2015 Archived Content
The RNAi for Functional Genomics Screening will cover the latest in the use of RNA interference (RNAi) screens for identifying and validating drug targets and exploring unknown cellular pathways. It will cover everything from assay design to data analysis for the use of in vitro and in vivo siRNA (small interfering RNA) and shRNA (short hairpin RNA) screens. There is growing interest in combining RNAi screens with gene editing, chemical genomics, overexpression studies and phenotypic screens, which will all be discussed here. Screening experts from industry and academia will share their experiences leveraging the utility of these diverse screening platforms for a wide range of applications.
Final Agenda
Day 1 | Day 2 | Download Brochure
Tuesday, September 22
7:00 am Registration and Morning Coffee
8:00 Chairperson’s Opening Remarks
Scott Martin, Ph.D., Group Lead, Functional Genomics, Genentech, Inc.
8:10 Harnessing High-Throughput RNAi for Target Identification Utilizing 3D Cell Culture Models
Geoffrey Bartholomeusz, Ph.D., Associate Professor and Director, siRNA Core Facility, Department of Experimental Therapeutics, Division of Cancer Medicine, The University of Texas M.D. Anderson Cancer Center
It is now well accepted that 3D cell culture models although varied and complex, replicate clinically relevant outcomes associated with the tumor microenvironment. They are thus relevant models for target discovery and drug screens. It is imperative to select a relevant 3D model when performing high-throughput screens. The development of 3D models used in high-throughput screening to address relevant questions in cancer biology will be discussed.
8:40 Development of Physiologically Relevant Assay Platforms to Probe Various Disease Pathologies
Madhu Lal-Nag, Ph.D., Team Leader, RNAi Screening, National Center for Advancing Translational Sciences, National Institutes of Health
There is a tremendous need for biologically relevant cancer models that can accurately predict the clinical efficaciousness of various drugs. Here we describe the development of In vitro model platforms that are designed to be biologically relevant to fill a critical gap between the cellular and animal model domains and offer the opportunity to study compounds that are therapeutically attractive.
9:10 Expanding the Screening Toolbox for the Interrogation of Gene Function
Scott Martin, Ph.D., Group Lead, Functional Genomics, Genentech, Inc.
It is increasingly evident that different approaches towards functional genomics screening (e.g., CRISPR, RNAi, and small molecules) have their own advantages and disadvantages. In that regard, a combined approach can be more informative than any one approach alone. This talk will attempt to highlight the need for a flexible approach towards functional genomics screening, and the contexts in which certain platforms may be more appropriate.
9:40 Grand Opening Coffee Break in the Exhibit Hall with Poster Viewing
10:25 Identifying Regulatory Interactions Among Genes Linked to Alzheimer’s Disease
Michael Ollmann, Ph.D., Principal Scientist, Genome Analysis Unit, Amgen, Inc.
Advances in genomic
technologies have helped reveal the network of genes involved in late-onset
Alzheimer’s disease (LOAD). High-throughput sequencing to catalog human genetic
variation, coupled with population studies to correlate genotype with disease
risk, has greatly expanded the list of genes linked to LOAD. Whole-genome gene
expression profiling from diseased and non-diseased brain tissue, integrated with
genotyping data and network analysis, has further developed our view of the
gene networks involved in LOAD. To help identify key
regulatory nodes and functional interactions among genes implicated in LOAD, we
developed a high-content assay quantifying the cell-surface protein expression
of TREM2 and CD33, two genes with nucleotide variants identified as LOAD risk
factors. We used this assay to screen a custom siRNA library targeting 940 genes
linked to the LOAD-gene network. This approach has helped reveal interactions
between TREM2, CD33, and other genes implicated in LOAD, and represents a
general method for using RNAi to reveal regulatory nodes in disease networks
defined by human genetics and genomics.
10:55 High-Throughput RNAi Screening for Immune Modulatory Ligands on Tumor Cells
Philipp Beckhove. Ph.D., Head, Translational Immunology Division, German Cancer Research Center Heidelberg; Director, Regensburg Center for Interventional Immunology, University of Regensburg
Immune checkpoint inhibition has lately revolutionized cancer therapy. We have established the first high-throughput screening approach to characterize the entities of immune modulatory molecules on different tumors - the immune modulatome of cancer. We identified many novel candidates derived from gene families that were hitherto not reported to play a role in the immune system and will report functional validation data for some of them.
