CN116808186A - Car-nk细胞联合心脏生理性肥大药物的用途 - Google Patents
Car-nk细胞联合心脏生理性肥大药物的用途 Download PDFInfo
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Abstract
本发明涉及CAR‑NK细胞联合心脏生理性肥大药物在制备治疗缺血相关的心脏疾病的药物中的用途。其中公开了可清除组织纤维化的CAR‑NK细胞的制备,还公开了AAV、LNP或VLP介导的心脏生理性肥大药物的制备;以及它们对缺血性心脏病相关疾病进行联合治疗。CAR‑NK细胞为靶向FAP或CD248的CAR‑NK细胞,缺血相关的心脏疾病包括急性或慢性心肌缺血、心肌梗死、冠心病、扩张型心肌病、HFrEF等所有与心肌缺血有关的心脏病。实验结果表明,CAR‑NK细胞的存活时间、增殖能力、杀伤能力显著增强,CAR‑NK细胞联合心脏生理性肥大药物使梗死面积占比减少,可更好的治疗多种缺血相关的心脏疾病。
Description
技术领域
本申请涉及医学免疫学技术领域,具体涉及CAR-NK细胞的用途,更具体涉及CAR-NK细胞联合心脏生理性肥大药物的用途,特别是在制备治疗缺血相关的心脏疾病的药物中的用途。
背景技术
自然杀伤细胞(NK细胞)是机体重要的免疫细胞,与抗肿瘤、抗病毒感染和免疫调节等多种过程有关,是与B、T细胞并列的第三类淋巴细胞。NK细胞对靶细胞的杀伤依靠NK细胞表面的多种受体,其分为激活性受体(KAR)和抑制性受体(KIR)。正常细胞的主要组织相容性复合体(MHC)可结合KIR,抑制NK细胞活性,避免误伤。但异己细胞和有害细胞的MHC会出现丢失或者不匹配,无法抑制NK细胞活性而成为了靶标。还有一部分异己细胞和有害细胞会大量表达KAR配体,直接激活了NK细胞的杀伤。NK细胞激活后产生2种免疫应答:与靶细胞特异性结合,释放穿孔素和颗粒酶,直接杀伤靶细胞;以及释放淋巴因子,增强免疫效应。
嵌合抗原受体NK细胞(CAR-NK细胞)指的是人工导入了CAR结构的NK细胞。CAR的结构包括抗原结合结构域、铰链结构域、跨膜结构域、胞内结构域,其能特异性识别靶细胞抗原,直接激活NK细胞并杀伤靶细胞。CAR的靶向目标由人工设计决定,因此CAR-NK细胞有精准定位并杀伤任何预期靶细胞的潜力。
缺血相关的心脏疾病指的是由心肌缺血引起的一类心脏病,其包括急性或慢性心肌缺血、心肌梗死、冠心病、扩张型心肌病、射血分数降低的心力衰竭(HFrEF)等所有与心肌缺血有关的心脏病。心肌缺血损伤后,内部的成纤维细胞、周细胞等被激活,分化为肌成纤维细胞,从而引起组织大量纤维化,使心肌变得僵硬,不利于收缩和舒张。成纤维细胞活化蛋白(FAP)和内皮唾液酸蛋白(CD248)是肌成纤维细胞的特异性标志物,仅在肌成纤维细胞中大量表达,不表达或很低表达于机体其他正常细胞中。因此,靶向FAP或者CD248的CAR-NK细胞具有特异性杀伤肌成纤维细胞,减少甚至清除组织纤维化,从而治疗缺血相关的心脏疾病的潜力。
但缺血相关的心脏疾病由于心肌细胞大量缺失,僵硬的纤维化疤痕同时有利于维持心脏结构。因此单方面的清除纤维化可能使薄弱的心室壁无法承受血压,导致心室壁破裂。心脏生理性肥大是一种常见的生理现象,特别在运动员当中。与心脏病理性肥大不同,生理性肥大可使心肌纤维增粗、心室壁增厚、收缩力增强、血管生成增多,提高了心脏功能,并且是可逆的。因此,CAR-NK细胞联合心脏生理性肥大的治疗,有利于在清除纤维化疤痕的同时增厚心室壁,促进血管生成,提高收缩力和心室壁的强度,防止心室壁破裂,从而治疗缺血相关的心脏疾病。
