september 8 – 10, 2011 the royal library copenhagen ... · 7 th annual meeting of the...

September 8 – 10, 2011The Royal Library

Copenhagen | Denmark

7th Annual Meeting of the

OligonucleotideTherapeutics Society

Meeting Abstracts Meeting Abstracts

Oligonucleotide Therapeutics Society

PROGRAM AGENDA

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7TH ANNUAL MEETING OF THE OLIGONUCLEOTIDE THERAPEUTICS SOCIETY

w w w . o l i g o t h e r a p e u t i c s . o r g2011 CONFERENCE PROGRAM

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Wednesday, September 7, 201115:00 – 18:00 Registration and Poster Set-Up

Thursday, September 8, 201108:00 – 09:00 Registration and Poster Set-Up

09:00 – 09:15 Welcome and Opening Remarks Gunther Hartmann, MD, PhD, Universitätsklinikum Bonn, OTS President

09:15 – 10:30 SESSION I: Hot Topics: Short Talks Selected from Abstracts Chair: Gunther Hartmann, MD, PhD, Universitätsklinikum 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 Lübeck

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 SESSION II: NUCLEIC ACID CHEMISTRY 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, Montreal12:00 – 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, Göttingen

14:30 – 16:00 SESSION III: RNA BIOLOGY: NEW FRONTIERS 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


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Copenhagen, Denmark8

16:30 – 18:00 SESSION IV: DELIVERY OF NUCLEIC ACIDS Chair: Jean-Jacques Toulmé, 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 Macromoléculaire, 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 Welcome Reception with Attended Posters

19:30 Meet the Experts End of Day 1

Friday, September 9, 2011

08:00 Registration Opens

08:30 – 10:00 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, Universitätsklinikum Bonn09:00 – 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


10:30 – 12:00 SESSION VI: TARGETING CODING 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 SESSION VII: TARGETING 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



w w w . o l i g o t h e r a p e u t i c s . o r g2011 CONFERENCE PROGRAM

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15:30 – 17:00 SESSION VIII: TARGETING LIVER AND TUMORS Chair: Henrik Ørum, 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 Ørum, PhD, Santaris Pharma A/S

17:15 – 19:15 Exhibitor Reception at the Børsen A reception featuring our meeting exhibitors.

End of Day 2

Saturday, September 10, 2011 08:00 Registration Opens

08:15 – 09:45 SESSION IX: TARGETING INFECTION 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 SESSION X: TARGETING THE CENTRAL NERVOUS SYSTEM 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


11:45 – 13:15 SESSION XI: TARGETING GENETIC DISEASES 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

SPEAKER ABSTRACTS

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Expanding thE Structural divErSity rEpErtoirE of sirnasDong-ki LeeGlobal Research Laboratory for RNAi Medicine, Department of Chemistry, Sungkyunkwan University, Suwon 440-746, Korea

Chemically synthesized small interfering RNAs (siRNAs) can mediate efficient gene silenc-ing 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 sequence-independent 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 strand-mediated 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 non-targeting long dsRNA. These novel siRNA structural variants exemplify the structural flex-ibility of gene silencing RNAs, and have the potential to become important alternatives to the current siRNA structure for future therapeutics development.

Dong-ki Lee, PhDGlobal Research Laboratory for RNAi MedicineDepartment of ChemistrySungkyunkwan UniversitySuwon 440-746 KoreaTelephone: 82-31-299-4565Email: [email protected]

Session 1: Hot Topics: Short Talks Selected from AbstractsDong-ki LeeThursday, September 8, 201109:15 – 09:30

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activation of rna intErfErancE in aniMalS With SinglE StrandEd oligonuclEotidESEric E. Swayze, Thazha P. Prakash, Walt F. Lima, and Stanley T. CrookeIsis Pharmaceuticals, Carlsbad, CA, USA

The use of double stranded oligonucleotides to inhibit gene expression via the RNA interfer-ence (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 rela-tionship (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 under-standing 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, PhDIsis Pharmaceuticals, Inc.2855 Gazelle CourtCarlsbad, CA 92010 USATelephone: 760-603-3825Email: [email protected]

Session 1: Hot Topics: Short Talks Selected from AbstractsEric SwayzeThursday, September 8, 201109:30 – 09:45

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liMitationS for rnai: intracEllular rElEaSE of sirna and cEll StrESSGeorg SczakielInstitute of Molecular Medicine, University of Lübeck, 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., Wünsche,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, PhDInstitute for Molecular MedicineUniversity of Lübeck Building: 50, 1F, Room 1319 Ratzeburger Allee 160 23538 Lübeck Germany Telephone: +49 451 500 2730 Email: [email protected]

Session 1: Hot Topics: Short Talks Selected from AbstractsGeorg SczakielThursday, September 8, 201109:45 – 10:00

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prEclinical Efficacy and SafEty of phoSphorodiaMidatE Morpholino oligoMErS (pMo) Peter SazaniAVI 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 phospho-rodiamidate morpholino oligomer (PMO) platform is a next generation of RNA-based thera-peutics 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 phos-phorodiamidate 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. AVI-4658 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 PMOplus™. 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, PhDAVI BioPharma, Inc.3450 Monte Villa ParkwayBothell, WA 98021 USATelephone: 425-354-5087 Email: [email protected]

Session 1: Hot Topics: Short Talks Selected from AbstractsPeter SazaniThursday, September 8, 201110:00 – 10:15

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5’-triphoSphatE dsrna-inducEd StiMulation of rig-i prEvEntS influEnza h1n1 infEction of priMary naSal EpithElial cEll culturE and protEctS MicE froM infEction in vivoCoch C.1*, Stehle V.1*, Kümmerer B.3, Stabenow D.4, Stümpel J.P.1, Daßler J.1, Herberhold S.2, Lüdenbach B.1, Kochs G.5, Stäheli 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 ‘Retinoic acid inducible gene-I’ (RIG-I) detects viral 5´-triphosphate dsRNA (3p-dsRNA) as contained in viral genomes leading to an antiviral response that protects the host. To circumvent antivi-ral 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 epi-thelial cells in vitro and in a mouse model in vivo. Methods: Primary cultures of nasal epithelial cells were generated from conchotomy speci-men 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 gener-ated or synthetic 3p-dsRNA induced antiviral cytokines directly in nasal epithelial cells (IP-10, 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 epi-thelium. 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 po-tential use of 3p-dsRNA as immunotherapeutic agent in Influenza virus infection.

Christoph Coch, MDInstitute of Clinical Chemistry and Clinical Pharmacology, University Hospital BonnSigmund-Freud-Str. 25, 53105 Bonn, Germany Telephone: 0049/228/287-14018 or 16080Email: [email protected]

Session 1: Hot Topics: Short Talks Selected from AbstractsChristoph CochThursday, September 8, 201110:15 – 10:30

NUCLEOTIDE CHEMISTRY AND GENE SILENCINGJesper WengelUniversity 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, PhDUniversity of Southern DenmarkNucleic Acid CenterCampusvej 55DK-5230 Odense MDenmarkTelephone: +45 65502510Email: [email protected]

Session 2: Nucleic Acid ChemistryJesper WengelThursday, September 8, 201111:00 – 11:30

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araBinoSE ModifiEd antiSEnSE and SirnaS: Biological applicationS, Structural conSidErationS, and dElivEry StratEgiESMasad 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, PhDDepartment of Chemistry, McGill University801 Sherbrooke St. WestMontreal, QC, CANADA, H3A-2K6Telephone: 514-398-7552 Email: [email protected]

Session 2: Nucleic Acid ChemistryMasad J. DamhaThursday, September 8, 201111:30 – 12:00

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sirna activity, StaBility and 3d-Structural propErtiES of 2’-fluoro rna and gnaMartin EgliDepartmentof 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 right-handed 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/

referencesM. 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, PhDDepartmentof Biochemistry, Vanderbilt UniversityNashville, TN 37232-0146 USATelephone: 1-615-343-8070Email: [email protected]

Session 2: Nucleic Acid ChemistryMartin EgliThursday, September 8, 201112:00 – 12:30

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oligonuclEotidE thErapEuticS rEviEWEd in MEMory of alan gEWirtzEckstein, F. Max-Planck-Institute for Experimental Medicine, Göttingen, 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 oligo-nucleotide 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 appli-cation 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 im-mune response or similarly binding to the retinoic acid-inducible gene (RIG-1). Also aptam-ers 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, PhDMax-Planck-Institute for Experimental MedicineHermann-Rein-Str. 337075 Göttingen, GermanyTelephone: (49) 551-3899 274Email: [email protected]

Keynote Session: Oligonucleotides reviewed in memory of Alan GewirtzFritz EcksteinThursday, September 8, 201114:00 – 14:30

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priMary microrna function in targEt rEcognition and rEprESSion Chang-Zheng Chen, Si-Biao Yue, Robin Deis Trujillo, Yujie Tang, & William E. O’GormanDepartment 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 re-garding 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 essen-tial 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 regu-latory information encoded in the structural and sequence elements of pri-miRNAs can be translated into activity through direct interactions between target mRNAs and structured pri-miRNAs and may have broad implications in understanding the mechanisms by which miR-NAs recognize and repress their cognate targets.

Chang-Zheng Chen, PhDDepartment of Microbiology and Immunology, Baxter Laboratory for Stem Cell Biology Stanford University School of MedicineStanford, CA 94305 USATelephone: 650-725-1667Email: [email protected]

Session 3: RNA Biology: New FrontiersChang-Zheng ChenThursday, September 8, 201114:30 – 15:00

rEgulation of SMall rna functionGunter MeisterUniversity 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 phosphoryla-tion 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 phospho-tyrosine 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, PhDUniversity of RegensburgUniversitaetsstrasse 31Regensburg, 93053GermanyTelephone: 0049 941 943 2847Email: [email protected]

Session 3: RNA Biology: New FrontiersGunter MeisterThursday, September 8, 201115:00 – 15:30

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thE MEchaniSM of mirna and sirna argonautE loading in MaMMalSMark A. Kay, Shuo Gu, and Lan Jin Depts of Pediatrics and GeneticsStanford University School of MedicineStanford, 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 per-formed various genetic analyses in living cells to study the molecular process of RISC as-sembly and activation. Ago loading is the process of duplex RNA association followed by re-moval 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 coun-terparts. 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, PhDDennis Farrey Family Professor Departments of Pediatrics and GeneticsVice Chair for Basic Research (Pediatrics) Stanford University School of Medicine269 Campus Drive CCSR Bldg., Suite 2105Stanford, CA USA 94305Telephone: 650-498-6531Email: [email protected]

Session 3: RNA Biology: New FrontiersMark KayThursday, September 8, 201115:30 – 16:00

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chEMical StratEgiES for dElivEry of rnai drugS Muthiah ManoharanAlnylam 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.

referencesChemical Modifications of RNAiI.

Manoharan, M. “RNA interference and chemically modified small interfering RNAs.” 1. Curr. Opin. Chem. Biol. 2004, 8, 570-579.Bumcrot, D. et al. “RNAi therapeutics: a potential new class of pharmaceutical drugs.”2. Nature Chemical Biology 2006, 2, 711-719. Manoharan, M. and Rajeev, K. G. “Utilizing chemistry to harness RNA interference path-3. ways for therapeutics: chemically modified siRNAs and antagomirs.” Antisense Drug Technology (2nd Ed.), 2008, 437-464.Zlatev, I. et al. “Efficient solid-phase chemical synthesis of 5’-triphosphates of DNA, 4. RNA and their analogs.” Organic Letters 2010, 12, 2190-2193.Watts, J5. . et al. “Effect of chemical modifications on modulation of gene expression by duplex antigene RNAs that are complementary to non-coding transcripts at gene promot-ers.” Nucleic Acids Res. 2010, 38, 5242-5259.Addepalli, H. et al. “Modulation of thermal stability can enhance the potency of siRNA.” 6. Nucleic Acids Res. 2010, 38, 7320-7331. Manoharan, M. et al. “Unique gene-silencing and structural properties of 2’-F modified 7. siRNAs.” Angewandte Chemie, (International Edition) 2011, 50, 2284-2288.Pallan, P. S. et al.;. 8. Unexpected origins of the enhanced pairing affinity of 2’-fluoro-modified RNA. Nucleic Acids Res. 2011, 39, 3482-3495.

