WO2010048585A2 - Oligomeric compounds and methods - Google Patents

Oligomeric compounds and methods Download PDF

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Publication number
WO2010048585A2
WO2010048585A2 PCT/US2009/061959 US2009061959W WO2010048585A2 WO 2010048585 A2 WO2010048585 A2 WO 2010048585A2 US 2009061959 W US2009061959 W US 2009061959W WO 2010048585 A2 WO2010048585 A2 WO 2010048585A2
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Prior art keywords
oligomeric compound
alkyl
substituted
nucleoside
och
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PCT/US2009/061959
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French (fr)
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WO2010048585A3 (en
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Eric E. Swayze
Balkrishen Bhat
Walter F. Lima
Thazha P. Prakash
Garth A. Kinberger
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Isis Pharmaceuticals, Inc.
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Priority to US13/125,751 priority Critical patent/US8987435B2/en
Priority to EP09744287A priority patent/EP2358398A2/en
Publication of WO2010048585A2 publication Critical patent/WO2010048585A2/en
Publication of WO2010048585A3 publication Critical patent/WO2010048585A3/en
Priority to US14/618,853 priority patent/US9738895B2/en

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    • 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
    • C12N15/1137—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 against enzymes
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    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/11—Antisense
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12N2310/141—MicroRNAs, miRNAs
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/31—Chemical structure of the backbone
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
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    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • modified nucleosides and oligomeric compounds prepared therefrom.
  • modified nucleosides are provided having at least one 5'-substituent and a 2'-substituent, oligomeric compounds comprising at least one of these modified nucleosides and compositions comprising at least one of these oligomeric compounds.
  • the oligomeric compounds provided herein are expected to hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.
  • the oligomeric compounds are also expected to be useful as primers and/or probes in diagnostic applications.
  • Antisense compounds have been used to modulate target nucleic acids. Antisense compounds comprising a variety of modifications and motifs have been reported. In certain instances, such compounds are useful as research tools and as therapeutic agents. Certain double-stranded RNA-like compounds
  • RNA molecules are known to inhibit protein expression in cells. Such double-stranded RNA compounds function, at least in part, through the RNA-inducing silencing complex (RISC). Certain single-stranded RNA-like compounds (ssRNAs) have also been reported to function at least in part through RISC.
  • RISC RNA-inducing silencing complex
  • antisense technology in the treatment of a disease or condition that stems from a disease-causing gene is that it is a direct genetic approach that has the ability to modulate (increase or decrease) the expression of specific disease-causing genes.
  • Another advantage is that validation of a therapeutic target using antisense compounds results in direct and immediate discovery of the drug candidate; the antisense compound is the potential therapeutic agent.
  • RNAi RNA interference
  • MicroRNAs are small non-coding RNAs that regulate the expression of protein-coding RNAs.
  • the binding of an antisense compound to a microRNA prevents that microRNA from binding to its messenger RNA targets, and thus interferes with the function of the microRNA. Regardless of the specific mechanism, this sequence-specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of malignancies and other diseases.
  • Antisense technology is an effective means for reducing the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications.
  • Chemically modified nucleosides are routinely used for incorporation into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target RNA.
  • the antisense compound, Vitravene® was the first antisense drug to achieve marketing clearance from the U.S. Food and Drug Administration (FDA), and is currently a treatment of cytomegalovirus (CMV)-induced retinitis in ADDS patients.
  • FDA U.S. Food and Drug Administration
  • New chemical modifications have improved the potency and efficacy of antisense compounds, uncovering the potential for oral delivery as well as enhancing subcutaneous administration, decreasing potential for side effects, and leading to improvements in patient convenience.
  • Chemical modifications increasing potency of antisense compounds allow administration of lower doses, which reduces the potential for toxicity, as well as decreasing overall cost of therapy. Modifications increasing the resistance to degradation result in slower clearance from the body, allowing for less frequent dosing.
  • Different types of chemical modifications can be combined in one compound to further optimize the compound's efficacy.
  • the synthesis of 5'-substituted DNA and RNA derivatives and their incorporation into oligomeric compounds has been reported in the literature (Saha et al, J. Org.
  • Amide linked nucleoside dimers have been prepared for incorporation into oligonucleotides wherein the 3' linked nucleoside in the dimer (5 1 to 3') comprises a 2'-OCH 3 and a 5'-(S)-CH 3 (Mesmaeker et al, Synlett, 1997, 1287-1290).
  • oligomeric compounds such as antisense compounds useful for modulating gene expression pathways, including those relying on mechanisms of action such as RNaseH, RNAi and dsRNA enzymes, as well as other antisense mechanisms based on target degradation or target occupancy.
  • modified nucleosides having at least one 2' substituent group and either a 5' substituent group, a 5' phosphorus moiety or both a 5' substituent group and a 5' phosphorus moiety, oligomeric compounds that include such modified nucleosides and methods of using the oligomeric compounds. Also provided herein are intermediates and methods for preparing these modified nucleosides and oligomeric compounds. In certain embodiments, modified nucleosides are provided that are 5'-mono (R, S or mixed) or bis substituted and 2'-O-substituted, that can be incorporated into oligomeric compounds.
  • the modified nucleosides provided herein are expected to be useful for enhancing one or more properties of the oligomeric compounds they are incorporated into such as for example nuclease resistance.
  • the oligomeric compounds and compositions provided herein that incorporate one or more of these modified nucleosides are expected to hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.
  • the oligomeric compounds are also expected to be useful as primers and probes in diagnostic applications.
  • the invention provides oligomeric compounds comprising an oligonucleotide comprising a nucleoside having Formula VII:
  • Bx is a heterocyclic base moiety
  • T 3 is a phosphorus moiety
  • T 4 is an internucleoside linking group attaching the nucleoside of Formula I to the remainder of the oligonucleotide; and each of qi and q 2 is, independently selected from H, Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -
  • Xi is S, NRi 6 , or CRioR ⁇ wherein each Ri 0 and R n is, independently, H, F, Ci-C 6 haloalkyl , or Ci-C 6 alkyl; and
  • Ri is selected from a halogen, X 2 -V, and 0-X 4 ; or each of qi and q 2 is, independently, selected from H, Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 - C 6 alkynyl, substituted Ci-C 6 alkyl, substituted Ci-C 6 alkenyl and substituted C 2 -C 6 alkynyl;
  • Xi is O, S, NRi 6 Rn, or CRi 0 Ri 1 wherein each Ri 0 and R n is, independently, H, F, Q- C 6 haloalkyl , or Ci-C 6 alkyl; and R, is X 2 -V; or each of qi and q 2 is, independently, selected from Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-C 6 alkyl, substituted CpC 6 alkenyl and substituted C 2 -C 6 alkynyl;
  • Xi is O, S, NRi 6 Rn, or CRioRn wherein each Ri 0 and Rn is, independently, H, F, Q- C 6 haloalkyl , or Q-C 6 alkyl;
  • Ri is selected from halogen, X 2 -V, and 0-X 4 ; wherein: X 2 is O, S or CR 7 Rg wherein each R 7 and R 8 is, independently, H or Ci-C 6 alkyl; V is selected from cholesterol, (CH 2 ) 2 [O(CH 2 ) 2 ] t OCH 3 , where t is from 1-3, (CH 2 ) 2 F, CH 2 COOH, CH 2 CONH 2 , CH 2 CONR 5 R 61 CH 2 COOCH 2 CH 3 , CH 2 CONH(CH 2 ) ⁇ S-R 4 where i is from 1 to 10, CH 2 CONH(CH 2 ) J NR 5 R 6 where j is from 1 to 6, CH 2 CONH[(CH 2 ) k i-N(H)] k r(CH2)kiNH2 where each Ic 1 is independently from 2 to 4 and k 2 is from 2 to 10, and aryl;
  • R 4 is selected from H, Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-C 6 alkyl, substituted Ci-C 6 alkenyl, substituted C 2 -C 6 alkynyl, C 6 -C M aryl and athio protecting group;
  • R 5 and R 6 are each, independently, selected from H, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, and substituted C 2 -C 6 alkynyl;
  • Ri 6 is selected from H, Ci-C 6 alkyl, or substituted C]-C 6 alkyl;
  • the invention provides oligomeric compounds comprising an oligonucleotide comprising a nucleoside having Formula VII:
  • Bx is a heterocyclic base moiety
  • Xi is O, S, NRi 6 Ri 7 , or CRioR ⁇ wherein each Ri 0 and Rn is, independently, H, F, or Ci-C 6 alkyl, and each Ri 6 and R n is independently, H, CpC 6 alkyl, substituted Ci-C 6 alkyl, T 3 is a phosphorus moiety; T 4 is an internucleoside linking group attaching the nucleoside of Formula I to the remainder of the oligonucleotide;
  • Ri is selected from halogen, trifluoroalkoxy, azido, aminooxy, O-alkyl, S-alkyl, N(J 4 )-alkyl, O- alkenyl, S-alkenyl, N(J 4 )-alkenyl, O-alkynyl, S-alkynyl or N(J 4 )-alkynyl, O-alkoxy, and X 2 -V, wherein: X 2 is O, S or CR 7 R 8 wherein each R 7 and R 8 is, independently, H or C 1 -C 6 alkyl;
  • V is selected from (CH 2 ) 2 F, CH 2 COOH, CH 2 CONH 2 , CH 2 COOCH 2 CH 3 , CH 2 CONH(CH 2 )J-S-R 4 where i is from 1 to 10, CH 2 CONH(CH 2 )JNR 5 R 6 where j is from 1 to 6, and CH 2 CONH ⁇ (CH 2 ) k i-N(H) ⁇ k2 - (CH 2 XiNH 2 where each ki is independently from 2 to 4 and k 2 is from 2 to 10;
  • R 4 is selected from H, CpC 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-C 6 alkyl, substituted Cj-C 6 alkenyl, substituted C 2 -C 6 alkynyl, C 6 -Ci 4 aryl and a thio protecting group;
  • R 5 and R 6 are each, independently, selected from H, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, and substituted C 2 -C 6 alkynyl; and each of q ! and q 2 is, independently, selected from H, Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-C 6 alkyl, substituted Ci-C 6 alkenyl and substituted C 2 -C 6 alkynyl; provided that if each of qi and q 2 is H, then: either:
  • Ri is selected from, trifluoroalkoxy, azido, aminooxy, S-alkyl, N(J 4 )-alkyl, O-alkenyl, S- alkenyl, N(J 4 )-alkenyl, O-alkynyl, S-alkynyl and N(J 4 )-alkynyl, and X 2 -V; and Xi selected from O, S, N, and CRi 0 Ri 1; or
  • Ri is selected from halogen, O-alkyl, O-haloalkyl, O-alkoxy. In certain embodiments, Ri is F. In certain embodiments, R 1 is 0-C 2 -C 4 alkyl or haloalkyl. In certain embodiments,Ri is OCH 3 . In certain embodiments, Ri is O(CH 2 ) 2 OCH 3 In certain embodiments, Ri is FCH 2 CH 3 . In certain embodiments,Ri is (CH 2 ) 2 [O(CH 2 ) 2 ] t OCH 3 , where t is from 1-3.
  • Ri is selected from, trifluoroalkoxy, azido, aminooxy, S-alkyl, N(J 4 )-alkyl, O-alkenyl, S-alkenyl, N(J 4 )-alkenyl, O-alkynyl, S- alkynyl, N(J 4 )-alkynyl, and X 2 -V.
  • Ri is X 2 -V.
  • V is (CH 2 ) 2 F.
  • V is CH 2 CONH(CH 2 ) J -S-R 4 .
  • V is CH 2 CONH-(CH 2 ) 3 -N(H)-(CH 2 ) 4 -N(H)-(CH 2 ) 3 NH 2 .
  • V is CH 2 CONH(CH 2 )JNR 5 R 6 .
  • at least one of R 5 and R 6 is other than H.
  • at least one of R 5 and R 6 is methyl.
  • R 5 is methyl and R 6 is methyl.
  • X 2 is O. In certain embodiments, X 2 is S.
  • X 2 is CR 7 R 8 . In certain embodiments, R 7 and R 8 are both H. In certain embodiments, at least one of qi and q 2 is Ci-C ⁇ alkyl or substituted Ci-C 6 alkyl. In certain embodiments, at least one of qi and q 2 is CpC 6 alkyl. In certain embodiments, at least one of qi and q 2 is methyl. In certain embodiments, at least one of qi and q 2 is H. In certain embodiments, one of qi and q 2 is methyl and the other of qi and q 2 is H. In certain embodiments, qi and q 2 are each CpC 6 alkyl or substituted Ci-C 6 alkyl.
  • Xi is O. In certain embodiments,Xi is S. In certain embodiments, Xi is CRioRn. In certain embodiments, Ri 0 and Rn are both H. In certain embodiments, the phosphorus moiety is P(Y a )(Y b )(Y C5 ) where Y a is O or S and each Yb and Y 0 is, independently, selected from OH, SH, alkyl, alkoxy, substituted C r C 6 alkyl and substituted CpC 6 alkoxy. In certain embodiments, Y a is O and Y b and Y c are each OH.
  • the invention provides such oligomeric compounds comprising a nucleoside of Formula VIII:
  • such oligomeric compounds have the configuration:
  • qi is methyl and q 2 is H. In certain embodiments, qi is H and q 2 is methyl.
  • the invention provides oligomeric compounds comprising a di-nucleoside of Formula IX: wherein: each Bx is independently a heterocyclic base moiety; T 4 is an internucleoside linking group attaching the nucleoside of Formula IV to the remainder of the oligonucleotide; each of qi and q 2 is, independently selected from H, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 - C 6 alkynyl, substituted C 1 -C 6 alkyl, substituted C]-C 6 alkenyl and substituted C 2 -C 6 alkynyl;
  • Xi is S, NRi 6 , or CRi 0 Ri i wherein each Ri 0 and R n is, independently, H, F, Q-C 6 haloalkyl , or Ci-C 6 alkyl; and
  • Ri is selected from a halogen, X 2 -V, and 0-X 4 ; or each of qi and q 2 is, independently, selected from H, Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 - C 6 alkynyl, substituted C]-C 6 alkyl, substituted Q-C 6 alkenyl and substituted C 2 -C 6 alkynyl;
  • Xi is O, S, NRi 6 Ri 7 , or CRi oRn wherein each R ⁇ and Rn is, independently, H, F, Q-
  • R] is X 2 -V; or each of qi and q 2 is, independently, selected from Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-C 6 alkyl, substituted Ci-C 6 alkenyl and substituted C 2 -C 6 alkynyl;
  • Xi is O, S, NR J6 R 17 , or CR] 0 Rii wherein each Rj 0 and Rn is, independently, H, F, Q- C 6 haloalkyl , or Q-C 6 alkyl; and
  • Ri is selected from halogen, X 2 -V, and 0-X 4 ; wherein: X 2 is O, S or CR 7 R 8 wherein each R 7 and Rg is, independently, H or Q-C 6 alkyl; V is selected from cholesterol, (CH 2 ) 2 [O(CH 2 ) 2 ] t OCH 3 , where t is from 1-3, (CH 2 ) 2 F, CH 2 COOH, CH 2 CONH 2 , CH 2 CONR 5 R 61 CH 2 COOCH 2 CH 3 , CH 2 CONH(CH 2 ) J -S-R 4 where i is from 1 to 10, CH 2 CONH(CH 2 )JNR 5 R 6 where j is from 1 to 6, and CH 2 CONHt(CH 2 X 1 -N(H)] I a-(CH 2 X 1 NH 2 where each k, is independently from 2 to 4 and k 2 is from 2 to 10; R 4 is selected from H, Ci-C 6 alkyl, C 2 -C
  • Ci-C 6 alkenyl substituted C 2 -C 6 alkynyl, C 6 -Cj 4 aryl and a thio protecting group
  • R 5 and R 6 are each, independently, selected from H, Ci-C 6 alkyl, substituted C r C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, and substituted C 2 -C 6 alkynyl;
  • Ri 6 is selected from H, Ci-C 6 alkyl, or substituted Ci-C 6 alkyl;
  • X c is O, S, or N(E 1 );
  • Rd is H, Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-C 6 alkyl, substituted Q-C 6 alkenyl and substituted C 2 -C 6 alkynyl or NE 2 E 3 ; each Ei, E 2 , and E 3 is independently H, C]-C 6 alkyl, or substituted Ci-C 6 alkyl; n is 1 to 6; m is O or 1; and k is O or 1; and wherein X 3 is OH or SH; Y a is O or S; each Y b and Y c is, independently, selected from OH, SH, alkyl, alkoxy, substituted C]-C 6 alkyl and substituted Ci-C 6 alkoxy;
  • Rj is F.
  • Ri is OCH 3 .
  • Ri is 0-C 2 -C 4 alkyl or haloalkyl.
  • Rj is O(CH 2 ) 2 OCH 3 .
  • Ri is FCH 2 CH 3 .
  • Ri is (CH 2 ) 2 [O(CH 2 ) 2 ] t OCH 3 , where t is from 1-3.
  • Ri is selected from, trifluoroalkoxy, azido, aminooxy, S-alkyl, N(J 4 )-alkyl, O-alkenyl, S- alkenyl, N(J 4 )-alkenyl, O-alkynyl, S-alkynyl, N(J 4 )-alkynyl, and X 2 -V.
  • Rj is X 2 -V.
  • V is (CH 2 ) 2 F.
  • V is CH 2 CONH(CH 2 ) J -S-R 4 .
  • V is CH 2 CONHf(CH 2 Xi-N(H)] I a-(CH 2 XiNH 2 . In certain embodiments, V is CH 2 CONH- (CH 2 ) 3 -N(H)-(CH 2 ) 4 -N(H)-(CH 2 ) 3 NH 2 . In certain embodiments, V is CH 2 CONH(CH 2 )JNR 5 R 6 . In certain such embodiments, j is 2. In certain embodiments, at least one of R 5 and R 6 is other than H. In certain embodiments, at least one of R 5 and R 6 is methyl. In certain embodiments, R 5 is methyl and Re is methyl. In certain embodiments, X 2 is O.
  • X 2 is S. In certain embodiments, X 2 is CR7R8. In certain embodiments, R 7 and R 8 are both H. In certain embodiments, at least one of qi and q 2 is Ci-Ce alkyl or substituted C r C 6 alkyl. In certain embodiments, at least one of qi and q 2 is Q-C 6 alkyl. In certain embodiments, at least one of qi and q 2 is methyl. In certain embodiments, at least one of qi and q 2 is H. In certain embodiments, one of qi and q 2 is methyl and the other of qi and q 2 is H.
  • qi and q 2 are each Ci-C 6 alkyl or substituted Ci-C 6 alkyl.
  • Xi is O.
  • Xi is S.
  • Xi is CRi 0 Ri i-
  • Ri 0 and Rn are both H.
  • R 9 is selected from F, OCH 3 and O(CH 2 ) 2 OCH 3 .
  • Rg is OCH 3 .
  • R 9 is F.
  • R 9 is O(CH 2 ) 2 ⁇ CH 3 .
  • such di-nucleoside having Formula IX has Formula X:
  • qi is methyl and q 2 is H. In certain embodiments, qi is H and q 2 is methyl.
  • the invention provides oligomeric compounds comprising an oligonucleotide comprising a nucleoside having Formula II:
  • Bx is a heterocyclic base moiety;
  • A is O, S or N(Ri);
  • Ri is H, Ci-C 6 alkyl or substituted Ci-C 6 alkyl
  • T 3 and T 4 is an internucleoside linking group linking the monomer to the remainder of the oligomeric compound and the other of T 3 and T 4 is H, a protecting group, a phosphorus moiety, a 5' or 3'- terminal group or an internucleoside linking group linking the monomer to the oligomeric compound
  • one of Qi and Q 2 is H, Ci-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 and the other of Qi and Q 2 is Ci-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
  • X is O, S orN(Ei);
  • Z is H, halogen, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 2 -C 6 alkynyl or N(E 2 )(E 3 );
  • each Bx is, independently, uracil, 5-thiazolo-uracil, 2-thio-uracil, 5- propynyl-uracil, thymine, 2'-thio-thymine, cytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl- cytosine, adenine, guanine, 2,6-diaminopurine, lH-pyrimido[5,4-b][l,4benzoxazin-2(3H)-one), IH- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one, 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)- one, 2H-pyrimido[4,5-b]indol-2-one or H-pyrido[3',2':4,5]
  • each Qi is H. In certain embodiments, each Q 2 is H. In certain embodiments, each Qi and each Q 2 are other than H. In certain embodiments, at least one of Qi and Q 2 is substituted Q-C 6 alkyl. In certain embodiments, such substituted Ci-C 6 alkyl comprises at least one substituent group independently selected from halogen, C 2 -C 6 alkenyl, OJi, NJiJ 2 and CN, wherein each Ji and J 2 is, independently, H or Ci-C 6 alkyl. In certain embodiments, substituted Ci-C 6 alkyl comprises at least one substituent group independently selected from fluoro and OCH 3 .
  • At least one of Qi and Q 2 is Ci-C 6 alkyl.
  • Qi is methyl.
  • Q 2 is methyl.
  • Gj is F.
  • T 3 is a phosphorus moiety.
  • said phosphorus moiety has the formula:
  • R 3 and R 0 are each, independently, OH, SH, C r C 6 alkyl, substituted CpC 6 alkyl, Ci-C 6 alkoxy, substituted Ci-C 6 alkoxy, amino or substituted amino; and R b is O or S.
  • R a and R 0 are each OH. In certain embodiments, R a and R 0 are each OCH 3 . In certain embodiments, R a and R 0 are each OCH 2 CH 3 . In certain embodiments, R b is O. In certain embodiments, R b is S. In certain embodiments, each monomer of Formula II has the configuration:
  • such monomer of Formula II is at the 5'end of an oligomeric compound.
  • the invention provides oligomeric compounds comprising an oligonucleotide comprising a nucleoside having Formula IV: wherein independently for each monomer of Formula IV: Bx is a heterocyclic base moiety; one ofT 7 and T 8 is an internucleoside linking group linking the monomer to the remainder of the oligomeric compound and the other of T 7 and T 8 is H, a hydroxyl protecting group, a phosphorus moiety, a 5' or 3 '-terminal group or an internucleoside linking group linking the monomer to the oligomeric compound; Qi, Q 2 , Q 3 and Q 4 are each, independently, H, halogen, C 1 -Ce alkyl, substituted C r 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; G, is O-[C(R 2 -C 6 al
  • Z is H, halogen, C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 2 -C 6 alkynyl or N(E 2 )(E 3 );
  • Bx is, independently, uracil, 5-thiazolo-uracil, 2-thio-uracil, 5- propynyl-uracil, thymine, 2'-thio-thymine, cytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl- cytosine, adenine, guanine, 2,6-diaminopurine, lH-pyrimido[5,4-b][l,4benzoxazin-2(3H)-one), IH- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one, 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)- one, 2H-pyrimido[4,5-b]indol-2-one or H-pyrido[3',2':4,5]
  • Gi is F.
  • T 8 is a 3 '-terminal group. In certain embodiments, at least one of T 7 and T 8 is a conjugate group. In certain embodiments, one T 7 is a phosphorus moiety. In certain embodiments, said phosphorus moiety has the formula:
  • R 3 and R 0 are each, independently, OH, SH, CpC 6 alkyl, substituted Q-C 6 alkyl, Ci-C 6 alkoxy, substituted Ci-C 6 alkoxy, amino or substituted amino; and R b is O or S.
  • R 3 and R 0 are each OH. In certain embodiments, R a and R 0 are each OCH 3 . In certain embodiments, R 3 and R 0 are each OCH 2 CH 3 . In certain embodiments, R t is O. In certain embodiments, R 1 , is S.
  • each monomer of Formula IV one of Qi, Q 2 , Q 3 and Q 4 is substituted Ci-C 6 alkyl.
  • each monomer of Formula FV one of Qi, Q 2 , Q 3 and Q 4 is substituted Ci-C 6 alkyl and the other three of Qi, Q 2 , Q 3 and Q 4 are H.
  • said substituted C r C 6 alkyl comprises at least one substituent group selected from halogen, C 2 -C 6 alkenyl, OJi, NJ 1 J 2 and CN, wherein each Ji and J 2 is, independently, H or Ci-C 6 alkyl.
  • said substituted Ci-C 6 alkyl comprises at least one substituent group selected from fluoro and OCH 3 .
  • each monomer of Formula IV one of Qi, Q 2 , Q 3 and Q 4 is Ci-C 6 alkyl.
  • one of Qi and Q 2 is CpC 6 alkyl.
  • one of Q 3 and Q 4 is Ci-C 6 alkyl.
  • said C 1 -C 6 alkyl group is methyl.
  • monomer of Formula IV one of Q b Q 2 , Q 3 and Q 4 is F.
  • two of Qi, Q 2 , Q 3 and Q 4 are F.
  • monomer of Formula IV Qi and Q 2 are each F. In certain embodiments, for each monomer of Formula IV Q 3 and Q 4 are each F. In certain embodiments, for each monomer of Formula IV, Q 1 , Q 2 , Q 3 and Q 4 are each F or H. hi certain embodiments, each monomer of Formula IV has the configuration:
  • the invention provides an oligomeric compound comprising a monomer of Formula IV at the 5 'end.
  • the invention provides oligomeric compounds comprising an oligonucleotide comprising a nucleoside at the 5'-end having Formula XIII:
  • Bx is a heterocyclic base moiety
  • A is O, S OrN(R 1 );
  • Ri is H, Ci-C 6 alkyl or substituted Q-C 6 alkyl; T 3 is a phosphorus moiety;
  • T 4 is an internucleoside linking group linking the monomer to the remainder of the oligomeric compound;
  • one of Qi and Q 2 is H, CpC 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 and the other of Qi and Q 2 is Ci-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;
  • X is O, S Or N(E 1 );
  • Z is H, halogen, C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 2 -C 6 alkynyl or N(E 2 )(E 3 );
  • Bx is uracil, 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil, thymine, 2'-thio-thymine, cytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl-cytosine, adenine, guanine, 2,6-diaminopurine, lH-pyrimido[5,4-b][l,4benzoxazin-2(3H)-one), lH-pyrimido[5,4- b] [ 1 ,4]benzothiazin-2(3H)-one, 9-(2-aminoethoxy)-H-pyrimido[5,4-b] [ 1 ,4]benzoxazin-2(3H)-one, 2H- pyrimido[4,5-b]indol-2-one or H-pyrido[3',2':4,5]
  • Bx is uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine.
  • ⁇ is H.
  • Q 2 is H.
  • Qi and Q2 are each other than H.
  • at least one OfQ 1 and Q 2 is substituted Ci-C 6 alkyl.
  • each substituted Ci-C 6 alkyl comprises at least one substituent group independently selected from halogen, C 2 -C 6 alkenyl, OJi, NJ]J 2 and CN, wherein each Ji and J 2 is, independently, H or C r C 6 alkyl.
  • each substituted Ci-C 6 alkyl comprises at least one substituent group independently selected from fluoro and OCH 3 .
  • at least one of Qi and Q 2 is Ci-C 6 alkyl.
  • Qi is methyl.
  • ⁇ is methyl.
  • said phosphorus moiety has the formula:
  • R a and R 0 are each, independently, OH, SH, C r C 6 alkyl, substituted C r C 6 alkyl, C r C 6 alkoxy, substituted Ci-C 6 alkoxy, amino or substituted amino; and R b is O or S.
  • R a and R 0 are each OH. In certain embodiments, R a and R 0 are each OCH 3 . In certain embodiments, R a and R 0 are each OCH 2 CH 3 . In certain embodiments, R b is O. In certain embodiments, R b is S.
  • Gi is F.
  • Gi is 0-Ci-C 6 alkyl, 0-Ci-C 6 substituted alkyl, O-aryl.
  • Gi is -0-Ci-C 6 substituted alkyl.
  • Gi is - OCH 2 CH 2 OCH 3 .
  • Gi is -OCH 2 CH 2 F.
  • the monomer of Formula XIII has the configuration:
  • the invention provides oligomeric compounds comprising an oligonucleotide comprising a nucleoside at the 5'-end having Formula XTV:
  • Bx is a heterocyclic base moiety
  • T 7 is a phosphorus moiety
  • Tg is an internucleoside linking group linking the monomer to the remainder of the oligomeric compound
  • L is O, S orNJ 3 ; each Ji, h and J 3 is, independently, H or Ci-C 6 alkyl; when j is 1 then Z is other than halogen or N(E 2 )(E 3 ); and when Qi, Q 2 , Q 3 and Q 4 are each H or when Q 1 and Q 2 are H and Q 3 and Q 4 are each F or when Qi and Q 2 are each H and one OfQ 3 and Q 4 is H and the other OfQ 3 and Q 4 is R 9 then Gi is other than H, hydroxyl, OR 9 , halogen, CF 3 , CCl 3 , CHCl 2 and CH 2 OH wherein R 9 is alkyl, alkenyl, alkynyl, aryl or alkaryl.
  • Bx is uracil, 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil, thymine, 2'-thio-thymine, cytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl-cytosine, adenine, guanine, 2,6-diaminopurine, lH-pyrimido[5,4-b][l,4benzoxazin-2(3H)-one), lH-pyrimido[5,4- b] [ 1 ,4]benzothiazin-2(3H)-one, 9-(2-aminoethoxy)-H-pyrimido[5,4-b] [ 1 ,4]benzoxazin-2(3H)-one, 2H- pyrimido[4,5-b]indol-2-one or H-pyrido[3',2':4,5]
  • one of Qi, Q 2 , Q 3 and Q 4 is substituted C 1 -C 6 alkyl.
  • one OfQ 1 , Q 2 , Q 3 and Q 4 is substituted Ci-C 6 alkyl and the other three Of Q 1 , Q 2 , Q 3 and Q 4 are H.
  • said substituted Ci-C 6 alkyl comprises at least one substituent group selected from halogen, C 2 - C 6 alkenyl, OJi, NJiJ 2 and CN, wherein each Ji and J 2 is, independently, H or Ci-C 6 alkyl.
  • said substituted Q-C 6 alkyl comprises at least one substituent group selected from fluoro and OCH 3 .
  • one of Qi, Q 2 , Q 3 and Q 4 is Ci-C 6 alkyl. In certain embodiments, one of Qi and Q 2 is Ci-C 6 alkyl. In certain embodiments, one OfQ 3 and Q 4 is C 1 -C 6 alkyl. In certain embodiments, said C 1 -C 6 alkyl group is methyl. In certain embodiments, three of Qi, Q 2 , Q 3 and Q 4 are H. In certain embodiments, one of Qi, Q 2 , Q 3 and Q 4 is F. In certain embodiments, two of Qi, Q 2 , Q 3 and Q 4 are F. In certain embodiments, Qi and Q 2 are each F. In certain embodiments, Q 3 and Q 4 are each F.
  • Qi, Q 2 , Q 3 and Q 4 are each F or H.
  • Qi is H.
  • Q 2 is H.
  • Q 1 and Q 2 are each other than H.
  • at least one Of Q 1 and Q 2 is substituted C 1 -C 6 alkyl.
  • at least one Of Q 1 and Q 2 is Ci-C 6 alkyl.
  • Q 1 is methyl.
  • Q 2 is methyl.
  • said phosphorus moiety has the formula:
  • R a and R 0 are each, independently, OH, SH, CpC 6 alkyl, substituted CpC 6 alkyl, Ci-C 6 alkoxy, substituted Ci-C 6 alkoxy, amino or substituted amino; and R b is O or S.
  • R a and R 0 are each OH. In certain embodiments, R a and R 0 are each OCH 3 . In certain embodiments, R a and R 0 are each OCH 2 CH 3 . In certain embodiments, R b is O. In certain embodiments, R b is S. In certain embodiments,G, is OCH 3 , OCH 2 F, OCHF 2 , OCF 3 , OCH 2 CH 3 , O(CH 2 ) 2 F, OCH 2 CHF 2 ,
  • Gi is F.
  • Gi is 0-Ci-C 6 alkyl, 0-CpC 6 substituted alkyl, O-aryl.
  • Gi is -0-C]-C 6 substituted alkyl.
  • Gi is - OCH 2 CH 2 OCH 3 .
  • G] is -OCH 2 CH 2 F.
  • a monomer of Formula XTV has the configuration:
  • the invention provides an oligomeric compound comprising an oligonucleotide comprising a phosphate stabilizing nucleoside at the 5 '-end, wherein the phosphate stabilizing nucleoside comprises: a 5 '-terminal modified or unmodified phosphate; a modified sugar moiety comprising: a 5'- modification; or a 2 '-modification; or both a 5'-modificaton and a 2 '-modification; and a linking group linking the phosphate stabilizing nucleoside to the remainder of the oligonucleotide.
  • the 5 '-terminal modified phosphate is selected from: phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphorothioate, phosphoramidate, alkylphosphonothioate, substituted alkylphosphonothioate, phosphorodithioate, thiophosphoramidate, and phosphotriester;
  • the 5 '-modification of the sugar moiety of the phosphate stabilizing nucleoside is selected from 5'- alkyl and 5 '-halogen;
  • the 2'-modification of the sugar moiety of the phosphate stabilizing nucleoside is selected from: halogen, allyl, amino, azido, thio, O-allyl, -0-C 1 -Ci 0 alkyl, -0-C 1 -Ci 0 substituted alkyl, -OCF 3 , -O-(CH 2
  • the modified phosphate is selected from: phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphotriester, phosphorothioate, phosphorodithioate, thiophosphoramidate, and phosphoramidate.
  • the modified phosphate is selected from phosphonate, alkylphosphonate, and substituted alkylphosphonate.
  • the 5 '-phosphate is selected from 5'-deoxy-5'-thio phosphate, phosphoramidate, methylene phosphonate, mono-fluoro methylene phosphonate and di-fluoro methylene phosphonate.
  • the sugar moiety of the phosphate stabilizing nucleoside comprises a 5'- modificaton and a 2'-modification.
  • the remainder of the oligonucleotide comprises at least one modified nucleoside.
  • the oligomeric compound comprises a modified base.
  • the oligomeric compound comprises a sugar surrogate.
  • the sugar surrogate is a tetrahydropyran.
  • the tetrahydropyran is F-HNA.
  • the remainder of the oligonucleotide comprises at least one nucleoside comprising a modified sugar.
  • the at least one modified nucleoside comprising a modified sugar is selected from a bicyclic nucleoside and a 2'-modified nucleoside.
  • the at least one modified nucleoside is a bicyclic nucleoside.
  • the bicyclic nucleoside is a (4'-CH 2 -O-2') BNA nucleoside.
  • the bicyclic nucleoside is a (4'-(CH 2 ) 2 -O-2') BNA nucleoside.
  • the bicyclic nucleoside is a (4'-C(CH 3 )H-O-2') BNA nucleoside.
  • the at least one modified nucleoside is a 2'-modifed nucleoside.
  • the at least one 2'-modified nucleoside is selected from a 2'-F nucleoside, a 2'-OCH 3 nucleoside, and a 2'-O(CH 2 ) 2 OCH 3 nucleoside.
  • the at least one 2'-modified nucleoside is a 2'-F nucleoside.
  • the at least one 2'-modif ⁇ ed nucleoside is a T- OCH 3 nucleoside. In certain embodiments, the at least one 2'-modified nucleoside is a 2'-O(CH 2 ) 2 OCH 3 nucleoside.
  • the remainder of the oligonucleotide comprises at least one unmodified nucleoside.
  • the unmodified nucleoside is a ribonucleoside. In certain embodiments, the unmodified nucleoside is a deoxyribonucleoside.
  • the remainder of the oligomeric oligonucleotide comprises at least two modified nucleosides.
  • the at least two modified nucleosides comprise the same modification.
  • the at least two modified nucleosides comprise different modifications.
  • at least one of the at least two modified nucleosides comprises a sugar surrogate.
  • at least one of the at least two modified nucleosides comprises a 2'-modification.
  • each of the at least two modified nucleosides is independently selected from 2'-F nucleosides, 2'-OCH 3 nucleosides and 2'-O(CH 2 ) 2 OCH 3 nucleosides.
  • each of the at least two modified nucleosides is a 2'-F nucleoside. In certain embodiments, each of the at least two modified nucleosides is a 2'-OCH 3 nucleosides. In certain embodiments, each of the at least two modified nucleosides is a 2'-O(CH 2 ) 2 OCH 3 nucleoside. In certain embodiments, essentially every nucleoside of the oligomeric compound is a modified nucleoside. In certain embodiments, every nucleoside of the oligomeric compound is a modified nucleoside.
  • the remainder of the oligonucleotide comprises:
  • each first-type region independently comprising 1-20 contiguous nucleosides wherein each nucleoside of each first-type region comprises a first-type modification
  • each second-type region independently comprising 1-20 contiguous nucleosides wherein each nucleoside of each second-type region comprises a second-type modification
  • each third-type region independently comprising 1-20 contiguous nucleosides wherein each nucleoside of each third-type region comprises a third-type modification; wherein the first-type modification, the second-type modification, and the third-type modification are each independently selected from 2'-F, 2'-OCH 3 , 2'-O(CH 2 ) 2 OCH 3 , BNA, F-HNA, 2'-H and 2'-OH; provided that the first-type modification, the second-type modification, and the third-type modification are each different from one another.
  • the oligonucleotide comprises 2-20 first-type regions; 3-20 first-type regions; 4-20 first-type regions; 5-20 first-type regions; or 6-20 first-type regions. In certain embodiments, the oligonucleotide comprises 1-20 second-type regions; 2-20 second-type regions; 3-20 second-type regions; 4-20 second-type regions; or 5-20 second-type regions. In certain embodiments, the oligonucleotide comprisesl-20 third-type regions; 2-20 third-type regions; 3-20 third-type regions; 4-20 third-type regions; or 5-20 third-type regions.
  • the oligomeric compound comprises a third-type region at the 3 '-end of the oligomeric compound
  • the oligomeric compound comprises a third-type region at the 3 '-end of the oligomeric compound
  • the third-type region contains from 1 to 3 modified nucleosides and the third-type modification is 2'-O(CH 2 ) 2 OCH 3 .
  • the third same type region contains two modified nucleosides and the third-type modification is 2'-O(CH 2 ) 2 ⁇ CH 3 .
  • each first-type region contains from 1 to 5 modified nucleosides.
  • each first-type region contains from 6 to 10 modified nucleosides.
  • each first-type region contains from 11 to 15 modified nucleosides.
  • each first-type region contains from 16 to 20 modified nucleosides.
  • the first-type modification is 2'-F. In certain embodiments, the first-type modification is 2'-OMe. In certain embodiments, the first-type modification is DNA. In certain embodiments, the first-type modification is 2'-O(CH 2 ) 2 ⁇ CH 3 . In certain embodiments, the first-type modification is 4'-CH 2 - O-2'. In certain embodiments, the first-type modification is 4'-(CH 2 ) 2 -O-2'. In certain embodiments, the first- type modification is 4'-C(CH 3 )H-O-2'. In certain embodiments, each second-type region contains from 1 to 5 modified nucleosides. In certain embodiments, each second-type region contains from 6 to 10 modified nucleosides.
  • each second-type region contains from 11 to 15 modified nucleosides. In certain embodiments, each second-type region contains from 16 to 20 modified nucleosides. In certain embodiments, the second-type modification is 2'-F. In certain embodiments, the second-type modification is 2'-OMe. In certain embodiments, the second-type modification is DNA. In certain embodiments, the second - type modification is 2'-O(CH 2 ) 2 OCH 3 . In certain embodiments, the second -type modification is 4'-CH 2 -O- 2'. In certain embodiments, the second -type modification is 4'-(CH 2 ) 2 -O-2'. In certain embodiments, the second -type modification is 4'-C(CH 3 )H-O-2'. In certain embodiments, the oligomeric compound has an alternating motif wherein the first-type regions alternate with the second-type regions.
  • the invention provides oligomeric compounds wherein the remainder of the oligonucleotide comprises at least one region of nucleosides having a nucleoside motif: (A) n -(B) n -(A) n -(B) n , wherein:
  • a an B are differently modified nucleosides; and each n is independently selected from 1, 2, 3, 4, and 5.
  • a and B are each independently selected from a bicyclic and a 2 '-modified nucleoside. In certain embodiments, at least one of A and B is a bicyclic nucleoside. In certain embodiments, at least one of A and B is a (4'-CH 2 -O-2') BNA nucleoside. In certain embodiments, at least one of A and B is a (4'-(CH 2 ) 2 -O-2') BNA nucleoside. In certain embodiments, at least one of A and B is a (4'-C(CH 3 )H-O- 2') BNA nucleoside. In certain embodiments, at least one of A and B is a 2'-modified nucleoside.
  • the 2'-modified nucleoside is selected from: a 2'-F nucleoside, a 2'-OCH 3 nucleoside, and a 2'-O(CH 2 ) 2 OCH 3 nucleoside.
  • a and B are each independently selected from: a 2'-F nucleoside, a 2'-OCH 3 nucleoside, a 2'-O(CH 2 ) 2 OCH 3 nucleoside, a (4'-CH 2 -O-2') BNA nucleoside, a (4'- (CH 2 ) 2 -O-2') BNA nucleoside, a (4'-C(CH 3 )H-O-2') BNA nucleoside, a DNA nucleoside, an RNA nucleoside, and an F-HNA nucleoside.
  • a and B are each independently selected from: a 2'-F nucleoside, a 2'-OCH 3 nucleoside, a (4'-CH 2 -O-2') BNA nucleoside, a (4'-(CH 2 ) 2 -O-2') BNA nucleoside, a (4'-C(CH 3 )H-O-2') BNA nucleoside, and a DNA nucleoside.
  • one of A and B is a 2'-F nucleoside.
  • one of A and B is a 2'-OCH 3 nucleoside.
  • one of A and B is a T- O(CH 2 ) 2 OCH 3 nucleoside.
  • A is a 2'-F nucleoside and B is a 2'-OCH 3 nucleoside. In certain embodiments, A is a 2'-OCH 3 nucleoside and B is a 2'- F nucleoside. In certain embodiments, one of A and B is selected from a (4'-CH 2 -O-2') BNA nucleoside, a (4'-(CH 2 ) 2 -O-2 5 ) BNA nucleoside, and a (4'-C(CH 3 )H-O-2') BNA nucleoside and the other of A and B is a DNA nucleoside.
  • the invention provides oligomeric compounds wherein the remainder of the oligonucleotide comprises a nucleoside motif: (A) X -(B) 2 -(A) Y -(B) 2 -(A) Z -(B) 3 wherein
  • A is a nucleoside of a first type
  • B is a nucleoside of a second type
  • X is 0-10
  • Y is 1-10;
  • Z is 1-10.
  • X is selected from 0, 1, 2 and 3. In certain embodiments, X is selected from
  • Y is selected from 1, 2 and 3. In certain embodiments, Y is selected from 4, 5, 6 and 7. In certain embodiments, Z is selected from 1, 2 and 3. In certain embodiments, Z is selected from 4, 5, 6 and 7. In certain embodiments, A is a 2'-F nucleoside. In certain embodiments, B is a T- OCH 3 nucleoside.
  • the invention provides oligomeric compounds of comprising a 3 '-region consisting of from 1 to 5 nucleosides at the 3 '-end of the oligomeric compound wherein: the nucleosides of the 3 '-region each comprises the same modification as one another; and the nucleosides of the 3'-region are modified differently than the last nucleoside adjacent to the 3'- region.
  • the modification of the 3 '-region is different from any of the modifications of any of the other nucleosides of the oligomeric compound.
  • the nucleosides of the 3 '-region are 2'-O(CH 2 ) 2 ⁇ CH 3 nucleosides.
  • the 3 '-region consists of 2 nucleosides. In certain embodiments, the 3 '-region consists of 3 nucleosides. In certain embodiments, each nucleoside of the 3'-region comprises a uracil base. In certain embodiments, each nucleoside of the 3'-region comprises an adenine base. In certain embodiments, each nucleoside of the 3'-region comprises a thymine base.
  • the remainder of the oligonucleotide comprises a region of uniformly modified nucleosides.
  • the region of uniformly modified nucleosides comprises 2-20 contiguous uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 3-20 contiguous uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 4-20 contiguous uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 5-20 contiguous uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 6-20 contiguous uniformly modified nucleosides.
  • the region of uniformly modified nucleosides comprises 5-15 contiguous uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 6-15 contiguous uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 5-10 contiguous uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 6-10 contiguous uniformly modified nucleosides. In certain embodiments, the remainder of the oligonucleotide comprises a region of alternating modified nucleosides and a region of uniformly modified nucleosides.
  • the region of alternating nucleotides is 5' of the region of fully modified nucleosides. In certain embodiments, the region of alternating nucleotides is 3' of the region of fully modified nucleosides. In certain embodiments, the alternating region and the fully modified region are immediately adjacent to one another. In certain embodiments, the oligomeric compound has additional nucleosides between the alternating region and the fully modified region.
  • the remainder of the oligonucleotide comprises at least one region of nucleosides having a motif I:
  • N m is a 2'-OCH 3 nucleoside
  • PS is a phosphorothioate linking group
  • PO is a phosphodiester linking group.
  • the oligomeric compound comprises at least 2, or 3, or 4, or 6, or 7, or 8, or 9, or 10 separate regions of nucleosides having the motif I.
  • the invention provides oligomeric compounds comprising at least one region having a nucleoside motif selected from: AABBAA;
  • A is a nucleoside of a first type and B is a nucleoside of a second type.
  • oligomeric compounds of the invention comprise one or more conjugate groups. In certain embodiments, oligomeric compounds of the invention consist of the oligonucleotide.
  • the invention provides oligomeric compounds comprising an oligonucleotide comprising a contiguous sequence of linked nucleosides wherein the sequence has the formula: 5'-(ZV(L-Q 1 -L-Q 2 X-(L-Q 1 ) U -(L-Q 3 ) V -(GVS' wherein: each L is an internucleoside linking group;
  • Z is a 5' stabilizing nucleoside.
  • w is 1. In certain embodiments, w is 0.
  • Qi and Q 2 is, independently, a 2'-modified nucleoside having a 2'-substituent group selected from halogen and 0-Ci-C 6 alkyl. In certain embodiments, each Qi and Q 2 is, independently, a 2'-modified nucleoside having a 2'- substituent group selected from F and O-methyl. In certain embodiments, each Q 3 is a 2'-modified nucleoside having a 2 '-substituent group of 0-(CH 2 ⁇ -OCH 3 . In certain embodiments, a is O. In certain embodiments, v is 2. In certain embodiments, u is 0. In certain embodiments, u is 1.
  • the oligonucleotide consists of 8-80 linked nucleoside; 8-26 linked nucleosides; 10-24 linked nucleosides; 16-22 linked nucleosides; 16-18 linked nucleosides; 19-22 linked nucleosides.
  • the second nucleoside from the 5 '-end comprises a sugar moiety comprising a 2'-substituent selected from OH and a halogen.
  • the second nucleoside from the 5'-end is a 2'-F modified nucleoside.
  • the oligomeric compound comprises at least one modified linking group.
  • each internucleoside linking group is, independently, phosphodiester or phosphorothioate.
  • the 5 '-most internucleoside linking group is a phosphorothioate linking group.
  • At least one phosphorothioate region comprising at least two contiguous phosphorothioate linking groups. In certain embodiments, the at least one phosphorothioate region comprises from 3 to 12 contiguous phosphorothioate linking groups. In certain embodiments, the at least one phosphorothioate region comprises from 6 to 8 phosphorothioate linking groups. In certain embodiments, the at least one phosphorothioate region is located at the 3 '-end of the oligomeric compound. In certain embodiments, the at least one phosphorothioate region is located within 3 nucleosides of the 3 '-end of the oligomeric compound.
  • the 7-9 internucleoside linkages at the 3 'end of the oligonucleotide are phosphorothioate linkages and the internucleoside linkage at the 5 '-end is a phosphorothioate linkage.
  • the invention provides oligomeric compounds comprising an oligonucleotide consisting of 10 to 30 linked nucleosides wherein:
  • the nucleoside at the 5' end is a phosphate stabilizing nucleoside comprising: a 5 '-terminal modified or unmodified phosphate; and a modified sugar moiety comprising: a 5'- modification; or a 2 '-modification; or both a 5'-modificaton and a 2'-modification;
  • the sugar moiety of the second nucleoside from the 5 '-end is selected from an unmodified 2'-OH sugar, and a modified sugar comprising a modification selected from: 2'-halogen, 2'O-alkyl, and 2'-O- substituted alkyl; and
  • At least one internucleoside linkage is other than a phosphorothioate linkage.
  • the 5'-terminal modified phosphate is selected from: phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphorothioate, phosphoramidate, alkylphosphonothioate, substituted alkylphosphonothioate, phosphorodithioate, thiophosphoramidate, and phosphotriester;
  • the 5 '-modification of the sugar moiety of the phosphate stabilizing nucleoside is selected from 5'- alkyl and 5 '-halogen;
  • the 2'-modification of the sugar moiety of the phosphate stabilizing nucleoside is selected from: halogen, allyl, amino, azido, thio, O-allyl, -O-C r C 10 alkyl, -O-C r Ci 0 substituted alkyl, -OCF 3 , -O-(CH 2 ) 2
  • O(CH 2 ) n ONH 2 , -OCH 2 C( O)N(H)CH 3i -O(CH 2 ) n ON[(CH 2 ) n CH 3 ] 2 , where n and m are from 1 to about 10;
  • C to Cio alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl.
  • the modified phosphate is selected from: phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphotriester, phosphorothioate, phosphorodithioate, thiophosphoramidate, and phosphoramidate.
  • the modified phosphate is selected from: phosphonate, alkylphosphonate, and substituted alkylphosphonate. In certain embodiments, the modified phosphate is selected from 5'-deoxy-5'-thio phosphate, phosphoramidate, methylene phosphonate, mono-fluoro methylene phosphonate and di-fluoro methylene phosphonate. In certain embodiments, the sugar moiety of the phosphate stabilizing nucleoside comprises a 5'-modificaton and a 2'-modification.
  • the oligomeric compound is an antisense compound.
  • the antisense compound is an RNAi compound.
  • the antisense compound is an siRNAi compound.
  • the antisense compound is a microRNA mimic.
  • the antisense compound is an RNase H antisense compound.
  • the antisense compound modulates splicing.
  • the nucleobase sequence of the oligonucleotide is complementary to a portion of a target nucleic acid, wherein the target nucleic acid is selected from: a target mRNA, a target pre-mRNA, a target microRNA, and a target non-coding RNA.
  • the nucleobase sequence of the oligonucleotide a region of 100% complementarity to the target nucleic acid and wherein the region of 100% complementarity is at least 10 nucleobases. In certain embodiments, the region of 100% complementarity is at least 15 nucleobases. In certain embodiments, the region of 100% complementarity is at least 20 nucleobases.
  • the oligonucleotide is at least 85% complementary to the target nucleic acid. In certain embodiments, the oligonucleotide is at least 90% complementary to the target nucleic acid. In certain embodiments, the oligonucleotide is at least 95% complementary to the target nucleic acid. In certain embodiments, the oligonucleotide is at least 98% complementary to the target nucleic acid. In certain embodiments, the oligonucleotide is 100% complementary to the target nucleic acid.
  • the antisense compound is a microRNA mimic having a nucleobase sequence comprising a portion that is at least 80% identical to the seed region of a microRNA and that has overall identity with the microRNA of at least 70%.
  • the nucleobase sequence of the microRNA mimic has a portion that is at least 80% identical to the sequence of the seed region of a microRNA and has overall identity with the microRNA of at least 75%.
  • the nucleobase sequence of the microRNA mimic has a portion that is at least 80% identical to the sequence of the seed region of a microRNA and has overall identity with the microRNA of at least 80%.
  • the nucleobase sequence of the microRNA mimic has a portion that is at least 100% identical to the sequence of the seed region of a microRNA and has overall identity with the microRNA of at least 80%. In certain embodiments, the nucleobase sequence of the microRNA mimic has a portion that is at least 100% identical to the sequence of the seed region of a microRNA and has overall identity with the microRNA of at least 85%. In certain embodiments, the nucleobase sequence of the microRNA mimic has a portion that is 100% identical to the sequence of the microRNA. In certain embodiments, nucleobase sequence of the oligonucleotide comprises a region of 100% complementarity to a seed match segment of a target nucleic acid.
  • the antisense compound is a microRNA mimic having a nucleobase sequence comprising a portion that is at least 80% identical to the seed region of a microRNA and that has overall identity with the microRNA of at least 50%. In certain embodiments, the antisense compound is a microRNA mimic having a nucleobase sequence comprising a portion that is at least 80% identical to the seed region of a microRNA and that has overall identity with the microRNA of at least 55%. In certain embodiments, the antisense compound is a microRNA mimic having a nucleobase sequence comprising a portion that is at least 80% identical to the seed region of a microRNA and that has overall identity with the microRNA of at least 60%.
  • the antisense compound is a microRNA mimic having a nucleobase sequence comprising a portion that is at least 80% identical to the seed region of a microRNA and that has overall identity with the microRNA of at least 65%.
  • the oligomeric compound comprises a nucleobase sequence selected from a microRNA sequence found in miRBase. In certain embodiments, the oligomeric compound consists of a nucleobase sequence selected from a microRNA sequence found in miRBase.
  • the target nucleic acid is a target mRNA. In certain embodiments, the target nucleic acid is a target pre-mRNA. In certain embodiments, the target nucleic acid is a non-coding RNA. In certain embodiments, the target nucleic acid is a microRNA. In certain embodiments, the target nucleic acid is a pre-mir. In certain embodiments, the target nucleic acid is a pri-mir.
  • the nucleobase sequence of the oligonucleotide comprises a region of 100% complementarity to the target nucleic acid and wherein the region of 100% complementarity is at least 10 nucleobases. In certain embodiments, the nucleobase sequence of the oligonucleotide comprises a region of 100% complementarity to the target nucleic acid and wherein the region of 100% complementarity is at least
  • the nucleobase sequence of the oligonucleotide comprises a region of 100% complementarity to the target nucleic acid and wherein the region of 100% complementarity is at least
  • the target nucleic acid is a mammalian target nucleic acid. In certain embodiments, the mammalian target nucleic acid is a human target nucleic acid.
  • oligomeric compounds comprise from 1 to 3 terminal group nucleosides on at least one end of the oligonucleotide. In certain embodiments, oligomeric compound comprise from 1 to 3 terminal group nucleosides at the 3 '-end of the oligonucleotide. In certain embodiments, oligomeric compound comprise from 1 to 3 terminal group nucleosides at the 5'-end of the oligonucleotide. In certain embodiments, oligomeric compounds of the invention are single stranded.
  • oligomeric compounds of the invention are double stranded.
  • the invention provides pharmaceutical compositions comprising an oligomeric compounds and a pharmaceutically acceptable diluent or carrier.
  • the pharmaceutically acceptable diluent or carrier is pharmaceutical grade sterile saline.
  • the invention provides methods comprising contacting a cell with an oligomeric compound described herein. In certain embodiments, such methods comprise detecting antisense activity. In certain embodiments, the detecting antisense activity comprises detecting a phenotypic change in the cell. In certain embodiments, the detecting antisense activity comprises detecting a change in the amount of target nucleic acid in the cell.
  • the detecting antisense activity comprises detecting a change in the amount of a target protein.
  • the cell is in vitro.
  • the cell is in an animal.
  • animal is a mammal.
  • the mammal is a human.
  • the invention provides methods of modulating a target mRNA in a cell comprising contacting the cell with an oligomeric compound of the invention and thereby modulating the mRNA in a cell.
  • such methods comprise detecting a phenotypic change in the cell.
  • methods comprise detecting a decrease in mRNA levels in the cell.
  • methods comprise detecting a change in the amount of a target protein.
  • the cell is in vitro.
  • the cell is in an animal.
  • the animal is a mammal.
  • the mammal is a human.
  • the invention provides methods of administering to an animal a pharmaceutical composition of the invention.
  • the animal is a mammal. In certain embodiments, the mammal is a human. In certain embodiments, the methods comprise detecting antisense activity in the animal. In certain embodiments, the methods comprise detecting a change in the amount of target nucleic acid in the animal. In certain embodiments, the methods comprise detecting a change in the amount of a target protein in the animal. In certain embodiments, the methods comprise detecting a phenotypic change in the animal. In certain embodiments, the phenotypic change is a change in the amount or quality of a biological marker of activity.
  • the invention provides use of an oligomeric compound of the invention for the manufacture of a medicament for the treatment of a disease characterized by undesired gene expression.
  • the invention provides use of an oligomeric compound of the invention for the manufacture of a medicament for treating a disease by inhibiting gene expression.
  • the invention provides methods of comprising detecting antisense activity wherein the antisense activity is microRNA mimic activity.
  • the detecting microRNA mimic activity comprises detecting a change in the amount of a target nucleic acid in a cell.
  • the detecting microRNA mimic activity comprises detecting a change in the amount of a target protein in cell.
  • the invention provides a nucleoside having the Formula I:
  • Bx is a heterocyclic base moiety
  • T 1 is H, a hydroxyl protecting group, or a phosphorous moiety
  • T 2 is H, a hydroxyl protecting group, or a reactive phosphorous group
  • each of qi and q 2 is, independently selected from Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-C 6 alkyl, substituted Q-C 6 alkenyl and substituted C 2 -C 6 alkynyl
  • Xi is S, NRi 6 , or CRioRn wherein each Ri 0 and Rn is, independently, H, F, Ci-C 6 haloalkyl , or Ci-C 6 alkyl
  • Xi is S, NRi 6 , or CRioRn wherein each Ri 0 and Rn is, independently, H, F, Ci-C 6 haloalkyl , or Ci-C 6 alkyl
  • Ri is selected from a halogen, X 2 -V, and 0-X 4 ; or each of qi and q 2 is, independently, selected from H, C r C 6 alkyl, C 2 -C 6 alkenyl, C 2 - C 6 alkynyl, substituted Q-C 6 alkyl, substituted Q-C 6 alkenyl and substituted C 2 -C 6 alkynyl;
  • Xi is S, NRi 6 Rn, or CRioRn wherein each R 10 and Rn is, independently, H, F, Ci-Ce haloalkyl , or Ci-C 6 alkyl; and
  • Ri is X 2 -V; or each of qi and q 2 is, independently, selected from Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-C 6 alkyl, substituted C 1 -C 6 alkenyl and substituted C 2 -C 6 alkynyl;
  • Xi is O, S, NRi 6 Ri 7 , or CRi 0 Ri i wherein each Ri 0 and Rn is, independently, H, F, Q- C 6 haloalkyl , or Ci-C 6 alkyl; and
  • Ri is X 2 -V; wherein:
  • X 2 is O, S or CR 7 R 8 wherein each R 7 and Rg is, independently, H or Ci-C 6 alkyl;
  • V is selected from cholesterol, (CH 2 ) 2 [O(CH 2 ) 2 ] t OCH 3 , where t is from 1-3, (CH 2 ) 2 F, CH 2 COOH, CH 2 CONH 2 , CH 2 CONR 5 R 61 CH 2 COOCH 2 CH 3 , CH 2 CONH(CH 2 ) I -S-R 4 where i is from 1 to 10, CH 2 CONH(CH 2 )JNR 5 R 6 where j is from 1 to 6, and CH 2 CONHt(CH 2 Xi-N(H)] ⁇ -(CH 2 XiNH 2 where each ki is independently from 2 to 4 and k 2 is from 2 to 10;
  • R 4 is selected from H, Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-C 6 alkyl, substituted C]-C 6 alkenyl, substituted C 2 -C 6 alkynyl, C 6 -Ci 4 aryl and a thio protecting group;
  • R 5 and R 6 are each, independently, selected from H, CpC 6 alkyl, substituted Ci-C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, and substituted C 2 -C 6 alkynyl;
  • Ri 6 is selected from H, Ci-C 6 alkyl, or substituted Ci-C 6 alkyl;
  • At least one of qj and q 2 is C]-C 6 alkyl or substituted Cj-C 6 alkyl. In certain embodiments, at least one of qi and q 2 is Ci-C 6 alkyl. In certain embodiments, at least one of q ! and q 2 is methyl. In certain embodiments, one of qi and q 2 is H. In certain embodiments, one of qi and q 2 is methyl and the other of qi and q 2 is H. In certain embodiments, Z is O. In certain embodiments, Y is CH 2 CONH(CH 2 )JNR 5 R 6 . In certain embodiments, j is 2. In certain embodiments, one of R 5 and R 6 Is other than H.
  • At least one of R 5 and R 6 is selected from Ci-C 6 alkyl, substituted Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C r C 6 alkyl, substituted Ci-C 6 alkenyl, and substituted C 2 -C 6 alkynyl.
  • at least one of R 5 and R 6 is Ci-C 6 alkyl or substituted Q-C 6 alkyl.
  • at least one of R 5 and R 6 is methyl.
  • both R 5 and R 5 are other than H.
  • Xi is O. In certain embodiments, Xi is S.
  • Xi is CRioRn.
  • R 10 and Rn are both H.
  • Ti is a phosphorus moiety.
  • the phosphorus moiety is selected from P(Y a )Y b Y c , where Y a is O or S and each Y b and Y c is, independently, OH, SH, alkyl, substituted C r C 6 alkyl, alkoxyl and substituted Ci-C 6 alkoxyl.
  • Y a is O and Y b and Y 0 are each OH.
  • Y a is 0; ii. Y b and Y c are each OH; and iii. T 2 is diisopropylcyanoethoxy phosphoramidite.
  • T 2 is a reactive phosphorus group.
  • the reactive phosphorous group is diisopropylcyanoethoxy phosphoramidite or H-phosphonate.
  • a nucleoside has Formula XII:
  • both R 5 and R 6 are other than H. In certain embodiments, both R 5 and R 6 are methyl. In certain embodiments, qi is methyl and q 2 is H. In certain embodiments, qi is H and q 2 is methyl. In certain embodiments, such nucleosides have the configuration:
  • the oligomeric compound has a microRNA sequence associated with an accession number from miRBase version 10.1 released December 2007 selected from:
  • the invention provides oligomeric compounds having a nucleobase sequence selected from among SEQ ID NOs 20, 21, 23, 24, 25, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
  • the present invention provides oligomeric compounds having a nucleobase sequence selected from the table below.
  • 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 nucleosides having mimetic bases and/or sugar groups. Nucleosides may be modified with any of a variety of substituents. Nucleosides may include a phosphate moiety.
  • sugar moiety means a natural or modified sugar ring or sugar surrogate.
  • 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, 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 phosphate linking group.
  • 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. 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.
  • bicyclic nucleoside or “BNA” refers to a nucleoside wherein the sugar moiety of the nucleoside comprises a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic sugar moiety.
  • 4 '-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.
  • 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'-F refers to a nucleoside comprising a sugar comprising a fluoro group at the 2' position.
  • 2'-0Me or "2'-OCH 3 " or “2'-O-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 OCH 3 " or “2'-O-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.
  • phosphorous moiety refers to a to monovalent P v phosphorus radical group.
  • a phosphorus moiety is selected from: a phosphate, phosphonate, alkylphosphonate, aminoalkyl phosphonate, phosphorothioate, phosphoramidite, alkylphosphonothioate, phosphorodithioate, thiophosphoramidate, phosphotriester and the like.
  • modified phosphorous moieties have the following structural formula:
  • R a and R 0 are each, independently, OH, SH, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, Ci-C 6 alkoxy, substituted Ci-C 6 alkoxy, amino or substituted amino; and R b is O or S.
  • phosphate moiety refers to a terminal phosphate group that includes phosphates as well as modified phosphates.
  • the phosphate moiety can be located at either terminus but is preferred at the 5'-terminal nucleoside.
  • the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R) or alkyl where R is H, an amino protecting group or unsubstituted or substituted alkyl.
  • the 5' and or 3' terminal group can comprise from 1 to 3 phosphate moieties that are each, independently, unmodified or modified.
  • phosphate stabilizing modification refers to a nucleoside modification that results in stabilization of a 5 '-phosphate group of nucleoside, relative to the stability of a 5 '-phosphate of an unmodified nucleoside under biologic conditions. Such stabilization of a 5'-phophate group includes but is not limit to resistance to removal by phosphatases.
  • phosphate stabilizing nucleoside refers to a nucleoside comprising at least one phosphate stabilizing modification.
  • the phosphate stabilizing modification is a T- modification.
  • the phosphate stabilizing modification is at the 5' position of the nucleoside.
  • a phosphate stabilizing modification is at the 5' position of the nucleoside and at the 2' position of the nucleoside.
  • 5 '-stabilizing nucleoside refers to a nucleoside that, when placed at the 5'-end of an oligonucleotide, results in an oligonucleotide that is more resistant to exonuclease digestion, and/or has a stabilized phosphate group.
  • oligonucleotide refers to a compound comprising a plurality of linked nucleosides.
  • an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).
  • oligonucleoside refers to an oligonucleotide in which none of the internucleoside linkages contains a phosphorus atom.
  • oligonucleotides include oligonucleosides.
  • modified oligonucleotide refers to an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
  • internucleoside linkage refers to a covalent linkage between adjacent nucleosides.
  • naturally occurring internucleoside linkage refers to a 3' to 5 1 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 substructures.
  • an oligomeric compound is an oligonucleotide.
  • an oligomeric compound is a single-stranded oligonucleotide.
  • an oligomeric compound is a double-stranded duplex comprising two oligonucleotides.
  • an oligomeric compound is a single-stranded or double-stranded oligonucleotide comprising one or more conjugate groups and/or terminal groups.
  • duplex refers to two separate oligomeric compounds that are hybridized together.
  • terminal group refers to one or more atom attached to either, or both, the 3' end or the 5' end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more additional nucleosides.
  • conjugate refers to an atom or group of atoms bound to an oligonucleotide or oligomeric compound.
  • 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.
  • 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.
  • protecting group refers to a labile chemical moiety which is known in the art to protect reactive groups including without limitation, hydroxyl, amino and thiol groups, against undesired reactions during synthetic procedures.
  • Protecting groups are typically used selectively and/or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group as is or available for further reactions.
  • Protecting groups as known in the art are described generally in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999).
  • orthogonal protection refers to functional groups which are protected with different classes of protecting groups, wherein each class of protecting group can be removed in any order and in the presence of all other classes (see, Barany et al, J. Am. Chem. Soc, 1977, 99, 7363-7365; Barany et ah, J. Am. Chem. Soc, 1980, 102, 3084-3095).
  • Orthogonal protection is widely used in for example automated oligonucleotide synthesis.
  • a functional group is deblocked in the presence of one or more other protected functional groups which is not affected by the deblocking procedure. This deblocked functional group is reacted in some manner and at some point a further orthogonal protecting group is removed under a different set of reaction conditions. This allows for selective chemistry to arrive at a desired compound or oligomeric compound. This allows for selective chemistry to arrive at a desired compound or oligomeric compound.
  • 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. In certain embodiments, an antisense compound modulates (increases or decreases) expression or amount of a target nucleic acid. In certain embodiments, an antisense compound alters splicing of a target pre-mRNA resulting in a different splice variant. In certain embodiments, an antisense compound modulates expression of one or more different target proteins.
  • Antisense mechanisms contemplated herein include, but are not limited to an RNase H mechanism, RNAi mechanisms, splicing modulation, translational arrest, altering RNA processing, inhibiting microRNA function, or mimicking microRNA function.
  • 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.
  • RNAi refers to a mechanism by which certain antisense compounds effect expression or amount of a target nucleic acid. RNAi mechanisms involve the RISC pathway.
  • RNAi compound refers to an oligomeric compound that acts, at least in part, through an RNAi mechanism to modulate a target nucleic acid and/or protein encoded by a target nucleic acid. RNAi compounds include, but are not limited to double-stranded short interfering RNA (siRNA), single-stranded RNA (ssRNA), and microRNA, including microRNA mimics.
  • 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 detecting and/or measuring the amount of target protein or the relative amounts of splice variants of a target protein.
  • antisense activity is assessed by detecting and/or measuring the amount of target nucleic acids and/or cleaved target nucleic acids and/or alternatively spliced target nucleic acids. In certain embodiments, 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.
  • 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 mRNA refers to a pre-selected RNA molecule that encodes a protein.
  • target pre-mRNA refers to a pre-selected RNA transcript that has not been fully processed into mRNA. Notably, pre-RNA includes one or more intron.
  • target microRNA refers to a pre-selected non-coding RNA molecule about 18-30 nucleobases in length that modulates expression of one or more proteins or to a precursor of such a non- coding molecule.
  • target pdRNA refers to refers to a pre-selected RNA molecule that interacts with one or more promoter to modulate transcription.
  • microRNA refers to a naturally occurring, small, non-coding RNA that represses gene expression at the level of translation.
  • a microRNA represses gene expression by binding to a target site within a 3 ' untranslated region of a target nucleic acid.
  • a microRNA has a nucleobase sequence as set forth in miRBase, a database of published microRNA sequences found at http://microrna.sanger.ac.uk/sequences/.
  • a microRNA has a nucleobase sequence as set forth in miRBase version 10.1 released December 2007, which is herein incorporated by reference in its entirety.
  • a microRNA has a nucleobase sequence as set forth in miRBase version 12.0 released September 2008, which is herein incorporated by reference in its entirety.
  • microRNA mimic refers to an oligomeric compound having a sequence that is at least partially identical to that of a microRNA.
  • a microRNA mimic comprises the microRNA seed region of a microRNA.
  • a microRNA mimic modulates translation of more than one target nucleic acids.
  • seed region refers to a region at or near the 5 'end of an antisense compound having a nucleobase sequence that is import for target nucleic acid recognition by the antisense compound.
  • a seed region comprises nucleobases 2-8 of an antisense compound.
  • a seed region comprises nucleobases 2-7 of an antisense compound.
  • a seed region comprises nucleobases 1-7 of an antisense compound. In certain embodiments, a seed region comprises nucleobases 1-6 of an antisense compound. In certain embodiments, a seed region comprises nucleobases 1-8 of an antisense compound.
  • microRNA seed region refers to a seed region of a microRNA or microRNA mimic.
  • a microRNA seed region comprises nucleobases 2-8 of a microRNA or microRNA mimic.
  • a microRNA seed region comprises nucleobases 2-7 of a microRNA or microRNA mimic.
  • a microRNA seed region comprises nucleobases 1- 7 of a microRNA or microRNA mimic.
  • a microRNA seed region comprises nucleobases 1-6 of a microRNA or microRNA mimic.
  • a microRNA seed region comprises nucleobases 1-8 of a microRNA or microRNA mimic.
  • seed match segment refers to a portion of a target nucleic acid having nucleobase complementarity to a seed region.
  • a seed match segment has nucleobase complementarity to nucleobases 2-8 of an siRNA, ssRNA, natural microRNA or microRNA mimic.
  • a seed match segment has nucleobase complementarity to nucleobases 2-7 of an siRNA, ssRNA, microRNA or microRNA mimic.
  • a seed match segment has nucleobase complementarity to nucleobases 1-6 of an siRNA, ssRNA, microRNA or microRNA mimic.
  • a seed match segment has nucleobase complementarity to nucleobases 1-7 of an siRNA, ssRNA, microRNA or microRNA mimic. In certain embodiments, a seed match segment has nucleobase complementarity to nucleobases 1-8 of an siRNA, ssRNA, microRNA or microRNA mimic.
  • seed match target nucleic acid refers to a target nucleic acid comprising a seed match segment.
  • microRNA family refers to a group of microRNAs that share a microRNA seed sequence. In certain embodiments, microRNA family members regulate a common set of target nucleic acids. In certain embodiments, the shared microRNA seed sequence is found at the same nucleobase positions in each member of a microRNA family. In certain embodiments, the shared microRNA seed sequence is not found at the same nucleobase positions in each member of a microRNA family. For example, a microRNA seed sequence found at nucleobases 1 -7 of one member of a microRNA family may be found at nucleobases 2-8 of another member of a microRNA family.
  • target non-coding RNA refers to a pre-selected RNA molecule that is not translated to generate a protein. Certain non-coding RNA are involved in regulation of expression.
  • target viral nucleic acid refers to a pre-selected nucleic acid (RNA or DNA) associated with a virus.
  • RNA or DNA a pre-selected nucleic acid associated with a virus.
  • viral nucleic acid includes nucleic acids that constitute the viral genome, as well as transcripts (including reverse-transcripts and RNA transcribed from RNA) of those nucleic acids, whether or not produced by the host cellular machinery.
  • viral nucleic acids also include host nucleic acids that are recruited by a virus upon viral infection.
  • 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 hybridization.
  • 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 complementary to the natural bases cytosine and 5-methyl cytosine. Hybridization can occur under varying circumstances.
  • oligomeric compound specifically hybridizes to more than one target site.
  • all identity refers to the nucleobase identity of an oligomeric compound relative to a particular nucleic acid or portion thereof, over the length of the oligomeric compound.
  • 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.
  • 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.
  • nucleoside comprising a 2'-0Me modified sugar and an adenine nucleobase and a nucleoside comprising a 2'-0Me modified sugar and a thymine nucleobase are not differently modified.
  • nucleoside having a modification of a first type may be an unmodified nucleoside.
  • 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.
  • alternating motif refers to an oligomeric compound or a portion thereof, having at least four separate regions of modified nucleosides in a pattern (AB) n A n , where A represents a region of nucleosides having a first type of modification; B represent a region of nucleosides having a different type of modification; n is 2-15; and m is 0 or 1.
  • alternating motifs include 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more alternating regions.
  • each A region and each B region independently comprises 1-4 nucleosides.
  • uniform modified or “uniformly modified” refer to oligomeric compounds or portions thereof that comprise the same modifications.
  • the nucleosides of a region of uniformly modified nucleosides all comprise the same 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.
  • mistigation refers to a lessening of at least one activity or one indicator of the severity of a condition or disease.
  • the severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art.
  • the condition may be a toxic effect of a therapeutic agent.
  • pharmaceutical agent refers to a substance that provides a therapeutic effect when administered to a subject.
  • a pharmaceutical agent provides a therapeutic benefit.
  • a pharmaceutical agent provides a toxic effect.
  • therapeutic index refers to the toxic dose of a drug for 50% of the population
  • TDsn TDsn divided by the minimum effective dose for 50% of the population
  • therapeutically effective amount refers to an amount of a pharmaceutical agent that provides a therapeutic benefit to an animal.
  • administering refers to providing a pharmaceutical agent to an animal, and includes, but is not limited to administering by a medical professional and self-administering.
  • co-administer refers to administering more than one pharmaceutical agent to an animal.
  • the more than one agent may be administered together or separately; at the same time or different times; through the same route of administration or through different routes of administration.
  • co-formulation refers to a formulation comprising two or more pharmaceutically active agents.
  • a co-formulation comprises two or more oligomeric compounds.
  • two or more oligomeric compound are oligomeric compounds of the present invention.
  • one or more oligomeric compound present in a co-formulation is not a compound of the present invention.
  • a co-formulation includes one or more non- oligomeric pharmaceutical agents.
  • route of administration refers to the means by which a pharmaceutical agent is administered to an animal.
  • a pharmaceutical composition refers to a mixture of substances suitable for administering to an animal.
  • a pharmaceutical composition may comprise an antisense oligonucleotide and a sterile aqueous solution.
  • a pharmaceutically acceptable carrier or diluent refers to any substance suitable for use in administering to an animal.
  • a pharmaceutically acceptable carrier or diluent is sterile saline.
  • such sterile saline is pharmaceutical grade saline.
  • animal refers to a human or a non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
  • parenteral administration refers to administration through injection or infusion.
  • Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.
  • subcutaneous administration refers to administration just below the skin.
  • Intravenous administration refers to administration into a vein.
  • active pharmaceutical ingredient refers to the substance in a pharmaceutical composition that provides a desired effect.
  • prodrug refers to a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
  • a dose refers to a specified quantity of a pharmaceutical agent provided in a single administration.
  • a dose may be administered in two or more boluses, tablets, or injections.
  • the desired dose requires a volume not easily accommodated by a single injection.
  • two or more injections may be used to achieve the desired dose.
  • a dose may be administered in two or more injections to minimize injection site reaction in an individual.
  • 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 (Ci-Ci 2 alkyl) with from 1 to about 6 carbon atoms (Ci-C 6 alkyl) being more preferred.
  • lower alkyl as used herein includes from 1 to about 6 carbon atoms (C r 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-Ci 2 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, l-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 Ci-Ci 2 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 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, «-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 refers to a radical comprising a mono- or polycyclic 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(O)-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 substituted 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 33 , Ry 3 and R 1x is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including 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.
  • 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.
  • 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 (O) 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.
  • modified nucleosides In certain embodiments, the invention provides modified nucleosides. Certain modified nucleosides comprise modified sugar moieties, modified heterocyclic bases, modified phosphorus moieties, or combinations of those modifications. In certain embodiments, modified oligonucleotides of the present invention comprise modified nucleosides comprising a modified sugar moiety. In certain embodiments, modified oligonucleotides of the present invention comprise modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides of the present invention comprise modified nucleosides comprising a modified sugar moiety and a modified nucleobase.
  • nucleoside may comprise a modified phosphate or phosphorus moiety at the 5 '-end (note that in certain embodiments, nucleoside that are linked to another nucleoside at the 5 '-end may also comprise modified phosphates or phosphorus moieties, though such circumstance is typically refered to herein as a modified internucleoside linkage).
  • the invention provides nucleosides comprising a modification at the 5 '-position of the sugar.
  • modifications at the 5 '-position of the sugar or its substituents are typically referred to as modified sugars and modifications distal to that position are referred to as modified phosphates.
  • modified phosphates modifications distal to that position.
  • the example below shows a modified nucleoside comprising an a sulfur atom in place of the oxygen that links the phosphorus moiety and the sugar of a natural nucleoside.
  • modifications are typically referred to as modified phosphates, however, one of skill in the art will recognize that such a modification could also be referred to as a modified sugar comprising a sulfer linked to the 5'- position of the sugar.
  • nucleosides of the present invention comprise modified phosphates. In certain embodiments, nucleosides of the present invention comprise 5 '-sugar modifications. In certain embodiments, nucleosides of the present invention comprise both modified phosphates and 5 '-sugar modifications. Examples of nucleosides having such modified phosphorus moieties and/or 5 '-modifications include, but are not limited to:
  • nucleosides comprising modified phosphate and/or 5 '-modified sugar groups may further comprise a modification at the 2'-position of the sugar. Many such 2'-modif ⁇ cations are known in the art.
  • Rx isselected from: -O-Methyl, -O-Ethyl, -O-Propyl, - O-Phenyl, O-methoxyethyl, S-Methyl, NMA, DMAEAc, DMAEOE, -0-CH 2 CH 2 F.
  • Rx is any substituents described herein or known in the art.
  • the nucleoside is not modified at the 2 '-position (i.e., Rx is H (DNA) or Rx is OH (RNA)). In certain embodiments, such nucleosides are at the 5 'end of an oligonucleotide.
  • nucleosides have Formula I:
  • Bx is a heterocyclic base moiety; A iS O 5 S Or N(R 1 );
  • Ri is H, Ci-C 6 alkyl or substituted Ci-C 6 alkyl
  • one of Ti and T 2 is H, a protecting group or a phosphorus moiety and the other of Ti and T 2 is H, a protecting group or a reactive phosphorus group
  • Z is H, halogen, Ci-C 6 alkyl, substituted C]-C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 2 -C 6 alkynyl or N(E 2 )(E 3 );
  • the compounds of Formula I are provided having the configuration:
  • nucleosides have Formula II:
  • Bx is a heterocyclic base moiety;
  • A is O, S or N(Ri);
  • Ri is H, Ci-C 6 alkyl or substituted C 1 -C 6 alkyl
  • one OfT 3 and T 4 is an internucleoside linking group linking the monomer to the oligomeric compound and the other of T 3 and T 4 is H, a protecting group, a phosphorus moiety, a 5 ' or 3 '-terminal group or an internucleoside linking group linking the monomer to the oligomeric compound
  • one of Qi and Q 2 is H, Q-C 6 alkyl, substituted C r 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 and the other of Qi and Q 2 is Ci-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
  • X is O, S OrN(E 1 );
  • Z is H, halogen, 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, substituted C 2 -C 6 alkynyl OrN(E 2 )(E 3 );
  • nucleosides of Formula II have the configuration:
  • nucleosides have Formula III:
  • Bx is a heterocyclic base moiety
  • T 5 is a phosphorus moiety or a reactive phosphorus group
  • Te is H, a protecting group or a reactive phosphorus group
  • Qi 5 Ch 5 Cb and Q 4 are each, independently, H, halogen, C 1 -C 6 alkyl, substituted C]-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;
  • X is O, S or N(Ei);
  • Z is H, halogen, C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 2 -C 6 alkynyl OrN(E 2 )(E 3 );
  • L is O, S or NJ 3 ; each J 1 , J 2 and J 3 is, independently, H or Ci-C 6 alkyl; when j is 1 then Z is other than halogen or N(E 2 )(E 3 ); and when Qi, Q 2 , Q 3 and Q 4 are each H or when Qi and Q 2 are H and Q 3 and Q 4 are each F or when Q 1 and Q 2 are each H and one of Q 3 and Q 4 is H and the other of Q 3 and Q 4 is R 9 then Gi is other than H, hydroxyl, OR 9 , halogen, CF 3 , CCl 3 , CHCl 2 and CH 2 OH wherein R 9 is alkyl, alkenyl, alkynyl, aryl or alkaryl.
  • the compounds of Formula III have the configuration:
  • nucleosides have Formula IV: wherein independently for each monomer of Formula IV:
  • Bx is a heterocyclic base moiety
  • one ofT 7 and T 8 is an internucleoside linking group linking the monomer to the oligomeric compound and the other of T 7 and T 8 is H, a hydroxyl protecting group, a phosphorus moiety, a 5' or 3'- terminal group or an internucleoside linking group linking the monomer to the oligomeric compound;
  • Q b Q 2 , Q 3 and Q 4 are each, independently, H, halogen, C]-C 6 alkyl, substituted C 1 -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;
  • Z is H, halogen, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 2 -C 6 alkynyl or N(E 2 )(E 3 );
  • L is O, S or NJ 3 ; each Ji, J 2 and J 3 is, independently, H or Ci-C 6 alkyl; when j is 1 then Z is other than halogen or N(E 2 )(E 3 ); and when Qi, Q 2 , Q 3 and Q 4 are each H or when Qi and Q 2 are H and Q 3 and Q 4 are each F or when Qi and Q 2 are each H and one of Q 3 and Q 4 is H and the other OfQ 3 and Q 4 is R 9 then Gi is other than H, hydroxyl, OR 9 , halogen, CF 3 , CCl 3 , CHCl 2 and CH 2 OH wherein R 9 is alkyl, alkenyl, alkynyl, aryl or alkaryl.
  • oligomeric compounds are provided comprising at least one monomer having Formula IV, wherein each monomer of Formula IV has the configuration:
  • the present invention provides nucleosides comprising a modification at the 2'-position of the sugar. In certain embodiments, the invention provides nucleosides comprising a modification at the 5'-positin of the sugar. In certain embodiments, the invention provides nucleosides comprising modifications at the 2'-position and the 5'-position of the sugar. In certain embodiments, modified nucleosides may be useful for incorporation into oligonucleotides. In certain embodiment, modified nucleosides are incorporated into oligonucleosides at the 5 '-end of the oligonucleotide. In certain embodiments, modified nucleosides of the present invention have Formula VII:
  • Bx is a heterocyclic base moiety
  • Xi is O, S, N, or CRi 0 Ri i wherein each Ri 0 and Rn is, independently, H or Ci-C 6 alkyl; T 3 is a phosphorus moiety;
  • T 4 is an internucleoside linking group attaching the nucleoside to the remainder of the oligonucleotide
  • Ri is selected from halogen, amino, trifluoroalkyl, trifiuoroalkoxy, azido, aminooxy, Ci-C 6 alkyl, C 2 - C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-C 6 alkyl, substituted Ci-C 6 alkenyl, substituted C 2 -C 6 alkynyl, O- alkyl, S-alkyl, N(J 4 )-alkyl, O-alkenyl, S-alkenyl, N(J 4 )-alkenyl, O-alkynyl, S-alkynyl or N(J 4 )-alkynyl, O- aryl, S-aryl, N-aryl, O-aralkyl, S-aralkyl, N(J 4 )-aralkyl and -X 2 -V, wherein:
  • X 2 is O, S or CR 7 R 8 wherein each R 7 and R 8 is, independently, H or Ci-C 6 alkyl;
  • V is selected from -CH 2 COOH, -CH 2 CONH 2 , -CH 2 COOCH 2 CH 3 , -CH 2 CONH(CHz) 1 -S-R 4 where i is from 1 to 10, -CH 2 CONH(CH 2 ) J NR 5 R 6 where j is from 1 to 6, and -CH 2 CONH ⁇ (CH 2 ) k ,-N(H) ⁇ k2 -(CH 2 ) kl NH 2 where each kl is independently from 2 to 4 and k2 is from 2 to 10;
  • R 4 is selected from H, CpC 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C]-C 6 alkyl, substituted Ci-C 6 alkenyl, substituted C 2 -C 6 alkynyl, C 6 -C] 4 aryl and a thio protecting group;
  • R 5 and R 6 are each, independently, selected from H, Q-C 6 alkyl, substituted Cj-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Q-C 6 alkyl, substituted C]-C 6 alkenyl and substituted C 2 -C 6 alkynyl; and each qi and q 2 is, independently, selected from H, Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Q-C 6 alkyl, substituted Q-C 6 alkeny
  • the present invention provides modified nucleosides.
  • modified nucleosides of the present invention have Formula XI.
  • Bx is a heterocyclic base moiety
  • Xi is O, S, N, or CRioR ⁇ wherein each Ri 0 and Rn is, independently, H or Q-C 6 alkyl; Ti is H, a hydroxyl protecting group, or a phosphorus moiety; T 2 is H, a hydroxyl protecting group, or a reactive phosphorus group; each qi and q 2 is, independently selected from H, C] -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-C 6 alkyl, substituted Cj-C 6 alkenyl, and substituted C 2 -C 6 alkynyl, provided that at least one of qi and q 2 is other than H;
  • X 2 is O, S or CR 7 R 8 wherein each R 7 and Rg is, independently, H, Q-C 6 alkyl, or substituted Ci-C 6 ;
  • V is selected from Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-C 6 alkyl, substituted Q- C 6 alkenyl, substituted C 2 -C 6 alkynyl, CH 2 COOH, CH 2 COONH 2 , CH 2 COOEt, -CH 2 CONH(CH 2 ) J -S-R 4 where i is from 1 to 10, CH 2 CONH(CH 2 )JNR 5 R 6 where j is from 1 to 6, and (CH 2 XiNH 2 where each kl is independently from 2 to 4 and k2 is from 2 to 10;
  • R 4 Is selected from H, Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-C 6 alkyl, substituted Ci-C 6 alkenyl, substituted C 2 -C 6 alkynyl, C 6 -Ci 4 aryl and a thio protecting group;
  • nucleosides of the present invention include, but are not limited to any of the following:
  • nucleosides are incorporated into oligomeric compounds. In certain embodiments, such nucleosides are incorporated at the 5 '-terminal end of an oligonucleotide or oligomeric compound.
  • the present invention provides modified oligonucleotides comprising one or more nucleosides comprising one or more previously described modification.
  • such previously described modification is a modified sugar moiety.
  • a modified sugar moiety is a bicyclic sugar moiety.
  • a modified sugar moiety is a non-bicyclic modified sugar moiety.
  • modified sugar moiety moieties are known and can be used to alter, typically increase, the affinity of the antisense compound for its target and/or increase nuclease resistance.
  • a representative list of preferred modified sugar moieties includes but is not limited to bicyclic modified sugar moieties (BNA's), including methyleneoxy (4'-CH 2 -O-2') BNA, ethyleneoxy (4'-(CH 2 ) 2 -O-2') BNA and methyl(methyleneoxy) (4'-C(CH 3 )H-O-2') BNA; substituted sugar moieties, especially 2'-substituted sugar moieties having a 2'-F, 2'-OCH 3 or a 2'-O(CH 2 ) 2 -OCH 3 substituent group; and 4'-thio modified sugar moieties.
  • BNA's bicyclic modified sugar moieties
  • substituted sugar moieties especially 2'-substituted sugar moieties having a 2'-F, 2'-OCH
  • Sugar moieties can also be replaced with sugar moiety mimetic groups among others.
  • Methods for the preparations of modified sugar moieties are well known to those skilled in the art. Some representative patents and publications that teach the preparation of such modified sugar moieties include, but are not limited to, U.S.
  • Patents 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; 6,172,209; 6,271,358; and 6,600,032; and WO 2005/121371.
  • the present invention provides modified nucleosides comprising a bicyclic sugar moiety.
  • 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 one of the formulae: 4'-(CH 2 )-O-2' (LNA); 4'-(CH 2 )-S-2'; 4'-(CH 2 ) 2 -O-2' (ENA); 4'-CH(CH 3 )-O-2' and 4'-CH(CH 2 OCH 3 )-O-2' (and analogs thereof see U.S.
  • Patent 1, All, 612, issued on September 23, 2008); 4'-CH 2 -C(H)(CH 3 ) ⁇ ' (see Chattopadhyaya, et a!., J. Org. Chem.,2009, 74, 118-134); and 4'-CH 2 -C( CH 2 )-2' (and analogs thereof see published International Application WO 2008/154401, published on December 8, 2008).
  • Certain such sugar moieties have been described. 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.
  • Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example ⁇ -L-ribofuranose and ⁇ -D-ribofuranose (see PCT international application PCT/DK98/00393, published on March 25, 1999 as WO 99/14226).
  • nucleosides comprising a bicyclic sugar moiety have increased affinity for a complementary nucleic acid.
  • nucleosides comprising a bicyclic sugar moiety provide resistance to nuclease degradation of an oligonucleotide in which they are incorporated.
  • Antisense oligonucleotides comprising BNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 5633-5638).
  • Certain bicyclic-sugar moiety containing nucleosides comprise a bridge linking the 4' carbon and the 2' carbon of the sugar moiety.
  • the bridging group is a methyleneoxy (4'-CH 2 -O-2').
  • the bridging group is an ethyleneoxy (4'-CH 2 CH 2 -O- 2') (Singh et al., Chem. Commun., 1998, 4, 455-456: Morita et al, Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226).
  • the bridge of a bicyclic sugar moiety is , -[C(R a )(R b )] n -, -[C(R a )(R b )] n -O-,
  • the bridge is 4'-CH 2 -2', 4'-(CH 2 ) 2 -2', 4'- (CH 2 ) 3 -2' 5 4'-CH 2 -O-2', 4'-(CH 2 ) 2 -O-2', 4'-CH 2 -O-N(R a )-2' and 4'-CH 2 -N(R a )-O-2 I - wherein each R a is, independently, H, a protecting group or Ci-Ci 2 alkyl.
  • bicyclic nucleosides are further defined by isomeric configuration.
  • a nucleoside comprising a 4'-2' methylenoxy bridge may be in the ⁇ -L configuration or in the ⁇ -D configuration.
  • alpha-L- methyleneoxy (4'-CH 2 -O-2 5 ) BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).
  • bicyclic nucleosides include, but are not limited to, (A) ⁇ -L-Methyleneoxy (4'-CH 2 -O-2') BNA , (B) ⁇ -D-Methyleneoxy (4'-CH 2 -O-2') BNA , (C) Ethyleneoxy (4'-(CH 2 ) 2 -O-2') BNA , (D) Aminooxy (4'-CH 2 -O-N(R)-2') BNA, (E) Oxyamino (4'-CH 2 -N(R)-O-2') BNA, and (F) Methyl(methyleneoxy) (4'-C(CH 3 )H-O-2 ! ) BNA, as depicted below.
  • bicyclic nucleosides include, but are not limited to, the structures below:
  • Bx is the base moiety
  • bicyclic nucleoside having the formula:
  • Bx is a heterocyclic base moiety
  • R 0 is C 1 -Cn alkyl or an amino protecting group;
  • T a and T b are each, independently, hydroxyl, a protected hydroxyl, a conjugate group, an activated phosphorus moiety or a covalent attachment to a support medium.
  • bicyclic nucleoside having the formula:
  • Bx is a heterocyclic base moiety
  • T c is H or a hydroxyl protecting group
  • T d is H, a hydroxyl protecting group or a reactive phosphorus group
  • Z a is Q-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-C 6 alkyl, substituted C 2 -C 6 alkenyl, substituted C 2 -C 6 alkynyl, acyl, substituted acyl, or substituted amide.
  • the Z a group is C 1 -C 6 alkyl substituted with one or more X x , wherein each X x is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH 3 O-), substituted alkoxy or azido.
  • X x is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH 3 O-), substituted alkoxy or azido.
  • bicyclic nucleoside having the formula:
  • Bx is a heterocyclic base moiety; one of T e and T f is H or a hydroxyl protecting group and the other of T e and T f is H, a hydroxyl protecting group or a reactive phosphorus group;
  • Bx is a heterocyclic base moiety; one of T g and T h is H or a hydroxyl protecting group and the other of T g and T h is H, a hydroxyl protecting group or a reactive phosphorus group;
  • R f is Ci-C 6 alkyl, substituted C]-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;
  • BNA methyleneoxy (4'-CH 2 -O-2') BNA 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.
  • the present invention provides modified nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. Certain such modified nucleosides are known.
  • the sugar ring of a nucleoside may be modified at any position.
  • sugar modifications useful in this invention include, but are not limited to compounds comprising a sugar substituent group selected from: OH, F, O-alkyl, S-alkyl, N-alkyl, or O-alkyl-0-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cj 0 alkyl or C 2 to Ci 0 alkenyl and alkynyl. In certain such embodiments, such substituents are at the 2' position of the sugar.
  • modified nucleosides comprise a substituent at the 2' position of the sugar.
  • modified nucleosides suitable for use in the present invention are: 2- methoxyethoxy, 2'-O-methyl (2'-O- CH 3 ), 2'-fluoro (2'-F).
  • 2'-sugar substituent groups include: Ci to C 10 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , 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.
  • modifed nucleosides comprise a 2'-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000).
  • 2'-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as T- O- methyl, O-propyl, and 0-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, P., HeIv. Chim.
  • 2'-Sugar substituent groups are in either the arabino (up) position or ribo (down) position.
  • a 2'-arabino modification is 2'-F arabino (FANA). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • nucleosides suitable for use in the present invention have sugar surrogates such as cyclobutyl in place of the pentofuranosyl sugar.
  • sugar surrogates such as cyclobutyl in place of the pentofuranosyl sugar.
  • Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;
  • nucleosides suitable for use in the present invention comprise sugar surrogates, which replace the pentafuranose ring of an unmodified nucleoside.
  • sugar surrogates include but are not limited to substituted or unsubstituted tetrahydropyran rings, such as F- HNA.
  • modified tetrahydropyran nucleoside having the formula:
  • Bx is a heterocyclic base moiety; T k is a hydroxyl protecting group; L a is H, halogen, Ci-C 6 alkyl or substituted CpCe alkyl; Z c is O " or OE a ; Z d is OH, OE a or N(E a )(E b ); each E a and E b is, independently, alkyl or substituted alkyl; q» qj, qk, qi, q m> q n and q p are each, independently, H, Ci-C 6 alkyl, substituted C 1 -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; wherein each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ m , NJ 1n J n
  • tetrahydropyran nucleoside analogs having Formula F wherein q « qj 5 qi» qi > q m , q n and q p are each H. In certain embodiments, at least one of q,, c ⁇ , q k , q ls q m , q n and q p is other than H. In certain embodiments, at least one of q ; , q j , q k , q 1 ⁇ q m , q n and q p is methyl. In certain embodiments, tetrahydropyran nucleoside analogs having Formula F are provided wherein L a is F. In certain embodiments, L a is H.
  • tetrahydropyran nucleoside analogs having Formula F wherein Z c is O " and Z d is OH.
  • Z 0 is O(CH 2 ) 2 CN
  • Z d is N[CH 2 (CH 3 ) 2 ] 2
  • T k is 4,4'- dimethoxytrityl.
  • Z c is O(CH 2 ) 2 CN
  • Z d is N[CH 2 (CH 3 ) 2 ] 2
  • T k is 4,4'-dimethoxytrityl which provides a phosphoramidite at the 3 '-position.
  • 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, Swayze, E.E. andBhat, B., The medicinal Chemistry of Oligonucleotides in ANTISENSE DRUG TECHNOLOGY, Chapter 6, pages 143-182 (Crooke, S.T., ed., 2008); U.S. Patent No.
  • 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 al, 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.
  • 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.
  • 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; 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. III. Certain oligonucleotides
  • the present invention provides modified oligonucleotides.
  • modified oligonucleotides of the present invention comprise modified nucleosides.
  • modified oligonucleotides of the present invention comprise modified internucleoside linkages.
  • modified oligonucleotides of the present invention comprise modified nucleosides and modified internucleoside linkages.
  • nucleosides may be linked together using any internucleoside linkage.
  • the two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom.
  • Non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH 2 -N(CH 3 )-O-CH 2 -), thiodiester (-O-C(O)-S-), thionocarbamate (-O- C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH 2 -N(CH 3 )-N(CH 3 )-).
  • Oligonucleotides having non-phosphorus internucleoside 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.
  • internucleoside 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 internucleoside linkages are well known to those skilled in the art.
  • 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), ⁇ 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.
  • internucleoside linkage or "internucleoside linking group” is meant to include all manner of internucleoside linking groups known in the art including but not limited to, phosphorus containing internucleoside linking groups such as phosphodiester and phosphorothioate, and non-phosphorus containing internucleoside linking groups such as formacetyl and methyleneimino.
  • neutral internucleoside linkage is intended to include internucleoside linkages that are non-ionic.
  • Further neutral internucleoside 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 internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH 2 component parts.
  • the invention provides oligomeric compounds comprising oligonucleotides. In certain embodiments, the present invention provides oligomeric compounds including oligonucleotides of any of a variety of ranges of lengths. In certain embodiments, the invention provides oligomeric compounds comprising oligonucleotides consisting of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range.
  • X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X ⁇ Y.
  • the invention provides oligomeric compounds which comprise oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 22,
  • the oligomeric compound or oligonucleotide may, nonetheless further comprise additional other substituents.
  • an oligonucleotide consisting of 8-30 nucleosides excludes oligonucleotides having 31 nucleosides, but, unless otherwise indicated, such an oligonucleotide may further comprise, for example one or more conjugates, terminal groups, or other substituents.
  • terminal groups include, but are not limited to, terminal group nucleosides.
  • the terminal group nucleosides are differently modified than the terminal nucleoside of the oligonucleotide, thus distinguishing such terminal group nucleosides from the nucleosides of the oligonucleotide.
  • the present invention provides oligonucleotides comprising one or more regions having a particular nucleoside motif.
  • the 5 '-terminal nucleoside of a modified oligonucleotide of the present invention comprises a phosphorous moiety at the 5'-end.
  • the 5'-terminal nucleoside comprises a 2 '-modification.
  • the 2 '-modification of the 5'-terminal nucleoside is a cationic modification.
  • the 5'-terminal nucleoside comprises a 5'-modif ⁇ cation.
  • the 5 '-terminal nucleoside comprises a 2'-modification and a 5 '-modification.
  • the 5 '-terminal nucleoside of an oligonucleotide is a nucleoside of Formula ⁇ . In certain embodiments, the 5 '-terminal nucleoside is a nucleoside of Formula IV. In certain embodiments, the 5 '-terminal nucleoside is a nucleoside of Formula VI. In certain embodiments, the 5'- terminal nucleoside is a nucleoside of Formula VII. In certain embodiments, the 5 '-terminal nucleoside is a nucleoside of Formula VIII. In certain embodiments, the 5'-terminal nucleoside is a nucleoside of Formula XIII.
  • the 5'-terminal nucleoside is a nucleoside of Formula XTV. In certain embodiments, the two 5'-terminal nucleosides have Formula IX. In certain embodiments, the two 5'-terminal nucleosides have Formula X. In certain embodiments, the 5'-terminal nucleoside is a 5 '-stabilizing nucleoside. In certain embodiments, the modifications of the 5'-terminal nucleoside stabilize the 5'-phosphate. In certain embodiments, oligonucleotides comprising modifications of the 5 '-terminal nucleoside are resistant to exonucleases.
  • oligonucleotides comprising modifications of the 5 '-terminal nucleoside have improved antisense properties. In certain such embodiments, oligonucleotides comprising modifications of the 5 '-terminal nucleoside have improved association with members of the RISC pathway. In certain embodiments, oligonucleotides comprising modifications of the 5 '-terminal nucleoside have improved affinity for Ago2.
  • the 5 'terminal nucleoside is attached to a plurality of nucleosides by a modified linkage. In certain such embodiments, the 5 'terminal nucleoside is a plurality of nucleosides by a phosphorothioate linkage.
  • oligonucleotides of the present invention comprise one or more regions of alternating modifications. In certain embodiments, oligonucleotides comprise one or more regions of alternating nucleoside modifications. In certain embodiments, oligonucleotides comprise one or more regions of alternating linkage modifications. In certan embodiments, oligonucleotides comprise one or more regions of alternating nucleoside and linkage modifications.
  • oligonucleotides of the present invention comprise one or more regions of alternating 2'-F modified nucleosides and 2'-0Me modified nucleosides.
  • regions of alternating 2'F modified and 2'0Me modified nucleosides also comprise alternating linkages.
  • the linkages at the 3' end of the 2'-F modified nucleosides are phosphorothioate linkages.
  • the linkages at the 3 'end of the 2'OMe nucleosides are phosphodiester linkages.
  • such alternating regions are:
  • oligomeric compounds comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 such alternatig regions. Such regions may be contiguous or may be interupted by differently modified nucleosides or linkages.
  • one or more alternating regions in an alternating motif include more than a single nucleoside of a type.
  • oligomeric compounds of the present invention may include one or more regions of any of the following nucleoside motifs:
  • ABBABAABB ABBABAABB; ABABAA;
  • BABBAABBABABAA or ABABBAABBABABAA; wherein A is a nucleoside of a first type and B is a nucleoside of a second type.
  • a and B are each selected from 2'-F, 2'-OMe, BNA, DNA, and MOE.
  • A is DNA. In certain embodiments, B is 4'-CH 2 O-2'-BNA. In certain embodiments, A is DNA and B is 4'-CH 2 O-2'-BNA. In certain embodiments A is 4'-CH 2 O-2'-BNA. In 5 certain embodiments, B is DNA. In certain embodiments A is 4'-CH 2 O-2'-BNA and B is DNA. In certain embodiments, A is 2'-F. In certain embodiments, B is 2'-0Me. In certain embodiments, A is 2'-F and B is 2'-OMe. In certain embodiemtns, A is 2'-OMe. In certain embodiments, B is 2'-F.
  • A is 2'-0Me and B is 2'-F. In certain embodiments, A is DNA and B is 2'-0Me. In certain embodiments, A is 2'-0Me and B is DNA. 0 In certain embodiments, oligomeric compounds having such an alternating motif also comprise a 5' terminal nucleoside comprising a phosphate stabilizing modification. In certain embodiments, oligomeric compounds having such an alternating motif also comprise a 5' terminal nucleoside comprising a T- cationic modification. In certain embodiments, oligomeric compounds having such an alternating motif also comprise a 5' terminal nucleoside of formula II, IV, VI, VII, VIII, XIII, or XTV. In certain embodiments, oligomeric 5 compounds having such an alternating motif comprise a 5 ' terminal di-nucleoside of formula DC or X.
  • oligonucleotides of the present invention comprise a region having a 2-2-3 motif. Such regions comprises the following motif:
  • A is a first type of modifed nucleosde
  • B, C, D, and E are nucleosides that are differently modified than A, however, B, C, D, and E may have the same or different modifications as one another; w and z are from O to 15; x and y are from 1 to 15. 5
  • A is a 2'-0Me modified nucleoside.
  • B, C, D, and E are nucleosides that are differently modified than A, however, B, C, D, and E may have the same or different modifications as one another; w and z are from O to 15; x and y are from 1 to 15. 5
  • A is a 2'-0Me modified nucleoside.
  • A is a 2'-0Me modified nucleoside and B, C, D, and E are all 2'-F modified nucleosides.
  • the linkages of a 2-2-3 motif are all modifed linkages. In certain embodiments, the linkages are all phosphorothioate linkages. In certain embodiemtns, the linkages at the 3'- O end of each modification of the first type are phosphodiester.
  • Z is O.
  • the region of three nucleosides of the first type are at the 3'-end of the oligonucleotide. In certain embodiments, such region is at the 3'-end of the oligomeric compound, with no additional groups attached to the 3' end of the region of three nucleosides of the first type.
  • an oligomeric compound comprising an oligonucleotide where Z is O may comprise a terminal group attached to the 3 '-terminal nucleoside. Such terminal groups may include additional nucleosides. Such additional nucleosides are typically non-hybridizing nucleosides.
  • oligonucleotide may comprise two or more motifs.
  • oligomeric compounds may have nucleoside motifs as described in the table below.
  • the term “None” indicates that a particular feature is not present in the oligonucleotide.
  • “None” in the column labeled "5' motif/modification” indicates that the 5' end of the oligonucleotide comprises the first nucleoside of the central motif.
  • Oligomeric compounds having any of the various nucleoside motifs described herein may have any linkage motif.
  • the oligomeric compounds including but not limited to those described in the above table, may have a linkage motif selected from non-limiting the table below:
  • the lengths of the regions defined by a nucleoside motif and that of a linkage motif need not be the same.
  • the 3 'region in the nucleoside motif table above is 2 nucleosides
  • the 3 '-region of the linkage motif table above is 6-8 nucleosides.
  • Combining the tables results in an oligonucleotide having two 3 '-terminal MOE nucleosides and six to eight 3 '-terminal phosphorothioate linkages (so some of the linkages in the central region of the nucleoside motif are phosphorothioate as well).
  • nucleoside motifs and sequence motifs are combined to show five non-limiting examples in the table below.
  • the first column of the table lists nucleosides and linkages by position from Nl (the first nucleoside at the 5 '-end) to N20 (the 20 th position from the 5 '-end).
  • oligonucleotides of the present invention are longer than 20 nucleosides (the table is merely exemplary). Certain positions in the table recite the nucleoside or linkage "none" indicating that the oligonucleotide has no nucleoside at that position.
  • Column A represent an oligomeric compound consisting of 20 linked nucleosides, wherein the oligomeric compound comprises: a modified 5 '-terminal nucleoside of Formula II, IV, VI, VII, VIII, DC, X, X ⁇ i, or XTV; a region of alternating nucleosides; a region of alternating linkages; two 3 '-terminal MOE nucleosides, each of which comprises a uracil base; and a region of six phosphorothioate linkages at the 3'- end.
  • Column B represents an oligomeric compound consisting of 18 linked nucleosides, wherein the oligomeric compound comprises: a modified 5 '-terminal nucleoside of Formula II, IV, VI, VII, VIII, IX, X, XIII, or XTV; a 2-2-3 motif wherein the modified nucleoside of the 2-2-3 motif are 2 O-Me and the remaining nucleosides are all 2'-F; two 3 '-terminal MOE nucleosides, each of which comprises a uracil base; and a region of six phosphorothioate linkages at the 3 '-end.
  • Column C represents an oligomeric compound consisting of 20 linked nucleosides, wherein the oligomeric compound comprises: a modified 5'-terminal nucleoside of Formula II, IV, VI, VII, VIII, IX, X, XIII, or XTV; a region of uniformly modified 2'-F nucleosides; two 3 '-terminal MOE nucleosides, each of which comprises a uracil base; and wherein each internucleoside linkage is a phosphorothioate linkage.
  • Column D represents an oligomeric compound consisting of 20 linked nucleosides, wherein the oligomeric compound comprises: a modified 5 '-terminal nucleoside of Formula II, IV, VI, VII, VIII, EX, X, Xi ⁇ , or XTV; a region of alternating 2'-OMe/2'-F nucleosides; a region of uniform 2'F nucleosides; a region of alternating phosphorothioate/phosphodiester linkages; two 3 '-terminal MOE nucleosides, each of which comprises an adenine base; and a region of six phosphorothioate linkages at the 3 '-end.
  • Column E represents an oligomeric compound consisting of 17 linked nucleosides, wherein the oligomeric compound comprises: a modified 5 '-terminal nucleoside of Formula II, IV, VI, VII, VIII, IX, X, XIII, or XTV; a 2-2-3 motif wherein the modified nucleoside of the 2-2-3 motif are 2'F and the remaining nucleosides are all 2'-OMe; three 3'-terminal MOE nucleosides.
  • Column F represents an oligomeric compound consisting of 18 linked nucleosides, wherein the oligomeric compound comprises: a region of alternating 2'-OMe/2'-F nucleosides; a region of uniform 2'F nucleosides; a region of alternating phosphorothioate/phosphodiester linkages; two 3 '-terminal MOE nucleosides, one of which comprises a uracil base and the other of which comprises an adenine base; and a region of six phosphorothioate linkages at the 3 '-end.
  • lengths of oligomeric compounds can be easily manipulated by lengthening or shortening one or more of the described regions, without disrupting the motif.
  • oligomeric compounds comprise an oligonucleotide.
  • an oligomeric compound comprises an oligonucleotide and one or more conjugate and/or terminal groups. Such conjugate and/or terminal groups may be added to oligonucleotides having any of the chemical motifs discussed above.
  • an oligomeric compound comprising an oligonucleotide having region of alternating nucleosides may comprise a terminal group.
  • oligomeric compounds are modified by attachment of one or more conjugate groups.
  • conjugate groups modify one or more properties of the attached oligomeric compound including but not limited to pharmacodynamics, pharmacokinetics, stability, 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 conjugate linking moiety or conjugate linking group to a parent compound such as an oligomeric compound, such as an oligonucleotide.
  • 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.
  • Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci.
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
  • a conjugate group comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, ( ⁇ S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • Oligonucleotide-drug conjugates and their preparation are described in U.S. Patent Application 09/334,130.
  • U.S. patents that teach the preparation of oligonucleotide conjugates include, but are not limited to, U.S.: 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
  • conjugate groups are directly attached to oligonucleotides in oligomeric compounds.
  • conjugate groups are attached to oligonucleotides by a conjugate linking group.
  • conjugate linking groups including, but not limited to, bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein.
  • Conjugate linking groups are useful for attachment of conjugate groups, such as chemical stabilizing groups, functional groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound.
  • a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups.
  • One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group.
  • the conjugate linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units.
  • functional groups that are routinely used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups.
  • bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.
  • conjugate linking moieties include pyrrolidine, 8-amino-3,6- dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and 6- aminohexanoic acid (AHEX or AHA).
  • ADO 8-amino-3,6- dioxaoctanoic acid
  • SMCC succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate
  • AHEX or AHA 6- aminohexanoic acid
  • linking groups include, but are not limited to, substituted Cl- ClO alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
  • Conjugate groups may be attached to either or both ends of an oligonucleotide (terminal conjugate groups) and/or at any internal position.
  • conjugate groups are at the 3 '-end of an oligonucleotide of an oligomeric compound. In certain embodiments, conjugate groups are near the 3 '-end. In certain embodiments, conjugates are attached at the 3 'end of an oligomeric compound, but before one or more terminal group nucleosides. In certain embodiments, conjugate groups are placed within a terminal group. Solely to illustrate such groups at a 3 '-end, and not to limit such groups, the following examples are provided.
  • conjugate groups are attached to a nucleoside.
  • a nucleoside may be incorporated into an oligomeric compound or oligonucleotide.
  • conjugated nucleotides may be incorporated into an oligonucleotide at the 5' terminal end.
  • conjugated nucleotides may be incorporated into an oligonucleotide at the 3' terminal end.
  • conjugated nucleotides may be incorporated into an oligonucleotide internally. Solely for illustration, and not to limit the conjugate or its placement, the following example shows oligonucleotides where each uracil nucleoside is, separately replaced with a conjugated thymidine nucleoside:
  • oligomeric compounds comprise terminal groups at one or both ends.
  • a terminal group may comprise any of the conjugate groups discussed above.
  • terminal groups may comprise additional nucleosides and/or inverted abasic nucleosides.
  • a terminal group is a stabilizing group.
  • oligomeric compounds comprise one or more terminal stabilizing group that enhances properties such as for example nuclease stability. Included in stabilizing groups are cap structures.
  • the terms "cap structure" or “terminal cap moiety,” as used herein, refer to chemical modifications, which can be attached to one or both of the termini of an oligomeric compound.
  • the cap can be present at the 5'-terminus (5'-cap) or at the 3'-terminus (3'-cap) or can be present on both termini.
  • the 5'-cap includes inverted abasic residue (moiety), 4',5'-methylene nucleotide; l-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L- nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl riboucleotide, 3 '-3 '-inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted nucleotide
  • Particularly suitable 3'-cap structures of the present invention include, for example 4',5'-methylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; l,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2- aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha- nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide;
  • one or more additional nucleosides is added to one or both terminal ends of an oligonucleotide of an oligomeric compound.
  • Such additional terminal nucleosides are referred to herein as terminal-group nucleosides.
  • terminal-group nucleosides are terminal (3' and/or 5') overhangs.
  • terminal-group nucleosides may or may not be complementary to a target nucleic acid.
  • terminal-group nucleosides are typically non- hybridizing.
  • the terminal-group nucleosides are typically added to provide a desired property other than hybridization with target nucleic acid. Nonetheless, the target may have complementary bases at the positions corresponding with the terminal-group nucleosides. Whether by design or accident, such complementarity of one or more terminal-group nucleosides does not alter their designation as terminal-group nucleosides.
  • the bases of terminal-group nucleosides are each selected from adenine (A), uracil (U), guanine (G), cytosine (C), thymine (T), and analogs thereof.
  • the bases of terminal-group nucleosides are each selected from adenine (A), uracil (U), guanine (G), cytosine (C), and thymine (T). In certain embodiments, the bases of terminal-group nucleosides are each selected from adenine (A), uracil (U), and thymine (T). In certain embodiments, the bases of terminal-group nucleosides are each selected from adenine (A) and thymine (T). In certain embodiments, the bases of terminal-group nucleosides are each adenine (A). In certain embodiments, the bases of terminal-group nucleosides are each thymine (T).
  • the bases of terminal-group nucleosides are each uracil (U). In certain embodiments, the bases of terminal-group nucleosides are each cytosine (C). In certain embodiments, the bases of terminal-group nucleosides are each guanine (G).
  • terminal-group nucleosides are sugar modified. In certain such embodiments, such additional nucleosides are 2'-modifed. In certain embodiments, the 2 '-modification of terminal-group nucleosides are selected from 2'-F, 2'-OMe, and 2'-MOE. In certain embodiments, terminal- group nucleosides are 2'-MOE modified. In certain embodiments, terminal-group nucleosides comprise 2'- MOE sugar moieties and adenine nucleobases (2'-MOE A nucleosides). In certain embodiments, terminal- group nucleosides comprise 2'-MOE sugar moieties and uracil nucleobases (2'-MOE U nucleosides).
  • terminal-group nucleosides comprises 2'-MOE sugar moieties and guanine nucleobases (2'-MOE G nucleosides). In certain embodiments, terminal-group nucleosides comprises 2'-MOE sugar moieties and thymine nucleobases (2'-MOE T nucleosides). In certain embodiments, terminal-group nucleosides comprises 2'-MOE sugar moieties and cytosine nucleobases (2'-MOE C nucleosides).
  • terminal-group nucleosides comprise bicyclic sugar moieties. In certain such embodiments, terminal-group nucleosides comprise LNA sugar moieties. In certain embodiments, terminal-group nucleosides comprise LNA sugar moieties and adenine nucleobases (LNA A nucleosides). In certain embodiments, terminal-group nucleosides comprise LNA sugar moieties and uracil nucleobases (LNA nucleosides). In certain embodiments, terminal-group nucleosides comprise LNA sugar moieties and guanine nucleobases (LNA G nucleosides).
  • terminal-group nucleosides comprise LNA sugar moieties and thymine nucleobases (LNA T nucleosides). In certain embodiments, terminal-group nucleosides comprise LNA sugar moieties and cytosine nucleobases (LNA C nucleosides). In certain embodiments, oligomeric compounds comprise 1-4 terminal-group nucleosides at the
  • oligomeric compounds comprise 1-3 terminal- group nucleosides at the 3 'end of the oligomeric compound. In certain embodiments, oligomeric compounds comprise 1-2 terminal-group nucleosides at the 3 'end of the oligomeric compound. In certain embodiments, oligomeric compounds comprise 2 terminal-group nucleosides at the 3 'end of the oligomeric compound. In certain embodiments, oligomeric compounds comprise 1 terminal-group nucleoside at the 3 'end of the oligomeric compound. In certain embodiments having two or more terminal-group nucleosides, the two or more terminal-group nucleosides all have the same modification type and the same base.
  • oligomeric compounds comprise a 3'-terminal group comprising 2 terminal- group nucleosides, wherein each terminal group nucleoside is a 2'-MOE T. In certain embodiments, oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a 2'-MOE A. In certain embodiments, oligomeric compounds comprise a 3'- terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a 2'- MOE U.
  • oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a 2'-MOE C. In certain embodiments, oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a 2'-MOE G.
  • oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal- group nucleosides, wherein each terminal group nucleoside is a LNA T. In certain embodiments, oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a LNA A. In certain embodiments, oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a LNA U. In certain embodiments, oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a LNA C. In certain embodiments, oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a LNA G.
  • oligomeric compounds of the present invention are antisense compounds.
  • the oligomeric compound is complementary to a target nucleic acid.
  • a target nucleic acid is an RNA.
  • a target nucleic acid is a non-coding RNA.
  • a target nucleic acid encodes a protein.
  • a target nucleic acid is selected from a mRNA, a pre-mRNA, a microRNA, a non-coding RNA, including small non- coding RNA, and a promoter-directed RNA.
  • oligomeric compounds are at least partially complementary to more than one target nucleic acid.
  • oligomeric compounds of the present invention may be microRNA mimics, which typically bind to multiple targets.
  • Antisense mechanisms include any mechanism involving the hybridization of an oligomeric compound with target nucleic acid, wherein the hybridization results in a biological effect. In certain embodiments, such hybridization results in either target nucleic acid degradation or occupancy with concomitant inhibition or stimulation of the cellular machinery involving, for example, translation, transcription, or splicing of the target nucleic acid.
  • RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like" elicit RNase H activity in mammalian cells. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of DNA-like oligonucleotide-mediated inhibition of gene expression.
  • Antisense mechanisms also include, without limitation RNAi mechanisms, which utilize the RISC pathway. Such RNAi mechanisms include, without limitation siRNA, ssRNA and microRNA mechanisms. Such mechanism include creation of a microRNA mimic and/or an anti-microRNA. Antisense mechanisms also include, without limitation, mechanisms that hybridize or mimic non- coding RNA other than microRNA or mRNA. Such non-coding RNA includes, but is not limited to promoter-directed RNA and short and long RNA that effects transcription or translation of one or more nucleic acids.
  • antisense compounds specifically hybridize when there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
  • stringent hybridization conditions or “stringent conditions” refers to conditions under which an antisense compound will hybridize to its target sequence, but to a minimal number of other sequences.
  • Stringent conditions are sequence-dependent and will be different in different circumstances, and "stringent conditions" under which antisense compounds hybridize to a target sequence are determined by the nature and composition of the antisense compounds and the assays in which they are being investigated. It is understood in the art that incorporation of nucleotide affinity modifications may allow for a greater number of mismatches compared to an unmodified compound. Similarly, certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences.
  • T m melting temperature
  • T m or ⁇ T m can be calculated by techniques that are familiar to one of ordinary skill in the art. For example, techniques described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allow one of ordinary skill in the art to evaluate nucleotide modifications for their ability to increase the melting temperature of an RNA:DNA duplex.
  • oligomeric compounds of the present invention are RNAi compounds. In certain embodiments, oligomeric compounds of the present invention are ssRNA compounds. In certain embodiments, oligomeric compounds of the present invention are paired with a second oligomeric compound to form an siRNA. In certain such embodiments, the second oligomeric compound is also an oligomeric compound of the present invention. In certain embodiments, the second oligomeric compound is any modified or unmodified nucleic acid. In certain embodiments, the oligomeric compound of the present invention is the antisense strand in an siRNA compound. In certain embodiments, the oligomeric compound of the present invention is the sense strand in an siRNA compound.
  • oligomeric compounds of the present invention are particularly suited for use as single-stranded antisense compounds.
  • such oligomeric compounds are single-stranded RNAi compounds.
  • such oligomeric compounds are ssRNA compounds or microRNA mimics.
  • Certain 5 '-terminal nucleosides described herein are suited for use in such single-stranded oligomeric compounds.
  • such 5 '-terminal nucleosides stabilize the 5 '-phosphorous moiety.
  • 5 '-terminal nucleosides of the present invention are resistant to nucleases.
  • the motifs of the present invention are particularly suited for use in single-stranded oligomeric compounds.
  • RNAi compounds Use of single-stranded RNAi compounds has been limited. In certain instances, single stranded RNAi compounds are quickly degraded and/or do not load efficiently into RISC. Certain compounds of the present invention possess properties superior to previously described ssRNAi compounds.
  • oligomeric compounds of the present invention are superior ssRNAi compounds in vitro.
  • the 5 '-terminal phosphorous moiety is stabilized.
  • the 5 '-nucleoside is resistant to nuclease cleavage.
  • the 5 '-terminal end loads efficiently into RISC.
  • the motif stabilizes the oligomeric compound.
  • the 3 '-terminal end of the oligomeric compound is stabilized.
  • Design of single-stranded RNAi compounds for use in cells and/or for use in vivo presents several challenges.
  • the compound must be chemically stable, resistant to nuclease degradation, capable of entering cells, capable of loading into RISC (e.g., binding Agol or Ago2), capable of hybridizing with a target nucleic acid, and not toxic to cells or animals.
  • RISC e.g., binding Agol or Ago2
  • a modification or motif that improves one such feature may worsen another feature, rendering a compound having such modification or motif unsuitable for use as an RNAi compound.
  • oligomeric compounds of the present invention combine modifications to provide single-stranded RNAi compounds that are active as single-stranded RNAi compounds.
  • a single-stranded oligomeric compound comprising a 5 '-phosphorous moiety is desired.
  • such 5 '-phosphorous moiety is necessary or useful for RNAi compounds, particularly, single-stranded RNAi compounds.
  • oligonucleotides in which the 5 '-phosphorous moiety and the 5 '-nucleoside have been stabilized are desired.
  • the present invention provides modified nucleosides that may be placed at the 5 '-end of an oligomeric compound, resulting in stabilized phosphorous and stabilized nucleoside.
  • the phosphorous moiety is resistant to removal in biological systems, relative to unmodified nucleosides and/or the 5 '-nucleoside is resistant to cleavage by nucleases.
  • such nucleosides are modified at one, at two or at all three of: the 2'-position, the 5'-position, and at the phosphorous moiety.
  • Such modified nucleosides may be incorporated at the 5 '-end of an oligomeric compound.
  • oligomeric compounds of the present invention may also be paired with a second strand to create a double-stranded oligomeric compound.
  • the second strand of the double-stranded duplex may or may not also be an oligomeric compound of the present invention.
  • oligomeric compounds of the present invention bind and/or activate one or more nucleases. In certain embodiments, such binding and/or activation ultimately results in antisense activity.
  • an oligomeric compound of the invention interacts with a target nucleic acid and with a nuclease, resulting in activation of the nuclease and cleavage of the target nucleic acid.
  • an oligomeric compound of the invention interacts with a target nucleic acid and with a nuclease, resulting in activation of the nuclease and inactivation of the target nucleic acid.
  • an oligomeric compound of the invention forms a duplex with a target nucleic acid and that duplex activates a nuclease, resulting in cleavage and/or inactivation of one or both of the oligomeric compound and the target nucleic acid.
  • an oligomeric compound of the invention binds and/or activates a nuclease and the bound and/or activated nuclease cleaves or inactivates a target nucleic acid.
  • Nucleases include, but are not limited to, ribonucleases (nucleases that specifically cleave ribonucleotides), double-strand nucleases (nucleases that specifically cleave one or both strands of a double- stranded duplex), and double-strand ribonucleases.
  • nucleases include, but are not limited to RNase H, an argonaute protein (including, but not limitied to Ago2), and dicer.
  • oligomeric compounds of the present invention activate RNase H.
  • an oligomeric compound of the present invention is sufficiently DNA-like to activate RNase H, resulting in cleavage of an RNA nucleic acid target.
  • the oligomeric compound comprises at least one region comprised of DNA or DNA-like nucleosides and one or more regions comprised of nucleosides that are otherwise modified.
  • such otherwise modified nucleosides increase stability of the oligomeric compound and/or its affinity for the target nucleic acid. Certain such oligomeric compounds posses a desirable combination of properties.
  • certain such compounds by virtue of the DNA or DNA-like region, are able to support RNase H activity to cleave a target nucleic acid; and by virtue of the otherwise modified nucleosides, have enhanced affinity for the target nucleic acid and/or enhanced stability (including resistance to single- strand-specific nucleases).
  • such otherwise modified nucleosides result in oligomeric compounds having desired properties, such as metabolic profile and/or pharmacologic profile.
  • oligomeric compounds of the present invention interact with an argonaute protein (Ago).
  • Ago argonaute protein
  • such oligomeric compounds first enter the RISC pathway by interacting with another member of the pathway (e.g., dicer).
  • oligomeric compounds first enter the RISC pathway by interacting with Ago.
  • such interaction ultimately results in antisense activity.
  • the invention provides methods of activating Ago comprising contacting Ago with an oligomeric compound.
  • such oligomeric compounds comprise a modified 5 '-phosphate group.
  • the invention provides methods of modulating the expression or amount of a target nucleic acid in a cell comprising contacting the cell with an oligomeric compound capable of activating Ago, ultimately resulting in cleavage of the target nucleic acid.
  • the cell is in an animal.
  • the cell is in vitro.
  • the methods are performed in the presence of manganese.
  • the manganese is endogenous.
  • the methods are performed in the absence of magnesium.
  • the Ago is endogenous to the cell.
  • the cell is in an animal.
  • the Ago is human Ago.
  • the Ago is Ago2.
  • the Ago is human Ago2.
  • oligomeric compounds of the present invention interact with the enzyme dicer. In certain such embodiments, oligomeric compounds bind to dicer and/or are cleaved by dicer. In certain such embodiments, such interaction with dicer ultimately results in antisense activity. In certain embodiments, the dicer is human dicer. In certain embodiments, oligomeric compounds that interact with dicer are double-stranded oligomeric compounds. In certain embodiments, oligomeric compounds that interact with dicer are single-stranded oligomeric compounds.
  • any oligomeric compound described herein may be suitable as one or both strands of a dicer duplex.
  • each strand of the dicer duplex is an oligomeric compound of the present invention.
  • one strand of the dicer duplex is an oligomeric compound of the present invention and the other strand is any modified or unmodified oligomeric compound.
  • one or both strands of a dicer duplex comprises a nucleoside of Formula II, IV, VI, VII, VIII, IX, X, XHI, or XTV at the 5' end.
  • one strand of a dicer duplex is an antisense oligomeric compound and the other strand is its sense complement.
  • the dicer duplex comprises a 3 '-overhang at one or both ends. In certain embodiments, such overhangs are additional nucleosides. In certain embodiments, the dicer duplex comprises a 3 ' overhang on the sense oligonucleotide and not on the antisense oligonucleotide. In certain embodiments, the dicer duplex comprises a 3' overhang on the antisense oligonucleotide and not on the sense oligonucleotide. In certain embodiments, 3 Overhangs of a dicer duplex comprise 1-4 nucleosides. In certain embodiments, such overhangs comprise two nucleosides.
  • the nucleosides in the 3'- overhangs comprise purine nucleobases. In certain embodiments, the nucleosides in the 3' overhangs comprise adenine nucleobases. In certain embodiments, the nucleosides in the 3' overhangs comprise pyrimidines. In certain embodiments, dicer duplexes comprising 3 '-purine overhangs are more active as antisense compounds than dicer duplexes comprising 3' pyrimidine overhangs. In certain embodiments, oligomeric compounds of a dicer duplex comprise one or more 3' deoxy nucleosides. In certain such embodiments, the 3' deoxy nucleosides are dT nucleosides.
  • each strand of a dicer duplex comprises a phosphate moiety.
  • the antisense strand of a dicer duplex comprises a phosphate moiety and the sense strand of the dicer duplex does not comprise a phosphate moiety.
  • the sense strand of a dicer duplex comprises a phosphate moiety and the antisense strand of the dicer duplex does not comprise a phosphate moiety.
  • a dicer duplex does not comprise a phosphate moiety at the 3' end.
  • a dicer duplex is cleaved by dicer. In such embodiments, dicer duplexes do not comprise 2'-OMe modifications on the nucleosides at the cleavage site. In certain embodiments, such cleavage site nucleosides are RNA.
  • interaction of an oligomeric compound with dicer ultimately results in antisense activity.
  • dicer cleaves one or both strands of a double-stranded oligomeric compound and the resulting product enters the RISC pathway, ultimately resulting in antisense activity.
  • dicer does not cleave either strand of a double-stranded oligomeric compound, but nevertheless facilitates entry into the RISC pathway and ultimately results in antisense activity.
  • dicer cleaves a single-stranded oligomeric compound and the resulting product enters the RISC pathway, ultimately resulting in antisense activity.
  • dicer does not cleave the single- stranded oligomeric compound, but nevertheless facilitates entry into the RISC pathway and ultimately results in antisense activity.
  • the invention provides methods of activating dicer comprising contacting dicer with an oligomeric compound.
  • the dicer is in a cell.
  • the cell is in an animal. Dicer
  • oligomeric compounds of the present invention interact with the enzyme dicer. In certain such embodiments, oligomeric compounds bind to dicer and/or are cleaved by dicer. In certain such embodiments, such interaction with dicer ultimately results in antisense activity. In certain embodiments, the dicer is human dicer. In certain embodiments, oligomeric compounds that interact with dicer are double-stranded oligomeric compounds. In certain embodiments, oligomeric compounds that interact with dicer are single-stranded oligomeric compounds.
  • any oligomeric compound described herein may be suitable as one or both strands of a dicer duplex.
  • each strand of the dicer duplex is an oligomeric compound of the present invention.
  • one strand of the dicer duplex is an oligomeric compound of the present invention and the other strand is any modified or unmodified oligomeric compound.
  • one or both strands of a dicer duplex comprises a nucleoside of Formula II, IV, VI, VII, VIII, IX, X, XIH, or XIV at the 5'.
  • one strand of a dicer duplex is an antisense oligomeric compound and the other strand is its sense complement.
  • a dicer duplex comprises a first and second oligomeric compound wherein each oligomeric compound comprises an oligonucleotide consisting of 25 to 30 linked nucleosides. In certain such embodiments, each oligonucleotide of the dicer duplex consists of 27 linked nucleosides. In certain embodiments, the dicer duplex comprises a 3 '-overhang at one or both ends. In certain embodiments, such overhangs are additional nucleosides. In certain embodiments, the dicer duplex comprises a 3' overhang on the sense oligonucleotide and not on the antisense oligonucleotide.
  • the dicer duplex comprises a 3' overhang on the antisense oligonucleotide and not on the sense oligonucleotide.
  • 3 Overhangs of a dicer duplex comprise 1-4 nucleosides. In certain embodiments, such overhangs comprise two nucleosides.
  • 3 '-overhangs comprise purine nucleobases. In certain embodiments, 3 '-overhangs comprise adenine overhangs. In certain embodiments, 3 '-overhangs are pyrimidines.
  • dicer duplexes comprising 3 '-purine overhangs are more active as antisense compounds than dicer duplexes comprising 3'-pyrimidine overhangs.
  • oligomeric compounds of a dicer duplex comprise 3'-deoxy nucleosides.
  • the 3'-deoxy nucleosides are dT nucleosides.
  • each strand of a dicer duplex comprises phosphate moiety.
  • the antisense strand of a dicer duplex comprises a phosphate moiety and the sense strand of the dicer duplex does not comprises a phosphate moiety.
  • the sense strand of a dicer duplex comprises a phosphate moiety and the antisense strand of the dicer duplex does not comprises a phosphate moiety.
  • a dicer duplex does not comprise a phosphate moiety at the 3'- end.
  • a dicer duplex is cleaved by dicer. In such embodiments, dicer duplexes do not comprise 2'-OMe modifications at the nucleosides at the cleavage site. In certain embodiments, such cleavage site nucleosides are RNA.
  • a dicer duplex comprises a first oligomeric compound comprising an antisense oligonucleotide and a second oligomeric compound comprising a sense oligonucleotide; wherein the sense oligonucleotide comprises a 3' overhang consisting of two purine nucleosides and the antisense oligonucleotide comprises a 3 Overhang consisting of two adenosine or modified adenosine nucleosides; each of the sense and antisense oligonucleotides consists of 25 to 30 linked nucleosides, the 5'end of the antisense oligonucleotide comprises a phosphorous moiety, and wherein the dicer cleavage sites of the dicer duplex are not O-Me modified nucleosides.
  • the invention provides single-stranded oligomeric compounds that interact with dicer.
  • such single-stranded dicer compounds comprise a nucleoside of Formula II, IV, VI, V ⁇ , Vi ⁇ , IX, X, Xi ⁇ , or XrV.
  • single-stranded dicer compounds do not comprise a phosphorous moiety at the 3 '-end.
  • such single-stranded dicer compounds may comprise a 3 '-overhangs. In certain embodiments, such 3 '-overhangs are additional nucleosides.
  • such 3 '-overhangs comprise 1-4 additional nucleosides that are not complementary to a target nucleic acid and/or are differently modified from the adjacent 3' nucleoside of the oligomeric compound.
  • a single-stranded oligomeric compound comprises an antisense oligonucleotide having two 3 '-end overhang nucleosides wherein the overhang nucleosides are adenine or modified adenine nucleosides.
  • single stranded oligomeric compounds that interact with dicer comprise a nucleoside of Formula II, IV, VI, VII, VIII, IX, X, XIII, or XTV.
  • interaction of an oligomeric compound with dicer ultimately results in antisense activity.
  • dicer cleaves one or both strands of a double-stranded oligomeric compound and the resulting product enters the RISC pathway, ultimately resulting in antisense activity.
  • dicer does not cleave either strand of a double-stranded oligomeric compound, but nevertheless facilitates entry into the RISC pathway and ultimately results in antisense activity.
  • dicer cleaves a single-stranded oligomeric compound and the resulting product enters the RISC pathway, ultimately resulting in antisense activity.
  • dicer does not cleave the single- stranded oligomeric compound, but nevertheless facilitates entry into the RISC pathway and ultimately results in antisense activity.
  • the invention provides methods of activating dicer comprising contacting dicer with an oligomeric compound.
  • the dicer is in a cell.
  • the cell is in an animal. Ago
  • oligomeric compounds of the present invention interact with Ago.
  • such oligomeric compounds first enter the RISC pathway by interacting with another member of the pathway (e.g., dicer), hi certain embodiments, oligomeric compounds first enter the RISC pathway by interacting with Ago.
  • such interaction ultimately results in antisense activity, hi certain embodiments, the invention provides methods of activating Ago comprising contacting Ago with an oligomeric compound.
  • the Ago is in a cell. In certain such embodiments, the cell is in an animal.
  • a portion of an oligomeric compound is 100% identical to the nucleobase sequence of a microRNA, but the entire oligomeric compound is not fully identical to the microRNA.
  • the length of an oligomeric compound having a 100% identical portion is greater than the length of the microRNA.
  • a microRNA mimic consisting of 24 linked nucleosides, where the nucleobases at positions 1 through 23 are each identical to corresponding positions of a microRNA that is 23 nucleobases in length, has a 23 nucleoside portion that is 100% identical to the nucleobase sequence of the microRNA and has approximately 96% overall identity to the nucleobase sequence of the microRNA.
  • the nucleobase sequence of oligomeric compound is fully identical to the nucleobase sequence of a portion of a microRNA.
  • a single-stranded microRNA mimic consisting of 22 linked nucleosides, where the nucleobases of positions 1 through 22 are each identical to a corresponding position of a microRNA that is 23 nucleobases in length, is fully identical to a 22 nucleobase portion of the nucleobase sequence of the microRNA.
  • Such a single-stranded microRNA mimic has approximately 96% overall identity to the nucleobase sequence of the entire microRNA, and has 100% identity to a 22 nucleobase portion of the microRNA.
  • Oligomerization of modified and unmodified nucleosides and nucleotides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).
  • Oligomeric compounds provided herein can be conveniently and routinely made through the well- known technique of solid phase synthesis.
  • Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
  • the invention is not limited by the method of antisense compound synthesis.
  • 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 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.
  • employed in the methods described herein is a pharmaceutical composition comprising an antisense compound and a pharmaceutically acceptable diluent.
  • the pharmaceutically acceptable diluent is PBS.
  • 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.
  • preparations are made that include a polyamine compound or a lipid moiety complexed with a nucleic acid.
  • such preparations comprise one or more compounds each individually having a structure defined by formula (I) or a pharmaceutically acceptable salt thereof,
  • Such preparations are described in PCT publication WO/2008/042973, which is herein incorporated by reference in its entirety.
  • the invention provides oligomeric compounds comprising or consisting of antisense oligonucleotides.
  • an antisense oligonucleotide comprises a phosphate stabilizing nucleoside.
  • an antisense oligonucleotide comprises a phosphate stabilizing nucleoside at the 5 '-end.
  • a phosphate stabilizing nucleoside comprises a modified phosphate group and/or a modified sugar moiety.
  • an antisense oligonucleotide comprises a 5 '-stabilizing nucleotide.
  • the 5 '-stabilizing nucleoside comprises a modified sugar moiety.
  • the 5 '-end of an antisens compound comprises a phosphate stabilizing modification and a 5 '-stabilizing nucleoside.
  • a single modification results in both phosphate stabilization and nucleoside stabilization.
  • the phosphate stabilizing modification and the nucleoside stabilizing modification are different modifications.
  • tow or more modifications at the 5 '-end of an oligomeric compound together provide phosphate stabilization and nucleoside stabilization.
  • an antisense oligomeric compound comprises the following features selected from: a 5'-phosphate or 5'-modifed phosphate; a 5'-most nucleoside (position 1 nucleoside); a nucleoside second from the 5 '-end (position 2 nucleoside); a nucleoside third from the 5 '-end (position 3 nucleoside); a region having a nucleoside motif; a region having a linkage motif; a terminal group.
  • the 5 '-phosphate is selected from: unmodified phosphate, modified phosphate, phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphorothioate, phosphoramidate, alkylphosphonothioate, substituted alkylphosphonothioate, phosphorodithioate, thiophosphoramidate, and phosphotriester.
  • the 5'-phosphate is selected from: modified phosphate, phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphotriester, phosphorothioate, phosphorodithioate, thiophosphoramidate, and phosphoramidate.
  • the 5 '-phosphate is selected from: modified phosphate, phosphonate, alkylphosphonate, and substituted alkylphosphonate. In certain embodiments, the 5 '-phosphate is selected from 5'-deoxy-5'-thio phosphate, phosphoramidate, methylene phosphonate, mono-fluoro methylene phosphonate and di-fluoro methylene phosphonate.
  • the position 1 nucleoside comprises a modified sugar.
  • the sugar comprises a 5 '-modification.
  • the sugar of the position 1 nucleoside comprises a 2'-modification.
  • the sugar of the position 1 nucleoside comprises a 5 '-modification and a 2 '-modification.
  • the 5 '-modification of the sugar of the position 1 nucleoside is selected from 5 '-alkyl,5 '-substituted alkyl, 5'-olkoxy, 5'-substitued alkoxy, and 5 '-halogen.
  • the 5' modification of the sugar at position 1 is selected from 5'- alkyl and 5 '-substituted alkyl.
  • the modification is selected from methyl and ethyl.
  • the T- modification of the sugar of the position 1 nucleoside is selected from: F, -0-C 1 -C 1 O alkyl, -0-Ci-C 10 substituted alkyl, -O-(CH 2 ) 2 -O-CH 3 , -O(CH 2 ) 2 SCH 3 , -O-(CH 2 ) 2 -O-N(R m )(R n ), -0-Cm-C(O)-N(R n O(R n ), where each R n , and R n is, independently, H or substituted or unsubstituted Ci-Ci 0 alkyl, -O[(CH 2 ) n O] m CH 3 , - O(CH 2 ) n NH 2 , -O(CH 2 ) n CH 3 , -O(CH 2 ) n ONH 2 , -OCH 2 C(O)N(H)CH 3, -O(CH
  • the position 2 nucleoside comprises a 2'-modification.
  • the 2'-modification of the position 2 nucleoside is selected from halogen, alkyl, and substituted alkyl.
  • the 2 '-modification of the position 2 nucleoside is selected from 2'-F and T- alkyl.
  • the 2'-modification of the position 2 nucleoside is 2'-F.
  • the 2'-substitued of the position 2 nucleoside is an unmodified OH (as in naturally occurring RNA).
  • the position 3 nucleoside is a modified nucleoside. In certain embodiments, the position 3 nucleoside is a bicyclic nucleoside. In certain embodiments, the position 3 nucleoside comprises a sugar surrogate. In certain such embodiments, the sugar surrogate is a tetrahydropyran. In certain embodiments, the sugar of the position 3 nucleoside is a F-HNA.
  • an antisense oligomeric compound comprises an oligonucleotide comprising 10 to 30 linked nucleosides wherein the oligonucleotide comprises: a 5 '-terminal phosphate or modified phosphate: a position 1 modified nucleoside comprising a modified sugar moiety comprising: a 5'- modification; or a 2 '-modification; or both a 5'-modificaton and a 2'-modification; a position 2 nucleoside comprising a sugar moiety which is differently modified compared to the sugar moiety of the position 1 modified nucleoside; and from 1 to 4 3 '-terminal group nucleosides each comprising a 2'-modification; and wherein at least the seven 3 '-most internucleoside linkages are phosphorothioate linkages.
  • the 5 '-terminal modified phosphate is selected from: phosphonate, alkylphosphonate, aminoalkyl phosphonate, phosphorothioate, phosphoramidite, alkylphosphonothioate, phosphorodithioate, thiophosphoramidate, phosphotriester;
  • the5' -modification of the sugar moiety of the position 1 modified nucleoside is selected from 5'-alkyl and 5 '-halogen;
  • the 2'-modification of the sugar moiety of the position 1 modified nucleoside is selected from: halogen (including, but not limited to F), allyl, amino, azido, thio, O-allyl, -O-Q-Cio alkyl, -0-Ci-C 1O substituted alkyl, -OCF 3 , -O-(CH 2 ) 2 -O-CH 3 , -O(CH 2 ) 2 SCH 3 , -O-(CH 2 )
  • the sugar moiety of the position 2 nucleoside comprises a 2'-F.
  • such oligonucleotides comprises 8 to 20, 10 to 15, 11 to 14, or 12 to 13 phosphorothioate interaucleoside linkages overall. In certain embodiments, the remaining internucleoside linkages are phosphodiester. In certain embodiments, the eighth internucleoside linkage from the 3 'end of the oligonucleotide is a phosphodiester. In certain embodiments, the ninth internucleoside linkage from the 3' end is a phosphodiester. In certain embodiments, each internucleoside linkage is either a phosphorothioate or a phosphodiester linkage.
  • antisense oligomeric compounds have the features described in the following non-limiting table:
  • the third nucleoside from the 5'-end (position 3) is a modified nucleoside.
  • the nucleoside at position 3 comprises a sugar modification.
  • the sugar moiety of the position 3 nucleoside is a bicyclic nucleoside.
  • the position 3 nucleoside is a modified non-bicyclic nucleoside.
  • the position 3 nucleoside is selected from: F-HNA and 2'-OMe.
  • the present invention provides compounds and methods for reducing the amount or activity of a target nucleic acid.
  • the invention provides antisense compounds and methods.
  • the invention provides antisense compounds and methods based on activation of RNase H.
  • the invention provides RNAi compounds and methods.
  • an antisense compound that functions at least in part through RISC.
  • unmodified RNA whether single-stranded or double stranded is not suitable.
  • Single-stranded RNA is relatively unstable and double-stranded RNA does not easily enter cells.
  • the challenge has been to identify modifications and motifs that provide desirable properties, such as improved stability, without interfering with (and possibly even improving upon) the antisense activity of RNA through RNAi.
  • the present invention provides oligonucleotides having motifs (nucleoside motifs and/or linkage motifs) that result in improved properties. Certain such motifs result in single-stranded oligonucleotides with improved stability and/or cellular uptake properties while retaining antisense activity. For example, oligonucleotides having an alternating nucleoside motif and seven phosphorothioate linkages at to 3 '-terminal end have improved stability and activity.
  • RNAi compounds having motifs herein result in single-stranded RNAi compounds having desirable properties.
  • such oligonucleotides may be paired with a second strand to form a double-stranded RNAi compound.
  • the second strand of such double-stranded RNAi compounds may comprise a motif of the present invention, may comprise another motif of modifications or may be unmodified.
  • RNAi activity if but has much less RNAi activity if it lacks such 5 '-phosphate group.
  • the present inventors have recognized that in certain circumstances unmodified 5'-phophate groups may be unstable (either chemically or enzymatically). Accordingly, in certain circumstances, it is desirable to modify the oligonucleotide to stabilize the 5 '-phosphate. In certain embodiments, this is achieved by modifying the phosphate group. In certain embodiments, this is achieved by modifying the sugar of the 5 '-terminal nucleoside. In certain embodiments, this is achieved by modifying the phosphate group and the sugar.
  • the sugar is modified at the 5'-position, the 2'-position, or both the 5'-position and the 2'-position.
  • a phosphate stabilizing modification must not interfere with the ability of the oligonucleotide to interact with RISC pathway components (e.g., with Ago).
  • the invention provides oligonucleotides comprising a phosphate-stabilizing modification and a motif described herein.
  • such oligonucleotides are useful as single-stranded RNAi compounds having desirable properties.
  • such oligonucleotides may be paired with a second strand to form a double-stranded RNAi compound.
  • the second strand may comprise a motif of the present invention, may comprise another motif of modifications or may be unmodified RNA.
  • the target for such antisense compounds comprising a motif and/or 5 '-phosphate stabilizing modification of the present invention can be any naturally occurring nucleic acid.
  • the target is selected from: pre-mRNA, mRNA, non-coding RNA, small non-coding RNA, pd-RNA, and microRNA.
  • a target nucleic acid is a pre-RNA or a mRNA
  • the target may be the same as that of a naturally occurring micro-RNA (i.e., the oligonucleotide may be a microRNA mimic). In such embodiments, there may be more than one target mRNA.
  • the invention provides compounds and methods for antisense activity in a cell.
  • the cell is in an animal.
  • the animal is a human.
  • the invention provides methods of administering a compound of the present invention to an animal to modulate the amount or activity or function of one or more target nucleic acid.
  • oligonucleotides comprise one or more motifs of the present invention, but do not comprise a phosphate stabilizing modification. In certain embodiments, such oligonucleotides are useful for in vitro applications. In certain embodiments, such oligonucleotides are useful for in vivo applications where RISC activity is not required. For example, in certain embodiments, such oligonucleotides alter splicing of pre-mRNA.
  • RNA as required, in reality, those sequences may be modified with any combination of chemical modifications.
  • RNA or "DNA” to describe modified oligonucleotides is, in certain instances, arbitrary.
  • 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).
  • 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 is 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 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 is 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.
  • 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.
  • 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.
  • an antisense oligomeric compound having two non-hybridizing 3 '-terminal 2'-MOE modified nucleosides, but otherwise fully complementary to a target nucleic acid may be described as an oligonucleotide comprising a region of 2'- MOE-modif ⁇ ed nucleosides, wherein the oligonucleotide is less than 100% complementary to its target.
  • an oligomeric compound comprising: (1) an oligonucleotide that is 100% complementary to its nucleic acid target and (2) a terminal group wherein the terminal group comprises two 2'-MOE modified terminal-group nucleosides.
  • nucleoside phosphoramidites The preparation of nucleoside phosphoramidites is performed following procedures that are illustrated herein and in the art such as but not limited to US Patent 6,426,220 and published PCT WO 02/36743.
  • oligomeric compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.
  • Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as alkylated derivatives and those having phosphorothioate linkages.
  • the oligomeric compounds are recovered by precipitating with greater than 3 volumes of ethanol from a 1 M NH 4 OAc solution.
  • Phosphinate internucleoside linkages can be prepared as described in U.S. Patent 5,508,270.
  • Alkyl phosphonate internucleoside linkages can be prepared as described in U.S. Patent 4,469,863.
  • 3 '-Deoxy-3 '-methylene phosphonate internucleoside linkages can be prepared as described in U.S. Patents 5,610,289 or 5,625,050.
  • Phosphoramidite internucleoside linkages can be prepared as described in U.S. Patent, 5,256,775 or U.S. Patent 5,366,878.
  • Alkylphosphonothioate internucleoside linkages can be prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively).
  • 3'-Deoxy-3'-amino phosphoramidate internucleoside linkages can be prepared as described in U.S. Patent 5,476,925.
  • Phosphotriester internucleoside linkages can be prepared as described in U.S. Patent 5,023,243.
  • Borano phosphate internucleoside linkages can be prepared as described in U.S. Patents 5,130,302 and 5,177,198.
  • Formacetal and thioformacetal internucleoside linkages can be prepared as described in U.S. Patents 5,264,562 and 5,264,564.
  • Ethylene oxide internucleoside linkages can be prepared as described in U.S. Patent 5,223,618.
  • the oligomeric compounds including without limitation oligonucleotides and oligonucleosides, are recovered by precipitation out of 1 M NH 4 OAc with >3 volumes of ethanol. Synthesized oligomeric compounds are analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis is determined by the ratio of correct molecular weight relative to the -16 amu product (+/-32 +/-48).
  • oligomeric compounds are purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material are generally similar to those obtained with non-HPLC purified material.
  • Oligomeric compounds can be synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format.
  • Phosphodiester internucleoside linkages are afforded by oxidation with aqueous iodine.
  • Phosphorothioate internucleoside linkages are generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile.
  • Standard base-protected beta- cyanoethyl-diiso-propyl phosphoramidites can be purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, CA, or Pharmacia, Piscataway, NJ).
  • Non-standard nucleosides are synthesized as per standard or patented methods and can be functionalized as base protected beta-cyanoethyldiisopropyl phosphoramidites.
  • Oligomeric compounds can be cleaved from support and deprotected with concentrated NH 4 OH at elevated temperature (55-60 0 C) for 12-16 hours and the released product then dried in vacuo. The dried product is then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.
  • the concentration of oligomeric compounds in each well can be assessed by dilution of samples and UV absorption spectroscopy.
  • the full-length integrity of the individual products can be evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACETM MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACETM 5000, ABI 270). Base and backbone composition is confirmed by mass analysis of the oligomeric compounds utilizing electrospray- mass spectroscopy. All assay test plates are diluted from the master plate using single and multi-channel robotic pipettors. Plates are judged to be acceptable if at least 85% of the oligomeric compounds on the plate are at least 85% full length.
  • oligomeric compounds on target nucleic acid expression is tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. Cell lines derived from multiple tissues and species can be obtained from American Type Culture Collection (ATCC, Manassas, VA).
  • b.END cells The mouse brain endothelial cell line b.END was obtained from Dr. Werner Risau at the Max Plank Institute (Bad Nauheim, Germany). b.END cells are routinely cultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, CA).
  • Cells are routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Cells are seeded into 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, MA) at a density of approximately 3000 cells/well for uses including but not limited to oligomeric compound transfection experiments.
  • oligomeric compounds When cells reached 65-75% confluency, they are treated with one or more oligomeric compounds.
  • the oligomeric compound is mixed with LIPOFECTINTM Invitrogen Life Technologies, Carlsbad, CA) in Opti-MEMTM-1 reduced serum medium (Invitrogen Life Technologies, Carlsbad, CA) to achieve the desired concentration of the oligomeric compound(s) and a LIPOFECTINTM concentration of 2.5 or 3 ⁇ g/mL per 100 nM oligomeric compound(s).
  • This transfection mixture is incubated at room temperature for approximately 0.5 hours. For cells grown in 96-well plates, wells are washed once with 100 ⁇ L OPTI-MEMTM-1 and then treated with 130 ⁇ L of the transfection mixture.
  • Cells grown in 24-well plates or other standard tissue culture plates are treated similarly, using appropriate volumes of medium and oligomeric compound(s). Cells are treated and data are obtained in duplicate or triplicate. After approximately 4-7 hours of treatment at 37°C, the medium containing the transfection mixture is replaced with fresh culture medium. Cells are harvested 16-24 hours after treatment with oligomeric compound(s).
  • transfection reagents known in the art include, but are not limited to, CYTOFECTINTM, LIPOFECTAMINETM, OLIGOFECTAMINETM, and FUGENETM.
  • Other suitable transfection methods known in the art include, but are not limited to, electroporation.
  • Quantitation of target mRNA levels is accomplished by real-time quantitative PCR using the ABI PRISMTM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to manufacturer's instructions.
  • ABI PRISMTM 7600, 7700, or 7900 Sequence Detection System PE-Applied Biosystems, Foster City, CA
  • This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time.
  • PCR polymerase chain reaction
  • products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes.
  • a reporter dye e.g., FAM or JOE, obtained from either PE- Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA
  • a quencher dye e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA
  • TAMRA obtained from either PE-Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA
  • annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5'- exonuclease activity of Taq polymerase.
  • cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated.
  • additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISMTM Sequence Detection System.
  • a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.
  • primer-probe sets specific to the target gene being measured are evaluated for their ability to be "multiplexed" with a GAPDH amplification reaction.
  • multiplexing both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample.
  • mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only ("single-plexing"), or both (multiplexing).
  • RT and PCR reagents are obtained from Invitrogen Life Technologies (Carlsbad, CA).
  • RT real-time PCR is carried out by adding 20 ⁇ L PCR cocktail (2.5x PCR buffer minus MgCl 2 , 6.6 mM MgCl 2 , 375 ⁇ M each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5x ROX dye) to 96-well plates containing 30 ⁇ L total RNA solution (20-200 ng).
  • PCR cocktail 2.5x PCR buffer minus MgCl 2 , 6.6 mM MgCl 2 , 375 ⁇ M each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125
  • the RT reaction is carried out by incubation for 30 minutes at 48°C. Following a 10 minute incubation at 95°C to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol are carried out: 95°C for 15 seconds (denaturation) followed by 60 0 C for 1.5 minutes (annealing/extension).
  • Gene target quantities obtained by RT, real-time PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RIBOGREENTM (Molecular Probes, Inc. Eugene, OR).
  • GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately.
  • Total RNA is quantified using RiboGreenTM RNA quantification reagent (Molecular Probes, Inc. Eugene, OR). Methods of RNA quantification by RIBOGREENTM are taught in Jones, L.J., et al, (Analytical Biochemistry, 1998, 265, 368-374). In this assay, 170 ⁇ L of RIBOGREENTM working reagent (RIBOGREENTM reagent diluted 1 :350 in
  • 1OmM Tris-HCl, 1 mM EDTA, pH 7.5 is pipetted into a 96-well plate containing 30 ⁇ L purified, cellular RNA.
  • the plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485nm and emission at 530nm.
  • Antisense modulation of a target expression can be assayed in a variety of ways known in the art.
  • a target mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR.
  • Real-time quantitative PCR is presently desired.
  • RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.
  • One method of RNA analysis of the present disclosure is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art.
  • Northern blot analysis is also routine in the art.
  • Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISMTM 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, CA and used according to manufacturer's instructions.
  • Protein levels of a target can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS).
  • Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp.
  • Enzyme-linked immunosorbent assays are standard in the art and can be found at, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.
  • the oligomeric compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition.
  • Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of a target in health and disease.
  • phenotypic assays which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, OR; PerkinElmer, Boston, MA), protein-based assays including enzymatic assays (Panvera, LLC, Madison, WI; BD Biosciences, Franklin Lakes, NJ; Oncogene Research Products, San Diego, CA), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, MI), triglyceride accumulation (Sigma-Aldrich, St.
  • MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies are treated with a target inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above.
  • treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.
  • Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest. Measurement of the expression of one or more of the genes of the cell after treatment is also used as an indicator of the efficacy or potency of the a target inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells. In vivo studies The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.
  • PoIy(A)+ mRNA is isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium is removed from the cells and each well is washed with 200 ⁇ L cold PBS. 60 ⁇ L lysis buffer ( 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) is added to each well, the plate is gently agitated and then incubated at room temperature for five minutes.
  • lysis buffer 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex
  • lysate is transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine CA). Plates are incubated for 60 minutes at room temperature, washed 3 times with 200 ⁇ L of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate is blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 ⁇ L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70 0 C, is added to each well, the plate is incubated on a 90 0 C hot plate for 5 minutes, and the eluate is then transferred to a fresh 96-well plate.
  • wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate is blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes.
  • Total RNA is isolated using an RNEASY 96TM kit and buffers purchased from Qiagen Inc. (Valencia, CA) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium is removed from the cells and each well is washed with 200 ⁇ L cold PBS. 150 ⁇ L Buffer RLT is added to each well and the plate vigorously agitated for 20 seconds. 150 ⁇ L of 70% ethanol is then added to each well and the contents mixed by pipetting three times up and down. The samples are then transferred to the RNEASY 96TM well plate attached to a QIA VACTM manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum is applied for 1 minute.
  • RNA is then eluted by pipetting 140 ⁇ L of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.
  • the repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia CA). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.
  • Probes and primers may be designed to hybridize to a target sequence, using published sequence information.
  • primer-probe set was designed using published sequence information (GENBANKTM accession number U92436.1, SEQ ID NO: 1).
  • FAM-TTGCAGCAATTCACTGTAAAGCTGGAAAGG-TAMRA (SEQ ID NO: 4), where FAM is the fluorescent dye and TAMRA is the quencher dye.
  • Western blot analysis is carried out using standard methods.
  • Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ⁇ l/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting.
  • Appropriate primary antibody directed to a target is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGERTM (Molecular Dynamics, Sunnyvale CA).
  • the residue obtained was purified by silica gel column chromatography and eluted first with 50% EtOAc in hexanes and then with 5% methanol in CH 2 Cl 2 .
  • the product obtained was dissolved in a mixture of triethylamine trihydrofluoride (1.38 mL, 8.44 mmol) and triethylamine (0.58 mL, 4.22 mmol) in THF (8.4 mL). After 72 h the mixture was diluted with EtOAc (60 mL), washed with water (40 mL), saturated aqueous NaHCO 3 (40 mL) and brine (40 mL) then dried over Na 2 SO 4 , filtered and evaporated.
  • Compound 8 is prepared as per the procedures illustrated in Example 15.
  • Compound 22 is prepared according to the scheme illustrated above.
  • Compound 22 is incorporated into oligonucleotides according to standard solid phase synthesis procedures. Phosphorylation at the 5' end of oligonucleotides is achieved during synthesis by using Glen Research (Sterling, VA) chemical phosphorylation reagent.
  • Compound 14 is prepared as per the procedures illustrated in Example 16.
  • Compound 43 is prepared as per the procedures illustrated in Example 21.
  • Compound 43 is prepared as per the procedures illustrated in Example 21.
  • Compound 43 is prepared as per the procedures illustrated in Example 21.
  • Compound 42 is prepared as per the procedures illustrated in Example 21.
  • Compound 58 was prepared as per the procedures illustrated in Example 26.
  • Compound 61, diethyl-(difluoromethane)phosphonate is commercially available.
  • the preparation of Compound 67 was achieved as per the procedures illustrated in Example 27 and confirmed by spectral analysis, 1 HNMR and mass spectroscopy.
  • Example 28
  • Compound 65 was prepared as per the procedures illustrated in Example 27.
  • the preparation of Compound 69 was achieved as per illustrated in Example 28 and confirmed by spectral analysis, 1 HNMR and mass spectroscopy.
  • Compound 65 is prepared as per the procedures illustrated in Example 27.
  • Compound 65 is prepared as per the procedures illustrated in Example 27.
  • Compound 64 is prepared as per the procedures illustrated in Example 27.
  • Compound 80 is prepared according to the procedures illustrated in published U.S. Patent
  • Compound 87, 5'-amino-deoxythymidine is commercially available.
  • Compound 88 is prepared according to the method of Mag and Engels (Mag, M.; Engles, J. W. Nucleic Acids Res. 1989, i 7, 5973-5988).
  • Compound 93 is prepared according to the method of Jahn-Hofmann and Engels (Jahn- Hofmann, K.; Engles, J. W. Helvetica Chimica Acta 2004, 87, 2812-2828).
  • Compound 39 is prepared as per the procedures illustrated in Example 21.
  • Compound 100 is prepared according to the method published by Inoue, H. et al. Nucleic Acids Research 1987, 15, 6131-6148.
  • Compound 97 is prepared as per the procedures illustrated in Example 36.
  • Compound 80 is prepared according to the procedures published in U.S. Patent 5,969,116.
  • Compound 68 is prepared as per the procedures illustrated in Example 28.
  • Compound 80 is prepared according to the procedures published in U.S. Patent 5,969,116.
  • Compound 66 is prepared as per the procedures illustrated in Example 27.
  • Compound 100 is prepared according to the method published by Inoue, H. et al. Nucleic Acids Research 1987, 15, 6131-6148.
  • Compound 78 is prepared as per the procedures illustrated in Example 31.
  • Compound 114 is prepared according to procedures published by Ikeda, H. et al. Nucleic Acids Research 1998, 26, 2237-2244.
  • Compounds 96 and 98 are prepared as per the procedures illustrated in Example 36.
  • Compound 103 is prepared as per the procedures illustrated in Example 37.
  • Compounds 119, 120 and 121 are prepared as per the procedures illustrated in Example 41.
  • Compound 38 is prepared as per the procedures illustrated in Example 21.
  • Compound 126 is prepared as per the procedures illustrated in Example 43.
  • Compound 127 is prepared as per the procedures illustrated in Example 43.
  • Compound 132 is prepared as per the procedures illustrated in Example 44.
  • Compound 132 is prepared as per the procedures illustrated in Example 44.
  • Compound 140 is prepared as per the procedures illustrated in Example 45.
  • Example 53
  • the antisense activity of oligomeric compounds can be tested in vivo.
  • Five- to six-week old Balb/c mice (Jackson Laboratory, Bar Harbor, ME) are injected with modified ssRNA targeted to PTEN at doses of 80 mg/kg daily, 60 mg/kg daily, or 40 mg/kg twice daily for several days.
  • the mice are sacrificed 72 hours following the last administration.
  • Liver tissues are homogenized and mRNA levels are quantitated using real-time PCR using procedures illustrated herein for comparison to untreated control levels (%UTC).
  • Other modifications and motifs as disclosed herein are also amenable to in vivo testing.
  • liver transaminase levels alanine aminotranferease (ALT) and aspartate aminotransferase (AST), in serum are also measured relative to saline injected mice.
  • ALT alanine aminotranferease
  • AST aspartate aminotransferase
  • Each nucleoside is connected to the following nucleoside by a phosphodiester intemucleoside linkage except underlined nucleosides which are connected to the following nucleoside by a phosphorothioate intemucleoside linkage (going 5' to 3').
  • a "P" at the 5'-end indicates a 5'-phosphate group.
  • a "P s " at the 5'- end indicates a 5'-thiophosphate group.
  • Nucleosides followed by a subscript d, ef, f, m, e or x are sugar modified nucleosides.
  • a subscript "d” indicates a 2'-OCH 2 (CO)NH(CH 2 ) 2 N(CH3) 2 (DMAEAc)
  • subscript "ef ' indicates a 2'-OCH 2 CH 2 F (FEt) modified nucleoside
  • a subscript "f ' indicates a 2'-fluoro modified nucleoside
  • a subscript "m” indicates 2'-O-methyl modified nucleoside
  • a subscript "e” indicates a 2'- O(CH 2 ) 2 OCH 3 (MOE) modified nucleoside
  • a subscript R or S or Rd or Sd or x indicates one of the 5'- modif ⁇ ed nucleosides (R or S) or one of the 2', 5'-bis modified nucleosides listed below (Rd, Sd, Rb, Sb, Rc or Sc).
  • each modified nucleoside having an x after it will have the same sugar modification.
  • Gapped oligomeric compounds targeted to PTEN in vivo study
  • oligomeric compounds are synthesized and tested for their ability to reduce PTEN expression in vivo at doses of 20 and 60 mg/kg.
  • Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) are administered a single intraperitoneal (i.p) injection at either 20 or 60 mg/kg of a 2-10-2 gapped oligomer.
  • i.p intraperitoneal
  • a 5-10-5 gapped oligomer having 2'-O-MOE modified nucleosides or other modified nucleosides as provided herein in the wings is also included for comparison.
  • Other motifs as disclosed herein are also amenable to in vivo testing.
  • Each dose group will include four animals.
  • the mice are sacrificed 48 hours following the final administration to determine the PTEN mRNA levels in liver using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols.
  • PTEN mRNA levels are determined relative to total RNA (using Ribogreen), prior to normalization to saline-treated control.
  • the average % inhibition of mRNA expression for each treatment group, normalized to saline-injected control is determined.
  • ALT and AST Liver transaminase levels, alanine aminotranferease (ALT) and aspartate aminotransferase (AST), in serum are measured relative to saline injected mice.
  • Each unmodified nucleoside is a ⁇ -D-2'-deoxyribonucleoside.
  • Each internucleoside linkage is a phosphorothioate intemucleoside linkage.
  • a "P" at the 5'-end indicates a 5'-phosphate group.
  • me C indicates a 5 '-methyl cytosine nucleoside.
  • Each nucleoside having a subscript x is selected from the list at the end of Example 57, e.g., Rb, Sb, Rc, Sc, Rd and Sd. In general, each modified nucleoside having an x after it will have the same sugar modification but can have different bases.
  • oligomeric compounds were synthesized and tested for their ability to reduce PTEN expression over a range of doses.
  • Human HeLa cells were treated with either ISIS 447581 or ISIS 404320.
  • a dose comparison was evaluated with dose concentrations of .20, .62, 1.9, 5.5, 16.7 and 50 nM using methods described herein.
  • Expression levels of PTEN were determined using real- time PCR and normalized to RIBOGREENTM using methods described herein. The percent inhibition of
  • PTEN mRNA was determined. Resulting dose-response curves were used to determine the EC 50 . Tm's were assessed in 100 mM phosphate buffer, 0.1 mM EDTA, pH 7, at 260 nm using 4 ⁇ M modified oligomers and 4 ⁇ M complementary RNA. The EC 50 S are listed below.
  • Each nucleoside is connected to the following nucleoside by a phosphodiester internucleoside linkage except underlined nucleosides which are connected to the following nucleoside by a phosphorothioate internucleoside linkage (going 5' to 3')-
  • a "P" at the 5'-end indicates a 5'-phosphate group.
  • Nucleosides followed by a subscript f, m or e are sugar modified nucleosides.
  • a subscript "f ' indicates a 2'-fluoro modified nucleoside
  • a subscript "m” indicates 2'-O-methyl modified nucleoside
  • a subscript "e” indicates a 2'-O(CH 2 ) 2 ⁇ CH 3 (MOE) modified nucleoside
  • a subscript Rc indicates the 2', 5'-bis modified nucleoside listed in Example 57.
  • a serum stability assay is useful for evaluating the stability of oligomeric compounds in the presences of nucleases and other enzymes found in serum. For example, the stability of a 5 '-terminal phosphate group of an oligomeric compound can be evaluated by assessing the ability of the 5 '-terminal phosphate group to remain attached to the oligomeric compound in the presence of serum. Accordingly, a serum stability assay was employed to evaluate the stability of modified ssRNAs having a 5 '-terminal phosphate group.
  • ssRNAs having a 5 '-terminal phosphate group (10 ⁇ M) were dissolved in 95% of fresh mouse serum and incubated at 37 0 C. Aliquots of serum (100 ⁇ L) were removed after 0, 1, 3, 6 or 24 hours of incubation times. The serum samples were immediately quenched and snap frozen. The samples were extracted by the strong anion exchange (SAX) and octadecylsilyl (C- 18) columns. For each incubation time, the amount of full length modified ssRNA having a 5 '-terminal phosphate group was determined by LC/MS, and the half-life of the full length modified ssRNA having a 5' terminal phosphate group was calculated. The results are expressed as half-time (Ti /2 ) in the table below. These data demonstrate that modifications to oligomeric compounds can improve the stability of the 5 '-terminal phosphate group.
  • SAX strong anion exchange
  • C- 18 octadecylsilyl
  • Each nucleoside is connected to the following nucleoside by a phosphodiester internucleoside linkage except underlined nucleosides which are connected to the following nucleoside by a phosphorothioate internucleoside linkage (going 5' to 3 1 ).
  • a "P" at the 5'-end indicates a 5'-phosphate group.
  • Nucleosides followed by a subscript d, e, f, R or Sf are sugar modified nucleosides.
  • a subscript "d” indicates a 2'-O- dimethylaminoethyl acetamide (DMAEAc) modified nucleoside
  • a subscript "e” indicates a 2'-0(CH 2 ⁇ OCH 3 (MOE) modified nucleoside
  • a subscript "f indicates a 2'-fluoro modified nucleoside
  • a subscript "R” indicates ( ⁇ ?)-5'-methyl-2 I -deoxyribonucleoside
  • a subscript Sf indicates the T, 5'-bis modified nucleoside listed below.
  • a series of nucleic acid duplexes comprising the compounds of the present invention and their complements can be designed.
  • the nucleobase sequence of the antisense strand of the duplex comprises at least a portion of an antisense oligonucleotide targeted to a target sequence as described herein.
  • the ends of the strands may be modified by the addition of one or more natural or modified nucleosides to form an overhang.
  • the sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus.
  • both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini.
  • a duplex comprising an antisense strand having the sequence
  • a duplex comprising an antisense strand having the same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 10) may be prepared with blunt ends (no single stranded overhang) as shown:
  • RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, CO). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 ⁇ M. Once diluted, 30 ⁇ L of each strand is combined with 15 ⁇ L of a 5X solution of annealing buffer. The final concentration of the buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 ⁇ L. This solution is incubated for 1 minute at 90 0 C and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37 0 C at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 ⁇ M.
  • duplexed compounds are evaluated for their ability to modulate target mRNA levels.
  • they are treated with duplexed compounds of the invention.
  • OPTI-MEM- 1TM reduced-serum medium Gibco BRL
  • OPTI-MEM-1TM reduced-serum medium
  • LIPOFECTAMINE 2000TM Invitrogen Life Technologies, Carlsbad, CA
  • the medium is replaced with fresh medium.
  • Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by quantitative real-time PCR as described herein.
  • RNAs ssRNAs
  • AS antisense strands listed below were designed to target human PTEN, and each was also assayed as part of a duplex with the same sense strand (ISIS 341401, shown below) for their ability to reduce PTEN expression levels.
  • HeLa cells were treated with the single stranded or double stranded oligomeric compounds created with the antisense compounds shown below using methods described herein.
  • the IC 5O 1 S were calculated using the linear regression equation generated by plotting the normalized mRNA levels to the log of the concentrations used.
  • Each internucleoside linkage is a phosphodiester except that underlined nucleosides are linked to the following nucleoside by a phosphorothioate (going 5' to 3').
  • Each nucleoside not followed by a subscript is a ribonucleoside.
  • a "P" at the 5'-end indicates a 5'-phosphate group.
  • a “Py” at the 5'-end indicates a 5'- methylenephosphonate group, (PO(OH) 2 CH 2 -).
  • a "Pz” at the 5'-end indicates a 5'- difluoromethylenephosphonate group, (PO(OH) 2 CF 2 -).
  • Nucleosides followed by a subscript indicate modification as follows: subscript “d” indicates a 2'-O-dimethylaminoethyl acetamide (DMAEAc) modified nucleoside; subscript “e” indicates a 2'-O(CH 2 ) 2 OCH 3 (MOE) modified nucleoside, subscript “f indicates a 2'-fluoro modified nucleoside; subscript “m” indicates 2'-O-methyl modified nucleoside; and subscript “R” indicates a ( ⁇ ? y )-5'-methyl-2'-deoxyribonucleoside. Superscript “me” indicates a 5-methyl group on the pyrimidine base of the nucleoside. Nucleosides with subscripts "Rc” or “Sc” are shown below.
  • Modified ssRNAs and dsRNAs targeted to PTEN were designed as shown below.
  • Phosphorothioate internucleoside linkages are indicated by underlining. Modified nucleosides are indicated by a subscripted letter following the capital letter indicating the nucleoside. In particular, subscript "f” indicates 2'-fluoro; subscript “m” indicates 2'-O-methyl; and subscript “e” indicates 2'-O-methoxyethyl (MOE). For example U n , is a modified uridine having a 2'-OCH 3 group. Some of the strands have a 5'- phosphate group designated as "P-".
  • a dose response experiment was performed targeting PTEN in human HeLa cells to determine the effects of placement of sugar and internucleoside linkages within ssRNAs. More specifically, the modified ssRNAs were tested for their ability to reduce PTEN mRNA in cultured cells.
  • the modified ssRNAs are shown below, and contain2'-OMe and 2'-fluoro modified nucleosides, two 2'-0-MOE modified nucleosides at the 3'-terminus, and seven phosphorothioate linkages at the 3 '-terminus of the ssRNAs.
  • HeLa cells were treated with ssRNAs shown below at concentrations of 1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM, 20 nM and 50 nM using methods described herein. Levels of mRNA were determined using realtime PCR methods as described herein. The IC 5O for each ssRNA was determined. These data demonstrate that these modified ssRNA exhibit similar activity in decreasing target mRNA levels.
  • Phosphorothioate internucleoside linkages are indicated by underlining. Modified nucleosides are indicated by a subscripted letter following the capital letter indicating the nucleoside. In particular, subscript "f ' indicates 2'-fluoro; subscript “m” indicates 2'-O-methyl; and subscript “e” indicates 2'-O-methoxyethyl (MOE). For example, U f is a modified uridine having a 2'-fluoro group. Some of the strands have a 5'- phosphate group designated as "P-".

