Transcriptomic analysis of of SNX-5422-treated human primary tracheobronchial epithelial (TBE) cells.

Purpose: In addition to developing antivirals targeting specific SARS-CoV-2 viral proteins, there has been a growing effort in developing therapeutics targeting host proteins exploited by SARS-CoV-2 for their replication or implicated in viral pathogenesis. As such therapeutics can impair normal functioning of host proteins, a clear understanding of their impact on cellular mechanisms is critically important. Here, we characterized the interactions of a host-targeted anti-SARS-CoV-2 therapeutic, SNX-5422, with the cellular machinery using a physiologically relevant ex vivo human primary tracheobronchial epithelial (TBE) cell model. Methods: Fully differentiated human TBE cells (EpiAirwayTM) from 3 independent donors with no reported respiratory disease or smoking history were obtained from MatTek (Ashland, MA). The cells were cultured at the air-liquid interface in 1 ml of AIR-100-MM culture medium (MatTek) in 6 well plates at 37°C in 5% CO2. Upon receipt of cells, the cultures were acclimated for 16-24hr prior to the start of experiments. Human TBE cells from each donor were treated with 1µM SNX-5422 or 0.1% DMSO added to the media on the basolateral side of the culture, in three biological replicates. After 48hrs, cells were resuspended in TRIzol reagent (Thermo Fisher) and total RNA from the cells was extracted by phase separation with chloroform and subsequently using the RNeasy Mini Kit (Qiagen). RNA-Seq libraries were prepared using TruSeq RNA library Prep Kit v2 (Illumina, Inc. USA). Before pooling and sequencing, fragment length distribution and library quality were assessed on a TapeStation 2200 (Agilent Technologies), and the libraries were validated by Qubit Fluorometers (Thermo Fisher). All libraries were then pooled in a concentration at 4nM and sequenced in quadruplets on a NextSeq 500 Illumina sequencing platform system using NextSeq 500/550 High Output Kit v2.0 (150 cycles) (Illumina, Inc. USA). Conclusion: This study identified differentially expressed genes between DMSO-treated and SNX-5422-treated human TBE cells. Bulk RNA-seq analysis of human TBE cells from 3 independent donors with no reported respiratory disease or smoking history treated with 1μM SNX-5422 or 0.1%DMSO (drug-vehicle)

实验目的：除开发靶向特定SARS-CoV-2病毒蛋白的抗病毒药物外，针对靶向宿主蛋白的抗SARS-CoV-2治疗手段的研发投入亦与日俱增——此类宿主蛋白或被病毒利用以完成复制周期，或与病毒致病机制密切相关。由于此类靶向宿主的治疗药物可能干扰宿主蛋白的正常生理功能，清晰阐明其对细胞机制的影响至关重要。本研究采用具有生理相关性的离体原代人气管支气管上皮（tracheobronchial epithelial, TBE）细胞模型，对靶向宿主的抗SARS-CoV-2治疗药物SNX-5422与细胞机制的相互作用进行了表征。
实验方法：从美国马萨诸塞州阿什兰市MatTek公司获取3名无呼吸道疾病史、无吸烟史的独立供体来源的完全分化人TBE细胞（EpiAirway™）。将细胞以气液界面培养模式接种于6孔板中，每孔加入1mL AIR-100-MM培养基（MatTek），于37℃、5%CO₂培养箱中培养。细胞到货后，需先适应性培养16~24小时方可启动实验。将每位供体来源的人TBE细胞设置3个生物学重复，分别向培养基的基底侧添加1μM SNX-5422，或0.1%二甲基亚砜（DMSO）作为溶剂对照。处理48小时后，将细胞重悬于TRIzol试剂（赛默飞世尔科技）中，通过氯仿相分离法提取总RNA，随后采用RNeasy Mini试剂盒（凯杰公司）完成RNA纯化。使用TruSeq RNA文库制备试剂盒v2（美国Illumina公司）构建RNA测序（RNA-Seq）文库。在混合文库与测序前，采用TapeStation 2200生物分析仪（安捷伦科技）评估文库的片段长度分布与质量，并通过Qubit荧光计（赛默飞世尔科技）验证文库浓度。将所有文库统一调整至4nM浓度后混合，采用NextSeq 500/550 High Output试剂盒v2.0（150个循环）（美国Illumina公司），在NextSeq 500 Illumina测序平台上以四重重复方式完成测序。
实验结论：本研究鉴定出经DMSO处理与经SNX-5422处理的人TBE细胞之间的差异表达基因。本研究对3名无呼吸道疾病史、无吸烟史的独立供体来源的人TBE细胞开展了批量RNA-seq分析，这些细胞分别经1μM SNX-5422或0.1%DMSO（药物载体）处理。