CN112218622B - 包含核壳结构的微粒作为活性成分的用于增加凝血因子基因表达的组合物 - Google Patents
包含核壳结构的微粒作为活性成分的用于增加凝血因子基因表达的组合物 Download PDFInfo
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Abstract
本发明涉及用于增加凝血因子基因表达的组合物,所述组合物包含核壳结构的微粒作为活性成分。当与凝血因子VIII基因或其变体基因一起体内施用时,根据本发明的用于增加凝血因子基因表达的组合物可以使基因的表达增加至少30%或大于30%。当与基因治疗剂一起施用时,即使具有少量的基因,组合物也可以实现治疗效果,因此组合物是有用的。
Description
技术领域
本公开涉及用于增加凝血因子基因表达的组合物,其包含核壳结构的微粒作为活性成分。
背景技术
血友病是一种严重的遗传紊乱。血友病A是X基因相关疾病,每5000名男性出现1例。它是由人凝血因子VIII(也称为因子VIII、因子8、FVIII或F8)的突变引起的,人凝血因子VIII是血液凝固过程中重要的血浆糖蛋白。
20世纪80年代以前,血友病患者接受从他人血浆中提取的凝血因子VIII进行治疗。但是,该方法存在例如病毒感染等严重问题。为了克服该缺点,自20世纪80年代以来,基于重组蛋白研究,将在CHO细胞等中产生的全长因子VIII用于治疗。
本公开的发明人已经研究开发了即使使用少量基因也能够实现治疗效果的基因治疗剂。通过这样做,他们已经确定,当将组成为卤代烃和/或卤化硫作为核且脂质成分作为外壳的核壳结构微粒与凝血因子VIII基因一起在体内施用时,凝血因子基因的表达显著增加,从而完成了本公开。
相关技术的参考
专利文献
(专利文献001)KR 10-1542752 B。
(专利文献002)KR 10-2018-0118659 A。
发明内容
技术问题
本公开旨在提供用于增加凝血因子基因表达的组合物,其包含核壳结构的微粒作为活性成分。
本公开还旨在提供用于预防或治疗出血紊乱或出血的药物组合物,其包含上述组合物。
技术方案
本公开提供了用于增加凝血因子基因表达的组合物,其包含核壳结构的微粒作为活性成分,其中核为卤代烃、卤化硫或其混合物作为生物相容性气体,壳是脂质或其衍生物,凝血因子基因是选自人凝血因子VIII基因或其变体基因的一种或多于一种基因。
在本公开的一个示例性实施方案中,生物相容性气体可以选自六氟化硫、八氟丙烷、溴氯二氟甲烷、一氯二氟甲烷、二氯二氟甲烷、溴三氟甲烷、氯三氟甲烷、氯五氟乙烷、二氯四氟乙烷及其混合物。
在本公开的一个示例性实施方案中,卤代烃可以是全氟烃。
在本公开的一个示例性实施方案中,全氟烃可以是全氟甲烷、全氟乙烷、全氟丙烷、全氟丁烷、全氟戊烷、全氟己烷、全氟庚烷、全氟丙烯、全氟丁烯、全氟丁二烯、全氟丁-2-烯、全氟环丁烷、全氟甲基环丁烷、全氟二甲基环丁烷、全氟三甲基环丁烷、全氟环戊烷、全氟甲基环戊烷、全氟二甲基环戊烷、全氟甲基环己烷、全氟甲基环己烷、全氟甲基环己烷或其混合物。
在本公开的一个示例性实施方案中,脂质可以是选自单脂、磷脂、甘油糖脂、鞘糖脂、胆固醇和阳离子脂质中的一种或多于一种。
在本公开的一个示例性实施方案中,磷脂可以选自磷脂酰胆碱衍生物、磷脂酰乙醇胺衍生物、磷脂酰丝氨酸衍生物、二乙酰化磷脂、L-α-二油醇磷脂酰乙醇胺、二油精、磷脂酸、磷脂酰甘油、磷脂酰肌醇、溶血磷脂酰胆碱、鞘磷脂、聚乙二醇化磷脂、蛋黄卵磷脂、大豆卵磷脂和氢化磷脂。
在本公开的一个示例性实施方案中,甘油糖脂可以选自磺氧基核糖基甘油酯、二糖基二甘油酯、二半乳糖基二甘油酯、半乳糖基二甘油酯和糖基二甘油酯。
在本公开的一个示例性实施方案中,鞘糖脂可以是半乳糖基脑苷脂、乳糖基脑苷脂或神经节苷脂。
