CN111742049A - 转化的人细胞及其用途 - Google Patents
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Abstract
本发明涉及转化的人细胞及其用途,更具体地,涉及一种通过使用借助向导RNA的基因表达抑制***,用编码MHC I细胞膜受体和MHC II细胞膜受体的基因转化的细胞,及其用途。这种转化细胞即使在体内也可以有效地表现出细胞的治疗效果,并且不会被体内免疫应答除去。因此,期望包含该免疫细胞作为活性成分的组合物可以有效地用于癌症、传染病、退行性疾病、或免疫性疾病的治疗。
Description
技术领域
本发明涉及转化的人细胞及其用途,并且更具体地,涉及通过向导RNA转化的人细胞及其用途。
背景技术
作为用于治疗癌症或传染病的方法,利用患者的免疫功能的免疫疗法引起了关注。免疫疗法意指通过免疫细胞(例如,NK细胞、T细胞、树突细胞等)的相互作用而用于疾病的治疗方法。在这些治疗方法中,出现了免疫疗法,其使用了表达对抗原具有特异性的嵌合抗原受体的基因修饰的T细胞。另外,据报道,通过离体活化而具有高细胞毒性的NK细胞显示出对血液癌(例如白血病)的优异治疗作用(Blood Cells Molecules&Disease,33:p261-266,2004)。
同时,尽管如上所述免疫细胞有可能用作癌症或传染病的治疗剂,但是与那些健康个体相比,存在于患者体内的免疫细胞在功能和数量方面明显较低。因此,利用同种异体免疫细胞的移植比使用自体免疫细胞更有效。然而,在移植同种异体免疫细胞的情况下,可能会发生一些问题,例如移植排斥或由于体内非己识别而引起的免疫学消除。因此,为了克服这些缺点,需要将同种异体免疫细胞制成细胞库同时允许同种异体免疫细胞被识别为自身的替代方案。
发明内容
技术问题
为了解决上述问题,发明人已合成了向导RNA,该向导RNA在细胞中靶向编码MHC I细胞膜受体的基因和编码MHC II细胞膜受体的基因的向导RNA。另外,发明人已使用抑制基因表达的组合物制备了一种细胞,在该细胞中MHC I细胞膜受体和MHC II细胞膜受体的表达受到抑制,该抑制基因表达的组合物包含向导RNA和RNA引导的核酸内切酶作为活性成分,其中可以将HLA-E引入组合物中,从而防止对细胞的体内免疫学消除。
因此,本发明的目的是提供靶向编码MHC I细胞膜受体的基因和编码MHC II细胞膜受体的基因的向导RNA,并提供使用该向导RNA转化的细胞。
解决方案
为了实现上述目的,本发明提供了:与编码β2-微球蛋白(B2M)的核酸序列互补结合的向导RNA,该向导RNA包含选自由SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:17、以及SEQID NO:26组成的组的任何一个核酸序列;与编码HLA-DQ的核酸序列互补结合的向导RNA,该向导RNA包含选自由SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:87、以及SEQ ID NO:90组成的组的任何一个核酸序列;与编码HLA-DP的核酸序列互补结合的向导RNA,该向导RNA包含SEQ ID NO:123或SEQ ID NO:129的核酸序列;以及与编码HLA-DR的核酸序列互补结合的向导RNA,该向导RNA包含选自由SEQ ID NO:186、SEQ ID NO:188、和SEQ ID NO:225组成的组的任何一个核酸序列。
另外,本发明提供了用于抑制基因表达的组合物,该组合物包含作为活性成分的向导RNA或编码该向导RNA的核苷酸序列、以及RNA引导的核酸内切酶或编码该RNA引导的核酸内切酶的核苷酸序列。
另外,本发明提供了转化细胞,在所述转化细胞中MHC I细胞膜受体和MHC II细胞膜受体的表达受到抑制。
另外,本发明提供了用于治疗癌症、传染病、退行性疾病、遗传性疾病、或免疫性疾病的药物组合物,该药物组合物包含作为活性成分的转化细胞;以及用于治疗癌症、传染病、退行性疾病、遗传性疾病、或免疫性疾病的方法,该方法包括向对象施用该组合物。
另外,本发明提供了转化细胞在用于治疗癌症、传染病、退行性疾病、遗传性疾病、或免疫性疾病中的用途,其中MHC I细胞膜受体和MHC II细胞膜受体的表达在该转化细胞中受到抑制,并且该转化细胞在细胞膜表面上表达结合至修饰的MHC I细胞膜受体的肽抗原,例如G肽。
本发明的有益效果
通过使用利用根据本发明的向导RNA的基因表达抑制***,可以制备这样的细胞:在该细胞中编码MHC I细胞膜受体的基因和编码MHC II细胞膜受体的基因进行了修饰。另外,可以将与肽抗原(例如G肽)结合的HLA-E额外引入细胞中。如上所述的转化细胞甚至在体内也可以有效地显示其治疗功效,并且不会被体内免疫应答消除。因此,期望包含该细胞作为有效成分的组合物可以有效地用于癌症、传染病、退行性疾病、遗传性疾病、或免疫性疾病的治疗。
附图说明
图1示出了通过使用流式细胞术分析使用靶向B2M的gRNA制备的细胞中的HLA-ABC阴性细胞得到的结果;
图2示出了通过使用流式细胞术分析使用靶向HLA-DRA的gRNA制备的细胞中的HLA-DR阴性细胞得到的结果;
图3示出了通过使用流式细胞术分析通过使用靶向HLA-DQA的gRNA制备的细胞中的HLA-DQ阴性细胞得到的结果;
图4示出了通过使用流式细胞术分析使用靶向HLA-DPA的gRNA制备的细胞中的HLA-DP阴性细胞得到的结果;
图5示出了依赖于靶向B2M的gRNA的HLA-ABC阴性细胞系的产率;
图6示出了依赖于靶向DRA的gRNA的HLA-DR阴性细胞系的产率;
图7示出了依赖于靶向DQA的gRNA的HLA-DQ阴性细胞系的产率;
图8示出了依赖于靶向DPA的gRNA的HLA-DP阴性细胞系的产率;
图9示出了用靶向B2M的gRNA制备的细胞系中编码B2M的核酸中的突变;
图10示出了用靶向HLA-DRA的gRNA制备的细胞系中编码HLA-DRA的核酸中的突变;
图11示出了用靶向HLA-DQA的gRNA制备的细胞系中编码HLA-DQA的核酸中的突变;
图12示出了用靶向HLA-DPA的gRNA制备的细胞系中编码HLA-DPA的核酸中的突变;
图13示出了细胞分离后的HLA-1阳性NK-92MI细胞系和HLA-1阴性NK-92MI细胞系;
图14示出了对HLA-1阳性NK-92MI细胞系和HLA-1阴性NK-92MI细胞系的细胞杀伤能力的评估结果;
图15示出了通过使用gRNA对CD4T细胞、CD8T细胞和NK细胞进行转化然后用流式细胞术进行分析得到的结果;
图16示出了单个gRNA转化的细胞和多个gRNA转化的细胞中靶标的缺失效率;
图17比较了单个gRNA转化的细胞、多个gRNA转化的细胞、以及对照组细胞之间的细胞生长率;
图18比较了HLA-I阳性T细胞和HLA-I阴性T细胞之间的细胞因子生产能力;
图19比较了HLA-I阳性NK细胞和HLA-I阴性NK细胞之间的细胞因子生产能力;
图20示出了NK细胞对HLA-I阳性Raji细胞系和HLA-I阴性Raji细胞系的细胞杀伤能力的评估结果;
图21示出了负载有G肽的HLA-E的示意图以及用于表达其的蛋白质的结构;
图22示出了对通过转导而在K562细胞系中表达的HLA-E进行分析而得到的结果;
图23示出了NK细胞对表达HLA-E的K562细胞系(K562 G-B2M-HLA-E)和对照组K562细胞系(K562)的细胞杀伤能力的评估结果。
具体实施方式
在本发明的一个方面,提供与编码β2-微球蛋白(B2M)的核酸序列互补结合的向导RNA,该向导RNA包含选自由SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:17、以及SEQ ID NO:26组成的组的任何一个核酸序列。
如本文所用,术语“B2M”是指β2-微球蛋白,其为MHC I的一种组分。B2M对于在细胞表面表达MHC I细胞膜受体至关重要;并且当移除或修饰B2M时,MHC I细胞膜受体在细胞表面上的表达难以发生。因此,可以通过修饰B2M的基因来出除MHC I细胞膜受体的功能。
与编码B2M的核酸序列互补结合的向导RNA可以是选自由SEQ ID NOs:1-58组成的组的任何一个,并且可以特别地是任何一个选自由SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:17、以及SEQ ID NO:26组成的组的任何一个。
另外,在本发明的一个方面,提供与编码HLA-DQ的核酸序列互补结合的向导RNA,该向导RNA包含选自由SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:87、以及SEQ ID NO:90组成的组的任何一个核酸序列。
如本文所用,术语“HLA”是指人白细胞抗原,其是MHC基因的产物。HLA由HLA I和HLA II组成。HLA I可以包括HLA-A、HLA-B和HLA-C;并且HLA II可以包括HLA-DQ、HLA-DP和HLA-DR。
如本文所用,术语“HLA-DQ”是指构成MHC II的αβ异二聚体。DQ由HLA-DQA1和HLA-DQB1组成。α亚基由HLA-DQA1基因编码,β亚基由HLA-DQB1基因编码。可以通过修饰DQ的基因来抑制MHC II细胞膜受体的表达。
与编码DQ的核酸序列互补结合的向导RNA可以是选自由SEQ ID NOs:59-116组成的组的任何一个,并且可以特别地是选自由SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:87、以及SEQ ID NO:90组成的组的任何一个。
在本发明的另一个方面,提供与编码HLA-DP的核酸序列互补结合的向导RNA,该向导RNA包含SEQ ID NO:123或SEQ ID NO:129的核酸序列。
如本文所用,术语“HLA-DP”是指由DPα亚基和DPβ亚基组成的编码的MHC II细胞表面受体。DPα由HLA-DPA1编码,DPβ由HLA-DPB1编码。可以通过修饰DP的基因来抑制MHC II细胞膜受体的表达。
与编码DP的核酸序列互补结合的向导RNA可以是选自由SEQ ID NOs:117-175组成的组的任何一个,并且可以特别地是SEQ ID NO:123或SEQ ID NO:129。
另外,在本发明的又一个方面中,提供与编码HLA-DR的核酸序列互补结合的向导RNA,该向导RNA包含选自由SEQ ID NO:186、SEQ ID NO:188、和SEQ ID NO:225组成的组的任何一个核酸序列。
如本文所用,术语“HLA-DR”是指MHC II细胞表面受体,特别是构成MHC II细胞表面受体的αβ异二聚体。HLA-DR的每个亚基都包含两个胞外结构域:跨膜结构域和胞质尾区。可以通过修饰DR的基因来抑制MHC II细胞膜受体的表达。
与编码DR的核酸序列互补结合的向导RNA可以是选自由SEQ ID NOs:176-234组成的组的任何一个,并且可以优选为选自由SEQ ID NO:186、SEQ ID NO:188、和SEQ ID NO:225组成的组的任何一个。
如本文所用,术语“向导RNA(gRNA)”是指特异性识别靶DNA并与核酸酶形成复合物从而将该核酸酶引导至靶DNA的RNA分子。
向导RNA可以是衍生自原核的成簇的规律间隔短回文重复(Clustered regularlyinterspaced short palindromic repeat,CRISPR)***的向导RNA。
向导RNA可以包含非天然存在的嵌合crRNA序列,并且crRNA序列可以包含能够与靶序列杂交的可变靶向结构域。
此外,向导RNA包含B2M、HLA-DQ、HLA-DP、以及HLA-DR基因中每个基因的互补序列。递送入细胞后,向导RNA能够识别靶序列并与RNA引导的核酸内切酶形成复合物。
在本发明的又一个方面,提供了用于抑制基因表达的组合物,该组合物包含作为活性成分的向导RNA或编码该向导RNA的核苷酸序列、以及RNA引导的核酸内切酶或编码该RNA引导的核酸内切酶的核苷酸序列。
RNA引导的核酸内切酶可以以mRNA或蛋白质的形式递送,或者可以通过使用负载有编码该mRNA或蛋白质的DNA的载体的转化而递送至靶细胞。当使用蛋白质形式的核酸内切酶时,该核酸内切酶可以充当通过与向导RNA形成复合物而得到的RNP复合物。
如本文所用,术语“RNP复合物”是指包含作为活性成分的向导RNA和RNA引导的核酸内切酶的复合物,其中该复合物能够识别并结合至靶序列,从而选择性地切割或裂解靶序列。例如,RNA复合物可以是Cas9-gRNA复合物,但不限于此。
在本发明的一个实施方式中,RNA引导的核酸内切酶可以是选自由以下组成的组的任何一个:Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9、Cas10、Cas12a、Cas12b、Cas12c、Cas12d、Cas12e、Cas13a、Cas13b、Cas13c、Cas13d、Cpf1、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、以及Csf4,并且可以特别地为Cas9。
在本发明的一个方面,提供了转化细胞,在该转化细胞中MHC I细胞膜受体和MHCII细胞膜受体的表达受到抑制。
如本文所用,术语“表达抑制”意指核苷酸序列上的修饰,由于这种表达抑制,该修饰导致靶基因的功能降低,并且优选地意指使得靶基因的表达不可检测或靶基因表达至无意义的水平。
在本发明的一个实施方式中,转化细胞可以在细胞膜表面上表达肽抗原。肽抗原的实例包括但不限于HLA-A、HLA-B、HLA-C、以及HLA-G的信号肽,并且该肽抗原特别地为HLA-G的信号肽(G肽)。肽抗原可以结合至修饰的MHC I细胞膜受体。
在本发明的一个实施方式中,修饰的MHC I细胞膜受体具有HLA-E与B2M连接的结构。具体而言,在修饰的MHC I细胞膜受体中,B2M的C端可以经由第一接头连接至HLA-E的α1的N端,并且G肽的C端可以经由第二接头连接至B2M的N端。修饰的MHC I细胞膜受体可以具有HLA-G与B2M连接的结构。
在本发明的一个实施方式中,G肽可以具有SEQ ID NO:236的序列;HLA-E可以具有SEQ ID NO:240的序列;B2M可以具有SEQ ID NO:237的序列;以及在一个实施方案中,第一接头可以为(G4S)n(n为1至5的整数)并且可以具有SEQ ID NO:238的序列。第二接头可以为(G4S)n(n为2至6的整数)并且可以具有SEQ ID NO:241的序列。
