Fifth International

RNAi-2007-Boston Meeting
on
RNA interference: Biochemistry to Drugs & Therapeutics

May 2-4, 2007
Venue: Hilton Garden Inn, 420 Totten Pond Road, Waltham, MA, 02451 USA

The Most Popular and The Best Global Event in the RNAi Field!

Target Audience: 200
Total Speaker Presentations: 30
Total Poster Presentations: 30
Total Exhibit Booths: 30

AGENDA/SPEAKERS

Click Here For Final Agenda

Wednesday, May 2, 2007
7:00 – 8:30 A.M: Registration Open
7:30 – 8:45 A.M: Breakfast
9:00 - 12:30 P.M: Scientific Session
2:00 – 6:00 P.M: Technology Session I (consists of 6 lectures)

Thursday, May 3, 2007
7:00 – 8:30 A.M: Registration Open
7:30 – 8:00 A.M: Breakfast
8:00 - 9:00 A.M: Technology Session II
9:00 - 1:00 P.M: Scientific Session
2:00 – 6:00 P.M: Scientific Session

Friday, May 4, 2007
7:00 – 8:00 A.M: Registration Open
7:30 – 8:00 AM: Breakfast
8:00 - 9:00 A.M: Technology Session III
9:00 - 1:00 P.M: Scientific Session
1:00 P.M Meeting Ends

The actual agenda will be updated. Please visit again.

Scientific Organizing Committee:

	Krishnarao Appasani, PhD., MBA (Chair) Founder & CEO GeneExpression Systems, Inc. Waltham, MA USA
	Christopher Burge, PhD. Professor of Biology Massachusetts Institute of Technology, Cambridge, MA, USA
	Dr. Anjana Rao, PhD. Senior Investigator at The CBR Institute for Biomedical Research Professor of Pathology, Harvard Medical School Boston, MA, USA
	Roberto Weinmann, PhD. Director of Oncology Discovery Bristol Myers Squibb, Princeton, NJ
	Eric Lader Ph.D. Associate Director, R&D QIAGEN Sciences, Germantown, MD, USA
	Richard Schultz, PhD. Patricia Williams Term Professor and Department Chair University of Pennsylvania Philadelphia PA, USA

Inaugural Speaker on May 2, 2007 AM (Lifetime Achievement Award)

	Dudley R. Herschbach, PhD. 1986 Chemistry Nobel Laureate Frank B. Baird, Jr. Professor of Science Department of Chemistry and Chemical Biology Harvard University Cambridge, MA USA Title:Action of DNA Polymerase: Importance of Nuptial Alignment (Molecular dynamics modeling of replication rates)

	Alexander, Rich, MD. William Thompson Sedgwick Professor of Biophysics Massachusetts Institute of Technology Cambridge, MA, USA Title: Discovery of double stranded RNA, hybridization and an early RNAi/microRNAs Prediction

Keynote Speaker on May 2, 2007 (RNAi Innovator Award)

Phillip D. Zamore, PhD. Gretchen Stone Cook Professor of Biomedical Sciences Department of Biochemistry & Molecular Pharmacology University of Massachusetts Medical School Worcester, MA, USA Title:Small RNAs: Past and Future

Troels Koch, PhD. Vice President of Drug Discovery and Manufacturing Santaris Pharma, Horshol, Denmark Title:Single Stranded RNA Interference

Other Speakers:

Richard Schultz, PhD.
Patricia Williams Term Professor and Department Chair
Department of Biology
University of Pennsylvania
Philadelphia, PA, USA
Title: RNAi, Dicer, and mouse oocyte development

Hristo B. Houbaviy, PhD.
Research Assistant Professor
Department of Genetics
Rutgers University
Piscataway, NJ 08854-8009
Title: MicroRNAs in stem cells.

Christopher Burge, PhD.
Professor
Department of Biology
Massachusetts Institute of Technology
Cambridge, MA, USA

Anjana Rao, PhD.
Senior Investigator at The CBR Institute for Biomedical Research
Professor of Pathology,Harvard Medical School
Boston, MA 02115, USA
Title: Drosophila RNAi screens applied to signaling pathways and to human disease

Jesse S. Boehm, Ph.D.
Broad Institute of Harvard and MIT
Cambridge, MA, USA
Title: Integrating functional approaches to identify cancer targets

Andrei Goga, M.D., Ph.D.
Assistant Professor of Medicine
University of California, San Francisco
San Francisco, CA, USA
Title: miRNA regulation of oncogenes and tumor suppressors

Eric Lader Ph.D.
Associate Director, R&D
QIAGEN Sciences, Germantown, MD, USA
Title: Innovative siRNA design and flexible plate layout for successful customized RNAi experiments

