Sixth International

RNAi, MicroRNAs -2008-Boston Meeting

on

RNA interference & MicroRNAs: Biochemistry to Drugs & Therapeutics

April 7 - 8, 2008

Venue: Hilton Garden Inn, 420 Totten Pond Road, Waltham, MA, 02451 USA

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

This Year RNAi Meeting Will Be in Conjunction with Stem Cells-2008 Meeting. Two Tracks, One Venue

AGENDA/SPEAKERS

Click Here For Detailed Agenda

Monday, April 7, 2008

7:00 – 8:30 A.M: Registration Open
7:30 – 8:45 A.M: Continental breakfast
9.00 AM – 6.00 PM Scientific Sessions

Tuesday April 8, 2008

7:00 – 8:30 A.M: Registration Open
7:30 – 8:45 AM: Continental breakfast
8.00 AM – 4.00 PM Scientific Sessions

	Krishnarao Appasani, PhD., MBA (Chair) Founder & CEO GeneExpression Systems, Inc Waltham, MA USA
	Joanne B. Weidhaas, M.D., Ph.D. Assistant Professor of Therapeutic Radiology Yale University School of Medicine New Haven, CT, USA Title: MicroRNAs in Cancer Therapy
	Roberto Weinmann, PhD. Director of Oncology Discovery Bristol Myers Squibb Princeton, NJ, USA
	Mehmet Fatih Yanik, PhD. Assistant Professor of Electrical Engineering Massachusetts Institute of Technology Cambridge, MA, USA Title: Microfluidic chip based large-scale screening for RNAi

Other Speakers:

	Keynote Speaker on April 7 Craig P. Hunter, PhD. Professor of Molecular and Cellular Biology Harvard University Cambridge MA, USA Title: Double strand RNA delivery and the mechanism of miRNA inhibition of translation
	Keynote Speaker on April 8 PM Harvey F. Lodish, PhD. Member, Whitehead Institute Professor of Biology & Bioengineering Massachusetts Institute of Technology Cambridge, MA, USA Title: microRNAs that regulate hematopoiesis and adipocyte differentiation
	Industry Keynote Speaker on April 8th AM Kieth McCullagh, PhD. President & CEO Santaris Pharma Denmark
	Tod Woolf, PhD. President & CEO RXi Pharmaceuticals, Inc. Worcester, MA USA
	Rachel Meyers, PhD. Senior Director of RNAi Lead Development Alnylam Pharmaceuticals, Inc. Cambridge, MA, USA
	Klaus Rajewsky, M.D. Senior Investigator at CBR Institute Fred S. Rosen Professor of Pediatrics and Professor of Pathology Harvard Medical School Boston, MA, USA Title: MicroRNAs in Immunity
	James J. Collins, PhD. University Professor & Professor of Biomedical Engineering Co-Director, Center for BioDynamics Boston University Boston, MA, USA Title: RNAi Synthetic Biology & Systems Biology
	Feng Guo, Ph.D. Assistant Professor of Biological Chemistry UCLA School of Medicine, Los Angeles, CA, USA Title: Heme is require for efficient microRNA processing in human cells
	Thomas F. Duchaine PhD. Assistant Professor of Biochemistry McGill University & McGill Cancer Center  Montreal, Quebec, Canada
	Dong-ki Lee, PhD. Associate Professor Sungkyunkwan University Suwon, South Korea Title: Efficient and specific gene silencing triggered by siRNAs shorter than 19 base pairs
	Li Ma, PhD. Life Sciences Research Foundation Fellow Dr. Robert Weinberg’s Lab, Whitehead Institute Massachusetts Institute of Technology Cambridge, MA, USA Title: miRNAs in malignant progression
	Clark Chen, MD., PhD. Post Doctoral Fellow in the Lab of Prof. Alan D’Andrea Department of Radiation Oncology Dana-Farber Cancer Institute & Harvard Medical School Boston, MA, USA Title: TBA
	Claes Wahlestedt, M.D., Ph.D. Professor & Director of Neuroscience Discovery The Scripps Research Institute - Scripps Florida Jupiter, FL, USA Title: Knockdown Strategies to Study the Functions of Non-Coding RNAs
	Sven Diederichs. Ph.D Massachusetts General Hospital Cancer Center Charlestown MA, USA Title: Argonaute proteins in microRNA processing and regulation
	R. Chris Wilkins, PhD. President & CEO Genovis Inc. USA Title: TBA
	Robyn Hickerson, PhD. Senior Scientist TransDerm, Inc., Santa Cruz, CA, USA Title: Development of mutation-specific therapeutic siRNAs for treatment of skin disorders
	David H. Dreyfus M.D., Ph.D. Founder & Medical Director Keren Pharmaceuticals, Inc.& Assistant Clinical Professor in Pediatrics Yale School of Medicine, New Haven, CT, USA Title: Potential synergy between gene silencing with RNAse P and siRNA
	Anders Høgset, PhD. Research Director PCI Biotech AS, Oslo, Norway Title: TBA
	Attila Seyhan, PhD. Open Biosystems, Inc Huntsville, AL, USA Title: Lentiviral human primary microRNA library for in vitro and in vivo gain-of-function studies
	Jianzhu Chen, PhD. Cottrell Professor of Biology & Immunology Center for Cancer Research Massachusetts Institute of Technology Cambridge, MA, USA Title: RNAi in influenza and mechanisms of siRNA delivery

