METHODS IN MOLECULAR MEDICINE
`
`Antisense
`Therapeutics
`
`Second Edition
`
`Edited by
`M. lan Phillips, php, psc
`Vice President for Research
`University of South Florida, Tampa, FL
`
`Foreword by
`Stanley T. Crooke, mb, PhD
`Isis Pharmaceuticals Inc., Carlsbad, CA
`
`Humana Press xK Totowa, New Jersey
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`© 2005 Humana Press Inc.
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`Cover illustration: “The principle of antisense inhibition,” Figure 1 from chapter 1, Antisense Therapeutics: A
`Promise Waiting to be Fulfilled, by M. Ian Philips
`
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`Library of Congress Cataloging in Publication Data
`Antisense therapeutics / edited by M. Ian Phillips.— 2nd ed.
`p. ; cm. — (Methods in molecular medicine ; 106)
`Includes bibliographical references and index.
` ISBN 1-58829-205-3 (alk. paper); eISBN 1-59259-854-4
`1. Antisense nucleic acids—Therapeutic use. [DNLM: 1. Oligonucleotides, Antisense—
`therapeutic use. 2. Oligonucleotides, Antisense—pharmacology. QU 57 A6332 2005] I.
`Phillips, M. Ian. II. Series.
`RM666.A564A585 2005
` 615'.31—dc22 2004006680
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`Foreword
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`We are now more than 15 years into a large-scale experiment to deter-
`mine the viability of antisense technology. The challenges of creating a new
`pharmacological drug discovery platform are prodigious, requiring sizeable
`investments, long-term commitment, insight, and perseverance. For antisense
`technology to progress, advances in understanding the behavior of the recep-
`tor, RNA, and the behavior of the drugs, oligonucleotide analogs, were neces-
`sary. A new medicinal industry, the medicinal industry of oligonucleotides,
`had to be invented, and numerous drug development challenges—such as creat-
`ing efficient manufacturing and analytical processes and formulations—had to be
`overcome. All of those advances then needed to be focused in drug candidates
`designed to interact with specific targets and to be effective in patients with spe-
`cific diseases. This has taken time and a good bit of money and although the
`progress in the technology has been gratifying, there have, of course, been failures
`of individual clinical trials and individual drugs along the way.
`What have we learned? Antisense technology works. Oligonucleotide
`analogs with a reasonable drug-dependent property can be synthesized and used
`to inhibit gene function through a variety of antisense mechanisms. Antisense
`drugs distribute to a wide range of tissues and reduce the expression of targets in
`a dose fashion consistent with the pharmaceutics of the drugs. First-generation
`antisense drugs are sufficient for relatively severe indications and second-genera-
`tion drugs are performing significantly better. Moreover, these drugs are effec-
`tive by a wide variety of routes including intravenous, subcutaneous, intradermal,
`rectal, and aerosol, and progress in oral delivery has been reported. Today numer-
`ous clinical trials in a wide range of diseases using a variety of oligonucleotide
`chemistries and antisense mechanisms are in progress.
`In this year alone, positive clinical data in rheumatoid arthritis, diabetes,
`hyperlipidemia, cancer, and other diseases have been reported.
`In this edition of Antisense Therapeutics, a number of approaches to anti-
`sense and therapeutic areas are discussed, as well as specific diagnostic oppor-
`tunities. That the breadth of activities presented in this volume is as impressive
`as it is and yet does not begin to cover all of the work in progress, underscores
`the range of utility and potential value of antisense technology.
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`vi
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`Foreword
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`Nevertheless, despite antisense being an accepted tool that has facili-
`tated better understanding of biological systems, much remains to be done
`before the true potential of the technology for therapeutic purposes can be
`defined. What this volume emphasizes, however, is that exponential progress
`in defining the long-term roles and value of antisense-based therapeutics is
`being made.
`We look forward to the continued evolution of the technology.
