`
`(19) (19)
`
`
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`(12) (cid:9)(12) (cid:9)
`
`
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`Europaisches Patentamt Europaisches Patentamt
`
`
`
`European Patent Office European Patent Office
`
`
`
`Office europeen des brevets Office europeen des brevets
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`
`
`11 11
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`
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`(11) (cid:9)(11) (cid:9)
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`EP 0 586 520 131 EP 0 586 520 131
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`EUROPEAN PATENT SPECIFICATION EUROPEAN PATENT SPECIFICATION
`
`
`(45) Date of publication and mention (45) Date of publication and mention
`
`of the grant of the patent: of the grant of the patent:
`
`19.04.2000 Bulletin 2000/16 19.04.2000 Bulletin 2000/16
`
`
`
`(21) Application number: 92912190.3 (21) Application number: 92912190.3
`
`
`
`(22) Date of filing: 21.05.1992 (22) Date of filing: 21.05.1992
`
`
`(51) Int. ci.7: C120 1/68, A61K 31/70, (51) Int. ci.7: C120 1/68, A61K 31/70,
`
`CO7H 21/00 CO7H 21/00
`
`
`(86) International application number: (86) International application number:
`
`PCT/US92/04305 PCT/US92/04305
`
`
`(87) International publication number: (87) International publication number:
`
`WO 92/20823 (26.11.1992 Gazette 1992/29) WO 92/20823 (26.11.1992 Gazette 1992/29)
`
`
`
`(54) BACKBONE MODIFIED OLIGONUCLEOTIDE ANALOGS (54) BACKBONE MODIFIED OLIGONUCLEOTIDE ANALOGS
`
`
`IN DER HAUPTKETTE MODIFIZIERTE OLIGONUKLEOTID-ANALOGE IN DER HAUPTKETTE MODIFIZIERTE OLIGONUKLEOTID-ANALOGE
`
`ANALOGUES D'OLIGONUCLEOTIDES A SQUELETTE MODIFIE ANALOGUES D'OLIGONUCLEOTIDES A SQUELETTE MODIFIE
`
`
`(84) Designated Contracting States: (84) Designated Contracting States:
`
`AT BE CH DE DK ES FR GB GRIT LI LU MC NL AT BE CH DE DK ES FR GB GRIT LI LU MC NL
`
`SE SE
`
`
`
`(30) Priority: 21.05.1991 US 703619 (30) Priority: 21.05.1991 US 703619
`
`
`(43) Date of publication of application: (43) Date of publication of application:
`
`16.03.1994 Bulletin 1994/11 16.03.1994 Bulletin 1994/11
`
`
`(73) Proprietors: (73) Proprietors:
`
`• ISIS PHARMACEUTICALS, INC. • ISIS PHARMACEUTICALS, INC.
`
`Carlsbad, CA 92008 (US) Carlsbad, CA 92008 (US)
`
`Novartis AG Novartis AG
`
`4058 Basel (CH) 4058 Basel (CH)
`
`
`(72) Inventors: (72) Inventors:
`
`• DE MESMAEKER, Alain • DE MESMAEKER, Alain
`
`CH-4447 Kaenerkinden (CH) CH-4447 Kaenerkinden (CH)
`
`LEBRETON, Jacques LEBRETON, Jacques
`
`F-68100 Mulhouse (FR) F-68100 Mulhouse (FR)
`
`WALDNER, Adrian WALDNER, Adrian
`
`CH-4123 Basel (CH) CH-4123 Basel (CH)
`
`• COOK, Phillip, Dan • COOK, Phillip, Dan
`
`Carlsbad, CA 92009 (US) Carlsbad, CA 92009 (US)
`
`
`(74) Representative: (74) Representative:
`
`Hallybone, Huw George Hallybone, Huw George
`
`CARPMAELS AND RANSFORD CARPMAELS AND RANSFORD
`
`43 Bloomsbury Square 43 Bloomsbury Square
`
`London WC1A 2RA (GB) London WC1A 2RA (GB)
`
`
`(56) References cited: (56) References cited:
`
