"RNA interference: Biology to Drugs & Therapeutics"

Target Audience:	300
Total Speaker Presentations:	30
Total Poster Presentations:	30
Total Exhibit Booths:	30

 

AGENDA/SPEAKERS(click here to view the detailed agenda)

Sunday, May 2, 2004
3:00 – 7:30 P.M: Registration Open

4:00 – 8:00 P.M: Technology Session I (consists of 6 lectures)

Monday, May 3, 2004
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 8:45 A.M and Ends at 5:30 P.M on (May 3-4) both days.

Tuesday, May 4, 2004
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:

K. Appasani, PhD. GeneExpression Systems, Inc.
J. Lieberman, MD., PhD. Associate Professor, Harvard Medical School,  Boston, MA
J. Rossi, PhD. Professor and Chair of Molecular Biology, Beckman Research
 Institute of the City of Hope, Duarte, CA., USA
R. Weinmann, PhD. Director of Oncology and Oncopharmacogenomics,
 Bristol-Myers Squibb Company, Princeton, NJ
W. Marshall, PhD. Executive VP, Dharmacon, Inc.
L. Van Parijs, PhD. Assistant Professor, Massachusetts Institute of  Technology, Cambridge, MA
D. Samarsky, PhD. Director of Technology Development, Sequitur, Inc.
Mr. S. Kriegsman, President and CEO, CytRx Corporation, Los Angeles, CA

Keynote Speakers on May 3rd Monday:

Craig Mello, PhD. Professor and HHMI Investigator
 University of Massachusetts Medical School, Worcester, MA
Title: RNAi and Development in C. elegans

Gary Ruvkun, PhD. Professor of Molecular Biology
 Massachusetts General Hospital, Harvard Medical School, Boston, MA
Title: Functional Genomic and Genetic Analysis of RNAi and MicroRNA Pathaways

Keynote Speaker on May 4th Tuesday:

Ken C. Reed, PhD. Director of Research and Technology
  Benitec Australia Ltd. St Lucia, Australia

Key presentations: (See below for abstracts).

Michael Butros, PhD. Boveri-Group Leader & Principle Investigator, German Cancer Research Center, Heidelberg, Germany
Title: "Genome-wide RNAi to Dissect Cellular Pathways in Drosophila Cells"

Jen-Tsan Ashley Chi, PhD. Research Fellow with Professor Patrick O. Brown, Stanford University Medical
School, Stanford, CA
Title: The monitor of RNA interference activities in mammalian cells

Antonin deFougerolles, PhD. Director of Research, Alnylam Pharmaceuticals, Cambridge, MA
Title: siRNA Therapeutics – The Path To Products

David Dorris, PhD. Director of RNAi Technologies, Ambion, Inc.
Title: Target Validation Experiments of Kinases Using Highly Effective siRNAs

Boro Dropulic, PhD. CSO, VIRxSYS Corporation, Gaithersburg, MD
Title: Highly efficient delivery of an inhibitory anti-HIV RNA in primary T lymphocytes by a HIV
based lentiviral vector

Thomas Grunfeld, MD. CEO, Interagon AS, Oslo, Norway
Title: Improved identification of optimal siRNAs by deployment of extreme compute capacity technology

Klaus Giese, PhD. CSO and VP Research, atugen AG, Germany
Title: Novel siRNA Structures
Aimee Jackson, PhD. Senior Research Biologist, Rosetta Inpharmatics/Merck, Kirkland, WA
Title: What can expression profiling tell us about siRNAs?

Lata Jayaraman, PhD. Research Investigator, Bristol-Myers Squibb, Princeton, NJ
Title: Validation of Novel Drug Targets using RNAi

Mark A. Kay, M.D., PhD. Professor of Pediatrics and Genetics and Director of the Program in Human Gene
Therapy, Stanford University School of Medicine, Stanford, CA, C0-Founder, Avocel, Inc.
Title: In vivo pre-clinical studies for RNA interference directed therapies for human viral hepatitis infection

Steven A. Kriegsman, President and CEO, CytRx Corporation, Los Angeles, CA
Title: Strategy for developing RNAi as a therapeutic

Eric Lader, PhD. Associate Director, Business Development, Qiagen, Germantown, MD
Title: Genome-wide siRNA: Development of an Automated, High Throughput siRNA Design and Synthesis Platform

Judith Lieberman, MD., PhD. Associate Professor, Harvard Medical School, Boston, MA
Title: Silencing the Messenger: Towards In Vivo Delivery

William Marshall,PhD. Vice President, R & D, Dharmacon, Lafayette, CO
Title: Recent Enabling Developments in the Application of siRNA Based Gene Silencing Methods

