RNA interference: Chemical Biology to Bio-Drug & Therapeutic Development

May 1-3, 2005

Venue: Doubletree Guest Suites, Waltham, MA, USA

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AGENDA/SPEAKERS (click here for agenda)

Sunday, May 1, 2005
1:00 – 7:30 P.M: Registration Open
2:00 – 7:00 P.M: Technology Session I (consists of 6-8 lectures)
Monday, May 2, 2005
7:00 – 8:30 A.M: Registration Open
7:30 – 8:45 A.M: Continental breakfast

8:00 - 9:00 A.M: Technology Session II
Scientific Sessions Start at 9:00 A.M and Ends at 5:30 P.M on (May 2nd and 3rd) both days.
Tuesday, May 3, 2005
7:00 – 8:30 A.M: Registration Open
7:30 – 8:45 AM: Continental breakfast

8:00 - 9:00 A.M: Technology Session III

The actual agenda will be updated. Please visit again.

Scientific Advisory Committee:

Krishnarao Appasani, PhD., MBA.
Founder & CEO, GeneExpression Systems, Inc.
Waltham, MA

Connie Cepko, PhD.
Professor of Genetics and Investigator
Howard Hughes Medical Institute
National Academy Member
Department of Genetics
Harvard Medical School, Boston, MA

Kazunari Taira, PhD.
Professor of Chemistry & Biotechnology
The University of Tokyo, Tokyo, Japan

Carl D. Novina, MD., PhD.
Assistant Professor
Department of Immunology and AIDS
Dana-Farber Cancer Institute
Harvard Medical School, Boston, MA

Inder Verma, PhD.
Professor of Molecular Biology
Laboratory of Genetics
National Academy Member
The Salk Institute, La Jolla, CA

Esther Stoeckli, PhD.
Professor of Developmental Neuroscience
University of Zurich
Zürich, Switzerland

Roberto Weinmann, PhD.
Director of Oncology Discovery, Bristol Myers Squibb, Princeton, NJ

William Marshall, PhD.
Vice President R&D, Dharmacon, Inc., Lafayette, CO

Inaugural Speaker: May 2nd

Sidney Altman, PhD. Nobel Laureate 1989 (Chemistry) Yale University, New Haven, CT Title: A useful RNase P-external guide sequence (EGS) technology

Marshall W. Nirenberg, PhD. Nobel Laureate 1968 (Medicine & Physiology) Laboratory Chief of Biochemical Genetics National Heart, Lung and Blood Institute National Institutes of Health, Bethesda, MD Title: Screening Drosophila genes by RNAi

Keynote Speaker on RNAi:

Ronald H.A. Plasterk, PhD. Director, Netherlands Institute for Developmental Biology Utrecht, The Netherlands Title: RNAi and transposon silencing in C. elegans

Keynote Speaker on miRNAs:

David P. Bartel, PhD. Professor of Biology & Member Whitehead Institute for Biomedical Research Massachusetts Institute of Technology Cambridge, MA Title: MicroRNAs and their targets in plants and animals

Keynote Speaker on Therapeutics:

Barry Polisky , PhD. Vice President & CSO Sirna Therapeutics Boulder, CO Title: Preclinical and Clinical Development of siRNA Therapeutics

Other Speakers:

Connie Cepko, PhD. Professor of Genetics and Investigator of HHMI, Harvard Medical School, Boston, MA
Title: RNAi in the Vertebrate retina

Esther T. Stoeckli, PhD. Professor of Developmental Neuroscience, Institute of Zoology, University of Zurich, Zürich, Switzerland
Title: In ovo RNAi turns the chicken embryo into an (even better) model organims for developmental studies

Carl D. Novina MD., PhD. Assistant Professor, Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA
Title: Killing the Messenger: Mechanisms and Applications of Mammalian RNAi

Mark Behlke MD, PhD. Vice President, Molecular Genetics, Integrated DNA Technologies, Inc., Coralville, IA
Title: Increased potency of 27mer siRNA Duplexes

Kerry E. Lowrie, PhD. Assoc. Business Area Manager- RNAi Technologies, Invitrogen Corporation, Carlsbad, CA
Title: Next Generation RNAi: Technologies and Applications

Kathy Latham, PhD. Manager of RNAi Technologies, Ambion, Inc., Austin, TX
Title: RNAi Screening in Human Cells Facilitated by High-throughput Electroporation and Transfection


Jorg F. Rippmann, PhD., Head, Genomics Group, Boehringer Ingelheim Pharma GmbH, Biberach an der Riss, Germany

Maen Abdelrahim, Ph.D. Research Scientist, Center for Environmental and Genetic Medicine, Texas A&M University, Houston, TX
Title: Role of Sp Proteins in Regulation of Vascular Endothelial Growth Factor Expression and Proliferation of Pancreatic Cancer Cells