Alex Meissner, Ph.D.
Whitehead Institute for Biomedical Research
Massachusetts Inst. Of Technology
Cambridge, MA, USA
Title: TBA

Graham Brock, Ph.D.
Director of Target and Drug Discovery
Ordway Research Institute, Albany, NY USA
Title: Over-expression of the microRNA hsa-miR-200c leads to reduced expression of the transcription factor TCF8 and increased expression of E-Cadherin

Siew P Ho, PhD.
Oncology Discovery, Bristol Myers Squibb
Princeton, NJ 08543-4000
Title: Validation of drug targets through in vivo application of siRNA oligonucleotides

Daniel Anderson, Ph.D.
Fellow, Center for Cancer Research
Massachusetts Institute of Technology, Cambridge, MA USA

Paul D. Kassner, Ph.D.
Principal Scientist
Amgen Inc., South San Francisco, CA, USA
Title: siRNA Library Screening: Understanding the Problems to Realize the Potential

Zachary Zimmerman, PhD.
Director of External Alliances
Alnylam Pharmaceuticals
Cambridge, MA, USA
Title: RNAi therapeutics for emerging infectious disease and biodefense

Pascale Belguise, Ph.D.
Business Development Manager
PolyPlus-transfection, Illkirch, France
Title: Efficient and selective gene silencing in mammalian cells using picomolar siRNA concentrations

Nelson Lau, PhD.
Department of Molecular Biology
Massachusetts General Hospital
Boston, MA 02114, USA
Title: The Piwi-interacting RNA complex in animals

Caifu Chen, PhD.
Director, Assays/Arrays R&D
Applied Biosystems, Foster City, CA, USA
Title: Single ES cell profiling reveals unique microRNA expression signatures and heterogeneities

Stephanie Uder
Functional Genomics Product Manager
Sigma-Aldrich Company, St. Louis, MO, USA
Title: A Pilot Screen using viral delivery of shRNAs To Identify Factors Involved in Drug Sensitivity

Himanshu Brahmbhatt, Ph.D.
Joint-CEO & Director
EnGeneIC, Sydney, Australia
Title: Turning siRNA into a cancer therapeutic: Bacterially-derived non-living nanoparticles for the targeted delivery of siRNA and shRNA for potent anti-tumor effects in vivo

Troy Moore
Chief Technology Officer
Open Biosystems Inc., Huntsville, AL, USA
Title: A Validated Library of shRNAmir Clones Targeting Cancer Genes

Kathy L. Fosnaugh, PhD.
Scientist
Nastech Pharmaceuticals, Inc., Bothell, WA, USA
Title: Development and Delivery of RNAi Therapeutics Targeting Influenza

Brenton R. Graveley, PhD.
Associate Professor of Genetics and Developmental Biology
University of Connecticut Heath Center, Farmington, CT 06030-3705, USA
Title: MicroRNAs in the Planarian Schmidtea mediterranea: A Model System for Stem Cell Biology

Tod Woolf, PhD.
CEO & President, RXi Pharmaceuticals, Worcester, MA, USA
Title: Delivery of Intact RNAi in vivo

Meehyein Kim, Ph.D.
Scientist, Immunology and Virology group
Mogam Biotechnology Research Institute
Yongin-si, Kyonggi-do, 449-913, South Korea
Title: A novel approach to systemically deliver siRNA to the liver mediated by apolipoprotein A-I

Alexander Aristarkhov, PhD.
Director of Science & Technology
Exiqon, Woburn, MA, USA
Title: Novel principles of regulation of gene expression and unprecedented targets for detection

Amy C. Seila, PhD.
Postdoctoral fellow in the lab of Professor Phil Sharp
Center for Cancer Research, Massachusetts Institute of Technology
Cambridge, MA, USA
Title: Cloning of short RNAs from murine embryonic stem cells

Johannes Fruehauf, MD.
GI Cancer Laboratory, Beth Israel Deaconess Medical center
Harvard Medical School, Boston, MA, USA
Title: Bacteria deliver RNA Interference in vitro and in vivo for cancer therapy

And many more from Biotech and Large pharma…..

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

Panel Discussion on May 4 th 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.

Key Sessions:
The most-up-to-date developments will be addressed:
The Biochemistry and Genetics of RNAi and microRNAs
RNAi-their Molecular Mechanisms in various organisms
New vectors for RNAi delivery in vivo, in vitro
microRNA discovery and their diverse roles in biology
Computational and Disease Biology of miRNAs
Drug Target identification and Validation
High throughput genome-wide screenings
Innovative strategies to develop anti-viral, ocular and cancer therapeutics

Exhibitors are welcome to reserve their booth space early!

Please contact if you are interested in speaking in the scientific or Technology workshops of this meeting.