Luk Van Parijs, PhD. Assistant Professor, Massachusetts Institute of Technology, Cambridge, MA
Title: Studying Disease Process Using RNAi

John Rossi, PhD. Professor and Chair of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte,
CA., USA
Title: Optimizing RNAi in mammalian cells

Dmitry Samarsky, PhD. Director of Technology Development, Invitrogen Corporation, Natick, MA
Title: RNAi in drug discovery

Bradley J. Scherer, Ph.D. BDClontech, Palo Alto, CA
Title: Recent Advances in Viral RNAi Delivery

Ronald Scheule, PhD. Director, Genzyme Corporation, Framingham, MA
Title: Small Interfering RNA Transiently Attenuates Liver Expression from Exogenous Genes and Reduces Subsequent Immune Responses to the Transgene Product in Mice

Oliver Steinbach, PhD. Director, Functional Cloning, ALTANA Research Institute, Waltham, MA
Title: RNAi for Target Identification and Validation in Drug Development

Mario Stevenson, PhD. Professor of Molecular Medicine, University of Massachusetts Medical School,
Worcester, MA
Title: Manipulating HIV by RNAi

Kazunari Taira, PhD. Professor of Chemistry and Biotechnology, University of Tokyo, Japan
Title: Lessons from the Small RNA World in the Bio-Medical Revolution

Kader Thiam, PhD. Senior Scientific Consultant and Director of Transgenic Technologies, GenOWay,
Cedex, France
Title: Essential Requirements for successful Use of RNAi in Transgenic Mice

Nassim Usman, PhD.VP & COO, Sirna Therapeutics, Inc. Boulder, CO

Martin Woodle, PhD. CEO, Intradigm Corporation, Rockville, MD
Title: In Vivo siRNA: Drug Target Validation and Therapeutics

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

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

Panel Discussion on May 4th 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.

Exhibitors are welcome to reserve their booth space.

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


Abstracts

Novel siRNA Structures
Klaus Giese, Ph.D. Chief Scientific Officer and Vice President Research, Atugen AG, Berlin, Germany

RNA interference (RNAi) using siRNA-mediated gene silencing is a powerful tool for gene function analysis. The short bio-availability of conventional siRNA molecules is, however, an impediment for functional in vivo applications. Non-modified siRNA molecules are rapidly degraded by nucleases in serum and other body fluids. Atugen has processed towards in vivo applications of siRNA molecules by the development of novel stabilized siRNA structures, which differ significantly from the conventional siRNA molecules in terms of nucleotide composition, length of the molecules and covalent modifications. This advancement opens the potential to develop therapeutic siRNA molecules.

Small Interfering RNA Transiently Attenuates Liver Expression from Exogenous Genes and Reduces Subsequent Immune Responses to the Transgene Product in Mice
Ronald Scheule, PhD. Director, Genzyme Corporation, Framingham, MA

Hepatocytes have been shown to function as an effective depot for the production of secreted proteins from gene therapy vectors. When these vectors or their delivery induces hepatic inflammation, adaptive immune responses against the transgene product can ensue. In the context of hydrodynamic delivery of a plasmid DNA bearing a foreign transgene to the liver, we have used small interfering RNA (siRNA) to overcome the antibody response against the exogenous transgene product in both wild type mice and mice knocked out for the transgene product. This strategy also resulted in a long term immune status approximating “tolerance” to the transgene product.

In vivo pre-clinical studies for RNA interference directed therapies for human viral hepatitis infection
Mark A. Kay, M.D., Ph.D. Professor of Pediatrics, Genetics & Director of the Program in Human Gene Therapy, Stanford University School of Medicine, Stanford, CA, and Co-Founder, Avocel, Inc.

RNA interference (RNAi) has great potential for the treatment of viral infections. Our laboratory has focused on liver directed transfer of short-inhibitory RNAs and the expression of short-hairpin RNAs from the livers of living animals to inhibit transgene expression. To do this, we injected either short-inhibitory RNAs or plasmids expressing the short-hairpin RNAs along with a plasmid expressing the luciferase transgene, luciferase-HCV fusion gene, or HBV genome into the livers of mice by a hydrodynamic transfection procedure. We established that: (1) RNAi was functional in whole mammals; (2) Expressed shRNAs were efficient at down-regulating gene expression, suggesting that RNAi-mediated therapies can be adapted to a gene therapy approach; (3) RNAi against hepatitis C virus sequences were inhibitory; and (4) shRNAs directed against hepatitis B virus inhibited viral HBV DNA replication in the liver. To further pursue a gene therapy approach against hepatitis virus infection in animal models, we are pursuing different clinically relevant gene transfer strategies that can be implemented into a clinical trial within the next few years.

