CA2265917C - Method for using oligonucleotides having modified cpg dinucleosides - Google Patents

Abstract

The invention relates to modified oligonucleotides that are useful for studies of gene expression and for the antisense therapeutic approach. The invention provides modified oligonucleotides that inhibit gene expression and that produce fewer side effects than conventional phosphorothioate oligonucleotides. In particular, the invention provides modified CpG-containing oligonucleotides that result in reduced splenomegaly and platelet depletion when administered to a mammal, relative to conventional CpG-containing phosphorothioate oligonucleotides. The ivnention further provides methods for using such oligonucleotides to modulate gene expression in vivo, including such use for therapeutic treatment of diseases caused by aberrant gene expression.

Description

20253035CA 02265917 1999-03-10W0 98/11211 PCT/US97Il60l71METHOD FOR USING OLIGONUCLEOTIDES HAVINGMODIFIED CpG DINUCLEOSIDESBACKGROUND OF THE INVENTION Field of the inventionThe invention relates to modified oligonucleotides thatare useful for studies of gene expression and for theantisense therapeutic approach.Summary of the related artThe potential for using oligonucleotides as inhibitorsof specific gene expression in an antisense therapeuticapproach was first suggested in three articles published in1977 and 1978. Paterson et al., Proc. Natl. Acad. Sci. USA14: 4370-4374 (1977) discloses that cell-free translationof mRNA can be inhibited by binding a complementaryoligonucleotide to the mRNA. Zamecnik and Stephenson,Proc. Natl. Acad. Sci. USA 1;: 280-284 and 285-288 (1978)disclose that a 13-mer synthetic oligonucleotide that iscomplementary to a part of the Rous sarcoma virus (RSV)genome can inhibit RSV replication in infected cellcultures and can inhibit RSV—mediated transformation ofprimary chick fibroblasts into malignant sarcoma cells.Since these early studies, the ability of antisenseoligonucleotides to inhibit virus propagation has becomefirmly established. US Patent No. 4,806,463 teaches thathuman immunodeficiency virus propagation can be inhibitedby oligonucleotides that are complementary to any ofUS Patent No. 5,194,428discloses inhibition of influenza virus replication byvarious regions of the HIV genome.phosphorothioate oligonucleotides complementary to theinfluenza virus polymerase 1 gene. Agrawal, Trends inBiotechnology lg: 152-158 (1992) reviews the use ofantisense oligonucleotides as antiviral agents.Antisense oligonucleotides have also been developed as 4-4 ¢............».....u..«.....«..........«...........«.....m ,.. . ,.1020253035CA 02265917 1999-03-10W0 98/1 121 1 PCT/U S97/ 160172PCT publication no. W093/13740discloses the use of antisense oligonucleotides to inhibitanti—parasitic agents.propagation of drug—resistant malarial parasites. Tao et123-129 (1995)teaches inhibition of propagation of a schistosome parasiteal., Antisense Research and Development 5:by antisense oligonucleotides.More recently, antisense oligonucleotides have shownpromise as candidates for therapeutic applications fordiseases resulting from expression of cellular genes. PCTpublication no. W095/09236 discloses reversal of betaamyloid—induced neuronal cell line morphologicalabnormalities by oligonucleotides that inhibit beta amyloidW094/26887 disclosesreversal of aberrant splicing of a globin gene transcriptexpression. PCT publication no.by oligonucleotides complementary to certain portions ofPCT application no. PCT/US94/13685discloses inhibition of tumorigenicity by oligonucleotidesthat transcript.complementary to the gene encoding DNA methyltransferase.The development of various antisense oligonucleotides astherapeutic and diagnostic agents has recently beenreviewed by Agrawal and Iyer, Current Opinion in12-19 (1995).As interest in the antisense therapeutic approach hasBiotechnology Q:grown, various efforts have been made to improve thepharmacologic properties of oligonucleotides by modifyingUS Patent No. 5,149,797describes chimeric oligonucleotides having athe sugar—phosphate backbone.phosphorothioate core region interposed between -methylphosphonate or phosphoramidate flanking regions. PCTpublication no. W094/02498 discloses hybridoligonucleotides having regions of 2'—O—substitutedribonucleotides flanking a DNA core region.Much is currently being discovered about thepharmacodynamic properties of oligonucleotides.al., 7-16al., Clinical Pharmacology and Therapeutics §§;(1995) disclose pharmacokinetics of anti—HIVAgrawal et(1995) and Zhang et44-53Clinical Pharmacokinetics gg:10CA 02265917 1999-03-10W0 98/1 121 l PCT/US97/160173oligonucleotides in human patients. Some of these newdiscoveries have led to new challenges to be overcome forthe optimization of oligonucleotides as therapeutic agents.For example, Kniep et al., Nature ;14: 546~549 (1995)discloses that oligonucleotides containing the CGdinucleotide flanked by certain other sequences have amitogenic effect. We have discovered that many sideeffects produced by phosphorothioate oligonucleotides are aconsequence of the phosphorothioate—linked CpGdinucleotide. There is, therefore, a need for modifiedoligonucleotides that retain gene expression inhibitionproperties while producing fewer side effects thanconventional phosphorothioate oligonucleotides. 