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 March 6, 2008

ALL ABSTRACTS

MicroRNAs in Cancer Therapy
Joanne B. Weidhaas, M.D., Ph.D., Assistant Professor, Department of Therapeutic Radiology
Yale University School of Medicine, New Haven, CT, USA

MicroRNAs are commonly misregulated in cancer, however, the impact of this misregulation in cancer is just being understood. Several studies show that microRNA levels can predict outcome in certain cancer types, and we have found that microRNAs are also involved in the cellular response to cancer therapy, and can be manipulated to alter this response to enhance tumor cure.

RNAi Synthetic Biology & Systems Biology
James J. Collins, PhD. University Professor & Professor of Biomedical Engineering
Co-Director, Center for BioDynamics Boston University, Boston, MA 02215, USA

In this talk, we describe how we developed within the context of synthetic biology a tunable, modular mammalian genetic switch that can be used to effectively turn any gene off. We engineered the switch by creating a synthetic gene network that couples repressor proteins with a novel RNAi design. We discuss how this work establishes a system for tight, tunable control of mammalian gene expression that can be used to explore the functional role of various genes as well as to determine whether a phenotype is the result of a threshold response to changes in gene expression. In addition, we present integrated computational-experimental approaches that enable construction of quantitative models of gene-protein regulatory networks using expression measurements and no prior information on the network structure or function. We discuss how the reverse-engineered network models, coupled to experiments, can be used to identify (1) the pathways and gene products targeted by RNAi and/or therapeutic compounds and (2) the genetic mediators of different diseases.

Heme is require for efficient microRNA processing in human cells
Feng Guo, Ph.D., Assistant Professor, Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA 90095, USA

During development and in tumors, the processing efficiency of miRNA primary transcripts (pri-miRNAs) is decreased. However, little is known about how this processing step is regulated. We previously reported a surprising finding that DGCR8, an RNA-binding protein required for cleavage of pri-miRNAs by the Drosha nuclease, is a heme-binding protein. The heme-bound DGCR8 is more active in pri-miRNA processing in vitro than the heme-free monomer. Here we show that heme-binding deficient DGCR8 mutants are incapable of supporting efficient pri-miRNA processing in HeLa cells, using a knockdown-and-rescue approach. Our data suggest that heme is required for efficient miRNA maturation in humans.

Argonaute proteins in microRNA processing and regulation
Dr. Sven Diederichs, Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown MA, USA

From my perspective, the discovery of novel mechanisms in microRNA processing and regulation is of immediate interest to the entire field of RNA interference since these mechanisms also allow conclusions and a deeper understanding of experimental RNAi and processing of synthetic shRNA.

Efficient and specific gene silencing triggered by siRNAs shorter than 19 base pairs
Dong-ki Lee, PhD. Associate Professor, Sungkyunkwan University Suwon, South Korea

Chan Il Chang1, Jae Wook Yoo1, Sun Woo Hong2, Hye Suk Kang2, Changill Ban1, Soyoun Kim3, and Dong-ki Lee2,*
1Department of Chemistry, Pohang University of Science and Technology, Pohang, Korea.
2Department of Chemistry, Sungkyunkwan University, Suwon, Korea
3Department of Chemistry, Dongguk University, Seoul, Korea.

Specific gene silencing by RNA interference can be readily achieved with the use of small interfering RNAs (siRNAs). An earlier study with Drosophila extracts revealed that the most effective siRNA structure consists of a 19—base pair duplex and 2-nucleotide 3’-overhangs (the 19+2 siRNAs), whereas siRNAs shorter than 19 base pairs (bp) are inefficient silencers. Here we report on the identification of siRNA structures with duplexes shorter than 19 bp that can efficiently trigger gene silencing in human cell lines. We also show that the shorter siRNA structures ameliorate the non-specific effects triggered by the 19+2 siRNAs, such as sense strand-mediated off-target silencing and saturation of RNAi machineries. Therefore, these novel siRNA structures have advantages over currently used 19+2 siRNA structures in functional genomics and therapeutic applications.

