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7th Annual Meeting of the
Oligonucleotide
Therapeutics Society
September 8 – 10, 2011
The Royal Library
Copenhagen | Denmark
Meeting Abstracts
Oligonucleotide Therapeutics Society
PROGRAM AGENDA
Every effort has been made to ensure the meeting agenda listed in this booklet
is accurate. However it’s possible that some changes occurred after printing.
We will notify you of any changes to the agenda from the main stage.
2011 CONFERENCE PROGRAM
w w w .ol i goth e rapeut ics.org
Wednesday, September 7, 2011
15:00 – 18:00
Registration and Poster Set-Up
Thursday, September 8, 2011
08:00 – 09:00
09:00 – 09:15
Registration and Poster Set-Up
Welcome and Opening Remarks
Gunther Hartmann, MD, PhD, Universitätsklinikum Bonn, OTS President
09:15 – 10:30
S ESSION 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
S ESSION II: N UCLEIC A CID C HEMISTRY
Chair: Jesper Wengel, PhD, University of Southern Denmark, Odense
11:00 – 11:30 Nucleotide Chemistry and Gene Silencing
Jesper Wengel, PhD, University of Southern Denmark, Odense
11:30 – 12:00 Arabinose Modified Antisense and siRNAs: Biological Applications, Structural Considerations,
and Delivery Strategies
Masad J. Damha, PhD, McGill University, Montreal
12:00 – 12:30 siRNA Activity, Stability and 3D-Structural Properties of 2’-Fluoro RNA and GNA
Martin Egli, PhD, Vanderbilt University Medical Center, Nashville
12:30 – 14:00
Lunch with Attended Poster Session
12:30 – 13:15 Standing Lunch Buffet
13:15 – 14:00 Attended Poster Session
14:00 – 14:30
Keynote Session: Oligonucleotide Therapeutics reviewed in memory of Alan Gewirtz
Keynote Speaker: Fritz Eckstein, PhD
Max-Planck-Institute for Experimental Medicine, Göttingen
14:30 – 16:00
S ESSION III: RNA B IOLOGY: N EW F RONTIERS
Chair: Mark Kay, MD, PhD, Stanford University
14:30 – 15:00 Primary microRNA Function in Target Recognition and Repression
Chang-Zheng Chen, Stanford University
15:00 – 15:30 Regulation of small RNA function
Gunter Meister, PhD, University of Regensburg
15:30 – 16:00 The mechanism of miRNA and siRNA Argonaute loading in mammals
Mark Kay, MD, PhD, Stanford University
16:00 – 16:30
Refreshment Break
7 ANNUAL MEETING OF THE OLIGONUCLEOTIDE THERAPEUTICS SOCIETY
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7 ANNUAL MEETING OF THE OLIGONUCLEOTIDE THERAPEUTICS SOCIETY
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16:30 – 18:00
S ESSION IV: D ELIVERY OF N UCLEIC A CIDS
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
19:30
Welcome Reception with Attended Posters
Meet the Experts
End of Day 1
Friday, September 9, 2011
08:00
08:30 – 10:00
Registration Opens
SESSION V: IMMUNOBIOLOGY of NUCLEIC ACIDS
Chair: Arthur Krieg, MD, Atlas Venture
08:30 – 09:00 RIG-I and tolerance of self-RNA
Gunther Hartmann, MD, PhD, Universitätsklinikum Bonn
09:00 – 09:30 Regulation of the Innate Immune Response Using Oligonucleotide-based Inhibitors of Toll-Like Receptors
Franck Barrat, PhD, Dynavax Technologies
09:30 – 10:00 Intracellular trafficking of TLR ligands in human plasmacytoid dendritic cells
Patricia Fitzgerald-Bocarsly, PhD, UMDNJ – New Jersey Medical School
10:00 – 10:30
Refreshment Break
10:30 – 12:00
S ESSION VI: T ARGETING C ODING RNA
Chair: David Corey, PhD, UT Southwestern Medical Center, Dallas
10:30 – 11:00 Gymnotic Delivery of Antisense Oligonucleotides
Cy Stein, MD, PhD, Albert Einstein College of Medicine, Bronx
11:00 – 11:30 Modulation of Gene Expression by Oligonucleotide Chemistry-Dependent Recruitment of
Proteins to RNA Transcripts
Frank Rigo, PhD, Isis Pharmaceuticals
11:30 – 12:00 Clinical development of mipomersen
Erik Stroes, MD, PhD, AMC Research Institute, Amsterdam
12:00 – 13:30
Lunch with Attended Poster Session
12:00 – 12:45 Standing Lunch Buffet
12:45 – 13:30 Attended Poster Session
13:30 – 15:00
S ESSION VII: T ARGETING microRNA
Chair: Markus Stoffel, MD, PhD, ETH Zurich
13:30 – 14:00 Therapeutic Targeting of microRNAs
Neil Gibson, PhD, Regulus Therapeutics
14:00 – 14:30 Therapeutic targeting of individual cardiac cell types by miRNA antagonists
Thomas Thum, MD, PhD, Integrated Research Center, Medical School Hannover, Germany
14:30 – 15:00 Oncomir-1 in cancer and development: a tale of mice and men
Andrea Ventura, MD, PhD, Sloan-Kettering Institute, New York City
15:00 – 15:30
8 Copenhagen, Denmark
Refreshment Break
2011 CONFERENCE PROGRAM
w w w .ol i goth e rapeut ics.org
15:30 – 17:00
S ESSION VIII: T ARGETING L IVER AND T UMORS
Chair: Henrik 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
08:15 – 09:45
Registration Opens
S ESSION IX: T ARGETING I NFECTION
Chair: John Rossi, PhD, City of Hope, Duarte
08:15 – 08:45 The development of aptamers as candidate antiviral agents: towards a polyvalent microbicide
William James, PhD, Sir William Dunn School of Pathology, University of Oxford
08:45 – 09:15 Developing the next generation of expressed RNAi modalities against rapidly-evolving viral infections
Marc Weinberg, PhD, University of the Witwatersrand Medical School, Parktown
09:15 – 09:45 Dual function therapeutic Aptamer siRNA combinations
John Rossi, PhD, City of Hope, Duarte
09:45 – 11:15
S ESSION X: T ARGETING THE C ENTRAL N ERVOUS S YSTEM
Sponsored by International Society of Neurochemistry
Chair: Hermona Soreq PhD, The Hebrew University of Jerusalem
09:15 – 10:15 Systemic versus CNS Delivery of MOE Antisense Oligonucleotide to Correct Defective Splicing in a
Severe Mouse Model of Spinal Muscular Atrophy
Adrian Krainer, PhD, Cold Spring Harbor Laboratory
10:15 – 10:45 Micro-RNAs in the Neuroimmune Interface: From Inflammation to Lethal Poisoning Protection
Hermona Soreq, PhD, The Hebrew University of Jerusalem
10:45 – 11:15 The role of microRNA in regulating the central stress response
Alon Chen, PhD, MBA, Weizmann Institute of Science, Rehovot, Israel
11:15 – 11:45
Refreshment Break
11:45 – 13:15
S ESSION XI: T ARGETING G ENETIC D ISEASES
Chair: Dieter Gruenert PhD, University of California, San Francisco
11:45 – 12:15 Exploring and exploiting designer nucleases for targeted genome engineering
Toni Cathomen, PhD, Hannover Medical School
12:15 – 12:45 Oligonucleotide-directed gene modification in mouse Embryonic Stem Cells to study variants of
cancer-related genes
Hein te Riele, PhD, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital
12:45 – 13:15 siDNA, a new strategy for inhibiting DNA repair pathways
Marie Dutriex, PhD, Institut Curie, Centre Universitaire, Orsay
13:15
Closing Session
End of Conference
7 ANNUAL MEETING OF THE OLIGONUCLEOTIDE THERAPEUTICS SOCIETY
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SPEAKER ABSTRACTS
Session 1: Hot Topics: Short Talks Selected from Abstracts
Dong-ki Lee
Thursday, September 8, 2011
09:15 – 09:30
Expanding the Structural Diversity Repertoire
of siRNAs
Dong-ki Lee
Global Research Laboratory for RNAi Medicine, Department of Chemistry, Sungkyunkwan
University, Suwon 440-746, Korea
Chemically synthesized small interfering RNAs (siRNAs) can mediate efficient gene silencing in a sequence-specific manner by utilizing the endogenous RNA interference (RNAi)
pathway. The early structure-activity relationship study suggested the existence of strict rules
of siRNA structural design to achieve optimal gene silencing. These include the presence
of 3’-overhang, fixed duplex length, and structural symmetry, which defined the structure
of the current standard siRNA (the so-called 19+2 form). However, several recent studies
demonstrated that the gene silencing RNA structure could be much more flexible than what
was originally proposed. In addition, the synthetic 19+2 siRNA structure exhibits sequenceindependent non-specific effects and poor cellular delivery by cationic polymers, which
justifies the effort to develop alternative RNAi-triggering structures. Here we introduce
three siRNA structural variants with improved functionalities over current siRNA structures.
First, we introduce asymmetric shorter-duplex siRNAs (asiRNAs) that can efficiently trigger
gene silencing without non-specific effects triggered by 19+2 siRNAs, such as sense strandmediated off-target gene silencing, saturation of the cellular RNAi machinery, and reduced
TLR3 activation. Second, we present multi-armed gene silencing RNA structures, which not
only mediate multiple gene silencing, but also show enhanced cellular delivery by cationic
polymers such as polyethyleneimine (PEI). Third, we show a long dsRNA structure that can
trigger RNAi as well as immunostimulation. The structure, termed long interfering dsRNA
(liRNA), show enhanced anticancer activity compared with conventional siRNA or nontargeting long dsRNA. These novel siRNA structural variants exemplify the structural flexibility of gene silencing RNAs, and have the potential to become important alternatives to the
current siRNA structure for future therapeutics development.
Dong-ki Lee, PhD
Global Research Laboratory for RNAi Medicine
Department of Chemistry
Sungkyunkwan University
Suwon 440-746 Korea
Telephone: 82-31-299-4565
Email: dklee@skku.edu
11
Session 1: Hot Topics: Short Talks Selected from Abstracts
Eric Swayze
Thursday, September 8, 2011
09:30 – 09:45
ACTIVATION OF RNA INTERFERANCE IN ANIMALS WITH SINGLE
STRANDED OLIGONUCLEOTIDES
Eric E. Swayze, Thazha P. Prakash, Walt F. Lima, and Stanley T. Crooke
Isis Pharmaceuticals, Carlsbad, CA, USA
The use of double stranded oligonucleotides to inhibit gene expression via the RNA interference (RNAi) pathway has generally required a complex (usually lipid based) formulation
for delivery of the oligonucleotide to the desired compartment of a cell. Unfortunately, this
requirement has extended from cell culture to animals, which severely limits the potential
of harnessing the RNAi pathway for therapeutic approaches. Unlike double stranded
oligonucleotides, single stranded antisense oligonucleotides have shown activity in multiple
species (including humans) without formulation vehicles. Furthermore, the double stranded
RNA (dsRNA) structure is not required for RNAi, as single stranded RNA (ssRNA) delivered
to cells using cationic lipids has been shown to activate the RNAi pathway. \This suggested
to us that the dsRNA structure could be simplified to a single stranded oligonucleotide that
would activate RNAi in cells and in animals, and provide significant benefits over double
stranded structures for the potential development of human therapeutic agents.
To achieve this objective, we have engaged in an extensive chemical structure activity relationship (SAR) study of ssRNAs, and coupled this to biochemical studies on the mechanism
of activation of the RNA induced silencing complex (RISC). This SAR has led to an understanding of key structural features required for a ssRNA activity, and allowed us to achieve
potency within 5-fold of the corresponding double stranded structures in cells with multiple
fully modified, partially phosphorothioated ssRNA oligonucleotide designs. Our initial
attempts to show activity in animals with these early designs failed due to pharmacokinetic
limitations. These limitations were overcome with further chemical stabilization achieved
via an in vivo SAR optimization cycle. Sequence optimization of the optimal chemical
motif provided highly potent compounds in cell culture which were shown to function via an
argonaute-2 dependent mechanism. These ssRNAs are active in cell culture without cationic
lipids, and this activity translated to activity in animals at pharmacologically relevant doses
with subcutaneous administration in saline formulations. These studies provide a framework
for further optimization of the ssRNA structure for the potential development of human
therapeutic agents.
Eric E. Swayze, PhD
Isis Pharmaceuticals, Inc.
2855 Gazelle Court
Carlsbad, CA 92010 USA
Telephone: 760-603-3825
Email: eswayze@isisph.com
12
Session 1: Hot Topics: Short Talks Selected from Abstracts
Georg Sczakiel
Thursday, September 8, 2011
09:45 – 10:00
Limitations for RNAi: intracellular release of siRNA
and cell stress
Georg Sczakiel
Institute of Molecular Medicine, University of 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, PhD
Institute for Molecular Medicine
University of Lübeck
Building: 50, 1F, Room 1319
Ratzeburger Allee 160
23538 Lübeck Germany
Telephone: +49 451 500 2730
Email: sczakiel@imm.uni-luebeck.de
13
Session 1: Hot Topics: Short Talks Selected from Abstracts
Peter Sazani
Thursday, September 8, 2011
10:00 – 10:15
Preclinical Efficacy and Safety of
Phosphorodiamidate Morpholino OligOmers (PMO)
Peter Sazani
AVI BioPharma, Inc., Bothell WA, USA
The development of RNA-based therapeutics has been driven largely by the incorporation of
chemical modifications that improve the drug-like character of these agents. AVI’s phosphorodiamidate morpholino oligomer (PMO) platform is a next generation of RNA-based therapeutics with two key modifications; the replacement of ribose with a 6-membered morpholine
ring, and the replacement of the natural phosphodiester linkage with a charge-neutral phosphorodiamidate moiety. The biological efficacy of AVI-4658, our lead PMO for the treatment
of Duchenne Muscular Dystrophy (DMD), has been demonstrated, and will be discussed in
relation to its preclinical efficacy and toxicity profile. In clinical trials in patients, AVI-4658
dosed at up to 20 mg/kg intravenously for 12 weeks yielded dystrophin expression increases
of up to 55% of healthy control muscle. These doses were also well tolerated clinically. AVI4658 was also well tolerated in mice (both dystrophic and healthy) and cynomolgus monkeys
at up to the maximum feasible doses of 960 mg/kg and 320 mg/kg, respectively. In further
modifications, selective addition of positive charges into the PMO backbone have generated
a new class of PMOs, called 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, PhD
AVI BioPharma, Inc.
3450 Monte Villa Parkway
Bothell, WA 98021 USA
Telephone: 425-354-5087
Email: psazani@avibio.com
14
Session 1: Hot Topics: Short Talks Selected from Abstracts
Christoph Coch
Thursday, September 8, 2011
10:15 – 10:30
5’-triphosphate dsRNA-induced stimulation of RIG-I
prevents influenza H1N1 infection of primary nasal
epithelial cell culture and protects mice from
infection in vivo
Coch C.1*, Stehle V.1*, 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 antiviral immune activation the Influenza virus has evolved mechanisms to inhibit recognition of
its genome by RIG-I. Nasal mucosa represents the first site of infection of the upper airways.
As a new immunotherapeutic strategy against infection with Influenza A (H1N1) virus we
investigated the antiviral effect of RIG-I activation by 3p-dsRNA in human primary nasal epithelial cells in vitro and in a mouse model in vivo.
Methods: Primary cultures of nasal epithelial cells were generated from conchotomy specimen obtained during surgery. Cells were stimulated with enzymatically generated or synthetic
3p-dsRNA and were subsequently infected with Influenza virus type A (H1N1).
IP-10 and IL-6 were analyzed by ELISA, type I IFN with a reporter-assay. Viral load was
determined by RT PCR, apoptosis by FACS. Mice were intranasally infected with Influenza
with or without 3p-dsRNA application and survival and body weight were monitored.
Results: RIG-I was expressed in primary human nasal epithelial cells. Enzymatically generated or synthetic 3p-dsRNA induced antiviral cytokines directly in nasal epithelial cells (IP10, IL-6, type I IFN) in the absence of immune cell subsets. Compared to untreated controls,
viral load in nasal epithelial cells exposed to 3p-dsRNA was reduced by 97 %.
In comparison, the antiviral activity of recombinant IFN-b was substantially lower. More
over, 3p-dsRNA induced antiviral cytokines in mice in vivo and protected mice from infection
with Influenza virus.
Conclusion: We demonstrate expression and function of RIG-I in primary human nasal epithelium. Stimulation of RIG-I by 3p-dsRNA leads to significant reduction of viral load
in vitro and to protection from infection with Influenza virus in vivo. Our results suggest a potential use of 3p-dsRNA as immunotherapeutic agent in Influenza virus infection.
Christoph Coch, MD
Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn
Sigmund-Freud-Str. 25, 53105 Bonn, Germany
Telephone: 0049/228/287-14018 or 16080
Email: ccoch@uni-bonn.de
15
Session 2: Nucleic Acid Chemistry
Jesper Wengel
Thursday, September 8, 2011
11:00 – 11:30
NUCLEOTIDE CHEMISTRY AND GENE SILENCING
Jesper Wengel
University of Southern Denmark, Odense
Like with any class of drug molecules, chemistry has a distinct influence on key properties
of oligonucleotides. The effect can be on pharmacokinetic or pharmacodynamic features, but
in fact relatively few oligonucleotide chemistries have been evaluated carefully and only few
in vivo head-to-head comparisons have been published. The importance of chemistry will be
pointed out from selected examples, and prospects for oligonucleotide medicinal chemistry
will be discussed.
Jesper Wengel, PhD
University of Southern Denmark
Nucleic Acid Center
Campusvej 55
DK-5230 Odense M
Denmark
Telephone: +45 65502510
Email: jwe@ifk.sdu.dk
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Session 2: Nucleic Acid Chemistry
Masad J. Damha
Thursday, September 8, 2011
11:30 – 12:00
ARABINOSE MODIFIED ANTISENSE AND siRNAs: BIOLOGICAL
APPLICATIONS, STRUCTURAL CONSIDERATIONS, AND DELIVERY
STRATEGIES
Masad J. Damha
McGill University, Montreal, Quebec, Canada
This presentation will provide an overview of recent work in my laboratory and those of
collaborators concerning the properties of oligonucleotides carrying 2’-F sugar modifications,
particularly 2’-fluoroarabinonucleic acids (2’-F ANA). We found that the characteristics of
2’-F RNA (C3’-endo) and 2’-fluoroarabinonucleic acid (2’-F ANA; C2’/O4’-endo) make it
particularly suitable for the design of highly effective siRNAs. This modification approach
also benefits from retention of an A-form dsRNA-like helical conformation. Furthermore,
it allows for tuning of duplex thermodynamics, reduced immunostimulation, and increased
nuclease resistance without impairment of gene silencing activity against reporter constructs
and endogenous genes. In the case of 2’-F ANA:RNA hybrids, a pseudohydrogen bond
between the 2’-F and H8 of the nucleobase plays an important role in determining duplex
stability, as supported by NMR, molecular modeling, and UV melting experiments. Structural
and synthetic aspects of novel fluorinated nucleotide analogues will also be presented.
Applications of both modified
antisense and siRNAs towards
novel endogenous targets
will be described, with a
focus on ongoing anticancer
applications, e.g., treatment
of malignant gliomas. To
date, a means for effective
delivery of both unmodified
and modified antisense and
siRNA oligonucleotides to
target cells and tissues presents
a major challenge impeding the
development of therapeutic oligonucleotides. To this end, we will also discuss early advances
towards new delivery strategies for these compounds, with and without delivery vehicles to
facilitate cell uptake.
Masad J. Damha, PhD
Department of Chemistry, McGill University
801 Sherbrooke St. West
Montreal, QC, CANADA, H3A-2K6
Telephone: 514-398-7552
Email: masad.damha@mcgill.ca
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Session 2: Nucleic Acid Chemistry
Martin Egli
Thursday, September 8, 2011
12:00 – 12:30
siRNA Activity, Stability and 3D-Structural Properties
of 2’-Fluoro RNA and GNA
Martin Egli
Departmentof Biochemistry, Vanderbilt University, Nashville, TN 37232-0146, USA
Various characteristics of 2’-F RNA make it particularly suitable for the design of highly effective
siRNAs. The small size of fluorine enables position-independent incorporation of modified nucleotides
into both strands, and its high electronegativity locks the sugar in the RNA-compatible C3’endo conformation. We also found that 2’F-modified siRNAsexhibited increased nuclease stability,
significantly decreased immune stimulation in an in vitro model, and, in some cases, improved in
vitro and in vivo activity relative to that of the unmodified control RNA.Detailed calorimetric
and UV melting experiments revealed that the higher thermal stability of 2’-F-modified duplexes was
predominantly due to increased enthalpy rather than entropic effects. This is a surprising finding given
the analog’s reputation to conformationally preorganize the backbone for an RNA target. We hypothesize
that the favorable enthalpy exhibited by 2’-F RNA is due to strengthening of Watson-Crick
base pairing and/or enhanced stacking interactions. We have tested these possibilities using high-field
NMR and further thermodynamic measurements.
Glycol nucleic acid(GNA) is arguably the simplest artificial nucleic acid pairing system with a
phosphodiester backbone. Despite its acyclic backbone GNA is capable of stable self-pairing and
the (S)-GNA enantiomercross-pairs with RNA. Neither (S)- nor (R)-GNA exhibit stable pairing
with DNA. Meggers and coworkers reported crystal structures of (S)-GNA duplexes whose righthanded geometry differed substantially from that of a canonical
A-RNA duplex. However, despite a large helical pitch andan increased number of residues per turn,
(S)-GNA shares with RNA a strong negative inclination between the backbone and base axes. This
latter feature may provide a conformational basis for the formation of stable (S)-GNA:RNA duplexes.
We hypothesize that (S)-GNA, similar to DNA, is able to adapt to the conformational constraints of
RNA and that the geometry of the hybrid duplex resembles the A-form. Ideally, an analysis of GNA’s
conformational properties will allow insights into the structural underpinnings of the pairing with
RNA and its inability to pair with DNA. Moreover, one would expect structural data to provide an
explanation for the stereoselective pairing with RNA [i.e. (S)-GNA pairs and (R)-GNA does not]. We
recently determined the crystal structure of an RNA dodecamer duplex with incorporated (S)-GNA-Ts
at high resolution and the pairing properties of GNA will be discussed in light of the structural data.
Supported by the US NIH, R01GM055237; Lab URL http://structbio.vanderbilt.edu/~eglim/
References
M. Manoharan, A. Akinc, R. K. Pandey, J. Qin, P. Hadwiger, M. John, K. Mills, K. Charisse, M. A. Maier, L. Nechev, E. M.
Greene, P. S. Pallan, E. Rozners, K. G. Rajeev, and M. Egli (2011) Unique gene-silencing and structural properties of 2’-F
modified siRNAs. Angew. Chem. Int. Ed. 50: 2284-2288.
P. S. Pallan, E. Greene, P. Jicman, R. Pandey, M. Manoharan, E. Rozners, and M. Egli (2011) Unexpected origins of the
enhanced pairing affinity of 2′-fluoro-modifiedRNA. Nucleic Acids Res. 39: 3482-3495.
P. S. Pallan, P. Lubini, M. Bolli, and M. Egli (2007) Backbone-base Inclination as a fundamental determinant of nucleic acid
self- and cross-pairing.Nucleic Acids Res. 35:
6611-6624.
Martin Egli, PhD
Departmentof Biochemistry, Vanderbilt University
Nashville, TN 37232-0146 USA
Telephone: 1-615-343-8070
Email: martin.egli@vanderbilt.edu
18
Keynote Session: Oligonucleotides reviewed in memory of Alan Gewirtz
Fritz Eckstein
Thursday, September 8, 2011
14:00 – 14:30
OLIGONUCLEOTIDE THERAPEUTICS REVIEWED IN MEMORY OF
ALAN GEWIRTZ
Eckstein, F.
Max-Planck-Institute for Experimental Medicine, 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 oligonucleotide field to be developed for medical application. As a practising clinician he was one
of the few of us to combine in an admirable way wide ranging basic research with the application in the clinic. Early on he administered his c-myb antisense oligonucleotide to chronic
myeloid leukaemia (CML) patients. The commitment to his patients stimulated his strength
with never ending dedication to his work. His strong belief that the oligonucleotide approach
will eventually bring the expected result for the patients was contagious and stimulated us,
his colleagues, to look for improvements of our strategies.
The oligonuclotide field has moved forward considerably since Alan began his work. Thus
RNase-H dependent cleavage of mRNA is now one of several strategies, followed by various
RNAi approaches with great potential. Another powerful area with no cleavage of RNA is the
correction of faulty splicing, a major cause for diseases. Oligonucleotide-protein interaction
rather than interaction with RNA is yet another wide area of interest. This is exemplified by
oligonucleotides binding to Toll Like Receptors (TLRs) as agonists for stimulation of the immune response or similarly binding to the retinoic acid-inducible gene (RIG-1). Also aptamers as alternatives to antibodies fall into the category of oligonucleotides binding to proteins.
Thus, the field has come a long way opening up great potentials. We will miss Alan’s strong
commitment, dedication, stimulation and help.
Fritz Eckstein, PhD
Max-Planck-Institute for Experimental Medicine
Hermann-Rein-Str. 3
37075 Göttingen, Germany
Telephone: (49) 551-3899 274
Email: eckstein@em.mpg.de
19
Session 3: RNA Biology: New Frontiers
Chang-Zheng Chen
Thursday, September 8, 2011
14:30 – 15:00
PRIMARY microRNA FUNCTION IN TARGET RECOGNITION AND
REPRESSION
Chang-Zheng Chen, Si-Biao Yue, Robin Deis Trujillo, Yujie Tang, & William E. O’Gorman
Department of Microbiology and Immunology, Baxter Laboratory for Stem Cell Biology,
Stanford University School of Medicine, Stanford, CA 94305 USA
MicroRNA (miRNA) genes encode small regulatory RNAs that control gene expression
at the post-transcriptional level. Despite progress in the field, many fundamental questions regarding mechanisms of action during target recognition and repression remain unresolved. In
particular, one critical problem that has largely been overlooked is whether precursor miRNA
molecules, including primary (pri-) and precursor (pre-) miRNAs, have direct roles in target
recognition and repression. Both pri-miRNAs and pre-miRNAs contain the mature miRNA
sequences and in theory can interact with target mRNAs. Addressing this question is essential in order to define the regulatory information used in target recognition and the molecular
steps required for target repression. We found that structural and sequence elements outside
the mature miRNA region control the activities of miRNA genes and that pri-miRNAs can
have a direct role in target recognition and repression. These findings demonstrate that regulatory information encoded in the structural and sequence elements of pri-miRNAs can be
translated into activity through direct interactions between target mRNAs and structured primiRNAs and may have broad implications in understanding the mechanisms by which miRNAs recognize and repress their cognate targets.
Chang-Zheng Chen, PhD
Department of Microbiology and Immunology, Baxter Laboratory for Stem Cell Biology
Stanford University School of Medicine
Stanford, CA 94305 USA
Telephone: 650-725-1667
Email: czchen@stanford.edu
20
Session 3: RNA Biology: New Frontiers
Gunter Meister
Thursday, September 8, 2011
15:00 – 15:30
Regulation of small RNA function
Gunter Meister
University of Regensburg and Max-Planck-Institute of Biochemistry, Munich
Argonaute (Ago) proteins are highly conserved between species and constitute a direct
binding platform for small RNAs including short interfering RNAs (siRNAs), microRNAs
(miRNAs) and Piwi interacting RNAs (piRNAs). Small RNAs function as guides whereas
Ago proteins are the actual mediators of gene-silencing. Although extensive research has
elucidated the major steps in small RNA guided gene silencing, not much is known about
Ago protein regulation. We have performed a comprehensive analysis of Ago2 phosphorylation in human cells. We find that the highly conserved tyrosine Y529, located in the small
RNA 5’ end-binding pocket of Ago proteins can be phosphorylated. By substituting Y529
with a negatively charged glutamate mimicking a phosphorylated tyrosine, we show that
small RNA-binding is strongly reduced. Our data suggest that a negatively charged phosphotyrosine generates a repulsive force that prevents efficient binding of the negatively charged
5’ phosphate of the small RNA.
In addition, we have identified another phosphorylation site located in the PIWI domain of
Ago2. Phosphorylation of a specific residue does not alter small RNA binding. However, it
interferes with Ago2 cleavage activity by affecting the catalytic center.
In summary, we have found that different phosphorylation events influence Ago activities by
different mechanisms.
Gunter Meister, PhD
University of Regensburg
Universitaetsstrasse 31
Regensburg, 93053
Germany
Telephone: 0049 941 943 2847
Email: gunter.meister@vkl.uni-regensburg.de
21
Session 3: RNA Biology: New Frontiers
Mark Kay
Thursday, September 8, 2011
15:30 – 16:00
The mechanism of miRNA and siRNA Argonaute loading
in mammals
Mark A. Kay, Shuo Gu, and Lan Jin
Depts of Pediatrics and Genetics
Stanford University School of Medicine
Stanford, CA USA
The mammalian RNA induced silencing complex (RISC) contains a single-stranded RNA
derived from a duplex miRNA/siRNA and one-of-four Argonaute (Ago) proteins. Argonaute
proteins contain four domains referred to as: N-terminal, Paz, MID and PIWI. We have performed various genetic analyses in living cells to study the molecular process of RISC assembly and activation. Ago loading is the process of duplex RNA association followed by removal of the inactive passenger strand RNA. We have established that mammalian Agos bind
duplex RNAs without discrimination and the passenger strand removal is the rate-limiting
step during non-cleavage RISC activation. The degree of RISC activation correlated with the
thermodynamic instability of the duplex RNAs rather than their structure. Interestingly, Ago
loading of siRNAs was less sensitive to the thermostability compared to their shRNA counterparts. By studying RISC loading in Paz-deficient Ago mutants, we established that the PAZ
domain plays a critical role in the unwinding of duplex RNAs. We will discuss the process
and provide a model for RISC loading. Taken together, these results provide the rationale for
future strategies in siRNA and shRNA designs including insights into how to devise potent si/
shRNAs with minimal off-target effects for therapeutic applications.
Mark A. Kay, MD, PhD
Dennis Farrey Family Professor
Departments of Pediatrics and Genetics
Vice Chair for Basic Research (Pediatrics)
Stanford University School of Medicine
269 Campus Drive
CCSR Bldg., Suite 2105
Stanford, CA USA 94305
Telephone: 650-498-6531
Email: markay@stanford.edu
22
Session 4: Delivery of Nucleic Acids
Muthiah Manoharan
Thursday, September 8, 2011
16:30 – 17:00
CHEMICAL STRATEGIES FOR DELIVERY OF RNAi DRUGS
Muthiah Manoharan
Alnylam Pharmaceuticals, 300 Third Street, Cambridge MA 02493 USA
At Alnylam Pharmaceuticals, we have developed and applied multiple chemistry strategies
to address the challenge of cellular delivery of drugs that function through RNAi pathways.
These include chemical modifications of oligonucleotides, molecular conjugates and delivery
systems based on liposomal nanoparticles (LNPs).
Our progress in these areas will be summarized.
References
I. Chemical Modifications of RNAi
1. Manoharan, M. “RNA interference and chemically modified small interfering RNAs.” Curr. Opin. Chem. Biol. 2004, 8, 570-579.
2. Bumcrot, D. et al. “RNAi therapeutics: a potential new class of pharmaceutical drugs.”
Nature Chemical Biology 2006, 2, 711-719.
3. Manoharan, M. and Rajeev, K. G. “Utilizing chemistry to harness RNA interference pathways for therapeutics: chemically modified siRNAs and antagomirs.” Antisense Drug
Technology (2nd Ed.), 2008, 437-464.
4. Zlatev, I. et al. “Efficient solid-phase chemical synthesis of 5’-triphosphates of DNA,
RNA and their analogs.” Organic Letters 2010, 12, 2190-2193.
5. Watts, J. et al. “Effect of chemical modifications on modulation of gene expression by
duplex antigene RNAs that are complementary to non-coding transcripts at gene promoters.” Nucleic Acids Res. 2010, 38, 5242-5259.
6. Addepalli, H. et al. “Modulation of thermal stability can enhance the potency of siRNA.”
Nucleic Acids Res. 2010, 38, 7320-7331.
7. Manoharan, M. et al. “Unique gene-silencing and structural properties of 2’-F modified
siRNAs.” Angewandte Chemie, (International Edition) 2011, 50, 2284-2288.
8. Pallan, P. S. et al.;. Unexpected origins of the enhanced pairing affinity of 2’-fluoromodified RNA. Nucleic Acids Res. 2011, 39, 3482-3495.
II. Conjugates for siRNA and Antagomir Delivery
9. Soutschek, J. et al. “Therapeutic silencing of an endogenous gene by systemic
administration of modified siRNAs.” Nature, 2004, 432, 173-178.
10.Kruetzfeldt, J. et al. “Silencing of microRNAs in vivo with ‘antagomirs’.”
Nature, 2005, 438, 685-689.
11. Wolfrum, C. et al., “Mechanisms and optimization of in vivo delivery of lipophilic
siRNAs.” Nature Biotech. 2007, 25, 1149-1157.
12.Wu, Y. et al. “Durable protection from herpes simplex virus-2 transmission following
intravaginal application of siRNAs targeting both a viral and host gene.” Cell Host &
Microbe 2009, 5, 84-94.
13.Querbes, W. et al. “Direct CNS delivery of siRNA mediates robust silencing in
oligodendrocytes.” Oligonucleotides 2009, 19, 23-30.
Continued on the next page
23
Session 4: Delivery of Nucleic Acids
Muthiah Manoharan
Thursday, September 8, 2011
16:30 – 17:00
Continued from the previous page
14.Chen, Q. et al. “Lipophilic siRNAs mediate efficient gene silencing in oligodenrocytes
with direct CNS delivery.” Journal of Controlled Release 2010, 144: 227-232.
15.DiFiglia, M. et al. “Therapeutic silencing of mutant huntingtin with siRNA attenuates
striatal and cortical neuropathology and behavioral deficits.” PNAS, 2007 104,
17204-17209.
16.Alam, Md. R. et al. Multivalent Cyclic RGD Conjugates for Targeted Delivery of Small
Interfering RNA. Bioconjugate Chemistry, 2011, (in press).
17.Jayaprakash, K. N. et al. “Non-nucleoside building blocks for copper-assisted and copper-free click chemistry for the efficient synthesis of RNA conjugates.” Organic Letters
2010, 12, 5410-5413.
18.Yamada, T. et al. “Versatile site-specific conjugation of small molecules to siRNAs using
click chemistry.” Org. Chem., 2011, 76, 1198-1211.
III.Liposomal Nanoparticles (LNPs)
19.Zimmermann, T. et al. “RNAi-mediated gene silencing in non-human primates.” Nature,
2006, 441, 111-114.
20.Akinc, A. et al. “A combinatorial library of lipid-like materials for delivery of RNAi
therapeutics.” Nature Biotech. 2008, 26, 561-569.
