13th Annual Meeting of the Oligonucleotide Therapeutics Society

MEETINGTHE OLIGO September 24-27, 2017

P a l a i s d e l a B o u r s e | B o r d e a u x , F r a n c e

Palais de la Bourse | Bordeaux | France

Poster Abstracts

13th Annual Meeting of the Oligonucleotide Therapeutics Society Poster Presentation Index

Every effort has been made to ensure the information contained here is accurate, although some changes may occur.

Poster # Last Name First Name Affiliation Abstract Title

001 Anderson Brooke The Scripps Research Institute, Ramanarayanan Krishnamurthy Lab

Heterogeneous Pyrophosphate Linked DNA-Oligonucleotides: Aversion for DNA but Affinity for RNA

002 Kamatkar Nachiket Albert Einstein College of Medicine Aptamers for the FGF Receptor Family to Modulate Activation of Astrocytes

003 Andersson Patrik AstraZeneca R&D Gothenburg Safety of chronic Kras ASO knockdown

004

005 Ashush Hagit Quark Pharmaceuticals Inc Topical Application of siRNA for Treatment of Hair Follicle Pathologies

006 Aupy Philippine University of Versailles Saint QuentinEvaluation of the Phosphorothioate content within

tricyclo-DNA splice-switching oligonucleotides in DMD mouse model

007 Benizri Sébastien ChemBioPharm, INSERM U1212 Delivery of nucleic acids and anticancer drugs using Lipid-Oligonucleotides technology

008 Brand Emily Ionis PharmaceuticalsGeneration 2.5 Constrained Ethyl Antisense

Oligonucleotide Renal Sub-Organ Pharmacodynamics in the Healthy Rodent Kidney

009 Brill Wolfgang Brill Chemistry Recycling of Nucleosides and Reagents from Oligonucleotide Synthesis

010 Brown Jonathan MPEG LA, LLC Defined Multimeric Oligonucleotides for Enhanced Therapeutic Effect

011 Brown Christopher Alnylam PharmaceuticalsMechanistic Insights and Progress on the GalNAc-siRNA Conjugate Platform for Targeted Delivery of

RNAi Therapeutics to the Liver

012 Chernolovskaya Elena Institute of Chemical Biology and Fundamental Medicine SB RAS

Targeting multiple drug resistance gene with small interfering RNAs

013 Christou Melina The Cyprus Institute of Neurology and Genetics, The Cyprus School of Molecular Medicine

Novel oligonucleotides for the therapy of Myotonic Dystrophy

014 Conneely Michael University of Dundee Delivering Gene Silencing Therapy to the Epidermis in an Ex Vivo Model

015 Croft LauraQueensland University of Technology, Institute

of Health and Biomedical Innovation, Translational Research Institute

Targeting DNA damage response pathways with antisense oligonucleotides

13th Annual Meeting of the Oligonucleotide Therapeutics Society Poster Presentation Index

Every effort has been made to ensure the information contained here is accurate, although some changes may occur.

Poster # Last Name First Name Affiliation Abstract Title

016 Dallas Anne SomaGenics, IncmiR-Direct®: A Sensitive and Specific Method for

Quantification of miRNAs Directly from Plasma and other Biofluids

017 Daujotyte Dalia Lexogen Recovering pre-PCR numbers of fragment copies in

RNA-Seq from statistics of Universal Molecular Identifiers

018 de Franciscis Vittorio Istituto per l’Endocrinologia e Oncologia Sperimentale "G. Salvatore" STAT3 AsiC Aptamer- for GBM targeted therapy

019 de Jong Annika Leiden University Medical Center Allele-specific inhibition of von Willebrand factor p.Cys2773Ser restores a severe multimerization defect

020 de Mollerat du Jeu Xavier Thermo Fisher ScientificInvivofectamine™ Rx Reagent: Novel Lipid

Nanoparticles (LNP) for Therapeutic In Vivo mRNA and RNAi delivery to the various tissue and organs

021 Debacker Alexandre RNA Therapeutics Institute University of Massachusetts Medical School

PNAs and catalytic oligonucleotides: the impossible marriage?

022 Delpy Laurent CNRS UMR7276 - CRIBL, Centre Biologie et Recherche en Santé

Antisense oligonucleotide-mediated exon skipping of immunoglobulin transcripts as an innovative anticancer

approach for the treatment of multiple myeloma and lymphoproliferative disorders

023 Dobbs Craig TriLink Biotechnologies Innate Immune Focused Approaches to Maximize Messenger RNA Therapeutic Activity

024 Epps Elizabeth Beckman Research Insitute of City of Hope Enhancing Lentiviral Production Using Splice-Inhibiting Antisense Oligonucleotides

025 Fernstrum Grant ThermoFisher Scientific Therapeutic Oligo Quality: Profiling and Controlling for Raw Material Impurities

026 Frandsen Niels Exiqon Targeting nascent primary transcripts with antisense LNA™ GapmeRs

027 Fujii Masayuki Kindai University A Facile Synthesis of DNA/RNA Multiple Conjugates by Chemo-enzymatic Approach

028 Gagnon Keith Southern Illinois University Compatibility of Nucleic Acid Chemical Modification with CRISPR-Cas9

029 Ghavami Mahdi University of Copenhagen Effect of mismatch sequence on antisense activity of PNA

030 Gilles Maud-Emmanuelle

Institute for RNA Medicine, BIDMC, Harvard Medical School

miR-21-5p as an RNA-therapeutic target for personalized medicine in pancreatic cancer

13th Annual Meeting of the Oligonucleotide Therapeutics Society Poster Presentation Index

Every effort has been made to ensure the information contained here is accurate, although some changes may occur.

Poster # Last Name First Name Affiliation Abstract Title

031 Giorgio Elisa University of TorinoAllele-specific silencing as therapeutic strategy for

disorders due to gene duplication: a proof-of-principle in Autosomal Dominant LeukoDystrophy (ADLD)

032 Gonzalez-Torres Alejandro CINVESTAV Mir-143/145 Expression In Keratinocyte Differentiation And Its Role In Cervical Cancer Progression

033 Habib Nagy Imperial College London, Department of Surgery and Cancer, Hammersmith Hospital

MTL-CEBPA has efficacy in a broad range of liver disease models and encouraging early clinical data in a

Phase 1 trial in HCC

034 Hagopian Jonathon Advanced Analytical Technologies, Inc

Scalable Nucleic Acid Quality Assessments for Illumina Next-Generation Sequencing Library Prep:

Simultaneous qualification and quantification of nucleic acids with the Fragment Analyzer™

035 Halloy François ETH Zürich Oligonucleotide therapy for treatment of erythropoeitic protoporphyria

036 Heo Roun Sungkyunkwan University Enhanced cancer immunotherapy based on tumor cell foreignization with PD-L1 blockade

037 Heyes James Arbutus Biopharma Corporation Development of Clinically Viable Lipid Nanoparticles for mRNA

038 Horiba Masahiko Osaka UniversitySynthesis and Evaluation of a Tricyclic Nucleic Acid

Analogue Bearing the Locked Sugar Moiety and Torsion Angle ε

039 Hoshino Hidekazu National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN)

The selection system of the sugar modified nucleic acid aptamer by KOD polymerase mutants

040 Hwang Larn Marina BiotechPreclinical Development of siRNA Therapeutics for

Inflammatory Bowel Disease (IBD) using Transkingdom RNA Interference (tkRNAi) Bacterial Delivery Platform

041 Hwang Larn Marina BiotechDose-Exposure and Dose-Response Analysis for

Trabedersen − A TGF-β2-Specific Antisense Oligonucleotide in Cancer Patients

042 Hwang Larn Marina BiotechSTART-FAP Phase I Study of CEQ508 RNA

Interference in Patients with Familial Adenomatous Polyposis (FAP)

043 Issa Hasan Wolfson Childhood Cancer Research Centre, Newcastle University

Therapeutic Targeting of the Leukaemic Fusion Gene RUNX1/ETO

044 Jadhav Vasant Alnylam Pharmaceuticals REVERSIR™ Platform for the Tailored Control of GalNAc-siRNA Conjugate Pharmacology

045 Jansson-Löfmark Rasmus AstraZeneca Dose-response time modelling of circulating biomarkers for nucleotides

13th Annual Meeting of the Oligonucleotide Therapeutics Society Poster Presentation Index

Every effort has been made to ensure the information contained here is accurate, although some changes may occur.

Poster # Last Name First Name Affiliation Abstract Title

046 Järver Peter Stockholm University, Department of Molecular Biosciences, The Wenner-Gren Institute

Single stranded nucleic acids regulate endocytic uptake and downstream Toll-like receptor signalling

047 Jaschinski Frank Secarna Pharmaceuticals GmbH & Co. KGTargeting hIDO1 with 3rd generation antisense

oligonucleotides for modulation of the tumor microenvironment

048 Jaschinski Frank Secarna Pharmaceuticals GmbH & Co. KGInhibition of immune suppressive CD39 and CD73 by

3rd generation antisense oligonucleotides for treatment of cancer

049 Jirka Silvana Centre for Human Drug Research Characterization of TLR7/8-induced skin responses using a standardized dermatological toolbox

050 Johnston Brian SomaGenics, IncRealSeq™, a novel method that greatly reduces

sequence bias in construction of small-RNA sequencing libraries

051 Kabilova Tatyana Institute of Chemical Biology and Fundamental Medicine SB RAS

Molecular mechanisms of antiproliferative and interferon-inducing activity of immunostimulating

dsRNA

052 Kanazawa Takanori Nihon University Nose-to-brain siRNA delivery combined with cell-penetrating peptide modified polymer micelles

053 Kasahara Yuuya National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN)

Evaluation of antisense activity under the conditions of CEM method focused on base composition

and Tm value

054 Kielpinski Lukasz Jan Roche Innovation Center Copenhagen A/SRNase H sequence preferences influence antisense

oligonucleotide efficiency and have shaped the HIV-1 genome

055 Kitade Yukio Aichi Institute of Technology Preparation of dsRNAs library possessing biomolecule-ligands and their properties

056 Knight John University of Alabama at Birmingham Investigational RNAi Mediated Oxalate Reduction Therapy

057 Kupryushkin Maxim Novosibirsk Institute of Chemical Biology and Fundamental Medicine SB RAS

Dodecyl-modified oligodeoxyribonucleotides as platform for oligonucleotide delivery

into eukaryotic cells

058 Lee Sang Deuk BioInfra Co., LtdEstablishment of a Method for the Determination of

ISIS2503 in Plasma Using LC-MS/MS Detection and Comparison with ELISA Method

059 Linnane Emily AstraZeneca Insights into the Kinetics of Antisense Oligonucleotide Uptake in Non Small Cell Lung Cancer

060 Llamusi Beatriz University of Valencia Derepressing muscleblind expression by miRNA

sponges ameliorates myotonic dystrophy-like phenotypes in Drosophila

13th Annual Meeting of the Oligonucleotide Therapeutics Society Poster Presentation Index

Every effort has been made to ensure the information contained here is accurate, although some changes may occur.

Poster # Last Name First Name Affiliation Abstract Title

061 Lobos-Gonzalez Lorena Andes Biotechnologies and Fundacion Ciencia y Vida

Lactadherin and VE-cadherin Incorporated in to exosomes secreted post-interference of ASncmtRNA decrease tumorigenic property in breast cancer cell

062 Lobos-Gonzalez Lorena Andes Biotechnologies and Fundacion Ciencia y Vida

Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors

063 Ly Socheata University of Massachusetts Medical School, RNA Therapeutics Institute Intracellular Trafficking of Chemically Modified siRNA

064 Machielse Jürgen Zeochem AG Enhanced Oligonucleotide Purification via Novel Orthogonal, Doped Reverse Phase Chromatography

065 Maier Martin Alnylam PharmaceuticalsImpact of Enhanced Metabolic Stability on In Vivo

Performance of GalNAc-siRNA Conjugates

066 Mallikaratchy Prabodhika Lehman College for City University of New YorkLigand-Guided Selection (LIGS): A Screening

Technology to Identify Specific Aptamers Against Cell-surface Markers

067 Masaki Yoshiaki Tokyo Institute of Technology Relationship between deformability of sugar-modified RNA duplexes and their melting temperatures

068 Matos Liliana National Health Institute Dr. Ricardo JorgeUse of an antisense-mediated exon skipping approach

as a therapeutic option for a common Mucolipidosis type II causing mutation

069 McCampbell Alexander Biogen, IncPreclinical efficacy of SOD1 antisense oligonucleotide

in phase 1 testing for SOD1 amyotrophic lateral sclerosis

070 Milstein Stuart Alnylam Pharmaceuticals Preclinical Development of RNAi Therapeutic Drug Candidate for Targeting Hepatitis B Virus

071 Miyatake Shouta National Center of Neurology and Psychiatry, Department of Molecular Therapy

Scavenger receptor class A1 mediates physiological uptake of morpholino oligomer with negative zeta

potential into dystrophic skeletal muscles

072 Moelling Karin University of Zürich RT and RNase H: from Viruses to antiviral Defense

073 Monine Michael Biogen, IncExperimental and model-based characterization of antisense oligonucleotide (ASO) distribution in the

cerebral spinal fluid (CSF)

074 Murray Sue Ionis Pharmaceuticals Cellular Fractionation and Evaluation of Hepatic Cells after Antisense Oligonucleotide Treatment in Mice

075 Narayan Chandan Pohang University of Science and Technology (POSTECH)

Development of Rapid Influenza Diagnostic test using aptamers

13th Annual Meeting of the Oligonucleotide Therapeutics Society Poster Presentation Index

Every effort has been made to ensure the information contained here is accurate, although some changes may occur.

Poster # Last Name First Name Affiliation Abstract Title

076 Osborn Maire RNA Therapeutics Institute, University of Massachusetts Medical School

Harnessing Endogenous Lipoproteins to Drive Systemic siRNA Delivery

077 Ozeri-Galai Efrat Splisense Restoration of CFTR function by Antisense Oligonucleotide splicing modulation

078 Paneda Covadonga Sylentis Targeting NRARP with siRNA based compounds for the treatment of retinal neovascularization

079 Paneda Covadonga Sylentis Clinical and preclinical correlation analysis for SYL1001, a new treatment for dry eye disease

080 Panzner Steffen Lipocalyx GmbH Viromer – Enabling mRNA Delivery

081 Patzel Volker National University of Singapore, Dept. of Microbiology & Immunology

An Efficient, Scalable Mitochondrial Delivery Vector System for Ribonucleic Acids

082 Pendergraff Hannah Roche Innovation Center Copenhagen A/S Intracellular quantification of unmodified LNA oligonucleotides

083 Porciani David University of Missouri – Columbia A modular cell-internalizing aptamer nanostructure to deliver large functional RNAs

084 Prins Jurriën Leiden University Medical Center Quaking Modulates Cortical Mechanical Properties of Monocytes

085 Puri Sanyogitta AstraZeneca Tumour targeting Chitosan nanoparticles: Key design parameters influencing siRNA and mRNA delivery

086

087 Relizani Karima University of Versailles St-Quentin en Yvelines, SQY Therapeutics

Efficacy and safety profile of tricyclo-DNA antisense oligonucleotides in Duchenne muscular dystrophy

mouse model

088 Rocchi Palma Marseille Cancerology Research CenterTCTP inhibition by specific antisense oligonucleotide lipid moiety-modified for the treatment of castration-

resistant prostate cancer

089 Romero-Palomo Fernando F. Hoffmann-La Roche LtdBiodistribution, metabolism and in situ accumulation of naked and GalNAc-conjugated locked nucleic acids in

rat liver and kidney

090 Rossiello Francesca IFOM, the FIRC Institute of Molecular OncologySequence specific inhibition of DNA damage response at double-strand breaks and deprotected telomeres by

antisense oligonucleotides

13th Annual Meeting of the Oligonucleotide Therapeutics Society Poster Presentation Index

Every effort has been made to ensure the information contained here is accurate, although some changes may occur.

Poster # Last Name First Name Affiliation Abstract Title

091 Sapag Amalia Universidad de Chile

Rational Truncation of RNA Aptamer G70 Leads to Improved Inhibition of the Sphingomyelinase Activity of

Two Recombinant Isoforms of the Main Toxin of the Loxosceles laeta Spider Venom

092 Sasaki Shruti Ionis Pharmaceuticals

Antisense oligonucleotide-mediated upregulation of CFTR via steric block of post-transcriptional control

elements: A novel approach for Cystic Fibrosis therapeutics

093 Sawamoto Hiroaki Mitsubishi Tanabe Pharma Corporation Effective Synthesis of 2′-Amino-LNAs Bearing Any of the Four Nucleobases

094 Schlegel Mark Alnylam Pharmaceuticals Impact of Glycol Nucleic Acid (GNA) on siRNA Structure and Function

095 Schlegel Mark Alnylam PharmaceuticalsImproved Specificity and Therapeutic Index with ESC+

siRNA Conjugates Utilizing Seed-Pairing Destabilization via Novel Chemical Modifications

096 Schmidt Karyn Dana-Farber Cancer Institute

Inhibiting the Androgen Receptor Interaction with the Long Non-Coding RNA SLNCR using 2’-FANA-Modified

Oligonucleotides Decreases Melanoma Invasion and Proliferation

097 Schnell Frederick Sarepta Therapuetics Novel PMO-Based Chemistries for CNS and Muscle Targeting

098 Setten Ryan City of Hope: Irell & Manella Graduate School of Biological Science

Characterizing a Non-Coding RNA of the +9kb CEBPA Enhancer

099 Shimo Takenori Osaka University A Novel Human Muscle Cell Model of Duchenne Muscular Dystrophy Created by CRISPR/Cas9

100 Shin Hye Su Sungkyunkwan UniversityAlkaline Phosphatase Placental like 2 as a Diagnostic

Biomarker for Detection of Pancreatic Ductal Adenocarcinoma

101 Smith C. I. Edvard Karolinska InstitutetAnti-gene oligonucleotides targeting pathological triplet

repeat expansions in Huntington’s disease and Friedreich’s ataxia

102 Sugai Hiroka Tohoku University

Development of Cancer-Cell-Specific Drug Delivery System Using MMP-Activatable PEG-Conjugated

Oligoarginine Peptide and Its Application for MicroRNA Inhibition

103 Svrzikapa Nenad WAVE Life Sciences

An Investigational Assay Using Next-Generation Sequencing Technology to Detect Single Nucleotide Polymorphisms (SNPs) Associated with the Mutant

Huntingtin Allele

104 Tachikawa Kiyoshi Arcturus Therapeutics LUNAR-mRNA for protein replacement and gene editing therapies

105 Tagnouti Nadia Precision NanoSystems, IncA Scalable Microfluidic Platform for Oligonucleotides

Lipid Nanoparticles (LNPs) Manufacturing from Microliters to Liters

13th Annual Meeting of the Oligonucleotide Therapeutics Society Poster Presentation Index

Every effort has been made to ensure the information contained here is accurate, although some changes may occur.

Poster # Last Name First Name Affiliation Abstract Title

106 Tago Nobuhiro Ludwig-Maximilians-Universität München Synthesis of RNA containing the epigenetically relevant 5- hydroxymethyl -, 5-formyl-, and 5- carboxycytidine

107 Takahashi Mayumi US Food and Drug AdministrationDesign and Synthesis of Positively Charged

Oligonucleotides Functionalized with Thermolytic Masking Groups

108 Tanaka Keisuke Osaka University Generation and PPI inhibition analysis of artificial nucleic acid aptamers targeting Ebola virus protein

109 Theler Dominik NCCR RNA & Disease NCCR RNA & Disease: A multi-disciplinary Swiss-wide research initiative

110 van Putten Maaike Leiden University Medical Center A dystrophic mouse model for testing human specific antisense oligonucleotides

111 Villegas Jaime Fundación Ciencia & VidaMitochondrial ASncmtRNA-1 and ASncmtRNA-2 as

potent targets to inhibit tumor growth and metastasis in the RenCa murine renal adenocarcinoma model

112 Villegas Jaime Fundación Ciencia & VidaEvaluation of the efficacy of antisense therapy in a patient derived xenograft model (PDX) of advanced

cervical cancer

113 Villiet Pierre ChemGenes Corporation

Polythiol Probes & Microarrays for DNA Hybridization & Highly Sensitive Detection. Improved DNA Microarray

Hybridization Using Polythiol Probes and Their Immobilization by Thiol-Ene Chemistry** & ***

114 Wada Takehiko IMRAM, Tohoku University

Remarkable Enhancement of RNaseH Cleavage Activities of RNA Complexed with Peptide Ribonucleic Acid (PRNA) – DNA Chimera for Effective Cancer Cell

Specific Oligonucleotide Therapeutics

115 Whisenand Josh TriLink Biotechnologies Considerations for the Design and cGMP Manufacturing of mRNA Therapeutics

116 Wilds Christopher Concordia University Influence of O4-Alkylthymidine Lesion Orientation on Replication by Human DNA Polymerase η

117 Woodward Caroline Biogen, IncApplication of Liposomal Formulation for the Delivery of

Antisense Oligonucleotide into Brain after Intrathecal Administration

118 Woodward Caroline Biogen, Inc

Development of a Robust, Sensitive and Selective Liquid Chromatography High Resolution Mass Spectrometry Method for the Quantitation of

Encapsulated Antisense Oligonucleotide in Rat Brain after Intrathecal Administration

119 Wu Karen Lucerna, Inc. SpinachTM Splice Sensor: A Fluorescent Drug Screening Platform

120 Yavin Eylon The Hebrew University of Jerusalem Peptide Nucleic Acids (PNAs) as Diagnostic Molecules for the Early Detection of Cancer

13th Annual Meeting of the Oligonucleotide Therapeutics Society Poster Presentation Index

Every effort has been made to ensure the information contained here is accurate, although some changes may occur.

Poster # Last Name First Name Affiliation Abstract Title

121 Yoon Sorah Beckman Research Institute of City of Hope Blind SELEX to discover biomarkers and therapeutic aptamers

122 Yoshida Tokuyuki National Institute of Health Sciences Evaluation of Off-target Effects of Splice-switching Oligonucleotides

123 Zarrouki Faouzi Université Versailles St-Quentin Cerebral therapeutic exon skipping in the mdx mouse using tricyclo-DNA antisense oligonucleotides

124 Zhang Jason WAVE Life Sciences Pharmacologic Properties of Stereopure Oligonucleotides

125 Zhou Haiyan University College London

Gapmer antisense oligonucleotides selectively suppress the mutant allele of COL6A3 gene and

restore functional protein production in dominant Ullrich congenital muscular dystrophy

126 Zhou Jiehua Beckman Research Institute of City of Hope Targeting HIV reservoirs with cell-specific aptamer-based conjugates

127 Wood Matthew University of Oxford

Preclinical Studies of WVE-210201, an Investigational Stereopure Antisense Oligonucleotide for the

Treatment of Patients with Duchenne Muscular Dystrophy Amenable to Exon 51 Skipping

Heterogeneous Pyrophosphate Linked DNA-Oligonucleotides: Aversion for DNA but Affinity for RNA Brooke A. Anderson, Ramanarayanan Krishnamurthy The Scripps Research Institute Pyrophosphate linkages are important in extant biology and are hypothesized to have played a role in prebiotic chemistry and in the origination of oligonucleotides. Inspired by the possible role of pyrophosphate as backbones of primordial oligomers, we synthesized DNA oligomers with varying amounts of pyrophosphate inserts (ppDNA), as a model system, and investigated their base-pairing properties. With increasing amounts of pyrophosphate inserts in the backbone the thermal stability of ppDNA-DNA duplexes was compromised. In contrast, the ppDNA-RNA duplex exhibited, remarkably, duplex stability even with accumulation of pyrophosphate linkages. The favorable accommodation of a pyrophosphate linkage within a ppDNA-RNA (vis-à-vis a ppDNA-DNA) duplex structure seems to be a consequence of the higher inclination of the base-pair-axis with respect to the backbone in RNA (A-form), as compared to DNA (B-form). These results suggest that pyrophosphate linkages could function as alternative backbone linkers for similarly inclined systems such as RNA. Brooke A. Anderson, Ph.D Postdoctoral Research Associate The Scripps Research Institute 10550 North Torrey Pines Rd La Jolla, CA USA brookea@scripps.edu

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Aptamers for the FGF Receptor Family to Modulate Activation of Astrocytes Nachiket Kamatkar1, Wenfei Kang1,2, Sayan Nandi1,2, Matthew Levy3, Jean Hebert1,2

1. Dominick P. Purpura Department of Neuroscience 2. Department of Genetics 3. Department of Biochemistry Astrogliosis is the process by which astrocytes become activated after brain injury. Astrocyte activation takes place after virtually any insult to the central nervous system including trauma, infection, and cell death. It is a complex and graded process that relies on multiple cellular signals for not only astrocyte activation but also to inactivate astrocytes. In severe instances, astrocyte activation can result in the formation of a glial scar that can be both beneficial in terms of restricting inflammation and detrimental since it inhibits axon regeneration. Recently, our lab has discovered that the fibroblast growth factor (FGF) signaling pathway plays a critical role in the inhibition of astrocyte activation in the normal and injured brain. Moreover, we have also demonstrated that both gain-of-function and loss-of-function studies of FGF signaling result in a decrease in glial scar size. The FGFs are a family of secreted proteins used for angiogenesis, wound healing, and multiple other roles in the developing organism and also play an important role in numerous pathological processes. To date, there are limited options for people who suffer from FGF signaling related diseases since there are no specific agonists or antagonists for the receptors of FGF signaling. I will develop novel molecular tools specific for all three fibroblast growth factor receptors (FGFRs) expressed in the brain and ultimately test them as therapeutics in glial scar formation. To achieve this goal, I will develop aptamers, nucleic acid ligands that have the potential to be specific antagonists and agonists to modulate signaling through the FGFRs. To date, I have identified a number of nuclease stabilized ligands to FGFR3. One aptamer specifically, NK01, demonstrates high affinity for FGFR3 and inhibits FGF2 from binding FGFR3. Another aptamer, C20, stimulates signaling from FGFR3 when presented to the cells as a multimer. I am currently testing these and other aptamers for specificity to FGFR3 using a neurosphere model. In following experiments, I will characterize these molecules further and then systemically/locally deliver the drugs to the injured cortex to modulate astrocyte activation. Nachiket Kamatkar, MSc Graduate Student Albert Einstein College of Medicine 230 Kennedy Center 1410 Pelham Pkwy S Bronx, NY 10461 USA nachiket.kamatkar@phd.einstein.yu.edu 574.303.8441

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Aptamers for the FGF Receptor Family to Modulate Activation of Astrocytes Nachiket Kamatkar1, Wenfei Kang1,2, Sayan Nandi1,2, Matthew Levy3, Jean Hebert1,2

1. Dominick P. Purpura Department of Neuroscience 2. Department of Genetics 3. Department of Biochemistry Astrogliosis is the process by which astrocytes become activated after brain injury. Astrocyte activation takes place after virtually any insult to the central nervous system including trauma, infection, and cell death. It is a complex and graded process that relies on multiple cellular signals for not only astrocyte activation but also to inactivate astrocytes. In severe instances, astrocyte activation can result in the formation of a glial scar that can be both beneficial in terms of restricting inflammation and detrimental since it inhibits axon regeneration. Recently, our lab has discovered that the fibroblast growth factor (FGF) signaling pathway plays a critical role in the inhibition of astrocyte activation in the normal and injured brain. Moreover, we have also demonstrated that both gain-of-function and loss-of-function studies of FGF signaling result in a decrease in glial scar size. The FGFs are a family of secreted proteins used for angiogenesis, wound healing, and multiple other roles in the developing organism and also play an important role in numerous pathological processes. To date, there are limited options for people who suffer from FGF signaling related diseases since there are no specific agonists or antagonists for the receptors of FGF signaling. I will develop novel molecular tools specific for all three fibroblast growth factor receptors (FGFRs) expressed in the brain and ultimately test them as therapeutics in glial scar formation. To achieve this goal, I will develop aptamers, nucleic acid ligands that have the potential to be specific antagonists and agonists to modulate signaling through the FGFRs. To date, I have identified a number of nuclease stabilized ligands to FGFR3. One aptamer specifically, NK01, demonstrates high affinity for FGFR3 and inhibits FGF2 from binding FGFR3. Another aptamer, C20, stimulates signaling from FGFR3 when presented to the cells as a multimer. I am currently testing these and other aptamers for specificity to FGFR3 using a neurosphere model. In following experiments, I will characterize these molecules further and then systemically/locally deliver the drugs to the injured cortex to modulate astrocyte activation. Nachiket Kamatkar, MSc Graduate Student Albert Einstein College of Medicine 230 Kennedy Center 1410 Pelham Pkwy S Bronx, NY 10461 USA nachiket.kamatkar@phd.einstein.yu.edu 574.303.8441

Safety of chronic Kras ASO knockdown Patrik Andersson1, Stephanie Klein1, Stefan Öhlin1, Anna Björnson-Granqvist2, Lena William-Olsson2, Barbro Basta2, Christine Ahlström2, Alex Bell3, Alexey Revenko3, Rob MacLeod3, Mark Anderton1, 2 Drug Safety and Metabolism1, CVMD IMED2, AstraZeneca R&D and Ionis Pharmaceuticals3

The Ras family of GTPases are central signal mediators of many cellular signaling pathways. Together with Ionis Pharmaceuticals, the highly selective Kras antisense oligo nucleotide (ASO) AZD4785 was identified and is now in Phase 1 for oncology indications. Six weeks toxicity studies in mice and monkeys showed a good safety profile. Published data using an ASO against Kras showed promising effects in pre-clinical models of acute kidney injury. Given its central role in signaling, would a Kras ASO approach be safe for prolonged use treating chronic kidney disease? Kras knockout mice show cardiac malformations, anemia and increased apoptosis in liver and CNS. Using subcutaneous injections, we performed a 3 months toxicity study (30 and 100 mg/kg/week) in Balb/c mice using a mouse active Kras ASO with histopathological analysis of 13 tissues, plasma chemistry and hematology. At 3 months termination, liver and kidney Kras mRNA were reduced by 98% and 81% respectively. Despite this significant mRNA reduction, the only noticeable finding was a mild effect on hematopoiesis. This indicates a favorable on-target safety profile for chronic therapeutic use of ASOs targeting Kras.

Patrik Andersson, PhD, ERT Principle Scientist Drug Safety and Metabolism AstraZeneca R&D Gothenburg Pepparedsleden 1 431 50 Mölndal Sweden Patrik.J.Andersson@astrazeneca.com

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Physico-chemical studies of diastereomeric oligonucleotides (Rp and Sp) modified with phosphoryl guanidine group Valeriia S. Apukhtina1,2, Andrey V. Shernuykov1,3, Alexander A. Lomzov1,2, Maxim S. Kupryshkin2, Dmitrii V. Pyshnyi1,2 Novosibirsk State University, Novosibirsk, Russia1, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia2, Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of the Sciences, Novosibirsk, Russia3 Oligonucleotides with neutral-charged groups, such as phosphoryl guanidine (PG) oligonucleotides, can be a promise as therapeutics agents due to their improved cell uptake and biological stability. Recently it has been shown that PG oligonucleotide derivatives can be synthesized using a standard solid-phase phosphoramidite DNA protocol; thus, PG oligonucleotides can be easily applied in different areas of medicine and molecular biology. Addition of one PG group (1,3-dimethylimidazolidine-2-imino, see below) into oligonucleotide structure yields pair of diastereomers, which have different physico-chemical and biological properties. In current research we examined the properties of each diastereomer. Two DNA duplexes with diastereomeric oligonucleotide and native, complementary sequence were obtained. NMR-studies and molecular dynamic simulations of DNA duplexes allowed us to determine the configuration of a substituted phosphorus atom for two separated diastereomers. Enzymatic resistance experiments were carried out. Both of diastereomers demonstrate chemical stability and high binding affinity with DNA in a sequence-specific manner.

This work was supported by RFBR (No. 16-34-01219), RSF (No. 16-15-10238 to M.K.) Valeriia Apukhtina, Student Novosibirsk State University 2 Pirogov Street Novosibirsk, 630090 Russia valeriya.apukhtina@gmail.com

Topical Application of siRNA for Treatment of Hair Follicle Pathologies Hagit Ashush1, Shan Gao2, Sharon Avkin1, Hagar Kalinski1, Igor Spivak1, Anat Brafman1, Jinyu Huang2, Naidan Luo2, Hongyan Zhang2, Elena Feinstein1 Affiliation(s) 1 Quark Pharmaceuticals Inc, Fremont, CA/Ness Ziona, Israel; 2 Suzhou Ribo Life Sciences Co. Ltd, Kunshan, People’s Republic of China There are a wide variety of diseases (or conditions) associated with hair follicles, alopecia (e.g. male or female pattern or chemotherapy-induced hair loss) and acne vulgaris being the most common of them. Here we demonstrate the possibility of productive delivery of siRNA compounds into hair follicles following topical application on mouse skin in vivo as well as on human skin explants. The penetration of siRNA into various hair follicle substructures was confirmed using both fluorescence-labeled compounds and their detection by in situ hybridization on histological sections of mouse and human skin. In human scalp skin explants, topical treatment with siRNA produced significant dose-dependent up-to 60% target gene mRNA knockdown via the RNA interference mechanism as confirmed by RLM-RACE. Significant reduction of the target protein expression was demonstrated by both immunoblotting and immunohistochemistry analyses. Topically administered siRNA does not leak into systemic circulation regardless of whether the applications were single or repeated as shown both in vivo in mice and ex vivo in Franz diffusing cells (with human skin). No skin irritation following repeated topical applications of siRNA was observed either.

The efficacy of topical siRNA treatment in ameliorating hair loss was tested in the mouse model of chemotherapy-induced alopecia (CIA). The backs of C57BL/6 mice were depilated to synchronize hair growth in this area in anagen VI active hair shaft growth stage. At this stage, matrix keratinocytes become susceptible to cyclophosphamide-induced apoptosis that eventually leads to massive hair loss specifically in the previously depilated area. The mice received 4 topical applications of siRNA or vehicle on the depilated area on four consecutive days – two before and two after intraperitoneal cyclophosphamide injection. The results indicated that siRNA-treated mice were partially rescued from hair loss while demonstrating significantly accelerated (by 8-9 days) hair re-growth in the affected area compared to control. Altogether, the data support the possibility of development of oligonucleotide-based therapeutics (or cosmeceuticals) for treatment of hair follicle-associated conditions. Hagit Ashush, PhD VP Research Quark Pharmaceuticals Inc Weizmann Science Park, POB 4071 Ness Ziona, 70400 Israel hashush@quarkpharma.com +972-8-9305203

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Evaluation of the Phosphorothioate content within tricyclo-DNA splice-switching oligonucleotides in DMD mouse model Philippine Aupy1, Lucia Echevarria1, Karima Relizani², Florence Blandel1, Graziella Griffith², Fedor Svinartchouk², Branislav Dugovic3, Luis Garcia1 and Aurelie Goyenvalle1

1 Université de Versailles St-Quentin, UFR des Sciences de la Santé – INSERM U1179, France. ² SQY Therapeutics, Université de Versailles St-Quentin, France 3 SYNTHENA, University of Bern, Switzerland

Antisense Oligonucleotides (AONs) hold promise for therapeutic splice switching correction for genetic diseases and in particular for Duchenne Muscular Dystrophy (DMD), for which AON-exon skipping represents one of the most advanced therapeutic strategy. We have previously reported the therapeutic potential of tricyclo-DNA (tcDNA) in mouse models of DMD, highlighting their unique pharmaceutical properties and unprecedented uptake in many tissues after systemic delivery, including the heart and central nervous system. However, a typical phosphorothioate (PS) associated toxicity due to PS-tcDNA accumulation was also observed, albeit very mild. In this study, we investigate the influence of the content in PS linkages on exon skipping efficacy and toxicity. Mdx mice were injected intravenously once weekly for 4 weeks with tcDNA carrying various amounts of PS linkages (0, 25%, 33%, 50%, 66%, 83% and 100%). The results indicate that the level of exon 23 skipping and dystrophin rescue correlates directly and increases with the number of PS linkage in most skeletal tissues but not in the heart. Interestingly, the AONs-tcDNA containing 10PS (i.e. 83%) induces similar levels of exon 23 skipping compared to the full-PS counterpart. Moreover, C3 complement activation as well as coagulation studies show that the decrease in PS content minimizes the well-known PS-AON associated toxicity. Concurring with these results, we show that tcDNA - protein binding in serum is directly proportional to the number of PS linkages on the tcDNA backbone. Altogether these data contribute to establish the appropriate sulphur content within tcDNA backbone for maximal efficacy and minimal toxicity of the oligonucleotide.

Philippine Aupy PhD student University of Versailles Saint Quentin UFR des Sciences de la santé 78180 Montigny le Bretonneux France Email : philippine.aupy2@uvsq.fr

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Evaluation of the Phosphorothioate content within tricyclo-DNA splice-switching oligonucleotides in DMD mouse model Philippine Aupy1, Lucia Echevarria1, Karima Relizani², Florence Blandel1, Graziella Griffith², Fedor Svinartchouk², Branislav Dugovic3, Luis Garcia1 and Aurelie Goyenvalle1

1 Université de Versailles St-Quentin, UFR des Sciences de la Santé – INSERM U1179, France. ² SQY Therapeutics, Université de Versailles St-Quentin, France 3 SYNTHENA, University of Bern, Switzerland

Antisense Oligonucleotides (AONs) hold promise for therapeutic splice switching correction for genetic diseases and in particular for Duchenne Muscular Dystrophy (DMD), for which AON-exon skipping represents one of the most advanced therapeutic strategy. We have previously reported the therapeutic potential of tricyclo-DNA (tcDNA) in mouse models of DMD, highlighting their unique pharmaceutical properties and unprecedented uptake in many tissues after systemic delivery, including the heart and central nervous system. However, a typical phosphorothioate (PS) associated toxicity due to PS-tcDNA accumulation was also observed, albeit very mild. In this study, we investigate the influence of the content in PS linkages on exon skipping efficacy and toxicity. Mdx mice were injected intravenously once weekly for 4 weeks with tcDNA carrying various amounts of PS linkages (0, 25%, 33%, 50%, 66%, 83% and 100%). The results indicate that the level of exon 23 skipping and dystrophin rescue correlates directly and increases with the number of PS linkage in most skeletal tissues but not in the heart. Interestingly, the AONs-tcDNA containing 10PS (i.e. 83%) induces similar levels of exon 23 skipping compared to the full-PS counterpart. Moreover, C3 complement activation as well as coagulation studies show that the decrease in PS content minimizes the well-known PS-AON associated toxicity. Concurring with these results, we show that tcDNA - protein binding in serum is directly proportional to the number of PS linkages on the tcDNA backbone. Altogether these data contribute to establish the appropriate sulphur content within tcDNA backbone for maximal efficacy and minimal toxicity of the oligonucleotide.

Philippine Aupy PhD student University of Versailles Saint Quentin UFR des Sciences de la santé 78180 Montigny le Bretonneux France Email : philippine.aupy2@uvsq.fr

Delivery of nucleic acids and anticancer drugs using Lipid-Oligonucleotides technology Sébastien BENIZRI1, Arnaud GISSOT1, Brune VIALET1, Sara KARAKI2, Palma ROCCHI2, Philippe BARTHELEMY1 1ARN : régulations naturelle et artificielle INSERM U1212, UMR5320 CNRS - Université de Bordeaux, 33076 Bordeaux - France

2 Centre de Recherche en Cancérologie de Marseille (CRCM) - Institut Paoli-Calmettes, Aix Marseille Université, INSERM U1068, UMR7258 CNRS - 13009 Marseille – France

Bio-inspired amphiphilic molecules like lipid conjugated nucleotides or oligonucleotides are a promising tool for drug delivery. Indeed, these molecules can self-assemble in aqueous media to form nano-objects, which can then be uptaken by cells. Nucleolipids can vectorise silencing systems like antisenses or siRNAs. Moreover, lipid chains of the nucleolipids create a lipophilic environment and allow the formulation of hydrophobic anticancer drugs. The physico-chemical properties and / or colloidal stability of nucleolipids based nano-system can be modulated to fit various applications. For example, their structure can be turn from liposomes to lamellar phases depending of pH due to the orthoester linkage between the nucleotides and the lipid chains. In contrast to nucleolipids, the conjugation of lipid chains on oligonucleotides displays smaller particles organized in micelles. The micellar core offers high opportunity to encapsulate hydrophobic compounds. Furthermore, antisense sequences of lipid-oligonucleotides confer dual activity in pathologies. The presence of the hydrophobic part does not affect antisense activities. To conclude, amphiphilic molecules based on nucleotide sequence or a derivative of nucleotide allowed to vectorise a multitude of hydrophobic or hydrophilic molecules without toxicity. In perspective, this nucleolipids / Lipid-OligoNucleotides platform could be loaded with fluorescent molecules or contrast agent together with the therapeutic activity of antisense to create a theranostic platform. Sébastien BENIZRI, PhD Student ChemBioPharm, INSERM U1212 ARN Régulation Naturelle et Artificielle 146 rue Léo Saignat 33076 Bordeaux FRANCE sebastien.benizri@inserm.fr Tel: 06 11 01 15 01

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Generation 2.5 Constrained Ethyl Antisense Oligonucleotide Renal Sub-Organ Pharmacodynamics in the Healthy Rodent Kidney Emily Brand, Emily Brand, Aaron Donner, Rosanne Crooke, Mark Graham, and Thomas Bell Ionis Pharmaceuticals Inc. In healthy subjects, the liver and kidney are the principal sites of antisense oligonucleotide (ASO) accumulation. This preferential distribution of ASOs to the liver has enabled the development of an array of antisense therapeutics for the treatment of many cardio-metabolic disorders. Drug discovery in the kidney presents additional challenges due to the structural complexity of the nephron and the increased diversity of cell types. In order to help facilitate the development of ASOs for the treatment of renal diseases, we have performed detailed sub-organ pharmacodynamic analyses of generation 2.5 3-10-3 constrained ethyl (cEt) gapmers in healthy mouse kidney. A single injection, dose-response study was performed in naive FVB mice that were administered ASOs targeting the long, non-coding RNA, MALAT-1. Target reduction in the whole kidney was determined by rt-PCR 72 hours post injection. MALAT-1 RNA was reduced by 81, 70, and 68% after administration of 67, 22, and 7 mg/kg of drug, respectively. To evaluate ASO activity in key regions of the kidney, glomerular, cortical, and medullary samples were collected by laser-capture microdissection from mice administered either PBS or a 67 mg/kg dose of the MALAT-1 ASO. Analysis of these samples revealed that the decrease in MALAT-1 RNA was the greatest in the cortex, at 75%, and less significant in glomeruli (42%) and medulla (58%). Additionally, ASO distribution and activity were evaluated by immunohistochemistry and in situ hybridization (ISH) assays. Using these techniques, the cortex, specifically the proximal convoluted tubule, was the primary site of ASO accumulation and activity in the healthy kidney, confirming data from previous reports. Since glomerular damage and dysfunction is a crucial step in the pathogenesis of many types of glomerulonephritis (GN), we sought to determine efficacy in the different cell types that comprise glomeruli. To do so, we utilized an ISH fluorescent multiplex assay with probes for MALAT-1, along with podocyte and mesangial cell-specific markers. Results from this experiment revealed ASO mediated reductions in both key glomerular cell types. Due to the broad efficacy of ASOs observed across a majority of renal cell types, these observations set the stage for future in-depth experiments to compare and contrast PK/PD in both normal and diseased kidney states. Emily Brand, M.S. Senior Research Associate Ionis Pharmaceuticals 2855 Gazelle Ct Carlsbad, CA 92010 USA ebrand@ionisph.com

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Generation 2.5 Constrained Ethyl Antisense Oligonucleotide Renal Sub-Organ Pharmacodynamics in the Healthy Rodent Kidney Emily Brand, Emily Brand, Aaron Donner, Rosanne Crooke, Mark Graham, and Thomas Bell Ionis Pharmaceuticals Inc. In healthy subjects, the liver and kidney are the principal sites of antisense oligonucleotide (ASO) accumulation. This preferential distribution of ASOs to the liver has enabled the development of an array of antisense therapeutics for the treatment of many cardio-metabolic disorders. Drug discovery in the kidney presents additional challenges due to the structural complexity of the nephron and the increased diversity of cell types. In order to help facilitate the development of ASOs for the treatment of renal diseases, we have performed detailed sub-organ pharmacodynamic analyses of generation 2.5 3-10-3 constrained ethyl (cEt) gapmers in healthy mouse kidney. A single injection, dose-response study was performed in naive FVB mice that were administered ASOs targeting the long, non-coding RNA, MALAT-1. Target reduction in the whole kidney was determined by rt-PCR 72 hours post injection. MALAT-1 RNA was reduced by 81, 70, and 68% after administration of 67, 22, and 7 mg/kg of drug, respectively. To evaluate ASO activity in key regions of the kidney, glomerular, cortical, and medullary samples were collected by laser-capture microdissection from mice administered either PBS or a 67 mg/kg dose of the MALAT-1 ASO. Analysis of these samples revealed that the decrease in MALAT-1 RNA was the greatest in the cortex, at 75%, and less significant in glomeruli (42%) and medulla (58%). Additionally, ASO distribution and activity were evaluated by immunohistochemistry and in situ hybridization (ISH) assays. Using these techniques, the cortex, specifically the proximal convoluted tubule, was the primary site of ASO accumulation and activity in the healthy kidney, confirming data from previous reports. Since glomerular damage and dysfunction is a crucial step in the pathogenesis of many types of glomerulonephritis (GN), we sought to determine efficacy in the different cell types that comprise glomeruli. To do so, we utilized an ISH fluorescent multiplex assay with probes for MALAT-1, along with podocyte and mesangial cell-specific markers. Results from this experiment revealed ASO mediated reductions in both key glomerular cell types. Due to the broad efficacy of ASOs observed across a majority of renal cell types, these observations set the stage for future in-depth experiments to compare and contrast PK/PD in both normal and diseased kidney states. Emily Brand, M.S. Senior Research Associate Ionis Pharmaceuticals 2855 Gazelle Ct Carlsbad, CA 92010 USA ebrand@ionisph.com

Recycling of Nucleosides and Reagents from Oligonucleotide Synthesis Wolfgang K.-D. Brill, Chemistry Consultant Therapeutic oligonucleotides often need modified nucleotide components to improve their ADME properties and improve their efficacy and potency. Particularly sugar and base modifications often require multistep partial or total synthesis of the building blocks rendering them significantly more expensive than naturally occurring building blocks. In turn, chemical solid phase synthesis of oligonucleotides requires access of nucleotide building blocks to assure almost quantitative coupling yields. Access of building blocks assures a pseudo first order reaction kinetics and efficient quenching of reactive impurities on the solid support. During large-scale synthesis of modified oligonucleotides, wasting nucleotide becomes a cost factor. I present a method for the recycling of nucleotides and reagents to compensate this problem, enabling a more economical use of modified nucleotides and rendering the production of more efficacious oligonucleotide drugs possible. This poster presents some chemical methods for recycling and a 5'-protective group facilitating its recovery. An example flow diagram of a recycling process is shown below.

Wolfgang K.-D. Brill PhD Feststraße 2 D-63165 Mühlheim am Main Germany E-mail: w.brill@hotmail.com Tel. +49 (0) 15253389997

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Mechanistic Insights and Progress on the GalNAc-siRNA Conjugate Platform for Targeted Delivery of RNAi Therapeutics to the Liver Christopher R. Brown Alnylam Pharmaceuticals, Cambridge, MA 02142, USA The past few years have seen major advances toward achieving safe and effective siRNA delivery, moving the field closer to the realization of RNAi-based therapies. We have developed a siRNA conjugate platform to achieve targeted delivery to hepatocytes utilizing a synthetic multivalent N-acetylgalactosamine (GalNAc) ligand. Progress in design features and chemistry has led to GalNAc-Enhanced Stabilization Chemistry (ESC) siRNA conjugates, which have demonstrated potent and durable activity against multiple liver targets in investigational clinical studies across multiple programs. Ongoing efforts to maximize the potential therapeutic benefit of GalNAc-siRNA conjugates focus on improving our mechanistic understanding of the interaction of conjugates with the RNAi pathway and the effects of chemical modifications on conjugate stability, potency and duration of effect by examining liver exposure, intracellular trafficking, Ago2 loading and target gene silencing. Christopher R. Brown, PhD Scientist, Research Alnylam Pharmaceuticals 300 Third Street Cambridge, MA 02142 USA cbrown@alnylam.com

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Mechanistic Insights and Progress on the GalNAc-siRNA Conjugate Platform for Targeted Delivery of RNAi Therapeutics to the Liver Christopher R. Brown Alnylam Pharmaceuticals, Cambridge, MA 02142, USA The past few years have seen major advances toward achieving safe and effective siRNA delivery, moving the field closer to the realization of RNAi-based therapies. We have developed a siRNA conjugate platform to achieve targeted delivery to hepatocytes utilizing a synthetic multivalent N-acetylgalactosamine (GalNAc) ligand. Progress in design features and chemistry has led to GalNAc-Enhanced Stabilization Chemistry (ESC) siRNA conjugates, which have demonstrated potent and durable activity against multiple liver targets in investigational clinical studies across multiple programs. Ongoing efforts to maximize the potential therapeutic benefit of GalNAc-siRNA conjugates focus on improving our mechanistic understanding of the interaction of conjugates with the RNAi pathway and the effects of chemical modifications on conjugate stability, potency and duration of effect by examining liver exposure, intracellular trafficking, Ago2 loading and target gene silencing. Christopher R. Brown, PhD Scientist, Research Alnylam Pharmaceuticals 300 Third Street Cambridge, MA 02142 USA cbrown@alnylam.com

Defined Multimeric Oligonucleotides for Enhanced Therapeutic Effect Jonathan Miles Brown1, James Dahlman1, Kristin Neuman1, Carla Prata2, Monika Krampert2, Philipp Hadwiger2, and Hans-Peter Vornlocher2 1MPEG LA, LLC; 2Axolabs GmbH, Kulmbach, Germany Modulating gene expression using therapeutic oligonucleotides has the potential to improve disease treatment and in vivo studies of complex biological processes. However, it remains difficult to i) to target several genes at once, and ii) to functionally deliver oligonucleotides to a particular cell or tissue type. To address both issues, we developed a method to manufacture defined “multimeric” oligonucleotides, where a defined number of oligonucleotides (e.g., 2 or more siRNAs) are conjugated to a single targeting ligand. The multimeric oligonucleotides are synthesized via a novel chemical intermediate that provides advantages over current methods of chemical linking and solid phase synthesis. The linking methodology is robust. It is compatible with a wide range of targeting ligands such as peptides, carbohydrates and aptamers, and can be used to deliver any combination of siRNAs, shRNAs, micro-RNAs, and ASOs. We previously demonstrated that the in vivo potencies of a series of GalNAc-conjugated “homodimers” of the same siRNA (two identical siRNAs linked together and conjugated to GalNAc) prepared via this approach were approximately double that of the corresponding single GalNAc siRNA monomer per mole of GalNAc ligand. We further demonstrated that the bioactivity of each siRNA in a GalNAc-conjugated “heterotrimer” of siRNAs targeting three separate mRNAs was essentially equal to that of a pool of the corresponding GalNAc-conjugated siRNA monomers. Taken together, these data indicate that the bioavailability of oligonucleotides can be improved by increasing the number of oligonucleotides delivered per targeting ligand. Experiments are now underway to further increase siRNA bioavailability by increasing the size of the multimeric siRNAs such that serum half-life and target tissue availability is significantly increased, due to reduced glomerular filtration by the kidney. This approach will be particularly relevant for cell-targeting ligand systems other than GalNAc where receptor biology, safety, and/or cost are limiting factors Jonathan Miles Brown, PhD FRSC Biotechnology Licensing MPEG LA, LLC 5425 Wisconsin Avenue, Suite 801 Chevy Chase, MD 20815 USA jmbrown@mpegla.com +1.240.406.2746

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Targeting multiple drug resistance gene with small interfering RNAs E. L. Chernolovskaya, I. V. Chernikov, O. V. Gvozdeva, D. V. Gladkih, M. I. Meschaninova, A. G. Ven`yaminova, M. A. Zenkova, V. V. Vlassov Institute of Chemical Biology and Fundamental Medicine SB RAS

Challenges associated with nuclease stability, selecting effective sequences and achieving efficient delivery to target cells and tissues have significantly limited the biomedical applications of siRNA. Many of these limitations could be resolved with the use of chemical modifications improving the siRNA properties.