101520253035CA 02265917 1999-03-10WO 98/11211 PCT/US97/16017BRIEF SUMMARY OF THE INVENTIONThe invention relates to modified oligonucleotides thatare useful for studies of gene expression and for theantisense therapeutic approach. The invention providesmodified oligonucleotides that inhibit gene expression andthat produce fewer side effects than conventionalphosphorothioate oligonucleotides. In particular, theinvention provides methods for using CpG—containingphosphorothioate oligonucleotides to modulate geneexpression with reduced splenomegaly and reduced depletionof platelets, relative to conventional CpG-containingphosphorothioate oligonucleotides.In a first aspect, the invention provides modified CpG-containing phosphorothioate oligonucleotides andcompositions of matter for inhibiting specific geneexpression with reduced side effects. Such inhibition ofgene expression can be used as an alternative to mutantanalysis for determining the biological function ofspecific genes in cell or animal models. Such inhibitionof gene expression can also be used to therapeuticallytreat diseases that are caused by expression of the genesof a virus or a pathogen, or by the inappropriateexpression of cellular genes. In one preferred embodimentaccording to this aspect of the invention, the compositionof matter comprises phosphorothioate oligonucleotideshaving one or more modified CpG dinucleosides. In certainparticularly preferred embodiments, all CpG dinucleosidespresent in the oligonucleotide are modified. According tothis aspect of the invention, a CpG dinucleoside ismodified so that it confers upon the oligonucleotide areduced ability to cause splenomegaly and plateletdepletion when administered to a mammal, relative to anotherwise identical oligonucleotide having an unmodifiedphosphorothioate CpG dinucleoside.102025CA 02265917 1999-03-10W0 98/ 1 121 1 PCT/US97/160175In a second aspect, the invention provides a method formodulating gene expression in a mammal with reduced sideeffects. In the method according to this aspect of theinvention, a composition of matter according to the firstaspect of the invention is administered to the mammal,wherein the oligonucleotide is complementary to a gene thatis being expressed in the mammal.In a third aspect, the invention provides a method fortherapeutically treating, with reduced side effects, adisease caused by aberrant gene expression, the methodcomprising administering to an individual having thedisease a composition of matter according to the firstaspect of the invention, wherein the oligonucleotide iscomplementary to a gene that is aberrantly expressed,wherein such aberrant expression causes the disease. Inthis context, aberrant gene expression means expression ina host organism of a gene required for the propagation of aVirus or a prokaryotic or eukaryotic pathogen, orinappropriate expression of a host cellular gene.Inappropriate host cellular gene expression includesexpression of a mutant allele of a cellular gene, orunderexpression or overexpression of a normal allele of acellular gene, such that disease results from suchinappropriate host cellular gene expression.,.,.,..,..u...,............w.«........u. ., ,, W . .10152025CA 02265917 1999-03-10W0 98/ l 1211 PCT/US97/16017BRIEF DESCRIPTION OF THE DRAWINGSFigure 1 shows results of platelet counts of CD1 miceintraperitoneally administered saline, conventional(91),modified CpG oligonucleotide(255),(256),phosphorothioate oligonucleotide methylphosphonate—inverted CpGoligonucleotide(257).and 5-methylC CpG oligonucleotideFigure 2 shows results of spleen weight analysis of CD1mice intraperitoneally administered saline, conventionalphosphorothioate oligonucleotide (91), methylphosphonate—modified CpG oligonucleotide(255),(256),inverted CpGoligonucleotide(257).and 5—methylC CpG oligonucleotideFigure 3 shows results of analysis of platelet counts(Panel A), ALT levels (Panel B), and AST levels (Panel C)of Fisher rats intraperitoneally