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Antisense technology and prospects for therapy of viral infections and cancer
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Antisense technology and prospects for therapy of viral infections and cancer Eighteen years ago, antisense oligonucleotide therapeutics that can selectively knock out disease-causing genes could easily have been viewed as science fiction. Yet today, through much persistence and focused investment, the technology has nearly evolved to the point of realization. A number of first-generation antisense compounds have entered human clinical trials. Some of these compounds appear to work by an antisense mechanism to inhibit the expression of disease-causing genes, while others probably work by unanticipated, yet clinically beneficial, mechanisms. In this review, the current status of antisense oligonucleotide development will be described as it relates to two areas of concentrated effort: antiviral and anticancer applications. ANTISENSE oligonucleotides (ONs) are short stretches (7-30 nucleotides in length) of DNA or modified DNA that contain a complementary base sequenceto a target RNA. The ONs are added extracellularly to cells and are expected to enter the cell and selectively hybridize to their target by Watson-Crick hydrogen-bonded base pairs. This hybridization should interfere with the function of the RNA by blocking RNA transport, splicing or translation. In some cases,the RNA-ON hybrid forms a substratefor RNase H, a cellular enzyme that selectively hydrolyzes the RNA portion of the RNA-DNA hybrid and inactivates the RNA (Fig. 1). However, severalfundamental barriers have been encounteredduring development of antisenseONs asbroadly applicable therapeutics. (1) Unmodified ONs are susceptible to degradation by cellular and extracellular nucleases. Chemical modification of each of the phosphodiester linkages in an ON by a variety of methods (phosphorothioate being the most commonly used) confersresistanceto degradation in cell culture (Fig. 2). Alternatively, modification of the ribose moiety can also result in increased resistance to nucleases. Nevertheless, ONs are still metabolized in animals more rapidly than predicted by cell culture studies and improvements in ON pharmacokinetics are still needed’. (2) Cellular penetration by ONs has been shown in cell culture studies to be inefficient. For example, phosphorothioate ONs added directly to the cellular growth media failed to demonstrate antisense activity, yet when these same ONs were microinjected or delivered with cationic lipids, expression of the targeted proteins wasefficiently inhibitedz4. Cationic liposomes, microinjection and electroporation have been used to enhance cellular delivery and the potency of ONs in cell culture23Z5. In animal studies, several interesting and provocative reports have suggestedthat ONs can penetrate cells in at least some tissues in the absenceof cationic lipidsa. The implications of these results offer a tremendous boost to the technology, but their general applicability Copyright
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emerge. In some cases, these non-antisense mechanisms might have clinical benefit (see below). However, most of these problems now have solutions, resulting in very convincing demonstrations of specific antisense effects using highly controlled assays.This evolution of the technology for use in human diseases,such as Crohn’s disease, arthritis, ulcerative colitis, viral infections and cancer, is reflected in the compounds that have entered clinical trials. In this review, we focus on the antiviral and anticancer applications of ONs.
Antiviral applications of antiseme oligonucleotides
1. Mechanism of action of antisense oliiucleotides. Antisense oliiucleotidaa (0N.s) are small nucleotidesin length) syntheticnucleicacids resemblingsingle-strandedDNA AMkense ONs h@idize to the coding (sense) sequence in a speckally Urge&d mRNAby Walscn-Cridc base+Mg rules.AntisenseONscanbindtothetargetedRNAinthenudeusorcytophsm.Whentheybindtheir$rgetmRNAin the nucleus,the mRNAportion of the anlisense ON-RNA complex is degraded by RNase H (not shorn). In the cytoplasm,the bound antkerw ON inhibits expressionof the mRNAby interferingwith protein bans&n. In both cases,the diww-cwsi ng proteinfailsto be synthesized.A major advantageof ankensa techrwkgy over traditional phannaceuti& is that antisense ONs can be ratio&y designed to target any dkeasecausinggene within the entire human genome as long as a partial sequenceof the gene is known. Figure (7-30
Viruses are attractive targets for antisense technology because they harbor genetic information that is distinct from that of the host cell. Some of the first antisense compounds to enter controlled clinical trials have been developed to inhibit viral pathogens: HIV, cytomegalovirus (CMV) and human papillomavirus (I-W). In general, the data presented so far for these compounds suggest that antisense plays a minor, if not insignificant, role in the mechanism of inhibition of these viruses. Below we provide a critical evaluation of the data supporting the mechanism of action for the antiviral ONs along with their clinical status.
HIV
GEM91, a 25 nucleotide (nt) phosphorothioate ON, is complementary in sequenceto the gag mRNA of HIV and is undergoing testing in Phase I/II clinical trials sponsored by Hybridon, Inc. (Worcester,MA, USA). Its antiviral activity has been demonstrated in will have to await fnrther examples in a variety of systemsto demon- acutely and chronically infected CEM cells (a T lymphoblastoid cell strate a convincing proof of concept. line), human peripheral blood lymphocytes and macrophages, and (3) Much effort has been expended using ONs that do not bind isolates from HIV-l-infected patients. GEM91 was found to be sixwell to their target. In general, this is due to the poor hybridization of fold more active than a control ON (a 25 nt random mixture of ONs ONs to RNA in cells. A number of chemical modifications have been synthesized from a mixture of all four bases) at inhibiting HIV-l developed that increase the affinity of ONs for RNA, resulting in replication in MOLT-3 cells (a T lymphoblastoid cell line). This more potent antisense effects9(Fig. 2). Also, much is now known result implied a sequence-dependentcomponent of antiviral activity about how to target the mBNA encoding a specific protein. It is and led to the conclusion that antisense was a component of the necessaryto test multiple antisense sequencescomplementary to a antiviral mechanism of action for GEM91 (Ref. 11). variety of regions along an mBNA molecule to find sequencesthat Sequence dependence does not necessarily imply an antisense are effective. The common practice of testing one antisensesequence mechanism of action, however.Phosphorothioate ONs, in addition to and its sensecontrol targeted to the AUG initiation codon of a mes- their ability to bind RNA, bind a variety of proteins (Fig. 3), includsage is not necessarily doomed to failure, but a great deal of luck is ing HIV reverse transcriptase, CD4 (the cell-surface receptor to which HIV binds) and gp120, an I-D&envelope protein that binds to required for this approach to work’. Many of these barriers have, at times, appeared quite daunting, CD4 (Ref. 10). Many of thesenon-antisenseeffectsare dependent on leading some to speculate as to whether antisense can ever work as a the sequenceand length of the ON. For example, a 28 nt phosphorotherapeutic strategylo. However, this is a surprisingly pessimistic thioate ON containing only cytosine inhibits HIV adsorption to its view during a time when major advanceshave been made. Clearly, target cells. The activity of this molecule is dependent on sequence, problems existed with the interpretation of early results, and mecha- becausea 28 nt phosphorothioate ON containing only adenosine was nisms underlying the non-antisense effects of ONs continue to much less active’*. The activity of the 28 nt cytosine-only ON as
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compared with GEM9 1 in the above cell culture studies is not known. Thus, alternative mechanisms, such as inhibition of viral adsorption to a cell, or cellular toxicity, could be responsible for at least part of the anti-HIV activity of GEM91 (Fig. 3)13*14. A significant amount of pharmacological data has been made available from studies of GEM91 in rats, mice and humans15.GEM91 was administered by intravenous infusion to humans at 0.1 mg kg-’ for 2 h. The slow infusion was necessaryto avoid complement activation and related hypotension, and increased clotting times caused by nonspecific ON binding to thrombin. These undesirable effectshave been observed in studies of monkeys’6. Data using GEM91 in humans showedthat the 35S-labeledON was rapidly cleared from plasma, with biphasic t,, values of 0.18 f 0.04 h and 26.71 f 1.67 h for plasma-derived radioactivity”. The B-phase of elimination was, however, somewhat misleading because it reflected radioactivity and not intact GEM91. GEM91 was rapidly metabolized in humans; the majority of the plasma-derived GEM9 1 radioactivity was associated with intact ON only up to 2 h after infusion. If high and prolonged plasma concentrations of GEM91 are required for anti-HIV activity in viva, then these data suggest that frequent (and inconvenient) 2 h dosing regimens might be needed. Efficacy data for GEM91 have not yet been published.
O=P-o-
Figure 2. Structure and chemical modificationsof oligonucleotidee. The general structure of an oligonucleotide is shown on the left. Regionsthat can be modified to enhance the affinityand nuclease resistance of an digonudeotide include (a) the heterocycle,(b) the backbone and (c) the dbose moiety.Examples of heterocycleanalogs (a) include: top, propyne modificationat the C-7 position of 7-deazaguanosine; center, propyne modificationat the C-5 position of uracil; and bottom, a phenoxazine-modified cytosine.Backbone modifications (b) include: top, a phosphorothioate analog; center, a phosphorsmidate analog; and bottom, a peptide-nucleic-acid linkage. Modiiications of the ribose sugar (c) include: top, changing ribose to hexose; center, modfting the 2’ hydroxylto a methoxygroup; and bottom, modifyingthe 2’ hydroxylto a methoxyethoxymoiety.Modified from Ref.9.
