Table Of ContentHandbook of
Experimental Pharmacology
Volume 131
Editorial Board
G. V.R. Born, London
P. Cuatrecasas, Ann Arbor, MI
D. Ganten, Berlin
H. Herken, Berlin
K. Starke, Freiburg i. Br.
Springer-Verlag Berlin Heidelberg GmbH
Antisense Research
and Application
Contributors
S. Agrawal, T. Akiyama, C.F. Bennett, M. Butler,
B.J. Chiasson, T.P. Condon, P.D. Cook, S.J. Craig,
R.M. Crooke, S.T. Crooke, G. Davidkova, N.M. Dean,
F.A. Dorr, D. Fabbro, R.S. Geary, T. Geiger, A.M. Gewirtz,
M.O. Hebb, S.P. Henry, M. Hogan, S.L. Hutcherson,
F. Kalkbrenner, D.L. Kisner, A.M. Krieg, J.M. Leeds,
A.A. Levin, R.R. Martin, B.P. Monia, D.K. Monteith,
M. Müller, P.L. Nicklin, P.E. Nielsen, J.A. Phillips,
H.A. Robertson, P.J. Schechter, G. Schultz, W.R. Shanahan,
Jr., M.V. Templin, B. Weiss, B . Wittig, R. Zhang
Editor
Stanley T. Crooke
fi» Springer
STANLEY T. Crooke, M.D., Ph.D.
ISIS Pharmaceuticals, Inc.
Carlsbad Research Center
2292 Faraday Avenue
Carlsbad, CA 92008
USA
With 118 Figures and 42 Tables
ISBN 978-3-642-63730-8 ISBN 978-3-642-58785-6 (eBook)
DOI 10.1007/978-3-642-58785-6
Library of Congress Cataloging-in-Publication Data
Antisense research and application/contributors, S. Agrawel... [et al.]; editor, Stanley T. Crooke. p. cm. —
(Handbook of experimental pharmacology; v. 131) Includes bibliographical references and index.
ISBN 978-3-642-63730-8 1. Antisense nucleic acids—Therapeutic use. I. Agrawal, Sudhir.
II. Crooke, Stanley T. III. Series. [DNLM: 1. Antisense Elements (Genetics)—phermacology.
2. Antisense Elements (Genetics)—therapeutic use. Wl HA51L v. 131 1998/QV 185 A833 1998]
QP905.H3 vol.131 [RM666.A564] 815M s—dc21 [815'.31] DNLM/DLC for Library of Congress
97-48588
CIP
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Preface
For nearly two decades advances in molecular biology have resulted in ex
traordinary insights into normal and pathophysiological processes. Indeed, I
believe we are in the midst of a process that will redefine diseases in molecular
pathological terms. This process has already shown the central role that a
relatively few complex multigene families play in physiological processes and
in pathophysiological events. In the next few years the rate at which new
molecular targets will be identified will increase exponentially as the sequenc
ing of the human and other genomes proceeds.
As exciting as the progress in the elucidation of molecular physiological
process is, the identification of potential molecular targets is only the very first
step in the discovery and development of improved therapeutic modalities for
the vast array of diseases that remain undertreated. In fact, the identification
of multigene families, homologies between individual members of these fami
lies, and the likely roles of individual isotypes in disease states raises an
important question: How will we create drugs of sufficient specificity to selec
tively interact with individual isotypes of multigene families?
That antisense technology has arguably the best potential to rationally
design isotype selective inhibitors was obvious from its conception and led to
the excitement about the technology, both as a tool to dissect the roles of
various genes and to create new therapeutics. The question about antisense
was: Would it work?
The chapters in this volume, recent publications, and recent symposia -
such as the meeting sponsored recently by Nature Biotechnology - provide
reasonably compelling answers to the questions about the technology. All in
all, the data provide ample justification for cautious optimism. This technology
is clearly a remarkably valuable tool for dissecting pharmacological processes
and confirming the roles of various genes. Perhaps more importantly, it ap
pears that even the first-generation compounds, the phosphorothioates, may
have properties that support their use as drugs for selected indications, and
new generations of antisense drugs may broaden the therapeutic utility of
drugs based on this technology.
