中性脂质及胆固醇的情况下,上述阳离子脂质、中性脂质及胆固醇的重量比可以为1至9.5:9至0.5:0.05至3、3至8:7至1:0.45至7.0或1至3.5:1至3.5:0.5至3,但不限定于此。
若脱离上述重量比例,则会显著降低信使核糖核酸递送效率。
例如,在还包含胆固醇的情况下,对于DOTAP:DOPE=1:1,能够以0.2至0.85或0.5至0.85的重量比的比例混合胆固醇来制备脂质体。
进而,本发明的包含基于阳离子脂质的脂质体的信使核糖核酸递送用组合物还可以包含一种以上鱼精蛋白(protamine)、白蛋白(albumin)、转铁蛋白(transferrin)、蛋白转到结构域(PTD,protein transduction domains)、细胞穿膜肽(CPP,cell penetratingpeptide)及巨噬细胞靶向部分(Macrophage targeting moiety)等递送用因子。
进而,本发明的包含基于阳离子脂质的脂质体的信使核糖核酸递送用组合物还可以包含免疫增强剂。
上述免疫增强剂相当于病原体相关分子模式(Pathogen-associated molecularpattern,PAMP),可以为与病原体识别受体(Pathogen recognition receptor,PRR)反应的物质组、非毒性脂寡糖(detoxified lipooligosaccharide,dLOS)、非甲基化的胞嘧啶鸟嘌呤二核苷酸(CpG DNA)、脂蛋白(lipoprotein)、鞭毛(flagella)、聚肌胞苷酸(poly I:C)、皂苷、角鲨烯(Squalene)、癸酸甘油三酯(tricaprin)和/或3D-MPL,但不限定于此。
具体地,上述非毒性脂寡糖(dLOS)可以为韩国授权专利第1509456号或韩国授权专利第2042993号中公开的物质,但不限定于此。
在本发明的信使核糖核酸递送用组合物中,以脂质体为代表的信使核糖核酸运载体与信使核糖核酸的混合比例可以表示为N:P比,表达及运载体的稳定性受N:P比的影响。
上述脂质体与信使核糖核酸的N:P比可以为0.23至1.39:1、0.3至1.3:1、0.4至1.2:1、0.5至1.1:1、0.6至1.0:1、0.6至0.9:1、0.6至0.8:1或0.7:1,但不限定于此。
在本发明的信使核糖核酸递送用组合物中,上述信使核糖核酸的特征可以为,编码能够起到免疫原的作用的肽或蛋白质。
典型地,上述信使核糖核酸可以为具有与信使核糖核酸一同提供的通过能够被细胞或有机体翻译的至少一个开放阅读框(Open reading frame,ORF)。该翻译的产物为抗原,优选地,为能够起到免疫原的作用的肽或蛋白质。并且,该产物可以为由两个以上免疫原形成的融合蛋白,例如,由源自相同或不同蛋白质的两个以上表位、肽或蛋白质形成的融合蛋白质,在此情况下,表位、肽或蛋白质可以通过接头序列连接。
并且,上述信使核糖核酸可以为人造信使核糖核酸,即,可以理解为非自然产生的信使核糖核酸分子。人造信使核糖核酸分子可以理解为非天然信使核糖核酸分子。这样的信使核糖核酸分子(非自然产生的)可以为个别序列和/或非自然产生的其他变更,例如,可以为通过核苷酸的结构性变更的非天然。人造信使核糖核酸分子可以在核苷酸的所希望的人造序列(异种序列)通过相应的基因操作方法来设计和/或生成。
进而,上述信使核糖核酸可以表现出增加对生物体内分解(例如通过核酸外切酶或核酸内切酶的分解)和/或生物体外分解(例如通过疫苗给药前的制备过程或者在给药的疫苗溶液的制备过程中)的耐性的表型。核糖核酸的稳定化可以通过例如5'-CAP结构、聚-A-环或人以其他UTR变形的提供来实现。并且,核糖核酸的稳定化可以通过化学变形或核酸的G/C含量的变形来实现。多种其他方法在本发明所属技术领域中使公知的,都适用于本发明。
根据本发明的再一实施方式,本发明提供包含上述信使核糖核酸递送用组合物作为有效成分的用于预防或治疗选自由癌症、肿瘤、自身免疫性疾病、遗传疾病、炎症疾病、病毒感染及细菌感染组成的组中的疾病的药物组合物。
在本发明中,“预防”是指通过给药本发明的组合物来抑制上述疾病或延迟其病程的所有行为。
在本发明,“治疗”是指通过给药本发明的药物组合物来使上述疾病的症状好转或变更为痊愈的所有行为。
本发明的药物组合物可以包含一种以上制药上或生理学上可接受的载体、稀释剂或赋形剂以及它们的组合。上述药物组合物可以包含:例如中性缓冲盐水、磷酸盐缓冲盐水、柠檬酸缓冲溶液等缓冲剂;例如葡萄糖、甘露糖、蔗糖、糊精、甘露醇等碳水化合物;蛋白质;多肽或氨基酸,例如甘氨酸;抗氧化剂;螯合剂,例如乙二胺四乙酸(EDTA)或谷胱甘肽;佐剂(例如氢氧化铝);以及防腐剂。
本发明的药物组合物可以口服或胃肠外给药,例如,可以通过静脉内给药、皮下给药、皮内给药、肌肉内给药、腹腔内给药、肿瘤内给药、脑内给药、颅骨内给药、肺内给药及直肠内给药等来给药,但不限定于此。
本发明的药物组合物以药学上有效的量来给药。在本发明中,“药学上有效的量”是指以能够适用于医学治疗的合理的收益/风险比例来治疗疾病的充分的量,有效量可以根据包括患者所患疾病的种类、疾病的严重程度、药物的活性、对药物的敏感度、给药时间、给药途径及代谢比例、治疗期间、同时使用的药物在内的因素及其他医学领域中广为人知的因素来决定。
本发明的药物组合物能够以单独的治疗剂来给药或者与其他治疗剂联合来给药,可以与现有的治疗剂依次或同时给药,可以单次或多次给药。重点在于,考虑上述因素后以没有副作用的最小的量来获得最大的效果,这可以通过本发明所属技术领域的普通技术人员轻松地确定。
具体地,本发明的药物组合物的有效量可以随患者的年龄、性别、状态、体重、体内活性成分的吸收度、灭活率及代谢速度、疾病种类、联合使用的药物等的不同而不同。
本发明的药物组合物包含上述信使核糖核酸递送用组合物作为有效成分,为避免说明书过度的复杂性,将省略重复内容的说明。
根据本发明的还一实施方式,本发明提供预防或治疗癌症、肿瘤、自身免疫性疾病、炎症疾病、病毒感染及细菌感染的方法,包括向需要预防或治疗癌症、肿瘤、自身免疫性疾病、炎症疾病、病毒感染及细菌感染的患者给药本发明的信使核糖核酸递送用组合物的步骤。