`WO-A-92/02534 WO-A-92/02534
`
`
`
`WO-A-92/05186 WO-A-92/05186
`
`
`• NUCLEIC ACIDS RESEARCH., vol.19, no.3, 1991, • NUCLEIC ACIDS RESEARCH., vol.19, no.3, 1991,
`
`ARLINGTON, VIRGINIA US pages 427 - 433 ARLINGTON, VIRGINIA US pages 427 - 433
`
`U.HEINEMANN ET AL. 'Effect of a Single 3.-U.HEINEMANN ET AL. 'Effect of a Single 3.-
`
`Methylene Phosphonate Linkage on the Methylene Phosphonate Linkage on the
`
`Conformation of an A-DNA Octamer Double Conformation of an A-DNA Octamer Double
`
`Helix' Helix'
`
`• GBF MONOGRAPHS, vol.8, 1987 pages 107 -113 • GBF MONOGRAPHS, vol.8, 1987 pages 107 -113
`
`M.MORR ET AL. 'Building Blocks for the M.MORR ET AL. 'Building Blocks for the
`
`Chemical Synthesis of DNA Containing C(3')-Chemical Synthesis of DNA Containing C(3')-
`
`CH2-P Bonds' CH2-P Bonds'
`
`• JOURNAL OF THE AMERICAN CHEMICAL • JOURNAL OF THE AMERICAN CHEMICAL
`
`SOCIETY., vol.114, no.10, 6 May 1992, GASTON, SOCIETY., vol.114, no.10, 6 May 1992, GASTON,
`
`PA US pages 4006 - 4007 J-J. VASSEUR ET AL. PA US pages 4006 - 4007 J-J. VASSEUR ET AL.
`
`ET AL. 'Oligonucleosides : Synthesis of a Novel ET AL. 'Oligonucleosides : Synthesis of a Novel
`
`Methylhydroxylamine-Linked Nucleoside Dimer Methylhydroxylamine-Linked Nucleoside Dimer
`
`and its Incorporation into Antisense Sequences' and its Incorporation into Antisense Sequences'
`
`• JOURNAL OF ORGANIC CHEMISTRY., vol.56, • JOURNAL OF ORGANIC CHEMISTRY., vol.56,
`
`no.12, 7 June 1991, EASTON US pages 3869 -no.12, 7 June 1991, EASTON US pages 3869 -
`
`3882 Z.HUANG ET AL. 'Building Blocks for 3882 Z.HUANG ET AL. 'Building Blocks for
`
`Oligonucleotide Analogues with Dimethylene Oligonucleotide Analogues with Dimethylene
`
`Sulfide, Sulfoxide, and Sulfone Groups Sulfide, Sulfoxide, and Sulfone Groups
`
`Replacing Phosphodiester Linkages' Replacing Phosphodiester Linkages'
`
`• COHEN, ed., "Non-ionic Antisense • COHEN, ed., "Non-ionic Antisense
`
`Oligonucleotides" in Oligonucleotides, Oligonucleotides" in Oligonucleotides,
`
`published 1989, by CRC Press, Inc. (USA), see published 1989, by CRC Press, Inc. (USA), see
`
`pages 79 and 80. pages 79 and 80.
`
`• JOHN GOODCHILD, "Inhibition of Gene • JOHN GOODCHILD, "Inhibition of Gene
`
`Expression by Oligonucleotides" in Expression by Oligonucleotides" in
`
`Oligonucleotides, published 1989, by CRC Press Oligonucleotides, published 1989, by CRC Press
`
`Inc., (USA), see pages 53-78. Inc., (USA), see pages 53-78.
`
`• Tetrahedron Letters, Volume 31, Number 17, • Tetrahedron Letters, Volume 31, Number 17,
`
`issued 22 May 1990 (London), MARK issued 22 May 1990 (London), MARK
`
`MATTEUCCI, "Deoxyoligonucleotide Analogs MATTEUCCI, "Deoxyoligonucleotide Analogs
`
`Based on Formacetal Linkages", see pages Based on Formacetal Linkages", see pages
`
`2385-2388. 2385-2388.
`
`
`Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give
`
`notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in
`
`a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art.
`
`99(1) European Patent Convention). 99(1) European Patent Convention).