21.Frank-Kamenetsky, M. et al. “Therapeutic RNAi targeting PCSK9 acutely lowers plasma
cholesterol in rodents and LDL cholesterol in nonhuman primates.” PNAS, 2008, 105,
11915-11920.
22.Akinc, A. et al., “Development of lipidoid-siRNA formulations for systemic delivery to
the liver.” Molecular Therapy, 2009, 17, 872-879.
23.Love, K. T. et al., “Lipid-like materials for low-dose, in vivo gene silencing.”PNAS 2010,
107, 9915.
24.Akinc, A. et al. “Targeted delivery of RNAi therapeutics.” Molecular Therapy 2010, 18,
1357-1364.
25.Semple, S. et al. “Rational design of cationic lipids for siRNA delivery.”
Nature Biotechnology 2010, 28, 172-176.
Muthiah Manoharan, PhD
Alnylam Pharmaceuticals
300 Third Street
Cambridge MA 02493 USA
Telephone: 617-551-8200
Email: mmanoharan@alnylam.com
24
Session 4: Delivery of Nucleic Acids
Gilles Divita
Thursday, September 8, 2011
17:00 – 17:30
NON-COVALENT PEPTIDE-BASED DELIVERY SYSTEMS
G. Divita, A. Rydstrom, S. Deshayes, K. Konate
CRBM-CNRS-UMR5237, Dpt-Molecular Biophysics & Therapeutics, 1919 route de Mende,
Montpellier, France
The development of siRNA, has provided great hope for therapeutic targeting of specific
genes responsible of patholological disorders. However their clinical application remains
limited by their poor cellular uptake and insufficient capability to reach targets in vivo.
We have designed the “NANOVEPEP” strategy based on short amphipathic peptides that
forms stable positively-charged nanoparticles with siRNA, through non-covalent electrostatic
and hydrophobic interactions. Self assembly of NANOVEPEP molecules around siRNA
leads to a “raspberry”-like nanoparticle associating several peptide/siRNA complexes of
15-25 nm, cemented together by a matrix of free peptides. NANOVEPEP promotes efficient
targeted-delivery of siRNA or small oligonucleotides into a wide variety of cell lines. Cellular
uptake mechanism of NANOVEPEP/siRNA nanoparticles is dependent on the size of the
particle and involves membrane dynamic. When applied by systemic intravenous injection,
NANOVEPEP promotes the delivery of siRNAs in most of the tissues without triggering
any nonspecific inflammatory response. The surface of NANOVEPEP particles can easily be
functionalized and we showed that addition of cholesterol-, peptide- or antibody-moiety significantly improves stability in vivo and enables lung, brain, ganglion or tumor targeting. The
therapeutic potential of this strategy have been validated for cancer treatment by targeting
major cell cycle regulatory proteins in various mouse tumour models. We demonstrated that
NANOVEPEP-mediated delivery of Cyclin B1 siRNA (0.1 mg/Kg) prevents tumour growth
in vivo following systemic intravenous injections. Given the robustness of the biological
response achieved through this approach, we propose that non-covalent, NANOVEPEP based
technologies hold a strong promise for therapeutic administration of siRNA.
Gilles Divita, PhD
Centre de Recherches de Biochimie Macromoléculaire
UMR 5237, CNRS, UM-1, UM-2
CRBM-Department of Molecular Biophysics and Therapeutics
Montpellier, France
Telephone: 33 (0) 4 67613392
Email: gilles.divita@crbm.cnrs.fr
25
Session 4: Delivery of Nucleic Acids
Ken Clark
Thursday, September 8, 2011
17:30 – 18:00
INVESTIGATING THE POTENTIAL OF THERAPEUTIC
OLIGONUCLEOTIDES FOR PULMONARY DISEASES
Ken Clark, Stephen Hughes, Jeremy Kitson, Jill Coates, Joel Parry, Chris Gruenloh,
Daren Levin, Pallav Bulsara, Scott Summerfield & Mark Edbrooke
Respiratory Therapeutics and Platform Technology & Science, GlaxoSmithKline, Stevenage,
United Kingdom & Research Triangle Park, USA
• Respiratory disease is an attractive opportunity for oligonucleotide therapeutics given
the ability to deliver molecules locally by the inhaled or intranasal route. Such local delivery to the airways should achieve high concentrations in the target tissue while minimising systemic exposure, thereby potentially enhancing efficacy and therapeutic index.
• Nevertheless, the airways are adept at responding to and preventing the cellular entry of
oligonucleotides and so new delivery technologies may well be required to address the
significant challenge of turning the promise of therapeutic oligonucleotides in respiratory disease into a reality for patients.
• Significant progress has being made by many groups and several inhaled oligonucleotide
programmes have now advanced to clinical evaluation. This presentation will review
progress in the field with both inhaled siRNA and anti-sense oligonucleotide therapeutics, and discuss the remaining key research and development challenges.
Ken Clark, PhD
Respiratory Therapeutics, GlaxoSmithKline
Stevenage SG1 2NY, United Kingdom
Telephone: +44 7920 567189
Email: Kennth.L.Clark@GSK.com
26
Session 5: Immunobiology of Nucleic Acids
Gunther Hartmann
Friday, September 9, 2011
08:30 – 09:00
RIG-I and tolerance of self-RNA
Gunther Hartmann
Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, Bonn, Germany
The innate immune system detects viruses based on the recognition of viral nucleic acids.
Immunorecognition of viral nucleic acids leads to the initiation of early antiviral immune
responses that limit viral replication and are essential for eliciting acquired immune responses to virus specific antigens. Detection of viral nucleic acids in the cytosol is based on the
family of the so-called RIG-I-like receptors (RLRs). The cytosolic helicase RIG-I is highly
conserved in vertebrates, including mammals, birds and fish, and is ubiquitously expressed in
all cell types and tissues. In previous work we identified the ligand of RIG-I (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 structure which allows the innate virus sensor RIG-I to discriminate self from pathogenic RNA.
We found that a highly conserved amino acid in RIG-I controls immune tolerance of
2′O-methylated cap1 self RNA. Our results demonstrate for the first time a novel principle in
innate immunity: that an immunoreceptor specifically evolved to tolerate defined self
structures rather than to molecularly adapt to foreign molecular structures. RIG-I is the first
example of this new principle, and the same may apply to other innate immunoreceptors.
The structural requirements for RIG-I ligands now led us to new highly active and selective
RIG-I oligonucleotide ligands. We demonstrate that local intratumoral injection of such a
novel synthetic RIG-I oligonucleotide ligand shows superior anti-tumor activity in otherwise
difficult-to treat mouse models of pre-established melanoma. Thus, RIG-I is an extremely
interesting target for the development of new treatments for cancer and viral infection. High
conservation between species allows much better prediction of therapeutic activities
compared to the diverse family of Toll-like receptors with its inter-species differences.
1. Hornung V, … and Hartmann G. 5’-Triphosphate RNA Is the Ligand for RIG-I. Science
2006; Nov 10; 314:994-7.
2. Schlee M, … and Hartmann G. Recognition of 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, PhD
Institute of Clinical Chemistry and Clinical Pharmacology
University Hospital
Sigmund-Freud-Str. 25, 53127 Bonn, Germany
Telephone: +49-22816080
Email: gunther.hartmann@uni-bonn.de
27
Session 5: Immunobiology of Nucleic Acids
Franck Barrat
Friday, September 9, 2011
09:00 – 09:30
Regulation of the Innate Immune Response Using
Oligonucleotide-based Inhibitors of Toll-Like
Receptors
Franck J. Barrat
Dynavax Technologies, Berkeley, California, USA
SLE is an autoimmune disease where the innate tolerance to self nucleic acids is broken with
devastating consequences. The hallmark of the disease is an increased IFN-α signature in the
blood which is tightly associated with levels of autoantibodies and disease activities. Selfnucleic acid recognition by like receptors (TLR)7 and TLR9 on B cells and PDC is believed
to be key in the pathogenesis of SLE promoting immune complexes (IC) and the production
of type I IFN, both of which are associated with the severity of the disease. We have generated oligonucleotide-based bi-functional inhibitors of TLR7&9 (called ImmunoRegulatory
Sequences, IRS) and have shown that these can block IFN production by PDC as well as B
cell activation. In addition, IRS are active in vivo and treatment of lupus-prone mice lead to
reduced disease symptoms and end-organ damage. SLE patient are often treated with glucocorticoids (GC) but under maintenance levels often suffer from disease flares that necessitate high dose pulse therapy. We have shown that PDC were significantly more resistant
to GC induced death in lupus-prone mice, a phenomenon that was completely reversed by
pre-treatment with TLR7&9 inhibitor. These data provide a new understanding of the role of
self recognition of DNA and RNA by TLR as an important parameter during inflammatory
response. These data also stress the potential utilization of TLR7&9 specific inhibitors as cortico-sparing drugs which would be open new possibilities with respect to therapeutic applications. Finally, we have shown that IRS can prevent skin lesions following mechanical injury
by blocking PDC activation in the skin environment. The lead IRS inhibitor, called DV1179,
has recently entered a human clinical trial and its safety will be assessed in multiple ascending doses. The pharmacokinetics and pharmacodynamics properties of DV1179 in rodents
and non-human primates have been key to the design of the trial.
Franck J. Barrat, PhD
Dynavax Technologies
2929 Seventh Street
Berkeley CA 94710 USA
Telephone: +1.510.665.7266
Email: fbarrat@dynavax.com
28
Session 5: Immunobiology of Nucleic Acids
Patricia Fitzgerald-Bocarsly
Friday, September 9, 2011
09:30 – 10:00
INTRACELLULAR TRAFFICKING OF TLR LIGANDS IN HUMAN
PLASMACYTOID DENDRITIC CELLS
Patricia Fitzgerald-Bocarsly
UMDNJ- New Jersey Medical School, Newark, New Jersey, USA
Human plasmacytoid dendritic cells (pDC) are the most potent producers of both type I and
type III interferons in the body, signaling through TLR7 and TLR9. We have demonstrated
that in addition to responding to cell-free virus and CpG A stimuli, pDC also respond vigorously to virus-infected cells with the production of these IFNs. Using conventional
flow cytometry, Amnis ImageStream imaging flow cytometry and confocal microscopy, we
have investigated the uptake and intracellular trafficking of labeled CpG oligonucleotides,
GFP-expressing HSV-1 and membrane and cytoplasm from HSV-infected and uninfected
Raji cells by pDC. Additionally, the preferential uptake of portions of virus-infected cells vs.
uninfected cells by pDC but not conventional (cDC) and the subsequent distinctive trafficking of material from early to late endosomal compartments, IFN production and maturation in
these DC sub-populations will be discussed. Data demonstrating that the autophagy pathway,
including the formation of amphisomes, is activated by virus (HSV, Influenza and HIV) or
CpG A interaction with pDC, and that IFN-a production in these cells is inhibited by inhibitors of the PI-3 kinase pathway/autophagy will also be presented.
Patricia Fitzgerald-Bocarsly, PhD
UMDNJ – New Jersey Medical School
185 So. Orange Ave.
Newark, NJ 07103 USA
Telephone: 973-972-5233
Email: Bocarsly@umdnj.edu
29
Session 6: Targeting Coding RNA
Cy Stein
Friday, September 9, 2011
10:30 – 11:00
Gymnotic Delivery of Antisense Oligonucleotides
Abstract unavailable at time of printing.
30
Session 6: Targeting Coding RNA
Frank Rigo
Friday, September 9, 2011
11:00 – 11:30
Abstract available for review at the meeting.
31
Session 6: Targeting Coding RNA
Erik Stroes
Friday, September 9, 2011
11:30 – 12:00
Clinical development of mipomersen
Abstract unavailable at time of printing.
32
Session 7: Targeting microRNA
Neil Gibson
Friday, September 9, 2011
13:30 – 14:00
THERAPEUTIC TARGETING OF microRNAs
Neil W. Gibson, Balkrishen Bhat, Christine C. Esau, Scott Davis, Jia Tay, Eric Marcusson,
Hubert Chen, Aimee L. Jackson, Lars Karlsson, 1Kathryn J. Moore, 2Andrei Goga
Regulus Therapeutics, San Diego, CA, 1Dept of Cell Biology, NYU School of Medicine,
New York, NY, 2Dept of Medicine, UCSF, San Francisco, CA
microRNAs act as master regulators in biological pathways, and are dysregulated in disease
areas including cancer, metabolism, fibrosis, and inflammation. Their ability to modulate
disease pathways makes targeting microRNAs an exciting new approach for drug discovery.
Oligonucleotides that inhibit microRNA function have been termed anti-miRs. Critical to the
development of anti-miRs as a therapeutic modality are chemical modifications to enhance
stability and target affinity, and an understanding of functional biodistribution of anti-miRs
to cells and tissues of therapeutic interest. Systemic and local delivery of unformulated antimiRs enables broad distribution for targeting microRNA function in a diverse range of tissues
and cell types. We present recent advances in our use of anti-miRs against two specific
targets – miR-33 in metabolic disease and miR-21 in oncology.
miR-33a and b are intronic microRNAs located within the SREBF2 and SREBF1 genes,
respectively. This microRNA family suppresses the expression of the ABCA1 cholesterol
transporter and lowers HDL levels. LDL receptor-deficient mice treated with anti-miR-33
showed an increase in circulating HDL levels as well as enhanced reverse cholesterol
transport to the plasma, liver, and feces. The anti-miR-33-treated mice had reduced plaque
size and lipid content, increased markers of plaque stability, and decreased inflammatory gene
expression. The systemic delivery of an antisense oligonucleotide that targets both miR-33a
and miR-33b increases hepatic expression of ABCA1 and induces a sustained increase in
plasma HDL cholesterol in African green monkeys. These data suggest the modulation of
microRNA function as a promising strategy to treat atherosclerotic vascular disease.
miR-21 is frequently over-expressed and has been shown to correlate with poor outcome in
multiple cancer types. We have used a publically available data set from 86 patients to show
that miR-21 is over-expressed in hepatocellular carcinoma (HCC). Short term treatment with
the anti-miR-21 oligonucleotide in a genetically engineered mouse model of HCC led to a
reduction in tumor formation and an increased survival advantage. Furthermore, inhibition of
miR-21 was clearly demonstrated by analysis of genome wide mRNA expression data from
treated versus untreated tumors. Our findings suggest that miR-21 is a promising candidate
for the therapeutic intervention of liver cancer and further highlights the potential of antimiR-mediated inhibition of microRNAs in cancer.
Overall our data suggest the therapeutic utility of anti-miRs targeting microRNAs involved in
human disease pathogenesis.
Neil W. Gibson, PhD
3545 John Hopkins Ct
San Diego, CA 92130 USA
Telephone: 858-202-6303
Email: ngibson@regulusrx.com
33
Session 7: Targeting microRNA
Thomas Thum
Friday, September 9, 2011
14:00 – 14:30
Therapeutic targeting of individual cardiac cell
types by miRNA antagonists
T. Thum
Institute for Molecular and Translational Thereapeutic Strategies (IMTTS), Hannover
Medical School, Hannover, Germany
Cardiac stress leads to remodeling and development of heart failure. At the cellular level,
cardiac cells are very sensitive to environmental changes and common pathophysiological responses include development of cardiomyocyte hypertrophy, cardiac fibrosis and insufficient
vascularization. In the last years, it was shown that specific microRNAs (miRNA) contribute
to cellular dysfunction in the heart and first miRNA-based therapeutic strategies have been
successfully developed. We here will summarize recent work of our laboratory about the use
of miRNA inhibitors to target various miRNAs and downstream target networks to result in
beneficial cardiac healing and improved function.
Thomas Thum, MD, PhD
Director, Institute for Molecular and Translational Therapeutic Strategies (IMTTS)
Integrated Research Center (IFB-Tx)
Medical School Hannover (MHH)
Carl-Neuberg-Straße 1, 30625 Hannover, Germany
Telephone: +49 511 532-5272 (office)
Email: Thum.Thomas@mh-hannover.de
34
Session 7: Targeting microRNA
Andrea Ventura
Friday, September 9, 2011
14:30 – 15:00
Oncomir-1 in cancer and development: a tale of mice
and men
Andrea Ventura
Memorial Sloan Kettering Cancer Center, New York, NY, USA
MiR-17~92 has emerged as the prototypical oncogenic microRNA cluster in humans
and mice. It encodes six distinct miRNAs that can be grouped into four “seed families”
(miR17/20, miR-18, miR-19a/b and miR-92). We have previously reported the generation
and characterization of mice carrying a targeted deletion of the entire cluster. Homozygous
mutant mice are significantly smaller than their wild type counterpart, die soon after birth
and display a complex array of defects involving heart lungs and lymphocyte development.
One limitation of such an analysis is that these results were obtained by deleting the entire
miR-17~92 locus. As such, little is known with respect to the relative role of each of the six
microRNAs encoded by miR-17~92. I will discuss the results of two parallel lines of investigation that our laboratory is undertaking to address this important issue:
a) We have carried out a systematic genetic analysis by creating an allelic series of knockin mice, each lacking only one of the four “seed families” encoded by miR-17~92. To our
knowledge, this is the first time such an analysis is being carried out in mammals.
b) In a parallel line of investigation, we have used a conditional miR-17~92 knockout allele to
determine the role of this cluster in Myc-induced B cell lymphomas. By using this approach
we show that endogenous miR-17~92 is required in lymphoma to suppress apoptosis via the
concerted action on a number of genes and that this effect is largely, if not exclusively attributable to the miR-19 seed family.
Andrea Ventura, MD, PhD
Dept. of Cancer Biology and Genetics
Memorial Sloan Kettering Cancer Center
408 East 69th, ZRC-1201
New York, NY, 10065 USA
Telephone: 646-888-3068
Email: venturaa@mskcc.org
35
Session 8: Targeting Liver and Tumors
Aby Buchbinder
Friday, September 9, 2011
15:30 – 16:00
EVALUATION OF LOCKED NUCLEIC ACID (LNA)-BASED mRNA
ANTAGONISTS IN CANCER PATIENTS
Buchbinder A, Kalambakas S, Huq N, Berkowitz N
Enzon Pharmaceuticals, Inc., Piscataway, NJ, USA
Background: HIF-1 is a transcription factor that is a critical mediator of angiogenesis, cell proliferation, metabolism, survival, and adaptive responses to stress. EZN-2968 is a potent locked
nucleic acid antisense oligonucleotide that downmodulates HIF-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, tolerability, pharmacokinetics (PK), and pharmacodynamics.
Results: EZN-2968: 49 pts received weekly EZN-2968 doses for 3, 4, or 5 of 6 weeks. Doselimiting toxicities (DLT) were intracerebral bleeding (n=1, 3.5 mg/kg), Grade 3 fatigue (n=2,
8 mg/kg, 18 mg/kg for 5 of 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. EZN2968 was generally well tolerated in previously treated pts with advanced tumors who received
up to 10 cycles of EZN-2968. The best response was SD. Evidence for down-regulation of the
HIF-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-407
2. Tolcher AW, Patnaik A, Papadopoulos KP, et al; AACR 2011 Annual Meeting, Abs.
LB-409
Aby Buchbinder, MD
Enzon Pharmaceuticals, Inc.
20 Kingsbridge Rd
Piscataway, NJ, 08854 USA
Telephone: 1-(732)-980-4817
Email: aby.buchbinder@Enzon.com
36
Session 8: Targeting Liver and Tumors
Dinah Sah
Friday, September 9, 2011
16:00 – 16:30
ALN-TTR, AN RNAi THERAPEUTIC FOR THE TREATMENT OF
TRANSTHYRETIN AMYLOIDOSIS
Dinah W. Y. Sah1, Qingmin Chen1, Susete Costelha2, Jim Butler1, Shannon Fishman1,
Anthony Rossomando1, Lubomir Nechev1, Maria Joao Saraiva2, Teresa Coelho3, Ole B.
Suhr4, David Adams5, Pierre Lozeron5, Philip Hawkins6, Timothy Mant7, Renta Hutabarat1,
Rick Falzone1, Jeff Cehelsky1, Yaysie Figueroa1, Akshay Vaishnaw1, Jared Gollob1
1
Alnylam Pharmaceuticals, Cambridge, MA 02142, USA
2
Molecular Neurobiology Group, Institute for Molecular and Cell Biology, Porto, Portugal
3
Unidade Clinica de Paramiloidose, Porto, Portugal
4
Department of Medicine, Umeå University, Umeå, Sweden
5
National Referral Center for FAP, Le Kremlin Bicêtre, France
6
National Amyloidosis Centre, London, United Kingdom
7
Quintiles Drug Research Unit, London, United Kingdom
Transthyretin amyloidosis (ATTR) is a fatal, autosomal dominant, multisystem disease
caused by abnormal extracellular deposits of transthyretin (TTR) amyloid that lead to
familial amyloidotic polyneuropathy (FAP) and/or familial amyloidotic cardiomyopathy
(FAC), depending on the sites of deposition. More than 100 TTR mutations have been
reported, with the vast majority resulting in amyloid pathology. Wild-type TTR also contributes to pathology and clinical progression. There is a high unmet medical need for new therapies, with liver transplantation being the only available treatment for a subset of FAP patients.
ALN-TTR is a systemically administered lipid nanoparticle-formulation of a small interfering
RNA (siRNA) targeting wild-type and all mutant forms of TTR. This formulation delivers
the siRNA predominantly to the liver, thereby inhibiting TTR synthesis at the primary site
of production. In transgenic mice expressing the human V30M transgene in a heat shock
transcription factor 1 null background, ALN-TTR01 administration led to robust reduction
of TTR mRNA levels in the liver and TTR protein levels in the circulation, and significant
regression of TTR protein in tissues known to be affected by ATTR, including the peripheral
nervous system and gut. These results demonstrate the potential therapeutic benefit of ALNTTR01 for the treatment of ATTR. A Phase 1 randomized, single-blind, placebo-controlled
clinical trial of ALN-TTR01 is underway in Portugal, Sweden, the United Kingdom and
France. The primary objective is to evaluate the safety and tolerability of a single intravenous
dose of ALN-TTR01 in patients with ATTR. Secondary objectives include characterization
of plasma and urine drug pharmacokinetics, and assessment of pharmacodynamic activity
based on measurements of circulating TTR serum levels. In this presentation, an update on
the clinical development of ALN-TTR01 will be provided, as well as an update on the status
of a second generation RNAi therapeutic targeting TTR.
Dinah Sah, PhD
Alnylam Pharmaceuticals
300 Third Street
Cambridge, MA 02142 USA
Phone: +1-617-551-8357
Email: dsah@alnylam.com
37
Session 8: Targeting Liver and Tumors
Henrik Ørum
Friday, September 9, 2011
16:30 – 17:00
TARGETING miRNA-122 FOR THE TREATMENT OF HCV
Abstract unavailable at time of printing.
38
Session 9: Targeting Infection
William James
Saturday, September 10, 2011
08:15 – 08:45
THE DEVELOPMENT OF APTAMERS AS CANDIDATE ANTIVIRAL
AGENTS: TOWARDS A POLYVALENT MICROBICIDE
William James1, Michael D. Moore1, D.H.J. Bunka2, P.G. Stockley2, P.G. Spear3, J. Cookson1,
L. Rabe4, R.D. Cranston4, Brian Sproat5 and I. McGowan4
1. Sir William Dunn School of Pathology, University of Oxford, OX1 3RE, UK
2. Astbury Centre for Structural Molecular Biology, University of Leeds, LS2 9JT, UK
3. Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University,
Chicago, IL 60611, USA
4. Magee-Women’s Research Institute, 204 Craft Ave, Room B505, Pittsburgh, PA
15213, USA
5. Chemconsilium GCV, Jaarmarktstraat 48, 2221 Booischot, Belgium
Since the mid-1980s, we and others have sought to exploit artificial RNAs in order to inhibit
virus replication for therapeutic or preventive purposes. Gene inhibition therapies that seek
to express antisense, ribozyme, decoy and others sequences within the target cell have faced
the formidable challenge of safe and efficient vector delivery in vivo. Fully synthetic virusinhibitory nucleic acids have the advantage of avoiding inefficient and potentially dangerous
gene vectors, and can be modified chemically to enhance their resistance to degradation,
pharmacokinetic properties and cellular delivery. A third approach avoids the challenge of
delivering antiviral RNAs into target cells by using them as smart chemical barriers at the
sites of potential virus entry into the body. Here we describe the development of aptamers
that neutralize the infectivity of the causative viruses of AIDS and genital herpes as potential
multivalent microbicides.
We used the recombinant envelope glycoproteins, gp120 and gD, of HIV-1 and HSV-2,
respectively, as targets for selection of specific ligands from libraries of random-sequence
2’F-pyrimidine-substituted RNA. The pool of high-affinity aptamers were cloned and
screened for neutralization of virus in cell culture, and those with IC50 < 50nM were further
characterized. The secondary structures of selected aptamers were determined and mutational
analysis undertaken to identify the functional domains. In the case of the HIV-1 aptamers,
the precise binding surface on gp120 was identified, and short, fully synthetic versions were
produced that were suitable for translational scale-up. Preclinical studies using short-term
culture of human rectal and cervical explants confirmed the antiviral efficacy of these
lead agents ex vivo.We uncovered a number of nucleases that are able to rapidly degrade
2’-F-modified RNA in rectal and vaginal secretions. We demonstrate that the combination of
selected 2’-O-Me-modifications and Zn2+ cations can fully protect antiviral aptamers under
these circumstances.
William James, PhD
Sir William Dunn School of Pathology
University of Oxford, OX1 3RE, UK
Telephone: +44 1865 275548
Email: William.james@path.ox.ac.uk
39
Session 9: Targeting Infection
Marc Weinberg
Saturday, September 10, 2011
08:45 – 09:15
DEVELOPING THE NEXT GENERATION OF EXPRESSED RNAi
MODALITIES AGAINST RAPIDLY-EVOLVING VIRAL INFECTIONS
Marc S. Weinberg
Antiviral Gene Therapy Research Unit, University of the Witwatersrand, Johannesburg,
South Africa
Expressed precursor mimics of the mammalian RNAi pathway have been extensively exploited
for efficient gene knockdown and for the development of novel therapeutics. However,
to consistently suppress highly mutable viruses such as HIV, a combination of effective antiviral-RNAi moieties, referred to as combinatorial RNAi (co-RNAi), is required to
prevent the emergence of escape mutants that are refractory to targeting. In this talk, I will
elaborate on efforts aimed at improving expressed RNAi precursors for therapeutic application against viral infections, with the emphasis on their function and utility in co-RNAi
applications. Here novel findings in the development of next-generation approaches for antiviral short hairpin (sh) RNAs, long hairpin (lh)RNAs and primary microRNA (pri-miRNA)
mimics are presented.
Marc S. Weinberg, PhD
Department of Molecular Medicine and Haematology,
University of the Witwatersrand Medical School
7 York Rd.,Parktown
Johannesburg 2193
South Africa
Telephone: +27117172561
Email: marc.weinberg@wits.ac.za
40
Session 9: Targeting Infection
John Rossi
Saturday, September 10, 2011
09:15 – 09:45
Dual function therapeutic Aptamer siRNA combinations
John J. Rossi1, Jiehua Zhou1, Katrin Tiemann1, Pritsana Chomchan1, Preston Neff2 and
Ramesh Akkina2
1. Beckman Research Institute of the City of Hope, Duarte, CA; 2. Colorado State University,
Fort Collins, CO
A goal of our research is the application of small RNA based therapeutics for the treatment
of disease. We have developed dual inhibitory RNA aptamers that function as specific inhibitory agents and delivery vehicles for therapeutic siRNAs. The first aptamer targets the HIV
envelope protein gp 120. This aptamer neutralizes the virus and blocks infection. The same
aptamer can deliver an attached siRNA into HIV infected cells via binding to envelope
expressed on the cell surface, resulting in internalization of the aptamer and delivery of a
dicer substrate siRNA to RISC. In vivo delivery of the aptamer and aptamer-siRNA conjugates into a humanized mouse model for HIV infection resulted in one million fold inhibition
of the virus, demonstrated siRNA directed cleavage of the target mRNA and complete protection of T-cells from HIV mediated T-cell killing. These results represent the first such small
RNA applications for the successful treatment of HIV-1 infection, and could potentially be
used in HIV-1 eradication strategies.
The second aptamer we have evolved binds with high affinity to the BAFFR1 receptor
expressed on B-cells. This receptor is a target for therapeutic treatment of autoimmune diseases
and lymphomas since binding of the Baff ligand to the receptor initiates a B-cell proliferative response. The evolved BAFFR1 aptamer blocks Baff ligand mediated proliferation
of lymphoma cells in culture as well as blocking intracellular signaling pathways. Binding
to BAFFR1 results in internalization of the aptamer allowing the delivery of therapeutic
siRNAs. The specificity of this aptamer for B-cells and its ability to serve as a delivery
vehicle for siRNAs opens up many therapeutic possibilities. The application of this aptamer
in targeting human tumors in humanized mice will be described.
John J. Rossi, PhD
Department of Molecular and Cellular Biology
Beckman Research Institute of the City of Hope
1500 East Duarte Road
Duarte, CA 91010 USA
Telephone: (626) 301-8360
Email: jrossi@coh.org
41
Session 10: Targeting the Central Nervous System
Sponsored by International Society of Neurochemistry
Adrian Krainer
Saturday, September 10, 2011
09:45 – 10:15
Abstract available for review at the meeting.
42
Session 10: Targeting the Central Nervous System
Sponsored by International Society of Neurochemistry
Hermona Soreq
Saturday, September 10, 2011
10:15 – 10:45
Micro-RNAs in the Neuroimmune Interface: From
Inflammation to Lethal poisoning protection
Hermona Soreq
The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Rapidly converging lines of evidence document active involvement of micro-RNAs (miR)
in both neuronal and immune functions. However, the role of miRs in the communication between the nervous and the immune system and its functional consequences
remained uncovered. Here, I report that miRs involved in either neuronal or immune
cells (e.g. miR-132, -212, -155 and -146a), are likely to participate in the corresponding
neuroinflammatory dialogue. This implies that in response to inflammatory insults, such miRs
may alter cognitive processes by targeting numerous mRNA participants of diverse signaling pathways. Of note, brain penetration of peripheral miR-regulated cytokines and other
inflammatory agents can affect neurotransmission, plasticity, cognitive and stress responses.
Specifically, the miR-132-/212 cluster targets, among other mRNAs the transcriptional
activator CREB, the viral infection regulator p300 and the acetylcholine hydrolyzing
enzyme acetylcholinesterase (AChE). When over-expressed under TLR4 or TLR9 insults,
miR-132 reduces AChE activity in immune cells, enhancing parasympathetic control over
the resolution of inflammation. At the same time, up-regulation of the miR-132/-212 cluster in the dorsal striatum limits the risk of drug addiction through modulating CREB, but
the suppressed inflammatory response also reduces neuronal protection from viral infection
(e.g.encephalitis). Thus, miR-132/-212 mal-functioning may co-modify the risk of many
inter-related syndromes with distinct neuro-inflammatory characteristics (e.g. drug addiction,
Alzheimer’s and Parkinson’s diseases, multiple sclerosis, amyotrophic lateral sclerosis).
The future promise of miR-based studies therefore depends on in-depth understanding of
their role in the finely tuned, multi-leveled balance of body-to-brain communication.
Hermona Soreq, PhD
The Hebrew University of Jerusalem
Dept. of Biological Chemistry
Edmond J. Safra Campus
Jerusalem 91904 Israel
Telephone: 972-2-6585109
Email: soreq@cc.huji.ac.il
43
Session 10: Targeting the Central Nervous System
Alon Chen
Sponsored by International Society of Neurochemistry
Saturday, September 10, 2011
10:45 – 11:15
THE ROLE OF microRNA IN REGULATING THE CENTRAL STRESS
RESPONSE
Chen A.
Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel
The etiology and pathophysiology of anxiety and mood disorders is linked to inappropriate
regulation of the central stress response. To determine if microRNAs have a functional role
in the regulation of the stress response, we inactivated microRNA processing by a lentiviral
induced local ablation of the Dicer gene in the central amygdala (CeA) of adult mice.
CeA Dicer ablation induced a robust increase in anxiety-like behavior, while manipulated
neurons survive and appear to exhibit normal gross morphology in the period examined.
We also observed that acute stress in wild type mice induced a differential expression profile
of microRNAs in the amygdala. Bioinformatic analysis identified putative gene targets for
these stress-responsive microRNAs, some of which are known to be associated with stress.
One of the prominent stress-induced microRNAs found in this screen, miR-34c, induced
anxiolytic behavior following challenge when lentivirally over-expressed in the adult CeA.
Of particular interest, one of miR-34c targets is the stress-related corticotropin releasing
factor receptor type 1 (CRFR1) mRNA, regulated via a single evolutionary conserved seed
complementary site on its 3’UTR. Additional in vitro studies demonstrated that miR-34c
reduces cells’ responsiveness to CRF in neuronal cells endogenously expressing CRFR1.
Our results suggest a physiological role for microRNAs in regulating the central stress
response, and position them as potential targets for treatment of stress-related disorders.
Alon Chen, PhD
Department of Neurobiology, Weizmann Institute of Science
Rehovot 76100, Israel
Telephone: +972-8-9344490
Email: alon.chen@weizmann.ac.il
44
Session 11: Targeting Genetic Diseases
Toni Cathomen
Saturday, September 10, 2011
11:45 – 12:15
EXPLORING AND EXPLOITING DESIGNER NUCLEASES FOR
TARGETED GENOME ENGINEERING
Toni Cathomen
Dept. of Experimental Hematology, Hannover Medical School, Hannover, Germany
Designer nucleases have developed into powerful tools to edit the genomes of complex
organisms at will. The most widely used class of tailored nucleases are zinc-finger nucleases
(ZFNs), which consist of an engineered, highly specific DNA-binding domain and a nonspecific endonuclease domain. ZFNs have been employed to trigger the targeted editing of
genomes at over 50 different gene loci in more than 10 organisms, including model organisms,
such as fruitfly, zebrafish, rat, mouse, and pig, as well as human stem cells. In my talk, I will
summarize the technological innovations that have successfully catapulted ZFNs into the
human gene therapy arena and provide an overview of parameters that determine ZFN
activity and ZFN-associated toxicity, both key qualities in any therapeutic application
involving designer nucleases. As a final point, I will present data that introduce TALe-based
nucleases (TALeNs) as a valuable alternative to ZFNs.
Toni Cathomen, PhD
Department of Experimental Hematology
Hannover Medical School
Carl-Neuberg-Str. 1
30625 Hannover, Germany
Telephone: +49 511 532 5170
Email: Cathomen.toni@mh-hannover.de
45
Session 11: Targeting Genetic Diseases
Hein te Riele
Saturday, September 10, 2011
12:15 – 12:45
Oligonucleotide-directed gene modification in
mouse embryonic stem cells to study variants of
cancer-related genes
Hein te Riele, Marieke Aarts, Eva Wielders, Marleen Dekker, Sandra de Vries, Rob Dekker
The Netherlands Cancer Institute, Amsterdam, The Netherlands
Gene targeting by single-stranded oligodeoxyribonucleotides (ssODN) is emerging as a
powerful tool for site-specific modification of the mammalian genome. We have focused
on developing protocols for ssODN-directed base-pair substitution in mouse embryonic
stem cells (ESC) since subtle modification rather than full ablation of genes may yield better mouse models for human disease. As readout for successful ‘oligo targeting’ in ESC, we
measure reactivation of chromosomally-located, disabled neomycin (neo) or EGFP reporter
genes. We demonstrated the feasibility of ssODN-directed gene reactivation in ESC, but
found that its efficacy was strongly suppressed by the DNA mismatch repair system (MMR).
