CN117460542A - 包含用于将寡核苷酸递送至靶细胞内的肽-脂质缀合物的纳米颗粒以及包含其的药物组合物 - Google Patents
包含用于将寡核苷酸递送至靶细胞内的肽-脂质缀合物的纳米颗粒以及包含其的药物组合物 Download PDFInfo
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Abstract
本发明涉及一种用于将寡核苷酸递送至细胞内用于治疗用途的载体。为了在细胞中抑制靶蛋白或促进靶蛋白的表达,制备了肽‑脂质缀合物,并提供了由包含寡核苷酸的肽‑脂质缀合物组成的纳米颗粒和包含该纳米颗粒的药物组合物。证实了通过使用根据本发明的由肽‑脂质缀合物组成的纳米颗粒,寡核苷酸被有效地递送至细胞内。此外,通过证实寡核苷酸降低了致癌蛋白的表达并且在发生癌症的动物模型中表现出抗癌效果,发现由肽‑脂质缀合物组成的纳米颗粒在递送寡核苷酸方面是有效的。
Description
技术领域
本发明涉及包含载有寡核苷酸的肽-脂质缀合物的纳米颗粒,用于抑制细胞中的靶蛋白或促进靶蛋白的表达,以及包含其的药物组合物。
背景技术
开发了寡核苷酸,特别是siRNA和mRNA形式的寡核苷酸,作为控制导致癌症和传染病的基因的治疗剂。由于以mRNA的形式注射以诱导免疫反应的含有编码COVID 19病毒刺突蛋白的基因的RNA疫苗最近已被FDA批准,mRNA的应用引起了极大的关注。mRNA是无毒的,因为它是由体内的mRNA人工合成的。然而,siRNA和mRNA在体内被核酸酶快速降解,带负电荷,并且不能靶向特定的病变。此外,尽管siRNA和mRNA只有在细胞质中起作用时才能发挥其作用,但由于其带负电荷和较大的尺寸,它们自身不能透过细胞膜。因此,为了克服这些限制,需要能够将寡核苷酸稳定递送至细胞内的载体。
最广泛使用的载体是脂质纳米颗粒,并且使用各种阳离子脂质的寡核苷酸载体是已知的(US2006/0083780、US2006/0240554、US2008/0020058、US2009/0263407、US2009/0285881、以及PCT专利公开号WO 2009/086558、WO 2009/127060、WO 2009/132131、WO2010/042877、WO 2010/054384、WO 2010/054401、WO 2010/054405、WO 2010/054406和WO2010/105209)。传统的阳离子脂质诸如CLinDMA和DLinDMA已经用于将寡核苷酸递送至肝脏,但是已知在高剂量下具有非最佳的递送效率并具有肝毒性。
脂质纳米颗粒是使用阳离子脂质、胆固醇和聚乙二醇(PEG)制备的。阳离子脂质使得寡核苷酸穿过细胞膜,胆固醇使得寡核苷酸保持其形状,以及PEG使得脂质纳米颗粒的长循环。然而,据报道PEG会生成抗PEG的抗体,从而诱导过敏反应。因此,需要能够安全有效地将寡核苷酸递送至细胞内的新型寡核苷酸载体。
因此,为了产生包含能够安全有效地将寡核苷酸递送至细胞内的肽-脂质缀合物的纳米颗粒,本发明人使用包含用于识别靶细胞表面的序列和具有细胞穿透功能、促进从内体中逃逸并与寡核苷酸结合的序列的肽来产生肽-脂质缀合物,并且发现,当使用该包含该肽-脂质缀合物的纳米颗粒时,寡核苷酸被有效地递送至细胞内,致癌蛋白的表达被所装载的寡核苷酸减少,并且在诱发有癌症的动物模型中获得了抗癌效果。基于此,完成了本发明。
在该背景技术部分中公开的信息仅仅是为了增强对本发明的背景的理解而提供的,因此它可能不包括形成对本领域技术人员来说已经显而易见的现有技术的信息。
发明内容
本发明的一个目的是提供一种能够安全有效地将寡核苷酸递送至细胞内的肽-脂质缀合物。
本发明的另一个目的是提供用于将寡核苷酸递送至细胞内的纳米颗粒,该纳米颗粒中肽-脂质缀合物和寡核苷酸彼此结合。
本发明的又一个目的是提供一种包含该纳米颗粒的药物组合物。
本发明的再一个目的是提供一种预防或治疗疾病的方法,包括向受试者给药该纳米颗粒。
本发明的还有一个目的是提供纳米颗粒用于预防或治疗疾病的用途。
本发明的还有另一个目的是提供纳米颗粒在制备用于预防或治疗疾病的药物中的用途。
为了实现上述目的,本发明提供了一种肽-脂质缀合物,其具有下式1作为基本单元的肽-脂质缀合物:
[式1]
A-B-C-D-E
其中,A是具有12至20个碳原子的脂肪酸或阳离子脂质;
B是促进内体逃逸的肽;
C是RNA结合肽;
D是具有细胞穿透功能的肽;以及
E是识别靶细胞表面的肽。
此外,本发明提供了其中肽-脂质缀合物和寡核苷酸彼此结合的纳米颗粒。
此外,本发明提供了一种用于将寡核苷酸递送至细胞内的组合物,其包含其中肽-脂质缀合物和寡核苷酸彼此结合的纳米颗粒。
此外,本发明提供了一种包含其中肽-脂质缀合物和寡核苷酸彼此结合的纳米颗粒的药物组合物。
此外,本发明提供了一种预防或治疗疾病的方法,包括向受试者给药该纳米颗粒。
此外,本发明提供了纳米颗粒用于预防或治疗疾病的用途。
此外,本发明提供了纳米颗粒在制备用于预防或治疗疾病的药物中的用途。
附图说明
图1是说明在寡核苷酸与肽-脂质缀合物的RNA结合肽结合后,通过自组装形成纳米颗粒的过程的示意图。
图2是说明其中使用化学交联剂将具有识别靶细胞表面和细胞穿透功能的肽与mRNA结合的构型的示意图。
图3A示出了包含mRNA和肽-脂质缀合物的纳米颗粒在琼脂糖凝胶上以各自的反应比率、通过TEM形成的纳米颗粒以及纳米颗粒的大小分布进行电泳的结果。图3B示出了包含siRNA和肽-脂质缀合物的纳米颗粒在琼脂糖凝胶上以各自的反应比率、通过TEM形成的纳米颗粒以及纳米颗粒的大小分布进行电泳的结果。
图4A示出了当用单独包含mRNA的纳米颗粒以及包含mRNA和肽-脂质缀合物的组合的纳米颗粒处理细胞时,穿透到细胞内的程度的观察结果,并且图4B示出了了当用单独包含siRNA的纳米颗粒以及包含siRNA和肽-脂质缀合物的组合的纳米颗粒处理细胞时,穿透到细胞内的程度的观察结果。
图5是示出用包含mRNA和肽-脂质缀合物的纳米颗粒进行用以检测细胞中蛋白质表达水平的蛋白质印迹结果的图。