Brent A. Rupnow, PhD. Senior Research Investigator, Oncology Drug Discovery, Bristol-Myers Squibb, Princeton, NJ
Title: RNAi in cancer target discovery: opportunities and challenges

Biao Luo, PhD. Group Leader, RNAi Consortium, Broad Institute of MIT and Harvard, Cambridge MA
Title: Construction and application of mammalian genome-wide shRNA libraries

Tarif Awad, PhD. Senior Scientist, Affymetrix Genomics Collaborations, Affymetrix, Inc. Santa Clara, CA
Title: A Genome-wide Look at the Actions of siRNA

Eric Lader, PhD. Associate Director of R &D, QIAGEN, Inc., Germantown, MD
Title: Advances in High Throughput Design, Synthesis, and Screening using RNAi

Ron Herzig, PhD. Director of RNAi and Innovation Labs, Upstate Biotechnology & Chemicon
T itle: Control of Cellular Genes with shRNA Expression Systems

Sakari Kauppinen, M.Sc., Ph.D., Director of Functional Genomics, Exiqon, Vedbaek,Denmark
Title: Detection and analysis of microRNAs using LNA probes

Takayuki Mizutani, Director for Business Development, B-Bridge International, Inc., Sunnyvale, CA
Title: Avoiding Off-Target Responses in RNAi Studies

Zvi Bentwich,M.D., Professor of Medicine & Chief Scientist, Rosetta Genomics, Rehovot, Israel
Title: Hundreds of Conserved and Nonconserved Human MicroRNAs Detected by a Novel Approach

Michelle Lyles, PhD. Vice President, Sales and Marketing, Genospectra, Inc., Fremont, CA
Title: Innovative Tools for RNAi Research: An integrated approach for siRNA delivery, light controllable mRNA-knockdown, and quantification of knockdown and downstream expression events

Steven Haney, PhD. Group Leader, Oncology Genomics, Department of Biological Technologies, Wyeth Research, Cambridge, MA
Title: Incorporating High Content Screens into RNAi-Based Target Validation

Yukikazu Natori, RNAi Co. Ltd. Japan through Proligo, LLC, Boulder, CO
Title: Gene function analysis by genome-wide siRNA libraries with high knock-down efficiency and low off-target effects

Kader Thiam, PhD. Director of Transgenic Technologies, genOway, Lyon, France
Title: Efficient shRNA gene knock down in genetically modified mice for in vivo target validation

Anna Krichevsky, Ph.D. Instructor in Neuroscience, Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA
Title: MicroRNA is an anti-apoptotic factor in human brain tumors

Steffen Panzner, PhD. Cheif Executive Officer, novosom AG, Halle, Germany
Title: Systemic low dose antisense delivery and anti-inflammatory effect using Smarticles® vectors

Kazunari Taira, PhD. Professor, Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo; and Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan
Title: It’s a small RNA world that makes a big revolution in the bio-medical field

Zachary Zimmermann, PhD. Director of External Alliances, Alnylam Pharmaceuticals Inc., Cambridge, MA
Title: Silence as Therapy: Translating RNAi into Drugs

Troy Moore, PhD. Chief Technology Officer, Open Biosystems, Inc., Huntsville, AL
Title: Expression ArrestTM shRNA libraries: Solutions for whole genome RNAi screening

Inder M. Verma, PhD. Professor of Molecular Biology, Laboratory of Genetics, The Salk Institute, La Jolla, CA
Title: siRNA Delivery by Lentiviral Vectors

William S. Marshall, Ph.D. Executive Vice President Research & Operations and Site Manager, Dharmacon Inc. Lafayette, CO
Title: Optimizing the Properties of siRNA to Maximize Their Utility in Therapeutic Discovery

Abstracts

A useful RNase P-external guide sequence (EGS) technology
Sidney Altman, PhD. Nobel Laureate 1989 (Chemistry), Sterling Professor of Molecular, Cellular & Developmental Biology, Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT USA

A brief history of RNase P and the external guide sequence (EGS) technology will be presented. The cases studied with the EGS technology and its current status will be reviewed and some data that compares RNAi with the EGS technology will be presented.

Screening Drosophila genes by RNAi
Marshall Nirenberg, PhD. Chief, Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD

During the last few years the functions of many Drosophila genes have been screened by RNAi. Both slow, laborious RNAi studies with intact embryos and rapid high-throughput studies with tissue culture cells, will be discussed. The high throughput RNAi studies are mostly those of Norbert Perrimon and his co-workers.


