eBriefing

RNAi-based Genetic Screening: Past, Present & Future

RNAi-based Genetic Screening: Past, Present & Future

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Overview

RNA interference (RNAi) screening methodologies have revolutionized the fields of basic biological research, functional genomics, and drug development and discovery. These methods provide complex and extremely powerful tools to investigate mechanisms underlying human disease and to accelerate therapy development. On April 11, 2011, the Non-coding RNA Biology Discussion Group at the New York Academy of Sciences convened senior and junior investigators to explore lessons, challenges, new discoveries and future directions in the development of RNAi screening technologies and delivery processes. The successful application of these technologies in various areas of biology and translational science such as vesicular transport, cancer, and infectious diseases was a focus of this meeting.

Norbert Perrimon, from Harvard Medical School and Howard Hughes Medical Institute, began the symposium with an overview of the reagents and approaches of RNAi screening in Drosophila. Perrimon described the reagents that his lab has developed for both tissue culture and in vivo RNAi screens in Drosophila. He highlighted two recent studies that exemplified how RNAi screens can be combined with mass spectrometry to analyze the structure of signaling networks and cellular mechanisms in 1) the building a kinome network (the collection of all kinases) in early Drosophila eggs and 2) the regulation of autophagy in muscle cells.

Marino Zerial, from the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, Germany, presented the quantitative image analysis system that his group has developed to measure cellular parameters from automatically-acquired, high-resolution microscopy images. Zerial discussed the application of this system to an image-based RNAi screening of HeLa cells for receptor-mediated endocytosis. As Zerial pointed out, this image analysis platform could yield new generations of RNAi-based technologies and improve the specificity of phenotypic assessments of gene function.

Joining the symposium from New York University School of Medicine, Ramanuj DasGupta reported on the use of RNAi-based "modifier screen" approaches to identify novel and specific chemical and miRNA (microRNA) modulators of β-Catenin-Responsive Transcription (CRT). CRT yields one of the most important effectors of the Wnt pathway and has been implicated in the genesis of many cancers. DasGupta described the results of applying a novel methodology that combines a "sensitized" chemical genetic high-throughput screen with RNAi technology for the identification of specific small molecule inhibitors of CRT. These inhibitors antagonize CRT activity in a wide range of cell types, including Drosophila, mouse and human cells, and a variety of Wnt/β-catenin-responsive cancer cell lines.

Ken Koblan of Alnylam Pharmaceuticals, presented a comprehensive progress report on where the company is now in terms of their efforts to develop synthetic RNAi as a new approach to drug development, and previewed the direction of the company's research and development in the upcoming years. According to Koblan, the variety of RNAi-accessible targets bodes well for our ability to manipulate almost any cell function and even to tackle targets once-thought-to-be invulnerable to pharmaceutical intervention.

Dan Anderson, of the Massachusetts Institute of Technology, described his work on high-throughput methods for developing and characterizing biomaterials, and in particular his work on siRNA (small interfering RNA) delivery systems. Anderson explained how 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. He also reviewed the successful development of siRNA delivery mechanisms that function in vivo and that have the potential for therapeutic application for the treatment of genetic disease, viral infection, and cancer.

After lunch, Serena Silver, from the Broad Institute of Massachusetts Institute of Technology and Harvard University, presented a highly effective lentiviral shRNA (small, hairpin RNA) library produced by the RNAi Consortium for high-throughput screening. Silver described best practices for screening with the library, which includes almost 250,000 individual clones targeting over 30,000 mouse and human genes in both arrayed and pooled formats. She also shared several examples of successful screens.

Craig S. Mickanin, investigator at the Novartis Institutes for Biomedical Research (NIBR), brought a pharmaceutical company perspective on the use of RNAi screening technologies. Mickanin offered a summary of his team's efforts over the past 8 years to develop a robust, comprehensive RNAi screening platform within the NIBR and underscored the crucial role of these studies as a gateway to identifying essential mediators of signal transduction pathways and potential drug targets.

New York University School of Medicine's Eva M. Hernando explained how her lab has used 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 melanoma. Her results suggest that relevant miRNA alterations occur at early stages of melanoma development and that these alterations can be predictive of the progression from a treatable, primary tumor to an invasive and ultimately metastatic disease.