发明内容
为了解决现有技术中存在的至少一个技术问题,本申请提供了CAR-NK细胞联合心脏生理性肥大药物在制备治疗缺血相关的心脏疾病的药物中的用途。
为了达到上述目的,本申请提供的技术方案如下所述:
一方面,本发明提供了CAR-NK细胞联合心脏生理性肥大药物在制备治疗缺血相关的心脏疾病的药物中的用途。
优选地,所述CAR-NK细胞为靶向FAP或CD248的CAR-NK细胞。
优选地,所述心脏生理性肥大药物通过AAV、LNP、VLP三种方式的其中一种制备而成。
另一方面,本发明提供了CAR-NK细胞的CAR结构的设计:CAR的抗原结合结构域由FAP、CD248中的一种的scFv构成,可特异性识别并结合表达相应抗原的靶细胞。铰链结构域由CD8、CD28中的一种构成。跨膜结构域由CD8、CD28、NKG2D、程序化膜蛋白(proMP)中的一种构成。胞内结构域包括由CD28、肿瘤坏死因子受体超家族成员9重组蛋白(4-1BB)、2B4中的一种构成的共刺激域和由CD3 zeta构成的信号传导域。其中CD28能带来短期、强效的杀伤能力;而4-1BB能带来持久的杀伤能力。
CAR-NK细胞的制备方法: NK细胞来源于外周血、脐带血、NK-92、NK92MI等细胞系或iPSC。
使用慢病毒或逆转录病毒转染,将能够编码CAR的核酸导入NK细胞,可得到CAR-NK细胞。
使用慢病毒或逆转录病毒转染,将能够编码白细胞介素15(IL15)、白细胞介素7(IL7)、CC趋化因子受体2(CCR2)、B细胞淋巴瘤因子2(Bcl-2)、髓样细胞白血病蛋白1(MCL1)、CD16的其中一种核酸导入CAR-NK细胞,可增强其存活时间、增殖和杀伤能力。
使用慢病毒或逆转录病毒转染,将能够编码单纯疱疹病毒胸苷激酶(HSV-TK)、诱导性半胱天冬酶9(iCasp-9)、利妥昔单抗结合表位的其中一种核酸导入CAR-NK细胞,可作为自杀基因。
使用CRISPR/Cas 9系统可敲除雌激素受体结合片段相关抗原9(EBAG9)、RAS GTP酶激活蛋白2(RASA2)。
作为优选,本发明提供了生理性肥大药物的3种制备方法。
作为优选,在制备之前,还需要获取诱导心脏生理性肥大的相关基因,相关基因如下:心肌特异性过表达胰岛素样生长因子1受体(IGF1R)、磷脂酰肌醇-3-激酶(PI3K)、Cbp/P300与富含Glu/Asp的羧基末端结构域4相互作用的反式激活子(CITED4)、microRNA-222、lncRNA CPhar中的一种或敲低甲基转移酶14(METTL14)、CCAAT增强子结合蛋白β(C/EBPβ)、叉头盒O3(FOXO3)、糖原合成酶激酶3β(GSK3β)、lncRNA ExACT1中的一种,可诱导心脏生理性肥大。
第一种制备方法是:使用AAV转染,将其中一种过表达基因的DNA导入心肌,或将其中一种敲低基因的shRNA导入心肌。上述核酸序列前需要添加心肌特异性启动子(心肌肌钙蛋白(cTNT))。
第二种制备方法是:使用LNP转染,将其中一种过表达基因的mRNA导入心肌,或将其中一种敲低基因对应的siRNA导入心肌。LNP的包膜上需要添加心肌特异性靶标(JP2或Cx43)。
第三种制备方法是:使用VLP转染,将其中一种过表达基因的mRNA导入心肌,或将其中一种敲低基因对应的siRNA导入心肌。VLP的包膜上需要添加心肌特异性靶标(JP2或Cx43)。
优选地,本发明还提供了CAR-NK细胞联合心脏生理性肥大药物在治疗急性或慢性心肌缺血、心肌梗死、冠心病、扩张型心肌病、射血分数降低的心力衰竭的用途。