RNAi and transposon silencing in C. elegans
Ronald Plasterk, PhD. Professor and Director, Hubrecht Laboratory / Netherlands Institute for Developmental Biology, Utrecht, The Netherlands

While the genome of the nematode C. elegans contains multiple copies of the transposons Tc1, Tc3, Tc5 and others, none of them are mobile in the germline. We previously performed genetic screens to find mutants in which transposition was activated (Ketting et al., 1999). Surprisingly most of these mutator-mutants were found to be defective also in RNA interference or RNAi; it seems plausible that a natural function of RNAi is to protect the genome against transposon activity. We used a combination of genetics and biochemistry to define the molecular basis of RNAi and transposon silencing. Our current model is that the mechanistic link between RNAi and transposon silencing is that double stranded Tc1 transcripts induce silencing of transposase expression in the germline. In line with this hypothesis we observe dsRNA of Tc1, and also siRNAs that corresponds to the inverted terminal repeats and also the internal sequences of Tc1. Addition of a Tc1 terminal repeat sequence to a reporter gene results in silencing in the germ line.

A second line of research in the laboratory addresses the role of miRNAs in zebrafish development. Injection of let-7 and other miRNAs into one-cell embryos was used to define requirements for let-7 miRNA activity. A complete mutant spectrum of this miRNA was derived. We also determined the expression pattern of all 120 miRNAs that are conserved between zebrafish and human, and surprisingly found a large fraction to be entirely organ-specific.

In ovo RNAi turns the chicken embryo into an (even better) model organisms for developmental studies
Esther T. Stoeckli, PhD. Professor of Developmental Neuroscience, Institute of Zoology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland

Chicken embryos have been widely used for developmental studies for more than a century. However, recently their importance has suffered due to the lack of genetic tools. The establishment of in ovo RNAi has changed that and turned the chicken embryo into an efficient model organism for functional genomics. The power of in ovo RNAi is the temporal and spatial control of gene silencing during embryonic development, a feature that is unique among vertebrates. We have taken advantage of the possibility to silence genes in a temporally controlled manner to study the role of morphogens during later stages of development, when neural circuits are formed. Such studies would not have been possible using classical genetic tools in mice.

Role of Sp Proteins in Regulation of Vascular Endothelial Growth Factor Expression and Proliferation of Pancreatic Cancer Cells
Maen Abdelrahim, Ph.D. Research Scientist, Center for Environmental and Genetic Medicine, Texas A&M University, Houston, TX

Maen Abdelrahim1, Robert Burghardt1, Devin Leake2 and Stephen Safe1
1 Texas A&M University. Health Science Center. Center for Environmental and Genetic Medicine. 2121 W. Holcombe Blvd. Houston, TX 77030
2 Dharmacon Research Inc. 2650 Crescent Drive #100. Lafayette, Colorado 80026

Sp proteins play an important role in angiogenesis and growth of cancer cells. RNA interference was used to investigate the role of Sp family proteins on regulation of VEGF expression and proliferation of pancreatic cancer cells. It was initially shown that Sp1 and Sp3 were required for transactivation of VEGF promoter inserts, and this was primarily dependent on proximal GC-rich motifs. We also showed for the first time that Sp4 was expressed in different pancreatic cancer cells, and RNA interference experiments suggested that Sp4 cooperatively interacted with Sp1 and Sp3 to activate VEGF promoter constructs in these cells. Small inhibitory RNAs for Sp3, but not Sp1 or Sp4, inhibited phosphorylation of retinoblastoma protein, blocked G0/G1 ? S phase progression, and upregulated p27 protein/promoter activity of several pancreatic cancer cells, suggesting that Sp3-dependent growth of pancreatic cancer cells is due to inhibition of p27 expression. In vivo experiments are in progress to target Sp proteins in pancreatic cancer cells using in vivo stable siRNAs in immuno-deficient athymic nude mice.

RNAi Screening in Human Cells Facilitated by High-throughput Electroporation and Transfection
Kathy Latham, PhD. Manager of RNAi Technologies, Ambion, Inc. Austin, TX

siRNA delivery is critical to mammalian RNA interference experiments. We have developed two siRNA delivery procedures specifically for high throughput RNAi applications in mammalian cell systems. The first, developed for immortalized cells, involves simultaneously plating and transfecting cells to reduce time and improve transfection efficiency. The second method was developed for primary, neuronal, and suspension cells and features high throughput electroporation. It overcomes problems with poor cell viability inherent in mammalian cell electroporation and provides up to 95% target gene reduction in primary cell populations. In addition, we have measured how siRNA concentration, siRNA pooling, cell type, and time between transfection and analysis influence the final screening data. Our results provide a framework for setting up functional screens with siRNA libraries and show the power of using such an approach to correlate genes with cellular functions.

