RNA Interference Technology FROM BASIC SCIENCE TO DRUG DEVELOPMENT ppt

Since RNAi also can be used to regulate gene expression in specific cell types, the possibility that RNAi can be used therapeutically to treat diseases or certain viral infections by targ

RNA INTERFERENCE TECHNOLOGY

RNA Interference (RNAi) technology has rapidly become one of the keymethods used in functional genomics RNAi is used to block the expression

of genes and create phenotypes that can potentially yield clues about thefunction of these genes In the postgenomic era, the elucidation of the phys-iological function of genes has become the rate-limiting step in the quest

to develop “gene-based drugs” and RNAi could potentially play a pivotalrole in the validation of such novel drugs In this cutting-edge overview,the basic concepts of RNAi biology are discussed, as well as the current andpotential applications Leading experts from both academia and industryhave contributed to this invaluable reference for graduate students, post-docs, and researchers from academia wanting to initiate RNAi research intheir own labs, as well as for those working in research and development

in biotech and pharmaceutical companies who need to understand thisemerging technology

Krishnarao Appasani is the Founder and Chief Executive Officer of Expression Systems, a gene discovery company focusing on functional ge-nomics in cancer research

Gene-RNA Interference Technology

FROM BASIC SCIENCE TO DRUG DEVELOPMENT

National Institutes of Health

Winner of the Nobel Prize in Physiology or Medicine, 1968

Cambridge University Press

The Edinburgh Building, Cambridge cb2 2ru, UK

First published in print format

isbn-13 978-0-521-83677-7

isbn-13 978-0-511-08221-4

© Cambridge University Press 2005

Information on this title: www.cambridge.org/9780521836777

This book is in copyright Subject to statutory exception and to the provision of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press.

isbn-10 0-511-08221-5

isbn-10 0-521-83677-8

Cambridge University Press has no responsibility for the persistence or accuracy of urls for external or third-party internet websites referred to in this book, and does not guarantee that any content on such websites is, or will remain, accurate or appropriate.

Published in the United States of America by Cambridge University Press, New York

www.cambridge.org

hardback

eBook (NetLibrary) eBook (NetLibrary) hardback

In memory of my parents

For my teachers, family members

and especially my wife Shyamala and sons Raakish and Raghu

2 Dicer in RNAi: Its roles in vivo and utility in vitro 29

Jason W Myers and James E Ferrell, Jr.

