Oligonucleotide therapy Stanley 1. Crooke Isis Pharmaceuticals, Carlsbad, California, USA Rapid progress in oligonucieotide therapeutics has continued over the past year as major programs established in the past four years have grown and begun to be productive. Important advances were reported in the medicinal chemistry of oligonucleotides and in understanding their pharmacodynamic properties. Significant progress was made in understanding the pharmacokinetic and toxicologic properties of first generation analogs, particularly phosphorothioates and one oligonucleotide, ISIS 2105, entered clinical trials. Additionally, combinatorial approaches designed to identify oligonucleotides that may bind to a variety of targets were reported. Current Opinion in Biotechnology 1992, 3:656-661
Introduction 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 b e e n selected for review primarily on the basis of their potential generic importance.
Therapeutic activities The activities of a n u m b e r of oligonucleotides against a wide variety of cellular targets in tissue culture continue to be reported. In general, recent studies include appropriate controls that support the theory that the effects observed are due to the mechanisms proposed. Additionally, in m a n y studies effective dose response curves have b e e n reported. A recent review summarizes i n v i t r o activities and derives several generalizations [1"]. A n u m b e r of laboratories have reported unmodified phosphodiester oligonucleotide activity in cells incubated in the absence of serum, even though phosphodiesters are degraded relatively rapidly. The concentrations required to display activity were typically greater than 10btM. A variety of modified oligonucleotides have b e e n reported to be active. Methylphosphonates a p p e a r to be less potent than phosphorothioates, but considerable variation has b e e n observed, d e p e n d i n g on the system. Conjugation of alkylators and intercalators to phosphodiesters and methylphosphates increases potency. Lipophilic and poly-lysine conjugates have also displayed e n h a n c e d activities. Oligonucleotides have demonstrated a broad array of activities against viral targets, oncogenes, normal host gene products and various transfected genes. Thus, there is clear evidence supporting the broad poten-
tial applicability of these drugs. Although the data from studies incorporated in Table 1 are limited, w h e n combined with i n v i t r o toxicologic data, the therapeutic index of phosphorothioates appears to be quite high i n vitro. Methytphosphonates a p p e a r to have lower therapeutic indexes, but too few data are available to draw conclusions about other classes of oligonucleotides. Very few data are available to support putative mechanisms of action, and generalizations concerning optimization of mechanisms of action are not possible. Nevertheless, a variety of mechanisms of action may be employed by oligonucleotides resulting in significant biological activity. In addition to the preliminary report of anti-herpes activity of a phosphorothioate oligonucleotide applied topically to the cornea of mice infected with herpes virus type 1 (reviewed in [2]), two reports of i n v i v o activity have been published. A phosphodiester oligonucleotide targeted to a site in N - m y c RNA inhibited expression of the gene and growth of the tumor w h e n administered to animals [3]. Multiple intraperitoneal doses of a phosphorothioate oligonucleotide have also b e e n s h o w n to inhibit the growth of an intraperitoneal tumor that expresses the p120 oncogene [4].
Advances in the medicinal chemistry of oligonucleotides A recent review [5"'] documents the potential of oligonucleotides in medicinal chemistry. Modifications of the bases and sugars of the b a c k b o n e have b e e n incorporated into oligonucleotides and are reported to improve pharmacokinetic and pharmacodynamic properties. T w o major advances in b a c k b o n e chemistry were reported in the past year. Neilsen and colleagues [6"']
Abbreviation PNA--peptide nucleic acids.
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Oligonucleotide therapy Crooke 657 -able 1. Antisense oligonucleotides: summary of activities in vitro (continued on page 568). Target
Cell type
Serum
Oligo types
Length (nt)
Concentration
P PS PS PS, others PS CH3P PS
12-26 14 28 18-24 20 18-28 7 28 12 12 21 9 11 10-15 11 variable 6-9 variable 15 4-30
5-50 mg ml- 1 0.5-1 BM 1-10 ltM 4-20btgm1-1 10 J.tM 50-100 ptM 1-10 I.tM (non-antisense) 20-50 ~tM 5.ttM 0.2-4p.M 25-50gtM 50-150 FtM 0.1 pM 50pM 0.1-1 ~M 25 ItM 10 ~M 8,5 p-M 0.01 1 gM
15 15 21 18 20 15 9
30 p.M 10 pM 100 p.M 40 mg ml- 1 25-150u.M 1-5 ~tM inactive
Viruses HTLV-III HIV H IV (gag/pol) HIV HIV Herpes simplex Herpes simplex Herpes simplex Herpes simplex Herpes simplex Vesicular stomatitis Vesicular stomatitis Vesicular stomatitis Influenza Tick born encephalitis SV40 Rous sarcoma Hepatitis B Bovine papilloma
H9 cells H-T cells H-T cells H9 cells CZM cells Vero cells HeLa cells
+ + + + + +
Vero cells Vero cells HeLa cells L929 cells L929 cells L929 cells MDCK cells MDCK cells Chicken fibroblasts Alexander C-127 cells
+ + + + + + + + + + + +
T lymphocytes HL-60 cells Bu rkitt cells PMBC L697 cells Neuroblastoma cells T15 cells
+ +
CH3P CH3P psoralen PS CH3P P-lipid p-poly I-lysine P-acridine various CH3P various P PS
Oncogenes c-myc c-myc c-myc c-myb BCL-2 N-myc N-ras
+ + +
P P, PS P P P, PS P CH3P
[ CH3P, methylphosphonate oligonucleotides; HTLV, human T-cell lymphocytic virus; P, phosphodiester oligonucleotides; P-acridine, phosphodiester oligonucleotide conjugated with an acridine moiety; P-lipid, phosphodiester oligonucleotide conjugated with a lipid moiety; PS, phosphorothioate oligonucleotides; nt, nucleotides; SV40, simian virus 40.
reported the synthesis and preliminary characterization of oligonucleotides in which the s u g a r - p h o s p h a t e b a c k b o n e was replaced entirely with a polyamide backbone. They coined the term 'peptide nucleic acids' (PNA) to desc.n'be this chemical class. Several factors m a k e the PNA backbone potentially quite important. First, it demonstrates that by maintaining the spatial relationships b e t w e e n oligonucleotide bases, the whole sugar-phosphate unit m a y be replaced with entirely different chemicals. This opens up the oligonucleotide b a c k b o n e to a m u c h broader range of potential modifications than was previously believed possible. Second, the modification resulted in oligonucleotides that display a dramatically increased affinity for DNA and high specificity. Third, the data derived using double-stranded h o m o p o l y m e r s suggest that PNA oligonucleotides can invade double-stranded DNA. If this is confirmed in mixed sequences, it could m e a n that transcriptional arrest is feasible without the requirement for triple-strand strategies dependent on non-Watson-Crick hybridization. More recent studies at Isis Pharmaceuticals, in collaboration with Neilsen and colleagues, have confirmed the earlier observations, demonstrating excellent affinity for RNA and double-stranded nucleic acids with mixed sequences. The second group of backbone analogs with exciting properties was reported by Vasseur et al. [7-].