Abstract

The present invention provides oligomeric compounds and uses thereof. In certain embodiments, such oligomeric compounds are useful as antisense compounds. Certain such antisense compounds are useful as RNase H antisense compounds or as RNAi compounds.

Description

OLIGOMERIC COMPOUNDS AND METHODS
STATEMENT OF GOVERNMENT SUPPORT
This invention was made with United States Government support under contract #5R44GM076793- 03 awarded by the NIH. The United States Government may have certain rights in the invention.
SEQUENCE LISTBNfG
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 20091023_CHEM0055USWOSEQ.txt, created on October 23, 2009, which is 20 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
Field of the Invention
The present invention provides compounds and methods for modulating nucleic acids and proteins. Provided herein are modified nucleosides and oligomeric compounds prepared therefrom. In certain embodiments, modified nucleosides are provided having at least one 5'-substituent and a 2'-substituent, oligomeric compounds comprising at least one of these modified nucleosides and compositions comprising at least one of these oligomeric compounds. In some embodiments, the oligomeric compounds provided herein are expected to hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA. The oligomeric compounds are also expected to be useful as primers and/or probes in diagnostic applications.
Background of the Invention
Antisense compounds have been used to modulate target nucleic acids. Antisense compounds comprising a variety of modifications and motifs have been reported. In certain instances, such compounds are useful as research tools and as therapeutic agents. Certain double-stranded RNA-like compounds
(siRNAs) are known to inhibit protein expression in cells. Such double-stranded RNA compounds function, at least in part, through the RNA-inducing silencing complex (RISC). Certain single-stranded RNA-like compounds (ssRNAs) have also been reported to function at least in part through RISC.
Targeting disease-causing gene sequences was first suggested more than thirty years ago (Belikova et al., Tet. Lett., 1967, 37, 3557-3562), and antisense activity was demonstrated in cell culture more than a decade later (Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A., 1978, 75, 280-284). One advantage of antisense technology in the treatment of a disease or condition that stems from a disease-causing gene is that it is a direct genetic approach that has the ability to modulate (increase or decrease) the expression of specific disease-causing genes. Another advantage is that validation of a therapeutic target using antisense compounds results in direct and immediate discovery of the drug candidate; the antisense compound is the potential therapeutic agent.
Generally, the principle behind antisense technology is that an antisense compound hybridizes to a target nucleic acid and modulates gene expression activities or function, such as transcription or translation. The modulation of gene expression can be achieved by, for example, target degradation or occupancy-based inhibition. An example of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi generally refers to antisense- mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of targeted endogenous mRNA levels. An additional example of modulation of RNA target function by an occupancy-based mechanism is modulation of microRNA function. MicroRNAs are small non-coding RNAs that regulate the expression of protein-coding RNAs. The binding of an antisense compound to a microRNA prevents that microRNA from binding to its messenger RNA targets, and thus interferes with the function of the microRNA. Regardless of the specific mechanism, this sequence-specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of malignancies and other diseases. Antisense technology is an effective means for reducing the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications. Chemically modified nucleosides are routinely used for incorporation into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target RNA. In 1998, the antisense compound, Vitravene® (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, CA) was the first antisense drug to achieve marketing clearance from the U.S. Food and Drug Administration (FDA), and is currently a treatment of cytomegalovirus (CMV)-induced retinitis in ADDS patients.
New chemical modifications have improved the potency and efficacy of antisense compounds, uncovering the potential for oral delivery as well as enhancing subcutaneous administration, decreasing potential for side effects, and leading to improvements in patient convenience. Chemical modifications increasing potency of antisense compounds allow administration of lower doses, which reduces the potential for toxicity, as well as decreasing overall cost of therapy. Modifications increasing the resistance to degradation result in slower clearance from the body, allowing for less frequent dosing. Different types of chemical modifications can be combined in one compound to further optimize the compound's efficacy. The synthesis of 5'-substituted DNA and RNA derivatives and their incorporation into oligomeric compounds has been reported in the literature (Saha et al, J. Org. Chem., 1995, 60, 788-789; Wang et al, Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890; and Mikhailov et al, Nucleosides & Nucleotides, 1991, 10(1-3), 339-343; Leonid et al, 1995, 14(3-5), 901-905; and Eppacher et al, Helvetica Chimica Acta, 2004, 87, 3004-3020). The 5'-substituted monomers have also been made as the monophosphate with modified bases (Wang et al, Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337).
A genus of modified nucleosides including optional modification at a plurality of positions including the 5'-position and the 2'-position of the sugar ring and oligomeric compounds incorporating these modified nucleosides therein has been reported (see International Application Number: PCT/US94/02993, Published on October 13, 1994 as WO 94/22890).
The synthesis of 5'-CH2 substituted 2'-O-protected nucleosides and their incorporation into oligomers has been previously reported (see Wu et ah, Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al. Bioconjugate Chem. 1999, 10, 921-924).
Amide linked nucleoside dimers have been prepared for incorporation into oligonucleotides wherein the 3' linked nucleoside in the dimer (51 to 3') comprises a 2'-OCH3 and a 5'-(S)-CH3 (Mesmaeker et al, Synlett, 1997, 1287-1290).
A genus of 2'-substituted 5'-CH2 (or O) modified nucleosides and a discussion of incorporating them into oligonucleotides has been previously reported (see International Application Number: PCT/US92/01020, published on February 07, 1992 as WO 92/13869). The synthesis of modified 5'-methylene phosphonate monomers having 2'-substitution and their use to make modified antiviral dimers has been previously reported (see US Patent Application Number: 10/418,662, published on April 6, 2006 as US 2006/0074035).
There remains a long-felt need for agents that specifically regulate gene expression via antisense mechanisms. Disclosed herein are oligomeric compounds such as antisense compounds useful for modulating gene expression pathways, including those relying on mechanisms of action such as RNaseH, RNAi and dsRNA enzymes, as well as other antisense mechanisms based on target degradation or target occupancy. One having skill in the art, once armed with this disclosure will be able, without undue experimentation, to identify, prepare and exploit antisense compounds for these uses.
Summary of the Invention
In certain embodiments, provided herein are modified nucleosides having at least one 2' substituent group and either a 5' substituent group, a 5' phosphorus moiety or both a 5' substituent group and a 5' phosphorus moiety, oligomeric compounds that include such modified nucleosides and methods of using the oligomeric compounds. Also provided herein are intermediates and methods for preparing these modified nucleosides and oligomeric compounds. In certain embodiments, modified nucleosides are provided that are 5'-mono (R, S or mixed) or bis substituted and 2'-O-substituted, that can be incorporated into oligomeric compounds. The modified nucleosides provided herein are expected to be useful for enhancing one or more properties of the oligomeric compounds they are incorporated into such as for example nuclease resistance. In certain embodiments, the oligomeric compounds and compositions provided herein that incorporate one or more of these modified nucleosides are expected to hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA. The oligomeric compounds are also expected to be useful as primers and probes in diagnostic applications.
The variables are defined individually in further detail herein. It is to be understood that the modified nucleosides and oligomeric compounds provided herein include all combinations of the embodiments disclosed and variables defined herein.
In certain embodiments, the invention provides oligomeric compounds comprising an oligonucleotide comprising a nucleoside having Formula VII:
Figure imgf000005_0001
Vϋ wherein:
Bx is a heterocyclic base moiety; T3 is a phosphorus moiety;
T4 is an internucleoside linking group attaching the nucleoside of Formula I to the remainder of the oligonucleotide; and each of qi and q2 is, independently selected from H, Ci-C6 alkyl, C2-C6 alkenyl, C2-
C6 alkynyl, substituted CpC6 alkyl, substituted Q-C6 alkenyl and substituted C2-C6 alkynyl;
Xi is S, NRi6, or CRioRπ wherein each Ri0 and Rn is, independently, H, F, Ci-C6 haloalkyl , or Ci-C6 alkyl; and
Ri is selected from a halogen, X2-V, and 0-X4; or each of qi and q2 is, independently, selected from H, Ci-C6 alkyl, C2-C6 alkenyl, C2- C6 alkynyl, substituted Ci-C6 alkyl, substituted Ci-C6 alkenyl and substituted C2-C6 alkynyl;
Xi is O, S, NRi6Rn, or CRi0Ri 1 wherein each Ri0 and Rn is, independently, H, F, Q- C6 haloalkyl , or Ci-C6 alkyl; and R, is X2-V; or each of qi and q2 is, independently, selected from Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted CpC6 alkenyl and substituted C2-C6 alkynyl;
Xi is O, S, NRi6Rn, or CRioRn wherein each Ri0 and Rn is, independently, H, F, Q- C6 haloalkyl , or Q-C6 alkyl; and
Ri is selected from halogen, X2-V, and 0-X4; wherein: X2 is O, S or CR7Rg wherein each R7 and R8 is, independently, H or Ci-C6 alkyl; V is selected from cholesterol, (CH2)2[O(CH2)2]tOCH3, where t is from 1-3, (CH2)2F, CH2COOH, CH2CONH2, CH2CONR5R61 CH2COOCH2CH3, CH2CONH(CH2)ΓS-R4 where i is from 1 to 10, CH2CONH(CH2)JNR5R6 where j is from 1 to 6, CH2CONH[(CH2)ki-N(H)]kr(CH2)kiNH2 where each Ic1 is independently from 2 to 4 and k2 is from 2 to 10, and aryl;
R4 is selected from H, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted Ci-C6 alkenyl, substituted C2-C6 alkynyl, C6-CM aryl and athio protecting group;
R5 and R6 are each, independently, selected from H, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, and substituted C2-C6 alkynyl; Ri6 is selected from H, Ci-C6 alkyl, or substituted C]-C6 alkyl;
X4 is [C(Ra)(Rb)]n-[(C=O)mXc]k-Rd wherein each Ra and Rb is independently H or halogen; Xc is O, S, orN(Ei);
Rd is H, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted CrC6 alkenyl and substituted C2-C6 alkynyl or NE2E3; each Ei, E2, and E3 is independently H, Ci-C6 alkyl, or substituted CpC6 alkyl; n is 1 to 6; m is O or 1; and k is O or 1 ; and wherein each substituted group is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, OJi, NJiJ2, SJi, N3, OC(=O)Ji and CN, wherein each Ji and J2 is, independently, H or CrC6 alkyl; and J4 is hydrogen, or a protecting group.
In certain embodiments, the invention provides oligomeric compounds comprising an oligonucleotide comprising a nucleoside having Formula VII:
Figure imgf000006_0001
wherein:
Bx is a heterocyclic base moiety; Xi is O, S, NRi6Ri7, or CRioRπ wherein each Ri0 and Rn is, independently, H, F, or Ci-C6 alkyl, and each Ri6 and Rn is independently, H, CpC6 alkyl, substituted Ci-C6 alkyl, T3 is a phosphorus moiety; T4 is an internucleoside linking group attaching the nucleoside of Formula I to the remainder of the oligonucleotide;
Ri is selected from halogen, trifluoroalkoxy, azido, aminooxy, O-alkyl, S-alkyl, N(J4)-alkyl, O- alkenyl, S-alkenyl, N(J4)-alkenyl, O-alkynyl, S-alkynyl or N(J4)-alkynyl, O-alkoxy, and X2-V, wherein: X2 is O, S or CR7R8 wherein each R7 and R8 is, independently, H or C1-C6 alkyl;
V is selected from (CH2)2F, CH2COOH, CH2CONH2, CH2COOCH2CH3, CH2CONH(CH2)J-S-R4 where i is from 1 to 10, CH2CONH(CH2)JNR5R6 where j is from 1 to 6, and CH2CONH{(CH2)ki-N(H)}k2- (CH2XiNH2 where each ki is independently from 2 to 4 and k2 is from 2 to 10;
R4 is selected from H, CpC6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted Cj-C6 alkenyl, substituted C2-C6 alkynyl, C6-Ci4 aryl and a thio protecting group;
R5 and R6 are each, independently, selected from H, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, and substituted C2-C6 alkynyl; and each of q! and q2 is, independently, selected from H, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted Ci-C6 alkenyl and substituted C2-C6 alkynyl; provided that if each of qi and q2 is H, then: either:
Ri is selected from, trifluoroalkoxy, azido, aminooxy, S-alkyl, N(J4)-alkyl, O-alkenyl, S- alkenyl, N(J4)-alkenyl, O-alkynyl, S-alkynyl and N(J4)-alkynyl, and X2-V; and Xi selected from O, S, N, and CRi0Ri 1; or
Ri is selected from halogen, trifluoroalkoxy, azido, aminooxy, O-alkyl, S-alkyl, N(J4)-alkyl, O-alkenyl, S-alkenyl, N(J4)-alkenyl, O-alkynyl, S-alkynyl or N(J4)-alkynyl, O-alkoxy, and X2-V; and Xi is selected from S, N, or CRi0Ri 1, and wherein each substituted group is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, OJi, NJiJ2, SJi, N3, OC(=O)Ji and CN, wherein each Ji and J2 is, independently, H or Ci-C6 alkyl; and J4 is hydrogen, or a protecting group.
In certain such embodiments, Ri is selected from halogen, O-alkyl, O-haloalkyl, O-alkoxy. In certain embodiments, Ri is F. In certain embodiments, R 1 is 0-C2-C4 alkyl or haloalkyl. In certain embodiments,Ri is OCH3. In certain embodiments, Ri is O(CH2)2OCH3 In certain embodiments, Ri is FCH2CH3. In certain embodiments,Ri is (CH2)2[O(CH2)2]tOCH3, where t is from 1-3. In certain embodiments,Ri is selected from, trifluoroalkoxy, azido, aminooxy, S-alkyl, N(J4)-alkyl, O-alkenyl, S-alkenyl, N(J4)-alkenyl, O-alkynyl, S- alkynyl, N(J4)-alkynyl, and X2-V. In certain embodiments, Ri is X2-V. In certain embodiments, V is (CH2)2F. In certain embodiments, V is CH2CONH(CH2)J-S-R4. In certain embodiments,CH2CONH[(CH2)ki- N(H)]Ic2-(CH2XiNH2. In certain embodiments, V is CH2CONH-(CH2)3-N(H)-(CH2)4-N(H)-(CH2)3NH2. In certain embodiments, V is CH2CONH(CH2)JNR5R6. In certain embodiments, at least one of R5 and R6 is other than H. In certain embodiments, at least one of R5 and R6 is methyl. In certain embodiments, R5 is methyl and R6 is methyl. In certain embodiments, X2 is O. In certain embodiments, X2 is S. In certain embodiments, X2 is CR7R8. In certain embodiments, R7 and R8 are both H. In certain embodiments, at least one of qi and q2 is Ci-Cβ alkyl or substituted Ci-C6 alkyl. In certain embodiments, at least one of qi and q2 is CpC6 alkyl. In certain embodiments, at least one of qi and q2 is methyl. In certain embodiments, at least one of qi and q2 is H. In certain embodiments, one of qi and q2 is methyl and the other of qi and q2 is H. In certain embodiments, qi and q2 are each CpC6 alkyl or substituted Ci-C6 alkyl. In certain embodiments, Xi is O. In certain embodiments,Xi is S. In certain embodiments, Xi is CRioRn. In certain embodiments, Ri0 and Rn are both H. In certain embodiments, the phosphorus moiety is P(Ya)(Yb)(YC5) where Ya is O or S and each Yb and Y0 is, independently, selected from OH, SH, alkyl, alkoxy, substituted CrC6 alkyl and substituted CpC6 alkoxy. In certain embodiments, Ya is O and Yb and Yc are each OH.
In certain embodiments, the invention provides such oligomeric compounds comprising a nucleoside of Formula VIII:
Figure imgf000008_0001
In certain embodiments, such oligomeric compounds have the configuration:
Figure imgf000008_0002
In certain such embodiments, qi is methyl and q2 is H. In certain embodiments, qi is H and q2 is methyl.
In certain embodiments, the invention provides oligomeric compounds comprising a di-nucleoside of Formula IX:
Figure imgf000009_0001
wherein: each Bx is independently a heterocyclic base moiety; T4 is an internucleoside linking group attaching the nucleoside of Formula IV to the remainder of the oligonucleotide; each of qi and q2 is, independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2- C6alkynyl, substituted C1-C6 alkyl, substituted C]-C6 alkenyl and substituted C2-C6 alkynyl;
Xi is S, NRi6, or CRi0Ri i wherein each Ri0 and Rn is, independently, H, F, Q-C6 haloalkyl , or Ci-C6 alkyl; and
Ri is selected from a halogen, X2-V, and 0-X4; or each of qi and q2 is, independently, selected from H, Ci-C6 alkyl, C2-C6 alkenyl, C2- C6 alkynyl, substituted C]-C6 alkyl, substituted Q-C6 alkenyl and substituted C2-C6 alkynyl; Xi is O, S, NRi6Ri7, or CRi oRn wherein each R^ and Rn is, independently, H, F, Q-
C6 haloalkyl , or Cj-C6 alkyl; and
R] is X2-V; or each of qi and q2 is, independently, selected from Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted Ci-C6 alkenyl and substituted C2-C6 alkynyl;
Xi is O, S, NRJ6R17, or CR]0Rii wherein each Rj0 and Rn is, independently, H, F, Q- C6 haloalkyl , or Q-C6 alkyl; and
Ri is selected from halogen, X2-V, and 0-X4; wherein: X2 is O, S or CR7R8 wherein each R7 and Rg is, independently, H or Q-C6 alkyl; V is selected from cholesterol, (CH2)2[O(CH2)2]tOCH3, where t is from 1-3, (CH2)2F, CH2COOH, CH2CONH2, CH2CONR5R61 CH2COOCH2CH3, CH2CONH(CH2)J-S-R4 where i is from 1 to 10, CH2CONH(CH2)JNR5R6 where j is from 1 to 6, and CH2CONHt(CH2X1-N(H)]Ia-(CH2X1NH2 where each k, is independently from 2 to 4 and k2 is from 2 to 10; R4 is selected from H, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted
Ci-C6 alkenyl, substituted C2-C6 alkynyl, C6-Cj4 aryl and a thio protecting group;
R5 and R6 are each, independently, selected from H, Ci-C6 alkyl, substituted CrC6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, and substituted C2-C6 alkynyl;
Ri6 is selected from H, Ci-C6 alkyl, or substituted Ci-C6 alkyl; X4 is [C(Ra)(Rb)]n-[(C=O)mXc]k-Rd wherein each Ra and Rb is independently H or halogen; Xc is O, S, or N(E1);
Rd is H, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted Q-C6 alkenyl and substituted C2-C6 alkynyl or NE2E3; each Ei, E2, and E3 is independently H, C]-C6 alkyl, or substituted Ci-C6 alkyl; n is 1 to 6; m is O or 1; and k is O or 1; and wherein X3 is OH or SH; Ya is O or S; each Yb and Yc is, independently, selected from OH, SH, alkyl, alkoxy, substituted C]-C6 alkyl and substituted Ci-C6 alkoxy;
R9 is selected from is selected from a halogen, X2-V, and 0-X4; wherein each substituted group is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, OJi, NJ]J2, SJj, N3, OC(=O)Ji and CN, wherein each Ji and J2 is, independently, H or Q-C6 alkyl; and J4 is hydrogen, or a protecting group.
In certain embodiments, Rj is F. In certain embodiments, Ri is OCH3. In certain embodiments, Ri is 0-C2-C4 alkyl or haloalkyl. In certain embodiments, Rj is O(CH2)2OCH3. In certain embodiments, Ri is FCH2CH3. In certain embodiments, Ri is (CH2)2[O(CH2)2]tOCH3, where t is from 1-3. In certain embodiments, Ri is selected from, trifluoroalkoxy, azido, aminooxy, S-alkyl, N(J4)-alkyl, O-alkenyl, S- alkenyl, N(J4)-alkenyl, O-alkynyl, S-alkynyl, N(J4)-alkynyl, and X2-V. In certain embodiments, Rj is X2-V. In certain embodiments, V is (CH2)2F. In certain embodiments, V is CH2CONH(CH2)J-S-R4 . In certain embodiments, V is CH2CONHf(CH2Xi-N(H)]Ia-(CH2XiNH2. In certain embodiments, V is CH2CONH- (CH2)3-N(H)-(CH2)4-N(H)-(CH2)3NH2. In certain embodiments, V is CH2CONH(CH2)JNR5R6. In certain such embodiments, j is 2. In certain embodiments, at least one of R5 and R6 is other than H. In certain embodiments, at least one of R5 and R6 is methyl. In certain embodiments, R5 is methyl and Re is methyl. In certain embodiments, X2 is O. In certain embodiments, X2 is S. In certain embodiments, X2 is CR7R8. In certain embodiments, R7 and R8 are both H. In certain embodiments, at least one of qi and q2 is Ci-Ce alkyl or substituted CrC6 alkyl. In certain embodiments, at least one of qi and q2 is Q-C6 alkyl. In certain embodiments, at least one of qi and q2 is methyl. In certain embodiments, at least one of qi and q2 is H. In certain embodiments, one of qi and q2 is methyl and the other of qi and q2 is H. In certain embodiments, qi and q2 are each Ci-C6 alkyl or substituted Ci-C6 alkyl. In certain embodiments, Xi is O. In certain embodiments, Xi is S. In certain embodiments, Xi is CRi0Ri i- In certain embodiments, Ri0 and Rn are both H. In certain embodiments, R9 is selected from F, OCH3 and O(CH2)2OCH3. In certain embodiments, Rg is OCH3. In certain embodiments, R9 is F. In certain embodiments, R9 is O(CH2)2θCH3. In certain embodiments, such di-nucleoside having Formula IX has Formula X:
Figure imgf000011_0001
In certain such embodiments, qi is methyl and q2 is H. In certain embodiments, qi is H and q2 is methyl.
In certain embodiments, the invention provides oligomeric compounds comprising an oligonucleotide comprising a nucleoside having Formula II:
Figure imgf000011_0002
wherein independently for each monomer of Formula II: Bx is a heterocyclic base moiety; A is O, S or N(Ri);
Ri is H, Ci-C6 alkyl or substituted Ci-C6 alkyl; one of T3 and T4 is an internucleoside linking group linking the monomer to the remainder of the oligomeric compound and the other of T3 and T4 is H, a protecting group, a phosphorus moiety, a 5' or 3'- terminal group or an internucleoside linking group linking the monomer to the oligomeric compound; one of Qi and Q2 is H, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl and the other of Qi and Q2 is Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
G, is O-[C(R2)(R3)]n-[(C=O)m-X]rZ or halogen; each R2 and R3 is, independently, H or halogen;
X is O, S orN(Ei);
Z is H, halogen, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(E2)(E3);
Ei, E2 and E3 are each, independently, H, Ci-C6 alkyl or substituted Ci-C6 alkyl; n is from 1 to about 6; m is 0 or 1; j is 0 or 1 ; each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJb N(Ji)(J2), =NJb SJb N3, CN, OC(=L)Ji, OC(=L)N(J,)(J2) and C(=L)N(Ji)(J2); L is O, S or NJ3; each Ji, J2 and J3 is, independently, H or Ci-C6 alkyl; and when j is 1 then Z is other than halogen or N(E2)(E3) and when A is O then Gi is other than halogen. In certain embodiments, each Bx is, independently, uracil, 5-thiazolo-uracil, 2-thio-uracil, 5- propynyl-uracil, thymine, 2'-thio-thymine, cytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl- cytosine, adenine, guanine, 2,6-diaminopurine, lH-pyrimido[5,4-b][l,4benzoxazin-2(3H)-one), IH- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one, 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)- one, 2H-pyrimido[4,5-b]indol-2-one or H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one. In certain embodiments, each Bx is, independently, uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine.
In certain embodiments, each Qi is H. In certain embodiments, each Q2 is H. In certain embodiments, each Qi and each Q2 are other than H. In certain embodiments, at least one of Qi and Q2 is substituted Q-C6 alkyl. In certain embodiments, such substituted Ci-C6 alkyl comprises at least one substituent group independently selected from halogen, C2-C6 alkenyl, OJi, NJiJ2 and CN, wherein each Ji and J2 is, independently, H or Ci-C6 alkyl. In certain embodiments, substituted Ci-C6 alkyl comprises at least one substituent group independently selected from fluoro and OCH3. In certain embodiments, at least one of Qi and Q2 is Ci-C6 alkyl. In certain embodiments, Qi is methyl. In certain embodiments, Q2 is methyl. In certain embodiments,^ is OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, O(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-SCH3, O(CH2)2-OCF3, O(CH2)3-N(R4)(R5), O(CH2)2- ON(R4)(R5), O(CH2)2-O(CH2)2-N(R4)(R5), OCH2Ct=O)-N(R4)(R5), OCH2C(=O)-N(R6)-(CH2)2-N(R4)(R5) or O(CH2)2-N(R6)-C(=NR7)[N(R4)(R5)] wherein R4, R5, R6 and R7 are each, independently, H or CrC6 alkyl. In certain embodiments^ is OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2- O(CH2)2-N(CH3)2, OCH2C(=O)-N(H)CH3, OCH2C(=O)-N(H)-(CH2)2-N(CH3)2 or OCH2-N(H)-C(=NH)NH2. In certain embodiments, is OCH3, O(CH2)2-OCH3, OCH2C(=O)-N(H)CH3 or OCH2C(=O)-N(H)-(CH2)2- N(CH3)2. In certain embodiments, Gj is F.
In certain embodiments, T3 is a phosphorus moiety. In certain embodiments, said phosphorus moiety has the formula:
Figure imgf000013_0002
wherein:
R3 and R0 are each, independently, OH, SH, CrC6 alkyl, substituted CpC6 alkyl, Ci-C6 alkoxy, substituted Ci-C6 alkoxy, amino or substituted amino; and Rb is O or S.
In certain embodiments, Ra and R0 are each OH. In certain embodiments, Ra and R0 are each OCH3. In certain embodiments, Ra and R0 are each OCH2CH3. In certain embodiments, Rb is O. In certain embodiments, Rb is S. In certain embodiments, each monomer of Formula II has the configuration:
Figure imgf000013_0001
In certain embodiments, such monomer of Formula II is at the 5'end of an oligomeric compound.
In certain embodiments, the invention provides oligomeric compounds comprising an oligonucleotide comprising a nucleoside having Formula IV:
Figure imgf000014_0001
wherein independently for each monomer of Formula IV: Bx is a heterocyclic base moiety; one ofT7 and T8 is an internucleoside linking group linking the monomer to the remainder of the oligomeric compound and the other of T7 and T8 is H, a hydroxyl protecting group, a phosphorus moiety, a 5' or 3 '-terminal group or an internucleoside linking group linking the monomer to the oligomeric compound; Qi, Q2, Q3 and Q4 are each, independently, H, halogen, C1-Ce alkyl, substituted CrC6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; G, is O-[C(R2)(R3)]n-[(C=O)m-X]j-Z or halogen; each R2 and R3 is, independently, H or halogen; X is O, S Or N(E1);
Z is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(E2)(E3);
E1, E2 and E3 are each, independently, H, Ci-C6 alkyl or substituted Ci-C6 alkyl; n is from 1 to about 6; m is O or l; j is O or 1; each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ1, N(J1)(J2), =NJU SJ,, N3, CN, OC(=L)Jb OC(=L)N(Ji)(J2) and Q=L)N(Ji)(J2); L is O, S or NJ3; each Ji, J2 and J3 is, independently, H or CpC6 alkyl; when j is 1 then Z is other than halogen or N(E2)(E3); and when Qi, Q2, Q3 and Q4 are each H or when Qi and Q2 are H and Q3 and Q4 are each F or when Q1 and Q2 are each H and one of Q3 and Q4 is H and the other of Q3 and Q4 is R9 then Gi is other than H, hydroxyl, OR9, halogen, CF3, CCl3, CHCl2 and CH2OH wherein R9 is alkyl, alkenyl, alkynyl, aryl or alkaryl.
In certain such embodiments, Bx is, independently, uracil, 5-thiazolo-uracil, 2-thio-uracil, 5- propynyl-uracil, thymine, 2'-thio-thymine, cytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl- cytosine, adenine, guanine, 2,6-diaminopurine, lH-pyrimido[5,4-b][l,4benzoxazin-2(3H)-one), IH- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one, 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)- one, 2H-pyrimido[4,5-b]indol-2-one or H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one. In certain embodiments, Bx is, independently, uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine.
In certain embodiments, Gi is OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, O(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-SCH3, O(CH2)2-OCF3, 0(CH2)J-N(R4)(R5), O(CH2)2- ON(R4)(R5), O(CH2)2-O(CH2)2-N(R4)(R5), OCH2CC=O)-N(R4)(R5), OCH2C(=O)-N(R6)-(CH2)2-N(R4)(R5) or O(CH2)2-N(R6)-C(=NR7)[N(R4)(R5)] wherein R4, R5, R6 and R7 are each, independently, H or C1-C6 alkyl. In certain embodiments, G1 is OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2- O(CH2)2-N(CH3)2, OCH2C(=O)-N(H)CH3, OCH2C(=O)-N(H)-(CH2)2-N(CH3)2 or OCH2-N(H)-C(=NH)NH2. In certain embodiments, G1 is OCH3, O(CH2)2-OCH3, OCH2C(=O)-N(H)CH3 or OCH2C(=O)-N(H)-(CH2)2- N(CH3)2. In certain embodiments,Gi is F.
In certain embodiments,T8 is a 3 '-terminal group. In certain embodiments, at least one of T7 and T8 is a conjugate group. In certain embodiments, one T7 is a phosphorus moiety. In certain embodiments, said phosphorus moiety has the formula:
Figure imgf000015_0001
wherein:
R3 and R0 are each, independently, OH, SH, CpC6 alkyl, substituted Q-C6 alkyl, Ci-C6 alkoxy, substituted Ci-C6 alkoxy, amino or substituted amino; and Rb is O or S.
In certain embodiments, R3 and R0 are each OH. In certain embodiments, Ra and R0 are each OCH3. In certain embodiments, R3 and R0 are each OCH2CH3. In certain embodiments, Rt is O. In certain embodiments, R1, is S.
In certain embodiments, each monomer of Formula IV one of Qi, Q2, Q3 and Q4 is substituted Ci-C6 alkyl. In certain embodiments, each monomer of Formula FV one of Qi, Q2, Q3 and Q4 is substituted Ci-C6 alkyl and the other three of Qi, Q2, Q3 and Q4 are H. In certain embodiments, said substituted CrC6 alkyl comprises at least one substituent group selected from halogen, C2-C6 alkenyl, OJi, NJ1J2 and CN, wherein each Ji and J2 is, independently, H or Ci-C6 alkyl. In certain embodiments, said substituted Ci-C6 alkyl comprises at least one substituent group selected from fluoro and OCH3.
In certain embodiments, each monomer of Formula IV one of Qi, Q2, Q3 and Q4 is Ci-C6 alkyl. In certain embodiments, for each monomer of Formula TV one of Qi and Q2 is CpC6 alkyl. In certain embodiments, one of Q3 and Q4 is Ci-C6 alkyl. In certain embodiments, said C1-C6 alkyl group is methyl. In certain embodiments, for each monomer of Formula IV three of Qi, Q2, Q3 and Q4 are H. In certain embodiments, monomer of Formula IV one of Qb Q2, Q3 and Q4 is F. In certain embodiments, two of Qi, Q2, Q3 and Q4 are F. In certain embodiments, monomer of Formula IV Qi and Q2 are each F. In certain embodiments, for each monomer of Formula IV Q3 and Q4 are each F. In certain embodiments, for each monomer of Formula IV, Q1, Q2, Q3 and Q4 are each F or H. hi certain embodiments, each monomer of Formula IV has the configuration:
Figure imgf000016_0001
In certain embodiments, the invention provides an oligomeric compound comprising a monomer of Formula IV at the 5 'end.
In certain embodiments, the invention provides oligomeric compounds comprising an oligonucleotide comprising a nucleoside at the 5'-end having Formula XIII:
Figure imgf000016_0002
wherein: Bx is a heterocyclic base moiety;
A is O, S OrN(R1);
Ri is H, Ci-C6 alkyl or substituted Q-C6 alkyl; T3 is a phosphorus moiety;
T4 is an internucleoside linking group linking the monomer to the remainder of the oligomeric compound; one of Qi and Q2 is H, CpC6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl and the other of Qi and Q2 is Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
Gi is O-[C(R2)(R3)]n-[(C=O)m-X]j-Z, 0-C1-C6 alkyl, O-CrC6 substituted alkyl, O-aryl, or halogen; each R2 and R3 is, independently, H or halogen;
X is O, S Or N(E1);
Z is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(E2)(E3);
E], E2 and E3 are each, independently, H, Q-C6 alkyl or substituted Ci-C6 alkyl; n is from 1 to about 6; m is 0 or 1; j is O or 1; each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ1, N(J1)(J2), =NJb SJ1, N3, CN, OCX=L)J1, OQ=L)N(J1)(J2) and CX=L)N(J1)(J2); L is O, S orNJ3; each J1, J2 and J3 is, independently, H or C1-C6 alkyl; and when j is 1 then Z is other than halogen or N(E2)(E3) and when A is O then G1 is other than halogen. In certain such embodiments, Bx is uracil, 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil, thymine, 2'-thio-thymine, cytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl-cytosine, adenine, guanine, 2,6-diaminopurine, lH-pyrimido[5,4-b][l,4benzoxazin-2(3H)-one), lH-pyrimido[5,4- b] [ 1 ,4]benzothiazin-2(3H)-one, 9-(2-aminoethoxy)-H-pyrimido[5,4-b] [ 1 ,4]benzoxazin-2(3H)-one, 2H- pyrimido[4,5-b]indol-2-one or H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one. In certain embodiments, Bx is uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine. In certain embodiments^ is H. In certain embodiments, Q2 is H. In certain embodiments, Qi and Q2 are each other than H. In certain embodiments, at least one OfQ1 and Q2 is substituted Ci-C6 alkyl. In certain embodiments, each substituted Ci-C6 alkyl comprises at least one substituent group independently selected from halogen, C2-C6 alkenyl, OJi, NJ]J2 and CN, wherein each Ji and J2 is, independently, H or CrC6 alkyl. In certain such embodiments, each substituted Ci-C6 alkyl comprises at least one substituent group independently selected from fluoro and OCH3. In certain embodiments, at least one of Qi and Q2 is Ci-C6 alkyl. In certain embodiments,Qi is methyl. In certain embodiments^ is methyl. In certain embodiments, said phosphorus moiety has the formula:
Figure imgf000017_0001
wherein: Ra and R0 are each, independently, OH, SH, CrC6 alkyl, substituted CrC6 alkyl, CrC6 alkoxy, substituted Ci-C6 alkoxy, amino or substituted amino; and Rb is O or S.
In certain embodiments, Ra and R0 are each OH. In certain embodiments, Ra and R0 are each OCH3. In certain embodiments, Ra and R0 are each OCH2CH3. In certain embodiments, Rb is O. In certain embodiments, Rb is S.
In certain embodimentsA is OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, O(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-SCH3, O(CH2)2-OCF3, 0(CH2VN(R4)(R5), O(CH2)2- ON(R4)(R5), O(CH2)2-O(CH2)2-N(R4)(R5), OCH2C(=O)-N(R,)(R5), OCH2C(=O)-N(R6)-(CH2)2-N(R4)(R5) or O(CH2)2-N(R6)-C(=NR7)[N(R4)(R5)] wherein R4, R5, R6 and R7 are each, independently, H or CrC6 alkyl. In certain embodiments, G1 is OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2- O(CH2)2-N(CH3)2, OCH2CC=O)-N(H)CH3, OCH2C(=O)-N(H)-(CH2)2-N(CH3)2 or OCH2-N(H)-C(=NH)NH2. In certain embodiments, G1 is OCH3, O(CH2)2-OCH3, OCH2C(=O)-N(H)CH3 or OCH2C(=O)-N(H)-(CH2)2- N(CH3)2. In certain embodiments, Gi is F. In certain embodiments,Gi is 0-Ci-C6 alkyl, 0-Ci-C6 substituted alkyl, O-aryl. In certain embodiments, Gi is -0-Ci-C6 substituted alkyl. In certain embodiments, Gi is - OCH2CH2OCH3. In certain embodiments, Gi is -OCH2CH2F.
In certain embodiments, the monomer of Formula XIII has the configuration:
Figure imgf000018_0001
In certain embodiments, the invention provides oligomeric compounds comprising an oligonucleotide comprising a nucleoside at the 5'-end having Formula XTV:
Figure imgf000018_0002
wherein:
Bx is a heterocyclic base moiety; T7 is a phosphorus moiety;
Tg is an internucleoside linking group linking the monomer to the remainder of the oligomeric compound;
Qi5 Q2, Q3 and Q4 are each, independently, H, halogen, CrC6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; Gi is O-[C(R2)(R3)]n-[(C=O)m-X]j-Z, 0-Ci-C6 alkyl, O-CrC6 substituted alkyl, O-aryl, or halogen; each R2 and R3 is, independently, H or halogen; each R2 and R3 is, independently, H or halogen; X is O, S OrN(E1); Z is H, halogen, CrC6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(E2)(E3);
Ei, E2 and E3 are each, independently, H, Cχ-C6 alkyl or substituted C1-C6 alkyl; n is from 1 to about 6; m is 0 or 1 ; j is 0 or 1 ; each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ1, N(Ji)(J2), =NJj, SJ1, N3, CN, OC(=L)Jl5 OC(=L)N(Ji)(J2) and C^L)N(J1)(J2);
L is O, S orNJ3; each Ji, h and J3 is, independently, H or Ci-C6 alkyl; when j is 1 then Z is other than halogen or N(E2)(E3); and when Qi, Q2, Q3 and Q4 are each H or when Q1 and Q2 are H and Q3 and Q4 are each F or when Qi and Q2 are each H and one OfQ3 and Q4 is H and the other OfQ3 and Q4 is R9 then Gi is other than H, hydroxyl, OR9, halogen, CF3, CCl3, CHCl2 and CH2OH wherein R9 is alkyl, alkenyl, alkynyl, aryl or alkaryl. In certain such embodiments, Bx is uracil, 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil, thymine, 2'-thio-thymine, cytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl-cytosine, adenine, guanine, 2,6-diaminopurine, lH-pyrimido[5,4-b][l,4benzoxazin-2(3H)-one), lH-pyrimido[5,4- b] [ 1 ,4]benzothiazin-2(3H)-one, 9-(2-aminoethoxy)-H-pyrimido[5,4-b] [ 1 ,4]benzoxazin-2(3H)-one, 2H- pyrimido[4,5-b]indol-2-one or H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one. In certain embodiments, Bx is uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine.
In certain embodiments, one of Qi, Q2, Q3 and Q4 is substituted C1-C6 alkyl. In certain embodiments, one OfQ1, Q2, Q3 and Q4 is substituted Ci-C6 alkyl and the other three Of Q1, Q2, Q3 and Q4 are H. In certain embodiments, said substituted Ci-C6 alkyl comprises at least one substituent group selected from halogen, C2- C6 alkenyl, OJi, NJiJ2 and CN, wherein each Ji and J2 is, independently, H or Ci-C6 alkyl. In certain embodiments, said substituted Q-C6 alkyl comprises at least one substituent group selected from fluoro and OCH3. In certain embodiments, one of Qi, Q2, Q3 and Q4 is Ci-C6 alkyl. In certain embodiments, one of Qi and Q2 is Ci-C6 alkyl. In certain embodiments, one OfQ3 and Q4 is C1-C6 alkyl. In certain embodiments, said C1-C6 alkyl group is methyl. In certain embodiments, three of Qi, Q2, Q3 and Q4 are H. In certain embodiments, one of Qi, Q2, Q3 and Q4 is F. In certain embodiments, two of Qi, Q2, Q3 and Q4 are F. In certain embodiments, Qi and Q2 are each F. In certain embodiments, Q3 and Q4 are each F. In certain embodiments, Qi, Q2, Q3 and Q4 are each F or H. In certain embodiments, Qi is H. In certain embodiments, Q2 is H. In certain embodiments, Q1 and Q2 are each other than H. In certain embodiments, at least one Of Q1 and Q2 is substituted C1-C6 alkyl. In certain embodiments, at least one Of Q1 and Q2 is Ci-C6 alkyl. In certain embodiments, Q1 is methyl. In certain embodiments, Q2 is methyl. In certain embodiments, said phosphorus moiety has the formula:
Figure imgf000020_0001
wherein:
Ra and R0 are each, independently, OH, SH, CpC6 alkyl, substituted CpC6 alkyl, Ci-C6 alkoxy, substituted Ci-C6 alkoxy, amino or substituted amino; and Rb is O or S.
In certain embodiments, Ra and R0 are each OH. In certain embodiments, Ra and R0 are each OCH3. In certain embodiments, Ra and R0 are each OCH2CH3. In certain embodiments, Rb is O. In certain embodiments, Rb is S. In certain embodiments,G, is OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, O(CH2)2F, OCH2CHF2,
OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-SCH3, O(CH2)2-OCF3, 0(CHz)3-N(R4)(R5), O(CH2)2- ON(R4)(R5), O(CH2)2-O(CH2)2-N(R4)(R5), OCH2C(=O)-N(R4)(R5), OCH2C(=O)-N(R6)-(CH2)2-N(R4)(R5) or O(CH2)2-N(R6)-C(=NR7)[N(R4)(R5)] wherein R4, R5, R6 and R7 are each, independently, H or C1-C6 alkyl. In certain embodiments, d is OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2- O(CH2)2-N(CH3)2, OCH2C(=O)-N(H)CH3, OCH2C(=O)-N(H)-(CH2)2-N(CH3)2 or OCH2-N(H)-C(=NH)NH2. In certain embodiments, d is OCH3, O(CH2)2-OCH3, OCH2C(=O)-N(H)CH3 or OCH2C(=O)-N(H)-(CH2)2- N(CH3)2. In certain embodiments, Gi is F. In certain embodiments, Gi is 0-Ci-C6 alkyl, 0-CpC6 substituted alkyl, O-aryl. In certain embodiments, Gi is -0-C]-C6 substituted alkyl. In certain embodiments, Gi is - OCH2CH2OCH3. In certain embodiments, G] is -OCH2CH2F. In certain embodiments, a monomer of Formula XTV has the configuration:
Figure imgf000020_0002
In certain embodiments, the invention provides an oligomeric compound comprising an oligonucleotide comprising a phosphate stabilizing nucleoside at the 5 '-end, wherein the phosphate stabilizing nucleoside comprises: a 5 '-terminal modified or unmodified phosphate; a modified sugar moiety comprising: a 5'- modification; or a 2 '-modification; or both a 5'-modificaton and a 2 '-modification; and a linking group linking the phosphate stabilizing nucleoside to the remainder of the oligonucleotide.
In certain such embodiments, the 5 '-terminal modified phosphate is selected from: phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphorothioate, phosphoramidate, alkylphosphonothioate, substituted alkylphosphonothioate, phosphorodithioate, thiophosphoramidate, and phosphotriester; the 5 '-modification of the sugar moiety of the phosphate stabilizing nucleoside is selected from 5'- alkyl and 5 '-halogen; the 2'-modification of the sugar moiety of the phosphate stabilizing nucleoside is selected from: halogen, allyl, amino, azido, thio, O-allyl, -0-C1-Ci0 alkyl, -0-C1-Ci0 substituted alkyl, -OCF3, -O-(CH2)2-O-CH3, - O(CH2)2SCH3, -0-(CHz)2-O-N(R1n)(R11), -O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted CrCio alkyl, -O[(CH2)nO]mCH3, -O(CH2)nNH2, -O(CH2)nCH3, - O(CH2)nONH2, -OCH2C(K))N(H)CH3, -O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10; Ci to Cio alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl.
In certain embodiments, the modified phosphate is selected from: phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphotriester, phosphorothioate, phosphorodithioate, thiophosphoramidate, and phosphoramidate.
In certain embodiments, the modified phosphate is selected from phosphonate, alkylphosphonate, and substituted alkylphosphonate. In certain embodiments, the 5 '-phosphate is selected from 5'-deoxy-5'-thio phosphate, phosphoramidate, methylene phosphonate, mono-fluoro methylene phosphonate and di-fluoro methylene phosphonate. In certain embodiments, the sugar moiety of the phosphate stabilizing nucleoside comprises a 5'- modificaton and a 2'-modification.
In certain of any of the above embodiments, the remainder of the oligonucleotide comprises at least one modified nucleoside. In certain embodiments, the oligomeric compound comprises a modified base. In certain embodiments, the oligomeric compound comprises a sugar surrogate. In certain embodiments, the sugar surrogate is a tetrahydropyran. In certain embodiments, the tetrahydropyran is F-HNA.
In certain embodiments, the remainder of the oligonucleotide comprises at least one nucleoside comprising a modified sugar. In certain embodiments, the at least one modified nucleoside comprising a modified sugar is selected from a bicyclic nucleoside and a 2'-modified nucleoside. In certain embodiments, the at least one modified nucleoside is a bicyclic nucleoside. In certain embodiments, the bicyclic nucleoside is a (4'-CH2-O-2') BNA nucleoside. In certain embodiments, the bicyclic nucleoside is a (4'-(CH2)2-O-2') BNA nucleoside. In certain embodiments, the bicyclic nucleoside is a (4'-C(CH3)H-O-2') BNA nucleoside. In certain embodiments, the at least one modified nucleoside is a 2'-modifed nucleoside. In certain embodiments, the at least one 2'-modified nucleoside is selected from a 2'-F nucleoside, a 2'-OCH3 nucleoside, and a 2'-O(CH2)2OCH3 nucleoside. In certain embodiments, the at least one 2'-modified nucleoside is a 2'-F nucleoside. In certain embodiments, the at least one 2'-modifϊed nucleoside is a T- OCH3 nucleoside. In certain embodiments, the at least one 2'-modified nucleoside is a 2'-O(CH2)2OCH3 nucleoside.
In certain embodiments, the remainder of the oligonucleotide comprises at least one unmodified nucleoside. In certain embodiments, the unmodified nucleoside is a ribonucleoside. In certain embodiments, the unmodified nucleoside is a deoxyribonucleoside.
In certain embodiments, the remainder of the oligomeric oligonucleotide comprises at least two modified nucleosides. In certain embodiments, the at least two modified nucleosides comprise the same modification. In certain embodiments, the at least two modified nucleosides comprise different modifications. In certain embodiments, at least one of the at least two modified nucleosides comprises a sugar surrogate. In certain embodiments, at least one of the at least two modified nucleosides comprises a 2'-modification. In certain embodiments, each of the at least two modified nucleosides is independently selected from 2'-F nucleosides, 2'-OCH3 nucleosides and 2'-O(CH2)2OCH3 nucleosides. In certain embodiments, each of the at least two modified nucleosides is a 2'-F nucleoside. In certain embodiments, each of the at least two modified nucleosides is a 2'-OCH3 nucleosides. In certain embodiments, each of the at least two modified nucleosides is a 2'-O(CH2)2OCH3 nucleoside. In certain embodiments, essentially every nucleoside of the oligomeric compound is a modified nucleoside. In certain embodiments, every nucleoside of the oligomeric compound is a modified nucleoside.
In certain embodiments, the remainder of the oligonucleotide comprises:
1-20 first-type regions, each first-type region independently comprising 1-20 contiguous nucleosides wherein each nucleoside of each first-type region comprises a first-type modification;
0-20 second-type regions, each second-type region independently comprising 1-20 contiguous nucleosides wherein each nucleoside of each second-type region comprises a second-type modification; and
0-20 third-type regions, each third-type region independently comprising 1-20 contiguous nucleosides wherein each nucleoside of each third-type region comprises a third-type modification; wherein the first-type modification, the second-type modification, and the third-type modification are each independently selected from 2'-F, 2'-OCH3, 2'-O(CH2)2OCH3, BNA, F-HNA, 2'-H and 2'-OH; provided that the first-type modification, the second-type modification, and the third-type modification are each different from one another.
In certain embodiments, the oligonucleotide comprises 2-20 first-type regions; 3-20 first-type regions; 4-20 first-type regions; 5-20 first-type regions; or 6-20 first-type regions. In certain embodiments, the oligonucleotide comprises 1-20 second-type regions; 2-20 second-type regions; 3-20 second-type regions; 4-20 second-type regions; or 5-20 second-type regions. In certain embodiments, the oligonucleotide comprisesl-20 third-type regions; 2-20 third-type regions; 3-20 third-type regions; 4-20 third-type regions; or 5-20 third-type regions. In certain embodiments, the oligomeric compound comprises a third-type region at the 3 '-end of the oligomeric compound, the oligomeric compound comprises a third-type region at the 3 '-end of the oligomeric compound the third-type region contains from 1 to 3 modified nucleosides and the third-type modification is 2'-O(CH2)2OCH3. In certain embodiments, the third same type region contains two modified nucleosides and the third-type modification is 2'-O(CH2)2θCH3. In certain embodiments, each first-type region contains from 1 to 5 modified nucleosides. In certain embodiments, each first-type region contains from 6 to 10 modified nucleosides. In certain embodiments, each first-type region contains from 11 to 15 modified nucleosides. In certain embodiments, each first-type region contains from 16 to 20 modified nucleosides.
In certain embodiments, the first-type modification is 2'-F. In certain embodiments, the first-type modification is 2'-OMe. In certain embodiments, the first-type modification is DNA. In certain embodiments, the first-type modification is 2'-O(CH2)2θCH3. In certain embodiments, the first-type modification is 4'-CH2- O-2'. In certain embodiments, the first-type modification is 4'-(CH2)2-O-2'. In certain embodiments, the first- type modification is 4'-C(CH3)H-O-2'. In certain embodiments, each second-type region contains from 1 to 5 modified nucleosides. In certain embodiments, each second-type region contains from 6 to 10 modified nucleosides. In certain embodiments, each second-type region contains from 11 to 15 modified nucleosides. In certain embodiments, each second-type region contains from 16 to 20 modified nucleosides. In certain embodiments, the second-type modification is 2'-F. In certain embodiments, the second-type modification is 2'-OMe. In certain embodiments, the second-type modification is DNA. In certain embodiments, the second - type modification is 2'-O(CH2)2OCH3. In certain embodiments, the second -type modification is 4'-CH2-O- 2'. In certain embodiments, the second -type modification is 4'-(CH2)2-O-2'. In certain embodiments, the second -type modification is 4'-C(CH3)H-O-2'. In certain embodiments, the oligomeric compound has an alternating motif wherein the first-type regions alternate with the second-type regions.
In certain embodiments, the invention provides oligomeric compounds wherein the remainder of the oligonucleotide comprises at least one region of nucleosides having a nucleoside motif: (A)n-(B)n-(A)n-(B)n, wherein:
A an B are differently modified nucleosides; and each n is independently selected from 1, 2, 3, 4, and 5.
In certain embodiments, A and B are each independently selected from a bicyclic and a 2 '-modified nucleoside. In certain embodiments, at least one of A and B is a bicyclic nucleoside. In certain embodiments, at least one of A and B is a (4'-CH2-O-2') BNA nucleoside. In certain embodiments, at least one of A and B is a (4'-(CH2)2-O-2') BNA nucleoside. In certain embodiments, at least one of A and B is a (4'-C(CH3)H-O- 2') BNA nucleoside. In certain embodiments, at least one of A and B is a 2'-modified nucleoside. In certain embodiments, the 2'-modified nucleoside is selected from: a 2'-F nucleoside, a 2'-OCH3 nucleoside, and a 2'-O(CH2)2OCH3 nucleoside. In certain embodiments, A and B are each independently selected from: a 2'-F nucleoside, a 2'-OCH3 nucleoside, a 2'-O(CH2)2OCH3 nucleoside, a (4'-CH2-O-2') BNA nucleoside, a (4'- (CH2)2-O-2') BNA nucleoside, a (4'-C(CH3)H-O-2') BNA nucleoside, a DNA nucleoside, an RNA nucleoside, and an F-HNA nucleoside. In certain embodiments, A and B are each independently selected from: a 2'-F nucleoside, a 2'-OCH3 nucleoside, a (4'-CH2-O-2') BNA nucleoside, a (4'-(CH2)2-O-2') BNA nucleoside, a (4'-C(CH3)H-O-2') BNA nucleoside, and a DNA nucleoside. In certain embodiments, one of A and B is a 2'-F nucleoside. In certain embodiments, one of A and B is a 2'-OCH3 nucleoside. In certain embodiments, one of A and B is a T- O(CH2)2OCH3 nucleoside. In certain embodiments, A is a 2'-F nucleoside and B is a 2'-OCH3 nucleoside. In certain embodiments, A is a 2'-OCH3 nucleoside and B is a 2'- F nucleoside. In certain embodiments, one of A and B is selected from a (4'-CH2-O-2') BNA nucleoside, a (4'-(CH2)2-O-25) BNA nucleoside, and a (4'-C(CH3)H-O-2') BNA nucleoside and the other of A and B is a DNA nucleoside.
In certain embodiments, the invention provides oligomeric compounds wherein the remainder of the oligonucleotide comprises a nucleoside motif: (A)X-(B)2-(A)Y-(B)2-(A)Z-(B)3 wherein
A is a nucleoside of a first type; B is a nucleoside of a second type;
X is 0-10;
Y is 1-10; and
Z is 1-10.
In certain embodiments, X is selected from 0, 1, 2 and 3. In certain embodiments, X is selected from
4, 5, 6 and 7. hi certain embodiments, Y is selected from 1, 2 and 3. In certain embodiments, Y is selected from 4, 5, 6 and 7. In certain embodiments, Z is selected from 1, 2 and 3. In certain embodiments, Z is selected from 4, 5, 6 and 7. In certain embodiments, A is a 2'-F nucleoside. In certain embodiments, B is a T- OCH3 nucleoside. hi certain embodiments, the invention provides oligomeric compounds of comprising a 3 '-region consisting of from 1 to 5 nucleosides at the 3 '-end of the oligomeric compound wherein: the nucleosides of the 3 '-region each comprises the same modification as one another; and the nucleosides of the 3'-region are modified differently than the last nucleoside adjacent to the 3'- region. In certain embodiments, the modification of the 3 '-region is different from any of the modifications of any of the other nucleosides of the oligomeric compound. In certain embodiments, the nucleosides of the 3 '-region are 2'-O(CH2)2θCH3 nucleosides. In certain embodiments, the 3 '-region consists of 2 nucleosides. In certain embodiments, the 3 '-region consists of 3 nucleosides. In certain embodiments, each nucleoside of the 3'-region comprises a uracil base. In certain embodiments, each nucleoside of the 3'-region comprises an adenine base. In certain embodiments, each nucleoside of the 3'-region comprises a thymine base.
In certain embodiments, the remainder of the oligonucleotide comprises a region of uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 2-20 contiguous uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 3-20 contiguous uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 4-20 contiguous uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 5-20 contiguous uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 6-20 contiguous uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 5-15 contiguous uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 6-15 contiguous uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 5-10 contiguous uniformly modified nucleosides. In certain embodiments, the region of uniformly modified nucleosides comprises 6-10 contiguous uniformly modified nucleosides. In certain embodiments, the remainder of the oligonucleotide comprises a region of alternating modified nucleosides and a region of uniformly modified nucleosides. In certain embodiments, the region of alternating nucleotides is 5' of the region of fully modified nucleosides. In certain embodiments, the region of alternating nucleotides is 3' of the region of fully modified nucleosides. In certain embodiments, the alternating region and the fully modified region are immediately adjacent to one another. In certain embodiments, the oligomeric compound has additional nucleosides between the alternating region and the fully modified region.
In certain embodiments, the remainder of the oligonucleotide comprises at least one region of nucleosides having a motif I:
Nf(PS)N1n(PO), wherein: Nf is a 2 ' -F nucleoside,
Nm is a 2'-OCH3 nucleoside PS is a phosphorothioate linking group; and PO is a phosphodiester linking group.
In certain embodiments, the oligomeric compound comprises at least 2, or 3, or 4, or 6, or 7, or 8, or 9, or 10 separate regions of nucleosides having the motif I. In certain embodiments, the invention provides oligomeric compounds comprising at least one region having a nucleoside motif selected from: AABBAA;
ABBABB;
AABAAB;
ABBABAABB;
ABABAA; AABABAB;
ABABAA;
ABBAABBABABAA;
BABBAABBABABAA; or
ABABBAABBABABAA; wherein A is a nucleoside of a first type and B is a nucleoside of a second type.
In certain embodiments, oligomeric compounds of the invention comprise one or more conjugate groups. In certain embodiments, oligomeric compounds of the invention consist of the oligonucleotide.
In certain embodiments, the invention provides oligomeric compounds comprising an oligonucleotide comprising a contiguous sequence of linked nucleosides wherein the sequence has the formula: 5'-(ZV(L-Q1-L-Q2X-(L-Q1)U-(L-Q3)V-(GVS' wherein: each L is an internucleoside linking group;
G is a conjugate or a linking group; a is 0 or 1; each OfQ1, Q2 and Q3 is, independently, a 2'-modified nucleoside having a 2'-substituent group selected from halogen, allyl, amino, azido, O-allyl, 0-Ci-C6 alkyl, OCF3, O-(CH2)2-O-CH3, O(CH2)2SCH3, 0- (CHy2-O-N(J5)(J6) and O-CH2-C(=O)-N(J5)(J6), where each J5 and J6 is, independently, H, an amino protecting group or substituted or unsubstituted Ci-C6 alkyl; provided that Qi, Q2 and Q3 are different from one another; t is from 4 to 8; u is 0 or 1;
Figure imgf000026_0001
3; w is 0 or 1; and
Z is a 5' stabilizing nucleoside.
In certain embodiments, w is 1. In certain embodiments, w is 0. In certain embodiments, Qi and Q2 is, independently, a 2'-modified nucleoside having a 2'-substituent group selected from halogen and 0-Ci-C6 alkyl. In certain embodiments, each Qi and Q2 is, independently, a 2'-modified nucleoside having a 2'- substituent group selected from F and O-methyl. In certain embodiments, each Q3 is a 2'-modified nucleoside having a 2 '-substituent group of 0-(CH2^-OCH3. In certain embodiments, a is O. In certain embodiments, v is 2. In certain embodiments, u is 0. In certain embodiments, u is 1.
In certain of any of the above embodiments, the oligonucleotide consists of 8-80 linked nucleoside; 8-26 linked nucleosides; 10-24 linked nucleosides; 16-22 linked nucleosides; 16-18 linked nucleosides; 19-22 linked nucleosides.
In certain of any of the above embodiments, the second nucleoside from the 5 '-end comprises a sugar moiety comprising a 2'-substituent selected from OH and a halogen. In certain embodiments, the second nucleoside from the 5'-end is a 2'-F modified nucleoside. In certain of any of the above embodiments, the oligomeric compound comprises at least one modified linking group. In certain embodiments, each internucleoside linking group is, independently, phosphodiester or phosphorothioate. In certain embodiments, the 5 '-most internucleoside linking group is a phosphorothioate linking group. In certain embodiments, at least one phosphorothioate region comprising at least two contiguous phosphorothioate linking groups. In certain embodiments, the at least one phosphorothioate region comprises from 3 to 12 contiguous phosphorothioate linking groups. In certain embodiments, the at least one phosphorothioate region comprises from 6 to 8 phosphorothioate linking groups. In certain embodiments, the at least one phosphorothioate region is located at the 3 '-end of the oligomeric compound. In certain embodiments, the at least one phosphorothioate region is located within 3 nucleosides of the 3 '-end of the oligomeric compound. In certain embodiments, the 7-9 internucleoside linkages at the 3 'end of the oligonucleotide are phosphorothioate linkages and the internucleoside linkage at the 5 '-end is a phosphorothioate linkage.
In certain embodiments, the invention provides oligomeric compounds comprising an oligonucleotide consisting of 10 to 30 linked nucleosides wherein:
(a) the nucleoside at the 5' end is a phosphate stabilizing nucleoside comprising: a 5 '-terminal modified or unmodified phosphate; and a modified sugar moiety comprising: a 5'- modification; or a 2 '-modification; or both a 5'-modificaton and a 2'-modification;
(b) the sugar moiety of the second nucleoside from the 5 '-end is selected from an unmodified 2'-OH sugar, and a modified sugar comprising a modification selected from: 2'-halogen, 2'O-alkyl, and 2'-O- substituted alkyl; and
(c) the first internucleoside linkage at the 5 '-end and the last seven internucleoside linkages at the 3 '-end are phosphorothioate linkages; and
(d) at least one internucleoside linkage is other than a phosphorothioate linkage.
In certain embodiments, the 5'-terminal modified phosphate is selected from: phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphorothioate, phosphoramidate, alkylphosphonothioate, substituted alkylphosphonothioate, phosphorodithioate, thiophosphoramidate, and phosphotriester; the 5 '-modification of the sugar moiety of the phosphate stabilizing nucleoside is selected from 5'- alkyl and 5 '-halogen; and the 2'-modification of the sugar moiety of the phosphate stabilizing nucleoside is selected from: halogen, allyl, amino, azido, thio, O-allyl, -O-CrC10 alkyl, -O-CrCi0 substituted alkyl, -OCF3, -O-(CH2)2-O-CH3, - O(CH2)2SCH3, -O-(CH2)2-O-N(Rm)(Rn), -O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted Ci-Ci0 alkyl, -O[(CH2)nO]mCH3, -O(CH2)nNH2, -O(CH2)nCH3, -
O(CH2)nONH2, -OCH2C(=O)N(H)CH3i -O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10; C, to Cio alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl. In certain embodiments, the modified phosphate is selected from: phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphotriester, phosphorothioate, phosphorodithioate, thiophosphoramidate, and phosphoramidate.
In certain embodiments, the modified phosphate is selected from: phosphonate, alkylphosphonate, and substituted alkylphosphonate. In certain embodiments, the modified phosphate is selected from 5'-deoxy-5'-thio phosphate, phosphoramidate, methylene phosphonate, mono-fluoro methylene phosphonate and di-fluoro methylene phosphonate. In certain embodiments, the sugar moiety of the phosphate stabilizing nucleoside comprises a 5'-modificaton and a 2'-modification.
In certain embodiments, the oligomeric compound is an antisense compound. In certain embodiments, the antisense compound is an RNAi compound. In certain embodiments, the antisense compound is an siRNAi compound. In certain embodiments, the antisense compound is a microRNA mimic. In certain embodiments, the antisense compound is an RNase H antisense compound. In certain embodiments, the antisense compound modulates splicing.
In certain embodiments, at least a portion of the nucleobase sequence of the oligonucleotide is complementary to a portion of a target nucleic acid, wherein the target nucleic acid is selected from: a target mRNA, a target pre-mRNA, a target microRNA, and a target non-coding RNA. In certain embodiments, the nucleobase sequence of the oligonucleotide a region of 100% complementarity to the target nucleic acid and wherein the region of 100% complementarity is at least 10 nucleobases. In certain embodiments, the region of 100% complementarity is at least 15 nucleobases. In certain embodiments, the region of 100% complementarity is at least 20 nucleobases. In certain embodiments, the oligonucleotide is at least 85% complementary to the target nucleic acid. In certain embodiments, the oligonucleotide is at least 90% complementary to the target nucleic acid. In certain embodiments, the oligonucleotide is at least 95% complementary to the target nucleic acid. In certain embodiments, the oligonucleotide is at least 98% complementary to the target nucleic acid. In certain embodiments, the oligonucleotide is 100% complementary to the target nucleic acid.
In certain embodiments, the antisense compound is a microRNA mimic having a nucleobase sequence comprising a portion that is at least 80% identical to the seed region of a microRNA and that has overall identity with the microRNA of at least 70%. In certain embodiments, the nucleobase sequence of the microRNA mimic has a portion that is at least 80% identical to the sequence of the seed region of a microRNA and has overall identity with the microRNA of at least 75%. In certain embodiments, the nucleobase sequence of the microRNA mimic has a portion that is at least 80% identical to the sequence of the seed region of a microRNA and has overall identity with the microRNA of at least 80%. In certain embodiments, the nucleobase sequence of the microRNA mimic has a portion that is at least 100% identical to the sequence of the seed region of a microRNA and has overall identity with the microRNA of at least 80%. In certain embodiments, the nucleobase sequence of the microRNA mimic has a portion that is at least 100% identical to the sequence of the seed region of a microRNA and has overall identity with the microRNA of at least 85%. In certain embodiments, the nucleobase sequence of the microRNA mimic has a portion that is 100% identical to the sequence of the microRNA. In certain embodiments, nucleobase sequence of the oligonucleotide comprises a region of 100% complementarity to a seed match segment of a target nucleic acid. In certain embodiments, the antisense compound is a microRNA mimic having a nucleobase sequence comprising a portion that is at least 80% identical to the seed region of a microRNA and that has overall identity with the microRNA of at least 50%. In certain embodiments, the antisense compound is a microRNA mimic having a nucleobase sequence comprising a portion that is at least 80% identical to the seed region of a microRNA and that has overall identity with the microRNA of at least 55%. In certain embodiments, the antisense compound is a microRNA mimic having a nucleobase sequence comprising a portion that is at least 80% identical to the seed region of a microRNA and that has overall identity with the microRNA of at least 60%. In certain embodiments, the antisense compound is a microRNA mimic having a nucleobase sequence comprising a portion that is at least 80% identical to the seed region of a microRNA and that has overall identity with the microRNA of at least 65%. In certain embodiments, the oligomeric compound comprises a nucleobase sequence selected from a microRNA sequence found in miRBase. In certain embodiments, the oligomeric compound consists of a nucleobase sequence selected from a microRNA sequence found in miRBase.
In certain embodiments, the target nucleic acid is a target mRNA. In certain embodiments, the target nucleic acid is a target pre-mRNA. In certain embodiments, the target nucleic acid is a non-coding RNA. In certain embodiments, the target nucleic acid is a microRNA. In certain embodiments, the target nucleic acid is a pre-mir. In certain embodiments, the target nucleic acid is a pri-mir.
In certain embodiments, the nucleobase sequence of the oligonucleotide comprises a region of 100% complementarity to the target nucleic acid and wherein the region of 100% complementarity is at least 10 nucleobases. In certain embodiments, the nucleobase sequence of the oligonucleotide comprises a region of 100% complementarity to the target nucleic acid and wherein the region of 100% complementarity is at least
6 nucleobases. In certain embodiments, the nucleobase sequence of the oligonucleotide comprises a region of 100% complementarity to the target nucleic acid and wherein the region of 100% complementarity is at least
7 nucleobases. In certain embodiments, the target nucleic acid is a mammalian target nucleic acid. In certain embodiments, the mammalian target nucleic acid is a human target nucleic acid.
In certain embodiments, oligomeric compounds comprise from 1 to 3 terminal group nucleosides on at least one end of the oligonucleotide. In certain embodiments, oligomeric compound comprise from 1 to 3 terminal group nucleosides at the 3 '-end of the oligonucleotide. In certain embodiments, oligomeric compound comprise from 1 to 3 terminal group nucleosides at the 5'-end of the oligonucleotide. In certain embodiments, oligomeric compounds of the invention are single stranded.
In certain embodiments, oligomeric compounds of the invention are double stranded. In certain embodiments, the invention provides pharmaceutical compositions comprising an oligomeric compounds and a pharmaceutically acceptable diluent or carrier. In certain embodiments, the pharmaceutically acceptable diluent or carrier is pharmaceutical grade sterile saline. In certain embodiments, the invention provides methods comprising contacting a cell with an oligomeric compound described herein. In certain embodiments, such methods comprise detecting antisense activity. In certain embodiments, the detecting antisense activity comprises detecting a phenotypic change in the cell. In certain embodiments, the detecting antisense activity comprises detecting a change in the amount of target nucleic acid in the cell. In certain embodiments, the detecting antisense activity comprises detecting a change in the amount of a target protein. In certain embodiments, the cell is in vitro. In certain embodiments, the cell is in an animal. In certain embodiments, animal is a mammal. In certain embodiments, the mammal is a human.
In certain embodiments, the invention provides methods of modulating a target mRNA in a cell comprising contacting the cell with an oligomeric compound of the invention and thereby modulating the mRNA in a cell. In certain embodiments, such methods comprise detecting a phenotypic change in the cell. In certain embodiments, methods comprise detecting a decrease in mRNA levels in the cell. In certain embodiments, methods comprise detecting a change in the amount of a target protein. In certain embodiments, the cell is in vitro. In certain embodiments, the cell is in an animal. In certain embodiments, the animal is a mammal. In certain embodiments, the mammal is a human. In certain embodiments, the invention provides methods of administering to an animal a pharmaceutical composition of the invention. In certain embodiments, the animal is a mammal. In certain embodiments, the mammal is a human. In certain embodiments, the methods comprise detecting antisense activity in the animal. In certain embodiments, the methods comprise detecting a change in the amount of target nucleic acid in the animal. In certain embodiments, the methods comprise detecting a change in the amount of a target protein in the animal. In certain embodiments, the methods comprise detecting a phenotypic change in the animal. In certain embodiments, the phenotypic change is a change in the amount or quality of a biological marker of activity.
In certain embodiments, the invention provides use of an oligomeric compound of the invention for the manufacture of a medicament for the treatment of a disease characterized by undesired gene expression.
In certain embodiments, the invention provides use of an oligomeric compound of the invention for the manufacture of a medicament for treating a disease by inhibiting gene expression.