Nathaniel R Dudley, Ahmad Z Amin, and Bob Goldstein

Amy E Pasquinelli

5 miRNAs in the brain and the application of RNAi

Anna M Krichevsky, Shih-Chu Kao, Li-Huei Tsai, and Kenneth S Kosik

Section two Design, synthesis of siRNAs

6 Design and synthesis of small interfering RNA (siRNA) 103

Queta Boese, William S Marshall, and Anastasia Khvorova

7 Automated design and high throughput chemical synthesis

Yerramilli V B K Subrahmanyam and Eric Lader

8 Rational design of siRNAs with the Sfold software 129

Ye Ding and Charles E Lawrence

9 Enzymatic production of small interfering RNAs 139

Muhammad Sohail and Graeme Doran

vii

Section three Vector development and in vivo, in vitro and in ovo

delivery methods

10 Six methods of inducing RNAi in mammalian cells 147

Kathy Latham, Vince Pallotta, Lance Ford, Mike Byrom, Mehdi Banan,

Po-Tsan Ku, and David Brown

Ying Mao, Chris Mello, Laurence Lamarcq, Brad Scherer, Thomas Quinn,

Patty Wong, and Andrew Farmer

12 siRNA delivery by lentiviral vectors: Design and applications 174

Oded Singer, Gustavo Tiscornia, and Inder Verma

Mouldy Sioud and Dag R Sørensen

14 Chemical modifications to achieve increased stability and sensitive

Philipp Hadwiger and Hans-Peter Vornlocher

15 RNA interference in postimplantation mouse embryos 207

Frank Buchholz, Federico Calegari, Ralf Kittler, and Wieland B Huttner

16 In ovo RNAi opens new possibilities for functional genomics in

Dimitris Bourikas, Thomas Baeriswyl, Rejina Sadhu, and Esther T Stoeckli

Section four Gene silencing in model organisms

17 Practical applications of RNAi in C elegans 235

Karen E Stephens, Olivier Zugasti, Nigel J O’Neil, and Patricia E Kuwabara

18 Inducible RNAi as a forward genetic tool in Trypanosoma brucei 247

Mark E Drew, Shawn A Motyka, James C Morris, Zefeng Wang,

and Paul T Englund

19 RNA-mediated gene silencing in fission yeast 257

Section five Drug target validation

22 Delivering siRNA in vivo for functional genomics and novel

Patrick Y Lu and Martin C Woodle

23 The role of RNA interference in drug target validation: Application

Antje Ostareck-Lederer, Sandra Clauder-M¨ unster, Rolf Thermann, Maria

Polycarpou-Schwarz, Marc Gentzel, Matthias Wilm, and Joe D Lewis

Steven A Haney, Peter Lapan, Jeff Aalfs, Chris Childs, Paul Yaworsky, and

Chris Miller

Contents ix

25 RNA interference technology in the discovery and validation of

Neil J Clarke, John E Bisi, Caretha L Creasy, Michael K Dush, Kris J.

Fisher, John M Johnson III, Christopher J A Ring, and Mark R Edbrooke

Section six Therapeutic and drug development

26 RNAi-mediated silencing of viral gene expression and replication 363

Derek M Dykxhoorn

27 RNAi in drug development: Practical considerations 384

Dmitry Samarsky, Margaret Taylor, Mark A Kay, and Anton P McCaffrey

Lars Zender, Michael P Manns, and Stefan Kubicka

29 RNAi applications in living animal systems 406

Lisa Scherer and John J Rossi

Section seven High-throughput genome-wide RNAi analysis

30 High-throughput RNAi by soaking in Caenorhabtis elegans 419

Asako Sugimoto

31 Tools for integrative genomics: Genome-wide RNAi and expression

Michael Boutros and Marc Hild

32 Microarray analysis and RNA silencing to determine genes

Maria E Ramos-Nino and Brooke T Mossman

Howard Y Chang, Nancy N Wang, and Jen-Tsan Chi

34 Generation of highly specific vector-based shRNAi libraries

Makoto Miyagishi, Sahohime Matsumoto, Takashi Futami, Hideo Akashi,

Krishnarao Appasani, Yasuomi Takagi, Shizuyo Sutou, Takashi Kadowaki,

Ryozo Nagai, and Kazunari Taira

Andrew Fire

It has been a privilege to watch the growth of RNA interference technology over

the last ten years Starting with a mixture of curiosity and chagrin, the field has

grown into a substantial enterprise which impacts (and utilizes resources from)

virtually every field of biomedical research Research in RNAi derives from a set of

apparently unconnected observations: strange pigment patterns in plants,

unex-pected failures and successes in antisense and overexpression studies, small

regu-latory RNAs in bacteria If there is an underlying and recurring scientific lesson,

it has been: “Pursue the unexpected.” Basic and applied research each advance as

a consequence of this pursuit; certainly this has been no better illustrated than

in the last ten years of RNAi

The work of hundreds of researchers in different fields that is reported in this

book should provide the reader with both solid information (needed for

experi-mental design and evaluation) and a lively and hopeful scientific story (needed to

keep us all going through the long haul of scientific research) Our knowledge of

the realm of genetic regulation by small RNAs has grown with remarkable speed

Starting in 1981 with a single known example of a modulatory short RNA

(regu-lating copy number of the ColE1 plasmid), small RNAs are now known to regulate