在本公开的一个示例性实施方案中,阳离子脂质可以选自1,2-二油酰基-3-三甲基铵丙烷(DOTAP)、N-(2,3-二油基氧基丙-1-基)-N,N,N-三甲基氯化铵(DOTMA)、2,3-二油基氧基-N-[2-(精胺酰胺基)乙基]-N,N-二甲基-1-丙三氟乙酸铵(DOSPA)、1,2-二肉豆蔻基氧基丙基-3-二甲基羟乙基溴化铵(DMRIE)、1,2-二油酰基氧基丙基-3-二乙基羟乙基溴化铵(DORIE)和3β-[N-(N′,N′-二甲基氨基乙基)氨基甲酰基]胆固醇(DC-Chol)。
在本公开的一个示例性实施方案中,凝血因子VIII可以由SEQ ID NO 1表示的氨基酸序列组成,凝血因子VIII的变体可以由SEQ ID NO 3表示的氨基酸序列组成。
在本公开的一个示例性实施方案中,组合物可以使凝血因子基因的表达增加30%或大于30%。
本公开还提供了用于预防或治疗出血紊乱或出血的药物组合物,其包含上述组合物和人凝血因子VIII基因或其变体基因。
在本公开的一个示例性实施方案中,出血紊乱可以是血友病A、由针对凝血因子VIII或凝血因子VIIIa的抑制性抗体诱导或并发的血友病、或血友病B。
在本公开的一个示例性实施方案中,出血紊乱或出血可以选自新生儿凝血病、严重肝病、血小板减少症、因子V、VII、X或XI的先天性缺乏和具有针对血管性假血友病因子的抑制因子的血管性假血友病。
在本公开的一个示例性实施方案中,出血可以是由于高风险外科手术引起的失血、创伤性失血、由骨髓移植引起的失血或由脑出血引起的失血。
有益效果
当与凝血因子VIII基因或其变体基因(例如编码该基因的多核苷酸或包含该基因的载体)一起体内施用时,本公开的用于增加凝血因子基因的表达的组合物可以使凝血因子基因的表达增加至少30%。因此,这是有用的,因为当将组合物与基因治疗剂一起施用时,可以用非常少量的基因获得治疗效果。
附图说明
图1示意性地示出了根据本公开的一个示例性实施方案设计凝血因子VIII变体(F8M)的方法。
图2示出了根据本公开的一个示例性实施方案的pGP载体的酶切图。
图3比较了根据施用pGP-F8M(仅基因)、对照组的药物组合物(F8M-JetPEI)和根据实施例的药物组合物(F8M-MP1和F8M-MP2)的小鼠中F8蛋白的表达水平。
图4比较了根据施用pGP-F9(仅基因)和根据对比例的药物组合物(F9-MP1和F9-MP2)的小鼠中F9蛋白的表达水平。
图5示意性地示出了根据本公开的一个示例性实施方案构成人凝血因子VIII的结构域结构。
最佳实施方式
在下文中,更详细地描述了本公开内容。
在一方面,本公开提供了用于增加凝血因子基因的表达的组合物,其包含核壳结构的微粒作为活性成分,其中核为卤代烃、卤化硫或其混合物作为生物相容性气体,壳是脂质或其衍生物,凝血因子基因是选自人凝血因子VIII基因或其变体基因的一种或多于一种基因。
核
在本公开中,“核”可以由作为生物相容性气体的卤代烃、卤化硫或其混合物组成。
生物相容性气体可以是六氟化硫、八氟丙烷、溴氯二氟甲烷、一氯二氟甲烷、二氯二氟甲烷、溴三氟甲烷、氯三氟甲烷、氯五氟乙烷、二氯四氟乙烷或其混合物。
具体地,卤代烃可以是全氟烃。
全氟烃可以是全氟甲烷、全氟乙烷、全氟丙烷、全氟丁烷、全氟戊烷、全氟己烷、全氟庚烷、全氟丙烯、全氟丁烯、全氟丁二烯、全氟丁-2-烯、全氟环丁烷、全氟甲基环丁烷、全氟二甲基环丁烷、全氟三甲基环丁烷、全氟环戊烷、全氟甲基环戊烷、全氟二甲基环戊烷、全氟甲基环己烷、全氟甲基环己烷、全氟甲基环己烷或其混合物。
具体地,本公开的生物相容性气体可以是六氟化硫或全氟丁烷。
壳
在本公开中,“壳”可以由脂质或其衍生物组成。
脂质可以是选自单脂、磷脂、甘油糖脂、鞘糖脂、胆固醇和阳离子脂质中的一种或多于一种。具体而言,其可以是磷脂。
磷脂可以是磷脂酰胆碱衍生物、磷脂酰乙醇胺衍生物、磷脂酰丝氨酸衍生物、二乙酰化磷脂、L-α-二油基磷脂酰乙醇胺、二油精、磷脂酸、磷脂酰甘油、磷脂酰肌醇、溶血磷脂酰胆碱、鞘磷脂、聚乙二醇化磷脂、蛋黄卵磷脂、大豆卵磷脂、氢化磷脂等。
甘油糖脂可以是磺氧基核糖基甘油酯、二糖基二甘油酯、二半乳糖基二甘油酯、半乳糖基二甘油酯、糖基二甘油酯等。
