WO2013022966A1 - Linkage modified gapped oligomeric compounds and uses thereof - Google Patents

Linkage modified gapped oligomeric compounds and uses thereof Download PDF

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WO2013022966A1
WO2013022966A1 PCT/US2012/049987 US2012049987W WO2013022966A1 WO 2013022966 A1 WO2013022966 A1 WO 2013022966A1 US 2012049987 W US2012049987 W US 2012049987W WO 2013022966 A1 WO2013022966 A1 WO 2013022966A1
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certain embodiments
oligomeric compound
nucleoside
modified
nucleosides
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PCT/US2012/049987
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French (fr)
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Michael OESTERGAARD
Thazha P. Prakash
Punit P. Seth
Eric E. SWAYZ
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Isis Pharmaceuticals, Inc.
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Application filed by Isis Pharmaceuticals, Inc. filed Critical Isis Pharmaceuticals, Inc.
Priority to US14/237,967 priority Critical patent/US20140303235A1/en
Priority to EP12748812.0A priority patent/EP2742135B2/en
Priority to DK12748812.0T priority patent/DK2742135T4/en
Priority to ES12748812T priority patent/ES2635866T5/en
Publication of WO2013022966A1 publication Critical patent/WO2013022966A1/en
Priority to US15/407,037 priority patent/US20170130224A1/en

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Definitions

  • the present invention pertains generally to chemically-modified oligonucleotides for use in research, diagnostics, and/or therapeutics.
  • Antisense compounds have been used to modulate target nucleic acids. Antisense compounds comprising a variety of chemical modifications and motifs have been reported. In certain instances, such compounds are useful as research tools, diagnostic reagents, and as therapeutic agents. In certain instances antisense compounds have been shown to modulate protein expression by binding to a target messenger R A (mR A) encoding the protein. In certain instances, such binding of an antisense compound to its target mRNA results in cleavage of the mRNA. Antisense compounds that modulate processing of a pre-mRNA have also been reported. Such antisense compounds alter splicing, interfere with polyadenlyation or prevent formation of the 5 ' -cap of a pre-mRN A.
  • mR A target messenger R A
  • DNA or RNA containing oligonucleotides comprising alkylphosphonate internucleoside linkage backbone have been disclosed (see US patents 5,264,423 and 5,286,717).
  • Gogoi et al presented the synthesis of thioacetamido nucleic acids (TANA) backbone and thermal stability studies with complementary DNA and RNA sequences (Gogoi et al, Chem. Commun., 2006, 2373-2375).
  • nitrogen containing backbones include oxime (Sanghvi et al, In Nucleosides and Nucleotides as Antitumor and
  • HMIM methyliminomethylene
  • Oligomeric compounds have been prepared using Click chemistry wherein alkynyl phosphonate intemucleoside linkages on an oligomeric compound attached to a solid support are converted into the 1,2,3-triazolylphosphonate intemucleoside linkages and then cleaved from the solid support (Krishna et al, J. Am. Chem. Soc, DOI: 101021/ja3026714, published online May 21, 2012).
  • gapped oligomeric compounds comprising at least one modified intemucleoside linkage having Formula I in the gap region.
  • the oligomeric compounds provided herein hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.
  • the oligomeric compounds disclosed herein provide improved selectivity for a target RNA.
  • the oligomeric compounds provide improved potency for a target RNA.
  • the oligomeric compounds provided herein provide an improvement in the toxicity profile.
  • the oligomeric compounds provided herein provide an improvement in the proinflammatory profile.
  • the oligomeric compounds provide improved potency and selectivity for a target RNA.
  • the oligomeric compounds provide improved potency, selectivity and an improvement in the proinflammatory profile.
  • the variables are defined individually in further detail herein. It is to be understood that the oligomeric compounds provided herein include all combinations of the embodiments disclosed and variables defined herein.
  • gapped oligomeric compounds comprising a contiguous sequence of linked monomer subunits having a gap region located between a 5 '-region and a 3 '-region wherein the 5' and 3'-regions each, independently, have from 2 to 8 contiguous modified nucleosides wherein essentially each modified nucleoside in the 5' and 3 '-regions are RNA-like and the gap region has from 6 to 14 contiguous monomer subunits selected from ⁇ - ⁇ -2'- deoxyribonucleosides and modified nucleosides that are DNA-like and wherein at least one of the intemucleoside linking groups in the gap region or linking the gap region and the 5'-region or the 3'- region has Formula I:
  • X is O or S
  • each J] is, independently, H or C C 6 alkyl.
  • X is O or S
  • each J] is, independently, H or Cj-C 6 alkyl.
  • gapped oligomeric compounds consisting of a contiguous sequence of linked monomer subunits having a gap region located between a 5 '-region and a 3'-region wherein the 5' and 3'-regions each, independently, have from 2 to 8 contiguous modified nucleosides wherein each modified nucleoside in the 5' and 3 '-regions are RNA-like and the gap region has from 6 to 14 contiguous monomer subunits selected from P-D-2'-deoxyribo- nucleosides and modified nucleosides that are DNA-like and wherein at least one of the
  • internucleoside linking groups in the gap region or linking the gap region and the 5'-region or the 3'- region has Formula I:
  • X is O or S
  • gapped oligomenc compounds consisting of a contiguous sequence of linked monomer subunits having a gap region located between a 5'-region and a 3 '-region wherein the 5' and 3 '-regions each, independently, have from 2 to 8 contiguous modified nucleosides wherein each modified nucleoside in the 5' and 3 '-regions are RNA-like and the gap region has from 6 to 14 contiguous monomer subunits selected from P-D-2'-deoxyribo- nucleosides and wherein at least one of the intemucleoside linking groups in the gap region or linking the gap region and the 5'-region or the 3'-region has Formula I:
  • X is O or S
  • each Ji is, independently, H or Q-C6 alkyl.