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

Poster Abstract Submission by April 20, 2007

All Abstracts

Action of DNA Polymerase: Importance of Nuptial Alignment
Dudley Herschbach, PhD. Department of Chemistry & Chemical Biology, Harvard University, Cambridge, MA, USA

Optical tweezer experiments have examined how tension applied to a partly single-stranded, partly double-stranded DNA chain affects the replication rate as a polymerase moves along the chain. The customary interpretation of such experiments had concluded that more than one nucleotide changed from ss to ds geometry in the transition state, even though only a single nucleotide is added to the primer strand in each catalytic cycle. That interpretation is not compatible with x-ray structural data for ternary complexes of a DNA polymerase with DNA primer/template and incoming nucleotide. This talk describes a simpler interpretation, consistent with the structural data and supported by an extensive molecular dynamics simulation. A key feature is the restricted range of angular orientations of the DNA segments adjacent to the active site of the enzyme.

Over-expression of the microRNA hsa-miR-200c leads to reduced expression of the transcription factor TCF8 and increased expression of E-Cadherin
Gregory J Hurteau1, J. Andrew Carlson2, Simon D Spivack3 & Graham J Brock1
1.Ordway Research Institute, 150 New Scotland Ave. Albany NY 12208
2. Department of Pathology, Albany Medical College, Albany, NY 12208
3. Human Toxicology & Molecular Epidemiology, NYS Dept of Health, Albany, NY 12201
MicroRNAs are ~22 nucleotide (nt) sequences that interact with multiple mRNAs resulting in either translational repression or degradation (1, 2). We previously reported that miR-200c, had variable expression in mammalian cell lines (3). Using a combination of bioinformatics and qRT-PCR we identified potential targets including the zinc finger transcription factor TCF8 (ZEB1, dEF-1) with expression levels inversely proportional to miR-200c (3). Knockout experiments using Anti-MicroRNA Oligonucleotides (AMOs) increased TCF8 levels but also had non-specific effects (3).Therefore to verify these target predictions we used ectopic or over-expression of miR-200c in select cells lines. The expression level of miR-200c in A549 (non-small cell lung cancer) cells is very low in contrast to normal human bronchial epithelial cells (3). Over-expression of miR-200c in these cells results in a loss of TCF8, an increase in expression of its regulatory target, E-cadherin (4) and altered cell morphology. In MCF7 (ER positive breast cancer) cells there is endogenous expression of miR-200c and E-cadherin but TCF8 is absent. Conversely, MDA-MB-231 (ER negative breast cancer) cells lack miR-200c and E-cadherin (reportedly due to promoter region methylation) but express TCF8. The ectopic expression of miR-200c in this cell line reduced levels of TCF8, restored E-cadherin expression and altered cell morphology. As the down regulation of E-cadherin is a crucial event in epithelial to mesenchymal transition (EMT) the loss of miR-200c expression could play a significant role in initiation of an invasive phenotype, and equally over-expression holds potential for its reversal.

1.Ambros V. microRNAs: tiny regulators with great potential. Cell 2001;107(7):823-6.
2.Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science 2001;294(5543):853-8.
3.Hurteau GJ, Spivack SD, Brock GJ. Potential mRNA degradation targets of hsa-miR-200c, identified using informatics and qRT-PCR. Cell Cycle 2006;5(17):1951-6.
4.Williams TM, Montoya G, Wu Y, Eddy RL, Byers MG, Shows TB. The TCF8 gene encoding a zinc finger protein (Nil-2-a) resides on human chromosome 10p11.2. Genomics 1992;14(1):194-6.

Innovative siRNA design and flexible plate layout for successful customized RNAi experiments & Efficient and specific quantification of mammalian micro RNAs by a novel real-time PCR approach
Eric Lader Ph.D. Associate Director, R&D, QIAGEN Sciences, Germantown, MD

Tobias Bergauer, Christian Bergmann, Martin Gossen, Ute Krueger, Bettina Haedrich, and Eric Lader*
QIAGEN GmbH, Hilden, Germany; *QIAGEN Sciences, Germantown, MD, USA

In recent years, RNAi has become the technology of choice for pathway analysis and drug discovery. The availability of siRNA design tools which provide siRNA that is both potent, effectively inducing silencing at low concentrations, and specific, resulting in minimal off-target effects, is critical for the success of RNAi experiments particularly when multiple siRNAs are used to knockdown groups of genes. In addition, increased use of RNAi has led to a demand for highly customized sets of siRNAs to suit specific research interests. We have used advances in the knowledge of the RNAi mechanism to improve our siRNA design algorithm to ensure design of potent and specific siRNA. siRNA is designed using a neural network and new design features include 3' UTR/seed region analysis, SNP avoidance, and interferon motif avoidance. We have created a user-friendly Web interface for the creation of fully customized sets of siRNAs at various scales to suit individual requirements. This enables creation of sets containing exactly the siRNAs and controls required for specific projects or follow-up screens. We describe the siRNA design process and the generation of customized sets.

