RNAi-based genetic screening: Past, Present & Future

RNAi-based genetic screening: Past, Present & Future

Monday, April 11, 2011

The New York Academy of Sciences

Presented By

 

RNA interference (RNAi) screening methodologies have revolutionized the fields of basic biological research, functional genomics, and drug development and discovery, providing complex and sometime challenging but yet extremely powerful tools to approach mechanisms underlying human disease and accelerate the development of treatments. This 1-day symposium will explore lessons, challenges, new discoveries and future directions in the development of RNAi screening technologies and delivery processes.

Participants will 1) look back on the lessons that have been learned from the first wave of technology developments and applications in this exciting field, 2) overview current status of the various kinds of RNAi screens, 3) explore the challenges regarding design and optimization, quality control, and normalization strategies, and 4) examine what new technologies and delivery methods promise to bring in a near future. Featuring leading speakers in the field of RNAi delivery and screening, the agenda will address various types of RNAi constructs for library building, cell lines, screening paradigms, reagent types, and read-out methodologies, and will emphasize the translational aspects of these technologies by presenting successful applications in various areas of biology such as cancer, AIDS, vesicular transport, and other disorders.

This event will also be broadcast as a webinar.

Please note: Transmission of presentations via the webinar is subject to individual consent by the speakers. Therefore, we cannot guarantee that every speaker's presentation will be broadcast in full via the webinar. To access all speakers' presentations in full, we invite you to attend the live event in New York City, where possible.

Networking reception to follow

Agenda

*Presentation times are subject to change.


8:00 AM

Registration and Continental Breakfast

9:00 AM

Opening Remarks

9:15 AM

RNAi Screening in Drosophila: Reagents and Approaches
Norbert Perrimon, PhD, Harvard Medical School

10:00 AM

Quantitative Multiparametric Image Analysis for RNAi Induced On- and Off-target Effects
Marino Zerial, PhD, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden

10:45 AM

Coffee Break

11:15 AM

Screening For Novel Chemical and Genetic Regulators of the Wnt/Wg Pathway
Ramanuj DasGupta, PhD, NYU School of Medicine

11:45 AM

Delivery of siRNAs to Cultured Cells
Ken Koblan, PhD, Alnylam Pharmaceuticals

12:15 PM

Combinatorial Development of Biomaterials and Synthetic siRNA Delivery Systems
Dan Anderson, PhD, Massachusetts Institute of Technology

12:45 PM

Lunch

1:45 PM

Lentiviral RNAi Libraries and Viral-mediated Delivery
Serena Silver, PhD, The Broad Institute

2:15 PM

Industrializing RNAi Screens: Go Big or Go Home
Craig S. Mickanin, PhD, Novartis Institutes for Biomedical Research

2:45 PM

Elucidating the Functions of miRNAs in Melanoma Progression
Eva M Hernando-Monge, PhD, NYU Langone Medical-Center

3:15 PM

Genetic Synthetic Lethality: Genetic Approaches For Next Generation Personalized Breast Cancer Therapies
Jose M. Silva, PhD, Columbia University

3:45 PM

Coffee Break

4:15 PM

Genetic Strategies to Investigate Host-virus Interactions
Abe L. Brass, MD, PhD, The Ragon Institute, Massachusetts General Hospital

4:45 PM

Whole Genome RNAi Screen to Identify Host Factors Required For Early Events of HIV-1 Infection
Jason Rodriguez, PhD, Columbia University

5:15 PM

Networking Reception

Organizers

José M. Silva, PhD

Columbia University

Thomas Tuschl, PhD

Rockefeller University

Marta Murcia, PhD

The New York Academy of Sciences

Speakers

Dan Anderson, PhD

Massachusetts Institute of Technology

Dr. Daniel G. Anderson is an Associate Professor in the departments of chemical engineering and the division of health science technology, and is an intramural member of the David H. Koch Institute for Integrative Cancer Research at the Massachusetts Institute of Technology. He received his PhD in molecular genetics from the University of California at Davis. At MIT, he pioneered the use of robotic methods for the development of smart biomaterials for drug delivery and tissue engineering. He has developed methods allowing rapid synthesis, formulation, analysis, and biological testing of large libraries of biomaterials for use in medical devices, cell therapy and drug delivery. In particular, the advanced drug delivery systems he has developed provide new methods for nanoparticulate and microparticulate drug delivery, non-viral gene therapy, siRNA delivery, and vaccines. His work has resulted in the publication of over 140 papers, patents and patent applications. These patents have led to a number of licenses to pharmaceutical, chemical and biotechnology companies.