In this series of compounds, the phosphate was replaced b y a synthetically versatile four-atom synthon. In the first series of molecules, oximes, aminohydroxy and N-methylhydrazino backbones w e r e studied. Interesting insights into structure-activity relationships were reported and several of the b a c k b o n e s displayed high affinity and specificity for DNA and RNA targets. These molecules are nuclease resistant and can be synthesized to have a variety of charges at neutral pH.
Several other b a c k b o n e modifications were also reported in the past year. A sulfonamide moiety was used to replace the phosphate and its 5' oxygen atom in a dimer [8] and a dinucleotide was synthesized with a methyl sulfonyl moiety [9]. Additionally, a dimethylene sulfonate moiety was used to replace 3' and 5' oxygens and the phosphate in a thymidine tetramer [10] which was reported to produce stable duplcxes with oligodeoxynucleotides.
The exciting advances reported during the review period together with modifications reported previously demonstrate very substantial progress in creating a repertoire of backbones that may replace first generation backbones, such as phosphorothioates and methylphosphonates.
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Pharmaceuticalapplications Table 1. Antisense oligonucleotides: summary of activities in vitro (continued from page 657). Target
Cell type
Serum
Oligo types
Length (nt)
Concentration
Host genes
Multiple drug resistance PCNA (cyclin) Prothymosin T-cell receptor GM-CSF CSF-1 EGF receptor BFGF [3 globin TAU cAMP-protein kinase II [3 Myeloblastin Phospholipase A2-activating protein ICAM-1 IL-2 IL-1 cc IL-1 [3 IGF-1 Perforin
MCF 1 cells 3T3 Human myeloma cells T cells Endothelial cells FL-ras/myc cells Human astrocytes Rabbit reticulocytes Neurons HL-60 cells HL-60 cells BC3H 1 A549, HUVEC, lymphocytes T lymphocytes H UVEC Monocytes Myoblasts T lymphocytes
+ + + + + + + + + + + + -
PS P P P P P P P CH3P P P P P
15 18 1621 22 15, 18 ? 13 15 9 20-25 21 18 25
10-SM ? 30 ~tM 10-75 ~M 100 ~M 3-50 ~M 15 ~M ? 25 btM
PS P P PS P P
18-20 15 18 15 15 18
0.01-1 btM 5 btM 10 btM 0.1-2.5 ~tM 10 ~M 5 35 ~M
21 18-28
5-30~M 0.25-5 btM
14-20
1-10btM
30~M 40 ~tM
Other
Chloramphenicol acetyl transferase Placental alkaline phosphatase driven by HIV TAR Chloramphenicol acetyl transferase driven by human papilloma virus E2-responsive element
CV-1 cells SK-mel-2 cells O127 and CV-1 cells
+ + +
P, PS, CH3P PS PS
CH3P, methylphosphonate oligonucleotides; EGF, epidermal growth factor; GM-CSF, macrophage colony-stimulating factor; P, phosphodiester oligonucleotides; PS, phosphorothioate oligonucleotides; PCNA, proliferating cell nuclear antigen; PMA, phorbol mysteric acid; TAR, Tat response element; nt, nucleotides; CSF, colony-stimulating factor; IL, interleukin; ICAM, intercellular growth factor; IGF, insulin-like growth factor.
Pentofuranase sugar modifications
Modifications at other positions
The most important advances in this area are the 2' modifications which have recently been shown to produce RNA-like oligonucleotides that are much more stable than RNA (AM Kawasaki et al., abstract 71, International Union of Biochemistry Conference on Nucleic Acid Therapeutics, Clearwater Beach, 1991) [5"',11]. A number of these modifications were shown to enhance the affinity of oligonucleotides for RNA. This effort also demonstrates that the 2' position is an excellent site at which to introduce substituents, in that a wide range of ethyl and alkylamino substituents were shown to be tolerated without significant loss of affinity. Thus, modifications in the 2' position can be introduced to increase affinity for RNA and/or alter other properties of oligonucleotides, e.g. lipophilicity or cleavage of RNA targets.
Work is continuing on base, pendant and combinations of various modifications to create chimeric oligonucleotides, and has been reviewed recently in [5"'].
Another area of considerable interest are the c~-anomer oligonucleotides. These molecules, in which the configuration of the sugar is inverted, have been shown to hybridize to DNA and RNA and to have a degree of nuclease resistance [12]. Chimeric molecules, in which each sugar is in the (x configuration and each phosphate is a phosphorothioate, have been shown to possess enhanced nuclease resistance [12].
Advances in understanding pharmacodynamic properties of oligonucleotides Occupancy-only mediated mechanisms Classic competitive antagonists are thought to alter biological activity because they bind to receptors, thereby preventing natural agonists from binding and inducing normal biological processes. Binding of oligonucleotides to specific sequences may inhibit the interaction of the RNA or DNA with proteins, other nucleic acids, or other factors required for essential steps in the intermediary metabolism of the RNA or its utilization by the cell.
Transcriptional arrest Oligonucleotides may bind to DNA and prevent either initiation or elongation of transcription b y preventing effective binding of factors required for transcription, thus causing transcriptional arrest. Progress in
Oligonucleotide therapy Crooke 659 triplex strategies has b e e n reviewed recently [13"']. In 1991, advances were made in developing strategies that partially circumvent the requirements f o r polypyrimidine-polypurine motifs [14-16]. Also, triplestrand binding has b e e n shown to b e enhanced by the use of intercalators attached covalently to oligonucleotides [17]. Additionally, a n u m b e r of triplexing oligonucleotides w e r e reported to have biological activity. A 13-mer phosphodiester homopyrimidine was reported to inhibit the transcription of E s c h e r i c h i a coli RNA polymerase [18]. An oligonucleotide at a concentration of 20 btM targeted to the promoter region of the c~ subunit of the interleukin-2 receptor was reported to reduce mRNA content by 32% [19].
eukaryotic cells. Multiple enzymes with RNase H activity have b e e n observed in prokaryotes [20"]. Perhaps the most important theme to emerge with regard to RNase H is an increased understanding of the structure-activity relationships of oligonucleotides with regard to their ability to support RNase H cleavage. Oligonucleotides that are not negatively charged or that are RNA-like have not b e e n s h o w n to support RNase H cleavage [11,23]. RNase cleavage of most, if not all, sequences is not s u p p o r t e d by (x oligonucleotides [12]. Very recently, chimeric oligonucleotides have b e e n synthesized which contain modifications that enhance binding to RNA and, in some positions in the molecules, modifications that support RNase H activity.