Human cytomegalovirus ISIS 2922 (Isis Pharmaceuticals, Carlsbad, CA, USA) is a 21 nt phosphorothioate ON that is complementary to sequencesin the major immediate early region of human cytomegalovirus (HCMV) mRNA. This compound is in PhaseIII clinical trials for the treatment of HCMV retinitis, an opportunistic eye infection that occurs in nearly 30% of AIDS patients. Cell culture studies using ISIS 2922 have shownthat the agent is a sequence-dependentinhibitor of HCMV viral infection’*. In these studies, the effect of mismatch and length wasevaluated for the compound. A two-basemismatch control ON that had a thermal denaturation temperature below physiological temperature when bound to RNA (-35°C vs. -55°C for ISIS 2922) retained 88% antiviral activity compared with ISIS 2922. However, a single base deletion from the 3’ end of ISIS 2922 retained its thermal melting temperature (-52”(J), but retained only 40% of its antiviral activity. Treatment of cells with ISIS 2922 before infection was absolutely essential for antiviral activity”. These data do not, therefore, correlate well with an antisense mechanism and strongly suggestthat ISIS 2922 inhibits HCMV viral infection by a sequence-dependentbut non-antisense mechanism of action that probably involves inhibition of viral adsorption, penetration or uncoating (Fig. 3). Although the mechanism of action of ISIS 2922 is poorly understood, it appearsto be clinically beneficial and might become a suc-
cessful ON-based therapeutic. ISIS 2922 has been administered by intravitreal injection every other week to AIDS patients in a Phase I./II study. The results showed a dose response for activity in an open-label trial that evaluated HCMV progression using a blinded evaluation of retinal photographs”. These encouraging data prompted a Phase III study, the results of which should be reported in early 1997. Human papillomavirus ISIS 2105 (Isis Pharmaceuticals, Carlsbad, CA, USA) is a 20 nt phosphorothioate ON that is complementary to the E2 gene of HPV. ISIS 2105 has been evaluated in Phase I/II human clinical trials for the treatment of genital warts as an adjuvant to cryotherapy; however,the compound wasdiscontinued owing to lack of efficacy in the tlidS.
I-W is a difficult virus to screen compounds against because it cannot be reliably cultured in any significant quantity and the in vivo models do not truly reflect the human diseases caused by I-IF?? Bovine papillomavirus, for which there are cell culture models, was initially usedto validate inhibition of the E2 gene product, a papillomavirus-encoded transcription factor, by antisense ONszO. Positive results in this model prompted the development of ISIS 2105, which was complementary to the E2 mRNA of HPV-6 and HPV- 11. This compound inactivated E2-dependent transactivation of a reporter 33
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guanosine quartet have antiproliferative activity. Also, ONs can bind to growth factors and their receptors,which could interfere with cellular proliferation10*23.Furthermore, Virus ONs containing cytosine-phosphate-guanosine dimers can activate B cells and natural killer cells; this immune stimulation might z result in antitumor activity irt vid+. This activity can be mitigated by methylation of a Binding and fusion cytosine. Thus, many nonspecific effects of ONs might have interfered with the interpretation of experiments designed to assesstheir antisenseactivityzvz6. The ultimate successand value of antisense technology depend on the ability to design potent, sequence-selective ON inhibitors, and to demonstrate that the biological and clinical benefits of the ON can be attributed to an antisense mechanism of virus particles action. Sequence selectivity and potency of antisense ONs is dependent on chemical Translation of modifications of the ON that affect the affinity of the ON for its target, sequencecontext and ON length*‘. In fact, phosphorotbioate ONs have demonstrated selective targeting of a mutant rus gene containing a single base w Viral mRNA change in codon 12 (Ref. 28). The maximum selectivity between the mutant and normal 0 Transcription rus genes was obtained with a 17 nt antisense of viral genes and ON and was about 5-fold. In general, anti\ viral replication sense ONs have a 5-30-fold window of Nucleus specificityin tissuecultnre3~4272g. The parameters that affectnonspecificinhibition of nontargeted Figure 3. Overview of a generic virus-replication cycle and the targets for oligonucleotides (ONs) during RNA by ONs are not clear, but it is probably viral replication. (a) The virion binds to a specificcell-surface receptor and fuses with the plasma membrane. (b) The viral capsid is transported to the nucleus (during the retrovirus replication cycle the viral due to partially complementary binding of RNA is first reverse transcribed to DNA). Once bound to the nucleus, the viral DNA is released into the the ON to a nontargeted RNA and RNase H nucleus, where it is (c) transcribed by cellular enzymes and replicated. (d) Viral proteins are synthesized cleavage of the nontargeted transcript. using the cellular translation apparatus and (e) new viral particles are assembled and released. Sites of Now that these limitations have been recantisense ON action are indicated by boxes. Phosphorothioates can bind to a varietyof proteins on the cellular surface by a sequence-dependent, non-antisense mechanism of action, and block virion binding ognized, improved ONs, methods and conand fusion (a). Phosphorothioate ONs have also been shown to inhibit reverse transcriptase in a trols have been used to assessthe antisense sequence-independent manner (b). Although phosphorothioate ONs can act nonspecificallyto inhibit viral activity of anticancer ONs, resulting in conreplication, phosphorothioate ONs have also been suggested to inhibit viral gag gene expression during vincing data that showselective inhibition of HIV-1 infections (for example, GEM91) and immediate early gene expression in human cytomegalovirus gene expression in cell culture. Furthermore, (for example, ISIS 2922). See text for details. in viva data, while more difficult to control rigorously, strongly suggestthat antisenseONs construct in cells; however,inhibition of HPV infection in cell cul- might be effective against xenograft tumor transplants. This work has ture or in viva models has not been reported. The pharmacokinetics, laid the foundation for clinical trials that are now under way. distribution and stability of ISIS 2 105 have been evaluated following intradermal injection in rat?‘. These studies suggestedthat ISIS 2105 bcl-2 would be bio-available at sufficient concentrations following once or In ~85% of follicular B-cell lymphomas, bd-2 is overexpressedas a twice weekly intralesional dosing in patients with genital warts. result of a trauslocation of the bcl-2 gene to the immunoglobulin heavy-chain locus. bcl-2 is a member of a related multi-gene family that regulates programmed cell death by protein-protein interactions AntisenseONs for the treatment of cancer Antisense ONs have also been extensively studied for their use as (Fig. 4). Bcl-2 homodimers repress apoptosis while homodimers of anti-neoplastic agents”. Much cell culture and animal data has been Bax, a 21 kDa protein with extensive amino acid homology to Bcl-2, published that describes the anti-growth activity of ONs. Similar to induce apoptosis. Thus, according to the model, cell fate can depend the data from the antiviral ON studies, many of the anticancer ONs on the ratio of the ‘death inducer’ to the ‘death suppressor’“.31. An 18 nt phosphorothioate antisense ON complementary to bcl-2 have been shown to exhibit their effectsthrough sequence-dependent but non-antisense mechanisms‘o-23 . For example, ONs containing a (G3 139) is being developed by Genta (San Diego, CA, USA) to treat
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Glossary Survival
Athymic nude mice - Hairless mice that have a rudimentary thymus in which T-cell maturation cannot occur normally.As a result, these mice have no cell-mediated immune reactions, including xenograft rejection. W-2 - The B-cell lymphomalleukemia2 gene, a gene that has been implicatedin the etiology of many common human lymphoidtumors. bc-2 is a member of a large gene family whose protein products can homo- and heterodimerizeto inhibit apoptosis (Bcl-2) or accelerate apoptosis @ax). Biphasic t,n values -A term that describes a two-compartment pharmacokinetic model in which a rapid plasma clearance time (the first phase or a phase) for a drug is followed by a slower clearance time (the second phase or p phase). t,, describes the time it takes for 50% of the drug to clear the plasma in each phase. Cytoslne-phosphatequanosine dimer - A motif in oligonucleotides that, when placed in proper sequence context, is mitcgenic by a nonantisense mechanismfor B cells and natural killer cells. Methylationof cytosine at the C-5 position blocks the mitogeniceffect. Folllcular lymphoma - A malignantdisorder of lymphoidtissue that originates in identifiablenodules (follicles) of a lymph node. gag-An HIV gene that encodes the nucleocapsidprotein. Major immedlate early reglon - A region of cytomegalovirus from which the first genes are transcribed after infection of a permissive cell. Transcription of the immediateearly genes does not require preceding viral protein synthesis. 2’0methoxyethylribonucleosides - A chemical modificationat the 2’-hydroxy position of the ribose sugar of nucleosides, which makes them less susceptible to nuclease attack and enhances the affinity of the antisense oligonucleotidefor its target. Phosphorothloate - An intemucleotidelinkage in DNA or RNA in which one oxygen atom in the phosphate moiety is replaced with sulfur, making the bond resistant to nucleases. RNase H - A cellular enzyme that hydrolyzes the RNA potion of RNA-DNA hybrids. Severe combined immunodeticisncy @CID) - A genetic disease of inbred mice in which both humoral and cell-mediated immunity fail to develop owing to a defect in the ability to rearrange Tcell receptorand immunoglobulin genes.B and T cellsare absentor foundat very lowlevels.SCIDmiceare usedto evaluatethe efficacy of pre-clinical therapeutics in the absenceof manynormalimmune responses. Thermaldenaturationtemperature- The temperature at whicha nucleicacid duplexmeltsby 50%underequilibrium conditionsat a givensaltandbufferconcentration. It is a qualitativemeasureof the affinityof an antisense compoundfor its target RNAandallowsthe relativerankingof thestabilityof nucleicacidduplexes.
