CA2102804A1 - 3'-end blocked oligonucleotides - Google Patents

3'-end blocked oligonucleotides


Publication number
CA2102804A1 CA 2102804 CA2102804A CA2102804A1 CA 2102804 A1 CA2102804 A1 CA 2102804A1 CA 2102804 CA2102804 CA 2102804 CA 2102804 A CA2102804 A CA 2102804A CA 2102804 A1 CA2102804 A1 CA 2102804A1
Prior art keywords
therapeutic composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Application number
CA 2102804
Other languages
French (fr)
Sudhir Agrawal
Jamal Temsamani
Jin-Yan Tang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Idera Pharmaceuticals Inc
Original Assignee
Sudhir Agrawal
Jamal Temsamani
Jin-Yan Tang
Hybridon, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US69856891A priority Critical
Priority to US698,568 priority
Application filed by Sudhir Agrawal, Jamal Temsamani, Jin-Yan Tang, Hybridon, Inc. filed Critical Sudhir Agrawal
Publication of CA2102804A1 publication Critical patent/CA2102804A1/en
Application status is Abandoned legal-status Critical



    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids



The invention provides oligonucleotides having 3'-ends blocked by cap structures and having one or more artificial internucleoside linkage. Such oligonucleotides are resistant to in vivo degradation and extension, and thus have superior half-life in vivo.


WO 92/21K9 / 2 1 0 2 8 0 9 P~f/US92/038b7 3'-E~D BLQCKljD Ql,IGQNUÇLEOTIDES

Field of th~ Invention The invention relates to antisense oligonucleotide therapy. More particularly, the invention relates to the production of oli8onucleotides suitable for in viVO therapeutic use, and to the use of such oligonucleotides intherapeutic treatment of human disease.
Summarv of the Related Art The use of an ansisense oligonucleotide approach for the treatmcnt of human disease is a promising developmcnt in the fields of medicine relased to antiviral therapy and therapy for genetic disorders.
In the last several years, it has been demonstrated that oligonucleotides are capable of iDhibiting the replication of certain viruses in tissue culture systems.
Zamecnik and Stephenson, Proc. Natl. Acad. Sci. U.S.A. 75: 280-284 (1978), first showed oligonucleotide-mediated inhibition of virus replication in tissue culture, using Rous Sarcoma Virus.
Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A. 83: 4143-4146 (1986~, demonstrates inhibition in tissue culture of the HTLV-III ~drus (now called HIV-I) associated with AIDS.
More receotly, it has been shown that modified oligonucleotides, having altered internucleoside linkages, proYide greater efficacy in viruS
inhibition-in in v~rrotissue culture systems.
Agrawal es al., Proc. Natl. Acad. Sci. U.S.A. 85: 7079-7083~(1988), teaches inhibition in tissue culture of HIV-I with increased efficacy, using oligonucleoside phosphoramidates and phosphorothioates.
Sarin et al., Proc. Natl. Acad. Sci. U.S.A.85: 7448~7451 (1988), teaches ;nhibitioo in tissue culture of HIV-I with increased efficacy, using oligonucleoside methylphosphonates.
Agrawal et al., Proc. Natl. Acad. Sci. U.S.A. ~i: 7790-7794 (198 teaches nucieotide sequence specific inhibition of HIV-I in both early~
infected and chronically infecsed cell cultures, using oligonucleotide phosphorothioates.
Leiter et al., Proe. Natl. Acad. Sci. U.S.A. ~ Z: 3430-3434 ( I 990), teaches inhibition in tissue culture of influenza ~rirus replicasion by oligonucleolide phosphorothioates.

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WO 92/20~972 ~ ~ 2 ~ CI/US92/03867 : '~