鞘糖脂可以是半乳糖基脑苷脂、乳糖基脑苷脂、神经节苷脂等。
并且,阳离子脂质可以是1,2-二油酰基-3-三甲基铵丙烷(DOTAP)、N-(2,3-二油基氧基丙-1-基)-N,N,N-三甲基氯化铵(DOTMA)、2,3-二油基氧基-N-[2-(精胺酰胺基)乙基]-N,N-二甲基-1-丙三氟乙酸铵(DOSPA)、1,2-二肉豆蔻基氧基丙基-3-二甲基羟乙基溴化铵(DMRIE)、1,2-二油酰基氧基丙基-3-二乙基羟乙基溴化铵(DORIE)、3β-[N-(N′,N′-二甲基氨基乙基)氨基甲酰基]胆固醇(DC-Chol)等。
微粒
本公开的微粒通过包围核气体的壳而稳定。壳阻止气体向附近液体的扩散,并防止微粒之间的融合。
当在体内施用时,微粒保持其形状,直到其到达靶细胞或组织,并在靶细胞或组织附近被破坏时释放出气体。释放的气体可引起靶细胞的细胞膜变化,并可通过气体的喷射力促进生长因子基因进入靶细胞的细胞质环境。
微粒的平均直径可以为1μm至10μm,特别是2μm至8μm,更特别是2μm至4μm。
用于增加基因表达的组合物
近年来,对以气体为核的核壳微粒进行了许多研究。在先前的研究中,除非与微粒一起超声辐照,否则无法达到增加基因表达的效果。
具体而言,Sang-Chol Lee等人,Korean Circulation J 2006;36:32-38;″Enhancement of Gene Delivery into Mouse Skeletal Muscle with MicrobubbleDestruction by Low-Frequency Ultrasound″公开了当仅注射荧光素酶基因-微粒混合物而不进行超声辐照时无法实现增加基因表达的效果。
ZP Shen等人,Gene Therapy(2008)15,1147-1155;″Ultrasound withmicrobubbles enhances gene expression of plasmid DNA in the liver viaintraportal delivery″也公开了当仅注射荧光素酶基因-微粒混合物而不进行超声辐照时无法实现增加基因表达的效果。
另外,Xingsheng Li等人,J Ultrasound Med 2008;27:453-460;″ExperimentalResearch on Therapeutic Angiogenesis Induced by Hepatocyte Growth FactorDirected by Ultrasound-Targeted Microbubble Destruction in Rats″公开了当仅注射HGF基因-脂质体微粒混合物而不进行超声辐照时无法达到增加基因表达的效果。
然而,本公开的发明人已经发现,在研究微粒的使用期间,当基因与本公开的微粒一起注射时,即使在无超声辐照的情况下,凝血因子基因,特别是凝血因子VIII基因的表达水平也显著提高,并且完成了本公开。同时,如稍后描述的测试实施例中具体描述的那样,除了凝血因子VIII基因或其变体基因之外的凝血因子基因(例如,凝血因子IX(也称为因子IX、因子9、FIX或F9))的微粒无法实现增加基因表达的效果。
当与凝血因子VIII基因或其变体基因一起体内施用时,本公开的用于增加基因表达的组合物可以使凝血因子基因的表达水平增加至少30%。
在以下实施例中证实了当与人凝血因子VIII变体基因一起施用于小鼠时,本公开的用于增加基因表达的组合物显著提高凝血因子VIII基因的表达水平。更具体地,证实了当将本公开的用于增加基因表达的组合物和凝血因子VIII变体基因一起施用到动物模型中时,凝血因子VIII基因的表达水平增加40%或大于40%。相反,对于凝血因子IX基因,即使将本公开的用于增加基因表达的组合物一起施用给小鼠,该基因的表达也几乎不增加。
也就是说,似乎本公开的用于增加基因表达的组合物仅在与选自凝血因子中的人凝血因子VIII基因及其变体基因的一种或多于一种基因一起施用时才有效地特异性地提高人凝血因子基因的表达水平。
