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 313 (Part A) of Methods in Enzymology and its companion Volume 314 (Part B) 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. This volume, Part A: General Methods, Methods of Delivery, and RNA Studies, includes several methods of antisense design and construction, general methods of delivery, and antisense used in RNA studies. In Part B: Applica- tions, chapters cover methods in which antisense 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, complete the volume. 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 xiii Contributors to Volume 313 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. SURESH ALAHARI (19), Department of Phar- macology, School of Medicine, University of North Carolina, Chapel Hill, North Caro- lina 27599 SIDNEY ALTMAN (26), Department of Molecu- lar, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520 ANNA ASTRIAB (19), Department of Pharma- cology, School of Medicine, University of North Carolina, Chapel Hill, North Caro- lina 27599 DAVID BELLIDO (14), Unitat de Biologia Cel- lular, Departament de Bioqulmica i Fisio- logia, Universitat de Barcelona, E-08028 Barcelona, Spain LYUBA BENIMETSKAYA (16), Columbia Uni- versity, New York, New York 10032 ECKHART BUDDECKE (15), Division of Molec- ular Cardiology, Institute for Arteriosclero- sis Research, University of Miinster, D- 48149 Miinster, Germany JEFFREY S. BUZBY (22), Hematology Research Laboratory, Children's Hospital of Orange County, Orange, California 92868 ALAN CARLETON (7), Institut Alfred Fessard, CNRS, 91198 Gif-sur-Yvette Cedex, France DANIELA CASTANOTI'O (23), Department of Molecular Biology, Beckman Research In- stitute of the City of Hope, Duarte, Califor- nia 91010 DOUGLAS L. COLE (12), Manufacturing Pro- cess Department, ISIS Pharmaceuticals, Inc., Carlsbad, California 92008 STANLEY T. CROOKE (1), ISIS Pharmaceuti- cals, Inc., Carlsbad, California 92008 JOHN M. DAGLE (24), Department of Pediat- rics, University of Iowa, Iowa City, Iowa 52242 CHARLOTI'E DARRAH (29), Department of Human Anatomy and Genetics, Oxford University, Oxford 051 3QX,, United Kingdom SCOTT F. DEAMOND (17), Department of BiD- chemistry, The Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland 21225 RANJIT R. DESHMUKH (12), Manufacturing Process Department, ISIS Pharmaceuticals, Inc., Carlsbad, California 92008 DAVID DESMAISONS (7), Institut Alfred Fes- sard, CNRS, 91198 Gif-sur-Yvette Cedex, France SONIA DHEUR (3), Laboratoire de Biophy- sique, INSERM U201, CNRS URA481, Museum National d'Histoire Naturelle, 75005 Paris, France BEIHUA DONG (31), Department of Cancer, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 ROBERT J. DUFF (17), Department of BiD- chemistry, The Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland 21225 GEORGE L. ELICEIRI (25), Department of Pa- thology, Saint Louis University School of Medicine, St. Louis, Missouri 63104-1028 RAMON ERITJA (14), European Molecular Bi- ology Laboratory, D-69012 Heidelberg, Germany LOUISE EVERATT (29), Department of Human Anatomy and Genetics, Oxford University, Oxford OS1 3QX, United Kingdom JEAN-CHRISTOPHE FRANCOIS (4), Laboratoire de Biophysique, INSERM U201, CNRS UMR8646, Museum National d'Histoire Naturelle, 75005 Paris, France CHANDRAMALLIKA GHOSH (6), AVI Bio- Pharma, Inc., Corvallis, Oregon 97333 x CONTRIBUTORS TO VOLUME 313 RICHARD V. GILES (5), Department of Haem- atology, The University of Liverpool, Royal Liverpool University Hospital, Liverpool L7 8XP, United Kingdom LINDA GORMAN (30), Lineberger Cancer Cen- ter, University of North Carolina, Chapel Hill, North Carolina 27599 VLADIMIR V. GORN (9), Epoch Pharmaceuti- cals, Inc., Redmond, Washington 98052 CECILIA GUERRIER-TAKADA (26), Depart- ment