Method Article

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

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DOI:

10.3791/56913

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April 3rd, 2018

 ,  ,  ,  , 
1Children's Hospital of Eastern Ontario (CHEO) Research Institute, 2Department of Biology, Microbiology and Immunology, University of Ottawa, 3Department of Pediatrics, University of Ottawa, 4Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary

In This Article

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 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 IRE1α (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 IRE1α 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.

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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 or several to test. It may be necessary to test several transfection reagents, as some may be more toxic or efficient than others in a given cell line. Also, keep in mind that some may impact on virus infection or may generate high background signal upon imaging.
    3. Decide on positive [e.g. siRNA targeting PLK-1(Polo Like Kinase 1) mRNA, which induces cell death] and negative transfection controls (e.g. non-targeting siRNA).
    4. Follow the reverse transfection protocol for the particular transfection reagent, but vary the amount of transfection reagent (e.g. 0.05, 0.1, 0.15 and 0.2 μL/well) and cell seeding densities (e.g. 625, 1250, 2500 cells/well). Include replicates of the following conditions: untreated cells, mock transfected cells (i.e. cells plus transfection reagent), and cells transfected with positive and negative transfection control siRNA (see Figure 2A for a sample plate layout).
      1. In general, prepare 80 nM dilutions of each siRNA for a final concentration in the well of 10 nM (depending on the library, a higher concentration of siRNA may be required). Unless otherwise specified, dilute the siRNA with the transfection reagent diluent. Prepare dilutions of the transfection reagent.
      2. Pipet 5 μL of siRNA per well followed by 5 μL of diluted transfection reagent. To facilitate this step, distribute along a column of a U-bottom 96-well plate, the siRNA mixes and the siRNA diluent alone (for untreated and mock transfected cells). Distribute along a second column, the diluted transfection reagent and transfection reagent diluent alone (for untreated cells).
        1. Using an electronic pipet, pipet 5 μL/well of the contents of the first column containing siRNA or siRNA diluent into the corresponding wells of the 384-well plate followed by 5 μL/well of the diluted transfection reagent or transfection reagent diluent from the second column.
      3. Centrifuge the plates at 1026 x g for 1 min at room temperature, and incubate for the time prescribed for the transfection reagent (e.g. 5-20 min).
      4. While the plates are incubating, prepare cell suspensions of different densities. Pipet 30 μL of cell suspension per well. Before placing the plates in the incubator, let the plates sit for 45-60 min at room temperature to allow for the uniform settling of cells19.
        Note: If the incubation period prescribed for the transfection reagent with siRNA is short, prepare the cell suspensions before the incubation.
      5. Incubate for 48 to 72 h depending on the desired length of knockdown chosen for the screen.
    5. Prepare a fixing and staining solution consisting of formaldehyde, Hoechst 33342 and PBS (phosphate-buffered saline) for a final concentration in each well of 4% formaldehyde and 5 ug/mL of Hoechst 33342. As needed, try a higher (e.g. 7.5 ug/mL) or lower concentration (e.g. 2 ug/mL) of Hoechst if the signal is too weak or too strong, respectively.
    6. Dispense 30 μL of the fixing and staining solution directly into all wells. Incubate the plates for 30 min at 37 °C. Store the plates at 4 °C. If using a non-confocal microscope, wash the plates with PBS using a plate washer; in most cases, washing is unnecessary when using a confocal microscope.
    7. Develop a script to quantify the number of cells per well.
      NOTE: There are several software suites available for this purpose and, while each may have different ways of generating scripts, they all generally have a means of identifying nuclei, cells and regions of interest as well as calculating intensities and morphological properties. When developing a screening script, it is important to consider the time required to run the script per plate and to perhaps include only essential measurements to minimize processing time. For example, to quantify cell number, one might calculate the Hoechst area per well above a specified intensity; and to quantify virus spread, one might calculate the GFP (green fluorescent protein) area per well above a specified intensity. The "pared down" script will need to be compared to the more elaborate script to ensure no essential information is lost.
    8. Analyze the results to determine the optimal transfection conditions, that is the transfection reagent amount and the cell seeding density that results in significant cell death (0-30% cell survival) and is minimally toxic to the cells (e.g. 90-100% cell survival). Note: It may take longer to observe the cell death phenotype in cell lines with long doubling times. See Figure 2B and 2C for representative results. Also, ensure that the cells transfected with non-targeting siRNA are approximately 90-100% confluent at the time of fixation as later on one will want to infect cells that are of this confluency.
  2. Determination of the optimal amount of virus and length of infection
    1. Perform the same reverse transfection steps as described above (see 1.1.4.1 to 1.1.4.4); however, use only the optimal transfection conditions (see 1.1.8) (e.g. for 786-O cells: 1500 cells/well and 0.05 μL/well of RNAiMAX) and, in addition, include extra wells to be infected with virus [e.g. wells with untreated cells, mock transfected cells, cells transfected with non-targeting siRNA, and cells transfected with positive virus controls if ones are known (i.e. siRNA targeting host genes that will inhibit or enhance replication)].
      1. See Figure 3A for a sample plate layout. Incubate for 48-72 h.
    2. Infect the additional virus wells with an oncolytic virus expressing a fluorescent reporter protein (e.g. GFP) with a range of multiplicities of infection (MOIs) (e.g. 0.001 to 10; The range chosen will be depend on the level of resistance or susceptibility of the cell line.). Include uninfected wells as well. Pipet 40 μL of each virus dilution per well for a final volume of 80 μL. Centrifuge each plate at 400 x g for 30 min at room temperature.