Conjugates for siRNA and Antagomir DeliveryII. Soutschek, J. et al. “Therapeutic silencing of an endogenous gene by systemic 9. administration of modified siRNAs.” Nature, 2004, 432, 173-178. Kruetzfeldt, J. et al. “Silencing of microRNAs in vivo with ‘antagomirs’.” 10. Nature, 2005, 438, 685-689.Wolfrum, C. et al., “Mechanisms and optimization of11. in vivo delivery of lipophilic siRNAs.” Nature Biotech. 2007, 25, 1149-1157.Wu, Y. et al.12. “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.Querbes, W. et al.13. “Direct CNS delivery of siRNA mediates robust silencing in oligodendrocytes.” Oligonucleotides 2009, 19, 23-30.

Session 4: Delivery of Nucleic AcidsMuthiah ManoharanThursday, September 8, 201116:30 – 17:00

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Chen, Q. et al. “Lipophilic siRNAs mediate efficient gene silencing in oligodenrocytes 14. with direct CNS delivery.” Journal of Controlled Release 2010, 144: 227-232.DiFiglia, M. et al. “Therapeutic silencing of mutant huntingtin with siRNA attenuates 15. striatal and cortical neuropathology and behavioral deficits.” PNAS, 2007 104, 17204-17209. Alam, Md. R. et al. Multivalent Cyclic RGD Conjugates for Targeted Delivery of Small 16. Interfering RNA. Bioconjugate Chemistry, 2011, (in press). Jayaprakash, K. N. et al. “Non-nucleoside building blocks for copper-assisted and cop-17. per-free click chemistry for the efficient synthesis of RNA conjugates.” Organic Letters 2010, 12, 5410-5413.Yamada, T. et al. “Versatile site-specific conjugation of small molecules to siRNAs using 18. click chemistry.” Org. Chem., 2011, 76, 1198-1211.

Liposomal Nanoparticles (LNPs)III. Zimmermann, T. et al. “RNAi-mediated gene silencing in non-human primates.” 19. Nature, 2006, 441, 111-114.Akinc, A. et al. “A combinatorial library of lipid-like materials for delivery of RNAi 20. therapeutics.” Nature Biotech. 2008, 26, 561-569. Frank-Kamenetsky, M. et al. “Therapeutic RNAi targeting PCSK9 acutely lowers plasma 21. cholesterol in rodents and LDL cholesterol in nonhuman primates.” PNAS, 2008, 105, 11915-11920. Akinc, A. et al.,22. “Development of lipidoid-siRNA formulations for systemic delivery to the liver.” Molecular Therapy, 2009, 17, 872-879.Love, K. T. et al., “Lipid-like materials for low-dose, in vivo gene silencing.”23. PNAS 2010, 107, 9915. Akinc, A. et al. “Targeted delivery of RNAi therapeutics.” 24. Molecular Therapy 2010, 18, 1357-1364.Semple, S. et al. “Rational design of cationic lipids for siRNA delivery.” 25. Nature Biotechnology 2010, 28, 172-176.

Muthiah Manoharan, PhDAlnylam Pharmaceuticals300 Third StreetCambridge MA 02493 USATelephone: 617-551-8200Email: [email protected]

Session 4: Delivery of Nucleic AcidsMuthiah ManoharanThursday, September 8, 201116:30 – 17:00

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non-covalEnt pEptidE-BaSEd dElivEry SyStEMSG. 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 sig-nificantly 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, PhDCentre de Recherches de Biochimie MacromoléculaireUMR 5237, CNRS, UM-1, UM-2 CRBM-Department of Molecular Biophysics and TherapeuticsMontpellier, France Telephone: 33 (0) 4 67613392Email: [email protected]

Session 4: Delivery of Nucleic AcidsGilles DivitaThursday, September 8, 201117:00 – 17:30

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invEStigating thE potEntial of thErapEutic oligonuclEotidES for pulMonary diSEaSESKen Clark, Stephen Hughes, Jeremy Kitson, Jill Coates, Joel Parry, Chris Gruenloh, Daren Levin, Pallav Bulsara, Scott Summerfield & Mark EdbrookeRespiratory 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 de-livery to the airways should achieve high concentrations in the target tissue while mini-mising 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 respira-tory 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 therapeu-tics, and discuss the remaining key research and development challenges.

Ken Clark, PhDRespiratory Therapeutics, GlaxoSmithKlineStevenage SG1 2NY, United KingdomTelephone: +44 7920 567189Email: [email protected]

Session 4: Delivery of Nucleic AcidsKen ClarkThursday, September 8, 201117:30 – 18:00

rig-i and tolErancE of SElf-rnaGunther HartmannInstitute 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 respon-ses 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 (5´-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 struc-ture 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 5´-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 T°, Hartmann G°, Patel DJ°. 2010. Structural and functional insights into 5‘-ppp RNA pattern recognition by the innate immune receptor RIG-I. Nat Struct Mol Biol 2010;17:781-7. °equally contributed

Gunther Hartmann, MD, PhDInstitute of Clinical Chemistry and Clinical PharmacologyUniversity Hospital Sigmund-Freud-Str. 25, 53127 Bonn, GermanyTelephone: +49-22816080Email: [email protected]

Session 5: Immunobiology of Nucleic AcidsGunther HartmannFriday, September 9, 201108:30 – 09:00

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rEgulation of thE innatE iMMunE rESponSE uSing oligonuclEotidE-BaSEd inhiBitorS of toll-likE rEcEptorSFranck J. BarratDynavax 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-α signature in the blood which is tightly associated with levels of autoantibodies and disease activities. Self-nucleic 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 gener-ated 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 glu-cocorticoids (GC) but under maintenance levels often suffer from disease flares that neces-sitate 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 cor-tico-sparing drugs which would be open new possibilities with respect to therapeutic applica-tions. 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 ascend-ing 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, PhDDynavax Technologies2929 Seventh StreetBerkeley CA 94710 USATelephone: +1.510.665.7266 Email: [email protected]

Session 5: Immunobiology of Nucleic AcidsFranck BarratFriday, September 9, 201109:00 – 09:30

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intracEllular trafficking of tlr ligandS in huMan plaSMacytoid dEndritic cEllSPatricia Fitzgerald-BocarslyUMDNJ- 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 vigor-ously 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 traffick-ing 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 inhibi-tors of the PI-3 kinase pathway/autophagy will also be presented.

Patricia Fitzgerald-Bocarsly, PhDUMDNJ – New Jersey Medical School185 So. Orange Ave.Newark, NJ 07103 USATelephone: 973-972-5233Email: [email protected]

Session 5: Immunobiology of Nucleic AcidsPatricia Fitzgerald-BocarslyFriday, September 9, 201109:30 – 10:00

gyMnotic dElivEry of antiSEnSE oligonuclEotidES

Abstract unavailable at time of printing.

Session 6: Targeting Coding RNACy SteinFriday, September 9, 201110:30 – 11:00

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Abstract available for review at the meeting.

Session 6: Targeting Coding RNAFrank RigoFriday, September 9, 201111:00 – 11:30

clinical dEvElopMEnt of MipoMErSEn


Session 6: Targeting Coding RNAErik StroesFriday, September 9, 201111:30 – 12:00

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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 GogaRegulus 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 anti-miRs 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 anti-miR-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, PhD3545 John Hopkins CtSan Diego, CA 92130 USATelephone: 858-202-6303Email: [email protected]

Session 7: Targeting microRNANeil GibsonFriday, September 9, 201113:30 – 14:00

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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 re-sponses 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, PhDDirector, Institute for Molecular and Translational Therapeutic Strategies (IMTTS)Integrated Research Center (IFB-Tx)Medical School Hannover (MHH)Carl-Neuberg-Straße 1, 30625 Hannover, GermanyTelephone: +49 511 532-5272 (office)Email: [email protected]

Session 7: Targeting microRNAThomas ThumFriday, September 9, 201114:00 – 14:30

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oncoMir-1 in cancEr and dEvElopMEnt: a talE of MicE and MEnAndrea VenturaMemorial 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 investi-gation 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 knock-in 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 attrib-utable to the miR-19 seed family.

Andrea Ventura, MD, PhDDept. of Cancer Biology and GeneticsMemorial Sloan Kettering Cancer Center408 East 69th, ZRC-1201New York, NY, 10065 USATelephone: 646-888-3068 Email: [email protected]

Session 7: Targeting microRNAAndrea VenturaFriday, September 9, 201114:30 – 15:00

Evaluation of lockEd nuclEic acid (lna)-BaSEd mrna antagoniStS in cancEr patiEntS Buchbinder A, Kalambakas S, Huq N, Berkowitz NEnzon Pharmaceuticals, Inc., Piscataway, NJ, USA

Background: HIF-1 is a transcription factor that is a critical mediator of angiogenesis, cell pro-liferation, metabolism, survival, and adaptive responses to stress. EZN-2968 is a potent locked nucleic acid antisense oligonucleotide that downmodulates HIF-1α 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, toler-ability, pharmacokinetics (PK), and pharmacodynamics. results: EZN-2968: 49 pts received weekly EZN-2968 doses for 3, 4, or 5 of 6 weeks. Dose-limiting 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 6 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 >90 days. Objective tumor shrinkage was observed in several pts. HIF-1α expression was evaluated pretreatment and on-treatment in tumor (n=6) and skin (n=41) samples. Tumor biopsy HIF-1α mRNA decreased in 4 pts, increased in 1 pt, and did not change in 1 pt. Skin biopsy HIF-1α 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 8 mg/kg (Grade 3 increased aspartate or alanine aminotransferase [AST or ALT]). The MTD for EZN-3042 is 6.5 mg/kg. Drug-related adverse events (AEs) (in >15% 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 in 5 pts.conclusions: The MTD of EZN-2968 is 18 mg/kg given weekly for 4 out of 6 weeks. EZN-2968 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-1α 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-4072. Tolcher AW, Patnaik A, Papadopoulos KP, et al; AACR 2011 Annual Meeting, Abs. LB-409

Aby Buchbinder, MDEnzon Pharmaceuticals, Inc.20 Kingsbridge RdPiscataway, NJ, 08854 USATelephone: 1-(732)-980-4817Email: [email protected]

Session 8: Targeting Liver and TumorsAby BuchbinderFriday, September 9, 201115:30 – 16:00

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aln-ttr, an rnai thErapEutic for thE trEatMEnt of tranSthyrEtin aMyloidoSiSDinah 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, USA2Molecular Neurobiology Group, Institute for Molecular and Cell Biology, Porto, Portugal3Unidade Clinica de Paramiloidose, Porto, Portugal4Department of Medicine, Umeå University, Umeå, Sweden5National Referral Center for FAP, Le Kremlin Bicêtre, France6National Amyloidosis Centre, London, United Kingdom7Quintiles 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 contrib-utes to pathology and clinical progression. There is a high unmet medical need for new thera-pies, 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 ALN-TTR01 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, PhDAlnylam Pharmaceuticals300 Third StreetCambridge, MA 02142 USAPhone: +1-617-551-8357Email: [email protected]

Session 8: Targeting Liver and TumorsDinah SahFriday, September 9, 201116:00 – 16:30

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targEting mirna-122 for thE trEatMEnt of hcv


Session 8: Targeting Liver and TumorsHenrik ØrumFriday, September 9, 201116:30 – 17:00

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thE dEvElopMEnt of aptaMErS aS candidatE antiviral agEntS: toWardS a polyvalEnt MicroBicidEWilliam 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

Sir William Dunn School of Pathology, University of Oxford, OX1 3RE, UK1. Astbury Centre for Structural Molecular Biology, University of Leeds, LS2 9JT, UK2. Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, 3. Chicago, IL 60611, USAMagee-Women’s Research Institute, 204 Craft Ave, Room B505, Pittsburgh, PA 4. 15213, USAChemconsilium GCV, Jaarmarktstraat 48, 2221 Booischot, Belgium5.

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 virus-inhibitory 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]

Session 9: Targeting Infection William JamesSaturday, September 10, 201108:15 – 08:45

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dEvEloping thE nExt gEnEration of ExprESSEd rnai ModalitiES againSt rapidly-Evolving viral infEctionSMarc S. WeinbergAntiviral 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 effec-tive 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 ap-plication 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 anti-viral short hairpin (sh) RNAs, long hairpin (lh)RNAs and primary microRNA (pri-miRNA) mimics are presented.

Marc S. Weinberg, PhDDepartment of Molecular Medicine and Haematology,University of the Witwatersrand Medical School7 York Rd.,ParktownJohannesburg 2193South AfricaTelephone: +27117172561Email: [email protected]

Session 9: Targeting Infection Marc WeinbergSaturday, September 10, 201108:45 – 09:15

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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 inhibi-tory 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 conju-gates 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 protec-tion 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 prolifera-tive 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 Hope1500 East Duarte Road Duarte, CA 91010 USA Telephone: (626) 301-8360Email: [email protected]

Session 9: Targeting Infection John RossiSaturday, September 10, 201109:15 – 09:45

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Abstract available for review at the meeting.

Session 10: Targeting the Central Nervous System Sponsored by International Society of NeurochemistryAdrian KrainerSaturday, September 10, 201109:45 – 10:15

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Micro-rnas in thE nEuroiMMunE intErfacE: froM inflaMMation to lEthal poiSoning protEction Hermona SoreqThe 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 communi-cation 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 signal-ing 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 clus-ter 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, PhDThe Hebrew University of JerusalemDept. of Biological ChemistryEdmond J. Safra CampusJerusalem 91904 IsraelTelephone: 972-2-6585109Email: [email protected]

Session 10: Targeting the Central Nervous System Sponsored by International Society of NeurochemistryHermona SoreqSaturday, September 10, 201110:15 – 10:45

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thE rolE of microrna in rEgulating thE cEntral StrESS rESponSEChen 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, PhDDepartment of Neurobiology, Weizmann Institute of ScienceRehovot 76100, IsraelTelephone: +972-8-9344490Email: [email protected]

Session 10: Targeting the Central Nervous System Sponsored by International Society of NeurochemistryAlon ChenSaturday, September 10, 201110:45 – 11:15

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Exploring and Exploiting dESignEr nuclEaSES for targEtEd gEnoME EnginEEringToni CathomenDept. 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 non-specific 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, PhDDepartment of Experimental HematologyHannover Medical SchoolCarl-Neuberg-Str. 130625 Hannover, GermanyTelephone: +49 511 532 5170Email: [email protected]

Session 11: Targeting Genetic DiseasesToni CathomenSaturday, September 10, 201111:45 – 12:15

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oligonuclEotidE-dirEctEd gEnE Modification in MouSE EMBryonic StEM cEllS to Study variantS of cancEr-rElatEd gEnESHein te Riele, Marieke Aarts, Eva Wielders, Marleen Dekker, Sandra de Vries, Rob DekkerThe 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 bet-ter mouse models for human disease. As readout for successful ‘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 tran-siently 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 dis-carded 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 ‘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 integ-rity. 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 ca-pacity. In cases where the functional implications are less clear, or when a mutation only par-tially 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]

Session 11: Targeting Genetic DiseasesHein te RieleSaturday, September 10, 201112:15 – 12:45

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sidna, a nEW StratEgy for inhiBiting dna rEpair pathWaySM. Dutreix1, J-S. Sun2

1Institut Curie research center, Institut National de la Santé et de la Recherche Médicale U1021, Centre National de la Recherche Scientifique UMR3427, Paris-Orsay, France;2DNA 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, DNA-PK, 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 phospho-rylation after Dbait treatment. Surprisingly, the DHDR did not disrupt cell cycle and prolif-eration 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 radio-therapy with Dbait.

Marie Dutreix, PhDUMR ETIC, Institut Curie, Bat110Centre Universitaire91405, Orsay, FranceTelephone : +33(0)169867186 Email : [email protected]

Session 11: Targeting Genetic DiseasesMarie DutreixSaturday, September 10, 201112:45 – 13:15

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POSTER ABSTRACTS

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siDNA, A NEW STRATEGY FOR INHIBITING DNA REPAIR PATHWAYSM. Dutreix1, J-S. Sun2

1Institut Curie research center, Institut National de la Santé et de la Recherche Médicale U1021, Centre National de la Recherche Scientifique UMR3427, Paris-Orsay, France;2DNA 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, DNA-PK, 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 phospho-rylation after Dbait treatment. Surprisingly, the DHDR did not disrupt cell cycle and prolif-eration 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 radio-therapy with Dbait.

Marie Dutreix, PhDUMR ETIC, Institut Curie, Bat110Centre Universitaire91405, Orsay, FranceTelephone : +33(0)169867186 Email : [email protected]

DEvElOPmENT OF SYSTEmIc ANTISENSE TREATmENT IN DYSTROPHIc mOuSE mODElS FOR DucHENNE muSculAR DYSTROPHYAnnemieke Aartsma-Rus1, Christa de Winter1, Maaike van Putten1, Tatyana Karnaoek2, Judith van Deutekom2 and Gert-Jan van Ommen1

1Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands2Prosensa BV, Leiden, the Netherlands

Antisense-mediated reading frame restoration is one of the most promising therapeutic ap-proaches 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 +/-; inter-mediate 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 de-creased 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, PhDAlbinusdreef 22333 ZA LeidenThe NetherlandsTelephone: +31 71 5269436Email: [email protected]

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AuTO-vEcTORIZED microRNA mImIc-BASED NANOSYSTEm FOR HEPATOcEllulAR cARcINOmA THERAPYAimé 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 nano-systems 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 Aimé, 2nd year PhD studentINSER U869 Université Victor Segalen Bordeaux II1er étage, porte 12 146, Rue Léo Saignat 33 076 Bordeaux Cedex - FranceTelephone: 0033 6 65 24 96 94Email: [email protected] / [email protected]

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DEvElOPmENT OF A mIcRO-RNA-34 THERAPEuTIc: PHARmAcOlOGIcAl DRuG DElIvERY & PREDIcTING THE FuTuRE PATIENT POPulATIONAndreas G. Bader, David Brown, Jason Wiggins, Jane Zhao, Christopher Daige, Michael Omotola, Kevin Kelnar, Neil Leatherbury, Jay Stoudemire, Paul LammersMirna 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 in-tegral 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, PhDAssociate Director, Research2150 Woodward Street, Suite 100Austin, TX 78744 USATelephone: 512-681-5241Email: [email protected]

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PTPIP51 – A POSSIBlE TARGET FOR NOvEl DIABETES AND OBESITY THERAPIESBobrich MA1, Schwabe SA1, Kamm M1, Brobeil AM1, Krüger K2, Mooren FC2, Steger K3, Tag C1, Wimmer M1

1Institute of Anatomy and Cell Biology, Justus-Liebig-University, Giessen, Germany2Department of Sports Medicine, Justus-Liebig-University, Giessen, Germany3Department 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 14-3-3beta 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, StudentAulweg 123, 35392 Gießen, GermanyTelephone: +49641 99 47012 Email: [email protected]

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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 mono-nuclear cells (PBMC), plasmacytoid dendritic cells and cells expressing recombinant human TLR8, all of these could be activated with imiquimod or resiquimod and phophorothioate-ssRNA40 (Heil et al. 2004), but not with functional Spiegelmers (L-ss/dsRNA) or phosphoro-thioate-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 un-likely 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 303:1526Robbins et al. (2009) Oligonucleotides 19:89

Klaus Buchner, PhDNOXXON Pharma AGMax-Dohrn-Str. 8-1010589 Berlin, GermanyTelephone: +49-30-726247-146Email: [email protected]

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A uNIquE mOE-DNA cHImERIc OlIGONuclEOTIDE INDucES mDA-5 DEPENDENT INDucTION OF TYPE I INTERFERON RE-SPONSESebastien A Burel1, Hiroki Sato2, Todd Machemer1, Patrick Cauntay1, Scott P Henry1 1ISIS Pharmaceuticals, Carlsbad, California, USA 2Institute for Virus Research, Kyoto University, Kyoto, Japan

Second generation ‘Gapmer’ antisense oligonucleotides (ASO) containing 2’-O-methoxyeth-ylribose (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 gener-ally 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 recep-tors, 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, PhD2855 Gazelle Court, Carlsbad, CA 92010 USATelephone: 1-760-603-2717 Email: [email protected]

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INcORPORATION OF lNA INTO APTAmER SElEcTIONMeghan A. Campbell, Birte Vester, Jesper WengelNucleic 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 en-richment (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 modi-fications 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 ss-LNA can then be used for the next round of selection and the cycle continues. Following nu-merous 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, PhDInstitute for Physics and ChemistryUniversity of Southern DenmarkCampusvej 555230 Odense M, DenmarkTelephone: +45 6550 2548Email: [email protected]

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[3H]-RADIOlABElING OF SmAll INTERFERING RNA (siRNA)Christensen J1,2, Andres H1, Natt F1, Hunziker J1, Krauser J1, Swart P1

1Novartis Institutes of Biomedical Research, Basel, Switzerland2Nucleic 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]-radi-olabeling 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 un-changed. 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 2´-O-methyluridine. Either a 5-bromouridine or 5-bromo-2´-O-methyluridine phosphoramidite building block was incorporated into the middle of the antisense strand which was synthesized on a DNA/RNA-synthesizer using standard phos-phoramidite chemistry. Because the method is solely dependent upon the existence of a uridine or a 2´-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, fluores-cence), the use of radiolabeled oligonucleotides to perform (pre)clinical in vitro/in vivo studies of-fers a distinct advantage of avoiding chemical modification of these compounds.

Jesper Christensen, MScNovartis Campus, WSJ-153.1.08 CH-4056 Basel, Switzerland Telephone: +41 79 593 9171Email: [email protected]

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cOmPuTER-GuIDED OPTImIZATION OF AN RNA APTAmER FOR clINIcAl APPlIcATIONS§Justin P Dassie, ‡William M Rockey, ¶Frank J Hernandez, ¥,±,†,€Sheng-You Huang, ¥,±,€Song Cao, ¶Craig A Howell, ¶Luiza I Hernandez, §Gregory S Thomas, ¶Xiu Ying Liu, ≠Natalia Lapteva, ≠David M Spencer, ¶James O McNamara II, ¥,±,†,€Xiaoqin Zou, ¥,±,€Shi-Jie Chen, ‡,¶,§Paloma H Giangrande‡Department of Radiation Oncology, ¶Department of Internal Medicine, §Molecular & Cellular Biology Program, University of Iowa, Iowa City, IA, USA. ¥Department of Physics and Astronomy, ±Department of Biochemistry, †Dalton Cardiovascular Research Center, and €Informatics Institute, University of Missouri, Columbia, MO, USA. ≠Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA

RNA aptamers represent an emerging class of pharmaceuticals with great potential for tar-geted cancer diagnostics and therapy. Several RNA aptamers that bind cancer cell-surface antigens with high affinity and specificity have been described. However, their clinical po-tential 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 ap-plications 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 PSMA-expressing prostate cancer cells. Finally, we demonstrate the potential of this reagent for im-aging 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 func-tion. Furthermore, these studies have resulted in a PSMA RNA aptamer that due to its opti-mized in vivo profile and shorter sequence length is now one step closer to the clinic.

