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工程化改造哺乳动物细胞用于疾病诊断和治疗的设计策略

发布: 2019-03-02 898阅读 178评论

原文以 Engineering mammalian cells for disease diagnosis and treatment发表在期刊 《Current Opinion in Biotechnology》。 本文内容节选自原文, 有删改。
作者：高元力, 中国科学院大学生物系本科生
全文约2200字，阅读时间约为6分钟

合成生物学旨在重新编程细胞以使其感受各种输入信号, 并在不同的条件下选择地表达用户设定的基因以作出响应。哺乳动物细胞, 作为底盘, 可以被改造以感受人类疾病信号,进行计算, 输出易检测的信号, 或实现适当的治疗功能。本文总结了用于构建检测可溶性分子或细胞表面结合分子的哺乳细胞的元件及设计策略。

可溶性分子(soluble-molecules)的检测-响应回路
1) 天然受体
检测病理水平浓度的可溶性物质的最简单的策略就是利用细胞自身的配体-受体结合机制, 并且通过对下游信号级联通路的改造实现理想的响应。 G蛋白耦联受体(GPCR) 和离子通道受体都可以用作这种信号的检测与传递, 并通过下游的环磷酸腺苷(cAMP)通路和Ca2+通路激活下游的启动子。

Figure 1. 天然受体

2) Tango
Tango是一种基于正交的、依赖于蛋白酶的细胞转导模块的回路设计策略。 Tango由天然的或经人工进化的受体,跨膜且含有蛋白酶切位点的连接子(linker)和胞内的转录因子组成。 配体与受体结合后, 招募与蛋白酶融合的信号分子,蛋白酶剪切连接子并释放转录因子, 激活特定基因的表达。 采用Tango策略, 不同的G蛋白偶联受体(GPCR), 受体酪氨酸激酶(RTK) 和类固醇激素受体都可以与下游不同功能的基因相接合。另外, Kipniss等人将Tango的设计结构与dCas9-TF结合, 将蛋白酶与GPCR的C末端融合, 而将信号蛋白与dCas9-TF融合, 获得了优于原Tango设计的功能[2]。

Figure 2. Tango

3) MESA
MESA受体包含2个与合成抗体相融合的抗体单链可变片段(ScFv), 一条ScFv链与转录因子通过含有含有蛋白酶切位点的肽链连接, 另一条ScFv链与匹配的蛋白酶融合。 当配体与受体结合后, 两条链发生二聚作用, 蛋白酶水解肽链使转录因子释放。与前两种方法相比, MESA利用合成受体可以检测更多种类、甚至天然受体未知的分子, 但是MESA在没有配体的时候有显著的本底活性, 而且在配体结合后的激活水平并不理想。

Figure 3. MESA

4) GEMS
GEMS (Generalized Extracellular Molecule Sensor)的结构基于与不同配体结合的结构域融合的促红细胞生成素(erythropoietin)受体, 可以响应胞外多种类的分子。 GEMS受体的胞质部分含有可以特异性激活下游4个信号通路(JAK/STAT,ERK/MAPK, PLC 和 PI3K/AKT)的多个结构域,可以同时驱动多个转录程序。 相比基于MESA和Tango的方法, GEMS充分利用了细胞内级联通路的信号放大作用, 且不再使系统的效果过度依赖于某个启动子的选择。另一方面, 由于GEMS可以较容易的改造以适应新的可溶性抗原, 其有望成为开发基于细胞的诊断或治疗多种疾病的有效工具。

Figure 4. GEMS

细胞表面结合分子(surface-bound molecules)的检测-响应回路
1) CAR
CAR (chimeric antigen receptors, 嵌合抗原受体)由胞外的抗体衍生的scFv(抗体单链可变片段) 或纳米抗体和胞内的调控结构域组成。 CAR是目前临床上应用的最为先进的T细胞工程平台, 表达CAR的T细胞能够检测并攻击表面带有目标抗原的癌细胞。