图6示出了蛋白质印迹的结果,其通过mRNA中表达的蛋白质示出了用包含mRNA和肽-脂质缀合物的纳米颗粒处理细胞后活性KRAS的减少和亚信号pERK的减少。
图7是示出细胞中KRAS mRNA的量被包含KRAS siRNA和肽-脂质缀合物的纳米颗粒改变的图。
图8示出了在具有H358诱导的肺癌的异种移植物模型中检测包含mRNA和肽-脂质复合物的纳米颗粒的抗癌效果的结果。
具体实施方式
除非另有定义,否则本文使用的所有技术和科学术语具有与本发明所属领域的技术人员所理解的相同含义。通常,本文所用的术语是本领域公知的,并且是通常使用的。
在本发明中,产生了包含能够安全有效地将功能性寡核苷酸递送至细胞内的肽-脂质缀合物的纳米颗粒。首先,使用包含用于识别靶细胞表面的序列和具有细胞穿透功能、促进从内体中逃逸并与寡核苷酸结合的序列的肽产生肽-脂质缀合物,并且通过将寡核苷酸结合到肽-脂质缀合物上而产生纳米颗粒。发现当使用该包含肽-脂质缀合物的纳米颗粒时,寡核苷酸被有效地递送至细胞内,致癌蛋白的表达被所装载的寡核苷酸减少,并且在诱发有癌症的动物模型中获得了抗癌效果。
因此,在一个方面,本发明涉及一种具有下式1的肽-脂质缀合物作为基本单元的肽-脂质缀合物:
[式1]
A-B-C-D-E
其中,A是具有12至20个碳原子的脂肪酸或阳离子脂质;
B是促进内体逃逸的肽;
C是RNA结合肽;
D是具有细胞穿透功能的肽;以及
E是识别靶细胞表面的肽。
在本发明中,
A是脂质,更具体地,是具有12至20个碳原子的饱和脂肪酸、不饱和脂肪酸或阳离子脂质。所述阳离子脂质包括但不限于3β-[N-(N',N'-二甲基氨基乙烷)氨基甲酰基]胆固醇(DC-Chol);1,2-二油酰基-3-三甲基铵-丙烷(DOTAP);1,2-二油酰基-3-二甲基铵-丙烷(DODAP);双十烷基二甲基溴化铵(DDAB);1,2-二月桂酰基-sn-甘油基-3-乙基磷酸胆碱氯化物(DL-EPC);N-[1-(2,3-二油酰氧基)丙基]-N-N-N-三甲基氯化铵(DOTMA);N-[1-(2,3-二油酰氧基)丙基]-N-N-N-二甲基氯化铵(DODMA);1,2-二月桂酰基-sn-甘油基-3-乙基磷酸胆碱氯化物(DOTMA);N,N-双十二烷基-N,N-二甲基氯化铵(DODAC);N-(1-(2,3-二油基氧基)丙基)-N-2-(精胺甲酰胺基)乙基)-N,N-二甲基三氟乙酸铵盐(DOSPA);1,2-二肉豆蔻基氧基丙基-3-二甲基羟乙基溴化铵(DMRIE);二十八烷基酰胺基甘氨酰精胺(DOGS);与阳离子修饰剂缀合的中性脂质;及其组合。此外,许多阳离子脂质可用于市售制剂中,诸如,Lipofectin(GIBCO)、Lipofectamine(GIBCO)和Transfectam(Promega)。
在本发明中,脂质部分可以以式1的0至约60.0摩尔百分比,或制剂中脂质的约5.0至约50.0摩尔百分比范围内的浓度存在。
在本发明中,B可以包含促进内体逃逸的肽序列。C可以包含RNA结合的肽序列。D可以包含具有细胞穿透功能的肽序列。E可以包含识别靶细胞表面的肽序列。
在本发明中,B是由4至12个组氨酸组成的肽,D是选自由4至12个精氨酸、4至12个赖氨酸和2个半胱氨酸或其组合组成的组的肽,但不限于此。
在本发明中,C可以是由SEQ ID NO:1至SEQ ID NO:15的任一氨基酸序列表示的肽,但不限于此。
SEQ ID No.1:LKKLLKLLKKLLKLAG
SEQ ID No.2:IKKILIKIIKKLIKLAG
SEQ ID No.3:LRRLLRLLRRLLRLAG
SEQ ID No.4:LKKLLKLLOrnKLLDapLAG
SEQ ID No.5:LRRLLRLLOrnRLLDapLAG
SEQ ID No.6:LRKIIRLIOrnKLLDapLAG
SEQ ID No.7:LKKLLKLLOrnKLLKLAG
SEQ ID No.8:WKKLLKLLKKLLKLAG
SEQ ID No.9:WRRLLRLLRRLLRLAG
SEQ ID No.10:WRKLLRLLKKLLKLAG
SEQ ID No.11:LKKLLDabLLKKLLKWAG
SEQ ID No.12:LRRLLDabLLRRLLRWAG
SEQ ID No.13:LKRLIDabIIKKLIKWAG
SEQ ID No.14:LKKLLKWLOrnKLLDapLAG
SEQ ID No.15:LRRLLRWLOrnRLLDabLAG
Orn=鸟氨酸,Dab=1,4-二氨基丁酸,Dap=1,3-二氨基丙酸。
在本发明中,E是与靶细胞表面结合的序列,并且可以通过噬菌体展示技术而被最新发现和应用。
在本发明的一个实施方式中,E可以是由SEQ ID NO:16的氨基酸序列代表的肽,但不限于此。
SEQ ID NO:16:CAIYPRH
在本发明中,式1的A-B可以通过酰胺键连接,并且A-B的酰胺键可以是基于化学合成的。
在本发明中,式1的B-C-D-E可以通过化学合成或重组表达方法形成。
在本发明的一个实施方式中,B-C-D-E可以通过基于固相肽合成的合成来形成,然后可以化学形成与A连接的酰胺键。
另一方面,本发明涉及其中肽-脂质缀合物和寡核苷酸彼此结合的纳米颗粒。
在本发明中,寡核苷酸可以是DNA、siRNA、miRNA或mRNA,并且优选具有抑制或增加靶基因表达的功能。所述siRNA可以具有与编码肿瘤诱导蛋白或致病蛋白的核苷酸序列互补的核苷酸序列,但不限于此。
在本发明的一个实施方式中,突变KRAS的mRNA用于抑制突变KRAS的表达。具有编码降解KRAS的蛋白质的序列的mRNA可以是具有SEQ ID NO:17和SEQ ID NO:18的核苷酸序列的mRNA,但不限于此。
SEQ ID NO:17:编码降解KRAS的蛋白质的mRNA序列1
5'帽-5'UTR-
