WO2011097614A1 - Mehods and compositions useful in diseases or conditions related to repeat expansion - Google Patents

Mehods and compositions useful in diseases or conditions related to repeat expansion Download PDF

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WO2011097614A1
WO2011097614A1 PCT/US2011/024019 US2011024019W WO2011097614A1 WO 2011097614 A1 WO2011097614 A1 WO 2011097614A1 US 2011024019 W US2011024019 W US 2011024019W WO 2011097614 A1 WO2011097614 A1 WO 2011097614A1
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substituted
oligomeric compound
region
nucleosides
central
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PCT/US2011/024019
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French (fr)
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C. Frank Bennett
Eric E. Swayze
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Isis Pharmaceuticals, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===

Definitions

  • the present invention pertains generally to chemically-modified oligomers for use in research, diagnostics, and/or therapeutics.
  • RNA molecules are known to include repeat regions consisting essentially of repeating units of 3-5 nucleotides. Depending on the particular gene, the repeat region a normal wild-type RNA molecule may comprise from about 5 up to about 40 copies of the repeating unit. In certain instances, the number of such repeating units can become increased and the resulting nucleotide repeat-containing RNA molecule may disruptive to the cell. Certain diseases can result.
  • the present invention provides oligomeric compounds comprising an oligonucleotide consisting of 13 to 30 linked nucleosides and having a nucleobase sequence complementary to a repeat region of a nucleotide repeat-containing RNA.
  • the oligonucleotide contains:
  • a 5 '-region consisting of 1-5 linked 5 '-region nucleosides, wherein the 5 '-region nucleosides each have the same modification as one another;
  • each nucleoside of the central region either comprises a 2'-fluoro sugar moiety or is a non-2 '-fluoro central- region nucleoside, provided that at least one nucleoside of the central region comprises a 2'-fluoro sugar moiety, and wherein the non-2 '-fluoro central-region nucleosides each have the same modification as one another; and
  • 3'-region nucleosides consisting of 1-5 linked 3'-region nucleosides, wherein the 3'-region nucleosides each have the same modification as one another.
  • the oligonucleotide is 85% complementary to the repeat region of the nucleotide repeat containing RNA. In certain embodiments, the oligonucleotide is 90% complementary to the repeat region of the nucleotide repeat containing RNA. In certain embodiments, the oligonucleotide is 95% complementary to the repeat region of the nucleotide repeat containing RNA. In certain embodiments, the oligonucleotide is 100% complementary to the repeat region of the nucleotide repeat containing RNA.
  • the oligonucleotide is complementary to the nucleotide repeat containing RNA within the repeat region and is not complementary to the nucleobases adjacent to the repeat region of the nucleotide repeat containing RNA.
  • no more than four contiguous nucleosides of the central region are non-2 '-fluoro central-region nucleosides.
  • the 5 '-region nucleosides comprise a modified 2' -sugar moiety.
  • the 5 '-region nucleosides comprise a bicyclic sugar moiety.
  • the bicyclic sugar moiety of the 5'-region nucleosides comprise a 4' to 2' bridge.
  • the 4' to 2' bridge of the 5 '-region nucleosides is ,
  • each Rc and R ⁇ i is independently hydrogen, halogen, substituted or unsubstituted C C 6 alkyl; and each Re is independently hydrogen or substituted or unsubstituted Ci-Ce alkyl.
  • the 4' to 2' bridge of the 5'-region nucleosides is 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-0-2 ⁇ 4'-CH(CH3)-0-2', 4'-(CH2)2-0-2', 4*-CH2-0-N(Re)-2' and 4'-CH2- N(Re)-0-2'- bridge.
  • the bridge of the 5 '-region nucleosides is 4'- CH(CH 3 )-0-2 ⁇
  • non-2'-fluoro central-region nucleosides are 2'- deoxyribonucleosides.
  • the non-2'-fluoro central-region nucleosides comprise a modified 2'-sugar moiety.
  • the non-2 '-fluoro central-region nucleosides comprise a bicyclic sugar moiety.
  • the bicyclic sugar moiety of the non-2'-fluoro central- region nucleosides comprises a 4' to 2' bridge.
  • 4' to 2' bridge of the non-2'-fluoro central-region nucleosides is -[C(Rc)(Rd)] n -, -[C(Rc)(Rd)] n -0-, -C(RcRd)-N(Re)-0- or -C(RcRd)-0-N(Re)-, wherein: each Rc and Rd is independently hydrogen, halogen, substituted or unsubstituted C]-C 6 alkyl; and each Re is independently hydrogen or substituted or unsubstituted CrC 6 alkyl.
  • the 4' to 2' bridge of the non-2 '-fluoro central-region nucleosides is 4 , -(CH 2 )2-2', 4 , -(CH 2 )3-2 , J 4'-CH 2 -0-2', 4'-CH(CH 3 )-0-2*, 4 , -(CH 2 ) 2 -0-2', 4'-CH 2 - 0- ⁇ (3 ⁇ 4)-2' and 4'-CH 2 -N(Re)-0-2'- bridge.
  • the bridge of the non-2 '- fluoro central-region nucleosides is 4'-CH(CH3)-0-2'.
  • the 3 '-region nucleosides comprise a modified 2 '-sugar moiety.
  • the 3 '-region nucleosides comprise a bicyclic sugar moiety.
  • the bicyclic sugar moiety of the 3 '-region nucleosides comprises a 4' to 2' bridge.
  • x 0, 1, or 2;
  • y is 1, 2, 3, or 4;
  • the 4' to 2' bridge of the 3'-region nucleosides is, -[C(Rc)(Rd)] n -, -[C(Rc)(Rd)] n -0-, -C(R c R ⁇ i)-N(Re)-0- or wherein: each 3 ⁇ 4 and 3 ⁇ 4 is independently hydrogen, halogen, substituted or unsubstituted -Ce alkyl; and each 3 ⁇ 4 is independently hydrogen or substituted or unsubstituted Q-Q alkyl.
  • the, 4' to 2' bridge of the 3 '-region nucleosides is 4'-(CH 2 ) 2 -2', 4'-(CH2)3-2*, 4'-CH2-0-2', 4'-CH(CH 3 )-0-2', 4'-(CH2) 2 -0-2', 4*-CH 2 -0-N(R e )-2' and 4'-CH 2 - NCR ⁇ -CW- bridge.
  • the bridge of the 3 '-region nucleosides is 4'- CH(CH 3 )-0-2 ⁇
  • the 5 '-region nucleosides and the 3' -region nucleosides comprise the same modification as one another. In certain embodiments, the 5 '-region nucleosides and the non-2' -fluoro central-region nucleosides comprise the same modification as one another. In certain embodiments, the 5 '-region nucleosides and the 3 '-region nucleosides and the non-2' -fluoro central-region nucleosides all comprise the same modification as one another. In certain embodiments, the 5 '-region nucleosides and the 3 '-region nucleosides and the non-2 '-fluoro central-region nucleosides each comprises a 2'-(CH2) 2 OCH 3 modification.
  • the central region comprises 1 to 10 blocks of non-2 '-fluoro central-region nucleosides, wherein each block independently consists of 1 to 4 non-2 '-fluoro central-region nucleosides. In certain embodiments, the central region comprises 1 to 5 blocks of non-2'-fluoro central-region nucleosides. In certain embodiments, the central region comprises 1 to 2 blocks of non-2'-fluoro central-region nucleosides. In certain embodiments, the central region comprises 1 block of non-2'-fluoro central-region nucleosides. In certain embodiments, the blocks of non-2 '-fluoro central-region nucleosides independently consist of 1-3 non-2'-fluoro central-region nucleosides.
  • the blocks of non-2'-fluoro central-region nucleosides independently consist of 1 or 2 non-2'-fluoro central-region nucleosides. In certain embodiments, the blocks of non-2'-fluoro central-region nucleosides independently consist of 3 or 4 non-2'-fluoro central-region nucleosides. In certain embodiments, the central region consists of linked 2'-fluoro modified nucleosides.
  • the oligonucleotide has the Formula:
  • each uj is a 5 '-region nucleoside
  • each Nu 2 and each Nu 4 is a 2'-fluoro nucleoside
  • each Nu 3 is a non-2 '-fluoro central-region nucleoside
  • each Nu 5 is a 3 '-region nucleoside
  • nl is from 1 to 5;
  • n5 is from 0 to 5;
  • n2 is from 1 to 24 and n4 is from 1 to 24, provided that the sum of n2 and n4 is from 10 to 25; and n3 is from 0 to 4.
  • n3 is from 1 to 4. In certain embodiments, n3 is 0.
  • the oligonucleotide comprises one or more modified
  • each internucleoside linkage is modified. In certain embodiments, each internucleoside linkage of the oligonucleotide is either a
  • the oligonucleotide comprises at least one modified nucleobase.
  • the modified oligonucleotide is a 5-methylcytosine.
  • the repeat region of the nucleotide repeat-containing RNA is a repeating quintet. In certain embodiments, the repeat region of the nucleotide repeat-containing RNA is a repeating quartet. In certain embodiments, the repeat region of the nucleotide repeat- containing RNA is a repeating CCUG or AUUCU.
  • the repeat region of the nucleotide repeat-containing RNA is a repeating triplet.
  • the repeating triplet is selected from: CAG, CUG, CGG, GCC, and GAA.
  • the repeating triplet is CAG.
  • the repeating triplet is CUG.
  • the nucleotide repeat-containing RNA is associated with a disease.
  • the disease is selected from among: ataxin 8, atrophin 1, fragile X syndrome, Friedrich's ataxia, Huntington's disease, Huntington's disease-like 2, myotonic dystrophy, spinal and bulbar muscular atrophy, and spinocerebellar ataxia
  • the disease is Huntington's disease.
  • the disease is myotonic dystrophy.
  • the myotome dystrophy is myotonic dystrophy type 1.
  • the myotome dystrophy is myotonic dystrophy type 2.
  • the disease is spinocerebellar ataxia.
  • the spinocerebellar ataxia is spinocerebellar ataxia 10.
  • the oligomeric compound is a mutant selective compound. In certain embodiments, the oligomeric compound is capable of reducing the activity or amount of a nucleotide repeat-containing RNA at least two fold more than it reduces the activity or amount of a corresponding wild type RNA.
  • the invention provides methods of selectively reducing the activity or amount of a nucleotide repeat-containing RNA in a cell, comprising contacting a cell having a nucleotide repeat-containing RNA with any of the above oligomeric compounds; and thereby selectively reducing the activity or amount of the nucleotide repeat-containing RNA in the cell.
  • the amount or activity of the nucleotide repeat-containing RNA is reduced at least two-fold more than that of a corresponding wild-type RNA.
  • the cell is in vitro. In certain embodiments, the cell is in an animal.
  • the invention provides pharmaceutical compositions comprising one or more oligomeric compound and a pharmaceutical carrier or diluent.
  • the invention provides methods of treating a patient having a disease associated with a nucleotide repeat-containing RNA comprising administering to the patient a pharmaceutical composition comprising any of the above oligomeric compounds.
  • the disease is selected from among: ataxin 8, atrophin 1, fragile X syndrome, Friedrich's ataxia, Huntington's disease, Huntington's disease-like 2, myotonic dystrophy, spinal and bulbar muscular atrophy, and spinocerebellar ataxia.
  • the disease is Huntington's disease.
  • the disease is myotonic dystrophy.
  • the myotonic dystrophy is myotonic dystrophy type 1.
  • the myotonic dystrophy is myotonic dystrophy type 2.
  • the disease is spinocerebellar ataxia.
  • the spinocerebellar ataxia is spinocerebellar ataxia 10.
  • the pharmaceutical composition is administered by injection.
  • the pharmaceutical composition is injected into the central nervous system.
  • the pharmaceutical composition is injected into the brain.
  • the injection is a bolus injection.
  • the injection is an infusion.
  • nucleotide repeat-containing RNA means a mutant RNA molecule having a nucleobase sequence that includes a repeat region consisting essentially of repeating units of 3-5 nucleobases that repeat at least 10 times in the repeating region, and wherein the presence or length of the repeat region affects the normal processing, function, or activity of the RNA.
  • corresponding wild type RNA means the non-mutant version of the nucleotide repeat-containing RNA having normal function and activity.
  • corresponding wild type RNA molecules comprise a repeat region which is shorter than that of a nucleotide repeat-containing RNA.
  • nucleoside refers to a compound comprising a heterocyclic base moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA), abasic nucleosides, modified nucleosides, and sugar-modified nucleosides. Nucleosides may be modified with any of a variety of substituents.
  • sugar moiety means a natural (furanosyl), a modified sugar moiety or a sugar surrogate.
  • modified sugar moiety means a chemically-modified furanosyl sugar or a non-furanosyl sugar moiety. Also, embraced by this term are furanosyl sugar analogs and derivatives including bicyclic sugars, tetrahydropyrans, morpholinos, 2'-modified sugars, 4'- modified sugars, 5 '-modified sugars, and 4'-subsituted sugars.
  • sugar-modified nucleoside means a nucleoside comprising a modified sugar moiety.
  • sugar surrogate refers to a structure that is capable of replacing the furanose ring of a naturally occurring nucleoside.
  • sugar surrogates are non-furanose (or 4'-substituted furanose) rings or ring systems or open systems.
  • Such structures include simple changes relative to the natural furanose ring, such as a six membered ring or may be more complicated as is the case with the non-ring system used in peptide nucleic acid.
  • Sugar surrogates includes without limitation morpholinos and cyclohexenyls and cyclohexitols. In most nucleosides having a sugar surrogate group the heterocyclic base moiety is generally maintained to permit hybridization.
  • nucleotide refers to a nucleoside further comprising a modified or unmodified phosphate linking group or a non-phosphate internucleoside linkage.
  • linked nucleosides may or may not be linked by phosphate linkages and thus includes “linked nucleotides.”
  • nucleobase refers to the heterocyclic base portion of a nucleoside. Nucleobases may be naturally occurring or may be modified and therefore include, but are not limited to adenine, cytosine (including, a 5-methylcytosine), guanidine, uracil, thymidine and analogues thereof. In certain embodiments, a nucleobase may comprise any atom or group of atoms capable of hydrogen bonding to a base of another nucleic acid.
  • modified nucleoside refers to a nucleoside comprising at least one modification compared to naturally occurring RNA or DNA nucleosides. Such modification may be at the sugar moiety and/or at the nucleobases.
  • non-2 '-fiuoro nucleoside refers to any nucleoside other than one having fluorine at the 2 '-position of the sugar.
  • non-2 '-fiuoro nucleosides are modified nucleosides, provided the modification is other than 2'-fluoro.
  • non-2'-fluoro nucleosides are unmodified nucleosides, such as DNA.
  • T m means melting temperature which is the temperature at which the two strands of a duplex nucleic acid separate. T m is often used as a measure of duplex stability or the binding affinity of an antisense compound toward a complementary RNA molecule.
  • a "high-affinity sugar modification” is a modified sugar moiety which when it is included in a nucleoside and said nucleoside is incorporated into an antisense oligonucleotide, the stability (as measured by T m ) of said antisense oligonucleotide: RNA duplex is increased as compared to the stability of a DNA:RNA duplex.
  • a "high-affinity sugar-modified nucleoside” is a nucleoside comprising a modified sugar moiety that when said nucleoside is incorporated into an antisense compound, the binding affinity (as measured by T m ) of said antisense compound toward a complementary RNA molecule is increased.
  • at least one of said sugar- modified high-affinity nucleosides confers a AT m of at least 1 to 4 degrees per nucleoside against a complementary RNA as determined in accordance with the methodology described in Freier et al., Nucleic Acids Res., 1997, 25, 4429-4443, which is incorporated by reference in its entirety.
  • At least one of the high-affinity sugar modifications confers about 2 or more, 3 or more, or 4 or more degrees per modification.
  • examples of sugar-modified high affinity nucleosides include, but are not limited to, (i) certain 2 '-modified nucleosides, including 2'-subtstituted and 4' to 2' bicyclic nucleosides, and (ii) certain other non-ribofuranosyl nucleosides which provide a per modification increase in binding affinity such as modified tetrahydropyran and tricycloDNA nucleosides.
  • modifications that are sugar-modified high-affinity nucleosides see Freier et al., Nucleic Acids Res., 1997, 25, 4429-4443.
  • nuclease resistant nucleotide means a chemically modified nucleotide comprising one or both of a modified sugar or modified internucleoside linkage which, when incorporated into an oligonucleotide, makes said oligonucleotide more stable to degradation under cellular nucleases (exo- or endo-nucleases).
  • nuclease resistant nucleotides but are not limited to, phosphorothioate nucleotides, bicyclic sugar nucleotides, 2 '-modified nucleotides
  • bicyclic nucleoside refer to a modified nucleoside comprising a bicyclic sugar moiety.
  • examples of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4' and the 2' ribosyl ring atoms.
  • oligomeric compounds provided herein include one or more bicyclic nucleosides wherein the bridge comprises a 4' to 2' bicyclic nucleoside.
  • 4' to 2' bicyclic nucleosides include but are not limited to one of the formulae: 4'-(CH 2 )-0-2' (LNA); 4'-(CH 2 )-S-2'; 4'- (CH 2 ) 2 -0-2' (ENA); 4 * -CH(CH 3 )-0-2' and 4'-CH(CH 2 OCH 3 )-0-2' (and analogs thereof see U.S.
  • Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example a-L-ribofuranose and ⁇ -D-ribofuranose (see PCT international application
  • x 0, 1, or 2;
  • n 1, 2, 3, or 4;
  • the bridge of a bicyclic sugar moiety is , -[C(R a )(Rb)] n -,
  • the bridge is 4'-CH 2 -2', 4 * -(CH 2 ) 2 -2', 4 * -(CH 2 ) 3 -2', 4'-CH 2 -0-2', 4'-(CH 2 ) 2 -0-2', 4'-CH 2 -0-N(R)-2' and 4'- CH2-N(R)-0-2'- wherein each Ris, independently, H, a protecting group or C 1 -C 12 alkyl.
  • bicyclic nucleosides are further defined by isomeric configuration.
  • a nucleoside comprising a 4'-2' methylene-oxy bridge may be in the a-L configuration or in the ⁇ -D configuration.
  • a-L-methyleneoxy (4'- ⁇ 3 ⁇ 4-0-2') BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et ah, Nucleic Acids Research, 2003, 21, 6365-6372).
  • bicyclic nucleosides include, but are not limited to, (A) a-L- Methyleneoxy (4'-CH 2 -0-2') BNA , (B) ⁇ -D-Methyleneoxy (4'-CH 2 -0-2') BNA ("LNA") , (C) Ethyleneoxy (4 , -(CH 2 ) 2 -0-2') BNA ("ENA”) , (D) Aminooxy (4'-CH 2 -0-N(R)-2') BNA, (E) Oxyamino (4'-CH 2 -N(R)-0-2') BNA, and (F) Methyl(methyleneoxy) (4'-CH(CH 3 )-0-2') BNA (“cEt”), (G) methylene-thio (4'-CH 2 -S-2') BNA, (H) methylene-amino (4'-CH2-N(R)-2') BNA, (I) methyl carbocyclic (4
  • Bx is the base moiety and R is independently H, a protecting group or C 1 -C 12 alkyl.
  • bicyclic nucleoside having Formula I having Formula I:
  • Bx is a heterocyclic base moiety
  • Rc is d-Ci2 alkyl or an amino protecting group
  • T a and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.
  • bicyclic nucleoside having Formula II having Formula II:
  • Bx is a heterocyclic base moiety
  • T a and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
  • Z a is Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Q-C6 alkyl, substituted C 2 -C 6 alkenyl, substituted C 2 -C 6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio.
  • bicyclic nucleoside having Formula III having Formula III:
  • Bx is a heterocyclic base moiety
  • T a and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
  • bicyclic nucleoside having Formula IV having Formula IV:
  • Bx is a heterocyclic base moiety
  • T a and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
  • Rd is C!-C 6 alkyl, substituted Ci-C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl or substituted C 2 -C 6 alkynyl;
  • each q a , qb, q c and qd is, independently, H, halogen, d-C6 alkyl, substituted Q-Q alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl or substituted C 2 -C 6 alkynyl, d-C6 alkoxyl, substituted C C 6 alkoxyl, acyl, substituted acyl, C]-C aminoalkyl or substituted d-C 6 aminoalkyl;
  • Bx is a heterocyclic base moiety
  • T a and T3 ⁇ 4 are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
  • q g and qi are each, independently, H, halogen, C1-C12 alkyl or substituted Cj-Cn alkyl.
  • Bx is a heterocyclic base moiety
  • 4'-2' bicyclic nucleoside or “4' to 2' bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2' carbon atom and the 4' carbon atom of the sugar ring.
  • nucleosides refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties.
  • sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.
  • 2 '-modified sugar means a furanosyl sugar modified at the 2' position.
  • such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl.
  • 2' modifications are selected from substituents including, but not limited to:
  • 2 - substituent groups can also be selected from: C1-C12 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O- alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, CF 3 , OCF 3 , SOCH 3 , S0 2 CH 3 , ON0 2 , N0 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving
  • modifed nucleosides comprise a 2'-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2 -MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2'- O- methyl, O-propyl, and O-aminopropyl.
  • Oligonucleotides having the 2 -MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, ?., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al, Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).
  • a "modified tetrahydropyran nucleoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran "sugar” substituted in for the pentofuranosyl residue in normal nucleosides.
  • Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) ⁇ see Leumann, CJ. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA) or those compounds having Formula X:
  • Bx is a heterocyclic base moiety
  • T 3 and T 4 are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the oligomeric compound or one of T 3 and T 4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the oligomeric compound and the other of T3 and T 4 is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3'-terminal group;
  • qi > ⁇ > 3 ⁇ 43, 3 ⁇ 44 > q 5 , q 6 a d q 7 are each independently, H, Ci-C alkyl, substituted Ci-C ⁇ alkyl, C2- 5 alkenyl, substituted C2-C 6 alkenyl, C 2 -C 6 alkynyl or substituted C 2 -C 6 alkynyl; and
  • R] and R2 is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ1J2, SJi, N 3 , and CN, wherein X is O, S or NJi and each Ji, J 2 and J3 is, independently, H or Ci-C 6 alkyl.
  • the modified ⁇ nucleosides of Formula X are provided wherein q m , q cicl, q p , q r , q s , q t and q u are each H. In certain embodiments, at least one of q m , q n , q p , q r , q s , q t and q u is other than H. In certain embodiments, at least one of q m , q n , q p , q r , q s , qt and q u is methyl.
  • THP nucleosides of Formula X are provided wherein one of R] and R 2 is F.
  • Rt is fluoro and R2 is H; Ri is methoxy and R2 is H, and R] is methoxyethoxy and R 2 is H.
  • 2'-modified or “2 '-substituted” refers to a nucleoside comprising a sugar comprising a substituent at the 2' position other than H or OH.
  • 2'-modifed nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.
  • 2'-F refers to a nucleoside comprising a sugar comprising a fluoro group at the 2' position. Unless otherwise indicated, the sugar of a 2'-F nucleoside is a furanose in which the 2'-OH has been replace with a F.
