7th Annual Meeting of the Oligonucleotide Therapeutics Society September 8 – 10, 2011 The Royal Library Copenhagen | Denmark Meeting Abstracts Oligonucleotide Therapeutics Society PROGRAM AGENDA Every effort has been made to ensure the meeting agenda listed in this booklet is accurate. However it’s possible that some changes occurred after printing. We will notify you of any changes to the agenda from the main stage. 2011 CONFERENCE PROGRAM w w w .ol i goth e rapeut ics.org Wednesday, September 7, 2011 15:00 – 18:00 Registration and Poster Set-Up Thursday, September 8, 2011 08:00 – 09:00 09:00 – 09:15 Registration and Poster Set-Up Welcome and Opening Remarks Gunther Hartmann, MD, PhD, UniversitГ¤tsklinikum Bonn, OTS President 09:15 – 10:30 S ESSION I: Hot Topics: Short Talks Selected from Abstracts Chair: Gunther Hartmann, MD, PhD, UniversitГ¤tsklinikum Bonn 09:15 – 09:30 Expanding the Structural Diversity Repertoire of siRNAs Dong-ki Lee, PhD, Sungkyunkwan University 09:30 – 09:45 Activation of RNA Interferance in Animals with Single Stranded Oligonucleotides Eric Swayze, PhD, Isis Pharmaceuticals 09:45 – 10:00 Limitations for RNAi: intracellular release of siRNA and cell stress Georg Sczakiel, PhD, Institute for Molecular Medicine, University of LГјbeck 10:00 – 10:15 Preclinical Efficacy and Safety of Phosphorodiamidate Morpholino Oligomers (PMO) Peter Sazani, PhD, AVI BioPharma, Inc. 10:15 – 10:30 5’-triphosphate dsRNA-induced stimulation of RIG-I prevents influenza H1N1 infection of primary nasal epithelial cell culture and protects mice from infection in vivo Christoph Coch, MD, Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn 10:30 – 11:00 Refreshment Break and Poster Set Up 11:00 – 12:30 S ESSION II: N UCLEIC A CID C HEMISTRY Chair: Jesper Wengel, PhD, University of Southern Denmark, Odense 11:00 – 11:30 Nucleotide Chemistry and Gene Silencing Jesper Wengel, PhD, University of Southern Denmark, Odense 11:30 – 12:00 Arabinose Modified Antisense and siRNAs: Biological Applications, Structural Considerations, and Delivery Strategies Masad J. Damha, PhD, McGill University, Montreal 12:00 – 12:30 siRNA Activity, Stability and 3D-Structural Properties of 2’-Fluoro RNA and GNA Martin Egli, PhD, Vanderbilt University Medical Center, Nashville 12:30 – 14:00 Lunch with Attended Poster Session 12:30 – 13:15 Standing Lunch Buffet 13:15 – 14:00 Attended Poster Session 14:00 – 14:30 Keynote Session: Oligonucleotide Therapeutics reviewed in memory of Alan Gewirtz Keynote Speaker: Fritz Eckstein, PhD Max-Planck-Institute for Experimental Medicine, GГ¶ttingen 14:30 – 16:00 S ESSION III: RNA B IOLOGY: N EW F RONTIERS Chair: Mark Kay, MD, PhD, Stanford University 14:30 – 15:00 Primary microRNA Function in Target Recognition and Repression Chang-Zheng Chen, Stanford University 15:00 – 15:30 Regulation of small RNA function Gunter Meister, PhD, University of Regensburg 15:30 – 16:00 The mechanism of miRNA and siRNA Argonaute loading in mammals Mark Kay, MD, PhD, Stanford University 16:00 – 16:30 Refreshment Break 7 ANNUAL MEETING OF THE OLIGONUCLEOTIDE THERAPEUTICS SOCIETY TH 7 7 ANNUAL MEETING OF THE OLIGONUCLEOTIDE THERAPEUTICS SOCIETY TH ww w.oligo t h e ra p e u t i c s . o r g 16:30 – 18:00 S ESSION IV: D ELIVERY OF N UCLEIC A CIDS Chair: Jean-Jacques ToulmГ©, PhD, INSERM, University of Bordeaux, Paris 16:30 – 17:00 Chemical Strategies for Delivery of RNAi Drugs Muthiah Manoharan, PhD, Alnylam Pharmaceuticals 17:00 – 17:30 Non covalent peptide-based delivery systems Gilles Divita, PhD, Centre de Recherches de Biochimie MacromolГ©culaire, Department of Molecular Biophysics and Therapeutics, Montpellier 17:30 – 18:00 Investigating the Potential of Therapeutic Oligonucleotides for Pulmonary Diseases Ken Clark, GlaxoSmithKline 18:00 – 19:30 19:30 Welcome Reception with Attended Posters Meet the Experts End of Day 1 Friday, September 9, 2011 08:00 08:30 – 10:00 Registration Opens SESSION V: IMMUNOBIOLOGY of NUCLEIC ACIDS Chair: Arthur Krieg, MD, Atlas Venture 08:30 – 09:00 RIG-I and tolerance of self-RNA Gunther Hartmann, MD, PhD, UniversitГ¤tsklinikum Bonn 09:00 – 09:30 Regulation of the Innate Immune Response Using Oligonucleotide-based Inhibitors of Toll-Like Receptors Franck Barrat, PhD, Dynavax Technologies 09:30 – 10:00 Intracellular trafficking of TLR ligands in human plasmacytoid dendritic cells Patricia Fitzgerald-Bocarsly, PhD, UMDNJ – New Jersey Medical School 10:00 – 10:30 Refreshment Break 10:30 – 12:00 S ESSION VI: T ARGETING C ODING RNA Chair: David Corey, PhD, UT Southwestern Medical Center, Dallas 10:30 – 11:00 Gymnotic Delivery of Antisense Oligonucleotides Cy Stein, MD, PhD, Albert Einstein College of Medicine, Bronx 11:00 – 11:30 Modulation of Gene Expression by Oligonucleotide Chemistry-Dependent Recruitment of Proteins to RNA Transcripts Frank Rigo, PhD, Isis Pharmaceuticals 11:30 – 12:00 Clinical development of mipomersen Erik Stroes, MD, PhD, AMC Research Institute, Amsterdam 12:00 – 13:30 Lunch with Attended Poster Session 12:00 – 12:45 Standing Lunch Buffet 12:45 – 13:30 Attended Poster Session 13:30 – 15:00 S ESSION VII: T ARGETING microRNA Chair: Markus Stoffel, MD, PhD, ETH Zurich 13:30 – 14:00 Therapeutic Targeting of microRNAs Neil Gibson, PhD, Regulus Therapeutics 14:00 – 14:30 Therapeutic targeting of individual cardiac cell types by miRNA antagonists Thomas Thum, MD, PhD, Integrated Research Center, Medical School Hannover, Germany 14:30 – 15:00 Oncomir-1 in cancer and development: a tale of mice and men Andrea Ventura, MD, PhD, Sloan-Kettering Institute, New York City 15:00 – 15:30 8 Copenhagen, Denmark Refreshment Break 2011 CONFERENCE PROGRAM w w w .ol i goth e rapeut ics.org 15:30 – 17:00 S ESSION VIII: T ARGETING L IVER AND T UMORS Chair: Henrik Г�rum, PhD, Santaris Pharma A/S 15:30 – 16:00 Evaluation of locked nucleic acid (LNA)-based mRNA antagonists in cancer patients Aby Buchbinder, Enzon Pharmaceuticals 16:00 – 16:30 ALN-TTR, an RNAi Therapeutic for the Treatment of Transthyretin Amyloidosis Dinah Sah, PhD, Alnylam Pharmaceuticals 16:30 – 17:00 Targeting miRNA-122 for the Treatment of HCV Henrik Г�rum, PhD, Santaris Pharma A/S 17:15 – 19:15 Exhibitor Reception at the BГёrsen A reception featuring our meeting exhibitors. End of Day 2 Saturday, September 10, 2011 08:00 08:15 – 09:45 Registration Opens S ESSION IX: T ARGETING I NFECTION Chair: John Rossi, PhD, City of Hope, Duarte 08:15 – 08:45 The development of aptamers as candidate antiviral agents: towards a polyvalent microbicide William James, PhD, Sir William Dunn School of Pathology, University of Oxford 08:45 – 09:15 Developing the next generation of expressed RNAi modalities against rapidly-evolving viral infections Marc Weinberg, PhD, University of the Witwatersrand Medical School, Parktown 09:15 – 09:45 Dual function therapeutic Aptamer siRNA combinations John Rossi, PhD, City of Hope, Duarte 09:45 – 11:15 S ESSION X: T ARGETING THE C ENTRAL N ERVOUS S YSTEM Sponsored by International Society of Neurochemistry Chair: Hermona Soreq PhD, The Hebrew University of Jerusalem 09:15 – 10:15 Systemic versus CNS Delivery of MOE Antisense Oligonucleotide to Correct Defective Splicing in a Severe Mouse Model of Spinal Muscular Atrophy Adrian Krainer, PhD, Cold Spring Harbor Laboratory 10:15 – 10:45 Micro-RNAs in the Neuroimmune Interface: From Inflammation to Lethal Poisoning Protection Hermona Soreq, PhD, The Hebrew University of Jerusalem 10:45 – 11:15 The role of microRNA in regulating the central stress response Alon Chen, PhD, MBA, Weizmann Institute of Science, Rehovot, Israel 11:15 – 11:45 Refreshment Break 11:45 – 13:15 S ESSION XI: T ARGETING G ENETIC D ISEASES Chair: Dieter Gruenert PhD, University of California, San Francisco 11:45 – 12:15 Exploring and exploiting designer nucleases for targeted genome engineering Toni Cathomen, PhD, Hannover Medical School 12:15 – 12:45 Oligonucleotide-directed gene modification in mouse Embryonic Stem Cells to study variants of cancer-related genes Hein te Riele, PhD, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital 12:45 – 13:15 siDNA, a new strategy for inhibiting DNA repair pathways Marie Dutriex, PhD, Institut Curie, Centre Universitaire, Orsay 13:15 Closing Session End of Conference 7 ANNUAL MEETING OF THE OLIGONUCLEOTIDE THERAPEUTICS SOCIETY TH 9 SPEAKER ABSTRACTS Session 1: Hot Topics: Short Talks Selected from Abstracts Dong-ki Lee Thursday, September 8, 2011 09:15 – 09:30 Expanding the Structural Diversity Repertoire of siRNAs Dong-ki Lee Global Research Laboratory for RNAi Medicine, Department of Chemistry, Sungkyunkwan University, Suwon 440-746, Korea Chemically synthesized small interfering RNAs (siRNAs) can mediate efficient gene silencing in a sequence-specific manner by utilizing the endogenous RNA interference (RNAi) pathway. The early structure-activity relationship study suggested the existence of strict rules of siRNA structural design to achieve optimal gene silencing. These include the presence of 3’-overhang, fixed duplex length, and structural symmetry, which defined the structure of the current standard siRNA (the so-called 19+2 form). However, several recent studies demonstrated that the gene silencing RNA structure could be much more flexible than what was originally proposed. In addition, the synthetic 19+2 siRNA structure exhibits sequenceindependent non-specific effects and poor cellular delivery by cationic polymers, which justifies the effort to develop alternative RNAi-triggering structures. Here we introduce three siRNA structural variants with improved functionalities over current siRNA structures. First, we introduce asymmetric shorter-duplex siRNAs (asiRNAs) that can efficiently trigger gene silencing without non-specific effects triggered by 19+2 siRNAs, such as sense strandmediated off-target gene silencing, saturation of the cellular RNAi machinery, and reduced TLR3 activation. Second, we present multi-armed gene silencing RNA structures, which not only mediate multiple gene silencing, but also show enhanced cellular delivery by cationic polymers such as polyethyleneimine (PEI). Third, we show a long dsRNA structure that can trigger RNAi as well as immunostimulation. The structure, termed long interfering dsRNA (liRNA), show enhanced anticancer activity compared with conventional siRNA or nontargeting long dsRNA. These novel siRNA structural variants exemplify the structural flexibility of gene silencing RNAs, and have the potential to become important alternatives to the current siRNA structure for future therapeutics development. Dong-ki Lee, PhD Global Research Laboratory for RNAi Medicine Department of Chemistry Sungkyunkwan University Suwon 440-746 Korea Telephone: 82-31-299-4565 Email: [email protected] 11 Session 1: Hot Topics: Short Talks Selected from Abstracts Eric Swayze Thursday, September 8, 2011 09:30 – 09:45 ACTIVATION OF RNA INTERFERANCE IN ANIMALS WITH SINGLE STRANDED OLIGONUCLEOTIDES Eric E. Swayze, Thazha P. Prakash, Walt F. Lima, and Stanley T. Crooke Isis Pharmaceuticals, Carlsbad, CA, USA The use of double stranded oligonucleotides to inhibit gene expression via the RNA interference (RNAi) pathway has generally required a complex (usually lipid based) formulation for delivery of the oligonucleotide to the desired compartment of a cell. Unfortunately, this requirement has extended from cell culture to animals, which severely limits the potential of harnessing the RNAi pathway for therapeutic approaches. Unlike double stranded oligonucleotides, single stranded antisense oligonucleotides have shown activity in multiple species (including humans) without formulation vehicles. Furthermore, the double stranded RNA (dsRNA) structure is not required for RNAi, as single stranded RNA (ssRNA) delivered to cells using cationic lipids has been shown to activate the RNAi pathway. \This suggested to us that the dsRNA structure could be simplified to a single stranded oligonucleotide that would activate RNAi in cells and in animals, and provide significant benefits over double stranded structures for the potential development of human therapeutic agents. To achieve this objective, we have engaged in an extensive chemical structure activity relationship (SAR) study of ssRNAs, and coupled this to biochemical studies on the mechanism of activation of the RNA induced silencing complex (RISC). This SAR has led to an understanding of key structural features required for a ssRNA activity, and allowed us to achieve potency within 5-fold of the corresponding double stranded structures in cells with multiple fully modified, partially phosphorothioated ssRNA oligonucleotide designs. Our initial attempts to show activity in animals with these early designs failed due to pharmacokinetic limitations. These limitations were overcome with further chemical stabilization achieved via an in vivo SAR optimization cycle. Sequence optimization of the optimal chemical motif provided highly potent compounds in cell culture which were shown to function via an argonaute-2 dependent mechanism. These ssRNAs are active in cell culture without cationic lipids, and this activity translated to activity in animals at pharmacologically relevant doses with subcutaneous administration in saline formulations. These studies provide a framework for further optimization of the ssRNA structure for the potential development of human therapeutic agents. Eric E. Swayze, PhD Isis Pharmaceuticals, Inc. 2855 Gazelle Court Carlsbad, CA 92010 USA Telephone: 760-603-3825 Email: [email protected] 12 Session 1: Hot Topics: Short Talks Selected from Abstracts Georg Sczakiel Thursday, September 8, 2011 09:45 – 10:00 Limitations for RNAi: intracellular release of siRNA and cell stress Georg Sczakiel Institute of Molecular Medicine, University of LГјbeck, Germany The successful application of short silencing RNA (siRNA) in vivo is crucially dependent on efficient cellular uptake. Even after physical cellular internalization of siRNA it remains to be a major hurdle to direct exogenous siRNA to the sub-cellular site of action, i.e. the location at which the RNA-induced silencing complex (RISC) catalyzes the siRNA-mediated destruction of target RNA. The phosphorothioate (PS)-stimulated cellular uptake of naked extra-cellular siRNA seems to be promising because (i) it makes use of a caveosomal rather than an endosomal pathway and (ii) the PS-stimulated mode delivers large amounts of siRNA primarily into the perinuclear space which is related to measurable though moderate target suppression [1]. The observed limited efficacy, however, seems to be related to intracellular trapping of siRNA. Argonaute 2 (Ago2) is a key component of RISC and provides its catalytic activity that cleaves target RNA. Our recent observations indicate that the sub-cellular localization of Ago2 and/or its post-translational modification are affected by cell stress induced by heat, NaAsO2, or Lipofectamine-based transfection protocols [2]. All of these kinds of cell stress also affect the efficiency of microRNA- and siRNA-induced RNA interference. [1] Detzer,A. & Sczakiel,G. Phosphorothioate-stimulated uptake of siRNA by mammalian cells: a novel route for delivery.Curr. Top. Med. Chem., 9, 1109-1116 (2009). [2] Detzer,A., Engel,C., WГјnsche,W. & Sczakiel,G., Cell stress is related to re-localization of Argonaute 2 and to decreased RNA interference in human cells. Nucleic Acids Res. 39, 2727-2741 (2011). Georg Sczakiel, PhD Institute for Molecular Medicine University of LГјbeck Building: 50, 1F, Room 1319 Ratzeburger Allee 160 23538 LГјbeck Germany Telephone: +49 451 500 2730 Email: [email protected] 13 Session 1: Hot Topics: Short Talks Selected from Abstracts Peter Sazani Thursday, September 8, 2011 10:00 – 10:15 Preclinical Efficacy and Safety of Phosphorodiamidate Morpholino OligOmers (PMO) Peter Sazani AVI BioPharma, Inc., Bothell WA, USA The development of RNA-based therapeutics has been driven largely by the incorporation of chemical modifications that improve the drug-like character of these agents. AVI’s phosphorodiamidate morpholino oligomer (PMO) platform is a next generation of RNA-based therapeutics with two key modifications; the replacement of ribose with a 6-membered morpholine ring, and the replacement of the natural phosphodiester linkage with a charge-neutral phosphorodiamidate moiety. The biological efficacy of AVI-4658, our lead PMO for the treatment of Duchenne Muscular Dystrophy (DMD), has been demonstrated, and will be discussed in relation to its preclinical efficacy and toxicity profile. In clinical trials in patients, AVI-4658 dosed at up to 20 mg/kg intravenously for 12 weeks yielded dystrophin expression increases of up to 55% of healthy control muscle. These doses were also well tolerated clinically. AVI4658 was also well tolerated in mice (both dystrophic and healthy) and cynomolgus monkeys at up to the maximum feasible doses of 960 mg/kg and 320 mg/kg, respectively. In further modifications, selective addition of positive charges into the PMO backbone have generated a new class of PMOs, called PMOplusв„ў. This platform modification enabled the selection of several preclinically effective antiviral compounds, including a potent influenza lead compound, AVI-7100. In studies in the ferret model of H1N1 disease, AVI-7100 reduced the combined average daily viral titer in nasal wash through peak viral load (days 1 - 3) versus saline and oseltamivir controls, at systemic doses of up to 30 mg/kg. These data show that AVI-7100 is active against a fully virulent and non-adapted strain of pandemic H1N1 virus in the ferret model. In toxicity studies AVI-7100 was also very well tolerated in both rodents and monkeys up to 240 mg/kg. The data presented support that PMOs constitute a platform oligomer chemistry with good drug like properties and therapeutic safety window across several disease areas and chemical modifications. Peter Sazani, PhD AVI BioPharma, Inc. 3450 Monte Villa Parkway Bothell, WA 98021 USA Telephone: 425-354-5087 Email: [email protected] 14 Session 1: Hot Topics: Short Talks Selected from Abstracts Christoph Coch Thursday, September 8, 2011 10:15 – 10:30 5’-triphosphate dsRNA-induced stimulation of RIG-I prevents influenza H1N1 infection of primary nasal epithelial cell culture and protects mice from infection in vivo Coch C.1*, Stehle V.1*, KГјmmerer B.3, Stabenow D.4, StГјmpel J.P.1, DaГџler J.1, Herberhold S.2, LГјdenbach B.1, Kochs G.5, StГ¤heli P.5, Bootz F.2, Schlee M.1, Hartmann E.2*, Hartmann G.1* 1 Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Germany. 2 ENT Department, University Hospital Bonn, Germany. 3 Institute of Virology, University Hospital Bonn, Germany. 4 Institute of Molecular Medicine and Experimental Immunology, University Hospital Bonn, Germany. 5 Institute of Virology, University Hospital Freiburg, Germany * authors contributed equally Background: Causing seasonal epidemic or pandemic outbreaks, Influenza type A represents a severe global health problem. There are limited therapeutic options with virustatic agents due to antigenic shift and evolution of new viral subtypes. The cytosolic helicase вЂ�Retinoic acid inducible gene-I’ (RIG-I) detects viral 5Вґ-triphosphate dsRNA (3p-dsRNA) as contained in viral genomes leading to an antiviral response that protects the host. To circumvent antiviral immune activation the Influenza virus has evolved mechanisms to inhibit recognition of its genome by RIG-I. Nasal mucosa represents the first site of infection of the upper airways. As a new immunotherapeutic strategy against infection with Influenza A (H1N1) virus we investigated the antiviral effect of RIG-I activation by 3p-dsRNA in human primary nasal epithelial cells in vitro and in a mouse model in vivo. Methods: Primary cultures of nasal epithelial cells were generated from conchotomy specimen obtained during surgery. Cells were stimulated with enzymatically generated or synthetic 3p-dsRNA and were subsequently infected with Influenza virus type A (H1N1). IP-10 and IL-6 were analyzed by ELISA, type I IFN with a reporter-assay. Viral load was determined by RT PCR, apoptosis by FACS. Mice were intranasally infected with Influenza with or without 3p-dsRNA application and survival and body weight were monitored. Results: RIG-I was expressed in primary human nasal epithelial cells. Enzymatically generated or synthetic 3p-dsRNA induced antiviral cytokines directly in nasal epithelial cells (IP10, IL-6, type I IFN) in the absence of immune cell subsets. Compared to untreated controls, viral load in nasal epithelial cells exposed to 3p-dsRNA was reduced by 97 %. In comparison, the antiviral activity of recombinant IFN-b was substantially lower. More over, 3p-dsRNA induced antiviral cytokines in mice in vivo and protected mice from infection with Influenza virus. Conclusion: We demonstrate expression and function of RIG-I in primary human nasal epithelium. Stimulation of RIG-I by 3p-dsRNA leads to significant reduction of viral load in vitro and to protection from infection with Influenza virus in vivo. Our results suggest a potential use of 3p-dsRNA as immunotherapeutic agent in Influenza virus infection. Christoph Coch, MD Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn Sigmund-Freud-Str. 25, 53105 Bonn, Germany Telephone: 0049/228/287-14018 or 16080 Email: [email protected] 15 Session 2: Nucleic Acid Chemistry Jesper Wengel Thursday, September 8, 2011 11:00 – 11:30 NUCLEOTIDE CHEMISTRY AND GENE SILENCING Jesper Wengel University of Southern Denmark, Odense Like with any class of drug molecules, chemistry has a distinct influence on key properties of oligonucleotides. The effect can be on pharmacokinetic or pharmacodynamic features, but in fact relatively few oligonucleotide chemistries have been evaluated carefully and only few in vivo head-to-head comparisons have been published. The importance of chemistry will be pointed out from selected examples, and prospects for oligonucleotide medicinal chemistry will be discussed. Jesper Wengel, PhD University of Southern Denmark Nucleic Acid Center Campusvej 55 DK-5230 Odense M Denmark Telephone: +45 65502510 Email: [email protected] 16 Session 2: Nucleic Acid Chemistry Masad J. Damha Thursday, September 8, 2011 11:30 – 12:00 ARABINOSE MODIFIED ANTISENSE AND siRNAs: BIOLOGICAL APPLICATIONS, STRUCTURAL CONSIDERATIONS, AND DELIVERY STRATEGIES Masad J. Damha McGill University, Montreal, Quebec, Canada This presentation will provide an overview of recent work in my laboratory and those of collaborators concerning the properties of oligonucleotides carrying 2’-F sugar modifications, particularly 2’-fluoroarabinonucleic acids (2’-F ANA). We found that the characteristics of 2’-F RNA (C3’-endo) and 2’-fluoroarabinonucleic acid (2’-F ANA; C2’/O4’-endo) make it particularly suitable for the design of highly effective siRNAs. This modification approach also benefits from retention of an A-form dsRNA-like helical conformation. Furthermore, it allows for tuning of duplex thermodynamics, reduced immunostimulation, and increased nuclease resistance without impairment of gene silencing activity against reporter constructs and endogenous genes. In the case of 2’-F ANA:RNA hybrids, a pseudohydrogen bond between the 2’-F and H8 of the nucleobase plays an important role in determining duplex stability, as supported by NMR, molecular modeling, and UV melting experiments. Structural and synthetic aspects of novel fluorinated nucleotide analogues will also be presented. Applications of both modified antisense and siRNAs towards novel endogenous targets will be described, with a focus on ongoing anticancer applications, e.g., treatment of malignant gliomas. To date, a means for effective delivery of both unmodified and modified antisense and siRNA oligonucleotides to target cells and tissues presents a major challenge impeding the development of therapeutic oligonucleotides. To this end, we will also discuss early advances towards new delivery strategies for these compounds, with and without delivery vehicles to facilitate cell uptake. Masad J. Damha, PhD Department of Chemistry, McGill University 801 Sherbrooke St. West Montreal, QC, CANADA, H3A-2K6 Telephone: 514-398-7552 Email: [email protected] 17 Session 2: Nucleic Acid Chemistry Martin Egli Thursday, September 8, 2011 12:00 – 12:30 siRNA Activity, Stability and 3D-Structural Properties of 2’-Fluoro RNA and GNA Martin Egli Departmentof Biochemistry, Vanderbilt University, Nashville, TN 37232-0146, USA Various characteristics of 2’-F RNA make it particularly suitable for the design of highly effective siRNAs. The small size of fluorine enables position-independent incorporation of modified nucleotides into both strands, and its high electronegativity locks the sugar in the RNA-compatible C3’endo conformation. We also found that 2’F-modified siRNAsexhibited increased nuclease stability, significantly decreased immune stimulation in an in vitro model, and, in some cases, improved in vitro and in vivo activity relative to that of the unmodified control RNA.Detailed calorimetric and UV melting experiments revealed that the higher thermal stability of 2’-F-modified duplexes was predominantly due to increased enthalpy rather than entropic effects. This is a surprising finding given the analog’s reputation to conformationally preorganize the backbone for an RNA target. We hypothesize that the favorable enthalpy exhibited by 2’-F RNA is due to strengthening of Watson-Crick base pairing and/or enhanced stacking interactions. We have tested these possibilities using high-field NMR and further thermodynamic measurements. Glycol nucleic acid(GNA) is arguably the simplest artificial nucleic acid pairing system with a phosphodiester backbone. Despite its acyclic backbone GNA is capable of stable self-pairing and the (S)-GNA enantiomercross-pairs with RNA. Neither (S)- nor (R)-GNA exhibit stable pairing with DNA. Meggers and coworkers reported crystal structures of (S)-GNA duplexes whose righthanded geometry differed substantially from that of a canonical A-RNA duplex. However, despite a large helical pitch andan increased number of residues per turn, (S)-GNA shares with RNA a strong negative inclination between the backbone and base axes. This latter feature may provide a conformational basis for the formation of stable (S)-GNA:RNA duplexes. We hypothesize that (S)-GNA, similar to DNA, is able to adapt to the conformational constraints of RNA and that the geometry of the hybrid duplex resembles the A-form. Ideally, an analysis of GNA’s conformational properties will allow insights into the structural underpinnings of the pairing with RNA and its inability to pair with DNA. Moreover, one would expect structural data to provide an explanation for the stereoselective pairing with RNA [i.e. (S)-GNA pairs and (R)-GNA does not]. We recently determined the crystal structure of an RNA dodecamer duplex with incorporated (S)-GNA-Ts at high resolution and the pairing properties of GNA will be discussed in light of the structural data. Supported by the US NIH, R01GM055237; Lab URL http://structbio.vanderbilt.edu/~eglim/ References M. Manoharan, A. Akinc, R. K. Pandey, J. Qin, P. Hadwiger, M. John, K. Mills, K. Charisse, M. A. Maier, L. Nechev, E. M. Greene, P. S. Pallan, E. Rozners, K. G. Rajeev, and M. Egli (2011) Unique gene-silencing and structural properties of 2’-F modified siRNAs. Angew. Chem. Int. Ed. 50: 2284-2288. P. S. Pallan, E. Greene, P. Jicman, R. Pandey, M. Manoharan, E. Rozners, and M. Egli (2011) Unexpected origins of the enhanced pairing affinity of 2′-fluoro-modifiedRNA. Nucleic Acids Res. 39: 3482-3495. P. S. Pallan, P. Lubini, M. Bolli, and M. Egli (2007) Backbone-base Inclination as a fundamental determinant of nucleic acid self- and cross-pairing.Nucleic Acids Res. 35: 6611-6624. Martin Egli, PhD Departmentof Biochemistry, Vanderbilt University Nashville, TN 37232-0146 USA Telephone: 1-615-343-8070 Email: [email protected] 18 Keynote Session: Oligonucleotides reviewed in memory of Alan Gewirtz Fritz Eckstein Thursday, September 8, 2011 14:00 – 14:30 OLIGONUCLEOTIDE THERAPEUTICS REVIEWED IN MEMORY OF ALAN GEWIRTZ Eckstein, F. Max-Planck-Institute for Experimental Medicine, GГ¶ttingen, Germany The passing away of Alan Gewirtz fills us with great sorrow as we have lost a great colleague and a good friend. He was one of the founding fathers of OTS and a pioneer in the oligonucleotide field to be developed for medical application. As a practising clinician he was one of the few of us to combine in an admirable way wide ranging basic research with the application in the clinic. Early on he administered his c-myb antisense oligonucleotide to chronic myeloid leukaemia (CML) patients. The commitment to his patients stimulated his strength with never ending dedication to his work. His strong belief that the oligonucleotide approach will eventually bring the expected result for the patients was contagious and stimulated us, his colleagues, to look for improvements of our strategies. The oligonuclotide field has moved forward considerably since Alan began his work. Thus RNase-H dependent cleavage of mRNA is now one of several strategies, followed by various RNAi approaches with great potential. Another powerful area with no cleavage of RNA is the correction of faulty splicing, a major cause for diseases. Oligonucleotide-protein interaction rather than interaction with RNA is yet another wide area of interest. This is exemplified by oligonucleotides binding to Toll Like Receptors (TLRs) as agonists for stimulation of the immune response or similarly binding to the retinoic acid-inducible gene (RIG-1). Also aptamers as alternatives to antibodies fall into the category of oligonucleotides binding to proteins. Thus, the field has come a long way opening up great potentials. We will miss Alan’s strong commitment, dedication, stimulation and help. Fritz Eckstein, PhD Max-Planck-Institute for Experimental Medicine Hermann-Rein-Str. 3 37075 GГ¶ttingen, Germany Telephone: (49) 551-3899 274 Email: [email protected] 19 Session 3: RNA Biology: New Frontiers Chang-Zheng Chen Thursday, September 8, 2011 14:30 – 15:00 PRIMARY microRNA FUNCTION IN TARGET RECOGNITION AND REPRESSION Chang-Zheng Chen, Si-Biao Yue, Robin Deis Trujillo, Yujie Tang, & William E. O’Gorman Department of Microbiology and Immunology, Baxter Laboratory for Stem Cell Biology, Stanford University School of Medicine, Stanford, CA 94305 USA MicroRNA (miRNA) genes encode small regulatory RNAs that control gene expression at the post-transcriptional level. Despite progress in the field, many fundamental questions regarding mechanisms of action during target recognition and repression remain unresolved. In particular, one critical problem that has largely been overlooked is whether precursor miRNA molecules, including primary (pri-) and precursor (pre-) miRNAs, have direct roles in target recognition and repression. Both pri-miRNAs and pre-miRNAs contain the mature miRNA sequences and in theory can interact with target mRNAs. Addressing this question is essential in order to define the regulatory information used in target recognition and the molecular steps required for target repression. We found that structural and sequence elements outside the mature miRNA region control the activities of miRNA genes and that pri-miRNAs can have a direct role in target recognition and repression. These findings demonstrate that regulatory information encoded in the structural and sequence elements of pri-miRNAs can be translated into activity through direct interactions between target mRNAs and structured primiRNAs and may have broad implications in understanding the mechanisms by which miRNAs recognize and repress their cognate targets. Chang-Zheng Chen, PhD Department of Microbiology and Immunology, Baxter Laboratory for Stem Cell Biology Stanford University School of Medicine Stanford, CA 94305 USA Telephone: 650-725-1667 Email: [email protected] 20 Session 3: RNA Biology: New Frontiers Gunter Meister Thursday, September 8, 2011 15:00 – 15:30 Regulation of small RNA function Gunter Meister University of Regensburg and Max-Planck-Institute of Biochemistry, Munich Argonaute (Ago) proteins are highly conserved between species and constitute a direct binding platform for small RNAs including short interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi interacting RNAs (piRNAs). Small RNAs function as guides whereas Ago proteins are the actual mediators of gene-silencing. Although extensive research has elucidated the major steps in small RNA guided gene silencing, not much is known about Ago protein regulation. We have performed a comprehensive analysis of Ago2 phosphorylation in human cells. We find that the highly conserved tyrosine Y529, located in the small RNA 5’ end-binding pocket of Ago proteins can be phosphorylated. By substituting Y529 with a negatively charged glutamate mimicking a phosphorylated tyrosine, we show that small RNA-binding is strongly reduced. Our data suggest that a negatively charged phosphotyrosine generates a repulsive force that prevents efficient binding of the negatively charged 5’ phosphate of the small RNA. In addition, we have identified another phosphorylation site located in the PIWI domain of Ago2. Phosphorylation of a specific residue does not alter small RNA binding. However, it interferes with Ago2 cleavage activity by affecting the catalytic center. In summary, we have found that different phosphorylation events influence Ago activities by different mechanisms. Gunter Meister, PhD University of Regensburg Universitaetsstrasse 31 Regensburg, 93053 Germany Telephone: 0049 941 943 2847 Email: [email protected] 21 Session 3: RNA Biology: New Frontiers Mark Kay Thursday, September 8, 2011 15:30 – 16:00 The mechanism of miRNA and siRNA Argonaute loading in mammals Mark A. Kay, Shuo Gu, and Lan Jin Depts of Pediatrics and Genetics Stanford University School of Medicine Stanford, CA USA The mammalian RNA induced silencing complex (RISC) contains a single-stranded RNA derived from a duplex miRNA/siRNA and one-of-four Argonaute (Ago) proteins. Argonaute proteins contain four domains referred to as: N-terminal, Paz, MID and PIWI. We have performed various genetic analyses in living cells to study the molecular process of RISC assembly and activation. Ago loading is the process of duplex RNA association followed by removal of the inactive passenger strand RNA. We have established that mammalian Agos bind duplex RNAs without discrimination and the passenger strand removal is the rate-limiting step during non-cleavage RISC activation. The degree of RISC activation correlated with the thermodynamic instability of the duplex RNAs rather than their structure. Interestingly, Ago loading of siRNAs was less sensitive to the thermostability compared to their shRNA counterparts. By studying RISC loading in Paz-deficient Ago mutants, we established that the PAZ domain plays a critical role in the unwinding of duplex RNAs. We will discuss the process and provide a model for RISC loading. Taken together, these results provide the rationale for future strategies in siRNA and shRNA designs including insights into how to devise potent si/ shRNAs with minimal off-target effects for therapeutic applications. Mark A. Kay, MD, PhD Dennis Farrey Family Professor Departments of Pediatrics and Genetics Vice Chair for Basic Research (Pediatrics) Stanford University School of Medicine 269 Campus Drive CCSR Bldg., Suite 2105 Stanford, CA USA 94305 Telephone: 650-498-6531 Email: [email protected] 22 Session 4: Delivery of Nucleic Acids Muthiah Manoharan Thursday, September 8, 2011 16:30 – 17:00 CHEMICAL STRATEGIES FOR DELIVERY OF RNAi DRUGS Muthiah Manoharan Alnylam Pharmaceuticals, 300 Third Street, Cambridge MA 02493 USA At Alnylam Pharmaceuticals, we have developed and applied multiple chemistry strategies to address the challenge of cellular delivery of drugs that function through RNAi pathways. These include chemical modifications of oligonucleotides, molecular conjugates and delivery systems based on liposomal nanoparticles (LNPs). Our progress in these areas will be summarized. References I. Chemical Modifications of RNAi 1. Manoharan, M. “RNA interference and chemically modified small interfering RNAs.” Curr. Opin. Chem. Biol. 2004, 8, 570-579. 2. Bumcrot, D. et al. “RNAi therapeutics: a potential new class of pharmaceutical drugs.” Nature Chemical Biology 2006, 2, 711-719. 3. Manoharan, M. and Rajeev, K. G. “Utilizing chemistry to harness RNA interference pathways for therapeutics: chemically modified siRNAs and antagomirs.” Antisense Drug Technology (2nd Ed.), 2008, 437-464. 4. Zlatev, I. et al. “Efficient solid-phase chemical synthesis of 5’-triphosphates of DNA, RNA and their analogs.” Organic Letters 2010, 12, 2190-2193. 5. Watts, J. et al. “Effect of chemical modifications on modulation of gene expression by duplex antigene RNAs that are complementary to non-coding transcripts at gene promoters.” Nucleic Acids Res. 2010, 38, 5242-5259. 6. Addepalli, H. et al. “Modulation of thermal stability can enhance the potency of siRNA.” Nucleic Acids Res. 2010, 38, 7320-7331. 7. Manoharan, M. et al. “Unique gene-silencing and structural properties of 2’-F modified siRNAs.” Angewandte Chemie, (International Edition) 2011, 50, 2284-2288. 8. Pallan, P. S. et al.;. Unexpected origins of the enhanced pairing affinity of 2’-fluoromodified RNA. Nucleic Acids Res. 2011, 39, 3482-3495. II. Conjugates for siRNA and Antagomir Delivery 9. Soutschek, J. et al. “Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.” Nature, 2004, 432, 173-178. 10.Kruetzfeldt, J. et al. “Silencing of microRNAs in vivo with вЂ�antagomirs’.” Nature, 2005, 438, 685-689. 11. Wolfrum, C. et al., “Mechanisms and optimization of in vivo delivery of lipophilic siRNAs.” Nature Biotech. 2007, 25, 1149-1157. 12.Wu, Y. et al. “Durable protection from herpes simplex virus-2 transmission following intravaginal application of siRNAs targeting both a viral and host gene.” Cell Host & Microbe 2009, 5, 84-94. 13.Querbes, W. et al. “Direct CNS delivery of siRNA mediates robust silencing in oligodendrocytes.” Oligonucleotides 2009, 19, 23-30. Continued on the next page 23 Session 4: Delivery of Nucleic Acids Muthiah Manoharan Thursday, September 8, 2011 16:30 – 17:00 Continued from the previous page 14.Chen, Q. et al. “Lipophilic siRNAs mediate efficient gene silencing in oligodenrocytes with direct CNS delivery.” Journal of Controlled Release 2010, 144: 227-232. 15.DiFiglia, M. et al. “Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits.” PNAS, 2007 104, 17204-17209. 16.Alam, Md. R. et al. Multivalent Cyclic RGD Conjugates for Targeted Delivery of Small Interfering RNA. Bioconjugate Chemistry, 2011, (in press). 17.Jayaprakash, K. N. et al. “Non-nucleoside building blocks for copper-assisted and copper-free click chemistry for the efficient synthesis of RNA conjugates.” Organic Letters 2010, 12, 5410-5413. 