Martin Kreutz, James Qin, Holger Engel, Po-Jen Shih, Subu Yerramilli, Eric Lader*
QIAGEN GmbH, Hilden, Germany; *QIAGEN Sciences, Germantown, MD, USA

We have developed an efficient and accurate method for automated, transcriptome-wide miRNA quantification by real time PCR using a SYBR green-based detection system. This method is very specific, sensitive and requires very small amounts of input RNA.  This method also allows researchers to simultaneously quantify miRNAs as well as mRNAs using the same cDNA preparation. A single cDNA preparation per sample is sufficient to quantify several miRNAs of interest, there by avoiding the need to prepare multiple cDNA preparations. Application of this technology to miRNA expression profiling will be discussed.

siRNA Library Screening: Understanding the Problems to Realize the Potential Paul D. Kassner, Ph.D. Principal Scientist, Amgen Inc., South San Francisco, CA USA

I will be using examples from screens we have performed at Amgen to illustrate some of the difficulties in developing screens, and the methods we use to overcome these issues.

Using RNAi screening to tackle malaria: from assay nightmare to therapeutic "golden nuggets"
Christophe J. Echeverri, Ph. D. Cheif Executive and Scientific Officer, Cenix BioScience GmbH, Dresden, Germany

The asymptomatic liver stage of Plasmodium infection offers a unique intervention point for the development of prophylactic anti-malarial agents that stop the disease before it reaches the devastating blood stage. A HT-RNAi screen was carried out to identify human liver genes that are non-essential to the host but necessary for infection. Over 800 human genes were assayed for their involvement in the infection of Huh7 hepatoma cells by Plasmodium berghei sporozoites freshly isolated from infected mosquitoes. This led to the identification of several target candidates, two of which were further investigated, and will be discussed at the meeting.

Integrating functional approaches to identify cancer targets
Jesse S. Boehm, Ph.D. Research Scientist, Broad Institute of Harvard and MIT, Cambridge, MA, USA

Nearly all epithelial malignancies exhibit numerous karyotypic abnormalities. This complexity of genome structure hampers efforts to identify relevant mutations important for cancer development. However, the recent development of several complementary experimental approaches now provides the tools necessary to systematically deconvolute the complexity of somatic cancer genetics. We have initiated a pilot project to identify and validate cancer targets with high promise for clinical translation in a comprehensive, genome-wide manner. By integrating data from RNA interference screens with analysis of whole genome structure and the unbiased identification of transforming kinases we have identified IKBKE as an oncogene that activates the NF-kB pathway in breast cancer.

Efficient and selective gene silencing in mammalian cells using picomolar siRNA concentrations
Pascale Belguise, PhD. Business Development Manager, Polyplus-transfection SA, BIOPARC, Illkirch, France

Prevention of unwanted side effects induced by siRNA can be done by lowering the siRNA concentration used. Polyplus-transfection has developed a novel delivery reagent which offers perspectives to silence genes within the picomolar range of active siRNA. Furthermore, this siRNA transfection reagent is perfectly suitable for reverse transfection procedure and automated RNAi experiments both in 96- and 384-well-plates. Moreover, our delivery system, compatible with serum and antibiotics, shows excellent cell viability. A summary of the various data obtained with this product and the technical advantages will be presented.

Single ES cell profiling reveals unique microRNA expression signatures and heterogeneities
Caifu Chen, PhD. Director, Assays/Arrays R&D, Applied Biosystems, Foster City, CA, USA

We describe a new method for simultaneously quantifying 237 mouse microRNAs (miRNAs) and 21 messenger RNAs (mRNAs) from single embryonic stem (ES) and differentiated cells. The method is based on multiplex RT, multiplex preamplification, and singleplex real-time TaqMan® PCR assays.  Assays are quantitative for a dynamic range of at least three logs.  Single cell expression signatures could classify individual ES, embryoid body (EB), or somatic cells. Significant inter-cell variations of both miRNA and mRNA expression were observed within ES cell lines, indicating the heterogeneity of ES cells. The highest variability was observed among EB cells (CV = 139%), demonstrating that EB cells undergo differentiation at different stages.  Interestingly, ES marker gene Oct4 and signaling gene Tdgf1 were co-expressed. Both were absent in 3T3 and splenocyte cells, highly expressed in ES cells, and significantly reduced in EB cells.  Furthermore, there was no correlation in the expression levels between miRNAs and their predicted target mRNAs, thereby supporting a translational repression model.  The total number of expressed miRNA genes in ES, EB, and somatic cells remained constant. However, their expression levels were significantly elevated during differentiation, further suggesting the involvement of miRNAs in cellular development and specification.  Our results provide new insight into both miRNA and mRNA expression patterns at the single cell level.