在本发明的一个实施方案中,编码MHC I细胞膜受体的基因的修饰可以使用与编码B2M的核酸序列互补结合的向导RNA(例如,SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:17、或SEQ ID NO:26)来进行。具体而言,MHC I的修饰可以通过使用单个向导RNA的单个缺失来进行。
在本发明的一个实施方案中,编码MHC II细胞膜受体的DQ、DP和DR基因的修饰可以使用与编码DQ的核酸序列互补结合的向导RNA(例如,SEQ ID NO:64、SEQ ID NO:65、SEQID NO:87、或SEQ ID NO:90)、与编码DP的核酸序列互补结合的向导RNA(例如,SEQ ID NO:123或SEQ ID NO:129)、与编码DR的核酸序列互补结合的向导RNA(例如,SEQ ID NO:186、SEQ ID NO:188、或SEQ ID NO:225)来进行。MHC II的修饰与MHC I的修饰一起进行,这可以通过使用多个向导RNA(例如包含SEQ ID NO:1、SEQ ID NO:64、SEQ ID NO:129、以及SEQ IDNO:188中的全部)的多重缺失来进行。
在本发明的一个实施方式中,转化细胞可以为治疗性同种异体细胞。如本文所用,术语“治疗性同种异体细胞”是指为了抑制疾病的发展、治疗疾病、或减轻疾病的症状而注射进对象的非自体同种异体细胞,其实例包括但不限于,免疫细胞和干细胞。
如本文所用,术语“免疫细胞”是指参与人体免疫应答的细胞,其实例包括NK细胞、T细胞、B细胞、树突细胞和巨噬细胞。
在本发明的一个实施方式中,免疫细胞可以为NK细胞或T细胞。
如本文所用,术语“干细胞”是指能够分化成各种细胞的多能细胞。干细胞的实例可以包括人胚胎干细胞、骨髓干细胞、间充质干细胞、人神经干细胞、口腔粘膜细胞等。具体而言,干细胞可以为间充质干细胞。
另外,在本发明的一个方面,提供了用于治疗癌症、传染病、退行性疾病、遗传性疾病、或免疫性疾病的药物组合物,该药物组合物包含转化细胞作为活性成分。
在本发明的一个实施方式中,癌症可以为选自由以下组成的组中的任何一种:慢性淋巴细胞白血病(Chronic lymphocytic leukemia,CLL)、B细胞急性淋巴细胞白血病(B-cell acute lymphocytic leukemia,B-ALL)、急性淋巴细胞白血病、急性骨髓性白血病、淋巴瘤、非霍奇金淋巴瘤(Non-Hodgkin's lymphoma,NHL)、多发性骨髓瘤、血液癌、胃癌、肝癌、胰腺癌、结肠直肠癌、肺癌、乳腺癌、卵巢癌、皮肤癌、黑色素瘤、肉瘤、***癌、食道癌,肝细胞癌、星形细胞瘤、间皮瘤、头颈癌、以及髓母细胞瘤。
在本发明的一个实施方式中,传染病可以为选自由以下组成的组中的任何一种:乙型肝炎、丙型肝炎、人***瘤病毒(Human papilloma virus,HPV)感染、巨细胞病毒感染、爱泼斯坦巴尔病毒(Epstein Barr virus,EBV)感染、病毒性呼吸道疾病、以及流行性感冒。
如本文所用,术语“退行性疾病”是指由于定量的组织不可逆亏损而使组织丧失其原始功能的病理状况。退行性疾病的例子包括但不限于脑神经疾病、缺血性疾病、皮肤损伤、骨病、以及退行性关节炎。
如本文所用,术语“遗传性疾病”是指由于对基因或染色体有害的突变而发生的病理状况。遗传性疾病的实例包括但不限于血友病、白化病、法布里病(Fabry disease)、亨特综合征(Hunter syndrome)、以及糖原贮积症。
如本文所用,术语“免疫性疾病”是指任何由于过度或不期望的免疫反应而使组织受损的病理状况。因此,术语“免疫性疾病”与“过度活跃的免疫性疾病”含义相同,并且术语“用于预防或治疗免疫性疾病的组合物”与“免疫抑制剂”含义相同。
免疫性疾病的实例包括但不限于移植物抗宿主病、移植排斥、慢性炎性疾病、炎性疼痛、神经性疼痛、慢性阻塞性肺病(Chronic obstructive pulmonary disease,COPD)、以及自身免疫性疾病。
术语“自身免疫性疾病”是指当免疫细胞不能将自身与异物区分开并从而攻击自身时发生的病理状况。自身免疫性疾病的实例可以包括但不限于:类风湿性关节炎、全身性红斑狼疮、桥本氏甲状腺炎(Hashimoto's thyroiditis)、格雷夫斯病(Grave's disease)、多发性硬化症、硬皮病、重症肌无力、I型糖尿病、过敏性脑脊髓炎、肾小球性肾炎、白癜风、白塞病(Behcet's disease)、克罗恩病(Crohn's disease)、强直性脊柱炎、血小板减少性紫癜、寻常天疱疮、自身免疫性溶血性贫血、肾上腺脑白质营养不良(Adrenoleukodystrophy,ALD)、以及全身性红斑狼疮(Systemic lupus erythematosus,SLE)。
在本发明的一个方面,提供了用于治疗癌症、传染病、退行性疾病、遗传性疾病、或免疫性疾病的方法,该方法包括向对象施用该药物组合物。
在本发明的一个实施方式中,施用可以是通过选自由以下组成的组中的任何一种途径进行的:静脉内的、肌内的、皮内的、皮下的、腹膜内的、小动脉内的、心室内的、病灶内的、鞘内的、局部的、及其组合。
在本发明的另一个实施方式中,提供了转化细胞在用于治疗癌症、传染病、退行性疾病、遗传性疾病、或免疫性疾病中的用途,其中MHC I细胞膜受体和MHC II细胞膜受体的表达在该转化细胞中受到抑制,并且该转化细胞在细胞膜表面上表达结合至修饰的MHC I细胞膜受体的G肽。
在本发明的又一个方面,提供了用于修饰MHC I细胞膜受体的基因和MHC II细胞膜受体的基因的试剂盒,该试剂盒包含向导RNA或编码向导RNA的核苷酸序列、以及RNA引导的核酸内切酶或编码该RNA引导的核酸内切酶的核苷酸序列。
本发明的方式方式
下文将通过以下实施例更为详细地描述本发明。然而,以下实施例仅用于说明本发明,并且本发明的范围不受以下实施例的限制。
实施例1.靶向HLA的gRNA的合成和选择
实施例1.1.gRNA序列的搜索与合成
为了寻找gRNA序列,使用了NCBI(https://www.ncbi.nlm.nih.gov/)提供的基因的完整核苷酸序列。作为gRNA的设计工具,使用了基于Web的***CHOPCHOP(http://chopchop.cbu.uib.no/)、E-CRISP(http://www.e-crisp.org/E-CRISP/designcrispr.html)、CRISPR-ERA(http://crispr-era.stanford.edu/)、RGEN工具(http://www.rgenome.net/cas-designer/)。在设计的gRNA中,每个所需的靶标均获得了约60个最适合基因敲除的gRNA。基于这些序列,使用GeneArt精密gRNA合成试剂盒(GeneArtPrecision gRNA Synthesis Kit;Thermo Fisher Scientific,A29377)根据制造商的说明来合成gRNA。
也就是说,合成了合成编码每个gRNA的DNA模板所需的正向和反向寡核苷酸引物,然后使用合成的引物和GeneArt精密gRNA合成试剂盒(Thermo Fisher Scientific,A29377)中包含的Tracr片段+T7引物混合物,用PCR热循环仪(FlexCycler2,AnalytikJena)进行PCR。使用以下PCR参数:在98℃下进行预变性10秒,随后在98℃下5秒和在55℃下15秒的条件下进行变性和退火,32个循环,最后在72℃下延伸1分钟。使用得到的PCR产物作为模板,在37℃进行体外转录反应4小时;然后纯化所得物以得到gRNA。
得到的gRNA显示在下表1-4中。具体而言,HLA-ABC(B2M)的gRNA序列显示在表1中;HLA-DQ的gRNA序列示于表2中;HLA-DP的gRNA序列显示在下表3中;HLA-DR的gRNA序列示在下表4中。
[表1]
[表2]
[表3]
[表4]
实施例1.2.通过转染到Raji细胞系中选择gRNA
将7.5μg得到的gRNA在65℃下孵育10分钟以形成单链。然后,向其中加入7.5μg的Cas9蛋白(Toolgen,TGEN_CP3或Clontech,M0646T),并在25℃下孵育10分钟来制备Cas9-gRNA复合物(RNP复合物)。使用SG Cell LineX Kit S(Lonza,V4XC-3032)用4D-NucleofectorTM X Unit(Lonza,AAF-1002X)将RNP复合物转染到具有4×105个细胞的Raji细胞系中。将转染的细胞孵育7天,然后鉴定细胞表面上HLA的表达水平和基因组DNA中突变的存在。
实施例1.3.使用流式细胞仪鉴定HLA的表达水平
将RNP复合物转染的Raji细胞系和对照组Raji细胞系各2×105个细胞悬浮在100μL FACS缓冲液(1%FBS/鞘缓冲液)中,并在5-mL管中制备。对细胞进行抗体处理。然后,遮光30分钟,并在4℃下进行孵育。关于抗体,使用了PE抗HLA-ABC(Miltenyi Biotec,130-101-448)、PE抗HLA-DR(Biolegend,361605)、PE抗HLA-DQ(Biolegend,318106)、以及PE抗HLA-DP(Leinco Technologies,H130)。之后,向其中加入3mL FACS缓冲液,并在4℃以2,000rpm离心3分钟。然后,去除上清液以得到样品,并通过LSR Fortessa分析该样品。测试了共计60个gRNA三次,每次使用20个gRNA。通过从各gRNA的“HLA阴性%”值中减去对照组的“HLA阴性%”值而计算出的值(标准化的HLA阴性%)的结果示于图1-4中。另外,通过立即对值为10或更高的gRNA(然而,在靶向B2M的gRNA的情况下,对值为1或更高的gRNA)再次进行实验而得到的结果示于图5-8中。
根据图5-8的结果,总共选择了能够有效降低每种HLA的表达的13个gRNA,其中针对各自的靶标(HLA-ABC、HLA-DQ、HLA-DP、以及HLA-DR)选择了2至4个gRNA。具体而言,HLA-ABC选择了B2M-01gRNA、B2M-07gRNA、B2M-18gRNA和B2M-27gRNA;HLA-DQ选择了DQA-14、DQA-15、DQA-37和DQA-40;HLA-DP选择了DPA-07和DPA-13;HLA-DR选择了DRA-18、DRA-20和DRA-58。
实施例1.4鉴定靶基因的基因组DNA中的突变
为了鉴定使用流式细胞术选择的靶向HLA的gRNA是否引起基因组DNA突变,根据制造商的说明,使用Guide-it突变检测试剂盒(Guide-it Mutation Detection Kit,Clontech,631443)对基因组DNA进行了分析。
也就是说,将RNP复合物转染的Raji细胞系和对照组Raji细胞系各5×105个细胞以1200rpm离心5分钟,然后除去上清液。然后,向其中加入90μL包含在Guide-it突变检测试剂盒(Clontech,631443)中的提取缓冲液1,并在95℃下孵育10分钟。然后,向其中加入10μL包含在Guide-it突变检测试剂盒(Clontech,631443)中的提取缓冲液2,并将通过移液得到的DNA裂解物在纯水中以1:8的比例稀释以进行PCR。使用稀释的DNA裂解物用PCR热循环仪(FlexCycler2,Analytik Jena)进行PCR,用于靶基因组的选择的gRNA和DNA的分析PCR引物如下表5所示。
使用以下PCR参数来生产PCT产物:在98℃下进行预变性2分钟,随后在98℃下10秒、在60℃下15秒、以及在68℃下1分钟的条件下进行变性和退火,35个循环,随后在68℃延伸5分钟。为了使得到的PCR产物变性和再杂交,将5μL用于PCR的纯水添加到10μL PCR产物中。随后,在95℃下孵育5分钟,然后在温度从95℃每秒降低2℃至85℃和从85℃每秒降低0.1℃至25℃的情况下改变温度。最后,向其中加入1μL的Guide-it Resolvase,并在37℃进行30分钟孵育。然后,在1.5%琼脂糖凝胶上进行电泳。结果在图9-12中示出。
在电泳结果中鉴定了靶向HLA的gRNA转染的细胞的PCT产物中的Guide-itResolvase切割的DNA片段。从这些结果中发现,选择的13种靶向HLA的gRNA诱导了其靶基因组DNA的突变。
[表5]
这样,选择出能够通过在Raji细胞中转染而有效降低各个HLA的表达的gRNA。在实施例2和3中,使用选择的gRNA来制备转化的NK细胞,然后鉴定其功效。
实施例2.缺失HLA-I的细胞的制备与鉴定
实施例2.1.NK-92MI细胞系中HLA-1的缺失
将37.5μg B2M-01gRNA在65℃下孵育10分钟以形成单链。然后,向其中加入37.5μg的Cas9蛋白(Toolgen,TGEN_CP3),并在25℃下进行10分钟孵育来制备Cas9-gRNA复合物(RNP复合物)。使用细胞系核转染试剂盒R(Cell Line nucleofector Kit R,Lonza,VCA-1001)用NucleofectorTM 2b(Lonza,AAB-1001)将RNP复合物转染到具有2×106个细胞的NK-92MI细胞系中。将转染的细胞孵育3天,然后使用细胞分离器进行细胞分离。
实施例2.2.HLA I阴性细胞的分离
将B2M-01RNP复合物转染的NK-92MI细胞系转移到5-mL试管中,然后用PE抗HLA-ABC(Miltenyi Biotec,130-101-448)和7-AAD(Beckman Coulter,Inc.,A07704)处理。然后,遮光30分钟,并在4℃下进行孵育。使用顶部过滤FACS试管(Falcon,352235)过滤染色的细胞,然后使用FACS Aria II(BD)分离HLA-1阳性细胞和HLA-1阴性细胞。结果示出在图13中。鉴定出HLA-1阴性细胞的纯度为95.9%,HLA-1阳性细胞的纯度为97.2%。
实施例2.3.缺失HLA-I的NK-92MI细胞系的细胞杀伤能力的评估
使用在细胞分离后各孵育4天的HLA-1阳性细胞和HLA-1阴性细胞,比较两者对K562细胞系的细胞杀伤能力。根据制造商的说明,将K562细胞系用30μM钙黄绿素-AM(Calcein-AM;Invitrogen,C3099)染色,然后与NK-92MI细胞系在U型底板上以10:1、3:1、1:1、或0.3:1的E:T比进行孵育。4小时后,每个孵育物取100μL,并用荧光计(VictorTMX3,PerkinElmer)测量细胞死亡分泌的钙黄绿素-AM的量。结果如图14所示,鉴定出HLA-1阳性细胞和HLA-1阴性细胞对K562细胞系的细胞杀伤能力是相当的。从这些结果发现,HLA-1的缺失不影响细胞杀伤能力。