18.Yamada, T. et al. “Versatile site-specific conjugation of small molecules to siRNAs using click chemistry.” Org. Chem., 2011, 76, 1198-1211. III.Liposomal Nanoparticles (LNPs) 19.Zimmermann, T. et al. “RNAi-mediated gene silencing in non-human primates.” Nature, 2006,В 441,В 111-114. 20.Akinc, A. et al. “A combinatorial library of lipid-like materials for delivery of RNAi therapeutics.” Nature Biotech. 2008, 26, 561-569. 21.Frank-Kamenetsky, M. et al. “Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates.” PNAS, 2008, 105, 11915-11920. 22.Akinc, A. et al., “Development of lipidoid-siRNA formulations for systemic delivery to the liver.” Molecular Therapy, 2009, 17, 872-879. 23.Love, K. T. et al., “Lipid-like materials for low-dose, in vivo gene silencing.”PNAS 2010, 107, 9915. 24.Akinc, A. et al. “Targeted delivery of RNAi therapeutics.” Molecular Therapy 2010, 18, 1357-1364. 25.Semple, S. et al. “Rational design of cationic lipids for siRNA delivery.” Nature Biotechnology 2010, 28, 172-176. Muthiah Manoharan, PhD Alnylam Pharmaceuticals 300 Third Street Cambridge MA 02493 USA Telephone: 617-551-8200 Email: [email protected] 24 Session 4: Delivery of Nucleic Acids Gilles Divita Thursday, September 8, 2011 17:00 – 17:30 NON-COVALENT PEPTIDE-BASED DELIVERY SYSTEMS G. Divita, A. Rydstrom, S. Deshayes, K. Konate CRBM-CNRS-UMR5237, Dpt-Molecular Biophysics & Therapeutics, 1919 route de Mende, Montpellier, France The development of siRNA, has provided great hope for therapeutic targeting of specific genes responsible of patholological disorders. However their clinical application remains limited by their poor cellular uptake and insufficient capability to reach targets in vivo. We have designed the “NANOVEPEP” strategy based on short amphipathic peptides that forms stable positively-charged nanoparticles with siRNA, through non-covalent electrostatic and hydrophobic interactions. Self assembly of NANOVEPEP molecules around siRNA leads to a “raspberry”-like nanoparticle associating several peptide/siRNA complexes of 15-25 nm, cemented together by a matrix of free peptides. NANOVEPEP promotes efficient targeted-delivery of siRNA or small oligonucleotides into a wide variety of cell lines. Cellular uptake mechanism of NANOVEPEP/siRNA nanoparticles is dependent on the size of the particle and involves membrane dynamic. When applied by systemic intravenous injection, NANOVEPEP promotes the delivery of siRNAs in most of the tissues without triggering any nonspecific inflammatory response. The surface of NANOVEPEP particles can easily be functionalized and we showed that addition of cholesterol-, peptide- or antibody-moiety significantly improves stability in vivo and enables lung, brain, ganglion or tumor targeting. The therapeutic potential of this strategy have been validated for cancer treatment by targeting major cell cycle regulatory proteins in various mouse tumour models. We demonstrated that NANOVEPEP-mediated delivery of Cyclin B1 siRNA (0.1 mg/Kg) prevents tumour growth in vivo following systemic intravenous injections. Given the robustness of the biological response achieved through this approach, we propose that non-covalent, NANOVEPEP based technologies hold a strong promise for therapeutic administration of siRNA. Gilles Divita, PhD Centre de Recherches de Biochimie MacromolГ©culaire UMR 5237, CNRS, UM-1, UM-2 CRBM-Department of Molecular Biophysics and Therapeutics Montpellier, France Telephone: 33 (0) 4 67613392 Email: [email protected] 25 Session 4: Delivery of Nucleic Acids Ken Clark Thursday, September 8, 2011 17:30 – 18:00 INVESTIGATING THE POTENTIAL OF THERAPEUTIC OLIGONUCLEOTIDES FOR PULMONARY DISEASES Ken Clark, Stephen Hughes, Jeremy Kitson, Jill Coates, Joel Parry, Chris Gruenloh, Daren Levin, Pallav Bulsara, Scott Summerfield & Mark Edbrooke Respiratory Therapeutics and Platform Technology & Science, GlaxoSmithKline, Stevenage, United Kingdom & Research Triangle Park, USA • Respiratory disease is an attractive opportunity for oligonucleotide therapeutics given the ability to deliver molecules locally by the inhaled or intranasal route. Such local delivery to the airways should achieve high concentrations in the target tissue while minimising systemic exposure, thereby potentially enhancing efficacy and therapeutic index. • Nevertheless, the airways are adept at responding to and preventing the cellular entry of oligonucleotides and so new delivery technologies may well be required to address the significant challenge of turning the promise of therapeutic oligonucleotides in respiratory disease into a reality for patients. • Significant progress has being made by many groups and several inhaled oligonucleotide programmes have now advanced to clinical evaluation. This presentation will review progress in the field with both inhaled siRNA and anti-sense oligonucleotide therapeutics, and discuss the remaining key research and development challenges. Ken Clark, PhD Respiratory Therapeutics, GlaxoSmithKline Stevenage SG1 2NY, United Kingdom Telephone: +44 7920 567189 Email: [email protected] 26 Session 5: Immunobiology of Nucleic Acids Gunther Hartmann Friday, September 9, 2011 08:30 – 09:00 RIG-I and tolerance of self-RNA Gunther Hartmann Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, Bonn, Germany The innate immune system detects viruses based on the recognition of viral nucleic acids. Immunorecognition of viral nucleic acids leads to the initiation of early antiviral immune responses that limit viral replication and are essential for eliciting acquired immune responses to virus specific antigens. Detection of viral nucleic acids in the cytosol is based on the family of the so-called RIG-I-like receptors (RLRs). The cytosolic helicase RIG-I is highly conserved in vertebrates, including mammals, birds and fish, and is ubiquitously expressed in all cell types and tissues. In previous work we identified the ligand of RIG-I (5Вґ-triphosphate RNA) and characterized the molecular interaction with its ligand (1-3). Since viral and self RNA are located in the same compartment (cytosol) discrimination of self versus viral RNA is molecularly challenging. Like viral RNA, cytosolic self mRNA harbors a triphosphate moiety at its 5’end; however, self mRNA contains a 5’triphosphate linked N7-methylguanidine (m7G) mRNA cap (cap0). In higher eukaryotes mRNA additionally possess 2’O-methylations at the 5’-penultimate residues N1 or N1+N2 (cap1 or cap2). It is well-known that N7-methylation is essential for the attachment of cap binding proteins and translation; however, the role of 2’O-methylations is poorly defined. Our recent work now provides evidence that 2’O-methylation of mRNA cap1 is the key structure which allows the innate virus sensor RIG-I to discriminate self from pathogenic RNA. We found that a highly conserved amino acid in RIG-I controls immune tolerance of 2′O-methylated cap1 self RNA. Our results demonstrate for the first time a novel principle in innate immunity: that an immunoreceptor specifically evolved to tolerate defined self structures rather than to molecularly adapt to foreign molecular structures. RIG-I is the first example of this new principle, and the same may apply to other innate immunoreceptors. The structural requirements for RIG-I ligands now led us to new highly active and selective RIG-I oligonucleotide ligands. We demonstrate that local intratumoral injection of such a novel synthetic RIG-I oligonucleotide ligand shows superior anti-tumor activity in otherwise difficult-to treat mouse models of pre-established melanoma. Thus, RIG-I is an extremely interesting target for the development of new treatments for cancer and viral infection. High conservation between species allows much better prediction of therapeutic activities compared to the diverse family of Toll-like receptors with its inter-species differences. 1. Hornung V, … and Hartmann G. 5’-Triphosphate RNA Is the Ligand for RIG-I. Science 2006; Nov 10; 314:994-7. 2. Schlee M, … and Hartmann G. Recognition of 5Вґ-triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative strand virus. Immunity 2009, 31:25-34. 3. Wang Y, Ludwig J, ...Tuschl TВ°, Hartmann GВ°, Patel DJВ°. 2010. Structural and functional insights into 5вЂ�-ppp RNA pattern recognition by the innate immune receptor RIG-I. Nat Struct Mol Biol 2010;17:781-7. В°equally contributed Gunther Hartmann, MD, PhD Institute of Clinical Chemistry and Clinical Pharmacology University Hospital Sigmund-Freud-Str. 25, 53127 Bonn, Germany Telephone: +49-22816080 Email: [email protected] 27 Session 5: Immunobiology of Nucleic Acids Franck Barrat Friday, September 9, 2011 09:00 – 09:30 Regulation of the Innate Immune Response Using Oligonucleotide-based Inhibitors of Toll-Like Receptors Franck J. Barrat Dynavax Technologies, Berkeley, California, USA SLE is an autoimmune disease where the innate tolerance to self nucleic acids is broken with devastating consequences. The hallmark of the disease is an increased IFN-О± signature in the blood which is tightly associated with levels of autoantibodies and disease activities. Selfnucleic acid recognition by like receptors (TLR)7 and TLR9 on B cells and PDC is believed to be key in the pathogenesis of SLE promoting immune complexes (IC) and the production of type I IFN, both of which are associated with the severity of the disease. We have generated oligonucleotide-based bi-functional inhibitors of TLR7&9 (called ImmunoRegulatory Sequences, IRS) and have shown that these can block IFN production by PDC as well as B cell activation. In addition, IRS are active in vivo and treatment of lupus-prone mice lead to reduced disease symptoms and end-organ damage. SLE patient are often treated with glucocorticoids (GC) but under maintenance levels often suffer from disease flares that necessitate high dose pulse therapy. We have shown that PDC were significantly more resistant to GC induced death in lupus-prone mice, a phenomenon that was completely reversed by pre-treatment with TLR7&9 inhibitor. These data provide a new understanding of the role of self recognition of DNA and RNA by TLR as an important parameter during inflammatory response. These data also stress the potential utilization of TLR7&9 specific inhibitors as cortico-sparing drugs which would be open new possibilities with respect to therapeutic applications. Finally, we have shown that IRS can prevent skin lesions following mechanical injury by blocking PDC activation in the skin environment. The lead IRS inhibitor, called DV1179, has recently entered a human clinical trial and its safety will be assessed in multiple ascending doses. The pharmacokinetics and pharmacodynamics properties of DV1179 in rodents and non-human primates have been key to the design of the trial. Franck J. Barrat, PhD Dynavax Technologies 2929 Seventh Street Berkeley CA 94710 USA Telephone: +1.510.665.7266 Email: [email protected] 28 Session 5: Immunobiology of Nucleic Acids Patricia Fitzgerald-Bocarsly Friday, September 9, 2011 09:30 – 10:00 INTRACELLULAR TRAFFICKING OF TLR LIGANDS IN HUMAN PLASMACYTOID DENDRITIC CELLS Patricia Fitzgerald-Bocarsly UMDNJ- New Jersey Medical School, Newark, New Jersey, USA Human plasmacytoid dendritic cells (pDC) are the most potent producers of both type I and type III interferons in the body, signaling through TLR7 and TLR9. We have demonstrated that in addition to responding to cell-free virus and CpG A stimuli, pDC also respond vigorously to virus-infected cells with the production of these IFNs. Using conventional flow cytometry, Amnis ImageStream imaging flow cytometry and confocal microscopy, we have investigated the uptake and intracellular trafficking of labeled CpG oligonucleotides, GFP-expressing HSV-1 and membrane and cytoplasm from HSV-infected and uninfected Raji cells by pDC. Additionally, the preferential uptake of portions of virus-infected cells vs. uninfected cells by pDC but not conventional (cDC) and the subsequent distinctive trafficking of material from early to late endosomal compartments, IFN production and maturation in these DC sub-populations will be discussed. Data demonstrating that the autophagy pathway, including the formation of amphisomes, is activated by virus (HSV, Influenza and HIV) or CpG A interaction with pDC, and that IFN-a production in these cells is inhibited by inhibitors of the PI-3 kinase pathway/autophagy will also be presented. Patricia Fitzgerald-Bocarsly, PhD UMDNJ – New Jersey Medical School 185 So. Orange Ave. Newark, NJ 07103 USA Telephone: 973-972-5233 Email: [email protected] 29 Session 6: Targeting Coding RNA Cy Stein Friday, September 9, 2011 10:30 – 11:00 Gymnotic Delivery of Antisense Oligonucleotides Abstract unavailable at time of printing. 30 Session 6: Targeting Coding RNA Frank Rigo Friday, September 9, 2011 11:00 – 11:30 Abstract available for review at the meeting. 31 Session 6: Targeting Coding RNA Erik Stroes Friday, September 9, 2011 11:30 – 12:00 Clinical development of mipomersen Abstract unavailable at time of printing. 32 Session 7: Targeting microRNA Neil Gibson Friday, September 9, 2011 13:30 – 14:00 THERAPEUTIC TARGETING OF microRNAs Neil W. Gibson, Balkrishen Bhat, Christine C. Esau, Scott Davis, Jia Tay, Eric Marcusson, Hubert Chen, Aimee L. Jackson, Lars Karlsson, 1Kathryn J. Moore, 2Andrei Goga Regulus Therapeutics, San Diego, CA, 1Dept of Cell Biology, NYU School of Medicine, New York, NY, 2Dept of Medicine, UCSF, San Francisco, CA microRNAs act as master regulators in biological pathways, and are dysregulated in disease areas including cancer, metabolism, fibrosis, and inflammation. Their ability to modulate disease pathways makes targeting microRNAs an exciting new approach for drug discovery. Oligonucleotides that inhibit microRNA function have been termed anti-miRs. Critical to the development of anti-miRs as a therapeutic modality are chemical modifications to enhance stability and target affinity, and an understanding of functional biodistribution of anti-miRs to cells and tissues of therapeutic interest. Systemic and local delivery of unformulated antimiRs enables broad distribution for targeting microRNA function in a diverse range of tissues and cell types. We present recent advances in our use of anti-miRs against two specific targets – miR-33 in metabolic disease and miR-21 in oncology. miR-33a and b are intronic microRNAs located within the SREBF2 and SREBF1 genes, respectively. This microRNA family suppresses the expression of the ABCA1 cholesterol transporter and lowers HDL levels. LDL receptor-deficient mice treated with anti-miR-33 showed an increase in circulating HDL levels as well as enhanced reverse cholesterol transport to the plasma, liver, and feces. The anti-miR-33-treated mice had reduced plaque size and lipid content, increased markers of plaque stability, and decreased inflammatory gene expression. The systemic delivery of an antisense oligonucleotide that targets both miR-33a and miR-33b increases hepatic expression of ABCA1 and induces a sustained increase in plasma HDL cholesterol in African green monkeys. These data suggest the modulation of microRNA function as a promising strategy to treat atherosclerotic vascular disease. miR-21 is frequently over-expressed and has been shown to correlate with poor outcome in multiple cancer types. We have used a publically available data set from 86 patients to show that miR-21 is over-expressed in hepatocellular carcinoma (HCC). Short term treatment with the anti-miR-21 oligonucleotide in a genetically engineered mouse model of HCC led to a reduction in tumor formation and an increased survival advantage. Furthermore, inhibition of miR-21 was clearly demonstrated by analysis of genome wide mRNA expression data from treated versus untreated tumors. Our findings suggest that miR-21 is a promising candidate for the therapeutic intervention of liver cancer and further highlights the potential of antimiR-mediated inhibition of microRNAs in cancer. Overall our data suggest the therapeutic utility of anti-miRs targeting microRNAs involved in human disease pathogenesis. Neil W. Gibson, PhD 3545 John Hopkins Ct San Diego, CA 92130 USA Telephone: 858-202-6303 Email: [email protected] 33 Session 7: Targeting microRNA Thomas Thum Friday, September 9, 2011 14:00 – 14:30 Therapeutic targeting of individual cardiac cell types by miRNA antagonists T. Thum Institute for Molecular and Translational Thereapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany Cardiac stress leads to remodeling and development of heart failure. At the cellular level, cardiac cells are very sensitive to environmental changes and common pathophysiological responses include development of cardiomyocyte hypertrophy, cardiac fibrosis and insufficient vascularization. In the last years, it was shown that specific microRNAs (miRNA) contribute to cellular dysfunction in the heart and first miRNA-based therapeutic strategies have been successfully developed. We here will summarize recent work of our laboratory about the use of miRNA inhibitors to target various miRNAs and downstream target networks to result in beneficial cardiac healing and improved function. Thomas Thum, MD, PhD Director, Institute for Molecular and Translational Therapeutic Strategies (IMTTS) Integrated Research Center (IFB-Tx) Medical School Hannover (MHH) Carl-Neuberg-StraГџe 1, 30625 Hannover, Germany Telephone: +49 511 532-5272 (office) Email: [email protected] 34 Session 7: Targeting microRNA Andrea Ventura Friday, September 9, 2011 14:30 – 15:00 Oncomir-1 in cancer and development: a tale of mice and men Andrea Ventura Memorial Sloan Kettering Cancer Center, New York, NY, USA MiR-17~92 has emerged as the prototypical oncogenic microRNA cluster in humans and mice. It encodes six distinct miRNAs that can be grouped into four “seed families” (miR17/20, miR-18, miR-19a/b and miR-92). We have previously reported the generation and characterization of mice carrying a targeted deletion of the entire cluster. Homozygous mutant mice are significantly smaller than their wild type counterpart, die soon after birth and display a complex array of defects involving heart lungs and lymphocyte development. One limitation of such an analysis is that these results were obtained by deleting the entire miR-17~92 locus. As such, little is known with respect to the relative role of each of the six microRNAs encoded by miR-17~92. I will discuss the results of two parallel lines of investigation that our laboratory is undertaking to address this important issue: a) We have carried out a systematic genetic analysis by creating an allelic series of knockin mice, each lacking only one of the four “seed families” encoded by miR-17~92. To our knowledge, this is the first time such an analysis is being carried out in mammals. b) In a parallel line of investigation, we have used a conditional miR-17~92 knockout allele to determine the role of this cluster in Myc-induced B cell lymphomas. By using this approach we show that endogenous miR-17~92 is required in lymphoma to suppress apoptosis via the concerted action on a number of genes and that this effect is largely, if not exclusively attributable to the miR-19 seed family. Andrea Ventura, MD, PhD Dept. of Cancer Biology and Genetics Memorial Sloan Kettering Cancer Center 408 East 69th,В ZRC-1201 New York, NY, 10065 USA Telephone: 646-888-3068 Email: [email protected] 35 Session 8: Targeting Liver and Tumors Aby Buchbinder Friday, September 9, 2011 15:30 – 16:00 EVALUATION OF LOCKED NUCLEIC ACID (LNA)-BASED mRNA ANTAGONISTS IN CANCER PATIENTS Buchbinder A, Kalambakas S, Huq N, Berkowitz N Enzon Pharmaceuticals, Inc., Piscataway, NJ, USA Background: HIF-1 is a transcription factor that is a critical mediator of angiogenesis, cell proliferation, metabolism, survival, and adaptive responses to stress. EZN-2968 is a potent locked nucleic acid antisense oligonucleotide that downmodulates HIF-1О± mRNA and protein in vitro (IC50 <1 nM and <5 nM, respectively). Survivin, the smallest member of the inhibitors of apoptosis protein gene family, functions as a key regulator of mitosis and apoptosis. EZN-3042 is a potent locked nucleic acid antisense oligonucleotide that down-modulates survivin mRNA and consequently protein in vitro (IC50 <5 nM). The LNA based mRNA antagonists EZN-2968 and EZN-3042 were evaluated in patients with cancer in Phase 1 clinical trials (1,2). Methods: The primary objective of the studies was to determine the maximum tolerated doses (MTD) and recommended Phase 2 dose. Secondary objectives were to evaluate safety, tolerability, pharmacokinetics (PK), and pharmacodynamics. Results: EZN-2968: 49 pts received weekly EZN-2968 doses for 3, 4, or 5 of 6 weeks. Doselimiting toxicities (DLT) were intracerebral bleeding (n=1, 3.5 mg/kg), Grade 3 fatigue (n=2, 8 mg/kg, 18 mg/kg for 5 of 6В weeks), and Grade 3 increased AST (n=1, 18 mg/kg for 5 of 6 weeks). The MTD for EZN-2968 is 18 mg/kg given weekly for 4 out of 6 weeks. Drug-related adverse events (AEs) reported for >10% of pts were fatigue (29%) and headache (14%). Grade 3 drug-related AEs (fatigue [n=3], AST increase [n=3], ALT increase [n=2], and hyponatremia [n=1]) were reported for 7 patients (14%); no drug-related AE was Grade 4 or 5. The current best response for EZN-2968 is stable disease (SD by RECIST) in 16 pts; 7 pts were treated for >90В days. Objective tumor shrinkage was observed in several pts. HIF-1О± expression was evaluated pretreatment and on-treatment in tumor (n=6) and skin (n=41) samples. Tumor biopsy HIF-1О± mRNA decreased in 4 pts, increased in 1 pt, and did not change in 1 pt. Skin biopsy HIF-1О± mRNA decreased in 63% (26/41) of pts, remained the same in 10% (4/41) of pts, and increased in 27% (11/41) of pts.EZN-3042: 24 pts received weekly EZN3042 doses for 4 out of 4 weeks. DLT was observed in 3 pts at 8В mg/kg (Grade 3 increased aspartate orВ alanine aminotransferase [AST or ALT]). The MTD for EZN-3042 is 6.5В mg/kg. Drug-related adverse events (AEs) (in >15%В pts) were AST increase (42%), ALT increase (38%), fatigue (33%), and diarrhea (17%). Most AEs were Grade 1 or 2. The best response was stable disease inВ 5 pts. Conclusions: The MTD of EZN-2968 is 18 mg/kg given weekly for 4 out of 6 weeks. EZN2968 was generally well tolerated in previously treated pts with advanced tumors who received up to 10 cycles of EZN-2968. The best response was SD. Evidence for down-regulation of the HIF-1О± target is supported by observations in tumor and skin biopsies. The MTD of EZN-3042 is 6.5 mg/kg. EZN-3042 was well tolerated in previously treated pts with advanced tumors. 1. Cohen RB, Olszanski A, Figueroa J, et al; AACR 2011 Annual Meeting, Abs. LB-407 2. Tolcher AW, Patnaik A, Papadopoulos KP, et al; AACR 2011 Annual Meeting, Abs. LB-409 Aby Buchbinder, MD Enzon Pharmaceuticals, Inc. 20 Kingsbridge Rd Piscataway, NJ, 08854 USA Telephone: 1-(732)-980-4817 Email: [email protected] 36 Session 8: Targeting Liver and Tumors Dinah Sah Friday, September 9, 2011 16:00 – 16:30 ALN-TTR, AN RNAi THERAPEUTIC FOR THE TREATMENT OF TRANSTHYRETIN AMYLOIDOSIS Dinah W. Y. Sah1, Qingmin Chen1, Susete Costelha2, Jim Butler1, Shannon Fishman1, Anthony Rossomando1, Lubomir Nechev1, Maria Joao Saraiva2, Teresa Coelho3, Ole B. Suhr4, David Adams5, Pierre Lozeron5, Philip Hawkins6, Timothy Mant7, Renta Hutabarat1, Rick Falzone1, Jeff Cehelsky1, Yaysie Figueroa1, Akshay Vaishnaw1, Jared Gollob1 1 Alnylam Pharmaceuticals, Cambridge, MA 02142, USA 2 Molecular Neurobiology Group, Institute for Molecular and Cell Biology, Porto, Portugal 3 Unidade Clinica de Paramiloidose, Porto, Portugal 4 Department of Medicine, UmeГҐ University, UmeГҐ, Sweden 5 National Referral Center for FAP, Le Kremlin BicГЄtre, France 6 National Amyloidosis Centre, London, United Kingdom 7 Quintiles Drug Research Unit, London, United Kingdom Transthyretin amyloidosis (ATTR) is a fatal, autosomal dominant, multisystem disease caused by abnormal extracellular deposits of transthyretin (TTR) amyloid that lead to familial amyloidotic polyneuropathy (FAP) and/or familial amyloidotic cardiomyopathy (FAC), depending on the sites of deposition. More than 100 TTR mutations have been reported, with the vast majority resulting in amyloid pathology. Wild-type TTR also contributes to pathology and clinical progression. There is a high unmet medical need for new therapies, with liver transplantation being the only available treatment for a subset of FAP patients. ALN-TTR is a systemically administered lipid nanoparticle-formulation of a small interfering RNA (siRNA) targeting wild-type and all mutant forms of TTR. This formulation delivers the siRNA predominantly to the liver, thereby inhibiting TTR synthesis at the primary site of production. In transgenic mice expressing the human V30M transgene in a heat shock transcription factor 1 null background, ALN-TTR01 administration led to robust reduction of TTR mRNA levels in the liver and TTR protein levels in the circulation, and significant regression of TTR protein in tissues known to be affected by ATTR, including the peripheral nervous system and gut. These results demonstrate the potential therapeutic benefit of ALNTTR01 for the treatment of ATTR. A Phase 1 randomized, single-blind, placebo-controlled clinical trial of ALN-TTR01 is underway in Portugal, Sweden, the United Kingdom and France. The primary objective is to evaluate the safety and tolerability of a single intravenous dose of ALN-TTR01 in patients with ATTR. Secondary objectives include characterization of plasma and urine drug pharmacokinetics, and assessment of pharmacodynamic activity based on measurements of circulating TTR serum levels. In this presentation, an update on the clinical development of ALN-TTR01 will be provided, as well as an update on the status of a second generation RNAi therapeutic targeting TTR. Dinah Sah, PhD Alnylam Pharmaceuticals 300 Third Street Cambridge, MA 02142 USA Phone: +1-617-551-8357 Email: [email protected] 37 Session 8: Targeting Liver and Tumors Henrik Г�rum Friday, September 9, 2011 16:30 – 17:00 TARGETING miRNA-122 FOR THE TREATMENT OF HCV Abstract unavailable at time of printing. 38 Session 9: Targeting Infection William James Saturday, September 10, 2011 08:15 – 08:45 THE DEVELOPMENT OF APTAMERS AS CANDIDATE ANTIVIRAL AGENTS: TOWARDS A POLYVALENT MICROBICIDE William James1, Michael D. Moore1, D.H.J. Bunka2, P.G. Stockley2, P.G. Spear3, J. Cookson1, L. Rabe4, R.D. Cranston4, Brian Sproat5 and I. McGowan4 1. Sir William Dunn School of Pathology, University of Oxford, OX1 3RE, UK 2. Astbury Centre for Structural Molecular Biology, University of Leeds, LS2 9JT, UK 3. Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA 4. Magee-Women’s Research Institute, 204 Craft Ave, Room B505, Pittsburgh, PA 15213, USA 5. Chemconsilium GCV, Jaarmarktstraat 48, 2221 Booischot, Belgium Since the mid-1980s, we and others have sought to exploit artificial RNAs in order to inhibit virus replication for therapeutic or preventive purposes. Gene inhibition therapies that seek to express antisense, ribozyme, decoy and others sequences within the target cell have faced the formidable challenge of safe and efficient vector delivery in vivo. Fully synthetic virusinhibitory nucleic acids have the advantage of avoiding inefficient and potentially dangerous gene vectors, and can be modified chemically to enhance their resistance to degradation, pharmacokinetic properties and cellular delivery. A third approach avoids the challenge of delivering antiviral RNAs into target cells by using them as smart chemical barriers at the sites of potential virus entry into the body. Here we describe the development of aptamers that neutralize the infectivity of the causative viruses of AIDS and genital herpes as potential multivalent microbicides. We used the recombinant envelope glycoproteins, gp120 and gD, of HIV-1 and HSV-2, respectively, as targets for selection of specific ligands from libraries of random-sequence 2’F-pyrimidine-substituted RNA. The pool of high-affinity aptamers were cloned and screened for neutralization of virus in cell culture, and those with IC50 < 50nM were further characterized. The secondary structures of selected aptamers were determined and mutational analysis undertaken to identify the functional domains. In the case of the HIV-1 aptamers, the precise binding surface on gp120 was identified, and short, fully synthetic versions were produced that were suitable for translational scale-up. Preclinical studies using short-term culture of human rectal and cervical explants confirmed the antiviral efficacy of these lead agents ex vivo.We uncovered a number of nucleases that are able to rapidly degrade 2’-F-modified RNA in rectal and vaginal secretions. We demonstrate that the combination of selected 2’-O-Me-modifications and Zn2+ cations can fully protect antiviral aptamers under these circumstances. William James, PhD Sir William Dunn School of Pathology University of Oxford, OX1 3RE, UK Telephone: +44 1865 275548 Email: [email protected] 39 Session 9: Targeting Infection Marc Weinberg Saturday, September 10, 2011 08:45 – 09:15 DEVELOPING THE NEXT GENERATION OF EXPRESSED RNAi MODALITIES AGAINST RAPIDLY-EVOLVING VIRAL INFECTIONS Marc S. Weinberg Antiviral Gene Therapy Research Unit, University of the Witwatersrand, Johannesburg, South Africa Expressed precursor mimics of the mammalian RNAi pathway have been extensively exploited for efficient gene knockdown and for the development of novel therapeutics. However, to consistently suppress highly mutable viruses such as HIV, a combination of effective antiviral-RNAi moieties, referred to as combinatorial RNAi (co-RNAi), is required to prevent the emergence of escape mutants that are refractory to targeting. In this talk, I will elaborate on efforts aimed at improving expressed RNAi precursors for therapeutic application against viral infections, with the emphasis on their function and utility in co-RNAi applications. Here novel findings in the development of next-generation approaches for antiviral short hairpin (sh) RNAs, long hairpin (lh)RNAs and primary microRNA (pri-miRNA) mimics are presented. Marc S. Weinberg, PhD Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School 7 York Rd.,Parktown Johannesburg 2193 South Africa Telephone: +27117172561 Email: [email protected] 40 Session 9: Targeting Infection John Rossi Saturday, September 10, 2011 09:15 – 09:45 Dual function therapeutic Aptamer siRNA combinations John J. Rossi1, Jiehua Zhou1, Katrin Tiemann1, Pritsana Chomchan1, Preston Neff2 and Ramesh Akkina2 1. Beckman Research Institute of the City of Hope, Duarte, CA; 2. Colorado State University, Fort Collins, CO A goal of our research is the application of small RNA based therapeutics for the treatment of disease. We have developed dual inhibitory RNA aptamers that function as specific inhibitory agents and delivery vehicles for therapeutic siRNAs. The first aptamer targets the HIV envelope protein gp 120. This aptamer neutralizes the virus and blocks infection. The same aptamer can deliver an attached siRNA into HIV infected cells via binding to envelope expressed on the cell surface, resulting in internalization of the aptamer and delivery of a dicer substrate siRNA to RISC. In vivo delivery of the aptamer and aptamer-siRNA conjugates into a humanized mouse model for HIV infection resulted in one million fold inhibition of the virus, demonstrated siRNA directed cleavage of the target mRNA and complete protection of T-cells from HIV mediated T-cell killing. These results represent the first such small RNA applications for the successful treatment of HIV-1 infection, and could potentially be used in HIV-1 eradication strategies. The second aptamer we have evolved binds with high affinity to the BAFFR1 receptor expressed on B-cells. This receptor is a target for therapeutic treatment of autoimmune diseases and lymphomas since binding of the Baff ligand to the receptor initiates a B-cell proliferative response. The evolved BAFFR1 aptamer blocks Baff ligand mediated proliferation of lymphoma cells in culture as well as blocking intracellular signaling pathways. Binding to BAFFR1 results in internalization of the aptamer allowing the delivery of therapeutic siRNAs. The specificity of this aptamer for B-cells and its ability to serve as a delivery vehicle for siRNAs opens up many therapeutic possibilities. The application of this aptamer in targeting human tumors in humanized mice will be described. John J. Rossi, PhD Department of Molecular and Cellular Biology Beckman Research Institute of the City of Hope 1500 East Duarte Road Duarte, CA 91010 USA Telephone: (626) 301-8360 Email: [email protected] 41 Session 10: Targeting the Central Nervous System Sponsored by International Society of Neurochemistry Adrian Krainer Saturday, September 10, 2011 09:45 – 10:15 Abstract available for review at the meeting. 42 Session 10: Targeting the Central Nervous System Sponsored by International Society of Neurochemistry Hermona Soreq Saturday, September 10, 2011 10:15 – 10:45 Micro-RNAs in the Neuroimmune Interface: From Inflammation to Lethal poisoning protection Hermona Soreq The Hebrew University of Jerusalem, Jerusalem 91904, Israel Rapidly converging lines of evidence document active involvement of micro-RNAs (miR) in both neuronal and immune functions. However, the role of miRs in the communication between the nervous and the immune system and its functional consequences remained uncovered. Here, I report that miRs involved in either neuronal or immune cells (e.g. miR-132, -212, -155 and -146a), are likely to participate in the corresponding neuroinflammatory dialogue. This implies that in response to inflammatory insults, such miRs may alter cognitive processes by targeting numerous mRNA participants of diverse signaling pathways. Of note, brain penetration of peripheral miR-regulated cytokines and other inflammatory agents can affect neurotransmission, plasticity, cognitive and stress responses. Specifically, the miR-132-/212 cluster targets, among other mRNAs the transcriptional activator CREB, the viral infection regulator p300 and the acetylcholine hydrolyzing enzyme acetylcholinesterase (AChE). When over-expressed under TLR4 or TLR9 insults, miR-132 reduces AChE activity in immune cells, enhancing parasympathetic control over the resolution of inflammation. At the same time, up-regulation of the miR-132/-212 cluster in the dorsal striatum limits the risk of drug addiction through modulating CREB, but the suppressed inflammatory response also reduces neuronal protection from viral infection (e.g.encephalitis). Thus, miR-132/-212 mal-functioning may co-modify the risk of many inter-related syndromes with distinct neuro-inflammatory characteristics (e.g. drug addiction, Alzheimer’s and Parkinson’s diseases, multiple sclerosis, amyotrophic lateral sclerosis). The future promise of miR-based studies therefore depends on in-depth understanding of their role in the finely tuned, multi-leveled balance of body-to-brain communication. Hermona Soreq, PhD The Hebrew University of Jerusalem Dept. of Biological Chemistry Edmond J. Safra Campus Jerusalem 91904 Israel Telephone: 972-2-6585109 Email: [email protected] 43 Session 10: Targeting the Central Nervous System Alon Chen Sponsored by International Society of Neurochemistry Saturday, September 10, 2011 10:45 – 11:15 THE ROLE OF microRNA IN REGULATING THE CENTRAL STRESS RESPONSE Chen A. Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel The etiology and pathophysiology of anxiety and mood disorders is linked to inappropriate regulation of the central stress response. To determine if microRNAs have a functional role in the regulation of the stress response, we inactivated microRNA processing by a lentiviral induced local ablation of the Dicer gene in the central amygdala (CeA) of adult mice. CeA Dicer ablation induced a robust increase in anxiety-like behavior, while manipulated neurons survive and appear to exhibit normal gross morphology in the period examined. We also observed that acute stress in wild type mice induced a differential expression profile of microRNAs in the amygdala. Bioinformatic analysis identified putative gene targets for these stress-responsive microRNAs, some of which are known to be associated with stress. One of the prominent stress-induced microRNAs found in this screen, miR-34c, induced anxiolytic behavior following challenge when lentivirally over-expressed in the adult CeA. Of particular interest, one of miR-34c targets is the stress-related corticotropin releasing factor receptor type 1 (CRFR1) mRNA, regulated via a single evolutionary conserved seed complementary site on its 3’UTR. Additional in vitro studies demonstrated that miR-34c reduces cells’ responsiveness to CRF in neuronal cells endogenously expressing CRFR1. Our results suggest a physiological role for microRNAs in regulating the central stress response, and position them as potential targets for treatment of stress-related disorders. Alon Chen, PhD Department of Neurobiology, Weizmann Institute of Science Rehovot 76100, Israel Telephone: +972-8-9344490 Email: [email protected] 44 Session 11: Targeting Genetic Diseases Toni Cathomen Saturday, September 10, 2011 11:45 – 12:15 EXPLORING AND EXPLOITING DESIGNER NUCLEASES FOR TARGETED GENOME ENGINEERING Toni Cathomen Dept. of Experimental Hematology, Hannover Medical School, Hannover, Germany Designer nucleases have developed into powerful tools to edit the genomes of complex organisms at will. The most widely used class of tailored nucleases are zinc-finger nucleases (ZFNs), which consist of an engineered, highly specific DNA-binding domain and a nonspecific endonuclease domain. ZFNs have been employed to trigger the targeted editing of genomes at over 50 different gene loci in more than 10 organisms, including model organisms, such as fruitfly, zebrafish, rat, mouse, and pig, as well as human stem cells. In my talk, I will summarize the technological innovations that have successfully catapulted ZFNs into the human gene therapy arena and provide an overview of parameters that determine ZFN activity and ZFN-associated toxicity, both key qualities in any therapeutic application involving designer nucleases. As a final point, I will present data that introduce TALe-based nucleases (TALeNs) as a valuable alternative to ZFNs. Toni Cathomen, PhD Department of Experimental Hematology Hannover Medical School Carl-Neuberg-Str. 1 30625 Hannover, Germany Telephone: +49 511 532 5170 Email: [email protected] 45 Session 11: Targeting Genetic Diseases Hein te Riele Saturday, September 10, 2011 12:15 – 12:45 Oligonucleotide-directed gene modification in mouse embryonic stem cells to study variants of cancer-related genes Hein te Riele, Marieke Aarts, Eva Wielders, Marleen Dekker, Sandra de Vries, Rob Dekker The Netherlands Cancer Institute, Amsterdam, The Netherlands Gene targeting by single-stranded oligodeoxyribonucleotides (ssODN) is emerging as a powerful tool for site-specific modification of the mammalian genome. We have focused on developing protocols for ssODN-directed base-pair substitution in mouse embryonic stem cells (ESC) since subtle modification rather than full ablation of genes may yield better mouse models for human disease. As readout for successful вЂ�oligo targeting’ in ESC, we measure reactivation of chromosomally-located, disabled neomycin (neo) or EGFP reporter genes. We demonstrated the feasibility of ssODN-directed gene reactivation in ESC, but found that its efficacy was strongly suppressed by the DNA mismatch repair system (MMR). We have shown that wild-type ESC can be rendered permissive for oligo targeting by transiently suppressing MSH2 or MLH1 protein levels by RNA interference. This raised the gene correction efficiency 200-500-fold up to a frequency of 10-5-10-4. Furthermore, we have discarded transcription and homologous recombination as critical mediators, but found evidence that the ssODN becomes integrated into the genome in the context of replication. Finally, we have shown that successfully targeted ESC retained pluripotency, allowing ssODN-modified alleles to be introduced into the mouse germ line. We use вЂ�oligo targeting’ to reconstruct in murine ESC unclassified variant alleles of MMR genes that are frequently found in the human population. While large deletions, premature stop codons and frameshifts completely abrogating gene function are obviously pathogenic and predispose to cancer (HNPCC/Lynch syndrome), missense mutations affecting only a single amino acid are more difficult to interpret. ESC in which such “variants of uncertain significance” (VUS) or “unclassified variants” (UV) have been introduced, are studied by functional assays that address main MMR functions relevant for maintaining genome integrity. Our approach allows variant alleles to be expressed from the endogenous locus, which is crucial as both higher and lower levels of MMR proteins are notorious for affecting MMR capacity. In cases where the functional implications are less clear, or when a mutation only partially affects MMR capacity, mice can be generated from mutant ESCs to investigate whether attenuated MMR activity promotes tumorigenesis in vivo. We will present results of several MSH2 VUS that have been found in (suspected) HNPCC families. Hein te Riele, PhD The Netherlands Cancer Institute, Division of Molecular Biology Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Telephone: 31-20-512-2084 Email: [email protected] 46 Session 11: Targeting Genetic Diseases Marie Dutreix Saturday, September 10, 2011 12:45 – 13:15 siDNA, A NEW STRATEGY FOR INHIBITING DNA REPAIR PATHWAYS M. Dutreix1, J-S. Sun2 1 Institut Curie research center, Institut National de la SantГ© et de la Recherche MГ©dicale U1021, Centre National de la Recherche Scientifique UMR3427, Paris-Orsay, France; 2 DNA Therapeutics, Genopole, Evry, France Enhanced DNA repair activity is often associated with tumor resistance to radiotherapy (RT) or chemotherapy (CT). Various strategies combining RT and CT with in situ gene therapies or molecular therapies inhibiting DNA repair have been developped to maximize the selective pressure against cancer cell growth while minimizing treatment associated toxicity. All these therapies target one main enzyme of a specific DNA repair pathway such as the PARP, DNAPK, CHK2... However, tumors can escape to their effect by modifying the target or enhancing activity of another alternative repair pathway. Here we propose a new strategy that consists in hyperactivating the DNA damage signaling by introducing a false damage signal in cells. This activation results in the lost of the spatio-temporal organization of the cellular response to DNA damage (DDR) and the disabling of all the repair machinery. We designed short DNA bait molecules that mimic DNA double-strand breaks (called Dbait). In transfected cells, Dbait binds DNA-PK complex and PARP enzymes and triggers their activities in cytoplasm as well as nucleus in absence of DNA damage on chromosomes. This Dbait hijacked damage response (DHDR) prevents further detection of damage and thereby inhibits repair of most DNA damage. In vitro, cells show extended parylation signal and DNA-PK targets phosphorylation after Dbait treatment. Surprisingly, the DHDR did not disrupt cell cycle and proliferation of non tumoral cells as primary or transformed fibroblasts. However, it was toxic for numerous tumoral cells line. This selective toxicity was confirmed on human xenografted tumours in which Dbait triggers a DHDR detected by immunohistology. Dbait administration reduces in a dose dependent manner the tumor growth in different animal models. Moreover, it acts in synergy with radiotherapy and increases significantly xenografted animal survival without adding toxicity on the irradiated healthy tissues. A “first-in-human” clinical trial (DRIIM) will start on September 2011 to assess tolerance of a combined treatment of radiotherapy with Dbait. Marie Dutreix, PhD UMR ETIC, Institut Curie, Bat110 Centre Universitaire 91405, Orsay, France TelephoneВ : +33(0)169867186В EmailВ : [email protected] 47 POSTER ABSTRACTS 001 siDNA, A NEW STRATEGY FOR INHIBITING DNA REPAIR PATHWAYS M. Dutreix1, J-S. Sun2 1 Institut Curie research center, Institut National de la SantГ© et de la Recherche MГ©dicale U1021, Centre National de la Recherche Scientifique UMR3427, Paris-Orsay, France; 2 DNA Therapeutics, Genopole, Evry, France Enhanced DNA repair activity is often associated with tumor resistance to radiotherapy (RT) or chemotherapy (CT). Various strategies combining RT and CT with in situ gene therapies or molecular therapies inhibiting DNA repair have been developped to maximize the selective pressure against cancer cell growth while minimizing treatment associated toxicity. All these therapies target one main enzyme of a specific DNA repair pathway such as the PARP, DNAPK, CHK2... However, tumors can escape to their effect by modifying the target or enhancing activity of another alternative repair pathway. Here we propose a new strategy that consists in hyperactivating the DNA damage signaling by introducing a false damage signal in cells. This activation results in the lost of the spatio-temporal organization of the cellular response to DNA damage (DDR) and the disabling of all the repair machinery. We designed short DNA bait molecules that mimic DNA double-strand breaks (called Dbait). In transfected cells, Dbait binds DNA-PK complex and PARP enzymes and triggers their activities in cytoplasm as well as nucleus in absence of DNA damage on chromosomes. This Dbait hijacked damage response (DHDR) prevents further detection of damage and thereby inhibits repair of most DNA damage. In vitro, cells show extended parylation signal and DNA-PK targets phosphorylation after Dbait treatment. Surprisingly, the DHDR did not disrupt cell cycle and proliferation of non tumoral cells as primary or transformed fibroblasts. However, it was toxic for numerous tumoral cells line. This selective toxicity was confirmed on human xenografted tumours in which Dbait triggers a DHDR detected by immunohistology. Dbait administration reduces in a dose dependent manner the tumor growth in different animal models. Moreover, it acts in synergy with radiotherapy and increases significantly xenografted animal survival without adding toxicity on the irradiated healthy tissues. A “first-in-human” clinical trial (DRIIM) will start on September 2011 to assess tolerance of a combined treatment of radiotherapy with Dbait. Marie Dutreix, PhD UMR ETIC, Institut Curie, Bat110 Centre Universitaire 91405, Orsay, France TelephoneВ : +33(0)169867186В EmailВ : [email protected] 49 002 Development of systemic antisense treatment in dystrophic mouse models for Duchenne Muscular Dystrophy Annemieke Aartsma-Rus1, Christa de Winter1, Maaike van Putten1, Tatyana Karnaoek2, Judith van Deutekom2 and Gert-Jan van Ommen1 1 Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands 2 Prosensa BV, Leiden, the Netherlands Antisense-mediated reading frame restoration is one of the most promising therapeutic approaches for Duchenne muscular dystrophy (DMD). It uses antisense oligonucleotides (AONs) to induce exon skipping during pre-mRNA splicing of mutated dystrophin transcripts. This is aimed to restore the disrupted open reading frame and allow synthesis of internally deleted, partly functional Becker-like dystrophin proteins. Proof of concept has been obtained in cultured cells and the mdx mouse model and this approach is currently tested in clinical trials by Prosensa/GSK (GSK2402968 and PRO044) and AVI Biopharma (AVI-4658). Dystrophic animal models allow detailed analysis of pharmacokinetic and pharmacodynamic effects of the 2’-O-methyl phosphorothioate (2OMePS) chemistry used in the Prosensa/ GSK trials AONs. Uptake by healthy muscle is low, but we have previously shown that after systemic delivery of 2OMePS AONs, the AON levels in dystrophic skeletal muscle are up to 10-fold higher, and that subcutaneous delivery of 2OMePS is possible. Here, we optimized dosing and maintenance regimes using subcutaneous 2OMePS AON injections in the mdx mouse model. In addition, we tested the safety and efficacy of high dose (200 mg/kg/week) AON treatment for up to 6 months in mouse models with varying levels of severity: mdx mice (mild phenotype) and mdx mice with one utrophin allele (mdx +/-; intermediate phenotype). This was well tolerated during treatment and liver and kidney weights and serum parameters were similar treated mice compared to saline treated controls at the end of treatment. Notably, in the more severely affected mdx +/- mice the therapeutic effect was larger: exon skip and dystrophin levels were higher, the creatine kinase levels were more decreased and rotarod running time was more increased. Preliminary results suggest that AON levels in the muscles of the more severely dystrophic mdx +/- are higher than in those in mdx mice, confirming the hypothesis that AON uptake is aided by the disease pathology. These results indicate that long term subcutaneous treatment with 2OMePS AONs is safe and efficient in dystrophic mouse models, which is encouraging for long term trials in patients, recently initiated by Prosensa Therapeutics/GSK. Annemieke Aartsma-Rus, PhD Albinusdreef 2 2333 ZA Leiden The Netherlands Telephone: +31 71 5269436 Email: [email protected] 50 003 AUTO-VECTORIZED microRNA MIMIC-BASED NANOSYSTEM FOR HEPATOCELLULAR CARCINOMA THERAPY AimГ© A., Bestzina N., Maurel M., Midoux P., Grosset C., Bestel I. INSERM U869, Victor Segalen University, Bordeaux, France MicroRNA (miR) are short endogenous, non coding RNA that regulate gene expression by controlling mRNA translational or degradation. Recent studies indicate that miR have essential roles in many basic biological processes including development, cell proliferation, differentiation and apoptosis but also in several pathologies. In carcinogenesis, the roles of miR as novel tumor suppressors or oncogenes has been clearly demonstrated pointing out miR as new therapeutic targets. Our project aims at developing new therapeutic molecules capable of modulating gene expression and blocking the growth of tumoral hepatic cells by using the regulatory and biological properties of miR. C. Grosset Team recently observed that miR-96 whose expression is profoundly altered in hepatocellular carcinoma (HCC), downregulates expression of glypican 3 (Gpc3). Gpc3 which is one of the most overexpressed pro-tumoral gene in HCC tissues plays a role in liver cancer as inducer of proliferation of malignant hepatic cells. Therefore, restoring high levels of miR-96 constitutes a very promising avenue in HCC therapy. Our strategy consists in the design, synthesis and delivery of amphiphilic auto-vectorized miR-96 mimics. These molecules deal with an oligonucleotide sequence which contains the biological activity and a hydrophobic vector which enables the formation of original nanosystems that can impact on cell penetration. These two parts have been extensively optimized leading to a small chemical miR-96 mimic library. In parallel to physico-chemical studies of amphiphilic miR-96 mimic self assemblies, the functional efficiency of these molecules, by using the FunREG method was evaluated. Moreover, to get further insights into the impact of the hydrophobic part on cell penetration, cellular uptake pathways were studied. The essential of the results will be presented here. Ahisan AimГ©, 2nd year PhD student INSER U869 UniversitГ© Victor Segalen Bordeaux II 1er Г©tage, porte 12 146, Rue LГ©o Saignat 33 076 Bordeaux Cedex - France Telephone: 0033 6 65 24 96 94 Email: [email protected] / [email protected] 51 004 DEVELOPMENT OF A MICRO-RNA-34 THERAPEUTIC: PHARMACOLOGICAL DRUG DELIVERY & PREDICTING THE FUTURE PATIENT POPULATION Andreas G. Bader, David Brown, Jason Wiggins, Jane Zhao, Christopher Daige, Michael Omotola, Kevin Kelnar, Neil Leatherbury, Jay Stoudemire, Paul Lammers Mirna Therapeutics, Inc., Austin TX Mirna Therapeutics is developing miRNA mimics that are modeled after naturally occurring tumor suppressor miRNAs. Among these is miR-34, a tumor suppressor miRNA that is lost or expressed at reduced levels in a broad range of human cancer types. miR-34 plays an integral role in the p53 tumor suppressor pathway and regulates genes to block cell cycle and induce apoptosis. A therapeutic benefit of miR-34 appears to be the ability to inhibit cancer stem cells. We have validated the concept of miRNA replacement therapy and demonstrated that the therapeutic delivery of miR-34 mimics inhibits cancer cell proliferation and tumor growth in multiple mouse models of cancer. The focus is now on pharmacological drug delivery to establish optimal PK/PD and tox parameters, as well as assessing the effects of miR-34 mimics in isogenic cancer lines to identify genotypes of cancer patients that are most likely to respond to therapy. Examples will be presented. Andreas G. Bader, PhD Associate Director, Research 2150 Woodward Street, Suite 100 Austin, TX 78744 USA Telephone: 512-681-5241 Email: [email protected] 52 005 PTPIP51 – A POSSIBLE TARGET FOR NOVEL DIABETES AND OBESITY THERAPIES Bobrich MA1, Schwabe SA1, Kamm M1, Brobeil AM1, KrГјger K2, Mooren FC2, Steger K3, Tag C1, Wimmer M1 1 Institute of Anatomy and Cell Biology, Justus-Liebig-University, Giessen, Germany 2 Department of Sports Medicine, Justus-Liebig-University, Giessen, Germany 3 Department of Urology and Pediatric Urology, Justus-Liebig-University, Giessen, Germany Our objective was to investigate the role of Protein Tyrosine Phosphatase Interacting Protein 51 (PTPIP51) in the development of obesity and Insulin resistance. We included a total of 60 mice in the study. Eighteen control animals were fed a standard diet, sixteen were fed a high-fat diet, ten were submitted to a treadmill training parallel to the feeding of a normal diet and sixteen animals were submitted to a treadmill training parallel to the feeding of a high-fat diet. After 10 weeks, a glucose tolerance test was performed to determine the Insulin resistance of the animals. We investigated the collected adipose tissue samples of the animals by using immunohistochemistry, western blot, PCR and Duolink proximity ligation assay (DPLA). Our investigations revealed that PTPIP51 interacts with all included proteins in adipose tissue of different Insulin resistant states. In Insulin sensitive normal fed animals, the interaction between PTPIP51 and PTP1B was high, as also seen in trained high fat diet animals. Insulin resistant high fat diet fed animals displayed lower interaction levels of both proteins. The interaction profile of PTPIP51 with 14-3-3beta showed different patterns. Interaction was very high in trained high fat diet animals, whereas high fat animals displayed the lowest interaction of both proteins and normal fed animals displayed only slightly higher levels of interaction. The interaction profile with PTP1B, an inhibitor of the Insulin receptor, hints to a positive effect of the interaction of both proteins on the Insulin sensitivity. In addition to that, PTPIP51 interacts with the Insulin Receptor, independent of feeding status. Endurance training in normal fed animals led to a decrease of the interaction. We conclude that PTPIP51 could act as a docking agent for both inhibitors and activators of the Insulin receptor, hence being able to switch the Insulin signalling pathway on and off. The interaction with 14-33beta indicates that PTPIP51 is involved in modulation processes in adipocytes through the MAPK-pathway. All these facts make PTPIP51 an interesting protein in Insulin resistance, Diabetes and obesity. Further investigations will disclose the effect of inhibition of PTPIP51 with focus on pharmaceutical therapy of Diabetes. Manuel Anton Bobrich, Student Aulweg 123, 35392 GieГџen, Germany Telephone: +49641 99 47012 Email: [email protected] 53 006 TOLL-LIKE RECPTORS 3 AND 7/8 SHOW STEREO-SELECTIVITY AND ARE NOT STIMULATED BY SPIEGELMERS Buchner, K., Vater, A. and Klussmann, S. NOXXON Pharma AG, Berlin, Germany Spiegelmers are L-enantiomeric RNA aptamers showing biostability without any further chemical modification. Two Spiegelmers (inhibitors of the chemokines MCP-1 and SDF-1) have entered clinical development and demonstrated an excellent safety profile in phase I clinical trials. Since certain natural and synthetic RNAs, e.g. single stranded viral RNA (Heil et al., 2004), especially when stabilized as phosphorothioates, or some siRNA (for review see Robbins et al., 2009), have been shown to induce the innate immune response by activating toll-like receptors (TLRs), we investigated, whether Spiegelmers and L-RNA oligonucleotides in general are able to stimulate TLR7/8 (activated by single stranded RNA) or TLR3 (activated by double stranded RNA). In experiments with peripheral blood mononuclear cells (PBMC), plasmacytoid dendritic cells and cells expressing recombinant human TLR8, all of these could be activated with imiquimod or resiquimod and phophorothioatessRNA40 (Heil et al. 2004), but not with functional Spiegelmers (L-ss/dsRNA) or phosphorothioate-ssRNA40 in the enantiomeric (L-) configuration. In experiments with HUVEC which express TLR3, activation of NF-kB could be observed after stimulation with poly(I:C), but again, no activation was observed with Spiegelmers. The apparent stereo-selectivity of the TLRs investigated here and thus the lack of TLR activation by L-RNAs in vitro makes it unlikely that Spiegelmers induce an immunostimulatory response in vivo. This is corroborated by the lack of inflammatory cytokine expression in pre-clinical and clinical studies and adds a further point to the list of favorable characteristics of Spiegelmers. Heil et al. (2004) Science 303:1526 Robbins et al. (2009) Oligonucleotides 19:89 Klaus Buchner, PhD NOXXON Pharma AG Max-Dohrn-Str. 8-10 10589 Berlin, Germany Telephone: +49-30-726247-146 Email: [email protected] 54 007 A unique MOE-DNA chimeric oligonucleotide induces MDA-5 dependent induction of type I interferon response Sebastien A Burel1, Hiroki Sato2, Todd Machemer1, Patrick Cauntay1, Scott P Henry1 1 ISIS Pharmaceuticals, Carlsbad, California, USA 2 Institute for Virus Research, Kyoto University, Kyoto, Japan Second generation вЂ�Gapmer’ antisense oligonucleotides (ASO) containing 2’-O-methoxyethylribose (MOE) modifications have been shown to possess both excellent pharmacokinetic properties and robust pharmacological activity in several animal models of human disease. These beneficial properties have translated to human therapeutics. Gapmer ASOs are generally well tolerated displaying minimal to mild proinflammatory effect caused by the release of cytokines via the activation of monocyte and or dendritic cells compared to phosphorophioate deoxy oligonucleotides at doses far in excess of expected therapeutic doses. While the vast majority of 2’-MOE ASOs are largely void of hepatotoxicity as characterized by the absence of serum transaminases increase, a very small subset of ASO with a propensity for producing acute hepatotoxicity in mice has been identified. The mechanism for these findings is not clear at this point, but the effects are clearly sequence-specific. Intense effort has been made to understand the mechanism underlying these effects. One of those ASO, ISIS 147420, was found to cause profound hepatotoxicity characterized by increased ALT that was atypical of this class of oligonucleotides. In addition to increased ALT, subcutaneous injection of ISIS 147420 was associated with extensive hepatocyte apoptosis and necrosis, as well as mononuclear cell infiltrate in liver at 72 hours. Liver morphology and ALT levels were normal at 24 or 48 hours. Whole genome gene expression profiling was performed on livers collected at 8, 24, 48 and 72 hours. A large number of interferon stimulated genes (ISGs) were significantly upregulated as early as 24 hours. Administration of ISIS 147420 to Stat1 or IFNAR1 deficient mice showed no evidence of hepatotoxicity and no induction of ISGs up to 96 hours post treatment. We speculated that a specific sequence motif might cause ISIS 147420 to be mistaken for viral RNA or DNA thus triggering an innate immune response ultimately resulting in severe hepatotoxicity. ISIS 147420 toxicity was independent of Toll-like receptors as there was no decrease in ALT in TRIF or Myd88 deficient mice. The involvement of the cytosolic pattern recognition receptors, RIG-I and MDA-5, were also investigated. Pretreatment of mice with ASOs inhibitors of IPS-1, an adaptor protein critical to the function of RIG-I and MDA-5, reduced mRNA level down to 8% of control and prevented the toxicity induced by ISIS 147420 (reduced ALT and interferon-b levels). Unlike ASO pretreatment with RIG-I inhibitors that was unable to reduce the severity of hepatotoxicity, pretreatment with MDA-5 antisense inhibitors reduced MDA-5 mRNA level down to 7% of control and prevented the toxicity induced by ISIS 147420 (reduced ALT and interferon-b levels). These results revealed a novel mechanism of oligonucleotide mediated toxicity requiring both MDA-5 and IPS-1 and resulting in an adverse activation of the innate immune response. Sebastien Burel, PhD 2855 Gazelle Court, Carlsbad, CA 92010 USA Telephone: 1-760-603-2717 Email: [email protected] 55 008 Incorporation of LNA into aptamer selection Meghan A. Campbell, Birte Vester, Jesper Wengel Nucleic Acid Center, University of Southern Denmark, Odense, Denmark Aptamers continue to emerge as an important component of nucleic acid biotechnology and nanotechnology. These single-stranded DNA or RNA molecules can fold into unique tertiary structures that can bind with high affinity to small molecule or protein ligands. Aptamers are selected through a process known as Systematic Evolution of Ligands by EXponential enrichment (SELEX), where the best binding nucleic acid sequences are amplified and selected again. Typical aptamer structures can include stem-loop regions or G-quadruplex motifs, both of which can be stabilized by post-selection modification with Locked Nucleic Acid (LNA). Now, through the use of LNA-triphosphates, we are modifying the SELEX cycle to select LNA-containing aptamers. By including LNA in the selection process, the possibility of new or more stable aptamer structures is increased. Libraries have been created with LNA modifications at fixed or variable locations within the 40-mer random region. After the random library is bound to a ligand of interest, and non-binding sequences washed away, the selected ssLNA sequences are amplified into dsDNA. A dT20-tail is added at the 5’ end of the DNA strands to facilitate purification. This DNA then serves as a template for asymmetric PCR to regenerate ssLNA sequences. The shorter LNA strand is separable from the DNA strands by acrylamide gel electrophoresis and is purified in this manner. Purified ssLNA can then be used for the next round of selection and the cycle continues. Following numerous SELEX cycles, the selected LNA strands can be amplified into DNA and sequenced. Through this process we hope to produce higher affinity and more stabile aptamers for a wide variety of applications. Meghan A. Campbell, PhD Institute for Physics and Chemistry University of Southern Denmark Campusvej 55 5230 Odense M, Denmark Telephone: +45 6550 2548 Email: [email protected] 56 009 [3H]-RADIOLABELING OF SMALL INTERFERING RNA (siRNA) Christensen J1,2, Andres H1, Natt F1, Hunziker J1, Krauser J1, Swart P1 1 Novartis Institutes of Biomedical Research, Basel, Switzerland 2 Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, Odense, Denmark We describe a simple and efficient final step labeling procedure for single internal [3H]-radiolabeling of oligonucleotides. To our knowledge this is the first reported procedure for single internal final step radiolabeling of oligonucleotides, with no chemical modification (3H replacing a 1H), i.e. the reactivity and metabolism of the molecule should remain unchanged. The radiolabel is placed in a chemically stable and predetermined internal position, which decreases the likelihood of the label to be readily cleaved from the oligonucleotide in vivo. The radiolabeling takes place on the fully deprotected full length antisense strand by bromine/tritium exchange catalyzed by palladium on charcoal in the predetermined 5-position of either uridine or 2Вґ-O-methyluridine. Either a 5-bromouridine or 5-bromo-2ВґO-methyluridine phosphoramidite building block was incorporated into the middle of the antisense strand which was synthesized on a DNA/RNA-synthesizer using standard phosphoramidite chemistry. Because the method is solely dependent upon the existence of a uridine or a 2Вґ-O-methyluridine residue in the sequence, the method should be applicable to chemically modified as well as unmodified phosphodiester oligonucleotides. In order to develop siRNAs as pharmaceutical drugs, it is essential to study their biodistribution and biostability and this radiolabeling method of oligonucleotides provides a suitable research tool. Although a variety of non-isotopic labeling techniques exists (e.g. bioluminescence, fluorescence), the use of radiolabeled oligonucleotides to perform (pre)clinical in vitro/in vivo studies offers a distinct advantage of avoiding chemical modification of these compounds. Jesper Christensen, MSc Novartis Campus, WSJ-153.1.08 CH-4056 Basel, Switzerland Telephone: +41 79 593 9171 Email: [email protected] 57 010 COMPUTER-GUIDED OPTIMIZATION OF AN RNA APTAMER FOR CLINICAL APPLICATIONS Justin P Dassie, ‡William M Rockey, В¶Frank J Hernandez, ВҐ,В±,†,€Sheng-You Huang, ВҐ,В±,€Song Cao, В¶Craig A Howell, В¶Luiza I Hernandez, В§Gregory S Thomas, В¶Xiu Ying Liu, в‰ Natalia Lapteva, в‰ David M Spencer, В¶James O McNamara II, ВҐ,В±,†,€Xiaoqin Zou, ВҐ,В±,€Shi-Jie Chen, ‡,В¶,В§ Paloma H Giangrande ‡ Department of Radiation Oncology, В¶Department of Internal Medicine, В§Molecular & Cellular Biology Program, University of Iowa, Iowa City, IA, USA. ВҐDepartment of Physics and Astronomy, В±Department of Biochemistry, †Dalton Cardiovascular Research Center, and € Informatics Institute, University of Missouri, Columbia, MO, USA. в‰ Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA В§ RNA aptamers represent an emerging class of pharmaceuticals with great potential for targeted cancer diagnostics and therapy. Several RNA aptamers that bind cancer cell-surface antigens with high affinity and specificity have been described. However, their clinical potential has yet to be realized. An example of this, are RNA aptamers directed against prostate specific membrane antigen (PSMA), a prostate cancer-specific surface antigen. The therapeutic potential of the PSMA RNA aptamers has been demonstrated for many applications including inhibition of PSMA’s pro-carcinogenic properties, imaging and delivery of cytotoxic agents to prostate cancer cells. Given this, further optimization to improve their pharmacokinetic (PK)/pharmacodynamic (PD) profiles and to facilitate large-scale chemical synthesis of these RNAs is compelling. Toward this end, we have employed computational RNA structural modeling and RNA/protein docking models to guide the truncation and further chemical modification of the A9 PSMA RNA aptamer. These analyses resulted in a truncated derivative of the A9 aptamer (A9g, 43mer), which due to its reduced length is now amenable to large-scale chemical synthesis. The theoretical modeling has also enabled the identification of bases within A9g that when modified (with synthetic bases) impart increased resistance to nucleases present in human serum. Importantly, we show that the optimized A9g inhibits PSMA’s pro-carcinogenic activity and when directly applied to cells expressing PSMA, is effectively internalized, thus enabling the delivery of cytotoxic agents to PSMAexpressing prostate cancer cells. Finally, we demonstrate the potential of this reagent for imaging of prostate tumors using mouse models of metastatic prostate cancer. In summary, these studies demonstrate the utility of computational RNA structure models for guiding truncations and chemical modifications of RNA aptamers while retaining their function. Furthermore, these studies have resulted in a PSMA RNA aptamer that due to its optimized in vivo profile and shorter sequence length is now one step closer to the clinic. Justin P Dassie, BS 375 Newton Rd, 5241 MERF Iowa City, IA 52242 USA Telephone: (319) 384-3243 Email: [email protected] 58 011 RNA APTAMERS TO THE CANCER-ASSOCIATED PROTEINS UPA AND PAI-1 WITH THERAPEUTIC POTENTIAL Daniel M Dupont, Jeppe B. Madsen, Kenneth A. BГёtkjГ¦r, Bertrand Tavitian, FrГ©dГ©ric DucongГ©, JГёrgen Kjems and Peter A. Andreasen Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark The plasminogen activation system plays a role in a wide variety of pathological conditions such as cancer, inflammatory disorders and cardiovascular diseases. In order to develop novel potential therapeutics, we have screened libraries of 2Вґ-fluor-containing RNA oligonucleotides for aptamers targeting members of the system. Aptamers to the serine protease urokinase-type plasminogen activator (uPA) were identified with the ability to prevent the association of the protein with its receptor (uPAR) on cell-surfaces. In turn, the aptamer prevents cell-associated uPA-mediated plasminogen activation, which is important for cancer cell invasion and metastasis. While other uPA-uPAR targeting agents are directed towards the receptor and induce uPA-like proteolysis-independent agonistic downstream effects, the uPA aptamers are pure antagonists. Aptamers were also isolated against the primary physiological inhibitor of uPA, the serpin plasminogen activator inhibitor-1 (PAI-1), with ability to block PAI-1Вґs binding to its anchor point in the extracellular matrix, vitronectin, important for its proper localization in processes collectively known as tissue-remodeling and cell migration and invasion. In addition, the PAI-1 aptamers were able to stabilize the meta-stable active conformation of the protein, thereby increasing the half-life of the molecule at physiological conditions more than any known other ligand. One aptamer was even able to distinguish between different conformations of PAI-1, which could be explained by mapping the epitope. uPA and PAI-1 are established biomarkers for the malignant phenotype of cancers and potential targets for anticancer therapy. The aptamers are therefore potential agents for development of novel anticancer therapeutics and diagnostics. Daniel M. Dupont, PostDoc Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark Telephone: +45 89 42 50 79 Email: [email protected] 59 012 CLICK NUCLEIC ACID LIGATION A.H. El-Sagheer1, 2 and T. Brown1 1 School of Chemistry, University of Southampton SO17 1BJ. UK 2 Chemistry Branch, Dept. of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez Canal University, Suez, 43721, Egypt Click DNA ligation is a remarkably efficient method of joining together DNA and RNA strands. It utilizes the copper-catalyzed azide-alkyne cycloaddition (CuAAC reaction). The chemistry has been optimized for the synthesis of cyclic oligonucleotides,1,2 oligonucleotide catenanes,2 very stable cyclic mini-duplexes,1 duplexes that are linked across the major groove,3 covalently fixed DNA nanoconstructs4 and large RNA constructs.5 The method produces an unnatural DNA backbone that can be varied by changing the participating alkyne and azide. Careful design produces a linkage that can be read through by thermostable DNA polymerases during PCR.6,7 This has potential applications in nanotechnology, gene synthesis and genetic analysis. Figure 1. Overlay of triazole DNA (black) on normal DNA (grey) with the triazole ring bottom left. The largest difference between the two structures is the displacement of the 5’-carbon atom of the deoxyribose sugar (more than 3 angstroms, dotted line).Nevetheless, the triazole linkage does not affect the overall conformation of the DNA duplex; the changes are localized.. The model is from a highresolution NMR structure in collaboration with Andre Dallman (submitted). (1) El-Sagheer, A. H.; Kumar, R.; Findlow, S.; Werner, J. M.; Lane, A. N.; Brown, T. Chembiochem 2008, 9, 50. (2) Kumar, R.; El-Sagheer, A. H.; Tumpane, J.; Lincoln, P.; Wilhelmsson, L. M.; Brown, T. J. Am. Chem. Soc. 2007, 129, 6859. (3) Kocalka, P.; El-Sagheer, A. H.; Brown, T. Chembiochem 2008, 9, 1280. (4) Lundberg, E. P.; El-Sagheer, A. H.; Kocalka, P.; Wilhelmsson, L. M.; Brown, T.; Norden, B. Chem. Commun. 2010, 46, 3714. (5) El-Sagheer, A. H.; Brown, T. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 15329. (6) El-Sagheer, A. H.; Brown, T. J. Am. Chem. Soc. 2009, 131, 3958. (7) El-Sagheer, A. H.; Sanzone, A. P.; Gao, R.; Tavassolia, A.; Brown, T. Proc. Natl. Acad. Sci. U. S. A. in press. Afaf H. El-Sagheer, PhD Chemistry Department, University of Southampton High Field, Southampton, SO17 1BJ, UK Telephone: +44(0)2380596785 Email: [email protected] 60 013 NON-VIRAL DELIVERY OF STICKY-SIRNAв„ў TARGETING THE CELL CYCLE INHIBITS LUNG TUMOR METASTASIS Anne-Laure Bolcato-Bellemina, Marie-Elise Bonneta, Omar Zouniba, Elodie Benoita, Jean-Paul Behrb and Patrick Erbachera a Polyplus-transfection, Bioparc, Boulevard SГ©bastien Brandt, 67401 Illkirch, France b Laboratoire de Chimie GГ©nГ©tique, UniversitГ© de Strasbourg, FacultГ© de Pharmacie, 67401 Illkirch, France Cancer is a leading cause of death worldwide, accounting around 13% of all death. Among cancers, lung cancer is the most common in term of incidence and mortality. Metastasis, due to uncontrolled cell growth, is responsible for more than 90% of deaths from lung cancer. Thus, developing new treatments to inhibit cell metastasis will improve patient prognosis and quality of life. Since the discovery of RNA interference and its potency for therapeutic applications, this technology has been widely used to suppress target gene expression involved in many diseases such as cancer. However, to date, delivery still remains a major obstacle for siRNAbased drugs to be a success. Polyethylenimine (PEI) has been widely used for in vivo gene delivery. We have recently developed novel interfering RNA (STICKY-SIRNAв„ў) in order to improve the delivery and silencing efficiency of PEI polyplexes1. Our modified siRNA mimics the structure of long double-stranded nucleic acid through reversible concatemerization and shows higher silencing efficiency compared to non oligomerized siRNA. Moreover, no production of pro-inflammatory cytokines, including interferon-ОІ, nor hepatic enzymes were observed after systemic delivery of these modified siRNA2. Cyclin B1 is essential for the initiation of mitosis. Its deregulation is involved in neoplastic transformation. Recent papers have shown that suppression of cyclin B1 could be an attractive strategy for antiproliferative cancer therapy. We have analyzed the impact of suppression of cyclin B1 by delivering STICKY SIRNAв„ў with linear polyethylenimine in different cancer cell lines (TSA-Luc, B16-F10 and 3LLLuc). We have shown that cyclin B1 STICKY SIRNAв„ў arrested the cell cycle in G2/M phase in all cancer cell lines tested. Consequently, the ability of STICKY SIRNAв„ў to suppress cell metastasis in the lung was investigated. By injecting cyclin B1 STICKY SIRNAв„ў with linear polyethylenimine intravenously, tumor lung metastasis progression was blocked by more than 50%. The survival rate of the mice was also significantly increased. We have also shown that combination of STICKY SIRNAв„ў delivery with antiproliferative drugs (i.e. cis-platin) increase tumor growth inhibition by more than 70%. Taken together our data showed that the delivery of STICKY SIRNAв„ў with linear polyethylenimine could become a powerful tool for antitumor therapy. 