具体使用方法如下:使用AAV制备的心脏生理性肥大药物通过静脉注射的方式运输至体内:注射量为1*1013-1*1016GC/kg;注射次数为1-3次,每周注射一次。在首次注射后1-8周,采用静脉注射的方式将CAR-NK细胞运输至体内;注射量为1*104-1*108个/kg,注射次数为1-4次,注射周期为1-4周/次。
使用LNP或VLP制备的心脏生理性肥大药物通过静脉注射的方式运输至体内:注射量为100μg-10mg/kg(RNA质量/体重);注射次数为1-4次,每周注射一次。在首次注射后3天,采用静脉注射的方式将CAR-NK细胞运输至体内;注射量为1*104-1*108个/kg,注射次数为1-4次,注射周期为1-4周/次。
优选地,CAR-NK细胞的存活时间、增殖能力、杀伤能力显著增强;并且,使用AP1903等小分子诱导药物后,自杀基因诱导CAR-NK细胞凋亡,增强了CAR-NK细胞的可控性;CAR-NK细胞联合心脏生理性肥大药物可更好的治疗急性或慢性心肌缺血、心肌梗死、冠心病、扩张型心肌病、射血分数降低的心力衰竭等多种缺血相关的心脏疾病。
综上所述,本发明具有以下有益效果:
1、本发明提供了靶向FAP或者CD248的CAR-NK细胞的设计和制备方法,其可减少组织纤维化。
2、本发明还增强了CAR-NK细胞的存活时间、增殖和杀伤能力,并设计了CAR-NK细胞的自杀基因。
3、本发明提供了分别使用AAV、LNP、VLP制备心脏生理性肥大药物的方法,其可特异性诱导心肌生理性肥大。
4、本发明提供了靶向FAP或者CD248的CAR-NK细胞联合心脏生理性肥大药物治疗缺血相关的心脏疾病,其可有效清除心肌纤维化,增强心脏功能,并防止心脏破裂,改善缺血相关的心脏疾病。
附图说明
图1: CAR-NK细胞的设计和制备:
A:新型CAR的结构设计示意图;B:CAR-NK细胞的制备流程:使用CD56磁珠从淋巴细胞中分选出原代NK细胞;或直接使用NK-92等细胞系;并在培养基中使用IL2扩增NK细胞;再加入含CAR的核酸的慢病毒或逆转录转染NK细胞,经过进一步的培养、扩增最后得到CAR-NK细胞;
图2:心脏生理性肥大药物的制备:
A:AAV介导的心脏生理性肥大药物的制备:将目的基因DNA或shRNA片段克隆到AAV的基因组质粒中;将克隆好的AAV基因组质粒和AAV包膜质粒、AAV蛋白外壳质粒同时转染293T细胞;最后裂解细胞,浓缩纯化病毒后得到AAV介导的心脏生理性肥大药物;B:LNP介导的心脏生理性肥大药物的制备:将可电离阳离子脂质、磷脂、胆固醇和聚乙二充分混合后得到LNP;将目的基因mRNA或siRNA片段导入LNP中;经过纯化浓缩后再在LNP的包膜上添加心肌特异性靶点抗体,最终得到LNP介导的心脏生理性肥大药物;C:VLP介导的心脏生理性肥大药物的制备:将目的基因DNA或shRNA片段克隆到VLP的蛋白外壳质粒中;之后将所述质粒转染293T细胞,培养后裂解细胞,浓缩纯化病毒后再在VLP的包膜上添加心肌特异性靶点抗体,最终得到VLP介导的心脏生理性肥大药物;AAV:腺相关病毒;LNP:脂质纳米粒;VLP:病毒样颗粒;
图3: CAR-NK细胞联合心脏生理性肥大药物可显著改善缺血相关的心脏疾病:
马松染色的代表性图像(红色:细胞质,蓝色:胶原纤维);C:对照组;MI:心肌缺血组;CARNK:CAR-NK细胞治疗组;Hypertrophy:心脏生理性肥大治疗组;Combination: 联合治疗组;* P<0.05。
具体实施方式
本发明的具体实施方式主要包括CAR-NK细胞的制备、心脏生理性肥大药物的制备和CAR-NK细胞联合心脏生理性肥大药物治疗缺血相关的心脏疾病的方法。