Following with another example of cancer-related applications of RNAi screens, Jose M. Silva, from at the Institute for Cancer Genetics at Columbia University Medical Center, presented his research on genetic approaches to produce the next generation of personalized breast cancer therapies. Silva's work exploits the concept of genetic synthetic lethal interactions (GSL) and RNAi technology to identify genes that, when inhibited exclusively, reduce the viability of tumor cells carrying a preexisting genetic lesion. The identification of synthetic lethal interactions that involve major genetic alterations found in cancer opens up the possibility of developing novel, less toxic, and more efficient therapies based on the specific molecular changes that drive tumorigenesis in each patient.

Continuing with other disease-related applications of RNAi screening technologies, the symposium closed with a couple of talks featuring the application of these technologies to the investigation of host-virus interactions. Abraham L. Brass, at Massachusetts General Hospital in Boston, discussed his experience using RNAi screening to study the replication of HIV-1 and influenza A virus, while Jason Rodriguez from Columbia University, shared his work on a whole genome RNAi screen to identify host factors required for early events of HIV-1 infection.

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Presentations available from:
Abraham L. Brass MD, PhD (Massachusetts General Hospital)
Ramanuj DasGupta, PhD (NYU School of Medicine/Cancer Institute)
Ken Koblan, PhD (Alnylam Pharmaceuticals)
Eva M Hernando-Monge, PhD (NYU Langone Medical-Center)
Craig S. Mickanin, PhD (Novartis Institutes for Biomedical Research)
Norbert Perrimon, PhD (Harvard Medical School)
Jason Rodriguez, PhD (Columbia University)
José M. Silva, PhD (Columbia University)
Marino Zerial, PhD (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden)

Organizers

José M. Silva, PhD

Columbia University
e-mail | website | publications

Thomas Tuschl, PhD

Rockefeller University
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Marta Murcia, PhD

The New York Academy of Sciences
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Speakers

Dan Anderson, PhD

Massachusetts Institute of Technology
e-mail | website | publications

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
e-mail | website | publications

Abraham Brass is an Investigator at the Ragon Institute at Massachusetts General Hospital (MGH) in Boston, MA where he studies viral-host interactions. Brass obtained his MD and PhD from the University of Chicago. After a residency in Internal Medicine at the Brigham and Women's Hospital in Boston, MA, Brass completed his clinical subspecialty training in Gastroenterology at MGH while working as a postdoctoral research fellow in the Genetics Department of Harvard Medical School.

Ramanuj DasGupta, PhD

NYU School of Medicine/Cancer Institute
e-mail | website | publications

Ramanuj 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 Norbert Perrimon's lab, 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.

Ken Koblan, PhD

Alnylam Pharmaceuticals
e-mailwebsite | publications

Ken Koblan joined Alnylam in April 2010 with a proven 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, 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. Koblan also led the implementation of a significant restructuring initiative that serves as a foundation for sustainable growth within Merck Research Laboratories. Koblan received his PhD in Biochemistry from Johns Hopkins University and his BS in Biology from the Massachusetts Institute of Technology (MIT).

Eva M. Hernando-Monge, PhD

NYU Langone Medical Center
e-mail | website | publications

Eva Hernando obtained her PhD 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, and does so by analyzing patient samples and using a variety of in vitro and in vivo models.

Craig S. Mickanin, PhD

Novartis Institutes for Biomedical Research
e-mail | publications

Craig Mickanin obtained his BS 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 industry, including the industrialized registration, distribution, and screening of a variety of genome-based sample classes.

Norbert Perrimon, PhD

Harvard Medical School
e-mail | website | publications

Norbert 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.

Jason Rodriguez, PhD

Columbia University
e-mail | website | publications

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, Rodriguez is a post-doctoral associate in the HHMI laboratory of Stephen Goff at Columbia University. 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
e-mail | website | publications

José Silva has been an Assistant Professor of Pathology at the Institute for Cancer Genetics (HICCC) at Columbia University Medical Center, New York, since 2008. 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, and this in turn could lead to more efficient and less harmful breast cancer therapies. Silva received a BS from the Complutense University of Madrid in 1995 and a PhD from the Autonoma University of Madrid in 2000. In 2001, he began his postdoctoral studies at 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, 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
e-mailpublications

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. 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. Silver conducted her graduate work in the Rebay lab at MIT.

Marino Zerial, PhD

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden
e-mail | website | publications

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 work at Institute J. Monod, Paris and at the European Molecular Biology Laboratory EMBL, Heidelberg. In addition, 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. Currently 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 has led to an increased 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.

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