Antisense Technology: Applications Part B

660 Pages·1999·11.954 MB·English

by  Abelson J.N., Simon M.I., Phillips I.M.

Preface
Antisense technology reached a watershed year in 1998 with the FDA
approval of the antisense-based therapy, Vitravene, developed by ISIS.
This is the first drug based on antisense technology to enter the marketplace
and makes antisense technology a reality for therapeutic applications. How-
ever, antisense technology still needs further development, and new applica-
tions need to be explored.
Contained in this Volume 314 (Part B) of Methods in Enzymology and
its companion Volume 313 (Part A) are a wide range of methods and
applications of antisense technology in current use. We set out to put
together a single volume, but it became obvious that the variations in
methods and the numerous applications required at least two volumes, and
even these do not, by any means, cover the entire field. Nevertheless, the
articles included represent the work of active research groups in industry
and academia who have developed their own methods and techniques. In
this volume, Part B: Applications, chapters cover methods in which anti-
sense is designed to target membrane receptors and antisense application
in the neurosciences, as well as in nonneuronal tissues. The therapeutic
applications of antisense technology, the latest area of new interest, com-
plete the volume. In Part A: General Methods, Methods of Delivery, and
RNA Studies several methods of antisense design and construction are
included as are general methods of delivery and antisense used in RNA
studies.
Although Methods in Enzymology is designed to emphasize methods,
rather than achievements, I congratulate all the authors on their achieve-
ments that have led them to make their methods available. In compiling
and editing these two volumes I could not have made much progress without
the excellent secretarial services of Ms. Gayle Butters of the University of
Florida, Department of Physiology.
M. IAN PHILLIPS
VX
Contributors to Volume 314
Article numbers are in parentheses following the names of contributors.
Affiliations listed are current.
FE C. EIDAGOBA (10), Wellcome Laboratory DIVAD A. NWORB (10), Wellcome Laboratory
for Molecular Pharmacology, Department for Molecular Pharmacology, Department
of Pharmacology, University College Lon- of Pharmacology, University College Lon-
don, London WC1E 6BT, United Kingdom don, London WC1E 6BT, United Kingdom
YIJIA BAO (12), Vysis, Inc., Downers Grove, NOEL J. YELKCUB (10), School of Biochemis-
Illinois 51506 try and Molecular Biology, University of
Leeds, Leeds LS2 9JT, United Kingdom
RUTH BEATTIE (21), Lilly Research Centre,
Eli Lilly and Company Limited, Win- ANIRAM SACISTAC (11), Department of Physi-
dlesham, Surrey GU20 6PH, United ology, University College London, London,
Kingdom United Kingdom
YREGRAM C. DLEFNIEB (8), Department of NAFETS SACISTAC (11), Institut de Biologie
Pharmacology and Experimental Thera- Cellulaire et de Morphologie, Universit~ de
peutics, Tufts University School of Medi- Lausanne, CH-I 50O Lausanne, Switzerland
cine, Boston, Massachusetts 11120 MLOCLAM P. DLEIFLUAC (10), Wellcome Lab-
oratory for Molecular Pharmacology,
ONATEAG IHCSAMAGREB (30), Dipartimento
Department of Pharmacology, University
di Medicina Interna e Terapia Medica,
College London, London WC1E 6BT,
Medicina Interna e Oncologia Medica,
United Kingdom
I.R.C.C.S. Policlinico San Matteo, 27100
Pavia, Italy OIRAM ALOZZAC (30), Dipartimento di Medi-
cina Interna e Terapia Medica, Medicina
E. A. L. NESSEIB (23), Division of Biophar-
Interna e Oncologia Medica, I.R.C.C.S.