Justin P Dassie, BS375 Newton Rd, 5241 MERFIowa City, IA 52242 USATelephone: (319) 384-3243Email: [email protected]

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RNA APTAmERS TO THE cANcER-ASSOcIATED PROTEINS uPA AND PAI-1 WITH THERAPEuTIc POTENTIAl Daniel M Dupont, Jeppe B. Madsen, Kenneth A. Bøtkjær, Bertrand Tavitian, Frédéric Ducongé, Jørgen Kjems and Peter A. AndreasenDepartment 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 2´-fluor-containing RNA oligo-nucleotides 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 pre-vents 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-1´s 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 physiologi-cal 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 poten-tial targets for anticancer therapy. The aptamers are therefore potential agents for develop-ment of novel anticancer therapeutics and diagnostics.

Daniel M. Dupont, PostDocDepartment of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, DenmarkTelephone: +45 89 42 50 79 Email: [email protected]

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clIcK NuclEIc AcID lIGATIONA.H. El-Sagheer1, 2 and T. Brown1

1School of Chemistry, University of Southampton SO17 1BJ. UK2Chemistry 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 pro-duces 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 de-oxyribose 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 high-resolution 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, PhDChemistry Department, University of SouthamptonHigh Field, Southampton, SO17 1BJ, UKTelephone: +44(0)2380596785Email: [email protected]

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NON-vIRAl DElIvERY OF STIcKY-SIRNA™ TARGETING THE cEll cYclE INHIBITS luNG TumOR mETASTASISAnne-Laure Bolcato-Bellemina, Marie-Elise Bonneta, Omar Zouniba, Elodie Benoita, Jean-Paul Behrb and Patrick Erbachera

a Polyplus-transfection, Bioparc, Boulevard Sébastien Brandt, 67401 Illkirch, Franceb Laboratoire de Chimie Génétique, Université de Strasbourg, Faculté de Pharmacie, 67401 Illkirch, France

Cancer is a leading cause of death worldwide, accounting around 13% of all death. Among can-cers, lung cancer is the most common in term of incidence and mortality. Metastasis, due to uncon-trolled 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 siRNA-based drugs to be a success. Polyethylenimine (PEI) has been widely used for in vivo gene delivery. We have recently developed novel interfering RNA (STICKY-SIRNA™) 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-β, 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 attrac-tive strategy for antiproliferative cancer therapy.We have analyzed the impact of suppression of cyclin B1 by delivering STICKY SIRNA™ with linear polyethylenimine in different cancer cell lines (TSA-Luc, B16-F10 and 3LL-Luc). We have shown that cyclin B1 STICKY SIRNA™ arrested the cell cycle in G2/M phase in all cancer cell lines tested. Consequently, the ability of STICKY SIRNA™ to sup-press cell metastasis in the lung was investigated. By injecting cyclin B1 STICKY SIRNA™ 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 SIRNA™ 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 SIRNA™ with linear polyethyl-enimine could become a powerful tool for antitumor therapy.1 Bolcato-Bellemin et al., PNAS, 2007, 104, 16050-5.2 Bonnet et al., Pharm Res, 2008, 25, 2972-82.

Anne-Laure Bolcato-Bellemin, PhDPolyplus-transfection BioparcBoulevard Sébastien Brandt67401 Illkirch, FranceTelephone: +33 3 90 40 61 80Email: [email protected]

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SElF-DElIvERING cATIONIc siRNAs mEDIATE EFFIcIENT AND SPEcIFIc GENE SIlENcING IN HumAN cEllSPatrick Erbacher, Valérie Moreau, Clément Paris, Gaëlle Deglane, Bernard Couturier, Anne-Laure Bellemin, Marie-Elise Bonnet, Jean-Serge Remy*, Jean-Paul Behr*, Nathalie LennePolyplus-transfection, Illkirch, France*Laboratoire de Chimie Génétique, Faculté 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 cul-ture. 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 mech-anism 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, PhDPolyplus-transfection SA, BIOPARC, Boulevard Sébastien Brant, BP 90018F-67401 Illkirch, FranceTelephone: +33 (0)390 406 180Email: [email protected]

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REcEPTOR-mEDIATED uPTAKE OF OlIGONuclEOTIDE NANO-cOmPlExES WITH cEll-PENETRATING PEPTIDES AND THEIR FORmulATION INTO A PHARmAcEuTIcAllY STABlE SOlID DOSAGE FORmKariem Ezzat, Henrik Helmfors and Ülo LangelDepartment of Neurochemistry, Stockholm University, Stockholm, Sweden

A new generation of cell-penetrating peptides (CPPs), PepFects, is capable of forming non-covalent 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 scav-enger 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 ͦ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 CPP-based delivery systems and demonstrate a novel technology to formulate such delivery sys-tems into active and stable pharmaceutical dosage forms.

Kariem Ezzat, PhD studentDepartment of Neurochemistry, Stockholm University, SE-106 91Stockholm, SwedenTelephone: 0046700674381Email: [email protected]

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INHIBITION OF cOmPlEmENT c6 SYNTHESIS IN THE lIvER uSING ANTISENSE OlIGONuclEOTIDES AFFEcTS NEuRO-REGENERATIONKees Fluiter, Valeria Ramaglia, Frank BaasAcademic 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 oligonucle-otides 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 en-cephalomyelitis (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, PhDAMC, K2-210Meibergdreef 9, 1105 AZ AmsterdamThe NetherlandsTelephone: +31205663746Email: [email protected]

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SuPPRESSION OF BcR/ABl GENE ExPRESSION BY SIRNA cONjuGATESMasayuki Fujii, Diala IrminaDepartment 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-3‘siRNA (anti bcr/abl mRNA361-381)sense: NES-5’-GCAGAGUUCAAAAGCCCUUTT-3’antisense: 3’-TTCGUCUCAAGUUUUCGGGAA-5’NES Peptides:c1: βALVLDKLTI-OH (TFIIIA NES)c2: βALPPLERLTL-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 hu-man 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, siRNA-NES conjugates could not penetrate the cellular membrane by itself and required a transfec-tion 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.

Reference1. 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, PhD11-6 KayanomoriIizuka, Fukuoka 820-8555 JapanTelephone: +81-948-22-5655-210Email: [email protected]

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INFluENcE OF THE TERmINAl BASE PAIRS OF 5’-PPP-RNA ON RIG-I lIGAND AcTIvITYM. 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 antivi-ral 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 re-quirement 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 hu-man 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 structur-al 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 studentInstitute for Clinical Chemistry and Clinical PharmacologyUniversity Hospital BonnSigmund-Freud-Str. 2553105 Bonn, GermanyTelephone: +49-0228-287-51157Email: [email protected]

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ANTISENSE OlIGONuclEOTIDES: PROmISING TOOlS FOR mOlEculAR THERAPY OF mYOTONIc DYSTROPHYAnchel 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, Bé 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 progres-sion of the disease. DMPK RNA transcripts with long (CUG)n repeats are retained in the nu-cleus, where they form hairpin-like structures which are involved in the entrapment and deple-tion 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, con-tributing 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 de-rived 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 tran-scripts 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 produc-tion 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, MScDept. of Cell BiologyRadboud University Nijmegen Medical Centre Nijmegen Centre for Molecular Life Sciences Geert Grooteplein 26/286525 GA NijmegenThe NetherlandsTelephone: +31.24.36 13664Email: [email protected]

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REScuE OF SEvERElY AFFEcTED DYSTROPHIN/uTROPHIN DEFIcIENT mIcE BY vEcTORIZED ANTISENSE mEDIATED ExON SKIPPINGGoyenvalle 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. Antisense-mediated 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 treat-ment results in near-normal levels of dystrophin expression in all muscles examined, includ-ing 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 exon-skipping in systemic treatment of the DMD phenotype.

Aurelie Goyenvalle, PhDMRC Functional Genomics UnitDepartment of Physiology, Anatomy and GeneticsUniversity of OxfordSouth Parks RoadOxford OX1 3QX, UKTelephone: +44 (0)1865 285 864Email: aurelie.goyenvalle @dpag.ox.ac.uk

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Photograph of a dKO control mouse (left) and a scAAV9-U7dtex23 treated dKO mouse (right) at 12 weeks of age

TARGETING PIm-1 BY mIR-33a IN cOlON cARcINOmA THROuGH PEI-mEDIATED mIRNA REPlAcEmENT THERAPY Arnold Grünweller, Ahmed Fawzy Ibrahim, Maren Thomas, Kerstin Lange-Grünweller, 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 muta-genesis 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. Nota-bly, 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-145-mediated 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 Grünweller, PhDPharmazeutische Chemie, Marbacher Weg 6, 35037 Marburg, GermanyTelephone: 0049-6421-2825849Email: [email protected]

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SElEcTIvE INTERSTRAND cROSS-lINKING THROuGH IN SITu OxIDATION OF FuRAN-mODIFIED OlIGODEOxYNuclEOTIDESE. Gyssels, M. Op de Beeck, A. MadderLaboratorium 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 oligo-nucleotides. 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 GysselsLaboratorium for organic and biomimetic chemistryKrijgslaan 281, S4, 9000 Gent Belgium Telephone: +3292644479Email: [email protected]

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THE uSE OF ExPRESSION PROFIlES TO ASSESS NON- HYBRIDISATION mEDIATED EFFEcTS OF PHOSPHOROTHIOATE ANTISENSE OlIGONuclEOTIDESPeter H Hagedorn, Andreas Petri, Sakari Kauppinen, Morten LindowSantaris Pharma A/S, Kogle Alle 6, DK-2970 Hørsholm, Denmark

We have initiated systematic investigations of general class effects of LNA-modified phos-phorothioate 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 trans-fection, 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 sub-sets of experiments and correlate these to experimental setup and oligonucleotide design. Ul-timately 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, PhDSantaris Pharma A/SKogle Allé 6DK 2970 HørsholmDanmarkTelephone: +45 4517 9919Email: [email protected]

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A mIcRORNA-mEDIATED REGulATORY FEEDBAcK lOOP IN A NEW mEcHANISm OF RESISTANcE TO TGF-BETA INDucED GROWTH INHIBITIONJonathan Hall, Afzal M. Dogar, Harry TowbinInstitute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 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-β and yet they are usually resistant to TGF-beta-induced growth inhibition. We found that suppression of TGF-β1 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 in-cluded included transiently increased SMAD2 and AKT phosphorylation. MicroRNA expres-sion 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 fu-rin 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, PhDInstitute of Pharmaceutical SciencesHCI H 437Wolfgang-Pauli-Str. 108093 Zürich, SwitzerlandTelephone: 044 633 74 35Email: [email protected]

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NOvEl cEll PENETRATING PEPTIDES FOR SKElETAl AND cARDIAc muSclE DElIvERY OF PmO ANTISENSE OlIGONuclEOTIDES FOR THE TREATmENT OF DucHENNE muSculAR DYSTROPHYSuzan 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

1Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; 2Tian-jin Research Centre of Basic Medical Science, Tianjin Medical University, Tianjin, China; 3Medical 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 respira-tory 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 tran-scripts. Dystrophin is unique in that the central portion of the gene encodes a large rod do-main made up of twenty four large spectrin-like repeats and four flexible hinge regions. Dele-tions 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 pep-tides. 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 fi-bres 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 de-velopment 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 ad-vancing 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, PhDOxford UniversityDepartment of Physiology, Anatomy and GeneticsLe Gros Clark BuildingSouth Parks RoadOxford OX3 1QX United KingdomTelephone: 01865 272167Email: [email protected]

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RETINOIc AcID- INDucIBlE GENE-I (RIG-I) DETEcTS (TRIPHOSPHORYlATED) RNA OF Listeria monocytogenes DuRING INFEcTIONAnna 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-531272. 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 recep-tors (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 bacte-rial DNA or second messengers such as cyclo-di-AMP. We hypothesized that besides bacte-rial 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 studentSigmund-Freud-Str 2553127 Bonn, GermanyTelephone: +49 (0)228 287 51157Email: [email protected]

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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 phar-macological effect of miravirsen (as miR-122’s major function is the fine-tuning of lipid metab-olism), 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 treatment-free 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/sKogle Allé 6, 2970 Hoersholm, DenmarkTelephone: +45 4517 9800Email: [email protected]

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DNA-cONTROllED ASSEmBlY OF lIPOSOmESUlla Jakobsen and Stefan VogelNucleic 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, comple-mentary 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 re-versible, and the aggregates break down to the individual liposomes when heated to above the melting 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.1Jakobsen, U.; Simonsen, A. C.; Vogel, S. J. Am. Chem. Soc. 2008, 130, 10462-10463.2Jakobsen, U.; Vogel, S. Methods Enzymol. 2009, 464, 233-248.

Ulla Jakobsen, PhDCampusvej 55, 5230 Odense M, DenmarkTelephone: +45 65502599Email: [email protected]

Figure 1 Schematic representation of assembly of liposomes controlled by triple helix formation. TFO (red ribbon), double helix (blue ribbons) and liposomes (grey spheres) are not drawn to scale.

ENZYmATIc SYNTHESIS OF OlIGONuclEOTIDES WITH 2’-AmINO-lNA TRIPHOSPHATES AND TEmPlATESMarie 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 par-tially 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 aptam-ers 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 re-ceived 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 oli-gonucleotides 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 increas-ing the chemical diversity of DNAzymes and aptamers.

1 Doessing, H.; Vester, B. Locked and Unlocked Nucleosides in Functional Nucleic Acids. Molecules 2011, 16, 4511-4526.

2 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

Marie Willaing Johannsen, MChem, MSc.University of Southern DenmarkCampusvej 555230 Odense MDenmarkTelephone: +45 6550 2548Email: [email protected]

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lIPID NANOPARTIclE FORmulATIONS OF mINImAl-lENGTH shRNAS SHOW POTENT INHIBITION OF Hcv-DRIvEN, lIvER-SPEcIFIc GENE ExPRESSION IN mIcEB. H. Johnstona,e, A. Dallasa, H. Ilvesa, J. Shorensteina, M. A. Behlkeb, SP Wonge, R. Harbottlec, and I. MacLachland aSomaGenics, Inc., 2161 Delaware Ave., Santa Cruz, CA 95060, USA bIDT, Coralville, IA 52241, USA cImperial College London, UK dTekmira Pharmaceuticals, Burnaby, B.C., Canada eDept. 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 subsequent-ly 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 previ-ously 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 inhi-bition 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.

1Q. Ge, et al. (2010) RNA 16(1):106-17; 2Q. Ge, et al. (2010) RNA 16(1):118-30.

Brian Johnston, PhDSomaGenics, Inc.2161 Delaware AvenueSanta Cruz, CA 95060 USATelephone: (831) 426-7700 ext. 12Email: [email protected]

HIGHlY POTENT, mINImAl-lENGTH shRNAS TARGETING HEPATITIS c vIRuS HAvE NOvEl mEcHANISTIc FEATuRESB. H. Johnstona,d, A. Dallasa, H. Ilvesa, P. Kumara, S. A. Kazakova, J. Shorensteina, M. A. Behlkeb, M. McManusc, and Q. Gea aSomaGenics, Inc., 2161 Delaware Ave., Santa Cruz, CA 95060, USA bIDT, Coralville, IA 52241, USA cUCSF Diabetes Center, University of California, San Francisco, CA 94143, USA dDept. 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 non-nucleotide 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 posi-tion 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.

Q. Ge, H. Ilves, A. Dallas, P. Kumar, J. Shorenstein, S. A. Kazakov, and B. H. Johnston (2010) 1. Minimal-length short hairpin RNAs: The Relationship of Structure and RNAi Activity. RNA 16(1):106-17.Q. Ge, A. Dallas, H. Ilves, J. Shorenstein, M. A. Behlke, and B. H. Johnston (2010) Effects of 2. Chemical Modification on the Potency, Serum Stability, and Immunostimulatory Properties of Short shRNAs. RNA 16(1):118-30.

Brian Johnston, PhDSomaGenics, Inc.2161 Delaware AvenueSanta Cruz, CA 95060 USATelephone: (831) 426-7700 ext. 12Email: [email protected]

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EPATOcYTE SPEcIFIc TARGETING AND DElIvERY OF siRNA-cARBOHYDRATE cONjuGATESKallanthottathil 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. ZimmermannAlnylam Pharmaceuticals, Cambridge, MA, USA

The asialoglycoprotein receptor (ASGPR) is a transmembrane receptor that mediates clear-ance 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, PhD300 Third Street, Cambridge, MA 02142 USATelephone: 1 617 551 8313Email: [email protected]

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lNA ANTISENSE OlIGONuclEOTIDES - A STRAIGHTFORWARD cONcEPT FOR RNA THERAPEuTIcSSusanne Kammler¹, Niels Fisker Nielsen¹, Marie Lindholm¹, Robert E. Lanford², Andreas Petri¹, Sakari Kauppinen¹, Nathalie Uzcategui¹, Ellen Marie Straarup¹, Joacim Elmén¹, Troels Koch¹, Henrik Ørum¹, Maj Hedtjärn¹, Bo Rode Hansen¹ ¹Santaris Pharma A/S, Hørsholm, Denmark ²Department 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. Oligo-nucleotide 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 tis-sues. 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 regula-tion into tomorrow’s RNA therapeutics.

Susanne Kammler, Research Scientist, Technology & Partnered discoverySantaris Pharma A/SKogle Allé 6DK 2970 HørsholmDenmarkTelephone: +45 4517 9915Email: [email protected]

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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 mus-cular 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 signifi-cantly increased muscle mass relative to their normal counterparts. Several strategies are be-ing 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 (vi-vo-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 candidateSchool of Biological Sciences, Royal Holloway, University of London,Egham, Surrey, United Kingdom, TW20 0EXTelephone: 7895709958Email: [email protected]

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PREclINIcAl DEvElOPmENT OF AN SD-RXRNATm BASED THERAPY FOR TREATmENT OF FIBROSIS AND RETINAl DISORDERSAnastasia 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 identi-fied 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 silenc-ing 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, PhDRXi Pharmaceuticals60 Prescott StreetWorcester, MA 01605 USATelephone: 508-929-3616Email: [email protected]

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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, result-ing 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 hTERT-targeting 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 can-cer- 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 miR-NA181a 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 induc-tion. We observed that transgene induction by the ribozymes was specifically shown in can-cer 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 University126, Jukjeon-dong, Suji-gu Yongin 448-701, KoreaTelephone: 82-31-8005-3203Email: [email protected]

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HINT-1 HYDROlASE-ASSISTED clEAvAGE OF INTERNuclEOTIDE BONDS IN DINuclEOSIDE PHOSPHOROTHIOATES SuGGESTS ITS POSSIBlE PARTIcIPATION IN mETABOlISm OF OlIGONuclEOTIDE DRuGS AND PRODRuGSAgnieszka Krakowiak, Beata Rębowska, Milena Sobczak, Barbara Mikołajczyk,Wojciech J. StecCentre 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 cleav-age of internucleotide bond in dinucleotide phosphates and thiophosphates, and the products are corresponding nucleosides as well as nucleoside 5’-phosphates or –phospho-rothioates. 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, 709-7103 M. Ozga, Dolot R, Janicka M, Kaczmarek R, Krakowiak A. (2010) J Biol Chem. 285, 40809-18

Agnieszka Krakowiak, PhDPOLAND, 90-363 Lodz, Sienkiewicza 112Telephone: +4842 6803272 Email: [email protected]

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2’-BISPYRENE-mODIFIED OlIGO(2’-O mETHYlRIBONuclEOTIDE) PROBES AS TOOlS FOR THE SENSITIvE DETEcTION OF RNAKrasheninina 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 fluoro-phore 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'-O-methylribonucleotides) 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 «inverted» 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 66°C for DNA-targets and from 67 to 87°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'-bispyrene-labeled 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 con-stants 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 student630090, Lavrentiev ave. 8, Novosibirsk, Russia Telephone: 007(383)3635129 Email: [email protected]

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SYNTHESIS OF 2’-AmINO-uNA Niels Langkjær, Jesper WengelUniversity 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 (un-locked 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’-amino-UNA 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 previ-ously 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:Bramsen, J. B., Laursen, M. B., Nielsen, A. F., Hansen, T. B., Bus, C., Langkjaer, N., Babu, B. 1. R., Højland, 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., Müller, 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., Langkjær, N., Odadzic, D., Smicius, R., 2. Wengel, S. L., Chattopadhyaya, J., Engels, J. W., Herdewijn, P., Wengel, J., Kjems, J., Nucleic Acids Res. 2010, 38 (17), 5761-5773.Langkjær, N., Pasternak, A., Wengel, J., 3. Bioorg. Med. Chem. 2009, 17 (15), 5420-5425.

Niels Langkjær, Cand. Scient. ChemistryCampusvej 55, 5230 Odense M, DenmarkTelephone: +45 6550 2548Email: [email protected]

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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 ap-proach 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 in-hibiting 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 miR-122 target proteins were specifically stimulated only in HCV replicon Huh-7 cells, but not in naïve 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 repli-cating 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 BiotechnologyDankook University, Yongin 448-701, Republic KoreaTelephone: 82-31-8005-3203Fax: 82-31-8005-4058Email: [email protected]

*[email protected]

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ARE SHORT OlIGONuclEOTIDES lESS SPEcIFIc in vivo?Morten Lindow, Peter Hagedorn, Andreas PetriSantaris Pharma A/S, Kogle Alle 6, DK-2970 Hørsholm, 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 speci-ficity. 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 LNAphospho-rothioate oligonucleotides.

Morten Lindow, PhDSantaris Pharma A/SKogle Alle 6, DK2970-Horsholm, DenmarkTelephone: +45 2849 9682Email: [email protected]

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mEcHANISTIc INSIGHTS INTO lIPID NANOPARTIclE-mEDIATED DElIvERY OF siRNAMartin 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, USA2 Alcana Technologies, Vancouver, BC, Canada V6L 2A13 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 devel-opment 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 num-ber 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 nanopar-ticles, 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 perma-nent 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 siR-NA 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 facili-tate further rational optimization of this siRNA delivery platform.

Martin A. Maier, PhD300 Third StreetCambridge, MA 02142, USATelephone: +1-617-551-8274Email: [email protected]

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cHARAcTERISTIc ASPEcTS OF SKElETAl muSclE AS A TARGET ORGAN FOR siRNANaoki Makita1, Ichiro Fujimoto2, Sachiko Fukuda1, Takeshi Shimizudani1, Shunji Nagahara1

1DDS Research Group, Formulation Research & Development Laboratories, Technology Research & Development, Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan 2Research 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 acces-sible 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 atelocolla-gen 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 skel-etal 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 MakitaDDS Research Group, Formulation Research & Development Laboratories, Technology Research & Development, Dainippon Sumitomo Pharma Co., Ltd.3-45, Kurakakiuchi 1-Chome, Ibaraki-shi, Osaka 567-0878, JapanTelephone: +81-72-627-8146Email: [email protected]

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AcTIvATION OF cYclOOxYGENASE-2 (cOx-2) GENE ExPRESSION BY PROmOTER-TARGETED DuPlEx RNAsMasayuki 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, USA2 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 mecha-nism 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 prosta-glandin biosynthesis, and COX-2 gene expression is associated with tumorigenesis, inflam-mation, 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, du-plex 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 poly-merase 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 agR-NAs. We performed RACE and strand-specific RT-qPCR to examine whether similar non-coding 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 in-terferon 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 sug-gest 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, PhDUniversity of Texas Southwestern Medical Center at Dallas6001 Forest Park Road Dallas, TX 75390-9041 USATelephone: +1-214-645-6154Email: [email protected]

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Abstract and poster available for review at the meeting.