AUGGAAGUCCAAUUGUUGGAAUCUGGGGGUGGGCUUGUCCAACCUGGUGGUUCACUCCGUCUGAGCUGCGCCGCUUCCGGAUUCACUUUCUCCACCUUUUCUAUGAAUUGGGUUCGCCAGGCCCCAGGCAAGGGACUUGAGUGGGUUAGCUAUAUCAGCAGGACCAGUAAAACCAUCUACUACGCCGACAGCGUUAAGGGAAGGUUCACUAUAUCCCGGGACAAUUCUAAGAAUACUUUGUACUUGCAAAUGAACUCUUUGCGGGCAGAAGAUACUGCUGUCUACUAUUGUGCACGAGGUAGGUUUUUUGAUUAUUGGGGUCAGGGGACCCUCGUAACCGUUUCCAGUGGGGGCAGCGAGGGUAAGAGCUCCGGUUCAGGGUCAGAAUCUAAAAGCACUGGUGGCUCCGAUAUACAAAUGACCCAGAGUCCAAGCUCUCUUUCAGCCAGCGUGGGAGACAGGGUGACCAUCACCUGUCGAGCAUCUCAAUCCAUUUCAUCAUACCUCAAUUGGUAUCAACAGAAGCCUGGGGAAGCACCAAAGUUGCUUAUUUAUUCUGCCAGCGUACUGCAAAGUGGCGUACCUUCCCGCUUCUCCGGGUCUGGGAGUGGCACUGAUUUUACCCUCACAAUUAGCAGCCUGCAACCCGAGGACUUCGCUACAUACUACUGCCAACAAAGUGUAAUGAUACCCAUGACAUUCGGGCAAGGCACAAAAGUCGAGAUAAAGGGAAGUGGAGGAGGUGGCUCUAUGCCCCGCCGCGCUGAGAACUGGGAUGAGGCCGAAGUUGGUGCUGAAGAGGCCGGAGUUGAAGAAUAUGGGCCAGAAGAAGAUGGCGGUGAGGAAAGCGGGGCUGAAGAGAGCGGUCCAGAGGAGUCUGGUCCAGAGGAACUUGGAGCUGAGGAGGAGAUGGAGGCCGGACGUCCACGUCCUGUCCUGCGAUCAGUUAACUCAAGGGAGCCCUCACAGGUUAUCUUUUGUAAUCGAAGCCCUCGCGUGGUUUUGCCCGUCUGGCUCAACUUCGAUGGUGAACCCCAACCAUACCCAACCCUCCCCCCUGGUACCGGGCGGAGGAUUCAUUCCUAUCGGGGACACCUUUGGCUCUUCCGAGACGCUGGGACUCACGAUGGCCUGUUGGUCAAUCAGACCGAACUGUUUGUGCCCAGCCUGAACGUAGACGGGCAGCCAAUUUUCGCAAACAUAACACUGCCCGUAUAUACAUUGAAAGAAAGGUGUCUUCAGGUGGUACGCAGUCUGGUUAAACCAGAAAACUAUAGGCGUCUGGAUAUCGUGCGCAGCCUCUAUGAGGACCUUGAAGACCACCCUAACGUGCAAAAGGACCUCGAGCGGCUCACUCAGGAGCGCAUAGCUCAUCAGCGCAUGGGAGAUGAAAACCUUUACUUUCAGGGUGGGAGCGGCGGCAGCGGCGGGAGUCAUCAUCACCAUCAUCAUCACCACUGA-3'UTR-3'多聚A尾
SEQ ID NO:18:编码降解KRAS的蛋白质的mRNA序列2
5'帽-5'UTR
AUGGAAGUGCAACUUCUCGAAUCUGGAGGGGGGUUGGUACAACCCGGCGGCAGCUUGCGCUUGUCCUGUGCUGCCAGUGGGUUCACAUUUUCUACAUUCUCUAUGAACUGGGUUAGACAAGCCCCUGGCAAAGGGCUGGAAUGGGUGAGCUAUAUAAGUCGCACCUCCAAAACUAUUUACUAUGCAGAUAGUGUCAAGGGACGAUUUACUAUCAGCAGAGAUAACUCUAAGAACACUCUGUAUCUCCAGAUGAAUUCCCUUCGAGCCGAAGACACCGCAGUAUAUUACUGUGCUAGAGGGCGCUUCUUCGACUACUGGGGGCAAGGCACUCUGGUAACUGUCAGUAGCGGGGGUUCUGAGGGCAAAAGUUCAGGAUCUGGGUCCGAGUCUAAGUCAACUGGCGGGAGCGACAUACAAAUGACUCAGAGCCCCAGUAGCCUGAGCGCCUCCGUUGGUGACAGAGUCACUAUUACAUGUCGGGCAUCUCAAUCUAUUUCCUCUUAUCUUAACUGGUAUCAGCAAAAGCCAGGCGAAGCCCCCAAACUCCUGAUAUACUCCGCUAGUGUUCUUCAGAGUGGCGUUCCUAGUCGCUUCAGCGGAUCCGGAAGUGGCACUGAUUUUACACUUACAAUUAGUUCCUUGCAGCCUGAAGAUUUUGCCACAUACUACUGUCAGCAAUCAGUCAUGAUUCCCAUGACCUUUGGGCAAGGGACCAAAGUUGAGAUCAAAGGCUCCGGAGGAGGAGGGUCUAUGGAAAACAGAUGGCAAGUUAUGAUCGUUUGGCAAGUAGAUCGUAUGCGAAUACGCACAUGGAAAUCACUGGUAAAGCACCACAUGUAUGUGUCAGGCAAAGCACGAGGAUGGUUUUAUCGUCAUCACUAUGAGUCACCACAUCCCCGCAUCAGCUCUGAGGUGCACAUACCACUCGGUGAUGCUCGUCUGGUCAUAACUACCUACUGGGGCCUCCACACUGGUGAAAGAGAUUGGCACCUCGGCCAAGGAGUAAGUAUAGAAUGGCGCAAAAAGCGUUAUUCAACCCAAGUGGAUCCUGAGUUGGCAGAUCAGUUGAUCCACUUGUAUUACUUUGACUGCUUCAGCGAUAGUGCUAUUCGGAAGGCCCUCCUCGGGCACAUUGUAAGCCCACGGUGCGAGUAUCAAGCUGGUCAUAACAAGGUUGGUAGCCUCCAGUACUUGGCUUUGGCUGCACUUAUAACACCCAAAAAGAUAAAACCUCCACUCCCCUCAGUGACUAAACUCACUGAGGAUCGUUGGAACAAACCCCAGAAAACCAAGGGGCACAGGGGUUCACACACCAUGAAUGGCCACGAGAAUUUGUAUUUCCAAGGGGGAUCCGGGGGCAGUGGAGGGUCUCACCAUCAUCACCACCACCACCAUUGA-3'UTR-3'多聚A尾