11:25 Functional Genomic Screening to Accelerate the Discovery of Combination Therapies
Roderick Beijersbergen, Ph.D., Group Leader, Netherlands Cancer Institute and Head, NKI Robotics and Screening Center
Single drug cancer therapies meet with limited success due to drug resistance from reactivation or activation of redundant pathways. Due to the complexity of the targeted signaling networks in addition to tissue-specific characteristics, the prediction of the best combination therapy remains a major challenge. We apply large scale functional genomic screening in clinically relevant models to identify such interactions with the goal to accelerate the discovery of clinically active combinations.
11:55 High Content RNAi Screening with Persomics: Discover More Faster With Turnkey Printed Libraries
Neil Emans, Ph.D., CEO, Persomics USA, Inc.
RNA interference is routinely used in High Content and Phenotypic screening. However, set-up and operational costs are beyond the reach of individual labs and limit core facilities. Persomics technology miniaturizes, accelerates and de-industrializes RNAi screening. Preprinted libraries integrate with conventional HCS platforms and image analysis to enable off-the-shelf screening in individual labs and now allow core facilities to do more.
12:10 pm An In vivo Pooled RNAi Screen Reveals New Drivers of Breast Cancer Metastasis
Simon Knott, Ph.D., Research Investigator, Cold Spring Harbor Laboratory
Recently, we developed a mouse model of breast cancer heterogeneity that identified cells with differential capacities for performing each step of the metastatic cascade. Here, we found that genes more highly expressed in cells capable extravasation and colonization were clinically relevant in terms of their elevated expression in patients with metastatic relapse. By performing an in vivo pooled shRNA screen, focused on these targets, we were able to extract a subset of 28 genes that were further enriched for clinical significance, with Asparagine Synthetase (Asns) having the most relevance based on patient data. By manipulating its expression levels using next generation RNAi reagents, we were able to show that Asns and its human orthologue ASNS influence invasion and metastases formation. Intracellular Adhesion Molecule 1 (Icam1), also a member of the 28 gene subset, was found to be over-expressed in lung metastases from Asns silenced cells. Silencing of Asns and Icam1 in combination, using a dual shRNA vector strategy, resulted in an even greater reduction in metastases than silencing either gene alone. ICAM1 is a cell surface protein and ASNS is considered to be druggable. Thus, using our new shERWOOD UltramiR shRNA reagents, we have identified two targetable proteins that are likely to provide therapeutic benefit for patients with metastatic breast cancer.
12:25 Improving in vivo Delivery of RNAi and MessengerRNA to Enable and Accelerate Development of New Therapies
Nektaria Andronikou, Staff Scientist, Cell Biology, Thermo Fisher Scientific
There is now a great deal of interest in using siRNA and MessengerRNA in vivo to better understand diseases but also to be used as therapeutic molecules. In September 2010, we launched Invivofectamine® 2.0, the first in vivo delivery reagent commercially available, allowing researchers to utilize siRNA in vivo. Over the past few years, we have identified a new delivery reagent, Invivofectamine® 3.0, that is at least 10 times more potent than Invivofectamine 2.0, with an IC50 of 0.125 mg/kg compared to 1.2mg/kg. This fold improvement was the result of identifying new proprietary lipids, developing an improved formulation design by mixture DOE and incorporating an enhanced production method to control for the size of the particle. This new formulation dramatically cuts down the cost of an in vivo experiment, but also allows for a simpler and easier to use protocol where a single dilution step replaces a 2-hour dialysis step. Recently, we’ve also demonstrated that Invivofectamine 3.0 can successfully deliver mRNA to the liver and to other areas of the body that are of therapeutic interest, such as spleen, muscle and xenograft tumors. In addition, we are also continuing to identify new formulations that are able to deliver mRNA to the lung. In this study, we will present the discovery and development of Invivofectamine 3.0 as well as highlight the new applications utilizing different payloads and organs.
12:40 Session Break
12:45 Luncheon Presentation: The Future of RNAi Screening: Complementary Technologies and Advancements
Ryan Raver, Ph.D., Product Manager, Short hairpin RNA (shRNA) / LentiORFs, Sigma Life Science
RNAi screening has made it possible to identify new genes, or gene networks, that are involved in a wide vari¬ety of biological processes, including assays relevant to signal transduction, cell viability, cell morphology, protein localization and function, drug resistance, and responses of host cells to pathogens. As such, RNAi continues to help us gain critical insights into the mechanisms underlying human disease and accelerate the development of treatments for cancer and a host of other disorders. The intersection between RNAi screening and complementary approaches such as CRISPR-Cas9-mediated genome editing has opened up new opportunities for assay development, screening and validation. The successful implementation of genome-editing technologies in several species suggests this will serve as an important and relevant tool for validation studies in numerous cell lines and model systems. Additionally, RNAi rescue experiments using LentiORFs serve an important role in further validating and boosting confidence of screened hits. As we continue to develop new strategies to improve genome-wide RNAi screening and validation, the significance of RNAi as a research tool will remain for many years to come.
