rnai screening 2009 - RNAi SCREENS FOR TARGET IDENTIFICATION AND VALIDATION

RNAi SCREENS FOR TARGET IDENTIFICATION
AND VALIDATION
8:30 Chairperson’s Remarks
Paul D. Kassner, Ph.D., Principal Scientist, Amgen Inc.

8:40 RNAi Screening to Identify and Validate Cancer Targets
Xiaoyu Lin, Ph.D., Associate Research Investigator, siRNA Therapeutics, Abbott Laboratories
RNAi-Based large-scale screens have provided a powerful tool to gain new insight in critical biological processes and identify novel therapeutic targets. However, the impact of off-target effect in the context of large-scale gene knockdown experiments is still not fully appreciated. We will review the data from several different siRNA library screens in an effort to identify novel cancer targets. We will focus on a new siRNA library that has significantly reduced off-target effect. General siRNA library screen design and ‘hits-follow-up’ strategies will also be discussed.

9:10 Using siRNA Screening for Target Identification, Validation, and Compound Mechanism of Action Studies
Alex Gaither Ph.D., Research Investigator II, Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research
We have developed a siRNA screening optimization platform for identifying a compound’s mechanism of action as well as novel targets using disease model cell-based assays. Using a viral-based transduction system, we can rapidly optimize siRNA transfection conditions in most cell lines. We recently used a Smac mimetic compound (LBW242) targeting the Inhibitor of Apoptosis Proteins (IAP) in an apoptome siRNA screen to determine pathways that confer sensitivity and resistance across different cancer cell lines. Among the hits identified were multiple components of the TFNα signaling pathway as well as XIAP itself. Here we show that in a subset of highly sensitive tumor cell lines, activity of LBW242 is dependent on TFNα signaling. Mechanistic studies indicate that in this context XIAP is a positive modulator of TFNα induction whereas CIAP1 negatively regulates TFNα-mediated apoptosis.

9:40 1536-Well RNAi HTS Assays to Identify New Regulators of the Wnt Pathway
Marc Ferrer, Ph.D., Senior Research Fellow, Automated Biotechnology, Merck & Co.
The Wnt/α-Catenin cell signaling pathway is involved in development, cancer, tissue regeneration, bone and stem cell biology. Several genome-scale siRNA screens were carried out to better understand the genes regulating this pathway. A total of four cell lines were developed with a β-catenin activity reporter (BAR) driving luciferase expression. Two cell lines had an APC loss-of-function (LOF) and therefore had a constitutively active, Wnt-independent β-catenin activity. Two additional cell lines had Wnt-dependent b-catenin activity which was induced by conditioned media containing Wnt3a. In order to select genes that are independent of cell background, reduce data variation associated with prolonged operation of siRNA screen (typically several weeks), and to introduce robust statistical selection of primary hits, the assays were miniaturized to 1536w plate format and genome-wide siRNA screens were performed in triplicate (N=3), for each cell line. The ultra high throughput assay also allowed us to quickly test different siRNA collections to compare performance and identify possible siRNA activities due to off-target effects. The result is a very efficient siRNA screening paradigm that has identified new genes involved in the β-catenin pathway.

10:10 Grand Opening Coffee Break in the Exhibit Hall

10:40 Genome-Wide siRNA Screening and Data Integration to Determine Content Vulnerability for Human Tumors
Quick Que, Ph.D., Head of Target Validation, Cancer Drug Development Laboratory, Translational Genomics Research Institute
RNA interference has emerged as a powerful functional genomics tool to perform targeted gene knockdowns and study loss-of-function phenotypes. Here, we report a genome-wide, loss-of-function siRNA screen in cell lines representing different tumor types and genetic backgrounds. Genes selected as “vulnerable hits” from the primary screen were further validated by various approaches. Statistical methods along with integrative analyses of orthogonal datasets (array CGH, mutation, expression, knowledge mining) were employed to assign validated genes that upon knockdown caused significant loss of viability in a general or cell-specific context. Wherever possible, genomics data from primary tumor samples has been leveraged to establish clinical relevance. In conclusion, our study provides valuable data in understanding target or pathway dependence and functional consequences. The results also shed light on new targets as well as the development strategy for targeted agents.

