全基因组的 rna 干扰筛查以确定调节溶瘤病毒治疗的宿主因素
Summary
在这里 , 我们描述了一种使用高通量 rna 干扰筛选的协议 , 以发现宿主目标可以作来增强溶瘤病毒治疗 , 特别是 rhabodvirus 和痘苗病毒治疗 , 但它可以很容易地适应其他溶瘤病毒平台或发现宿主基因, 通常调制病毒复制。
Abstract
高通量全基因组 rna 干扰 (RNA 干涉) 筛选技术已被广泛用于发现影响病毒复制的宿主因素。在这里, 我们提出了这项技术的应用, 以揭露宿主目标, 专门调节复制的马拉巴病毒, 溶瘤 rhabdovirus, 和痘苗病毒的目的是加强治疗。虽然该协议已测试用于溶瘤马拉巴病毒和溶瘤痘苗病毒, 这种方法适用于其他溶瘤病毒, 也可以用来确定主机目标, 调节病毒复制的哺乳动物细胞在秘书长.本议定书介绍了哺乳动物细胞高通量 rna 干扰筛查的研究进展和验证, 主要的考虑因素和准备步骤对进行一次高通量的 rna 干扰屏幕, 以及一步一步指南为进行一次高通量的 rna 干扰屏幕;此外, 它概括地概述了进行二次屏幕验证和三级验证研究的方法。高通量 rna 干扰筛选的好处是, 它允许一个人以一种广泛而无偏见的方式对病毒复制的任何方面进行分类, 这些因素可以开发出一种体外检测方法, 如传染性、爆裂大小、和细胞毒性。它有能力揭露 biotherapeutic 的目标, 根据目前的知识。
Introduction
高通量全基因组 rna 检测已被证明是一个宝贵的工具, 以揭示生物洞察力在不同的研究领域, 包括病毒生物学。在过去的十年左右, 许多团体进行了基于细胞的大规模 rna 干扰筛查, 以广泛识别调节病毒复制的宿主基因 (在1,2,3,4,5,6, 7). 有趣的是, 大量的这些屏幕已经进行了癌症细胞和一些病毒, 目前溶瘤病毒候选包括痘苗病毒 8, 9, 10, 粘液瘤病毒11, 单纯疱疹病毒12, 水泡口炎病毒13,14和马拉巴病毒15。虽然大多数研究的重点是确定影响病毒复制的某些方面的宿主因素, 而不是确定增强溶瘤病毒治疗的 biotherapeutic 目标, 但有价值的数据可以从这些数据集中挖掘出来。这方面。很明显 , 确定可纵以加强溶瘤病毒治疗的宿主目标的强大潜力尚未得到充分利用。
目前, 大量的溶瘤病毒平台在临床前和临床研究中被测试, 其中包括单纯疱疹病毒、呼肠病毒和痘苗病毒, 这些都在后期试验中取得了明显的成功。对于大多数的这些平台, 已经致力于改变病毒基因组, 以加强治疗。例如, 为了增加肿瘤特异性, 特定的病毒基因已被删除或变异, 以显著限制病毒复制在正常细胞, 但不是在肿瘤细胞。在某些情况下, 转基因已被添加如 GM-脑脊液 (粒细胞巨噬素集落刺激因子), 以事实免疫应答或 NIS (碘化钠转运体), 使体内成像和细胞 radioiodide 吸收治疗目的.然而, 很少有工作专注于操纵宿主/肿瘤基因和探索宿主/肿瘤基因对溶瘤病毒复制的影响。随着高通量筛选技术的出现, 现在可以在基因组范围内探测宿主病毒相互作用, 并破译操作宿主基因组以增强溶瘤病毒治疗的机会 (16, 17,18)。
我们报告的第一项研究表明, 使用高通量基因筛选的概念证明, 以确定宿主目标, 可以操纵, 以加强溶瘤病毒治疗。为了发现调节马拉巴病毒裂癌的宿主基因, 该溶瘤 rhabdovirus 目前正在第一阶段和第二期试验中进行测试, 我们在三种不同的肿瘤细胞系中进行了全基因组 rna 检测, 并与 siRNA (短干扰 RNA) 重复。图书馆目标18120基因15。通过对筛查发现的通路进行分析, 揭示了 ER (内质网) 应力响应通路的富集。我们在这些路径中选择了10次命中, 以便使用 siRNA 与主屏幕中不同的目标序列进行二级验证。作为三级验证的一部分, 我们进行了 IRE1α (肌醇要求酶1α) 的抢救实验, 以确认命中目标。体外和体内使用 IRE1α的小分子抑制剂进行测试, 从而显著提高了溶瘤的功效。
Workenhe et 。12还使用一个聚集的慢病毒载体 shRNA (短发夹 RNA) 库对16056个基因进行了全基因组的 rna 干扰筛查, 以确定限制人疱疹病毒1型 (HSV-1) 突变体 KM100-mediated 裂癌的宿主因素癌细胞。在主屏幕上, 他们确定了343个基因的击倒, 导致 KM100-infected 细胞对模拟感染细胞的细胞毒性增强;他们选择了24这些基因进行二次验证。在24种基因中, 有8个基因在二次筛中被证实, 其中之一是 SRSF2 (丝氨酸/精氨酸-富含剪接因子 2), 随后在三级验证中使用基因抢救实验证实。该小组接着确定了一种 DNA 拓扑异构酶抑制剂化疗, 减少了 SRSF2 的磷酸化, 并表明 HSV-1 KM100 与此抑制剂的联合治疗导致了吐蕃肿瘤小鼠的延长生存。
在上述研究中, 遵循了类似的工作流程。每一个都是从一个主要的全基因组的 rna 干扰屏幕开始, 其次是在主屏幕上确定的点击次数中的辅助屏幕。其次是三级验证研究, 其中包括确认命中是在目标通过执行基因抢救实验在体外通过操纵目标基因产品在体内进行最终验证。在本文中, 我们首先提供了一个用于开发和验证高通量 siRNA 筛选试验的通用协议, 它涉及确定最佳转染条件、病毒数量和感染长度。我们还提供了一个详细的协议, 用于进行一个主要的高通量 siRNA 屏幕, 以及对执行次屏幕验证和三级验证的方法的总体描述。
Protocol
Representative Results
Discussion