上述信使核糖核酸的给药量可以为每剂量1μg至5μg、5μg至10μg、10μg至15μg、15μg至20μg、10μg至25μg、20μg至25μg、20μg至50μg、30μg至50μg、40μg至50μg、40μg至60μg、60μg至80μg、60μg至100μg、50μg至100μg、80μg至120μg、40μg至120μg、40μg至150μg、50μg至150μg、50μg至200μg、80μg至200μg、100μg至200μg、120μg至250μg、150μg至250μg、180μg至280μg、200-300μg、50μg至300μg、80μg至300μg、100μg至300μg、40μg至300μg、50μg至350μg、100μg至350μg、200μg至350μg、300μg至350μg、320μg至400μg、40μg至380μg、40μg至100μg、100μg至400μg、200μg至400μg或300μg至400μg,但不限定于此。
上述疫苗可以为对流感病毒、冠状病毒、带状疱疹病毒、人乳头瘤病毒、寨卡病毒、疱疹病毒、人获得性免疫缺陷病毒、发热伴血小板减少综合征(SFTS)病毒、麻疹病毒、水痘病毒、埃博拉病毒、中东呼吸综合征冠状病毒、肝炎病毒、禽流感病毒、狂犬病毒及口蹄疫病毒等可以在人类或动物中引起感染的病毒的疫苗,但不限定于此。
上述疫苗包含具有编码至少一种病毒抗原的多肽或其免疫原性片段的开放阅读框的至少一种信使核糖核酸。
本发明的治疗方法包含上述药物组合物作为有效成分,为避免说明书过度的复杂性,将省略重复内容的说明。
根据本发明的另一实施方式,本发明提供包含上述信使核糖核酸递送用组合物作为有效成分的疫苗。
上述疫苗在由上述信使核糖核酸能够起到免疫原的作用的肽或蛋白质引起的疾病的预防目的范围内考虑给药对象的体重、年龄、饮食阶段和/或免疫力来以适当的浓度包含上述信使核糖核酸递送用组合物。
上述疫苗还可以包含选自由载体、稀释剂、赋形剂及佐剂(adjuvant)组成的组中的一种以上。载体的种类不受特别限制,可以包括任意的所有溶剂、分散介质、涂层、稳定剂、保存剂、抗菌剂及抗真菌剂、等渗剂、吸收延迟剂等。
上述疫苗通过口服、胃肠外、皮下、肌肉内、皮内、舌下、经皮、直肠、经黏膜、通过吸入的表面积、颊侧给药或它们的组合来给药。
上述疫苗可以根据疫苗接种或治疗所希望的期间及有效性单次给药或多次给药,也可以间歇性给药,例如,可以在数天、数周或数月期间每天以相同的量或不同的给药量来给药。注射能够以所希望的量注射,或者通过向皮下或鼻腔喷雾来注入,或者作为替代方案,可以连续注入。
本发明的疫苗包含信使核糖核酸递送用组合物作为有效成分,为避免说明书过度的复杂性,将省略重复内容的说明。
根据本发明的又一实施方式,本发明提供包含信使核糖核酸递送用组合物作为有效成分的功能性化妆品组合物。
本发明的化妆品组合物可以包含化妆品组合物中通常使用的成分,例如,包含金属离子掩蔽剂、活性成分(例如,透明质酸钠(Sodium Hyaluronate)及生育酚乙酸酯(Tocopheryl Acetate)等)、防腐剂、增稠剂及香料等通常的辅助剂以及载体。
并且,本发明的化妆品组合物可以制备为本发明所属技术领域的通常的剂型,例如乳化剂型或可溶化剂型等形态。乳化剂型可以为例如营养化妆水、霜剂、精华素等,可溶化剂型可以为例如柔肤化妆水。并且,除化妆品以外,本发明的化妆品组合物还可以通过包含皮肤科学中可接受的介质或基质来制备为皮肤科学中通常使用的可以适用局部或适用全身的辅助剂形态。
适当的化妆品的剂型可以提供为例如溶液、凝胶、固体或或糊状无水产品、水包油的乳液、悬混液、微乳液、微胶囊、微型颗粒球或离子型(脂质体)、非离子型的囊泡分散剂的形态、霜剂、护肤水、乳剂、粉、软膏、喷剂或遮瑕棒(conceal stick)的形态。并且,还可以制备微泡沫(foam)的形态或还包含压缩的推进剂的气雾剂组合物的形态。
并且,本发明的化妆品组合物还可以包含脂肪物质、有机溶剂、溶解剂、浓缩剂及凝胶化机、软化剂、抗氧化剂、助悬剂、稳定剂、发泡剂、芳香剂、表面活性剂、水、离子型或非离子型乳化剂、填充剂、金属离子掩蔽剂及螯合剂、保存剂、维生素、阻断剂、湿润剂、香精油、染料、颜料、亲水性或亲油性活性剂、脂质囊泡或化妆品中通常使用的任意成分等化妆品学或皮肤科学领域中通常使用的辅助剂。而且,能够以皮肤科学领域中通常使用的量来导入上述成分。
能够添加本发明的化妆品组合物的制品有例如敛肤水、柔肤化妆水、营养化妆水、各种霜剂、精华素、面膜、粉底霜等化妆品以及洁面乳、洗眼剂、香皂、护肤水、美容液等。
本发明的化妆品组合物的具体剂型包括化妆水、柔肤水、爽肤水、收敛水、化妆水、乳液、保湿乳液、营养乳液、按摩霜、营养霜、保湿霜、护手霜、精华液、营养精华液、面膜、香皂、洗发水、洁面泡沫、洁面乳、洁面霜、润肤露、沐浴露、乳液、压粉、散粉、化妆贴、喷雾剂等剂型。
本发明的化妆品组合物包含上述信使核糖核酸递送用组合物作为有效成分,为避免说明书过度的复杂性,将省略重复内容的说明。
根据本发明的又一实施方式,本发明提供信使核糖核酸递送用组合物的制备方法,包括混合信使核糖核酸与脂质体的步骤。
上述信使核糖核酸和/或脂质体能够以冷冻干燥状态的粉末形态来提供,也能够以溶解在适当的溶液或缓冲液中的状态来提供。在信使核糖核酸和/或脂质体以冷冻干燥的状态来提供的情况下,可以溶解在适当的溶液或缓冲液来使用。
在本发明的具体实施例中,本发明通过调节脂质体及信使核糖核酸的混合顺序来分析制备的信使核糖核酸-脂质体复合物的特性,确认到生物体内信使核糖核酸随着脂质体及信使核糖核酸的混合顺序的表达。
在本发明中,上述信使核糖核酸及脂质体可以为以信使核糖核酸→脂质体的顺序来放入并混合。
通过以上述顺序混合信使核糖核酸及脂质体,可以增加制备的信使核糖核酸-脂质体复合物在生物体内的信使核糖核酸的递送率及表达率。
本发明的信使核糖核酸递送用组合物的制备方法还可以包括混合免疫增强剂的步骤。
上述免疫增强剂能够以冷冻干燥状态的粉末形态来提供,也能够以溶解在适当的溶液或缓冲液中的状态来提供。在免疫增强剂以冷冻干燥的状态来提供的情况下,可以溶解在适当的溶液或缓冲液来使用。