administered saline,inverted(3),conventional phosphorothioate oligonucleotide (1),(2),5—methylC CpG oligonucleotide (4),CpG oligonucleotide inverted CpG oligonucleotidemethylphosphonate-modified CpG oligonucleotide(5), 2'—O—substituted CpGoligonucleotide (6).203035CA 02265917 1999-03-10W0 98/1 1211 PCT/US97/16017DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTSThe invention relates to modified oligonucleotides thatare useful for studies of gene expression and for theAll US Patents,publications and scientific literature cited in thisspecification evidence the level of knowledge in this fieldantisense therapeutic approach. patentand are hereby incorporated by reference.The invention provides modified oligonucleotides thatinhibit gene expression and that produce fewer side effectsthan conventional phosphorothioate oligonucleotides. Inparticular, the invention provides modified CpG—containingoligonucleotides that result in reduced splenomegaly andplatelet depletion when administered to a mammal, relativeto conventional CpG~containing phosphorothioateoligonucleotides. The invention further provides methodsfor using such oligonucleotides to modulate gene expressionin vivo, including such use for therapeutic treatment ofdiseases caused by aberrant gene expression.In a first aspect, the invention provides modified CpG-containing phosphorothioate oligonucleotides andcompositions of matter for inhibiting specific geneexpression with reduced side effects. Such inhibition ofgene expression can be used as an alternative to mutantanalysis for determining the biological function ofspecific genes in cell or animal models. Such inhibitionof gene expression can also be used to therapeuticallytreat diseases that are caused by expression of the genesof a virus or a pathogen, or by the inappropriateexpression of cellular genes.In one preferred embodiment according to this aspect ofthe invention, the composition of matter comprisesphosphorothioate oligonucleotides having one or moremodified CpG dinucleoside. The CpG dinucleoside is 101520253035CA 02265917 1999-03-10W0 98/1 12 1 1 PCT/U S97/ 1601785'—CpG—3', in the 5' to 3'covalently linked to a G nucleoside through ani.e., direction, a C nucleosideinternucleoside linkage. For purposes of the invention,CpG dinucleoside is considered to be "unmodified" if theinternucleoside linkage is a racemic phosphorothioatelinkage and the 5-position of the C nucleoside is occupiedby a hydrogen atom. In certain particularly preferredembodiments, all CpG dinucleosides present in theoligonucleotide are modified. For purposes of the"modified"altered from the unmodified CpG dinucleoside such that itinvention, a CpG dinucleoside is if it isconfers upon the oligonucleotide a reduced ability to causesplenomegaly and platelet depletion when administered to amammal, relative to an otherwise identical oligonucleotidehaving an unmodified phosphorothioate CpG dinucleoside. Acomposition of matter for inhibiting specific geneexpression with reduced side effects, according to thisaspect of the invention, comprises a modified CpG-containing phosphorothioate oligonucleotide that iscomplementary to a portion of a genomic region or gene forwhich inhibition of expression is desired, or to RNAtranscribed from such a gene. For purposes of theinvention, the term oligonucleotide includes polymers ofor 2'-O-two or more deoxyribonucleotide, ribonucleotide,substituted ribonucleotide monomers, or any combinationthereof. The term oligonucleotide also encompasses suchpolymers having chemically modified bases or sugars and/ orhaving additional substituents, including withoutlimitation lipophilic groups, intercalating agents,diamines and adamantane. For purposes of the invention,the term "phosphorothioate oligonucleotide" means anoligonucleotide containing at least one phosphorothioateinternucleoside linkage, preferably from about 20% to about100% phosphorothioate internucleoside linkages, and mostpreferably from about 50% to about 100% phosphorothioatesuchinternucleoside linkages. Preferably,oligonucleotides will have from about 12 to about 50102030CA 02265917 1999-03-10W0 98/1 1211 PCT/US97/160179nucleotides, most preferably from about 17 to about 35nucleotides. For purposes of the invention the term“2'—O—substituted" means substitution of the 2' position ofthe pentose moiety with an —O—lower alkyl group containing1-6 saturated or unsaturated carbon atoms, or with an -0-aryl or allyl group having 2-6 carbon atoms, wherein suchalkyl, aryl or allyl group may be unsubstituted or may besubstituted, e.g., with halo, hydroxy, trifluoromethyl,cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl,or