genetic activity at virtually every level: DNA and chromosome structure,

transcrip-tion, RNA structure and stability, translatranscrip-tion, and protein stability Likewise, our

ability to experimentally alter cells using this system has advanced at an

unprece-dented rate As recently as 1990, the known examples of experimentally-induced

silencing were a few unusual and accidental plant pigmentation patterns; now

there are extensive menus of silencing-based methods as part of the “standard”

molecular biology toolkit

Work in this field is by no means finished We still don’t understand all of

the modalities of RNA-triggered genetic regulation, why these modalities exist,

and how they interact with each other We don’t have a clear picture the full

extent of RNA-based regulation As these questions are further investigated and

understood, and as the underlying mechanisms are understood in detail, it will

become possible to carry out more and more sophisticated experimental

manipu-lations of genetic function More questions: How do some organisms encapsulate

xi

RNA triggers to produce a systemic response? How are long term RNAi effectsperpetuated? What is the link between RNAi and immunity? What biological ef-fects will come from the selective or global inactivation or augmentation of theRNAi pathway? How can we best use RNAi to discover the most sensitive andcritical targets for biological investigation and drug development? Can we curediseases by specifically triggering the RNAi pathway to attack errant genes? Can

we treat other diseases by up- or down-regulating components of the RNAi chinery itself in specific cell types? How will cells and organisms respond in thelong term to continuous modulation or use of the RNAi machinery?

ma-We’ll all be busy for quite a while in addressing these questions Based on thefirst years of the field, one thing that can certainly be expected is a few moresurprises

Stanford, California, USAAugust 2, 2004

Marshall Nirenberg

RNA interference is a powerful tool that has been used to inhibit gene

func-tion either by increasing the destrucfunc-tion of mRNA corresponding to the gene,

or in some cases, by inhibiting the transcription of the gene or the translation

of mRNA to the corresponding protein Exploring gene function by the classical

approach of generating mutants of a gene often is much more laborious and time

consuming then silencing gene function by RNAi using double-stranded RNA or

double-stranded oligoribonucleotides about twenty two nucleotide residues in

length This book edited by Krishnarao Appasani is a timely and comprehensive

compendium of information on RNAi and will be useful to experts on RNAi as

well as investigators in many fields of research who may be interested in using

RNAi to explore problems they are studying

The RNAi field is only six years old Research on RNAi has been expanding

at an extraordinarily rapid rate, yet the field is in its infancy There is great

in-terest in using RNAi as a means of exploring gene function during embryonic

development and in the adult in many organisms Many aspects of RNAi remain

to be explored For example, the reactions and the molecules required for RNAi

targeted destruction of mRNA are incompletely known Similarly, the

mecha-nisms of RNAi targeted modification of DNA, which regulates, transcription of

DNA, as well as RNA targeted inhibition of mRNA translation are only partially

known Also, the functions of most micro RNA genes have not yet been explored

Since RNAi also can be used to regulate gene expression in specific cell types,

the possibility that RNAi can be used therapeutically to treat diseases or certain

viral infections by targeted gene silencing is an exciting, challenging possibility

However, difficult problems have to be overcome such as the problem of

deliv-ery of appropriate double-stranded oligoribonucleotides into cells, the stability,

concentration, and toxicity of the oligoribonucleotides, and the length of time

the oligoribonucleotides remain in the cells These are challenging research

prob-lems Nevertheless, the use of oligoribonucleotides as therapeutic agents to silence

gene expression has great potential for the future Libraries of small interfering

RNAs (siRNAs) or short hairpin RNAs (shRNA) have been constructed and have

been screened in cultured cells In addition, methods have been devised for high

xiii

throughput screening of siRNA or shRNA libraries RNAi has been used to inhibitreplication of viruses in cultured cells such as HIV, hepatitis C virus, and hepatitis