To prevent rapid degradation of siRNA we proposed an experimental algorithm for the site-specific modification of siRNAs based on the mapping of their nuclease-sensitive motives in the presence of serum followed by the incorporation of 2′-O-methyl analogs of ribonucleotides at the identified positions of cleavage. Modification of all nuclease sensitive sites protects siRNA from degradation in the presence of serum and significantly prolonged the duration of the silencing effect of the siRNA compared to non-modified, partially modified, or randomly modified siRNA of the same sequence.

Modification of siRNA duplex structure could be successfully used to increase the silencing activity and to hit difficult-to-silence targets. Favorable thermodynamic asymmetry of the duplex can be achieved by incorporation of mismatches into the 3’ part of the sense strand, providing fork-siRNAs with higher silencing activity, whereas chemical modification protects imperfect duplexes from accelerated nuclease degradation.

Novel type of interfering RNAs - trimeric-siRNA (tsiRNA) was proposed and studied. Selectively modified 63 bp tsiRNAs induced more effective RNAi at lower concentrations than classical 21 bp siRNA and act in a Dicer independent mode. This tsiRNA causes no undesirable side effects on immune system. These results remove the length limits for the design of RNAi effectors.

The conjugation of siRNA to the molecules, which can be internalized into the cell by natural transport mechanisms, is a promising approach for the delivery of siRNA into the cells. We show that the length of the linker between siRNA and lipophilic moiety is critical for their cellular accumulation and biological activity: for all studied conjugates, siRNA with longer linker display better accumulation. Chemically modified siRNAs conjugated with lipophilic molecules via specially designed linkers are able to enter cancer cells in a carrier-free mode and to silence the expression of target gene. The monitoring of biodistrubution of lipophilic siRNA in mice revealed enhanced accumulation of lipophilic siRNA in lungs, liver, kidney and KB-8-5 tumor as compared with canonical siRNA and their complexes with transfection reagents. Lipophilic siRNA silence the expression of MDR1 gene in vivo and increase the efficiency of the therapy of drug resistant tumors. The combination of these approaches can lead to the development of siRNAs with therapeutic value.

This work was supported by the Russian Scientific Foundation, grant # 14-14-00697.

Elena L. Chernolovskaya, Doctor of Sci. Principal Investigator Institute of Chemical biology and Fundamental Medicine SB RAS Lavrentiev ave., 8, Novosibirsk, 630090, Russia elena_ch@niboch.nsc.ru

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Novel oligonucleotides for the therapy of Myotonic Dystrophy Melina Christou1, Jesper Wengel2, Nikolaos Mastroyiannopoulos1, Leonidas Phylactou1 1Department of Molecular Genetics, Function and Therapy, The Cyprus Institute of Neurology and Genetics, 2Department of Physics, Chemistry and Pharmacy, University of Southern Denmark Myotonic dystrophy type 1 (DM1) is a slowly progressive, multisystemic disorder affecting primarily skeletal muscle tissue. The root cause of the disease is a CTG repeat expansion in the 3ʹ′UTR of the DMPK gene, which leads to the expression of a toxic gain-of-function RNA. The expanded CUG region in the mutant DMPK mRNA adopts a stable hairpin structure that recruits the splicing regulator muscleblind-like-1 (MBNL1) forming large, nuclear-retained ribonucleoprotein (RNAexp-MBNL1) foci. MBNL1 sequestration leads to its functional depletion in the nucleus and cytoplasm causing missplicing of its own pre-mRNA and other regulatory targets. The most promising approach towards DM1 therapy is the application of chemically modified antisense oligonucleotides (ASOs) that directly target the expanded CUG repeats in the mutant DMPK mRNA and mitigate their toxic effects. Several studies have tested the efficacy of different chemically modified ASOs to restore muscle function in DM1 mouse models. We report the first use of CAG repeat ASOs that incorporate two commonly used, high binding affinity modifications in a single antisense construct: the locked nucleic acid (LNA) chemistry, which exhibits unprecedented duplex stability, and the nontoxic, naturally occurring 2ʹ′-O-Methyl (2ʹ′OMe) chemistry, which shows high RNA binding affinity and inherent resistance to endonuclease degradation. We hypothesize that the combination of 2ʹ′OMe monomers with a limited number of LNA nucleotides will enhance ASO potency and minimize potential toxicity associated with the LNA chemistry. We designed LNA/2ʹ′OMe CAG repeat chimeras, varying in size and/or the number of incorporated LNA monomers, and tested their ability in mitigating CUGexp-mediated toxicity in proliferating human DM1 myoblasts. Our results demonstrate that LNA/2ʹ′OMe chimeras were able to almost completely eliminate nuclear RNAexp-MBNL1 foci in human DM1 myoblasts, without triggering degradation of mutant DMPK mRNA. Furthermore, LNA/2ʹ′OMe chimeras, exhibiting a high LNA distribution, significantly ameliorated MBNL1 exon 7 missplicing in human DM1 myoblasts compared to control LNA/DNA ASOs, suggesting that the combination of LNA and 2ʹ′OMe monomers in a single antisense construct greatly enhances ASO potency. We conclude that LNA/2ʹ′OMe mixmers show increased potency ex vivo compared to LNA-containing ASOs. Preliminary experiments to assess the efficacy of these ASOs after intramuscular delivery in DM1 transgenic mice have yielded some promising results. The main objective is to identify the most potent ASO and assess its biodistribution, safety and efficacy after systemic delivery in DM1 mice. Enhanced cellular uptake and increased ASO potency will potentially translate into lower therapeutic doses and reduced cost. Melina Christou PhD Student The Cyprus Institute of Neurology and Genetics | The Cyprus School of Molecular Medicine 6 International Airport Avenue, P.O. Box 23462 1683 Nicosia Cyprus christoum@cing.ac.cy

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Delivering Gene Silencing Therapy to the Epidermis in an Ex Vivo Model Michael J Conneely, Sylvain Roque, Yu-En Cheah, WH Irwin McLean, Robyn P Hickerson School of Life Science, University of Dundee, Dundee Scotland Despite great advances in identifying causative genes, the development of therapies for hereditary skin diseases remains slow, particularly in the more common dominant disorders where gene replacement therapy is not usually feasible. Gene silencing therapy, either in the form of antisense oligonucleotides (ASOs) or small interfering RNA (siRNA), represents a promising therapeutic avenue for the treatment of dominant-negative genetic disorders. In some conditions, where gene redundancy has been demonstrated, it should be possible to silence the mutated gene completely to achieve therapy. In other cases, where there is no gene redundancy, siRNAs (more so than typical ASOs) have offered the possibility of potently and specifically silencing mutant alleles, allowing the wild-type allele to function normally. However, in vivo siRNA delivery has proved to be extremely challenging, especially to skin, which is the primary barrier tissue responsible for protecting the body from chemical, antigenic and microbial attack. Recent advances in antisense oligonucleotide chemistry show promise for greatly increased ASO potency and importantly, the targeting of point mutations. With this advance, the major remaining obstacle impeding the translation of oligonucleotide therapy for dermatological disorders into the clinic is a lack of effective in vivo delivery strategies. To address this need, we have developed an ex vivo skin culture system in which skin is cultured under physiological tension resulting in markedly increased viability over the current known state-of-the-art. Preliminary data suggest that this model system maintains the physiological complexity, the metabolic activity, and the structural integrity of all skin compartments (i.e. the epidermis and dermis) – all due to the specific tension at which the skin is cultured. We have developed potent ASOs against the keratin 10 gene, which is expressed in all suprabasal cells of the epidermis. Delivering these “systemically” in our ex vivo skin model (i.e., in the medium in contact with the dermis), 70-80% knockdown of keratin 10 mRNA is achieved. We also demonstrate topical delivery of these ASOs using a simple formulation (20% transcutol and 2% Tween-20), which results in similar knockdown. These results are highly reproducible, exhibit dose dependence, and the rank order of potency in ex vivo skin is identical to that seen in vitro. These preliminary data show great promise for developing effective ASO therapy for disorders caused by defects in suprabasal keratins, such as pachyonychia congenita (keratins 6, 16 and 17), epidermolytic ichthyosis (keratin 10) and epidermolytic palmar plantar keratoderma (keratin 9). Michael Conneely, PhD Postdoctoral Research Associate University of Dundee James Black Centre (level 2) Dundee, DD1 5EH UK M.Conneely@Dundee.ac.uk

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Delivering Gene Silencing Therapy to the Epidermis in an Ex Vivo Model Michael J Conneely, Sylvain Roque, Yu-En Cheah, WH Irwin McLean, Robyn P Hickerson School of Life Science, University of Dundee, Dundee Scotland Despite great advances in identifying causative genes, the development of therapies for hereditary skin diseases remains slow, particularly in the more common dominant disorders where gene replacement therapy is not usually feasible. Gene silencing therapy, either in the form of antisense oligonucleotides (ASOs) or small interfering RNA (siRNA), represents a promising therapeutic avenue for the treatment of dominant-negative genetic disorders. In some conditions, where gene redundancy has been demonstrated, it should be possible to silence the mutated gene completely to achieve therapy. In other cases, where there is no gene redundancy, siRNAs (more so than typical ASOs) have offered the possibility of potently and specifically silencing mutant alleles, allowing the wild-type allele to function normally. However, in vivo siRNA delivery has proved to be extremely challenging, especially to skin, which is the primary barrier tissue responsible for protecting the body from chemical, antigenic and microbial attack. Recent advances in antisense oligonucleotide chemistry show promise for greatly increased ASO potency and importantly, the targeting of point mutations. With this advance, the major remaining obstacle impeding the translation of oligonucleotide therapy for dermatological disorders into the clinic is a lack of effective in vivo delivery strategies. To address this need, we have developed an ex vivo skin culture system in which skin is cultured under physiological tension resulting in markedly increased viability over the current known state-of-the-art. Preliminary data suggest that this model system maintains the physiological complexity, the metabolic activity, and the structural integrity of all skin compartments (i.e. the epidermis and dermis) – all due to the specific tension at which the skin is cultured. We have developed potent ASOs against the keratin 10 gene, which is expressed in all suprabasal cells of the epidermis. Delivering these “systemically” in our ex vivo skin model (i.e., in the medium in contact with the dermis), 70-80% knockdown of keratin 10 mRNA is achieved. We also demonstrate topical delivery of these ASOs using a simple formulation (20% transcutol and 2% Tween-20), which results in similar knockdown. These results are highly reproducible, exhibit dose dependence, and the rank order of potency in ex vivo skin is identical to that seen in vitro. These preliminary data show great promise for developing effective ASO therapy for disorders caused by defects in suprabasal keratins, such as pachyonychia congenita (keratins 6, 16 and 17), epidermolytic ichthyosis (keratin 10) and epidermolytic palmar plantar keratoderma (keratin 9). Michael Conneely, PhD Postdoctoral Research Associate University of Dundee James Black Centre (level 2) Dundee, DD1 5EH UK M.Conneely@Dundee.ac.uk

Targeting DNA damage response pathways with antisense oligonucleotides Laura Croft1, Didier Boucher1, Sam Beard1, Ken O’Byrne1,2 and Derek Richard1 1Cancer and Ageing Research Program, Queensland University of Technology, Faculty of Health, School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Translational Research Institute, Brisbane, QLD, Australia 2Medical Oncology Department, Princess Alexandra Hospital, Brisbane, QLD, Australia Cancer is one of the biggest clinical problems facing the world. It has been estimated that by 2030 half of all deaths worldwide will be from cancer. An enabling characteristic of cancer development is genomic instability, which can be caused by accumulation of DNA damage due to dysregulation of DNA damage repair pathways or defective cell cycle checkpoint signaling. Genome stability pathways offer one of the most promising areas of therapeutic intervention, indeed most chemotherapeutics function by causing DNA damage. More recently, specific inhibitors of key proteins in the DNA damage response (DDR) pathways have shown promising results with not only the potential to sensitize cells to these therapies, but also as single agent therapies. Using antisense technology, we have been able to inhibit an essential factor in the DDR pathway. We show that its depletion results in cancer cell death in vitro by increased H2AX phosphorylation and genomic fragmentation. We performed tumour xenograft models using metastatic prostate cancer cells (C4-2B) and lung cancer cells (H460) and in both models we observed a significantly lower tumour volume during a four-week treatment with antisense oligonucleotides (80 mg/kg) compared to PBS vehicle control. We are currently performing an in vivo dose escalation study and investigating the effect of our oligonucleotides on the immune system in human peripheral blood monocytes. These studies demonstrate that our antisense oligonucleotides may have potential as broad-spectrum cancer therapeutics. Laura Croft, PhD Title: Dr Queensland University of Technology, Faculty of Health, School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Translational Research Institute Level 6, 37 Kent Street, Woolloongabba, QLD, 4102, Australia Email: laura.croft@qut.edu.au Phone: +61415560686

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miR-Direct®: A Sensitive and Specific Method for Quantification of miRNAs Directly from Plasma and other Biofluids Anne Dallas, Colleen McLaughlin, Heini Ilves, Ryan E. Hogans, Catharina Casper-Lindley, Brian H. Johnston & Sergei A. Kazakov SomaGenics, Inc., Santa Cruz, CA, United States Circulating microRNAs (miRNAs) have great potential as biomarkers, but current methods for their accurate quantification are impeded by inefficient and inconsistent miRNA purification from biofluids, the very low concentrations of miRNAs in biofluids, and the difficulty in eliminating inhibitors of PCR reactions that co-purify with RNA. We have developed a novel method, called miR-Direct® for purification, concentration, and quantification of miRNAs from biofluid samples by RT-qPCR while avoiding procedures such as organic extraction, ethanol precipitation, and column adsorption/elution procedures that are known to produce inconsistencies in miRNA quantification. Samples are first treated to release miRNAs from their protein and lipid-containing complexes and vesicles while protecting them from degradation by RNases. A spike-in miRNA is added to serve as a positive control and a marker of experimental variability. Next, miRNAs of interest are hybridized in multiplex with miRNA-specific probes in solution (targeted capture). These hybridized complexes are then captured and concentrated on magnetic beads and stringently washed to remove PCR inhibitors and all other undesired solutes. Finally, the captured miRNAs are eluted from the beads, circularized and quantified using SomaGenics’ miR-ID® RT-qPCR assays. As a result, miR-Direct® provides higher sensitivity than other methods, allowing reliable and reproducible quantification of miRNAs with very low abundance. In addition, miR-ID® is uniquely capable of discriminating miRNA isoforms and isomiRs that vary in sequence or length by as little as one nucleotide along any position of the miRNA sequence, outperforming other qPCR methods in this respect. Sample processing up to the reverse transcription step is performed in a single tube. miRNA levels can be measured regardless of whether the samples were collected in EDTA, citrate, or heparin, whereas most commercially available purification methods are incompatible with heparin-containing plasma. Here we present data showing the application of miR-Direct® for quantification of miRNAs in plasma, serum, whole blood, and urine. miR-Direct® may be used for validating miRNA sequencing data and biomarker candidates as well as for developing diagnostic (e.g. liquid biopsy) and prognostic applications for circulating miRNAs, including canonical and non-canonical miRNA forms such as isomiRs. Anne Dallas, Ph.D. Principal Scientist SomaGenics, Inc. 2161 Delaware Avenue Santa Cruz, CA 95060-5706 USA adallas@somagenics.com (831) 426-7700 x16

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miR-Direct®: A Sensitive and Specific Method for Quantification of miRNAs Directly from Plasma and other Biofluids Anne Dallas, Colleen McLaughlin, Heini Ilves, Ryan E. Hogans, Catharina Casper-Lindley, Brian H. Johnston & Sergei A. Kazakov SomaGenics, Inc., Santa Cruz, CA, United States Circulating microRNAs (miRNAs) have great potential as biomarkers, but current methods for their accurate quantification are impeded by inefficient and inconsistent miRNA purification from biofluids, the very low concentrations of miRNAs in biofluids, and the difficulty in eliminating inhibitors of PCR reactions that co-purify with RNA. We have developed a novel method, called miR-Direct® for purification, concentration, and quantification of miRNAs from biofluid samples by RT-qPCR while avoiding procedures such as organic extraction, ethanol precipitation, and column adsorption/elution procedures that are known to produce inconsistencies in miRNA quantification. Samples are first treated to release miRNAs from their protein and lipid-containing complexes and vesicles while protecting them from degradation by RNases. A spike-in miRNA is added to serve as a positive control and a marker of experimental variability. Next, miRNAs of interest are hybridized in multiplex with miRNA-specific probes in solution (targeted capture). These hybridized complexes are then captured and concentrated on magnetic beads and stringently washed to remove PCR inhibitors and all other undesired solutes. Finally, the captured miRNAs are eluted from the beads, circularized and quantified using SomaGenics’ miR-ID® RT-qPCR assays. As a result, miR-Direct® provides higher sensitivity than other methods, allowing reliable and reproducible quantification of miRNAs with very low abundance. In addition, miR-ID® is uniquely capable of discriminating miRNA isoforms and isomiRs that vary in sequence or length by as little as one nucleotide along any position of the miRNA sequence, outperforming other qPCR methods in this respect. Sample processing up to the reverse transcription step is performed in a single tube. miRNA levels can be measured regardless of whether the samples were collected in EDTA, citrate, or heparin, whereas most commercially available purification methods are incompatible with heparin-containing plasma. Here we present data showing the application of miR-Direct® for quantification of miRNAs in plasma, serum, whole blood, and urine. miR-Direct® may be used for validating miRNA sequencing data and biomarker candidates as well as for developing diagnostic (e.g. liquid biopsy) and prognostic applications for circulating miRNAs, including canonical and non-canonical miRNA forms such as isomiRs. Anne Dallas, Ph.D. Principal Scientist SomaGenics, Inc. 2161 Delaware Avenue Santa Cruz, CA 95060-5706 USA adallas@somagenics.com (831) 426-7700 x16

Recovering pre-PCR numbers of fragment copies in RNA-Seq from statistics of Universal Molecular Identifiers Dalia Daujotyte, Andreas Tuerk, Michael Moldaschl Lexogen GmbH, Vienna PCR amplification ensures sufficient input material in RNA-Seq sequencing. However, efficiency variations in PCR also introduce bias in subsequent gene and isoform quantification. Unique molecular identifiers (UMIs) can identify PCR duplicates. Hence, the number of pre-PCR fragment copies is often estimated as the number of distinct UMIs. This approach fails if UMIs are not uniformly distributed or the number of fragment copies before PCR is too large. Here, we investigate computational methods for the correct estimation of pre-PCR fragment copies if the number of UMIs is not sufficiently large and potentially not uniform. We study two types of methods. The first is based on the number of distinct UMIs, the second can be summarized as duplication model estimates (DME). For DMEs the counts for each UMI after PCR are modelled by distributions parameterized by the number of pre-PCR copies, the efficiency and the number of PCR cycles. Parameterization depends on the duplication model which we choose to be linear or exponential. Efficiency and pre-PCR fragment number are estimated simultaneously for all UMIs by maximum likelihood optimization. We perform experiments on synthetic data from a Galton-Watson process with efficiencies of 0.5 and 0.8. Data were generated for 64 and 256 UMIs both evenly distributed and with bias. Simple UMI counting fails in most cases. We further investigate a corrected counting procedure which estimates the number of pre-PCR copies as the elements in a multinomial distribution with expected number of distinct elements equal to the distinct UMIs observed. This leads to improved results particularly for 256 UMIs and biased UMI distribution. However, corrected UMI counting fails to converge or seriously overestimates once almost all UMIs have been seen. DMEs, on the other hand, can be calculated on the full range of pre-PCR abundances and yield in the majority of cases the more accurate and repeatable results. In particular, we find that the DME with Galton-Watson duplication model and a mixture of delta and negative binomial or Gamma distribution for the UMI counts performs very well. Overall, DMEs outperform methods based on the number of distinct UMIs and yield reliable estimates of pre-PCR fragment copies also for small numbers of UMIs with potentially non-uniform distribution. Dalia Daujotyte, PhD Head of Business Development Lexogen Campus Vienna Biocenter 5 Vienna Austria dalia.daujotyte@lexogen.com

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STAT3 AsiC Aptamer- for GBM targeted therapy Vittorio de Franciscis, Silvia Nuzzo, Silvia Catuogno, Carla L. Esposito Istituto di Endocrinologia ed Oncologia Sperimentale “G. Salvatore”, CNR, Naples, Italy GBM (glioblastoma multiforme) is the most common primary brain tumour and one of the most lethal of all cancers, despite advances in surgical and medical neuro-oncology. Thus, the identification of new strategies able to target GBM represents a great challenge for oncological research. Oligonucleotide-based strategy revealed a great potential as anti-cancer therapeutics, but the development of safe, effective and selective approaches for their delivery remains a challenge. In this regard, aptamers exhibit many desirable properties for tumor-targeted drug delivery, such as ease of selection and synthesis, high binding affinity and specificity, low immunogenicity, and versatile synthetic accessibility. We have selected and characterized two aptamers (GL21.T and Gint4.T) that bind to, and inhibit the receptor tyrosine kinases Axl and PDGFRβ and able to selectively deliver miRNA/antimiR molecules to GBM cells. Notably, we showed that both aptamers as either single molecules or conjugates are transported through an in vitro blood-brain barrier (BBB) model, thus revealing a means for the selective and effective treatment of glioma tumours. Here, we took advantage from the developed strategy, for the targeted delivery of small interfering RNA antagonizing the signal transducer and activator of transcription-3 (STAT3) that has been reported as a key regulator of the highly aggressive glioblastoma subtype. We generated novel aptamer-STAT3 conjugates and demonstrated the selective delivery of the siRNA and the efficient silencing of STAT3 in aptamer target positive GBM cells. Importantly, conjugate treatment specifically results in reduced glioblastoma cell viability, thus indicating this molecule as a novel tool with great translational potential for glioma-targeted therapy. Vittorio de Franciscis PhD Research Director CNR Istituto per l’Endocrinologia e Oncologia Sperimentale "G. Salvatore" Via Tommaso De Amicis 95 Bdg C 3rd floor 80145 Naples Italy defranci@unina.it + 39 081 3722343 http://www.ieos.cnr.it/personale/index.php?id=1427899141

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STAT3 AsiC Aptamer- for GBM targeted therapy Vittorio de Franciscis, Silvia Nuzzo, Silvia Catuogno, Carla L. Esposito Istituto di Endocrinologia ed Oncologia Sperimentale “G. Salvatore”, CNR, Naples, Italy GBM (glioblastoma multiforme) is the most common primary brain tumour and one of the most lethal of all cancers, despite advances in surgical and medical neuro-oncology. Thus, the identification of new strategies able to target GBM represents a great challenge for oncological research. Oligonucleotide-based strategy revealed a great potential as anti-cancer therapeutics, but the development of safe, effective and selective approaches for their delivery remains a challenge. In this regard, aptamers exhibit many desirable properties for tumor-targeted drug delivery, such as ease of selection and synthesis, high binding affinity and specificity, low immunogenicity, and versatile synthetic accessibility. We have selected and characterized two aptamers (GL21.T and Gint4.T) that bind to, and inhibit the receptor tyrosine kinases Axl and PDGFRβ and able to selectively deliver miRNA/antimiR molecules to GBM cells. Notably, we showed that both aptamers as either single molecules or conjugates are transported through an in vitro blood-brain barrier (BBB) model, thus revealing a means for the selective and effective treatment of glioma tumours. Here, we took advantage from the developed strategy, for the targeted delivery of small interfering RNA antagonizing the signal transducer and activator of transcription-3 (STAT3) that has been reported as a key regulator of the highly aggressive glioblastoma subtype. We generated novel aptamer-STAT3 conjugates and demonstrated the selective delivery of the siRNA and the efficient silencing of STAT3 in aptamer target positive GBM cells. Importantly, conjugate treatment specifically results in reduced glioblastoma cell viability, thus indicating this molecule as a novel tool with great translational potential for glioma-targeted therapy. Vittorio de Franciscis PhD Research Director CNR Istituto per l’Endocrinologia e Oncologia Sperimentale "G. Salvatore" Via Tommaso De Amicis 95 Bdg C 3rd floor 80145 Naples Italy defranci@unina.it + 39 081 3722343 http://www.ieos.cnr.it/personale/index.php?id=1427899141

Allele-specific inhibition of von Willebrand factor p.Cys2773Ser restores a severe multimerization defect A. de Jong, R.J. Dirven, J.A. Oud, D.Tio and J. Eikenboom Department of internal medicine, division of Thrombosis and Hemostasis. Einthoven laboratory for vascular and regenerative medicine, Leiden University Medical Center, Leiden, the Netherlands Background von Willebrand disease (VWD) is the most common inherited bleeding disorder and most patients develop VWD because of dominant-negative mutations in the multimeric hemostatic protein von Willebrand factor (VWF). These dominant-negative mutations may lead to reduced VWF plasma levels or to a dysfunctional VWF protein. Treatment of VWD focusses on increasing VWF levels through administration of desmopressin which releases endogenously produced VWF, or administration of VWF concentrates. Both therapies usually raise VWF sufficiently, however the production of mutant VWF remains uninterrupted. Persistence of mutant VWF could itself lead to hemostatic problems like thrombocytopenia or the development of angiodysplasia. Inhibition of mutant VWF in dominant-negative VWD might overcome these effects. Aim Correct the abnormal multimer pattern of the dominant-negative VWF p.Cys2773Ser mutation applying allele-specific inhibition. Methods Allele-specific inhibition of VWF was performed by small interfering (si)RNAs. The siRNAs were not developed against the mutation itself, but to single nucleotide polymorphisms (SNPs) in VWF. This allows application of the siRNAs to different mutations, provided the patient is heterozygous for the specific SNP. siRNA mediated allele-specific inhibition was studied in HEK293 cells transiently transfected with two different VWF constructs that carried either of the two alleles of a SNP with one of the alleles also carrying VWF p.Cys2773Ser. Multimerization improvements were visualized by multimer analysis. Results Efficient and allele-specific siRNAs were identified for four different SNPs. Transfection of normal VWF and VWF p.Cys2773Ser resulted in an abnormal multimer pattern similar to the pattern in a heterozygous patient carrying this mutation. siRNA mediated allele-specific inhibition of the mutant allele by targeting the SNP allele linked to p.Cys2773Ser clearly improved, or for some siRNAs even restored, the multimer pattern. Conclusion siRNAs can discriminate VWF alleles by discrepancy of one nucleotide of a SNP. The clear phenotypic improvements observed after inhibition of the VWF p.Cys2773Ser allele show that siRNA mediated allele-specific inhibition of dominant VWF alleles is a promising therapeutic approach for dominant-negative VWD. Annika de Jong, MSc PhD candidate Leiden University Medical Center Albinusdreef 2 2333 ZA Leiden The Netherlands A.de_Jong.STOL@lumc.nl

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Invivofectamine™ Rx Reagent: Novel Lipid Nanoparticles (LNP) for Therapeutic In Vivo mRNA and RNAi delivery to the various tissue and organs Sean Essex1, Shikha Mishra1, Mu Li1, Nektaria Andronikou1, Xavier de Mollerat du Jeu1* 1Thermo Fisher Scientific, Carlsbad, CA, USA

The rapidly expanding utilization of nucleic acids as a therapeutic tool has presented the field with the task of optimizing and innovating delivery methods. We have developed novel lipid nanoparticles that are specifically optimized for use in vivo, and engineered to inherently target and deliver nucleic acid payloads (mRNA and siRNA) to liver, lungs and spleen without the use of biomolecular targeting. Our approach involves systematically designing an optimal DOE that takes into account both main and interaction effects, analyzing the experimental data using appropriate statistical techniques and generating response predictions from all potential combinations of the parameters. This iterative process is repeated till the optimal formulation is discovered. Adopting this approach provides us the flexibility to optimize formulations for organ specific delivery – Liver, Spleen, Lungs and Muscle. The process of formulating these LNPs is simple, scalable and results in uniform-sized LNPs with a narrow PDI. The LNPs efficiently encapsulate and protect the RNA from degradation and facilitate cellular uptake which translates into efficient delivery and reduced toxicity in vivo. We have made significant progress moving through several generations of formulations where our current generation liver delivery LNP is 15,000X more potent than its first generation predecessor. Upon in vivo administration of our LNPs complexed with a chemically modified, luciferase-encoding mRNA (Luc mRNA) we have demonstrated high luciferase activity in vivo - bioluminescence of 10^10 p/sec. and a significant boost in therapeutically relevant proteins - 4000X serum mouse erythropoietin (EPO) and high human Factor IX (hFIX) levels in mice with a single LNP dose. We also identified a novel formulation capable of exclusively targeting lung tissue and achieving highly efficient mRNA and RNAi transfection. This novel lipid nanoparticle is well tolerated in vivo, with no qualitative gross toxicity, and quantitatively analyzed by a comprehensive cytokine profiling performed on murine serum samples. Delivery of siRNA against an endothelial specific target, Tie2, using the lung targeted nanoparticles resulted in over 90% knockdown in isolated lung tissue. Lastly, protein expression was detected using immunohistochemistry, with expression seen in lung tissue sections using a LacZ expression system. . Ongoing work includes knockdown analysis of additional cell type specific targets using siRNA to validate transfection of specific populations of cells. Additionally, we have generated a comprehensive panel of elicited cytokines and liver-function related enzymes and shown that these LNPs are well-tolerated in a murine model Xavier de Mollerat du Jeu, PhD Director R&D, Life Science Group Thermo Fisher Scientific 5781 Van Allen Way|Carlsbad, CA 92008 Office: +1.760.476.6642 | Mobile: +1.858.204.3930 Xavier.demolleratdujeu@thermofisher.com

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Invivofectamine™ Rx Reagent: Novel Lipid Nanoparticles (LNP) for Therapeutic In Vivo mRNA and RNAi delivery to the various tissue and organs Sean Essex1, Shikha Mishra1, Mu Li1, Nektaria Andronikou1, Xavier de Mollerat du Jeu1* 1Thermo Fisher Scientific, Carlsbad, CA, USA

The rapidly expanding utilization of nucleic acids as a therapeutic tool has presented the field with the task of optimizing and innovating delivery methods. We have developed novel lipid nanoparticles that are specifically optimized for use in vivo, and engineered to inherently target and deliver nucleic acid payloads (mRNA and siRNA) to liver, lungs and spleen without the use of biomolecular targeting. Our approach involves systematically designing an optimal DOE that takes into account both main and interaction effects, analyzing the experimental data using appropriate statistical techniques and generating response predictions from all potential combinations of the parameters. This iterative process is repeated till the optimal formulation is discovered. Adopting this approach provides us the flexibility to optimize formulations for organ specific delivery – Liver, Spleen, Lungs and Muscle. The process of formulating these LNPs is simple, scalable and results in uniform-sized LNPs with a narrow PDI. The LNPs efficiently encapsulate and protect the RNA from degradation and facilitate cellular uptake which translates into efficient delivery and reduced toxicity in vivo. We have made significant progress moving through several generations of formulations where our current generation liver delivery LNP is 15,000X more potent than its first generation predecessor. Upon in vivo administration of our LNPs complexed with a chemically modified, luciferase-encoding mRNA (Luc mRNA) we have demonstrated high luciferase activity in vivo - bioluminescence of 10^10 p/sec. and a significant boost in therapeutically relevant proteins - 4000X serum mouse erythropoietin (EPO) and high human Factor IX (hFIX) levels in mice with a single LNP dose. We also identified a novel formulation capable of exclusively targeting lung tissue and achieving highly efficient mRNA and RNAi transfection. This novel lipid nanoparticle is well tolerated in vivo, with no qualitative gross toxicity, and quantitatively analyzed by a comprehensive cytokine profiling performed on murine serum samples. Delivery of siRNA against an endothelial specific target, Tie2, using the lung targeted nanoparticles resulted in over 90% knockdown in isolated lung tissue. Lastly, protein expression was detected using immunohistochemistry, with expression seen in lung tissue sections using a LacZ expression system. . Ongoing work includes knockdown analysis of additional cell type specific targets using siRNA to validate transfection of specific populations of cells. Additionally, we have generated a comprehensive panel of elicited cytokines and liver-function related enzymes and shown that these LNPs are well-tolerated in a murine model Xavier de Mollerat du Jeu, PhD Director R&D, Life Science Group Thermo Fisher Scientific 5781 Van Allen Way|Carlsbad, CA 92008 Office: +1.760.476.6642 | Mobile: +1.858.204.3930 Xavier.demolleratdujeu@thermofisher.com

PNAs and catalytic oligonucleotides:, the impossible marriage? Alexandre Debacker,1,2 Julia Alterman,1 Michael Moazami,1,2 Vivek Sharma,1 Bruno M. D. C. Godinho,1 Anastasia Khvorova,1 Jonathan Watts1 1RNA Therapeutics Institute, UMASS Medical School, Worcester, MA, USA 2University of Southampton, Southampton, UK The use of PNA in therapeutics is limited by its relatively poor bioavailability in vivo and its non-catalytic mechanism of action. PNA (peptide nucleic acid) acts as a steric blocker therefore one copy per target of these therapeutic oligonucleotides is needed. While this mechanism is compatible with splice-switching or anti-miRNA therapeutics, it is unable to engage Ago2 or RNase H dependent gene silencing. Although the failure of PNA to recruit these enzymes to cleave their target limits potency, the high nuclease stability, neutral backbone and high affinity of PNAs remain attractive features that could enhance efficacy and duration of action if they could be integrated within siRNAs and antisense oligonucleotides (ASOs). To address these issues, the combination of PNA with ASOs was first investigated by several groups in the late 1990s. These chimeras were shown to be able to recruit RNase H, but they showed relatively poor binding affinity for their RNA targets. This lack of affinity presumably stems from differences in the helical structure of PNA:RNA and DNA:RNA helices. To improve the affinity, we designed nine different junctions and investigated the affinity of the resulting chimeras for their RNA target as well as their ability to recruit RNase H to cleave structured and non-structured RNAs. We applied phosphorothioate DNA-PNA chimeras to silence MALAT1 in cultured cells and examined their in vivo distribution. We also investigated the combination of PNA with siRNA technology. We found that an siRNA containing a PNA sense strand shows silencing activity comparable to the silencing by a chemically modified ss-siRNA: thus the PNA strand does not block RISC entry but neither does it enhance RISC entry like an RNA sense strand. This observation spurred the development of a small PNA-peptide conjugate complementary to the 3' end of the antisense strand of an asymmetric siRNA. The PNA is being used as a carrier for delivery peptides facilitating the association of peptide and siRNAs. The silencing ability of these constructs has been investigated in cells by passive and lipid-mediated delivery. Alexandre Debacker PhD Student RNA Therapeutics Institute, University of Massachusetts Medical School AS4.1005, 368 Plantation St. Worcester, MA, 01605, USA Alexandre.Debacker@umassmed.edu 1-774-455-3765

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Antisense oligonucleotide-mediated exon skipping of immunoglobulin transcripts as an innovative anticancer approach for the treatment of multiple myeloma and lymphoproliferative disorders Laurent Delpy, Mohamad Omar Ashi, Jean-Marie Lambert, Michel Cogné and Nivine Srour1 1 CNRS UMR 7276 - Université de Limoges, Limoges, France

Multiple Myeloma (MM) is a currently incurable cancer of plasma cells (PCs) characterized by the presence of tumour clones in bone marrow niches. This PC neoplasm represents about 1% of all cancers and 10% of all hematological malignancies, making MM the second most common hematologic cancer after lymphomas. Roughly 86  000 new cases of MM are diagnosed annually worldwide and the risk of developing MM increases with age, with a median age at diagnosis > 65 years. Since the population is aging, the number of adults with MM is expected to double within the next 20 years. In almost all MM patients, the disease is characterized by a high level of monoclonal immunoglobulin (Ig) secretion. By studying alternative splicing of Ig transcripts in PCs, we previously demonstrated that exon skipping events can yield high amounts of internally deleted (variable domain-less) Ig polypeptides. Interestingly, the presence of these truncated-Ig chains causes a massive ER stress-associated apoptosis in PCs (Srour et al., J Exp Med 2016). In addition, we found that the treatment with antisense oligonucleotides (AON) hybridizing Ig pre-mRNAs enhanced exon skipping and the production of truncated-Ig chains. Altogether, these findings open new avenues for AON-mediated Ig exon skipping therapy development in MM and other PC disorders including AL-amyloidosis (Delpy et al., Patent: WO 2017/089359). As a proof of concept for this innovative AON-mediated anticancer approach, we targeted the monoclonal Ig lambda chain expressed by RPMI8226 myeloma cells. A massive cell death was observed after treatment with specific AON hybridizing Ig pre-mRNA, compared to irrelevant AON treatments. Experiments are ongoing to define optimal AON delivery conditions in pre-clinical studies. We will seek to determine whether specific AON treatments induce significant tumour cell death using MM patient cells and xenograft mouse models. To add value to this destructive AON-mediated exon skipping therapy, we also seek to establish industrial partnerships. Overall, the use of antisense technology to enforce the production of aberrant Ig chains and cause the destruction of malignant PCs is very innovative. By targeting specifically the monoclonal Ig component, this AON-mediated exon skipping therapy is highly specific to tumour PCs and almost innocuous for healthy cells. This pioneer approach should open new therapeutic perspectives for the treatment of PC neoplasms, including multiple myeloma and AL-amyloidosis. Laurent Delpy, PhD, Research Director at CNRS CNRS UMR 7276, CRIBL CBRS (Biology and Health Research Center) 2 rue du Dr Marcland, 87025 Limoges France laurent.delpy@unilim.fr +33 519 564 214

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Antisense oligonucleotide-mediated exon skipping of immunoglobulin transcripts as an innovative anticancer approach for the treatment of multiple myeloma and lymphoproliferative disorders Laurent Delpy, Mohamad Omar Ashi, Jean-Marie Lambert, Michel Cogné and Nivine Srour1 1 CNRS UMR 7276 - Université de Limoges, Limoges, France

Multiple Myeloma (MM) is a currently incurable cancer of plasma cells (PCs) characterized by the presence of tumour clones in bone marrow niches. This PC neoplasm represents about 1% of all cancers and 10% of all hematological malignancies, making MM the second most common hematologic cancer after lymphomas. Roughly 86  000 new cases of MM are diagnosed annually worldwide and the risk of developing MM increases with age, with a median age at diagnosis > 65 years. Since the population is aging, the number of adults with MM is expected to double within the next 20 years. In almost all MM patients, the disease is characterized by a high level of monoclonal immunoglobulin (Ig) secretion. By studying alternative splicing of Ig transcripts in PCs, we previously demonstrated that exon skipping events can yield high amounts of internally deleted (variable domain-less) Ig polypeptides. Interestingly, the presence of these truncated-Ig chains causes a massive ER stress-associated apoptosis in PCs (Srour et al., J Exp Med 2016). In addition, we found that the treatment with antisense oligonucleotides (AON) hybridizing Ig pre-mRNAs enhanced exon skipping and the production of truncated-Ig chains. Altogether, these findings open new avenues for AON-mediated Ig exon skipping therapy development in MM and other PC disorders including AL-amyloidosis (Delpy et al., Patent: WO 2017/089359). As a proof of concept for this innovative AON-mediated anticancer approach, we targeted the monoclonal Ig lambda chain expressed by RPMI8226 myeloma cells. A massive cell death was observed after treatment with specific AON hybridizing Ig pre-mRNA, compared to irrelevant AON treatments. Experiments are ongoing to define optimal AON delivery conditions in pre-clinical studies. We will seek to determine whether specific AON treatments induce significant tumour cell death using MM patient cells and xenograft mouse models. To add value to this destructive AON-mediated exon skipping therapy, we also seek to establish industrial partnerships. Overall, the use of antisense technology to enforce the production of aberrant Ig chains and cause the destruction of malignant PCs is very innovative. By targeting specifically the monoclonal Ig component, this AON-mediated exon skipping therapy is highly specific to tumour PCs and almost innocuous for healthy cells. This pioneer approach should open new therapeutic perspectives for the treatment of PC neoplasms, including multiple myeloma and AL-amyloidosis. Laurent Delpy, PhD, Research Director at CNRS CNRS UMR 7276, CRIBL CBRS (Biology and Health Research Center) 2 rue du Dr Marcland, 87025 Limoges France laurent.delpy@unilim.fr +33 519 564 214

Innate Immune Focused Approaches to Maximize Messenger RNA Therapeutic Activity Craig Dobbs, Krist T. Azizian, Dongwon Shin, Sabrina Shore, Jordana M. Henderson, Alexandre Lebedev, Anton P. McCaffrey and Richard I. Hogrefe TriLink BioTechnologies, San Diego, CA 92121, USA.

Messenger RNAs (mRNAs) are an exciting new class of nucleic acid therapeutics entering the clinic. mRNAs are useful in difficult to transfect, non-dividing cells. In contrast to plasmid or viral vectors, there is no risk of insertional mutagenesis or oncogenesis. Transient mRNA expression is also desirable for cellular reprogramming, genome editing (ZFNs, TALENs, CRISPR/Cas9) and vaccines.

For maximal expression in cells or target organs, transfected mRNAs must avoid detection by pattern recognition receptors (PRRs) that evolved to sense pathogenic non-self RNAs. These include PRRs that recognize improperly capped RNAs and double stranded RNA. PRR activation leads to cytokine production, translational arrest and cell toxicity or death. Mammalian mRNAs are modified post-transcriptionally to contain nucleotides with 2’-O-methyl residues, pseudouridine (Ψ) and N6-methyladenosine (m6A). These modifications can reduce activation of PRRs and allow maximal translation of the transfected mRNA. For vaccines, some immunogenicity may be desirable to serve as an adjuvant.

During mRNA capping, Cap0 (m7GpppN) is formed as an intermediate. Methylation of the 2’ position of the first and sometimes second nucleotide forms Cap1 and Cap2, respectively. Recombinant enzymes used to generate Cap1 mRNA are expensive, do not always go to completion and the RNA must be purified prior to capping. We developed a novel co-transcriptional capping method that yields Cap1 with high efficiency and lower costs in a “one pot” reaction. CleanCap™ also facilitates modification of the 5’ end of mRNAs with diverse functional groups, including m6A. We developed a capping assay that allows direct assessment of mRNA capping. Capping efficiencies as high as 99% were obtained.

We synthesized several Ψ derivatives to explore the impact on innate immune stimulation: N1-ethyl-Ψ (Et1Ψ), N1-fluoroethyl-Ψ (FE1Ψ), N1-propyl-Ψ (Pr1Ψ), N1- isopropyl-Ψ (iPr1Ψ) and N1-methoxylmethyl-Ψ (MOM1Ψ) 5’-triphosphates. As a control, we tested 5-methoxy-uridine alongside the pseudouridine derivatives because it previously gave good activity. Luciferase mRNAs were fully substituted with these modifications and translational potential was monitored in wheat germ extracts. Activity was also measured in the THP-1 monocyte cell line, which is a sensitive model for innate immune activation. A recent report showed that minimizing uridine content in mRNAs reduced immune stimulation by unmodified mRNAs. Here we show that incorporation of our modified uridine residues into uridine depleted luciferase resulted in equivalent activity relative to Ψ in THP-1 cells. Interestingly, translation in extracts did not mirror activity in THP-1 cells. Craig Dobbs Vice President TriLink Biotechnologies 9955 Mesa Rim Road San Diego, CA 92121 USA CDobbs@TriLinkBiotech.com

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Enhancing Lentiviral Production Using Splice-Inhibiting Antisense Oligonucleotides Elizabeth Epps1-2, Mayumi Takahashi2, John Rossi1-2, John Burnett1-2 Irell and Manella Graduate School of Biological Sciences1, City of Hope Beckman Research Institute2

Hematopoietic malignancies are characterized by an inability of the immune system

to function correctly and if left untreated, are fatal. Treatments to date include bone marrow transplants and in some cases like Human Immunodeficiency Virus (HIV)-lymphoma patients, techniques such as lentiviral transduction, electroporation, or zinc-finger nucleases have been used to genetically-modify transplant cells in order to also protect these patients from HIV infection. One pre-clinical therapy uses a lentivirus that incorporates a minichromosome maintenance complex component 7 (MCM7) gene cassette that contains artificial short-hairpin ribonucleic acids (shRNAs) in its intron that target different HIV genes.

MCM7 is a regulatory gene responsible for various replication events including pre-replication complex and replication fork formation and it contains three endogenous microRNAs (miRNAs) that assist in these processes. When knocked down or alternatively spliced, the MCM7 miRNAs are overexpressed and are indications of many cancers including breast, colon, and pancreatic cancers. These intronic miRNAs have also been shown to rely on spliceosomal activity for efficient miRNA biogenesis and their expression levels can be modulated using splice-inhibiting morpholinos.

Packaging efficiency of shRNA-containing vectors and especially the MCM7 cassette vectors is a known hurdle in clinical lentiviral production. In an attempt to improve viral titer, morpholino and 2’O-Methyl anti-sense oligonucleotides (ASOs) will be used to target the exon-intron and alternative splice sites of the lentiviral MCM7 gene cassette. These ASOs bind selectively to RNA and can affect splicing events. To test this, third generation MCM7 vectors have been packaged in the presence or absence of splice-inhibiting ASOs into HEK 293T cells to make lentivirus. Viral titer has been determined and the transduced cells tested for alternative splicing of the MCM7 cassette. ASO-treated viral titer increased along while alternative splice products decreased. These results indicate that clinical lentiviral production of MCM7 vectors would benefit from the incorporation of splice-inhibiting ASOs during packaging. Elizabeth Epps, B.S. PhD Candidate Irell and Manella Graduate School of Biological Sciences 1500 E Duarte Road, Fox North 2002H Duarte, CA 91010 United States eepps@coh.org

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Enhancing Lentiviral Production Using Splice-Inhibiting Antisense Oligonucleotides Elizabeth Epps1-2, Mayumi Takahashi2, John Rossi1-2, John Burnett1-2 Irell and Manella Graduate School of Biological Sciences1, City of Hope Beckman Research Institute2

Hematopoietic malignancies are characterized by an inability of the immune system

to function correctly and if left untreated, are fatal. Treatments to date include bone marrow transplants and in some cases like Human Immunodeficiency Virus (HIV)-lymphoma patients, techniques such as lentiviral transduction, electroporation, or zinc-finger nucleases have been used to genetically-modify transplant cells in order to also protect these patients from HIV infection. One pre-clinical therapy uses a lentivirus that incorporates a minichromosome maintenance complex component 7 (MCM7) gene cassette that contains artificial short-hairpin ribonucleic acids (shRNAs) in its intron that target different HIV genes.

MCM7 is a regulatory gene responsible for various replication events including pre-replication complex and replication fork formation and it contains three endogenous microRNAs (miRNAs) that assist in these processes. When knocked down or alternatively spliced, the MCM7 miRNAs are overexpressed and are indications of many cancers including breast, colon, and pancreatic cancers. These intronic miRNAs have also been shown to rely on spliceosomal activity for efficient miRNA biogenesis and their expression levels can be modulated using splice-inhibiting morpholinos.