1:25 Refreshment Break in the Exhibit Hall with Poster Viewing
1:50 Chairperson’s Remarks
Eugen Buehler, Ph.D., Group Leader, Informatics, National Center for Advancing Translational Sciences, National Institutes of Health
2:00 Development and Application of CARD, a Comprehensive Integrated Web-Based Platform for Analysis of RNAi Screening Data
Iain Fraser, Ph.D., Investigator, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health
We will describe a comprehensive web-application,
CARD, for integrated analysis and interactive visualization of RNAi screening
data. CARD combines both existing and novel algorithms for data pre-processing,
reducing false positive hits through gene expression and off-target filtering,
implementing network/pathway enrichment of high-confidence hits and predicting
active miRNAs. We will discuss the application of CARD to several screen
datasets and demonstrate both increased hit validation rates and improved hit
overlap between related screens.
2:30 RNAi for Rare Disease Drug Discovery: Signal or Noise?
Christopher Gibson, Ph.D., Co-Founder and CEO, Recursion Pharmaceuticals
RNAi has increasingly well-described off-target effects. High-content imaging assays, for which vast quantities of morphological data are collected, are particularly prone to convolution due to such effects. Findings will be presented from 1000+ feature high-content imaging data in various human cell types using up to 6 RNAi for each of more than 100 disease-related targets. A discussion of the usefulness and limitations of the data resulting from such RNAi-based results will also be discussed.
3:00 The Utility of CRISPR-Cas9 System Employing Synthetic RNAs to Validate Hits from an RNAi Functional Screen
Louise Baskin, Senior Product Manager, GE Healthcare Dharmacon, Inc.
We have previously performed an siRNA screen targeting 7,500 genes in a reporter cell line that enables high-content readout to identify genes involved in the ubiquitin-proteasome proteolytic pathway. Here we describe the use of the CRISPR-Cas9 system in this proteasome reporter cell line to assess previously identified hits. We modified the cell line to stably express Cas9 and then transfected synthetic tracrRNA and crRNAs that resulted in DNA cleavage of proteasome subunits. This model demonstrates that CRISPR-Cas9 can be an effective, orthogonal tool for hit validation of an siRNA screen.
3:30 Refreshment Break in the Exhibit Hall with Poster Viewing and Poster Winner Announced
4:10 3D Phenotypic Screening for Target Identification and Drug Discovery
Arvind Rao, Ph.D., Assistant Professor, Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center
A high-throughput kinome siRNA screen (880 kinases) was carried out to study their effects on tumor architecture and hypoxic response induced in 3D tumor spheroids. We present a workflow to identify and interpret gene function in such large scale 3D RNAi experiments by analyzing such image-derived data in the context of associated molecular data. Apart from describing the components of this integrative workflow, we will also share some “lessons learnt” during this process.
4:40 The Path from Arrayed RNAi to Arrayed CRISPR Screens: Lessons Learned and Challenges
Eugen Buehler, Ph.D., Group Leader, Informatics, National Center for Advancing Translational Sciences, National Institutes of Health
The use of CRISPR for whole genome functional screens has now been demonstrated by several groups. However, these screens have been performed only in a pooled format, which severely limits the range of biological functions that can be interrogated. We will detail our experiments in arrayed CRISPR screening and discuss the challenges and opportunities for future work.
5:10 Interactive Breakout Discussion Groups
This interactive session provides conference delegates and speakers an opportunity to choose a specific roundtable discussion group to join. Each group has a moderator to ensure focused discussions around key issues within the topic. This format allows participants to meet potential collaborators, share examples from their work, vet ideas with peers, and be part of a group problem-solving endeavor. The discussions provide an informal exchange of ideas and are not meant to be a corporate or specific product discussion.
Assay Design Considerations for RNAi Screens
Caroline Shamu, Ph.D., Director, ICCB-Longwood Screening Facility, Harvard Medical School
Scott Martin, Ph.D., Group Lead, Functional Genomics, Genentech Inc.