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ANTISENSE OlIGONuclEOTIDES FOR FuNcTIONAl STuDIES OF HumAN ARGONAuTE PROTEINSMescalchin A. and Innarella M.R.Institute of Molecular Medicine, University of Lübeck, Center for Structural and Cell Biology in Medicine (CSCM), Ratzeburger Allee 160, D-23538 Lübeck, Germany

The Argonaute proteins play important regulatory roles in the metabolism of cells and are in-volved 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 siRNA-mediated 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, PhDInstitute of Molecular Medicine, University of LübeckRatzeburger Allee 160, 23538 Lübeck, GermanyTelephone: +49 (0)451 500 2742 or +49 (0)451 500 2736 Email: [email protected]

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SENSE OlIGONuclEOTIDE-mEDIATED NATRE TEcHNOlOGY cONTROlS lEvElS OF cYTOKINE mESSENGER RNAMikio Nishizawa1, Emi Yoshigai1, Masaki Kaibori2, Tadayoshi Okumura2,3, A-Hon Kwon2 1Department 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 oligodeoxyribonucle-otides (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 Tran-script-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, PhD1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, JapanTelephone: 81-77-561-2876Email: [email protected]

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SIlENcING OF microRNA FAmIlIES BY SEED-TARGETING TINY lNAsSusanna Obad1, Andreas Petri1, Oliver Broom1, Markus Heidenblad1, Jan Stenvang1, Ellen Marie Straarup1, Troels Koch1, Henrik Frydenlund Hansen1 and Sakari Kauppinen1,2

1Santaris Pharma, Kogle Allé 6, DK-2970 Hørsholm, Denmark2Copenhagen 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 perturba-tions 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 disor-ders. 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 single-stranded 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 pharma-cological inhibition of disease-associated miRNAs.

Susanna Obad, PhDSantaris Pharma A/SKogle Allé 6DK 2970 HørsholmDanmarkTelephone: +45 45 179893Email: [email protected]

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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

1The 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 caus-ing 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 mo-lecular 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 func-tion 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 el-evates 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 (10.9±4.2 compared to 6.3±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-pro-tected 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, MScThe Hebrew UniversitySoreq Lab, Safra CampusJerusalem 91904, IsraelTelephone: 972-2-6585450Email: [email protected]

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PlASmA PHARmAcOKINETIcS OF mIRAvIRSEN IN HumANSPersson R, Bagger Y, Børgesen H, Hodges, MR, King BD, Levin AASantaris Pharma A/S, Hørsholm, 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 (t½=38 days). Comparison of AUCs following s.c. and i.v. doses indicated bioavailability was 100%. The terminal PK parameters (t½, 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 (± 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 (± 14) L/kg, indicating a distribution into a deeper compartment e.g. liver and kidney. Mean terminal plasma half-life was 38.3 (± 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, PhDSantaris Pharma A/SKogle Allé 6, 2970-Hørsholm, DenmarkTelephone: +45 4517 9947Email: [email protected]

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TOWARDS EvAluATION OF POINT-muTATIONS AND DRuG INTERFERENcE WITH miRNA REGulATED ExPRESSION OF BRcA1Christopher Polonyi, Sofi K.C. ElmrothBiochemistry 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 eukary-otic 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 in-terference. Potential drug candidates are used to in-vestigate 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 luciferase-based 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 studentDepartment of Biochemistry and Structural BiologyCenter for Molecular Protein Science, Lund UniversityPO Box 124, SE-22100 Lund, SwedenTelephone: +46 46 2228108 Email: [email protected]

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lEPIGENETIc mYOSTATIN BlOcKADE1Thomas Roberts, 1Graham McClorey, 1Samir EL-Andaloussi, 2Kevin Morris and 1Matthew Wood1 Department of Physiology, Anatomy and Genetics, University of Oxford, OX1 3QX, UK2 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA

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 myosta-tin 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 my-ostatin promoter. These were transfected in differentiated C2C12 mouse myotubes. Two siR-NAs resulted in significant myostatin silencing. Knock-down was observed in multiple trans-fection 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 allevi-ated 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, MBiochemDepartment of Physiology, Anatomy and GeneticsUniversity of OxfordLe Gros Clark BuildingSouth Parks RoadOxfordOX1 3QX, United KingdomTelephone: +44 (0)1865 272167Email: [email protected]

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A NEW TOOl FOR SITE-SPEcIFIc cHEmIcAl mODIFIcATION OF RNAShigeki 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 Biotin-modification 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.

Shigeki Sasaki, PhDGraduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, JapanTelephone and Fax: +80-92-642-6615Email: [email protected]____________________________1 (a) Bioconjugate Chem. 2009, 20, 799-803, (b) Nucleic Acids Res. 2010, 38, 1760-1766, (c) Biocon-jugate Chem. 2010, 21, 1508-1512, (d) Chem. Comm., 2011, 47, 5004-5006

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Fig. 1 The functionality transfer reaction (FTR) for modification of the 2-amino group of guanosine of the internal RNA

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. Kümmerer3, Gunther Hartmann1 and Martin Schlee1 1 Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Germany2 Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany3 Institute 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 ubiqui-tously 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 activa-tion at all. By contrast, a single 2′O-methyl group at the 5’-penultimate residue (N1), a charac-teristic 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 main-ly the 2’O-methylation at N1 mediated inhibition of binding of capped RNA to RIG-I. Stud-ies 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 triphosphate-containing RNA.

Christine Schuberth, Institute for Clinical Chemistry and Clinical PharmacologyUniversity Hospital BonnSigmund-Freud-Str. 2553105 Bonn, GermanyTelephone: +49-228-287-51157Email: [email protected]

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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 ge-nomic loci that contain the repressor element-1 (RE1) binding motif and recruits a series of epige-netic 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 treat-ment 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 admin-istration 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 Ingenuity™ Pathway Analysis (IPA). 416 genes were found to be up regulated and 119 genes down regulated following REST ASO treatment. Numerous novel genes were iden-tified 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, PhDISIS Pharmaceuticals Inc. 2855 Gazelle CourtCarlsbad, CA 92010 USATelephone: Brett Monia: 760-603-2350Email: [email protected]; [email protected]

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FuNcTIONAl ANAlYSIS OF microRNAs AS REGulATORS OF cARGO TRAFFIcKINGAndrius Serva, Sanchari Roy, Bettina Knapp, Lars Kaderali, Holger Erfle, Ursula Kummer, Roland Eils and Vytaute StarkuvieneBioQuant, 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 expres-sion of nearly 60% of protein-coding mammalian genes. Thereby, miRNAs affect a multitude of biological processes such as developmental transition, cell proliferation and apoptosis. Al-teration 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 oligonucle-otides 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 chang-es of cargo trafficking. Further examination of primary miRNA hits by combined microsco-py-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 com-prehensive understanding of how these networks coordinate the activity of secretory mem-brane trafficking as a global adaptive response system is needed to study miRNAs as promis-ing therapeutic agents and targets under particular pathological conditions.

Andrius Serva, MScBioQuant, University of HeidelbergIm Neuenheimer Feld 26769120 Heidelberg, GermanyTelephone: +49-6221-5451258Email: [email protected]

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SIlENcING THE cDK4 AND cDK6 GENES THROuGH RNA INTERFERENcE Małgorzata Sierant, Danuta Piotrzkowska and Barbara NawrotCentre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz, Poland

Alzheimer’s disease (AD), a disorder which affects millions people worldwide, is charac-terized 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 neu-ronal death in AD have been the objective of intense studies and several concepts were pro-posed [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 protei-ns expression, as cyclin dependent kinases, cyclins and cdk inhibitors was observed in patho-logically 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 low-ered. 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 se-lected cells transfected with these constructs the lowering of CDK4 and CDK6 genes expres-sion 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, PhDCMMS PAS, Lodz 90-363, Sienkiewicza 112, PolandTelephone: (+48 42) 680-32-72Email: [email protected]

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ImPROvED STABIlITY AND GENE SIlENcING AcTIvITY OF siRNA ANAlOGS cONTAINING PHOSPHORODITHIOATE lINKAGESMalgorzata Sierant 1, Magdalena Janicka 1, Xianbin Yang 2, Na Li 2 and Barbara Nawrot 1

1Centre 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 oli-gomers 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 β-secretase (BACE1) and enhanced green fluorescent protein (EGFP) transcripts [1,2]. CD spectra collected for PS2-siRNA duplexes are similar to the spectra of the unmodified duplex, consistent with the typi-cal 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 essen-tially 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 experi-ments 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 re-search 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, PhDCMMS PAS, Lodz 90-363, Sienkiewicza 112, PolandTelephone: (+48 42) 680 32 72Email: [email protected]

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DIcER-SuBSTRATE siRNA ExHIBIT ImPROvED GuIDE STRAND SElEcTION AND STRONGER RISc lOADING cOmPlEx FORmATION cOmPARED TO cANONIcAl siRNANicholas M. Snead, Kumi Sakurai, John J. RossiIrell 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 applica-tions, 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 cri-teria, to improve loading of the guide strand into the RNAi machinery, reduce off-target ef-fects, 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 as-says. 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’ triphos-phate 19+2 siRNAs generated by T7 in vitro transcription did not silence reporter gene ex-pression; 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, MSBeckman Research Institute at the City of Hope1500 E. Duarte Road, Fox N 2002HDuarte, CA, 91010 USATelephone: (626) 256-4673, ext. 64457Email: [email protected]

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FINDING cANcER THERAPEuTIc sgRNAs BASED ON TRuE GENE SIlENcINGM. Takahashi1, S. Matsuda1, Y. Watabe1, A. Goto1, D. Kamiya1, T. Sano1, N. Watanabe2, M. Narita2, M. Takahashi2, M. Tamura3, M. Nashimoto1

1Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan, 2Niigata University, Niigata, Japan, 3Hokkaido University, Sapporo, Japan

tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is one of the RNA-me-diated 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 sgR-NAs 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 xe-nograft 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, MasterHigashijima 265-1, Akihaku, Niigata, Niigata 956-8603, JapanTelephone: +81 250 25 5118Email: [email protected]

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KINETIc cOmPARISON OF GENE SIlENcING PROFIlE OF siRNA AND SHRNA-ExPRESSING PlASmID DNA IN vIvOYuki Takahashi, Yuriko Matsui, Makiya Nishikawa, Yoshinobu TakakuraGraduate 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 introduc-ing 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 develop-ment 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 RNAi-based 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 con-trol 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 shR-NA-expressing pDNA targeting Gaussia luciferase at varying doses by the hydrodynamic in-jection 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 quan-titative analysis of the gene silencing profiles based on a moment analysis confirmed the find-ing 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, PhD46-29 Yoshidashimoadachi-choSakyo-ku, Kyoto 606-8501, JapanTelephone: +81-75-753-4616Email: [email protected]

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EFFEcT OF GuANINE BASES mODIFIcATIONS AND l-DNA ON STABIlITY AND KINETIc OF FORmATION OF TETRAmOlEculAR quADRuPlExESPhong Lan Thao Tran1*, Antonella Virgilio2, Veronica Esposito2, Giuseppe Citarella2, Aldo Galeone2, Rui Moriyama3, Atsushi Maruyama3, Bernard Rayner1 and Jean-Louis Mergny1

1 Institut Européen de Chimie et Biologie, INSERM U869, Université de Bordeaux (France) 2 Università degli Studi di Napoli Federico II, Napoli, Italy3 Institute for Materials Chemistry and Engineering, Kyushu University, Fukuoka, Japan

Tetramolecular G-quadruplexes result from the association of four guanine-rich strands. These struc-tures 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 oligonucle-otide 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 8-methyl Guanine D-Guanine l-Guanine G-quadruplex

References:[1] J. Gros, et al., Chembiochem 2008, 2926-2928.[2] P.L.T. Tran, et al., Biochimie 2011, 93(3):399-408.[3] S.G. Sayyed, et al., Diabetologia 2009, 52, 2445-54.[4] P.L.T. Tran, et al. Chem. Commun. 2011, 47, 5437-5439.[5] B. Wlotzka, et al., Proc Natl Acad Sci USA 2002, 99, 8898-902.[6] Y. Kim, et al., Nucleic Acids Res 2007, 35, 7279-87. [7] M.S. Kaucher, et al., J. Am. Chem. Soc. 2006, 128, 38-39.[8] C. Lin, et al. Nano Lett. 2009, 9, 433-436.