组合物还可以包含药物佐剂,例如稳定剂、缓冲剂、控制渗透压的盐、赋形剂、防腐剂等或其他治疗上有用的物质,并且可以根据常见方法制备成用于口服或肠胃外施用,特别是用于肠胃外施用的各种制剂。具体地,用于肠胃外施用的制剂通常可以是等渗水溶液或悬浮液形式的注射制剂。或者,可将组合物制成粉末,然后在施用前立即悬浮在溶剂中。
在本公开的用于增加基因表达的组合物中,虽然没有特别限制,但微粒的含量可以为0.5μL/mL至2000μL/mL,特别是1μL/mL至1000μL/mL或5μL/mL至2000μg/mL,特别是10μL/mL至1000μg/mL。
如果微粒的含量在上述范围之外,则不能获得所需的效果。
具体地,可以将组合物与基因混合施用以获得更好的效果。
基因
当与以下基因一起施用时,根据本公开的用于增加基因表达的组合物可以进一步增加基因的表达和效率。
凝血因子VIII
在本公开中,术语“凝血因子VIII”、“因子VIII”、“FVIII”和“F8”可互换使用。成熟的人凝血因子VIII由2351个氨基酸(包括信号肽)组成,它们排列在图5所示的结构域结构中。
在图5中,a1、a2和a3是酸性结构域。已知酸性结构域a3参与凝血因子VIII分子与血管性假血友病因子(vWF)的结合,血管性假血友病因子在凝血中起重要作用。在分泌过程中,凝血因子VIII在B结构域和a3酸性结构域之间裂解,形成异二聚体多肽。凝血因子VIII异二聚体由轻链(包括A3、C1和C2)和大小可变的重链(包括A1、A2和B)组成。由于B结构域中的有限的蛋白水解,重链是异质的。在异二聚体的缺失B结构域的凝血因子VIII情况下,“重链”包括A1和A2,而B结构域部分或全部缺失。
成熟的野生型人凝血因子VIII的氨基酸序列示于SEQ ID NO 1。提及特定序列的氨基酸位置是指该氨基酸在VIII野生型蛋白中的位置,并且不排除在该序列的其他位置存在突变(例如,缺失、插入和/或置换)。编码SEQ ID NO 1的DNA序列由SEQ ID NO 2表示。
“凝血因子VIII”不仅包括野生型凝血因子VIII,而且包括具有野生型凝血因子VIII的促凝活性的野生型凝血因子VIII的衍生物。与野生型凝血因子VIII的氨基酸序列相比,该衍生物可以包括具有缺失、插入和/或添加的序列。具体地,该衍生物可以是缺失全部或部分的B结构域的凝血因子VIII的分子。在整个本公开中,氨基酸的位置始终是指全长成熟野生型凝血因子VIII(包括信号肽)中单个氨基酸的位置。
凝血因子VIII变体
在本公开中,术语“变体”包括氨基酸序列、核酸序列的保守或非保守的置换、插入或缺失等,并且这种改变基本上不改变赋予FVIII生物活性的活性位点或活性结构域。
根据本公开的凝血因子VIII变体是单链凝血因子VIII变体,其中从SEQ ID NO 1表示的凝血因子VIII中缺失氨基酸Asp784至Arg1671。凝血因子VIII变体是其中缺失了一部分B结构域(残基784至1667)和一部分a3结构域(残基1668至1671)的变体,该变体显示出与凝血因子VIII和缺失了B结构域的凝血因子VIII相比增强的蛋白质表达和改善的凝血活性和稳定性。凝血因子VIII变体具有由SEQ ID NO 3表示的氨基酸序列。
“单链凝血因子VIII”是指凝血因子VIII分子,其以单条多肽链的形式存在,而在表达该凝血因子VIII分子的细胞分泌过程中不被蛋白水解切割成两条链(例如,重链和轻链)。
另外,凝血因子VIII变体可以表达为编码氨基酸序列的多核苷酸。
多核苷酸
在本公开中,术语“多核苷酸”是指可以是未经修饰的RNA或DNA或经修饰的RNA或DNA的任何多核糖核苷酸或多脱氧核糖核苷酸。本发明的多核苷酸可以是单链DNA或RNA。如本文所使用的,术语“多核苷酸”包括包含经修饰的碱基和/或独特的碱基例如肌苷的DNA或RNA。显然,可以对DNA或RNA进行各种修饰以达到已知的有用目的。如本文所使用的,术语“多核苷酸”包括这些经化学修饰、酶促修饰或代谢修饰的多核苷酸。