We have shown that wild-type ESC can be rendered permissive for oligo targeting by transiently suppressing MSH2 or MLH1 protein levels by RNA interference. This raised the gene
correction efficiency 200-500-fold up to a frequency of 10-5-10-4. Furthermore, we have discarded transcription and homologous recombination as critical mediators, but found evidence
that the ssODN becomes integrated into the genome in the context of replication. Finally, we
have shown that successfully targeted ESC retained pluripotency, allowing ssODN-modified
alleles to be introduced into the mouse germ line.
We use ‘oligo targeting’ to reconstruct in murine ESC unclassified variant alleles of MMR
genes that are frequently found in the human population. While large deletions, premature
stop codons and frameshifts completely abrogating gene function are obviously pathogenic
and predispose to cancer (HNPCC/Lynch syndrome), missense mutations affecting only a
single amino acid are more difficult to interpret. ESC in which such “variants of uncertain
significance” (VUS) or “unclassified variants” (UV) have been introduced, are studied by
functional assays that address main MMR functions relevant for maintaining genome integrity. Our approach allows variant alleles to be expressed from the endogenous locus, which is
crucial as both higher and lower levels of MMR proteins are notorious for affecting MMR capacity. In cases where the functional implications are less clear, or when a mutation only partially affects MMR capacity, mice can be generated from mutant ESCs to investigate whether
attenuated MMR activity promotes tumorigenesis in vivo. We will present results of several
MSH2 VUS that have been found in (suspected) HNPCC families.
Hein te Riele, PhD
The Netherlands Cancer Institute, Division of Molecular Biology
Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
Telephone: 31-20-512-2084
Email: h.t.riele@nki.nl
46
Session 11: Targeting Genetic Diseases
Marie Dutreix
Saturday, September 10, 2011
12:45 – 13:15
siDNA, A NEW STRATEGY FOR INHIBITING DNA REPAIR PATHWAYS
M. Dutreix1, J-S. Sun2
1
Institut Curie research center, Institut National de la Santé et de la Recherche Médicale
U1021, Centre National de la Recherche Scientifique UMR3427, Paris-Orsay, France;
2
DNA Therapeutics, Genopole, Evry, France
Enhanced DNA repair activity is often associated with tumor resistance to radiotherapy (RT)
or chemotherapy (CT). Various strategies combining RT and CT with in situ gene therapies or
molecular therapies inhibiting DNA repair have been developped to maximize the selective
pressure against cancer cell growth while minimizing treatment associated toxicity. All these
therapies target one main enzyme of a specific DNA repair pathway such as the PARP, DNAPK, CHK2... However, tumors can escape to their effect by modifying the target or enhancing
activity of another alternative repair pathway. Here we propose a new strategy that consists
in hyperactivating the DNA damage signaling by introducing a false damage signal in cells.
This activation results in the lost of the spatio-temporal organization of the cellular response
to DNA damage (DDR) and the disabling of all the repair machinery. We designed short DNA
bait molecules that mimic DNA double-strand breaks (called Dbait). In transfected cells,
Dbait binds DNA-PK complex and PARP enzymes and triggers their activities in cytoplasm
as well as nucleus in absence of DNA damage on chromosomes. This Dbait hijacked damage
response (DHDR) prevents further detection of damage and thereby inhibits repair of most
DNA damage. In vitro, cells show extended parylation signal and DNA-PK targets phosphorylation after Dbait treatment. Surprisingly, the DHDR did not disrupt cell cycle and proliferation of non tumoral cells as primary or transformed fibroblasts. However, it was toxic for
numerous tumoral cells line. This selective toxicity was confirmed on human xenografted
tumours in which Dbait triggers a DHDR detected by immunohistology. Dbait administration
reduces in a dose dependent manner the tumor growth in different animal models. Moreover,
it acts in synergy with radiotherapy and increases significantly xenografted animal survival
without adding toxicity on the irradiated healthy tissues. A “first-in-human” clinical trial
(DRIIM) will start on September 2011 to assess tolerance of a combined treatment of radiotherapy with Dbait.
Marie Dutreix, PhD
UMR ETIC, Institut Curie, Bat110
Centre Universitaire
91405, Orsay, France
Telephone : +33(0)169867186 Email : marie.dutreix@curie.fr
47
POSTER ABSTRACTS
001
siDNA, A NEW STRATEGY FOR INHIBITING DNA REPAIR PATHWAYS
M. Dutreix1, J-S. Sun2
1
Institut Curie research center, Institut National de la Santé et de la Recherche Médicale
U1021, Centre National de la Recherche Scientifique UMR3427, Paris-Orsay, France;
2
DNA Therapeutics, Genopole, Evry, France
Enhanced DNA repair activity is often associated with tumor resistance to radiotherapy (RT)
or chemotherapy (CT). Various strategies combining RT and CT with in situ gene therapies or
molecular therapies inhibiting DNA repair have been developped to maximize the selective
pressure against cancer cell growth while minimizing treatment associated toxicity. All these
therapies target one main enzyme of a specific DNA repair pathway such as the PARP, DNAPK, CHK2... However, tumors can escape to their effect by modifying the target or enhancing
activity of another alternative repair pathway. Here we propose a new strategy that consists
in hyperactivating the DNA damage signaling by introducing a false damage signal in cells.
This activation results in the lost of the spatio-temporal organization of the cellular response
to DNA damage (DDR) and the disabling of all the repair machinery. We designed short DNA
bait molecules that mimic DNA double-strand breaks (called Dbait). In transfected cells,
Dbait binds DNA-PK complex and PARP enzymes and triggers their activities in cytoplasm
as well as nucleus in absence of DNA damage on chromosomes. This Dbait hijacked damage
response (DHDR) prevents further detection of damage and thereby inhibits repair of most
DNA damage. In vitro, cells show extended parylation signal and DNA-PK targets phosphorylation after Dbait treatment. Surprisingly, the DHDR did not disrupt cell cycle and proliferation of non tumoral cells as primary or transformed fibroblasts. However, it was toxic for
numerous tumoral cells line. This selective toxicity was confirmed on human xenografted
tumours in which Dbait triggers a DHDR detected by immunohistology. Dbait administration
reduces in a dose dependent manner the tumor growth in different animal models. Moreover,
it acts in synergy with radiotherapy and increases significantly xenografted animal survival
without adding toxicity on the irradiated healthy tissues. A “first-in-human” clinical trial
(DRIIM) will start on September 2011 to assess tolerance of a combined treatment of radiotherapy with Dbait.
Marie Dutreix, PhD
UMR ETIC, Institut Curie, Bat110
Centre Universitaire
91405, Orsay, France
Telephone : +33(0)169867186 Email : marie.dutreix@curie.fr
49
002
Development of systemic antisense treatment in
dystrophic mouse models for Duchenne Muscular
Dystrophy
Annemieke Aartsma-Rus1, Christa de Winter1, Maaike van Putten1, Tatyana Karnaoek2, Judith
van Deutekom2 and Gert-Jan van Ommen1
1
Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands
2
Prosensa BV, Leiden, the Netherlands
Antisense-mediated reading frame restoration is one of the most promising therapeutic approaches for Duchenne muscular dystrophy (DMD). It uses antisense oligonucleotides (AONs)
to induce exon skipping during pre-mRNA splicing of mutated dystrophin transcripts. This is
aimed to restore the disrupted open reading frame and allow synthesis of internally deleted,
partly functional Becker-like dystrophin proteins. Proof of concept has been obtained in
cultured cells and the mdx mouse model and this approach is currently tested in clinical trials
by Prosensa/GSK (GSK2402968 and PRO044) and AVI Biopharma (AVI-4658).
Dystrophic animal models allow detailed analysis of pharmacokinetic and pharmacodynamic
effects of the 2’-O-methyl phosphorothioate (2OMePS) chemistry used in the Prosensa/
GSK trials AONs. Uptake by healthy muscle is low, but we have previously shown that after
systemic delivery of 2OMePS AONs, the AON levels in dystrophic skeletal muscle are up to
10-fold higher, and that subcutaneous delivery of 2OMePS is possible.
Here, we optimized dosing and maintenance regimes using subcutaneous 2OMePS AON
injections in the mdx mouse model. In addition, we tested the safety and efficacy of high dose
(200 mg/kg/week) AON treatment for up to 6 months in mouse models with varying levels of
severity: mdx mice (mild phenotype) and mdx mice with one utrophin allele (mdx +/-; intermediate phenotype). This was well tolerated during treatment and liver and kidney weights
and serum parameters were similar treated mice compared to saline treated controls at the end
of treatment. Notably, in the more severely affected mdx +/- mice the therapeutic effect was
larger: exon skip and dystrophin levels were higher, the creatine kinase levels were more decreased and rotarod running time was more increased. Preliminary results suggest that AON
levels in the muscles of the more severely dystrophic mdx +/- are higher than in those in mdx
mice, confirming the hypothesis that AON uptake is aided by the disease pathology.
These results indicate that long term subcutaneous treatment with 2OMePS AONs is safe and
efficient in dystrophic mouse models, which is encouraging for long term trials in patients,
recently initiated by Prosensa Therapeutics/GSK.
Annemieke Aartsma-Rus, PhD
Albinusdreef 2
2333 ZA Leiden
The Netherlands
Telephone: +31 71 5269436
Email: a.m.rus@lumc.nl
50
003
AUTO-VECTORIZED microRNA MIMIC-BASED NANOSYSTEM FOR
HEPATOCELLULAR CARCINOMA THERAPY
Aimé A., Bestzina N., Maurel M., Midoux P., Grosset C., Bestel I.
INSERM U869, Victor Segalen University, Bordeaux, France
MicroRNA (miR) are short endogenous, non coding RNA that regulate gene expression
by controlling mRNA translational or degradation. Recent studies indicate that miR have
essential roles in many basic biological processes including development, cell proliferation,
differentiation and apoptosis but also in several pathologies. In carcinogenesis, the roles of
miR as novel tumor suppressors or oncogenes has been clearly demonstrated pointing out
miR as new therapeutic targets.
Our project aims at developing new therapeutic molecules capable of modulating gene
expression and blocking the growth of tumoral hepatic cells by using the regulatory and
biological properties of miR.
C. Grosset Team recently observed that miR-96 whose expression is profoundly altered in
hepatocellular carcinoma (HCC), downregulates expression of glypican 3 (Gpc3). Gpc3
which is one of the most overexpressed pro-tumoral gene in HCC tissues plays a role in liver
cancer as inducer of proliferation of malignant hepatic cells. Therefore, restoring high levels
of miR-96 constitutes a very promising avenue in HCC therapy.
Our strategy consists in the design, synthesis and delivery of amphiphilic auto-vectorized
miR-96 mimics. These molecules deal with an oligonucleotide sequence which contains the
biological activity and a hydrophobic vector which enables the formation of original nanosystems that can impact on cell penetration. These two parts have been extensively optimized
leading to a small chemical miR-96 mimic library.
In parallel to physico-chemical studies of amphiphilic miR-96 mimic self assemblies, the
functional efficiency of these molecules, by using the FunREG method was evaluated.
Moreover, to get further insights into the impact of the hydrophobic part on cell penetration,
cellular uptake pathways were studied. The essential of the results will be presented here.
Ahisan Aimé, 2nd year PhD student
INSER U869 Université Victor Segalen Bordeaux II
1er étage, porte 12 146, Rue Léo Saignat
33 076 Bordeaux Cedex - France
Telephone: 0033 6 65 24 96 94
Email: ahissan.aime@inserm.fr / audreyflora@yahoo.fr
51
004
DEVELOPMENT OF A MICRO-RNA-34 THERAPEUTIC:
PHARMACOLOGICAL DRUG DELIVERY & PREDICTING THE
FUTURE PATIENT POPULATION
Andreas G. Bader, David Brown, Jason Wiggins, Jane Zhao, Christopher Daige,
Michael Omotola, Kevin Kelnar, Neil Leatherbury, Jay Stoudemire, Paul Lammers
Mirna Therapeutics, Inc., Austin TX
Mirna Therapeutics is developing miRNA mimics that are modeled after naturally occurring
tumor suppressor miRNAs. Among these is miR-34, a tumor suppressor miRNA that is lost
or expressed at reduced levels in a broad range of human cancer types. miR-34 plays an integral role in the p53 tumor suppressor pathway and regulates genes to block cell cycle and
induce apoptosis. A therapeutic benefit of miR-34 appears to be the ability to inhibit cancer
stem cells. We have validated the concept of miRNA replacement therapy and demonstrated
that the therapeutic delivery of miR-34 mimics inhibits cancer cell proliferation and tumor
growth in multiple mouse models of cancer. The focus is now on pharmacological drug
delivery to establish optimal PK/PD and tox parameters, as well as assessing the effects of
miR-34 mimics in isogenic cancer lines to identify genotypes of cancer patients that are most
likely to respond to therapy. Examples will be presented.
Andreas G. Bader, PhD
Associate Director, Research
2150 Woodward Street, Suite 100
Austin, TX 78744 USA
Telephone: 512-681-5241
Email: abader@mirnarx.com
52
005
PTPIP51 – A POSSIBLE TARGET FOR NOVEL DIABETES AND
OBESITY THERAPIES
Bobrich MA1, Schwabe SA1, Kamm M1, Brobeil AM1, Krüger K2, Mooren FC2, Steger K3,
Tag C1, Wimmer M1
1
Institute of Anatomy and Cell Biology, Justus-Liebig-University, Giessen, Germany
2
Department of Sports Medicine, Justus-Liebig-University, Giessen, Germany
3
Department of Urology and Pediatric Urology, Justus-Liebig-University,
Giessen, Germany
Our objective was to investigate the role of Protein Tyrosine Phosphatase Interacting Protein
51 (PTPIP51) in the development of obesity and Insulin resistance.
We included a total of 60 mice in the study. Eighteen control animals were fed a standard
diet, sixteen were fed a high-fat diet, ten were submitted to a treadmill training parallel to the
feeding of a normal diet and sixteen animals were submitted to a treadmill training parallel
to the feeding of a high-fat diet. After 10 weeks, a glucose tolerance test was performed to
determine the Insulin resistance of the animals. We investigated the collected adipose tissue
samples of the animals by using immunohistochemistry, western blot, PCR and Duolink
proximity ligation assay (DPLA).
Our investigations revealed that PTPIP51 interacts with all included proteins in adipose tissue
of different Insulin resistant states. In Insulin sensitive normal fed animals, the interaction
between PTPIP51 and PTP1B was high, as also seen in trained high fat diet animals. Insulin
resistant high fat diet fed animals displayed lower interaction levels of both proteins. The
interaction profile of PTPIP51 with 14-3-3beta showed different patterns. Interaction was
very high in trained high fat diet animals, whereas high fat animals displayed the lowest
interaction of both proteins and normal fed animals displayed only slightly higher levels of
interaction. The interaction profile with PTP1B, an inhibitor of the Insulin receptor, hints to
a positive effect of the interaction of both proteins on the Insulin sensitivity. In addition to
that, PTPIP51 interacts with the Insulin Receptor, independent of feeding status. Endurance
training in normal fed animals led to a decrease of the interaction. We conclude that PTPIP51
could act as a docking agent for both inhibitors and activators of the Insulin receptor, hence
being able to switch the Insulin signalling pathway on and off. The interaction with 14-33beta indicates that PTPIP51 is involved in modulation processes in adipocytes through the
MAPK-pathway. All these facts make PTPIP51 an interesting protein in Insulin resistance,
Diabetes and obesity. Further investigations will disclose the effect of inhibition of PTPIP51
with focus on pharmaceutical therapy of Diabetes.
Manuel Anton Bobrich, Student
Aulweg 123, 35392 Gießen, Germany
Telephone: +49641 99 47012
Email: Manuel.Bobrich@anatomie.med.uni-giessen.de
53
006
TOLL-LIKE RECPTORS 3 AND 7/8 SHOW STEREO-SELECTIVITY
AND ARE NOT STIMULATED BY SPIEGELMERS
Buchner, K., Vater, A. and Klussmann, S.
NOXXON Pharma AG, Berlin, Germany
Spiegelmers are L-enantiomeric RNA aptamers showing biostability without any further
chemical modification. Two Spiegelmers (inhibitors of the chemokines MCP-1 and SDF-1)
have entered clinical development and demonstrated an excellent safety profile in phase I
clinical trials. Since certain natural and synthetic RNAs, e.g. single stranded viral RNA
(Heil et al., 2004), especially when stabilized as phosphorothioates, or some siRNA (for
review see Robbins et al., 2009), have been shown to induce the innate immune response
by activating toll-like receptors (TLRs), we investigated, whether Spiegelmers and L-RNA
oligonucleotides in general are able to stimulate TLR7/8 (activated by single stranded RNA)
or TLR3 (activated by double stranded RNA). In experiments with peripheral blood mononuclear cells (PBMC), plasmacytoid dendritic cells and cells expressing recombinant human
TLR8, all of these could be activated with imiquimod or resiquimod and phophorothioatessRNA40 (Heil et al. 2004), but not with functional Spiegelmers (L-ss/dsRNA) or phosphorothioate-ssRNA40 in the enantiomeric (L-) configuration. In experiments with HUVEC which
express TLR3, activation of NF-kB could be observed after stimulation with poly(I:C), but
again, no activation was observed with Spiegelmers. The apparent stereo-selectivity of the
TLRs investigated here and thus the lack of TLR activation by L-RNAs in vitro makes it unlikely that Spiegelmers induce an immunostimulatory response in vivo. This is corroborated
by the lack of inflammatory cytokine expression in pre-clinical and clinical studies and adds a
further point to the list of favorable characteristics of Spiegelmers.
Heil et al. (2004) Science 303:1526
Robbins et al. (2009) Oligonucleotides 19:89
Klaus Buchner, PhD
NOXXON Pharma AG
Max-Dohrn-Str. 8-10
10589 Berlin, Germany
Telephone: +49-30-726247-146
Email: kbuchner@noxxon.com
54
007
A unique MOE-DNA chimeric oligonucleotide induces
MDA-5 dependent induction of type I interferon response
Sebastien A Burel1, Hiroki Sato2, Todd Machemer1, Patrick Cauntay1, Scott P Henry1
1
ISIS Pharmaceuticals, Carlsbad, California, USA
2
Institute for Virus Research, Kyoto University, Kyoto, Japan
Second generation ‘Gapmer’ antisense oligonucleotides (ASO) containing 2’-O-methoxyethylribose (MOE) modifications have been shown to possess both excellent pharmacokinetic
properties and robust pharmacological activity in several animal models of human disease.
These beneficial properties have translated to human therapeutics. Gapmer ASOs are generally well tolerated displaying minimal to mild proinflammatory effect caused by the release of
cytokines via the activation of monocyte and or dendritic cells compared to phosphorophioate
deoxy oligonucleotides at doses far in excess of expected therapeutic doses. While the vast
majority of 2’-MOE ASOs are largely void of hepatotoxicity as characterized by the absence
of serum transaminases increase, a very small subset of ASO with a propensity for producing
acute hepatotoxicity in mice has been identified. The mechanism for these findings is not
clear at this point, but the effects are clearly sequence-specific. Intense effort has been made
to understand the mechanism underlying these effects.
One of those ASO, ISIS 147420, was found to cause profound hepatotoxicity characterized
by increased ALT that was atypical of this class of oligonucleotides. In addition to increased
ALT, subcutaneous injection of ISIS 147420 was associated with extensive hepatocyte
apoptosis and necrosis, as well as mononuclear cell infiltrate in liver at 72 hours. Liver
morphology and ALT levels were normal at 24 or 48 hours. Whole genome gene expression
profiling was performed on livers collected at 8, 24, 48 and 72 hours. A large number of
interferon stimulated genes (ISGs) were significantly upregulated as early as 24 hours.
Administration of ISIS 147420 to Stat1 or IFNAR1 deficient mice showed no evidence of
hepatotoxicity and no induction of ISGs up to 96 hours post treatment. We speculated that
a specific sequence motif might cause ISIS 147420 to be mistaken for viral RNA or DNA
thus triggering an innate immune response ultimately resulting in severe hepatotoxicity. ISIS
147420 toxicity was independent of Toll-like receptors as there was no decrease in ALT in
TRIF or Myd88 deficient mice. The involvement of the cytosolic pattern recognition receptors, RIG-I and MDA-5, were also investigated. Pretreatment of mice with ASOs inhibitors
of IPS-1, an adaptor protein critical to the function of RIG-I and MDA-5, reduced mRNA
level down to 8% of control and prevented the toxicity induced by ISIS 147420 (reduced ALT
and interferon-b levels). Unlike ASO pretreatment with RIG-I inhibitors that was unable to
reduce the severity of hepatotoxicity, pretreatment with MDA-5 antisense inhibitors reduced
MDA-5 mRNA level down to 7% of control and prevented the toxicity induced by ISIS
147420 (reduced ALT and interferon-b levels). These results revealed a novel mechanism
of oligonucleotide mediated toxicity requiring both MDA-5 and IPS-1 and resulting in an
adverse activation of the innate immune response.
Sebastien Burel, PhD
2855 Gazelle Court, Carlsbad, CA 92010 USA
Telephone: 1-760-603-2717
Email: sburel@isisph.com
55
008
Incorporation of LNA into aptamer selection
Meghan A. Campbell, Birte Vester, Jesper Wengel
Nucleic Acid Center, University of Southern Denmark, Odense, Denmark
Aptamers continue to emerge as an important component of nucleic acid biotechnology and
nanotechnology. These single-stranded DNA or RNA molecules can fold into unique tertiary
structures that can bind with high affinity to small molecule or protein ligands. Aptamers are
selected through a process known as Systematic Evolution of Ligands by EXponential enrichment (SELEX), where the best binding nucleic acid sequences are amplified and selected
again. Typical aptamer structures can include stem-loop regions or G-quadruplex motifs, both
of which can be stabilized by post-selection modification with Locked Nucleic Acid (LNA).
Now, through the use of LNA-triphosphates, we are modifying the SELEX cycle to select
LNA-containing aptamers. By including LNA in the selection process, the possibility of new
or more stable aptamer structures is increased. Libraries have been created with LNA modifications at fixed or variable locations within the 40-mer random region. After the random
library is bound to a ligand of interest, and non-binding sequences washed away, the selected
ssLNA sequences are amplified into dsDNA. A dT20-tail is added at the
5’ end of the DNA strands to facilitate purification. This DNA then serves as a template for
asymmetric PCR to regenerate ssLNA sequences. The shorter LNA strand is separable from
the DNA strands by acrylamide gel electrophoresis and is purified in this manner. Purified ssLNA can then be used for the next round of selection and the cycle continues. Following numerous SELEX cycles, the selected LNA strands can be amplified into DNA and sequenced.
Through this process we hope to produce higher affinity and more stabile aptamers for a wide
variety of applications.
Meghan A. Campbell, PhD
Institute for Physics and Chemistry
University of Southern Denmark
Campusvej 55
5230 Odense M, Denmark
Telephone: +45 6550 2548
Email: mcam@ifk.sdu.dk
56
009
[3H]-RADIOLABELING OF SMALL INTERFERING RNA (siRNA)
Christensen J1,2, Andres H1, Natt F1, Hunziker J1, Krauser J1, Swart P1
1
Novartis Institutes of Biomedical Research, Basel, Switzerland
2
Nucleic Acid Center, Department of Physics and Chemistry, University of Southern
Denmark, Odense, Denmark
We describe a simple and efficient final step labeling procedure for single internal [3H]-radiolabeling of oligonucleotides. To our knowledge this is the first reported procedure for single
internal final step radiolabeling of oligonucleotides, with no chemical modification
(3H replacing a 1H), i.e. the reactivity and metabolism of the molecule should remain unchanged. The radiolabel is placed in a chemically stable and predetermined internal position,
which decreases the likelihood of the label to be readily cleaved from the oligonucleotide
in vivo. The radiolabeling takes place on the fully deprotected full length antisense strand
by bromine/tritium exchange catalyzed by palladium on charcoal in the predetermined
5-position of either uridine or 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 phosphoramidite 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, fluorescence), the use of radiolabeled oligonucleotides to perform (pre)clinical in vitro/in vivo studies offers a distinct advantage of avoiding chemical modification of these compounds.
Jesper Christensen, MSc
Novartis Campus, WSJ-153.1.08
CH-4056 Basel, Switzerland
Telephone: +41 79 593 9171
Email: jesper.christensen@novartis.com
57
010
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 targeted cancer diagnostics and therapy. Several RNA aptamers that bind cancer cell-surface
antigens with high affinity and specificity have been described. However, their clinical potential has yet to be realized. An example of this, are RNA aptamers directed against prostate
specific membrane antigen (PSMA), a prostate cancer-specific surface antigen.
The therapeutic potential of the PSMA RNA aptamers has been demonstrated for many applications including inhibition of PSMA’s pro-carcinogenic properties, imaging and delivery
of cytotoxic agents to prostate cancer cells. Given this, further optimization to improve their
pharmacokinetic (PK)/pharmacodynamic (PD) profiles and to facilitate large-scale chemical
synthesis of these RNAs is compelling. Toward this end, we have employed computational
RNA structural modeling and RNA/protein docking models to guide the truncation and
further chemical modification of the A9 PSMA RNA aptamer. These analyses resulted in a
truncated derivative of the A9 aptamer (A9g, 43mer), which due to its reduced length is now
amenable to large-scale chemical synthesis. The theoretical modeling has also enabled the
identification of bases within A9g that when modified (with synthetic bases) impart increased
resistance to nucleases present in human serum. Importantly, we show that the optimized
A9g inhibits PSMA’s pro-carcinogenic activity and when directly applied to cells expressing
PSMA, is effectively internalized, thus enabling the delivery of cytotoxic agents to PSMAexpressing prostate cancer cells. Finally, we demonstrate the potential of this reagent for imaging of prostate tumors using mouse models of metastatic prostate cancer.
In summary, these studies demonstrate the utility of computational RNA structure models for
guiding truncations and chemical modifications of RNA aptamers while retaining their function. Furthermore, these studies have resulted in a PSMA RNA aptamer that due to its optimized in vivo profile and shorter sequence length is now one step closer to the clinic.
Justin P Dassie, BS
375 Newton Rd, 5241 MERF
Iowa City, IA 52242 USA
Telephone: (319) 384-3243
Email: justin-dassie@uiowa.edu
58
011
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. Andreasen
Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C,
8000 Aarhus C, Denmark
The plasminogen activation system plays a role in a wide variety of pathological conditions
such as cancer, inflammatory disorders and cardiovascular diseases. In order to develop
novel potential therapeutics, we have screened libraries of 2´-fluor-containing RNA oligonucleotides for aptamers targeting members of the system. Aptamers to the serine protease
urokinase-type plasminogen activator (uPA) were identified with the ability to prevent the
association of the protein with its receptor (uPAR) on cell-surfaces. In turn, the aptamer prevents cell-associated uPA-mediated plasminogen activation, which is important for cancer
cell invasion and metastasis. While other uPA-uPAR targeting agents are directed towards the
receptor and induce uPA-like proteolysis-independent agonistic downstream effects, the uPA
aptamers are pure antagonists. Aptamers were also isolated against the primary physiological
inhibitor of uPA, the serpin plasminogen activator inhibitor-1 (PAI-1), with ability to block
PAI-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 physiological conditions more than any known other ligand. One aptamer was even able to distinguish
between different conformations of PAI-1, which could be explained by mapping the epitope.
uPA and PAI-1 are established biomarkers for the malignant phenotype of cancers and potential targets for anticancer therapy. The aptamers are therefore potential agents for development of novel anticancer therapeutics and diagnostics.
Daniel M. Dupont, PostDoc
Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C,
8000 Aarhus C, Denmark
Telephone: +45 89 42 50 79
Email: dmd@mb.au.dk
59
012
CLICK NUCLEIC ACID LIGATION
A.H. El-Sagheer1, 2 and T. Brown1
1
School of Chemistry, University of Southampton SO17 1BJ. UK
2
Chemistry Branch, Dept. of Science and Mathematics, Faculty of Petroleum and Mining
Engineering, Suez Canal University, Suez, 43721, Egypt
Click DNA ligation is a remarkably efficient method of joining together DNA and RNA
strands. It utilizes the copper-catalyzed azide-alkyne cycloaddition (CuAAC reaction). The
chemistry has been optimized for the synthesis of cyclic oligonucleotides,1,2 oligonucleotide
catenanes,2 very stable cyclic mini-duplexes,1 duplexes that are linked across the major
groove,3 covalently fixed DNA nanoconstructs4 and large RNA constructs.5 The method produces an unnatural DNA backbone that can be varied by changing the participating alkyne
and azide. Careful design produces a linkage that can be read through by thermostable DNA
polymerases during PCR.6,7 This has potential applications in nanotechnology, gene synthesis
and genetic analysis.
Figure 1. Overlay of triazole DNA (black) on normal DNA (grey) with the triazole ring bottom left.
The largest difference between the two structures is the displacement of the 5’-carbon atom of the deoxyribose sugar (more than 3 angstroms, dotted line).Nevetheless, the triazole linkage does not affect
the overall conformation of the DNA duplex; the changes are localized.. The model is from a highresolution NMR structure in collaboration with Andre Dallman (submitted).
(1) El-Sagheer, A. H.; Kumar, R.; Findlow, S.; Werner, J. M.; Lane, A. N.; Brown, T.
Chembiochem 2008, 9, 50.
(2) Kumar, R.; El-Sagheer, A. H.; Tumpane, J.; Lincoln, P.; Wilhelmsson, L. M.; Brown, T.
J. Am. Chem. Soc. 2007, 129, 6859.
(3) Kocalka, P.; El-Sagheer, A. H.; Brown, T. Chembiochem 2008, 9, 1280.
(4) Lundberg, E. P.; El-Sagheer, A. H.; Kocalka, P.; Wilhelmsson, L. M.; Brown, T.; Norden, B.
Chem. Commun. 2010, 46, 3714.
(5) El-Sagheer, A. H.; Brown, T. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 15329.
(6) El-Sagheer, A. H.; Brown, T. J. Am. Chem. Soc. 2009, 131, 3958.
(7) El-Sagheer, A. H.; Sanzone, A. P.; Gao, R.; Tavassolia, A.; Brown, T. Proc. Natl. Acad. Sci.
U. S. A. in press.
Afaf H. El-Sagheer, PhD
Chemistry Department, University of Southampton
High Field, Southampton, SO17 1BJ, UK
Telephone: +44(0)2380596785
Email: ahes@soton.ac.uk
60
013
NON-VIRAL DELIVERY OF STICKY-SIRNA™ TARGETING THE
CELL CYCLE INHIBITS LUNG TUMOR METASTASIS
Anne-Laure Bolcato-Bellemina, Marie-Elise Bonneta, Omar Zouniba, Elodie Benoita,
Jean-Paul Behrb and Patrick Erbachera
a
Polyplus-transfection, Bioparc, Boulevard Sébastien Brandt, 67401 Illkirch, France
b
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 cancers, lung cancer is the most common in term of incidence and mortality. Metastasis, due to uncontrolled cell growth, is responsible for more than 90% of deaths from lung cancer. Thus, developing
new treatments to inhibit cell metastasis will improve patient prognosis and quality of life.
Since the discovery of RNA interference and its potency for therapeutic applications, this
technology has been widely used to suppress target gene expression involved in many
diseases such as cancer. However, to date, delivery still remains a major obstacle for siRNAbased drugs to be a success.
Polyethylenimine (PEI) has been widely used for in vivo gene delivery. We have recently
developed novel interfering RNA (STICKY-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 attractive 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 3LLLuc). 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 suppress 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 polyethylenimine could become a powerful tool for antitumor therapy.
1
2
Bolcato-Bellemin et al., PNAS, 2007, 104, 16050-5.
Bonnet et al., Pharm Res, 2008, 25, 2972-82.
Anne-Laure Bolcato-Bellemin, PhD
Polyplus-transfection
Bioparc
Boulevard Sébastien Brandt
67401 Illkirch, France
Telephone: +33 3 90 40 61 80
Email: albellemin@polyplus-transfection.com
61
014
SELF-DELIVERING CATIONIC siRNAs MEDIATE EFFICIENT AND
SPECIFIC GENE SILENCING IN HUMAN CELLS
Patrick Erbacher, Valérie Moreau, Clément Paris, Gaëlle Deglane, Bernard Couturier,
Anne-Laure Bellemin, Marie-Elise Bonnet, Jean-Serge Remy*, Jean-Paul Behr*,
Nathalie Lenne
Polyplus-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 culture. While offering great therapeutic promises, the siRNAs approach has been facing strong
difficulties, among which the primary requirement: entering cells when administered in vivo.
siRNAs are commonly formulated with cationic polymers or lipids, to form supramolecular
particles capable of crossing the negatively charged cell membrane. However, particles size
limits their diffusion through organs when administered in vivo.
Considering the need for diffusible drugs for in vivo use, we are developing cationic siRNAs,
composed of an antisense sequence annealed to an oligospermine conjugated sense strand.
Such cationic molecules have been previously shown to display gene silencing activity in
cell culture (Nothisen et al., J. Am. Chem. Soc. 2009). Since, methods for synthesizing and
characterizing oligospermine-oligonucleotides conjugates have been improved, providing
molecules compatible with a drug development process and with enhanced biological activity
in vitro.
Here, we report a complete in vitro study demonstrating that cationic siRNAs enter cells in
absence of carrier and specifically suppress gene expression through RNAi – mediated mechanism in human cells in presence of serum. Our preliminary data confirm also the promising
perspectives for the in vivo use of cationic siRNAs and future preclinical developments.
Patrick Erbacher, PhD
Polyplus-transfection SA, BIOPARC, Boulevard Sébastien Brant, BP 90018
F-67401 Illkirch, France
Telephone: +33 (0)390 406 180
Email: perbacher@polyplus-transfection.com
62
015
Receptor-mediated uptake of oligonucleotide nanocomplexes with cell-penetrating peptides and their
formulation into a pharmaceutically stable solid
dosage form
Kariem Ezzat, Henrik Helmfors and Ülo Langel
Department of Neurochemistry, Stockholm University, Stockholm, Sweden
A new generation of cell-penetrating peptides (CPPs), PepFects, is capable of forming noncovalent nanocomplexes with oligonucleotides (ONs), which are then delivered with great
efficiency to different cell lines in-vitro and different tissues in vivo. However, the detailed
uptake mechanism of such nanocomplexes is not clear. Here, we show that PepFect14 forms
negatively charged nanocomplexes with ONs and their uptake is mediated by class-A scavenger receptors (SCARA). Specific inhibitory ligands of SCARA totally inhibit the activity
of PepFect14-ON nanocomplexes in the HeLa pLuc705 splice-correction cell model, while
nonspecific, chemically related molecules do not. Fetuin, which mediates uptake via SCARA,
significantly enhances the activity. In addition, RNAi knock-down of SCARA subtypes
(SCARA3 & SCARA5) that are expressed this cell-line led to a significant reduction of the
activity to less than 50%.