在本发明的具体实施例中,本发明人对于通过调节脂质体、信使核糖核酸及免疫增强剂的混合顺序来制备的信使核糖核酸-脂质体复合物,确认到生物体内信使核糖核酸随脂质体、信使核糖核酸及免疫增强剂的混合顺序的表达。
在本发明中,上述信使核糖核酸、脂质体及免疫增强剂能够以免疫增强剂→信使核糖核酸→脂质体的顺序放入并混合。
通过以上述顺序混合信使核糖核酸、脂质体以及免疫增强剂,可以增加制备的信使核糖核酸-脂质体复合物在生物体内的信使核糖核酸的递送率及表达率。
本发明的信使核糖核酸及脂质体包含上述信使核糖核酸递送用组合物作为有效成分,为避免说明书过度的复杂性,将省略重复内容的说明。
【发明的效果】
本发明的包含阳离子脂质体的信使核糖核酸递送用组合物的保管稳定性优秀,在生物体内表现出高的细胞内递送率和表达率,可以提高癌症治疗/预防用信使核糖核酸疫苗或者病毒或细菌感染预防用信使核糖核酸疫苗等的稳定性及效率。
【附图说明】
图1为确认以不同使用量的信使核糖核酸制备的脂质体-信使核糖核酸复合物在小鼠内的信使核糖核酸表达的结果。
图2为确认使用以不同的DOTAP:DOPE的比例制备的脂质体的脂质体-信使核糖核酸复合物在小鼠内的信使核糖核酸表达的结果。
图3为确认使用以不同的DOTAP:DOPE:胆固醇的比例制备的脂质体的脂质体-信使核糖核酸复合物在小鼠内的信使核糖核酸表达的结果。
图4为分析以不同的信使核糖核酸与脂质体的混合顺序来制备的信使核糖核酸-脂质体复合物的试样大小、分散度、ZETA电位的结果。
图5为在生物体内确认随着信使核糖核酸与脂质体的混合顺序的信使核糖核酸的表达差异的结果。
图6a至图6c为使用以不同的信使核糖核酸与脂质体的混合顺序制备的信使核糖核酸-脂质体复合物使小鼠免疫化后,确认细胞免疫反应(图6a)、抗体免疫反应(图6b)及中和抗体效价(图6c)的结果。
图7为确认以不同的非毒性脂寡糖使用量制备的脂质体-信使核糖核酸复合物在小鼠的信使核糖核酸的表达的结果。
图8为确认以信使核糖核酸、脂质体及非毒性脂寡糖的不同混合顺序制备信使核糖核酸-脂质体-非毒性脂寡糖复合物并向小鼠注射时确认信使核糖核酸的表达的结果(LP:脂质体;R:信使核糖核酸;dL:非毒性脂寡糖)。
图9a至图9c是为测定以不同N/P比制备的信使核糖核酸-脂质体复合物的稳定性而以不同时间测量尺寸(图9a)、分散度(图9b)、ZETA电位(图9c)的结果。
【具体实施方式】
以下,通过实施例更为详细地说明本发明。本发明所属技术领域的普通技术人员应该自明的是,这些实施例仅用于更为具体地说明本发明,本发明的范围不应解释为限定于这些实施例。
【制备例1.制备信使核糖核酸-脂质体复合物(mRNA-liposome)】
【制备脂质体(薄膜方法(Film method))】
分别向DOTAP(Merck&Cie公司/CH2900014)、DOPE(Avanti Polar Lipid公司)和/或胆固醇(Avanti Polar Lipid公司)混合三氯甲烷后,在37℃的温度下完全溶解10分钟来制备液体溶液。
在圆底烧瓶中以规定的重量比混合上述液体溶液来制备脂质混合物(lipidmixture),在旋转蒸发器(Rotary evaporator)(Buchi公司/B491_R200)中以60℃的温度挥发30分钟挥发掉三氯甲烷来在烧瓶壁面制备脂质薄膜。
向上述制备脂质薄膜的烧瓶中放入含有4%(w/v)的蔗糖的20mM的HEPES缓冲液(pH7.4),在60℃的温度下融化脂质薄膜来以脂质体的浓度为7.5mg/mL的方式形成脂质体。使用动态光散射分析设备测量形成的脂质体离子的尺寸、ZETA电位、分散度。剩余的制备的脂质体在4℃的温度下保管直至试验前。
【制备信使核糖核酸-脂质体复合物】
在含有4%的蔗糖的20mM的HEPES缓冲液(pH7.4)中混合上述制备的脂质体(LP-DOTAP/DOPE/胆固醇(Chol)(40:40:20,w/w/w)或LP-DOTAP/DOPE(50:50,w/w))与有关海肾荧光素酶(Renilla Luciferase)的信使核糖核酸(序列1)来制备信使核糖核酸-脂质体复合物。
在此情况下,7.5mg/mL的脂质体溶液(LP-DOTAP/DOPE/胆固醇(40:40:20,w/w/w)制备后马上使用或者使用制备后2周内冷藏保管的试样。在-70℃的温度下保管1mg/mL的海肾荧光素酶信使核糖核酸(序列1)溶液,在使用前即刻在冰块上融化来使用。含有4%的蔗糖的20nM的HEPES缓冲液(pH7.4)在实验前即刻制备来使用或者在实验前一天制备后冷藏保管。
作为脂质体与信使核糖核酸的混合比例的N/P比(N/P ratio)通过如下计算式计算。
【实施例1.确认信使核糖核酸随信使核糖核酸-脂质体复合物的信使核糖核酸含量的表达】
在上述制备例1的脂质体-信使核糖核酸复合物的制备中,对于信使核糖核酸的量分别为5μg、10μg及20μg的情况,制备分别混合18.75μg、37.5μg、75μg的量的DOTAP:DOPE:胆固醇(40:40:20,w/w/w)的脂质体(N/P比=0.7)后,在生物体内确认表达率。
具体地,向去毛的小鼠(7周龄的雌性C57BL/6N,Orientbio公司,中央实验动物)的大腿部三角肌肌肉注射(intramuscular injection;I.M.injection)100μl剂量的各脂质体-信使核糖核酸复合物。
给药6小时后,使用阿佛丁(avertin)(250mg/kg)麻醉小鼠后,静脉注射(intravenous injection;I.V.injection)200μl的向荧光素酶底物储备溶液(ViviRenTM生物体内海参荧光素酶底物储备溶液(in vivo Renilla Luciferase substrate stocksolution))中放入2.4mL的1X磷酸盐缓冲溶液(PBS)来制备为0.15μg/μl的底物(substrate)。
给药后,马上将小鼠固定在IVIS设备(Ami-HTX,美国(USA))内并将拍摄曝光时间设定为60秒钟来拍摄小鼠(xenogen IVIS-200),使用Aura成像软件(Imaging Software)(Spectral Instruments Imaging公司,美国)将给药部位的荧光素酶的表达程度(感兴趣区(Region of interest,ROI))数值化。