Translational arrest In 1991, significant additional results elucidating the activity of oligonucleotides designed to bind to the translational initiation c o d o n have b e e n reported and are reviewed in [5"']. Most importantly, these experiments were well controlled and high quality dose response curves were reported [20"]. Additionally, it is clear that agents that induce translational arrest may also rely on other mechanisms, e.g. RNase H, an enzyme which specifically cleaves the RNA in RNA-DNA duplexes.
Disruption of RNA structure Oligonucleotides that disrupt the Tat responsive element, TAR, and therefore affect the activity of HIV have b e e n reported by Vickers et al. [21]. This publication represents an initial effort to design oligonucleotides that bind to and disrupt RNA stems and loops. This is likely to be an important focus of study in the future.
Occupancy-activated destabilization RNA molecules regulate their own metabolism. A number of structural features of RNA are k n o w n to influence stability, various processing events, subcellular distribution, and transport. As RNA intermediary metabolism b e c o m e s better understood, m a n y other regulatory features and mechanisms will probably be identified. One mechanism to destabilize RNA and to interfere with translation is to bind to the 5' cap and either cleave the it or inhibit the binding of cap-binding proteins. A recent publication demonstrating that oligonucleotides may bind to the 5' cap and alter binding of cap proteins was reported by Baker, Miraglia and H a g e d o r n [22]. Interactions in the 3' untranslated region m a y also destabilize RNA structures and this was demonstrated clearly by Chiang et al. [20"].
RNase H mediated cleavage of RNA RNase H is an ubiquitous enzyme which degrades the RNA strand of RNA-DNA duplexes. It has b e e n identified in organisms as diverse as viruses and humans. At least two classes of RNase H have b e e n identified in
Pharmacokinetics of phosphorothioates The in vitro pharmacokinetics of phosphorothioates have recently b e e n summarized [24"']. The basic conclusions supported b y the data are described here. Uptake, as assessed b y the intracellular accumulation of a radiolabeled or fluorescently labeled compound, was d e p e n d e n t on time, temperature, and concentration and appeared to require cellular energy. Specific characteristics of uptake differed according to oligonucleotide class. It appears that some oligomers are concentrated in some cells. Studies that quantified the amount of oligonucleotide taken up showed that the amount internalized ranged from 1-11%. The extent and rate of oligonucleotide uptake varied depending on the specific cell type. Some data also suggested that uptake in vivo may differ from that determined in vitro. Nevertheless, different types of oligomers were taken up in pharmacologically relevant concentrations by a variety of cells. The stability of oligonucleotides taken into cells also varied with oligonucleotide class and dep e n d e d on whether or not oligomers w e r e modified by pendant groups. Modification of oligonucleotides with substituents, such as acridine, poly(L-lysine), or various hydrophobic groups, enhanced the uptake but not necessarily the activity of the compounds. Although it was not demonstrated specifically, intracellular distribution probably differs depending u p o n specific pendant modifications [24"]. The mechanism of uptake varied according to oligonucleotide type [24"]. The data suggest that methylphosphonates, which are non-ionic and lipophilic, cross cellular m e m b r a n e s b y passive diffusion. Several groups have data suggesting that phosphodiester and phosphorothioate oligonucleotides are taken up by receptor-mediated endocytosis. Localization studies using fluorescently labeled oligonucleotides support this notion. In these localization experiments, however, possible quenching of e n h a n c e m e n t of fluorescence of the acridine or fluorescein substituents within ceils was not investigated. Many more experiments must be done to prove unequivocally that receptor-mediated endocytosis is the only m e c h a n i s m of uptake of charged oligonucleotides. Direct isolation and characterization of a phosphorothioate-specific receptor has not yet b e e n done, nor has anyone p r o v e d that uptake
660
Pharmaceuticalapplications of phosphodiester h o m o p o l y m e r s is equivalent to uptake of heterosequence phosphodiesters or phosphorothioates. Data obtained from studies using fluorescently labeled c o m p o u n d s suggest that oligomers are mainly localized in the cytoplasm within vesicular structures. Encapsulation within liposomes e n h a n c e d uptake of oligonucleotides. Given what is k n o w n about the uptake of liposomes and the fate of various pharmacological agents encapsulated by these vesicles, the mechanism of uptake and intracetlular fate of the oligomers was probably altered, though it has not b e e n specifically shown. Additionally, cationic lipids have recently b e e n shown to enhance the u p t a k e of phosphorothioate oligonucleotides into some cells in tissue culture [25]. Although substantial progress has been m a d e in understanding the i n vivo pharmacokinetics of phosphorothioate oligonucleotides, few publications have emerged as yet.
In vivo
pharmacokinetics
Preliminary in vivo pharmacokinetic data are n o w available on m e t h y l p h o s p h o n a t e and phosphorothioate oligonucleotides. A 12-met 3H-labeled methylphosphonate injected in the tail vein of mice was cleared rapidly as intact oligonucleotide and distributed widely to all tissues except the brain [26]. More extensive studies have b e e n performed on 35S-labeled phosphorothioates in rats. A true distribution phase of 15-25 minutes was observed after a single intravenous dose of a 27-met followed by a prolonged elimination phase of 20-40 hours [26]. The prolonged elimination phase may result from the binding of phosphorothioates to serum proteins. Phosphorothioates were distributed to all tissues except the brain and eliminated intact in the urine. Phosphorothioates were rapidly and extensively absorbed after intramuscular and intraperitoneal administration [26]. Repeated daily doses of 50 mg kg- 1 of a 27-mer phosphorothioate to mice resulted in similar distribution and elimination kinetics to, but slight differences in tissue concentrations from, single dose studies. The liver, kidney, spleen and lung were the organs with the highest concentrations. Again, the drug was excreted intact in the urine [26]. Continuous subcutaneous osmotic p u m p administration of the same c o m p o u n d for four weeks at doses of 50-150 mg resulted in similar pharmacokinetics [26]. Studies with ISIS 1082, a 21-met phosphorothioate, in mice s h o w e d that w h e n applied to the cornea in a sodium acetate buffer, significant adsorption to the cornea and absorption into the aqueous and vitreous humors occurred. Moreover, significant systemic bioavailability was observed [25]. In the rabbit, as much as 25% of an applied ocular dose was systemically bioavailable (ST Crooke, unpublished data). Post-absorption pharmacokinetics were equivalent to intravenous pharmacokinetics.