`
`Stanley T. Crooke, MD, PhD
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`Preface
`
`This is the second edition of Antisense Therapeutics. The first edition
`was edited by Sudhir Agrawal and published in 1996. At that time there was
`no therapy based on antisense, but plenty of promise for the highly specific
`targeting of genes that cause disease. Antisense oligonucleotides were first
`reported as viral replication inhibitors by Paul Zamecnik and Mary Stephen-
`son in 1978. Although this was excellent work, nothing much happened until
`new procedures for synthesizing DNA sequences were developed. Once oli-
`gonucleotides were easy to make, more and more studies were published in
`the 1980s, most of which were directed to cells in culture. In the early 1990s
`antisense oligonucleotides were increasingly tested in vivo. There were many
`controversies and a great deal of concern about backbone modification of the
`phosphodiester bridges that link the DNA bases. To protect against break-
`down by nucleases in cells or blood, phosphorothioate oligonucleotides were
`adopted. In 1998 a phosphorothioated antisense agent was the first FDA-
`approved antisense therapy. Vitravene™, developed by Isis Pharmaceuticals,
`made antisense therapeutics a reality.
`Since then, the complete sequencing of the human genome in April, 2003
`has demonstrated the presence of a vast number of targets for antisense oligo-
`nucleotides. So we now have thousands of targets, hundreds of preclinical
`animal studies, and some 20 clinical trials ongoing. Any successful trial with
`an antisense compound will open a floodgate of new therapies for a panoply
`of diseases.
`This second edition of Antisense Therapeutics deals less with the basic
`science of antisense and more with the actual therapeutic applications. For
`that reason it is organized into disease states.
`I thank the authors for their patience and their strong contributions. Since
`this book was being edited at a time when I moved from the University of
`Florida to the University of South Florida, I ended up with two secretaries. I
`would like to thank Ms. Gayle Butters at the University of Florida and Mr.
`Eric J. Wheeler at the University of South Florida for their essential help. I
`am also grateful to Craig Adams at Humana Press for his patience.
`
`M. Ian Phillips, PhD, DSc
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`Contents
`
`Foreword ...........................................................................................................v
`Preface ........................................................................................................... vii
`Contributors .....................................................................................................xi
`
`PART I. INTRODUCTION
`1 Antisense Therapeutics: A Promise Waiting to be Fulfilled
`M. Ian Phillips.........................................................................................3
`2 Antisense Inhibition: Oligonucleotides, Ribozymes, and siRNAs
`Y. Clare Zhang, Meghan M. Taylor, Willis K. Samson,
`and M. Ian Phillips...........................................................................11
`
`PART II. CARDIOVASCULAR
`3 Local Application of Antisense for Prevention of Restenosis
`Patrick L. Iversen, Nicholas Kipshidze, Jeffrey W. Moses,
`and Martin B. Leon..........................................................................37
`4 Antisense Therapeutics for Hypertension:
`Targeting the Renin–Angiotensin System
`M. Ian Phillips and Birgitta Kimura ...................................................51
`5 Antisense Strategies for the Treatment of Heart Failure
`Sian E. Harding, Federica del Monte, and Roger J. Hajjar.............69
`
`PART III. CANCER
`6 Clinical Studies of Antisense Oligonucleotides for Cancer Therapy
`Rosanne M. Orr and F. Andrew Dorr.................................................85
`7 Antisense Therapy in Clinical Oncology:
`Preclinical and Clinical Experiences
`Ingo Tamm..........................................................................................113
`8 Radionuclide–Peptide Nucleic Acid Diagnosis and Treatment
`of Pancreatic Cancer
`Eric Wickstrom, Xiaobing Tian, Nariman V. Amirkhanov,