Apoptosis (death)
Figure 4. Model for the action of Bcl-2 and Bax in regulating apoptosis. Bcl-2 homodimers repress apoptosis while Bax homodimers accelerate it. Whether or not a cell enters apoptosis depends on the ratio of Bax to Bcl-2. Antisense digonucleotiies have been designed to induce apoptosis by inhibiting the translation of Bcl-2, thereby increasing the ratio of Bax to Bcl-2.
in this model. These results appear promising, although still quite preliminary3’. In a pilot toxicology study of G3139, cynomolgus monkeys were dosed with 0.125, 1.0 and 10 mg kg-‘&y-’ of G3139 by subcutaneous infusion for 14 days. No deaths or treatment-related clinical symptoms were observed, although histopathological changes were seen in the kidney cortex (slight brown pigmentation) andthe spleen (which was enlarged) at the highest ON dose. A Phase I/II trial for follicular lymphoma was initiated in November 1995 at the Royal Marsden Hospital (London, UK). G3 139 is also in pre-clinical development for use in chemoresistant solid tumors, which often aberrantly expressbcl-2 (Ref. 32). c-Raf-I
c&f-l is a serinejthreonine kinase that participates in the stimulation of the mitogen-activated protein kinase (MAPK) signaling cascade. c-Raf-1 is activated by Ras, a GTP-regulated molecular switch that is mutatedin nearly a third of all human cancers.In addition to Ras, other signals emanating from cell-surface receptors are integrated by c-Raf-1 and transmitted by phosphorylation to downstream targets, leading to the proliferation or differentiation of the cell (Fig. 5). An activated form of c-Raf-1 is oncogenic and has been associatedwith human malignancies. Recently, a 20 nt phosphorothioate ON (ISIS 5132; Isis Pharmaceuticals, Carlsbad, CA, USA) complementary to human c-ruf-1 follicular lymphoma. In a pre-clinical model, administration of entered clinical trials. This followed the demonstration of potent G3139 at 5 mg kg-’ day-’ for 21 days to severecombined immuno- antiproliferative properties in cultured cells and also antitumor activdeficient (SCID) mice inoculated with follicular lymphoma cells ity in xenografts with human lung, human bladder and human breast resulted in the elimination of lymphoma cells in 6/6 mice. This was tumors7. When a cationic lipid wasused to transfect ISIS 5132 into determined by PCR and fluorescence-activated cell sorter (FACS) cultured cells, this c-@-l antisense ON was demonstrated to be analysis of blood and tissue samples. A sense-ON control failed to sequencespecific. Control ONs containing l-7 mismatches showed cure the lymphoma, but a more stringent mismatch control ON con- reduced antisense activity. c-ruf-I antisense ONs also demonstrated taining the same basecomposition as the antisenseON wasnot tested isotype-specific inhibition becausethey had no effect on RNA levels 35
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Growth factor binding and receptor
Growth factor receptor
Figure 5. Signal transduction pathways that control cellular proliferation and differentiation.Binding of a growth factor to its receptor causes autophosphorylation of tyrosine residues on the cytoplasmicside of the receptor.The phosphorylatedreceptor activatesvarioussignal transduction pathways,including the mitogen-activated protein kinase (MAPK) pathway (left) and the phosphoinositide pathway (right). In the MAPK pathway, adaptor molecules such as GRB2 bind to the phosphorylated receptor and activateRas, a small GTP-binding protein. This leads to the activationof c-Raf-l ,a serine/threonine kinase,triggering a phosphorylatiin cascade involving MAPK and its kinase, MEK. In the phosphoinositide pathway, phospholipase C (PLC) binds to the phosphotylated receptor and catalysesthe hydrolysisof a membrane phospholipid, phosphatidylinositol (43)~biiphosphate [Ptdlns(4,5)P,] to inositol (1,4b)-trisphosphate [lns(l,4,5)PJ and 1,2diacylglycerol (DAG). lns(l,4,5)P, causes an increase in the cellular Ca2+ concentration, which synergizes with DAG to activate protein kinase C-O!(PKCa), another serine/threonine kinase. Substrates of PKC-a include c-Raf-l and MAPK. Antisense oligonucleotides (ONs) targeting the mRNA for c-Raf-1and PKC-a should inhibit these pathways, which are oftenconstitutivelyactivein cancer cells.
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of another member of the Raf-1 family, A-Raf. The results of these well-controlled experiments strongly suggest that the c-raf-I antisenseONs are functioning by a sequence-specificantisense mechanism of action’. In a pre-clinical study, mice bearing tumors were administered with 0.00640 mg kg-’ of ISIS 5132 (without the use of a cationic lipid) by intravenous injection once daily for 11 days’. Growth of the tumor xenograft, as determined by measuring tumor volume, was inhibited at an ON concentration of 0.0646 mg kg-‘. The mismatch control ON had no effect on tumor growth. In this study, c-~$1 mRNA levels were shown to be significantly reduced in the tumors dosed with the antisense ON. Maximal inhibition of c-r&l RNA levels (15% of control RNA levels) occurred after 13 days of a 6.0 mg kg-’ day-’ ON dose. Although c-ruf-1 mRNA levels were reduced by antisense ONs, the effect on c-r&l protein levels in the tumors was not investigated. In addition, A-raf mRNA and protein levels were not analyzed, as they had been in the studies described above, making it difficult to determine the specificity of the c-r&l antisense ONs in viva. Nevertheless, the findings are encouraging becausethey demonstrate inhibition of human c-ruf-1 mRNA levels and potent antitumor effectsin viva. Phase1 clinical trials to determine the safetyof ISIS 5132 are being initiated by Isis Pharmaceuticals in collaboration with Ciba-Geigy Ltd (Basel, Switzerland). However, the use of c-ruf-1 antisenseONs could be limited by their associatedtoxicity in normal cells; in nondiseased cells, c-r&l is a critical component for cellular proliferation and differentiation. Despite this, preliminary results using an antisense ON complementary to murine c-ruf-1 failed to show any ON-associated toxicities in normal mice’, suggesting that ONs might be preferentially targeted to tumors (although previous data indicate that ONs are widely distributed in mice’). Alternatively, other members of the ruf gene family might compensate for c-Raf-1 activity in normal cells. Results from Phase1 trials should help to addresssafety and pharmacokinetic concernsassociatedwith these ONs. Protein kinase C-a
Protein kinase C-cw(PKC-cl) is a member of a family of homologous serine/threonine protein kinases involved in proliferation and differentiation (Fig. 5). PKC-cx is the most widely expressedPKC and the major intracellular receptor for tumor-promoting phorbol esters,agents that mimic the action of diacylglycerol. Because of the tremendous homology among the PKC isozymes, it has so far been difficult to develop isotype-specific small molecules that inhibit specific PKC isoforms and have favorable clinical properties. PKC-(Y has been successfullyinhibited using antisense ONs both in vitro using cationic lipids and in viva without cationic lipids33. When a 20 nt phosphorothioate ON complementary to murine PKC-a wasadministered to mice, it demonstrated a 64% reduction in PKC-(u mRNA levels in the liver following a single 50 mg kg-’ intraperitoneal dose. A control ON with a sequencethat was unrelated to PKC-(w had little effect on PKC-(r mRNA levels. The decreasein PKC-(x mRNA was,however,limited to the liver, consistent with tissue distribution of phosphorothioateONs, and wasisozyme specific33. Human PKC-(u has also been targeted by phosphorothioate ONs. Treatment of tumor xenografts in athymic nude mice with a 20 ntlong human PKC-cx antisense ON (ISIS 3521) suppressedtumor growth, whereasthe control ON had no effect”. Second-generation antisense ONs have now been synthesized and analyzed35.These
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chimeric ONs contain six phosphodiester-linked 2’-0-methoxyethyl ribonucleosides on the 5’ and 3’ ends of the ON, and have an 8 nt phosphorothioate center that functions as an RNase H target site36.In tissue culture, these ONs were sixfold more potent than ISIS 3521 yet, remarkably, when administered to tumor-bearing nude mice, the chimeric ONs were about lOO-fold more potent?. The pharmacokinetics of the chimeric ONs were also quite different in mice from ISIS 3521, displaying a more rapid clearance. It is therefore surprising that the biological potency as compared with ISIS 3521 was superior. Furthermore, the chimeric ONs showa reduction in adverse effects on clotting time of human plasma. Based on these data, second-generation antisense compounds should demonstrate increasedbiological potency and decreasedtoxicity in viva A Phase 1 clinical trial using first-generation PKC-a antisense ONs began in January 1996 for the treatment of cancer.The introduction of secondgeneration ONs into the clinic is still, however,severalyears away. Several phosphorothioate ONs have already entered clinical trials for antiviral and anticancer indications. In each case,the candidates were selected on the basis of cell culture and pharmacokinetic results. For antisenseONs targeting HIV and CMV, the cell culture data demonstrated the antiviral activity of the ONs and showed that the effects were sequence dependent. However, sequencedependence does not prove an antisensemechanism of action, and it is likely that the ONs act by non-antisense mechanisms. Such mechanisms do not undermine the clinical effectivenessor ultimate successof the drugs, but they do not validate antisense therapeutics as a broadly applicable genetic targeting technology. However, the demonstration of the clinical benefit of ONs is important for the technology becauseit will indicate that such agents have the appropriate pharmacodynamic properties to be useful therapeutics. The genes for Bcl-2, c-Raf-1 and PKC-cwhave been targeted by antisense ONs and these ONs have entered clinical trials as anticancer compounds. Both c-r&l and PKC-a ONs target the same signal transduction pathway (Fig. 5) and many tumor types might be sensitive to both c-ruf-I and PKC-a antisenseONs. However, several lines of evidence suggest that PKC-a might also function in parallel to the c-Raf-l-activated MAPK pathway.Therefore, differential sensitivities of tumors to c-ruf--l and PKC-cx ONs would be expected. Synergism betweenthe ONs might also be possible in specific tumor types. These anti-cancer ONs should prove to be valuable therapeutic agents as well as powerful researchtools for elucidating the function
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of important signal transduction pathways in the development and maintenance of human cancers.