在本发明的一个实施方式中,KRAS的表达通过使用siRNA而被抑制,并且siRNA可以具有SEQ ID NO:19和SEQ ID NO:20的核苷酸序列,但不限于此。
SEQ ID NO:19和SEQ ID NO:20:抑制KRAS的表达的siRNA序列
SEQ ID NO:19(正义):CAGCUAAUUCAGAAUCAUU
SEQ ID NO:20(反义):AAUGAUUCUGAAUUAGCUG
在本发明中,siRNA可以是天然的或经化学修饰的siRNA,或者使用在5'端被巯基或胺基取代的siRNA,与细胞穿透和靶识别肽中的半胱氨酸的巯基化学结合。
在本发明中,mRNA可以编码需要增加表达的靶蛋白、重组嵌合蛋白或病毒抗原。mRNA可以是具有3'帽和5'多聚A尾的天然mRNA,或者可以在巯基或胺基结合到5'端后,使用化学交联剂结合到细胞穿透和靶识别肽中的半胱氨酸的巯基上。
在本发明中,在制备式1之后,寡核苷酸可以键合至式1,特别是式1中的C,并静置预定的时间以允许通过自组装形成纳米颗粒。纳米颗粒的大小可以是10至200nm。
在本发明中,siRNA或mRNA与肽-脂质缀合物的分子量比可以是1∶1至1∶100。
在本发明中,siRNA和mRNA可以用作抗癌药物,但不限于此。
又一方面,本发明涉及一种用于将寡核苷酸递送至细胞内的组合物,其包含其中肽-脂质缀合物和寡核苷酸彼此结合的纳米颗粒。
再一方面,本发明涉及一种包含其中肽-脂质缀合物和寡核苷酸彼此结合的纳米颗粒的药物组合物。
在本发明中,药物组合物可用于治疗或预防癌症或炎性疾病,并且癌症包括鳞状细胞癌、基底细胞癌、腺癌、肝细胞癌、肾细胞癌、膀胱癌、肠癌、乳腺癌、宫颈癌、子宫癌、结肠癌、食道癌、头癌、肾癌、肝癌、肺癌、卵巢癌、胰腺癌、前列腺癌、胃癌、白血病、良性和恶性淋巴瘤,特别是伯基特淋巴瘤和非霍奇金淋巴瘤、良性和恶性黑色素瘤、骨髓增生性疾病、肉瘤包括尤文氏肉瘤、血管肉瘤、卡波西肉瘤、脂肪肉瘤、肌瘤、神经上皮肉瘤、滑膜肉瘤、神经肉瘤、星形细胞瘤、少突胶质细胞瘤、脑细胞瘤、胶质母细胞瘤、神经母细胞瘤、神经节细胞瘤、神经母细胞瘤、松果体瘤、脑膜瘤、脑膜肉瘤、神经纤维瘤和神经鞘瘤、睾丸癌、甲状腺癌、癌肉瘤、霍金斯病、维尔姆斯瘤或畸胎癌,但不限于此。
在本发明中,所述药物组合物用于治疗或预防哮喘、自身免疫性疾病、类风湿性关节炎、多发性硬化、睫状病、腭裂、糖尿病、心脏病、高血压、炎性肠病、精神发育迟滞、情绪障碍、肥胖症、屈光不正、不孕症、安格尔曼综合征、卡纳万氏病、慢性消化系统疾病、恰克-马利-杜斯氏(CMT)病、囊性纤维化、杜氏肌营养不良、血色素沉着症、血友病、克氏综合征、神经纤维瘤病、苯丙酮尿症、常染色体显性多囊肿瘤(PKD1或PKD2)、普拉德-威利综合征、镰状细胞贫血、泰-萨克斯病、特纳综合征、HIV感染或HCV感染,但不限于此。
根据本发明,药物组合物以选自由以下组成的任一种制剂来制备:注射剂、口服制剂、贴剂、溶液、胶囊、颗粒剂、片剂、粉剂、喷雾剂、软膏剂、凝胶剂、粘膜制剂和栓剂,但不限于此。这些制剂可以通过本领域用于制剂的常规方法或在Remington's PharmaceuticalScience(最新版),Mack Publishing Company,Easton PA中公开的方法制备,并且可以根据每种疾病或成分制备成各种制剂。然而,该描述是说明性的,适用于本发明的制剂不限于上述那些。
在本发明中,药物组合物还可以包含可接受的佐剂,并且佐剂可以是,例如,载体。药学上可接受的载体可以是盐水、无菌水、林格氏液、缓冲盐水、葡萄糖溶液、麦芽糖糊精溶液、甘油、乙醇或这些成分中的一种或多种的混合物,并且如果需要,可以进一步包含其他常规添加剂,例如抗氧化剂、缓冲剂或抑菌剂。此外,可以另外加入稀释剂、分散剂、表面活性剂、粘合剂和润滑剂来制备注射制剂,诸如水性溶液、悬浮液或乳液、丸剂、胶囊、颗粒或片剂。然而,该描述是说明性的,可用于本发明的佐剂或载体不限于上述那些。
还有一方面,本发明涉及一种预防或治疗疾病的方法,包括向受试者给药该纳米颗粒。
还有另一方面,本发明涉及纳米颗粒用于预防或治疗疾病的用途。
还有又一方面,本发明涉及纳米颗粒在制备用于预防或治疗疾病的药物中的用途。
在本发明中,疾病可以是癌症、炎性疾病、哮喘、自身免疫性疾病、类风湿性关节炎、多发性硬化、睫状病、腭裂、糖尿病、心脏病、高血压、炎性肠病、精神发育迟滞、情绪障碍、肥胖症、屈光不正、不孕症、安格尔曼综合征、卡纳万氏病、慢性消化系统疾病、恰克-马利-杜斯氏(CMT)病、囊性纤维化、杜氏肌营养不良、血色素沉着症、血友病、克氏综合征、神经纤维瘤病、苯丙酮尿症、常染色体显性多囊肿瘤(PKD1或PKD2)、普拉德-威利综合征、镰状细胞贫血、泰-萨克斯病、特纳综合征、HIV感染或HCV感染,但不限于此。
在本发明中,包含根据本发明的纳米颗粒的方法、用途和用法涉及包含纳米颗粒的药物组合物,并且省略了与上述纳米颗粒和药物组合物重复的描述。
在下文中,将参考实施例更详细地描述本发明。然而,对于本领域技术人员来说,显然这些实施例仅用于说明本发明,而不应被解释为限制本发明的范围。
实施例1:肽和肽-脂质缀合物的合成