ln addition, oligonucleotjdes have been used to modulate normal cellular processes, su8gesting a potential use in the treatm~nt of genetic disorders.
Goodchild et al., Arch. Bioclhem. Biophys. 264: 401-409 (1988), teaches 5inhibition of rabbit ~-globin synthesis by oligonucleotides in a cell free systsm.
Temsamani et al., Journal of' Bjological Chemistry (USA) 266: 468-472 (1991), teaches inhibition of spliccosome assembly by oligonucleoside methylphosphonates.
10The inhibition of viruseS and modulation of normal cellular processes indicates some promise for the use of an antisense oligonucleotide approach for the treatment of viral disease and 8enetic disorders. However, antisense oligonucleotide therapy depends upon the in vivo specificity and efficacy of oligonucleotides, both of which aro related to oligonucleotide length, base 15composition and hybridization propertics. Thus, if oligonucleotides are rapidly de~raded in Yivo to produce shortcr degradation products, decreased efficacy may result, and the loss of specificity could lead to toxic side effects. Consequently, concerns arise about developing oligonucleotides that are resistant to degradation in vivo. Oligonucleotides having modified 20internucleoside linkages have been used by the previously-cited investigators toward this end.
Agrawal and Sarin, Advanced Drug Del;very Revicws, Elsevier Press, in press (1990), teaches that unmodified oligonucleotides are poor inhibitors of virus replication relstive to modified (resistant) oligonucleotides~
25Shaw et al., Nucleic Acids Res. 19: 747-750 tl991), teaches that otherwise unmodified oligonucleotides become more resistant to nucleases in vi~ro when they are blocked at the 3' end by certain capping structures and that une~apped oligonucleoside phosphorothioates also are not de8raded in vitro.
30Unfortunately, virtually nothing is known about the stability or biodistribution of modified or unmodified oligonucleotides in ViYo, which is parlicularly relevant if oligonucleotides are to be used for human therapy.
Since in Yitro models cannot be made to predict the stability and bioavailability of oligonucleotides in vivo, there is a need for systems that can 35directly provide such information. Moreover, if such in vivo data shows that existing oligonucleotides are not sufficiently stable, there will be a need for oligonucleotides that can resist the degradative influences of intrinsic ,, .
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. 3 enzymatic activities in vil~o. Ideally, structural motifs associated with increascd stability in vi~o should be identified, and in vivo systems should bc developed which allow simplc and convcnient comparisons for the optimization of in viYo stability.

Figute 1. Structurc of oligonucleotides uscd in ~xamples 1-7.
Figure 2. E~amples of 3'-hydroxyl cap structures useful in forming thc oligonucleotides of the present invention. R ~ an organic ~roup, _&.
alkyl, aryl, cyclic, cholesteryl, etc.; X ~ O, S, Sc or NHR; Y ~ O, S, Se or NHR;
Z ~ (;1, S, or NH; and B - purinc or pyrimidine base.
Figure 3. Comparative stability of uncapped, 5'-capped, 3~-capped and 3',~'-cappcd oligonucleosidc phosphorothioate in monkey serum.
Oligonucleotidcs were incubated in monkey serum at 37C and at time points (shown above the lanes in hour), aliquots wcrc withdrawn, extracted and analyzed on gel electrophoresis. In larle 6 hr. from ~'-capped, most of the incubation mi:cture was lost during handlin~.
Figure 4. Stability of oligonucleoside phosphorothioate in urine, collected from the mice rcceiving 30 mg/kg of oli8onucleotide intravenously. ~- ;
The urine was collected up to 24 hour post-dosin8~ e~tracted and analyzed by - - ` `
gel electrophoresis. The lanes under control are oligonucleotides before administration and lanes under urine (0-24 hours) are oligonucleotides ; ~ ~ `
recovered from urinc. Diffused band of 3'.-cappcd oligonucleotidc is due to the lo v specific activity ~Table 1).
Figure 5. Status of the oligonucleoside phosphorothioate ill mice kidncy after 24 hour post-dosing intravenously. The lanes shown control are oligonucleoside before administration and lanes under kidney (24 hours) are the oligonucleoside e~ctracted from kidney. `~ p Figure 6. Gel electrophoresis of oligonucleoside phosphorothioate e~tracted from the liver after 24 hours post-dosing in mice~ intravenously.
The lanes under control are oligonucleoside phosphorothioate before administration and lancs under liver (~4 hours) arc oligonucleoside ~ -phosphorothioate e~tracted from liver. ~ ~-`; ~ ~'-: '`
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~'WO g2/2~697 PC~ r US92/03867 ~RIEE SUMMARY OF TIIE I~VENTIQN
The invontion relates to oligonucleotides that are useful in antisense oli~onucleotide-based therapeutic approaches. More particularly the invention relates to oligonucleotides that have sufficient specificity and efficacy in Vil~O to be useful in therapeutic treatment of human discase. Thc invention provides oligonucleotides that possess grcater in vivo specif icity a nd efficacy than oligonucleotides kno-vn in thc art. The 8reater specificity and efficacy of oligonucleotides according to the invention arises from thcir inherent resistance to exonuclcolytic digestion by intrinsic nucleases. This resistance is thc product of two ssructural ~catures of oligonucleotides according to the invention: (I) the presence of one or more artificial internucleoside linkage and (2) the presencc of a particular cap structure at the 3' end of the molecule.
For the first time, the invention provides in vivo pharmacokinetic information about modified and unmodified oligonucleotides. This information cannot be extrapolated from in vitro results, and indeed surprising differences are observed between in viYo and in vi~ro results. The invention provides such informatioo throu~h the use of:a convenient method for asssssing whether any given oligonucleotide has the resistance to nucleolytic degradation necessary to provide it with the specificity and efficacy required to use as an antisense oligonucleotide in a therapeutic approach to the treatment of human disease. This mcthod uses a mouse model to assess resistancè of a radiolabelled oligonuclcotide to il vivo nucleases by examining the status of oligonucleotides present in urine and hornogenized tissues of organs. The method also allows assessment of bioavailability of oligonucleotides by measuring thc oligonucleotide content of various organs. By using the method of the invention, one skilled in the art can readily determine whether any oligonucleotide having the structural fcatures of oligonucleotides according to the invention aiso is resistant to nucleolytic degradation in ~ivo. This method allows the skilled artisan to makc such assessments by protocol, without undue e~cperimentation.