amino groups; or with a hydroxy, an amino or a halogroup, but not with a 2'—H group. The term "complementary"means having the ability to hybridize to a genomic region,a gene, or an RNA transcript thereof under physiologicalconditions. Such hybridization is ordinarily the result ofbase—specific hydrogen bonding between complementarystrands, preferably to form Watson-Crick or Hoogsteen basepairs,although other modes of hydrogen bonding, as well asbase stacking can also lead to hybridization. As apractical matter, such hybridization can be inferred fromthe observation of specific gene expression inhibition.The gene sequence or RNA transcript sequence to which themodified oligonucleotide sequence is complementary willdepend upon the biological effect that is sought to bemodified. or RNAIn some cases, the genomic region, gene,transcript thereof may be from a virus. Preferred virusesinclude, without limitation, human immunodeficiency virus(tYPerespiratory(type 1 or 2), influenza virus, herpes simplex virus1 or 2),syncytial virus,Epstein—Barr virus, cytomegalovirus,influenza virus, hepatitis B virus,hepatitis C virus and papilloma virus. theor RNA transcript thereof may be fromIn other cases,genomic region, gene,endogenous mammalian(including human) chromosomal DNA.Preferred examples of such genomic regions, genes or RNAtranscripts thereof include, without limitation, sequences(VEGF), betaamyloid, DNA methyltransferase, protein kinase A, ApoE4encoding vascular endothelial growth factorprotein, p-glycoprotein, c—MYC protein, BCL—2 protein and101520253035CA 02265917 1999-03-10W0 98/11211 PCT/US97Il601710CAPL. or RNAtranscript thereof may be from a eukaryotic or prokaryoticIn yet other cases, the genomic region, gene,pathogen including, without limitation, Plasmodiumfalciparum, Plasmodium malarie, Plasmodium ovale,Schistosoma spp., and Mycobacterium tuberculosis.In addition to the modified oligonucleotide according tothe invention, the composition of matter for inhibitinggene expression with reduced side effects may optionallycontain any of the well known pharmaceutically acceptablecarriers or diluents. This composition of matter mayfurther contain one or more additional oligonucleotidesthiscomposition may contain one or more traditional antisenseaccording to the invention. Alternatively,oligonucleotide, such as an oligonucleotidephosphorothioate, a hybrid oligonucleotide, or a chimericoligonucleotide, or it may contain any otherpharmacologically active agent.In one preferred embodiment according to this aspect ofthe invention, the modified CpG dinucleotide is selectedfrom alkylphosphonate CpG,CpG, 2'—O—substituted CpG,CpG, phosphotriester CpG, phosphoramidate CpG and 2'—5'CpG.An alkylphosphonate CpG is a CpG dinucleoside in whichinverted CpG, 5—methylcytosinestereospecific phosphorothioatethe C nucleoside and the G nucleoside are covalently linkedto each other through an alkylphosphonate internucleosidelinkage. Alkylphosphonate CpG-containing oligonucleotidesare conveniently prepared by using any conventional solidphase synthesis protocol to produce the CpG—containingoligonucleotide, except that the alkylphosphonate CpGdinucleoside is prepared using any standard procedure forintroducing alkylphosphonate internucleoside linkages intooligonucleotides. One particularly preferred procedure forthis step is described in Iyer et al., Bioorganic and1393-1398 (1996).the alkyl moiety of the alkylphosphonateMedicinal Chemistry Letters 6:Preferably,linkage is a lower alkyl moiety of l~6 carbon atoms, which203035CA 02265917 1999-03-10W0 98/11211 PCTIUS97/1601711may optionally be unsaturated and/or substituted.preferably,CpG.An inverted CpG is a 5'—GpC-3'Mostthe alkylphosphonate CpG is a methylphosphonatedinucleoside. InvertedCpG-containing oligonucleotides are conveniently preparedby using any conventional solid phase synthesis protocol toproduce the oligonucleotide, except that a G monomersynthon is used in place of the C monomer synthon and visa-versa.A 5-methylC CpG is a CpG dinucleoside in which the Cnucleoside is methylated at the 5 position of the cytosinebase. 