of Molecular, Cellular and Develop- mental Biology, Yale University, New Ha- ven, Connecticut 06520 TROY O. HARASYM (18), Inex Pharmaceuti- cals Corporation, Burnaby, British Colum- bia, Canada V5J 5J8 KAIZHANG HE (13), Department of Chemis- try, Duke University, Durham, North Caro- lina 27708-0346 MICHAEL J. HOPE (18), Inex Pharmaceuticals Corporation, Burnaby, British Columbia, Canada V5J 5J8 JEFF HUGHES (19), Department of Pharma- ceutics, University of Florida, Gainesville, Florida 32610 MASAYORI INOUYE (28), Department of Bio- chemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 PATRICK IVERSEN (6), A VI BioPharma, Inc., Corvallis, Oregon 97333 EMMA R. JAKOX (27), Department of Physiol- ogy, Medical College of Virginia~Virginia Commonwealth University, Richmond, Vir- ginia 23298 R. L. JULIANO (19), Department of Pharma- cology, School of Medicine, University of North Carolina, Chapel Hill, North Caro- lina 27599 SHIN-HONG KANG (30), Lineberger Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599 HARUKO KATAYAMA (20), Department of Neurosurgery, Teikyo University Ichihara Hospital, lchihara City, Chiba 299-0111, Japan MICHAEL W. KILPATRICK (29), Department of Pediatrics, University of Connecticut Health Center, Farmington, Connecticut 06030 SANDRA K. KLIMUK (18), Department of Bio- chemistry and Molecular Biology, The Uni- versity of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3 RYSZARD KOLE (30), Lineberger Cancer Cen- ter and Department of Pharmacology, Uni- versity of North Carolina, Chapel Hill, North Carolina 27599 IGOR KUTYAVlN (9), Epoch Pharmaceuticals, Inc., Redmond, Washington 98052 JI~ROME LACOSTE (4), Plasticit~ et expression des g~nomes microbiens, CNRS EP2029, CEA LRC12, CERMO, Universit~ Joseph Fourier, 38041 Grenoble, France LAURENT LACROIX (4), Laboratoire de Bio- physique, INSERM U201, CNRS UMR8646, Museum National d'Histoire Naturelle, 75005 Paris, France BERNARD LEBLEU (11), Institut de G~n~tique Mol~culaire de Montpellier, UMR5535, CNRS, F-34293 Montpellier, France EARVIN LIANG (19), Department of Pharma- ceutics, University of Florida, Gainesville, Florida 32610 PIERRE-MARIE LLEDO (7), InstitutAlfred Fes- sard, CNRS, 91198 Gif-sur-Yvette Cedex, France EUGENE A. LUKHTANOV (9), Epoch Pharma- ceuticals, Inc., Redmond, Washington 98052 RATAN K. MAITRA (31), HIV Core Facility, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 DESPINA MANIOTIS (29), Department of Hu- man Anatomy and Genetics, Oxford Uni- versity, Oxford OS1 3QX, United Kingdom AKIRA MATSUNO (20), Department of Neuro- surgery, Teikyo University lchihara Hospi- tal, Ichihara City, Chiba 299-0111, Japan JEAN-LOuiS MERGNY (4), Laboratoire de Bio- physique, 1NSERM U201, CNRS UMR8646, Museum National d'Histoire Naturelle, 75005 Paris, France DAVID MILESI (9), Epoch Pharmaceuticals, Inc., Redmond, Washington 98052 CONTRIBUTORS TO VOLUME 313 xi OLEG MIROCHNITCHENKO (28), Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 PAUL A. MORCOS (10), Gene Tools, LLC, Corvallis, Oregon 97333 TADASHI NAGASHIMA (20), Department of Neurosurgery, Teikyo University Ichihara Hospital, Ichihara City, Chiba 299-0111, Japan PETER E. NIELSEN (8), Department of Medical Biochemistry and Genetics, The Panum In- stitute, University of Copenhagen, D K-2200 Copenhagen N, Denmark ANVSCH PEYMAN (15), Chemical Research G 838, Hoechst Marion Roussel Deutschland GmbH, D-65926 Frankfurt am Main, Germany M. IAn PHILLIPS (2), Department of Physiol- ogy, University of Florida College of Medi- cine, Gainesville, Florida 32610 LEONIDAS A. PHYLACTOU (29), Cyprus Insti- tute of Neurology and Genetics, 1683 Ni- cosia, Cyprus JAUME PIULATS (14), Laboratorio de Bioin- vestigaci6n, Merck Farma y Qufmica, S.A., E-08010 Barcelona, Spain