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.

<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>&lt;strong&gt;Consumables&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</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 ...

genome-wide-rnai-screening-to-identify-host-factors-that-modulate

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

9.0K Views.  Children's Hospital of Eastern Ontario (CHEO) Research Institute. 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.

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

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

Immunology and Infection

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

Medicine

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

Genome-Scale Screening to Identify and Compare Therapeutic Targets in Tumors

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

Author Spotlight: Finding New Therapeutic Targets for Malignant Peripheral Nerve Sheath Tumor Through Genome-Scale shRNA Screens 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Biology

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

MicroRNA-based Regulation of Picornavirus Tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

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

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RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

DNA Vector-based RNA Interference to Study Gene Function in Cancer

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

MISSION esiRNA for RNAi Screening in Mammalian Cells

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

MicroRNA-based Regulation of Picornavirus Tropism

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells