Wednesday, February 1

7:00-8:00 Conference Registration

8:00-8:15 Chairperson’s Opening Remarks

RNAI FOR TARGET VALIDATION

8:15-8:45 In Vivo Target Validation of Liver-X-Receptor Alpha with the Use of Adenoviral hsRNA Delivery 
Dr. Jörg F. Rippmann, Head, Genomics Group, Boehringer Ingelheim Pharma GbmB & Co. KG
The presentation will briefly describe the RNAi portfolio for target validation at BI Germany and will further focus on the presentation of the experimental design and results of an in vivo target validation study with the use of adenovirus vectors delivered by Galadeno. The liver-X-receptor alpha is a liver specific member of the nuclear hormone receptor family and involved in the transcriptional control of lipid metabolism and transport. In vitro characterized knock-down vectors were injected into the mice at different dosages and the target gene expression as well as the physiological consequence was analyzed. The results will be critically discussed and future directions will be highlighted.

8:45-9:15 siRNA Technology to Validate Novel Drug Targets from Genetics/Genomics Screens
Dr. Cristina M. Rondinone, Director of Research, Metabolic Diseases, Hoffmann-La Roche Inc. 
siRNA technology holds extraordinary promise for the identification of novel signal transduction pathways and potential drug targets. siRNAi can be useful to understand and infer the function of novel protein targets that can emerge from genetic linkage studies and genomics. This talk will cover the use of siRNA to validate, select and prioritize the hits from these genetic/genomic screens and obtained the best targets for metabolic diseases.

9:15-10:15 Coffee Break with Exhibit and Poster Viewing

GENOME-WIDE SIRNA HIGH-CONTENT SCREENING

10:15-10:45 Statistical and Functional Analysis of a Genome-Wide siRNA Cell Cycle Screen
Dr. Yan Feng, Lab Head, Genome and Proteome Sciences, Novartis Institutes for BioMedical Research
We used a multi-parameter imaging cytometry-based assay and genome-wide siRNA knockdown to characterize genes and pathways that regulate cell cycle progression. Cells from various cell cycle stages were categorized by a combination of clustering and machine learning algorithms. A multi-parameter cytometry fingerprint was then generated for each gene. System level analysis was performed by putting the cell cycle fingerprint into a function genomic context.

10:45-11:15 Genome-Wide RNAi Screen Identifying Novel Cell Cycle and Cancer Targets
Dr. Daniel R. Rines, Institute Fellow, Lead Discovery, Genomics Institute of the Novartis Research Foundation (GNF)
The application of highly parallelized methods to examine cellular phenotypes can enable the study of gene activities at the level of the genome. Toward this end, we have assembled genome-wide collections of siRNAs and cDNAs. Combining these libraries with high-throughput methodologies for parallel transduction of various cell-types and high speed microscopy platforms, we have executed cell-based assays to examine a diverse range of activities. In particular, we have recently used a RNA library of 49,000 double-stranded (ds)RNAs, targeting approximately 24,000 genes, in a genome-wide loss-of-function screen for essential mitotic chromosome segregation genes. Multi-parametric quantitative analysis of the image-based data has allowed us to isolate over 200 known and novel genes. The application of this functional profiling technology has led to the elucidation of several novel gene activities, and will likely play an integral role in the understanding of gene function on a global scale.

11:15-11:45 Genome-Wide High-Content RNAi Screening to Identify Regulators of Endocytotic Pathways
Dr. Eberhard Krausz, HT-Technology Development Studio, Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG)
We are applying siRNA libraries to multi-parametric high-content assays using an automated high-throughput microscope. In primary viral infection screens, kinases were identified to regulate two independent endocytotic pathways each specifically hijacked by a virus. Primary ‘hits’ were further characterized in a set of six secondary assays that allow pathway dissection. Complex regulative networks were discovered. A subsequent genome-wide screen has been implemented.

11:45-12:15 Rapid, High-Content Genome-Wide Assays Using Cell Microarrays 
Dr. Anne E. Carpenter, Novartis Fellow of the Life Sciences Research Foundation, Sabatini Laboratory, Whitehead Institute for Biomedical Research
Cell-based microarrays allow the preparation of thousands of individual cell samples on a single microscope slide using conventional microarrays. This allows testing an entire genome for high-content phenotypes on 4-8 slides. Each spot on the slide is a cluster of several hundred cells that are perturbed by a single gene expression plasmid, small molecule, or RNA interference reagent (RNAi), and can be imaged using high resolution microscopy. Because existing commercial software had limitations, we developed CellProfiler cell image analysis software to measure a variety of interesting phenotypes of cells on these arrays. By knocking down each gene in Drosophila by RNAi and analyzing cells with CellProfiler, we are obtaining a high-quality, high-content readout of the effects of knocking down each gene on a variety of cellular phenotypes.