Recently, a 20-mer phosphodiester was administered intravenously to rabbits. Clearance from the b l o o d was rapid and after 90 minutes 16% of the dose was found intact in the urine. In the blood, at least 17% of the drug was estimated to b e completed degraded within five minutes [27].
Toxicologic properties of phosphorothioate oligonucleotides The toxicological properties of phosphorothioate oligonucleotides have recently b e e n reviewed [24,26]. I n vitro, the conclusions are that phosphorothioate oligonucleotides display a very substantial therapeutic index. Although too little in vivo information has b e e n published to draw firm conclusions, within the next year additional publications should a p p e a r on the several oligonucleotides being studied in animals and on ISIS 2105 which is currently being administered to humans.
Combinatorial strategies Combinatorial methods to identify oligonucleotides that m a y bind to either nucleic acid or non-nucleic acid targets have b e e n developed. The first report of the identification of an analog nucteotide with potential therapeutic utility a p p e a r e d this year [28]. Two phosphodiester deoxyoligonucleotides w e r e s h o w n to slow clotting time and bind to thrombin w h e n incubated at 20 btM and assayed within two minutes.
Conclusions Very substantial progress has taken place over the past year. This sets the stage for even more significant advances in the future. Based on data generated to date, there is cause for continued optimism about the therapeutic utility of oligonucleotides.
Acknowledgement The excellent technical assistance of Mrs Colleen Matzinger is greatly~ appreciated. ,
#
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest •. of outstanding interest 1. CROOKE ST: T h e r a p e u t i c A p p l i c a t i o n s o f O l i g o n u •. cleotides. A n n u Rev Pharmacol Toxicol 1992, 32:329-376. This review places oligonucleotide therapeutics in the context of modern molecular pharmacology and summarizes the status of the area. 2.
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NEILSENFE, EGItOLM M, BERG RH, BUCHARDT O: S e q u e n c e Selective R e c o g n i t i o n o f DNA b y S t r a n d D i s p l a c e m e n t w i t h a T h y m i n e - s u b s t i t u t e d P o l y a m i d e . Science 1991, 254:1497-1500. This paper reports synthesis and initial characterization of an entirely n e w a n d potentially very important backbone modification. 6. •.
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SUN JS, GIOVANNANGELI C, FRGNCOIS JC, KURFURST R, MONTENAY-GARESTIER T, ASSELINE V, SAISON-BEHMOARAS T, THUNOG NT, HELENE C: T r i p l e H e l i x F o r m a t i o n b y c~ O l l g o d e o x y n u c l e o t i d e s a n d c~ O l i g o d e o x y n u c l e o t i d e i n t e r c a l a t o r C o n j u g a t e s . Proc Natl A c a d Sci USA 1991, 88:6023.
B O C K LC, GRIFFIN LC, LATHAM JA, VERMAAS EH, TOOLE JJ: S e l e c t i o n o f S i n g l e - s t r a n d e d D N A M o l e c u l e s t h a t B i n d a n d I n h i b i t H u m a n T h r o m b i n . Nature 1992, 355:564-566.
ST Crooke, Isis Pharmaceuticals, 2280 Faraday Avenue, Carlsbad, California 92008, USA.
MORVKNF, RAYNER B, IMBACHJ-L: ~ - O l i g o n u c l e o t i d e s : A U n i q u e Class o f M o d i f i e d C h i m e r i c Nucleic Acids. Anticancer Drug Des 1991, 6:521-529.
HELENE C: T h e A n t i - g e n e Strategy: C o n t r o l o f G e n e E x p r e s s i o n b y T r i p l e x - f o r m i n g O l i g o n u d e o t i d e s . Anticancer Drug Des 1991, 6:569-584. This review provides an excellent s u m m a r y of published results on triple-strand research a n d additional u n p u b l i s h e d data.
15.
CHIANG MY, CHAN H, ZOUNES m a , FREIER SM, LIMA -'vVF, BENNETt CF: A n t i s e n s e O l i g o n u c l e o t i d e s I n h i b i t I n t e r c e l h d a r A d h e s i o n M o l e c u l e 1 E x p r e s s i o n b y T w o Dist i n c t M e c h a n i s m s . J Biol Chem 1991, 266:18162-18171. This review provides an excellent s u m m a r y of published results on triple-strand research and additional u n p u b l i s h e d data.
25.
13. •.
14.
20.
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Introduction 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 b e e n selected for review primarily on the basis of their potential generic importance.
Therapeutic activities The activities of a n u m b e r of oligonucleotides against a wide variety of cellular targets in tissue culture continue to be reported. In general, recent studies include appropriate controls that support the theory that the effects observed are due to the mechanisms proposed. Additionally, in m a n y studies effective dose response curves have b e e n reported. A recent review summarizes i n v i t r o activities and derives several generalizations [1"]. A n u m b e r of laboratories have reported unmodified phosphodiester oligonucleotide activity in cells incubated in the absence of serum, even though phosphodiesters are degraded relatively rapidly. The concentrations required to display activity were typically greater than 10btM. A variety of modified oligonucleotides have b e e n reported to be active. Methylphosphonates a p p e a r to be less potent than phosphorothioates, but considerable variation has b e e n observed, d e p e n d i n g on the system. Conjugation of alkylators and intercalators to phosphodiesters and methylphosphates increases potency. Lipophilic and poly-lysine conjugates have also displayed e n h a n c e d activities. Oligonucleotides have demonstrated a broad array of activities against viral targets, oncogenes, normal host gene products and various transfected genes. Thus, there is clear evidence supporting the broad poten-
tial applicability of these drugs. Although the data from studies incorporated in Table 1 are limited, w h e n combined with i n v i t r o toxicologic data, the therapeutic index of phosphorothioates appears to be quite high i n vitro. Methytphosphonates a p p e a r to have lower therapeutic indexes, but too few data are available to draw conclusions about other classes of oligonucleotides. Very few data are available to support putative mechanisms of action, and generalizations concerning optimization of mechanisms of action are not possible. Nevertheless, a variety of mechanisms of action may be employed by oligonucleotides resulting in significant biological activity. In addition to the preliminary report of anti-herpes activity of a phosphorothioate oligonucleotide applied topically to the cornea of mice infected with herpes virus type 1 (reviewed in [2]), two reports of i n v i v o activity have been published. A phosphodiester oligonucleotide targeted to a site in N - m y c RNA inhibited expression of the gene and growth of the tumor w h e n administered to animals [3]. Multiple intraperitoneal doses of a phosphorothioate oligonucleotide have also b e e n s h o w n to inhibit the growth of an intraperitoneal tumor that expresses the p120 oncogene [4].
