Antisense technology Stanley T. Crooke ISIS Pharmaceuticals, Carlsbad, California, USA During the past 12 months, significant advances have been reported in the medicinal chemistry, and the pharmacodynamic and pharmacokinetic characterization of oligonucleotides. Advances in medicinal chemistry suggest that the scope for additional modifications as a means of developing therapeutic agents is substantial. This is confirmed by our clearer understanding of the pharmacodynamic and pharmacokinetic properties of oligonucleotides and the increasing number of molecular targets against which they have been shown to be active. Current Opinion in Biotechnology 1991, 2:282-287 Introduction Because of the explosion in interest and, therefore, the number of scientists involved, it is fair to say that the vast majority of the total research performed on antisense technology has been completed in the past year. Consequently, summarizing results published in 1990 is very much like reporting on the tip of an enormous iceberg of progress that will manifest itself in publications in the next few years. In this review, I will focus strictly on oligonucleotide therapeutics, excluding antisense gene therapy and plant biology, as these endeavors represent substantially different approaches. Publications have been selected for review primarily on the basis of their potential generic importance. A table summarizing the molecular targets against which oligonucleotides have been reported to be effective is also provided (Table 1). As with any other drug-based research, important discoveries are derived from studies on the structure and function of the receptor for the drug, as well as the medicinal chemistry and pharmacodynamic and pharmacokinetic properties of the drug. In the case of oligonucleotide therapeutics, it is helpful to consider the task of drug discovery as a problem in creating selective inhibitors of RNA intermediary metabolism, beginning with transcription and ending with degradation of the mRNA.
Nucleic acid structure and function The ability to practice etficient rational drug design is an important potential advantage of oligonucleotide therapeutics. The criteria required are the ability to predict binding constants for oligonucleotides, and knowledge of the secondary structure of RNA targets based on a variety of hybridization and nearest neighbor-derived algorithms. However, predictions of RNA secondary s t r u c -
ture and potential oligonucleotide binding constants are only approximations based on a number of assumptions, some of which are unproven. Consequently, studies that enhance the predictive precision of such algorithms are extremely important. Jaeger et al. [1.-] have carried o u t denaturation and chemical modifications of a well characterized RNA, the Tetrahymena thermophila large subunit ribosomal RNA, containing a seN-splicing intron, to determine the validity of a number of the underlying assumptions. The major conclusions drawn from the study are that most of the assumptions appear to be valid and, in particular, studies in 1 M NaCl provide data similar to those in 0.05M Na + and 10mM Mg2+. Moreover, they suggest that programs that take a more realistic approach to multi-branch loops are more precise, and that tertiary interactions appear to play a relatively small role in determining RNA structure. Finally, their data support the inclusion of favorable free energy terms for terminal GU base pairs, and stacking interactions in loops. Important advances in experimental characterization of RNA secondary and tertiary structures employing physical and chemical methods have also been reported. The conformation of an RNA pseudoknot was defined using enzymatic methods and nuclear magnetic resonance (NMR) spectroscopy [2]. This study demonstrated that pseudoknots are in equilibrium with hairpins and that the stems and loops of a pseudoknot differ in size, position and contribution to the stability of the structure. A minor distortion of helical stacking at the junctions and stems was also demonstrated. Furthermore, the solution structure of an unusually stable hairpin was solved using NMR spectroscopy [3]. A second major theme that has emerged in recent years is the ability of RNA to behave enzymatically. This is important because it encourages examination of RNA from an enzymatically functional perspective in addition to studies of its behavior as a substrate in information transfer processes. Moreover, it focuses attention on RNA-specitic
Abbreviations NMR--nuclear magnetic resonance; TAR--transactivator response element; TAT--transactivating protein. 282
© Current Biology Ltd ISSN 0958-1669
Antisense technology Crooke
Table 1. A summary of the activities of antisense oligonucleotides in vitro.
Target Viruses HIV HIV (gag/pol) HIV HSV HSV VSV VSV IV TBE SV40 RSV HB Oncogenes c-myc c-myc c-myc c-myb n-myc Host genes Multiple drug resistance PCNA (cyclin) T cell receptor GM-CSF CSF-1 EGF receptor [3-Globin Other Chloramphenicol acetyltransferase
Cell type
Serum
Oligotypes
Length (nucleotides)
Concentration
Reference
H-T cells H-T cells H9 cells Vero cells Vero cells L929 cells L929 cells MDCK cells MDCK cells Chicken fibroblasts Alexander
+ + + + + + + + + + +
PS PS PS, others CH3-P CH3-P CH3P P-poly 1 P-acridine Various CH3-P Various
14-28 18-24 20 7 12 9 10-15 11 Variable 6.9 Variable
0.5-1 ~tM 1-10~M 4-20 I~g/ml- 1 50-100 ~tM 20-50 I~M 25-50 I.tM 0.1 ~tM 50 ~tM 0.1-1 ~tM 25 IIM 10 I~M
[22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]
+
P
15
8.5 I.tM
[33]
T-lymphocytes HL-60 Burkitt cells PMBC Neu roblastoma
+ + + +
P P, PS P P P
15 15 21 18 15
30 IIM 10 I~M 100 I*M 40 mg m l - 1 1-5 I*M
[34] [35] [36] [37] [38]
MCF-1 3T3 T cells Endothelial FL-ras/myc Rabbit reticulocytes
+ + + + + +
PS P P P P P CH3P
15 18 22 15, 18 ? 13 9
101~M ? 30 p.M 100 p.M
[39] [40] [41] [42] [43] [44] [45]
CV-1
+
P, PS, CH3P
21
5-30 I~M
[46]
30 p.M
CSF, colony-stimulating factor; EGF, epidermal growth factor; GM-CSF, granulocyte macrophage CSF; HB, hepatitis B; HIV, human immunodeficiency virus; HSV, herpes simplex virus; IV, influenza virus; MDCK, Madian-Darby canine kidney; P, phosphodiester; PCNA, proliferating cell nuclear antigen; PS, phosphorothioate; RSV, Rous sarcoma virus; SV40, simian virus 40; TBE, tick-borne encephalitis; VSV, vesicular stomatitis
chemistry involving the 2' hydroxyl group. It also suggests the potential of modified ribozymes themselves as drugs. During 1990, a large number of important studies on ribozymes were published. These dealt with either the enzymology of Tetrahymena or hammerhead ribozymes, or the biological activities of ribozymes. The enzymology of Tetrahymena ribozymes has been particularly well characterized by Cech and colleagues [4*%5°" ]. The effects of modification of a ribozyme by substitution of phosphorothioates have also been reported [4°° ]. Studies on hammerhead ribozymes have defined the minimum structure required for enzymologic properties [6,7]. Importantly, minimized ribozymes have been shown to cleave sequences on a complementary oligonucleotide [6].