1 2 Bolcato-Bellemin et al., PNAS, 2007, 104, 16050-5. Bonnet et al., Pharm Res, 2008, 25, 2972-82. Anne-Laure Bolcato-Bellemin, PhD Polyplus-transfection Bioparc Boulevard SГ©bastien Brandt 67401 Illkirch, France Telephone: +33 3 90 40 61 80 Email: [email protected] 61 014 SELF-DELIVERING CATIONIC siRNAs MEDIATE EFFICIENT AND SPECIFIC GENE SILENCING IN HUMAN CELLS Patrick Erbacher, ValГ©rie Moreau, ClГ©ment Paris, GaГ«lle Deglane, Bernard Couturier, Anne-Laure Bellemin, Marie-Elise Bonnet, Jean-Serge Remy*, Jean-Paul Behr*, Nathalie Lenne Polyplus-transfection, Illkirch, France *Laboratoire de Chimie GГ©nГ©tique, FacultГ© de Pharmacie, Illkirch, France Discovered 10 years ago, small interfering RNA technology has developed increasingly fast, thanks to its unprecedented efficiency to turn-off the expression of a target gene in tissue culture. While offering great therapeutic promises, the siRNAs approach has been facing strong difficulties, among which the primary requirement: entering cells when administered in vivo. siRNAs are commonly formulated with cationic polymers or lipids, to form supramolecular particles capable of crossing the negatively charged cell membrane. However, particles size limits their diffusion through organs when administered in vivo. Considering the need for diffusible drugs for in vivo use, we are developing cationic siRNAs, composed of an antisense sequence annealed to an oligospermine conjugated sense strand. Such cationic molecules have been previously shown to display gene silencing activity in cell culture (Nothisen et al., J. Am. Chem. Soc. 2009). Since, methods for synthesizing and characterizing oligospermine-oligonucleotides conjugates have been improved, providing molecules compatible with a drug development process and with enhanced biological activity in vitro. Here, we report a complete in vitro study demonstrating that cationic siRNAs enter cells in absence of carrier and specifically suppress gene expression through RNAi – mediated mechanism in human cells in presence of serum. Our preliminary data confirm also the promising perspectives for the in vivo use of cationic siRNAs and future preclinical developments. Patrick Erbacher, PhD Polyplus-transfection SA, BIOPARC, Boulevard SГ©bastien Brant, BP 90018 F-67401 Illkirch, France Telephone: +33 (0)390В 406 180 Email: [email protected] 62 015 Receptor-mediated uptake of oligonucleotide nanocomplexes with cell-penetrating peptides and their formulation into a pharmaceutically stable solid dosage form Kariem Ezzat, Henrik Helmfors and Гњlo Langel Department of Neurochemistry, Stockholm University, Stockholm, Sweden A new generation of cell-penetrating peptides (CPPs), PepFects, is capable of forming noncovalent nanocomplexes with oligonucleotides (ONs), which are then delivered with great efficiency to different cell lines in-vitro and different tissues in vivo. However, the detailed uptake mechanism of such nanocomplexes is not clear. Here, we show that PepFect14 forms negatively charged nanocomplexes with ONs and their uptake is mediated by class-A scavenger receptors (SCARA). Specific inhibitory ligands of SCARA totally inhibit the activity of PepFect14-ON nanocomplexes in the HeLa pLuc705 splice-correction cell model, while nonspecific, chemically related molecules do not. Fetuin, which mediates uptake via SCARA, significantly enhances the activity. In addition, RNAi knock-down of SCARA subtypes (SCARA3 & SCARA5) that are expressed this cell-line led to a significant reduction of the activity to less than 50%. Furthermore, we demonstrate the feasibility of incorporating such nanocomplexes into stable and active solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique was utilized by drying the nanocomplexes at 55-60 Н¦C under vacuum in the presence of water soluble excipients. The formed solid formulations were as active as the freshly prepared nanocomplexes in solution even when stored at elevated temperatures for several weeks. This shows that using PepFect14 is a very promising translational approach for the delivery of ONs in different pharmaceutical forms. In conclusion, the results presented here shed the light on a novel uptake mechanism of CPPbased delivery systems and demonstrate a novel technology to formulate such delivery systems into active and stable pharmaceutical dosage forms. Kariem Ezzat, PhD student Department of Neurochemistry, Stockholm University, SE-106 91 Stockholm, Sweden Telephone: 0046700674381 Email: [email protected] 63 016 INHIBITION OF COMPLEMENT C6 SYNTHESIS IN THE LIVER USING ANTISENSE OLIGONUCLEOTIDES AFFECTS NEURO-REGENERATION Kees Fluiter, Valeria Ramaglia, Frank Baas Academic Medical Center, Dept. of Neurogenetics, Amsterdam, The Netherlands Introduction: The complement system is always activated in neurodegenerative diseases. We previously identified a role of the terminal complement pathway in neuro-degeneration. Rats deficient for C6 showed faster recovery after nerve crush and inhibiting complement activation using a C1 esterase inhibitor reduced damage after traumatic brain injury. Method: RNase H recruiting antisense oligonucleotides against C6 were developed to test whether pharmacological intervention in the terminal complement pathway can be used to modulate neuro-degeneration and regeneration. These antisense oligonucleotides were tested in vivo in two mouse models. First, in a model of acute neuronal damage and second in a model of chronic nerve degeneration. Results: Administration of C6 antisense oligonucleotides to mice results in a substantial and specific knock-down of complement C6 mRNA in liver and C6 protein in circulation. The compounds appear to be well tolerated and provide a potent and long-lived reduction in C6 levels that lasts for weeks after administration. Mice receiving the antisense oligonucleotides in a model of acute nerve degeneration (nerve crush) show better recovery from injury. These compounds were also effective in an antibody dependent experimental autoimmune encephalomyelitis (AD-EAE) mouse, a model of chronic nerve degeneration. The treated mice showed substantially less disease severity. Favorable clinical scores were associated with less nerve inflammation and demyelination according to standard histological assays. Conclusion: We expect that antisense oligonucleotides targeting the terminal complement pathway are promising drugs for neurodegenerative disorders. Kees Fluiter, PhD AMC, K2-210 Meibergdreef 9, 1105 AZ Amsterdam The Netherlands Telephone: +31205663746 Email: [email protected] 64 017 Suppression of bcr/abl gene expression by siRNA conjugates Masayuki Fujii, Diala Irmina Department of Biological & Environmental Chemistry, School of Humanity Oriented Science and Technology, Kinki University Herein we described the synthesis of siRNA-peptide conjugates by solid phase fragment coupling (SPFC) and application of them to silencing of bcr/abl chimeric gene in human leukemia cell line K562. Synthesis of siRNA-NES conjugates was achieved by SPFC as described previously.(1) Target sequence of bcr/abl mRNA(355-390) 5’-ggauuuaagcagaguucaa/aagcccuucagcggcca-3вЂ� siRNA (anti bcr/abl mRNA361-381) sense: NES-5’-GCAGAGUUCAAAAGCCCUUTT-3’ antisense: 3’-TTCGUCUCAAGUUUUCGGGAA-5’ NES Peptides: C1: ОІALVLDKLTI-OH (TFIIIA NES) C2: ОІALPPLERLTL-OH (HIV-1 Rev NES) As a result, two types of siRNA-NES conjugates C1 and C2 were prepared in which 5’-end of sense strand was covalently linked to N-terminus of the NES peptides derived from TFIIIA and HIV-1 rev, respectively. Silencing effects of C1 and C2 against bcr/abl mRNA in human leukemia cell line K562 were evaluated by quantitative PCR. The expression of bcr/abl gene was suppressed to 30.2 % at 200nM and 36.3 % at 50 nM by native siRNA. Significant enhancement of silencing efficiency was observed with C1 and C2. siRNA TFIIIA NES (C1) suppressed the expression of bcr/abl gene to 8.3% at 200 nM and 11.6 % at 50nM and siRNA-HIV-1rev NES (C2) suppressed to 4.0 % at 200 nM and 6.3 % at 50nM. Previously, we reported that DNA-HIV-1 rev NES peptide conjugate was localized in cytoplasm of Jurkat cell. (2) The large enhancement of the silencing efficiency of siRNA-NES conjugates could be reasonably ascribed to the localization of siRNA-NES conjugates in cytoplasm. It can be also pointed out that modification of 5’-endo of sense strand reduced off-target effect by minimizing the extent of the sense strand incorporation into RISC. (3) Unfortunately, siRNANES conjugates could not penetrate the cellular membrane by itself and required a transfection reagent to be taken up into cells, while oligodeoxyribonucleotide-NES conjugates were internalized into cells without any transfection reagents. (2) It can be speculated the double stranded structure of siRNA retarded the penetration through cellular membrane. Reference 1. T. Kubo, M. Morikawa, H. Ohba, M. Fujii, Org. Lett., 2003, 5, 2623-2626. 2. T. Kubo, Z. Zhelev, B. Rumiana, H. Ohba, K. Doi and M. Fujii, Org.Biomol.Chem., 2005, 3, 3257-3259. 3. Y. Ueno, K. Yoshikawa, Y. Kitamura, Y. Kitade, Bioorg. Med. Chem. Lett., 2009, 19, 875-877. Masayuki Fujii, PhD 11-6 Kayanomori Iizuka, Fukuoka 820-8555 Japan Telephone: +81-948-22-5655-210 Email: [email protected] 65 018 Influence of the terminal base pairs of 5’-PPP-RNA on RIG-I ligand activity M. Goldeck1, C. Schuberth1, M. Schlee1, G. Hartmann1 and J. Ludwig1 1. Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, University of Bonn, Bonn, Germany The innate immune response to viruses is based on pattern recognition receptors (PRR) that detect a characteristic structure, modification or an unusual location of viral nucleic acids. Most of the highly pathogenic viruses are RNA genome-based viruses. The cytosolic helicase RIG-I senses double-stranded 5’triphosphorylated RNA (5’-ppp-dsRNA) as contained in the genomic or replicative form of RNA viruses. Binding and recognition of 5’-ppp-dsRNA is mediated by the RIG-I regulatory domain (RD). Stimulation of the RD activates the RIG-I CARD domain, which binds and activates downstream adaptor molecules inducing an antiviral immune response, including production of type I interferons. As revealed by the crystal structure of the RIG-I RD bound to a blunt end 5’-ppp-dsRNA, the triphosphate moiety interacts with a lysine-rich basic cleft. The crystal structure furthermore unravels stacking of phenylalanine F853 with the terminal base pair, which is essential for RIG-I function. The elucidation of this key intermolecular interaction supports the strict requirement for an intact base pair at the 5’-ppp terminal site of the ligand. Using synthetic 5’-pppRNAs we performed a systematic analysis of all 16 possible base pairs of naturally occurring nucleosides at the first two 5’ppp-terminal positions of the duplex. The stimulatory potency of these duplexes was assessed by type I interferon induction in human monocytes. Besides the analysis of varying Watson-Crick base pairing these data allow an extended definition of RIG-I ligand requirements concerning the acceptance of alternate hydrogen bonding occurring in wobble or Hoogsteen base pairing or in other noncanonical base-base interactions. The data demonstrate a high sensitivity of RIG-I for terminal structural elements and emphasizes the requirement for using well-defined, chemically synthesized 5’-pppRNA for the unequivocal characterization of RIG-I ligand interactions. Marion Goldeck, PhD student Institute for Clinical Chemistry and Clinical Pharmacology University Hospital Bonn Sigmund-Freud-Str. 25 53105 Bonn, Germany Telephone: +49-0228-287-51157 Email: [email protected] 66 019 ANTISENSE OLIGONUCLEOTIDES: PROMISING TOOLS FOR MOLECULAR THERAPY OF MYOTONIC DYSTROPHY Anchel Gonzalez1,2, Susan A.M. Mulders1,2, Walther J.A.A. van den Broek2, Ingeborg van Kessel2, Jeroen van de Giessen1, Judith C.T. van Deutekom1, BГ© Wieringa2 and Derick G. Wansink2 1 Prosensa Therapeutics B.V., Leiden, The Netherlands. 2 Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, The Netherlands Myotonic Dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults. DM1 patients carry an unstable (CTG)n repeat in the 3’ UTR of the DM Protein Kinase (DMPK) gene. The length of this repeat is correlated to the severity of symptoms and progression of the disease. DMPK RNA transcripts with long (CUG)n repeats are retained in the nucleus, where they form hairpin-like structures which are involved in the entrapment and depletion of splice factors like MNBL (muscleblind) and in stabilized expression of CUG-binding protein-1. In turn, this causes aberrant splicing of pre-mRNAs from several other genes, contributing to the complex multisystemic manifestation of disease in patients. There is no cure available for DM1 yet. However, recent experiments in cell and animal models have shown that expanded DMPK transcripts can be silenced by the use of antisense (CAG)n oligonucleotides (AONs) directed towards the toxic (CUG)n repeat segment, offering a new therapeutic approach. Here, we report on further therapeutic developments in pre-clinical in vitro studies on AONs of different chemistry, length and sequence composition in myoblast-myotube cell models derived from patients and from a DM1 mouse model with a human (CTG)500 DMPK transgene. Northern blotting and real time RT-qPCR analysis showed a significant reduction of expanded DMPK mRNA after AON transfection while endogenous (CUG)n- or (CAG)n-bearing transcripts were not affected. The working mechanism of our AONs is presently unknown. We are currently monitoring the fate of DMPK (pre)mRNAs by quantitative measurements of production rate and steady state levels in different cellular compartments, in presence and absence of different AONs. Fluorescence microscopy and real time RT-qPCR experiments are used to investigate the uptake pathway of AONs and to assess the stoichiometry of AONs and mRNA targets and timing and place of degradation of the expanded DMPK mRNA. Anchel Gonzalez, MSc Dept. of Cell Biology Radboud University Nijmegen Medical Centre Nijmegen Centre for Molecular Life Sciences Geert Grooteplein 26/28 6525 GA Nijmegen The Netherlands Telephone: +31.24.36 13664 Email: [email protected] 67 020 RESCUE OF SEVERELY AFFECTED DYSTROPHIN/UTROPHIN DEFICIENT MICE BY VECTORIZED ANTISENSE MEDIATED EXON SKIPPING Goyenvalle A, Babbs A, Wilkins V, Wright J, Powell D and Davies KE MRC Functional Genomics Unit, University of Oxford, Oxford, UK Duchenne Muscular Dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisensemediated exon skipping is one of the most promising approaches for the treatment of DMD because of its capacity to correct the reading frame and restore dystrophin expression which has been demonstrated in vitro and in vivo. This approach has been tested in clinical trials in the Netherlands and the UK with Antisense Oligonucleotides (AO) against exon 51 and has demonstrated encouraging results. However, AO-mediated exon-skipping for DMD still faces major hurdles such as extremely low efficacy in the cardiac muscle, poor cellular uptake and relative rapid clearance from circulation, which impose repeated administration to achieve some therapeutic efficacy. To overcome these limitations, we proposed to use small nuclear RNAs (snRNA), especially U7snRNA to shuttle the antisense sequences and to vectorize them into Adeno-Associated Virus (AAV) vectors. In this study, we have investigated for the first time the therapeutic potential of AAV-mediated exon skipping in the utrophin/dystrophin double-knockout mouse (dKO) which is a much more severe and progressive mouse model of DMD. A single intravenous injection of scAAV9 vector encoding a specific U7snRNA targeted to exon 23 of dystrophin pre-mRNA in dKO mice induces widespread high levels of exon-skipping in the treated mice. This treatment results in near-normal levels of dystrophin expression in all muscles examined, including the heart, leading to a considerable improvement of their muscle function and dystrophic pathology. ScAAV9-U7dtex23 injection strikingly prevented kyphosis and contractures in dKO mice and remarkably improved their motility, as assessed by activity monitoring. Treated dKO mice showed almost return to normalcy for most of the examined parameters as well as an impressive extended lifespan (over 36 weeks compared to an average of 12 weeks for untreated dKO controls). These results suggest a great potential for AAV-mediated exonskipping in systemic treatment of the DMD phenotype. Aurelie Goyenvalle, PhD MRC Functional Genomics Unit Department of Physiology, Anatomy and Genetics University of Oxford South Parks Road Oxford OX1 3QX, UK Telephone: +44 (0)1865 285 864 Email: aurelie.goyenvalle @dpag.ox.ac.uk Photograph of a dKO control mouse (left) and a scAAV9U7dtex23 treated dKO mouse (right) at 12 weeks of age 68 021 TARGETING PIM-1 BY MIR-33a IN COLON CARCINOMA THROUGH PEI-MEDIATED MIRNA REPLACEMENT THERAPY Arnold GrГјnweller, Ahmed Fawzy Ibrahim, Maren Thomas, Kerstin Lange-GrГјnweller, Ulrike Weirauch, Achim Aigner, and Roland K. Hartmann Philipps-University Marburg, Pharmazeutische Chemie, Germany The oncogenic kinase Pim-1 is an important target for tumor therapy because Pim kinases are generally not expressed in healthy cells. However, upregulation of Pim-1 causes cancer in several cell types. So far, a regulation of Pim-1 by miRNAs has not been reported. Here, we establish miR-33a as a miRNA with potential tumor suppressor activity due to its inhibitory effect on Pim-1. A screen for miRNA expression in several cancer cell lines revealed general low endogenous miR-33a levels relative to other miRNAs. We observed substantially reduced Pim-1 levels upon transfection of K562 and LS174T cells with miR-33a mimics. Seed mutagenesis of the Pim-1 3’-UTR in a luciferase reporter construct and in a genuine Pim-1 cDNA demonstrated the specificity of the miR-33a-dependent downregulation. The persistence of this effect was comparable to that of a siRNA-mediated knockdown of Pim-1 and results in decelerated cell proliferation. We further establish miRNA replacement therapy through polyethylenimine (PEI-)-mediated systemic application of miR-33a in s.c. colon carcinoma xenograft mouse models as a novel cancer treatment strategy. The systemic PEI/miR-33a treatment resulted in Pim-1 downregulation and significant reduction of tumor growth. Notably, similar results were obtained upon PEI-mediated delivery of Pim-1 specific siRNA. We further extended our in vivo strategy for replacement of miR-145. We also observed miR-145mediated tumor inhibition through reduced proliferation and increased apoptosis. In this case c-Myc and Erk5 could be validated as in vivo targets for miR-145. Arnold GrГјnweller, PhD Pharmazeutische Chemie, Marbacher Weg 6, 35037 Marburg, Germany Telephone: 0049-6421-2825849 Email: [email protected] 69 022 SELECTIVE INTERSTRAND CROSS-LINKING THROUGH IN SITU OXIDATION OF FURAN-MODIFIED OLIGODEOXYNUCLEOTIDES E. Gyssels, M. Op de Beeck, A. Madder Laboratorium for Organic and Biomimetic Chemistry, University Ghent, Belgium The ease of customizing the reaction cycles of automated, solid-phase DNA synthesis has allowed for the efficient and site-specific introduction of chemical modifications in oligonucleotides. In our lab we became interested in techniques for crosslinking nucleic acids, whereby a covalent bond is introduced between two non-covalently hybridized strands. A novel crosslinking method was developed, where a customized oligodeoxynucleotide (ODN) is modified with a furan unit. Conversion of the furan to a reactive ketoaldehyde leads to site selective crosslinking to specific DNA-sequences. Several furan modified nucleosides have been synthesized for incorporation in reactive nucleic acids, allowing for a variable positioning of the furan unit in the duplex.[1-5] The position and linker by which the furan moiety is attached were shown to significantly influence the crosslinking properties of the furan-modified ODN and the characteristics of the duplex. Furthermore, crosslink possibilities were tested in triplex DNA by modifying the triplex forming oligonucleotide, TFO, with a furan moiety. The crosslinking characteristics of these furan modified oligonucleotides will be presented together with an outlook on various applications. References: 1.Stevens, K., et al., Furan-oxidation triggered inducible DNA cross-linking: acyclic versus cyclic furan containing building blocks. On the benefit of restoring the cyclic sugar backbone. Chemistry- A European Journal, 2011. accepted. 2.Op de Beeck, M. and A. Madder, Unprecedented C-Selective Interstrand Cross-Linking through in Situ Oxidation of Furan-Modified Oligodeoxynucleotides. Journal of the American Chemical Society, 2011. 133(4): p. 796-807. 3.Jawalekar, A.M., et al., Synthesis and incorporation of a furan-modified adenosine building block for DNA interstrand crosslinking. Chemical Communications, 2011. 47(10): p. 2796-2798. 4.Stevens, K. and A. Madder, Furan-modified oligonucleotides for fast, high-yielding and site-selective DNA inter-strand cross-linking with non-modified complements. Nucleic Acids Research, 2009. 37(5): p. 1555-1565. 5.Halila, S., et al., Fine-tuning furan toxicity: fast and quantitative DNA interchain crosslink formation upon selective oxidation of a furan containing oligonucleotide. Chemical Communications, 2005(7): p. 936-938. Ellen Gyssels Laboratorium for organic and biomimetic chemistry Krijgslaan 281, S4, 9000 Gent Belgium Telephone: +3292644479 Email: [email protected] 70 023 THE USE OF EXPRESSION PROFILES TO ASSESS NONHYBRIDISATION MEDIATED EFFECTS OF PHOSPHOROTHIOATE ANTISENSE OLIGONUCLEOTIDES Peter H Hagedorn, Andreas Petri, Sakari Kauppinen, Morten Lindow Santaris Pharma A/S, Kogle Alle 6, DK-2970 HГёrsholm, Denmark We have initiated systematic investigations of general class effects of LNA-modified phosphorothioate antisense oligonucleotides. To this end, we have created a reference set of gene-expression profiles from cultured cells, rodents, and non-human primates treated with different oligonucleotides, designated here as the Oligo Effect Map. Currently, this collection comprises profiles from 28 LNA oligonucleotides contrasted to saline vehicle or mock transfection, and integrates more than 500 samples (in vivo from liver and kidney and in vitro from cultured HuH-7, PC3, and HeLa cells) from 19 independent experiments. For each profile, we have catalogued detailed experimental information (for example oligonucleotide sequence, including positions with chemical modifications, delivery method, dosing regimen and dose level/concentration used), as well as phenotypic observations for the animals representing the profile. Using this resource we are able to detect gene expression patterns that occur in subsets of experiments and correlate these to experimental setup and oligonucleotide design. Ultimately this may provide us with useful information that enables the selection of conditions (experimental or oligochemistry) avoid of unwanted effects, facilitating the design of efficient and safe RNA drugs. Peter Hagedorn, PhD Santaris Pharma A/S Kogle AllГ© 6 DK 2970 HГёrsholm Danmark Telephone: +45 4517 9919 Email: [email protected] 71 024 A microRNA-mediated regulatory feedback loop in a new mechanism of resistance to TGF-beta induced growth inhibition Jonathan Hall, Afzal M. Dogar, Harry Towbin Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093В Zurich, Switzerland In an effort to develop new methods to identify RNAs which play dominant roles in disease mechanisms, we examined the function of several oncomirs in TGF-beta-mediated growth suppression. TGF-beta signaling is a complex biology and its dysregulation is implicated in several forms of cancer. Cancer cells usually secrete large amounts of latent TGF-ОІ and yet they are usually resistant to TGF-beta-induced growth inhibition. We found that suppression of TGF-ОІ1 mRNA by RNAi caused apoptosis in several cell lines with features in HeLa cells that indicated, paradoxically, a restoration of growth-inhibitory TGF-beta signaling. These included included transiently increased SMAD2 and AKT phosphorylation. MicroRNA expression profiling in Hela cells revealed that RNAi against TGF-beta-1 led to globally decreased oncomir expression including miR-18a and miR-24 which accounted for the derepression of TGF-beta-1 processing factors, thrombospondin-1 and furin, respectively. A detailed investigation led us to conclude that latent TGF-beta-1, thrombospondin-1 and furin form a microRNA-controlled regulatory feedback loop. For cells secreting high levels of latent TGF-beta, this loop offers a potentially widespread mechanism of escape from TGF-beta mediated growth inhibition at the earliest point in the signaling pathway – latent TGF-beta processing (1). Pharmacological targeting of TGF-beta mRNA by antisense or RNAi mechanisms may be of value in pathological mechanisms characterized by loss of the cytostatic TGF-beta signaling. (1) A. M. Dogar, H. Towbin, J. Hall. J. Biol. Chem. (2011) 286, 16447-16458. Jonathan Hall, PhD Institute of Pharmaceutical Sciences HCI H 437 Wolfgang-Pauli-Str. 10 8093 ZГјrich, Switzerland Telephone: 044 633 74 35 Email: [email protected] 72 025 NOVEL CELL PENETRATING PEPTIDES FOR SKELETAL AND CARDIAC MUSCLE DELIVERY OF PMO ANTISENSE OLIGONUCLEOTIDES FOR THE TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY Suzan M Hammond1, HaiFang Yin1,2, Amer F Saleh3, Corinne Betts1, Patrizia Camelliti1, Yiqi Seow1, Shirin Ashraf1, Andrey Arzumanov3, Thomas Merritt, Michael J Gait3 and Matthew JA Wood1 1 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; 2Tianjin Research Centre of Basic Medical Science, Tianjin Medical University, Tianjin, China; 3 Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Duchenne muscular dystrophy (DMD) is a severe recessive X-linked muscular dystrophy in which patients experience severe progressive muscle wasting, loss of ambulation and respiratory weakness. Greater than 90% of DMD patients also present with cardiac abnormalities. Aberrant missense mutations and deletions in the dystrophin gene (DMD) cause a variety of defects including the generation of premature termination codons and out-of-frame transcripts. Dystrophin is unique in that the central portion of the gene encodes a large rod domain made up of twenty four large spectrin-like repeats and four flexible hinge regions. Deletions which cause out-of-frame transcripts can be modified with controlled skipping of exons surrounding the mutation to restore in-frame DMD transcripts. Recent progress in antisense oligonucleotide (AON) therapy has advanced treatment of DMD into clinical trials. AONs are made more effective when combined with conjugated or non-conjugated cell penetrating peptides. We have developed a series of PNA/PMO internalizing peptides (Pip) called the Pip5 series. The Pip5 series enhances systemic delivery of exon skipping PMO AONs, particularly for delivery to the heart. The lead peptide conjugated PMO, Pip5e-PMO, is able to efficiently correct exon skipping and restore dystrophin production in mdx mice. With a single low dose administration of 25mg/kg, Pip5e restores dystrophin to 50-100% of the skeletal muscle fibres tested. Unique to the Pip5e peptide is the ability to also restore greater than 50% of the normal dystrophin level in the heart. To understand the elements of Pip5e which are important for its function we have begun development on a new series of Pip peptides conjugated to PMO (Pip6-PMO). With the Pip6 series we will be able to elucidate the important elements of the peptides which are important for dystrophin production in the heart. The Pip series derivatives have the potential for advancing the use of AON for treating DMD and may allow for further AON delivery toward the treatment of other cardiac related diseases. Suzan Michelle Hammond, PhD Oxford University Department of Physiology, Anatomy and Genetics Le Gros Clark Building South Parks Road Oxford OX3 1QX United Kingdom Telephone: 01865 272167 Email: [email protected] 73 026 Retinoic acid- inducible gene-I (RIG-I) detects (triphosphorylated) RNA of Listeria monocytogenes during infection Anna Maria Herzner1, Cristina Amparo Hagmann1, Christine Schuberth1, Christoph Coch1, Paul G. Higgins2, Hilmar Wisplinghoff2, Veit Hornung1, Gunther Hartmann1 and Martin Schlee1 1. Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, University of Bonn, Bonn, Germany D-53127 2. Institute of Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany The facultative intracellular bacterium L. monocytogenes induces cell-autonomous innate immune responses that are not yet fully understood. The innate immune system senses pathogens by pattern recognition receptors (PRR) located in different cell compartments. Components of the bacterial cell wall and conserved proteins are detected by Toll-like receptors (TLRs) on the cell surface, and by NODs or NOD like receptors (NLRs) in the cytosol of cells. Viruses are recognized via their nucleic acids by TLRs in the endosome and RIG-I like helicases in the cytosol. However, like the genetic material of viruses, nucleic acids of bacteria are also recognized by TLRs in the endosome. In contrast to exclusively extracellular bacteria, intracellular bacteria have evolved mechanisms to escape the endosome and enter the cytosol, where they are accessible to cytosolic PRRs. L. monocytogenes infection has been linked to cytosolic nucleic acid sensor-dependent type I IFN induction. Current knowledge indicates that the immune cell type I IFN response to L. monocytogenes depends on the signalling adaptor MITA downstream of recognition of bacterial DNA or second messengers such as cyclo-di-AMP. We hypothesized that besides bacterial DNA, bacterial RNA may enter the cytosol and contribute to innate immune responses to L. monocytogenes. In this study a novel in vivo RNA labelling technique applying click chemistry allowed us to visualize immediate cytosolic delivery of L. monocytogenes RNA upon infection. Furthermore, transfection of bacterial RNA into the cytosol of human monocytes, epithelial cells or hepatocytes resulted in a type I IFN response. In contrast to monocytes, the type I IFN response of epithelial cells and hepatocytes was not triggered by bacterial DNA. However, these cells still produced type I IFN during L. monocytogenes infection, demonstrating that a distinct DNA-independent nucleic acid recognition pathway must exist in these cells. SiRNA mediated knock-down of RIG-I or MAVS in epithelial cells abolished the IFN response to L. monocytogenes, but did not affect the immune response of monocytic cells. By contrast, knockdown of MITA in monocytic cells reduced cytosolic L. monocytogenes-mediated type I IFN induction indicating a DNA/ cyclo-di-AMP dependent recognition. Together these results provide evidence that RIG-I is required for immunorecognition of L. monocytogenes in non-immune cells such as epithelia or hepatocytes that lack a functional MITA dependent pathway for a direct response to DNA or cyclo-di-AMP. Anna Maria Herzner, PhD student Sigmund-Freud-Str 25 53127 Bonn, Germany Telephone: +49 (0)228 287 51157 Email: [email protected] 74 027 THIRTEEN WEEK NON-CLINCIAL TESTING OF MIRAVIRSEN IN CYNOMOLGUS MONKEYS Hildebrandt-Eriksen L, Persson R, Foy J, Tessier Y, Levin AA Santaris Pharma A/S, Hoersholm, Denmark The safety and tolerability of miravirsen sodium (SPC3649), currently in phase 2 clinical trials, was evaluated in a 13-week study with cynomolgus monkeys (Macaca fascicularis). Miravirsen is an antagonist to microRNA-122 (miR-122), which in turn has been shown to be a host factor in hepatitis C infection. The subcutaneous dosing regimen was divided into a loading phase with 18, 40, 80 and 180 mg/kg/week respectively over 2 doses the first week and a maintenance phase with 2.5, 5, 10 and 25 mg/kg/week once weekly for 12 weeks followed by 12 weeks off treatment. Miravirsen was well tolerated systemically and locally. Treatment-related changes in clinical pathology parameters mainly consisted of decreased serum cholesterol level, an expected pharmacological effect of miravirsen (as miR-122’s major function is the fine-tuning of lipid metabolism), transient, mild aPTT and PT prolongation and activation of the alternative complement pathway. Although some of these changes remained present through the 12-week treatment-free period, they were considered not to be adverse owing to their small magnitude and/or since they had no clinical or histopathological correlate. Microscopic findings were noted at all dose levels and were considered to be related to the known class-related effects of oligonucleotides. The kidneys were the main target organ. A dose-related accumulation of eosinophilic granules in the proximal convoluted tubules associated with a minor level of degeneration accounted for some functional disturbances in animals given ≥10 mg/kg/week and correlated with slightly increased serum urea and creatinine levels. Changes in the kidney resolved at the end of the treatmentfree period in most animals. The other histopathological effects (eg accumulation of enlarged macrophages principally in the lymph nodes and liver (Kupffer cells) but also in injection sites) were considered not to be adverse since they were not associated with any degenerative changes. At the end of the treatment-free period, the vacuolated macrophages in the lymph nodes showed a significant reduction in extent. Although some histopathological changes were seen at 5 mg/kg/week, they were at a minimal level, in a few animals, and were not expected to result in functional changes, including in the kidneys. Liver and kidney samples were analyzed for miravirsen content after 13 weeks treatment and 12 weeks recovery, and in a small cohort of animals receiving 80mg/kg for the first week only, on study days 5, 22, 43 and 70. The plasma pharmacokinetic evaluation confirmed the long tissue half-life time of miravirsen, accounting for the incomplete reversibility of some of the observed changes in tissues. The good safety profile of miravirsen sodium supports further clinical testing. Lisa Hildebrandt-Eriksen, PhD Santaris Pharma a/s Kogle AllГ© 6, 2970 Hoersholm, Denmark Telephone: +45 4517 9800 Email: [email protected] 75 028 DNA-CONTROLLED ASSEMBLY OF LIPOSOMES Ulla Jakobsen and Stefan Vogel Nucleic Acid Center, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark Oligonucleotide strands modified at both ends with alkyl chains have previously been reported to be able to induce assembly of liposomes in the presence of unmodified, complementary strands.1,2 Here, the assembly of liposomes caused by triple helix formation as well as the effect of varying the length of the alkyl chains in the membrane anchors are described. Liposome assembly controlled by triple helix formation is based on the same principle as the assembly controlled by double helix formation, albeit the steric and structural requirements are considerably more demanding. The assembly requires one triplex forming oligonucleotide (TFO) modified at both ends with lipophilic substituents, which will adhere reversibly to the surface of liposomes when mixed with these. In the presence of a duplex target capable of forming a triplex with the modified TFO, the for-med triple helix will be too rigid for both of the ends of a TFO to be anchored to the same liposome, and one of the ends will be released and adhere to another liposome, resulting in the formation of liposome aggregates (Figure 1). The assembly is reFigure 1 Schematic representation of assembly of liposomes versible, and the aggregates break controlled by triple helix formation. TFO (red ribbon), double down to the individual liposomes helix (blue ribbons) and liposomes (grey spheres) are not when heated to above the melting drawn to scale. temperature of the triple helix, but are reformed by cooling. As the liposome aggregates scatter the light considerably more than the individual liposomes, the assembly and disassembly process can be monitored by optical methods, e.g. dynamic light scattering (DLS) and UV spectroscopy. The membrane anchor previously described for assembly controlled by duplex formation consisted of two palmityl chains attached to a crown ether scaffold,1,2 and shorter (dodecyl) or longer (icosanyl) chains failed to function as membrane anchors, presumably because the former is attached to weakly and the latter to strongly to the liposomes. Jakobsen, U.; Simonsen, A. C.; Vogel, S. J. Am. Chem. Soc. 2008, 130, 10462-10463. Jakobsen, U.; Vogel, S. Methods Enzymol. 2009, 464, 233-248. 1 2 Ulla Jakobsen, PhD Campusvej 55, 5230 Odense M, Denmark Telephone: +45 65502599 Email: [email protected] 76 029 ENZYMATIC SYNTHESIS OF OLIGONUCLEOTIDES WITH 2’-AMINOLNA TRIPHOSPHATES AND TEMPLATES Marie W. Johannsen, Rakesh N. Veedu, Birte Vester, Jesper Wengel Nucleic Acid Center, University of Southern Denmark, Odense, Denmark Two very interesting classes of functional oligonucleotides are aptamers and DNAzymes. These have potential uses in both therapy and diagnostics, and rely either exclusively or partially on 3-dimensional structure rather than base pairing for activity. It is therefore not trivial to select an aptamer or DNAzyme for a specific target. Both aptamers and DNAzymes can be selected using SELEX (Systematic Evolution of Ligands by Exponential Enrichment), a method that relies on PCR to amplify the active molecules in the selection pool. Most aptamers and DNAzymes are modified to some degree after the selection process is complete, but there are obvious advantages to direct selection of oligonucleotides from modified selection pools. This requires that modified triphosphates are tolerated as substrates for enzymatic synthesis and that modified oligonucleotide templates can be copied enzymatically. LNA has previously been used in post-SELEX modification of aptamers and DNAzymes, and has received a lot of interest for its improvement of target binding as well as nuclease stability of oligonucleotides.1 A similar modification, 2’-amino-LNA, combines increased stability with the opportunity for conjugation to amino acids, fluorophores, etc.2 LNA triphosphates have previously been synthesised by our group as well as others and have been incorporated in oligonucleotides by enzymatic template directed primer extension. We present the synthesis of 2’-amino-LNA triphosphates with and without modification of the 2’-nitrogen as well as their incorporation into ONs by primer extension. We also show how oligonucleotides modified with 2’-amino-LNA-T can be used as templates for primer extension. This is the first step on the way to carrying out SELEX from 2’-amino-LNA modified selection pools, thus increasing the chemical diversity of DNAzymes and aptamers. Doessing, H.; Vester, B. Locked and Unlocked Nucleosides in Functional Nucleic Acids. Molecules 2011, 16, 4511-4526. 1 Johannsen, M.; Crispino, L.; Wamberg, M.; Kalra, N.; Wengel, J. Amino acids attached to 2’-amino-LNA: synthesis and excellent duplex stability. Organic & Biomolecular Chemistry, 2011, 9, 243-252 2 Marie Willaing Johannsen, MChem, MSc. University of Southern Denmark Campusvej 55 5230 Odense M Denmark Telephone: +45 6550 2548 Email: [email protected] 77 030 LIPID NANOPARTICLE FORMULATIONS OF MINIMAL-LENGTH shRNAS SHOW POTENT INHIBITION OF HCV-DRIVEN, LIVER-SPECIFIC GENE EXPRESSION IN MICE B. H. Johnstona,e, A. Dallasa, H. Ilvesa, J. Shorensteina, M. A. Behlkeb, SP Wonge, R. Harbottlec, and I. MacLachland a SomaGenics, Inc., 2161 Delaware Ave., Santa Cruz, CA 95060, USA b IDT, Coralville, IA 52241, USA c Imperial College London, UK d Tekmira Pharmaceuticals, Burnaby, B.C., Canada e Dept. of Pediatrics, Stanford Univ., Stanford, CA, USA We have identified short shRNAs (sshRNAs) that target a highly conserved HCV sequence and potently inhibit expression in reporter systems.1 Chemical modifications were subsequently introduced to increase nuclease stability while minimizing immunostimulatory activity and maintaining potency.2 In the current study, we assessed the efficiency of liver delivery and in vivo efficacy of these sshRNAs when formulated into lipid nanoparticles (LNP). Single doses of HCV-targeting sshRNAs formulated into LNP were injected i.v. into mice that had previously been treated with a plasmid providing long-term expression of an HCV-firefly luciferase fusion protein from a liver-specific promoter. In vivo imaging showed a dose-dependent inhibition of luciferase expression (>90% at 2.5 mg/kg sshRNA), with a half-time of recovery of about 3 weeks (see Figure; expression normalized to Day 0 = 100%). No inhibition was seen with a scrambled control, saline, or a mock treatment. These results demonstrate the ability to provide durable knockdown of an HCV target by systemic delivery of formulated sshRNAs. Q. Ge, et al. (2010) RNA 16(1):106-17; 2Q. Ge, et al. (2010) RNA 16(1):118-30. 