`
`
`Printed by Xerox (UK) Business Services Printed by Xerox (UK) Business Services
`
`2.16.7 (HRS)13.6 2.16.7 (HRS)13.6
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`EP 0 586 520 B1
`EP 0 586 520 B1
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`Alnylam Exh. 1062
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`1
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`(cid:9)
`(cid:9)
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`EP 0 586 520 B1
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`Description
`
`BACKGROUND OF THE INVENTION
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`5 (cid:9)
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`[0001] (cid:9)
`It is well known that most of the bodily states in mammals, including most disease states, are effected by
`proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to
`many diseases in animals and man.
`[0002] (cid:9)
`Classical therapeutics has generally focused upon interactions with such proteins in an effort to moderate
`their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the
`actual production of such proteins by interactions with the molecules (i.e., intracellular RNA) that direct their synthesis.
`These interactions have involved the hybridization to RNA of complementary "antisense" oligonucleotides or certain
`analogs thereof. Hybridization is the sequence-specific hydrogen bonding of oligonucleotides or oligonucleotide ana-
`logs to RNA or to single stranded DNA. By interfering with the production of proteins, it has been hoped to effect ther-
`apeutic results with maximum effect and minimal side effects. Oligonucleotide analogs also may modulate the
`production of proteins by an organism by a similar mechanism.
`[0003] (cid:9)
`The pharmacological activity of antisense oligonucleotides and oligonucleotide analogs, like other therapeu-
`tics, depends on a number of factors that influence the effective concentration of these agents at specific intracellular
`targets. One important factor for oligonucleotides is the stability of the species in the presence of nucleases. It is unlikely
`that unmodified oligonucleotides will be useful therapeutic agents because they are rapidly degraded by nucleases.
`20 (cid:9) Modifications of oligonucleotides to render them resistant to nucleases therefore are greatly desired.
`[0004] (cid:9)
`Modifications of oligonucleotides to enhance nuclease resistance have generally taken place on the phos-
`phorus atom of the sugar-phosphate backbone. Phosphorothioates, methyl phosphonates, phosphoramidates, and
`phosphorotriesters have been reported to confer various levels of nuclease resistance. However, phosphate-modified
`oligonucleotides of this type generally have suffered from inferior hybridization properties. Cohen, J.S., ed. Oligonucle-
`otides: Antisense Inhibitors of Gene Expression, (CRC Press, Inc., Boca Raton FL, 1989).
`[0005] (cid:9)
`Another key factor is the ability of antisense compounds to traverse the plasma membrane of specific cells
`involved in the disease process. Cellular membranes consist of lipid-protein bilayers that are freely permeable to small,
`nonionic, lipophilic compounds yet inherently impermeable to most natural metabolites and therapeutic agents. Wilson,
`D.B. Ann. Rev. Biochem. 47:933-965 (1978). The biological and antiviral effects of natural and modified oligonucle-
`otides in cultured mammalian cells have been well documented. Thus, it appears that these agents can penetrate mem-
`branes to reach their intracellular targets. Uptake of antisense compounds by a variety of mammalian cells, including
`HL-60, Syrian Hamster fibroblast, U937, L929, CV-1 and ATH8 cells, has been studied using natural oligonucleotides
`and certain nuclease resistant analogs, such as alkyl triesters. Miller, P.S., Braiterman, L.T. and Ts'O, P.O.R,
`Biochemistry 16:1988-1996 (1977); methyl phosphonates, Marcus-Sekura, C.H., Woerner, A.M., Shinozuka, K., Zon,
`35 (cid:9) G., and Quinman, G.V., Nuc. Acids Res. 15:5749-5763 (1987) and Miller, P.S., McParland, K.B., Hayerman, K. and
`Ts'O, P.O.P., Biochemistry 16: 1988-1996 (1977) and Loke, S.K., Stein, C., Zhang, X.H. Avigan, M., Cohen, J. and
`Neckers, L.M. Top. Microbiol. Immunol. 141: 282:289 (1988).
`[0006] (cid:9)
`Modified oligonucleotides and oligonucleotide analogs often are less readily internalized than their natural
`counterparts. As a result, the activity of many previously available antisense oligonucleotides has not been sufficient for
`practical therapeutic, research or diagnostic purposes. Two other serious deficiencies of prior art oligonucleotides that
`have been designed for antisense therapeutics are inferior hybridization to intracellular RNA and the lack of a defined
`chemical or enzyme-mediated event to terminate essential RNA functions.