What can expression profiling tell us about siRNAs?
Aimee Jackson, Ph.D. Sr. Research Biologist, Rosetta Inpharmatics/Merck, Kirkland, WA

RNA interference is a powerful approach for the identification of targets by functional genomics. However, the success of this approach is dependent upon consistently high and specific gene silencing by siRNAs. We have used gene expression profiling to characterize the efficacy, potency, and specificity of gene silencing by siRNAs in cultured human cells. We discuss the implications of these findings to the design of effective siRNAs, and the application of siRNA library screens to target identification.

RNAi for Target Identification and Validation in Drug Development
Oliver C Steinbach, PhD. Director Functional Cloning, ALTANA Research Institute, Waltham, MA

Zaklina Strezoska1, Jonathan M Gilbert1, Raghu L Visvanath1, Haiying Yu1, Don J Misumi1, Jian Wang1, Hans-Peter Hofmann2, Mathias Schmidt2, Volker Gekeler2 , Klaus Melchers1, and Oliver C Steinbach1
1 ALTANA Research Institute, 610 Lincoln Street, Waltham, MA 02466; 2 ALTANA Pharma AG, Department RDR/ON, Byk-Gulden Straße 2, 78467 Konstanz, Germany

The use of RNAi is proven to be a valid approach for sequence-specific suppression of gene expression and hence inhibition of their corresponding functions in a cellular context. This "loss of function" approach, therefore, has been widely used in the last couple of years for analysis of gene function, and in drug discovery, for identification and validation of potential drug target candidates. We use RNAi for functional screens in order to identify disease associated signals and targets whose specific inhibition could potentially result in a therapeutic effect for treatment of complex human diseases. The focus of these screens performed in our laboratory is on druggable gene families rather than genome wide approaches. The functional linkage of members of these selected gene families to disease related pathways or phenotypes on the cellular level provides unique inroads to novel drug discovery efforts. One area of interest is on kinases as a source of druggable targets for development of novel cancer therapies. We have successfully developed and employed a focused RNAi-based “synthetic lethality” screening process to unravel relevant tumor associated kinase targets.

Highly efficient delivery of an inhibitory anti-HIV RNA in primary T lymphocytes by a HIV
based lentiviral vector
Boro Dropulic, PhD. Founder and Chief scientific Officer, VIRxSYS Corporation, Gaithersburg, MD

Boro Dropulic1,2, Laurent Humeau1, Qiao Yu1, Ziping Chen1, Tatiana Slepushkina1, Mario Pereira1, Patricia Echeagaray1, Gwendolyn Binder1, and Xiaobin Lu1.
1VIRxSYS Corporation, Gaithersburg, Maryland 20877 2Sydney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231

Control of gene expression through RNA is a promising approach for genetic and infectious disease alike. The first major breakthrough in this area was with antisense, followed later by the discovery of RNAi in eukaryotic cells, which is presently an intense area of research. Two major advantages to using RNAi are the high sequence specificity of the targeting siRNAs, as well as the amplifying effect of RNAi. These qualities make an RNAi a good approach for gene therapy involving diseases resulting from single-base changes in the involved gene. However, for genetic diseases involving genes that are polymorphic across the population, or infectious disease involving a microorganism with a high mutation rate like HIV, long antisense is a better approach. This statement is supported by a recent study by Boden et al. that demonstrated that escape HIV mutants quickly evolve in response to RNAi. Since antisense is generally much longer than siRNA, small variations in sequence do not affect the efficacy of the antisense effect. We have investigated this in detail for HIV infection. We have constructed an HIV-1 based lentiviral vector expressing long antisense of 937 bases, VRX496, directed against the HIV envelope gene. VRX496 efficiently delivers antisense to target CD4+ T lymphocytes at a frequency greater than 94%, and subsequently inhibits HIV replication several logs. These data established the feasibility of antisense as a therapeutic option for HIV treatment. To investigate the mechanism of antisense-meditated inhibition and any development of viral escape mutants, we serially passaged virus through highly permissive the Sup-T1 T cell line in the presence of antisense to select for mutants capable of escape, then cloned the resulting viruses to sequence genomic changes within and surrounding the target region. We observed a significant increase in the number of deletions in the envelope antisense target region (91% over the baseline 27.5%), demonstrating mutations developing as a direct result of antisense-mediated selection. In clones where point mutations instead of deletions were observed, there was a high rate of A-G transitions within the antisense target region, but not in the flanking regions. This is consistent with mutations that are predicted as a result of antisense-mediated modification of double-stranded RNA-specific adenosine deaminase (dsRNA/DRADA), but not cysteine deaminases such as APOBEC-3G. The replicative ability of antisense-selected mutants were severely attenuated or destroyed in the absence of antisense; those clones capable of replication were not true escape mutants as they could not replicate in the presence of antisense. Since dsRAD/DRADA is a nuclear enzyme that modifies adenosines to inosines to cause nuclear retention and degradation of dsRNA in the nucleus, activation of the interferon response is avoided. This is in contrast to RNAi, which is a cytoplasmic pathway, recently reported to induce interferon in certain cell types. Therefore, depending upon the cell type, target gene, and polymorphisms in the target gene, antisense remains an important approach for investigations into new therapeutics, in particular those enabled by gene therapy.