  • 2'-F RNA refers to a 2'-F nucleoside, wherein the fluoro group is in the ribo position.
  • 2'-F ANA refers to a 2'-F substituted nucleoside, wherein the fluoro group is in the arabino position.
  • ANA'Other than in the context of "2'F ANA” refers to altritol nucleic acid, which is a modified tetrahydropyran nucleoside as desrcribed above. Unless further modified or otherwise indicated, "ANA" nucleosides have the following structure:
  • 2'-OMe or “2'-OCH 3 " or “2'-0-methyl” each refers to a nucleoside comprising a sugar comprising an -OCH 3 group at the 2' position of the sugar ring.
  • MOE or "2'-MOE” or “2'-OCH 2 CH 2 OCH3" or “2'-0-methoxyethyl” each refers to a nucleoside comprising a sugar comprising a -OCH 2 CH 2 OCH 3 group at the 2' position of the sugar ring.
  • oligonucleotide refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA). As used herein, “modified oligonucleotide” refers to an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
  • nucleoside linkage refers to a covalent linkage between adjacent nucleosides.
  • naturally occurring internucleoside linkage refers to a 3' to 5' phosphodiester linkage.
  • modified internucleoside linkage refers to any internucleoside linkage other than a naturally occurring internucleoside linkage.
  • oligomeric compound refers to a polymeric structure comprising two or more sub-structures.
  • an oligomeric compound comprises an
  • an oligomeric compound comprises a single-stranded oligonucleotide. In certain embodiments, an oligomeric compound is a double-stranded duplex comprising two oligonucleotides. In certain embodiments, an oligomeric compound is a single- stranded or double-stranded oligonucleotide comprising one or more conjugate groups and/or terminal groups.
  • conjugate refers to an atom or group of atoms bound to an
  • conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to
  • Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to the parent compound such as an oligomeric compound.
  • conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.
  • conjugates are terminal groups.
  • conjugates are attached to a 3' or 5' terminal nucleoside or to an internal nucleosides of an oligonucleotide.
  • conjugate linking group refers to any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound. Linking groups or
  • bifunctional linking moieties such as those known in the art are amenable to the present invention.
  • antisense compound refers to an oligomeric compound, at least a portion of which is at least partially complementary to a target nucleic acid to which it hybridizes and modulates the activity, processing or expression of said target nucleic acid.
  • expression refers to the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, splicing, post-transcriptional modification, and translation.
  • antisense oligonucleotide refers to an antisense compound that is an oligonucleotide.
  • antisense activity refers to any detectable and/or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid.
  • such activity may be an increase or decrease in an amount of a nucleic acid or protein.
  • such activity may be a change in the ratio of splice variants of a nucleic acid or protein.
  • Detection and/or measuring of antisense activity may be direct or indirect.
  • antisense activity is assessed by observing a phenotypic change in a cell or animal.
  • detecting or “measuring” in connection with an activity, response, or effect indicate that a test for detecting or measuring such activity, response, or effect is performed.
  • detection and/or measuring may include values of zero.
  • a test for detection or measuring results in a finding of no activity (activity of zero)
  • the step of detecting or measuring the activity has nevertheless been performed.
  • the present invention provides methods that comprise steps of detecting antisense activity, detecting toxicity, and/or measuring a marker of toxicity. Any such step may include values of zero.
  • target nucleic acid refers to any nucleic acid molecule the expression, amount, or activity of which is capable of being modulated by an antisense compound.
  • the target nucleic acid is DNA or RNA.
  • the target RNA is mRNA, pre-mRNA, non-coding RNA, pri-microRNA, pre-microRNA, mature microRNA, promoter-directed RNA, or natural antisense transcripts.
  • the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent.
  • target nucleic acid is a viral or bacterial nucleic acid.
  • target mR A refers to a pre-selected RNA molecule that encodes a protein.
  • selectivity refers to the ability of an antisense compound to exert an antisense activity on a target nucleic acid to a greater extent than on a non-target nucleic acid.
  • mutant selective refers to a compound that has a greater effect on a mutant nucleic acid than on the corresponding wild-type nucleic acid.
  • the effect of a mutant selective compound on the mutant nucleic acid is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 100 times greater than the effect of the mutant selective compound on the
  • selectivity results from greater affinity of the mutant selective compound for the mutant nucleic acid than for the corresponding wild type nucleic acid.
  • selectivity results from a difference in the structure of the mutant compared to the wild-type nucleic acid.
  • selectivity results from differences in processing or sub-cellular distribution of the mutant and wild-type nucleic acids.
  • some selectivity may be attributable to the presence of additional target sites in a mutant nucleic acid compared to the wild-type nucleic acid.
  • a target mutant allele comprises an expanded repeat region comprising additional copies of a target sequence, while the wild-type allele has fewer copies of the repeat and, thus, fewer sites for hybridization of an antisense compound targeting the repeat region.
  • a mutant selective compound has selectivity equal to or greater than the selectivity predicted by the increased number of target sites. In certain embodiments, a mutant selective compound has selectivity greater than the selectivity predicted by the increased number of target sites.
  • the ratio of inhibition of a mutant allele to a wild type allele is equal to or greater than the ratio of the number of repeats in the mutant allele to the wild type allele. In certain embodiments, the ratio of inhibition of a mutant allele to a wild type allele is greater than the ratio of the number of repeats in the mutant allele to the wild type allele.
  • targeting or “targeted to” refers to the association of an antisense compound to a particular target nucleic acid molecule or a particular region of nucleotides within a target nucleic acid molecule.
  • An antisense compound targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions.
  • target site refers to a region of a target nucleic acid that is bound by an antisense compound.
  • a target site is at least partially within the 3' untranslated region of an RNA molecule.
  • a target site is at least partially within the 5' untranslated region of an RNA molecule.
  • a target site is at least partially within the coding region of an RNA molecule.
  • a target site is at least partially within an exon of an RNA molecule.
  • a target site is at least partially within an intron of an RNA molecule.
  • a target site is at least partially within a microRNA target site of an RNA molecule.
  • a target site is at least partially within a repeat region of an RNA molecule.
  • target protein refers to a protein, the expression of which is modulated by an antisense compound.
  • a target protein is encoded by a target nucleic acid.
  • expression of a target protein is otherwise influenced by a target nucleic acid.
  • complementary nucleobase refers to a nucleobase that is capable of base pairing with another nucleobase.
  • adenine (A) is complementary to thymine (T).
  • adenine (A) is complementary to uracil (U).
  • complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid.
  • nucleobases at a certain position of an antisense compound are capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid
  • the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.
  • Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.
  • non-complementary in reference to nucleobases refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support
  • complementary in reference to linked nucleosides, oligonucleotides, or nucleic acids, refers to the capacity of an oligomeric compound to hybridize to another oligomeric compound or nucleic acid through nucleobase complementarity.
  • an antisense compound and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bond with each other to allow stable association between the antisense compound and the target.
  • nucleobases that can bond with each other to allow stable association between the antisense compound and the target.
  • antisense compounds may comprise up to about 20% nucleotides that are mismatched (i.e., are not nucleobase complementary to the corresponding nucleotides of the target).
  • the antisense compounds contain no more than about 15%, more preferably not more than about 10%, most preferably not more than 5% or no mismatches.
  • the remaining nucleotides are nucleobase complementary or otherwise do not disrupt hybridization (e.g., universal bases).
  • One of ordinary skill in the art would recognize the compounds provided herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to a target nucleic acid.
  • hybridization refers to the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases).
  • nucleobases complementary nucleoside or nucleotide bases
  • the natural base adenine is nucleobase complementary to the natural nucleobases thymidine and uracil which pair through the formation of hydrogen bonds.
  • the natural base guanine is nucleobase
  • Hybridization can occur under varying circumstances.
  • oligomeric compound specifically hybridizes to more than one target site.
  • modulation refers to a perturbation of amount or quality of a function or activity when compared to the function or activity prior to modulation.
  • modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression.
  • modulation of expression can include perturbing splice site selection of pre-mRNA processing, resulting in a change in the amount of a particular splice-variant present compared to conditions that were not perturbed.
  • modulation includes perturbing translation of a protein.
  • motif refers to a pattern of modifications in an oligomeric compound or a region thereof. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligomeric compound.
  • nucleoside motif refers to a pattern of nucleoside modifications in an oligomeric compound or a region thereof.
  • the linkages of such an oligomeric compound may be modified or unmodified.
  • motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.
  • linkage motif refers to a pattern of linkage modifications in an oligomeric compound or region thereof.
  • the nucleosides of such an oligomeric compound may be modified or unmodified.
  • motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.
  • the same modifications refer to modifications relative to naturally occurring molecules that are the same as one another, including absence of modifications.
  • two unmodified DNA nucleoside have “the same modification,” even though the DNA nucleoside is unmodified.
  • nucleoside having a modification of a first type may be an unmodified nucleoside.
  • separate regions refers to a portion of an oligomeric compound wherein the nucleosides and internucleoside linkages within the region all comprise the same
  • nucleosides and/or the internucleoside linkages of any neighboring portions include at least one different modification.
  • pharmaceutically acceptable salts refers to salts of active compounds that retain the desired biological activity of the active compound and do not impart undesired toxicological effects thereto.
  • cap structure or “terminal cap moiety” refers to chemical modifications incorporated at either terminus of an antisense compound.
  • alkyl refers to a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.
  • Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C1-C12 alkyl) with from 1 to about 6 carbon atoms (CrC 6 alkyl) being more preferred.
  • lower alkyl as used herein includes from 1 to about 6 carbon atoms (Ci- C 6 alkyl).
  • Alkyl groups as used herein may optionally include one or more further substituent groups.
  • alkyl without indication of number of carbon atoms means an alkyl having 1 to about 12 carbon atoms (Ci-C 12 alkyl).
  • alkenyl refers to a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond.
  • alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 1-methyl- 2-buten-l-yl, dienes such as 1,3 -butadiene and the like.
  • Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred.
  • Alkenyl groups as used herein may optionally include one or more further substituent groups.
  • alkynyl refers to a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond.
  • alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, and the like.
  • Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred.
  • Alkynyl groups as used herein may optionally include one or more further substituent groups.
  • aminoalkyl refers to an amino substituted alkyl radical. This term is meant to include C1-C12 alkyl groups having an amino substituent at any position and wherein the alkyl group attaches the aminoalkyl group to the parent molecule. The alkyl and/or amino portions of the aminoalkyl group can be further substituted with substituent groups.
  • aliphatic refers to a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond.
  • An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred.
  • the straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus.
  • Such aliphatic groups interrupted by heteroatoms include without limitation polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.
  • alicyclic or “alicyclyl” refers to a cyclic ring system wherein the ring is aliphatic.
  • the ring system can comprise one or more rings wherein at least one ring is aliphatic.
  • Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring.
  • Alicyclic as used herein may optionally include further substituent groups.
  • alkoxy refers to a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule.
  • alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, w-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like.
  • Alkoxy groups as used herein may optionally include further substituent groups.
  • halo and “halogen,” refer to an atom selected from fluorine, chlorine, bromine and iodine.
  • aryl and “aromatic,” refer to a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings.
  • aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like.
  • Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings.
  • Aryl groups as used herein may optionally include further substituent groups.
  • aralkyl and arylalkyl refer to a radical formed between an alkyl group and an aryl group wherein the alkyl group is used to attach the aralkyl group to a parent molecule. Examples include, but are not limited to, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.
  • heterocyclic radical refers to a radical mono-, or poly-cyclic ring system that includes at least one heteroatom and is unsaturated, partially saturated or fully saturated, thereby including heteroaryl groups. Heterocyclic is also meant to include fused ring systems wherein one or more of the fused rings contain at least one heteroatom and the other rings can contain one or more heteroatoms or optionally contain no heteroatoms.
  • a heterocyclic group typically includes at least one atom selected from sulfur, nitrogen or oxygen.
  • heterocyclic groups include, [l,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and the like.
  • Heterocyclic groups as used herein may optionally include further substituent groups.
  • heteroaryl and “heteroaromatic,” refer to a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatom. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms.
  • Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen.
  • heteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.
  • Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom.
  • Heteroaryl groups as used herein may optionally include further substituent groups.
  • heteroarylalkyl refers to a heteroaryl group as previously defined having an alky radical that can attach the heteroarylalkyl group to a parent molecule. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl, napthyridinylpropyl and the like. Heteroarylalkyl groups as used herein may optionally include further substituent groups on one or both of the heteroaryl or alkyl portions.
  • mono or poly cyclic structure refers to any ring systems that are single or polycyclic having rings that are fused or linked and is meant to be inclusive of single and mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic, heteroarylalkyl.
  • Such mono and poly cyclic structures can contain rings that are uniform or have varying degrees of saturation including fully saturated, partially saturated or fully unsaturated.
  • Each ring can comprise ring atoms selected from C, N, O and S to give rise to heterocyclic rings as well as rings comprising only C ring atoms which can be present in a mixed motif such as for example benzimidazole wherein one ring has only carbon ring atoms and the fused ring has two nitrogen atoms.
  • mono or poly cyclic structures can be attached to a parent molecule directly through a ring atom, through a substituent group or a bifunctional linking moiety.
  • acyl refers to a radical formed by removal of a hydroxyl group from an organic acid an d has the general formula -C(0)-X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.
  • hydrocarbyl refers to any group comprising C, O and H. Included are straight, branched and cyclic groups having any degree of saturation. Such hydrocarbyl groups can include one or more heteroatoms selected from N, O and S and can be further mono or poly substituted with one or more substituent groups.
  • substituted and substituteduent group include groups that are typically added to other groups or parent compounds to enhance desired properties or give desired effects. Substituent groups can be protected or unprotected and can be added to one available site or to many available sites in a parent compound. Substituent groups may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.
  • each R aa , Rbb and Roc is, independently, H, an optionally linked chemical functional group or a further substituent group which may be selected from, without limitation: H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl.
  • Selected substituents within the compounds described herein are present to a recursive degree.
  • "recursive substituent” means that a substituent may recite another instance of itself. Because of the recursive nature of such substituents, theoretically, a large number may be present in any given claim.
  • Recursive substituents are an intended aspect of the invention.
  • One of ordinary skill in the art of medicinal and organic chemistry understands the versatility of such substituents.
  • stable compound and “stable structure” as used herein are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Only stable compounds are contemplated herein.
  • a zero (0) in a range indicating number of a particular unit means that the unit may be absent.
  • an oligomeric compound comprising 0-2 regions of a particular motif means that the oligomeric compound may comprise one or two such regions having the particular motif, or the oligomeric compound may not have any regions having the particular motif.
  • the portions flanking the absent portion are bound directly to one another.
  • the term "none" as used herein indicates that a certain feature is not present.
  • analogue or derivative means either a compound or moiety similar in structure but different in respect to elemental composition from the parent compound regardless of how the compound is made. For example, an analogue or derivative compound does not need to be made from the parent compound as a chemical starting material.
  • the present invention provides compounds and methods for modulating the amount, activity or function of a nucleotide repeat-containing RNA.
  • nucleotide repeat-containing RNA molecules have been associated with a number of diseases or disorders.
  • Certain normal wild-type RNA molecules comprise repeat regions, which, in certain instances can become expanded.
  • the shorter repeat regions of wild type transcripts not associated with disease typically have secondary structure, making them relatively inaccessible for base pairing with a complementary nucleic acid.
  • the number of repeats in the expanded repeat region of a nucleotide repeat-containing RNA is typically at least 2 fold normal and often more (e.g., 3, 5, 10 fold, up to 100 or even more than 1000 fold). This expansion increases the likelihood that part of the repeat is, at least temporarily, more accessible to base pairing with a complementary nucleic acid molecule, relative to the wild type allele.
  • oligomeric compounds of the present invention comprise oligonucleotides are complementary to a repeat sequence present in both wild-type and repeat-expanded transcripts, in certain embodiments, such compounds selectively hybridize to the disease-associated repeat- expanded transcript. Such selectivity is beneficial for treating diseases associated with nucleotide repeat-containing RNA irrespective of the mechanism of reduction of the aberrant transcript.
  • nucleotide repeat-containing RNA have been referred to in the art as "gain-of- function RNAs" for their ability to sequester hnRNPs and impair the normal action of RNA processing in the nucleus (see e.g., Cooper, T. (2009) Cell 136, 777-793; O'Rourke, JR (2009) J. Biol. Chem. 284 (12), 7419-7423, which are herein incorporated by reference in the entirety).
  • Several disease states are associated with nucleotide repeat-containing RNA, some of which only occur once a threshold number of repeats within the nucleotide repeat-containing RNA is reached.
  • the present invention provides methods of reducing the activity, function, or amount of a nucleotide repeat-containing RNA having at least 10, 15, 20, , 25, 30, 35, 40, 45, 50, 55, 60, 80, 90, 100, 200, 300, 400, 500, 1000, or more than 1000 copies of a repeating nucleotide unit.
  • the present invention provides compounds and methods for ;eting or treating any of the disorders in the following none limiting table:
  • compounds of the present invention are used to alter the activity or amount of nucleotide repeat-containing RNA.
  • compounds of the present invention are mutant selective. Accordingly, certain such compounds reduce the amount or activity of nucleotide repeat-containing RNA to a greater extent than they reduce the amount or activity of the corresponding wild-type RNA.
  • the present invention provides oligomeric compounds useful for studying, diagnosing, and/or treating a disease or disorder associtaed with a nucleotide repeat- containing RNA.
  • oligomeric compounds of the present invention comprise an oligonucleotide and a conjugate or terminal group.
  • oligomeric compounds consist of an oligonucleotide.
  • oligonucleotide of the present invention have a nucleobase sequence comprising a region that is complementary to a nucleotide repeat-containing RNA. In certain embodiments, oligonucleotide of the present invention have a nucleobase sequence comprising a region that is complementary to a repeat region of a nucleotide repeat-containing RNA.
  • oligonucleotides of the present invention comprise one or more modification. In certain embodiments, oligonucleotides of the present invention comprise one or more modifed nucleoside. In certain embodiments, modified nucleosides of the present invention comprise a modifed nucleobase. In certain embodiments, nucleosides of the present invention comprise one or more modified sugar. In certain embodiments, oligonucleotides of the present invention comprise one or more high-affinity sugar modified nucleoside. In certain embodiments, oligonucleotides of the present invention comprise one or more modfied internucleoside linkage. In certain embodiments, oligonucleotides of the present invention comprise one or more nuclease resistant nucleotide. a. Certain Nucleobases
  • nucleosides of the present invention comprise unmodified nucleobases. In certain embodiments, nucleosides of the present invention comprise modifed nucleobases.
  • nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to the oligomeric compounds.
  • "unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases also referred to herein as heterocyclic base moieties include other synthetic and natural nucleobases, many examples of which such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, 7-deazaguanine and 7-deazaadenine among others.
  • Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone.
  • Certain modified nucleobases are disclosed in, for example, Sw yze, E.E. and Bhat, B., The Medicinal Chemistry of Oligonucleotides in ANTISENSE DRUG TECHNOLOGY, Chapter 6, pages 143-182 (Crooke, S.T., ed., 2008); U.S. Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858- 859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al.,
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2
  • nucleobases comprise polycyclic heterocyclic compounds in place of one or more heterocyclic base moieties of a nucleobase.
  • a number of tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand. The most studied modifications are targeted to guanosines hence they have been termed G- clamps or cytidine analogs.
  • cytosine analogs that make 3 hydrogen bonds with a guanosine in a second strand include l,3-diazaphenoxazine-2-one (Kurchavov, et ah, Nucleosides and
  • oligonucleotides The T m data indicate an even greater discrimination between the perfect match and mismatched sequences compared to dC5 me . It was suggested that the tethered amino group serves as an additional hydrogen bond donor to interact with the Hoogsteen face, namely the 06, of a complementary guanine thereby forming 4 hydrogen bonds. This means that the increased affinity of G-clamp is mediated by the combination of extended base stacking and additional specific hydrogen bonding.
  • Tricyclic heterocyclic compounds and methods of using them that are amenable to the present invention are disclosed in U.S. Patent 6,028,183, and U.S. Patent 6,007,992, the contents of both are incorporated herein in their entirety.
  • Modified polycyclic heterocyclic compounds useful as heterocyclic bases are disclosed in but not limited to, the above noted U.S. 3,687,808, as well as U.S.: 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187; 5,459,255; 5,484,908;
  • oligonucleotides of the present invention comprise one or more modified nucleoside comprising a modified sugar moiety.
  • modified sugar moieties are known in the art to improve certain properties of oligonucleotides.
  • oligonucleotides having certain modified sugar moieties have increased resistance to digestion by ribonucleases.
  • nuclease resistance sugar modifications are suitable for nucleosides for use in the present invention.
  • Oligonucleotides incorporating certain modified sugar moieties have increased affinity for a target nucleic acid. In certain embodiments, such high affinity sugar modifications are suitable for use in the present invention.
  • the present invention includes oligonucleotides having one or more high-affinity sugar modified nucleoside. In certain embodiments, the present invention includes oligonucleotides having one or more bicyclic nucleoside. In certain embodiments, the present invention includes oligonucleotides having one or more nucleoside comprising a 2 '-modified sugar. In certain embodiments, the present invention includes oligonucleotides having one or more modified tetrahydropyran nucleoside.
  • RNA duplexes exist in what has been termed "A Form” geometry while DNA duplexes exist in “B Form” geometry.
  • RNA:RNA duplexes are more stable, or have higher melting temperatures (T m ) than DNA:DNA duplexes (Sanger et al, Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, NY.; Lesnik et al, Biochemistry, 1995, 34, 10807- 10815; Conte et al, Nucleic Acids Res., 1997, 25, 2627-2634).
  • RNA duplex The increased stability of RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et al, Nucleic Acids Res., 1993, 21, 2051-2056).
  • the presence of the 2' hydroxyl in RNA biases the sugar toward a C3' endo pucker, i.e., also designated as Northern pucker, which causes the duplex to favor the A-form geometry.
  • the 2' hydroxyl groups of RNA can form a network of water mediated hydrogen bonds that help stabilize the RNA duplex (Egli et al, Biochemistry, 1996, 35, 8489-8494).
  • deoxy nucleic acids prefer a C2' endo sugar pucker, i.e., also known as Southern pucker, which is thought to impart a less stable B-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York, NY).
  • the relative ability of a chemically-modified oligomeric compound to bind to complementary nucleic acid strands, as compared to natural oligonucleotides, is measured by obtaining the melting temperature of a hybridization complex of said chemically-modified oligomeric compound with its complementary unmodified target nucleic acid.
  • the melting temperature (T m ) a characteristic physical property of double helixes, denotes the temperature in degrees centigrade at which 50% helical versus coiled (unhybridized) forms are present.
  • T m also commonly referred to as binding affinity
  • Base stacking which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently a reduction in UV absorption indicates a higher T m .
  • RNA target duplex can be modulated through incorporation of chemically-modified nucleosides into the antisense compound.
  • Sugar-modified nucleosides have provided the most efficient means of modulating the T m of an antisense compound with its target RNA.