11:10 Beneficial Use of siRNA Off-Target Effects for Assay Development, Screening Quality Control, and Hit-Selection
Paul D. Kassner, Ph.D., Principal Scientist, Amgen Inc.
RNA interference library screens have enormous potential to identify genes involved in phenotypes and pathways of interest. However, the execution and interpretation of high quality screens requires overcoming the challenges of off-target effects, problematic cell biology, and intricate informatics. During assay development (AD) and high-throughput screening (HTS) campaigns we model off-target effects existing in our library with the large-scale use of non-targeting (NT) control siRNAs. Our panel of NT siRNAs is used during AD for identification of ideal transfection conditions, during HTS for monitoring reproducibility between runs, and eventually for selection of high-confidence hits.

11:40 Strategies for Effective RNAi Screens
Moderator: Paul D. Kassner, Ph.D., Principal Scientist, Amgen Inc
Target discovery campaigns have been performed in disease-relevant cells across a wide range of diseases using an arrayed adenoviral shRNA collection targeting the human drugable genome. For diseases such as asthma, osteoporosis and osteoarthritis, human primary cells were screened using assays that mimic the biology of the disease identifying known as well as novel targets. High-Content screens are currently underway to identify proteins that play a critical role in cancer and neurological disorders. These screens are performed in disease-relevant cell types, such as primary cells and patient-derived cell lines, which are difficult to transfect using conventional siRNA methods. Here data will be presented on RNAi screens for novel targets in cancer and Huntington Disease with a focus on further validation of identified targets.

12:25pm New Technologies for RNAi Screening with Pooled Lentiviral shRNA Libraries
Alex Chenchik, Ph.D., President/CEO, Cellecta, Inc.
We have developed a high throughput technology for the functional discovery and validation of drug targets associated with viability of cancer cells. A second generation of Tet-regulated Functionally Validated bar-coded lentiviral pooled 30K shRNA library designed for the set of 10,000 druggable and cell signaling genes was developed by validation of more than 100,000 shRNA constructs. An optimal shRNA cassette design was developed by screening over 60 different shRNA cassettes that contain combinations of the most widely used structural features. The FV shRNA library is transduced in several cancer cell lines, and cytotoxic shRNAs and corresponding target genes are identified by high throughput sequence analysis. Data will be presented that demonstrate the application, problems, and solutions for genome-wide functional analysis with arrayed and pooled siRNA libraries for several cancer cell models. We will present the following:

Comparison of pooled and array formats
Lessons and solutions for RNAi screens
High throughput validation of functional shRNAs
Optimization of the shRNA expression cassette
Negative and positive RNAi screens for cancer models

12:40 Target Identification and Validation Using High Content Screening in Combination
with Adenoviral shRNA Technology
Richard Janssen, Ph.D., Senior Director, Target Discovery, BioFocus DPI, A Galapagos Company
Target discovery campaigns have been performed in disease-relevant cells across a wide range of diseases using an arrayed adenoviral shRNA collection targeting the human drugable genome. For diseases such as asthma, osteoporosis and osteoarthritis, human primary cells were screened using assays that mimic the biology of the disease identifying known as well as novel targets. High-Content screens are currently underway to identify proteins that play a critical role in cancer and neurological disorders. These screens are performed in disease-relevant cell types, such as primary cells and patient-derived cell lines, which are difficult to transfect using conventional siRNA methods. Here data will be presented on RNAi screens for novel targets in cancer and Huntington Disease with a focus on further validation of identified targets.

12:55 Luncheon Technology Workshop Sponsored by
Next Generation siRNAs: Combining Innovative Design With Novel Chemical Modifications, For Superior Consistency of Phenotypic Results
Michel Cannieux, Ph.D., MBA, Product Manager, RNA Interference, Applied Biosystems
Poor specificity and efficacy of siRNA prediction too often combine into poor consistency and reproducibility of RNAi experiments. We will describe the experimental studies and results leading to the discovery of a novel arrangement of chemical modifications in siRNAs that significantly reduce off-target effects as measured by microarray and cell based assays.








