CN116549480B - 针对HIF1α的shRNA在制备治疗肿瘤的药物中的应用 - Google Patents
针对HIF1α的shRNA在制备治疗肿瘤的药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种提高NK细胞在低氧条件下的增殖能力和存活能力的方法,本发明首次发现抑制HIF1α的表达能够显著提高NK细胞在低氧条件下的增殖能力和存活能力,进而提高NK细胞在肿瘤免疫治疗中的治疗效果,本发明为本领域提供了一种优化NK细胞肿瘤免疫治疗的新思路和新策略,为肿瘤免疫治疗的推广和效果的提高起到了推动作用。
Description
技术领域
本发明属于生物医药技术领域,具体地,本发明涉及一种提高NK细胞在低氧条件下的增殖能力和存活能力的方法。
背景技术
低氧诱导因子(Hypoxia-inducible factors,HIFs)是介导下游基因表达以应对低氧应激的中心因子。HIF家族包含两个不同的亚基:α亚基和β亚基。α部分由HIF-1α、HIF-2α和HIF-3α组成;β部分包含一个蛋白质(HIF-1β)。HIF-1α广泛表达于所有人体组织中,而HIF-2α和HIF-3α仅在一些特定的组织中可检测到。α亚基蛋白受细胞氧水平的调节,而β亚基则是组成型表达。在常氧条件下,HIF-α蛋白(HIF-1α、HIF-2α和HIF-3α)通过脯氨酰基残基的羟化作用迅速泛素化并被蛋白酶体依次降解。HIF-α蛋白包含一个氧依赖的降解域,两个脯氨酸位点被氧依赖的脯氨酸羟化酶家族(Proline hydroxylase domains,PHDs)羟基化,有趣的是,这种酶活性需要氧、铁和2-氧-戊二酸。HIF-α羟化后与希林佩尔林道基因产物(product of von Hippel-Lindau gene,pVHL)相互作用,促进HIF-α泛素-蛋白酶体降解。然而,在缺氧条件下,PHDs的酶活性受到抑制,这阻止了HIF-α羟基化和泛素介导的蛋白酶体降解。随后,HIF-α亚基与HIF-1β相互作用形成转录复合体二聚体,然后进入细胞核,与缺氧响应元件(HREs)结合,诱导大量下游基因的表达。
目前,如何增强肿瘤中的免疫细胞的功能仍然是肿瘤免疫治疗领域所面临的主要挑战之一。缺氧是实体瘤的共同特征,而且缺氧这种特征与肿瘤的增殖、分化、血管生成、能量代谢以及癌症的耐药性发生、患者预后较差等密切相关,细胞通过上调转录因子HIF1α来适应肿瘤的低氧微环境。NK92细胞是一种来源于人恶性非霍奇金淋巴瘤患者的自然杀伤细胞(Natural killer cell,NK),其免疫表型为CD56brightCD16-,无ADCC效应,具有高效广谱的抗肿瘤作用。NK-92是NK细胞系中唯一进入临床研究的细胞系,其对不同来源肿瘤细胞系,例如白血病、乳腺癌等均表现出高效的杀伤活性。其表面表达大量活化受体,例如NKp30、NKp46、NKG2D、NKG2E、CD28;抑制受体的表达极少,只有NKGA/B和低水平的K1112DIA与ILT-2,缺乏大多数正常NK细胞克隆表达的KIRs,却保留穿孔素/颗粒酶介导的细胞毒作用。NK92细胞可无限增殖,具有高效的细胞毒性和稳定的免疫特性,与原始NK细胞相比,NK92细胞更容易获取和扩增。NK92细胞在肿瘤免疫治疗领域发挥着重要的作用,本发明所要解决的技术问题是如何提高NK细胞在低氧条件下的增殖能力和存活能力,进一步提高其对肿瘤细胞的杀伤效果。
迄今为止,尚未见HIF1α敲降对在低氧条件下NK92细胞的增殖速度和存活能力的影响的相关研究或报道。
发明内容
针对现有技术中存在的上述技术问题,本发明的目的在于提供一种提高NK细胞在低氧条件下的增殖能力和存活能力的方法。
为了实现上述目的,本发明采用了如下技术方案:
本发明的第一方面提供了抑制HIF1α表达的试剂在提高NK细胞在低氧条件下的增殖能力、存活能力和/或杀伤效果中的应用。
进一步,所述抑制HIF1α表达的试剂包括针对HIF1α的shRNA、RNA反义分子、DNA反义分子、siRNA、dsRNA、miRNA、和/或核酶;
优选地,所述抑制HIF1α表达的试剂为针对HIF1α的shRNA;
更优选地,所述shRNA的序列为如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQID NO:4所示的序列中的任意一种;
优选地,所述NK细胞包括NK92细胞、NK-92MI细胞、KHYG-1细胞、YT细胞、CIML-NK细胞、NKG细胞、NKL细胞、NK-YS细胞、SNK-6细胞、IMC-1细胞、PB-NK细胞、iPSC-NK细胞、和/或UCB-NK细胞;
更优选地,所述NK细胞为NK92细胞;
优选地,所述低氧条件为(0.1-10)%O2浓度的条件;
更优选地,所述低氧条件为5% O2浓度的条件。
进一步,通过如下任一种或多种方式组合在NK细胞中表达所述shRNA:慢病毒载体、脂质转染法、微注射、电穿孔、DNA载体、逆转录病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、腺相关病毒载体、TALEN、ZFN、和/或CRISPR/Cas9;
优选地,通过慢病毒载体在NK细胞中表达所述shRNA。
在一些实施方案中,本发明所述shRNA的序列如CCGGGTGATGAAAGA ATTACCGAATCTCGAGATTCGGTAATTCTTTCATCACTTTTT(SEQ ID NO:1)所示或与SEQ ID NO:1具有至少90%同源性的核苷酸序列,该序列对应的sh RNA能够显著提高NK细胞在低氧条件下的增殖能力、存活能力和/或杀伤效果。