B virus The oncogenic fusion protein p210 in chronic myelogenous leukemiacells promotes cell division in these cells Both siRNA and a lentivirus vector con-taining shRNA have been shown to reduce the levels of p210 protein in cell linesand thereby inhibit cell division In addition, RNAi has been used in intact mice

to reduce the function of a mutant gene which results in the movement disorder,spinocerebellar ataxia type one Treatment of mice by RNAi resulted in improvedmotor coordination and the cellular changes in the brain characteristic of thedisease were no longer visible RNAi also is being investigated as a therapy forocular diseases

It is too early to say how successful RNAi therapy will be However, it is clear thatRNAi is a powerful tool that has revolutionized basic research and that the ability

of RNAi to down-regulate almost any gene affords remarkable opportunities toexplore the use of duplex oligoribonucleotides as therapeutic agents for manydiseases

Laboratory of Biochemical GeneticsNational Heart, Lung, and Blood InstituteNational Institutes of Health

Bethesda, MD

Krishnarao Appasani, PhD., MBA

GeneExpression Systems, Inc

Johnson & Johnson Research

Level 4, 1 Central Avenue

Eveleigh, NSW 1430

Sydney, Australia

E-mail: garndt@medau.jnj.com

xv

Department of Molecular Oncology

Serono Reproductive Biology InstituteOne Technology Place

Contributors xvii

Frank Buchholz

Max Plank Institute of Molecular Cell Biology and Genetics

Pfotenhauer Strasse 108 Dresden

Max Plank Institute of Molecular Cell Biology and Genetics

Pfotenhauer Strasse 108 Dresden

Germany

E-mail: Calegari@mpi-cbg.de

Howard Y Chang

Departments of Biochemistry and Dermatology

Stanford University School of Medicine

Departments of Biochemistry and Dermatology

Stanford University School of Medicine

Sandra Clauder-M¨ unster

Anadys Pharmaceuticals Europe GmbH

Dept of Mol Microbiology, Rm 9210

Washington University School of Medicine

Box 8230, 4940 Parkview Place

St Louis, MO 63110

USA

E-mail: drew@borcim.wustl.edu

The Center for Blood Research

Harvard Medical School

Department of Biological Chemistry

Johns Hopkins Medical School

Department of Molecular Pharmacology

Stanford University School of Medicine

269 Campus Drive, CCSR Rm 3160

Stanford, CA 94305-5174

USA

Andrew Fire, PhD.

Departments of Pathology and Genetics

Stanford University School of Medicine

300 Pasteur Drive, Room L235

Center for Cancer Research

Massachusetts Institute of Technology

Max Plank Institute of Molecular Cell Biology and Genetics

Pfotenhauer Strasse 108 Dresden

Takashi Kadowaki

Department of Internal Medicine

Graduate School of Medicine

The University of Tokyo

Harvard Medical School

4Blackfan Circle, HIM 760

Boston, MA 02115

USA

Mark A Kay, MD., PhD.

Stanford University School of Medicine

Departments of Pediatrics and Genetics

Program in Human Gene Therapy

Harvard Medical School

4Blackfan Circle, HIM 760

Boston, MA 02115

USA

Contributors xxiii

Anna M Krichevsky, PhD.

Department of Neurology and Center for Neurologic Diseases

Brigham and Women’s Hospital

Harvard Medical School

4Blackfan Circle, HIM 760

The School of Medical Sciences

University Walk, Bristol BS8 1TD

Gene Function Research Center

National Institute of Advanced Industrial Science and Technology (AIST)

Central 4, 1-1-1 Higashi

Tsukuba Science City 305-8562

Japan

and

Department of Internal Medicine

Graduate School of Medicine

The University of Tokyo

Hongo, Tokyo 113-8655

Japan

Anton P McCaffrey, PhD.