实施例3缺失HLA-I的细胞和缺失HLA-II的细胞的制备与鉴定
实施例3.1.NK细胞的制备和孵育
将冷冻保存的外周血单核细胞(Peripheral blood mononuclear cell,PBMC)快速溶于37℃水浴中,然后转移至50-mL锥形管中。随着管的摇动,将解冻培养基(RPMI,11875-093+10%FBS+55μMβ-ME)滴加到管中并混合。随后,在4℃以1,200rpm离心10分钟以除去上清液,并重悬于10mL CellGro SCGM(CELLGENIX,2001)培养基中。然后,对细胞数量进行定量。将细胞以1×106个细胞/mL的浓度重悬于培养基(CellGro SCGM+10ng/mL OKT3+500IU/mL IL-2+5%人血浆)中,然后放入培养袋(NIPRO,87-352)。在37℃,CO2培养箱中进行24小时孵育,然后进行转染。
实施例3.2.T细胞的制备和孵育方法
将冷冻保存的外周血单核细胞(Peripheral blood mononuclear cell,PBMC)快速溶于37℃水浴中,然后转移至50-mL锥形管中。随着管的摇动,将解冻培养基(RPMI,11875-093+10%FBS+55μMβ-ME)滴加到管中并混合。随后,在4℃下以1200rpm离心10分钟以除去上清液,并重悬于40mL MACS缓冲液(PBS+0.5%FBS+2mM EDTA)中。然后,对细胞数量进行定量。每107个细胞用20μL CD3微珠(Miltenyi Biotec,130-050-101)进行处理。然后,遮光15分钟,并在4℃孵育。随后,在4℃以1,350rpm离心8分钟以除去上清液,并将所得物重悬于500μL MACS缓冲液中。然后,将重悬液加载到安装在QuadroMACS分离器(MiltenyiBiotec,130-090-976)上的LS柱(Miltenyi Biotec,130-042-401)上。将LS柱用MACS缓冲液洗涤3次,然后从QuadroMACS分离器中移出。然后,用柱塞挤压移出的LS柱以得到CD3阳性细胞。将细胞以1×106个细胞/mL的浓度重悬于T细胞培养基(X-VIVO15(Lonza,BE02-060Q)+40μL/mL Dynabeads人T-活化剂CD3/CD28(Gibco,111.31D)+200IU/mL IL-2+5%人血浆)中,然后放入培养袋(NIPRO,87-352)。在37℃,CO2培养箱中进行24小时孵育,然后进行转染。
实施例3.3.使用选择的gRNA制备缺失HLA的NK细胞和T细胞
将37.5μg的各gRNA在65℃下孵育10分钟以形成单链。然后,向其中加入37.5μg的Cas9蛋白(Clontech,M0646T),并在25℃下孵育10分钟来制备Cas9-gRNA复合物(RNP复合物)。在多重缺失的情况下,将相应的gRNA的量的总和设为37.5μg。使用P3 Primary CellX Kit L(Lonza,V4XP-3024)用4D-NucleofectorTMX Unit(Lonza,AAF-1002X)将RNP复合物转染到2×106个细胞中。将转染的细胞孵育3天,然后观察到细胞因子的产生。将转染的细胞孵育14天,然后通过流式细胞术鉴定HLA表达是否降低。
实施例3.4.使用流式细胞术鉴定HLA的表达降低
将RNP复合物转染的细胞和对照组细胞各2×105个细胞悬浮在100μL FACS缓冲液(1%FBS/鞘缓冲液)中,并在5-mL管中制备。进行3次以上细胞染色。对于初次染色,使用了抗HLA-DP(Abcam,ab20897);对于二次染色,使用了PE山羊抗小鼠IgG(eBioscience,12-4010-82);对于三次染色,使用了V450抗CD4(BD,560345)、APC-Cy7抗CD8(BD,557834)、BV510抗HLA-ABC(Biolegend,311436)、PE-Cy7抗HLA-DR(eBioscience,25-9952-42)、Alexa647抗HLA-DQ(BD,564806)。另一方面,在NK细胞的情况下,使用BV421抗CD56(Biolegend,318328)代替V450抗CD4。每次进行抗体处理后,遮光30分钟,并在4℃下进行孵育。此后,向其中加入3mL FACS缓冲液,并在4℃以2,000rpm离心3分钟以除去上清液。获得所有染色的样品并用LSR Fortessa进行分析。结果在图15-17中示出。
使用B2M-01、DRA-20、DQA-14和DPA-13作为用于缺失相应的HLA的gRNA。从图15的结果中发现,在用单个gRNA进行转染时,以至少70%和至多99%的高效率实现了靶HLA的缺失。从图16的结果中发现,与单个缺失的效率相比,多重缺失的效率没有显著降低。当将多重缺失的效率与单个缺失的效率进行比较时,效率通过用这样得到的值乘以100来表示:将单个缺失的“负%”值除以多重缺失的“负%”值。另外,参考图17中的结果发现,RNP复合物转染的细胞的14天孵化率与对照组细胞的14天孵化率相似。特别地,鉴定出单个gRNA(DPA-13)转染的细胞与多个gRNA转染的细胞之间在孵化率方面没有差异。
实施例3.5.缺失HLA的T细胞和NK细胞的活性分析
用PMA、离子霉素(细胞刺激混合物,eBioscience,00-4970-03)和APC抗CD107α(BD,560664)对RNP复合物转染的细胞和对照细胞各1×106个细胞进行处理,随后进行孵育,或与APC抗CD107α和2×105个K562细胞一起孵育。5小时后,用PerCP-Cy5.5抗CD3(Tonbo,65-0038-T100)、BV421抗CD56(Biolegend,318328)、FITC抗B2M(Biolegend,316304)、APC-Cy7抗HLA-ABC(Biolegend,311426)、以及PE抗-HLA-DR/DP/DQ(MiltenyiBiotec,130-104-827)进行处理。然后,遮光30分钟,并在4℃下进行孵育以便进行表面染色。
此后,向其中加入3mL FACS缓冲液,并在4℃下以2,000rpm离心3分钟以除去上清液。然后,使用BD Cytofix/CytopermTM缓冲液(BD,554722)进行30分钟固定和渗透。用1XPerm/Wash缓冲液(BD,554723)洗涤两次,并用PE-Cy7抗TNF-α(eBioscience,25-7349-82)和V500抗IFN-γ(BD,554701)进行处理。遮光30分钟,并在4℃下进行孵育以便进行胞内染色。随后,向其中加入3mL FACS缓冲液,并在4℃以2,000rpm离心3分钟以除去上清液。然后,用1X Perm/Wash缓冲液洗涤两次,并用流式细胞术分析T细胞和NK细胞中的细胞因子产生。结果在图18和图19中示出。
在图18中,已鉴定出即使缺失了HLA,T细胞活化时分泌的TNF-α、IFN-γ和CD107α的量也与HLA阳性细胞并无不同。同样在图19中,已鉴定出即使缺失了HLA,NK细胞活化时分泌的TNF-α、IFN-γ和CD107α的量也与HLA阳性细胞并无不同。根据这些结果发现,即使缺失了HLA-1和HLA-II,NK细胞的活性也得以保留。
实施例4.表达HLA-E的载体的合成及表达鉴定
实施例4.1.NK细胞对缺失HLA-I的Raji细胞系的细胞杀伤能力的评估
为了鉴定NK细胞在缺失HLA-1的细胞中的细胞杀伤能力是否增加,使用B2M-01RNP复合物对Raji细胞系进行转染,并使用细胞分离器分离HLA-1阳性细胞和HLA-1阴性细胞。根据制造商的说明,将各细胞用钙黄绿素-AM染色,然后与NK-92MI细胞系在U型底板上以10:1、3:1、1:1、或0.3:1的E:T比进行孵育。5小时后,用荧光计测量细胞死亡分泌的钙黄绿素-AM的量。如图20所示,已鉴定出NK细胞在HLA-1阴性细胞中相比在HLA-1阳性细胞中的细胞杀伤能力增加。
实施例4.2.HLA-E载体的合成
为了避免如实施例4.1中的当用B2M RNP复合物对细胞进行转染以缺失HLA-1时发生的由NK细胞引起的细胞杀伤现象,合成了用于将HLA-E引入细胞的HLA-E载体。也就是说,合成了转化的HLA-E(G-B2M-HLA-E),其中B2M(SEQ ID NO:237)经由三个第一G4S接头(SEQID NO:238)连接至与B2M信号肽(B2M SS;SEQ ID NO:235)相连的G肽(SEQ ID NO:236),并且B2M经由四个第二G4S接头(SEQ ID NO:241)连接至附有HA标签(SEQ ID NO:239)的HLA-E(SEQ ID NO:240)。相应的序列示于下表6中。通过***pLVX-EF1α-IRES-Puro载体(Clontech,631988)中,克隆合成的转化的HLA-E,其结构如图21所示。
[表6]
实施例4.3.HLA-E通过转导进入K562细胞系的表达及其鉴定
将***有转化的HLA-E的pLVX-EF1α-IRES-Puro载体与慢病毒包装载体一起转染到293T细胞系中。3天后,通过0.45-μm过滤器得到慢病毒上清液。用慢病毒上清液对K562细胞系进行处理,并在32℃以3,000rpm离心1小时。3天后,将1×106个细胞转移到5-mL管中,并用PE-Cy7抗HLA-E(Biolegend,342608)和APC抗B2M(Biolegend,316312)进行细胞表面染色30分钟。用FACS缓冲液洗涤,然后用流式细胞术检查HLA-E和B2M的表达。从图22可以看出,已鉴定出在表达转化的HLA-E的K562细胞系中HLA-E和B2M以高水平表达。
实施例4.4.在引入HLA-E的细胞中的细胞杀伤能力评估
根据制造商的说明,用钙黄绿素-AM对表达转化的HLA-E的K562细胞系和对照组K562细胞系分别进行染色,然后将1×104个细胞与NK细胞在U型底板上以10:1、3:1、1:1、或0.3:1的E:T比进行孵育。5小时后,从各培养物中取出100μL,并用荧光计(VictorTMX3,PerkinElmer)测量由细胞死亡分泌的钙黄绿素-AM的量。
从图23可以看出,已鉴定出与对照组K562细胞系(K562)相比,就表达了转化的HLA-E的K562细胞系(K562G-B2M-HLA-E)而言,由NK细胞导致的细胞死亡现象明显减少。从这些结果发现,HLA-E的表达可以防止由NK细胞导致的细胞死亡。
实施例5.将HLA-E引入缺失HLA-I的NK细胞和缺失HLA-II的NK细胞
使用如实施例4.2中制备的HLA-E载体,将与G肽结合的HLA-E引入在实施例3中制备的缺失HLA-I的NK细胞和缺失HLA-II的NK细胞,以制备转化的NK细胞。
<110> 财团法人牧岩生命科学研究所
<120> 转化的人细胞及其用途
<130> PCB711063MOG
<150> US 62/587,068
<151> 2017-11-16
<160> 259
<170> KoPatentIn 3.0
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<213> 人工序列
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<400> 2
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<212> RNA
<213> 人工序列
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<223> gRNA of B2M-04
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<212> RNA
<213> 人工序列
<220>
<223> gRNA of B2M-05
<400> 4
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<210> 5
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<213> 人工序列
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ggccacggag cgagacaucu 20
<210> 7
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<212> RNA
<213> 人工序列
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<400> 7
ggccgagaug ucucgcuccg 20
<210> 8
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<213> 人工序列
<220>
<223> gRNA of B2M-09
<400> 8
ucacgucauc cagcagagaa 20
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<213> 人工序列
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<400> 9
acaaagucac augguucaca 20
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<213> 人工序列
<220>
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<400> 10
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<213> 人工序列
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<400> 11
aagucaacuu caaugucgga 20
<210> 12
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<213> 人工序列
<220>
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<400> 12