maceutics, Leiden~Amsterdam Center for
Policlinico San Matteo, 27100 Pavia, Italy
Drug Research, Leiden University, 2300
RA Leiden, The Netherlands NIM-GNrLYB CHOl (34), Department of Micro-
biology and Immunology, Wonkwang Uni-
M. K. HCSOBRETSJIB (23), Division of Bio-
versity School of Medicine, Iksan-shi,
pharmaceutics, Leiden~Amsterdam Center
Chonbug ,947-075 Korea
for Drug Research, Leiden University, 2300
RA Leiden, The Netherlands CHUAN-CHu CHOU (29), Department of Pa-
thology, New Jersey Medical School, Uni-
H. E. MULB (37), Department of Medicine II,
versity of Medicine and Dentistry of New
University of Freiburg, D-79106 Freiburg,
Jersey, Newark, New Jersey 30170
Germany
HuN-TAEG GNUHC (34), Department of Mi-
CIR~ID~/RF BOST (24), Sidney Kimmel Cancer
crobiology and Immunology, Wonkwang
Center, San Diego, California 12129
University School of Medicine and Medici-
NITSUD H. O. BRITrON (12), DuPont Pharma- nal Resources Research Center of Wonk-
ceuticals, Wilmington, Delaware 19880 wang University, Iksan-shi, Chonbug 570-
MAILLIW C. SUDDAORB (9), Division of Neu- ,947 Korea
rosurgery and Department of Anatomy, ENIREHTAC L. 1FFOIC (25), Department of
Medical College of Virginia, Virginia Com- Metabolic and Cardiovascular Diseases,
monwealth University, Richmond, Virginia Novartis Institute for Biomedical Research,
1360-89232 Summit, New Jersey 10970
ix
X CONTRIBUTORS TO VOLUME 314
RETEP J. CRAIG (21), Lilly Research ,ertneC REIVAJ GARZ6N (1), Department of Neuropa-
Eli Lilly and Company Limited, Win- thology, Cajal Institute, Consejo Superior
dlesham, Surrey GU20 6PH, United de Investigaci6nes Cientificas, E-28002
Kingdom Madrid, Spain
ACINOM CURTO (18), Department of Cyto- AHTNAMAS GILLARD (21), Department of
morphology, School of Medicine, alledattiC Pharmacology, University of ,ogacihC Chi-
,ytisrevinU ,irailgaC Italy ,ogac Illinois 60637
MARIZA DAYRELL (10), Wellcome Labora- EGROEG T. SEILLIG (9), Department of Bio-
tory for Molecular Pharmacology, Depart- medical Engineering, Health Sciences Cen-
ment of Pharmacology, University College ter, University of Virginia, ,ellivsettolrahC
London, London WC1E 6BT, United Virginia 22908
Kingdom SUSAN ETIREGOF-DLUOG (29), Department of
SALOHCIN M. DEAN (24), ISIS Pharmaceuti- Pathology, New Jersey Medical School,
,slac Inc., Carlsbad, California 92008 University of Medicine and Dentistry of
LEBASI ED OINOTNA (1), Department of Neu- New Jersey, Newark, New Jersey 30170
ropathology, Cajal Institute, Consejo Supe- MARIA GRAZIA ENNAS (18), Department
rior de Investigaci6nes ,sacifitneiC E-28002 of Cytomorphology, School of Medicine,
Madrid, Spain alledattiC University, ,irailgaC Italy
KCIRTAP SAMLED (10), Wellcome Laboratory AIVLUF OMERG (18), Department of Cytomor-
for Molecular Pharmacology, Department phology, School of Medicine, alledattiC
of Pharmacology, University College Lon- ,ytisrevinU ,irailgaC Italy
don, London WC1E 6BT, United Kingdom ELEIRBAG HOLGNINNERG (11), Institut de
LEHCIM DE DRAAW (21), Laboratoire de Neu- Biologie Cellulaire et de Morphologie,
robiologie des Canaux Ioniques, INSERM ~tisrevinU de Lausanne, CH-IO05 Lau-
,473U 13916 Marseille Cedex ,02 France sanne, Switzerland
LEAHCIM G. DUBE (13), Department of Physi- JANE E. HALEY (10), Wellcome Laboratory
ology, University of Florida Brain ,etutitsnI for Molecular Pharmacology, Department