结果如图1所示,可以确认在5μg、10μg的信使核糖核酸中没有表达率的差异,但在包含20μg的信使核糖核酸的情况与包含5μg、10μg的信使核糖核酸的情况相比,表达率分别增加135%、170%。若信使核糖核酸增加到规定水平以上,则增加生物体内表达率。
【制备例2.调节脂质体的阳离子脂质:中性脂质:胆固醇(DOTAP:DOPE:胆固醇(cholesterol))的混合比例来制备信使核糖核酸-脂质体复合物】
【2-1.调节DOTAP:DOPE的混合比例】
以下述表1所示的DOTAP:DOPE的比例(w/w)制备脂质体后,与信使核糖核酸混合来制备脂质体-信使核糖核酸复合物。以脂质体-信使核糖核酸复合物的NP比(摩尔比(molarratio))为0.7:1的方式对于20μg的信使核糖核酸(海肾荧光素酶信使核糖核酸,序列1)使用包含30μg的DOTAP的脂质体。在此情况下,将根据生产公司推荐的指导意见使用NanoAssemblr Ignite(Precision NanoSystems公司)以50:10:38.5:1.5的比例混合C12-200、DSPC、胆固醇及DMG-PEG2000的LNP(C12-200:DSPC:胆固醇:DMG-PEG2000=50:10:38.5:1.5)用作脂质体对照组。
【表1】
【2-2.调节DOTAP:DOPE:胆固醇的混合比例】
以下述表2所示的DOTAP:DOPE:胆固醇的比例(w/w)制备脂质体后,与信使核糖核酸混合来制备脂质体-信使核糖核酸复合物。以脂质体-信使核糖核酸复合物的NP比(摩尔比(molar ratio))为0.7:1的方式对于20μg的信使核糖核酸(海肾荧光素酶信使核糖核酸,序列1)使用包含30μg的DOTAP的脂质体。在此情况下,使用invivofectamine(赛默飞世尔公司(Thermo Fisher Scientific))作为脂质体对照组。
【表2】
实验组 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
DOTAP | 50 | 45 | 40 | 35 | 30 | 20 | 40 | 60 | 80 |
DOPE | 50 | 45 | 40 | 35 | 30 | 60 | 40 | 20 | 0 |
胆固醇 | 0 | 10 | 20 | 30 | 40 | 20 | 20 | 20 | 20 |
【实施例2.确认信使核糖核酸随脂质体的阳离子脂质:中性脂质:胆固醇(DOTAP:DOPE:胆固醇)的混合比例的表达】
【2-1.随DOTAP:DOPE的混合比例的生物体内表达率】
对于上述制备例2-1中制备的脂质体-信使核糖核酸复合物,以与上述实施例1相同的方法确认生物体内表达。
结果如图2所示,可以确认使用以40:60、50:50及70:30的DOTAP:DOPE的比例制备的脂质体的脂质体-信使核糖核酸复合物在小鼠中表现出高的信使核糖核酸表达率。
【2-2.随DOTAP:DOPE:胆固醇的混合比例的生物体内表达率】
对于上述制备例2-2中制备的各脂质体-信使核糖核酸复合物,以与上述实施例1相同的方法确认生物体内表达。
结果如图3所示,可以确认,在将DOTAP:DOPE的比例固定为1:1后调节胆固醇的比例时,使用以40:40:20至35:35:30的DOTAP:DOPE:胆固醇的比例制备的脂质体的脂质体-信使核糖核酸复合物在小鼠内表现出高的信使核糖核酸表达率,在固定胆固醇的比例时,使用以20:60:20至40:40:20的DOTAP:DOPE:胆固醇的比例制备的脂质体的脂质体-信使核糖核酸复合物在小鼠内表现出高的信使核糖核酸表达率。
【实施例3.分析随脂质体的阳离子脂质:中性脂质:胆固醇(DOTAP:DOPE:胆固醇)的混合比例的特性】
使用含有4%的蔗糖的20nM的HEPES缓冲液(pH7.4)将上述制备例2-2中制备的脂质体及信使核糖核酸-脂质体复合物稀释为1/10。使用Zetasizer Nano ZSP(MalvernPnanlytical公司)进行动态光散射(DLS,Dynamic Light Scattering)分析来测量复合物的大小(size)、分散度(PDI)、ZETA电位(Zeta potential)。
结果如下述表3(脂质体)及表4(脂质体-信使核糖核酸复合物)所示,可以确认脂质体的大小为100nm-200nm,分散度小于0.4。观察到当DOTAP与DOPE为同比时,胆固醇的含有比例越大,粒子的尺寸和分散度越大,当胆固醇的含有比同为20时,表现出DOPE相对于DOATP的含量越多,粒子大小和分散度越大的倾向(表3)。大体上,脂质体-信使核糖核酸复合物的大小为180nm-240nm,分散度小于0.3,这些数值比未混合信使核糖核酸的脂质体有所增加。在混合信使核糖核酸的情况下,没有观察到粒子大小随胆固醇含有比例变化而变化的状态。
【表3】
【表4】
【制备例3.通过调节脂质体及信使核糖核酸的混合顺序来制备信使核糖核酸-脂质体复合物】
使用上述制备例1的在-70℃的温度下保管的1mg/mL的海肾荧光素酶信使核糖核酸溶液、冷藏保管的脂质体溶液(LP-DOTAP/DOPE/胆固醇(40:40:20,w/w/w))以及含有4%的蔗糖的20nM的HEPES缓冲液(pH7.4)。
下述制备方法例示400μL的脂质体-信使核糖核酸复合物的制备,根据需要以规定比例增量来制备,溶液的混合使用包括吹打在内的搅拌等已知的方法。
【3-1.信使核糖核酸→脂质体的顺序】
使用核糖核酸专用200P针头(tip)取340μL的含有4%的蔗糖的20nM的HEPES缓冲液(pH7.4)分注到微型试管中。使用200P针头取40μL的完全解冻的1mg/mL的海肾荧光素酶信使核糖核酸溶液(40μg的信使核糖核酸)并放入分注有缓冲液的微型试管中后,吹打约10次。利用200P针头取20μL的7.5mg/mL的脂质体溶液(LP-DOTAP/DOPE/胆固醇(40:40:20,w/w/w)(150μg的脂质体)放入上述分注有上述缓冲液及信使核糖核酸溶液的微型试管中后吹打约30次。最后,使用1000P的针头吹打混合上述缓冲液、信使核糖核酸溶液及脂质体溶液的试样约10次来追加混合。