`[0007] (cid:9)
`Modifications to enhance the effectiveness of the antisense oligonucleotides and overcome these problems
`have taken many forms. These modifications include base ring modifications, sugar moiety modifications, and sugar-
`phosphate backbone modifications. Prior sugar-phosphate backbone modifications, particularly on the phosphorus
`atom, have effected various levels of resistance to nucleases. However, while the ability of an antisense oligonucleotide
`to bind to specific DNA or RNA with fidelity is fundamental to antisense methodology, modified phosphorus oligonucle-
`otides have generally suffered from inferior hybridization properties.
`[0008] (cid:9)
`Replacement of the phosphorus atom has been an alternative approach in attempting to avoid the problems
`associated with modification on the pro-chiral phosphate moiety. Some modifications in which replacement of the phos-
`phorus atom has been achieved are disclosed by: Matteucci, M. Tetrahedron Letters 31:2385-2388 (1990), wherein
`replacement of the phosphorus atom with a methylene group is limited by available methodology which does not pro-
`vide for uniform insertion of the formacetal linkage throughout the backbone, and its instability, making it unsuitable for
`work; Cormier, et al. Nucleic Acids Research 16:4583-4594 (1988), wherein replacement of the phosphorus moiety
`55 (cid:9) with a diisopropylsilyl moiety is limited by methodology, solubility of the homopolymers and hybridization properties;
`Stirchak, et al. Journal of Organic chemistry 52:4202-4206 (1987), wherein replacement of the phosphorus linkage by
`short homopolymers containing carbamate or morpholino linkages is limited by methodology, the solubility of the result-
`ing molecule, and hybridization properties; Mazur, et al. Tetrahedron 40:3949-3956 (1984), wherein replacement of the
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`EP 0 586 520 B1
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`phosphorus linkage with a phosphonic linkage has not been developed beyond the synthesis of a homotrimer molecule;
`and Goodchild, J., Bioconjugate Chemistry 1:165-187 (1990), wherein ester linkages are enzymatically degraded by
`esterases and are therefore unsuitable to replace the phosphate bond in antisense applications.
`[0009] (cid:9)
`The limitations of the available methods for modification of the phosphorus backbone have led to a continu-
`ing and long felt need for other modifications which provide resistance to nucleases and satisfactory hybridization prop-
`erties for antisense oligonucleotide diagnostics, therapeutics, and research.
`
`OBJECTS OF THE INVENTION
`
`[0010] (cid:9)
`It is an object of the invention to provide oligonucleotide analogs for use in antisense oligonucleotide diag-
`nostics, research reagents, and therapeutics.
`[0011] (cid:9)
`It is a further object of the invention to provide oligonucleotide analogs which possess enhanced cellular
`uptake.
`[0012] (cid:9)
`Another object of the invention is to provide such oligonucleotide analogs which have greater efficacy than
`unmodified antisense oligonucleotides.
`[0013] (cid:9)
`It is yet another object of the invention to provide methods for synthesis and use of such oligonucleotide ana-
`logs.
`[0014] (cid:9)
`These and other objects will become apparent to persons of ordinary skill in the art from a review of the
`present specification and the appended claims.
`
`SUMMARY OF THE INVENTION
`
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`[0015] (cid:9)
`Compositions useful for modulating the activity of an RNA or DNA molecule in accordance with this invention
`generally comprise oligonucleotide analogs having at least portions of their backbone linkages modified. In these mod-
`if ications, the phosphorodiester linkage of the sugar phosphate backbone found in wild type nucleic acids has been
`replaced with various four atom linking groups. Such four atom linking groups maintain a desired four atom spacing
`between the 3'-carbon of one sugar or sugar analog and the 4'-carbon of the adjacent sugar or sugar analog. Oligonu-
`cleotide analogs made in accordance with the teachings of the invention are comprised of a selected sequence which
`is specifically hybridizable with a preselected nucleotide sequence of single stranded or double stranded DNA or RNA.