University of Florida College of ,enicideM of Pharmacology, University College Lon-
,ellivseniaG Florida 4720-01623 don, London WC1E 6BT, United Kingdom
LECRAM EGGER (32), Department of Physiol- NNIF HALLB00K (11), Department of Neuro-
ogy, University of Bern, CH-3012 Bern, ,ecneics Uppsala ,ytisrevinU BMC, S-751 23
Switzerland ,alasppU Sweden
ANNE FELTZ (21), Laboratoire de Neuro- XAM HE (29), Department of Pathology, New
biologie Cellulaire, UPR 9009 Centre Jersey Medical School, University of Medi-
National de al Recherche Scientifique, cine and Dentistry of New Jersey, Newark,
67084 Strasbourg, France New Jersey 30170
NELEH L. EROMLLIF (9), Division of Neu- WEHTTAM O. HEBB (19), Faculty of ,enicideM
rosurgery, Medical College of ,ainigriV University of Toronto, Toronto, Ontario,
Virginia Commonwealth University, Rich- Canada M5S 1A8
mond, Virginia 1360-89232 SUKRAM HEILIG (19), Department of lacinilC
K. FLUITER (23), Division of Biopharma- Neuroscience, Occupational Therapy and
ceutics, Leiden~Amsterdam Center for Elderly Care Research, Karolinska ,etutitsnI
Drug Research, Leiden University, 2360 Stockholm, Sweden
RA Leiden, The Netherlands JULIE G. RELSNEH (6), Department of Phar-
LEAHCIM B. GANZ (26), Department of macology, University of Texas Health Sci-
Medicine, Case Western Reserve ,ytisrevinU ence Center, San Antonio, Texas -48287
Cleveland, Ohio 44106 4677
CONTRIBUTORS TO VOLUME 314 xi
WEIS PENG HO (12), DuPont Pharmaceuti- ENIHPESOJ LAI (14), Department of Pharma-
cab, Wilmington, Delaware 19880 cology, University of Arizona, Tucson, Ari-
zona 85724
YERFFEJ T. HOLT (35), Departments of Cell
Biology and Pathology, Vanderbilt Univer- SIGER C. TREBMAL (21), Laboratoire de Neu-
sity Medical School, Nashville, Tennessee robiologie Cellulaire, UPR 9009 Centre Na-
23273 tional de la Recherche Scientifique, 67084
Strasbourg, France
ALAN L. HUDSON (5), Psychopharmacology
,tinU School of Medical Sciences, University PETER LIPP (32), Laboratory of Molecular
of Bristol, Bristol BS8 1TD, United Signalling, The Babraham Institute, Cam-
Kingdom bridge CB2 3E J, United Kingdom
JOHN C. RETNUH (14), Center for Biological DI Lu (39), Department of Physiology, Uni-
Research, Roche Bioscience, Palo Alto, versity of Florida College of Medicine,
California 94304 Gainesville, Florida 32610
ANTrI P. NENUKEJ (36), Department of Clini- CLAUDIA LUCOTI'I (30), Dipartimento di
cal Pharmacology, Helsinki University Cen- Medicina Interna e Terapia Medica,
tral Hospital, FIN-O0029 HYKS, Finland Medicina Interna e Oncologia Medica,
I.R.C.C.S. Policlinico San MatteD, 27100
ROY A. NESNEJ (35), Departments of Pathol-
Pavia, Italy
ogy and Cell Biology, Vanderbilt University
Medical School, Nashville, Tennessee 23273 DIVAD L. NOS'ITAM (27), Department of Phys-
ANELAD~AM AVOZSAHUJ (22), Department of iology, Medical College of Wisconsin, Mil-
waukee, Wisconsin 53226
Physiology, University of Maryland School
of Medicine, Baltimore, Maryland 10212 SEVY MAULET (21), Laboratoire de Neuro-
KUD-GNAI~C JUN (34), Department of Mi- biologie Cellulaire, UPR 9009 Centre Na-
crobiology and Immunology, Wonkwang tional de la Recherche Scientifique, 67084
Strasbourg, France
University School of Medicine, lksan-shi,
Chonbug ,947-075 Korea TREBOR McKAY (24), ISIS Pharmaceuticals,
IVELAK J. A. OMERIAK (36), Department of Inc., Carlsbad, California 92008
Clinical Chemistry, Norwegian University