    3. Following infection, image the cells live with a high-content, automated microscope at regular intervals (e.g. every 4-8 h). See Figure 3B and 3C.
    4. Develop a script to quantify virus spread (e.g. GFP area per well) (see 1.1.7). Analyze the images and select a time-point and an MOI that allow for the detection of increases and decreases in spread. Ensure the time-point and MOI fall within a linear range to facilitate detection of small changes in spread. Estimate the Z-factor (or Z') for the given time-point and MOI.
      NOTE: Estimated Z-factor = 1 - 3(σp + σn)/|μp-μn|, where σp is the sample standard deviation of the positive virus controls and σn is the sample standard deviation of the negative virus controls; and μp is the sample mean of the positive virus controls and μn the sample mean of the negative controls. An estimated Z-factor of 0.5 - 1.0 is considered to be an excellent assay and suitable for screening20,21.
    5. Confirm that the negative virus control siRNA has no impact on virus replication by comparing it to the mock transfected and untreated wells. It is often necessary to test multiple negative controls as some negative siRNA controls can impact on virus replication.
  3. Final validation of the high-throughput screening assay
    1. Repeat steps 1.2.1 and 1.2.2 above, but only this time infect cells with the optimal MOI. Instead of live imaging, fix and stain the plate (as described in 1.1.5 and 1.1.6) at the optimal time-point (see 1.2.4).
    2. Image the plate. Analyze the results using the same script that will be used for the screen (see 1.1.7 and 1.2.4). Confirm the transfection conditions (i.e. good knockdown in wells transfected with positive transfection control and minimal toxicity in wells transfected with negative transfection control). Estimate the Z-factor to determine the robustness of the assay (see 1.2.4).
  4. Further testing and considerations for conducting high-throughput screening
    1. Test the durability of the microtiter plates as some plates may crack during centrifugation, especially when the lids are kept on and multiple plates are centrifuged at once. To reduce cracking, centrifuge without the lids (provided the plates have seals), or decrease the number of plates centrifuged per bucket.
    2. Test the stability of the transfection reagent mix over time. Once the transfection reagent is added to the plates, there may be a significant delay before the addition of cells; therefore, it is important to know the period of time for which the transfection reagent mix will remain effective. For the screen, it may be necessary to prepare cells in advance of adding the transfection reagent mix to the plates.
    3. Prepare for using the liquid dispenser. Determine the volumes required to dispense transfection reagent, cells, virus, and fix taking into account the volume that the tubing holds, the volume lost to pre-dispensing (if the unit has this feature), and the residual volume required in the bottles used for dispensing.
      1. Set and test the programs that will be used for dispensing transfection reagent, cells, and virus. To minimize the loss of transfection reagent, limit, if possible, the number of pre-dispenses. Establish a protocol for calibrating the liquid dispenser and testing its accuracy and precision.
    4. Prepare for the bulk dispensing of transfection reagent, cells and virus
      1. First determine the number of plates to be processed in one batch considering the number of plates of cells that can feasibly be trypsinized at once, the number of microtiter plates that can be centrifuged at once, the length of time required to process one batch of plates with the liquid dispenser, and also the time required for imaging. Note: Generally, processing approximately 30 plates at a time is quite feasible and processing 3 batches of plates is manageable in one day.
      2. Empirically verify the volume of transfection reagent required to process one batch of plates. Test that this volume is adequate by dispensing 5 μL of distilled water multiple times across one plate the equivalent number of times that will be required to process one batch.
      3. To ensure the even distribution of cells, estimate the time necessary to dispense cells across one batch of plates. Prepare a bottle containing the volume of cells required for a batch, and dispense the cells into multiple columns of a plate at a time, at regular intervals over the same course of time it will take to dispense across a batch of plates. Test how often it will be necessary to mix the cells to maintain an even distribution of cells.
      4. To ensure the even distribution of virus, repeat the same procedure as above (see 1.4.4.3), but this time dispensing virus onto confluent wells. If the virus is not evenly distributed, consider preparing the virus in conical tubes and vortexing each tube prior to dispensing.
    5. Track the growth of cells to determine the timeline for growing enough cells for the screen. Also, determine the average number of cells that can be harvested per plate of cells growing in log phase in order to be able to estimate the number required for the screen.
  5. Final preparation steps for high-throughput screening
    1. Up to one month prior to commencing screening, determine all of the materials necessary for the screen. Order any required reagents (see Materials). Ensure the required amount of virus is on hand (Preferably, use the same stock that was used during optimization.).
    2. Up to two weeks prior to commencing screening, thaw and grow up the cells required for the screen. Ensure the availability of the centrifuge and square centrifuge buckets.
    3. During the week prior to commencing screening, prepare media bottles for the reverse transfection and infection, a stock of 70% ethanol, 2X transfection reagent diluent (e.g. 2X Opti-MEM), if necessary (e.g. if the siRNA library is diluted in RNase-free water, one will want to use 2X to transfect cells), and aliquots of virus (one per batch of plates). Verify the condition of the cells.
      1. Prepare a stock solution of Hoechst 33342 stain (e.g. 5 mg/mL). Prepare a balance plate if one will be required for centrifuging.
      2. Ensure the plate washer is in good working order if it will be used. Ensure the liquid dispenser is properly calibrated and dispensing accurately and precisely. Also, ensure the programs are properly set on the liquid dispenser.