合成所需的氨基酸和试剂购自GL Biochem和Sigma-Aldrich。使用肽合成仪,通过Fmoc固态化学合成方法从C-末端合成肽。也就是说,使用结合有Fmoc-(9-芴基甲氧羰基)作为封闭基团的rink树脂(0.075mmol/g,100至200目,1% DVB交联)来合成肽,将50mg的rink树脂注入肽合成仪中,用DMF溶胀树脂,然后使用20%哌啶/DMF溶液除去Fmoc-基团。从C-末端开始,按顺序以5、10和5当量加入0.5M氨基酸溶液(溶剂:二甲基甲酰胺,DMF)、1.0MDIPEA(溶剂:二甲基甲酰胺&n-甲基吡咯烷酮,DMF&NMP)和0.5M HBTU(溶剂:二甲基甲酰胺,DMF),并在氮气流下反应1至2小时。在每个去保护和偶联步骤后,用DMF和异丙醇洗涤两次。在偶联最后一个氨基酸后,进行去保护以除去Fmoc-基团。从N-末端最后一个氨基酸上除去Fmoc保护基团后,在DMF存在下,通过加入DIPEA(10当量)、HBTU(5当量)和棕榈酸(5当量)进行合成。使用卡瑟测试溶液(Kaiser test solution)进行反应,直到获得阴性结果。反应完成后,用DMF和MeOH洗涤树脂,并在真空烘箱中干燥。以每1g的树脂20ml的速率加入三氟乙酸(TFA)裂解混合物(cocktail),振荡3小时,并通过过滤分离其中溶解了树脂和肽的混合物。使用旋转蒸发器除去经过滤的溶液后,加入冷乙醚或直接将过量的冷乙醚加入溶解有肽-脂质缀合物的TFA混合物溶液中,以使肽-脂质缀合物结晶成固相,并通过离心分离肽-脂质缀合物。此时,通过用乙醚洗涤几次并离心,完全除去TFA混合物。将如此获得的肽-脂质缀合物溶解在蒸馏水中并冻干。冻干完成后,通过高效液相色谱(岛津公司(Shimadzu),日本)进行分离和纯化。使用直径为4.6mm的C18柱,使0.1% TFA/H2O和0.092% TFA/乙腈以1ml/min的流量从0%到60%流动30分钟。此时,紫外检测器的波长为220nm。在与溶剂和检测波长相同的条件下,使用直径为2.2cm的柱以20ml/min的流速进行纯化。通过质谱测定纯化肽的分子量。
实施例2:通过肽-脂质缀合物和mRNA的缀合产生纳米颗粒
将编码降解抑制KRAS表达的突变KRAS和siRNA(SEQ ID NO:19和SEQ ID NO:20)的蛋白质的基因的mRNA(SEQ ID NO:17或SEQ ID NO:18)与实施例1中制备的肽-脂质缀合物结合,以产生纳米颗粒。
对于共价键合,使用化学交联剂将被mRNA的5'端被巯基或胺基取代的siRNA与细胞穿透和靶识别肽中的半胱氨酸的巯基结合,并通过自组装产生纳米颗粒。
将100μg的肽-脂质缀合物溶解在100μL的作为溶剂的90% EtOH和10%10mM柠檬酸钠缓冲液的混合物中。将mRNA和siRNA各100μg溶解在100μL的10mM柠檬酸钠缓冲液(RNase,无酶无菌&PCR认证水(RNase,DNase Free&PCR certified water)(科技与创新公司(Tech&Innovation),BWA-8000))中。将mRNA或siRNA溶液和肽-脂质缀合物以1∶10、1∶20、1∶50和1∶100的比例少量缓慢混合。
所形成的纳米颗粒通过凝胶阻滞试验进行鉴定。将RNA和肽-脂质缀合物以1:10、1:20、1:50和1:100的比例上样到2%(w/v)琼脂糖凝胶上,上样量为0.1μg RNA,然后进行电泳。观察到单个mRNA和siRNA到正(+)电荷减少(图3),而肽-脂质纳米颗粒从1∶10的比例没有减少。这意味着颗粒表面被肽-脂质变成中性。此外,使用透射电子显微镜(TEM,JEM-1230,JEOL)观察包含mRNA和肽-脂质缀合物(1∶10)的纳米颗粒。使用动态光散射(DLS,马尔文仪器有限公司(Malvern Instruments,Ltd.),UK)测量颗粒大小(图3)。对于mRNA来说,具有100nm或更大的大小的颗粒更多,对于siRNA来说,具有大约100nm大小的颗粒更多。
实施例3:对于将纳米颗粒递送至细胞内的确认
将实施例2中产生的纳米颗粒施加到H358细胞的培养液中,并且观察到纳米颗粒被递送至细胞内。使用荧光(Cy5.5)标记的KRAS降解蛋白mRNA和荧光(Cy5.5)标记的KRASsiRNA形成肽-脂质缀合物,并且在共聚焦显微镜下观察到纳米颗粒被递送至细胞内(图4)。
为了测试包含mRNA和肽-脂质缀合物的纳米颗粒的细胞渗透性,用Cy5.5(英杰公司(Invitrogen))标记mRNA或siRNA,并且以与实施例2中相同的方式生产纳米颗粒。以1×104的密度接种H358(人肺癌细胞,CRL-5807,ATCC)。24小时后,用含有Cy5.5-mRNA和肽-脂质缀合物或含有Cy5.5-siRNA和肽-脂质缀合物的100nM纳米颗粒处理培养基1小时。然后,用10%中性福尔马林溶液固定细胞,并用共聚焦差示扫描显微镜测量。
结果,在H358细胞的细胞质中观察到显著增加的纳米颗粒的量(图4)。因此,发现包含载有mRNA或siRNA的肽-脂质缀合物的纳米颗粒可以有效地穿透细胞。
实施例4:通过递送至细胞内的mRNA对KARS降解蛋白的表达的鉴定
此外,通过蛋白质印迹法检测包含mRNA和肽-脂质缀合物的纳米颗粒在细胞中表达的蛋白质的量(图5)。
将H358细胞以70%的密度接种在6孔板中。16小时后,用含0.5% FBS的DMEM进行过夜细胞饥饿处理。用含有纳米颗粒的培养基处理细胞16小时,所述纳米颗粒包含浓度为10nM和20nM的mRNA和肽-脂质缀合物。将10nM的混合有lipofectamine的mRNA用作对照。使用含有酶抑制和磷酸酶抑制剂的RIPA裂解缓冲液(25mM Tris HCl pH 7.6,150mM NaCl,1%NP-40,1%脱氧胆酸钠,0.1%SDS)裂解蛋白质。通过BCA蛋白测定法(23227,赛默飞世尔(Thermo Scientific))对蛋白质进行定量,通过蛋白质印迹法测定KRAS降解蛋白和肌动蛋白的表达水平。通过将相同量的样品与大小标志物一起上样在8% SDS PAGE凝胶上,进行约2小时的电泳,然后将其转移到硝酸纤维素膜上,进行蛋白质印迹法。用5%脱脂乳封闭转移的膜1小时,并与一抗(GST,Abcam,ab19256)以1∶1000的比例反应过夜。然后,用含有0.1%吐温-20的TBST洗涤膜,并与HRP连接的二抗(抗兔,A90-116P,贝瑟实验室(BETHYLLab.);抗鼠,A120-101P,贝瑟实验室)反应1小时,并且用ECL底物识别化学发光。
结果,如图5所示,与脂质纳米颗粒诸如lipofectamine相比,包含根据本发明的肽-脂质缀合物的纳米颗粒表现出在细胞中由mRNA表达的KRAS降解蛋白的量高得多,并且在1∶10的比例下的表达最高(*p<0.5,**p<0.01)。