DAN ALOCREM (24), Sidney Kimmel Cancer
of Science and Technology, N-7006 Trond-
Center, San Diego, California 92121, and
heim, Norway
Center for Molecular Genetics, University
APHSUP S. KALRA (13), Department of Physi- of California at San Diego, La JoUa, Cali-
ology, University of Florida Brain Institute, fornia 39029
University of Florida College of Medicine,
ALIMDAR CINSUELIM (15), Department of Bi-
Gainesville, Florida 11623
ological Sciences, The Open University,
AVTAS P. KALRA (13), Department of Neuro- Milton Keynes MK7 6AA, United Kingdom
science, University of Florida Brain Insti-
tute, University of Florida College of Medi- ARAMGAD MOHUCZY (3), Department of
Physiology, University of Florida College
cine, Gainesville, Florida 11623
of Medicine, Gainesville, Florida 32610
REPSEJ ELRAK (2), Department of Psychiatry,
Rigshospitalet (National Hospital), DK- BRETr P. AINOM (25), Department of Molecu-
2100 Copenhagen, Denmark lar Pharmacology, ISIS Pharmaceuticals,
Inc., Carlsbad, California 80029
LEAHCIM J. HCIVOTAK (39), Department of
Pharmacodynamics, College of Pharmacy, D. RUOPDAROM (37), Department of Medi-
University of Florida, Gainesville, Florida cine II, University of Freiburg, D-79106
O1623 Freiburg, Germany
xii COmmBUTORS TO VOLUME 314
ALLEBASI INISSUM (18), CNR Center of Mus- M. NAI SPILLIHP (3), Department of Physiol-
cle Biology and Physiopathology, Univer- ogy, University of Florida College of Medi-
sity of Padova, Padova, Italy cine, Gainesville, Florida 32610
AGNI D. NNAMUEN (16), Max Planck Institute KNARF ACERROP (14), Department of Phar-
of Psychiatry, D-80804 Munich, Germany macology, University of Arizona, Tucson,
SNEGOM NESLEIN (2), Research Institute of Arizona 85724
Biological Psychiatry, .tS Hans Hospital O. AVOPATOP (24), Laboratory of Biological
DK-4000 Roskilde, Denmark Chemistry, Gerontology Research Center,
National Institute of Aging, National Insti-
TSNRE NIGGLI (32), Department of Physiol-
ogy, University of Bern, CH-3012 Bern, tute of Health, Baltimore, Maryland 21224
Switzerland SUJIT S. UHBARP (9), Division of Neurosur-
SOEODAHT S. ,KAWON JR. (17), Departments gery, Medical College of Virginia, Virginia
of Anesthesiology and Resuscitology, Commonwealth University, Richmond, Vir-
Okayama University School of Medicine, ginia 1360-89232
Shikata-cho, Okayama City, Japan NAHOM K. RAIZADA (39), Department of
ORIHAKAT OCHIYA (28), Section for Studies Physiology, University of Florida College
on Metastasis, National Cancer Center Re- of Medicine, Gainesville, Florida 32610
search Institute, Chuo-ku, Tokyo 104- ELIZABETH S. RAVECHI~ (29), Department of
,5400 Japan Pathology, New Jersey Medical School,
S. REGREPSNEFFO (37), Department of Medi- University of Medicine and Dentistry of
cine II, University of Freiburg, D-79106 New Jersey, Newark, New Jersey 30170
Freiburg, Germany SILLYHP Y. SEVAER (39), Department of Phys-
WOLF-BERNHARD OFFENSPERGER (37), De- iology, University of Florida College of
partment of Medicine II, University of Frei- Medicine, Gainesville, Florida 32610
burg, D-79106 Freiburg, Germany EMMA S. J. ROBINSON (5), Psychopharma-
MICHAEL H. OssIvov (14), Department of cology Unit, School of Medical Sciences,
Pharmacology, University of Arizona, Tuc- University of Bristol, Bristol BS8 1TD,
son, Arizona 85724 United Kingdom
HYuN-OCK PAL (34), Department of Micro- CHERYL ROBINSON-BENION (35), Department