Single Stranded RNA Interference
Troels Koch, MSc, Ph.D., Vice President Drug Discovery & Manufacturing, Santaris Pharma A/S, Denmark

In recent years the interest for oligonucleotide drugs has increased significantly. New and improved oligonucleotide analogues - like Locked Nucleic Acid - have reached the clinical stage and opened new perspectives for the interference with RNA expression. Lately, microRNA has been recognised as a new important class of targets in pathogenesis and that has further expanded the therapeutic scope of oligonucleotides. In contrast to siRNA, single stranded LNA can potently interfere with the expression of both mRNA and microRNA. The presentation will cover: (1). An introduction to LNA, (2). Comparisons with other chemistries, (3). Uptake, distribution and tox profile of LNA, (4). Potency and efficacy in targeting mRNA and microRNAs.

Turning siRNA into a cancer therapeutic: Bacterially-derived non-living nanoparticles for the targeted delivery of siRNA and shRNA for potent anti-tumor effects in vivo
Himanshu Brahmbhatt, PhD. Joint CEO, EnGeneIC Pty Ltd, 105 Delhi Road, North Ryde, Sydney, Australia,

Jennifer A. MacDiarmid, Jocelyn C. Weiss, Nancy B. Mugridge, Alison Ginn, Leo Phillips, Kristie-Ann Dickson, Stephanie Wetzel, Vatsala N. Brahmbhatt, Alexander C. James, Scott T. Pattison, Himanshu Brahmbhatt.
EnGeneIC Pty Ltd, 105 Delhi Road, North Ryde, Sydney, Australia,

One of the major hurdles facing the development and therapeutic application of siRNA and small molecule therapeutics in oncology is tumor cell-specific delivery in-vivo. Additionally, the current systemic administration of chemotherapeutic drugs results in indiscriminate drug distribution and severe toxicity. siRNAs directed against intracellular tumor targets such as polo kinase 1 as well as drug resistance mediating proteins such as Multidrug resistance protein 1 (MDR1) have been shown to exhibit direct anti-tumor effects and reverse drug resistance in-vitro respectively.

Here we report a technology potentially overcoming these shortcomings through encapsulation and cancer cell-specific targeting of siRNAs, shRNAs, molecularly targeted drugs and chemotherapeutic drugs in bacterially-derived 400nm sized nanoparticles abbreviated as EDVs (EnGeneIC Delivery Vehicle). We discovered that EDVs can be packaged with therapeutically significant concentrations of siRNAs as well as chemotherapeutic drugs of differing charge, hydrophobicity and solubility. Targeting of EDVs via bispecific antibodies (BsAbs) to receptors on cancer cell membranes results in endocytosis, intracellular degradation and release of the payload. Intact and fully functional siRNAs are released intracellularly to achieve target mRNA knockdown resulting in highly significant anti-tumor effects as well as drug resistance reversal. Similar in-vivo results have also been obtained following delivery of shRNAs. Mouse xenograft studies show that i.v. administration of 109 bispecific antibody targeted EDVs packaged with siRNAs directed against proteins such as Kinesin spindle protein (KSP) or polo kinase 1 (Plk1) results in highly significant anti-tumor effects.

Additionally, xenografts established with drug resistant human tumors could be effectively treated using a dual treatment protocol where the initial dose carries BsAb-targeted EDVs packaged with siRNA against MDR1 protein followed by BsAb-targeted EDVs packaged with a chemotherapeutic drug to which the tumors were initially highly resistant. This effect was achieved with several thousand-fold less drug administered via EDVs compared to free drug therapy. Various doses of monkey EGFR-targeted, doxorubicin-packaged EDVs have been tested in two separate rhesus monkey toxicity trials and the results show no signs of toxicity despite five repeat doses. The antibody response to the major EDV-surface exposed antigen, the O-polysaccharide, was also found to be very weak and short lived. The versatility, safety and efficacy of the EDV potentially paves the way for tailor-made cancer therapy where targeted EDVs carrying siRNAs and/or drugs can be mixed and matched for individual patient needs.