WO 92/2069/ PCI/US')2/03867 0 2 g ~

In a first aspect the inverltion provides oligonucleotides that are resistant in vivo to both degradation and extension by intrinsic enzymatic activities. These oligonucleotides according to the invention have greater specificity and increased half-life, relative to e~isting oligonucleotides. Sucholigonucleotides are well-suised for use in th~ treatment of virus infecsions or disorders of gene e~cpression. Virus infections include infections by DNA, RNA, and retroviruses. Disorders of 8ene e~pression include inherited genetic defects and disorders resultin8 from abnormal 8ene expression, or from e~cpression of abnormal 8enes~ e ~., oncogene expression Issociated with neoplasia. .
The therapeutic approach using antisense oli~onucleotides is based on the principle that an appropriate length of oligonucleotide complementary to the tar8et can disrupt the function of the target, which could be a viral or cellular gene. The specificity of antisense oligonucleotides results from the formation of Watson-Crick base pairing between the heterocyclic bases on the oligonucleotide and complementary bases on the target nucleic acid. A
nucleotide sequence of 16 nucleotides in length will be expected to occur randomly at abous every 416 or 4~c109 nucleotides. Thus such a sequence might be e~cpected to occur only once in the human genome. In contrast, a nucleotide sequence of 10 nucleotidos in length would occur randomly at about every 410 or l~clO~ nucleotides. Thus such a sequence might be present thousands of times in the human ~enome. Consequently, oligonucleotides o~ - -greater length are more specific than oligonucleotides of Iesser length and are :
less likely to lead to any toxic complications that mi8h~ resul,t from nonspecific hybridization. In addition, longer oligonucleotides show greater inhibitory effects upon HIV in tissue culture, within certain limits (i.e.. 25~
mer ~ 20-mer > 15-mer ~ 10-mer). Thus oligonucleotide length should exceed certain limits for purposes of specificity and effectiveness.
In vivo degradation of oligonucleotides produces oligonucleotide breakdown products of reduced length. Such breakdown products are morc - ~-Iikely to engage in non-specific hybridization and are less likely to be effective, relative to their full-length counterparts. Thus, it is desirable to produce oligonucleotides that are resistant to degradation in the body.
Preferably, such oligonucleolides should be bioavailable to the various orgalls and tissues of the body as well. Oligonucleotides according to the invention - resist degradation by intrinsic nucleolytic activities and are bioavailable to . . . :~
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WO 92/~697 2 :L O 2 ~ PCI, ~JS92t03867 many organs and tissues. Thus in this aspect, the invcntion provides oligonucleotides that are well suitcd to act effectively and specifically in antisense oligonucleotide therapeutic approaches. In addition, in YiVo metabolism of oligonucleotides rcsults in e~cterision of oligonucleotides in certain tissues, including at least liver, kidney, small intcstine and large intestine. This can lead to reduced bioavailability of particular oligonucleotides and can lead to reduced specificity and to potentially mutagcnic sidc effects.
Oligonucleotides accordin2 to the invention are resistant to both in vi1~0 degradation and e~tension due to two structural features. The first feature is the presence of one or more in2crnal artificial internucleoside linkages. E~amples of such linkages that may be substituted for phosphodiester linkages include phosphorothioates, methylphosphonates, sulfone, sulfate, ketyl, phosphorodithioates, various phosphoramidates, phosphate esters, bridged phosphorothioates and bridged phosphoramidates.
Such examplcs are illustrative, rather than limiting, since other internucleoside linkages are kDown in the art. See, ~,&. Cohen, Trends in Biotechnology, ~1990). The synthesis of oligonucleotides having one or more of these substituted for phosphodiester internucleosidc linkages is well known in the art, which includes synthetic pathways for the production of oligonucleotides having mixed internucleoside linkages. The second feature of oligonucleotides according to the invention is the prescnce of a cap structure at the 3'-OH of the molccule. This cap blocks access to the 3' hydroxyl functional group, thus rendering the molecule more resistalnt to both extension 3' exonucleolytic activity, which is the primary intrinsic media~or of in vivo oligonucleotide degradation. Cap struclures according to the in~ention include N-Fmoc-O'-DMTr-3-amino-1,2-propanediol, as wcll ~s the structures shown in Figure 2. Such examples are merely illustrative, however, since rnany blocking ~roups are known in the art and those skilled in the art will recognize how to attach such groups to the 3' end of the oligonucleotide. Thus, for purposes of the invention, a cap structure is construcd to encompass any blocking group that restricts access to the 3' hydro~tyl of an oligonucleotide, thereby rendering the oligonucleotide res;stant to i~1 vivo degradation or extension. For purposes of the inven~ion.
an oligonucleotide is considered to be rendered resistant to In YiVo degradation if its in vi~o half life is longer than that of an oligonucleotide having all phosphodiester internucleoside linkages, but otherwise being oî