Ribozymes were also shown to cleave single- and doublestranded DNA, thus expanding the repertoire of potential substrates beyond intramolecular RNA cleavage [8,9]. Furthermore, a synthetic ribozyme comprised of deoxyribonucleotides and ribonucleotides was shown to be active [ 10]. Finally, microinjection of a ribozyme into Xenc> pus oocytes was shown to cleave U7 small nuclear RNA
[11]. A third important series of observations are derived from studies on the human immunodeficiency virus that focus on a stem-loop structure that binds a transactivating protein (TAT) to the transactivator response element (TAR). Thus, this structure and its role are now well understood and demonstrate the importance of specific RNA
283
284
Pharmaceuticalapplications Several potentially important modifications of RNA oligonucleotides have been evaluated more extensively in 1990. Modifications at the 2' position are likely to be important because they enhance the stability of duplexes formed with RNA, relative to those formed between DNA oligonucleotides and RNA. These modifications may also enhance nuclease stability. To date, the most extensively studied are the 2'-0-methyl analogs [14.',15]. Another interesting modification is the fusion of lipids to oligonucleotides [16]. The resulting increase in lipophilicity may enhance cellular uptake of the oligonucleotide. The basic message emphasized by these and other studies is that ollgonucleotide therapeutics offer great scope for medicinal chemistry because numerous sites appear to tolerate substantial modification without dramatic reduction in atFmity for receptor sequences and with little loss in specificity.
structures in regulation of biological activities [12..]. TAR is a very stable stem-loop structure. A cellular protein binds to the loop and TAT binds to a bulge in the stem. These interactions result in an increase in transcription and translation of mRNAs that contain the TAR element. Thus, this particular structural motif in RNA plays an important biological role and is a useful model for learning how to destabilize RNA secondary structure for the purpose of creating desired pharmacologic effects.
Medicinal chemistry In a particularly useful review, Uhlmann and Peyman [13"] have provided a valuable summary of the methods available for both synthesizing and modifying oligonucleotides. Although a significant number of modifications has been reported, numerous others remain to be studied, and structure-activity relationships are just being defined.
Table 2. Summary Length nucleotides)
of antisense pharmacokinetics.
Label
Modifications
Serum nuclease
Cell types
Percentage uptake
Kinetics
Distribution
Metabolism
Reference
Phosphodiesters 15 5'35S
None
Heat inactivated
K562
1~
4h
-
Degradation in medium bylh
[47]
10-30
5'32p
None
-
T cell
5
Rapid, reaching plateau in 4h
-
Intact at 15 min and 4 h
[48]
3-20
None
5' acridine None
+ +
HL60 HL60
11
Saturable; temperature-dependent 50 h; temperatureindependent ~ 9 0 min
-
[21]
5'32p
5'32p
None
+
HeLa and others
0.3-2.3
Linear for 24 h
Degradation in serum within 2 h
[49]
-
-
[50]
-
Stable to serum
[49]
Rapid for 30 min, then plateau
Nucleus
-
[51]
18
Oligo-T n
None
5' acridine
+
-
-
0.25-1.2
Phosphorothioates
Saturable PS inhibitors but not CH3P * Linear for 2h
-
18
5'32p
None
-
HeLa and others
28
None
Fluorescein
+
H9, U937
12-20
5'35S
None
+
HL60
1-2
Active
-
Stable for more than 3 days
[52]
None
+
CVl
-
Linear for 2 h
-
Stable
[46]
Methylphosphonates 21 5'32p
AntisensetechnologyCrooke Pharmacodynamics Among the more important advances of the past year was the identification of a number of terminating events or proximal mechanisms of action in addition to RNaseH and translation arrest. Further evidence that inhibition of splicing reactions and modification to 3' and 5' untranslational regions disrupt RNA secoridary structures was presented [17]. Inhibition of key RNA activities has also been described. For example, oligonucleotides complementary to a conserved sequence in 5.8S RNA were reported to inhibit protein synthesis [18]. In some cases, inhibition of transcription may be required to achieve desired therapeutic effects. DNA triplex formation may provide a means of inhibiting the production of certain proteins. Before this mechanism can be routinely employed, however, oligonucleotides with enhanced affinity for DNA, together with the ability to interact in a sequence-specific fashion independently of polypurine or polypyrimidine tracts must be developed. During 1990, Strobel and Dervan [19"] reported that an oligonucleotide coupled to EDTA-Fe2+ bound and cleaved a complementary polypurine sequence inserted in a 340 kb chromosome embedded in an agarose gel. Research into phosphorothioates has demonstrated that they may have the necessary attributes to be first generation oligonucelotide drugs. However, a number of studies suggest that they might bind to proteins non-specifically via interactions involving the thiols, so that, in some cases, the observed effects are not dependent on nucleic acid interactions. Gao et al. [20.'] have demonstrated that a 28-mer phosphorothioate inhibits herpes virus replication by interacting with a number of herpes virus proteins. Despite the potential of phosphorothioates to produce non-specific effects, studies in a number of laboratories have demonstrated that cellular toxicity only occurs at very high concentrations.
Pharmacokinetics Phosphorothioates, methylphosphonates and T-0-methyl-substituted oligonucleotides have been shown to be highly resistant to various nucleases [14"',15]. Cellular uptake of phosphorothioates and methylphosphonates has been studied in vitro [13"]. Surprisingly high uptake of phosphorothioates by cells in tissue culture has been reported, and the compound is clearly shown to be intact intracellularly. Uptake of phosphorothioates appears to involve several active mechanisms and, in particular, a 90 kD membrane protein has been reported to bind phosphorothioate oligonucleotides [21]. Table 2 summarizes recent data on in vitro pharmacokinetics.
that oligonucleotide therapeutics may offer important advantages over conventional drugs and may indeed be the most direct route to the next phase in therapeutics: rational design of isotype-selective therapeutics.
Acknowledgements The excellent typographical assistance of Mrs Coleen Matzinger and the critical reviews by Drs Christopher Mirabelli, David Ecker and P Dan Cook are much appreciated.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •, of outstanding interest 1. •.
JAEGERJA, ZUKERM, TURNERDH: Melting and Chemical Modification of a Cyclized Self-splicing Group I Intron: Similarity of Structure in 1M Na +, in 10mM Mg2+, and in the Presence of Substrate. Biochemistry 1990, 29:10147-10158. This report extends the study of RNA folding programs, demonstrating reasonable predictiveness with confirmed and enhanced assumptions. 2.
PUGLISIJD, WYATt JR, TINOCO I JR: Conformation of a RNA Pseudoknot. J Mol Biol 1990, 214:437-453.
3.
CHEONGC, VAEANIG, TINOCO I JR: Solution Structure of an Unusually Stable RNA Hairpin, 5'GGAC(UUG GUCC). Nature 1990, 346:680-682.
McSWIGGENJA, CECH TR: Stereochemistry of RNA Cleavage by the Tetrahymena Ribozyme and Evidence that the Chemical Step is not Rate-limiting. Science 1989, 244:679-683. This article summarizes a series of studies describing the biochemical and kinetic properties of the Tetrahymena ribozyme. 4.
•,
5. •.