  • gapped oligomenc compounds are provided having only one intemucleoside linking group of Formula I in the gap region. In certain embodiments, gapped oligomenc compounds are provided having only two intemucleoside linking groups of Formula I in the gap region. In certain embodiments, gapped oligomenc compounds are provided having only three intemucleoside linking groups of Formula I in the gap region. In certain embodiments, the intemucleoside linking groups having Formula I are contiguous. In certain embodiments, gapped oligomenc compounds are provide having at least two intemucleoside linking groups of Formula I in the gap region that are separated by at least one phosphorothioate or phosphodiester
  • gapped oligomeric compounds are provided wherein the intemucleoside linking group linking the 5 '-region and the gap region has Formula I. In certain embodiments, gapped oligomeric compounds are provided wherein the intemucleoside linking group linking the 5'-region and the gap region and the adjacent intemucleoside linkage in the gap region has Formula I. In certain embodiments, the intemucleoside linking group linking the 3'- region and the gap region has Formula I. In certain embodiments, gapped oligomeric compounds are provided wherein the intemucleoside linking group linking the 3 -region and the gap region and the adjacent intemucleoside linkage in the gap region has Formula I.
  • the intemucleoside linking group linking the 5 -region and the gap region has Formula I and the intemucleoside linking group linking the 3'-region and the gap region has Formula I. In certain embodiments, only one intemucleoside linking group of Formula I is located in the gap region.
  • each intemucleoside linking group in the 5' and 3'-regions and each intemucleoside linking group in the gap region other than intemucleoside linking groups having Formula I is a phosphodiester or a phosphorothioate intemucleoside linking group.
  • each intemucleoside linking group in the 5' and 3 '-regions and each intemucleoside linking group in the gap region other than intemucleoside linking groups having Formula I is a phosphorothioate intemucleoside linking group.
  • each intemucleoside linking group in the 5' and 3 '-regions and each intemucleoside linking group in the gap region other than intemucleoside linking groups having Formula I is a phosphodiester intemucleoside linking group.
  • each monomer subunit comprises a heterocyclic base independently selected from an optionally protected purine, substituted purine, pyrimidine or substituted pyrimidine.
  • each monomer subunit comprises a heterocyclic base independently selected from uracil, thymine, cytosine, 4-N- benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine or 2-N-isobutyrylguanine.
  • each Q is C3 ⁇ 4. In certain embodiments, each Q is C ⁇ CH.
  • gapped oligomeric compounds are provided wherein each X is O. In certain embodiments, each X is S.
  • gapped oligomeric compounds are provided wherein the chirality of each intemucleoside linking group having Formula I is Rp. In certain embodiments, the chirality of each intemucleoside linking group having Formula I is Sp. In certain embodiments, each modified nucleoside in the 5' and 3'-regions provides enhanced hybridization affinity for an RNA target as compared to an unmodified nucleoside. In certain embodiments, each modified nucleoside in the 5' and 3 -regions comprises a modified sugar moiety.
  • each modified nucleoside in the 5' and 3'-regions is, independently, a bicyclic nucleoside comprising a bicyclic furanosyl sugar moiety or a modified nucleoside comprising a furanosyl sugar moiety having at least one substituent group.
  • each bridging group is 4'-CH[(S)-(CH 3 )]-0-2'.
  • gapped oligomeric compounds are provided wherein the sugar moieties of each modified nucleoside in the 5' and 3'-regions are the same. In certain embodiments, gapped oligomeric compounds are provided comprising at least two different types of modified nucleosides in the 5' and 3'-regions wherein the different types of modified nucleosides have at least different modified sugar moieties. In certain embodiments, the different types of modified nucleosides include bicyclic nucleosides comprising bicyclic furanosyl sugar moieties and modified nucleosides comprising furanosyl sugar moieties having at least one substituent group.
  • the different types of modified nucleosides include 4'-CH[(S)-(CH3)]-0-2' bicyclic nucleosides and 2'-0(CH2) 2 -OCH3 substituted nucleosides.
  • the 5' and 3'- regions include only 4'-CH[(S)-(CH 3 )]-0-2' bicyclic nucleosides and 2'-0(CH 2 )2-OCH 3 substituted nucleosides.
  • gapped oligomeric compounds wherein one or more modified nucleosides in the 5' and 3 -regions comprise a sugar surrogate.
  • gapped oligomeric compounds are provided wherein each monomer subunit in the gap region is a p-D-2'-deoxyribonucleoside. In certain embodiments, at least one monomer subunit in the gap region is a modified nucleoside that is DNA-like. In certain embodiments,
  • each modified nucleoside that is DNA-like is a 2'-(ara)-F modified nucleoside.
  • gapped oligomeric compounds are provided wherein the 5' and 3'- regions each, independently, have from 2 to 8 monomer subunits. In certain embodiments, the 5' and 3 -regions each, independently, have from 3 to 6 monomer subunits. In certain embodiments, the gap region has from 6 to 14 monomer subunits. In certain embodiments, the gap region has from 8 to 10 monomer subunits. In certain embodiments, the 5' and 3'-regions each, independently, have from 3 to 6 monomer subunits and the gap region has from 8 to 14 monomer subunits. In certain embodiments, the 5' and 3'-regions each, independently, have from 3 to 6 monomer subunits and the gap region has from 6 to 10 monomer subunits.
  • the 5' and 3'- regions each, independently, have from 3 to 6 monomer subunits and the gap region has from 6 to 8 monomer subunits. In certain embodiments, the 5' and 3'-regions each, independently, have from 4 to 5 monomer subunits and the gap region has from 7 to 8 monomer subunits.
  • gapped oligomeric compounds wherein at least one modified nucleoside in the 5' and 3 -regions is other than a 2'-OCH 3 substituted nucleoside or a 2'-0- CH 2 -4' bridged bicyclic nucleoside.
  • gapped oligomeric compounds are provided that include one 5'- terminal group. In certain embodiments, gapped oligomeric compounds are provided that include one 3 '-terminal group. In certain embodiments, gapped oligomeric compounds are provided that include at least one 5' or 3'-terminal group.
  • the gapped oligomeric compounds provided herein are other than the gapped oligomeric compounds listed below:
  • each internucleoside linkage is a phosphorothioate.