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iden~ical length and sequence. Preferably, the resistant oligonucleotidc will have an in vivo half life th~s is longer tha~ that of an uncapped oligonucleoside phosphorothioate of idcntical length and sequence.
In a second aspect, the invention provides a convenicnt method for assessing whcthcr any partil:ular oligonuclcotide constitutcs an oligonuoleotide according to thc invention. More particularly, thc invention provides a convenicnt method for dctermining whcthcr an oligonucleotide having one or more internal internucleosidc linkage that is not a phosphodiester linkage, as wcll as having a 3' cap structurc, is resistant to invivo degradation. The method of the inventioD also allows convenient assessment of bioavailability of such oli~onucleotides, which is preferable in certain embodiments of the oligonucleotides of the invention. Thus the invention provides a convenient method for assessing, without undue e~perimentation, whether a particular oligonuclcotide possesses ] 5 characteristics that make it desirable for use in therapeutic approaches involving antisense oligonucleotides.
In this aspect, the invention utilizcs methods known in the art for synthesis of radioactively labclled oligonucleotides having one or more internal interDucleoside linkage that is not a phosphodiester linkage, as well as having a cap structure attached to the 3' end. Such oligonucleotides are administered to mice in a physiologically acceptable carrier by either intravenous or intraperitoneal injection. After an appropriate interval, urine is collected from the treated mouse and the status of the oligonuclcotides present therein is determined by PAGE and autoradiography. Bioavailability is determined by homogenization of organs and measurement of radioaltivity therein. Finally, status of oligonucleotides in various organs is determincd by e~traction of the oligonucleotides from the homogenized organ tissues, followed by analysis using PAGE and autoradiography.
The invention further provides an even simpler assay which provides some preliminary information aboùt oligonucleotide stability. This assay involves incubation of the oligonucleotides in the presence of monkey serum, followed by e~traction of the oligonucleotides and analysis of degradation using PAGE and autoradiography.
The following e~camplcs are provided to further illustrate aspcc~s of the invention and are not limiting in nature.

