Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

Summary

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

Abstract

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Introduction

High-throughput genome-wide RNAi screening has proven to be an invaluable tool for revealing biological insights in diverse areas of study including virus biology. Over the past decade or so, numerous groups have undertaken cell-based large-scale RNAi screening to extensively identify host genes that modulate virus replication (Reviewed in^{1,2,3,4,5,6,7}). Interestingly, a large number of these screens have been conducted in cancer cells an....

Protocol

1. Development and validation of a high-throughput siRNA screening assay

NOTE: For all experiments, use Hepes-buffered growth media appropriate for each cell line. See Figure 1 for an overview of the workflow from assay optimization to tertiary validation.

1. Determination of the optimal transfection conditions
   1. Select a tumor cell line or ideally multiple tumor cells lines with which to optimize transfection conditions (e.g. 786-O, NCI-H226, SF268, U373, OVCAR-8, U20S; see Discussion for more on the selection of cell lines).
   2. Select a transfection reagent....

Results

An overview of the workflow for identifying host targets to enhance oncolytic virus therapy is presented in Figure 1.

As illustrated in Figure 1, the critical first step to conducting a high-throughput RNAi screen is assay development. Figure 2A provides a sample plate layout for the transfection optimization assay. Fig.......

Discussion

Here we present a protocol for employing high-throughput RNAi screening to identify host targets that can be manipulated to enhance oncolytic virus therapy. This has been tested successfully with oncolytic Maraba virus and oncolytic vaccinia virus, but, as noted, it can be adapted for use with other oncolytic viruses or other viruses generally to identify host genes that modulate virus replication. The screening protocol is also designed to identify genes that increase or decrease spread, but it can be readily modified f.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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