Development of mutation-specific therapeutic siRNAs for treatment of skin disorders
Robyn P. Hickerson, PhD., Senior Scientist, TransDerm, Inc., Santa Cruz, CA, USA

We have identified a siRNA (TD101) that is highly specific for a single nucleotide mutation (N171K) in the keratin 6a (K6a) gene in tissue culture and in animal studies. This mutation is responsible for the dominant negative skin disorder pachyonychia congenita (PC), characterized by thickened and dystrophic nails and hyperkeratosis/blistering on the soles of the feet. A double-blind Phase 1b clinical trial (to determine safety) treating PC foot lesions of eligible and willing U.S. patients harboring K6a(N171K) has begun. This siRNA clinical trial is the “first-in-man” for skin treatment as well as the first to target a mutant gene.

Potential synergy between gene silencing with RNAse P and siRNA
David H. Dreyfus M.D., Ph.D. Founder & Medical Director, Keren Pharmaceuticals, Inc. & Assistant Clinical Professor in Pediatrics, Yale University School of Medicine, New Haven, CT, USA

David H. Dreyfus, MD/PhD. Founder & Medical Director,
Lucy Ghoda, PhD, Scientific Director Keren Pharmaceuticals, Inc. New Haven, CT

RNAse P, a ubiquitous RNA enzyme can be used for targeted cleavage of messenger RNA or other RNA targets in combination with a small nucleotide molecule termed an external guide sequence (EGS). An EGS is designed to form a structure resembling transfer RNA precursor, the natural substrate of RNAse P. EGS can be expressed in the cell as RNA, or introduced in small chemically synthesized forms including nuclease resistant RNA, or DNA. In this presentation, the biology of RNAse P will be reviewed briefly with particular emphasis on use of RNAse P in synergy with SiRNA for therapy of respiratory diseases such as influenza and asthma.

Another potential application of RNAse P gene silencing is anticipated to be for knockdown of endogenous micro-RNA. Since RNAse P is resident in the cellular nucleus potentially micro-RNAs could be intercepted before export and processing in the cytoplasm. Preliminary efforts to target micro-RNA MIR-155, a micro-RNA playing a critical role in the immune inflammatory response are described.

Keren pharmaceutical (kerenpharm.com), a seed stage biotechnology company is interested in cooperative and collaborative ventures with researchers and other biotechnology ventures with developing platforms in gene silencing. Gene silencing with RNAse P and EGS may have significant advantages relative to other gene silencing technologies including speed of onset and duration of therapy or in novel applications such as silencing of micro RNA.

Photochemical Internalisation – light-induced siRNA delivery.
Anders Høgset, PhD., Research Director, PCI Biotech AS, Oslo, Norway

Photochemical internalisation (PCI) is a technology for light-induced release of endocytosed molecules into the cell cytosol, and can be used for site-specific in vivo delivery of many different molecules. The therapeutic efficiency of PCI has been demonstrated in animal tumour models with cytotoxic drugs, proteins and genes, and the first PCI clinical study will start in 2008. PCI can significantly improve siRNA delivery mediated by various vectors. In this presentation recent results on the use of PCI for siRNA delivery, and the future potential for PCI in delivery of RNA therapeutics, will be discussed.

Argonaute proteins in microRNA processing and regulation
Sven Diederichs, PhD. Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown MA 02129-2020, USA

Argonaute proteins are well known as effector proteins of the RNA interference and microRNA pathways. Human Ago2 is the RNase slicer of the RISC cleaving the siRNA-targeted mRNA. Here, two novel functions of human Argonaute proteins are identified: First, all human Argonaute proteins enhance mature microRNA expression and thus are post-transcriptional regulators of microRNA expression. In addition, Ago2 actively participates in microRNA processing and cleaves the pre-miRNA hairpin in the passenger strand generating a novel nicked precursor, the ac-pre-miRNA. In summary, Ago proteins play a role in microRNA processing, regulation and in their effector phase and hence are prime candidates to coordinate microRNA biogenesis and function. Diederichs & Haber, Cell (2007) 131: 1097-1108.