cauacucauc uuuuucagug 20
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<213> 人工序列
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<213> 人工序列
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<400> 15
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<400> 16
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<213> 人工序列
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<213> 人工序列
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<400> 20
aagaaaagga aacugaaaac 20
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<213> 人工序列
<220>
<223> gRNA of B2M-22
<400> 21
aagaaggcau gcacuagacu 20
<210> 22
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<213> 人工序列
<220>
<223> gRNA of B2M-23
<400> 22
acauguaagc agcaucaugg 20
<210> 23
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of B2M-24
<400> 23
acccagacac auagcaauuc 20
<210> 24
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of B2M-25
<400> 24
acuugucuuu cagcaaggac 20
<210> 25
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of B2M-26
<400> 25
caagccagcg acgcagugcc 20
<210> 26
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of B2M-27
<400> 26
cacagcccaa gauaguuaag 20
<210> 27
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<213> 人工序列
<220>
<223> gRNA of B2M-29
<400> 27
caucacgaga cucuaagaaa 20
<210> 28
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<212> RNA
<213> 人工序列
<220>
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<400> 28
cgcagugcca gguuagagag 20
<210> 29
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<212> RNA
<213> 人工序列
<220>
<223> gRNA of B2M-31
<400> 29
cuaaccuggc acugcgucgc 20
<210> 30
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<213> 人工序列
<220>
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<400> 30
gaaagucccu cucucuaacc 20
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<213> 人工序列
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<400> 31
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<210> 32
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<213> 人工序列
<220>
<223> gRNA of B2M-34
<400> 32
gagucucgug auguuuaaga 20
<210> 33
<211> 20
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<213> 人工序列
<220>
<223> gRNA of B2M-35
<400> 33
gcagugccag guuagagaga 20
<210> 34
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<213> 人工序列
<220>
<223> gRNA of B2M-36
<400> 34
uaagaaggca ugcacuagac 20
<210> 35
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<213> 人工序列
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<400> 35
ucgaucuaug aaaaagacag 20
<210> 36
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<213> 人工序列
<220>
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<400> 36
uucagacuug ucuuucagca 20
<210> 37
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<212> RNA
<213> 人工序列
<220>
<223> gRNA of B2M-40
<400> 37
uuccugaauu gcuauguguc 20
<210> 38
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<212> RNA
<213> 人工序列
<220>
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<400> 38
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<210> 39
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<213> 人工序列
<220>
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<400> 39
cuggcacugc gucgcuggcu 20
<210> 40
<211> 20
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<213> 人工序列
<220>
<223> gRNA of B2M-43
<400> 40
ugcgucgcug gcuuggagac 20
<210> 41
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<213> 人工序列
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<223> gRNA of B2M-44
<400> 41
gcuggcuugg agacagguga 20
<210> 42
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<212> RNA
<213> 人工序列
<220>
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<400> 42
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<210> 43
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<212> RNA
<213> 人工序列
<220>
<223> gRNA of B2M-46
<400> 43
caaucaggac aaggcccgca 20
<210> 44
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<213> 人工序列
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<400> 44
ccugcgggcc uuguccugau 20
<210> 45
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<213> 人工序列
<220>
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<400> 45
ccaaucagga caaggcccgc 20
<210> 46
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<213> 人工序列
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<400> 46
cgggccuugu ccugauuggc 20
<210> 47
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<213> 人工序列
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<400> 47
gggccuuguc cugauuggcu 20
<210> 48
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<213> 人工序列
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<400> 48
gugcccagcc aaucaggaca 20
<210> 49
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<400> 49
aaacgcgugc ccagccaauc 20
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<400> 50
gggcacgcgu uuaauauaag 20
<210> 51
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<400> 51
cacgcguuua auauaagugg 20
<210> 52
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<400> 52
uauaagugga ggcgucgcgc 20
<210> 53
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<400> 53
aaguggaggc gucgcgcugg 20
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aguggaggcg ucgcgcuggc 20
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uuccugaagc ugacagcauu 20
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<400> 56
uccugaagcu gacagcauuc 20
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<400> 57
ugggcuguga caaagucaca 20
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acucucucuu ucuggccugg 20
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uuaggaucau ccucuuccca 20
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<400> 60
aacucuaccg cugcuaccaa 20
<210> 61
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acaaugucuu caccuccaca 20
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accaccguga ugagccccug 20
<210> 63
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<400> 63
acccaguguc acgggagacu 20
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<400> 64
accuccacag gggcucauca 20
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<400> 65
caaugucuuc accuccacag 20
<210> 66
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<400> 66