Using high performance computing to predict siRNA and miRNA off-target effects
Pal Saetrom, PhD. Research Scientist, Interagon AS, Trondheim, Norway

Due to sequence complementarity with mRNA besides the intended target, siRNAs may induce sequence-related off-target effects. Whether this results in mRNA cleavage (siRNA effect) or translational suppression (miRNA effect) seems to depend solely on the degree of complementarity between probe and target. We present a high performance search system that uses sequence similarity searches to identify potential siRNA off-target sites, and a machine learning solution that uses our search system to predict microRNA target sites. Together, these tools represent a basis for identifying sequence-related off-target effects.


Increased potency of 27mer siRNA Duplexes
Mark Behlke MD., PhD. Vice President, Molecular Genetics, Integrated DNA Technologies, Coralville, IA
Mark A. Behlke1, Scott D. Rose1, Dongho Kim2, and John J. Rossi2
1. Integrated DNA Technologies, Inc. (IDT), Coralville, IA.
2. Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA.

In attempts to identify RNAi triggers that effectively function at lower concentrations, we found that synthetic RNA duplexes of ~27 nucleotides in length can be up to 100 fold more potent than corresponding 21mer siRNAs. The enhanced potency of the longer duplexes is attributed to the fact that they are Dicer substrates, directly linking the production of siRNAs to incorporation into RISC. Not all 27mers show this increased potency, mostly due to variation in dicing patterns. We have defined methods to direct dicing to produce specific, desired products and thereby make design of high-potency 27mer siRNAs more predictable.

Preclinical and Clinical Development of siRNA-Based Therapeutics
Barry Polisky, Ph.D.Vice President and Chief Scientific Officer, Sirna Therapeutics, Boulder, CO

The RNAi pathway is a naturally occurring process used by cells to down-regulate gene expression that can be harnessed to prevent the expression of virtually any RNA target. Chemically synthesized small interfering RNA molecules (siRNAs) may be used to mediate this process. Alternatively they may be expressed from a number of vectors. The key challenges for the development of siRNAs as therapeutics include extra- and intracellular stability, improved specificity, delivery and pharmacokinetics (PK). Sirna has successfully overcome many of these challenges using chemical modification and formulation. Preclinical animal efficacy has been demonstrated in several areas including ocular neovascularization, viral infection, neurodegeneration, diabetes, asthma and tumor models using therapeutically relevant routes of administration. A complete proof of concept has been demonstrated in Age-Related Macular Degeneration, including a reduction in molecular endpoints, using a chemically modified siRNA targeting VEGFR-1, Sirna-027. An update on several preclinical programs and the clinical development of Sirna-027 will be presented.

RNAi in the Vertebrate Retina
Connie Cepko, PhD. Professor of Genetics, Investigator, HHMI, Harvard Medical School, Boston, MA
Constance Cepko, Takahiko Matsuda, Sunjay Harparvat
The availability of a large number of candidate genes made available by the comprehensive analysis of genomes requires that relatively rapid techniques for the study of function be developed. A rapid and convenient electroporation method for both gain- and loss-of-function studies in vivo and in vitro in the rodent and chick retina will be presented. Plasmid DNA directly injected into the subretinal space of neonatal rodent pups or embryonic chicks is taken up by a significant fraction of exposed cells following several pulses of high voltage. Using this technique, GFP expression vectors and/or RNAi hairpin vectors are efficiently transfected into retinal cells with little damage to the operated animals. In addition, for stable transduction of hairpins, retroviral vectors based upon Moloney Murine Leukemia Virus or Avian Leukemia Virus have been used. The hairpin efficacy is first tested using in vitro cell based assays. Subsequently, efficacy is followed in vivo by co-introduction of hairpins with a GFP encoding target. Hairpiins directed against two transcription factors important in photoreceptor development led to photoreceptor phenotypes similar to those of the corresponding knock-out mice. Other applications will be discussed.

Detection and analysis of microRNAs using LNA probes
Sakari Kauppinen, M.Sc., Ph.D., Director of Functional Genomics, Exiqon, Vedbaek, Denmark

To exploit the significantly improved hybridization properties of LNA oligonucleotides against complementary RNA targets, we have designed several LNA-modified DNA probes for detection of different microRNAs in animals and plants by northern blot analysis and in situ hybridization. We will describe the results obtained from detection and analysis of different miRNAs in C. elegans, zebrafish, mouse, and plants. In addition, we will describe a novel LNA-based method for expression profiling of mature miRNAs by quantitative RT-PCR.

A Genome-wide Look at the Actions of siRNA
Tarif Awad, Ph.D. Senior Scientist, Affymetrix Genomics Collaborations, Affymetrix, Inc. Santa Clara, CA

siRNA gene silencing and array-based expression profiling are a powerful combination of technologies useful for studying complex biological processes. The arrays provide a highly detailed 'readout' for siRNA manipulations, helping test specificity as well as generate a molecular phenotype reflecting the downstream effects of gene silencing on living cells. This presentation will provide an overview on the experimental design and data analysis strategies for using the two technologies together, drawing on examples from recent collaborations between Affymetrix and partners in the field.