Stanford University School of Medicine

Departments of Pediatrics and Genetics

Program in Human Gene Therapy

Gene Function Research Center

National Institute of Advanced Industrial Science and Technology (AIST)Central 4, 1-1-1 Higashi

Tsukuba Science City 305-8562

Environmental Pathology Program, Department of Pathology

University of Vermont, College of Medicine

Department of Biological Chemistry

Johns Hopkins Medical School

Department of Molecular Pharmacology

Stanford University School of Medicine

269 Campus Drive, CCSR Rm 3160

Stanford, CA 94305-5174

USA

E-mail: jmyers@stanford.edu

Contributors xxvii

Ryozo Nagai

Department of Internal Medicine

Graduate School of Medicine

The University of Tokyo

Hongo, Tokyo 113-8655

Japan

Nigel J O’Neil

The Wellcome Trust Sanger Institute

Hinxton, Cambridge CB10 1SA

Contributors xxix

Lisa Scherer, PhD.

Division of Molecular Biology, Graduate School of Biological Sciences

Beckman Research Institute of the City of Hope

City of Hope, Duarte, CA 91010

USA

Oded Singer, PhD.

Laboratory of Genetics

The Salk Institute

10010 North Torrey Pines Road

La Jolla, CA 92037

USA

E-mail: singer@salk.edu

Mouldy Sioud, DEA Pharm, PhD.

Department of Immunology, Molecular Medicine Group

The Norwegian Radium Hospital

Department of Immunology, Molecular Medicine Group

The Norwegian Radium Hospital

Montebello, 0310

Norway

Karen E Stephens

The Wellcome Trust Sanger Institute

Hinxton, Cambridge CB10 1SA

Laboratory for Developmental Genomics

RIKEN Center for Developmental Biology

Department of Chemistry and Biotechnology

School of Engineering, The University of Tokyo

Hongo, Tokyo 113-8656

Japan

E-mail: taira@chembio.t.u-tokyo.ac.jp

and

Gene Function Research Center

National Institute of Advanced Industrial Science and Technology (AIST)Central 4, 1-1-1 Higashi

Tsukuba Science City 305-8562

The Salk Institute

10010 North Torrey Pines Road

La Jolla, CA 92037

USA

E-mail: coyne@salk.edu

Li-Huei Tsai

Department of Neurology and Center for Neurologic Diseases

Brigham and Women’s Hospital

Harvard Medical School

4Blackfan Circle, HIM 760

Boston, MA 02115

USA

The Salk Institute

10010 North Torrey Pines Road

Departments of Biochemistry and Dermatology

Stanford University School of Medicine

Stanford, CA 94305

USA

Zefeng Wang

Dept of Mol Microbiology, Rm 9210

Washington University School of Medicine

Box 8230, 4940 Parkview Place

St Louis, MO 63110

USA

and

Department of Biological Chemistry

Johns Hopkins Medical School

Contributors xxxiii

Patty Wong

BD Biosciences Clontech

1020 East Meadow Circle

Palo Alto, CA 94303 USA

The Wellcome Trust Sanger Institute

Hinxton, Cambridge CB10, 1SA

UK

E-mail: omz@sanger.ac.uk

Krishnarao Appasani

“If the RNAi technology can be made to work, there’s a long list of diseases it can be

applied to.”

–Phillip A Sharp, Nobel laureate, MIT From CBSNews.com July 18, 2003

Gene Expression (genomics) is a new discipline within molecular biology that

narrates the functional organization of genes Most of you are aware of the terms

Genome (study of the expression of all the genes in an organism known as

Ge-nomics), Proteome (study of the expression of all the proteins in an organism

known as Proteomics), and Glycome (study of the expression of all the

glycopro-teins in an organism known as Glycomics) Another scientific buzz word that is

spreading fast in the research community these days is RNome, the RNA

equiv-alent of the ‘proteome,’ ‘genome’ or ‘glycome,’ with the subject referred to as