Furthermore, we demonstrate the feasibility of incorporating such nanocomplexes into stable
and active solid formulations that could be suitable for several therapeutic applications. Solid
dispersion technique was utilized by drying the nanocomplexes at 55-60 ͦC under vacuum in
the presence of water soluble excipients. The formed solid formulations were as active as the
freshly prepared nanocomplexes in solution even when stored at elevated temperatures for
several weeks. This shows that using PepFect14 is a very promising translational approach
for the delivery of ONs in different pharmaceutical forms.
In conclusion, the results presented here shed the light on a novel uptake mechanism of CPPbased delivery systems and demonstrate a novel technology to formulate such delivery systems into active and stable pharmaceutical dosage forms.
Kariem Ezzat, PhD student
Department of Neurochemistry, Stockholm University, SE-106 91
Stockholm, Sweden
Telephone: 0046700674381
Email: kariem@neurochem.su.se
63
016
INHIBITION OF COMPLEMENT C6 SYNTHESIS IN THE LIVER
USING ANTISENSE OLIGONUCLEOTIDES AFFECTS
NEURO-REGENERATION
Kees Fluiter, Valeria Ramaglia, Frank Baas
Academic Medical Center, Dept. of Neurogenetics, Amsterdam, The Netherlands
Introduction: The complement system is always activated in neurodegenerative diseases.
We previously identified a role of the terminal complement pathway in neuro-degeneration.
Rats deficient for C6 showed faster recovery after nerve crush and inhibiting complement
activation using a C1 esterase inhibitor reduced damage after traumatic brain injury.
Method: RNase H recruiting antisense oligonucleotides against C6 were developed to test
whether pharmacological intervention in the terminal complement pathway can be used to
modulate neuro-degeneration and regeneration. These antisense oligonucleotides were tested
in vivo in two mouse models. First, in a model of acute neuronal damage and second in a
model of chronic nerve degeneration.
Results: Administration of C6 antisense oligonucleotides to mice results in a substantial and
specific knock-down of complement C6 mRNA in liver and C6 protein in circulation. The
compounds appear to be well tolerated and provide a potent and long-lived reduction in C6
levels that lasts for weeks after administration. Mice receiving the antisense oligonucleotides in a model of acute nerve degeneration (nerve crush) show better recovery from injury.
These compounds were also effective in an antibody dependent experimental autoimmune encephalomyelitis (AD-EAE) mouse, a model of chronic nerve degeneration. The treated mice
showed substantially less disease severity. Favorable clinical scores were associated with less
nerve inflammation and demyelination according to standard histological assays.
Conclusion: We expect that antisense oligonucleotides targeting the terminal complement
pathway are promising drugs for neurodegenerative disorders.
Kees Fluiter, PhD
AMC, K2-210
Meibergdreef 9, 1105 AZ Amsterdam
The Netherlands
Telephone: +31205663746
Email: k.fluiter@amc.uva.nl
64
017
Suppression of bcr/abl gene expression by siRNA
conjugates
Masayuki Fujii, Diala Irmina
Department of Biological & Environmental Chemistry, School of Humanity Oriented Science
and Technology, Kinki University
Herein we described the synthesis of siRNA-peptide conjugates by solid phase fragment
coupling (SPFC) and application of them to silencing of bcr/abl chimeric gene in human
leukemia cell line K562.
Synthesis of siRNA-NES conjugates was achieved by SPFC as described previously.(1)
Target sequence of bcr/abl mRNA(355-390)
5’-ggauuuaagcagaguucaa/aagcccuucagcggcca-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 human leukemia cell line K562 were evaluated by quantitative PCR. The expression of bcr/abl
gene was suppressed to 30.2 % at 200nM and 36.3 % at 50 nM by native siRNA. Significant
enhancement of silencing efficiency was observed with C1 and C2. siRNA TFIIIA NES
(C1) suppressed the expression of bcr/abl gene to 8.3% at 200 nM and 11.6 % at 50nM and
siRNA-HIV-1rev NES (C2) suppressed to 4.0 % at 200 nM and 6.3 % at 50nM. Previously,
we reported that DNA-HIV-1 rev NES peptide conjugate was localized in cytoplasm of Jurkat
cell. (2) The large enhancement of the silencing efficiency of siRNA-NES conjugates could
be reasonably ascribed to the localization of siRNA-NES conjugates in cytoplasm. It can
be also pointed out that modification of 5’-endo of sense strand reduced off-target effect by
minimizing the extent of the sense strand incorporation into RISC. (3) Unfortunately, siRNANES conjugates could not penetrate the cellular membrane by itself and required a transfection reagent to be taken up into cells, while oligodeoxyribonucleotide-NES conjugates were
internalized into cells without any transfection reagents. (2) It can be speculated the double
stranded structure of siRNA retarded the penetration through cellular membrane.
Reference
1. T. Kubo, M. Morikawa, H. Ohba, M. Fujii, Org. Lett., 2003, 5, 2623-2626.
2. T. Kubo, Z. Zhelev, B. Rumiana, H. Ohba, K. Doi and M. Fujii, Org.Biomol.Chem., 2005, 3, 3257-3259.
3. Y. Ueno, K. Yoshikawa, Y. Kitamura, Y. Kitade, Bioorg. Med. Chem. Lett., 2009, 19, 875-877.
Masayuki Fujii, PhD
11-6 Kayanomori
Iizuka, Fukuoka 820-8555 Japan
Telephone: +81-948-22-5655-210
Email: mfujii@fuk.kindai.ac.jp
65
018
Influence of the terminal base pairs of 5’-PPP-RNA on
RIG-I ligand activity
M. Goldeck1, C. Schuberth1, M. Schlee1, G. Hartmann1 and J. Ludwig1
1. Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn,
University of Bonn, Bonn, Germany
The innate immune response to viruses is based on pattern recognition receptors (PRR) that
detect a characteristic structure, modification or an unusual location of viral nucleic acids.
Most of the highly pathogenic viruses are RNA genome-based viruses. The cytosolic helicase
RIG-I senses double-stranded 5’triphosphorylated RNA (5’-ppp-dsRNA) as contained in the
genomic or replicative form of RNA viruses. Binding and recognition of 5’-ppp-dsRNA is
mediated by the RIG-I regulatory domain (RD). Stimulation of the RD activates the RIG-I
CARD domain, which binds and activates downstream adaptor molecules inducing an antiviral immune response, including production of type I interferons.
As revealed by the crystal structure of the RIG-I RD bound to a blunt end 5’-ppp-dsRNA, the
triphosphate moiety interacts with a lysine-rich basic cleft. The crystal structure furthermore
unravels stacking of phenylalanine F853 with the terminal base pair, which is essential for
RIG-I function. The elucidation of this key intermolecular interaction supports the strict requirement for an intact base pair at the 5’-ppp terminal site of the ligand.
Using synthetic 5’-pppRNAs we performed a systematic analysis of all 16 possible base pairs
of naturally occurring nucleosides at the first two 5’ppp-terminal positions of the duplex.
The stimulatory potency of these duplexes was assessed by type I interferon induction in human monocytes. Besides the analysis of varying Watson-Crick base pairing these data allow
an extended definition of RIG-I ligand requirements concerning the acceptance of alternate
hydrogen bonding occurring in wobble or Hoogsteen base pairing or in other noncanonical
base-base interactions. The data demonstrate a high sensitivity of RIG-I for terminal structural elements and emphasizes the requirement for using well-defined, chemically synthesized
5’-pppRNA for the unequivocal characterization of RIG-I ligand interactions.
Marion Goldeck, PhD student
Institute for Clinical Chemistry and Clinical Pharmacology
University Hospital Bonn
Sigmund-Freud-Str. 25
53105 Bonn, Germany
Telephone: +49-0228-287-51157
Email: mgoldeck@uni-bonn.de
66
019
ANTISENSE OLIGONUCLEOTIDES: PROMISING TOOLS FOR
MOLECULAR THERAPY OF MYOTONIC DYSTROPHY
Anchel Gonzalez1,2, Susan A.M. Mulders1,2, Walther J.A.A. van den Broek2,
Ingeborg van Kessel2, Jeroen van de Giessen1, Judith C.T. van Deutekom1, 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 progression of the disease. DMPK RNA transcripts with long (CUG)n repeats are retained in the nucleus, where they form hairpin-like structures which are involved in the entrapment and depletion of splice factors like MNBL (muscleblind) and in stabilized expression of CUG-binding
protein-1. In turn, this causes aberrant splicing of pre-mRNAs from several other genes, contributing to the complex multisystemic manifestation of disease in patients.
There is no cure available for DM1 yet. However, recent experiments in cell and animal
models have shown that expanded DMPK transcripts can be silenced by the use of antisense
(CAG)n oligonucleotides (AONs) directed towards the toxic (CUG)n repeat segment, offering
a new therapeutic approach.
Here, we report on further therapeutic developments in pre-clinical in vitro studies on AONs
of different chemistry, length and sequence composition in myoblast-myotube cell models derived from patients and from a DM1 mouse model with a human (CTG)500 DMPK transgene.
Northern blotting and real time RT-qPCR analysis showed a significant reduction of expanded
DMPK mRNA after AON transfection while endogenous (CUG)n- or (CAG)n-bearing transcripts were not affected. The working mechanism of our AONs is presently unknown. We are
currently monitoring the fate of DMPK (pre)mRNAs by quantitative measurements of production rate and steady state levels in different cellular compartments, in presence and absence
of different AONs. Fluorescence microscopy and real time RT-qPCR experiments are used to
investigate the uptake pathway of AONs and to assess the stoichiometry of AONs and mRNA
targets and timing and place of degradation of the expanded DMPK mRNA.
Anchel Gonzalez, MSc
Dept. of Cell Biology
Radboud University Nijmegen Medical Centre
Nijmegen Centre for Molecular Life Sciences
Geert Grooteplein 26/28
6525 GA Nijmegen
The Netherlands
Telephone: +31.24.36 13664
Email: a.gonzalez@ncmls.ru.nl
67
020
RESCUE OF SEVERELY AFFECTED DYSTROPHIN/UTROPHIN
DEFICIENT MICE BY VECTORIZED ANTISENSE MEDIATED
EXON SKIPPING
Goyenvalle A, Babbs A, Wilkins V, Wright J, Powell D and Davies KE
MRC Functional Genomics Unit, University of Oxford, Oxford, UK
Duchenne Muscular Dystrophy (DMD) is a severe neuromuscular disorder caused by
mutations in the dystrophin gene that result in the absence of functional protein. Antisensemediated exon skipping is one of the most promising approaches for the treatment of DMD
because of its capacity to correct the reading frame and restore dystrophin expression which
has been demonstrated in vitro and in vivo. This approach has been tested in clinical trials in
the Netherlands and the UK with Antisense Oligonucleotides (AO) against exon 51 and has
demonstrated encouraging results. However, AO-mediated exon-skipping for DMD still faces
major hurdles such as extremely low efficacy in the cardiac muscle, poor cellular uptake and
relative rapid clearance from circulation, which impose repeated administration to achieve
some therapeutic efficacy. To overcome these limitations, we proposed to use small nuclear
RNAs (snRNA), especially U7snRNA to shuttle the antisense sequences and to vectorize
them into Adeno-Associated Virus (AAV) vectors.
In this study, we have investigated for the first time the therapeutic potential of AAV-mediated
exon skipping in the utrophin/dystrophin double-knockout mouse (dKO) which is a much
more severe and progressive mouse model of DMD. A single intravenous injection of
scAAV9 vector encoding a specific U7snRNA targeted to exon 23 of dystrophin pre-mRNA
in dKO mice induces widespread high levels of exon-skipping in the treated mice. This treatment results in near-normal levels of dystrophin expression in all muscles examined, including the heart, leading to a considerable improvement of their muscle function and dystrophic
pathology. ScAAV9-U7dtex23 injection strikingly prevented kyphosis and contractures
in dKO mice and remarkably improved their motility, as assessed by activity monitoring.
Treated dKO mice showed almost return to normalcy for most of the examined parameters as
well as an impressive extended lifespan (over 36 weeks compared to an average of 12 weeks
for untreated dKO controls). These results suggest a great potential for AAV-mediated exonskipping in systemic treatment of the DMD phenotype.
Aurelie Goyenvalle, PhD
MRC Functional Genomics Unit
Department of Physiology,
Anatomy and Genetics
University of Oxford
South Parks Road
Oxford OX1 3QX, UK
Telephone: +44 (0)1865 285 864
Email: aurelie.goyenvalle @dpag.ox.ac.uk
Photograph of a dKO control mouse (left) and a scAAV9U7dtex23 treated dKO mouse (right) at 12 weeks of age
68
021
TARGETING PIM-1 BY MIR-33a IN COLON CARCINOMA THROUGH
PEI-MEDIATED MIRNA REPLACEMENT THERAPY
Arnold 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 mutagenesis of the Pim-1 3’-UTR in a luciferase reporter construct and in a genuine Pim-1 cDNA
demonstrated the specificity of the miR-33a-dependent downregulation. The persistence of
this effect was comparable to that of a siRNA-mediated knockdown of Pim-1 and results
in decelerated cell proliferation. We further establish miRNA replacement therapy through
polyethylenimine (PEI-)-mediated systemic application of miR-33a in s.c. colon carcinoma
xenograft mouse models as a novel cancer treatment strategy. The systemic PEI/miR-33a
treatment resulted in Pim-1 downregulation and significant reduction of tumor growth. Notably, similar results were obtained upon PEI-mediated delivery of Pim-1 specific siRNA. We
further extended our in vivo strategy for replacement of miR-145. We also observed miR-145mediated tumor inhibition through reduced proliferation and increased apoptosis. In this case
c-Myc and Erk5 could be validated as in vivo targets for miR-145.
Arnold Grünweller, PhD
Pharmazeutische Chemie, Marbacher Weg 6, 35037
Marburg, Germany
Telephone: 0049-6421-2825849
Email: gruenwel@staff.uni-marburg.de
69
022
SELECTIVE INTERSTRAND CROSS-LINKING THROUGH IN SITU
OXIDATION OF FURAN-MODIFIED OLIGODEOXYNUCLEOTIDES
E. Gyssels, M. Op de Beeck, A. Madder
Laboratorium for Organic and Biomimetic Chemistry, University Ghent, Belgium
The ease of customizing the reaction cycles of automated, solid-phase DNA synthesis has
allowed for the efficient and site-specific introduction of chemical modifications in oligonucleotides. In our lab we became interested in techniques for crosslinking nucleic acids,
whereby a covalent bond is introduced between two non-covalently hybridized strands. A
novel crosslinking method was developed, where a customized oligodeoxynucleotide (ODN)
is modified with a furan unit. Conversion of the furan to a reactive ketoaldehyde leads to site
selective crosslinking to specific DNA-sequences.
Several furan modified nucleosides have been synthesized for incorporation in reactive
nucleic acids, allowing for a variable positioning of the furan unit in the duplex.[1-5]
The position and linker by which the furan moiety is attached were shown to significantly
influence the crosslinking properties of the furan-modified ODN and the characteristics of
the duplex. Furthermore, crosslink possibilities were tested in triplex DNA by modifying the
triplex forming oligonucleotide, TFO, with a furan moiety.
The crosslinking characteristics of these furan modified oligonucleotides will be presented
together with an outlook on various applications.
References:
1.Stevens, K., et al., Furan-oxidation triggered inducible DNA cross-linking: acyclic versus cyclic furan
containing building blocks. On the benefit of restoring the cyclic sugar backbone. Chemistry- A European
Journal, 2011. accepted.
2.Op de Beeck, M. and A. Madder, Unprecedented C-Selective Interstrand Cross-Linking through in Situ
Oxidation of Furan-Modified Oligodeoxynucleotides. Journal of the American Chemical Society, 2011.
133(4): p. 796-807.
3.Jawalekar, A.M., et al., Synthesis and incorporation of a furan-modified adenosine building block for DNA
interstrand crosslinking. Chemical Communications, 2011. 47(10): p. 2796-2798.
4.Stevens, K. and A. Madder, Furan-modified oligonucleotides for fast, high-yielding and site-selective DNA
inter-strand cross-linking with non-modified complements. Nucleic Acids Research, 2009. 37(5): p. 1555-1565.
5.Halila, S., et al., Fine-tuning furan toxicity: fast and quantitative DNA interchain crosslink formation upon
selective oxidation of a furan containing oligonucleotide. Chemical Communications, 2005(7): p. 936-938.
Ellen Gyssels
Laboratorium for organic and biomimetic chemistry
Krijgslaan 281, S4, 9000 Gent Belgium
Telephone: +3292644479
Email: Ellen.Gyssels@UGent.be
70
023
THE USE OF EXPRESSION PROFILES TO ASSESS NONHYBRIDISATION MEDIATED EFFECTS OF PHOSPHOROTHIOATE
ANTISENSE OLIGONUCLEOTIDES
Peter H Hagedorn, Andreas Petri, Sakari Kauppinen, Morten Lindow
Santaris Pharma A/S, Kogle Alle 6, DK-2970 Hørsholm, Denmark
We have initiated systematic investigations of general class effects of LNA-modified phosphorothioate antisense oligonucleotides. To this end, we have created a reference set of
gene-expression profiles from cultured cells, rodents, and non-human primates treated with
different oligonucleotides, designated here as the Oligo Effect Map. Currently, this collection
comprises profiles from 28 LNA oligonucleotides contrasted to saline vehicle or mock transfection, and integrates more than 500 samples (in vivo from liver and kidney and in vitro from
cultured HuH-7, PC3, and HeLa cells) from 19 independent experiments. For each profile, we
have catalogued detailed experimental information (for example oligonucleotide sequence,
including positions with chemical modifications, delivery method, dosing regimen and dose
level/concentration used), as well as phenotypic observations for the animals representing the
profile. Using this resource we are able to detect gene expression patterns that occur in subsets of experiments and correlate these to experimental setup and oligonucleotide design. Ultimately this may provide us with useful information that enables the selection of conditions
(experimental or oligochemistry) avoid of unwanted effects, facilitating the design of efficient
and safe RNA drugs.
Peter Hagedorn, PhD
Santaris Pharma A/S
Kogle Allé 6
DK 2970 Hørsholm
Danmark
Telephone: +45 4517 9919
Email: pha@santaris.com
71
024
A microRNA-mediated regulatory feedback loop in
a new mechanism of resistance to TGF-beta induced
growth inhibition
Jonathan Hall, Afzal M. Dogar, Harry Towbin
Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences,
ETH Zurich, 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 included included transiently increased SMAD2 and AKT phosphorylation. MicroRNA expression profiling in Hela cells revealed that RNAi against TGF-beta-1 led to globally decreased
oncomir expression including miR-18a and miR-24 which accounted for the derepression of
TGF-beta-1 processing factors, thrombospondin-1 and furin, respectively.
A detailed investigation led us to conclude that latent TGF-beta-1, thrombospondin-1 and furin form a microRNA-controlled regulatory feedback loop. For cells secreting high levels of
latent TGF-beta, this loop offers a potentially widespread mechanism of escape from
TGF-beta mediated growth inhibition at the earliest point in the signaling pathway – latent
TGF-beta processing (1). Pharmacological targeting of TGF-beta mRNA by antisense or
RNAi mechanisms may be of value in pathological mechanisms characterized by loss of the
cytostatic TGF-beta signaling.
(1) A. M. Dogar, H. Towbin, J. Hall. J. Biol. Chem. (2011) 286, 16447-16458.
Jonathan Hall, PhD
Institute of Pharmaceutical Sciences
HCI H 437
Wolfgang-Pauli-Str. 10
8093 Zürich, Switzerland
Telephone: 044 633 74 35
Email: jonathan.hall@pharma.ethz.ch
72
025
NOVEL CELL PENETRATING PEPTIDES FOR SKELETAL AND
CARDIAC MUSCLE DELIVERY OF PMO ANTISENSE
OLIGONUCLEOTIDES FOR THE TREATMENT OF DUCHENNE
MUSCULAR DYSTROPHY
Suzan M Hammond1, HaiFang Yin1,2, Amer F Saleh3, Corinne Betts1, Patrizia Camelliti1,
Yiqi Seow1, Shirin Ashraf1, Andrey Arzumanov3, Thomas Merritt, Michael J Gait3 and
Matthew JA Wood1
1
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; 2Tianjin Research Centre of Basic Medical Science, Tianjin Medical University, Tianjin, China;
3
Medical Research Council Laboratory of Molecular Biology, Cambridge, UK
Duchenne muscular dystrophy (DMD) is a severe recessive X-linked muscular dystrophy in
which patients experience severe progressive muscle wasting, loss of ambulation and respiratory weakness. Greater than 90% of DMD patients also present with cardiac abnormalities.
Aberrant missense mutations and deletions in the dystrophin gene (DMD) cause a variety
of defects including the generation of premature termination codons and out-of-frame transcripts. Dystrophin is unique in that the central portion of the gene encodes a large rod domain made up of twenty four large spectrin-like repeats and four flexible hinge regions. Deletions which cause out-of-frame transcripts can be modified with controlled skipping of exons
surrounding the mutation to restore in-frame DMD transcripts. Recent progress in antisense
oligonucleotide (AON) therapy has advanced treatment of DMD into clinical trials. AONs are
made more effective when combined with conjugated or non-conjugated cell penetrating peptides. We have developed a series of PNA/PMO internalizing peptides (Pip) called the Pip5
series. The Pip5 series enhances systemic delivery of exon skipping PMO AONs, particularly
for delivery to the heart. The lead peptide conjugated PMO, Pip5e-PMO, is able to efficiently
correct exon skipping and restore dystrophin production in mdx mice. With a single low dose
administration of 25mg/kg, Pip5e restores dystrophin to 50-100% of the skeletal muscle fibres tested. Unique to the Pip5e peptide is the ability to also restore greater than 50% of the
normal dystrophin level in the heart.
To understand the elements of Pip5e which are important for its function we have begun development on a new series of Pip peptides conjugated to PMO (Pip6-PMO). With the Pip6
series we will be able to elucidate the important elements of the peptides which are important
for dystrophin production in the heart. The Pip series derivatives have the potential for advancing the use of AON for treating DMD and may allow for further AON delivery toward
the treatment of other cardiac related diseases.
Suzan Michelle Hammond, PhD
Oxford University
Department of Physiology, Anatomy and Genetics
Le Gros Clark Building
South Parks Road
Oxford OX3 1QX United Kingdom
Telephone: 01865 272167
Email: suzan.hammond@dpag.ox.ac.uk
73
026
Retinoic acid- inducible gene-I (RIG-I) detects
(triphosphorylated) RNA of Listeria monocytogenes
during infection
Anna Maria Herzner1, Cristina Amparo Hagmann1, Christine Schuberth1, Christoph Coch1,
Paul G. Higgins2, Hilmar Wisplinghoff2, Veit Hornung1, Gunther Hartmann1 and
Martin Schlee1
1. Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn,
University of Bonn, Bonn, Germany D-53127
2. Institute of Medical Microbiology, Immunology and Hygiene, University of Cologne,
Cologne, Germany
The facultative intracellular bacterium L. monocytogenes induces cell-autonomous innate
immune responses that are not yet fully understood. The innate immune system senses
pathogens by pattern recognition receptors (PRR) located in different cell compartments.
Components of the bacterial cell wall and conserved proteins are detected by Toll-like receptors (TLRs) on the cell surface, and by NODs or NOD like receptors (NLRs) in the cytosol
of cells. Viruses are recognized via their nucleic acids by TLRs in the endosome and RIG-I
like helicases in the cytosol. However, like the genetic material of viruses, nucleic acids of
bacteria are also recognized by TLRs in the endosome. In contrast to exclusively extracellular
bacteria, intracellular bacteria have evolved mechanisms to escape the endosome and enter
the cytosol, where they are accessible to cytosolic PRRs.
L. monocytogenes infection has been linked to cytosolic nucleic acid sensor-dependent type
I IFN induction. Current knowledge indicates that the immune cell type I IFN response to L.
monocytogenes depends on the signalling adaptor MITA downstream of recognition of bacterial DNA or second messengers such as cyclo-di-AMP. We hypothesized that besides bacterial DNA, bacterial RNA may enter the cytosol and contribute to innate immune responses
to L. monocytogenes. In this study a novel in vivo RNA labelling technique applying click
chemistry allowed us to visualize immediate cytosolic delivery of L. monocytogenes RNA
upon infection. Furthermore, transfection of bacterial RNA into
the cytosol of human monocytes, epithelial cells or hepatocytes resulted in a type I IFN
response. In contrast to monocytes, the type I IFN response of epithelial cells and hepatocytes
was not triggered by bacterial DNA. However, these cells still produced type I IFN during
L. monocytogenes infection, demonstrating that a distinct DNA-independent nucleic acid
recognition pathway must exist in these cells. SiRNA mediated knock-down of RIG-I or
MAVS in epithelial cells abolished the IFN response to L. monocytogenes, but did not affect
the immune response of monocytic cells. By contrast, knockdown of MITA in monocytic
cells reduced cytosolic L. monocytogenes-mediated type I IFN induction indicating a DNA/
cyclo-di-AMP dependent recognition.
Together these results provide evidence that RIG-I is required for immunorecognition of L.
monocytogenes in non-immune cells such as epithelia or hepatocytes that lack a functional
MITA dependent pathway for a direct response to DNA or cyclo-di-AMP.
Anna Maria Herzner, PhD student
Sigmund-Freud-Str 25
53127 Bonn, Germany
Telephone: +49 (0)228 287 51157
Email: aherzner@uni-bonn.de
74
027
THIRTEEN WEEK NON-CLINCIAL TESTING OF MIRAVIRSEN IN
CYNOMOLGUS MONKEYS
Hildebrandt-Eriksen L, Persson R, Foy J, Tessier Y, Levin AA
Santaris Pharma A/S, Hoersholm, Denmark
The safety and tolerability of miravirsen sodium (SPC3649), currently in phase 2 clinical
trials, was evaluated in a 13-week study with cynomolgus monkeys (Macaca fascicularis).
Miravirsen is an antagonist to microRNA-122 (miR-122), which in turn has been shown to be
a host factor in hepatitis C infection.
The subcutaneous dosing regimen was divided into a loading phase with 18, 40, 80 and 180
mg/kg/week respectively over 2 doses the first week and a maintenance phase with 2.5, 5, 10
and 25 mg/kg/week once weekly for 12 weeks followed by 12 weeks off treatment.
Miravirsen was well tolerated systemically and locally. Treatment-related changes in clinical
pathology parameters mainly consisted of decreased serum cholesterol level, an expected pharmacological effect of miravirsen (as miR-122’s major function is the fine-tuning of lipid metabolism), transient, mild aPTT and PT prolongation and activation of the alternative complement
pathway. Although some of these changes remained present through the 12-week treatment-free
period, they were considered not to be adverse owing to their small magnitude and/or since they
had no clinical or histopathological correlate. Microscopic findings were noted at all dose levels
and were considered to be related to the known class-related effects of oligonucleotides. The
kidneys were the main target organ. A dose-related accumulation of eosinophilic granules in the
proximal convoluted tubules associated with a minor level of degeneration accounted for some
functional disturbances in animals given ≥10 mg/kg/week and correlated with slightly increased
serum urea and creatinine levels. Changes in the kidney resolved at the end of the treatmentfree period in most animals. The other histopathological effects (eg accumulation of enlarged
macrophages principally in the lymph nodes and liver (Kupffer cells) but also in injection
sites) were considered not to be adverse since they were not associated with any degenerative
changes. At the end of the treatment-free period, the vacuolated macrophages in the lymph
nodes showed a significant reduction in extent. Although some histopathological changes were
seen at 5 mg/kg/week, they were at a minimal level, in a few animals, and were not expected to
result in functional changes, including in the kidneys.
Liver and kidney samples were analyzed for miravirsen content after 13 weeks treatment and
12 weeks recovery, and in a small cohort of animals receiving 80mg/kg for the first week
only, on study days 5, 22, 43 and 70. The plasma pharmacokinetic evaluation confirmed the
long tissue half-life time of miravirsen, accounting for the incomplete reversibility of some of
the observed changes in tissues.
The good safety profile of miravirsen sodium supports further clinical testing.
Lisa Hildebrandt-Eriksen, PhD
Santaris Pharma a/s
Kogle Allé 6, 2970 Hoersholm, Denmark
Telephone: +45 4517 9800
Email: LHE@santaris.com
75
028
DNA-CONTROLLED ASSEMBLY OF LIPOSOMES
Ulla Jakobsen and Stefan Vogel
Nucleic Acid Center, University of Southern Denmark, Campusvej 55, 5230 Odense M,
Denmark
Oligonucleotide strands modified at both ends with alkyl chains have previously been
reported to be able to induce assembly of liposomes in the presence of unmodified, complementary strands.1,2 Here, the assembly of liposomes caused by triple helix formation as well
as the effect of varying the length of the alkyl chains in the membrane anchors are described.
Liposome assembly controlled by triple helix formation is based on the same principle as the
assembly controlled by double helix formation, albeit the steric and structural requirements
are considerably more demanding. The assembly requires one triplex forming oligonucleotide
(TFO) modified at both ends with lipophilic substituents, which will adhere reversibly to the
surface of liposomes when mixed
with these. In the presence of a
duplex target capable of forming
a triplex with the modified TFO,
the for-med triple helix will be
too rigid for both of the ends of a
TFO to be anchored to the same
liposome, and one of the ends
will be released and adhere to
another liposome, resulting in the
formation of liposome aggregates
(Figure 1). The assembly is reFigure 1 Schematic representation of assembly of liposomes versible, and the aggregates break
controlled by triple helix formation. TFO (red ribbon), double down to the individual liposomes
helix (blue ribbons) and liposomes (grey spheres) are not
when heated to above the melting
drawn to scale.
temperature of the triple helix, but
are reformed by cooling.
As the liposome aggregates scatter the light considerably more than the individual liposomes,
the assembly and disassembly process can be monitored by optical methods, e.g. dynamic
light scattering (DLS) and UV spectroscopy.
The membrane anchor previously described for assembly controlled by duplex formation
consisted of two palmityl chains attached to a crown ether scaffold,1,2 and shorter (dodecyl)
or longer (icosanyl) chains failed to function as membrane anchors, presumably because the
former is attached to weakly and the latter to strongly to the liposomes.
Jakobsen, U.; Simonsen, A. C.; Vogel, S. J. Am. Chem. Soc. 2008, 130, 10462-10463.
Jakobsen, U.; Vogel, S. Methods Enzymol. 2009, 464, 233-248.
1
2
Ulla Jakobsen, PhD
Campusvej 55, 5230 Odense M, Denmark
Telephone: +45 65502599
Email: uja@ifk.sdu.dk
76
029
ENZYMATIC SYNTHESIS OF OLIGONUCLEOTIDES WITH 2’-AMINOLNA TRIPHOSPHATES AND TEMPLATES
Marie W. Johannsen, Rakesh N. Veedu, Birte Vester, Jesper Wengel
Nucleic Acid Center, University of Southern Denmark, Odense, Denmark
Two very interesting classes of functional oligonucleotides are aptamers and DNAzymes.
These have potential uses in both therapy and diagnostics, and rely either exclusively or partially on 3-dimensional structure rather than base pairing for activity. It is therefore not trivial
to select an aptamer or DNAzyme for a specific target. Both aptamers and DNAzymes can
be selected using SELEX (Systematic Evolution of Ligands by Exponential Enrichment), a
method that relies on PCR to amplify the active molecules in the selection pool. Most aptamers and DNAzymes are modified to some degree after the selection process is complete, but
there are obvious advantages to direct selection of oligonucleotides from modified selection
pools. This requires that modified triphosphates are tolerated as substrates for enzymatic
synthesis and that modified oligonucleotide templates can be copied enzymatically. LNA has
previously been used in post-SELEX modification of aptamers and DNAzymes, and has received a lot of interest for its improvement of target binding as well as nuclease stability of
oligonucleotides.1 A similar modification, 2’-amino-LNA, combines increased stability with
the opportunity for conjugation to amino acids, fluorophores, etc.2 LNA triphosphates have
previously been synthesised by our group as well as others and have been incorporated in oligonucleotides by enzymatic template directed primer extension. We present the synthesis of
2’-amino-LNA triphosphates with and without modification of the 2’-nitrogen as well as their
incorporation into ONs by primer extension. We also show how oligonucleotides modified
with 2’-amino-LNA-T can be used as templates for primer extension. This is the first step on
the way to carrying out SELEX from 2’-amino-LNA modified selection pools, thus increasing the chemical diversity of DNAzymes and aptamers.
Doessing, H.; Vester, B. Locked and Unlocked Nucleosides in Functional Nucleic Acids.
Molecules 2011, 16, 4511-4526.
1
Johannsen, M.; Crispino, L.; Wamberg, M.; Kalra, N.; Wengel, J. Amino acids attached to
2’-amino-LNA: synthesis and excellent duplex stability. Organic & Biomolecular Chemistry,
2011, 9, 243-252
2
Marie Willaing Johannsen, MChem, MSc.
University of Southern Denmark
Campusvej 55
5230 Odense M
Denmark
Telephone: +45 6550 2548
Email: mwj@ifk.sdu.dk
77
030
LIPID NANOPARTICLE FORMULATIONS OF MINIMAL-LENGTH
shRNAS SHOW POTENT INHIBITION OF HCV-DRIVEN,
LIVER-SPECIFIC GENE EXPRESSION IN MICE
B. H. Johnstona,e, A. Dallasa, H. Ilvesa, J. Shorensteina, M. A. Behlkeb, SP Wonge,
R. Harbottlec, and I. MacLachland
a
SomaGenics, Inc., 2161 Delaware Ave., Santa Cruz, CA 95060, USA
b
IDT, Coralville, IA 52241, USA
c
Imperial College London, UK
d
Tekmira Pharmaceuticals, Burnaby, B.C., Canada
e
Dept. of Pediatrics, Stanford Univ., Stanford, CA, USA
We have identified short shRNAs (sshRNAs) that target a highly conserved HCV sequence
and potently inhibit expression in reporter systems.1 Chemical modifications were subsequently introduced to increase nuclease stability while minimizing immunostimulatory activity and
maintaining potency.2 In the current study, we assessed the efficiency of liver delivery and in
vivo efficacy of these sshRNAs when formulated into lipid nanoparticles (LNP). Single doses
of HCV-targeting sshRNAs formulated into LNP were injected i.v. into mice that had previously been treated with a plasmid providing long-term expression of an HCV-firefly luciferase
fusion protein from a liver-specific promoter. In vivo imaging showed a dose-dependent inhibition of luciferase expression (>90% at 2.5 mg/kg sshRNA), with a half-time of recovery of
about 3 weeks (see Figure; expression normalized to Day 0 = 100%). No inhibition was seen
with a scrambled control, saline, or a mock treatment. These results demonstrate the ability to
provide durable knockdown of an HCV target by systemic delivery of formulated sshRNAs.