In certain embodiments, the invention provides methods of comprising detecting antisense activity wherein the antisense activity is microRNA mimic activity. In certain embodiments, the detecting microRNA mimic activity comprises detecting a change in the amount of a target nucleic acid in a cell. In certain embodiments, the detecting microRNA mimic activity comprises detecting a change in the amount of a target protein in cell.
In certain embodiments, the invention provides a nucleoside having the Formula I:
Figure imgf000031_0001
wherein:
Bx is a heterocyclic base moiety;
T1 is H, a hydroxyl protecting group, or a phosphorous moiety; T2 is H, a hydroxyl protecting group, or a reactive phosphorous group; and each of qi and q2 is, independently selected from Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted Q-C6 alkenyl and substituted C2-C6 alkynyl; Xi is S, NRi6, or CRioRn wherein each Ri0 and Rn is, independently, H, F, Ci-C6 haloalkyl , or Ci-C6 alkyl; and
Ri is selected from a halogen, X2-V, and 0-X4; or each of qi and q2 is, independently, selected from H, CrC6 alkyl, C2-C6 alkenyl, C2- C6 alkynyl, substituted Q-C6 alkyl, substituted Q-C6 alkenyl and substituted C2-C6 alkynyl; Xi is S, NRi6Rn, or CRioRn wherein each R10 and Rn is, independently, H, F, Ci-Ce haloalkyl , or Ci-C6 alkyl; and
Ri is X2-V; or each of qi and q2 is, independently, selected from Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted C1-C6 alkenyl and substituted C2-C6 alkynyl;
Xi is O, S, NRi6Ri7, or CRi0Ri i wherein each Ri0 and Rn is, independently, H, F, Q- C6 haloalkyl , or Ci-C6 alkyl; and
Ri is X2-V; wherein:
X2 is O, S or CR7R8 wherein each R7 and Rg is, independently, H or Ci-C6 alkyl; V is selected from cholesterol, (CH2)2[O(CH2)2]tOCH3, where t is from 1-3, (CH2)2F, CH2COOH, CH2CONH2, CH2CONR5R61 CH2COOCH2CH3, CH2CONH(CH2)I-S-R4 where i is from 1 to 10, CH2CONH(CH2)JNR5R6 where j is from 1 to 6, and CH2CONHt(CH2Xi-N(H)]^-(CH2XiNH2 where each ki is independently from 2 to 4 and k2 is from 2 to 10;
R4 is selected from H, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted C]-C6 alkenyl, substituted C2-C6 alkynyl, C6-Ci4 aryl and a thio protecting group;
R5 and R6 are each, independently, selected from H, CpC6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, and substituted C2-C6 alkynyl; Ri6 is selected from H, Ci-C6 alkyl, or substituted Ci-C6 alkyl;
X4 is [C(Ra)(Rb)]n-[(C=O)mXc]k-Rd wherein each Ra and Rb is independently H or halogen; Xc is O, S5 OrN(E1);
Rd is H, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Q-C6 alkyl, substituted Ci-C6 alkenyl and substituted C2-C6 alkynyl or NE2E3; each Ei, E2, and E3 is independently H, Q-C6 alkyl, or substituted Ci-C6 alkyl; n is 1 to 6; m is O or 1; and k is O or 1; and wherein each substituted group is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, OJ1, NJiJ2, SJi, N3, OC(=O)Ji and CN, wherein each Ji and J2 is, independently, H or C]-C6 alkyl; and J4 is hydrogen, or a protecting group.
In certain embodiments, at least one of qj and q2 is C]-C6 alkyl or substituted Cj-C6 alkyl. In certain embodiments, at least one of qi and q2 is Ci-C6 alkyl. In certain embodiments, at least one of q! and q2 is methyl. In certain embodiments, one of qi and q2 is H. In certain embodiments, one of qi and q2 is methyl and the other of qi and q2 is H. In certain embodiments, Z is O. In certain embodiments, Y is CH2CONH(CH2)JNR5R6. In certain embodiments, j is 2. In certain embodiments, one of R5 and R6 Is other than H. In certain embodiments, at least one of R5 and R6 is selected from Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted CrC6 alkyl, substituted Ci-C6 alkenyl, and substituted C2-C6 alkynyl. In certain embodiments, at least one of R5 and R6 is Ci-C6 alkyl or substituted Q-C6 alkyl. In certain embodiments, at least one of R5 and R6 is methyl. In certain embodiments, both R5 and R5 are other than H. In certain embodiments, Xi is O. In certain embodiments, Xi is S. In certain embodiments, Xi is CRioRn. In certain embodiments, R10 and Rn are both H. In certain embodiments, Ti is a phosphorus moiety. In certain embodiments, the phosphorus moiety is selected from P(Ya)YbYc, where Ya is O or S and each Yb and Yc is, independently, OH, SH, alkyl, substituted CrC6 alkyl, alkoxyl and substituted Ci-C6 alkoxyl. In certain embodiments, Ya is O and Yb and Y0 are each OH.
In certain embodiments: i. Ya is 0; ii. Yb and Yc are each OH; and iii. T2 is diisopropylcyanoethoxy phosphoramidite.
In certain embodiments, In certain embodiments, T2 is a reactive phosphorus group. In certain embodiments, the reactive phosphorous group is diisopropylcyanoethoxy phosphoramidite or H-phosphonate. In certain embodiments, a nucleoside has Formula XII:
Figure imgf000033_0001
In certain embodiments, both R5 and R6 are other than H. In certain embodiments, both R5 and R6 are methyl. In certain embodiments, qi is methyl and q2 is H. In certain embodiments, qi is H and q2 is methyl. In certain embodiments, such nucleosides have the configuration:
Figure imgf000033_0002
In certain embodiments, the oligomeric compound has a microRNA sequence associated with an accession number from miRBase version 10.1 released December 2007 selected from:
MIMAT0000062, MMAT0004481, MIMAT0000063, MIMAT0004482, MMAT0000064, MIMAT0004483, MIMAT0000065, MMAT0004484, MMAT0000066, MIMAT0004485, MIMAT0000067, MIMAT0004486, MMAT0004487, MIMAT0000414, MIMAT0004584, MIMAT0000415, MIMAT0004585, MIMAT0000416, MIMAT0000098, MIMAT0004512, MIMAT0000099, MIMAT0004513, MIMATOOOO 101, MIMATOOOO 102, MIMAT0004516, MIMATOOOO 103, MIMAT0004517, MIMAT0000680, MIMAT0004672, MIMATOOOO 104, MIMAT0000253, MIMAT0004555, MIMAT0000254, MIMAT0004556, MIMAT0000421, MIMAT0004590, MIMAT0005459, MIMAT0005458, MIMAT0005573, MIMAT0005572, MIMAT0005577, MIMAT0005576, MIMAT0005580, MCMAT0005583, MIMAT0005582, MIMAT0005584, MIMAT0005586, MIMAT0005588, MIMAT0005589, MIMAT0005591, MIMAT0005592, MIMAT0005593, MIMAT0000422, MIMAT0004591, MIMAT0004602, MIMAT0000443, MIMAT0000423, MIMAT0000423, MIMAT0004592, MIMAT0004603, MIMAT0000445, MIMAT0000444, MIMAT0000446, MIMAT0004604, MIMAT0000424, MIMAT0004548, MIMAT0004605, MIMAT0000242, MDVLAT0000425, MIMAT0004593, MIMAT0000691, MIMAT0004680, MIMAT0000426, MIMAT0004594, MIMAT0000427, MIMAT0000770, MIMAT0000447, MIMAT0000428, MIMAT0004595, MIMAT0000758, MIMAT0004698, MIMAT0000448, MIMAT0004606, MEVLAT0000429, MIMAT0000430, MIMAT0004607, MIMAT0004596, MIMAT0004552, MIMAT0000250, MIMAT0004597, MIMAT0000431, MIMAT0000432, MIMAT0004598, MIMAT0000434, MIMAT0000433, MIMAT0000435, MDV1AT0004599, MIMAT0000436, MIMAT0004600, MIMAT0000437, MIMAT0004601, MIMAT0000449, MIMAT0004608, MIMAT0004766, MIMAT0002809, MIMAT0000251, MIMAT0004928, MIMAT0000243, MIMAT0004549, MIMAT0000759, MIMAT0004699, MIMAT0000450, MIMAT0004609, MIMAT0000451 , MIMAT0004610, MIMAT0000757, MIMAT0004697, MIMAT0000438, MIMAT0000439, MIMAT0000439, MIMAT0000452, MIMAT0000453, MIMAT0000646, MIMAT0004658, MIMAT0000068, MIMAT0004488, MDMAT0000417, MIMAT0004586, MIMAT0000069, MIMAT0004489, MIMAT0004518, MIMAT0000070, MIMAT0000071, MIMAT0000256, MIMAT0000270, MIMAT0004558, MIMAT0000257, MDVLAT0000258, MIMAT0004559, MIMAT0002821, MIMAT0000259, MIMAT0000260, MIMAT0000261, MIMAT0004560, MIMAT0000454, MIMAT0004611, MIMAT0000456, MIMAT0004612, MIMAT0000262, MIMAT0004561, MEVLAT0004613, MIMAT0000457, MIMAT0000072, MEVLAT0002891, MEV1AT0001412, MIMAT0004751, MIMAT0000458, MIMAT0004929, MIMAT0000440, MIMATOOOl 618, MIMAT0000222, MIMAT0004543, MMAT0000459, MIMAT0004614, MIMAT0002819, MIMAT0004767, MIMAT0000460, MIMAT0004671, MEVLAT0000461, MIMAT0004615, MIMAT0000226, MIMAT0004562, MMAT0001080, MIMAT0000227, MIMAT0000228, MIMAT0000232, MIMAT0000231, MIMAT0004563, MIMAT0000263, MIMAT0000073, MIMAT0004490, MIMAT0000074, MIMAT0004491, MIMAT0004492, MIMAT0000682, MDVLATOOO 1620, MIMAT0000318, MEMAT0004571 , MIMAT0000617, MIMAT0004657, MIMAT0002811, MIMAT0002810, MIMAT0000264, MMAT0000265, MIMAT0000266, MIMAT0000462, MIMAT0000241, MIMAT0004960, MIMAT0000075, MHVLAT0004493, MMAT0001413, MIMAT0004752, MIMAT0000076, MIMAT0004494, MIMAT0000267, MIMAT0000268, MIMAT0000269, MIMAT0000271, MIMAT0004564, MIMAT0000272, MIMAT0000273, MIMAT0004959, MIMAT0000274, MIMAT0000275, MIMAT0004565, MIMAT0004566, MMAT0004567, MIMAT0004675, MIMAT0000276, MIMAT0000077, MIMAT0004495, MIMAT0000277, MIMAT0004908, MIMAT0004915, MIMAT0000278, MIMAT0004568, MIMAT0000279, MIMAT0004569, MMAT0000280, MIMAT0004570, MIMAT0000281, MIMAT0000078, MIMAT0004496, MLMAT0000418, MIMAT0004587, MIMAT0000080, MIMAT0000079, MMAT0004497, MIMAT0000081, MIMAT0004498, MIMAT0000082, MIMAT0004499, MMAT0004681, MIMAT0000083, MIMAT0004500, MIMAT0000084, MIMAT0004501, MMAT0000419, MIMAT0004588, MIMAT0004502, MIMAT0000085, MIMAT0004679, MMAT0000690, MIMAT0004450, MIMAT0004901, MEMAT0000687, MIMAT0002890, MDMAT0000086, MIMAT0004503, MIMATOOOO 100, MIMAT0004514, MIMAT0004515, MIMAT0000681, MIMAT0004673, MEVLAT0004903, MIMAT0000688, MIMAT0004958, MDVLAT0000684, MIMAT0000683, MIMAT0000715, MIMAT0000714, MIMAT0000717, MMAT0000716, MIMAT0000718, MIMAT0004685, MIMAT0000087, MIMAT0000088, MIMAT0000420, MIMAT0004589, MIMAT0000244, MIMAT0004674, MIMAT0004550, MIMAT0000245, MIMAT0004551, MIMAT0000692, MIMAT0000693, MMAT0000089, MIMAT0004504, MIMAT0000090, MIMAT0004505, MIMAT0000510, MIMAT0000755, MIMAT0004696, MIMAT0000762, MIMAT0000761, MIMAT0000771, MIMAT0000756, MIMAT0000752, MIMAT0001629, MIMAT0000751, MTMATO004693, MIMAT0000760, MIMAT0004700, MIMAT0000765, MIMAT0004703, MIMAT0000754, MIMAT0004695, MEV1AT0000763, MIMAT0004701, MIMAT0004702, MIMAT0000764, MDVLAT0000091, MIMAT0004506, MIMAT0003301, M3MAT0004811, MIMAT0004692, MIMAT0000750, MIMAT0000753, MIMAT0004694, MIMAT0000772, MIMAT0000773, MIMAT0000255, MIMAT0004557, MIMAT0004676, MIMAT0000685, MIMAT0004677, MEVLΛT0000686, MMAT0004682, MIMAT0000703, MIMAT0004683, MIMAT0000705, MIMAT0000707, MIMAT0003385, MIMAT0000710, MIMAT0000719, MIMAT0004686, MIMAT0000721, MIMAT0001621, MIMAT0000722, MIMAT0000723, MIMAT0004687, MIMAT0000724, MTMAT0000726, MIMAT0000725, MIMAT0000727, MIMAT0004688, MIMAT0004955, MIMAT0004956, MIMAT0000728, MIMAT0000729, MIMAT0003386, MIMAT0002172, MIMAT0000720, MMAT0000730, MIMAT0004689, MIMAT0000732, MIMAT0000731, MIMAT0000733, MIMAT0004690, MIMAT0000735, MIMAT0000734, MIMAT0000736, MIMAT0000737, MIMAT0000738, MIMATOOO 1075, MIMAT0001639, MIMAT0001638, MIMAT0002171, MIMAT0003329, MIMAT0004813, MIMAT0002170, MIMAT0003339, MIMAT0001339, MMAT0001340, MIMAT0004748, MIMAT0001341, MIMAT0004749, MMAT0003393, MIMAT0001343, MIMAT0001536, MIMAT0001625, MIMAT0004757, MIMAT0002814, MIMAT0002815, MIMAT0001627, MIMAT0001532, MMAT0001541, MIMAT0003327, MIMAT0001545, MIMAT0004910, MIMAT0004909, MIMAT0001631, MIMAT0001635, MIMAT0001636, MIMAT0001630, MIMAT0003885, MIMAT0003884, MIMAT0004784, MIMAT0003150, MIMAT0002173, MIMAT0004761, MIMAT0002174, MIMAT0002176, MIMAT0002175, MIMAT0004762, MIMAT0002177, MIMAT0002178, MIMAT0003180, MIMAT0004763, MIMAT0002804, MIMAT0002805, MIMAT0002806, MIMAT0004764, MIMAT0004765, MIMAT0002807, MIMAT0002812, MIMAT0003161, MIMAT0002813, MIMAT0002816, MIMAT0002817, MIMAT0002818, MIMAT0002820, MBS1AT0004768, MIMAT0002824, MIMAT0004772, MIMAT0002870, MIMAT0004773, MIMAT0002871, MIMAT0004774, MIMAT0002872, MMAT0004775, MIMAT0002873, MIMAT0002874, MIMAT0002875, MIMAT0002876, MIMAT0004776, MIMAT0002878, MIMAT0002879, MIMAT0002880, MIMAT0004778, MIMAT0004975, MIMAT0002881, MIMAT0004779, MDMAT0002882, MIMAT0002808, MIMAT0002823, MIMAT0002822, MIMAT0004777, MIMAT0002877, MIMAT0005788, MIMAT0005789, MIMAT0002883, MIMAT0002827, MMAT0002826, MIMAT0002860, MIMAT0004770, MIMAT0002859, MIMAT0002851, MIMAT0002852, MIMAT0002857, MIMAT0002866, MIMAT0002863, MIMAT0005457, MMAT0002844, MIMAT0002848, MCMAT0002847, MIMAT0002864, MIMAT0005456, MIMAT0002861, MEMAT0005450, MIMAT0002842, MIMAT0002841, MIMAT0002869, MIMAT0005452, MIMAT0002837, MIMAT0005454, MEVLAT0002832, MIMAT0002831, MIMAT0002853, MIMAT0002829, MIMAT0002828, MIMAT0002834, MIMAT0002833, MMAT0002843, MIMAT0002846, MIMAT0005455, MMAT0002856, MIMAT0002855, MIMAT0002825, MIMAT0002830, MIMAT0002858, MIMAT0002867, MIMAT0002854, MIMAT0002868, MIMAT0005451, MIMAT0002840, MIMAT0005449, MIMAT0002850, MIMAT0002849, MIMAT0002839, MIMAT0002838, MIMAT0002845, MIMAT0002835, MIMAT0002836, MIMAT0002862, MMAT0004780, MIMAT0002888, MMAT0003163, MIMAT0004920, MIMAT0004919, MIMAT0003389, MIMAT0003340, MMAT0004954, MIMAT0003164, MIMAT0003165, MIMAT0004785, MIMAT0003251, MMAT0004803, MIMAT0003254, MIMAT0004798, MIMAT0003285, MIMAT0004806, MMAT0003323, MIMAT0003323, MIMAT0004812, MIMAT0003333, MMAT0004800, MIMAT0003257, MIMAT0003214, MIMAT0003233, MIMAT0004794, MMAT0003215, MIMAT0003216, MIMAT0003217, MIMAT0003219, MIMAT0004793, MMAT0003220, MIMAT0003221, MIMAT0003222, MIMAT0003223, MIMAT0003225, MMAT0003226, MMAT0003227, MIMAT0003228, MIMAT0003230, MIMAT0003231, MIMAT0003232, MIMAT0003234, MIMAT0003235, MIMAT0003236, MIMAT0003237, MIMAT0003238, MEMAT0003239, MIMAT0004795, MIMAT0003240, MIMAT0004796, MIMAT0003241, MMAT0003242, MIMAT0003243, MMAT0003244, MEMAT0003245, MIMAT0003246, MIMAT0004797, MIMAT0003247, MIMAT0003248, MEMAT0003249, MIMAT0003250, MMAT0003252, MIMAT0003253, MIMAT0003255, MIMAT0004799, MIMAT0003256, MMAT0004801, MIMAT0003258, MEMAT0003259, MIMAT0003260, MIMAT0004802, MIMAT0003261, MIMAT0003263, MIMAT0003264, MIMAT0003265, MIMAT0003266, MIMAT0003267, MIMAT0003268, MMAT0003269, MIMAT0003270, MIMAT0003271, MIMAT0003272, MIMAT0003273, MIMAT0003274, MMAT0003275, MIMAT0003276, MDV1AT0003277, MIMAT0003278, MEV1AT0003279, MIMAT0003280, MIMAT0003281, MMAT0003282, MEMAT0003283, MIMAT0004804, MIMAT0004805, MIMAT0003284, MIMAT0003286, MIMAT0003287, MIMAT0003288, MIMAT0003289, MIMAT0003290, MMAT0003291, MIMAT0003292, MIMAT0004807, MIMAT0003293, MIMAT0003294, MIMAT0004808, MEV1AT0003295, MIMAT0003296, MIMAT0003297, MIMAT0004809, MIMAT0004810, MIMAT0003298, MIMAT0003299, MIMAT0003300, MIMAT0003302, MIMAT0003303, MEViAT0003304, MIMAT0003305, MIMAT0003306, MIMAT0003307, MEVIAT0003308, MIMAT0003309, MIMAT0003310, MIMAT0003311, MIMAT0003312, MEV1ATO003313, MIMAT0003314, MIMAT0003315, MIMAT0003316, MIMAT0003317, MMAT0003318, MIMAT0003319, MEV1AT0003320, MIMAT0003321, MIMAT0003322, MIMAT0003328, MIMAT0004814, MIMAT0003330, MIMAT0003331, MIMAT0003332, MMAT0003335, MIMAT0003336, MIMAT0003337, MIMAT0003338, JVHMAT0003324, MMAT0003325, MIMAT0003326, MIMAT0004952, MIMAT0003881, MDV1AT0004819, MIMAT0003880, MIMAT0004284, MIMAT0000252, MIMAT0004926, MIMAT0004927, MIMAT0004553, MEMAT0004554, MIMAT0004945, MIMAT0004946, MMAT0003879, MIMAT0004957, MIMAT0003945, MIMAT0003888, MIMAT0003883, MIMAT0003882, MIMAT0003947, MIMAT0003946, MIMAT0003887, MMAT0003886, MIMAT0003948, MIMAT0004209, MDVIAT0004185, MIMAT0004953, MIMAT0004911, MIMAT0004923, MIMAT0004922, MIMAT0004925, MIMAT0004924, MMAT0004949, MIMAT0004950, MIMAT0004948, MIMAT0004947, MIMAT0004906, MEV1AT0004905, MIMAT0004951, MIMAT0004916, MIMAT0004917, MIMAT0004921, MEMAT0004912, MIMAT0004902, MIMAT0004913, MIMAT0004907, MIMAT0004918, MIMAT0000441, MIMAT0000442, MMAT0004970, MIMAT0004971, MMAT0004972, MCMAT0004973, MIMAT0004974, MIMAT0000092, MTMAT0004507, MEMAT0004508, MIMAT0003218, MIMAT0004792, MIMAT0000093, MIMAT0004509, MIMAT0004976, MIMAT0004977, MIMAT0004978, MIMAT0004979, MIMAT0004980, MIMAT0004981, MIMAT0004982, MIMAT0004983, MIMAT0004984, MIMAT0004985, MMAT0004986, MIMAT0004987, MIMAT0000094, MMAT0000095, MMAT0004510, MIMAT0000096, MIMAT0000097, MMAT0004511, MIMAT0000689, and MIMAT0004678.
In certain embodiments the invention provides oligomeric compounds having a nucleobase sequence selected from among SEQ ID NOs 20, 21, 23, 24, 25, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, and 91. In certain embodiments, the present invention provides oligomeric compounds having a nucleobase sequence selected from the table below.
Figure imgf000037_0001
Figure imgf000038_0001
Detailed description of the Invention
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. Each of the following patent applications is hereby incorporated by reference in its entirety: US Provisional Applications 61/108,457, filed 2008-10-24; 61/108,464, filed 2008-10-24; 61/149,297, filed 2009-02-02; 61/150,492, filed 2009-02-06; 61/163,217,filed 2009-03-25; 61/174,137, filed 2009-04-30; 61/239,672, filed 2009-09-03; and PCT/US2009/061913 and PCT/US2009/061916 each filed 2009-10-23 (the same day as the present application).
I. 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, "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 nucleosides having mimetic bases and/or sugar groups. Nucleosides may be modified with any of a variety of substituents. Nucleosides may include a phosphate moiety.
As used herein, "sugar moiety" means a natural or modified sugar ring or sugar surrogate. 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, 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 phosphate linking group. 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. 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, "bicyclic nucleoside" or "BNA" refers to a nucleoside wherein the sugar moiety of the nucleoside comprises a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic sugar moiety.
As used herein, "4 '-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, "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-CrCi0 alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-O- N(R1n)(Rn), or O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-CiO 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.
As used herein, "2'-0Me" or "2'-OCH3" or "2'-O-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'-O-methoxyethyl" each refers to a nucleoside comprising a sugar comprising a -OCH2CH2OCH3 group at the 2' position of the sugar ring.
As used herein, "phosphorous moiety" refers to a to monovalent Pv phosphorus radical group. In certain embodiments, a phosphorus moiety is selected from: a phosphate, phosphonate, alkylphosphonate, aminoalkyl phosphonate, phosphorothioate, phosphoramidite, alkylphosphonothioate, phosphorodithioate, thiophosphoramidate, phosphotriester and the like. In certain embodiments, modified phosphorous moieties have the following structural formula:
Figure imgf000040_0001
wherein: Ra and R0 are each, independently, OH, SH, Ci-C6 alkyl, substituted Ci-C6 alkyl, Ci-C6 alkoxy, substituted Ci-C6 alkoxy, amino or substituted amino; and Rb is O or S.
The term "phosphate moiety" as used herein, refers to a terminal phosphate group that includes phosphates as well as modified phosphates. The phosphate moiety can be located at either terminus but is preferred at the 5'-terminal nucleoside. In one aspect, the terminal phosphate is unmodified having the formula -0-P(=0)(0H)0H. In another aspect, the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R) or alkyl where R is H, an amino protecting group or unsubstituted or substituted alkyl. In certain embodiments, the 5' and or 3' terminal group can comprise from 1 to 3 phosphate moieties that are each, independently, unmodified or modified.
As used herein, "phosphate stabilizing modification" refers to a nucleoside modification that results in stabilization of a 5 '-phosphate group of nucleoside, relative to the stability of a 5 '-phosphate of an unmodified nucleoside under biologic conditions. Such stabilization of a 5'-phophate group includes but is not limit to resistance to removal by phosphatases.
As used herein, "phosphate stabilizing nucleoside" refers to a nucleoside comprising at least one phosphate stabilizing modification. In certain embodiments the phosphate stabilizing modification is a T- modification. In certain embodiments, the phosphate stabilizing modification is at the 5' position of the nucleoside. In certain embodiments, a phosphate stabilizing modification is at the 5' position of the nucleoside and at the 2' position of the nucleoside.
As used herein, "5 '-stabilizing nucleoside" refers to a nucleoside that, when placed at the 5'-end of an oligonucleotide, results in an oligonucleotide that is more resistant to exonuclease digestion, and/or has a stabilized phosphate group. 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 "oligonucleoside" refers to an oligonucleotide in which none of the internucleoside linkages contains a phosphorus atom. As used herein, oligonucleotides include oligonucleosides. 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 51 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 substructures. In certain embodiments, an oligomeric compound is an oligonucleotide. In certain embodiments, an oligomeric compound is 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, "duplex" refers to two separate oligomeric compounds that are hybridized together. As used herein, "terminal group" refers to one or more atom attached to either, or both, the 3' end or the 5' end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more additional nucleosides.
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, "protecting group," as used herein, refers to a labile chemical moiety which is known in the art to protect reactive groups including without limitation, hydroxyl, amino and thiol groups, against undesired reactions during synthetic procedures. Protecting groups are typically used selectively and/or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group as is or available for further reactions. Protecting groups as known in the art are described generally in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999).
The term "orthogonally protected" refers to functional groups which are protected with different classes of protecting groups, wherein each class of protecting group can be removed in any order and in the presence of all other classes (see, Barany et al, J. Am. Chem. Soc, 1977, 99, 7363-7365; Barany et ah, J. Am. Chem. Soc, 1980, 102, 3084-3095). Orthogonal protection is widely used in for example automated oligonucleotide synthesis. A functional group is deblocked in the presence of one or more other protected functional groups which is not affected by the deblocking procedure. This deblocked functional group is reacted in some manner and at some point a further orthogonal protecting group is removed under a different set of reaction conditions. This allows for selective chemistry to arrive at a desired compound or oligomeric compound. This allows for selective chemistry to arrive at a desired compound or oligomeric compound.
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. In certain embodiments, an antisense compound modulates (increases or decreases) expression or amount of a target nucleic acid. In certain embodiments, an antisense compound alters splicing of a target pre-mRNA resulting in a different splice variant. In certain embodiments, an antisense compound modulates expression of one or more different target proteins. Antisense mechanisms contemplated herein include, but are not limited to an RNase H mechanism, RNAi mechanisms, splicing modulation, translational arrest, altering RNA processing, inhibiting microRNA function, or mimicking microRNA function. 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, "RNAi" refers to a mechanism by which certain antisense compounds effect expression or amount of a target nucleic acid. RNAi mechanisms involve the RISC pathway. As used herein, "RNAi compound" refers to an oligomeric compound that acts, at least in part, through an RNAi mechanism to modulate a target nucleic acid and/or protein encoded by a target nucleic acid. RNAi compounds include, but are not limited to double-stranded short interfering RNA (siRNA), single-stranded RNA (ssRNA), and microRNA, including microRNA mimics.
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. For example, in certain embodiments, antisense activity is assessed by detecting and/or measuring the amount of target protein or the relative amounts of splice variants of a target protein. In certain embodiments, antisense activity is assessed by detecting and/or measuring the amount of target nucleic acids and/or cleaved target nucleic acids and/or alternatively spliced target nucleic acids. 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 mRNA" refers to a pre-selected RNA molecule that encodes a protein. As used herein, "target pre-mRNA" refers to a pre-selected RNA transcript that has not been fully processed into mRNA. Notably, pre-RNA includes one or more intron.
As used herein, "target microRNA" refers to a pre-selected non-coding RNA molecule about 18-30 nucleobases in length that modulates expression of one or more proteins or to a precursor of such a non- coding molecule.
As used herein, "target pdRNA" refers to refers to a pre-selected RNA molecule that interacts with one or more promoter to modulate transcription.
As used herein, "microRNA" refers to a naturally occurring, small, non-coding RNA that represses gene expression at the level of translation. In certain embodiments, a microRNA represses gene expression by binding to a target site within a 3 ' untranslated region of a target nucleic acid. In certain embodiments, a microRNA has a nucleobase sequence as set forth in miRBase, a database of published microRNA sequences found at http://microrna.sanger.ac.uk/sequences/. In certain embodiments, a microRNA has a nucleobase sequence as set forth in miRBase version 10.1 released December 2007, which is herein incorporated by reference in its entirety. In certain embodiments, a microRNA has a nucleobase sequence as set forth in miRBase version 12.0 released September 2008, which is herein incorporated by reference in its entirety.
As used herein, "microRNA mimic" refers to an oligomeric compound having a sequence that is at least partially identical to that of a microRNA. In certain embodiments, a microRNA mimic comprises the microRNA seed region of a microRNA. In certain embodiments, a microRNA mimic modulates translation of more than one target nucleic acids. As used herein, "seed region" refers to a region at or near the 5 'end of an antisense compound having a nucleobase sequence that is import for target nucleic acid recognition by the antisense compound. In certain embodiments, a seed region comprises nucleobases 2-8 of an antisense compound. In certain embodiments, a seed region comprises nucleobases 2-7 of an antisense compound. In certain embodiments, a seed region comprises nucleobases 1-7 of an antisense compound. In certain embodiments, a seed region comprises nucleobases 1-6 of an antisense compound. In certain embodiments, a seed region comprises nucleobases 1-8 of an antisense compound.
As used herein, "microRNA seed region" refers to a seed region of a microRNA or microRNA mimic. In certain embodiments, a microRNA seed region comprises nucleobases 2-8 of a microRNA or microRNA mimic. In certain embodiments, a microRNA seed region comprises nucleobases 2-7 of a microRNA or microRNA mimic. In certain embodiments, a microRNA seed region comprises nucleobases 1- 7 of a microRNA or microRNA mimic. In certain embodiments, a microRNA seed region comprises nucleobases 1-6 of a microRNA or microRNA mimic. In certain embodiments, a microRNA seed region comprises nucleobases 1-8 of a microRNA or microRNA mimic.
As used herein, "seed match segment" refers to a portion of a target nucleic acid having nucleobase complementarity to a seed region. In certain embodiments, a seed match segment has nucleobase complementarity to nucleobases 2-8 of an siRNA, ssRNA, natural microRNA or microRNA mimic. In certain embodiments, a seed match segment has nucleobase complementarity to nucleobases 2-7 of an siRNA, ssRNA, microRNA or microRNA mimic. In certain embodiments, a seed match segment has nucleobase complementarity to nucleobases 1-6 of an siRNA, ssRNA, microRNA or microRNA mimic. In certain embodiments, a seed match segment has nucleobase complementarity to nucleobases 1-7 of an siRNA, ssRNA, microRNA or microRNA mimic. In certain embodiments, a seed match segment has nucleobase complementarity to nucleobases 1-8 of an siRNA, ssRNA, microRNA or microRNA mimic.
As used herein, "seed match target nucleic acid" refers to a target nucleic acid comprising a seed match segment. As used herein, "microRNA family" refers to a group of microRNAs that share a microRNA seed sequence. In certain embodiments, microRNA family members regulate a common set of target nucleic acids. In certain embodiments, the shared microRNA seed sequence is found at the same nucleobase positions in each member of a microRNA family. In certain embodiments, the shared microRNA seed sequence is not found at the same nucleobase positions in each member of a microRNA family. For example, a microRNA seed sequence found at nucleobases 1 -7 of one member of a microRNA family may be found at nucleobases 2-8 of another member of a microRNA family.
As used herein, "target non-coding RNA" refers to a pre-selected RNA molecule that is not translated to generate a protein. Certain non-coding RNA are involved in regulation of expression.
As used herein, "target viral nucleic acid" refers to a pre-selected nucleic acid (RNA or DNA) associated with a virus. Such viral nucleic acid includes nucleic acids that constitute the viral genome, as well as transcripts (including reverse-transcripts and RNA transcribed from RNA) of those nucleic acids, whether or not produced by the host cellular machinery. In certain instances, viral nucleic acids also include host nucleic acids that are recruited by a virus upon viral infection.
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, "overall identity" refers to the nucleobase identity of an oligomeric compound relative to a particular nucleic acid or portion thereof, over the length of the oligomeric compound.
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, "different modifications" or "differently modified" refer to modifications relative to naturally occurring molecules that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are "differently modified," even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are "differently modified," even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nuclcobascs arc not differently modified, unless otherwise indicated. For example, a nucleoside comprising a 2'-0Me modified sugar and an adenine nucleobase and a nucleoside comprising a 2'-0Me modified sugar and a thymine nucleobase are not differently modified.
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, "alternating motif refers to an oligomeric compound or a portion thereof, having at least four separate regions of modified nucleosides in a pattern (AB)nAn, where A represents a region of nucleosides having a first type of modification; B represent a region of nucleosides having a different type of modification; n is 2-15; and m is 0 or 1. Thus, in certain embodiments, alternating motifs include 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more alternating regions. In certain embodiments, each A region and each B region independently comprises 1-4 nucleosides.
As used herein, "fully modified" refers to an oligomeric compound or portion thereon wherein each nucleoside is a modified nucleoside. The modifications of the nucleosides of a fully modified oligomeric compound may all be the same or one or more may be different from one another.
As used herein, "uniform modified" or "uniformly modified" refer to oligomeric compounds or portions thereof that comprise the same modifications. The nucleosides of a region of uniformly modified nucleosides all comprise the same 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, "mitigation" refers to a lessening of at least one activity or one indicator of the severity of a condition or disease. The severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art. In certain embodiments, the condition may be a toxic effect of a therapeutic agent.
As used herein, "pharmaceutical agent" refers to a substance that provides a therapeutic effect when administered to a subject. In certain embodiments, a pharmaceutical agent provides a therapeutic benefit. In certain embodiments, a pharmaceutical agent provides a toxic effect. As used herein, "therapeutic index" refers to the toxic dose of a drug for 50% of the population
(TDsn) divided by the minimum effective dose for 50% of the population (ED50). A high therapeutic index is preferable to a low one: this corresponds to a situation in which one would have to take a much higher amount of a drug to cause a toxic effect than the amount taken to cause a therapeutic benefit..
As used herein, "therapeutically effective amount" refers to an amount of a pharmaceutical agent that provides a therapeutic benefit to an animal. As used herein, "administering" refers to providing a pharmaceutical agent to an animal, and includes, but is not limited to administering by a medical professional and self-administering.
As used herein, "co-administer" refers to administering more than one pharmaceutical agent to an animal. The more than one agent may be administered together or separately; at the same time or different times; through the same route of administration or through different routes of administration.
As used herein, "co-formulation" refers to a formulation comprising two or more pharmaceutically active agents. In certain embodiments, a co-formulation comprises two or more oligomeric compounds. In certain such embodiments, two or more oligomeric compound are oligomeric compounds of the present invention. In certain embodiments, one or more oligomeric compound present in a co-formulation is not a compound of the present invention. In certain embodiments, a co-formulation includes one or more non- oligomeric pharmaceutical agents.
As used herein, "route of administration" refers to the means by which a pharmaceutical agent is administered to an animal.
As used herein, "pharmaceutical composition" refers to a mixture of substances suitable for administering to an animal. For example, a pharmaceutical composition may comprise an antisense oligonucleotide and a sterile aqueous solution.
As used herein, "pharmaceutically acceptable carrier or diluent" refers to any substance suitable for use in administering to an animal. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline. As used herein, "animal" refers to a human or a non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
As used herein, "parenteral administration," refers to administration through injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.
As used herein, "subcutaneous administration" refers to administration just below the skin. "Intravenous administration" refers to administration into a vein.
As used herein, "active pharmaceutical ingredient" refers to the substance in a pharmaceutical composition that provides a desired effect. As used herein, "prodrug" refers to a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
As used herein, the term "dose" refers to a specified quantity of a pharmaceutical agent provided in a single administration. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual.
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 (Ci-Ci2 alkyl) with from 1 to about 6 carbon atoms (Ci-C6 alkyl) being more preferred. The term "lower alkyl" as used herein includes from 1 to about 6 carbon atoms (CrC6 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-Ci2 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, l-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 Ci-Ci2 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, «-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 polycyclic 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 =O 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(O)-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)R33), carboxyl (-C(O)O-Raa), aliphatic groups, alicyclic groups, alkoxy, substituted oxo (-0-R33), aryl, aralkyl, heterocyclic, heteroaryl, hetero- arylalkyl, amino (-NR^R^), imino(=NRbb), amido (-C(O)NRbbRccor -N(Rbb)C(0)R33), azido (-N3), nitro (- NO2), cyano (-CN), carbamido (-OC(O)NRbbRcc or -N(R1111)C(O)OR33), ureido (-N(Rbb)C(0)NRbbRcc), thioureido (-N(Rbb)C(S)NRbbRcc), guanidinyl (-N(Rbb)C(=NRbb)NRbbRcc), amidinyl (-C(=NRbb)NRbbRcc or -N(RbD)C(NRb15)R33), thiol (-SRM,), sulfmyl (-S(O)Rbb), sulfonyl (-S(O)2Rbb), sulfonamidyl (-S(O)2NR1115R0COr -N(R1Ji))S(O)2RhI,) and conjugate groups. Wherein each R33, Ry3 and R1x is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including 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 (O) 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.
II. Certain modified nucleosides In certain embodiments, the invention provides modified nucleosides. Certain modified nucleosides comprise modified sugar moieties, modified heterocyclic bases, modified phosphorus moieties, or combinations of those modifications. In certain embodiments, modified oligonucleotides of the present invention comprise modified nucleosides comprising a modified sugar moiety. In certain embodiments, modified oligonucleotides of the present invention comprise modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides of the present invention comprise modified nucleosides comprising a modified sugar moiety and a modified nucleobase.
A. Modified sugar and phosphorous moieties In certain embodiments in which a nucleoside is not linked to another nucleoside at its 5 '-end (e.g., it is a monomer or it is the 5'-terminal nucleoside of an oligonucleotide), the nucleoside may comprise a modified phosphate or phosphorus moiety at the 5 '-end (note that in certain embodiments, nucleoside that are linked to another nucleoside at the 5 '-end may also comprise modified phosphates or phosphorus moieties, though such circumstance is typically refered to herein as a modified internucleoside linkage). In certain embodiments, the invention provides nucleosides comprising a modification at the 5 '-position of the sugar. Herein, modifications at the 5 '-position of the sugar or its substituents are typically referred to as modified sugars and modifications distal to that position are referred to as modified phosphates. One of skill in the art will appreciate that the boundary between these terms, particularly once modifications are introduced, becomes arbitrary. The example below shows a modified nucleoside comprising an a sulfur atom in place of the oxygen that links the phosphorus moiety and the sugar of a natural nucleoside. Herein, such modifications are typically referred to as modified phosphates, however, one of skill in the art will recognize that such a modification could also be referred to as a modified sugar comprising a sulfer linked to the 5'- position of the sugar.
Figure imgf000054_0001
In certain embodiments, nucleosides of the present invention comprise modified phosphates. In certain embodiments, nucleosides of the present invention comprise 5 '-sugar modifications. In certain embodiments, nucleosides of the present invention comprise both modified phosphates and 5 '-sugar modifications. Examples of nucleosides having such modified phosphorus moieties and/or 5 '-modifications include, but are not limited to:
Figure imgf000055_0001
The above examples are intended to illustrate and not to limit the invention as regards modifications at the 5'- phosphate and the 5'-position of the sugar. In the above illustrative examples, the 2'-position of the sugar is labeled Rx. However, in certain embodiments of the present invention, nucleosides comprising modified phosphate and/or 5 '-modified sugar groups may further comprise a modification at the 2'-position of the sugar. Many such 2'-modifϊcations are known in the art. In certain embodiments, Rx in any of the above examples may be selected from: a halogen (including, but not limited to F), allyl, amino, azido, thio, O-allyl, -O-Ci-Cio alkyl, -0-C1-C10 substitued alkyl, -OCF3, -O-(CH2)2-O-CH3, -O(CH2)2SCH3, -O-(CH2)2-O- N(R1n)(Rn), -0-CH2-C(=0)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl, -O[(CH2)nO]mCH3, -O(CH2)nNH2, -O(CH2)nCH3, -O(CH2)nONH2, - OCH2C(=O)N(H)CH3,-O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10; C1 to C10 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, poly- alkylamino, substituted silyl. In certain embodiments, Rx isselected from: -O-Methyl, -O-Ethyl, -O-Propyl, - O-Phenyl, O-methoxyethyl, S-Methyl, NMA, DMAEAc, DMAEOE, -0-CH2CH2F. In certain embodiments, Rx is any substituents described herein or known in the art. In certain embodiments, the nucleoside is not modified at the 2 '-position (i.e., Rx is H (DNA) or Rx is OH (RNA)). In certain embodiments, such nucleosides are at the 5 'end of an oligonucleotide.
In certain embodiments, nucleosides have Formula I:
Figure imgf000056_0001
wherein:
Bx is a heterocyclic base moiety; A iS O5 S Or N(R1);
Ri is H, Ci-C6 alkyl or substituted Ci-C6 alkyl; one of Ti and T2 is H, a protecting group or a phosphorus moiety and the other of Ti and T2 is H, a protecting group or a reactive phosphorus group; one of Qi and Q2 is H, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl and the other of Qi and Q2 is Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; G, is O-[C(R2)(R3)]n-[(C=O)m-X]j-Z or halogen; each R2 and R3 is, independently, H or halogen; X is O, S or N(E1);
Z is H, halogen, Ci-C6 alkyl, substituted C]-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(E2)(E3);
E1, E2 and E3 are each, independently, H, C1-C6 alkyl or substituted Ci-C6 alkyl; n is from 1 to about 6; m is O or 1; j is O or 1; each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ1, N(Ji)(J2), =NJb SJ1, N3, CN, OC(=L)Jl5 OC(=L)N(J0(J2) and C(=L)N(J0(J2); L is O, S orNJ3; each Ji, J2 and J3 is, independently, H or Q-C6 alkyl; and when j is 1 then Z is other than halogen or N(E2)(E3) and when A is O then Gi is other than halogen.
In certain embodiments, the compounds of Formula I are provided having the configuration:
Figure imgf000057_0001
In certain embodiments, nucleosides have Formula II:
Figure imgf000057_0002
wherein independently for each monomer of Formula II:
Bx is a heterocyclic base moiety; A is O, S or N(Ri);
Ri is H, Ci-C6 alkyl or substituted C1-C6 alkyl; one OfT3 and T4 is an internucleoside linking group linking the monomer to the oligomeric compound and the other of T3 and T4 is H, a protecting group, a phosphorus moiety, a 5 ' or 3 '-terminal group or an internucleoside linking group linking the monomer to the oligomeric compound; one of Qi and Q2 is H, Q-C6 alkyl, substituted CrC6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl and the other of Qi and Q2 is Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
Gi is O-[C(R2)(R3)]n-[(C=O)m-X]j-Z or halogen; each R2 and R3 is, independently, H or halogen;
X is O, S OrN(E1);
Z is H, halogen, C]-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl OrN(E2)(E3);
Ei, E2 and E3 are each, independently, H, C1-C6 alkyl or substituted Ci-C6 alkyl; n is from 1 to about 6; m is 0 or 1 ; j is 0 or 1; each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ,, N(J1)(J2), =NJ,, SJ1, N3, CN, OC(=L)Ji, OC(=L)N(Jj)(J2) and C(=L)N(J0(J2); L is O, S orNJ3; each J], J2 and J3 is, independently, H or CrC6 alkyl; and when j is 1 then Z is other than halogen or N(E2)(E3) and when A is O then Gi is other than halogen.
In certain embodiments, nucleosides of Formula II have the configuration:
Figure imgf000058_0001
In certain embodiments, nucleosides have Formula III:
Figure imgf000058_0002
wherein:
Bx is a heterocyclic base moiety; T5 is a phosphorus moiety or a reactive phosphorus group;
Te is H, a protecting group or a reactive phosphorus group;
Qi5 Ch5 Cb and Q4 are each, independently, H, halogen, C1-C6 alkyl, substituted C]-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; G1 is O-[C(R2)(R3)]n-[(C=O)m-X]j-Z or halogen; each R2 and R3 is, independently, H or halogen;
X is O, S or N(Ei);
Z is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl OrN(E2)(E3); Ei, E2 and E3 are each, independently, H, Ci-C6 alkyl or substituted C1-C6 alkyl; n is from 1 to about 6; m is 0 or 1; j is O or 1; each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJi, N(Ji)(J2), =NJb SJ1, N3, CN, OC(=L)Jl5 OC(=L)N(Ji)(J2) and Q=L)N(J1)(J2);
L is O, S or NJ3; each J1, J2 and J3 is, independently, H or Ci-C6 alkyl; when j is 1 then Z is other than halogen or N(E2)(E3); and when Qi, Q2, Q3 and Q4 are each H or when Qi and Q2 are H and Q3 and Q4 are each F or when Q1 and Q2 are each H and one of Q3 and Q4 is H and the other of Q3 and Q4 is R9 then Gi is other than H, hydroxyl, OR9, halogen, CF3, CCl3, CHCl2 and CH2OH wherein R9 is alkyl, alkenyl, alkynyl, aryl or alkaryl.
In certain embodiments, the compounds of Formula III have the configuration:
Figure imgf000059_0001
In certain embodiments, nucleosides have Formula IV:
Figure imgf000060_0001
wherein independently for each monomer of Formula IV:
Bx is a heterocyclic base moiety; one ofT7 and T8 is an internucleoside linking group linking the monomer to the oligomeric compound and the other of T7 and T8 is H, a hydroxyl protecting group, a phosphorus moiety, a 5' or 3'- terminal group or an internucleoside linking group linking the monomer to the oligomeric compound;
Qb Q2, Q3 and Q4 are each, independently, H, halogen, C]-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
G1 is O-[C(R2)(R3)]n-[(C=O)m-X]j-Z or halogen; each R2 and R3 is, independently, H or halogen; X is O, S or N(E1);
Z is H, halogen, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(E2)(E3);
E], E2 and E3 are each, independently, H, Ci-C6 alkyl or substituted Ci-C6 alkyl; n is from 1 to about 6; m is O or l; j is 0 or 1; each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ1, N(J1)(J2), =NJl5 SJ1, N3, CN, OC(=L)Jls OCt=L)N(J1)(J2) and C(=L)N(Jj)(J2);
L is O, S or NJ3; each Ji, J2 and J3 is, independently, H or Ci-C6 alkyl; when j is 1 then Z is other than halogen or N(E2)(E3); and when Qi, Q2, Q3 and Q4 are each H or when Qi and Q2 are H and Q3 and Q4 are each F or when Qi and Q2 are each H and one of Q3 and Q4 is H and the other OfQ3 and Q4 is R9 then Gi is other than H, hydroxyl, OR9, halogen, CF3, CCl3, CHCl2 and CH2OH wherein R9 is alkyl, alkenyl, alkynyl, aryl or alkaryl. In certain embodiments, oligomeric compounds are provided comprising at least one monomer having Formula IV, wherein each monomer of Formula IV has the configuration:
Figure imgf000061_0001
In certain embodiments, the present invention provides nucleosides comprising a modification at the 2'-position of the sugar. In certain embodiments, the invention provides nucleosides comprising a modification at the 5'-positin of the sugar. In certain embodiments, the invention provides nucleosides comprising modifications at the 2'-position and the 5'-position of the sugar. In certain embodiments, modified nucleosides may be useful for incorporation into oligonucleotides. In certain embodiment, modified nucleosides are incorporated into oligonucleosides at the 5 '-end of the oligonucleotide. In certain embodiments, modified nucleosides of the present invention have Formula VII:
Figure imgf000061_0002
wherein:
Bx is a heterocyclic base moiety;
Xi is O, S, N, or CRi0Ri i wherein each Ri0 and Rn is, independently, H or Ci-C6 alkyl; T3 is a phosphorus moiety;
T4 is an internucleoside linking group attaching the nucleoside to the remainder of the oligonucleotide;
Ri is selected from halogen, amino, trifluoroalkyl, trifiuoroalkoxy, azido, aminooxy, Ci-C6 alkyl, C2- C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted Ci-C6 alkenyl, substituted C2-C6 alkynyl, O- alkyl, S-alkyl, N(J4)-alkyl, O-alkenyl, S-alkenyl, N(J4)-alkenyl, O-alkynyl, S-alkynyl or N(J4)-alkynyl, O- aryl, S-aryl, N-aryl, O-aralkyl, S-aralkyl, N(J4)-aralkyl and -X2-V, wherein:
X2 is O, S or CR7R8 wherein each R7 and R8 is, independently, H or Ci-C6 alkyl; V is selected from -CH2COOH, -CH2CONH2, -CH2COOCH2CH3, -CH2CONH(CHz)1-S-R4 where i is from 1 to 10, -CH2CONH(CH2)JNR5R6 where j is from 1 to 6, and -CH2CONH{(CH2)k,-N(H)}k2-(CH2)klNH2 where each kl is independently from 2 to 4 and k2 is from 2 to 10;
R4 is selected from H, CpC6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C]-C6 alkyl, substituted Ci-C6 alkenyl, substituted C2-C6 alkynyl, C6-C]4 aryl and a thio protecting group; R5 and R6 are each, independently, selected from H, Q-C6 alkyl, substituted Cj-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Q-C6 alkyl, substituted C]-C6 alkenyl and substituted C2-C6 alkynyl; and each qi and q2 is, independently, selected from H, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Q-C6 alkyl, substituted Q-C6 alkenyl and substituted C2-C6 alkynyl; provided that if qi and q2 are both H, then:
R1 is selected from, trifluoroalkoxy, azido, aminooxy, S-alkyl, N(J4)-alkyl, O-alkenyl, S-alkenyl, N(J4)-alkenyl, O-alkynyl, S-alkynyl or N(J4)-alkynyl, and X2-V; or X1 is S, N, or CR7R8; wherein each substituted group is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, OJi, NJiJ2, SJi, N3, OC(=O)Ji and CN, wherein each Ji and J2 is, independently, H or Ci-C6 alkyl; and J4 is hydrogen, or a protecting group.
In certain embodiments, the present invention provides modified nucleosides. In certain embodiments, modified nucleosides of the present invention have Formula XI.
Figure imgf000062_0001
XI wherein:
Bx is a heterocyclic base moiety;
Xi is O, S, N, or CRioRπ wherein each Ri0 and Rn is, independently, H or Q-C6 alkyl; Ti is H, a hydroxyl protecting group, or a phosphorus moiety; T2 is H, a hydroxyl protecting group, or a reactive phosphorus group; each qi and q2 is, independently selected from H, C] -C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted Cj-C6 alkenyl, and substituted C2-C6 alkynyl, provided that at least one of qi and q2 is other than H;
X2 is O, S or CR7R8 wherein each R7 and Rg is, independently, H, Q-C6 alkyl, or substituted Ci-C6; V is selected from Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted Q- C6 alkenyl, substituted C2-C6 alkynyl, CH2COOH, CH2COONH2, CH2COOEt, -CH2CONH(CH2)J-S-R4 where i is from 1 to 10, CH2CONH(CH2)JNR5R6 where j is from 1 to 6, and
Figure imgf000062_0002
(CH2XiNH2 where each kl is independently from 2 to 4 and k2 is from 2 to 10;
R4 Is selected from H, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted Ci-C6 alkenyl, substituted C2-C6 alkynyl, C6-Ci4 aryl and a thio protecting group; R5 and R6 are each, independently selected from H, C] -C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted Ci-C6 alkenyl and substituted C2-C6 alkynyl; and wherein each substituted group is, independently selected from mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, OJ1, NJiJ2, SJi, N3, OC(=O)Ji and CN, wherein each Ji and J2 is independently H or Ci-C6 alkyl.
In certain embodiments, nucleosides of the present invention include, but are not limited to any of the following:
Figure imgf000063_0001
Figure imgf000063_0002
Figure imgf000064_0001
Figure imgf000064_0002
Figure imgf000065_0001
In certain embodiments, such nucleosides are incorporated into oligomeric compounds. In certain embodiments, such nucleosides are incorporated at the 5 '-terminal end of an oligonucleotide or oligomeric compound.
In certain embodiments, the present invention provides modified oligonucleotides comprising one or more nucleosides comprising one or more previously described modification. In certain embodiments, such previously described modification is a modified sugar moiety. In certain embodiments, a modified sugar moiety is a bicyclic sugar moiety. In certain embodiments a modified sugar moiety is a non-bicyclic modified sugar moiety.
Certain modified sugar moiety moieties are known and can be used to alter, typically increase, the affinity of the antisense compound for its target and/or increase nuclease resistance. A representative list of preferred modified sugar moieties includes but is not limited to bicyclic modified sugar moieties (BNA's), including methyleneoxy (4'-CH2-O-2') BNA, ethyleneoxy (4'-(CH2)2-O-2') BNA and methyl(methyleneoxy) (4'-C(CH3)H-O-2') BNA; substituted sugar moieties, especially 2'-substituted sugar moieties having a 2'-F, 2'-OCH3 or a 2'-O(CH2)2-OCH3 substituent group; and 4'-thio modified sugar moieties. Sugar moieties can also be replaced with sugar moiety mimetic groups among others. Methods for the preparations of modified sugar moieties are well known to those skilled in the art. Some representative patents and publications that teach the preparation of such modified sugar moieties include, but are not limited to, U.S. Patents: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; 6,172,209; 6,271,358; and 6,600,032; and WO 2005/121371.
Certain Bicyclic sugar moieties
In certain embodiments, the present invention provides modified nucleosides 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 one of the formulae: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' and 4'-CH(CH2OCH3)-O-2' (and analogs thereof see U.S. Patent 7,399,845, issued on July 15, 2008); 4'-C(CH3)(CH3)-O-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-O-N(CH3)-2' (see published U.S. Patent Application US2004-0171570, published September 2, 2004 ); 4'-CH2-N(R)-O-2', wherein R is H, C1-C12 alkyl, or a protecting group (see U.S. Patent 1, All, 612, issued on September 23, 2008); 4'-CH2-C(H)(CH3)^' (see Chattopadhyaya, et a!., 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). Certain such sugar moieties have been described. 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. A., 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) 8362-79 (JuI. 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 Inverts. Drugs, 2001, 2, 558-561; Braasch et al, Chem. Biol, 2001, 8 1-7; and Oram et al, Curr. Opinion MoI 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 α-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, nucleosides comprising a bicyclic sugar moiety have increased affinity for a complementary nucleic acid. In certain embodiments, nucleosides comprising a bicyclic sugar moiety provide resistance to nuclease degradation of an oligonucleotide in which they are incorporated. For example, methyleneoxy (4'-CH2-O-2') BNA and other bicyclic sugar moiety analogs display duplex thermal stabilities with complementary DNA and RNA (Tm = +3 to +10° C), stability towards 3'-exonucleolytic degradation and good solubility properties. Antisense oligonucleotides comprising BNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 5633-5638).
Certain bicyclic-sugar moiety containing nucleosides (or BNA nucleosides) comprise a bridge linking the 4' carbon and the 2' carbon of the sugar moiety. In certain embodiments, the bridging group is a methyleneoxy (4'-CH2-O-2'). In certain embodiments, the bridging group is an ethyleneoxy (4'-CH2CH2-O- 2') (Singh et al., Chem. Commun., 1998, 4, 455-456: Morita et al, Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226).
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 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(R1,)-, -C(Ra)=N-, -C(=NRa)-, -C(=O)-, -C(=S)-, -O-, -Si(Ra)r, -S(=O)X-, and -N(R1)-; 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, C1-Cj2 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C]2 alkenyl, C2-Ci2 alkynyl, substituted C2-Ci2 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, OJi, NJ1J2, SJh N3, COOJb acyl (C(=O)- H), substituted acyl, CN, sulfonyl (S(=O)2-Ji), or sulfoxyl (S(=O)-Ji); and each Ji and J2 is, independently, H, CrCi2 alkyl, substituted Ci-Ci2 alkyl, C2-Ci2 alkenyl, substituted C2-Ci2 alkenyl, C2-Ci2 alkynyl, substituted C2-Ci2 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)- H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, Ci-Ci2 aminoalkyl, substituted CpCi2 aminoalkyl or a protecting group. In certain embodiments, the bridge of a bicyclic sugar moiety is , -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]n-O-,
-C(R3Rb)-N(Ri)-O- or -C(R2Rb)-O-N(R3)-. In certain embodiments, the bridge is 4'-CH2-2', 4'-(CH2)2-2', 4'- (CH2)3-2'5 4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-O-N(Ra)-2' and 4'-CH2-N(Ra)-O-2I- wherein each Ra is, independently, H, a protecting group or Ci-Ci2 alkyl.
In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4'-2' methylenoxy bridge, may be in the α-L configuration or in the β-D configuration. Previously, alpha-L- methyleneoxy (4'-CH2-O-25) BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).
In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) α-L-Methyleneoxy (4'-CH2-O-2') BNA , (B) β-D-Methyleneoxy (4'-CH2-O-2') BNA , (C) Ethyleneoxy (4'-(CH2)2-O-2') BNA , (D) Aminooxy (4'-CH2-O-N(R)-2') BNA, (E) Oxyamino (4'-CH2-N(R)-O-2') BNA, and (F) Methyl(methyleneoxy) (4'-C(CH3)H-O-2!) BNA, as depicted below.
Figure imgf000068_0001
wherein Bx is the base moiety. In certain embodiments, bicyclic nucleosides include, but are not limited to, the structures below:
Figure imgf000068_0002
Figure imgf000068_0003
wherein Bx is the base moiety.
In certain embodiments, bicyclic nucleoside having the formula:
Figure imgf000068_0004
wherein Bx is a heterocyclic base moiety;
-Qa-Qb-Qc- is -CH2-N(Rc)-CH2-, -Q=O)-N(Rc)-CH2-, -CH2-O-N(Rc)- OrN(Rc)-O-CH2-; R0 is C1-Cn alkyl or an amino protecting group; and
Ta and Tb are each, independently, hydroxyl, a protected hydroxyl, a conjugate group, an activated phosphorus moiety or a covalent attachment to a support medium.
In certain embodiments, bicyclic nucleoside having the formula:
Figure imgf000069_0001
wherein:
Bx is a heterocyclic base moiety; Tc is H or a hydroxyl protecting group; Td is H, a hydroxyl protecting group or a reactive phosphorus group;
Za is Q-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amide.
In one embodiment, each of the substituted groups, is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, hydroxyl, OJC, NJcJd, SJ0, N3, OC(=X)JC, OC(=X)NJcJd, NJeC(=X)NJcJd and CN, wherein each J0, Jd and J6 is, independently, H or C1-C6 alkyl, and X is O, S or NJC.
In one embodiment, each of the substituted groups, is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ0, NJJd, SJC, N3, OC(=X)JC, and NJeC(=X)NJJd, wherein each Jc, Jd and Je is, independently, H, CpC6 alkyl, or substituted Ci-C6 alkyl and X is O orNJc.
In one embodiment, the Za group is CrC6 alkyl substituted with one or more Xx, wherein each X" is independently OJC, NJJd, SJC, N3, OC(=X)JC, OC(=X)NJJd, NJeC(=X)NJJdor CN; wherein each J0, Jd and Je is, independently, H or Q-C6 alkyl, and X is O, S or NJC. In another embodiment, the Za group is C1-C6 alkyl substituted with one or more Xx, wherein each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O-), substituted alkoxy or azido.
In certain embodiments, bicyclic nucleoside having the formula:
Figure imgf000069_0002
wherein:
Bx is a heterocyclic base moiety; one of Te and Tfis H or a hydroxyl protecting group and the other of Te and Tf is H, a hydroxyl protecting group or a reactive phosphorus group;
Zb is Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl or substituted acyl (C(=O)-); wherein each substituted group is mono or poly substituted with substituent groups independently selected from halogen, C]-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, OJ1, SJb NJfJ8, N3, COOJ6 CN, O-C(=O)NJfJg, N(H)CC=NH)NRdRe or N(H)C(=X)N(H)Jg wherein X is O or S; and each Jf and Jg is, independently, H, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2- C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, Cj-C6 aminoalkyl, substituted Ci-C6 aminoalkyl or a protecting group. In certain embodiments, bicyclic nucleoside having the formula:
Figure imgf000070_0001
wherein:
Bx is a heterocyclic base moiety; one of Tg and Th is H or a hydroxyl protecting group and the other of Tg and Th is H, a hydroxyl protecting group or a reactive phosphorus group;
Rf is Ci-C6 alkyl, substituted C]-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; qa and qb are each independently, H, halogen, CpC6 alkyl, substituted Cj-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, Cj-C6 alkoxyl, substituted Q-C6 alkoxyl, acyl, substituted acyl, Ci-C6 aminoalkyl or substituted Ci-C6 aminoalkyl; qc and qd are each independently, H, halogen, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, Cj-C6 alkoxyl, substituted C]-C6 alkoxyl, acyl, substituted acyl, Cj-C6 aminoalkyl or substituted Cj-C6 aminoalkyl; wherein each substituted group is, independently, mono or poly substituted with substituent groups independently selected from halogen, OJh, SJh, NJhJj, N3, COOJh, CN, 0-CC=O)NJhJ1, N(H)C(=NH)NJhJ, or N(H)CC=X)N(H)J1WhCrCin X is O or S; and each Jh and J; is, independently, H, CpC6 alkyl, C2-Ce alkenyl, C2-CO alkynyl, C1-C6 aminoalkyl or a protecting group. In certain embodiments, bicyclic nucleoside having the formula:
Figure imgf000071_0001
wherein:
Bx is a heterocyclic base moiety; one of T, and Tj is H or a hydroxyl protecting group and the other of T1 and Tj is H, a hydroxyl protecting group or a reactive phosphorus group; qe and qf are each, independently, halogen, Ci-Cj2 alkyl, substituted Ci-Ci2 alkyl, C2-Ci2 alkenyl, substituted C2-Ci2 alkenyl, C2-Ci2 alkynyl, substituted C2-Ci2 alkynyl, Ci-Ci2 alkoxy, substituted Ci-Ci2 alkoxy, OJj, SJJ, SOJJ, SO2JJ, NJjJk, N3, CN, C(=O)OJJ5 C(=O)NJjJk, C(=O)JJ5 O-C(=O)NJjJk, N(H)C(=NH)NJjJk, N(H)C(=O)NJjJk or N(H)C(=S)NJjJk; or qe and qf together are =C(qg)(qh); qg and qh are each, independently, H, halogen, Ci-Ci2 alkyl or substituted CrCi2 alkyl; each substituted group is, independently, mono or poly substituted with substituent groups independently selected from halogen, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, OJj, SJj, NJjJfc, N3, CN, C(=O)OJj, C(=O)NJjJk, C(=O)Jj, O-C(=O)NJjJk, N(H)C(=O)NJjJkorN(H)C(=S)NJjJk; and each Jj and Jk is, independently, H, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C6 aminoalkyl or a protecting group.
The synthesis and preparation of the methyleneoxy (4'-CH2-O-2') BNA 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-O-2') BNA, methyleneoxy (4'-CH2-O-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.
Certain Non-Bicyclic Modified Sugar Moieties
In certain embodiments, the present invention provides modified nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. Certain such modified nucleosides are known. In certain embodiments, the sugar ring of a nucleoside may be modified at any position. Examples of sugar modifications useful in this invention include, but are not limited to compounds comprising a sugar substituent group selected from: OH, F, O-alkyl, S-alkyl, N-alkyl, or O-alkyl-0-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cj0 alkyl or C2 to Ci0 alkenyl and alkynyl. In certain such embodiments, such substituents are at the 2' position of the sugar.
In certain embodiments, modified nucleosides comprise a substituent at the 2' position of the sugar. In certain embodiments, such substituents are selected from: a halide, including, but not limited to F, allyl, amino, azido, thio, O-allyl, 0-Ci-C10 alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-O- N(R1n)(Rn), or O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted Ci-Cio alkyl.
In certain embodiments, modified nucleosides suitable for use in the present invention are: 2- methoxyethoxy, 2'-O-methyl (2'-O- CH3), 2'-fluoro (2'-F).
In certain embodiments, modified nucleosides having a substituent group at the 2'-position selected from: O[(CH2)nO]mCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, OCH2C(=O)N(H)CH3, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. Other 2'-sugar substituent groups include: Ci to C10 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, 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 T- O- methyl, O-propyl, and 0-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, P., HeIv. 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).
In certain embodiments, 2'-Sugar substituent groups are in either the arabino (up) position or ribo (down) position. In certain such embodiments, a 2'-arabino modification is 2'-F arabino (FANA). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
In certain embodiments, nucleosides suitable for use in the present invention have sugar surrogates such as cyclobutyl in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;
5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, each of which is herein incorporated by reference in its entirety.
In certain embodiments, nucleosides suitable for use in the present invention comprise sugar surrogates, which replace the pentafuranose ring of an unmodified nucleoside. In certain embodiments, such sugar surrogates include but are not limited to substituted or unsubstituted tetrahydropyran rings, such as F- HNA.
In certain embodiments, modified tetrahydropyran nucleoside (F-HNA) having the formula:
Figure imgf000073_0001
wherein:
Bx is a heterocyclic base moiety; Tk is a hydroxyl protecting group; La is H, halogen, Ci-C6 alkyl or substituted CpCe alkyl; Zc is O" or OEa; Zd is OH, OEa or N(Ea)(Eb); each Ea and Eb is, independently, alkyl or substituted alkyl; q» qj, qk, qi, qm> qnand qp are each, independently, H, Ci-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; wherein each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJm, NJ1nJn, SJm, N3, OC(=X)Jm, OC(=X)NJmJn, NJpC(=X)NJmJn and CN, wherein each Jm, Jn and Jp is, independently, H or Q-C6 alkyl, and X is O, S or NJm.
In certain embodiments, tetrahydropyran nucleoside analogs having Formula F are provided wherein q« qj5 qi» qi> qm, qnand qp are each H. In certain embodiments, at least one of q,, c^, qk, qls qm, qnand qp is other than H. In certain embodiments, at least one of q;, qj, qk, q1} qm, qn and qp is methyl. In certain embodiments, tetrahydropyran nucleoside analogs having Formula F are provided wherein La is F. In certain embodiments, La is H.
In certain embodiments, tetrahydropyran nucleoside analogs having Formula F are provided wherein Zc is O" and Zd is OH. In certain embodiments, Z0 is O(CH2)2CN, Zd is N[CH2(CH3)2]2 and Tk is 4,4'- dimethoxytrityl. In certain embodiments, Zc is O' and Zd is OH which provides an H phosphonate group at the 4' position of the tetrahydropyran nucleoside analog which can also be written as 3'-O-P(=O)(H)(OH or O"amine+). In certain embodiments, Zc is O(CH2)2CN, Zd is N[CH2(CH3)2]2 and Tk is 4,4'-dimethoxytrityl which provides a phosphoramidite at the 3 '-position.
B. 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, Swayze, E.E. andBhat, 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 O- 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 al, 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 ΔTm 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 Tn, 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, JJ.; 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. III. Certain oligonucleotides
In certain embodiments, the present invention provides modified oligonucleotides. In certain embodiments, modified oligonucleotides of the present invention comprise modified nucleosides. In certain embodiments, modified oligonucleotides of the present invention comprise modified internucleoside linkages. In certain embodiments, modified oligonucleotides of the present invention comprise modified nucleosides and modified internucleoside linkages.
A. Certain Internucleoside Linkages
In such embodiments, nucleosides may be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters (P=O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P=S). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-O- C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Oligonucleotides having non-phosphorus internucleoside 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, internucleoside 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 internucleoside 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), α 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 "internucleoside linkage" or "internucleoside linking group" is meant to include all manner of internucleoside linking groups known in the art including but not limited to, phosphorus containing internucleoside linking groups such as phosphodiester and phosphorothioate, and non-phosphorus containing internucleoside linking groups such as formacetyl and methyleneimino. Internucleoside linkages also includes neutral non-ionic internucleoside linkages such as amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5') and methylphosphonate wherein a phosphorus atom is not always present.
As used herein the phrase "neutral internucleoside linkage" is intended to include internucleoside linkages that are non-ionic. Neutral internucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MM (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5')5 amide-4 (3'-CH2-N(H)- C(=0)-5'), formacetal (3'-O-CH2-O-5'), and thioformacetal (3'-S-CH2-O-5'). Further neutral internucleoside 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 internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 component parts.
B. Certain Lengths