Advances in the medicinal chemistry of oligonucleotides A recent review [5"'] documents the potential of oligonucleotides in medicinal chemistry. Modifications of the bases and sugars of the b a c k b o n e have b e e n incorporated into oligonucleotides and are reported to improve pharmacokinetic and pharmacodynamic properties. T w o major advances in b a c k b o n e chemistry were reported in the past year. Neilsen and colleagues [6"']
Abbreviation PNA--peptide nucleic acids.
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© Current Biology Ltd ISSN 0958-1669
Oligonucleotide therapy Crooke 657 -able 1. Antisense oligonucleotides: summary of activities in vitro (continued on page 568). Target
Cell type
Serum
Oligo types
Length (nt)
Concentration
P PS PS PS, others PS CH3P PS
12-26 14 28 18-24 20 18-28 7 28 12 12 21 9 11 10-15 11 variable 6-9 variable 15 4-30
5-50 mg ml- 1 0.5-1 BM 1-10 ltM 4-20btgm1-1 10 J.tM 50-100 ptM 1-10 I.tM (non-antisense) 20-50 ~tM 5.ttM 0.2-4p.M 25-50gtM 50-150 FtM 0.1 pM 50pM 0.1-1 ~M 25 ItM 10 ~M 8,5 p-M 0.01 1 gM
15 15 21 18 20 15 9
30 p.M 10 pM 100 p.M 40 mg ml- 1 25-150u.M 1-5 ~tM inactive
Viruses HTLV-III HIV H IV (gag/pol) HIV HIV Herpes simplex Herpes simplex Herpes simplex Herpes simplex Herpes simplex Vesicular stomatitis Vesicular stomatitis Vesicular stomatitis Influenza Tick born encephalitis SV40 Rous sarcoma Hepatitis B Bovine papilloma
H9 cells H-T cells H-T cells H9 cells CZM cells Vero cells HeLa cells
+ + + + + +
Vero cells Vero cells HeLa cells L929 cells L929 cells L929 cells MDCK cells MDCK cells Chicken fibroblasts Alexander C-127 cells
+ + + + + + + + + + + +
T lymphocytes HL-60 cells Bu rkitt cells PMBC L697 cells Neuroblastoma cells T15 cells
+ +
CH3P CH3P psoralen PS CH3P P-lipid p-poly I-lysine P-acridine various CH3P various P PS
Oncogenes c-myc c-myc c-myc c-myb BCL-2 N-myc N-ras
+ + +
P P, PS P P P, PS P CH3P
[ CH3P, methylphosphonate oligonucleotides; HTLV, human T-cell lymphocytic virus; P, phosphodiester oligonucleotides; P-acridine, phosphodiester oligonucleotide conjugated with an acridine moiety; P-lipid, phosphodiester oligonucleotide conjugated with a lipid moiety; PS, phosphorothioate oligonucleotides; nt, nucleotides; SV40, simian virus 40.
reported the synthesis and preliminary characterization of oligonucleotides in which the s u g a r - p h o s p h a t e b a c k b o n e was replaced entirely with a polyamide backbone. They coined the term 'peptide nucleic acids' (PNA) to desc.n'be this chemical class. Several factors m a k e the PNA backbone potentially quite important. First, it demonstrates that by maintaining the spatial relationships b e t w e e n oligonucleotide bases, the whole sugar-phosphate unit m a y be replaced with entirely different chemicals. This opens up the oligonucleotide b a c k b o n e to a m u c h broader range of potential modifications than was previously believed possible. Second, the modification resulted in oligonucleotides that display a dramatically increased affinity for DNA and high specificity. Third, the data derived using double-stranded h o m o p o l y m e r s suggest that PNA oligonucleotides can invade double-stranded DNA. If this is confirmed in mixed sequences, it could m e a n that transcriptional arrest is feasible without the requirement for triple-strand strategies dependent on non-Watson-Crick hybridization. More recent studies at Isis Pharmaceuticals, in collaboration with Neilsen and colleagues, have confirmed the earlier observations, demonstrating excellent affinity for RNA and double-stranded nucleic acids with mixed sequences. The second group of backbone analogs with exciting properties was reported by Vasseur et al. [7-].
In this series of compounds, the phosphate was replaced b y a synthetically versatile four-atom synthon. In the first series of molecules, oximes, aminohydroxy and N-methylhydrazino backbones w e r e studied. Interesting insights into structure-activity relationships were reported and several of the b a c k b o n e s displayed high affinity and specificity for DNA and RNA targets. These molecules are nuclease resistant and can be synthesized to have a variety of charges at neutral pH.
Several other b a c k b o n e modifications were also reported in the past year. A sulfonamide moiety was used to replace the phosphate and its 5' oxygen atom in a dimer [8] and a dinucleotide was synthesized with a methyl sulfonyl moiety [9]. Additionally, a dimethylene sulfonate moiety was used to replace 3' and 5' oxygens and the phosphate in a thymidine tetramer [10] which was reported to produce stable duplcxes with oligodeoxynucleotides.
The exciting advances reported during the review period together with modifications reported previously demonstrate very substantial progress in creating a repertoire of backbones that may replace first generation backbones, such as phosphorothioates and methylphosphonates.