HERSHLAGD, CECIl TR: Catalysis of RNA Cleavage by the Tetrahymena thermophila Ribozyme. 1. Kinetic Description of the Reaction of an RNA Substrate Complementary to the Active Site. Biochemistry 1990, 29:10159-10171. Detailed enzyme kinetic studies demonstrating the mechanisms of cleavage by the Tetrahymena ribozyme are presented. 6.
UHLENBECKOC: A Small Catalytic Oligoribonucleotide. Nature 1987, 328:596-600.
7.
JEFFmESHC, SYMONSRH: A Catalytic 13-met Ribozyme. Nucleic Acid Res 1989, 17:1371-1375.
8.
ROBERTSONDL, JOYCE G:F Selection In Vitro of an RNA Enzyme that Specifically Cleaves Single-stranded DNA. Nature 1990, 344:467468.
9.
HERSHLA6D, CECIl TR: DNA Cleavage Catalysed by the Ribozyme from Tetrahymenat Nature 1990, 344:405-409.
10.
PERREAULTJ-P, WU T, COUSINEAUB, OGILVIE KK, CEDERGREN R:Mixed Deoxyribo- and Ribo-oligonucleotides with Catalytic Activity. Nature 1990, 344:565-567.
11.
COTTENM, BIRNSTIELML: Ribozyme Mediated Destruction of RNA In Viva EMBO J 1989, 8:3861-3866.
12. ~.
Conclusion
CULLERBR~ The HIV-1 TAT Protein: an RNA Sequence-specific Processivity Factor? Cell 1990, 63:655-657. s brief review summarizes the data on TAR and TAT interactions. Because TAR is an important RNA structure and target for antisense drugs, this review is valuable.
Substantial progress in validating the antisense paradigm and enhancing our understanding of a number of the key elements of oligonudeotide therapeutics has been reported during 1990. The progress continues to suggest
13. UHLMANNE, PEYMANA: Antisense Oligonucleotides: a New ,, Therapeutic Principle. Chem Rev 1990, 90:544-584. A detailed review of the antisense research of the past decade and its importance in therapeutic studies.
285
286
Pharmaceutical applications 14. oo
BEIJERB, SULSTONL, SPROATBS, RIDm P, LAMONDAI, NEUNERP: Synthesis and Applications of Oligoribonucleotides with Selected 2'-O-methylation Using the 2'-O-[1-(2-fluorophenyl)4-methoxypiperidin-4-ylk] Protecting Group. Nucleic Acid Res 1990, 18:5143-5151. The synthesis and preliminary characterization of 2'0 methyl oligonucleotides is presented. These compounds may be important as they might provide nuclease resistance and enhanced binding potential for RNA targets.
Infection by Sequence-specific Oligodeoxyribonucleoside Methylphosphonates. Biochemistry 1986, 25:626845275. 28.
LEMAITREM, BAYARDB, LEBLEUB: Specific Antiviral Activity of a Poly(L-lysine)-conjugated Oligodeoxyribonucleotide Sequence Complementary to Vesicular Stomatitis Virus N Protein mRNA Initiation Site. Biochemistry 1987, 84:648-452.
29.
ZERIALA~ THUONG NT, HELENE C: Selective Inhibition of the Cytopathic Effect of Type A Influenza Viruses by Oligodeoxynucleotides Covalently Linked to an Intercalating Agent. Nucleic Acids Res 1987, 15:9909-9919.
30.
VLASSOVVV: Inhibition of Tick-borne Viral Encephalitis Expression Using Covalently Linked Oligonucleotide Analogs. Meeting on Oligodeoxynucleotides as Ant~ense Inhibitors of Gene Expression: Therapeutic Implications' [abstract]. June 18-21, 1989, Rockville, Maryland, USA.
15.
IRIBARRENAM, SPROAT BS, NEUNER P, SULSTON I, RYDER U, LAMOND AI: 2'-O-Alkyl Oligoribonucleotides as Antisense Probes. Proc Natl Acad Sci USA 1990, 87:7747-7751.
16.
SHEARG, MARSTERSJC, BISCHOFBERGERN: Synthesis, Hybridization Properties and Antiviral Activity of Lipid-oligodeoxynucleotide Conjugates. Nucleic Acid Res 1990 18:3777-3783.
17.
SAXENASK, ACKERMANEJ: Microinjected Oligonucleotides Complementary to the ct-Sarcin Loop of 28 S RNA Abolish Protein Synthesis in X e n o p u s Oocytes. J Biol Chem 1990, 265:3263-3269.
31.
MILLERPS, AGRISCH, AUREIJANL, BLAKEKR, MURAKAMIA, REDDY M_P, SPITZ S& TS'O POP: Control of Ribonucleic Acid Function by Oligonucleoside Methylphosphonates. Biochimie 1985, 67:769-776.
18.
WALKERK, ELELASA, NAZARRN: Inhibition of Protein Synthesis by Anti-5.8 S rRNA Oligodeoxyribonucleotides. J Biol Chem 1990, 265:2428-2430.
32.
ZAMECNIKPC, STEPHENSON M: Inhibition of Rous Sarcoma Virus Replication and Cell Transformation by a Specific Oligodeoxynucleotide. Proc Natl Acad Sci USA 1978, 75:280-284.
33.
GOODARZIG, GROSS SC, TEWARI A, WATABE K: Antisense Oligodeoxyribonucleotides Inhibit the Expression of the Gene for Hepatitis B Virus Surface Antigen. J Gen Virol 1990, 71:3021-3025.
34.
HEIKKI~R, SCHWABG, WICKSTROME, LOKE SL, PLUZNmDH, WATT R, NECKERS LM: A c-myc Antisense Oligodeoxynucleotide Inhibits Entry into S Phase but not Progress from GO to G 1. Nature 1987, 328:445-449.
35.
WICKSTROMEL, BACONTA, GONZALEZ& LYMANGH, WICKSTROM E: Anti-c-myc DNA Increases Differentiation and Decreases Colony Formation by HL-60 Cells. In Vitro Cell Devel Biol 1989, 25:297.
36.
MCMANAWAY ME, NECKERSLM, LOKESL, AL-NASSERAA~ REDNER RL, S~RAMIZU BT, GOLDSCHMIDTS WL, HUBER BE, BHATIA K, MAGRATH IT: Tumor-specific Inhibition of Lymphoma Growth by an Antisense Oligodeoxynucleotide. Lancet 1990, 335:808-811.
37.
GEWmTZAM, ANFOSSIG, VENTURELLID, VALPREDAS, SIMS R, CALABRETrAB: A c - m y b Antisense Oligodeoxynucleoside Inhibits Normal Human Hematopoiesis I n Vitro. Science 1989, 245:1303.
19. •.
STROBELSA, DERVANPB: Site Specific Cleavage of a Yeast Chromosome by Oligonucleotide-directed Triple-helix Formation. Science 1990, 249:73-75. The cleavage of a polypurine insert in a 340 kb chromosome by a triplex-forming oligonucleotide modified with EDTA-Fe2+ is reported, This implies that cleavage of DNAwith sequence specifidty may be possible. 20. •.
