The RNA Summit: Research, Diagnostics & Therapeutics Agenda

Conference Registration, Materials Pick-Up, Morning Coffee and Pastries

Coffee Break and Networking

Networking Lunch

Technology Spotlight:
Studying the Diversity and “Signature” of Small RNA in Different Bodily Fluids using Next Generation Sequencing and the Impact on Biomarker Discovery
Bernard Lam, Senior Research Scientist, Norgen Biotek Corporation

In this presentation, we will discuss the impact of various steps in the Next Generation Sequencing workflow, including RNA extraction, enhancement of target signal by abundant transcript removal and library preparation, on obtaining valuable small RNA expression profile from a specific bodily fluid. The use of bodily fluids such as whole blood, plasma, serum, urine or saliva for biomarker discovery has attracted tremendous attention in recent years. However, the characteristics of RNA (and DNA) present in bodily fluids are quite different from traditional samples such as cells and tissues. First, many of the RNAs in bodily fluids are either free-circulating or are within extra-cellular vesicles such as exosomes. More importantly, the RNAs present in bodily fluids are usually of very low abundance and small in sizes. This presents challenges for detection and studies using many next-generation gene expression technologies. We will present data on the “signature” or small RNA characteristics of different bodily fluids. We will present examples, using such RNA “signature” of a bodily fluid, where removing an abundant RNA transcript can significantly improve the small RNA diversity detected by sequencing. Finally, we will discuss different RNA extraction technologies and their ability to recover microRNA with low GC contents from bodily fluids with low RNA content.

Long Non-Coding RNA (lncRNA) Genes as Causes of, and Post-Genomic Therapeutic Targets in, Human Disease
Leonard Lipovich, Associate Professor, Center for Molecular Medicine and Genetics, Wayne State University, United States of America

A summary of lncRNAs as an abundant, multifunctional class of human genes in post-genomic gene catalogs, and our work on specific lncRNAs as direct causal candidates in cancer and diabetes.

Regulating the Regulator: lncRNAs in the p53 Network
Ashish Lal, Investigator and Head, Regulatory RNAs and Cancer Section, Genetics Branch, National Cancer Institute (NCI), United States of America

Thousands of long noncoding RNAs (lncRNAs) have been discovered, yet the function of the vast majority remains to be investigated. We have identified and functionally characterized two nuclear-retained lncRNAs that we termed PINCR (p53 induced noncoding RNA) and PURPL (p53-upregulated regulator of p53 levels). Our results show that both PINCR and PURPL function as pro-survival lncRNAs in vitro and in vivo. Mechanistically, PINCR and PURPL mediate the effect of p53 in colorectal cancer cells by regulating p53 itself or by modulating the expression of a subset of p53 targets via distinct mechanisms. These studies reveal critical roles of PINCR and PURPL in the p53 network and uncover unique modes by which lncRNAs function.

Standardizing Extracellular Small RNASeq for Biomarker Discovery
Yaoyu Wang, Associate Director, Dana-Farber Cancer Institute, United States of America

The presence and relative stability of extracellular RNAs (exRNAs) in biofluids has led to an emerging recognition of their promise as ‘liquid biopsies’ for diseases. The recent application of next-generation sequencing to discovery of exRNA biomarkers has revealed the presence of potential novel miRNAs as well as other RNA species such as tRNAs, snoRNAs, piRNAs and lncRNAs in biofluids. At the same time, the use of RNA sequencing for biofluids poses unique challenges, including low amounts of input RNAs, the presence of exRNAs in different compartments with varying degrees of vulnerability to isolation techniques, and the high abundance of specific RNA species (thereby limiting the sensitivity of detection of less abundant species). Moreover, discovery in human diseases often relies on archival biospecimens of varying age and limiting amounts of samples. In this study, we have tested RNA isolation methods to optimize profiling exRNAs by RNA sequencing in individuals without any known diseases. Our findings are consistent with other recent studies that detect microRNAs and ribosomal RNAs as the major exRNA species in plasma. Similar to other recent studies, we found that the landscape of biofluid microRNA transcriptome is dominated by several abundant microRNAs that appear to comprise conserved extracellular miRNAs. This study highlights the challenges in detecting and quantifying less abundant plasma miRNAs in health and disease using RNA sequencing platforms.