【3-2.脂质体→信使核糖核酸的顺序】
使用核糖核酸专用200P针头取340μL的含有4%的蔗糖的20nM的HEPES缓冲液(pH7.4)分注到微型试管中。利用200P针头取20μL的7.5mg/mL的脂质体溶液(LP-DOTAP/DOPE/胆固醇(40:40:20,w/w/w)(150μg脂质体)放入上述分注有缓冲液的微型试管中后吹打约10次。使用200P针头取40μL的完全解冻的1mg/mL的海肾荧光素酶信使核糖核酸溶液(40μg的信使核糖核酸)并放入上述分注有缓冲液及脂质体溶液的微型试管中后吹打约30次。最后,使用1000P的针头吹打混合上述缓冲液、信使核糖核酸溶液及脂质体溶液的试样约10次来追加混合。
【实施例4.分析随脂质体及信使核糖核酸的混合顺序的特性】
对上述制备例3中制备的脂质体-信使核糖核酸复合物进行动态光散射分析来导出复合物的大小、分散度、ZETA电位的平均值和标准差。
结果如图4所示,可以确认,脂质体-信使核糖核酸复合物的物理特性为示出显著差异。
【实施例5.确认信使核糖核酸随脂质体及信使核糖核酸的混合顺序的表达】
对于上述制备例3中以不同混合顺序制备的脂质体-信使核糖核酸复合物,以与上述实施例1相同的方法分析生物体内表达。
结果如图5所示,在以信使核糖核酸→脂质体的顺序混合来制备的复合物中,信使核糖核酸的表达显著高。
【实施例6.确认随脂质体及信使核糖核酸的混合顺序的免疫原性】
除使用SARS-CoV-2S信使核糖核酸(序列3)替代对于用海肾荧光素酶的信使核糖核酸以外,以与上述制备例3相同的方法,对于以与不同混合顺序制备的脂质体-信使核糖核酸复合物进行如下实验。
首先,向6周龄的雌性小鼠(B6C3F1/slc,中央实验动物)的后腿大腿部通过肌肉注射的途径以0.1HD(人体剂量(human dose))间隔3周给药2次以不同混合顺序制备的脂质体-信使核糖核酸复合物。
【6-1.细胞因子】
【脾细胞再刺激(Splenocyte restimulation)】
最后给药2周后,以颈椎错位的方式使小鼠安乐死并摘出脾脏后,将脾脏池化(pooling)并移到分注有加入1%的青霉素/链霉素的磷酸盐缓冲溶液(以下称为PBS w/antibiotics)的24孔培养板中。使用的培养基如下述表5及表6所示。
【表5】
【表6】
在超净工作台内使用镊子夹起脾脏组织在PBS w/antibiotics中洗涤后,移到装有3mL的基础培养基的60mm培养皿中,使用40μm的细胞过滤网(cell strainer)压碎组织来分离脾细胞(splenocyte)。将分离的脾细胞移到15mL的试管中后,以4℃、3000rpm的条件离心分离5分钟并去除上清液后,使用3mL的红细胞(RBC)裂解缓冲液使细胞悬浮并在常温下静置3分钟后,以4℃、3000rpm的条件离心分离5分钟。去除上清液并使用3ml的PBS w/antibiotics使细胞悬浮后以4℃、3000rpm的条件离心分离5分钟,去除上清液后使用10mL的完全培养基使细胞悬浮。使用完全培养基将上述细胞悬浮液稀释至2×107cells/mL后,以100μl/孔的量分注到96孔细胞培养板中。
另外,分别向1小瓶的PepMix SARS-CoV-2-S1肽池(pool,嘉华特公司(JPT))及SARS-CoV-2-S2肽池(嘉华特公司)放入50μl的二甲基亚砜(DMSO)并溶解后,混合完全培养基使最终浓度为2.5μg/mL来制备SARS CoV-2刺突肽刺激剂(stimulant)。
向上述装有细胞悬浮液的96孔中加入40μg/孔的上述刺激剂及60μl/孔的完全培养基后在37℃、5%的CO2条件下反应72小时。
【确认γ干扰素(IFN-γ)浓度】
使用试剂稀释剂(reagent diluent,1%的牛血清白蛋白(BSA))将被刺激的脾细胞的培养液稀释为1/5后,以100μl/孔的量分注到包被有抗小鼠γ干扰素捕获抗体(Jackson公司)的酶标板并覆盖密封膜在常温下静置2小时后,使用洗板机(ELISA washer)(帝肯公司(Tecan)/Hydroflexelisa)去除各孔的溶液并使用洗涤缓冲液洗涤3次。
使用试剂稀释剂将γ干扰素酶联免疫吸附测定试剂盒(IFN-γELISA kit)(MouseIFN-γDuoset ELISA,R&D systems公司)内的链霉亲和素-辣根过氧化物酶(Streptavidin-HRP)稀释为1/40后,以100μl/孔的量分注到免疫板并覆盖密封膜在常温下静置20分钟后,使用洗板机去除各孔的溶液并使用洗涤缓冲液洗涤3次。
使用试剂稀释剂将试剂盒内的抗小鼠γ干扰素酶(anti-mouse IFN-γ)检测抗体稀释为200ng/mL后,以100μl/孔的量分注到免疫板并覆盖密封膜在常温下静置1小时后,使用洗板机(帝肯公司/Hydroflexelisa)去除各孔的溶液并使用洗涤缓冲液洗涤3次。
向免疫板的每孔分注100μl的3,3',5,5'-四甲基联苯胺(TMB)底物(KPL sureblue3,3',5,5'-四甲基联苯胺微孔过氧化物酶底物(TMB microwell peroxidase substrate),Seracare公司)溶液并在常温的暗室里反应15分钟后,向免疫板的每孔中分注100μl的1N的硫酸(H2SO4)溶液终止反应并使用酶标仪(ELISA reader)在450nm的波长中测定吸光度。
结果如图6a所示,以信使核糖核酸→脂质体的顺序制备的复合物中的γ干扰素的浓度最高。
【6-2.抗体效价(IgG滴度(IgG titers))】
最后给药2周后,通过腹腔给药250mg/kg的阿佛丁工作溶液(Avertin workingsolution)来麻醉后,通过心脏采血来采取全血。将采取的全血移到微型试管中在常温下放置3小时后,以4℃、15000rpm的条件离心分离10分钟,将上清液移到新的微型试管获得血清后在-20℃的温度下保管直至分析前。
