M Ian Phillips PhD dsc

Vice President for Research University of South Florida, Tampa, FL

Foreword by

Stanley T. Crooke, md, phd

Isis Pharmaceuticals Inc., Carlsbad, CA

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Promise Waiting to be Fulfilled, by M. Ian Philips

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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

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Foreword

We are now more than 15 years into a large-scale experiment to determine 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 receptor, RNA, and the behavior of the drugs, oligonucleotide analogs, were necessary. A new medicinal industry, the medicinal industry of oligonucleotides, had to be invented, and numerous drug development challenges—such as creating 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 specific 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-generation drugs are performing significantly better. Moreover, these drugs are effective by a wide variety of routes including intravenous, subcutaneous, intradermal, rectal, and aerosol, and progress in oral delivery has been reported. Today numerous 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 antisense and therapeutic areas are discussed, as well as specific diagnostic opportunities. 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.

Foreword

Nevertheless, despite antisense being an accepted tool that has facilitated 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

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 breakdown 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 oligonucleotides. 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

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

Contents

9 Suppression of Pancreatic and Colon Cancer Cells by Antisense K-ras RNA Expression Vectors KazunoriAoki, 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

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

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

Introduction

Antisense 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 a, 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 control treatments. The results nevertheless proved that antisense was well tolerated 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 arthritis, hepatitis, skin diseases, polycystic kidney disease, and chronic cardiovas-

From: Methods in Molecular Medicine, Vol. 106: Antisense Therapeutics, Second Edition Edited by: I. Phillips © Humana Press Inc., Totowa, NJ

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 causing 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 cardiovascular 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 produced. 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 translating the message of the mRNA into protein. Therefore, there are at least two

Methoxyethyl Ribose

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 endogenous copies of RNA.

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 endogenous 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 endogenous 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 advantage 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 effectively is reduce overexpression of proteins, and it is the overexpression of proteins that can cause disease states.

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 numerous 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 phosphorate 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'-0-methyl modification. Hybridon favors its IMO™ backbone modification, 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 optimal 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 optimal length of the oligonucleotide, the optimal concentration for effective treatment, 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 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 P-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 p-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 hybridize. 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 difficult. 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 hydrogen 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 inhibition 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 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 without 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 mammalian 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 systemic 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 strategies 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. Several siRNA molecules targeting the same mRNA can be used in combination to achieve greater effects and to avoid cellular resistance to siRNA. An independent combinatorial effect of AS-ODNs and siRNAs has also been observed

Imo Immunomodulatory Oligonucleotides

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.

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 injection into mice, the highest inhibition of target mRNA was found in liver, kidney, 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 mechanism, or if one approach is associated with more side effects than the other.

7. Conclusion

The brief history of antisense therapeutics has been characterized by cycles of success and disappointment. However, through it all, the promise of antisense therapy has been so appealing that hope remains for that blockbuster breakthrough that will open the doors for so many potential treatments. There are now thousands of targets available with known genomic sequences. There are hundreds of preclinical studies pointing to new treatments with antisense. And there are a score of human trials that are paving the way. Once one major treatment is accepted, each new antisense therapy will be more easily and quickly brought to those who suffer from diseases that are not yet satisfactorily treated with drugs.

References

1 Crooke, S. T. (2004) Progress in antisense technology. Annu. Rev. Med. 55, 61-95.

2 Crooke, S. T. (1998) Molecular mechanisms of antisense drugs: RNase H. Antisense Nucleic Acid Drug Dev. 8(2), 133-134.

3. Wickstrom, E. and Smith, J. B. (1998) DNA combination therapy to stop tumor growth. Cancer J. Sci. Am. 4(Suppl. 1), S43-S47.

4 Agrawal, S., Kandimalla, E. R., Yu, D., et al. (2002) GEM 231, a second-generation antisense agent complementary to protein kinase A alpha subunit, potentiate antitumor activity of irinotecan in human colon, pancreas, prostrate and lung cancer xenografts. Int. J. Oncol. 21(1), 65-72.

5 Li, B., Hughes, J. A., and Phillips, M. I. (1997) Uptake and efflux of intact antisense phosphorothioate deoxyoligonucleotide directed against angiotensin receptors in bovine adrenal cells. Neurochem. Int. 31(3), 393-403.

6 Gyurko, R., Wielbo, D., and Phillips, M. I. (1993) Antisense inhibition of AT1 receptor mRNA and angiotensinogen mRNA in the brain of spontaneously hypertensive rats reduces hypertension of neurogenic origin. Regul. Pept. 49(2), 167-174.

7 Phillips, M. I. (2001) Gene therapy for hypertension: sense and antisense strategies. Expert Opin. Biol. Ther. (4), 655-662.

8 Zhang, Y. C., Bui, J. D., Shen, L., and Phillips, M. I. (2000) Antisense inhibition of beta(1)-adrenergic receptor mRNA in a single dose produces a profound and prolonged reduction in high blood pressure in spontaneously hypertensive rats. Circulation 101(6), 682-688.