658
Pharmaceuticalapplications Table 1. Antisense oligonucleotides: summary of activities in vitro (continued from page 657). Target
Cell type
Serum
Oligo types
Length (nt)
Concentration
Host genes
Multiple drug resistance PCNA (cyclin) Prothymosin T-cell receptor GM-CSF CSF-1 EGF receptor BFGF [3 globin TAU cAMP-protein kinase II [3 Myeloblastin Phospholipase A2-activating protein ICAM-1 IL-2 IL-1 cc IL-1 [3 IGF-1 Perforin
MCF 1 cells 3T3 Human myeloma cells T cells Endothelial cells FL-ras/myc cells Human astrocytes Rabbit reticulocytes Neurons HL-60 cells HL-60 cells BC3H 1 A549, HUVEC, lymphocytes T lymphocytes H UVEC Monocytes Myoblasts T lymphocytes
+ + + + + + + + + + + + -
PS P P P P P P P CH3P P P P P
15 18 1621 22 15, 18 ? 13 15 9 20-25 21 18 25
10-SM ? 30 ~tM 10-75 ~M 100 ~M 3-50 ~M 15 ~M ? 25 btM
PS P P PS P P
18-20 15 18 15 15 18
0.01-1 btM 5 btM 10 btM 0.1-2.5 ~tM 10 ~M 5 35 ~M
21 18-28
5-30~M 0.25-5 btM
14-20
1-10btM
30~M 40 ~tM
Other
Chloramphenicol acetyl transferase Placental alkaline phosphatase driven by HIV TAR Chloramphenicol acetyl transferase driven by human papilloma virus E2-responsive element
CV-1 cells SK-mel-2 cells O127 and CV-1 cells
+ + +
P, PS, CH3P PS PS
CH3P, methylphosphonate oligonucleotides; EGF, epidermal growth factor; GM-CSF, macrophage colony-stimulating factor; P, phosphodiester oligonucleotides; PS, phosphorothioate oligonucleotides; PCNA, proliferating cell nuclear antigen; PMA, phorbol mysteric acid; TAR, Tat response element; nt, nucleotides; CSF, colony-stimulating factor; IL, interleukin; ICAM, intercellular growth factor; IGF, insulin-like growth factor.
Pentofuranase sugar modifications
Modifications at other positions
The most important advances in this area are the 2' modifications which have recently been shown to produce RNA-like oligonucleotides that are much more stable than RNA (AM Kawasaki et al., abstract 71, International Union of Biochemistry Conference on Nucleic Acid Therapeutics, Clearwater Beach, 1991) [5"',11]. A number of these modifications were shown to enhance the affinity of oligonucleotides for RNA. This effort also demonstrates that the 2' position is an excellent site at which to introduce substituents, in that a wide range of ethyl and alkylamino substituents were shown to be tolerated without significant loss of affinity. Thus, modifications in the 2' position can be introduced to increase affinity for RNA and/or alter other properties of oligonucleotides, e.g. lipophilicity or cleavage of RNA targets.
Work is continuing on base, pendant and combinations of various modifications to create chimeric oligonucleotides, and has been reviewed recently in [5"'].
Another area of considerable interest are the c~-anomer oligonucleotides. These molecules, in which the configuration of the sugar is inverted, have been shown to hybridize to DNA and RNA and to have a degree of nuclease resistance [12]. Chimeric molecules, in which each sugar is in the (x configuration and each phosphate is a phosphorothioate, have been shown to possess enhanced nuclease resistance [12].
Advances in understanding pharmacodynamic properties of oligonucleotides Occupancy-only mediated mechanisms Classic competitive antagonists are thought to alter biological activity because they bind to receptors, thereby preventing natural agonists from binding and inducing normal biological processes. Binding of oligonucleotides to specific sequences may inhibit the interaction of the RNA or DNA with proteins, other nucleic acids, or other factors required for essential steps in the intermediary metabolism of the RNA or its utilization by the cell.
Transcriptional arrest Oligonucleotides may bind to DNA and prevent either initiation or elongation of transcription b y preventing effective binding of factors required for transcription, thus causing transcriptional arrest. Progress in
Oligonucleotide therapy Crooke 659 triplex strategies has b e e n reviewed recently [13"']. In 1991, advances were made in developing strategies that partially circumvent the requirements f o r polypyrimidine-polypurine motifs [14-16]. Also, triplestrand binding has b e e n shown to b e enhanced by the use of intercalators attached covalently to oligonucleotides [17]. Additionally, a n u m b e r of triplexing oligonucleotides w e r e reported to have biological activity. A 13-mer phosphodiester homopyrimidine was reported to inhibit the transcription of E s c h e r i c h i a coli RNA polymerase [18]. An oligonucleotide at a concentration of 20 btM targeted to the promoter region of the c~ subunit of the interleukin-2 receptor was reported to reduce mRNA content by 32% [19].
eukaryotic cells. Multiple enzymes with RNase H activity have b e e n observed in prokaryotes [20"]. Perhaps the most important theme to emerge with regard to RNase H is an increased understanding of the structure-activity relationships of oligonucleotides with regard to their ability to support RNase H cleavage. Oligonucleotides that are not negatively charged or that are RNA-like have not b e e n s h o w n to support RNase H cleavage [11,23]. RNase cleavage of most, if not all, sequences is not s u p p o r t e d by (x oligonucleotides [12]. Very recently, chimeric oligonucleotides have b e e n synthesized which contain modifications that enhance binding to RNA and, in some positions in the molecules, modifications that support RNase H activity.
Translational arrest In 1991, significant additional results elucidating the activity of oligonucleotides designed to bind to the translational initiation c o d o n have b e e n reported and are reviewed in [5"']. Most importantly, these experiments were well controlled and high quality dose response curves were reported [20"]. Additionally, it is clear that agents that induce translational arrest may also rely on other mechanisms, e.g. RNase H, an enzyme which specifically cleaves the RNA in RNA-DNA duplexes.
Disruption of RNA structure Oligonucleotides that disrupt the Tat responsive element, TAR, and therefore affect the activity of HIV have b e e n reported by Vickers et al. [21]. This publication represents an initial effort to design oligonucleotides that bind to and disrupt RNA stems and loops. This is likely to be an important focus of study in the future.
Occupancy-activated destabilization RNA molecules regulate their own metabolism. A number of structural features of RNA are k n o w n to influence stability, various processing events, subcellular distribution, and transport. As RNA intermediary metabolism b e c o m e s better understood, m a n y other regulatory features and mechanisms will probably be identified. One mechanism to destabilize RNA and to interfere with translation is to bind to the 5' cap and either cleave the it or inhibit the binding of cap-binding proteins. A recent publication demonstrating that oligonucleotides may bind to the 5' cap and alter binding of cap proteins was reported by Baker, Miraglia and H a g e d o r n [22]. Interactions in the 3' untranslated region m a y also destabilize RNA structures and this was demonstrated clearly by Chiang et al. [20"].