  • Each " X T" is a 2-thio-thymidine modified nucleoside.
  • Nucleosides not followed by a subscript are ⁇ - ⁇ -2'- deoxyribonucleosides.
  • Nucleosides followed by a subscript “e” are 2'-0-methoxyethyl (MOE) modified nucleosides.
  • Nucleosides followed by a subscript "k” are 6'-(2> -03 ⁇ 4 (cEt) bicyclic modified nucleosides.
  • Each " m C” is a 5-methyl cytosine modified nucleoside.
  • the gapped oligomeric compounds provided herein are other than gapped oligomeric compounds complementary to at least a region of a nucleic acid that is a
  • the gapped oligomeric compounds provided herein are other than gapped oligomeric compounds complementary to at least a region of a nucleic acid comprising a single-nucleotide polymorphism. In certain embodiments, the gapped oligomeric compounds provided herein are other than gapped oligomeric compounds complementary to at least a region of a nucleic acid comprising a single-nucleotide polymorphism-containing-target nucleic acid of a Huntingtin gene transcript.
  • the gapped oligomeric compounds provided herein are other than gapped oligomeric compounds complementary to at least a region of a nucleic acid comprising a single-nucleotide polymorphism-containing-target nucleic acid of a gene transcript other than Huntingtin.
  • methods of inhibiting gene expression comprising contacting one or more cells, a tissue or an animal with an oligomeric compound as provided herein wherein said oligomeric compound is complementary to a target RNA.
  • the cells are in a human.
  • the target RNA is human mRNA.
  • the target RNA is cleaved thereby inhibiting its function.
  • in vitro methods of inhibiting gene expression comprising contacting one or more cells or a tissue with an oligomeric compound as provided herein.
  • oligomeric compound as provided herein are used in an in vivo method of inhibiting gene expression said method comprising contacting one or more cells, a tissue or an animal with an oligomeric compound as provided herein.
  • oligomeric compounds as provided herein are used in medical therapy.
  • Such gapped oligomeric compounds comprise a contiguous sequence of linked monomer subunits having a gap region located between a 5 '-region and a 3 '-region wherein the 5' and 3 '-regions each, independently, have from 2 to 8 contiguous modified nucleosides wherein essentially each modified nucleoside in the 5' and 3 '-regions are RNA-like and the gap region has from 6 to 14 contiguous monomer subunits selected from P-D-2'-deoxyribonucleosides and modified nucleosides that are DNA-like and wherein at least one of the intemucleoside linking groups in the gap region or between the gap region and the 5 '-region or the 3 '-region has Formula I:
  • X is O or S
  • each J] is, independently, H or Ci-Ce alkyl.
  • the gapped oligomeric compounds provided herein have been shown to have improved properties.
  • the activity of an otherwise unmodified gapped oligomeric compound against a target nucleic acid is enhanced by incorporation of at least one intemucleoside linking group having Formula I in the gap region.
  • at least one intemucleoside linking group having Formula I is added to the gap region.
  • intemucleoside linking group having Formula I is located at the gap junction on the 5' side wherein the internucleoside linkage separates the gap region from the wing region.
  • at least one internucleoside linking group having Formula I is located at the gap junction on the 3' side.
  • a gapped oligomeric compound of interest is identified and then a series of identical oligomeric compounds are prepared with a single internucleoside linking group having Formula I walked across the gap region. If there are 8 monomer subunits in the gap then there will be 8 oligomeric compounds prepared having the internucleoside linking group having Formula I located at a different position in each of the oligomeric compounds which are
  • additional internucleoside linking groups having Formula I are incorporated into the gap region of the lead oligomeric compound and assayed in one or more assays as illustrated herein.
  • the lead compound is further functionalized with one or more terminal groups such as for example a conjugate group.
  • a gapped oligomeric compound of interest is identified and then a series of identical oligomeric compounds are prepared with blocks of at least two internucleoside linking group having Formula I walked across the gap region.
  • gapped oligomeric compounds are provided having a reduced proinflammatory response when compared to unmodified gapped oligomeric compounds.
  • gapped oligomeric compounds are provided having enhanced potency (IC 50 ) and selectivity when compared to unmodified gapped oligomeric compounds.
  • IC 50 enhanced potency
  • selectivity when compared to unmodified gapped oligomeric compounds.
  • gapped oligomeric compounds are provided having enhanced selectivity with comparable potency (ICso) when compared to unmodified gapped oligomeric compounds.
  • ICso comparable potency
  • gapped oligomeric compounds are provided having enhanced selectivity with comparable potency (IC 50 ) when compared to an unmodified gapped oligomeric compound.
  • IC 50 comparable potency
  • gapped oligomeric compounds are provided having improved hepatotoxicity profiles when compared to unmodified gapped oligomeric compounds.
  • the activity and organ weights are similar for the SRB-1 assay. The activity is reduced for the modified oligomeric compound in the PTEN assay.
  • the liver is slightly elevated for the modified gapped oligomeric compound whereas for the unmodified oligomeric compound the liver and the spleen weights are both elevated.
  • chemical modification means a chemical difference in a compound when compared to a naturally occurring counterpart.
  • Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence.
  • furanosyl means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.
  • naturally occurring sugar moiety means a ribofuranosyl as found in naturally occurring RNA or a 2'-deoxyribofuranosyl as found in naturally occurring DNA.
  • sugar moiety means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.
  • modified sugar moiety means a substituted sugar moiety or a sugar surrogate.
  • substituted sugar moiety means a furanosyl that is not a naturally occurring sugar moiety.
  • Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2'-position, the 3'-position, the 5'-position and/or the 4'-position.
  • Certain substituted sugar moieties are bicyclic sugar moieties.
  • 2 '-substituted sugar moiety means a furanosyl comprising a substituent at the 2 '-position other than H or OH. Unless otherwise indicated, a 2 '-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2 '-substituent of a 2 '-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring.
  • MOE means -OCH 2 CH 2 OCH 3 .
  • 2'-F nucleoside refers to a nucleoside comprising a sugar comprising fluorine at the 2' position. Unless otherwise indicated, the fluorine in a 2'-F nucleoside is in the ribo position (replacing the OH of a natural ribose).