Packaging efficiency of shRNA-containing vectors and especially the MCM7 cassette vectors is a known hurdle in clinical lentiviral production. In an attempt to improve viral titer, morpholino and 2’O-Methyl anti-sense oligonucleotides (ASOs) will be used to target the exon-intron and alternative splice sites of the lentiviral MCM7 gene cassette. These ASOs bind selectively to RNA and can affect splicing events. To test this, third generation MCM7 vectors have been packaged in the presence or absence of splice-inhibiting ASOs into HEK 293T cells to make lentivirus. Viral titer has been determined and the transduced cells tested for alternative splicing of the MCM7 cassette. ASO-treated viral titer increased along while alternative splice products decreased. These results indicate that clinical lentiviral production of MCM7 vectors would benefit from the incorporation of splice-inhibiting ASOs during packaging. Elizabeth Epps, B.S. PhD Candidate Irell and Manella Graduate School of Biological Sciences 1500 E Duarte Road, Fox North 2002H Duarte, CA 91010 United States eepps@coh.org

Therapeutic Oligo Quality: Profiling and Controlling for Raw Material Impurities

Grant Fernstrum, Indra Pal, Chandrashekar Gudise, and Gary Held Thermo Fisher Scientific As a growing number of therapeutic oligonucleotide compounds continue to be introduced into the clinical pipeline, and advancing into larger, late phase clinical trials, an increasingly stringent demand is placed upon the phosphoramidite supply chain. With global raw material suppliers scaling up production to meet market demands, heightened concern surrounds the increased potential for generating novel, as well as previously identified, impurities. The impurities found within the material supply chain can directly impact the quality of phosphoramidite synthesis, and thus potentially affect the quality of a therapeutic oligo. Therefore, an increased focus has been placed upon the controlling incoming raw materials, understanding the impact to phosphoramidite chemistry, including impurity profile, and subsequent effects to oligo purity. Thermo Fisher Scientific Milwaukee continues to undertake a comprehensive approach to supply chain management through partnerships with raw material suppliers, as well as customers, to define raw material specifications, including control for impurity levels to satisfy the dynamic quality requirements. Recently, the Process Development team performed a deep investigation into the quality of an integral raw material, 4-4'-Dimethoxytrityl Chloride (DMTr-Cl). The team capably identified the role and potential deleterious impact of two potential impurities, 4-Acetoxy-4'-methoxytrityl Chloride (AMTr-Cl) and 4-Hydroxy-4'-methoxytrityl Chloride (HMTr-Cl), in the synthesized phosphoramidite (illustrated in Synthetic Schemes). Grant Fernstrum Process Development Scientist ThermoFisher Scientific 2202 N Bartlett Ave. Milwaukee, WI 53202 USA Grant.fernstrum@thermofisher.com 1 (414) 227-2364

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Targeting nascent primary transcripts with antisense LNA™ GapmeRs Niels Montano Frandsen, Johnathan Lai, Jesper Culmsee Tholstrup, Dhany Saputra & Niels Tolstrup Exiqon, Denmark Exiqon has developed an empirically derived design algorithm to provide Antisense LNA™ GapmeRs that achieve potent target knockdown with a high hit-rate (available at www.exiqon.com/gapmers). As a result for the past four years LNA™ GapmeRs have become readily available as research tools to thousands of researchers around the world in academia and pharma companies. Today LNA™ GapmeRs have become one of the preferred tools for KD of lncRNA in cell cultures and animal models and have played an important role in the discovery of several promising therapeutic lncRNA targets We report the latest results of our on-going efforts to improve our design tool. Despite considerable bioinformatics efforts to ensure target specificity, we observed that about a third of our LNA™ GapmeRs had detectable effects on cell viability when delivered at very high concentration. By analysing large data sets we have found that we can significantly reduce these problems by narrowing the window of one of our design parameters. As a result our latest version of design tool now provides LNA™ GapmeRs with overall greatly reduced toxicity and probably improved target specificity. Finally we report further evidence that gapmers target nascent primary transcripts in the nucleus prior to splicing. We show that gapmers targeting introns and exons are equally efficient at knocking down mature spliced RNA. In addition we present different examples of gapmers that by virtue of targeting a single primary transcript silence multiple mature RNAs, which normally derive from this transcript. These observations have a number of practical implications for gapmer design and interesting new gapmer applications. Niels Montano Frandsen, PhD Global Product Manager Exiqon Skelstedet 16 2950 Vedbaek Denmark e-mail: nmf@exiqon.com

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Targeting nascent primary transcripts with antisense LNA™ GapmeRs Niels Montano Frandsen, Johnathan Lai, Jesper Culmsee Tholstrup, Dhany Saputra & Niels Tolstrup Exiqon, Denmark Exiqon has developed an empirically derived design algorithm to provide Antisense LNA™ GapmeRs that achieve potent target knockdown with a high hit-rate (available at www.exiqon.com/gapmers). As a result for the past four years LNA™ GapmeRs have become readily available as research tools to thousands of researchers around the world in academia and pharma companies. Today LNA™ GapmeRs have become one of the preferred tools for KD of lncRNA in cell cultures and animal models and have played an important role in the discovery of several promising therapeutic lncRNA targets We report the latest results of our on-going efforts to improve our design tool. Despite considerable bioinformatics efforts to ensure target specificity, we observed that about a third of our LNA™ GapmeRs had detectable effects on cell viability when delivered at very high concentration. By analysing large data sets we have found that we can significantly reduce these problems by narrowing the window of one of our design parameters. As a result our latest version of design tool now provides LNA™ GapmeRs with overall greatly reduced toxicity and probably improved target specificity. Finally we report further evidence that gapmers target nascent primary transcripts in the nucleus prior to splicing. We show that gapmers targeting introns and exons are equally efficient at knocking down mature spliced RNA. In addition we present different examples of gapmers that by virtue of targeting a single primary transcript silence multiple mature RNAs, which normally derive from this transcript. These observations have a number of practical implications for gapmer design and interesting new gapmer applications. Niels Montano Frandsen, PhD Global Product Manager Exiqon Skelstedet 16 2950 Vedbaek Denmark e-mail: nmf@exiqon.com

A Facile Synthesis of DNA/RNA Multiple Conjugates by Chemo-enzymatic Approach Masayuki Fujii1, Yasuhiro Shinkai1, Alesya A. Fokina2, Dmitry A. Stetsenko2,3 Kindai University1, Fukuoka, Japan, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia2, Novosibirsk State University, Novosibirsk, Russia3 Oligonucleotide conjugates have been attracting intensive attentions for nucleic acid

therapeutics. [1] Recently we reported that siRNA-NES conjugates showed drastically enhanced silencing of BCR/ABL chimeric gene in human chronic myelogenous leukemia cell line K562 [2]. Although many approaches to the synthesis of oligonucleotide conjugates have been

reported [3], none of them allows us to conjugate an oligonucleotide with multiple peptides, sugars and other molecules at the same time. In the present study, we successfully demonstrated that enzymatic ligation of short

oligonucleotide fragments which was chemically conjugated with one or two functional molecules gave oligonucleotide multiple conjugates. As an example, DNA fragment C1 (5’-TCAGX1GGT-3’) bearing galactose and DNA

fragment C2 (5’-pCAGCX2TTG-3’) bearing nuclear localization signal (NLS) peptide (GPKKKRKVG) derived from SV40 large T antigen were reacted in the presence of T4 DNA ligase and DNA template at 22oC. After 2 hours, substrates C1 and C2 disappeared completely and a product was obtained in quantitative yield. The product was characterized by RP HPLC and MALDI TOF-MS and gave satisfactory results (m/z = 6384.45 (M + H+), calculated 6383.84). We could also synthesize multiple conjugates of oligoDNA, oligoRNA and chemically

modified oligonucleotides including 2’-OMe RNAs and phosphorothioate oligonucleotides. 1. Winkler J. Oligonucleotide conjugates for therapeutic applications. Ther. Deliv. 2013, 4, 791–809. 2. Shinkai Y., Kashihara S., Minematsu G., Fujii H., Naemura M., Kotake Y., Morita Y., Ohnuki K., Fokina A.A., Stetsenko D.A., Filichev V.V., Fujii M. Silencing of BCR/ABL Chimeric Gene in Human Chronic Myelogenous Leukemia Cell Line K562 by siRNA-Nuclear Export Signal Peptide Conjugates. Nucleic Acid Ther. 2017, 27, 168-175. 3. Juliano R.L., Ming X., Nakagawa O. The Chemistry and Biology of Oligonucleotide Conjugates. Acc. Chem. Res. 2012, 45, 1067–1076. M.F. is grateful for financial support by JSPS KAKEN Grant Number 22550159 and Kindai Research Grant KD201704. D.A.S. is supported by RSF grant No. 14-44-00068. A.A.F. acknowledges support from RFBR grant No. 16-03-01055. Masayuki Fujii, PhD Kindai University 11-6 Kayanomori, IIzuka, Fukuoka 820-8555, Japan mfujii@fuk.kindai.ac.jp +81-948-22-5655

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Compatibility of Nucleic Acid Chemical Modification with CRISPR-Cas9 Zachary J. Kartje1, Daniel O'Reilly2, Maryam Habibian2, Elise Malek-Adamian2, Austin Weigle1, Lauren DeRossett1, Masad J. Damha2, and Keith T. Gagnon1, 3 1Department of Chemistry & Biochemistry, Southern Illinois University 2Department of Chemistry, McGill University 3Department of Biochemistry & Molecular Biology, School of Medicine, Southern Illinois University Certain therapeutic applications of CRISPR are likely to benefit from chemical modification of the RNA guide. These may include CRISPR-Cas9 ribonucleoprotein (RNP) formulations or hybrid viral and synthetic CRISPR RNA (crRNA) delivery approaches. In anticipation of these future therapeutic approaches, we have systematically modified the crRNA component of CRISPR-Cas9 and have established general mechanistic rules for modification. We find that several modifications, depending on position in the crRNA sequence, are compatible with catalytic activity and genome editing. These are generally modifications that do not introduce bulky adducts. These include DNA, 2',5'-linked RNA, 2'F-RNA, and to some extent 2'F-ANA and LNA. The placement of modifications is critical. RNA nucleotides must be retained in or very near the seed region, or else modifications that strongly mimic RNA structural and functional properties must be used. The 3' end of the crRNA that base-pairs with tracrRNA can tolerate DNA substitutions very well, as well as 2'F-ANA nucleotides to some degree, as can the 5'-most 10-12 nucleotides of the guide. 2',5'-linked RNA can also be mixed with RNA (3',5'-linked) and yield high activity. Phosphorothioate has thus far been tolerated in a few of the crRNAs tested. This analysis demonstrates that A-form-like helical structure must be maintained in or around the seed region of the crRNA. However, certain modifications can substitute for RNA residues. Examples of crRNAs that lack any RNA nucleotides have also been characterized, demonstrating that substantial modification is possible with the correct choice of chemistry. The high compatibility of DNA and poor compatibility of bulky modifications suggests that flexibility and Cas9 dynamics are a critical factor in successful modification schemes. With the growing array of modifications available and the general rules we have uncovered, we expect greater exploration of crRNA modification. This study indicates that nucleic acid chemical modification of CRISPR systems is a promising approach that opens new possibilities for therapeutic development. Keith T. Gagnon, Ph.D. Biochem. & Mol. Biol., School of Medicine Chem. & Biochem Southern Illinois University 1245 Lincoln Dr. Carbondale, IL 62901 USA ktgagnon@siu.edu

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Compatibility of Nucleic Acid Chemical Modification with CRISPR-Cas9 Zachary J. Kartje1, Daniel O'Reilly2, Maryam Habibian2, Elise Malek-Adamian2, Austin Weigle1, Lauren DeRossett1, Masad J. Damha2, and Keith T. Gagnon1, 3 1Department of Chemistry & Biochemistry, Southern Illinois University 2Department of Chemistry, McGill University 3Department of Biochemistry & Molecular Biology, School of Medicine, Southern Illinois University Certain therapeutic applications of CRISPR are likely to benefit from chemical modification of the RNA guide. These may include CRISPR-Cas9 ribonucleoprotein (RNP) formulations or hybrid viral and synthetic CRISPR RNA (crRNA) delivery approaches. In anticipation of these future therapeutic approaches, we have systematically modified the crRNA component of CRISPR-Cas9 and have established general mechanistic rules for modification. We find that several modifications, depending on position in the crRNA sequence, are compatible with catalytic activity and genome editing. These are generally modifications that do not introduce bulky adducts. These include DNA, 2',5'-linked RNA, 2'F-RNA, and to some extent 2'F-ANA and LNA. The placement of modifications is critical. RNA nucleotides must be retained in or very near the seed region, or else modifications that strongly mimic RNA structural and functional properties must be used. The 3' end of the crRNA that base-pairs with tracrRNA can tolerate DNA substitutions very well, as well as 2'F-ANA nucleotides to some degree, as can the 5'-most 10-12 nucleotides of the guide. 2',5'-linked RNA can also be mixed with RNA (3',5'-linked) and yield high activity. Phosphorothioate has thus far been tolerated in a few of the crRNAs tested. This analysis demonstrates that A-form-like helical structure must be maintained in or around the seed region of the crRNA. However, certain modifications can substitute for RNA residues. Examples of crRNAs that lack any RNA nucleotides have also been characterized, demonstrating that substantial modification is possible with the correct choice of chemistry. The high compatibility of DNA and poor compatibility of bulky modifications suggests that flexibility and Cas9 dynamics are a critical factor in successful modification schemes. With the growing array of modifications available and the general rules we have uncovered, we expect greater exploration of crRNA modification. This study indicates that nucleic acid chemical modification of CRISPR systems is a promising approach that opens new possibilities for therapeutic development. Keith T. Gagnon, Ph.D. Biochem. & Mol. Biol., School of Medicine Chem. & Biochem Southern Illinois University 1245 Lincoln Dr. Carbondale, IL 62901 USA ktgagnon@siu.edu

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miR-21-5p as an RNA-therapeutic target for personalized medicine in pancreatic cancer

Maud-Emmanuelle Gilles1, Liang Hao2, Ling Huang3, Rajesha Rupaimoole1, Pedro Lopez4, Emilia Pulver2, Jong Cheol Jeong3, Sentil Muthuswamy3, Manuel Hidalgo3,4, Sangeeta N Bhatia2, Frank J Slack1 1Institute for RNA Medicine at Beth Israel Deaconess Medical Center, Harvard Medical School, 2Koch Institute for Integrative Cancer Research, MIT, 3Beth Israel Deaconess Medical Center, Harvard Medical School, 4Spanish National Cancer Research Centre (CNIO)

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a very poor prognosis that progresses rapidly. Many therapeutic trials, such as with chemotherapy, radiation, and anti-stromal therapies developed to date have only resulted in small advances, and survival after diagnosis is generally less than a year. Clearly, novel therapeutics based on a better understanding of this disease combined with new drug delivery system are desperately needed

In this study, we show the utility of RNA-based therapy for personalized medicine by using, PDAC 3D-models, patient-derived-organoid (PDO) and patient-derived-xenograft (PDX) models. We have performed miRNA profiling on PDX samples to determine the status of miRNA deregulation in individual pancreatic ductal adenocarcinoma (PDAC) patients, which revealed a common miR-21 over-expression signature. miR-21 is an established oncomiR and a promising target for therapy.

PDAC is hypovascularized and largely not penetrable making therapeutic drug delivery a challenge. RNAs are often large and charged, preventing them from easy internalization in the cell. In addition, instability in circulation and rapid renal clearance result in their short half-life make the delivery of small RNAs to the tumor site particularly difficult. To deliver anti-miR-21 therapy to PDAC we coupled anti-miR-21 LNAs with tumor-penetrating nanocomplexes (TPN) targeting PDAC tumors. As a validation for our pre-clinical strategy, the therapeutic potential of our nano-drug (TPN-21) was first shown in PDO avatars for individual patients, then in their PDX avatars. Our results show that the TPN-21 is delivered in PDAC and limits the growth of PDAC 3D-models, PDO and PDX avatars. Since drug responses vary between patients it is crucial to develop pre-clinical or co-clinical strategies that forecast patient response. The general approach performed in our study, seems suitable for pre-clinical validation of personalized RNA medicine and to pave the way to a prospectively identify patients expressing high miR-21 levels, eligible for the TPN-21 therapy.

Maud-Emmanuelle Gilles, PhD Institute for RNA Medicine, BIDMC, Harvard Medical school Center for Life Sciences (CLS – 417-H5) 3 Blackfan Circle, 02115, Boston, MA USA mgilles1@bidmc.harvard.edu

 

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miR-21-5p as an RNA-therapeutic target for personalized medicine in pancreatic cancer

Maud-Emmanuelle Gilles1, Liang Hao2, Ling Huang3, Rajesha Rupaimoole1, Pedro Lopez4, Emilia Pulver2, Jong Cheol Jeong3, Sentil Muthuswamy3, Manuel Hidalgo3,4, Sangeeta N Bhatia2, Frank J Slack1 1Institute for RNA Medicine at Beth Israel Deaconess Medical Center, Harvard Medical School, 2Koch Institute for Integrative Cancer Research, MIT, 3Beth Israel Deaconess Medical Center, Harvard Medical School, 4Spanish National Cancer Research Centre (CNIO)

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a very poor prognosis that progresses rapidly. Many therapeutic trials, such as with chemotherapy, radiation, and anti-stromal therapies developed to date have only resulted in small advances, and survival after diagnosis is generally less than a year. Clearly, novel therapeutics based on a better understanding of this disease combined with new drug delivery system are desperately needed

In this study, we show the utility of RNA-based therapy for personalized medicine by using, PDAC 3D-models, patient-derived-organoid (PDO) and patient-derived-xenograft (PDX) models. We have performed miRNA profiling on PDX samples to determine the status of miRNA deregulation in individual pancreatic ductal adenocarcinoma (PDAC) patients, which revealed a common miR-21 over-expression signature. miR-21 is an established oncomiR and a promising target for therapy.

PDAC is hypovascularized and largely not penetrable making therapeutic drug delivery a challenge. RNAs are often large and charged, preventing them from easy internalization in the cell. In addition, instability in circulation and rapid renal clearance result in their short half-life make the delivery of small RNAs to the tumor site particularly difficult. To deliver anti-miR-21 therapy to PDAC we coupled anti-miR-21 LNAs with tumor-penetrating nanocomplexes (TPN) targeting PDAC tumors. As a validation for our pre-clinical strategy, the therapeutic potential of our nano-drug (TPN-21) was first shown in PDO avatars for individual patients, then in their PDX avatars. Our results show that the TPN-21 is delivered in PDAC and limits the growth of PDAC 3D-models, PDO and PDX avatars. Since drug responses vary between patients it is crucial to develop pre-clinical or co-clinical strategies that forecast patient response. The general approach performed in our study, seems suitable for pre-clinical validation of personalized RNA medicine and to pave the way to a prospectively identify patients expressing high miR-21 levels, eligible for the TPN-21 therapy.

Maud-Emmanuelle Gilles, PhD Institute for RNA Medicine, BIDMC, Harvard Medical school Center for Life Sciences (CLS – 417-H5) 3 Blackfan Circle, 02115, Boston, MA USA mgilles1@bidmc.harvard.edu

 

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Mir-143/145 Expression In Keratinocyte Differentiation And Its Role In Cervical Cancer Progression Alejandro Gonzalez-Torres1, Natalia Martinez-Acuña1, Juana Virginia Tapia-Vieyra1, José Arellano-Galindo2, Oliver Antelmo Toledo-Sánchez3, Eric Sulpice4,5,6, Xavier Gidrol4,5,6, Luis Marat Alvarez-Salas1 1 Laboratorio de Terapia Génica. Departamento de Genética y Biología Molecular. Centro de Investigación y de Estudios Avanzados del IPN 2 Laboratorio de Infectología. Hospital Infantil de Mexico Federico Gomez 3 Servicio de cirugía. Hospital Pediátrico Moctezuma 4 Université Grenoble-Alpes, BIG-BGE, F-3800 5 CEA, BIG-BGE, F-38000 6 INSERM, BIG-BGE, F-38000 MicroRNAs (miRNAs) are small non-coding RNAs which regulate the expression of mRNAs containing complementary sequences. The disruption of miRNA function has been associated to many human diseases, including cancer. Cervical cancer is a major public health problem in Mexico and the world. Infection with high-risk human papilloma virus (HPV) is the major risk factor for the subsequent development of cervical cancer. However, HPV infection alone is not enough to produce a malignant phenotype. Therefore, it has been proposed that aberrations at the miRNome could contribute to malignant progression. The miR-143/145 cluster has shown to be down-regulated in a wide range of cancers, including cervical cancer. We analyzed the miR-143/145 expression in keratinocytes (primary and HPV-immortalized) and cervical cancer cell lines, showing that only primary keratinocytes expressed miR-143 but no miR145, even though both miRNAs raised from the same primary transcript. Interestingly, miR-145 was expressed after calcium-induced keratinocyte differentiation in primary culture but no in HPV-immortalized keratinocytes. Furthermore, when miR-143/145 mimics were overexpressed in HeLa cells, a cervical cancer cell line, these inhibited the anchorage-independent growth, impaired 3D-culture growth and decreased cell migration. Notably, the highest effect was showed by miR-145 while miR-143 showed a smaller or no effect. Thus, these findings suggest that miR-143/145 expression would be regulated by HPV infection and when the miR-143/145 is restored in cancer cells, this miRNA cluster functions as a tumor suppressor. Further bioinformatics analysis and luciferase based reporter constructs experiments may elucidate the targets involved in the miR-143/145 tumor suppressor role in cervical cancer. Alejandro Gonzalez-Torres, MCs PhD Student Centro de Investigación y de Estudios Avanzados del IPN 2508 IPN Avenue 07360 Mexico City, CDMX Mexico agonzalezt@cinvestav.mx

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Mir-143/145 Expression In Keratinocyte Differentiation And Its Role In Cervical Cancer Progression Alejandro Gonzalez-Torres1, Natalia Martinez-Acuña1, Juana Virginia Tapia-Vieyra1, José Arellano-Galindo2, Oliver Antelmo Toledo-Sánchez3, Eric Sulpice4,5,6, Xavier Gidrol4,5,6, Luis Marat Alvarez-Salas1 1 Laboratorio de Terapia Génica. Departamento de Genética y Biología Molecular. Centro de Investigación y de Estudios Avanzados del IPN 2 Laboratorio de Infectología. Hospital Infantil de Mexico Federico Gomez 3 Servicio de cirugía. Hospital Pediátrico Moctezuma 4 Université Grenoble-Alpes, BIG-BGE, F-3800 5 CEA, BIG-BGE, F-38000 6 INSERM, BIG-BGE, F-38000 MicroRNAs (miRNAs) are small non-coding RNAs which regulate the expression of mRNAs containing complementary sequences. The disruption of miRNA function has been associated to many human diseases, including cancer. Cervical cancer is a major public health problem in Mexico and the world. Infection with high-risk human papilloma virus (HPV) is the major risk factor for the subsequent development of cervical cancer. However, HPV infection alone is not enough to produce a malignant phenotype. Therefore, it has been proposed that aberrations at the miRNome could contribute to malignant progression. The miR-143/145 cluster has shown to be down-regulated in a wide range of cancers, including cervical cancer. We analyzed the miR-143/145 expression in keratinocytes (primary and HPV-immortalized) and cervical cancer cell lines, showing that only primary keratinocytes expressed miR-143 but no miR145, even though both miRNAs raised from the same primary transcript. Interestingly, miR-145 was expressed after calcium-induced keratinocyte differentiation in primary culture but no in HPV-immortalized keratinocytes. Furthermore, when miR-143/145 mimics were overexpressed in HeLa cells, a cervical cancer cell line, these inhibited the anchorage-independent growth, impaired 3D-culture growth and decreased cell migration. Notably, the highest effect was showed by miR-145 while miR-143 showed a smaller or no effect. Thus, these findings suggest that miR-143/145 expression would be regulated by HPV infection and when the miR-143/145 is restored in cancer cells, this miRNA cluster functions as a tumor suppressor. Further bioinformatics analysis and luciferase based reporter constructs experiments may elucidate the targets involved in the miR-143/145 tumor suppressor role in cervical cancer. Alejandro Gonzalez-Torres, MCs PhD Student Centro de Investigación y de Estudios Avanzados del IPN 2508 IPN Avenue 07360 Mexico City, CDMX Mexico agonzalezt@cinvestav.mx

MTL-CEBPA has efficacy in a broad range of liver disease models and encouraging early clinical data in a Phase 1 trial in HCC Nagy Habib1, Vikash Reebye1, Jon Voutila2, Robert Habib2, David Collin2, Joanna Nicholls1, Pinelopi Andrikakou1, Pantelitsa Protopapa2, Pål Sætrom3, Siv Anita Hegre3, Hans Huber4, Kai-Wen Huang5, John Rossi6 and David Blakey2

1Department of Surgery and Cancer, Imperial College London, London, UK. 2MiNA Therapeutics Ltd, London, UK. 3Department of Cancer Research and Molecular Medicine, Trondheim, Norway. 4BioTD Strategies, Lansdale, PA, USA. 5Graduate Institute of Clinical Medicine, National Taiwan University, Taipei, Taiwan. 6Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, California, USA.

Small activating RNAs (saRNAs) are designed to selectively up-regulate therapeutic proteins by recruiting endogenous transcriptional complexes to a target gene, leading to increased expression of naturally processed mRNA. Transcription factor C/EBPα (CCAAT/enhancer-binding protein alpha) is a leucine zipper protein which acts as a master regulator of liver homeostasis and multiple oncogenic processes including cell cycle control, proliferation and angiogenesis. MTL-CEBPA comprises a double stranded RNA payload (CEBPA-51) formulated inside a SMARTICLES® liposomal nanoparticle to specifically target the CEBPA gene.

In vitro transfection of CEBPA-51 in Hepatocellular carcinoma (HCC) cell lines leads to up-regulation of CEBPA mRNA, CEBPA protein and inhibition of tumour cell growth. In vivo MTL-CEBPA treatment in a diethylnitrosamine induced rat model of cirrhotic HCC leads to up-regulation of CEBPA mRNA and 80-90% inhibition of tumour growth accompanied by improved liver function. MTL-CEBPA has shown therapeutic activity, including disease regression and improved survival, in a range of other liver disease models including high fat diet, MCD diet, CCl4 liver fibrosis and cirrhosis and liver regeneration models. Increased levels (2 fold) of CEBPA mRNA have been seen in the liver in these models post treatment with MTL-CEBPA. Based on this encouraging preclinical data a Phase 1 trial (standard 3+3 dose escalation study) in patients with HCC or secondary liver cancers has been initiated (Clinical trial information: NCT02716012). MTL-CEBPA is administered as a 1-hr IV infusion on Day 1, 8 and 15 of a 28-day cycle. RECIST tumor response is assessed after every 2 cycles. The primary objective is to determine safety and tolerability; secondary objectives include PK, PD, liver function improvement and anti-tumour activity. The initial starting dose was 28mg/m2 (approximately 0.8mg/kg) which is a dose at which we have seen therapeutic activity in pre-clinical models. Initial findings have confirmed target engagement in the clinic by comparing CEBPA mRNA levels in patient white blood cells before and after administration of MTL-CEBPA. Dose escalation in the trial is continuing to define a RP2D for an expansion cohort in cirrhotic HCC and early clinical results are encouraging.

Prof Nagy Habib MD, ChM, FRCS Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital London, UK Nagy.habib@imperial.ac.uk

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Scalable Nucleic Acid Quality Assessments for Illumina Next-Generation Sequencing Library Prep: Simultaneous qualification and quantification of nucleic acids with the Fragment Analyzer™ Jonathon Hagopian Advanced Analytical Technologies, Inc. Assessing nucleic acid quality during library preparation is essential for the success of next-generation sequencing (NGS) applications. Verifying the integrity of nucleic acid samples before library preparation identifies samples likely to produce suboptimal libraries with poor sequencing performance due to degradation, fragmentation, or low purity. Similarly, the accurate assessment of both quality and quantity of prepared libraries can be used to optimize cluster generation during sequencing, resulting in cost- and time-savings while maximizing both sequencing data quality and output.

The Fragment Analyzer Automated Capillary Electrophoresis (CE) System from Advanced Analytical Technologies, Inc. accelerates nucleic acid analysis workflows and provides a method that has been tested by Illumina, for accurate quality assessment with multiple sample types. This poster provides an overview of the quality control (QC) workflow using the Fragment Analyzer during library preparation and provides representative data plots for different sample and library types. Jonathon Hagopian, PhD Director – Business Development Advanced Analytical Technologies, Inc. 2450 SE Oak Tree Ct. Ankeny, IA USA jhagopian@aati-us.com

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Scalable Nucleic Acid Quality Assessments for Illumina Next-Generation Sequencing Library Prep: Simultaneous qualification and quantification of nucleic acids with the Fragment Analyzer™ Jonathon Hagopian Advanced Analytical Technologies, Inc. Assessing nucleic acid quality during library preparation is essential for the success of next-generation sequencing (NGS) applications. Verifying the integrity of nucleic acid samples before library preparation identifies samples likely to produce suboptimal libraries with poor sequencing performance due to degradation, fragmentation, or low purity. Similarly, the accurate assessment of both quality and quantity of prepared libraries can be used to optimize cluster generation during sequencing, resulting in cost- and time-savings while maximizing both sequencing data quality and output.

The Fragment Analyzer Automated Capillary Electrophoresis (CE) System from Advanced Analytical Technologies, Inc. accelerates nucleic acid analysis workflows and provides a method that has been tested by Illumina, for accurate quality assessment with multiple sample types. This poster provides an overview of the quality control (QC) workflow using the Fragment Analyzer during library preparation and provides representative data plots for different sample and library types. Jonathon Hagopian, PhD Director – Business Development Advanced Analytical Technologies, Inc. 2450 SE Oak Tree Ct. Ankeny, IA USA jhagopian@aati-us.com

Oligonucleotide therapy for treatment of erythropoeitic protoporphyria François Halloy, Paulina Ćwiek, Daniel Schümperli and Jonathan Hall Institute for Pharmaceutical Sciences, ETH Zürich, Zürich, Switzerland

Erythropoeitic protoporphyria (EPP) is a rare genetic disease where patients suffer from extreme skin irritation under natural and artificial light. EPP is caused by genetic variations on both alleles of the ferrochelatase (FECH) gene: in one, a non-sense or missense mutation prevents synthesis of the FECH enzyme; in the other, an intronic single nucleotide polymorphism (SNP) causes missplicing of the pre-mRNA by enhancing the use of a cryptic splice site. Low levels of FECH leads to accumulation of its photoreactive substrate protoporphyrin IX (PPIX) in erythroid cells in the blood, in bone marrow and in the liver, which in turn triggers photosensitivity. One strategy to treat EPP is to use splice-switching oligonucleotides (SSOs) - oligonucleotides complementary to the FECH pre-mRNA which bind close to the cryptic splice site and “switch” its splicing back to the functional FECH mRNA. For this purpose, we are using SSOs composed of 2’-O-methoxyethyl (MOE) ribose units linked by a phosphorothioate (PS) backbone, the same chemistry employed in recently approved drugs mipomirsen and nusinersen. A lead SSO sequence able to redirect splicing was identified by screening a stretch of FECH intron 3 in a minigene reporter assay. It is presently being investigated in a mouse model for its distribution to various tissues, including bone marrow. Besides, we have further developed the MOE-PS chemistry with the introduction of stereochemically-pure phosphorothioate linkages. As delivery to organs other than liver and kidney is a fundamental limitation for oligonucleotide therapeutics, we are also investigating the conjugation of various short peptides to the SSO for enhanced uptake into hematopoietic compartments. The peptide library comprises sequences that are known to bind to receptors on erythroid cells, or to locate to the cell nucleus. Conjugates are being tested in erythroid cells and we are quantifying delivery using a technique developed for chemically modified oligonucleotides - chemical-ligation qPCR (CL-qPCR). François Halloy PhD Student ETH Zürich Vladimir Prelog Weg 4 8093 Zurich Switzerland francois.halloy@pharma.ethz.ch

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Enhanced cancer immunotherapy based on tumor cell foreignization with PD-L1 blockade Roun Heo1, Jung Min Shin2, Jae Hyung Park1,2* 1 Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University 2 School of Chemical Engineering, Sungkyunkwan University Cytotoxic T lymphocytes (CTLs) are key elements to induce immunological rejection via non-self antigen recognition. However, most tumors are capable of avoiding CTL recognition by presenting self-antigens. Also, they express programmed death ligand-1 (PD-L1), a well-known immune checkpoint, which paralyzes the cytotoxic activity of T cells toward tumors. Therefore, both issues should be considered for successful T-cell mediated immunotherapy. In this regards, we prepared PD-L1 siRNA/polyethyleneimine polyplexes (PPLs) coated with polymeric conjugates, composed of PEGylated hyaluronic acid (PEG-HA) as the CD44 tumor-targeting ligand and ovalbumin (OVA) as a foreign antigen. We hypothesized that the PEG-HA-OVA/PPLs can foreignize tumor cells by delivering non-self antigen specifically to tumor cells and their siRNA-mediated knockdown of PD-L1 can synergistically boost cancer immunotherapy. The PEG-HA-OVA/PPLs were selectively taken up by CD44-positive tumor cells while allowing for antigenic OVA peptide (OVA257-264) presentation at MHC class I molecules on the cell surface. In addition, the PEG-HA-OVA/PPLs effectively accumulated in tumor tissues after their systemic administration into the tail vein of tumor-bearing mice. Through these results, we verified the possibilities of PEG-HA-OVA/PPLs for effective cancer immunotherapy with their high tumor targetability. Further, in vivo therapy is in progress to confirm the tumor growth inhibition ability of PEG-HA-OVA/PPLs.

Schematic illustration depicting working mechanism of the PEG-HA-OVA/PPLs

Roun Heo, Ph.D. student Sungkyunkwan University 81716 Seoburo2066, Jangangu Suwon /Gyeonggido South Korea ggobie@skku.edu

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Enhanced cancer immunotherapy based on tumor cell foreignization with PD-L1 blockade Roun Heo1, Jung Min Shin2, Jae Hyung Park1,2* 1 Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University 2 School of Chemical Engineering, Sungkyunkwan University Cytotoxic T lymphocytes (CTLs) are key elements to induce immunological rejection via non-self antigen recognition. However, most tumors are capable of avoiding CTL recognition by presenting self-antigens. Also, they express programmed death ligand-1 (PD-L1), a well-known immune checkpoint, which paralyzes the cytotoxic activity of T cells toward tumors. Therefore, both issues should be considered for successful T-cell mediated immunotherapy. In this regards, we prepared PD-L1 siRNA/polyethyleneimine polyplexes (PPLs) coated with polymeric conjugates, composed of PEGylated hyaluronic acid (PEG-HA) as the CD44 tumor-targeting ligand and ovalbumin (OVA) as a foreign antigen. We hypothesized that the PEG-HA-OVA/PPLs can foreignize tumor cells by delivering non-self antigen specifically to tumor cells and their siRNA-mediated knockdown of PD-L1 can synergistically boost cancer immunotherapy. The PEG-HA-OVA/PPLs were selectively taken up by CD44-positive tumor cells while allowing for antigenic OVA peptide (OVA257-264) presentation at MHC class I molecules on the cell surface. In addition, the PEG-HA-OVA/PPLs effectively accumulated in tumor tissues after their systemic administration into the tail vein of tumor-bearing mice. Through these results, we verified the possibilities of PEG-HA-OVA/PPLs for effective cancer immunotherapy with their high tumor targetability. Further, in vivo therapy is in progress to confirm the tumor growth inhibition ability of PEG-HA-OVA/PPLs.

Schematic illustration depicting working mechanism of the PEG-HA-OVA/PPLs

Roun Heo, Ph.D. student Sungkyunkwan University 81716 Seoburo2066, Jangangu Suwon /Gyeonggido South Korea ggobie@skku.edu

Development of Clinically Viable Lipid Nanoparticles for mRNA J. Heyes, K. Lam, A. Judge, L. Palmer, H. Yuen, P. Schreiner Arbutus Biopharma, Corp. Arbutus’ Lipid Nanoparticle (LNP) platform is the leading nucleic acid delivery technology platform, enabling a number of early and late stage clinical trials. They are designed to deliver their payloads to sites of disease and have been used to target both viral and endogenous gene targets. LNP technology is particularly well suited to delivery of mRNA payloads. However appropriate care must be taken when formulating these large, fragile payloads to ensure their integrity and suitable long term shelf life of the product. Here we show that commonly used formulating buffers can have a deleterious effect on payload integrity. This can be addressed by careful selection of buffers and process conditions. In addition, we have established successful methodology for lyophilisation of mRNA-LNP. Lyophilization has no impact on biological performance of the LNP and considerably improves shelf life. James Heyes, PhD VP Drug Delivery Arbutus Biopharma, Corp. 8900 Glenlyon Parkway Burnaby, BC V5J 5J8 Canada jheyes@arbutusbio.com

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Synthesis and Evaluation of a Tricyclic Nucleic Acid Analogue Bearing the Locked Sugar Moiety and Torsion Angle ε Masahiko Horiba, Satoshi Obika Graduate School of Pharmaceutical Sciences, Osaka University Oligonucleotides modified with 2’,4’-BNA/LNA exhibit high binding affinity toward complementary DNA and RNA. 1 Therefore, 2’,4’-BNA/LNA have been utilized for many biological tools along with medical application. In contrast, D2-CNA-dT, in which torsion angle ε, ζ and the sugar conformation are locked, was developed by Chattopadhyaya et al. to acquire high binding affinity with complementary RNA. However, D2-CNA-dT showed low binding affinity because the covalent bond between bridge structure and phosphate moiety of D2-CNA-dT would cause the distortion of the duplex structure. For achieving higher binding affinity than 2’,4’-BNA/LNA, we designed a novel tricyclic nucleic acid analogue, where torsion angle ε is locked in a different way. The amidite block was synthesized in 14 steps from a known starting compound with an overall yield of 13%. The amidite block was incorporated into oligonucleotide to evaluate the binding affinity of the tricyclic analogue toward complementary DNA and RNA. Unexpectedly, the oligonucleotide modified with the tricyclic analogue showed lower binding affinity than the natural DNA oligonucleotide. Evaluations of thermodynamic parameters and CD spectra suggested that the worse binding affinity is caused by the loss of entropy during the duplex forming step. [1] Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.; Imanishi, T. Tetrahedron Lett. 1997, 38, 8735–8738. [2] Zhou, C.; Plashkevych, O.; Chattopadhyaya, J. J. Org. Chem. 2009, 74, 3248–3265. Masahiko Horiba, PhD student Graduate School of Pharmaceutical Sciences, Osaka University 565-0871 1-6 Yamadaoka, Suita, Osaka Japan horiba-m@phs.osaka-u.ac.jp

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Synthesis and Evaluation of a Tricyclic Nucleic Acid Analogue Bearing the Locked Sugar Moiety and Torsion Angle ε Masahiko Horiba, Satoshi Obika Graduate School of Pharmaceutical Sciences, Osaka University Oligonucleotides modified with 2’,4’-BNA/LNA exhibit high binding affinity toward complementary DNA and RNA. 1 Therefore, 2’,4’-BNA/LNA have been utilized for many biological tools along with medical application. In contrast, D2-CNA-dT, in which torsion angle ε, ζ and the sugar conformation are locked, was developed by Chattopadhyaya et al. to acquire high binding affinity with complementary RNA. However, D2-CNA-dT showed low binding affinity because the covalent bond between bridge structure and phosphate moiety of D2-CNA-dT would cause the distortion of the duplex structure. For achieving higher binding affinity than 2’,4’-BNA/LNA, we designed a novel tricyclic nucleic acid analogue, where torsion angle ε is locked in a different way. The amidite block was synthesized in 14 steps from a known starting compound with an overall yield of 13%. The amidite block was incorporated into oligonucleotide to evaluate the binding affinity of the tricyclic analogue toward complementary DNA and RNA. Unexpectedly, the oligonucleotide modified with the tricyclic analogue showed lower binding affinity than the natural DNA oligonucleotide. Evaluations of thermodynamic parameters and CD spectra suggested that the worse binding affinity is caused by the loss of entropy during the duplex forming step. [1] Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.; Imanishi, T. Tetrahedron Lett. 1997, 38, 8735–8738. [2] Zhou, C.; Plashkevych, O.; Chattopadhyaya, J. J. Org. Chem. 2009, 74, 3248–3265. Masahiko Horiba, PhD student Graduate School of Pharmaceutical Sciences, Osaka University 565-0871 1-6 Yamadaoka, Suita, Osaka Japan horiba-m@phs.osaka-u.ac.jp

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The selection system of the sugar modified nucleic acid aptamer by KOD polymerase mutants Hidekazu Hoshino1, Yuuya Kasahara1, 2, Masayasu Kuwahara3, Satoshi Obika1, 2 National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN)1 Graduate School of Pharmaceutical Sciences, Osaka University2 Graduate School of Science and Technology, Gunma University3 Systematic Evolution of Ligands by EXponential enrichment (SELEX) method using a modified nucleic acid library makes it possible to generate modified nucleic acid aptamers without post-modifications. The modified nucleic acid aptamers will show high nuclease resistance and high affinity to the target molecule derived from characteristics of modified nucleotides. This method includes steps of transcription and reverse transcription, in other words, it encodes DNA into modified nucleic acids and decodes the modified nucleic acids back into DNA. However, ordinary polymerase can't recognize the modified nucleotide triphosphate as the substrate because of the strict selectivity. To resolve the problem, the development of novel polymerase mutants is required. Then, we attempted to create the novel polymerase mutants that can use sugar modified nucleotide triphosphate as the substrate. Over 100 kinds of polymerase mutants were generated by means of point mutation into KOD polymerase derived from Thermococcus kodakaraensis KOD1, having superiority in fidelity and extension rate. We performed primer extension experiments to evaluate the ability of KOD polymerase mutants to transcription and reverse transcription. Several polymerase mutants were able to transcript and reverse transcript successfully. Further, sugar modified nucleic acid libraries made by the polymerase mutants were exposed to serum to assess the nuclease resistance. As a result, significant nuclease resistance was observed. These results indicate the possibility and usability of modified nucleic acid aptamers. Hidekazu Hoshino Ph.D. National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN) 7-6-8 Saito Asagi Ibaraki Japan h-hoshino@nibiohn.go.jp

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Preclinical Development of siRNA Therapeutics for Inflammatory Bowel Disease (IBD) using Transkingdom RNA Interference (tkRNAi) Bacterial Delivery Platform Larn Hwang1, Kevin Ng2, Wen Wang2, David Nam2, Joseph Ramelli1, Falguni Trieu1, Mihir Munsif1, Osmond J. D’Cruz2 1 Marina Biotech, 2Autotelic Inc. Background: The balance between pro-inflammatory and anti-inflammatory cytokines in the mucosa regulates the development and potential perpetuation of mucosal inflammation in patients with IBD. We evaluated live attenuated bacterial delivery of small interfering RNA (siRNA) against 8 IBD gene targets to achieve specificity, efficacy, and safety. Methods: An optimum pair of sequences for 8 relevant IBD epithelial cell gene targets (IL-­‐6Rα, IL-­‐7, IL-­‐13Rα1, IL-­‐18, Chitinase 3-­‐like-­‐1, Claudin-­‐2, MIP3α, and TNF-α) were selected from 169 mouse-­‐specific siRNAs and screened for efficacy at the mRNA level. Two siRNA sequences for each target were encoded into cDNA for producing a short hairpin (shRNA) in a transkingdom RNA interference (tkRNAi) bacterial delivery platform. These cDNAs were cloned into an expression vector that also encodes for invasin and listeriolysin and subsequently transformed into the appropriate carrier strain of Escherichia coli (CEQ600 strains). The in vitro efficacy was assessed by an invasion assay using the CMT-93 mouse colon epithelial cells (or RAW264.7 macrophages for TNF-α) and qRT-PCR measurement of mRNA reduction vs β-­‐actin control. Three gene targets (IL-­‐6Rα, Claudin-­‐2, and MIP3α) and two tkRNAi strains were tested in vivo using an oxazolone or dextran sulfate sodium (DSS) acute murine colitis model. The primary endpoint was to target and silence the gene of interest in the colonic mucosa (reduction in mRNA and/or protein). Secondary endpoints were improvement in the disease phenotype (body weight loss, colon length and weight, endoscopy and histologic scoring). Results: Oral delivery of IL-­‐6Rα tkRNAi strains (CEQ608/CEQ609) led to a significant reduction in colon length and abolished IL-­‐6Rα message in proximal ileum in DSS exposed groups. Claudin-­‐2 strains (CEQ621/CEQ626) caused a significant reduction in Claudin-­‐2 mRNA expression and protein levels in the colon as well as attenuation of the disease phenotype. Treatment with MIP3α therapeutic strains CEQ631/CEQ632 also resulted in a significant reduction in sum pathology scores and reduction in MIP3α mRNA expression. Conclusion: These findings suggest that tkRNAi-­‐mediated gene silencing of pro-inflammatory targets represents a potential therapeutic development avenue for IBD therapy. Larn Hwang, PhD CSO Marina Biotech 940 South Coast Drive Suite 100, Costa Mesa, CA 92626 USA lhwang@autotelicinc.com  

040

Preclinical Development of siRNA Therapeutics for Inflammatory Bowel Disease (IBD) using Transkingdom RNA Interference (tkRNAi) Bacterial Delivery Platform Larn Hwang1, Kevin Ng2, Wen Wang2, David Nam2, Joseph Ramelli1, Falguni Trieu1, Mihir Munsif1, Osmond J. D’Cruz2 1 Marina Biotech, 2Autotelic Inc. Background: The balance between pro-inflammatory and anti-inflammatory cytokines in the mucosa regulates the development and potential perpetuation of mucosal inflammation in patients with IBD. We evaluated live attenuated bacterial delivery of small interfering RNA (siRNA) against 8 IBD gene targets to achieve specificity, efficacy, and safety. Methods: An optimum pair of sequences for 8 relevant IBD epithelial cell gene targets (IL-­‐6Rα, IL-­‐7, IL-­‐13Rα1, IL-­‐18, Chitinase 3-­‐like-­‐1, Claudin-­‐2, MIP3α, and TNF-α) were selected from 169 mouse-­‐specific siRNAs and screened for efficacy at the mRNA level. Two siRNA sequences for each target were encoded into cDNA for producing a short hairpin (shRNA) in a transkingdom RNA interference (tkRNAi) bacterial delivery platform. These cDNAs were cloned into an expression vector that also encodes for invasin and listeriolysin and subsequently transformed into the appropriate carrier strain of Escherichia coli (CEQ600 strains). The in vitro efficacy was assessed by an invasion assay using the CMT-93 mouse colon epithelial cells (or RAW264.7 macrophages for TNF-α) and qRT-PCR measurement of mRNA reduction vs β-­‐actin control. Three gene targets (IL-­‐6Rα, Claudin-­‐2, and MIP3α) and two tkRNAi strains were tested in vivo using an oxazolone or dextran sulfate sodium (DSS) acute murine colitis model. The primary endpoint was to target and silence the gene of interest in the colonic mucosa (reduction in mRNA and/or protein). Secondary endpoints were improvement in the disease phenotype (body weight loss, colon length and weight, endoscopy and histologic scoring). Results: Oral delivery of IL-­‐6Rα tkRNAi strains (CEQ608/CEQ609) led to a significant reduction in colon length and abolished IL-­‐6Rα message in proximal ileum in DSS exposed groups. Claudin-­‐2 strains (CEQ621/CEQ626) caused a significant reduction in Claudin-­‐2 mRNA expression and protein levels in the colon as well as attenuation of the disease phenotype. Treatment with MIP3α therapeutic strains CEQ631/CEQ632 also resulted in a significant reduction in sum pathology scores and reduction in MIP3α mRNA expression. Conclusion: These findings suggest that tkRNAi-­‐mediated gene silencing of pro-inflammatory targets represents a potential therapeutic development avenue for IBD therapy. Larn Hwang, PhD CSO Marina Biotech 940 South Coast Drive Suite 100, Costa Mesa, CA 92626 USA lhwang@autotelicinc.com  

Dose-Exposure and Dose-Response Analysis for Trabedersen − A TGF-β2-Specific Antisense Oligonucleotide in Cancer Patients Larn Hwang1, Wen Wang1, Sanjive Qazi2, Kevin Ng2, David Nam2, and Vuong Trieu1 1Oncotelic Inc, 2Autotelic Inc Background:  Trabedersen is a phosphorothioate antisense oligodeoxynucleotide specifically inhibiting the expression of human transforming growth factor-beta2 (TGF-β2), whose overexpression is a pivotal factor for malignant progression in solid tumors. In the clinical Phase I/II study, pharmacokinetic (PK) profile, safety and efficacy of trabedersen administered intravenously were evaluated in patients with advanced tumors. Dose-exposure and dose-response analysis was performed to determine the dose of trabedersen in the future clinical studies. Methods: A total of 61 patients with pancreatic cancer (n=37), malignant melanoma (n=19), or colorectal carcinoma (n=5) were treated with trabedersen with escalating doses in 2 treatment schedules (7-day-on/7-day-off and 4-day-on/10-day-off). PK parameters, including AUC and Cmax were calculated using Phoenix WinNonlin 7.0. The correlation of dose and plasma drug exposure (AUC), dose and adverse events (AEs), and dose and overall survival (OS) were analyzed using JMP Clinical 5.0. Generalized Linear Models (GLM) were fitted to binomial response model (proportion of NCI-CTC grade 1 relative to combined grade 2,3,4 AEs) to investigate the effect of clinical parameters and treatment schedule on proportion of grade 1 AEs in cancer patients. Results: The drug exposure (AUC) of trabedersen is dose-proportional for 7-day-on/7-day-off treatment regimen, but not clear for 4-day-on/10-day-off. The 7-day-on/7-day-off treatment regimen reached maximum tolerated dose (MTD) at 160 mg/m2/day, while the 4-day-on/10-day-off treatment regimen didn’t reach MTD with dose up to 330 mg/m2/day. GLM analysis showed that the most significant effect comparing intensity of AEs for the 2 schedules was shown in gastrointestinal disorders. Total dose administered is a significant factor for the proportion of grade 1 AE (P = 0.0098); with increase of dose, the proportion of grade 1 AE reduced. Similarly, AUC is a significant factor for the proportion of grade 1 AE (p=0.0144); with increase of AUC, the proportion of grade 1 AE reduced. For pancreatic cancer patients treated with 4-day-on/10-day-off regimen, there is no clear correlation of dose with OS. Conclusion: Increase of dose and AUC of trabedersen is associated with increased toxicity, but not OS of cancer patients, therefore the lowest safe and effective dose, 140/mg/m2/day (4-day-on/10-day-off), will be used as the dosing regimen in the future clinical study. Larn Hwang, Ph.D. CSO Oncotelic Inc. 29397 Agoura Road, #107 Agoura Hills, CA 91301 USA lhwang@autotelicinc.com

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START-FAP Phase I Study of CEQ508 RNA Interference in Patients with Familial Adenomatous Polyposis (FAP)

Larn Hwang 1, Osmond D’Cruz2, Kevin Ng2, Sanjive Qazi2, Wen Wang2, David Nam2, Annie Fong2, Vuong Trieu1 Marina Biotech Inc.1, Autotelic Inc.2 Objectives: FAP is a rare inherited GI disorder that patients if left untreated would lead to early-onset of colorectal cancer. Mutations in the Adenomatous Polyposis Coli gene result in accumulation of β-catenin which contributes to formation of adenomatous polyps in the GI mucosa of FAP patients. CEQ508 is a novel therapeutic agent using live-attenuated Escherichia coli genetically engineered to produce and deliver β-catenin suppressing short-hairpin RNA into the GI mucosa. START-FAP Phase I study aimed to evaluate the safety, tolerability, and efficacy in response to CEQ508 in FAP patients. Methods: CEQ508 was produced by transKingdom RNA interference technology. For the dose escalation study, 6 patients with FAP were orally administered (3 each in Cohort 1 and 2) with CEQ508 (108 and 109 colony forming units [CFU]/day for 28 days). The primary objective was to establish general safety of orally administered CEQ508 and to determine the maximum tolerated dose (MTD). The secondary objective was the effectiveness of CEQ508 on the β-­‐catenin gene expression levels in GI tissues measured using qPCR and normalized to housekeeping genes. A mixed Nested-ANOVA model was used to evaluate β-catenin knockdown in normal GI mucosa and polyps. Results: Daily oral dosing of 108 and 109 CFU of CEQ508 for 28 days was well-tolerated. Immunohistological evaluation of polyps and normal mucosa at baseline and end-of-treatment (EOT) indicated no changes in tissue morphology or inflammation within GI tissues in Cohort 1 patients. A slight inflammation (from score of 0 to 1 at EOT) was noted in colon normal mucosa of Cohort 2 patients. Daily oral dosing of 109 CFU of CEQ508 for 28 days was well-tolerated with targeted β-catenin knockdown in polyps. Significant reduction was observed in overall β-catenin expression in polyps at EOT (P = 0.0005). Reduction was observed primarily in the duodenum (39.3% decrease, P< 0.0001) and ileum (28.8% decrease, P = 0.012). Conclusion: START-FAP study of bacterial delivery of RNAi in FAP patients demonstrated an acceptable safety profile at the two dose levels tested. Without hitting MTD, START-FAP achieved both the primary endpoint of safety and secondary endpoint of β-catenin knockdown. Larn Hwang, Ph.D. CSO Marina Biotech 940 South Coast Drive Suite100, Costa Mesa, CA 92626 USA lhwang@autotelicinc.com

042

START-FAP Phase I Study of CEQ508 RNA Interference in Patients with Familial Adenomatous Polyposis (FAP)

Larn Hwang 1, Osmond D’Cruz2, Kevin Ng2, Sanjive Qazi2, Wen Wang2, David Nam2, Annie Fong2, Vuong Trieu1 Marina Biotech Inc.1, Autotelic Inc.2 Objectives: FAP is a rare inherited GI disorder that patients if left untreated would lead to early-onset of colorectal cancer. Mutations in the Adenomatous Polyposis Coli gene result in accumulation of β-catenin which contributes to formation of adenomatous polyps in the GI mucosa of FAP patients. CEQ508 is a novel therapeutic agent using live-attenuated Escherichia coli genetically engineered to produce and deliver β-catenin suppressing short-hairpin RNA into the GI mucosa. START-FAP Phase I study aimed to evaluate the safety, tolerability, and efficacy in response to CEQ508 in FAP patients. Methods: CEQ508 was produced by transKingdom RNA interference technology. For the dose escalation study, 6 patients with FAP were orally administered (3 each in Cohort 1 and 2) with CEQ508 (108 and 109 colony forming units [CFU]/day for 28 days). The primary objective was to establish general safety of orally administered CEQ508 and to determine the maximum tolerated dose (MTD). The secondary objective was the effectiveness of CEQ508 on the β-­‐catenin gene expression levels in GI tissues measured using qPCR and normalized to housekeeping genes. A mixed Nested-ANOVA model was used to evaluate β-catenin knockdown in normal GI mucosa and polyps. Results: Daily oral dosing of 108 and 109 CFU of CEQ508 for 28 days was well-tolerated. Immunohistological evaluation of polyps and normal mucosa at baseline and end-of-treatment (EOT) indicated no changes in tissue morphology or inflammation within GI tissues in Cohort 1 patients. A slight inflammation (from score of 0 to 1 at EOT) was noted in colon normal mucosa of Cohort 2 patients. Daily oral dosing of 109 CFU of CEQ508 for 28 days was well-tolerated with targeted β-catenin knockdown in polyps. Significant reduction was observed in overall β-catenin expression in polyps at EOT (P = 0.0005). Reduction was observed primarily in the duodenum (39.3% decrease, P< 0.0001) and ileum (28.8% decrease, P = 0.012). Conclusion: START-FAP study of bacterial delivery of RNAi in FAP patients demonstrated an acceptable safety profile at the two dose levels tested. Without hitting MTD, START-FAP achieved both the primary endpoint of safety and secondary endpoint of β-catenin knockdown. Larn Hwang, Ph.D. CSO Marina Biotech 940 South Coast Drive Suite100, Costa Mesa, CA 92626 USA lhwang@autotelicinc.com

Therapeutic Targeting of the Leukaemic Fusion Gene RUNX1/ETO Hasan Issa, Helen Blair, Alessandro Dal Porto, Nidhi Jyotsana, Michael Heuser, Olaf Heidenreich The t(8,21) translocation is the most prevalent chromosomal translocation in Acute myeloid leukaemia (AML), which leads to the expression of chimaeric RUNX1/ETO oncogene protein. Previous work done by our group showed that RNAi mediated RUNX1/ETO knockdown in vitro inhibits cell proliferation, reduces clonogenicity, promotes cell cycle arrest as well as delays transplantation in immunocompromised host. Dlin-MC3-DMA lipid nanoparticles have been reported previously to have high encapsulation efficiency of siRNA, low toxicity and desirable diameter and charge for oligonucleotide delivery. We have silenced RUNX1/ETO expression by siRNA/LNP system in vitro and in vivo. To enhance RUNX1/ETO targeted siRNA we introduced several chemical modifications on the 2’-OH of the ribose and on the phosphodiester backbone. In vitro studies demonstrated that lower dose of chemically modified siRNA significantly inhibited RUNX1/ETO and provided prolonged phenotypic effect on AML cell lines in comparison with unmodified siRNA. Single treatment with modified siRNA/LNP induced irreversible inhibition of RUNX1/ETO in vitro and in t(8,21) AML primary cells. The efficacy siRNA/LNP system was also confirmed in a xenotransplantation model. Harvested human leukaemic cells from Rag2-/- ɣc-/- treated mice showed more than 40% reduction of RUNX1/ETO level. Knockdown of RUNX1/ETO in vivo led to significant and substantial downregulation of RUNX1/ETO target genes such as CCND2 and TERT. RUNX1/ETO depletion also severely impaired the clonogenic potential of the harvested leukaemic cells from siRNA/LNP treated mice and triggered senescence. Currently, we are investigating the effect of siRNA/LNP mediated RUNX1/ETO depletion on survival of immunocompromised mice. Taken together, we have demonstrated that liposomal delivery of chemically modified RUNX1/ETO siRNA impairs RUNX1/ETO-driven transcriptional networks and therewith associated leukaemic self-renewal function, which may have a therapeutic potential. Hasan Issa, MSc Wolfson Childhood Cancer Research Centre Northern Institute for Cancer Research Herschel Building, Level 6 Newcastle University Brewery Lane Newcastle upon Tyne NE1 7RU United Kingdom H.issa1@newcastle.ca.uk +44 - 7451984204

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REVERSIR™ Platform for the Tailored Control of GalNAc-siRNA Conjugate Pharmacology Vasant Jadhav Alnylam Pharmaceuticals, Cambridge, MA 02142, USA

One of the hallmarks of the 3′-tri-N-acetylgalactosamine (GalNAc) short interfering RNA (siRNA) conjugates currently in investigational clinical studies is their extended duration of activity. To enhance their therapeutic profile, we describe an approach that offers the ability to control GalNAc-siRNA duration of silencing. By developing REVERSIR, a platform utilizing a GalNAc-conjugated single-stranded high affinity oligonucleotide complementary to the siRNA guide strand, we achieved effective reversal of siRNA activity. A single dose of REVERSIR (lower than the GalNAc-siRNA conjugate) with short length and dispersed locked nucleic acid (LNA) content allowed for full reversal of target gene silencing in vivo within four days. We will present design insights including length and optimal positioning of LNA that may be applicable for other oligonucleotide therapeutics, such as anti-miRs. While many of the ESC GalNAc-siRNA conjugates in our pipeline are being developed for chronic indications where infrequent dosing is desired, we believe that the ability to achieve tailored pharmacology is a highly desirable attribute for all precision medicines.