`Atis Chakrabarti, Mohan R. Aruva, Ponugoti S. Rao,
`Wenyi Qin, Weizhu Zhu, Edward R. Sauter,
`and Mathew L. Thakur...................................................................135
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`Contents
`
`9 Suppression of Pancreatic and Colon Cancer Cells
`by Antisense K-ras RNA Expression Vectors
`Kazunori Aoki, Shumpei Ohnami, and Teruhiko Yoshida............193
`10 Induction of Tumor Cell Apoptosis and Chemosensitization
`by Antisense Strategies
`Manuel Rieber and Mary Strasberg-Rieber....................................205
`11 Utility of Antioncogene Ribozymes and Antisense
`Oligonucleotides in Reversing Drug Resistance
`Tadao Funato.....................................................................................215
`
`PART IV. BLOOD–BRAIN BARRIER
`12 Transport of Antisense Across the Blood–Brain Barrier
`Laura B. Jaeger and William A. Banks...........................................237
`
`PART V. DERMAL
`13 Transdermal Delivery of Antisense Oligonucletoides
`Rhonda M. Brand and Patrick L. Iversen........................................255
`
`PART VI. DRUGS
`14 Antisense Strategies for Redirection of Drug Metabolism:
`Using Paclitaxel as a Model
`Vikram Arora......................................................................................273
`
`PART VII. GASTROINTESTINAL
`15 Antisense Oligonucleotide Treatment
`of Inflammatory Bowel Diseases
`Bruce R. Yacyshyn............................................................................295
`
`PART VIII. HEPATITIS
`16 Optimizing Electroporation Conditions for the Intracellular Delivery
`of Morpholino Antisense Oligonucleotides Directed Against the
`Hepatitis C Virus Internal Ribosome Entry Site
`Ronald Jubin......................................................................................309
`Index ............................................................................................................ 323
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`Contributors
`
`NARIMAN V. AMIRKHANOV • Departments of Biochemistry and Molecular
`Pharmacology, Kimmel Cancer Center, Thomas Jefferson University,
`Philadelphia, PA
`KAZUNORI AOKI • Section for Studies on Host-Immune Response, National
`Cancer Center Research Institute, Tokyo, Japan
`VIKRAM ARORA • Research and Development, AVI BioPharma, Corvallis, OR
`MOHAN R. ARUVA • Department of Radiology, Kimmel Cancer Center,
`Thomas Jefferson University, Philadelphia, PA
`WILLIAM A. BANKS • GRECC, VA Medical Center St. Louis, Department of
`Internal Medicine, St. Louis University, St. Louis, MO
`RHONDA M. BRAND • Division of Emergency Medicine, Evanston Northwestern
`Healthcare, and Department of Medicine, Feinberg School of Medicine,
`Northwestern University, Evanston, IL
`ATIS CHAKRABARTI • Departments of Biochemistry and Molecular Pharmacology,
`Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA
`STANLEY T. CROOKE • Chairman and CEO, ISIS Pharmaceuticals Inc.,
`Carlsbad, CA
`FEDERICA DEL MONTE • Cardiovascular Research Center, Massachusetts
`General Hospital and Harvard Medical School, Boston, MA
`F. ANDREW DORR • Salmedix Inc., San Diego, CA
`TADAO FUNATO • Division of Molecular Diagnostics, Tohoku University
`School of Medicine, Sendai, Japan
`ROGER J. HAJJAR • Cardiovascular Research Center, Massachusetts General
`Hospital and Harvard Medical School, Boston, MA
`SIAN E. HARDING • National Heart and Lung Institute, Imperial College,
`London, UK
`PATRICK L. IVERSEN • AVI BioPharma, Corvallis, OR
`LAURA B. JAEGER • Department of Pharmacological and Physiological
`Science, St. Louis University, St. Louis, MO
`RONALD JUBIN • Department of Antiviral Therapy, Schering Plough Research
`Institute, Kenilworth, NJ
`BIRGITTA KIMURA • Department of Anthropology, University of Florida,
`Gainesville, FL
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`xii
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`Contributors
`
`NICHOLAS KIPSHIDZE • Lenox Hill Heart and Vascular Institute, Cardiovascular
`Research Foundation, Lenox Hill Hospital, New York, NY
`MARTIN B. LEON • Lenox Hill Heart and Vascular Institute, Cardiovascular
`Research Foundation, Lenox Hill Hospital, New York, NY
`JEFFREY W. MOSES • Lenox Hill Heart and Vascular Institute, Cardiovascular
