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Recommended
Short Coarse
SC3: RNAi for Beginners -
Oct. 23, 2007
- Beginners Course on RNAi Knockdown
Technologies
- Tutors from Industry and Academia
- For Those Considering or Have Just
Begun to Use RNAi
Tutors:
Anastasia Khuorova, Ph.D., Director of Biology, R&D, Dharmacon
Christophe J. Echeverri, Ph.D., CEO & CSO, Cenix BioScience GmbH
Bernard Mathey-Prevot, Ph.D., Genetics, Harvard Medical School
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Wednesday is a shared
program with Preclinical Disease Models
7:00 am Registration & Morning Coffee
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7:30 Breakfast Workshop
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Sponsored
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Advanced RNAi Technology for Genome-Wide
Screening
Dr. Eric Lader, Associate Director, R&D, QIAGEN
Virtually any mammalian gene can be targeted in cell culture for silencing by short interfering RNA (siRNA), efficiently delivered into cells using lipid based transfection reagents. Synthetic siRNA libraries are ideal tools for genome-wide high throughput screening in cell models. We will present latest technology and product solutions developed at QIAGEN, specifically on genome-wide library design and synthesis, stringent homology search tools to avoid off-target effects, best siRNA controls to standardize all cellular assays, and high throughput siRNA delivery using reverse transfection approaches. |
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| KEYNOTE SESSION |
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8:30 Keynote Introduction
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8:40 Target Discovery: Seeking Innovation and the Role of Collaboration with Biotech and Academia
Jeremy Levin, D. Phil, MB. Bchir, Global Head, Business
Development and Strategic Alliances, Novartis
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9:10
Reducing Clinical Attrition through Efficiencies in Discovery
Joseph Bolen, Ph.D., Senior Vice President, Research & Drug Discovery,
Millennium Pharmaceuticals |
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9:40 Coffee Break in the Exhibit Hall
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IN VIVO RNAI FOR FUNCTIONAL ANALYSIS, TARGET VALIDATION & THERAPEUTIC DEVELOPMENT:
DELIVERY AND STABILITY OF RNAI IN VIVO
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10:20 Introduction and Welcome to RNAi: From Target Discovery &
Validation to Therapeutic Development
10:30 Cell-Type and Tissue-Specific RNAi (Genes & Development
Jan. 2006)
Miles Wilkinson, Ph.D., Professor, Department of Biochemistry and
Molecular Biology, MD Anderson Cancer Center
We present a simple approach to stably knockdown the expression of
genes in specific cell types in vivo. The approach mimics the way
naturally occurring microRNAs down regulate their targets. This microRNA-based
approach can be used to determine the tissue- or cell type-specific
function of genes in experimental animals or be used as a therapeutic
agent to silence deleterious genes in specific target tissues or cell
types in humans.
11:00 siRNA Targeting TNF Modulates Host Resistance to Herpes
Simplex Virus (Nature Protocols in press)
Edouard Cantin, Ph.D., Professor and Associate Chairman, Division
of Virology, Beckman Research Institute, City of Hope Medical Center
Data on in vivo application of siRNA targeting TNF in a mouse model of
HSV encephalitis will be presented. Peritoneal macrophages were
efficiently transfected with 27-mer siRNAs in vivo using Mirus TransIT TKO
transfection reagent and these cells migrated rapidly to the brainstem of
HSV infected mice. Using this in vivo siRNA delivery system, we have been
able to confirm that TNF signals independently of the known TNF receptors
to mediate resistance to fatal HSV encephalitis. Additionally, we have
shown trafficking of in vivo transfected macrophages into tumors,
illustrating the potential utility of the procedure for targeting
macrophages in diverse disease states.