Vasant Jadhav, PhD Senior Director, Research Alnylam Pharmaceuticals 300 Third Street Cambridge, MA 02142 USA Email: vjadhav@alnylam.com

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Dose-response time modelling of circulating biomarkers for nucleotides Rasmus Jansson Löfmark, Peter Gennemark Drug Metabolism and Pharmacokinetics, Cardiovascular and Metabolic Diseases IMED Biotech Unit, AstraZeneca R&D, 431 83 Gothenburg, Sweden Oligonucleotides and small interfering RNAs (siRNAs) have a distinct plasma pharmacokinetic pattern compared to small molecule drugs with a clear distribution phase followed by a long terminal half-life. For nucleotides the terminal half-life reflects the half-life in tissue, which usually corresponds to the site of action. It may be challenging to estimate the terminal half-life of nucleotide drugs since the plasma concentration levels are relatively low, in particular for novel potent chemistries. However, there is often a circulating protein for the encoded mRNA which can be continuously measured throughout early clinical trials and which directly reflects drug target engagement. There are mathematical methods to estimate the drug biophase half-life based on biomarker data, but to our knowledge, such methodologies are not routinely applied in the nucleotide area. The objective of this work was to demonstrate the use of dose-response time modelling to predict the pharmacokinetics and pharmacodynamics of oligonucleotides. Clinical public data consisting of multiple dose-levels and target engagement biomarker measured over time were collated and digitized. A total of nine clinical drugs were used in this assessment (IONIS ANGPTL3, Apo(a), Apo(a)-LICA, ApoB (Mipomersen), APOCIII, TTRRx, FXIRx, and Alnylam Inclisiran, Alnylam PCSK9 iv). A mathematical dose-response model was fitted to each dataset. The model consisted of an apparent pharmacokinetic part with an uptake rate to, and an elimination rate from, the drug biophase. The targeted protein was represented by an indirect response model where the drug inhibited the production rate via a Hill function of the protein which was assumed to be cleared by a first order elimination rate. If clinical pharmacokinetic data were available these data were also compiled and the terminal plasma pharmacokinetic half-life was compared to the estimated half-life from the pharmacodynamic dose-response time data. For all nine drugs, we could estimate the biophase kinetic half-life with acceptable accuracy. Besides, the effective drug biophase half-lifes were closely aligned to the reported pharmacokinetic terminal half-lives, when those data were available. Finally, the apparent drug potencies were adequately estimated in all cases, and the turnover rate was relatively consistent cross drugs. We conclude that dose-response time modelling for nucleotides can be effectively used to assess the drug kinetic biophase half-life when temporal target-engagement biomarker data are available. Such modelling is also valuable to predict subsequent clinical trials offering potential for efficient clinical trial design. Rasmus Jansson Löfmark, PhD Team Leader PKPD and Modelling & Simulation AstraZeneca Pepparedsleden 1, SE-431 83 Mölndal Sweden Rasmus.Jansson.Lofmark@astrazeneca.com

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Single stranded nucleic acids regulate endocytic uptake and downstream Toll-like receptor signalling Peter Järver1, Aleksandra Dondalska1, Annette Sköld1, AnnSofi Sandberg, Eman Zaghloul, David Brodin, Candice Poux1, Janne Lehtiö, Kim Lennox3, Mark Behlke3, Roger LeGrand, Samir EL-Andaloussi2,4, Anna-Lena Spetz1 1 Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Sweden 2 Department of Laboratory Medicine, Karolinska Institute, Huddinge, Sweden 3 Integrated DNA Technologies Inc, Coralville, Iowa, USA 4 Department of Physiology, Anatomy and Genetics, University of Oxford, UK Endocytic pathways need to be tightly regulated in order to maintain fundamental cellular functions and control uptake of molecules destined for endosomes. However, there is a lack of knowledge about extracellular molecules that regulate endocytic uptake. In the present study we show that single stranded oligonucleotides (ssON) inhibit certain endocytic pathways in primary human monocyte derived cells (moDC), while leaving other pathways unaffected. Both single stranded DNA and RNA conferred the endocytic inhibition, it was concentration dependent, and required certain ssON length. The ssON-mediated inhibition modulated cell signalling downstream of pattern recognition receptors that localize within the affected endosomal pathways both in vitro and in non-human primates. Introduction of non-natural linkages and/or modified nucleosides was not essential for inhibition, but was tolerated and might be necessary in order to retain the effect over longer periods of time. These studies reveal a regulatory role for extracellular ssONs in the endocytic uptake of Toll-like receptor ligands and further provide a mechanistic explanation of immunomodulation by nucleic acids. Peter Järver, PhD E-mail: peter.jarver@su.se Stockholm University Department of Molecular Biosciences, The Wenner-Gren Institute SE-106 91 Stockholm

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Single stranded nucleic acids regulate endocytic uptake and downstream Toll-like receptor signalling Peter Järver1, Aleksandra Dondalska1, Annette Sköld1, AnnSofi Sandberg, Eman Zaghloul, David Brodin, Candice Poux1, Janne Lehtiö, Kim Lennox3, Mark Behlke3, Roger LeGrand, Samir EL-Andaloussi2,4, Anna-Lena Spetz1 1 Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Sweden 2 Department of Laboratory Medicine, Karolinska Institute, Huddinge, Sweden 3 Integrated DNA Technologies Inc, Coralville, Iowa, USA 4 Department of Physiology, Anatomy and Genetics, University of Oxford, UK Endocytic pathways need to be tightly regulated in order to maintain fundamental cellular functions and control uptake of molecules destined for endosomes. However, there is a lack of knowledge about extracellular molecules that regulate endocytic uptake. In the present study we show that single stranded oligonucleotides (ssON) inhibit certain endocytic pathways in primary human monocyte derived cells (moDC), while leaving other pathways unaffected. Both single stranded DNA and RNA conferred the endocytic inhibition, it was concentration dependent, and required certain ssON length. The ssON-mediated inhibition modulated cell signalling downstream of pattern recognition receptors that localize within the affected endosomal pathways both in vitro and in non-human primates. Introduction of non-natural linkages and/or modified nucleosides was not essential for inhibition, but was tolerated and might be necessary in order to retain the effect over longer periods of time. These studies reveal a regulatory role for extracellular ssONs in the endocytic uptake of Toll-like receptor ligands and further provide a mechanistic explanation of immunomodulation by nucleic acids. Peter Järver, PhD E-mail: peter.jarver@su.se Stockholm University Department of Molecular Biosciences, The Wenner-Gren Institute SE-106 91 Stockholm

Targeting hIDO1 with 3rd generation antisense oligonucleotides for modulation of the tumor microenvironment Frank Jaschinski1, Richard Klar1, Sandra M. Kallert2, Tamara Hilmenyuk1, Monika Schell1, Lisa Hinterwimmer1, Marina Van Ark2, Alfred Zippelius2 1Secarna pharmaceuticals GmbH & Co. KG; 2Department of Biomedicine, University Hospital and University of Basel, Switzerland Targeting the immunosuppressive microenvironment of tumors has emerged as a promising treatment option for oncologic indications in the last years. However, despite long lasting remissions in a small subset of tumor patients, the majority of patients does not respond to the currently available immunotherapies. One possible reason is the fact that tumor cells and suppressive immune cells can express a plethora of immunosuppressive factors like indoleamin-2,3-dioxygenase 1 (IDO1) leading to immune evasion of the tumor. The degradation of tryptophan in the tumor microenvironment which is catalyzed by IDO1 leads on the one hand to tryptophan starvation and on the other hand to the generation of kynurenines both resulting in a suppression of immune effector cells. We designed antisense oligonucleotides (ASOs) as locked-nucleic-acid (LNA) GapmeRs with specificity for human IDO1 (hIDO1) and murine IDO1 (mIDO1). We tested knockdown efficacy in vitro by unformulated ASOs in cancer cell lines and primary immune cells. We identified a subset of IDO1-ASOs that resulted in an IDO1 mRNA knockdown of >90% with IC50 values in the low nanomolar range in cancer cell lines. The treatment of cancer cell lines as well as primary human cells resulted in reduction of IDO protein levels by >85%. Importantly, we observed a complete block in the production of immunosuppressive L-Kynurenine in ASO treated cells and a strongly increased proliferation of T cells when hIDO1 was knocked down in cocultured tumor cells. Preliminary in vivo experiments suggest that treatment of tumor bearing mice with unformulated mIDO1-ASOs results in the knockdown of mIDO1 in tumor cells as well as tumor infiltrating myeloid cells. In conclusion, we selected IDO1-ASOs that potently reduce the immunosuppressive capacity of IDO-expressing cells. Potent mouse specific IDO1 ASOs have been identified and will be used for in vivo efficacy studies in tumor-bearing mice. Taken together, we developed an innovative immunotherapeutic tool to block the expression of IDO1 that will potentially improve treatment options for cancer patients in the future. Frank Jaschinski, (PhD) CSO Secarna Pharmaceuticals GmbH & Co. KG Am Klopferspitz 19 82152 Planegg/Martinsried Germany frank.jaschinski@secarna.com

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Inhibition of immune suppressive CD39 and CD73 by 3rd generation antisense oligonucleotides for treatment of cancer Frank Jaschinski1, Tamara Hilmenyuk1, Sandra M. Kallert2, Richard Klar1, Monika Schell1, Lisa Hinterwimmer1, Marina Van Ark2, Alfred Zippelius2 1Secarna Pharmaceuticals GmbH & Co. KG, IZB, Planegg/Martinsried, Germany, 2University Hospital and University of Basel, Department of Biomedicine, Basel, Switzerland During the last decades it became obvious that the immune system can be utilized to evoke effective anti-tumor responses. However, cancer cells develop mechanisms to circumvent this. The two ectonucleotidases CD39 and CD73 are promising drug targets, as they act in concert to convert extracellular immune-stimulating ATP to immunosuppressive adenosine. CD39 and CD73 are expressed on different immune cells as well as on a range of cancer cells and the latter are recognized to co-opt the two ectonucleotidases for circumventing anti-tumor immune responses. Accordingly, in order to enhance immunity against tumors, it would be favorable to increase extracellular ATP- and to simultaneously reduce adenosine concentrations in the tumor microenvironment. Therefore, as therapeutic strategy we designed Antisense Oligonucleotides (ASOs) to target CD39 and CD73 expression in tumor- and immune cells. The application of CD39- or CD73-specific ASOs led to a successful target knockdown on mRNA and protein level in different cancer cell lines and in primary human CD4+ and CD8+

T cells without the need of any transfection reagent. Furthermore, degradation of extracellular ATP was significantly blocked by CD39-specific ASOs, while accumulation of adenosine was suppressed by CD73-ASOs. Supplementation of cell culture medium with ATP or AMP significantly impaired proliferation and viability of CD39- or CD73- expressing primary human T cells, presumably due to formation of degradation products such as adenosine. Strikingly, these effects were reversed by treatment with CD39- or CD73-specific ASOs. In vivo, target downregulation in different tissues was seen after treatment of mice with murine-specific CD39 or CD73 ASOs. Moreover, CD39-ASO treatment of tumor-bearing mice significantly reduced CD39 expression on tumor infiltrating macrophages and Tregs and resulted in diminished intratumoral Treg levels. The impact of CD39- and CD73 ASOs on immune-mediated anti-tumor activity in vivo is currently being investigated. Altogether, our data reveal that targeting CD39 and CD73 by ASOs represents a promising state-of-the art approach to improve anti-tumor immune responses and has great potential to ameliorate current established immunotherapies for the treatment of cancers. Frank Jaschinski, (PhD) CSO Secarna Pharmaceuticals GmbH & Co. KG Am Klopferspitz 19 82152 Planegg/Martinsried Germany frank.jaschinski@secarna.com

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Inhibition of immune suppressive CD39 and CD73 by 3rd generation antisense oligonucleotides for treatment of cancer Frank Jaschinski1, Tamara Hilmenyuk1, Sandra M. Kallert2, Richard Klar1, Monika Schell1, Lisa Hinterwimmer1, Marina Van Ark2, Alfred Zippelius2 1Secarna Pharmaceuticals GmbH & Co. KG, IZB, Planegg/Martinsried, Germany, 2University Hospital and University of Basel, Department of Biomedicine, Basel, Switzerland During the last decades it became obvious that the immune system can be utilized to evoke effective anti-tumor responses. However, cancer cells develop mechanisms to circumvent this. The two ectonucleotidases CD39 and CD73 are promising drug targets, as they act in concert to convert extracellular immune-stimulating ATP to immunosuppressive adenosine. CD39 and CD73 are expressed on different immune cells as well as on a range of cancer cells and the latter are recognized to co-opt the two ectonucleotidases for circumventing anti-tumor immune responses. Accordingly, in order to enhance immunity against tumors, it would be favorable to increase extracellular ATP- and to simultaneously reduce adenosine concentrations in the tumor microenvironment. Therefore, as therapeutic strategy we designed Antisense Oligonucleotides (ASOs) to target CD39 and CD73 expression in tumor- and immune cells. The application of CD39- or CD73-specific ASOs led to a successful target knockdown on mRNA and protein level in different cancer cell lines and in primary human CD4+ and CD8+

T cells without the need of any transfection reagent. Furthermore, degradation of extracellular ATP was significantly blocked by CD39-specific ASOs, while accumulation of adenosine was suppressed by CD73-ASOs. Supplementation of cell culture medium with ATP or AMP significantly impaired proliferation and viability of CD39- or CD73- expressing primary human T cells, presumably due to formation of degradation products such as adenosine. Strikingly, these effects were reversed by treatment with CD39- or CD73-specific ASOs. In vivo, target downregulation in different tissues was seen after treatment of mice with murine-specific CD39 or CD73 ASOs. Moreover, CD39-ASO treatment of tumor-bearing mice significantly reduced CD39 expression on tumor infiltrating macrophages and Tregs and resulted in diminished intratumoral Treg levels. The impact of CD39- and CD73 ASOs on immune-mediated anti-tumor activity in vivo is currently being investigated. Altogether, our data reveal that targeting CD39 and CD73 by ASOs represents a promising state-of-the art approach to improve anti-tumor immune responses and has great potential to ameliorate current established immunotherapies for the treatment of cancers. Frank Jaschinski, (PhD) CSO Secarna Pharmaceuticals GmbH & Co. KG Am Klopferspitz 19 82152 Planegg/Martinsried Germany frank.jaschinski@secarna.com

Characterization of TLR7/8-induced skin responses using a standardized dermatological toolbox S. Jirka1, T. van der Kolk1, G. Feiss2, M.B.A. van Doorn3, E. P. Prens3, , M.Moerland1, J. Burggraaf1, R. Rissmann1

1Centre for Human Drug Research, Leiden, the Netherlands, 2Cutanea Life Science, Wayne, Pennsylvania, USA, 3Department of Dermatology Erasmus Medical Centre, Rotterdam, the Netherlands

When administered subcutaneously, therapeutic oligonucleotides cause a specific local reaction originating around the injection site, usually characterized by erythema, itching, induration, discomfort, pain and sometimes also more severe manifestations such as ulceration or necrosis. These injection site reactions (ISRs) are common, but rather poorly and only seldom described in literature ( van Meer et al., Br J Clin Pharmacol. 2016). We aimed to develop a standardized set of dermatological assessments allowing systematic characterization of such responses in human subjects.

We conducted a randomized, open-label, vehicle-controlled, parallel-cohort, dose ranging study in 16 healthy male subjects using imiquimod (IMQ), a known TLR 7/8 agonist causing skin inflammation upon topical administration as a trigger. IMQ (5 mg) was administered topically once daily for 3 consecutive days under occlusion to the upper back by a 12 mm Finn Chamber. Subjects were randomized 1:1 to receive i) IMQ over a fully competent skin barrier or ii) IMQ over a perturbed barrier by tape stripping of the skin. Dermal assessments included visual erythema grading, erythema by colorimetry, erythema by 2D photography analysis, perfusion by laser speckle contrast imaging, skin surface biomarkers by transdermal analysis patch and skin biopsy biomarkers (mRNA expression, histology and immunohistochemistry). IMQ showed a dose-dependently induced skin inflammation in terms of erythema and perfusion, when applied on tape stripped skin (p<0.05 versus negative control, i.e. cetomacrogol cream). The application of IMQ resulted in dose-dependent elevations in CXCL10, hBD-2, ICAM-1 and MX-A in the biopsies.

To conclude we compiled a set of dermal assessments to systematically and objectively quantify the response induced by topical administration of IMQ . Since oligonucleotides may activate TLR 7/8 signaling, this standardized dermatological toolbox may be advantageous in better documentation of oligonucleotide-induced skin responses in human subjects, and as such increase the etiology of such responses. Silvana Jirka, PhD Scientist Centre for Human Drug Research Zernikedreef 8 2333 CL, Leiden the Netherlands SJirka@CHDR.nl

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RealSeq™, a novel method that greatly reduces sequence bias in construction of small-RNA sequencing libraries Brian H. Johnston, Ryan E. Hogans, Jenny M. Vo, Sergei A. Kazakov, Sergio Barberan-Soler SomaGenics, Inc., 2161 Delaware Ave., Santa Cruz, CA 95060, USA Accurate expression profiling of all microRNAs (miRNA) is important for understanding miRNA biology and for development of new biomarkers and therapeutic targets. Small RNA-Seq is currently the most comprehensive approach for discovery and expression profiling of small RNAs. However, current methods for small RNA-Seq underestimate the abundance of most miRNAs in a sample, some by as much as 10,000-fold. Most of this misrepresentation results from bias in the enzymatic ligation of adapters to the ends of the miRNA during preparation of sequencing libraries. To address this problem, we have developed a new approach, called RealSeq™-AC, for preparing small-RNA sequencing libraries. By using a novel single adapter design and altered enzymatic steps, the method greatly reduces library preparation bias. In sequencing a reference pool containing equimolar concentrations of 962 synthetic miRNAs, we found that the percentage of miRNAs that appeared in their correct proportions (within 2-fold) in Illumina sequencing was 77% for RealSeq™-AC, compared with only 28.4% for the widely-used NEBNext® Small RNA library preparation kit (NEB). This reduction in bias allows for accurate quantification of small RNAs and, by being more sensitive to the presence of otherwise poorly-detected or undetected miRNAs, enables the discovery of novel miRNAs, the sequencing of lower RNA inputs and more samples in parallel, and a reduction in cost. Profiling of miRNAs from a reference human brain RNA sample by Illumina sequencing also showed that, among the tested small RNA library preparation kits, the relative quantification provided by RealSeq™-AC provided the best correlation with RT-qPCR. RealSeq-AC is likely to be applicable to a wide range of RNA sizes, including the fragmented large RNAs used in standard RNA-Seq as well as small RNAs. RealSeq has also been adapted to targeted RNA sequencing (RealSeq™-T), which among other applications is useful in following the effects of anti-miR treatment. Brian Johnston, Ph.D. President and CEO SomaGenics, Inc. 2161 Delaware Avenue Santa Cruz, CA 95060 USA bjohnston@somagenics.com Tel: 1 (831) 426-7700 ext. 12

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RealSeq™, a novel method that greatly reduces sequence bias in construction of small-RNA sequencing libraries Brian H. Johnston, Ryan E. Hogans, Jenny M. Vo, Sergei A. Kazakov, Sergio Barberan-Soler SomaGenics, Inc., 2161 Delaware Ave., Santa Cruz, CA 95060, USA Accurate expression profiling of all microRNAs (miRNA) is important for understanding miRNA biology and for development of new biomarkers and therapeutic targets. Small RNA-Seq is currently the most comprehensive approach for discovery and expression profiling of small RNAs. However, current methods for small RNA-Seq underestimate the abundance of most miRNAs in a sample, some by as much as 10,000-fold. Most of this misrepresentation results from bias in the enzymatic ligation of adapters to the ends of the miRNA during preparation of sequencing libraries. To address this problem, we have developed a new approach, called RealSeq™-AC, for preparing small-RNA sequencing libraries. By using a novel single adapter design and altered enzymatic steps, the method greatly reduces library preparation bias. In sequencing a reference pool containing equimolar concentrations of 962 synthetic miRNAs, we found that the percentage of miRNAs that appeared in their correct proportions (within 2-fold) in Illumina sequencing was 77% for RealSeq™-AC, compared with only 28.4% for the widely-used NEBNext® Small RNA library preparation kit (NEB). This reduction in bias allows for accurate quantification of small RNAs and, by being more sensitive to the presence of otherwise poorly-detected or undetected miRNAs, enables the discovery of novel miRNAs, the sequencing of lower RNA inputs and more samples in parallel, and a reduction in cost. Profiling of miRNAs from a reference human brain RNA sample by Illumina sequencing also showed that, among the tested small RNA library preparation kits, the relative quantification provided by RealSeq™-AC provided the best correlation with RT-qPCR. RealSeq-AC is likely to be applicable to a wide range of RNA sizes, including the fragmented large RNAs used in standard RNA-Seq as well as small RNAs. RealSeq has also been adapted to targeted RNA sequencing (RealSeq™-T), which among other applications is useful in following the effects of anti-miR treatment. Brian Johnston, Ph.D. President and CEO SomaGenics, Inc. 2161 Delaware Avenue Santa Cruz, CA 95060 USA bjohnston@somagenics.com Tel: 1 (831) 426-7700 ext. 12

Molecular mechanisms of antiproliferative and interferon-inducing activity of immunostimulating dsRNA Tatyana Kabilova, Mihail Zhorov, Aleksandra Sen’kova, Valentin Vlassov, Marina Zenkova, Elena Chernolovskaya Institute of Chemical Biology and Fundamental Medicine SB RAS

Small interfering RNAs, depending on their structure, sequence, and mode of delivery, can stimulate innate and adaptive immunity. Recently, we designed a series of immunostimulating 19-bp RNA duplexes with 3-nucleotid overhangs in the 3’-ends (here and after isRNA) that possess pronounced antiproliferative, antitumor, and antimetastatic activities. isRNA under study did not have substantial homology to any human or mouse mRNAs. The aim of this study was to evaluate the mechanisms of antiproliferative activity of selected isRNA in human carcinoma KB-3-1 cells and its immunostimulating activity in human PBMC. For the delivery of isRNA we used cationic liposomes 2X3-DOPE consisting of the recently created polycationic lipid 2X3 (1,26-Bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride) and the lipid-helper DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), which enhance interferon-inducing activity of isRNA in comparison with Lipofectamine2000.

We isolated the individual types of immune cells (NK, T cells, B cells, dendritic cells, monocytes/macrophages) by immunomagnetic sorption to identify the effector cells of isRNA-mediated immunostimulation. We showed that dendritic cells, monocytes and B cells produce IFN-α in response to the isRNA, while other cell types do not secrete IFN-α. Thus, the effector cells, which mediate immunostimulatory activity of isRNA, are antigen presenting cells of innate and adaptive immunity. We analysed changes in gene expression in response to isRNA in PBMC, using PCR Array, which covered 84 genes related to innate and adaptive immune response. We identified 26 up-regulated genes, and the highest up-regulation was observed for IFNα, IFNβ and IP10 genes. Based on these results we selected regulators, which could be involved in isRNA signalling, namely genes of cellular sensors (TLR3, TLR7/8, NOD2, RIG-I, MDA5, PKR) and master regulators (IRF3, IRF7), and used them as the targets for shRNAs. The results show that selective silencing of RIG-I, MDA5 and PKR in KB-3-1 cells blocks antiproliferative effect of isRNA. The involvement of PKR-mediated pathway in intracellular signaling activated by isRNA was confirmed by the data obtained using specific inhibitor of PKR 2-aminopurine.

Thus, we identified cellular sensors, which responsible for antiproliferative activity of isRNA in cancer cells and immunostimulating activity in PBMC. This work was supported by the Russian Scientific Foundation (grant No 16-15-10105)

Tatyana O. Kabilova, PhD Institute of Chemical biology and Fundamental Medicine SB RAS 8, Lavrentiev ave, Novosibirsk, 630090, Russia kabilova@niboch.nsc.ru

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Nose-to-brain siRNA delivery combined with cell-penetrating peptide modified polymer micelles Takanori Kanazawa1,2, Kazuki Morisaki2, Shohei Suzuki2, Fuminari Akiyama2, Shino Kakizaki2, Yuuki Takashima2, Toyofumi Suzuki1,Yasuo Seta2 School of Pharmacy, Nihon University1, Tokyo University of Pharmacy and Life Sciences2 The potential for RNA-based agents to serve as effective therapeutics for central nerve systems (CNS) disorders has been successfully demonstrated in vitro. However, the blood-brain barrier limits the distribution of systemically administered therapeutics to the CNS, posing a major challenge for drug development aimed at combatting CNS disorders. Therefore, the development of effective strategies to enhance siRNA delivery to the brain is of great interest in clinical and pharmaceutical fields. In this study, to improve the efficiency of small interfering RNA (siRNA) delivery to the brain, we developed a nose-to-brain delivery system combined with cell-penetrating peptide (CPP) modified nano-micelles comprising polyethylene glycol-polycaprolactone (PEG-PCL) copolymers conjugated with the CPP, Tat (MPEG-PCL-Tat). We first demonstrated that the intranasal delivery with MPEG-PCL-Tat improved siRNA delivery to the brain compared to either intravenous delivery of siRNA with MPEG-PCL-Tat or intranasal delivery of siRNA without MPEG-PCL-Tat. We also studied the intranasal transfer of MPEG-PCL-Tat to the brain via the olfactory, trigeminal nerves, and cerebrospinal fluid (CSF), which are main pathways of nose-to-brain. As a results, we found that MPEG-PCL-Tat accelerated transport along the olfactory, trigeminal nerve, and CSF pathway because of its high permeation across the nasal mucosa. In addition, we determined the therapeutic effects in cerebral ischemia-reperfusion injuries in t-MCAO model rats by siRNA targeted for TNF-α (siTNF-α) with combination of nose-to-brain delivery and MPEG-PCL-Tat. As a result, the nose-to-delivery of siTNF-α with MPEG-PCL-Tat micelles suppressed the infarction area, TNF-α production, and neurological score, demonstrating the efficacy of this treatment approach for ischemia-reperfusion injuries in the brain. Furthermore, We applied a nose-to-brain delivery system with MPEG-PCL-Tat micelles to investigate its therapeutic effects on a rat model of malignant glioma using siRNA with a Raf-1 (siRaf-1)/camptothecin (CPT) co-delivery system. As a results, siRaf-1/ CPT co-loaded MPEG-PCL-Tat micelles showed a marked prolongation of the mean survival period. These results indicate that nose-to-brain delivery with MPEG-PCL-Tat micelles is an excellent therapeutic approach by nucleic acid medicine for brain disorders. Takanori Kanazawa, PhD Senior Lecturer School of Pharmacy, Nihon University 7-7-1 Narashinodai, Funabashi Chiba Japan kanazawa.takanori02@nihon-u.ac.jp +81-47-465-6587

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Nose-to-brain siRNA delivery combined with cell-penetrating peptide modified polymer micelles Takanori Kanazawa1,2, Kazuki Morisaki2, Shohei Suzuki2, Fuminari Akiyama2, Shino Kakizaki2, Yuuki Takashima2, Toyofumi Suzuki1,Yasuo Seta2 School of Pharmacy, Nihon University1, Tokyo University of Pharmacy and Life Sciences2 The potential for RNA-based agents to serve as effective therapeutics for central nerve systems (CNS) disorders has been successfully demonstrated in vitro. However, the blood-brain barrier limits the distribution of systemically administered therapeutics to the CNS, posing a major challenge for drug development aimed at combatting CNS disorders. Therefore, the development of effective strategies to enhance siRNA delivery to the brain is of great interest in clinical and pharmaceutical fields. In this study, to improve the efficiency of small interfering RNA (siRNA) delivery to the brain, we developed a nose-to-brain delivery system combined with cell-penetrating peptide (CPP) modified nano-micelles comprising polyethylene glycol-polycaprolactone (PEG-PCL) copolymers conjugated with the CPP, Tat (MPEG-PCL-Tat). We first demonstrated that the intranasal delivery with MPEG-PCL-Tat improved siRNA delivery to the brain compared to either intravenous delivery of siRNA with MPEG-PCL-Tat or intranasal delivery of siRNA without MPEG-PCL-Tat. We also studied the intranasal transfer of MPEG-PCL-Tat to the brain via the olfactory, trigeminal nerves, and cerebrospinal fluid (CSF), which are main pathways of nose-to-brain. As a results, we found that MPEG-PCL-Tat accelerated transport along the olfactory, trigeminal nerve, and CSF pathway because of its high permeation across the nasal mucosa. In addition, we determined the therapeutic effects in cerebral ischemia-reperfusion injuries in t-MCAO model rats by siRNA targeted for TNF-α (siTNF-α) with combination of nose-to-brain delivery and MPEG-PCL-Tat. As a result, the nose-to-delivery of siTNF-α with MPEG-PCL-Tat micelles suppressed the infarction area, TNF-α production, and neurological score, demonstrating the efficacy of this treatment approach for ischemia-reperfusion injuries in the brain. Furthermore, We applied a nose-to-brain delivery system with MPEG-PCL-Tat micelles to investigate its therapeutic effects on a rat model of malignant glioma using siRNA with a Raf-1 (siRaf-1)/camptothecin (CPT) co-delivery system. As a results, siRaf-1/ CPT co-loaded MPEG-PCL-Tat micelles showed a marked prolongation of the mean survival period. These results indicate that nose-to-brain delivery with MPEG-PCL-Tat micelles is an excellent therapeutic approach by nucleic acid medicine for brain disorders. Takanori Kanazawa, PhD Senior Lecturer School of Pharmacy, Nihon University 7-7-1 Narashinodai, Funabashi Chiba Japan kanazawa.takanori02@nihon-u.ac.jp +81-47-465-6587

Evaluation of antisense activity under the conditions of CEM method focused on base composition and Tm value Yuuya Kasahara1,2, Harumi Yamaguma1, Futaba Makimura1, Satoshi Obika1, 2 National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN)1 Graduate School of Pharmaceutical Science, Osaka University2 For a design of antisense oligonucleotide (ASO), the selection of the target area of mRNA along with the physical properties of ASO are of great importance. The target mRNA has sometimes several thousands nucleotide bases and is much longer than ASO. In addition, mRNA forms a higher ordered structure. It is easy to imagine that ASO is more accessible to the loop region than the stem region of the target mRNA. Therefore, selection of the target area is very important to obtain highly active ASO to knock down the target mRNA efficiently. Similarly, it is necessary to pay attention to the physical properties of ASO (e.g., base composition (GC%), Tm value, length, intramolecular interaction and dimer formation) for efficient ASO acquisition. We carefully considered the above points and designed gapmer-type ASOs containing 2’,4’-BNA/LNA in “wing region” for various target mRNAs and evaluated the knockdown efficiency of the designed ASOs in vitro. The Ca2+ enrichment of medium (CEM) method was used for the transfection step of ASO. Based on the obtained results, we examined the correlation between the knockdown efficiency and the physical properties of ASO. Here, we will report the effect of base composition and Tm value of the ASO on the knockdown efficiency under the conditions of CEM method. Yuuya Kasahara Sub-group Leader National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN) 7-6-8 Saito Asagi Ibaraki, Osaka Japan y-kasahara@nibiohn.go.jp

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Preparation of dsRNAs library possessing biomolecule-ligands and their properties Y. Kitade [a,b] *, N. Miyamoto[a], R. Nakashima[a], Mi. Kawade[b], Mo. Kawade[b], T. Takahashi[b], T. Sobajima[b], M. S. Higashi[b], A. Shibata[b], Y. Kitamura[b],, Y. Akao[b] [a] Aichi Institute of Technology, Toyota 470-0392, Japan and [b] Gifu University, Gifu 501-1193, Japan Small interfering RNA (siRNA) and microRNA (miRNA) are short double-stranded RNAs containing a 2-nucleotide overhand at the 3’-end (dangling end) of each strand. One of these strands is loaded onto the RNA-induced silencing complex (RISC). Argonaute 2 (Ago2), catalytic component of RISC, plays a central role in this gene silencing process. The PAZ domain of Ago2 specially recognizes the antisense (guide) strand of dsRNA through binding to the dangling end. Recently, we designed and synthesized chemically modified siRNAs and miRNAs possessing aromatic residues instead of the naturally occurring dinucleotide at the dangling end. We prepared 5-ethynyl-1,3-benzenedimethanol (BE) previously described as a versatile probe for labeling of oligonucleotides through a click reaction.

In this paper, RNA molecules possessing various biomolecule-ligands such as various sugars and aminosugars were prepared via the click reaction. We prepared the dsRNA consisting with modified sense strands possessing ligands and antisense strands and the delivery property of these dsRNAs, such as siRNAs and miRNAs, into the target cells was also evaluated. Yukio Kitade, PhD Professor Aichi Institute of Technology Yachigusa 1247, Yakusa, Toyota 470-0392 Japan ykkitade@aitech.ac.jp

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Investigational RNAi Mediated Oxalate Reduction Therapy John Knight1, Abigail Liebow2, Kyle Wood1, Sonia Fargue1, W. Todd Lowther3, David Erbe2, Ross Holmes1 1University of Alabama at Birmingham, Birmingham AL 2Alnylam Pharmaceuticals ,Cambridge MA 3Wake Forest School of Medicine, Wake Forest NC Primary Hyperoxaluria Type 1 is an autosomal recessive disorder of glyoxylate metabolism from loss of function of alanine-glyoxylate aminotransferase in hepatocyte peroxisomes of affected individuals, resulting in profound oxalate overproduction. End stage renal disease is common at an early age with necessary treatment via dual liver-kidney transplantation. siRNA-GalNAc conjugate technology has provided a way to specifically target this defective pathway in hepatocyte peroxisomes by silencing the mRNA for the upstream enzyme, glycolate oxidase, and starving the substrate for oxalate production. Evaluation of such an approach is ongoing in an early stage clinical trial in patients with the investigational RNAi therapeutic ALN-GO1. To expand the potential application of substrate reduction therapy to other diseases of oxalate excess, the current study targeted the downstream enzyme in oxalate synthesis – lactate dehydrogenase (LDH). LDH was shown as early as 1973 to be the key enzyme in oxidizing glyoxylate to oxalate in human livers. Thus, its inhibition could have the potential to treat the full array of patients with primary hyperoxaluria (types, 1, 2, 3, and idiopathic) and even common stone forming diseases. Of course, LDH and its metabolites are also well established regulators of energetic flux in liver cells (e.g., Cori Cylcle, Krebs Cycle) and the safety of its inhibition must be explored carefully to understand any impact on hepatic and whole body metabolism. In mice, liver-specific knockdown of LDH did result in profound oxalate lowering in both healthy and diseased animals. However, additional findings contradicted expectations - instead of the predicted increase in glycolate levels that was anticipated, treated mice showed profound whole-body decreases in glycolate levels – in their livers, plasma, and urine. Additionally, substantial changes in lactate and pyruvate levels were seen in the livers of treated animals, consistent with the known role of LDH in their conversion. Thus, the importance of these and other potential changes will need to be investigated fully, including in chronic toxicology studies, to help define the potential of LDH-based therapy in hyperoxalurias while ensuring the safety of this approach. John Knight, PhD Assistant Professor University of Alabama at Birmingham Dept of Urology, Kaul Building, Room 816B 720 20th Street S, Birmingham, AL 35294 U.S.A. knight74@uab.edu (205) 996-2295

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Investigational RNAi Mediated Oxalate Reduction Therapy John Knight1, Abigail Liebow2, Kyle Wood1, Sonia Fargue1, W. Todd Lowther3, David Erbe2, Ross Holmes1 1University of Alabama at Birmingham, Birmingham AL 2Alnylam Pharmaceuticals ,Cambridge MA 3Wake Forest School of Medicine, Wake Forest NC Primary Hyperoxaluria Type 1 is an autosomal recessive disorder of glyoxylate metabolism from loss of function of alanine-glyoxylate aminotransferase in hepatocyte peroxisomes of affected individuals, resulting in profound oxalate overproduction. End stage renal disease is common at an early age with necessary treatment via dual liver-kidney transplantation. siRNA-GalNAc conjugate technology has provided a way to specifically target this defective pathway in hepatocyte peroxisomes by silencing the mRNA for the upstream enzyme, glycolate oxidase, and starving the substrate for oxalate production. Evaluation of such an approach is ongoing in an early stage clinical trial in patients with the investigational RNAi therapeutic ALN-GO1. To expand the potential application of substrate reduction therapy to other diseases of oxalate excess, the current study targeted the downstream enzyme in oxalate synthesis – lactate dehydrogenase (LDH). LDH was shown as early as 1973 to be the key enzyme in oxidizing glyoxylate to oxalate in human livers. Thus, its inhibition could have the potential to treat the full array of patients with primary hyperoxaluria (types, 1, 2, 3, and idiopathic) and even common stone forming diseases. Of course, LDH and its metabolites are also well established regulators of energetic flux in liver cells (e.g., Cori Cylcle, Krebs Cycle) and the safety of its inhibition must be explored carefully to understand any impact on hepatic and whole body metabolism. In mice, liver-specific knockdown of LDH did result in profound oxalate lowering in both healthy and diseased animals. However, additional findings contradicted expectations - instead of the predicted increase in glycolate levels that was anticipated, treated mice showed profound whole-body decreases in glycolate levels – in their livers, plasma, and urine. Additionally, substantial changes in lactate and pyruvate levels were seen in the livers of treated animals, consistent with the known role of LDH in their conversion. Thus, the importance of these and other potential changes will need to be investigated fully, including in chronic toxicology studies, to help define the potential of LDH-based therapy in hyperoxalurias while ensuring the safety of this approach. John Knight, PhD Assistant Professor University of Alabama at Birmingham Dept of Urology, Kaul Building, Room 816B 720 20th Street S, Birmingham, AL 35294 U.S.A. knight74@uab.edu (205) 996-2295

Dodecyl-modified oligodeoxyribonucleotides as platform for oligonucleotide delivery into eukaryotic cells Maxim Kupryushkin1, Oleg Markov1, Marina Zenkova1, Inna Pyshnaya1, Dmitrii Pyshnyi1,2 Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia1, Novosibirsk State University, Novosibirsk, Russia2

Classical ways of transfection as formation cationic complexes between oligonucleotides and polycations/lipid cations or conjugation with lipophilic agents (cholesterol, bile or fatty acids and etc.) cannot be apply to some classes of modified oligonucleotides, especially for uncharged analogues. We developed universal platform for delivery of different kind of nucleic acids.

A nonnucleosidic phosphoramidite containing dodecyl moiety as sidechain functionality was synthesized, and corresponding oligodeoxyribonucleotides bearing modified units in single or multiple positions along the chain were prepared on a commercial DNA/RNA synthesizer.

Cell uptake of synthetized oligonucleotides were demonstrated by confocal microscopy. It was shown oligonucleotides containing dodecyl moieties able to penetrate across cell membrane and accumulate in cytosol. Efficiency of cell transfection was estimated by flow cytometry. Oligonucleotide containing three nonnucleosidic units demonstrates significant increase of transfection efficacy comparing with corresponding oligonucleotides bearing one or two dodecyl modifications. Cell accumulation of three-dodecyl-modifed oligonucleotide was higher even than with Lipofectamine 2000™.

Synthetized oligonucleotide derivatives were tested as transporter molecules for complementary oligonucleotides. Double-stranded complexes were prepared using modified oligonucleotide and target sequence with different backbone nature – native oligonucleotide (PO), phosphorotoate analogue (PS) and uncharged phosphoryl guanidine analogue (PGO) containing 1,3-dimethylimidazolidine-2-imino groups. In all cases target sequence was efficiently delivered. Thus developed molecules bearing three dodecyl moieties can be promising tools in a field of nucleic acid delivery.

This work was supported by RFBR (No. 16-34-01219) and RSF (No. 14-44-00068) to M.K., RSF (No. 16-15-10238) to I.P., and Ministry of Education and Science of the Russian Federation (Agreement No. 14.607.21.0125 from 27.10.2015), unique identifier of the project -RFMEFI60715X0125 to D.P.

Maxim Kupryushkin, PhD Junior Research Associate Novosibirsk Institute of Chemical Biology and Fundamental Medicine SB RAS. Lavrentiev ave. 8 Novosibirsk, 630090 Russia kuprummax@gmail.com

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Establishment of a Method for the Determination of ISIS2503 in Plasma Using LC-MS/MS Detection and Comparison with ELISA Method Sang Deuk Lee1, Kwang Kyu Park1, Nam Hee Kim1, Kyung Jin Jung2 1BioInfra Co., Ltd., 2Korea Institute of Toxicology The lack of reproducibility and sensitivity of LC-MS/MS method for the assay of oligonucleotide therapeutics remains as a barrier for application to preclinical and clinical studies. In this study, we established a reproducible and sensitive LC-MC/MS method for the assay of ISIS2503, a model compound of oligonucleotide drug, in human plasma over the concentration range of 0.2~50 ng/mL. ISIS2503 in plasma sample was stabilized by adding 25% ammonia solution to human plasma sample and ISIS2503-F5, (fluorinated ISIS2503) was used as internal standard. ISIS2503 and IS were extracted from the stabilized sample with phenol-chloroform solution. Adsorption of ISIS2503 from the supernatent was prevented by adding 25% ammonia solution. Phenol in the supernatent was removed by rinsing with chloroform twice and 10 uL of processed sample was injected onto UPLC-MS/MS system. Chromatographic separation was carried out on an Acquity UPLC system (Waters, MA, USA), installed with Acquity C18 BEH 50x2.1mm i.d., 1.7um (Waters, MA, USA). A gradient elution profile using the mobile phase composed of 1,1,1,3,3,3,-hexafluoro-2-propanol, N,N-diisopropylethylamine, acetonitrile and water was employed at a flow rate of 0.15 mL/min for 13.5 min. Detection was achieved by Waters XevoTM TQ-S mass spectrophotometer equipped with an electrospray ion source working in negative ion mode. The respective mass transition used for quantification of the nonavalent ion of IS2503 and IS2503-F5

were 704.67→95.13 and 714.67→95.13. The intra-batch and inter-batch precision (%CV) across four quality control levels was less than 9.0% and accuracy was -5.3~+11.8%. CV of the slope of six calibration curves measured over 92 days was 6.0%. Stability of solutions, matrix samples and processed samples were also confirmed. This method was modified with the calibration range of 2~500 ng/mL for application to pharmacokinetic study in monkey. The validation results were succesful and LC-MS/MS method was more precise and accurate when compared to ELISA method. The plasma concentrations of ISIS2503 after intravenous administration of 1 mg/kg of ISIS2503 were sucessfully determined up to 72 hours. This study provides a reproducible and sensitive LC-MS/MS method for the determination of oligonucleotide therapeutics applicable to preclinical and clinical studies. Sang Deuk Lee, PhD President BioInfra Co., Ltd. Gyunggi Bio-Center, 147 Gwanggyo-ro, Yeongtong-gu, Suwon-si, Gyunggi-do 16229 Republic of Korea sdlee@bioinfrakorea.co.kr

058

Establishment of a Method for the Determination of ISIS2503 in Plasma Using LC-MS/MS Detection and Comparison with ELISA Method Sang Deuk Lee1, Kwang Kyu Park1, Nam Hee Kim1, Kyung Jin Jung2 1BioInfra Co., Ltd., 2Korea Institute of Toxicology The lack of reproducibility and sensitivity of LC-MS/MS method for the assay of oligonucleotide therapeutics remains as a barrier for application to preclinical and clinical studies. In this study, we established a reproducible and sensitive LC-MC/MS method for the assay of ISIS2503, a model compound of oligonucleotide drug, in human plasma over the concentration range of 0.2~50 ng/mL. ISIS2503 in plasma sample was stabilized by adding 25% ammonia solution to human plasma sample and ISIS2503-F5, (fluorinated ISIS2503) was used as internal standard. ISIS2503 and IS were extracted from the stabilized sample with phenol-chloroform solution. Adsorption of ISIS2503 from the supernatent was prevented by adding 25% ammonia solution. Phenol in the supernatent was removed by rinsing with chloroform twice and 10 uL of processed sample was injected onto UPLC-MS/MS system. Chromatographic separation was carried out on an Acquity UPLC system (Waters, MA, USA), installed with Acquity C18 BEH 50x2.1mm i.d., 1.7um (Waters, MA, USA). A gradient elution profile using the mobile phase composed of 1,1,1,3,3,3,-hexafluoro-2-propanol, N,N-diisopropylethylamine, acetonitrile and water was employed at a flow rate of 0.15 mL/min for 13.5 min. Detection was achieved by Waters XevoTM TQ-S mass spectrophotometer equipped with an electrospray ion source working in negative ion mode. The respective mass transition used for quantification of the nonavalent ion of IS2503 and IS2503-F5

were 704.67→95.13 and 714.67→95.13. The intra-batch and inter-batch precision (%CV) across four quality control levels was less than 9.0% and accuracy was -5.3~+11.8%. CV of the slope of six calibration curves measured over 92 days was 6.0%. Stability of solutions, matrix samples and processed samples were also confirmed. This method was modified with the calibration range of 2~500 ng/mL for application to pharmacokinetic study in monkey. The validation results were succesful and LC-MS/MS method was more precise and accurate when compared to ELISA method. The plasma concentrations of ISIS2503 after intravenous administration of 1 mg/kg of ISIS2503 were sucessfully determined up to 72 hours. This study provides a reproducible and sensitive LC-MS/MS method for the determination of oligonucleotide therapeutics applicable to preclinical and clinical studies. Sang Deuk Lee, PhD President BioInfra Co., Ltd. Gyunggi Bio-Center, 147 Gwanggyo-ro, Yeongtong-gu, Suwon-si, Gyunggi-do 16229 Republic of Korea sdlee@bioinfrakorea.co.kr

Insights into the Kinetics of Antisense Oligonucleotide Uptake in Non Small Cell Lung Cancer

Emily Linnane1, Mark Edbrooke1, Sanyogitta Puri2, Jim Norman3, Alexey Revenko4, Robert Macleod4 and Sarah Ross1 1 AstraZeneca R&D, Oncology iMed, Chesterford Research Park, Saffron Walden, CB10 1XL 2AstraZeneca R&D, Advanced Drug Delivery, Pharmaceutical Sciences Gawsworth, Macclesfield, SK10 2NA 3 Cancer Research UK Beatson Institute, Bearsden Glasgow, G61 1BD 4Ionis Pharmaceuticals, Carlsbad, CA 92010, USA.