In certain embodiments, the invention provides oligomeric compounds comprising oligonucleotides. In certain embodiments, the present invention provides oligomeric compounds including oligonucleotides of any of a variety of ranges of lengths. In certain embodiments, the invention provides oligomeric compounds comprising oligonucleotides consisting of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X≤Y. For example, in certain embodiments, the invention provides oligomeric compounds which comprise oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22,
10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26,
11 to 27, 11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14,
13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to
26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21,
14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to
27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides. In embodiments where the number of nucleosides of an oligomeric compound or oligonucleotide is limited, whether to a range or to a specific number, the oligomeric compound or oligonucleotide may, nonetheless further comprise additional other substituents. For example, an oligonucleotide consisting of 8-30 nucleosides excludes oligonucleotides having 31 nucleosides, but, unless otherwise indicated, such an oligonucleotide may further comprise, for example one or more conjugates, terminal groups, or other substituents. In certain embodiments, terminal groups include, but are not limited to, terminal group nucleosides. In such embodiments, the terminal group nucleosides are differently modified than the terminal nucleoside of the oligonucleotide, thus distinguishing such terminal group nucleosides from the nucleosides of the oligonucleotide. C. Certain motifs In certain embodiments, the present invention provides oligonucleotides comprising one or more regions having a particular nucleoside motif.
L Certain 5 '-terminal nucleosides
In certain embodiments, the 5 '-terminal nucleoside of a modified oligonucleotide of the present invention comprises a phosphorous moiety at the 5'-end. In certain embodiments the 5'-terminal nucleoside comprises a 2 '-modification. In certain such embodiments, the 2 '-modification of the 5'-terminal nucleoside is a cationic modification. In certain embodiments, the 5'-terminal nucleoside comprises a 5'-modifϊcation. In certain embodiments, the 5 '-terminal nucleoside comprises a 2'-modification and a 5 '-modification.
In certain embodiments, the 5 '-terminal nucleoside of an oligonucleotide is a nucleoside of Formula π. In certain embodiments, the 5 '-terminal nucleoside is a nucleoside of Formula IV. In certain embodiments, the 5 '-terminal nucleoside is a nucleoside of Formula VI. In certain embodiments, the 5'- terminal nucleoside is a nucleoside of Formula VII. In certain embodiments, the 5 '-terminal nucleoside is a nucleoside of Formula VIII. In certain embodiments, the 5'-terminal nucleoside is a nucleoside of Formula XIII. In certain embodiments, the 5'-terminal nucleoside is a nucleoside of Formula XTV. In certain embodiments, the two 5'-terminal nucleosides have Formula IX. In certain embodiments, the two 5'-terminal nucleosides have Formula X. In certain embodiments, the 5'-terminal nucleoside is a 5 '-stabilizing nucleoside. In certain embodiments, the modifications of the 5'-terminal nucleoside stabilize the 5'-phosphate. In certain embodiments, oligonucleotides comprising modifications of the 5 '-terminal nucleoside are resistant to exonucleases. In certain embodiments, oligonucleotides comprising modifications of the 5 '-terminal nucleoside have improved antisense properties. In certain such embodiments, oligonucleotides comprising modifications of the 5 '-terminal nucleoside have improved association with members of the RISC pathway. In certain embodiments, oligonucleotides comprising modifications of the 5 '-terminal nucleoside have improved affinity for Ago2.
In certain embodiments, the 5 'terminal nucleoside is attached to a plurality of nucleosides by a modified linkage. In certain such embodiments, the 5 'terminal nucleoside is a plurality of nucleosides by a phosphorothioate linkage.
2. Certain alternating regions
In certain embodiments, oligonucleotides of the present invention comprise one or more regions of alternating modifications. In certain embodiments, oligonucleotides comprise one or more regions of alternating nucleoside modifications. In certain embodiments, oligonucleotides comprise one or more regions of alternating linkage modifications. In certan embodiments, oligonucleotides comprise one or more regions of alternating nucleoside and linkage modifications.
In certain embodiments, oligonucleotides of the present invention comprise one or more regions of alternating 2'-F modified nucleosides and 2'-0Me modified nucleosides. In certain such embodiments, such regions of alternating 2'F modified and 2'0Me modified nucleosides also comprise alternating linkages. In certan such embodiments, the linkages at the 3' end of the 2'-F modified nucleosides are phosphorothioate linkages. In certain such embodiments, the linkages at the 3 'end of the 2'OMe nucleosides are phosphodiester linkages. In certain embodiments, such alternating regions are:
(2'-F)-(PS)-(2'-OMe)-(PO) In certain embodiments, oligomeric compounds comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 such alternatig regions. Such regions may be contiguous or may be interupted by differently modified nucleosides or linkages.
In certan embodiments, one or more alternating regions in an alternating motif include more than a single nucleoside of a type. For example, oligomeric compounds of the present invention may include one or more regions of any of the following nucleoside motifs:
AABBAA;
ABBABB;
AABAAB;
ABBABAABB; ABABAA;
AABABAB;
ABABAA;
ABBAABBABABAA;
BABBAABBABABAA; or ABABBAABBABABAA; wherein A is a nucleoside of a first type and B is a nucleoside of a second type. In certain embodiments, A and B are each selected from 2'-F, 2'-OMe, BNA, DNA, and MOE.
In certain embodiments, A is DNA. In certain embodiments, B is 4'-CH2O-2'-BNA. In certain embodiments, A is DNA and B is 4'-CH2O-2'-BNA. In certain embodiments A is 4'-CH2O-2'-BNA. In 5 certain embodiments, B is DNA. In certain embodiments A is 4'-CH2O-2'-BNA and B is DNA. In certain embodiments, A is 2'-F. In certain embodiments, B is 2'-0Me. In certain embodiments, A is 2'-F and B is 2'-OMe. In certain embodiemtns, A is 2'-OMe. In certain embodiments, B is 2'-F. In certain embodiments, A is 2'-0Me and B is 2'-F. In certain embodiments, A is DNA and B is 2'-0Me. In certain embodiments, A is 2'-0Me and B is DNA. 0 In certain embodiments, oligomeric compounds having such an alternating motif also comprise a 5' terminal nucleoside comprising a phosphate stabilizing modification. In certain embodiments, oligomeric compounds having such an alternating motif also comprise a 5' terminal nucleoside comprising a T- cationic modification. In certain embodiments, oligomeric compounds having such an alternating motif also comprise a 5' terminal nucleoside of formula II, IV, VI, VII, VIII, XIII, or XTV. In certain embodiments, oligomeric 5 compounds having such an alternating motif comprise a 5 ' terminal di-nucleoside of formula DC or X.
3. Two-Two-Three Motifs
In certain embodiments, oligonucleotides of the present invention comprise a region having a 2-2-3 motif. Such regions comprises the following motif:
5'- (E)w-(A)2-(B)x-(A)2-(C)y-(A)3-(D)z 0 wherein: A is a first type of modifed nucleosde;
B, C, D, and E are nucleosides that are differently modified than A, however, B, C, D, and E may have the same or different modifications as one another; w and z are from O to 15; x and y are from 1 to 15. 5 In certain embodiments, A is a 2'-0Me modified nucleoside. In certain embodiments, B, C, D, and
E are all 2'-F modified nucleosides. In certain embodiments, A is a 2'-0Me modified nucleoside and B, C, D, and E are all 2'-F modified nucleosides.
In certain embodiments, the linkages of a 2-2-3 motif are all modifed linkages. In certain embodiments, the linkages are all phosphorothioate linkages. In certain embodiemtns, the linkages at the 3'- O end of each modification of the first type are phosphodiester.
In certain embodiments, Z is O. In such embodiments, the region of three nucleosides of the first type are at the 3'-end of the oligonucleotide. In certain embodiments, such region is at the 3'-end of the oligomeric compound, with no additional groups attached to the 3' end of the region of three nucleosides of the first type. In certain embodiments, an oligomeric compound comprising an oligonucleotide where Z is O, may comprise a terminal group attached to the 3 '-terminal nucleoside. Such terminal groups may include additional nucleosides. Such additional nucleosides are typically non-hybridizing nucleosides.
In certain embodiments, Z is 1-3. In certain embodiments, Z is 2. In certain embodiments, the nucleosides of Z are 2'-MOE nucleosides. In certain embodiments, Z represents non-hybridizing nucleosides. To avoid confussion, it is noted that such non-hybridizing nucleosides might also be described as a 3 '-terminal group with Z=O.
D. Combinations of Motifs
It is to be understood, that certain of the above described motifs and modifications may be combined. Since a motif may comprises only a few nucleosides, a particular oligonucleotide may comprise two or more motifs. By way of non-limiting example, in certain embodiments, oligomeric compounds may have nucleoside motifs as described in the table below. In the table below, the term "None" indicates that a particular feature is not present in the oligonucleotide. For example, "None" in the column labeled "5' motif/modification" indicates that the 5' end of the oligonucleotide comprises the first nucleoside of the central motif.
Figure imgf000081_0001
Oligomeric compounds having any of the various nucleoside motifs described herein, may have any linkage motif. For example, the oligomeric compounds, including but not limited to those described in the above table, may have a linkage motif selected from non-limiting the table below:
Figure imgf000082_0001
As is apparent from the above, non-limiting tables, the lengths of the regions defined by a nucleoside motif and that of a linkage motif need not be the same. For example, the 3 'region in the nucleoside motif table above is 2 nucleosides, while the 3 '-region of the linkage motif table above is 6-8 nucleosides. Combining the tables results in an oligonucleotide having two 3 '-terminal MOE nucleosides and six to eight 3 '-terminal phosphorothioate linkages (so some of the linkages in the central region of the nucleoside motif are phosphorothioate as well). To further illustrate, and not to limit in any way, nucleoside motifs and sequence motifs are combined to show five non-limiting examples in the table below. The first column of the table lists nucleosides and linkages by position from Nl (the first nucleoside at the 5 '-end) to N20 (the 20th position from the 5 '-end). In certain embodiments, oligonucleotides of the present invention are longer than 20 nucleosides (the table is merely exemplary). Certain positions in the table recite the nucleoside or linkage "none" indicating that the oligonucleotide has no nucleoside at that position.
Figure imgf000082_0002
Figure imgf000083_0001
In the above, non-limiting examples:
Column A represent an oligomeric compound consisting of 20 linked nucleosides, wherein the oligomeric compound comprises: a modified 5 '-terminal nucleoside of Formula II, IV, VI, VII, VIII, DC, X, Xπi, or XTV; a region of alternating nucleosides; a region of alternating linkages; two 3 '-terminal MOE nucleosides, each of which comprises a uracil base; and a region of six phosphorothioate linkages at the 3'- end.
Column B represents an oligomeric compound consisting of 18 linked nucleosides, wherein the oligomeric compound comprises: a modified 5 '-terminal nucleoside of Formula II, IV, VI, VII, VIII, IX, X, XIII, or XTV; a 2-2-3 motif wherein the modified nucleoside of the 2-2-3 motif are 2 O-Me and the remaining nucleosides are all 2'-F; two 3 '-terminal MOE nucleosides, each of which comprises a uracil base; and a region of six phosphorothioate linkages at the 3 '-end.
Column C represents an oligomeric compound consisting of 20 linked nucleosides, wherein the oligomeric compound comprises: a modified 5'-terminal nucleoside of Formula II, IV, VI, VII, VIII, IX, X, XIII, or XTV; a region of uniformly modified 2'-F nucleosides; two 3 '-terminal MOE nucleosides, each of which comprises a uracil base; and wherein each internucleoside linkage is a phosphorothioate linkage.
Column D represents an oligomeric compound consisting of 20 linked nucleosides, wherein the oligomeric compound comprises: a modified 5 '-terminal nucleoside of Formula II, IV, VI, VII, VIII, EX, X, Xiπ, or XTV; a region of alternating 2'-OMe/2'-F nucleosides; a region of uniform 2'F nucleosides; a region of alternating phosphorothioate/phosphodiester linkages; two 3 '-terminal MOE nucleosides, each of which comprises an adenine base; and a region of six phosphorothioate linkages at the 3 '-end.
Column E represents an oligomeric compound consisting of 17 linked nucleosides, wherein the oligomeric compound comprises: a modified 5 '-terminal nucleoside of Formula II, IV, VI, VII, VIII, IX, X, XIII, or XTV; a 2-2-3 motif wherein the modified nucleoside of the 2-2-3 motif are 2'F and the remaining nucleosides are all 2'-OMe; three 3'-terminal MOE nucleosides.
Column F represents an oligomeric compound consisting of 18 linked nucleosides, wherein the oligomeric compound comprises: a region of alternating 2'-OMe/2'-F nucleosides; a region of uniform 2'F nucleosides; a region of alternating phosphorothioate/phosphodiester linkages; two 3 '-terminal MOE nucleosides, one of which comprises a uracil base and the other of which comprises an adenine base; and a region of six phosphorothioate linkages at the 3 '-end.
The above examples are provided solely to illustrate how the described motifs may be used in combination and are not intended to limit the invention to the particular combinations or the particular modifications used in illustrating the combinations. Further, specific examples herein, including, but not limited to those in the above table are intended to encompass more generic embodiments. For example, column A in the above table exemplifies a region of alternating 2'-0Me and 2'-F nucleosides. Thus, that same disclosure also exemplifies a region of alternating different 2'-modifications. It also exemplifies a region of alternating 2'-0-alkyl and 2 '-halogen nucleosides. It also exemplifies a region of alternating differently modified nucleosides. All of the examples throughout this specification contemplate such generic interpretation.
It is also noted that the lengths of oligomeric compounds, such as those exemplified in the above tables, can be easily manipulated by lengthening or shortening one or more of the described regions, without disrupting the motif.
IV. Oligomeric Compounds In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, oligomeric compounds comprise an oligonucleotide. In certain embodiments, an oligomeric compound comprises an oligonucleotide and one or more conjugate and/or terminal groups. Such conjugate and/or terminal groups may be added to oligonucleotides having any of the chemical motifs discussed above. Thus, for example, an oligomeric compound comprising an oligonucleotide having region of alternating nucleosides may comprise a terminal group.
A. Certain Conjugate Groups In certain embodiments, oligomeric compounds are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligomeric compound including but not limited to pharmacodynamics, pharmacokinetics, stability, 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 conjugate linking moiety or conjugate linking group to a parent compound such as an oligomeric compound, such as an oligonucleotide. 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. Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
In certain embodiments, a conjugate group comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (<S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. Patent Application 09/334,130.
Representative U.S. patents that teach the preparation of oligonucleotide conjugates include, but are not limited to, U.S.: 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.
In certain embodiments, conjugate groups are directly attached to oligonucleotides in oligomeric compounds. In certain embodiments, conjugate groups are attached to oligonucleotides by a conjugate linking group. In certain such embodiments, conjugate linking groups, including, but not limited to, bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein. Conjugate linking groups are useful for attachment of conjugate groups, such as chemical stabilizing groups, functional groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound. In general a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group. In some embodiments, the conjugate linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups that are routinely used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.
Some nonlimiting examples of conjugate linking moieties include pyrrolidine, 8-amino-3,6- dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and 6- aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted Cl- ClO alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
Conjugate groups may be attached to either or both ends of an oligonucleotide (terminal conjugate groups) and/or at any internal position.
In certain embodiments, conjugate groups are at the 3 '-end of an oligonucleotide of an oligomeric compound. In certain embodiments, conjugate groups are near the 3 '-end. In certain embodiments, conjugates are attached at the 3 'end of an oligomeric compound, but before one or more terminal group nucleosides. In certain embodiments, conjugate groups are placed within a terminal group. Solely to illustrate such groups at a 3 '-end, and not to limit such groups, the following examples are provided.
Figure imgf000087_0002
Figure imgf000087_0001
In certain embodiments, conjugate groups are attached to a nucleoside. Such a nucleoside may be incorporated into an oligomeric compound or oligonucleotide. In certain embodiments conjugated nucleotides may be incorporated into an oligonucleotide at the 5' terminal end. In certain embodiments conjugated nucleotides may be incorporated into an oligonucleotide at the 3' terminal end. In certain embodiments conjugated nucleotides may be incorporated into an oligonucleotide internally. Solely for illustration, and not to limit the conjugate or its placement, the following example shows oligonucleotides where each uracil nucleoside is, separately replaced with a conjugated thymidine nucleoside:
Figure imgf000087_0003
Figure imgf000088_0002
Figure imgf000088_0001
B. Terminal Groups
In certain embodiments, oligomeric compounds comprise terminal groups at one or both ends. In certain embodiments, a terminal group may comprise any of the conjugate groups discussed above. In certain embodiments, terminal groups may comprise additional nucleosides and/or inverted abasic nucleosides. In certain embodiments, a terminal group is a stabilizing group. In certain embodiments, oligomeric compounds comprise one or more terminal stabilizing group that enhances properties such as for example nuclease stability. Included in stabilizing groups are cap structures. The terms "cap structure" or "terminal cap moiety," as used herein, refer to chemical modifications, which can be attached to one or both of the termini of an oligomeric compound. These terminal modifications protect the oligomeric compounds having terminal nucleic acid moieties from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap) or at the 3'-terminus (3'-cap) or can be present on both termini. In non-limiting examples, the 5'-cap includes inverted abasic residue (moiety), 4',5'-methylene nucleotide; l-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L- nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl riboucleotide, 3 '-3 '-inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3 '-phosphate; 3'-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for more details see Wincott et al., International PCT publication No. WO 97/26270).
Particularly suitable 3'-cap structures of the present invention include, for example 4',5'-methylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; l,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2- aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha- nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxy-pentyl nucleotide, 5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety; 5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino; bridging and/or non-bridging 5'-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties (for more details see Beaucage and Tyer, 1993, Tetrahedron 49, 1925 and Published U.S. Patent Application Publication No. US 2005/0020525 published on January 27, 2005). Further 3' and 5'-stabilizing groups that can be used to cap one or both ends of an oligomeric compound to impart nuclease stability include those disclosed in WO 03/004602.
1. Terminal-group Nucleosides
In certain embodiments, one or more additional nucleosides is added to one or both terminal ends of an oligonucleotide of an oligomeric compound. Such additional terminal nucleosides are referred to herein as terminal-group nucleosides. In a double-stranded compound, such terminal-group nucleosides are terminal (3' and/or 5') overhangs. In the setting of double-stranded antisense compounds, such terminal-group nucleosides may or may not be complementary to a target nucleic acid.
In a single-stranded antisense oligomeric compound, terminal-group nucleosides are typically non- hybridizing. The terminal-group nucleosides are typically added to provide a desired property other than hybridization with target nucleic acid. Nonetheless, the target may have complementary bases at the positions corresponding with the terminal-group nucleosides. Whether by design or accident, such complementarity of one or more terminal-group nucleosides does not alter their designation as terminal-group nucleosides. In certain embodiments, the bases of terminal-group nucleosides are each selected from adenine (A), uracil (U), guanine (G), cytosine (C), thymine (T), and analogs thereof. In certain embodiments, the bases of terminal-group nucleosides are each selected from adenine (A), uracil (U), guanine (G), cytosine (C), and thymine (T). In certain embodiments, the bases of terminal-group nucleosides are each selected from adenine (A), uracil (U), and thymine (T). In certain embodiments, the bases of terminal-group nucleosides are each selected from adenine (A) and thymine (T). In certain embodiments, the bases of terminal-group nucleosides are each adenine (A). In certain embodiments, the bases of terminal-group nucleosides are each thymine (T). In certain embodiments, the bases of terminal-group nucleosides are each uracil (U). In certain embodiments, the bases of terminal-group nucleosides are each cytosine (C). In certain embodiments, the bases of terminal-group nucleosides are each guanine (G).
In certain embodiments, terminal-group nucleosides are sugar modified. In certain such embodiments, such additional nucleosides are 2'-modifed. In certain embodiments, the 2 '-modification of terminal-group nucleosides are selected from 2'-F, 2'-OMe, and 2'-MOE. In certain embodiments, terminal- group nucleosides are 2'-MOE modified. In certain embodiments, terminal-group nucleosides comprise 2'- MOE sugar moieties and adenine nucleobases (2'-MOE A nucleosides). In certain embodiments, terminal- group nucleosides comprise 2'-MOE sugar moieties and uracil nucleobases (2'-MOE U nucleosides). In certain embodiments, terminal-group nucleosides comprises 2'-MOE sugar moieties and guanine nucleobases (2'-MOE G nucleosides). In certain embodiments, terminal-group nucleosides comprises 2'-MOE sugar moieties and thymine nucleobases (2'-MOE T nucleosides). In certain embodiments, terminal-group nucleosides comprises 2'-MOE sugar moieties and cytosine nucleobases (2'-MOE C nucleosides).
In certain embodiments, terminal-group nucleosides comprise bicyclic sugar moieties. In certain such embodiments, terminal-group nucleosides comprise LNA sugar moieties. In certain embodiments, terminal-group nucleosides comprise LNA sugar moieties and adenine nucleobases (LNA A nucleosides). In certain embodiments, terminal-group nucleosides comprise LNA sugar moieties and uracil nucleobases (LNA nucleosides). In certain embodiments, terminal-group nucleosides comprise LNA sugar moieties and guanine nucleobases (LNA G nucleosides). In certain embodiments, terminal-group nucleosides comprise LNA sugar moieties and thymine nucleobases (LNA T nucleosides). In certain embodiments, terminal-group nucleosides comprise LNA sugar moieties and cytosine nucleobases (LNA C nucleosides). In certain embodiments, oligomeric compounds comprise 1-4 terminal-group nucleosides at the
3 'end of the oligomeric compound. In certain embodiments, oligomeric compounds comprise 1-3 terminal- group nucleosides at the 3 'end of the oligomeric compound. In certain embodiments, oligomeric compounds comprise 1-2 terminal-group nucleosides at the 3 'end of the oligomeric compound. In certain embodiments, oligomeric compounds comprise 2 terminal-group nucleosides at the 3 'end of the oligomeric compound. In certain embodiments, oligomeric compounds comprise 1 terminal-group nucleoside at the 3 'end of the oligomeric compound. In certain embodiments having two or more terminal-group nucleosides, the two or more terminal-group nucleosides all have the same modification type and the same base. In certain embodiments having two or more terminal-group nucleosides, the terminal-group nucleosides differ from one another by modification and/or base. In certain embodiments, oligomeric compounds comprise a 3'-terminal group comprising 2 terminal- group nucleosides, wherein each terminal group nucleoside is a 2'-MOE T. In certain embodiments, oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a 2'-MOE A. In certain embodiments, oligomeric compounds comprise a 3'- terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a 2'- MOE U. In certain embodiments, oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a 2'-MOE C. In certain embodiments, oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a 2'-MOE G.
In certain embodiments, oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal- group nucleosides, wherein each terminal group nucleoside is a LNA T. In certain embodiments, oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a LNA A. In certain embodiments, oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a LNA U. In certain embodiments, oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a LNA C. In certain embodiments, oligomeric compounds comprise a 3 '-terminal group comprising 2 terminal-group nucleosides, wherein each terminal group nucleoside is a LNA G.
V. Antisense Compounds In certain embodiments, oligomeric compounds of the present invention are antisense compounds. In such embodiments, the oligomeric compound is complementary to a target nucleic acid. In certain embodiments, a target nucleic acid is an RNA. In certain embodiments, a target nucleic acid is a non-coding RNA. In certain embodiments, a target nucleic acid encodes a protein. In certain embodiments, a target nucleic acid is selected from a mRNA, a pre-mRNA, a microRNA, a non-coding RNA, including small non- coding RNA, and a promoter-directed RNA. In certain embodiments, oligomeric compounds are at least partially complementary to more than one target nucleic acid. For example, oligomeric compounds of the present invention may be microRNA mimics, which typically bind to multiple targets.
Antisense mechanisms include any mechanism involving the hybridization of an oligomeric compound with target nucleic acid, wherein the hybridization results in a biological effect. In certain embodiments, such hybridization results in either target nucleic acid degradation or occupancy with concomitant inhibition or stimulation of the cellular machinery involving, for example, translation, transcription, or splicing of the target nucleic acid.
One type of antisense mechanism involving degradation of target RNA is RNase H mediated antisense. RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like" elicit RNase H activity in mammalian cells. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of DNA-like oligonucleotide-mediated inhibition of gene expression.
Antisense mechanisms also include, without limitation RNAi mechanisms, which utilize the RISC pathway. Such RNAi mechanisms include, without limitation siRNA, ssRNA and microRNA mechanisms. Such mechanism include creation of a microRNA mimic and/or an anti-microRNA. Antisense mechanisms also include, without limitation, mechanisms that hybridize or mimic non- coding RNA other than microRNA or mRNA. Such non-coding RNA includes, but is not limited to promoter-directed RNA and short and long RNA that effects transcription or translation of one or more nucleic acids. In certain embodiments, antisense compounds specifically hybridize when there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays. As used herein, "stringent hybridization conditions" or "stringent conditions" refers to conditions under which an antisense compound will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances, and "stringent conditions" under which antisense compounds hybridize to a target sequence are determined by the nature and composition of the antisense compounds and the assays in which they are being investigated. It is understood in the art that incorporation of nucleotide affinity modifications may allow for a greater number of mismatches compared to an unmodified compound. Similarly, certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences. One of ordinary skill in the art is capable of determining an appropriate number of mismatches between oligonucleotides, or between an oligonucleotide and a target nucleic acid, such as by determining melting temperature (Tm). Tm or ΔTm can be calculated by techniques that are familiar to one of ordinary skill in the art. For example, techniques described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allow one of ordinary skill in the art to evaluate nucleotide modifications for their ability to increase the melting temperature of an RNA:DNA duplex.
In certain embodiments, oligomeric compounds of the present invention are RNAi compounds. In certain embodiments, oligomeric compounds of the present invention are ssRNA compounds. In certain embodiments, oligomeric compounds of the present invention are paired with a second oligomeric compound to form an siRNA. In certain such embodiments, the second oligomeric compound is also an oligomeric compound of the present invention. In certain embodiments, the second oligomeric compound is any modified or unmodified nucleic acid. In certain embodiments, the oligomeric compound of the present invention is the antisense strand in an siRNA compound. In certain embodiments, the oligomeric compound of the present invention is the sense strand in an siRNA compound.
L Single-stranded antisense compounds
In certain embodiments, oligomeric compounds of the present invention are particularly suited for use as single-stranded antisense compounds. In certain such embodiments, such oligomeric compounds are single-stranded RNAi compounds. In certain embodiments, such oligomeric compounds are ssRNA compounds or microRNA mimics. Certain 5 '-terminal nucleosides described herein are suited for use in such single-stranded oligomeric compounds. In certain embodiments, such 5 '-terminal nucleosides stabilize the 5 '-phosphorous moiety. In certain embodiments, 5 '-terminal nucleosides of the present invention are resistant to nucleases. In certain embodiments, the motifs of the present invention are particularly suited for use in single-stranded oligomeric compounds.
Use of single-stranded RNAi compounds has been limited. In certain instances, single stranded RNAi compounds are quickly degraded and/or do not load efficiently into RISC. Certain compounds of the present invention possess properties superior to previously described ssRNAi compounds. In certain embodiments, oligomeric compounds of the present invention are superior ssRNAi compounds in vitro. In certain such embodiments, the 5 '-terminal phosphorous moiety is stabilized. In certain such embodiments, the 5 '-nucleoside is resistant to nuclease cleavage. In certain embodiments, the 5 '-terminal end loads efficiently into RISC. In certain embodiments, the motif stabilizes the oligomeric compound. In certain embodiments the 3 '-terminal end of the oligomeric compound is stabilized. Design of single-stranded RNAi compounds for use in cells and/or for use in vivo presents several challenges. For example, the compound must be chemically stable, resistant to nuclease degradation, capable of entering cells, capable of loading into RISC (e.g., binding Agol or Ago2), capable of hybridizing with a target nucleic acid, and not toxic to cells or animals. In certain instances, a modification or motif that improves one such feature may worsen another feature, rendering a compound having such modification or motif unsuitable for use as an RNAi compound. For example, certain modifications, particularly if placed at or near the 5 '-end of an oligomeric compound, may make the compound more stable and more resistant to nuclease degradation, but may also inhibit or prevent loading into RISC by blocking the interaction with RISC components, such as Agol or Ago2. Despite its improved stability properties, such a compound would be unsuitable for use in RNAi. Thus, the challenge is to identify modifications and combinations and placement of modifications that satisfy each parameter at least sufficient to provide a functional single- stranded RNAi compound. In certain embodiments, oligomeric compounds of the present invention combine modifications to provide single-stranded RNAi compounds that are active as single-stranded RNAi compounds.
In certain instances, a single-stranded oligomeric compound comprising a 5 '-phosphorous moiety is desired. For example, in certain embodiments, such 5 '-phosphorous moiety is necessary or useful for RNAi compounds, particularly, single-stranded RNAi compounds. In such instances, it is further desirable to stabilize the phosphorous moiety against degradation or de-phosphorolation, which may inactivate the compound. Further, it is desirable to stabilize the entire 5 '-nucleoside from degradation, which could also inactivate the compound. Thus, in certain embodiments, oligonucleotides in which the 5 '-phosphorous moiety and the 5 '-nucleoside have been stabilized are desired. In certain embodiments, the present invention provides modified nucleosides that may be placed at the 5 '-end of an oligomeric compound, resulting in stabilized phosphorous and stabilized nucleoside. In certain such embodiments, the phosphorous moiety is resistant to removal in biological systems, relative to unmodified nucleosides and/or the 5 '-nucleoside is resistant to cleavage by nucleases. In certain embodiments, such nucleosides are modified at one, at two or at all three of: the 2'-position, the 5'-position, and at the phosphorous moiety. Such modified nucleosides may be incorporated at the 5 '-end of an oligomeric compound.
Although certain oligomeric compounds of the present invention have particular use as single- stranded compounds, such compounds may also be paired with a second strand to create a double-stranded oligomeric compound. In such embodiments, the second strand of the double-stranded duplex may or may not also be an oligomeric compound of the present invention.
In certain embodiments, oligomeric compounds of the present invention bind and/or activate one or more nucleases. In certain embodiments, such binding and/or activation ultimately results in antisense activity. In certain embodiments, an oligomeric compound of the invention interacts with a target nucleic acid and with a nuclease, resulting in activation of the nuclease and cleavage of the target nucleic acid. In certain embodiments, an oligomeric compound of the invention interacts with a target nucleic acid and with a nuclease, resulting in activation of the nuclease and inactivation of the target nucleic acid. In certain embodiments, an oligomeric compound of the invention forms a duplex with a target nucleic acid and that duplex activates a nuclease, resulting in cleavage and/or inactivation of one or both of the oligomeric compound and the target nucleic acid. In certain embodiments, an oligomeric compound of the invention binds and/or activates a nuclease and the bound and/or activated nuclease cleaves or inactivates a target nucleic acid. Nucleases include, but are not limited to, ribonucleases (nucleases that specifically cleave ribonucleotides), double-strand nucleases (nucleases that specifically cleave one or both strands of a double- stranded duplex), and double-strand ribonucleases. For example, nucleases include, but are not limited to RNase H, an argonaute protein (including, but not limitied to Ago2), and dicer. In certain embodiments, oligomeric compounds of the present invention activate RNase H. RNase
H is a cellular nuclease that cleaves the RNA strand of a duplex comprising an RNA strand and a DNA or DNA-like strand. In certain embodiments, an oligomeric compound of the present invention is sufficiently DNA-like to activate RNase H, resulting in cleavage of an RNA nucleic acid target. In certain such embodiments, the oligomeric compound comprises at least one region comprised of DNA or DNA-like nucleosides and one or more regions comprised of nucleosides that are otherwise modified. In certain embodiments, such otherwise modified nucleosides increase stability of the oligomeric compound and/or its affinity for the target nucleic acid. Certain such oligomeric compounds posses a desirable combination of properties. For example, certain such compounds, by virtue of the DNA or DNA-like region, are able to support RNase H activity to cleave a target nucleic acid; and by virtue of the otherwise modified nucleosides, have enhanced affinity for the target nucleic acid and/or enhanced stability (including resistance to single- strand-specific nucleases). In certain embodiments, such otherwise modified nucleosides result in oligomeric compounds having desired properties, such as metabolic profile and/or pharmacologic profile.
In certain embodiments, oligomeric compounds of the present invention interact with an argonaute protein (Ago). In certain embodiments, such oligomeric compounds first enter the RISC pathway by interacting with another member of the pathway (e.g., dicer). In certain embodiments, oligomeric compounds first enter the RISC pathway by interacting with Ago. In certain embodiments, such interaction ultimately results in antisense activity. In certain embodiments, the invention provides methods of activating Ago comprising contacting Ago with an oligomeric compound. In certain embodiments, such oligomeric compounds comprise a modified 5 '-phosphate group. In certain embodiments, the invention provides methods of modulating the expression or amount of a target nucleic acid in a cell comprising contacting the cell with an oligomeric compound capable of activating Ago, ultimately resulting in cleavage of the target nucleic acid. In certain embodiments, the cell is in an animal. In certain embodiments, the cell is in vitro. In certain embodiments, the methods are performed in the presence of manganese. In certain embodiments, the manganese is endogenous. In certain embodiment the methods are performed in the absence of magnesium. In certain embodiments, the Ago is endogenous to the cell. In certain such embodiments, the cell is in an animal. In certain embodiments, the Ago is human Ago. In certain embodiments, the Ago is Ago2. In certain embodiments, the Ago is human Ago2.
In certain embodiments, oligomeric compounds of the present invention interact with the enzyme dicer. In certain such embodiments, oligomeric compounds bind to dicer and/or are cleaved by dicer. In certain such embodiments, such interaction with dicer ultimately results in antisense activity. In certain embodiments, the dicer is human dicer. In certain embodiments, oligomeric compounds that interact with dicer are double-stranded oligomeric compounds. In certain embodiments, oligomeric compounds that interact with dicer are single-stranded oligomeric compounds.
In embodiments in which a double-stranded oligomeric compound interacts with dicer, such double- stranded oligomeric compound forms a dicer duplex. In certain embodiments, any oligomeric compound described herein may be suitable as one or both strands of a dicer duplex. In certain embodiments, each strand of the dicer duplex is an oligomeric compound of the present invention. In certain embodiments, one strand of the dicer duplex is an oligomeric compound of the present invention and the other strand is any modified or unmodified oligomeric compound. In certain embodiments, one or both strands of a dicer duplex comprises a nucleoside of Formula II, IV, VI, VII, VIII, IX, X, XHI, or XTV at the 5' end. In certain embodiments, one strand of a dicer duplex is an antisense oligomeric compound and the other strand is its sense complement.
In certain embodiments, the dicer duplex comprises a 3 '-overhang at one or both ends. In certain embodiments, such overhangs are additional nucleosides. In certain embodiments, the dicer duplex comprises a 3 ' overhang on the sense oligonucleotide and not on the antisense oligonucleotide. In certain embodiments, the dicer duplex comprises a 3' overhang on the antisense oligonucleotide and not on the sense oligonucleotide. In certain embodiments, 3 Overhangs of a dicer duplex comprise 1-4 nucleosides. In certain embodiments, such overhangs comprise two nucleosides. In certain embodiments, the nucleosides in the 3'- overhangs comprise purine nucleobases. In certain embodiments, the nucleosides in the 3' overhangs comprise adenine nucleobases. In certain embodiments, the nucleosides in the 3' overhangs comprise pyrimidines. In certain embodiments, dicer duplexes comprising 3 '-purine overhangs are more active as antisense compounds than dicer duplexes comprising 3' pyrimidine overhangs. In certain embodiments, oligomeric compounds of a dicer duplex comprise one or more 3' deoxy nucleosides. In certain such embodiments, the 3' deoxy nucleosides are dT nucleosides. In certain embodiments, the 5' end of each strand of a dicer duplex comprises a phosphate moiety. In certain embodiments the antisense strand of a dicer duplex comprises a phosphate moiety and the sense strand of the dicer duplex does not comprise a phosphate moiety. In certain embodiments the sense strand of a dicer duplex comprises a phosphate moiety and the antisense strand of the dicer duplex does not comprise a phosphate moiety. In certain embodiments, a dicer duplex does not comprise a phosphate moiety at the 3' end. In certain embodiments, a dicer duplex is cleaved by dicer. In such embodiments, dicer duplexes do not comprise 2'-OMe modifications on the nucleosides at the cleavage site. In certain embodiments, such cleavage site nucleosides are RNA.
In certain embodiments, interaction of an oligomeric compound with dicer ultimately results in antisense activity. In certain embodiments, dicer cleaves one or both strands of a double-stranded oligomeric compound and the resulting product enters the RISC pathway, ultimately resulting in antisense activity. In certain embodiments, dicer does not cleave either strand of a double-stranded oligomeric compound, but nevertheless facilitates entry into the RISC pathway and ultimately results in antisense activity. In certain embodiments, dicer cleaves a single-stranded oligomeric compound and the resulting product enters the RISC pathway, ultimately resulting in antisense activity. In certain embodiments, dicer does not cleave the single- stranded oligomeric compound, but nevertheless facilitates entry into the RISC pathway and ultimately results in antisense activity.
In certain embodiments, the invention provides methods of activating dicer comprising contacting dicer with an oligomeric compound. In certain such embodiments, the dicer is in a cell. In certain such embodiments, the cell is in an animal. Dicer
In certain embodiments, oligomeric compounds of the present invention interact with the enzyme dicer. In certain such embodiments, oligomeric compounds bind to dicer and/or are cleaved by dicer. In certain such embodiments, such interaction with dicer ultimately results in antisense activity. In certain embodiments, the dicer is human dicer. In certain embodiments, oligomeric compounds that interact with dicer are double-stranded oligomeric compounds. In certain embodiments, oligomeric compounds that interact with dicer are single-stranded oligomeric compounds.
In embodiments in which a double-stranded oligomeric compound interacts with dicer, such double-stranded oligomeric compound forms a dicer duplex. In certain embodiments, any oligomeric compound described herein may be suitable as one or both strands of a dicer duplex. In certain embodiments, each strand of the dicer duplex is an oligomeric compound of the present invention. In certain embodiments, one strand of the dicer duplex is an oligomeric compound of the present invention and the other strand is any modified or unmodified oligomeric compound. In certain embodiments, one or both strands of a dicer duplex comprises a nucleoside of Formula II, IV, VI, VII, VIII, IX, X, XIH, or XIV at the 5'. In certain embodiments, one strand of a dicer duplex is an antisense oligomeric compound and the other strand is its sense complement.
In certain embodiments, a dicer duplex comprises a first and second oligomeric compound wherein each oligomeric compound comprises an oligonucleotide consisting of 25 to 30 linked nucleosides. In certain such embodiments, each oligonucleotide of the dicer duplex consists of 27 linked nucleosides. In certain embodiments, the dicer duplex comprises a 3 '-overhang at one or both ends. In certain embodiments, such overhangs are additional nucleosides. In certain embodiments, the dicer duplex comprises a 3' overhang on the sense oligonucleotide and not on the antisense oligonucleotide. In certain embodiments, the dicer duplex comprises a 3' overhang on the antisense oligonucleotide and not on the sense oligonucleotide. In certain embodiments, 3 Overhangs of a dicer duplex comprise 1-4 nucleosides. In certain embodiments, such overhangs comprise two nucleosides. In certain embodiments, 3 '-overhangs comprise purine nucleobases. In certain embodiments, 3 '-overhangs comprise adenine overhangs. In certain embodiments, 3 '-overhangs are pyrimidines. In certain embodiments, dicer duplexes comprising 3 '-purine overhangs are more active as antisense compounds than dicer duplexes comprising 3'-pyrimidine overhangs. In certain embodiments, oligomeric compounds of a dicer duplex comprise 3'-deoxy nucleosides. In certain such embodiments, the 3'-deoxy nucleosides are dT nucleosides.
In certain embodiments, the 5' end of each strand of a dicer duplex comprises phosphate moiety. In certain embodiments the antisense strand of a dicer duplex comprises a phosphate moiety and the sense strand of the dicer duplex does not comprises a phosphate moiety. In certain embodiments the sense strand of a dicer duplex comprises a phosphate moiety and the antisense strand of the dicer duplex does not comprises a phosphate moiety. In certain embodiments, a dicer duplex does not comprise a phosphate moiety at the 3'- end. In certain embodiments, a dicer duplex is cleaved by dicer. In such embodiments, dicer duplexes do not comprise 2'-OMe modifications at the nucleosides at the cleavage site. In certain embodiments, such cleavage site nucleosides are RNA.
One of skill will appreciate that the above described features of dicer duplexes may be combined. For example, in certain embodiments, a dicer duplex comprises a first oligomeric compound comprising an antisense oligonucleotide and a second oligomeric compound comprising a sense oligonucleotide; wherein the sense oligonucleotide comprises a 3' overhang consisting of two purine nucleosides and the antisense oligonucleotide comprises a 3 Overhang consisting of two adenosine or modified adenosine nucleosides; each of the sense and antisense oligonucleotides consists of 25 to 30 linked nucleosides, the 5'end of the antisense oligonucleotide comprises a phosphorous moiety, and wherein the dicer cleavage sites of the dicer duplex are not O-Me modified nucleosides.
Li certain embodiments, the invention provides single-stranded oligomeric compounds that interact with dicer. In certain embodiments, such single-stranded dicer compounds comprise a nucleoside of Formula II, IV, VI, VΠ, Viπ, IX, X, Xiπ, or XrV. In certain embodiments, single-stranded dicer compounds do not comprise a phosphorous moiety at the 3 '-end. In certain embodiments, such single-stranded dicer compounds may comprise a 3 '-overhangs. In certain embodiments, such 3 '-overhangs are additional nucleosides. In certain embodiments, such 3 '-overhangs comprise 1-4 additional nucleosides that are not complementary to a target nucleic acid and/or are differently modified from the adjacent 3' nucleoside of the oligomeric compound. In certain embodiments, a single-stranded oligomeric compound comprises an antisense oligonucleotide having two 3 '-end overhang nucleosides wherein the overhang nucleosides are adenine or modified adenine nucleosides. In certain embodiments, single stranded oligomeric compounds that interact with dicer comprise a nucleoside of Formula II, IV, VI, VII, VIII, IX, X, XIII, or XTV.
In certain embodiments, interaction of an oligomeric compound with dicer ultimately results in antisense activity. In certain embodiments, dicer cleaves one or both strands of a double-stranded oligomeric compound and the resulting product enters the RISC pathway, ultimately resulting in antisense activity. In certain embodiments, dicer does not cleave either strand of a double-stranded oligomeric compound, but nevertheless facilitates entry into the RISC pathway and ultimately results in antisense activity. In certain embodiments, dicer cleaves a single-stranded oligomeric compound and the resulting product enters the RISC pathway, ultimately resulting in antisense activity. In certain embodiments, dicer does not cleave the single- stranded oligomeric compound, but nevertheless facilitates entry into the RISC pathway and ultimately results in antisense activity. hi certain embodiments, the invention provides methods of activating dicer comprising contacting dicer with an oligomeric compound. In certain such embodiments, the dicer is in a cell. In certain such embodiments, the cell is in an animal. Ago
In certain embodiments, oligomeric compounds of the present invention interact with Ago. In certain embodiments, such oligomeric compounds first enter the RISC pathway by interacting with another member of the pathway (e.g., dicer), hi certain embodiments, oligomeric compounds first enter the RISC pathway by interacting with Ago. In certain embodiments, such interaction ultimately results in antisense activity, hi certain embodiments, the invention provides methods of activating Ago comprising contacting Ago with an oligomeric compound. In certain such embodiments, the Ago is in a cell. In certain such embodiments, the cell is in an animal.
2. Oligomeric compound identity In certain embodiments, a portion of an oligomeric compound is 100% identical to the nucleobase sequence of a microRNA, but the entire oligomeric compound is not fully identical to the microRNA. In certain such embodiments, the length of an oligomeric compound having a 100% identical portion is greater than the length of the microRNA. For example, a microRNA mimic consisting of 24 linked nucleosides, where the nucleobases at positions 1 through 23 are each identical to corresponding positions of a microRNA that is 23 nucleobases in length, has a 23 nucleoside portion that is 100% identical to the nucleobase sequence of the microRNA and has approximately 96% overall identity to the nucleobase sequence of the microRNA.
In certain embodiments, the nucleobase sequence of oligomeric compound is fully identical to the nucleobase sequence of a portion of a microRNA. For example, a single-stranded microRNA mimic consisting of 22 linked nucleosides, where the nucleobases of positions 1 through 22 are each identical to a corresponding position of a microRNA that is 23 nucleobases in length, is fully identical to a 22 nucleobase portion of the nucleobase sequence of the microRNA. Such a single-stranded microRNA mimic has approximately 96% overall identity to the nucleobase sequence of the entire microRNA, and has 100% identity to a 22 nucleobase portion of the microRNA.
E. Synthesis. Purification and Analysis
Oligomerization of modified and unmodified nucleosides and nucleotides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).
Oligomeric compounds provided herein can be conveniently and routinely made through the well- known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. The invention is not limited by the method of antisense compound synthesis.
Methods of purification and analysis of oligomeric compounds are known to those skilled in the art. Analysis methods include capillary electrophoresis (CE) and electrospray-mass spectroscopy. Such synthesis and analysis methods can be performed in multi-well plates. The method of the invention is not limited by the method of oligomer purification. 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, employed in the methods described herein is a pharmaceutical composition comprising an antisense compound and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS.
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.
In certain methods, preparations are made that include a polyamine compound or a lipid moiety complexed with a nucleic acid. In certain embodiments, such preparations comprise one or more compounds each individually having a structure defined by formula (I) or a pharmaceutically acceptable salt thereof,
Figure imgf000101_0001
wherein each Xa and Xb, for each occurrence, is independently Ci-6 alkylene; n is 0, 1, 2, 3, 4, or 5; each R is independently H, wherein at least n + 2 of the R moieties in at least about 80% of the molecules of the compound of formula (I) in the preparation are not H; m is 1, 2, 3 or 4; Y is O, NR2, or S; R1 is alkyl, alkenyl, or alkynyl; each of which is optionally substituted with one or more substituents; and R2 is H, alkyl, alkenyl, or alkynyl; each of which is optionally substituted each of which is optionally substituted with one or more substituents; provided that, if n = 0, then at least n + 3 of the R moieties are not H. Such preparations are described in PCT publication WO/2008/042973, which is herein incorporated by reference in its entirety.
Certain preparations, some of which are shown below, are described in Akinc et al., Nature Biotechnology 26, 561 - 569 (01 May 2008), which is herein incorporated by reference in its entirety.
Figure imgf000101_0002
Figure imgf000102_0001
Figure imgf000103_0001
Certain Antisense Oligomeric compounds
In certain embodiments, the invention provides oligomeric compounds comprising or consisting of antisense oligonucleotides. In certain embodiments, an antisense oligonucleotide comprises a phosphate stabilizing nucleoside. In certain embodiments, an antisense oligonucleotide comprises a phosphate stabilizing nucleoside at the 5 '-end. hi certain embodiments, a phosphate stabilizing nucleoside comprises a modified phosphate group and/or a modified sugar moiety.
In certain embodiments, an antisense oligonucleotide comprises a 5 '-stabilizing nucleotide. In certain embodiments, the 5 '-stabilizing nucleoside comprises a modified sugar moiety. In certain embodiments, the 5 '-end of an antisens compound comprises a phosphate stabilizing modification and a 5 '-stabilizing nucleoside. In certain embodiments, a single modification results in both phosphate stabilization and nucleoside stabilization. In certain embodiments, the phosphate stabilizing modification and the nucleoside stabilizing modification are different modifications. In certain embodiments, tow or more modifications at the 5 '-end of an oligomeric compound together provide phosphate stabilization and nucleoside stabilization. In certain embodiments, an antisense oligomeric compound comprises the following features selected from: a 5'-phosphate or 5'-modifed phosphate; a 5'-most nucleoside (position 1 nucleoside); a nucleoside second from the 5 '-end (position 2 nucleoside); a nucleoside third from the 5 '-end (position 3 nucleoside); a region having a nucleoside motif; a region having a linkage motif; a terminal group.
In certain embodiments, the 5 '-phosphate is selected from: unmodified phosphate, modified phosphate, phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphorothioate, phosphoramidate, alkylphosphonothioate, substituted alkylphosphonothioate, phosphorodithioate, thiophosphoramidate, and phosphotriester.
In certain embodiments, the 5'-phosphate is selected from: modified phosphate, phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphotriester, phosphorothioate, phosphorodithioate, thiophosphoramidate, and phosphoramidate.
In certain embodiments, the 5 '-phosphate is selected from: modified phosphate, phosphonate, alkylphosphonate, and substituted alkylphosphonate. In certain embodiments, the 5 '-phosphate is selected from 5'-deoxy-5'-thio phosphate, phosphoramidate, methylene phosphonate, mono-fluoro methylene phosphonate and di-fluoro methylene phosphonate.
In certain embodiments, the position 1 nucleoside comprises a modified sugar. In certain such embodiments, the sugar comprises a 5 '-modification. In certain embodiments, the sugar of the position 1 nucleoside comprises a 2'-modification. In certain embodiments, the sugar of the position 1 nucleoside comprises a 5 '-modification and a 2 '-modification. In certain embodiments, the 5 '-modification of the sugar of the position 1 nucleoside is selected from 5 '-alkyl,5 '-substituted alkyl, 5'-olkoxy, 5'-substitued alkoxy, and 5 '-halogen. In certain embodiments, the 5' modification of the sugar at position 1 is selected from 5'- alkyl and 5 '-substituted alkyl. In certain such embodiments, the modification is selected from methyl and ethyl. In certain embodiments, the 2' modification is selected from: halogen (including, but not limited to F), allyl, amino, azido, thio, O-allyl, -0-C1-Ci0 alkyl, -0-Ci-Ci0 substituted alkyl, -OCF3, -O-(CH2)2-O-CH3, - O(CH2)2SCH3, -O-(CH2)2-O-N(Rm)(Rn), -O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted Ci-C10 alkyl, -O[(CH2)nO]mCH3, -O(CH2)nNH2, -O(CH2)nCH3, - O(CH2)nONH2, -OCH2C(=O)N(H)CH3)-O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10; Ci to C10 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl. In certain embodiments, the 2'-modification of the sugar of the position 1 nucleoside is selected from: F, -0-C1-C1O alkyl, -0-Ci-C10 substituted alkyl, -OCF3, -O- (CH2)2-O-CH3, -O(CH2)2SCH3, -O-(CH2)2-O-N(Rm)(Rn), -0-Cm-CC=O)-N(R1n)(Rn), where each R31 and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl, -O[(CH2)nO]mCH3, -O(CH2)nNH2, - 0(CHz)nCH3, -O(CH2)nONH2, -OCH2C(O)N(H)CH3, -O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10; -O-aryl, S-alkyl, NMA, DMAEAc, DMAEOE, and -O-alkyl-F. In certain embodiments, the T- modification of the sugar of the position 1 nucleoside is selected from: F, -0-C1-C1O alkyl, -0-Ci-C10 substituted alkyl, -O-(CH2)2-O-CH3, -O(CH2)2SCH3, -O-(CH2)2-O-N(Rm)(Rn), -0-Cm-C(O)-N(RnO(Rn), where each Rn, and Rn is, independently, H or substituted or unsubstituted Ci-Ci0 alkyl, -O[(CH2)nO]mCH3, - O(CH2)nNH2, -O(CH2)nCH3, -O(CH2)nONH2, -OCH2C(O)N(H)CH3, -O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10; -O-aryl, S-alkyl, NMA, DMAEAc, DMAEOE, and -O-alkyl-F.
In certain embodiments, the position 2 nucleoside comprises a 2'-modification. In certain such embodiments, the 2'-modification of the position 2 nucleoside is selected from halogen, alkyl, and substituted alkyl. In certain embodiments, the 2 '-modification of the position 2 nucleoside is selected from 2'-F and T- alkyl. In certain embodiments, the 2'-modification of the position 2 nucleoside is 2'-F. In certain embodiments, the 2'-substitued of the position 2 nucleoside is an unmodified OH (as in naturally occurring RNA).
In certain embodiments, the position 3 nucleoside is a modified nucleoside. In certain embodiments, the position 3 nucleoside is a bicyclic nucleoside. In certain embodiments, the position 3 nucleoside comprises a sugar surrogate. In certain such embodiments, the sugar surrogate is a tetrahydropyran. In certain embodiments, the sugar of the position 3 nucleoside is a F-HNA.
In certain embodiments, an antisense oligomeric compound comprises an oligonucleotide comprising 10 to 30 linked nucleosides wherein the oligonucleotide comprises: a 5 '-terminal phosphate or modified phosphate: a position 1 modified nucleoside comprising a modified sugar moiety comprising: a 5'- modification; or a 2 '-modification; or both a 5'-modificaton and a 2'-modification; a position 2 nucleoside comprising a sugar moiety which is differently modified compared to the sugar moiety of the position 1 modified nucleoside; and from 1 to 4 3 '-terminal group nucleosides each comprising a 2'-modification; and wherein at least the seven 3 '-most internucleoside linkages are phosphorothioate linkages.
In certain such embodiments, the 5 '-terminal modified phosphate is selected from: phosphonate, alkylphosphonate, aminoalkyl phosphonate, phosphorothioate, phosphoramidite, alkylphosphonothioate, phosphorodithioate, thiophosphoramidate, phosphotriester; the5' -modification of the sugar moiety of the position 1 modified nucleoside is selected from 5'-alkyl and 5 '-halogen; the 2'-modification of the sugar moiety of the position 1 modified nucleoside is selected from: halogen (including, but not limited to F), allyl, amino, azido, thio, O-allyl, -O-Q-Cio alkyl, -0-Ci-C1O substituted alkyl, -OCF3, -O-(CH2)2-O-CH3, -O(CH2)2SCH3, -O-(CH2)2-O-N(Rm)(Rn), -O-CH2-C(=O> N(R1n)(Rn), where each R1n and Rn is, independently, H or substituted or unsubstituted Ci-Ci0 alkyl, - O[(CH2)nO]mCH3, -O(CH2)nNH2, -O(CH2)nCH3, -O(CH2)nONH2, -OCH2C(=O)N(H)CH3, - O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10; Ci to C]0 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl; and the sugar moiety of the position 2 nucleoside is selected from unmodified 2'-OH (RNA) sugar, and a modified sugar comprising a modification selected from: 2'-halogen, 2'0-alkyl, 2'-alkyl, 2 '-substituted alkyl.
In certain embodiments, the sugar moiety of the position 2 nucleoside comprises a 2'-F.
In certain embodiments, such oligonucleotides comprises 8 to 20, 10 to 15, 11 to 14, or 12 to 13 phosphorothioate interaucleoside linkages overall. In certain embodiments, the remaining internucleoside linkages are phosphodiester. In certain embodiments, the eighth internucleoside linkage from the 3 'end of the oligonucleotide is a phosphodiester. In certain embodiments, the ninth internucleoside linkage from the 3' end is a phosphodiester. In certain embodiments, each internucleoside linkage is either a phosphorothioate or a phosphodiester linkage.
In certain such embodiments, antisense oligomeric compounds have the features described in the following non-limiting table:
Figure imgf000106_0001
Figure imgf000107_0001
In certain embodiments, the third nucleoside from the 5'-end (position 3) is a modified nucleoside. In certain embodiments, the nucleoside at position 3 comprises a sugar modification. In certain such embodiments, the sugar moiety of the position 3 nucleoside is a bicyclic nucleoside. In certain embodiments the position 3 nucleoside is a modified non-bicyclic nucleoside. In certain embodiments, the position 3 nucleoside is selected from: F-HNA and 2'-OMe.
Certain Methods/Uses
In certain embodiments, the present invention provides compounds and methods for reducing the amount or activity of a target nucleic acid. In certain embodiments, the invention provides antisense compounds and methods. In certain embodiments, the invention provides antisense compounds and methods based on activation of RNase H. In certain embodiments, the invention provides RNAi compounds and methods.
In certain instances it is desirable to use an antisense compound that functions at least in part through RISC. In certain such instances unmodified RNA, whether single-stranded or double stranded is not suitable. Single-stranded RNA is relatively unstable and double-stranded RNA does not easily enter cells. The challenge has been to identify modifications and motifs that provide desirable properties, such as improved stability, without interfering with (and possibly even improving upon) the antisense activity of RNA through RNAi.
In certain embodiments, the present invention provides oligonucleotides having motifs (nucleoside motifs and/or linkage motifs) that result in improved properties. Certain such motifs result in single-stranded oligonucleotides with improved stability and/or cellular uptake properties while retaining antisense activity. For example, oligonucleotides having an alternating nucleoside motif and seven phosphorothioate linkages at to 3 '-terminal end have improved stability and activity. Similar compounds that comprise phosphorothioate linkages at each linkage have further improved stability, but are not active as RNAi compounds, presumably because the additional phosphorothioate linkages interfere with the interaction of the oligonucleotide with the RISC pathway components (e.g., with Ago). In certain embodiments, the oligonucleotides having motifs herein result in single-stranded RNAi compounds having desirable properties. In certain embodiments, such oligonucleotides may be paired with a second strand to form a double-stranded RNAi compound. In such embodiments, the second strand of such double-stranded RNAi compounds may comprise a motif of the present invention, may comprise another motif of modifications or may be unmodified.
It has been shown that in certain circumstances for single-stranded RNA comprising a 5 '-phosphate group has RNAi activity if but has much less RNAi activity if it lacks such 5 '-phosphate group. The present inventors have recognized that in certain circumstances unmodified 5'-phophate groups may be unstable (either chemically or enzymatically). Accordingly, in certain circumstances, it is desirable to modify the oligonucleotide to stabilize the 5 '-phosphate. In certain embodiments, this is achieved by modifying the phosphate group. In certain embodiments, this is achieved by modifying the sugar of the 5 '-terminal nucleoside. In certain embodiments, this is achieved by modifying the phosphate group and the sugar. In certain embodiments, the sugar is modified at the 5'-position, the 2'-position, or both the 5'-position and the 2'-position. As with motifs, above, in embodiments in which RNAi activity is desired, a phosphate stabilizing modification must not interfere with the ability of the oligonucleotide to interact with RISC pathway components (e.g., with Ago).
In certain embodiments, the invention provides oligonucleotides comprising a phosphate-stabilizing modification and a motif described herein. In certain embodiments, such oligonucleotides are useful as single-stranded RNAi compounds having desirable properties. In certain embodiments, such oligonucleotides may be paired with a second strand to form a double-stranded RNAi compound. In such embodiments, the second strand may comprise a motif of the present invention, may comprise another motif of modifications or may be unmodified RNA.
The target for such antisense compounds comprising a motif and/or 5 '-phosphate stabilizing modification of the present invention can be any naturally occurring nucleic acid. In certain embodiments, the target is selected from: pre-mRNA, mRNA, non-coding RNA, small non-coding RNA, pd-RNA, and microRNA. In embodiments, in which a target nucleic acid is a pre-RNA or a mRNA, the target may be the same as that of a naturally occurring micro-RNA (i.e., the oligonucleotide may be a microRNA mimic). In such embodiments, there may be more than one target mRNA.