MARX( R. PLACER (31), 3-Dimensional Phar- maceutical, Inc., Extort, Pennsylvania 19341 KEN PORXER (13), Department of Chemistry, Duke University, Durham, North Carolina 27708-0346 VLADIMIR RAIT (13), Department of Chemis- try, Duke University, Durham, North Caro- lina 27708-0346 MICHAEL W. REED (9), Epoch Pharmaceuti- cals, Inc., Redmond, Washington 98052 IAN ROBBINS (11), Institut de G~n~tique Mo- l~culaire de MontpeUier, UMR5535, CNRS, F-34293 Montpellier, France CLINTON ROBV (17), Department of Biochem- istry, The Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland 21225 JoNN J. RossI (23), Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010 ANTONINA RYTE (15), Lombardi Cancer Cen- ter, Georgetown University Medical Center, Washington, DC 20007-2197 E. TULA SAISON-BEHMOARAS (3), Labora- toire de Biophysique, INSERM U201, CNRS URA481, Museum National d'His- toire NatureUe, 75005 Paris, France YOGESH S. SANGHVI (12), Manufacturing Process Department, ISIS Pharmaceuticals, Inc., Carlsbad, California 92008 MICHAELA SCHERR (23), Abteilung Haemato- logie und Onkologie, Medizinische Hoch- schule Hannover, D-30625 Hannover, Germany ANNETTE SCHMIDT (15), Division of Molecu- lar Cardiology, Institute for Arteriosclerosis Research, University of Manster, D-48149 Miinster, Germany lEAN C. SEMPLE (18), Inex Pharmaceuticals Corporation, Burnaby, British Columbia, Canada VIJ 5J8 DMITRI SERGUEEV (13, 19), Department of Chemistry, Duke University, Durham, North Carolina 27708-0346 ZINAIDA SERGUEEVA (13), Department of Chemistry, Duke University, Durham, North Carolina 27708-0346 W. L. SEVERT (27), Department of Physiology, Medical College of Virginia~Virginia Com- monwealth University, Richmond, Vir- ginia 23298 BARBARA RAMSAY SHAW (13, 19), Depart- ment of Chemistry, Duke University, Dur- ham, North Carolina 27708-0346 HALINA SIERAKOWSKA (30), Lineberger Can- cer Center, University of North Carolina, Chapel Hill, North Carolina 27599 ROBERT H. IlLVERMAN (31), Department of Cancer, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 DAVID G. SPILLER (5), School of Biological Sciences, The University of Liverpool, Liv- erpool L69 7ZB, United Kingdom C. A. STEIN (16), Columbia University, New York, New York 10032 xii CONTRIBUTORS TO VOLUME 313 DAVID STEIN (6), AVI BioPharma, Inc., Cor- vallis, Oregon 97333 JACK SUMMERS (13), Department of Chemis- try, Duke University, Durham, North Caro- lina 27708-0346 AKIRA TAMURA (20), Department of Neuro- surgery, Tokyo University Hospital, Ita- bashi-ku, Tokyo 173-0003, Japan ANA M. TARI (21), Department of Bioimmu- notherapy, University of Texas MD Ander- son Cancer Center, Houston, Texas 77030 GEMMA TARRAS6N (14), Laboratorio de Bio- investigaci6n, Merck Farina y Qu[mica, S.A., E-08010 Barcelona, Spain DAVID M. TIDD (5), School of Biological Sci- ences, The University of Liverpool, Liv- erpool L69 7ZB, United Kingdom JOHN TONKINSON (16), Columbia University, New York, New York 10032 PAUL F. TORRENCE (31), Section on Biomedi- cal Chemistry, Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Na- tional Institutes of Health, Bethesda, Mary- land 20892-0805 PAUL O. P. Ts'o (17), Department of Bio- chemistry, The Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland 21225 EUGEN UHLMANN (15), Chemical Research G 838, Hoechst Marion Roussel Deutschland GmbH, D-65926 Frankfurt am Main, Germany SEN~N VILAR6 (14), Unitat de Biologia Cellu- lar, Departament de Bioqu[mica i Fisio- logia, Universitat de Barcelona, E-08028 Barcelona, Spain JEAN-DIDIER VINCENT (7), Institut Alfred Fes- sard, CNRS, 91198 Gif-sur-Yvette Cedex, France DANIEL L. WEEKS (24), Department of Bio- chemistry, University of Iowa, Iowa City, Iowa 52242 DWIGHT WELLER (6), A VI BioPharma, Inc., Corvallis, Oregon 