Phong Lan Thao Tran2, rue Robert Escarpit, 33607 Pessac FranceTelephone: +33 6 27 12 15 57Email: [email protected]

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TARGETING TumOR ANTIGENS TO DENDRITIc cEllS in vivo uSING REcEPTOR-SPEcIFIc APTAmERS cONjuGATED TO TumOR ANTIGENJohannes Urban, David Boczkowski, Kyle Kok Loong Phua, Bruce Sullenger and Smita NairDepartment 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 antibod-ies, 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 2´Fluoro-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, PhDDuke University Medical Center106 Research Drive, Durham, NC 27701 USATelephone: 919-684-6780Email: [email protected]

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DESIGNED ANKYRIN REPEAT PROTEINS FOR TARGETED DElIvERY OF siRNA OlIGONuclEOTIDESJohannes Winkler1, Claudia Böhme1, Nikolas Stefan2, Andreas Plückthun2, Uwe Zangemeister-Wittke2, 3

1University of Vienna, Department of Medicinal Chemistry, Vienna, Austria2University of Zürich, Department of Biochemistry, Zürich, Switzerland3University of Bern, Institute of Pharmacology, Bern, Switzerland

Designed Ankyrin Repeat Proteins (DARPins) are a novel class binding proteins with prom-ising 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 fu-sion 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 expres-sion 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 de-livery 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, PhDUniversity of Vienna, Department of Medicinal ChemistryAlthanstraße 14, 1090 Vienna, AustriaTelephone: +43 1 4277 55058Email: [email protected]

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POTENcY cOmPARISON OF SERIES OF BNA-BASED ANTISENSES IN DYSlIPIDEmIc ANImAlSTsuyoshi 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 phosphorothioat-ed 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 ef-ficacious 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 student1-6 Yamadaoka, Suita, Osaka 565-0871, JapanTelephone: +81-6-6879-8202Email: [email protected]

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OlIGONuclEOTIDE cONjuGATES AS INHIBITORS OF HIv-1 INTEGRASE AND REvERSE TRANScRIPTASET. Zatsepin,1,2 J. Agapkina,1 S. Korolev,1 M. Gottikh1 1Department of Chemistry and Belozersky Institute of Physical and Chemical Biology, Lomonosov Moscow State University, Moscow, Russia2Central 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 activ-ity 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 11-mer 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 (IC50 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, PhDDepartment of Chemistry, Lomonosov Moscow State UniversityLeninskie gory 1-3, 119991 Moscow, RussiaTelephone: +7-495-939-3148Email: [email protected]

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INFluENcE OF RNA SEcONDARY STRucTuRE ON THE AcTIvATION OF TOll-lIKE REcEPTORS 7 AND 8Thomas Zillinger1*, Vera Wimmenauer1*, Eicke Latz2, Gunther Hartmann1, Winfried Barchet1

1Institute 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 en-coded 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 sti-mulatory 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 ac-tivators 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 re-cognition 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]

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RIG-I lIGAND TRIPHOSPHATE RNA (3pRNA) POTENTIAllY INHIBITS YEllOW FEvER vIRuS REPlIcATION IN TYPE 1 INTERFERON DEPENDENT mANNER1Mohammad Intakhab Alam, 1Christine Schuberth, 1Thomas Zillinger, 1Christoph Coch 1Win-fried Barchet, 1Jasper van den Boorn, 1Debby van den Boorn-Konijnenberg, 2Beate Kuemmerer, 1Martin Schlee and 1Gunther Hartmann1) Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Germany and 2) Institute for Virology, University Hospital Bonn, Germany

RIG-I has been shown to be involved in sensing every members of the flavivirus genus. Yel-low Fever Virus (YFV) possesses mechanism to overcome innate immunity. Here we pro-pose 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 replica-tion 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 defi-cient Vero cells. However, addition of rec-IFNβ 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 upregu-lated 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 depen-dent. 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-IFNβ with 3pRNA induced IFN-β 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, PhDInstitute for Clinical Chemistry and Clinical PharmacologySigmund-Freud-Str. 25D-53127 Bonn, GermanyTelephone: +49-228-28751150 Email: [email protected]

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GlucAN PARTIclES FOR SElEcTIvE DElIvERY OF siRNA TO PHAGOcYTIc cEllS IN mIcEMyriam 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. CzechProgram in Molecular Medicine. University of Massachusetts Medical School, Worcester, MA, USA

Phagocytic macrophages and dendritic cells are desirable targets for potential RNAi thera-peutics 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 dis-covered 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 mac-rophages, but not other cell types, and released the labeled siRNA into the macrophage cyto-plasm. 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 cata-lyze escape of siRNA from phagosomes. Thus we have developed a simplified siRNA deliv-ery system that effectively and specifically targets phagocytes in culture or in intact mice.

Myriam Aouadi, PhD373 Plantation st, Biotech II, suite 100Worcester, MA, 01605 USATelephone: 1-508-856-6858Email: [email protected]

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DEvElOPmENT OF AN RNA APTAmER FOR HumAN TOll-lIKE REcEPTOR 2 EcTODOmAINJonghoe Byun, Ji-hae YoonDepartment 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 im-munity 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, PhD126 Jukjeon-dong, Suji-gu, Yongin-si, Gyeonggi-do 448-701, KoreaTelephone: +82-31-8005-3194Email: [email protected]

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ARREST OF THE ElONGATION OF TRANSlATION BY PEPTIDE NuclEIc AcIDS Cordier Céline, Lecosnier Sabine, François 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. We have shown that it is possible to successfully target mixed sequences in structured regions of mes-senger 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 elon-gation. 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. Stud-ies 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 in-hibits 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 trans-formation4. 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.

1Dias et al. RNA hairpin invasion and ribosome elongation arrest by mixed base PNA oligo-mer. J. Mol. Biol. 2002; 320:489-501. 2Sénamaud-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, Si-mon P, François JC, Saison-Behmoaras TE. A steric blocker of translation elongation inhibits IGF-1R expression and cell transformation. FASEB J. 2011 Mar 14.

Céline Cordier, PhD studentMNHN, RDDM, INSERM U565-CNRS UMR 7196, 43 rue Cuvier CP26Paris 75005, FRANCE Telephone: 33 1 40 79 36 89Email: [email protected]

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RNAi- BASED PANcREATIc cANcER THERAPY BY lOcAl DElIvERY OF ANTI K-RASmT siRNAEithan 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 tu-mors. 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 LODER® (Local Drug EltueR), a miniature biodegradable polymeric matrix encompassing anti KRASG12D siRNA drug and preserving it against degradation. The LODER® 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 prolifera-tion 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. Further-more, 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]

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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, Germany2 Max-Delbrück-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 can-didate 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.

Claudia Haftmann, MSc Cell Biology Group (Prof. Dr. Andreas Radbruch) German Rheumatism Research Center Berlin Charitéplatz 1 10117 Berlin, Germany Telephone: +49-30-28460 731 Email: [email protected]

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IDENTIFIcATION OF PROcESS RElATED ImPuRITIES IN DNA/RNA BY lcmS ANAlYSISKenneth W. HillAgilent 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, PhD5555 Airport BlvdBoulder, Colorado, USA 80301Telephone: 303 222 4945Email: [email protected]

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DESIGN OF PROTEIN-RESPONSIvE shRNA SYSTEm FOR TRANSlATIONAl cONTROl IN HumAN cEllTan Inoue, Shunichi Kashida, Yoshihiko Fujita, Hirohide SaitoGraduate 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 kink-turn 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 mo-tifs consisting of U1A or NFκB 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, PhDGraduate School of Biostudies, Kyoto UniversityKyoto 606-8502, JapanTelephone: +81 75 753 3995 Email: [email protected]

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PmOplus™ APPROAcHES TO RAPID RESPONSE THERAPEuTIcS FOR EmERGING INFEcTIOuS DISEASESPatrick 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 impor-tant and novel application of AVI BioPharma’s RNA therapeutic platform. AVI’s phospho-rodiamidate 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 prob-ability 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 pro-gressed 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, PhD3450 Monte Villa ParkwayBothell, WA 98021 USATelephone: 425-354-5038Email: [email protected]

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INTEGRATED mIcROFluIDIc PlATFORm FOR mulTIPlEx SElEx ON A cHIPSoyoun KimDepartment 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 high-speed 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 cross-contamination 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, PhDAssociate Professor, Dongguk University26,3 Pil-dong, Chung-Gu, Seoul 100-715, KoreaTelephone: 822-2260-3840Email: [email protected]

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DEvElOPmENT OF POTENT RNAi THERAPEuTIcS uSING lONG ANTISENSE siRNAsSera Kim, Seul Gi Jo, Chang Han Lee, Pooja Dua, and Dong Ki LeeGlobal 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 repres-sion 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 anti-sense 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 candidate330517, Chemistry BuildingSungkyunkwan University, Chunchundong, Suwon, Korea Telephone: 82-31-299-4889Email: [email protected]

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STEAlTH DElIvERY OF OlIGONuclEOTIDES Akira Komoriya and Beverly Z. PackardOncoImmunin, Inc., Gaithersburg, MD, USA

Nucleic acid delivery into live cells has been one of the major hurdles for oligonucleotide-based therapeutics. Although various approaches and methods have been developed and are currently being utilized, a new platform, Xyton Stealth DeliveryTM (XSD), has been devel-oped for intracellular delivery of oligonucleotides with the aim of expanding therapeutic ap-plications. 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, cellu-lar 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 trans-port 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 sum-mary, 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, PhDOncoImmunin, Inc.207A Perry Parkway, Suite #6Gaithersburg, MD 20877 USATelephone: (01) 301-987-7881Email: [email protected]

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lONG dsRNA-mEDIATED RNA INTERFERENcE AND ImmuNOSTImulATION: lONG INTERFERING dsRNA (liRNA) AS A POTENT ANTIcANcER THERAPEuTIcSTae Yeon Lee1, Chan Il Chang1,2, and Dong-ki Lee1,*

1 Global Research Laboratory (GRL) for RNAi Medicine, Department of Chemistry, Sungkyunkwan University (SKKU), Suwon, Korea2Skip Ackerman Center for Molecular Therapeutics, Beth Israel Deconness Medical Center, Harvard Medical School, Boston USA*Corresponding author:[email protected]

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 inhibi-tion of cancer cell growth. In this study, we report an immunostimulatory long dsRNA struc-ture with ability to trigger RNAi-mediated specific target gene silencing, termed long interfer-ing 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 Survivin-targeting liRNA to inhibit cancer cell growth was superior over conventional siRNA or non-targeting dsRNA structures. Our results thus provide a simple, efficient dual function immu-nostimulatory RNAi-triggering structure, which is potentially applicable for potent anticancer therapeutics development.

Tae Yeon Lee330-517,Chemistry Building, Sungkyunkwan UniversitySuwon Republic of Korea 440-746Email: [email protected]

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DEPENDENcY OF THE cROSS-lINKING REAcTIvITY WITH 2-AmINO-6-vINYlPuRINE ON THE NEIGHBORING BASES Fumi Nagatsugi, Shinya Hagihara, Shuhei Kusano, Chao Xiao GuangInstitute 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 be-cause 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 sta-bility. 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 previous-ly 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 cross-linking 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.