GAO W-Y, JAROSZEWSKIJW, COHENJS, CHENGY-C: Mechanisms of Inhibition of Herpes Simplex Virus Type 2 Growth by 28-mer Phosphorothioate Oligodeoxycytidine. J Biol Chem 1990, 265:20172-20178. The activities of a 28-mer phosphorothioate that inhibit growth of herpes viruses are described. 21.
Lo~ SL, STEIN CA, Z ~ G XH, Mova K, NAKAI~SHI M, SUBASlGNHE C, COHEN JS, NECKERS LM: Characterization of Oligonucleotide Transport into Living Cells. Proc Natl Acad Sci USA 1989, 86:3474-3478.
22.
MATSUKURAM, SHINOZUKA K, ZON G, MITSUYA H, RE1TZ M, COHEN JS, BRODER S: Phosphorothioate Analogs of Oligodeoxynucleotides: Inhibitors of Replication and Cytopathic Effects of Human lmmunodeficiency Virus. Proc Natl Acad Sci USA 1987, 84:7706-7710.
23.
KINCHINGTON D, GALPIN S: GAG and POL Antisense Oligodeoxynucleotides as Inhibitors of HIV-1. Meeting on "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression: Therapeutic Implications [abstract]. June 18-21, 1989, Rockvflle, Maryland, USA.
38.
ROSOLENA, WHITESELL L, POPLACKD, ZAJAKM, KENNET RH, IKEGAKI N, BASSO G, NECKERS LM: Antisense Inhibition of n - m y c Translation in a Celi-free System and a Human Neuroblastoma Cell Line.Proc A m Assoc Cancer Res A n n u a l Meeting [abstract] 1989, 30:20.
24.
AGRAWALS, GOODCHILDJ, CIVEIRAMP, THORNTON AH, SARIN PS, ZEMECNIK PC: Oligodeoxynucleoside Phosphoamidates and Phosphorothioates as Inhibitors of Human Immunodeficiency Virus. Proc Natl A c a d Sci USA 1988, 85:7079-7083.
39.
JAROSZEWSKIJW, KAPLANO, SY1J-L, SEHESTEDM, FAUSTINOPJ, COHEI~ JS: Concerning Antisense Inhibition of the Multiple Drug Resistance Gene. Cancer Communications 1989, 2:287-294.
25.
SMITHCC, AURELIAN L, REDDY MP, M[ILER PS, TS'O P: Antiviral Effect of an Oligo(Nucleoside Methylphosphonate) Complementary to the Splice Junction of Herpes Simplex Virus Type 1 Immediate Early Pre-mRNAs 4 and 5. Proc Natl Acad Sci USA 1986, 83:2787-2791.
40.
JASKULSKI D, DERIEL JK, MERCER ~0~E, CALABRETFA B, BASERGA R: Inhibition of Cellular Proliferation by Antisense Oligodeoxynucleotides to PCNA Cyclin. Science 1989, 240:1544-1546.
41.
26.
KULKAM, SMITH C, AURELIAN L, FISHELEVICH tL MEADE K, MILLER P, TS'O P: Site Specificity of the Inhibitory Effects of Oligo(Nucleoside Methylphosphonate)s Complementary to the Acceptor Splice Junction of Herpes Simplex Virus Type 1 Immediate Early mRNA 4. Proc Natl Acad Sci USA 1989, 86:6868~5872.
ZHENGH, SAHAIBM, KILGANNONP, FOTEDARA~ GREENDR: Specific Inhibition of Cell-surface T-cell Receptor Expression by Antisense Oligodeoxynucleotides and its Effect on the Production of an Antigen-specific Regulatory T-cell Factor. Proc Natl Acad Sci USA 1989, 86:3758-3762.
42.
SEGALGM, FORTE S, GROVER CB: Selective Inhibition of Interleukin- 1 (IL-1)-induced Granulocyte-macrophage (GMCSF) and Granulocyte Colony-stimulating Factor (G-CSF) Gene Expression in Endothelial Cells (ECs) by Antisense
27.
AGmSCH, BLAKE K, MMER P, REDDY M, TS'O POP: Inhibition of Vesicular Stomatitis Virus Protein Synthesis and
Antisense technology Oligonucleotides: Expression of GM-CSF and G-CSF are not Linked. Meeting on "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression: Therapeutic Implications' [ab~ stract]. June 18-21, 1989, Rockville, Maryland, USA_ 43.
44.
BIRCHENALL-ROBERTSMC, FAI.KLA, FERRERC, RUSCETrI FW: A CSF-1 Antisense Oligodeoxynucleotide Inhibits Proliferation of Immortalized Murine Monocytes Establishment of Autocrine Regulation. J Cell Biochem Suppl 13, (Part C) 1989, 18. YEOMANLC, DANELSY'J, LYNCH MJ: Inhibition of Colon Tumor Cell Growth by Direct Addition of anti-EGF Receptor Oligodeoxyribonucleotides. Meeting on "Oligodeoxynucle& tides as Antisense Inhibitors of Gene Expression: Therapeutic Implications' [abstract]. June 18-21, 1989, Rockvflle, Maryland, USA.
45.
BLAKEKR, MURAKAMIA, MUiR PS: Inhibition of Rabbit Globin mRNA Translation by Sequence-specific Oligodeoxyribonucleotides. Biochemistry 1985, 24:61324138.
46.
MARCUS-SEKURA C, WOERNEREA, SHINOZUKAK, ZON G, QU1NNAS G: Comparative Inhibition of Chloramphenicol Acetyltransferase Gene Expression by Antisense Oligonucleotide Analogues Having Alkyl Phosphotriester, Methylphosphonate and Phosphorothioate Linkages. Nucleic Acids Res 1987, 15:5749-5763.
47.
WICKSTROMEL, BACONTA, GONZALEZA, FREEMANDL, LYMAN GH, WICKSTROM E: Human Promyelocytic Leukemia HL60 Cell Proliferation and c-myc Protein Expression are Inhibited by an Antisense Pentadecadeoxynucleotide Target Against c-myc mRNA. Proc Natl Acad Sci USA 1988, 85:1028-1032.
Crooke
48.
ZAMECNIKPC, GOODCHILDJ, YAGUCHIY, SARINP: Inhibition of Replication and Expression of Human T-cell Lymphotropic Virus Type HI in Cultured Cells by Exogenous Synthetic Oligonucleotides Complementary to Viral RNA. Proc Natl Acad Sci USA 1986, 83:4143--4146.
49.
CERUZZIM, DRAPER K: The Intracellular and Extracellular Fate of Oligodeoxyribonucleotides in Tissue Culture Systems. Nucleosides Nucleotides 1989, 8:815.
50.
NECKERSLM: Characterization of Oligonucleotide Transport into Living Cells. Meeting on 'Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression: Therapeutic Implications' [abstract]. June 18-21, 1989, Rockville, Maryland, USA.
51.