然后,使用1X的磷酸盐缓冲溶液将RBD(SARS-CoV-2受体结合结构域(receptorbinding domain))抗原(Mybiosource公司,美国)稀释为1μg/mL后,以100μl/孔的量分注到免疫板(immunoplate)并覆盖密封膜在4℃的温度下静置一夜。使用洗板机(帝肯公司/Hydroflexelisa)去除各孔的溶液并使用洗涤缓冲液(在使用纯水稀释20X的磷酸盐缓冲溶液的1L的1X磷酸盐缓冲溶液中放入500μl的吐温(tween)20)洗涤5次。以200μl/孔的量向免疫板分注试剂稀释剂(reagent diluent,1%的牛血清白蛋白,将1g的牛血清白蛋白溶解到100mL的磷酸盐缓冲溶液来制备)并覆盖密封膜在37℃的反应器中静置1小时。使用洗板机去除各孔的溶液并使用洗涤缓冲液洗涤5次。以100μl/孔的量向免疫板分注试剂稀释剂。
利用试剂稀释剂(1%的牛血清白蛋白(BSA))以1:50的比例稀释获得的血清后,分注100μl到免疫板的B~G的一列,在每孔内吹打数次来混合试样后,按照从1列取100μl放入2号列的方法在酶标板中1/2连续稀释(serial dilution)直至12列。在此情况下,为了评估试验的适当性,利用试剂稀释剂以1:200的比例稀释高血清(hyper serum)后在各免疫板的H的一列分注100μl并以上述方法进行1/2连续稀释。
使用密封膜覆盖免疫板在37℃的反应器中反应2小时。使用洗板机去除各孔的溶液并使用洗涤缓冲液洗涤5次。利用试剂稀释剂以1:5000的比例稀释山羊抗小鼠(goatanti-mouse)IgG抗体(Jackson Laboratory公司)后,向免疫板的每孔分注100μl并覆盖密封膜在37℃的反应器中反应1小时。使用洗板机去除各孔的溶液并使用洗涤缓冲液洗涤5次。
向免疫板的每孔中分注100μl的在常温下平衡的3,3',5,5'-四甲基联苯胺底物溶液并在常温的暗室中反应5分钟。向免疫板的每孔中分注100μl的1N的硫酸(H2SO4)溶液终止反应并使用酶标仪在450nm的波长中测定吸光度。
结果如图6b所示,可以确认在以信使核糖核酸→脂质体的顺序混合来制备的复合物中,抗体性免疫反应最优秀。
【6-3.分析中和抗体形成能力(替代性中和)(%)】
在1.5mL的微型试管中分别将60μL的阴性对照组(negative control)(杜氏改良伊戈尔培养基(DMEM))或上述实施例6-2中获得的在-20℃的温度下保管的免疫化的小鼠血清样品与60μL的以1:1000的比例连续稀释的结合辣根过氧化物酶的RBD(1:1000diluted-HRP conjugated RBD)混合后在37℃的温度下反应30分钟,向微量滴定测试条板(Microtiter test strip plate)分注100μl后,覆盖密封膜在37℃的温度下反应15分钟。使用洗板机去除各孔的溶液并使用1X的洗涤缓冲液洗涤4次。以100μl/孔分注3,3',5,5'-四甲基联苯胺溶液(TMB solution)并覆盖密封膜在常温的暗室中反应15分钟后,以50μl/孔分注终止溶液来终止反应后,使用酶标仪在405nm波长中测定光学密度(opticaldensity)。
结果如图6c所示,可以确认以信使核糖核酸→脂质体的顺序混合来制备的复合物示出最高水平的中和抗体效价诱导能力。
【小结】
在制备信使核糖核酸与脂质体的复合物时,生物体内表达及免疫原性诱导能力随制备时的混合方法的不同而不同,在以信使核糖核酸→脂质体的顺序混合时,所表现的效果最为优秀。
【制备例4.制备通过调节脂质体、信使核糖核酸及免疫增强剂的混合顺序来制备的信使核糖核酸-脂质体复合物】
【4-1.包含脂质体、信使核糖核酸及免疫增强剂的信使核糖核酸-脂质体复合物】
在上述制备例1中追加混合免疫增强剂dLOS(非毒性脂寡糖)(TLR4激动剂(agonist);(株)EYEGENE公司,韩国)来制备信使核糖核酸-脂质体复合物。
【4-2.调节脂质体、信使核糖核酸及免疫增强剂的混合顺序】
使用上述制备例1的在-70℃的温度下保管的1mg/mL的海肾荧光素酶信使核糖核酸溶液、冷藏保管的脂质体溶液(LP-DOTAP/DOPE/胆固醇(40:40:20,w/w/w))以及含有4%的蔗糖的20nM的HEPES缓冲液(pH7.4)。追加地,使用免疫增强剂非毒性脂寡糖。以表示能够调节上述3种溶液(信使核糖核酸、脂质体及非毒性脂寡糖)的混合顺序的情况的数字,即,使用下述6种方法混合试样。
a:脂质体→信使核糖核酸→非毒性脂寡糖
b:脂质体→非毒性脂寡糖→信使核糖核酸
c:信使核糖核酸→脂质体→非毒性脂寡糖
d:使核糖核酸→非毒性脂寡糖→脂质体
e:非毒性脂寡糖→脂质体→信使核糖核酸
f:非毒性脂寡糖→信使核糖核酸→脂质体
具体地,使用核糖核酸专用200P针头取定量的含有4%的蔗糖的20nM的HEPES缓冲液(pH7.4)分注到微型试管中。利用200P针头取定量的第一溶液放入分注有缓冲液的微型试管中后吹打约10次。利用200P针头取定量的第而溶液放入分注有缓冲液的微型试管中后吹打约10次。利用200P针头取定量的第三溶液放入分注有缓冲液的微型试管中后吹打约10次。最后,使用1000P的针头吹打约30次来混合。
【实施例7:确认信使核糖核酸随免疫增强剂含量的不同的表达】
在上述制备例4-1中加入不同含量的免疫增强剂非毒性脂寡糖来确认脂质体-信使核糖核酸复合物在生物体内的表达率。在此情况下,分别加入0.25μg、0.5μg、0.75μg及1μg的非毒性脂寡糖,以与上述实施例1相同的方法确认生物体内表达。
结果如图7所示,在非毒性脂寡糖的添加量为0.25μg以上且1μg以下的情况下,表达量增加。
【实施例8:确认信使核糖核酸随脂质体、信使核糖核酸及免疫增强剂的混合顺序的表达】
对于上述制备例4-2中制备的各脂质体-信使核糖核酸复合物,以与上述实施例1相同的方法确认生物体内表达。
结果如图8所示,在给药以非毒性脂寡糖→信使核糖核酸→脂质体的顺序混合的组合物的小鼠中确认到更高的表达量。
【实施例9:确认信使核糖核酸-脂质体复合物的稳定性(物理化学性质)随时间的变化】
【EGFP】
以EGFP信使核糖核酸(序列2)与脂质体(DOTAP:DOPE:胆固醇=40:40:20,w/w/w)的复合物为对象,通过动态光散射测定确认制备后随时间经过的物理化学性质变化。
具体地,在冷藏保管上述制备的EGFP信使核糖核酸-脂质体复合物以冷冻干燥剂型的9周期间,每周测定大小、ZETA电位及分散度。
结果如图9所示,可以确认在NP比为1.39:1以下时,9周期间稳定。