Figure 5. CAR

2) SynNotch
SynNotch受体由相互正交的胞外和胞内结构域组成, 可以对输入信号进行编程。SynNotch受体与临近细胞中的表面抗原结合而被激活, 从而触发合成的转录因子的蛋白水解释放, 从而调控目标基因的表达。 这种嵌合抗体曾被用以检测来自免疫抑制性的肿瘤微环境的抗原, 并表达免疫刺激分子以进行响应[3]。

Figure 6. SynNotch

SynNotch和CAR受体可以联合起来限制T细胞的活化, 使得只有携带有两种靶抗原的肿瘤细胞存在时T细胞才会发生活化。 通过如下图所示的AND逻辑门, 第一种抗原激活synNotch受体, 进而激活CAR的表达, 而CAR可以检测第二种抗原激活下游通路, 从而实现体内环境肿瘤识别的高选择性。

Figure 7. synNotch 与CAR组成的与门

3) JAK/STAT-CD45 cell-cell contact pathway:
膜受体IL4与IL13的胞外结构域与scFv融合。 当与细胞表面抗原结合时, IL4和IL13受体部分发生异源二聚, 解除磷酸酶CD45胞内结构域对JAK-STAT信号传导的阻断, 通过STAT6诱导的信号转导, 激活响应STAT6的启动子表达选定的基因。 采用这种策略编辑的人间充质干细胞, 可以特异的杀死表达有HER2(人表皮生长因子受体, 乳腺癌标志物)的SKBR3乳腺癌细胞。

Figure 8. JAK/STAT-CD45 cell-cell contact pathway

前景展望与面临的挑战
1) 工程细胞拥有着在根本上改变现有的诊断、预防和治疗疾病的方法的潜力。T细胞疗法是第一个应用于临床的合成生物学方法, 而这注定只是工程细胞疗法浪潮的第一朵浪花。而且最近随着靶向癌细胞表面抗原的限制被打破, CAR T疗法的应用范围被拓展至可溶性抗原的检测。这将允许T细胞识别实体瘤微环境中的可溶性蛋白质, 并对其产生治疗性响应。

2) 对其他造成血液中生物标志物浓度改变的疾病, 临床前的数据中主要是通过将工程细胞包封入藻酸盐微囊(alginate beads), 然后植入鼠模型的血管外部采集的。一些更大尺度上包封细胞的技术(macroencapsulation)正在研发之中, 并且有可能在维持和发挥被包封细胞的功能和活力方面超过藻酸盐封装, 并且由于体积更大, 在有需要的时候检查起来更加容易。 由于包封可以保护治疗细胞免受患者免疫系统的侵害，因此非自体细胞与疾病的临床相关性应当与患者来源的细胞相同。 使用“现成的”(off-the-shelf)同种异体细胞可能是更具成本效益的疗法。

Figure 9. 基因工程改造的哺乳细胞疗法的体内实验

3) 临床经验的不断增加不可避免的带来更多挑战。例如，至今仍缺乏具有快速响应特性的基因回路，这种基因回路将造福于患有糖尿病的患者。 为了实现几分钟的响应时间，这样的基因回路必须绕过基于转录的慢响应，而依赖于转录后水平的作用。 随着工程细胞疗法越来越接近患者的需求，将不断会有令人兴奋的新挑战产生。

参考文献:
[1]Ana P T , Martin F .Engineering mammalian cells for disease diagnosis and treatment[J]. CurrentOpinion in Biotechnology, 2019, 55:87-94.
[2] Kipniss N H , Dingal P C D P , Abbott T R , etal. Engineering cell sensing and responses using a GPCR-coupled CRISPR-Cassystem[J]. Nature Communications, 2017, 8(1):2212.
[3] Roybal K T , Rupp L J , Morsut L , et al.Precision Tumor Recognition by T Cells With Combinatorial Antigen-SensingCircuits[J]. Cell, 2016, 164(4):770-779.

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