Q. Ge, et al. (2010) RNA 16(1):106-17; 2Q. Ge, et al. (2010) RNA 16(1):118-30.
1
Brian Johnston, PhD
SomaGenics, Inc.
2161 Delaware Avenue
Santa Cruz, CA 95060 USA
Telephone: (831) 426-7700 ext. 12
Email: bjohnston@somagenics.com
78
031
HIGHLY POTENT, MINIMAL-LENGTH shRNAS TARGETING
HEPATITIS C VIRUS HAVE NOVEL MECHANISTIC FEATURES
B. H. Johnstona,d, A. Dallasa, H. Ilvesa, P. Kumara, S. A. Kazakova, J. Shorensteina,
M. A. Behlkeb, M. McManusc, and Q. Gea
a
SomaGenics, Inc., 2161 Delaware Ave., Santa Cruz, CA 95060, USA
b
IDT, Coralville, IA 52241, USA
c
UCSF Diabetes Center, University of California, San Francisco, CA 94143, USA
d
Dept. of Pediatrics, Stanford University, Stanford, CA 94305, USA
Current therapies for hepatitis C, which afflicts some 200 million people world-wide, have
severe side-effects and are of limited efficacy. The recently approved protease inhibitors are
more effective when given as a combination therapy with interferon, but due to the latter’s
severe side-effects, there is an urgent need for alternatives with an independent mechanism
of action that can replace interferon. We have identified short hairpin RNAs (shRNAs) that
target a highly conserved HCV sequence and potently inhibit expression in reporter systems,
with IC50 < 10 pM in human 293FT cells.1 shRNAs are of similar or greater potency than
cognate siRNAs in human hepatocytes, HCV replicon systems, and mouse models of HCV.
A connecting (loop) sequence between the sense and antisense sequences of 2 nt (or a nonnucleotide linker of similar length) gives optimal potency, but a loop of 1 nt or even a direct
connection between the sense and antisense sequences also provide good activity. The position of the shRNA loop is important: unlike right-hand (R) loop shRNAs, which require a 3’
overhang for optimal activity, left-hand (L) loop shRNAs can be effective with no overhang,
and are generally more potent than R-type shRNAs. Blunt-ended molecules require some
2’-modification to avoid inducing a RIG-I-mediated immune response. Modification patterns
that increase nuclease stability while minimizing immunostimulatory activity and maintaining
potency have been systematically identified.2 shRNAs with stem lengths of 16–19 bp are not
substrates for recombinant Dicer and maintain their potency in conditional Dicer-knockout
cells, yet can be highly potent. 5’-RACE analysis shows a normal target cleavage position
regardless of Dicer cleavability. Chemical modification to block nuclease cleavage at various
sites, Ago-immunoprecipitation results, and other evidence indicates that the mechanism of
action of shRNAs need not involve cleavage of the loop and may differ from the mechanism
of siRNAs or Dicer-substrate RNAs in some key respects. Our findings are encouraging for
the prospects of the therapeutic use of direct-delivered shRNAs.
1. Q. Ge, H. Ilves, A. Dallas, P. Kumar, J. Shorenstein, S. A. Kazakov, and B. H. Johnston (2010)
Minimal-length short hairpin RNAs: The Relationship of Structure and RNAi Activity. RNA
16(1):106-17.
2. Q. Ge, A. Dallas, H. Ilves, J. Shorenstein, M. A. Behlke, and B. H. Johnston (2010) Effects of
Chemical Modification on the Potency, Serum Stability, and Immunostimulatory Properties of
Short shRNAs. RNA 16(1):118-30.
Brian Johnston, PhD
SomaGenics, Inc.
2161 Delaware Avenue
Santa Cruz, CA 95060 USA
Telephone: (831) 426-7700 ext. 12
Email: bjohnston@somagenics.com
79
032
EPATOCYTE SPECIFIC TARGETING AND DELIVERY OF siRNACARBOHYDRATE CONJUGATES
Kallanthottathil G. Rajeev, K. NarayanannairJayaprakash, Gang Wang, Ligang Zhang, Chang
GengPeng, Jennifer Sherman, Mariano Severgnini, Amy Chan, Klaus Charisse,
Renta Hutabarat, Martin Maier, Kevin Fitzgerald,Dinah W.Y. Sah, Muthiah Manoharan,
Kenneth Koblan and Tracy S. Zimmermann
Alnylam Pharmaceuticals, Cambridge, MA, USA
The asialoglycoprotein receptor (ASGPR) is a transmembrane receptor that mediates clearance of extracellular glycoproteins with exposed terminal galactose residues. Thisreceptor
is highly expressed on the surface of liver hepatocytes and facilitates multiple rounds of
uptake and clearance of glycoproteins. These features of the ASGPR along with its ligand
specificity make it an attractive strategy for liver-specific delivery of galactosylated-drugs.
We have shown that conjugation of a triantennary N-acetylgalactosamine ligand to a siRNA
(siRNA-GalNAc) has nM binding affinity to the ASGPR and facilitates uptake and gene
silencing both in vitro and in vivo. We will describe further improvements ondelivery of
siRNA-GalNAc conjugates that lead to enhanced siRNA pharmacology. The results to
be presented include the impact of mode of administration on efficacy, application across
multiple targets in hepatocytes, and demonstration of liver-specific gene silencing at low mg/
kg siRNA-GalNAc doses in mouse, rat and non-human primate.
Gopalan Rajeev Kallanthottathil, PhD
300 Third Street, Cambridge, MA 02142 USA
Telephone: 1 617 551 8313
Email: rajeevk@alnylam.com
80
033
LNA ANTISENSE OLIGONUCLEOTIDES - A STRAIGHTFORWARD
CONCEPT FOR RNA THERAPEUTICS
Susanne 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. Oligonucleotide mode of action spans from RNaseH mediated mRNA degradation to microRNA
inhibition and modulation of RNA splicing. Locked Nucleic Acid (LNA) oligonucleotides,
epitomise this new generation of effective RNA therapeutics, combining high binding affinity,
target specificity, regulatory potency and nuclease stability with unmatched short length of
the antisense oligonucleotide. It has been shown that LNA oligonucleotides readily enter cells
via un-assisted uptake (gymnosis), in an active form allowing in vitro prediction of potent
therapeutically active compounds as well as in vivo distribution to manifold cell types and tissues. This makes Santaris Pharma A/S’ Locked Nucleic Acid (LNA) drug discovery platform
a straightforward tool to transform today’s knowledge about disease associated gene regulation into tomorrow’s RNA therapeutics.
Susanne Kammler, Research Scientist, Technology & Partnered discovery
Santaris Pharma A/S
Kogle Allé 6
DK 2970 Hørsholm
Denmark
Telephone: +45 4517 9915
Email: suk@santaris.com
81
034
ANTISENSE-INDUCED MYOSTATIN EXON SKIPPING LEADS TO
MUSCLE HYPERTROPHY IN MICE FOLLOWING IN-VIVO
TREATMENT WITH CONJUGATED OLIGONUCLEOTIDES
Kang JK, Malerba A, Popplewell L, Foster K, Dickson J
Royal Holloway, University Of London, Egham, Surrey, United Kingdom, TW20 0EX
Inadequate intake of energy could lead to a reversible loss of muscle mass but in certain other
conditions this loss cannot be reverted nutritionally. Patients suffering from cancer, chronic
obstructive pulmonary disease (COPMD), AIDS etc, tend to lose body weight, a condition
called cachexia. Cachexia is responsible for increased risk of death due to the underlying
condition in such patients. Degenerative muscular disorders such as various forms of muscular dystrophies could actually be fatal at an early age of mid to late twenties. Muscle mass
loss resulting from ageing and obesity also has a very high impact on public health.
Myostatin is a negative regulator of muscle mass, and natural mutations in myostatin in
whippets, Belgian blue bull, myostatin mull mice and even in humans have resulted in significantly increased muscle mass relative to their normal counterparts. Several strategies are being developed to knock down the expression of the myostatin gene as a means to bring about
improvements in the muscle wasting conditions including Duchenne muscular dystrophy.
In this study the use of antisense oligonucleotides (AOs) to manipulate pre-mRNA splicing
and knockdown of myostatin has been reported. Previously we have demonstrated the use
of AOs of three different chemistries: 2’O-methyl RNA (2’OMePS-with a phosphorothioate
backbone), phosphorodiamidate morpholino (PMO) and octa-guanidine conjugated PMO (vivo-PMO) to induce myostatin exon skipping. Systemic administration of vivo-PMO showed
that myostatin exon skipping led to a significant increase in the mass and cross sectional area
of soleus muscle of treated mice. In order to examine the longevity of PMO action in treated
muscles, exon skipping and muscle mass was evaluated in mice across an 8 week period in
response to a single intramuscular administration. Following a single intramuscular injection
of vivo-PMO in mice, exon skipping in the myostatin mRNA was recorded from week 1
through to week 8 at high levels. In addition, a significant increase in muscle mass was
observed 4 weeks following treatment, and this weight gain was maintained and enhanced
out to 8 weeks. In conclusion, myostatin knock-down by exon skipping Vivo-PMOs exhibits
a long half-life of action in treated skeletal muscle, and thus provides a potential therapeutic
strategy to counter muscular dystrophy, muscular atrophy, cachexia and sarcopenia.
Jagjeet Kaur Kang, PhD candidate
School of Biological Sciences, Royal Holloway, University of London,
Egham, Surrey, United Kingdom, TW20 0EX
Telephone: 7895709958
Email: Jagjeet.Kang@gmail.com
82
035
Preclinical Development of an sd-rxRNATM Based Therapy
for Treatment of Fibrosis and Retinal Disorders
Anastasia Khvorova, Karen Bulock, James Cardia, Lyn Libertine, Kehlen Flannery-Rossi,
Jake Metterville, Tim Drew, Michael Byrne, Christine Resmini, Nick Palumbo, Glenna Ford,
Katherine Holton, Amanda Rodgers, Jessica Lam, William Stanney, Kevin Fettes,
Pamela Pavco
RXi Pharmaceuticals, Worcester, MA, USA, 01605
Fibrosis is a widespread reparative or reactive process resulting in the accumulation of excess
fibrous tissue. This biological process is involved in a large number of clinical indications
with significant unmet medical need. We have identified a novel class of RNAi compounds
termed “self-delivering rxRNA” (sd-rxRNA) that demonstrates robust cellular uptake and
silencing of target genes without the requirement for a delivery vehicle. sd-rxRNAs combine
features of conventional RNAi and antisense technology and are heavily chemically modified
to improve stability and lipophilicity. These RNAi compounds are rapidly and efficiently
taken up by cells and demonstrate potent activity, enhanced stability, and reduced immune
stimulation. sd-rxRNAs with in vitro EC50 values in the low nM range have been identified for several genes known to be involved in fibrosis. Using intra-dermal injection in a
rodent model of dermal wound healing, we have demonstrated statistically significant, dose
dependent silencing of the anti-fibrotic target, CTGF. The data presented show the efficacy
and longevity (>2 weeks) of locally-administered sd-rxRNAs targeting CTGF. CTGF silencing in vivo results in potent reduction in myofibroblast infiltration and collagen deposition,
indicating that anti-CTGF sd-rxRNA (RXi-109) is a promising anti-fibrotic agent. An IND
supporting evaluation of RXi-109 safety, tolerability and indication of efficacy is expected to
be filed later in the year.
In addition, data on uptake, dose response, and duration of silencing (up to 21 days) and
initial safety of sd-rxRNAs in rodent retina will be presented.
Anastasia Khvorova, PhD
RXi Pharmaceuticals
60 Prescott Street
Worcester, MA 01605 USA
Telephone: 508-929-3616
Email: akhvorova@rxipharma.com
83
036
Anti- cancer approach through microRNA-regulated
RNA replacement
Ju-hyun Kim, You Sub Won, Ranhui Won, and Seong-Wook Lee*
Department of Molecular Biology, Institute of Nanosensor and Biotechnology,
Dankook University, Yongin 448-701, Korea (*, SWL0208@dankook.ac.kr)
We have previously presented that the group I intron from Tetrahymena could induce new
gene activity selectively in target cells by recognizing the cell-specific RNA and catalyzing
the in-frame fusion of desired RNA sequences with trans-splicing reaction. Moreover, the
ribozymes trigger cytotoxin activity only in cancer cells expressing the target RNA, resulting in specific regression of the cells. Particularly, the specific trans-splicing reaction worked
well by the delivery of the specific ribozyme using the adenovirus into various xenograft
carcinomatosis nude mouse model, inducing highly selective expression of a reporter gene
in the human tumors but not in normal mouse organs, and moreover, causing the efficient
regression of hTERT-positive tumors in the mice. However, one concern for the hTERTtargeting anti-cancer approach includes potential side effects to hTERT+ normal cells such
as germ line, stem cells, and highly proliferating normal cells. In this study, to improve cancer- specific expression of the ribozyme, we have attempted to combine miRNA regulation
via using miRNA target site. To overcome the side effects to hTERT(+) bone-marrow derived
blood cells, we have attempted to insert perfect target sites against blood cell-specific miRNA181a into 3’UTR downstream of 3’ exon of the trans-splicing ribozyme. Moreover, we
have used target site of liver-specific miRNA122a for liver cancer specific transgene induction. We observed that transgene induction by the ribozymes was specifically shown in cancer cell. Furthermore, retardation of cell growth was specifically induced in hTERT(+) and
miRNA(miR181(-) or miR122(-)) cancer cells when infected adenovirus with miRNA181a
or miRNA122a target site-containing ribozyme with HSVtk gene as 3’ exon and treated with
GCV, but not in miRNA(miR181(+) or miR122(+)) cells. Moreover, specificity of cancer
regression in xenograft mouse model was also highly increased without compromising its
anti-cancer efficacy. These observations provide the basis for a novel approach to cancer gene
therapy and demonstrate that targeted and miRNA-regulated transgene induction can be used
as powerful anti-cancer approach.
Ju-hyun Kim, MS
Department of Molecular Biology, Dankook University
126, Jukjeon-dong, Suji-gu Yongin 448-701, Korea
Telephone: 82-31-8005-3203
Email: a8327447@gmail.com
84
037
HINT-1 HYDROLASE-ASSISTED CLEAVAGE OF INTERNUCLEOTIDE
BONDS IN DINUCLEOSIDE PHOSPHOROTHIOATES SUGGESTS
ITS POSSIBLE PARTICIPATION IN METABOLISM OF
OLIGONUCLEOTIDE DRUGS AND PRODRUGS
Agnieszka Krakowiak, Beata Rębowska, Milena Sobczak, Barbara Mikołajczyk,
Wojciech J. Stec
Centre of Molecular and Macromolecular Studies of the Polish Academy of Science,
Department of Bioorganic Chemistry, Lodz, Poland
Therapeutic oligonucleotides, e.g. siRNA or antisense constructs, are degraded in vivo by
numerous endo- and exonucleases. Phosphorothioate oligonucleotides (PS-oligos) containing
a sulfur atom attached to the phosphorus at each internucleotide bond are considered as
prospective therapeutic molecules because they are much more stable in blood, plasma and
various cellular systems than their natural unmodified counterparts [1]. However, under
certain nucleolytic conditions these oligomers undergo slow degradation, which proceeds
mainly from the 3’-end and generates mononucleoside 5’-phosphorothioates (NMPS or
dNMPS). The products of degradation may alter cell proliferation, DNA and RNA synthesis
as well as induce other unknown effects. In 2007, the phosphorothioate DNA segments have
been identified in bacterial DNA [2], which makes investigations on the PS-oligo metabolism
even more important.
Enzymes able to metabolize nucleoside 5’-phosphorothioates are poorly known: only
5’-nucleotidase (ecto-5’-NT) and alkaline phosphatase have been taken into account so far.
We found that Hint-1, which belongs to histidine triad superfamily (HIT), catalyzes the cleavage of internucleotide bond in dinucleotide phosphates and thiophosphates, and
the products are corresponding nucleosides as well as nucleoside 5’-phosphates or –phosphorothioates. Moreover, Hint-1 enzyme is able to desulfurate NMPS and dNMPS under in vitro
conditions [3].
These results indicate that Hint-1 protein, which homologues have been found in all forms of
life, may play important role in metabolism of therapeutic oligonucleotides.
1 Dias N., Stein CA, (2002) Mol. Cancer Ther. 1, 347–355.
2 Wang, L., Chen, S., Xu, T., Taghizadeh, K., Wishnok, J.S., Zhou, X., You, D., Deng, Z., Dedon,
P.C. (2007) Nature Chem. Biol. 3, 709-710
3 M. Ozga, Dolot R, Janicka M, Kaczmarek R, Krakowiak A. (2010) J Biol Chem. 285, 40809-18
Agnieszka Krakowiak, PhD
POLAND, 90-363 Lodz, Sienkiewicza 112
Telephone: +4842 6803272
Email: akrakow@bio.cbmm.lodz.pl
85
038
2’-BISPYRENE-MODIFIED OLIGO
(2’-O METHYLRIBONUCLEOTIDE) PROBES AS TOOLS FOR THE
SENSITIVE DETECTION OF RNA
Krasheninina O.A., Novopashina D.S., Venyaminova A.G.
Institute of Chemical Biology and Fundamental Medicine SB RAS
To date pyrene-modified oligonucleotides have considered as challenging tools in molecular
biology, genomics and diagnostics. It is related to remarkable properties of pyrene as fluorophore such as high quantum yields, abilities to form p-stacking dimers and to intercalate into
nucleic acids duplex.
Conjugates of octadeca(2'-O-methylribonucleotides) which contained from one to three
2'-bispyrenylmethylphosphorodiamidate pyrimidine or purine nucleosides were obtained
by analogy with [1]. The properties of new type of the 2'-bispyrene-modified oligo(2'-Omethylribonucleotides) as fluorescent probes for RNA detection were investigated. The
sequences of probes were complementary to two available regions of MDR1 mRNA. In order
to increase nuclease resistance and duplex stability of pyrene-modifed probes, we introduced
into probes 3’-terminal «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'-bispyrenelabeled probes varied depending on their sequences and structures. In all cases the binding of
pyrene-labeled probes to the model NA targets resulted in enhancement of the pyrene eximer
fluorescence. The hybridization of the most sensitive probes to model 25-mer RNA and DNA
targets and 5'-terminal fragment of MDR1 mRNA (nucleotides 1-678) was investigated by
fluorescence-monitored titration with an increasing target concentration. The association constants obtained from fluorescence titration data using the modified Shtern-Folmer equation
were calculated for model 25-mer RNA targets and 678-mer fragment of MDR1 mRNA. The
results obtained in this study indicate that the new fluorescent 2'-bispyrene-labeled oligo(2'O-methylribonucleotide) probes are sensitive tools for RNA detection.
1. Novopashina D.S., Stetsenko D.A., Totskaya O.S., Repkova M.N., Venyaminova A.G.
Nucleosides Nucleotides Nucleic Acids, 24, 729 (2005).
This work is supported by Federal Target Program “Scientific and science educational
personnel of innovative Russia“ (State contract N P1334).
Olga Krasheninina, PhD student
630090, Lavrentiev ave. 8, Novosibirsk, Russia
Telephone: 007(383)3635129
Email: OKrasheninina@gmail.com
86
039
Synthesis of 2’-Amino-UNA
Niels Langkjær, Jesper Wengel
University of Southern Denmark, Odense, Denmark
Over the last decade chemicaly modified nucleosides have recived a renewed focus with
the development novel techniques in biochemistry such as RNAi. Especially UNA (unlocked nucleic acid) has been demonstrated to perform well in siRNA applications with
lowered off-target effects and enhanced activity.1,2 We herein introduce the 2’-aminoUNA derivative with an amino functionality at the 2’-position of UNA and present the
synthesis of the 2’-amino-UNA phosphoramidite (9) from uridine (1) using the previously published selective benzoylation of the 2’-hydroxylgroup of UNA.3
Figure 1 - UNA, 2-Amino UNA and RNA.
Scheme 1 - Synthesis of 2’-Amino-UNA.
References:
1.
2.
3.
Bramsen, J. B., Laursen, M. B., Nielsen, A. F., Hansen, T. B., Bus, C., Langkjaer, N., Babu, B.
R., 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.,
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., Bioorg. Med. Chem. 2009, 17 (15), 5420-5425.
Niels Langkjær, Cand. Scient. Chemistry
Campusvej 55, 5230 Odense M, Denmark
Telephone: +45 6550 2548
Email: nlk@ifk.sdu.dk
87
040
Allosteric ribozyme capable of specifically
inhibiting miR-122 functions through HCV NS5B protein
sensing
Chang Ho Lee, Ji Hyun Kim, Hee Won Kim, Seong-Wook Lee*
Department of Molecular Biology, Institute of Nanosensor and Biotechnology
Dankook University, Yongin 448-701, Republic Korea
Functional sequestration of microRNA 122 (miR-122) by treatment with an oligonucleotide
complementary to the microRNA results in long-lasting suppression of hepatitis C virus
(HCV) viremia in primates. However, the safety of the constitutive miR-122 silencing approach to HCV inhibition is unclear, since miR-122 can modulate the expression of many
host genes. Here, we try to develop a genetic regulation system capable of specifically inhibiting miR-122 activity only upon HCV infection. With an in vitro selection technique we
developed an allosteric self-cleavable Hammerhead ribozyme, whose activity was specifically
induced by HCV NS5B replicase such as to release the most efficacious antisense sequence
to miR-122 only in the presence of HCV NS5B. The activity of the reporter construct with
miR-122 target sequences at its 3’ untranslated region and the expression of endogenous miR122 target proteins were specifically stimulated only in HCV replicon Huh-7 cells, but not
in 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 replicating cells. Noticeably, HCV replicon replication was efficiently inhibited by the allosteric
ribozyme. This ribozyme could be useful for the specific, safe, and efficacious anti-HCV
therapeutic tool.
Chang Ho Lee, PhD candidate
Department of Molecular Biology, Institute of Nanosensor and Biotechnology
Dankook University, Yongin 448-701, Republic Korea
Telephone: 82-31-8005-3203
Fax: 82-31-8005-4058
Email: chang-ho79@hotmail.com
*swl0208@dankook.ac.kr
88
041
Are short oligonucleotides less specific in vivo?
Morten Lindow, Peter Hagedorn, Andreas Petri
Santaris 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 specificity. By comparison, hybridization conditionsin live animals or humans cannot be adjusted,
instead altering the length, design and chemical modifications of a given oligonucleotide
must be utilized to achieve adequate specificity.
Here, we present and compare different oligonucleotide specificity prediction algorithms.
We show that different methods can produce qualitatively very different results. Algorithms
based on minimizing mismatches predict that short oligonucleotides are less specific, whereas
methods that maximize the number of basepairs or binding affinity to putative targets predict
that longer oligonucleotides are less specific.We assess the aforementioned approaches using
transcriptomal expression profiling of mice livers after treatment with different LNAphosphorothioate oligonucleotides.
Morten Lindow, PhD
Santaris Pharma A/S
Kogle Alle 6, DK2970-Horsholm, Denmark
Telephone: +45 2849 9682
Email: mol@santaris.com
89
042
MECHANISTIC INSIGHTS INTO LIPID NANOPARTICLE-MEDIATED
DELIVERY OF siRNA
Martin A. Maier1, Jerome Gilleron3, William Querbes1, Xuemei Zhang1, Valerie Clausen1,
Renta Hutabarat1, Yosef Landesman1, Nenad Svrzikapa1, June Qin1, Undine Schubert3,
Muthusamy Jayaraman1, Michael J. Hope2, Dinah W. Y. Sah1, Kevin Fitzgerald1,
Kallanthottathil G. Rajeev1, Mark A. Tracy1, Rachel Meyers1, Muthiah Manoharan1,
Marino Zerial3, Akin Akinc1
1
Alnylam Pharmaceuticals, Cambridge, MA 02142, USA
2
Alcana Technologies, Vancouver, BC, Canada V6L 2A1
3
Max Planck Institute of Molecular Cell Biology and Genetics, 01309 Dresden, Germany
Safe and efficient delivery of short interfering RNAs (siRNAs) is a prerequisite for the development and advancement of RNAi therapeutics as a new class of innovative medicines. Lipid
nanoparticles (LNPs) are among the most efficient carriers for systemic delivery of siRNA
and have been successfully employed to silence therapeutically relevant genes in a number of species from rodents to non-human primates. In recent years, multiple liver-targeted
RNAi programs utilizing LNP-formulated siRNAs have advanced into human clinical trials.
Meanwhile, the discovery of novel lipid materials and the recent progress on the mechanistic
understanding of the LNP platform has enabled the development of the next generation LNPs
with improved therapeutic indices. Comparing two distinct classes of lipid-based nanoparticles, we have previously shown that apolipoprotein E acts as an endogenous targeting ligand
for ionizable lipid nanoparticles but not for cationic lipid nanoparticles, which carry a permanent positive charge at physiologic pH. At the cellular level, functional delivery requires the
release of siRNA from the endosomal compartment into the cytoplasm. Electron microscopy
(EM) was used to track the cellular uptake and intracellular trafficking of siRNA labeled with
gold nanoparticles and formulated in LNPs. Based on EM analysis of cultured cells as well as
liver sections from treated animals, we estimate that a low percentage of the Au-labeled siRNA escapes the endosomal pathway and reaches the cytosol, which is in good agreement with
time course studies using stem-loop PCR for siRNA quantification. These results help provide
insights into the intracellular mechanism of LNP-mediated siRNA delivery, which may facilitate further rational optimization of this siRNA delivery platform.
Martin A. Maier, PhD
300 Third Street
Cambridge, MA 02142, USA
Telephone: +1-617-551-8274
Email: mmaier@alnylam.com
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043
Characteristic aspects of skeletal muscle as a target
organ for siRNA
Naoki Makita1, Ichiro Fujimoto2, Sachiko Fukuda1, Takeshi Shimizudani1, Shunji Nagahara1
1
DDS Research Group, Formulation Research & Development Laboratories, Technology
Research & Development, Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan
2
Research Institute, Koken Co., Ltd., Tokyo, Japan
Skeletal muscle is one of major organ and in which some severe disorders, including muscle
dystrophy, are known to occur due to various factors abnormality. Skeletal muscle is accessible organ and therefore is thought to be good target for treatment by siRNA, however few
approaches have been reported so far.
Atelocollagen is the first vehicle which succeeded to deliver siRNA into tumor by systemic
administration.[1] After that, a lot of studies which demonstrate usefulness of atelocollagen
as a delivery vehicle for siRNA by a local and systemic administration have been reported in
terms of efficacy and safety. In 2008, it was reported that siRNA formulated with atelocollagen suppressed its target gene expression and led to morphological changes in skeletal muscle
via not only local but also systemic injection.[2] So, we utilized atelocollagen as a vehicle for
introducing siRNA into skeletal muscle in order to clarify characteristics of skeletal muscle as
a target organ for siRNA treatment. In this session, we will discuss unique properties of skeletal muscle, based on our result of a series of experiments to investigate relationship between
therapeutic efficacy and experimental conditions, such as administration procedures, dosage
amount and so on.
[1] Takeshita, F. et.al. Proc Natl Acad Sci USA., 2005, 102, 12177-12182.
[2] Kinouchi, N. et.al. Gene Therapy, 2008, 15, 1126-1130.
Naoki Makita
DDS Research Group, Formulation Research & Development Laboratories, Technology
Research & Development, Dainippon Sumitomo Pharma Co., Ltd.
3-45, Kurakakiuchi
1-Chome, Ibaraki-shi, Osaka 567-0878, Japan
Telephone: +81-72-627-8146
Email: naoki-makita@ds-pharma.co.jp
91
044
ACTIVATION OF CYCLOOXYGENASE-2 (COX-2) GENE
EXPRESSION BY PROMOTER-TARGETED DUPLEX RNAs
Masayuki Matsui1, Huiying Zhang1, Yougjun Chu1, Klaus Charisse2, Muthiah Manoharan2,
David R. Corey1, and Bethany A. Janowski1
1
Departments of Pharmacology and Biochemistry, University of Texas Southwestern
Medical Center, 6001 Forest Park Road, Dallas, TX 75390-9041, USA
2
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142, USA
RNA-mediated gene activation has been investigated over several years in some genes such
as progesterone receptor (PR), LDL receptor (LDLR), p21, and E-cadherin, but its mechanism is not fully understood yet. To obtain more insights into mechanism of RNA-mediated
gene activation, we targeted cyclooxygenase-2 (COX-2) gene using promoter-targeted duplex
RNAs (antigene RNAs (agRNA)). COX-2 is a rate-limiting enzyme responsible for prostaglandin biosynthesis, and COX-2 gene expression is associated with tumorigenesis, inflammation, and apoptosis. Small RNAs that modulate COX-2 gene expression would be useful
as tools to investigate biological functions of COX-2 in cells.
To investigate whether agRNAs have the potential to modulate COX-2 gene expression, duplex RNAs targeting the promoter were designed and tested in A549 lung cancer cells for
gene activation. After transfecting agRNAs, western blot and qPCR analysis were performed
to check expression levels of COX-2 protein and mRNA. We found that some of the duplex
RNAs (agRNA-12 etc; 25 nM) can activate COX-2 gene expression by more than 20-fold
relative to mismatch controls. The fold activation was similar with that for IL-1b (10 ng/mL),
a natural activator for COX-2 gene. We observed more than 100-fold activation in COX-2
mRNA in cells treated with both the activating agRNA and IL-1b, which indicates the two
activators can work synergistically. ChIP analysis showed increased occupancy of RNA polymerase II by 3-5 fold and NFkB by 2-4 fold in the promoter, suggesting that this activation
occurs at the level of transcription. In our previous studies with PR and LDLR, we identified
non-coding transcripts overlapping their promoters, which could be potential targets for agRNAs. We performed RACE and strand-specific RT-qPCR to examine whether similar noncoding transcripts are expressed in the COX-2 gene promoter. Those analyses revealed that
sense and antinsense transcripts are expressed at the COX-2 gene promoter. These transcripts
might be an endogenous mediator for regulating COX-2 gene expression that can be targeted
by agRNAs. Transfection of activating agRNAs did not substantially change expression of interferon responsive genes such as OAS1, OAS2, MX1, IFITM1, and ISGF3g, suggesting that
the activation of COX-2 gene by agRNAs is not due to interferon response. These data suggest that COX-2 gene can be induced by promoter-targeted duplex RNAs and the gene can be
used as a model for research of RNA-mediated gene activation.
Masayuki Matsui, PhD
University of Texas Southwestern Medical Center at Dallas
6001 Forest Park Road
Dallas, TX 75390-9041 USA
Telephone: +1-214-645-6154
Email: Masayuki.Matsui@UTsouthwestern.edu
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045
Abstract and poster available for review at the meeting.
93
046
Antisense oligonucleotides for Functional Studies of
Human Argonaute Proteins
Mescalchin A. and Innarella M.R.
Institute of Molecular Medicine, University of 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 involved in RNA interference (RNAi) mechanisms. In Caenorhabditis elegans and Arabidopsis
thaliana, creation of Argonaute (Ago) mutant strains has allowed the study of their biological
function, while in mammals this approach has been so far unsuccessful due to lethality of
such mutations.
In human cells four different Ago paralogs are present (hAgo1-4) and the functional studies
of these proteins relied on phenotypic suppression using small interfering RNA (siRNA).
The knockdown is achieved by the activity of the Ago proteins themselves which are
involved in the RNA interference mechanism. By suppressing the expression of hAgos using
these tools, the obvious consequence is a dying out of the silencing effect, which could lead
to interference between observed functional phenotypes and the mechanism of inhibition.
Thus, alternative methods acting by different regulatory mechanisms would be advantageous
in order to exclude unspecific effects. The knockdown may also be achieved by using specific
antisense oligonucleotides (asONs) which act via an RNase H-dependent mechanism, not
thought to interfere with the natural processes in which Agos are involved.
Functional observations in the use of hAgo-specific asONs indicate the relevance of this
assumption and confirm that hAgo2 is involved in microRNA (miRNA) and in siRNAmediated silencing pathways while the other hAgos play a role only in miRNA-based gene
regulation (Mescalchin et al. RNA 2010 16:2529-2536).
Here, we describe the effects of hAgo-specific downregulation by asONs and compared
them to those obtained by siRNA and shRNA. Moreover, we describe a novel strategy which
allows functional studies of hAgos and it is based on the simultaneous downregulation of the
endogenous proteins and overexpression of recombinant tagged and codon optimized hAgos.
Alessandra Mescalchin, PhD
Institute of Molecular Medicine, University of Lübeck
Ratzeburger Allee 160, 23538 Lübeck, Germany
Telephone: +49 (0)451 500 2742 or +49 (0)451 500 2736
Email: mescalchin@imm.uni-luebeck.de
94
047
SENSE OLIGONUCLEOTIDE-MEDIATED NATRE TECHNOLOGY
CONTROLS LEVELS OF CYTOKINE MESSENGER RNA
Mikio Nishizawa1, Emi Yoshigai1, Masaki Kaibori2, Tadayoshi Okumura2,3, A-Hon Kwon2
1
Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University,
Kusatsu, 2Department of Surgery, Kansai Medical University, Hirakata, 3Research
Organization of Science and Technology, Ritsumeikan University, Kusatsu, Japan
Natural antisense transcripts (asRNAs) are transcribed from many genes, and we recently
found asRNA transcribed from rat and mouse genes encoding inducible nitric oxide synthase
(iNOS), which is involved in inflammation [1,2]. The iNOS asRNA stabilizes iNOS mRNA
by interactions with the mRNA and RNA-binding proteins, and SENSE oligodeoxyribonucleotides (ODNs), which have the same sequences as that of mRNA, decrease levels of iNOS
mRNA [1]. In the presence of asRNA, the sense ODN functions as an effector to regulate
mRNA stability. In contrast, a sense ODN of the antisense technology is a negative control
that does not affect mRNA levels. We applied this novel method, i.e. Natural Antisense Transcript-targeted REgulation (NATRE; pronounces /nature/) technology, to cytokine genes.
1. Sequences of the iNOS sense ODNs were optimized by transfection of hepatocytes.
Each effective sense ODN included single-stranded portion(s) of stem-loops of the
mRNA.When the sense ODNs were compared with siRNA, the effects on iNOS mRNA
levels were comparative.
2. Tumor necrosis factor (TNF) alpha asRNA was induced by interleukin 1beta in
hepatocytes. Sense ODNs to TNF-alpha mRNA increased levels of TNF-alpha mRNA.
3. Rat asRNAs were transcribed from several other cytokine genes that are involved in
inflammation. When the NATRE technology was applied to them, some sense ODNs
decreased and the others increased the mRNA levels. It is possible that annealing of a sense ODN changes conformation of mRNA and accessibility of RNA-binding proteins.
4. Human asRNAs from cytokine genes were detected in various tissues.
Together, it implies that human asRNA may be a target of the sense oligonucleotide-mediated
NATRE technology to regulate mRNA stability.