5-methylC CpG-containing oligonucleotides are‘conveniently prepared by using any conventional solid phasesynthesis protocol to produce the oligonucleotide, exceptthat a 5-methylC monomer synthon is used in place of the Cmonomer synthon.A 2'—O—substituted CpG is a CpG dinucleoside in whichthe 2‘ position of the pentose moiety is substituted,having an -O—lower alkyl group containing 1-6 saturated orunsaturated carbon atoms, or an —O—aryl or allyl grouphaving 2-6 carbon atoms, wherein such alkyl, aryl or allylgroup may be unsubstituted or may be substituted, e.g.,with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl,acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; orwith a hydroxy, but not with athe 2'—O—substituted CpG is a2'—O—methyl cytosine containing CpG,an amino or a halo group,2'—H group. Most preferably,or a 2'—O—methyl2'—O—substituted CpG-containing oligonucleotides are conveniently prepared byguanosine containing CpG or both.using any conventional solid phase synthesis protocol toproduce the oligonucleotide, except that a 2'—O—substitutedmonomer synthon is used in place of the monomer synthon.A phosphotriester CpG is a CpG dinucleoside in which theC nucleoside and the G nucleoside are covalently linked toeach other through a phosphotriester internucleosidelinkage. Phosphotriester CpG-containing oligonucleotidesare conveniently prepared by using any conventional solid.... ...........«.......-.ua...u...................<.»../..« ~. ~ - 1020253035CA 02265917 1999-03-10W0 98/ 1 1211 PCT/US97/1601712phase synthesis protocol to produce the CpG—containingoligonucleotide, except that the phosphotriester CpGdinucleoside is prepared using any standard procedure forintroducing phosphotriester internucleoside linkages intooligonucleotides. One particularly preferred procedure forthis step is described in Iyer et al.,;1: 1539-1542 (1996).linkage is a methylphosphotriester linkage.A phosphoramidate CpG is a CpG dinucleoside in which theC nucleoside and the G nucleoside are covalently linked toeach other through a phosphoramidate internucleosideTetrahedron LettersPreferably, the phosphotriesterlinkage. Phosphoramidate CpG—containing oligonucleotidesare conveniently prepared by using any conventional solidphase synthesis protocol to produce the CpG—containingoligonucleotide, except that the phosphoramidate CpGdinucleoside is prepared using any standard procedure forintroducing phosphoramidate internucleoside linkages intooligonucleotides. One particularly preferred procedure forthis step is described in Iyer et al., Tetrahedron Letters11: 1539-1542 (1996).internucleoside linkage is a primary phosphoramidateMost preferably, the phosphoramidateinternucleoside linkage.A stereospecific phosphorothioate CpG is a CpGdinucleoside in which the C nucleoside and the G nucleosideare covalently linked to each other through astereospecific phosphorothioate internucleoside linkage.stereospecific phosphorothioate CpG—containingoligonucleotides are conveniently prepared by using anyconventional solid phase synthesis protocol to produce theCpG—containing oligonucleotide, except that thephosphoramidate CpG dinucleoside is prepared using aprocedure for introducing stereospecific phosphorothioateinternucleoside linkages into oligonucleotides, preferablyas described in Iyer et al., Tetrahedron Asymmetry Q: 1051-1054 (1995).A 2'—5' CpG is a CpG dinucleoside in which the Cnucleoside and the G nucleoside are covalently linked to2030CA 02265917 1999-03-10W0 98/11211 PCT/US97/1601713each other through a 2'-5‘ internucleoside linkage. Theinternucleoside linkage may be of any type, and ispreferably a phosphorothioate or phosphodiester linkage.2‘-5' CpG—containing oligonucleotides are convenientlyprepared by using any conventional solid phase synthesisprotocol to produce the CpG—containing oligonucleotide,except that the 2‘-5' CpG dinucleoside is prepared using aprocedure for introducing stereospecific phosphorothioateinternucleoside linkages into oligonucleotides, for exampleJ. Am. Chem. Soc. ;;g:or Hashimoto and Switzer,as described in Dougherty et al.,6254 (1992)Other modifications of the CpG dinucleoside includesubstitution of the phosphorothioate internucleosidelinkage with any other internucleoside linkage, includingwithout limitation phosphorodithioate,alkylphosphonothioate, siloxane, carbonate,carboxymethylester, acetamidate, carbamate, thioether,amide (PNA), bridged phosphoramidate, bridged methylenephosphonate, bridged phosphorothioate and sulfoneinternucleotide linkages.In certain preferred embodiments of compositionsthe"chimeric" oraccording to this aspect of the invention,oligonucleotides will be configured as"hybrid" oligonucleotides, for example as described5,149,797 and PCT publicationchimeric oligonucleotides containrespectively in US Patent No.no. W094/02498.oligonucleotide regions having ionic internucleosideBriefly,linkages as well as oligonucleotide regions having nonionicinternucleoside linkages. Hybrid oligonucleotides haveoligonucleotide regions containing DNA as well asoligonucleotide regions containing RNA or 2'—O-substitutedRNA.elements of these preferred embodiments can be combined andThose skilled in the art will recognize that thethe inventor does contemplate such combination. Forexample, 2'—O—substituted ribonucleotide regions may wellinclude from one to all nonionic internucleoside linkages.Alternatively, nonionic regions may have from one to all.......... .....