2. Conducting the primary high throughput screen

NOTE: Prior to commencing the RNAi screen, microtiter plates (e.g. 384-well plates) need to be arrayed with the siRNA library and screening controls. The following steps are best conducted by two persons with one person (i.e. Person A) being primarily responsible for dispensing the transfection reagent and cells across the plates, and the second person (i.e. Person B) being responsible for centrifuging the plates immediately prior to transfection and for preparing the cells. Included in parentheses are some specifics for processing one batch of 33 plates (i.e. half of the library) with the 786-O cell line.

  1. Preparation for reverse transfection
    1. Person A: Place enough media, trypsin and PBS necessary to process one batch of plates in a 37 °C water bath. Optional: Trypsinize a plate of cells, and count the number of cells per plate in order to provide a real-time estimate of the number of plates required per batch.
    2. Obtain a batch of plates (e.g. 33 plates) from the freezer and wait 20-30 min to allow the plates to defrost. While the plates are defrosting, continue with the following steps.
      1. Person A: Pipet into 50 mL conical tubes the amount of transfection reagent diluent necessary for processing one batch of plates (e.g. 2 x 37.125 mL) and place them in a 37 °C water bath. Fill two bottles with 70% ethanol (e.g. 250-500 mL bottles). Fill two 50 mL conical tubes with PBS and one with distilled water.
      2. Person A: Immerse the liquid dispenser tubing into a bottle of 70% ethanol. If all of the tips will not be used for dispensing the transfection reagent (e.g. if the outer wells of the plate are not being used), detach the unnecessary tubing first. Place a piece of masking tape around the tubing to mark the point below which the tubing can be considered sterile. Prime with approximately 25 mL. Pour additional ethanol into the bottle to replace the lost volume. Let the tubing sit in ethanol for a minimum of 5 min.
      3. Person B: Prepare to trypsinize cells. Spray the tissue culture (TC) hood with 70% ethanol. Spray and place the necessary pipets, tips, bottles and microfuge tubes for preparing cells in the hood (see 2.2.1.1 and 2.2.1.2). Centrifuge the sealed plates in sets at 1026 x g for 2 min at room temperature. Remove the lids from the plates in a TC hood, if previously determined that the plates might crack during centrifugation when the lids are kept on (see 1.4.1).
    3. Person A: Remove the seals from the plates. Before, during or after the seals have been removed (depending on the length of incubation required), pipet the required amount of transfection reagent (e.g. 375 μL/tube for a final concentration of 0.05 μl of RNAiMAX/well) into the conical tubes containing the pre-warmed transfection reagent diluent (e.g. 37.125 mL/tube).
    4. Mix by pipetting up and down 10 times. Incubate at room temperature for the time prescribed for the transfection reagent being used (e.g. 0-10 min).
  2. Reverse transfection
    1. Have Person B complete the following tasks.
      1. Begin trypsinizing the cells (e.g. 3 plates of 786-O cells). Aspirate the media from each plate. Rinse with 9 mL of PBS. Pipet 3 mL of trypsin per plate. Incubate at 37 °C for approximately 5 min. Resuspend the cells with 22 mL of media. Pipet up and down 10 times.
      2. Combine the cell suspension from each tissue culture plate into a sterile bottle. Mix the cell suspension by inverting. Pipet 1 mL aliquots of the cell suspension into two separate microfuge tubes. Remove a sample from each microfuge tube and count the cells. Calculate the volume of cell suspension required for the density required (e.g. 1500 cells/well), and prepare enough diluted cell suspension (e.g. 500 mL) to dispense across one batch of plates.
    2. Have Person A complete the following tasks in parallel to Person B.
      1. Empty the liquid dispenser tubing of ethanol. Prime the tubing with approximately 25 mL of PBS, and then empty the tubing. Be careful when handling the tubing to ensure that the section of tubing below the masking tape 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.
      2. Pipet the transfection reagent solutions up and down 10 times. Prime the tubing with the transfection reagent solution. To minimize the loss of transfection reagent, prime with just enough solution to barely clear the tips. Select the correct program on the liquid dispenser and dispense 5 μl of transfection reagent per well.
        CAUTION: Closely monitor the level of the transfection reagent so that it is replenished before it runs out. The completion of the plates that are set apart should provide a cue to add more transfection reagent solution.
      3. Centrifuge the plates at 1026 x g for 1 min at room temperature. This time the lids will be on, so it may be necessary to centrifuge less plates at a time to prevent cracking (e.g. 2 plates per bucket). Incubate the plates at room temperature for the time prescribed for the transfection reagent being used (e.g. 10-20 min).