References:
1) Matsui K, et al. (2008) Hepatology. 47, 686–697.
2) Yoshida H. et al. (2008) Shock. 30, 734–739.
Mikio Nishizawa, MD, PhD
1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan
Telephone: 81-77-561-2876
Email: nishizaw@sk.ritsumei.ac.jp
95
048
SILENCING OF microRNA FAMILIES BY SEED-TARGETING
TINY LNAs
Susanna Obad1, Andreas Petri1, Oliver Broom1, Markus Heidenblad1, Jan Stenvang1,
Ellen Marie Straarup1, Troels Koch1, Henrik Frydenlund Hansen1 and Sakari Kauppinen1,2
1
Santaris Pharma, Kogle Allé 6, DK-2970 Hørsholm, Denmark
2
Copenhagen Institute of Technology, Aalborg University, Lautrupvang 15, DK-2750
Ballerup, Denmark
MicroRNAs act as important post-transcriptional regulators of gene expression by mediating
mRNA degradation or translational repression. There is now ample evidence that perturbations in the levels of individual or entire families of miRNAs are prevalent in and strongly
associated with the development of a variety of human diseases. Apart from cancer, miRNAs
have also been implicated in viral infections, cardiovascular disease and neurological disorders. Hence, disease-associated miRNAs could represent a potential new class of targets for
oligonucleotide-based therapeutics, which may yield patient benefits unobtainable by other
therapeutic approaches.
LNAs comprise a class of bicyclic high-affinity RNA analogues, in which the ribose ring
in the sugar-phosphate backbone is locked in an RNA-like, C3’-endo conformation by the
introduction of a 2’-O,4’-C methylene bridge. This results in high binding affinity of singlestranded LNA-modified oligonucleotides to their complementary miRNA targets. Moreover,
LNAs combined with a phosphorothioate backbone show high biostability and enhanced
pharmacokinetic properties in vivo. Here, we describe an approach that enables miRNA
knock-down using 8 nucleotide fully LNA-modified phosphorothioate oligonucleotides,
termed tiny LNAs, complementary to the miRNA seed region. Transfection of tiny LNAs
into cells resulted in simultaneous inhibition of miRNAs within families sharing the same
seed with concomitant up-regulation of direct targets. In addition, systemically delivered,
unconjugated tiny LNAs showed uptake in many normal tissues, and pharmacological effect
in different mouse models, coinciding with long-term miRNA silencing. Considered together,
these data support the utility of tiny LNAs in elucidating the functions of miRNA families
with important implications for the development of therapeutic strategies aiming at pharmacological inhibition of disease-associated miRNAs.
Susanna Obad, PhD
Santaris Pharma A/S
Kogle Allé 6
DK 2970 Hørsholm
Danmark
Telephone: +45 45 179893
Email: suo@santaris.com
96
049
MICRORNA-132 REGULATES THE MAINTENANCE OF
NEUROMUSCULAR JUNCTIONS
Keren Ofek1, Geula Hanin1, Adi Gilboa-Geffen1, Amit Berson1, David S. Greenberg,1
Steve Wilton2 and Hermona Soreq1
1
The Hebrew University of Jerusalem/Israel 91904, 2Molecular Genetic Therapy Group,
Australian Neuromuscular Research Institute, Nedlands 6009/Western Australia
Neuromuscular junctions (NMJ), the synapses linking axon terminals of motor neurons with
the highly-excitable region of muscle fiber plasma membrane are responsible for initiating,
in response to acetylcholine action potentials across the muscle’s surface, ultimately causing the muscle to contract. Failed maintenance of NMJ functioning occurs in neuromuscular
diseases (e.g. Duchenne’s muscular dystrophy) or under exposure to auto-immune antibodies
(e.g. in myasthenia gravis) or to poisonous anti-cholinesterases) [1], but the underlying molecular mechanism(s) are incompletely understood. Given that the Acetylcholine hydrolyzing
enzyme acetylcholinesterase (AChE) is subject to blockade by microRNA-132 (miR-132)
[2], we explored the possibility that dynamic changes in miR-132 levels modulate NMJ function and that manipulating this miR levels in vivo would facilitate NMJ functioning under
insults. Here, we report that within 24 hours, intravenous (IV) injection of 3.3 mg/kg AM132,
a 22-mer 2’-O-methyl modified antisense oligonucleotide on a phosphorothioate backbone elevates the RNA levels of the miR-132 targets AChE, MeCP2 and p250GAP, increases AChE
activity in the diaphragm and gastrocnemius muscles and facilitates histochemical staining
of AChE in diaphragm NMJ (10.9±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-protected mice exhibited less poisoning symptoms (Mann-Whitney U-test, P=0.028, N=4/group),
better recovery and survival (ANOVA P=6.62E-12, N=4-7/group) or extended survival time
compared to controls (ANOVA P=6.8E-9, N=5/group). Our findings highlight the role of
miR-132 as a master controller of NMJ functioning and open new venues for manipulating
NMJ function under disease and/or environmental and war insults.
References
[1] Soreq & Seidman. Acetylcholinesterase-new roles for an old actor. Nat Rev Neurosci.
2(4):294-302. (2001).
[2] Shaked et al. MicroRNA-132 potentiates cholinergic anti-inflammatory signaling by
targeting acetylcholinesterase. Immunity. 2009 ;31(6):965-73
Keren Ofek, MSc
The Hebrew University
Soreq Lab, Safra Campus
Jerusalem 91904, Israel
Telephone: 972-2-6585450
Email: Keren.ailon@mail.huji.ac.il
97
050
PLASMA PHARMACOKINETICS OF MIRAVIRSEN IN HUMANS
Persson R, Bagger Y, Børgesen H, Hodges, MR, King BD, Levin AA
Santaris 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, PhD
Santaris Pharma A/S
Kogle Allé 6, 2970-Hørsholm, Denmark
Telephone: +45 4517 9947
Email: rop@santaris.com
98
051
Towards evaluation of point-mutations and drug
interference with miRNA regulated expression of
BRCA1
Christopher Polonyi, Sofi K.C. Elmroth
Biochemistry and Structural Biology, PO Box 124 SE-22100 Lund, Sweden
It is a challenge to try to understand how gene expression is regulated in various eukaryotic organisms, including the human body. The cells in eukaryotic organisms contain small
double-stranded hairpin-like RNA structures coded for by microRNA (miRNA) genes in the
cell nucleus. By use of the RNA induced silencing complex (RISC), the miRNAs interfere
with translation by targeting mRNA. This kind of gene silencing can also arise if a small
double-stranded exogenous RNA, so called small interfering RNA (siRNA), is introduced
into a eukaryotic cell1. The aim in the present project is to further increase the knowledge
concerning the sensitivity of the siRNA and miRNA machinery towards interference caused
by point-mutations and drugs. Strategies that are used involve in-house design of siRNAs and
miRNAs and construction
of drug-miRNA adducts
for evaluation of drug interference. Potential drug
candidates are used to investigate the expression of
the BRCA1 gene, which
codes for proteins that
are associated with breast
cancer development2. In
the initial studies, BRCA1
mRNA targeting has been
evaluated in a luciferasebased assay (see Figure 1)
and on protein level.
References:
1. Carthew RW, Sontheimer EJ. Origins and mechanisms of miRNAs and siRNAs.
Cell. 2009 Feb 20; 136(4):642-55.
2. Shen J, Ambrosone CB, DiCioccio RA, Odunsi K, Lele SB, Zhao H. A functional
polymorphism in the miR-146a gene and age of familial breast/ ovarian cancer diagnosis. Carcinogenesis. 2008 Oct;29(10):1963-6.
Christopher Polonyi, PhD student
Department of Biochemistry and Structural Biology
Center for Molecular Protein Science, Lund University
PO Box 124, SE-22100 Lund, Sweden
Telephone: +46 46 2228108
Email: Christopher.Polonyi@biochemistry.lu.se
99
052
lEPIGENETIC MYOSTATIN BLOCKADE
Thomas Roberts, 1Graham McClorey, 1Samir EL-Andaloussi, 2Kevin Morris and
1
Matthew Wood
1 Department of Physiology, Anatomy and Genetics, University of Oxford, OX1 3QX, UK
2 Department of Molecular and Experimental Medicine, The Scripps Research Institute,
La Jolla, CA, USA
1
Myostatin is a secreted growth factor that acts to negatively regulate muscle mass. Myostatin
null mutations result in increased musculature in cattle, mice and humans and thus myostatin blockade is being considered as a putative therapy for muscle wasting disorders such as
Duchenne muscular dystrophy (DMD). Various myostatin blockade strategies have been
shown to induce functional improvement in the mdx mouse model of DMD. Similarly, the
combination of myostatin blockade with dystrophin splice correction has results in greater
functional improvement than either strategy alone. Transcriptional Gene Silencing (TGS)
can be induced by small RNA molecules complementary to target gene promoter sequences.
TGS effectors target chromatin modifying activities to low copy number transpromoter RNAs
which induce silent state chromatin structure and, in some cases, promoter DNA methylation.
Consequently, TGS may be permanent and heritable. In order to investigate the possibility of
silencing myostatin by TGS we designed small interfering RNAs (siRNAs) targeting the myostatin promoter. These were transfected in differentiated C2C12 mouse myotubes. Two siRNAs resulted in significant myostatin silencing. Knock-down was observed in multiple transfection experiments, and with both conventional and nuclear-targeting transfection reagents.
The results were replicated in the disease relevant H2K-mdx cell line indicating that the effect
is not unique to C2C12 myoblasts. Myostatin silencing was also found to be independent of
the interferon response. Treatment with the histone deacetylase inhibitor Trichostatin A alleviated the silencing effect of promoter-targeting siRNA suggesting that epigenetic remodelling
at the myostatin promoter is responsible for the observed silencing. Similarly, enrichment of
the silent state histone mark H3K9me2 was observed at the myostatin promoter following
treatment with promoter-targeting siRNA.
We also report progress towards achieving myostatin transcriptional silencing in vivo.
Thomas Roberts, MBiochem
Department of Physiology, Anatomy and Genetics
University of Oxford
Le Gros Clark Building
South Parks Road
Oxford
OX1 3QX, United Kingdom
Telephone: +44 (0)1865 272167
Email: thomas.roberts@dpag.ox.ac.uk
100
053
A New tool for Site-Specific Chemical Modification
of RNA
Shigeki Sasaki, Atsushi Shibata, Natsuki Inaba, Kazumitsu Onizuka, Yosuke Taniguchi
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan
The chemical modification of RNA has a strong impact on RNA research; the fluorescent
labelling of RNA is useful to monitor RNA production, processing, relocation in the cell,
interaction with other intracellular components and degradation, etc. Modified nucleotides
in RNA play an important role in cellular functions and the progression of diseases. Thus,
chemical tools that can modify nucleotides in RNA with high specificity and efficiency in
situ may specifically alter the function of the target RNA, and thereby will be valuable for an
RNA study as well as for manipulation of the gene expression at the RNA level.
We recently developed the efficient functionality-transfer reaction (FTR) for site-specific
modification of RNA by using the ODN probe involving the 6-thioguanosine functionalized
with the transfer group.RNA modification occurs at the 4-amino group of the cytosine at pH
7 or at the 2-amino group of the guanine base under alkaline conditions or in the presence of
NiCl2. The FTR was applied to pin-point labeling of RNA with a variety of functional groups
such as biotin, fluorescent dyes and PEG, etc. through “click chemistry” (Fig. 1).1d Biotinmodification of RNA may have broad utility, because biotin can bring avidin-fused proteins
to the specific site of RNA. In this paper, we discuss whether the functionality-transfer ODN
can modify large RNA molecules such as mRNA, and what effects may be induced on the
biological functions of the target RNA such as for polymerization and translation.
Fig. 1 The functionality transfer reaction (FTR) for modification of the
2-amino group of guanosine of the internal RNA
Shigeki Sasaki, PhD
Graduate School of Pharmaceutical Sciences, Kyushu University,
3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Telephone and Fax: +80-92-642-6615
Email: sasaki@phar.kyushu-u.ac.jp
____________________________
1 (a) Bioconjugate Chem. 2009, 20, 799-803, (b) Nucleic Acids Res. 2010, 38, 1760-1766, (c) Bioconjugate Chem. 2010, 21, 1508-1512, (d) Chem. Comm., 2011, 47, 5004-5006
101
054
mRNA Cap 2′O-methylation prevents immune
recognition of self-RNA by RIG-I
Christine Schuberth1, Janos Ludwig1, Anna Maria Herzner1, Thomas Zillinger1,
Romy Kerber2, Marion Goldeck1, Christoph Coch1, Veit Hornung1, Christian Drosten3,
Winfried Barchet1, Beate M. Kümmerer3, Gunther Hartmann1 and Martin Schlee1
1Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Germany
2Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany
3Institute of Virology, University of Bonn Medical Centre, Bonn, Germany
The innate immune response to viruses is based on pattern recognition receptors (PRR) that
detect a characteristic structure or an unusual location of viral nucleic acids within the cell.
As viral RNA is located in the same compartment as host RNA, PRRs in the cytosol have to
discriminate between viral and self RNA by structure or modification. This challenging task
is taken on by the cytosolic DExD/H-box family helicases RIG-I and MDA-5. The ubiquitously expressed receptor RIG-I detects 5’triphosphorylated RNA (pppRNA) occurring during
viral RNA replication thereby controlling the immune response to most RNA viruses. Like
viral RNA, eukaryotic mRNA harbours a triphosphate at its 5’end. So far it is presumed, that
the N7-methylguanine (m7G) cap structure, as present on all eukaryotic mRNAs, shields the
5’-triphosphate of self mRNA from RIG-I-mediated immune recognition. Here we synthesized
RNA containing different cap structures and demonstrated that a m7G cap could only partially
reduce RIG-I stimulation, while a G cap (without N7 methylation) did not impair RIG-I activation at all. By contrast, a single 2′O-methyl group at the 5’-penultimate residue (N1), a characteristic feature of vertebrate mRNA, was sufficient to abrogate RIG-I activation completely.
Interaction assays of recombinant RIG-I with modified 3P-RNA ligands confirmed that mainly the 2’O-methylation at N1 mediated inhibition of binding of capped RNA to RIG-I. Studies on the yellow fever virus-encoded 2′O-methyltransferase indicated that viruses mimic cap
2′O-methylation at N1 to escape RIG-I recognition. Thus, not the m7G cap but the 2′O-methyl
group at N1 is the critical determinant for RIG-I to distinguish self from foreign triphosphatecontaining RNA.
Christine Schuberth,
Institute for Clinical Chemistry and Clinical Pharmacology
University Hospital Bonn
Sigmund-Freud-Str. 25
53105 Bonn, Germany
Telephone: +49-228-287-51157
Email: christine.schuberth@uni-bonn.de
102
055
ANTISENSE APPROACHES TO STUDY REST/NRSF
TRANSCRIPTIONAL REGULATION AS A THERAPEUTIC
TARGET FOR NEURODEGENERATIVE DISORDERS
Yalda Sedaghat, Curt Mazur, Brett P. Monia
ISIS pharmaceuticals, Carlsbad, CA, USA, 92010
The repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/
NRSF) is a master regulator of neuronal gene expression. REST functions through binding the genomic loci that contain the repressor element-1 (RE1) binding motif and recruits a series of epigenetic and regulatory cofactors to its N- and C-terminal domains. Various studies have characterized
the specific roles played by REST during neuronal lineage specification and maturation. Among
the REST targets BDNF has emerged as a candidate of interest as a potential neuroprotective and
functionally restorative treatment in psychiatric and neurological disorders, including Huntington’s
disease (HD). In HD, BDNF levels are significantly attenuated as a result of impaired BDNF
transport from the cortex to the striatum in the presence of mutant HTT. Wild-type HTT also
regulates transcription of BDNF through REST, by sequestering REST in the cytoplasm, therefore
restricting its access to the nucleus, which leads to the transcription of target genes such as BDNF.
In this study, we were interested in down-regulation of REST as a potential therapeutic strategy
to both slow down cell death and maintain the functional state of remaining neurons. This
could be possible through the neuroprotective and functional effects of BDNF by activation of
signaling in neuronal pro-survival pathways. In order to specifically target REST, we applied
2nd generation Antisense Oligonucleotides (ASOs) to reduce levels of REST mRNA in the liver
and the CNS in both normal (wild type) and HD mice and characterized the effects on BDNF
expression and gene expression more globally, as determined by microarray analysis.
We have demonstrated specific reduction in REST levels in both liver and CNS following treatment by ASOs, which resulted in the induction of a number of neuronal genes including BDNF
and Synapsin1 at the mRNA and protein levels. Gene array expression analysis was performed on
BALB/c mouse liver and R6/2 brain, a mouse model of HD, following systemic and ICV administration of REST ASO, respectively. Samples were analyzed by hybridization to the MouseWG-6
v2 Expression BeadChip array (Illumina), to gain insight into putative pathways affected by REST
suppression. Array data analysis was performed using GeneSpring, followed by Gene network
prediction using 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 identified that were affected by REST suppression that are predicted to play a role in cancer, genetic
disorders, neurological diseases, cell-to-cell signaling, and tissue development.
Our findings suggest that REST may be an important target for neurodegenerative diseases
like HD, that it is also involved in the regulation of a broad range of cellular pathways outside
of the CNS, and that the antisense approach is a viable strategy for selectively modulating
REST activity in both the CNS and in the periphery.
Yalda Sedaghat, PhD/Brett P. Monia, PhD
ISIS Pharmaceuticals Inc.
2855 Gazelle Court
Carlsbad, CA 92010 USA
Telephone: Brett Monia: 760-603-2350
Email: bmonia@isisph.com; ysedaghat@isisph.com
103
056
FUNCTIONAL ANALYSIS OF microRNAs AS REGULATORS OF
CARGO TRAFFICKING
Andrius Serva, Sanchari Roy, Bettina Knapp, Lars Kaderali, Holger Erfle, Ursula Kummer,
Roland Eils and Vytaute Starkuviene
BioQuant, University of Heidelberg, Heidelberg, Germany
MicroRNAs (miRNAs) are small non-coding RNAs emerged as important translational
gene expression regulators in eukaryotic cells. miRNAs are predicted to modulate the expression of nearly 60% of protein-coding mammalian genes. Thereby, miRNAs affect a multitude
of biological processes such as developmental transition, cell proliferation and apoptosis. Alteration of miRNA expression patterns leads to tumorigenesis, diabetes, neurodevelopmental
disorders and many other diseases.
In the present study we demonstrate an unbiased integrative approach to identify miRNAs
and their functionally relevant target genes involved in the regulation of secretory membrane
trafficking. By applying (i) molecular biology, (ii) fluorescence microscopy, (iii) statistical
data analysis, (iv) bioinformatics and (v) miRNA and mRNA expression profiling methods
we are enabled to identify miRNAs of interest. A high-throughput screening of oligonucleotides mimicking 470 human miRNAs led to the identification of 44 miRNAs with an effect
on the model cargo protein ts-O45-G secretion rate. In parallel, a large-scale library screening
of 875 miRNA inhibitors yielded 68 miRNAs, which inhibition also caused significant changes of cargo trafficking. Further examination of primary miRNA hits by combined microscopy-based and automated image analysis-based Golgi complex integrity assay revealed that
perturbation of 13 miRNAs resulted in quantitative changes of Golgi complex morphology.
Moreover, we uncovered an active role of oncogenic miR-17~92 cluster in the regulation of
cargo trafficking and identified TBC1D2 and LDLR genes as novel targets of miR-17.
In perspective, the results of detailed analysis of identified miRNAs and their target genes
will allow us to model the posttranscriptional miRNA-mediated regulatory networks. A comprehensive understanding of how these networks coordinate the activity of secretory membrane trafficking as a global adaptive response system is needed to study miRNAs as promising therapeutic agents and targets under particular pathological conditions.
Andrius Serva, MSc
BioQuant, University of Heidelberg
Im Neuenheimer Feld 267
69120 Heidelberg, Germany
Telephone: +49-6221-5451258
Email: andrius.serva@bioquant.uni-heidelberg.de
104
057
SILENCING THE CDK4 AND CDK6 GENES THROUGH RNA
INTERFERENCE
Małgorzata Sierant, Danuta Piotrzkowska and Barbara Nawrot
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences,
Lodz, Poland
Alzheimer’s disease (AD), a disorder which affects millions people worldwide, is characterized by progressive memory loss and a spectrum of behavioral abnormalities leading to
dementia. An effective treatment of AD is still not available. Preventing or slowing down
progression of neurodegeneration will considerably reduce the number of severely affected
patients and improve the quality of life of the aging population. The biological causes of neuronal death in AD have been the objective of intense studies and several concepts were proposed [1]. Several investigators observed close association between the neuronal cell death
and reactivation of the cell cycle in post-mitotic neurons. Abnormal cell-cycle-related proteins expression, as cyclin dependent kinases, cyclins and cdk inhibitors was observed in pathologically affected parts of AD brains [2]. Usually, in postmitotic and terminally differentiated
neurons, the cell-cycle activity is arrested by the enrichment of cdk inhibitors, neurons are
“locked” in the G0 phase. At the beginning of neuropathogenesis, neurons leave the G0 phase
and progress until the S phase and die at the G1-M transition. The aim of our studies was to
down-regulate the expression of two cyclin dependent kinases CDK4 and CDK6 in neural
cells through RNA interference-based gene silencing. Such approach could be the proposal
for the new neuroprotective gene therapy in AD and the other neurodegenerative disorders.
Thus, sixteen siRNAs with sequences complementary to human, mouse and rat transcripts of
CDK4/6 genes were designed and synthesized in house. Their silencing activity was screened
in human (HeLa, SH-SY5Y), mouse (Neuro2a, RAW 264.7) and rat (PC12) cell lines and
assessed by quantitative RT-PCR (target mRNA level) and by Western blot (target proteins
level) analyses. Some of the silencers dowregulated the expression of both target genes up to
90%. Activity of siRNA was also valuated by flow cytometry, where we counted the number
of cells in G1/G0, S and G2/M phases. Analysis has shown that for cells transfected with
active siRNA molecules, which result in down-regulation of CDK4/6 genes expression the
number of cells in G1/G0 phaseis is increased, while the number of cells in S phase is lowered. This effect dependented on concentration of siRNA. Selected sequences of the most
active siRNAs were used for construction of shRNA and micro RNA based shRNA inserts
cloned into two type of plasmids: with U6 promoter (Pol III) (pSilencer-2.0 U6, Ambion) and
CMV promoter (Pol II) (pEGFP-C1, Clontech and pcDNA 6.2-GW/miR, Invitrogen). For selected cells transfected with these constructs the lowering of CDK4 and CDK6 genes expression was observed in the range 10-70% (mRNA) and 20-70% (protein).
[1] B. Nawrot, et al. RSC Drug Discovery 3, 230, 2009; [2] Y. Yang, et. at. J. Neurosci. 23,
2557, 2003.
Malgorzata Sierant, PhD
CMMS PAS, Lodz 90-363, Sienkiewicza 112, Poland
Telephone: (+48 42) 680-32-72
Email: msierant@bio.cbmm.lodz.pl
105
058
IMPROVED STABILITY AND GENE SILENCING ACTIVITY OF siRNA
ANALOGS CONTAINING PHOSPHORODITHIOATE LINKAGES
Malgorzata Sierant 1, Magdalena Janicka 1, Xianbin Yang 2, Na Li 2 and Barbara Nawrot 1
1
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences,
Lodz, Poland; 2AM Biotechnologies LLC, Houston, Texas, USA
Short interfering RNAs (siRNAs) are widely used to identify gene functions and hold great
potential in providing a new class of therapeutics. Although unmodified siRNAs are used
with success for a gene silencing, chemical modifications of one or both strands are desired
for pharmaceutical applications to enhance a nuclease stability, potency and to improve
pharmacokinetic properties of duplexes. Modification of the internucleotide bond involving
substitution of a single non-bridging phosphate oxygen atom with e.g. an sulfur atom, leads
to the achiral modified phosphorus center and to mixture of unresolvable diastereomeric oligomers possibly having variable biochemical, biophysical, and biological properties.
The substitution of both non-bridging phosphate oxygen atoms with sulfurs gives rise to
a phosphorodithioate (PS2) internucleotide linkage which, like natural RNA, is achiral at
phosphorus. The PS2 linkage is isosteric and isopolar with the normal phosphodiester and
should have other biochemical and biophysical properties similar to natural RNA. Here we
describe the application of RNA molecules containing the PS2 linkages as components of the
novel type of the PS2-siRNA duplexes. In our studies, for tests of new PS2-siRNA molecules,
we chose previously identified active siRNA sequences for the β-secretase (BACE1) and
enhanced green fluorescent protein (EGFP) transcripts [1,2]. CD spectra collected for PS2siRNA duplexes are similar to the spectra of the unmodified duplex, consistent with the typical A-type structure of double-stranded RNA. The UV-monitored thermal dissociation studies
have shown that the duplexes with up to five PS2 linkages exhibit transition curves essentially identical in shape to the unmodified siRNA. We did not observe remarkable fluctuations
in Tm values in comparison to the unmodified reference duplex. The gene silencing experiments performed with several siRNA duplexes containing the PS2 linkages either in the sense
or antisense strand indicate that the extent of silencing effect depends on not only the number
of the PS2 modifications but expecially on the strand and position within an siRNA duplex. In
some cases we observed enhanced gene knockdown activity in comparison with unmodified
duplex. Given the straightforward synthesis of PS2 linkages, as well as their known nuclease
resistance and lack of chirality, PS2 modifications are promising candidates for siRNA in research and therapeutic applications.
[1] Sipa K. et al. RNA 13, 1301, 2007
[2] Sierant M. et al. Int. J. Alzheimers Dis. 2009, Jul 14, 2009
Malgorzata Sierant, PhD
CMMS PAS, Lodz 90-363, Sienkiewicza 112, Poland
Telephone: (+48 42) 680 32 72
Email: msierant@bio.cbmm.lodz.pl
106
059
DICER-SUBSTRATE siRNA EXHIBIT IMPROVED GUIDE STRAND
SELECTION AND STRONGER RISC LOADING COMPLEX
FORMATION COMPARED TO CANONICAL siRNA
Nicholas M. Snead, Kumi Sakurai, John J. Rossi
Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute at the
City of Hope, Duarte, CA USA
As small interfering RNA (siRNA) continues making headway toward therapeutic applications, researchers are looking to optimize the efficacy of both a suitable delivery vehicle
and the therapeutic RNA itself. We are interested in maximizing the efficacy of siRNAs at
the RNA level through relatively minor chemical and structural modifications to canonical
siRNA. The rationale behind modifying the canonical siRNA structure is, among other criteria, to improve loading of the guide strand into the RNAi machinery, reduce off-target effects, and possibly reduce competition with endogenous miRNAs, ultimately leading to better
efficacy. Here, we present a parallel analysis of four siRNA variants (the canonical 21mer, a
commonly used 19merTT, a Dicer substrate interfering RNA (dsiRNA), and a short trigger
16merTT)) in gene silencing, RNAi protein binding, and miRNA competition disruption assays. The dsiRNA consistently demonstrated better gene silencing by the guide strand and
worse gene silencing of the passenger strand compared to its analogous 21mer or 19merTT.
The dsiRNA also showed more robust formation of a high molecular weight complex known
to contain Dicer and TRBP (two primary members of the RISC-loading complex) compared
to its analogous 21mer and 19merTT. Small RNA deep sequencing results reveal that (1) cells
transfected with dsiRNA have a global miRNA profile more similar to untransfected cells
compared to 21mer- or 19merTT transfected cells, and (2) at low transfection concentrations,
the guide strand of the dsiRNA is more abundant than the 21mer or 19merTT. The 16merTT
did not perform comparably in any of the assays. Also, we are interested in re-exploring the
5’ triphosphate group on siRNAs that would trigger RIG-I-mediated interferon production as
a bifunctional way to ablate diseased cells. Interestingly, preliminary results using 5’ triphosphate 19+2 siRNAs generated by T7 in vitro transcription did not silence reporter gene expression; we are exploring this further. While delivery of therapeutic small RNAs is arguably
the largest hurdle to overcome, our studies show that relatively small changes to canonical
siRNAs can also improve potency.
Nicholas M. Snead, MS
Beckman Research Institute at the City of Hope
1500 E. Duarte Road, Fox N 2002H
Duarte, CA, 91010 USA
Telephone: (626) 256-4673, ext. 64457
Email: nsnead@coh.org
107
060
FINDING CANCER THERAPEUTIC sgRNAs BASED ON TRUE GENE
SILENCING
M. Takahashi1, S. Matsuda1, Y. Watabe1, A. Goto1, D. Kamiya1, T. Sano1, N. Watanabe2,
M. Narita2, M. Takahashi2, M. Tamura3, M. Nashimoto1
1
Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan,
2
Niigata University, Niigata, Japan, 3Hokkaido University, Sapporo, Japan
tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is one of the RNA-mediated gene expression control technologies that have therapeutic potential.1,2 This technology
is based on the properties of tRNase ZL that it can cleave any target RNA at any desired site
under the direction of an appropriate artificial small guide RNA (sgRNA) and that cytosolic
tRNase ZL can modulate gene expression by cleaving mRNA under the direction of cellular
5′-half-tRNA or microRNA as sgRNA (Figures 1 and 2).3,4
We have shown that heptamer-type sgRNA with simple chemical modifications can be taken
up by human cells without any transfection reagents and can exist in the cells stably. We have
also demonstrated that a “naked” heptamer-type sgRNA targeting the human Bcl-2 mRNA
can induce apoptosis of leukemia cells without inducing the interferon response.
In order to find candidate cancer therapeutic sgRNAs, we designed 160 heptamer-type sgRNAs targeting various human mRNAs, and examined them for inducing apoptosis in human
leukemia and myeloma cell lines. We found that 40 of the 160 naked sgRNAs can efficiently
induce apoptosis in at least one of the cancer cell lines. We also showed that a subset of the
sgRNA library can significantly decrease levels of targeted cellular mRNAs. Furthermore, we
demonstrated that 10 of the 40 effective sgRNAs can reduce tumor growth rates in mouse xenograft models. These results show that naked sgRNAs can induce apoptosis in leukemia and
myeloma cells through targeting various mRNAs.
1. A. Nakashima, et al. (2007) Gene Therapy, 14, 78–85.
2. R. A. Elbarbary, et al. (2009) Biochem. Biophys. Res. Commun., 379, 924–927.
3. R. A. Elbarbary, et al. (2009) PLoS ONE, 4, e5908.
4. R. A. Elbarbary, et al. (2009) FEBS Lett., 583, 3241–3246.
Masayuki Takahashi, Master
Higashijima 265-1, Akihaku, Niigata, Niigata 956-8603, Japan
Telephone: +81 250 25 5118
Email: s09m13u@st.nupals.ac.jp
108
061
KINETIC COMPARISON OF GENE SILENCING PROFILE OF siRNA
AND SHRNA-EXPRESSING PLASMID DNA IN VIVO
Yuki Takahashi, Yuriko Matsui, Makiya Nishikawa, Yoshinobu Takakura
Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
RNA interference (RNAi) is a gene silencing event in which small interfering RNA (siRNA)
degrades target mRNA in a sequence-specific manner. RNAi can be also induced by introducing plasmid DNA (pDNA) that expresses short-hairpin RNA (shRNA) into cells. Because of
the specificity and potency of RNAi-mediated gene silencing, the research for the development of RNAi-based therapy has been an exciting field since the discovery of RNAi. For
therapeutic application of RNAi, the time course of gene silencing is one of the important
factors to be considered. However, little attention has been paid to the duration of RNAibased gene silencing in vivo. There has been no report of gene silencing 1 week or longer
after the induction of in vivo RNAi. In the present study, we evaluated and compared the
profiles of gene silencing induced by siRNA and shRNA-expressing pDNA in mice. A newly
developed reporter pDNA (pROSA-gLuc) encoding Gaussia luciferase cDNA under the control of human ROSA26 promoter was used to obtain a prolonged expression of the reporter
protein over three months. We administered this pDNA to mice together with siRNA or shRNA-expressing pDNA targeting Gaussia luciferase at varying doses by the hydrodynamic injection method. The serum level of Gaussia luciferase was greatly but transiently reduced by
siRNA, whereas a sustained reduction was obtained with shRNA-expressing pDNA. A quantitative analysis of the gene silencing profiles based on a moment analysis confirmed the finding that shRNA-expressing pDNA suppresses the target gene expression longer than siRNA.
These results provide important information about the time-courses of RNAi in vivo.
Yuki Takahashi, PhD
46-29 Yoshidashimoadachi-cho
Sakyo-ku, Kyoto 606-8501, Japan
Telephone: +81-75-753-4616
Email: ytakahashi@pharm.kyoto-u.ac.jp
109
062
Effect of guanine bases modifications and L-DNA
on stability and kinetic of formation of
tetramolecular quadruplexes
Phong Lan Thao Tran1*, Antonella Virgilio2, Veronica Esposito2, Giuseppe Citarella2,
Aldo Galeone2, Rui Moriyama3, Atsushi Maruyama3, Bernard Rayner1 and
Jean-Louis Mergny1
1 Institut Européen de Chimie et Biologie, INSERM U869, Université de Bordeaux (France)
2 Università degli Studi di Napoli Federico II, Napoli, Italy
3 Institute for Materials Chemistry and Engineering, Kyushu University, Fukuoka, Japan
Tetramolecular G-quadruplexes result from the association of four guanine-rich strands. These structures are formed by the interaction of four guanines organized in a cyclic Hoogsteen hydrogen bonding
arrangement termed a G-quartet, and by the stacking of several G-quartets. G-quadruplexes can be very
stable under physiological conditions and the evidence for quadruplex formation in vivo is compelling.
Two quadruplex related molecules are currently in phase II clinical trials and a number of aptamers
adopt a quadruplex fold. Modification of the backbone strand or the guanine bases of the oligonucleotide may improve stability or introduce new functionalities. In this regard, position 8 of the guanosine
base is particularly suitable for introduction of modifications since as it is positioned in the groove of
the quadruplex structure [1]. In this study, we investigated the effect of an 8-methyl-2’-deoxyguanosine
residue (M) on the structure and stability of tetramolecular parallel G-quadruplexes [2].
We also studied the mirror image of natural DNA that may form structures of opposite chirality. One of
the advantages of L-nucleic acids is their nuclease resistance, allowing one spiegelmer (from “Spiegel”
meaning mirror in German) to enter clinical trials [3].
We demonstrate here that a short guanine-rich L-DNA strand may form a tetramolecular quadruplex
with the same properties as a D-DNA strand of identical sequence, and L- and D- strands self-exclude
when mixed together [4].
These structures have already found a number of interesting applications in biotechnology [5, 6] and
nanotechnology [7, 8] and we are currently working on new developments involving
G-quadruplexes.
Tetramolecular
G-quadruplex
8-methyl Guanine
D-GuanineL-Guanine
References:
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
J. Gros, et al., Chembiochem 2008, 2926-2928.
P.L.T. Tran, et al., Biochimie 2011, 93(3):399-408.