9 Summerton, J. (1999) Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim. Biophys. Acta. 1489(1), 141-158.

10 Zamore, P. D. and Aronin, N. (2003) siRNAs knocks down hepatitis. Nat. Med. 9(3), 266-267.

11 Fedor, M. J. and Westhof, E. (2002) Ribozymes: the first 20 years. Mol. Cell. 10(4), 703-704.

12. Vickers, T. A., Koo, S., Bennett, C. F., Crooke, S. T., Dean, N. M., and Baker, B. F. (2003) Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents: a comparative analysis. J. Biol. Chem. 278(9), 7108-7118.

Antisense Inhibition

Oligonucleotides, Ribozymes, and siRNAs

Y. Clare Zhang, Meghan M. Taylor, Willis K. Samson, and M. Ian Phillips

1. Introduction

Over a span of more than two decades, antisense strategies for gene therapy have expanded from antisense oligonucleotides (AS-ODNs) solely, to the addition of ribozymes and, more recently, to the inclusion of small interfering RNAs (siRNAs). Antisense therapeutics has also experienced its phases of high expectation, sudden disappointment, and meticulous rediscovery, while maintaining its status as a viable and effective gene therapy approach. With the discovery of RNA interference (RNAi) and development in delivery of these gene drugs, more preclinical and clinical investigations are anticipated to take place in the near future to finally fulfill the promise of antisense therapeutics in humans.

2. Antisense Oligonucleotides

AS-ODNs are typically 18-25 bases in length, consisting of sequences that are complementary to the target RNA. They can be injected directly into tissues or delivered systemically. Once delivered into cells, oligonucleotide binds to its RNA counterpart and suppresses expression of the proteins encoded by target RNA. The specificity of this approach is based on the probability that any sequence longer than a minimal number of nucleotides (nt)—13 for RNA and 17 for DNA—occurs only once within the human genome. The idea of antisense therapy for inhibiting disease-associated proteins has become par-

From: Methods in Molecular Medicine, Vol. 106: Antisense Therapeutics, Second Edition Edited by: I. Phillips © Humana Press Inc., Totowa, NJ

ticularly appealing since Zamecnik and Stephenson (1) first demonstrated in 1978 the reduction of Rous sarcoma viral RNA translation by a specific oligonucleotide.

2.1. Mechanisms of Antisense Inhibition

Gene expression can be altered by oligonucleotides by means of either posttranscriptional inhibition or splicing shift. Posttranscriptional inhibition is accomplished by several mechanisms including sterical blockade of ribosomal access to the target mRNA, induction of RNase H cleavage of mRNA, and inhibition of ribosomal assembly. The net outcome of this process is the diminished translation of target proteins. Oligonucleotides chemically modified by phosphorothioation are especially effective in activating RNase H, resulting in sequence-specific digestion of the target mRNA molecules. This destruction of RNA while leaving the DNA oligonucleotide intact allows the oligonucleotide to be recycled, which makes AS-ODNs long lasting. A majority of antisense studies so far, including most clinical trials, are aimed at reducing undesired disease-associated proteins by virtue of translational inhibition. Alternatively, oligonucleotides that are RNase H inactive and designed toward a certain exon-intron junction can prevent the pre-mRNA splicing at the targeted site and redirect the splicing to a more favored site. The therapeutic potential of this approach has been exemplified in the correction of the expression of P-globin and the breast cancer gene BCL-X in related diseases. Certain forms of p-thalassemia are caused by aberrant splicing of p-globin pre-mRNA that leads to abrogation of the protein production (2). AS-ODNs designed to the untoward splice site have been proven effective at inhibiting aberrant splicing and at restoring p-globin expression in thalassemic patients (3). Likewise, alternative splicing of BCL-X pre-mRNA gives rise to two isoforms, BCL-XL and BCL-XS, with opposing antiapoptotic and proapoptotic activities. Targeting the BCL-XL splice site with oligonucleotides favored production of the proapoptotic BCL-XS protein that enhances cell death in prostate and breast tumor cells (4).

2.2. Targeting Antisense

Although antisense can be designed against any region of the target RNA in theory, different sequences vary markedly in efficiency of gene inhibition. The accessibility of oligonucleotides to RNA is considered the most important factor in choosing the optimal antisense sequences. Computational analysis of the secondary structure of RNA by programs such as mfold or RNAstructure has been used to facilitate selection of target sites for antisense action (5); however, it does not take into account the three-dimensional structures as well as the instant interaction of RNA molecules with other factors. More commonly taken routes involve evaluation of accessible sites by use of RNase H mapping (6) or scanning oligonucleotide arrays for the best hybridization signals (7). Nevertheless, in general, targeting the start codon AUG, where mRNA is supposedly open for ribosomal entry, has been a successful strategy, although in many cases other sequences turned out to be more effective. Despite these predictive approaches, the selection of optimal antisense sequences still requires trial-and-error testing initially and, in the end, needs to be confirmed in vivo.