RNomics RNomics is a newly emerging sub-discipline that categorically studies

the structure, function and processes of noncoding RNAs and the mechanism of

RNA interference in a cell

RNA Interference Technology: From Basic Science to Drug Development is primarily

intended for readers in the molecular cell biology and genomics fields but may

be useful in more advanced graduate level courses Much of the text should be

of interest to those in applied sciences such as molecular medicine, genome

sci-ence, and biotechnology This book, which focuses on the concepts of basic RNAi

biology and applications in drug development, consists of thirty four chapters,

grouped into seven sections Most of the chapters are written by the original

discoverers or their associated scientists from academia, biotech and pharma,

ex-clusively from the RNAi field This is the first book of its kind that integrates

the academic science with industry applications in drug validation and

thera-peutic development This book will serve as a reference for graduate students,

post-docs, and professors from academic research institutions who wish to

initi-ate RNAi and siRNA research in their own laboratories This book will also serve

as a descriptive and in-depth analysis for executives, directors and scientists in

1

research and development from biotech and pharmaceutical companies This willprovide a valuable executive summary for investors and those responsible for busi-ness development in the life sciences, who need to keep abreast with the RNAirevolution.

In the post-genomic era, elucidation of the physiological function of geneshas become a major rate-limiting step in the quest to develop gene-based-

drugs As we advance in the ‘functional omics’ arena, with the hope of

discov-ering novel drug targets and therapies, their validation is a pivotal step beforetheir use in clinical practice Such an endeavor can be tested using small interfer-ence RNAs (siRNAs) or RNA-mediated genetic interference (RNAi) This elegantand revolutionary reverse genetic approach has tremendous commercial promisewith regard to developing new drugs and therapeutics for human diseases Thisbook provides an in-depth summary of the field of RNAi by international expertsand predicts some of the potential applications of gene suppression strategies inthe pre-clinical drug discovery process within the biotech and pharmaceuticalindustries

We are now beginning to exploit the information gleaned from the genomesequencing projects of humans and other organisms However, this wealth ofgenetic information opens new challenges in deciphering the complete list ofprotein-encoding genes In addition, transcriptional events such as RNA splicingand post-translational modifications make it difficult to predict the exact num-ber of genes or proteins With this degree of complexity, monitoring the entireproteome expression levels as a means of elucidating the functions of genes andproteins and developing them as potential drug targets are significant challengesfor the biotech industry Despite the “proteome” sequencing efforts, the “RNome”also has to be studied in depth to fully understand and tally the number of genesencoded by a genome and their regulation The challenge for scientists in bothacademia and industry is to identify the whole complement of non-coding RNAsand elucidate their functions in gene expression and regulation From a health-care point of view, it is important to identify the disease relevant genes from thesefunctional “OMES.”

Six years ago, Mello and his colleagues discovered the phenomenon of RNAi.Since then, this technique has become widely used within the cell and molecu-lar biology communities as a tool to aid understanding of gene expression and

regulation In addition, scientists have begun to take an RNomics approach to

un-derstanding the nature and function of microRNAs and siRNAs in order to utilize

them as a mechanism of gene silencing In broad terms,“post-transcriptional gene silencing,” “co-suppression,” “quelling” and “siRNA” are collectively included in

the phenomenon of “RNA interference.”

The RNAi mechanism and siRNAs have tremendous commercial potential in thebio-industry This molecular tool will allow investigators to routinely implement

“loss-of-function” screens and enable the development of rapid tests for geneticinteractions in mammalian cells, which until now have been difficult to performquickly Many of the current and potential applications are described within thevarious themes of the book, which are summarized below

