6月 9日 (一)
7:30am – 6:00 pm Registration Open
7:30 Morning Coffee
8:45 Chairperson’s Opening Remarks
Christopher Miller, Ph.D., Director, Applied Genomics, Bristol-Myers Squibb Co.
8:50 RNAi-Based Functional Screens in Drug Discovery
Christopher Miller, Ph.D., Director, Applied Genomics, Bristol-Myers Squibb Co.
We are using a variety of RNAi-based screening approaches, including libraries of chemically-synthesized siRNAs and lentivirus-expressed shRNAs in combination with single endpoint and multi-parametric high-content cell-based assays. Applications include screening RNAi libraries for new candidate target genes, drug response modulators and biomarkers. Examples will be given of these approaches and will include discussion of their strengths and weaknesses.
9:15 RNAi Screening to Identify and Validate Therapeutic Targets in
Metabolic Diseases
John Reidhaar-Olson, Ph.D., Research Leader, Functional Genomics, F. Hoffmann-La Roche Ltd.
Metabolic pathways in hepatocytes represent opportunities for new target discovery for metabolic diseases such as obesity and type 2 diabetes. We have used in vitro cellular models to mimic the fat accumulation in the liver that arises from elevated plasma free fatty acid levels in diabetic and obese individuals. Using this model system, we have developed RNAi-based screening methods that have yielded novel candidate targets.
9:45 RNAi Screens of Oncogenic Signaling Pathways
David Davis, Ph.D., Scientist, Molecular Biology, Genentech Inc.
The ability to perform reverse genetic screens in mammalian cells has made RNAi a powerful tool to dissect signaling pathways relevant to human disease. Two distinct library formats have been established. Arrayed RNAi libraries (comprised of synthetic siRNA oligonucleotides or viral based shRNA) provide a means for high-throughput screens. The second library format consists of pooled shRNA vectors targeting hundreds to thousands of genes within the same experimental sample. When applied in parallel, both formats provide a powerful method for mechanistic studies. The advantages and considerations for such a multi-pronged screening approach will be presented.
10:15 Networking Coffee Break
10:45 RNAi: From Cancer Target Identification and Validation to Cancer Therapy
Yu Shen, Ph.D., Senior Group Leader, siRNA Therapeutics, Abbott Laboratories
We systematically compared several siRNA libraries and identified one with dramatically reduced off-target effect and comparable degrees of target knockdown. Screening this advanced-siRNA library against the “druggable genome” with a unique assay, we identified a number of novel cancer targets. We also explored RNAi-based methodology for target validation in animal models.
11:15 RNAi Technology Reveals a Novel Target for Obesity and Diabetes
Madhu Dhar, Ph.D., Research Associate Professor, Large Animal Clinical Sciences, University of Tennessee
Mice carrying two pink-eyed dilution locus heterozygous deletions on mouse chromosome 7 are hyperglycemic, hyperinsulinemic, hyperlipidemic, insulin resistant, and obese. A single gene, ATP10C, a novel type 4 P-type ATPase, maps to this region. Ablation of ATP10C via transient transfection of ATP10C-specific siRNA duplexes in 3T3-L1 mouse adipocytes shows a signifi-cant increase in insulin-stimulated glucose uptake.
11:45 A Systems Biology Approach Towards the Identification of Human Host Factors Required for HIV-1 Replication Using Genome-Wide siRNA Libraries
Renate Koenig, Ph.D., Staff Scientist, Infectious & Inflammatory Disease Center, Burnham Institute for Medical Research
Human immunodeficiency virus (HIV)-1 is dependent on the host cell machinery to replicate. We conducted a genome-wide RNAi screen in mammalian cells to comprehensively identify those cellular factors that facilitate HIV-1 replication, the gammaretrovirus (MLV) or parvovirus (AAV). This study revealed a number of shared, as well as pathogen-specific host factors and led to the identification of 40 novel genes that support HIV-1 infection at distinct steps of the life cycle.
12:15 pm Close of Morning Session
12:30 Luncheon Technology Workshops
(Sponsorships Available) or Lunch on Your Own
2:00 Chairperson’s Remarks
John Reidhaar-Olson, Ph.D., Research Leader, Functional Genomics, F. Hoffmann-La Roche Ltd.
2:05 Discovery of Novel Targets with siRNA Library Screening
Paul D. Kassner, Ph.D., Principal Scientist, Amgen Inc.
RNA interference has great potential to elucidate gene function and is widely used for target discovery and validation. However, with this potential come numerous challenges involved in performing high-quality siRNA library screens. Using data from viability-based and phenotypic pathway-based screens in mammalian cells, I will demonstrate methods to overcome some of the problems encountered during assay development (AD) and high-throughput screening (HTS).
2:35 High-Content Genome-Wide siRNA HTS-Target Identification through Functional Genomics
Lazlo Kiss, Ph.D., Research Fellow, Automated Biotechnology, Merck & Co. Inc.
3:05 High-Throughput Cell-Based RNAi Screen for Drug Profiling
Holly Yin, Ph.D., Head of Cellular Genomics, Cancer Drug Development Laboratory, Translational Genomics Research Institute
We have applied advanced genomic technologies and data integration strategies to discover cancer specific genetic determinants of clinical drug’s response. The outcome of this study can help to improve and accelerate decisions regarding clinical indications, patient stratification and identify combination therapeutics. Case studies on profiling of the late-clinical stage cancer
drugs will be presented.
3:35 Technology Spotlight
(Sponsorship Available)
3:50 Networking Refreshment Break
4:15 From Cell-based HT-RNAi Screen to Systemic siRNA Testing In Vivo: Streamlined Discovery and Validation of Novel Anti-Malarial Targets and Leads
Christophe J. Echeverri, Ph.D., CEO/CSO, Cenix BioScience GmbH
In collaboration with the group of Maria Mota (IMM, Lisbon), we have carried out the first genome-scale HT-RNAi screen to identify novel host factors needed for liver stage malaria infection. Using a focused library of siRNAs covering the entire human kinome and genes involved in lipoprotein uptake and metabolism pathways, we applied a high-content microscopy-based assay for Plasmodium infection in human hepatoma cells to investigate the role of these genes in this process. The 3-phase systematic screening, which used at least 3 individual siRNAs for each tested gene, yielded 6 top hits including scavenger receptor BI (SR-BI), atypical PKCzeta and 4 other kinases. While the RNAi-induced loss-of-function phenotypes for all of these genes consistently showed significant drops in Plasmodium infection rates with multiple distinct siRNAs in Huh7 hepatoma cells, we sought to add further patho-physiological validation of these results both in vitro and in vivo. This was indeed achieved in Huh7 and primary hepatocytes using synthetic inhibitor compounds, blocking antibody, and pseudo-substrate inhibitors for SR-BI and aPKCzeta. This unusual multiplicity of independent inhibitor molecules enabled us to unambiguously validate the screening results, and add further depth of phenotypic characterization for both of these hits. Finally, these findings were also further confirmed by in vivo analyses using a mouse model of malaria infection, through systemic administration of liposome-formulated siRNAs (with the assistance of Alnylam Pharmaceuticals) as well as genetic knock-outs. Overall, this represents an unusually comprehensive dataset illustrating the power of RNAi-based discovery and validation to accelerate key, early stages of preclinical pipelines.
High-Throughput RNAi Screening
5:15 Welcoming Reception in the Exhibit Hall
6:30 Close of Day