在一些实施方案中,本发明所述shRNA的序列如CCGGCCGCTGGAGACA CAATCATATCTCGAGATATGATTGTGTCTCCAGCGGTTTTT(SEQ ID NO:2)所示或与SEQ ID NO:2具有至少90%同源性的核苷酸序列,所述序列对应的shRNA能够显著提高NK细胞在低氧条件下的增殖能力、存活能力和/或杀伤效果。
在一些实施方案中,本发明所述shRNA的序列如CCGGCCAGTTATGATT GTGAAGTTACTCGAGTAACTTCACAATCATAACTGGTTTTT(SEQ ID NO:3)所示或与SEQ ID NO:3具有至少90%同源性的核苷酸序列,所述序列对应的shRNA能够显著提高NK细胞在低氧条件下的增殖能力、存活能力和/或杀伤效果。
在一些实施方案中,本发明所述shRNA的序列如CCGGCGGCGAAGTAA AGAATCTGAACTCGAGTTCAGATTCTTTACTTCGCCGTTTTT(SEQ ID NO:4)所示或与SEQ ID NO:4具有至少90%同源性的核苷酸序列,所述序列对应的shRNA能够显著提高NK细胞在低氧条件下的增殖能力、存活能力和/或杀伤效果。
在本发明中,所述NK细胞并不局限于NK92细胞,还包括但不限于:NK-92MI细胞、KHYG-1细胞、YT细胞、CIML-NK细胞、NKG细胞、NKL细胞、NK-YS细胞、SNK-6细胞、IMC-1细胞、PB-NK细胞、iPSC-NK细胞、和/或UCB-NK细胞,任何来源或类型的NK细胞均在本发明的保护范围内。
在本发明的具体实施方案中,本发明通过实验验证首次发现抑制HIF1α的表达能够显著提高NK细胞在低氧条件下的增殖能力和存活能力,进而能够提高其在肿瘤低氧微环境下杀伤肿瘤细胞的效果。
本发明的第二方面提供了用于基因改造NK细胞以提高NK细胞在低氧条件下的增殖能力、存活能力和/或杀伤效果的构建体。
进一步,所述构建体包含编码抑制HIF1α表达的试剂的核苷酸;
优选地,所述抑制HIF1α表达的试剂包括针对HIF1α的shRNA、RNA反义分子、DNA反义分子、siRNA、dsRNA、miRNA、和/或核酶;
更优选地,所述抑制HIF1α表达的试剂为针对HIF1α的shRNA;
最优选地,所述shRNA的序列为如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQID NO:4所示的序列中的任意一种;
优选地,所述NK细胞包括NK92细胞、NK-92MI细胞、KHYG-1细胞、YT细胞、CIML-NK细胞、NKG细胞、NKL细胞、NK-YS细胞、SNK-6细胞、IMC-1细胞、PB-NK细胞、iPSC-NK细胞、和/或UCB-NK细胞;
更优选地,所述NK细胞为NK92细胞;
优选地,所述低氧条件为(0.1-10)%O2浓度的条件;
更优选地,所述低氧条件为5% O2浓度的条件。
本发明的第三方面提供了一种用于提高NK细胞在低氧条件下的增殖能力、存活能力和/或杀伤效果的表达载体。
进一步,所述表达载体包含本发明第二方面所述的构建体;
优选地,所述表达载体包括病毒载体、DNA载体;
更优选地,所述病毒载体包括慢病毒载体、逆转录病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、和/或腺相关病毒载体;
更优选地,所述DNA载体包括DNA质粒载体、结合DNA质粒的脂质体、结合DNA质粒的分子耦联体、和/或结合DNA质粒的多聚物;
最优选地,所述表达载体为慢病毒载体;
优选地,所述NK细胞包括NK92细胞、NK-92MI细胞、KHYG-1细胞、YT细胞、CIML-NK细胞、NKG细胞、NKL细胞、NK-YS细胞、SNK-6细胞、IMC-1细胞、PB-NK细胞、iPSC-NK细胞、和/或UCB-NK细胞;
更优选地,所述NK细胞为NK92细胞;
优选地,所述低氧条件为(0.1-10)%O2浓度的条件;
更优选地,所述低氧条件为5% O2浓度的条件。
本发明的第四方面提供了一种经基因改造的NK细胞。
进一步,所述经基因改造的NK细胞低表达或不表达HIF1α;
优选地,所述经基因改造的NK细胞包含本发明第二方面所述的构建体、和/或本发明第三方面所述的表达载体;
更优选地,所述NK细胞包括NK92细胞、NK-92MI细胞、KHYG-1细胞、YT细胞、CIML-NK细胞、NKG细胞、NKL细胞、NK-YS细胞、SNK-6细胞、IMC-1细胞、PB-NK细胞、iPSC-NK细胞、和/或UCB-NK细胞;
最优选地,所述NK细胞为NK92细胞。
在本发明的具体实施方案中,采用慢病毒载体表达所述针对HIF1α的shRNA,在优选的实施方案中,首先,以慢病毒载体例如pLenti-TGFBR2 shRNA-GFP-puro质粒为骨架载体,插入如前所述的调控HIF1α的shRNA序列,构建含有调控HIF1α基因的慢病毒载体(pLenti-HIF1αshRNA-GFP-puro);然后,将载体pLenti-HIF1αshRNA-GFP-puro通过慢病毒转染方式转入NK92细胞中,进行puro药杀,筛选含有调控HIF1α基因的NK92细胞;最后,将药杀后的NK92细胞进行基因组PCR插入鉴定以及qPCR表达鉴定,确认获得HIF1αshRNA-NK92细胞(即为经基因改造的NK细胞)。
本发明的第五方面提供了一种提高NK细胞在低氧条件下的增殖能力、存活能力和/或杀伤效果的方法。
进一步,所述方法包括如下步骤:抑制NK细胞中HIF1α的表达;
优选地,采用抑制HIF1α表达的试剂抑制NK细胞中HIF1α的表达;