`They are synthesized through, for example, known solid state synthetic methodology to be complementary to or at least
`to be specifically hybridizable with the preselected nucleotide sequence of the RNA or DNA. Nucleic acid synthesizers
`are commercially available and their use is generally understood by persons of ordinary skill in the art as being effective
`in generating nearly any oligonucleotide or oligonucleotide analog of reasonable length which may be desired.
`[0016] (cid:9)
`In the context of this invention, the term "nucleoside" refers to the unit made up of a heterocyclic base and
`its sugar. The term "nucleotide" refers to a nucleoside having a phosphate group on its 3' or 5' sugar hydroxyl group.
`Thus, nucleosides, unlike nucleotides, have no phosphate group. "Oligonucleotide" refers to a plurality of joined nucle-
`otide units formed in a specific sequence from naturally occurring bases and pentofuranosyl groups joined through a
`sugar group by native phosphodiester bonds. These nucleotide units may be nucleic acid bases such as guanine, ade-
`nine, cytosine, thymine or uracil. The sugar group can be a deoxyribose or ribose. This term refers to both naturally
`occurring and synthetic species formed from naturally occurring subunits.
`[0017] (cid:9)
`The term "oligonucleotide analog", as used in connection with this invention, refers to moieties which func-
`tion similarly to oligonucleotides but which have non-naturally occurring portions. Oligonucleotide analogs can have
`altered sugar moieties, altered base moieties or altered inter-sugar linkages. For the purposes of this invention, an oli-
`gonucleotide analog having non-phosphodiester bonds, i.e., an altered inter-sugar linkage, can alternately be consid-
`erect as an "oligonucleoside." Such an oligonucleoside thus refers to a plurality of joined nucleoside units joined by
`linking groups other than native phosphodiester linking groups. Additionally, for the purposes of this invention, the ter-
`minology "oligomers" can be considered to encompass oligonucleotides, oligonucleotide analogs or oligonucleosides.
`Thus, in speaking of "oligomers" reference is made to a series of nucleosides or nucleoside analogs that are joined
`together via either natural phosphodiester bonds or via other linkages including the four atom linkers of this invention.
`50 (cid:9) Generally, while the linkage is from the 3' carbon of one nucleoside to the 5' carbon of a second nucleoside, the term
`"oligomer" can also include other linkages such as a 2' - 5' linkage.
`[0018] (cid:9)
`Oligonucleotide analogs can also comprise other modifications consistent with the spirit of this invention,
`particularly that increase nuclease resistance and, thus, facilitate antisense therapeutic, diagnostic, or research reagent
`use of a particular oligonucleotide. For example, when the sugar portion of a nucleoside or nucleotide is replaced by a
`carbocyclic or other moiety, it is no longer a sugar. Moreover, when other substitutions, such a substitution for the inter-
`sugar phosphorodiester linkage are made, the resulting material is no longer a true nucleic acid species. The com-
`pounds that result from such substitutions all are denominated as analogs. Throughout this specification, reference to
`the sugar portion of a nucleic acid species shall be understood to refer to either a true sugar or to a species taking the
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`EP 0 586 520 B1
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`traditional space of the sugar of wild type nucleic acids. Moreover, reference to inter-sugar linkages shall be taken to
`include moieties serving to join the sugar or sugar analog portions together in the fashion of wild type nucleic acids.
`[0019] (cid:9)
`In accordance with the present invention, novel types of antisense oligonucleotide analogs are provided
`which are modified to enhance cellular uptake, nuclease resistance, and hybridization properties and to provide a
`defined chemical or enzymatically mediated event to terminate essential RNA functions.
`[0020] (cid:9)
`It has been found that certain classes of oligonucleotide analog compositions can be useful in therapeutics
`and for other objects of this invention. Such oligonucleotide analogs are formed from subunits, at least some of which
`have the structure:
`
`wherein 13, is a variable base moiety; Q is 0, CH2, CHF or CF2 and X is H; OH; C1 to C10 lower alkyl, substituted lower
`alkyl, alkaryl or arally1; F; CI; Br; CN; CF3; OCF3; OCN; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2;
`NO2, N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino or substituted silyl.