本领域技术人员将理解,由于遗传密码的简并性,凝血因子VIII变体可以由几种多核苷酸编码。换句话说,可以在本发明中使用的编码凝血因子VIII变体的多核苷酸序列被解释为包括显示出与氨基酸序列基本同一的核苷酸序列。
质粒
当将根据本公开的用于增加基因表达的组合物与含有编码基因的单链多核苷酸的质粒一起施用时,可以进一步提高基因的表达效率和功效。
在本公开中,术语“质粒”通常是指通过与载体可操作地连接而形成的环状DNA分子,使得外源基因可以在宿主细胞中表达。然而,质粒可以用作通过基因重组被特定限制酶降解以掺入所需基因的载体。因此,术语质粒和载体在本文中可互换使用,即使没有按名称区分,基因工程领域的技术人员也将充分理解它们的含义。
在本公开中,术语“载体”是指可以稳定地将外源基因转运到宿主细胞中的作为载体的DNA分子。作为有用的载体,它必须是可复制的,具有进入宿主细胞的方式,并配备有检测其存在的机制。
表达
表达载体
当与包括编码基因的单链多核苷酸的表达载体一起施用时,根据本公开的用于增加基因表达的组合物可以进一步增加基因的表达和效率。
在本公开中,术语“表达”是指基因在细胞中的产生。
在本公开中,术语“表达载体”是指能够在合适的宿主中表达靶基因的载体,并且是指包括为了表达基因插入物可操作地连接了必需调控元件的基因构建体。
在本公开中,术语“可操作地连接”是指将核酸表达调节序列和编码靶基因的多核苷酸功能性连接以执行一般功能。例如,启动子和编码基因的多核苷酸可以可操作地连接以影响多核苷酸的表达。可以使用本领域众所周知的遗传重组技术来实现与重组载体的可操作的连接,并且可以使用本领域通常已知的酶来实现位点特异性的DNA切割和连接。
尽管不限于此,但是可以使用质粒、载体或病毒载体来制备本公开的表达载体。合适的表达载体可以包括表达调节元件、例如启动子、操纵子、起始密码子、终止密码子、多聚腺苷酸信号、增强子等,并且可以根据目的进行不同的制备。载体的启动子可以是组成型的或可诱导的。由于质粒是目前最常用的载体形式,因此有时术语“质粒”和“载体”在本公开中可互换使用。为了本公开的目的,优选使用质粒载体。可用于此目的的典型质粒载体具有的结构包括(a)复制起点,用于根据宿主细胞有效复制出数个至上百个的质粒载体;以及(b)限制性内切酶位点,可将外源DNA片段插入该位点。即使不存在适当的限制性内切酶位点,也可以根据常规方法使用合成的寡核苷酸衔接子或接头轻松连接载体和外源DNA。
用于过表达根据本公开的基因的载体可以是本领域已知的表达载体。可以在本公开中使用的框架载体可以选自pCDNA3.1、pGP、pEF、pVAX、pUDK、pCK、pQE40、pT7、pET/Rb、pET28a、pET-22b(+)和pGEX,尽管不特别限于此。具体而言,就效果而言,优选使用选自pGP、pEF、pCK、pUDK和pVAX的载体。
在一个具体的示例性实施方案中,本公开的表达载体可以是包括具有图2的酶切图的pGP载体的表达载体。
药物组合物
在另一方面,本公开提供了用于预防或治疗出血紊乱或出血的药物组合物,其包含本公开的用于增加基因表达的组合物和凝血因子VIII基因或其变体基因。
出血紊乱可以是血友病A、由针对凝血因子VIII或凝血因子VIIIa的抑制性抗体诱导或并发的血友病、或血友病B。另外,出血紊乱或出血可以选自新生儿凝血病、严重肝病、血小板减少症、因子V、VII、X或XI的先天性缺乏和具有针对血管性假血友病因子的抑制因子的血管性假血友病。
并且,出血可以是由于高风险外科手术引起的失血、创伤性失血、由骨髓移植引起的失血或由脑出血引起的失血。
用于预防或治疗出血紊乱或出血的药物组合物可以有效地用于预防或治疗出血紊乱或出血,因为它具有优异的治疗效果,即使使用少量的凝血因子VIII基因或其变体基因,体内的基因表达也会增加。
在本公开的药物组合物中,可以以1∶0.5至30(重量/体积)的体积比包含用于增加基因表达的组合物和凝血因子VIII基因或其变体基因。
本公开的药物组合物可以用于基因治疗。