cacaaugucu ucaccuccac 20
<210> 67
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<400> 67
caguacaccc augaauuuga 20
<210> 68
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<400> 68
cucugugagc ucugacauag 20
<210> 69
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<400> 69
cuguggaggu gaagacauug 20
<210> 70
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<400> 70
ggcuggaauc ucaggcucug 20
<210> 71
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<400> 71
guugggcuga cccaguguca 20
<210> 72
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<400> 72
ucaugggugu acuggccaga 20
<210> 73
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<400> 73
uccaagucuc ccgugacacu 20
<210> 74
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<400> 74
uccacagggg cucaucacgg 20
<210> 75
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<400> 75
uguggaggug aagacauugu 20
<210> 76
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<213> 人工序列
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<400> 76
uuccaagucu cccgugacac 20
<210> 77
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-27
<400> 77
uugggcugac ccagugucac 20
<210> 78
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-28
<400> 78
aacaucacau ggcugagcaa 20
<210> 79
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-29
<400> 79
acaucacaug gcugagcaau 20
<210> 80
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<213> 人工序列
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<400> 80
agccauguga uguugaccac 20
<210> 81
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<400> 81
aggaaugauc acucuuggag 20
<210> 82
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<213> 人工序列
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<400> 82
aucacucuug gagaggaagc 20
<210> 83
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<400> 83
augacugcaa gguggagcac 20
<210> 84
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<400> 84
caagguggag cacuggggcc 20
<210> 85
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<400> 85
caucaaauuc auggguguac 20
<210> 86
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<400> 86
caugugaugu ugaccacagg 20
<210> 87
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<400> 87
ccucaccaca gagguuccug 20
<210> 88
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<213> 人工序列
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<400> 88
cucaucucca ucaaauucau 20
<210> 89
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<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-39
<400> 89
cuccuguggu caacaucaca 20
<210> 90
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-40
<400> 90
gaagaaggaa ugaucacucu 20
<210> 91
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-41
<400> 91
gacugcaagg uggagcacug 20
<210> 92
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-42
<400> 92
gagguaacug aucuugaaga 20
<210> 93
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-43
<400> 93
ggacaacauc uuuccuccug 20
<210> 94
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-44
<400> 94
gugcuguuuc cucaccacag 20
<210> 95
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-45
<400> 95
ucuucugaaa cacuggggua 20
<210> 96
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-47
<400> 96
uucaugggug uacuggccag 20
<210> 97
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-48
<400> 97
agagacugug gucugcgccc 20
<210> 98
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-49
<400> 98
gacauagggg cuggaaucuc 20
<210> 99
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-50
<400> 99
gagacugugg ucugcgcccu 20
<210> 100
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-51
<400> 100
ggccucgugg gcauuguggu 20
<210> 101
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-52
<400> 101
gggccucgug ggcauugugg 20
<210> 102
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-53
<400> 102
gucagagcuc acagagacug 20
<210> 103
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-54
<400> 103
gucucuguga gcucugacau 20
<210> 104
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-55
<400> 104
gugagcucug acauaggggc 20
<210> 105
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-56
<400> 105
guuggugcuu ccagacacca 20
<210> 106
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-57
<400> 106
ucucugugag cucugacaua 20
<210> 107
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-58
<400> 107
ugacugcaag guggagcacu 20
<210> 108
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-59
<400> 108
ugcccaccac aaugcccacg 20
<210> 109
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-60
<400> 109
uggaagcacc aacugaacgc 20
<210> 110
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-61
<400> 110
ugugggccuc gugggcauug 20
<210> 111
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-62
<400> 111
uuaccccagu guuucagaag 20
<210> 112
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-63
<400> 112
uuggaaaaca cugugaccuc 20
<210> 113
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-64
<400> 113
uuggugcuuc cagacaccaa 20
<210> 114
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-65
<400> 114
aaacaaagcu cugcugcugg 20
<210> 115
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-66
<400> 115
aaaucucauc agcagaaggg 20
<210> 116
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DQA-67
<400> 116
cuaaacaaag cucugcugcu 20
<210> 117
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-01
<400> 117
ucuaugcguc uguacaaacg 20
<210> 118
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-02
<400> 118
guacagacgc auagaccaac 20
<210> 119
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-03
<400> 119
uacagacgca uagaccaaca 20
<210> 120
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-04
<400> 120
gaaggagacc gucuggcauc 20
<210> 121
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-05
<400> 121
ggagaccguc uggcaucugg 20
<210> 122
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-06
<400> 122
gucuggcauc uggaggaguu 20
<210> 123
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-07
<400> 123
gugguuggaa cgcuggauca 20
<210> 124
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-08
<400> 124
gucuucaggg cgcauguugu 20
<210> 125
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-09
<400> 125
ugucuucagg gcgcauguug 20
<210> 126