2~ 02~
WO 92/~06g7 PCI, ~IS92/03867 ~ a~iD!e I " ,~
Svnthcsis o~ Oli~Qnu~l~Qside PhosohQrothio~
Oligonucleoside phosphorothioates were synthesized on a Model 8700 au~iomaled synthesizer (Mill;gen-Biosearch, Burlington, MA) using H-phosphonate chemistry on controlled pore glass (CPG), followed by oxidation ` ~ :
with 0.2~ sulfur in carbon disulfide/pyridinc/triethylaminc (9:9:1, v/v~
Synthesis was carried out on a 5~clO micromolar scale. Oligonucleosidc phosphorothioates were purified by low prcssure ion e~cchange chromatography (DEAE-cellulose, DE-50 Whatruan), followed by reverse phase chromatography (C~8) and dialysis. A detailed description of the H-phosphonate approach to synthesizing oligonuclcosidc phosphorothioates is given in Agrawal and Tan8~ Tetrahedron Letters ~: 7541-7544 (1990). In ~ d addition, synthesis of oligonucleoside mcthylphosphonates, phosphorodithioatcs, phosphoramidases, phosphatc cstcrs, bridged ~ -phosphoramidates and bridged phosphorothioatcs is known in the art. See ~ ~ `
e.R.. Agrawal and Goodchild, l ctrahedron Lcttcrs 2&: 3539 (1987); Nielsen ct al., Tctrahedron Letters 29: 2911 (1988); ~a8er et al., Biochemistry 27: 7237 (1988j;Uznanskietal.,TetrahedronLetters28:3401 (1987);1~annwarth,HclY, Chim. Acta 71:1517 (1988); Crosstick and Vyle, Tetrahedron Letters 30: 4693 ~ -(1989); Agrawal et al., Proc. Natl. Acad. Sci. USA 87: 1401-1405 (1990).
E~amDle 2 SYnthesi~ of ~a~Rçd Oli~onPçleoside Phosr~horothioates 5'-capped oligonucleosido phosphorothiostes were prepared by carrying out thc last coupling, after the assembly of the required scqu~ence, with N-Fmoc-O'-DMTr-3-amino-1,2-propanediol-H-phosphonatc. The S'-capped oli~onucleosidc H-phosphonate was thcn o~tidizcd with sulfur. 3'-capped oligonucleosidc phosphorothioates were assembled on N-Fmoc-O'-DMTr-3-amino-1,2-propanediol-CPG, followed by sulfur oxidation.
Combina~ion of thcse procedures was uscd to produce 3',5'-capped oligonucleoside phosphorothioates. See Figure 1.
Alternativcly, oligonucleosidc phosphorothioates having other 3' or 5' cap structurcs, (scc, ~.L Fi~urc 2), are prepared by substituting the phosphonate or CPG-derivatized cap struclurcs for the N-Fmoc-O'-DMTr-3-arnino-l,2-propanediol-H phosphonate or CPG in the capping procedure.
Similarly, capped, modified oligonuclcotidcs other than oligonuclcosidc phosphorothioates arc prcparcd in an analogous manncr by appcnding ~he capping procedure to the appropriate synthetic procedure.

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~. WO92/20~. 2 ~ ~/US92/03867 4 ~ _9_ l~xarnr~l~ 3 Prena~ation of 35S-labellecl Q!i~Qnuçleosid~ PhosDhorothioate :
Five milligrams of CPG-bound oli80nucleoside H-phosphonate was oxidized with a mixture of ~SS~I (5mCi, lCi/mg, Amcrsham, Arlington Heights, lllinois) in 40 microlitres carbon disulfid-/pyridine/trieshylamine (9:9:i). Aftor 30 minutes, 100 mictolitres cold Sa in the same solvcnt mixture ~;
was addcd and the rcaction was allowed to COQtinue for 60 minutes. The solution was removcd and the support was washed thrce t;mes with 500 microlitrcs carbon disulfidc and tllree times with 700 microlitres acetonitrile.The product was deprotected in concentrated ammonia at 55C for 14 hours, evaporated, and desalted using Sep paiCTM C18 column (Waters, Milford, MA).
The resultant product was purified by PAGE (20% polyacrylamidc, 7M urea).
The appropriate band was C:Ut under UV shadowing, e7~tracIed from the 8cl and desaltcd. Yield was fivc A2~0 units or 150 micrograms. Specific activi~y -was 5x109 cpm/micromole or 440 nanocuries/microgram. ~ `: -- .
Other modified oligonucleotidescan be labelled according to standard procedures, using ~H or I~C as label.

