Monitoring RNA for Diagnosis and Prognosis: Visualization of RNA in Tissues and Monitoring RNA in Circulation

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Monitoring RNA for Diagnosis and Prognosis: Visualization of RNA in Tissues and Monitoring RNA in Circulation

Monday, April 23, 2012

The New York Academy of Sciences

Presented By

 

Monitoring changes in the abundance of protein-coding and non-coding RNAs in tissues is valuable for diagnosis and prognosis of various disease states. RNA in circulation has also received significant interest in biomarker studies. Though the detection and quantification of RNA molecules appears within reach for such applications, technical concerns related to the preservation of RNA in tissues or its abundance and fixation in tissue are just some of the issues that have interfered with the establishment of routine diagnostic and prognostic RNA assays in comparison to protein-detection-based approaches. This symposium will review the current state of RNA methodologies and how these will impact the future development of RNA diagnostics.

Reception to follow.

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Agenda

* Presentation times are subject to change.


Monday April 23, 2012

12:30 PM

Registration

1:00 PM

Welcome and Introduction
Jennifer Henry, PhD, The New York Academy of Sciences
Tom Tuschl, PhD, The Rockefeller University

1:10 PM

A Transgenic Mouse Model for Imaging Single RNA Dynamics in Tissue
Hye Yoon Park, PhD, Albert Einstein College of Medicine

1:45 PM

Development of Quantitative Multiplex RNA in situ Hybridization for Diagnostic Applications
Pavol Cekan, PhD, The Rockefeller University

2:20 PM

RNA Mimics of Green Fluorescent Protein
Samie R. Jaffrey, MD, PhD, Weill Medical College of Cornell University

2:55 PM

Coffee Break

3:30 PM

Deep Sequence Profiles of Circulating MicroRNA
Iddo Ben-Dov, MD, PhD, The Rockefeller University

4:05 PM

Massive Expansion of RNA Species in Tissues and in Circulation Revealed from RNA-Sequencing
Christopher E. Mason, PhD, Weill Medical College of Cornell University

4:40 PM

Quantitation of Tissue-Specific Target Gene Modulation using Circulating RNA
Alfica Sehgal, PhD, Alnylam Pharmaceuticals, Inc.

5:15 PM

Networking Reception

6:00 PM

Close

Speakers

Organizers

Tom Tuschl, PhD

The Rockefeller University

Dr. Tuschl received his PhD in chemistry from the University of Regensburg, in Germany, in 1995. He went to the Max Planck Institute for Experimental Medicine in Göttingen, Germany, pursuing research with Fritz Eckstein. He next joined the biology department at the Massachusetts Institute of Technology and the Whitehead Institute for Biomedical Research, where he worked with Phillip A. Sharp and David P. Bartel. Dr. Tuschl was a junior investigator at the Max Planck Institute for Biophysical Chemistry before coming to Rockefeller in 2003 as associate professor. He was named professor in 2009. Dr. Tuschl's most recent honors include the Ernst Jung Prize for Medicine in 2008 and the Max Delbrück Medal and the Karl Heinz Beckurtz Award in 2007. In 2006 he received the Molecular Bioanalytics Prize from Roche Diagnostics. In 2005 he was named a fellow of the New York Academy of Sciences and received the Meyenburg Prize, the Irma T. Hirschl Trust Career Scientist Award and the Ernst Schering Award. In 2003 he received the Wiley Prize in Biomedical Sciences, the New York City Mayor's Award for Excellence in Science and Technology and the Newcomb Cleveland Prize from the American Association for the Advancement of Science. Dr. Tuschl was the recipient of the European Molecular Biology Organization Young Investigator Award in 2001 and the Biofuture Award from the German government in 1999. He is also a Howard Hughes Medical Institute investigator.

Jennifer Henry, PhD

The New York Academy of Sciences

Speakers

Pavol Cekan, PhD

The Rockefeller University

Pavol Cekan is a post-doctoral researcher in Thomas Tuschl’s laboratory at the Rockefeller University. Pavol received his B.Sc. and Ph.D. from the University of Iceland. As a PhD candidate, Pavol trained as nucleic acid chemist/biochemist and structural biologist with a focus on modified oligonucleotide synthesis and spectroscopic analysis of nucleic acid structure and function. After joining the Tuschl lab in 2009, he has focused on developing novel processes for improving the retention of nucleic acids in tissue sections during RNA in situ hybridization as well as preparation of specific oligonucleotide probes and fluorescent signal amplification processes. Currently, he is establishing conditions for diagnostic microRNA/mRNA in situ hybridization.