S.G. Sayyed, et al., Diabetologia 2009, 52, 2445-54.
P.L.T. Tran, et al. Chem. Commun. 2011, 47, 5437-5439.
B. Wlotzka, et al., Proc Natl Acad Sci USA 2002, 99, 8898-902.
Y. Kim, et al., Nucleic Acids Res 2007, 35, 7279-87.
M.S. Kaucher, et al., J. Am. Chem. Soc. 2006, 128, 38-39.
C. Lin, et al. Nano Lett. 2009, 9, 433-436.
Phong Lan Thao Tran
2, rue Robert Escarpit, 33607 Pessac France
Telephone: +33 6 27 12 15 57
Email: plt.tran@iecb.u-bordeaux.fr
110
063
Targeting tumor antigens to dendritic cells in vivo
using receptor-specific aptamers conjugated to
tumor antigen
Johannes Urban, David Boczkowski, Kyle Kok Loong Phua, Bruce Sullenger and Smita Nair
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
The success of immunotherapy depends on the induction of effective primary immune
responses to treat existing cancers and the generation of subsequent memory responses to
prevent recurrence at a new site. Dendritic cells (DC), recognized as major antigen presenting
cells, are uniquely equipped to initiate and regulate immune responses, making them a key
target for developing new therapies. Immunizing with antigen-loaded DC is a powerful
method of inducing CD4+ and CD8+ T cell responses and antibodies (Abs). Although ex
vivo DC therapy has shown promise, it is a customized, complex, patient-specific cell therapy
that reduces its universal applicability for cancer. Thus there is a critical need to develop
strategies that circumvent the need for ex vivo DC loading. Directly targeting antigens to
DC in vivo will facilitate the development of a more effective “off-the-shelf” tumor vaccine,
which will be accessible to large numbers of patients.
DC express a number of specialized endocytic receptors of the C-type lectin family enabling
capture of antigen by receptor-mediated endocytosis at very low concentrations. Studies
exploiting the endocytic properties of DC receptors demonstrated that antigens conjugated to
receptor targeting moieties, such as the natural receptor-ligands or receptor-specific antibodies, increased antigen capture and presentation 100-1000-fold as compared to soluble antigen.
Moreover, receptor targeting facilitates cross-presentation to induce Ab and CD4+ T cell
immunity as well as elicit MHC-class-I-restricted CD8+ or cytotoxic T lymphocytes (CTL),
critical for cancer immunotherapy.
To deliver antigens to DC in vivo, we developed artificial receptor ligands to the macrophage
mannose receptor (MMR or CD206), an endocytic C-type lectin receptor expressed on DC
and macrophages. Herein, a RNA aptamer library comprised of 40 randomized nucleotides
and modified with 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, PhD
Duke University Medical Center
106 Research Drive, Durham, NC 27701 USA
Telephone: 919-684-6780
Email: ju6@duke.edu
111
064
Designed Ankyrin Repeat Proteins for Targeted
Delivery of siRNA Oligonucleotides
Johannes Winkler1, Claudia Böhme1, Nikolas Stefan2, Andreas Plückthun2,
Uwe Zangemeister-Wittke2, 3
1
University of Vienna, Department of Medicinal Chemistry, Vienna, Austria
2
University of Zürich, Department of Biochemistry, Zürich, Switzerland
3
University of Bern, Institute of Pharmacology, Bern, Switzerland
Designed Ankyrin Repeat Proteins (DARPins) are a novel class binding proteins with promising biochemical properties with regard to tissue-specific siRNA delivery. Ankyrin repeat
proteins form an extended interaction interface which is able to bind to structural epitodes on
the target protein surface. Their small size, high stability and the absence of post-translation
modifications facilitate protein engineering and results in high-yield recombinant production
in E. coli. By introducing a C-terminal cystein, a unique binding site for conjugations can be
created, enabling the introduction of modifications for attachment or complexation of siRNA
oligonucleotides. DARPins targeted at the extracellular domain of the cancer protein EpCAM
were selected from ankyrin repeat protein libraries using ribosome display. We generated fusion proteins of an EpCAM-binding DARPin and highly basic peptides which were expressed
in E. coli in high yields and after purification efficiently complexed the siRNA cargo.
DARPin fusion protein mediated delivery resulted in specifically enhanced cellular uptake of
siRNA in antigen-positive cells. A bcl-2 targeted siRNA silenced the respective gene expression in EpCAM-positive MCF-7 breast cancer cells and resulted in enhanced susceptibility to
doxorubicin treatment.
Dimerization of the DARPin increased binding affinity, but had only minor effects on delivery efficiency. Initial experiments indicated that the loading capacity for siRNA is a more
important characteristic for efficient gene silencing than the antigen affinity of the targeting
moiety.
The obtained results demonstrate the usefulness of DARPins for targeted siRNA delivery and
give insight into important attributes of siRNA delivery devices.
Johannes Winkler, PhD
University of Vienna, Department of Medicinal Chemistry
Althanstraße 14, 1090 Vienna, Austria
Telephone: +43 1 4277 55058
Email: johannes.winkler@univie.ac.at
112
065
POTENCY COMPARISON OF SERIES OF BNA-BASED ANTISENSES
IN DYSLIPIDEMIC ANIMALS
Tsuyoshi Yamamoto1,5, Mariko Harada-Shiba1, Moeka Nakatani1,5, Hidenori Yasuhara1,5,
Shunsuke Wada1,3, Keisuke Narukawa5, Kiyomi Sasaki3, Hidetaka Torigoe3, Tetsuji Yamaoka2,
Takeshi Imanishi4 and Satoshi Obika5
Department of Molecular Innovation in Lipidology1 and Department of Biomedical Engineering2,
National Cerebral and Cardiovascular Center Research Institute, Faculty of Science, Tokyo
University of Science3, BNA Inc.4 and Graduate School of Pharmaceutical Sciences, Osaka
University5, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
Development of high-performance nucleic acids (HiPerNA) and innovative design of
antisense oligonucleotide (AON) have still been required to more safely and effectively treat
patients by using antisense drugs [1]. We have presented several types of HiPerNA such
as bridged nucleic acids (BNAs) that provides AON with better abilities to stably circulate
through the body and selectively capture the exact target [2]. Recently, short phosphorothioated LNA gapmers (≤ 16-mer) have reported to show higher potency than conventional 20-mer
AON both in vitro and in vivo [3]. We here evaluated a potency of novel BNAs incorporated
into various lengths of gapmers.
PCSK9 plays a key role in maintaining cholesterol homeostasis. PCSK9 is known as a
potential therapeutic target for the treatment of hyperchcolesterolemia. To develop PCSK9
inhibitor with enough potency and safety to withstand a clinical application, we screened efficacious target sites on PCSK9 mRNA and optimized strand lengths and chemistries in vivo
as well as in vitro.
Collectively, our results would let us suggest better concepts of design of HiPerNA and AON
for clinical application of antisense drugs.
Reference:
1. Yamamoto, T, Nakatani, M, Narukawa, K, and Obika, S (2011). Antisense drug discovery
and development. Future Med Chem 3: 339-365.
2. Obika, S, Rahman, SMA, Fujisaka, A, Kawada, Y, Baba, T, and Imanishi, T (2010).
Bridged Nucleic Acids: Development, Synthesis and Properties.
Heterocycles 81: 1347-1392.
3. Straarup, EM, et al. (2010). Short locked nucleic acid antisense oligonucleotides potently
reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates.
Nucleic Acids Res 38: 7100-7111.
Tsuyoshi Yamamoto, PhD student
1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
Telephone: +81-6-6879-8202
Email: t-yam@phs.osaka-u.ac.jp
113
066
oligonucleotide coNjugates as inhibitors of
HIV-1 integrase and reverse transcriptase
T. Zatsepin,1,2 J. Agapkina,1 S. Korolev,1 M. Gottikh1
1
Department of Chemistry and Belozersky Institute of Physical and Chemical Biology,
Lomonosov Moscow State University, Moscow, Russia
2
Central Research Institute of Epidemiology, Moscow, Russia
Reverse transcription of human immunodeficiency virus type 1 RNA and integration of
resultant DNA into genome of the infected cell are two key steps of the viral replication
cycle. Therefore viral enzymes reverse transcriptase and integrase, which realizes the reverse
transcription and integration, respectively, represent attractive and validated targets for the
development of new antiviral drugs. In this report at first we studied the anti-integrase activity in vitro of a series of conjugates of single stranded oligonucleotides with hydrophobic
molecules, and the structure-activity relationships were also analyzed. Both oligonucleotide
and hydrophobic parts of the conjugates influenced the inhibitory potency. Conjugates of 11mer phosphorothioate oligonucleotides with 6-carboxy-4,7,2’,4’,5’,7’-hexachlorofluorescein
(HEX) were found to be the most efficient inhibitors (IC50 20 nM), and might be considered
as lead compounds for further development of integrase inhibitors. Moreover, several
conjugates were tested as potential inhibitors of RNA-dependent DNA polymerization,
DNA-dependent DNA polymerization and RNase H activities of HIV-1 reverse transcriptase.
Conjugates of 2’-OMe oligonucleotide with 2’,4’,5’,7’-tetrabromofluorescein (eosin) were
found to be the most efficient inhibitor of RHase H activity of the reverse transcriptase
(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, PhD
Department of Chemistry, Lomonosov Moscow State University
Leninskie gory 1-3, 119991 Moscow, Russia
Telephone: +7-495-939-3148
Email: tsz@yandex.ru
114
067
Influence of RNA Secondary Structure on the
Activation of Toll-like Receptors 7 and 8
Thomas Zillinger1*, Vera Wimmenauer1*, Eicke Latz2, Gunther Hartmann1,
Winfried Barchet1
1
Institute of Clinical Chemistry and Clinical Pharmacology and 2Institute of Innate Immuniy, University of Bonn, Germany
* These authors contributed equally.
Immunorecognition of viruses hinges on the detection of viral nucleic acids by germline encoded receptors at specific cellular locations. Human Toll like receptors (TLR) 7 and 8 are
able to detect single stranded RNA in the endosome of distinct immune cells.While most stimulatory RNAs activate both TLR7 and TLR8, short Oligoribonucleotides (ORN) and small
molecule agonists can show preferential engagement of either TLR7 or TLR8. A structural
basis for this distinction is unknown. Here we identify RNA stem structures in the secondary
structures commonly assumed by single stranded RNA, as a potent and highly selective activators of TLR7. Such stem structures induced type I IFN in plasmacytoid dendritic cells
(PDC), but did not activate TLR8 in monocytes. TLR7 selectivity was maintained even when
TLR7 and TLR8 were expressed in the same cell type. We further provide evidence that recognition of viral and bacterial RNAs via TLR7 is mediated by RNA secondary structure,
rather than the linear sequence motifs required for the activation of TLR8. We idenfied and
developed TLR7 agonistic RNA stems that show activity without a requirement for accessory
components such as cationic polymers.
Thomas Zillinger
Emmy Noether Group
“Immunorecognition of Viral Nucleic Acids in the Cytosol”
Institute of Clinical Chemistry and Clinical Pharmacology
BMZ, University Hospital, University of Bonn
Sigmund-Freud-Strasse 25
53127 Bonn, Germany
Telephone: +49-228-287 -51146
Email: Thomas.Zillinger@uni-bonn.de
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068
RIG-I LIGAND TRIPHOSPHATE RNA (3pRNA) POTENTIALLY
INHIBITS YELLOW FEVER VIRUS REPLICATION IN TYPE 1
INTERFERON DEPENDENT MANNER
Mohammad Intakhab Alam, 1Christine Schuberth, 1Thomas Zillinger, 1Christoph Coch 1Winfried Barchet, 1Jasper van den Boorn, 1Debby van den Boorn-Konijnenberg,
2
Beate Kuemmerer, 1Martin Schlee and 1Gunther Hartmann
1) Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn,
Germany and 2) Institute for Virology, University Hospital Bonn, Germany
1
RIG-I has been shown to be involved in sensing every members of the flavivirus genus. Yellow Fever Virus (YFV) possesses mechanism to overcome innate immunity. Here we propose the potent antiviral activity of RIG-I ligand (3pRNA). We used a sub genomic Yellow
Fever Renilla Replicon (YFRR) model. Our Luciferase data showed that, 3pRNA inhibits
one log replication of YFRR in HepG2 cells at non-toxic concentration (800ng/ml). MTT
assay ruled out the detrimental effect of 3pRNA on HepG2 cells and the inhibition of replication is 3pRNA specific in dose dependent manner. We served poly A RNA in HepG2 cells as
negative control and we did not observe inhibition of replication that, supports the antiviral
activity of 3pRNA. 3pRNA additionally induces type 1 IFN in addition to the YFRR itself
in HepG2 cells that leads to the inhibition of replication. Type 1 IFN and IP-10 were highly
expressed in 3pRNA treated cells. 3pRNA did not show inhibition of replication in IFN deficient Vero cells. However, addition of rec-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 upregulated RIG-I and IRF3 and strongly inhibited NS4B protein of YFRR.
Our real time-PCR data showed that 3pRNA potentially upregulated IFN dependent antiviral
genes (OAS, RNAse L, MxA, IFIT1 but not IFIT2). Small form of PARP13 (ZAPs) is highly
upregulated by 3pRNA which sparks the RIG-I signaling. 3pRNA did not inhibit replication
in RIG-I-/- MEFs that clearly indicates that, inhibition of replication is RIG-I ligand dependent. RIG-I-/- MEFs showed enhanced replication compared to WT-MEFs.
Over expression of Flag-tagged RIG-I showed non significant inhibitory effect on replication
that also support our idea that 3pRNA is potent immune stimulator. Plaque assay showed that,
3pRNA inhibited about 65% virus particles of 17-D in infected HepG2 cells. Equivalent unit
of rec-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, PhD
Institute for Clinical Chemistry and Clinical Pharmacology
Sigmund-Freud-Str. 25
D-53127 Bonn, Germany
Telephone: +49-228-28751150
Email: malam@uni-bonn.de
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Glucan Particles for Selective Delivery of siRNA to
Phagocytic Cells in Mice
Myriam Aouadi, Gregory J. Tesz, Matthieu Prot, Sarah M. Nicoloro, Shinya U. Amano,
Emilie Boutet, Anca Goller, Mengxi Wang, Chang-An Guo, William E. Salomon,
Joseph V. Virbasius, Rebecca A. Baum, Mark J. O’Connor, Jr., Gary R. Ostroff and
Michael P. Czech
Program in Molecular Medicine. University of Massachusetts Medical School,
Worcester, MA, USA
Phagocytic macrophages and dendritic cells are desirable targets for potential RNAi therapeutics because they often mediate pathogenic inflammation and autoimmune responses. We
recently engineered a complex, 5 component glucan-based encapsulation system for siRNA
delivery to phagocytes. In experiments designed to simplify this original formulation, we discovered that the amphipathic peptide Endo-Porter forms stable nanocomplexes with siRNA
that can mediate potent gene silencing in multiple cell types. In order to restrict such gene
silencing to phagocytes, a method was developed to entrap siRNA/Endo-Porter complexes
in glucan shells of 2-4 microns diameter in the absence of other components. The resulting
glucan particles containing fluorescently labeled siRNA were readily internalized by macrophages, but not other cell types, and released the labeled siRNA into the macrophage cytoplasm. Intra-peritoneal administration of such glucan particles containing siRNA/Endo-Porter
complexes to mice caused gene silencing specifically in macrophages that internalized the
particles. These data indicate that specific targeting to phagocytes is mediated by the glucan,
while Endo-Porter peptide serves both to anchor siRNA within glucan particles and to catalyze escape of siRNA from phagosomes. Thus we have developed a simplified siRNA delivery system that effectively and specifically targets phagocytes in culture or in intact mice.
Myriam Aouadi, PhD
373 Plantation st, Biotech II, suite 100
Worcester, MA, 01605 USA
Telephone: 1-508-856-6858
Email: Myriam.aouadi@umassmed.edu
117
070
Development of an RNA Aptamer for Human Toll-like
Receptor 2 Ectodomain
Jonghoe Byun, Ji-hae Yoon
Department of Molecular Biology, Dankook University, Yongin-si, Gyeonggi-do, Korea
Currently, there is a growing interest in the biology of toll-like receptors (TLRs). These
pattern recognition receptors (PRRs), expressed on various immune and non-immune cell
types, recognize pathogen-associated molecular patterns and play a crucial role in innate immunity by mediating the activation of macrophages. They also recruit phagocytic cells and
produce proinflammatory cytokine/chemokine. Recently, it was suggested that activation of
TLRs contributes to the development of certain cardiovascular diseases like atherosclerosis,
congestive heart failure and cardiac dysfunction in sepsis. The cDNA encoding TLR2 was
cloned from U937 cells using RT-PCR procedure. Human TLR2 (hTLR2) has 23 leucine
rich repeats (LRRs) including a C-terminal LRR and two N-terminal LRRs. LRR9-LRR12
domain binds to ligands, whereas LRR11-LRR14 domain make contacts with hTLR1. To
develop an RNA aptamer for hTLR2, LRR8-LRR15 domain was chosen as target protein and
its coding sequences were cloned into pQE-80L vector that can express membrane protein
with accompanying 6 his-tag sequences. Origami strain of E.coli that can efficiently express
membrane protein was used for overexpression of LRR 8-15 domain protein. The purified
protein was then used for in vitro selection of RNA aptamer(s) from the RNA library using
SELEX procedure. Characterizations of the obtained aptamers are currently underway and
preliminary results including electrophoretic mobility shift assay and minimization data will
be presented at the meeting.
Jonghoe Byun, PhD
126 Jukjeon-dong, Suji-gu, Yongin-si, Gyeonggi-do 448-701, Korea
Telephone: +82-31-8005-3194
Email: jonghoe@dankook.ac.kr
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071
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 messenger RNA with short PNAs that form duplex and triplex structures that arrest elongating
ribosomes and induce truncated proteins synthesis1, 2, 3. PNA is unique among
the investigated steric blocking agents in its ability to specifically inhibit the translation elongation. In cells, dominant negatives could be thus generated thanks to PNAs.
Type I insulin-like growth factor receptor (IGF-IR) has been implicated in the progression of
a large number of cancers and represents an important target for anti-cancer strategies. Studies showed that transfected dominant negatives of IGF-IR are able to decrease tumor growth
and metastasis formation. We have shown that a PNA sequence forming a partial triplex on
the coding region of IGFIR’s mRNA induces a truncated IGF-IR in cell free system and inhibits the receptor expression in cultured prostate cancer cells. Therefore, we have shown that
a steric blocker can stop the translation elongation in cells. In addition, IGF-1R knockdown
by PNA led to an attenuation of phosphorylation of downstream signaling pathways, PI3K/
AKT and MAPK, involved in survival and mitogenesis and also to a decrease in cell transformation4. Of the steric blockers tested, which included phosphorodiamidate morpholino
oligomers and locked nucleic acids, PNAs was unique in
its ability to form triplex structures with mRNA and to arrest translation elongation.
Dias et al. RNA hairpin invasion and ribosome elongation arrest by mixed base PNA oligomer. J. Mol. Biol. 2002; 320:489-501. 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, Simon P, François JC, Saison-Behmoaras TE. A steric blocker of translation elongation inhibits
IGF-1R expression and cell transformation. FASEB J. 2011 Mar 14.
1
Céline Cordier, PhD student
MNHN, RDDM, INSERM U565-CNRS UMR 7196, 43 rue Cuvier CP26
Paris 75005, FRANCE
Telephone: 33 1 40 79 36 89
Email: celinecordier@mnhn.fr
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072
RNAi- BASED PANCREATIC CANCER THERAPY BY LOCAL
DELIVERY OF ANTI K-RASMT siRNA
Eithan Galun*, Racheli Gabai-Malka**, Gili Focht**, Amotz Shemi** and
Elina Zorde Khvalevsky**
* Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital,
Jerusalem, Israel
** Silenseed LTD, Israel
One of the major challenges for oligonucleotides based therapeutics is delivery. Obstacles in
systemic delivery include targeting, over-dosing and dose fluctuations, liver aggregation and
immune-stimulation of the drug carriers, renal clearance and cost. Direct injection doesn’t
solve such issues in many cases. Local and prolonged delivery overcomes these hurdles and
offer an attractive solution at least for a sub group of regional diseases, amongst are solid tumors. Pancreatic cancer is an aggressive disease, being the 4th leading cause of cancer–related
death in the western world. Genetic alterations in KRAS signaling pathway are involved in
95% of cases, while the majority of KRAS mutations are gain-of-function mutations at codon
12 (KRASG12D) of the oncogene. The tumor is addicted to KRAS activity. We developed an
implanted controlled drug delivery system called 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 proliferation and induction of cell death. In vivo the growth of human pancreatic cancer cell lines
(PANC1 and CAPAN1) was retarded, both in subcutaneous and orthotopic models. Furthermore, the survival of mice implanted with siG12D LODERs was significantly improved in
the two cell lines tested in the orthotopic mouse model. Based on these results, Silenseed has
initiated a phase I study with siG12D LODER implanted into patients with a locally advanced
pancreatic tumor: http://clinicaltrials.gov/ct2/results?term=silenseed.
Eithan Galun
Goldyne Savad Institute of Gene Therapy
Hadassah Hebrew University Hospital,
Jerusalem, 91120, Israel
Telephone: 972-2-6778589 or 77762
Email: eithang@hadassah.org.il
120
073
REGULATION OF PATHOGENIC EFFECTOR MEMORY T HELPER
1 CELL SURVIVAL BY microRNA
Claudia Haftmann1*; Anna-Barbara Stittrich1*; Gitta Heinz1; Hyun-Dong Chang1, Nikolaus
Rajewsky2, Andreas Radbruch1* and Mir-Farzin Mashreghi1*
1
German Rheumatism Research Center (DRFZ) Berlin, Germany
2
Max-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 candidate miRNA being induced in pathogenic effector Th1 cells. Computational target analysis
via TargetScan and PicTar predicted BIM and Pten as putative targets. Overexpression of this
miRNA in activated Th1 cells results in 50 % down-regulation of endogenous Bim and Pten
expression. Conversely, inhibition of the candidate miRNA in chronically activated memory
Th1 cells by antagomirs results in induced protein levels of BIM and significantly enhanced
cell death and reduced proliferation. With its targets having important roles for survival and
proliferation of pathogenic effector memory Th1 cells, this candidate miRNA represents a
promising molecular target for the treatment of immune mediated disease. 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: haftmann@drfz.de
121
074
Abstract and poster available for review at the meeting.
122
075
IDENTIFICATION OF PROCESS RELATED IMPURITIES IN
DNA/RNA BY LCMS ANALYSIS
Kenneth W. Hill
Agilent Technologies, Boulder, Colorado, USA
LCMS is a powerful tool for identification of impurities in DNA/RNA based API’s.
Replacing ammonia with methyl amine in the deprotection process of a DNA thiolate gave
rise to a new set of impurities. LCMS analysis showed that these impurities were formed by
transamination of cytidine by methyl amine. The progress of methyl amine deprotection of
acetyl protected dC/rC containing sequences was followed by LCMS. The data presented here
show that the transaminated impurities arise from the deprotected cytidine and not the N-acetyl
protected species and that this reaction is general to other alkyl amine based deprotections.
Kenneth W. Hill, PhD
5555 Airport Blvd
Boulder, Colorado, USA 80301
Telephone: 303 222 4945
Email: Kenneth_hill@agilent.com
123
076
Design of protein-responsive shRNA system for
translational control in human cell
Tan Inoue, Shunichi Kashida, Yoshihiko Fujita, Hirohide Saito
Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan
(ICORP project, JST, Tokyo, Japan)
We have developed an artificial protein-responsive shRNA switch by employing short
hairpin RNA (shRNA) that serves as a powerful tool for RNAi in human cell. This artificially
designed molecular systems for responding to particular gene products and rewire gene
expression networks in living cells are highly useful.
As a pilot system, we have developed a synthetic shRNA (Kt-shRNA) that combine target
sequences (double strand region) for target mRNA with Box C/D kink-turn RNA motif set in
its loop region. An Archaeal ribosomal protein L7Ae and its binding motif, Box C/D kinkturn RNA motif, were employed as the RNP interaction motif for protecting Kt-shRNA from
Dicer cleavage. Kt-shRNA responds to cellularly expressed L7Ae and controls target gene
expression to promote apoptosis of human cancer cells.
(Saito H. et al, Nat. commun., 2 : 160 (2011) ).
To develop new protein-responsive shRNA switches, we employed 3D molecular modeling
software for adequately positioning the protein-binding motif in order to properly control
Dicer activity. We were able to build new protein responsive shRNA switches with RNP motifs consisting of U1A or 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, PhD
Graduate School of Biostudies, Kyoto University
Kyoto 606-8502, Japan
Telephone: +81 75 753 3995
Email: tan@kuchem.kyoto-u.ac.jp
124
077
PMOplus™ Approaches to Rapid Response Therapeutics
for Emerging Infectious Diseases
Patrick Iversen1, Fred Schnell2, Laura Hauck2, Stacey Crumley2 and Dan Mourich2
AVI BioPharma, Inc., 1Bothell, WA and 2Corvallis, OR
Rapid response to emerging infectious disease and laboratory accidents represents an important and novel application of AVI BioPharma’s RNA therapeutic platform. AVI’s phosphorodiamidate morpholino oliogmer (PMO) approach to rapid response relies upon multiple
favorable properties of these compounds and a growing database of positive studies with a
wide variety of infectious disease agents. AVI has demonstrated efficacy for our synthetic
oligomers in cell culture for 19 different viral families and in vivo efficacy in 11 of those
families. This experience has reinforced the value of our therapeutic design strategies. The
exceptionally high success rate in animal infection challenge studies indicates a high probability of therapeutic development success. In 2001 we responded to lethal outbreaks of
feline calicivirus in Atlanta, GA and Eugene, OR resulting in 94% (47/50) survival in treated
kittens compared to 9.7% (3/31) survival untreated kittens. In 2002, within 14 days we progressed from concept to the successful treatment of Humboldt Penguins infected with West
Nile (100% survival of treated birds). In 2004 we responded to an accident with Ebola Zaire
to a laboratory worker which progressed from concept to delivery of clinical supply in 7 days.
Studies confirming PMOplus efficacy have been conducted in lethal challenge animal models
involving mouse, guinea pig and nonhuman primates. More recently, AVI participated in a
rapid response exercise involving H1N1 influenza in 2009 in which progress from concept to
compound took 7 days. PMOplus efficacy was demonstrated in both mouse and ferret animal
models. In 2010, AVI undertook a rapid response exercise involving Dengue virus where the
time from request to an effective PMOplus compound was 11 days. Supportive nonclincial
programs have led to open INDs for AVI-6002 for Ebola Zaire, AVI-6003 for Marburgvirus
and AVI-7100 for influenza A. Studies are currently in progress to expand the rapid response
capability to include multidrug resistant bacterial infections. We conclude that AVI’s RNA
therapeutic technology is well suited to rapid therapeutic response.
Patrick Iversen, PhD
3450 Monte Villa Parkway
Bothell, WA 98021 USA
Telephone: 425-354-5038
Email: piversen@avibio.com
125
078
Integrated microfluidic platform for Multiplex
SELEX on a Chip
Soyoun Kim
Department of Biomedical Engineering, Dongguk University, South Korea
Multi-target aptamer selection called Multiplex SELEX (Systematic Evolution of Ligands
by EXponential enrichment) gains its interests rapidly because of its potential to highspeed and high-throughput aptamer selection capacity. Parallelization of chemical process
by integration of microfluidic components can be a key technique to develop a multiplex
SELEX process. One of the potential problems in multplexing chemical processes on-chip
is cross-contamination. In order to avoid this consideration, we propose a microfluidic
network platform with a pneumatic valving strategy to allow serial loading and incubation of
aptamers with sol-gel entrapped target proteins. After target binding inside the sol-gels crosscontamination free parallel elution of specifically bound aptamers is performed. The platform
allows selective binding against five different targets immobilized in sol-gel spots. When
eluting bound species liquid flow across nearby reaction chamber are sealed by integrated
pneumatically driven rubber microvalves. We demonstrate specific aptamer binding to its
respective protein target and subsequent aptamer elution without any cross contamination.
The current proof of concept opens the route to increased automation and microscale parallel
processing of the SELEX methodology.
Figure. Concept of parallel process of aptamer binding and partitioning. Five different targets included sol-gel
were spotted on the surface (Heater electrodes). Then, RNA mixture (Aptamers) were injected and incubated by
fluidic operation for binding aptamers to their target proteins. Bound aptamers were collected by applying heat.
(Pneumatic valves are operated during heat process in order to remove cross contamination).
Soyoun Kim, PhD
Associate Professor, Dongguk University
26,3 Pil-dong, Chung-Gu, Seoul 100-715, Korea
Telephone: 822-2260-3840
Email: skim@dongguk.edu
126
079
Development of potent RNAi therapeutics using long
antisense siRNAs
Sera Kim, Seul Gi Jo, Chang Han Lee, Pooja Dua, and Dong Ki Lee
Global Research Laboratory (GRL) for RNAi medicine, BK21, Department of Chemistry,
Sungkyunkwan University (SKKU), Suwon, Korea
Corresponding author: dklee@skku.edu
Specific gene silencing using RNA interference (RNAi) has proven to be a useful tool in the
fields of biotechnology and a promising future therapeutic modality. Small interfering RNAs
(siRNAs) are used as a molecular trigger of RNAi to specifically block the expression of
the genes causing various diseases including cancer. In this study, we chose KRAS (ki-ras2
Kirsten rat sarcoma viral oncogene homolog) as a target gene to develop anticancer siRNA
therapeutics. KRAS belongs to a group of small GTP-binding proteins, and involved in cell
signaling and growth. Therefore, by targeting KRAS through the RNAi, we expect repression of tumor survival and proliferation. The conventional siRNA structure shows several
limitations such as off-targeting effects and inefficient gene silencing for certain genes. To
overcome this, we developed various long antisense siRNA structural variants which harbor
long antisense 3’ overhang sequence complementary to KRAS mRNA. We show that long
antisense siRNAs can trigger improved gene silencing over conventional siRNAs. Long antisense structure also improved gene silencing triggered by asymmetric shorter-duplex siRNAs
(asiRNAs). Our results demonstrate that long antisense siRNA can be an alternative structure
to conventional siRNA with improved efficacy and specificity in gene silencing.
Sera Kim, MS candidate
330517, Chemistry Building
Sungkyunkwan University, Chunchundong, Suwon, Korea
Telephone: 82-31-299-4889
Email: fanybang@naver.com
127
080
Stealth Delivery of Oligonucleotides
Akira Komoriya and Beverly Z. Packard
OncoImmunin, Inc., Gaithersburg, MD, USA
Nucleic acid delivery into live cells has been one of the major hurdles for oligonucleotidebased therapeutics. Although various approaches and methods have been developed and are
currently being utilized, a new platform, Xyton Stealth DeliveryTM (XSD), has been developed for intracellular delivery of oligonucleotides with the aim of expanding therapeutic applications. Unique attributes of this new methodology include the formation of molecularly
dispersed solutions and nearly maximal cellular uptake in the presence of serum within 90
minutes by a variety of cell types including primary hematopoetic cells. Furthermore, cellular entry of both single strands and duplexes of either DNA or RNA by this mode of delivery
is nontoxic, homogeneous, and sequence-independent. When XSD was applied to transport of 24-mer antisense oligonucleotides, intracellular hybridization with complimentary
sequences was shown to be saturable. Furthermore, Dicer substrates designed by the XSD
technology were processed by the recombinant human enzyme; upon addition of a Dicer
substrate targeting the eGFP gene to primary murine transgenic eGFP PBMC in serum, the
expected siRNA effects were observed after 3 days as judged by flow cytometry. In summary, XSD delivery into the cytosol of both antisense DNA and RNA duplexes was uniform
and rapid in a variety of target cells with mRNA hybridization and Dicer processing followed
by specific gene knockdown, respectively. The delivery method described herein utilizes a
totally unique class of delivery vehicles relative to what has been utilized in the RNAi and
microRNA communities to date.
Akira Komoriya, PhD
OncoImmunin, Inc.
207A Perry Parkway, Suite #6
Gaithersburg, MD 20877 USA
Telephone: (01) 301-987-7881
Email: Komoriya@PhiPhiLux.com
128
081
Long dsRNA-mediated RNA interference and
immunostimulation: long interfering dsRNA (liRNA)
as a potent anticancer therapeutics
Tae Yeon Lee1, Chan Il Chang1,2, and Dong-ki Lee1,*
Global Research Laboratory (GRL) for RNAi Medicine, Department of Chemistry,
Sungkyunkwan University (SKKU), Suwon, Korea
2
Skip Ackerman Center for Molecular Therapeutics, Beth Israel Deconness Medical Center,
Harvard Medical School, Boston USA
*Corresponding author:dklee@skku.edu
1
In most applications, small interfering RNAs are designed to execute specific gene
silencing via RNA interference (RNAi) without triggering non-specific responses such as
immunostimulation. However, in anticancer therapeutics development, immunostimulation
combined with specific oncogene silencing could be beneficial, resulting in synergistic inhibition of cancer cell growth. In this study, we report an immunostimulatory long dsRNA structure with ability to trigger RNAi-mediated specific target gene silencing, termed long interfering dsRNA (liRNA). liRNA targeting Survivin mRNA not only triggered efficient and specific
target gene silencing via RNA interference, but also stimulated protein kinase
R (PKR) pathway to induce expression of interferon. As a result, the ability of Survivintargeting liRNA to inhibit cancer cell growth was superior over conventional siRNA or nontargeting dsRNA structures. Our results thus provide a simple, efficient dual function immunostimulatory RNAi-triggering structure, which is potentially applicable for potent anticancer
therapeutics development.
Tae Yeon Lee
330-517,Chemistry Building, Sungkyunkwan University
Suwon Republic of Korea 440-746
Email: kakaross52@naver.com
129
082
DEpendency of the Cross-linking reactivity with
2-Amino-6-vinylpurine on the Neighboring Bases
Fumi Nagatsugi, Shinya Hagihara, Shuhei Kusano, Chao Xiao Guang
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University
An oligonucleotide (ON) with a sequence complementary to a specific mRNA can inhibit its
expression. The use of unmodified ON for applications in cells, however, is very limited because of the poor cellular uptake efficiency, targeted delivery, its low specificity and affinity
for the target sequence, and susceptibility to degradation by nucleases. Many modified ONs
containing unnatural bases or modified sugars have been prepared for improving the efficacy
of the antisense method. One such example is ONs incorporating a 2′-modified nucleotide.
These analogues are known to exhibit high binding affinity to target RNA and metabolic stability. Furthermore, cross-linking reactions are expected to enhance the antisense activity by
irreversibly binding to the target RNA, based on the steric blocking mechanism. We previously demonstrated that the 2′-O-methyl (2′-OMe) analogue of 2-amino-6-vinylpurine (2-AVP)
(1) reacted selectively to thymine in DNA, but did not react effectively to uracil in RNA
under neutral conditions. 1 In this study, we investigated about the effect of adjacent bases
with 2-AVP on the cross-linking reactivity. In consequence, we found that ON incorporating
(1) reacted more effectively to uracil under neutral conditions in the cases where the ON had
the sequences 3’ G (1) C5’. Herein, we describe the neighboring bases effect on the crosslinking reactivity of 2′-OMe analog of 2-AVP, and evaluation of the antisense effect with the
use of 1 in non-cell translation assay and in cell experiments.