RNase H mediated cleavage of RNA RNase H is an ubiquitous enzyme which degrades the RNA strand of RNA-DNA duplexes. It has b e e n identified in organisms as diverse as viruses and humans. At least two classes of RNase H have b e e n identified in
Pharmacokinetics of phosphorothioates The in vitro pharmacokinetics of phosphorothioates have recently b e e n summarized [24"']. The basic conclusions supported b y the data are described here. Uptake, as assessed b y the intracellular accumulation of a radiolabeled or fluorescently labeled compound, was d e p e n d e n t on time, temperature, and concentration and appeared to require cellular energy. Specific characteristics of uptake differed according to oligonucleotide class. It appears that some oligomers are concentrated in some cells. Studies that quantified the amount of oligonucleotide taken up showed that the amount internalized ranged from 1-11%. The extent and rate of oligonucleotide uptake varied depending on the specific cell type. Some data also suggested that uptake in vivo may differ from that determined in vitro. Nevertheless, different types of oligomers were taken up in pharmacologically relevant concentrations by a variety of cells. The stability of oligonucleotides taken into cells also varied with oligonucleotide class and dep e n d e d on whether or not oligomers w e r e modified by pendant groups. Modification of oligonucleotides with substituents, such as acridine, poly(L-lysine), or various hydrophobic groups, enhanced the uptake but not necessarily the activity of the compounds. Although it was not demonstrated specifically, intracellular distribution probably differs depending u p o n specific pendant modifications [24"]. The mechanism of uptake varied according to oligonucleotide type [24"]. The data suggest that methylphosphonates, which are non-ionic and lipophilic, cross cellular m e m b r a n e s b y passive diffusion. Several groups have data suggesting that phosphodiester and phosphorothioate oligonucleotides are taken up by receptor-mediated endocytosis. Localization studies using fluorescently labeled oligonucleotides support this notion. In these localization experiments, however, possible quenching of e n h a n c e m e n t of fluorescence of the acridine or fluorescein substituents within ceils was not investigated. Many more experiments must be done to prove unequivocally that receptor-mediated endocytosis is the only m e c h a n i s m of uptake of charged oligonucleotides. Direct isolation and characterization of a phosphorothioate-specific receptor has not yet b e e n done, nor has anyone p r o v e d that uptake
660
Pharmaceuticalapplications of phosphodiester h o m o p o l y m e r s is equivalent to uptake of heterosequence phosphodiesters or phosphorothioates. Data obtained from studies using fluorescently labeled c o m p o u n d s suggest that oligomers are mainly localized in the cytoplasm within vesicular structures. Encapsulation within liposomes e n h a n c e d uptake of oligonucleotides. Given what is k n o w n about the uptake of liposomes and the fate of various pharmacological agents encapsulated by these vesicles, the mechanism of uptake and intracetlular fate of the oligomers was probably altered, though it has not b e e n specifically shown. Additionally, cationic lipids have recently b e e n shown to enhance the u p t a k e of phosphorothioate oligonucleotides into some cells in tissue culture [25]. Although substantial progress has been m a d e in understanding the i n vivo pharmacokinetics of phosphorothioate oligonucleotides, few publications have emerged as yet.
In vivo
pharmacokinetics
Preliminary in vivo pharmacokinetic data are n o w available on m e t h y l p h o s p h o n a t e and phosphorothioate oligonucleotides. A 12-met 3H-labeled methylphosphonate injected in the tail vein of mice was cleared rapidly as intact oligonucleotide and distributed widely to all tissues except the brain [26]. More extensive studies have b e e n performed on 35S-labeled phosphorothioates in rats. A true distribution phase of 15-25 minutes was observed after a single intravenous dose of a 27-met followed by a prolonged elimination phase of 20-40 hours [26]. The prolonged elimination phase may result from the binding of phosphorothioates to serum proteins. Phosphorothioates were distributed to all tissues except the brain and eliminated intact in the urine. Phosphorothioates were rapidly and extensively absorbed after intramuscular and intraperitoneal administration [26]. Repeated daily doses of 50 mg kg- 1 of a 27-mer phosphorothioate to mice resulted in similar distribution and elimination kinetics to, but slight differences in tissue concentrations from, single dose studies. The liver, kidney, spleen and lung were the organs with the highest concentrations. Again, the drug was excreted intact in the urine [26]. Continuous subcutaneous osmotic p u m p administration of the same c o m p o u n d for four weeks at doses of 50-150 mg resulted in similar pharmacokinetics [26]. Studies with ISIS 1082, a 21-met phosphorothioate, in mice s h o w e d that w h e n applied to the cornea in a sodium acetate buffer, significant adsorption to the cornea and absorption into the aqueous and vitreous humors occurred. Moreover, significant systemic bioavailability was observed [25]. In the rabbit, as much as 25% of an applied ocular dose was systemically bioavailable (ST Crooke, unpublished data). Post-absorption pharmacokinetics were equivalent to intravenous pharmacokinetics.
Recently, a 20-mer phosphodiester was administered intravenously to rabbits. Clearance from the b l o o d was rapid and after 90 minutes 16% of the dose was found intact in the urine. In the blood, at least 17% of the drug was estimated to b e completed degraded within five minutes [27].
Toxicologic properties of phosphorothioate oligonucleotides The toxicological properties of phosphorothioate oligonucleotides have recently b e e n reviewed [24,26]. I n vitro, the conclusions are that phosphorothioate oligonucleotides display a very substantial therapeutic index. Although too little in vivo information has b e e n published to draw firm conclusions, within the next year additional publications should a p p e a r on the several oligonucleotides being studied in animals and on ISIS 2105 which is currently being administered to humans.
Combinatorial strategies Combinatorial methods to identify oligonucleotides that m a y bind to either nucleic acid or non-nucleic acid targets have b e e n developed. The first report of the identification of an analog nucteotide with potential therapeutic utility a p p e a r e d this year [28]. Two phosphodiester deoxyoligonucleotides w e r e s h o w n to slow clotting time and bind to thrombin w h e n incubated at 20 btM and assayed within two minutes.
Conclusions Very substantial progress has taken place over the past year. This sets the stage for even more significant advances in the future. Based on data generated to date, there is cause for continued optimism about the therapeutic utility of oligonucleotides.