  • 2'-(ara)-F refers to a 2'-F substituted nucleoside, wherein the fluoro group is in the arabino position.
  • sucrose surrogate means a structure that does not comprise a furanosyl ring and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside sub-units or monomer subunits are capable of linking together and/or linking to other nucleosides or other monomer subunits to form an oligomeric compound which is capable of hybridizing to a complementary oligomeric compound such as a nucleic acid target.
  • Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen atom of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen, wherein replacement of the oxygen atom with sulfur in furanose is generally considered a modified nucleoside as opposed to a sugar surrogate but can be considered both); or both a change in the number of atoms and a replacement of the oxygen.
  • Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents).
  • Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid).
  • Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols.
  • bicyclic sugar moiety means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure.
  • the 4 to 7 membered ring is a sugar ring.
  • the 4 to 7 membered ring is a furanosyl.
  • the bridge connects the 2'-carbon and the 4'-carbon of the furanosyl.
  • nucleoside means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.
  • nucleotide means a nucleoside further comprising a phosphate linking group.
  • linked nucleosides may or may not be linked by phosphate linkages and thus includes, but is not limited to “linked nucleotides.”
  • linked nucleosides are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked).
  • nucleobase generally refers to the nucleobase of a nucleoside or modified nucleoside.
  • heterocyclic base moiety is broader than the term nucleobase in that it includes any heterocyclic base that can be attached to a sugar to prepare a nucleoside or modified nucleoside.
  • heterocyclic base moieties include but are not limited to naturally occurring nucleobases (adenine, guanine, thymine, cytosine and uracil) and protected forms of unmodified nucleobases (4-N-benzoylcytosine, 6-N-benzoyladenine and 2-N-isobutyrylguanine) as well as modified (5 -methyl cytosine) or non-naturally occurring heterocyclic base moieties and synthetic mimetics thereof (such as for example phenoxazines).
  • nucleobases adenine, guanine, thymine, cytosine and uracil
  • protected forms of unmodified nucleobases (4-N-benzoylcytosine, 6-N-benzoyladenine and 2-N-isobutyrylguanine)
  • modified (5 -methyl cytosine) or non-naturally occurring heterocyclic base moieties and synthetic mimetics thereof such as for example phenoxazines
  • modified nucleoside refers to a nucleoside comprising a modified heterocyclic base and or a sugar moiety other than ribose and 2'-deoxyribose.
  • a modified nucleoside comprises a modified heterocyclic base moiety.
  • a modified nucleoside comprises a sugar moiety other than ribose and 2'-deoxyribose.
  • a modified nucleoside comprises a modified heterocyclic base moiety and a sugar moiety other than ribose and 2'-deoxyribose.
  • the term "modified nucleoside” is intended to include all manner of modified nucleosides that can be incorporated into an oligomeric compound using standard oligomer synthesis protocols. Modified nucleosides include abasic nucleosides but in general a heterocyclic base moiety is included for hybridization to a complementary nucleic acid target.
  • modified nucleosides include a furanose or modified furanose sugar group such as a 4'-S analog (4'-S-modified nucleoside and 4'-S-ribonucleoside refer to replacement of the furanose oxygen atom with S).
  • modified nucleosides include without limitation, substituted nucleosides (such as 2 5', and/or 4' substituted nucleosides) 4'-S-modified nucleosides, (such as 4'-S-ribonucleosides, 4'-S-2'-deoxyribonucleosides and 4'-S-2'-substituted ribonucleosides), bicyclic modified nucleosides (such as 2 * -0-CH(CH 3 )-4*, 2'-0-CH 2 -4' or 2'-0-(CH 2 ) 2 -4* bridged furanose analogs) and base modified nucleosides.
  • substituted nucleosides such as 2 5', and/or 4' substituted nucleosides
  • 4'-S-modified nucleosides such as 4'-S-ribonucleosides, 4'-S-2'-deoxyribonucleosides and 4'-S-2'-substitute
  • the sugar can be modified with more than one of these modifications listed such as for example a bicyclic modified nucleoside further including a 5'- substitution or a 5' or 4' substituted nucleoside further including a 2' substituent.
  • modified nucleoside also includes combinations of these modifications such as base and sugar modified nucleosides. These modifications are meant to be illustrative and not exhaustive as other modifi- cations are known in the art and are also envisioned as possible modifications for the modified nucleosides described herein.
  • modified nucleosides comprise a sugar surrogate wherein the furanose ring has been replaced with a mono or polycyclic ring system or a non-cyclic sugar surrogate such as that used in peptide nucleic acids.
  • sugar moieties for such modified nucleosides includes without limitation morpholino, hexitol, cyclohexenyl, 2.2.2 and 3.2.1 cyclohexose and open non-cyclic groups.
  • modified nucleosides comprise a non-naturally occurring sugar moiety and a modified heterocyclic base moiety.
  • modified nucleosides include without limitation modified nucleosides wherein the heterocyclic base moiety is replaced with a
  • phenoxazine moiety for example the 9-(2-aminoethoxy)-l,3-diazaphenoxazine-2-one group, also referred to as a G-clamp which forms four hydrogen bonds when hybridized with a guanosine base
  • a sugar surrogate group such as for example a morpholino, a cyclohexenyl or a bicyclo[3.1.0]hexyl.
  • bicyclic nucleoside or "BNA” means a nucleoside comprising a bicyclic sugar moiety.
  • constrained ethyl nucleoside or “cEt” means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH 3 )-0-2' bridge.
  • the cEt comprises a comprising a 4'-CH((5)-CH3)-0-2' bridge.
  • the cEt comprises a comprising a 4'-CH((i?)-CH 3 )-0-2' bridge.
  • locked nucleic acid nucleoside or "LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH 2 -0-2'bridge.
  • 2 '-substituted nucleoside means a ribofuranosyl nucleoside comprising a substituent at the 2'-position other than H or OH. Unless otherwise indicated, a 2 '-substituted nucleoside is not a bicyclic nucleoside.