Coffee Break and Networking

Physiologic Role of microRNAs in Skeletal Development and Homeostasis
Tatsuya Kobayashi, Assistant Professor, Massachusetts General Hospital, United States of America

Despite the plethora of knowledge on many microRNAs accumulated to date, at the organismal level, their physiologic roles have been demonstrated only in limited number of microRNAs.  As a skeletal biologist, I will discuss about the physiologic roles and cellular and molecular mechanisms of microRNA genes in skeletal development and homeostasis.

Modification, Multimerization, and New Directions in Therapeutic Oligonucleotides
Jonathan Watts, Associate Professor, RNA Therapeutics Institute, UMass Medical School, United States of America

Current design principles of therapeutic oligonucleotides can be categorized into three “dimensions,” based on mechanism (e.g., RNA interference, RNase H-mediated silencing, or splice switching), oligonucleotide chemistry (modification of phosphates, sugars, and nucleobases), and bioactive conjugates (i.e., ligands that target a tissue of interest or improve pharmacokinetic properties). We will present evidence that oligonucleotide multimerization may constitute a fourth dimension of oligonucleotide design that influences both potency and tissue distribution in vivo.  We will describe synthetic methods to access these multimeric structures as well as in vivo gene silencing data.

Networking Cocktail Reception with Beer, Wine and Appetizers. Enjoy the Boston Skyline, Engage with Colleagues and Discuss Collaborations and Partnerships

Close of Day 1 of the Conference

Professor Laurent Dinner Training Course on "Circulating RNAs for Liquid Biopsies" [separate registration required to attend this dinner training course]

Morning Coffee, Breakfast Pastries, and Networking

Therapeutic Targeting of MicroRNAs in Cardio-Metabolic Diseases
Anders Näär, Professor, Department of Cell Biology, Harvard Medical School and Massachusetts General Hospital Cancer Center, United States of America

A discussion regarding microRNAs as crucial regulators of metabolism and as therapeutic targets in cardiovascular disease, Type 2 diabetes, obesity, and non-alcoholic fatty liver diseases.

Using RNA Signatures For Therapeutic Development in Amyotrophic Lateral Sclerosis (ALS) and Fronto-Temporal Dementia
Clotilde Lagier-Tourenne, Assistant Professor of Neurology, Massachusetts General Hospital and Harvard Medical School, United States of America

Expanded GGGGCC repeats in a non-coding region of the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Several pathogenic mechanisms have been proposed including loss of function from reduced expression of C9ORF72 and/or toxicity derived from the expansion-containing RNAs. Accumulation of nuclear RNA foci and cytoplasmic dipeptide repeat proteins (DPRs) translated from the repeat-containing RNAs are pathological hallmarks of disease in patient cells and mice expressing C9ORF72 RNAs with up to 450 repeats. Indeed, hexanucleotide expansions caused age-, repeat length- and expression level-dependent accumulation of RNA foci and DPRs, accompanied by loss of hippocampal neurons, increased anxiety, and impaired cognitive function in transgenic mice. The presence of pathological hallmarks of C9ORF72 disease and behavioral abnormalities establishes this mouse model as an essential tool to develop novel therapeutic approaches, including RNA-targeting antisense oligonucleotides and immunotherapies for patients with ALS/FTD. Antisense oligonucleotides (ASOs) were identified which reduce GGGGCC-containing nuclear foci without altering overall C9ORF72 RNA levels in patient cells. By contrast, siRNAs failed to reduce nuclear RNA foci despite marked reduction in overall C9ORF72 RNAs. In mice, single dose intracerebroventricular injection of ASOs that target repeat-containing RNAs produced sustained reductions in RNA foci and dipeptide-repeat proteins, and ameliorated behavioral deficits. These findings establish ASO-mediated degradation of repeat-containing RNAs as a significant therapeutic approach for ALS/FTD linked to C9ORF72 expansions.

