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Nature:更快、更便宜、更精准!“魔剪”CRISPR或改写细胞疗法

2018-07-16 佚名 生物探索

无需病毒载体,实现对人类T细胞高效、精准的基因改造,这是科学家们最新发表在Nature杂志上的突破成果。而“魔剪”CRISPR又在其中发挥了至关重要的作用。研究人员认为,这一成果对科研、医学和工业领域具有重大意义,将大大加快下一代细胞疗法的发展,为癌症等疾病的治疗带来新希望。

病毒引发感染是通过注射它们自己的遗传物质。自上世纪70年代以来,科学家们就已经开始运用病毒的这一特性:利用病毒载体运送DNA到细胞中,用于研究、基因疗法等。

然而,构建病毒载体其实是一个非常艰苦、昂贵的过程,临床级载体(clinical-grade vectors)的短缺还导致了基因疗法和细胞疗法的生产瓶颈。此外,即使是可用的,病毒载体也远不是一种理想的工具,因为它们会将基因随意插入到细胞基因组中,这将损害现有的健康基因或使新引入的基因不受确保细胞正常运作的调节机制的控制。这些局限性可能会导致严重的副作用,已经引起了人们对基因疗法和细胞疗法(如CAR-T)的担忧。


图片来源:Nature

摆脱“病毒”的新技术

7月11日,发表在Nature杂志上题为“Reprogramming human T cell function and specificity with non-viral genome targeting”的研究中,来自加州大学旧金山分校(UCSF)的科学家们开发出了一种在不使用病毒插入DNA的前提下就能“重写”人类T细胞基因组的强大技术。

具体来说,该技术依赖于电穿孔:将一个电场应用于细胞,使得细胞膜暂时具有更大的渗透性。经过一年的探索,UCSF的Alex Marson博士带领的团队发现,当一定数量的T细胞、DNA和CRISPR被混合在一起,然后暴露于适当的电场后,T细胞能够精准整合特定的基因序列到基因组中被CRISPR切割的位点。

Alex Marson, MD, PhD, UCSF immunologist whose group has developed an approach to edit T cell genomes without using viruses, speaks with MD/PhD student Theo Roth. Credit: Noah Berger.

事实上,Marson博士实验室的成员先前已经在利用电穿孔和CRISPR插入少量遗传物质到T细胞中取得了一定程度的成功。但大量尝试将长的DNA序列插入到T细胞中的实验都失败了:导致了细胞死亡。这使得很多研究人员认为,大的(长的)DNA序列对T细胞毒性过大。

为了展示新技术的多功能性和实力,Marson博士等利用它修复了T细胞中的一种致病基因突变,并且创造出了能够找到和杀死人类黑色素瘤细胞的“定制T细胞”。

据论文第一作者Theodore L. Roth介绍,科学家们尝试将新基因插入T细胞已有30年之久。在他们的研究中,经过近一年的反复试验,Roth确定了T细胞数量、DNA数量和CRISPR丰度(CRISPR abundance)的比值,结合拥有适当参数的电场,实现了对T细胞基因组高效、准确的编辑。


The research team created CRISPR guides that would cause green fluorescent protein to be expressed in only certain cellular locations and structures. Credit: Alex Marson's Lab.

“实力”验证

研究中,Roth首先设计CRISPR用绿色荧光蛋白(green fluorescent protein,GFP)标记一组不同的T细胞蛋白,结果证实,新技术具有高度的特异性,且脱靶效应水平非常低:GFP只在特定的细胞位置和结构中表达。

接着,他们开始验证新技术的治疗前景。在第一个实验中,Roth及其同事使用了由耶鲁医学院Kevan Herold提供给Marson博士实验室的T细胞。这些细胞来自患罕见、严重自身免疫性疾病的三个兄弟姐妹。基因组测序显示,这些儿童的T细胞携带着IL2RA基因的突变。而IL2RA基因编码的是对调节性T细胞(Tregs,可抑制其他免疫细胞,阻止自身免疫)发育至关重要的一种细胞表面受体。

利用基于CRISPR的新技术,UCSF的团队快速修复了儿童T细胞中的IL2RA缺陷,以及因突变受损的细胞信号。研究人员希望,就像利用CAR-T疗法抗癌一样,类似的技术能够有效治疗自身免疫性疾病。

在第二个实验中,借助新技术,科学家们用“被专门设计用于识别人类黑色素瘤细胞特殊亚型的新受体”完全取代了“正常人类T细胞中的天然T细胞受体(T cell receptors,细胞用于检测疾病和感染的传感器)”。在培养皿中,被改造过的T细胞表现出了特异性:不仅能够有效地找到目标黑色素瘤细胞,同时还会忽略其他细胞。这对于癌症精准治疗是非常重要的。

研究人员强调,在不使用病毒载体的情况下,他们能够生产出大量经CRISPR改造后表达新T细胞受体的工程细胞。而将这些CRISPR工程细胞(CRISPR-engineered cells)移植给携带人类黑色素瘤的小鼠后,它们能够进入肿瘤部位,并显示出抗癌活性。


Set of test tubes in Alex Marson's lab. Credit: Noah Berger.

前景广阔

Roth表示,由于新技术使得“在一周多一点的时间内创建可用的定制T细胞系”成为了可能,因此,它已经改变了Marson博士实验室的研究环境。先前一些受限于病毒载体的实验现在可以进行了。

“这是一种能够用于改变、增强以及重编程T细胞的快速、灵活的方法。与速度和易用性一样重要的是,新技术使得‘将大量的DNA片段插入T细胞中’成为了可能,这将赋予T细胞强大的新特性。”Marson博士评价道。

总结来说,研究人员希望,他们的新技术——一种基于CRISPR的快速、全能、经济的方法能够在新兴的细胞治疗领域得到广泛应用,加速开发新的、更安全的癌症、自身免疫性疾病和其他疾病(包括罕见的遗传性疾病)的治疗方法。

原始出处:

Theodore L. Roth, Cristina Puig-Saus, Alexander Marson, et.al.  Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 11 July 2018

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    2019-06-16 liye789132251
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    2018-07-20 GZphD9

    学习

    0

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    2018-07-18 yuandd
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    2018-07-17 神功盖世

    学习

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    2018-07-17 sunfeifeiyang

    学习

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    2018-07-16 神功盖世

    0

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