«-...~..u............w.....w...... . .10202530CA 02265917 1999-03-10W0 98/ 1 1211 PCT/US97/16017142'-O—substituted ribonucleotides. Moreover,oligonucleotides according to the invention may contain 2'-O—substituted or nonionic regions in the core region of theoligonucleotide flanked by phosphorothioate—containing DNAregions, or visa-versa, and further may containcombinations of one or more 2'—O—substituted ribonucleotideeither or bothbeing flanked by phosphorothioate regions.& Nucleotides 14; 1031-1035 (1995)region and one or more nonionic region,(See Nucleosidesfor relevant synthetictechniques).In a second aspect, the invention provides a method formodulating gene expression in a mammal with reduced sideeffects. In the method according to this aspect of theinvention, a composition of matter according to the firstaspect of the invention is administered to the mammal,wherein the oligonucleotide is complementary to a gene thatsuchis being expressed in the mammal. Preferably,administration may be parenteral, oral, intranasal orintrarectal. Preferably, a total dosage of oligonucleotidewill range from about 0.1 mg oligonucleotide per kg bodyweight per day to about 200 mg oligonucleotide per kg bodyafter theweight per day. In a preferred embodiment,composition of matter is administered, the biologicaleffects of splenomegaly and platelet depletion are reduced,relative to the same effects obtained upon administrationof an otherwise identical composition containing the samequantity of an otherwise identical oligonucleotide, exceptthat such oligonucleotide contains an unmodified CpGdinucleoside in place of the modified CpG dinucleoside.This preferred biological effect can be monitored bymeasuring blood levels of platelets before and afteroligonucleotide administration. Preferably, platelets willbe depleted by less than about 20%,than about 10%.most preferably by lessThe biological effect may also be observedby measuring serum alanine aminotransferase (ALT) and serumaspartate aminotransferase (AST) levels following10202530CA 02265917 1999-03-10W0 98/ 1 1211 PCT/US97l160l7I5oligonucleotide administration. Preferably, serum ALT andAST levels will increase by less than 2.5 fold, mostpreferably by less than 2.0 fold.In a third aspect, the invention provides a method fortherapeutically treating, with reduced side effects, adisease caused by aberrant gene expression, the methodcomprising administering to an individual having thedisease a composition of matter according to the firstaspect of the invention, wherein the oligonucleotide iscomplementary to a gene that is aberrantly expressed,wherein such aberrant expression causes the disease. Thus,this is a preferred example of a method for modulating geneexpression in a mammal, as discussed above for the secondaspect of the invention. In this context, aberrant geneexpression means expression in a host organism of a generequired for the propagation of a virus or a prokaryotic oreukaryotic pathogen, or inappropriate expression of a hostcellular gene. Inappropriate host cellular gene expressionincludes expression of a mutant allele of a cellular gene,or underexpression or overexpression of a normal allele ofa cellular gene, such that disease results from suchinappropriate host cellular gene expression. Preferably,such administration should be parenteral, oral, sublingual,transdermal, topical, intranasal or intrarectal.Administration of the therapeutic compositions can becarried out using known procedures at dosages and forperiods of time effective to reduce symptoms or surrogatemarkers of the disease. When administered systemically,the therapeutic composition is preferably administered at asufficient dosage to attain a blood level ofoligonucleotide from about 0.01 micromolar to about 10micromolar. For localized administration, much lowerconcentrations than this may be effective, and much highera totaldosage of oligonucleotide will range from about 0.1 mgconcentrations may be tolerated. Preferably,oligonucleotide per patient per day to about 200 mg1020CA 02265917 1999-03-10W0 98/11211 PCT/US97/1601716oligonucleotide per kg body weight per day. It maydesirable to administer simultaneously, or sequentially atherapeutically effective amount of one or more of thetherapeutic compositions of the invention to an individualas a single treatment episode. In a preferredembodiment, after the composition of matter