`Research Foundation, Lenox Hill Hospital, New York, NY
`SHUMPEI OHNAMI • Central RI Laboratory, National Cancer Center Research
`Institute, Tokyo, Japan
`ROSANNE M. ORR • Cancer Research UK Centre for Cancer Therapeutic,
`The Institute of Cancer Research, Sutton, Surrey, UK
`M. IAN PHILLIPS • Vice President for Research, Office of Research, University
`of South Florida, Tampa, FL
`WENYI QIN • Department of Surgery, University of Missouri, Columbia, MO
`PONUGOTI S. RAO • Department of Radiology, Kimmel Cancer Center,
`Thomas Jefferson University, Philadelphia, PA
`MANUEL RIEBER • Tumor Cell Biology Laboratory, Center of Microbiology
`and Cell Biology, IVIC, Caracas, Venezuela
`WILLIS K. SAMSON • Department of Pharmacological and Physiological
`Science, St. Louis University, St. Louis, MO
`EDWARD R. SAUTER • Department of Surgery, University of Missouri, Columbia, MO
`MARY STRASBERG-RIEBER • Tumor Cell Biology Laboratory, Center of
`Microbiology and Cell Biology, IVIC, Caracas, Venezuela
`INGO TAMM • Department of Hematology and Oncology, Charite, Campus
`Virchow, Humboldt University of Berlin, Berlin, Germany
`MEGHAN M. TAYLOR • Department of Pharmacological and Physiological
`Science, St. Louis University, St. Louis, MO
`MATHEW L. THAKUR • Department of Radiology, Kimmel Cancer Center,
`Thomas Jefferson University, Philadelphia, PA
`XIAOBING TIAN • Departments of Biochemistry and Molecular Pharmacology,
`Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA
`ERIC WICKSTROM • Departments of Biochemistry and Molecular Pharmacology,
`Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA
`BRUCE R. YACYSHYN • Louis Stokes VA Hospital and Case Western Reserve
`University, Cleveland, OH
`TERUHIKO YOSHIDA • Genetics Division, National Cancer Center Research
`Institute, Tokyo, Japan
`Y. CLARE ZHANG • Department of Pediatrics, University of South Florida, St.
`Petersburg, FL
`WEIZHU ZHU • Department of Surgery, University of Missouri, Columbia, MO
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`I I
`I I
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`NTRODUCTION
`NTRODUCTION
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`
`
`1 A
`
`ntisense Therapeutics
`A Promise Waiting to Be Fulfilled
`
`M. Ian Phillips
`
`1. Introduction
`During the past decade, only one antisense-based therapy has received full
`Food and Drug Administration (FDA) approval. Vitravene™, developed by
`Isis Pharmaceuticals, was the first drug based on antisense technology to be
`successfully commercialized and used in treatment (1). The therapeutic area it
`is used in is a small niche related to the treatment of preventing blindness in
`acquired immunodeficiency syndrome (AIDS) patients by inhibiting cytome-
`galovirus-induced retinitis. The success of Vitravene, however, showed that
`antisense could be taken all the way through the FDA approval process and
`provide those patients taking it with a vitally important effect. With Vitravene
`we saw the first breakthrough in antisense therapy, and, yet, euphoria has turned
`to disappointment without a second breakthrough. Subsequent trials of
`Affinitak (Isis), an antisense inhibitor of protein kinase C (cid:95), failed to show
`statistically significant benefits as an antisense therapy for the treatment of
`non–small cell carcinoma of the lung better than the median survival with con-
`trol treatments. The results nevertheless proved that antisense was well toler-
`ated and tended toward greater benefit to the survival of patients (p < 0.054).
`The promise of antisense therapy is so attractive that some 20 trials continue.
`The appeal of antisense is that it potentially provides highly specific, nontoxic
`effects for safe and effective therapeutics of an enormous number of diseases
`including AIDS, Crohn’s disease, pouchitis, psoriasis, cancers, diabetes,
`mulitiple sclerosis, muscular dystrophy, restenosis, asthma, rheumatoid arthri-
`tis, hepatitis, skin diseases, polycystic kidney disease, and chronic cardiovas-
`
`F r o m : Methods in Molecular Medicine, Vol. 106: Antisense Therapeutics, Second Edition
`E d i
`t e d b y :
`
`I
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` P h i
`l
`l
`i p s © H u m a n a P r e s s
`I n c .