1 Brian Johnston, PhD SomaGenics, Inc. 2161 Delaware Avenue Santa Cruz, CA 95060 USA Telephone: (831) 426-7700 ext. 12 Email: [email protected] 78 031 HIGHLY POTENT, MINIMAL-LENGTH shRNAS TARGETING HEPATITIS C VIRUS HAVE NOVEL MECHANISTIC FEATURES B. H. Johnstona,d, A. Dallasa, H. Ilvesa, P. Kumara, S. A. Kazakova, J. Shorensteina, M. A. Behlkeb, M. McManusc, and Q. Gea a SomaGenics, Inc., 2161 Delaware Ave., Santa Cruz, CA 95060, USA b IDT, Coralville, IA 52241, USA c UCSF Diabetes Center, University of California, San Francisco, CA 94143, USA d Dept. of Pediatrics, Stanford University, Stanford, CA 94305, USA Current therapies for hepatitis C, which afflicts some 200 million people world-wide, have severe side-effects and are of limited efficacy. The recently approved protease inhibitors are more effective when given as a combination therapy with interferon, but due to the latter’s severe side-effects, there is an urgent need for alternatives with an independent mechanism of action that can replace interferon. We have identified short hairpin RNAs (shRNAs) that target a highly conserved HCV sequence and potently inhibit expression in reporter systems, with IC50 < 10 pM in human 293FT cells.1 shRNAs are of similar or greater potency than cognate siRNAs in human hepatocytes, HCV replicon systems, and mouse models of HCV. A connecting (loop) sequence between the sense and antisense sequences of 2 nt (or a nonnucleotide linker of similar length) gives optimal potency, but a loop of 1 nt or even a direct connection between the sense and antisense sequences also provide good activity. The position of the shRNA loop is important: unlike right-hand (R) loop shRNAs, which require a 3’ overhang for optimal activity, left-hand (L) loop shRNAs can be effective with no overhang, and are generally more potent than R-type shRNAs. Blunt-ended molecules require some 2’-modification to avoid inducing a RIG-I-mediated immune response. Modification patterns that increase nuclease stability while minimizing immunostimulatory activity and maintaining potency have been systematically identified.2 shRNAs with stem lengths of 16–19 bp are not substrates for recombinant Dicer and maintain their potency in conditional Dicer-knockout cells, yet can be highly potent. 5’-RACE analysis shows a normal target cleavage position regardless of Dicer cleavability. Chemical modification to block nuclease cleavage at various sites, Ago-immunoprecipitation results, and other evidence indicates that the mechanism of action of shRNAs need not involve cleavage of the loop and may differ from the mechanism of siRNAs or Dicer-substrate RNAs in some key respects. Our findings are encouraging for the prospects of the therapeutic use of direct-delivered shRNAs. 1. Q. Ge, H. Ilves, A. Dallas, P. Kumar, J. Shorenstein, S. A. Kazakov, and B. H. Johnston (2010) Minimal-length short hairpin RNAs: В The Relationship of Structure and RNAi Activity. RNA 16(1):106-17. 2. Q. Ge, A. Dallas, H. Ilves, J. Shorenstein, M. A. Behlke, and B. H. Johnston (2010) Effects of Chemical Modification on the Potency, Serum Stability, and Immunostimulatory Properties of Short shRNAs. RNA 16(1):118-30. Brian Johnston, PhD SomaGenics, Inc. 2161 Delaware Avenue Santa Cruz, CA 95060 USA Telephone: (831) 426-7700 ext. 12 Email: [email protected] 79 032 EPATOCYTE SPECIFIC TARGETING AND DELIVERY OF siRNACARBOHYDRATE CONJUGATES Kallanthottathil G. Rajeev, K. NarayanannairJayaprakash, Gang Wang, Ligang Zhang, Chang GengPeng, Jennifer Sherman, Mariano Severgnini, Amy Chan, Klaus Charisse, Renta Hutabarat, Martin Maier, Kevin Fitzgerald,Dinah W.Y. Sah, Muthiah Manoharan, Kenneth Koblan and Tracy S. Zimmermann Alnylam Pharmaceuticals, Cambridge, MA, USA The asialoglycoprotein receptor (ASGPR) is a transmembrane receptor that mediates clearance of extracellular glycoproteins with exposed terminal galactose residues. Thisreceptor is highly expressed on the surface of liver hepatocytes and facilitates multiple rounds of uptake and clearance of glycoproteins. These features of the ASGPR along with its ligand specificity make it an attractive strategy for liver-specific delivery of galactosylated-drugs. We have shown that conjugation of a triantennary N-acetylgalactosamine ligand to a siRNA (siRNA-GalNAc) has nM binding affinity to the ASGPR and facilitates uptake and gene silencing both in vitro and in vivo. We will describe further improvements ondelivery of siRNA-GalNAc conjugates that lead to enhanced siRNA pharmacology. The results to be presented include the impact of mode of administration on efficacy, application across multiple targets in hepatocytes, and demonstration of liver-specific gene silencing at low mg/ kg siRNA-GalNAc doses in mouse, rat and non-human primate. Gopalan Rajeev Kallanthottathil, PhD 300 Third Street, Cambridge, MA 02142 USA Telephone: 1 617 551 8313 Email: [email protected] 80 033 LNA ANTISENSE OLIGONUCLEOTIDES - A STRAIGHTFORWARD CONCEPT FOR RNA THERAPEUTICS Susanne KammlerВ№, Niels Fisker NielsenВ№, Marie LindholmВ№, Robert E. LanfordВІ, Andreas PetriВ№, Sakari KauppinenВ№, Nathalie UzcateguiВ№, Ellen Marie StraarupВ№, Joacim ElmГ©nВ№, Troels KochВ№, Henrik Г�rumВ№, Maj HedtjГ¤rnВ№, Bo Rode HansenВ№ В№Santaris Pharma A/S, HГёrsholm, Denmark ВІDepartment of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, TX 78227, USA In the last decade, due to innovative oligonucleotide chemistry and designs, single stranded antisense oligonucleotides have emerged as a highly promising therapeutic modality. Oligonucleotide mode of action spans from RNaseH mediated mRNA degradation to microRNA inhibition and modulation of RNA splicing. Locked Nucleic Acid (LNA) oligonucleotides, epitomise this new generation of effective RNA therapeutics, combining high binding affinity, target specificity, regulatory potency and nuclease stability with unmatched short length of the antisense oligonucleotide. It has been shown that LNA oligonucleotides readily enter cells via un-assisted uptake (gymnosis), in an active form allowing in vitro prediction of potent therapeutically active compounds as well as in vivo distribution to manifold cell types and tissues. This makes Santaris Pharma A/S’ Locked Nucleic Acid (LNA) drug discovery platform a straightforward tool to transform today’s knowledge about disease associated gene regulation into tomorrow’s RNA therapeutics. Susanne Kammler, Research Scientist, Technology & Partnered discovery Santaris Pharma A/S KogleВ AllГ© 6 DK 2970 HГёrsholm Denmark Telephone: +45В 4517 9915 Email: [email protected] 81 034 ANTISENSE-INDUCED MYOSTATIN EXON SKIPPING LEADS TO MUSCLE HYPERTROPHY IN MICE FOLLOWING IN-VIVO TREATMENT WITH CONJUGATED OLIGONUCLEOTIDES Kang JK, Malerba A, Popplewell L, Foster K, Dickson J Royal Holloway, University Of London, Egham, Surrey, United Kingdom, TW20 0EX Inadequate intake of energy could lead to a reversible loss of muscle mass but in certain other conditions this loss cannot be reverted nutritionally. Patients suffering from cancer, chronic obstructive pulmonary disease (COPMD), AIDS etc, tend to lose body weight, a condition called cachexia. Cachexia is responsible for increased risk of death due to the underlying condition in such patients. Degenerative muscular disorders such as various forms of muscular dystrophies could actually be fatal at an early age of mid to late twenties. Muscle mass loss resulting from ageing and obesity also has a very high impact on public health. Myostatin is a negative regulator of muscle mass, and natural mutations in myostatin in whippets, Belgian blue bull, myostatin mull mice and even in humans have resulted in significantly increased muscle mass relative to their normal counterparts. Several strategies are being developed to knock down the expression of the myostatin gene as a means to bring about improvements in the muscle wasting conditions including Duchenne muscular dystrophy. In this study the use of antisense oligonucleotides (AOs) to manipulate pre-mRNA splicing and knockdown of myostatin has been reported. Previously we have demonstrated the use of AOs of three different chemistries: 2’O-methyl RNA (2’OMePS-with a phosphorothioate backbone), phosphorodiamidate morpholino (PMO) and octa-guanidine conjugated PMO (vivo-PMO) to induce myostatin exon skipping. Systemic administration of vivo-PMO showed that myostatin exon skipping led to a significant increase in the mass and cross sectional area of soleus muscle of treated mice. In order to examine the longevity of PMO action in treated muscles, exon skipping and muscle mass was evaluated in mice across an 8 week period in response to a single intramuscular administration. Following a single intramuscular injection of vivo-PMO in mice, exon skipping in the myostatin mRNA was recorded from week 1 through to week 8 at high levels. In addition, a significant increase in muscle mass was observed 4 weeks following treatment, and this weight gain was maintained and enhanced out to 8 weeks. In conclusion, myostatin knock-down by exon skipping Vivo-PMOs exhibits a long half-life of action in treated skeletal muscle, and thus provides a potential therapeutic strategy to counter muscular dystrophy, muscular atrophy, cachexia and sarcopenia. Jagjeet Kaur Kang, PhD candidate School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, United Kingdom, TW20 0EX Telephone: 7895709958 Email: [email protected] 82 035 Preclinical Development of an sd-rxRNATM Based Therapy for Treatment of Fibrosis and Retinal Disorders Anastasia Khvorova, Karen Bulock, James Cardia, Lyn Libertine, Kehlen Flannery-Rossi, Jake Metterville, Tim Drew, Michael Byrne, Christine Resmini, Nick Palumbo, Glenna Ford, Katherine Holton, Amanda Rodgers, Jessica Lam, William Stanney, Kevin Fettes, Pamela Pavco RXi Pharmaceuticals, Worcester, MA, USA, 01605 Fibrosis is a widespread reparative or reactive process resulting in the accumulation of excess fibrous tissue. This biological process is involved in a large number of clinical indications with significant unmet medical need. We have identified a novel class of RNAi compounds termed “self-delivering rxRNA” (sd-rxRNA) that demonstrates robust cellular uptake and silencing of target genes without the requirement for a delivery vehicle. sd-rxRNAs combine features of conventional RNAi and antisense technology and are heavily chemically modified to improve stability and lipophilicity. These RNAi compounds are rapidly and efficiently taken up by cells and demonstrate potent activity, enhanced stability, and reduced immune stimulation. sd-rxRNAs with in vitro EC50 values in the low nM range have been identified for several genes known to be involved in fibrosis. Using intra-dermal injection in a rodent model of dermal wound healing, we have demonstrated statistically significant, dose dependent silencing of the anti-fibrotic target, CTGF. The data presented show the efficacy and longevity (>2 weeks) of locally-administered sd-rxRNAs targeting CTGF. CTGF silencing in vivo results in potent reduction in myofibroblast infiltration and collagen deposition, indicating that anti-CTGF sd-rxRNA (RXi-109) is a promising anti-fibrotic agent. An IND supporting evaluation of RXi-109 safety, tolerability and indication of efficacy is expected to be filed later in the year. In addition, data on uptake, dose response, and duration of silencing (up to 21 days) and initial safety of sd-rxRNAs in rodent retina will be presented. Anastasia Khvorova, PhD RXi Pharmaceuticals 60 Prescott Street Worcester, MA 01605 USA Telephone: 508-929-3616 Email: [email protected] 83 036 Anti- cancer approach through microRNA-regulated RNA replacement Ju-hyun Kim, You Sub Won, Ranhui Won, and Seong-Wook Lee* Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University, Yongin 448-701, Korea (*, [email protected]) We have previously presented that the group I intron from Tetrahymena could induce new gene activity selectively in target cells by recognizing the cell-specific RNA and catalyzing the in-frame fusion of desired RNA sequences with trans-splicing reaction. Moreover, the ribozymes trigger cytotoxin activity only in cancer cells expressing the target RNA, resulting in specific regression of the cells. Particularly, the specific trans-splicing reaction worked well by the delivery of the specific ribozyme using the adenovirus into various xenograft carcinomatosis nude mouse model, inducing highly selective expression of a reporter gene in the human tumors but not in normal mouse organs, and moreover, causing the efficient regression of hTERT-positive tumors in the mice. However, one concern for the hTERTtargeting anti-cancer approach includes potential side effects to hTERT+ normal cells such as germ line, stem cells, and highly proliferating normal cells. In this study, to improve cancer- specific expression of the ribozyme, we have attempted to combine miRNA regulation via using miRNA target site. To overcome the side effects to hTERT(+) bone-marrow derived blood cells, we have attempted to insert perfect target sites against blood cell-specific miRNA181a into 3’UTR downstream of 3’ exon of the trans-splicing ribozyme. Moreover, we have used target site of liver-specific miRNA122a for liver cancer specific transgene induction. We observed that transgene induction by the ribozymes was specifically shown in cancer cell. Furthermore, retardation of cell growth was specifically induced in hTERT(+) and miRNA(miR181(-) or miR122(-)) cancer cells when infected adenovirus with miRNA181a or miRNA122a target site-containing ribozyme with HSVtk gene as 3’ exon and treated with GCV, but not in miRNA(miR181(+) or miR122(+)) cells. Moreover, specificity of cancer regression in xenograft mouse model was also highly increased without compromising its anti-cancer efficacy. These observations provide the basis for a novel approach to cancer gene therapy and demonstrate that targeted and miRNA-regulated transgene induction can be used as powerful anti-cancer approach. Ju-hyun Kim, MS Department of Molecular Biology, Dankook University 126, Jukjeon-dong, Suji-gu Yongin 448-701, Korea Telephone: 82-31-8005-3203 Email: [email protected] 84 037 HINT-1 HYDROLASE-ASSISTED CLEAVAGE OF INTERNUCLEOTIDE BONDS IN DINUCLEOSIDE PHOSPHOROTHIOATES SUGGESTS ITS POSSIBLE PARTICIPATION IN METABOLISM OF OLIGONUCLEOTIDE DRUGS AND PRODRUGS Agnieszka Krakowiak, Beata RД™bowska, Milena Sobczak, Barbara MikoЕ‚ajczyk, Wojciech J. Stec Centre of Molecular and Macromolecular Studies of the Polish Academy of Science, Department of Bioorganic Chemistry, Lodz, Poland Therapeutic oligonucleotides, e.g. siRNA or antisense constructs, are degraded in vivo by numerous endo- and exonucleases. Phosphorothioate oligonucleotides (PS-oligos) containing a sulfur atom attached to the phosphorus at each internucleotide bond are considered as prospective therapeutic molecules because they are much more stable in blood, plasma and various cellular systems than their natural unmodified counterparts [1]. However, under certain nucleolytic conditions these oligomers undergo slow degradation, which proceeds mainly from the 3’-end and generates mononucleoside 5’-phosphorothioates (NMPS or dNMPS). The products of degradation may alter cell proliferation, DNA and RNA synthesis as well as induce other unknown effects. In 2007, the phosphorothioate DNA segments have been identified in bacterial DNA [2], which makes investigations on the PS-oligo metabolism even more important. Enzymes able to metabolize nucleoside 5’-phosphorothioates are poorly known: only 5’-nucleotidase (ecto-5’-NT) and alkaline phosphatase have been taken into account so far. We found that Hint-1, which belongs to histidine triad superfamily (HIT), catalyzes the cleavage of internucleotide bond in dinucleotide phosphates and thiophosphates, and the products are corresponding nucleosides as well as nucleoside 5’-phosphates or –phosphorothioates. Moreover, Hint-1 enzyme is able to desulfurate NMPS and dNMPS under in vitro conditions [3]. These results indicate that Hint-1 protein, which homologues have been found in all forms of life, may play important role in metabolism of therapeutic oligonucleotides. 1 Dias N., Stein CA, (2002) Mol. Cancer Ther. 1, 347–355. 2 Wang, L., Chen, S., Xu, T., Taghizadeh, K., Wishnok, J.S., Zhou, X., You, D., Deng, Z., Dedon, P.C. (2007) Nature Chem. Biol. 3, 709-710 3 M. Ozga, Dolot R, Janicka M, Kaczmarek R, Krakowiak A. (2010) J Biol Chem. 285, 40809-18 Agnieszka Krakowiak, PhD POLAND, 90-363 Lodz, Sienkiewicza 112 Telephone: +4842 6803272 Email: [email protected] 85 038 2’-BISPYRENE-MODIFIED OLIGO (2’-O METHYLRIBONUCLEOTIDE) PROBES AS TOOLS FOR THE SENSITIVE DETECTION OF RNA Krasheninina O.A., Novopashina D.S., Venyaminova A.G. Institute of Chemical Biology and Fundamental Medicine SB RAS To date pyrene-modified oligonucleotides have considered as challenging tools in molecular biology, genomics and diagnostics. It is related to remarkable properties of pyrene as fluorophore such as high quantum yields, abilities to form p-stacking dimers and to intercalate into nucleic acids duplex. Conjugates of octadeca(2'-O-methylribonucleotides) which contained from one to three 2'-bispyrenylmethylphosphorodiamidate pyrimidine or purine nucleosides were obtained by analogy with [1]. The properties of new type of the 2'-bispyrene-modified oligo(2'-Omethylribonucleotides) as fluorescent probes for RNA detection were investigated. The sequences of probes were complementary to two available regions of MDR1 mRNA. In order to increase nuclease resistance and duplex stability of pyrene-modifed probes, we introduced into probes 3’-terminal В«invertedВ» thymidine. The structures of pyrene-modified probes were confirmed by mass-spectrometry, UV- and fluorescent spectroscopy. Thermal stability of duplexes of probes with model NA targets corresponding to MDR1 mRNA nucleotides 113-137 and 315-336 was investigated by thermal denaturation method. The incorporation of 2’-bispyrene-modified nucleotides led to the slight decrease of the thermal stability of probes duplexes, with Tm ranged from 54 to 66В°C for DNA-targets and from 67 to 87В°C for RNA-targets. It’s worth noting that the thermal stability of these duplexes decreased upon increasing quantity of 2'-bispyrene nucleotides. The fluorescent properties of 2'-bispyrenelabeled probes varied depending on their sequences and structures. In all cases the binding of pyrene-labeled probes to the model NA targets resulted in enhancement of the pyrene eximer fluorescence. The hybridization of the most sensitive probes to model 25-mer RNA and DNA targets and 5'-terminal fragment of MDR1 mRNA (nucleotides 1-678) was investigated by fluorescence-monitored titration with an increasing target concentration. The association constants obtained from fluorescence titration data using the modified Shtern-Folmer equation were calculated for model 25-mer RNA targets and 678-mer fragment of MDR1 mRNA. The results obtained in this study indicate that the new fluorescent 2'-bispyrene-labeled oligo(2'O-methylribonucleotide) probes are sensitive tools for RNA detection. 1. Novopashina D.S., Stetsenko D.A., Totskaya O.S., Repkova M.N., Venyaminova A.G. Nucleosides Nucleotides Nucleic Acids, 24, 729 (2005). This work is supported by Federal Target Program “Scientific and science educational personnel of innovative Russia“ (State contract N P1334). Olga Krasheninina, PhD student 630090, Lavrentiev ave. 8, Novosibirsk, Russia Telephone: 007(383)3635129 Email: [email protected] 86 039 Synthesis of 2’-Amino-UNA Niels LangkjГ¦r, Jesper Wengel University of Southern Denmark, Odense, Denmark Over the last decade chemicaly modified nucleosides have recived a renewed focus with the development novel techniques in biochemistry such as RNAi. Especially UNA (unlocked nucleic acid) has been demonstrated to perform well in siRNA applications with lowered off-target effects and enhanced activity.1,2 We herein introduce the 2’-aminoUNA derivative with an amino functionality at the 2’-position of UNA and present the synthesis of the 2’-amino-UNA phosphoramidite (9) from uridine (1) using the previously published selective benzoylation of the 2’-hydroxylgroup of UNA.3 Figure 1 - UNA, 2-Amino UNA and RNA. Scheme 1 - Synthesis of 2’-Amino-UNA. References: 1. 2. 3. Bramsen, J. B., Laursen, M. B., Nielsen, A. F., Hansen, T. B., Bus, C., Langkjaer, N., Babu, B. R., HГёjland, T., Abramov, M. , Van Aerschot, A., Odadzic, D., Smicius, R., Haas, J., Andree, C., Barman, J., Wenska, M., Srivastava, P., Zhou, C., Honcharenko, D., Hess, S., MГјller, E., Bobkov, G. V., Mikhilov, S. N., Fava, E., Meyer, T. F., Chattopadhyaya, J., Zerial, M., Engels, J. W., Herdewijn, P., Wengel, J., Kjems, J., Nucleic Acids Res., 2009, 37, 2867-2881. Bramsen, J. B., Pakula, M. M., Hansen, T. B., Bus, C., LangkjГ¦r, N., Odadzic, D., Smicius, R., Wengel, S. L., Chattopadhyaya, J., Engels, J. W., Herdewijn, P., Wengel, J., Kjems, J., Nucleic Acids Res. 2010, 38 (17), 5761-5773. LangkjГ¦r, N., Pasternak, A., Wengel, J., Bioorg. Med. Chem. 2009, 17 (15), 5420-5425. Niels LangkjГ¦r, Cand. Scient. Chemistry Campusvej 55, 5230 Odense M, Denmark Telephone: +45 6550 2548 Email: [email protected] 87 040 Allosteric ribozyme capable of specifically inhibiting miR-122 functions through HCV NS5B protein sensing Chang Ho Lee, Ji Hyun Kim, Hee Won Kim, Seong-Wook Lee* Department of Molecular Biology, Institute of Nanosensor and Biotechnology Dankook University, Yongin 448-701, Republic Korea Functional sequestration of microRNA 122 (miR-122) by treatment with an oligonucleotide complementary to the microRNA results in long-lasting suppression of hepatitis C virus (HCV) viremia in primates. However, the safety of the constitutive miR-122 silencing approach to HCV inhibition is unclear, since miR-122 can modulate the expression of many host genes. Here, we try to develop a genetic regulation system capable of specifically inhibiting miR-122 activity only upon HCV infection. With an in vitro selection technique we developed an allosteric self-cleavable Hammerhead ribozyme, whose activity was specifically induced by HCV NS5B replicase such as to release the most efficacious antisense sequence to miR-122 only in the presence of HCV NS5B. The activity of the reporter construct with miR-122 target sequences at its 3’ untranslated region and the expression of endogenous miR122 target proteins were specifically stimulated only in HCV replicon Huh-7 cells, but not in naГЇve Huh-7 cells, when transfected with the specific allosteric ribozyme, indicating that miR-122 activity can be specifically silenced by the allosteric ribozyme only in HCV replicating cells. Noticeably, HCV replicon replication was efficiently inhibited by the allosteric ribozyme. This ribozyme could be useful for the specific, safe, and efficacious anti-HCV therapeutic tool. Chang Ho Lee, PhD candidate Department of Molecular Biology, Institute of Nanosensor and Biotechnology Dankook University, Yongin 448-701, Republic Korea Telephone: 82-31-8005-3203 Fax: 82-31-8005-4058 Email: [email protected] *[email protected] 88 041 Are short oligonucleotides less specific in vivo? Morten Lindow, Peter Hagedorn, Andreas Petri Santaris Pharma A/S, Kogle Alle 6, DK-2970 HГёrsholm, Denmark The challenge of developing effective and safe RNA-targeted therapeutics calls for robust in silico design of target-specific oligonucleotides with minimal effect on unintended targets. In this process, many molecular biologists and statisticians often ask how many perfect match recognition sequencesa given oligo-nucleotide has in the transcriptome. This, in turn, leads to the assumption that the longer a given oligonucleotide, the more specific it will be, whereas short oligonucleotides areoften considered unspecific and to have more off-target effects. However, this assumptionis only correct if the hybridization stringencycan be controlled in such a way that a single mismatch, an insertion, or a deletion will abrogate effective binding to unintendedtargets.This is indeed the case in most experiments such as PCR, Northern blot analysis, and in situ hybridization. The primary goal is to find experimental settings in which the oligonucleotidepreferably hybridizes to the intended target. In the lab, hybridization conditions such as temperature and ionic strength can be controlled to achieve optimal specificity. By comparison, hybridization conditionsin live animals or humans cannot be adjusted, instead altering the length, design and chemical modifications of a given oligonucleotide must be utilized to achieve adequate specificity. Here, we present and compare different oligonucleotide specificity prediction algorithms. We show that different methods can produce qualitatively very different results. Algorithms based on minimizing mismatches predict that short oligonucleotides are less specific, whereas methods that maximize the number of basepairs or binding affinity to putative targets predict that longer oligonucleotides are less specific.We assess the aforementioned approaches using transcriptomal expression profiling of mice livers after treatment with different LNAphosphorothioate oligonucleotides. Morten Lindow, PhD Santaris Pharma A/S Kogle Alle 6, DK2970-Horsholm, Denmark Telephone: +45 2849 9682 Email: [email protected] 89 042 MECHANISTIC INSIGHTS INTO LIPID NANOPARTICLE-MEDIATED DELIVERY OF siRNA Martin A. Maier1, Jerome Gilleron3, William Querbes1, Xuemei Zhang1, Valerie Clausen1, Renta Hutabarat1, Yosef Landesman1, Nenad Svrzikapa1, June Qin1, Undine Schubert3, Muthusamy Jayaraman1, Michael J. Hope2, Dinah W. Y. Sah1, Kevin Fitzgerald1, Kallanthottathil G. Rajeev1, Mark A. Tracy1, Rachel Meyers1, Muthiah Manoharan1, Marino Zerial3, Akin Akinc1 1 Alnylam Pharmaceuticals, Cambridge, MA 02142, USA 2 Alcana Technologies, Vancouver, BC, Canada V6L 2A1 3 Max Planck Institute of Molecular Cell Biology and Genetics, 01309 Dresden, Germany Safe and efficient delivery of short interfering RNAs (siRNAs) is a prerequisite for the development and advancement of RNAi therapeutics as a new class of innovative medicines. Lipid nanoparticles (LNPs) are among the most efficient carriers for systemic delivery of siRNA and have been successfully employed to silence therapeutically relevant genes in a number of species from rodents to non-human primates. In recent years, multiple liver-targeted RNAi programs utilizing LNP-formulated siRNAs have advanced into human clinical trials. Meanwhile, the discovery of novel lipid materials and the recent progress on the mechanistic understanding of the LNP platform has enabled the development of the next generation LNPs with improved therapeutic indices. Comparing two distinct classes of lipid-based nanoparticles, we have previously shown that apolipoprotein E acts as an endogenous targeting ligand for ionizable lipid nanoparticles but not for cationic lipid nanoparticles, which carry a permanent positive charge at physiologic pH. At the cellular level, functional delivery requires the release of siRNA from the endosomal compartment into the cytoplasm. Electron microscopy (EM) was used to track the cellular uptake and intracellular trafficking of siRNA labeled with gold nanoparticles and formulated in LNPs. Based on EM analysis of cultured cells as well as liver sections from treated animals, we estimate that a low percentage of the Au-labeled siRNA escapes the endosomal pathway and reaches the cytosol, which is in good agreement with time course studies using stem-loop PCR for siRNA quantification. These results help provide insights into the intracellular mechanism of LNP-mediated siRNA delivery, which may facilitate further rational optimization of this siRNA delivery platform. Martin A. Maier, PhD 300 Third Street Cambridge, MA 02142, USA Telephone: +1-617-551-8274 Email: [email protected] 90 043 Characteristic aspects of skeletal muscle as a target organ for siRNA Naoki Makita1, Ichiro Fujimoto2, Sachiko Fukuda1, Takeshi Shimizudani1, Shunji Nagahara1 1 DDS Research Group, Formulation Research & Development Laboratories, Technology Research & Development, Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan 2 Research Institute, Koken Co., Ltd., Tokyo, Japan Skeletal muscle is one of major organ and in which some severe disorders, including muscle dystrophy, are known to occur due to various factors abnormality. Skeletal muscle is accessible organ and therefore is thought to be good target for treatment by siRNA, however few approaches have been reported so far. Atelocollagen is the first vehicle which succeeded to deliver siRNA into tumor by systemic administration.[1] After that, a lot of studies which demonstrate usefulness of atelocollagen as a delivery vehicle for siRNA by a local and systemic administration have been reported in terms of efficacy and safety. In 2008, it was reported that siRNA formulated with atelocollagen suppressed its target gene expression and led to morphological changes in skeletal muscle via not only local but also systemic injection.[2] So, we utilized atelocollagen as a vehicle for introducing siRNA into skeletal muscle in order to clarify characteristics of skeletal muscle as a target organ for siRNA treatment. In this session, we will discuss unique properties of skeletal muscle, based on our result of a series of experiments to investigate relationship between therapeutic efficacy and experimental conditions, such as administration procedures, dosage amount and so on. [1] Takeshita, F. et.al. Proc Natl Acad Sci USA., 2005, 102, 12177-12182. [2] Kinouchi, N. et.al. Gene Therapy, 2008, 15, 1126-1130. Naoki Makita DDS Research Group, Formulation Research & Development Laboratories, Technology Research & Development, Dainippon Sumitomo Pharma Co., Ltd. 3-45, Kurakakiuchi 1-Chome, Ibaraki-shi, Osaka 567-0878, Japan Telephone: +81-72-627-8146 Email: [email protected] 91 044 ACTIVATION OF CYCLOOXYGENASE-2 (COX-2) GENE EXPRESSION BY PROMOTER-TARGETED DUPLEX RNAs Masayuki Matsui1, Huiying Zhang1, Yougjun Chu1, Klaus Charisse2, Muthiah Manoharan2, David R. Corey1, and Bethany A. Janowski1 1 Departments of Pharmacology and Biochemistry, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390-9041, USA 2 Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142, USA RNA-mediated gene activation has been investigated over several years in some genes such as progesterone receptor (PR), LDL receptor (LDLR), p21, and E-cadherin, but its mechanism is not fully understood yet. To obtain more insights into mechanism of RNA-mediated gene activation, we targeted cyclooxygenase-2 (COX-2) gene using promoter-targeted duplex RNAs (antigene RNAs (agRNA)). COX-2 is a rate-limiting enzyme responsible for prostaglandin biosynthesis, and COX-2 gene expression is associated with tumorigenesis, inflammation, and apoptosis. Small RNAs that modulate COX-2 gene expression would be useful as tools to investigate biological functions of COX-2 in cells. To investigate whether agRNAs have the potential to modulate COX-2 gene expression, duplex RNAs targeting the promoter were designed and tested in A549 lung cancer cells for gene activation. After transfecting agRNAs, western blot and qPCR analysis were performed to check expression levels of COX-2 protein and mRNA. We found that some of the duplex RNAs (agRNA-12 etc; 25 nM) can activate COX-2 gene expression by more than 20-fold relative to mismatch controls. The fold activation was similar with that for IL-1b (10 ng/mL), a natural activator for COX-2 gene. We observed more than 100-fold activation in COX-2 mRNA in cells treated with both the activating agRNA and IL-1b, which indicates the two activators can work synergistically. ChIP analysis showed increased occupancy of RNA polymerase II by 3-5 fold and NFkB by 2-4 fold in the promoter, suggesting that this activation occurs at the level of transcription. In our previous studies with PR and LDLR, we identified non-coding transcripts overlapping their promoters, which could be potential targets for agRNAs. We performed RACE and strand-specific RT-qPCR to examine whether similar noncoding transcripts are expressed in the COX-2 gene promoter. Those analyses revealed that sense and antinsense transcripts are expressed at the COX-2 gene promoter. These transcripts might be an endogenous mediator for regulating COX-2 gene expression that can be targeted by agRNAs. Transfection of activating agRNAs did not substantially change expression of interferon responsive genes such as OAS1, OAS2, MX1, IFITM1, and ISGF3g, suggesting that the activation of COX-2 gene by agRNAs is not due to interferon response. These data suggest that COX-2 gene can be induced by promoter-targeted duplex RNAs and the gene can be used as a model for research of RNA-mediated gene activation. Masayuki Matsui, PhD University of Texas Southwestern Medical Center at Dallas 6001 Forest Park Road Dallas, TX 75390-9041 USA Telephone: +1-214-645-6154 Email: [email protected] 92 045 Abstract and poster available for review at the meeting. 93 046 Antisense oligonucleotides for Functional Studies of Human Argonaute Proteins Mescalchin A. and Innarella M.R. Institute of Molecular Medicine, University of LГјbeck, Center for Structural and Cell Biology in Medicine (CSCM), Ratzeburger Allee 160, D-23538 LГјbeck, Germany The Argonaute proteins play important regulatory roles in the metabolism of cells and are involved in RNA interference (RNAi) mechanisms. In Caenorhabditis elegans and Arabidopsis thaliana, creation of Argonaute (Ago) mutant strains has allowed the study of their biological function, while in mammals this approach has been so far unsuccessful due to lethality of such mutations. In human cells four different Ago paralogs are present (hAgo1-4) and the functional studies of these proteins relied on phenotypic suppression using small interfering RNA (siRNA). The knockdown is achieved by the activity of the Ago proteins themselves which are involved in the RNA interference mechanism. By suppressing the expression of hAgos using these tools, the obvious consequence is a dying out of the silencing effect, which could lead to interference between observed functional phenotypes and the mechanism of inhibition. Thus, alternative methods acting by different regulatory mechanisms would be advantageous in order to exclude unspecific effects. The knockdown may also be achieved by using specific antisense oligonucleotides (asONs) which act via an RNase H-dependent mechanism, not thought to interfere with the natural processes in which Agos are involved. Functional observations in the use of hAgo-specific asONs indicate the relevance of this assumption and confirm that hAgo2 is involved in microRNA (miRNA) and in siRNAmediated silencing pathways while the other hAgos play a role only in miRNA-based gene regulation (Mescalchin et al. RNA 2010 16:2529-2536). Here, we describe the effects of hAgo-specific downregulation by asONs and compared them to those obtained by siRNA and shRNA. Moreover, we describe a novel strategy which allows functional studies of hAgos and it is based on the simultaneous downregulation of the endogenous proteins and overexpression of recombinant tagged and codon optimized hAgos. Alessandra Mescalchin, PhD Institute of Molecular Medicine, University of LГјbeck Ratzeburger Allee 160, 23538 LГјbeck, Germany Telephone: +49 (0)451 500 2742 or +49 (0)451 500 2736 Email: [email protected] 94 047 SENSE OLIGONUCLEOTIDE-MEDIATED NATRE TECHNOLOGY CONTROLS LEVELS OF CYTOKINE MESSENGER RNA Mikio Nishizawa1, Emi Yoshigai1, Masaki Kaibori2, Tadayoshi Okumura2,3, A-Hon Kwon2 1 Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, Kusatsu, 2Department of Surgery, Kansai Medical University, Hirakata, 3Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, Japan Natural antisense transcripts (asRNAs) are transcribed from many genes, and we recently found asRNA transcribed from rat and mouse genes encoding inducible nitric oxide synthase (iNOS), which is involved in inflammation [1,2]. The iNOS asRNA stabilizes iNOS mRNA by interactions with the mRNA and RNA-binding proteins, and SENSE oligodeoxyribonucleotides (ODNs), which have the same sequences as that of mRNA, decrease levels of iNOS mRNA [1]. In the presence of asRNA, the sense ODN functions as an effector to regulate mRNA stability. In contrast, a sense ODN of the antisense technology is a negative control that does not affect mRNA levels. We applied this novel method, i.e. Natural Antisense Transcript-targeted REgulation (NATRE; pronounces /nature/) technology, to cytokine genes. 1. Sequences of the iNOS sense ODNs were optimized by transfection of hepatocytes. Each effective sense ODN included single-stranded portion(s) of stem-loops of the mRNA.When the sense ODNs were compared with siRNA, the effects on iNOS mRNA levels were comparative. 2. Tumor necrosis factor (TNF) alpha asRNA was induced by interleukin 1beta in hepatocytes. Sense ODNs to TNF-alpha mRNA increased levels of TNF-alpha mRNA. 3. Rat asRNAs were transcribed from several other cytokine genes that are involved in inflammation. When the NATRE technology was applied to them, some sense ODNs decreased and the others increased the mRNA levels. It is possible that annealing of a sense ODN changes conformation of mRNA and accessibility of RNA-binding proteins. 4. Human asRNAs from cytokine genes were detected in various tissues. Together, it implies that human asRNA may be a target of the sense oligonucleotide-mediated NATRE technology to regulate mRNA stability. References: 1) Matsui K, et al. (2008) Hepatology. 47, 686–697. 2) Yoshida H. et al. (2008) Shock. 