      4. During the incubation, empty the tubing of the transfection reagent solution, and place it into a full 50 mL conical tube containing PBS. Rinse the tubing with approximately 25 mL of PBS by priming and emptying.
        1. If more of the tips are going to be used for dispensing cells than were used for dispensing transfection reagent, re-attach the unused tubing, and re-sterilize all the tubing with 70% ethanol before rinsing the tubing with PBS (see 2.1.2.2).
      5. Mix the bottle containing the cell suspension once it has been prepared. Place the tubing in the cell suspension, and prime enough to see that each tip is dispensing properly. Dispense 30 μL per well of the cell suspension across all the plates. If necessary, mix the cell suspension at regular intervals to avoid settling.
        CAUTION: Sometimes droplets of suspension containing media may stick to the sides of the tips. When this is observed, aspirate as soon as possible or wipe with lint-free tissue doused in 70% ethanol.
      6. Empty the tubing of the cell suspension. Rinse the tubing with approximately 25 mL of PBS, followed by approximately 50 mL of distilled water.
    3. Allow the plates to sit for 45-60 min from the time of cell seeding at room temperature before returning the plates to the incubator. Incubate the plates in an undisturbed incubator for the desired length of gene silencing (e.g. 48 h) (see 1.1.4.5).
  3. Infection
    1. To save time, the day prior to infecting the cells, pipet into sterile bottles the precise amount of media required to infect each batch of plates including media for uninfected wells (e.g. 2 x 350 mL and 2 x 50 mL for 33 plates). In addition, test the liquid dispenser for precision and accuracy, and re-calibrate if necessary.
    2. Warm the media at 37 °C. Fill two bottles with 70% ethanol, two 50 mL conical tubes with PBS and one with distilled water. Verify that the centrifuge is at room temperature.
    3. Sterilize the tubing with 70% ethanol, and rinse with PBS as previously described (see 2.1.2.2 and 2.2.2.1).While the tubing is sitting in the ethanol, pipet the precise volume of virus required into the bottle containing the media previously prepared for infection (see 2.3.1).Rinse the tubing with PBS by priming with 25 mL and then emptying.
    4. Remove the plates from the incubator and place them into the TC hood. Prime the tubing with media. Dispense 40 μL of media into the uninfected control wells. Empty the tubing of media and rinse with 25 mL PBS. (see 2.2.2.5 for CAUTION)
    5. After mixing the virus, place the tubing into the bottle containing the virus. Dispense 40 μl of virus across the plates. Centrifuge the plates for 30 min at 400 x g at room temperature. Return the plates to the incubator. Incubate the plates for the previously determined length of time (e.g. 24 h) (see 1.2.4).
  4. Fixing and staining cells
    1. To save time, the day prior to fixing, prepare a bottle of fixing and staining solution containing formaldehyde and PBS (e.g. 179 mL of 37% formaldehyde and 270 mL of PBS for a final concentration of 4% formaldehyde) for each batch of plates, but omit the Hoechst stain. Store in the flammable cabinet away from light.
    2. Rinse the tubing with 70% ethanol and PBS as previously described (see 2.1.2.2 and 2.2.2.1). Add the required volume of Hoechst (e.g. 2.5 mL of a 5 mg/mL of Hoechst for a final concentration in the well of 7.5 μg/mL) to the fixing and staining solution and mix.
    3. Dispense 30 μL of fixing and staining solution across all the plates. Place the plates in the incubator for 30 min. Following incubation, apply the seals to the plates, and store the plates at 4°C protected from light. Wash the plates if necessary (see 1.1.6).
  5. Imaging and image analysis
    1. Image the plates with an automated, high-content microscope. Use the previously determined settings, but first test the settings to ensure no adjustments need to be made.
    2. Quantify the GFP (or other fluorescent reporter) area and Hoechst area per well using the previously developed script (see 1.3.2 ). Test the script with a handful of plates first to determine if any slight modifications need to be made before batch analyzing all of the plates.
    3. Identify hits. Note: The robust Z-score/MAD (median absolute deviation) method is a method that is often used towards this end. Typically, scores of >2 or <-2 are set as cut-offs. This method is best used when the siRNA samples are randomly distributed across the plates and not, for example, grouped by function as the samples are used as de facto negative controls. Filter out cytotoxic siRNAS as these would be expected to non-specifically decrease virus spread (see Discussion for further details on filtering out cytotoxic hits).