Genome-wide RNAi to Dissect Cellular Pathways in Drosophila Cells
Michael Butros, PhD. Boveri-Group Leader & Principle Investigator, German Cancer Research Center, Heidelberg, Germany

A key challenge in the functional exploration of sequenced genomes is the integration of diverse experimental approaches and data sets into a coherent map of cellular processes. During the past years, model organisms, such as yeast, C. elegans and Drosophila have been at the frontier to establish widely applied genomic technologies and have contributed significantly to our understanding of conserved disease-relevant cellular pathways. Genome-wide functional screens have become one of the next challenges to systematically discover gene function. The availability of whole genome sequence of man and major model organisms has opened many new avenues for approaches that test the expression, interaction and depletion of gene products not only through one-gene-at-a-time paradigms but through high-throughput technologies that collect thousands of experimental observations at once or in very short time-frames. Following pilot experiments based on a limited number of dsRNAs, we decided to generate a set of genome-wide dsRNAs to conduct high-throughput cell-based screen by silencing every gene in the Drosophila genome. Using a versatile genome-wide amplicon approach (GAA), we synthesized a total of 21,306 dsRNA covering all 13,600 gene predicted by the Drosophila Genome Project plus additional open reading discovered through genome-wide expression profiling. We will present results that show how genome-wide RNAi screens can be efficiently performed in a high-throughput format, allowing the comprehensive identification of components of cellular pathways in few days time.
References: Boutros et al. (2004). Genome-wide RNAi Analysis of Growth and Viability in Drosophila Cells. Science 306, 832-835

Genome-wide siRNA: Development of an Automated, High Throughput siRNA Design and Synthesis Platform
Eric Lader, PhD. Associate Director, Business Development, Qiagen, Germantown, MD

The application of RNAi to target validation has the potential to revolutionize the drug discovery process. Increasingly, interest is on developing the ability to perform genome-wide screening using RNAi. The flexibility, reliability, and throughput of chemically synthesized siRNA make it the reagent of choice for high-throughput screening approaches for target validation. However, two significant challenges had to be overcome in order to succeed at these endeavors: First was the development and application of high throughput tools for genome-wide siRNA design. QIAGEN’s high throughput tool incorporates both an advanced algorithm for effective siRNA design and a comprehensive whole genome homology analysis to insure specificity of effect. Second, the manufacturing infrastructure for routine, robust, automated synthesis of tens of thousands of siRNAs was developed. QIAGEN has directly applied both of these solutions in the first two genome-wide siRNA projects, undertaken as collaborative efforts with major pharmaceutical partners.

Strategy for developing RNAi as a therapeutic
Steven A. Kriegsman, President and CEO, CytRx Corporation, Los Angeles, CA

CytRx has implemented a dual strategy in its RNAi program. It has a focus on using RNAi both as a therapeutic and as a drug discovery tool. To minimize the long development timelines associated with developing RNAi as a therapeutic, CytRx has focused on ALS as is initial disease target. This orphan drug opportunity offers the potential for expedited regulatory review, and the typical issues related to RNAi drug delivery can be circumvented in the case of ALS. This initial model will open the door to utilizing RNAi to target other motor neuron diseases. As part of its overall strategy, CytRx also expects to utilize RNAi as both a drug discovery tool (short term) and as a possible therapeutic (longer term) in the type 2 diabetes and obesity areas which are important therapeutic areas for CytRx. By focusing in on both short term and longer term applications as well as using an outsourcing model with sponsored research agreements, CytRx has positioned itself for a cost effective and a rapid drug development program.