30, 734–739. Mikio Nishizawa, MD, PhD 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan Telephone: 81-77-561-2876 Email: [email protected] 95 048 SILENCING OF microRNA FAMILIES BY SEED-TARGETING TINY LNAs Susanna Obad1, Andreas Petri1, Oliver Broom1, Markus Heidenblad1, Jan Stenvang1, Ellen Marie Straarup1, Troels Koch1, Henrik FrydenlundВ Hansen1 and Sakari Kauppinen1,2 1 Santaris Pharma, Kogle AllГ© 6, DK-2970 HГёrsholm, Denmark 2 Copenhagen Institute of Technology, Aalborg University, Lautrupvang 15, DK-2750 Ballerup, Denmark MicroRNAs act as important post-transcriptional regulators of gene expression by mediating mRNA degradation or translational repression. There is now ample evidence that perturbations in the levels of individual or entire families of miRNAs are prevalent in and strongly associated with the development of a variety of human diseases. Apart from cancer, miRNAs have also been implicated in viral infections, cardiovascular disease and neurological disorders. Hence, disease-associated miRNAs could represent a potential new class of targets for oligonucleotide-based therapeutics, which may yield patient benefits unobtainable by other therapeutic approaches. LNAs comprise a class of bicyclic high-affinity RNA analogues, in which the ribose ring in the sugar-phosphate backbone is locked in an RNA-like, C3’-endo conformation by the introduction of a 2’-O,4’-C methylene bridge. This results in high binding affinity of singlestranded LNA-modified oligonucleotides to their complementary miRNA targets. Moreover, LNAs combined with a phosphorothioate backbone show high biostability and enhanced pharmacokinetic properties in vivo. Here, we describe an approach that enables miRNA knock-down using 8 nucleotide fully LNA-modified phosphorothioate oligonucleotides, termed tiny LNAs, complementary to the miRNA seed region. Transfection of tiny LNAs into cells resulted in simultaneous inhibition of miRNAs within families sharing the same seed with concomitant up-regulation of direct targets. In addition, systemically delivered, unconjugated tiny LNAs showed uptake in many normal tissues, and pharmacological effect in different mouse models, coinciding with long-term miRNA silencing. Considered together, these data support the utility of tiny LNAs in elucidating the functions of miRNA families with important implications for the development of therapeutic strategies aiming at pharmacological inhibition of disease-associated miRNAs. Susanna Obad, PhD Santaris Pharma A/S Kogle AllГ© 6 DK 2970 HГёrsholm Danmark Telephone: +45 45 179893 Email: [email protected] 96 049 MICRORNA-132 REGULATES THE MAINTENANCE OF NEUROMUSCULAR JUNCTIONS Keren Ofek1, Geula Hanin1, Adi Gilboa-Geffen1, Amit Berson1, David S. Greenberg,1 Steve Wilton2 and Hermona Soreq1 1 The Hebrew University of Jerusalem/Israel 91904, 2Molecular Genetic Therapy Group, Australian Neuromuscular Research Institute, Nedlands 6009/Western Australia Neuromuscular junctions (NMJ), the synapses linking axon terminals of motor neurons with the highly-excitable region of muscle fiber plasma membrane are responsible for initiating, in response to acetylcholine action potentials across the muscle’s surface, ultimately causing the muscle to contract. Failed maintenance of NMJ functioning occurs in neuromuscular diseases (e.g. Duchenne’s muscular dystrophy) or under exposure to auto-immune antibodies (e.g. in myasthenia gravis) or to poisonous anti-cholinesterases) [1], but the underlying molecular mechanism(s) are incompletely understood. Given that the Acetylcholine hydrolyzing enzyme acetylcholinesterase (AChE) is subject to blockade by microRNA-132 (miR-132) [2], we explored the possibility that dynamic changes in miR-132 levels modulate NMJ function and that manipulating this miR levels in vivo would facilitate NMJ functioning under insults. Here, we report that within 24 hours, intravenous (IV) injection of 3.3 mg/kg AM132, a 22-mer 2’-O-methyl modified antisense oligonucleotide on a phosphorothioate backbone elevates the RNA levels of the miR-132 targets AChE, MeCP2 and p250GAP, increases AChE activity in the diaphragm and gastrocnemius muscles and facilitates histochemical staining of AChE in diaphragm NMJ (10.9В±4.2 compared to 6.3В±2.7 stained NMJs/ 100 um field in control mice, t test, P=0.0069, N=5 mice/group and 18 fields/specimen). In the rotarod test, AM132-treated mice predictably showed a certain level of muscle fatigue (t test, P=0.025); however, when exposed to increasingly lethal doses of the anti-AChE paraoxon, AM132-protected mice exhibited less poisoning symptoms (Mann-Whitney U-test, P=0.028, N=4/group), better recovery and survival (ANOVA P=6.62E-12, N=4-7/group) or extended survival time compared to controls (ANOVA P=6.8E-9, N=5/group). Our findings highlight the role of miR-132 as a master controller of NMJ functioning and open new venues for manipulating NMJ function under disease and/or environmental and war insults. References [1] Soreq & Seidman. Acetylcholinesterase-new roles for an old actor. Nat Rev Neurosci. 2(4):294-302. (2001). [2] Shaked et al. MicroRNA-132 potentiates cholinergic anti-inflammatory signaling by targeting acetylcholinesterase. Immunity. 2009 ;31(6):965-73 Keren Ofek, MSc The Hebrew University Soreq Lab, Safra Campus Jerusalem 91904, Israel Telephone: 972-2-6585450 Email: [email protected] 97 050 PLASMA PHARMACOKINETICS OF MIRAVIRSEN IN HUMANS Persson R, Bagger Y, BГёrgesen H, Hodges, MR, King BD, Levin AA Santaris Pharma A/S, HГёrsholm, DK, San Diego CA USA MiR-122, an abundant hepatic microRNA is an obligate host factor for HCV propagation and represents a unique target for Hepatitis C Virus (HCV) therapy. The pharmacokinetics of a LNA-modified antisense oligonucleotide complementary to miR-122, (miravirsen sodium), was studied after single or multiple doses (i.v.) or (s.c.) to healthy subjects. Subjects were divided into 6 groups (receiving 5 doses between 1.0 and 5.0 mg/kg/week). Each group consisted of 5 individuals (4 active and 1 placebo). Plasma concentration of miravirsen was quantitated using a validated hybridization-dependent ELISA. Plasma pharmacokinetics (e.g. AUC values) did not substantially change moving from a single dose to multiple doses and exposures after i.v. and s.c. administrations were similar. The plasma PK for miravirsen were multiphasic with distribution phase(s) and a terminal phase. The distribution phase was completed within 24 hours (t½≈1-2 hours) and the plasma compartment was almost freed of miravirsen (to > 99%). The terminal phase lasted for weeks (tВЅ=38 days). Comparison of AUCs following s.c. and i.v. doses indicated bioavailability was 100%. The terminal PK parameters (tВЅ, Vz and Cl) were similar independent of route. AUC0-96 h and Cmax after s.c. administration increased dose –proportionally for AUC0-96 h, and less than dose-proportionally for Cmax on both Days 1 and 29. The mean ratio of AUCday29/AUCday1 was 1.0, indicating that there was no accumulation after 5 doses. Terminal PK parameters were estimated from extended sampling (up to 140 days) after the last dose on Day 29. Total body clearance, Cl/F, was low and independent of dose, with a mean value of 36.0 (В± 8.5) mL/h/kg (all s.c. doses combined). The apparent volume of distribution, Vz/F, was large and independent of dose, with a mean value of 45.4 (В± 14) L/kg, indicating a distribution into a deeper compartment e.g. liver and kidney. Mean terminal plasma half-life was 38.3 (В± 16) days (all s.c. dose levels combined). While there was no significant accumulation of miravirsen in plasma, equilibrium or trough plasma concentrations increased linearly with dose and with number of doses, reflecting accumulation in deeper compartments. These data will be used to design dosing schedules for future clinical trials. Robert Persson, PhD Santaris Pharma A/S Kogle AllГ© 6, 2970-HГёrsholm, Denmark Telephone: +45 4517 9947 Email: [email protected] 98 051 Towards evaluation of point-mutations and drug interference with miRNA regulated expression of BRCA1 Christopher Polonyi, Sofi K.C. Elmroth Biochemistry and Structural Biology, PO Box 124 SE-22100 Lund, Sweden It is a challenge to try to understand how gene expression is regulated in various eukaryotic organisms, including the human body. The cells in eukaryotic organisms contain small double-stranded hairpin-like RNA structures coded for by microRNA (miRNA) genes in the cell nucleus. By use of the RNA induced silencing complex (RISC), the miRNAs interfere with translation by targeting mRNA. This kind of gene silencing can also arise if a small double-stranded exogenous RNA, so called small interfering RNA (siRNA), is introduced into a eukaryotic cell1. The aim in the present project is to further increase the knowledge concerning the sensitivity of the siRNA and miRNA machinery towards interference caused by point-mutations and drugs. Strategies that are used involve in-house design of siRNAs and miRNAs and construction of drug-miRNA adducts for evaluation of drug interference. Potential drug candidates are used to investigate the expression of the BRCA1 gene, which codes for proteins that are associated with breast cancer development2. In the initial studies, BRCA1 mRNA targeting has been evaluated in a luciferasebased assay (see Figure 1) and on protein level. References: 1. Carthew RW, Sontheimer EJ. Origins and mechanisms of miRNAs and siRNAs. Cell. 2009 Feb 20; 136(4):642-55. 2. Shen J, Ambrosone CB, DiCioccio RA, Odunsi K, Lele SB, Zhao H. A functional polymorphism in the miR-146a gene and age of familial breast/ ovarian cancer diagnosis. Carcinogenesis. 2008 Oct;29(10):1963-6. Christopher Polonyi, PhD student Department of Biochemistry and Structural Biology Center for Molecular Protein Science, Lund University PO Box 124, SE-22100 Lund, Sweden Telephone: +46 46 2228108 Email: [email protected] 99 052 lEPIGENETIC MYOSTATIN BLOCKADE Thomas Roberts, 1Graham McClorey, 1Samir EL-Andaloussi, 2Kevin Morris and 1 Matthew Wood 1 Department of Physiology, Anatomy and Genetics, University of Oxford, OX1 3QX, UK 2 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA 1 Myostatin is a secreted growth factor that acts to negatively regulate muscle mass. Myostatin null mutations result in increased musculature in cattle, mice and humans and thus myostatin blockade is being considered as a putative therapy for muscle wasting disorders such as Duchenne muscular dystrophy (DMD). Various myostatin blockade strategies have been shown to induce functional improvement in the mdx mouse model of DMD. Similarly, the combination of myostatin blockade with dystrophin splice correction has results in greater functional improvement than either strategy alone. Transcriptional Gene Silencing (TGS) can be induced by small RNA molecules complementary to target gene promoter sequences. TGS effectors target chromatin modifying activities to low copy number transpromoter RNAs which induce silent state chromatin structure and, in some cases, promoter DNA methylation. Consequently, TGS may be permanent and heritable. In order to investigate the possibility of silencing myostatin by TGS we designed small interfering RNAs (siRNAs) targeting the myostatin promoter. These were transfected in differentiated C2C12 mouse myotubes. Two siRNAs resulted in significant myostatin silencing. Knock-down was observed in multiple transfection experiments, and with both conventional and nuclear-targeting transfection reagents. The results were replicated in the disease relevant H2K-mdx cell line indicating that the effect is not unique to C2C12 myoblasts. Myostatin silencing was also found to be independent of the interferon response. Treatment with the histone deacetylase inhibitor Trichostatin A alleviated the silencing effect of promoter-targeting siRNA suggesting that epigenetic remodelling at the myostatin promoter is responsible for the observed silencing. Similarly, enrichment of the silent state histone mark H3K9me2 was observed at the myostatin promoter following treatment with promoter-targeting siRNA. We also report progress towards achieving myostatin transcriptional silencing in vivo. Thomas Roberts, MBiochem Department of Physiology, Anatomy and Genetics University of Oxford Le Gros Clark Building South Parks Road Oxford OX1 3QX, United Kingdom Telephone: +44 (0)1865 272167 Email: [email protected] 100 053 A New tool for Site-Specific Chemical Modification of RNA Shigeki Sasaki, Atsushi Shibata, Natsuki Inaba, Kazumitsu Onizuka, Yosuke Taniguchi Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan The chemical modification of RNA has a strong impact on RNA research; the fluorescent labelling of RNA is useful to monitor RNA production, processing, relocation in the cell, interaction with other intracellular components and degradation, etc. Modified nucleotides in RNA play an important role in cellular functions and the progression of diseases. Thus, chemical tools that can modify nucleotides in RNA with high specificity and efficiency in situ may specifically alter the function of the target RNA, and thereby will be valuable for an RNA study as well as for manipulation of the gene expression at the RNA level. We recently developed the efficient functionality-transfer reaction (FTR) for site-specific modification of RNA by using the ODN probe involving the 6-thioguanosine functionalized with the transfer group.RNA modification occurs at the 4-amino group of the cytosine at pH 7 or at the 2-amino group of the guanine base under alkaline conditions or in the presence of NiCl2. The FTR was applied to pin-point labeling of RNA with a variety of functional groups such as biotin, fluorescent dyes and PEG, etc. through “click chemistry” (Fig. 1).1d Biotinmodification of RNA may have broad utility, because biotin can bring avidin-fused proteins to the specific site of RNA. In this paper, we discuss whether the functionality-transfer ODN can modify large RNA molecules such as mRNA, and what effects may be induced on the biological functions of the target RNA such as for polymerization and translation. Fig. 1 The functionality transfer reaction (FTR) for modification of the 2-amino group of guanosine of the internal RNA Shigeki Sasaki, PhD Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Telephone and Fax: +80-92-642-6615 Email: [email protected] ____________________________ 1 (a) Bioconjugate Chem. 2009, 20, 799-803, (b) Nucleic Acids Res. 2010, 38, 1760-1766, (c) Bioconjugate Chem. 2010, 21, 1508-1512, (d) Chem. Comm., 2011, 47, 5004-5006 101 054 mRNA Cap 2′O-methylation prevents immune recognition of self-RNA by RIG-I Christine Schuberth1, Janos Ludwig1, Anna Maria Herzner1, Thomas Zillinger1, Romy Kerber2, Marion Goldeck1, Christoph Coch1, Veit Hornung1, Christian Drosten3, Winfried Barchet1, Beate M. KГјmmerer3, Gunther Hartmann1 and Martin Schlee1 1Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Germany 2Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany 3Institute of Virology, University of Bonn Medical Centre, Bonn, Germany The innate immune response to viruses is based on pattern recognition receptors (PRR) that detect a characteristic structure or an unusual location of viral nucleic acids within the cell. As viral RNA is located in the same compartment as host RNA, PRRs in the cytosol have to discriminate between viral and self RNA by structure or modification. This challenging task is taken on by the cytosolic DExD/H-box family helicases RIG-I and MDA-5. The ubiquitously expressed receptor RIG-I detects 5’triphosphorylated RNA (pppRNA) occurring during viral RNA replication thereby controlling the immune response to most RNA viruses. Like viral RNA, eukaryotic mRNA harbours a triphosphate at its 5’end. So far it is presumed, that the N7-methylguanine (m7G) cap structure, as present on all eukaryotic mRNAs, shields the 5’-triphosphate of self mRNA from RIG-I-mediated immune recognition. Here we synthesized RNA containing different cap structures and demonstrated that a m7G cap could only partially reduce RIG-I stimulation, while a G cap (without N7 methylation) did not impair RIG-I activation at all. By contrast, a single 2′O-methyl group at the 5’-penultimate residue (N1), a characteristic feature of vertebrate mRNA, was sufficient to abrogate RIG-I activation completely. Interaction assays of recombinant RIG-I with modified 3P-RNA ligands confirmed that mainly the 2’O-methylation at N1 mediated inhibition of binding of capped RNA to RIG-I. Studies on the yellow fever virus-encoded 2′O-methyltransferase indicated that viruses mimic cap 2′O-methylation at N1 to escape RIG-I recognition. Thus, not the m7G cap but the 2′O-methyl group at N1 is the critical determinant for RIG-I to distinguish self from foreign triphosphatecontaining RNA. Christine Schuberth, Institute for Clinical Chemistry and Clinical Pharmacology University Hospital Bonn Sigmund-Freud-Str. 25 53105 Bonn, Germany Telephone: +49-228-287-51157 Email: [email protected] 102 055 ANTISENSE APPROACHES TO STUDY REST/NRSF TRANSCRIPTIONAL REGULATION AS A THERAPEUTIC TARGET FOR NEURODEGENERATIVE DISORDERS Yalda Sedaghat, Curt Mazur, Brett P. Monia ISIS pharmaceuticals, Carlsbad, CA, USA, 92010 The repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/ NRSF) is a master regulator of neuronal gene expression. REST functions through binding the genomic loci that contain the repressor element-1 (RE1) binding motif and recruits a series of epigenetic and regulatory cofactors to its N- and C-terminal domains. Various studies have characterized the specific roles played by REST during neuronal lineage specification and maturation. Among the REST targets BDNF has emerged as a candidate of interest as a potential neuroprotective and functionally restorative treatment in psychiatric and neurological disorders, including Huntington’s disease (HD). In HD, BDNF levels are significantly attenuated as a result of impaired BDNF transport from the cortex to the striatum in the presence of mutant HTT. Wild-type HTT also regulates transcription of BDNF through REST, by sequestering REST in the cytoplasm, therefore restricting its access to the nucleus, which leads to the transcription of target genes such as BDNF. In this study, we were interested in down-regulation of REST as a potential therapeutic strategy to both slow down cell death and maintain the functional state of remaining neurons. This could be possible through the neuroprotective and functional effects of BDNF by activation of signaling in neuronal pro-survival pathways. In order to specifically target REST, we applied 2nd generation Antisense Oligonucleotides (ASOs) to reduce levels of REST mRNA in the liver and the CNS in both normal (wild type) and HD mice and characterized the effects on BDNF expression and gene expression more globally, as determined by microarray analysis. We have demonstrated specific reduction in REST levels in both liver and CNS following treatment by ASOs, which resulted in the induction of a number of neuronal genes including BDNF and Synapsin1 at the mRNA and protein levels. Gene array expression analysis was performed on BALB/c mouse liver and R6/2 brain, a mouse model of HD, following systemic and ICV administration of REST ASO, respectively. Samples were analyzed by hybridization to the MouseWG-6 v2 Expression BeadChip array (Illumina), to gain insight into putative pathways affected by REST suppression. Array data analysis was performed using GeneSpring, followed by Gene network prediction using Ingenuityв„ў Pathway Analysis (IPA). 416 genes were found to be up regulated and 119 genes down regulated following REST ASO treatment. Numerous novel genes were identified that were affected by REST suppression that are predicted to play a role in cancer, genetic disorders, neurological diseases, cell-to-cell signaling, and tissue development. Our findings suggest that REST may be an important target for neurodegenerative diseases like HD, that it is also involved in the regulation of a broad range of cellular pathways outside of the CNS, and that the antisense approach is a viable strategy for selectively modulating REST activity in both the CNS and in the periphery. Yalda Sedaghat, PhD/Brett P. Monia, PhD ISIS Pharmaceuticals Inc. 2855 Gazelle Court Carlsbad, CA 92010 USA Telephone: Brett Monia: 760-603-2350 Email: [email protected]; [email protected] 103 056 FUNCTIONAL ANALYSIS OF microRNAs AS REGULATORS OF CARGO TRAFFICKING Andrius Serva, Sanchari Roy, Bettina Knapp, Lars Kaderali, Holger Erfle, Ursula Kummer, Roland Eils and Vytaute Starkuviene BioQuant, University of Heidelberg, Heidelberg, Germany MicroRNAs (miRNAs) are small non-coding RNAs emerged as important translational gene expression regulators in eukaryotic cells. miRNAs are predicted to modulate the expression of nearly 60% of protein-coding mammalian genes. Thereby, miRNAs affect a multitude of biological processes such as developmental transition, cell proliferation and apoptosis. Alteration of miRNA expression patterns leads to tumorigenesis, diabetes, neurodevelopmental disorders and many other diseases. In the present study we demonstrate an unbiased integrative approach to identify miRNAs and their functionally relevant target genes involved in the regulation of secretory membrane trafficking. By applying (i) molecular biology, (ii) fluorescence microscopy, (iii) statistical data analysis, (iv) bioinformatics and (v) miRNA and mRNA expression profiling methods we are enabled to identify miRNAs of interest. A high-throughput screening of oligonucleotides mimicking 470 human miRNAs led to the identification of 44 miRNAs with an effect on the model cargo protein ts-O45-G secretion rate. In parallel, a large-scale library screening of 875 miRNA inhibitors yielded 68 miRNAs, which inhibition also caused significant changes of cargo trafficking. Further examination of primary miRNA hits by combined microscopy-based and automated image analysis-based Golgi complex integrity assay revealed that perturbation of 13 miRNAs resulted in quantitative changes of Golgi complex morphology. Moreover, we uncovered an active role of oncogenic miR-17~92 cluster in the regulation of cargo trafficking and identified TBC1D2 and LDLR genes as novel targets of miR-17. In perspective, the results of detailed analysis of identified miRNAs and their target genes will allow us to model the posttranscriptional miRNA-mediated regulatory networks. A comprehensive understanding of how these networks coordinate the activity of secretory membrane trafficking as a global adaptive response system is needed to study miRNAs as promising therapeutic agents and targets under particular pathological conditions. Andrius Serva, MSc BioQuant, University of Heidelberg Im Neuenheimer Feld 267 69120 Heidelberg, Germany Telephone: +49-6221-5451258 Email: [email protected] 104 057 SILENCING THE CDK4 AND CDK6 GENES THROUGH RNA INTERFERENCE MaЕ‚gorzata Sierant, Danuta Piotrzkowska and Barbara Nawrot Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz, Poland Alzheimer’s disease (AD), a disorder which affects millions people worldwide, is characterized by progressive memory loss and a spectrum of behavioral abnormalities leading to dementia. An effective treatment of AD is still not available. Preventing or slowing down progression of neurodegeneration will considerably reduce the number of severely affected patients and improve the quality of life of the aging population. The biological causes of neuronal death in AD have been the objective of intense studies and several concepts were proposed [1]. Several investigators observed close association between the neuronal cell death and reactivation of the cell cycle in post-mitotic neurons. Abnormal cell-cycle-related proteins expression, as cyclin dependent kinases, cyclins and cdk inhibitors was observed in pathologically affected parts of AD brains [2]. Usually, in postmitotic and terminally differentiated neurons, the cell-cycle activity is arrested by the enrichment of cdk inhibitors, neurons are “locked” in the G0 phase. At the beginning of neuropathogenesis, neurons leave the G0 phase and progress until the S phase and die at the G1-M transition. The aim of our studies was to down-regulate the expression of two cyclin dependent kinases CDK4 and CDK6 in neural cells through RNA interference-based gene silencing. Such approach could be the proposal for the new neuroprotective gene therapy in AD and the other neurodegenerative disorders. Thus, sixteen siRNAs with sequences complementary to human, mouse and rat transcripts of CDK4/6 genes were designed and synthesized in house. Their silencing activity was screened in human (HeLa, SH-SY5Y), mouse (Neuro2a, RAW 264.7) and rat (PC12) cell lines and assessed by quantitative RT-PCR (target mRNA level) and by Western blot (target proteins level) analyses. Some of the silencers dowregulated the expression of both target genes up to 90%. Activity of siRNA was also valuated by flow cytometry, where we counted the number of cells in G1/G0, S and G2/M phases. Analysis has shown that for cells transfected with active siRNA molecules, which result in down-regulation of CDK4/6 genes expression the number of cells in G1/G0 phaseis is increased, while the number of cells in S phase is lowered. This effect dependented on concentration of siRNA. Selected sequences of the most active siRNAs were used for construction of shRNA and micro RNA based shRNA inserts cloned into two type of plasmids: with U6 promoter (Pol III) (pSilencer-2.0 U6, Ambion) and CMV promoter (Pol II) (pEGFP-C1, Clontech and pcDNA 6.2-GW/miR, Invitrogen). For selected cells transfected with these constructs the lowering of CDK4 and CDK6 genes expression was observed in the range 10-70% (mRNA) and 20-70% (protein). [1] B. Nawrot, et al. RSC Drug Discovery 3, 230, 2009; [2] Y. Yang, et. at. J. Neurosci. 23, 2557, 2003. Malgorzata Sierant, PhD CMMS PAS, Lodz 90-363, Sienkiewicza 112, Poland Telephone: (+48 42) 680-32-72 Email: [email protected] 105 058 IMPROVED STABILITY AND GENE SILENCING ACTIVITY OF siRNA ANALOGS CONTAINING PHOSPHORODITHIOATE LINKAGES Malgorzata Sierant 1, Magdalena Janicka 1, Xianbin Yang 2, Na Li 2 and Barbara Nawrot 1 1 Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz, Poland; 2AM Biotechnologies LLC, Houston, Texas, USA Short interfering RNAs (siRNAs) are widely used to identify gene functions and hold great potential in providing a new class of therapeutics. Although unmodified siRNAs are used with success for a gene silencing, chemical modifications of one or both strands are desired for pharmaceutical applications to enhance a nuclease stability, potency and to improve pharmacokinetic properties of duplexes. Modification of the internucleotide bond involving substitution of a single non-bridging phosphate oxygen atom with e.g. an sulfur atom, leads to the achiral modified phosphorus center and to mixture of unresolvable diastereomeric oligomers possibly having variable biochemical, biophysical, and biological properties. The substitution of both non-bridging phosphate oxygen atoms with sulfurs gives rise to a phosphorodithioate (PS2) internucleotide linkage which, like natural RNA, is achiral at phosphorus. The PS2 linkage is isosteric and isopolar with the normal phosphodiester and should have other biochemical and biophysical properties similar to natural RNA. Here we describe the application of RNA molecules containing the PS2 linkages as components of the novel type of the PS2-siRNA duplexes. In our studies, for tests of new PS2-siRNA molecules, we chose previously identified active siRNA sequences for the ОІ-secretase (BACE1) and enhanced green fluorescent protein (EGFP) transcripts [1,2]. CD spectra collected for PS2siRNA duplexes are similar to the spectra of the unmodified duplex, consistent with the typical A-type structure of double-stranded RNA. The UV-monitored thermal dissociation studies have shown that the duplexes with up to five PS2 linkages exhibit transition curves essentially identical in shape to the unmodified siRNA. We did not observe remarkable fluctuations in Tm values in comparison to the unmodified reference duplex. The gene silencing experiments performed with several siRNA duplexes containing the PS2 linkages either in the sense or antisense strand indicate that the extent of silencing effect depends on not only the number of the PS2 modifications but expecially on the strand and position within an siRNA duplex. In some cases we observed enhanced gene knockdown activity in comparison with unmodified duplex. Given the straightforward synthesis of PS2 linkages, as well as their known nuclease resistance and lack of chirality, PS2 modifications are promising candidates for siRNA in research and therapeutic applications. [1] Sipa K. et al. RNA 13, 1301, 2007 [2] Sierant M. et al. Int. J. Alzheimers Dis. 2009, Jul 14, 2009 Malgorzata Sierant, PhD CMMS PAS, Lodz 90-363, Sienkiewicza 112, Poland Telephone: (+48 42) 680 32 72 Email: [email protected] 106 059 DICER-SUBSTRATE siRNA EXHIBIT IMPROVED GUIDE STRAND SELECTION AND STRONGER RISC LOADING COMPLEX FORMATION COMPARED TO CANONICAL siRNA Nicholas M. Snead, Kumi Sakurai, John J. Rossi Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute at the City of Hope, Duarte, CA USA As small interfering RNA (siRNA) continues making headway toward therapeutic applications, researchers are looking to optimize the efficacy of both a suitable delivery vehicle and the therapeutic RNA itself. We are interested in maximizing the efficacy of siRNAs at the RNA level through relatively minor chemical and structural modifications to canonical siRNA. The rationale behind modifying the canonical siRNA structure is, among other criteria, to improve loading of the guide strand into the RNAi machinery, reduce off-target effects, and possibly reduce competition with endogenous miRNAs, ultimately leading to better efficacy. Here, we present a parallel analysis of four siRNA variants (the canonical 21mer, a commonly used 19merTT, a Dicer substrate interfering RNA (dsiRNA), and a short trigger 16merTT)) in gene silencing, RNAi protein binding, and miRNA competition disruption assays. The dsiRNA consistently demonstrated better gene silencing by the guide strand and worse gene silencing of the passenger strand compared to its analogous 21mer or 19merTT. The dsiRNA also showed more robust formation of a high molecular weight complex known to contain Dicer and TRBP (two primary members of the RISC-loading complex) compared to its analogous 21mer and 19merTT. Small RNA deep sequencing results reveal that (1) cells transfected with dsiRNA have a global miRNA profile more similar to untransfected cells compared to 21mer- or 19merTT transfected cells, and (2) at low transfection concentrations, the guide strand of the dsiRNA is more abundant than the 21mer or 19merTT. The 16merTT did not perform comparably in any of the assays. Also, we are interested in re-exploring the 5’ triphosphate group on siRNAs that would trigger RIG-I-mediated interferon production as a bifunctional way to ablate diseased cells. Interestingly, preliminary results using 5’ triphosphate 19+2 siRNAs generated by T7 in vitro transcription did not silence reporter gene expression; we are exploring this further. While delivery of therapeutic small RNAs is arguably the largest hurdle to overcome, our studies show that relatively small changes to canonical siRNAs can also improve potency. Nicholas M. Snead, MS Beckman Research Institute at the City of Hope 1500 E. Duarte Road, Fox N 2002H Duarte, CA, 91010 USA Telephone: (626) 256-4673, ext. 64457 Email: [email protected] 107 060 FINDING CANCER THERAPEUTIC sgRNAs BASED ON TRUE GENE SILENCING M. Takahashi1, S. Matsuda1, Y. Watabe1, A. Goto1, D. Kamiya1, T. Sano1, N. Watanabe2, M. Narita2, M. Takahashi2, M. Tamura3, M. Nashimoto1 1 Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan, 2 Niigata University, Niigata, Japan, 3Hokkaido University, Sapporo, Japan tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is one of the RNA-mediated gene expression control technologies that have therapeutic potential.1,2 This technology is based on the properties of tRNase ZL that it can cleave any target RNA at any desired site under the direction of an appropriate artificial small guide RNA (sgRNA) and that cytosolic tRNase ZL can modulate gene expression by cleaving mRNA under the direction of cellular 5′-half-tRNA or microRNA as sgRNA (Figures 1 and 2).3,4 We have shown that heptamer-type sgRNA with simple chemical modifications can be taken up by human cells without any transfection reagents and can exist in the cells stably. We have also demonstrated that a “naked” heptamer-type sgRNA targeting the human Bcl-2 mRNA can induce apoptosis of leukemia cells without inducing the interferon response. In order to find candidate cancer therapeutic sgRNAs, we designed 160 heptamer-type sgRNAs targeting various human mRNAs, and examined them for inducing apoptosis in human leukemia and myeloma cell lines. We found that 40 of the 160 naked sgRNAs can efficiently induce apoptosis in at least one of the cancer cell lines. We also showed that a subset of the sgRNA library can significantly decrease levels of targeted cellular mRNAs. Furthermore, we demonstrated that 10 of the 40 effective sgRNAs can reduce tumor growth rates in mouse xenograft models. These results show that naked sgRNAs can induce apoptosis in leukemia and myeloma cells through targeting various mRNAs. 1. A. Nakashima, et al. (2007) Gene Therapy, 14, 78–85. 2. R. A. Elbarbary, et al. (2009) Biochem. Biophys. Res. Commun., 379, 924–927. 3. R. A. Elbarbary, et al. (2009) PLoS ONE, 4, e5908. 4. R. A. Elbarbary, et al. (2009) FEBS Lett., 583, 3241–3246. Masayuki Takahashi, Master Higashijima 265-1, Akihaku, Niigata, Niigata 956-8603, Japan Telephone: +81 250 25 5118 Email: [email protected] 108 061 KINETIC COMPARISON OF GENE SILENCING PROFILE OF siRNA AND SHRNA-EXPRESSING PLASMID DNA IN VIVO Yuki Takahashi, Yuriko Matsui, Makiya Nishikawa, Yoshinobu Takakura Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan RNA interference (RNAi) is a gene silencing event in which small interfering RNA (siRNA) degrades target mRNA in a sequence-specific manner. RNAi can be also induced by introducing plasmid DNA (pDNA) that expresses short-hairpin RNA (shRNA) into cells. Because of the specificity and potency of RNAi-mediated gene silencing, the research for the development of RNAi-based therapy has been an exciting field since the discovery of RNAi. For therapeutic application of RNAi, the time course of gene silencing is one of the important factors to be considered. However, little attention has been paid to the duration of RNAibased gene silencing in vivo. There has been no report of gene silencing 1 week or longer after the induction of in vivo RNAi. In the present study, we evaluated and compared the profiles of gene silencing induced by siRNA and shRNA-expressing pDNA in mice. A newly developed reporter pDNA (pROSA-gLuc) encoding Gaussia luciferase cDNA under the control of human ROSA26 promoter was used to obtain a prolonged expression of the reporter protein over three months. We administered this pDNA to mice together with siRNA or shRNA-expressing pDNA targeting Gaussia luciferase at varying doses by the hydrodynamic injection method. The serum level of Gaussia luciferase was greatly but transiently reduced by siRNA, whereas a sustained reduction was obtained with shRNA-expressing pDNA. A quantitative analysis of the gene silencing profiles based on a moment analysis confirmed the finding that shRNA-expressing pDNA suppresses the target gene expression longer than siRNA. These results provide important information about the time-courses of RNAi in vivo. Yuki Takahashi, PhD 46-29 Yoshidashimoadachi-cho Sakyo-ku, Kyoto 606-8501, Japan Telephone: +81-75-753-4616 Email: [email protected] 109 062 Effect of guanine bases modifications and L-DNA on stability and kinetic of formation of tetramolecular quadruplexes Phong Lan Thao Tran1*, Antonella Virgilio2, Veronica Esposito2, Giuseppe Citarella2, Aldo Galeone2, Rui Moriyama3, Atsushi Maruyama3, Bernard Rayner1 and Jean-Louis Mergny1 1В Institut EuropГ©en de Chimie et Biologie, INSERM U869, UniversitГ© de Bordeaux (France) 2В UniversitГ degli Studi di Napoli Federico II, Napoli, Italy 3В Institute for Materials Chemistry and Engineering, Kyushu University, Fukuoka, Japan Tetramolecular G-quadruplexes result from the association of four guanine-rich strands. These structures are formed by the interaction of four guanines organized in a cyclic Hoogsteen hydrogen bonding arrangement termed a G-quartet, and by the stacking of several G-quartets. G-quadruplexes can be very stable under physiological conditions and the evidence for quadruplex formation in vivo is compelling. Two quadruplex related molecules are currently in phase II clinical trials and a number of aptamers adopt a quadruplex fold. Modification of the backbone strand or the guanine bases of the oligonucleotide may improve stability or introduce new functionalities. In this regard, position 8 of the guanosine base is particularly suitable for introduction of modifications since as it is positioned in the groove of the quadruplex structure [1]. In this study, we investigated the effect of an 8-methyl-2’-deoxyguanosine residue (M) on the structure and stability of tetramolecular parallel G-quadruplexes [2]. We also studied the mirror image of natural DNA that may form structures of opposite chirality. One of the advantages of L-nucleic acids is their nuclease resistance, allowing one spiegelmer (from “Spiegel” meaning mirror in German) to enter clinical trials [3]. We demonstrate here that a short guanine-rich L-DNA strand may form a tetramolecular quadruplex with the same properties as a D-DNA strand of identical sequence, and L- and D- strands self-exclude when mixed together [4]. These structures have already found a number of interesting applications in biotechnology [5, 6] and nanotechnology [7, 8] and we are currently working on new developments involving G-quadruplexes. Tetramolecular G-quadruplex 8-methyl Guanine D-GuanineL-Guanine References: [1] [2] [3] [4] [5] [6] [7] [8] J. Gros, et al., Chembiochem 2008, 2926-2928. P.L.T. Tran, et al., Biochimie 2011, 93(3):399-408. S.G. Sayyed, et al., Diabetologia 2009, 52, 2445-54. P.L.T. Tran, et al. Chem. Commun. 2011, 47, 5437-5439. B. Wlotzka, et al., Proc Natl Acad Sci USA 2002, 99, 8898-902. Y. Kim, et al., Nucleic Acids Res 2007, 35, 7279-87. M.S. Kaucher, et al., J. Am. Chem. Soc.