Abraham L. Brass MD, PhD

Massachusetts General Hospital

Abe Brass is an Investigator at the Ragon Institute at Massachusetts General Hospital (MGH) in Boston, Mass. studying viral-host interactions. Abe obtained his M.D. and Ph.D. from the University of Chicago, IL. After a residency in Internal Medicine at the Brigham and Women’s Hospital in Boston Mass., Abe completed his clinical subspecialty training in Gastroenterology at MGH, while working as a postdoctoral research fellow in the Genetics Dept. of Harvard Medical School.

Ramanuj DasGupta, PhD

NYU School of Medicine/Cancer Institute

Dr. DasGupta is Assistant Professor of Pharmacology at the New York University School of Medicine and New York University Cancer Institute. He is also the Director of the RNAi Screening Facility of New York University School of Medicine and member of the Helen and Martin Kimmel Center for Stem Cell Biology, NYUSoM. Trained in Dr. Norbert Perrimon’s lab, Dr. Dasgupta has a unique expertise in state-of-the-art functional genomic approaches, such as RNAi based high-throughput screen in Drosophila and mammalian cultured cells. His lab uses cutting-edge technology to answer basic biological questions in cellular signaling with the specific goal of defining mechanisms of the regulation of the Wnt pathway, both in development and in disease. Recently, Dr. DasGupta has developed an RNAi-based integrated screening technology to identify novel small molecule and microRNA modifiers of the Wnt pathway. The novel and innovative use of combining RNAi with chemical genetics, as well as novel RNAi based enhancer-suppressor screens has allowed his lab to develop very targeted and innovative methods to screen for modifiers of key components of the Wnt signaling pathway. More recently, he has also developed protocols for RNAi-screens aimed at addressing the function of cell-signaling pathways in stem cell maintenance/differentiation in mouse/human embryonic stem cells (ESCs). Since 2006, Dr. DasGupta is the Director of the NYU RNAi screening core facility, which he established within the first few years of arriving at NYU Medical School. As such, he has extensive experience with all facets of siRNA and chemical genomic screening procedures, which he routinely applies to whole genome screens. He also has all of the necessary experience in assay execution, data collection and interpretation along with the requisite reagents, detection equipment, and analysis software for a wide variety of cell-based assays.

Ken Koblan, PhD

Alnylam Pharmaceuticals

Dr. Koblan joined Alnylam in April 2010 with a proven track record of success in the pharmaceutical industry. During his 18 years with Merck Research Laboratories, he held several positions of increasing responsibility culminating in his role as Vice President and site head of Rahway Basic Research. In this role, Dr. Koblan was responsible for leading basic biology and medicinal chemistry research at Merck's largest research center, resulting in two Phase III registration programs, two phase IIb development programs, and the advancement of key pre-clinical drug discovery programs. Dr. Koblan also led the implementation of a significant restructuring initiative that serves as a foundation for sustainable growth within Merck Research Laboratories. Dr. Koblan received his Ph.D. in Biochemistry from Johns Hopkins University and his B.S. in Biology from the Massachusetts Institute of Technology (MIT).

Eva M Hernando-Monge, PhD

NYU Langone Medical-Center

Eva Hernando obtained her PhD degree in Molecular Biology from the University Autonoma (Madrid, Spain) in 1999. She conducted her postdoctoral studies at Memorial Sloan-Kettering Cancer Center and Cold Spring Harbor Laboratories. She became an Assistant Professor at the Department of Pathology of NYU School of Medicine in 2006. Her laboratory studies the molecular basis of sarcoma and melanoma pathogenesis, by analyzing patient samples and using a variety of in vitro and in vivo models.

Craig S. Mickanin, PhD

Novartis Institutes for Biomedical Research

Craig Mickanin obtained his BS degree from the College of William and Mary in 1992. He held positions at the Children’s Hospital of Philadelphia and Onyx Pharmaceuticals prior to joining Novartis Pharamceuticals in 1998. He currently holds the title of Senior Investigator II in the Target Discovery Technologies Unit of the Developmental and Molecular Pathways Department at the Novartis Institutes for Biomedical Research, Inc. in Cambridge, MA. Over the past 10 years, he has pioneered the use of large-scale genome based libraries in the pharmaceutical industy, including the industrialized registration, distribution, and screening of a variety of genome-based sample classes.