LUNCHEON TECHNOLOGY SHOWCASE

12:45-1:00 Recent Developments in siRNA-Based RNA Interference

Sponsored by

Mr. Kirk Brown, Territory Manager, Dharmacon One of the keys to achieving potent gene silencing is the selection of well-designed, highly functional siRNAs. We have identified thermodynamic and sequence-related characteristics that affect key steps in RISC-mediated mRNA degradation. These parameters have been combined into a weighted rational design SMARTselectionTM algorithm for the identification of highly functional siRNAs. Silencing efficiency is further improved by pooling selected siRNA duplexes into one SMARTpool reagent. Together, SMARTselection design and SMARTpool technologies enable functional siRNAs to be designed against any gene target. Recently, Dharmacon introduced ON-TARGETTM siRNA which further increases the specificity of RNAi by introducing modifications to the siRNA sense strand that eliminate sense stand mediated off-target effects as determined by microarray-based genome wide expression profiling. Continuing innovations at Dharmacon are directed toward enabling researchers to expand studies beyond cultured cells to whole animal systems. In particular, Dharmacon developed siSTABLETM, a chemical modification strategy that increases siRNA half-life in serum from minutes to days, increases the duration of silencing after a single treatment and increases the specificity of certain siRNA sequences. Rational siRNA design and the use of stabilized duplexes provide a generally useful mechanism for reducing expression of any target gene in biological systems and accelerates critical investigations across a broad range of biomedical and biological research.

 

1:00-1:30 Application of Ingenuity Pathways Analysis and RNAi technology in drug discovery programs

Sponsored by

Dr. Keith Joho, Senior Vice President Research and Product Management, Ingenuity Systems, Inc Ingenuity Pathways Analysis (IPA) enables biologists and bioinformaticians to model, analyze and understand the complex biological systems at the core of life science research. IPA 3.1 allows scientists to elucidate and customize biological pathways for particular targets, biomarkers, disease areas, and processes, leveraging Ingenuity's broad knowledge base of biological relationships between genes and proteins, cells, tissues, and diseases. This web-delivered application enables effective interpretation of critical pathways that are activated or disrupted upon RNAi knockdown. Target identification studies in which Ingenuity Pathways Analysis has been used in conjunction with RNAi to elucidate pathways for potential drug targets and to assess genome-wide effects of a knockdown of that target will be presented.

1:30-1:45 RNAi Goes Genomic: Elucidating Gene Function with siRNA Libraries 
Dr. David Dorris, Vice President RNAi Technologies, Ambion, Inc
Four keys to successful RNAi screens in human cells are: using highly effective siRNAs, reproducibly and efficiently delivering those siRNAs, ascertaining RNAi effects with a robust assay, and carefully controlling experiments. We will discuss the latest developments in siRNA design and delivery as they relate to RNAi screening, and present data from experiments using siRNA libraries to identify genes involved in apoptosis, cell proliferation and cell cycle progression. We will also present data on use of siRNA pools versus multiple individual siRNAs, as well as data illustrating the need for multiple carefully chosen siRNA controls for siRNA screening experiments

1:45-2:00 Technology Short Talks
Additional Sponsorship Available. Contact: Carol Dinerstein at 781-972-5471 or dinerstein@healthtech.com.

CELLULAR ASSAYS FOR PATHWAY ANALYSIS

2:15-2:45 Phenotypic Fingerprinting of shRNA Effects on Mitotic Progression Using High-Content Imaging
To Be Announced
Successful application of RNAi at scale combined with high-content imaging has the potential to greatly increase our understanding of a wide range of cellular processes. Lentiviral-based shRNA delivery is a particularly effective method that allows for RNAi evaluation in a wide range of cell types, including non-dividing primary cells. This presentation focuses on high-content analysis of both on and off targets effects of lentiviral delivered shRNAs on mitotic progression in human cells with the aim of identifying phenotypic fingerprints derived from protein knockdown. Analysis of high-content data captured using Cellomics VTI technology, with an emphasis on the mitotic markers cyclinB1 and phospho-histoneH3, will be presented. RNAi technology comparison data (shRNA vs. siRNA) will also be included.