  • 2'-deoxynucleoside means a nucleoside comprising 2'-H(H) furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA).
  • a 2'-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).
  • RNA-like nucleoside means a modified nucleoside other than a ⁇ -D-ribose nucleoside that provides an A-form (northern) duplex when incorporated into an oligomeric compound and duplexed with a complementary RNA.
  • RNA-like nucleosides are used as replacements for RNA nucleosides in oligomeric compounds to enhance one or more properties such as, for example, nuclease resistance and or hybridization affinity.
  • RNA-like nucleosides include, but are not limited to modified furanosyl nucleosides that adopt a 3'-endo conformational geometry when put into an oligomeric compound.
  • RNA-like nucleosides also include RNA surrogates such as F-HNA.
  • RNA-like nucleosides include but are not limited to modified nucleosides comprising a 2'-substituent group selected from F, 0(CH2) 2 OCH3 (MOE) and OCH3.
  • RNA-like nucleosides also include but are not limited to modified nucleosides comprising bicyclic furanosyl sugar moiety comprising a 4*-CH 2 -0-2' 5 4'-(CH 2 ) 2 -0-2*, 4'-C(H)[(R)-CH 3 ]-0-2* or 4'- C(H)[(S)-CH 3 ]-0-2* bridging group.
  • DNA-like nucleoside means a modified nucleoside other than a ⁇ - ⁇ -2'- doxyribose nucleoside that provides a B-form (southern) duplex when incorporated into an oligomeric compound and duplexed with a complementary DNA.
  • DNA-like nucleosides provide an intermediate duplex when incorporated into an oligomeric compound and duplexed with a complementary RNA that is between A-form and B-form.
  • DNA -like nucleosides are used as replacements for DNA nucleosides in oligomeric compounds to enhance one or more properties.
  • DNA-like nucleosides include, but are not limited to modified nucleosides that adopt a 2'-endo conformational geometry when put into an oligomeric compound.
  • oligonucleotide means a compound comprising a plurality of linked nucleosides.
  • an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.
  • oligonucleoside means an oligonucleotide in which none of the
  • oligonucleotides include oligonucleosides.
  • modified oligonucleotide means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
  • nucleoside linkage means a covalent linkage between adjacent nucleosides in an oligonucleotide.
  • naturally occurring internucleoside linkage means a 3' to 5' phosphodiester linkage.
  • modified internucleoside linkage means any internucleoside linkage other than a naturally occurring internucleoside linkage.
  • a monomer subunit is meant to include all manner of monomers that are amenable to oligomer synthesis.
  • a monomer subunit includes at least a sugar moiety having at least two reactive sites that can form linkages to further monomer subunits.
  • all monomer subunits include a heterocyclic base moiety that is hybridizable to a complementary site on a nucleic acid target.
  • Reactive sites on monomer subunits located on the termini of an oligomeric compound can be protected or unprotected (generally OH) or can form an attachment to a terminal group (conjugate or other group).
  • Monomer subunits include, without limitation, nucleosides and modified nucleosides.
  • monomer subunits include nucleosides such as ⁇ -D- ribonucleosides and p-D-2'-deoxyribnucleosides and modified nucleosides including but not limited to substituted nucleosides (such as 2', 5' and bis substituted nucleosides), 4'-S-modified nucleosides (such as 4'-S-ribonucleosides, 4'-S-2'-deoxyribonucleosides and 4'-S-2'-substituted ribonucleosides), bicyclic modified nucleosides (such as bicyclic nucleosides wherein the sugar moiety has a 2'-0-
  • CHR a -4' bridging group wherein R a is H, alkyl or substituted alkyl), other modified nucleosides and nucleosides having sugar surrogates.
  • conjugate means 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 pharmacodynamic,
  • conjugate linking group means any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound.
  • antisense compound means a compound comprising or consisting of an oligonucleotide or oligomeric compound wherein at least a portion of which is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity.
  • antisense activity means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid.
  • detecting or “measuring” means that a test or assay for detecting or measuring is performed. Such detection and/or measuring may result in a value 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.
  • detecttable and/or measureable activity means a statistically significant activity that is not zero.
  • essentially unchanged means little or no change in a particular parameter, particularly relative to another parameter which changes much more.
  • a parameter is essentially unchanged when it changes less than 5%.
  • a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold.
  • an antisense activity is a change in the amount of a target nucleic acid.
  • the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target nucleic acid does, but the change need not be zero.
  • expression means the process by which a gene ultimately results in a protein.
  • Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenlyation, addition of 5 '-cap), and translation.
  • target nucleic acid means a nucleic acid molecule to which an antisense compound hybridizes.
  • mRNA means an RNA molecule that encodes a protein.
  • pre-mRNA means an RNA transcript that has not been fully processed into mRNA. Pre-RNA includes one or more intron.
  • object RNA means an RNA molecule other than a target RNA, the amount, activity, splicing, and/or function of which is modulated, either directly or indirectly, by a target nucleic acid.
  • a target nucleic acid modulates splicing of an object RNA.
  • an antisense compound modulates the amount or activity of the target nucleic acid, resulting in a change in the splicing of an object RNA and ultimately resulting in a change in the activity or function of the object RNA.
  • microRNA means a naturally occurring, small, non-coding RNA that represses gene expression of at least one mRNA.
  • a microRNA represses gene expression by binding to a target site within a 3' untranslated region of an mRNA.
  • a microRNA has a nucleobase sequence as set forth in miRBase, a database of published microRNA sequences found at http://microrna.sanger.ac.ulc/sequences/.
  • 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 means 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.
  • a microRNA mimic is double-stranded.
  • differentiating nucleobase means a nucleobase that differs between two nucleic acids.
  • a target region of a target nucleic acid differs by 1-4 nucleobases from a non-target nucleic acid. Each of those differences is referred to as a differentiating nucleobase.
  • a differentiating nucleobase is a single-nucleotide polymorphism.
  • target-selective nucleoside means a nucleoside of an antisense compound that corresponds to a differentiating nucleobase of a target nucleic acid.