RNAi in cancer target discovery: opportunities and challenges
Brent A. Rupnow, PhD. Senior Research Investigator, Oncology Drug Discovery, Bristol-Myers Squibb, Princeton, NJ

RNAi is a powerful tool for understanding the functional consequences of target inactivation in mammalian cells and model organisms. RNAi technology has great potential for the identification and validation of novel drug targets. However, our understanding of the mechanisms of RNAi in mammalian cells continues to evolve and the potential for off-target toxicity using siRNAs has been realized. This can be problematic in the validation of oncology targets as tumor cell death is often a desired phenotype. In this presentation, I will describe our approach using RNAi and other tools to discover and validate oncology targets, provide examples of success and failure and highlight lessons learned.

Avoiding Off-Target Responses in RNAi Studies
Takayuki Mizutani, Director for Business Development, B-Bridge International, Inc., Sunnyvale, CA

RNAi technology has become an essential tool to study gene function. However, when using siRNA, it is critical to avoid off-target effects that may distort or mask the actual effects resulting from knocking down a particular gene. Thus, it is essential to accurately predict the effectiveness and ensure the specificity of a particular siRNA sequence. By exploiting sequence variations across different species to provide a more varied pool of potential off-target candidates, we have been able to generate a large set of data to analyze potential off-target knockdowns. Using this data, we have developed novel approaches to predict off-target effects of siRNA molecules and help ensure the design of more effectively targeted siRNA. In this presentation, we will review these results and discuss how to design effective siRNA sequences that maximize knockdown while minimizing the potential problems from off-target effects.

Killing the messenger: mechanisms and applications of mammalian RNAi
Carl D. Novina, MD., PhD. Assistant Professor, Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA

The regulation of gene expression by short RNAs is a field of immense biological importance. Short RNAs are implicated in setting the precise tempo of gene expression for a number of processes necessary for normal cell function and are critical for proper organism development. However, the exact mechanism(s) by which short RNAs silence their target genes remains unknown. In addition to controlling important cellular processes, short RNAs can be used as a tool to precisely silence genes leading to an understanding of gene function. Because it is easy to use, RNAi has been applied widely to the discovery of gene function. While understanding the basic mechanisms of RNAi makes it better tool, its widespread use has not always translated into insights into the mechanism(s) of RNAi. Data will be presented on the use of short RNAs both to dissect the molecular mechanisms of virus infectivity and to decipher the mechanisms of mammalian RNAi.

Innovative Tools for RNAi Research: An integrated approach for siRNA delivery, light controllable mRNA-knockdown, and quantification of knockdown and downstream expression events
Michelle Lyles, PhD. Vice President, Sales and Marketing, Genospectra, Inc., Fremont, CA

An integrated platform for siRNA research will be presented in which a novel delivery reagent, light-controllable siRNA (csiRNATM), and precise mRNA quantification are utilized to characterize the impact of hNuf2 knockdown in difficult-to-transfect, Jurkat, cells. Cells are transfected with inactive hNuf2-csiRNA and allowed to recover. hNuf2-csiRNA is photoactivated by brief light exposure in the UCOMTM microplate-format instrument. Knockdown is quantified directly from cell lysate using QuantiGeneTM, a precise assay for gene expression powered by Branched DNA. Similarly, apoptosis-related gene expression is profiled using QuantiGeneTM Plex, the multiplexed version of QuantiGene in which expression of 3 to 30 genes are quantified simultaneously on Luminex platform. Together, these innovative tools provide precise control and accurate results for cutting-edge siRNA research.

Incorporating High Content Screens into RNAi-Based Target Validation
Steven Haney, PhD. Group Leader, Oncology Genomics, Department of Biological Technologies, Wyeth Research, Cambridge, MA

High Content Screening, or automated microscopy and image analysis, has been used successfully in the drug discovery process for several years in late preclinical studies, especially toxicology. HCS is currently being used in target ID and validation approaches, largely as a result of the increased power of recent image analysis algorithms. HCS allows novel and highly complex assays to be used for characterizing new targets, and is readily compatible with RNAi screens. However, challenges inherent to any RNAi screening platform exist for HCS-based RNAi screens as well. These challenges can be addressed during the development of the assay itself, as well as in the data analysis stage.