In Vivo siRNA: Drug Target Validation and Therapeutics
Martin Woodle, PhD. CEO, Intradigm Corporation, Rockville, MD

RNAi has rapidly become a powerful tool for drug target discovery and validation in cell culture. Intradigm has developed effective means to utilize siRNA in vivo to knockdown endogenous genes and as a result induce phenotypic changes in pathology. Application of the approach as an efficient means for drug target validation within preclinical disease models will be presented. Also, development of efficient and clinically feasible means for systemic administration of siRNA for therapeutic applications will be presented. Product development for tissue targeted siRNA inhibitors of angiogenesis will be described as well as results from proof of concept studies on local treatments for head and neck cancer, rheumatoid arthritis and SARS Corona virus infection.

Development of siRNA-Based Therapeutics
Nassim Usman, PhD. Senior Vice President and Chief Operating 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. The key challenges for the development of siRNAs as therapeutics include extra- and intracellular stability, improved specificity, delivery and pharmacokinetics (PK). Stability in serum and tissue on the order of days has been achieved by chemical modification with little or no loss of biological activity. These chemical modifications also improve the specificity, PK and tissue distribution of siRNA. Modified siRNA can be detected in both tissue and plasma up to 96 hours post single administration. These stable, potent and targeted siRNAs are currently being developed for three indications; age-related macular degeneration (AMD), hepatitis and oncology. Preclinical animal efficacy has been demonstrated in all three areas including systemic efficacy by therapeutic routes of administration in viral infection and tumor models. In the case of the AMD program, a complete proof of concept has been demonstrated, including reductions in molecular endpoints, which proves the mechanism of action of a chemically modified siRNA targeting VEGFR-1, Sirna-027. Sirna-027 has entered preclinical development with an IND scheduled for late 2004.

Target Validation Experiments of Kinases Using Highly Effective siRNAs
David Dorris, PhD. Director of RNAi Technologies, Ambion, Inc.

Critical parameters for RNAi experimental success include siRNA design, siRNA validation, and siRNA delivery. Here we present results demonstrating the power of RNAi when these parameters are successfully optimized. In brief, siRNAs were designed and synthesized against nearly 600 human kinases. Multiple siRNAs per gene for over 300 of these kinase genes were tested for their ability to reduce target mRNA levels by a quantitative RT-PCR assay, resulting in the identification of at least one siRNA that reduces the target mRNA by greater than 70% for each gene. These validated siRNAs are effective for mRNA reduction in each human cell line tested and the maximum concentration for effective knockdown is 10 nM, which reduces off-target effects. Reduction of kinase mRNA levels using these siRNAs showed a significant alteration of growth rates for >25% of the nearly 200 kinase siRNAs tested. Additionally, RNAi-induced knockdown of these kinases resulted in greater than 25% showing a significant change in the mitotic index for rapidly growing cells. The correlation between growth rate and mitotic index can be put into multiple phenotypic categories. Together, these data highlight the usefulness of siRNAs for the identification of potential kinase targets for future drug development.

Essential Requirements for successful Use of RNAi in Transgenic Mice
Kader Thiam, PhD. Senior Scientific Consultant and Director of Transgenic Technologies, genOway, Cedex, France

Over the last decade, the use of siRNA and its mechanisms to inhibit in vivo gene expression have been well documented in a variety of organisms, including mammalian cells. Based on the latest scientific breakthroughs, genOway has developed an innovative and attractive technology using transgenically supplied RNAi for efficient and stable in vivo gene knockdown: Safe RNAi Transgenesis™. In addition, the recent advances demonstrating the possibility to control gene repression in a time and tissue-specific manner further confirm that this new approach represents a powerful tool in the field of genetically modified animal models.

Minimizing preanalytical variability in RNA based clinical studies through integrated collection, stabilization and purification
Lynne Rainen, Ph.D. Scientific Director, PreAnalytiX GmbH and BD Preanalytical Systems, Franklin Lakes, NJ

Gene transcription profiling has gained importance as a tool for diagnosing and monitoring human disease and drug response. Accurate analysis, however, of in vivo gene expression in whole blood may be complicated by post-phlebotomy changes in cellular transcript patterns. We have developed the PAXgene™ Blood RNA System for the collection of whole blood and the stabilization and purification of cellular RNA. The PAXgene Blood RNA System stabilizes the transcript profile at the time of phlebotomy and produces high quality intracellular RNA from a whole blood sample. The effects of sample handling on gene transcription profiles from blood samples collected in conventional methods vs. the PAXgene system will be discussed. The PAXgene Blood RNA System is for research use only and not for use in diagnostic test methods.