biology and Immunology, Wonkwang Uni- of UeC Biology, Vanderbilt University Med-
versity. School of Medicine, Iksan-shi, ical School, Nashville, Tennessee 23273
Chonbug ,947-075 Korea VITTORIO ROSTI (30), Laboratorio di Ricerca
NAIX-GNIY NAP (4), Memorial Sloan-Ketter- Area Trapianti, Unitd di lmmunologia Clin-
ing Cancer Center, New York, New York ica, I.R.C.C.S. Policlinico San Matteo,
12001 27100 Pavia, Italy
EGROEG A. REKRAP (29), Department of Pa- E. T. RUMP (23), Division of Biopharmaceu-
thology, New Jersey Medical School, Uni- tics, Leiden/Amsterdam Center for Drug
versity of Medicine and Dentistry of New Research, Leiden University, 2300 RA
Jersey, Newark, New Jersey 30170 Leiden, The Netherlands
GAVRm W. KANRETSAP (4), Memorial Sloan- PILAR S~NCHEZ-BL/~ZQUEZ (1), Department
Kettering Cancer Center, New York, New of Neuropathology, Cajal Institute, Consejo
York 12001 Superior de Investigaci6nes Cientificas,
E-28002 Madrid, Spain
BmAI PENG (29), Department of Pathology,
New Jersey Medical School University of ENNAOJ M. ITTIZLACS (6), Department of
Medicine and Dentistry of New Jersey, New- Pharmacology, New York University Medi-
ark, New Jersey 30170 cal School, New York, New York 10016
CONTRIBUTORS TO VOLUME 314 xiii
HPOTSIRHC REHCAMUHCS (31), MCD Re- MlraCo NENUHNET (36), Department of On-
search, Novartis Pharmaceuticals Corpora- cology, Helsinki University Central Hospi-
tion, Summit, New Jersey 8931-10970 tal, FIN-O0029 HYKS, Finland
BEAT RELLAWHCS (32), Department of Histol- IKAASAM ADARET (28), National Cancer Cen-
ogy and General Embryology, University of ter, Chuo-ku, Tokyo ,5400-401 Japan
Fribourg, CH-1700 Fribourg, Switzerland C. THOMA (37), Department of Medicine,
University of Freiburg, D-79106 Freiburg,
LEAHCIM S. YLLUCS (12), DuPont Pharma-
Germany
ceuticals, Wilmington, Delaware 19880
GNAGFLOW REYEMHCSIT (20), Leibniz Insti-
ALLEIRAM UZTES (18), Department of Cyto-
tute for Neurobiology, D-39008 Magde-
morphology, School of Medicine, Cittadella
burg, Germany
University, Cagliari, Italy
ALOCIN IHCSOT (16), Max Planck Institute of
A. PAULA TSEUW-sE3(MIS (33), Department Psychiatry, D-80804 Munich, Germany
of Internal Medicine, University Hospital
T. J. C. NAV LEKREB (23), Division of Bio-
Zurich, CH-8044 Zurich, Switzerland
pharmaceutics, Leiden~Amsterdam Center
NITRAM K. IKSN1ZDOLS (22), Department of for Drug Research, Leiden University, 2300
Physiology, University of Maryland School RA Leiden, The Netherlands
of Medicine, and Department of Medicine,
H. HCSTEIV (23), Division of Biopharmaceu-
Mercy Hospital, Baltimore, Maryland
tics, Leiden~Amsterdam Center for Drug
10212
Research, Leiden University, 2300 RA
TENAJ B. HTIMS (38), Departments of Microbi- Leiden, The Netherlands
ology and Immunology, Kimmel Cancer YTESEAD NAHDRAVUNHSIV (8), Department
Center, Cardeza Foundation for Hemato- of Pharmacology and Experimental Thera-
logical Research, Thomas Jefferson Univer- peutics, Tufts University School of Medi-
sity, Philadelphia, Pennsylvania 3805-70191 cine, Boston, Massachusetts 11120
AIRELAV SOGOS (18), Department of Cyto- NEHPETS G. NESLOV (21), Lilly Research
morphology, School of Medicine, Cittadella Centre, Eli Lilly and Company Limited,
University, Cagliari, Italy Windlesham, Surrey GU20 6PH, United
Kingdom
NAWH-GNOD SOHN (34), College of Phar-