97333 SHIRLEY A. WILLIAMS (22), Hematology Re- search Laboratory, Children's Hospital of Orange County, Orange, California 92868 HooN Yoo (19), Department of Pharmacol- ogy, School of Medicine, University of North Carolina, Chapel Hill, North Caro- lina 27599 Y. CLARE ZHANG (2), Department of Physiol- ogy, University of Florida College of Medi- cine, Gainesville, Florida 32610 YUANZHONG ZHOU (17), Cell Works, Inc., Baltimore, Maryland 21227 [ 1] PROGRESS IN ANTISENSE TECHNOLOGY 3 [ I] Progress in Antisense Technology: The End of the Beginning By STANLEY T. CROOKE Introduction During the past decade, intense efforts to develop and exploit antisense technology have been mounted. With the recent FDA approval of Vitra- vene, the first drug based on antisense technology to be commercialized, the technology has achieved an important milestone. Nevertheless, the technology is still in its infancy. Although the basic questions have been answered, there are still many more unanswered than answered questions. The objectives of this article are to provide an overview of the progress in converting the antisense concept into broad therapeutic reality and to provide advice about appropriate experimental design and interpretation of data with regard to the therapeutic potential of the technology. Proof of Mechanism Factors That May Influence Experimental Interpretations Clearly, the ultimate biological effect of an oligonucleotide will be influ- enced by the local concentration of the oligonucleotide at the target RNA, the concentration of the RNA, the rates of synthesis and degradation of the RNA, the type of terminating mechanism, and the rates of the events that result in termination of the activity of RNA. At present, we understand essentially nothing about the interplay of these factors. Oligonucleotide Purity. Currently, phosphorothioate oligonucleotides can be prepared consistently and with excellent purity. 1 However, this has only been the case since the mid-1990s. Prior to that time, synthetic methods were evolving and analytical methods were inadequate. In fact, our labora- tory reported that different synthetic and purification procedures resulted in oligonucleotides that varied in cellular toxicity 2 and that potency varied from batch to batch. Although there are no longer synthetic problems with phosphorothioates, they undoubtedly complicated earlier studies. More 1 S. T. Crooke and C. K. Mirabelli, "Antisense Resarch and Applications." CRC Press, Boca Raton, FL, 1993. 2 R. M. Crooke, Anti-Cancer Drug Design 6, 609 (1991). Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. METHODS IN ENZYMOLOGY, VOL. 313 0076-6879/99 $30.00 4 GENERAL METHODS [ II importantly, with each new analog class, new synthetic, purification, and analytical challenges are encountered. Oligonucleotide Structure. Antisense oligonucleotides are designed to be single stranded. We now understand that certain sequences, e.g., stretches of guanosine residues, are prone to adopt more complex structures. 3 The potential to form secondary and tertiary structures also varies as a function of the chemical class. For example, higher affinity 2'-modified oligonucleo- tides have a greater tendency to self-hybridize, resulting in more stable oligonucleotide duplexes than would be expected based on rules derived from oligodeoxynucleotides. 3a RNA Structure. RNA is structured. The structure of the RNA has a profound influence on the affinity of the oligonucleotide and on the rate of binding of the oligonucleotide to its RNA target. 