Future prospectsof ONs as therapeutics
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15 Agmwal, S., Temsamani, J., Galbraith, W. and Tang, J. (1995) Pharmacokinetics of antisenseoligonudeotides, Clin. Phartnacokiner. 28,7-16 16 Galbraith, W.M. et al. (1994) Complement activation and hemodynamic changes following intravenous administration of phosphorothioate oligonudeothles in the monkey,AntisenseRex Dev. 4.201-206 17 Zhang, R. er al. (1995) Pharmacokktics of an anti-human hmmmodeficiency virus antisense oligodeoxynudeotide phosphorothioate (GEM91) in HIVinfected subjects,Clin. Pharmacol. Thex 58,4k53 18 Azad, R.F. et al. (1993) Antiviral activityof a phosphorothioate oliionucleotide complementary to RNA of the human cytomegalovirus major immediateearly region, Antimicrob. Agents Chemorher: 37.1945-1954 19 Hutcherson, S.L. et al. (1995) Antisense oligonucleotide safetyand efficacyfor CMV rethdtis in AIDS patients, Proceedings of rhe 35rh Inrernarional Confer-
Antisense ONs can be rationally designed to inhibit the expressionof disease-causinggenes by binding specifically to their mRNAs, yet the non-antisense effectsof ONs have, so far, complicated the interpretation of results. Nevertheless, researchers in the antisensetherapeutics field are increasingly optimistic. In recent years, many of the challenges facing the development of antisenseONs as therapeutic compounds have been overcome: successful inhibition of therapeutic targets is being observed in animal models; and several clinical trials are now in progress to evaluate antisense ONs in various medical conditions, including viral infections (HCMV, HIV), ence on Antimicmbial Agenrs and Chemorherapy, San Francisco, CA, USA, p. 204, cancer (solid tumors and leukemia) and inflammation (not described American Society for Microbiology in this review). The results from clinical trials using first-generation 20 Cowsert, L.M., Fox, MC., Zen, G. and Miibelli, C.K. (1993) In vitro evaluation antisense compounds will soon be known. Second-generation antiofphosphorotbioateoUgon~targdedtotheE2mRNAofpapiUorua~ sensecompounds have decreasedproduction costs,and showenhanced potential treatment for genital warts, Antimicrob Agenrs Chemorhel: 37,171-177 potency, improved pharmacokinetics and reduced ON-associated tox- 21 Cossum,P.A.et al. (1994) Pharmacotietics of a “C-labeled phosphorothioate icities. These are in pre-clinical development and clinical trials are oligonudeotide, ISIS 2105, after intradennal admhdskation to rats, J. Phan expected in the next two years. It now appearsthat 18 years of antiExp. Thel: 269,89-94 senseresearchwith high-profile successesand failures have resulted 22 Tonkinson, J.L. and Skin, CA. (1996) Antisem oligodeoxynucleotide as diniin the evolution of an exciting new classof therapeutic compounds. cd therapeutic agents,Cancer Znvesr. 14,54-65
23 Stein, CA. (1996) Phosphorothioate antisense oligonucleotides: questions of Acknowledgements. We thankMary Hogsett for figure and manuscript preparation. References 1 Crooke, ST. er al. (1996) Pharmacoldnetic properties of severalnovel oligonucleotide analogs in mice, .I.Pharmacol. Exp. The,: 211,923-937 2 Bennett, CF. er al. (1992) Cationic lipids enhance cellular uptake and activity of phosphorothioate antisenseoligooucleotides, Mol. Phonnncol. 41.1023-1033 3 Wagner, R.W. et al. (1993) A&ease gew inhibition by oligonocleotides containing C-5 propyne pyrimidines, Science 260,1510-1513 4 Flanagan, W.M., Su, L.L. and Wagner, R.W. (1996) Elucidation of gene function using C-5 propyne antisenseoligonucleotides, Nar. BiorechnoL 14.1139-l 145 5 Bergan, R., Connell, Y., Fahmy, B. and Neckers, L. (1993) -ration enhan= c-my antisenseoligodeoxynudeotide eflicacy,Nucleic Acids Res. 21,3567-3573 6 McKay, R. and Dean, M.N. (1994) Inhibition of protein kinase C-a expression in mice after systemic&nin%ration of phosphorothioate, Pmt. Natl. Acad. Sci. U. S. A. 91.1762-1766
spec&ity, Trena!~ Biotechnol. 14,147-149 24 Krieg, A.M. et al. (1995) CpG motifs in bacterial DNA trigger direct B-cell activation, Nature 374,546-549 25 Stein, C.A. and Krieg, A.M. (1994) Problems in inteqretation of data derived from in vitro and in viw use of antisenseoligod~xynucleotide, Anrisense Res. Dev. 4,67-69 26 Heidenreich, 0.. Kang. S-H., Xu, X. and Nerenberg, M. (1995) Application of
antisensetedmology to therapeutics, Mol. Med. Today 1,128-133 27 Flanagan,
28 29 30
7 Monia, B.P. et al. (1996) Antitumor activity of a phosphorothioate antisense oligodeoxynuckotide targeted against c&f kinase, Nat.Mea! 2,668-675 8 Plenat, F. (1996) Animal models of antisenseoligonucleotides: lessonsfor use in humans, Mol. Med. To&y 2.250-257 9 Matteucci, M.D. and Wagner, R.W. (1996) In pursuit of antisense,Nature 384 (Suppl.) 20-22
10 Stein, CA. (1995)Dnesa&ense~? Nor. Med. 1,1119-1121 11 Lisziewicz, J. et al. (1994) Antisense oligodeoxynuckotide phosphorothioate complementary to gag mRNA blocks replication of human immunodetkiency virus type 1 in human peripheral blood ceils, Proc. Narl. Acad. Sci. U. S. A. 91,
31
W.M., Kothavale,
A. and Wagner, R.W. (1996) Effects
of oligonu-
de&de length, mismatches,and mRNA levelson C-5 propyne-modiikd antisense potency,Nucleic Acids Res. 24,2936-2941 Monia, BP. et al. (1992) Selectiveinhibition of mutant Ha-rus mRNA expression by antknse oligonucleotides. J. Biol. Chem 267,19954-19962 Bennett, CF. et al. (1994) Inhibition of endothelial cell adhesion molecule expressionwith antisenseoligonnckotides, J. Immunol. 152,3S3C-3540 White, E. (1996) Life, death, and the pursuit of apoptosis, GenesDev.lO, l-15 Oltvai, Z.N., MiUiman, CL. and Korsmeyer, S.J. (1993) Bcl-2 heterodimerixes in viw with a conservedhomolog, Bax, that accelerates programed cell death, Ceil 74,609-619
32 Arnold, L.J. (1996) High affinity antisense oligonucleotides and their application to disease targets, Oligonucleotiak- and Gene Thempy-based Antisense Therapeutics, Inremational Business Communications, San Diego, CA, USA, pp. l-22, International Business Communications 33 Dean, N.M., McKay, R., Condon, T.P. and Bennett, CF. (1994) Inhibition of pro-
tein kinase Ca expression in human A549 cells by antisenseoligonudeotides inhibits induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by 7942-1949 phorbol esters, J. Biol. Chem. 269.1641616424 12 Matsukura, M. et al. (1987) Phosphorothioate analogs of oligodeoxynu- 34 Dean, N. et al. (1996) Inhibition of growth of human tumor cell lines in nude ckotides: inhibitors of replication and cytopathic effectsof human hnmunomice by an antisense oligooucleotide inhibitor of protein kinase C-a expresdeficiencyvirus, Proc. Narl. Acad. Sci. U. S. A. S4,7706-7710 sion, CancerRes. 56,3499-3507 13 Lisziewicz, J. er al. (1993) Long-term treahnent of human bmmmodefEeiency 35 McKay, R.A. et al. (1996) Enhanced activity of an antisense oligonudeotide virus-infected cells with antisense oligonucleotide phospborothioates, Pnx. targeting mmine protein kinase-c~by the incorporation of 2’-O-propyl modiNarl. Acad. Sci. U. S. A. 90,386C-3864 14 Milligan, J.F., Matteucci, M.D. and Martin, J.C. (1993) sensedrug design,.l. Med. Chem. 36.1923-1937
38
fieatioos, Nucleic Acids Res. ti, 411417
Current conceptsin anti-
36 Altmann, K.H. et al. (1996) Second generation of antisense oligonudeotides: from nucleaseresistanceto biological efficacyin animals, Chemia 50,168-176
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Antisense technology and prospects for therapy of viral infections and cancer Eighteen years ago, antisense oligonucleotide therapeutics that can selectively knock out disease-causing genes could easily have been viewed as science fiction. Yet today, through much persistence and focused investment, the technology has nearly evolved to the point of realization. A number of first-generation antisense compounds have entered human clinical trials. Some of these compounds appear to work by an antisense mechanism to inhibit the expression of disease-causing genes, while others probably work by unanticipated, yet clinically beneficial, mechanisms. In this review, the current status of antisense oligonucleotide development will be described as it relates to two areas of concentrated effort: antiviral and anticancer applications. ANTISENSE oligonucleotides (ONs) are short stretches (7-30 nucleotides in length) of DNA or modified DNA that contain a complementary base sequenceto a target RNA. The ONs are added extracellularly to cells and are expected to enter the cell and selectively hybridize to their target by Watson-Crick hydrogen-bonded base pairs. This hybridization should interfere with the function of the RNA by blocking RNA transport, splicing or translation. In some cases,the RNA-ON hybrid forms a substratefor RNase H, a cellular enzyme that selectively hydrolyzes