Introduction 3

Section 1: Basic biology of RNAi, siRNA, microRNAs and gene

silencing mechanisms

The seminal work of Sydney Brenner, Robert Horovitz and John Sulston paved

the way for the development of Caenorhabditis elegans as a model organism for

studying development and behavior, for which they received the Nobel Prize for

Medicine in 2002 C elegans is the same organism in which the RNA interference

was first demonstrated The term “RNA interference” (hereafter RNAi) was coined

by Andrew Fire and Craig Mello to describe a sequence-specific gene silencing

phenomenon in C elegans In this book, Chapter 1 by Alla Grishok, one of the

scientists involved in the discovery of the RNAi phenomenon in the laboratory of

Craig Mello, describes the historical beginnings and genetics of RNAi, transgene

silencing, and the systemic nature of the phenomenon in C elegans RNAi shares

a remarkable degree of similarity with the gene silencing phenomenon observed

in other organisms, and this supports the notion that they were derived from an

ancient, conserved pathway used to regulate gene expression

The discovery of RNAi in C elegans initiated a flurry of biochemical and

ge-netic experiments aimed at identifying the molecular components involved in

this amazing phenomenon The biochemistry of RNAi is well described here in

Chapter 2 written by Myers and Ferrell This chapter also includes the role and

utility of Dicer enzyme both in vivo and in vitro and details its application in high

throughput in vitro dicing, split-pool screening and small hairpin RNA expression

library construction Most importantly, this chapter highlights the advantages of

double-stranded siRNAs (d-siRNAs) in the silencing of multiple targets

The cell biology of RNAi is detailed by Dudley et al in Chapter 3 in this section,

giving the details of the two-step model for RNAi, and the genes required for

RNAi (including initiators, effectors, RISC components and RNA-dependent RNA

polymerases) This chapter also details the roles for chromatin-modifying proteins

in RNAi The continued identification of genes required for RNAi will further our

understanding of the mechanisms of RNAi and its biology in general

The emergence of RNAi has helped to clarify another enigma of non-coding

temporal RNAs or microRNAs (miRNA) that were thought to antagonize gene

ex-pression by binding to the 3-untranslated region Genetic studies in C elegans

revealed the existence of miRNA genes which are now recognized to permeate the

genomes of all multi-cellular organisms These tiny RNA regulators are being

im-plicated in diverse biological pathways, ranging from development to neuronal

differentiation to fat metabolism Chapter 4written by Pasquinelli summarizes

the historical aspects and the birth of tiny non-coding temporal RNAs or miRNAs

from the laboratories of Victor Ambros and Gary Ruvkun that include lin-4 and

let-7 respectively Her chapter also details the connection between miRNAs and

RNAi In contrast to siRNAs, miRNAs are derived from the processing of

endoge-nously encoded short hairpin RNAs However, miRNAs are dependent on dicer

for processing and associate with a complex that shares components present in

RISC, suggesting that a mechanistic link between the RNAi and miRNA pathways

exists

Several hundred microRNAs have been cloned to date from a wide range oforganisms The discovery of predominantly brain-specific miRNAs suggests a rolefor RNAi-mediated mechanisms of gene regulation in neuronal development andfunctioning In Chapter 5 Krichevsky et al detail the recent findings concerningthe discovery of miRNAs in mammalian brain development This chapter also

highlights the ‘miRNA oligonucleotide array’ method for the display of

expres-sion profiles that uniquely correspond to developmental epochs of brain opment, and for the study of cell lineages during stem cell differentiation Thediscovery of extensively regulated predominantly brain-specific miRNAs, sharingprocessing pathways with siRNAs, suggests a role of RNAi-related mechanisms ofgene regulation in neuronal development

devel-Section 2: Design and synthesis of siRNAs

The fundamental challenge for successfully implementing RNAi in mammaliansystems rests with designing specific and potent siRNAs The rules that governsiRNA design and target silencing are largely undefined Some targets are easy

to silence while others are more difficult, requiring multiple screens of siRNAs

to identify a single potent duplex Therefore, various siRNA selection strategieshave been developed and summarized in this section Boese et al describe theconventional and functional siRNA design methods and specificity studies inChapter 6