In certain embodiments, the invention provides compounds and methods for antisense activity in a cell. In certain embodiments, the cell is in an animal. In certain embodiments, the animal is a human. In certain embodiments, the invention provides methods of administering a compound of the present invention to an animal to modulate the amount or activity or function of one or more target nucleic acid.
In certain embodiments oligonucleotides comprise one or more motifs of the present invention, but do not comprise a phosphate stabilizing modification. In certain embodiments, such oligonucleotides are useful for in vitro applications. In certain embodiments, such oligonucleotides are useful for in vivo applications where RISC activity is not required. For example, in certain embodiments, such oligonucleotides alter splicing of pre-mRNA.
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. Likewise, one of skill will appreciate that in certain circumstances using the conventions described herein, the same compound may be described in more than one way. For example, an antisense oligomeric compound having two non-hybridizing 3 '-terminal 2'-MOE modified nucleosides, but otherwise fully complementary to a target nucleic acid may be described as an oligonucleotide comprising a region of 2'- MOE-modifϊed nucleosides, wherein the oligonucleotide is less than 100% complementary to its target. Or that same compound may be described as an oligomeric compound comprising: (1) an oligonucleotide that is 100% complementary to its nucleic acid target and (2) a terminal group wherein the terminal group comprises two 2'-MOE modified terminal-group nucleosides. Such descriptions are not intended to be exclusive of one another or to exclude overlapping subject matter.
Examples (General)
1 1HH and 13C NMR spectra were recorded on a 300 MHz and 75 MHz Bruker spectrometer, respectively.
Example 1
Synthesis of Nucleoside Phosphoramidites
The preparation of nucleoside phosphoramidites is performed following procedures that are illustrated herein and in the art such as but not limited to US Patent 6,426,220 and published PCT WO 02/36743.
Example 2
Synthesis of Oligomeric Compounds
The oligomeric compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as alkylated derivatives and those having phosphorothioate linkages. Oligomeric compounds: Unsubstituted and substituted phosphodiester (P=O) oligomeric compounds, including without limitation, oligonucleotides can be synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.
In certain embodiments, phosphorothioate internucleoside linkages (P=S) are synthesized similar to phosphodiester internucleoside linkages with the following exceptions: thiation is effected by utilizing a 10% w/v solution of 3,H-l,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time is increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55°C (12-16 hr), the oligomeric compounds are recovered by precipitating with greater than 3 volumes of ethanol from a 1 M NH4OAc solution. Phosphinate internucleoside linkages can be prepared as described in U.S. Patent 5,508,270.
Alkyl phosphonate internucleoside linkages can be prepared as described in U.S. Patent 4,469,863. 3 '-Deoxy-3 '-methylene phosphonate internucleoside linkages can be prepared as described in U.S. Patents 5,610,289 or 5,625,050.
Phosphoramidite internucleoside linkages can be prepared as described in U.S. Patent, 5,256,775 or U.S. Patent 5,366,878. Alkylphosphonothioate internucleoside linkages can be prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively).
3'-Deoxy-3'-amino phosphoramidate internucleoside linkages can be prepared as described in U.S. Patent 5,476,925.
Phosphotriester internucleoside linkages can be prepared as described in U.S. Patent 5,023,243.
Borano phosphate internucleoside linkages can be prepared as described in U.S. Patents 5,130,302 and 5,177,198.
Oligomeric compounds having one or more non-phosphorus containing internucleoside linkages including without limitation methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone oligomeric compounds having, for instance, alternating MMI and P=O or P=S linkages can be prepared as described in U.S. Patents 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289.
Formacetal and thioformacetal internucleoside linkages can be prepared as described in U.S. Patents 5,264,562 and 5,264,564.
Ethylene oxide internucleoside linkages can be prepared as described in U.S. Patent 5,223,618.
Example 3
Isolation and Purification of Oligomeric Compounds
After cleavage from the controlled pore glass solid support or other support medium and deblocking in concentrated ammonium hydroxide at 55°C for 12-16 hours, the oligomeric compounds, including without limitation oligonucleotides and oligonucleosides, are recovered by precipitation out of 1 M NH4OAc with >3 volumes of ethanol. Synthesized oligomeric compounds are analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis is determined by the ratio of correct molecular weight relative to the -16 amu product (+/-32 +/-48). For some studies oligomeric compounds are purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material are generally similar to those obtained with non-HPLC purified material.
Example 4
Synthesis of Oligomeric Compounds using the 96 Well Plate Format Oligomeric compounds, including without limitation oligonucleotides, can be synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleoside linkages are afforded by oxidation with aqueous iodine. Phosphorothioate internucleoside linkages are generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta- cyanoethyl-diiso-propyl phosphoramidites can be purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, CA, or Pharmacia, Piscataway, NJ). Non-standard nucleosides are synthesized as per standard or patented methods and can be functionalized as base protected beta-cyanoethyldiisopropyl phosphoramidites. Oligomeric compounds can be cleaved from support and deprotected with concentrated NH4OH at elevated temperature (55-60 0C) for 12-16 hours and the released product then dried in vacuo. The dried product is then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.
Example 5
Analysis of Oligomeric Compounds using the 96- Well Plate Format
The concentration of oligomeric compounds in each well can be assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products can be evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition is confirmed by mass analysis of the oligomeric compounds utilizing electrospray- mass spectroscopy. All assay test plates are diluted from the master plate using single and multi-channel robotic pipettors. Plates are judged to be acceptable if at least 85% of the oligomeric compounds on the plate are at least 85% full length.
Example 6
In Vitro Treatment of Cells with Oligomeric Compounds
The effect of oligomeric compounds on target nucleic acid expression is tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. Cell lines derived from multiple tissues and species can be obtained from American Type Culture Collection (ATCC, Manassas, VA).
The following cell type is provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays or RT-PCR. b.END cells: The mouse brain endothelial cell line b.END was obtained from Dr. Werner Risau at the Max Plank Institute (Bad Nauheim, Germany). b.END cells are routinely cultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, CA). Cells are routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Cells are seeded into 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, MA) at a density of approximately 3000 cells/well for uses including but not limited to oligomeric compound transfection experiments.
Experiments involving treatment of cells with oligomeric compounds:
When cells reach appropriate confluency, they are treated with oligomeric compounds using a transfection method as described.
LIPOFECTIN™
When cells reached 65-75% confluency, they are treated with one or more oligomeric compounds. The oligomeric compound is mixed with LIPOFECTIN™ Invitrogen Life Technologies, Carlsbad, CA) in Opti-MEM™-1 reduced serum medium (Invitrogen Life Technologies, Carlsbad, CA) to achieve the desired concentration of the oligomeric compound(s) and a LIPOFECTIN™ concentration of 2.5 or 3 μg/mL per 100 nM oligomeric compound(s). This transfection mixture is incubated at room temperature for approximately 0.5 hours. For cells grown in 96-well plates, wells are washed once with 100 μL OPTI-MEM™-1 and then treated with 130 μL of the transfection mixture. Cells grown in 24-well plates or other standard tissue culture plates are treated similarly, using appropriate volumes of medium and oligomeric compound(s). Cells are treated and data are obtained in duplicate or triplicate. After approximately 4-7 hours of treatment at 37°C, the medium containing the transfection mixture is replaced with fresh culture medium. Cells are harvested 16-24 hours after treatment with oligomeric compound(s).
Other suitable transfection reagents known in the art include, but are not limited to, CYTOFECTIN™, LIPOFECTAMINE™, OLIGOFECTAMINE™, and FUGENE™. Other suitable transfection methods known in the art include, but are not limited to, electroporation.
Example 7
Real-time Quantitative PCR Analysis of target mRNA Levels
Quantitation of target mRNA levels is accomplished by real-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE- Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA) is attached to the 5' end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA) is attached to the 3' end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3' quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5'- exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.
Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be "multiplexed" with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only ("single-plexing"), or both (multiplexing).
Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.
RT and PCR reagents are obtained from Invitrogen Life Technologies (Carlsbad, CA). RT, real-time PCR is carried out by adding 20 μL PCR cocktail (2.5x PCR buffer minus MgCl2, 6.6 mM MgCl2, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5x ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction is carried out by incubation for 30 minutes at 48°C. Following a 10 minute incubation at 95°C to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol are carried out: 95°C for 15 seconds (denaturation) followed by 600C for 1.5 minutes (annealing/extension).
Gene target quantities obtained by RT, real-time PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RIBOGREEN™ (Molecular Probes, Inc. Eugene, OR). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, OR). Methods of RNA quantification by RIBOGREEN™ are taught in Jones, L.J., et al, (Analytical Biochemistry, 1998, 265, 368-374). In this assay, 170 μL of RIBOGREEN™ working reagent (RIBOGREEN™ reagent diluted 1 :350 in
1OmM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485nm and emission at 530nm.
Example 8
Analysis of oligonucleotide inhibition of target expression
Antisense modulation of a target expression can be assayed in a variety of ways known in the art. For example, a target mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR. Real-time quantitative PCR is presently desired. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. One method of RNA analysis of the present disclosure is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, CA and used according to manufacturer's instructions.
Protein levels of a target can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997. Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.
Example 9 Design of phenotypic assays and in vivo studies for the use of target inhibitors
Phenotypic assays
Once target inhibitors have been identified by the methods disclosed herein, the oligomeric compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition. Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of a target in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, OR; PerkinElmer, Boston, MA), protein-based assays including enzymatic assays (Panvera, LLC, Madison, WI; BD Biosciences, Franklin Lakes, NJ; Oncogene Research Products, San Diego, CA), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, MI), triglyceride accumulation (Sigma-Aldrich, St. Louis, MO), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, CA; Amersham Biosciences, Piscataway, NJ). In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay
(i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with a target inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.
Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest. Measurement of the expression of one or more of the genes of the cell after treatment is also used as an indicator of the efficacy or potency of the a target inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells. In vivo studies The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.
Example 10 RNA Isolation
PoIy(A)+ mRNA isolation
PoIy(A)+ mRNA is isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium is removed from the cells and each well is washed with 200 μL cold PBS. 60 μL lysis buffer ( 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) is added to each well, the plate is gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate is transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine CA). Plates are incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate is blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 700C, is added to each well, the plate is incubated on a 900C hot plate for 5 minutes, and the eluate is then transferred to a fresh 96-well plate.
Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions. Total RNA Isolation
Total RNA is isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, CA) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium is removed from the cells and each well is washed with 200 μL cold PBS. 150 μL Buffer RLT is added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol is then added to each well and the contents mixed by pipetting three times up and down. The samples are then transferred to the RNEASY 96™ well plate attached to a QIA VAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum is applied for 1 minute. 500 μL of Buffer RWl is added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum is again applied for 1 minute. An additional 500 μL of Buffer RWl is added to each well of the RNEASY 96™ plate and the vacuum is applied for 2 minutes. 1 mL of Buffer RPE is then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash is then repeated and the vacuum is applied for an additional 3 minutes. The plate is then removed from the QIA V AC™ manifold and blotted dry on paper towels. The plate is then re-attached to the QIA V AC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA is then eluted by pipetting 140 μL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.
The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia CA). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.
Example 11
Target-specific primers and probes
Probes and primers may be designed to hybridize to a target sequence, using published sequence information.
For example, for human PTEN, the following primer-probe set was designed using published sequence information (GENBANK™ accession number U92436.1, SEQ ID NO: 1).
Forward primer: AATGGCTAAGTGAAGATGACAATCAT (SEQ ID NO: 2) Reverse primer: TGCACATATCATTACACCAGTTCGT (SEQ ID NO: 3) And the PCR probe:
FAM-TTGCAGCAATTCACTGTAAAGCTGGAAAGG-TAMRA (SEQ ID NO: 4), where FAM is the fluorescent dye and TAMRA is the quencher dye.
Example 12 Western blot analysis of target protein levels
Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to a target is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale CA).
Example 13
Preparation of Compound 3
Figure imgf000120_0001
a) Preparation of 5'-0-(4,4'-dimethoxytrityl)-2'-0-(2-N-[2-(dimethylamino)ethyl]- acetamide)-5-methyluridine (Compound 2) Compound 1 was prepared according to published literature (Prakash et ah, Org. Let. 2003,
5, 403-406) using ethyl-2-bromoacetate for alkylation. Compound 1 (5.378 g, 8.50 mmol) was dissolved in anhydrous THF (66 mL). To this was added Ny/V-dimethylethylenediamine (18.7 mL, 170 mmol) and the reaction mixture was stirred at ambient temperature. After 6 h, toluene (80 mL) was added and the solvent was evaporated in vacuo to give Compound 2 as a white foam (6.12 g, 95%). 1H ΝMR (CDCl3): δ 7.64 (s, 3H), 7.41-6.79 (m, 13H), 5.94 (d, IH, J v,τ = 2.4 Hz), 4.41 (m, IH), 4.31 (q ab, 2H), 4.19 (m, IH), 3.95 (m, IH), 3.75 (s, 6H), 3.52 (m, 2H), 2.75 (m, 2H), 2.48 (m, 2H), 2.24 (s, 6H), 1.36 (s, 3H). 13C ΝMR (CDCl3): δ 170.1, 164.7, 158.7, 151.0, 144.4, 135.5, 135.3, 134.9, 130.1, 129.0, 128.1, 127.7, 127.1, 113.3, 110.9, 88.5, 86.7, 84.8, 83.3, 70.7, 68.2, 61.8, 58.4, 45.4, 36.0, 12.0. HRMS (MALDI) calcd for C37H44N4O9 + Na+: 711.3006. Found: 711.3001. TLC: CH2Cl2-EtOAc-MeOH-NEt3, 64:21 :21 :5, v/v/v/v; Rf 0.4.
b) Preparation of 5'-0-(4,4'-dimethoxytrityI)-2'-0-(2-7V-[2-(dimethylaniino)ethyl]- acetamide)-5-methyluridine-3'-(2-cyanoethyl-7VyV-diisopropylphosphoramidite) (Compound 3) Compound 2 (5.754 g, 8.35 mmol) was dried by coevaporation with anhydrous pyridine (2 x 75 mL) and then dissolved in CH2Cl2 (60 mL). To this solution, diisopropylamine tetrazolide (715 mg, 4.18 mmol) and 2-cyanoethyl-iV^V,iV,iV-tetraisopropylphosphordiamidite (3.18 mL, 10.02 mmol) were added. After 13 h, EtOAc (420 mL) was added and about 60 mL of solvent was evaporated in vacuo. The organic was washed with half-saturated NaHCO3 (3 x 80 mL), then with brine (2 x 40 mL), dried over MgSO4, filtered and evaporated in vacuo at 270C to give an oil. The resulting residue was coevaporated with toluene (2 x 300 mL) to give a foam which was then dissolved in CH2Cl2 (20 mL). Hexanes (1000 mL) were slowly added to the rapidly stirred solution via an addition funnel to yield a wax and the supernatant was decanted. The wax was washed with hexanes thrice and the washes were decanted. The precipitation was repeated one more time to give a white wax which was dried in vacuo at ambient temperature to give Compound 3 as a foam (6.60 g, 89%). LRMS (ES): m/z 889 (M + H+), 911 (M + Na+). 31P NMR (CDCl3): δ 151.5, 151.0. Compound 3 was incorporated into oligonucleotides according to standard solid phase synthesis procedures. Phosphorylation at the 5' end of oligonucleotides was achieved during synthesis by using Glen Research (Sterling, VA) chemical phosphorylation reagent.
Example 14
Preparation of Compound 4
Figure imgf000121_0001
Compound 4 was prepared according to the procedures described in published patent application WO 94/22890. Compound 4 was incorporated into oligonucleotides according to standard solid phase synthesis procedures. Phosphorylation at the 5' end of oligonucleotides was achieved during synthesis by using Glen Research (Sterling, VA) chemical phosphorylation reagent. Example 15
Preparation of Compound 13
Figure imgf000122_0001
a) Preparation of 5-O-Benzyol-3-O-(2-methylnaphthalene)-l,2-O-bis(acetyl)-5-(R)-methyl- ribose (Compound 6) Compound 5 was prepared according to the method of De Mesmaeker wherein NapBr was used instead of BnBr (Mesmaeker et al, Synlett, 1997, 1287-1290). Dried Compound 5 (21.1 g, 47.04 mmol) was dissolved in a mixture of glacial acetic acid (104 mL) and acetic anhydride (17.2 mL). To this solution was added 14 drops of concentrated H2SO4. After 1.5 h, the resulting light brown solution was diluted in EtOAc (600 mL), washed with sat. NaHCO3 (5 x 600 mL), dried over anhydrous Na2SO4, filtered, evaporated and dried under high vacuum to yield Compound 6 (22.7 g, 99%) as a pale oil. ES MS mlz 515.1 [M + Na] +.
b) Preparation of 5'-O-Benzyol-3'-O-(2-methylnaphthalene)-5'-(R)-methyl-5- methyluridine (Compound 7)
A mixture of Compound 6 (23.3 g, 46.70 mmol) and thymine (10.01 g, 79.40 mmol) was suspended in anhydrous CH3CN (233 mL). To this mixture was added Λf ø-bis-trimethylsilyl- acetamide (41.06 mL, 167.94 mmol), followed by heating at 55 0C for 1 h. The mixture was cooled to 0 0C, then trimethylsilyl trifluoromethanesulfonate (19.07 mL, 105.54 mmol) was added dropwise over 15 min. The mixture was subsequently heated at 55 0C. After 3 hours the mixture was cooled to 0 0C and quenched with the dropwise addition of saturated aqueous NaHCO3 (20 mL). The mixture was poured into EtOAc, washed with brine (4 x 0.8 mL), dried over anhydrous Na2SO4, filtered, evaporated and dried under high vacuum. The residue was purified by silica gel column chromatography and eluted with 20% to 50% EtOAc in hexanes to yield Compound 7 (22.27 g, 85%) as a white foam. ES MS mlz 559.2 [M + H] +.
c) Preparation of 3'-0-(2-methyInaphthalene)-5'-(/f )-methyl-3-W-(benyloxymethyl)-5- methyluridine (Compound 8)
Compound 7 (11.71 g, 20.98 mmol) was dissolved in anhydrous DMF (115 mL). To this was added l,8-diazabicycl-[5-4-0] undec-7-ene (DBU, 9.30 mL, 62.41 mmol). The reaction mixture was cooled in an ice bath. To this was added benzyl chloromethyl ether (4.36 mL, 31.47 mmol), and stirred at 0 0C for 1 hour. The mixture was diluted with EtOAc (200 mL), washed with saturated aqueous NaHCO3 (200 mL) and brine (200 mL) then dried (Na2SO4), filtered and evaporated. The residue obtained was dissolved in methanol (89 mL) and K2CO3 (8.76 g, 63.40 mmol). The reaction mixture was stirred at room temperature for 1 h. The mixture was poured into EtOAc (200 mL), washed with water (200 mL) and brine (200 mL), dried over anhydrous Na2SO4, filtered and evaporated. The residue was purified by silica gel column chromatography and eluted with 5% methanol in CH2Cl2 to yield Compound 8 (8.93 g, 80%) as a white foam. ES MS mlz 533.2 [M + H] +.
d) Preparation of 2'-O-(2-methoxyethyl)-3'-O-(2-methyInaphthaIene)-5'-(R)-methyl-3-iV- (benyIoxymethyl)-5-methyluridine (Compound 9)
Compound 8 (4.30 g, 8.07 mmol) was dried over P2O5 under reduced pressure and dissolved in anhydrous DMF (24 mL). The mixture was cooled to -20 0C. To this was added NaH (0.48 g, 12.11 mmol, 60% dispersion in mineral oil) with stirring for 30 minutes followed by addition of 1- methoxy-2-iodoethane (2.25 g, 12.11 mmol). The reaction mixture was warmed up to 0 0C. After stirring for 1.5 h at 0 0C the reaction mixture was cooled to -20 0C and additional NaH (0.48 g, 12.11 mmol, 60% dispersion in mineral oil) was added. Stirring was continued at -20 0C for 30 minutes and l-methoxy-2-iodoethane (2.25 g, 12.11 mmol) was added. The reaction mixture was warmed to 0 0C and with stirring for an additional 1.5 h. The reaction was quenched with methanol (5 mL), diluted with EtOAc (100 mL), washed with water (100 mL) and brine (100 mL), dried over Na2SO4, filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with 5% methanol in CH2Cl2 to yield Compound 9 (2.95 g, 62%). ES MS mlz 591.2 [M + H] +.
e) Preparation of 5'-0-Benzoyl-2'-0-(2-methoxyethyl)-5'-(R)-methyl-5-methyluridine (Compound 10)
Compound 9 (2.2 g, 3.73 mmol) was dissolved in anhydrous pyridine (7 mL) and cooled in an ice bath. To this benzoyl chloride (0.88 mL, 7.61 mmol) was added and once the addition was over, reaction mixture was allowed to come to room temperature. The reaction mixture was stirred at room temperature for 4 h under an argon atmosphere and subsequently cooled the reaction mixture in an ice bath and quenched by adding saturated aqueous NaHCO3 (5 mL). Diluted the reaction mixture with EtOAc (50 mL) and washed with saturated aqueous NaHCO3 (2 x 50 mL), brine (50 mL), dried over Na2SO4, filtered and concentrated. The residue obtained was dissolved in CH2Cl2 (40 mL) and added 2,4-dichloro-5,6-dicyano-l,4-benzoquinone (DDQ, 1.93 g, 8.5 mmol) and H2O (0.15 mL, 8.5 mmol) and stirred at room temperature. After 18 h, diluted the reaction mixture with EtOAc (60 mL), washed with saturated aqueous NaHCO3 (2 x 80 mL), brine (50 mL), dried over Na2SO4, filtered and evaporated under reduced pressure. The residue was dissolved in MeOH (30 mL) and palladium hydroxide (1.1 g, 20 wt% Pd on carbon dry base) and stirred under H2 atmosphere for 6 h. To this acetic acid (0.56 mL) was added and stirred for 5 min. The reaction mixture was filtered through a pad of celite 545, and washed the celite with copious amount of MeOH. The combined filtrate and washing were concentrated under reduced pressure and the residue was purified by silica gel column chromatography and eluted with 5% methanol in CH2Cl2 to yield Compound 10 (1.43 g, 88%). ES MS mlz 435.1 [M + H] +.
f) Preparation of 2'-0-(2-methoxyethyl)-5'-(R)-methyI-3'-0-tert-butyldimethylsilyl-5- methyluridine (Compound 11)
A mixture of Compound 10 (1.33 g, 3.06 mmol) and imidazole (2.09, 30.70 mmol) was dissolved in anhydrous DMF (11.4 mL). To this solution tert-butyldimethylsilyl chloride (2.31 g, 15.33 mmol) was added with stirring at room temperature for 16 h under an atmosphere of argon. The reaction mixture was diluted with EtOAc (75 mL) and washed with saturated aqueous NaHCO3 (2 x 60 mL) and brine (50 mL), dried over Na2SO4, filtered and concentrated. The residue obtained was dissolved in methanolic ammonia (20 mL, 7M) and stirred for 24 h at 55 0C. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography and eluted with 50% EtOAc in hexanes to yield Compound 11 (1.21 g, 89%). ES MS mlz 455.2 [M + H] +.
g) Preparation of 5'-0-(4,4'-dimethoxytrityI)-2'-0-(2-methoxyethyl)-5'-(Jf)-methyl-5- methyluridine (Compound 12)
Compound 11 (0.42 g, 0.96 mmol) was mixed with 4,4'-dimethoxytrityl chloride (0.82 g, 2.41 mmol) and dried over P2O5 under reduced pressure. The mixture was dissolved in anhydrous pyridine (3 mL) and stirred at 45 0C for 18 h under an atmosphere of argon. The reaction mixture was cooled to room temperature and diluted with EtOAc (40 mL) and washed with saturated aqueous NaHCO3 (60 mL) and brine (40 mL), dried over Na2SO4, filtered and concentrated. The residue obtained was purified by silica gel column chromatography and eluted first with 50% EtOAc in hexanes and then with 5% methanol in CH2Cl2. The product obtained was dissolved in a mixture of triethylamine trihydrofluoride (1.38 mL, 8.44 mmol) and triethylamine (0.58 mL, 4.22 mmol) in THF (8.4 mL). After 72 h the mixture was diluted with EtOAc (60 mL), washed with water (40 mL), saturated aqueous NaHCO3 (40 mL) and brine (40 mL) then dried over Na2SO4, filtered and evaporated. The residue obtained was purified by silica gel column chromatography and eluted with 70% EtOAc in hexanes to yield Compound 12 (0.44 g, 73%). ES MS mlz 631.2 [M + H] +. h) Preparation of 5'-0-(4,4'-dimethoxytrityl)-2'-0-(2-methoxyethyl)-5'-(if)-methyl-5- methyluridine -3'-(2-cyanoethyl-7V^V-diisopropylphosphoramidite (Compound 13)
Compound 12 (0.35 g, 0.55 mmol) was dried over P2Os under reduced pressure then dissolved in anhydrous DMF (1.8 niL). To this 1-H-tetrazole (0.033 mg, 0.48 mmol), N-methyl- imidazole (0.012 mL, 0.15 mmol) and 2-cyanoethyl-N;jV)N'r/V'-tetraisopropylphosphordiamidite (0.27 mL, 0.86mmol) were added. After 3 h, EtOAc (40 mL) was added and the mixture was washed with saturated NaHCO3 (30 mL) and brine (40 mL), dried over anhydrous Na2SO4, filtered and evaporated in vacuo to give an oil. The oily residue was purified by silica gel column chromatography by eluting with EtOAc/hexane (1 :1) to yield Compound 13 (0.38 g, 83%) as a white foam. MS (ES): m/z 831 [M + H]+; 31P NMR (121 MHz, CDCl3): δ 150.2, 149.
Example 16
Preparation of Compound 22
Figure imgf000127_0001
Figure imgf000128_0001
Compound 8 is prepared as per the procedures illustrated in Example 15. Compound 22 is prepared according to the scheme illustrated above. Compound 22 is incorporated into oligonucleotides according to standard solid phase synthesis procedures. Phosphorylation at the 5' end of oligonucleotides is achieved during synthesis by using Glen Research (Sterling, VA) chemical phosphorylation reagent.
Example 17
Preparation of Compound 26
Figure imgf000129_0001
Figure imgf000129_0002
Scheme 1. (i) 4-nitrobenzoic acid, triphenylphosphine, diisopropyl azodicarboxylate, rt; (ii) NH3, MeOH, 55 0C; (iii) a. DMTCl, pyridine, 450C, b. THF.3HF, TEA, THF; (iv) 2-cyanoethyl- iV.iV.N'N-tetraisopropylphosphordiamidite, 1-H-tetrazole, 7V-methyl-imidazole, DMF. Compound 11 is prepared as per the procedures illustrated in Example 15.
Example 18
Preparation of Compound 30
Figure imgf000130_0001
Scheme 2. Nap: 2-methylnaphthalene; Bz: benzoyl; TBDMS: tert-butyldimethylsilyl; (i) DMF, 2- bromoethyl acetate, NaH; (ii) a. aqueous CH3NH2, THF, b. BzCl, pyridine, rt, c. DDQ, CH2Cl2, H2O, rt, c. Pd(OH)2, MeOH, H2, AcOH; (iii) a. TBDMSCl, Im, DMF, rt, b. NH3, MeOH, 55 0C; (iv) a. DMTCl, Py, 45 0C, b. TEA.3HF, TEA, THF; (v) 2-cyanoethyl-iV,iV,iV'iV'-tetraisopropyl- phosphordiamidite, 1-H-tetrazole, iV-methylimidazole, DMF.
Compound 14 is prepared as per the procedures illustrated in Example 16.
Example 19
Preparation of Compound 34
Figure imgf000131_0001
Figure imgf000131_0002
Scheme 3. (i) 4-nitrobenzoic acid, triphenylphosphine, diisopropyl azodicarboxylate, it; (ii) NH3, MeOH, 55 0C; (iii) a. DMTCl, pyridine, 45 0C, b. TEA.3HF, TEA, THF; (iv) 2-cyanoethyl- ΛζΛζN'N'-tetraisopropylphosphordiamidite, 1-H-tetrazole, 7V-methylimidazole, DMF. Compound 28 is prepared as per the procedures illustrated in Example 18
Example 20 Preparation of Compound 37
Figure imgf000131_0003
a) Preparation of Compound 36 Commercially available l,2;5,6-di-O-isopropylidene-α-D-allofuranose, Compound 35, (135 g, 519.0 mmol) and 2-(bromomethyl)-naphthalene (126 g, 570.0 mmol) were dissolved in DMF (500 mL) in a three-necked flask (500 mL) and the reaction was cooled in an ice bath. Sodium hydride (60% w/w, 29 g, 727.0 mmol) was carefully added (6 g portions every 10 minutes) to the reaction and the stirring was continued for another 60 minutes after the addition was complete. At this time TLC analysis showed no more sugar (Compound 35). The reaction was carefully poured onto crushed ice (ca. 500 g) and the resulting slurry was stirred vigorously until all the ice melted. The resulting off-white solid was collected by filtration and suspended in water. The suspension was stirred vigorously using a mechanical stirrer for 30 minutes after which the solid was collected by filtration and suspended in hexanes. The suspension was stirred vigorously for 30 minutes after which the solid was collected by filtration and air dried for 4-6 hours and then dried under high vacuum over P2O5 for 16 hours to provide Compound 36 (206.0 g, 99%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δ: 7.85 (m, 4H), 7.48 (m, 3H), 5.74 (s, IH), 4.92 (d, IH, J= 11.7), 4.75 (d, IH, J= 11.6), 4.58 (m, IH), 4.36 (m, IH), 4.15 (m, IH), 4.03-3.86 (m, 3H), 1.61 (s, 3H), 1.36 (s, 9H).
b) Preparation of Compound 37
Compound 36 (200.0 g, 0.5 moles) was added in small portions to a solution of acetic acid (2.2 L) and water (740 mL). The reaction was stirred at room temperature for 16 h after which, TLC analysis (30% EtOAc/hexanes) indicated complete consumption of Compound 36. The reaction was then concentrated under reduced pressure until most of the acetic acid was removed. The remaining solution was poured into a stirred mixture of EtOAc (IL) and water (IL). Solid KOH was then added to the above mixture until the aqueous layer was strongly basic (pH>12). The organic layer was then separated, washed with saturated sodium bicarbonate solution and brine then dried (Na2SO4), filtered and concentrated under reduced pressure to provide Compound 37 as a yellow foam, which was used without any further purification. Example 21
Preparation of Compound 45
Figure imgf000133_0001
Compound 37 is prepared as per the procedures illustrated in Example 20. Example 22
Preparation of Compound 47
Figure imgf000134_0001
Compound 43 is prepared as per the procedures illustrated in Example 21.
Example 23
Preparation of compound 50
Figure imgf000134_0002
Compound 43 is prepared as per the procedures illustrated in Example 21.
Example 24 Preparation of compound 53
Figure imgf000135_0001
Compound 43 is prepared as per the procedures illustrated in Example 21.
Example 25
Preparation of Compound 57
Figure imgf000136_0001
Compound 42 is prepared as per the procedures illustrated in Example 21.
Example 26
Preparation of Compound 58
Figure imgf000136_0002
Compound 37 was prepared as per the procedures illustrated in Example 20. A solution of
NaIO4 (107.0 g) in water (3 L) was added over 40 minutes to a stirred (mechanical stirrer) solution of Compound 37 (crude from above) in dioxane (1.5 L). After 60 minutes the reaction mixture was poured into EtOAc (1.5 L) and the organic layer was separated, washed with water (IL) and brine (IL) then dried (Na2SO4) and concentrated to provide Compound 58 as a yellow oil, which was used without any further purification. Example 27
Preparation of Compound 67
Figure imgf000137_0001
Compound 58 was prepared as per the procedures illustrated in Example 26. Compound 61, diethyl-(difluoromethane)phosphonate is commercially available. The preparation of Compound 67 was achieved as per the procedures illustrated in Example 27 and confirmed by spectral analysis, 1HNMR and mass spectroscopy. Example 28
Preparation of Compound 69
Figure imgf000138_0001
Compound 65 was prepared as per the procedures illustrated in Example 27. The preparation of Compound 69 was achieved as per illustrated in Example 28 and confirmed by spectral analysis, 1HNMR and mass spectroscopy.
Example 29
Preparation of Compound 72
Figure imgf000139_0001
Compound 65 is prepared as per the procedures illustrated in Example 27.
Example 30
Preparation of Compound 75
Figure imgf000139_0002
Compound 65 is prepared as per the procedures illustrated in Example 27.
Example 31
Preparation of Compound 79
Figure imgf000140_0001
Compound 64 is prepared as per the procedures illustrated in Example 27.
Example 32
Preparation of Compound 86
Figure imgf000141_0001
Compound 80 is prepared according to the procedures illustrated in published U.S. Patent
5,969,116. Example 33
Preparation of 5'-iV-(4-methoxytrityI)-5'-amino-5'deoxy-thymidme-3'-(2-cyanoethyl-7V>iV- diisopropylphosphoramidite) (Compound 89)
Figure imgf000142_0001
a) Preparation of 5'-iV-(4-methoxytrityl)-5'-amino-5'deoxy-thymidine (Compound 88)
Compound 87, 5'-amino-deoxythymidine is commercially available. Compound 88 is prepared according to the method of Mag and Engels (Mag, M.; Engles, J. W. Nucleic Acids Res. 1989, i 7, 5973-5988).
b) Preparation of 5'-JV-(4-methoxytrityl)-5'-amino-5'deoxy-thymidine-3'-(2-cyanoethyl- ΛyV-diisopropylphosphoramidite) (Compound 89)
To the solution of Compound 88 (1.05 g, 1.88 mmol) and tetrazole (0.11 g, 1.5 mmol) in anhydrous DMF (9 mL) was added 1-methylimidazole (0.039 mL, 0.5 mmol) while stirring under a nitrogen atmosphere. The reaction mixture was cooled to 0 0C and 2-cyanoethyl-iV,iV,7V JV- tetraisopropylphosphordiamidite (0.89 mL, 2.8 mmol) was added. After 3.5 h, the reaction was quenched with butanol (2 mL) and the reaction volume was reduced to 50% by volume under reduced pressure. The reaction mixture was diluted with EtOAc (50 mL), washed with saturated N-1HCO3 (35 mL), then with brine (50 mL) and dried briefly over anhydrous Na2SO4. The organic phase was filtered and concentrated under reduced pressure. The resulting residue was dissolved in diethyl ether:CH2Cl2 (1 :1, 2.25 mL) and was added drop-wise into an ice cold pentane (300 mL) solution. The resulting solid was filtered to afford Compound 89 (1.24 g, 86.7%). 31P NMR (121 MHz, CD3CN): δ 148.31 and 148.08. Example 34
Preparation of Compound 92
Figure imgf000143_0001
Compounds 81 and 84 are prepared as per the procedures illustrated in Example 32.
Example 35
Preparation of 5 '-S-(4,4'-dimethoxytrityl)-5'-thio thymidine 3'-(2-cyanoethyl- diisopropylphosphoramidite) (Compound 93)
Figure imgf000144_0001
Compound 93 is prepared according to the method of Jahn-Hofmann and Engels (Jahn- Hofmann, K.; Engles, J. W. Helvetica Chimica Acta 2004, 87, 2812-2828).
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000146_0002
Compound 39 is prepared as per the procedures illustrated in Example 21. Compound 100 is prepared according to the method published by Inoue, H. et al. Nucleic Acids Research 1987, 15, 6131-6148.
Example 37
Preparation of Compound 106 
Figure imgf000147_0001
Compound 97 is prepared as per the procedures illustrated in Example 36. Compound 80 is prepared according to the procedures published in U.S. Patent 5,969,116.
Example 38
Preparation of Compound 109
Figure imgf000148_0001
Compound 68 is prepared as per the procedures illustrated in Example 28. Compound 80 is prepared according to the procedures published in U.S. Patent 5,969,116.
Figure imgf000150_0001
Compound 66 is prepared as per the procedures illustrated in Example 27. Compound 100 is prepared according to the method published by Inoue, H. et al. Nucleic Acids Research 1987, 15, 6131-6148.
5
Figure imgf000152_0001
Compound 78 is prepared as per the procedures illustrated in Example 31. Compound 114 is prepared according to procedures published by Ikeda, H. et al. Nucleic Acids Research 1998, 26, 2237-2244.
Example 41
Preparation of Compounds 119, 120 and 121
Figure imgf000153_0001
Compounds 96 and 98 are prepared as per the procedures illustrated in Example 36. Compound 103 is prepared as per the procedures illustrated in Example 37.
Figure imgf000154_0001
Compounds 119, 120 and 121 are prepared as per the procedures illustrated in Example 41.
Example 43 Preparation of Compounds 126 and 127
Figure imgf000155_0001
Compound 38 is prepared as per the procedures illustrated in Example 21.
Figure imgf000156_0001
Compound 126 is prepared as per the procedures illustrated in Example 43.
Figure imgf000158_0001
Compound 127 is prepared as per the procedures illustrated in Example 43.
Example 46
Preparation of Compounds 147 and 149
Figure imgf000159_0001
Compound 141 is prepared as per the procedures illustrated in Example 45.
Example 47
Preparation of Compounds 151 and 153
Figure imgf000160_0001
Compound 132 is prepared as per the procedures illustrated in Example 44.
Example 48
Preparation of Compounds 155 and 157
Figure imgf000161_0001
Compound 141 is prepared as per the procedures illustrated in Example 45.
Example 49
Preparation of Compounds 159 and 161
Figure imgf000162_0001
Compound 132 is prepared as per the procedures illustrated in Example 44.
Example 50
Preparation of Compounds 163 and 165
Figure imgf000163_0001
Compound 132 is prepared as per the procedures illustrated in Example 44. 9
Figure imgf000164_0001
Compound 141 is prepared as per the procedures illustrated in Example 45.
Example 52
Preparation of Compounds 172 and 174
Figure imgf000165_0001
Compound 140 is prepared as per the procedures illustrated in Example 45. Example 53
Preparation of Compounds 177 and 179
Figure imgf000166_0001
Compound 131 is prepared as per the procedures illustrated in Example 44. Example 54
General procedure for the preparation of compounds of Formula Ha and Hb
Figure imgf000167_0001
The preparation of compounds of Formula Ia, Ib, Ha and lib are illustrated in Examples 21-
25, 27-35 and 44-53. Example 55
General procedure for the preparation of compounds of Formula Ilia
Figure imgf000168_0001
The preparation of compounds of Formula Ha, Hc, and Ilia are illustrated in Examples 13,
15-19, 21-25 and 27-53.
Example 56
General procedure for the preparation of compounds of Formula IHb and HIc
Figure imgf000169_0001
The preparation of compounds of Formula lib, Hd, He, Hf, IHb and HIc are illustrated in Examples 13, 15-19, 21-25 and 27-53. Example 57
Chemically modified ssRNAs targeting PTEN - in vivo study
The antisense activity of oligomeric compounds can be tested in vivo. Five- to six-week old Balb/c mice (Jackson Laboratory, Bar Harbor, ME) are injected with modified ssRNA targeted to PTEN at doses of 80 mg/kg daily, 60 mg/kg daily, or 40 mg/kg twice daily for several days. The mice are sacrificed 72 hours following the last administration. Liver tissues are homogenized and mRNA levels are quantitated using real-time PCR using procedures illustrated herein for comparison to untreated control levels (%UTC). Other modifications and motifs as disclosed herein are also amenable to in vivo testing. Liver transaminase levels, alanine aminotranferease (ALT) and aspartate aminotransferase (AST), in serum are also measured relative to saline injected mice. At the end of the study, liver and spleen tissues are harvested from animals treated with the modified ssRNAs, the tissues are weighed to assess gross organ alterations.
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
Figure imgf000173_0002
Each nucleoside is connected to the following nucleoside by a phosphodiester intemucleoside linkage except underlined nucleosides which are connected to the following nucleoside by a phosphorothioate intemucleoside linkage (going 5' to 3'). A "P" at the 5'-end indicates a 5'-phosphate group. A "Ps" at the 5'- end indicates a 5'-thiophosphate group. Nucleosides followed by a subscript d, ef, f, m, e or x are sugar modified nucleosides. A subscript "d" indicates a 2'-OCH2(CO)NH(CH2)2N(CH3)2 (DMAEAc), subscript "ef ' indicates a 2'-OCH2CH2F (FEt) modified nucleoside, a subscript "f ' indicates a 2'-fluoro modified nucleoside, , a subscript "m" indicates 2'-O-methyl modified nucleoside, a subscript "e" indicates a 2'- O(CH2)2OCH3 (MOE) modified nucleoside, and a subscript R or S or Rd or Sd or x indicates one of the 5'- modifϊed nucleosides (R or S) or one of the 2', 5'-bis modified nucleosides listed below (Rd, Sd, Rb, Sb, Rc or Sc). In general, each modified nucleoside having an x after it will have the same sugar modification.
Figure imgf000173_0001
Figure imgf000174_0001
Figure imgf000174_0002
Example 58
Gapped oligomeric compounds targeted to PTEN: in vivo study In accordance with the present disclosure, oligomeric compounds are synthesized and tested for their ability to reduce PTEN expression in vivo at doses of 20 and 60 mg/kg. Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) are administered a single intraperitoneal (i.p) injection at either 20 or 60 mg/kg of a 2-10-2 gapped oligomer. A 5-10-5 gapped oligomer having 2'-O-MOE modified nucleosides or other modified nucleosides as provided herein in the wings is also included for comparison. Other motifs as disclosed herein are also amenable to in vivo testing.
Each dose group will include four animals. The mice are sacrificed 48 hours following the final administration to determine the PTEN mRNA levels in liver using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. PTEN mRNA levels are determined relative to total RNA (using Ribogreen), prior to normalization to saline-treated control. The average % inhibition of mRNA expression for each treatment group, normalized to saline-injected control is determined.
Liver transaminase levels, alanine aminotranferease (ALT) and aspartate aminotransferase (AST), in serum are measured relative to saline injected mice.
Figure imgf000175_0001
Each unmodified nucleoside is a β-D-2'-deoxyribonucleoside. Each internucleoside linkage is a phosphorothioate intemucleoside linkage. A "P" at the 5'-end indicates a 5'-phosphate group. meC indicates a 5 '-methyl cytosine nucleoside. Each nucleoside having a subscript x is selected from the list at the end of Example 57, e.g., Rb, Sb, Rc, Sc, Rd and Sd. In general, each modified nucleoside having an x after it will have the same sugar modification but can have different bases.
Example 59 Oligomeric compounds targeted to PTEN: in vitro study
In accordance with the present disclosure, oligomeric compounds were synthesized and tested for their ability to reduce PTEN expression over a range of doses. Human HeLa cells were treated with either ISIS 447581 or ISIS 404320. A dose comparison was evaluated with dose concentrations of .20, .62, 1.9, 5.5, 16.7 and 50 nM using methods described herein. Expression levels of PTEN were determined using real- time PCR and normalized to RIBOGREEN™ using methods described herein. The percent inhibition of
PTEN mRNA was determined. Resulting dose-response curves were used to determine the EC50. Tm's were assessed in 100 mM phosphate buffer, 0.1 mM EDTA, pH 7, at 260 nm using 4μM modified oligomers and 4μM complementary RNA. The EC50S are listed below.
Figure imgf000175_0002
Each nucleoside is connected to the following nucleoside by a phosphodiester internucleoside linkage except underlined nucleosides which are connected to the following nucleoside by a phosphorothioate internucleoside linkage (going 5' to 3')- A "P" at the 5'-end indicates a 5'-phosphate group. Nucleosides followed by a subscript f, m or e are sugar modified nucleosides. A subscript "f ' indicates a 2'-fluoro modified nucleoside, a subscript "m" indicates 2'-O-methyl modified nucleoside, a subscript "e" indicates a 2'-O(CH2)2θCH3 (MOE) modified nucleoside and a subscript Rc indicates the 2', 5'-bis modified nucleoside listed in Example 57.
Example 60 Modifed ssRNA 5'-phosphate serum stability assay
A serum stability assay is useful for evaluating the stability of oligomeric compounds in the presences of nucleases and other enzymes found in serum. For example, the stability of a 5 '-terminal phosphate group of an oligomeric compound can be evaluated by assessing the ability of the 5 '-terminal phosphate group to remain attached to the oligomeric compound in the presence of serum. Accordingly, a serum stability assay was employed to evaluate the stability of modified ssRNAs having a 5 '-terminal phosphate group.
Various modifed ssRNAs, shown below, having a 5 '-terminal phosphate group (10 μM) were dissolved in 95% of fresh mouse serum and incubated at 37 0C. Aliquots of serum (100 μL) were removed after 0, 1, 3, 6 or 24 hours of incubation times. The serum samples were immediately quenched and snap frozen. The samples were extracted by the strong anion exchange (SAX) and octadecylsilyl (C- 18) columns. For each incubation time, the amount of full length modified ssRNA having a 5 '-terminal phosphate group was determined by LC/MS, and the half-life of the full length modified ssRNA having a 5' terminal phosphate group was calculated. The results are expressed as half-time (Ti/2) in the table below. These data demonstrate that modifications to oligomeric compounds can improve the stability of the 5 '-terminal phosphate group.
Figure imgf000176_0001
Each nucleoside is connected to the following nucleoside by a phosphodiester internucleoside linkage except underlined nucleosides which are connected to the following nucleoside by a phosphorothioate internucleoside linkage (going 5' to 31). A "P" at the 5'-end indicates a 5'-phosphate group. Nucleosides followed by a subscript d, e, f, R or Sf are sugar modified nucleosides. A subscript "d" indicates a 2'-O- dimethylaminoethyl acetamide (DMAEAc) modified nucleoside, a subscript "e" indicates a 2'-0(CH2^OCH3 (MOE) modified nucleoside, a subscript "f indicates a 2'-fluoro modified nucleoside, a subscript "R" indicates (ϊ?)-5'-methyl-2I-deoxyribonucleoside and a subscript Sf indicates the T, 5'-bis modified nucleoside listed below.
Figure imgf000177_0001
Example 61
Design and screening of duplexed antisense compounds
In accordance with the present invention, a series of nucleic acid duplexes comprising the compounds of the present invention and their complements can be designed. The nucleobase sequence of the antisense strand of the duplex comprises at least a portion of an antisense oligonucleotide targeted to a target sequence as described herein. The ends of the strands may be modified by the addition of one or more natural or modified nucleosides to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini. For example, a duplex comprising an antisense strand having the sequence
CGAGAGGCGGACGGGACCG (SEQ ID NO: 11) and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure:
Figure imgf000177_0002
In another embodiment, a duplex comprising an antisense strand having the same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 10) may be prepared with blunt ends (no single stranded overhang) as shown:
Figure imgf000177_0003
RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, CO). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 μM. Once diluted, 30 μL of each strand is combined with 15 μL of a 5X solution of annealing buffer. The final concentration of the buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 μL. This solution is incubated for 1 minute at 90 0C and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37 0C at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 μM.
Once prepared, the duplexed compounds are evaluated for their ability to modulate target mRNA levels. When cells reach 80% confluency, they are treated with duplexed compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 μL OPTI-MEM- 1™ reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM-1™ containing 5 μg/mL LIPOFECTAMINE 2000™ (Invitrogen Life Technologies, Carlsbad, CA) and the duplex antisense compound at the desired final concentration. After about 4 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by quantitative real-time PCR as described herein.
Example 62
5' and 2' bis-substituted modified oligomeric compounds targeting PTEN - in vitro study (ssRNAs vs siRNAs) A series of 5' and 2' bis-substituted modified oligomeric compounds were prepared as single strand
RNAs (ssRNAs). The antisense (AS) strands listed below were designed to target human PTEN, and each was also assayed as part of a duplex with the same sense strand (ISIS 341401, shown below) for their ability to reduce PTEN expression levels. HeLa cells were treated with the single stranded or double stranded oligomeric compounds created with the antisense compounds shown below using methods described herein. The IC5O 1S were calculated using the linear regression equation generated by plotting the normalized mRNA levels to the log of the concentrations used.
Figure imgf000178_0001
Figure imgf000179_0002
Each internucleoside linkage is a phosphodiester except that underlined nucleosides are linked to the following nucleoside by a phosphorothioate (going 5' to 3'). Each nucleoside not followed by a subscript is a ribonucleoside. A "P" at the 5'-end indicates a 5'-phosphate group. A "Py" at the 5'-end indicates a 5'- methylenephosphonate group, (PO(OH)2CH2-). A "Pz" at the 5'-end indicates a 5'- difluoromethylenephosphonate group, (PO(OH)2CF2-). Nucleosides followed by a subscript indicate modification as follows: subscript "d" indicates a 2'-O-dimethylaminoethyl acetamide (DMAEAc) modified nucleoside; subscript "e" indicates a 2'-O(CH2)2OCH3 (MOE) modified nucleoside, subscript "f indicates a 2'-fluoro modified nucleoside; subscript "m" indicates 2'-O-methyl modified nucleoside; and subscript "R" indicates a (ϊ?y)-5'-methyl-2'-deoxyribonucleoside. Superscript "me" indicates a 5-methyl group on the pyrimidine base of the nucleoside. Nucleosides with subscripts "Rc" or "Sc" are shown below.
Figure imgf000179_0001
Example 63 Modified ssRNAs targeting PTEN - in vivo study
Modified ssRNAs and dsRNAs targeted to PTEN were designed as shown below.
Figure imgf000179_0003
Phosphorothioate internucleoside linkages are indicated by underlining. Modified nucleosides are indicated by a subscripted letter following the capital letter indicating the nucleoside. In particular, subscript "f" indicates 2'-fluoro; subscript "m" indicates 2'-O-methyl; and subscript "e" indicates 2'-O-methoxyethyl (MOE). For example Un, is a modified uridine having a 2'-OCH3 group. Some of the strands have a 5'- phosphate group designated as "P-".
Example 64
Effect of modified internucleoside linkages on modified ssRNAs targeting PTEN - in vitro study
A dose response experiment was performed targeting PTEN in human HeLa cells to determine the effects of placement of sugar and internucleoside linkages within ssRNAs. More specifically, the modified ssRNAs were tested for their ability to reduce PTEN mRNA in cultured cells. The modified ssRNAs are shown below, and contain2'-OMe and 2'-fluoro modified nucleosides, two 2'-0-MOE modified nucleosides at the 3'-terminus, and seven phosphorothioate linkages at the 3 '-terminus of the ssRNAs.
HeLa cells were treated with ssRNAs shown below at concentrations of 1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM, 20 nM and 50 nM using methods described herein. Levels of mRNA were determined using realtime PCR methods as described herein. The IC5O for each ssRNA was determined. These data demonstrate that these modified ssRNA exhibit similar activity in decreasing target mRNA levels.
Figure imgf000180_0001
Phosphorothioate internucleoside linkages are indicated by underlining. Modified nucleosides are indicated by a subscripted letter following the capital letter indicating the nucleoside. In particular, subscript "f ' indicates 2'-fluoro; subscript "m" indicates 2'-O-methyl; and subscript "e" indicates 2'-O-methoxyethyl (MOE). For example, Uf is a modified uridine having a 2'-fluoro group. Some of the strands have a 5'- phosphate group designated as "P-".
Example 65 ssRNAs Stability in Hepatocyte Cell Homogenate Assay - in vivo study The stability of oligomeric compounds can be evaluated in a cell homogenate assay.
Hepatocytes were harvested from bal/c mice in ice-cold hepatocyte wash media (William E Media) with fetal bovine serum, sedimented by centrifiigationat lOOOg for 8 minutes and then washed with hepatocyte wash media. Hepatocytes were homogenized with RIPA buffer (50 mM Tris pH 7.5, 10 niM MgCl2, 150 mM NaCl, 0.5 % NP-40 alternative, one tablet of Roche protease inhibitor #11836170001), and centrifuged at 1400Og for 15 minutes at 4°C and the supernatant was removed and stored in ice. Protein concentration (BSA mg/mL) was determined with Bradford assay and adjusted to a final protein concentration of 2 mg/mL by addition of Ripa buffer volume or cell homogenate volume.
Phenol/Choroform Extraction. ssRNA (1 mL, 20 μL) were homogenized in a homogenation buffer (20 mM Tris, pH 8, 20 mM EDTA and 0.1 M NaCl in 0.5% NP-40) at time points 0, 5, 10, 20, 30, 40 and 60 minutes (Exception: 06/408877 at time points 0, 15, 30, 60, 120 and 240 mins, 06/409044, at time points 0, 0.5, 1, 2, 4, 8, and 18 hours). An internal standard (18/355868, a 27-mer, 2'-<9-methoxyethyl-modified phosphorothioate oligonucleotide, or 19/116847, a 5-10-5 gappmer, 2'-0-methoxyethyl-modifϊed phosphorothioate oligonucleotide) with concentration at 20ug/g was added prior to extraction. Tissue samples were extracted with 70 μL of NH4OH and 240 μL of phenol/chloroform/isoamyl alcohol (25:24:1). The supernatant was removed after centrifugation at 14000 rpm for 2 min. The remaining extractant was vortexed with an additional 500μL of water and the aqueous layer was removed and combined with the supernatant after centrifugation at 14000 rpm for 2 minutes.
Solid Phase Extraction. Triethylammonium acetate solution at IM (500μL) was added to the supernatant. The aqueous layer of the mixture was loaded onto the pre-conditioned Biotage™ Phenyl Solid Phase
Extraction Plate (SPE plate) after centrifugation at 9000 rpm for 20 minutes. The SPE plate was washed several times with water. The sample was then eluted with 1.5 mL of 1% TEA in 90% MeOH and filtered through the Protein Precipitation Plate (Phenomenex™). The elutent was evaporated to dryness and diluted to 200 μL with 50% quenching buffer (8 M urea, 50 mM EDTA) and water before sample injection.
LC-MS. An Agilent 1100 Series LC/MSD system was connected in-line to a mass spectrometry. Mass spectrometer was operated in the electrospray negative ionization mode. The nebulizer nitrogen gas was set at 325 psi and the drying nitrogen gas was set at 12 L/min. The drying temperature was 325 0C. Samples (25 μL/well) were introduced via an auto sampler and reversed-phase chromatography was carried out with an XBridge OST C18 2.5 μm 2.1 mm x 50 mm HPLC column using a flow rate of 300 μL/min at 55 0C. The ion pair buffers consisted of A: 5mM tributylammonium acetate (TBAA) in 20% acetonitrile and B: 5nM TBAA in 90% acetonitrile and the loading buffer was 25 mM TBAA in 25% Acetonitrile. Separation was performed on a 30% to 70% B in 9 min and then 80% B in 11 min gradient.
Quantitative analysis of oligonucleotide and internal standard by extracted ion chromatograms of the most abundant ions was performed using MSD ChemStation software. The results are expressed as half-time (Ti/2) in the table below. These data demonstrate that modifications to oligomeric compounds improve their stability in a cell homogenate assay.
Figure imgf000182_0001
Internal standards:
Figure imgf000182_0002
Each internucleoside linkage is a phosphorothioate internucleoside linkage indicated by underlining
(going 5' to 3'). Each unmodified nucleoside is a β-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicates a 2'-0(CH2^OCH3 (MOE) modified nucleoside. Superscript "me" indicates a 5- methyl group on the pyrimidine base of the nucleoside.
Example 66
MicroRNA mimics: cell cycle assay
Oligomeric compounds comprising the nucleobase sequence of a microRNA were synthesized to have certain modifications described herein. These microRNA mimics were tested for their ability to imitate microRNA activity. A cell cycle assay was used to evaluate the activity of microRNA mimics. A549 cells were plated at a density of approximately 45,000 cells per well of a 24-well plate. The following day, cells were transfected with microRNA mimics and control oligomeric compounds, using RNAIMAX as the transfection reagent. Oligomeric compounds were tested at concentrations ranging from 0.1 nM to 100 nM. Control oligomeric compounds were also tested. Approximately 24 hours following transfection, nocodazole was added to the cells at a concentration ranging from 0.5 to 2.0 μg/ml. Approximately 16 hours later, the cells were harvested, washed, ethanol-fixed and stained with propidium iodide. Cells cycle profiles were generated by subjecting the stained cells to flow cytometry (FACSCAN).
miR-16 mimics: cell cycle assay
A cell cycle assay was used to test the activity of miR-16 mimics (shown in table below). The addition of a double-stranded miR-16 mimic blocked cells in the Gl phase of the cell cycle. The single stranded miR-16 mimic produced the same phenotype as the double-stranded mimic, blocking cells in the Gl phase of the cell cycle. The single stranded miR-16 mimic exhibited similar efficacy as the double-stranded miR-16 mimic.
Figure imgf000183_0001
Internucleoside linkage and sugar modifications are indicated as described in previous examples.
miR-34 mimics: cell cycle assay A cell cycle assay was used to test the activity of miR-34 mimics. The addition of a double-stranded miR-34 mimic blocked cells in the Gl phase of the cell cycle. The above single stranded miR-34 mimic produced the same phenotype as the double-stranded mimic, blocking cells in the Gl phase of the cell cycle. The single stranded miR-34 mimic exhibited similar efficacy as the double-stranded miR-34 mimic.
In addition to measuring cell cycle progression, cells treated with miR-34 mimics were subjected to microarray analysis to compare the profile of gene expression changes following treatment with microRNA mimics. The microarray analysis is used to evaluate the enrichment of target nucleic acids that comprise a seed match segment in their 3' untranlated regions from among the pool of nucleic acids that are down- regulated following treatment with a microRNA mimic.
Both the double-stranded miR-34 mimic and single-stranded miR-34 mimic down-regulated miR-34 seed-matched nucleic acids. However, also observed was an enrichment of nucleic acids comprising a seed match segment of the microRNA complement strand (the "passenger strand") of the double-stranded mimic, thus the microRNA complement strand was also acting an antisense compound. This activity is not specific to miR-34. Accordingly, a single-strand microRNA mimic can provide improved specificity relative to a double-stranded mimic. These data demonstrate that the oligomeric compounds described herein can be designed as microRNA mimics. Further, single-stranded mimics are effective at imitating microRNA activity.
Figure imgf000184_0001
Internucleoside linkage and sugar modifications are indicated as described in previous examples.
Additional miR-34 mimics: cell cycle assay
Additional single-stranded miR-34 mimics were tested in a cell cycle assay. Each of these oligomeric compounds resulted in a block in the Gl phase of the cell cycle, indicating that these single-stranded microRNA mimics are effective at imitating microRNA activity.
Figure imgf000184_0002
Internucleoside linkage and sugar modifications are indicated as described in previous examples.
Example 67
MicroRNA mimics: cytokine signaling assay Oligomeric compounds comprising the nucleobase sequence of a microRNA were synthesized to have certain modifications described herein. These oligomeric compounds were tested for their ability to mimic microRNA activity. A cytokine signaling assay was used to evaluate the activity of microRNA mimics. miR-146 mimics miR-146 is known to stimulate the release of cytokines such as IL-8, thus the following assay can be used to measure the activity of miR-146 mimics. A549 cells were treated with the miR-146 mimics shown below. Cells were treated with EL-IB at a concentration ranging from 0.1 to 2.0 ng/ml. After 8 hours and 24 hours, samples were collected for ELISA analysis to measure the release of the cytokine IL-8. Measurement of DL-8 in the cell culture supernatant revealed that single-strand miR-146 mimics decreased the release of IL-8 in a dose-responsive manner in this assay. Accordingly, the single-strand miR-146 mimics shown below exhibit an activity of miR-146.
Figure imgf000185_0001
Additional oligomeric compounds were designed and comprise the nucleobase sequence of miR-146. These oligomeric compounds were shown to mimic miR-146 activity in the IL-8 release assay described above.
Figure imgf000185_0002
Additional oligomeric compounds were designed and comprise the nucleobase sequence of miR-146.
Figure imgf000185_0003
miR-155 mimics
Additional oligomeric compounds were designed and comprise the nucleobase sequence of miR-155.
Figure imgf000185_0004
Internucleoside linkage and sugar modifications are indicated as described in previous examples.
Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference (including, but not limited to, journal articles, U.S. and non- U.S. patents patent application publications, international patent application publications, gene back accession numbers, and the like) cited in the present application is incorporated herein by reference in its entirety.
Example 68
Phosphate stability in Mouse serum Single-stranded oligomeric compounds were tested for stability in mouse serum. The single stranded oligomeric compounds and the half lives of full compound with intact phosphorous moiety are provided in the table below.
Figure imgf000186_0002
Subscripts in the Table above: d = DMAEAc; i = N-methoxyamino BNA; J = tcDNA; k = (R) 5 '-methyl
Separately, four oligomeric compounds were tested for stability in mouse serum, as summarized in the table below.
Figure imgf000186_0003
Figure imgf000186_0001
Example 69 Modified oligomeric compounds targeting PTEN: in vitro study
In accordance with the present disclosure, oligomeric compounds were synthesized and tested for their ability to reduce PTEN expression over a range of doses. Human HeLa cells were treated with either ISIS 447581, 467074, 418046 or 467076 . A dose comparison was evaluated with dose concentrations of 0.067, 0.2, 0.62, 1.9, 5.5, 16.7 and 50 nM using methods described herein. Expression levels of PTEN were determined using real-time PCR and normalized to RIBOGREEN™ using methods described herein. The percent inhibition of PTEN mRNA was determined and the resulting dose-response curves were used to determine the EC50. The EC50S are listed below.
Figure imgf000187_0002
Each internucleoside linkage is a phosphodiester except that underlined nucleosides are linked to the following nucleoside by a phosphorothioate (going 5' to 3'). A "P" at the 5'-end indicates a 5'-phosphate group. A "Py" at the 5'-end indicates a 5'-methylenephosphonate group, (PO(OH)2CH2-). Nucleosides followed by a subscript e, f or m indicate modification as follows: subscript "e" indicates a 2'-0(CH2^OCHa (MOE) modified nucleoside, subscript "P indicates a 2'-fluoro modified nucleoside; subscript "m" indicates 2'-O-methyl modified nucleoside. Superscript "me" indicates a 5-methyl group on the pyrimidine base of the nucleoside. Nucleosides with subscript "Rc" or "Sc" are shown below.
Figure imgf000187_0001
Example 70
5'- modified oligomeric compounds targeting PTEN: in vivo study
Three oligomeric compounds (ISIS 467074, ISIS 467076, ISIS 116847) were synthesized as described above. Sequence and chemistry of the three oligomeric compounds are provided in the table, below. The nucleobase sequence of each oligomeric compound is complementary to PTEN.
Figure imgf000187_0003
Each internucleoside linkage is a phosphodiester except that underlined nucleosides are linked to the following nucleoside by a phosphorothioate (going 5' to 3'). "Py" at the 5'-end indicates a 5'- methylenephosphonate group, (PO(OH)2CH2-). Each unmodified nucleoside is a β-D-2'- deoxyribonucleosides. Nucleosides followed by a subscript e, f or m indicate modification as follows: subscript "e" indicates a 2'-O(CH2)2θCH3 (MOE) modified nucleoside, subscript "F indicates a 2'-fluoro modified nucleoside; subscript "m" indicates 2'-O-methyl modified nucleoside. Superscript "me" indicates a 5-methyl group on the pyrimidine base of the nucleoside. Nucleoside with subscript "Sc" is shown below.
Figure imgf000188_0001
Six-week-old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected intraperitenially with a single dose of 75 mg/kg of one of the three oligomeric compounds above or with saline control. Each dose group consisted of four animals. The mice were sacrificed 48 hours following administration. Livers were collected and PTEN mRNA levels were assessed using real-time PCR and RDBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. PTEN mRNA levels were determined relative to total RNA (using Ribogreen), and normalized to the saline-treated control. Results are listed below as the average % inhibition of PTEN mRNA expression for each treatment group, normalized to saline-injected control.
Figure imgf000188_0002
Example 71
Stability of 5'- modified oligomeric compounds targeting PTEN: in vivo study
The in vivo stability of the three oligomeric compounds in Example 70 was evaluated. The tissue samples were obtained from the animals in which PTEN was assessed. Tissue samples were collected and prepared using the same technique described in Example 65. Quantitative analysis of the oligonucleotides standard were performed by extracted ion chromatograms in the most abundant charge state (-4) using Chemstation software. The tissue level (μg/g) of intact compound of ISIS 116847, 467074 and 467076 was measured and are provided below:
Figure imgf000189_0001
The 5-10-5 MOE gapmer compound was present at high levels and was a potent inhibitor of PTEN. Intact 467076 was present at a lower concentration and resulted in smaller inhibition of PTEN. Intact 467074 was not detected and resulted in the lowest amount of PTEN reduction. Some 467074 lacking the 5'- phosphate was detected.
Example 72
Effect of modified internucleoside linkages on modified oligomeric compounds targeting PTEN- in vitro study
In accordance with the present disclosure, oligomeric compounds were synthesized and tested for their ability to reduce PTEN expression over a range of doses. Human HeLa cells were treated with the following oligomeric compounds. A dose comparison was evaluated with dose concentrations of 0.167, 0.5, 1.5, 5, 15 and 50 nM using methods described herein. Expression levels of PTEN were determined using real-time PCR and normalized to RIBOGREEN™ using methods described herein. The percent inhibition of PTEN mRNA was determined and the resulting dose-response curves were used to determine the IC5O. The IC50S are listed below.
Figure imgf000189_0002
Figure imgf000190_0001
Each internucleoside linkage is a phosphodiester except that underlined nucleosides are linked to the following nucleoside by a phosphorothioate (going 5' to 3'). A "P" at the 5'-end indicates a 5'-phosphate group. Each unmodified nucleoside is a β-D-2'-deoxyribonucleoside. Nucleosides followed by a subscript d, e, f, m or x indicate modification as follows: a subscript "d" indicates a 2'-OCH2(CO)NH(CH2)2N(CH3)2 (DMAEAc), subscript "e" indicates a 2'-O(CH2)2θCH3 (MOE) modified nucleoside, subscript "f ' indicates a 2'-fluoro modified nucleoside subscript "m" indicates 2'-O-methyl modified nucleoside and subscript "ef indicates a 2'-OCH2CH2F (FEt) modified nucleoside.