- Key decision points in assay development for high throughput siRNA screens
- Evaluating reagents, libraries and assay platforms
- Recommended strategies for follow-up and validating RNAi on-target performance
Optimizing Data Analysis and Interpretation
Eugen Buehler, Ph.D., Group Leader, Informatics, National Center for Advancing Translational Sciences, National Institutes of Health
Arvind Rao, Ph.D., Assistant Professor, Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center
- Data handing tools and best practices for RNAi and CRISPR screens
- Considerations for hit prioritization and validation
- Tackling off-target effects
- Avoiding confounding viability and transcription effects
- Image analysis pipelines for 3D screens
- Managing big image data and data mining infrastructures, possibly on the cloud
- Integration across biological scale and with genomics data
How to Best Utilize 3D Cell Culture-based Screens
Geoffrey A. Bartholomeusz, Ph.D., Associate Professor and Director, siRNA Core Facility, Department of Experimental Therapeutics, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center
Madhu Lal-Nag, Ph.D., Team Leader, RNAi Screening, National Center for Advancing Translational Sciences, National Institutes of Health
- Design and use of 3D spheroid models
- Selecting appropriate 3D cell models for high throughput screening
- Standard operation procedures for performing HTS using 3D models
- Drawback of utilizing immortalized cells for 3D cultures
- The next generation 3D models using tumor biopsy samples
6:10 Welcome Reception in the Exhibit Hall with Poster Viewing
7:15 Close of Day
Day 1 | Day 2 | Download Brochure
Wednesday, September 23
7:30 am Registration and Morning Coffee
8:00 Chairperson’s Remarks
Ralph Garippa, Ph.D., Director, RNAi Core Facility, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center
8:10 Pooled shRNA, Arrayed siRNA and CRISPR-Cas9: Three Essential Tools towards Understanding Gene Function in Cancer and Disease Biology
Ralph Garippa, Ph.D., Director, RNAi Core Facility, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center
The addition of CRISPR-Cas9 systems to the incumbent technologies of arrayed siRNA and pooled shRNA have created an unparalleled gene perturbation toolbox for investigators to deploy. Specific examples, published and unpublished, of each of these types of experimental designs will attest to the power of these endeavors, particularly for cancer biology. Respective strengths and weaknesses of each technology will be presented.
8:40 Parallel shRNA and CRISPR/Cas9 Screens Reveal Biology of Stress Pathways and Identify Novel Drug Targets
Michael Bassik, Ph.D., Assistant Professor, Department of Genetics, Stanford University
We have developed high-complexity shRNA libraries (25 shRNAs/gene) that greatly reduce false negatives/false positives, and have adapted these libraries to knock down gene pairs to perform systematic genetic interaction maps in mammalian cells. We have used these maps to study ER-trafficking toxins, and identified novel protein complexes as well as insights into retrograde trafficking. We are now using this strategy together with the CRISPR/Cas9 system to study stress signaling and identify novel drug targets.
9:10 TECHNOLOGY PANEL: Finding the Right Functional Genomics Tool to Address Your Biological Question
This panel will bring together 4-5 technical experts from leading technology and service companies to discuss screening trends and improvements in assay platforms and reagents that users can expect to see in the near future.
Panelists:
- Louise Baskin, Senior Product Manager, GE Healthcare Dharmacon,
Inc.
- Neil Emans, Ph.D., CEO, Persomics USA, Inc.
- Shawn Shafer, Ph.D., Market Segment Manager, Functional Genomics, Sigma-Aldrich
- Ross Whittaker, Ph.D., Product Manager, Synthetic Biology, Thermo Fisher Scientific
- Gwen Fewell, Ph.D., Chief Commercial Officer, transOMIC technologies inc.
- Paul Diehl, Ph.D., Director, Business Development, Cellecta, Inc.
9:40 Coffee Break in the Exhibit Hall with Poster Viewing
10:25 Screening the Kinome: Lessons from Using Functional Screens in Glioblastoma Stem Cells
Brent Cochran, Ph.D., Professor, Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine
It is possible to isolate and maintain in culture glioblastoma cell lines with stem cell properties. We have conducted shRNA screens of three different GBM stem cell lines in both arrayed and pooled screening format for growth and survival of these cells, under normoxia and hypoxia. We have found that there are considerable differences in the kinase requirements between these cell lines. These results argue for a personalized therapeutic strategy for glioblastoma.
10:55 From Model Systems to Mammalian Applications: Learning from Functional Genomics Analyses in Drosophila
Stephanie Mohr, Ph.D., Lecturer, Genetics & Director of the Drosophila RNAi Screening Center, Harvard Medical School
Drosophila cell and in vivo systems are exemplary platforms for functional genomics. We have developed algorithms for the design of RNAi and CRISPR reagents; platforms for efficient RNAi reagent production; and mature methods for large-scale screening and data analysis. Our workflows for CRISPR knockout and RNAi screens in human disease-relevant sensitized backgrounds will be discussed. Emphasis will be given to approaches, algorithms and analyses relevant to mammalian systems.
11:25 Enjoy Lunch on Your Own
12:55 pm Plenary Keynote Program
2:40 Refreshment Break in the Exhibit Hall with Poster Viewing
3:25 Close of Conference
Day 1 | Day 2 | Download Brochure