  • Sugar-modified nucleosides that increase the population of or lock the sugar in the C3 1 -endo (Northern, RNA-like sugar pucker) configuration have predominantly provided a per modification T m increase for antisense compounds toward a complementary RNA target.
  • Sugar-modified nucleosides that increase the population of or lock the sugar in the C2'-endo (Southern, DNA-like sugar pucker) configuration predominantly provide a per modification Tm decrease for antisense compounds toward a complementary RNA target.
  • the sugar pucker of a given sugar-modified nucleoside is not the only factor that dictates the ability of the nucleoside to increase or decrease an antisense compound's T m toward complementary RNA.
  • the sugar-modified nucleoside tricycloDNA is predominantly in the Q -endo conformation, however it imparts a 1.9 to 3° C per modification increase in T m toward a complementary RNA.
  • Another example of a sugar- modified Wgh-affinity nucleoside that does not adopt the CV-endo conformation is a-L-LNA.
  • nucleosides are linked together using any intemucleoside linkage.
  • the two main classes of intemucleoside linking groups are defined by the presence or absence of a phosphorus atom.
  • non-phosphorus containing intemucleoside linking groups include, but are not limited to, methylenemethylimino (-CH 2 - N(CH 3 )-0-CH 2 -), thiodiester (-O-C(O)-S-), thionocarbamate (-0-C(0)(NH)-S-); siloxane (-0- Si(H)2-0-); and ⁇ , ⁇ '-dimethylhydrazine (-CH 2 -N(CH 3 )-N(CH 3 )-).
  • Oligonucleotides having non-phosphorus intemucleoside linking groups may be referred to as oligonucleosides.
  • Modified linkages compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligomeric compound.
  • intemucleoside linkages having a chiral atom can be prepared a racemic mixture, as separate enantomers.
  • chiral linkages include, but are not limited to, alkylphosphonates and
  • oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), a or ⁇ such as for sugar anomers, or as (D) or (L) such as for amino acids et al. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
  • intemucleoside linkage or "intemucleoside linking group” is meant to include all manner of intemucleoside linking groups known in the art including but not limited to, phosphorus containing intemucleoside linking groups such as phosphodiester and phosphorothioate, and non-phosphorus containing intemucleoside linking groups such as formacetyl and memyleneimino.
  • neutral intemucleoside linkage is intended to include intemucleoside linkages that are non-ionic.
  • thioformacetal (3'-S-CH -0-5').
  • Further neutral intemucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65).
  • Further neutral intemucleoside linkages include nonionic linkages comprising mixed N, O, S and CH 2 component parts.
  • the intemucleoside linkage found in native nucleic acids is a phosphodiester linkage. This linkage has not been the linkage of choice for synthetic oligonucleotides that are for the most part targeted to a portion of a nucleic acid such as mRNA because of stability problems e.g. degradation by nucleases.
  • Preferred intemucleoside linkages or intemucleoside linkages as is the case for non phosphate ester type linkages include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates,
  • oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3 '-most intemucleoside linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof).
  • Various salts, mixed salts and free acid forms are also included.
  • Preferred modified intemucleoside linkages that do not include a phosphorus atom therein include short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • siloxane siloxane, sulfide, sulfoxide, sulfone, formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl, alkenyl, sulfamate, methyleneimino, methylenehydrazino, sulfonate, sulfonamide, amide and others having mixed N, O, S and CH 2 component parts.
  • oligonucleosides include, but are not limited to, U.S.: 5,034,506; 5,166,315; 5,185,444;
  • the invention provides oligomeric compounds with
  • oligonucleotides have a motif that is useful in modulating nucleotide repeat-containing RNA.
  • the motif is a nucleoside motif.
  • the motif is a linkage motif.
  • the motif includes a nucleoside motif and a linkage motif.
  • Certain such oligonucleotides are antisense compounds.
  • Certain specific motifs disclosed herein are useful broadly. For example, certain motifs having activity for a particular nucleotide repeat-containing RNA have similar activity for other nucleotide repeat-containing RNA. In certain such embodiments, the activity is mutant selective activity.
  • oligonucleotides comprise one or more conjugates or additional groups, particularly at the 3' position of the sugar on the 3' terminal nucleotide and/or the 5' position of 5' terminal nucleotide.
  • oligonucleotides of the present invention may comprise ligand or non-ligand moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.
  • a thioether e.g., hexyl-S-tritylthiol
  • a thiocholesterol Olet al., Nucl.
  • a phospholipid e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl.
  • Oligomeric compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations.
  • compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • Oligomeric compounds including antisense compounds, can be utilized in any combination.
  • compositions by combining such oligomeric compounds with a suitable pharmaceutically acceptable diluent or carrier.
  • a pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS).
  • PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in certain embodiments, the invention provides a
  • the pharmaceutical composition comprising an antisense compound and a pharmaceutically acceptable diluent.
  • the pharmaceutically acceptable diluent is sterile pharmaceutical grade PBS.
  • the pharmaceutically acceptable diluent is sterile pharmaceutical grade water.
  • the pharmaceutically acceptable diluent is sterile pharmaceutical grade saline.
  • compositions comprising oligomeric compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters.
  • pharmaceutical compositions comprising oligomeric compounds comprise one or more oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
  • Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • a prodrug can include the incorporation of additional nucleosides at one or both ends of an oligomeric compound which are cleaved by endogenous nucleases within the body, to form the active oligomeric compound.
  • Lipid-based vectors have been used in nucleic acid therapies in a variety of methods.
  • the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids.
  • DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid.
  • the present invention provides methods of contacting a cell with an oligomeric compound described herein.
  • the cell is in vitro.
  • the cell is in an animal (e.g., rodent, primate, monkey or human).
  • antisense activity is detected.
  • an oligomeric compound comprising an oligonucleotide consisting of 13 to 30 linked nucleosides and having a nucleobase sequence 100% complementary to a repeat region of a nucleotide repeat-containing RNA, wherein the oligonucleotide contains: a. a 5'-region consisting of 1-5 linked 5'-region nucleosides, wherein the 5'-region nucleosides each have the same modification as one another;
  • a central region consisting of 5 to 20 linked nucleosides, wherein each nucleoside of the central region either comprises a 2'-fluoro sugar moiety or is a non-2'-fiuoro central-region nucleoside, provided that at least one nucleoside of the central region comprises a 2'-fluoro sugar moiety, and wherein the non-2 '-fluoro central-region nucleosides each have the same modification as one another; and
  • a 3'-region consisting of 1-5 linked 3'-region nucleosides, wherein the 3'-region nucleosides each have the same modification as one another; for the treatment of a disease associated with a C AG nucleotide repeat-containing RNA.
  • the disease is any of Atrophin 1, Huntington's Disease,
  • Huntington disease-like 2 (HDL2), spinal and bulbar muscular atrophy, Kennedy disease, spinocerebellar ataxia 1, spinocerebellar ataxia 12, spinocerebellar ataxia 17, Huntington disease-like 4 (HDL4), spinocerebellar ataxia 2, spinocerebellar ataxia 3, Machado- Joseph disease, spinocerebellar ataxia 6, and spinocerebellar ataxia 7.
  • compounds of the present invention are administered to an animal (e.g., a human) to provide a therapeutic effect.
  • an animal e.g., a human
  • Certain diseases or disorders have been identified to be associated with nucleotide repeat-containing RNA. Any such disease or disorder might be treated with compounds of the present invention.
  • the disease is selected from among: ataxin 8, atrophin 1, fragile X syndrome, Friedrich's ataxia, Huntington's disease, Huntington's disease-like 2, myotonic dystrophy, spinal and bulbar muscular atrophy, and spinocerebellar ataxia.
  • the disease is Huntington's disease.
  • the disease is myotonic dystrophy.
  • the myotonic dystrophy is myotonic dystrophy type 1. In certain embodiments, the myotonic dystrophy is myotonic dystrophy type 2. In certain embodiments, the disease is spinocerebellar ataxia. In certain embodiments, the spinocerebellar ataxia is spinocerebellar ataxia 10.
  • One of skill in the art may choose a formulation and route of administration based on the needs particular disease or disorder, for example, one may tailor a formulation and route of administration to result in delivery of the oligomeric compound to the tissue or cell in need.
  • compositions of the present invention are administered to a subject.
  • such pharmaceutical compositions are administered by injection.
  • such pharmaceutical compositions are administered by infusion.
  • compositions are administered by injection or infusion into the CSF. In certain such embodiments, pharmaceutical compositions are administered by direct injection or infusion into the spine. In certain embodiments,
  • compositions are administered by injection or infusion into the brain.
  • pharmaceutical compositions are administered by intrathecal injection or infusion rather than into the spinal cord tissue itself.
  • the antisense compound released into the surrounding CSF may penetrate into the spinal cord parenchyma.
  • intrathecal delivery is that the intrathecal route mimics lumbar puncture administration (i.e., spinal tap) already in routine use in humans.
  • compositions are administered by:
  • Intracerebroventricular or intraventricular delivery of a pharmaceutical composition comprising one or more oligomeric compound may be performed in any one or more of the brain's ventricles, which are filled with cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • CSF is a clear fluid that fills the ventricles, is present in the subarachnoid space, and surrounds the brain and spinal cord.
  • CSF is produced by the choroid plexuses and via the weeping or transmission of tissue fluid by the brain into the ventricles.
  • the choroid plexus is a structure lining the floor of the lateral ventricle and the roof of the third and fourth ventricles.
  • compositions are administered
  • compositions are administered systemically.
  • pharmaceutical compositions are administered subcutaneously.
  • pharmaceutical compositions are administered intravenously.
  • pharmaceutical compositions are administered by intramuscular injection.
  • compositions are administered both directly to the CSF (e.g., IT and/or ICV injection and/or infusion) and systemically.
  • compounds of the present invention have one or more desirable properties making them suitable for such administration.
  • Drug design typically requires a balance of several variables, including, but not limited to: potency, toxicity, stability, tissue distribution, convenience, and cost of a candidate compound. Such balancing is influenced by a number of factors, including the severity and typical duration of the disease treated. For example, greater drug-related toxicity is tolerated for use in treating acute lethal diseases than chronic sub-lethal diseases.
  • compounds of the present invention will have one or more improved properties compared to similar compounds that lack certain features of the present invention.
  • the compounds of the present invention may, in certain embodiments, have improved potency or may have similar potency but reduced toxicity and consequently improved therapeutic index.
  • compounds of the present invention may have improved pharmecokinetics or distribution to a particular desired target tissue.
  • suitablility of the present compounds for a particular indication may be assessed based on a number of variables specific to each such particular indication.
  • oligomeric compounds of the present invention are used in cells in vitro. In certain such embodiments, such uses are to identify and/or study nucleotide repeat- containing nucleic acids and mechanisms surrounding them and associated diseases.
  • RNA Ribonucleotide
  • DNA DNA having a modified sugar moiety and a thymine base
  • RNA having a modified base thymine (methylated uracil) for natural uracil of RNA
  • nucleic acid sequences provided herein are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases.
  • an oligomeric compound having the nucleobase sequence "ATCGATCG” encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence
  • AUCGAUCG and those having some DNA bases and some RNA bases such as
  • AUCGATCG and oligomeric compounds having other modified bases, such as
  • AT me CGAUCG wherein me C indicates a cytosine base comprising a methyl group at the 5- position.
  • oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high- affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.
  • Example 1 Effect of LN A-modified oligonucleotides, targeting human huntingtin (hit) mRNA, on huntingtin (Htt) protein
  • Antisense oligonucleotides targeted to the CAG repeat sequence of mutant huntingtin mRNA and with LNA modifications were tested for their effect on Htt protein levels in vitro.
  • the GM04281 fibroblast cell line (Coriell Institute for Medical Research, NJ, USA) containing 69 CAG repeats in the mutant htt allele and 17 CAG repeats in the wild-type allele, was utilized in this assay.
  • Cells were cultured at a density of 60,000 cells per well in 6-well plates and were transfected using LipofectamineTM RNAiMAX reagent (Invitrogen, CA) with 100 nM antisense oligonucleotide for 24 hours. The wells were then aspirated and fresh culture medium was added to each well.
  • the antisense oligonucleotides utilized in the assay are described in Table 1.
  • the antisense oligonucleotides were obtained from either Sigma Aldrich or ISIS Pharmaceuticals.
  • DNA22 is an unmodified oligonucleotide (DNA nucleosides with phosphodiester linkages).
  • the negative control is a scrambled oligonucleotide sequence.
  • the LNA modifications in each oligonucleotide are indicated by the subscript 'L' after each base.
  • oligonucleotides reduced nucleotide repeat-containing RNA more than they reduced the corresponding wild-type.
  • Example 2 in vitro dose-dependent effect of LNA-modified nucleotides on human Htt protein
  • Antisense oligonucleotides from Example 1 were tested at various doses in patient fibroblast cells.
  • GM04281 fibroblast cells were plated at a density of 60,000 cells per well in 6-well plates and transfected using
  • RNAiMAX reagent (Invitrogen, CA) reagent with increasing concentrations of antisense oligonucleotide for 24 hours, as specified in Tables 2 and 3.
  • the cell samples were processed for protein analysis utilizing the procedure outlined in Example 1.
  • Results are presented in Tables 2 and 3 as percent inhibition of wild-type and mutant Htt protein, relative to untreated control cells.
  • the data presented is an average of several independent assays performed with each antisense oligonucleotide.
  • Htt mutant protein levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.
  • the IC 50 (nM) values for each oligonucleotide for inhibition of the mutant protein and wild-type protein is also shown and indicates that each oligonucleotide preferentially targets the mutant htt mRNA compared to the wild-type.
  • the oligonucleotides listed in Table 3 demonstrate low in vitro potency and or little or no preferential lowering of the mutant mRNA compared to the wild-type.
  • Example 3 Effect of chemically modified oligonucleotides, targeting human huntingtin (hit) mRNA, on huntingtin (Htt) protein
  • Antisense oligonucleotides targeted to the CAG repeat sequence of mutant huntingtin nucleic acid and with various chemical modifications were tested for their effects on Htt protein levels in vitro.
  • GM04281 fibroblast cells were cultured at a density of 60,000 cells per well in 6- well plates and transfected using LipofectamineTM RNAi AX reagent (Invitrogen, C A) with 100 nM antisense oligonucleotide for 24 hours.
  • the cell samples were processed for protein analysis utilizing the procedure outlined in Example 1.
  • the percentage inhibition of the protein samples was calculated relative to the negative control sample and presented in Table 4.
  • the comparative percent inhibitions of the wild-type Htt protein and the mutant Htt protein are also presented.
  • the T m value for each oligonucleotide, determined by DSC, is also shown.
  • the antisense oligonucleotides utilized in the assay are described in Table 4.
  • the antisense oligonucleotides were obtained from Sigma Aldrich, ISIS Pharmaceuticals, Glen Research (Virginia, USA), or the M.J. Damha laboratory (McGill Univeristy, Montreal, Cancada), and are indicated as such.
  • Example 4 in vitro dose-dependent effect of chemically modified oligonucleotides on human Htt protein
  • Antisense oligonucleotides from Example 3 were tested at various doses in patient fibroblast cells.
  • GM04281 fibroblast cells were plated at a density of 60,000 cells per well in 6-well plates and transfected using
  • RNAiMAX reagent (Invitrogen, CA) reagent with increasing concentrations of antisense oligonucleotide for 24 hours, as specified in Tables 5 and 6.
  • the cell samples were processed for protein analysis utilizing the procedure outlined in Example 1.
  • Results are presented in Tables 5 and 6 as percent inhibition of wild-type and mutant Htt protein, relative to untreated control cells.
  • the data presented is an average of several independent assays performed with each antisense oligonucleotide.
  • Htt mutant protein levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.
  • the IC50 (nM) values for each oligonucleotide for inhibition of the mutant protein and wild-type protein is also shown and indicates that each oligonucleotide preferentially targets the mutant htt mRNA compared to the wild-type.
  • the oligonucleotides listed in Table 6 demonstrate low in vitro potency and/or little to no preferential reduction of the mutant mRNA compared to the wild-type.
  • Table 5 Table 5:
  • Example 5 Effect of oligonucleotides having phosphorothioate backbone, targeting human huntingtin (htt) mRNA, on huntingtin (Htt) protein
  • Antisense oligonucleotides targeted to the C AG repeat sequence of mutant huntingtin nucleic acid and with uniform phosphorothioate backbone were tested for their effects on Htt protein levels in vitro.
  • GM04281 fibroblast cells were cultured at a density of 60,000 cells per well in 6-well plates and transfected using LipofectamineTM RNAiMAX reagent (Invitrogen, CA) with 100 nM antisense oligonucleotide for 24 hours. The cell samples were processed for protein analysis utilizing the procedure outlined in Example 1.
  • the percentage inhibition of the protein samples was calculated relative to the negative control sample and presented in Table 7.
  • the comparative percent inhibitions of the wild-type Htt protein and the mutant Htt protein are also presented.
  • the T m value for each oligonucleotide, determined by DSC is also shown.
  • the antisense oligonucleotides utilized in the assay are described in Table 7.
  • Example 6 in vitro dose-dependent effect of oligonucleotides having phosphorothioate backbone on human Htt protein
  • Antisense oligonucleotides from Example 5 were tested at various doses in patient fibroblast cells.
  • GM04281 fibroblast cells were plated at a density of 60,000 cells per well in 6-well plates and transfected using
  • RNAiMAX reagent (Invitrogen, CA) reagent with increasing concentrations of antisense oligonucleotide for 24 hours, as specified in Tables 8 and 9.
  • the cell samples were processed for protein analysis utilizing the procedure outlined in Example 1.
  • Results are presented in Tables 8 and 9 as percent inhibition of wild-type and mutant Htt protein, relative to untreated control cells.
  • the data presented is an average of several independent assays performed with each antisense oligonucleotide.
  • Htt mutant protein levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.
  • the IC 50 (nM) values for each oligonucleotide for inhibition of the mutant protein and wild-type protein is also shown and indicates that each oligonucleotide preferentially targets the mutant htt mRNA compared to the wild-type.
  • the oligonucleotides listed in Table 9 do not show preferential targeting of the mutant mRNA compared to the wild-type.
  • Example 7 Effect of chemically modified oligonucleotides targeting CAG repeats on human huntingtin ⁇ hit) mRNA levels
  • Antisense oligonucleotides from Example 1, Example 3, and Example 5 were tested in GM04281 cells. Antisense oligonucleotides targeted to the CAG repeat sequence of mutant huntingtin nucleic acid and with various chemical modifications were tested for their effects on htt mRNA levels in vitro.
  • GM04281 cells were cultured at a density of 60,000 cells per well in 6- well plates and transfected using LipofectamineTM RNAiMAX reagent (Invitrogen, CA) with 50 nM antisense oligonucleotide for 24 hours. The wells were then aspirated and fresh culture medium was added to each well. After a post-transfection period of 3 days, the cells were harvested with trypsin solution (0.05% Trypsin-EDTA, Invitrogen) and lysed.
  • trypsin solution 0.05% Trypsin-EDTA, Invitrogen
  • RNA from treated and untreated fibroblast cells was extracted using TRIzol reagent (Invitrogen). Samples were then treated with DNase I (Worthington Biochemical Corp.) at 25°C for 10 min. Reverse transcription reactions were carried out using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol.
  • results are presented in Table 10 and indicate the percent inhibition of htt mRNA compared to untreated cells. The results indicate that mRNA levels were unaffected by treatment with the antisense oligonucleotides.
  • Example 8 Time-dependent effect of an LNA-modified antisense oligonucleotide, targeting mutant htt mRNA, on human huntingtin (Htt) protein levels
  • the ISIS antisense oligonucleotide with LNA modifications at the thymine bases and which demonstrated significant selective inhibition of mutant huntingtin protein compared to wild-type protein was further studied.
  • the time-dependent effect of this oligonucleotide was tested.
  • GM04281 fibroblast cells were cultured at a density of 60,000 cells per well in 6-well plates and transfected using LipofectamineTM RNAiMAX reagent (Invitrogen, CA) with 100 nM antisense oligonucleotide for 24 hours.
  • the cell samples were processed for protein analysis at 2 days, 3 days, 4 days, 5 days, and 6 days post-transfection, utilizing the procedure outlined in Example 1.
  • RNAiMAX LipofectamineTM RNAiMAX (Invitrogen, USA), according to the manufacturer's instructions. Media was exchanged 1 day after transfection with fresh supplemented media. Cells were washed with PBS and harvested 4 days after transfection for protein analysis. Protein analysis was undertaken utilizing the procedure outlined in Example 1.
  • the antisense oligonucleotides tested were LNA (T) (described in Example 1) and cET (described in Example 3).
  • the results are presented in Table 12 as the IC 50 for each antisense oligonucleotide.
  • each of the antisense oligonucleotides demonstrates three- to seven-fold selectivity for the mutant allele versus the wild-type allele. This data indicates that allele-specific antisense oligonucleotides can discriminate between the wild-type allele and mutant allele of htt, even when the numbers of CAG repeats are 41 and 44 in number.
  • Table 12 :
  • RNAiMAX LipofectamineTM RNAiMAX (Invitrogen, CA, USA), OligofectamineTM (Invitrogen, CA, USA), TriFECTin (Integrated DNA Technologies, CA, USA), 7ra>wIT®-01igo (Minis Bio LLC, WI, USA), and PepMuteTM (Signagen Laboratories, MD, USA) were utilized in this study.
  • the antisense oligonucleotides tested were LNA (T) (described in Example 1), MOE (described in Example 4), 2'F-RNA full and 2'F-ANA full (described in Example 4).
  • a negative control LNA oligonucleotide and a positive control siRNA (siHdhl siR A) were also included in the assay.
  • the antisense oligonucleotides were transfected into GM04281 cell and protein analysis of htt was done in a procedure similar to that described in Example 1. The results are presented in Table 13, below and are expressed as percent inhibition compared to the negative control.
  • the LNA(T) oligonucleotide demonstrated potency and allele- specificity, regardless of the transfection reagent used, whereas the 2'F-RNA full and 2'F-ANA full oligonucleotides demonstrated poor inhibition and were largely non-specific for the mutant versus the wild-type allele.
  • the performance of the all lipid-based transfection reagents (LipofectamineTM R AiMAX, OligofectamineTM, TriFECTin, and 7>awIT®-01igo) were therefore similar.
  • transfection reagent PepMuteTM
  • MOE oligonucleotide transfected into cells with this transfection reagent, demonstrated both potency and allele-specificity.
  • the choice of transfection reagent may affect comparisons between oligonucleotide chemistries and may be the reason for an antisense oligonucleotide underperforming in a particular cellular assay.

Abstract

The present invention relates to compounds and methods useful for the treatment and investigation of diseases and disorders associated with nucleotide repeat-containing RNA molecules.

Description

METHODS AND COMPOSITIONS USEFUL IN DISEASES OR CONDITIONS
RELATED TO REPEAT EXPANSION
FIELD OF THE INVENTION
The present invention pertains generally to chemically-modified oligomers for use in research, diagnostics, and/or therapeutics.
SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled CORE0090WOSEQ.txt, created on February 7, 2011 , which is 2.45 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
Certain RNA molecules are known to include repeat regions consisting essentially of repeating units of 3-5 nucleotides. Depending on the particular gene, the repeat region a normal wild-type RNA molecule may comprise from about 5 up to about 40 copies of the repeating unit. In certain instances, the number of such repeating units can become increased and the resulting nucleotide repeat-containing RNA molecule may disruptive to the cell. Certain diseases can result.
SUMMARY OF THE INVENTION
In certain embodiments, the present invention provides oligomeric compounds comprising an oligonucleotide consisting of 13 to 30 linked nucleosides and having a nucleobase sequence complementary to a repeat region of a nucleotide repeat-containing RNA. In certain such embodiments, the oligonucleotide contains:
a 5 '-region consisting of 1-5 linked 5 '-region nucleosides, wherein the 5 '-region nucleosides each have the same modification as one another;
a central region consisting of 5 to 20 linked nucleosides, wherein each nucleoside of the central region either comprises a 2'-fluoro sugar moiety or is a non-2 '-fluoro central- region nucleoside, provided that at least one nucleoside of the central region comprises a 2'-fluoro sugar moiety, and wherein the non-2 '-fluoro central-region nucleosides each have the same modification as one another; and
a 3'-region consisting of 1-5 linked 3'-region nucleosides, wherein the 3'-region nucleosides each have the same modification as one another.
In certain embodiments, the oligonucleotide is 85% complementary to the repeat region of the nucleotide repeat containing RNA. In certain embodiments, the oligonucleotide is 90% complementary to the repeat region of the nucleotide repeat containing RNA. In certain embodiments, the oligonucleotide is 95% complementary to the repeat region of the nucleotide repeat containing RNA. In certain embodiments, the oligonucleotide is 100% complementary to the repeat region of the nucleotide repeat containing RNA. In certain of any of the above embodiments, the oligonucleotide is complementary to the nucleotide repeat containing RNA within the repeat region and is not complementary to the nucleobases adjacent to the repeat region of the nucleotide repeat containing RNA.
In certain embodiments, no more than four contiguous nucleosides of the central region are non-2 '-fluoro central-region nucleosides.
In certain embodiments, the 5 '-region nucleosides comprise a modified 2' -sugar moiety. In certain such embodiments, the 5'-region nucleosides comprise a 2'-substituent selected from: OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, 0(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, 0(CH2)2-OCH3, 0(CH2)2-SCH3, 0(CH2)2-OCF3, 0(CH2)3-N(R4)(R5), 0(CH2)2-ON(R4)(R5), 0(CH2)2-0(CH2)2-N(R4)(R5), OCH2C(=0)-N(R4)(R5), OCH2C(=0)-N(R6)-(CH2)2-N(R4)(Rs) and 0(CH2)2-N(R6)-C(=NR7)[N(R4)(R5)] wherein R4, R5, Re and R7 are each, independently, H or Q-C6 alkyl. In certain embodiments, the 5 '-region nucleosides comprise a 2'- 0(CH2)2-OCH3.
In certain embodiments, the 5 '-region nucleosides comprise a bicyclic sugar moiety. In certain embodiments, the bicyclic sugar moiety of the 5'-region nucleosides comprise a 4' to 2' bridge. In certain such embodiments, the 4' to 2' bridge of the 5'-region nucleosides comprise from 2 to 4 linked groups independently selected from -[C(Ra)(Rb)]y-, -C(Ra)=C(Rb)-, -C(Ra)=N- , -C(=NRa)-, -C(=0)-, -C(=S)-, -0-, -Si(Ra)2-, -S(=0)x-, and -Ν(¾)-;
wherein: x is 0, 1, or 2; y is 1, 2, 3, or 4; each Ra and Rb is, independently, H, a protecting group, hydroxyl, C C6 alkyl, substituted d-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C alkynyl, C5-C9 aryl, substituted C5-C2o aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJiJ2, SJls N3, COOJl5 acyl (C(=0)-H), substituted acyl, CN, sulfonyl (S(=0)2-Ji), or sulfoxyl (S(=0)-J1); and each Ji and J2 is, independently, H, Ci-C6 alkyl, substituted Ci-C alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C5-C20 aryl, substituted C5-C9 aryl, acyl (C(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C C6 aminoalkyl, substituted d-C6 aminoalkyl or a protecting group.
In certain embodiments, the 4' to 2' bridge of the 5 '-region nucleosides is ,
-[C(Rc)(Rd)]n-, -[C(Rc)(Rd)]n-0-, -C(RcRd)-N(Re)-0- or -C(RcRd)-0-N(Re)-, wherein: each Rc and R<i is independently hydrogen, halogen, substituted or unsubstituted C C6 alkyl; and each Re is independently hydrogen or substituted or unsubstituted Ci-Ce alkyl.
In certain embodiments, the 4' to 2' bridge of the 5'-region nucleosides is 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-0-2\ 4'-CH(CH3)-0-2', 4'-(CH2)2-0-2', 4*-CH2-0-N(Re)-2' and 4'-CH2- N(Re)-0-2'- bridge. In certain embodiments, the bridge of the 5 '-region nucleosides is 4'- CH(CH3)-0-2\
In certain embodiments, the non-2'-fluoro central-region nucleosides are 2'- deoxyribonucleosides.
In certain embodiments, the non-2'-fluoro central-region nucleosides comprise a modified 2'-sugar moiety. In certain such embodiments, the non-2' -fluoro central-region nucleosides comprise a 2'-substituent selected from: OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, 0(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, 0(CH2)2-OCH3, 0(CH2)2-SCH3, 0(CH2)2- OCF3, 0(CH2)3-N(R4)(R5), 0(CH2)2-ON(R4)(R5), 0(CH2)2-0(CH2)2-N(R4)(R5), OCH2C(=0)- N(R4)(R5), OCH2C(=0)-N(R6)-(CH2)2-N(R4)(R5) and 0(CH2)2-N(R6)-C(=NR7)[N(R4)(R5)] wherein R4, R5, Re and R7 are each, independently, H or C C6 alkyl. In certain embodiments, the 2'-substituent of the non-2'-fluoro central-region nucleosides is 0(CH2)20CH3.
In certain embodiments, the non-2 '-fluoro central-region nucleosides comprise a bicyclic sugar moiety. In certain embodiments, the bicyclic sugar moiety of the non-2'-fluoro central- region nucleosides comprises a 4' to 2' bridge. In certain embodiments, the 4' to 2' bridge of the non-2 '-fluoro central-region nucleosides comprises from 2 to 4 linked groups independently selected from -[C(Ra)(Rb)]y-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=0)-, -C(=S)-, -0-, - Si(Ra)2-, -S^O^ and -NCR -; wherein: x is 0, 1, or 2; y is 1 , 2, 3, or 4; each Ra and Rb is, independently, H, a protecting group, hydroxyl, Q-Q alkyl, substituted Ci-Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C5-C9 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJ1J2, SJi, N3, COOJl 5 acyl (C(=0)-H), substituted acyl, CN, sulfonyl
Figure imgf000005_0001
and each 31 and J2 is, independently, H, C C6 alkyl, substituted Ci-Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C5-C20 aryl, substituted C5-C aryl, acyl (C(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C\-Ce aminoalkyl, substituted C C6 aminoalkyl or a protecting group.
In certain embodiments, 4' to 2' bridge of the non-2'-fluoro central-region nucleosides is -[C(Rc)(Rd)]n-, -[C(Rc)(Rd)]n-0-, -C(RcRd)-N(Re)-0- or -C(RcRd)-0-N(Re)-, wherein: each Rc and Rd is independently hydrogen, halogen, substituted or unsubstituted C]-C6 alkyl; and each Re is independently hydrogen or substituted or unsubstituted CrC6 alkyl.
In certain embodiments, the 4' to 2' bridge of the non-2 '-fluoro central-region nucleosides is 4,-(CH2)2-2', 4,-(CH2)3-2, J 4'-CH2-0-2', 4'-CH(CH3)-0-2*, 4,-(CH2)2-0-2', 4'-CH2- 0-Ν(¾)-2' and 4'-CH2-N(Re)-0-2'- bridge. In certain embodiments, the bridge of the non-2 '- fluoro central-region nucleosides is 4'-CH(CH3)-0-2'.
In certain embodiments, the 3 '-region nucleosides comprise a modified 2 '-sugar moiety. In certain embodiments, the 2 '-modified sugar moiety of the 3 '-region nucleosides comprises a 2'-substituent selected from: OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, 0(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, 0(CH2)2-OCH3, 0(CH2)2-SCH3, 0(CH2)2-OCF3, 0(CH2)3- Ν(¾)(¾), 0(CH2)2-ON(R4)(R5), 0(CH2)2-0(CH2)2-N(R4)(R5), OCH2C(=0)-N(R4)(R5), OCH2C(=0)-N(R6)-(CH2)2-N(R4)(R5) and 0(CH2)2-N(R6)-C(=NR7)[N(R4)(R5)] wherein R4, R5, Re and R7 are each, independently, H or C C6 alkyl. In certain embodiments, the 2'-substituent of the 3 '-region nucleosides is 0(CH2)2OCH3.
In certain embodiments, the 3 '-region nucleosides comprise a bicyclic sugar moiety. In certain embodiments, the bicyclic sugar moiety of the 3 '-region nucleosides comprises a 4' to 2' bridge. In certain embodiments, the 4' to 2' bridge of the 3 '-region nucleosides comprises from 2 to 4 linked groups independently selected from -[C(Ra)(Rb)]y-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=0)-, -C(=S , -0-, -Si(Ra)2-, -S(=0)x-, and -N(R -;
wherein:
x is 0, 1, or 2;
y is 1, 2, 3, or 4;
each Ra and Rb is, independently, H, a protecting group, hydroxyl, Ci-C6 alkyl, substituted Cj-C alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C5-C9 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ], NJiJ2, SJ1} N3, COOJl5 acyl (C(=0)-H), substituted acyl, CN, sulfonyl (S(=O)2-J , or sulfoxyl (S(=0)-J1); and
each J] and J2 is, independently, H, Q-C6 alkyl, substituted Q-Q alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C5-C2o aryl, substituted C5-C aryl, acyl (C(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, CrC6 aminoalkyl, substituted C]-C6 aminoalkyl or a protecting group. In certain embodiments, the 4' to 2' bridge of the 3'-region nucleosides is, -[C(Rc)(Rd)]n-, -[C(Rc)(Rd)]n-0-, -C(RcR<i)-N(Re)-0- or
Figure imgf000007_0001
wherein: each ¾ and ¾ is independently hydrogen, halogen, substituted or unsubstituted -Ce alkyl; and each ¾ is independently hydrogen or substituted or unsubstituted Q-Q alkyl.
In certain embodiments, the, 4' to 2' bridge of the 3 '-region nucleosides is 4'-(CH2)2-2', 4'-(CH2)3-2*, 4'-CH2-0-2', 4'-CH(CH3)-0-2', 4'-(CH2)2-0-2', 4*-CH2-0-N(Re)-2' and 4'-CH2- NCR^-CW- bridge. In certain embodiments, the bridge of the 3 '-region nucleosides is 4'- CH(CH3)-0-2\
In certain embodiments, the 5 '-region nucleosides and the 3' -region nucleosides comprise the same modification as one another. In certain embodiments, the 5 '-region nucleosides and the non-2' -fluoro central-region nucleosides comprise the same modification as one another. In certain embodiments, the 5 '-region nucleosides and the 3 '-region nucleosides and the non-2' -fluoro central-region nucleosides all comprise the same modification as one another. In certain embodiments, the 5 '-region nucleosides and the 3 '-region nucleosides and the non-2 '-fluoro central-region nucleosides each comprises a 2'-(CH2)2OCH3 modification.
In certain embodiments, the central region comprises 1 to 10 blocks of non-2 '-fluoro central-region nucleosides, wherein each block independently consists of 1 to 4 non-2 '-fluoro central-region nucleosides. In certain embodiments, the central region comprises 1 to 5 blocks of non-2'-fluoro central-region nucleosides. In certain embodiments, the central region comprises 1 to 2 blocks of non-2'-fluoro central-region nucleosides. In certain embodiments, the central region comprises 1 block of non-2'-fluoro central-region nucleosides. In certain embodiments, the blocks of non-2 '-fluoro central-region nucleosides independently consist of 1-3 non-2'-fluoro central-region nucleosides. In certain embodiments, the blocks of non-2'-fluoro central-region nucleosides independently consist of 1 or 2 non-2'-fluoro central-region nucleosides. In certain embodiments, the blocks of non-2'-fluoro central-region nucleosides independently consist of 3 or 4 non-2'-fluoro central-region nucleosides. In certain embodiments, the central region consists of linked 2'-fluoro modified nucleosides.
In certain embodiments, the oligonucleotide has the Formula:
(Nu1)nl-(Nu2)n2-(Nu3)n3-(Nu4)n4-(Nu5)n5 wherein:
each uj is a 5 '-region nucleoside;
each Nu2 and each Nu4 is a 2'-fluoro nucleoside;
each Nu3 is a non-2 '-fluoro central-region nucleoside;
each Nu5 is a 3 '-region nucleoside;
nl is from 1 to 5;
n5 is from 0 to 5;
n2 is from 1 to 24 and n4 is from 1 to 24, provided that the sum of n2 and n4 is from 10 to 25; and n3 is from 0 to 4.
In certain embodiments, n3 is from 1 to 4. In certain embodiments, n3 is 0.
In certain embodiments, the oligonucleotide comprises one or more modified
internucleoside linkage. In certain embodiments, each internucleoside linkage is modified. In certain embodiments, each internucleoside linkage of the oligonucleotide is either a
phosphodiester or a phosphorothioate linkage.
In certain embodiments, the oligonucleotide comprises at least one modified nucleobase. In certain embodiments, the modified oligonucleotide is a 5-methylcytosine.
In certain embodiments, the repeat region of the nucleotide repeat-containing RNA is a repeating quintet. In certain embodiments, the repeat region of the nucleotide repeat-containing RNA is a repeating quartet. In certain embodiments, the repeat region of the nucleotide repeat- containing RNA is a repeating CCUG or AUUCU.
In certain embodiments, the repeat region of the nucleotide repeat-containing RNA is a repeating triplet. In certain embodiments, the repeating triplet is selected from: CAG, CUG, CGG, GCC, and GAA. In certain embodiments, the repeating triplet is CAG. In certain embodiments, the repeating triplet is CUG.
In certain embodiments, the nucleotide repeat-containing RNA is associated with a disease. In certain embodiments, the disease is selected from among: ataxin 8, atrophin 1, fragile X syndrome, Friedrich's ataxia, Huntington's disease, Huntington's disease-like 2, myotonic dystrophy, spinal and bulbar muscular atrophy, and spinocerebellar ataxia In certain embodiments, the disease is Huntington's disease. In certain embodiments, the disease is myotonic dystrophy. In certain embodiments, the myotome dystrophy is myotonic dystrophy type 1. In certain embodiments, the myotome dystrophy is myotonic dystrophy type 2. In certain embodiments, the disease is spinocerebellar ataxia. In certain embodiments, the spinocerebellar ataxia is spinocerebellar ataxia 10.
In certain embodiments, the oligomeric compound is a mutant selective compound. In certain embodiments, the oligomeric compound is capable of reducing the activity or amount of a nucleotide repeat-containing RNA at least two fold more than it reduces the activity or amount of a corresponding wild type RNA.
In certain embodiments, the invention provides methods of selectively reducing the activity or amount of a nucleotide repeat-containing RNA in a cell, comprising contacting a cell having a nucleotide repeat-containing RNA with any of the above oligomeric compounds; and thereby selectively reducing the activity or amount of the nucleotide repeat-containing RNA in the cell.
In certain embodiments, the amount or activity of the nucleotide repeat-containing RNA is reduced at least two-fold more than that of a corresponding wild-type RNA. In certain embodiments, the cell is in vitro. In certain embodiments, the cell is in an animal.
In certain embodiments, the invention provides pharmaceutical compositions comprising one or more oligomeric compound and a pharmaceutical carrier or diluent.
In certain embodiments, the invention provides methods of treating a patient having a disease associated with a nucleotide repeat-containing RNA comprising administering to the patient a pharmaceutical composition comprising any of the above oligomeric compounds. In certain embodiments, the disease is selected from among: ataxin 8, atrophin 1, fragile X syndrome, Friedrich's ataxia, Huntington's disease, Huntington's disease-like 2, myotonic dystrophy, spinal and bulbar muscular atrophy, and spinocerebellar ataxia. In certain embodiments, the disease is Huntington's disease. In certain embodiments, the disease is myotonic dystrophy. In certain embodiments, the myotonic dystrophy is myotonic dystrophy type 1. In certain embodiments, the myotonic dystrophy is myotonic dystrophy type 2. In certain embodiments, the disease is spinocerebellar ataxia. In certain embodiments, the spinocerebellar ataxia is spinocerebellar ataxia 10.
In certain embodiments, the pharmaceutical composition is administered by injection. In certain embodiments, the pharmaceutical composition is injected into the central nervous system. In certain embodiments, the pharmaceutical composition is injected into the brain. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is an infusion.
DETAILED DESCRIPTION
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.
A. Definitions
Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Certain such techniques and procedures may be found for example in "Carbohydrate
Modifications in Antisense Research" Edited by Sangvi and Cook, American Chemical Society , Washington D.C., 1994; "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 18th edition, 1990; and "Antisense Drug Technology, Principles, Strategies, and Applications" Edited by Stanley T. Crooke, CRC Press, Boca Raton, Florida; and Sambrook et al., "Molecular Cloning, A laboratory Manual," 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure herein are incorporated by reference in their entirety.
Unless otherwise indicated, the following terms have the following meanings:
As used herein, the term "nucleotide repeat-containing RNA" (NRR) means a mutant RNA molecule having a nucleobase sequence that includes a repeat region consisting essentially of repeating units of 3-5 nucleobases that repeat at least 10 times in the repeating region, and wherein the presence or length of the repeat region affects the normal processing, function, or activity of the RNA.
As used herein, the term "corresponding wild type RNA" means the non-mutant version of the nucleotide repeat-containing RNA having normal function and activity. Typically, corresponding wild type RNA molecules comprise a repeat region which is shorter than that of a nucleotide repeat-containing RNA.
As used herein, "nucleoside" refers to a compound comprising a heterocyclic base moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA), abasic nucleosides, modified nucleosides, and sugar-modified nucleosides. Nucleosides may be modified with any of a variety of substituents.
As used herein, "sugar moiety" means a natural (furanosyl), a modified sugar moiety or a sugar surrogate.
As used herein, "modified sugar moiety" means a chemically-modified furanosyl sugar or a non-furanosyl sugar moiety. Also, embraced by this term are furanosyl sugar analogs and derivatives including bicyclic sugars, tetrahydropyrans, morpholinos, 2'-modified sugars, 4'- modified sugars, 5 '-modified sugars, and 4'-subsituted sugars.
As used herein, "sugar-modified nucleoside" means a nucleoside comprising a modified sugar moiety.
As used herein the term "sugar surrogate" refers to a structure that is capable of replacing the furanose ring of a naturally occurring nucleoside. In certain embodiments, sugar surrogates are non-furanose (or 4'-substituted furanose) rings or ring systems or open systems. Such structures include simple changes relative to the natural furanose ring, such as a six membered ring or may be more complicated as is the case with the non-ring system used in peptide nucleic acid. Sugar surrogates includes without limitation morpholinos and cyclohexenyls and cyclohexitols. In most nucleosides having a sugar surrogate group the heterocyclic base moiety is generally maintained to permit hybridization.
As used herein, "nucleotide" refers to a nucleoside further comprising a modified or unmodified phosphate linking group or a non-phosphate internucleoside linkage.
As used herein, "linked nucleosides" may or may not be linked by phosphate linkages and thus includes "linked nucleotides."
As used herein, "nucleobase" refers to the heterocyclic base portion of a nucleoside. Nucleobases may be naturally occurring or may be modified and therefore include, but are not limited to adenine, cytosine (including, a 5-methylcytosine), guanidine, uracil, thymidine and analogues thereof. In certain embodiments, a nucleobase may comprise any atom or group of atoms capable of hydrogen bonding to a base of another nucleic acid.
As used herein, "modified nucleoside" refers to a nucleoside comprising at least one modification compared to naturally occurring RNA or DNA nucleosides. Such modification may be at the sugar moiety and/or at the nucleobases.
As used herein, "non-2 '-fiuoro nucleoside" refers to any nucleoside other than one having fluorine at the 2 '-position of the sugar. In certain embodiments, non-2 '-fiuoro nucleosides are modified nucleosides, provided the modification is other than 2'-fluoro. In certain embodiments, non-2'-fluoro nucleosides are unmodified nucleosides, such as DNA.
As used herein, "Tm" means melting temperature which is the temperature at which the two strands of a duplex nucleic acid separate. Tm is often used as a measure of duplex stability or the binding affinity of an antisense compound toward a complementary RNA molecule.
As used herein, a "high-affinity sugar modification" is a modified sugar moiety which when it is included in a nucleoside and said nucleoside is incorporated into an antisense oligonucleotide, the stability (as measured by Tm) of said antisense oligonucleotide: RNA duplex is increased as compared to the stability of a DNA:RNA duplex.
As used herein, a "high-affinity sugar-modified nucleoside" is a nucleoside comprising a modified sugar moiety that when said nucleoside is incorporated into an antisense compound, the binding affinity (as measured by Tm) of said antisense compound toward a complementary RNA molecule is increased. In certain embodiments of the invention at least one of said sugar- modified high-affinity nucleosides confers a ATm of at least 1 to 4 degrees per nucleoside against a complementary RNA as determined in accordance with the methodology described in Freier et al., Nucleic Acids Res., 1997, 25, 4429-4443, which is incorporated by reference in its entirety. In another aspect, at least one of the high-affinity sugar modifications confers about 2 or more, 3 or more, or 4 or more degrees per modification. In the context of the present invention, examples of sugar-modified high affinity nucleosides include, but are not limited to, (i) certain 2 '-modified nucleosides, including 2'-subtstituted and 4' to 2' bicyclic nucleosides, and (ii) certain other non-ribofuranosyl nucleosides which provide a per modification increase in binding affinity such as modified tetrahydropyran and tricycloDNA nucleosides. For other modifications that are sugar-modified high-affinity nucleosides see Freier et al., Nucleic Acids Res., 1997, 25, 4429-4443.