制剂
本公开的药物组合物可以被制备为用于治疗目的的药物制剂。
药物载体和赋形剂以及合适的药物制剂是本领域已知的(例如,″PharmaceuticalFormulation Development of Peptides and Proteins″,Frokjaer等人,Taylor&Francis(2000)或″Handbook of Pharmaceutical Excipients″,3rd edition,Kibbe等人,Pharmaceutical Press(2000))。特别地,本公开的药物组合物可以配制为冻干形式或稳定的液体形式。本公开的组合物可以通过本领域已知的各种方法冷冻干燥。通过添加一种或多于一种药学上可接受的稀释剂例如注射用无菌水或无菌生理盐水来重构冻干制剂。
组合物的制剂通过任何药学上合适的施用方式递送给对象。各种已知的递送系统可用于通过任何方便的途径递送组合物。本公开的组合物主要是全身施用的。对于全身施用,本公开的组合物根据常规方法配制为用于肠胃外(例如静脉内、皮下、肌内、腹膜内、脑内、肺内、鼻内或透皮)递送或肠内(例如口服、阴道或直肠)递送。最优选的施用途径是静脉内和肌内途径。这些制剂可以通过输注连续施用或推注施用。一些制剂包括缓释系统。
本公开的组合物以治疗有效剂量施用给患者,所述剂量足以产生所需效果,其预防或减轻所治疗的病症或适应症的严重性或扩散,而不会达到产生无法忍受的不良反应。确切剂量取决于许多因素,例如适应症、制剂、施用方式等,并且必须通过临床前和临床试验针对每种适应症进行确定。
本公开的药物组合物可以单独施用或与其他治疗剂组合施用。这些试剂可以作为相同药物的一部分掺入。
治疗方法
本公开还涉及用于治疗患有例如血友病A、血友病B或获得性血友病的出血紊乱的对象或患有慢性疾病或肝病的对象的方法。该治疗方法可以包括将有效量的本公开的药物组合物施用于对象的步骤。
根据本公开的一个示例性实施方案,可以以10ng至100mg的剂量施用本公开的基因,例如凝血因子VIII基因或其变体基因。当重复施用凝血因子VIII、其变体或编码其的多核苷酸多于一次时,每次施用的施用剂量可以相同或不同。
在下文中,通过具体实施例更详细地描述本公开内容。然而,这些实施例仅用于更详细地说明本公开,并且对于本领域的普通技术人员明显的是,本公开的范围不受这些实施例的限制。
实施例
材料准备
基因
凝血因子VIII(F8)
由Genscript(美国)合成了SEQ ID 1表示的基因全长凝血因子VIII(F8)。
凝血因子VIII的变体(F8M)
以SEQ ID NO 1表示的全长凝血因子VIII基因为模板,使用引物1和引物2通过聚合酶链反应(PCR)制备SEQ ID NO 4表示的片段1。通过混合2μL模板DNA、1μL的10pmol/μL引物、2.5μL的2.5mM dNTP、1μL的pfu酶混合物(Enzynomics,Korea)、2.5μL的10x缓冲液和50μL的无菌三重蒸馏水进行PCR。溶液在95℃下反应30秒,在60℃下反应30秒,在72℃下反应30秒,共进行40个循环。使用引物3和引物4,以与上述相同的方式通过PCR制备由SEQ ID NO 5表示的片段2。随后,使用引物5和引物6对片段1和片段2进行重叠PCR,从而制备单链凝血因子VIII变体。在重叠PCR中,每种DNA片段添加2μL,反应时间和方法与上述PCR中所述相同。图1示意性地示出了根据本公开的示例性实施方案的设计凝血因子VIII的变体(F8M)的过程。
引物总结于下表1中。
[表1]
凝血因子IX(F9)
由Bionics(韩国)合成了由SEQ ID NO 6表示的全长凝血因子IX的基因(参见GenBank碱基序列FR846239.1)。
质粒(pGP)