MARTIGE, ZON G, EGANW, NOGUCHIP, MATSUKURAM, BRODER S: Flow Cytometric Analysis of the Cellular Uptake and Efflux of a Fluorescein-labelled 'Antisense' Oligonucleotide Phosphorothioate: Light Microscopic Determination of the Intracellular Localization of the Fluorochrome. Meeting on "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expre~ sion: Therapeutic Implications' [abstract]. June 18-21, 1989, Rockx~e, Maryland, USA.
52.
SHAWJ-P, KENT K, FISHBACKJ, FROEHLERB: Uptake and Stability of Deoxyoligonucleotides in Mammalian Cells. Meeting on Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression: Therapeutic Implications' [abstract]. June, 18-21, 1989, Rockville, Maryland, USA.
ST Crooke, ISIS Pharmaceuticals, 2280 Faraday Avenue, Carlsbad, California 92008, USA.
287
Nucleic acid structure and function The ability to practice etficient rational drug design is an important potential advantage of oligonucleotide therapeutics. The criteria required are the ability to predict binding constants for oligonucleotides, and knowledge of the secondary structure of RNA targets based on a variety of hybridization and nearest neighbor-derived algorithms. However, predictions of RNA secondary s t r u c -
ture and potential oligonucleotide binding constants are only approximations based on a number of assumptions, some of which are unproven. Consequently, studies that enhance the predictive precision of such algorithms are extremely important. Jaeger et al. [1.-] have carried o u t denaturation and chemical modifications of a well characterized RNA, the Tetrahymena thermophila large subunit ribosomal RNA, containing a seN-splicing intron, to determine the validity of a number of the underlying assumptions. The major conclusions drawn from the study are that most of the assumptions appear to be valid and, in particular, studies in 1 M NaCl provide data similar to those in 0.05M Na + and 10mM Mg2+. Moreover, they suggest that programs that take a more realistic approach to multi-branch loops are more precise, and that tertiary interactions appear to play a relatively small role in determining RNA structure. Finally, their data support the inclusion of favorable free energy terms for terminal GU base pairs, and stacking interactions in loops. Important advances in experimental characterization of RNA secondary and tertiary structures employing physical and chemical methods have also been reported. The conformation of an RNA pseudoknot was defined using enzymatic methods and nuclear magnetic resonance (NMR) spectroscopy [2]. This study demonstrated that pseudoknots are in equilibrium with hairpins and that the stems and loops of a pseudoknot differ in size, position and contribution to the stability of the structure. A minor distortion of helical stacking at the junctions and stems was also demonstrated. Furthermore, the solution structure of an unusually stable hairpin was solved using NMR spectroscopy [3]. A second major theme that has emerged in recent years is the ability of RNA to behave enzymatically. This is important because it encourages examination of RNA from an enzymatically functional perspective in addition to studies of its behavior as a substrate in information transfer processes. Moreover, it focuses attention on RNA-specitic
Abbreviations NMR--nuclear magnetic resonance; TAR--transactivator response element; TAT--transactivating protein. 282
© Current Biology Ltd ISSN 0958-1669
Antisense technology Crooke
Table 1. A summary of the activities of antisense oligonucleotides in vitro.
Target Viruses HIV HIV (gag/pol) HIV HSV HSV VSV VSV IV TBE SV40 RSV HB Oncogenes c-myc c-myc c-myc c-myb n-myc Host genes Multiple drug resistance PCNA (cyclin) T cell receptor GM-CSF CSF-1 EGF receptor [3-Globin Other Chloramphenicol acetyltransferase
Cell type
Serum
Oligotypes
Length (nucleotides)
Concentration
Reference
H-T cells H-T cells H9 cells Vero cells Vero cells L929 cells L929 cells MDCK cells MDCK cells Chicken fibroblasts Alexander
+ + + + + + + + + + +
PS PS PS, others CH3-P CH3-P CH3P P-poly 1 P-acridine Various CH3-P Various
14-28 18-24 20 7 12 9 10-15 11 Variable 6.9 Variable
0.5-1 ~tM 1-10~M 4-20 I~g/ml- 1 50-100 ~tM 20-50 I~M 25-50 I.tM 0.1 ~tM 50 ~tM 0.1-1 ~tM 25 IIM 10 I~M
[22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]
+
P
15
8.5 I.tM
[33]
T-lymphocytes HL-60 Burkitt cells PMBC Neu roblastoma
+ + + +
P P, PS P P P
15 15 21 18 15
30 IIM 10 I~M 100 I*M 40 mg m l - 1 1-5 I*M
[34] [35] [36] [37] [38]
MCF-1 3T3 T cells Endothelial FL-ras/myc Rabbit reticulocytes
+ + + + + +
PS P P P P P CH3P
15 18 22 15, 18 ? 13 9
101~M ? 30 p.M 100 p.M
[39] [40] [41] [42] [43] [44] [45]
CV-1
+
P, PS, CH3P
21
5-30 I~M
[46]
30 p.M
CSF, colony-stimulating factor; EGF, epidermal growth factor; GM-CSF, granulocyte macrophage CSF; HB, hepatitis B; HIV, human immunodeficiency virus; HSV, herpes simplex virus; IV, influenza virus; MDCK, Madian-Darby canine kidney; P, phosphodiester; PCNA, proliferating cell nuclear antigen; PS, phosphorothioate; RSV, Rous sarcoma virus; SV40, simian virus 40; TBE, tick-borne encephalitis; VSV, vesicular stomatitis
chemistry involving the 2' hydroxyl group. It also suggests the potential of modified ribozymes themselves as drugs. During 1990, a large number of important studies on ribozymes were published. These dealt with either the enzymology of Tetrahymena or hammerhead ribozymes, or the biological activities of ribozymes. The enzymology of Tetrahymena ribozymes has been particularly well characterized by Cech and colleagues [4*%5°" ]. The effects of modification of a ribozyme by substitution of phosphorothioates have also been reported [4°° ]. Studies on hammerhead ribozymes have defined the minimum structure required for enzymologic properties [6,7]. Importantly, minimized ribozymes have been shown to cleave sequences on a complementary oligonucleotide [6].
Ribozymes were also shown to cleave single- and doublestranded DNA, thus expanding the repertoire of potential substrates beyond intramolecular RNA cleavage [8,9]. Furthermore, a synthetic ribozyme comprised of deoxyribonucleotides and ribonucleotides was shown to be active [ 10]. Finally, microinjection of a ribozyme into Xenc> pus oocytes was shown to cleave U7 small nuclear RNA
[11]. A third important series of observations are derived from studies on the human immunodeficiency virus that focus on a stem-loop structure that binds a transactivating protein (TAT) to the transactivator response element (TAR). Thus, this structure and its role are now well understood and demonstrate the importance of specific RNA
283
284
Pharmaceuticalapplications Several potentially important modifications of RNA oligonucleotides have been evaluated more extensively in 1990. Modifications at the 2' position are likely to be important because they enhance the stability of duplexes formed with RNA, relative to those formed between DNA oligonucleotides and RNA. These modifications may also enhance nuclease stability. To date, the most extensively studied are the 2'-0-methyl analogs [14.',15]. Another interesting modification is the fusion of lipids to oligonucleotides [16]. The resulting increase in lipophilicity may enhance cellular uptake of the oligonucleotide. The basic message emphasized by these and other studies is that ollgonucleotide therapeutics offer great scope for medicinal chemistry because numerous sites appear to tolerate substantial modification without dramatic reduction in atFmity for receptor sequences and with little loss in specificity.
structures in regulation of biological activities [12..]. TAR is a very stable stem-loop structure. A cellular protein binds to the loop and TAT binds to a bulge in the stem. These interactions result in an increase in transcription and translation of mRNAs that contain the TAR element. Thus, this particular structural motif in RNA plays an important biological role and is a useful model for learning how to destabilize RNA secondary structure for the purpose of creating desired pharmacologic effects.