As used herein, a "nuclease resistant nucleotide" means a chemically modified nucleotide comprising one or both of a modified sugar or modified internucleoside linkage which, when incorporated into an oligonucleotide, makes said oligonucleotide more stable to degradation under cellular nucleases (exo- or endo-nucleases). Examples of nuclease resistant nucleotides, but are not limited to, phosphorothioate nucleotides, bicyclic sugar nucleotides, 2 '-modified nucleotides
As used herein, "bicyclic nucleoside" refer to a modified nucleoside comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4' and the 2' ribosyl ring atoms. In certain embodiments, oligomeric compounds provided herein include one or more bicyclic nucleosides wherein the bridge comprises a 4' to 2' bicyclic nucleoside. Examples of such 4' to 2' bicyclic nucleosides, include but are not limited to one of the formulae: 4'-(CH2)-0-2' (LNA); 4'-(CH2)-S-2'; 4'- (CH2)2-0-2' (ENA); 4*-CH(CH3)-0-2' and 4'-CH(CH2OCH3)-0-2' (and analogs thereof see U.S. Patent 7,399,845, issued on July 15, 2008); 4'-C(CH3)(CH3)-0-2' (and analogs thereof see published International Application WO/2009/006478, published January 8, 2009); 4'-CH2- N(OCH3)-2' (and analogs thereof see published International Application WO/2008/150729, published December 11, 2008); 4'-CH2-0-N(CH3)-2' (see published U.S. Patent Application US2004-0171570, published September 2, 2004 ); 4'-CH2-N(R)-0-2', wherein R is H, C C12 alkyl, or a protecting group (see U.S. Patent 7,427,672, issued on September 23, 2008); 4'-CH2- C(H)(CH3)-2' (see Chattopadhyaya, et al, J. Org. Chem.,2009, 74, 118-134); and 4*-CH2-C- (=CH2)-2' (and analogs thereof see published International Application WO 2008/154401, published on December 8, 2008). See, for example: Singh et al., Chem. Commun., 1998, 4, 455- 456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U. S. , 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J Am. Chem. Soc, 129(26) 8362-8379 (Jul. 4, 2007); U.S. Patent Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499;
7,034,133; and 6,525,191; Elayadi et al, Curr. Opinion Invens. Drugs, 2001, 2, 558-561;
Braasch et al, Chem. Biol, 2001, 8, 1-7; and Oram et al, Curr. Opinion Mol Ther., 2001, 3, 239-243; and U.S. 6,670,461; International applications WO 2004/106356; WO 94/14226; WO 2005/021570; U.S. Patent Publication Nos. US2004-0171570; US2007-0287831; US2008- 0039618; U.S. Patent Nos. 7,399,845; U.S. Patent Serial Nos. 12/129,154; 60/989,574;
61/026,995; 61/026,998; 61/056,564; 61/086,231; 61/097,787; 61/099,844; PCT International Applications Nos. PCT/US2008/064591; PCT/US2008/066154; PCT/US2008/068922; and Published PCT International Applications WO 2007/134181. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example a-L-ribofuranose and β-D-ribofuranose (see PCT international application
PCT/DK98/00393, published on March 25, 1999 as WO 99/14226).
In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4' and the 2' position of the pentofuranosyl sugar moiety wherein such bridges independently comprises 1 or from 2 to 4 linked groups independently selected from -[C(Ra)(Rb)]n-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)- , -C(=0)-, -C(=S)-, -0-, -Si(Ra)2-, -S(=0)x-, and -N(Ra)-; wherein:
x is 0, 1, or 2;
n is 1, 2, 3, or 4;
each Ra and Rb is, independently, H, a protecting group, hydroxyl, C\-Cn alkyl, substituted CrC12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C2o aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJl5 NJj J2, SJj, N3, COOJ acyl (C(=0)-H), substituted acyl, CN, sulfonyl (S(=O)2-J , or sulfoxyl (S(=O)-J ; and
each J\ and J2 is, independently, H, CrC12 alkyl, substituted CrC12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C2o aryl, substituted C5-C20 aryl, acyl (C(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, CrC12 aminoalkyl, substituted Q-Cn aminoalkyl or a protecting group.
In certain embodiments, the bridge of a bicyclic sugar moiety is , -[C(Ra)(Rb)]n-,
-[C(Ra)(Rb)]n-0-, -C(RaRb)-N(R)-0- or -C(RaRb)-0-N(R)-. In certain embodiments, the bridge is 4'-CH2-2', 4*-(CH2)2-2', 4*-(CH2)3-2', 4'-CH2-0-2', 4'-(CH2)2-0-2', 4'-CH2-0-N(R)-2' and 4'- CH2-N(R)-0-2'- wherein each Ris, independently, H, a protecting group or C1-C12 alkyl.
In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4'-2' methylene-oxy bridge, may be in the a-L configuration or in the β-D configuration. Previously, a-L-methyleneoxy (4'-Ο¾-0-2') BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et ah, Nucleic Acids Research, 2003, 21, 6365-6372).
In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) a-L- Methyleneoxy (4'-CH2-0-2') BNA , (B) β-D-Methyleneoxy (4'-CH2-0-2') BNA ("LNA") , (C) Ethyleneoxy (4,-(CH2)2-0-2') BNA ("ENA") , (D) Aminooxy (4'-CH2-0-N(R)-2') BNA, (E) Oxyamino (4'-CH2-N(R)-0-2') BNA, and (F) Methyl(methyleneoxy) (4'-CH(CH3)-0-2') BNA ("cEt"), (G) methylene-thio (4'-CH2-S-2') BNA, (H) methylene-amino (4'-CH2-N(R)-2') BNA, (I) methyl carbocyclic (4'-CH2-CH(CH3)-2') BNA, (J) propylene carbocyclic (4'-(CH2)3-2') BNA, and (K) ethylene carbocyclic (4'-CH2-CH2-2') (carba LNA or "cLNA") as depicted below.
Figure imgf000016_0001
Figure imgf000016_0002
Figure imgf000016_0003
wherein Bx is the base moiety and R is independently H, a protecting group or C1-C12 alkyl.
In certain embodiments, bicyclic nucleoside having Formula I:
Figure imgf000016_0004
wherein: Bx is a heterocyclic base moiety;
-Qa-Qb-Qc- is -CH2-N(Rc)-CH2-, -C(=0)-N(Rc)-CH2-, -CH2-0-N(Rc)-, -CH2-N(Rc)-0- or -N(Rc)-0-CH2;
Rc is d-Ci2 alkyl or an amino protecting group; and
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.
In certain embodiments, bicyclic nucleoside having Formula II:
Figure imgf000017_0001
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Za is Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Q-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio.
In one embodiment, each of the substituted groups, is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJc, NJJd, SJC, N3, OC(=X)Jc, and NJeC(=X)NJcJd, wherein each Jc, Jd and Je is, independently, H, C C6 alkyl, or substituted Cj-Q alkyl and X is O or NJC.
In certain embodiments, bicyclic nucleoside having Formula III:
Figure imgf000018_0001
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Zb is C]-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Q-Q alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl or substituted acyl (C(=0)-).
In certain embodiments, bicyclic nucleoside having Formula IV:
Figure imgf000018_0002
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Rd is C!-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
each qa, qb, qc and qd is, independently, H, halogen, d-C6 alkyl, substituted Q-Q alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, d-C6 alkoxyl, substituted C C6 alkoxyl, acyl, substituted acyl, C]-C aminoalkyl or substituted d-C6 aminoalkyl; In certain embodiments, bicyclic nucleoside having Formula V:
Figure imgf000019_0001
wherein:
Bx is a heterocyclic base moiety;
Ta and T¾ are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; qe and qf are each, independently, hydrogen, halogen, Ci-C12 alkyl, substituted
Figure imgf000019_0002
alkyl, C2-C12 alkenyl, substituted C2-Ci2 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, Cj- C12 alkoxy, substituted d-C12 alkoxy, OJj, SJjs SOJj, S02Jj, NJjJk, N3, CN, C(=0)OJj,
C(=0)NJjJk, C(=0)Jj, 0-C(=0)NJjJk, N(H)C(=NH)NJjJk, N(H)C(=0)NJjJkorN(H)C(=S)NJjJk; or qe and qf together are =C(qg)(qh);
qg and qi, are each, independently, H, halogen, C1-C12 alkyl or substituted Cj-Cn alkyl.
The synthesis and preparation of the methyleneoxy (4'-CH2-0-2') B A monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their
oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
Analogs of methyleneoxy (4'-CH2-0-2') BNA, methyleneoxy (4'-CH2-0-2') BNA and 2'-thio-BNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett, 1998, 8, 2219- 2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226 ). Furthermore, synthesis of 2'-amino-BNA, a novel comformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2 -Amino- and 2'-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported. In certain embodiments bicyclic nucleoside having Formula VI:
Figure imgf000020_0001
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; each q,, ¾, qk and ¾ is, independently, H, halogen, C1-Q2 alkyl, substituted Q-C12 alkyl, C2-C12 alkenyl, substituted C2-Q2 alkenyl, C2-C]2 alkynyl, substituted C2-Q2 alkynyl, C1-C12 alkoxyl, substituted CrC12 alkoxyl, OJj, SJj, SOJj, S02Jj, NJjJk, N3, CN, C(=0)OJj, C(=0)NJjJk, C(=0)Jj, 0-C(=0)NJjJk, N(H)C(=NH)NJjJk5 N(H)C(=0)NJjJkorN(H)C(=S)NJjJu; and
wherein when one of qk or qi is CH3 then at least one of the other of qk or qi or one of φ and qj is other than H.
One carbocyclic bicyclic nucleoside having a 4'-(CH2)3-2' bridge and the alkenyl analog bridge 4'-CH=CH-CH2-2' have been described (Frier et al, Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al, J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al, J. Am. Chem. Soc. 2007, 129(26), 8362- 8379).
As used herein, "4'-2' bicyclic nucleoside" or "4' to 2' bicyclic nucleoside" refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2' carbon atom and the 4' carbon atom of the sugar ring.
As used herein, "monocyclic nucleosides" refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.
As used herein, "2 '-modified sugar" means a furanosyl sugar modified at the 2' position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2' modifications are selected from substituents including, but not limited to:
0[(CH2)nO]mCH3, 0(CH2)„NH2, 0(CH2)nCH3, 0(CH2)nONH2, OCH2C(=0)N(H)CH3, and
0(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. Other 2 - substituent groups can also be selected from: C1-C12 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O- alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving
pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an oligomeric compound, and other substituents having similar properties. In certain embodiments, modifed nucleosides comprise a 2'-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2 -MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2'- O- methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2 -MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, ?., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al, Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).
As used herein, a "modified tetrahydropyran nucleoside" or "modified THP nucleoside" means a nucleoside having a six-membered tetrahydropyran "sugar" substituted in for the pentofuranosyl residue in normal nucleosides. Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) {see Leumann, CJ. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA) or those compounds having Formula X:
Formula X:
Figure imgf000021_0001
X
wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula X:
Bx is a heterocyclic base moiety;
T3 and T4 are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the oligomeric compound or one of T3 and T4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the oligomeric compound and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3'-terminal group;
qi> φ> ¾3, ¾4> q5, q6 a d q7 are each independently, H, Ci-C alkyl, substituted Ci-C^ alkyl, C2- 5 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; and
one of R] and R2 is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ1J2, SJi, N3,
Figure imgf000022_0001
and CN, wherein X is O, S or NJi and each Ji, J2 and J3 is, independently, H or Ci-C6 alkyl.
In certain embodiments, the modified ΊΉΡ nucleosides of Formula X are provided wherein qm, q„, qp, qr, qs, qtand qu are each H. In certain embodiments, at least one of qm, qn, qp, qr, qs, qt and qu is other than H. In certain embodiments, at least one of qm, qn, qp, qr, qs, qt and qu is methyl. In certain embodiments, THP nucleosides of Formula X are provided wherein one of R] and R2 is F. In certain embodiments, Rt is fluoro and R2 is H; Ri is methoxy and R2 is H, and R] is methoxyethoxy and R2 is H.
As used herein, "2'-modified" or "2 '-substituted" refers to a nucleoside comprising a sugar comprising a substituent at the 2' position other than H or OH. 2'-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2' carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2'substituents, such as allyl, amino, azido, thio, O-allyl, O-Cj-Ci0 alkyl, -OCF3, 0-(CH2)2-0-CH3, 2'-0(CH2)2SCH3, 0-(CH2)2-0-N(Rm)(Rn), or 0-CH2-C(=0)- N(Rm)(Rn), where each Rm and R„ is, independently, H or substituted or unsubstituted CrQo alkyl. 2'-modifed nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase. As used herein, "2'-F" refers to a nucleoside comprising a sugar comprising a fluoro group at the 2' position. Unless otherwise indicated, the sugar of a 2'-F nucleoside is a furanose in which the 2'-OH has been replace with a F.
As used herein, "2'-F RNA" refers to a 2'-F nucleoside, wherein the fluoro group is in the ribo position.
As used herein, "2'-F ANA" refers to a 2'-F substituted nucleoside, wherein the fluoro group is in the arabino position.
Figure imgf000023_0001
2'F-RNA 2'F-ANA
As used herein, "ANA'Other than in the context of "2'F ANA" refers to altritol nucleic acid, which is a modified tetrahydropyran nucleoside as desrcribed above. Unless further modified or otherwise indicated, "ANA" nucleosides have the following structure:
Figure imgf000023_0002
As used herein, "2'-OMe" or "2'-OCH3" or "2'-0-methyl" each refers to a nucleoside comprising a sugar comprising an -OCH3 group at the 2' position of the sugar ring.
As used herein, "MOE" or "2'-MOE" or "2'-OCH2CH2OCH3" or "2'-0-methoxyethyl" each refers to a nucleoside comprising a sugar comprising a -OCH2CH2OCH3 group at the 2' position of the sugar ring.
As used herein, "oligonucleotide" refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA). As used herein, "modified oligonucleotide" refers to an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
As used herein "internucleoside linkage" refers to a covalent linkage between adjacent nucleosides.
As used herein "naturally occurring internucleoside linkage" refers to a 3' to 5' phosphodiester linkage.
As used herein, "modified internucleoside linkage" refers to any internucleoside linkage other than a naturally occurring internucleoside linkage.
As used herein, "oligomeric compound" refers to a polymeric structure comprising two or more sub-structures. In certain embodiments, an oligomeric compound comprises an
oligonucleotide. In certain embodiments, an oligomeric compound comprises a single-stranded oligonucleotide. In certain embodiments, an oligomeric compound is a double-stranded duplex comprising two oligonucleotides. In certain embodiments, an oligomeric compound is a single- stranded or double-stranded oligonucleotide comprising one or more conjugate groups and/or terminal groups.
As used herein, "conjugate" refers to an atom or group of atoms bound to an
oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to
pharmakodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to the parent compound such as an oligomeric compound. In certain embodiments, conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. In certain embodiments, conjugates are terminal groups. In certain embodiments, conjugates are attached to a 3' or 5' terminal nucleoside or to an internal nucleosides of an oligonucleotide.
As used herein, "conjugate linking group" refers to any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound. Linking groups or
bifunctional linking moieties such as those known in the art are amenable to the present invention.
As used herein, "antisense compound" refers to an oligomeric compound, at least a portion of which is at least partially complementary to a target nucleic acid to which it hybridizes and modulates the activity, processing or expression of said target nucleic acid.
As used herein, "expression" refers to the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, splicing, post-transcriptional modification, and translation.
As used herein, "antisense oligonucleotide" refers to an antisense compound that is an oligonucleotide.
As used herein, "antisense activity" refers to any detectable and/or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, such activity may be an increase or decrease in an amount of a nucleic acid or protein. In certain embodiments, such activity may be a change in the ratio of splice variants of a nucleic acid or protein. Detection and/or measuring of antisense activity may be direct or indirect. In certain embodiments, antisense activity is assessed by observing a phenotypic change in a cell or animal.
As used herein "detecting" or "measuring" in connection with an activity, response, or effect indicate that a test for detecting or measuring such activity, response, or effect is performed. Such detection and/or measuring may include values of zero. Thus, if a test for detection or measuring results in a finding of no activity (activity of zero), the step of detecting or measuring the activity has nevertheless been performed. For example, in certain
embodiments, the present invention provides methods that comprise steps of detecting antisense activity, detecting toxicity, and/or measuring a marker of toxicity. Any such step may include values of zero.
As used herein, "target nucleic acid" refers to any nucleic acid molecule the expression, amount, or activity of which is capable of being modulated by an antisense compound. In certain embodiments, the target nucleic acid is DNA or RNA. In certain embodiments, the target RNA is mRNA, pre-mRNA, non-coding RNA, pri-microRNA, pre-microRNA, mature microRNA, promoter-directed RNA, or natural antisense transcripts. For example, the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In certain embodiments, target nucleic acid is a viral or bacterial nucleic acid.
As used herein, "target mR A" refers to a pre-selected RNA molecule that encodes a protein.
As used herein, "selectivity" refers to the ability of an antisense compound to exert an antisense activity on a target nucleic acid to a greater extent than on a non-target nucleic acid.
As used herein, "mutant selective" refers to a compound that has a greater effect on a mutant nucleic acid than on the corresponding wild-type nucleic acid. In certain embodiment, the effect of a mutant selective compound on the mutant nucleic acid is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 100 times greater than the effect of the mutant selective compound on the
corresponding wild-type nucleic acid. In certain embodiments, such selectivity results from greater affinity of the mutant selective compound for the mutant nucleic acid than for the corresponding wild type nucleic acid. In certain embodiments, selectivity results from a difference in the structure of the mutant compared to the wild-type nucleic acid. In certain embodiments, selectivity results from differences in processing or sub-cellular distribution of the mutant and wild-type nucleic acids. In certain embodiments, some selectivity may be attributable to the presence of additional target sites in a mutant nucleic acid compared to the wild-type nucleic acid. For example, in certain embodiments, a target mutant allele comprises an expanded repeat region comprising additional copies of a target sequence, while the wild-type allele has fewer copies of the repeat and, thus, fewer sites for hybridization of an antisense compound targeting the repeat region. In certain embodiments, a mutant selective compound has selectivity equal to or greater than the selectivity predicted by the increased number of target sites. In certain embodiments, a mutant selective compound has selectivity greater than the selectivity predicted by the increased number of target sites. In certain embodiments, the ratio of inhibition of a mutant allele to a wild type allele is equal to or greater than the ratio of the number of repeats in the mutant allele to the wild type allele. In certain embodiments, the ratio of inhibition of a mutant allele to a wild type allele is greater than the ratio of the number of repeats in the mutant allele to the wild type allele.
As used herein, "targeting" or "targeted to" refers to the association of an antisense compound to a particular target nucleic acid molecule or a particular region of nucleotides within a target nucleic acid molecule. An antisense compound targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions.
As used herein, "target site" refers to a region of a target nucleic acid that is bound by an antisense compound. In certain embodiments, a target site is at least partially within the 3' untranslated region of an RNA molecule. In certain embodiments, a target site is at least partially within the 5' untranslated region of an RNA molecule. In certain embodiments, a target site is at least partially within the coding region of an RNA molecule. In certain embodiments, a target site is at least partially within an exon of an RNA molecule. In certain embodiments, a target site is at least partially within an intron of an RNA molecule. In certain embodiments, a target site is at least partially within a microRNA target site of an RNA molecule. In certain embodiments, a target site is at least partially within a repeat region of an RNA molecule.
As used herein, "target protein" refers to a protein, the expression of which is modulated by an antisense compound. In certain embodiments, a target protein is encoded by a target nucleic acid. In certain embodiments, expression of a target protein is otherwise influenced by a target nucleic acid.
As used herein, "complementarity" in reference to nucleobases refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.
As used herein, "non-complementary" " in reference to nucleobases refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support
hybridization.
As used herein, "complementary" in reference to linked nucleosides, oligonucleotides, or nucleic acids, refers to the capacity of an oligomeric compound to hybridize to another oligomeric compound or nucleic acid through nucleobase complementarity. In certain embodiments, an antisense compound and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bond with each other to allow stable association between the antisense compound and the target. One skilled in the art recognizes that the inclusion of mismatches is possible without eliminating the ability of the oligomeric compounds to remain in association. Therefore, described herein are antisense compounds that may comprise up to about 20% nucleotides that are mismatched (i.e., are not nucleobase complementary to the corresponding nucleotides of the target). Preferably the antisense compounds contain no more than about 15%, more preferably not more than about 10%, most preferably not more than 5% or no mismatches. The remaining nucleotides are nucleobase complementary or otherwise do not disrupt hybridization (e.g., universal bases). One of ordinary skill in the art would recognize the compounds provided herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to a target nucleic acid.
As used herein, "hybridization" refers to the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases). For example, the natural base adenine is nucleobase complementary to the natural nucleobases thymidine and uracil which pair through the formation of hydrogen bonds. The natural base guanine is nucleobase
complementary to the natural bases cytosine and 5-methyl cytosine. Hybridization can occur under varying circumstances.
As used herein, "specifically hybridizes" refers to the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site. In certain embodiments, an antisense oligonucleotide specifically hybridizes to more than one target site.
As used herein, "modulation" refers to a perturbation of amount or quality of a function or activity when compared to the function or activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. As a further example, modulation of expression can include perturbing splice site selection of pre-mRNA processing, resulting in a change in the amount of a particular splice-variant present compared to conditions that were not perturbed. As a further example, modulation includes perturbing translation of a protein.
As used herein, "motif refers to a pattern of modifications in an oligomeric compound or a region thereof. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligomeric compound.
As used herein, "nucleoside motif refers to a pattern of nucleoside modifications in an oligomeric compound or a region thereof. The linkages of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.
As used herein, "linkage motif refers to a pattern of linkage modifications in an oligomeric compound or region thereof. The nucleosides of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.
As used herein, "the same modifications" refer to modifications relative to naturally occurring molecules that are the same as one another, including absence of modifications. Thus, for example, two unmodified DNA nucleoside have "the same modification," even though the DNA nucleoside is unmodified.
As used herein, "type of modification" in reference to a nucleoside or a nucleoside of a "type" refers to the modification of a nucleoside and includes modified and unmodified nucleosides. Accordingly, unless otherwise indicated, a "nucleoside having a modification of a first type" may be an unmodified nucleoside.
As used herein, "separate regions" refers to a portion of an oligomeric compound wherein the nucleosides and internucleoside linkages within the region all comprise the same
modifications; and the nucleosides and/or the internucleoside linkages of any neighboring portions include at least one different modification.
As used herein, "pharmaceutically acceptable salts" refers to salts of active compounds that retain the desired biological activity of the active compound and do not impart undesired toxicological effects thereto.
As used herein, "cap structure" or "terminal cap moiety" refers to chemical modifications incorporated at either terminus of an antisense compound.
As used herein, "alkyl," refers to a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C1-C12 alkyl) with from 1 to about 6 carbon atoms (CrC6 alkyl) being more preferred. The term "lower alkyl" as used herein includes from 1 to about 6 carbon atoms (Ci- C6 alkyl). Alkyl groups as used herein may optionally include one or more further substituent groups. Herein, the term "alkyl" without indication of number of carbon atoms means an alkyl having 1 to about 12 carbon atoms (Ci-C12 alkyl).
As used herein, "alkenyl," refers to a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 1-methyl- 2-buten-l-yl, dienes such as 1,3 -butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.
As used herein, "alkynyl," refers to a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.
As used herein, "aminoalkyl" refers to an amino substituted alkyl radical. This term is meant to include C1-C12 alkyl groups having an amino substituent at any position and wherein the alkyl group attaches the aminoalkyl group to the parent molecule. The alkyl and/or amino portions of the aminoalkyl group can be further substituted with substituent groups.