2:45-3:15 RNAi-Based Pathway Analysis Using HCA of Non-Adherent Primary Cells
Dr. Orian Shirihai, Professor, Pharmacology, Tufts University Medical School
To study cellular functions in heterogeneous populations of cells, such as tissue-derived cells, there is a need to continuously monitor functional parameters at a single cell resolution in a large number of cells. This is becoming a unique challenge when the cells of interest are non-adherent, as in the case of blood or bone marrow samples. To overcome this limitation, we used Molecular Cytomics’ Optical LiveCell™ Array, a device containing a densely packed array of transparent micron-sized wells. This technology enables real-time, ongoing observation of cellular events from thousands of individual adherent or non-adherent living cells, followed by structural and post-fixation studies on the same cells, using bright field and fluorescent microscopy. We employed this technology for investigating mitochondrial proteins involved in the heme biosynthetic pathway during differentiation of hematopoietic cells. We suggest this approach as a robust tool for HCA studies in heterogeneous populations of cells and as a revolutionary platform for imaging non-adherent cells.

3:15-3:45 Cytoskeletal and Cell Viability Assays for RNAi-Based Validation of Oncology Targets Associated with Rho/Rac Signaling
Dr. Robert Blake, Senior Scientist, Target Discovery, Exelixis Inc.
Conserved members of the Ras super-family Rho, Rac, and CDC42 are key regulators of the cytoskeleton that have roles in promoting cellular proliferation and are implicated in tumorigenesis. We have combined RNAi knockdown techniques and automated fluorescence imaging of cytoskeletal organization, cellular apoptosis and proliferation to investigate the role of candidate genes in these pathways. Knockdown analysis of Rho/Rac/CDC42 proteins or known pathway regulators / effectors, supports the view that Rho and Rac signaling are necessary for cell viability and proliferation and that knockdown of specific pathway components leads to well-defined cytoskeletal alterations. For example RhoA siRNA is associated with reduced myosin light chain phosphorylation. siRNAi of Rac1 or -3, or CDC42 is generally associate with increased actin content and in some cases with reduced cell/matrix adhesion. We discuss assay development for siRNA screens and target validation, and efforts to distinguish primary cytoskeletal alterations from those arising more indirectly from apoptosis or perturbations in other cellular processes.

3:45-5:00 Refreshment Break with Exhibit and Poster Viewing

STANDARDS FOR HIGH-CONTENT CELLULAR ASSAYS 

5:00-5:30 Controlling the Extracellular Matrix for Standardizing Cell Environment
Dr. John Elliott, Research Scientist, Biotechnology, National Institute of Standards and Technology (NIST)
Extracellular matrix (ECM) proteins provide adhesion sites and signaling cues to cells in culture. Even cells cultured on polystyrene are responding to ECM proteins adsorbed to the substrate surface. Since the molecular features of the adsorbed extracellular matrix proteins can have a dramatic effect on the adhesion and phenotype of adherent cells, it is important to control the tissue culture substrate for optimal intra-experimental and intra-laboratory comparison of high-content assay data. Self assembly of ECM protein, such as collagen, into films on alkanethiol monolayers is a controllable process that provides a highly reproducible, highly homogeneous matrix environment for cells, which can be independently validated by surface chemistry analytical techniques. Using this approach, we have studied how subtle variations in ECM preparations can alter cell phenotypic response in dramatic ways. Thus, control of the ECM environment is also critical for assuring the appropriateness of cell response in pharmacological screening, and for allowing correct interpretation of the assay results in terms of signaling pathways. This approach to fabricating ECM substrates can be used to benchmark cell response and may aid in the standardization of data that is available from high-content analysis.

5:30-6:00 Panel Discussion: Developing Standards for Cellular Assays: Reagents, Cells, Plateware, and Informatics

Joint Session with HCA 2006

6:00-7:00 ThinkTank Roundtable Discussions

The concurrent roundtable discussions (open to all delegates) provide a small-circle forum for discussing key issues and meeting potential partners. During the networking session, you are welcome to move freely between the roundtables. The discussion facilitators will present an update the following morning. (You must be a registered attendee to participate.)

Discussion Topics Include: 
• RNAi Validation and Off-Target Effects
• Genome-Wide siRNA Screening
• High-Content Cellular Assays for siRNA Screening
• RNAi for Pathway Analysis
• siRNA Delivery and Assay Development
• Integrating siRNA and Compound Library Screening

For more information please contact: 
Julia Boguslavsky, Conference Director, Cambridge Healthtech Institute
Phone: 781-972-5482 or E-mail: juliab@healthtech.com

For exhibit and sponsorship information, please contact:
Carol Dinerstein, Manager, Business Development
Phone: 781-972-5471 • E-mail: dinerstein@healthtech.com