11:30 Genetic Analysis of Autoimmunity by RNA Interference (Nature
Genetics April 2006)
Patrick Stern, Ph.D., Researcher, Center for Cancer Research, Massachusetts Institute of Technology
One of the challenges in identifying susceptibility genes in
multigenic diseases is the validation of individual candidate loci by
experimentation. We have made use of lentiviral RNA intereference by
transgenesis to test a candidate gene, Nramp1, in the type 1
diabetes-prone NOD mouse strain. We found that Nramp1 silencing reduced
disease frequency and confirmed this gene's involvement in disease, making
this approach a feasible strategy for the identification of susceptibility
genes in this and other complex diseases.
11:45 Reproducible and Inducible Knockdown of Gene Expression in
Mice (Genesis May 2006)
Jing Yu, Ph.D., Research Scientist, Andrew McMahon Laboratory,
Harvard University
In mammalian cells, vector-based expression of small hairpin RNAs (shRNA)
produces potent and stable gene knockdown effects. An inducible RNAi
system with reproducible levels of siRNA expression will extend the
usefulness of this methodology to the identification of gene functions
within the developing or adult mouse. We present evidence that an RNA
polymerase III driven U6 promoter with stuffer sequences flanked by loxP
sites inserted at three different sites within the promoter drives shRNA
expression in a Cre recombinase-dependent manner. We have utilized this
approach to develop a generic strategy for the reproducible knockdown of
gene expression in mice by placing the inducible shRNA cassette into the
ROSA26 locus of the mouse. This approach circumvents the pre-screening of
random integration in ES clones and further enables conditional gene
knockdown with temporal and/or tissue specificity. This methodology should
expedite large-scale functional studies.Technology Watch
12:00 Localized and Systemic In Vivo Delivery of RNAi
Kirk Brown, Ph.D., Field Application Scientist, Dharmacon
12:30 Lunch in the Exhibit Hall
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2:00
Roundtable: In Vivo Delivery Technology
Moderator: To be Announced
Discussion Points:
- Gene Specific Knockdown
- Localized and Systemic In Vivo Delivery of RNAi
- Comparison of Delivery Methodologies
Roundtable: RNAi Therapeutic Development
Moderator: Kevin Morris, Ph.D., Assistant Processor, Department of
Molecular and Experimental Medicine, The Scripps Research Institute
Discussion Points:
- Therapeutic Potential of siRNA-mediated Transcriptional Gene
Silencing
- Delivery – Make or Break
- Clinical Roadblocks
Roundtable: Target Effects & Other Technical Issues
Moderator: Michele Cleary, Ph.D., Research Fellow, Rosetta
Inpharmatics, LLC, a wholly owned subsidiary of Merck and Co.
Discussion Points:
- Incorporating Multiple shRNAs into Screening Platforms
- siRNA Controls
- siRNA Design
Roundtable: Validation of RNAi Knockdown
Moderator: Finbarr Murphy, Ph.D., Managing Director, Drug
Discovery, EiRx Therapeutics Ltd.
Discussion Points:
- Pro & Cons of Current Methodologies for Functional
Validation
- Is RT-QPCR the only Realistic High-throughput Method for Measuring
Endogenous Message Knockdown?
- Some Common Problems Associated with RT-QPCR in High-throughput
Screening
- How to Relate Message Knockdown with Protein Knockdown?
- What Level of Protein Knockdown Reflects Closest the Mode of Action
of Drug?
Roundtable: Building Better Disease Models
Moderator: Richard Roman, Ph.D., Founder, PhysioGenix; Head,
Renal Center, Medical College of Wisconsin
Discussion Points:
- Meeting the Challenges of Reproducing a Human Disease for Drug
Discovery
- Creating a Consistent System for Evaluating and Comparing the
Validity of Disease Models
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3:30 Refreshment Break in the Exhibit Hall
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EVALUATION OF IN VIVO DELIVERY AND STABILITY SOLUTIONS AVAILABLE
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Six or more companies will present their solutions to in vivo delivery
and stability in a series of concise presentations. Questions from
potential technology end-users from both larger pharma and biotech
companies and academic labs will be answered following each talk.
Discussions will follow.
4:00 Company One: Alnylam Pharmaceuticals Inc.
