This work was supported by grants from the Ontario Institute for Cancer Research, the Canada Foundation for Innovation, the Ottawa Regional Cancer Foundation, and the Terry Fox Research Institute. K.J.A. was supported by a Vanier Canada Graduate Scholarship, a Canadian Institute for Health Research-Master&#39;s Award, and an Ontario Graduate Scholarship.

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.
<ol>
	<li>Determination of the optimal transfection conditions
	<ol>
		<li>Select a tumor cell line or ideally multiple tumor cells lines with which to optimize transfection conditions (e....

<ol>
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The authors have nothing to disclose.

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...

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 in1,2,3,4,5,6,7). Interestingly, a large number of these screens have been conducted in cancer cells and several with viruses that are current oncolytic virus candidates including vaccinia virus8,9,10, Myxoma virus11, herpes simplex virus12, vesicular stomatitis virus13,14, and Maraba virus15. While the focus of the majority of these studies was on identifying host factors that influence some aspect of virus replication and not on identifying biotherapeutic targets for enhancing oncolytic virus therapy, valuable data could be mined from these data sets in this regard. It is clear that the powerful potential to identify host targets that can be manipulated to enhance oncolytic virus therapy has not been fully exploited.
A plethora of oncolytic virus platforms are currently being tested in preclinical and clinical studies including herpes simplex virus, reovirus and vaccinia virus, which have had demonstrable success in late-stage clinical trials. For the majority of these platforms, efforts have been directed toward altering the virus genomes to enhance therapy. For instance, to increase tumor specificity, specific virus genes have been deleted or mutated to significantly restrict viral replication in normal cells, but not in tumor cells. In some cases, transgenes have been added like GM-CSF (granulocyte macrophage colony-stimulating factor) to potentiate the immune response or NIS (sodium iodide symporter) to enable in vivo imaging and cellular radioiodide uptake for therapeutic purposes. However, there has been very little work focused on manipulating host/tumor genes and exploring the impact of host/tumor genes on oncolytic virus replication. With the advent of high-throughput screening technology, it is now possible to probe host-virus interactions on a genome scale and to decipher opportunities to manipulate the host genome to enhance oncolytic virus therapy (Reviewed in16,17,18).
We reported the first study demonstrating proof-of-concept for using high-throughput genetic screening to identify host targets that could be manipulated to enhance oncolytic virus therapy. To discover host genes that modulate Maraba virus oncolysis, an oncolytic rhabdovirus currently being tested in phase I and II trials, we conducted genome-wide RNAi screens across three different tumor cell lines in duplicate with an siRNA (short interfering RNA) library targeting 18,120 genes15. Pathway analysis of hits identified in the screens revealed enrichment of members of the ER (endoplasmic reticulum) stress response pathways. We selected 10 hits within these pathways for secondary validation using siRNA with different targeting sequences from those of the primary screen. As part of tertiary validation, we conducted a rescue experiment with IRE1&#945; (inositol-requiring enzyme 1 alpha) to confirm that the hit was on target. In vitro and in vivo testing using a small molecule inhibitor of IRE1&#945; resulted in dramatically enhanced oncolytic efficacy.
Workenhe et al.12 also conducted a genome-wide RNAi screen using a pooled lentiviral shRNA (short hairpin RNA) library targeting 16,056 genes to identify host factors restricting the human herpes simplex virus type 1 (HSV-1) mutant KM100-mediated oncolysis of breast cancer cells. In the primary screen, they identified 343 genes the knockdown of which lead to enhanced cytotoxicity of KM100-infected cells over mock-infected cells; they selected 24 of these genes for secondary validation. Out of the 24 genes, 8 genes were confirmed in the secondary screen with one of them being SRSF2 (serine/arginine-rich splicing factor 2) which was subsequently confirmed using a genetic rescue experiment during tertiary validation. The group went on to identify a DNA topoisomerase inhibitor chemotherapeutic that reduced the phosphorylation of SRSF2 and showed that the combination treatment of HSV-1 KM100 with this inhibitor lead to extended survival of TUBO tumor-bearing mice.
In the aforementioned studies, a similar workflow was followed. Each began with a primary genome-wide RNAi screen followed by a secondary screen on a select number of hits identified in the primary screen. This was followed by tertiary validation studies, which included confirming that the hit was on-target by performing genetic rescue experiments in vitro with ultimate validation performed by manipulating the target gene product in vivo. In this article, we first provide a general protocol for the development and validation of a high-throughput siRNA-screening assay, which involves determining the optimal transfection conditions, amount of virus, and length of infection. We also offer a detailed protocol for conducting a primary a high-throughput siRNA screen as well as an overall description of the method for performing secondary screen validation and tertiary validation.