3. Secondary screen validation of hits with the TRC (The RNAi Consortium) shRNA library

NOTE: See Discussion for details on selecting hits for secondary validation and possible screening technologies. If siRNA technology is selected for secondary validation, the same assay development and validation protocol can be used as was used for the primary screen (see 1).

  1. Obtain a custom TRC shRNA library targeting the candidate genes of interest as glycerol stocks.
  2. Prepare transfection quality plasmid DNA from the glycerol stocks. Note: A detailed protocol (i.e. "shRNA/sgRNA/ORF Glycerol and Plasmid DNA Preparation") can be found here: http://portals.broadinstitute.org/gpp/public/resources/protocols.
  3. Produce lentiviruses expressing shRNA targeting the genes of interest with the DNA plasmids. Note: A detailed protocol for lentivirus production in 96-well plates [i.e. "shRNA/sgRNA/ORF High Throughput Viral Production (96 well)"] can be found here: http://portals.broadinstitute.org/gpp/public/resources/protocols.
  4. Infect cells with the lentiviruses. Note: A detailed protocol for lentiviral infection in 96-well plates (i.e. "Lentiviral Infection") is available online at http://portals.broadinstitute.org/gpp/public/dir/download?dirpath=protocols/production&filename=TRC%20viral%20infection%20200909.pdf.
  5. Infect cells with oncolytic virus either following puromycin selection (see 3.4 for the selection protocol) of successfully transduced cells or immediately following transduction (without puromycin selection). To determine the optimal amount of virus and the length of incubation with virus, use the same method used for the primary screen.

4. Tertiary validation

NOTE: The purpose of tertiary validation is to primarily confirm that the hit is on target, tumor specific, and enhances efficacy in vivo. One can also test whether it modulates spread across a spectrum of tumor cell lines