Nevertheless, it is important to remember that we are less than a decade
into the aggressive creation and evaluation of antisense technology. Moreover,
we are attempting to create an entirely new branch of pharmacology: a new
chemical class, oligonucleotides; a new receptor, RNA; a new drug receptor
VI Preface
binding motif, hybridization; and new post-receptor binding mechanisms.
Thus there are still many more questions than answers. So arguably we are at
the end of the beginning of this technology. There is a great deal more to do
before we understand the true value and limits of antisense, but we are buoyed
by the progress to date and look forward to the challenges ahead.
STANLEY T. CROOKE
List of Contributors
AGRAWAL, S., Hybridon, Inc., 620 Memorial Drive, Cambridge, MA 02139,
USA
AKIYAMA, T., Department of Oncology, The Institute of Medical Science,
The University of Tokyo, 4-6-1 Shirokanedai Minato-ku, Tokyo 108,
Japan
BENNETT, c.F., Isis Pharmaceuticals, Inc., Carlsbad Research Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
BUTLER, M., Isis Pharmaceuticals, Inc., Carlsbad Research Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
CHIASSON, B.J., Neurobiology Research Group, Department of Anatomy
and Cell Biology, Medical Sciences Bldg., 1 King's College Circle,
University of Toronto, Toronto, Ontario, Canada M5S lA8
Current address: Novartis Pharmaceuticals Canada Inc.
525 University Avenue, Suite 1025, Toronto,
Ontario, M5G 2L3 Canada
CONDON, T.P., Isis Pharmaceuticals, Inc., Carlsbad Research Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
COOK, P.D., Isis Pharmaceuticals, Inc., Carlsbad Research Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
CRAIG, S.J., Novartis Horsham Research Centre, Wimblehurst Rd.,
Horsham, West Sussex RH12 4AB, Great Britain
CROOKE, R.M., Isis Pharmaceuticals, Inc., Carlsbad Research Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
CROOKE, S.T., Isis Pharmaceuticals, Inc., Carlsbad Research Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
DAVIDKOVA, G., Division of Neuropharmacology, Department of
Pharmacology, MCP Hahnemann School of Medicine,
Allegheny University of Health Sciences, 3200 Henry Avenue,
Philadelphia, PA 19129, USA
VIII List of Contributors
DEAN, N.M., Department of Pharmacology, Isis Pharmaceuticals, Inc.,
Carlsbad Research Center, 2292 Faraday Ave.,
Carlsbad, CA 92008, USA
DORR, F.A., Isis Pharmaceuticals, Inc., Carlsbad Research Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
FABBRO, D., Pharmaceuticals Research, Oncology, K-125.4.1O,
Unit: Growth Control, Novartis, Ltd., CH-4002 Basel, Switzerland
GEARY, RS., Isis Pharmaceuticals, Inc., Carlsbad Research Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
GEIGER, T., Pharmaceuticals Research, Oncology, K-125.2.12,
Unit: Growth Control, Novartis, Ltd., CH-4002 Basel, Switzerland
GEWIRTZ, A.M., Departments of Pathology and Laboratory Medicine and
Internal Medicine, University of Pennsylvania School of Medicine,
Room 513b Stellar Chance Building, 422 Curie Boulevard, Philadelphia,
PA 19104, USA
HEBB, M.O., Laboratory of Molecular, Neurobiology, Department of
Pharmacology, Dalhousie University, Halifax, Nova Scotia,
Canada B3H 4H7
HENRY, S.P., Isis Pharmaceuticals, Inc., Carlsbad Researh Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
HOGAN, M., Department of Molecular Physiology and Biophysics,
Baylor College of Medicine, T405, One Baylor Plaza, Houston,
TX 77030, USA
HUTCHERSON, S.L., Isis Pharmaceuticals, Inc., Carlsbad Research Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
KALKBRENNER, F., Institut fur Pharmakologie, Freie Universitat Berlin,
Thielallee 69173, D-14195 Berlin, FRG
KISNER, D.L., Isis Pharmaceuticals, Inc., Carlsbad Research Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
KRIEG, A.M., University of Iowa, Department of Internal Medicine,
540 EMRB, Iowa City, IA 52242, USA
LEEDS, 1.M., Isis Pharmaceuticals, Inc., Carlsbad Research Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