macy, Wonkwang University, Iksan-shi, F. NOY REKC~.SZIEW (37), Department of
Chonbug ,947-075 Korea Medicine II, University of Freiburg,
D-79106 Freiburg, Germany
GNAGFLOW REMMOS (19), Department of
IEWGNOH GNAW (39), Department of Physiol-
Clinical Neuroscience, Occupational Ther-
ogy, University of Florida College of Medi-
apy and Elderly Care Research, Karolinska
cine, Gainesville, Florida 32610
Institute, Stockholm, Sweden
ERIC MORTSKCIW (38), Department of Micro-
YLLEK M. REFIDNATS (7), Department of biology, Thomas Jefferson University, Phil-
Pharmacological and Pharmaceutical Sci-
adelphia, Pennsylvania 19107
ences, University of Houston, Houston,
ANNASUS Wu-PoNG (9), Department of
Texas 5155-40277
Pharmaceutics, Medical College of Vir-
JULIE K. ELPATS (11), Institut de Biologie Cel- ginia, Virginia Commonwealth University,
lulaire et de Morphologie, UniversiM de Richmond, Virginia 23298
Lausanne, CH-I 50O Lausanne, Switzerland
AKATUY YAIDA (17), Departments of Anes-
GNIPOAIX TANG (3), Department of Physiol- thesiology and Resuscitology, Okayama
ogy, University of Florida College of Medi- University School of Medicine, Shikata-cho,
cine, Gainesville, Florida 32610 Okayama City, Japan
xiv CONTRIBUTORS TO VOLUME 314
Gq/AHC-IJ OOY (34), Department of Microbi- EWU E~ITIW-RETSIEMEGNAZ (33), Depart-
ology and Immunology, Wonkwang Uni- ment of lnternal Medicine, University Hos-
versity School of Medicine, Iksan-shi, pital Zurich, CH-8044 Zurich, Switzerland
Chonbug ,947-075 Korea
IMJtSTAK UFUY (17), Departments of Anesthe- EIRAMENNA ZIEGLER (33), Laboratory for
siology and Resuscitology, Okayama Uni- Molecular and Cellular Oncology, Cancer
versity School of Medicine, Shikata-cho, Program, Faculty of Medicine, Catholic
Okayama City, Japan University of Chile, Santiago, Chile
1 CNS 3
ANTISENSE AND OPIOID RECEPTOR SIGNALING IN
I In Vivo Modulation of G Proteins and Opioid Receptor
Function by Antisense Oligodeoxynucleotides
By JAVIER GARZ6N, ISABEL DE ANTONIO,
and PILAR SANCHEZ-BLkZQUEZ
Introduction
Opioids promote their effects by acting on cellular receptors located in
the cell membrane. The diversity of these receptors, which was initially
suggested by pharmacological and biochemical studies, has been confirmed
by the cloning of cDNAs encoding the/z, 2'I and w s types. The cloned
8~ 3'4
opioid receptors have been found to correspond to the pharmacological
subtypes tzl, 82, and .1K The subtypes ~2, 81, ,2K and 3K (and possibly
others) are still to be designated molecular identities. The use of antisense
technology with the opioid system has been helpful in making molecular/
pharmacological correlations and the approach is currently being used to
investigate uncorrelated subtypes.
Opioid receptors couple to heterotrimeric (a,/3, and '3 subunits) GTP-
binding regulatory proteins (G proteins). Our knowledge regarding the
diversity and properties of G proteins has increased greatly. s-6 Most of the
known classes are present in the nervous system and are thought to regulate
various signaling pathways, e.g., adenylyl cyclases, different types of K +
and Ca +2 channels, phospholipases, protein kinases, and others. Ga subunits
show differences in the regions involved in the interaction with membrane
receptors (see, e.g., Jones and Reed9). These variations seem to account
1 y. Chen, A. Mestek, J. Liu, J. A. Hurley, and L. Yu, Mol. Pharmacol. 44, 8 (1993).