В 2006, 128, 38-39. C. Lin, et al. Nano Lett. 2009, 9, 433-436. Phong Lan Thao Tran 2, rue Robert Escarpit, 33607 Pessac France Telephone: +33 6 27 12 15 57 Email: [email protected] 110 063 Targeting tumor antigens to dendritic cells in vivo using receptor-specific aptamers conjugated to tumor antigen Johannes Urban, David Boczkowski, Kyle Kok Loong Phua, Bruce Sullenger and Smita Nair Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA The success of immunotherapy depends on the induction of effective primary immune responses to treat existing cancers and the generation of subsequent memory responses to prevent recurrence at a new site. Dendritic cells (DC), recognized as major antigen presenting cells, are uniquely equipped to initiate and regulate immune responses, making them a key target for developing new therapies. Immunizing with antigen-loaded DC is a powerful method of inducing CD4+ and CD8+ T cell responses and antibodies (Abs). Although ex vivo DC therapy has shown promise, it is a customized, complex, patient-specific cell therapy that reduces its universal applicability for cancer. Thus there is a critical need to develop strategies that circumvent the need for ex vivo DC loading. Directly targeting antigens to DC in vivo will facilitate the development of a more effective “off-the-shelf” tumor vaccine, which will be accessible to large numbers of patients. DC express a number of specialized endocytic receptors of the C-type lectin family enabling capture of antigen by receptor-mediated endocytosis at very low concentrations. Studies exploiting the endocytic properties of DC receptors demonstrated that antigens conjugated to receptor targeting moieties, such as the natural receptor-ligands or receptor-specific antibodies, increased antigen capture and presentation 100-1000-fold as compared to soluble antigen. Moreover, receptor targeting facilitates cross-presentation to induce Ab and CD4+ T cell immunity as well as elicit MHC-class-I-restricted CD8+ or cytotoxic T lymphocytes (CTL), critical for cancer immunotherapy. To deliver antigens to DC in vivo, we developed artificial receptor ligands to the macrophage mannose receptor (MMR or CD206), an endocytic C-type lectin receptor expressed on DC and macrophages. Herein, a RNA aptamer library comprised of 40 randomized nucleotides and modified with 2ВґFluoro-modified pyrimidines for improved nuclease resistance was selected for affinity binding to MMR. A complex selection scheme with alternating rounds on both, recombinant human and murine MMR, as well as on cells engineered to express human MMR enabled us to identify cross-reactive aptamers that recognize MMR with high affinity. A truncated, minimal version of the identified lead-aptamer with retained receptor-binding properties gets rapidly internalized into human monocyte-derived DC in vitro. Currently we are testing aptamer-tumor antigen chimeras and aptamer-decorated nanoparticles to deliver model tumor antigens to DC in vitro and in vivo and investigate their capability to elicit an antigen-specific immune response. Johannes Urban, PhD Duke University Medical Center 106 Research Drive, Durham, NC 27701 USA Telephone: 919-684-6780 Email: [email protected] 111 064 Designed Ankyrin Repeat Proteins for Targeted Delivery of siRNA Oligonucleotides Johannes Winkler1, Claudia BГ¶hme1, Nikolas Stefan2, Andreas PlГјckthun2, Uwe Zangemeister-Wittke2, 3 1 University of Vienna, Department of Medicinal Chemistry, Vienna, Austria 2 University of ZГјrich, Department of Biochemistry, ZГјrich, Switzerland 3 University of Bern, Institute of Pharmacology, Bern, Switzerland Designed Ankyrin Repeat Proteins (DARPins) are a novel class binding proteins with promising biochemical properties with regard to tissue-specific siRNA delivery. Ankyrin repeat proteins form an extended interaction interface which is able to bind to structural epitodes on the target protein surface. Their small size, high stability and the absence of post-translation modifications facilitate protein engineering and results in high-yield recombinant production in E. coli. By introducing a C-terminal cystein, a unique binding site for conjugations can be created, enabling the introduction of modifications for attachment or complexation of siRNA oligonucleotides. DARPins targeted at the extracellular domain of the cancer protein EpCAM were selected from ankyrin repeat protein libraries using ribosome display. We generated fusion proteins of an EpCAM-binding DARPin and highly basic peptides which were expressed in E. coli in high yields and after purification efficiently complexed the siRNA cargo. DARPin fusion protein mediated delivery resulted in specifically enhanced cellular uptake of siRNA in antigen-positive cells. A bcl-2 targeted siRNA silenced the respective gene expression in EpCAM-positive MCF-7 breast cancer cells and resulted in enhanced susceptibility to doxorubicin treatment. Dimerization of the DARPin increased binding affinity, but had only minor effects on delivery efficiency. Initial experiments indicated that the loading capacity for siRNA is a more important characteristic for efficient gene silencing than the antigen affinity of the targeting moiety. The obtained results demonstrate the usefulness of DARPins for targeted siRNA delivery and give insight into important attributes of siRNA delivery devices. Johannes Winkler, PhD University of Vienna, Department of Medicinal Chemistry AlthanstraГџe 14, 1090 Vienna, Austria Telephone: +43 1 4277 55058 Email: [email protected] 112 065 POTENCY COMPARISON OF SERIES OF BNA-BASED ANTISENSES IN DYSLIPIDEMIC ANIMALS Tsuyoshi Yamamoto1,5, Mariko Harada-Shiba1, Moeka Nakatani1,5, Hidenori Yasuhara1,5, Shunsuke Wada1,3, Keisuke Narukawa5, Kiyomi Sasaki3, Hidetaka Torigoe3, Tetsuji Yamaoka2, Takeshi Imanishi4 and Satoshi Obika5 Department of Molecular Innovation in Lipidology1 and Department of Biomedical Engineering2, National Cerebral and Cardiovascular Center Research Institute, Faculty of Science, Tokyo University of Science3, BNA Inc.4 and Graduate School of Pharmaceutical Sciences, Osaka University5, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan Development of high-performance nucleic acids (HiPerNA) and innovative design of antisense oligonucleotide (AON) have still been required to more safely and effectively treat patients by using antisense drugs [1]. We have presented several types of HiPerNA such as bridged nucleic acids (BNAs) that provides AON with better abilities to stably circulate through the body and selectively capture the exact target [2]. Recently, short phosphorothioated LNA gapmers (≤ 16-mer) have reported to show higher potency than conventional 20-mer AON both in vitro and in vivo [3]. We here evaluated a potency of novel BNAs incorporated into various lengths of gapmers. PCSK9 plays a key role in maintaining cholesterol homeostasis. PCSK9 is known as a potential therapeutic target for the treatment of hyperchcolesterolemia. To develop PCSK9 inhibitor with enough potency and safety to withstand a clinical application, we screened efficacious target sites on PCSK9 mRNA and optimized strand lengths and chemistries in vivo as well as in vitro. Collectively, our results would let us suggest better concepts of design of HiPerNA and AON for clinical application of antisense drugs. Reference: 1. Yamamoto, T, Nakatani, M, Narukawa, K, and Obika, S (2011). Antisense drug discovery and development. Future Med Chem 3: 339-365. 2. Obika, S, Rahman, SMA, Fujisaka, A, Kawada, Y, Baba, T, and Imanishi, T (2010). Bridged Nucleic Acids: Development, Synthesis and Properties. Heterocycles 81: 1347-1392. 3. Straarup, EM, et al. (2010). Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic Acids Res 38: 7100-7111. Tsuyoshi Yamamoto, PhD student 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan Telephone: +81-6-6879-8202 Email: [email protected] 113 066 oligonucleotide coNjugates as inhibitors of HIV-1 integrase and reverse transcriptase T. Zatsepin,1,2 J. Agapkina,1 S. Korolev,1 M. Gottikh1 1 Department of Chemistry and Belozersky Institute of Physical and Chemical Biology, Lomonosov Moscow State University, Moscow, Russia 2 Central Research Institute of Epidemiology, Moscow, Russia Reverse transcription of human immunodeficiency virus type 1 RNA and integration of resultant DNA into genome of the infected cell are two key steps of the viral replication cycle. Therefore viral enzymes reverse transcriptase and integrase, which realizes the reverse transcription and integration, respectively, represent attractive and validated targets for the development of new antiviral drugs. In this report at first we studied the anti-integrase activity in vitro of a series of conjugates of single stranded oligonucleotides with hydrophobic molecules, and the structure-activity relationships were also analyzed. Both oligonucleotide and hydrophobic parts of the conjugates influenced the inhibitory potency. Conjugates of 11mer phosphorothioate oligonucleotides with 6-carboxy-4,7,2’,4’,5’,7’-hexachlorofluorescein (HEX) were found to be the most efficient inhibitors (IC50 20 nM), and might be considered as lead compounds for further development of integrase inhibitors. Moreover, several conjugates were tested as potential inhibitors of RNA-dependent DNA polymerization, DNA-dependent DNA polymerization and RNase H activities of HIV-1 reverse transcriptase. Conjugates of 2’-OMe oligonucleotide with 2’,4’,5’,7’-tetrabromofluorescein (eosin) were found to be the most efficient inhibitor of RHase H activity of the reverse transcriptase (IC50В 250 nM). The work was supported by RFBR (grants 09-04-93108-CNRSL and 09-04-93112-CNRSL) and Russia President’s grant MK- 4821.2011.4. Timofei S. Zatsepin, PhD Department of Chemistry, Lomonosov Moscow State University Leninskie gory 1-3, 119991 Moscow, Russia Telephone: +7-495-939-3148 Email: [email protected] 114 067 Influence of RNA Secondary Structure on the Activation of Toll-like Receptors 7 and 8 Thomas Zillinger1*, Vera Wimmenauer1*, Eicke Latz2, Gunther Hartmann1, Winfried Barchet1 1 Institute of Clinical Chemistry and Clinical Pharmacology and 2Institute of Innate Immuniy, University of Bonn, Germany * These authors contributed equally. Immunorecognition of viruses hinges on the detection of viral nucleic acids by germline encoded receptors at specific cellular locations. Human Toll like receptors (TLR) 7 and 8 are able to detect single stranded RNA in the endosome of distinct immune cells.While most stimulatory RNAs activate both TLR7 and TLR8, short Oligoribonucleotides (ORN) and small molecule agonists can show preferential engagement of either TLR7 or TLR8. A structural basis for this distinction is unknown. Here we identify RNA stem structures in the secondary structures commonly assumed by single stranded RNA, as a potent and highly selective activators of TLR7. Such stem structures induced type I IFN in plasmacytoid dendritic cells (PDC), but did not activate TLR8 in monocytes. TLR7 selectivity was maintained even when TLR7 and TLR8 were expressed in the same cell type. We further provide evidence that recognition of viral and bacterial RNAs via TLR7 is mediated by RNA secondary structure, rather than the linear sequence motifs required for the activation of TLR8. We idenfied and developed TLR7 agonistic RNA stems that show activity without a requirement for accessory components such as cationic polymers. Thomas Zillinger Emmy Noether Group “Immunorecognition of Viral Nucleic Acids in the Cytosol” Institute of Clinical Chemistry and Clinical Pharmacology BMZ, University Hospital, University of Bonn Sigmund-Freud-Strasse 25 53127 Bonn, Germany Telephone: +49-228-287 -51146 Email: [email protected] 115 068 RIG-I LIGAND TRIPHOSPHATE RNA (3pRNA) POTENTIALLY INHIBITS YELLOW FEVER VIRUS REPLICATION IN TYPE 1 INTERFERON DEPENDENT MANNER Mohammad Intakhab Alam, 1Christine Schuberth, 1Thomas Zillinger, 1Christoph Coch 1Winfried Barchet, 1Jasper van den Boorn, 1Debby van den Boorn-Konijnenberg, 2 Beate Kuemmerer, 1Martin Schlee and 1Gunther Hartmann 1) Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Germany and 2) Institute for Virology, University Hospital Bonn, Germany 1 RIG-I has been shown to be involved in sensing every members of the flavivirus genus. Yellow Fever Virus (YFV) possesses mechanism to overcome innate immunity. Here we propose the potent antiviral activity of RIG-I ligand (3pRNA). We used a sub genomic Yellow Fever Renilla Replicon (YFRR) model. Our Luciferase data showed that, 3pRNA inhibits one log replication of YFRR in HepG2 cells at non-toxic concentration (800ng/ml). MTT assay ruled out the detrimental effect of 3pRNA on HepG2 cells and the inhibition of replication is 3pRNA specific in dose dependent manner. We served poly A RNA in HepG2 cells as negative control and we did not observe inhibition of replication that, supports the antiviral activity of 3pRNA. 3pRNA additionally induces type 1 IFN in addition to the YFRR itself in HepG2 cells that leads to the inhibition of replication. Type 1 IFN and IP-10 were highly expressed in 3pRNA treated cells. 3pRNA did not show inhibition of replication in IFN deficient Vero cells. However, addition of rec-IFNОІ showed inhibition of YFRR in Vero cells that suggests that, antiviral effect of 3pRNA is due to enhancement of IFN production and IFN signaling pathway mediated ISGs activation. WB and IF data showed that 3pRNA upregulated RIG-I and IRF3 and strongly inhibited NS4B protein of YFRR. Our real time-PCR data showed that 3pRNA potentially upregulated IFN dependent antiviral genes (OAS, RNAse L, MxA, IFIT1 but not IFIT2). Small form of PARP13 (ZAPs) is highly upregulated by 3pRNA which sparks the RIG-I signaling. 3pRNA did not inhibit replication in RIG-I-/- MEFs that clearly indicates that, inhibition of replication is RIG-I ligand dependent. RIG-I-/- MEFs showed enhanced replication compared to WT-MEFs. Over expression of Flag-tagged RIG-I showed non significant inhibitory effect on replication that also support our idea that 3pRNA is potent immune stimulator. Plaque assay showed that, 3pRNA inhibited about 65% virus particles of 17-D in infected HepG2 cells. Equivalent unit of rec-IFNОІ with 3pRNA induced IFN-ОІ showed almost similar negative effect on replication. Altogether, these findings raise the possibility that RIG-I ligand, a potent immune stimulator may be a promising antiviral therapeutic RNA molecule for YFV and other flavivirus. Mohammad Intakhab Alam, PhD Institute for Clinical Chemistry and Clinical Pharmacology Sigmund-Freud-Str. 25 D-53127 Bonn, Germany Telephone: +49-228-28751150 Email: [email protected] 116 069 Glucan Particles for Selective Delivery of siRNA to Phagocytic Cells in Mice Myriam Aouadi, Gregory J. Tesz, Matthieu Prot, Sarah M. Nicoloro, Shinya U. Amano, Emilie Boutet, Anca Goller, Mengxi Wang, Chang-An Guo, William E. Salomon, Joseph V. Virbasius, Rebecca A. Baum, Mark J. O’Connor, Jr., Gary R. Ostroff and Michael P. Czech Program in Molecular Medicine. University of Massachusetts Medical School, Worcester, MA, USA Phagocytic macrophages and dendritic cells are desirable targets for potential RNAi therapeutics because they often mediate pathogenic inflammation and autoimmune responses. We recently engineered a complex, 5 component glucan-based encapsulation system for siRNA delivery to phagocytes. In experiments designed to simplify this original formulation, we discovered that the amphipathic peptide Endo-Porter forms stable nanocomplexes with siRNA that can mediate potent gene silencing in multiple cell types. In order to restrict such gene silencing to phagocytes, a method was developed to entrap siRNA/Endo-Porter complexes in glucan shells of 2-4 microns diameter in the absence of other components. The resulting glucan particles containing fluorescently labeled siRNA were readily internalized by macrophages, but not other cell types, and released the labeled siRNA into the macrophage cytoplasm. Intra-peritoneal administration of such glucan particles containing siRNA/Endo-Porter complexes to mice caused gene silencing specifically in macrophages that internalized the particles. These data indicate that specific targeting to phagocytes is mediated by the glucan, while Endo-Porter peptide serves both to anchor siRNA within glucan particles and to catalyze escape of siRNA from phagosomes. Thus we have developed a simplified siRNA delivery system that effectively and specifically targets phagocytes in culture or in intact mice. Myriam Aouadi, PhD 373 Plantation st, Biotech II, suite 100 Worcester, MA, 01605 USA Telephone: 1-508-856-6858 Email: [email protected] 117 070 Development of an RNA Aptamer for Human Toll-like Receptor 2 Ectodomain Jonghoe Byun, Ji-hae Yoon Department of Molecular Biology, Dankook University, Yongin-si, Gyeonggi-do, Korea Currently, there is a growing interest in the biology of toll-like receptors (TLRs). These pattern recognition receptors (PRRs), expressed on various immune and non-immune cell types, recognize pathogen-associated molecular patterns and play a crucial role in innate immunity by mediating the activation of macrophages. They also recruit phagocytic cells and produce proinflammatory cytokine/chemokine. Recently, it was suggested that activation of TLRs contributes to the development of certain cardiovascular diseases like atherosclerosis, congestive heart failure and cardiac dysfunction in sepsis. The cDNA encoding TLR2 was cloned from U937 cells using RT-PCR procedure. Human TLR2 (hTLR2) has 23 leucine rich repeats (LRRs) including a C-terminal LRR and two N-terminal LRRs. LRR9-LRR12 domain binds to ligands, whereas LRR11-LRR14 domain make contacts with hTLR1. To develop an RNA aptamer for hTLR2, LRR8-LRR15 domain was chosen as target protein and its coding sequences were cloned into pQE-80L vector that can express membrane protein with accompanying 6 his-tag sequences. Origami strain of E.coli that can efficiently express membrane protein was used for overexpression of LRR 8-15 domain protein. The purified protein was then used for in vitro selection of RNA aptamer(s) from the RNA library using SELEX procedure. Characterizations of the obtained aptamers are currently underway and preliminary results including electrophoretic mobility shift assay and minimization data will be presented at the meeting. Jonghoe Byun, PhD 126 Jukjeon-dong, Suji-gu, Yongin-si, Gyeonggi-do 448-701, Korea Telephone: +82-31-8005-3194 Email: [email protected] 118 071 ARREST OF THE ELONGATION OF TRANSLATION BY PEPTIDE NUCLEIC ACIDS Cordier CГ©line, Lecosnier Sabine, FranГ§ois Jean-Christophe and Saison-Behmoaras Tula, Ester, INSERM U565– CNRS UMR7196, Paris 75005 Oligonucleotides targeting 5’UTR of mRNA are often used to inhibit translation initiation. In cell-free system, Peptide Nucleic Acids (PNAs) targeting the coding region and forming triplexes are able to stop the ribosomal machinery during the translation elongation.В We have shown that it is possible to successfully target mixed sequences in structured regions of messenger RNA with short PNAs that form duplex and triplex structures that arrest elongating ribosomes and induce truncated proteins synthesis1, 2, 3. PNA is unique among the investigated steric blocking agents in its ability to specifically inhibit the translation elongation. In cells, dominant negatives could be thus generated thanks to PNAs. Type I insulin-like growth factor receptor (IGF-IR) has been implicated in the progression of a large number of cancers and represents an important target for anti-cancer strategies. Studies showed that transfected dominant negatives of IGF-IR are able to decrease tumor growth and metastasis formation. We have shown that a PNA sequence forming a partial triplex on the coding region of IGFIR’s mRNA induces a truncated IGF-IR in cell free system and inhibits the receptor expression in cultured prostate cancer cells. Therefore, we have shown that a steric blocker can stop the translation elongation in cells. In addition, IGF-1R knockdown by PNA led to an attenuation of phosphorylation of downstream signaling pathways, PI3K/ AKT and MAPK, involved in survival and mitogenesis and also to a decrease in cell transformation4. Of the steric blockers tested, which included phosphorodiamidate morpholino oligomers and locked nucleic acids, PNAs was unique in its ability to form triplex structures with mRNA and to arrest translation elongation. Dias et al. RNA hairpin invasion and ribosome elongation arrest by mixed base PNA oligomer. J. Mol. Biol. 2002; 320:489-501. 2SГ©namaud-Beaufort et al. Short pyrimidine stretches containing mixed base PNAs are versatile tools to induce translation elongation arrest and truncated protein synthesis. Oligonucleotides. 2003; 13:465-478. 3Boutimah-Hamoudi et al. Cellular antisense activity of peptide nucleic acid (PNAs) targeted to HIV-1 polypurine tract (PPT) containing RNA. Nucleic Acids Res. 2007; 35:3907-3917. 4 Lecosnier S, Cordier C, Simon P, FranГ§ois JC, Saison-Behmoaras TE. A steric blocker of translation elongation inhibits IGF-1R expression and cell transformation. FASEB J. 2011 Mar 14. 1 CГ©line Cordier, PhD student MNHN, RDDM, INSERM U565-CNRS UMR 7196, 43 rue Cuvier CP26 Paris 75005, FRANCE Telephone: 33 1 40 79 36 89 Email: [email protected] 119 072 RNAi- BASED PANCREATIC CANCER THERAPY BY LOCAL DELIVERY OF ANTI K-RASMT siRNA Eithan Galun*, Racheli Gabai-Malka**, Gili Focht**, Amotz Shemi** and Elina Zorde Khvalevsky** * Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel ** Silenseed LTD, Israel One of the major challenges for oligonucleotides based therapeutics is delivery. Obstacles in systemic delivery include targeting, over-dosing and dose fluctuations, liver aggregation and immune-stimulation of the drug carriers, renal clearance and cost. Direct injection doesn’t solve such issues in many cases. Local and prolonged delivery overcomes these hurdles and offer an attractive solution at least for a sub group of regional diseases, amongst are solid tumors. Pancreatic cancer is an aggressive disease, being the 4th leading cause of cancer–related death in the western world. Genetic alterations in KRAS signaling pathway are involved in 95% of cases, while the majority of KRAS mutations are gain-of-function mutations at codon 12 (KRASG12D) of the oncogene. The tumor is addicted to KRAS activity. We developed an implanted controlled drug delivery system called LODERВ® (Local Drug EltueR), a miniature biodegradable polymeric matrix encompassing anti KRASG12D siRNA drug and preserving it against degradation. The LODERВ® is implanted by endoscopic ultrasound within the tumor and releases drug regionally, along 8-10 weeks. Treatment of pancreatic cell cultures with siG12D LODER resulted in a significant inhibition of KRAS expression both on mRNA and protein levels. This inhibition of expression was associated with a decrease in cell proliferation and induction of cell death. In vivo the growth of human pancreatic cancer cell lines (PANC1 and CAPAN1) was retarded, both in subcutaneous and orthotopic models. Furthermore, the survival of mice implanted with siG12D LODERs was significantly improved in the two cell lines tested in the orthotopic mouse model. Based on these results, Silenseed has initiated a phase I study with siG12D LODER implanted into patients with a locally advanced pancreatic tumor: http://clinicaltrials.gov/ct2/results?term=silenseed. Eithan Galun Goldyne Savad Institute of Gene Therapy Hadassah Hebrew University Hospital, Jerusalem, 91120, Israel Telephone: 972-2-6778589 or 77762 Email: [email protected] 120 073 REGULATION OF PATHOGENIC EFFECTOR MEMORY T HELPER 1 CELL SURVIVAL BY microRNA Claudia Haftmann1*; Anna-Barbara Stittrich1*; Gitta Heinz1; Hyun-Dong Chang1, Nikolaus Rajewsky2, Andreas Radbruch1* and Mir-Farzin Mashreghi1* 1 German Rheumatism Research Center (DRFZ) Berlin, Germany 2 Max-DelbrГјck-Center for Molecular Medicine (MDC) Berlin, Germany Chronically activated pathogenic effector memory type 1 T helper (Th1) cells participate in immune-mediated diseases such as autoimmunity or chronic inflammation. These cells are resistant against physiological regulation and conventional immunosuppressive therapy by probably reducing the expression of the pro-apoptotic proteins BIM and Pten. To identify microRNAs (miRNAs) which regulate the expression of BIM and Pten and therefore regulate the persistence of pathogenic Th1 cells we compared the miRNA expression profiles of either acutely (once) or chronically (repeatedly) activated memory Th1 cells. We identified a candidate miRNA being induced in pathogenic effector Th1 cells. Computational target analysis via TargetScan and PicTar predicted BIM and Pten as putative targets. Overexpression of this miRNA in activated Th1 cells results in 50 % down-regulation of endogenous Bim and Pten expression. Conversely, inhibition of the candidate miRNA in chronically activated memory Th1 cells by antagomirs results in induced protein levels of BIM and significantly enhanced cell death and reduced proliferation. With its targets having important roles for survival and proliferation of pathogenic effector memory Th1 cells, this candidate miRNA represents a promising molecular target for the treatment of immune mediated disease.В В Claudia Haftmann, MSc Cell Biology Group (Prof. Dr. Andreas Radbruch) German Rheumatism Research Center Berlin CharitГ©platz 1 10117 Berlin, Germany Telephone: +49-30-28460 731 Email: [email protected] 121 074 Abstract and poster available for review at the meeting. 122 075 IDENTIFICATION OF PROCESS RELATED IMPURITIES IN DNA/RNA BY LCMS ANALYSIS Kenneth W. Hill Agilent Technologies, Boulder, Colorado, USA LCMS is a powerful tool for identification of impurities in DNA/RNA based API’s. Replacing ammonia with methyl amine in the deprotection process of a DNA thiolate gave rise to a new set of impurities. LCMS analysis showed that these impurities were formed by transamination of cytidine by methyl amine. The progress of methyl amine deprotection of acetyl protected dC/rC containing sequences was followed by LCMS. The data presented here show that the transaminated impurities arise from the deprotected cytidine and not the N-acetyl protected species and that this reaction is general to other alkyl amine based deprotections. Kenneth W. Hill, PhD 5555 Airport Blvd Boulder, Colorado, USA 80301 Telephone: 303 222 4945 Email: [email protected] 123 076 Design of protein-responsive shRNA system for translational control in human cell Tan Inoue, Shunichi Kashida, Yoshihiko Fujita, Hirohide Saito Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan (ICORP project, JST, Tokyo, Japan) We have developed an artificial protein-responsive shRNA switch by employing short hairpin RNA (shRNA) that serves as a powerful tool for RNAi in human cell. This artificially designed molecular systems for responding to particular gene products and rewire gene expression networks in living cells are highly useful. As a pilot system, we have developed a synthetic shRNA (Kt-shRNA) that combine target sequences (double strand region) for target mRNA with Box C/D kink-turn RNA motif set in its loop region. An Archaeal ribosomal protein L7Ae and its binding motif, Box C/D kinkturn RNA motif, were employed as the RNP interaction motif for protecting Kt-shRNA from Dicer cleavage. Kt-shRNA responds to cellularly expressed L7Ae and controls target gene expression to promote apoptosis of human cancer cells. (Saito H. et al, Nat. commun., 2 : 160 (2011) ). To develop new protein-responsive shRNA switches, we employed 3D molecular modeling software for adequately positioning the protein-binding motif in order to properly control Dicer activity. We were able to build new protein responsive shRNA switches with RNP motifs consisting of U1A or NFОєB for the control of target gene expression. Thus, we succeeded in improving the ability of new shRNA switches by tuning their RNP interaction phases in silico. In conclusion, the protein-responsive-shRNA switch could serve as a useful tool for RNAi therapy or regenerative medical techniques, because a desired RNA motif binding to a disease or differentiation marker protein could be incorporated into the loop region of the synthetic shRNA for the control of a target mRNA translation. Tan Inoue, PhD Graduate School of Biostudies, Kyoto University Kyoto 606-8502, Japan Telephone: +81 75 753 3995 Email: [email protected] 124 077 PMOplusв„ў Approaches to Rapid Response Therapeutics for Emerging Infectious Diseases Patrick Iversen1, Fred Schnell2, Laura Hauck2, Stacey Crumley2 and Dan Mourich2 AVI BioPharma, Inc., 1Bothell, WA and 2Corvallis, OR Rapid response to emerging infectious disease and laboratory accidents represents an important and novel application of AVI BioPharma’s RNA therapeutic platform. AVI’s phosphorodiamidate morpholino oliogmer (PMO) approach to rapid response relies upon multiple favorable properties of these compounds and a growing database of positive studies with a wide variety of infectious disease agents. AVI has demonstrated efficacy for our synthetic oligomers in cell culture for 19 different viral families and in vivo efficacy in 11 of those families. This experience has reinforced the value of our therapeutic design strategies. The exceptionally high success rate in animal infection challenge studies indicates a high probability of therapeutic development success. In 2001 we responded to lethal outbreaks of feline calicivirus in Atlanta, GA and Eugene, OR resulting in 94% (47/50) survival in treated kittens compared to 9.7% (3/31) survival untreated kittens. In 2002, within 14 days we progressed from concept to the successful treatment of Humboldt Penguins infected with West Nile (100% survival of treated birds). In 2004 we responded to an accident with Ebola Zaire to a laboratory worker which progressed from concept to delivery of clinical supply in 7 days. Studies confirming PMOplus efficacy have been conducted in lethal challenge animal models involving mouse, guinea pig and nonhuman primates. More recently, AVI participated in a rapid response exercise involving H1N1 influenza in 2009 in which progress from concept to compound took 7 days. PMOplus efficacy was demonstrated in both mouse and ferret animal models. In 2010, AVI undertook a rapid response exercise involving Dengue virus where the time from request to an effective PMOplus compound was 11 days. Supportive nonclincial programs have led to open INDs for AVI-6002 for Ebola Zaire, AVI-6003 for Marburgvirus and AVI-7100 for influenza A. Studies are currently in progress to expand the rapid response capability to include multidrug resistant bacterial infections. We conclude that AVI’s RNA therapeutic technology is well suited to rapid therapeutic response. Patrick Iversen, PhD 3450 Monte Villa Parkway Bothell, WA 98021 USA Telephone: 425-354-5038 Email: [email protected] 125 078 Integrated microfluidic platform for Multiplex SELEX on a Chip Soyoun Kim Department of Biomedical Engineering, Dongguk University, South Korea Multi-target aptamer selection called Multiplex SELEX (Systematic Evolution of Ligands by EXponential enrichment) gains its interests rapidly because of its potential to highspeed and high-throughput aptamer selection capacity. Parallelization of chemical process by integration of microfluidic components can be a key technique to develop a multiplex SELEX process. One of the potential problems in multplexing chemical processes on-chip is cross-contamination. In order to avoid this consideration, we propose a microfluidic network platform with a pneumatic valving strategy to allow serial loading and incubation of aptamers with sol-gel entrapped target proteins. After target binding inside the sol-gels crosscontamination free parallel elution of specifically bound aptamers is performed. The platform allows selective binding against five different targets immobilized in sol-gel spots. When eluting bound species liquid flow across nearby reaction chamber are sealed by integrated pneumatically driven rubber microvalves. We demonstrate specific aptamer binding to its respective protein target and subsequent aptamer elution without any cross contamination. The current proof of concept opens the route to increased automation and microscale parallel processing of the SELEX methodology. Figure. Concept of parallel process of aptamer binding and partitioning. Five different targets included sol-gel were spotted on the surface (Heater electrodes). Then, RNA mixture (Aptamers) were injected and incubated by fluidic operation for binding aptamers to their target proteins. Bound aptamers were collected by applying heat. (Pneumatic valves are operated during heat process in order to remove cross contamination). Soyoun Kim, PhD Associate Professor, Dongguk University 26,3 Pil-dong, Chung-Gu, Seoul 100-715, Korea Telephone: 822-2260-3840 Email: [email protected] 126 079 Development of potent RNAi therapeutics using long antisense siRNAs Sera Kim, Seul Gi Jo, Chang Han Lee, Pooja Dua, and Dong Ki Lee Global Research Laboratory (GRL) for RNAi medicine, BK21, Department of Chemistry, Sungkyunkwan University (SKKU), Suwon, Korea Corresponding author: [email protected] Specific gene silencing using RNA interference (RNAi) has proven to be a useful tool in the fields of biotechnology and a promising future therapeutic modality. Small interfering RNAs (siRNAs) are used as a molecular trigger of RNAi to specifically block the expression of the genes causing various diseases including cancer. In this study, we chose KRAS (ki-ras2 Kirsten rat sarcoma viral oncogene homolog) as a target gene to develop anticancer siRNA therapeutics. KRAS belongs to a group of small GTP-binding proteins, and involved in cell signaling and growth. Therefore, by targeting KRAS through the RNAi, we expect repression of tumor survival and proliferation. The conventional siRNA structure shows several limitations such as off-targeting effects and inefficient gene silencing for certain genes. To overcome this, we developed various long antisense siRNA structural variants which harbor long antisense 3’ overhang sequence complementary to KRAS mRNA. We show that long antisense siRNAs can trigger improved gene silencing over conventional siRNAs. Long antisense structure also improved gene silencing triggered by asymmetric shorter-duplex siRNAs (asiRNAs). Our results demonstrate that long antisense siRNA can be an alternative structure to conventional siRNA with improved efficacy and specificity in gene silencing. Sera Kim, MS candidate 330517, Chemistry Building Sungkyunkwan University, Chunchundong, Suwon, Korea Telephone: 82-31-299-4889 Email: [email protected] 127 080 Stealth Delivery of Oligonucleotides Akira Komoriya and Beverly Z. Packard OncoImmunin, Inc., Gaithersburg, MD, USA Nucleic acid delivery into live cells has been one of the major hurdles for oligonucleotidebased therapeutics. Although various approaches and methods have been developed and are currently being utilized, a new platform, Xyton Stealth DeliveryTM (XSD), has been developed for intracellular delivery of oligonucleotides with the aim of expanding therapeutic applications. Unique attributes of this new methodology include the formation of molecularly dispersed solutions and nearly maximal cellular uptake in the presence of serum within 90 minutes by a variety of cell types including primary hematopoetic cells. Furthermore, cellular entry of both single strands and duplexes of either DNA or RNA by this mode of delivery is nontoxic, homogeneous, and sequence-independent. When XSD was applied to transport of 24-mer antisense oligonucleotides, intracellular hybridization with complimentary sequences was shown to be saturable. Furthermore, Dicer substrates designed by the XSD technology were processed by the recombinant human enzyme; upon addition of a Dicer substrate targeting the eGFP gene to primary murine transgenic eGFP PBMC in serum, the expected siRNA effects were observed after 3 days as judged by flow cytometry. In summary, XSD delivery into the cytosol of both antisense DNA and RNA duplexes was uniform and rapid in a variety of target cells with mRNA hybridization and Dicer processing followed by specific gene knockdown, respectively. The delivery method described herein utilizes a totally unique class of delivery vehicles relative to what has been utilized in the RNAi and microRNA communities to date. Akira Komoriya, PhD OncoImmunin, Inc. 207A Perry Parkway, Suite #6 Gaithersburg, MD 20877 USA Telephone: (01) 301-987-7881 Email: [email protected] 128 081 Long dsRNA-mediated RNA interference and immunostimulation: long interfering dsRNA (liRNA) as a potent anticancer therapeutics Tae Yeon Lee1, Chan Il Chang1,2, and Dong-ki Lee1,* Global Research Laboratory (GRL) for RNAi Medicine, Department of Chemistry, Sungkyunkwan University (SKKU), Suwon, Korea 2 Skip Ackerman Center for Molecular Therapeutics, Beth Israel Deconness Medical Center, Harvard Medical School, Boston USA *Corresponding author:[email protected] 1 In most applications, small interfering RNAs are designed to execute specific gene silencing via RNA interference (RNAi) without triggering non-specific responses such as immunostimulation. However, in anticancer therapeutics development, immunostimulation combined with specific oncogene silencing could be beneficial, resulting in synergistic inhibition of cancer cell growth. In this study, we report an immunostimulatory long dsRNA structure with ability to trigger RNAi-mediated specific target gene silencing, termed long interfering dsRNA (liRNA). liRNA targeting Survivin mRNA not only triggered efficient and specific target gene silencing via RNA interference, but also stimulated protein kinase R (PKR) pathway to induce expression of interferon. As a result, the ability of Survivintargeting liRNA to inhibit cancer cell growth was superior over conventional siRNA or nontargeting dsRNA structures. Our results thus provide a simple, efficient dual function immunostimulatory RNAi-triggering structure, which is potentially applicable for potent anticancer therapeutics development. Tae Yeon Lee 330-517,Chemistry Building, Sungkyunkwan University Suwon Republic of Korea 440-746 Email: [email protected] 129 082 DEpendency of the Cross-linking reactivity with 2-Amino-6-vinylpurine on the Neighboring Bases Fumi Nagatsugi, Shinya Hagihara, Shuhei Kusano, Chao Xiao Guang Institute of Multidisciplinary Research for Advanced Materials, Tohoku University An oligonucleotide (ON) with a sequence complementary to a specific mRNA can inhibit its expression. The use of unmodified ON for applications in cells, however, is very limited because of the poor cellular uptake efficiency, targeted delivery, its low specificity and affinity for the target sequence, and susceptibility to degradation by nucleases. Many modified ONs containing unnatural bases or modified sugars have been prepared for improving the efficacy of the antisense method. One such example is ONs incorporating a 2′-modified nucleotide. These analogues are known to exhibit high binding affinity to target RNA and metabolic stability. Furthermore, cross-linking reactions are expected to enhance the antisense activity by irreversibly binding to the target RNA, based on the steric blocking mechanism. We previously demonstrated that the 2′-O-methyl (2′-OMe) analogue of 2-amino-6-vinylpurine (2-AVP) (1) reacted selectively to thymine in DNA, but did not react effectively to uracil in RNA under neutral conditions. 