Recent Advances in Viral RNAi Delivery
Bradley J. Scherer, Ph.D. BD Biosciences Clontech, Palo Alto, CA

We have developed a series of vectors which efficiently express short hairpin RNAs driven by a pol III promoter. These vectors can be used to make recombinant retrovirus with a variety of tropisms depending on the packaging cell line used, or to make recombinant adenovirus using either a ligation or cre recombination-based method. A reproducible cotransfection assay using gene-of-interest enzymatic reporter constructs facilitates screening for efficacious shRNA sequences. Moreover, we have combined our vectors with a PCR-based method in order to rapidly screen for knockdown in transfected cells. Second generation vectors include fluorescent marker proteins which facilitate determination of transfection efficiency for shRNA introduction into cells.

Lentiviral delivery of inducible RNAi cassettes
Knut Madden, PhD. Invitrogen, 1600 Faraday Ave, Carlsbad CA

Knut Madden, Adam Harris, Ken Frimpong, Jennifer Kilzer, Beibei Wang, Heidi Welchin, Peter Welch and Rob Bennett

RNAi is a powerful loss-of-function tool for the manipulation of mammalian cells. To facilitate such experimentation, a rapid, versatile cloning system has been developed to create and transfer RNAi cassettes between multiple vectors. Select RNAi cassettes, once validated in a small ~3kb “Entry” vector, can easily be transferred to more complicated Adeno and Lentiviral vectors for delivery to “hard to transfect” and non-dividing cells, or to create transgenic animals. Additionally, a regulated promoter has been developed to control the expression of knock-down elements once a cassette is delivered and stably integrated into cells.

SMARTICLES® – in vitro and in vivo delivery of therapeutic nucleic acids
Dr. Steffen Panzner, novosom AG, Weinbergweg 22, 06120 Halle, Germany

SMARTICLES® define a novel class of fully charge-reversible liposomes. They are negatively charged under physiological conditions, but as the pH drops down to 5 or 4 during endocytosis, the particle surface becomes neutral and eventually positively charged. This unique property guarantees stable and aggregate-free travel within the bloodstream, but the acidification from endocytosis switches the charge of SMARTICLES®, leading to membrane fusion and the escape of the encapsulated cargo from the endosome. Once injected, SMARTICLES® are distributed in the body in the same manner as classic liposomes. Tiny openings in the capillaries are a necessary prerequisite for the escape from the bloodstream. Thus liver and spleen along with sites of inflammation and tumours are primary targets for SMARTICLES®. They provide a mechanism for endosome escape and thus deliver the cargo directly into the cytosol. The technology is therefore ideally suited for the delivery of therapeutic nucleic acids in vivo.
SMARTICLES® have shown themselves to be highly functional, and expression of GFP was demonstrated in liver, spleen or lungs after a single i.v. injection in a rat model. SMARTICLES® have also effectively transferred oligonucleotides and other small molecules to liver and cellular sites of inflammation.

Recently, we designed a centrifugal transfection procedure, allowing the use of those particles for in vitro transfection. Therefore, this reverse engineered process enables researchers in the field of antisense or siRNA first to optimize their drugs in vitro and second to verify the therapeutic power without any change in transfection agents.
SMARTICLES® technology offers significant advantages as compared to other delivery systems and is the only functional delivery method available for in vitro as well as in vivo delivery of therapeutic nucleic acids to targeted cells. SMARTICLES® are extremely easy to prepare and hence enjoy the benefit of low-cost. They do not require complicated procedures in their production, are not based on viral methods and can be scaled-up under GMP.

Citius, Altius, Fortius: Olympian Aspirations for Health Care with ddRNAi
Ken C Reed, PhD, Director, Research and Technology, Benitec Australia Ltd, PO Box 4193, St Lucia QLD 4067, Australia