Medicinal chemistry In a particularly useful review, Uhlmann and Peyman [13"] have provided a valuable summary of the methods available for both synthesizing and modifying oligonucleotides. Although a significant number of modifications has been reported, numerous others remain to be studied, and structure-activity relationships are just being defined.
Table 2. Summary Length nucleotides)
of antisense pharmacokinetics.
Label
Modifications
Serum nuclease
Cell types
Percentage uptake
Kinetics
Distribution
Metabolism
Reference
Phosphodiesters 15 5'35S
None
Heat inactivated
K562
1~
4h
-
Degradation in medium bylh
[47]
10-30
5'32p
None
-
T cell
5
Rapid, reaching plateau in 4h
-
Intact at 15 min and 4 h
[48]
3-20
None
5' acridine None
+ +
HL60 HL60
11
Saturable; temperature-dependent 50 h; temperatureindependent ~ 9 0 min
-
[21]
5'32p
5'32p
None
+
HeLa and others
0.3-2.3
Linear for 24 h
Degradation in serum within 2 h
[49]
-
-
[50]
-
Stable to serum
[49]
Rapid for 30 min, then plateau
Nucleus
-
[51]
18
Oligo-T n
None
5' acridine
+
-
-
0.25-1.2
Phosphorothioates
Saturable PS inhibitors but not CH3P * Linear for 2h
-
18
5'32p
None
-
HeLa and others
28
None
Fluorescein
+
H9, U937
12-20
5'35S
None
+
HL60
1-2
Active
-
Stable for more than 3 days
[52]
None
+
CVl
-
Linear for 2 h
-
Stable
[46]
Methylphosphonates 21 5'32p
AntisensetechnologyCrooke Pharmacodynamics Among the more important advances of the past year was the identification of a number of terminating events or proximal mechanisms of action in addition to RNaseH and translation arrest. Further evidence that inhibition of splicing reactions and modification to 3' and 5' untranslational regions disrupt RNA secoridary structures was presented [17]. Inhibition of key RNA activities has also been described. For example, oligonucleotides complementary to a conserved sequence in 5.8S RNA were reported to inhibit protein synthesis [18]. In some cases, inhibition of transcription may be required to achieve desired therapeutic effects. DNA triplex formation may provide a means of inhibiting the production of certain proteins. Before this mechanism can be routinely employed, however, oligonucleotides with enhanced affinity for DNA, together with the ability to interact in a sequence-specific fashion independently of polypurine or polypyrimidine tracts must be developed. During 1990, Strobel and Dervan [19"] reported that an oligonucleotide coupled to EDTA-Fe2+ bound and cleaved a complementary polypurine sequence inserted in a 340 kb chromosome embedded in an agarose gel. Research into phosphorothioates has demonstrated that they may have the necessary attributes to be first generation oligonucelotide drugs. However, a number of studies suggest that they might bind to proteins non-specifically via interactions involving the thiols, so that, in some cases, the observed effects are not dependent on nucleic acid interactions. Gao et al. [20.'] have demonstrated that a 28-mer phosphorothioate inhibits herpes virus replication by interacting with a number of herpes virus proteins. Despite the potential of phosphorothioates to produce non-specific effects, studies in a number of laboratories have demonstrated that cellular toxicity only occurs at very high concentrations.
Pharmacokinetics Phosphorothioates, methylphosphonates and T-0-methyl-substituted oligonucleotides have been shown to be highly resistant to various nucleases [14"',15]. Cellular uptake of phosphorothioates and methylphosphonates has been studied in vitro [13"]. Surprisingly high uptake of phosphorothioates by cells in tissue culture has been reported, and the compound is clearly shown to be intact intracellularly. Uptake of phosphorothioates appears to involve several active mechanisms and, in particular, a 90 kD membrane protein has been reported to bind phosphorothioate oligonucleotides [21]. Table 2 summarizes recent data on in vitro pharmacokinetics.
that oligonucleotide therapeutics may offer important advantages over conventional drugs and may indeed be the most direct route to the next phase in therapeutics: rational design of isotype-selective therapeutics.
Acknowledgements The excellent typographical assistance of Mrs Coleen Matzinger and the critical reviews by Drs Christopher Mirabelli, David Ecker and P Dan Cook are much appreciated.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •, of outstanding interest 1. •.
JAEGERJA, ZUKERM, TURNERDH: Melting and Chemical Modification of a Cyclized Self-splicing Group I Intron: Similarity of Structure in 1M Na +, in 10mM Mg2+, and in the Presence of Substrate. Biochemistry 1990, 29:10147-10158. This report extends the study of RNA folding programs, demonstrating reasonable predictiveness with confirmed and enhanced assumptions. 2.
PUGLISIJD, WYATt JR, TINOCO I JR: Conformation of a RNA Pseudoknot. J Mol Biol 1990, 214:437-453.
3.
CHEONGC, VAEANIG, TINOCO I JR: Solution Structure of an Unusually Stable RNA Hairpin, 5'GGAC(UUG GUCC). Nature 1990, 346:680-682.
McSWIGGENJA, CECH TR: Stereochemistry of RNA Cleavage by the Tetrahymena Ribozyme and Evidence that the Chemical Step is not Rate-limiting. Science 1989, 244:679-683. This article summarizes a series of studies describing the biochemical and kinetic properties of the Tetrahymena ribozyme. 4.
•,
5. •.
HERSHLAGD, CECIl TR: Catalysis of RNA Cleavage by the Tetrahymena thermophila Ribozyme. 1. Kinetic Description of the Reaction of an RNA Substrate Complementary to the Active Site. Biochemistry 1990, 29:10159-10171. Detailed enzyme kinetic studies demonstrating the mechanisms of cleavage by the Tetrahymena ribozyme are presented. 6.
UHLENBECKOC: A Small Catalytic Oligoribonucleotide. Nature 1987, 328:596-600.
7.
JEFFmESHC, SYMONSRH: A Catalytic 13-met Ribozyme. Nucleic Acid Res 1989, 17:1371-1375.
8.
ROBERTSONDL, JOYCE G:F Selection In Vitro of an RNA Enzyme that Specifically Cleaves Single-stranded DNA. Nature 1990, 344:467468.
9.