`,
` T o t o w a ,
` N J
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`4
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`Phillips
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`cular disease, such as hypertension, restenosis, and heart failure. Successes in
`phase I have shown that antisense therapy consistently has excellent safety
`results. With each trial we learn more, and this makes each new antisense drug
`candidate more easy to test. We are hampered by a lack of understanding of the
`theoretical considerations for optimal antisense inhibition. Failures in the past
`have been the result of incorrect design and use of unmodified backbones caus-
`ing instability, overly long oligonucleotides leading to unpredictable targeting,
`and aptermeric or nonantisense effects. However, with each experiment we
`learned more. For example, high doses of antisense in monkeys triggered car-
`diovascular collapse (2). This result was a setback until it was found that the
`reaction could be accounted for by the extremely high doses and a sensitivity
`to complement activation unique to nonhuman primates (3). Human trials, by
`contrast, have shown how well antisense is tolerated and how few side effects
`are encountered. The number of trials is increasing, and more than 2000 patients
`have received antisense. Isis is the leader with 11 phase I, 7 phase II, and 3 phase
`III trials. Genta is active with Genasense, and antisense to Bcl 2 for antitumor
`cell treatment is in phase III. AVI Biopharm has a third generation antisense
`platform, and around this it is testing four phase I, five phase II, and two phase
`III trials. Hybridon has conducted two phase I and has two phase II trials
`planned.
`
`2. Mechanism of Antisense Inhibition
`Antisense oligonucleotides (AS-ODNs) are designed to bind and inactivate
`specific mRNA sequences inside cells. The potential uses for AS-ODNs is vast
`because RNA is so ubiquitous and abundant. With the publication of the human
`genome sequence, we now have such a wide open access to the sequences of
`genes that antisense can in theory be applied to almost every known gene to
`inhibit its mRNA. Inhibiting mRNA prevents specific proteins from being pro-
`duced. Although routine human therapy may have been difficult to achieve, at
`a scientific level, antisense gene knockdown has become one of the fastest
`ways to study new therapeutic targets.
`AS-ODNs are synthetically made, single-stranded short sequences of DNA
`bases designed to hybridize to specific sequences of mRNA forming a duplex.
`This DNA-RNA coupling attracts an endogenous nuclease, RNase H, that
`destroys the bound RNA and frees the DNA antisense to rehybridize with
`another copy of mRNA (2). In this way, the effect is not only highly specific
`but prolonged because of the recycling of the antisense DNA sequence. The
`reduction in mRNA reduces the total amount of protein specified by mRNA. It
`is also theorized that hybridization sterically prevents ribosomes from translat-
`ing the message of the mRNA into protein. Therefore, there are at least two
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`Antisense Therapeutics
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`5
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`Fig. 1. Mechanism of AS-ODN posttranscriptional inhibition. AS-ODN enters the
`cell by an unknown uptake mechanism and hybridizes with a copy of a specific mRNA.
`The ODN-RNA duplex then prevents protein translation by (1) attracting RNase H to
`degrade the RNA and (2) steric hindrance of the ribosomal access and/or assembly.
`Note that the extent of inhibition depends on the AS-ODN competing with endog-
`enous copies of RNA.
`
`ways in which antisenses can work to effectively reduce the amount of protein
`being elaborated: RNase H degradation of RNA and hindering of ribosomal
`assembly and translation (Fig. 1). However, unless the antisense is designed to
`inhibit transcription, antisense would rarely be 100% inhibitory because the
`antisense inhibition of RNA does not shut down the transcription of endog-
`enous copies of mRNA. It competes with the RNA being produced by the cell,
`and the effect is a gene knockdown rather than knockout. This has the advan-
`tage of being more physiological as a therapeutic agent, since antisense does
`not cause a mutation and does not prevent a protein that is involved in normal
`physiology from assuming its role. What antisense therapy does very effec-
`tively is reduce overexpression of proteins, and it is the overexpression of pro-
`teins that can cause disease states.
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`Phillips
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`3. Stability
`One of the problems that dogged early attempts to achieve a therapy with
`antisense was the question of stability. This is largely being answered by numer-
`ous ways to modify backbones of the DNA sequence in an AS-ODN. Native
`DNA has a phosphodiester bridge between each successive base of the DNA
`sequence. It was quickly learned that unmodified AS-ODNs were very short
`lasting, because they were unprotected from breakdown by nucleases, which
`break apart the nuclear acids. A very successful modification was phos-
`phorothioate in which a sulfur atom replaces one oxygen atom in the phospho-
`rate group of the phosphodiester bond. Phosphorothioate oligonucleotides are
`resistant to nucleases and are stable. This extends the life of the AS-ODN to
`several days instead of a few hours. Many variations on this theme have been
`tested and patented so that there is now a range of second- and even third-
`generation backbone modifications available (2–4). Each company appears
`to favor its own particular modification. Isis uses phosphorothivates with
`2'-O-methyl modification. Hybridon favors its IMO™ backbone modifica-
`tion, which can increase or decrease immunomodulation. AVI Biopharm has
`used NeuGene® as a platform of third-generation antisense for its nine clinical
`trials. A factor in developing backbone modifications such as these and others,
`including peptide nucleic acid, is the cost.