As used herein, "aliphatic," refers to a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.
As used herein, "alicyclic" or "alicyclyl" refers to a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring.
Alicyclic as used herein may optionally include further substituent groups.
As used herein, "alkoxy," refers to a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, w-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.
As used herein, "halo" and "halogen," refer to an atom selected from fluorine, chlorine, bromine and iodine.
As used herein, "aryl" and "aromatic," refer to a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituent groups.
As used herein, "aralkyl" and "arylalkyl," refer to a radical formed between an alkyl group and an aryl group wherein the alkyl group is used to attach the aralkyl group to a parent molecule. Examples include, but are not limited to, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.
As used herein, "heterocyclic radical" refers to a radical mono-, or poly-cyclic ring system that includes at least one heteroatom and is unsaturated, partially saturated or fully saturated, thereby including heteroaryl groups. Heterocyclic is also meant to include fused ring systems wherein one or more of the fused rings contain at least one heteroatom and the other rings can contain one or more heteroatoms or optionally contain no heteroatoms. A heterocyclic group typically includes at least one atom selected from sulfur, nitrogen or oxygen. Examples of heterocyclic groups include, [l,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and the like.
Heterocyclic groups as used herein may optionally include further substituent groups.
As used herein, "heteroaryl," and "heteroaromatic," refer to a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatom. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms.
Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.
As used herein, "heteroarylalkyl," refers to a heteroaryl group as previously defined having an alky radical that can attach the heteroarylalkyl group to a parent molecule. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl, napthyridinylpropyl and the like. Heteroarylalkyl groups as used herein may optionally include further substituent groups on one or both of the heteroaryl or alkyl portions.
As used herein, "mono or poly cyclic structure" refers to any ring systems that are single or polycyclic having rings that are fused or linked and is meant to be inclusive of single and mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic, heteroarylalkyl. Such mono and poly cyclic structures can contain rings that are uniform or have varying degrees of saturation including fully saturated, partially saturated or fully unsaturated. Each ring can comprise ring atoms selected from C, N, O and S to give rise to heterocyclic rings as well as rings comprising only C ring atoms which can be present in a mixed motif such as for example benzimidazole wherein one ring has only carbon ring atoms and the fused ring has two nitrogen atoms. The mono or poly cyclic structures can be further substituted with substituent groups such as for example phthalimide which has two =0 groups attached to one of the rings. In another aspect, mono or poly cyclic structures can be attached to a parent molecule directly through a ring atom, through a substituent group or a bifunctional linking moiety.
As used herein, "acyl," refers to a radical formed by removal of a hydroxyl group from an organic acid an d has the general formula -C(0)-X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.
As used herein, "hydrocarbyl refers to any group comprising C, O and H. Included are straight, branched and cyclic groups having any degree of saturation. Such hydrocarbyl groups can include one or more heteroatoms selected from N, O and S and can be further mono or poly substituted with one or more substituent groups.
As used herein, "substituent" and "substituent group," include groups that are typically added to other groups or parent compounds to enhance desired properties or give desired effects. Substituent groups can be protected or unprotected and can be added to one available site or to many available sites in a parent compound. Substituent groups may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.
Unless otherwise indicated, the term substituted or "optionally substituted" refers to the following substituents: halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (-C(O)Raa), carboxyl (- C(O)O-Raa), aliphatic groups, alicyclic groups, alkoxy, substituted oxo (-O-Raa), aryl, aralkyl, heterocyclic, heteroaryl, heteroarylalkyl, amino (-NRbbRcc), imino(=NRbb), amido (-C(0)N- RbbRccOr -N(Rbb)C(0)Raa), azido (-N3), nitro (-N02), cyano (-CN), carbamido (-OC(0)NRbbRcc or -N(Rbb)C(0)ORaa), ureido (-N(Rbb)C(0)NRbbRcc), thioureido (-N(Rbb)C(S)NRbbRcc), guanidinyl (-N(Rbb)C(=NRbb)NRbbRcc), amidinyl (-C(=NRbb)NRbbRcc or -N(Rbb)C(NRbb)Raa), thiol (-SRbb), sulfinyl (-S(0)Rbb), sulfonyl (-S(0)2Rbb), sulfonamidyl (-S(0)2NRbbRcc or -N(Rbb)- S(0)2Rbb) and conjugate groups. Wherein each Raa, Rbb and Roc is, independently, H, an optionally linked chemical functional group or a further substituent group which may be selected from, without limitation: H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree. In this context, "recursive substituent" means that a substituent may recite another instance of itself. Because of the recursive nature of such substituents, theoretically, a large number may be present in any given claim. One of ordinary skill in the art of medicinal chemistry and organic chemistry understands that the total number of such substituents is reasonably limited by the desired properties of the compound intended. Such properties include, by way of example and not limitation, physical properties such as molecular weight, solubility or log P, application properties such as activity against the intended target and practical properties such as ease of synthesis.
Recursive substituents are an intended aspect of the invention. One of ordinary skill in the art of medicinal and organic chemistry understands the versatility of such substituents. To the degree that recursive substituents are present in a claim of the invention, the total number will be determined as set forth above.
The terms "stable compound" and "stable structure" as used herein are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Only stable compounds are contemplated herein.
As used herein, a zero (0) in a range indicating number of a particular unit means that the unit may be absent. For example, an oligomeric compound comprising 0-2 regions of a particular motif means that the oligomeric compound may comprise one or two such regions having the particular motif, or the oligomeric compound may not have any regions having the particular motif. In instances where an internal portion of a molecule is absent, the portions flanking the absent portion are bound directly to one another. Likewise, the term "none" as used herein, indicates that a certain feature is not present.
As used herein, "analogue" or "derivative" means either a compound or moiety similar in structure but different in respect to elemental composition from the parent compound regardless of how the compound is made. For example, an analogue or derivative compound does not need to be made from the parent compound as a chemical starting material.
B. Nucleotide Repeat-Containing RNA
In certain embodiments, the present invention provides compounds and methods for modulating the amount, activity or function of a nucleotide repeat-containing RNA. Such nucleotide repeat-containing RNA molecules have been associated with a number of diseases or disorders.
Certain normal wild-type RNA molecules comprise repeat regions, which, in certain instances can become expanded. The shorter repeat regions of wild type transcripts not associated with disease typically have secondary structure, making them relatively inaccessible for base pairing with a complementary nucleic acid. In contrast, the number of repeats in the expanded repeat region of a nucleotide repeat-containing RNA is typically at least 2 fold normal and often more (e.g., 3, 5, 10 fold, up to 100 or even more than 1000 fold). This expansion increases the likelihood that part of the repeat is, at least temporarily, more accessible to base pairing with a complementary nucleic acid molecule, relative to the wild type allele. Thus, even though certain oligomeric compounds of the present invention comprise oligonucleotides are complementary to a repeat sequence present in both wild-type and repeat-expanded transcripts, in certain embodiments, such compounds selectively hybridize to the disease-associated repeat- expanded transcript. Such selectivity is beneficial for treating diseases associated with nucleotide repeat-containing RNA irrespective of the mechanism of reduction of the aberrant transcript.
Certain nucleotide repeat-containing RNA have been referred to in the art as "gain-of- function RNAs" for their ability to sequester hnRNPs and impair the normal action of RNA processing in the nucleus (see e.g., Cooper, T. (2009) Cell 136, 777-793; O'Rourke, JR (2009) J. Biol. Chem. 284 (12), 7419-7423, which are herein incorporated by reference in the entirety). Several disease states are associated with nucleotide repeat-containing RNA, some of which only occur once a threshold number of repeats within the nucleotide repeat-containing RNA is reached. In certain embodiments, the present invention provides methods of reducing the activity, function, or amount of a nucleotide repeat-containing RNA having at least 10, 15, 20, , 25, 30, 35, 40, 45, 50, 55, 60, 80, 90, 100, 200, 300, 400, 500, 1000, or more than 1000 copies of a repeating nucleotide unit. In certain embodiments, the present invention provides compounds and methods for ;eting or treating any of the disorders in the following none limiting table:
Figure imgf000036_0001
Spinocerebellar CAG ATXN3 1996(NCBI/OMIM) ataxia 3
14 to 32 33 to 77 Wikipedia
(Machado- Joseph disease 10 to 51 55-87 Human Mol. Genet.
17: 2071, 2008 (NCBI/OMIM)
12 to 40 55 to 86 Wikipedia
Spinocerebellar CAG CACNA1A 4 to 18 21 to 30 Wikipedia ataxia 6
5 to 20 21 to 25 Am. J. Hum. Genet.
61: 336, 1997 (NCBI/OMIM)
Spinocerebellar CAG ATXN7 7 to 17 38-130 Nat. Genet. 17: 65, ataxia 7/OPCA3 1997
(NCBI/OMIM)
In certain embodiments, compounds of the present invention are used to alter the activity or amount of nucleotide repeat-containing RNA. In certain embodiments, compounds of the present invention are mutant selective. Accordingly, certain such compounds reduce the amount or activity of nucleotide repeat-containing RNA to a greater extent than they reduce the amount or activity of the corresponding wild-type RNA.
C. Certain Compounds of the Present Invention
In certain embodiments, the present invention provides oligomeric compounds useful for studying, diagnosing, and/or treating a disease or disorder associtaed with a nucleotide repeat- containing RNA. In certain embodiments, oligomeric compounds of the present invention comprise an oligonucleotide and a conjugate or terminal group. In certain embodiments, oligomeric compounds consist of an oligonucleotide.
In certain embodiments, oligonucleotide of the present invention have a nucleobase sequence comprising a region that is complementary to a nucleotide repeat-containing RNA. In certain embodiments, oligonucleotide of the present invention have a nucleobase sequence comprising a region that is complementary to a repeat region of a nucleotide repeat-containing RNA.
In certain embodiments, oligonucleotides of the present invention comprise one or more modification. In certain embodiments, oligonucleotides of the present invention comprise one or more modifed nucleoside. In certain embodiments, modified nucleosides of the present invention comprise a modifed nucleobase. In certain embodiments, nucleosides of the present invention comprise one or more modified sugar. In certain embodiments, oligonucleotides of the present invention comprise one or more high-affinity sugar modified nucleoside. In certain embodiments, oligonucleotides of the present invention comprise one or more modfied internucleoside linkage. In certain embodiments, oligonucleotides of the present invention comprise one or more nuclease resistant nucleotide. a. Certain Nucleobases
In certain embodiments, nucleosides of the present invention comprise unmodified nucleobases. In certain embodiments, nucleosides of the present invention comprise modifed nucleobases.
In certain embodiments, nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to the oligomeric compounds. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases also referred to herein as heterocyclic base moieties include other synthetic and natural nucleobases, many examples of which such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, 7-deazaguanine and 7-deazaadenine among others.
Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone. Certain modified nucleobases are disclosed in, for example, Sw yze, E.E. and Bhat, B., The Medicinal Chemistry of Oligonucleotides in ANTISENSE DRUG TECHNOLOGY, Chapter 6, pages 143-182 (Crooke, S.T., ed., 2008); U.S. Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858- 859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991 , 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B. , ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2
aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
In certain embodiments, nucleobases comprise polycyclic heterocyclic compounds in place of one or more heterocyclic base moieties of a nucleobase. A number of tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand. The most studied modifications are targeted to guanosines hence they have been termed G- clamps or cytidine analogs.
Representative cytosine analogs that make 3 hydrogen bonds with a guanosine in a second strand include l,3-diazaphenoxazine-2-one (Kurchavov, et ah, Nucleosides and
Nucleotides, 1997, 16, 1837-1846), l,3-diazaphenothiazine-2-one (Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117, 3873-3874) and 6,7,8,9-tetrafluoro-l,3- diazaphenoxazine-2-one (Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998, 39, 8385- 8388). When incorporated into oligonucleotides, these base modifications have been shown to hybridize with complementary guanine and the latter was also shown to hybridize with adenine and to enhance helical thermal stability by extended stacking interactions (also see U.S. Patent Application Publication 20030207804 and U.S. Patent Application Publication 20030175906, both of which are incorporated herein by reference in their entirety).
Helix-stabilizing properties have been observed when a cytosine analog/substitute has an aminoethoxy moiety attached to the rigid l,3-diazaphenoxazine-2-one scaffold (Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532). Binding studies demonstrated that a single incorporation could enhance the binding affinity of a model oligonucleotide to its complementary target DNA or RNA with a ATm of up to 18° relative to 5-methyl cytosine (dC5me), which is the highest known affinity enhancement for a single modification. On the other hand, the gain in helical stability does not compromise the specificity of the
oligonucleotides. The Tm data indicate an even greater discrimination between the perfect match and mismatched sequences compared to dC5me. It was suggested that the tethered amino group serves as an additional hydrogen bond donor to interact with the Hoogsteen face, namely the 06, of a complementary guanine thereby forming 4 hydrogen bonds. This means that the increased affinity of G-clamp is mediated by the combination of extended base stacking and additional specific hydrogen bonding.
Tricyclic heterocyclic compounds and methods of using them that are amenable to the present invention are disclosed in U.S. Patent 6,028,183, and U.S. Patent 6,007,992, the contents of both are incorporated herein in their entirety.
The enhanced binding affinity of the phenoxazine derivatives together with their sequence specificity makes them valuable nucleobase analogs for the development of more potent antisense-based drugs. The activity enhancement was even more pronounced in case of G-clamp, as a single substitution was shown to significantly improve the in vitro potency of a 20mer 2'-deoxyphosphorothioate oligonucleotides (Flanagan, W. M.; Wolf, J.J.; Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc. Natl. Acad. Sci. USA, 1999, 96, 3513-3518).
Modified polycyclic heterocyclic compounds useful as heterocyclic bases are disclosed in but not limited to, the above noted U.S. 3,687,808, as well as U.S.: 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187; 5,459,255; 5,484,908;
5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985;
5,646,269; 5,750,692; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S. Patent Application Publication 20030158403, each of which is incorporated herein by reference in its entirety.
b. Sugar Modifications
In certain embodiments, oligonucleotides of the present invention comprise one or more modified nucleoside comprising a modified sugar moiety. Certain such modified sugar moieties are known in the art to improve certain properties of oligonucleotides. For example, oligonucleotides having certain modified sugar moieties have increased resistance to digestion by ribonucleases. In certain embodiments, such nuclease resistance sugar modifications are suitable for nucleosides for use in the present invention.
Oligonucleotides incorporating certain modified sugar moieties have increased affinity for a target nucleic acid. In certain embodiments, such high affinity sugar modifications are suitable for use in the present invention. In certain embodiments, the present invention includes oligonucleotides having one or more high-affinity sugar modified nucleoside. In certain embodiments, the present invention includes oligonucleotides having one or more bicyclic nucleoside. In certain embodiments, the present invention includes oligonucleotides having one or more nucleoside comprising a 2 '-modified sugar. In certain embodiments, the present invention includes oligonucleotides having one or more modified tetrahydropyran nucleoside.
RNA duplexes exist in what has been termed "A Form" geometry while DNA duplexes exist in "B Form" geometry. In general, RNA:RNA duplexes are more stable, or have higher melting temperatures (Tm) than DNA:DNA duplexes (Sanger et al, Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, NY.; Lesnik et al, Biochemistry, 1995, 34, 10807- 10815; Conte et al, Nucleic Acids Res., 1997, 25, 2627-2634). The increased stability of RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et al, Nucleic Acids Res., 1993, 21, 2051-2056). The presence of the 2' hydroxyl in RNA biases the sugar toward a C3' endo pucker, i.e., also designated as Northern pucker, which causes the duplex to favor the A-form geometry. In addition, the 2' hydroxyl groups of RNA can form a network of water mediated hydrogen bonds that help stabilize the RNA duplex (Egli et al, Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleic acids prefer a C2' endo sugar pucker, i.e., also known as Southern pucker, which is thought to impart a less stable B-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York, NY).
The relative ability of a chemically-modified oligomeric compound to bind to complementary nucleic acid strands, as compared to natural oligonucleotides, is measured by obtaining the melting temperature of a hybridization complex of said chemically-modified oligomeric compound with its complementary unmodified target nucleic acid. The melting temperature (Tm), a characteristic physical property of double helixes, denotes the temperature in degrees centigrade at which 50% helical versus coiled (unhybridized) forms are present. Tm (also commonly referred to as binding affinity) is measured by using the UV spectrum to determine the formation and breakdown (melting) of hybridization. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently a reduction in UV absorption indicates a higher Tm.
It is known in the art that the relative duplex stability of an antisense compound:RNA target duplex can be modulated through incorporation of chemically-modified nucleosides into the antisense compound. Sugar-modified nucleosides have provided the most efficient means of modulating the Tm of an antisense compound with its target RNA. Sugar-modified nucleosides that increase the population of or lock the sugar in the C31 -endo (Northern, RNA-like sugar pucker) configuration have predominantly provided a per modification Tm increase for antisense compounds toward a complementary RNA target. Sugar-modified nucleosides that increase the population of or lock the sugar in the C2'-endo (Southern, DNA-like sugar pucker) configuration predominantly provide a per modification Tm decrease for antisense compounds toward a complementary RNA target. The sugar pucker of a given sugar-modified nucleoside is not the only factor that dictates the ability of the nucleoside to increase or decrease an antisense compound's Tm toward complementary RNA. For example, the sugar-modified nucleoside tricycloDNA is predominantly in the Q -endo conformation, however it imparts a 1.9 to 3° C per modification increase in Tm toward a complementary RNA. Another example of a sugar- modified Wgh-affinity nucleoside that does not adopt the CV-endo conformation is a-L-LNA. c. Certain Intemucleoside Linkages
In certain embodiments, nucleosides are linked together using any intemucleoside linkage. The two main classes of intemucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing intemucleoside linkages include, but are not limited to, phosphodiesters (P=0), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P=S). Representative non-phosphorus containing intemucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2- N(CH3)-0-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-0-C(0)(NH)-S-); siloxane (-0- Si(H)2-0-); and Ν,Ν'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Oligonucleotides having non-phosphorus intemucleoside linking groups may be referred to as oligonucleosides. Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligomeric compound. In certain embodiments, intemucleoside linkages having a chiral atom can be prepared a racemic mixture, as separate enantomers.
Representative chiral linkages include, but are not limited to, alkylphosphonates and
phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous- containing intemucleoside linkages are well known to those skilled in the art.
The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), a or β such as for sugar anomers, or as (D) or (L) such as for amino acids et al. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
As used herein the term "intemucleoside linkage" or "intemucleoside linking group" is meant to include all manner of intemucleoside linking groups known in the art including but not limited to, phosphorus containing intemucleoside linking groups such as phosphodiester and phosphorothioate, and non-phosphorus containing intemucleoside linking groups such as formacetyl and memyleneimino. Intemucleoside linkages also includes neutral non-ionic intemucleoside linkages such as amide-3 (3'-CH2-C(=0)-N(H)-5'), amide-4 (3'-CH2-N(H)- C(=0)-5') and methylphosphonate wherein a phosphorus atom is not always present.
As used herein the phrase "neutral intemucleoside linkage" is intended to include intemucleoside linkages that are non-ionic. Neutral intemucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI (3'-CH2-N(CH3)-0-5'), amide-3 (3'-CH2- C(=0)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=0)-5'), formacetal (3'-0-CH2-0-5,)5 and
thioformacetal (3'-S-CH -0-5'). Further neutral intemucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65).
Further neutral intemucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 component parts.
The intemucleoside linkage found in native nucleic acids is a phosphodiester linkage. This linkage has not been the linkage of choice for synthetic oligonucleotides that are for the most part targeted to a portion of a nucleic acid such as mRNA because of stability problems e.g. degradation by nucleases. Preferred intemucleoside linkages or intemucleoside linkages as is the case for non phosphate ester type linkages include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates,
thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3 '-5' linkages, 2 '-5' linked analogs of these, and those having inverted polarity wherein one or more intemucleoside linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3 '-most intemucleoside linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.
Representative United States patents that teach the preparation of the above phosphorus- containing linkages include, but are not limited to, U.S.: 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;
5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;
5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899;
5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.
Preferred modified intemucleoside linkages that do not include a phosphorus atom therein include short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include siloxane, sulfide, sulfoxide, sulfone, formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl, alkenyl, sulfamate, methyleneimino, methylenehydrazino, sulfonate, sulfonamide, amide and others having mixed N, O, S and CH2 component parts.
Representative United States patents that teach the preparation of the above
oligonucleosides include, but are not limited to, U.S.: 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677;
5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240;
5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608;
5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.
In certain embodiments, the invention provides oligomeric compounds with
phosphorothioate intemucleoside linkages and oligomeric compounds with heteroatom intemucleoside linkages, and in particular -CH2-NH-0-CH2-, -CH2-N(CH3)-0-CH2- [known as a methylene (methylimino) or MMI backbone], -CH2-0-N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)- CH2- and -0-N(CH3)-CH2-CH2- [wherein the native phosphodiester backbone is represented as - 0-P(=0)(OH)-0-CH2-] of the above referenced U.S. patent 5,489,677, and the amide internucleoside linkages of the above referenced U.S. patent 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. patent 5,034,506.
d. Certain Oligomeric Compounds
In certain embodiments, oligonucleotides have a motif that is useful in modulating nucleotide repeat-containing RNA. In certain embodiments, the motif is a nucleoside motif. In certain embodiments, the motif is a linkage motif. In certain embodiments, the motif includes a nucleoside motif and a linkage motif. Certain such oligonucleotides are antisense compounds. Certain specific motifs disclosed herein are useful broadly. For example, certain motifs having activity for a particular nucleotide repeat-containing RNA have similar activity for other nucleotide repeat-containing RNA. In certain such embodiments, the activity is mutant selective activity.
In certain embodiments, oligonucleotides comprise one or more conjugates or additional groups, particularly at the 3' position of the sugar on the 3' terminal nucleotide and/or the 5' position of 5' terminal nucleotide. For example, oligonucleotides of the present invention may comprise ligand or non-ligand moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison- Behmoaras et al., EMBO J., 1991, 10, 111 ; Kabanov et al., FEBS Lett., 1990, 259, 327;
Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or an octadecylamine or hexylamino- carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923). Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Patents Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;
5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046;
4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;
5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned, and each of which is herein incorporated by reference. e. Oligonucleotide Synthesis
Commercially available equipment routinely used for the support media based synthesis of oligomeric compounds and related compounds is sold by several vendors including, for example, Applied Biosystems (Foster City, CA), General Electric, as well as others. Suitable solid phase techniques, including automated synthesis techniques, are described in Scozzari and Capaldi, "Oligonucleotide Manufacturing and Analysic Processes for 2'-0-(2-methoxyethyl- Modified Oligonucleotides" in Crooke, ST (ed.) ANTISENSE THERAPEUTICS (2008). f. Compositions and Methods for Formulating Pharmaceutical Compositions Oligomeric compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations.
Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
Oligomeric compounds, including antisense compounds, can be utilized in
pharmaceutical compositions by combining such oligomeric compounds with a suitable pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in certain embodiments, the invention provides a
pharmaceutical composition comprising an antisense compound and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is sterile pharmaceutical grade PBS. In certain embodiments, the pharmaceutically acceptable diluent is sterile pharmaceutical grade water. In certain embodiments, the pharmaceutically acceptable diluent is sterile pharmaceutical grade saline.