Significant progress has been made in the development of Generation 2.5 antisense oligonucleotides (ASOs) as therapeutic agents across a number of clinical indications. Despite these advances in antisense technology and application, there are still many unanswered questions surrounding ASO trafficking and uptake within the cell, particularly in tumour cells where significant variability is observed across different tumour types. It is important to derive a strong understanding of the pathways that regulate trafficking of unformulated ASOs that would lead to robust target knockdown within cells (a process termed ‘productive uptake’), and why some cell lines and models respond better than others.

To address the unanswered questions surrounding ASO productive uptake, we have centred our study around the clinical candidate ASO, AZD4785/ Ionis 651987, which has shown effective knockdown of its target KRAS in multiple non small cell lung cancer cell lines (NSCLC). Therefore, a panel of NSCLC lines was selected to investigate differences in mechanisms of ASO trafficking within these cells. We found that the intracellular patterns of ASO distribution varied between cell lines but did not correlate with target knockdown. We also discovered that distribution of the ASO was dynamically regulated and was rapidly trafficked across the cell membrane. We observed via super resolution microscopy that ASO was associated with multi-vesicular bodies and accumulates in the cell over time. In addition, we analysed the kinetics of ASO trafficking including co-localisation with intracellular markers and how this was regulated between different cell lines. Taken together, these data highlight new insights into antisense trafficking within the cell which may ultimately impact patient selection strategies for this technology.

Emily Linnane, PhD Postdoctoral Researcher AstraZeneca Hodgkin Building Chesterford Research Park Saffron Walden, CB10 1XL emily.linnane@astrazeneca.com

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Derepressing muscleblind expression by miRNA sponges ameliorates myotonic dystrophy-like phenotypes in Drosophila

Llamusi B1,2,3, Cerro-Herreros E1,2,3, Fernandez-Costa JM1,2,3, Sabater-Arcis M1,2,3, Artero R1,2,3 1Translational Genomics Group, Incliva Health Research Institute, Valencia, Spain. 2Department of Genetics and Interdisciplinary Research Structure for Biotechnology and Biomedicine (ERI BIOTECMED), University of Valencia, Valencia, Spain. 3 CIPF-INCLIVA joint unit.

Introduction: Myotonic Dystrophy type 1 (DM1) originates from alleles of the DMPK gene with hundreds of CTG repeats in the 3′UTR. CUG repeat RNAs accumulate in foci that sequester Muscleblind-like (MBNL) proteins away from their functional target transcripts. Thus endogenous upregulation of MBNL proteins is a potential therapeutic approach to DM1. Using Drosophila model, we have studied endogenous regulation of Mbl (fly orthologue) by microRNAs which regulate protein synthesis.

Methods: Based on unpublished data, orthology with human miRNAs, and TargetScan predictions we selected a group of candidate miRNAs for Mbl regulation. miRNA sponge constructs were expressed in muscle of DM1 model flies to test the effect of the specific miRNA inhibition on the Mbl expression and on DM1-like phenotypes.

Results: Here we identified two miRNAs, dme-miR-277 and dme-miR-304, that differentially regulate Muscleblind RNA isoforms in miRNA sensor constructs. We also showed their sequestration by sponge constructs derepressing endogenous Mbl not only in a wild type background but also in a DM1 Drosophila model expressing non-coding CUG repeats throughout the musculature. Enhanced Mbl expression resulted in significant rescue of pathological phenotypes, including reversal of several mis-splicing events and reduced muscle atrophy in DM1 adult flies. Flies had also improved muscle function in climbing and flight assays, and had longer lifespan compared to disease controls. Given the success with the Drosophila model, we are currently extending these observations to mammalian DM1 experimental systems.

Conclusion: Our data support a relevant role of miRNAs in Mbl regulation and provide proof of concept for a similar potentially therapeutic approach to DM1 in humans.

Beatriz Llamusi Troísi, Ph.D. Postdoctoral senior researcher University of Valencia Dr. Moliner 50. 46100. Burjassot, Valencia. Spain mllatroi@uv.es

060

Derepressing muscleblind expression by miRNA sponges ameliorates myotonic dystrophy-like phenotypes in Drosophila

Llamusi B1,2,3, Cerro-Herreros E1,2,3, Fernandez-Costa JM1,2,3, Sabater-Arcis M1,2,3, Artero R1,2,3 1Translational Genomics Group, Incliva Health Research Institute, Valencia, Spain. 2Department of Genetics and Interdisciplinary Research Structure for Biotechnology and Biomedicine (ERI BIOTECMED), University of Valencia, Valencia, Spain. 3 CIPF-INCLIVA joint unit.

Introduction: Myotonic Dystrophy type 1 (DM1) originates from alleles of the DMPK gene with hundreds of CTG repeats in the 3′UTR. CUG repeat RNAs accumulate in foci that sequester Muscleblind-like (MBNL) proteins away from their functional target transcripts. Thus endogenous upregulation of MBNL proteins is a potential therapeutic approach to DM1. Using Drosophila model, we have studied endogenous regulation of Mbl (fly orthologue) by microRNAs which regulate protein synthesis.

Methods: Based on unpublished data, orthology with human miRNAs, and TargetScan predictions we selected a group of candidate miRNAs for Mbl regulation. miRNA sponge constructs were expressed in muscle of DM1 model flies to test the effect of the specific miRNA inhibition on the Mbl expression and on DM1-like phenotypes.

Results: Here we identified two miRNAs, dme-miR-277 and dme-miR-304, that differentially regulate Muscleblind RNA isoforms in miRNA sensor constructs. We also showed their sequestration by sponge constructs derepressing endogenous Mbl not only in a wild type background but also in a DM1 Drosophila model expressing non-coding CUG repeats throughout the musculature. Enhanced Mbl expression resulted in significant rescue of pathological phenotypes, including reversal of several mis-splicing events and reduced muscle atrophy in DM1 adult flies. Flies had also improved muscle function in climbing and flight assays, and had longer lifespan compared to disease controls. Given the success with the Drosophila model, we are currently extending these observations to mammalian DM1 experimental systems.

Conclusion: Our data support a relevant role of miRNAs in Mbl regulation and provide proof of concept for a similar potentially therapeutic approach to DM1 in humans.

Beatriz Llamusi Troísi, Ph.D. Postdoctoral senior researcher University of Valencia Dr. Moliner 50. 46100. Burjassot, Valencia. Spain mllatroi@uv.es

Lactadherin and VE-cadherin Incorporated in to exosomes secreted post-interference of ASncmtRNA decrease tumorigenic property in breast cancer cell Lobos-Gonzalez Lorena1,2,4, Bustos Rocio1,2,3*, Campos America 1,2,4, Silva Valeria1,2,3, Silva Veronica1,2, Varas-Godoy Manuel5, Caceres-Verchae Albano5, Villegas Jaime 1,2,3, Ezquer Fernando6, Ezquer Marcelo6, Burzio Veronica 1,2,3, Luis O. Burzio1,2,3 1Andes Biotechnologies S.A. 2Fundación Ciencia & Vida, 3Facultad de Ciencias Biológicas, Universidad Andrés Bello, 4 Facultad de Ciencias Químicas y Farmacéuticas Universidad de Chile, 5 Centro de Investigación Biomédica, Facultad de Medicina, Universidad de Los Andes, 6Centro de Medicina Regenerativa, Facultad de Medicina Clínica Alemana-Universidad del Desarrollo. Breast cancer is the most deadly female cancer worldwide; however, current therapies are unsuccesful in advanced stages of this disease due to tumor heterogeneity. Our group has proposed a new therapeutic target corresponding to a family of long non-coding mitochondrial RNAs (ncmtRNAs) called Sense (SncmtRNAs) and Antisense (ASncmtRNAs). Knockdown of ASncmtRNAs causes massive and selective cell death in tumor cells but not in normal cells. It's been known that during intercellular communication, cells release extracellular vesicles such as exosomes that contain proteins and RNAs with the potential to influence recipient cells proliferation and/or trigger apoptosis in a paracrine or autocrine manner. In this particular research, MDA-MB-231 cells were transfected with a therapeutic antisense oligonucleotide (ASO-1537S) complementary to ASncmtRNA or a non-related ASO (ASO-C) using Lipofectamine2000. At 24h post-transfection exosomes were purified from supernatants. Microparticle size and distribution was determined by transmission electron microscopy (TEM) and Nanoparticule Tracking Analysis (NTA). Exosome markers such as Alix, TSG101 were detected by Western blotting. Tumorigenic properties such as invasion, migration and anchorage-independent growth were analyzed. NTA and Transmition Electronic Microscopy revealed exosomes ranging from 50-120 nm in size. Exosomes released from ASO-1537S treated cells contained the exosome markers described above, with some differences in their expression. All tumorigenic properties analyzed were inhibited in cells treated with exosomes derived from ASO-1537S treated cells, compared to controls. All these experiments were corroborated with an incorporation assay and also a proliferation assay exposing exosomes to different conditions. Last, we did a proteomic analysis where we found 14 proteins expressed only in exosomes Exo-WT and Exo-C; of these, 6 have been described in literature as tumor promoters: HSP70, HSP90A, Lactadherin, VE-Cadherin, Protocadherin and S100A. These results suggest that the effect in the tumorigenic properties observed is possibly caused or modulated by exosomes loaded with Lactadherin or VE-Cadherin in particular. Lorena Lobos-Gonzalez (PhD and Mst Biochemistry) Andes Biotechnologies and Fundacion Ciencia y Vida Aveida Zzañartu 1482 Ñuñoa, Santiago Chile lorotae@gmail.com

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Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors. Lorena Lobos-González1,2,3, Verónica Silva1,2, Mariela Araya1,2, Franko Restovic1,2†, Javiera Echenique1,2, Luciana Oliveira-Cruz1,2, Christopher Fitzpatrick1,2,4, Macarena Briones1,2, Jaime Villegas1,2,4, Claudio Villota1,2,4, Soledad Vidaurre1,5, Vincenzo Borgna1,2,6, Miguel Socias7, Sebastián Valenzuela2, Constanza Lopez1,2, Teresa Socias1,2, Manuel Varas2, Jorge Díaz3, Luis O. Burzio1,2,4 and Verónica A. Burzio1,2,4*

1Andes Biotechnologies SpA, 2Fundación Ciencia & Vida, Av. Zañartu 1482, 3Facultad de Medicina, Universidad de Chile, Independencia 1027, 4Facultad de Ciencias Biológicas, Universidad Andrés Bello, República 252, 5Facultad de Salud, Deporte y Recreación, Universidad Bernardo O’Higgins, Gral. Gana 1702, 6Servicio de Urología, Hospital Barros-Lucco-Trudeau, Gran Av. JM Carrera 3122, 7Clínica Alemana, Av Vitacura 5951, Santiago, Chile. †Present address: Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Av. Libertador Bernardo O'Higgins, Santiago, Chile. ABSTRACT We reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, suggesting this approach for selective therapy against different types of cancer. In order to translate these results to a preclinical scenario, we characterized the murine noncoding mitochondrial RNAs (ncmtRNAs) and performed in vivo knockdown to syngeneic murine melanoma models. Mouse ncmtRNAs display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Knockdown of ASncmtRNAs with specific antisense oligonucleotides (ASO) reduces murine melanoma B16F10 cell proliferation and induces apoptosis in vitro through downregulation of pro-survival and metastasis markers, particularly survivin. For in vivo studies, subcutaneous B16F10 melanoma tumors in C57BL/6 mice were treated systemically with specific and control antisense oligonucleotides (ASO). For metastasis studies, tumors were resected, followed by systemic administration of ASOs and the presence of metastatic nodules in lungs and liver was assessed. Treatment with specific ASO inhibited tumor growth and metastasis after primary tumor resection. In a metastasis-only assay, mice inoculated intravenously with cells and treated with the same ASO displayed reduced number and size of melanoma nodules in the lungs, compared to controls. Our results suggest that ASncmtRNAs could be potent targets for melanoma therapy. To our knowledge, the ASncmtRNAs are the first potential non-nuclear targets for melanoma therapy. Lorena Lobos-Gonzalez (PhD and Mst Biochemistry) Andes Biotechnologies and Fundacion Ciencia y Vida Aveida Zzañartu 1482 Ñuñoa, Santiago Chile lorotae@gmail.com

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Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors. Lorena Lobos-González1,2,3, Verónica Silva1,2, Mariela Araya1,2, Franko Restovic1,2†, Javiera Echenique1,2, Luciana Oliveira-Cruz1,2, Christopher Fitzpatrick1,2,4, Macarena Briones1,2, Jaime Villegas1,2,4, Claudio Villota1,2,4, Soledad Vidaurre1,5, Vincenzo Borgna1,2,6, Miguel Socias7, Sebastián Valenzuela2, Constanza Lopez1,2, Teresa Socias1,2, Manuel Varas2, Jorge Díaz3, Luis O. Burzio1,2,4 and Verónica A. Burzio1,2,4*

1Andes Biotechnologies SpA, 2Fundación Ciencia & Vida, Av. Zañartu 1482, 3Facultad de Medicina, Universidad de Chile, Independencia 1027, 4Facultad de Ciencias Biológicas, Universidad Andrés Bello, República 252, 5Facultad de Salud, Deporte y Recreación, Universidad Bernardo O’Higgins, Gral. Gana 1702, 6Servicio de Urología, Hospital Barros-Lucco-Trudeau, Gran Av. JM Carrera 3122, 7Clínica Alemana, Av Vitacura 5951, Santiago, Chile. †Present address: Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Av. Libertador Bernardo O'Higgins, Santiago, Chile. ABSTRACT We reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, suggesting this approach for selective therapy against different types of cancer. In order to translate these results to a preclinical scenario, we characterized the murine noncoding mitochondrial RNAs (ncmtRNAs) and performed in vivo knockdown to syngeneic murine melanoma models. Mouse ncmtRNAs display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Knockdown of ASncmtRNAs with specific antisense oligonucleotides (ASO) reduces murine melanoma B16F10 cell proliferation and induces apoptosis in vitro through downregulation of pro-survival and metastasis markers, particularly survivin. For in vivo studies, subcutaneous B16F10 melanoma tumors in C57BL/6 mice were treated systemically with specific and control antisense oligonucleotides (ASO). For metastasis studies, tumors were resected, followed by systemic administration of ASOs and the presence of metastatic nodules in lungs and liver was assessed. Treatment with specific ASO inhibited tumor growth and metastasis after primary tumor resection. In a metastasis-only assay, mice inoculated intravenously with cells and treated with the same ASO displayed reduced number and size of melanoma nodules in the lungs, compared to controls. Our results suggest that ASncmtRNAs could be potent targets for melanoma therapy. To our knowledge, the ASncmtRNAs are the first potential non-nuclear targets for melanoma therapy. Lorena Lobos-Gonzalez (PhD and Mst Biochemistry) Andes Biotechnologies and Fundacion Ciencia y Vida Aveida Zzañartu 1482 Ñuñoa, Santiago Chile lorotae@gmail.com

Intracellular Trafficking of Chemically Modified siRNA Socheata Ly1,2, Anastasia Khvorova1,2 1Program in Molecular Medicine and 2RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA, 01605 siRNAs are a new class of drugs with promising clinical efficacy that require chemical modifications or formulation for delivery to the tissue and cell of interest. However, one of the major current limitations is that less than 1% of the siRNA internalized by the cell is biologically active, suggesting that there is a major non-productive pathway and minor productive pathway; therefore, the pathway used by each siRNA molecule may determine its fate. Previously, we demonstrated that asymmetric, chemically stabilized, cholesterol-conjugated siRNAs support rapid cellular uptake and efficient mRNA silencing without the need for formulation. Shortly after internalization, these modified siRNAs preferentially traffic through a pathway similar to that of epidermal growth factor and co-localize with EEA1-enriched endosomes, which is indicative of selective endosomal sorting. Here, we investigated the effects of different chemical modification patterns on intracellular siRNA trafficking, such as 2′-O-methyl and 2′-fluoro modifications, phosphorothioate bonds, and different conjugates. Using fluorescently-labeled siRNAs and immunofluorescence for cellular trafficking markers, we observed that the conjugate and phosphorothioate content of the siRNA affected its uptake efficiency into HeLa cells. Furthermore, internalized siRNA co-localized differently with several intracellular compartments including EEA1 (early endosomes), LBPA (late endosomes) and LAMP1 (lysosomes). A better understanding of the kinetics of chemically modified siRNA cellular uptake will assist in further optimization of siRNAs for therapeutic intervention. Socheata Ly PhD Candidate University of Massachusetts Medical School 368 Plantation Street, AS4-1016 Worcester, MA 01605 USA Socheata.Ly@umassmed.edu 774-455-3635

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Impact of Enhanced Metabolic Stability on In Vivo Performance of GalNAc-siRNA Conjugates Martin A. Maier Alnylam Pharmaceuticals, Cambridge, MA 02142, USA

The combination of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. However, even in the context of fully modified siRNAs, enhanced stabilization against nucleolytic degradation is essential for optimal in vivo performance of GalNAc-siRNA conjugates. Here we summarize a multi-pronged approach to further elucidate the relationship between siRNA chemistry and metabolic stability and to guide the development of advanced designs with improved potency and duration. This includes a screening paradigm for siRNA design optimization, which is based on a positional model for chemical modifications and relies on parallel screening across multiple sequences. These efforts led to the discovery of enhanced stability designs, which have fundamentally transformed the siRNA conjugate platform and helped to advance multiple RNAi therapeutics into pre-clinical and clinical development.

Martin A. Maier, PhD Vice President, Research Alnylam Pharmaceuticals 300 Third Street Cambridge, MA 02142 Email: mmaier@alnylam.com

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Ligand-Guided Selection (LIGS): A Screening Technology to Identify Specific Aptamers Against Cell-surface Markers Prabodhika Mallikaratchy1,2,3, Hasan Zumrut2, Rigzin Dekhang1, Sana Batool1 1Department of Chemistry, Lehman College, City University of New York, 250 Bedford Park Blvd. West, Bronx, NY 10468, USA 2Chemistry and Biochemistry Graduate Programs, City University of New York, 365 5th Avenue, New York, NY 10016 USA 3Molecular Cellular and Developmental Biology Graduate Program, City University of New York, 365 5th Avenue, New York, NY 10016 USA Aptamers are small DNA molecules with the ability to bind to proteins with high affinity and specificity. Aptamers are evolved using a combinatorial screening method called Systematic Evolution Exponential enrichment (SELEX). Recently, a number of new SELEX approaches were introduced to evolve aptamers against cell-surface markers. To increase the translational application of aptamers, it is necessary to develop methods to identify aptamers that could specifically recognize epitopes of proteins at their native state. To this end, we developed new strategy termed “Ligand-guided-Selection” (LIGS), which allows selection of aptamers against cell-surface proteins at their native state. The iterative process in conventional SELEX is designed to outcompete low-affinity binders through a competitive process whereby high affinity binders move on through the selection process. LIGS strategy exploits this step to isolate highly specific DNA aptamers against a predetermined target by introducing a naturally occurring stronger and highly specific bivalent binder, an antibody (Ab) interacting with its cognate epitope, to selectively out-compete specific aptamers from a partially enriched SELEX pool. Utilizing LIGS we have selected aptamers against membrane bound IgM (mIgM) expressed on B-cells and against Cluster of differentiation 3 (CD3) expressed on T-cells. This poster will present (1) the progress made on optimization of the aptamers selected using LIGS, (2) the progress made on optimization of the method to generate aptamers at physiological temperatures. Prabodhika Mallikaratchy PhD Assistant Professor of Chemistry New Science Hall-Office S-4404/Lab S-4401 Lehman College for City University of New York 250 Bedford Park Blvd. West Bronx, NY 10468 Prabodhika.Mallikaratchy@lehman.cuny.edu +1-347-577-4082

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Ligand-Guided Selection (LIGS): A Screening Technology to Identify Specific Aptamers Against Cell-surface Markers Prabodhika Mallikaratchy1,2,3, Hasan Zumrut2, Rigzin Dekhang1, Sana Batool1 1Department of Chemistry, Lehman College, City University of New York, 250 Bedford Park Blvd. West, Bronx, NY 10468, USA 2Chemistry and Biochemistry Graduate Programs, City University of New York, 365 5th Avenue, New York, NY 10016 USA 3Molecular Cellular and Developmental Biology Graduate Program, City University of New York, 365 5th Avenue, New York, NY 10016 USA Aptamers are small DNA molecules with the ability to bind to proteins with high affinity and specificity. Aptamers are evolved using a combinatorial screening method called Systematic Evolution Exponential enrichment (SELEX). Recently, a number of new SELEX approaches were introduced to evolve aptamers against cell-surface markers. To increase the translational application of aptamers, it is necessary to develop methods to identify aptamers that could specifically recognize epitopes of proteins at their native state. To this end, we developed new strategy termed “Ligand-guided-Selection” (LIGS), which allows selection of aptamers against cell-surface proteins at their native state. The iterative process in conventional SELEX is designed to outcompete low-affinity binders through a competitive process whereby high affinity binders move on through the selection process. LIGS strategy exploits this step to isolate highly specific DNA aptamers against a predetermined target by introducing a naturally occurring stronger and highly specific bivalent binder, an antibody (Ab) interacting with its cognate epitope, to selectively out-compete specific aptamers from a partially enriched SELEX pool. Utilizing LIGS we have selected aptamers against membrane bound IgM (mIgM) expressed on B-cells and against Cluster of differentiation 3 (CD3) expressed on T-cells. This poster will present (1) the progress made on optimization of the aptamers selected using LIGS, (2) the progress made on optimization of the method to generate aptamers at physiological temperatures. Prabodhika Mallikaratchy PhD Assistant Professor of Chemistry New Science Hall-Office S-4404/Lab S-4401 Lehman College for City University of New York 250 Bedford Park Blvd. West Bronx, NY 10468 Prabodhika.Mallikaratchy@lehman.cuny.edu +1-347-577-4082

Relationship between deformability of sugar-modified RNA duplexes and their melting temperatures Yoshiaki Masaki, Mitsuo Sekine, Kohji Seio Department of Life Science and Technology, Tokyo Institute of Technology Chemical modifications on RNA duplexes can change their functional, structural and biological properties. Among such properties, duplex stability is one of the most important factors. To modulate RNA duplex stability, 2´-O-modifications are often utilized. The effect of 2´-O-modifications on the RNA duplex stability is commonly explained by the so-called preorganization effect of the sugar moiety. This effect induces suppression of the entropy loss upon duplex formation. However, 2´-O-cyanoethyl-RNA duplexes were more stable than corresponding 2´-O-methyl-RNA duplexes, although the C2´-endo/C3´-endo ratio in the sugar puckering of 2´-O-cyanoethyl-nucleoside was quite similar to that of 2´-O-methyl-nucleoside. Other factors such as hydration, electrostatic, and stacking effects also play an important role on the RNA duplex stability. We envisioned that computational approaches to predict the effect of chemical modifications would be useful to develop more effective modifications. For this purpose, we have compared various kinds of calculated structural parameters with corresponding melting temperatures of the RNA duplexes actually synthesized. We found that the fluctuations of base-pair step parameters, especially deformability of base-pair step, were well correlated with experimentally determined melting temperatures. Based on this finding, we evaluated systematically selected 2´-modifications. In this poster, we will discuss the principle, applicability, and recent progress of this method. Yoshiaki Masaki, PhD Assistant Professor Department of Life Science and Technology, Tokyo Institute of Technology 4259-J2-16 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa JAPAN ymasaki@bio.titech.ac.jp +81-45-924-5750

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Use of an antisense-mediated exon skipping approach as a therapeutic option for a common Mucolipidosis type II causing mutation Matos L1, Vilela R1,2, Coutinho MF1, Gaspar P3, Alves S1

1 Research and Development Unit, Department of Human Genetics, INSA, Porto, Portugal; 2 Biology department, Faculty of Sciences, University of Porto, Portugal; 3 Newborn Screening, Metabolism and Genetics Unit, Department of Human Genetics, INSA, Porto, Portugal

Lysosomal Storage Disorders (LSDs) are a group of rare inherited diseases caused by the malfunction of the lysosomal system, resulting in the accumulation of undegraded substrates inside the lysosomes and leading to severe and progressive pathology. Among them is ML II, one of most severe LSDs, which is caused by the total or near total deficiency of the GlcNAc-phosphotransferase, a key in enzyme for the correct trafficking of lysosomal hydrolases to the lysosome. GlcNAc-phosphotransferase is a multimeric enzyme and is encoded by two genes: GNPTAB and GNPTG. One of the most frequent ML II causal mutations is a dinucleotide deletion on exon 19 of the GNPTAB gene that disrupts the reading frame and prevents the production of an active GlcNAc-phosphotransferase, which in turn impairs the proper targeting of lysosomal enzymes. Despite broad understanding of the molecular causes behind this and other LSDs, the same progress has not been observed in the development of new therapies, with current treatments still mostly symptomatic and presenting several limitations. Therefore, alternative options should be investigated in order to provide patients and families with better healthcare and more promising therapies. One possibility is the modulation of splicing by antisense oligonucleotides with the purpose of altering the splicing pattern, the mature mRNA and ultimately the final protein product. Acknowledging this, the present study intends to design and develop a RNA-based therapeutic agent through the use of antisense oligonucleotides capable of inducing the skipping of exon 19 of the GNPTAB gene and consequently circumvent the effects the most common ML II causal mutation. The approach is presently ongoing and different 2’O-Methyl AOs were designed and synthesized to target the GNPTAB exon 19 and promote its skipping. We have already succeed in inducing the skipping of exon 19 in control and ML II patient cell lines and we are now evaluating the effects of this therapeutic approach at biochemical levels.

Liliana Matos, PhD Research Fellow National Health Institute Dr. Ricardo Jorge Rua Alexandre Herculano, 321 4000-055, Porto Portugal liliana.matos@insa.min-saude.pt

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Preclinical efficacy of SOD1 antisense oligonucleotide in phase 1 testing for SOD1 amyotrophic lateral sclerosis. Alexander McCampbell1, Tracy Cole2, Sai Thankamony1, Jessica Amacker1, Brandon Farley1 Giulio Tomassy1; Linhong Sun1; Yi Luo1; Mingdi Zhang1; Holly Kordasiewicz2, Timothy Miller3, Hani Houshyar1, Toby Ferguson1, Eric Swayze2 1Biogen, Cambridge MA; 2Ionis Pharmaceuticals, Carlsbad, CA; 3Washington University, St. Louis, MO

Dominantly inherited amyotrophic lateral sclerosis (ALS) can be caused by mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene. Expression of these mutant forms of SOD1 in mice results in age-dependent motor neuron loss, paralysis, and premature death. Compelling evidence suggests that SOD1 toxicity requires protein expression. Therefore, reduction of SOD1 protein ought to be beneficial. A prior effort at Ionis led to the development of a clinical antisense oligonucleotides (ASOs) which completed phase 1 testing. In the interim, advances in screening and optimizing ASOs prompted an effort to identify a superior agent. BIIB067is an ASO capable of potently and safely suppressing SOD1 across a range of model systems. Administration of BIIB067 by intracerebroventricular injection lowered SOD1 in a dose-dependent manner, in mice expressing either WT or mutant SOD1-G93A. Efficacy of BIIB067 was assessed in the C57Bl6 high copy SOD1-G93A mouse. When administered at day 50 and 94, BIIB067 provided a significant improvement in rotarod performance, delayed body weight loss and disease onset, and prolonged survival, with the 100 µg and 300 µg dose group having comparable benefit. Subsequent studies show that survival benefit was independent of the timing of injection. BIIB067 preserved neuromuscular junction innervation, by both histological and electrophysiological measures, and prevented denervation-dependent changes in muscle fibers. To better quantify neuromuscular function, compound muscle action potentials (CMAP) in the tibialis anterior were measured. CMAP activity in SOD1-G93A is normal at week 5 in the model, but rapidly deteriorates by week 9. Treatment of SOD1 mice with ASO at week 5 prevents loss of CMAP. Treatment at week 9 leads to partial recovery of CMAP. After administration by lumbar puncture in cynomolgus monkeys, BIIB067 lowered SOD1 mRNA in the spinal cord and cortex in a dose-dependent manner. SOD1 protein was reduced in the CNS and by 50% in the cerebrospinal fluid. SOD1 mRNA and protein were also suppressed in multiple areas of the central nervous system of the NHPs. BIIB067 is currently in phase 1 testing for SOD1 ALS.

Alex McCampbell, PhD Principal Investigator Neurodegenerative Disease and Repair Biogen 225 Binney St Cambridge MA 02142. Ph # 617 914 0744

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Preclinical Development of RNAi Therapeutic Drug Candidate for Targeting Hepatitis B Virus Stuart Milstein, Tuyen Nguyen, Adam Castoreno, Abigail Liebow, Vasant Jadhav, Martin Maier, Laura Sepp-Lorenzino Alnylam Pharmaceuticals, Cambridge, MA 02142, USA

Current HBV therapies, including polymerase inhibitors, effectively reduce viral titers in chronic hepatitis B by inhibiting viral replication, but these therapies fail to eradicate the infection in ~90% of treated patients. Even in the absence of viral replication, high plasma levels of non-infectious, HBsAg-containing subviral particles are thought to mediate immunological tolerance. Reduction in HBsAg plasma levels is the single best predictor of immunological cure. An RNAi therapeutic targeting the HBV genome has the potential to achieve a “functional cure” by effectively decreasing expression of tolerogenic HBsAg, in addition to inhibiting all steps of the HBV life cycle. ALN-HBV, a therapeutic RNAi candidate consisting of a siRNA with Enhanced Stabilization Chemistry (ESC) design conjugated to a GalNAc-based targeting ligand and designed for SC administration has been developed. ALN-HBV effectively targets all viral transcripts as demonstrated by RNAseq. The sequence is pan-genotypic, targeting all major HBV genotypes and resides outside of an integration hotspot making it therefore unlikely to be disrupted by HBV genome integration. A single subcutaneous dose of ALN-HBV results in potent and durable silencing of tolerogenic HBsAg in preclinical models.

Stuart Milstein Associate Director Alnylam Pharmaceuticals 300 3rd St Cambridge MA 02142 USA smilstein@alnylam.com

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Preclinical Development of RNAi Therapeutic Drug Candidate for Targeting Hepatitis B Virus Stuart Milstein, Tuyen Nguyen, Adam Castoreno, Abigail Liebow, Vasant Jadhav, Martin Maier, Laura Sepp-Lorenzino Alnylam Pharmaceuticals, Cambridge, MA 02142, USA

Current HBV therapies, including polymerase inhibitors, effectively reduce viral titers in chronic hepatitis B by inhibiting viral replication, but these therapies fail to eradicate the infection in ~90% of treated patients. Even in the absence of viral replication, high plasma levels of non-infectious, HBsAg-containing subviral particles are thought to mediate immunological tolerance. Reduction in HBsAg plasma levels is the single best predictor of immunological cure. An RNAi therapeutic targeting the HBV genome has the potential to achieve a “functional cure” by effectively decreasing expression of tolerogenic HBsAg, in addition to inhibiting all steps of the HBV life cycle. ALN-HBV, a therapeutic RNAi candidate consisting of a siRNA with Enhanced Stabilization Chemistry (ESC) design conjugated to a GalNAc-based targeting ligand and designed for SC administration has been developed. ALN-HBV effectively targets all viral transcripts as demonstrated by RNAseq. The sequence is pan-genotypic, targeting all major HBV genotypes and resides outside of an integration hotspot making it therefore unlikely to be disrupted by HBV genome integration. A single subcutaneous dose of ALN-HBV results in potent and durable silencing of tolerogenic HBsAg in preclinical models.

Stuart Milstein Associate Director Alnylam Pharmaceuticals 300 3rd St Cambridge MA 02142 USA smilstein@alnylam.com

Scavenger receptor class A1 mediates physiological uptake of morpholino oligomer with negative zeta potential into dystrophic skeletal muscles Shouta Miyatake, Shin’ichi Takeda, Yoshitsugu Aoki Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry

Exon skipping therapy using phosphorodiamidate morpholino oligomer (PMO), which has a neutral chemical backbone, is a promising therapy for Duchenne muscular dystrophy (DMD) as recently approved by the US FDA as the first exon 51-skipping drug for DMD. The biggest limitation of the current drug is a lack of drug delivery system into muscles. Recently, we reported that the uptake of peptide-conjugated PMO into skeletal muscles was mediated by scavenger receptor class A (SR-A), which could bind to negatively-charged ligands. However, the mechanism of PMO uptake into dystrophin-deficient muscles has been poorly understood.

To elucidate the mechanism, we incubated dystrophin deficient H2K-mdx52 murine myotubes with endocytosis inhibitors and then transfected them with a 25-mer PMO or a Cy5-conjugated PMO (Cy5-PMO). We found that all inhibitors suppressed the uptake of Cy5-PMO up to 50% evaluated by Cy5 fluorescence. The zeta potential of PMO dissolved in saline, OptiMEM® or double-distilled water (DDW) showed +1.06, -13.34 and -19.6 mV, and also their conductivity was 19.7, 13.9 and 0.01 mS/cm, respectively. These data strongly suggest that the lower conductivity of solvent is correlated with the negative zeta potential of PMO, which is also found to be depending on the number of thymine subtracted by that of adenine in its sequence. To investigate the effect of the zeta potential of PMO on exon skipping, PMO dissolved in saline or DDW was injected into tibialis anterior muscle in mdx52 mice. Two weeks after the injection, the number of dystrophin positive fibers in DDW increased up to 2.3 fold compared with the ones in saline. Furthermore, the efficiency of PMO-based exon skipping in the H2K-mdx52 myotubes showed 20-23% reduction with SR inhibitors or siRNA-based SR-A1 knockdown. The equilibrium dissociation constant for binding of PMO to SR-A1 by Bio-Layer Interferometry assays was found to be 8.61 x10-1 µM. The number of dystrophin positive fibers after intravenous injection of PMO was significantly low in mdx52 mice with a heterozygous SR-A1 knockout compared with in mdx52 mice. In conclusion, PMO with negative zeta potential in culture medium with physiological conductivity interacts with SR-A1 in H2K-mdx52 myotubes. Also, PMO enhances the exon skipping in in vivo when its solvent-dependent zeta potential is negative. We believe that a development of negatively charged PMO or the usage of solvent with low conductivity could lead to a development of highly efficient exon skipping therapy for DMD. Shouta Miyatake, PhD Post Doctoral Fellow Department of Molecular Therapy, National Center of Neurology and Psychiatry 4-1-1 Ogawahigashi- Cho, Kodaira, Tokyo 187-8502, Japan. shouchiman5@gmail.com Tel.: +81 42 346 1720

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RT and RNase H: from Viruses to antiviral Defense Karin Moelling , Felix Broecker University of Zürich, Institute for Medical Microbiology and Max Planck Institute Molecular Genetis RT and RNAse H are enzymes essential for retroviral replication. However, they are much more ubiquitous. Both activities can function independently. They are among the most abundant proteins or protein-folds on our planet. The shaped various genomes including the human genomes contributing up to 50% of the genomic sequence. There they comprise Endogenous Retroviruses (ERVs) but also Line, Sine elements and solo LTRs. They are present in transposable elements (TEs) or retrotransposons. Furthermore, retrovirus composition resembles the antiviral defense machinery in several of their components suggesting an evolutionary relationship between viruses and antiviral defense. RNase H-like enzymes are present in siRNA defense and other systems, involving Ago, Integrases, Dicer, Cas9, transposases, IgG rearrangement enzymes (Rag), splicing. The "Ocean TARA" microbiome indicated that RT and RNAses H are among the most abundant proteins comprising up to 18%. The PIWI/RNase H activity is essential for semen fertility and safety for transgenerational inheritance by controlling TE activities. We are using ODNs to inhibit HIV replication with the goal of developing a microbicide or use compounds against the RNAse Has antivirals by dynamic molecular compound designs. Ann NY Acad Sci1341 (2015)126-135 Karin Moelling Prof Dr. University of Zürich Inst med Microbiol Gloriastr 32 Zurich, CH 8006 Switzerland moelling@molgen.mpg.de

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RT and RNase H: from Viruses to antiviral Defense Karin Moelling , Felix Broecker University of Zürich, Institute for Medical Microbiology and Max Planck Institute Molecular Genetis RT and RNAse H are enzymes essential for retroviral replication. However, they are much more ubiquitous. Both activities can function independently. They are among the most abundant proteins or protein-folds on our planet. The shaped various genomes including the human genomes contributing up to 50% of the genomic sequence. There they comprise Endogenous Retroviruses (ERVs) but also Line, Sine elements and solo LTRs. They are present in transposable elements (TEs) or retrotransposons. Furthermore, retrovirus composition resembles the antiviral defense machinery in several of their components suggesting an evolutionary relationship between viruses and antiviral defense. RNase H-like enzymes are present in siRNA defense and other systems, involving Ago, Integrases, Dicer, Cas9, transposases, IgG rearrangement enzymes (Rag), splicing. The "Ocean TARA" microbiome indicated that RT and RNAses H are among the most abundant proteins comprising up to 18%. The PIWI/RNase H activity is essential for semen fertility and safety for transgenerational inheritance by controlling TE activities. We are using ODNs to inhibit HIV replication with the goal of developing a microbicide or use compounds against the RNAse Has antivirals by dynamic molecular compound designs. Ann NY Acad Sci1341 (2015)126-135 Karin Moelling Prof Dr. University of Zürich Inst med Microbiol Gloriastr 32 Zurich, CH 8006 Switzerland moelling@molgen.mpg.de

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Cellular Fractionation and Evaluation of Hepatic Cells after Antisense Oligonucleotide Treatment in Mice Sue Murray, Heather Murray, Richard Lee, Raechel Peralta, Mark Graham, Thazha Prakash, Punit Seth, Wuxia Fu, and Shuling Guo IONIS Pharmaceuticals, Carlsbad CA Nonalcoholic Steatohepatitis (NASH) causes chronic liver disease and is most common in Western countries. Current treatments for NASH are difficult to maintain and combine drug therapy with lifestyle modification (Pearlman et al. Curr Opin Gastroenterol. 2014 May; 30(3):223-37). One reason NASH is complicated to treat is due to its effects on multiple cell populations within the liver. Still, current research is unclear which population is key to target for treatment of the disease. Antisense oligonucleotide (ASO) drugs are a likely therapy for NASH due to their preferential distribution to the liver. Moreover, conjugates can be attached to the ASO and enable targeted cell specific drug delivery to the liver. Conjugations to the ASO also facilitate enhanced potency in targeted cells types. Unconjugated ASOs preferentially accumulate in the non-parenchymal (NP) cells of the liver including liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC) and hepatic stellate cells (HSC). Conversely triantennary N-acetyl galactosamine (GalNAc)-conjugation of ASOs results in a shift of ASO accumulation to the hepatocytes (Prakash et al. NAR, 42: 8796, 2014). A recent study was designed to investigate the ASO activity in hepatocytes, LSEC, KC, and HSC cells after treatment with unconjugated ASO or GalNAc-conjugated ASO. Mice were treated for 2 or 6 weeks with 6 mM/kg/wk of unconjugated ASO or 2 mM/kg/wk of GalNAc-conjugated ASO. Following treatment, mouse livers were perfused, the hepatocytes were isolated by Percoll gradient and the NP cells by Optiprep (Lodixanol) gradient. NP cells were further isolated into enriched populations by fluorescence-activated cell sorting (FACS). Whole liver and isolated cell populations were evaluated for RNA reduction after treatment. As expected, and despite using 3-fold lower dose of GalNac-conjugated ASO, there was similar targeted mRNA reduction between unconjugated and conjugated ASOs in the hepatocyte cell fraction. Also, regardless of the duration of ASO treatment, there was targeted RNA reduction in all NP cell populations. However, longer dosing with GalNac-conjugated ASO achieved a 4-fold increase in stellate cell activity. These results suggest that GalNac-conjugated ASO can decrease targeted RNA not only in hepatocyte cells, but also in LSEC, KC and HSC cells. This finding is relevant to NASH treatment because it illustrates the ability of GalNac-conjugated ASO to enhance drug potency yet still allow drug delivery to all liver cell types.

Sue Murray, B.S. Director IONIS Pharmaceuticals 2855 Gazelle Court Carlsbad, CA 92010 USA smurray@ionisph.com 760-603-2472

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Cellular Fractionation and Evaluation of Hepatic Cells after Antisense Oligonucleotide Treatment in Mice Sue Murray, Heather Murray, Richard Lee, Raechel Peralta, Mark Graham, Thazha Prakash, Punit Seth, Wuxia Fu, and Shuling Guo IONIS Pharmaceuticals, Carlsbad CA Nonalcoholic Steatohepatitis (NASH) causes chronic liver disease and is most common in Western countries. Current treatments for NASH are difficult to maintain and combine drug therapy with lifestyle modification (Pearlman et al. Curr Opin Gastroenterol. 2014 May; 30(3):223-37). One reason NASH is complicated to treat is due to its effects on multiple cell populations within the liver. Still, current research is unclear which population is key to target for treatment of the disease. Antisense oligonucleotide (ASO) drugs are a likely therapy for NASH due to their preferential distribution to the liver. Moreover, conjugates can be attached to the ASO and enable targeted cell specific drug delivery to the liver. Conjugations to the ASO also facilitate enhanced potency in targeted cells types. Unconjugated ASOs preferentially accumulate in the non-parenchymal (NP) cells of the liver including liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC) and hepatic stellate cells (HSC). Conversely triantennary N-acetyl galactosamine (GalNAc)-conjugation of ASOs results in a shift of ASO accumulation to the hepatocytes (Prakash et al. NAR, 42: 8796, 2014). A recent study was designed to investigate the ASO activity in hepatocytes, LSEC, KC, and HSC cells after treatment with unconjugated ASO or GalNAc-conjugated ASO. Mice were treated for 2 or 6 weeks with 6 mM/kg/wk of unconjugated ASO or 2 mM/kg/wk of GalNAc-conjugated ASO. Following treatment, mouse livers were perfused, the hepatocytes were isolated by Percoll gradient and the NP cells by Optiprep (Lodixanol) gradient. NP cells were further isolated into enriched populations by fluorescence-activated cell sorting (FACS). Whole liver and isolated cell populations were evaluated for RNA reduction after treatment. As expected, and despite using 3-fold lower dose of GalNac-conjugated ASO, there was similar targeted mRNA reduction between unconjugated and conjugated ASOs in the hepatocyte cell fraction. Also, regardless of the duration of ASO treatment, there was targeted RNA reduction in all NP cell populations. However, longer dosing with GalNac-conjugated ASO achieved a 4-fold increase in stellate cell activity. These results suggest that GalNac-conjugated ASO can decrease targeted RNA not only in hepatocyte cells, but also in LSEC, KC and HSC cells. This finding is relevant to NASH treatment because it illustrates the ability of GalNac-conjugated ASO to enhance drug potency yet still allow drug delivery to all liver cell types.

Sue Murray, B.S. Director IONIS Pharmaceuticals 2855 Gazelle Court Carlsbad, CA 92010 USA smurray@ionisph.com 760-603-2472

Development of Rapid Influenza Diagnostic test using aptamers Chandan Narayan1, Junyoung Kwon1, Kwang Ho Kim2, Ki Seok Kim2 and Sung Key Jang1 1Molecular Virology Laboratory, Postech Biotech Center, Department Of Life Science, Pohang University Of Science and Technology, San 31, Hyoja Dong, Nam-gu, Pohang, Gyeongbuk 37673, South Korea. 2Aptamer Science Inc., Postech Biotech Center, San 31, Hyoja Dong, Nam-gu, Pohang, Gyeongbuk 37673, South Korea. Influenza (flu) is a contagious respiratory illness caused by influenza viruses. It can cause mild to severe illness. Serious outcomes of flu infection can result in hospitalization or death. Some people, such as older people, young children, and people with certain health conditions, are at high risk for serious flu complications. Influenza occurs globally with an annual attack rate estimated at 5%–10% in adults and 20%–30% in children. Worldwide, these annual epidemics are estimated to result in about 3-5 million cases of severe illness, and about 250000 to 500000 deaths. Quick & simple kits with high sensitivity and specificity are the need of the hour. We are aiming to develop Rapid Influenza Diagnostic test using aptamers against nucleoprotein (NP), Hemagglutinin (HA) & Neuraminidase (NA). We are trying to obtain single stranded DNA aptamers specific to the influenza HA and NA proteins through “Systematic Evolution of Ligands by Exponential Enrichment” (SELEX) procedure. In the later stages, we are planning to check the Proof-of-concept by Lateral Flow assay. We used Purified recombinant HA and NA proteins and recombinant Baculoviruses expressing the above proteins on their envelopes derived from cDNA clones of Type A influenza virus subtypes A/Thailand/2004 (H5N1), A/Puerto Rico/8/1934 (H1N1) , A/Korea pandemic/2009 (H1N1), A/Hong Kong/1968 (H3N2), A/Anhui/1/2013 (H7N9) as the starting material. SELEX process for obtaining aptamers against HA and NA proteins are under process. Chandan Balaganchi Narayan, MSc Graduate Student Pohang University of Science and Technology (POSTECH) San 31, Hyoja Dong, Nam-gu, Pohang, Gyeongbuk 37673, South Korea. Chandannarayan12@gmail.com Ph. No: +82 10 3680021

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Harnessing Endogenous Lipoproteins to Drive Systemic siRNA Delivery Maire F. Osborn, Andrew H. Coles, Annabelle Biscans, Bruno M.D.C. Godinho, Reka A. Haraszti, Anastasia Khvorova RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA The primary obstacles facing the clinical advancement of therapeutic siRNAs are efficient delivery and immunogenicity. Chemical modifications installed throughout the RNA backbone and ribose sugar can preclude recognition by endo- and exonucleases and immune receptors, lengthening the molecule’s half-life in vivo. However, without the use of a targeting ligand or formulation, even highly modified siRNAs are rapidly cleared with little tissue penetrance. To address this issue, we have synthesized a panel of fully chemically stabilized, lipid-conjugated siRNAs (hsiRNAs). After systemic administration of these constructs, we observe distinct patterns of tissue accumulation and silencing efficacy in several tissues, including the liver, kidney, adrenal gland, spleen, and bone marrow. We directly measured the interactions of these lipid conjugated-siRNAs with serum albumin and circulating lipoproteins in vivo in wild-type mice. hsiRNAs lacking a lipid conjugate associate with albumin, and have a very short half-life following subcutaneous administration. Conversely, lipid-conjugated hsiRNAs are capable of leveraging VLDL, LDL, and/or HDL as endogenous carriers, greatly improving half-life, increasing tissue penetrance, and diverting them from inevitable clearance via the kidneys. These modifications may improve the pharmacokinetic and pharmacodynamic behavior of established tissue-targeting platforms. Maire F. Osborn, Ph.D. Ruth L. Kirschstein NRSA Postdoctoral Fellow RNA Therapeutics Institute University of Massachusetts Medical School 368 Plantation Street Worcester, MA USA maire.osborn@umassmed.edu (508) 654-6215

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Harnessing Endogenous Lipoproteins to Drive Systemic siRNA Delivery Maire F. Osborn, Andrew H. Coles, Annabelle Biscans, Bruno M.D.C. Godinho, Reka A. Haraszti, Anastasia Khvorova RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA The primary obstacles facing the clinical advancement of therapeutic siRNAs are efficient delivery and immunogenicity. Chemical modifications installed throughout the RNA backbone and ribose sugar can preclude recognition by endo- and exonucleases and immune receptors, lengthening the molecule’s half-life in vivo. However, without the use of a targeting ligand or formulation, even highly modified siRNAs are rapidly cleared with little tissue penetrance. To address this issue, we have synthesized a panel of fully chemically stabilized, lipid-conjugated siRNAs (hsiRNAs). After systemic administration of these constructs, we observe distinct patterns of tissue accumulation and silencing efficacy in several tissues, including the liver, kidney, adrenal gland, spleen, and bone marrow. We directly measured the interactions of these lipid conjugated-siRNAs with serum albumin and circulating lipoproteins in vivo in wild-type mice. hsiRNAs lacking a lipid conjugate associate with albumin, and have a very short half-life following subcutaneous administration. Conversely, lipid-conjugated hsiRNAs are capable of leveraging VLDL, LDL, and/or HDL as endogenous carriers, greatly improving half-life, increasing tissue penetrance, and diverting them from inevitable clearance via the kidneys. These modifications may improve the pharmacokinetic and pharmacodynamic behavior of established tissue-targeting platforms. Maire F. Osborn, Ph.D. Ruth L. Kirschstein NRSA Postdoctoral Fellow RNA Therapeutics Institute University of Massachusetts Medical School 368 Plantation Street Worcester, MA USA maire.osborn@umassmed.edu (508) 654-6215

Restoration of CFTR function by Antisense Oligonucleotide splicing modulation Efrat Ozeri-Galai1,2, Yifat S. Oren1,2 , Ofra Avizur1,2 , Michal Irony-Tur Sinai1, Steve Wilton3 , Steven M. Rowe4, Eylon Yavin5 and Batsheva Kerem1 1Department of Genetics, The Hebrew University, Jerusalem, Israel 2 SpliSense Givaat Ram, Jerusalem, Israel. 3 Australian Neuromuscular Research Institute, University of Western Australia, Perth, Australia 4Cystic Fibrosis Research Center,University of Alabama at Birmingham, USA 5The Institute for Drug Research, The School of Pharmacy, The Hebrew University of Jerusalem, Hadassah Ein-Kerem, Jerusalem, Israel A significant proportion (20–30%) of disease-causing mutations in humans affects pre-mRNA splicing. In Cystic fibrosis (CF), 10-15% of the mutations affect the correct splicing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Disease severity in patients carrying these mutations is highly variable, correlated with the level of aberrantly spliced transcripts. Antisense oligonucleotides (ASOs) were found to be highly efficient in modulation of the splicing pattern in several genetic diseases in which a significant clinical benefit was demonstrated. Here we aim to develop an antisense oligonucleotides (ASOs) based therapy to modulate the levels of correctly spliced CFTR. We first focuse on a common splicing mutation, the 3849+10kb C-to-T, which leads to inclusion of an 84 bp cryptic exon between exons 22-23 in the mature mRNA. This cryptic exon contains an in-frame stop codon that leads to degradation of a significant fraction of the mRNA by the NMD pathway as well as to the production of prematurely truncated nonfunctional proteins. We designed 2'-O-methyl phosphorothioate-modified ASOs, targeted to prevent the recognition of enhancer splice motifs in the cryptic exon or to mask the junctions between this exon and its flanking sequences. Screening of these AOSs led to the identification of several ASOs that significantly decrease the level of aberrantly spliced CFTR mRNA and incease the level of correctly apliced CFTR mRNA. Importantly we demonstrate that these ASOs restore the CFTR channel function. Our results indicate that ASOs targeted to mask splicing motifs in the CFTR gene can increase the correct splicing of CFTR leading to improvement of channel function. Efrat Ozeri Galai, PhD CTO SpliSense Edmond J Safra Campus The Hebrew University of Jerusalem Givat Ram Jerusalem Israel 91904 Efrat@Splisense.com