2.3. Chemical Modifications

Stability and efficient delivery, prerequisites for oligonucleotides to achieve observable therapeutic effects, have been obstacles due to their macromolecu-lar nature. Numerous chemical modifications and delivery approaches have been developed to overcome this problem (Fig. 1). The first generation of antisense agents contains backbone modifications such as replacement of oxygen atom of the phosphate linkage by sulfur (phosphorothioates), methyl group (methylphosphonates), or amines (phosphoramidates). Of these, the phosphor-othioates have been the most successful and used for gene silencing because of their sufficient resistance to nucleases and ability to induce RNase H functions. However, their profiles of binding affinity to the target sequences, specificity, and cellular uptake are less satisfactory. The second generation of antisense modifications was aimed at improving these properties, among which substitutions of position 2' of ribose with an alkoxyl group (e.g., methyl or methoxyethyl groups) were most successful. 2'-O-methyl and 2'-O-methoxyethyl derivatives can be further combined with phosphorothioate linkage (8). The third generation contains structural elements, such as zwitterionic oligonucleotides (possessing both positive and negative charges in the molecule); locked nucleic acids (LNAs)/bridged nucleic acids (BNAs) (9); morpholino (10); peptide nucleic acids (PNAs) (with a pseudopeptide backbone) (11); and, more recently, hexitol nucleic acids (HNA) (12). All of the modifications enhanced AA-ODNs in terms of nuclease resistance; specific binding; and with agents such as PNA and morpholino, cellular uptake. However, the ability of oligonucleotides to induce RNase H cleavage was abolished by these alterations. Therefore, chimeric oligonucleotides with an unmodified RNase H-susceptible core flanked by modified nuclease-resistant nucleotides have recently been proposed to address this issue and applied in a number of investigations (13), including clinical trials.

2.4. Delivery of Antisense

Oligonucleotides are primarily taken up by cells via endocytosis. Only a portion of oligonucleotides are able to escape endosome/lysome, enter the nucleus, and bind to its RNA complement. Because of the hydrophilic and

Phosphodiester Phosphorothioate

Fig. 1. Structures of synthetic oligonucleotides: (A) phosphorothioate. (B) 2'-O-methyl phosphorothioate; (C) 2'-O-aminopropyl ^ phosphodiester; (D) locked/bridged nucleic acids (LNA/BNA); (E) phosphoramidate; (F) morpholino; (G) peptide nucleic acid ^ (PNA); (H) hexitol nucleic acid (HNA). ^

macromolecular nature, permeation of oligonucleotides across cell membrane is relatively difficult. Even after two decades of research, safe and efficient delivery of oligonucleotides in vivo still remains a major barrier to the clinical success of antisense therapies. Cationic liposomes and electroporation are commonly used carriers. A large variety of liposomal formulas have been developed to facilitate antisense delivery, some of which have entered clinical trials (14). More recently, nanoparticles and oligonucleotide conjugates have shown improved cellular uptake, biodistribution, and targeted delivery, especially in cancer treatment (15,16). A hydrodynamic tail vein injection has proven very effective in delivering oligonucleotides into liver of rodents (17). Inhalable and topical applications of oligonucleotides in patients have shown satisfactory profiles of uptake and distribution (18,19). However, interestingly, most AS-ODNs that are therapeutically valuable in animal models and in patients have been administered in the form of naked compounds, despite the progress in antisense delivery.

2.5. Antisense in Therapies

Antisense therapeutics has seen its ups and downs since the first antisense trial was planned in leukemia in 1992 (20), followed by the excitement over the FDA approval of the first antisense drug, Fomivirsen, for the treatment of cytomeglovirus (CMV) retinitis in 1998 (21). In addition, more recently, a phase III trial reported disappointing results for Affinitak (an antisense inhibitor of protein kinase C-a [PKC-a]) for the treatment of non-small cell lung cancer (NSCLC). Cancer is the major target of ongoing clinical trials using antisense therapies, followed by human immunodeficiency virus (HIV) and other immune-related diseases (Table 1). The targets of antisense for cancer treatment include genes involved in cell growth, apoptosis, angiogenesis, and metastasis. A limitation for antisense as a therapy for cancer may be the singletarget approach. Even if the target is successfully inhibited by antisense, other targets may be activated and compensate for the antisense inhibition. Another potential problem is that for successful suppression of cancer growth, the inhibition should be 100%. However, the mechanism of antisense inhibition is always in competition with constitutive copies of mRNA, making a 100% knockdown difficult to achieve. It is noteworthy that after extensive efforts at endogenous expression of antisense RNA by plasmids and viral vectors in a variety of disease models, viral delivery of antisense has recently advanced to human patients; VRX 496 (a lentivirus vector encoding antisense to HIV-1 env protein) started its phase I trial in 2003. Cancer vaccine, a cell therapy using NSCLC cell lines genetically engineered to express transforming growth fac-tor-p (TGF-P) antisense, has also been tested in patients with lung cancer. With the emergence of new generations of modified oligonucleotides and delivery

Ongoing Clinical Trials for Antisense Therapy

mRNA target

Drug

Company

Diseases"

Phase

Notes6

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