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-10
<400> 126
ucuucagggc gcauguugug 20
<210> 127
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-11
<400> 127
guugcauacc ccagugcuug 20
<210> 128
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-12
<400> 128
gaccuuugug cccucagcag 20
<210> 129
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-13
<400> 129
gagacucagc aggaaagcca 20
<210> 130
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-14
<400> 130
gagccucaaa ggaaaaggcu 20
<210> 131
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-15
<400> 131
gaucuugaga gcccucuccu 20
<210> 132
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-16
<400> 132
gccaucaagg gugagugcuc 20
<210> 133
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-17
<400> 133
gccaugaccc ccgggcccag 20
<210> 134
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-18
<400> 134
gcccagcucc acaggcuccu 20
<210> 135
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-19
<400> 135
gcccugagcc ucaaaggaaa 20
<210> 136
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-20
<400> 136
gccuuuuccu uugaggcuca 20
<210> 137
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-21
<400> 137
gcguucuggc caugaccccc 20
<210> 138
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-22
<400> 138
gcuuuccugc ugagucuccg 20
<210> 139
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-23
<400> 139
ggaaacacgg ucaccucagg 20
<210> 140
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-24
<400> 140
ggacuucuau gacugcaggg 20
<210> 141
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-25
<400> 141
ggagacugug cucugugccc 20
<210> 142
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-26
<400> 142
ggccaugacc cccgggccca 20
<210> 143
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-27
<400> 143
ggccuagucg gcaucaucgu 20
<210> 144
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-29
<400> 144
gggaaacacg gucaccucag 20
<210> 145
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-30
<400> 145
gggccuaguc ggcaucaucg 20
<210> 146
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-31
<400> 146
gucauagaag uccucugcug 20
<210> 147
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-32
<400> 147
guccucugcu gagggcacaa 20
<210> 148
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-33
<400> 148
guggaagcug uaaucuguuc 20
<210> 149
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-34
<400> 149
gugggaagaa cuugucaaug 20
<210> 150
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-35
<400> 150
guugguggcc ugaguguggu 20
<210> 151
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-36
<400> 151
guugucucag gcaucuggau 20
<210> 152
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-37
<400> 152
gcugagucuc cgaggagcug 20
<210> 153
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-38
<400> 153
ucucuacugu cuuuaugcag 20
<210> 154
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-39
<400> 154
uauggaacau ucugucuuca 20
<210> 155
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-40
<400> 155
ucaagaucac agcucugaua 20
<210> 156
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-41
<400> 156
ucaaacauaa acuccccugu 20
<210> 157
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-42
<400> 157
uaccguuggu ggccugagug 20
<210> 158
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-43
<400> 158
uccugagcac ucacccuuga 20
<210> 159
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-44
<400> 159
ugaggugacc guguuuccca 20
<210> 160
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-45
<400> 160
ugcguucugg ccaugacccc 20
<210> 161
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-46
<400> 161
uuuccuuuga ggcucagggc 20
<210> 162
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-47
<400> 162
ugccgacuag gcccagcacc 20
<210> 163
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-48
<400> 163
ucagcaggaa agccaaggag 20
<210> 164
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-49
<400> 164
ugaagaugag auguucuaug 20
<210> 165
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-50
<400> 165
ugcugagucu ccgaggagcu 20
<210> 166
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-51
<400> 166
ugagauguuc uauguggauc 20
<210> 167
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-52
<400> 167
ugggaaacac ggucaccuca 20
<210> 168
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-53
<400> 168
uggaagcugu aaucuguucu 20
<210> 169
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-54
<400> 169
uggacaagaa ggagaccguc 20
<210> 170
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-55
<400> 170
ugcccacgau gaugccgacu 20
<210> 171
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-56
<400> 171
uggccaagcc uuuuccuuug 20
<210> 172
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-57
<400> 172
guggcugugc aacggggagc 20
<210> 173
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-58
<400> 173
uccccugggc ccggggguca 20
<210> 174
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-59
<400> 174
ucaccucagg gggaucugga 20
<210> 175
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DPA-60
<400> 175
ucuccuucca gaucccccug 20
<210> 176
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-08
<400> 176
aagaagaaaa uggccauaag 20
<210> 177
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-09
<400> 177
aaucaugggc uaucaaaggu 20
<210> 178
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-10
<400> 178
agcugugcug augagcgcuc 20
<210> 179
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-11
<400> 179
auaaguggag ucccugugcu 20
<210> 180
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-12
<400> 180
acuuauggcc auuuucuucu 20
<210> 181
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-13
<400> 181
augaugaaaa auccuagcac 20
<210> 182
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-14
<400> 182
cagagcgccc aagaagaaaa 20
<210> 183
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-15
<400> 183
caggaaucau gggcuaucaa 20
<210> 184
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-16
<400> 184
cuuauggcca uuuucuucuu 20
<210> 185
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-17
<400> 185
gacugucucu gacacuccug 20
<210> 186
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-18
<400> 186
gagccucuuc ucaagcacug 20
<210> 187