There is no longer any doubt that RNA interference (RNAi) is an intrinsic characteristic of mammalian cells. Publications citing its characteristics and utility now number in the thousands and already it is established as an essential tool in molecular and cell biology. Indeed it is the perfect investigative tool, a capability for universal gene knockdown that we would have been compelled to invent if we hadn’t stumbled upon the natural phenomenon. The impact of RNAi on definitive and high throughput functional genomics is well understood, but in this presentation we are concerned not so much with molecular dissection of cell biology as with the prospects afforded by RNAi for future modes of disease treatment. We believe that this technology, and more specifically its delivery by DNA vectors (ddRNAi), has the potential to revolutionize ameliorative, curative and preventive health care. For the first time, disease modelling and target validation in vivo becomes universally practical with lentivirus-mediated ddRNAi transgenics, applicable to all genes and apparently to all species. ddRNAi provides knockdowns and knockouts that are dominant, definitive, rapid and controllable in time and space. Significantly, the bioactive agent used for target validation can itself become a therapeutic agent and systems biology analysis afforded by ddRNAi knockouts can simultaneously provide preclinical therapeutic data. ddRNAi constructs further allow delivery of multiple bioactives in a single agent, formulated such that no cell can receive less than the full complement, virtually eliminating the possibility of selection for resistance in highly mutable diseases such as HIV and cancers. It appears to be ideally suited to treating such diseases but its potential applications range far wider. Most importantly, it is a generic therapy that eliminates the crippling quest for small molecules, offering hope for many orphan diseases and the promise of personalized therapies. Effective delivery of ddRNAi depends on a suite of gene transfer vectors and promoters that have yet to realize their full potential, but it must be noted that induction of RNAi is a far simpler proposition than controlled expression of a functional protein, the goal of current gene therapies. No clinical trials of ddRNAi have yet commenced and it would be foolish to suggest that no unforeseen problems will arise. But the astounding progress in understanding and implementing ddRNAi in mammals in the six short years since it was first described by Michael Graham provides assurance that it will indeed lead us to faster, higher, stronger health care delivery.
NeoPhectin-AT™: A Unique Ready-to-Use In Vivo Delivery System for siRNA
Imran Ahmad, PhD. Chief Scientific Officer and Senior Vice President, Research & Development, NeoPharm, Inc. Lake Forest, IL

We have developed NeoPhectin-AT™, a novel well characterized and ready-to-use liposome-based delivery system for systemic administration. Intravenous administration with c-raf specific siRNA using NeoPhectin-AT™ inhibits growth of well established s.c. implanted human prostate tumor in SCID mice. The potential of NeoPhectin-AT™ mediated delivery of siRNA will be discussed.

Optimizing RNAi in mammalian cells.
John J. Rossi, PhD. 1, Mark Behlke2 Nancy Lee1 and Dongho Kim1
1. Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA; 2. Integrated DNA Technologies, Inc. (IDT), Coralville, Iowa.

RNAi is one of the most powerful sequence specific knockdown techniques available for use in mammalian cells. Despite the potency of RNAi for some targets, it is not always possible to achieve significant mRNA knockdowns with synthetic or expressed siRNAs. Our lab has been investigating aspects of the RNAi mechanism as well as methods for enhancing RNAi efficacy. We have identified the well-known RNA binding protein, La as an essential component of RNAi in human cells. Since the RNAse III like enzyme Dicer produces 21 to 23mer siRNAs with 2 base 3’ overhangs, we believe that La binds to these ends and protects them from nuclease degradation. La is part of a multiprotein complex binds siRNAs and an associated helicase that unwinds the two strands with a handoff to the RNA induced silencing complex (RISC). Experiments from our lab suggest that the concerted action of Dicer cleavage, followed by unwinding and handoff to RISC produces more effective RNAi. An example of a synthetic duplex RNA design that allows full participation in this pathway and is highly active in subnanomolar concentrations will be given. We have termed these RNAs as dicer substrate RNAs or disRNAs to differentiate them from small interfering RNAs that are the products of Dicer. We have also been investigating approaches for enhanced intracellular functioning of promoter expressed shRNAs and siRNAs. Comparisons of separately expressed sense and antisense, Pol III transcribed short hairpin RNAs and a chimeric VA1-shRNA will be presented. The data from these experiments further support a concerted pathway for RNAi in which linking the various steps results in more potent RNAi.

Customized high compute power technology for state of the art siRNA design
Thomas Grunfeld, MD. Chief Executive Officer, Interagon AS, Oslo Norway

The use of small interfering RNA (siRNA) to induce sequence specific gene silencing is fast becoming a standard tool in functional genomics. Progress in the field is rapid, but there are still uncertainties regarding how to design optimal siRNAs with respect to both efficacy and specificity. Existing bioinformatics tools have clear limitations comprising both applicability and compute power requirements when addressing these issues. These limitations are becoming increasingly evident in genome-wide, high-throughput applications. Interagon AS has addressed these issues deploying a dedicated application specific integrated circuit (ASIC) allowing for a thousand-fold acceleration of traditional PCs. This compute power has enabled us to develop state of the art predictors for siRNA efficacy and specificity, working at unprecedented speed, performing mammalian transcriptome-wide screens in hours. On efficacy, machine learning with genetic programming accelerated by the PMC creates predictors that outperform known efficacy algorithms and predictors created by support vector machines, which are currently regarded as “state of the art” in machine learning technology. On specificity, all potential siRNAs from the transcriptome are screened for potential off-target hits in the transcriptome or genome. This is a formidable task in terms of compute requirements, and the process outputs the most specific siRNAs available for any gene in the transcriptome in hours. These specificity screens will find all homologies with any desired number of mismatches or G-U wobbles, thus overcoming the technical limitations of BLAST (and similar algorithms), and the compute capacity requirement limitations of the Smith-Waterman algorithm. Simultaneous review of the efficacy and specificity allows for qualified trade offs in the final choice of siRNAs in experiments and for drug design. Interagon will demonstrate the high compute power siRNA design technology with relevant benchmarks. The technology provides the next generation of dedicated bioinformatics tools for the RNA interference field; both with respect to improved design of siRNAs, as well as strongly improved analysis and interpretation of biological data.