更优选地,所述抑制HIF1α表达的试剂包括针对HIF1α的shRNA、RNA反义分子、DNA反义分子、siRNA、dsRNA、miRNA、和/或核酶;
优选地,所述抑制HIF1α表达的试剂为针对HIF1α的shRNA;
更优选地,所述shRNA的序列为如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQID NO:4所示的序列中的任意一种;
优选地,所述NK细胞包括NK92细胞、NK-92MI细胞、KHYG-1细胞、YT细胞、CIML-NK细胞、NKG细胞、NKL细胞、NK-YS细胞、SNK-6细胞、IMC-1细胞、PB-NK细胞、iPSC-NK细胞、和/或UCB-NK细胞;
更优选地,所述NK细胞为NK92细胞;
优选地,所述低氧条件为(0.1-10)%O2浓度的条件;
更优选地,所述低氧条件为5% O2浓度的条件。
进一步,通过如下任一种或多种方式组合在NK细胞中表达所述shRNA:慢病毒载体、脂质转染法、微注射、电穿孔、DNA载体、逆转录病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、和/或腺相关病毒载体;
优选地,通过慢病毒载体在NK细胞中表达所述shRNA。
本发明的第六方面提供了一种用于治疗肿瘤的药物组合物。
进一步,所述药物组合物包含本发明第二方面所述的构建体、本发明第三方面所述的表达载体、和/或本发明第四方面所述的经基因改造的NK细胞;
优选地,所述药物组合物中还可包含具有抗肿瘤活性的药物、和/或细胞毒剂;
更优选地,所述具有抗肿瘤活性的药物包括化疗药物、抗肿瘤血管生成药物、和/或免疫检查点抑制剂;
更优选地,所述细胞毒剂包括生物碱类细胞毒剂、和/或抗生素类细胞毒剂。
在一些实施方案中,所述化疗药物包括紫杉烷类化疗药物、长春花碱类化疗药物、金属铂类化疗药物、蒽环类化疗药物、抗叶酸类化疗药物、氮芥类化疗药物、鬼臼生物碱类化疗药物。
在一些实施方案中,所述紫杉烷类化疗药物包括紫杉醇、紫杉醇脂质体、白蛋白结合型紫杉醇、多西他赛;所述长春花碱类化疗药物包括长春新碱、长春地辛、长春花碱;所述金属铂类化疗药物包括顺铂、卡铂、奥沙利铂、奈达铂、洛铂;所述蒽环类化疗药物包括表柔比星、吡柔比星、阿霉素、表阿霉素;所述抗叶酸类化疗药物包括甲氨蝶呤、培美曲塞二钠;所述氮芥类化疗药物包括环磷酰胺、苯达莫司汀;所述鬼臼生物碱类化疗药物包括依托泊苷、替尼泊苷。
在一些实施方案中,所述抗肿瘤血管生成药物包括小分子多靶点血管生成抑制剂、大分子单靶点血管生成抑制剂、内源性泛靶点血管生成抑制剂。
在一些实施方案中,所述小分子多靶点血管生成抑制剂包括索拉非尼、舒尼替尼、培唑帕尼、凡德他尼、卡博替尼、瑞戈非尼、阿昔替尼、尼达尼布、乐伐替尼、阿帕替尼、安罗替尼、呋喹替尼、厄达替尼;所述大分子单靶点血管生成抑制剂包括贝伐珠单抗、雷莫芦单抗、阿柏西普;所述内源性泛靶点血管生成抑制剂包括恩度。
在一些实施方案中,所述免疫检查点抑制剂包括伊匹木单抗、帕博利株单抗、纳武利尤单抗、阿替利珠单抗、度伐利尤单抗、阿维鲁单抗、信迪利单抗。
在一些实施方案中,所述生物碱类细胞毒剂包括氢溴酸山莨菪碱、高三尖杉酯碱;所述抗生素类细胞毒剂包括伊达比星、盐酸多柔比星。
进一步,所述药物组合物还可包含药学上可接受的载体和/或辅料。
在一些实施方案中,所述药学上可接受的载体和/或辅料的具体示例性例子包括但不限于:糖类,如乳糖、葡萄糖和蔗糖;淀粉,如玉米淀粉和土豆淀粉;纤维素及其衍生物,如羧甲基纤维素钠、乙基纤维素和甲基纤维素;西黄蓍胶粉末;麦芽;明胶;滑石;固体润滑剂,如硬脂酸和硬脂酸镁;硫酸钙;植物油,如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油;多元醇,如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇;海藻酸;乳化剂,如润湿剂,如月桂基硫酸钠;着色剂;调味剂;压片剂、稳定剂;抗氧化剂;防腐剂;无热原水;等渗盐溶液;和磷酸盐缓冲液等。所述药学上可接受的载体和/或辅料在Remington'sPharmaceutical Sciences(19th ed.,1995)中有详细的记载。
此外,本发明还提供了本发明第二方面所述的构建体、本发明第三方面所述的表达载体、本发明第四方面所述的经基因改造的NK细胞、和/或本发明第六方面所述的药物组合物在制备用于治疗肿瘤的生物制剂中的应用。
在一些实施方案中,所述生物制剂中包含治疗有效量的本发明第四方面所述的经基因改造的NK细胞。
本发明的第七方面提供了抑制HIF1α表达的试剂在制备治疗肿瘤的药物中的应用。
进一步,所述抑制HIF1α表达的试剂包括针对HIF1α的shRNA、RNA反义分子、DNA反义分子、siRNA、dsRNA、miRNA、和/或核酶;
优选地,所述抑制HIF1α表达的试剂为针对HIF1α的shRNA;
更优选地,所述shRNA的序列为如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQID NO:4所示的序列中的任意一种;
优选地,所述抑制HIF1α表达的试剂通过提高NK细胞在低氧条件下的增殖能力、存活能力和/或杀伤效果发挥治疗肿瘤的作用。
此外,本发明还提供了一种治疗肿瘤的免疫治疗方法,所述方法包括如下步骤:给有需要的受试者施用有效量的本发明第六方面所述的药物组合物、和/或本发明提供的生物制剂。