the RNA portion of the RNA-DNA hybrid and inactivates the RNA (Fig. 1). However, severalfundamental barriers have been encounteredduring development of antisenseONs asbroadly applicable therapeutics. (1) Unmodified ONs are susceptible to degradation by cellular and extracellular nucleases. Chemical modification of each of the phosphodiester linkages in an ON by a variety of methods (phosphorothioate being the most commonly used) confersresistanceto degradation in cell culture (Fig. 2). Alternatively, modification of the ribose moiety can also result in increased resistance to nucleases. Nevertheless, ONs are still metabolized in animals more rapidly than predicted by cell culture studies and improvements in ON pharmacokinetics are still needed’. (2) Cellular penetration by ONs has been shown in cell culture studies to be inefficient. For example, phosphorothioate ONs added directly to the cellular growth media failed to demonstrate antisense activity, yet when these same ONs were microinjected or delivered with cationic lipids, expression of the targeted proteins wasefficiently inhibitedz4. Cationic liposomes, microinjection and electroporation have been used to enhance cellular delivery and the potency of ONs in cell culture23Z5. In animal studies, several interesting and provocative reports have suggestedthat ONs can penetrate cells in at least some tissues in the absenceof cationic lipidsa. The implications of these results offer a tremendous boost to the technology, but their general applicability Copyright
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emerge. In some cases, these non-antisense mechanisms might have clinical benefit (see below). However, most of these problems now have solutions, resulting in very convincing demonstrations of specific antisense effects using highly controlled assays.This evolution of the technology for use in human diseases,such as Crohn’s disease, arthritis, ulcerative colitis, viral infections and cancer, is reflected in the compounds that have entered clinical trials. In this review, we focus on the antiviral and anticancer applications of ONs.
Antiviral applications of antiseme oligonucleotides
1. Mechanism of action of antisense oliiucleotides. Antisense oliiucleotidaa (0N.s) are small nucleotidesin length) syntheticnucleicacids resemblingsingle-strandedDNA AMkense ONs h@idize to the coding (sense) sequence in a speckally Urge&d mRNAby Walscn-Cridc base+Mg rules.AntisenseONscanbindtothetargetedRNAinthenudeusorcytophsm.Whentheybindtheir$rgetmRNAin the nucleus,the mRNAportion of the anlisense ON-RNA complex is degraded by RNase H (not shorn). In the cytoplasm,the bound antkerw ON inhibits expressionof the mRNAby interferingwith protein bans&n. In both cases,the diww-cwsi ng proteinfailsto be synthesized.A major advantageof ankensa techrwkgy over traditional phannaceuti& is that antisense ONs can be ratio&y designed to target any dkeasecausinggene within the entire human genome as long as a partial sequenceof the gene is known. Figure (7-30
Viruses are attractive targets for antisense technology because they harbor genetic information that is distinct from that of the host cell. Some of the first antisense compounds to enter controlled clinical trials have been developed to inhibit viral pathogens: HIV, cytomegalovirus (CMV) and human papillomavirus (I-W). In general, the data presented so far for these compounds suggest that antisense plays a minor, if not insignificant, role in the mechanism of inhibition of these viruses. Below we provide a critical evaluation of the data supporting the mechanism of action for the antiviral ONs along with their clinical status.
HIV
GEM91, a 25 nucleotide (nt) phosphorothioate ON, is complementary in sequenceto the gag mRNA of HIV and is undergoing testing in Phase I/II clinical trials sponsored by Hybridon, Inc. (Worcester,MA, USA). Its antiviral activity has been demonstrated in will have to await fnrther examples in a variety of systemsto demon- acutely and chronically infected CEM cells (a T lymphoblastoid cell strate a convincing proof of concept. line), human peripheral blood lymphocytes and macrophages, and (3) Much effort has been expended using ONs that do not bind isolates from HIV-l-infected patients. GEM91 was found to be sixwell to their target. In general, this is due to the poor hybridization of fold more active than a control ON (a 25 nt random mixture of ONs ONs to RNA in cells. A number of chemical modifications have been synthesized from a mixture of all four bases) at inhibiting HIV-l developed that increase the affinity of ONs for RNA, resulting in replication in MOLT-3 cells (a T lymphoblastoid cell line). This more potent antisense effects9(Fig. 2). Also, much is now known result implied a sequence-dependentcomponent of antiviral activity about how to target the mBNA encoding a specific protein. It is and led to the conclusion that antisense was a component of the necessaryto test multiple antisense sequencescomplementary to a antiviral mechanism of action for GEM91 (Ref. 11). variety of regions along an mBNA molecule to find sequencesthat Sequence dependence does not necessarily imply an antisense are effective. The common practice of testing one antisensesequence mechanism of action, however.Phosphorothioate ONs, in addition to and its sensecontrol targeted to the AUG initiation codon of a mes- their ability to bind RNA, bind a variety of proteins (Fig. 3), includsage is not necessarily doomed to failure, but a great deal of luck is ing HIV reverse transcriptase, CD4 (the cell-surface receptor to which HIV binds) and gp120, an I-D&envelope protein that binds to required for this approach to work’. Many of these barriers have, at times, appeared quite daunting, CD4 (Ref. 10). Many of thesenon-antisenseeffectsare dependent on leading some to speculate as to whether antisense can ever work as a the sequenceand length of the ON. For example, a 28 nt phosphorotherapeutic strategylo. However, this is a surprisingly pessimistic thioate ON containing only cytosine inhibits HIV adsorption to its view during a time when major advanceshave been made. Clearly, target cells. The activity of this molecule is dependent on sequence, problems existed with the interpretation of early results, and mecha- becausea 28 nt phosphorothioate ON containing only adenosine was nisms underlying the non-antisense effects of ONs continue to much less active’*. The activity of the 28 nt cytosine-only ON as
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compared with GEM9 1 in the above cell culture studies is not known. Thus, alternative mechanisms, such as inhibition of viral adsorption to a cell, or cellular toxicity, could be responsible for at least part of the anti-HIV activity of GEM91 (Fig. 3)13*14. A significant amount of pharmacological data has been made available from studies of GEM91 in rats, mice and humans15.GEM91 was administered by intravenous infusion to humans at 0.1 mg kg-’ for 2 h. The slow infusion was necessaryto avoid complement activation and related hypotension, and increased clotting times caused by nonspecific ON binding to thrombin. These undesirable effectshave been observed in studies of monkeys’6. Data using GEM91 in humans showedthat the 35S-labeledON was rapidly cleared from plasma, with biphasic t,, values of 0.18 f 0.04 h and 26.71 f 1.67 h for plasma-derived radioactivity”. The B-phase of elimination was, however, somewhat misleading because it reflected radioactivity and not intact GEM91. GEM91 was rapidly metabolized in humans; the majority of the plasma-derived GEM9 1 radioactivity was associated with intact ON only up to 2 h after infusion. If high and prolonged plasma concentrations of GEM91 are required for anti-HIV activity in viva, then these data suggest that frequent (and inconvenient) 2 h dosing regimens might be needed. Efficacy data for GEM91 have not yet been published.
O=P-o-
Figure 2. Structure and chemical modificationsof oligonucleotidee. The general structure of an oligonucleotide is shown on the left. Regionsthat can be modified to enhance the affinityand nuclease resistance of an digonudeotide include (a) the heterocycle,(b) the backbone and (c) the dbose moiety.Examples of heterocycleanalogs (a) include: top, propyne modificationat the C-7 position of 7-deazaguanosine; center, propyne modificationat the C-5 position of uracil; and bottom, a phenoxazine-modified cytosine.Backbone modifications (b) include: top, a phosphorothioate analog; center, a phosphorsmidate analog; and bottom, a peptide-nucleic-acid linkage. Modiiications of the ribose sugar (c) include: top, changing ribose to hexose; center, modfting the 2’ hydroxylto a methoxygroup; and bottom, modifyingthe 2’ hydroxylto a methoxyethoxymoiety.Modified from Ref.9.