Fig.1 Design of the 2-Amino-6-vinylpurine (2-AVP) (1) and Dependency of the Reactivity on the
Neighboring Bases of (1)
1 Imoto, S.; Hori, T.; Hagihara, S.; Taniguchi, Y.; Sasaki, S.; Nagatsugi, F., Bioorg. Med. Chem. Lett.,
20, 6121-6124 (2010)
Fumi Nagatsugi, PhD
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University
2-1-1 Katahira, Aoba-ku Sendai-shi, Miyagi, 980-8577, Japan
Telephone: 001- 81-22-(217)-5633
Email: nagatugi@tagen.tohoku.ac.jp
130
083
ATTENUATION OF AUTOIMMUNITY BY
ANTI-HUMAN-INTERLEUKIN-17 APTAMER IN MOUSE MODELS
Yoshikazu Nakamura, Hironori Adachi, Akira Ishiguro
Institute of Medical Science, University of Tokyo, Tokyo, Japan
Interleukin-17 (IL-17) is a pro-inflammatory cytokine produced primarily by a subset
of CD4+ T cells, called Th17 cells, that is involved in host defense, inflammation and
autoimmune disorders. Two IL-17 family members, IL-17A and IL-17F, are most structurally
related, and form homodimeric (IL-17A/A, IL-17F/F) and heterodimeric (IL-17A/F)
structures. We have isolated RNA aptamers against human IL-17A. Aptamers are short
single-stranded nucleic acid sequences that are selected in vitro based on their high affinity to
a target molecule. One such aptamer, Apt21-2, blocked the interaction between IL-17A and
its receptor IL-17R in vitro, and inhibited the production of interleukin-6 from mouse and
human cells. A PEGylated form of Apt21-2 inhibited the development of GPI-induced rheumatoid arthritis and MOG-induced experimental autoimmune encephalomyelitis in a dosedependent manner, showing its therapeutic potential in autoimmune diseases.
Among several dimeric conformers of IL-17, Apt21-2 recognizes both IL-17A/A and
IL-17A/F as does the known anti-IL-17A antibody. Although the biological significance
of IL-17A and IL-17F have been investigated using respective antibodies or gene knockout
mice, the functional study of IL-17A/F heterodimeric form has been hampered by the lack
of an inhibitory tool specific to IL-17A/F. Hence, we aimed to develop an RNA aptamer
that specifically inhibits IL-17A/F. One such aptamer against human IL-17A/F, AptAF42, was
isolated through the subtractive SELEX procedure employing counter-selection against
IL-17A/A. AptAF42 and its optimized derivative AptAF42dope1 blocked the binding of
IL-17 receptor to IL-17A/F, but not to IL-17A/A or IL-17F/F, in the surface plasmon
resonance assay in vitro. Consistently, AptAF42dope1 did not affect cytokine GRO- 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 Professor
Institute of Medical Science, University of Tokyo
4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Telephone: +81(JPN) 3 5449 5307
Email: nak@ims.u-tokyo.ac.jp
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A NOVEL ASSAY FOR THE DETECTION OF miRNA MATURATION
Saskia 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 human disease formation and progression.[1] In order to investigate the activity of potential
inhibitors and modulators of Dicer-mediated miRNA maturation, a facile and accurate assay
is needed.[2, 3] Here we provide a novel rapid and reliable assay of Dicer-mediated miRNA
maturation, without the need of tedious and expensive labeling of pre-miRNAs. The assay is
based on the rolling circle amplification (RCA) method,[4] wherein only the mature miRNA
but not the pre-miRNA can initiate isothermal amplification of a cyclic DNA template leading
to the formation of a linear single stranded DNA (ssDNA) product. A secondary primer binds
the product sequence and initiates the formation of a secondary DNA strand resulting in an
enhanced fluorescence signal of SYBR gold. So far, we have established the assay for three
different miRNAs and showed that each miRNA binds specifically to its target circular DNA
and leads to specific amplification. Moreover, inhibitors can easily be screened for selectivity
in the presence of other pre-miRNAs.[5] The use of unmodified pre-miRNAs reduces the risk
of generating assay derived artifacts. Thus, we are optimistic that this assay provides a useful
tool for the investigation of miRNA maturation and helps accelerating the search for specific
inhibitors of miRNA maturation.
C. Arenz, Angew Chem Int Ed 2006, 45, 5048-5050. [2] C. M. Klemm, A. Berthelmann, S. Neubacher,
C. Arenz, Eur J Org Chem 2009, 17, 2788-2794. [3] B. P. Davies, C. Arenz, Angew Chem Int Ed, 2006,
45, 5550-5552. [4] S. Neubacher, C. Arenz, ChemBioChem 2009, 10, 1289-1291. [5] S. Neubacher, C.
Arenz, Angew Chem, submitted.
[1]
Saskia Neubacher, Dipl.Chem.
Humboldt-Universität zu Berlin
Brook-Taylor-Str. 2, 12489 Berlin, Germany
Telephone: +49 30 2093 7511
Email: saskianeubacher@arcor.de
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DEVELOPMENT OF DNA NANOGELS AND HYDROGELS AS
INJECTABLE DELIVERY SYSTEMS FOR CPG DNA AND OTHER
NUCLEIC ACID-BASED DRUGS
Makiya Nishikawa, Kohta Mohri, Kohei Ogawa, Natsuki Takahashi, Yuki Takahashi,
Yoshinobu Takakura
Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
DNA can be a building block for constructing a wide variety of unique structures, including
but not limited to DNA cubes, DNA origami, DNA polyhedra and DNA hydrogels. In
addition to the excellent property as a block, DNA is a biologically active compound that can
be and is being used in the treatment of human diseases. One of such compounds is the DNA
containing unmethylated CpG dinucleotides with particular flanking sequences (CpG motif),
the ligand for Toll-like receptor-9 (TLR9). The ligation of TLR9 triggers the induction of
proinflammatory cytokines, and CpG DNA has been developed for the treatment of cancer,
allergic diseases, and as adjuvants for vaccines against cancer and infectious diseases.
Although the phosphodiester bond of CpG DNA is generally replaced by phosphorothioate
one to increase the enzymatic stability, this modification is associated with high levels
of cytotoxicity. We hypothesized that the use of DNA nanotechnology can be a new and
promising approach to increasing the activity of CpG DNA without changing its chemical
structure. Based on this, we have developed a series of DNA assemblies having sticky ends
through which the assembled DNA units are connected to each other. The immunostimulatory
activity of CpG DNA was significantly increased by the formation of tripod- or tetrapodlike structures. Furthermore, the development of DNA dendrimer by connecting the tripodlike DNA assemblies further increased the activity. Then, large DNA hydrogels were
successfully developed using several types of assembled DNA units. We demonstrated that
CpG DNA hydrogels activate dendritic cells, continuously release intercalated doxorubicin,
an anticancer agent, and was highly effective in inhibiting subcutaneous tumor growth in
mice after in vivo administration. Then, we have developed injectable sol-gel transition DNA
hydrogels utilizing the difference in salt concentrations between test tubes and biological
fluids. We confirmed that the administration of solutions containing DNA assemblies with
proper sequences resulted in the formation of DNA hydrogels at the administration sites, such
as dermis and nasal cavity. These findings suggest that DNA nanogels (DNA dendrimer) and
DNA hydrogels are promising delivery systems for nucleic acid-based drugs, such as CpG
DNA, as well as for anticancer agents, antigens and other bioactive compounds.
Makiya Nishikawa, PhD
Graduate School of Pharmaceutical Sciences, Kyoto University
46-29 Yoshidashimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
Telephone: +81-75-753-4580
Email: makiya@pharm.kyoto-u.ac.jp
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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-Peter
Roche Kulmbach GmbH, D-95326 Kulmbach, Germany
The process of impurity formation during annealing of siRNA was investigated. siRNAs containing common chemical RNA modifications (2’-O-methyl-, 2’-deoxy-2’-fluoro-,
2’-deoxy-ribose and phosphorothioate linkages) were used to determine major side reactions
- such as 2’,3’-isomerisation, strand scission and HF elimination. Individual impurities were
characterized using analytical SEC, denaturing and non-denaturing IP-RP-HPLC, DSC and
UV-spectrometry. The degradation pathways described in this work can lead to significantly
reduced product quality and compromised drug activity. Based on our data, incubation temperature and time as well as the presence of divalent cations are the most critical factors in
controlling impurity formation. Without metal-ion catalysts, formation of 2’,5’-isomers was
the major side reaction of the two RNA single strands containing 2’-unmodified nucleotides.
When Mg2+-ions were present, metal-ion-catalyzed degradation of all three investigated RNA
strands was observed to a significant extent. For 2’-deoxy-2’-fluoro-modified strands HFelimination at the modified pyrimidine-nucleotides was the dominant side reaction leading to
the formation of 2,2’-O-anhydroribonucleotides and arabinosylnucleotides.All side reactions
(isomerisation, elimination and strand scission) were suppressed by duplex formation. Since
impurity formation was most pronounced at elevated temperatures, heating of the siRNA solution should be limited to the lowest temperature required for substantial dissociation of the
duplex. The optimal annealing temperature may therefore be close to the Tm of the duplex.
Divalent cations such as Mg2+ catalyze strand scission and are best avoided in annealing solutions. The strong concentration dependency of the Tm in salt-free solutions allows for adjustment of the Tm by changing siRNA concentration.
Taken together, annealing in salt-free solutions allows for lower annealing temperatures,
minimizes side reactions and simplifies lyophilisation of the resulting siRNA molecule. Our
data provides background to successfully address challenges associated with the manufacture
of siRNAs and other nucleic acid therapeutics, such as aptamers, spiegelmers, decoy and antisense oligonucleotides.
Bernhard Noll, Associate Director Analytics CMC
Fritz-Hornschuch-Strasse 9
D-95326 Kulmbach, Germany
Telephone: +49 (0) 9221 827 62 8546
Email: Bernhard_noll@gmx.de
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087
Abstract and poster available for review at the meeting.
135
088
EXPERIMENTAL ASSESSMENT OF ANTIMIR SPECIFICITY
Andreas Petri1,Susanna Obad2, Darryl Pappin3, Sakari Kauppinen1,2, and Morten Lindow1
1) Informatics, Santaris Pharma A/S, Hørsholm, Denmark
2) MicroRNA Drug Discovery, Santaris Pharma A/S, Hørsholm, Denmark
3) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA
The use of short 8-mer antimiR seed targeting tiny LNAs has recently been shown to provide
an efficient means to silence single miRNAs or entire miRNA families. Due to their short
length, this class of antimiRs will have many perfect match binding sites in the transcriptome.
We wanted to assess whether or not tiny LNA oligos affect transcripts harboring binding sites
or proteins translated from such transcripts. To this end, we used Sylamer to perform an unbiased and exhaustive analysis of sequence motifs associated with differentially expressed
genes following tiny LNA administration to cell lines or mice. In all experiments with antimiRs, Sylamer analysis showed significant overrepresentation among the up-regulated genes
of motifs corresponding to binding sites of the cognate seed. However, sequence motifs corresponding to direct binding sites for the tiny LNAs were not overrepresented. The effect on
protein level was analyzed using both small scale iTRAQ analyses of a subset of the samples
used for transcription profiling as well as an in vitro luciferase reporter assay of tiny LNAs
targeting various parts of the luciferase transcript. The proteomics data also showed up-regulation of predicted miRNA targets following miRNA silencing, while no significant effect
was seen on proteins encoded by transcripts harboring a direct binding site. The latter finding
was corroborated by data from the luciferase reporter assay. Collectively, our data show that
while the transcriptome harbors many perfect match binding sites for short 8-mer oligonucleotides, the presence of high affinity tiny LNAs generally has no effect on the transcripts containing perfect match binding sites or on the levels of protein translated from such transcripts.
Andreas Petri, Ph.d.
Santaris Pharma A/S
KogleAllé 6
DK 2970 Hørsholm
Danmark
Telephone: +45 4517 9876
Email: anp@santaris.com
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An RNA aptamer is a potential biomarker for pancreatic cancer
Partha Ray, Bruce A. Sullenger, Rebekah R. White
Department of Surgery, Duke University Medical Center, Durham, NC
Introduction: The symptoms of pancreatic cancer are usually non-specific and late. Existing tumor
markers (such as CA19-9 and CEA) are neither sensitive nor specific enough to serve as useful
screening tests. Tools for earlier detection could increase the proportion of patients who are cured
with surgical resection. Aptamers are oligonucleotide ligands that are identified from large libraries
of RNA or DNA molecules using an iterative selection process termed as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Aptamers bind their specific targets with high affinity
and specificity and can be used for the dual purpose of identifying novel biomarkers and detecting
them in biological specimens.
Methods: In vitro “positive/negative” SELEX was performed using the conditioned media (the
“secretome”) of the MiaPaCa2 human pancreatic ductal adenocarcinoma cell line (positive selection)
and the immortalized, non-cancerous human pancreatic ductal cell line HPDE (negative selection).
The rationale for “positive/negative” selection was to select for aptamers against molecules that are
selectively secreted by cancerous cells but not by normal cells. Standard radioactive filter-based
binding assays were used to measure binding.
Results/Conclusion: We have successfully selected several nuclease resistant (2’-fluoro-modified)
RNA aptamers that differentially bind the secretome of pancreatic cancer cells compared to noncancerous cells. One of the aptamers (M9-5) binds pancreatic cancer patient serum with high affinity
compared to control serum collected from healthy donors. Using a maximal fraction bound (FB) of
11% as a cutoff, the aptamer discriminated between untreated pancreatic cancer patients (N=19) and
healthy volunteers (N=20) with 95% sensitivity and 100% specificity, and the difference in aggregate
binding values between the groups is highly significant (P<0.0001, Mann-Whitney U test). In addition, paired serum samples obtained from two patients pre- and post-chemoradiation therapy and
pre- and two months post-resection have demonstrated a decrease in M9-5 binding with decreased
tumor burden. Notably, serum CA19-9 levels were normal (<40 U/ml) in 62% of cancer patients,
and CEA levels were normal (<2.5 ng/ml) in 57% of cancer patients, indicating that our biomarker
might be more sensitive than these existing serum biomarkers for pancreatic cancer. For the M9-5
target identification, several steps of biochemical fractionation were performed. At each step of the
fractionation, the M9-5 binding activity was assayed to follow the purification scheme. At the end
of final purification step the fraction was resolved by gel electrophoresis and after Coomassie staining a ~20 kDa major polypeptide band was visualized. Cyclophilin B (CypB) was identified upon
mass spectrometric analysis of the band. To verify the M9-5 target identification, the biotinylated
M9-5 aptamer was used for affinity purification. CypB was detected upon Western blot analysis of
the purified proteins. Additionally, to confirm that CypB is the protein target of aptamer M9-5, we
measured CypB levels in these same serum samples using ELISA. Similar differential expression
between cancer patients and healthy volunteers was observed using the ELISA assay as with the
M9-5 binding assay. Also, there was a statistically strong correlation between M9-5 binding and
CypB levels by ELISA. The implication of these finding will be further discussed.
Partha Ray, PhD
DUMC Box 103035
Durham, NC 27710 USA
Telephone: 919-684-4864
Email: partha.ray@duke.edu
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090
Endosomal Escape of liposomal siRNA via membrane
fusion and in vivo delivery to tumor
Yu Sakurai, Hiroto Hatakeyama, Yusuke Sato, Hidetaka Akita, Hideyoshi Harashima
Hokkaido University, Sapporo, Hokkaido, Japan
To deliver siRNA to tumor tissue, we have developed liposome type carrier, a multi-functional envelope type nano device (MEND). While PEGylation is the most popular strategy to
stabilize liposome in the circulation, reduction of cellular uptake and endosomal escape by
PEGylation is a crucial problem. We previously reported that pH-sensitive fusogenic peptide,
GALA, modification onto the MEND circumvented this barrier in vitro cultured cell (Sakurai
et al., Biol Pharm Bull, 2009). GALA-MEND was destabilized in an acidic compartment endosome because the structure of GALA changed into alpha-helix at low pH (~5.5), and then
GALA on the surface of GALA-MEND induced membrane fusion between MEND and endosome. As a result, encapsulated siRNA was released into cytosol efficiently. Although GALAmodified PEGylated MEND (GALA-MEND) elicits an siRNA delivery efficiency, it was
clarified that GALA-MEND intrabenously injected into mice was subject to a rapid clearance
from blood circulation. Therefore, we designed a shorter version GALA (shGALA). In this
study, we would like to introduce in vivo siRNA delivery with shGALA-modified PEGylated
MEND (shGALA-MEND).
shGALA-MEND exhibited a higher knockdown effect (60% reduction, 480 nM) than
only PEGylated MEND (PEG-MEND) in vitro. Microscopy and flowcytometry analyses
indicated that the result was caused by increasing endosomal escape of siRNA, not enhanced
cellular uptake. Because inhibition of acidification of endosome/lysosome compartments
by ammonium chloride suppressed siRNA efficiencyof shGALA-MEND, endosomal
escape of siRNA by shGALA depended on acidification of endosome. Then, to evaluate
the biodistribution of both siRNA and lipid envelope of systemically injected MENDs,
we labeled these components with radio isotope. While free siRNA showed a very rapid
blood clearance, siRNA assembled with PEG-MEND and shGALA-MEND demonstrated a
high blood concentration (>10% ID/mL blood) even 6 hr after injection, and no significant
difference was observed between PEG- and shGALA-MEND Next, we investigated the in
vivo knockdown and the anti-tumor effect of shGALA-MEND. We administered MENDs
into tumor-bearing mice via tail vein 4 times every 24 hr at a dose of 4 mg siRNA/kg body
weight, and then measured target gene expression and tumor volume change. A significant
mRNA reduction (60%) and inhibition of tumor growth were observed in the group of mice
treated with shGALA-MEND. In addition, shGALA-MEND showed no notable somatic and
hepatic toxicity. In conclusion, we succeeded in developing an in vivo siRNA delivery system
to tumor tissue (Sakurai et al., Biomaterials, in press).
Yu Sakurai, doctoral course
Kita-12, Nishi-6, Kita-ku, Sapporo, Hokkaido, Japan
Telephone: +81-11-706-2197
Email: yu-m@pharm.hokudai.ac.jp
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IN VIVO siRNA DELIVERY WITH PH-SENSITIVE LIPOSOME
Yusuke Sato, Hiroto Hatakeyama, Yu Sakurai, Mamoru Hyodo, Hidetaka Akita,
Hideyoshi Harashima
Faculty of Pharmaceutical Science Hokkaido University, Sapporo/Hokkaido, Japan
Small interfering RNA (siRNA) is the biofunctional nucleic acid inducing RNA interference
(RNAi) and is emphasized as the tool for gene therapy. The physicochemical characteristics
of siRNA, such as high molecular weight and hydrophilicity prevent passive diffusion across
plasma membrane. So, delivery carriers are required which allow siRNA to arrive at target organs and enter target cells. In addition, it is necessary for siRNA to localize cytoplasm which
is the place for working and avoid lisosomal compartmentalization. To date, we developed
a multifunctional envelope-type nano device (MEND) for siRNA delivery carrier to tumour
tissue. The most of conventional liposomal carriers are composed of cationic lipids and have
high cationic charge on their surface for the purpose of high affinity for cells but lead to nonspecific interaction with anionic species in the blood and exhibit rapid clearance from blood
circulation by the reticulo-endothelial system (RES). This problem can be overcome by using
polyethylene glycol (PEG) attached on the particle’s surface. Even though, the modification
of a lot of PEG actually prolonged circulation time, it dramatically inhibits intracellular trafficking. To solve this ‘PEG dilemma’, we previously developed a cleavable PEG-lipid in response to tumour environment1) and a novel endosomal escape peptide device2). In the present
study, we picked on pH-sensitive lipid, which exhibits neutral at physiological pH and cationic at acidic pH. First, we constructed pH-sensitive MEND by using DODAP, which is known
as a pH-sensitive aminolipid but it resulted in lower knockdown activity than conventional
cationic MEND using DOTAP, which is known as a cationic lipid. Next, we synthesized a
novel pH-sensitive lipid, YSK05 and optimized lipid composition to improve the intracellular
trafficking of pH-sensitive MEND. As a result, the optimized pH-sensitive MEND successfully showed 100-fold higher knockdown activity than the conventional cationic MEND.
Then, we tried to confirm whether pH-sensitive MEND actually facilitated endosomal escape
by depending on endosomal acidification by using ammonium chloride, which buffers lysosomes. Expectively, the knockdown activity of pH-sensitive MEND was dramatically decreased depending on ammonium chloride concentration, which suggested endosomal acidification was important process for pH-sensitive MEND to escaping from endosome. Finally,
we evaluated knockdown activity of pH-sensitive MEND in subcutaneous OSRC-II tumour
model. Both single local and systemic administration of pH-sensitive MEND-formulated human polo-like kinase 1 (hPLK1) caused reduction in tumour-derived hPLK1 mRNA. RNAispecific mRNA cleavage products in tumour were found by 5’-RACE PCR, which suggested
that mRNA reduction resulted from RNAi and was sequence specific. These results suggest
that pH-sensitive MEND is a useful tool for siRNA delivery to tumour tissue and has potent
carrier for therapeutics.
1) Hatakeyama H et. al. Biomaterials, 32, 4306-4316 (2011)
2) Sakurai Y et. al. Biomaterials, in press
Yusuke Sato, Doctor Course
Telephone: +81-11-706-2197
Email: yukke_overture@mail.sci.hokudai.ac.jp
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092
OTS 2011: PMOs – The Clinical Story
Stephen Shrewsbury
AVI BioPharma, Bothell, WA USA
Phosphorodiamidate Morpholino oligomers (PMOs) differ from the older Antisense
OligoNucleotides (AONs) by the substitution of a synthetic 6-membered morpholine ring
for the ribose and linking the monomeric backbone with phosphorodiamidates rather than
phosphorothioates moieties. AVI has pioneered PMO development for 25 years and has tested
5 PMOs in 18 clinical studies in several diseases – neuroinvasive West Nile Virus, Hepatitis
C, polycystic kidney disease, cytochrome induction and the lead PMO (AVI-4658, eteplirsen)
recently completed phase 2 testing in Duchenne Muscular Dystrophy (DMD) at individual
doses of up to 900mg IV (20mg/kg).
Maximum Feasible Doses (MFD) of PMOs were well tolerated in 2 mammalian species over
12 weeks to open an eteplirsen IND allowing evaluation of doses of up to 100mg/kg, and
supporting plans for further clinical testing. Recently, 219 clinical doses of eteplirsen were
well tolerated in a 12-week of dosing, open label, dose escalation, phase 1b/2 clinical study of
eteplirsen with a typical pediatric trial AE profile superimposed on the chronic DMD disease.
No significant drug related AEs or SAEs were reported and no AE was deemed probably
or definitely drug related. Of particular note, no local injection site reactions were reported.
Clinical hematology, coagulation and chemistry (renal function especially) remained stable,
and encouraging but variable biological efficacy was demonstrated, especially at the 10 and
20 mg/kg dose levels. A further placebo-controlled, phase 2 study of eteplirsen at higher
doses (30 and 50 mg/kg) is due to start dosing in June 2011.
Advanced generation PMO based chemistries are now evolving, based on the neutral and
stable PMO backbone, by highly specific positioning of limited positive charges (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, PhD
3450 Monte Villa Parkway
Bothell, WA 99021 USA
Telephone: 425-354-5038
Email: sshrewsbury@avibio.com
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AMPHIPHILIC PEPTIDYL-RNA CONJUGATES ANCHORED TO
AND ENRICHED ON PHOSPHO- AND GLYCOLIPID BILAYERS
Peter Strazewski,1 Alexandra Le Chevalier Isaad,1 Kollappillil S. Krishnakumar,1
Dominique Lafont,2
(1) Laboratoire de Synthèse de Biomolécules, (2) Laboratoire de Chimie Organique 2 ; Institut 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 immersed into the lipophilic interior of membrane bilayers. Different vesicles, which assembled
spontaneously from various lipids and glycolipids, were used to study RNA anchoring. In
order to visualize by confocal fluorescence microscopy peptidyl-RNA being anchored onto
lipid vesicles, the conjugate and the lipids were labeled with suitable fluorophores.
A hydrolytically stable peptidyl-RNA conjugate was synthesized in a step-by-step fashion by
a solid support procedure using an orthogonally protected derivative of 3’-l-alanylamino-3’deoxyadenosine (a puromycin analogue) as a first synthetic building block that was immobilized through its 2’-hydroxyl function to an appropriate resin. A solid support peptide synthesis using Fmoc strategy was carried out departing from the 3’ position of the puromycin
analogue. After DMT cleavage, an oligoribonucleotide strand was grown on the same solid
support beginning at the 5’ position of the puromycin analogue. After the final cleavage of all
protective groups, the solubilised amphiphilic conjugate was purified by HPLC and identified
by ESI-MS. The target molecule was composed of a lipophilic 20-meric oligopeptide being
covalently linked through a C-terminal amide bond to a 22-meric oligoribonucleotide hairpin
that was inspired by the acceptor stem of E. coli tRNAAla and closed by a stable UUXG
tetraloop (Figure).
We could unequivocally demonstrate by
confocal fluorescence microscopy that the
peptidyl-RNA conjugate, when externally
added to giant uni- and multilamellar lipidic vesicles, spontaneously interacted with
their outer bilayer membranes. At initial
Figure. L = l-leucine, A = l-alanine, 3'-A = 3'-amino-3'- lipid-to-conjugate molar ratios between
deoxyadenylate, ACGU = adenylate, cytidylate, guanylate, 150:1 and 1500:1 they visibly and strongly
uridylate, X = fluorescein linked to thymidylate.
enriched on the readily accessible lipidic
surfaces. These studies were supplemented
by control experiments and quantitative permeability measurements on large unilamellar vesicles, as well as by FTIR and CD spectroscopic studies of the peptide interacting with lipids.
Prof. Peter Strazewski
Laboratoire de Synthèse de Biomolécules, Bât. Chevreul (5ème étage)
Université Claude Bernard Lyon 1
43 boulevard du 11 novembre 1918, 69622 Villeurbanne Cedex, France
Email: strazewski@univ-lyon1.fr
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094
Therapeutic Application of DNAzymes for
Nasopharyngeal Carcinomas
Sun L-Q, Yang L, Cao Y
Xiangya Hospital and Center for Molecular Medicine, Central South University; Cancer
Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan
410078, China
DNAzymes are synthetic, single-stranded DNA catalysts that can be engineered to bind and
cleave the target mRNA of a disease-causing gene. The ability of the 10-23 DNAzyme to
specifically cleave RNA with high efficiency under simulated physiological conditions has
fuelled expectation that this agent may have useful biological application in a gene inactivation strategy. To explore this potential, we used DNAzymes to validate therapeutic targets for
therapeutic approaches to nasopharyngeal carcinomas (NPC).
Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has been known to
have oncogenic properties during latent infection in NPC. Genetic manipulation of LMP1
expression may provide a novel strategy for the treatment of NPC. By targeting the LMP1
mRNA, we successfully obtained a phosphorothioate-modified ‘‘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, PhD
Xiangya Hospital and Center for Molecular Medicine, Central South University
Changsha, Hunan 410078, China
Telephone: +86 731 84327646
Email: lq-sun@hotmail.com
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INHIBITION OF MICRORNA ACTIVITY BY 2ʹ-O-METHYL-4ʹTHIORIBONUCLEOSIDE MODIFIED ANTI-microRNA
OLIGONUCLEOTIDE (AMO)
Mayumi Takahashi,1 Noriaki Minakawa2 and Akira Matsuda1
Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku,
Sapporo 060-0812, Japan, 2Graduate School of Pharmaceutical Sciences, The University of
Tokushima, Shomachi 1-78-1, Tokushima 770-8505, Japan
1
MicroRNAs (miRNAs) are a class of endogenously expressed small non-cording RNA
molecules which regulate the gene expression post-transcriptionally. Recent advances in understanding of the role of miRNAs in the biological process revealed that most of them are
implicated in the human diseases, such as cancer, viral infection and cardiovascular disorders.
Therefore, miRNAs are considered to be a potential new class of therapeutic target. Among
the approaches to modulate the function of miRNA, anti-miRNA oligonucleotide (AMO)based inhibition has been the most widely used not only to exploit the biological function of
miRNAs but also as candidates for therapeutic agents. Although AMOs represent sequence
specific inhibition based on the Watson-Crick base pairing, they are single-stranded oligonucleotides and easily degraded in biological fluid. Chemical modification on AMOs can
be beneficial to improve hybridization ability for target miRNA, resistance toward nuclease
degradation, and pharmacological properties such as tissue uptake for in vivo delivery. A wide
variety of chemically modified AMOs was developed to date, including 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-4ʹthioRNA, 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 stability 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 University
Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
Telephone: +81 11 706 3763
Email: m-tab@pharm.hokudai.ac.jp
143
096
RNAi in humans: Phase I dose-escalation study of
ALN-VSP02, a novel RNAi therapeutic for solid tumors
with liver involvement
A.K. Vaishnaw9, A. Cervantes1, M. Alsina2, J. Tabernero2, J.R. Infante3, P. LoRusso4, G.I. Shapiro5, L.
Paz-Ares6, G. Schwartz7, G. Weiss8, R. Falzone9, J. Hill9, J. Cehelsky9, A. White9, I. Toudjarska9, D.
Bumcrot9, R. Meyers9, G. Hinkle9, N. Svrzikapa9, D.W. Sah9, H.A. Burris3, J.A. Gollob9
1
Hospital 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,
8
TGen Clinical Research Services at Scottsdale Healthcare, Scottsdale, AZ, USA,
9
Alnylam Pharmaceuticals, Inc., Cambridge, MA, USA
Background: ALN-VSP02 is a RNA interference (RNAi) therapeutic comprised of lipid
nanoparticle-formulated small interfering RNAs (siRNAs) targeting the expression of vascular endothelial growth factor (VEGF)-A and kinesin spindle protein (KSP).
Methods: A multi-center, open label, Phase 1 dose-escalation trial of ALN-VSP02 administered as a 15-minute iv infusion q2 wks was initiated in patients (pts) with advanced solid
tumors and at least one measurable liver lesion. Main objectives included evaluation of
safety/tolerability and assessment of PK/PD.
Results: Forty-one pts were enrolled across 7 dose levels (0.1-1.5 mg/kg); median age 57 yrs,
all with multiple prior therapies. A total of 182 doses have been administered to date, mean of
4.4 (range 1-24). Treatment was generally well-tolerated; the most common AEs were lowgrade nausea, fatigue and fever with no clear dose-dependence. One on-study death (liver
failure in a pt with near complete replacement of the liver by tumor) deemed possibly related
to treatment occurred at 0.7 mg/kg. Grade 2 infusion-related reactions occurred in 15% of
pts or 3% of total doses administered and were managed with slowing of infusion. Grade 1-2
chills/rigors were seen in 3 of 11 pts at 1.25 mg/kg. Dose-limiting toxicities included reversible grade 3 thrombocytopenia (2 pts at1.25 mg/kg) and hypokalemia (1 pt at 1.5 mg/kg).
Plasma PK showed dose-proportional AUC and Cmax. Post-treatment biopsies from 10 pts (7
liver and 3 extrahepatic tumors) showed pharmacologically relevant concentrations (2.2-142
ng/g tissue) of both siRNAs. Molecular evidence of RNAi-mediated VEGF mRNA cleavage
was shown in liver tumor biopsies (n=2 at 0.4 mg/kg) and in an extrahepatic tumor biopsy
(ovarian cancer at 1.25 mg/kg) through use of the 5’ RACE assay. Additional evidence for
an anti-VEGF effect with ALN-VSP02 included a decrease in Ktrans of at least 40% by
DCE-MRI in 46% of evaluable pts. Among 37 pts evaluable for response, 8% (1 of 13) at
doses ≤ 0.4 mg/kg had stable disease (SD) for at least 2 mo compared to 50% (12/24) with
SD (n=11) or PR (n=1, endometrial cancer with liver metastases) at doses ≥ 0.7 mg/kg.
Conclusions: ALN-VSP02 is well-tolerated and has antitumor activity. Pharmacodynamic
data are consistent with an anti-VEGF effect, and 1.0 mg/kg q2wks is the recommended
Phase II dose.
Akshay K. Vaishnaw, MD, PhD
Alnylam Pharmaceuticals Inc.
300 Third Street, Cambridge, MA 02142 USA
Telephone: 617-551-8360
Email: avaishnaw@alnylam.com
144
097
microRNA turnover and AntagomiR-mediated inhibition
of inflammation
Bryan R.G. Williams,1 Michael P. Gantier1, H. James Stunden1, Claire E. McCoy1, and
Mark A. Behlke2
Centre for Cancer Research, Monash Institute of Medical Research, Monash University,
Clayton, Victoria, Australia; 2Integrated DNA Technologies, Coralville, Iowa, USA
microRNAs (miRNAs) are key regulators of gene expression and critical for normal cell
function. However, little is known of their overall persistence in the cell following processing. Characterization of this is key to the full appreciation of their regulatory roles. We have
devised a system to allow inducible genetic ablation of Dicer, the enzyme required for production of mature miRNAs. Using this system we measured miRNA decay rates in mouse
embryonic fibroblasts following loss of Dicer1 enzymatic activity. The results confirm the
inherent stability of miRNAs, the intracellular levels of which were mostly affected by cell
division. Using the decay rates of a panel of six miRNAs representative of the global trend of
miRNA decay, we have established a model of miRNA turnover and determine an average
miRNA half-life of 119 h, (i.e. ~5 days). This study constitutes the first in-depth characterization of miRNA decay in mammalian cells and our findings indicate that miRNAs are up to 10
times more stable than messenger RNA. Interestingly some miRNAs turn over more rapidly
than others suggesting the existence of novel mechanism(s) controlling selective miRNA cellular concentration and function. We have also used the inducible genetic ablation of Dicer1
and subsequent miRNA depletion to uncover an important role for miRNAs in the control
of pro-inflammatory cytokine production in cells. Antisense-mediated inhibition of miRNA
function by antagomiR confirmed the involvement of select miRNAs in the positive control
of NFkB signalling. Through the use of in silico predictions and reporter assays we show that
these miRNAs negatively control expression of A20 (TNFAIP3), resulting in increased NFkB
signalling. AntagomiR-mediated inhibition of these miRNAs results in inhibition of proinflammatory cytokine production. These results suggest that antagomiRs targeting negative
regulators of NFkB signalling could offer novel therapeutic avenues to control inflammatory
disorders and infections.
Bryan R.G. Williams PhD
Centre for Cancer Research, Monash Institute of Medical Research
Monash University Clayton, Victoria, 3168, Australia
Email: bryan.williams@monash.edu
145
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