A Pilot Screen using viral delivery of shRNAs To Identify Factors Involved in Drug Sensitivity
Stephanie Uder, Functional Genomics Product Manager, Sigma-Aldrich Chemical Company, St. Louis, MO, USA

RNA interference methodologies have emerged as extremely effective tools for dissecting and understanding gene function. Through the utilization of a recombinant lentivirus delivery system, the RNAi Consortium (TRC) shRNA library targets cell types that are often not amenable to synthetic siRNA transfection, and facilitates screens and experiments that require long-term knockdown that cannot be achieved by synthetic siRNA. We will discuss current validation efforts of this library to demonstrate its applicability in a broad array of cell types. To further explore its utility in screening, we set out to develop strategies using the lentiviral-based shRNA libraries in larger scale silencing projects. Pilot screens using a tumor suppressor gene family set were used to screen for genes that could enhance cell sensitivity to the widely used cancer therapy drug, Paclitaxel. This screen was performed to address biologically relevant questions while simultaneously developing screening strategies that could be used by researchers in the field.

RNAi, Dicer, and mouse oocyte development
Richard Schultz, PhD. Patricia Williams Term Professor and Department Chair, Department of Biology, University of Pennsylvania, Philadelphia PA, USA

The RNAi pathway operates in mouse oocytes, as assessed by the ability of long dsRNA to target endogenous mRNAs, including dormant maternal mRNAs, in a highly specific manner. In contrast to most somatic cells that mount an interferon response initiated by long dsRNA, oocytes (and pre-implantation embryos) lack an interferon response because they do not express genes in the interferon response pathway. Moreover, there appears little off targeting in response to the siRNA generated by Dicer-mediated processing of long dsRNA. Oocyte-specific ablation of Dicer has unmasked an essential function for Dicer in oocyte maturation, because Dicer-deficient oocytes exhibit multiple disorganized spindles and severe chromosome congression defects. In addition, Dicer function is critical for degradation of a population of maternal mRNAs that are normally degraded during the course of maturation.

MicroRNAs in the Planarian Schmidtea mediterranea: A Model System for Stem Cell Biology
Brenton R. Graveley, PhD. Associate Professor of Genetics and Developmental Biology
University of Connecticut Heath Center, Farmington, CT, USA

Planarians are bilaterally symmetric metazoans that have the unique ability to completely regenerate lost tissues or organs. This regenerative capacity is facilitated by a population of stem cells known as neoblasts. Planarians are therefore an excellent model system for studying many aspects of stem cell biology. We have recently reported the cloning and characterization of 71 miRNAs from the planarian Schmidtea mediterranea. To identify miRNAs that are regulated during regeneration, we have used high-throughput pyrosequencing to examine the miRNA expression profiles in intact and regenerating animals. I will discuss our progress in this area.  This work lays the foundation for functional studies aimed at addressing the role of these miRNAs in regeneration, cell lineage decisions, and basic stem cell biology.

Development and Delivery of RNAi Therapeutics Targeting Influenza
Kathy Fosnaugh, PhD.Scientists, Nastech Pharmaceutical Company, Bothell, WA, USA 

Kathy Fosnaugh, Shaguna Seth, Mary Prieve, Diane Frank, Michael Houston, Sharin Roth, Mohammed Ahmadian, Dong Liang, Greg Severson, Brian Granger, Oleksandr Baturevych, Kunyuan Cui, Mike Templin, Paul H. Johnson and Steven C. Quay
Nastech Pharmaceutical Company, Bothell, WA, USA 

Virulent influenza strains such as H5N1 pose a significant worldwide health risk. We are developing RNAi therapeutics to treat influenza A viral infections by selecting potent siRNAs that target 89-99% of the published influenza strains.   Five potent siRNAs (IC50 20-500 pM), directed against the NP, PB1, PB2, or PA genes, have been selected and shown to reduce viral replication in vitro and in vivo.  Treatment with a candidate siRNA before or after infection resulted in a 10-fold or greater reduction in viral titer demonstrating its prophylactic and therapeutic treatment potential. Nasal administration of formulated siRNA significantly reduced virus production in mice.  Formulation optimization will enable lower doses, reduced dosing frequency and longer duration of effect.

Validation of drug targets through in vivo application of siRNA oligonucleotides
Siew Peng Ho, PhD., Principal Scientist, Applied Genomics, Bristol Myers Squibb, Princeton, NJ, USA

RNA interference has proven to be a rapid, reliable and robust method for conducting knockdown studies in cell-based systems. The ability to extend this powerful technology to animal models will facilitate rapid validation of disease related drug targets, and help identify potential mechanism-based toxicities related to these drug targets. In vivo experiments conducted using two different approaches, cholesterol-conjugated siRNAs versus siRNAs encapsulated in a liposomal formulation will be described.

Delivery of Intact RNAi in vivo
Tod Woolf, PhD. CEO & President, RXi Pharmaceuticals< Worcester, MA, USA
 
Data will be presented demonstrating the delivery of active RNAi to the mouse liver and other tissues with nanotransporters.  RNAi compounds are readily formulated with the nanotransporters and activity is obtained at doses of 1mg/kg.  Data will also be presented demonstrating knock down of SOD1 in the spinal column in a mouse ALS model.  Lastly, the RNAi validation of RIP140 as a target gene for obesity will be described.  RIP140 is a transcription repressor, that when inhibited by RNAi, activates metabolism in adipocytes.