Delivery Technology: siRNA design, modifications of siRNA for improved
stability, systemic delivery, anti-viral applications
Presenter: TBA, Alnylam Pharmaceuticals Inc.
4:20 Company Two: Nastech Pharmaceutical Company
Inc.
Delivery Technology: siRNA delivery via intra-nasal, intra-pulmonary and
systemic routes, peptide and lipid based delivery, anti-viral applications
Presenter: Paul H. Johnson, Ph. D., Senior Vice President
of Research and Development, Chief Scientific Officer, Nastech
Pharmaceutical Company Inc.
4:40 Company Three: Atugen AG
Delivery Technology: siRNA design, modifications of siRNA for improved
stability, oncology applications
Presenter: Klaus Giese, Ph.D., Chief Scientific Officer,
Atugen AG / SR Pharma plc
5:00 Company Four: Artemis Pharmaceuticals GmbH
Delivery Technology: RNAi transgenics (constitutive and inducible)
Presenter: Holger Kissel, PhD., Senior Manager,
Scientific Project Management, Artemis Pharmaceuticals-an Exelixis Company
5:20 Company Five: Protiva Biotherapeutics Inc.
Delivery Technology: siRNA Design, Modifications of siRNA for Improved
Stability, SNALP Based Delivery
Presenter: Ian Maclachlan, Ph.D., Chief Scientific
Officer, Protiva Biotherapeutics Inc.
5:40 Company Six: Sirna Therapeutics, Inc.
Delivery Technology: siRNA design, modifications of siRNA for improved
stability, anti-viral (HCV/HBV), dermal applications
Presenter: Barry Polisky, Senior Vice President & CSO,
Sirna Therapeutics, Inc.
6:00 End-User Panelists
- Moitreyee Chatterjee-Kishore, Ph.D., Inflammation Team Leader,
Biological Technologies, Wyeth Research
- Joanne Kamens, Ph.D., Group Leader, Molecular & Cellular
Biology, Abbott Bioresearch Center
- Miles Wilkinson, Ph.D., Professor, Department of Biochemistry and
Molecular Biology, M.D. Anderson Cancer Center
- Edouard Cantin, Ph.D., Professor and Associate Chairman, Division of
Virology, Beckman Research Institute, City of Hope Medical Center
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6:30 In Vivo Networking Reception (Sponsorship Available)
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INTEGRATING METHODOLOGIES TO INCREASE EFFICIENCY -
THE TARGET VALIDATION TOOL BOX OF TODAY
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RNAi had become a very powerful tool in target discovery and validation
but is becoming one of the tools in the tool kit rather than the tool of
choice. The current overall strategy for validation is to continue to
integrate a wide variety of methodologies to increase efficiency. The
following group of experts will highlight the tools and technologies they
are currently being employed to get the job done and will mention the
maturing of RNAi as a technology. Some time will be dedicated to
discussing when to use RNAi technology as an entry point for a project or
as follow up from another entry point or how to know when it is the not
the tool for the job at all! The key to target validation is a platform
approach that brings multiple, system relevant approaches to bear in a
variety of systematic industrialized approaches. Case Studies will be
given by the following experts in the Target Validation arena.
8:30 Chairperson’s Remarks
Michael R. Cancilla, Ph.D., Associate Director, Translational
Medicine, Exelixis, Inc.
8:40 Target ID & Validation Toolbox Case Study #1:
Mark A. Labow, Ph.D., Executive Director, Platform and Chemical
Biology Department, Genome and Proteome Sciences, Novartis Institutes
for BioMedical Research
The changing behavior of cells in health and disease is largely
governed by the activation, repression and interaction of signal
transduction pathways. We will describe a platform of technologies for
systematic gain-of-function and loss-of-function analysis in mammalian
cells. The application of these approaches to identification of molecular
pathways and individual proteins which regulate disease-related processes
will be presented.
9:10 Target ID & Validation Toolbox Case Study #2:
Michele Cleary, Ph.D., Research Fellow, Rosetta Inpharmatics, LLC,
a wholly owned subsidiary of Merck and Co.