Human cytomegalovirus ISIS 2922 (Isis Pharmaceuticals, Carlsbad, CA, USA) is a 21 nt phosphorothioate ON that is complementary to sequencesin the major immediate early region of human cytomegalovirus (HCMV) mRNA. This compound is in PhaseIII clinical trials for the treatment of HCMV retinitis, an opportunistic eye infection that occurs in nearly 30% of AIDS patients. Cell culture studies using ISIS 2922 have shownthat the agent is a sequence-dependentinhibitor of HCMV viral infection’*. In these studies, the effect of mismatch and length wasevaluated for the compound. A two-basemismatch control ON that had a thermal denaturation temperature below physiological temperature when bound to RNA (-35°C vs. -55°C for ISIS 2922) retained 88% antiviral activity compared with ISIS 2922. However, a single base deletion from the 3’ end of ISIS 2922 retained its thermal melting temperature (-52”(J), but retained only 40% of its antiviral activity. Treatment of cells with ISIS 2922 before infection was absolutely essential for antiviral activity”. These data do not, therefore, correlate well with an antisense mechanism and strongly suggestthat ISIS 2922 inhibits HCMV viral infection by a sequence-dependentbut non-antisense mechanism of action that probably involves inhibition of viral adsorption, penetration or uncoating (Fig. 3). Although the mechanism of action of ISIS 2922 is poorly understood, it appearsto be clinically beneficial and might become a suc-
cessful ON-based therapeutic. ISIS 2922 has been administered by intravitreal injection every other week to AIDS patients in a Phase I./II study. The results showed a dose response for activity in an open-label trial that evaluated HCMV progression using a blinded evaluation of retinal photographs”. These encouraging data prompted a Phase III study, the results of which should be reported in early 1997. Human papillomavirus ISIS 2105 (Isis Pharmaceuticals, Carlsbad, CA, USA) is a 20 nt phosphorothioate ON that is complementary to the E2 gene of HPV. ISIS 2105 has been evaluated in Phase I/II human clinical trials for the treatment of genital warts as an adjuvant to cryotherapy; however,the compound wasdiscontinued owing to lack of efficacy in the tlidS.
I-W is a difficult virus to screen compounds against because it cannot be reliably cultured in any significant quantity and the in vivo models do not truly reflect the human diseases caused by I-IF?? Bovine papillomavirus, for which there are cell culture models, was initially usedto validate inhibition of the E2 gene product, a papillomavirus-encoded transcription factor, by antisense ONszO. Positive results in this model prompted the development of ISIS 2105, which was complementary to the E2 mRNA of HPV-6 and HPV- 11. This compound inactivated E2-dependent transactivation of a reporter 33
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guanosine quartet have antiproliferative activity. Also, ONs can bind to growth factors and their receptors,which could interfere with cellular proliferation10*23.Furthermore, Virus ONs containing cytosine-phosphate-guanosine dimers can activate B cells and natural killer cells; this immune stimulation might z result in antitumor activity irt vid+. This activity can be mitigated by methylation of a Binding and fusion cytosine. Thus, many nonspecific effects of ONs might have interfered with the interpretation of experiments designed to assesstheir antisenseactivityzvz6. The ultimate successand value of antisense technology depend on the ability to design potent, sequence-selective ON inhibitors, and to demonstrate that the biological and clinical benefits of the ON can be attributed to an antisense mechanism of virus particles action. Sequence selectivity and potency of antisense ONs is dependent on chemical Translation of modifications of the ON that affect the affinity of the ON for its target, sequencecontext and ON length*‘. In fact, phosphorotbioate ONs have demonstrated selective targeting of a mutant rus gene containing a single base w Viral mRNA change in codon 12 (Ref. 28). The maximum selectivity between the mutant and normal 0 Transcription rus genes was obtained with a 17 nt antisense of viral genes and ON and was about 5-fold. In general, anti\ viral replication sense ONs have a 5-30-fold window of Nucleus specificityin tissuecultnre3~4272g. The parameters that affectnonspecificinhibition of nontargeted Figure 3. Overview of a generic virus-replication cycle and the targets for oligonucleotides (ONs) during RNA by ONs are not clear, but it is probably viral replication. (a) The virion binds to a specificcell-surface receptor and fuses with the plasma membrane. (b) The viral capsid is transported to the nucleus (during the retrovirus replication cycle the viral due to partially complementary binding of RNA is first reverse transcribed to DNA). Once bound to the nucleus, the viral DNA is released into the the ON to a nontargeted RNA and RNase H nucleus, where it is (c) transcribed by cellular enzymes and replicated. (d) Viral proteins are synthesized cleavage of the nontargeted transcript. using the cellular translation apparatus and (e) new viral particles are assembled and released. Sites of Now that these limitations have been recantisense ON action are indicated by boxes. Phosphorothioates can bind to a varietyof proteins on the cellular surface by a sequence-dependent, non-antisense mechanism of action, and block virion binding ognized, improved ONs, methods and conand fusion (a). Phosphorothioate ONs have also been shown to inhibit reverse transcriptase in a trols have been used to assessthe antisense sequence-independent manner (b). Although phosphorothioate ONs can act nonspecificallyto inhibit viral activity of anticancer ONs, resulting in conreplication, phosphorothioate ONs have also been suggested to inhibit viral gag gene expression during vincing data that showselective inhibition of HIV-1 infections (for example, GEM91) and immediate early gene expression in human cytomegalovirus gene expression in cell culture. Furthermore, (for example, ISIS 2922). See text for details. in viva data, while more difficult to control rigorously, strongly suggestthat antisenseONs construct in cells; however,inhibition of HPV infection in cell cul- might be effective against xenograft tumor transplants. This work has ture or in viva models has not been reported. The pharmacokinetics, laid the foundation for clinical trials that are now under way. distribution and stability of ISIS 2 105 have been evaluated following intradermal injection in rat?‘. These studies suggestedthat ISIS 2105 bcl-2 would be bio-available at sufficient concentrations following once or In ~85% of follicular B-cell lymphomas, bd-2 is overexpressedas a twice weekly intralesional dosing in patients with genital warts. result of a trauslocation of the bcl-2 gene to the immunoglobulin heavy-chain locus. bcl-2 is a member of a related multi-gene family that regulates programmed cell death by protein-protein interactions AntisenseONs for the treatment of cancer Antisense ONs have also been extensively studied for their use as (Fig. 4). Bcl-2 homodimers repress apoptosis while homodimers of anti-neoplastic agents”. Much cell culture and animal data has been Bax, a 21 kDa protein with extensive amino acid homology to Bcl-2, published that describes the anti-growth activity of ONs. Similar to induce apoptosis. Thus, according to the model, cell fate can depend the data from the antiviral ON studies, many of the anticancer ONs on the ratio of the ‘death inducer’ to the ‘death suppressor’“.31. An 18 nt phosphorothioate antisense ON complementary to bcl-2 have been shown to exhibit their effectsthrough sequence-dependent but non-antisense mechanisms‘o-23 . For example, ONs containing a (G3 139) is being developed by Genta (San Diego, CA, USA) to treat
-r
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Glossary Survival
Athymic nude mice - Hairless mice that have a rudimentary thymus in which T-cell maturation cannot occur normally.As a result, these mice have no cell-mediated immune reactions, including xenograft rejection. W-2 - The B-cell lymphomalleukemia2 gene, a gene that has been implicatedin the etiology of many common human lymphoidtumors. bc-2 is a member of a large gene family whose protein products can homo- and heterodimerizeto inhibit apoptosis (Bcl-2) or accelerate apoptosis @ax). Biphasic t,n values -A term that describes a two-compartment pharmacokinetic model in which a rapid plasma clearance time (the first phase or a phase) for a drug is followed by a slower clearance time (the second phase or p phase). t,, describes the time it takes for 50% of the drug to clear the plasma in each phase. Cytoslne-phosphatequanosine dimer - A motif in oligonucleotides that, when placed in proper sequence context, is mitcgenic by a nonantisense mechanismfor B cells and natural killer cells. Methylationof cytosine at the C-5 position blocks the mitogeniceffect. Folllcular lymphoma - A malignantdisorder of lymphoidtissue that originates in identifiablenodules (follicles) of a lymph node. gag-An HIV gene that encodes the nucleocapsidprotein. Major immedlate early reglon - A region of cytomegalovirus from which the first genes are transcribed after infection of a permissive cell. Transcription of the immediateearly genes does not require preceding viral protein synthesis. 2’0methoxyethylribonucleosides - A chemical modificationat the 2’-hydroxy position of the ribose sugar of nucleosides, which makes them less susceptible to nuclease attack and enhances the affinity of the antisense oligonucleotidefor its target. Phosphorothloate - An intemucleotidelinkage in DNA or RNA in which one oxygen atom in the phosphate moiety is replaced with sulfur, making the bond resistant to nucleases. RNase H - A cellular enzyme that hydrolyzes the RNA potion of RNA-DNA hybrids. Severe combined immunodeticisncy @CID) - A genetic disease of inbred mice in which both humoral and cell-mediated immunity fail to develop owing to a defect in the ability to rearrange Tcell receptorand immunoglobulin genes.B and T cellsare absentor foundat very lowlevels.SCIDmiceare usedto evaluatethe efficacy of pre-clinical therapeutics in the absenceof manynormalimmune responses. Thermaldenaturationtemperature- The temperature at whicha nucleicacid duplexmeltsby 50%underequilibrium conditionsat a givensaltandbufferconcentration. It is a qualitativemeasureof the affinityof an antisense compoundfor its target RNAandallowsthe relativerankingof thestabilityof nucleicacidduplexes.