isadministered, the biological effects of splenomegaly,platelet depletion, are reduced, relative to the sameeffects obtained upon administration of an otherwiseidentical composition containing the same quantity of anotherwise identical oligonucleotide, except that sucholigonucleotide contains an unmodified CpG dinucleoside inplace of the modified CpG dinucleoside. This preferredbiological effect can be monitored by measuring bloodlevels of platelets before and after oligonucleotideplatelets will be depleted bymost preferably by less than aboutadministration. Preferably,less than about 20%,10%.by measuring serum alanine aminotransferase(AST)oligonucleotide administration. Preferably,The preferred biological effect may also be observed(ALT) and serumaspartate aminotransferase levels followingserum ALT andAST levels will increase by less than 2.5 fold, mostpreferably by less than 2.0 fold.1020253035CA 02265917 1999-03-10W0 98/11211 PCT/US97/1601717The following examples are intended to furtherillustrate certain preferred embodiments of the inventionand are not intended to limit the scope of the invention.Example 1Synthesis, Deprotection And Purification OfoligonucleotidesOligonucleotide phosphorothioates were synthesized using(Model 8700,using a beta—cyanoethy1 phosphoramiditean automated DNA synthesizerBedford, MA)approach on a 10 micromole scale.Biosearch,To generate thephosphorothioate linkages, the intermediate phosphitelinkage obtained after each coupling was oxidized using 3H,1,2—benzodithiole—3H-one—1,1—dioxide (See Beaucage, InProtocols for oligonucleotides and Analogs: Synthesis andProperties, Agrawal33-62 (1993).)generate phosphodiester linkages,(editor), Humana Press, Totowa, NJ, pp.Similar synthesis was carried out toexcept that a standardoxidation was carried out using standard iodine reagent.Synthesis of methylphosphonate CpG—containingOligonucleotide was carried out in the same manner, exceptthat methylphosphonate linkages were assembled usingnucleoside methylphosphonamiditeVA).tetrahydrofuran/2,6—lutidine/waterAgrawal & Goodchild, Tet. Lett. 28:(Glen Research, Sterling,followed by oxidation with 0.1 M iodine in(75:25:0.2S) (see3539-3542 (1987).Deprotection and purification of oligonucleotides wascarried out according to standard procedures,& Dev. 4:(Seel85—l99except for oligonucleotides containingPadmapriya et al., Antisense Res.(1994)).methylphosphonate—containing regions. For thoseoligonucleotides, the CPG—bound oligonucleotide was treatedwith concentrated ammonium hydroxide for 1 hour at roomtemperature, and the supernatant was removed and evaporatedto obtain a pale yellow residue, which was then treated101520253035CA 02265917 1999-03-10W0 98/ 1 1211 PCT/US97/1601718with a mixture of ethylenediamine/ethanol (lzl v/v) for 6hours at room temperature and dried again under reducedpressure.Examle 2Reduced In Vivo Splenomegaly Using ModifiedQp§—Containing OligonucleotidesCD-1 mice and Fischer rats (Charles River Laboratories,Raleigh, NC) were injected intravenously daily for sevendays with a dose ranging from 3-30 mg/kg body weight ofCpG—containing phosphorothioate oligonucleotide,methylphosphonate CpG—containing phosphorothioateoligonucleotide, inverted CpG-containing phosphorothioateoligonucleotide, 5—methylC CpG—containing phosphorothioateoligonucleotide, 2‘—O—substituded CpG, or saline as acontrol. On day 8, the animals were euthanized and thespleens were removed and weighed. Animals treated withmethylphosphonate CpG—containing phosphorothioateoligonucleotide, inverted CpG—containing phosphorothioateoligonucleotide, 5—methylC CpG—containing phosphorothioateoligonucleotide, or 2—O—substituded CpG, showedsignificantly less increase in spleen weight than thosetreated with CpG—containing oligonucleotidephosphorothioates. Similar results are expected to beobserved for phosphotriester CpG—containingphosphorothioate oligonucleotides, phosphoramidate CpG-and 2--5‘CpG—containing phosphorothioate oligonucleotides.containing phosphorothioate oligonucleotidesExamle 3Reduced In Vivo Platelet Depletion Using ModifiedQQG—Containing OligonucleotidesCD-1 mice and Fischer rats were injected intravenouslydaily for seven days with a dose ranging from 3-30 mg/kgbody weight of CpG—containing phosphorothioate10203035CA 02265917 1999-03-10W0 98/1 121 1 PCT/U S97/ 1601719oligonucleotide, methylphosphonate CpG-containingphosphorothioate oligonucleotide, inverted CpG-containingphosphorothioate oligonucleotide, 5—methylC CpG-containingphosphorothioate oligonucleotide, 2'—O~substituted CpG, orsaline as a control. At day 8, blood was taken from theanimals and platelet counts were taken. Animals treatedwith