在一些实施方案中,本发明所述的肿瘤包括各种类型的恶性肿瘤和良性肿瘤,其中,所述恶性肿瘤包括但不限于:肺癌、乳腺癌、大肠癌、肝癌、脑癌、骨癌、食管癌、胃癌、鼻咽癌、甲状腺癌、胰腺癌、子宫内膜癌、卵巢癌、宫颈癌、肾细胞癌、结直肠癌、前列腺癌、膀胱癌、胰腺癌、胶质母细胞瘤、黑色素瘤、白血病、淋巴瘤、骨髓瘤等;所述良性肿瘤包括但不限于:乳腺纤维瘤、囊肿、脂肪瘤、胆囊息肉、结节等。
相对于现有技术,本发明具有的优点和有益效果:
本发明首次发现抑制HIF1α的表达能够显著提高NK细胞在低氧条件下的增殖能力和存活能力,可通过抑制NK细胞中HIF1α的表达提高NK细胞在肿瘤低氧微环境中的增殖能力和存活能力,从而提高NK细胞在肿瘤免疫治疗中的治疗效果,本发明为本领域提供了一种优化NK细胞肿瘤免疫治疗的新思路和新策略,为肿瘤免疫治疗的推广和效果的提高起到了推动作用,具有广阔的应用前景。
附图说明
图1为pLenti-HIF1αshRNA-GFP-puro载体结构示意图;
图2为HIF1αshRNA-NK92细胞的基因组插入鉴定结果图,其中,M:DNA marker,T:HIF1αshRNA-NK92,C:WT-NK92,N:阴性对照,P:阳性对照;
图3为RT-qPCR检测HIF1αshRNA-NK92细胞中目的基因HIF1α表达水平的结果图,其中,A图:结果统计图,B图:CT值的平均值,C图:扩增曲线图;
图4为低氧条件(5%的O2浓度)对HIF1αshRNA-NK92细胞增殖能力影响的结果图,其中,A图:HIF1αshRNA-NK92细胞在低氧条件(5%的O2浓度)下持续培养4天,同时设置未经改造的NK92细胞作为对照组,分别在第0天、第2天、第4天采集得到的细胞图片,B图:添加了0μg/mL Na2SO3和NaHSO3的混合物(9:1质量比)的低氧条件下HIF1αshRNA-NK92细胞数量变化的结果图,C图:添加了50μg/mL Na2SO3和NaHSO3的混合物(9:1质量比)的低氧条件下HIF1αshRNA-NK92细胞数量变化的结果图,D图:添加了100μg/mL Na2SO3和NaHSO3的混合物(9:1质量比)的低氧条件下HIF1αshRNA-NK92细胞数量变化的结果图,E图:CCK8定量分析细胞增殖状况的结果图;
图5为正常氧气(21%的O2浓度)条件对HIF1αshRNA-NK92细胞增殖能力影响的结果图;
图6为低氧条件(5%的O2浓度)对HIF1αshRNA-NK92细胞存活能力影响的结果图,其中,A图:检测HIF1αshRNA-NK92细胞存活情况的结果图,B图:检测HIF1αshRNA-NK92细胞存活情况的结果统计图。
具体实施方式
除非另有定义,否则本文所用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常所理解的相同含义。为了促进对本发明的理解,此处对本发明中涉及到的如下术语进行解释:
在本文中使用,术语“包括”或“包含”,是指包括所陈述的元件或组成部分中的任意一种或多种,而并未排除其它元件或其它组成部分。
在本文中使用,术语“抑制HIF1α表达”,包括在DNA水平(通过抑制HIF1α基因产物的形成,即通过阻止或干扰转录)、在RNA水平上(通过中和或使mRNA稳定以防止或干扰翻译)或在蛋白水平上(通过中和或抑制HIF1α,或在翻译过程中靶向新生蛋白质)干扰HIF1α的基因产物的功能。可以在细胞表面或在蛋白质在表面上表达之前(例如通过将蛋白质保留在细胞内细胞器中)实现蛋白质水平的中和。通常,抑制HIF1α的最终功能作用是通过抑制HIF1α的表达提高NK细胞在低氧条件下的增殖能力、存活能力和/或杀伤效果。
在一些实施方案中,抑制HIF1α的表达不一定意味着HIF1α的完全消融,尽管也可以设想。众所周知,特别是对于反义RNA和siRNA,但对于抗体也是如此,抑制通常是部分抑制而不是完全抑制。但是,降低功能性HIF1α的水平会产生有益的效果,即使无法完全抑制也是如此。因此,根据特定的实施方案,所述抑制将导致HIF1α基因产物减少10%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%或多达100%。测量HIF1α基因产物水平的方法是本领域所属技术人员已知的,并且这些可以在添加抑制HIF1α表达的试剂之前和之后进行测量以评估功能基因产物水平的降低,或者可以与HIF1α不被抑制的合适对照细胞进行比较。类似地,与未抑制HIF1α的细胞相比,该抑制作用可显著提高NK细胞在低氧条件下的增殖能力、存活能力和/或杀伤效果。
在一些实施方案中,可以在三个层面上实现NK细胞中HIF1α的功能性抑制。首先,在DNA水平,例如通过在所述免疫细胞中去除或破坏基因(在本发明中具体为HIF1α基因),或防止转录发生(在两种情况下均阻止HIF1α基因产物的合成)。其次,在RNA水平,例如通过阻止发生有效的翻译-这可能是通过使mRNA不稳定来使其在转录本上发生翻译之前被降解,或者是与mRNA杂交。最后,在蛋白质水平,例如通过与蛋白质结合,抑制其功能,将蛋白质保留在不同的细胞位置和/或标记蛋白质进行降解。