【SARS-CoV-2S】
以液体剂型和冷冻干燥剂型的SARS-CoV-2S信使核糖核酸(序列3)与脂质体(DOTAP:DOPE:胆固醇=40:40:20,w/w/w)的复合物为对象,通过动态光散射测定确认制备后随时间经过的物理化学性质变化。
具体地,在冷藏保管上述制备的SARS-CoV-2S信使核糖核酸-脂质体复合物的16周期间以规定时间(0周、2周、4周、8周、12周、16周)测定大小、ZETA电位及分散度。
结果如表7(液体剂型)及表8(冷冻干燥剂型)所示,可以确认液体剂型和冷冻干燥剂型在16周时间内都稳定。
【表7】
区分 | 0周 | 2周 | 4周 | 8周 | 12周 | 16周 |
粒子大小(nm) | 216.0 | 225.8 | 227.1 | 222.9 | 199.9 | 196.5 |
分散度 | 0.187 | 0.194 | 0.175 | 0.219 | 0.187 | 0.159 |
ZETA电位(mV) | -62.7 | -56.4 | -52.3 | -55.0 | -72.3 | -68.7 |
【表8】
区分 | 0周 | 2周 | 4周 | 8周 | 12周 | 16周 |
粒子大小(nm) | 339.1 | 316.1 | 322.4 | 329.2 | 280.5 | 272.0 |
分散度 | 0.284 | 0.215 | 0.245 | 0.264 | 0.243 | 0.228 |
ZETA电位(mV) | -45.4 | -43.7 | -42.8 | -43.8 | -60.7 | -60.2 |
【产业上的可利用性】
本发明涉及信使核糖核酸体内递送用组合物,具体地,涉及包含基于阳离子脂质的脂质体的信使核糖核酸体内递送用组合物及其制备方法。
序列表
<110> 艾金株式会社
<120> 核糖核酸的体内递送用组合物及其制备方法
<130> PP220013
<150> KR 10-2021-0029929
<151> 2021-03-08
<160> 3
<170> KoPatentIn 3.0
<210> 1
<211> 936
<212> RNA
<213> 人工序列
<220>
<223> Renilla mRNA
<400> 1
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cagcacgacu ucuucaaguc cgccaugccc gaaggcuacg uccaggagcg caccaucuuc 300
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gugaaccgca ucgagcugaa gggcaucgac uucaaggagg acggcaacau ccuggggcac 420
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gaccacuacc agcagaacac ccccaucggc gacggccccg ugcugcugcc cgacaaccac 600
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720
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<212> RNA
<213> 人工序列
<220>
<223> CoV2-F004 mRNA
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aagguguuca gauccagcgu gcugcacucu acccaggacc uguuccugcc uuucuucagc 180
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aaccccgugc ugcccuucaa cgacggggug uacuuugcca gcaccgagaa guccaacauc 300
aucagaggcu ggaucuucgg caccacacug gacagcaaga cccagagccu gcugaucgug 360
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cugggcgucu acuaccacaa gaacaacaag agcuggaugg aaagcgaguu ccggguguac 480
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ggcaagcagg gcaacuucaa gaaccugcgc gaguucgugu uuaagaacau cgacggcuac 600
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gagaacggca ccaucaccga cgccguggau ugugcucugg auccucugag cgagacaaag 900
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cagcccaccg aauccaucgu gcgguucccc aauaucacca aucugugccc cuucggcgag 1020
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guguucgccc aagugaagca gaucuacaag accccuccua ucaaggacuu cggcggcuuc 2400
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aagcugaucg ccaaccaguu caacagcgcc aucggcaaga uccaggacag ccugagcagc 2820
acagcaagcg cccugggaaa gcugcaggac guggucaacc agaaugccca ggcacugaac 2880