Determinants of Targeting by Endogenous and Exogenous MicroRNAs
Christopher Burge, PhD., Associate Professor, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

miRNAs frequently target mRNAs for repression through mechanisms involving pairing to miRNA bases 2-7. We found that 7mers matching miRNA bases 2-8 or matching miRNA bases 2-7 followed by an adenosine were associated with stronger mRNA downregulation than 6mer seed matches, and that 8mers matching miRNA bases 2-8 followed by adenosine had roughly twice the downregulation effect of these 7mers. This ordering and the relative magnitudes of 7mer and 8mer seed match effects were observed consistently in data from miRNA and siRNA over-expression studies and from experiments reducing endogenous miRNA levels by knockout of Dicer. Evolutionarily conserved miRNA target genes are enriched for additional targeting determinants. Presence of adenosine or uridine opposite miRNA base 9 (t9W) as such a determinant is supported by analyses of both miRNA/siRNA transfection data and UTR sequence conservation. These results aid in prediction of miRNA targets in the absence of sequence conservation.

A Validated Library of shRNAmir Clones Targeting Cancer Genes
Troy Moore, Chief Technology Officer, Open Biosystems Inc., Huntsville, AL, USA

From a whole genome library of human lentiviral shRNAmir clones, we have derived a cancer specific array of 135 genes represented by 3 shRNAmir clones per gene.  Lentivirus was prepared in a multi-well format and the knock-down efficiency measured via quantitative PCR in two cancer cell lines, MCF-7 & OVCAR-8.  While cell line differences are readily apparent, results indicate the shRNAmir lentiviral clones have a high level of knock-down activity.  The validation process and analysis of the resulting data will be presented.

Novel principles of regulation of gene expression and unprecedented targets for detection
Alexander Aristarkhov, PhD. Director of Science & Technology, Exiqon, Woburn, MA, USA

The regulation of gene expression in humans has unexpectedly become an unknown field. The breadth and importance of global regulators is overlooked in Drug Discovery and Development. We will discuss accuracy and precision requirements in the detection of emerging and unprecedented targets – small regulatory RNAs and their interaction with target mRNAs. Clearly those requirements are critical for reliable data collection. LNA is a high affinity nucleic acid analogue that enables the development of short oligonucleotide probes with required affinities for miRNAs. The application of LNA-based probes in cross-validating platforms has been truly enabling for the exploration of the microRNA field, from discovery to function in cells, in a step-by-step manner.

MicroRNAs in Stem Cells
Hristo B. Houbaviy, PhD., Assistant Professor, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Stratford, NJ, USA

At the molecular level, stem cell maintenance and differentiation depend on a complex network of mechanisms that control gene expression. While transcription factors that govern key aspects of stem cell biology (e.g., Oct-4 and Nanog in embryonic stem (ES) cells) have been identified, very little is known about the role of microRNA (miRNA) dependent post-transcriptional control. Direct short RNA cloning led to the identification of miR-290-295 as the most abundant miRNAs in mouse ES cells. These miRNAs are encoded by a cluster of six pre-miRNA hairpins with related sequences and appear to be ES cell and pre-implantation embryo-specific by the following criteria: (1) their sequences are unrelated to any previously described miRNAs, including the extensive collection of miRNAs cloned from adult mouse organs; (2) they can not be detected in adult mouse organs with northern blots; (3) they are repressed during ES cell differentiation in vitro; and (4) all ESTs that map within the miR-290-295 cluster are derived from ES cells and pre-implantation embryos. Sequence analysis shows that this Early Embryonic miRNA Cluster (EEmiRC) is remarkably variable in different mammalian orders and can only be identified bioinformatically in placental (eutherian) mammals. This result fits nicely with the fact that pre-implantation development is a uniquely eutherian feature and suggests that EEmiRC may be involved in the differentiation and/or maintenance of the extraembryonic lineages. Consistent with eutherian specific function we have detected the EEmiRC miRNAs in trophoblastic stem (TS) cells. However, preliminary data indicate that EEmiRC is not expressed in mature placenta. Thus, EEmiRC is likely to play a role very early in development, perhaps as early as pre- or peri- implantation. Since ES and TS cells are derived from the blastocyst inner cell mass and trophoblast, respectively, it is tempting to speculate that EEmiRC controls the initial differentiation of these lineages or of cell types directly derived from them.