We have optimized high throughput lentivirus-mediated delivery of
short hairpin RNAs (shRNAs) for use in screens for novel cancer gene
targets. We are using this approach to identify genes whose silencing
enhances the effects of existing chemotherapeutic compounds. The hits from
our screens are useful in two key ways: First, these genes, or members of
their respective pathways, may serve as ideal targets for small molecule
inhibitors that could be administered in combination with lower doses of
standard chemotherapies. Second, these hits may provide better predictions
of patient response to the treatment studied. Along with screening for
novel drug targets, we are using vector-mediated RNAi to explore cancer
gene networks through microarray profiling. Previously, we reported that
transfection of small interfering RNAs (siRNAs) can result in the cleavage
of unintended targets. Although less prevalent, we find that shRNAs can
also result in silencing of transcripts other than that for which the
shRNA was designed. We see that such off-target mRNAs are enriched for
transcripts containing the shRNA antisense strand equivalents of microRNA
seed-region hexamers. This observation suggests that, like siRNAs, shRNAs
are not exquisitely specific, and very short regions of homology within a
core shRNA sequence are sufficient to produce undesired effects. These
results emphasize the need to use multiple RNAi sequences to ensure that
observed phenotypes correspond directly to silencing of intended target
genes. Because of this, we have incorporated the use of multiple shRNAs
into our screening platform.
9:40 Target ID & Validation Toolbox Case Study #3:
Speaker to Be Announced |
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10:10 Coffee Break in the Exhibit Hall
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RNAI - GETTING DOWN TO THE DETAILS & DOING IT RIGHT
PRACTICAL TECHNICAL TIPS SESSION
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RNAi has established itself as a powerful technology to identify and
validate drug targets. Many technical issues have been addressed in the
past five years and many lessons have been learned along the way. This
session will highlight how to do careful and well thought out RNAi
experiments through a series of case studies straight from the bench.
- How Good Are Your Cell Lines for RNAi-Based Screens?- Know Your
Resources?
- How Well Are You Validating Your Knockdowns?
- How Are You Dealing with Off-Target Effects?
- Primary Cell Transfection – Facing Facts
- Data Interpretation – Making Sense of the Data
- Dealing with Partial Knockdown
11:05 Know Your Resources: Cell Line
Characterization Prior to Target Discovery and Validation with RNAi
Michael R. Cancilla, Ph.D., Associate Director, Translational
Medicine, Exelixis, Inc.
Small molecule and RNAi-based screening with cell lines has expedited
the process of target discovery and validation. The multiple sources of
cell lines used for screening and limited molecular characterization can
lead to the use of inappropriate cell lines. This presentation will
illustrate how we have used genotyping, RNA expression analysis and
immunohistochemistry coupled with the accessible public databases to
characterize our collection of cell lines before they are used for in
vitro and/or in vivo experiments.