Apoptosis (death)
Figure 4. Model for the action of Bcl-2 and Bax in regulating apoptosis. Bcl-2 homodimers repress apoptosis while Bax homodimers accelerate it. Whether or not a cell enters apoptosis depends on the ratio of Bax to Bcl-2. Antisense digonucleotiies have been designed to induce apoptosis by inhibiting the translation of Bcl-2, thereby increasing the ratio of Bax to Bcl-2.
in this model. These results appear promising, although still quite preliminary3’. In a pilot toxicology study of G3139, cynomolgus monkeys were dosed with 0.125, 1.0 and 10 mg kg-‘&y-’ of G3139 by subcutaneous infusion for 14 days. No deaths or treatment-related clinical symptoms were observed, although histopathological changes were seen in the kidney cortex (slight brown pigmentation) andthe spleen (which was enlarged) at the highest ON dose. A Phase I/II trial for follicular lymphoma was initiated in November 1995 at the Royal Marsden Hospital (London, UK). G3 139 is also in pre-clinical development for use in chemoresistant solid tumors, which often aberrantly expressbcl-2 (Ref. 32). c-Raf-I
c&f-l is a serinejthreonine kinase that participates in the stimulation of the mitogen-activated protein kinase (MAPK) signaling cascade. c-Raf-1 is activated by Ras, a GTP-regulated molecular switch that is mutatedin nearly a third of all human cancers.In addition to Ras, other signals emanating from cell-surface receptors are integrated by c-Raf-1 and transmitted by phosphorylation to downstream targets, leading to the proliferation or differentiation of the cell (Fig. 5). An activated form of c-Raf-1 is oncogenic and has been associatedwith human malignancies. Recently, a 20 nt phosphorothioate ON (ISIS 5132; Isis Pharmaceuticals, Carlsbad, CA, USA) complementary to human c-ruf-1 follicular lymphoma. In a pre-clinical model, administration of entered clinical trials. This followed the demonstration of potent G3139 at 5 mg kg-’ day-’ for 21 days to severecombined immuno- antiproliferative properties in cultured cells and also antitumor activdeficient (SCID) mice inoculated with follicular lymphoma cells ity in xenografts with human lung, human bladder and human breast resulted in the elimination of lymphoma cells in 6/6 mice. This was tumors7. When a cationic lipid wasused to transfect ISIS 5132 into determined by PCR and fluorescence-activated cell sorter (FACS) cultured cells, this c-@-l antisense ON was demonstrated to be analysis of blood and tissue samples. A sense-ON control failed to sequencespecific. Control ONs containing l-7 mismatches showed cure the lymphoma, but a more stringent mismatch control ON con- reduced antisense activity. c-ruf-I antisense ONs also demonstrated taining the same basecomposition as the antisenseON wasnot tested isotype-specific inhibition becausethey had no effect on RNA levels 35
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Growth factor binding and receptor
Growth factor receptor
Figure 5. Signal transduction pathways that control cellular proliferation and differentiation.Binding of a growth factor to its receptor causes autophosphorylation of tyrosine residues on the cytoplasmicside of the receptor.The phosphorylatedreceptor activatesvarioussignal transduction pathways,including the mitogen-activated protein kinase (MAPK) pathway (left) and the phosphoinositide pathway (right). In the MAPK pathway, adaptor molecules such as GRB2 bind to the phosphorylated receptor and activateRas, a small GTP-binding protein. This leads to the activationof c-Raf-l ,a serine/threonine kinase,triggering a phosphorylatiin cascade involving MAPK and its kinase, MEK. In the phosphoinositide pathway, phospholipase C (PLC) binds to the phosphotylated receptor and catalysesthe hydrolysisof a membrane phospholipid, phosphatidylinositol (43)~biiphosphate [Ptdlns(4,5)P,] to inositol (1,4b)-trisphosphate [lns(l,4,5)PJ and 1,2diacylglycerol (DAG). lns(l,4,5)P, causes an increase in the cellular Ca2+ concentration, which synergizes with DAG to activate protein kinase C-O!(PKCa), another serine/threonine kinase. Substrates of PKC-a include c-Raf-l and MAPK. Antisense oligonucleotides (ONs) targeting the mRNA for c-Raf-1and PKC-a should inhibit these pathways, which are oftenconstitutivelyactivein cancer cells.
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of another member of the Raf-1 family, A-Raf. The results of these well-controlled experiments strongly suggest that the c-raf-I antisenseONs are functioning by a sequence-specificantisense mechanism of action’. In a pre-clinical study, mice bearing tumors were administered with 0.00640 mg kg-’ of ISIS 5132 (without the use of a cationic lipid) by intravenous injection once daily for 11 days’. Growth of the tumor xenograft, as determined by measuring tumor volume, was inhibited at an ON concentration of 0.0646 mg kg-‘. The mismatch control ON had no effect on tumor growth. In this study, c-~$1 mRNA levels were shown to be significantly reduced in the tumors dosed with the antisense ON. Maximal inhibition of c-r&l RNA levels (15% of control RNA levels) occurred after 13 days of a 6.0 mg kg-’ day-’ ON dose. Although c-ruf-1 mRNA levels were reduced by antisense ONs, the effect on c-r&l protein levels in the tumors was not investigated. In addition, A-raf mRNA and protein levels were not analyzed, as they had been in the studies described above, making it difficult to determine the specificity of the c-r&l antisense ONs in viva. Nevertheless, the findings are encouraging becausethey demonstrate inhibition of human c-ruf-1 mRNA levels and potent antitumor effectsin viva. Phase1 clinical trials to determine the safetyof ISIS 5132 are being initiated by Isis Pharmaceuticals in collaboration with Ciba-Geigy Ltd (Basel, Switzerland). However, the use of c-ruf-1 antisenseONs could be limited by their associatedtoxicity in normal cells; in nondiseased cells, c-r&l is a critical component for cellular proliferation and differentiation. Despite this, preliminary results using an antisense ON complementary to murine c-ruf-1 failed to show any ON-associated toxicities in normal mice’, suggesting that ONs might be preferentially targeted to tumors (although previous data indicate that ONs are widely distributed in mice’). Alternatively, other members of the ruf gene family might compensate for c-Raf-1 activity in normal cells. Results from Phase1 trials should help to addresssafety and pharmacokinetic concernsassociatedwith these ONs. Protein kinase C-a
Protein kinase C-cw(PKC-cl) is a member of a family of homologous serine/threonine protein kinases involved in proliferation and differentiation (Fig. 5). PKC-cx is the most widely expressedPKC and the major intracellular receptor for tumor-promoting phorbol esters,agents that mimic the action of diacylglycerol. Because of the tremendous homology among the PKC isozymes, it has so far been difficult to develop isotype-specific small molecules that inhibit specific PKC isoforms and have favorable clinical properties. PKC-(Y has been successfullyinhibited using antisense ONs both in vitro using cationic lipids and in viva without cationic lipids33. When a 20 nt phosphorothioate ON complementary to murine PKC-a wasadministered to mice, it demonstrated a 64% reduction in PKC-(u mRNA levels in the liver following a single 50 mg kg-’ intraperitoneal dose. A control ON with a sequencethat was unrelated to PKC-(w had little effect on PKC-(r mRNA levels. The decreasein PKC-(x mRNA was,however,limited to the liver, consistent with tissue distribution of phosphorothioateONs, and wasisozyme specific33. Human PKC-(u has also been targeted by phosphorothioate ONs. Treatment of tumor xenografts in athymic nude mice with a 20 ntlong human PKC-cx antisense ON (ISIS 3521) suppressedtumor growth, whereasthe control ON had no effect”. Second-generation antisense ONs have now been synthesized and analyzed35.These
Reviews
chimeric ONs contain six phosphodiester-linked 2’-0-methoxyethyl ribonucleosides on the 5’ and 3’ ends of the ON, and have an 8 nt phosphorothioate center that functions as an RNase H target site36.In tissue culture, these ONs were sixfold more potent than ISIS 3521 yet, remarkably, when administered to tumor-bearing nude mice, the chimeric ONs were about lOO-fold more potent?. The pharmacokinetics of the chimeric ONs were also quite different in mice from ISIS 3521, displaying a more rapid clearance. It is therefore surprising that the biological potency as compared with ISIS 3521 was superior. Furthermore, the chimeric ONs showa reduction in adverse effects on clotting time of human plasma. Based on these data, second-generation antisense compounds should demonstrate increasedbiological potency and decreasedtoxicity in viva A Phase 1 clinical trial using first-generation PKC-a antisense ONs began in January 1996 for the treatment of cancer.The introduction of secondgeneration ONs into the clinic is still, however,severalyears away. Several phosphorothioate ONs have already entered clinical trials for antiviral and anticancer indications. In each case,the candidates were selected on the basis of cell culture and pharmacokinetic results. For antisenseONs targeting HIV and CMV, the cell culture data demonstrated the antiviral activity of the ONs and showed that the effects were sequence dependent. However, sequencedependence does not prove an antisensemechanism of action, and it is likely that the ONs act by non-antisense mechanisms. Such mechanisms do not undermine the clinical effectivenessor ultimate successof the drugs, but they do not validate antisense therapeutics as a broadly applicable genetic targeting technology. However, the demonstration of the clinical benefit of ONs is important for the technology becauseit will indicate that such agents have the appropriate pharmacodynamic properties to be useful therapeutics. The genes for Bcl-2, c-Raf-1 and PKC-cwhave been targeted by antisense ONs and these ONs have entered clinical trials as anticancer compounds. Both c-r&l and PKC-a ONs target the same signal transduction pathway (Fig. 5) and many tumor types might be sensitive to both c-ruf-I and PKC-a antisenseONs. However, several lines of evidence suggest that PKC-a might also function in parallel to the c-Raf-l-activated MAPK pathway.Therefore, differential sensitivities of tumors to c-ruf--l and PKC-cx ONs would be expected. Synergism betweenthe ONs might also be possible in specific tumor types. These anti-cancer ONs should prove to be valuable therapeutic agents as well as powerful researchtools for elucidating the function
Reviews
MOLECULAR
of important signal transduction pathways in the development and maintenance of human cancers.