`
`4. Cellular Uptake
`Another area that has required time (and money) to investigate is the opti-
`mal conditions for uptake and distribution. This is particularly important when
`it comes to systemic injection as opposed to the early experiments in which
`antisenses were simply applied to cells in culture. There is both uptake and
`efflux of intact AS-ODNs in cells (5).The backbone modifications become
`extremely important when systemic injections are used because of nucleases
`and the binding of oligonucleotides to proteins. The backbone modification
`can alter cell uptake, distribution, metabolism, and excretion. Nonantisense
`effects are a concern because they may alter the interpretation of whether the
`antisense effect is truly through an antisense mechanism or not. Mechanisms
`for the uptake of oligonucleotides into cells are still not clearly understood.
`The lack of a theory of the uptake and kinetic effects on oligonucleotides has
`required a lot of trial-and-error studies. This affects how to determine the opti-
`mal length of the oligonucleotide, the optimal concentration for effective treat-
`ment, and the frequency of treatments to maintain constant therapy. Despite
`these complications and holes in the study of antisense, phosphorothioated oli-
`gonucleotides are surprisingly easy to work with. In our own studies, which
`were in vivo applications of AS-ODN, we aimed injections into the brain and
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`Antisense Therapeutics
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`7
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`into the blood at receptor targets involved in cardiovascular disease. We found
`highly significant effects using AS-ODNs of 15–18 bases in length delivered
`in the brain without any vehicle (6) and in the blood delivered with liposomes
`(7). Call it science or dumb luck, we nevertheless were able to show significant
`physiological effects of antisense delivery in models of hypertension. Because
`hypertension is a chronic disease, the findings were remarkable because of the
`long-lasting efficacy of a single antisense treatment. Reductions in blood
`pressure lasted weeks with a single systemic injection of antisense targeting
`(cid:96)-1 receptors (8).
`The distribution of AS-ODNs injected systemically is to all parts of the body
`except the brain. The lipophobicity and/or negative change appear to prevent
`AS-ODNs from crossing the blood-brain barrier. However, the oligonucle-
`otides accumulate in liver, kidney, and spleen. The lack of entry into the brain
`probably translates into few side effects. With the antisense to (cid:96)-1 receptors,
`this could be a definite advantage (8). For treating liver or kidney disease,
`however, AS-ODNs might have a built-in advantage in terms of delivery.
`
`5. The Target
`Clearly, the target protein for antisense inhibition is crucially important for
`a therapeutic effect. To reach the target, the antisense therapy must enter the
`cell through an uptake mechanism and escape from endosomes and lysosomes
`within the cell in sufficient amounts to avoid intracellular degradation. If the
`target mRNA is shielded or coiled, it may be difficult for AS-ODNs to hybrid-
`ize. DNA and RNA are folded and studded with regulated proteins. Predicting
`how RNA folds and its secondary structures in a living cell is still very diffi-
`cult. Once again, trial and error must be used. The stability of the oligos also
`depends on the interactions of the G-C proportions because of the three hydro-
`gen bonds instead of the two hydrogen bonds that are in the A-T interaction.
`Having sufficient length of bases is necessary to make a specific match, but
`having too long a sequence can overlap the coding regions and inhibit more
`than single-target RNA.
`Even when everything is successful and there is good uptake—good inhibi-
`tion of the target—it does not necessarily lead to a therapeutic effect, because
`the target may not be the only player in the disease. If knocking down one gene
`leads to an increase in a compensatory gene, there may be little or no effect.