在这里, 我们提出了一个使用高通量 rna 干扰筛选的协议, 以确定宿主目标, 可以操作, 以加强溶瘤病毒治疗。这已成功地通过溶瘤马拉巴病毒和溶瘤痘苗病毒进行了测试, 但是, 正如所指出的, 它可以适应其他溶瘤病毒或其他病毒的使用, 通常用于识别调节病毒复制的宿主基因。该筛选协议还旨在确定增加或减少传播的基因, 但它可以很容易地修改其他读数。例如, 我们的马拉巴病毒的屏幕被设计用来…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了安大略省癌症研究研究所、加拿大创新基金会、渥太华区域癌症基金会和特里福克斯研究所的资助。K.J.A. 得到了加拿大 Vanier 研究生奖学金、加拿大卫生研究所硕士奖和安大略省研究生奖学金的支持。
Materials
| Consumables | |||
| siRNA library | GE Dharmacon | G-005005-02 | |
| Corning 384-well plate | Corning | 3985 | |
| Falcon 384-well plate | Becton Dickinson | 353962 | |
| Water | Sigma | W4502 | |
| siGenome SMARTpool PLK1 | GE Dharmacon | M-003290-01 | |
| AllStars Negative Control siRNA | Qiagen | SI03650318 | |
| siGenome Non-targeting siRNA Pool #2 | GE Dharmacon | D-001206-14-20 | |
| DMEM/HIGH Glucose | GE Healthcare Life Sciences | SH30022.01 | |
| Fetal Bovine Serum | Sigma | F15051-500ML | |
| HEPES solution (1M) | Sigma | H3535-100ML | |
| Penicillin-Streptomycin 100X solution | GE Healthcare Life Sciences | SV30010 | |
| Trypsin-EDTA (0.25%) | Thermo Fisher Scientific | 2500056 | |
| DPBS/Modified | GE Healthcare Life Sciences | SH30028.02 | |
| Lipofectamine RNAiMAX | Thermo Fisher Scientific | 13778100 | |
| Oligofectamine | Thermo Fisher Scientific | 1225011 | |
| Opti-MEM | Thermo Fisher Scientific | 22600-050 | |
| Resazurin sodium salt | Sigma | R7017-5G | |
| Hoechst 33342 | Thermo Fisher Scientific | H21492 | |
| Formaldehyde (37% by weight) | Fisher Scientific | F79-4 | |
| 500 ml bottles | Corning | 430282 | |
| Adhesive Sealing Film For Microplates | Excel Scientific | 361006007 | |
| Peelable Heat Sealing Foil | Thermo Fisher Scientific | AB-3720 | |
| BioTek MicroFlo Select Dispenser cassette | Fisher Scientific | 11-120-625 | |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| MicroFlo Select (liquid dispenser) | Fisher Scientific | 11120621 | |
| BioTek Synergy HT plate reader | Biotek | N/A | |
| Opera Imaging and Analysis Instrument | PerkinElmer | HH10000115 | |
| KiNEDx Robotic Arm | Peak Analysis and Automation (formerly Peak Robotics) | KX-300-660 | |
| Cytomat 24C Series Automated Incubator | Thermo Fisher Scientific | 50080227 | |
| JANUS Automated Workstation | PerkinElmer | AJI4M01 | |
| Plate Carousel | Peak Analysis and Automation (formerly Peak Robotics) | N/A | |
| Alps 3000 plate sealer | Thermo Fisher Scientific | AB-3000 |
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