`[0021] (cid:9)
`In accordance with other embodiments, the oligonucleotide analogs of this invention preferably have one of
`the following formulas:
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`EP 0 586 520 B1
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`H N
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`HN
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`R —y15
`x
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`[0022] (cid:9)
`L1-L2-L3-L4 represents a four-atom linker and is selected from NR-C(0)-CH2-CH2, NR-C(S)-CH2-CH2, CH2-
`NR-C(0)-CH2, CH2-NR-C(S)-CH2, CH2-CH2-NR-C(0), CH2-CH2-NR-C(S), C(0)-NR-CH2-CH2, C(S)-NR-CH2-CH2,
`CH2-C(0)-NR-CH2, and CH2-C(S)-NR-CH2 where R is hydrogen, alkyl, substituted alkyl, aralkyl, alkenyl, alkaryl, ami-
`noalkyl, hydroxyalkyl, hetercycloalkyl or heterocycloaralkyl. The remaining subunits of the oligonucleotides are natural
`or synthetic subunits as will be appreciated by persons of ordinary skill in the art.
`[0023] (cid:9)
`It is preferred that the oligonucleotide analogs be such tat X is H or OH, or, alternatively F, 0-alkyl or 0-alke-
`nyl, especially where Q is O. The group BX is preferably adenine, guanine, uracil, thymine, cystosine, 2-aminoadenosine
`or 5-methylcytosine, although other non-naturally occurring species can be employed.
`[0024] (cid:9)
`It is preferred that the oligonucleotide analogs of the invention comprise from about 4 to about 50 subunits,
`and even more preferably from about 5 to about 20 subunits having the given structure. While substantially each subunit
`of the oligonucleotide analogs can have said structure, it is also desirable for substantially alternating or substantially
`random subunits to have said structure.
`[0025] (cid:9)
`The oligonucleotide analogs of this invention are preferably prepared in a pharmaceutically acceptable car-
`rier for therapeutic administration to patients. The analogs are believed to exhibit improved nuclease resistance as com-
`50 pared to corresponding wild type oligonucleotides. This invention is also directed to use of the above-described
`oligonucleotide analogs in the manufacture of a composition for modulating the production or activity of a protein in an
`organism, the oligonucleotide analogs being hybridizable with at least a portion of a nucleic acid sequence coding for
`said protein.
`[0026] (cid:9)
`Additionally, the invention is directed to use of the above-described oligonucleotide analogs in the manufac-
`ture of a composition for treating an organism having a disease characterized by the undesired production of a protein.
`[0027] (cid:9)
`In another aspect, the present invention provides compounds useful in preparing the above-described oligo-
`nucleotide analogs. In certain embodiments, these compounds have a structure:
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`EP 0 586 520 B1
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`B
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`wherein RH pi and RH p2 are, independently, H or hydroxyl protecting groups. In certain embodiments, these compounds
`are prepared by providing a first synthon having structure (I) and second synthon having structure (II):
`
`X
`
`( I )
`
`wherein:
`
`(a) RT is NH2 and RB is RA-CH2-CH2;
`(b) RT is CH2-NH2 and RB is RA-CH2;
`(C) RT is CH2-CH2-NH2 and RB is RA;
`(d) RT is RA and RB is NH2-CH2-CH2; or
`(e) RT is CH2-RA and RB is NH2-CH2;
`
`where RA is C(0)0H, C(S)OH, or an activated derivative thereof; and coupling the first and second synthons to form an
`amide or thioamide linkage through the RT and RB groups.
`[0028] (cid:9)
`It is useful to formulate therapeutic compositions where at least one portion of said oligonucleotide analog
`is incorporated into a further oligonucleotide species to provide said further oligonucleotide analog with wild type phos-
`phodiester bonds substantially alternating with areas so coupled. The incorporation is preferably achieved by phos-
`phodiester linkage of a desired sequence of dinucleotides, said dinucleotides having been previously so coupled.