  • allelic pair means one of a pair of copies of a gene existing at a particular locus or marker on a specific chromosome, or one member of a pair of nucleobases existing at a particular locus or marker on a specific chromosome, or one member of a pair of nucleobase sequences existing at a particular locus or marker on a specific chromosome.
  • each allelic pair will normally occupy corresponding positions (loci) on a pair of homologous chromosomes, one inherited from the mother and one inherited from the father.
  • the organism or cell is said to be “homozygous” for that allele; if they differ, the organism or cell is said to be “heterozygous” for that allele.
  • Wild-type allele refers to the genotype typically not associated with disease or dysfunction of the gene product.
  • Melt allele refers to the genotype associated with disease or dysfunction of the gene product.
  • allelic variant means a particular identity of an allele, where more than one identity occurs.
  • an allelic variant may refer to either the mutant allele or the wild-type allele.
  • single nucleotide polymorphism or "SNP” means a single nucleotide variation between the genomes of individuals of the same species.
  • a SNP may be a single nucleotide deletion or insertion.
  • SNPs occur relatively frequently in genomes and thus contribute to genetic diversity. The location of a SNP is generally flanked by highly conserved sequences. An individual may be homozygous or heterozygous for an allele at each SNP site.
  • single nucleotide polymorphism site or “SNP site” refers to the nucleotides surrounding a SNP contained in a target nucleic acid to which an antisense compound is targeted.
  • targeting means the association of an antisense compound to a particular target nucleic acid molecule or a particular region of 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.
  • nucleobase complementarity or “complementarity” when in reference to nucleobases means 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 means 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 means a pair of nucleobases that do not form hydrogen bonds with one another.
  • complementary in reference to oligomeric compounds (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity under stringent conditions.
  • Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated.
  • complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary). In certain embodiments, complementary oligomeric compounds or regions are 80% complementary.
  • complementary oligomeric compounds or regions are 90% complementary. In certain embodiments, complementary oligomeric compounds or regions are 95% complementary. In certain embodiments, complementary oligomeric compounds or regions are 100%
  • mismatch means a nucleobase of a first oligomeric compound that is not capable of pairing with a nucleobase at a corresponding position of a second oligomeric compound, when the first and second oligomeric compound are aligned.
  • first and second oligomeric compounds may be oligonucleotides.
  • hybridization means 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 nucleobases.
  • oligomeric compound specifically hybridizes to more than one target site.
  • oligonucleotide or portion thereof means that each nucleobase of the oligonucleotide or portion thereof is capable of pairing with a nucleobase of a complementary nucleic acid or contiguous portion thereof.
  • complementary region comprises no mismatches or unhybridized nucleobases in either strand.
  • percent complementarity means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.
  • percent identity means the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.
  • modulation means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of a molecule, 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 a change in splice site selection of pre-mR A processing, resulting in a change in the absolute or relative amount of a particular splice- variant compared to the amount in the absence of modulation.
  • modification motif means a pattern of chemical 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 means a pattern of nucleoside modifications in an oligomeric compound or a region thereof.
  • the linkages of such an oligomeric compound may be modified or unmodified.
  • motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.
  • sugar motif means a pattern of sugar modifications in an oligomeric compound or a region thereof.
  • linkage motif means 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.
  • nucleobase modification motif means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence.
  • sequence motif means a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modifications and thus may have any combination of chemical modifications, including no chemical modifications.
  • nucleoside having a modification of a first type may be an unmodified nucleoside.
  • telomeres As used herein, “differently modified” mean chemical modifications or chemical substituents 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 nucleobases are not differently modified.
  • nucleoside comprising a 2'-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2'-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.
  • the same type of modifications refers to modifications that are the same as one another, including absence of modifications.
  • two unmodified DNA nucleoside have “the same type of modification,” even though the DNA nucleoside is unmodified.
  • Such nucleosides having the same type modification may comprise different nucleobases.
  • pharmaceutically acceptable carrier or diluent means 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.
  • substituted nucleoside and “substituent group,” means an atom or group that replaces the atom or group of a named parent compound.
  • a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2'-substuent is any atom or group at the 2'-position of a nucleoside other than H or OH).
  • Substituent groups can be protected or unprotected.
  • compounds of the present invention have substituents at one or at more than one position of the parent compound. Substituents 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.
  • substituted in reference to a chemical functional group means an atom or group of atoms differs from the atom or a group of atoms normally present in the named functional group.
  • a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group).
  • each Raa, R bb and Rcc is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, 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.
  • alkyl means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms.
  • alkyl groups include without limitation, 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 (C]-C 12 alkyl) with from 1 to about 6 carbon atoms being more preferred.
  • alkenyl means 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 without limitation, 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 means 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, without limitation, 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.
  • acyl means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula -C(0)-X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.
  • alicyclic means 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.
  • aliphatic means 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.
  • alkoxy means 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 without limitation, methoxy, ethoxy, propoxy, isopropoxy, rc-butoxy, sec- butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like.
  • Alkoxy groups as used herein may optionally include further substituent groups.
  • aminoalkyl means an amino substituted Ci-C alkyl radical.
  • the alkyl portion of the radical forms a covalent bond with a parent molecule.
  • the amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.
  • aralkyl and arylalkyl mean an aromatic group that is covalently linked to a C ! -Ci 2 alkyl radical.
  • the alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, 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.
  • aryl and aromatic mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings.
  • aryl groups include without limitation, 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.
  • heteroaryl and “heteroaromatic,” mean a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. 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 without limitation, 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.
  • oligomeric compound refers to a contiguous sequence of linked monomer subunits. Each linked monomer subunit normally includes a heterocyclic base moiety but monomer subunits also includes those without a heterocyclic base moiety such as abasic monomer subunits. At least some and generally most if not essentially all of the heterocyclic bases in an oligomeric compound are capable of hybridizing to a nucleic acid molecule, normally a preselected RNA target.
  • the term “oligomeric compound” therefore includes oligonucleotides, oligonucleotide analogs and oligonucleosides. It also includes polymers having one or a plurality of nucleosides having sugar surrogate groups.