HERSHLA6D, CECIl TR: DNA Cleavage Catalysed by the Ribozyme from Tetrahymenat Nature 1990, 344:405-409.
10.
PERREAULTJ-P, WU T, COUSINEAUB, OGILVIE KK, CEDERGREN R:Mixed Deoxyribo- and Ribo-oligonucleotides with Catalytic Activity. Nature 1990, 344:565-567.
11.
COTTENM, BIRNSTIELML: Ribozyme Mediated Destruction of RNA In Viva EMBO J 1989, 8:3861-3866.
12. ~.
Conclusion
CULLERBR~ The HIV-1 TAT Protein: an RNA Sequence-specific Processivity Factor? Cell 1990, 63:655-657. s brief review summarizes the data on TAR and TAT interactions. Because TAR is an important RNA structure and target for antisense drugs, this review is valuable.
Substantial progress in validating the antisense paradigm and enhancing our understanding of a number of the key elements of oligonudeotide therapeutics has been reported during 1990. The progress continues to suggest
13. UHLMANNE, PEYMANA: Antisense Oligonucleotides: a New ,, Therapeutic Principle. Chem Rev 1990, 90:544-584. A detailed review of the antisense research of the past decade and its importance in therapeutic studies.
285
286
Pharmaceutical applications 14. oo
BEIJERB, SULSTONL, SPROATBS, RIDm P, LAMONDAI, NEUNERP: Synthesis and Applications of Oligoribonucleotides with Selected 2'-O-methylation Using the 2'-O-[1-(2-fluorophenyl)4-methoxypiperidin-4-ylk] Protecting Group. Nucleic Acid Res 1990, 18:5143-5151. The synthesis and preliminary characterization of 2'0 methyl oligonucleotides is presented. These compounds may be important as they might provide nuclease resistance and enhanced binding potential for RNA targets.
Infection by Sequence-specific Oligodeoxyribonucleoside Methylphosphonates. Biochemistry 1986, 25:626845275. 28.
LEMAITREM, BAYARDB, LEBLEUB: Specific Antiviral Activity of a Poly(L-lysine)-conjugated Oligodeoxyribonucleotide Sequence Complementary to Vesicular Stomatitis Virus N Protein mRNA Initiation Site. Biochemistry 1987, 84:648-452.
29.
ZERIALA~ THUONG NT, HELENE C: Selective Inhibition of the Cytopathic Effect of Type A Influenza Viruses by Oligodeoxynucleotides Covalently Linked to an Intercalating Agent. Nucleic Acids Res 1987, 15:9909-9919.
30.
VLASSOVVV: Inhibition of Tick-borne Viral Encephalitis Expression Using Covalently Linked Oligonucleotide Analogs. Meeting on Oligodeoxynucleotides as Ant~ense Inhibitors of Gene Expression: Therapeutic Implications' [abstract]. June 18-21, 1989, Rockville, Maryland, USA.
15.
IRIBARRENAM, SPROAT BS, NEUNER P, SULSTON I, RYDER U, LAMOND AI: 2'-O-Alkyl Oligoribonucleotides as Antisense Probes. Proc Natl Acad Sci USA 1990, 87:7747-7751.
16.
SHEARG, MARSTERSJC, BISCHOFBERGERN: Synthesis, Hybridization Properties and Antiviral Activity of Lipid-oligodeoxynucleotide Conjugates. Nucleic Acid Res 1990 18:3777-3783.
17.
SAXENASK, ACKERMANEJ: Microinjected Oligonucleotides Complementary to the ct-Sarcin Loop of 28 S RNA Abolish Protein Synthesis in X e n o p u s Oocytes. J Biol Chem 1990, 265:3263-3269.
31.
MILLERPS, AGRISCH, AUREIJANL, BLAKEKR, MURAKAMIA, REDDY M_P, SPITZ S& TS'O POP: Control of Ribonucleic Acid Function by Oligonucleoside Methylphosphonates. Biochimie 1985, 67:769-776.
18.
WALKERK, ELELASA, NAZARRN: Inhibition of Protein Synthesis by Anti-5.8 S rRNA Oligodeoxyribonucleotides. J Biol Chem 1990, 265:2428-2430.
32.
ZAMECNIKPC, STEPHENSON M: Inhibition of Rous Sarcoma Virus Replication and Cell Transformation by a Specific Oligodeoxynucleotide. Proc Natl Acad Sci USA 1978, 75:280-284.
33.
GOODARZIG, GROSS SC, TEWARI A, WATABE K: Antisense Oligodeoxyribonucleotides Inhibit the Expression of the Gene for Hepatitis B Virus Surface Antigen. J Gen Virol 1990, 71:3021-3025.
34.
HEIKKI~R, SCHWABG, WICKSTROME, LOKE SL, PLUZNmDH, WATT R, NECKERS LM: A c-myc Antisense Oligodeoxynucleotide Inhibits Entry into S Phase but not Progress from GO to G 1. Nature 1987, 328:445-449.
35.
WICKSTROMEL, BACONTA, GONZALEZ& LYMANGH, WICKSTROM E: Anti-c-myc DNA Increases Differentiation and Decreases Colony Formation by HL-60 Cells. In Vitro Cell Devel Biol 1989, 25:297.
36.
MCMANAWAY ME, NECKERSLM, LOKESL, AL-NASSERAA~ REDNER RL, S~RAMIZU BT, GOLDSCHMIDTS WL, HUBER BE, BHATIA K, MAGRATH IT: Tumor-specific Inhibition of Lymphoma Growth by an Antisense Oligodeoxynucleotide. Lancet 1990, 335:808-811.
37.
GEWmTZAM, ANFOSSIG, VENTURELLID, VALPREDAS, SIMS R, CALABRETrAB: A c - m y b Antisense Oligodeoxynucleoside Inhibits Normal Human Hematopoiesis I n Vitro. Science 1989, 245:1303.
19. •.
STROBELSA, DERVANPB: Site Specific Cleavage of a Yeast Chromosome by Oligonucleotide-directed Triple-helix Formation. Science 1990, 249:73-75. The cleavage of a polypurine insert in a 340 kb chromosome by a triplex-forming oligonucleotide modified with EDTA-Fe2+ is reported, This implies that cleavage of DNAwith sequence specifidty may be possible. 20. •.
GAO W-Y, JAROSZEWSKIJW, COHENJS, CHENGY-C: Mechanisms of Inhibition of Herpes Simplex Virus Type 2 Growth by 28-mer Phosphorothioate Oligodeoxycytidine. J Biol Chem 1990, 265:20172-20178. The activities of a 28-mer phosphorothioate that inhibit growth of herpes viruses are described. 21.
Lo~ SL, STEIN CA, Z ~ G XH, Mova K, NAKAI~SHI M, SUBASlGNHE C, COHEN JS, NECKERS LM: Characterization of Oligonucleotide Transport into Living Cells. Proc Natl Acad Sci USA 1989, 86:3474-3478.
22.
