CRISPR女神Jennifer再发重量级Reviews:CRISPR-Cas系统引领药物发现途径和疾病治疗方案的革新

2016-12-28 生物谷 不详



Jennifer Doudna

CRISPR-Cas系统作为基因组编辑和调节的编程工具,可以在各种细胞中(包括人类细胞)进行遗传操作。虽然目前科学家们的注意力主要集中在CRISPR-Cas系统治疗孟德尔遗传疾病方面的潜力,但是该技术还有望为复杂的体细胞疾病提供新的治疗方法,同时CRISPR-Cas通过加速药物靶点的鉴定和验证,成为下一代药物研发的有利工具。

近日CRISPR先驱——“女神”Jennifer在nature再发新成果,她的研究小组发现了两个CRISPR新系统,十分振奋人心。由此可见CRISPR自问世以来总是带给人们惊喜不断,可谓引发了遗传学等生物学科研究和医药领域的革命。12月23日《Nature Reviews Drug Discovery》在线发表的文章,Jennifer也参与其中,此次她们将目光投向CRISPR-Cas系统带来的药物发现途径和疾病治疗方案的革新。



CRISPR-Cas系统指的是聚簇规则间隔短回文重复序列(clustered regularlyinterspaced short palindromic repeat ,CRISPR)和CRISPR相关蛋白(CRISPR-associated protein,Cas),能够在哺乳动物模型系统和人体组织中快速而准确地改变基因组信息。通过对患者的体细胞进行直接编辑,引入校正突变或修饰调节元件,使几乎所有靶点的药物开发都成为可能。

CRISPR-Cas系统简要地概括就是sgRNA指导Cas9核酸内切酶在同源位点诱导双链断裂,然后通过细胞DNA修复机制进行修复,包括NHEJ和HDR两种途径。 NHEJ最常用于破坏基因序列,而HDR可以用于通过设计的修复模板在特定基因座引入或改变遗传信息。催化失活的突变体Cas9可以融合到各种效应结构域以活化或抑制靶基因的转录,分别被称为CRISPRa和CRISPRi的策略。

┃CRISPR-Cas作为药物发现的工具

1建立精确的细胞模型(应用于药物靶点发现)

DNA测序的发展及其大规模应用刺激了“个性化”或“精准”医学的发展。虽然可在细胞之间比较某个基因(例如TP53,MYC或KRAS)的突变状态,但通常会有许多其它混杂的特征掩盖了基因型和疾病类型之间的直接关系。研究人员可以使用来自患者和正常人群组织样品进行比较,或者通过过度表达适当cDNA的技术,但是这些原始的技术会耗费大量劳动力和时间,这阻碍了它们广泛应用于药物开发。

CRISPR-Cas基因编辑的出现戏剧性地改变了这个状况。通过CRISPR-Cas进行基因敲除已被证明用于几乎所有的细胞类型,包括诱导多能干细胞/">干细胞ipsCs),癌症特异性免疫细胞。这种基因敲除允许研究人员在确定的背景下快速确定致癌基因、肿瘤抑制因子和其他因子的致病作用。类似地,通过HDR“敲入”突变等位基因,研究人员可以测试疾病相关等位基因突变的作用,如与多种癌症相关的KRAS等位基因的研究,能够分析突变体对疾病发展的影响或验证突变体靶向治疗候选物的特异性。

HDR需要以病毒载体、质粒(编码Cas9和sgRNA)或Cas9-sgRNA核糖核蛋白(RNP)复合物的形式递送Cas9-sgRNA复合物以及DNA修复模板。虽然CRISPR-Cas敲除在几乎任何细胞中都有效,但是效率随细胞类型而变化。在非传染性人类细胞(包括神经元)中实现中等水平的HDR是困难的。这些障碍使人沮丧,于是科学家们绕过HDR途径,使用非同源或微生物介导的方式实现了在非有丝分裂人细胞中的应用。另一个令人兴奋的发展是应用具有额外功能的Cas酶,可以直接改变靶碱基,来实现无模板的精确特异性突变的引入。实现这一目标的重要方法是各种胞苷脱氨酶与Cas9的融合。

2快速生成动物模型(推动新药动物实验发展)

除了细胞培养应用之外,基因编辑已经极大地改变了产生疾病的动物模型的能力。在CRISPR-Cas工具最初发展之后不久,它就被用于产生具有多个遗传突变的小鼠。CRISPR-Cas可以在单个步骤中靶向多个基因,快速产生双突变和多突变小鼠,但是必须注意的是这些等位基因要在育种后不依赖孟德尔分离。通常通过在受精卵中进行显微注射或简单电穿孔实现有效的CRISPR-Cas编辑技术(包括NHEJ 和HDR),而不需要使用传统的胚胎干细胞操作的方式。在受精卵中进行基因编辑省去了分离、培养和编辑ES细胞的步骤,节省了时间,且加速了在现有动物模型中产生额外突变的进程。然而,通过受精卵编辑引入大量转基因或复杂的多组分系统仍然是低效的,所以ES细胞中的基因靶向仍然是产生具有这种突变的动物所选择的方法。