在参考Lee等人的文献(Lee Y,et al.Improved expression of vascularendothelial growth factor by naked DNA in mouse skeletal muscles:implicationfor gene therapy of ischemic diseases.Biochem.Biophys.Res.Commun.2000;272(1):230-235)合成pCK质粒后,使用下表2的引物7和引物8,以文献中描述的相同方式进行PCR。使获得的片段与EcoRI酶在37℃下反应1小时后,使用Expin Gel SV(GeneAll,Korea)试剂盒纯化DNA。然后,在使用T4连接酶进行连接30分钟后,将DNA与大肠杆菌一起孵育过夜。第二天,通过mini-prep从菌落中分离出DNA后,获得了SEQ ID NO 7所示的pGP质粒。图2示出了根据本公开的示例性实施方案的pGP载体的酶切图。
[表2]
制备例
包含基因的质粒DNA的制备
将以上制备的每个基因和每个pGP质粒用NheI酶和NotI酶切割1小时,并通过在琼脂糖凝胶上进行电泳来分离片段。使用T4连接酶将分离的片段连接30分钟,然后与大肠杆菌一起孵育过夜。第二天,通过mini-prep从菌落中分离DNA,然后用NheI和NotI消化。在存在卡那霉素的情况下,将克隆的DNA与用限制性酶消化的大肠杆菌上清液在4L烧瓶中孵育过夜。将使用Endofree Giga制备试剂盒(美国Qiagen)产生的质粒DNA用于动物实验。制备的质粒DNA总结在表3中。
[表3]
基因 | 质粒DNA |
凝血因子VIII(F8) | pGP-F8 |
凝血因子VIII变体(F8M) | pGP-F8M |
凝血因子IX(F9) | pGP-F9 |
实施例
实施例:包含用于增加基因表达的组合物和F8M(F8M-MP1和F8M-MP2)的药物组合
物的制备
用于增加基因表达的组合物
参考号为621400210的核壳结构微粒,其平均直径约为2.5μm,由六氟化硫为核,脂质为壳,购自Bracco Imaging Korea(MP1)。另外,从GE Healthcare Korea(MP2)购买了参考号为646300210的核壳结构微粒,其平均直径为约2.4μm至3.6μm,由作为核的全氟丁烷和包含脂质和表面活性剂的壳组成。
包含用于增加基因表达的组合物和F8M的药物组合物的制备
通过分别将15μL的用于增加基因表达的组合物(MP1和MP2)与以上制备的pGP-F8M(70μg/35μL)混合来制备药物组合物F8M-MP1和F8M-MP2。
对比例:包含用于增加基因表达的组合物和F9(F9-MP1和F9-MP2)的药物组合物的
制备
用于增加基因表达的组合物
以与实施例相同的方式制备用于增加基因表达的组合物。
包含用于增加基因表达的组合物和F9的药物组合物的制备
通过分别将15μL的用于增加基因表达的组合物(MP1和MP2)与以上制备的pGP-F9(70μg/35μL)混合来制备药物组合物F9-MP1和F9-MP2。
对照组
用于增加基因表达的组合物
根据制造商的手册,通过将128μL体内JetPEI(美国,Polyplus)与2mL的5%葡萄糖混合制备混悬剂(对应于增加基因表达的组合物)。
包含用于增加基因表达的组合物和每种基因的药物组合物的制备
通过将15μL的组合物与以上制备的pGP-F8M(70μg/35μL)混合来制备药物组合物F8M-JetPEI。
测试实施例
测试实施例:蛋白质在小鼠中的表达水平
将根据对照组、实施例和对比例的每种药物组合物以75μg/50μL/腿注射到C57bl/6小鼠的下小腿肌肉中。
施用后第7天,处死小鼠并切下注射区域的肌肉。然后,使用液氮和蛋白质提取试剂盒(Cell Biolabs,美国)研磨切下的肌肉后,分离出总蛋白质。使用DC蛋白质测定试剂盒(美国Bio-Rad实验室)测量分离的总蛋白质的量。
为了测量F8蛋白,使用ELISA试剂盒(Stago Asserchrom VIII:Ag,法国)测量对于相同量的蛋白的每个基因的表达水平。结果如图3所示。