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Targeting NRARP with siRNA based compounds for the treatment of retinal neovascularization Covadonga Pañeda1, Tamara Martínez1, Ingo Roehl2, Susana Monteiro1, Amor Guerra1, Cristian Salvador1, and Ana Isabel Jiménez1

1Sylentis S.AU. Madrid, Spain 2Axolabs, Kulmbach, Germany Deregulation of angiogenesis is one of the hallmarks of wet AMD. In pathological angiogenesis there is an unbalance of VEGF and Notch signalling in the endothelium. High levels of VEGF and low expression of Notch makes the endothelium invasive and unstable. NRARP (Notch-regulated ankyrin repeat protein) is induced by Notch at newly formed branch points, where it differently modulates the activity of Notch and Wnt signalling to balance stalk proliferation and vessel stability. We have found that down regulation of NRARP in HUVEC cells reduces the formation of VEGF-induced tube-like structures, proliferation and cell migration. In vivo, NRARP down regulation correlates with an increase in Notch in stalk cells which translates into vessel regression. A selection of siRNAs targeting NRARP were designed, optimized and tested in vitro and subsequently tested in a model of laser-induced neovascularization in rats. The results of the efficacy study indicate that NRARP is a potential target for the treatment of angiogenic retinal diseases and that reduction of NRARP with specific siRNAs reduces the integrated density and area of the neovascularization site compared to PBS. Covadonga Pañeda, Ph.D. R&D Manager Sylentis c/Santiago Grisolía, 2 Madrid Spain cpaneda@sylentis.com +34 91 804 3817

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Targeting NRARP with siRNA based compounds for the treatment of retinal neovascularization Covadonga Pañeda1, Tamara Martínez1, Ingo Roehl2, Susana Monteiro1, Amor Guerra1, Cristian Salvador1, and Ana Isabel Jiménez1

1Sylentis S.AU. Madrid, Spain 2Axolabs, Kulmbach, Germany Deregulation of angiogenesis is one of the hallmarks of wet AMD. In pathological angiogenesis there is an unbalance of VEGF and Notch signalling in the endothelium. High levels of VEGF and low expression of Notch makes the endothelium invasive and unstable. NRARP (Notch-regulated ankyrin repeat protein) is induced by Notch at newly formed branch points, where it differently modulates the activity of Notch and Wnt signalling to balance stalk proliferation and vessel stability. We have found that down regulation of NRARP in HUVEC cells reduces the formation of VEGF-induced tube-like structures, proliferation and cell migration. In vivo, NRARP down regulation correlates with an increase in Notch in stalk cells which translates into vessel regression. A selection of siRNAs targeting NRARP were designed, optimized and tested in vitro and subsequently tested in a model of laser-induced neovascularization in rats. The results of the efficacy study indicate that NRARP is a potential target for the treatment of angiogenic retinal diseases and that reduction of NRARP with specific siRNAs reduces the integrated density and area of the neovascularization site compared to PBS. Covadonga Pañeda, Ph.D. R&D Manager Sylentis c/Santiago Grisolía, 2 Madrid Spain cpaneda@sylentis.com +34 91 804 3817

Clinical and preclinical correlation analysis for SYL1001, a new treatment for dry eye disease Covadonga Pañeda1, Victoria González1, Verónica Ruz1, Beatriz Vargas1, Anne-Marie Bleau1, and Ana Isabel Jiménez1

1Sylentis S.AU. Madrid, Spain SYL1001 is a siRNA targeting Transient Receptor Protein Vanilloid 1 (TRPV1) under development for the treatment of signs and symptoms of dry eye disease (DED). Biodistribution studies carried out in New Zealand white rabbits with different topical doses (0.375%, 0.75%, 1.125% and 1.5%) of the compound showed that SYL1001 is efficiently taken up by the cells of the ocular tissues and that only trace levels of the product are detected in plasma and systemic tissues. These studies demonstrated that absorption of SYL1001 is dose-dependent up to the dose of 1.125%. Concentrations above 1.125% resulted in lower amounts of SYL1001 in tissues, most likely due to saturation of the entrance mechanisms. The above mentioned doses were assessed in two phase 2 trials (NCT01776658, EudraCT No: 2012-001177-93 & NCT02455999, EudraCT No: 2014-004857-15). In these trials 127 patients were randomized into five arms (0.375%, 0.75%, 1.125% and 2.25% of SYL1001 and placebo (vehicle) (q.d). The dose of 1.125% was significant better than the other doses and placebo (p=0.016) in the treatment of patients with DED in terms of reducing the ocular pain/discomfort (VAS scale). Corneal staining and hyperemia were also improved with this dose. In conclusion, the results of the animal biodistribution studies correlate with the results of the phase II clinical trial and provide mechanistic explanation for the results of the dose-finding study. Covadonga Pañeda, Ph.D. R&D Manager Sylentis c/Santiago Grisolía, 2 Madrid Spain cpaneda@sylentis.com +34 91 804 3817

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Viromer – Enabling mRNA Delivery Steffen Panzner, Christian Reinsch and Patrick Kreideweiss Lipocalyx GmbH Viromers are highly efficient synthetic carriers for the delivery of mRNA, siRNA or pDNA. Mechanistically, Viromers mimic the active endosome escape described for viral particles. Viromers were constructed from PEI backbones and alkyl and carboxyalkyl side chains. Large libraries were synthesized and tested for their ability to transfect siRNA, plasmid DNA or mRNA. Lead compounds were promoted to in vivo models and mediate delivery of mRNA in spleen, muscle, dermis, lung and brain. Dr. Steffen Panzner CEO Lipocalyx GmbH Weinbergweg 23 Halle Germany Steffen.panzner@lipocalyx.de +49 345 55 59 845

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Viromer – Enabling mRNA Delivery Steffen Panzner, Christian Reinsch and Patrick Kreideweiss Lipocalyx GmbH Viromers are highly efficient synthetic carriers for the delivery of mRNA, siRNA or pDNA. Mechanistically, Viromers mimic the active endosome escape described for viral particles. Viromers were constructed from PEI backbones and alkyl and carboxyalkyl side chains. Large libraries were synthesized and tested for their ability to transfect siRNA, plasmid DNA or mRNA. Lead compounds were promoted to in vivo models and mediate delivery of mRNA in spleen, muscle, dermis, lung and brain. Dr. Steffen Panzner CEO Lipocalyx GmbH Weinbergweg 23 Halle Germany Steffen.panzner@lipocalyx.de +49 345 55 59 845

An Efficient, Scalable Mitochondrial Delivery Vector System for Ribonucleic Acids Volker Patzel1,2, Kaustav Chatterjee1, Han Yu1 National University of Singapore1, University of Cambridge2 Mitochondria are the cellular organelles harboring the respiratory chain and thirteen mitochondrial genes code for subunits of respiratory enzymes. Defects of all protein-coding mitochondrial genes have been associated with human, mainly neurodegenerative disorders, and with aging. Cells of patients with mitochondrial diseases or of elder patients suffering from other diseases or injuries, often do not harbour sufficient ‘healthy’ mitochondria. That affects the function of somatic cells, and of iPSCs generated thereof for therapeutic purpose. Mitochondrial gene therapy represents a promising solution to these problems but is hampered by the lack of efficient mitochondrial delivery vectors. The long non-coding ß2.7 RNA (~2500 nt) of the human cytomegalovirus was described to localize to the mitochondria of mammalian cells and to bind to mitochondrial complex I. We identified four conserved structural subdomains (D1-D4) within the ß2.7 RNA which govern its mitochondrial targeting activity. Subdomains D1 and D2 (100 nt) protected human SH-SY5Y dopaminergic neuroblastoma cells against 6-hydroxydopamine treatment just as well as the full-length ß2.7 RNA. Subdomains D1 and D4 exhibited a high structural and functional symmetry within a hypothetical antisense ß2.7 RNA transcript which hasn't been described yet. Subdomain D3 resembled the mitochondrial targeting activity of the full-length ß2.7 RNA. D3 tetramer (D3)4 was twice as active and (D3)4 followed by D2 tetramer (D2)4 showed three-fold higher activity, depicting the modularity and scalability of this mitochondria-targeting vector system. We used structurally stabilized ß2.7 RNA-derived sequences to co-deliver recombinant nucleic acids into mitochondria in order to trigger mitochondria-specific phenotypes: Firstly, a mRNA furnished with mitochondria-specific start and stop codons lead to recombinant GFP expression in the mitochondria; secondly, targeted mitochondrial delivery of antisense RNA triggered efficient knockdown of mitochondrial genes mtATP6 or mtATP8 leading to a reduction of mitochondrial ATP levels and cell viability. This technology can be explored towards mitochondrial gene therapy, e.g. by mitochondrial delivery of functional RNA or of ‘healthy’ mitochondrial genomes, or for mitochondrial targeting of non-nucleic acid compounds thus paving the way for the therapy of yet incurable human diseases. Volker Patzel, PhD, MBA Asst. Professor & PI National University of Singapore Dept. of Microbiology & Immunology, 5 Science Drive 2, Blk MD4, Level 5 Singapore 117597 Singapore micvp@nus.edu.sg +65 65163318

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Intracellular quantification of unmodified LNA oligonucleotides Hannah Pendergraff1, Jonas Vikeså1, Steffen Schmidt1, Christian Weile1, Jeppe Vinther2, Marie W. Lindholm1* Roche Innovation Center Copenhagen A/S1, Department of Biology, University of Copenhagen2

This project focuses on developing novel and innovative techniques to detect nuclear localized LNA oligonucleotides at low concentrations in live cultured cells and in vivo. So far, the field of oligonucleotide research has to rely on covalently attached fluorescent tags to study intracellular location of oligonucleotides. The alternative to fluorescent tags is to infer entry into the productive nuclear compartment indirectly through target transcript reduction. Target transcript analysis after LNA oligonucleotide treatment is essential for finding good drug candidates, but cannot provide information on how much compound escapes endosomes and enters the nuclei. We propose alternative detection methods in this project that can be used to determine if LNA oligonucleotides can have innate properties that affect their ability to enter into the cytosol and nucleus.

We have been optimizing a protocol to proximity label LNA oligonucleotides in living cells. Our approach uses a novel stable cell line which produces a fusion protein containing a heme peroxidase, APEX2, a fluorescent eGFP protein, and a cellular localization signal, adapted from previously published article using APEX2 to biotinylate proteins1,2. With this technology, we could selectively biotinylate LNA oligonucleotides in either the nuclear or cytoplasmic cellular compartments. We have made great progress towards having a working protocol, and have developed methods of detection of the biotinylated LNA oligonucleotides.

Alternatively, we have also optimized subcellular fractionation protocols in order to isolate LNA oligonucleotides within specific cellular compartments. We are currently in the final stages of developing methods which can identify the concentration of nuclear retained LNA oligonucleotides. Using this information, we can determine specific properties of the LNA oligonucleotides themselves with affect cellular localization and endosomal escape.

1Lam, S.S., et al. 2015. Nat. Methods 12, 51-54.2Hung, V., et a. 2016. Nat. Protoc. 11, 456-475.

Hannah M. Pendergraff, Ph.D. Postdoctoral Research Scientist Roche Innovation Center Copenhagen A/S Fremtidsvej 3 2970 HørsholmDenmark Hannah.pendergraff@roche.com +45 61638586

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A modular cell-internalizing aptamer nanostructure to deliver large functional RNAs David Porciani1,2,3, Kwaku D. Tawiah1,2, Leah N. Cardwell3, Khalid K. Alam1,2,5 Margaret J. Lange3, Mark A. Daniels3 & Donald H. Burke1,2,3,4 1 Department of Biochemistry, University of Missouri, Columbia, MO 65211, United States; 2 Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, United States; 3 Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, MO 65212, United States; 4 Department of Bioengineering, University of Missouri, Columbia, MO 65211, United States; 5 Department of Chemical & Biological Engineering, Northwestern University, Evanston, IL 60208 United States Cell-internalizing aptamers are nucleic acid ligands that bind cell-surface markers and internalize through ligand-induced or receptor-mediated endocytosis. They have been built into various RNA nanoparticles to perform targeted delivery of siRNAs (<15 KDa, 19-21 nt) and small chemotherapeutics. Aptamers, CRISPR guide RNAs, and large regulatory RNAs such as messenger RNAs or long non-coding RNAs represent a class of highly powerful potential therapeutics. However, effective tools to perform their targeted delivery are still lacking. Here, we demonstrate a modular nanostructure for the aptamer-mediated cellular delivery of large functional RNA cargoes (~50-80 kDa). Our strategy exploits (i) targeting and delivery abilities of cell-internalizing aptamers that recognize human transferrin receptor-expressing cells and B-cell tumor cells, and (ii) reporter RNA payloads in the ~175-250 nt range that include improved variations of the fluorogenic Broccoli aptamer. The use of reporter cargoes accelerates the testing of platform designs and enables rapid evaluation of the impact of assembly and internalization on the aptamer. Flow cytometry and confocal microscopy showed that targeting and cargo aptamers both retained their original functionality when assembled into a more complex structure, and were both internalized by malignant B cell leukemia cells. We demonstrated modularity of the nanostructure design by redirecting the targeting specificity of the fluorogenic RNAs to different cancer cell lines by using the appropriate aptamer for those cell types. This proof-of-principle is the first demonstration of aptamer-mediated, cell-internalizing delivery of large functional RNAs and may represent a useful starting point for further targeted delivery applications of large regulatory RNAs. David Porciani, PhD Postdoctoral fellow University of Missouri – Columbia Bond Life Sciences Center 1201 E. Rollins St. Columbia, Missouri United States porcianid@missouri.edu

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Quaking Modulates Cortical Mechanical Properties of Monocytes Jurriën Prins1, Alexander Cartagena-Rivera2, Richard Chadwick2, Anton Jan van Zonneveld1, Eric van der Veer1 1Department of Internal Medicine (Nephrology) and Einthoven Laboratory for Vascular Medicine, Leiden University Medical Center, The Netherlands 2Laboratory of Cell Biology, Section on Auditory Mechanics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland Background: Modulation of cellular function necessitates a versatile utilization of the transcriptome, thereby altering the proteome. These functional adaptations are post-transcriptionally guided by RNA-binding proteins (RBP), which confer cells with the capacity to rapidly respond to various stimuli and stressors. Recently, we described a pivotal role for the RBP Quaking (QKI) in monocyte and macrophage biology. Computational pathway analysis revealed that cytoskeleton reorganization was particularly affected by QKI-mediated alternative splicing of pre-mRNAs. Since the (cortical) actin cytoskeleton is crucial for maintaining cell shape and mechanical properties, this prompted us to focus our attention on a C-terminal exclusion event of exon 13 in the actin-capping protein γ-Adducin (ADD3). At present, the functional consequences of ADD3 isoform switching are unknown. Methods and Results: We have previously shown that decreased QKI expression is characterized by an increase in ADD3 exon 13 inclusion. To pinpoint whether QKI specifically regulates this splicing event, we mutated the QKI binding site proximal to exon 13 in a minigene. These studies revealed that disrupting the capacity for QKI to bind at this intronic sequence abolished ADD3 splicing (wild type: 2.0% versus mutated: 94.9% exon inclusion; p<0.0001). Using a predictive bioinformatics tool, inclusion of exon 13 was found to induce conformational changes in ADD3 protein structure. We hypothesized that ADD3 isoform switching would impact cortical actin organization and function, and result in altered cell mechanics. Therefore, we utilized atomic force microscopy (AFM) to study the cortical mechanical properties of THP-1 monocytes. In short, shRNA-mediated abrogation of QKI expression in monocytes resulted in a 0.94-fold decrease in cell diameter (p = 0.06), accompanied by an increase in both cortical tension (control versus KD = 216.1 ± 12.3 versus 255.3 ± 14.3 pN/μm; p = 0.05) and intracellular pressure (control versus KD = 67.14 ± 4.46 vs 84.44 ± 5.15 Pa; p = 0.02). These findings suggest a potential role for QKI in maintaining cell morphology and function of the cortical actin cytoskeleton. Further analyses of AFM experiments with monocytes expressing different ADD3 isoforms will show if these mechanical changes are initiated by isoform switching of ADD3. Jurrien Prins, MSc PhD Candidate Leiden University Medical Center Albinusdreef 2, 2333 ZA Leiden, Zuid-Holland The Netherlands j.prins@lumc.nl +31715265370

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Quaking Modulates Cortical Mechanical Properties of Monocytes Jurriën Prins1, Alexander Cartagena-Rivera2, Richard Chadwick2, Anton Jan van Zonneveld1, Eric van der Veer1 1Department of Internal Medicine (Nephrology) and Einthoven Laboratory for Vascular Medicine, Leiden University Medical Center, The Netherlands 2Laboratory of Cell Biology, Section on Auditory Mechanics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland Background: Modulation of cellular function necessitates a versatile utilization of the transcriptome, thereby altering the proteome. These functional adaptations are post-transcriptionally guided by RNA-binding proteins (RBP), which confer cells with the capacity to rapidly respond to various stimuli and stressors. Recently, we described a pivotal role for the RBP Quaking (QKI) in monocyte and macrophage biology. Computational pathway analysis revealed that cytoskeleton reorganization was particularly affected by QKI-mediated alternative splicing of pre-mRNAs. Since the (cortical) actin cytoskeleton is crucial for maintaining cell shape and mechanical properties, this prompted us to focus our attention on a C-terminal exclusion event of exon 13 in the actin-capping protein γ-Adducin (ADD3). At present, the functional consequences of ADD3 isoform switching are unknown. Methods and Results: We have previously shown that decreased QKI expression is characterized by an increase in ADD3 exon 13 inclusion. To pinpoint whether QKI specifically regulates this splicing event, we mutated the QKI binding site proximal to exon 13 in a minigene. These studies revealed that disrupting the capacity for QKI to bind at this intronic sequence abolished ADD3 splicing (wild type: 2.0% versus mutated: 94.9% exon inclusion; p<0.0001). Using a predictive bioinformatics tool, inclusion of exon 13 was found to induce conformational changes in ADD3 protein structure. We hypothesized that ADD3 isoform switching would impact cortical actin organization and function, and result in altered cell mechanics. Therefore, we utilized atomic force microscopy (AFM) to study the cortical mechanical properties of THP-1 monocytes. In short, shRNA-mediated abrogation of QKI expression in monocytes resulted in a 0.94-fold decrease in cell diameter (p = 0.06), accompanied by an increase in both cortical tension (control versus KD = 216.1 ± 12.3 versus 255.3 ± 14.3 pN/μm; p = 0.05) and intracellular pressure (control versus KD = 67.14 ± 4.46 vs 84.44 ± 5.15 Pa; p = 0.02). These findings suggest a potential role for QKI in maintaining cell morphology and function of the cortical actin cytoskeleton. Further analyses of AFM experiments with monocytes expressing different ADD3 isoforms will show if these mechanical changes are initiated by isoform switching of ADD3. Jurrien Prins, MSc PhD Candidate Leiden University Medical Center Albinusdreef 2, 2333 ZA Leiden, Zuid-Holland The Netherlands j.prins@lumc.nl +31715265370

Tumour targeting Chitosan nanoparticles: Key design parameters influencing siRNA and mRNA delivery Sanya Puri1, Marianne Ashford1, E. Lallana2, Annalisa Tirella,2 Nicola Tirelli2 Affiliation(s) AstraZeneca Pharmaceutical Sciences, UK1 University of Manchester, UK2 Systematic investigations into the physicochemical properties of the nanoparticle carrier components is critical for rational design and optimal functional behaviour of RNA delivery systems. Here we present data on the influence of Chitosan macromolecular (molecular weight; degree of deacetylation, i.e., charge density) and nanoparticle variables (complexation strength, i.e., stability; nucleic acid protection; internalization rate) on their ability to achieve either mRNA silencing (anti luc siRNA delivery) or protein expression (luc encoding mRNA delivery) to CD44 overexpressing HCT116 tumour cells.

Fig 1a : Chitosan/HA Nanoparticles Feature HA (Green) on the Surface and a Core Where the Two Polymers Produce a Ternary Complex with RNA (Blue). L,UL,H denote Low, Ultralow and High Mol weight chitosan whilst 51, 91 and 84 are the degree of deacetylation (amine groups on chitosan) (b) Relationship between uptake in HCT cells and functional behavior, high mol weight/high amine content showed optimal functional performance across both payloads. The RNA avidity for chitosan showed—as expected—an inverse relationship: higher avidity–higher polyplex stability–lower transfection efficiency. The avidity of chitosan for RNA appears to lead to opposite effects: higher avidity–higher polyplex stability but also higher transfection efficiency, although less amenable to decomplex from the RNA, overall we think the positive functional performance of this variant can attributed to improved endoctyic escape and protective properties of this variant. Hence, a better understanding of the nanoparticle/endosomal environment interactions is absolutely critical for a more accurate prediction of the nanoparticle behavior. Dr Sanyogitta Puri Team Leader AstraZeneca Macclesfield, Cheshire Sk10 2NA UK Sanyogitta.puri@astrazeneca.com 0044 1625515847

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Enzymatic synthesis of random sequences of RNA analogues by a DNA polymerase mutant: a versatile way for RNA functionalization and Aptamer design. Irina Randrianjatovo-Gbalou and Marc Delarue Unité de Dynamique Structurale des Macromolécules, Institut Pasteur, UMR 3528 du C.N.R.S., 25 Rue du Docteur Roux, 75015 Paris, France

Nucleic acids (RNA or DNA) aptamers exhibit valuable advantages and properties compared to protein therapeutics in terms of synthetic accessibility, affinity, specificity and size. As these molecules can be selected from pools of random-sequence oligo- or poly-nucleotides, the engineering of DNA or RNA polymerases capable to generate random libraries of RNA or DNA remains at the basis of the success of such synthesis of functional nucleic acids analogues.

We describe here the design of a A-family DNA polymerase that can efficiently

incorporate natural or modified ribonucleotides, resulting in long polymers that could serve as a library for the selection of aptamers or ribozymes. Five mutants of DNA polymerase were generated.

The functional characterization of each mutant has been performed and one promising candidate was shown to be able to display an enhanced efficiency to incorporate the four natural ribonucleotides (ATP, UTP, CTP and GTP) compared to the wild-type. As a result, long homo- or heteropolymers of ribonucleotides were obtained whose length (20-300 nt) can be controlled by the running time of the reaction, which necessitates Mn2+ ions. HPLC analysis of the resulting ribonucleosides obtained after enzymatic digestion of the newly synthesized RNA showed equal probability of incorporation of the four ribonucleotides and the randomness of the sequences was confirmed after RNA-sequencing.

Moreover, the incorporation of modified deoxy- and ribonucleotides has been also investigated. The following analogues were successfully accepted by at least one promising mutant: 2’-Fluoro-dATP, 2’-Fluoro-dUTP, 2’-Fluoro-dCTP, 2’-Fluoro-dGTP, 2’-Fluoro-dTTP, 2’-Amino-dATP, 2’-Amino-dTTP, Ara-ATP (Vidarabine triphosphate), Ara-CTP (Cytarabine triphosphate), 2’-O-methyl-ATP, 2’-O-methyl-CTP, 2’ O-CH3-ddTTP, ⍷-ATP, 2-Aminopurine.

These properties of DNA polymerase mutants will contribute to broaden the

applicability of chemically modified nucleic acids in RNA biology, medical diagnosis, and molecular recognition strategies. By this work, a versatile toolbox for RNA and DNA functionalization and for aptamer design may come into being. Irina Volahasina Randrianjatovo-Gbalou, PhD Postdoctoral fellow Institut Pasteur vrandria@pasteur.fr Paris, 75015 France +33 6 66 09 69 93

Efficacy and safety profile of tricyclo-DNA antisense oligonucleotides in Duchenne muscular dystrophy mouse model Karima Relizani1,2*, Graziella Griffith1,2*, Lucía Echevarría1, Faouzi Zarrouki1,3, Patricia Facchinetti1, Cyrille Vaillend3, Christian Leumann4, Luis Garcia1 and Aurelie Goyenvalle1 1 Université de Versailles St- Quentin, U1179 INSERM, UFR des Sciences de la Santé – LIA

BAHN CSM, France. 2 SQY Therapeutics, UFR des Sciences de la Santé, Montigny-le-Bretonneux, France ;

Université de Versailles Saint-Quentin en Yvelines. 3 Neuro-PSI, UMR 9197, Université Paris Sud, CNRS, Université Paris Saclay, Orsay,

France. 4 Departments of Chemistry & Biochemistry, University of Bern, Switzerland. *K. R and G.G contributed equally to this study. Antisense oligonucleotides (AONs) hold promise for therapeutic splice-switching correction in many genetic diseases. However, despite advances in AON chemistry and design, systemic use of AONs is limited due to poor tissue uptake and sufficient therapeutic efficacy is still difficult to achieve. A novel class of AONs made of tricyclo-DNA (tcDNA) is considered very promising for the treatment of Duchenne muscular dystrophy (DMD), a neuromuscular disease typically caused by frame-shifting deletions or nonsense mutations in the gene encoding dystrophin and characterized by progressive muscle weakness, cardiomyopathy and respiratory failure in addition to cognitive impairment. Herein, we report the efficacy and toxicology profile of a 13mer tcDNA in mdx mice. We show that systemic delivery of 13mer tcDNA allows restoration of dystrophin in skeletal muscles and to a lower extent in the brain, leading to muscle function improvement and correction of behavioural features linked to the emotional/cognitive deficiency. More importantly tcDNA treatment was generally limited to minimal glomerular changes and few cell necrosis in proximal tubules, with only slight variation in serum/urinary kidney toxicity biomarkers levels and coagulation parameters, which is typical and well-known profile of phosphorothioate AONs. These results demonstrate an encouraging safety profile for tcDNA and confirm their therapeutic potential for the systemic treatment of DMD patients. Karima Relizani, PhD Post-doctoral researcher University of Versailles St-Quentin en Yvelines, SQY Therapeutics 02, Avenue de la source de la bièvre Montigny-le bretonneux France sonia.relizani-karima@uvsq.fr Tel: 0033 642 66 51 64

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TCTP inhibition by specific antisense oligonucleotide lipid moiety-modified for the treatment of castration-resistant prostate cancer Palma Rocchi1-3

1Marseille Cancerology Research Center (CRCM) INSERM UMR1068, 2Paoli-Calmettes Institute, 3 Aix-Marseille University Prostate cancer (PC) is one of the most common causes of death by cancer in the Western World. Once diagnosed, androgen ablation is the first line therapy. However, the disease progresses to a castration-resistant state (CRPC), and chemotherapy used prolongs life span of a few months. In order to improve therapy for CRPC, our strategy is to target genes amplified after androgen deprivation. It has been shown that Translationally controlled Tumor protein (TCTP) is overexpressed and p53 expression and function are lost in CRPC. Analysis showed that TCTP’s expression was found to be significantly down regulated after androgen ablation to become uniformly highly expressed in 75% of castration-resistant prostate cancer. We developed and patented a first generation oligonucleotide antisense (ASO) to target and inhibit TCTP expression. We choose the ASO strategy since ASO can be used for human therapy and can inhibit specifically target genes especially whose that are difficult to target with small molecules inhibitors or neutralizing antibodies. We have shown that TCTP-ASO significantly inhibits CRPC pre-clinical models and restores p53 expression and function (Baylot et al, 2012; Patent PCT 10306447.3, 2010). In order to improve stability, delivery and biodisponibility of TCTP-ASO, we developed a third generation ASO by using a lipid-conjugated oligonucleotide modification (LASO) (Patent PCT/IB2013/001517/2013). Interestingly, transfection with TCTP-LASO leads to rapid and prolonged internalisation via micropinocytosis with no transfecting agent. TCTP-LASO down-regulates TCTP protein expression and is associated with significant decreased viability and increase in apoptosis of CRPC cells. These results were confirmed in vivo where we showed that lipid-modification leads to TCTP-tumour down regulation and enhances TCTP-ASO efficiency to down-regulate tumour growth in CRPC xenografts with no systemic toxicity for the animals (Karaki et al, 2017).

Palma Rocchi, PhD-HDR Head of the Prostate Cancer Research Project Molecular Oncology Department Marseille Cancerology Research Center UMR 1068 Inserm-Paoli-Calmettes Institute 27 Bd. Leï Roure, BP 30059 13273 Marseille Cedex 09, France Email : palma.rocchi@inserm.fr

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TCTP inhibition by specific antisense oligonucleotide lipid moiety-modified for the treatment of castration-resistant prostate cancer Palma Rocchi1-3

1Marseille Cancerology Research Center (CRCM) INSERM UMR1068, 2Paoli-Calmettes Institute, 3 Aix-Marseille University Prostate cancer (PC) is one of the most common causes of death by cancer in the Western World. Once diagnosed, androgen ablation is the first line therapy. However, the disease progresses to a castration-resistant state (CRPC), and chemotherapy used prolongs life span of a few months. In order to improve therapy for CRPC, our strategy is to target genes amplified after androgen deprivation. It has been shown that Translationally controlled Tumor protein (TCTP) is overexpressed and p53 expression and function are lost in CRPC. Analysis showed that TCTP’s expression was found to be significantly down regulated after androgen ablation to become uniformly highly expressed in 75% of castration-resistant prostate cancer. We developed and patented a first generation oligonucleotide antisense (ASO) to target and inhibit TCTP expression. We choose the ASO strategy since ASO can be used for human therapy and can inhibit specifically target genes especially whose that are difficult to target with small molecules inhibitors or neutralizing antibodies. We have shown that TCTP-ASO significantly inhibits CRPC pre-clinical models and restores p53 expression and function (Baylot et al, 2012; Patent PCT 10306447.3, 2010). In order to improve stability, delivery and biodisponibility of TCTP-ASO, we developed a third generation ASO by using a lipid-conjugated oligonucleotide modification (LASO) (Patent PCT/IB2013/001517/2013). Interestingly, transfection with TCTP-LASO leads to rapid and prolonged internalisation via micropinocytosis with no transfecting agent. TCTP-LASO down-regulates TCTP protein expression and is associated with significant decreased viability and increase in apoptosis of CRPC cells. These results were confirmed in vivo where we showed that lipid-modification leads to TCTP-tumour down regulation and enhances TCTP-ASO efficiency to down-regulate tumour growth in CRPC xenografts with no systemic toxicity for the animals (Karaki et al, 2017).

Palma Rocchi, PhD-HDR Head of the Prostate Cancer Research Project Molecular Oncology Department Marseille Cancerology Research Center UMR 1068 Inserm-Paoli-Calmettes Institute 27 Bd. Leï Roure, BP 30059 13273 Marseille Cedex 09, France Email : palma.rocchi@inserm.fr

Biodistribution, metabolism and in situ accumulation of naked and GalNAc-conjugated locked nucleic acids in rat liver and kidney Fernando Romero-Palomo, Annamaria Braendli-Baiocco, Pawel Dzygiel, Matthias Festag, Guy Fischer, Christophe Husser , Erich Koller, Barbara Lenz, Rachel Neff, Simone Schadt, Bernd Steinhuber, Andreas Brink Roche Pharma Research and Early Development (pRED), Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd Locked nucleic acids (LNAs) are chemically modified RNA nucleotides with the potential to be used as therapeutic anti-sense oligonucleotides. Despite their proved therapeutic potential, some LNAs have also showed toxic effects mainly associated with their accumulation in liver and kidney. We performed a rat toxicity study to further characterize mechanisms of toxicity after single and multiple doses of selected tool LNAs with three different sequences and safety profiles (safe/medium/high toxicity based on previous studies). Four rats per group and time point were dosed subcutaneously every 7 days for up to 6 weeks and sacrificed at either 4, 18 or 39 days after first dose, including a vehicle control group. These LNAs were used both unconjugated (naked) or conjugated with N-acetyl galactosamine (GalNAc), which is a high-affinity ligand for the asialoglycoprotein receptor expressed on the hepatocyte cell surface. LNA exposure in liver and kidney was quantified and subjected to metabolite identification by means of LC-MS/MS. The presence of LNAs was evaluated in situ by immunohistochemistry (IHC) and additionally by MALDI-FTICR mass spectrometry imaging. An antibody targeting phosphorothioate (PS) internucleotide modifications present in all LNA compounds was used for the immunohistochemical assessment. For MALDI imaging, LNAs were detected independently of their sequence based on a diagnostic fragment originating from the PS backbone. Presence of naked LNAs was demonstrated mainly in Kupffer cells, whereas GalNAc-conjugated LNAs accumulated in both Kupffer cells and hepatocytes. All tool LNAs accumulated primarily in proximal renal tubules. Quantitative exposure assessment greatly correlated with IHC in the kidney, and to a lesser extent in the liver, probably due to the different target cells of accumulation in this organ. Interestingly, both naked and GalNAc compounds with high toxicity profile showed several major metabolites of different length, unlike the rest of compounds (safe/medium toxicity), which showed one major metabolite per compound. The distribution and metabolism of naked and GalNAc LNAs provide a useful starting point for additional investigations on the pathogenesis of LNA-induced toxicity. Fernando Romero-Palomo, DVM, PhD Postdoc Scientist F. Hoffmann-La Roche Ltd 4070 Basel Switzerland fernando.romero_palomo@roche.com

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Sequence specific inhibition of DNA damage response at double-strand breaks and deprotected telomeres by antisense oligonucleotides Francesca Rossiello1, Flavia Michelini1, Julio Aguado1, Valerio Vitelli1, Sara Sepe1, Fabio Pessina1, Sethuramasundaram Pitchiaya2, Fabrizio d’Adda di Fagagna1,3. 1IFOM, the FIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy. 2Michigan Center for Translational Pathology, University of Michigan Cancer Center, MI 48109, Ann Arbor, USA. 3Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (IGM-CNR), Via Abbiategrasso 207, 27100 Pavia, Italy. Site-specific small non-coding RNAs generated by DICER and DROSHA (we named them DDRNAs) have been shown to play a role in the DNA damage response (DDR) signalling and in DNA repair (Francia et al, Nature, 2012; Wei et al, Cell, 2012; Gao et al, Cell Research, 2014; Qi et al, Mut Res, 2016). We showed that when a double-strand break occurs, RNA polymerase II is recruited to the damaged site and synthesizes damage-induced long non-coding RNAs (dilncRNAs) that act both as DDRNA precursors and by recruiting DDRNAs through RNA:RNA pairing. Interference with dilncRNA:DDRNA interactions by antisense oligonucleotides (ASOs) enables site-specific DDR inhibition. We extended these observations to dysfunctional telomeres, showing that telomeric DDRNAs play a role in the full DDR activation at deprotected telomeres. Indeed inhibition of telomeric DDRNAs functions by sequence-specific ASOs prevented the activation of DDR markers specifically at telomeres in cultured cells and in mouse tissues (Rossiello et al, Nat Comm, 2017). These data strongly support the evidence that ASO approach can be used as a powerful tool to silence DDR activation in a sequence-specific manner. Francesca Rossiello, PhD Postdoctoral fellow IFOM, the FIRC Institute of Molecular Oncology Via Adamello 16 Milan, 20139 Italy francesca.rossiello@ifom.eu

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Sequence specific inhibition of DNA damage response at double-strand breaks and deprotected telomeres by antisense oligonucleotides Francesca Rossiello1, Flavia Michelini1, Julio Aguado1, Valerio Vitelli1, Sara Sepe1, Fabio Pessina1, Sethuramasundaram Pitchiaya2, Fabrizio d’Adda di Fagagna1,3. 1IFOM, the FIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy. 2Michigan Center for Translational Pathology, University of Michigan Cancer Center, MI 48109, Ann Arbor, USA. 3Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (IGM-CNR), Via Abbiategrasso 207, 27100 Pavia, Italy. Site-specific small non-coding RNAs generated by DICER and DROSHA (we named them DDRNAs) have been shown to play a role in the DNA damage response (DDR) signalling and in DNA repair (Francia et al, Nature, 2012; Wei et al, Cell, 2012; Gao et al, Cell Research, 2014; Qi et al, Mut Res, 2016). We showed that when a double-strand break occurs, RNA polymerase II is recruited to the damaged site and synthesizes damage-induced long non-coding RNAs (dilncRNAs) that act both as DDRNA precursors and by recruiting DDRNAs through RNA:RNA pairing. Interference with dilncRNA:DDRNA interactions by antisense oligonucleotides (ASOs) enables site-specific DDR inhibition. We extended these observations to dysfunctional telomeres, showing that telomeric DDRNAs play a role in the full DDR activation at deprotected telomeres. Indeed inhibition of telomeric DDRNAs functions by sequence-specific ASOs prevented the activation of DDR markers specifically at telomeres in cultured cells and in mouse tissues (Rossiello et al, Nat Comm, 2017). These data strongly support the evidence that ASO approach can be used as a powerful tool to silence DDR activation in a sequence-specific manner. Francesca Rossiello, PhD Postdoctoral fellow IFOM, the FIRC Institute of Molecular Oncology Via Adamello 16 Milan, 20139 Italy francesca.rossiello@ifom.eu

Rational Truncation of RNA Aptamer G70 Leads to Improved Inhibition of the Sphingomyelinase Activity of Two Recombinant Isoforms of the Main Toxin of the Loxosceles laeta Spider Venom Amalia Sapag, Marcela Cueto Laboratory of Gene Pharmacotherapy, Department of Pharmacological and Toxicological Chemistry, Faculty of Chemical and Pharmaceutical Sciences, Universidad de Chile Loxoscelism, the envenomation caused by the bite of Loxosceles spiders, is characterized by dermonecrosis, haemolysis and even death. The venom contains several isoforms of a toxic sphingomyelinase D (SMD). There are no diagnostic methods and treatment is mainly palliative: horse antisera, of unproven efficacy, may elicit anaphylactic reactions. Aptamers targeted to recombinant SMD isoforms may provide safe, specific and effective theranostic means. SELEX based on binding to SMD isoform Ll2 of Loxosceles laeta has allowed the identification of several promising RNA aptamers (107 nt long, 60 variable nucleotides in the center). Five aptamers were truncated with the dual aim of improving aptamer function and making large scale chemical synthesis affordable (<60 nucleotides). These aptamers were the three most highly represented in the selected cohort, G17, G27 and G37, and the two that exert the highest inhibition of the Ll2 sphingomyelinase activity, G62 and G70. Part of a structural element common to all, arm C, formed by the constant sequence segments flanking the variable center, was deleted. Aptamers were synthesized by in vitro transcription. Binding to Ll2 was measured using nitrocellulose filters and inhibition of the sphingomyelinase activity of three Loxosceles laeta SMD isoforms (Ll1, Ll2, LlPLD1) was determined fluorimetrically. Binding of each of the five aptamers partially deleted in arm C (78 nt) to Ll2 was shown to be specific and, in most cases, did not differ greatly from that of full length versions. Similarly, truncation did not affect inhibition of the sphingomyelinase activity of any of the three SMD isoforms tested, except in the case of G70. The short version of G70 (150 nM) proved capable of significant inhibition of Ll1 (30%) and Ll2 (24%), a substantial improvement over the inhibition attained with the full length aptamer, 24% and 8% respectively. Thus, the inhibitory activity of additional G70 variants bearing deletions in each of its three arms of predicted secondary structure (A-B-C) was measured. G70 lacking arm B (83 nt) results in 18% inhibition of both Ll1 and Ll2, while partial and full deletions of arm A are clearly detrimental to inhibition of Ll1 and Ll2. When tested individually, arms A, B and C were not capable of supporting strong inhibitions of either Ll1 or Ll2. Overall, a 54 nt variant of G70 having arm A, but lacking arm B and part of arm C, may constitute the molecular basis of novel therapeutic and diagnostic agents for loxoscelism. (FONDECYT 1100209, LFTG-UCH 14-04, LFTG-UCH 17-05, PEEI-FCQF-2017) Amalia Sapag (PhD) Associate Professor, Universidad de Chile Sergio Livingstone 1007, Independencia Santiago, 838-0492, Chile asapag@uchile.cl

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Antisense oligonucleotide-mediated upregulation of CFTR via steric block of post-transcriptional control elements: A novel approach for Cystic Fibrosis therapeutics Shruti Sasaki, Chenguang Zhao, Jeff Crosby, Brett P. Monia, Shuling Guo Ionis Pharmaceuticals

Cystic fibrosis (CF) is an autosomal-recessive genetic disease that affects approximately 70,000 individuals worldwide. CF is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene that result in decreased protein production and function of the chloride channel. Mutations in CFTR most severely affect the lung, causing a build-up of thick mucus and increased susceptibility to infection. As a result, lung disease is the leading cause of morbidity and mortality in CF patients. While many symptomatic treatments exist, quality of life and life expectancy for CF patients are low, and better therapies to improve CFTR expression and function are in need. Thus, we have developed an approach to upregulate CFTR expression using antisense oligonucleotide (ASO) treatment. ASOs are a therapeutically proven approach to drug discovery for diseases with a genetic basis. They are often used to decrease protein expression via targeted RNase H-mediated degradation of mRNA. More recently, ASOs have been used as steric blocks of splicing factors and regulatory elements to increase mRNA and protein expression (Liang et al., Nat. Biotech. 2016, Finkel et al., Lancet 2017). ASOs can be effectively delivered to various lung cell types by aerosol inhalation (Crosby et al., J Pharmacol Exp Ther. 2007).

The human CFTR mRNA is a 6 kb transcript with many putative regulatory sites. In various cell types and tissues, wild type CFTR mRNA and protein expression are not correlated, suggesting that CFTR may be tightly regulated at the mRNA level and could therefore be a good target for ASO-mediated upregulation. We have identified potential structural elements, RNA-binding elements and miRNA sites in the CFTR transcript that may affect its translation. ASOs to block these sites and other previously identified regulatory elements that potentially inhibit CFTR protein expression have been designed, and we have developed high-throughput assays to monitor the effects of ASO treatment on CFTR expression and function. ASO inhibition of 5’ structural elements and a single uORF increases CFTR protein expression by 2- to 4-fold, and functional assays and mechanistic analyses are ongoing. Thus, ASO-mediated upregulation of CFTR presents a promising and novel approach to the development of therapeutics for cystic fibrosis.

Shruti Sasaki, PhD Postdoctoral Fellow Ionis Pharmaceuticals 2855 Gazelle Court Carlsbad, CA USA ssasaki@ionisph.com

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Effective Synthesis of 2′-Amino-LNAs Bearing Any of the Four Nucleobases Hiroaki Sawamoto1, Yuuki Arai1, Shuhei Yamakoshi1, Satoshi Obika2, Eiji Kawanishi1 Mitsubishi Tanabe Pharma Corporation 1, Osaka University 2 2′-Amino-LNA was synthesized by Prof. Wengel in 1998, [1] and its unique properties and functions have been reported until now. [2] However, the synthesis of those nucleosides bearing purine bases was so difficult, [3] and the development of the convenient synthetic method was desired. Since a common intermediate obtained at later steps will allow reducing the number of total steps, we selected 2′-amino-LNA-T as the intermediate for the synthesis of any other 2′-amino-LNA monomers. After several attempts, we found that 2′-amino-LNAs bearing A (adenine), G (guanine) and mC (5-methylcytosine) were successfully synthesized from 2′-amino-LNA-T via transglycosylation reaction with high yield and β-selectivity. The mild reaction condition enable the coexistence of acid-labile protecting groups including a 4,4′-dimethoxytrytyl (DMTr) group. Thus, 2′-amino-LNAs bearing any of the four nucleobase are able to be synthesized easily and effectively by this discovery.

References [1] Singh, S. K.; Kumar, R.; Wengel, J. J. Org. Chem. 1998, 63, 6078. [2] Astakhova, I. K.; Wengel, J. Acc. Chem. Res. 2014, 70, 8617. [3] Ravn, J.; Rosenbohm, C.; Christensen, M. S.; Koch, T. Nucleosides, Nucleotides, and Nucleic Acids, 2006, 25, 845. Hiroaki Sawamoto, PhD Senior Research Scientist Mitsubishi Tanabe Pharma Corporation 1000, Kamoshida-cho, Aoba-ku Yokohama 227-0033 Japan sawamoto.hiroaki@mp.mt-pharma.co.jp

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Impact of Glycol Nucleic Acid (GNA) on siRNA Structure and Function Mark K. Schlegel, Donald J. Foster, Alexander V. Kel’in, Ivan Zlatev, Anna Bisbe, Muthusamy Jayaraman, Jeremy G. Lackey, Kallanthottathil G. Rajeev, Klaus Charissé, Joel Harp, Pradeep S. Pallan, Martin A. Maier, Martin Egli, Muthiah Manoharan Alnylam Pharmaceuticals, Cambridge, MA 02142, USA Glycol nucleic acid (GNA), a nucleic acid analog comprised of a flexible acyclic, three-carbon backbone with only one stereocenter, has previously been shown to be thermally destabilizing when incorporated into a DNA duplex. Here we evaluated the effect of GNA on RNA duplex structure, nuclease resistance and siRNA activity in vitro and in vivo. Crystal structure data indicate that, when incorporated into an RNA duplex, the (S)-GNA nucleotide backbone adopts a conformation that has little impact on the overall duplex structure, while the (R)-nucleotide disrupts the phosphate backbone and hydrogen bonding of an adjacent base pair. Consistent with the structural data, modification of an siRNA with (S)-GNA resulted in greater in vitro potencies over identical sequences containing (R)-GNA. A walk of (S)-GNA along the guide and passenger strands of a GalNAc-conjugated siRNA targeting mouse transthyretin (TTR) indicated that GNA is well tolerated at multiple positions of both strands. To some extent, the potency benefits observed in vitro translated in vivo with several GNA-modified siRNAs showing comparable or improved efficacy in mice. Mark K. Schlegel, Ph.D. Sr. Scientist, RNAi Discovery Alnylam Pharmaceuticals 300 Third Street Cambridge, MA 02142 USA mschlegel@alnylam.com

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Impact of Glycol Nucleic Acid (GNA) on siRNA Structure and Function Mark K. Schlegel, Donald J. Foster, Alexander V. Kel’in, Ivan Zlatev, Anna Bisbe, Muthusamy Jayaraman, Jeremy G. Lackey, Kallanthottathil G. Rajeev, Klaus Charissé, Joel Harp, Pradeep S. Pallan, Martin A. Maier, Martin Egli, Muthiah Manoharan Alnylam Pharmaceuticals, Cambridge, MA 02142, USA Glycol nucleic acid (GNA), a nucleic acid analog comprised of a flexible acyclic, three-carbon backbone with only one stereocenter, has previously been shown to be thermally destabilizing when incorporated into a DNA duplex. Here we evaluated the effect of GNA on RNA duplex structure, nuclease resistance and siRNA activity in vitro and in vivo. Crystal structure data indicate that, when incorporated into an RNA duplex, the (S)-GNA nucleotide backbone adopts a conformation that has little impact on the overall duplex structure, while the (R)-nucleotide disrupts the phosphate backbone and hydrogen bonding of an adjacent base pair. Consistent with the structural data, modification of an siRNA with (S)-GNA resulted in greater in vitro potencies over identical sequences containing (R)-GNA. A walk of (S)-GNA along the guide and passenger strands of a GalNAc-conjugated siRNA targeting mouse transthyretin (TTR) indicated that GNA is well tolerated at multiple positions of both strands. To some extent, the potency benefits observed in vitro translated in vivo with several GNA-modified siRNAs showing comparable or improved efficacy in mice. Mark K. Schlegel, Ph.D. Sr. Scientist, RNAi Discovery Alnylam Pharmaceuticals 300 Third Street Cambridge, MA 02142 USA mschlegel@alnylam.com

Improved Specificity and Therapeutic Index with ESC+ siRNA Conjugates Utilizing Seed-Pairing Destabilization via Novel Chemical Modifications Mark K. Schlegel, Maja M. Janas, I. Ramesh Babu, Lauren E. Blair, Christopher R. Brown, Adam Castoreno, Carole E. Harbison, Gregory Hinkle, Muthusamy Jayaraman, Yongfeng Jiang, Alexander V. Kel’in, Philip Kretschmer, Muthiah Manoharan, Shigeo Matsuda, Stuart Milstein, Rubina Parmar, Kallanthottathil G. Rajeev, Sally Schofield, Svetlana Shulga Morskaya, Christopher S. Theile, Vedat O. Yilmaz, Ivan Zlatev, Vasant Jadhav, Martin A. Maier Alnylam Pharmaceuticals, Cambridge, MA 02142, USA The typical lead selection process for enhanced stabilization chemistry (ESC) GalNAc-siRNAs begins with an in-house algorithm, which predicts sequences with high on-target activity and low complementarity to the rest of the transcriptome. These in silico designed sequences are screened for RNAi activity, and the most potent molecules are then advanced for safety assessment in rats, a key step in the process of development candidate selection. Typically, a subset of these compounds exhibit hepatotoxicity at dose and exposure multiples significantly above therapeutically relevant ranges. Potential mechanisms of hepatotoxicity include chemical toxicity, perturbation of endogenous RNAi pathways and/or RNAi-mediated off-target effects. In the present work, we demonstrate that rat hepatotoxicity observed with a subset of GalNAc-siRNAs is the consequence of RNAi-mediated miRNA-like off-target effects, with little to no apparent contribution of siRNA chemistry or endogenous RNAi pathway perturbation. To mitigate miRNA-like off-target effects, we utilized novel chemical modifications incorporated into the antisense seed region to destabilize the seed-mediated off-target recognition. This ESC+ design strategy maintains on-target potency while minimizing miRNA-like off-target effects and rat hepatotoxicity. These findings support the development of ESC+ siRNA conjugates with improved specificity and therapeutic index. Mark K. Schlegel, Ph.D. Sr. Scientist, RNAi Discovery Alnylam Pharmaceuticals 300 Third Street Cambridge, MA 02142 USA mschlegel@alnylam.com

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Inhibiting the Androgen Receptor Interaction with the Long Non-Coding RNA SLNCR using 2’-FANA-Modified Oligonucleotides Decreases Melanoma Invasion and Proliferation Karyn Schmidt, Carl D. Novina Dana-Farber Cancer Institute, Harvard Medical School, Broad Institute of MIT and Harvard Long non-coding RNAs (lncRNAs) are critical regulators of numerous physiological processes and diseases, especially cancers. The tissue- and disease-specific expression of lncRNAs, as well as their gene-regulatory functions, makes lncRNAs ideal therapeutic targets. However, development of lncRNA-based cancer therapies is limited because the mechanisms of many lncRNAs are obscure. We identified a novel lncRNA, SLNCR, whose expression is associated with worse overall melanoma survival. SLNCR contains a highly conserved sequence that binds to the androgen receptor (AR) and mediates increased melanoma invasion and proliferation in an androgen-independent manner. Thorough biochemical characterization of the AR-RNA interaction reveals that the N-terminal regulatory domain of AR binds to single-stranded RNA in a sequence-specific manner. To develop candidate novel therapeutics inhibiting the SLNCR- and AR-mediated invasion and proliferation, we designed short (21-28 nucleotide) oligos that are either (i) reverse complement to SLNCR’s AR binding sequence, which bind to SLNCR to generate double stranded RNA incapable of AR binding, or (ii) mimics of the SLNCR AR binding sequence, which bind directly to AR to preclude SLNCR binding. Both SLNCR- and AR-binding oligos are capable of sterically blocking the AR-SLNCR association. Moreover, gymnotic delivery (i.e. delivery of naked oligos independent of any agents) of 2′-deoxy-2′-fluoro-D-arabinonucleic acid (2′-FANA) modified oligos to patient-derived melanoma cells significantly reduces melanoma invasion and proliferation. Thus, these SLNCR- and AR-binding oligos represent novel therapeutic approaches for inhibition of melanoma growth and metastasis. Moreover, these studies show that characterization of protein-lncRNA interactions is critical for the development of lncRNA-targeted therapeutics. Karyn Schmidt, PhD Postdoctoral Fellow Dana-Farber Cancer Institute 450 Brookline Ave, Room D1424 Boston, MA 02215 USA KarynA_Schmidt@DFCI.harvard.com 716-622-7613