1 Imoto, S.; Hori, T.; Hagihara, S.; Taniguchi, Y.; Sasaki, S.; Nagatsugi, F., Bioorg. Med. Chem. Lett., 20, 6121-6124 (2010)

Fumi Nagatsugi, PhDInstitute of Multidisciplinary Research for Advanced Materials, Tohoku University2-1-1 Katahira, Aoba-ku Sendai-shi, Miyagi, 980-8577, JapanTelephone: 001- 81-22-(217)-5633 Email: [email protected]

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Fig.1 Design of the 2-Amino-6-vinylpurine (2-AVP) (1) and Dependency of the Reactivity on the Neighboring Bases of (1)

ATTENuATION OF AuTOImmuNITY BY ANTI-HumAN-INTERlEuKIN-17 APTAmER IN mOuSE mODElSYoshikazu Nakamura, Hironori Adachi, Akira IshiguroInstitute 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 rheu-matoid arthritis and MOG-induced experimental autoimmune encephalomyelitis in a dose-dependent 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- α production induced by IL-17A/A or IL-17F/F, but blocked IL-17A/F-induced GRO- α 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 ProfessorInstitute of Medical Science, University of Tokyo 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, JapanTelephone: +81(JPN) 3 5449 5307Email: [email protected]

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A NOvEl ASSAY FOR THE DETEcTION OF miRNA mATuRATIONSaskia Neubacher, Christoph Arenz*

Humboldt-Universität 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 hu-man 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.

[1] 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.

Saskia Neubacher, Dipl.Chem.Humboldt-Universität zu BerlinBrook-Taylor-Str. 2, 12489 Berlin, GermanyTelephone: +49 30 2093 7511Email: [email protected]

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DEvElOPmENT OF DNA NANOGElS AND HYDROGElS AS INjEcTABlE DElIvERY SYSTEmS FOR cPG DNA AND OTHER NuclEIc AcID-BASED DRuGSMakiya Nishikawa, Kohta Mohri, Kohei Ogawa, Natsuki Takahashi, Yuki Takahashi, Yoshinobu TakakuraGraduate 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 tetrapod-like structures. Furthermore, the development of DNA dendrimer by connecting the tripod-like 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, PhDGraduate School of Pharmaceutical Sciences, Kyoto University46-29 Yoshidashimoadachi-cho, Sakyo-ku, Kyoto 606-8501, JapanTelephone: +81-75-753-4580Email: [email protected]

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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-PeterRoche Kulmbach GmbH, D-95326 Kulmbach, Germany

The process of impurity formation during annealing of siRNA was investigated. siRNAs con-taining 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 tem-perature 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 HF-elimination 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 so-lution 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 solu-tions. The strong concentration dependency of the Tm in salt-free solutions allows for adjust-ment 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 anti-sense oligonucleotides.

Bernhard Noll, Associate Director Analytics CMC Fritz-Hornschuch-Strasse 9 D-95326 Kulmbach, GermanyTelephone: +49 (0) 9221 827 62 8546 Email: [email protected]

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ExPERImENTAl ASSESSmENT OF ANTImIR SPEcIFIcITYAndreas Petri1,Susanna Obad2, Darryl Pappin3, Sakari Kauppinen1,2, and Morten Lindow1

1) Informatics, Santaris Pharma A/S, Hørsholm, Denmark2) MicroRNA Drug Discovery, Santaris Pharma A/S, Hørsholm, Denmark3) 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 un-biased and exhaustive analysis of sequence motifs associated with differentially expressed genes following tiny LNA administration to cell lines or mice. In all experiments with anti-miRs, Sylamer analysis showed significant overrepresentation among the up-regulated genes of motifs corresponding to binding sites of the cognate seed. However, sequence motifs cor-responding 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-reg-ulation 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 oligonucle-otides, the presence of high affinity tiny LNAs generally has no effect on the transcripts con-taining perfect match binding sites or on the levels of protein translated from such transcripts.

Andreas Petri, Ph.d.Santaris Pharma A/SKogleAllé 6DK 2970 HørsholmDanmarkTelephone: +45 4517 9876Email: [email protected]

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AN RNA APTAmER IS A POTENTIAl BIOmARKER FOR PANcRE-ATIc 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 Evolu-tion 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 non-cancerous 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 ad-dition, 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 stain-ing 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, PhDDUMC Box 103035Durham, NC 27710 USATelephone: 919-684-4864Email: [email protected]

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ENDOSOmAl EScAPE OF lIPOSOmAl siRNA vIA mEmBRANE FuSION AND in vivo DElIvERY TO TumORYu Sakurai, Hiroto Hatakeyama, Yusuke Sato, Hidetaka Akita, Hideyoshi HarashimaHokkaido University, Sapporo, Hokkaido, Japan

To deliver siRNA to tumor tissue, we have developed liposome type carrier, a multi-func-tional 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 en-dosome 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 endo-some. As a result, encapsulated siRNA was released into cytosol efficiently. Although GALA-modified 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 courseKita-12, Nishi-6, Kita-ku, Sapporo, Hokkaido, JapanTelephone: +81-11-706-2197 Email: [email protected]

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IN vIvO siRNA DElIvERY WITH PH-SENSITIvE lIPOSOmEYusuke Sato, Hiroto Hatakeyama, Yu Sakurai, Mamoru Hyodo, Hidetaka Akita, Hideyoshi HarashimaFaculty 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 or-gans 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 non-specific 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 traf-ficking. To solve this ‘PEG dilemma’, we previously developed a cleavable PEG-lipid in re-sponse 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 cation-ic 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 success-fully 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 lyso-somes. Expectively, the knockdown activity of pH-sensitive MEND was dramatically de-creased depending on ammonium chloride concentration, which suggested endosomal acidi-fication 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 hu-man polo-like kinase 1 (hPLK1) caused reduction in tumour-derived hPLK1 mRNA. RNAi-specific 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 CourseTelephone: +81-11-706-2197Email: [email protected]

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OTS 2011: PmOS – THE clINIcAl STORYStephen ShrewsburyAVI 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 (PMOplus™ chemistry) for viral diseases (Ebola, Marburg and Junin hemorrhagic fevers, Dengue and Influenza), arginine based peptide conjugates (PPMO), or novel internal/external additions (PMO-X™), 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, PhD3450 Monte Villa ParkwayBothell, WA 99021 USATelephone: 425-354-5038Email: [email protected]

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AmPHIPHIlIc PEPTIDYl-RNA cONjuGATES ANcHORED TO AND ENRIcHED ON PHOSPHO- AND GlYcOlIPID BIlAYERSPeter Strazewski,1 Alexandra Le Chevalier Isaad,1 Kollappillil S. Krishnakumar,1 Dominique Lafont,2 (1) Laboratoire de Synthèse de Biomolécules, (2) Laboratoire de Chimie Organique 2 ; Insti-tut de Chimie et Biochimie Moléculaires et Supramoléculaires Université 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 ‘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 immer-sed 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 immo-bilized through its 2’-hydroxyl function to an appropriate resin. A solid support peptide syn-thesis 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 lip-idic vesicles, spontaneously interacted with their outer bilayer membranes. At initial lipid-to-conjugate molar ratios between 150:1 and 1500:1 they visibly and strongly enriched on the readily accessible lipidic surfaces. These studies were supplemented

by control experiments and quantitative permeability measurements on large unilamellar ve-sicles, as well as by FTIR and CD spectroscopic studies of the peptide interacting with lipids.

Prof. Peter StrazewskiLaboratoire de Synthèse de Biomolécules, Bât. Chevreul (5ème étage)Université Claude Bernard Lyon 143 boulevard du 11 novembre 1918, 69622 Villeurbanne Cedex, France Email: [email protected]

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Figure. L = l-leucine, A = l-alanine, 3'-A = 3'-amino-3'-deoxyadenylate, ACGU = adenylate, cytidylate, guanylate, uridylate, X = fluorescein linked to thymidylate.

THERAPEuTIc APPlIcATION OF DNAZYmES FOR NASOPHARYNGEAl cARcINOmASSun L-Q, Yang L, Cao YXiangya 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 inactiva-tion 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 ‘‘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-κB, 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, PhDXiangya Hospital and Center for Molecular Medicine, Central South UniversityChangsha, Hunan 410078, ChinaTelephone: +86 731 84327646Email: [email protected]

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iNhiBitiON OF miCRORNA ACtivity By 2ʹ-o-methyl-4ʹ-THIORIBONuclEOSIDE mODIFIED ANTI-microRNA OlIGONuclEOTIDE (AmO)Mayumi Takahashi,1 Noriaki Minakawa2 and Akira Matsuda1

1Faculty 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

MicroRNAs (miRNAs) are a class of endogenously expressed small non-cording RNA molecules which regulate the gene expression post-transcriptionally. Recent advances in un-derstanding 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 oligo-nucleotides 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 2ʹ-OMe, 2ʹ-OMOE and LNA, and successfully inhibits miRNA function in vitro as well as in vivo.

We have recently developed a novel chemically modified oligonucleotide, 2ʹ-OMe-4ʹ-thioRNA, which can be considered as a hybrid chemical modification based on 2ʹ-OMe and 4ʹ-thioribonucleoside. In its hybridization property, 2ʹ-OMe-4ʹ-thioRNA preferred RNA as a complementary partner rather than DNA. In addition, investigation of their biological stabil-ity revealed that 2ʹ-OMe-4ʹ-thioRNA was extremely stable in 50% human plasma compared with 2ʹ-OMeRNA and 4ʹ-thioRNA. Prompted by such favorable properties, we planed to utilize 2ʹ-OMe-4ʹ-thioribonucleosides to develop novel and effective chemically modified AMOs. We evaluated a several 2ʹ-OMe-4ʹ-thioribonucleoside modified AMO for their ability to inhibit miR-21 and miR-122 activity by dual-luciferase reporter assay. We found that fully 2ʹ-OMe-4ʹ-thionucleoside modified AMOs were significantly more potent than corresponding 2ʹ-OMe modified AMOs. In addition, the prolonged inhibition activity of AMO was achieved by 2ʹ-OMe-4ʹ-thioribonucleoside modifications.

Mayumi Takahashi, doctoral student (D3)Hokkaido UniversityKita-12, Nishi-6, Kita-ku, Sapporo 060-0812, JapanTelephone: +81 11 706 3763Email: [email protected]

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RNAi IN HumANS: PHASE I DOSE-EScAlATION STuDY OF AlN-vSP02, A NOvEl RNAi THERAPEuTIc FOR SOlID TumORS WITH lIvER INvOlvEmENTA.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 1Hospital Clínico 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 Rocío, Seville, Spain, 7Memorial Sloan-Kettering Cancer Center, New York, NY, USA, 8TGen Clinical Research Services at Scottsdale Healthcare, Scottsdale, AZ, USA,9Alnylam 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 vascu-lar endothelial growth factor (VEGF)-A and kinesin spindle protein (KSP). methods: A multi-center, open label, Phase 1 dose-escalation trial of ALN-VSP02 admin-istered 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 low-grade 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 revers-ible 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, PhDAlnylam Pharmaceuticals Inc.300 Third Street, Cambridge, MA 02142 USATelephone: 617-551-8360Email: [email protected]

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microRNA TuRNOvER AND ANTAGOmIR-mEDIATED INHIBITION OF INFlAmmATIONBryan 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 process-ing. 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 pro-duction 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 characteriza-tion 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 cel-lular 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 pro-inflammatory 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 ResearchMonash University Clayton, Victoria, 3168, Australia Email: [email protected]

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The Oligonucleotide Therapeutics Society (OTS) has become recognized as a premier organization fostering the research and drug development in all areas of oligonucleotide science. The OTS, formed in 2004, is a nonpro�t, educational organization to provide a neutral forum for the dissemination of information and scienti�c exchange in all aspects of oligonucleotide research and therapeutic development. Our annual meetings are highly regarded in the �eld of oligonucleotide research for their unsurpassed scienti�c quality and breadth.

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OTS membership is open to all individuals from industry, academia and other institutions worldwide who are interested in research and development of oligonucleotide therapeutics (RNAi, CpG, antisense, ribozymes, and others).

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• A network of experts covering all areas of nucleic-acid drug development (chemistry, physics, mechanisms, biology, model systems, clinical trials, market analysis, patent issues)

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