总体而言,CRISPR-Cas通过减少生成目标模型所需的时间不断革新小鼠遗传学。目前,使用基于病毒或转座子的载体,CRISPR-Cas已经可以在某些组织中直接引入体细胞突变,例如肺和肝组织。这种方法被用于创建癌症和其他疾病模型。

靶向的CRISPR-Cas是具有巨大的前景的。传统的基因靶向在小鼠以外的临床前模型中仍然困难,而 CRISPR-Cas编辑已经在大鼠、狗和猴中进行,这些动物在临床前药物发现和开发期间都是常用的。灵长类动物中疾病模型的产生,例如恒河猴Duchenne肌营养不良的模型,进一步强调基因编辑可用于测试治疗化合物的功效和安全性。



应用CRISPR建立动物模型过程

CRISPR-Cas提供新的治疗方案

1应用于CAR-T细胞治疗

随着癌症免疫治疗领域的快速扩大,基因编辑在体细胞疾病的应用集中在下一代嵌合抗原受体(CAR)T细胞的生产。

CAR包含细胞外结合结构域(目前为单链可变片段),能识别在肿瘤细胞上强烈表达和特异的抗原,激活T细胞的细胞内信号传导,促进T细胞介导的肿瘤细胞杀伤。第一批CAR-T细胞介导的治疗靶向CD19,一种B细胞和相关癌细胞表达的抗原,几种这些疗法已经进入临床试验(Juno Therapeutics:NCT02535364和NCT02631044; Kite Pharma:NCT02601313和NCT02348216;Novartis:NCT02030834和NCT02445248)。目前,大多数CAR-T细胞使用每个患者自身的T细胞产生,涉及为每个新患者分离、修饰和扩增T细胞。因此,CAR-T细胞的经济性比基于抗体的检查点的癌症免疫疗法(例如ipilimumab,pembrolizumab和nivolumab)更不好。如果可以产生通用供体CAR-T细胞,则CAR-T细胞治疗可以变得更快,更便宜。然而, CAR-T细胞识别受体细胞引起的移植物宿主排斥,仍然是现有方法的主要障碍。

CRISPR-Cas可以用于敲除内源性T细胞受体基因防止宿主反应性,也可以用于消除或减少供体T细胞上的组织相容性抗原的表达来预防或延迟受试者免疫系统对CAR-T细胞的排斥。此外,基因编辑还可以用通过敲除编码T细胞抑制性受体或信号分子的基因,如细胞毒性T淋巴细胞相关蛋白4(CTLA4)或程序性细胞死亡蛋白1(PD-1)来促进CAR-T细胞功效。

美国国家卫生研究院(NIH)重组DNA咨询委员会(RAC)最近批准了将在宾夕法尼亚大学进行的黑色素瘤靶向CAR-T临床试验,其中Cas9将用于敲除细胞编码PD1和内源性T细胞受体基因。中国最近开始了CRISPR-Cas的第一次临床试验。该试验使用Cas9敲除患有肺癌的个体的T细胞中的PD-1。而类似的试验,用于前列腺和膀胱癌以及肾细胞癌的PD-1敲除T细胞也正在开始。

2离体基因编辑治疗

向细胞或组织递送药物是非常挑战性的,是药物治疗的主要限制。CRISPR-Cas的应用,对靶细胞进行体外操作,则规避了这个问题。造血系统是离体基因编辑的极好的靶标,因为细胞容易从外周血样品获得,并且可以在操作和扩增后重新注射。例如研究人员使用ZFNs破坏从HIV患者中分离的T细胞中的CCR5基因,随后进行扩增,再重新回输编辑的T细胞,以在患者体内产生HIV抗性的自体T细胞。这种方法的I / II期临床试验正在进行中。尽管T细胞中CCR5基因的突变是永久的,但T细胞本身不是。研究人员最近正致力于破坏HSCs中的CCR5,以产生长期自我更新的HIV抗性细胞。



总之,CRISPR-Cas编辑可以加速功能基因组学发展,揭示细胞机制并确证新的药物靶点,开发更好的用安全性测试的模型,以及改善治疗方案等。快速基因编辑也可以生成定制的自体细胞治疗,包括癌症治疗的T细胞和重新编程的ipsC,应用于非遗传性疾病创新疗法。虽然CRISPR-Cas系统将进一步改进,但我们相信基因编辑已开始对全世界的药物发现和开发产生直接的影响。

原文来源:

Cornerstones ofCRISPR–Cas in drug discovery and therapy

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    2017-06-19 xugumin
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    2016-12-30 yuandd
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    2016-12-30 仁心济世

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