HIGH-THROUGHPUT FUNCTIONAL SCREENS
2:20 Chairperson’s Remarks
Christophe J. Echeverri, Ph.D., Chief Executive Officer / Chief Scientific Officer, Cenix BioScience GmbH

2:25 The RNAi Strategy in Cancer Research
Roderick L. Beijersbergen, Ph.D., Group Leader, Division of Molecular Carcinogenesis, The Netherlands Cancer Institute
The development of the RNA interference (RNAi) technology has changed the way we approach target discovery and validation in cancer research. The potential to study the consequence of the inactivation of each individual gene is a very effective tool to identify novel targets, elucidate the mechanism of action of novel therapeutics and to identify potential mechanisms of resistance. We have developed bar code technology which is based on the use of large collections of shRNA vectors in a pooled format combined with the quantification of the relative abundance of each individual shRNA vector by DNA micro-array analysis. We have used this technology to identify important cellular components required for the effect of a novel anti-cancer drug and for the identification of genes involved in the response to Trastuzumab in breast cancer cells. These studies demonstrate that bar code screening is a rapid and powerful technology for the identification of genes associated with important cellular pathways in disease and will accelerate the development of novel cancer drugs.

2:55 Cancer Proliferation Gene Discovery Through Functional Genomics
Stephen J. Elledge, Ph.D., Department of Genetics, Center for Genetics and Genomics, Harvard Medical School
Retroviral short hairpin RNA (shRNA)-mediated genetic screens in mammalian cells are powerful tools for discovering loss-of-function phenotypes. We have developed a highly parallel multiplex methodology for screening large pools of shRNAs using half-hairpin barcodes for microarray deconvolution. We carried out dropout screens for shRNAs that affect cell proliferation and viability in cancer cells and normal cells. We identified many shRNAs to be anti-proliferative that target core cellular processes, such as the cell cycle and protein translation, in all cells examined. Moreover, we identified genes that are selectively required for proliferation and survival in different cell lines. Our platform enables rapid and cost-effective genome-wide screens to identify cancer proliferation and survival genes for target discovery. Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells.

3:25 Tools and Strategies for High Confidence RNAi Screens
Kirk Brown, Field Application Specialist, Thermo Scietific
Successful merging of RNA interference (RNAi) into high throughput applications requires implementing sound strategies and tools that address factors which can distort the interpretation of gene silencing studies. To that end, this presentation will review the importance of assay design and assay metrics, the quality of silencing reagent(s), and positive and negative controls highlighting the contribution that each parameter makes to data quality and false positive/negative rates. Lastly, hit validation strategies will also be discussed.

3:55 Networking Refreshment Break in the Exhibit Hall

4:30 Genome-Wide RNAi Screen for Host Cell Factors Involved in Influenza Infection Cycle
Nikolaus Machuy, Ph.D., Head of the Screening Unit, Max-Planck Institute for Infection Biology
Influenza is responsible for 250,000 to 500,000 deaths per year worldwide. Even more frightening are the recurring influenza pandemics. Due to the high mutation rates of viruses, drugs directed against viral determinants have limited reach, raising the necessity to identify host cell factors amenable to act as flu drug targets. In order to identify host cell factors affecting influenza virus infectious cycles, we performed a genome-wide systematic RNAi screen. Due to a two-step readout, we can distinguish genes, which are necessary for the virus entry or which play an essential role in the completion of the life cycle.

5:00 HT-RNAi Screening Using High Content Assays in Human Cells
Christophe J. Echeverri, Ph.D., Chief Executive Officer / Chief Scientific Officer, Cenix BioScience GmbH

5:30 Dissecting Signaling Networks by Systematic RNAi Screens
Michael Boutros, Ph.D., Division of Signaling and Functional Genomics, German Cancer Research Center
While an increasing number of genomes are sequenced, the function of many genes remains unknown. Genetic screens for phenotypes on the level of the organism have been successfully used to characterize the function of genes and order their action into cellular pathways. RNAi screens now allow a phenotypic characterization of genes on a genome- wide scale and support a systems-level understanding of cellular processes. We have developed approaches to rapid screen through large libraries of siRNA to identify genes that are required for particular biological processes. We show how strategies derived from classical genetic screens in combination with quantitative HTS and HCS phenotypes can be used to dissect cellular signaling networks.

6:00 Happy Hour in Exhibit Hall

7:30 End of Day