4,5 Moreover, the RNA structure produces asymmetrical binding sites that then result in very diver- gent affinity constants depending on the position of oligonucleotide in that structure:: This in turn influences the optimal length of an oligonucleotide needed to achieve maximal affinity. We understand very little about how RNA structure and RNA protein interactions influence antisense drug action. Variations in in Vitro Cellular Uptake and Distribution. Studies in several laboratories have clearly demonstrated that cells in tissue culture may take up phosphorothioate oligonucleotides via an active process and that the uptake of these oligonucleotides is highly variable depending on many conditions. 2'8 Cell type has a dramatic effect on total uptake, kinetics of uptake, and pattern of subcellular distribution. At present, there is no unifying hypothesis to explain these differences. Tissue culture conditions, such as the type of medium, degree of confluence, and the presence of serum, can all have enormous effects on uptake. 8 The oligonucleotide chem- ical class obviously influences the characteristics of uptake as well as the mechanism of uptake. Within the phosphorothioate class of oligonucleo- 3 j. R. Wyatt, T. A. Vickers, J. L. Roberson, R. W. Buckheit, Jr., T. Klimkait, E. DeBaets, P. W. Davis, B. Rayner, J. L. Imbach, and D, J. Ecker, Proc. Natl. Acad. Sci. U.S.A. 91, 1356 (1994). 3a S. M. Freier, unpublished results. 4 S. M. Freier, in "Antisense Research and Applications" (S. T. Crooke and B. Lebleu, eds0, p. 67. CRC Press, Boca Raton, FL, 1993. 5 D. J. Ecker, in "Antisense Research and Applications" (S. T. Crooke and R. Lebleu, eds.) p. 387. CRC Press, Boca Raton, FL, 1993. 6 W. F. Lima, B. P. Monia, D. J. Ecker, and S. M. Freier, Biochemistry 31, 12055 (1992). 7 D. J. Ecker, T. A. Vickers, T. W. Bruice, S. M. Freier, R. D. Jenison, M. Manoharan, and M. Zounes, Science 257, 958 (1992). 8 S. T. Crooke, L. R. Grillone, A. Tendolkar, A. Garrett, M. J. Fratkin, J. Leeds, and W. H. Barr, Clin. Pharmacol. Ther. 56, 641 (1994). [ 1 ] PROGRESS IN ANTISENsE TECHNOLOGY 5 tides, uptake varies as a function of length, but not linearly. Uptake varies as a function of sequence and stability in cells is also influenced by the se- quence. 8,9 Given the foregoing, it is obvious that conclusions about in vitro uptake must be made very carefully and generalizations are virtually impossible. Thus, before an oligonucleotide could be said to be inactive in vitro, it should be studied in several cell lines. Furthermore, while it may be abso- lutely correct that receptor-mediated endocytosis is a mechanism of uptake of phosphorothioate oligonucleotides, 1° it is obvious that a generalization that all phosphorothioates are taken up by all cells in vitro primarily by receptor-mediated endocytosis is simply unwarranted. Finally, extrapolations from in vitro uptake studies to predictions about in vivo pharmacokinetic behavior are entirely inappropriate and, in fact, there are now several lines of evidence in animals and humans that demon- strate that even after careful consideration of all in vitro uptake data, one cannot predict in vivo pharmacokinetics of the compounds. 8,11-~3 Binding to and Effects of Binding to Nonnucleic Acid Targets. Phospho- rothioate oligonucleotides tend to bind to many proteins and those interac- tions are influenced by many factors. The effects of binding can influence cell uptake, distribution, metabolism, and excretion. They may induce non- antisense effects that can be mistakenly interpreted as antisense or compli- cate the identification of an antisense mechanism. By inhibiting RNase H, protein binding may inhibit the antisense activity of some oligonucleotides. Finally, binding to proteins can certainly have toxicological consequences. In addition to proteins, oligonucleotides may interact with other biologi- cal molecules, such as lipids or carbohydrates, and such interactions, like those with proteins, will be influenced by the chemical class of oligonucleo- tide studied. Unfortunately, essentially no data bearing on such interactions are currently available. An especially complicated experimental situation is encountered in many in vitro antiviral assays. In these assays, high concentrations of drugs, viruses, and cells are often coincubated. The sensitivity of each virus to 9 S. T. Crooke, in "Burger's Medicinal Chemistry and Drug Discovery" (M. E. Wolff, ed.), vol. 1, p. 863. Wiley, New York, 1995. 