LEVIN, A.A., Isis Pharmaceuticals, Inc., Carlsbad Research Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
MARTIN, RR, Drug Development, Hybridon, Inc., 620 Memorial Drive,
Cambridge, MA 02139, USA
List of Contributors IX
MONIA, B.P., Department of Molecular Pharmacology, Isis Pharmaceuticals,
Inc., Carlsbad Research Center, 2292 Faraday Ave., Carlsbad,
CA 92008, USA
MONTEITH, D.K., Isis Pharmaceuticals, Inc., Carlsbad Research Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
MULLER, M., Pharmaceuticals Research, Oncology, K-125.4.43,
Unit: Growth Control, Novartis, Ltd., CH-4002 Basel, Switzerland
NICKLIN, P.L., Novartis Horsham Research Centre, Wimblehurst Rd.,
Horsham, West Sussex RH12 4AB, Great Britain
NIELSEN, P.E., Center for Biomolecular Recognition, Department of
Medical Biochemistry and Genetics, Biochemical Laboratory B, The
Panum Institute, Blegdamsvej 3c, DK-2200 Copenhagen N, Denmark
PHILLIPS, J.A., Novartis Horsham Research Centre, Wimblehurst Rd.,
Horsham, West Sussex RH12 4AB, Great Britain
ROBERTSON, H.A., Laboratory of Molecular, Neurobiology, Department of
Pharmacology, Dalhousie University, Halifax, Nova Scotia,
Canada B3H 4H7
SCHECHTER, P.J., Drug Development, Hybridon, Inc., 620 Memorial Drive,
Cambridge, MA 02139, USA
SCHULTZ, G., Institut flir Pharmakologie, Freie Universitat Berlin,
Thielallee 69173, D-14195 Berlin, FRG
SHANAHAN, W.R., Jr., Isis Pharmaceuticals, Inc., Carlsbad Research Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
TEMPLIN, M.Y., Isis Pharmaceuticals, Inc., Carlsbad Research Center,
2292 Faraday Ave., Carlsbad, CA 92008, USA
WEISS, B., Division of Neuropharmacology, Department of Pharmacology,
MCP Hahnemann School of Medicine, Allegheny University of Health
Sciences, 3200 Henry A venue, Philadelphia, P A 19129, USA
WITTIG, B., Institut flir Molekularbiologie und Biochemie,
Freie UniversWit Berlin, Arminallee 22-24, D-14195 Berlin, FRG
ZHANG, R., Division of Clinical Pharmacology and Department of
Pharmacology and Toxicology, University of Alabama at Birmingham,
Volker Hall 113, Box 600, 1670 University Boulevard, Birmingham,
AL 35294-0019, USA
Contents
CHAPTER 1
Basic Principles of Antisense Therapeutics
S.T. CROOKE. With 3 Figures.. ..... .... .... ..... ......... .... .... 1
A. Introduction ............................................ 1
B. Proof of Mechanism ..................................... 1
I. Factors that May Influence Experimental
Interpretations ..................................... 1
1. Oligonucleotide Purity. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Oligonucleotide Structure . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. RNA Structure .................................. 2
4. Variations in In Vitro Cellular Uptake and
Distribution .................................... 2
5. The Binding to and Effects of Binding to
Nonnucleic Acid Targets ......................... 3
6. Terminating Mechanisms ......................... 3
7. Effects of "Control Oligonucleotides" .............. 4
8. Kinetics of Effects ............................... 4
II. Recommendations ................................. 4
1. Positive Demonstration of Antisense
Mechanism and Specificity ........................ 4
C. Molecular Mechanisms of Antisense Drugs ................. 5
I. Occupancy Only Mediated Mechanisms ............... 5
1. Inhibition of Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2. Translational Arrest ............................. 6
3. Disruption of Necessary RNA Structure ............ 7
II. Occupancy Activated Destabilization ................. 8
1. 5' Capping ...................................... 8
2. Inhibition of 3' Polyadenylation . . . . . . . . . . . . . . . . . . . . 9
III. Other Mechanisms ................................. 9
IV. Activation of RNase H . ......... ..... ......... ..... . 10
V. Activation of Double-Strand RNase .................. 12
D. Characteristics of Phosphorothioate Oligodeoxynucleotides ... 12
I. Introduction ....................................... 12
II. Hybridization ...................................... 13