Norbert Perrimon, PhD

Harvard Medical School

Dr. Perrimon has 30 years of experience in the fields of developmental genetics, signal transduction and genomics. By developing, improving, and applying a number of genetic techniques (germline clones, FLP/FRT, Gal4/UAS, etc.), his group identified many key components of the Receptor Tyrosine Kinases, JAK/STAT, Wnt, Hedgehog and Notch signaling pathways. In recent years, his group established high-throughout genome-wide RNAi screens to systematically interrogate the entire Drosophila genome in various cell-based assays. In 2003 he created the Drosophila RNAi Screening Center at Harvard Medical School to make this technology available to the community. Currently, his laboratory is applying large-scale RNAi and proteomic methods to obtain a global understanding to the structure of a number of signaling pathways and their cross-talks. In addition, he is studying the roles of signaling pathways in homeostasis and tissue remodeling in Drosophila muscles and gut stem cells. Dr. Perrimon has trained more than 80 students and postdoctoral fellows, with most of them currently holding academic positions.

Jason Rodriguez, PhD

Columbia University

Dr. Jason Rodriguez obtained his Bachelor of Science in Biochemistry from the State University of New York at Geneseo in 1999 and received his doctorate in Biomedicine at Mount Sinai School of Medicine in 2005. His dissertation work discovered how specific paramyxoviruses subvert the Interferon-mediated innate immune system.

Currently, Dr. Jason Rodriguez is a post-doctoral associate in the HHMI laboratory of Dr. Stephen Goff at Columbia University. Dr. Rodriguez’s research focuses on characterizing virus-host interactions of the retroviruses XMRV and HIV. He is actively pursuing whole-genome RNAi technology to identify genes required for HIV infection to gain a better understanding between HIV and its host. Knowledge gained from this work will not only discover key aspects of HIV biology but also a better understanding of host gene function and will open opportunities for therapeutics.

José M. Silva, PhD

Columbia University

Dr. Silva, is an Assistant Professor of Pathology at the Institute for Cancer Genetics, HICCC, at Columbia University Medical Center, New York, since 2008. Dr. Silva’s research centers on breast cancer biology, in particular on the identification of tumor targets for personalized breast cancer therapies. His work focuses on identifying genes that are essential for the viability of breast cancer cells that carry cancer-specific genetic alterations (synthetic lethality) using genome-wide RNAi screens. Blocking the function of these genes will reduce the viability of tumor cells without affecting normal cells, which could lead to more efficient and less harmful breast cancer therapies.

Dr. Silva received a Bachelor of Science degree from the Complutense University of Madrid in 1995, and his doctorate from the Autonoma University of Madrid in 2000. In 2001, he began his postdoctoral studies at Dr. Greg Hannon’s lab at Cold Spring Harbor Laboratory (CHLS), New York, where he was promoted to senior fellow in 2007. During his time at CHSL, Dr. Silva developed shRNA plasmid libraries to target every gene in the human genome and developed a microarray-based platform that facilitates genome-wide RNAi screens.

Serena Silver, PhD

The Broad Institute

Dr. Serena Silver leads the RNAi Screening group within the RNAi Platform at the Broad Institute, where she works with collaborators from the Harvard and MIT community and beyond to design and execute RNAi and overexpression screens.

Dr. Silver and her colleagues have used high throughput RNAi screens to elucidate the pathways and genes important for many developmental processes and disease states, including altered response of an immune cell to pathogens, increased growth of axons from neurons, or increased resistance or susceptibility of a cancer cell to a chemotherapy agent.

Marino Zerial, PhD

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden

Marino Zerial graduated in biology at the University of Trieste in 1982 with a thesis on applied biochemistry of lysosomal storage disorders. He conducted post-doctoral experiences at Institute J. Monod, Paris and at the European Molecular Biology Laboratory EMBL, Heidelberg. He became EMBL Research Group Leader in 1991 when he started his work on the molecular regulation of endocytosis. In 1998 he became Max Planck Director and co-founder of the Max Planck Institute of Molecular Cell Biology and Genetics, MPI-CBG, Dresden. He is honorary Professor at the Medical Faculty, University of Technology, Dresden.
Marino Zerial's research group investigates the molecular mechanisms underlying organelle biogenesis and intracellular transport, focusing on endocytosis, a central process in all eukaryotic cells. His work led to the understanding of the molecular mechanisms underlying endosomal membrane tethering and fusion, especially the role of Rab GTPases in this process, and endosome biogenesis. More recently, he has been combining quantitative image analysis with functional genomics to conduct a system analysis of endocytosis and integrate it with other cellular functions such as signal transduction and cell polarity, in the context of tissue morphogenesis.