实施例5:通过递送至细胞内的KRAS降解蛋白mRNA对活性KARS的减少的确认
蛋白质印迹的结果显示,在实施例4中,在注射了包含mRNA和肽-脂质缀合物的纳米颗粒的细胞中,活性KRAS减少,亚信号pERK减少(图6)。
将H358细胞以70%的密度接种在6孔板中。16小时后,用含0.5% FBS的DMEM进行过夜细胞饥饿处理。用含有纳米颗粒的培养基处理细胞16小时,所述纳米颗粒包含浓度为10nM和20nM的mRNA和肽-脂质缀合物。作为对照,使用10nM的混合有lipofectamine的mRNA。使用含有酶抑制和磷酸酶抑制剂的RIPA裂解缓冲液(25mM Tris HCl pH 7.6,150mM NaCl,1% NP-40,1%脱氧胆酸钠,0.1% SDS)裂解蛋白质。通过BCA蛋白测定法(23227,赛默飞世尔(Thermo Scientific))对蛋白质进行测定,通过蛋白质印迹法检测FER酪氨酸激酶和肌动蛋白的表达水平。通过将相同量的样品与大小标志物一起上样在8% SDS PAGE凝胶上,进行约2小时的电泳,然后将其转移到硝酸纤维素膜上,进行蛋白质印迹法。用5%脱脂乳封闭转移的膜1小时,并与一抗(GST,Abcam,ab19256)以1∶1000的比例反应过夜。然后,用含有0.1%吐温-20的TBST洗涤膜,并与HRP连接的二抗(抗兔,A90-116P,贝瑟实验室(BETHYLLab.);抗鼠,A120-101P,贝瑟实验室)反应1小时,并且用ECL底物识别化学发光。
结果,如图6所示,与脂质纳米颗粒诸如lipofectamine相比,包含根据本发明的肽-脂质缀合物的纳米颗粒有效降低了活性KRAS和亚信号pMEK。
实施例6:通过递送至细胞内的KRAS siRNA对信使KARS的减少的确认
将H358细胞以70%的密度接种在6孔板中。16小时后,用含0.5% FBS的DMEM进行过夜细胞饥饿处理。用含有纳米颗粒的培养基处理细胞16小时,所述纳米颗粒包含浓度为100nM的KRAS siRNA和肽-脂质缀合物。作为对照,使用100nM的混合有Lipofectamine的siRNA。
以下述方式进行定量实时PCR。对于mRNA定量,使用Qiagen RNeasy试剂盒分离总RNA。使用Verso cDNA试剂盒(赛默飞世尔)使用500ng RNA合成cDNA。用基于SYBR Green的7500快速实时PCR系统(Applied Biosystems)测量mRNA值。用基于SYBR Green的7500快速实时PCR系统(应用生物系统(Applied Biosystems))测量mRNA值。
这里使用的引物如下。
KRAS:
F-TGACCTGCTGTGTCGAGAAT(SEQ ID NO:21),
R-TTGTGGACGAATATGATCCAA(SEQ ID NO:22);
18S:
F-CGCCGCTAGAGGTGAAATTC(SEQ ID NO:23),
R-TTGGCAAATGCTTTCGCTC(SEQ ID NO:24)
18S rRNA用作持家基因(对照基因)。使用逆转录的RNA进行PCR,并使用100ng/μL的正义和反义引物(总体积为20μL)。进行40个循环,每个循环包括在95℃变性15秒,退火1分钟,以及在60℃下延伸。结果示出于图7。用KRAS siRNA和肽-脂质缀合物(肽-脂质NC)处理表现出最大程度的KRAS mRNA降低,而单独的siRNA或对照siRNA没有显示出作用。
实施例7:肺癌动物模型中抗癌效果的确认
将实施例2中制备的包含mRNA和肽-脂质缀合物的纳米颗粒给药于具有由H358细胞系诱导的肺癌的异种移植小鼠模型,并鉴定其抗癌效果。将100μL的1X106 H358细胞和Matrigel(BD生物科学(BD Bioscience),圣地亚哥,CA,USA)的混合物植入5-6周龄雌性Balb/c裸鼠(5-6周龄;日本SLC公司,日本滨松)以形成大小为100mm3的肿瘤。包含mRNA和肽-脂质缀合物的纳米颗粒和由mRNA和lipofectamine制备的脂质纳米颗粒(LNP)分别以1μg/kg的剂量每周两次腹膜内注射30天。每3至4天用游标卡尺测量肿瘤的大小,在第30天,处死小鼠以切除肿瘤,并对切除的肿瘤进行成像。
结果,如图8所示,在给药包含肽-脂质缀合物的纳米颗粒的实验组中,肿瘤大小没有增加,并且用Lipofectamine制备的脂质颗粒的肿瘤大小持续增加。
工业实用性
根据本发明,由与寡核苷酸连接的肽-脂质缀合物组成的纳米颗粒有效地将治疗性寡核苷酸递送至细胞内,并通过寡核苷酸改变靶蛋白的表达,因此可用作治疗剂。
尽管已经详细描述了本发明的具体配置,但是本领域的技术人员将会理解,提供该描述是为了说明的目的而阐述优选实施方式,并且不应该被解释为限制本发明的范围。因此,本发明的实质范围由所附权利要求及其等同物来限定。
文本的序列表
附上电子文件。
<110> 纳米智能生物医学工程有限公司
首尔大学校产学协力团
<120> 包含用于将寡核苷酸递送至靶细胞内的肽-脂质缀合物的纳米颗粒以及包含其的药物组合物
<130> PF-B2963
<150> KR 2021-0075838
<151> 2021-06-11
<160> 24
<170> KoPatentIn 3.0
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1 5 10
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Cys Ala Ile Tyr Pro Arg His
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uacgccgaca gcguuaaggg aagguucacu auaucccggg acaauucuaa gaauacuuug 240
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gguaagagcu ccgguucagg gucagaaucu aaaagcacug guggcuccga uauacaaaug 420