methylphosphonate CpG-containing phosphorothioateoligonucleotide, inverted CpG-containing phosphorothioateoligonucleotide, or 5—methylC CpG-containingphosphorothioate oligonucleotide showed significantly lessdepletion of platelets than those treated with CpG—containing oligonucleotide phosphorothioates. Similarresults are expected to be observed for phosphotriesterCpG-containing phosphorothioate oligonucleotides,phosphoramidate CpG-containing phosphorothioateoligonucleotides and 2'—5' CpG-containing phosphorothioateoligonucleotides.Examle 4Reduced In Vivo Increase in Serum ALT and AST Levels UsingModified gQ§—Containing OligonucleotidesCD-1 mice and Fischer rats were injected intravenouslydaily for seven days with a dose ranging from 3-30 mg/kgbody weight of CpG-containing phosphorothioateoligonucleotide, methylphosphonate CpG—containingphosphorothioate oligonucleotide, inverted CpG-containingphosphorothioate oligonucleotide, 5—methylC CpG-containingphosphorothioate oligonucleotide, 2‘—O-substituted CpG, orAt day 8, blood was taken from theanimals and serum ALT and AST levels were measured using asaline as a control.Roche Cobas Fara Chemistry Analyzer (Roche DiagnosticSystems, Branchburg, NJ). Animals treated withmethylphosphonate CpG—containing phosphorothioateoligonucleotide, inverted CpG-containing phosphorothioateoligonucleotide, 5—methylC CpG-containing phosphorothioateoligonucleotide, or 2'-O—substituted CpG-containing.,,..._.._., >A’.4I , ,.t.,,...,m..«..,...n..,~mM........«...........‘....»a..~.,....,.. , V§0Vn‘|‘¢4IVI1*lI~<5Ck‘4“UIUk‘7>vA4>»‘>I~r\‘- .,,.. 4171*!“ 10CA 02265917 1999-03-10W0 98/11211 PCT/US97/1601720 .phosphorothioate oligonucleotide, showed a significantreduction in the increase of serum ALT and AST levels ascompared those treated with CpG—containing oligonucleotidephosphorothioates. Similar results are expected to beobserved for phosphotriester CpG—containingphosphorothioate oligonucleotides, phosphoramidate CpG-and 2'—5'CpG—containing phosphorothioate oligonucleotides.containing phosphorothioate oligonucleotides

Claims (6)

THE EMBODIMENTS OF THE INVENTION FOR WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A modified CpG-containing phosphorothioate oligonucleotide for inhibiting specific gene expression with reduced side effects relative to an unmodified CpG-containing oligonucleotide, said oligonucleotide being complementary to a portion of a genomic region or gene for which inhibition of expression is desired, or to RNA
transcribed from such a gene, wherein said modified CpG is alkylphosphonate CpG, inverted CpG, 2'-O-substituted CpG, stereospecific phosphorothioate CpG, phosphotriester CpG, phosphoramidate CpG or 2'-5' CpG, and wherein said reduced side effects comprise reduced splenomegaly and reduced platelet depletion.

2. Use of an effective amount of the oligonucleotide according to claim 1 for modulation of gene expression with reduced side effects relative to an unmodified CpG-containing oligonucleotide in a mammal, wherein the oligonucleotide is complementary to a gene that is being expressed in the mammal, and wherein said reduced side effects comprise reduced splenomegaly and reduced platelet depletion.

3. Use of the oligonucleotide according to claim 1 in the manufacture of a medicament for modulation of gene expression with reduced side effects relative to an unmodified CpG-containing oligonucleotide in a mammal, wherein the oligonucleotide is complementary to a gene that is being expressed in the mammal, and wherein said reduced side effects comprise reduced splenomegaly and reduced platelet depletion.

4. Use of a therapeutically effective amount of the oligonucleotide according to claim 1 for treatment, with reduced side effects relative to an unmodified CpG-containing oligonucleotide, of a disease caused by aberrant gene expression, wherein the oligonucleotide is complementary to a gene that is aberrantly expressed, wherein such aberrant expression causes the disease, and wherein the reduced side effects comprise reduced splenomegaly and reduced platelet depletion.

5. Use of the oligonucleotide according to claim 1 in the manufacture of a medicament for treatment, with reduced side effects relative to an unmodified CpG-containing oligonucleotide, of a disease caused by aberrant gene expression, wherein the oligonucleotide is complementary to a gene that is aberrantly expressed, wherein such aberrant expression causes the disease, and wherein the reduced side effects comprise reduced splenomegaly and reduced platelet depletion.

6. A composition comprising the oligonucleotide of claim 1 and a pharmaceutically acceptable carrier.