  • oligomeric compounds comprise a plurality of monomer subunits independently selected from naturally occurring nucleosides, non-naturally occurring nucleosides, modified nucleosides and nucleosides having sugar surrogate groups.
  • oligomeric compounds are single stranded.
  • oligomeric compounds are double stranded comprising a double-stranded duplex.
  • oligomeric compounds comprise one or more conjugate groups and/or terminal groups.
  • the oligomeric compounds as provided herein can be modified by covalent attachment of one or more terminal groups to the 5' or 3'-terminal groups.
  • a terminal group can also be attached at any other position at one of the terminal ends of the oligomeric compound.
  • the terms "5'-terminal group”, “3'-terminal group”, “terminal group” and combinations thereof are meant to include useful groups known to the art skilled that can be placed on one or both of the terminal ends, including but not limited to the 5' and 3'-ends of an oligomeric compound respectively, for various purposes such as enabling the tracking of the oligomeric compound (a fluorescent label or other reporter group), improving the pharmacokinetics or pharmacodynamics of the oligomeric compound (such as for example: uptake and/or delivery) or enhancing one or more other desirable properties of the oligomeric compound (a group for improving nuclease stability or binding affinity).
  • 5' and 3'-terminal groups include without limitation, modified or unmodified nucleosides; two or more linked nucleosides that are independently, modified or unmodified; conjugate groups; capping groups; phosphate moieties; and protecting groups.
  • the present invention provides oligomeric compounds.
  • such oligomeric compounds comprise oligonucleotides optionally comprising one or more conjugate and/or terminal groups.
  • an oligomeric compound consists of an oligonucleotide.
  • oligonucleotides comprise one or more chemical modifications. Such chemical modifications include modifications of one or more nucleoside (including modifications to the sugar moiety and/or the nucleobase) and/or modifications to one or more internucleoside linkage.
  • modified nucleosides comprise a modified sugar moeity, a modified nucleobase, or both a modifed sugar moiety and a modified nucleobase.
  • compounds of the invention comprise one or more modifed nucleosides comprising a modifed sugar moiety.
  • Such compounds comprising one or more sugar- modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to an oligonucleotide comprising only nucleosides comprising naturally occurring sugar moieties.
  • modified sugar moieties are substitued sugar moieties.
  • modified sugar moieties are sugar surrogates.
  • Such sugar surogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.
  • modified sugar moieties are substituted sugar moieties comprising one or more non-bridging sugar substituents, including but not limited to substituents at the 2' and/or 5' positions.
  • sugar substituents suitable for the 2 '-position include, but are not limited to: 2'-F, 2'-OCH 3 ("OMe” or "O-methyl"), and 2'-0(CH 2 ) 2 OCH 3 (“MOE").
  • sugar substituents at the 5'-position include, but are not limited to:, 5'-methyl (R or S); 5 -vinyl, and 5'-methoxy.
  • substituted sugars comprise more than one non-bridging sugar substituent, for example, 2'-F-5'-methyl sugar moieties (see,e.g., PCT International Application WO 2008/101157, for additional 5', 2'-bis substituted sugar moieties and nucleosides).
  • Nucleosides comprising 2' -substituted sugar moieties are referred to as 2 '-substituted nucleosides.
  • These 2 -substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (N0 2 ), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
  • a 2'- substituted nucleoside comprises a sugar moiety comprising a 2'-substituent group selected from F, OCF 3 , 0-CH 3 , OCH 2 CH 2 OCH 3 , 0(CH 2 ) 2 SCH 3 , 0-(CH 2 ) 2 -0- N(CH 3 ) 2 , -0(CH 2 ) 2 0(CH 2 ) 2 N(CH 3 ) 2 , and 0-
  • a 2'- substituted nucleoside comprises a sugar moiety comprising a 2'-substituent group selected from F, 0-CH 3 , and OCH 2 CH 2 OCH 3 .
  • Certain modifed sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety.
  • the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms.
  • Examples of such 4' to 2' sugar substituents include, but are not limited to: -[C(Ra)(R b )] n -, -[C(Ra)(Rb)] n -0-, -C(R a R b )-N(R)-0- or, -C(R a Rb)-0-N(R)-; 4 , -CH 2 -2', 4'-(CH 2 ) 2 -2', 4'-(CH 2 ) 3 -2 , 4'-(CH 2 )-0-2* (LNA); 4'-(CH 2 )-S-2'; 4'- (CH 2 ) 2 -0-2' (ENA); 4'-CH(CH 3 )-0-2* (cEt) and 4'-CH(CH 2 OCH 3 )-0-2 , ,and analogs thereof (see, e.g., U.S.
  • x 0, 1, or 2;
  • n 1, 2, 3, or 4;
  • each R a and R b is, independently, H, a protecting group, hydroxyl, Cj-C 12 alkyl, substituted d-Ci 2 alkyl, C 2 -C 12 alkenyl, substituted C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, substituted C 2 -C 12 alkynyl, C 5 -C 20 aryl, substituted C 5 -C 2 o aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C 5 -C 7 alicyclic radical, substituted C 5 -C 7 alicyclic radical, halogen, OJi, NJ !
  • Bicyclic nucleosides include, but are not limited to, (A) a-L-Methyleneoxy (4'-CH 2 -0-2') BNA , (B) ⁇ -D-Methyleneoxy (4'-CH 2 -0-2') BNA (also referred to as locked nucleic acid or LNA), (C) Ethyleneoxy (4'-(CH 2 ) 2 -0-2') BNA , (D) Aminooxy (4'-CH 2 -0-N(R)-2') BNA, (E) Oxyamino (4'-CH 2 -N(R)-0-2') BNA, (F) Methyl(methyleneoxy) (4'-CH(CH 3 )-0-2') BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4'-CH 2 -
  • Bx is a nucleobase moiety and R is, independently, H, a protecting group, or C1-C12 alkyl.
  • bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration.
  • a nucleoside comprising a 4 '-2' methylene-oxy bridge may be in the a-L configuration or in the ⁇ -D configuration.