如果在DNA水平上实现抑制,可以通过使用基因治疗敲除或破坏靶标基因来进行。如本文中所使用的,“敲降”可以是基因敲低,或可以将基因直接敲除,通过使用本领域所熟知的技术,包括但不限于:逆转录病毒基因转移,造成突变,例如点突变、插入、删除、移码、或错义突变。可以将基因敲除的另一方式是,使用锌指核酸酶(ZFN)。锌指核酸酶是通过将锌指DNA结合结构域与DNA切割结构域融合而产生的人工限制性酶。可以对锌指结构域进行改造以靶向目的DNA序列,这可使锌指核酸酶靶向复杂基因组中的独特序列。通过利用内源的DNA修复机制,这些试剂可用于精确改变高等生物体的基因组。其它可用来敲除基因的基因组定制技术有Meganuclease和TAL效应物核酸酶(TALENs,Cellectis bioresearch)。由用于序列特异性识别的TALE DNA结合结构域与引入双链断裂(DSB)的核酸内切酶的催化结构域融合组成。Meganuclease是序列特异性核酸内切酶,是天然产生的“DNA剪刀”,其源自各种单细胞生物体例如细菌、酵母、藻类和某些植物细胞器。Meganuclease具有12至30个碱基对的长识别位点。可以改变天然Meganuclease的识别位点以使其靶向天然的基因组DNA序列(例如内源基因)。
可以将基因敲除的另一方式是,基因组编辑技术-CRISPR/Cas系统,其可以被用来实现RNA引导的基因组改造。CRISPR干扰是允许序列特异性控制原核和真核细胞中基因表达的遗传技术。其基于源自细菌免疫系统的CRISPR(规律成簇的间隔短回文重复)通路。
在本文中使用,术语“shRNA”,是指短发夹RNA(short hairpin RNA,shRNA),具有发夹结构,经加工而生成miRNA、siRNA(small interfering RNA,小干扰RNA)。在一些实施方案中,shRNA在细胞内从shRNA表达载体转录。
在本文中使用,术语“siRNA”,是指小干扰RNA(small interfering RNA,siRNA),是由15-30个碱基对构成的低分子双链RNA。siRNA参与被称为RNA干扰的现象,通过靶核酸的破坏,序列特异性地抑制核酸的表达。在本发明中,siRNA是指作为破坏HIF1α并抑制HIF1α的表达的拮抗剂发挥功能的分子。本申请中的siRNA除了天然核酸分子外还可以包括人工核酸分子。
在本文中使用,术语“dsRNA”,是指双链RNA(double-stranded RNA,dsRNA),在本发明中,是指任何双链RNA分子,其能抑制或下调HIF1α的表达,例如以序列特异性方式通过促进RNA干扰(“RNAi”或“iRNA”)或基因沉默。本发明的dsRNA可以是适合Dicer的底物,或是适合与RISC结合以通过RNAi来介导基因沉默的底物。dsRNA的一条或两条链可以进一步包括末端磷酸基团,如5’-磷酸或5’,3’-二磷酸。在本发明中,dsRNA分子,除了至少一个核糖核苷酸之外,可以进一步包括取代、化学修饰的核苷酸和非核苷酸。
在本文中使用,术语“miRNA”,是指小分子核糖核酸(microRNA,miRNA),是真核生物中广泛存在的一种长约21到23个核苷酸的RNA分子,可调节其他基因的表达,miRNA来自一些从DNA转录而来,但无法进一步翻译成蛋白质的RNA(属于非编码RNA)。miRNA通过与目标mRNA结合,进而抑制转录后的基因表达,在调控基因表达、细胞周期、生物体发育时序等方面发挥着重要作用。
在本文中使用,术语“同源性”,是指与需要比对的核苷酸序列的序列相似性。“同源性”包括与本发明提供的shRNA对应的核苷酸序列具有75%或更高,或85%或更高,或90%或更高,或95%或更高同源性的核苷酸序列。同源性可以用肉眼或计算机软件进行评价。使用计算机软件,两个或多个序列之间的同一性可以用百分比(%)表示,其可以用来评价相关序列之间的同源性。所述75%或75%以上同源性,可为75%、80%、85%、90%或95%以上的同源性。
在本文中使用,术语“表达载体”,是指任何重组表达系统,该系统目的为在体外或体内、组成型或诱导型的、在任何细胞包括原核、酵母、真菌、植物、昆虫或哺乳动物细胞中表达本发明的核酸序列。该术语包括线状或环状的表达系统。该术语包括保持附加形或整合入宿主细胞基因组中的表达系统。该表达系统可具有自身复制的能力或者没有,即仅在细胞中驱动瞬时的表达。该术语包括仅含有重组核酸转录所需的最少元件的重组表达盒。
在一些实施方案中,本发明对载体没有特别的限制,其选择取决于所期望的功能。载体的非限制性实例包括质粒载体、病毒来源的载体、噬菌体载体和其他常规用于例如遗传工程的载体。可基于本领域技术人员众所周知的方法构建各种质粒和载体。
在一些实施方案中,根据本发明的表达载体能够指导本发明所述的核酸分子在宿主中的复制和表达,并因此保证由此编码的本发明所述的HIF1αshRNA在选择的宿主或宿主细胞中的表达。表达载体可以例如是克隆载体、双元载体或整合型载体。表达包括核酸分子的转录,例如转录成可翻译的mRNA。
载体的非限制性实例包括pQE-12、pUC-系列、pBluescript(Stratagene)、pET-系列表达载体(Novagen)或pCRTOPO(Invitrogen)、λgt11、pJOE、pBBR1-MCS系列、pJB861、pBSMuL、pBC2、pUCPKS、pTACT1、pTRE、pCAL-n-EK、pESP-1、pOP13CAT、E-027pCAG Kosak-Cherry(L45a)载体系统、pREP(Invitrogen)、pCEP4(Invitrogen)、pMC1neo(Stratagene)、pXT1(Stratagene)、pSG5(Stratagene)、EBO-pSV2neo、pBPV-1、pdBPVMMTneo、pRSVgpt、pRSVneo、pSV2-dhfr、pIZD35、Okayama-Berg cDNA表达载体pcDV1(Pharmacia)、pRc/CMV、pcDNA1、pcDNA3(Invitrogen)、pcDNA3.1、pSPORT1(GIBCO BRL)、pGEMHE(Promega)、pLXIN、pSIR(Clontech)、pIRES-EGFP(Clontech)、pEAK-10(EdgeBiosystems)pTriEx-Hygro(Novagen)和pCINeo(Promega)。适合于巴斯德毕赤氏酵母(Pichia pastoris)的质粒载体的非限制性实例包括例如质粒pAO815、pPIC9K和pPIC3.5K(全部为Invitrogen)。适合于在爪蟾属(Xenopus)胚胎、斑马鱼胚胎以及各种各样的哺乳动物和禽类细胞中表达蛋白质的另一种载体是多用途表达载体pCS2+。