一、CAR-NK细胞的制备(如图1所示)
1.NK细胞的获取
从人或动物外周血、脐带血提取出淋巴细胞。使用CD56磁珠从淋巴细胞中分选出NK细胞。也可诱导iPSC(诱导型多能干细胞)分化成NK细胞,或直接使用NK-92、NK-92MI等细胞系。将细胞放入含0.2mM肌醇、0.1mM β-巯基乙醇、0.02mM、200 U/mL白介素2(IL2)、12.5%马血清、12.5%胎牛血清、1%青-链霉素的MEMα培养基中扩增。培养环境为37 ℃、95%空气、5%CO2。
2. 编码CAR的核酸的获得
CAR的抗原结合结构域由纤维细胞活化蛋白(FAP)、内皮唾液酸蛋白(CD248)中的一种的单链抗体(scFv)构成。连接肽为甘氨酸和丝氨酸的多聚体。铰链结构域由CD8、CD28中的一种构成。CD8、CD28、NKG2D、程序化膜蛋白(proMP)中的一种构成。CAR的共刺激域由CD28、4-1BB、2B4中的一种构成。CAR的信号传导域由CD3 zeta构成;
3.内源基因表达的上调以及自杀基因的导入
使用PCR技术得到上调基因和自杀基因的核酸后,通过酶切连接技术将其接入到CAR核酸序列之后,并使用T2A、P2A序列将CAR、上调基因、自杀基因单独翻译和表达,得到新型的编码CAR的核酸。上调基因包括:白细胞介素15(IL15)、白细胞介素7(IL7)、CC趋化因子受体2(CCR2)、B细胞淋巴瘤因子2(Bcl-2)、髓样细胞白血病蛋白1(MCL1)、CD16。自杀基因包括:单纯疱疹病毒胸苷激酶(HSV-TK)、诱导性半胱天冬酶9(iCasp-9)、利妥昔单抗结合表位。
4.将编码CAR的核酸导入NK细胞
将NK细胞接种到RetroNectin(5μg/cm)包被的24孔板中。之后加入含新型的CAR的核酸的病毒和Polybrene(7μg/ml)转染NK细胞;在室温下1000 g离心1小时后将细胞放入培养箱中。孵育过夜后,更换新鲜培养基扩增NK细胞,最终获得CAR-NK细胞。
5.下调CAR-NK细胞内源基因表达
通过CRISPR/Cas 9技术下调CAR-NK细胞内源基因的表达。设计好下调基因的gRNA序列后,使用人工基因合成的方法得到gRNA序列对应的DNA片段,并使用PCR技术得到Cas9的核酸。通过酶切连接技术将其中一种下调基因的gRNA的DNA片段与Cas9的核酸相连,再将其通过慢病毒或者逆转录病毒导入CAR-NK细胞,从而实现CAR-NK细胞内源基因表达的下调。下调基因包括:雌激素受体结合片段相关抗原9(EBAG9)、RAS GTP酶激活蛋白2(RASA2)。
二、心脏生理性肥大药物的制备(如图2所示)
1.诱导心脏生理性肥大的相关基因的获取
通过人工合成的方式获得心肌特异性过表达胰岛素样生长因子1受体(IGF1R)、磷脂酰肌醇-3-激酶(PI3K)、Cbp/P300与富含Glu/Asp的羧基末端结构域4相互作用的反式激活子(CITED4)、microRNA-222和lncRNA CPhar的DNA片段以及敲低甲基转移酶14(METTL14)、CCAAT增强子结合蛋白β(C/EBPβ)、叉头盒O3(FOXO3)、糖原合成酶激酶3β(GSK3β)和lncExACT1对应的shRNA片段。再通过体外转录,得到IGF1R、PI3K、CITED4、microRNA-222和lncRNA CPhar的mRNA片段(microRNA-222和lncRNA CPhar分别为转录好的microRNA和lncRNA)以及METTL14、C/EBPβ、FOXO3、GSK3β和lncExACT1对应的siRNA片段。