Gene functional analysis by genome-wide siRNA with high knock-down efficiency and less off-target effect
Yukikazu Natori, Chairman of RNAi Co., Ltd, Tokyo, 101-0047, Japan

Everyone of the researcher worried about the siRNA sequence design talks about that in the past. Using our siRNA sequence design system named siDirectTM which has been invented by Prof. Kaoru Saigo, Prof. Shinichi Morishita and others at University of Tokyo, it is straight forward to design and select the most effective siRNA with less off-target for all of human, mouse and rat genes published. We have never had any failure to knock-down a target gene even though we have done experiments on hundreds of endogenous genes. One single siRNA is enough for each target. siDirectTM allows us to study functional analysis of each variant and/or combination of variants with minimum off-target effect. Genome-wide siRNA (siPerfectTM) for all of human genes is Now available to researchers through collaboration with Proligo.

Efficient shRNA gene knock down in genetically modified mice for in vivo target validation
Kader Thiam, Ph.D. Director of Transgenic Technologies, genOway, Immeuble Chateaubriand, Lyon, France

The recent demonstration that RNAi-mediated gene silencing can occur in rodents have opened great opportunities for in vivo target validation programs. genOway has developed innovative technologies enabling the control of key parameters critical for getting RNAi working in mouse. “Safe RNAi transgenesis” allows the control of shRNA copy number and gives access to high number of founders. “Quick RNAi” secures shRNA expression using targeted insertion in a permissive locus and allows the generation of animal model displaying one single copy of shRNA expressing construct. This technology bypasses the drawbacks associated with random integration of shRNA expressing constructs and only one mouse line is needed. Combining this technology with Cre recombinase based approach; the gene knockdown could be controlled in a tissue- and time-specific manner. Furthermore, the company has developed proprietary technologies enabling the in vivo validation of shRNA against human target gene and evaluation of the off target effect. Based on validated and optimized tools, these approaches enable efficient and stable in vivo gene knock down and are time saving and cost effective.

MicroRNA is an anti-apoptotic factor in human brain tumors
Anna Krichevsky, Ph.D. Instructor in Neuroscience, Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA

Roles of microRNAs (miRNAs) in lineage determination and proliferation as well as location of several miRNA genes at sites of translocation breakpoints or deletions has led to a speculation that miRNAs could be important factors in development or maintenance of the neoplastic state. Using oligonucleotide array, we analyzed expression of 180 miRNAs in highly malignant human brain tumor, glioblastoma. We demonstrate that the glioblastomas strongly overexpress a specific miRNA. Knockdown of this miRNA in cultured glioblastoma cells triggers activation of caspases and leads to increased apoptotic cell death. Our data validate a functional role of miRNA in human neoplasia and suggest that aberrantly expressed miRNA may function as a 'micro-oncogene' blocking expression of
critical apoptosis-related genes.

Systemic low dose antisense delivery and anti-inflammatory effect using Smarticles® vectors
Steffen Panzner, PhD. Cheif Executive Officer, novosom AG, Halle, Germany

novosom AG is developing carrier-based drugs for the treatment of inflammatory disorders. A pipeline will be presented including small molecules as well as two different antisense candidates under development. Proof of principle data in therapeutic models will support the claim of delivery –enhanced product development opportunities using our Smarticles® platform for targeted intracellular delivery of various classes of drug substances.

It’s a small RNA world that makes a big revolution in the bio-medical field
Kazunari Taira, PhD. Professor, Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo; and Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan

Hammerhead ribozymes were engineered to enhance their intracellular activities. Then, libraries were made of ribozymes with randomized binding arms. The libraries were then introduced into cells either by transfection or by viral vectors. This procedure made it possible to readily identify the relevant genes associated with phenotype in the apoptosis, cancer metastasis, and/or cell differentiation pathways. Importantly, the identified functional genes originated not only from the coding region but also from the non-coding region. The discovery of small modulatory dsRNAs (smRNAs) extends the important contribution of non-coding RNAs as key regulators of cell behavior at both transcriptional and post-transcriptional levels. As an alternative library, siRNA library for the entire human genome is attractive. Indeed, such an siRNA library enabled us to identify important components involved in RNAi mechanism, including slicer (eIF2C2) and an RNA helicase. Our siRNA expression system was designed such that the sense strand was inactivated and the interferon response was avoided. Same technology can be used to express long dsRNA without inducing interferon responses.

Hundreds of Conserved and Non-conserved Human MicroRNAs Detected by a Novel Approach
Zvi Bentwich, M.D., Professor of Medicine & Chief Scientist, Rosetta Genomics, Rehovot, Israel

MicroRNAs are a class of ~22-nuleotide non-coding RNAs that have emerged as an evolutionary conserved group with important regulatory roles, especially during development, but also in several physiological systems in health and disease. To prove our hypothesis that the total number of microRNAs may be much larger and that several have emerged only in primates, we have developed an integrative approach that combines bioinformatic predictions, with the usage of microarrays and sequence directed cloning. Here we report on application of this approach to the human genome that resulted in the cloning and sequencing of 84 novel human microRNAs (almost doubling the current number of truly sequenced human microRNAs), 54 of which are not conserved beyond primates, and in the projection that the total number of human microRNAs is at least 770 and probably much larger.