通常,载体可含有一种或多种用于克隆或表达的复制起点(ori)和遗传系统、一种或多种用于在宿主中选择的标记(例如,抗生素抗性)和一种或多种表达盒。另外,可以使用已确立的方法将载体中包含的编码序列与转录调控元件和/或与其他氨基酸编码序列连接。这种调控序列是本领域技术人员所熟知的,并且包括但不限于确保转录起始的调控序列、内部核糖体进入位点(IRES)和任选的确保转录终止和转录物稳定的调控元件。确保转录起始的这种调控元件的非限制性实例包括启动子、翻译起始密码子、增强子、绝缘子和/或确保转录终止的调控元件,其包括在本发明提供的HIF1αshRNA的下游。进一步的例子包括Kozak序列和侧翼为用于RNA剪接的供体和受体位点的间插序列,编码分泌信号的核苷酸序列,或取决于所用的表达系统的信号序列,其能够将表达的蛋白质引导至细胞区室或培养基。载体也可含有编码一种或多种蛋白伴侣的另外的可表达多核苷酸,以促进正确的蛋白质折叠。
在本文中使用,术语“构建体”,是指人工组装或分离的核酸分子,其可包含一个或更多个核酸序列,其中核酸序列可以是编码序列(即,编码终产物的序列)、调节序列、非编码序列或其任何组合。术语“构建体”包括,例如,载体、质粒,但不应被视为限于此。在一些实施方案中,术语“调节序列”指的是DNA序列,其对于实现与其可操作地连接(link)(连接(connect)/连接(ligate))的编码序列的表达是必需的。调节序列的性质取决于宿主细胞而不同。例如,在原核生物中,调节/控制序列可以包括启动子、核糖体结合位点和/或终止子。例如,在真核生物中,调节/控制序列可以包括启动子、终止子、增强子、反式激活子和/或转录因子。与编码序列“可操作地连接”的调节序列以使得在合适的条件下实现编码序列的表达的方式连接。在一些实施方案中,“构建体”是指人工组装或分离的核酸分子,其包含感兴趣的编码区和任选的另外的调节或非编码序列。
在本文中使用,术语“药物组合物”或“生物制剂”,可具有选自如下的任一种制剂:片剂、丸剂、粉剂、颗粒剂、胶囊、悬浮液、溶液剂、乳剂、糖浆、灭菌水溶液剂、非水溶液剂、悬浮剂、乳剂、冻干制剂和栓剂。此外,可以一次或多次施用。此时,以液体制剂、粉剂、气雾剂、胶囊、阴道片剂、胶囊或栓剂的形式施用所述药物组合物和/或生物制剂。
所述药物组合物和/或生物制剂的施用途径包括但不限于:腹膜内、静脉内、肌内、皮下、皮内、经口、局部、鼻内、肺内、直肠内等。当口服施用时,可配制成保护药物组合物中的活性成分以防止其在胃中降解的包衣。此外,可通过任何能够转移至靶组织的装置施用活性成分。在具体实施方案中,本发明提供的药物组合物可根据实际需要制成各种剂型,并可由临床医师根据受试者的种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用注射或其它本领域技术人员公知的任何合适的给药方式。
在本文中使用,术语“受试者”,包括人类和非人类动物。非人动物包括所有脊椎动物(例如哺乳动物和非哺乳动物)例如非人灵长类(例如,食蟹猴)、绵羊、狗、牛、鸡、两栖动物和爬行动物。在某些实施方案中,所述“受试者”优选为人。
下面结合具体实施例,进一步阐述本发明,仅用于解释本发明,而不能理解为对本发明的限制。本领域的普通技术人员可以理解为:在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由权利要求及其等同物限定。下列实施例中未注明具体条件的实验方法,通常按照常规条件或按照厂商所建议的条件实施检测。
实施例1HIF1αshRNA质粒载体构建
1、实验方法
以pLenti-TGFBR2 shRNA-GFP-puro质粒(由通用生物(安徽)股份有限公司提供)为低氧诱导因子1α(HIF1α)的shRNA序列表达的骨架载体,酶切位点是BamHI;基因合成shRNA序列,所述shRNA序列如SEQ ID NO:1所示,合成公司为通用生物(安徽)股份有限公司,将所述shRNA序列插入骨架质粒的BamHI酶切位点之间,载体命名为pLenti-HIF1αshRNA-GFP-puro。
shRNA核酸序列:CCGGGTGATGAAAGAATTACCGAATCTCGAGATTCGGTAATTCTTTCATCACTTTTT(SEQ ID NO:1)。
2、实验结果
构建得到的pLenti-HIF1αshRNA-GFP-puro质粒载体的结构示意图如图1所示。
实施例2慢病毒转染获得稳转的HIF1αshRNA-NK92细胞
1、慢病毒包装
(1)细胞接种:10cm dish培养瓶接种1.5×107个293T细胞。加20mL含10% FBS的DMEM培养基,37℃,5% CO2培养箱过夜培养,16-24h后转染。
(2)细胞转染:细胞生长的交汇度达到80-90%,准备转染。转染体系见下表1。
表1转染体系
B液逐滴加入A液中,边加边摇匀,室温22-26℃条件下静置15min。逐滴加到培养皿中,轻轻摇匀,5% CO2,37℃过夜培养。
(3)转染换液:16-18h后,去掉含转染试剂的培养基,加入10mL含10%FBS的DMEM,5% CO2、37℃条件下继续培养。