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Inhibiting the Androgen Receptor Interaction with the Long Non-Coding RNA SLNCR using 2’-FANA-Modified Oligonucleotides Decreases Melanoma Invasion and Proliferation Karyn Schmidt, Carl D. Novina Dana-Farber Cancer Institute, Harvard Medical School, Broad Institute of MIT and Harvard Long non-coding RNAs (lncRNAs) are critical regulators of numerous physiological processes and diseases, especially cancers. The tissue- and disease-specific expression of lncRNAs, as well as their gene-regulatory functions, makes lncRNAs ideal therapeutic targets. However, development of lncRNA-based cancer therapies is limited because the mechanisms of many lncRNAs are obscure. We identified a novel lncRNA, SLNCR, whose expression is associated with worse overall melanoma survival. SLNCR contains a highly conserved sequence that binds to the androgen receptor (AR) and mediates increased melanoma invasion and proliferation in an androgen-independent manner. Thorough biochemical characterization of the AR-RNA interaction reveals that the N-terminal regulatory domain of AR binds to single-stranded RNA in a sequence-specific manner. To develop candidate novel therapeutics inhibiting the SLNCR- and AR-mediated invasion and proliferation, we designed short (21-28 nucleotide) oligos that are either (i) reverse complement to SLNCR’s AR binding sequence, which bind to SLNCR to generate double stranded RNA incapable of AR binding, or (ii) mimics of the SLNCR AR binding sequence, which bind directly to AR to preclude SLNCR binding. Both SLNCR- and AR-binding oligos are capable of sterically blocking the AR-SLNCR association. Moreover, gymnotic delivery (i.e. delivery of naked oligos independent of any agents) of 2′-deoxy-2′-fluoro-D-arabinonucleic acid (2′-FANA) modified oligos to patient-derived melanoma cells significantly reduces melanoma invasion and proliferation. Thus, these SLNCR- and AR-binding oligos represent novel therapeutic approaches for inhibition of melanoma growth and metastasis. Moreover, these studies show that characterization of protein-lncRNA interactions is critical for the development of lncRNA-targeted therapeutics. Karyn Schmidt, PhD Postdoctoral Fellow Dana-Farber Cancer Institute 450 Brookline Ave, Room D1424 Boston, MA 02215 USA KarynA_Schmidt@DFCI.harvard.com 716-622-7613

Novel PMO-Based Chemistries for CNS and Muscle Targeting Frederick J. Schnell, Li Gan, Nelsa L. Estrella, Jenna A. Wood, Monica Yao, Gunnar J. Hanson, Marco A. Passini Sarepta Therapeutics, Inc., 215 First Street, Cambridge, MA USA Phosphorodiamidate morpholino oligomers (PMO) are single stranded oligonucleotides bearing uncharged internucleoside linkages capable of binding to target RNAs to modulate activity. PMOs must enter cells to be effective, and the delivery into cells and overall activity of these oligonucleotides may be improved by chemically modifying the PMO internucleoside linkages or by adding cell penetrating peptides. Sarepta has generated a series of novel and proprietary classes of compounds that includes PPMO, PMO-X® and PMOplus®. The goal of this report is to investigate the efficacies of these classes in the CNS and skeletal muscle of mice. Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in SMN1 that encodes SMN. The onset of disease and degree of severity are determined by SMN2, which encodes for SMN but due to a splicing point mutation the majority of SMN2 transcripts are missing exon-7 that results in a nonfunctional protein. One therapeutic approach is to increase SMN levels by re-directing SMN2 pre-mRNA splicing. A screen of chemically-modified PMOs identified a series of potent compounds that increase exon-7 inclusion and SMN levels in a dose-dependent manner in SMA patient fibroblasts. A subset of these compounds were injected into the cerebrospinal fluid (CSF) of a severe mouse model of SMA. Treatment with the selected compounds significantly increased median survival, increased body weight, and improved grip strength scores compared to saline treatment. Overall, PMO-X® provided the highest level of efficacy in SMA mice and suggests that this class of compounds may be amenable for brain and spinal cord targeting via CSF delivery. Myostatin naturally interferes with muscle growth and development in mammals; thus, reducing the levels of myostatin may increase muscle mass in neuromuscular diseases, metabolic diseases, muscular dystrophies, and cachexia. An in vitro screen identified PMO sequences that reduced myostatin levels in muscle cells in culture. The top sequences were then conjugated with a cell-penetrating peptide to form PPMOs and injected into the tail vein of wild-type mice. The results show that the levels of myostatin exon skipping correlates with myostatin protein reduction in the skeletal muscle and heart in a dose dependent manner. Taken together, these findings suggest that PMO-X® and PPMO antisense agents are effective for CNS and muscle targeting, respectively. Frederick J. Schnell, PhD Senior Scientist, Translational Development Sarepta Therapeutics 215 First Street, Cambridge MA, 02142 USA fschnell@sarepta.com

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Characterizing a Non-Coding RNA of the +9kb CEBPA Enhancer Ryan L Setten1, John J. Rossi1,2, John C. Burnett1,2

City of Hope: Irell and Manella Graduate School of Biological Sciences1

City of Hope: Dept. of Molecular & Cellular Biology2 CCAAT/enhancer binding protein alpha (CEBPA) is involved in the regulation of cell cycle inhibition, metabolism, and differentiation of certain cell lineages. Mutation or altered regulation of CEBPA contributes to the development of acute myeloid leukemia, hepatocellular carcinoma (HCC), chronic myeloid leukemia, and lung cancer. CEBPA transcription appears to be under the regulation of an enhancer region located ~9kb downstream of the CEBPA transcriptional start site (TSS) in several cell lines and tissues. Using publically available CAGE-seq data we inferred that an enhancer RNA (eRNA) is transcribed from this region. We confirmed this transcript in HEP3B by 5’ RACE PCR and RT-qPCR. We are currently determining whether this eRNA is physiologically relevant or merely a product of transcriptional noise from open chromatin. Additionally, we are characterizing how this enhancer region might affect transcription of both CEBPA mRNA and non-coding RNAs transcribed from the CEBPA locus. We anticipate the findings from this study will contribute to the growing understanding of eRNA function. Ryan Lowell Setten, B.S. PhD Candidate City of Hope: Irell & Manella Graduate School of Biological Science 1500 E. Duarte Rd., Fox North 2nd fl. Duarte, CA USA rsetten@coh.org

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Characterizing a Non-Coding RNA of the +9kb CEBPA Enhancer Ryan L Setten1, John J. Rossi1,2, John C. Burnett1,2

City of Hope: Irell and Manella Graduate School of Biological Sciences1

City of Hope: Dept. of Molecular & Cellular Biology2 CCAAT/enhancer binding protein alpha (CEBPA) is involved in the regulation of cell cycle inhibition, metabolism, and differentiation of certain cell lineages. Mutation or altered regulation of CEBPA contributes to the development of acute myeloid leukemia, hepatocellular carcinoma (HCC), chronic myeloid leukemia, and lung cancer. CEBPA transcription appears to be under the regulation of an enhancer region located ~9kb downstream of the CEBPA transcriptional start site (TSS) in several cell lines and tissues. Using publically available CAGE-seq data we inferred that an enhancer RNA (eRNA) is transcribed from this region. We confirmed this transcript in HEP3B by 5’ RACE PCR and RT-qPCR. We are currently determining whether this eRNA is physiologically relevant or merely a product of transcriptional noise from open chromatin. Additionally, we are characterizing how this enhancer region might affect transcription of both CEBPA mRNA and non-coding RNAs transcribed from the CEBPA locus. We anticipate the findings from this study will contribute to the growing understanding of eRNA function. Ryan Lowell Setten, B.S. PhD Candidate City of Hope: Irell & Manella Graduate School of Biological Science 1500 E. Duarte Rd., Fox North 2nd fl. Duarte, CA USA rsetten@coh.org

A Novel Human Muscle Cell Model of Duchenne Muscular Dystrophy Created by CRISPR/Cas9 Takenori Shimo 1,2, Kana Hosoki 2, Yusuke Nakatsuji 1, Toshifumi Yokota 2,3 and Satoshi Obika 1 1 Graduate School of Pharmaceutical Sciences, Osaka University, 1–6, Yamadaoka, Suita, Osaka, 565–0871, Japan, 2 Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, 8-31 Medical Science Building, Edmonton, AB T6G 2H7, Canada

3 Muscular Dystrophy Canada Research Chair, University of Alberta, Edmonton, Alberta, Canada Oligonucleotide-mediated splicing modulation is an attractive therapeutic approach for Duchenne muscular dystrophy (DMD). In 2016, the U.S. Food and Drug Administration approved eteplirsen, a phosphorodiamidate morpholino oligomer (PMO)-based splice-switching oligonucleotide (SSO) targeting DMD exon 51 for DMD patients. It is expected to emerge the application of SSOs targeting other exons. Patient-derived myogenic cells are usually used for evaluation of SSOs because they can be used to detect the restoration of dystrophin expression. However, it is difficult to obtain patient cells that have minor mutations in the DMD gene. Thus, we sought to establish a novel DMD model cell line that has minor mutations of DMD patients. We revealed the differentiated Human Rhabdomyosarcoma (RD) cell line expresses both DMD mRNA and dystrophin protein. Therefore RD cell line was used for cell establishment. We selected one of minor mutation patterns, DMD exons 51-57 deletion (approximately 0.3 Mb), that only three patients have been reported according to the database [1]. We removed the region in the RD cell line using the CRISPR/Cas9 system. In the cell line, we evaluated the exon skipping activity of previously reported SSOs at both mRNA and protein levels [2]. In theory, this methodology can be applied to other deletion mutations in the DMD gene. Overall, these DMD model cells can contribute to the evaluation of exon skipping efficiency of SSOs before in vivo examinations or clinical trials. [1] Bladen, C. L., et al. (2015), Hum Mutat, 36 (4), 395-402. [2] Aartsma-Rus, A., et al. (2002), Neuromuscul Disord, 12, S71-S77. Takenori Shimo Msc PhD student Osaka University 1-6 Yamadaoka, Suita, Osaka 565-0871 Japan shimo-t@phs.osaka-u.ac.jp +81-6-6879-8203

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Alkaline Phosphatase Placental like 2 as a Diagnostic Biomarker for Detection of Pancreatic Ductal Adenocarcinoma Hye Su Shin, Pooja Dua, Dong-Ki Lee Sungkyunkwan University Pancreatic cancer is one of the most common causes of cancer-related deaths. Pancreatic ductal adenocarcinoma (PDAC), especially, is the most common malignancy of the pancreas with difficulty in diagnosis and management. However, the research on PDAC associated biomarkers is still far behind the requirements of clinical translation. Using Cell-SELEX (Systematic evolution of ligands by exponential enrichment) and AptaBiD technology, we developed an aptamer for pancreatic cancer [1] and identified alkaline phosphatase placental-like 2 (ALPPL2) as a novel putative biomarker associated with PDAC. ALPPL2 is a glycosylphosphatidylinositol (GPI)-anchored protein and our work shows its involvement in pancreatic cancer cell proliferation and metastasis [2].

In the current study, we validate ALPPL2 as a PDAC biomarker and develop an aptamer based diagnostic platform using pancreatic cancer derived extracellular vesicles (EVs). We derived EVs from secretomes of ALPPL2 positive pancreatic cancer cell lines (PANC-1+ve and Capan-1) and ALPPL2 negative cell line (MIAPaCa-2). Using western blot analysis we confirmed that ALPPL2 is present on exosomes in significant amounts. Further we developed AptaELISA (aptamer-based enzyme-linked immunosorbent assay) for ALPPL2 quantitative analysis in these exosomes and found that exosome based ALPPL2 diagnosis is far sensitive and specific than the whole secretome. Currently we are optimizing the system for complex samples like serum and plasma and intend to use this AptaELISA set-up for clinical diagnosis of pancreatic cancer using exosome. We belive that that our ALPPL2- aptamer based diagnosis of pancreatic cancer, using exosome has a great potential both clinically and commercially.

[1] P. Dua et al., Cancer Res 73, 1934-1945 (2013) [2] P. Dua et al., Nucleic Acid Ther 25, 180-186 (2015)

Hye Su Shin Sungkyunkwan University 2066, Seobu-ro, Jangan-gu Suwon-si, Gyeonggi-do Republic of Korea hyesu_1221@nate.com

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Alkaline Phosphatase Placental like 2 as a Diagnostic Biomarker for Detection of Pancreatic Ductal Adenocarcinoma Hye Su Shin, Pooja Dua, Dong-Ki Lee Sungkyunkwan University Pancreatic cancer is one of the most common causes of cancer-related deaths. Pancreatic ductal adenocarcinoma (PDAC), especially, is the most common malignancy of the pancreas with difficulty in diagnosis and management. However, the research on PDAC associated biomarkers is still far behind the requirements of clinical translation. Using Cell-SELEX (Systematic evolution of ligands by exponential enrichment) and AptaBiD technology, we developed an aptamer for pancreatic cancer [1] and identified alkaline phosphatase placental-like 2 (ALPPL2) as a novel putative biomarker associated with PDAC. ALPPL2 is a glycosylphosphatidylinositol (GPI)-anchored protein and our work shows its involvement in pancreatic cancer cell proliferation and metastasis [2].

In the current study, we validate ALPPL2 as a PDAC biomarker and develop an aptamer based diagnostic platform using pancreatic cancer derived extracellular vesicles (EVs). We derived EVs from secretomes of ALPPL2 positive pancreatic cancer cell lines (PANC-1+ve and Capan-1) and ALPPL2 negative cell line (MIAPaCa-2). Using western blot analysis we confirmed that ALPPL2 is present on exosomes in significant amounts. Further we developed AptaELISA (aptamer-based enzyme-linked immunosorbent assay) for ALPPL2 quantitative analysis in these exosomes and found that exosome based ALPPL2 diagnosis is far sensitive and specific than the whole secretome. Currently we are optimizing the system for complex samples like serum and plasma and intend to use this AptaELISA set-up for clinical diagnosis of pancreatic cancer using exosome. We belive that that our ALPPL2- aptamer based diagnosis of pancreatic cancer, using exosome has a great potential both clinically and commercially.

[1] P. Dua et al., Cancer Res 73, 1934-1945 (2013) [2] P. Dua et al., Nucleic Acid Ther 25, 180-186 (2015)

Hye Su Shin Sungkyunkwan University 2066, Seobu-ro, Jangan-gu Suwon-si, Gyeonggi-do Republic of Korea hyesu_1221@nate.com

Anti-gene oligonucleotides targeting pathological triplet repeat expansions in Huntington’s disease and Friedreich’s ataxia C. I. Edvard Smith1, Eman M. Zaghloul1, Olof Gissberg1, Helen Bergquist1, Cristina S. J. Rocha1, L. Good2, Jesper Wengel3, Karin E. Lundin1, Rula Zain1,4. 1Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden. 2Pathology and Infectious Diseases, Royal Veterinary College, University of London, United Kingdom. 3Physics and Chemistry, Nucleic Acid Centre, University of Southern Denmark, Odense, Denmark. 4Clinical Genetics, Centre for Rare Diseases, Karolinska University, Sweden Expansion of DNA trinucleotide repeats (TNR) is associated with more than 30 human hereditary neurological disorders including fragile X syndrome, myotonic dystrophy type 1, Huntington’s diseases (HD) and Friedreich’s ataxia (FRDA). HD and FRDA are fatal neurodegenerative diseases and existing therapeutics are only symptomatic and do not alter the disease progression or increase life expectancy. Toxic mutant HTT transcripts (muHTT), and protein, are the result of a (CAG)n expansion in exon 1 of the Huntingtin gene, whereas (GAA)n expanded repeats in the first intron of the Frataxin gene are associated with reduced mRNA and protein levels and the development of FRDA. TNR are predisposed for genomic instability and hence their correlation with disease severity. This is mainly attributed to the ability of the repeat sequences to adopt non-canonical DNA structures, so-called non-B-DNA, including triplex DNA / H-DNA, cruciform and quadruplex structures. We have developed a new therapeutic approach by targeting the higher order DNA structures within pathological TNR in FRDA and HD, respectively, using anti-gene oligonucleotides (AGOs). We found that oligomers based on peptide nucleic acids and locked nucleic acids are able to disrupt DNA triplex formation in FRDA expanded TNR using chemical probing and atomic force microscopy (Bergquist et al., PLoS One 2016). We also found that AGOs directly targeting the HTT CTG repeat DNA resulted in partial but significant and possibly long-term, HTT knock-down of both mRNA and protein in HD patient derived fibroblasts (Zaghloul et al., Nucl Acids Res in press). Diminished phosphorylation of HTT gene-associated RNA-polymerase II was observed, suggestive of altered chromatin and reduced transcription downstream the AGO-targeted repeat. Different backbone chemistries were found to have a strong impact on the AGO efficiency. We also successfully used delivery vehicles and naked uptake of the AGOs, demonstrating versatility and providing insights for in vivo treatment. Thus, by targeting TNR at the DNA level, modified AGOs can be developed as a novel concept for efficient treatment of several neurological triplet-repeat diseases. C. I. Edvard Smith, MD, PhD, Professor Clinical Research Centre Dep. of Laboratory Medicine Karolinska Institutet SE-141 86, Huddinge Sweden edvard.smith@ki.se

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Development of Cancer-Cell-Specific Drug Delivery System Using MMP-Activatable PEG-Conjugated Oligoarginine Peptide and Its Application for MicroRNA Inhibition Hiroka Sugai1, Moeka Matsushima1, Seiji Sakamoto1, Yasuyuki Araki1, Ikuhiko Nakase2, Satoru Ishibashi3, Takanori Yokota3, Takehiko Wada1 Institute of Multidisciplinary Research for Advanced Materials, Tohoku Univesity1, Nanoscience and Nanotechnology Research Center, Osaka Prefecture University.2, Tokyo Medical and Dental University3 In last a couple of decades, oligonucleotide therapeutics has received much attention as promising therapeutic strategy for various kinds of diseases including cancer and cerebral infraction, due to increased knowledge about gene regulation. A number of oligonucleotide therapeutics has reported under clinical trials and some of them have already been approved for practical stage. For efficient therapeutics, one of the most critical requirements for the molecule is high affinity to the target RNA. However, the high affinity molecules often formed complex with similar sequenced non-target RNAs and induced serious side effects named off-target effects. For the practical application of oligonucleotide therapeutics, target-cell-specific intracellular delivery has been pointed out as one of the critical issues to avoid off-target effects. Thus, such target-cell-specific delivery carriers have been intensively investigated. Even so, an effective and realistic methodology has not been reported. Here, we have designed cancer-cell-specific intracellular delivery system using three functional modules, i.e., oligo-arginine (Arg) peptide, polyethylene glycol (PEG), and matrix metalloproteinase (MMP) substrate peptide (Fig. 1). Oligo-Arg, one of the most famous cell penetrating peptide, has been utilizing as a improving cellular uptake of macromolecules including oligonucleotides. In order to provide cancer-cell-specificity, oligo-Arg was connected with PEG via MMPs substrate that exerts as cancer-responsive cleavable linker, because MMPs specifically overexpress in cancer cells. In this design, PEG chain was utilized to decrease non-specific protein adsorption and opsonization. Cellular uptake of PEG-conjugated peptide was scarcely observed in contrast to that of unPEGylated oligo Arg. In this presentation, we will discuss design and synthesis of MMP-activatable PEG-conjugated oligo-Arg peptide, and its properties such as MMP cleavage, cellular uptake, and cytotoxicity. In addition, we will report intracellular delivery of anti-miR-21 by PEG-conjugated oligo-Arg peptide for miRNA inhibition. Hiroka Sugai Ph.D. student Tohoku University 2-1-1, Katahira, Aoba-ku Sendai, 980-8577 Japan h.sugai@dc.tohoku.ac.jp

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Development of Cancer-Cell-Specific Drug Delivery System Using MMP-Activatable PEG-Conjugated Oligoarginine Peptide and Its Application for MicroRNA Inhibition Hiroka Sugai1, Moeka Matsushima1, Seiji Sakamoto1, Yasuyuki Araki1, Ikuhiko Nakase2, Satoru Ishibashi3, Takanori Yokota3, Takehiko Wada1 Institute of Multidisciplinary Research for Advanced Materials, Tohoku Univesity1, Nanoscience and Nanotechnology Research Center, Osaka Prefecture University.2, Tokyo Medical and Dental University3 In last a couple of decades, oligonucleotide therapeutics has received much attention as promising therapeutic strategy for various kinds of diseases including cancer and cerebral infraction, due to increased knowledge about gene regulation. A number of oligonucleotide therapeutics has reported under clinical trials and some of them have already been approved for practical stage. For efficient therapeutics, one of the most critical requirements for the molecule is high affinity to the target RNA. However, the high affinity molecules often formed complex with similar sequenced non-target RNAs and induced serious side effects named off-target effects. For the practical application of oligonucleotide therapeutics, target-cell-specific intracellular delivery has been pointed out as one of the critical issues to avoid off-target effects. Thus, such target-cell-specific delivery carriers have been intensively investigated. Even so, an effective and realistic methodology has not been reported. Here, we have designed cancer-cell-specific intracellular delivery system using three functional modules, i.e., oligo-arginine (Arg) peptide, polyethylene glycol (PEG), and matrix metalloproteinase (MMP) substrate peptide (Fig. 1). Oligo-Arg, one of the most famous cell penetrating peptide, has been utilizing as a improving cellular uptake of macromolecules including oligonucleotides. In order to provide cancer-cell-specificity, oligo-Arg was connected with PEG via MMPs substrate that exerts as cancer-responsive cleavable linker, because MMPs specifically overexpress in cancer cells. In this design, PEG chain was utilized to decrease non-specific protein adsorption and opsonization. Cellular uptake of PEG-conjugated peptide was scarcely observed in contrast to that of unPEGylated oligo Arg. In this presentation, we will discuss design and synthesis of MMP-activatable PEG-conjugated oligo-Arg peptide, and its properties such as MMP cleavage, cellular uptake, and cytotoxicity. In addition, we will report intracellular delivery of anti-miR-21 by PEG-conjugated oligo-Arg peptide for miRNA inhibition. Hiroka Sugai Ph.D. student Tohoku University 2-1-1, Katahira, Aoba-ku Sendai, 980-8577 Japan h.sugai@dc.tohoku.ac.jp

An Investigational Assay Using Next-Generation Sequencing Technology to Detect Single Nucleotide Polymorphisms (SNPs) Associated with the Mutant Huntingtin Allele Nenad Svrzikapa, Kenneth Longo, Meena, Susovan Mohapatra, Pallavi Lonkar, Hailin Yang, Naoki Iwamoto, David Butler, Keith Bowman, Stephany Standley, Mamoru Shimizu, Michael Panzara, Zhong Zhong, Chris Francis, Chandra Vargeese Wave Life Sciences, Cambridge MA 02138

Huntington’s disease (HD) is a genetic disease associated with the expansion of a cytosine-adenine-guanine (CAG) triplet repeat in the huntingtin (HTT) gene, which results in the production of mutant HTT (mHTT) protein. Accumulation of mHTT protein leads to the progressive loss of neurons in the brain. Previous nonclinical studies have suggested that silencing of mHTT ameliorates or reverses the symptoms of HD. Wild type HTT (wtHTT) protein is critical for neuronal development. While the purpose of wtHTT in adults is not completely understood, some studies have shown that it may play an important role in neuronal function. Therefore, selective lowering of mHTT gene expression, while leaving wtHTT relatively intact, may be desirable.

Wave Life Sciences is developing two stereopure antisense oligonucleotides (ASOs) that are intended to selectively target the mHTT transcript of the U variant of single nucleotide polymorphism rs362307 (SNP1) and rs362331 (SNP2), respectively, for degradation; these SNPs are present in roughly two-thirds of HD patients. Both investigational compounds, WVE-120101 and WVE-120102, selectively decreased mHTT mRNA and protein levels compared with wtHTT in multiple cells lines including patient-derived fibroblasts. To enable clinical research of these investigational compounds, we have developed an investigational assay to identify HD patients who carry one of the targeted SNPs. Next-generation long-read sequencing technology is used to determine if patients diagnosed with HD are heterozygous for one of the targeted SNPs, and if the thymine (T) variant of the SNP is on the same allele as the pathogenic CAG triplet repeat expansion. This test provides strong posterior inference for phasing, and high specificity, sensitivity and accuracy. The design and methods of the investigational assay will be presented. Nenad Svrzikapa Bioinformatics Wave Life Sciences 733 Concord Avenue Cambridge, MA 02138 USA nsvrzikapa@wavelifesci.com

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LUNAR-mRNA for protein replacement and gene editing therapies Kiyoshi Tachikawa1, Suvasini Ramaswamy2, Pattraranee Limphong1, Jerel Vega1, Marciano Sablad1, Carlos Perez-Garcia1, Hari Bhaskaran1, Rachel Wilkie-Grantham1,Priya Karmali1, Christine Esau1, Nina Tonnu2, Pad Chivukula1, Inder Verma2 1Arcturus Therapeutics, San Diego, CA, USA 2Salk Institute for Biological Studies, La Jolla, CA, USA Messenger RNA (mRNA) plays a fundamental role as a blueprint of protein synthesis within a living cells. Recently, mRNA has increased in importance for de novo protein expression in cells and become a new class of drugs. Applications of mRNA-based therapies include cancer vaccines, antibodies, protein replacement treatments and gene editing. mRNA also has advantages in simplicity of manufacturing compared to protein therapeutics. However, there are still some fundamental challenges with the use of mRNA as a therapeutics, including stability, duration of action, immunogenicity, and delivery to the target cells or tissues. Arcturus’ LUNAR platform technology provides safe and effective delivery of mRNA to the liver, lung, and other target tissues. We have reported the application of LUNAR-mRNA for the treatment of hemophilia due to Factor IX (FIX) deficiency. A single dose of 2 mg/kg LUNAR-hFIX mRNA to the FIX knockout mouse model is able to restore circulating FIX protein to normal levels and rescue the clotting defect for at least six days. LUNAR-hFIX was well-tolerated up to 10mg/kg, with no liver enzyme elevations after a single dose. Repeat dosing was also well-tolerated with sustained production of Factor IX protein. We have also demonstrated durable activity of LUNAR-OTC mRNA in the spf/ash mouse model of the urea cycle disorder OTC deficiency. A single dose of LUNAR-OTC suppressed the disease biomarker urinary orotate for more than one week and prevented hyperammonemia after protein challenge. We show that co-encapsulation of multiple RNA strands in single nanoparticle is possible. This characteristic makes possible to generate LUNAR-Gene Editing (GE) for in vivo gene correction or modification. One example for the LUNAR-GE is to co-encapsulate mRNA encoding Cas9 and guide RNA. Administration of LUNAR-GE which targets transthyretin (TTR) resulted in up to 95% reduction in serum TTR in mice. In conclusion, LUNAR platform enables delivery of not only short RNAs but also long RNAs such as mRNA for protein replacement and gene editing therapies in patients. Kiyoshi Tachikawa, Ph.D. Director of Frontier Science Arcturus Therapeutics 10628 Science Center Drive, Suite 200 San Diego, CA 92121 USA kiyoshi@arcturusrx.com

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LUNAR-mRNA for protein replacement and gene editing therapies Kiyoshi Tachikawa1, Suvasini Ramaswamy2, Pattraranee Limphong1, Jerel Vega1, Marciano Sablad1, Carlos Perez-Garcia1, Hari Bhaskaran1, Rachel Wilkie-Grantham1,Priya Karmali1, Christine Esau1, Nina Tonnu2, Pad Chivukula1, Inder Verma2 1Arcturus Therapeutics, San Diego, CA, USA 2Salk Institute for Biological Studies, La Jolla, CA, USA Messenger RNA (mRNA) plays a fundamental role as a blueprint of protein synthesis within a living cells. Recently, mRNA has increased in importance for de novo protein expression in cells and become a new class of drugs. Applications of mRNA-based therapies include cancer vaccines, antibodies, protein replacement treatments and gene editing. mRNA also has advantages in simplicity of manufacturing compared to protein therapeutics. However, there are still some fundamental challenges with the use of mRNA as a therapeutics, including stability, duration of action, immunogenicity, and delivery to the target cells or tissues. Arcturus’ LUNAR platform technology provides safe and effective delivery of mRNA to the liver, lung, and other target tissues. We have reported the application of LUNAR-mRNA for the treatment of hemophilia due to Factor IX (FIX) deficiency. A single dose of 2 mg/kg LUNAR-hFIX mRNA to the FIX knockout mouse model is able to restore circulating FIX protein to normal levels and rescue the clotting defect for at least six days. LUNAR-hFIX was well-tolerated up to 10mg/kg, with no liver enzyme elevations after a single dose. Repeat dosing was also well-tolerated with sustained production of Factor IX protein. We have also demonstrated durable activity of LUNAR-OTC mRNA in the spf/ash mouse model of the urea cycle disorder OTC deficiency. A single dose of LUNAR-OTC suppressed the disease biomarker urinary orotate for more than one week and prevented hyperammonemia after protein challenge. We show that co-encapsulation of multiple RNA strands in single nanoparticle is possible. This characteristic makes possible to generate LUNAR-Gene Editing (GE) for in vivo gene correction or modification. One example for the LUNAR-GE is to co-encapsulate mRNA encoding Cas9 and guide RNA. Administration of LUNAR-GE which targets transthyretin (TTR) resulted in up to 95% reduction in serum TTR in mice. In conclusion, LUNAR platform enables delivery of not only short RNAs but also long RNAs such as mRNA for protein replacement and gene editing therapies in patients. Kiyoshi Tachikawa, Ph.D. Director of Frontier Science Arcturus Therapeutics 10628 Science Center Drive, Suite 200 San Diego, CA 92121 USA kiyoshi@arcturusrx.com

A Scalable Microfluidic Platform for Oligonucleotides Lipid Nanoparticles (LNPs) Manufacturing from Microliters to Liters Nadia Tagnaouti, K. Ou, A. Thomas, SM. Garg , RJ. Taylor and EC. Ramsay Precision NanoSystems Inc. The use of nucleic acids Lipid Nanoparticles (LNPs) to manipulate gene expression has been limited due to the lack of reproducible and scalable LNPs manufacturing methods. Here we describe the microfluidic and non-viral NanoAssemblrTM platform for fast, robust and scalable manufacture of LNPs from microliters to liters solving all major bottle necks of genetic medicines development: speed, reproducibility and scalability. We will describe the safe and effective delivery of a siRNA LNPs both in vitro and in vivo and the possibility to fine-tune their size, composition and surface properties. Finally, we will conclude on the scalability of this platform for seamless scale up manufacturing of LNPs in cGMP environment to support all stages of genetic medicines development, from early discovery, screening and up to preclinical and clinical trials. Nadia Tagnaouti, PhD Regional Manager Europe and Middle East Precision NanoSystems Inc. 50-655 West Kent Ave. N. Vancouver, British Columbia CANADA ntagnaouti@precision-nano.com +491706210868

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Synthesis of RNA containing the epigenetically relevant 5- hydroxymethyl -, 5-formyl-, and 5- carboxycytidine Nobuhiro Tago, Iacovos N. Michaelides, Bastien Viverge, Thomas Carell Fakultät für Chemie und Pharmazie, Ludwig-Maximilians-Universität München

5-Hydroxymethyl-, 5-formyl-, and 5-carboxycytidine are new epigenetic bases (hmdC, fdC, cadC) that were recently discovered in the DNA of higher eukaryotes. In some cancer, the level of hmdC, fdC and cadC as well as the related enzymes (Ten-eleven translocation, TET enzymes) are either up/down regulated.1,2 The same bases (hmC, fC and caC) have now also been detected in mammalian RNA with a high abundance in mRNA.3 Furthermore, it has been reported that TET enzymes can mediate the oxidation of hmC, fC and caC in RNA as the same manner as DNA.4,5 This implies that the level of these modifications in RNA might be differed in some diseases. In this poster, we report novel RNA phosphoramidites (PAs) for hmC, fC and caC that can be used in routine RNA synthesis. The new building blocks are compatible with the canonical PAs and also with themselves, which enabled even the synthesis of RNA strands containing all three of these bases. The study will pave the way for detailed physical, biochemical and biological studies to unravel the function of these non-canonical modifications in RNA as well as the related enzymes. Reference: 1. S. Ito, et al. Science 2011, 333, 1300-1303. 2. C. G. Spruijt, et al. Cell 2013, 152, 1146-1159. 3. W. Huang, et al. Chemical Science 2016, 7, 5495-5502. 4. L. Fu, et al. J. Am. Chem. Soc 2014, 136, 11582-11585. 5. M. Basanta‐Sanchez, et al. ChemBioChem 2017, 18, 72-76. Nobuhiro Tago, Ph.D Post-doctoral researcher Ludwig-Maximilians-Universität München Butenandtstr. 5-13, 81377 Munich, Bavaria Germany nobuhiro.tago@cup.lmu.de +49-89-2180-77745

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Design and Synthesis of Positively Charged Oligonucleotides Functionalized with Thermolytic Masking Groups Mayumi Takahashi, Harsh V. Jain and Serge L. Beaucage US Food and Drug Administration, Silver Spring, MD, USA Despite all successful pre-clinical and early-stage clinical studies involving the use of oligonucleotide therapeutics, a number of issues still hamper their clinical translations. For example, inherent physicochemical characteristics of nucleic acids are among those issues. Because of the highly negatively charged nature of nucleic acids, native oligonucleotides cannot easily pass through the plasma membrane and most microinjected oligonucleotides are unable to escape cellular endosomal compartments, thereby resulting in lysosomal degradation. We hypothesized that masking the negatively charged phosphodiester functions of oligonucleotides with lipophilic groups would improve permeability to cell membranes and prevent endosomal sequestration. We have previously demonstrated that the use of thermolytic 2-(N-formyl-N-methylamino)ethyl (fma) groups, for protection of the negatively charged thiophosphate diester functions of immunostimulatory CpG-containing oligonucleotides, was able to induce an immune response in mice to an extent similar to that induced by unmodified CpG-containing oligonucleotides. Here, we postulate that the incorporation of positively charged functional groups with thermolytic properties may facilitate cellular uptake and endosomal escape of antisense oligonucleotides (ASOs). We have designed positively charged thermolytic groups functionalized with imidazolyl or N,N-dimethylamino groups for incorporation into synthetic nucleic acid sequences. The synthesis of thermolytic positively charged nucleic acid sequences along with their cellular uptake and functional properties will be presented. Mayumi Takahashi, PhD IOTF Fellow US Food and Drug Administration 10903 New Hampshire Ave Silver Spring, MD 20993 USA mayumi.takahashi@fda.hhs.gov +1(240)402-3411

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Generation and PPI inhibition analysis of artificial nucleic acid aptamers targeting Ebola virus protein Keisuke Tanaka1, Yuuya Kasahara1,2, Yoichi Miyamoto2, Tatsuro Kasai3, Kentaro Onodera3, Masayasu Kuwahara3, Masahiro Oka2, Satoshi Obika1,2

Graduate School of Pharmaceutical Sciences, Osaka University1

National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN)2

Graduate School of Science and Technology, Gunma University3

Aptamers are single stranded nucleic acids with well ordered structures and interact with a specific target molecule. They are expected to be molecular-targeting drugs like antibodies. Compared with antibodies, one important feature or advantage of aptamers is ease of chemical modifications. By introducing chemical modifications, they get high affinity to target molecules and stability against nuclease degradation. They are selected from libraries by Systematic Evolution of Ligands by EXponential enrichment (SELEX). In order to generate chemically modified aptamers, many researchers developed SELEX with libraries containing various artificial nucleic acids. A target of this study is one of the Ebola virus proteins, which inhibits immune response of human cells. This inhibition is one of the reasons that Ebola hemorrhagic fever makes patients worse. We suppose aptamers targeting the Ebola virus protein could be a novel cure for Ebola hemorrhagic fever. We prepared an initial library containing a 5-substituted pyrimidine nucleobase in order to get high-affinity aptamers. Using this library, we generated 10 artificial nucleic acid aptamers with Capillary Electrophoresis (CE)-SELEX, and inhibition of Protein-Protein Interaction (PPI) of the Ebola virus protein by the aptamers was analyzed using CE and bio-layer interferometry. Keisuke Tanaka Graduate student Graduate School of Pharmaceutical Sciences, Osaka University Grad. Sch. of Pharm. Sci. Bioorganic Chemistry Yamadaoka 1-6 Osaka, Suita City Japan keisuke060306@gmail.com

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Generation and PPI inhibition analysis of artificial nucleic acid aptamers targeting Ebola virus protein Keisuke Tanaka1, Yuuya Kasahara1,2, Yoichi Miyamoto2, Tatsuro Kasai3, Kentaro Onodera3, Masayasu Kuwahara3, Masahiro Oka2, Satoshi Obika1,2

Graduate School of Pharmaceutical Sciences, Osaka University1

National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN)2

Graduate School of Science and Technology, Gunma University3

Aptamers are single stranded nucleic acids with well ordered structures and interact with a specific target molecule. They are expected to be molecular-targeting drugs like antibodies. Compared with antibodies, one important feature or advantage of aptamers is ease of chemical modifications. By introducing chemical modifications, they get high affinity to target molecules and stability against nuclease degradation. They are selected from libraries by Systematic Evolution of Ligands by EXponential enrichment (SELEX). In order to generate chemically modified aptamers, many researchers developed SELEX with libraries containing various artificial nucleic acids. A target of this study is one of the Ebola virus proteins, which inhibits immune response of human cells. This inhibition is one of the reasons that Ebola hemorrhagic fever makes patients worse. We suppose aptamers targeting the Ebola virus protein could be a novel cure for Ebola hemorrhagic fever. We prepared an initial library containing a 5-substituted pyrimidine nucleobase in order to get high-affinity aptamers. Using this library, we generated 10 artificial nucleic acid aptamers with Capillary Electrophoresis (CE)-SELEX, and inhibition of Protein-Protein Interaction (PPI) of the Ebola virus protein by the aptamers was analyzed using CE and bio-layer interferometry. Keisuke Tanaka Graduate student Graduate School of Pharmaceutical Sciences, Osaka University Grad. Sch. of Pharm. Sci. Bioorganic Chemistry Yamadaoka 1-6 Osaka, Suita City Japan keisuke060306@gmail.com

NCCR RNA & Disease: A multi-disciplinary Swiss-wide research initiative Dominik Theler1,2,3, Jonathan Hall1,2

NCCR RNA & Disease1 , ETH Zurich2, University of Bern3

The National Center of Competence in Research (NCCR) RNA & Disease consists of over forty research groups addressing a wide range of scientific questions applying a broad portfolio of approaches creating added value through collaboration. NCCRs represent the Swiss academic funding scheme through which areas deemed of strategic importance are promoted on a long-term basis. In addition to pursuing research at an internationally competitive level, NCCRs are mandated to be active in areas such as knowledge and technology transfer, communication, equal opportunities and education, which in return benefit as well its research activities. Research topics range from the development of novel oligonucleotide chemistries to the generation of mouse models for diseases. Members have access to dedicated technology platforms and are supported for stays in other laboratories to acquire technological expertise. Thematically, research is grouped into three interconnected focal areas: Non-coding RNAs, RNA metabolism and translation. The NCCR RNA & Disease is seeking collaborations with pharmaceutical companies as well as clinicians. Recent results generated by NCCR RNA & Disease member groups will be presented illustrating the added value created by the network.

Dominik Theler, PhD Scientific Officer NCCR RNA & Disease Otto Stern Weg 5 8093 Zurich Switzerland dtheler@ethz.ch

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A dystrophic mouse model for testing human specific antisense oligonucleotides Maaike van Putten1, Marcel Veltrop1, Laura van Vliet1, Margriet Hulsker1, Jill Claassens2, Conny Brouwers2, Cor Breukel2, Jos van der Kaa2, Margot M. Linssen2, Johan T. Den Dunnen1,3, Sjef Verbeek1,2, Annemieke Aartsma-Rus1 1Department of Human Genetics, Leiden University Medical Center, Leiden, 2300 RC, The Netherlands 2Transgenic Facility, Leiden University Medical Center, Leiden, 2300 RC, The Netherlands 3Department of Clinical Genetics, Leiden University Medical Center, Leiden, 2300 RC, The Netherlands Duchenne muscular dystrophy (DMD) is characterized by severe and progressive muscle-wasting leading to death in the third decade in the Western world. The disease is caused by mutations in the DMD gene that disrupt the reading frame resulting in the absence of functional dystrophin protein. Mutations in the same gene which maintain the reading frame lead to the synthesis of shortened but partly functional dystrophin protein, present in milder affected Becker muscular dystrophy (BMD) patients. Antisense oligonucleotide mediated exon skipping aims to restore the disrupted reading frame allowing the synthesis of BMD-like dystrophin proteins. Since exon skipping is a sequence specific approach, the lack of a humanized mouse model with a muscular dystrophy hampered pre-clinical research. To overcome this, we generated a unique humanized mouse model; the del52hDMD/mdx mouse by targeting vector and Transcription Activator-like Effector Nucleases (TALENs). This mouse model carries a deletion of exon 52 in the human DMD gene, and a stop mutation in exon 23 of the murine Dmd gene. Consequently, these mice lack both human and mouse dystrophin. Like mdx mice, del52hDMD/mdx mice have mild functional impairment, elevated creatine kinase levels and on a histological level fibrosis, central nucleation and inflammation. We confirmed the absence of both human and mouse dystrophin protein in the del52hDMD/mdx mouse by western blot and immunofluorescence. Local intramuscular treatment with exon 51 or 53 specific AONs in del52hDMD/mdx mice resulted in pronounced exon skipping in both gastrocnemius and triceps muscles. Reading frame restoration resulted in restoration of human specific dystrophin protein. The del52hDMD/mdx mouse allows, for the first time, in vivo testing and optimization of human specific AONs targeting either exon 51 or 53 not only on RNA and protein levels, but also on muscle function and pathology. This makes this model unique and instrumental in further pre-clinical development of human specific AONs for DMD. Maaike van Putten, PhD Researcher Leiden University Medical Center Department of Human Genetics, DMD genetic therapy group Einthovenweg 20 Postzone S4-P; P.O. Box 9600 2300 RC Leiden; the Netherlands m.van_putten@lumc.nl Tel: +31 71 526 94 39

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A dystrophic mouse model for testing human specific antisense oligonucleotides Maaike van Putten1, Marcel Veltrop1, Laura van Vliet1, Margriet Hulsker1, Jill Claassens2, Conny Brouwers2, Cor Breukel2, Jos van der Kaa2, Margot M. Linssen2, Johan T. Den Dunnen1,3, Sjef Verbeek1,2, Annemieke Aartsma-Rus1 1Department of Human Genetics, Leiden University Medical Center, Leiden, 2300 RC, The Netherlands 2Transgenic Facility, Leiden University Medical Center, Leiden, 2300 RC, The Netherlands 3Department of Clinical Genetics, Leiden University Medical Center, Leiden, 2300 RC, The Netherlands Duchenne muscular dystrophy (DMD) is characterized by severe and progressive muscle-wasting leading to death in the third decade in the Western world. The disease is caused by mutations in the DMD gene that disrupt the reading frame resulting in the absence of functional dystrophin protein. Mutations in the same gene which maintain the reading frame lead to the synthesis of shortened but partly functional dystrophin protein, present in milder affected Becker muscular dystrophy (BMD) patients. Antisense oligonucleotide mediated exon skipping aims to restore the disrupted reading frame allowing the synthesis of BMD-like dystrophin proteins. Since exon skipping is a sequence specific approach, the lack of a humanized mouse model with a muscular dystrophy hampered pre-clinical research. To overcome this, we generated a unique humanized mouse model; the del52hDMD/mdx mouse by targeting vector and Transcription Activator-like Effector Nucleases (TALENs). This mouse model carries a deletion of exon 52 in the human DMD gene, and a stop mutation in exon 23 of the murine Dmd gene. Consequently, these mice lack both human and mouse dystrophin. Like mdx mice, del52hDMD/mdx mice have mild functional impairment, elevated creatine kinase levels and on a histological level fibrosis, central nucleation and inflammation. We confirmed the absence of both human and mouse dystrophin protein in the del52hDMD/mdx mouse by western blot and immunofluorescence. Local intramuscular treatment with exon 51 or 53 specific AONs in del52hDMD/mdx mice resulted in pronounced exon skipping in both gastrocnemius and triceps muscles. Reading frame restoration resulted in restoration of human specific dystrophin protein. The del52hDMD/mdx mouse allows, for the first time, in vivo testing and optimization of human specific AONs targeting either exon 51 or 53 not only on RNA and protein levels, but also on muscle function and pathology. This makes this model unique and instrumental in further pre-clinical development of human specific AONs for DMD. Maaike van Putten, PhD Researcher Leiden University Medical Center Department of Human Genetics, DMD genetic therapy group Einthovenweg 20 Postzone S4-P; P.O. Box 9600 2300 RC Leiden; the Netherlands m.van_putten@lumc.nl Tel: +31 71 526 94 39

Mitochondrial ASncmtRNA-1 and ASncmtRNA-2 as potent targets to inhibit tumor growth and metastasis in the RenCa murine renal adenocarcinoma model. Jaime Villegas1,2,4, Vincenzo Borgna1,2,3,Verónica A. Burzio1,2,4, Sebastián Belmar2, Mariela Araya1,2,4, Emanuel Jeldes1,2,4, Lorena Lobos-González1,2, Verónica Silva1,2, Claudio Villota1,2,5, Luciana Oliveira-Cruz1,2, Constanza Lopez1,2, Teresa Socias1,2, Octavio Castillo5,6 and Luis O. Burzio1,2,4 1Andes Biotechnologies SpA, 2Fundación Ciencia & Vida, 3Servicio de Urología, Hospital Barros Luco-Trudeau, 4Facultad de Ciencias Biológicas, Universidad Andrés Bello, 5Facultad de Salud, Universidad Bernardo O´Higgins, 6 Centro de Cirugía Robótica, Clínica Indisa, Santiago, Chile. Knockdown of antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptosis in several human and mouse tumor cell lines, but not normal cells, suggesting this approach for a selective therapy against different types of cancer. Here we show that in vitro knockdown of murine ASncmtRNAs induces apoptotic death of mouse renal adenocarcinoma RenCa cells, but not normal murine kidney epithelial cells. In a syngeneic subcutaneous RenCa model, treatment delayed and even reversed tumor growth. Since the subcutaneous model does not reflect the natural microenviroment of renal cancer, we used an orthotopic model of RenCa cells inoculated under the renal capsule. These studies showed inhibition of tumor growth and metastasis. Direct metastasis assessment by tail vein injection of RenCa cells also showed a drastic reduction in lung metastatic nodules. In vivo treatment reduces survivin, N-cadherin and P-cadherin levels, providing a molecular basis for metastasis inhibition. In consequence, the treatment significantly enhanced mouse survival in these models. Our results suggest that the ASncmtRNAs could be potent and selective targets for therapy against human renal cell carcinoma Jaime Villegas O. PhD Senior Research Fundación Ciencia & Vida Fac. Ciencias Biológicas – Universidad Andrés Bello Av. Zañartu 1482 – Ñuñoa Santiago – Chile jvillegas@bioschile.cl

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Evaluation of the efficacy of antisense therapy in a patient derived xenograft model (PDX) of advanced cervical cancer Villegas, J1,2., Guevara, F1,2., Silva, V1., Lobos-Gonzales, L1., Villota, C1,4., Carrasco, M1., Sanhueza, N1,2., Reyes, C1,2., Castillo, J3., Bustamante, E4. and Burzio, L.O.1,2 1Fundación Ciencia & Vida – Andes Biotechnologies SpA; 2 Facultad de Ciencias Biológicas, Universidad Andrés Bello; 3 Unidad de Anatomía Patológica, Hospital Barros Luco-Trudeau; 4Instituto Oncológico Fundación Arturo López Pérez, 4Departamento de Ciencias Químicas y Biológicas, Facultad de Salud, Universidad Bernardo O’Higgins, Santiago, Chile. Carcinoma of the cervix remains a significant health problem for women worldwide and new treatments are urgently required. In previous works we have described that the interference of Antisense non-coding mitochondrial RNA (AsncmtRNA) with antisense oligonucleotides (ASO) induces a massive cell death mediated by apoptosis in primary cultures of human advanced cervical cancer. The aim of this study was to evaluate the efficacy of the ASO treatment using patient derived xenograft (PDX) models of advanced cervical cancer. Therefore, fresh human biopsies were surgically implanted into the cervix of immuno-compromised mice (F0). One hundred days after the engrafted, tumor was harvested and fragmented to expand subcutaneously into a new group of mice, referred as F1. Animals were treated with 200 µg of AS0 1537S, every other day until complete 10 doses. Primary biopsies and tumor growth in the different generations, were HPV-genotyped by PCR and, a broad analysis of proliferation, anti-apoptotic, epithelial and angiogenesis biomarkers were carried out by western blot and immunohistochemistry. The characterization of tumor samples determined the presence of HPV 16 and no changes in expression of Cytokeratin 17, PCNA and Ki67 were observed through the different generations. The evaluation of the ASO therapy do not show a delay on tumor growth but shows an important inhibition of the metastatic spread, associated with a drop in the expression of Survivin, Rac and Bcl-xL. Grants: FONDEF D10E1090 and CCTE-PFB16. CONICYT. Jaime Villegas O. PhD Senior Research Fundación Ciencia & Vida Fac. Ciencias Biológicas – Universidad Andrés Bello Av. Zañartu 1482 – Ñuñoa Santiago – Chile jvillegas@bioschile.cl

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Evaluation of the efficacy of antisense therapy in a patient derived xenograft model (PDX) of advanced cervical cancer Villegas, J1,2., Guevara, F1,2., Silva, V1., Lobos-Gonzales, L1., Villota, C1,4., Carrasco, M1., Sanhueza, N1,2., Reyes, C1,2., Castillo, J3., Bustamante, E4. and Burzio, L.O.1,2 1Fundación Ciencia & Vida – Andes Biotechnologies SpA; 2 Facultad de Ciencias Biológicas, Universidad Andrés Bello; 3 Unidad de Anatomía Patológica, Hospital Barros Luco-Trudeau; 4Instituto Oncológico Fundación Arturo López Pérez, 4Departamento de Ciencias Químicas y Biológicas, Facultad de Salud, Universidad Bernardo O’Higgins, Santiago, Chile. Carcinoma of the cervix remains a significant health problem for women worldwide and new treatments are urgently required. In previous works we have described that the interference of Antisense non-coding mitochondrial RNA (AsncmtRNA) with antisense oligonucleotides (ASO) induces a massive cell death mediated by apoptosis in primary cultures of human advanced cervical cancer. The aim of this study was to evaluate the efficacy of the ASO treatment using patient derived xenograft (PDX) models of advanced cervical cancer. Therefore, fresh human biopsies were surgically implanted into the cervix of immuno-compromised mice (F0). One hundred days after the engrafted, tumor was harvested and fragmented to expand subcutaneously into a new group of mice, referred as F1. Animals were treated with 200 µg of AS0 1537S, every other day until complete 10 doses. Primary biopsies and tumor growth in the different generations, were HPV-genotyped by PCR and, a broad analysis of proliferation, anti-apoptotic, epithelial and angiogenesis biomarkers were carried out by western blot and immunohistochemistry. The characterization of tumor samples determined the presence of HPV 16 and no changes in expression of Cytokeratin 17, PCNA and Ki67 were observed through the different generations. The evaluation of the ASO therapy do not show a delay on tumor growth but shows an important inhibition of the metastatic spread, associated with a drop in the expression of Survivin, Rac and Bcl-xL. Grants: FONDEF D10E1090 and CCTE-PFB16. CONICYT. Jaime Villegas O. PhD Senior Research Fundación Ciencia & Vida Fac. Ciencias Biológicas – Universidad Andrés Bello Av. Zañartu 1482 – Ñuñoa Santiago – Chile jvillegas@bioschile.cl

Polythiol Probes & Microarrays for DNA Hybridization & Highly Sensitive Detection. Improved DNA Microarray Hybridization Using Polythiol Probes and Their Immobilization by Thiol-Ene Chemistry** & *** Suresh C. Srivastava & Pierre Villiet ChemGenes Corp., 33 Industrial Way, Wilmington , MA 01887 The development of high-performance methods for the sensitive and selective detection of DNA/RNA targets has become a key point in biomedical studies. Nucleic Acid microarrays are largely used for diagnostic to determine the type of infection due to a bacteria or a virus. A major issue to have a high reproducibility, reliability and analytical sensivity is correlated to the controlled and efficient immobilization of the probe that should hybridize the PCR product for detection. In the present technology, a key component, monothiol mono-DMT-phosphoramidite was developed, which was then coupled to synthesize a variety of oligonucleotide probes. Such probes are amenable for detecting viruses and bacteria. The monothiol mono-DMT-phosphoramidite could be coupled to an oligonucleotide sequence mutliple time to improve probe capture and detection efficiency. The immobilization of the mono thiol or polythiol oligonucleotide probes on a surface was performed using thiol-ene or thiol-yne chemistry for detection of PCR products from Salmonella or Campylobacteria to test the capture and detection efficiency. The data with a number of examples will be presented. The detection was found to be quite sensitive and PCR products were detected at as low as 20 pM. ** The technology was developed by F.Morvan, M-J Banuls, P.Jimenez-Meneses, A. Meyer, J.-J. Vasseur, J.Escorihuela, R.Puchades, A.Maquieira; Universitat Politecnica De Valencia, IBMM, France. *** ChemGenes Corp. holds worldwide license on this technology.