Transfection using Magnetic Nanostructures
Chris Wilkins, PhD., President & CEO, Genovis Inc.
  The characteristics of supermagnetic nanoparticles make them well suited for biomedical applications. They have been used for cell sorting, macromolecular separation, diagnostics, transfection, hyperthermia, drug delivery, and imaging. Genovis has developed a series of nanoparticles of various sizes and coatings specially designed for highly efficient delivery of siRNA and DNA. The nanostructures exhibit low cytotoxicity and serve as an excellent cellular labeling agent, allowing cells transfected ex vivo to be easily traced after animal injection using MRI. The ability to vary particle coatings to include multiple functionalities further expands the possibilities to customize particles to meet specific delivery application needs.

MicroRNAs in Malignant Progression
Li Ma, PhD., Life Sciences Research Foundation Fellow, Dr. Robert Weinberg’s Lab, Whitehead Institute, Massachusetts Institute of Technology, Cambridge, MA, USA

We have recently identified miR-10b as a miRNA that is highly expressed in metastatic cells and positively regulates cell migration and invasion. When overexpressed in otherwise non-metastatic breast cancer cells, miR-10b can trigger invasion and distant metastasis from the primary mammary tumor. Expression of this miRNA is regulated by the pleiotropic transcription factor Twist. miR-10b targets HOXD10, and this in turn derepresses RHOC, a well-established metastasis gene. Significantly, miR-10b level is elevated in metastatic breast tumors from cancer patients. We hypothesize that miR-10b may enable proper cell migration during embryogenesis, and that its physiologic functions are co-opted by carcinoma cells during the course of malignant progression. To test this hypothesis, we are currently combining genetic and pharmacological approaches to study miR-10b loss-of-function effects in breast cancer metastasis.

Development of mutation-specific therapeutic siRNAs for treatment of skin disorders
Robyn P. Hickerson, PhD., Senior Scientist, TransDerm, Inc., Santa Cruz, CA, USA

We have identified a siRNA (TD101) that is highly specific for a single nucleotide mutation (N171K) in the keratin 6a (K6a) gene in tissue culture and in animal studies. This mutation is responsible for the dominant negative skin disorder pachyonychia congenita (PC), characterized by thickened and dystrophic nails and hyperkeratosis/blistering on the soles of the feet. A double-blind Phase 1b clinical trial (to determine safety) treating PC foot lesions of eligible and willing U.S. patients harboring K6a(N171K) has begun. This siRNA clinical trial is the “first-in-man” for skin treatment as well as the first to target a mutant gene.

High-throughput on-chip small-animal screening at sub-cellular resolution using microfluidics and femtosecond laser microsurgery
Mehmet Fatih Yanik, PhD., Assistant Professor of Electrical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

We report a suite of key microfluidic devices for complex high-throughput whole-animal genetic and drug screens. We demonstrate a high-speed microfluidic sorter that can isolate and immobilize Caenorhabditis elegans in a well defined geometry for screening phenotypic features at subcellular resolution in physiologically active animals. We show an integrated chip containing individually addressable screening-chamber devices for incubation and exposure of individual animals to biochemical compounds and high-resolution time-lapse imaging of many animals on a single chip without the need for anesthesia. We describe a design for delivery of compound libraries in standard multi-well plates to microfluidic devices and also for rapid dispensing of screened animals into multi-well plates. When used in various combinations, these devices will facilitate a variety of high-throughput assays using whole animals, including mutagenesis and RNAi and drug screens at subcellular resolution, as well as high-throughput high-precision manipulations such as femtosecond laser microsurgery for large-scale in vivo neural degeneration and regeneration studies.

Double strand RNA delivery and the mechanism of miRNA inhibition of translation
Craig P. Hunter, Ph.D., Professor of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA

One of the more fascinating aspects of RNAi in C. elegans is that silencing information spreads between cells and is even transmitted to the progeny.   This systemic property of RNAi does not appear to be restricted to nematodes and some of the machinery that transports dsRNA in C. elegans is broadly conserved among animals.  We are using the powerful experimental tools available in C. elegans to identify and characterize genes required for the process and characterizing the activities of the vertebrate homologues.
 
Development of a novel RNAi therapeutics platform
Tod Woolf, PhD., President & CEO, RXi Pharmaceuticals, Inc., Worcester, MA, USA

The design and effective delivery of synthetic RNAi compounds are important factors for therapeutic applications. We will present data using our proprietary rxRNA™ compounds.  rxRNAs can be up to 100 times more potent than conventional siRNAs, demonstrate nuclease resistance, and are potentially more specific for their intended targets.