Studying Disease Process Using RNAi
Luk Van Parijs, PhD. Assistant Professor in Immunology, Massachusetts Institute of technology, Center for Cancer Research, Cambridge MA 02139

RNA interference provides a powerful new approach to study the genetics of complex biological processes and disease. We have developed lentivirus-based RNAi systems that can silence gene expression in primary cells, tissues, and in animals. We have used this technology to perform reverse and forward screens for genes that control the development of autoimmunity and immune cell cancers in the mouse. How these experiments serve to identify and validate targets for drug development will be discussed.

Validation of Novel Drug Targets Using RNAi
L. Jayaraman, M. Lorenzi & M. Gottardis
Bristol-Myers Squibb Oncology, Princeton, NJ

RNA interference has gained increasing importance as a powerful tool to evaluate the contribution of genes to signaling pathways. We have incorporated post-transcriptional gene silencing by siRNA and the analysis of physiologically relevant endpoints resulting as a consequence of it into our validation efforts to validate novel targets for oncology drug discovery. Our results show that gene manipulation of siRNA can be used not only to study the inhibition of transformed phenotypes in vitro but also to generate cancer relevant models.

RNAi Technology Development and Applications in Drug Discovery
Ruitang Deng, Ph.D., Senior Research Investigator, Genetic Technology, Genomic and Proteomic Sciences, Exploratory Medicinal Sciences Department, Groton Laboratories, Pfizer Global Research and Development, Pfizer Inc. Groton, CT

RNA interference (RNAi) has recently emerged as a powerful molecular tool for drug discovery. To meet diversified needs from therapeutic areas and various drug discovery lines including target validation, gene functional studies, assay tool development, disease model construction, ADME and toxicity studies; we have developed a universal RNAi screening system called combined short target sequence (CSTS)-GFP fusion system in HEK 293 cell. The system can be effectively used to screen and identify potent RNAi reagents for gene targets. The system was validated by a strong correlation of gene silencing activities of siRNAs with target mRNA derived from CSTS-GFP fusion and full-length gene-GFP fusion. The data showed that a fragment of 21 bases, a preferred size of a siRNA, is enough to function as an efficient and specific target site for gene silencing. Our data suggest that the primary sequence rather than the content or local secondary structure of an RNAi target site in a mRNA plays the critical role in determining RNAi potency. We have applied the system to identify functional RNAi reagents including siRNAs, shRNAs and vectored shRNAs for a number of gene targets.

RNAi-2003-Boston Meeting Testimonials

“Gave an update on the RNAi field. Presentations were informative. Exhibits gave a good overview of the companies working on RNAi. The turnout was excellent.” – Prof. Frank Ruddle, Yale University.

“The meeting was stimulating as well as encouraging. It was interactive and had breakthrough data that helps us all.” - Dr. Ken Reed, Business development, Benitec Australia Ltd.

“The meeting was very well organized. I liked that many talks were relatively brief, and covered so much ground. An excellent job overall.” - Malorye Branca, Editor, Bio-ITWorld magazine

“It was very interesting meeting for me to attend and know the newest situation of advanced technology in RNAi field. Totally, it was very well organized and arranged.” - Takuji Sekine, Nagase & Co., Ltd. Japan

"This was the best RNAi meeting I went to this year, well organized and stimulating scientific atmosphere.” - Dr. E. Lader, Global Business Manager, Qiagen

“Opened possibilities for everyone, informative and useful overall.” - Dr. Xinzhong Wang, Biogen

“Since I am a graduate student in academia the meeting was a great way to network with scientists in
biotech/pharma and get some insight into the way research is conducted at those institutions.“ – M. Vella, Yale

To view the details of 2003 meeting Sponsors, Posters, Awards, and Surveys please visit our main page and click After RNAi-2003 Meeting information.