11:35 siRNA and the Problem of Getting What You
Want
Finbarr Murphy, Ph.D., Managing Director, Drug Discovery, EiRx
Therapeutics Ltd
The gold standard for validation of siRNA knockdown is demonstration
of a reduction in protein expression.However, given the prohibitive cost
and time of generating antibodies, the limited commercial availability of
antibodies to novel target proteins, and the move by many target discovery
departments towards high through put analysis, this is not always
practicable. To overcome this bottleneck, most practitioners monitor the
effect of siRNA oligonucleotides by examining the level of message RNA
using RT-QPCR. Although scientists at EiRx have demonstrated that
meaningful results can be achieved by this approach, we have uncovered
examples of apparent message knockdown (up to90% reduction in target
message levels) without knockdown of cognate protein. We present a case
study of our anomalous RT-QPCR analysis findings and discuss the steps
that we have put in place to ensure that RT-QPCR results accurately
reflect changes at the protein level, in order to enable accurate
interpretation of functional assays and ultimately reduce the danger of
discarding potentially exciting targets.12:05 RNAi and Functional HTS
Assay Development and Validation Glenn Cowley, Ph.D., Senior Scientist,
Abbott Bioresearch CenterTechnology Watch
12:05 TBA
Technology Watch
12:35 RNAi Goes Genomic: Elucidating Gene
Function with siRNA Libraries
Christina Buchanan, Ph.D., Technical Applications Specialist,
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. |
12:50 Technology Workshop (Sponsorship
Available) or Lunch on Your Own
2:00 Chairperson’s Remarks
2:05 Technology Watch (Sponsorship
Available)
2:20 HT siRNA Screen for Genes that Regulate
Amyloidogenic Processing of ß-amyloid Precursor Protein
Shane Marine, Research Biochemist, Department of Automated
Biotechnology, Merck Research Laboratories
Proteolysis of the ß-amyloid precursor protein (APP) into the amyloid
ß peptide is a critical step in the pathogenesis of Alzheimer’s
Disease. Although the enzymes that process APP have been identified, the
physiological pathways regulating their expression and function have not
been fully described. Here we describe the implementation of a genome-wide
HT siRNA screen for the unbiased identification of human genes that
regulate the amyloidogenic processing of APP.
2:50 Phenotyping RNAi Libraries through Imaging
and Localization Studies
Bahram Parvin, Ph.D., Staff Scientist, Information & Computing
Sciences, Lawrence Berkeley National Laboratory
High-content screening of imaging assays is a promising and
complementary approach for evaluating RNAi in support of model generation.
One main technological barrier has been the imaging bioinformatics
component for constructing phenotypic responses of each end-point as a
result of a specific gene knockdown. Our presentation will outline
operation computational and informatics modules in support of such assays
for simple to a more realistic model system.3:20 Refreshment Break in the
Exhibit HallRNAi Therapeutics – Full Speed Ahead
3:50 RNAi Therapeutics: Advancements in
Delivery and In Vivo Efficacy (Nature May 2006)
Tracy Stage Zimmermann, Ph.D., Senior Scientist, Alnylam
Pharmaceuticals Inc.
Since many diseases are caused by the inappropriate activity of
specific genes, the ability to down modulate genes selectively through
RNAi provides a powerful pharmacologic approach to treat a wide range of
human diseases. A major hurdle to achieving the therapeutic potential of
RNAi therapeutics is the delivery of siRNA to the target tissue at a
clinically relevant dose and manner. Results demonstrating efficient siRNA
delivery and potent in vivo efficacy at clinically relevant doses of siRNA
will be presented.
4:20 Promoter RNAs Guide siRNA Mediated
Transcriptional Gene Silencing in Human Cells
Kevin Morris, Ph.D., Assistant Processor, Department of Molecular
and Experimental Medicine, The Scripps Research Institute
Small interfering RNAs (siRNAs) targeting promoter regions can direct
silent chromatin modifications that engender transcriptional gene
silencing (TGS) in human cells. Whether siRNAs can recognize and bind
directly to DNA or to an uncharacterized RNA transcript across the
promoter has remained unclear. Here we provide evidence that an RNA is
transcribed through the RNA polymerase II (RNAPII) promoters for
elongation factor 1 alpha (EF1-alpha) and chemokine receptor CCR5. These
promoter specific RNAs (pRNAs) are detected by the antisense strand of
siRNA and do not exhibit significant reduction in copy number when
targeted by siRNAs. Our results suggest that a low-level pRNA is
transcribed through RNAPII promoters, and furthermore, that pRNAs
facilitate TGS in human cells by possibly acting as a scaffold for the
antisense strand of the siRNA to bind and mediate TGS.
4:50 Informal Q&A
5:00 Close of Conference
For more information, please contact:
Edel O'Regan, Senior Conference Director, Cambridge Healthtech Institute
Phone: 617-630-6323 E-mail: eoregan@healthtech.com
For exhibits and sponsorship information, please contact:
David Karp at 781-972-5483 or dkarp@healthtech.com
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