2.通过AAV制备的心脏生理性肥大药物
在上述DNA片段或shRNA片段前添加心肌特异性启动子序列(cTNT),之后将核酸片段通过酶切连接的方式导入AAV的基因组质粒中,再联合AAV的包膜质粒和蛋白外壳质粒一起转染293T细胞,最后得到AAV介导的心脏生理性肥大药物。
3.通过LNP制备的心脏生理性肥大药物
将可电离阳离子脂质:磷脂:胆固醇:聚乙二醇按50:10:38.5:1.5的比例通过微流体混合设备混合,之后得到制备好的LNP。将上述mRNA片段(lncRNA CPhar为转录好的lncRNA)或siRNA片段导入LNP中,LNP:RNA为10:1的比例。经过纯化浓缩后再在LNP的包膜上添加心肌特异性靶标(Junctophilin-2(JP2)或Cx43),最终得到LNP介导的心脏生理性肥大药物。
4.通过VLP制备的心脏生理性肥大药物
得到病毒衣壳蛋白的质粒后,将上述mRNA片段(lncRNA CPhar为转录好的lncRNA)或siRNA片段导入所述质粒中。之后将所述质粒转染293T细胞,培养后裂解细胞,得到VLP。经过纯化浓缩后再在VLP的包膜上添加心肌特异性靶标(JP2或Cx43),最终得到VLP介导的心脏生理性肥大药物。
三、CAR-NK细胞联合心脏生理性肥大药物治疗缺血相关的心脏疾病的方法(如图3所示)
将AAV介导的心脏生理性肥大药物通过静脉注射的方式运输至体内:注射量为1*1013-1*1016GC/kg;注射次数为1-3次,每周注射一次。在首次注射后1-8周,采用静脉注射的方式将CAR-NK细胞运输至体内;注射量为1*104-1*108个/kg,注射次数为1-4次,注射周期为1-4周/次。
将LNP或VLP介导的心脏生理性肥大药物通过静脉注射的方式运输至体内:注射量为100μg-10mg/kg(RNA质量/体重);注射次数为1-4次,每周注射一次。在首次注射后3天,采用静脉注射的方式将CAR-NK细胞运输至体内;注射量为1*104-1*108个/kg,注射次数为1-4次,注射周期为1-4周/次。
结果分析
原代CAR-NK细胞由于其存活时间、增殖能力有限,导致其治疗成本较大;细胞系和iPSC衍生的CAR-NK细胞则杀伤能力较低,并由于其无限增殖而存在安全隐患。而我们的结果显示,本发明的CAR-NK细胞的存活时间、增殖能力、杀伤能力显著增强。并且,使用AP1903等小分子诱导药物后,自杀基因诱导CAR-NK细胞凋亡,增强了CAR-NK细胞的可控性。
在缺血相关的心脏疾病患者和动物中,都出现了显著的组织纤维化,梗死面积占比达到了30%-50%,因心肌梗死的死亡率达到20%以上。我们的结果显示,靶向FAP和CD248的CAR-NK细胞显著减少了心肌纤维化,梗死面积占比减少了30%以上,增强了心功能。但也导致20%的动物因心室壁破裂而死亡。此外,心脏生理性肥大药物的单独使用显著增加了心肌纤维面积和心室壁厚度,心肌梗死的死亡率减少到10%以内,但梗死面积占比只减少了10%。而CAR-NK细胞与心脏生理性肥大药物的联合治疗使梗死面积占比减少了60%以上,同时心肌梗死的死亡率减少到10%以内,并且没有动物因心室壁破裂而死亡,表现出对缺血相关的心脏疾病的良好治疗效果。
总之,本发明成功制备了可减少组织纤维化的CAR-NK细胞和可诱导心脏生理性肥大的药物,并且它们的联合使用对缺血相关的心脏疾病有着良好的治疗效果。而且,相比申请人先前申请的CAR-NK细胞在心肌纤维化的治疗更好。
另外,CD8信号肽、连接肽和T2A等的序列如下:
CD8信号肽(SEQ ID NO:1):