tRNA-Derived Fragments: From Biology to Technology and Therapeutics
Pavel Ivanov, Associate Member, Broad Institute of Harvard and MIT; Associate Immunologist at Brigham and Women’s Hospital, United States of America

tRNA-derived fragments are novel class of small non-coding RNAs with diverse biological functions. I will give an overview on the basic molecular mechanisms underlying their functions in cell physiology and proposed functions in various pathologies. I will discuss a translational potential of tRNA-derived fragments as disease biomarkers and therapeutic targets for cancer and neurodegenerative diseases.

FolamiRs: Ligand-Targeted, Vehicle-Free microRNA Replacement Therapy
Andrea Kasinski, William and Patty Miller, Assistant Professor of Biological Sciences, Purdue University, United States of America

MicroRNAs are small non-coding RNAs that negatively regulate gene expression at the posttranscriptional level. Because elevations or reductions in microRNA levels can promote or maintain disease states, microRNA-based therapeutics are being evaluated extensively. Unfortunately, the therapeutic potential of microRNA replacement is limited by deficient delivery vehicles. In this work, we introduce a novel delivery platform that delivers microRNAs in the absence of a protective vehicle. The method relies on the direct attachment of a microRNA to folate (FolamiR). Folate mediates the delivery of the conjugated microRNA into cells that overexpress the folate receptor (FR), a feature commonly found in multiple cancers.  Using this strategy, we show that a specific tumor suppressive FolamiR, FolamiR-34a, is quickly taken up by human triple-negative breast cancer cells, in culture and in vivo, and slows their progression, and demonstrate efficacy in an aggressive Kras;p53 non-small cell lung cancer mouse model. These findings suggest that this first-in-class method of delivering microRNAs directly to tumors in vivo, without the use of toxic delivery vehicles, represents an exciting advance in the pursuit to develop non-toxic, targeted therapeutics with promising clinical efficacy.

Coffee Break and Networking

MicroRNAs in Airway Smooth Muscle Function and Asthma
Quan Lu, Associate Professor, Harvard School of Public Health, United States of America

I will discuss the versatile regulatory roles of microRNAs in airway smooth muscle function and asthma.

RNA Cleavage at the Leading Edge of the Innate Immune Response
Alexei Korennykh, Associate Professor, Princeton University, United States of America

Why do cells activate RNA cleavage during the interferon (IFN) response? Whereas many intuitive explanations converge on a simple model of RNA degradation as a strategy to slow down and eliminate damaged/infected cells, recent data from others and from our group point to signaling mechanisms that reshape cellular programs. Using structural biology we are learning about regulation of this pathway, whereas a variety of RNA-Seq methods tell us about its specific coding and non-coding RNA targets, leading to the elusive cellular roles of the attack on cell’s RNA from within.

Networking Lunch

Decoding the Functions of Long Non-Coding RNAs in Innate Immunity
Maninjay Atianand, Research Fellow, University of Massachusetts Medical School, United States of America

A discussion regarding the emerging role of long non-coding RNAs in gene expression programs associated with innate immunity and inflammation will be provided.

Using CRISPR Screens to Find Drug Targets in Liver Cancer
Suet Yan Kwan, Research Fellow, University of Massachusetts Medical School, United States of America

Hepatocellular carcinoma is the third-leading cause of cancer-death worldwide. The current treatment for hepatocellular carcinoma is limited to surgery and sorafenib depending on the stage of the disease. However, the majority of patients are not eligible for surgery and the prognosis for patients receiving sorafenib treatment is poor. Therefore, additional drug targets are desperately needed for treatment of hepatocellular carcinoma. To identify additional drug targets in liver cancer, we performed a kinome CRISPR/sgRNA screen in three hepatocellular carcinoma cell lines and compared sgRNA representation between early and late passages. By finding sgRNAs that are depleted at late passages, we can identify potential kinases that are required for cell growth in hepatocellular carcinoma.