<table>
	<tbody>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>siRNA library</td>
			<td>GE Dharmacon</td>
			<td>G-005005-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 384-well plate</td>
			<td>Corning</td>
			<td>3985</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 384-well plate</td>
			<td>Becton Dickinson</td>
			<td>353962</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr>
			<td>siGenome SMARTpool PLK1</td>
			<td>GE Dharmacon</td>
			<td>M-003290-01</td>
			<td></td>
		</tr>
		<tr>
			<td>AllStars Negative Control siRNA</td>
			<td>Qiagen</td>
			<td>SI03650318</td>
			<td></td>
		</tr>
		<tr>
			<td>siGenome Non-targeting siRNA Pool #2</td>
			<td>GE Dharmacon</td>
			<td>D-001206-14-20</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/HIGH Glucose</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F15051-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES solution (1M)</td>
			<td>Sigma</td>
			<td>H3535-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 100X solution</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Thermo Fisher Scientific</td>
			<td>2500056</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS/Modified</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>13778100</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligofectamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>1225011</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>22600-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin sodium salt</td>
			<td>Sigma</td>
			<td>R7017-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H21492</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde (37% by weight)</td>
			<td>Fisher Scientific</td>
			<td>F79-4</td>
			<td></td>
		</tr>
		<tr>
			<td>500 ml bottles</td>
			<td>Corning</td>
			<td>430282</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Sealing Film For Microplates</td>
			<td>Excel Scientific</td>
			<td>361006007</td>
			<td></td>
		</tr>
		<tr>
			<td>Peelable Heat Sealing Foil</td>
			<td>Thermo Fisher Scientific</td>
			<td>AB-3720</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek MicroFlo Select Dispenser cassette</td>
			<td>Fisher Scientific</td>
			<td>11-120-625</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MicroFlo Select (liquid dispenser)</td>
			<td>Fisher Scientific</td>
			<td>11120621</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Synergy HT plate reader</td>
			<td>Biotek</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Opera Imaging and Analysis Instrument</td>
			<td>PerkinElmer</td>
			<td>HH10000115</td>
			<td></td>
		</tr>
		<tr>
			<td>KiNEDx Robotic Arm</td>
			<td>Peak Analysis and Automation (formerly Peak Robotics)</td>
			<td>KX-300-660</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytomat 24C Series Automated Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>50080227</td>
			<td></td>
		</tr>
		<tr>
			<td>JANUS Automated Workstation</td>
			<td>PerkinElmer</td>
			<td>AJI4M01</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Carousel</td>
			<td>Peak Analysis and Automation (formerly Peak Robotics)</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Alps 3000 plate sealer</td>
			<td>Thermo Fisher Scientific</td>
			<td>AB-3000</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome-wide rnai screening to identify host factors that modulate oncolytic virus therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;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.

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...

Watch this Scientific Journal Video about Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy at JoVE.com

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

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.