  1. Confirmation that knockdown of the candidate gene is on-target
    1. First confirm that there is a correlation between the phenotype and gene silencing. Use the same assay that was developed for the screen or modify it for a 6-well format to assess the enhancement or inhibition of spread in a larger format. Conduct a time-course with cells transfected with siRNA targeting the gene(s) of interest plus and minus virus.
    2. Image the wells containing virus-infected cells, and collect the cell lysates from the uninfected wells at regular time intervals. Determine whether there is a correlation between gene silencing and enhancement or inhibition of spread. Assess gene silencing by Quantitative Polymerase Chain Reaction (qPCR) and/or Western blotting.
    3. Perform a genetic rescue experiment to confirm the hit is on-target. Note: In a typical rescue experiment, the candidate gene is knocked-down with RNAi while at the same time cells are transfected transiently with RNAi resistant cDNA (e.g. ortholog of the gene) expressing the candidate gene to determine whether the phenotype of the gene is restored or "rescued." Stable cell lines overexpressing RNAi resistant cDNA of the candidate gene of interest can also be employed.
    4. Alternatively, instead of a genetic rescue experiment, use multiple orthogonal assays to confirm that the hit is on-target [e.g. determine whether the same phenotype is obtained upon knockdown of the target gene with multiple siRNAs, upon knockdown of the target gene in multiple stable cell lines expressing shRNA against the target gene, and upon knockout with CRISPR (Clustered regularly interspaced short palindromic repeat)-Cas9 technology in multiple cell lines.].
  2. Confirmation that the hit is tumor-specific and modulates spread across a range of tumor cell lines
    1. Conduct a similar assay as in 4.1.1, but with normal cells as well as with other tumor cell lines.
  3. Confirmation that manipulation of the candidate gene enhances efficacy in vivo
    NOTE: Described below are three possible methods for manipulating the gene target in vivo.
    1. Find a drug to manipulate the gene target. If there is a drug such as a small molecule inhibitor that targets the same gene, administer it with the oncolytic virus in vivo. It is important to determine the optimal timing for administering the drug.
    2. Engineer the virus to inhibit or overexpress the gene target depending on whether one desires to inhibit or enhance the gene target. To inhibit the gene, engineer the oncolytic virus to express a dominant negative of the gene or to express shRNA targeting the gene. To enhance the expression of the gene target, clone the oncolytic virus with a transgene overexpressing the gene.
    3. Engineer cell lines with the gene knocked-down or knocked-out using shRNA or CRISPR-Cas9 technology, respectively. Implant the engineered cell line and the wild-type cell line in vivo and subsequently infect with the oncolytic virus. Note: This method serves as a proof-of-principle and can help determine whether investing additional time and resources to develop a drug to manipulate the gene in vivo, for example, would be worthwhile.

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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...

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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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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's Award, and an Ontario Graduate Scholarship.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Consumables
siRNA libraryGE DharmaconG-005005-02
Corning 384-well plateCorning3985
Falcon 384-well plateBecton Dickinson353962
WaterSigmaW4502
siGenome SMARTpool PLK1GE DharmaconM-003290-01
AllStars Negative Control siRNAQiagenSI03650318
siGenome Non-targeting siRNA Pool #2GE DharmaconD-001206-14-20
DMEM/HIGH GlucoseGE Healthcare Life SciencesSH30022.01
Fetal Bovine SerumSigmaF15051-500ML
HEPES solution (1M)SigmaH3535-100ML
Penicillin-Streptomycin 100X solutionGE Healthcare Life SciencesSV30010
Trypsin-EDTA (0.25%)Thermo Fisher Scientific2500056
DPBS/ModifiedGE Healthcare Life SciencesSH30028.02
Lipofectamine RNAiMAXThermo Fisher Scientific13778100
OligofectamineThermo Fisher Scientific1225011
Opti-MEMThermo Fisher Scientific22600-050
Resazurin sodium saltSigmaR7017-5G
Hoechst 33342Thermo Fisher ScientificH21492
Formaldehyde (37% by weight)Fisher ScientificF79-4
500 ml bottlesCorning430282
Adhesive Sealing Film For MicroplatesExcel Scientific361006007
Peelable Heat Sealing FoilThermo Fisher ScientificAB-3720
BioTek MicroFlo Select Dispenser cassetteFisher Scientific11-120-625
NameCompanyCatalog NumberComments
Equipment
MicroFlo Select (liquid dispenser)Fisher Scientific11120621
BioTek Synergy HT plate readerBiotekN/A
Opera Imaging and Analysis InstrumentPerkinElmerHH10000115
KiNEDx Robotic ArmPeak Analysis and Automation (formerly Peak Robotics)KX-300-660
Cytomat 24C Series Automated IncubatorThermo Fisher Scientific50080227
JANUS Automated WorkstationPerkinElmerAJI4M01
Plate CarouselPeak Analysis and Automation (formerly Peak Robotics)N/A
Alps 3000 plate sealerThermo Fisher ScientificAB-3000

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Tags

Host Factor IdentificationVirus Replication AssayHigh throughput ScreeningTransfection Reagent PrimingCell Suspension PreparationVirus Infection ProtocolSecondary Validation StudiesTertiary Validation Methods

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