`[0029] (cid:9)
`Oligonucleotide analogs having modified sugar linkages have been found to be effective in accomplishing
`these goals. The oligonucleotide analogs preferably range from about 4 to about 50 nucleic acid base subunits in
`length, with from about 5 to about 20 being more preferred. Oligonucleotide analogs described in this invention are
`hybridizable with preselected nucleotide sequences of single stranded or double stranded DNA and RNA. The nucleic
`acid bases which comprise this invention can be pyrimidines such as thymine, uracil or cytosine or purines such as gua-
`nine or adenine, or modifications thereof such as 5-methylcytosine, arranged in a selected sequence. The sugar moiety
`can be of the ribose or deoxyribose type or a sugar mimic such as a carbocylic ring. In accordance with one preferred
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`embodiment of this invention, the oligonucleotide analogs or oligonucleosides hybridize to HIV mRNA encoding the tat
`protein, or to the TAR region of HIV mRNA. The oligonucleotide analogs or oligonucleosides can mimic the secondary
`structure of the TAR region of HIV mRNA to bind the tat protein. Other preferred antisense oligonucleotide analog or
`oligonucleoside sequences include complementary sequences for herpes, papilloma and other viruses.
`[0030] (cid:9)
`The modified linkages of this invention use a four atom linking group to replace the naturally occurring phos-
`phodiester-5'-methylene linkage. Replacement of the naturally occurring linkage by four atom linkers of the present
`invention confers nuclease resistance and enhanced cellular uptake upon the resulting oligonucleotide analog. Prefer-
`ably included within the four atom linker is a 3'-deoxy function on one of the linked sugars. The four atom linker is of the
`structure -L1-L2-L3-L4-. It is preferred that the modified linkage occurs at substantially each linkage location. Alterna-
`tively, modification can occur at less that every location such as at alternating linkage locations or substantially ran-
`domly. The linkage can be neutral or can be positively or negatively charged.
`[0031] (cid:9)
`There are described herein methods for synthesizing such oligonucleosides; for example, the coupling of a
`3'-deoxy-3'-substituted, especially methyl substituted, nucleoside with 5'-deoxy-5'-substituted nucleoside through the
`addition of a two atom fragment or substituted two atom fragment. The addition reaction can occur through a stepwise
`procedure involving the activation of the 3' and 5' positions of respective nucleosides to a variety of suitable electrophilic
`moieties, followed by the addition of a suitable linking group to react with the electrophiles. In the alternative, the proce-
`dure can occur in a concerted manner. Such methods can employ solid supports via a DNA synthesizer, by manual
`manipulation of the support, or otherwise.
`[0032] (cid:9)
`This invention is also directed to use of the above-described oligonucleotide analogs in the manufacture of
`a composition for modulating the production of proteins by an organism. It is preferred that the RNA or DNA portion
`which is to be modulated be preselected to comprise that portion of DNA or RNA which codes for the protein whose
`formation or activity is to be modulated. The targeting portion of the composition to be employed is, thus, selected to be
`complementary to the preselected portion of DNA or RNA that is to be an antisense oligonucleotide for that portion.
`[0033] (cid:9)
`This invention is also directed to use of the above-described oligonucleotide analogs in the manufacture of
`a composition for treating an organism having a disease characterized by the undesired production of a protein. The
`composition is preferably one which is designed to specifically bind with messenger RNA which codes for the protein
`whose production or activity is to be modulated. Diagnostic methods for detecting the presence or absence of abnormal
`RNA molecules or abnormal or inappropriate expression of normal RNA molecules in organisms or cells are also
`described.
`[0034] (cid:9)
`Methods for the selective binding of RNA for research and diagnostic purposes are also described. Such
`selective, strong binding is accomplished by interacting such RNA or DNA with compositions of the invention which are
`resistant to degradative nucleases and which hybridize more strongly and with greater fidelity than known oligoncucle-
`otides or oligonucleotide analogs.
`
`5 (cid:9)
`
`10 (cid:9)
`
`15 (cid:9)
`
`20 (cid:9)
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`25 (cid:9)
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`30 (cid:9)
`
`35 (cid:9) BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0035]
`
`Figure 1 is a schematic, synthetic scheme in accordance with certain embodiments of the invention; and
`Figure 2 is a schematic, synthetic scheme in accordance with further embodiments of the invention.
`
`40 (cid:9)
`
`DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
`
`45 (cid:9)
`
`[0036] (cid:9)
`The biological activity of the antisense oligonucleotides previously available has not generally been sufficient
`for practical therapeutic research or diagnostic use. This invention is directed to modified oligonucleotides, i.e. oligonu-
`cleotide analogs or oligonucleosides, and to methods for effecting useful modifications. These modified oligonucle-
`otides and oligonucleotide analogs exhibit increased stability relative to their naturally occurring counterparts.