`Alternatively, a target gene may have been involved in starting the disease, but
`once the disease is present that target is no longer necessary, and, therefore,
`inhibiting it does not alter the disease state. Targeting transcription factors or
`signaling pathway proteins important in regulating cells may not be specific
`enough. If the target protein is overexpressed only in the disease state, then
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`Phillips
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`antisense should be efficacious, but if the target is similarly expressed in both
`normal and malignant cells, antisense treatment may cause both types of cells
`to undergo apoptosis. Then the therapy becomes a question of benefit vs risk.
`Because of the competition for RNA inhibition with antisense vs endogenous
`production of copies of mRNA in a cell, antisense for cancer is not a cell killer
`and, therefore, will not destroy all cancerous cells. However, it can be used
`with other treatments for cancer, and that is the protocol proposed for Affinitak
`and for Genasense.
`
`6. Alternative to Oligonucleotides
`In recent years, there has been a tremendous increase in interest in mor-
`pholinos (9), small inhibitory RNA (siRNA) (10), as well as ribozymes (11).
`Morpho-linos are assembled from four different morpholino subunits each of
`which contains one of the four genetic bases linked to a six-sided morpholine
`ring. Morpholinos are supposed to have complete resistance to nucleases,
`high sequence specificity, and predictable targeting because they invade the
`RNA secondary structure and are fast and easy to deliver to the nucleus with-
`out liposome delivery systems. siRNAs are double-stranded RNA (dsRNA)
`molecules of 21–25 bp in length. They mediate RNA interference, an antiviral
`response initially identified in Caenorhabditis elegans and subsequently found
`active in specific gene silencing in many other organisms including mamma-
`lian cells. The sense and antisense strands of an siRNA first unwind, and the
`antisense strand binds to the target mRNA and recruits RNA-induced silencing
`complex (RISC) (Fig. 2). The sense strand is released from RISC, and RISC
`catalyzes the mRNA cleavage. The gene silencing efficiency of siRNA has
`reportedly been greater than antisense in general, typically reaching 80–90%.
`However, the maximal effects of optimal AS-ODNs and siRNAs targeting the
`same mRNA sequence are comparable. siRNAs are being used because of their
`stability and specificity, but it is not clear how effective they will be in sys-
`temic injections or oral delivery. Vickers et al. (12) conducted a comparative
`study of single-stranded AS-ODNs vs siRNA. Examination of 80 siRNA oli-
`gonucleotide duplexes designed to bind human RNA showed that both strate-
`gies are valid in terms of potency, maximal effects, specificity, and duration of
`action, at least in vitro.
`The design of AS-ODNs and siRNAs follows different rules. Unlike AS-
`ODNs, the selection of an effective siRNA does not depend on the secondary
`mRNA structure or sequence accessibility. Instead, nucleotide composition and
`the release rate of the sense strand from RISC seem to play major roles. Sev-
`eral siRNA molecules targeting the same mRNA can be used in combination to
`achieve greater effects and to avoid cellular resistance to siRNA. An indepen-
`dent combinatorial effect of AS-ODNs and siRNAs has also been observed
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`Antisense Therapeutics
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`Fig. 2. Mechanism of siRNA. Synthetic siRNA enters the cell as a dsRNA with
`sense and antisense strands. RISC multiprotein made up of helicase, RNase III, and an
`activating protein unwinds the two strands of RNA and uses the antisense to recognize
`the chosen sequence of RNA. The RNase cleaves the sequence of mRNA, which is
`degraded by cellular nucleases. The RISC-antisense complex can then recycle and
`silence more copies of mRNA.
`
`when siRNA was coadministered with nonhomologous AS-ODNs, targeting
`distant regions of the same mRNA. As alternative therapeutics, development
`of siRNA has covered a wide variety of disease models in a short time. The
`most studied fields of siRNA application are cancer and infectious diseases.
`siRNA has been administered in vivo in unmodified states. Following iv injec-
`tion into mice, the highest inhibition of target mRNA was found in liver, kid-
`ney, spleen, lung, and pancreas. If both strategies are equally effective, then
`the deciding factor in choosing one over the other would depend on the price of
`production. In addition, experience with AS-ODNs will count for some time
`against the newness of siRNA molecules. However, a lot will depend on
`whether there are side effects that are not due to the antisense mechani