Pharmaceutical compositions comprising oligomeric compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising oligomeric compounds comprise one or more oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
A prodrug can include the incorporation of additional nucleosides at one or both ends of an oligomeric compound which are cleaved by endogenous nucleases within the body, to form the active oligomeric compound.
Lipid-based vectors have been used in nucleic acid therapies in a variety of methods. In one method, the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In another method, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid.
Certain preparations are described in Akinc et al., Nature Biotechnology 26, 561 - 569 (01 May 2008), which is herein incorporated by reference in its entirety.
D. Certain Uses and Routes of Administration
In certain embodiments, the present invention provides methods of contacting a cell with an oligomeric compound described herein. In certain embodiments, the cell is in vitro. In certain embodiments, the cell is in an animal (e.g., rodent, primate, monkey or human). In certain embodiments, antisense activity is detected.
In certain embodiments, described herein is use of an oligomeric compound comprising an oligonucleotide consisting of 13 to 30 linked nucleosides and having a nucleobase sequence 100% complementary to a repeat region of a nucleotide repeat-containing RNA, wherein the oligonucleotide contains: a. a 5'-region consisting of 1-5 linked 5'-region nucleosides, wherein the 5'-region nucleosides each have the same modification as one another;
b. a central region consisting of 5 to 20 linked nucleosides, wherein each nucleoside of the central region either comprises a 2'-fluoro sugar moiety or is a non-2'-fiuoro central-region nucleoside, provided that at least one nucleoside of the central region comprises a 2'-fluoro sugar moiety, and wherein the non-2 '-fluoro central-region nucleosides each have the same modification as one another; and
c. a 3'-region consisting of 1-5 linked 3'-region nucleosides, wherein the 3'-region nucleosides each have the same modification as one another; for the treatment of a disease associated with a C AG nucleotide repeat-containing RNA.
In cetain embodiments, the disease is any of Atrophin 1, Huntington's Disease,
Huntington disease-like 2 (HDL2), spinal and bulbar muscular atrophy, Kennedy disease, spinocerebellar ataxia 1, spinocerebellar ataxia 12, spinocerebellar ataxia 17, Huntington disease-like 4 (HDL4), spinocerebellar ataxia 2, spinocerebellar ataxia 3, Machado- Joseph disease, spinocerebellar ataxia 6, and spinocerebellar ataxia 7.
In certain embodiments, compounds of the present invention are administered to an animal (e.g., a human) to provide a therapeutic effect. Certain diseases or disorders have been identified to be associated with nucleotide repeat-containing RNA. Any such disease or disorder might be treated with compounds of the present invention. In certain embodiments, the disease is selected from among: ataxin 8, atrophin 1, fragile X syndrome, Friedrich's ataxia, Huntington's disease, Huntington's disease-like 2, myotonic dystrophy, spinal and bulbar muscular atrophy, and spinocerebellar ataxia. In certain embodiments, the disease is Huntington's disease. In certain embodiments, the disease is myotonic dystrophy. In certain embodiments, the myotonic dystrophy is myotonic dystrophy type 1. In certain embodiments, the myotonic dystrophy is myotonic dystrophy type 2. In certain embodiments, the disease is spinocerebellar ataxia. In certain embodiments, the spinocerebellar ataxia is spinocerebellar ataxia 10. One of skill in the art may choose a formulation and route of administration based on the needs particular disease or disorder, for example, one may tailor a formulation and route of administration to result in delivery of the oligomeric compound to the tissue or cell in need.
In certain embodiments, pharmaceutical compositions of the present invention are administered to a subject. In certain embodiments, such pharmaceutical compositions are administered by injection. In certain embodiments, such pharmaceutical compositions are administered by infusion.
In certain embodiments, pharmaceutical compositions are administered by injection or infusion into the CSF. In certain such embodiments, pharmaceutical compositions are administered by direct injection or infusion into the spine. In certain embodiments,
pharmaceutical compositions are administered by injection or infusion into the brain. In certain embodiments, pharmaceutical compositions are administered by intrathecal injection or infusion rather than into the spinal cord tissue itself. Without being limited as to theory, in certain embodiments, the antisense compound released into the surrounding CSF and may penetrate into the spinal cord parenchyma. An additional advantage of intrathecal delivery is that the intrathecal route mimics lumbar puncture administration (i.e., spinal tap) already in routine use in humans.
In certain embodiments, pharmaceutical compositions are administered by
intracerebroventricular (ICV) injection or infusion. Intracerebroventricular or intraventricular delivery of a pharmaceutical composition comprising one or more oligomeric compound may be performed in any one or more of the brain's ventricles, which are filled with cerebrospinal fluid (CSF). CSF is a clear fluid that fills the ventricles, is present in the subarachnoid space, and surrounds the brain and spinal cord. CSF is produced by the choroid plexuses and via the weeping or transmission of tissue fluid by the brain into the ventricles. The choroid plexus is a structure lining the floor of the lateral ventricle and the roof of the third and fourth ventricles. Certain studies have indicated that these structures are capable of producing 400-600 ccs of fluid per day consistent with an amount to fill the central nervous system spaces four times in a day. In adult humans, the volume of this fluid has been calculated to be from 125 to 150 ml (4-5 oz). The CSF is in continuous formation, circulation and absorption. Certain studies have indicated that approximately 430 to 450 ml (nearly 2 cups) of CSF may be produced every day. Certain calculations estimate that production equals approximately 0.35 ml per minute in adults and 0.15 per minute in infant humans. The choroid plexuses of the lateral ventricles produce the majority of CSF. It flows through the foramina of Monro into the third ventricle where it is added to by production from the third ventricle and continues down through the aqueduct of Sylvius to the fourth ventricle. The fourth ventricle adds more CSF; the fluid then travels into the subarachnoid space through the foramina of Magendie and Luschka. It then circulates throughout the base of the brain, down around the spinal cord and upward over the cerebral hemispheres. The CSF empties into the blood via the arachnoid villi and intracranial vascular sinuses.
In certain embodiments, such pharmaceutical compositions are administered
systemically. In certain embodiments, pharmaceutical compositions are administered subcutaneously. In certain embodiments, pharmaceutical compositions are administered intravenously. In certain embodiments, pharmaceutical compositions are administered by intramuscular injection.
In certain embodiments, pharmaceutical compositions are administered both directly to the CSF (e.g., IT and/or ICV injection and/or infusion) and systemically. In certain such embodiments, compounds of the present invention have one or more desirable properties making them suitable for such administration. Drug design typically requires a balance of several variables, including, but not limited to: potency, toxicity, stability, tissue distribution, convenience, and cost of a candidate compound. Such balancing is influenced by a number of factors, including the severity and typical duration of the disease treated. For example, greater drug-related toxicity is tolerated for use in treating acute lethal diseases than chronic sub-lethal diseases. In certain embodiments, compounds of the present invention will have one or more improved properties compared to similar compounds that lack certain features of the present invention. For example, compared to other compounds, the compounds of the present invention, may, in certain embodiments, have improved potency or may have similar potency but reduced toxicity and consequently improved therapeutic index. In certain embodiments, compounds of the present invention may have improved pharmecokinetics or distribution to a particular desired target tissue. One of ordinary skill will appreciate that the suitablility of the present compounds for a particular indication may be assessed based on a number of variables specific to each such particular indication.
In certain embodiments, oligomeric compounds of the present invention are used in cells in vitro. In certain such embodiments, such uses are to identify and/or study nucleotide repeat- containing nucleic acids and mechanisms surrounding them and associated diseases. Nonlimiting disclosure and incorporation by reference
While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.
Although the sequence listing accompanying this filing identifies each sequence as either "RNA" or "DNA" as required, in reality, those sequences may be modified with any
combination of chemical modifications. One of skill in the art will readily appreciate that such designation as "RNA" or "DNA" to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2' -OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2' -OH for the natural 2'-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) for natural uracil of RNA).
Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligomeric compound having the nucleobase sequence "ATCGATCG" encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence
"AUCGAUCG" and those having some DNA bases and some RNA bases such as
"AUCGATCG" and oligomeric compounds having other modified bases, such as
"ATmeCGAUCG," wherein meC indicates a cytosine base comprising a methyl group at the 5- position.
EXAMPLES
The following examples illustrate certain embodiments of the present invention and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an
oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high- affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.
Example 1: Effect of LN A-modified oligonucleotides, targeting human huntingtin (hit) mRNA, on huntingtin (Htt) protein
Antisense oligonucleotides targeted to the CAG repeat sequence of mutant huntingtin mRNA and with LNA modifications were tested for their effect on Htt protein levels in vitro. The GM04281 fibroblast cell line (Coriell Institute for Medical Research, NJ, USA) containing 69 CAG repeats in the mutant htt allele and 17 CAG repeats in the wild-type allele, was utilized in this assay. Cells were cultured at a density of 60,000 cells per well in 6-well plates and were transfected using Lipofectamine™ RNAiMAX reagent (Invitrogen, CA) with 100 nM antisense oligonucleotide for 24 hours. The wells were then aspirated and fresh culture medium was added to each well.
After a post-transfection period of 4 days, the cells were harvested with trypsin solution (0.05% Trypsin-EDTA, Invitrogen) and lysed. The protein concentration in each sample was quantified with the micro-bicinchoninic acid (micro-BCA) assay (Thermo Scientific). An SDS- PAGE gel (Bio-Rad) was used to separate wild-type and mutant Htt proteins. Gels were run at 80 V for 15 min followed by 110 V for 5 hr. The electrophoresis apparatus was placed in an ice- water bath to prevent overheating. In parallel with analysis for Htt expression, portions of each protein lysate sample were also analyzed for β-actin expression by SDS-PAGE to ensure that there had been equal protein loading of each sample.
After electrophoresis, proteins in the gel were transferred to a nitrocellulose membrane (Hybond-C Extra; GE Healthcare Bio-Sciences). Primary antibodies specific for Htt
(MAB2166, Chemicon) and β-actin (Sigma) protein were used at 1 : 10,000 dilutions. HRP- conjugated anti-mouse secondary antibody (1:10,000, Jackson ImmunoResearch Laboratories) was used for visualizing proteins using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Protein bands were quantified using ImageJ software. The percentage inhibition was calculated relative to the negative control sample and presented in Table 1. The comparative percent inhibitions of the wild-type Htt protein and the mutant Htt protein are also presented. The Tm value for each oligonucleotide, determined by differential scanning calorimetry (DSC) is also shown.
The antisense oligonucleotides utilized in the assay are described in Table 1. The antisense oligonucleotides were obtained from either Sigma Aldrich or ISIS Pharmaceuticals. Of the antisense oligonucleotides presented in Table 1, DNA22 is an unmodified oligonucleotide (DNA nucleosides with phosphodiester linkages). The negative control is a scrambled oligonucleotide sequence. The LNA modifications in each oligonucleotide are indicated by the subscript 'L' after each base.
Table 1
Effect of LNA-modified antisense oligonucleotides on wild-type and mutant Htt protein
Figure imgf000053_0001
Several of the oligonucleotides reduced nucleotide repeat-containing RNA more than they reduced the corresponding wild-type.
Example 2: in vitro dose-dependent effect of LNA-modified nucleotides on human Htt protein
Antisense oligonucleotides from Example 1 (see Table 1 for description of chemical modifications) were tested at various doses in patient fibroblast cells. GM04281 fibroblast cells were plated at a density of 60,000 cells per well in 6-well plates and transfected using
Lipofectamine™ RNAiMAX reagent (Invitrogen, CA) reagent with increasing concentrations of antisense oligonucleotide for 24 hours, as specified in Tables 2 and 3. The cell samples were processed for protein analysis utilizing the procedure outlined in Example 1.
Results are presented in Tables 2 and 3 as percent inhibition of wild-type and mutant Htt protein, relative to untreated control cells. The data presented is an average of several independent assays performed with each antisense oligonucleotide. As illustrated in Table 2, Htt mutant protein levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The IC50 (nM) values for each oligonucleotide for inhibition of the mutant protein and wild-type protein is also shown and indicates that each oligonucleotide preferentially targets the mutant htt mRNA compared to the wild-type. The oligonucleotides listed in Table 3 demonstrate low in vitro potency and or little or no preferential lowering of the mutant mRNA compared to the wild-type.
Table 2:
Dose-dependent effect of LNA-modified oligonucleotides on wild-type versus mutant Htt protein
Figure imgf000054_0001
wild-type 9 14 17 23 25 28 >100
LNA(T)+22
mutant 10 24 34 43 58 69 37.2
Table 3:
Effect of LNA-modified oligonucleotides on wild-type versus mutant Htt protein
Figure imgf000055_0001
Example 3: Effect of chemically modified oligonucleotides, targeting human huntingtin (hit) mRNA, on huntingtin (Htt) protein
Antisense oligonucleotides targeted to the CAG repeat sequence of mutant huntingtin nucleic acid and with various chemical modifications were tested for their effects on Htt protein levels in vitro. GM04281 fibroblast cells were cultured at a density of 60,000 cells per well in 6- well plates and transfected using Lipofectamine™ RNAi AX reagent (Invitrogen, C A) with 100 nM antisense oligonucleotide for 24 hours. The cell samples were processed for protein analysis utilizing the procedure outlined in Example 1. The percentage inhibition of the protein samples was calculated relative to the negative control sample and presented in Table 4. The comparative percent inhibitions of the wild-type Htt protein and the mutant Htt protein are also presented. The Tm value for each oligonucleotide, determined by DSC, is also shown.
The antisense oligonucleotides utilized in the assay are described in Table 4. The antisense oligonucleotides were obtained from Sigma Aldrich, ISIS Pharmaceuticals, Glen Research (Virginia, USA), or the M.J. Damha laboratory (McGill Univeristy, Montreal, Cancada), and are indicated as such. The modifications in each oligonucleotide are indicated as follows: subscript Έ'= cET; subscript T=cLNA; subscript 'L'=LNA; bracketed base=ENA, italicized base=MOE; lowercase base=ANA; bolded base=2'F-RNA; and underlined base=2'F- ANA.
Table 4
Effect of chemically modified antisense oligonucleotides on wild-type and mutant Htt protein
Figure imgf000056_0001
2'F-RNA 8
GCUGCUGCUGCUGCUGCUG 19 98 5 0 full
2'F-ANA 9
GCTGCUGCTGCUGCTGCUG 19 0 0 0
Alt
2'F-ANA 8
GCUGCUGCUGCUGCUGCUG 19 0 0 0 full
2'F-ANA 3
GCTi GCTT GCT, GCT, GCT, GCTT G 19 0 0 0
LNA(T)
Example 4: in vitro dose-dependent effect of chemically modified oligonucleotides on human Htt protein
Antisense oligonucleotides from Example 3 (see Table 4 for description of chemical modifications) were tested at various doses in patient fibroblast cells. GM04281 fibroblast cells were plated at a density of 60,000 cells per well in 6-well plates and transfected using
Lipofectamine™ RNAiMAX reagent (Invitrogen, CA) reagent with increasing concentrations of antisense oligonucleotide for 24 hours, as specified in Tables 5 and 6. The cell samples were processed for protein analysis utilizing the procedure outlined in Example 1.
Results are presented in Tables 5 and 6 as percent inhibition of wild-type and mutant Htt protein, relative to untreated control cells. The data presented is an average of several independent assays performed with each antisense oligonucleotide. As illustrated in Table 4, Htt mutant protein levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The IC50 (nM) values for each oligonucleotide for inhibition of the mutant protein and wild-type protein is also shown and indicates that each oligonucleotide preferentially targets the mutant htt mRNA compared to the wild-type. The oligonucleotides listed in Table 6 demonstrate low in vitro potency and/or little to no preferential reduction of the mutant mRNA compared to the wild-type. Table 5:
Dose-dependent effect of chemically-modified oligonucleotides on wild-type versus mutant Htt protein
Figure imgf000058_0001
Table 6:
Effect of chemically modified oligonucleotides on wild-type versus mutant Htt protein
Figure imgf000058_0002
Alt2 mutant n.d. n.d. n.d. 1 13 21 >100
2'F-RNA wild-type 7 6 7 5 1 1 0 >100 full mutant 3 8 22 12 23 15 >100
2'F-ANA wild-type 18 18 29 33 28 22 >100 Alt mutant 21 24 36 38 32 30 >100
2'F-ANA wild-type 8 3 21 29 25 20 >100 full mutant 5 10 33 41 35 25 >100
2'F-ANA wild-type 15 18 26 28 34 31 >100 LNA(T) mutant 14 24 38 35 41 42 >100 n.d.= no data
Example 5: Effect of oligonucleotides having phosphorothioate backbone, targeting human huntingtin (htt) mRNA, on huntingtin (Htt) protein
Antisense oligonucleotides targeted to the C AG repeat sequence of mutant huntingtin nucleic acid and with uniform phosphorothioate backbone were tested for their effects on Htt protein levels in vitro. GM04281 fibroblast cells were cultured at a density of 60,000 cells per well in 6-well plates and transfected using Lipofectamine™ RNAiMAX reagent (Invitrogen, CA) with 100 nM antisense oligonucleotide for 24 hours. The cell samples were processed for protein analysis utilizing the procedure outlined in Example 1.
The percentage inhibition of the protein samples was calculated relative to the negative control sample and presented in Table 7. The comparative percent inhibitions of the wild-type Htt protein and the mutant Htt protein are also presented. The Tm value for each oligonucleotide, determined by DSC is also shown.
The antisense oligonucleotides utilized in the assay are described in Table 7. The antisense oligonucleotides were from ISIS Pharmaceuticals. The modifications in each oligonucleotide are indicated as follows: subscript Έ'= cET; subscript 'L'=LNA; italicized base=MOE; bolded base-2'F-RNA; and mC=5-methylcytosine. Table 7
Effect of antisense oligonucleotides with phosphorothioate backbone on wild-type and mutant
Htt protein
Figure imgf000060_0001
Example 6: in vitro dose-dependent effect of oligonucleotides having phosphorothioate backbone on human Htt protein
Antisense oligonucleotides from Example 5 (see Table 7 for description of chemical modifications) were tested at various doses in patient fibroblast cells. GM04281 fibroblast cells were plated at a density of 60,000 cells per well in 6-well plates and transfected using
Lipofectamine™ RNAiMAX reagent (Invitrogen, CA) reagent with increasing concentrations of antisense oligonucleotide for 24 hours, as specified in Tables 8 and 9. The cell samples were processed for protein analysis utilizing the procedure outlined in Example 1.
Results are presented in Tables 8 and 9 as percent inhibition of wild-type and mutant Htt protein, relative to untreated control cells. The data presented is an average of several independent assays performed with each antisense oligonucleotide. As illustrated in Table 8, Htt mutant protein levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The IC50 (nM) values for each oligonucleotide for inhibition of the mutant protein and wild-type protein is also shown and indicates that each oligonucleotide preferentially targets the mutant htt mRNA compared to the wild-type. The oligonucleotides listed in Table 9 do not show preferential targeting of the mutant mRNA compared to the wild-type. Table 8:
Dose-dependent effect of oligonucleotides with phosphorothioate backbone on wild-type versus mutant Htt protein
Figure imgf000061_0001
Table 9:
Effect of oligonucleotides with phosphorothioate backbone on wild-type versus mutant Htt protein
Figure imgf000061_0002
Example 7: Effect of chemically modified oligonucleotides targeting CAG repeats on human huntingtin {hit) mRNA levels
Antisense oligonucleotides from Example 1, Example 3, and Example 5 (see Table 1, Table 4, and Table 7, respectively, for description of chemical modifications) were tested in GM04281 cells. Antisense oligonucleotides targeted to the CAG repeat sequence of mutant huntingtin nucleic acid and with various chemical modifications were tested for their effects on htt mRNA levels in vitro. GM04281 cells were cultured at a density of 60,000 cells per well in 6- well plates and transfected using Lipofectamine™ RNAiMAX reagent (Invitrogen, CA) with 50 nM antisense oligonucleotide for 24 hours. The wells were then aspirated and fresh culture medium was added to each well. After a post-transfection period of 3 days, the cells were harvested with trypsin solution (0.05% Trypsin-EDTA, Invitrogen) and lysed.
Total RNA from treated and untreated fibroblast cells was extracted using TRIzol reagent (Invitrogen). Samples were then treated with DNase I (Worthington Biochemical Corp.) at 25°C for 10 min. Reverse transcription reactions were carried out using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol.
Quantitative PCR was performed on a BioRad CFX96 Real Time System using iTaq SYBR Green Supermix with ROX (Bio-rad). Data was normalized relative to GAPDH mRNA levels. Primer sequences specific for htt are as follows: forward primer, 5'- CGAC AGCGAGTC AGTGAATG-3 ' (SEQ ID NO: 9) and reverse primer, 5'- ACCACTCTGGCTTCACAAGG-3'(SEQ ID NO: 10). Primers specific for GAPDH were obtained from Applied Biosystems.
The results are presented in Table 10 and indicate the percent inhibition of htt mRNA compared to untreated cells. The results indicate that mRNA levels were unaffected by treatment with the antisense oligonucleotides.
Table 10:
Effect of antisense oligonucleotides targeting CAG repeats on htt mRNA levels
Figure imgf000062_0001
LNA(T)22 7
cET 16
LNA(T)-PS 0
cET-PS 0
MOE-2T-PS 0
Example 8: Time-dependent effect of an LNA-modified antisense oligonucleotide, targeting mutant htt mRNA, on human huntingtin (Htt) protein levels
The ISIS antisense oligonucleotide with LNA modifications at the thymine bases and which demonstrated significant selective inhibition of mutant huntingtin protein compared to wild-type protein (see Tables 1 and 2) was further studied. The time-dependent effect of this oligonucleotide was tested. GM04281 fibroblast cells were cultured at a density of 60,000 cells per well in 6-well plates and transfected using Lipofectamine™ RNAiMAX reagent (Invitrogen, CA) with 100 nM antisense oligonucleotide for 24 hours. The cell samples were processed for protein analysis at 2 days, 3 days, 4 days, 5 days, and 6 days post-transfection, utilizing the procedure outlined in Example 1.
The results are presented in Table 11 and indicate the preferential time-dependent decrease in mutant huntingtin protein levels. The effect was observed to be optimal at day 3 post- transfection.
Table 11:
Time-dependent effect of LNA-modified ISIS antisense oligonucleotide on mutant Htt protein levels
Figure imgf000063_0001
5 9 35
6 18 32
Example 9: Oligonucleotide selectivity of mutant huntingtin mR A containing 41 or 44 repeat lengths
Studies in Examples 1-8, describing oligonucleotide selectivity for the mutant allele versus the wild-type allele of the htt gene, were performed in the GM04281 fibroblast cell line, which contains 69 CAG repeats in the mutant htt allele. To determine whether the antisense oligonucleotides would selectively target mutant htt mRNA with shorter CAG repeats, two HD patient-derived fibroblast cell lines, GM04717 and GM04719 (Cornell Institute for Medical Research, NJ, USA), were utilized. The GM04717 fibroblast cell line contains 41 mutant repeats and 20 wild-type repeats. The GM04719 fibroblast cell line contains 44 mutant repeats and 15 wild-type repeats.