`Extracellular and intracellular nucleases generally do not recognize and, therefore, do not bind to the backbone modi-
`fied oligonucleotide analogs or oligonucleosides of the present invention. Any binding by a nuclease to the backbone
`50 (cid:9) will not result in cleavage of the nucleosidic linkages due to the lack of sensitive, phosphorus-oxygen bonds. In addition,
`the resulting, novel neutral or positively charged backbones of the present invention can be taken into cells by simple
`passive transport rather than requiring complicated protein mediated processes. Another advantage of the present
`invention is that the lack of a negatively charged backbone facilitates the sequence specific binding of the oligonucle-
`otide analogs or oligonucleosides to targeted RNA, which has a negatively charged backbone, and which will accord-
`ingly repel incoming similarly charged oligonucleotides. Still another advantage of the present invention is that sites for
`attaching functional groups which can initiate catalytic cleavage of targeted RNA are found in these structure types.
`[0037] (cid:9)
`In accordance with preferred embodiments, this invention is directed to replacing inter-sugar phosphate
`groups to yield oligonucleotides in which at least some of the subunits of the analogs have linkages as found in the
`
`55 (cid:9)
`
`7
`
`

`

`5
`
`10
`
`15
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`20
`
`25 (cid:9)
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`30
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`35 (cid:9)
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`40 (cid:9)
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`45 (cid:9)
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`50 (cid:9)
`
`55 (cid:9)
`
`structure:
`
`EP 0 586 520 B1
`
`L 1
`
`X
`
`2
`
`I 3— L 4
`
`X
`
`wherein:
`
`Bx is a variable base moiety;
`Q is 0, CH2, CHF or CF2;
`Xis H; OH; C1 to C10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; F; CI; Br; CN; CF3; OCF3; OCN; 0-, S-
`, or N-alkyl; 0-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; ami-
`noalkylamino; polyalkylamino; or substituted silyl.
`
`[0038] (cid:9)
`L1-L2-L3-L4 represents a four-atom linker and is selected from NR-C(0)-CH2-CH2, NR-C(S)-CH2-CH2, CH2-
`NR-C(0)-CH2, CH2-NR-C(S)-CH2, CH2-CH2-NR-C(0), CH2-CH2-NR-C(S), C(0)-NR-CH2-CH2, C(S)-NR-CH2-CH2,
`CH2-C(0)-NR-CH2, and CH2-C(S)-NR-CH2 where R is hydrogen, alkyl, substituted alkyl, aralkyl, alkenyl, alkaryl, ami-
`noalkyl, hydroxyalkyl, heterocycloalkyl or heterocycloaralkyl; and the remaining subunits are natural or synthetic.
`[0039] (cid:9)
`Methods are also described for the preparation of oligonucleosides with modified inter-sugar linkages.
`These modifications can be effected using solid supports which can be manually manipulated or used in conjunction
`with a DNA synthesizer using methodology commonly known to those skilled in DNA synthesizer arts. Generally, the
`procedure involves functionalizing the sugar moieties of two nucleosides which will be adjacent to one another in the
`selected sequence. In a 5' to 3' sense, the "upstream" nucleoside is generally modified at the 3' sugar site and is
`referred to hereinafter as "synthon 1". In one process, ribo- and 2'-deoxyribonucleosides of adenine, guanine, cytosine,
`uracil, thymine and their analogs are modified to give their 3'-deoxy-3-hydroxymethyl analogs. These 3'-hydroxymethyl
`groups are then convened into various types of electrophilic centers. This can be accomplished in a number of ways
`such as the following scheme.
`[0040] (cid:9)
`One class of starting materials, 3'-deoxy-3'-hydroxymethyl ribonucleosides, can be prepared as described
`by Townsend et at, Tetrahedron Letters, 31:3101-3104 (1990), Samano, V. and M.J. Morris, Journal of Organic
`Chemistry, 55:5186-5188 (1990) and Bergstrom, D.E., Nucleosides and Nucleotides 8(8):1529-1535 (1989). Appropri-
`ate, known, selective sugar hydroxyl protection of these nucleosides fo