Iddo Ben-Dov, MD, PhD

The Rockefeller University

Iddo Ben-Dov is a post-doctoral researcher in Thomas Tuschl's laboratory at the Rockefeller University. Iddo received his M.Sc., M.D. and Ph.D. from the Hebrew University in Jerusalem. For his Masters degree, he studied piezoelectric microbalance immunosensing of C. trachomatis in clinical specimens with Prof. Itamar Willner. His medical training in internal medicine and nephrology was combined with and followed by molecular biology studies of the parathyroid hormone mRNA in the laboratory of Profs. Tally Naveh and Justin Silver. Since 2009 he is an instructor in clinical investigation at the Rockefeller University's Clinical Scholars program and the Tuschl lab, where he conducts patient-oriented RNA research.

Samie R. Jaffrey, MD, PhD

Weill Medical College of Cornell University

Dr. Samie Jaffrey is an Associate Professor of Pharmacology at the Weill Medical College of Cornell University. He received and M.D. and Ph.D. from Johns Hopkins School of Medicine. Dr. Jaffrey's laboratory focuses on RNA regulatory mechanisms in neurons and how RNA processing pathways control neural circuit formation. A major focus is to identify RNA regulatory pathways that occur within growing axons, and to understand how defects in these pathways lead to neurodevelopmental disorders such as mental retardation and autism. His research uses novel viral, proteomic, microfluidic, and chemical biology approaches to address these questions. His group identified the first mRNAs that are locally translated in axons and are required for axon guidance. His laboratory has also developed a novel class of RNAs referred to RNA mimics of green fluorescent protein, which are used to image RNA localization and monitor RNA processing in living cells. The Jaffrey laboratory extended this technology to create a new type of genetically encoded biosensor composed of RNA that allows signaling molecules to be imaged in living cells. Dr. Jaffrey is a Klingenstein Neuroscience Fellow, Irma T. Hirschl Scholar, and recipient of the McKnight Technology development award and the NIH Director's Transformative R01 award.

Christopher E. Mason, PhD

Weill Medical College of Cornell University

Christopher E. Mason is an Assistant Professor of Computational Genomics at Weill Cornell Medical College of Cornell University in New York City, in the Department of Physiology and Biophysics and at the Institute for Computational Biomedicine. He also holds an appointment in the Tri-Institutional Program on Computational Biology and Medicine (Cornell, Memorial Sloan-Kettering Cancer Center and Rockefeller University) and the Weill Cornell Cancer Center. Professor Mason is also an affiliate fellow of Genomics, Ethics, and Law at Yale Law School, where he teaches classes and lectures on the implications of new genomics technology on the law. He has also serves as scientific adviser to the American Civil Liberties Union, faculty adviser the GenSpace community laboratory in Brooklyn, and is also the co-founder of the New York Synthetic Biology Association. Dr. Mason received his dual B.S. in Genetics and Biochemistry from University of Wisconsin-Madison in 2001, his Ph.D. in Genetics from Yale University in 2006, and spent three years as post-doc working at Yale University, focusing on mutations in genes implicated in patients with brain malformations and psychiatric disorders. Dr. Mason's current research focuses on using functional genomics and integrative molecular approaches to understand the genetic architecture of the human brain, and how the genome's elements change in human diseases such as neural tube defects and cancer.

Hye Yoon Park, PhD

Albert Einstein College of Medicine

Dr. Hye Yoon Park is a research fellow in Prof. Robert H. Singer's laboratory at Albert Einstein College of Medicine. Dr. Park received her B.S. in Physics from Seoul National University and her M.S. and Ph.D. in Applied Physics from Cornell University. For her M.S. degree, she performed research on the fabrication processes for polymeric microfluidic devices in Prof. Harold G. Craighead's group. During her Ph.D. research under the guidance of Prof. Lois Pollack and Prof. Watt W. Webb, she developed a laminar flow mixer for kinetic studies of protein and RNA folding on a microsecond time scale. In 2008, she joined the Singer lab as an NIH Ruth L. Kirschstein National Research Service Award (NRSA) Postdoctoral fellow, and has been working on single molecule imaging of RNA in live cells and tissues.