Mechanisms Underlying Functional siRNA Delivery
Jianzhu Chen, PhD., Cottrell Professor of Biology, Professor of Immunology, Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

A key challenge for therapeutic application of RNA interference is to efficiently deliver synthetic, small interfering RNAs (siRNAs) into target cells that will lead to the knockdown of the target transcript (functional siRNA delivery). We have shown that siRNA specific for conserved regions of influenza viral genome can potently inhibit influenza virus infection in cell cultures, chicken embryos and mice. We have now shown that although endocytosis is responsible for the uptake of the majority of siRNA lipoplexes, a minor pathway, likely mediated by fusion between siRNA lipoplexes and plasma membrane, is responsible for the functional siRNA delivery. Our findings illuminate directions for modifying cationic lipid carriers for improved siRNA delivery.

Development of an Antiviral for Respiratory Syncytial Virus (RSV) utilizing RNA Interference (RNAi)
Rachel Meyers, PhD., Senior Director of RNAi Lead Development, Alnylam Pharmaceuticals, Inc., Cambridge, MA 02142, USA

RSV is the leading cause of hospitalization of infants and is an important pathogen of the elderly and the immunosuppressed. Existing preventions and treatments are limited. ALN-RSV01 is an siRNA that effectively inhibits RSV replication both in vitro and in vivo. Preclinical safety and efficacy as well as progress in the clinical evaluation of ALN-RSV01 will be presented.

RNAi and the ERI proteins: the silence within
Thomas F. Duchaine PhD., Assistant Professor, McGill Cancer Center & Department of Biochemistry, McGill University, Montreal, Quebec, Canada

RNAi is now known to govern diverse gene regulation mechanisms. We are conducting a proteomic survey of the machineries involved in the endogenous functions of RNAi in C. elegans. We identified most and possibly all of the components in a ~850kDa complex, involving among others Dicer (DCR-1), the RNA-dependent RNA polymerase RRF-3, and the ERI proteins. One subunit of this complex exhibits nuclear localization, and specifically binds to histone H3 in vitro. Our results suggest a function in the triggering of RNAi responses from single-stranded RNAs, and suggest that some of these events take place on chromatin.

Lentiviral human primary microRNA library for in vitro and in vivo gain-of-function studies
Attila Seyhan, PhD. Senior Research Scientist, Virology, Open Biosystems, Huntsville, AL, USA

Attila A. Seyhan1, Patrick Mitchell2, John Wakefield1, Evan Kroh2, John Byon2, Stacia Wyman2, Muneesh Tewari2, and Troy Moore1

1 Open Biosystems, 601 Genome Way, Suite 2100, Huntsville, AL 35806, USA
2 Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North
Thomas Building, Room D4-235, Seattle, WA 98109, USA

MiRNAs are found across a broad range of eukaryotic organisms, and are often highly conserved through evolution. To date, ~556 human miRNAs have been identified and many more are predicted to be present. Since each miRNA is thought to regulate multiple genes, the potential regulatory network afforded by miRNAs is enormous. Additionally, their mis-regulation has been linked to cancer and other diseases such as viral infection.

However, relatively few miRNAs have been experimentally validated in cell culture or in animal models, and the functions of most miRNAs remain to be discovered.
Here we present the miR-express™ lentiviral-based human miRNA library to study the function of miRNAs in a wide range of cell types including non-dividing and difficult-to-transfect cell lines. To accomplish this, we have cloned all of the annotated human primary miRNAs (pri-miRNA) in constitutive (CMV) lentiviral expression vectors. The miRNA is co-expressed with TurboRed Fluorescent Protein (tRFP) and a puromycin resistance selectable marker, allowing easy tracking of miRNA expressing cells and generation of stable cell lines. The lentiviral-based human and mouse miRNA libraries will provide a valuable platform to study gain-of-function of these miRNAs in various functional assay systems, both in tissue culture and in animal models.

Clinical Applications of Small Scale SiRNA Screens in Cancer Therapy
Clark Chen, MD., PhD. Assistant Professor at the Harvard Medical School & Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

Historically, pharmaceutical development relies heavily on screening and modification of chemical inhibitors. While this approach has been fruitful, it suffers from the pleiotropic effect inherent within chemical inhibitors. That is, inhibitors often affect multiple gene targets. This complexity renders subsequent mechanistic investigations and rational drug refinements difficult. These difficulties are minimized with the development of Small Interference RNAs (SiRNA) screening technologies. Here we describe clinical applications of such screening technologies in identifying therapeutic strategies for targeting tumors with specific tumor suppressor gene inactivation (the Fanconi Anemia tumor suppressor gene) and for targeting tumors with oncogene over-expression (Epidermal Growth Factor Receptor Variant).