Role of microRNA Regulatory Networks for Leukotriene Modifier Response in Asthma
Amber Dahlin, Instructor of Medicine and Associate Epidemiologist, Channing Division of Network Medicine, Brigham and Women’s Hospital and Harvard Medical School, United States of America

Asthma is a major global public health burden, and severe asthma subtypes such as aspirin-exacerbated respiratory disease (AERD), represent a particular treatment challenge. AERD is characterized by excessive leukotriene (LT) production and end organ hyper-reactivity to cysteinyl LTs following aspirin ingestion. As a result, these patients may ideally benefit from leukotriene modifiers (LTMs) such as montelukast that block LT receptors to relieve symptoms. However, substantial inter-individual variability in montelukast response exists, which is partly due to genetic variation in LT regulatory pathways, resulting in unpredictable therapeutic outcomes. The activity of microRNAs also influences disease pathogenesis and treatment, as microRNAs have key roles in regulating gene expression. Interactions between genes and microRNAs are complex, since multiple microRNAs can regulate multiple genes and genes can be regulated by multiple miRNAs. These interactions can be explored computationally by integrating gene and microRNA expression data to construct networks of correlated microRNA-gene pairs (i.e. regulatory networks). Rather than analyzing microRNA-gene pairs separately, this approach enables analysis of multiple genes and microRNAs simultaneously. Using this approach to understand how AERD patients may respond to medication can promote development of personalized therapies, and obtaining insight into biological pathways will ultimately inspire development of more effective treatment regimens.

DIGIT is a Long Non-coding RNA that Regulates Endoderm Differentiation
Alan Mullen, Assistant Professor, Harvard Medical School and Massachusetts General Hospital, United States of America

DIGIT is a long noncoding RNA that is induced in response to activin signalling, which drives endoderm differentiation. DIGIT regulates endoderm differentiation of both human and murine embryonic stem cells and acts as a developmental regulator through control of the transcription factor Goosecoid.

Coffee Break and Networking

Defining Translation in “Noncoding” Regions Using Ribosome Profiling
Zhe Ji, Research Fellow, The Broad Institute and Harvard Medical School, United States of America

Characterizing protein coding regions across genomes is fundamentally important for understanding gene regulation and the regulation of RNA translation. Ribosome profiling reveals active translation genome-wide at single nucleotide resolution. We developed a computational pipeline named RibORF and revealed pervasive translation outside of canonical protein coding regions. Over 40% of annotated lncRNAs and pseudogene RNAs, and 35% of 5' UTRs in mRNAs are actively translated. Some peptides encoded by the non-canonical translation events are under stabilizing selection during evolution, and are likely to have biological functions. I will discuss their dynamic regulation and functional importance in cancers.

Visualizing Translation by Ensemble Cryo-EM
Andrei Korostelev, Associate Professor, RNA Therapeutics Institute, University of Massachusetts Medical School, United States of America

Translation of mRNA is a key step of gene expression regulation and a therapeutic target. Translation is governed by ribosome dynamics. To visualize the translational machinery in action, we determine high-resolution ensembles of structures by cryo-EM. I will discuss how this approach provides insights into the essential aspects of gene expression, such as translation initiation, translation fidelity and ribosome-induced stress signaling.

Nucleic Acid Scavenger Prevents Lung Injury in Rats Exposed to the Toxic Chemical 2-Chloroethyl Ethyl Sulfide
Aftab Ahmad, Associate Professor of Anesthesiology, Division of Molecular and Translational Biomedicine, University of Alabama at Birmingham, United States of America

Acute exposures to toxic chemicals can cause death and survivors often develop lung diseases later in life. The study explores the role of extracellular RNA in chemical-induced lung injury and provides evidence to support the role of nucleic acid scavengers as therapeutic targets for mitigating injury.

Close of Day 2 of the Conference