从图3中可以看出,与施用仅基因或对照组的组合物相比,施用本公开的药物组合物(实施例)的结果是统计学上显著更高的表达水平。具体地,与施用仅基因的组(pGP-F8M)相比,施用实施例的F8M-MP1的组表现出表达水平提高44%,并且与施用仅基因的组相比,施用F8M-MP2的组表现出表达水平提高116%。
为了测量F9蛋白,使用ELISA试剂盒(Abcam,USA)测量对于相同量的蛋白的每个基因的表达水平。结果如图4所示。
由图4可以看出,与施用仅基因相比,施用对比例的组合物几乎不使基因表达增加。
也就是说,从图3和图4中可以看出,与施用仅基因或施用对照组或对比例的组合物相比,施用本公开的组合物(实施例)获得明显更高的表达水平。
尽管利用上述特定示例性实施例示出了本公开,但是在不脱离本公开的主题和范围的情况下,可以对其进行各种修改或改变。另外,在本公开的主题内的这种修改或改变被包括在所附权利要求的范围内。
Claims (3)
1.一种用于增加凝血因子VIII基因表达的药物组合物,其包含核壳结构的微粒和凝血因子VIII的变体基因作为活性成分,其中
核是作为生物相容性气体的六氟化硫或全氟丁烷,壳是磷脂,
凝血因子VIII的变体由SEQ ID NO 3表示的氨基酸序列组成。
2.根据权利要求1所述的用于增加凝血因子VIII基因表达的药物组合物,其中磷脂选自磷脂酰胆碱衍生物、磷脂酰乙醇胺衍生物、磷脂酰丝氨酸衍生物、二乙酰化磷脂、L-α-二油醇磷脂酰乙醇胺、磷脂酸、磷脂酰甘油、磷脂酰肌醇、溶血磷脂酰胆碱、鞘磷脂、聚乙二醇化磷脂、蛋黄卵磷脂、大豆卵磷脂和氢化磷脂。
3.根据权利要求1所述的用于增加凝血因子VIII基因表达的药物组合物,其中所述药物组合物使凝血因子VIII基因表达增加30%或大于30%。
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AU2019217187A1 (en) | 2020-10-01 |
JP2021513544A (ja) | 2021-05-27 |
KR20190098060A (ko) | 2019-08-21 |
US20210113668A1 (en) | 2021-04-22 |
CN112218622A (zh) | 2021-01-12 |
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JP7236446B2 (ja) | 2023-03-09 |
WO2019156540A1 (ko) | 2019-08-15 |
JP7301860B2 (ja) | 2023-07-03 |
WO2019156541A1 (ko) | 2019-08-15 |
KR102230593B1 (ko) | 2021-03-23 |
AU2019217187B2 (en) | 2022-03-17 |
KR20190098073A (ko) | 2019-08-21 |
CA3093538C (en) | 2024-02-27 |
CA3093538A1 (en) | 2019-08-15 |
EP3753550A4 (en) | 2021-12-22 |
EP3753551A4 (en) | 2021-12-22 |
CN112203644A (zh) | 2021-01-08 |
KR102245539B1 (ko) | 2021-04-29 |
AU2019217186B2 (en) | 2022-03-17 |
US20230390211A1 (en) | 2023-12-07 |
JP2021513545A (ja) | 2021-05-27 |
US20210113480A1 (en) | 2021-04-22 |
EP3753551A1 (en) | 2020-12-23 |
AU2019217186A1 (en) | 2020-10-01 |
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