10 S. L. Loke, C. A. Stein, X. H. Zhang, K. Mori, M. Nakanishi, C. Subasinghe, J. S. Cohen, and L. M. Neckers, Proc. Natl. Acad. Sci. U.S.A. 86, 3474 (1989). alp. A. Cossum, H. Sasmor, D. Dellinger, L. Truong, L. Cummins, S. R. Owens, P. M. Markham, J. P. Shea, and S. Crooke, J. Pharmacol. Exp. Ther. 267, 1181 (1993). 12 p. A. Cossum, L. Truong, S. R. Owens, P. M. Markham, J. P. Shea, and S. T. Crooke, J. Pharmacol. Exp. Ther. 269, 89 (1994). 13 H. Sands, L. J. Gorey-Feret, S. P. Ho, Y. Bao, A. J. Cocuzza, D. Chidester, and F. W. Hobbs, Mol. Pharmacol. 47, 636 (1995). 6 GENERAL METHODS [ 11 nonantisense effects of oligonucleotides varies depending on the nature of the virion proteins and the characteristics of the oligonucleotides. 14,15 This has resulted in considerable confusion. In particular for human immune deficiency virus (HIV), herpes simplex viruses, cytomegaloviruses, and in- fluenza virus, the nonantisense effects have been so dominant that identi- fying oligonucleotides that work via an antisense mechanism has been difficult. Given the artificial character of such assays, it is difficult to know whether nonantisense mechanisms would be as dominant in vivo or result in antiviral activity. Terminating Mechanisms. It has been amply demonstrated that oligonu- cleotides may employ several terminating mechanisms. The dominant ter- minating mechanism is influenced by RNA receptor site, oligonucleotide chemical class, cell type, and probably many other factors. 16 Obviously, as variations in terminating mechanism may result in significant changes in antisense potency and studies have shown significant variations from cell type to cell type in vitro, it is essential that the terminating mechanism be well understood. Unfortunately, at present, our understanding of terminat- ing mechanisms remains rudimentary. Effects of "Control Oligonucleotides." A number of types of control oligonucleotides have been used, including randomized oligonucleotides. Unfortunately, we know little to nothing about the potential biological effects of such "controls," and the more complicated a biological system and test the more likely that "control" oligonucleotides may have activities that complicate interpretations. Thus, when a control oligonucleotide dis- plays a surprising activity, the mechanism of that activity should be explored carefully before concluding that the effects of the "control oligonucleotide" prove that the activity of the putative antisense oligonucleotide is not due to an antisense mechanism. Kinetics of Effects. Many rate constants may affect the activities of antisense oligonucleotides, e.g., the rate of synthesis and degradation of the target RNA and its protein, the rates of uptake into cells, the rates of distribution, extrusion, and metabolism of an oligonucleotide in cells, and similar pharmacokinetic considerations in animals. Despite this, relatively few time courses have been reported and in vitro studies have been reported that range from a few hours to several days. In animals, we have a growing body of information on pharmacokinetics, but in most studies reported to 14 L. M. Cowsert, in "Antisense Research and Applications" (S. T. Crooke and B. Lebleu, eds.), p. 521. CRC Press, Boca Raton, FL, 1993. 