Acknowledgement The excellent technical assistance of Mrs Colleen Matzinger is greatly~ appreciated. ,
#
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest •. of outstanding interest 1. CROOKE ST: T h e r a p e u t i c A p p l i c a t i o n s o f O l i g o n u •. cleotides. A n n u Rev Pharmacol Toxicol 1992, 32:329-376. This review places oligonucleotide therapeutics in the context of modern molecular pharmacology and summarizes the status of the area. 2.
CROOKEST: A n t i s e n s e T e c h n o l o g y . Curr Opin Biotechnol 1991, 2:282-287.
3.
WHITSELLL, ROSOLEN A, NECKERS LM: I n V i v o M o d u l a t i o n o f N-myc E x p r e s s i o n b y C o n t i n u o u s P e r f u s i o n w i t h
Oligonucleotide therapy Crooke a n A n t i s e n s e O l i g o n u c l e o t i d e . Antisense Res Dev 1991, 1:343.
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PERLAKYL, SAUO Y, BUSCH RK, BENNETT CF, MIRABELLI CK, CROOKE ST, BUSCH H: G r o w t h I n h i b i t i o n o f H u m a n T u m o r Ceil L i n e s b y A n t i s e n s e O l i g o n u c l e o t i d e s Des i g n e d to I n h i b i t P120 E x p r e s s i o n . Anticancer Drug Des 1992, 7:19-30.
ONO A, TS'O POP, KK\ LS: T r i p l e F o r m a t i o n o f O l i g o n u cleotides Containing 2/-O-Methylpseudoisocytidine in S u b s t i t u t i o n f o r 2 ' - D e o x y e i t i d i n e . J A m e r Chem Soc 1991, 113:4032.
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COOK PD: M e d i c i n a l C h e m i s t r y o f A n t i s e n s e O l i g o n u cleotides: F u t u r e O p p o r t u n i t i e s . Anticancer Drug Des 1991, 6:585-607. This review summarizes progress in the medicinal chemistry of oligonucleotides and demonstrates that a variety of strategies and sites of modification c a n be employed to enhance the pharmacological properties of oligonucleotides.
TAKASUGI M, GUENDOUZ A, CHASSIGNOL M, DECOUF JL, LHOMME J, THUONY NT, ttELENE C: S e q u e n c e - s p e c i f i c P h o t o - i n d u c e d C r o s s L i n k i n g o f t h e Two S t r a n d s o f D o u b l e - h e l i c a l DNA b y a P s o r a l e n C o v a l e n t l y L i n k e d t o a T r i p l e H e l i x - f o r m i n g O l i g o n u c l e o t i d e . Proc Natl A c a d Sci USA 1991, 88:5602.
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DUVAL-VALENTIN G, THUONG NT, HELENE C: Specific Inhibition of Transcription by Triple Helix-Formi n g O l i g o n u c l e o t i d e s . Proc Natl A c a d Sci USA 1991, 89:504-508.
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ORSON FM, THOMAS DW, McSI4AN WM, KESSLER DJ, HOGAN ME: O i l g o n u c l e o t i d e I n h i b i t i o n o f IL2Rc~ m R N A Transcription by Promoter Region Coilinear Triplex F o r m a t i o n i n L y m p h o c y t e s . N u c l Acids Res 1991, 19:3435.
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NEILSENFE, EGItOLM M, BERG RH, BUCHARDT O: S e q u e n c e Selective R e c o g n i t i o n o f DNA b y S t r a n d D i s p l a c e m e n t w i t h a T h y m i n e - s u b s t i t u t e d P o l y a m i d e . Science 1991, 254:1497-1500. This paper reports synthesis and initial characterization of an entirely n e w a n d potentially very important backbone modification. 6. •.
7. •.
VASSEURJJ, DEBAKT F, SANGHVI YS, COOK PD: O l l g o n u c l e osides: Synthesis of a Novel Methylhydroxylaminel i n k e d N u c l e o s i d e D i m e r a n d its I n c o r p o r a t i o n i n t o A n t i s e n s e S e q u e n c e s . J A m e r Chem Soc 1992, 114:4006~i007. A versatile approach to creating novel backbone structures which display valuable characteristics is reported. 8.
KIRSHENBAUMMR, HUIE EM, TRAINOR GL: Novel O l i g o n u c l e o t i d e A n a l o g u e s w i t h a s u l f u r B a s e d L i n k a g e . Colloq u i u m Gene Regulation by Antisense RNA a n d DNA 1991. CD210:19 Keystone Symposia on Molecular and Cellular Biology. Feb 2-7, 1991, Frisco, CO. J Cell Biochern 1991, 19.
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HUANGZ, SCHNEIDER KC, BENNER SA: B u i l d i n g B l o c k s f o r O i l g o n u d e o t i d e A n a l o g s w i t h D i m e t h y l e n e Sulfide, Sulfoxide and Sulfone Groups Replacing Phosphodie s t e r L i n k a g e s . J Org Chem 1991, 56:3869.
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GUINOSSOCJ, HOKE GD, ECKER DJ, MIRABELLI CK, CROOKE ST, COOK PS: S y n t h e s i s a n d B i o p h y s i c a l a n d B i o l o g i c a l E v a l u a t i o n o f 2"-Modified A n t i s e n s e O l i g o n u c l e o t i d e s . Nucleosides Nucleotides 1991, 10:259.
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VICKERST, BAKER BF, COOK PD, ZOUNES M, BUCKHEIT a w , GERMANY J, ECKER DJ: I n h i b i t i o n o f HIV-LTR G e n e Exp r e s s i o n b y O l i g o n u c l e o t i d e s T a r g e t e d to t h e TAR Ele m e n t . Nucleic Acids Res 1991, 19:3359-3368.
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BAKER BF, MIRAGLIA L, HAGEDORN CH: M o d u l a t i o n o f E u c a r y o t i c I n i t i a t i o n Factor-4E B i n d i n g to 5 ' - C a p p e d Oligoribonudeotides by Modified Antisense Oligonuc l e o t i d e s . J Biol Chem 1992, 267:11495-11499.
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AGRAWALS, MAYRAND SH, ZAMECNIK PC, PEDERSON T: SiteSpecific E x c i s i o n f r o m RNA b y R N a s e H a n d M i x e d p h o s p h a t e - b a c k b o n e O l i g o d e o x y n u c l e o t i d e s . Proc Natl A c a d Sci USA 1990, 87:1401.
24. •.
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