Alfica Sehgal, PhD

Alnylam

Alfica Sehgal is a Principal Scientist at Alnylam Pharmaceuticals. She has worked on different delivery projects since 2008 and is currently working on siRNA mediated therapeutics for fibrosis and hemophilia. She studied regulation of sterol and oxygen homeostasis during her post-doc at Johns Hopkins and Yale Univ. Her PhD was from Dept of Molecular Biology, TIFR, Mumbai and Masters in molecular biology and BS in microbiology from Delhi University.

Abstracts

Development of quantitative multiplex RNA in situ hybridization for diagnostic applications
Pavol Cekan, PhD, The Rockefeller University

We recently developed a multicolor miRNA-FISH method with the following innovations: (1) a novel carbodiimide-containing fixative for crosslinking the miRNA 5'-phosphate end to surrounding protein, (2) elimination of tissue permeabilization and enhanced antibody-based fluorescence signal amplification through the use of long linkers between the hapten and probe (3) prevention of RNA mishybridization through the use of short LNA-modified DNA (LNA/DNA) probes, and (4) directly-labeled fluorescent rRNA and poly(A) probes for assessing RNA retention and normalizing miRNA signals. We validated our method using two histologically similar skin tumors, namely basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC), which respectively express miR-205 and miR-375. After designing relevant probes, amplified miRNA signals were normalized against directly-labeled rRNA signals to correctly identify 16 BCC and MCC tumors. We are also optimizing mRNA-FISH, especially RNA retention and hybridization steps. We eliminated conventional signal amplification using multiple, short fluorescently-labeled LNA/DNA probes targeting different mRNA segments. We selected probe sequences to avoid RNA mishybridization and the length of each member of the probe pool was adjusted according to melting temperature. This approach requires 40-60 short probes per target mRNA, each carrying multiple fluorophores. mRNA-FISH signals are normalized using probes targeting poly(A), nuclear-encoded rRNA and mitochondrial rRNA; these probe sets also monitor RNA retention, integrity and enable subcellular localization. To date, we have successfully developed specific probes for ERBB2 in a breast cancer diagnostic system, where HER2+ (HCC1954), HER2- (MDA-MB231) paraffin-embedded tumor cell line pellets along with HER2+/HER2- mixed pellet were mounted on the same slide, processed and scanned in parallel.
 

Deep Sequence Profiles of Circulating MicroRNA
Iddo Ben-Dov, MD, PhD, The Rockefeller University

We profiled miRNA in cell-free serum and plasma samples from human volunteers using barcoded deep sequencing of small RNA cDNA libraries. By introducing calibrator synthetic oligonucleotides during library preparation we were able to calculate the total as well as specific concentrations of circulating miRNA. Studying trios of samples from newborn babies and their parents we were able to show placental-specific miRNA in maternal and newborn circulation. Furthermore, sequence variation in the miRNA profiles could be traced to the specific placenta of origin. These deep sequencing profiles, which may serve as a model for tumor or disease detection, along with additional studies of patients with kidney or heart disease, allow us to define the repertoire of miRNA abundance in the circulation and to discuss potential biomarker opportunities.
 

RNA Mimics of Green Fluorescent Protein
Samie R. Jaffrey, MD, PhD, Weill Medical College of Cornell University

Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various in vitro and in vivo applications, but a comparably robust and simple approach for fluorescently tagging RNA is lacking. We now describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNAs activate the fluorescence of these fluorophores, resulting in a palette of RNA-fluorophore complexes that span the visible spectrum. An RNA-fluorophore complex resembling enhanced GFP (EGFP), termed Spinach, emits a green fluorescence comparable in brightness to fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach to genetically encode fluorescent RNAs.
 