Sponsors

For sponsorship opportunities please contact Cristine Barreto at cbarreto@nyas.org  or 212.298.8610.

Abstracts

RNAi Screening in Drosophila: Reagents and Approaches

Norbert Perrimon, PhD, Harvard Medical School

I will describe the current state of the regents that we have developed for both tissue culture and in vivo RNAi in Drosophila. I will then present a number of recent studies that exemplify how we are combining RNAi screens with Mass Spectrometry to analyze the structure of signaling networks and cellular mechanisms. In particular I will discuss two ongoing projects: the building a kinome network in early Drosophila eggs and the regulation of autophagy in muscle cells.

Quantitative Multiparametric Image Analysis for RNAi Induced On- and Off-target Effects

Marino Zerial, PhD, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

RNA interference (RNAi) coupled to automated high-throughput high-resolution microscopy provide a technological platform for systematic genome-wide surveys of biological processes. Major obstacles to the elucidation of the function of genes based on silencing phenotypes are the large degree of morphological variation between cells and, especially, the variation between phenotypes of different siRNAs targeting the same gene. We have developed a quantitative, multi-parametric image analysis system for the measurement of cellular parameters from automatically acquired high-resolution microscopy images. This analysis was applied to an image-based RNAi screening of HeLa cells for receptor-mediated endocytosis (Collinet et al., Nature 2010). The analysis has provided a quantitative description of the phenotype induced by silencing each gene of the human genome (gene silencing phenotype profile). In addition, the reproducibility of individual phenotypic profiles between experimental replicates was tested and compared to the reproducibility between different siRNAs targeting the same gene. Statistical methods were applied to estimate the mean number of off-targets per siRNA and the minimum number of independent siRNAs which are required to infer a gene knockdown phenotype with given confidence for any particular RNAi library. This platform can help the development of new generations of RNAi-based technologies and improve the specificity in the phenotypic assessment of gene function.

Screening For Novel Chemical and Genetic Regulators of the Wnt/Wg Pathway

Ramanuj DasGupta, PhD, NYU School of Medicine

The Wnt/wingless (wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulate many aspects of metazoan development. Inappropriate activation of the Wnt pathway has been associated with tumorigenesis of the liver, colon, breast and skin. One of the most important effectors of the Wnt pathway is encoded by the transcription factor, beta-catenin (beta-cat). Since beta-Catenin Responsive Transcription (CRT) has been implicated in the genesis of many cancers, it makes an ideal target for developing therapeutics that could modulate the nuclear signaling activity of beta-cat. Recently, we employed a novel methodology of integrating a “sensitized” chemical genetic high-throughput screen (HTS) with RNA-interference (RNAi) technology in order to identify specific small molecule inhibitors of CRT in Drosophila and human cell lines. Our screening approach allows for the specific targeting of the activity of the signaling-competent beta-cat downstream of the Axin-mediated degradation complex. We report the identification of 3 lead small molecule inhibitors of CRT that antagonize CRT activity in a wide range of cell types, including Drosophila, mouse and human cells as well as a variety of Wnt/beta-cat-responsive cancer cell lines, such as the MCF7 human breast carcinoma cell line and HT-29/HCT-116 colon cancer cell lines. In my talk, I will elaborate on the use of RNAi-based “modifier screen” approaches in order to identify novel and specific chemical and miRNA modulators of CRT, and elucidate their mechanism of action.

Combinatorial Development of Biomaterials and Synthetic siRNA Delivery Systems

Dan Anderson, PhD, Massachusetts Institute of Technology

High throughput, combinatorial approaches have revolutionized small molecule drug discovery. Here we describe our work on high throughput methods for developing and characterizing biomaterials, and in particular siRNA delivery systems. Libraries of nanoparticles, degradable polymers and lipid-like materials have been synthesized, formulated and screened for their ability to deliver siRNA, both in vitro and in vivo. A number of siRNA delivery formulations have been developed with in vivo efficacy, and show potential therapeutic application for the treatment of genetic disease, viral infection, and cancer.

Lentiviral RNAi Libraries and Viral-mediated Delivery

Serena Silver, PhD, The Broad Institute

The RNAi Consortium (TRC) and RNAi Platform have developed a highly effective lentiviral shRNA library for high throughput screening, including almost 250,000 individual clones targeting over 30,000 mouse and human genes. I will describe best practices for screening with the library in both arrayed and pooled formats, and present several vignettes of successful screens.