Silence as Therapy: Translating RNAi into Drugs
John M. Maraganore, PhD. Chief Executive Officer, Alnylam Pharmaceuticals Inc., Cambridge, MA

RNA Interference (RNAi) holds significant promise as a therapeutic approach to silence disease-causing genes, particularly those not amenable to conventional drugs such as small molecule, protein, or monoclonal antibody therapeutics. The key hurdle for RNAi therapeutics is delivery needed to achieve in vivo gene silencing. A critical requirement for achieving safe and efficacious RNAi therapeutics is introduction of "drug-like" properties, such as stability, cellular delivery, and tissue bioavailability, into synthetic siRNAs. Chemically-stabilized siRNAs showed significantly enhanced resistance toward degradation by exo- and endonucleases without loss of biological activity. As compared with unmodified siRNAs, these chemically-stabilized, siRNAs have improved in vivo pharmacologic properties.

MicroRNA based prevention, diagnosis, prognosis and treatment of upper aerodigestive cancers
Carlo M. Croce, MD. Director, Human Cancer Genetics & Institute of Genetics, Chairman, Molecular Virology, Immunology and Medical Genetics, The Ohio State University Comprehensive Cancer Center, The Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Columbus, Ohio

We have shown the involvement of microRNA genes in the pathogenesis of human cancer. We have also shown that micro RNA gene expression profiling can be exploited for the diagnosis and prognosis of human leukemia. Most recently we have used the micro RNA gene chips we have developed to show alterations in microRNA gene expression in lung cancer and in other solid malignancies. This project will first focus on the development of nanochips for the assessment of microRNA gene expression in very small amounts of precancerous and cancerous tissues and on the development of high-throughput screening (HTS) nanotechnology for the mechanistic understanding, characterization, profiling and validation of microRNAs. The nanotechnology platform will comprise primarily three components:

A nanochip for the assessment of microRNA gene expression in precancerous and cancerous cells. Two different approaches will be used to build quantitative, label-free, high-density microRNA chips: One based on nanocantilever technology, and one based on conjugation detection based on conductivity changes or other physical signals (“e-chip”). A highly parallel fluidic system that will afford the high-throughput, automated transfection of a large number of cell microwells, their characterization by optical microscopy, the controlled supply of additional substances to individual cell wells, and the monitoring of their effects. In particular, this includes fluidic apparatus for that automated aliquoting of secretions from the cell population in the different microwells. Nanotextured substrates for TOF proteomic analysis that will permit the profiling and lead to the identification of proteomic signatures in molecular weight ranges that are currently inaccessible, and/or will afford advantages in the focusing on low-concentration molecular species.

These technologies will be integrated with existing approaches, and lead to higher efficiency in the microRNA profiling, signature, and target identification and validation, for all cancers of interest and their respective precancerous lesions in all projects undertaken within the CCNE, and in particular lung, esophageal, head and neck cancers, both from the animal models of interest and their human counterparts. These will be undertaken throughout the course of the project. The second component of the project will focus on the development of high-efficiency delivery systems for microRNA-based prevention and therapy in animal models, and in particular for the upper aerodigestive cancers of interest and the relative precancerous alterations in all center projects. Multiple delivery technologies will be comparatively developed and tested, drawing from the diverse platforms available to our CCNE.

HT RNAi phenotype profiling in human cancer cell lines
Spyro Mousses PhD. Senior Investigator & Director of Cancer Drug Development, Translational Genomics Research Institute, Gaithersburg MD

The ability to profile gene knockdowns in cell based screens has revolutionized the field of functional genomics, and accelerated the rate at which contextual vulnerabilities are discovered. The resources, infrastructure, and expertise that TGen has developed for HTS of large siRNA libraries that span the human genome will be discussed. This presentation will cover the state of the art in scanning the human genome with RNAi to discover genes that selectively kill cancer cells, targets that affect drug response, and genes that are synthetically lethal with cancer specific alterations.

Expression ArrestTM shRNA libraries: Solutions for whole genome RNAi screening
Troy Moore, PhD. Chief Technology Officer, Open Biosystems, Inc., Huntsville, AL
Expression ArrestTM whole genome human and mouse short hairpin RNA (shRNA) libraries from Open Biosystems are already cloned into retroviral vectors and are ready-to-use for gene silencing studies. shRNA constructs are expressed as microRNA-30 precursors which has been shown to greatly increase knockdown efficiency. This vector-based system is amenable to in vitro and in vivo applications such as the creation of stable knockdowns. Molecular barcodes are included in each shRNA vector enabling RNAi screens with pools of shRNA. Possible approaches for screening such whole genome RNAi collections will also be discussed. These include but are not limited to (1) screening individual shRNAs in multiwell format for activation or repression of a reporter or activity in a cell based or biochemical assay (2) Infecting cells with pools of shRNA followed by positive selection screens (3) Monitoring in mass transduced pools of cells, via molecular barcodes contained within the shRNAs, by microarrays containing the complement of barcode sequences to detect relative changes in shRNA representation following application of a selective stimulus.