Exarn~tle 4 Assçssment of Oli~Qrlucleotide Stabilitv in Monkev Plasma ~ -Ei~hty micrograms at 35S-labelled oligonucleoside phosphoro~hioate ~ ~ -(capped or uncapped, specific activity 1.3 mCi/mg) was incubated vith 50 microlitres monkey serum at 37C. Aliquots were removed at tirne points :
and treated with proteinase K, (2 mg/ml, final concentra~ion) in 0.5% SDS~
10m~ NaCI, 20mM TrisoCI (pH 7.6), 10mM EDTA for one hour a.t 37C, followed by phenol-chloroform extraction and ethanol prccipitation.
Rccovercd oligonucleotides were then analyzed by PAGE (20%
polyacrylamide/7M urea) followed by autoradio~raphy.
Re~ults are shown in Figure 3. Uncapped and 5'-capped - . -oligonucleoside phosphorothio~tes were de8raded extensively within 2 hours. In contrast, 3'-capped and 3',5'-cappcd oligonucleosjde phosphorothioatcs wcre stable after 24 hours. This indicates that de~radation in monkey serum is primarily due to 3' exonucleases. Ladder forma~ion indicates absence of significant levels of endonucleolytic activi~y.
When 3H or l~C-labelled oligonucleotides are used, autoradiography is carried out through thc use of an appropriate enhancing fluorophore.

WO 92/2~697 2 ~ ~ 2 ~ ~ ~ PC. JS92/038~7 ~ o In Vr~QOli~onuçleotjd~Sta~iLlitY Asse~5ed by IJrinary An~
Male CDC2FI mice (averaRo weight 20 grams) were treated by intravenous or intraperitoneal injcction with a 30 mg/kg dose of S oligonucleotides dissolvcd in 200 microlitres physiological saiine. Each capped or uncapped oligonucleot;de was administered to three mice. Urine was collccted ~eparately from each animal up to 24 hours post-dosing, chen c:ctracted with as in Example 4, and analyzed for radioactivity.
Radioactivity was also mcasured from cage rin~c to account for urine spill.
Analysis was by PAGE (20% polyacrylamide. 7M urea) followed by autoradiography. The results are shown in Tai)le I below.
Twenty-four hours after dosing, about 30% o~ oligonucleoside phosphorothioates were excreted, whether cappcd or uncapped. Excreted uncapped and 5'-capped oligonucleoside phosphorothioates were extensively I S dcgraded, as shown in Figure 4. Excreted 3'-capped and 3',5'-capped oligonucleoside phosphorothioatcs, in contrast, dcmonstratcd virtually no degradat;on. This indicates that in 9il~0 degradatioD of oligonucleoside phosphoro~hioates e~ccreted in urine is mediated by 3'-exonuclease activity which can be inhibited by addin8 a cap to ~hc 3' hydro~yl group of the oli~onucleotide.

Table l URINARY E~CP~ETION OF OLIGONUCLEOTIDES IN MICE' lq~c~nous AdmlDistrfiliQQ, O~lgo- DOS- % Ot DQSe ReCOYSred Mouse ~uclcotlde Admluistered ~c~vered In Urine ~m~t Num~er ~5 m~tkg-JCI) Urln$ Gn~e Rinse T~tal 1 uncapped 8.61 18.7 8.48 27.2 2 uncapped 8.6 1 26.4 8~55 35.0 3 uncapped 8.61 22.S 3.08 25.6 4 5'-capped 9.39 22.2 3.91 26.1 3S 5 S'-capped 9.39 16.4 8.42 24.8 6 5'-capped 9.39 11.4 11.9 23.3 7 3'-capped 4.99 25.9 5.75 3 17 8 3'-capped 4.99 18.1 7.70 25 8 9 3'-capped 4.99 23.3 4.89 28.2 3t,5'-capped 6.47 17.6 8.38 26.0 I l . 3',5'-capped 6.47 22.1 7.59 29.7 12 3',5'-cappcd 6.47 12.7 10.70 23.4 q ~WO 92t2069, 2 ~ ~ 2 ~ /US92/03867 Ex~mr~le 6 Biodistribution of Oli~onncleotidcs ~ -Following the urinary oligonucleotide analysis of E~amplc 5, the animals were sacrificed and au~opsied, and all organs wcre rcmoved. Each organ type was homogenized, Iysed in buffer and a3sayed for radioactivity.
Biodistribution of oligonucleotides is shown in Table 2 below. All types of capped and uncapped oligonucleosidc phosphorothioates tested were ~ :bioavailable in most of the tissues of the organs 24 hours post-dosing. The concentration of oligonucleoside phosphorothioate in eaeh tissue was ~:
independen~ of ~he presence, absence, or location of capping.
Oligonucleotide concentration ~as hi~hest in kidney, although ~otal mass of oligonucleotide was highest jD liver.