Subrahmanyam and Lader describe online design tools and rules based on servations and mechanisms Their Chapter 7 also deals about the TOM-Amiditechemistry based synthesis of siRNAs, as well as high-throughput and automa-tion strategies Chemical modification of siRNAs will lead to improved activity,duration of effect, efficacy of delivery, and minimization of off-target effects.The current advantages of using chemically synthesized siRNAs as well as po-tential future improvements are likely to ensure the continued dominance ofchemically synthesized siRNA as the reagent of choice for high-throughput targetidentification

ob-Ding and Lawrence summarize in Chapter 8 the Web-based software called

Sfold, used for the prediction of target accessibility based on RNA secondary

structure This method bypasses the long-standing difficulty of selecting a singlestructure for accessibility evaluation Integration of computational approaches formaximizing both potency and specificity will facilitate high-throughput applica-tions for functional genomics, drug validation, and the development of RNAi-based human therapeutics

Sohail and Doran from Edwin Southern’s research group summarize enzymatic

production of siRNAs by in vitro transcription in Chapter 9 Use of this method

not only gives huge quantities of siRNAs, but also extends to the production ofsingle or double-stranded RNAs of defined length and sequence This is a method

of choice for the production of long RNA sequences with defined ends for whichchemical synthesis of siRNA molecules may be difficult In addition, this chapteralso describes the use of the RNaseIII-based method, for which the initial optimal

Introduction 5

target site selection procedure do not need to be considered The combination

of well-defined, sophisticated design strategies coupled with reliable and

flexi-ble methods for siRNA synthesis makes siRNA-mediated RNAi among the most

promising reverse genetics tool available to date

Section 3: Vector development and in vivo, in vitro and in ovo

delivery methods

The choice of the siRNA production method will depend on a number of factors –

such as the amenability of the method to long-term studies or to siRNA labeling

Latham et al in this section describe six different methods commonly used to

induce gene silencing in mammalian cells (see Chapter 10), and demonstrate that

it is easier to produce cell lines that stably express shRNAs using RNA polymerase II

promoters than pol III The use of pol II promoters to express siRNAs may also

lead to the development of tissue-specific siRNA expression constructs in the

near future Retroviruses offer several advantages as vector delivery systems, for

example (i) they have relatively small and simple genomes, making it is easy

to clone DNA of interest into them; (ii) since they are enveloped viruses, it is

possible to change their target cell specificity simply by changing the envelope;

(iii) they can integrate stably into the genome of the host cell providing long-term

expression of any gene In Chapter 11 Mao et al describe the moloney murine

leukemia virus-based retroviral vectors for efficient delivery of siRNAs into the

mammalian cells

Lentiviral-based siRNA vectors will have the ability to generate transgenic

an-imals carrying an siRNA cassette in order to achieve silencing of endogenous

genes Singer et al in Chapter 12 detail the development of transgenic rodents by

in vitro transduction of fertilized eggs at different pre-implantation stages They

have also demonstrated the delivery of siRNA for green fluorescent protein into

the embryos, and measured the lentivirus integration copy number using

real-time PCR Thus, lentiviral vectors will be ideal for generating large number of cell

lines, tissues, and whole animals where the expression of targeted genes can be

reduced substantially to influence biological function

The benefits of stable silencing that are offered by lentiviral delivery systems are

balanced by the concern that the integration of the virus in particular genomic

locations could potentially lead to the activation of gene expression and the

possibility of oncogene transformation The ability to knock down specific genes

could mean that these lentiviral vectors are used to generate transgenics not only

in mice and rodents but also in other species

The success of siRNA as therapeutic agents largely depends on the development

of a delivery vehicle that can efficiently deliver them in vivo Because of the

sim-plicity and potent nature of cationic liposomes (positively charged lipid bilayers,

which could form a complex with negatively charged siRNA duplexes), Sioud and

Sørensen have adopted these molecules for the efficient in vivo delivery of siRNA

molecules In addition, they have evaluated the delivery of FITC-labeled siRNA

against tumor necrosis factor-α by both intravenous and intraperitoneal injection