5‘-ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCC-3’。
连接肽(SEQ ID NO:2):
5‘- GGCGGCGGAGGAAGCGGAGGCGGAGGATCTGGTGGTGGTGGATCT-3’。
T2A(SEQ ID NO:3):
5‘-AGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCC-3’。
P2A(SEQ ID NO:4):
5‘-GGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCTATG-3’。
EBAG9 gRNA(SEQ ID NO:5):5‘-UUUAGACAGAUGUUGAAGAG-3’。
RASA2 gRNA(SEQ ID NO:6):5‘-AUUUUGUGGGGUCCAAGAUA-3’。
CD7 gRNA(SEQ ID NO:7):5‘-CACGGGGACAGUCGUGCAGU-3’。
IL-1 gRNA(SEQ ID NO:8):5‘-GCCAUAGCUUACAUGAUAGA-3’。
IL-6 gRNA(SEQ ID NO:9):5‘-GGAGAAGGCAACUGGACCGA-3’。
GM-CSF gRNA(SEQ ID NO:10):5‘-GAUCUGCAAGGAGCGGGCAC-3’。
METTL14 shRNA(SEQ ID NO:11):5‘-GCATTGGTGCCGTGTTAAATA-3’。
C/EBPD NOhRN(SEQ ID NO:12):5‘-CCATGGAAGTGGCCAACTT-3’。
FOXO3 shRNA(SEQ ID NO:13):5‘-GCGAACGGACAGGAGTACATT-3’。
GSK3ID NONA(SEQ ID NO:14):5‘-GAGCCACTGATTATACCTCTA-3’。
FAP:参考期刊文献: Wang LC, Lo A, Scholler J, Sun J, Majumdar RS,Kapoor V, Antzis M, Cotner CE, Johnson LA, Durham AC, Solomides CC, June CH,Pure E and Albelda SM. Targeting fibroblast activation protein in tumorstroma with chimeric antigen receptor T cells can inhibit tumor growth andaugment host immunity without severe toxicity. Cancer Immunol Res. 2014;2:154-66.
CD248:IMGT INN Number: 9519。
uPAR:PDB DOI: 10.2210/pdb2FAT/pdb。
CD8铰链域:参考期刊文献:Alabanza L, Pegues M, Geldres C, Shi V,Wiltzius JJW, Sievers SA, Yang S and Kochenderfer JN. Function of Novel Anti-CD19 Chimeric Antigen Receptors with Human Variable Regions Is Affected byHinge and Transmembrane Domains. Mol Ther. 2017;25:2452-2465.