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

The overall goal of this protocol is to identify host targets that can be manipulated to enhance oncolytic virus therapy. This method can help answer key questions in the field of oncolytic virus therapy, such as what specific host factors and cellular pathways modulate oncolytic virus replication. The main advantage of this technique is that it allows one to identify host targets that can be manipulated to enhance oncolytic virus therapy in a comprehensive and unbiased manner.This protocol can also be utilized to identify host factors that modulate virus replication in general. After developing a high-throughput screening assay according to the text protocol, remove a batch of microtiter plates from the freezer and allow them to defrost for 20 to 30 minutes. In the meantime, fill two bottles with 70%ethanol, then fill two 50mL conical tubes with PBS and one with distilled water.Immerse liquid dispenser tubing into a bottle of 70%ethanol then place a piece of masking tape around the tubing to mark the point below which the tubing can be considered sterile. Use approximately 25mL of 70%ethanol to prime the tubing. Pour additional ethanol into the bottle to replace the lost volume, then let the tubing sit in ethanol for a minimum of five minutes.After preparing the cell culture hood, centrifuge the sealed plates and sets at 1026 times G in room temperature for two minutes. Next, remove the seals from the plates. Then pipette the required amount of transfection reagent into the conical tubes containing the pre-warmed transfection reagent diluent.Begin trypsinizing the cells by aspirating the medium from each plate. Use nine mL of PBS to rinse the cells, then pipette three mL of trypsin per plate. Incubate the cells at 37 degrees celsius for approximately five minutes.Then, use 22mL of medium to resuspend the cells pipetting up and down 10 times. Combine the cell suspension from each tissue culture plate into a sterile bottle and mix the cell suspension by inverting the bottle. Then pipette one mL aliquats in the cell suspension into two separate microfuge tubes.Remove the sample from each microfuge tube and count the cells. Then calculate the volume of cell suspension required for the density required, and prepare enough diluted cell suspension to dispense across one batch of plates. While the cells are being prepared, empty the liquid dispenser tubing of ethanol.Use approximately 25mL of PBS to prime the tubing, and then empty the tubing. Ensure that the portion of the tubing that will be immersed in transfection reagent, and later in the cell suspension, is kept sterile. Set apart plates that can safely be done with one conical tube of transfection reagent solution.Pipette the transfection reagent solutions up and down 10 times. Then use the solution to prime the tubing before dispensing 5 microliters per well. Centrifuge the plates at 1026 times G in room temperature for one minute.Then incubate the plates at room temperature for the time prescribed for the transfection reagent being used. During the incubation, empty the tubing of the transfection reagent solution and place it into a full 50mL conical tube containing PBS. Then use approximately 25mL of PBS to rinse the tubing by priming and then emptying it.Mix the bottle containing the cell suspension, then place the tubing in the suspension and prime the tubing enough to see that each tip is dispensing properly. Dispense 30 microliters per well of the cell suspension across all plates. Empty the cell suspension from the tubing, then rinse the tubing with approximately 25mL of PBS followed by approximately 50mL of distilled water.Allow the plates to sit at room temperature for 45 to 60 minutes from the time of cell seeding. Then incubate the plates in an undisturbed incubator for the desired length of gene silencing. To save time, the day prior to infecting the cells, pipette into sterile bottles the precise amount of medium required to infect each batch of plates, including medium for uninfected wells.Warm the medium at 37 degrees celsius, then fill two bottles with 70%ethanol, two 50mL conical tubes with PBS, and one with distilled water. Sterilize the tubing with 70%ethanol as demonstrated earlier. While the tubing is sitting in the ethanol, first vortex the virus and then pipette the precise volume of virus required into the bottle of infection medium.Then rinse the tubing by using 25mL of PBS to prime it, and then empty it. After removing the plates from the incubator, place them into the tissue culture hood. Use medium to prime the tubing, then dispense 40 microliters of medium into the uninfected control wells.Empty the tubing of medium, and use 25mL of PBS to rinse it. After mixing the virus, place the tubing into the bottle containing the virus, dispense 40 microliters of virus across the plates. Centrifuge the plates at 400 times G in room temperature for 30 minutes.Incubate the plates for the previously determined length of time. The day prior to fixing, prepare a bottle of fixing and standing solution containing four percent formaldehyde and PBS for each batch of plates, but omit the Hoechst stain. Store the solution in the flammable cabinet away from light.The following day, use 70%ethanol and PBS to rinse the tubing as demonstrated earlier. Dispense 30 microliters of the solution across all the plates, then place the plates in the incubator for 30 minutes. Following incubation, apply the seals to the plates and store them at four degrees celsius protected from light.Carry out imaging and analysis according to the text protocol. Shown here are 786 O-cells stained with Hoechst 33342 following a 72 hour incubation with transfection reagent diluent alone. Transfection reagent and diluent, non-targeting siRNA, and with siRNA targeting PLK1.The survival plot seen here indicates that 22%of cells transfected with PLK1 siRNA survived, and 94%of cells transfected with non-targeting siRNA survived. This graph depicts the results of the live time course of 786 O-cells infected with oncolytic vaccinia virus expressing enhanced GFP at various MOIs. Representative images of cells infected at an MOI of 0.05 over a live time course are shown here.An incubation period of 21 hours would allow for the detection of increases and decreases in spread as the time point is within a linear range. Following genome-wide RNAi screens across three tumor cell lines, 1008 hits common to at least two out of three cancer cell lines were identified using the MAD method. 10 hits within the UPR and ERAD pathways were selected for secondary validation via siRNA with different targeting sequences.When dispensing reagents, it is critical that you have the correct batch of plates, have selected the correct volume for dispensing, and that the tubing has been filled with the correct reagent and is always fully submerged in the reagent. After watching this video you should have a good understanding of how to conduct a high-throughput RNAi screen to identify host targets that can be manipulated to enhance oncolytic virus therapy, or to identify host factors that modulate virus replication in general.

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JoVE Journal

Cancer Research

9.0K Görüntüleme.  Children's Hospital of Eastern Ontario (CHEO) Research Institute. The overall goal of this protocol is to identify host targets that can be manipulated to enhance oncolytic virus therapy. This method can help answer key questions in the field of oncolytic virus therapy, such as what specific host factors and cellular pathways modulate oncolytic virus replication. The main advantage of this technique is that it allows one to identify host targets that can be manipulated to enhance oncolytic virus therapy in a comprehensive and unbiased manner.This pro...