(~e equiY~lerlts Of oligonucleotlde/gram of tissue) 3, ~
5~- 3~- 5~. -~! gnC.4DDed~:~IDDed~LP~ CaDD
Kidncy 195.00230.00184.67 200.33 Liver 27.3343.60 34.93 37.70 . ::
Lar8e Intestine 15.03 21.03 19.50 23.32 Small Intestine 10.68 14.20 12.87 12.91 Stomach 6.28 7.26 8.59 7.59 Spleen 6.03 12.40 9.41 1~3.13 Heart 4.16 7.02 5.23 6.31 Lung 3.81 5.41 4.33 5 90 Muscle 3.2g 4.38 3.39 4.61 Plasma 2.85 1.98 3.16 1.70 Testes 1.64 2.14 1.56 1.73 Brain 0.27 0.26 0.34 0.23 ~30 m8 oligonucleotide/kg body weight, intravenously. ~ -.~ ., ~1 ~9~n~
WO 92/20697 ~ 1 U ~ ~ V ~ PC~, US92/03867 ': `
-12- ` `
l~amDle 7 ~ ; -Status Qf Oli~onu~lçotides in Qr~ians Homogenjzed kidncy or t;ssue from E~ample 6 waS treated with ~ :
proteinase K (2 mg/ml final conccntration) in extraction buffer (0.5% SDS, . 10 mM NaCI, 20m~ Tris-HCI pH 7.6, lOm~ EDTA) for two hours at 37~C.
Samples were then extracted twice with phenol-chloroform and once with `
chloroform, followed by ethanol precipitation. Recovered oligonucleotides were fractionated by PAGE (20% polyacrylamide, 7~ urea). The gel was then fi~ed in 10% acetic acid, 10% methanol and subjected to autoradiography. The rcsults are shown for kidney in Figure 5. Uncapped oli~ionucleoside phosphorothioate extracted from kidney tissue was de8raded to about 50%, and slower migrating bands (2-23 nucleotidcs in len~th) were also dctected. Thus both degradation and extension of uncapped 8-oligonucleoside phosphotothioates occurs in kidncy. 5'-capped olieonucleosidc phosphorothioates produccd substantially identical results.
In contrast, the great majority of bioavailable oligonucleotide was undegraded when either 3'-capped or 3',5'-capped oligonucleoside was used, and neither showed any sign of e~tension. Similar results were obtained for liver, BS shown in Figure 6. No ex~ension Or 3' or 3',5'-capped oligonucleoside phosvhorothioate was vbscrved i= small inses~ine eilher.

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Claims (10)

1. An oligonucleotide, wherein one or more nucleotide linkage is a non-phosphodiester artificial linkage, said artificial linkage being resistant to nucleolytic degradation, and wherein the oligonucleotide has a cap structure at a 3' hydroxyl, said cap structure selected from the group consisting of the structure shown in Figure 1 and the structures shown in Figure 2, whereby the oligonucleotide is resistant to nucleolytic degradation in vivo.
2. An oligonucleotide according to claim 1, wherein one or more internucleoside linkage that is an artificial linkage is selected from the group consisting of phosphorothioate, methylphosphate, phosphorothioate, phosphate ester, bridged phosphoramide, and bridge phosphorothioate.
3. An oligonucleotide according to claim 1, wherein the cap structure is the structure shown in Figure 1.
4. A therapeutic composition comprising an oligonucleotide of claim 1, in a physiologically acceptable carrier.
5. A therapeutic composition comprising an oligonucleotide of claim 2, in a physiologically acceptable carrier.
6. A therapeutic composition comprising an oligonucleotide of claim 3, in a physiologically acceptable carrier.
7. A method of treating a mammal infected with a virus, comprising administering a therapeutic composition according to claim 4.
8. A method of treating a mammal infected with a virus, comprising administering a therapeutic composition according to claim 5.
9. A method of treating a mammal infected with a virus, comprising administering a therapeutic composition according to claim 5.
10. A method of treating a mammal having a disorder of gene expression, comprising administering a therapeutic composition according to claim 4.
CA 2102804 1991-05-10 1992-05-08 3'-end blocked oligonucleotides Abandoned CA2102804A1 (en)

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