CD28铰链域:参考期刊文献:Alabanza L, Pegues M, Geldres C, Shi V,Wiltzius JJW, Sievers SA, Yang S and Kochenderfer JN. Function of Novel Anti-CD19 Chimeric Antigen Receptors with Human Variable Regions Is Affected byHinge and Transmembrane Domains. Mol Ther. 2017;25:2452-2465.
CD8跨膜域:参考期刊文献:Alabanza L, Pegues M, Geldres C, Shi V,Wiltzius JJW, Sievers SA, Yang S and Kochenderfer JN. Function of Novel Anti-CD19 Chimeric Antigen Receptors with Human Variable Regions Is Affected byHinge and Transmembrane Domains. Mol Ther. 2017;25:2452-2465.
proMP跨膜域:参考期刊文献:Elazar A, Chandler NJ, Davey AS, WeinsteinJY, Nguyen JV, Trenker R, Cross RS, Jenkins MR, Call MJ, Call ME andFleishman SJ. De novo-designed transmembrane domains tune engineered receptorfunctions. Elife. 2022;11.
CD28跨膜域:参考期刊文献:Elazar A, Chandler NJ, Davey AS, WeinsteinJY, Nguyen JV, Trenker R, Cross RS, Jenkins MR, Call MJ, Call ME andFleishman SJ. De novo-designed transmembrane domains tune engineered receptorfunctions. Elife. 2022;11.
NKG2D跨膜域:参考期刊文献:Li Y, Hermanson DL, Moriarity BS and KaufmanDS. Human iPSC-Derived Natural Killer Cells Engineered with Chimeric AntigenReceptors Enhance Anti-tumor Activity. Cell Stem Cell. 2018;23:181-192 e5.
4-1BB共刺激域:NCBI NM_001561.6。
2B4共刺激域:参考期刊文献:Li Y, Hermanson DL, Moriarity BS and KaufmanDS. Human iPSC-Derived Natural Killer Cells Engineered with Chimeric AntigenReceptors Enhance Anti-tumor Activity. Cell Stem Cell. 2018;23:181-192 e5.
CD28共刺激域:参考期刊文献:Li Y, Hermanson DL, Moriarity BS andKaufman DS. Human iPSC-Derived Natural Killer Cells Engineered with ChimericAntigen Receptors Enhance Anti-tumor Activity. Cell Stem Cell. 2018;23:181-192 e5.
CD3 zeta信号传导域:参考期刊文献:Li Y, Hermanson DL, Moriarity BS andKaufman DS. Human iPSC-Derived Natural Killer Cells Engineered with ChimericAntigen Receptors Enhance Anti-tumor Activity. Cell Stem Cell. 2018;23:181-192 e5.
IL15:NCBI NM_000585.5。
IL7:NCBI NM_000880.4。
CCR2:NCBI NM_001123041.3。
Bcl-2:NCBI XM_047437733.1。
MCL1:NCBI NM_001197320.2。
CD16:NCBI NM_000569.8。
HSV- TK:GenBank: ACC91769.1。
iCasp-9:参考期刊文献:Wunderlich S, Haase A, Merkert S, Jahn K, DeestM, Frieling H, Glage S, Korte W, Martens A, Kirschning A, Zeug A, PonimaskinE, Gohring G, Ackermann M, Lachmann N, Moritz T, Zweigerdt R and Martin U.Targeted biallelic integration of an inducible Caspase 9 suicide gene iniPSCs for safer therapies. Mol Ther Methods Clin Dev. 2022;26:84-94.
利妥昔单抗结合表位:参考期刊文献:Philip B, Kokalaki E, Mekkaoui L,Thomas S, Straathof K, Flutter B, Marin V, Marafioti T, Chakraverty R, LinchD, Quezada SA, Peggs KS and Pule M. A highly compact epitope-based marker/suicide gene for easier and safer T-cell therapy. Blood. 2014;124:1277-87.