acccugguca agcagcuguc cuccaacuuc ggcgccauca gcucugugcu gaacgauauc 2940
cugagcagac uggacccucc ugaggccgag gugcagaucg acagacugau cacaggcaga 3000
cugcagagcc uccagacaua cgugacccag cagcugauca gagccgccga gauuagagcc 3060
ucugccaauc uggccgccac caagaugucu gagugugugc ugggccagag caagagagug 3120
gacuuuugcg gcaagggcua ccaccugaug agcuucccuc agucugcacc acacggcgug 3180
guguuucugc acgugaccua cgugcccgcu caagagaaga auuucaccac cgcuccagcc 3240
aucugccacg acggcaaagc ccacuuuccu agagaaggcg uguucguguc caacggcacc 3300
cauugguucg ugacacagcg gaacuucuac gagccccaga ucaucaccac cgacaacacc 3360
uucgugucug gcaacugcga cgucgugauc ggcauuguga acaauaccgu guacgacccu 3420
cugcagcccg agcuggacag cuucaaagag gaacuggaca aguacuuuaa gaaccacaca 3480
agccccgacg uggaccuggg cgauaucagc ggaaucaaug ccagcgucgu gaacauccag 3540
aaagagaucg accggcugaa cgagguggcc aagaaucuga acgagagccu gaucgaccug 3600
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Claims (19)
1.信使核糖核酸递送用组合物,其包含基于阳离子脂质的脂质体。
2.根据权利要求1所述的组合物,其中所述脂质体还包含中性脂质。
3.根据权利要求2所述的组合物,其中所述脂质体还包含胆固醇。
4.根据权利要求1所述的组合物,其中所述阳离子脂质为选自由二甲基双十八烷基溴化铵、1,2-二油酰基-3-三甲基丙烷铵、3β-[N-(N',N'-二甲氨基乙烷)氨基甲酰胆固醇、1,2-二油酰氧基-3-二甲基丙烷铵、1,2-二-O-十八烯基-3-三甲基丙烷铵、1,2-二肉豆蔻酰基-sn-甘油-3-乙基磷酸胆碱、1-棕榈酰-2-油酰-sn-甘油-3-乙基磷酸胆碱、1,2-二油酰-sn-甘油-3-乙基磷酸胆碱、1,2-二硬脂酰-sn-甘油-3-乙基磷酸胆碱、1,2-二棕榈酰-sn-甘油-3-乙基磷酸胆碱、1,2-二肉豆蔻酰-sn-甘油-3-乙基磷酸胆碱、1,2-二月桂酰-sn-甘油-3-乙基磷酸胆碱、N1-[2-((1S)-1-[(3-氨基丙基)氨基]-4-[二(3-氨基丙基)氨基]丁基甲酰氨基)乙基]-3,4-二[油氧基]-苯甲酰胺、1,2-二肉豆蔻酰基-3-二甲基丙烷铵、1,2-二棕榈酰基-3-二甲基丙烷铵、1,2-二硬脂酰基-3-二甲基丙烷铵、N-(4-羧基苄基)-N,N二甲基-2,3-双(油酰氧基)丙-1-铵、1,2-硬脂酰基-3-三甲基丙烷铵、1,2-二棕榈酰基-3-三甲基丙烷铵、1,2-二肉豆蔻酰基-3-三甲基丙烷铵及N4-胆固醇精胺组成的组中的一种以上。
5.根据权利要求2所述的组合物,其中所述中性脂质为选自由1,2-二肉豆蔻酰-sn-甘油-3-磷酸胆碱、1,2-二油酰-sn-甘油-3-磷酸胆碱、1,2-二油酰-sn-甘油-3-磷酸乙醇胺、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱、1,2-二硬脂酰-sn-甘油-3-磷酸胆碱、1,2-二亚油酰-sn-甘油-3-磷酸胆碱、磷酯酰丝氨酸、磷酸乙醇胺、磷脂酰甘油、磷酸及磷脂酰胆碱组成的组中的一种以上。
6.根据权利要求2所述的组合物,其中所述阳离子脂质与中性脂质的重量比为1:9~9.5:0.5。
7.根据权利要求3所述的组合物,其中所述阳离子脂质与胆固醇的重量比为6:1~1:3。
8.根据权利要求7所述的组合物,其中所述阳离子脂质、中性脂质及胆固醇的重量比为1.0~9.5:9.0~0.5:0.05~3.00。
9.根据权利要求1所述的组合物,其中所述信使核糖核酸编码能够起到免疫原作用的肽或蛋白质。
10.根据权利要求9所述的组合物,其中所述脂质体与信使核糖核酸的N:P比为0.23:1.00~1.39:1.00。
11.根据权利要求1所述的组合物,其中所述组合物还包含免疫增强剂。
12.根据权利要求11所述的组合物,其中所述免疫增强剂为选自由病原体相关分子模式、皂苷、非甲基化的胞嘧啶鸟嘌呤二核苷酸、脂蛋白、鞭毛、聚肌胞苷酸、角鲨烯、癸酸甘油三酯、3D-MPL及非毒性脂寡糖组成的组中的一种以上。
13.权利要求1~12之任一项所述的组合物在制造用于预防或治疗疾病的药物中的用途,
所述疾病选自由癌症、肿瘤、自身免疫性疾病、遗传疾病、炎症疾病、病毒感染及细菌感染组成的组中。
14.疫苗,其包含权利要求1~12之任一项所述的组合物作为有效成分。
15.功能性化妆品组合物,其包含权利要求1~12之任一项所述的组合物作为有效成分。
16.信使核糖核酸递送用组合物的制备方法,其包括混合信使核糖核酸与脂质体的步骤。
17.根据权利要求16所述的方法,其中所述方法为按照信使核糖核酸、脂质体的顺序放入来混合。
18.根据权利要求16所述的方法,其中所述方法还包括混合免疫增强剂的步骤。
19.根据权利要求18所述的方法,其中所述方法按照免疫增强剂、信使核糖核酸、脂质体的顺序放入来混合。
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AU2022232410A1 (en) | 2023-10-05 |
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