1 In this study, we investigated about the effect of adjacent bases with 2-AVP on the cross-linking reactivity. In consequence, we found that ON incorporating (1) reacted more effectively to uracil under neutral conditions in the cases where the ON had the sequences 3’ G (1) C5’. Herein, we describe the neighboring bases effect on the crosslinking reactivity of 2′-OMe analog of 2-AVP, and evaluation of the antisense effect with the use of 1 in non-cell translation assay and in cell experiments. Fig.1 Design of the 2-Amino-6-vinylpurine (2-AVP) (1) and Dependency of the Reactivity on the Neighboring Bases of (1) 1 Imoto, S.; Hori, T.; Hagihara, S.; Taniguchi, Y.; Sasaki, S.; Nagatsugi, F., Bioorg. Med. Chem. Lett., 20, 6121-6124 (2010) Fumi Nagatsugi, PhD Institute of Multidisciplinary Research for Advanced Materials, Tohoku University 2-1-1 Katahira, Aoba-ku Sendai-shi, Miyagi, 980-8577, Japan Telephone: 001- 81-22-(217)-5633 Email: [email protected] 130 083 ATTENUATION OF AUTOIMMUNITY BY ANTI-HUMAN-INTERLEUKIN-17 APTAMER IN MOUSE MODELS Yoshikazu Nakamura, Hironori Adachi, Akira Ishiguro Institute of Medical Science, University of Tokyo, Tokyo, Japan Interleukin-17 (IL-17) is a pro-inflammatory cytokine produced primarily by a subset of CD4+ T cells, called Th17 cells, that is involved in host defense, inflammation and autoimmune disorders. Two IL-17 family members, IL-17A and IL-17F, are most structurally related, and form homodimeric (IL-17A/A, IL-17F/F) and heterodimeric (IL-17A/F) structures. We have isolated RNA aptamers against human IL-17A. Aptamers are short single-stranded nucleic acid sequences that are selected in vitro based on their high affinity to a target molecule. One such aptamer, Apt21-2, blocked the interaction between IL-17A and its receptor IL-17R in vitro, and inhibited the production of interleukin-6 from mouse and human cells. A PEGylated form of Apt21-2 inhibited the development of GPI-induced rheumatoid arthritis and MOG-induced experimental autoimmune encephalomyelitis in a dosedependent manner, showing its therapeutic potential in autoimmune diseases. Among several dimeric conformers of IL-17, Apt21-2 recognizes both IL-17A/A and IL-17A/F as does the known anti-IL-17A antibody. Although the biological significance of IL-17A and IL-17F have been investigated using respective antibodies or gene knockout mice, the functional study of IL-17A/F heterodimeric form has been hampered by the lack of an inhibitory tool specific to IL-17A/F. Hence, we aimed to develop an RNA aptamer that specifically inhibits IL-17A/F. One such aptamer against human IL-17A/F, AptAF42, was isolated through the subtractive SELEX procedure employing counter-selection against IL-17A/A. AptAF42 and its optimized derivative AptAF42dope1 blocked the binding of IL-17 receptor to IL-17A/F, but not to IL-17A/A or IL-17F/F, in the surface plasmon resonance assay in vitro. Consistently, AptAF42dope1 did not affect cytokine GRO- О± production induced by IL-17A/A or IL-17F/F, but blocked IL-17A/F-induced GRO- О± production in human cells. RNA footprinting assay of AptAF42dope1 using ribonucleases in the presence or absence of IL-17A/F revealed that part of the predicted secondary structure fluctuates between alternate forms and that AptAF42dope1is globally protected from ribonuclease cleavage by IL-17A/F. These results suggest that the selected aptamer recognizes a global conformation specified by the heterodimeric surface of IL-17A/F. Yoshikazu Nakamura, PhD and Professor Institute of Medical Science, University of Tokyo 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Telephone: +81(JPN) 3 5449 5307 Email: [email protected] 131 084 A NOVEL ASSAY FOR THE DETECTION OF miRNA MATURATION Saskia Neubacher, Christoph Arenz* Humboldt-UniversitГ¤t zu Berlin, Berlin, Germany During the past years interest has increased in finding ways to manipulate miRNA function, not only in order to elucidate its mechanisms, but also with the perspective of affecting human disease formation and progression.[1] In order to investigate the activity of potential inhibitors and modulators of Dicer-mediated miRNA maturation, a facile and accurate assay is needed.[2, 3] Here we provide a novel rapid and reliable assay of Dicer-mediated miRNA maturation, without the need of tedious and expensive labeling of pre-miRNAs. The assay is based on the rolling circle amplification (RCA) method,[4] wherein only the mature miRNA but not the pre-miRNA can initiate isothermal amplification of a cyclic DNA template leading to the formation of a linear single stranded DNA (ssDNA) product. A secondary primer binds the product sequence and initiates the formation of a secondary DNA strand resulting in an enhanced fluorescence signal of SYBR gold. So far, we have established the assay for three different miRNAs and showed that each miRNA binds specifically to its target circular DNA and leads to specific amplification. Moreover, inhibitors can easily be screened for selectivity in the presence of other pre-miRNAs.[5] The use of unmodified pre-miRNAs reduces the risk of generating assay derived artifacts. Thus, we are optimistic that this assay provides a useful tool for the investigation of miRNA maturation and helps accelerating the search for specific inhibitors of miRNA maturation. C. Arenz, Angew Chem Int Ed 2006, 45, 5048-5050. [2] C. M. Klemm, A. Berthelmann, S. Neubacher, C. Arenz, Eur J Org Chem 2009, 17, 2788-2794. [3] B. P. Davies, C. Arenz, Angew Chem Int Ed, 2006, 45, 5550-5552. [4] S. Neubacher, C. Arenz, ChemBioChem 2009, 10, 1289-1291. [5] S. Neubacher, C. Arenz, Angew Chem, submitted. [1] Saskia Neubacher, Dipl.Chem. Humboldt-UniversitГ¤t zu Berlin Brook-Taylor-Str. 2, 12489 Berlin, Germany Telephone: +49 30 2093 7511 Email: [email protected] 132 085 DEVELOPMENT OF DNA NANOGELS AND HYDROGELS AS INJECTABLE DELIVERY SYSTEMS FOR CPG DNA AND OTHER NUCLEIC ACID-BASED DRUGS Makiya Nishikawa, Kohta Mohri, Kohei Ogawa, Natsuki Takahashi, Yuki Takahashi, Yoshinobu Takakura Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan DNA can be a building block for constructing a wide variety of unique structures, including but not limited to DNA cubes, DNA origami, DNA polyhedra and DNA hydrogels. In addition to the excellent property as a block, DNA is a biologically active compound that can be and is being used in the treatment of human diseases. One of such compounds is the DNA containing unmethylated CpG dinucleotides with particular flanking sequences (CpG motif), the ligand for Toll-like receptor-9 (TLR9). The ligation of TLR9 triggers the induction of proinflammatory cytokines, and CpG DNA has been developed for the treatment of cancer, allergic diseases, and as adjuvants for vaccines against cancer and infectious diseases. Although the phosphodiester bond of CpG DNA is generally replaced by phosphorothioate one to increase the enzymatic stability, this modification is associated with high levels of cytotoxicity. We hypothesized that the use of DNA nanotechnology can be a new and promising approach to increasing the activity of CpG DNA without changing its chemical structure. Based on this, we have developed a series of DNA assemblies having sticky ends through which the assembled DNA units are connected to each other. The immunostimulatory activity of CpG DNA was significantly increased by the formation of tripod- or tetrapodlike structures. Furthermore, the development of DNA dendrimer by connecting the tripodlike DNA assemblies further increased the activity. Then, large DNA hydrogels were successfully developed using several types of assembled DNA units. We demonstrated that CpG DNA hydrogels activate dendritic cells, continuously release intercalated doxorubicin, an anticancer agent, and was highly effective in inhibiting subcutaneous tumor growth in mice after in vivo administration. Then, we have developed injectable sol-gel transition DNA hydrogels utilizing the difference in salt concentrations between test tubes and biological fluids. We confirmed that the administration of solutions containing DNA assemblies with proper sequences resulted in the formation of DNA hydrogels at the administration sites, such as dermis and nasal cavity. These findings suggest that DNA nanogels (DNA dendrimer) and DNA hydrogels are promising delivery systems for nucleic acid-based drugs, such as CpG DNA, as well as for anticancer agents, antigens and other bioactive compounds. Makiya Nishikawa, PhD Graduate School of Pharmaceutical Sciences, Kyoto University 46-29 Yoshidashimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan Telephone: +81-75-753-4580 Email: [email protected] 133 086 CHARACTERIZATION OF SIDE REACTIONS DURING THE ANNEALING OF SMALL INTERFERING RNA Noll, Bernhard; Seiffert, Stephan; Debelak, Harald; Hadwiger, Philipp; Jahn-Hofmann, Kerstin; Roehl, Ingo; Vornlocher, Hans-Peter Roche Kulmbach GmbH, D-95326 Kulmbach, Germany The process of impurity formation during annealing of siRNA was investigated. siRNAs containing common chemical RNA modifications (2’-O-methyl-, 2’-deoxy-2’-fluoro-, 2’-deoxy-ribose and phosphorothioate linkages) were used to determine major side reactions - such as 2’,3’-isomerisation, strand scission and HF elimination. Individual impurities were characterized using analytical SEC, denaturing and non-denaturing IP-RP-HPLC, DSC and UV-spectrometry. The degradation pathways described in this work can lead to significantly reduced product quality and compromised drug activity. Based on our data, incubation temperature and time as well as the presence of divalent cations are the most critical factors in controlling impurity formation. Without metal-ion catalysts, formation of 2’,5’-isomers was the major side reaction of the two RNA single strands containing 2’-unmodified nucleotides. When Mg2+-ions were present, metal-ion-catalyzed degradation of all three investigated RNA strands was observed to a significant extent. For 2’-deoxy-2’-fluoro-modified strands HFelimination at the modified pyrimidine-nucleotides was the dominant side reaction leading to the formation of 2,2’-O-anhydroribonucleotides and arabinosylnucleotides.All side reactions (isomerisation, elimination and strand scission) were suppressed by duplex formation. Since impurity formation was most pronounced at elevated temperatures, heating of the siRNA solution should be limited to the lowest temperature required for substantial dissociation of the duplex. The optimal annealing temperature may therefore be close to the Tm of the duplex. Divalent cations such as Mg2+ catalyze strand scission and are best avoided in annealing solutions. The strong concentration dependency of the Tm in salt-free solutions allows for adjustment of the Tm by changing siRNA concentration. Taken together, annealing in salt-free solutions allows for lower annealing temperatures, minimizes side reactions and simplifies lyophilisation of the resulting siRNA molecule. Our data provides background to successfully address challenges associated with the manufacture of siRNAs and other nucleic acid therapeutics, such as aptamers, spiegelmers, decoy and antisense oligonucleotides. Bernhard Noll, Associate Director Analytics CMC Fritz-Hornschuch-Strasse 9 D-95326 Kulmbach, Germany Telephone: +49 (0) 9221 827 62 8546 Email: [email protected] 134 087 Abstract and poster available for review at the meeting. 135 088 EXPERIMENTAL ASSESSMENT OF ANTIMIR SPECIFICITY Andreas Petri1,Susanna Obad2, Darryl Pappin3, Sakari Kauppinen1,2, and Morten Lindow1 1) Informatics, Santaris Pharma A/S, HГёrsholm, Denmark 2) MicroRNA Drug Discovery, Santaris Pharma A/S, HГёrsholm, Denmark 3) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA The use of short 8-mer antimiR seed targeting tiny LNAs has recently been shown to provide an efficient means to silence single miRNAs or entire miRNA families. Due to their short length, this class of antimiRs will have many perfect match binding sites in the transcriptome. We wanted to assess whether or not tiny LNA oligos affect transcripts harboring binding sites or proteins translated from such transcripts. To this end, we used Sylamer to perform an unbiased and exhaustive analysis of sequence motifs associated with differentially expressed genes following tiny LNA administration to cell lines or mice. In all experiments with antimiRs, Sylamer analysis showed significant overrepresentation among the up-regulated genes of motifs corresponding to binding sites of the cognate seed. However, sequence motifs corresponding to direct binding sites for the tiny LNAs were not overrepresented. The effect on protein level was analyzed using both small scale iTRAQ analyses of a subset of the samples used for transcription profiling as well as an in vitro luciferase reporter assay of tiny LNAs targeting various parts of the luciferase transcript. The proteomics data also showed up-regulation of predicted miRNA targets following miRNA silencing, while no significant effect was seen on proteins encoded by transcripts harboring a direct binding site. The latter finding was corroborated by data from the luciferase reporter assay. Collectively, our data show that while the transcriptome harbors many perfect match binding sites for short 8-mer oligonucleotides, the presence of high affinity tiny LNAs generally has no effect on the transcripts containing perfect match binding sites or on the levels of protein translated from such transcripts. Andreas Petri, Ph.d. Santaris Pharma A/S KogleAllГ© 6 DK 2970 HГёrsholm Danmark Telephone: +45 4517 9876 Email: [email protected] 136 089 An RNA aptamer is a potential biomarker for pancreatic cancer Partha Ray, Bruce A. Sullenger, Rebekah R. White Department of Surgery, Duke University Medical Center, Durham, NC Introduction: The symptoms of pancreatic cancer are usually non-specific and late. Existing tumor markers (such as CA19-9 and CEA) are neither sensitive nor specific enough to serve as useful screening tests. Tools for earlier detection could increase the proportion of patients who are cured with surgical resection. Aptamers are oligonucleotide ligands that are identified from large libraries of RNA or DNA molecules using an iterative selection process termed as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Aptamers bind their specific targets with high affinity and specificity and can be used for the dual purpose of identifying novel biomarkers and detecting them in biological specimens. Methods: In vitro “positive/negative” SELEX was performed using the conditioned media (the “secretome”) of the MiaPaCa2 human pancreatic ductal adenocarcinoma cell line (positive selection) and the immortalized, non-cancerous human pancreatic ductal cell line HPDE (negative selection). The rationale for “positive/negative” selection was to select for aptamers against molecules that are selectively secreted by cancerous cells but not by normal cells. Standard radioactive filter-based binding assays were used to measure binding. Results/Conclusion: We have successfully selected several nuclease resistant (2’-fluoro-modified) RNA aptamers that differentially bind the secretome of pancreatic cancer cells compared to noncancerous cells. One of the aptamers (M9-5) binds pancreatic cancer patient serum with high affinity compared to control serum collected from healthy donors. Using a maximal fraction bound (FB) of 11% as a cutoff, the aptamer discriminated between untreated pancreatic cancer patients (N=19) and healthy volunteers (N=20) with 95% sensitivity and 100% specificity, and the difference in aggregate binding values between the groups is highly significant (P<0.0001, Mann-Whitney U test). In addition, paired serum samples obtained from two patients pre- and post-chemoradiation therapy and pre- and two months post-resection have demonstrated a decrease in M9-5 binding with decreased tumor burden. Notably, serum CA19-9 levels were normal (<40 U/ml) in 62% of cancer patients, and CEA levels were normal (<2.5 ng/ml) in 57% of cancer patients, indicating that our biomarker might be more sensitive than these existing serum biomarkers for pancreatic cancer. For the M9-5 target identification, several steps of biochemical fractionation were performed. At each step of the fractionation, the M9-5 binding activity was assayed to follow the purification scheme. At the end of final purification step the fraction was resolved by gel electrophoresis and after Coomassie staining a ~20 kDa major polypeptide band was visualized. Cyclophilin B (CypB) was identified upon mass spectrometric analysis of the band. To verify the M9-5 target identification, the biotinylated M9-5 aptamer was used for affinity purification. CypB was detected upon Western blot analysis of the purified proteins. Additionally, to confirm that CypB is the protein target of aptamer M9-5, we measured CypB levels in these same serum samples using ELISA. Similar differential expression between cancer patients and healthy volunteers was observed using the ELISA assay as with the M9-5 binding assay. Also, there was a statistically strong correlation between M9-5 binding and CypB levels by ELISA. The implication of these finding will be further discussed. Partha Ray, PhD DUMC Box 103035 Durham, NC 27710 USA Telephone: 919-684-4864 Email: [email protected] 137 090 Endosomal Escape of liposomal siRNA via membrane fusion and in vivo delivery to tumor Yu Sakurai, Hiroto Hatakeyama, Yusuke Sato, Hidetaka Akita, Hideyoshi Harashima Hokkaido University, Sapporo, Hokkaido, Japan To deliver siRNA to tumor tissue, we have developed liposome type carrier, a multi-functional envelope type nano device (MEND). While PEGylation is the most popular strategy to stabilize liposome in the circulation, reduction of cellular uptake and endosomal escape by PEGylation is a crucial problem. We previously reported that pH-sensitive fusogenic peptide, GALA, modification onto the MEND circumvented this barrier in vitro cultured cell (Sakurai et al., Biol Pharm Bull, 2009). GALA-MEND was destabilized in an acidic compartment endosome because the structure of GALA changed into alpha-helix at low pH (~5.5), and then GALA on the surface of GALA-MEND induced membrane fusion between MEND and endosome. As a result, encapsulated siRNA was released into cytosol efficiently. Although GALAmodified PEGylated MEND (GALA-MEND) elicits an siRNA delivery efficiency, it was clarified that GALA-MEND intrabenously injected into mice was subject to a rapid clearance from blood circulation. Therefore, we designed a shorter version GALA (shGALA). In this study, we would like to introduce in vivo siRNA delivery with shGALA-modified PEGylated MEND (shGALA-MEND). shGALA-MEND exhibited a higher knockdown effect (60% reduction, 480 nM) than only PEGylated MEND (PEG-MEND) in vitro. Microscopy and flowcytometry analyses indicated that the result was caused by increasing endosomal escape of siRNA, not enhanced cellular uptake. Because inhibition of acidification of endosome/lysosome compartments by ammonium chloride suppressed siRNA efficiencyof shGALA-MEND, endosomal escape of siRNA by shGALA depended on acidification of endosome. Then, to evaluate the biodistribution of both siRNA and lipid envelope of systemically injected MENDs, we labeled these components with radio isotope. While free siRNA showed a very rapid blood clearance, siRNA assembled with PEG-MEND and shGALA-MEND demonstrated a high blood concentration (>10% ID/mL blood) even 6 hr after injection, and no significant difference was observed between PEG- and shGALA-MEND Next, we investigated the in vivo knockdown and the anti-tumor effect of shGALA-MEND. We administered MENDs into tumor-bearing mice via tail vein 4 times every 24 hr at a dose of 4 mg siRNA/kg body weight, and then measured target gene expression and tumor volume change. A significant mRNA reduction (60%) and inhibition of tumor growth were observed in the group of mice treated with shGALA-MEND. In addition, shGALA-MEND showed no notable somatic and hepatic toxicity. In conclusion, we succeeded in developing an in vivo siRNA delivery system to tumor tissue (Sakurai et al., Biomaterials, in press). Yu Sakurai, doctoral course Kita-12, Nishi-6, Kita-ku, Sapporo, Hokkaido, Japan Telephone: +81-11-706-2197 Email: [email protected] 138 091 IN VIVO siRNA DELIVERY WITH PH-SENSITIVE LIPOSOME Yusuke Sato, Hiroto Hatakeyama, Yu Sakurai, Mamoru Hyodo, Hidetaka Akita, Hideyoshi Harashima Faculty of Pharmaceutical Science Hokkaido University, Sapporo/Hokkaido, Japan Small interfering RNA (siRNA) is the biofunctional nucleic acid inducing RNA interference (RNAi) and is emphasized as the tool for gene therapy. The physicochemical characteristics of siRNA, such as high molecular weight and hydrophilicity prevent passive diffusion across plasma membrane. So, delivery carriers are required which allow siRNA to arrive at target organs and enter target cells. In addition, it is necessary for siRNA to localize cytoplasm which is the place for working and avoid lisosomal compartmentalization. To date, we developed a multifunctional envelope-type nano device (MEND) for siRNA delivery carrier to tumour tissue. The most of conventional liposomal carriers are composed of cationic lipids and have high cationic charge on their surface for the purpose of high affinity for cells but lead to nonspecific interaction with anionic species in the blood and exhibit rapid clearance from blood circulation by the reticulo-endothelial system (RES). This problem can be overcome by using polyethylene glycol (PEG) attached on the particle’s surface. Even though, the modification of a lot of PEG actually prolonged circulation time, it dramatically inhibits intracellular trafficking. To solve this вЂ�PEG dilemma’, we previously developed a cleavable PEG-lipid in response to tumour environment1) and a novel endosomal escape peptide device2). In the present study, we picked on pH-sensitive lipid, which exhibits neutral at physiological pH and cationic at acidic pH. First, we constructed pH-sensitive MEND by using DODAP, which is known as a pH-sensitive aminolipid but it resulted in lower knockdown activity than conventional cationic MEND using DOTAP, which is known as a cationic lipid. Next, we synthesized a novel pH-sensitive lipid, YSK05 and optimized lipid composition to improve the intracellular trafficking of pH-sensitive MEND. As a result, the optimized pH-sensitive MEND successfully showed 100-fold higher knockdown activity than the conventional cationic MEND. Then, we tried to confirm whether pH-sensitive MEND actually facilitated endosomal escape by depending on endosomal acidification by using ammonium chloride, which buffers lysosomes. Expectively, the knockdown activity of pH-sensitive MEND was dramatically decreased depending on ammonium chloride concentration, which suggested endosomal acidification was important process for pH-sensitive MEND to escaping from endosome. Finally, we evaluated knockdown activity of pH-sensitive MEND in subcutaneous OSRC-II tumour model. Both single local and systemic administration of pH-sensitive MEND-formulated human polo-like kinase 1 (hPLK1) caused reduction in tumour-derived hPLK1 mRNA. RNAispecific mRNA cleavage products in tumour were found by 5’-RACE PCR, which suggested that mRNA reduction resulted from RNAi and was sequence specific. These results suggest that pH-sensitive MEND is a useful tool for siRNA delivery to tumour tissue and has potent carrier for therapeutics. 1) Hatakeyama H et. al. Biomaterials, 32, 4306-4316 (2011) 2) Sakurai Y et. al. Biomaterials, in press Yusuke Sato, Doctor Course Telephone: +81-11-706-2197 Email: [email protected] 139 092 OTS 2011: PMOs – The Clinical Story Stephen Shrewsbury AVI BioPharma, Bothell, WA USA Phosphorodiamidate Morpholino oligomers (PMOs) differ from the older Antisense OligoNucleotides (AONs) by the substitution of a synthetic 6-membered morpholine ring for the ribose and linking the monomeric backbone with phosphorodiamidates rather than phosphorothioates moieties. AVI has pioneered PMO development for 25 years and has tested 5 PMOs in 18 clinical studies in several diseases – neuroinvasive West Nile Virus, Hepatitis C, polycystic kidney disease, cytochrome induction and the lead PMO (AVI-4658, eteplirsen) recently completed phase 2 testing in Duchenne Muscular Dystrophy (DMD) at individual doses of up to 900mg IV (20mg/kg). Maximum Feasible Doses (MFD) of PMOs were well tolerated in 2 mammalian species over 12 weeks to open an eteplirsen IND allowing evaluation of doses of up to 100mg/kg, and supporting plans for further clinical testing. Recently, 219 clinical doses of eteplirsen were well tolerated in a 12-week of dosing, open label, dose escalation, phase 1b/2 clinical study of eteplirsen with a typical pediatric trial AE profile superimposed on the chronic DMD disease. No significant drug related AEs or SAEs were reported and no AE was deemed probably or definitely drug related. Of particular note, no local injection site reactions were reported. Clinical hematology, coagulation and chemistry (renal function especially) remained stable, and encouraging but variable biological efficacy was demonstrated, especially at the 10 and 20 mg/kg dose levels. A further placebo-controlled, phase 2 study of eteplirsen at higher doses (30 and 50 mg/kg) is due to start dosing in June 2011. Advanced generation PMO based chemistries are now evolving, based on the neutral and stable PMO backbone, by highly specific positioning of limited positive charges (PMOplusв„ў chemistry) for viral diseases (Ebola, Marburg and Junin hemorrhagic fevers, Dengue and Influenza), arginine based peptide conjugates (PPMO), or novel internal/external additions (PMO-Xв„ў), which have demonstrated better in-vivo cell penetration and potency Preclinical testing with these chemistries is well advanced and clinical testing of the PMOplus chemistry for two phase 1 programs (Ebola and Marburg) began in May 2011. Stephen B. Shrewsbury, MD, PhD 3450 Monte Villa Parkway Bothell, WA 99021 USA Telephone: 425-354-5038 Email: [email protected] 140 093 AMPHIPHILIC PEPTIDYL-RNA CONJUGATES ANCHORED TO AND ENRICHED ON PHOSPHO- AND GLYCOLIPID BILAYERS Peter Strazewski,1 Alexandra Le Chevalier Isaad,1 Kollappillil S. Krishnakumar,1 Dominique Lafont,2 (1) Laboratoire de SynthГЁse de BiomolГ©cules, (2) Laboratoire de Chimie Organique 2В ; Institut de Chimie et Biochimie MolГ©culaires et SupramolГ©culaires UniversitГ© Claude Bernard Lyon 1, France To elucidate RNA-controlled peptide synthesis, and ultimately the evolution of translation, anchoring studies between peptidyl-RNA and lipidic bilayers were carried out on lipidic vesicles. Such vesicles are вЂ�minimal cell’ mimics, viz. models for protocells that emerged in nature during some early stage of evolution. We synthesized amphiphilic peptidyl-RNA as model compounds in which the peptide part should be sufficiently hydrophobic to be immersed into the lipophilic interior of membrane bilayers. Different vesicles, which assembled spontaneously from various lipids and glycolipids, were used to study RNA anchoring. In order to visualize by confocal fluorescence microscopy peptidyl-RNA being anchored onto lipid vesicles, the conjugate and the lipids were labeled with suitable fluorophores. A hydrolytically stable peptidyl-RNA conjugate was synthesized in a step-by-step fashion by a solid support procedure using an orthogonally protected derivative of 3’-l-alanylamino-3’deoxyadenosine (a puromycin analogue) as a first synthetic building block that was immobilized through its 2’-hydroxyl function to an appropriate resin. A solid support peptide synthesis using Fmoc strategy was carried out departing from the 3’ position of the puromycin analogue. After DMT cleavage, an oligoribonucleotide strand was grown on the same solid support beginning at the 5’ position of the puromycin analogue. After the final cleavage of all protective groups, the solubilised amphiphilic conjugate was purified by HPLC and identified by ESI-MS. The target molecule was composed of a lipophilic 20-meric oligopeptide being covalently linked through a C-terminal amide bond to a 22-meric oligoribonucleotide hairpin that was inspired by the acceptor stem of E. coli tRNAAla and closed by a stable UUXG tetraloop (Figure). We could unequivocally demonstrate by confocal fluorescence microscopy that the peptidyl-RNA conjugate, when externally added to giant uni- and multilamellar lipidic vesicles, spontaneously interacted with their outer bilayer membranes. At initial Figure. L = l-leucine, A = l-alanine, 3'-A = 3'-amino-3'- lipid-to-conjugate molar ratios between deoxyadenylate, ACGU = adenylate, cytidylate, guanylate, 150:1 and 1500:1 they visibly and strongly uridylate, X = fluorescein linked to thymidylate. enriched on the readily accessible lipidic surfaces. These studies were supplemented by control experiments and quantitative permeability measurements on large unilamellar vesicles, as well as by FTIR and CD spectroscopic studies of the peptide interacting with lipids. Prof. Peter Strazewski Laboratoire de SynthГЁse de BiomolГ©cules, BГўt. Chevreul (5ГЁme Г©tage) UniversitГ© Claude Bernard Lyon 1 43 boulevard du 11 novembre 1918, 69622 Villeurbanne Cedex, France Email: [email protected] 141 094 Therapeutic Application of DNAzymes for Nasopharyngeal Carcinomas Sun L-Q, Yang L, Cao Y Xiangya Hospital and Center for Molecular Medicine, Central South University; Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan 410078, China DNAzymes are synthetic, single-stranded DNA catalysts that can be engineered to bind and cleave the target mRNA of a disease-causing gene. The ability of the 10-23 DNAzyme to specifically cleave RNA with high efficiency under simulated physiological conditions has fuelled expectation that this agent may have useful biological application in a gene inactivation strategy. To explore this potential, we used DNAzymes to validate therapeutic targets for therapeutic approaches to nasopharyngeal carcinomas (NPC). Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has been known to have oncogenic properties during latent infection in NPC. Genetic manipulation of LMP1 expression may provide a novel strategy for the treatment of NPC. By targeting the LMP1 mRNA, we successfully obtained a phosphorothioate-modified вЂ�вЂ�10–23’’ DNAzyme namely DZ1, through screening a series of DNAzymes. DZ1 could significantly down-regulate the expression of LMP1 in NPC cells, inhibit cell proliferation, metastasis, promote apoptosis and enhance radiosensitivity of NPC through interfering signal pathways which are abnormally activated by LMP1, including NF-ОєB, AP-1 and STAT3 signal pathways. In a clinical setting, we, for the first time, demonstrated that intratumoral administration of the DNAzyme in combination with a standard radiotherapy could increase the tumor regression rate in patients. Molecular imaging analysis (MRI) revealed that DNAzyme treatment could negatively impact on the tumor vasculatures. Together, interfering LMP1 signaling pathway could be a promising strategy to target the malignant phenotypes of NPC. Lun-Quan Sun, PhD Xiangya Hospital and Center for Molecular Medicine, Central South University Changsha, Hunan 410078, China Telephone: +86 731 84327646 Email: [email protected] 142 095 INHIBITION OF MICRORNA ACTIVITY BY 2К№-O-METHYL-4К№THIORIBONUCLEOSIDE MODIFIED ANTI-microRNA OLIGONUCLEOTIDE (AMO) Mayumi Takahashi,1 Noriaki Minakawa2 and Akira Matsuda1 Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan, 2Graduate School of Pharmaceutical Sciences, The University of Tokushima, Shomachi 1-78-1, Tokushima 770-8505, Japan 1 MicroRNAs (miRNAs) are a class of endogenously expressed small non-cording RNA molecules which regulate the gene expression post-transcriptionally. Recent advances in understanding of the role of miRNAs in the biological process revealed that most of them are implicated in the human diseases, such as cancer, viral infection and cardiovascular disorders. Therefore, miRNAs are considered to be a potential new class of therapeutic target. Among the approaches to modulate the function of miRNA, anti-miRNA oligonucleotide (AMO)based inhibition has been the most widely used not only to exploit the biological function of miRNAs but also as candidates for therapeutic agents. Although AMOs represent sequence specific inhibition based on the Watson-Crick base pairing, they are single-stranded oligonucleotides and easily degraded in biological fluid. Chemical modification on AMOs can be beneficial to improve hybridization ability for target miRNA, resistance toward nuclease degradation, and pharmacological properties such as tissue uptake for in vivo delivery. A wide variety of chemically modified AMOs was developed to date, including 2К№-OMe, 2К№-OMOE and LNA, and successfully inhibits miRNA function in vitro as well as in vivo. We have recently developed a novel chemically modified oligonucleotide, 2К№-OMe-4К№thioRNA, which can be considered as a hybrid chemical modification based on 2К№-OMe and 4К№-thioribonucleoside. In its hybridization property, 2К№-OMe-4К№-thioRNA preferred RNA as a complementary partner rather than DNA. In addition, investigation of their biological stability revealed that 2К№-OMe-4К№-thioRNA was extremely stable in 50% human plasma compared with 2К№-OMeRNA and 4К№-thioRNA. Prompted by such favorable properties, we planed to utilize 2К№-OMe-4К№-thioribonucleosides to develop novel and effective chemically modified AMOs. We evaluated a several 2К№-OMe-4К№-thioribonucleoside modified AMO for their ability to inhibit miR-21 and miR-122 activity by dual-luciferase reporter assay. We found that fully 2К№-OMe-4К№-thionucleoside modified AMOs were significantly more potent than corresponding 2К№-OMe modified AMOs. In addition, the prolonged inhibition activity of AMO was achieved by 2К№-OMe-4К№-thioribonucleoside modifications. Mayumi Takahashi, doctoral student (D3) Hokkaido University Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan Telephone: +81 11 706 3763 Email: [email protected] 143 096 RNAi in humans: Phase I dose-escalation study of ALN-VSP02, a novel RNAi therapeutic for solid tumors with liver involvement A.K. Vaishnaw9, A. Cervantes1, M. Alsina2, J. Tabernero2, J.R. Infante3, P. LoRusso4, G.I. Shapiro5, L. Paz-Ares6, G. Schwartz7, G. Weiss8, R. Falzone9, J. Hill9, J. Cehelsky9, A. White9, I. Toudjarska9, D. Bumcrot9, R. Meyers9, G. Hinkle9, N. Svrzikapa9, D.W. Sah9, H.A. Burris3, J.A. Gollob9 1 Hospital ClГnico Universitario de Valencia, Valencia, Spain, 2Hospital Universitario Vall d’Hebron, Barcelona, Spain, 3Sarah Cannon Research Institute, Nashville, TN, USA, 4Karmanos Cancer Center, Detriot, MI, USA, 5Dana-Farber Cancer Institute, Boston, MA, USA, 6Hospital Virgen del RocГo, Seville, Spain, 7Memorial Sloan-Kettering Cancer Center, New York, NY, USA, 8 TGen Clinical Research Services at Scottsdale Healthcare, Scottsdale, AZ, USA, 9 Alnylam Pharmaceuticals, Inc., Cambridge, MA, USA Background: ALN-VSP02 is a RNA interference (RNAi) therapeutic comprised of lipid nanoparticle-formulated small interfering RNAs (siRNAs) targeting the expression of vascular endothelial growth factor (VEGF)-A and kinesin spindle protein (KSP). Methods: A multi-center, open label, Phase 1 dose-escalation trial of ALN-VSP02 administered as a 15-minute iv infusion q2 wks was initiated in patients (pts) with advanced solid tumors and at least one measurable liver lesion. Main objectives included evaluation of safety/tolerability and assessment of PK/PD. Results: Forty-one pts were enrolled across 7 dose levels (0.1-1.5 mg/kg); median age 57 yrs, all with multiple prior therapies. A total of 182 doses have been administered to date, mean of 4.4 (range 1-24). Treatment was generally well-tolerated; the most common AEs were lowgrade nausea, fatigue and fever with no clear dose-dependence. One on-study death (liver failure in a pt with near complete replacement of the liver by tumor) deemed possibly related to treatment occurred at 0.7 mg/kg. Grade 2 infusion-related reactions occurred in 15% of pts or 3% of total doses administered and were managed with slowing of infusion. Grade 1-2 chills/rigors were seen in 3 of 11 pts at 1.25 mg/kg. Dose-limiting toxicities included reversible grade 3 thrombocytopenia (2 pts at1.25 mg/kg) and hypokalemia (1 pt at 1.5 mg/kg). Plasma PK showed dose-proportional AUC and Cmax. Post-treatment biopsies from 10 pts (7 liver and 3 extrahepatic tumors) showed pharmacologically relevant concentrations (2.2-142 ng/g tissue) of both siRNAs. Molecular evidence of RNAi-mediated VEGF mRNA cleavage was shown in liver tumor biopsies (n=2 at 0.4 mg/kg) and in an extrahepatic tumor biopsy (ovarian cancer at 1.25 mg/kg) through use of the 5’ RACE assay. Additional evidence for an anti-VEGF effect with ALN-VSP02 included a decrease in Ktrans of at least 40% by DCE-MRI in 46% of evaluable pts. Among 37 pts evaluable for response, 8% (1 of 13) at doses ≤ 0.4 mg/kg had stable disease (SD) for at least 2 mo compared to 50% (12/24) with SD (n=11) or PR (n=1, endometrial cancer with liver metastases) at doses ≥ 0.7 mg/kg. Conclusions: ALN-VSP02 is well-tolerated and has antitumor activity. Pharmacodynamic data are consistent with an anti-VEGF effect, and 1.0 mg/kg q2wks is the recommended Phase II dose. Akshay K. Vaishnaw, MD, PhD Alnylam Pharmaceuticals Inc. 300 Third Street, Cambridge, MA 02142 USA Telephone: 617-551-8360 Email: [email protected] 144 097 microRNA turnover and AntagomiR-mediated inhibition of inflammation Bryan R.G. Williams,1 Michael P. Gantier1, H. James Stunden1, Claire E. McCoy1, and Mark A. Behlke2 Centre for Cancer Research, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia; 2Integrated DNA Technologies, Coralville, Iowa, USA microRNAs (miRNAs) are key regulators of gene expression and critical for normal cell function. However, little is known of their overall persistence in the cell following processing. Characterization of this is key to the full appreciation of their regulatory roles. We have devised a system to allow inducible genetic ablation of Dicer, the enzyme required for production of mature miRNAs. Using this system we measured miRNA decay rates in mouse embryonic fibroblasts following loss of Dicer1 enzymatic activity. The results confirm the inherent stability of miRNAs, the intracellular levels of which were mostly affected by cell division. Using the decay rates of a panel of six miRNAs representative of the global trend of miRNA decay, we have established a model of miRNA turnover and determine an average miRNA half-life of 119 h, (i.e. ~5 days). This study constitutes the first in-depth characterization of miRNA decay in mammalian cells and our findings indicate that miRNAs are up to 10 times more stable than messenger RNA. Interestingly some miRNAs turn over more rapidly than others suggesting the existence of novel mechanism(s) controlling selective miRNA cellular concentration and function. We have also used the inducible genetic ablation of Dicer1 and subsequent miRNA depletion to uncover an important role for miRNAs in the control of pro-inflammatory cytokine production in cells. Antisense-mediated inhibition of miRNA function by antagomiR confirmed the involvement of select miRNAs in the positive control of NFkB signalling. Through the use of in silico predictions and reporter assays we show that these miRNAs negatively control expression of A20 (TNFAIP3), resulting in increased NFkB signalling. AntagomiR-mediated inhibition of these miRNAs results in inhibition of proinflammatory cytokine production. These results suggest that antagomiRs targeting negative regulators of NFkB signalling could offer novel therapeutic avenues to control inflammatory disorders and infections. Bryan R.G. Williams PhD Centre for Cancer Research, Monash Institute of Medical Research Monash University Clayton, Victoria, 3168, Australia Email: [email protected] 145 T he Oligonucleotide Therapeutics Society (OTS) has become recognized as a premier organization fostering the research and drug development in all areas of oligonucleotide science. 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