IGF1R:XM_047432442.1。
PI3K:NM_006218.4。
CITED4:NM_133467.3。
microRNA-222:NR_029636.1。
lncRNA Cphar:参考期刊文献:Gao R, Wang L, Bei Y, Wu X, Wang J, Zhou Q,Tao L, Das S, Li X and Xiao J. Long Noncoding RNA Cardiac PhysiologicalHypertrophy-Associated Regulator Induces Cardiac Physiological Hypertrophyand Promotes Functional Recovery After Myocardial Ischemia-ReperfusionInjury. Circulation. 2021;144:303-317.
lncExACT1 shRNA:参考期刊文献:Li H, Trager LE, Liu X, Hastings MH,Xiao C, Guerra J, To S, Li G, Yeri A, Rodosthenous R, Silverman MG, Das S,Ambardekar AV, Bristow MR, Gonzalez-Rosa JM and Rosenzweig A. lncExACT1 andDCHS2 Regulate Physiological and Pathological Cardiac Growth. Circulation.2022;145:1218-1233.
Claims (10)
1.CAR-NK细胞联合心脏生理性肥大药物在制备治疗缺血相关的心脏疾病的药物中的用途。
2.根据权利要求1所述的用途,其特征在于,所述CAR-NK细胞为靶向FAP或CD248的CAR-NK细胞。
3.根据权利要求1所述的用途,其特征在于,所述心脏生理性肥大药物通过AAV、LNP、VLP三种方式的其中一种制备而成。
4.根据权利要求3所述的用途,其特征在于,所述心脏生理性肥大药物的制备方法包括:获取诱导心脏生理性肥大的相关基因,步骤包括:通过人工合成的方式获得IGF1R、PI3K、CITED4、microRNA-222和lncRNA CPhar的DNA片段以及METTL14、C/EBPβ、FOXO3、GSK3β和lncExACT1对应的shRNA片段;再通过体外转录,得到IGF1R、PI3K、CITED4、microRNA-222和lncRNA CPhar的mRNA片段以及METTL14、C/EBP1、FOXO3、GSK33和lncExACT1对应的siRNA片段,其中microRNA-222和lncRNA CPhar分别为转录好的microRNA和lncRNA。
5.根据权利要求4所述的用途,其特征在于,通过AAV的方式制备所述心脏生理性肥大药物的步骤包括:制备包含过表达基因的DNA或敲低基因的shRNA的AAV;在核酸序列前添加心肌特异性启动子。
6.根据权利要求4所述的用途,其特征在于,通过LNP的方式制备所述心脏生理性肥大药物的步骤包括:制备包含过表达基因的mRNA或敲低基因的siRNA的LNP;在LNP的包膜上添加JP2或Cx43。
7.根据权利要求4所述的用途,其特征在于,通过VLP的方式制备所述心脏生理性肥大药物的步骤包括:制备包含过表达基因的mRNA或敲低基因的siRNA的VLP;在VLP的包膜上添加JP2或Cx43。
8.根据权利要求1所述的用途,其特征在于,所述缺血相关的心脏疾病包括急性或慢性心肌缺血、心肌梗死、冠心病、扩张型心肌病、射血分数降低的心力衰竭。
9.根据权利要求1所述的用途,其特征在于,所述的药物使用时,先静脉注射心脏生理性肥大药物,1-8周后静脉注射CAR-NK细胞。
10.根据权利要求1-9任一项所述的用途,其特征在于,CAR-NK细胞的存活时间、增殖能力、杀伤能力显著增强;并且,使用AP1903等小分子诱导药物后,自杀基因诱导CAR-NK细胞凋亡,增强了CAR-NK细胞的可控性;CAR-NK细胞联合心脏生理性肥大药物可更好的治疗急性或慢性心肌缺血、心肌梗死、冠心病、扩张型心肌病、射血分数降低的心力衰竭等多种缺血相关的心脏疾病。
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