acccagaguc caagcucucu uucagccagc gugggagaca gggugaccau caccugucga 480
gcaucucaau ccauuucauc auaccucaau ugguaucaac agaagccugg ggaagcacca 540
aaguugcuua uuuauucugc cagcguacug caaaguggcg uaccuucccg cuucuccggg 600
ucugggagug gcacugauuu uacccucaca auuagcagcc ugcaacccga ggacuucgcu 660
acauacuacu gccaacaaag uguaaugaua cccaugacau ucgggcaagg cacaaaaguc 720
gagauaaagg gaaguggagg agguggcucu augccccgcc gcgcugagaa cugggaugag 780
gccgaaguug gugcugaaga ggccggaguu gaagaauaug ggccagaaga agauggcggu 840
gaggaaagcg gggcugaaga gagcggucca gaggagucug guccagagga acuuggagcu 900
gaggaggaga uggaggccgg acguccacgu ccuguccugc gaucaguuaa cucaagggag 960
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gagaucaaag gcuccggagg aggagggucu auggaaaaca gauggcaagu uaugaucguu 780
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gugucaggca aagcacgagg augguuuuau cgucaucacu augagucacc acauccccgc 900
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ggccuccaca cuggugaaag agauuggcac cucggccaag gaguaaguau agaauggcgc 1020
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cagcuaauuc agaaucauu 19
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tgacctgctg tgtcgagaat 20
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Claims (22)
1.一种具有下式1的肽-脂质缀合物作为基本单元的肽-脂质缀合物:
[式1]
A-B-C-D-E
其中,A是具有12至20个碳原子的脂肪酸或阳离子脂质;
B是促进内体逃逸的肽;
C是RNA结合肽;
D是具有细胞穿透功能的肽;以及
E是识别靶细胞表面的肽。
2.根据权利要求1所述的肽-脂质缀合物,其中,B是由4到12个组氨酸组成的肽。
3.根据权利要求1所述的肽-脂质缀合物,其中,D是选自由4至12个精氨酸、4至12个赖氨酸和2个半胱氨酸或其组合组成的组的肽。
4.根据权利要求1所述的肽-脂质缀合物,其中,C是由SEQ ID NO:1至SEQ ID NO:15中任一氨基酸序列表示的肽。
5.根据权利要求1所述的肽-脂质缀合物,其中,E具有由SEQ ID NO:16表示的氨基酸序列。
6.根据权利要求1所述的肽-脂质缀合物,其中,式1的所述基本单元的A-B通过酰胺键连接。
7.根据权利要求6所述的肽-脂质缀合物,其中,A-B的所述酰胺键是基于化学合成形成的。
8.根据权利要求1所述的肽-脂质缀合物,其中,B-C-D-E通过化学合成或重组表达方法产生。
9.一种纳米颗粒,其中根据权利要求1至8中任一项所述的肽-脂质缀合物和寡核苷酸彼此结合。
10.根据权利要求9所述的纳米颗粒,其中,所述寡核苷酸选自由DNA、siRNA、miRNA和mRNA组成的组。
11.根据权利要求9所述的纳米颗粒,其中,所述寡核苷酸抑制或增加靶基因的表达。
12.根据权利要求10所述的纳米颗粒,其中,所述siRNA具有与编码肿瘤诱导蛋白或致病蛋白的核苷酸序列互补的核苷酸序列。
13.根据权利要求12所述的纳米颗粒,其中,所述siRNA是天然的或经化学修饰的siRNA,或者使用在5'端被巯基或胺基取代的siRNA,与细胞穿透和靶识别肽中的半胱氨酸的巯基化学结合。
14.根据权利要求10所述的纳米颗粒,其中,所述mRNA编码需要增加表达的靶蛋白、重组嵌合蛋白或病毒抗原。
15.根据权利要求9所述的纳米颗粒,其中,在所述寡核苷酸与式1中的C结合后,通过所述肽-脂质缀合物的自组装形成所述纳米颗粒。
16.根据权利要求15所述的纳米颗粒,其中,所述纳米颗粒的大小为10至200nm。
17.根据权利要求10所述的纳米颗粒,其中,所述寡核苷酸是siRNA或mRNA,并且所述siRNA或mRNA与肽-脂质缀合物的分子量比为1∶1至1∶100。
18.一种用于将寡核苷酸递送至细胞内的组合物,其包含其中根据权利要求9所述的肽-脂质缀合物和寡核苷酸彼此结合的所述纳米颗粒。
19.一种药物组合物,其包含其中根据权利要求9所述的肽-脂质缀合物和寡核苷酸彼此结合的所述纳米颗粒。
20.根据权利要求19所述的药物组合物,其用于治疗或预防癌症或炎性疾病。
21.根据权利要求20所述的药物组合物,其中,所述癌症选自由以下组成的组:鳞状细胞癌、基底细胞癌、腺癌、肝细胞癌、肾细胞癌、膀胱癌、肠癌、乳腺癌、宫颈癌、子宫癌、结肠癌、食道癌、头癌、肾癌、肝癌、肺癌、卵巢癌、胰腺癌、前列腺癌、胃癌、白血病、良性和恶性淋巴瘤、良性和恶性黑色素瘤、骨髓增生性疾病、肉瘤包括尤文氏肉瘤、血管肉瘤、卡波西肉瘤、脂肪肉瘤、肌瘤、神经上皮肉瘤、滑膜肉瘤、神经肉瘤、星形细胞瘤、少突胶质细胞瘤、脑细胞瘤、胶质母细胞瘤、神经母细胞瘤、神经节细胞瘤、神经母细胞瘤、松果体瘤、脑膜瘤、脑膜肉瘤、神经纤维瘤和神经鞘瘤、睾丸癌、甲状腺癌、癌肉瘤、霍金斯病、维尔姆斯瘤和畸胎癌。