Cells were plated in 6-well dishes at 60,000 cells/well 2 days before transfection. Stock solutions of modified antisense oligonucleotides were heated at 65 °C for 5 min prior to use to dissolve any aggregation and then transfected into cells using Lipofectamine™ RNAiMAX (Invitrogen, USA), according to the manufacturer's instructions. Media was exchanged 1 day after transfection with fresh supplemented media. Cells were washed with PBS and harvested 4 days after transfection for protein analysis. Protein analysis was undertaken utilizing the procedure outlined in Example 1.
The antisense oligonucleotides tested were LNA (T) (described in Example 1) and cET (described in Example 3). The results are presented in Table 12 as the IC50 for each antisense oligonucleotide. As presented in Table 12, each of the antisense oligonucleotides demonstrates three- to seven-fold selectivity for the mutant allele versus the wild-type allele. This data indicates that allele-specific antisense oligonucleotides can discriminate between the wild-type allele and mutant allele of htt, even when the numbers of CAG repeats are 41 and 44 in number. Table 12:
IC5o and allele-selectivity of antisense oligonucleotides in GM04717 and GM04719 fibroblasts
Figure imgf000065_0001
Example 10: Role of different transfection reagents in the efficacy of antisense
oligonucleotides targeting the CAG repeat sequence of htt mRNA
To test wheter the chemistries of the different antisense oligonucleotides affect the transfection efficiency of the oligonucleotides and, hence distort their efficacy to inhibit mutant htt mRNA, a side-by-side comparison of inhibition by the antisense oligonucleotides transfected with five different transfection reagents was performed. Transfection reagents Lipofectamine™ RNAiMAX (Invitrogen, CA, USA), Oligofectamine™ (Invitrogen, CA, USA), TriFECTin (Integrated DNA Technologies, CA, USA), 7ra>wIT®-01igo (Minis Bio LLC, WI, USA), and PepMute™ (Signagen Laboratories, MD, USA) were utilized in this study.
The antisense oligonucleotides tested were LNA (T) (described in Example 1), MOE (described in Example 4), 2'F-RNA full and 2'F-ANA full (described in Example 4). A negative control LNA oligonucleotide and a positive control siRNA (siHdhl siR A) were also included in the assay. The antisense oligonucleotides were transfected into GM04281 cell and protein analysis of htt was done in a procedure similar to that described in Example 1. The results are presented in Table 13, below and are expressed as percent inhibition compared to the negative control.
As presented in Table 13 the LNA(T) oligonucleotide demonstrated potency and allele- specificity, regardless of the transfection reagent used, whereas the 2'F-RNA full and 2'F-ANA full oligonucleotides demonstrated poor inhibition and were largely non-specific for the mutant versus the wild-type allele. The performance of the all lipid-based transfection reagents (Lipofectamine™ R AiMAX, Oligofectamine™, TriFECTin, and 7>awIT®-01igo) were therefore similar.
In case of the non-lipid peptide-based transfection reagent, PepMute™, it was observed that the MOE oligonucleotide, transfected into cells with this transfection reagent, demonstrated both potency and allele-specificity. Hence, the choice of transfection reagent may affect comparisons between oligonucleotide chemistries and may be the reason for an antisense oligonucleotide underperforming in a particular cellular assay.
Table 13:
Potency (% inhibition of htt mRNA) and allele-selectivity of antisense oligonucleotides with different transfection reagents
Figure imgf000066_0001

Claims

CLAIMS:
1. An oligomeric compound comprising an oligonucleotide consisting of 13 to 30 linked
nucleosides and having a nucleobase sequence 100% complementary to a repeat region of a nucleotide repeat-containing RNA, wherein the oligonucleotide contains: a. a 5'-region consisting of 1-5 linked 5'-region nucleosides, wherein the 5'-region
nucleosides each have the same modification as one another;
b. a central region consisting of 5 to 20 linked nucleosides, wherein each nucleoside of the central region either comprises a 2'-fluoro sugar moiety or is a non-2 '-fluoro central-region nucleoside, provided that at least one nucleoside of the central region comprises a 2 '-fluoro sugar moiety, and wherein the non-2 '-fluoro central-region nucleosides each have the same modification as one another; and
c. a 3'-region consisting of 1-5 linked 3'-region nucleosides, wherein the 3'-region
nucleosides each have the same modification as one another.
2. The oligomeric compound of claim 1, wherein no more than four contiguous nucleosides of the central region are non-2 '-fluoro central-region nucleosides.
3. The oligomeric compound of claim 1 or 2, wherein the 5'-region nucleosides comprise a modified 2'-sugar moiety.
4. The oligomeric compound of claim 3, wherein the 5 '-region nucleosides comprise a 2'- substituent selected from: OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, 0(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, 0(CH2)2-OCH3, 0(CH2)2-SCH3, 0(CH2)2-OCF3, 0(CH2)3- N(R4)(R5), 0(CH2)2-ON(R4)(R5), 0(CH2)2-0(CH2)2-N(R4)(R5), OCH2C(=0)-N(R4)(R5), OCH2C(=0)-N(R6)-(CH2)2-N(R4)(R5) and 0(CH2)2-N(R6)-C(=NR7)[N(R4)(R5)] wherein R4, R5, ¾ and R7 are each, independently, H or Q-C6 alkyl.
5. The oligomeric compound of claim 4, wherein the 5 '-region nucleosides comprise a 2'- 0(CH2)2-OCH3.
6. The oligomeric compound of claim 1 or 2, wherein the 5'-region nucleosides comprise a bicyclic sugar moiety.
7. The oligomeric compound of claim 6, wherein the bicyclic sugar moiety of the 5' -region nucleosides comprise a 4' to 2' bridge.
8. The oligomeric compound of claim 7, wherein the 4' to 2' bridge of the 5 '-region
nucleosides comprise from 2 to 4 linked groups independently selected from -[C(Ra)(Rb)]y-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=0)-, -C(=S)-, -0-, -Si(Ra)2-, -S(=0)x-, and -N(R -;
wherein:
x is 0, 1, or 2;
y is 1, 2, 3, or 4;
each Ra and Rj, is, independently, H, a protecting group, hydroxyl, C!-C6 alkyl, substituted -C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C5-C9 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5- C7 alicyclic radical, halogen, OJi, NJiJ2, SJj, N3, COOJl 5 acyl (C(=0)-H), substituted acyl, CN, sulfonyl (S(=O)2-J , or sulfoxyl (S(=0)-Ji); and
each J] and J2 is, independently, H, C!-C6 alkyl, substituted C\-Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, Cs-C20 aryl, substituted C5-C9 aryl, acyl (C(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, Ci-C6 aminoalkyl, substituted Ci-C6 aminoalkyl or a protecting group.
9. The oligomeric compound of claim 8, wherein the 4' to 2' bridge of the 5'-region
nucleosides is , -[ΰ(¾)^)]η-, -[C(Rc)(Rd)]n-0-, -C(ReRd)-N(Re)-0- or -C(RcRd)-0-N(Re)-, wherein:
each Rc and Rd is independently hydrogen, halogen, substituted or unsubstituted C]-C6 alkyl; and each Re is independently hydrogen or substituted or unsubstituted C C6 alkyl.
10. The oligomenc compound of claim 9, wherein the, 4' to 2' bridge of the 5'-region nucleosides is 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-0-2', 4'-CH(CH3)-0-2', 4'-(αί2)2-0-2·, 4'- CH2-0-N(Re)-2' and 4'-CH2-N(Re)-C-2'- bridge.
11. The oligomeric compound of claim 10, wherein the bridge of the 5 '-region nucleosides is 4'- CH(CH3)-0-2\
12. The oligomeric compound of any of claims 1-11, wherein the non-2'-fluoro central-region nucleosides are 2'-deoxyribonucleosides.
13. The oligomeric compound of any of claims 1-11, wherein the non-2 '-fluoro central-region nucleosides comprise a modified 2'-sugar moiety.
14. The oligomeric compound of claim 13, wherein the non-2 '-fluoro central-region nucleosides comprise a 2'-substituent selected from: OCH3, OCH2F, OCHF2, OCF3, OCH2CH3,
0(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, 0(CH2)2-OCH3, 0(CH2)2-SCH3, 0(CH2)2-OCF3, 0(CH2)3-N(R4)(R5), 0(CH2)2-ON(R4)(R5), 0(CH2)2-0(CH2)2-N(R4)(R5), OCH2C(=0)-N(R4)(R5), OCH2C(=0)-N(R6)-(CH2)2-N(R4)(R5) and 0( Η2)2-Ν^)- C(=NR7)[N(R4)(R5)] wherein R4, R5, R6 and R7 are each, independently, H or Ci-C6 alkyl.
15. The oligomeric compound of claim 14, wherein the 2'-substituent of the non-2 '-fluoro
central-region nucleosides is 0(CH2)2OCH3.
16. The oligomeric compound of claim 1-11, wherein the non-2'-fluoro central-region
nucleosides comprise a bicyclic sugar moiety.
17. The oligomeric compound of claim 16, wherein the bicyclic sugar moiety of the non-2'- fluoro central-region nucleosides comprises a 4' to 2' bridge.
18. The oligomeric compound of claim 17, wherein the 4' to 2' bridge of the non-2'-fluoro
central-region nucleosides comprises from 2 to 4 linked groups independently selected from - [C(Ra)(Rb)]y-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=0)-, -C(=S)-, -0-, -Si(Ra)2-, - S^O^ and -NCR -;
wherein:
x is 0, 1, or 2;
y is 1, 2, 3, or 4;
each Ra and Rb is, independently, H, a protecting group, hydroxyl, Q-Q alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C5-C9 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5- C7 alicyclic radical, halogen, OJi, NJ1J2, SJls N3, COOJi, acyl (C(=0)-H), substituted acyl, CN, sulfonyl (S(=O)2-J , or sulfoxyl (S(-O)-J ; and
each Ji and J2 is, independently, H, Q-Q alkyl, substituted C\-Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C5-C20 aryl, substituted C5-C aryl, acyl (C(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, d-C6 aminoalkyl, substituted Q-Q aminoalkyl or a protecting group.
19. The oligomeric compound of claim 18, wherein the 4' to 2' bridge of the non-2 '-fluoro
central-region nucleosides is -[C(Rc)(Rd)]n-, -[C(Rc)(Rd)]n-0-, -C(RcRd)-N(Re)-0- or - C(RcRd)-0-N(Re)-, wherein:
each Rc and R<j is independently hydrogen, halogen, substituted or unsubstituted C!-C6 alkyl; and
each Re is independently hydrogen or substituted or unsubstituted C[-C6 alkyl.
20. The oligomeric compound of claim 19, wherein the, 4' to 2' bridge of the non-2'-fluoro central-region nucleosides is 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-0-2*, 4'-CH(CH3)-0-2', 4'- (CH2)2-0-2', 4'-CH2-0-N(Re)-2' and 4'-CH2-N(Re)-0-2,- bridge.
21. The oligomeric compound of claim 20, wherein the bridge of the non-2'-fluoro central- region nucleosides is 4'-CH(CH3)-0-2'.
22. The oligomeric compound of any of claims 1-21, wherein the 3 '-region nucleosides comprise a modified 2'-sugar moiety.
23. The oligomeric compound of claim 22, wherein the 2'-modified sugar moiety of the 3'- region nucleosides comprises a 2'-substituent selected from: OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, 0(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, 0(CH2)2-OCH3, 0(CH2)2- SCH3, 0(CH2)2-OCF3, 0(CH2)3-N(R4)(R5), 0(CH2)2-ON(R4)(R5), 0(CH2)2-0(CH2)2- N(R4)(R5), OCH2C(=0)-N(R4)(R5), OCH2C(=0)-N(R6)-(CH2)2-N(R4)(R5) and 0(CH2)2- N(R6)-C(=NR7)[N(R4)(R5)] wherein R4, R5, e and R7 are each, independently, H or d-C6 alkyl.
24. The oligomeric compound of claim 23, wherein the 2'-substituent of the 3'-region
nucleosides is 0(CH2)20CH3.
25. The oligomeric compound of claim 1-21, wherein the 3'-region nucleosides comprise a
bicyclic sugar moiety.
26. The oligomeric compound of claim 25, wherein the bicyclic sugar moiety of the 3'-region nucleosides comprises a 4' to 2' bridge.
27. The oligomeric compound of claim 26, wherein the 4' to 2' bridge of the 3 '-region
nucleosides comprises from 2 to 4 linked groups independently selected from -[C(Ra)(Rb)]y-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=0)-, -C(=S)-, -0-, -Si(Ra)2-, -S(=0)x-, and -N(R -;
wherein:
x is 0, 1, or 2;
y is 1, 2, 3, or 4;
each Ra and R¾ is, independently, H, a protecting group, hydroxyl, C C alkyl, substituted Q-Q alkyl, C2-C alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C alkynyl, C5-C9 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5- C7 alicyclic radical, halogen, OJi, NJjJ2, SJ1? N3, COOJj, acyl (C(=0)-H), substituted acyl, CN, sulfonyl (S(=0)2-Ji), or sulfoxyl (S(=O)-J ; and each Ji and J2 is, independently, H, Cj-C alkyl, substituted CpC6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C5-C20 aryl, substituted C5-C9 aryl, acyl (C(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, Q-C6 aminoalkyl, substituted Q-Q aminoalkyl or a protecting group.
28. The oligomeric compound of claim 27, wherein the 4' to 2' bridge of the 3 '-region
nucleosides is, -[0(¾)(¾)]„-, -[C(Rc)(Rd)]n-0-, -CtRJLfrNQ y-O- or -C(RcRd)-0-N(Re)-, wherein:
each Rc and R<j is independently hydrogen, halogen, substituted or unsubstituted CrC6 alkyl; and
each Re is independently hydrogen or substituted or unsubstituted C C6 alkyl.
29. The oligomeric compound of claim 28, wherein the, 4' to 2' bridge of the 3 '-region
nucleosides is 4*-(CH2)2-2*, 4,-(CH2)3-2', 4'-CH2-0-2', ^-CHCC^-O^', 4'-(CH2)2-0-2', 4'- CH2-0-N(Re)-2' and 4'-CH2-N(Re)-0-2'- bridge.
30. The oligomeric compound of claim 29, wherein the bridge of the 3 '-region nucleosides is 4'- CH(CH3)-0-2'.
31. The oligomeric compound of any of claims 1-30, wherein the 5'-region nucleosides and the 3' -region nucleosides comprise the same modification as one another.
32. The oligomeric compound of any of claims 1-31, wherein the 5 '-region nucleosides and the non-2 '-fiuoro central-region nucleosides comprise the same modification as one another.
33. The oligomeric compound of 1-32, wherein the 5 '-region nucleosides and the 3 '-region nucleosides and the non-2' -fiuoro central-region nucleosides comprise the same modification as one another.
34. The oligomeric compound of claim 33, wherein the 5'-region nucleosides and the 3'-region nucleosides and the non-2 '-fluoro central-region nucleosides each comprise a 2'- (CH2)2OCH3 modification.
35. The oligomeric compound of any of claims 1-34, wherein the central region comprises 1 to 10 blocks of non-2 '-fluoro central-region nucleosides, wherein each block independently consists of 1 to 4 non-2'-fluoro central-region nucleosides.
36. The oligomeric compound of claim 35, wherein the central region comprises 1 to 5 blocks of non-2 '-fluoro central-region nucleosides.
37. The oligomeric compound of claim 36, wherein the central region comprises 1 to 2 blocks of non-2 '-fluoro central-region nucleosides.
38. The oligomeric compound of claim 37, wherein the central region comprises 1 block of non- 2 '-fluoro central-region nucleosides.
39. The oligomeric compound of any of claims 35-38, wherein the blocks of non-2'-fluoro
central-region nucleosides independently consist of 1-3 non-2 '-fluoro central-region nucleosides.
40. The oligomeric compound of any of claims 35-38, wherein the blocks of non-2'-fluoro
central-region nucleosides independently consist of 1 or 2 non-2 '-fluoro central-region nucleosides.
41. The oligomeric compound of any of claims 35-38, wherein the blocks of non-2'-fluoro
central-region nucleosides independently consist of 3 or 4 non-2'-fiuoro central-region nucleosides.
42. The oligomeric compound of any of claims 1-34, wherein the central region consists of linked 2'-fluoro modified nucleosides.
43. The oligomenc compound of any of claims 1-42, wherein the oligonucleotide has the Formula:
( ui)„i-( U2)^-(NU3)n3-( ll4)II4-(NU5)n5 wherein:
each Nui is a 5 '-region nucleoside;
each Nu2 and each Nu4 is a 2'-fluoro nucleoside;
each Nu3 is a non-2'-fluoro central-region nucleoside;
each Nu5 is a 3 '-region nucleoside;
nl is from 1 to 5;
n5 is from 0 to 5;
n2 is from 1 to 24 and n4 is from 1 to 24, provided that the sum of n2 and n4 is from 10 to 25; and
n3 is from 0 to 4.
44. The oligomeric compound of claim 43, wherein n3 is from 1 to 4.
45. The oligomeric compound of claim 43, wherein n3 is 0.
46. The oligomeric compound of any of claims 1-45, wherein the oligonucleotide comprises one or more modified intemucleoside linkage.
47. The oligomeric compound of claim 46, wherein each intemucleoside linkage is modified.
48. The oligomeric compound of either of claims 46 or 47 wherein each intemucleoside linkage of the oligonucleotide is either a phosphodiester or a phosphorothioate linkage.
49. The oligomeric compound of any of claims 1-48, wherein the repeat region of the nucleotide repeat-containing RNA consists of repeating units of CCUG or AUUCU.
50. The oligomeric compound of any of claims 1-48, wherein the repeat region of the nucleotide repeat-containing RNA consists of a repeating triplet.
51. The oligomeric compound of claim 50, wherein the repeating triplet is selected from: CAG, CUG, CGG, GCC, and GAA.
52. The oligomeric compound of claim 51, wherein the repeating triplet is CAG.
53. The oligomeric compound of claim 51, wherein the repeating triplet is CUG.
54. The oligomeric compound of any of claims 1-53 wherein the nucleotide repeat-containing RNA is associated with a disease.
55. The oligomeric compound of claim 54, wherein the disease is selected from among: ataxin 8, atrophin 1, fragile X syndrome, Friedrich's ataxia, Huntington's disease, Huntington's disease-like 2, myotonic dystrophy, spinal and bulbar muscular atrophy, and spinocerebellar ataxia.
56. The oligomeric compound of claim 55, wherein the disease is Huntington's disease.
57. The oligomeric compound of claim 55, wherein the disease is myotonic dystrophy.
58. The oligomeric compound of claim 57, wherein the myotonic dystrophy is myotonic
dystrophy type 1.
59. The oligomeric compound of claim 57, wherein the myotonic dystrophy is myotonic
dystrophy type 2.
60. The oligomeric compound of claim 55, wherein the disease is spinocerebellar ataxia.
61. The oligomeric compound of claim 60, wherein the spinocerebellar ataxia is spinocerebellar ataxia 10.
62. The oligomeric compound of any of claims 1-61, wherein the compound is a mutant selective compound.
63. The oligomeric compound of claim 62, wherein the compound is capable of reducing the activity or amount of a nucleotide repeat-containing RNA at least two fold more than it reduces the activity or amount of a corresponding wild type RNA.
64. The oligomeric compo und of any of 1-63, wherein the compound comprises at least one modified nucleobase.
65. The oligomeric compound of claim 64, wherein the modified nucleobase is a 5- methylcytosine.
66. A method of selectively reducing the activity or amount of a nucleotide repeat-containing RNA in a cell, comprising contacting a cell having a mutant repeat-containing RNA with an oligomeric compound of any of claims 1 to 65; and thereby selectively reducing the activity or amount of the mutant repeat-containing RNA in the cell.
67. The method of claim 66, wherein the amount or activity of the nucleotide repeat-containing RNA is reduced at least two-fold more than that of a corresponding wild-type RNA.
68. The method of claim 66 or 67, wherein the cell is in vitro.
69. The method of claim 66 or 67, wherein the cell is in an animal.
70. A pharmaceutical composition comprising at least one oligomeric compound of any of
claims 1-65 and a pharmaceutical carrier or diluents.
71. A method of treating patient having a disease associated with a nucleotide repeat-containing R A comprising administering to the patient the pharmaceutical composition of claim 70.
72. The method of claim 71, wherein the disease is selected from among: ataxin 8, atrophin 1, fragile X syndrome, Friedrich's ataxia, Huntington's disease, Huntington's disease-like 2, myotonic dystrophy, spinal and bulbar muscular atrophy, and spinocerebellar ataxia.
73. The method of claim 72, wherein the disease is Huntington's disease.
74. The method of claim 73, wherein the disease is myotonic dystrophy.
75. The method of claim 74, wherein the myotonic dystrophy is myotonic dystrophy type 1.
76. The method of claim 75, wherein the myotonic dystrophy is myotonic dystrophy type 2.
77. The method of claim 72, wherein the disease is spinocerebellar ataxia.
78. The method of claim 77, wherein the spinocerebellar ataxia is spinocerebellar ataxia 10.
79. The method of any of claims 72 to 78, wherein the pharmaceutical composition is
administered by injection.
80. The method of claim 79, wherein the pharmaceutical composition is injected into the central nervous system.
81. The method of claim 80, wherein the pharmaceutical composition is injected into the brain.
82. The method of any of claims 79-81, wherein the injection is a bolus injection.
83. The method of any of claims 79-81 , wherein the injection is an infusion.
84. Use of an oligomeric compound comprising an oligonucleotide consisting of 13 to 30 linked nucleosides and having a nucleobase sequence 100% complementary to a repeat region of a nucleotide repeat-containing RNA, wherein the oligonucleotide contains:
a. a 5'-region consisting of 1-5 linked 5'-region nucleosides, wherein the 5'-region nucleosides each have the same modification as one another;
b. a central region consisting of 5 to 20 linked nucleosides, wherein each nucleoside of the central region either comprises a 2'-fluoro sugar moiety or is a non-2 '-fluoro central-region nucleoside, provided that at least one nucleoside of the central region comprises a 2'-fluoro sugar moiety, and wherein the non-2'-fluoro central-region nucleosides each have the same modification as one another; and
c. a 3 '-region consisting of 1-5 linked 3 '-region nucleosides, wherein the 3 '-region nucleosides each have the same modification as one another;
for the treatment of a disease associated with a CAG nucleotide repeat-containing RNA.
85. The use of claim 84, wherein the disease is any of Atrophin 1, Huntington's Disease,
Huntington disease-like 2 (HDL2), spinal and bulbar muscular atrophy, Kennedy disease, spinocerebellar ataxia 1, spinocerebellar ataxia 12, spinocerebellar ataxia 17, Huntington disease-like 4 (HDL4), spinocerebellar ataxia 2, spinocerebellar ataxia 3, Machado- Joseph disease, spinocerebellar ataxia 6, and spinocerebellar ataxia 7.
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