15 R. F. Azad, V. B. Driver, K. Tanaka, R. M. Crooke, and K. P. Anderson, Antimicrob. Agents Chemother. 37, 1945 (1993). 16 S. T. Crooke, "Therapeutic Applications of Oligonucleotides." R. G. Landes Company, Austin, TX, 1995. [ 1] PROGRESS IN ANTISENSE TECHNOLOGY 7 date, the doses and schedules were chosen arbitrarily and, again, little information on duration of effect and onset of action has been presented. Clearly, more careful kinetic studies are required and rational in vitro and in vivo dose schedules must be developed. Recommendations Positive Demonstration of Antisense Mechanism and Specificity. Until more is understood about how antisense drugs work, it is essential to positively demonstrate effects consistent with an antisense mechanism. For RNase H-activating oligonucleotides, Northern blot analysis showing selec- tive loss of the target RNA is the best choice and many laboratories are publishing reports in vitro and in vivo of such activities) 7-2° Ideally, a demonstration that closely related isotypes are unaffected should be in- cluded. More recently, in our laboratories we have used RNA protection assays and DNA chip arrays. 2°a These assays provide a great deal of information about the levels of various RNA species. Coupled to careful kinetic analysis, such approaches can help assure that the primary mechanism of action of the drug is antisense and can identify events that are secondary to antisense inhibition of a specific target. This can then support the assignment of a target to a particular pathway, the analysis of the roles of a particular target, and the factors that regulate its activity. We have adapted all these methods for use in animals and will be determining their utility in clinical trials. In brief, then, for proof of mechanism, the following steps are recom- mended. Perform careful dose-response curves in vitro using several cell lines and methods of in vitro delivery. Correlate the rank order potency in vivo with that observed in vitro after thorough dose-response curves are generated in vivo. Perform careful "gene walks" for all RNA species and oligonucleotide chemical classes. Perform careful time courses before drawing conclusions about po- tency. 17 M. Y. Chiang, H. Chan, M. A. Zounes, S. M. Freier, W. F. Lima, and C. F. Bennett, J. Biol. Chem. 266, 18162 (1991). 18 N. M. Dean and R. McKay, Proc. Natl. Acad. Sci. U.S.A. 91, 11762 (1994). 19 T. Skorski, M. Nieborowska-Skorska, N. C. Nicolaides, C. Szczylik, P. Iversen, R. V. Iozzo, G. Zon, and B. Calabretta, Proc. Natl. Acad. Sci. U.S.A. 91, 4504 (1994). 20 N. Hijiya, J. Zhang, M. Z. Ratajezak, J. A. Kant, K. DeRiel, M. Hertyn, G. Zon, and A. M. Gewirtz, Proc. Natl. Acad. Sci. U.S.A. 91, 4499 (1994). 20a j. F. Taylor, Q. Q. Zhang, B. P. Monia, E. G. Marcusson, and N. M. Dean, Oncogene, in press. [...]... 11 Transpod Degradation Effects on Anabollsm of rnRNA ~ ; ", CAP "' ' ~ •"*"°°~'-.°° ~ CAP "~ AAAA [ Nucleus : ~ I .F ~AAAA + ~ ° .° r- ~ ° ° ."" °" "' Cytoplasm Effocts on ~.,91 -Catabolism of JAAAA CAP_ mRNA CAP ~- Translation t._~J AAAA FIG.1 R N A 9 Translational Arrest processing addition of long (hundreds of nucleotides) tracts of polyadenylate Polyadenylation stabilizes mRNA and may play other... bioavailability was observed, al 4a However, it seems likely that a principal limiting factor in the oral bioavailability of phosphorothioates may be degradation in the gut rather than absorption Studies using everted rat jejunum sacs demonstrated 112y Takakura, R I Mahato, M Yoshida, T Kanamaru, and M Hashida, Antisense Nucleic Acid Drug Del 6, 177 (1996) 113M Butler, K Stecker, and C F Bennett, Lab... Kitajima, T Shinohara, J Bilakovics, D A Brown, X Xiao, and M Nerenberg, Science 251t, 1792 (1992) 14oK