Future prospectsof ONs as therapeutics
MEDICINE
TODAY,
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1997
15 Agmwal, S., Temsamani, J., Galbraith, W. and Tang, J. (1995) Pharmacokinetics of antisenseoligonudeotides, Clin. Phartnacokiner. 28,7-16 16 Galbraith, W.M. et al. (1994) Complement activation and hemodynamic changes following intravenous administration of phosphorothioate oligonudeothles in the monkey,AntisenseRex Dev. 4.201-206 17 Zhang, R. er al. (1995) Pharmacokktics of an anti-human hmmmodeficiency virus antisense oligodeoxynudeotide phosphorothioate (GEM91) in HIVinfected subjects,Clin. Pharmacol. Thex 58,4k53 18 Azad, R.F. et al. (1993) Antiviral activityof a phosphorothioate oliionucleotide complementary to RNA of the human cytomegalovirus major immediateearly region, Antimicrob. Agents Chemorher: 37.1945-1954 19 Hutcherson, S.L. et al. (1995) Antisense oligonucleotide safetyand efficacyfor CMV rethdtis in AIDS patients, Proceedings of rhe 35rh Inrernarional Confer-
Antisense ONs can be rationally designed to inhibit the expressionof disease-causinggenes by binding specifically to their mRNAs, yet the non-antisense effectsof ONs have, so far, complicated the interpretation of results. Nevertheless, researchers in the antisensetherapeutics field are increasingly optimistic. In recent years, many of the challenges facing the development of antisenseONs as therapeutic compounds have been overcome: successful inhibition of therapeutic targets is being observed in animal models; and several clinical trials are now in progress to evaluate antisense ONs in various medical conditions, including viral infections (HCMV, HIV), ence on Antimicmbial Agenrs and Chemorherapy, San Francisco, CA, USA, p. 204, cancer (solid tumors and leukemia) and inflammation (not described American Society for Microbiology in this review). The results from clinical trials using first-generation 20 Cowsert, L.M., Fox, MC., Zen, G. and Miibelli, C.K. (1993) In vitro evaluation antisense compounds will soon be known. Second-generation antiofphosphorotbioateoUgon~targdedtotheE2mRNAofpapiUorua~ sensecompounds have decreasedproduction costs,and showenhanced potential treatment for genital warts, Antimicrob Agenrs Chemorhel: 37,171-177 potency, improved pharmacokinetics and reduced ON-associated tox- 21 Cossum,P.A.et al. (1994) Pharmacotietics of a “C-labeled phosphorothioate icities. These are in pre-clinical development and clinical trials are oligonudeotide, ISIS 2105, after intradennal admhdskation to rats, J. Phan expected in the next two years. It now appearsthat 18 years of antiExp. Thel: 269,89-94 senseresearchwith high-profile successesand failures have resulted 22 Tonkinson, J.L. and Skin, CA. (1996) Antisem oligodeoxynucleotide as diniin the evolution of an exciting new classof therapeutic compounds. cd therapeutic agents,Cancer Znvesr. 14,54-65
23 Stein, CA. (1996) Phosphorothioate antisense oligonucleotides: questions of Acknowledgements. We thankMary Hogsett for figure and manuscript preparation. References 1 Crooke, ST. er al. (1996) Pharmacoldnetic properties of severalnovel oligonucleotide analogs in mice, .I.Pharmacol. Exp. The,: 211,923-937 2 Bennett, CF. er al. (1992) Cationic lipids enhance cellular uptake and activity of phosphorothioate antisenseoligooucleotides, Mol. Phonnncol. 41.1023-1033 3 Wagner, R.W. et al. (1993) A&ease gew inhibition by oligonocleotides containing C-5 propyne pyrimidines, Science 260,1510-1513 4 Flanagan, W.M., Su, L.L. and Wagner, R.W. (1996) Elucidation of gene function using C-5 propyne antisenseoligonucleotides, Nar. BiorechnoL 14.1139-l 145 5 Bergan, R., Connell, Y., Fahmy, B. and Neckers, L. (1993) -ration enhan= c-my antisenseoligodeoxynudeotide eflicacy,Nucleic Acids Res. 21,3567-3573 6 McKay, R. and Dean, M.N. (1994) Inhibition of protein kinase C-a expression in mice after systemic&nin%ration of phosphorothioate, Pmt. Natl. Acad. Sci. U. S. A. 91.1762-1766
spec&ity, Trena!~ Biotechnol. 14,147-149 24 Krieg, A.M. et al. (1995) CpG motifs in bacterial DNA trigger direct B-cell activation, Nature 374,546-549 25 Stein, C.A. and Krieg, A.M. (1994) Problems in inteqretation of data derived from in vitro and in viw use of antisenseoligod~xynucleotide, Anrisense Res. Dev. 4,67-69 26 Heidenreich, 0.. Kang. S-H., Xu, X. and Nerenberg, M. (1995) Application of
antisensetedmology to therapeutics, Mol. Med. Today 1,128-133 27 Flanagan,
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7 Monia, B.P. et al. (1996) Antitumor activity of a phosphorothioate antisense oligodeoxynuckotide targeted against c&f kinase, Nat.Mea! 2,668-675 8 Plenat, F. (1996) Animal models of antisenseoligonucleotides: lessonsfor use in humans, Mol. Med. To&y 2.250-257 9 Matteucci, M.D. and Wagner, R.W. (1996) In pursuit of antisense,Nature 384 (Suppl.) 20-22
10 Stein, CA. (1995)Dnesa&ense~? Nor. Med. 1,1119-1121 11 Lisziewicz, J. et al. (1994) Antisense oligodeoxynuckotide phosphorothioate complementary to gag mRNA blocks replication of human immunodetkiency virus type 1 in human peripheral blood ceils, Proc. Narl. Acad. Sci. U. S. A. 91,
31
W.M., Kothavale,
A. and Wagner, R.W. (1996) Effects
of oligonu-
de&de length, mismatches,and mRNA levelson C-5 propyne-modiikd antisense potency,Nucleic Acids Res. 24,2936-2941 Monia, BP. et al. (1992) Selectiveinhibition of mutant Ha-rus mRNA expression by antknse oligonucleotides. J. Biol. Chem 267,19954-19962 Bennett, CF. et al. (1994) Inhibition of endothelial cell adhesion molecule expressionwith antisenseoligonnckotides, J. Immunol. 152,3S3C-3540 White, E. (1996) Life, death, and the pursuit of apoptosis, GenesDev.lO, l-15 Oltvai, Z.N., MiUiman, CL. and Korsmeyer, S.J. (1993) Bcl-2 heterodimerixes in viw with a conservedhomolog, Bax, that accelerates programed cell death, Ceil 74,609-619
32 Arnold, L.J. (1996) High affinity antisense oligonucleotides and their application to disease targets, Oligonucleotiak- and Gene Thempy-based Antisense Therapeutics, Inremational Business Communications, San Diego, CA, USA, pp. l-22, International Business Communications 33 Dean, N.M., McKay, R., Condon, T.P. and Bennett, CF. (1994) Inhibition of pro-
tein kinase Ca expression in human A549 cells by antisenseoligonudeotides inhibits induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by 7942-1949 phorbol esters, J. Biol. Chem. 269.1641616424 12 Matsukura, M. et al. (1987) Phosphorothioate analogs of oligodeoxynu- 34 Dean, N. et al. (1996) Inhibition of growth of human tumor cell lines in nude ckotides: inhibitors of replication and cytopathic effectsof human hnmunomice by an antisense oligooucleotide inhibitor of protein kinase C-a expresdeficiencyvirus, Proc. Narl. Acad. Sci. U. S. A. S4,7706-7710 sion, CancerRes. 56,3499-3507 13 Lisziewicz, J. er al. (1993) Long-term treahnent of human bmmmodefEeiency 35 McKay, R.A. et al. (1996) Enhanced activity of an antisense oligonudeotide virus-infected cells with antisense oligonucleotide phospborothioates, Pnx. targeting mmine protein kinase-c~by the incorporation of 2’-O-propyl modiNarl. Acad. Sci. U. S. A. 90,386C-3864 14 Milligan, J.F., Matteucci, M.D. and Martin, J.C. (1993) sensedrug design,.l. Med. Chem. 36.1923-1937
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fieatioos, Nucleic Acids Res. ti, 411417
Current conceptsin anti-
36 Altmann, K.H. et al. (1996) Second generation of antisense oligonudeotides: from nucleaseresistanceto biological efficacyin animals, Chemia 50,168-176