MATSUKURAM, SHINOZUKA K, ZON G, MITSUYA H, RE1TZ M, COHEN JS, BRODER S: Phosphorothioate Analogs of Oligodeoxynucleotides: Inhibitors of Replication and Cytopathic Effects of Human lmmunodeficiency Virus. Proc Natl Acad Sci USA 1987, 84:7706-7710.
23.
KINCHINGTON D, GALPIN S: GAG and POL Antisense Oligodeoxynucleotides as Inhibitors of HIV-1. Meeting on "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression: Therapeutic Implications [abstract]. June 18-21, 1989, Rockvflle, Maryland, USA.
38.
ROSOLENA, WHITESELL L, POPLACKD, ZAJAKM, KENNET RH, IKEGAKI N, BASSO G, NECKERS LM: Antisense Inhibition of n - m y c Translation in a Celi-free System and a Human Neuroblastoma Cell Line.Proc A m Assoc Cancer Res A n n u a l Meeting [abstract] 1989, 30:20.
24.
AGRAWALS, GOODCHILDJ, CIVEIRAMP, THORNTON AH, SARIN PS, ZEMECNIK PC: Oligodeoxynucleoside Phosphoamidates and Phosphorothioates as Inhibitors of Human Immunodeficiency Virus. Proc Natl A c a d Sci USA 1988, 85:7079-7083.
39.
JAROSZEWSKIJW, KAPLANO, SY1J-L, SEHESTEDM, FAUSTINOPJ, COHEI~ JS: Concerning Antisense Inhibition of the Multiple Drug Resistance Gene. Cancer Communications 1989, 2:287-294.
25.
SMITHCC, AURELIAN L, REDDY MP, M[ILER PS, TS'O P: Antiviral Effect of an Oligo(Nucleoside Methylphosphonate) Complementary to the Splice Junction of Herpes Simplex Virus Type 1 Immediate Early Pre-mRNAs 4 and 5. Proc Natl Acad Sci USA 1986, 83:2787-2791.
40.
JASKULSKI D, DERIEL JK, MERCER ~0~E, CALABRETFA B, BASERGA R: Inhibition of Cellular Proliferation by Antisense Oligodeoxynucleotides to PCNA Cyclin. Science 1989, 240:1544-1546.
41.
26.
KULKAM, SMITH C, AURELIAN L, FISHELEVICH tL MEADE K, MILLER P, TS'O P: Site Specificity of the Inhibitory Effects of Oligo(Nucleoside Methylphosphonate)s Complementary to the Acceptor Splice Junction of Herpes Simplex Virus Type 1 Immediate Early mRNA 4. Proc Natl Acad Sci USA 1989, 86:6868~5872.
ZHENGH, SAHAIBM, KILGANNONP, FOTEDARA~ GREENDR: Specific Inhibition of Cell-surface T-cell Receptor Expression by Antisense Oligodeoxynucleotides and its Effect on the Production of an Antigen-specific Regulatory T-cell Factor. Proc Natl Acad Sci USA 1989, 86:3758-3762.
42.
SEGALGM, FORTE S, GROVER CB: Selective Inhibition of Interleukin- 1 (IL-1)-induced Granulocyte-macrophage (GMCSF) and Granulocyte Colony-stimulating Factor (G-CSF) Gene Expression in Endothelial Cells (ECs) by Antisense
27.
AGmSCH, BLAKE K, MMER P, REDDY M, TS'O POP: Inhibition of Vesicular Stomatitis Virus Protein Synthesis and
Antisense technology Oligonucleotides: Expression of GM-CSF and G-CSF are not Linked. Meeting on "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression: Therapeutic Implications' [ab~ stract]. June 18-21, 1989, Rockville, Maryland, USA_ 43.
44.
BIRCHENALL-ROBERTSMC, FAI.KLA, FERRERC, RUSCETrI FW: A CSF-1 Antisense Oligodeoxynucleotide Inhibits Proliferation of Immortalized Murine Monocytes Establishment of Autocrine Regulation. J Cell Biochem Suppl 13, (Part C) 1989, 18. YEOMANLC, DANELSY'J, LYNCH MJ: Inhibition of Colon Tumor Cell Growth by Direct Addition of anti-EGF Receptor Oligodeoxyribonucleotides. Meeting on "Oligodeoxynucle& tides as Antisense Inhibitors of Gene Expression: Therapeutic Implications' [abstract]. June 18-21, 1989, Rockvflle, Maryland, USA.
45.
BLAKEKR, MURAKAMIA, MUiR PS: Inhibition of Rabbit Globin mRNA Translation by Sequence-specific Oligodeoxyribonucleotides. Biochemistry 1985, 24:61324138.
46.
MARCUS-SEKURA C, WOERNEREA, SHINOZUKAK, ZON G, QU1NNAS G: Comparative Inhibition of Chloramphenicol Acetyltransferase Gene Expression by Antisense Oligonucleotide Analogues Having Alkyl Phosphotriester, Methylphosphonate and Phosphorothioate Linkages. Nucleic Acids Res 1987, 15:5749-5763.
47.
WICKSTROMEL, BACONTA, GONZALEZA, FREEMANDL, LYMAN GH, WICKSTROM E: Human Promyelocytic Leukemia HL60 Cell Proliferation and c-myc Protein Expression are Inhibited by an Antisense Pentadecadeoxynucleotide Target Against c-myc mRNA. Proc Natl Acad Sci USA 1988, 85:1028-1032.
Crooke
48.
ZAMECNIKPC, GOODCHILDJ, YAGUCHIY, SARINP: Inhibition of Replication and Expression of Human T-cell Lymphotropic Virus Type HI in Cultured Cells by Exogenous Synthetic Oligonucleotides Complementary to Viral RNA. Proc Natl Acad Sci USA 1986, 83:4143--4146.
49.
CERUZZIM, DRAPER K: The Intracellular and Extracellular Fate of Oligodeoxyribonucleotides in Tissue Culture Systems. Nucleosides Nucleotides 1989, 8:815.
50.
NECKERSLM: Characterization of Oligonucleotide Transport into Living Cells. Meeting on 'Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression: Therapeutic Implications' [abstract]. June 18-21, 1989, Rockville, Maryland, USA.
51.
MARTIGE, ZON G, EGANW, NOGUCHIP, MATSUKURAM, BRODER S: Flow Cytometric Analysis of the Cellular Uptake and Efflux of a Fluorescein-labelled 'Antisense' Oligonucleotide Phosphorothioate: Light Microscopic Determination of the Intracellular Localization of the Fluorochrome. Meeting on "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expre~ sion: Therapeutic Implications' [abstract]. June 18-21, 1989, Rockx~e, Maryland, USA.
52.
SHAWJ-P, KENT K, FISHBACKJ, FROEHLERB: Uptake and Stability of Deoxyoligonucleotides in Mammalian Cells. Meeting on Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression: Therapeutic Implications' [abstract]. June, 18-21, 1989, Rockville, Maryland, USA.
ST Crooke, ISIS Pharmaceuticals, 2280 Faraday Avenue, Carlsbad, California 92008, USA.
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