A Higgins, J R Perez, T A Coleman, K Dorshkind, W A McComas, U M Sarmiento, C A Rosen, and R Narayan, Proc Natl Acad Sci U.S .A 9tl, 9901 (1993) 141N, Hijiya, J Zhang, M Z Ratajczak, J A Kant, K DeRiel, M Herlyn, G Zon, and A M Gewirtz, Proc Natl Acad Sci U.S .A 91, 4499 (1994) [1] PROGRESSIN ANTISENSETECHNOLOGY... designed, and data that directly support translation arrest as the mechanism have been lacking Target R N A species that have been reported to be inhibited by a translational arrest mechanism include HIV, vesicular stomatitis virus (VSV), n-myc, and a number of normal cellular genes, z7-33 In our laboratories, we have shown that a significant number of targets may be inhibited by binding to translation... substrates and Michaelis-Menten analyses, we were able to evaluate both binding and cleavage We showed that, in fact, E coli RNase H1 is a double-strand RNA-binding protein The Kd for R N A duplex was 1.6 /zM; the Kd for a D N A duplex was 176/.~M; and the Kd for single-strand D N A was 942 ~M In contrast, the enzyme could only cleave R N A in an R N A - D N A duplex Any 2' modification in the antisense. .. 2'-modified wings and a ribonucleotide center, we have shown that mammalian cells contain enzymes that can cleave double-stranded RNAs TM This is an important step forward because it adds to the repertoire of intracellular enzymes that may be used to cleave target RNAs and because chimeric oligonucleotide 2'modified wings and oligoribonucleotide gaps have a higher affinity for R N A targets than chimeras with... Ikegaki, R H Kennett, and L M Neckers, Cancer Res $0, 6316 (1990) 3o G Vasanthakumar and N K Ahmed, Cancer Commun 1, 225 (1989) 3x Sburlati, A R., R E Manrow, and S L Berger, Proc Natl Aead Sci U.S .A 88, 253 (1991) 32 H Zheng, B M Sahai, P Kilgannon, A Fotedar, and D R Green, Proc Natl Acad Sci U.S .A 86, 3758 (1989) 33 j A Maier, P Voulalas, D Roeder, and T Maciag, Science 249, 1570 (1990) 10 GENERAL... 5'-capping and 3'-adenylation, there are clearly other sequences in the 5'- and 3'-untranslated regions of mRNA that affect the stability of the molecules Again, there are a number of antisense drugs that may work by these mechanisms Zamecnik and Stephenson 42 reported that 13-mer targeted to untranslated 3'- and 5'-terminal sequences in Rous sarcoma viruses was active• Oligonucleotides conjugated to an acridine... our laboratory suggest that in rats, oligonucleotides administered intravenously at doses of 15-20 mg/kg saturate the serum protein-binding capacity, rosa Phosphorothioate oligonucleotides are absorbed rapidly and extensively after parenteral administration For example, in rats, after an intradermal dose of 3.6 mg/kg of [14C]ISIS 2105, a 20-mer phosphorothioate, approximately 70% of the dose was absorbed... 115j A Hughes, A V Avrutskaya, K L R Brouwer, E Wickstrom, and R L Juliano, Pharm Res 12, 817 (1995) 116S Agrawal, X Zhang, Z Lu, H Zhao, J M Tamburin, J Yan, H Cai, R B Diasio, I Habus, Z Jiang, R P Iyer, D Yu, and R Zhang, Biochem PharmacoL 50, 571 (1995) 117p D Cook, in "Antisense Research and Applications" (S T Crooke and B Lebleu, eds.), p 149 CRC Press, Boca Raton, FL, 1993 [1] PROGRESS IN ANTISENSE . ° ° CAP JAAAA CAP_ Degradation "~ r -~ CAP Translation ~- t._~J AAAA 9 FIG. 1. RNA processing. Transcriptional Arrest Effects on Anabollsm of rnRNA Effocts on ~.,91 Catabolism. target to a particular pathway, the analysis of the roles of a particular target, and the factors that regulate its activity. We have adapted all these methods for use in animals and will be determining. Carolina, Chapel Hill, North Carolina 27599 HARUKO KATAYAMA (20), Department of Neurosurgery, Teikyo University Ichihara Hospital, lchihara City, Chiba 299-0111, Japan MICHAEL W. KILPATRICK