Claims

Claims
1. An oligomeric compound comprising an oligonucleotide comprising a nucleoside having Formula VII:
Figure imgf000191_0001
wherein:
Bx is a heterocyclic base moiety; T3 is a phosphorus moiety; T4 is an internucleoside linking group attaching the nucleoside of Formula I to the remainder of the oligonucleotide; and each of qi and q2 is, independently selected from H, Ci-C6 alkyl, C2-C6 alkenyl, C2- C6alkynyl, substituted Ci-C6 alkyl, substituted Ci-C6 alkenyl and substituted C2-C6 alkynyl;
Xi is S, NRi6, or CRi0Ri 1 wherein each Ri0 and Rn is, independently, H, F, Ci-C6 haloalkyl , or C]-C6 alkyl; and
Ri is selected from a halogen, X2-V, and 0-X4; or each of qi and q2 is, independently, selected from H, CpC6 alkyl, C2-C6 alkenyl, C2- C6 alkynyl, substituted CpC6 alkyl, substituted Ci-C6 alkenyl and substituted C2-C6 alkynyl; Xi is O, S, NRi6Rn, or CRioRn wherein each Ri0 and Rn is, independently, H, F, Q-
C6 haloalkyl , or Ci-C6 alkyl; and
Ri is X2-V; or each of qi and q2 is, independently, selected from CpC6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted CpC6 alkyl, substituted CpC6 alkenyl and substituted C2-C6 alkynyl;
Xi is O, S, NR16Rn, or CRi0Rn wherein each Ri0 and Rn is, independently, H, F, C1- C6 haloalkyl , or C1-C6 alkyl; and
Ri is selected from halogen, X2-V, and 0-X4; wherein: X2 is O, S or CR7R8 wherein each R7 and R8 is, independently, H or C1-C6 alkyl;
V is selected from cholesterol, (CH2)2[O(CH2)2]tOCH3, where t is from 1-3, (CH2)2F, CH2COOH, CH2CONH2, CH2CONR5R61 CH2COOCH2CH3, CH2CONH(CH2)i-S-R4 where i is from 1 to 10, CH2CONH(CH2)JNR5R6 where j is from 1 to 6, CH2CONH[(CH2)ki -N(H)Jk2-(CH2)UNH2 where each ki is independently from 2 to 4 and k2 is from 2 to 10, and aryl;
R4 is selected from H, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted Ci-C6 alkenyl, substituted C2-C6 alkynyl, C6-Cu aryl and a thio protecting group; R5 and R6 are each, independently, selected from H, Q-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, and substituted C2-C6 alkynyl; Ri6 is selected from H, Q-C6 alkyl, or substituted Ci-C6 alkyl; X4 is [C(Ra)(Rb)]n-[(C=O)mXc]k-Rd wherein each Ra and Rb is independently H or halogen; Xc is O, S, or N(Ei);
Rd is H, Cx-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Q-C6 alkyl, substituted Q-C6 alkenyl and substituted C2-C6 alkynyl or NE2E3; each Ei, E2, and E3 is independently H, Ci-C6 alkyl, or substituted Ci-C6 alkyl; n is 1 to 6; m is 0 or 1 ; and k is 0 or 1; and wherein each substituted group is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, OJi, NJiJ2, SJi, N3, OC(=O)J! and CN, wherein each Ji and J2 is, independently, H or C]-C6 alkyl; and J4 is hydrogen, or a protecting group.
2. An oligomeric compound comprising an oligonucleotide comprising a nucleoside having Formula VII:
Figure imgf000192_0001
wherein:
Bx is a heterocyclic base moiety;
Xi is O, S, NRi6Ri7, or CRioRπ wherein each R^ and Rn is, independently, H, F, or Ci-C6 alkyl, and each Ri6 and Rn is independently, H, Ci-C6 alkyl, substituted Ci-C6 alkyl,
T3 is a phosphorus moiety; T4 is an internucleoside linking group attaching the nucleoside of Formula I to the remainder of the oligonucleotide; Ri is selected from halogen, trifluoroalkoxy, azido, aminooxy, O-alkyl, S-alkyl, N(J4)-alkyl, O- alkenyl, S-alkenyl, N(J4)-alkenyl, O-alkynyl, S-alkynyl or N(J4)-alkynyl, O-alkoxy, and X2-V, wherein: X2 is O, S or CR7Rg wherein each R7 and Rg is, independently, H or Ci-C6 alkyl; V is selected from (CH2)2F, CH2COOH, CH2CONH2, CH2COOCH2CH3, CH2CONH(CH2),-S-R4 where i is from 1 to 10, CH2CONH(CH2)JNR5R6 where j is from 1 to 6, and
Figure imgf000193_0001
(CH2)kiNH2 where each ki is independently from 2 to 4 and k2 is from 2 to 10;
R4 Is selected from H, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted C1-C6 alkenyl, substituted C2-C6 alkynyl, C6-Ci4 aryl and a thio protecting group;
R5 and R6 are each, independently, selected from H, Ci-C6 alkyl, substituted Q-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, and substituted C2-C6 alkynyl; and each of qi and q2 is, independently, selected from H, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted Ci-C6 alkenyl and substituted C2-C6 alkynyl; provided that if each of qi and q2 is H, then: either: Ri is selected from, trifluoroalkoxy, azido, aminooxy, S-alkyl, N(J4)-alkyl, O-alkenyl, S- alkenyl, N(J4)-alkenyl, O-alkynyl, S-alkynyl and N(J4)-alkynyl, and X2-V; and Xi selected from O, S, N, and CRi0Ri 1; or
Ri is selected from halogen, trifluoroalkoxy, azido, aminooxy, O-alkyl, S-alkyl, N(J4)-alkyl, O-alkenyl, S-alkenyl, N(J4)-alkenyl, O-alkynyl, S-alkynyl or N(J4)-alkynyl, O-alkoxy, and X2-V; and
Xi is selected from S, N, or CRioRπ; and wherein each substituted group is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, OJi, NJiJ2, SJi, N3, OC(=O)Ji and CN, wherein each Ji and J2 is, independently, H or Ci-C6 alkyl; and J4 is hydrogen, or a protecting group.
3. The oligomeric compound of claim 1 or 2 wherein Ri is selected from halogen, O-alkyl, O-haloalkyl, O-alkoxy.
4. The oligomeric compound of claim 1 or 2 wherein Ri is F.
5. The oligomeric compound of claim 1 or 2 wherein Ri is 0-C2-C4 alkyl or haloalkyl.
6. The oligomeric compound of claim 1 or 2 wherein Ri is OCH3.
7. The oligomeric compound of claim 1 or 2 wherein Ri is O(CH2)2OCH3
8. The oligomeric compound of claim 1 or 2 wherein Ri is FCH2CH3.
9. The oligomeric compound of claim 1 or 2 wherein Ri is (CH2)2[O(CH2)2]tOCH3, where t is from 1-3.
10. The oligomeric compound of claim 1 or 2 wherein Ri is selected from, trifluoroalkoxy, azido, aminooxy, S-alkyl, N(J4)-alkyl, O-alkenyl, S-alkenyl, N(J4)-alkenyl, O-alkynyl, S-alkynyl, N(J4)-alkynyl, and
X2-V.
11. The oligomeric compound of claim 10 wherein Ri is X2-V.
12. The oligomeric compound of claim 11 wherein V is (CH2)2F.
13. The oligomeric compound of claim 11 wherein V is CH2CONH(CH2)I-S-R4.
14. The oligomeric compound of claim 11 wherein V is CH2CONHt(CH2)IcI-N(H)Ik2-(CH2)ICiNH2.
15. The oligomeric compound of claim 11 wherein V is CH2CONH-(CH2)3-N(H)-(CH2)4-N(H)- (CH2)3NH2.
16. The oligomeric compound of claim 11 wherein V is CH2CONH(CH2)jNR5R5.
17. The oligomeric compound of claim 15 or 16 wherein at least one of R5 and R6 is other than H.
18. The oligomeric compound of any of claims 15-17 wherein at least one of R5 and Rs is methyl.
19. The oligomeric compound of any of claims 15-18 wherein R5 is methyl and R6 is methyl.
20. The oligomeric compound of any of claims 1-19 wherein X2 is O.
21. The oligomeric compound of any of claims 1-19 wherein X2 is S .
22. The oligomeric compound of any of claims 1-19 wherein X2 is CR7R8.
23. The oligomeric compound of any of claims 1 -22 wherein R7 and Rg are both H.
24. The oligomeric compound of any of claims 1-23 wherein at least one of qi and q2 is Ci-Ce alkyl or substituted CpC6 alkyl.
25. The oligomeric compound of claim 24 wherein at least one of qi and q2 is Ci-C6 alkyl.
26. The oligomeric compound of claim 25 wherein at least one of qi and q2 is methyl.
27. The oligomeric compound of any of claims 1-26 wherein at least one of qi and q2 is H.
28. The oligomeric compound of any of claims 1-27 wherein one of qi and q2 is methyl and the other of qi and q2 is H.
29. The oligomeric compound of any of claims 1-27 wherein qi and q2 are each Ci-C6 alkyl or substituted Ci-C6 alkyl.
30. The oligomeric compound of any of claims 1-29 wherein Xi is O.
31. The oligomeric compound of any of claims 1 -29 wherein Xi is S.
32. The oligomeric compound of any of claims 1-29 wherein Xi is CRi0Rn.
33. The oligomeric compound of any of claims 1-32 wherein RioandRn are both H.
34. The oligomeric compound of any of claims 1-33 wherein the phosphorus moiety is P(Ya)(Yb)(Yc,) where Ya is O or S and each Yb and Yc is, independently, selected from OH, SH, alkyl, alkoxy, substituted
Ci-C6 alkyl and substituted Ci-C6 alkoxy.
35. The oligomeric compound of claim 34 wherein Ya is O and Yb and Yc are each OH.
36. The oligomeric compound of any of claims 1-35 comprising a nucleoside of Formula VIII:
Figure imgf000195_0001
37. The oligomeric compound of claim 36 wherein the nucleoside of Formula VIII has the configuration:
Figure imgf000196_0001
38. The oligomeric compound of claim 36 or 37 wherein qi is methyl and q2 is H.
39. The oligomeric compound of claim 36 or 37 wherein qi is H and q2 is methyl.
40. An oligomeric compound comprising a di-nucleoside having Formula IX:
Figure imgf000196_0002
wherein: each Bx is independently a heterocyclic base moiety;
T4 is an internucleoside linking group attaching the nucleoside of Formula IV to the remainder of the oligonucleotide; each of qi and q2 is, independently selected from H, C]-C6 alkyl, C2-C6 alkenyl, C2- C6alkynyl, substituted CpC6 alkyl, substituted Q-C6 alkenyl and substituted C2-C6 alkynyl;
Xi is S, NR16, or CRi0Rn wherein each Ri0 and Rn is, independently, H, F, Ci-C6 haloalkyl , or Ci -C6 alkyl; and R1 is selected from a halogen, X2-V, and (D-X4; or each of qi and q2 is, independently, selected from H, C]-C6 alkyl, C2-C6 alkenyl, C2- C6 alkynyl, substituted Ci-C6 alkyl, substituted Ci-C6 alkenyl and substituted C2-C6 alkynyl; Xi is O, S, NRi6R]7, or CRioRn wherein each Rio and Rn is, independently, H, F, Q- C6 haloalkyl , or Q-C6 alkyl; and
Ri is X2-V; or each of qi and q2 is, independently, selected from C]-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted Ci-C6 alkenyl and substituted C2-C6 alkynyl;
Xi is O, S, NRi6Rn, or CR]0Rn wherein each Rj0 and Rn is, independently, H, F, Q- C6 haloalkyl , or C]-C6 alkyl; and
Ri is selected from halogen, X2-V, and 0-X4; wherein:
X2 is O, S or CR7Rg wherein each R7 and Rg is, independently, H or C]-C6 alkyl; V is selected from cholesterol, (CH2)2[O(CH2)2]tOCH3, where t is from 1-3, (CH2)2F, CH2COOH, CH2CONH2, CH2CONR5R61 CH2COOCH2CH3, CH2CONH(CH2X-S-R4 where i is from 1 to 10, CH2CONH(CH2)JNR5R6 where j is from 1 to 6, and CH2CONHt(CH2)H-N(H)Ik2-(CH2X1NH2 where each ki is independently from 2 to 4 and k2 is from 2 to 10;
R4 is selected from H, Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Cj-C6 alkyl, substituted Ci-C6 alkenyl, substituted C2-C6 alkynyl, C6-Ci4 aryl and a thio protecting group;
R5 and R6 are each, independently, selected from H, C]-C6 alkyl, substituted Cj-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, and substituted C2-C6 alkynyl; Ri6 is selected from H, Ci-C6 alkyl, or substituted Cj-C6 alkyl;
X4 is [C(RaχRb)]n-[(C=O)mXc]k-R<i wherein each Ra and Rb is independently H or halogen; XC iS O5 S5 OrN(E1);
Ra is H5 C]-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Cj-C6 alkyl, substituted C]-C6 alkenyl and substituted C2-C6 alkynyl or NE2E3; each Ei, E2, and E3 is independently H, Ci-C6 alkyl, or substituted CrC6 alkyl; n is 1 to 6; m is O or 1; and k is O or 1; and wherein X3 is OH or SH;
Ya is O or S; each Yb and Yc is, independently, selected from OH, SH, alkyl, alkoxy, substituted Ci-C6 alkyl and substituted Ci-C6 alkoxy;
R9 is selected from is selected from a halogen, X2-V, and Q-X4; wherein each substituted group is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, OJi, NJ]J2, SJ], N3, OC(=O)Ji and CN, wherein each Ji and J2 is, independently, H or Ci-C6 alkyl; and J4 is hydrogen, or a protecting group.
41. The oligomeric compound of claim 40 wherein Ri is F.
42. The oligomeric compound of claim 40 wherein Ri is OCH3.
43. The oligomeric compound of claim 40 wherein Ri is O-C2-C4 alkyl or haloalkyl.
44. The oligomeric compound of claim 40wherein Ri is O(CH2)2OCH3
45. The oligomeric compound of claim 40 wherein R] is FCH2CH3.
46. The oligomeric compound of claim 40 wherein Ri is (CH2)2[O(CH2)2]tOCH3, where t is from 1-3.
47. The oligomeric compound of claim 40 wherein Ri is selected from, trifluoroalkoxy, azido, aminooxy, S-alkyl, N(J4)-alkyl, O-alkenyl, S-alkenyl, N(J4)-alkenyl, O-alkynyl, S-alkynyl, N(J4)-alkynyl, and X2-V.
48. The oligomeric compound of claim 47 wherein Ri is X2-V.
49. The oligomeric compound of claim 48 wherein V is (CH2)2F.
50. The oligomeric compound of claim 48 wherein V is CH2CONH(CH2)J-S-R4
51. The oligomeric compound of claim 48 wherein V is CH2CONH[(CH2)ki -N(H)Jk2-(CH2XiNH2.
52. The oligomeric compound of claim 48 wherein V is CH2CONH-(CH2)3-N(H)-(CH2)4-N(H)- (CH2)3NH2.
53. The oligomeric compound of claim 48 wherein V is CH2CONH(CH2)JNR5R6.
54. The oligomeric compound of claim 53 wherein j is 2.
55. The oligomeric compound of claim 53 or 54 wherein at least one OfR5 and R6 is other than H.
56. The oligomeric compound of any of claims 53-55 wherein at least one OfR5 and R6 is methyl.
57. The oligomeric compound of any of claims 53-56 wherein R5 is methyl and R6 is methyl.
58. The oligomeric compound of any of claims 40-57 wherein X2 is O.
59. The oligomeric compound of any of claims 40-57 wherein X2 is S.
60. The oligomeric compound of any of claims 40-57 wherein X2 is CR7R8.
61. The oligomeric compound of any of claims 40-60 wherein R7 and R8 are both H.
62. The oligomeric compound of any of claims 40-61 wherein at least one of qi and q2 is Q-C6 alkyl or substituted Ci-C6 alkyl.
63. The oligomeric compound of claim 62 wherein at least one of qi and q2 is C1-C6 alkyl.
64. The oligomeric compound of claim 63 wherein at least one of qi and q2 is methyl.
65. The oligomeric compound of any of claims 40-64 wherein at least one of qi and q2 is H.
66. The oligomeric compound of any of claims 40-65 wherein one of qi and q2 is methyl and the other of qi and q2 is H.
67. The oligomeric compound of any of claims 40-66 wherein qi and q2 are each Ci-C6 alkyl or substituted Ci-C6 alkyl.
68. The oligomeric compound of any of claims 40-67 wherein X] is O.
69. The oligomeric compound of any of claims 40-67 wherein Xi is S.
70. The oligomeric compound of any of claims 40-67 wherein Xi is CRioRn-
71. The oligomeric compound of any of claims 40-70 wherein Ri0 and Ri i are both H.
72. The oligomeric compound of any of claims 40-71 wherein R9 is selected from F, OCH3 and
O(CH2)2OCH3.
73. The oligomeric compound of claim 72 wherein R9 is OCH3.
74. The oligomeric compound of claim 72 wherein R9 is F.
75. The oligomeric compound of claim 72 wherein R9 is 0(Ct^OCHs.
76. The oligomeric compound of claim 40 wherein the di-nucleoside having Formula IX has Formula X:
Figure imgf000200_0001
77. The oligomeric compound of claim 76 wherein qj is methyl and q2 is H.
78. The oligomeric compound of claim 76 wherein q] is H and q2 is methyl.
79. An oligomeric compound comprising an oligonucleotide comprising a nucleoside having Formula II:
Figure imgf000200_0002
wherein independently for each monomer of Formula II: Bx is a heterocyclic base moiety; A is O, S OrN(R1); R1 is H, Ci-C6 alkyl or substituted Q-C6 alkyl; one of T3 and T4 is an internucleoside linking group linking the monomer to the remainder of the oligomeric compound and the other of T3 and T4 is H, a protecting group, a phosphorus moiety, a 5' or 3'- terminal group or an internucleoside linking group linking the monomer to the oligomeric compound; one of Qi and Q2 is H, Ci-C6 alkyl, substituted Q-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl and the other of Qi and Q2 is Ci-C6 alkyl, substituted CrC6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
Gi is O-[C(R2)(R3)]n-[(C=O)m-X]j-Z or halogen; each R2 and R3 is, independently, H or halogen;
X is O, S OrN(E1); Z is H, halogen, Q-C6 alkyl, substituted Q-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(E2)(E3);
Ei, E2 and E3 are each, independently, H, Q-C6 alkyl or substituted Cx-C6 alkyl; n is from 1 to about 6; m is 0 or 1; j is O or l; each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJi, N(Ji)(J2), =NJ,, SJb N3, CN, OC(=L)Ji, OC(=L)N(Ji)(J2) and C(=L)N(J,)(J2);
L is O, S OrNJ3; each Ji, J2 and J3 is, independently, H or Ci-C6 alkyl; and when j is 1 then Z is other than halogen or N(E2)(E3) and when A is O then Gi is other than halogen.
80. The oligomeric compound of claim 79 wherein each Bx is, independently, uracil, 5-thiazolo-uracil, 2- thio-uracil, 5-propynyl-uracil, thymine, 2'-thio-thymine, cytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5- propynyl-cytosine, adenine, guanine, 2,6-diaminopurine, lH-pyrimido[5,4-b][l,4benzoxazin-2(3H)-one), IH- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one, 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)- one, 2H-pyrimido[4,5-b]indol-2-one or H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one.
81. The oligomeric compound of claim 79 wherein each Bx is, independently, uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine.
82. The oligomeric compound of any of claims 79 to 81 wherein each Qi is H.
83. The oligomeric compound of any one of claims 79 to 82 wherein each Q2 is H.
84. The oligomeric compound of any one of claims 79 to 81 wherein each Qi and each Q2 are other than
H.
85. The oligomeric compound of any one of claims 79 to 84 wherein for each monomer of Formula II at least one of Qi and Q2 is substituted Ci-C6 alkyl.
86. The oligomeric compound claim 85 wherein each substituted Ci-C6 alkyl comprises at least one substituent group independently selected from halogen, C2-C6 alkenyl, OJi, NJiJ2 and CN, wherein each Ji and J2 is, independently, H or Ci-C6 alkyl.
87. The oligomeric compound claim 85 wherein each substituted Ci-C6 alkyl comprises at least one substituent group independently selected from fluoro and OCH3.
88. The oligomeric compound of any one of claims 79 to 81 wherein for each monomer of Formula II at least one of Qi and Q2 is CpC6 alkyl.
89. The oligomeric compound of any one of claims 79 to 81 wherein for each monomer of Formula II Qi is methyl.
90. The oligomeric compound of any one of claims 79 to 81 wherein for each monomer of Formula II Q2 is methyl.
91. The oligomeric compound of any one of claims 79 to 90 wherein, independently for each monomer of Formula II, G, is OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, O(CH2)2F, OCH2CHF2, OCH2CF3, OCH2- CH=CH2, O(CH2)2-OCH3, O(CH2)2-SCH3, O(CH2)2-OCF3, O(CH2)3-N(R4)(R5), O(CH2)2-ON(R4)(R5), O(CH2)2-O(CH2)2-N(R4)(R5), OCH2CC=O)-N(R4)(R5), OCH2C(=O)-N(R6)-(CH2)2-N(R4)(R5) or O(CH2)2- N(R6)-C(=NR7)[N(R4)(R5)] wherein R4, R5, R6 and R7 are each, independently, H or C1-C6 alkyl.
92. The oligomeric compound of any one of claims 79 to 90 wherein, independently for each monomer of Formula II, G1 is OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-O(CH2)2- N(CH3)2, OCH2C(=O)-N(H)CH3, OCH2C(=O)-N(H)-(CH2)2-N(CH3)2 or OCH2-N(H)-C(=NH)NH2.
93. The oligomeric compound of any one of claims 79 to 90 wherein, independently for each monomer of Formula II, G, is OCH3, O(CH2)2-OCH3, OCH2C(=O)-N(H)CH3 or OCH2C(=O)-N(H)-(CH2)2-N(CH3)2.
94. The oligomeric compound of any one of claims 79 to 90 wherein for each monomer of Formula II Gi is F.
95. The oligomeric compound of any one of claims 79 to 94 wherein T3 is a phosphorus moiety.
96. The oligomeric compound of claim 95 wherein said phosphorus moiety has the formula:
Figure imgf000203_0001
wherein:
Ra and R0 are each, independently, OH, SH, C1-C6 alkyl, substituted Ci-C6 alkyl, C1-C6 alkoxy, substituted CpC6 alkoxy, amino or substituted amino; and Rb is O or S.
97. The oligomeric compound of claim 96 wherein Ra and R0 are each OH.
98. The oligomeric compound of claim 96 wherein R3 and R0 are each OCH3.
99. The oligomeric compound of claim 96 wherein Ra and R0 are each OCH2CH3.
100. The oligomeric compound of any one of claims 96 to 99 wherein Rb is O.
101. The oligomeric compound of any one of claims 96 to 99 wherein Rb is S.
102. The oligomeric compound of any one of claims 79 to 101 wherein each monomer of Formula II has the configuration:
Figure imgf000203_0002
103. The oligomeric compound of any one of claims 79 to 102 comprising one monomer of Formula II at the 5 'end.
104. An oligomeric compound comprising an oligonucleotide comprising a nucleoside having Formula IV:
Figure imgf000204_0001
wherein independently for each monomer of Formula IV: Bx is a heterocyclic base moiety; one OfT7 and T8 is an internucleoside linking group linking the monomer to the remainder of the oligomeric compound and the other of T7 and T8 is H, a hydroxyl protecting group, a phosphorus moiety, a 5' or 3 '-terminal group or an internucleoside linking group linking the monomer to the oligomeric compound; Qh Q2, Q3 and Q4 are each, independently, H, halogen, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-CO alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; Gi is O-[C(R2)(R3)]n-[(C=O)m-X]j-Z or halogen; each R2 and R3 is, independently, H or halogen; X is O, S or N(Ei);
Z is H, halogen, C1-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(E2)(E3);
Ei, E2 and E3 are each, independently, H, Ci-C6 alkyl or substituted Ci-C6 alkyl; n is from 1 to about 6; m is O or l; j is O or 1; each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJi, N(Ji)(J2), =NJb SJi, N3, CN, OC(=L)Ji, OC(=L)N(Ji)(J2) and CC=L)N(J1XJ2); L is O, S orNJ3; each Ji, J2 and J3 is, independently, H or Ci-C6 alkyl; when j is 1 then Z is other than halogen OrN(E2)(E3); and when Qi, Q2, Q3 and Q4 are each H or when Qi and Q2 are H and Q3 and Q4 are each F or when Qi and Q2 are each H and one of Q3 and Q4 is H and the other of Q3 and Q4 is R9 then Gi is other than H, hydroxyl, OR9, halogen, CF3, CCl3, CHCl2 and CH2OH wherein R9 is alkyl, alkenyl, alkynyl, aryl or alkaryl.
105. The oligomeric compound of claim 104 wherein each Bx is, independently, uracil, 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil, thymine, 2'-thio-thymine, cytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5- propynyl-cytosine, adenine, guanine, 2,6-diaminopurine, lH-pyrimido[5,4-b][l,4benzoxazin-2(3H)-one), IH- pyrimido[5,4-b] [ 1 ,4]benzothiazin-2(3H)-one, 9-(2-aminoethoxy)-H-pyrimido[5,4-b] [ 1 ,4]benzoxazin-2(3H)- one, 2H-pyrimido[4,5-b]indol-2-one or H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one.
106. The oligomeric compound of claim 104 wherein each Bx is, independently, uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine.
107. The oligomeric compound of any one of claims 104 to 106 wherein, independently for each monomer of Formula IV, G1 is OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, O(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-SCH3, O(CH2)2-OCF3, 0(CH2)S-N(R4)(R5), O(CH2)2-ON(R4)(R5); O(CH2)2-O(CH2)2-N(R4)(R5), OCH2C(=O)-N(R4)(R5), OCH2C(=O)-N(R6)-(CH2)2-N(R4)(R5) or O(CH2)2- N(R6)-C(=NR7)[N(R4)(R5)] wherein R4, R5, R6 and R7 are each, independently, H or C1-C6 alkyl.
108. The oligomeric compound of any one of claims 104 to 107 wherein, independently for each monomer of Formula IV, G1 is OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-O(CH2)2-N(CH3)2, OCH2C(=O)-N(H)CH3, OCH2C(=O)-N(H)-(CH2)2-N(CH3)2 or OCH2-N(H)- C(=NH)NH2.
109. The oligomeric compound of any one of claims 104 to 107 wherein, independently for each monomer of Formula IV, G1 is OCH3, O(CH2)2-OCH3, OCH2C(=O)-N(H)CH3 or OCH2C(=O)-N(H)-(CH2)2- N(CHj)2.
110. The oligomeric compound of any one of claims 104 to 107 wherein for each monomer of Formula IV Gi is F.
111. The oligomeric compound of any one of claims 104 to 110 wherein one T8 is a 3'-terminal group.
112. The oligomeric compound of any one of claims 104 to 111 wherein at least one of T7 and T8 is a conjugate group.
113. The oligomeric compound of any one of claims 104 to 112 wherein one T7 is a phosphorus moiety.
114. The oligomeric compound of claim 113 wherein said phosphorus moiety has the formula:
Figure imgf000205_0001
wherein: Ra and Rc are each, independently, OH, SH, Ci-C6 alkyl, substituted Ci-C6 alkyl, Ci-C6 alkoxy, substituted Ci-C6 alkoxy, amino or substituted amino; and Rb is O or S.
115. The oligomeric compound of claim 113 wherein Ra and R0 are each OH.
116. The oligomeric compound of claim 113 wherein Ra and R0 are each OCH3.
117. The oligomeric compound of claim 113 wherein Ra and R0 are each OCH2CH3.
118. The oligomeric compound of any one of claims 113 to 117 wherein Rb is O.
119. The oligomeric compound of any one of claims 113 to 117 wherein Rb is S.
120. The oligomeric compound of any one of claims 104 to 119 wherein for each monomer of Formula IV one of Qi, Q2, Q3 and Q4 is substituted C]-C6 alkyl.
121. The oligomeric compound of any one of claims 104 to 120 wherein for each monomer of Formula IV one of Qi, Q2, Q3 and Q4 is substituted Ci-C6 alkyl and the other three of Qi, Q2, Q3 and Q4 are H.
122. The oligomeric compound of any one of claims 120 or 121 wherein said substituted Ci-C6 alkyl comprises at least one substituent group selected from halogen, C2-C6 alkenyl, OJi, NJiJ2 and CN, wherein each Ji and J2 is, independently, H or C]-C6 alkyl.
123. The oligomeric compound of any one of claims 120 or 121 wherein said substituted Ci-C6 alkyl comprises at least one substituent group selected from fluoro and OCH3.
124. The oligomeric compound of any one of claims 104 to 123 wherein for each monomer of Formula IV one of Qi, Q2, Q3 and Q4 is CrC6 alkyl.
125. The oligomeric compound of claim 124 wherein for each monomer of Formula IV one of Qi and Q2 is Ci-C6 alkyl.
126. The oligomeric compound of claim 124 wherein for each monomer of Formula IV one OfQ3 and Q4 is Ci-C6 alkyl.
127. The oligomeric compound of any one of claims 123 to 126 wherein said C1-C6 alkyl group is methyl.
128. The oligomeric compound of any one of claims 104 to 123 wherein for each monomer of Formula IV three of Qi, Q2, Q3 and Q4 are H.
129. The oligomeric compound of any one of claims 104 to 123 and 125 to 127 wherein for each monomer of Formula IV one of Qi, Q2, Q3 and Q4 is F.
130. The oligomeric compound of any one of claims 104 to 128 wherein for each monomer of Formula IV two of Qi, Q2, Q3 and Q4 are F.
131. The oligomeric compound of any one of claims 104 to 128 wherein for each monomer of Formula IV Qi and Q2 are each F.
132. The oligomeric compound of any one of claims 104 to 128 wherein for each monomer of Formula IV Q3 and Q4 are each F.
133. The oligomeric compound of any one of claims 104 to 132 wherein for each monomer of Formula IV, Qi, Q2, Q3 and Q4 are each F or H.
134. The oligomeric compound of any one of claims 104 to 133 wherein each monomer of Formula IV has the configuration:
Figure imgf000207_0001
135. The oligomeric compound of any one of claims 104 to 134 comprising one monomer of Formula IV at the 5 'end.
136. An oligomeric compound comprising an oligonucleotide comprising a nucleoside at the 5'-end having Formula XIII:
Figure imgf000208_0001
wherein:
Bx is a heterocyclic base moiety; A is O, S or N(R,);
Ri is H, Ci-C6 alkyl or substituted Ci-C6 alkyl; T3 is a phosphorus moiety;
T4 is an internucleoside linking group linking the monomer to the remainder of the oligomeric compound; one of Qi and Q2 is H, C1-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl and the other of Qi and Q2 is Q-C6 alkyl, substituted Q-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
G1 is O-[C(R2)(R3)]n-[(C=O)m-X]j-Z, 0-Ci-C6 alkyl, O-CrC6 substituted alkyl, O-aryl, or halogen; each R2 and R3 is, independently, H or halogen; X iS O3 S OrN(E1);
Z is H, halogen, Q-C6 alkyl, substituted Q-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(E2)(E3);
E1, E2 and E3 are each, independently, H, C1-C6 alkyl or substituted Ci-C6 alkyl; n is from 1 to about 6; m is O or 1; j is O or 1 ; each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ1, N(J1)(J2), =NJb SJ1, N3, CN, OC(=L)Jb OC(=L)N(Ji)(J2) and C(=L)N(J,)(J2); L is O, S orNJ3; each J1, J2 and J3 is, independently, H or C1-C6 alkyl; and when j is 1 then Z is other than halogen or N(E2)(E3) and when A is O then Gi is other than halogen.
137. The oligomeric compound of claim 136 wherein Bx is uracil, 5-thiazolo-uracil, 2-thio-uracil, 5- propynyl-uracil, thymine, 2'-thio-thymine, cytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl- cytosine, adenine, guanine, 2,6-diaminopurine, lH-pyrimido[5,4-b][l,4benzoxazin-2(3H)-one), IH- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one, 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)- one, 2H-pyrimido[4,5-b]indol-2-one or H-pyrido[3',2':4,5]pyiτolo[2,3-d]pyrimidin-2-one.
138. The oligomeric compound of claim 137 wherein Bx is uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine.
139. The oligomeric compound of any of claims 136 to 138 wherein Q1 is H.
140. The oligomeric compound of any one of claims 136 to 139 wherein Q2 is H.
141. The oligomeric compound of any one of claims 136 to 138 wherein Qi and Q2 are each other than H.
142. The oligomeric compound of any one of claims 136 to 141 wherein at least one of Qi and Q2 is substituted Ci-C6 alkyl.
143. The oligomeric compound claim 142 wherein each substituted Ci-C6 alkyl comprises at least one substituent group independently selected from halogen, C2-C6 alkenyl, OJi, NJiJ2 and CN, wherein each Ji and J2 is, independently, H or Ci-C6 alkyl.
144. The oligomeric compound claim 143 wherein each substituted Q-C6 alkyl comprises at least one substituent group independently selected from fluoro and OCH3.
145. The oligomeric compound of any one of claims 136 to 144 wherein at least one of Qi and Q2 is Ci-C6 alkyl.
146. The oligomeric compound of any one of claims 136 to 145 wherein Qi is methyl.
147. The oligomeric compound of any one of claims 136 to 146 wherein Q2 is methyl.
148. The oligomeric compound of any of claims 136-147 wherein said phosphorus moiety has the formula:
Figure imgf000209_0001
wherein:
Ra and R0 are each, independently, OH, SH, Ci-C6 alkyl, substituted Q-C6 alkyl, Q-C6 alkoxy, substituted Ci-C6 alkoxy, amino or substituted amino; and Rb is O or S.
149. The oligomeric compound of claim 148 wherein Ra and R0 are each OH.
150. The oligomeric compound of claim 148 wherein Ra and R0 are each OCH3.
151. The oligomeric compound of claim 148 wherein Ra and R0 are each OCH2CH3.
152. The oligomeric compound of any one of claims 148 to 151 wherein Rb is O.
153. The oligomeric compound of any one of claims 148 to 152 wherein Rb is S.
154. The oligomeric compound of any one of claims 136 to 153 wherein, Gi is OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, O(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-SCH3, O(CH2)2-OCF3, 0(CH2VN(R4)(R5), 0(CH2)Z-ON(R4)(R5), O(CH2)2-O(CH2)2-N(R4)(R5), OCH2C(=O)-
N(R4)(R5), OCH2C(=O)-N(R6)-(CH2)2-N(R4)(R5) or O(CH2)2-N(R6)-C(=NR7)[N(R4)(R5)] wherein R4, R5, R5 and R7 are each, independently, H or Ci-C6 alkyl.
155. The oligomeric compound of any one of claims 136 to 153 wherein, Gi is OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-O(CH2)2-N(CH3)2, OCH2C(=O)-N(H)CH3,
OCH2C(=O)-N(H)-(CH2)2-N(CH3)2 or OCH2-N(H)-C(=NH)NH2.
156. The oligomeric compound of any one of claims 136 to 153 wherein, Gi is OCH3, O(CH2)2-OCH3, OCH2C(=O)-N(H)CH3 or OCH2C(=O)-N(H)-(CH2)2-N(CH3)2.
157. The oligomeric compound of any one of claims 136 to 153 wherein Gi is F.
158. The oligomeric compound of any one of claims 136 to 153 wherein Gi is 0-C1-C6 alkyl, 0-Ci-C6 substituted alkyl, O-aryl.
159. The oligomeric compound of any one of claims 136 to 153 wherein Gi is -0-Ci-C6 substituted alkyl.
160. The oligomeric compound of any one of claims 136 to 153 wherein Gi is -OCH2CH2OCH3.
161. The oligomeric compound of any one of claims 136 to 153 wherein Gi is -OCH2CH2F.
162. The oligomeric compound of any one of claims 136 to 161 wherein the monomer of Formula XIII has the configuration:
Figure imgf000211_0001
163. An oligomeric compound comprising an oligonucleotide comprising a nucleoside at the 5'-end having Formula XTV:
Figure imgf000211_0002
wherein:
Bx is a heterocyclic base moiety; T7 is a phosphorus moiety; T8 is an internucleoside linking group linking the monomer to the remainder of the oligomeric compound;
Qi> Q^5 Qi and Q4 are each, independently, H, halogen, C1-C6 alkyl, substituted CrC6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
G1 is O-[C(R2)(R3)]n-[(C=O)m-X]j-Z, 0-C1-C6 alkyl, 0-C1-C6 substituted alkyl, O-aryl, or halogen; each R2 and R3 is, independently, H or halogen; each R2 and R3 is, independently, H or halogen; X iS O5 S Or N(E1);
Z is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(E2)(E3); Ei, E2 and E3 are each, independently, H, CrC6 alkyl or substituted Ci-C6 alkyl; n is from 1 to about 6; m is 0 or 1 ; j is O or 1; each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ1, N(J1)(J2), =NJh SJ1, N3, CN, OQ=L)J1, OQ=L)N(J1)(J2) and CC=L)N(J1)(J2);
L is O, S or NJ3; each J], J2 and J3 is, independently, H or CpC6 alkyl; when j is 1 then Z is other than halogen or N(E2)(E3); and when Q1, Q2, Q3 and Q4 are each H or when Q1 and Q2 are H and Q3 and Q4 are each F or when Q1 and Q2 are each H and one of Q3 and Q4 is H and the other of Q3 and Q4 is R9 then Gi is other than H, hydroxyl, OR9, halogen, CF3, CCl3, CHCl2 and CH2OH wherein R9 is alkyl, alkenyl, alkynyl, aryl or alkaryl.
164. The oligomeric compound of claim 163 wherein Bx is uracil, 5-thiazolo-uracil, 2-thio-uracil, 5- propynyl-uracil, thymine, 2'-thio-thymine, cytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl- cytosine, adenine, guanine, 2,6-diaminopurine, lH-pyrimido[5,4-b][l,4benzoxazin-2(3H)-one), IH- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one, 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)- one, 2H-pyrimido[4,5-b]indol-2-one or H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one.
165. The oligomeric compound of claim 163 wherein Bx is uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine.
166. The oligomeric compound of any one of claims 163 to 165 wherein one OfQ1, Q2, Q3 and Q4 is substituted C1-C6 alkyl.
167. The oligomeric compound of any one of claims 163 to 166 wherein one of Qi, Q2, Q3 and Q4 is substituted Ci-C6 alkyl and the other three of Qi, Q2, Q3 and Q4 are H.
168. The oligomeric compound of any one of claims 166 or 167 wherein said substituted Ci-C6 alkyl comprises at least one substituent group selected from halogen, C2-C6 alkenyl, OJi, NJiJ2 and CN, wherein each Ji and J2 is, independently, H or Ci-C6 alkyl.
169. The oligomeric compound of any one of claims 166 or 167 wherein said substituted C1-C6 alkyl comprises at least one substituent group selected from fluoro and OCH3.
170. The oligomeric compound of any one of claims 163 to 169 wherein one of Qi, Q2, Q3 and Q4 is Ci-C6 alkyl.
171. The oligomeric compound of claim 170 wherein one of Qi and Q2 is Ci-C6 alkyl.
172. The oligomeric compound of claim 170 wherein one OfQ3 and Q4 is CpC6 alkyl.
173. The oligomeric compound of any one of claims 169 tol72 wherein said Ci-C6 alkyl group is methyl.
174. The oligomeric compound of any one of claims 163 to 173 wherein three of Qi, Q2, Q3 and Q4 are H.
175. The oligomeric compound of any one of claims 163 to 174 wherein one of Q1, Q2, Q3 and Q4 is F.
176. The oligomeric compound of any one of claims 163 to 173 wherein two of Qi, Q2, Q3 and Q4 are F.
177. The oligomeric compound of any one of claims 163 to 170 or 172 to 173 wherein Qi and Q2 are each F.
178. The oligomeric compound of any one of claims 163 to 171 or 173 wherein Q3 and Q4 are each F.
179. The oligomeric compound of any one of claims 163 to 178 wherein Q1, Q2, Q3 and Q4 are each F or H.
180. The oligomeric compound of any of claims 163 to 170 wherein Qi is H.
181. The oligomeric compound of any one of claims 163 to 170 wherein Q2 is H.
182. The oligomeric compound of any one of claims 163 to 170 wherein Q1 and Q2 are each other than H.
183. The oligomeric compound of any one of claims 163 to 170 wherein at least one of Qi and Q2 is substituted C1-C6 alkyl.
184. The oligomeric compound of any one of claims 163 to 170 wherein at least one of Qi and Q2 is Ci-C6 alkyl.
185. The oligomeric compound of any one of claims 163 to 170 wherein Qi is methyl.
186. The oligomeric compound of any one of claims 163 to 170 wherein Q2 is methyl.
187. The oligomeric compound of any of claims 163- 186 wherein said phosphorus moiety has the formula:
Figure imgf000214_0001
wherein:
Ra and R0 are each, independently, OH, SH, Ci-C6 alkyl, substituted Ci-Ce alkyl, Ci-Ce alkoxy, substituted Ci-C6 alkoxy, amino or substituted amino; and Rb is O or S.
188. The oligomeric compound of claim 187 wherein Ra and R0 are each OH.
189. The oligomeric compound of claim 187 wherein Ra and R0 are each OCH3.
190. The oligomeric compound of claim 187 wherein Ra and R0 are each OCH2CH3.
191. The oligomeric compound of any one of claims 187 to 190 wherein Rt, is O.
192. The oligomeric compound of any one of claims 187 to 190 wherein Rb is S.
193. The oligomeric compound of any one of claims 163 to 192 wherein, Gi is OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, O(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-SCH3, O(CH2)rOCF3, 0(CH2)3-N(R4)(R5), 0(CHz)2-ON(R4)(R5), O(CH2)2-O(CH2)2-N(R4)(R5), OCH2C(=O)- N(R4)(R5), OCH2C(=O)-N(R6)-(CH2)2-N(R4)(R5) or O(CH2)2-N(R6)-C(=NR7)[N(R4)(R5)] wherein R4, R5, R6 and R7 are each, independently, H or Ci-C6 alkyl.
194. The oligomeric compound of any one of claims 163 to 192 wherein, Gi is OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-O(CH2)2-N(CH3)2, OCH2C(=O)-N(H)CH3, OCH2C(=O)-N(H)-(CH2)2-N(CH3)2 or OCH2-N(H)-C(=NH)NH2.
195. The oligomeric compound of any one of claims 163 to 192 wherein, Gi is OCH3, O(CH2)2-OCH3, OCH2C(=O)-N(H)CH3 or OCH2C(=O)-N(H)-(CH2)2-N(CH3)2.
196. The oligomeric compound of any one of claims 163 to 192 wherein Gi is F.
197. The oligomeric compound of any one of claims 163 to 192 wherein Gi is 0-C]-C6 alkyl, 0-Ci-C6 substituted alkyl, O-aryl.
198. The oligomeric compound of any one of claims 163 to 192 wherein Gi is -O-CrC6 substituted alkyl.
199. The oligomeric compound of any one of claims 163 to 192 wherein Gi is -OCH2CH2OCH3.
200. The oligomeric compound of any one of claims 163 to 192 wherein Gi is -OCH2CH2F.
201. The oligomeric compound of any one of claims 163 to 192 the monomer of Formula XIV has the configuration:
Figure imgf000215_0001
202. An oligomeric compound comprising an oligonucleotide comprising a phosphate stabilizing nucleoside at the 5 '-end, wherein the phosphate stabilizing nucleoside comprises: a 5 '-terminal modified or unmodified phosphate; a modified sugar moiety comprising: a 5'- modification; or a 2 '-modification; or both a 5'-modificaton and a 2 '-modification; and a linking group linking the phosphate stabilizing nucleoside to the remainder of the oligonucleotide.
203. The oligomeric compound of claim 202, wherein : the 5 '-terminal modified phosphate is selected from: phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphorothioate, phosphoramidate, alkylphosphonothioate, substituted alkylphosphonothioate, phosphorodithioate, thiophosphoramidate, and phosphotriester; the 5 '-modification of the sugar moiety of the phosphate stabilizing nucleoside is selected from 5'-alkyl and 5 '-halogen; the 2'-modification of the sugar moiety of the phosphate stabilizing nucleoside is selected from: halogen, allyl, amino, azido, thio, O-allyl, -0-Ci-C10 alkyl, -O-CrCi0 substituted alkyl, -OCF3, -O-(CH2)2-O-CH3, -
O(CH2)2SCH3, -O-(CH2)2-O-N(Rm)(Rn), -0-Cm-CC=O)-N(R1n)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl, -O[(CH2)nO]mCH3, -O(CH2)nNH2, -O(CH2)nCH3, - O(CH2)nONH2, -OCH2C(=O)N(H)CH3,-O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10; Ci to C10 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl.
204. The oligomeric compound of claim 203 or 204, wherein the modified phosphate is selected from: phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphotriester, phosphorothioate, phosphorodithioate, thiophosphoramidate, and phosphoramidate.
205. The oligomeric compound of claim 203 or 204, wherein the modified phosphate is selected from: phosphonate, alkylphosphonate, and substituted alkylphosphonate.
206. The oligomeric compound of claim 203 or 204, wherein the 5'-phosphate is selected from: 5'-deoxy- 5'-thio phosphate, phosphoramidate, methylene phosphonate, mono-fluoro methylene phosphonate and di- fluoro methylene phosphonate.
207. The oligomeric compound of any of claims 203 to 206, wherein the sugar moiety of the phosphate stabilizing nucleoside comprises a 5'-modificaton and a 2 '-modification.
208. The oligomeric compound of any of claims 1-207 wherein the remainder of the oligonucleotide comprises at least one modified nucleoside.
209. The oligomeric compound of claim 208 wherein the at least one modified nucleoside comprises a modified base.
210. The oligomeric compound of claim 208 or 209 wherein the at least one modified nucleoside comprises a sugar surrogate.
211. The oligomeric compound of claim 210 wherein the sugar surrogate is a tetrahydropyran.
212. The oligomeric compound of any of claim 211 wherein the tetrahydropyran is F-HNA.
213. The oligomeric compound of any of claims 208-212 wherein the remainder of the oligonucleotide comprises at least one nucleoside comprising a modified sugar.
214. The oligomeric compound of claim 213 wherein the at least one modified nucleoside comprising a modified sugar is selected from a bicyclic nucleoside and a 2'-modified nucleoside.
215. The oligomeric compound of claim 214 wherein the at least one modified nucleoside is a bicyclic nucleoside.
216. The oligomeric compound of claim 215 wherein the bicyclic nucleoside is a (4'-CH2-(W) BNA nucleoside.
217. The oligomeric compound of claim 215 wherein the bicyclic nucleoside is a (4'-(CH2)2-O-2') BNA nucleoside.
218. The oligomeric compound of claim 215 wherein the bicyclic nucleoside is a (4 ' -C(CH3)H-O-2 ' ) BNA nucleoside.
219. The oligomeric compound of claim 214 wherein the at least one modified nucleoside is a 2 ' -modifed nucleoside.
220. The oligomeric compound of claim 219 wherein the at least one 2'-modified nucleoside is selected from a 2'-F nucleoside, a 2'-OCH3 nucleoside, and a 2'-0(CHO2OCH3 nucleoside.
221. The oligomeric compound of claim 220 wherein the at least one 2'-modifϊed nucleoside is a 2'-F nucleoside.
222. The oligomeric compound of claim 220 wherein the at least one 2'-modifϊed nucleoside is a T- OCH3 nucleoside.
223. The oligomeric compound of claim 220 wherein the at least one 2'-modified nucleoside is a T- O(CH2)2OCH3 nucleoside.
224. The oligomeric compound of any of claims 1-223 wherein the remainder of the oligonucleotide comprises at least one unmodified nucleoside.
225. The oligomeric compound of claim 224 wherein the unmodified nucleoside is a ribonucleoside.
226. The oligomeric compound of claim 224 wherein the unmodified nucleoside is a deoxyribonucleoside.
227. The oligomeric compound of any of claims 1-226 wherein the remainder of the oligomeric oligonucleotide comprises at least two modified nucleosides.
228. The oligomeric compound of claim 227 wherein the at least two modified nucleosides comprise the same modification.
229. The oligomeric compound of claim 227 wherein the at least two modified nucleosides comprise different modifications.
230. The oligomeric compound of any of claims 227-229 wherein at least one of the at least two modified nucleosides comprises a sugar surrogate.
231. The oligomeric compound of any of claims 227-230 wherein at least one of the at least two modified nucleosides comprises a 2'-modifϊcation.
232. The oligomeric compound of claim 231 wherein each of the at least two modified nucleosides is independently selected from 2'-F nucleosides, 2'-OCH nucleosides and 2'-O(CH2)2θCH3 nucleosides.
233. The oligomeric compound of claim 232 wherein each of the at least two modified nucleosides is a 2'- F nucleoside.
234. The oligomeric compound of claim 232 wherein each of the at least two modified nucleosides is a T- OCH3 nucleosides.
235. The oligomeric compound of claim 232 wherein each of the at least two modified nucleosides is a T- O(CH2)2OCH3 nucleoside.
236. The oligomeric compound of any of claims 1-235 wherein essentially every nucleoside of the oligomeric compound is a modified nucleoside.
237. The oligomeric compound of any of claims 1-223 or 227-235 wherein every nucleoside of the oligomeric compound is a modified nucleoside.
238. The oligomeric compound of any of claims 1-237 wherein the remainder of the oligonucleotide comprises:
1-20 first-type regions, each first-type region independently comprising 1-20 contiguous nucleosides wherein each nucleoside of each first-type region comprises a first-type modification;
0-20 second-type regions, each second-type region independently comprising 1-20 contiguous nucleosides wherein each nucleoside of each second-type region comprises a second-type modification; and 0-20 third-type regions, each third-type region independently comprising 1-20 contiguous nucleosides wherein each nucleoside of each third-type region comprises a third-type modification; wherein the first-type modification, the second-type modification, and the third-type modification are each independently selected from 2'-F, 2'-OCH3, 2'-0(CHa)2OCH3, BNA, F-HNA, 2'-H and 2'-OH; provided that the first-type modification, the second-type modification, and the third-type modification are each different from one another.
239. The oligomeric compound of claim 238 comprising 2-20 first-type regions.
240. The oligomeric compound of claim 238 comprising 3-20 first-type regions.
241. The oligomeric compound of claim 238 comprising 4-20 first-type regions.
242. The oligomeric compound of claim 238 comprising 5-20 first-type regions.
243. The oligomeric compound of claim 238 comprising 6-20 first-type regions.
244. The oligomeric compound of any of claims 238-243 comprising 1-20 second-type regions.
245. The oligomeric compound of any of claims 238-243 comprising 2-20 second-type regions.
246. The oligomeric compound of any of claims 238-243 comprising 3-20 second-type regions.
247. The oligomeric compound of any of claims 238-243 comprising 4-20 second-type regions.
248. The oligomeric compound of any of claims 238-243 comprising 5-20 second-type regions.
249. The oligomeric compound of any of claims 238-248 comprising 1-20 third-type regions.
250. The oligomeric compound of any of claims 238-248 comprising 2-20 third-type regions.
251. The oligomeric compound of any of claims 238-248 comprising 3-20 third-type regions.
252. The oligomeric compound of any of claims 238-248 comprising 4-20 third-type regions.
253. The oligomeric compound of any of claims 238-248 comprising 5-20 third-type regions.
254. The oligomeric compound of any of claims 238-253 wherein the oligomeric compound comprises a third-type region at the 3 '-end of the oligomeric compound.
255. The oligomeric compound of any of claims 238-253 wherein the third-type region contains from 1 to 3 modified nucleosides and the third-type modification is 2'-O(CH2)2θCH3.
256. The oligomeric compound of any of claims 238-253 wherein the third same type region contains two modified nucleosides and the third-type modification is 2'-O(CH2)2OCH3.
257. The oligomeric compound claim 238-256 wherein each first-type region contains from 1 to 5 modified nucleosides.
258. The oligomeric compound claim 238-256 wherein each first-type region contains from 6 to 10 modified nucleosides.
259. The oligomeric compound claim 238-256 wherein each first-type region contains from 11 to 15 modified nucleosides.
260. The oligomeric compound claim 238-256 wherein each first-type region contains from 16 to 20 modified nucleosides.
261. The oligomeric compound of any of claims 238-260 wherein the first-type modification is 2'-F.
262. The oligomeric compound of any of claims 238-260 wherein the first-type modification is 2'-OMe.
263. The oligomeric compound of any of claims 238-260 wherein the first-type modification is DNA.
264. The oligomeric compound of any of claims 238-260 wherein the first-type modification is T- O(CH2)2OCH3.
265. The oligomeric compound of any of claims 238-260 wherein the first-type modification is 4'-CH2-O-
2'.
266. The oligomeric compound of any of claims 238-260 wherein the first-type modification is 4'-(CH2)2- O-2'.
267. The oligomeric compound of any of claims 238-260 wherein the first-type modification is 4'- C(CH3)H-O-2\
268. The oligomeric compound any of claims 238-267 wherein each second-type region contains from 1 to 5 modified nucleosides.
269. The oligomeric compound any of claims 238-267 wherein each second-type region contains from 6 to 10 modified nucleosides.
270. The oligomeric compound any of claims 238-267 wherein each second-type region contains from 11 to 15 modified nucleosides.
271. The oligomeric compound any of claims 238-267 wherein each second-type region contains from 16 to 20 modified nucleosides.
272. The oligomeric compound of any of claims 238-260 or 262-271 wherein the second-type modification is 2'-F.
273. The oligomeric compound of any of claims 238-261 or 263-271 wherein the second-type modification is 2'-OMe.
274. The oligomeric compound of any of claims 238-262 or 263-271 wherein the second-type modification is DNA.
275. The oligomeric compound of any of claims 238-254, 257-263, or 265-271 wherein the second -type modification is 2'-0(CHb)2OCH3.
276. The oligomeric compound of any of claims 238-264 or 266-271 wherein the second -type modification is 4'-CH2-O-2'.
277. The oligomeric compound of any of claims 238-265 or 267-271 wherein the second -type modification is 4'-(CH2)2-O-2'.
278. The oligomeric compound of any of claims 238-266 or 268-271 wherein the second -type modification is 4'-C(CH3)H-O-2'.
279. The oligomeric compound of any of claims 238-278 comprising an alternating motif wherein the first-type regions alternate with the second-type regions.
280. The oligomeric compound of any of claims 1 -279 wherein the remainder of the oligonucleotide comprises at least one region of nucleosides having a nucleoside motif:
(A)n-(B)n-(A)n-(B)n, wherein: A an B are differently modified nucleosides; and each n is independently selected from 1, 2, 3, 4, and 5.
281. The oligomeric compound of claim 280 wherein A and B are each independently selected from a bicyclic and a 2'-modified nucleoside.
282. The oligomeric compound of claim 281 wherein at least one of A and B is a bicyclic nucleoside.
283. The oligomeric compound of claim 281 wherein at least one of A and B is a (4'-CH2-O-25) BNA nucleoside.
284. The oligomeric compound of claim 281 wherein at least one of A and B is a (4'-(CH2)2-O-2') BNA nucleoside.
285. The oligomeric compound of claim 281 wherein at least one of A and B is a (4'-C(CH3)H-O-2') BNA nucleoside.
286. The oligomeric compound of any of claims 280-285 wherein at least one of A and B is a 2'-modified nucleoside.
287. The oligomeric compound of claim 286 wherein the 2'-modifϊed nucleoside is selected from: a 2'-F nucleoside, a 2'-OCH3 nucleoside, and a 2'-O(CH2)2OCH3 nucleoside.
288. The oligomeric compound of claim 280 wherein A and B are each independently selected from: a T- F nucleoside, a 2'-OCH3 nucleoside, a 2'-O(CH2)2OCH3 nucleoside, a (4'-CH2-O-2') BNA nucleoside, a (4'- (CH2)2-O-2') BNA nucleoside, a (4'-C(CH3)H-O-2') BNA nucleoside, a DNA nucleoside, an RNA nucleoside, and an F-HNA nucleoside.
289. The oligomeric compound of claim 288 wherein A and B are each independently selected from: a 2'- F nucleoside, a 2'-OCH3 nucleoside, a (4'-CH2-O-2') BNA nucleoside, a (4'-(CH2)2-O-2') BNA nucleoside, a (4'-C(CH3)H-O-2') BNA nucleoside, and a DNA nucleoside.
290. The oligomeric compound of any of claims 280-289 wherein one of A and B is a 2'-F nucleoside.
291. The oligomeric compound of any of claims 280-290 wherein one of A and B is a 2'-OCH3 nucleoside.
292. The oligomeric compound of any of claims 280-291 wherein one of A and B is a 2'- O(CH2)2OCH3 nucleoside.
293. The oligomeric compound of claim 280 wherein A is a 2'-F nucleoside and B is a 2'-OCH3 nucleoside.
294. The oligomeric compound of claim 280 wherein A is a 2'-OCH3 nucleoside and B is a 2'-F nucleoside.
295. The oligomeric compound of claim 280 wherein one of A and B is selected from a (4'-CH2-O-2') BNA nucleoside, a (4'-(CH2)2-O-2') BNA nucleoside, and a (4'-C(CH3)H-O-2') BNA nucleoside and the other of A and B is a DNA nucleoside.
296. The oligomeric compound of any of claims 1-295 wherein the remainder of the oligonucleotide comprises a nucleoside motif: (A)x-(B)2-(A)Y-(B)2-(A)z-(B)3 wherein A is a nucleoside of a first type;
B is a nucleoside of a second type;
X is 0-10;
Y is 1-10; and
Z is 1-10.
297. The oligomeric compound of claim 296 wherein X is selected from 0, 1, 2 and 3.
298. The oligomeric compound of claim 296 wherein X is selected from 4, 5, 6 and 7.
299. The oligomeric compound of any of claims 295-298 wherein Y is selected from 1, 2 and 3.
300. The oligomeric compound of any of claims 295-298 wherein Y is selected from 4, 5, 6 and 7.
301. The oligomeric compound of any of claims 295-300 wherein Z is selected from 1, 2 and 3.
302. The oligomeric compound of any of claims 295-300 wherein Z is selected from 4, 5, 6 and 7.
303. The oligomeric compound of any of claims 295-302 wherein A is a 2'-F nucleoside.
304. The oligomeric compound of any of claims 295-303 wherein B is a 2'-OCH3 nucleoside.
305. The oligomeric compound of any of claims 1-304 comprising a 3 '-region consisting of from 1 to 5 nucleosides at the 3 '-end of the oligomeric compound wherein: the nucleosides of the 3 '-region each comprises the same modification as one another; and the nucleosides of the 3 '-region are modified differently than the last nucleoside adjacent to the 3'- region.
306. The oligomeric compound of claim 305 wherein the modification of the 3'-region is different from any of the modifications of any of the other nucleosides of the oligomeric compound.
307. The oligomeric compound of any of claims 305 or 306 wherein the nucleosides of the 3'-region are 2'-O(CH2)2OCH3 nucleosides.
308. The oligomeric compound of any of claims 305-307 wherein the 3 '-region consists of 2 nucleosides.
309. The oligomeric compound of any of claims 305-307 wherein the 3 '-region consists of 3 nucleosides.
310. The oligomeric compound of any of claims 305-309 wherein each nucleoside of the 3 '-region comprises a uracil base.
311. The oligomeric compound of any of claims 305-309 wherein each nucleoside of the 3'-region comprises an adenine base.
312. The oligomeric compound of any of claims 305-309 wherein each nucleoside of the 3 '-region comprises a thymine base.
313. The oligomeric compound of any of claims 1-312 wherein the remainder of the oligonucleotide comprises a region of uniformly modified nucleosides.
314. The oligomeric compound of claim 313 wherein the region of uniformly modified nucleosides comprises 2-20 contiguous uniformly modified nucleosides.
315. The oligomeric compound of claim 313 wherein the region of uniformly modified nucleosides comprises 3-20 contiguous uniformly modified nucleosides.
316. The oligomeric compound of claim 313 wherein the region of uniformly modified nucleosides comprises 4-20 contiguous uniformly modified nucleosides.
317. The oligomeric compound of claim 313 wherein the region of uniformly modified nucleosides comprises 5-20 contiguous uniformly modified nucleosides.
318. The oligomeric compound of claim 313 wherein the region of uniformly modified nucleosides comprises 6-20 contiguous uniformly modified nucleosides.
319. The oligomeric compound of claim 313 wherein the region of uniformly modified nucleosides comprises 5-15 contiguous uniformly modified nucleosides.
320. The oligomeric compound of claim 313 wherein the region of uniformly modified nucleosides comprises 6-15 contiguous uniformly modified nucleosides.
321. The oligomeric compound of claim 313 wherein the region of uniformly modified nucleosides comprises 5-10 contiguous uniformly modified nucleosides.
322. The oligomeric compound of claim 313 wherein the region of uniformly modified nucleosides comprises 6-10 contiguous uniformly modified nucleosides.
323. The oligomeric compound of any of claims 1-322 wherein the remainder of the oligonucleotide comprises a region of alternating modified nucleosides and a region of uniformly modified nucleosides.
324. The oligomeric compound of claim 323 wherein the region of alternating nucleotides is 5' of the region of fully modified nucleosides .
325. The oligomeric compound of claim 323 wherein the region of alternating nucleotides is 3' of the region of fully modified nucleosides.
326. The oligomeric compound of any of claims 323-325 wherein the alternating region and the fully modified region are immediately adjacent to one another.
327. The oligomeric compound of any of claims 323-326 having additional nucleosides between the alternating region and the fully modified region.
328. The oligomeric compound of any of claims 1-327 wherein the remainder of the oligonucleotide comprises at least one region of nucleosides having a motif I:
Nf(PS)N1n(PO), wherein: Nf is a 2'-F nucleoside, Nn, is a 2'-OCH3 nucleoside PS is a phosphorothioate linking group; and
PO is a phosphodiester linking group.
329. The oligomeric compound of claim 328 comprising at least 2 regions of nucleosides having the motif I.
330. The oligomeric compound of claim 328 comprising at least 3 regions of nucleosides having the motif I.
331. The oligomeric compound of claim 328 comprising at least 4 regions of nucleosides having the motif I.
332. The oligomeric compound of claim 328 comprising at least 5 regions of nucleosides having the motif I.
333. The oligomeric compound of claim 328 comprising at least 6 regions of nucleosides having the motif I.
334. The oligomeric compound of claim 328 comprising at least 7 regions of nucleosides having the motif I.
335. The oligomeric compound of claim 328 comprising at least 8 regions of nucleosides having the motif I.
336. The oligomeric compound of claim 328 comprising at least 9 regions of nucleosides having the motif I.
337. The oligomeric compound of claim 328 comprising 10 regions of nucleosides having the motif I.
338. The oligomeric compound of any of claims 1-337 comprising at least one region having a nucleoside motif selected from:
AABBAA;
ABBABB;
AABAAB;
ABBABAABB; ABABAA;
AABABAB;
ABABAA;
ABBAABBABABAA;
BABBAABBABABAA; or ABABBAABBABABAA; wherein A is a nucleoside of a first type and B is a nucleoside of a second type.
339. The oligomeric compound of any of claims 1-338 comprising one or more conjugate groups.
340. The oligomeric compound of any of claims 1-338 consisting of the oligonucleotide.
341. An oligomeric compound comprising an oligonucleotide comprising a contiguous sequence of linked nucleosides wherein the sequence has the formula:
5'-(Z)w-(L-QrL-Q2)t-(L-Q1)u-(L-Q3)v-(G)a-3' wherein: each L is an internucleoside linking group;
G is a conjugate or a linking group; a is 0 or 1; each of Qi, Q2 and Q3 is, independently, a 2'-modified nucleoside having a 2'-substituent group selected from halogen, allyl, amino, azido, O-allyl, 0-Ci-C6 alkyl, OCF3, O-(CH2)2-O-CH3, O(CH2)2SCH3, O- (CH2)2-O-N(J5)(J6) and 0-CH2-CC=O)-N(J5)(J6), where each J5 and J6 is, independently, H, an amino protecting group or substituted or unsubstituted Ci-C6 alkyl; provided that Qi, Q2 and Q3 are different from one another; t is from 4 to 8; u is 0 or 1; v is from 1 to 3; w is 0 or 1 ; and Z is a 5' stabilizing nucleoside.
342. The oligomeric compound of claim 341 wherein w is 1.
343. The oligomeric compound of claim 341 wherein w is 0.
344. The oligomeric compound of any one of claims 341-343 wherein each Q1 and Q2 is, independently, a 2'-modifϊed nucleoside having a 2'-substituent group selected from halogen and 0-Ci-C6 alkyl.
345. The oligomeric compound of any one of claims 341-343 wherein each Q1 and Q2 is, independently, a 2'-modifϊed nucleoside having a 2'-substituent group selected from F and O-methyl.
346. The oligomeric compound of any of claims 341-345 wherein each Q3 is a 2'-modified nucleoside having a 2'-substituent group of O-(CH2)2-OCH3.
347. The oligomeric compound of any of claims 341-346 wherein a is 0.
348. The oligomeric compound of any of claims 341-347 wherein v is 2.
349. The oligomeric compound of any of claims 341-348 wherein u is 0.
350. The oligomeric compound of any of claims 341-349 wherein u is 1.
351. The oligomeric compound of any of claims 1-350 wherein the oligonucleotide consists of 8-80 linked nucleosides.
352. The oligomeric compound of any of claims 1-350 wherein the oligonucleotide consists of 8-26 linked nucleosides.
353. The oligomeric compound of any of claims 1-350 wherein the oligonucleotide consists of 10-24 linked nucleosides.
354. The oligomeric compound of any of claims 1-350 wherein the oligonucleotide consists of 16-22 linked nucleosides.
355. The oligomeric compound of any of claims 1-350 wherein the oligonucleotide consists of 16-18 linked nucleosides.
356. The oligomeric compound of any of claims 1-350 wherein the oligonucleotide consists of 19-22 linked nucleosides.
357. The oligomeric compound of any of claims 1-356 wherein the second nucleoside from the 5 ' -end comprises a sugar moiety comprising a 2'-substituent selected from OH and a halogen.
358. The oligomeric compound of any of claims 1-357 wherein the second nucleoside from the 5 '-end is a 2'-F modified nucleoside.
359. The oligomeric compound of any of claims 1-358 comprising at least one modified linking group.
360. The oligomeric compound of claim 359 wherein each internucleoside linking group is, independently, phosphodiester or phosphorothioate.
361. The oligomeric compound of claim 359 wherein the 5'-most internucleoside linking group is a phosphorothioate linking group.
362. The oligomeric compound of any of claims 359-361 comprising at least one phosphorothioate region comprising at least two contiguous phosphorothioate linking groups.
363. The oligomeric compound of claim 362 wherein the at least one phosphorothioate region comprises from 3 to 12 contiguous phosphorothioate linking groups.
364. The oligomeric compound of claim 362 wherein the at least one phosphorothioate region comprises from 6 to 8 phosphorothioate linking groups.
365. The oligomeric compound of any of claims 362-364 wherein the at least one phosphorothioate region is located at the 3 '-end of the oligomeric compound.
366. The oligomeric compound of any of claims 362-365 wherein the at least one phosphorothioate region is located within 3 nucleosides of the 3 '-end of the oligomeric compound.
367. The oligomeric compound of any of claims 359-366 wherein the 7-9 internucleoside linkages at the 3 'end of the oligonucleotide are phosphorothioate linkages and the internucleoside linkage at the 5 '-end is a phosphorothioate linkage.
368. An oligomeric compound comprising an oligonucleotide consisting of 10 to 30 linked nucleosides wherein: (e) the nucleoside at the 5' end is a phosphate stabilizing nucleoside comprising: a 5 '-terminal modified or unmodified phosphate; and a modified sugar moiety comprising: a 5'- modification; or a 2'-modification; or both a 5'-modificaton and a 2'-modifϊcation;
(f) the sugar moiety of the second nucleoside from the 5 '-end is selected from an unmodified 2'-OH sugar, and a modified sugar comprising a modification selected from: 2'-halogen, 2'0-alkyl, and 2'-O- substituted alkyl; and
(g) the first internucleoside linkage at the 5 '-end and the last seven internucleoside linkages at the 3 '-end are phosphorothioate linkages; and
(h) at least one internucleoside linkage is other than a phosphorothioate linkage.
369. The oligomeric compound of claim 368, wherein: the 5 '-terminal modified phosphate is selected from: phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphorothioate, phosphoramidate, alkylphosphonothioate, substituted alkylphosphonothioate, phosphorodithioate, thiophosphoramidate, and phosphotriester; the 5 '-modification of the sugar moiety of the phosphate stabilizing nucleoside is selected from 5'- alkyl and 5 '-halogen; and the 2 '-modification of the sugar moiety of the phosphate stabilizing nucleoside is selected from: halogen, allyl, amino, azido, thio, O-allyl, -0-C1-Ci0 alkyl, -0-Ci-Ci0 substituted alkyl, -OCF3, -O-(CH2)2-O-CH3, - O(CH2)2SCH3, -O-(CH2)2-O-N(Rm)(Rn), -O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C,-Cio alkyl, -O[(CH2)nO]mCH3, -O(CH2)nNH2, -O(CH2)nCH3, - O(CH2)nONH2, -OCH2C(=O)N(H)CH3; -O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10; Ci to Cio alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl.
370. The oligomeric compound of claim 368 or 369, wherein the modified phosphate is selected from: phosphonate, alkylphosphonate, substituted alkylphosphonate, aminoalkyl phosphonate, substituted aminoalkyl phosphonate, phosphotriester, phosphorothioate, phosphorodithioate, thiophosphoramidate, and phosphoramidate.
371. The oligomeric compound of claim 368 or 369, wherein the modified phosphate is selected from: phosphonate, alkylphosphonate, and substituted alkylphosphonate.
372. The oligomeric compound of claim 368 or 369, wherein the modified phosphate is selected from 5'- deoxy-5'-thio phosphate, phosphoramidate, methylene phosphonate, mono-fluoro methylene phosphonate and di-fluoro methylene phosphonate.
373. The oligomeric compound of any of claims 368-372, wherein the sugar moiety of the phosphate stabilizing nucleoside comprises a 5'-modificaton and a 2'-modification.
374. The oligomeric compound of any of claims 1-373 wherein the oligomeric compound is an antisense compound.
375. The oligomeric compound of claim 374 wherein the antisense compound is an KNAi compound.
376. The oligomeric compound of any of claims 375 wherein the antisense compound is an siRNAi compound.
377. The oligomeric compound of any of claims 375 wherein the antisense compound is a microRNA mimic.
378. The oligomeric compound of any of claims 374 the antisense compound is an RNase H antisense compound.
379. The oligomeric compound of any of claims 374 wherein the antisense compound modulates splicing.
380. The oligomeric compound of any of claims 374 -379, wherein at least a portion of the nucleobase sequence of the oligonucleotide is complementary to a portion of a target nucleic acid, wherein the target nucleic acid is selected from: a target rnRNA, a target pre-mRNA, a target microRNA, and a target non- coding RNA.
381. The oligomeric compound of claim 380, wherein the nucleobase sequence of the oligonucleotide a region of 100% complementarity to the target nucleic acid and wherein the region of 100% complementarity is at least 10 nucleobases.
382. The oligomeric compound of claim 381, wherein the region of 100% complementarity is at least 15 nucleobases.
383. The oligomeric compound of claim 381, wherein the region of 100% complementarity is at least 20 nucleobases.
384. The oligomeric compound of any of claims 374-383 wherein the oligonucleotide is at least 85% complementary to the target nucleic acid.
385. The oligomeric compound of any of claims 374-383 wherein the oligonucleotide is at least 90% complementary to the target nucleic acid.
386. The oligomeric compound of any of claims 374-383 wherein the oligonucleotide is at least 95% complementary to the target nucleic acid.
387. The oligomeric compound of any of claims 374-383 wherein the oligonucleotide is at least 98% complementary to the target nucleic acid.
388. The oligomeric compound of any of claims 374-383 wherein the oligonucleotide is 100% complementary to the target nucleic acid.
389. The oligomeric compound of any of claims 374 or 380-388 wherein the antisense compound is a microRNA mimic having a nucleobase sequence comprising a portion that is at least 80% identical to the seed region of a microRNA and that has overall identity with the microRNA of at least 70%.
390. The oligomeric compound of claim 389 wherein the nucleobase sequence of the microRNA mimic has a portion that is at least 80% identical to the sequence of the seed region of a microRNA and has overall identity with the microRNA of at least 75%.
391. The oligomeric compound of claim 389 wherein the nucleobase sequence of the microRNA mimic has a portion that is at least 80% identical to the sequence of the seed region of a microRNA and has overall identity with the microRNA of at least 80%.
392. The oligomeric compound of claim 389 wherein the nucleobase sequence of the microRNA mimic has a portion that is at least 100% identical to the sequence of the seed region of a microRNA and has overall identity with the microRNA of at least 80%.
393. The oligomeric compound of claim 389 wherein the nucleobase sequence of the microRNA mimic has a portion that is at least 100% identical to the sequence of the seed region of a microRNA and has overall identity with the microRNA of at least 85%.
394. The oligomeric compound of claim 389 wherein the nucleobase sequence of the microRNA mimic has a portion that is 100% identical to the sequence of the microRNA.
395. The oligomeric compound of claim 377, wherein the nucleobase sequence of the oligonucleotide comprises a region of 100% complementarity to a seed match segment of a target nucleic acid.
396. The oligomeric compound of claim 377 wherein the antisense compound is a microRNA mimic having a nucleobase sequence comprising a portion that is at least 80% identical to the seed region of a microRNA and that has overall identity with the microRNA of at least 50%.
397. The oligomeric compound of claim 377 wherein the antisense compound is a microRNA mimic having a nucleobase sequence comprising a portion that is at least 80% identical to the seed region of a microRNA and that has overall identity with the microRNA of at least 55%.
398. The oligomeric compound of claim 377 wherein the antisense compound is a microRNA mimic having a nucleobase sequence comprising a portion that is at least 80% identical to the seed region of a microRNA and that has overall identity with the microRNA of at least 60%.
399. The oligomeric compound of claim 377 wherein the antisense compound is a microRNA mimic having a nucleobase sequence comprising a portion that is at least 80% identical to the seed region of a microRNA and that has overall identity with the microRNA of at least 65%.
400. The oligomeric compound of any of claims 1-399 wherein the oligomeric compound comprises a nucleobase sequence selected from a microRNA sequence found in miRBase.
401. The oligomeric compound of any of claims 1-399 wherein the oligomeric compound consists of a nucleobase sequence selected from a microRNA sequence found in miRBase.
402. The oligomeric compound of any of claims 374-401 wherein the target nucleic acid is a target mRNA.
403. The oligomeric compound of claim 374-401 wherein the target nucleic acid is a target pre-mRNA.
404. The oligomeric compound of claim 374-401 wherein the target nucleic acid is a non-coding RNA.
405. The oligomeric compound of claim 374-401 wherein the target nucleic acid is a microRNA.
406. The oligomeric compound of claim 374-401 wherein the target nucleic acid is a pre-mir.
407. The oligomeric compound of claim 374-401 wherein the target nucleic acid is a pri-mir.
408. The oligomeric compound of any of claims 374-407 wherein the nucleobase sequence of the oligonucleotide comprises a region of 100% complementarity to the target nucleic acid and wherein the region of 100% complementarity is at least 10 nucleobases.
409. The oligomeric compound of any of claims 374-407, wherein the nucleobase sequence of the oligonucleotide comprises a region of 100% complementarity to the target nucleic acid and wherein the region of 100% complementarity is at least 6 nucleobases.
410. The oligomeric compound of any of claims 374-407, wherein the nucleobase sequence of the oligonucleotide comprises a region of 100% complementarity to the target nucleic acid and wherein the region of 100% complementarity is at least 7 nucleobases. .
411. The oligomeric compound of any of claims 374-410, wherein the target nucleic acid is a mammalian target nucleic acid.
412. The oligomeric compound of claim 411, wherein the mammalian target nucleic acid is a human target nucleic acid.
413. The oligomeric compound of any of the claims 1-412 comprising from 1 to 3 terminal group nucleosides on at least one end of the oligonucleotide.
414. The oligomeric compound of claim 413 comprising from 1 to 3 terminal group nucleosides at the 3'- end of the oligonucleotide.
415. The oligomeric compound of claim 413 or 414 comprising from 1 to 3 terminal group nucleosides at the 5'-end of the oligonucleotide.
416. The oligomeric compound of any of claims 1-415, wherein the oligomeric compound is single stranded.
417. The oligomeric compound of any of claims 1-415 wherein the oligomeric compound is double stranded.
418. The oligomeric compound claim 417 wherein each strand of the double stranded oligomeric compound comprises an oligonucleotide according to any of claims 1-373.
419. The oligomeric compound claim 417 wherein the double stranded oligomeric compound comprises a first strand and a second strand wherein the first strand is an oligonucleotide according to any of claims 1-415 and the second strand is an oligonucleotide according to any of claims 1-373.
420. The oligomeric compound claim 417 wherein the double stranded oligomeric compound comprises a first strand and a second strand wherein the first strand is an oligonucleotide according to any of claims 1-415 and the second strand is an oligomeric compound.
421. The oligomeric compound of claim 420 wherein the second strand of the double stranded oligomeric compound comprises a modified oligonucleotide.
422. The oligomeric compound of any of claims 417-421 wherein the second strand consists of 8-40 linked nucleosides.
423. The oligomeric compound of any of claims 417-421 wherein the second strand consists of 10-30 linked nucleosides.
424. The oligomeric compound any of claims 417-421 wherein the second strand consists of 12-25 linked nucleosides.
425. The oligomeric compound of any of claims 417-424 wherein the second strand comprises at least one modified nucleoside having a 2'-substituent group of 0-(CH2VOCH3
426. The oligomeric compound of any of claims 417-425 wherein the second strand comprises at least one modified nucleoside having a 2'-substituent group of 2'F.
427. A pharmaceutical composition comprising an oligomeric compound according to any of claims 1-426 and a pharmaceutically acceptable diluent or carrier.
428. The pharmaceutical composition of claim 427 wherein the pharmaceutically acceptable diluent or carrier is pharmaceutical grade sterile saline.
429. A method comprising contacting a cell with an oligomeric compound according to any of claims 1-
426.
430. The method of claim 429 comprising detecting antisense activity.
431. The method of claim 430 wherein the detecting antisense activity comprises detecting a phenotypic change in the cell.
432. The method of claim 431 wherein the detecting antisense activity comprises detecting a change in the amount of target nucleic acid in the cell.
433. The method of claim 432 wherein the detecting antisense activity comprises detecting a change in the amount of a target protein.
434. The method of any of claims 430-433 wherein the cell is in vitro.
435. The method of claim 434 wherein the cell is in an animal.
436. The method of claim 435 wherein the animal is a mammal.
437. The method of claim 436 wherein the mammal is a human.
438. A method of modulating a target mRNA in a cell comprising contacting the cell with an oligomeric compound according to any of claims 1-426 and thereby modulating the mRNA in a cell.
439. The method of claim 438 comprising detecting a phenotypic change in the cell.
440. The method of claim 439 comprising detecting a decrease in mRNA levels in the cell.
441. The method of any of claims 439 or 440 comprising detecting a change in the amount of a target protein.
442. The method of any of claims 438-441 wherein the cell is in vitro.
443. The method of claim 442 wherein the cell is in an animal.
444. The method of claim 443 wherein the animal is a mammal.
445. The method of claim 444 wherein the mammal is a human.
446. A method comprising administering to an animal a pharmaceutical composition according to claim 427 or 428.
447. The method of claim 446 wherein the animal is a mammal.
448. The method of claim 447 wherein the mammal is a human.
449. The method of any of claims 446-448 comprising detecting antisensc activity in the animal.
450. The method of claim 449 wherein the detecting antisense activity comprises detecting a change in the amount of target nucleic acid in the animal.
451. The method of any of claims 449 or 450 wherein the detecting antisense activity comprises detecting a change in the amount of a target protein in the animal.
452. The method of any of claims 449-451 wherein the detecting antisense activity comprises detecting a phenotypic change in the animal.
453. The method of claim 452 wherein the phenotypic change is a change in the amount or quality of a biological marker of activity.
454. The use of an oligomeric compound of any one of claims 1-426 for the manufacture of a medicament for the treatment of a disease characterized by undesired gene expression.
455. The use of an oligomeric compound of any one of claims 1 -426 for the manufacture of a medicament for treating a disease by inhibiting gene expression.
456. The method of claim 430 wherein the antisense activity is microKNA mimic activity.
457. The method of claim 456 wherein the detecting microRNA mimic activity comprises detecting a change in the amount of a target nucleic acid in a cell.
458. The method of claim 457 wherein the detecting microRNA mimic activity comprises detecting a change in the amount of a target protein in cell.
459. A nucleoside having the Formula I:
Figure imgf000238_0001
wherein:
Bx is a heterocyclic base moiety;
Ti is H, a hydroxyl protecting group, or a phosphorous moiety; T2 is H, a hydroxyl protecting group, or a reactive phosphorous group; and each of qi and q2 is, independently selected from CpC6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C]-C6 alkyl, substituted CrC6 alkenyl and substituted C2-C6 alkynyl; Xi is S, NRi6, or CRioRn wherein each Rjoand Rn is, independently, H, F, Ci-C6 haloalkyl , or Ci-C6 alkyl; and Ri is selected from a halogen, X2-V, and O-X4; or each of qi and q2 is, independently, selected from H, Ci-C6 alkyl, C2-C6 alkenyl, C2- C6 alkynyl, substituted Ci-C6 alkyl, substituted Ci-C6 alkenyl and substituted C2-C6 alkynyl; Xi is S, NRi6R]7, or CRjoRn wherein each Rio and Rn is, independently, H, F, Ci-C6 haloalkyl , or C1-C6 alkyl; and
Ri is X2-V; or each of qi and q2 is, independently, selected from CpC6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted CpC6 alkyl, substituted CpC6 alkenyl and substituted C2-C6 alkynyl; Xi is O, S, NRi6Ri7, or CRi0Ri 1 wherein each Ri0 and Rn is, independently, H, F, Cp
C6 haloalkyl , or CpC6 alkyl; and
R1 Is X2-V; wherein:
X2 is O, S or CR7R8 wherein each R7 and Rg is, independently, H or CpC6 alkyl; V is selected from cholesterol, (CH2)2[O(CH2)2]tOCH3, where t is from 1-3, (CH2)2F, CH2COOH,
CH2CONH2, CH2CONR5R^ CH2COOCH2CH3, CH2CONH(CH2)J-S-R4 where i is from 1 to 10, CH2CONH(CH2)JNR5R6 where j is from 1 to 6, and CH2CONH[(CH2)kpN(H)]k2-(CH2)klNH2 where each ki is independently from 2 to 4 and k2 is from 2 to 10; R4 is selected from H, Q-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted CpC6 alkenyl, substituted C2-C6 alkynyl, C6-Cu aryl and athio protecting group;
R5 and R6 are each, independently, selected from H, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, and substituted C2-C6 alkynyl; Ri6 is selected from H, CrC6 alkyl, or substituted CrC6 alkyl;
X4 is [C(Ra)(Rb)]n-[(C=O)mXc]k-Rd wherein each Ra and Rt is independently H or halogen; X0 is O, S, or N(Ei);
Ra is H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted CrC6 alkyl, substituted Ci-C6 alkenyl and substituted C2-C6 alkynyl or NE2E3; each Ei, E2, and E3 is independently H, Ci-C6 alkyl, or substituted Q-C6 alkyl; n is 1 to 6; m is 0 or 1; and k is 0 or 1; and wherein each substituted group is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, OJi, NJiJ2, SJi, N3, OC(=O)Ji and CN, wherein each Ji and J2 is, independently, H or Ci-C6 alkyl; and J4 is hydrogen, or a protecting group.
460. The nucleoside of claim 459 wherein at least one of qi and q2 is Q-C6 alkyl or substituted Ci-C6 alkyl.
461. The nucleoside of any of claims 459 or 460 wherein at least one of qi and q2 is Ci-C6 alkyl.
462. The nucleoside of any of claims 459-461 wherein at least one of qi and q2 is methyl.
463. The nucleoside of any of claims 459-462 wherein one of qi and q2 is H.
464. The nucleoside of any of claims 459-463 wherein one of qi and q2 is methyl and the other of qi and q2 is H.
465. The nucleoside of any of claims 459-464 wherein Z is O.
466. The nucleoside of any of claims 459-465 wherein Y is CH2CONH(CH2)JNR5R6.
467. The nucleoside of claim 466 wherein j is 2.
468. The nucleoside of any of claims 459-467 wherein one of R5 and R6 is other than H.
469. The nucleoside of any of claims 459-468 wherein at least one of R5 and R6 is selected from Ci-C6 alkyl, substituted Cj-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted Ci-C6 alkenyl, and substituted C2-C6 alkynyl.
470. The nucleoside of any of claims 459-469 wherein at least one of R5 and R6 is C]-C6 alkyl or substituted Q-C6 alkyl.
471. The nucleoside of any of claims 459-470 wherein at least one of R5 and R6 is methyl.
472. The nucleoside of any one of claims any of claims 459-471 wherein both R5 and R6 are other than H.
473. The nucleoside of any one of claims 459-472 wherein Xi is O.
474. The nucleoside of any one of claims 459-472 wherein X] is S.
475. The nucleoside of any one of claims 459-472 wherein Xi is CRioRn.
476. The nucleoside of any one of claims 459-475 wherein Ri0 and R11 are both H.
477. The nucleoside of any one of claims 459-476 wherein Ti is a phosphorus moiety.
478. The nucleoside of claim 477 wherein the phosphorus moiety is selected from P(Y3)Yb Y0, where Ya is O or S and each Yb and Y0 is, independently, OH, SH, alkyl, substituted Ci-C6 alkyl, alkoxyl and substituted Ci-C6 alkoxyl.
479. The nucleoside of claim 478 wherein Ya is O and Yb and Yc are each OH.
480. The nucleoside of claim 479 wherein: i. Y3 is O; ii. Yb and Yc are each OH; and iii. T2 is diisopropylcyanoethoxy phosphoramidite.
481. The nucleoside of any one of claims 459-480 wherein T2 is a reactive phosphorus group.
482. The nucleoside of claim 481 wherein the reactive phosphorous group is diisopropylcyanoethoxy phosphoramidite or H-phosphonate.
483. The nucleoside of any one of claims 459-482 having Formula XII:
Figure imgf000241_0001
484. The nucleoside of claim 483 wherein both R5 and R6 are other than H.
485. The nucleoside of any one of claims 483 or 484 wherein both R5 and R6 are methyl.
486. The nucleoside of any one of claims 483-485 wherein qi is methyl and q2 is H.
487. The nucleoside of any one of claims 483-486 wherein qi is H and q2 is methyl.
488. The nucleoside of any one of claims 483-487 having the configuration:
Figure imgf000241_0002
489. The oligomeric compound of any of claims 1-426 wherein the oligomeric compound has a nucleobase sequence associated with an accession number from miRBase version 10.1 released December 2007 selected from:
MIMAT0000062, MIMAT0004481, MIMAT0000063, MEV1AT0004482, MIMAT0000064, MEV1AT0004483, MIMAT0000065, MEV1AT0004484, MEVIAT0000066, MEVIAT0004485, MDVIAT0000067, MEVIAT0004486, MBVIAT0004487, MEVIAT0000414, MIMAT0004584, MDVIAT0000415, MEVIAT0004585, MIMAT0000416, MIMAT0000098, MEV1AT0004512, MIMAT0000099, MIMAT0004513, MIMATOOOO 101, MEVIAT0000102, MIMAT0004516, MIMAT0000103, MIMAT0004517, MEVIAT0000680, MEVIAT0004672, MIMAT0000104, MEV1AT0000253, MIMAT0004555, MIMAT0000254, MEVIAT0004556, MEVIAT0000421, MIMAT0004590, MIMAT0005459, MIMAT0005458, MIMAT0005573, MDVIAT0005572, MEVIAT0005577, MEVIAT0005576, MEVIAT0005580, MDVIAT0005583, MDVIAT0005582, MDVIAT0005584, MBVIAT0005586, MIMAT0005588, MDV1AT0005589, MIMAT0005591, MDVIAT0005592, MIMAT0005593, MDVIAT0000422, MEVIAT0004591, MIMAT0004602, MEVIAT0000443, MDVIAT0000423, MDV1AT0000423, MDVIAT0004592, MEVIAT0004603, MIMAT0000445, MIMAT0000444, MEVIAT0000446, MLVIAT0004604, MIMAT0000424, MIMAT0004548, MEVIAT0004605, MIMAT0000242, MIMAT0000425, MIMAT0004593, MEVIAT0000691, MMAT0004680, MDVIAT0000426, MIMAT0004594, MIMAT0000427, MIMAT0000770, MIMAT0000447, MIMAT0000428, MBVIAT0004595, MIMAT0000758, MIMAT0004698, MIMAT0000448, MIMAT0004606, MIMAT0000429, MIMAT0000430, MIMAT0004607, MIMAT0004596, MIMAT0004552, MIMAT0000250, MIMAT0004597, MIMAT0000431, MIMAT0000432, MIMAT0004598, MIMAT0000434, MIMAT0000433, MIMAT0000435, MIMAT0004599, MIMAT0000436, MEMAT0004600, MIMAT0000437, MIMAT0004601, MMAT0000449, MDVLAT0004608, MIMAT0004766, MIMAT0002809, MIMAT0000251 , MIMAT0004928, MMAT0000243, MEVLAT0004549, MMAT0000759, MTMAT0004699, MIMAT0000450, MIMAT0004609, MIMAT0000451, MMAT0004610, MIMAT0000757, MIMAT0004697, MIMAT0000438, MIMAT0000439, MIMAT0000439, MIMAT0000452, MIMAT0000453, MIMAT0000646, MIMAT0004658, MMAT0000068, MIMAT0004488, MIMAT0000417, MIMAT0004586, MIMAT0000069, MMAT0004489, MDVIAT0004518, MIMAT0000070, MMAT0000071 , MIMAT0000256, MMAT0000270, MIMAT0004558, MIMAT0000257, MIMAT0000258, MMAT0004559, MMAT0002821, MIMAT0000259, MIMAT0000260, MIMAT0000261, MIMAT0004560, MIMAT0000454, MIMAT0004611, MIMAT0000456, MTMAT0004612, MIMAT0000262, MMAT0004561, MIMAT0004613, MIMAT0000457, MIMAT0000072, MMAT0002891, MMAT0001412, MIMAT0004751 , MIMAT0000458, MIMAT0004929, MIMAT0000440, MIMATOOO 1618, MIMAT0000222, MIMAT0004543, MIMAT0000459, MIMAT0004614, MIMAT0002819, MIMAT0004767, MIMAT0000460, MIMAT0004671, MIMAT0000461, MIMAT0004615, MIMAT0000226, MIMAT0004562, MIMAT0001080, MIMAT0000227, MIMAT0000228, MIMAT0000232, MIMAT0000231, MIMAT0004563, MIMAT0000263, MIMAT0000073, MIMAT0004490, MIMAT0000074, MDVLAT0004491 , MIMAT0004492, MIMAT0000682, MDMAT0001620, MIMAT0000318, MDVIAT0004571, MIMAT0000617, MIMAT0004657, MIMAT0002811, MIMAT0002810, MIMAT0000264, MIMAT0000265, MIMAT0000266, MIMAT0000462, MIMAT0000241, MEV1AT0004960, MIMAT0000075, MIMAT0004493, MIMAT0001413, MIMAT0004752, MIMAT0000076, MIMAT0004494, MEMAT0000267, MIMAT0000268, MIMAT0000269, MIMAT0000271, MIMAT0004564, MIMAT0000272, MIMAT0000273, MIMAT0004959, MIMAT0000274, MIMAT0000275, MIMAT0004565, MIMAT0004566, MIMAT0004567, MIMAT0004675, MIMAT0000276, MIMAT0000077, MIMAT0004495, MIMAT0000277, MIMAT0004908, MIMAT0004915, MIMAT0000278, MIMAT0004568, MIMAT0000279, MIMAT0004569, MIMAT0000280, MIMAT0004570, MIMAT0000281, MIMAT0000078, MIMAT0004496, MIMAT0000418, MIMAT0004587, MIMAT0000080, MIMAT0000079, MIMAT0004497, MIMAT0000081, MIMAT0004498, MIMAT0000082, MIMAT0004499, MIMAT0004681, MIMAT0000083, MIMAT0004500, MIMAT0000084, MIMAT0004501, MIMAT0000419, MIMAT0004588, MIMAT0004502, MIMAT0000085, MIMAT0004679, MIMAT0000690, MIMAT0004450, MIMAT0004901, MIMAT0000687, MIMAT0002890, MIMAT0000086, MIMAT0004503, MIMATOOOOIOO, MIMAT0004514, MIMAT0004515, MIMAT0000681, MMAT0004673, MIMAT0004903, MIMAT0000688, MIMAT0004958, MIMAT0000684, MIMAT0000683, MIMAT0000715, MIMAT0000714, MIMAT0000717, MIMAT0000716, MIMAT0000718, MIMAT0004685, MIMAT0000087, MIMAT0000088, MIMAT0000420, MIMAT0004589, MIMAT0000244, MIMAT0004674, MIMAT0004550, MIMAT0000245, MIMAT0004551, MIMAT0000692, MIMAT0000693, MIMAT0000089, MIMAT0004504, MIMAT0000090, MEMAT0004505, MIMAT0000510, MIMAT0000755, MIMAT0004696, MIMAT0000762, MIMAT0000761, MIMAT0000771, MIMAT0000756, MIMAT0000752, MIMATOOO 1629, MIMAT0000751, MDVLAT0004693, MIMAT0000760, MIMAT0004700, MEVLAT0000765, MIMAT0004703, MIMAT0000754, MIMAT0004695, MIMAT0000763, MIMAT0004701, MIMAT0004702, MIMAT0000764, MIMAT0000091, MIMAT0004506, MIMAT0003301, MIMAT0004811, MIMAT0004692, MIMAT0000750, MIMAT0000753, MMAT0004694, MMAT0000772, MIMAT0000773, MIMAT0000255, MIMAT0004557, MIMAT0004676, MMAT0000685, MIMAT0004677, MIMAT0000686, MIMAT0004682, MIMAT0000703, MMAT0004683, MEMAT0000705, MIMAT0000707, MIMAT0003385, MIMAT0000710, MIMAT0000719, MIMAT0004686, MIMAT0000721, MIMATOOO 1621, MIMAT0000722, MIMAT0000723, MIMAT0004687, MIMAT0000724, MIMAT0000726, MIMAT0000725, MIMAT0000727, MIMAT0004688, MIMAT0004955, MIMAT0004956, MIMAT0000728, MIMAT0000729, MIMAT0003386, MIMAT0002172, MIMAT0000720, MIMAT0000730, MIMAT0004689, MMAT0000732, MIMAT0000731, MIMAT0000733, MIMAT0004690, MIMAT0000735, MIMAT0000734, MIMAT0000736, MMAT0000737, MIMAT0000738, MIMAT0001075, MIMAT0001639, MIMAT0001638, MMAT0002171, MIMAT0003329, MIMAT0004813, MIMAT0002170, MMAT0003339, MIMAT0001339, MIMATOOO 1340, MIMAT0004748, MIMAT0001341, MMAT0004749, MIMAT0003393, MIMAT0001343, MEMAT0001536, MIMAT0001625, MIMAT0004757, MIMAT0002814, MIMAT0002815, MIMAT0001627, MIMAT0001532, MIMAT0001541, MIMAT0003327, MIMAT0001545, MIMAT0004910, MIMAT0004909, MIMATOOO 1631, MIMATOOO 1635, MIMATOOO 1636, MIMATOOO 1630, MMAT0003885, MIMAT0003884, MIMAT0004784, MIMAT0003150, MIMAT0002173, MIMAT0004761, MIMAT0002174, MDVLAT0002176, MIMAT0002175, MIMAT0004762, MIMAT0002177, MEMAT0002178, MIMAT0003180, MIMAT0004763, MIMAT0002804, MMAT0002805, MIMAT0002806, MIMAT0004764, MIMAT0004765, MIMAT0002807, MMAT0002812, MIMAT0003161, MIMAT0002813, MTMAT0002816, MIMAT0002817, MMAT0002818, MIMAT0002820, MIMAT0004768, MIMAT0002824, MIMAT0004772, MIMAT0002870, MIMAT0004773, MTMAT0002871, MIMAT0004774, MIMAT0002872, MEMAT0004775, MIMAT0002873, MIMAT0002874, MIMAT0002875, MIMAT0002876, MMAT0004776, MIMAT0002878, MIMAT0002879, MIMAT0002880, MIMAT0004778, MIMAT0004975, MIMAT0002881, MIMAT0004779, MIMAT0002882, MIMAT0002808, MIMAT0002823, MMAT0002822, MIMAT0004777, MIMAT0002877, MIMAT0005788, MIMAT0005789, MIMAT0002883, MIMAT0002827, MIMAT0002826, MIMAT0002860, MEVLAT0004770, MIMAT0002859, MIMAT0002851, MIMAT0002852, MEVLAT0002857, MMAT0002866, MMAT0002863, MIMAT0005457, MIMAT0002844, MIMAT0002848, MIMAT0002847, MMAT0002864, MIMAT0005456, MIMAT0002861, MIMAT0005450, MIMAT0002842, MEVLAT0002841 , MIMAT0002869, MDVLAT0005452, MIMAT0002837, MIMAT0005454, MIMAT0002832, MIMAT0002831, MIMAT0002853, MIMAT0002829, MIMAT0002828, MIMAT0002834, MIMAT0002833, MIMAT0002843, MIMAT0002846, MIMAT0005455, MIMAT0002856, MIMAT0002855, MIMAT0002825, MIMAT0002830, MIMAT0002858, MIMAT0002867, MIMAT0002854, MIMAT0002868, MIMAT0005451, MIMAT0002840, MIMAT0005449, MIMAT0002850, MIMAT0002849, MIMAT0002839, MMAT0002838, MIMAT0002845, MIMAT0002835, MMAT0002836, MEVLA.T0002862, MIMAT0004780, MIMAT0002888, MIMAT0003163, MDMAT0004920, MEMAT0004919, MIMAT0003389, MMAT0003340, MIMAT0004954, MMAT0003164, MIMAT0003165, MIMAT0004785, MIMAT0003251, MIMAT0004803, MIMAT0003254, MIMAT0004798, MIMAT0003285, MIMAT0004806, MIMAT0003323, MIMAT0003323, MEMAT0004812, MIMAT0003333, MIMAT0004800, MMAT0003257, MMAT0003214, MIMAT0003233, MIMAT0004794, MIMAT0003215, MIMAT0003216, MIMAT0003217, MTMAT0003219, MMAT0004793, MIMAT0003220, MMAT0003221, MIMAT0003222, MIMAT0003223, MIMAT0003225, MIMAT0003226, MIMAT0003227, MMAT0003228, MIMAT0003230, MMAT0003231, MEMAT0003232, MIMAT0003234, MIMAT0003235, MIMAT0003236, MIMAT0003237, MIMAT0003238, MIMAT0003239, MMAT0004795, MIMAT0003240, MMAT0004796, MIMAT0003241, MIMAT0003242, MIMAT0003243, MIMAT0003244, MMAT0003245, MIMAT0003246, MMAT0004797, MTMAT0003247, MIMAT0003248, MIMAT0003249, MIMAT0003250, MIMAT0003252, MMAT0003253, MIMAT0003255, MIMAT0004799, MMAT0003256, MIMAT0004801, MMAT0003258, MIMAT0003259, MIMAT0003260, MIMAT0004802, MIMAT0003261, MMAT0003263, MIMAT0003264, MIMAT0003265, MIMAT0003266, MIMAT0003267, MIMAT0003268, MTMAT0003269, MIMAT0003270, MIMAT0003271, MIMAT0003272, MIMAT0003273, MIMAT0003274, MIMAT0003275, MBVLAT0003276, MIMAT0003277, MMAT0003278, MIMAT0003279, MIMAT0003280, MMAT0003281, MIMAT0003282, MIMAT0003283, MIMAT0004804, MIMAT0004805, MMAT0003284, MDVLA.T0003286, MIMAT0003287, MIMAT0003288, MTMAT0003289, MIMAT0003290, MIMAT0003291, MIMAT0003292, MIMAT0004807, MIMAT0003293, MEMAT0003294, MIMAT0004808, MIMAT0003295, MIMAT0003296, MIMAT0003297, MIMAT0004809, MIMAT0004810, MIMAT0003298, MIMAT0003299, MIMAT0003300, MIMAT0003302, MIMAT0003303, MIMAT0003304, MIMAT0003305, MIMAT0003306, MIMAT0003307, MMAT0003308, MIMAT0003309, MIMAT0003310, MIMAT0003311, MIMAT0003312, MIMAT0003313, MIMAT0003314, MIMAT0003315, MIMAT0003316, MDMAT0003317, MIMAT0003318, MIMAT0003319, MIMAT0003320, MIMAT0003321, MMAT0003322, MIMAT0003328, MIMAT0004814, MIMAT0003330, MIMAT0003331, MMAT0003332, MIMAT0003335, MEMAT0003336, MIMAT0003337, MIMAT0003338, MIMAT0003324, MIMAT0003325, MIMAT0003326, MIMAT0004952, MIMAT0003881, MIMAT0004819, MMAT0003880, MIMAT0004284, MIMAT0000252, MIMAT0004926, MIMAT0004927, MIMAT0004553, MIMAT0004554, MIMAT0004945, MEMAT0004946, MIMAT0003879, MIMAT0004957, MIMAT0003945, MIMAT0003888, MIMAT0003883, MIMAT0003882, MMAT0003947, MIMAT0003946, MIMAT0003887, MIMAT0003886, MEMAT0003948, MMAT0004209, MIMAT0004185, MIMAT0004953, MIMAT0004911, MMAT0004923, MIMAT0004922, MIMAT0004925, MIMAT0004924, MIMAT0004949, MIMAT0004950, MMAT0004948, MMAT0004947, MIMAT0004906, MIMAT0004905, MIMAT0004951, MMAT0004916, MIMAT0004917, MIMAT0004921, MIMAT0004912, MIMAT0004902, MLV1AT0004913, MIMAT0004907, MMAT0004918, MIMAT0000441, MIMAT0000442, MIMAT0004970, MEV1AT0004971, MIMAT0004972, MIMAT0004973, MIMAT0004974, MIMAT0000092, MIMAT0004507, MIMAT0004508, MIMAT0003218, MEMAT0004792, MIMAT0000093, MEMAT0004509, MIMAT0004976, MIMAT0004977, MEMAT0004978, MIMAT0004979, MIMAT0004980, MIMAT0004981, MIMAT0004982, MIMAT0004983, MMAT0004984, MIMAT0004985, MIMAT0004986, MDVLAT0004987, MMAT0000094, MIMAT0000095, MIMAT0004510, MMAT0000096, MIMAT0000097, MIMAT0004511, MIMAT0000689, and MEVLAT0004678.
490. The oligomeric compound of claims 1-426 having a nucleobase sequence selected from SEQ ID NOs 20, 21, 23, 24, 25, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, and 91.
491. The method of any of claims 429, 456, 457, or 458, wherein the oligomeric compound comprises a nucleobase sequence selected from SEQ ID NOs 20, 21, 23, 24, 25, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, and 91.
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Cited By (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012027206A1 (en) 2010-08-24 2012-03-01 Merck Sharp & Dohme Corp. SINGLE-STRANDED RNAi AGENTS CONTAINING AN INTERNAL, NON-NUCLEIC ACID SPACER
US8202848B2 (en) 2008-03-17 2012-06-19 Board Of Regents, The University Of Texas System Identification of micro-RNAS involved in neuromuscular synapse maintenance and regeneration
WO2013022984A1 (en) * 2011-08-11 2013-02-14 Isis Pharmaceuticals, Inc. Selective antisense compounds and uses thereof
WO2013033223A1 (en) 2011-08-29 2013-03-07 Isis Pharmaceuticals, Inc. Methods and compounds useful in conditions related to repeat expansion
WO2013033230A1 (en) 2011-08-29 2013-03-07 Isis Pharmaceuticals, Inc. Oligomer-conjugate complexes and their use
US20130059902A1 (en) * 2010-02-08 2013-03-07 Isis Pharmaceuticals, Inc. Methods and compositions useful in treatment of diseases or conditions related to repeat expansion
US8440636B2 (en) 2007-07-31 2013-05-14 The Board Of Regents, The University Of Texas System Micro-RNA family that modulates fibrosis and uses thereof
WO2011139699A3 (en) * 2010-04-28 2013-07-04 Isis Pharmaceuticals, Inc. 5' modified nucleosides and oligomeric compounds prepared therefrom
US8481507B2 (en) 2007-07-31 2013-07-09 The Board Of Regents, The University Of Texas System Micro-RNAs that control myosin expression and myofiber identity
US8513209B2 (en) 2007-11-09 2013-08-20 The Board Of Regents, The University Of Texas System Micro-RNAS of the MIR-15 family modulate cardiomyocyte survival and cardiac repair
US8629119B2 (en) 2009-02-04 2014-01-14 The Board Of Regents, The University Of Texas System Dual targeting of MIR-208 and MIR-499 in the treatment of cardiac disorders
US8642751B2 (en) 2010-12-15 2014-02-04 Miragen Therapeutics MicroRNA inhibitors comprising locked nucleotides
WO2014131892A1 (en) * 2013-02-28 2014-09-04 Oligomer Sciences Ab Cell-penetrating oligonucleotides
WO2014172698A1 (en) * 2013-04-19 2014-10-23 Isis Pharmaceuticals, Inc. Compositions and methods for modulation nucleic acids through nonsense mediated decay
WO2014205451A2 (en) 2013-06-21 2014-12-24 Isis Pharmaceuticals, Inc. Compositions and methods for modulation of target nucleic acids
US8957040B2 (en) 2010-02-08 2015-02-17 Isis Pharmaceuticals, Inc. Selective reduction of allelic variants
US9127276B2 (en) 2013-05-01 2015-09-08 Isis Pharmaceuticals, Inc. Conjugated antisense compounds and their use
WO2015164693A1 (en) 2014-04-24 2015-10-29 Isis Pharmaceuticals, Inc. Oligomeric compounds comprising alpha-beta-constrained nucleic acid
US9382540B2 (en) 2014-05-01 2016-07-05 Isis Pharmaceuticals, Inc Compositions and methods for modulating angiopoietin-like 3 expression
US9388408B2 (en) 2012-06-21 2016-07-12 MiRagen Therapeutics, Inc. Oligonucleotide-based inhibitors comprising locked nucleic acid motif
US9428749B2 (en) 2011-10-06 2016-08-30 The Board Of Regents, The University Of Texas System Control of whole body energy homeostasis by microRNA regulation
US9885042B2 (en) 2015-01-20 2018-02-06 MiRagen Therapeutics, Inc. miR-92 inhibitors and uses thereof
US10087210B2 (en) 2010-04-28 2018-10-02 Ionis Pharmaceuticals, Inc. Modified nucleosides, analogs thereof and oligomeric compounds prepared therefrom
US10160969B2 (en) 2014-01-16 2018-12-25 Wave Life Sciences Ltd. Chiral design
US10167309B2 (en) 2012-07-13 2019-01-01 Wave Life Sciences Ltd. Asymmetric auxiliary group
US10246709B2 (en) 2016-03-07 2019-04-02 Arrowhead Pharmaceuticals, Inc. Targeting ligands for therapeutic compounds
US10280192B2 (en) 2011-07-19 2019-05-07 Wave Life Sciences Ltd. Methods for the synthesis of functionalized nucleic acids
US10280423B2 (en) 2014-05-01 2019-05-07 Ionis Pharmaceuticals, Inc. Compositions and methods for modulating complement factor B expression
US10294474B2 (en) 2016-09-02 2019-05-21 Arrowhead Pharmaceuticals, Inc. Targeting ligands
US10307434B2 (en) 2009-07-06 2019-06-04 Wave Life Sciences Ltd. Nucleic acid prodrugs and methods of use thereof
US10329318B2 (en) 2008-12-02 2019-06-25 Wave Life Sciences Ltd. Method for the synthesis of phosphorus atom modified nucleic acids
US10428019B2 (en) 2010-09-24 2019-10-01 Wave Life Sciences Ltd. Chiral auxiliaries
US10435430B2 (en) 2013-07-31 2019-10-08 Ionis Pharmaceuticals, Inc. Methods and compounds useful in conditions related to repeat expansion
US10557137B2 (en) 2015-11-06 2020-02-11 Ionis Pharmaceuticals, Inc. Modulating apolipoprotein (a) expression
JP2020522510A (en) * 2017-06-02 2020-07-30 ウェイブ ライフ サイエンシズ リミテッドＷａｖｅ Ｌｉｆｅ Ｓｃｉｅｎｃｅｓ Ｌｔｄ． Oligonucleotide composition and method of using the same
WO2020158910A1 (en) 2019-02-01 2020-08-06 国立大学法人大阪大学 5'-modified nucleoside and nucleotide using same
WO2020166551A1 (en) 2019-02-13 2020-08-20 国立大学法人大阪大学 5'-modified nucleoside and nucleotide using same
US11166976B2 (en) 2018-11-08 2021-11-09 Aligos Therapeutics, Inc. S-antigen transport inhibiting oligonucleotide polymers and methods
US11236335B2 (en) 2012-10-12 2022-02-01 Ionis Pharmaceuticals, Inc. Selective antisense compounds and uses thereof
EP3929293A3 (en) * 2015-04-03 2022-03-16 University Of Massachusetts Fully stabilized asymmetric sirna
US11400161B2 (en) 2016-10-06 2022-08-02 Ionis Pharmaceuticals, Inc. Method of conjugating oligomeric compounds
US11560563B2 (en) 2018-04-05 2023-01-24 Silence Therapeutics Gmbh SiRNAs with vinylphosphonate at the 5′ end of the antisense strand
US11597927B2 (en) 2017-06-02 2023-03-07 Wave Life Sciences Ltd. Oligonucleotide compositions and methods of use thereof
US11702659B2 (en) 2021-06-23 2023-07-18 University Of Massachusetts Optimized anti-FLT1 oligonucleotide compounds for treatment of preeclampsia and other angiogenic disorders
US11827882B2 (en) 2018-08-10 2023-11-28 University Of Massachusetts Modified oligonucleotides targeting SNPs
US11896669B2 (en) 2016-01-31 2024-02-13 University Of Massachusetts Branched oligonucleotides