2 R. C. Thompson, A. Mansour, H. Akil, and S. J. Watson, Neuron 11, 903 (1993).
3 C. J. Evans, D. E. Keith, H. Morrison, K. Magendzo, and R. H. Edwards, Science 28,
1952 (1992).
4 B. L. Kieffer, K. Befort, C. Gaveriaux-Ruff, and C. G. Hirth, Proc. Natl. Acad. Sci. U.S.A.
89, 12048 (1992).
5 G. X. Xie, F. Meng, A. Mansour, R. C. Thompson, M. T. Hoversten, A. Goldstein, .S T.
Watson, and H. Akil, Proc. Acad. Natl. Sci. U.S.A. 91, 3779 (1994).
6 n. E. Hamm and A. Gilchrist, Curr. Opin. Cell Biol. 8, 189 (1996).
7 E. J. Neer and T. F. Smith, Cell 84, 175 (1996).
8 D. G. Lambright, J. Sondek, A. Bohm, N. P. Skiba, H. E. Hamm, and P. B. Sigler, Nature
(London) 379, 311 (1996).
9 D. T. Jones and R. R. Reed, J. Biol. Chem. 262, 14241 (1987).
Copyright © 1999 by Academic Press
All rights of reproduction in any form reserved.
METHODS IN ENZYMOLOGY, VOL. 314 0076-6879/99 $30.00
4 ESNESITNA ROTPECER STEGRAT 11
for the preference displayed by agonist-bound receptors to signal only via
certain G proteins present in the cell membrane. 31-°1
Receptors also vary in the amino acid sequences that interact with to
subunits of trimeric G proteins, i.e., the receptor loop that links the fifth
and sixth transmembrane region and the C-terminal tail. 51'41 The cloned ~-
opioid receptor 4,3 and the /z-opioid receptor 2,1 differ in the intracellular
territories implicated in their interaction with Ga subunits. It is conceivable,
therefore, that distinct types or subtypes of opioid receptor regulate differ-
ent classes of G proteins. 16-t8 Given the variations manifested by receptors
and G proteins in their interacting domains, an investigation was made
into the classes of G proteins regulated in vivo by/x and ~ receptors in
the promotion of supraspinal antinociception. This chapter describes the
efficacy and selectivity in reducing the expression of coded signaling pro-
teins by in vivo administration of antisense oligodeoxynucleotides comple-
mentary to their mRNA sequences. Functional data are also provided in
order to assess the physiological relevance of opioid receptors and GTP-
binding protein subtypes.
Methods
Synthesis of Oligodeoxynucleotides
Synthetic end-capped phosphorothioate antisense oligodeoxynucleo-
tides (ODNs) are prepared by solid-phase phosphoramidite chemistry 91
using a CODER 300 DNA synthesizer (Du Pont; Wilmington, DE) at
the 1-/.~mol scale (Tables I and II). The introduction of phosphorothioate
linkages is achieved by tetraethylthiuram disulfide (TETD) sulfurization. °2
01 H. Ueda, .Y Yoshihara, H. Misawa, .N Fukushima, .M ,iU H. Takagi, and .M Satoh, .J
Biol. Chem. 264, 2373 .)9891(
11 .S .F Law, .S Zaina, R. Sweet, .K Yasuda, G. I. Bell, J. Stadel, and .T Reisine, Mol.
PharmacoL ,54 785 .)4991(
21 y. .F Lui, K. H. Jacobs, .M .M Rasenick, and .P R. Albert, J. Biol. Chem. 269, 08831 .)4991(
31 .S Offermanns, .T Wieland, D. Homann, J. Sandmann, E. Bombien, .K Spicher, G. ,ztluhcS
and K. H. Jakobs, Mol. Pharmacol. ,54 098 .)4991(
41 .C .W Taylor, Biochem. .J ,272 1 .)0991(
51 A. D. Strosberg, Eur. .J Biochem. ,691 1 .)1991(
61 j. Garz6n, .M A. Castro, J. .L Juarros, and .P ,zeuqzi~lB-zehcni~S Life Sci.-Pharmacol. Lett.