(4)病毒第一次收获:从转染开始算48h后,收获细胞上清,转移到50mL离心管中,3,000rpm离心10min,上清用0.45μm滤膜过滤,4℃保存。细胞加入10mL含10% FBS的DMEM,5% CO2、37℃条件下继续培养。
(5)病毒第二次收获:收获细胞上清,转移到50mL离心管中,3,000rpm离心10min,上清用0.45μm滤膜过滤,4℃保存。细胞用10%消毒液(84消毒液)处理后丢弃。
(6)病毒浓缩:将收集到的慢病毒组分用0.45μm滤器过滤去除细菌污染,将过滤后组分与PEG8000按照体积比4:1混合,轻轻颠倒混匀。
(7)4℃孵育过夜。
(8)4℃8000rpm离心90min,离心后会在管底看到白色沉淀。
(9)小心吸去上清液,不能破坏白色沉淀。
(10)用适当体积慢病毒保存液重悬沉淀,得到pLenti-HIF1αshRNA-GFP-puro慢病毒载体,并分装、保存于-80℃。
2、pLenti-HIF1αshRNA-GFP-puro载体转染到NK92细胞
(1)取30万NK92细胞置于C60培养皿,加入3mLα-MEM完全培养基。向NK92细胞加入1.8μL polybrene(母液浓度为10mg/mL),0.5μL BX795(母液浓度为10mM)和病毒感染复数(MOI)为20000的病毒浓缩液,轻轻摇晃均匀后放回培养箱培养2天。
(2)2天后,收集细胞悬液,离心,去除上清培养基,加入3mLα-MEM完全培养基。恢复NK92细胞状态。
(3)再次用pLenti-HIF1αshRNA-GFP-puro毒液感染NK92细胞,此次感染不再用BX795试剂。
(4)2天后,收集细胞悬液,离心,去除上清培养基,加入3mLα-MEM完全培养基。恢复NK92细胞状态。
(5)待细胞状态恢复好,开始使用1μg/mL嘌呤霉素(puro)筛选阳性细胞,直到细胞稳定为止。稳转细胞系命名为HIF1αshRNA-NK92。
实施例3HIF1αshRNA-NK92细胞的基因组插入鉴定
1、实验方法
收集实施例2中制备得到的200万个HIF1αshRNA-NK92细胞,提取基因组,用特异性引物PCR扩增目的片段(503bp),然后进行琼脂糖凝胶电泳。未转染目的基因的细胞作为负对照不能扩增出目的片段,构建好的细胞系可以扩增出目的片段则认为目的基因成功插入基因组。
其中,针对HIF1α的特异性引物序列如下:
正向引物序列:GGACTATCATATGCTTACCGTAACT(SEQ ID NO:5)
反向引物序列:GGCCATAACCCGTAAAGAGG(SEQ ID NO:6)
2、实验结果
结果如图2所示,用特异性引物PCR扩增目的片段,进行琼脂糖凝胶电泳实验,发现HIF1αshRNA-NK92细胞与阳性质粒产生了相同大小的目的条带,证明了HIF1αshRNA成功的插入NK92细胞的基因组,即本实施例成功构建HIF1αshRNA-NK92细胞。
实施例4HIF1αshRNA-NK92细胞的qPCR表达鉴定
1、实验方法
收集实施例2中制备得到的200万个HIF1αshRNA-NK92细胞,提取RNA,反转录cDNA。以此为模板,用特异性引物在RT-qPCR检测shRNA靶向的目的基因表达量是否比未构建的野生型细胞(WT-NK92)中表达量下调。如若这样,则证明HIF1αshRNA在NK92细胞中表达。
其中,针对目的基因HIF1α的特异性引物序列如下:
正向引物序列:GAACGTCGAAAAGAAAAGTCTCG(SEQ ID NO:7)
反向引物序列:CCTTATCAAGATGCGAACTCACA(SEQ ID NO:8)
2、实验结果
结果如图3A-3C所示,RT-qPCR检测的结果显示HIF1αshRNA-NK92细胞目的基因HIF1α的表达量显著低于未经改造的NK92细胞(WT-NK92),敲降了42.31%。证明了HIF1αshRNA在NK92细胞中成功地得到了表达。
实施例5低氧条件对HIF1αshRNA-NK92细胞增殖能力的影响
1、实验方法
将实施例2中制备得到的5万个HIF1αshRNA-NK92细胞在低氧培养箱(5%的O2浓度)持续培养4天,同时设置未经改造的NK92细胞(WT-NK92)作为对照组。分别在第0天、第2天、第4天采集细胞图片,并采用血球计数板以及CCK8技术手段进行定量分析细胞的增殖状况。为了更好的控制低氧条件,本实施例添加了Na2SO3和NaHSO3的混合物(9:1质量比),设置了终浓度为0μg/mL、50μg/mL和100μg/mL的Na2SO3和NaHSO3混合物(9:1质量比)的实验组。
其中,Na2SO3和NaHSO3具有较强的还原性,容易与氧气发生反应,以达到消耗氧气的目的。
2、实验结果
结果如图4A-4E所示,结果显示,在低氧条件下,HIF1αshRNA-NK92细胞的增殖速度显著高于未经改造的NK92细胞(WT-NK92)。其中,在第4天时,HIF1αshRNA-NK92细胞的增殖速度约为WT-NK92的1.78-1.99倍,表明了HIF1αshRNA能够显著提高NK92细胞的增殖能力。
实施例6正常氧气条件对HIF1αshRNA-NK92细胞增殖能力的影响1、实验方法
将实施例2中制备得到的5万个HIF1αshRNA-NK92细胞在正常氧气条件(21%的O2浓度)下持续培养4天,采用血球计数板计数的方式定量分析细胞的增殖状况,其余步骤同实施例5实验方法中所述。
2、实验结果
结果如图5所示,结果显示,在正常氧气条件下,HIF1αshRNA-NK92细胞的增殖速度显著低于未经改造的NK92细胞(WT-NK92)。其中,在第4天时,HIF1αshRNA-NK92细胞的增殖速度约是WT-NK92的0.499倍。