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-19
<400> 187
gauaguggaa cuugcggaaa 20
<210> 188
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-20
<400> 188
gaugagcgcu caggaaucau 20
<210> 189
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-21
<400> 189
gcuaucaaag guaggugcug 20
<210> 190
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-22
<400> 190
guuaccucug gagguacugg 20
<210> 191
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-23
<400> 191
uagcacaggg acuccacuua 20
<210> 192
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-24
<400> 192
ugaugaaaaa uccuagcaca 20
<210> 193
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-25
<400> 193
ugaugagcgc ucaggaauca 20
<210> 194
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-27
<400> 194
uuugccagcu uugaggcuca 20
<210> 195
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-28
<400> 195
aacuauacuc cgaucaccaa 20
<210> 196
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-29
<400> 196
agaagaacau gugaucaucc 20
<210> 197
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-30
<400> 197
agcagagagg gagguaccau 20
<210> 198
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-31
<400> 198
agcgcuuugu caugauuucc 20
<210> 199
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-32
<400> 199
agcuguggac aaagccaacc 20
<210> 200
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-33
<400> 200
agggagguac cauuggugau 20
<210> 201
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-34
<400> 201
auaaacucgc cugauugguc 20
<210> 202
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-35
<400> 202
auuggugauc ggaguauagu 20
<210> 203
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-36
<400> 203
ccauguggau auggcaaaga 20
<210> 204
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-37
<400> 204
cuuugaggcu caaggugcau 20
<210> 205
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-38
<400> 205
cuuugucaug auuuccaggu 20
<210> 206
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-39
<400> 206
ggauauggca aagaaggaga 20
<210> 207
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-40
<400> 207
uaucugaauc cugaccaauc 20
<210> 208
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-41
<400> 208
ugagauuuuc cauguggaua 20
<210> 209
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-42
<400> 209
ugaucacaug uucuucugaa 20
<210> 210
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-43
<400> 210
ugcaccuuga gccucaaagc 20
<210> 211
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-44
<400> 211
ugcauuggcc aacauagcug 20
<210> 212
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-45
<400> 212
uggacgauuu gccagcuuug 20
<210> 213
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-46
<400> 213
uggcaaagaa ggagacgguc 20
<210> 214
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-47
<400> 214
uggugaugag auuuuccaug 20
<210> 215
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-48
<400> 215
aaugucacgu ggcuucgaaa 20
<210> 216
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-49
<400> 216
agacaaguuc accccaccag 20
<210> 217
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-50
<400> 217
agacaaguuc accccaccag 20
<210> 218
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-51
<400> 218
gaacgcaggg ggccucugua 20
<210> 219
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-52
<400> 219
cugaggacgu uuacgacugc 20
<210> 220
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-53
<400> 220
gcggaaaagg uggucuuccc 20
<210> 221
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-54
<400> 221
ggacguuuac gacugcaggg 20
<210> 222
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-55
<400> 222
gucguaaacg uccucaguug 20
<210> 223
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-56
<400> 223
gugagcacag uuaccucugg 20
<210> 224
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-57
<400> 224
guguccccca guaccuccag 20
<210> 225
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-58
<400> 225
ugaggacguu uacgacugca 20
<210> 226
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-59
<400> 226
aauggaaaac cugucaccac 20
<210> 227
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-60
<400> 227
aguggaacuu gcggaaaagg 20
<210> 228
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-61
<400> 228
augaaacaga ugaggacguu 20
<210> 229
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-62
<400> 229
cagagacagu cuuccugccc 20
<210> 230
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-63
<400> 230
cgugacauug accacuggug 20
<210> 231
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-64
<400> 231
uaugaaacag augaggacgu 20
<210> 232
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-65
<400> 232
ucugacacuc cuguggugac 20
<210> 233
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-66
<400> 233
aaacguccuc aguugagggc 20
<210> 234
<211> 20
<212> RNA
<213> 人工序列
<220>
<223> gRNA of DRA-67
<400> 234
ucguaaacgu ccucaguuga 20
<210> 235
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> B2M 信号肽
<400> 235
Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser
1 5 10 15
Gly Leu Glu Ala
20
<210> 236
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> G肽
<400> 236
Val Met Ala Pro Arg Thr Leu Phe Leu
1 5
<210> 237
<211> 99
<212> PRT
<213> 人工序列
<220>
<223> B2M
<400> 237
Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu
1 5 10 15
Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro
20 25 30
Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys
35 40 45
Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu
50 55 60
Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys
65 70 75 80
Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp
85 90 95
Arg Asp Met
<210> 238
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> G4S 接头 1
<400> 238
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 239
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> HA 标签
<400> 239
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1 5
<210> 240
<211> 337
<212> PRT
<213> 人工序列
<220>
<223> HLA-E
<400> 240
Gly Ser His Ser Leu Lys Tyr Phe His Thr Ser Val Ser Arg Pro Gly
1 5 10 15
Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln
20 25 30
Phe Val Arg Phe Asp Asn Asp Ala Ala Ser Pro Arg Met Val Pro Arg
35 40 45
Ala Pro Trp Met Glu Gln Glu Gly Ser Glu Tyr Trp Asp Arg Glu Thr