Delivering RNAi Therapeutics
Zachary Zimmerman, PhD., Alnylam Pharmaceuticals Inc., Cambridge, MA, USA

RNA interference (RNAi) holds significant promise as a therapeutic approach to silence disease-causing genes. The key hurdle for RNAi therapeutics is delivery needed to achieve in vivo gene silencing. A critical requirement for RNAi therapy is the introduction of siRNAs into appropriate organs and/or tissues, targeting to specific cell types, and entry into the cytoplasm. To achieve delivery certain “drug-like” properties such as chemical modifications, formulations, etc., are being investigated. Our progress in achieving in vivo silencing with siRNAs will be discussed.

miRNA Regulation of Oncogenes and Tumor Suppressors
Andrei Goga, M.D., Ph.D., Assistant Professor of Medicine, University of California, San Francisco, San Francisco, CA, USA

Micro-RNAs are a class of small non-coding RNAs that can regulate cell differentiation and are deregulated in a variety of human cancers.  We are interested in identifying miRNAs that regulate oncogenes or tumor suppressor genes. We have generated a human miRNA retroviral expression library that permits functional screens for miRNAs that play a direct role in tumorigenesis. We have found that miR-125a/b can function as tumor suppressors by regulating ERBB2/3 expression.

The Piwi-interacting RNA complex in animals
Nelson C. Lau, PhD., Post doctoral Fellow from the Laboratory of Professor Robert Kingston, Dept. of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA

RNA interference pathways control gene expression by post-transcriptional and transcriptional gene silencing mechanisms. While somatic cells in animals utilize the Argonaute proteins and microRNAs to downregulate target mRNAs post-transcriptionally, the germ cells express a distinct subfamily of Argonaute proteins, the Piwi proteins, and an abundant, unique class of Piwi-interacting RNAs, or piRNAs. From rat testes, we identified and characterized a Piwi-interacting RNA complex (piRC), which includes piRNAs, Riwi, the rat homolog to Piwi, and rRecQ1, which is homologous to qde-3 from Neurospora. The biochemical activities and genetics link Piwi and rRecQ1 to gene silencing pathways. The piRNAs, frequently 29–30 nucleotides in length, map to 100 defined (<100 kb) genomic regions. Within these regions, the piRNAs generally distribute across only one genomic strand, or distribute on two strands but in a divergent, non-overlapping manner. piRNAs also contain a 3’ modification not present in microRNAs. Since piRC features appear distinct from Argonautes and microRNAs, the mechanism of action for this ribonucleoprotein may also be unique, perhaps functioning at the level of transcriptional silencing or genome surveillance. Although recent progress has suggested a role for piRC in silencing transposons, a direct mechanistic function for piRC remains to be determined. I will discuss our efforts to biochemically dissect piRC function by exploring in vitro systems containing piRC components.

s GI Cancer Laboratory, Beth Israel Deaconess Medical center, Harvard Medical School, Boston, MA, USA

Other Authors: J.H.Fruehauf, S.Xiang, H.Guo, T.Nguyen, Y.Yang, C. J. Li

Background: RNA Interference (RNAi) is a promising tool for both discovery and therapy. The first clinical trials are already being conducted with RNAi-based therapies. Targeting oncogenes required for cancer maintenance may become a successful tool for future cancer therapy. Delivery of RNAi remains a great challenge, which needs to be solved before the broad potential of this novel technology can be harnessed for clinical use.

Hypothesis: We hypothesized that bacteria could express hairpin RNA and deposit them in target cells, triggering RNA interference.

Methods: We designed a plasmid, called TRIP (Transkingdom RNA Interference Plasmid) which confers three novel properties to laboratory strains of E.Coli:
Hairpin expression cassette (with unique restriction sites allowing for the rapid switch between different siRNA targets)
Invasin (inv) expression cassette. This bacterial gene is derived from Yersinia enterocolitica and triggers the uptake of the bacteria into epithelial cells
Listeriolysin O (hly) expression cassette. This bacterial gene is derived from Listeria monocytogenes. It functions to destroy the endosome in which the bacteria are contained following invasion, to allow the bacterial content to reach the host cell cytoplasm

Results: We show that this method, called “transkingdom RNA Interference” (tkRNAi) is able to trigger specific and potent gene silencing in a variety of cultured cell lines. tkRNAi was used to knock down cancer specific oncogenes (beta-catenin and k-ras) and significantly inhibited colon cancer cell proliferation in vitro.
In a xenograft model of colon cancer, silencing of beta-catenin with tkRNAi lead to a significant decrease in the fraction of proliferating cells (PCNA stain). Beta-catenin expression was found suppressed in tumors of treated animals on both mRNA and protein levels, and tumor growth was decreased by more than 50%.

Conclusion: We conclude that tkRNAi is a useful method for the targeted delivery of therapeutic RNAi in vitro and in vivo and merits further research efforts.