Massive Expansion of RNA Species in Tissues and in Circulation Revealed from RNA-Sequencing
Christopher E. Mason, PhD, Weill Medical College of Cornell University

Here we present some surprising findings from the ongoing work of three large-scale efforts in RNA-Seq and transcriptomics. To perfect the detection of expression-based molecular signatures using RNA-Seq and define the site-to-site variance in measurement, the FDA’s Sequencing Quality Control (SEQC) Consortium has performed sequencing on three platforms (Illumina, LifeTech, and 454) using the SEQC standardized samples with synthetic RNA spike-ins. The ABRF’s SEQC has also performed experiments on these standardized samples, but with additional testing across more platforms (IonTorrent, PacBio) of variables such as sample quality, input amount, and chemistry. Finally, the Non-Human Primate Reference Transcritpome (NHPRT) Resource is performing deep sequencing of 21 tissues across 15 primates to improve gene models, accelerate genome builds, and enable novel evolutionary comparisons. To date, we have also discovered tens of thousands of tissue-specific, species-specific and cross-species transcriptionally active regions (TARs) beyond existing gene annotations, which highlight the utility of using unbiased methods for transcriptome characterization.
 

A Transgenic Mouse Model for Imaging Single RNA Dynamics in Tissue
Hye Yoon Park, PhD, Albert Einstein College of Medicine

We have developed a novel mouse model for studying the dynamics of mRNA in live cells and tissues. The MS2-GFP labeling technique has been widely used to visualize single RNA in living cells. We extend the utility of the technique for single molecule imaging in the context of a whole-animal system. The MS2-GFP technique exploits the high affinity binding of the MS2 bacteriophage capsid protein (MCP) to the MS2 RNA binding site (MBS). MCP is fused with green fluorescent protein (GFP), and 24 repeats of MBS are inserted into the RNA of interest. In order to apply the technique for live tissue imaging, we genetically engineered two mouse models: MCP-GFP transgenic mouse, Tg(MCP-GFP), and Actb-MBS mouse. In the Actb-MBS mouse, MBS cassette is knocked into the 3' untranslated region (3' UTR) of the essential β-actin gene (Lionnet et al., Nature Methods, 8, 165, 2011). By crossing Tg(MCP-GFP) mouse and Actb-MBS mouse, we generated a hybrid mouse expressing fluorescent RNA in every cell and tissue where β-actin is present. The homozygous mouse is viable and exhibits no gross abnormalities despite the addition of 1.2 kbp MBS cassette and approximately 1.2 MDa of MCP-GFP to the essential β-actin mRNA. These results suggest that this technology could be applied to a wide range of genes with minimal perturbation. Using this mouse, we are investigating the dynamics of β-actin mRNA transport in acute brain slices using multiphoton microscopy. In addition, single-molecule tracking of mRNA is employed to understand the mechanism of mRNA localization in hippocampal neuron cultures. The mouse model combined with high-resolution imaging can provide important insights into the dynamic regulation of an endogenous gene in its native tissue environment.
 

Quantitation of tissue-specific target gene modulation using circulating RNA
Alfica Sehgal, Alnylam Pharmaceuticals, Inc., Cambridge, MA, USA

Pharmacologic target gene modulation is the primary objective for RNA antagonist strategies, including small interfering RNA (siRNA), microRNA (miRNA) therapeutics, and antisense oligonucleotide therapeutics. In clinical applications, monitoring tissue-specific target mRNA modulation requires tissue procurement from patients that is highly limited in availability, if even justified. Here we show that circulating RNAs encoding tissue-specific gene transcripts can be detected in biological fluids of humans and experimental animals. Surprisingly, we demonstrate that RNAi-mediated target gene silencing in the liver by systemic administration of siRNA results in quantitative reductions in serum mRNA levels which closely corroborate the degree and kinetics of tissue mRNA silencing, including proof of the RNAi mechanism of action. Further, administration of an anti-miRNA oligonucleotide directed against a liver-specific miRNA was found to result in decreased levels of the miRNA in circulation. This technique was extended to a different tissue, where silencing of a brain-expressed mRNA was monitored and quantified in cerebrospinal fluid following intraparenchymal infusion of a specific siRNA. This non-invasive method for monitoring tissue-specific RNA modulation could greatly advance clinical development of gene therapy and RNA-based therapeutics.
 
*Additional abstracts to come.

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