22.根据权利要求20所述的药物组合物,其中,用于治疗或预防选自由以下组成的组的疾病:哮喘、自身免疫性疾病、类风湿性关节炎、多发性硬化、睫状病、腭裂、糖尿病、心脏病、高血压、炎性肠病、精神发育迟滞、情绪障碍、肥胖症、屈光不正、不孕症、安格尔曼综合征、卡纳万氏病、慢性消化系统疾病、恰克-马利-杜斯氏(CMT)病、囊性纤维化、杜氏肌营养不良、血色素沉着症、血友病、克氏综合征、神经纤维瘤病、苯丙酮尿症、常染色体显性多囊肿瘤(PKD1或PKD2)、普拉德-威利综合征、镰状细胞贫血、泰-萨克斯病、特纳综合征、HIV感染和HCV感染。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR20210075838 | 2021-06-11 | ||
KR10-2021-0075838 | 2021-06-11 | ||
PCT/KR2022/008229 WO2022260480A1 (ko) | 2021-06-11 | 2022-06-10 | 타겟 세포 내로 올리고뉴클레오티드를 전달하기 위한 펩타이드-지질의 결합체를 포함하는 나노입자 및 이를 포함하는 약학적 조성물 |
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EP1781593B1 (en) | 2004-06-07 | 2011-12-14 | Protiva Biotherapeutics Inc. | Cationic lipids and methods of use |
US7404969B2 (en) | 2005-02-14 | 2008-07-29 | Sirna Therapeutics, Inc. | Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules |
WO2007086883A2 (en) | 2005-02-14 | 2007-08-02 | Sirna Therapeutics, Inc. | Cationic lipids and formulated molecular compositions containing them |
JP5749494B2 (ja) | 2008-01-02 | 2015-07-15 | テクミラ ファーマシューティカルズ コーポレイション | 核酸の送達のための改善された組成物および方法 |
AU2009238175C1 (en) | 2008-04-15 | 2023-11-30 | Arbutus Biopharma Corporation | Novel lipid formulations for nucleic acid delivery |
US20090285881A1 (en) | 2008-04-16 | 2009-11-19 | Abbott Laboratories | Cationic lipids and uses thereof |
US20090263407A1 (en) | 2008-04-16 | 2009-10-22 | Abbott Laboratories | Cationic Lipids and Uses Thereof |
WO2009132131A1 (en) | 2008-04-22 | 2009-10-29 | Alnylam Pharmaceuticals, Inc. | Amino lipid based improved lipid formulation |
AU2009303345B2 (en) | 2008-10-09 | 2015-08-20 | Arbutus Biopharma Corporation | Improved amino lipids and methods for the delivery of nucleic acids |
HUE037082T2 (hu) | 2008-11-10 | 2018-08-28 | Arbutus Biopharma Corp | Új lipidek és készítmények terápiás hatóanyagok szállítására |
WO2010054384A1 (en) | 2008-11-10 | 2010-05-14 | Alnylam Pharmaceuticals, Inc. | Lipids and compositions for the delivery of therapeutics |
US20100267806A1 (en) | 2009-03-12 | 2010-10-21 | David Bumcrot | LIPID FORMULATED COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF Eg5 AND VEGF GENES |
KR101159296B1 (ko) * | 2009-10-22 | 2012-06-22 | 서울대학교산학협력단 | 표적 miRNA의 생성을 촉진하는 양면성 펩타이드 및 이를 이용한 표적 miRNA 생성 조절 방법 |
AU2019275071B2 (en) * | 2018-05-24 | 2022-12-15 | Sirnaomics, Inc. | Composition and methods of controllable co-coupling polypeptide nanoparticle delivery system for nucleic acid therapeutics |
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