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Remarkable Enhancement of RNaseH Cleavage Activities of RNA Complexed with Peptide Ribonucleic Acid (PRNA) – DNA Chimera for Effective Cancer Cell Specific Oligonucleotide Therapeutics R. Uematsu,1 H. Sugai1, M. Asai1, A, Nishio,2 J. Ariyoshi,3 S. Sakamoto,1 Y. Araki,1 A. Yamayoshi,3

S. Ishibashi,4 T. Yokota,3 Y. Inoue,2 and T. Wada1* 1Institute of Multidisciplinary Research for Advanced Materials (IMRAM), Tohoku Univ., Sendai, 980-8577, Japan; 2Osaka Univ., Japan; 3Kyoto Univ., JAPAN; 4Tokyo Medical & Dental Univ., Japan4

We have recently proposed a new strategy and a practical tool for cancer cell selective oligonucleotide therapeutics artificial nucleic acids, named Peptide Ribonucleic Acids (PRNAs) with active on-off control of functional RNA activities corresponding the cancer cell specific intracellular condition. The PRNAs can be actively off to on switching the complexation behaviour with target mRNA, miRNA, and siRNA induced by a low oxygen concentration of the cancer cellular cytoplasm. This strategy utilizes a new category of artificial nucleic acid that carries a ribonucleoside unit tethered to a peptide backbone as a recognition and stimulus-sensitive module. In this artificial nucleic acid called peptide ribonucleic acid (PRNA), the 5’-amino-pyrimidine ribonucleoside unit, which is in the syn conformation in normal cellular cytoplasm condition induced by intramolecular cyclic borate ester formation with incorporated phenylborate moiety, functions as a built-in switching moiety of syn to anti nucleobase orientation change function triggered by a the cancer cellular cytoplasm specific low oxygen condition, and is attached to the alpha-glutamine backbone as a pendant. The results are promising, validating that the original α-PRNAs with anti-oriented nucleobases form stable complexes with the target RNA under the low cytoplasm pH (pH = ca. 6.5) induced by a low oxygen concentration of the cancer cell, which are readily dissociated under normal cellular cytoplasm pH (pH = ca. 7.2). This means that the PRNA strategy can be used as a powerful tool for on-off switching the RNA complexation behaviour, which is potentially applicable to the cancer cell selective oligonucleotide therapeutics of the next generation.1

Meanwhile, RNase H activities of antisense molecules would be one of the most crucial factors for practical antisense, siRNA, and miRNA related drugs. Thus, in this study, we have been designed PRNA-DNA chimeras, in which both PRNA and DNA domains work as recognition sites for the complexation with complementary DNA/RNAs and PRNA moieties work as recognition control/switching devices, while DNA-RNA hybrids formed in the DNA domains of the chimera should be substrates of RNase H and then target RNAs hydrolysed by the enzyme. (Fig. 1). Efficient and very enhanced cleavage of target RNAs compared with those with DNAs was observed for PRNA-DNA chimera/RNA complex by RNaseH. Regulation of protein synthesis by PRNA-DNA chimera was also evaluated by in vitro cell-free protein synthesis system, and the effective regulation was observed. Moreover, RNAi inhibition with PRNA-DNA chimera was also demonstrated in vivo system.

We have designed and synthesized PRNA-DNA chimeras, which can form stable complex with RNA in high sequence specific manner. Remarkably enhanced efficient cleavage of target RNAs was observed for PRNA-DNA chimera/RNA complex by RNase H. These results strongly suggested PRNA-DNA chimera would be one of the most promising candidates and has high potential for cancer cell specific oligonucleotide therapeutics. Takehiko WADA, PhD; Professor; IMRAM, Tohoku University; Katahira, Aoba-ku, Sendai, 980-8577, Miyagi, Japan E-mail: hiko@tagen.tohoku.ac.jp; Tel: +81-22-217-5608

DNA

PNA

PRNA

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HN

O

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N

HN

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Fig.1 PRNA-PNA-DNA Chimera

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Remarkable Enhancement of RNaseH Cleavage Activities of RNA Complexed with Peptide Ribonucleic Acid (PRNA) – DNA Chimera for Effective Cancer Cell Specific Oligonucleotide Therapeutics R. Uematsu,1 H. Sugai1, M. Asai1, A, Nishio,2 J. Ariyoshi,3 S. Sakamoto,1 Y. Araki,1 A. Yamayoshi,3

S. Ishibashi,4 T. Yokota,3 Y. Inoue,2 and T. Wada1* 1Institute of Multidisciplinary Research for Advanced Materials (IMRAM), Tohoku Univ., Sendai, 980-8577, Japan; 2Osaka Univ., Japan; 3Kyoto Univ., JAPAN; 4Tokyo Medical & Dental Univ., Japan4

We have recently proposed a new strategy and a practical tool for cancer cell selective oligonucleotide therapeutics artificial nucleic acids, named Peptide Ribonucleic Acids (PRNAs) with active on-off control of functional RNA activities corresponding the cancer cell specific intracellular condition. The PRNAs can be actively off to on switching the complexation behaviour with target mRNA, miRNA, and siRNA induced by a low oxygen concentration of the cancer cellular cytoplasm. This strategy utilizes a new category of artificial nucleic acid that carries a ribonucleoside unit tethered to a peptide backbone as a recognition and stimulus-sensitive module. In this artificial nucleic acid called peptide ribonucleic acid (PRNA), the 5’-amino-pyrimidine ribonucleoside unit, which is in the syn conformation in normal cellular cytoplasm condition induced by intramolecular cyclic borate ester formation with incorporated phenylborate moiety, functions as a built-in switching moiety of syn to anti nucleobase orientation change function triggered by a the cancer cellular cytoplasm specific low oxygen condition, and is attached to the alpha-glutamine backbone as a pendant. The results are promising, validating that the original α-PRNAs with anti-oriented nucleobases form stable complexes with the target RNA under the low cytoplasm pH (pH = ca. 6.5) induced by a low oxygen concentration of the cancer cell, which are readily dissociated under normal cellular cytoplasm pH (pH = ca. 7.2). This means that the PRNA strategy can be used as a powerful tool for on-off switching the RNA complexation behaviour, which is potentially applicable to the cancer cell selective oligonucleotide therapeutics of the next generation.1

Meanwhile, RNase H activities of antisense molecules would be one of the most crucial factors for practical antisense, siRNA, and miRNA related drugs. Thus, in this study, we have been designed PRNA-DNA chimeras, in which both PRNA and DNA domains work as recognition sites for the complexation with complementary DNA/RNAs and PRNA moieties work as recognition control/switching devices, while DNA-RNA hybrids formed in the DNA domains of the chimera should be substrates of RNase H and then target RNAs hydrolysed by the enzyme. (Fig. 1). Efficient and very enhanced cleavage of target RNAs compared with those with DNAs was observed for PRNA-DNA chimera/RNA complex by RNaseH. Regulation of protein synthesis by PRNA-DNA chimera was also evaluated by in vitro cell-free protein synthesis system, and the effective regulation was observed. Moreover, RNAi inhibition with PRNA-DNA chimera was also demonstrated in vivo system.

We have designed and synthesized PRNA-DNA chimeras, which can form stable complex with RNA in high sequence specific manner. Remarkably enhanced efficient cleavage of target RNAs was observed for PRNA-DNA chimera/RNA complex by RNase H. These results strongly suggested PRNA-DNA chimera would be one of the most promising candidates and has high potential for cancer cell specific oligonucleotide therapeutics. Takehiko WADA, PhD; Professor; IMRAM, Tohoku University; Katahira, Aoba-ku, Sendai, 980-8577, Miyagi, Japan E-mail: hiko@tagen.tohoku.ac.jp; Tel: +81-22-217-5608

DNA

PNA

PRNA

O

HO OH

HN

O

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N

HN

OP

O

HO

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nNB = nucleobase

O

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Fig.1 PRNA-PNA-DNA Chimera

Considerations for the Design and cGMP Manufacturing of mRNA Therapeutics Josh M. Whisenand, Krist T. Azizian, Jordana M. Henderson, Sabrina Shore, Dongwon Shin, Alexandre Lebedev, Anton P. McCaffrey and Richard I. Hogrefe TriLink BioTechnologies, San Diego, CA 92121, USA. Recently, there has been significant interest in the use of messenger RNA (mRNA) as an ex vivo and in vivo therapeutic. Since mRNA is expressed in the cytoplasm it may be particularly useful for improving gene expression in difficult to transfect non-dividing cells. In contrast to plasmid or viral vectors, there is no risk of insertional mutagenesis or subsequent oncogenesis upon mRNA transfection and the transient nature of mRNA expression is desirable for genome editing (CRISPR/Cas9, ZFNs and TALENs) and vaccines. In each case, the goal is to produce a synthetic RNA that mimics a natural mRNA.

In anticipation of clinical testing, it is essential to use sequence designs, raw materials and manufacturing processes that are scalable. Early on, critical decisions must be made about 1) transcription template designs, 2) capping methodologies, 3) sequence optimization, 4) chemical modifications to evade innate immune responses, 5) polyadenylation methods and 6) purification methods.

For optimal expression in cells or target organs, transfected mRNAs must avoid detection by pattern recognition receptors (PRRs) that evolved to sense improperly capped RNAs and double stranded RNA. PRR activation leads to cytokine production, translational arrest and cell toxicity or death. Mammalian mRNAs are modified post-transcriptionally to contain modified nucleotides (e.g. pseudouridine and 5-methylcytidine). These modifications can reduce activation of PRRs and allow maximal translation.

During RNA capping, Cap0 (m7GpppN) is formed as an intermediate. Methylation of the 2’ position of the first nucleotide forms Cap1 (found in 100% of transcripts) and Cap2 (found in 50% of transcripts) of endogenous mRNAs. mRNAs generated with commercially available cap analogs (ARCA) contain Cap0 structures that can be immunogenic. Recombinant enzymes used to generate Cap1 mRNA are expensive, do not always go to completion and the RNA must be purified prior to capping. A novel co-transcriptional capping method that yields Cap1 or Cap2 with high efficiency and lower costs in a “one pot” reaction will be discussed.

First generation mRNAs were modified with pseudouridine or 5-methylcytidine/pseudouridine and had Cap0 structures. Data from improved second generation Cap1 mRNAs containing a combination of sequence engineering and chemical modifications will be presented. We will provide a broad roadmap for the application of these principles to the design and manufacturing of novel mRNA therapeutics.

Josh M. Whisenand Program manager TriLink Biotechnologies 9955 Mesa Rim Road San Diego, CA 92121 USA JWhisenand@TriLinkBiotech.com

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Influence of O4-Alkylthymidine Lesion Orientation on Replication by Human DNA Polymerase η Christopher J. Wilds1, Derek K. O’Flaherty1, Amritraj Patra2, Yan Su2, F. Peter Guengerich2, and Martin Egli2 Department of Chemistry and Biochemistry, Concordia University, Montréal, Québec H4B1R6, Canada1; Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA2 Alkylation of DNA can result from exposure to agents either endogenous or exogenous to the cell. These lesions can have adverse effects which include DNA polymerase (Pol) blockage and nucleotide misincorporation. Lesions that evade DNA repair can undergo translesion synthesis (TLS) by Y-family DNA Pols, which bypass modifications in an error-free or error-prone manner. Bypass activity of the human DNA Pol η (hPol η) has been correlated with resistance to chemotherapeutic agents such as cisplatin. The O4-atom of thymidine is susceptible to alkylation producing a lesion that results in high fidelity replicative DNA Pol misinserting dGTP in daughter strands. In the present study, the influence of O4-alkyl lesion orientation on hPol η bypass processivity was evaluated with oligonucleotides containing O4-methylthymidine (O4Me T), O4-ethylthymidine (O4Et T) and analogs where the C5 and O4 atoms are connected by a dimethylene (DFP) or trimethylene (TPP) linkage. The O4-methylene for the DFP and TPP analogs is constrained to adopt an anti-conformation. Single nucleotide incorporation studies demonstrated increased selectivity towards dATP over dGTP in the order O4Et T > DFP ≈ TPP whereas a slight preference for dGTP was observed for O4Me T. Primer extension reactions in the presence of all four dNTPs for templates containing the modifications revealed proficient nucleotide incorporation by hPol η across and past the modified site. However, it was observed that O4Me T and O4Et T exhibited greater accumulation of non-full length oligonucleotide products at reduced reaction times relative to DFP and TPP. LC-MS/MS analyses of the extension products from in vitro primer bypass studies revealed dGTP over dATP incorporation across the lesion was preferred except for the control (containing thymidine). The ratio of dGTP:dATP incorporation by hPol η decreased in the order TPP (4.6:1) > DFP (3.3:1) ≈ O4Et T (3.2:1) > O4Me T (2.1:1), demonstrating that increased size of the alkyl group (O4Et T vs O4Me T) or restriction of the O4-methylene group to an anti-conformation promotes dGTP misincorporation. The crystal structures of ternary hPol η•DNA•dATP and hPol η•DNA•dGTP complexes with the template strands containing O4Me T reveal that the lesion stacks atop a tryptophan residue near the ceiling of the active site instead of pairing with the incoming nucleotide. Together, these results provide mechanistic insights on the mutagenicity of O4-alkylated T when acted upon by hPol η. Christopher Wilds, PhD Associate Professor Concordia University 7141 Sherbrooke Street West Montréal, Québec Canada Chris.Wilds@concordia.ca (+1) 514-848-2424 ext. 5798

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Influence of O4-Alkylthymidine Lesion Orientation on Replication by Human DNA Polymerase η Christopher J. Wilds1, Derek K. O’Flaherty1, Amritraj Patra2, Yan Su2, F. Peter Guengerich2, and Martin Egli2 Department of Chemistry and Biochemistry, Concordia University, Montréal, Québec H4B1R6, Canada1; Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA2 Alkylation of DNA can result from exposure to agents either endogenous or exogenous to the cell. These lesions can have adverse effects which include DNA polymerase (Pol) blockage and nucleotide misincorporation. Lesions that evade DNA repair can undergo translesion synthesis (TLS) by Y-family DNA Pols, which bypass modifications in an error-free or error-prone manner. Bypass activity of the human DNA Pol η (hPol η) has been correlated with resistance to chemotherapeutic agents such as cisplatin. The O4-atom of thymidine is susceptible to alkylation producing a lesion that results in high fidelity replicative DNA Pol misinserting dGTP in daughter strands. In the present study, the influence of O4-alkyl lesion orientation on hPol η bypass processivity was evaluated with oligonucleotides containing O4-methylthymidine (O4Me T), O4-ethylthymidine (O4Et T) and analogs where the C5 and O4 atoms are connected by a dimethylene (DFP) or trimethylene (TPP) linkage. The O4-methylene for the DFP and TPP analogs is constrained to adopt an anti-conformation. Single nucleotide incorporation studies demonstrated increased selectivity towards dATP over dGTP in the order O4Et T > DFP ≈ TPP whereas a slight preference for dGTP was observed for O4Me T. Primer extension reactions in the presence of all four dNTPs for templates containing the modifications revealed proficient nucleotide incorporation by hPol η across and past the modified site. However, it was observed that O4Me T and O4Et T exhibited greater accumulation of non-full length oligonucleotide products at reduced reaction times relative to DFP and TPP. LC-MS/MS analyses of the extension products from in vitro primer bypass studies revealed dGTP over dATP incorporation across the lesion was preferred except for the control (containing thymidine). The ratio of dGTP:dATP incorporation by hPol η decreased in the order TPP (4.6:1) > DFP (3.3:1) ≈ O4Et T (3.2:1) > O4Me T (2.1:1), demonstrating that increased size of the alkyl group (O4Et T vs O4Me T) or restriction of the O4-methylene group to an anti-conformation promotes dGTP misincorporation. The crystal structures of ternary hPol η•DNA•dATP and hPol η•DNA•dGTP complexes with the template strands containing O4Me T reveal that the lesion stacks atop a tryptophan residue near the ceiling of the active site instead of pairing with the incoming nucleotide. Together, these results provide mechanistic insights on the mutagenicity of O4-alkylated T when acted upon by hPol η. Christopher Wilds, PhD Associate Professor Concordia University 7141 Sherbrooke Street West Montréal, Québec Canada Chris.Wilds@concordia.ca (+1) 514-848-2424 ext. 5798

Application of Liposomal Formulation for the Delivery of Antisense Oligonucleotide into Brain after Intrathecal Administration Caroline N Woodward1, Vishwesh Patil1, Kalyan Vasudevan1, Adnan Zunic2Jessica Stolee1 and Natalia Penner1 Biogen Inc.1, Takeda2, Antisense Oligonucleotides (ASOs) have shown potential as therapeutic agents for CNS disorders. The blood-brain barrier however, remains a major obstacle for the delivery of ASO’s to the CNS due in part to their molecular size. ASOs have to reach brain regions that are relevant to CNS diseases such as Amyotrophic lateral sclerosis, Alzheimer’s and Parkinson’s making intrathecal administration a viable option for ASO administration. After IT administration, drugs are rapidly drained through the dural venus sinuses thus reducing amount of drug available for brain distribution. Here we present the utility of liposomal encapsulation of a non-target specific ASO’s to prolong retention and potentially increase brain penetration after IT administration. To prepare the formulation, Spontaneous Vesicle Formation by Ethanol Dilution Method was used to prepare dosing solutions. Gel electrophoresis and LC-MS method were applied to measure efficiency of ASO encapsulation in the fluorescent dual labelled liposomal formulation. Two naked (non-encapsulated) dosing groups prepared at 180 and 65 µg and encapsulated ASO prepared at 180µg/per 30 µl dose were administered via IT to naïve rats. 99mTc-DTPA was co-injected with all formulations to control the success of the IT administration via SPECT. Brains were harvested at 8 hours post-dose and analyzed by LC-MS to compare residual brain concentrations for naked and liposomal ASOs. In addition, distribution of liposomal-ASO in rat brains was assessed after sectioning into slices and fluorescent imaging. The findings indicate that use of liposomal ASO’s resulted in significant increase of residual total brain concentration due to retention of encapsulated ASOS in subarachnoid space (SAS), in addition the experiment indicated that liposomal encapsulation of ASO’s slowed down the drainage of ASO from cerebrospinal fluid. Caroline Ngaara Woodward, MS Scientist II Biogen 300 Binney Street Cambridge, Ma 02412 USA Caroline.woodward@biogen.com (617)679-3380

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Development of a Robust, Sensitive and Selective Liquid Chromatography High Resolution Mass Spectrometry Method for the Quantitation of Encapsulated Antisense Oligonucleotide in Rat Brain after Intrathecal Administration Caroline Ngaara Woodward1, Vishwesh Patil1, Kalyan Vasudevan1, Adnan Zunic2, Natalia Penner1 1Biogen Inc, 2Takeda Antisense Oligonucleotides (ASOs) have shown promise as therapeutic agents due to their ability to specifically modulate disease pathways. In addition, manufacturing of ASO’s is simple, rugged and efficient which translates to quick identification, synthesis and testing of multiple gene-specific inhibitors in-vivo. Much like small molecules, pharmacokinetic properties of ASO’s is important for understanding species differences and in selecting safe and efficacious doses to be applied in a clinical setting. For small molecules, LC-MS/MS based bioanalytical assays have been adopted as the gold standard due to its robustness, selectivity and high-throughput capability. Quantitation of ASOs in biological matrices has traditionally relied on enzyme linked immunosorbent assay (ELISA) which is time consuming due to lengthy method development. LC-MS-based methods for ASO’s have been difficult to implement. Traditional extraction methods such as protein precipitation or liquid-liquid extraction provided poor recoveries, and also difficult to automate. In addition, LC-MS of ASOs has unique chromatography challenges as ion-pairing reagents are needed to retain and separate oligonucleotides. However, advances in separation techniques have made it possible to implement LC-MS of ASOs in a discovery setting where Go/No Go decisions are time-sensitive. Here we present the development of an LC-MS method for quantification of encapsulated non-target specific ASO in rat brain after intrathecal (IT) administration. Rat brain homogenate unknown and known (calibrations and Quality Control standards) were extracted using Phenomenex® Clarity Oligo Therapeutic Extraction (OTX™). Separation of the resulting supernatant was achieved using an Acquity Oligonucleotide Separation Technology (OST) column with Triethylamine and 1, 1, 1, 3,3,3-hexafluoro-2-propanol as ion-pairing reagents. Detection was achieved by electrospray ionization set to negative Selective Ion Monitoring mode on a high resolution QE Exactive plus Oribtrap (Thermo Scientific™, San Jose, CA). The assay had a linear range between 4-10000 ng/mL and was specific enough to discern between endogenous species and analytes of interest. This method was successfully applied to measure ASO in rat brain after IT administration. Caroline Ngaara Woodward, MS Scientist II Biogen 300 Binney Street Cambridge, Ma 02412 USA Caroline.woodward@biogen.com (617)679-3380

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Development of a Robust, Sensitive and Selective Liquid Chromatography High Resolution Mass Spectrometry Method for the Quantitation of Encapsulated Antisense Oligonucleotide in Rat Brain after Intrathecal Administration Caroline Ngaara Woodward1, Vishwesh Patil1, Kalyan Vasudevan1, Adnan Zunic2, Natalia Penner1 1Biogen Inc, 2Takeda Antisense Oligonucleotides (ASOs) have shown promise as therapeutic agents due to their ability to specifically modulate disease pathways. In addition, manufacturing of ASO’s is simple, rugged and efficient which translates to quick identification, synthesis and testing of multiple gene-specific inhibitors in-vivo. Much like small molecules, pharmacokinetic properties of ASO’s is important for understanding species differences and in selecting safe and efficacious doses to be applied in a clinical setting. For small molecules, LC-MS/MS based bioanalytical assays have been adopted as the gold standard due to its robustness, selectivity and high-throughput capability. Quantitation of ASOs in biological matrices has traditionally relied on enzyme linked immunosorbent assay (ELISA) which is time consuming due to lengthy method development. LC-MS-based methods for ASO’s have been difficult to implement. Traditional extraction methods such as protein precipitation or liquid-liquid extraction provided poor recoveries, and also difficult to automate. In addition, LC-MS of ASOs has unique chromatography challenges as ion-pairing reagents are needed to retain and separate oligonucleotides. However, advances in separation techniques have made it possible to implement LC-MS of ASOs in a discovery setting where Go/No Go decisions are time-sensitive. Here we present the development of an LC-MS method for quantification of encapsulated non-target specific ASO in rat brain after intrathecal (IT) administration. Rat brain homogenate unknown and known (calibrations and Quality Control standards) were extracted using Phenomenex® Clarity Oligo Therapeutic Extraction (OTX™). Separation of the resulting supernatant was achieved using an Acquity Oligonucleotide Separation Technology (OST) column with Triethylamine and 1, 1, 1, 3,3,3-hexafluoro-2-propanol as ion-pairing reagents. Detection was achieved by electrospray ionization set to negative Selective Ion Monitoring mode on a high resolution QE Exactive plus Oribtrap (Thermo Scientific™, San Jose, CA). The assay had a linear range between 4-10000 ng/mL and was specific enough to discern between endogenous species and analytes of interest. This method was successfully applied to measure ASO in rat brain after IT administration. Caroline Ngaara Woodward, MS Scientist II Biogen 300 Binney Street Cambridge, Ma 02412 USA Caroline.woodward@biogen.com (617)679-3380

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Peptide Nucleic Acids (PNAs) as Diagnostic Molecules for the Early Detection of Cancer Eylon Yavina, Dinah Hashoula, Ganeshkumar Moorthya, Abraham Rubinsteina, Rachel Shapirab, Vera Paviovb, and Aviram Nissanb aThe Institute for Drug Research, The School of Pharmacy, The Hebrew University of Jerusalem, Hadassah Ein-Kerem, Jerusalem 91120, Israel. bDepartment of General and Oncological Surgery, The Chaim Sheba Medical Center, Tel Hashomer, Israel.

Thiazole orange (TO), a cyanine dye and well-established DNA staining agent, has been previously developed by Seitz and co-workers as a surrogate base that when incorporated into a PNA (Peptide Nucleic Acid) sequence (a.k.a. FIT-PNA, forced intercalation-PNA), turns on its fluorescence upon hybridization to a complementary RNA/DNA sequence. We have recently developed FIT-PNAs with a new surrogate base (BisQ) that emits at the red-region (613nm) and shown that a fluorescent readout at SNP resolution in living cells is detected after 30 minutes by simple incubation of the FIT-PNA in cell culture. We have also targeted an oncogenic long non-coding RNA (lncRNA) termed CCAT1 (colon-cancer associated transcript 1) that has been found to be highly expressed in a variety of tumors. Herein we have designed a BisQ FIT-PNA for CCAT1 and show a clear fluorescent response in over-expressed CCAT1 in living cancer cells (HT-29 and Panc1 for colon and pancreatic cancer cells, respectively). Moreover, human fixed biopsies from colorectal cancer patients were highly fluorescent after CCAT1 FIT-PNA incubation. Lastly, fresh human tissues taken from cancerous regions of peritoneal post-surgery patients were tested with CCAT1 FIT-PNA. Following simple spraying (in PBS, 2.5 µM PNA) of the CCAT1 FIT-PNA probe directly on the tissue examined resulted in substantial fluorescence in a manner of minutes at regions suspected as cancerous ones (Figure). We envision that such PNA-FIT probes may be useful for non-invasive RNA biomarker detection in-vivo and for defining cancer margins during cytoreductive surgery.

Eylon Yavin, PhD Head of Nucleic Acid Laboratory School of Pharmacy, Faculty of Medicine The Hebrew University of Jerusalem Hadassah Ein-Kerem, Jerusalem, Israel 91120. eylony@ekmd.huji.ac.il

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Peptide Nucleic Acids (PNAs) as Diagnostic Molecules for the Early Detection of Cancer Eylon Yavina, Dinah Hashoula, Ganeshkumar Moorthya, Abraham Rubinsteina, Rachel Shapirab, Vera Paviovb, and Aviram Nissanb aThe Institute for Drug Research, The School of Pharmacy, The Hebrew University of Jerusalem, Hadassah Ein-Kerem, Jerusalem 91120, Israel. bDepartment of General and Oncological Surgery, The Chaim Sheba Medical Center, Tel Hashomer, Israel.

Thiazole orange (TO), a cyanine dye and well-established DNA staining agent, has been previously developed by Seitz and co-workers as a surrogate base that when incorporated into a PNA (Peptide Nucleic Acid) sequence (a.k.a. FIT-PNA, forced intercalation-PNA), turns on its fluorescence upon hybridization to a complementary RNA/DNA sequence. We have recently developed FIT-PNAs with a new surrogate base (BisQ) that emits at the red-region (613nm) and shown that a fluorescent readout at SNP resolution in living cells is detected after 30 minutes by simple incubation of the FIT-PNA in cell culture. We have also targeted an oncogenic long non-coding RNA (lncRNA) termed CCAT1 (colon-cancer associated transcript 1) that has been found to be highly expressed in a variety of tumors. Herein we have designed a BisQ FIT-PNA for CCAT1 and show a clear fluorescent response in over-expressed CCAT1 in living cancer cells (HT-29 and Panc1 for colon and pancreatic cancer cells, respectively). Moreover, human fixed biopsies from colorectal cancer patients were highly fluorescent after CCAT1 FIT-PNA incubation. Lastly, fresh human tissues taken from cancerous regions of peritoneal post-surgery patients were tested with CCAT1 FIT-PNA. Following simple spraying (in PBS, 2.5 µM PNA) of the CCAT1 FIT-PNA probe directly on the tissue examined resulted in substantial fluorescence in a manner of minutes at regions suspected as cancerous ones (Figure). We envision that such PNA-FIT probes may be useful for non-invasive RNA biomarker detection in-vivo and for defining cancer margins during cytoreductive surgery.

Eylon Yavin, PhD Head of Nucleic Acid Laboratory School of Pharmacy, Faculty of Medicine The Hebrew University of Jerusalem Hadassah Ein-Kerem, Jerusalem, Israel 91120. eylony@ekmd.huji.ac.il

Blind SELEX to discover biomarkers and therapeutic aptamers Sorah Yoon1, Brian Armstrong2, Nagy Habib3 and John J Rossi1 1Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA 2Light Microscopy core, City of Hope, Duarte, California, USA 3Department of Surgery and Cancer, Imperial College London, London, UK

Pathophysiological cell phenotype changes are common in cancer progression such as epithelial-mesenchymal transition (EMT). In the progress of the changes, intracellular proteins are translocated on the plasma membrane of cancer cells. These selectively translocated proteins have potential as biomarkers and therapeutic targets. For biomarker discovery, aptamers have great advantages as targeting agents; capturing low abundant antigens and no interfering the detection of peptide sequence. To improve therapeutic interventions via biomarker discovery, blind cell SELEX with RNA aptamers, untargeted SELEX, has been utilized against pancreatic cancer cells. Throughout the blind-SELEX, we have identified an aptamer, P15, which specifically bound to the human pancreatic adenocarcinoma cells. The tandem mass spectrometry (MASS-SPEC) was used to identify the P15 binding membrane protein. The results of MASS-SPEC identified the target of P15 as intermediate filament vimentin, biomarker of EMT. The vimentin is an intracellular protein but is specifically expressed on the plasma membrane of cancer cells. As EMT plays a pivotal role to transit cancer cells to invasive cells, tumor cell invasion assays were performed in vitro to determine the cancer cell metastasis. P15 treated pancreatic cancer cells showed the significant inhibition of tumor metastasis in wound healing and Boyden chamber assays. To investigate the downstream effects of P15, EMT related gene expression analysis was performed. In P15 treated cells, the expression of matrix metallopeptidase 3 (MMP3), which is involved in cancer invasion, was down-regulated. In this study for the first time, we showed that P15 binding to cell surface vimentin inhibits the tumor cell invasion and is associated with reduction of MMP3. Hence, P15 might be used as anti-metastatic drugs in pancreatic cancer. Additionally, we suggest that blind SELEX is a good strategy to identify biomarkers and to improve therapeutic intervention. Sorah Yoon (D.V.M., Ph.D) Staff scientist Beckman Research Institute of City of Hope 1500 E. Duarte Rd, Duarte, CA USA syoon@coh.org

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Evaluation of Off-target Effects of Splice-switching Oligonucleotides Tokuyuki Yoshida1,2, Kiyomi Sasaki1,2, Yuki Naito3, Makoto Koizumi4, Masakazu Tamura4, Hiroaki Miida4, Mitsuhiro Tagaya5, Kazuchika Takagaki5,Satoshi Obika2, Mikihiko Naito1,2, Takao Inoue1,2 1Division of Molecular Target and Gene Therapy Products, National Institute of Health Sciences 2Graduate School of Pharmaceutical Sciences, Osaka University 3Database Center for Life Science (DBCLS) 4 Daiichi Sankyo Co., Ltd 5 Nippon Shinyaku Co., Ltd

Recently, RNA targeting oligonucleotide therapeutics, such as Splice-switching

oligonucleotides (SSOs) which bind to target sequences in pre-mRNA and prevent the interaction of various splicing modulators, have been developed extensively. In 2016, the FDA has approved two SSOs, eteplirsen (Exondys51, SareptaTherapeutics) and nusinersen (Spinraza, Biogen and Ionis Pharmaceuticals). On the other hand, RNA targeting oligonucleotide therapeutics carry the risk of causing unintended toxicities. One of the potential toxicities is hybridization-dependent off-target effects which are caused by Watson and Crick base-pairing to unintended RNAs. However, methods for the evaluation of hybridization-dependent off-target effects of SSOs have not been fully discussed. In this study, we obtained experimental data for development of methods for the evaluation of hybridization-dependent off-target effects of SSOs. We would like to discuss the evaluation of off-target effects of SSOs in this meeting. Tokuyuki Yoshida, Ph.D. Researcher National Institute of Health Sciences Division of Molecular Target and Gene Therapy Products 1-18-1, Kamiyoga, Setagayaku Tokyo, 158-8501 Japan tokucox@nihs.go.jp

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Evaluation of Off-target Effects of Splice-switching Oligonucleotides Tokuyuki Yoshida1,2, Kiyomi Sasaki1,2, Yuki Naito3, Makoto Koizumi4, Masakazu Tamura4, Hiroaki Miida4, Mitsuhiro Tagaya5, Kazuchika Takagaki5,Satoshi Obika2, Mikihiko Naito1,2, Takao Inoue1,2 1Division of Molecular Target and Gene Therapy Products, National Institute of Health Sciences 2Graduate School of Pharmaceutical Sciences, Osaka University 3Database Center for Life Science (DBCLS) 4 Daiichi Sankyo Co., Ltd 5 Nippon Shinyaku Co., Ltd

Recently, RNA targeting oligonucleotide therapeutics, such as Splice-switching

oligonucleotides (SSOs) which bind to target sequences in pre-mRNA and prevent the interaction of various splicing modulators, have been developed extensively. In 2016, the FDA has approved two SSOs, eteplirsen (Exondys51, SareptaTherapeutics) and nusinersen (Spinraza, Biogen and Ionis Pharmaceuticals). On the other hand, RNA targeting oligonucleotide therapeutics carry the risk of causing unintended toxicities. One of the potential toxicities is hybridization-dependent off-target effects which are caused by Watson and Crick base-pairing to unintended RNAs. However, methods for the evaluation of hybridization-dependent off-target effects of SSOs have not been fully discussed. In this study, we obtained experimental data for development of methods for the evaluation of hybridization-dependent off-target effects of SSOs. We would like to discuss the evaluation of off-target effects of SSOs in this meeting. Tokuyuki Yoshida, Ph.D. Researcher National Institute of Health Sciences Division of Molecular Target and Gene Therapy Products 1-18-1, Kamiyoga, Setagayaku Tokyo, 158-8501 Japan tokucox@nihs.go.jp

Cerebral therapeutic exon skipping in the mdx mouse using tricyclo-DNA antisense oligonucleotides

Faouzi Zarrouki1,2, Cyrille Vaillend2, Luis Garcia1, Aurélie Goyenvalle1

1Université de Versailles St-Quentin, INSERM U1179, Université Paris-Saclay, Montigny-le-Bretonneux, France. 2Neuroscience Paris-Saclay Institute (Neuro-PSI), UMR9197, Université Paris-Sud, CNRS, Université Paris-Saclay, Orsay, France.

Duchenne muscular dystrophy (DMD) is caused by the absence of dystrophin, an important cytoskeletal protein in both muscle and brain. Loss of brain dystrophin in the mdx mouse model of DMD is associated with alterations in GABAA-receptor postsynaptic clustering, abnormal synaptic plasticity and a range of motor, emotional and cognitive disturbances. Concurring with cognitive deficits and psychiatric symptoms often observed in DMD patients, an enhanced defensive behavior in response to a threat has been reported in mdx mice, which has been shown to be independent of motor, respiratory or cardiac defect, but rather controlled by brain mechanisms. We have previously shown that tricyclo-DNA (tcDNA), a novel class of antisense oligonucleotides are able to induce skipping of the mutated exon 23 and rescue the expression of a truncated but functional dystrophin in both muscle and brain tissue after systemic injection. In order to investigate further the cognitive defects in the mdx mouse and determine the amount of dystrophin required to restore these altered cognitive functions, we used tcDNA-mediated exon skipping therapy as a tool. In preliminary experiments we performed intracerebroventricular (ICV) injections of tcDNA in mdx mice to validate the effectiveness of locoregional treatment and thus define the therapeutic threshold. Herein we report that five weeks after a single ICV injection of modified tcDNA, we can restore up to 40% of dystrophin in hippocampus, and approximately 8% in cortex and cerebellum. These levels allow the restoration of a normal defensive behavioral in the treated mice five weeks after treatment. This approach can therefore correct dystrophin-dependent brain mechanisms and help to better understand the neurobiology of DMD. Indeed, the correlation between the amount of cerebral dystrophin and the associated cognitive phenotype may allow us to better decipher the role of dystrophin in cognitive processes that are altered in DMD patients. The perspective of full treatment of the DMD syndrome can therefore be envisaged with tcDNA.

Faouzi Zarrouki PhD student Université Versailles St-Quentin 2 avenue de la source de la bièvre 78180 Montigny-le-Bx Faouzi.zarrouki@hotmail.fr +33614583103

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Pharmacologic Properties of Stereopure OligonucleotidesJason Zhang, Keith Bowman, David Butler, Young-Jin Cho, Chris Francis, Naoki Iwamoto, Mamoru Shimizu, Stephany Standley, Zhong Zhong, Chandra Vargeese WAVE Life Sciences, Cambridge, MA, USA

Phosphorothioate (PS) modification of oligonucleotides is a common alteration, where non-bridging oxygens are replaced with sulfur atoms at PS-modified linkages. Each PS linkage creates a chiral center that can either have an “Sp” configuration or an “Rp” configuration, depending on the three-dimensional arrangement of its atoms. In traditional PS oligonucleotide synthesis, chiral configurations of PS linkages are not purposefully designed and/or cannot be controlled, creating mixtures of thousands or even millions of stereoisomers, each having a different three-dimensional atomic arrangement. Such mixtures may lead to potentially undesired pharmacologic properties, with some constituent molecules producing therapeutic effects and others being less beneficial or even contributing to undesirable effects.

WAVE Life Sciences has developed proprietary technologies that enable the synthesis of PS-modified nucleic acid therapeutics in which stereochemistry at each PS position can be precisely controlled. This degree of control enables rational design and synthesis of optimized stereopure nucleic acid therapeutics, which show improved pharmacologic properties compared with mixture-based nucleic acid therapeutics. We believe our ability to selectively employ PS modifications as desired may provide further control over pharmacodynamics (stability, activity, duration of activity and selectivity) and may improve safety.

Experiments were conducted to determine whether PS chirality influences the effect of oligonucleotides on toll-like receptor 9 (TLR9) activity. Results showed that PS chirality impacts the TLR9-dependent activity of synthetic oligonucleotides in a species-specific manner, with distinct rules emerging for mouse and human. In both systems, we found that stereopure components of stereorandom mixtures can have dramatically distinct TLR9 activation profiles compared with sterorandom mixtures. We also provide evidence that while 2’-modifications are sufficient to abrogate TLR9 agonist activity in the mouse, 2’-modified synthetic oligonucleotides can still activate human TLR9. These findings support the hypothesis that well-designed stereopure oligonucleotides may have the potential for an improved profile over stereorandom oligonucleotides.

Jason Zhang, PhD Director, Biology WAVE Life Sciences 733 Concord Avenue Cambridge, MA 02138 USA jzhang@wavelifesci.com

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Gapmer antisense oligonucleotides selectively suppress the mutant allele of COL6A3 gene and restore functional protein production in dominant Ullrich congenital muscular dystrophy Haiyan Zhou, Elena Marrosu, Pierpaolo Ala and Francesco Muntoni Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neuroscience Programme, University College London Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. Dominant negative mutations in the genes that encode the three major α-chains of collagen type VI, COL6A1, COL6A2 and COL6A3, account for more than 50% of Ullrich congenital muscular dystrophy and nearly all Bethlem myopathy patients. Gapmer antisense oligonucleotides (AONs) are usually used for gene silencing by stimulating RNA cleavage through the recruitment of an endogenous endonuclease known as RNase H to cleave the RNA strand of a DNA-RNA duplex. In this study, we exploited the application of the allele-specific silencing approach by gapmer AON as a potential therapy for Collagen VI-related congenital muscular dystrophy (COL6-CMD). A series of AONs were designed to selectively target an 18 nucleotides heterozygous genomic deletion in exon 15 of COL6A3, at the mRNA and pre-mRNA level. We showed that gapmer AONs can selectively suppress the expression of mutant transcripts at both pre-mRNA and mRNA levels, and the latter strategy had a far stronger efficiency than the former. More importantly, we found that silencing of the mutant transcripts by gapmer AONs increased the deposition of collagen VI protein into the extracellular matrix, thus restoring functional protein production. Our findings provide proof-of-concept for AON allele-specific silencing as a therapeutic approach for COL6-CMD. Haiyan Zhou, PhD Senior Research Associate University College London, 30 Guilford Street London WC1N 1EH Haiyan.zhou@ucl.ac.uk Tel: 0044-20-79052136

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Targeting HIV reservoirs with cell-specific aptamer-based conjugates Jiehua Zhou1, Mayumi Takahashi2, Haitang Li1, Xin Xia1, Piotr Swiderski3, Nagy Habib4, Kevin Morris5, and John Rossi1, 6* 1 Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope; 2

Center for Drug Evaluation and Research (CDER), U.S. Food and Drug Administration; 3 Shared Resource-DNA/RNA Peptide, Department of Molecular Medicine, City of Hope; 4 Department of Surgery and Cancer, Imperial College London, London, UK; 5 Center for Gene Therapy, Hematologic Malignancies and Stem Cell Institute, City of Hope, Duarte, CA; 6 Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, CA, USA The HIV latent reservoir remains a formidable obstacle to eradication of HIV or sustained virology The HIV latent reservoir remains a formidable obstacle to eradication of HIV or sustained virologic remission after cessation of combinatorial anti-retroviral therapy (cART). Although reactivating latency while maintaining cART represents an opportunity to eliminate latent HIV-1 infection or restrict the reservoirs to a size bearable by the host immune system, safety concerns about global cellular activation and side effects associated with cART still remain. Thus, precise interventions involving cell-specific reactivation of latency and targeted clearance of reactivated reservoirs are needed to improve therapeutic specificity and efficiency. Nucleic acid aptamers, also called “chemical antibodies”, are in vitro-selected, single-stranded nucleic acids that assume specific and stable three-dimensional shapes, thereby providing exquisitely specific, tight binding to targeted ligands. We have previously capitalized on the exquisite specificity of nucleic acid aptamers against gp120 or CCR5 to deliver anti-HIV small interfering RNAs (siRNAs) into HIV+ or HIV susceptible cells. These dual-inhibitory function aptamer–siRNA conjugates knocked down target mRNA, inhibited viral entry by blocking viral envelope interactions with cellular receptors, and provided effective suppression of viral infection. This confirms that aptamers function as effective cell-specific delivery agents. Compared to antibody-drug conjugates that remain challenging, the use of nucleic acid aptamers is a more flexible and practical strategy for clinical translation. In this study, we engineered and evaluated the first cell-specific aptamer-based platform to deliver various latency-reversing agents (LRAs) to latent HIV reservoirs. CCR7, a member of the G protein-coupled receptor family, is also a well-known marker of central memory CD4+ T cells, the major latent HIV reservoir. We therefore hypothesize that the central memory CD4+ T cell HIV reservoir can be targeted using CCR7 aptamers. By functionalizing LRAs with cell-specific aptamers against CCR7, we expect to enhance cellular recognition, and induce selective reactivation of HIV reservoirs. Furthermore, through combination of CCR7 aptamer–LRA conjugate with HIV-1 gp120 specific aptamer conjugate designed to deliver siRNA against HIV long terminal repeat (LTR362) to reactivated infected cells, we expect to eliminate the reactivated reservoir. Jiehua Zhou, Ph.D., Assistant Research Professor Department of Molecular and Cellular Biology Beckman Research Institute of City of Hope 1500 E Duarte Rd, Duarte, CA 91010 USA Email: jzhou@coh.org Office: 626-218-0533

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Targeting HIV reservoirs with cell-specific aptamer-based conjugates Jiehua Zhou1, Mayumi Takahashi2, Haitang Li1, Xin Xia1, Piotr Swiderski3, Nagy Habib4, Kevin Morris5, and John Rossi1, 6* 1 Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope; 2

Center for Drug Evaluation and Research (CDER), U.S. Food and Drug Administration; 3 Shared Resource-DNA/RNA Peptide, Department of Molecular Medicine, City of Hope; 4 Department of Surgery and Cancer, Imperial College London, London, UK; 5 Center for Gene Therapy, Hematologic Malignancies and Stem Cell Institute, City of Hope, Duarte, CA; 6 Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, CA, USA The HIV latent reservoir remains a formidable obstacle to eradication of HIV or sustained virology The HIV latent reservoir remains a formidable obstacle to eradication of HIV or sustained virologic remission after cessation of combinatorial anti-retroviral therapy (cART). Although reactivating latency while maintaining cART represents an opportunity to eliminate latent HIV-1 infection or restrict the reservoirs to a size bearable by the host immune system, safety concerns about global cellular activation and side effects associated with cART still remain. Thus, precise interventions involving cell-specific reactivation of latency and targeted clearance of reactivated reservoirs are needed to improve therapeutic specificity and efficiency. Nucleic acid aptamers, also called “chemical antibodies”, are in vitro-selected, single-stranded nucleic acids that assume specific and stable three-dimensional shapes, thereby providing exquisitely specific, tight binding to targeted ligands. We have previously capitalized on the exquisite specificity of nucleic acid aptamers against gp120 or CCR5 to deliver anti-HIV small interfering RNAs (siRNAs) into HIV+ or HIV susceptible cells. These dual-inhibitory function aptamer–siRNA conjugates knocked down target mRNA, inhibited viral entry by blocking viral envelope interactions with cellular receptors, and provided effective suppression of viral infection. This confirms that aptamers function as effective cell-specific delivery agents. Compared to antibody-drug conjugates that remain challenging, the use of nucleic acid aptamers is a more flexible and practical strategy for clinical translation. In this study, we engineered and evaluated the first cell-specific aptamer-based platform to deliver various latency-reversing agents (LRAs) to latent HIV reservoirs. CCR7, a member of the G protein-coupled receptor family, is also a well-known marker of central memory CD4+ T cells, the major latent HIV reservoir. We therefore hypothesize that the central memory CD4+ T cell HIV reservoir can be targeted using CCR7 aptamers. By functionalizing LRAs with cell-specific aptamers against CCR7, we expect to enhance cellular recognition, and induce selective reactivation of HIV reservoirs. Furthermore, through combination of CCR7 aptamer–LRA conjugate with HIV-1 gp120 specific aptamer conjugate designed to deliver siRNA against HIV long terminal repeat (LTR362) to reactivated infected cells, we expect to eliminate the reactivated reservoir. Jiehua Zhou, Ph.D., Assistant Research Professor Department of Molecular and Cellular Biology Beckman Research Institute of City of Hope 1500 E Duarte Rd, Duarte, CA 91010 USA Email: jzhou@coh.org Office: 626-218-0533

Preclinical Studies of WVE-210201, an Investigational Stereopure Antisense Oligonucleotide for the Treatment of Patients with Duchenne Muscular Dystrophy Amenable to Exon 51 Skipping Matthew Wood,1 Jason Zhang,2 Carlo Rinaldi,1 Graham McClorey,1 Keith Bowman,2 David Butler,2 Nayantara Kothari,2 Naoki Iwamoto,2 Mamoru Shimizu,2 Hailin Yang,2 Zhong Zhong,2 Chandra Vargeese2

1Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK; 2Wave Life Sciences Ltd., Cambridge, MA 02138

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder caused predominantly by out-of-frame deletions in the DMD gene, resulting in absent or defective dystrophin protein. Exon-skipping technology has the potential to induce cellular machinery to ‘skip over’ a targeted exon and restore the reading frame, resulting in the production of internally truncated, but functional, dystrophin protein. WVE-210201 is an investigational stereopure antisense oligonucleotide (ASO) in development as a potential disease-modifying therapy intended to target DMD exon 51 for the treatment of patients with DMD. WVE-210201 was synthesized using a proprietary chemistry platform that enables the production of chirality-controlled (i.e. stereopure) ASOs, rather than stereorandom complex chemical mixtures conventionally seen with nucleic acid therapeutics.

In vitro experiments were conducted to determine the stability, potency, and immune stimulatory effects of WVE-210201 and stereorandom ASOs (a phosphorothioate [PS] and a PMO). Gymnotic delivery of 10 µM of WVE-210201 to DMD ∆48-50 patient cells induced dystrophin protein restoration to a greater extent than both the PS and the PMO ASOs; WVE-210201 showed a 75-fold increase in dystrophin protein restoration compared with the PMO. There was a dose-dependent increase in skipped mRNA transcript that translated to truncated dystrophin protein restoration. These results were further confirmed in a second DMD patient-derived cell line (Δ52 cells). Furthermore, WVE-210201 demonstrated enhanced stability in wild-type mouse muscle tissue (gastrocnemius) homogenates relative to comparator ASOs, and did not activate toll-like receptor 9 (TLR9) in a human reporter cell line (HEK- Blue™ hTLR9 cells, Invivogen).

The results of these preclinical studies suggest that WVE-210201 may have the potential to be an effective disease-modifying therapy, and support its further development, for the treatment of patients with DMD who are amenable to exon 51 skipping.

Matthew Wood Department of Physiology, Anatomy and Genetics University of Oxford Oxford OX1 3QX, UK matthew.wood@dpag.ox.ac.uk

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