50 55 60
Arg Ser Ala Arg Asp Thr Ala Gln Ile Phe Arg Val Asn Leu Arg Thr
65 70 75 80
Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln
85 90 95
Trp Met His Gly Cys Glu Leu Gly Pro Asp Gly Arg Phe Leu Arg Gly
100 105 110
Tyr Glu Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Leu Thr Leu Asn Glu
115 120 125
Asp Leu Arg Ser Trp Thr Ala Val Asp Thr Ala Ala Gln Ile Ser Glu
130 135 140
Gln Lys Ser Asn Asp Ala Ser Glu Ala Glu His Gln Arg Ala Tyr Leu
145 150 155 160
Glu Asp Thr Cys Val Glu Trp Leu His Lys Tyr Leu Glu Lys Gly Lys
165 170 175
Glu Thr Leu Leu His Leu Glu Pro Pro Lys Thr His Val Thr His His
180 185 190
Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe
195 200 205
Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Gln Asp Gly Glu Gly His
210 215 220
Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr
225 230 235 240
Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg
245 250 255
Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Val Thr Leu
260 265 270
Arg Trp Lys Pro Ala Ser Gln Pro Thr Ile Pro Ile Val Gly Ile Ile
275 280 285
Ala Gly Leu Val Leu Leu Gly Ser Val Val Ser Gly Ala Val Val Ala
290 295 300
Ala Val Ile Trp Arg Lys Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr
305 310 315 320
Ser Lys Ala Glu Trp Ser Asp Ser Ala Gln Gly Ser Glu Ser His Ser
325 330 335
Leu
<210> 241
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> G4S 接头 2
<400> 241
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 242
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 正向引物
<400> 242
ctggcttgga gacaggtgac 20
<210> 243
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 反向引物
<400> 243
gacgcttatc gacgccctaa 20
<210> 244
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 正向引物
<400> 244
cccaagtgaa ataccctggc a 21
<210> 245
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 反向引物
<400> 245
agcccttcct actagcctca 20
<210> 246
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 正向引物
<400> 246
aatttcttgg ggagggggtg 20
<210> 247
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 反向引物
<400> 247
agctggatag taggagaaga cagt 24
<210> 248
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 正向引物
<400> 248
gggttaaaga gtctgtccgt ga 22
<210> 249
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 反向引物
<400> 249
tgtcgagacc acataatacc tgt 23
<210> 250
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 正向引物
<400> 250
acctgacttg gcagggtttg 20
<210> 251
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 反向引物
<400> 251
cccaagatct accaccggag a 21
<210> 252
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 正向引物
<400> 252
tgctcccaag cagaaggtaa 20
<210> 253
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 反向引物
<400> 253
aacccatgaa gtgtggaaaa caag 24
<210> 254
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 正向引物
<400> 254
tccctccata ccagggttca 20
<210> 255
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 反向引物
<400> 255
aactcatcct taccccagtg t 21
<210> 256
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 正向引物
<400> 256
tgtgtcaact tatgccgcgt 20
<210> 257
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 反向引物
<400> 257
ttgggaaaca cggtcacctc 20
<210> 258
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 正向引物
<400> 258
tgtgaactgg agctctcttg a 21
<210> 259
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 反向引物
<400> 259
tatgagggcc agagggaaca t 21
Claims (28)
1.向导RNA,所述向导RNA与编码β2-微球蛋白(B2M)的核酸序列互补结合,所述向导RNA包含选自由SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:17、以及SEQ ID NO:26组成的组的任何一个核酸序列。
2.向导RNA,所述向导RNA与编码HLA-DQ的核酸序列互补结合,所述向导RNA包含选自由SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:87、以及SEQ ID NO:90组成的组的任何一个核酸序列。
3.向导RNA,所述向导RNA与编码HLA-DP的核酸序列互补结合,所述向导RNA包含SEQ IDNO:123或SEQ ID NO:129的核酸序列。
4.向导RNA,所述向导RNA与编码HLA-DR的核酸序列互补结合,所述向导RNA包含选自由SEQ ID NO:186、SEQ ID NO:188和SEQ ID NO:225组成的组的任何一个核酸序列。
5.用于抑制基因表达的组合物,所述组合物包含以下作为活性成分:
根据权利要求1至4中任一项所述的向导RNA或编码所述向导RNA的核苷酸序列;和
RNA引导的核酸内切酶或编码所述RNA引导的核酸内切酶的核苷酸序列。
6.根据权利要求5所述的组合物,其特征在于,所述RNA引导的核酸内切酶是任何一个选自由以下组成的组的RNA引导的核酸内切酶:Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9、Cas10、Cas12a、Cas12b、Cas12c、Cas12d、Cas12e、Cas13a、Cas13b、Cas13c、Cas13d、Cpf1、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、以及Csf4。
7.转化细胞,在所述转化细胞中MHC I细胞膜受体和MHC II细胞膜受体的表达受到抑制。
8.根据权利要求7所述的转化细胞,其特征在于,所述转化细胞在所述细胞膜表面上表达肽抗原。
9.根据权利要求8所述的转化细胞,其特征在于,所述肽抗原为G肽。
10.根据权利要求9所述的转化细胞,其特征在于,所述G肽结合至修饰的MHC I细胞膜受体。
11.根据权利要求10所述的转化细胞,其特征在于,所述修饰的MHC I细胞膜受体具有HLA-E与B2M连接的结构。
12.根据权利要求11所述的转化细胞,其特征在于,在所述修饰的MHC I细胞膜受体中,所述B2M的C端经由第一接头连接至所述HLA-E的α1的N端,并且所述G肽的C端经由第二接头连接至所述B2M的N端。
13.根据权利要求12所述的转化细胞,其特征在于,所述G肽具有SEQ ID NO:236的序列。
14.根据权利要求12所述的转化细胞,其特征在于,所述HLA-E具有SEQ ID NO:240的序列。
15.根据权利要求12所述的转化细胞,其特征在于,所述B2M具有SEQ ID NO:237的序列。
16.根据权利要求12所述的转化细胞,其特征在于,所述第一接头具有SEQ ID NO:238的序列。
17.根据权利要求12所述的转化细胞,其特征在于,所述第二接头具有SEQ ID NO:241的序列。
18.根据权利要求7所述的转化细胞,其特征在于,使用权利要求1所述的向导RNA对编码所述MHC I细胞膜受体的基因进行修饰。
19.根据权利要求7所述的转化细胞,其特征在于,分别使用权利要求2、3、4所述的向导RNA对编码所述MHC II细胞膜受体的DQ、DP、和DR基因进行修饰。
20.根据权利要求7所述的转化细胞,其特征在于,所述转化细胞是治疗性同种异体细胞。
21.根据权利要求20所述的转化细胞,其特征在于,所述治疗性同种异体细胞是免疫细胞或干细胞。
22.根据权利要求21所述的转化细胞,其特征在于,所述免疫细胞是NK细胞或T细胞。
23.用于治疗癌症、传染病、退行性疾病、遗传性疾病、或免疫性疾病的药物组合物,所述药物组合物包含权利要求7所述的转化细胞作为活性成分。
24.根据权利要求23所述的药物组合物,其特征在于,所述癌症是选自由以下组成的组中的任何一种:慢性淋巴细胞白血病(CLL)、B细胞急性淋巴细胞白血病(B-ALL)、急性淋巴细胞白血病、急性骨髓性白血病、淋巴瘤、非霍奇金淋巴瘤(NHL)、多发性骨髓瘤、血液癌、胃癌、肝癌、胰腺癌、结肠直肠癌、肺癌、乳腺癌、卵巢癌、皮肤癌、黑色素瘤、肉瘤、***癌、食道癌,肝细胞癌、星形细胞瘤、间皮瘤、头颈癌、以及髓母细胞瘤。
25.根据权利要求23所述的药物组合物,其特征在于,所述传染病是选自由以下组成的组中的任何一种:乙型肝炎、丙型肝炎、人***瘤病毒(HPV)感染、巨细胞病毒感染、爱泼斯坦巴尔病毒(EBV)感染、病毒性呼吸道疾病、以及流行性感冒。
26.一种用于治疗癌症、传染病、退行性疾病、遗传性疾病、或免疫性疾病的方法,所述方法包括向对象施用权利要求23至25中任一项所述的药物组合物。
27.根据权利要求26所述的方法,其特征在于,所述施用是通过选自由以下组成的组中的任何一种途径进行的:静脉内的、肌内的、皮内的、皮下的、腹膜内的、小动脉内的、心室内的、病灶内的、鞘内的、局部的、及其组合。
28.转化细胞在用于治疗癌症、传染病、退行性疾病、遗传性疾病、或免疫性疾病中的用途,其中MHC I细胞膜受体和MHC II细胞膜受体的表达在所述转化细胞中受到抑制,并且所述转化细胞在所述细胞膜表面上表达结合至修饰的MHC I细胞膜受体的肽抗原。
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KR20200074954A (ko) | 2020-06-25 |
IL274691A (en) | 2020-06-30 |
SG11202004529XA (en) | 2020-06-29 |
EP3712268A4 (en) | 2022-03-23 |
CA3082331A1 (en) | 2019-05-23 |
MX2020005235A (es) | 2020-08-24 |
US20200407713A1 (en) | 2020-12-31 |
JP2021503284A (ja) | 2021-02-12 |
EP3712268A2 (en) | 2020-09-23 |
WO2019098759A2 (ko) | 2019-05-23 |
AU2018367792A1 (en) | 2020-06-11 |
WO2019098759A3 (ko) | 2019-07-18 |
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