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8975389B2 (en) * 2009-03-02 2015-03-10 Alnylam Pharmaceuticals, Inc. Nucleic acid chemical modifications
WO2013173789A2 (en) 2012-05-17 2013-11-21 Isis Pharmaceuticals, Inc. Antisense oligonucleotide compositions
US9574193B2 (en) * 2012-05-17 2017-02-21 Ionis Pharmaceuticals, Inc. Methods and compositions for modulating apolipoprotein (a) expression
US9163235B2 (en) 2012-06-21 2015-10-20 MiRagen Therapeutics, Inc. Inhibitors of the miR-15 family of micro-RNAs
KR102493872B1 (en) 2016-09-02 2023-01-30 다이서나 파마수이티컬, 인크. 4´-phosphate analogs and oligonucleotides comprising the same
CN110088278B (en) * 2016-10-31 2023-08-11 e-NA生物科技公司 Double-stranded nucleic acid molecules and uses thereof
EP3548005A4 (en) 2016-11-29 2020-06-17 Puretech Health LLC Exosomes for delivery of therapeutic agents
JP2022544587A (en) * 2019-08-15 2022-10-19 アイオーニス ファーマシューティカルズ， インコーポレーテッド Bond-modified oligomeric compounds and uses thereof
WO2023178144A2 (en) 2022-03-16 2023-09-21 Empirico Inc. Galnac compositions for improving sirna bioavailability

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004295A1 (en) * 1994-07-29 1996-02-15 Amgen Inc. 5'-dithio-modified oligonucleotides
WO2001014400A1 (en) * 1999-08-19 2001-03-01 Isis Pharmaceuticals, Inc. 2'-o-acetamido modified monomers and oligomers

Family Cites Families (218)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2699808A (en) 1944-10-06 1955-01-18 Mark W Lowe Apparatus for peeling tomatoes
US2699508A (en) 1951-12-21 1955-01-11 Selectronics Inc Method of mounting and construction of mounting for low frequency piezoelectric crystals
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4500707A (en) 1980-02-29 1985-02-19 University Patents, Inc. Nucleosides useful in the preparation of polynucleotides
US5132418A (en) 1980-02-29 1992-07-21 University Patents, Inc. Process for preparing polynucleotides
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US4973679A (en) 1981-03-27 1990-11-27 University Patents, Inc. Process for oligonucleo tide synthesis using phosphormidite intermediates
US4668777A (en) 1981-03-27 1987-05-26 University Patents, Inc. Phosphoramidite nucleoside compounds
US4415732A (en) 1981-03-27 1983-11-15 University Patents, Inc. Phosphoramidite compounds and processes
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US4476301A (en) 1982-04-29 1984-10-09 Centre National De La Recherche Scientifique Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon
JPS5927900A (en) 1982-08-09 1984-02-14 Wakunaga Seiyaku Kk Oligonucleotide derivative and its preparation
FR2540122B1 (en) 1983-01-27 1985-11-29 Centre Nat Rech Scient NOVEL COMPOUNDS COMPRISING A SEQUENCE OF OLIGONUCLEOTIDE LINKED TO AN INTERCALATION AGENT, THEIR SYNTHESIS PROCESS AND THEIR APPLICATION
US4605735A (en) 1983-02-14 1986-08-12 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
US4948882A (en) 1983-02-22 1990-08-14 Syngene, Inc. Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis
US4824941A (en) 1983-03-10 1989-04-25 Julian Gordon Specific antibody to the native form of 2'5'-oligonucleotides, the method of preparation and the use as reagents in immunoassays or for binding 2'5'-oligonucleotides in biological systems
DE3329892A1 (en) 1983-08-18 1985-03-07 Köster, Hubert, Prof. Dr., 2000 Hamburg METHOD FOR PRODUCING OLIGONUCLEOTIDES
USRE34069E (en) 1983-08-18 1992-09-15 Biosyntech Gmbh Process for the preparation of oligonucleotides
US4587044A (en) 1983-09-01 1986-05-06 The Johns Hopkins University Linkage of proteins to nucleic acids
US5118800A (en) 1983-12-20 1992-06-02 California Institute Of Technology Oligonucleotides possessing a primary amino group in the terminal nucleotide
US5118802A (en) 1983-12-20 1992-06-02 California Institute Of Technology DNA-reporter conjugates linked via the 2' or 5'-primary amino group of the 5'-terminal nucleoside
US5550111A (en) 1984-07-11 1996-08-27 Temple University-Of The Commonwealth System Of Higher Education Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof
FR2567892B1 (en) 1984-07-19 1989-02-17 Centre Nat Rech Scient NOVEL OLIGONUCLEOTIDES, THEIR PREPARATION PROCESS AND THEIR APPLICATIONS AS MEDIATORS IN DEVELOPING THE EFFECTS OF INTERFERONS
US5430136A (en) 1984-10-16 1995-07-04 Chiron Corporation Oligonucleotides having selectably cleavable and/or abasic sites
US5367066A (en) 1984-10-16 1994-11-22 Chiron Corporation Oligonucleotides with selectably cleavable and/or abasic sites
US5258506A (en) 1984-10-16 1993-11-02 Chiron Corporation Photolabile reagents for incorporation into oligonucleotide chains
US4828979A (en) 1984-11-08 1989-05-09 Life Technologies, Inc. Nucleotide analogs for nucleic acid labeling and detection
FR2575751B1 (en) 1985-01-08 1987-04-03 Pasteur Institut NOVEL ADENOSINE DERIVATIVE NUCLEOSIDES, THEIR PREPARATION AND THEIR BIOLOGICAL APPLICATIONS
US5166315A (en) 1989-12-20 1992-11-24 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5235033A (en) 1985-03-15 1993-08-10 Anti-Gene Development Group Alpha-morpholino ribonucleoside derivatives and polymers thereof
US5185444A (en) 1985-03-15 1993-02-09 Anti-Gene Deveopment Group Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US5405938A (en) 1989-12-20 1995-04-11 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US4762779A (en) 1985-06-13 1988-08-09 Amgen Inc. Compositions and methods for functionalizing nucleic acids
US5317098A (en) 1986-03-17 1994-05-31 Hiroaki Shizuya Non-radioisotope tagging of fragments
JPS638396A (en) 1986-06-30 1988-01-14 Wakunaga Pharmaceut Co Ltd Poly-labeled oligonucleotide derivative
DE3788914T2 (en) 1986-09-08 1994-08-25 Ajinomoto Kk Compounds for cleaving RNA at a specific position, oligomers used in the preparation of these compounds and starting materials for the synthesis of these oligomers.
US5276019A (en) 1987-03-25 1994-01-04 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5264423A (en) 1987-03-25 1993-11-23 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US4904582A (en) 1987-06-11 1990-02-27 Synthetic Genetics Novel amphiphilic nucleic acid conjugates
AU598946B2 (en) 1987-06-24 1990-07-05 Howard Florey Institute Of Experimental Physiology And Medicine Nucleoside derivatives
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
US4924624A (en) 1987-10-22 1990-05-15 Temple University-Of The Commonwealth System Of Higher Education 2,',5'-phosphorothioate oligoadenylates and plant antiviral uses thereof
US5188897A (en) 1987-10-22 1993-02-23 Temple University Of The Commonwealth System Of Higher Education Encapsulated 2',5'-phosphorothioate oligoadenylates
US5525465A (en) 1987-10-28 1996-06-11 Howard Florey Institute Of Experimental Physiology And Medicine Oligonucleotide-polyamide conjugates and methods of production and applications of the same
DE3738460A1 (en) 1987-11-12 1989-05-24 Max Planck Gesellschaft MODIFIED OLIGONUCLEOTIDS
EP0348458B1 (en) 1987-11-30 1997-04-09 University Of Iowa Research Foundation Dna molecules stabilized by modifications of the 3'-terminal phosphodiester linkage and their use as nucleic acid probes and as therapeutic agents to block the expression of specifically targeted genes
US5403711A (en) 1987-11-30 1995-04-04 University Of Iowa Research Foundation Nucleic acid hybridization and amplification method for detection of specific sequences in which a complementary labeled nucleic acid probe is cleaved
US5082830A (en) 1988-02-26 1992-01-21 Enzo Biochem, Inc. End labeled nucleotide probe
WO1989009221A1 (en) 1988-03-25 1989-10-05 University Of Virginia Alumni Patents Foundation Oligonucleotide n-alkylphosphoramidates
US5278302A (en) 1988-05-26 1994-01-11 University Patents, Inc. Polynucleotide phosphorodithioates
US5109124A (en) 1988-06-01 1992-04-28 Biogen, Inc. Nucleic acid probe linked to a label having a terminal cysteine
US5216141A (en) 1988-06-06 1993-06-01 Benner Steven A Oligonucleotide analogs containing sulfur linkages
US5175273A (en) 1988-07-01 1992-12-29 Genentech, Inc. Nucleic acid intercalating agents
US5262536A (en) 1988-09-15 1993-11-16 E. I. Du Pont De Nemours And Company Reagents for the preparation of 5'-tagged oligonucleotides
US5194599A (en) 1988-09-23 1993-03-16 Gilead Sciences, Inc. Hydrogen phosphonodithioate compositions
US5512439A (en) 1988-11-21 1996-04-30 Dynal As Oligonucleotide-linked magnetic particles and uses thereof
US5457183A (en) 1989-03-06 1995-10-10 Board Of Regents, The University Of Texas System Hydroxylated texaphyrins
US5599923A (en) 1989-03-06 1997-02-04 Board Of Regents, University Of Tx Texaphyrin metal complexes having improved functionalization
US5391723A (en) 1989-05-31 1995-02-21 Neorx Corporation Oligonucleotide conjugates
US5256775A (en) 1989-06-05 1993-10-26 Gilead Sciences, Inc. Exonuclease-resistant oligonucleotides
US4958013A (en) 1989-06-06 1990-09-18 Northwestern University Cholesteryl modified oligonucleotides
US5451463A (en) 1989-08-28 1995-09-19 Clontech Laboratories, Inc. Non-nucleoside 1,3-diol reagents for labeling synthetic oligonucleotides
US5134066A (en) 1989-08-29 1992-07-28 Monsanto Company Improved probes using nucleosides containing 3-dezauracil analogs
US5254469A (en) 1989-09-12 1993-10-19 Eastman Kodak Company Oligonucleotide-enzyme conjugate that can be used as a probe in hybridization assays and polymerase chain reaction procedures
US5591722A (en) 1989-09-15 1997-01-07 Southern Research Institute 2'-deoxy-4'-thioribonucleosides and their antiviral activity
US5399676A (en) 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity
US5721218A (en) 1989-10-23 1998-02-24 Gilead Sciences, Inc. Oligonucleotides with inverted polarity
US5264562A (en) 1989-10-24 1993-11-23 Gilead Sciences, Inc. Oligonucleotide analogs with novel linkages
WO1991006556A1 (en) 1989-10-24 1991-05-16 Gilead Sciences, Inc. 2' modified oligonucleotides
US5264564A (en) 1989-10-24 1993-11-23 Gilead Sciences Oligonucleotide analogs with novel linkages
US5292873A (en) 1989-11-29 1994-03-08 The Research Foundation Of State University Of New York Nucleic acids labeled with naphthoquinone probe
US5177198A (en) 1989-11-30 1993-01-05 University Of N.C. At Chapel Hill Process for preparing oligoribonucleoside and oligodeoxyribonucleoside boranophosphates
US5130302A (en) 1989-12-20 1992-07-14 Boron Bilogicals, Inc. Boronated nucleoside, nucleotide and oligonucleotide compounds, compositions and methods for using same
US5486603A (en) 1990-01-08 1996-01-23 Gilead Sciences, Inc. Oligonucleotide having enhanced binding affinity
US6005087A (en) 1995-06-06 1999-12-21 Isis Pharmaceuticals, Inc. 2'-modified oligonucleotides
US5578718A (en) 1990-01-11 1996-11-26 Isis Pharmaceuticals, Inc. Thiol-derivatized nucleosides
US5623065A (en) 1990-08-13 1997-04-22 Isis Pharmaceuticals, Inc. Gapped 2' modified oligonucleotides
US5459255A (en) 1990-01-11 1995-10-17 Isis Pharmaceuticals, Inc. N-2 substituted purines
US5670633A (en) 1990-01-11 1997-09-23 Isis Pharmaceuticals, Inc. Sugar modified oligonucleotides that detect and modulate gene expression
US5587470A (en) 1990-01-11 1996-12-24 Isis Pharmaceuticals, Inc. 3-deazapurines
US5646265A (en) 1990-01-11 1997-07-08 Isis Pharmceuticals, Inc. Process for the preparation of 2'-O-alkyl purine phosphoramidites
US5587361A (en) 1991-10-15 1996-12-24 Isis Pharmaceuticals, Inc. Oligonucleotides having phosphorothioate linkages of high chiral purity
US5681941A (en) 1990-01-11 1997-10-28 Isis Pharmaceuticals, Inc. Substituted purines and oligonucleotide cross-linking
US5149797A (en) 1990-02-15 1992-09-22 The Worcester Foundation For Experimental Biology Method of site-specific alteration of rna and production of encoded polypeptides
US5220007A (en) 1990-02-15 1993-06-15 The Worcester Foundation For Experimental Biology Method of site-specific alteration of RNA and production of encoded polypeptides
US5214136A (en) 1990-02-20 1993-05-25 Gilead Sciences, Inc. Anthraquinone-derivatives oligonucleotides
AU7579991A (en) 1990-02-20 1991-09-18 Gilead Sciences, Inc. Pseudonucleosides and pseudonucleotides and their polymers
US5321131A (en) 1990-03-08 1994-06-14 Hybridon, Inc. Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling
US5470967A (en) 1990-04-10 1995-11-28 The Dupont Merck Pharmaceutical Company Oligonucleotide analogs with sulfamate linkages
GB9009980D0 (en) 1990-05-03 1990-06-27 Amersham Int Plc Phosphoramidite derivatives,their preparation and the use thereof in the incorporation of reporter groups on synthetic oligonucleotides
ES2116977T3 (en) 1990-05-11 1998-08-01 Microprobe Corp SOLID SUPPORTS FOR NUCLEIC ACID HYBRIDIZATION TESTS AND METHODS TO IMMOBILIZE OLIGONUCLEOTIDES IN A COVALENT WAY.
US5608046A (en) 1990-07-27 1997-03-04 Isis Pharmaceuticals, Inc. Conjugated 4'-desmethyl nucleoside analog compounds
US5610289A (en) 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5138045A (en) 1990-07-27 1992-08-11 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5688941A (en) 1990-07-27 1997-11-18 Isis Pharmaceuticals, Inc. Methods of making conjugated 4' desmethyl nucleoside analog compounds
US5677437A (en) 1990-07-27 1997-10-14 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
WO1992002258A1 (en) 1990-07-27 1992-02-20 Isis Pharmaceuticals, Inc. Nuclease resistant, pyrimidine modified oligonucleotides that detect and modulate gene expression
US5223618A (en) 1990-08-13 1993-06-29 Isis Pharmaceuticals, Inc. 4'-desmethyl nucleoside analog compounds
US5623070A (en) 1990-07-27 1997-04-22 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5378825A (en) 1990-07-27 1995-01-03 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs
US5618704A (en) 1990-07-27 1997-04-08 Isis Pharmacueticals, Inc. Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling
US5386023A (en) 1990-07-27 1995-01-31 Isis Pharmaceuticals Backbone modified oligonucleotide analogs and preparation thereof through reductive coupling
US5218105A (en) 1990-07-27 1993-06-08 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5602240A (en) 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
US5541307A (en) 1990-07-27 1996-07-30 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs and solid phase synthesis thereof
US5245022A (en) 1990-08-03 1993-09-14 Sterling Drug, Inc. Exonuclease resistant terminally substituted oligonucleotides
EP0541722B1 (en) 1990-08-03 1995-12-20 Sterling Winthrop Inc. Compounds and methods for inhibiting gene expression
US5177196A (en) 1990-08-16 1993-01-05 Microprobe Corporation Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof
US5512667A (en) 1990-08-28 1996-04-30 Reed; Michael W. Trifunctional intermediates for preparing 3'-tailed oligonucleotides
US5214134A (en) 1990-09-12 1993-05-25 Sterling Winthrop Inc. Process of linking nucleosides with a siloxane bridge
US5561225A (en) 1990-09-19 1996-10-01 Southern Research Institute Polynucleotide analogs containing sulfonate and sulfonamide internucleoside linkages
WO1992005186A1 (en) 1990-09-20 1992-04-02 Gilead Sciences Modified internucleoside linkages
US5432272A (en) 1990-10-09 1995-07-11 Benner; Steven A. Method for incorporating into a DNA or RNA oligonucleotide using nucleotides bearing heterocyclic bases
ATE198598T1 (en) 1990-11-08 2001-01-15 Hybridon Inc CONNECTION OF MULTIPLE REPORTER GROUPS ON SYNTHETIC OLIGONUCLEOTIDES
US5672697A (en) 1991-02-08 1997-09-30 Gilead Sciences, Inc. Nucleoside 5'-methylene phosphonates
US5371241A (en) 1991-07-19 1994-12-06 Pharmacia P-L Biochemicals Inc. Fluorescein labelled phosphoramidites
US5571799A (en) 1991-08-12 1996-11-05 Basco, Ltd. (2'-5') oligoadenylate analogues useful as inhibitors of host-v5.-graft response
EP0538194B1 (en) 1991-10-17 1997-06-04 Novartis AG Bicyclic nucleosides, oligonucleotides, their method of preparation and intermediates therein
US5594121A (en) 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
TW393513B (en) 1991-11-26 2000-06-11 Isis Pharmaceuticals Inc Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US5484908A (en) 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines
WO1993010820A1 (en) 1991-11-26 1993-06-10 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US5792608A (en) 1991-12-12 1998-08-11 Gilead Sciences, Inc. Nuclease stable and binding competent oligomers and methods for their use
US5359044A (en) 1991-12-13 1994-10-25 Isis Pharmaceuticals Cyclobutyl oligonucleotide surrogates
US20010044145A1 (en) * 1991-12-24 2001-11-22 Monia Brett P. Methods of using mammalian RNase H and compositions thereof
US5700922A (en) 1991-12-24 1997-12-23 Isis Pharmaceuticals, Inc. PNA-DNA-PNA chimeric macromolecules
US5565552A (en) 1992-01-21 1996-10-15 Pharmacyclics, Inc. Method of expanded porphyrin-oligonucleotide conjugate synthesis
US5595726A (en) 1992-01-21 1997-01-21 Pharmacyclics, Inc. Chromophore probe for detection of nucleic acid
FR2687679B1 (en) 1992-02-05 1994-10-28 Centre Nat Rech Scient OLIGOTHIONUCLEOTIDES.
US5633360A (en) 1992-04-14 1997-05-27 Gilead Sciences, Inc. Oligonucleotide analogs capable of passive cell membrane permeation
US5434257A (en) 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages
EP0577558A2 (en) 1992-07-01 1994-01-05 Ciba-Geigy Ag Carbocyclic nucleosides having bicyclic rings, oligonucleotides therefrom, process for their preparation, their use and intermediates
US5272250A (en) 1992-07-10 1993-12-21 Spielvogel Bernard F Boronated phosphoramidate compounds
US5652355A (en) 1992-07-23 1997-07-29 Worcester Foundation For Experimental Biology Hybrid oligonucleotide phosphorothioates
EP0652890B1 (en) 1992-07-27 1998-01-14 HYBRIDON, Inc. Oligonucleotide alkylphosphonothioates
EP0673559A1 (en) 1992-12-14 1995-09-27 Honeywell Inc. Motor system with individually controlled redundant windings
US5574142A (en) 1992-12-15 1996-11-12 Microprobe Corporation Peptide linkers for improved oligonucleotide delivery
CN1121721A (en) 1993-01-25 1996-05-01 海布里顿公司 Oligonucleotido alky phosphate and alky substituted phosphate
US5476925A (en) 1993-02-01 1995-12-19 Northwestern University Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups
GB9304618D0 (en) 1993-03-06 1993-04-21 Ciba Geigy Ag Chemical compounds
GB9304620D0 (en) 1993-03-06 1993-04-21 Ciba Geigy Ag Compounds
EP0691968B1 (en) 1993-03-30 1997-07-16 Sanofi Acyclic nucleoside analogs and oligonucleotide sequences containing them
AU6449294A (en) 1993-03-31 1994-10-24 Sterling Winthrop Inc. Novel 5'-substituted nucleosides and oligomers produced therefrom
ATE160572T1 (en) 1993-03-31 1997-12-15 Sanofi Sa OLIGONUCLEOTIDES WITH AMIDE CHAINS THAT USE PHOSPHOESTER CHAINS
DE4311944A1 (en) 1993-04-10 1994-10-13 Degussa Coated sodium percarbonate particles, process for their preparation and detergent, cleaning and bleaching compositions containing them
ES2128535T3 (en) 1993-05-12 1999-05-16 Novartis Ag NUCLEOSIDES AND OLIGONUCLEOTIDES WITH 2'-ETER GROUPS.
GB9311682D0 (en) 1993-06-05 1993-07-21 Ciba Geigy Ag Chemical compounds
US5502177A (en) 1993-09-17 1996-03-26 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US5457187A (en) 1993-12-08 1995-10-10 Board Of Regents University Of Nebraska Oligonucleotides containing 5-fluorouracil
EP0733059B1 (en) 1993-12-09 2000-09-13 Thomas Jefferson University Compounds and methods for site-directed mutations in eukaryotic cells
US5446137B1 (en) 1993-12-09 1998-10-06 Behringwerke Ag Oligonucleotides containing 4'-substituted nucleotides
US5519134A (en) 1994-01-11 1996-05-21 Isis Pharmaceuticals, Inc. Pyrrolidine-containing monomers and oligomers
US5596091A (en) 1994-03-18 1997-01-21 The Regents Of The University Of California Antisense oligonucleotides comprising 5-aminoalkyl pyrimidine nucleotides
US5627053A (en) 1994-03-29 1997-05-06 Ribozyme Pharmaceuticals, Inc. 2'deoxy-2'-alkylnucleotide containing nucleic acid
US5625050A (en) 1994-03-31 1997-04-29 Amgen Inc. Modified oligonucleotides and intermediates useful in nucleic acid therapeutics
US5646269A (en) 1994-04-28 1997-07-08 Gilead Sciences, Inc. Method for oligonucleotide analog synthesis
US5525711A (en) 1994-05-18 1996-06-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pteridine nucleotide analogs as fluorescent DNA probes
US5597696A (en) 1994-07-18 1997-01-28 Becton Dickinson And Company Covalent cyanine dye oligonucleotide conjugates
US5580731A (en) 1994-08-25 1996-12-03 Chiron Corporation N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith
US5597909A (en) 1994-08-25 1997-01-28 Chiron Corporation Polynucleotide reagents containing modified deoxyribose moieties, and associated methods of synthesis and use
US5681940A (en) 1994-11-02 1997-10-28 Icn Pharmaceuticals Sugar modified nucleosides and oligonucleotides
US5792747A (en) 1995-01-24 1998-08-11 The Administrators Of The Tulane Educational Fund Highly potent agonists of growth hormone releasing hormone
US5652356A (en) 1995-08-17 1997-07-29 Hybridon, Inc. Inverted chimeric and hybrid oligonucleotides
AU1430097A (en) 1996-01-16 1997-08-11 Ribozyme Pharmaceuticals, Inc. Synthesis of methoxy nucleosides and enzymatic nucleic acid molecules
GB9606158D0 (en) 1996-03-23 1996-05-29 Ciba Geigy Ag Chemical compounds
US5656408A (en) 1996-04-29 1997-08-12 Xerox Corporation Coated carrier particles
AU3340797A (en) 1996-06-28 1998-01-21 Novartis Ag Modified oligonucleotides
AU4815197A (en) 1996-10-09 1998-05-05 Pharmasset, Ltd. Tetraphosphonate bicyclic trisanhydrides
US6172209B1 (en) 1997-02-14 2001-01-09 Isis Pharmaceuticals Inc. Aminooxy-modified oligonucleotides and methods for making same
US6770748B2 (en) 1997-03-07 2004-08-03 Takeshi Imanishi Bicyclonucleoside and oligonucleotide analogue
JP3756313B2 (en) 1997-03-07 2006-03-15 武 今西 Novel bicyclonucleosides and oligonucleotide analogues
DE69829760T3 (en) 1997-09-12 2016-04-14 Exiqon A/S BI- AND TRI-CYCLIC-NUCLEOSIDE, NUCLEOTIDE AND OLIGONUCLEOTIDE ANALOG
US6794499B2 (en) 1997-09-12 2004-09-21 Exiqon A/S Oligonucleotide analogues
US6007992A (en) 1997-11-10 1999-12-28 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US6028183A (en) 1997-11-07 2000-02-22 Gilead Sciences, Inc. Pyrimidine derivatives and oligonucleotides containing same
HUP0102152A3 (en) 1998-05-26 2002-04-29 Icn Pharmaceuticals Inc Costa Nucleosid and oligo nucleotid analogues having bicyclic sugar derivative
US6271358B1 (en) 1998-07-27 2001-08-07 Isis Pharmaceuticals, Inc. RNA targeted 2′-modified oligonucleotides that are conformationally preorganized
US6043352A (en) 1998-08-07 2000-03-28 Isis Pharmaceuticals, Inc. 2'-O-Dimethylaminoethyloxyethyl-modified oligonucleotides
DK1178999T3 (en) 1999-05-04 2007-08-06 Santaris Pharma As L-ribo-LNA analogues
US6525191B1 (en) 1999-05-11 2003-02-25 Kanda S. Ramasamy Conformationally constrained L-nucleosides
US20050020525A1 (en) 2002-02-20 2005-01-27 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US6426220B1 (en) 2000-10-30 2002-07-30 Isis Pharmaceuticals, Inc. Antisense modulation of calreticulin expression
WO2002097134A2 (en) 2001-05-25 2002-12-05 Isis Pharmaceuticals, Inc. Modified peptide nucleic acid
US20030175906A1 (en) 2001-07-03 2003-09-18 Muthiah Manoharan Nuclease resistant chimeric oligonucleotides
WO2003004602A2 (en) 2001-07-03 2003-01-16 Isis Pharmaceuticals, Inc. Nuclease resistant chimeric oligonucleotides
US20030158403A1 (en) 2001-07-03 2003-08-21 Isis Pharmaceuticals, Inc. Nuclease resistant chimeric oligonucleotides
AU2003213628A1 (en) 2002-02-28 2003-09-16 Biota, Inc. Nucleoside 5'-monophosphate mimics and their prodrugs
US20060074035A1 (en) 2002-04-17 2006-04-06 Zhi Hong Dinucleotide inhibitors of de novo RNA polymerases for treatment or prevention of viral infections
US8101348B2 (en) 2002-07-10 2012-01-24 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. RNA-interference by single-stranded RNA molecules
EP1857547B2 (en) * 2002-08-05 2020-12-02 Silence Therapeutics GmbH Further novel forms of interfering RNA molecules
EP1562971B1 (en) 2002-11-05 2014-02-12 Isis Pharmaceuticals, Inc. Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
WO2004044132A2 (en) 2002-11-05 2004-05-27 Isis Pharmaceuticals, Inc. Modified oligonucleotides for use in rna interference
WO2005012371A2 (en) 2003-05-12 2005-02-10 Union Carbide Chemicals & Plastics Technology Corporation Process for control of polymer fines in a gas-phase polymerization
WO2005020885A2 (en) 2003-05-21 2005-03-10 Isis Pharmaceuticals, Inc. Compositions and methods for the treatment of severe acute respiratory syndrome (sars)
WO2004106356A1 (en) 2003-05-27 2004-12-09 Syddansk Universitet Functionalized nucleotide derivatives
JP2005060664A (en) 2003-07-31 2005-03-10 Asahi Glass Co Ltd Fluorine containing compound, fluorine containing polymer and production process for the same, and resist composition containing the same
US7683036B2 (en) * 2003-07-31 2010-03-23 Regulus Therapeutics Inc. Oligomeric compounds and compositions for use in modulation of small non-coding RNAs
WO2005021570A1 (en) 2003-08-28 2005-03-10 Gene Design, Inc. Novel artificial nucleic acids of n-o bond crosslinkage type
WO2005026356A1 (en) * 2003-09-12 2005-03-24 Commonwealth Scientific And Industrial Research Organisation Modified gene-silencing nucleic acid molecules and uses thereof
AU2005252663B2 (en) 2004-06-03 2011-07-07 Isis Pharmaceuticals, Inc. Double strand compositions comprising differentially modified strands for use in gene modulation
WO2006038865A1 (en) 2004-10-01 2006-04-13 Betagenon Ab Nucleotide derivatives for the treatment of type 2 diabetes and other disorders
DK2314594T3 (en) 2006-01-27 2014-10-27 Isis Pharmaceuticals Inc 6-modified bicyclic nucleic acid analogues
WO2007134181A2 (en) 2006-05-11 2007-11-22 Isis Pharmaceuticals, Inc. 5'-modified bicyclic nucleic acid analogs
MX363224B (en) 2006-10-03 2019-03-15 Alnylam Pharmaceuticals Inc Lipid containing formulations.
WO2008101157A1 (en) 2007-02-15 2008-08-21 Isis Pharmaceuticals, Inc. 5'-substituted-2'-f modified nucleosides and oligomeric compounds prepared therefrom
US8278425B2 (en) 2007-05-30 2012-10-02 Isis Pharmaceuticals, Inc. N-substituted-aminomethylene bridged bicyclic nucleic acid analogs
US8278426B2 (en) 2007-06-08 2012-10-02 Isis Pharmaceuticals, Inc. Carbocyclic bicyclic nucleic acid analogs
US8278283B2 (en) 2007-07-05 2012-10-02 Isis Pharmaceuticals, Inc. 6-disubstituted or unsaturated bicyclic nucleic acid analogs
US20090274686A1 (en) 2008-05-02 2009-11-05 Yat Sun Or Nucleoside phosphonate derivatives
EP2358397B1 (en) 2008-10-24 2020-01-01 Ionis Pharmaceuticals, Inc. 5' and 2' bis-substituted nucleosides and oligomeric compounds prepared therefrom
AT507215B1 (en) 2009-01-14 2010-03-15 Boehler Edelstahl Gmbh & Co Kg WEAR-RESISTANT MATERIAL
US9127033B2 (en) 2010-04-28 2015-09-08 Isis Pharmaceuticals, Inc. 5′ modified nucleosides and oligomeric compounds prepared therefrom
CN103154014B (en) 2010-04-28 2015-03-25 Isis制药公司 Modified nucleosides, modified nucleosides-like and oligomeric compounds prepared therefrom
US9984408B1 (en) 2012-05-30 2018-05-29 Amazon Technologies, Inc. Method, medium, and system for live video cooperative shopping
US9778708B1 (en) 2016-07-18 2017-10-03 Lenovo Enterprise Solutions (Singapore) Pte. Ltd. Dual sided latching retainer for computer modules

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004295A1 (en) * 1994-07-29 1996-02-15 Amgen Inc. 5'-dithio-modified oligonucleotides
WO2001014400A1 (en) * 1999-08-19 2001-03-01 Isis Pharmaceuticals, Inc. 2'-o-acetamido modified monomers and oligomers

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JAHN-HOFMANN, KERSTIN ET AL: "Efficient solid phase synthesis of cleavable oligodeoxynucleotides based on a novel strategy for the synthesis of 5'-S-(4,4'-dimethoxytrityl)-2'-deoxy-5'-th ionucleoside phosphoramidites" 2004, HELVETICA CHIMICA ACTA , 87(11), 2812-2828 CODEN: HCACAV; ISSN: 0018-019X , XP002580842 *
LIU X ET AL: "Uridylyl-(3' - 5')-(5'-thiouridine). An Exceptionally Base-labile Di-ribonucleoside Phosphate Analogue" TETRAHEDRON LETTERS, ELSEVIER, AMSTERDAM, NL LNKD- DOI:10.1016/0040-4039(95)00495-X, vol. 36, no. 19, 8 May 1995 (1995-05-08), pages 3413-3416, XP004028145 ISSN: 0040-4039 *
MATULIC-ADAMIC, JASENKA ET AL: "Synthesis and incorporation of 5'-amino- and 5'-mercapto-5'-deoxy-2'-O-methyl nucleosides into hammerhead ribozymes" NUCLEOSIDES & NUCLEOTIDES, vol. 16, 1997, - 1997 pages 1933-1950, XP9133026 *

Cited By (96)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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US8202848B2 (en) 2008-03-17 2012-06-19 Board Of Regents, The University Of Texas System Identification of micro-RNAS involved in neuromuscular synapse maintenance and regeneration
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US10329318B2 (en) 2008-12-02 2019-06-25 Wave Life Sciences Ltd. Method for the synthesis of phosphorus atom modified nucleic acids
US8629119B2 (en) 2009-02-04 2014-01-14 The Board Of Regents, The University Of Texas System Dual targeting of MIR-208 and MIR-499 in the treatment of cardiac disorders
US10307434B2 (en) 2009-07-06 2019-06-04 Wave Life Sciences Ltd. Nucleic acid prodrugs and methods of use thereof
US20130059902A1 (en) * 2010-02-08 2013-03-07 Isis Pharmaceuticals, Inc. Methods and compositions useful in treatment of diseases or conditions related to repeat expansion
US8957040B2 (en) 2010-02-08 2015-02-17 Isis Pharmaceuticals, Inc. Selective reduction of allelic variants
US10087210B2 (en) 2010-04-28 2018-10-02 Ionis Pharmaceuticals, Inc. Modified nucleosides, analogs thereof and oligomeric compounds prepared therefrom
US9127033B2 (en) 2010-04-28 2015-09-08 Isis Pharmaceuticals, Inc. 5′ modified nucleosides and oligomeric compounds prepared therefrom
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EP3372684A1 (en) * 2010-08-24 2018-09-12 Sirna Therapeutics, Inc. Single-stranded rnai agents containing an internal, non-nucleic acid spacer
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US9845466B2 (en) 2010-08-24 2017-12-19 Sirna Therapeutics, Inc. Single-stranded RNAi agents containing an internal, non-nucleic acid spacer
US9243246B2 (en) 2010-08-24 2016-01-26 Sirna Therapeutics, Inc. Single-stranded RNAi agents containing an internal, non-nucleic acid spacer
EP2609198A1 (en) * 2010-08-24 2013-07-03 Merck Sharp & Dohme Corp. SINGLE-STRANDED RNAi AGENTS CONTAINING AN INTERNAL, NON-NUCLEIC ACID SPACER
US10584335B2 (en) 2010-08-24 2020-03-10 Sirna Therapeutics, Inc. Single-stranded RNAi agents containing an internal, non-nucleic acid spacer
WO2012027206A1 (en) 2010-08-24 2012-03-01 Merck Sharp & Dohme Corp. SINGLE-STRANDED RNAi AGENTS CONTAINING AN INTERNAL, NON-NUCLEIC ACID SPACER
US10428019B2 (en) 2010-09-24 2019-10-01 Wave Life Sciences Ltd. Chiral auxiliaries
US8642751B2 (en) 2010-12-15 2014-02-04 Miragen Therapeutics MicroRNA inhibitors comprising locked nucleotides
US10280192B2 (en) 2011-07-19 2019-05-07 Wave Life Sciences Ltd. Methods for the synthesis of functionalized nucleic acids
WO2013022984A1 (en) * 2011-08-11 2013-02-14 Isis Pharmaceuticals, Inc. Selective antisense compounds and uses thereof
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US9752142B2 (en) 2011-08-11 2017-09-05 Ionis Pharmaceuticals, Inc. Gapped oligomeric compounds comprising 5′-modified deoxyribonucleosides in the gap and uses thereof
US10202599B2 (en) 2011-08-11 2019-02-12 Ionis Pharmaceuticals, Inc. Selective antisense compounds and uses thereof
US11732261B2 (en) 2011-08-11 2023-08-22 Ionis Pharmaceuticals, Inc. Selective antisense compounds and uses thereof
WO2013033223A1 (en) 2011-08-29 2013-03-07 Isis Pharmaceuticals, Inc. Methods and compounds useful in conditions related to repeat expansion
US9976138B2 (en) 2011-08-29 2018-05-22 Ionis Pharmaceuticals, Inc. Methods and compounds useful in conditions related to repeat expansion
US10023861B2 (en) 2011-08-29 2018-07-17 Ionis Pharmaceuticals, Inc. Oligomer-conjugate complexes and their use
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WO2013033230A1 (en) 2011-08-29 2013-03-07 Isis Pharmaceuticals, Inc. Oligomer-conjugate complexes and their use
US9428749B2 (en) 2011-10-06 2016-08-30 The Board Of Regents, The University Of Texas System Control of whole body energy homeostasis by microRNA regulation
US10337005B2 (en) 2012-06-21 2019-07-02 MiRagen Therapeutics, Inc. Oligonucleotide-based inhibitors comprising locked nucleic acid motif
US9388408B2 (en) 2012-06-21 2016-07-12 MiRagen Therapeutics, Inc. Oligonucleotide-based inhibitors comprising locked nucleic acid motif
US9803202B2 (en) 2012-06-21 2017-10-31 MiRagen Therapeutics, Inc. Oligonucleotide-based inhibitors comprising locked nucleic acid motif
US10167309B2 (en) 2012-07-13 2019-01-01 Wave Life Sciences Ltd. Asymmetric auxiliary group
US11236335B2 (en) 2012-10-12 2022-02-01 Ionis Pharmaceuticals, Inc. Selective antisense compounds and uses thereof
US9463200B2 (en) 2013-02-28 2016-10-11 Oligomer Sciences Ab Cell-penetrating oligonucleotides
WO2014131892A1 (en) * 2013-02-28 2014-09-04 Oligomer Sciences Ab Cell-penetrating oligonucleotides
WO2014172698A1 (en) * 2013-04-19 2014-10-23 Isis Pharmaceuticals, Inc. Compositions and methods for modulation nucleic acids through nonsense mediated decay
US10590412B2 (en) 2013-04-19 2020-03-17 Ionis Pharmaceuticals, Inc. Compositions and methods for modulation nucleic acids through nonsense mediated decay
US9957504B2 (en) 2013-05-01 2018-05-01 Ionis Pharmaceuticals, Inc. Compositions and methods for modulating apolipoprotein (a) expression
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US9181549B2 (en) 2013-05-01 2015-11-10 Isis Pharmaceuticals, Inc. Conjugated antisense compounds and their use
US11299736B1 (en) 2013-05-01 2022-04-12 Ionis Pharmaceuticals, Inc. Conjugated antisense compounds and their use
US9127276B2 (en) 2013-05-01 2015-09-08 Isis Pharmaceuticals, Inc. Conjugated antisense compounds and their use
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WO2014205451A2 (en) 2013-06-21 2014-12-24 Isis Pharmaceuticals, Inc. Compositions and methods for modulation of target nucleic acids
EP3564374A1 (en) 2013-06-21 2019-11-06 Ionis Pharmaceuticals, Inc. Compositions and methods for modulation of target nucleic acids
US10435430B2 (en) 2013-07-31 2019-10-08 Ionis Pharmaceuticals, Inc. Methods and compounds useful in conditions related to repeat expansion
US10160969B2 (en) 2014-01-16 2018-12-25 Wave Life Sciences Ltd. Chiral design
US10221416B2 (en) 2014-04-24 2019-03-05 Ionis Pharmaceuticals, Inc. Oligomeric compounds comprising alpha-beta-constrained nucleic acid
WO2015164693A1 (en) 2014-04-24 2015-10-29 Isis Pharmaceuticals, Inc. Oligomeric compounds comprising alpha-beta-constrained nucleic acid
US11732265B2 (en) 2014-05-01 2023-08-22 Ionis Pharmaceuticals, Inc. Compositions and methods for modulating complement factor B expression
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US10875884B2 (en) 2014-05-01 2020-12-29 Isis Pharmaceuticals, Inc. Compositions and methods for modulating angiopoietin-like 3 expression
US10280423B2 (en) 2014-05-01 2019-05-07 Ionis Pharmaceuticals, Inc. Compositions and methods for modulating complement factor B expression
US9382540B2 (en) 2014-05-01 2016-07-05 Isis Pharmaceuticals, Inc Compositions and methods for modulating angiopoietin-like 3 expression
US9885042B2 (en) 2015-01-20 2018-02-06 MiRagen Therapeutics, Inc. miR-92 inhibitors and uses thereof
US10280422B2 (en) 2015-01-20 2019-05-07 MiRagen Therapeutics, Inc. MiR-92 inhibitors and uses thereof
EP3929293A3 (en) * 2015-04-03 2022-03-16 University Of Massachusetts Fully stabilized asymmetric sirna
US11319536B2 (en) 2015-11-06 2022-05-03 Ionis Pharmacueticals, Inc. Modulating apolipoprotein (a) expression
US10557137B2 (en) 2015-11-06 2020-02-11 Ionis Pharmaceuticals, Inc. Modulating apolipoprotein (a) expression
US11896669B2 (en) 2016-01-31 2024-02-13 University Of Massachusetts Branched oligonucleotides
US10246709B2 (en) 2016-03-07 2019-04-02 Arrowhead Pharmaceuticals, Inc. Targeting ligands for therapeutic compounds
US10294474B2 (en) 2016-09-02 2019-05-21 Arrowhead Pharmaceuticals, Inc. Targeting ligands
US11174481B2 (en) 2016-09-02 2021-11-16 Arrowhead Pharmaceuticals, Inc. Targeting ligands
US11400161B2 (en) 2016-10-06 2022-08-02 Ionis Pharmaceuticals, Inc. Method of conjugating oligomeric compounds
JP2020522510A (en) * 2017-06-02 2020-07-30 ウェイブ ライフ サイエンシズ リミテッドＷａｖｅ Ｌｉｆｅ Ｓｃｉｅｎｃｅｓ Ｌｔｄ． Oligonucleotide composition and method of using the same
US11597927B2 (en) 2017-06-02 2023-03-07 Wave Life Sciences Ltd. Oligonucleotide compositions and methods of use thereof
US11560563B2 (en) 2018-04-05 2023-01-24 Silence Therapeutics Gmbh SiRNAs with vinylphosphonate at the 5′ end of the antisense strand
US11827882B2 (en) 2018-08-10 2023-11-28 University Of Massachusetts Modified oligonucleotides targeting SNPs
US11166976B2 (en) 2018-11-08 2021-11-09 Aligos Therapeutics, Inc. S-antigen transport inhibiting oligonucleotide polymers and methods
WO2020158910A1 (en) 2019-02-01 2020-08-06 国立大学法人大阪大学 5'-modified nucleoside and nucleotide using same
WO2020166551A1 (en) 2019-02-13 2020-08-20 国立大学法人大阪大学 5'-modified nucleoside and nucleotide using same
US11702659B2 (en) 2021-06-23 2023-07-18 University Of Massachusetts Optimized anti-FLT1 oligonucleotide compounds for treatment of preeclampsia and other angiogenic disorders

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