Control of Cellular Gene with shRNA Expression Systems
Ron Herzig, Ph.D. Director, RNAi and Innovation Labs, Upstate and Chemicon International Inc. Charlottesville, VA

RNA interference has had a profound impact not only on the understanding of eukaryotic gene regulation mechanisms but also on the fields of functional genomics and drug discovery. In the recent years, siRNA and shRNA have been used as research tools for connecting individual genes to cellular phenotypes. Essential for the successful execution of these experiments ultimately is an assay to determine the effectiveness and physiological relevance of the targeted gene knock-down. Upstate has developed PathwayProfiler™ for determining the targeted effects of a protein's elimination on a signaling pathway. Combining Upstate's broad line of extensively qPCR validated, gene target-specific shRNA expression clones with PathwayProfiler™ enables rapid, quantitative and multiplexed analysis of a signaling pathway in a single sample. In order to determine not only the efficacy of the siRNA-mediated, targeted knock-down, but also the relevant downstream consequence, PathwayProfiler™ not only determines the amount of specific proteins present, but also assesses the extent of their phosphorylation in a multi-analyte format.

siRNA Delivery by Lentiviral Vectors
Inder M. Verma, PhD. Professor of Molecular Biology, Laboratory of Genetics, The Salk Institute, La Jolla, CA

A major challenge in the post-genomic era of biology is to decipher the molecular function of over 30,000 genes. The gene knockout by homologous recombination has proven to be very useful but is laborious and expensive. RNAi interference has recently emerged as a novel pathway that allows modulation of gene expression.

The delivery of synthetic siRNAs to cells in culture is hampered by limitations in transfection efficiency for many cell types and the transient nature of the silencing effect. In vivo, delivering siRNAs to target cells is difficult due to lack of stability of siRNA and low uptake efficiency in the absence of transfection agents. Thus in order to apply this potent technique to both basic biological questions and therapeutic strategies, efficient siRNA delivery methods must be developed.

During the past decade gene delivery vehicles based on HIV-1, the best characterized of the lentiviruses, have been developed. Lentiviral vectors derived from HIV-1 are capable of infecting a wide variety of dividing and nondividing cells, and integrate stably into the host genome, resulting in long-term expression of the transgene.

We will describe the use of lentiviral silencing vectors (LV-siRNA vectors) in vitro and in vivo. We will also describe the use of LV-siRNA vectors for gene knockdown, transgenesis, gene knockdown in vivo, transgenic animals with LV-siRNA, regulated vectors, and delivery to specific tissues.

Optimizing the Properties of siRNA to Maximize Their Utility in Therapeutic Discovery
William S. Marshall, Ph.D. Executive Vice President Research & Operations and Site Manager, Dharmacon Inc. Lafayette, CO

Gene silencing by siRNAs has emerged as an extremely useful technology to knock down expression of specific genes and allow for assessment of gene function. In addition, RNAi technology possesses a level of potency and specificity that makes its implementation as a therapeutic intervention strategy very appealing. While the application of the technology as an in vitro functional genomics tool has been well established, there are several challenges that need to be overcome before it can be considered a broadly reliable tool for animal model studies or as a viable therapeutic approach. The challenges facing siRNA are similar to those that any potential drug candidate would face: (a) ensuring highly potent target inhibition, (b) achieving appropriate target specificity, (c) assuring stability of the active drug in biological fluids, (d) directing distribution to the appropriate target organ, and (e) minimizing target-based or chemical class-based toxicity. We have developed strategies to address some of these issues and examples of the utility of these strategies will be presented.
The importance of design in maximizing the potency of siRNA.
Rapid identification of hyper-functional siRNA
Employing advanced cell-profiling technologies to understand siRNA mediated gene silencing
Strategies that can optimize the selectivity of gene silencing
Progress in applying siRNA knockdown experimentation to animals

And A Speaker From Roche Applied Sciences

Each speaker will have 20 min for presentation and 5 min for discussion.

Panel Discussion on May 3rd with experts from:
- Venture Capital Firm
- Technology Transfer Office
- Professional Science/Business Journalists
- Patent Attorney from a Law Firm
and selected speakers from the conference.


For Speaker/Sponsor/Exhibitor opportunities please contact:

GeneExpression Systems, Inc.
P.O. Box 540170
Waltham, MA 02454-0170 USA
Tel: (781) 891-8181
Fax: (781-891-8234
E-Mail: Genexpsys@expressgenes.com