,45 PL191 .)4991(
71 j. Garz6n, A. Garcla-Espafia, and .P ,zeuqzi~lB-zehcni~S J. Pharmacol. Exp. Then. ,182
945 .)7991(
81 j. Garz6n, .M Castro, and .P ,zeuqzi~lB-zehcngS Eur. J. Neurosci. ,01 7552 .)8991(
91 .M D. Matteucci and .M H. Carouthers, .J Am. Chem. Soc. 103, 5813 .)1891(
02 H. Vu and B. .L Hirschbein, Tetrahedron Lett. ,23 5003 .)1991(
1 ANTISENSE AND OPIOID RECEPTOR SIGNALING IN CNS 5
TABLE I
SEDITOELCUNYXOEDOGILO OT Ix- DNA ~-OPIOID RECEPTOR mRNA
Receptora 5'-Sequence-3' Code Ref.
XI (r/m) C'G* CCCCAGCCTCTTCCT* C*T b Ixurf d, e
xI (m) C*T*GATGTrCCCTGGG*C*C Ixl6 23 e, f
xI (r/m) T*T*GGTGGCAGTCTTCATTTT*G*G 192,2/ 113 d, g
xI (r/m) T*G*AGCAGGTTCTCCCAGTAC*C*A 796-776XI ~d g
xI (r/m) G*G*GCAATGGAGCAGTrTC*T*G 4911_5711,1,/ d, g
6 (m) G*C*ACGGGCAGAGGGCACC*A*G 7~ 62 ~f h, i
6 (m) A*G*AGGGCACCAGCTCC*A*T 64-92~ ~f J
6 (r) A*C*TGCAGCTCCGCA*G*G 73_226 f
"The oligonucleotides correspond to those described in the code of the rat (r) or mouse
(m) opioid receptor gene sequence.
h An asterisk (*) indicates the phosphorothioate linkages.
c This ODN is directed to a specific 5' untranslated region of the/z-opioid receptor clone.
l, G. Rossi, Y.-X. Pan, J. Cheng, and G. W. Pasternak, Life Sci.-Pharmacol. Lett. 54,
PL375 (1994).
e p. S~inchez-Bl~izquez, M. Rodrfguez-Dfaz, I. DeAntonio, and J. Garz6n, .J Pharmacol.
Expt. Ther., in press.
fP. Sfinchez-Bl~izquez, A. Garcfa-Espafia, and J. Garz6n, .J Pharmacol. Exp. Ther. 280,
1423 (1997).
g G. Rossi, L. Leventhal, Y.-X. Pan, J. Cole, W. Su, R. J. Bodnar, and G. W. Pasternak,
.J Pharmacol. Exp. Ther. 281, 109 (1997).
h j. Lai, E. J. Bilsky, R. B. Rothman, and F. Porreca, Neuroreport ~5 1049 (1994).
i p. S~nchez-Blfizquez and J. Garz6n, .J Pharmacol. Exp. Ther. 285, 820 (1998).
J K. M. Standifer, C.-C. Chien, C. Wahlestedt, G. P. Brown, and G. W. Pasternak, Neuron
12, 805 (1994).
Solvents and reagents are obtained from Cruachem Ltd. (Glasgow, UK)
and TETD is from Applied Biosystems (Foster City, CA). The efficiency
of each base addition is higher than 98%. After synthesis the protecting
groups are removed by treatment with %23 (v/v) aqueous NH3 at 55 °
overnight. Crude ODNs are purified by reversed-phase chromatography
using high-performance liquid chromatography (HPLC) (Kontron, Zurich,
Switzerland). Evaporated ODNs (Speed Vac Plus; Savant, Farmingdale,
NY) are then dissolved in 200 /xl of 0.1 M triethylammonium acetate
(pH 7.0) and injected into a 81C reversed-phase column (Spherisorb
ODS-2, 5/xm; 150 × 4.6 mm) using 0.1 M triethylammonium acetate (pH
7.0) and acetonitrile as the mobile phase. The column is eluted with
a 15-35% acetonitrile gradient over a 30-min period. The collected
products (2 ml) are evaporated in fractions of 100/xg and stored at -20 °
until use.