实施例7低氧条件对HIF1αshRNA-NK92细胞存活能力的影响
1、实验方法
将实施例2中制备得到的5万个HIF1αshRNA-NK92细胞在低氧培养箱(5%的O2浓度)持续培养8天,同时设置未经改造的NK92细胞(WT-NK92)作为对照组。采用CalceinAM/PI活细胞/死细胞双染试剂盒检测HIF1αshRNA-NK92细胞的存活情况,并进行统计。
2、实验结果
结果如图6A和6B所示,结果显示,检测到约有77.09±5.40%的HIF1αshRNA-NK92细胞是活细胞,而对于WT-NK92而言,仅检测到33.31±4.79%的活细胞,表明了HIF1αshRNA可显著提高NK92细胞在低氧条件的存活率(存活能力)。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出,对于本领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也将落入本发明权利要求的保护范围内。
Claims (21)
1.用于基因改造NK细胞以提高NK细胞在低氧条件下的增殖能力、存活能力和/或杀伤效果的构建体,其特征在于,所述构建体包含编码抑制HIF1α表达的试剂的核苷酸;
所述低氧条件为5% O2浓度的条件;
所述抑制HIF1α表达的试剂为针对HIF1α的shRNA;
所述shRNA的序列如SEQ ID NO:1所示。
2.根据权利要求1所述的构建体,其特征在于,所述NK细胞包括NK92细胞、NK-92MI细胞、KHYG-1细胞、YT细胞、CIML-NK细胞、NKG细胞、NKL细胞、NK-YS细胞、SNK-6细胞、IMC-1细胞、PB-NK细胞、iPSC-NK细胞、和/或UCB-NK细胞。
3.根据权利要求2所述的构建体,其特征在于,所述NK细胞为NK92细胞。
4.一种用于提高NK细胞在低氧条件下的增殖能力、存活能力和/或杀伤效果的表达载体,其特征在于,所述表达载体包含权利要求1-3中任一项所述的构建体;
所述低氧条件为5% O2浓度的条件。
5.根据权利要求4所述的表达载体,其特征在于,所述表达载体包括病毒载体、DNA载体。
6.根据权利要求5所述的表达载体,其特征在于,所述病毒载体包括慢病毒载体、逆转录病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、和/或腺相关病毒载体。
7.根据权利要求5所述的表达载体,其特征在于,所述DNA载体包括DNA质粒载体、结合DNA质粒的脂质体、结合DNA质粒的分子耦联体、和/或结合DNA质粒的多聚物。
8.根据权利要求6所述的表达载体,其特征在于,所述表达载体为慢病毒载体。
9.根据权利要求4所述的表达载体,其特征在于,所述NK细胞包括NK92细胞、NK-92MI细胞、KHYG-1细胞、YT细胞、CIML-NK细胞、NKG细胞、NKL细胞、NK-YS细胞、SNK-6细胞、IMC-1细胞、PB-NK细胞、iPSC-NK细胞、和/或UCB-NK细胞。
10.根据权利要求9所述的表达载体,其特征在于,所述NK细胞为NK92细胞。
11.一种经基因改造的NK细胞,其特征在于,所述经基因改造的NK细胞低表达或不表达HIF1α;
所述经基因改造的NK细胞包含权利要求1-3中任一项所述的构建体、和/或权利要求4-10中任一项所述的表达载体。
12.根据权利要求11所述的经基因改造的NK细胞,其特征在于,所述NK细胞包括NK92细胞、NK-92MI细胞、KHYG-1细胞、YT细胞、CIML-NK细胞、NKG细胞、NKL细胞、NK-YS细胞、SNK-6细胞、IMC-1细胞、PB-NK细胞、iPSC-NK细胞、和/或UCB-NK细胞。
13.根据权利要求12所述的经基因改造的NK细胞,其特征在于,所述NK细胞为NK92细胞。
14.一种用于治疗肿瘤的药物组合物,其特征在于,所述药物组合物包含权利要求1-3中任一项所述的构建体、权利要求4-10中任一项所述的表达载体、和/或权利要求11-13中任一项所述的经基因改造的NK细胞。
15.抑制HIF1α表达的试剂在制备治疗肿瘤的药物中的应用,其特征在于,所述抑制HIF1α表达的试剂为针对HIF1α的shRNA;
所述shRNA的序列如SEQ ID NO:1所示。
16.根据权利要求15所述的应用,其特征在于,所述抑制HIF1α表达的试剂通过提高NK细胞在低氧条件下的增殖能力、存活能力和/或杀伤效果发挥治疗肿瘤的作用。
17.根据权利要求16所述的应用,其特征在于,所述低氧条件为5% O2浓度的条件。
18.根据权利要求16所述的应用,其特征在于,所述NK细胞包括NK92细胞、NK-92MI细胞、KHYG-1细胞、YT细胞、CIML-NK细胞、NKG细胞、NKL细胞、NK-YS细胞、SNK-6细胞、IMC-1细胞、PB-NK细胞、iPSC-NK细胞、和/或UCB-NK细胞。
19.根据权利要求18所述的应用,其特征在于,所述NK细胞为NK92细胞。
20.根据权利要求16所述的应用,其特征在于,通过如下任一种或多种方式组合在NK细胞中表达所述shRNA:慢病毒载体、脂质转染法、微注射、电穿孔、DNA载体、逆转录病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、腺相关病毒载体、TALEN、ZFN、和/或CRISPR/Cas9。
21.根据权利要求20所述的应用,其特征在于,通过慢病毒载体在NK细胞中表达所述shRNA。
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