Elucidating the Functions of Mirnas in Melanoma Progression

Douglas Hanniford1,5, Shulian Shang2, Miguel Segura1,5, Ting Tu3,5, Jan Zakrzewski3,5, Michelle Ma3,5, Holly Greenwald3,5, Anna C. Pavlick5,6, Russell S. Bermann4,5, Richard L. Shapiro4,5, Yongzhao Shao2, Iman Osman3,5,6, and Eva Hernando1,5

1 Department of Pathology, New York University School of Medicine, New York, NY
2 Division of Biostatistics, New York University School of Medicine, New York, NY
3 Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, NY
4 Department of Surgery, New York University School of Medicine, New York, NY
5 Interdisciplinary Melanoma Cooperative Group, New York University School of Medicine, New York, NY
6 Department of Medicine, New York University School of Medicine, New York, NY

Early-stage, localized melanoma is well treatable by surgical resection yielding 5-year survival rates of 98%. Patients with advanced melanoma, however, have progressively worse 5-year survival rates of 62% and 16% in those with regional lymph node involvement and distal metastasis, respectively. Understanding molecular events that occur early in melanoma development may inform clinical decision-making and uncover novel therapeutic targets. We are using a combination of microarray profiling and screening in a fluorescence-based in vitro invasion assay to find miRNA candidates that might confer a metastatic advantage to primary melanomas. These candidates are further characterized in vitro and in vivo for their ability to functionally affect melanoma progression. Our results suggest that relevant miRNA alterations occur at early stages of melanoma development and can be predictive of metastatic potential. Moreover, this study expands our understanding of the molecular defects that occur during melanomagenesis and how miRNA impact the progression from a treatable primary tumor to an invasive and ultimately metastatic disease.

Industrializing RNAi Screens: Go Big or Go Home

Craig S. Mickanin, PhD, Novartis Institutes for Biomedical Research

Interrogation of cellular processes using siRNA holds tremendous promise for the identification of essential mediators of signal transduction pathways and potential drug targets. This presentation will provide a summary of our efforts over the past 8 years to develop a robust, comprehensive RNAi screening platform within the Novartis Institutes for BioMedical Research.

Genetic Synthetic Lethality: Genetic Approaches For Next Generation Personalized Breast Cancer Therapies

Jose M. Silva, PhD, Columbia University

Cancer therapy has radically changed during the last decade. Novel therapies based on the specific molecular changes that drive tumorigenesis in every patient are emerging as less toxic and more efficient alternatives to classical treatments.

Genetic synthetic lethal interactions (GSL) occur when two genetic alterations that are individually innocuous appear in the same cell causing growth inhibition. This concept can be exploited to identify genes that, when inhibited, exclusively reduce the viability of tumor cells carrying a preexisting genetic lesion. RNA interference (RNAi) technology has emerged as a very powerful approach to attenuate the expression of any chosen gene. This technology represents a unique opportunity to identify synthetic lethal effects with major cancer alterations.

Genetic Strategies to Investigate Host-virus Interactions

Abraham L. Brass, MD, PhD, Massachusetts General Hospital

Viruses exploit host factors to replicate. In turn, the host cell combats viral infection with its own intrinsic immune system. Functional genomics represents a powerful means for investigating such viral-host interactions. I will discuss our experiences using RNAi screening to study the replication of HIV-1 and influenza A virus.

Whole Genome RNAi Screen to Identify Host Factors Required For Early Events of HIV-1 Infection

Jason Rodriguez, PhD, Columbia University

Infection of mammalian cells by HIV-1 is a complex process that requires specific host factors in addition to viral-encoded genes. Recent studies using RNAi technology have screened the human genome for genes required for HIV-1 infection and found genes that play critical roles during the HIV-1 life cycle. However, there is little overlap in the genes identified in different studies, suggesting further characterization of virus-host interactions of HIV-1 is needed. To this end, we screened for genes required for early events of HIV-1 infection by establishing a cell population stably infected with a lentiviral shRNA library covering greater than 90% of the annotated human genome. Cells that were resistant to repeated rounds of infection with a replication-incompetent HIV-mCherry reporter were sorted by flow cytometry and enriched shRNAs were found by microarray analysis. Genes identified in the study represent biological processes that may be important for early events of HIV-1 infection. Individual validation is revealing genes that play an important role in HIV biology and their impact on HIV-1 will be discussed.

*Additional abstracts coming soon

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