  • substituted sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5 '-substituted and 4'-2' bridged sugars), (see, PCT International Application WO 2007/134181, published on 11/22/07, wherein LNA is substituted with, for example, a 5'-methyl or a 5'-vinyl group).
  • bridging sugar substituent e.g., 5 '-substituted and 4'-2' bridged sugars
  • modified sugar moieties are sugar surrogates.
  • the oxygen atom of the naturally occuring sugar is substituted, e.g., with a sulfer, carbon or nitrogen atom.
  • such modified sugar moiety also comprises bridging and or non-bridging substituents as described above.
  • certain sugar surogates comprise a 4'-sulfer atom and a substitution at the 2 -position (see,e.g., published U.S. Patent Application US2005/0130923, published on June 16, 2005) and/or the 5' position.
  • carbocyclic bicyclic nucleosides having a 4 -2' bridge have been described (see, e.g., Freier et al, Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al, J. Org.
  • sugar surrogates comprise rings having other than 5-atoms.
  • a sugar surrogate comprises a six-membered tetrahydropyran.
  • Such tetrahydropyrans may be further modified or substituted.
  • Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) ⁇ see Leumann, CJ. Bioorg. & Med. Chem. (2002) 10:841- 854), fluoro H -HNA), and those compounds having Formula VII:
  • Bx is a nucleobase moiety
  • T 3 and T 4 are each, independently, an internucleoside linking group linking the
  • tetrahydropyran nucleoside analog to the antisense compound or one of T 3 and T 4 is an
  • internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T 3 and T 4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3'-terminal group;
  • qi > q2, q.3, q 4> q 5 , q 6 and q 7 are each, independently, H, Ci-C 6 alkyl, substituted 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 modified THP nucleosides of Formula VII are provided wherein qi > 2, q3, q4, q 5 , e and q 7 are each H. In certain embodiments, at least one of qi, q 2 , q 3 , q 4 , q 5 , q 6 and q 7 is other than H. In certain embodiments, at least one of q l5 q 2 , q 3 , q 4 , q 5 , q 6 and q 7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R ⁇ and R 2 is F.
  • R ⁇ is fluoro and R 2 is H
  • Ri is methoxy and R 2 is H
  • R ⁇ is
  • R 2 is H.
  • Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see, e.g., review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).
  • Patent Application US2005-0130923, published on June 16, 2005) or alternatively 5'-substitution of a bicyclic nucleic acid see PCT International Application WO 2007/134181, published on 11/22/07 wherein a 4'-CH 2 - 0-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group).
  • PCT International Application WO 2007/134181 published on 11/22/07 wherein a 4'-CH 2 - 0-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group.
  • carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al, J. Am. Chem. Soc. 2007, 129(26), 8362-8379).
  • the present invention provides oligonucleotides comprising modified nucleosides.
  • modified nucleotides may include modified sugars, modified nucleobases, and or modified linkages. The specific modifications are selected such that the resulting oligonucleotides possess desirable characteristics.
  • oligonucleotides comprise one or more RNA-like nucleosides.
  • oligonucleotides comprise one or more RNA-like nucleosides.
  • oligonucleotides comprise one or more DNA-like nucleosides.
  • the oligomeric compounds provided herein include RNA-like nucleosides that have been modified to influence the sugar conformation to have predominantly 3'- endo conformational geometry.
  • such modified nucleosides include synthetic modifications of the heterocyclic base moiety, the sugar moiety or both to induce a 3'-endo sugar conformation.
  • RNA-like nucleosides are selected from RNA surrogates such as including, but not limited to, F-HNA or cyclohexenyl nucleic acid. RNA-like nucleosides are used to replace and mimic RNA nucleosides in an oligomeric compound so that particular properties of the oligomeric compound can be enhanced.
  • RNA-like nucleosides are used in the 5' and 3 '-regions (wings) of gapped oligomeric compounds to improve stability in the presence of nucleases and also to increase the affinity for nucleic a nucleic acid target.
  • Other properties that can also be enhanced by using RNA-like nucleosides include but aren't limited to modulation of pharmacokinetic properties through modification of protein binding, protein off-rate, absorption and clearance as well as chemical stability and specificity of the oligomeric compound (affinity and specificity for enzymes as well as for complementary sequences); and increasing efficacy of RNA cleavage.
  • RNA-like nucleosides include modified nucleosides comprising one or more 2 3', 4' and 5' substituent groups, bicyclic nucleosides and RNA-surrogates.
  • RNA-like nucleosides also include but are not limited to modified nucleosides having a bicyclic furanosyl sugar moiety (bicyclic nucleosides) comprising a bridging group between the 4' and 2'-carbon atoms.
  • bicyclic nucleosides include, but are not limited to bridging groups consisting of from 1 to 3 linked biradical groups selected from O, S, NR a ,
  • each R a , R b and Rc is, independently, H, Ci-C 6 alkyl, Ci-Ce alkoxy, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • the bridging groups include, but are not limited to
  • the oligomeric compounds provided herein include DNA-like nucleosides that have been modified to influence the sugar conformation to have predominantly 2'- endo conformational geometry.
  • modified nucleosides can include synthetic modifications of the heterocyclic base moiety, the sugar moiety or both to induce the desired 2'-endo sugar conformation.
  • modified nucleosides are used to mimic RNA nucleosides so that particular properties of an oligomeric compound can be enhanced while maintaining the desirable 2'-endo conformational geometry.
  • DNA-like nucleosides include, but are not limited to 2 -substituted furanosyl nucleosides comprising: 2 -CH 2 , 2'-ara-CN, 2'-ara-F, 2'-ara-Br or 2'-ara-Cl, 2'-ara-N 3 , 2 - ara-OH, 2'-ara-0-CH 3 or 2'-dehydro-2'-ara-CH 3 .
  • nucleosides of the present invention comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present invention comprise one or more modifed nucleobases (heterocyclic base moieties).
  • a heterocyclic base moiety is any heterocyclic system that contains one or more atoms or groups of atoms capable of hydrogen bonding to a heterocyclic base of a nucleic acid.
  • nucleobase refers to purines, modified purines, pyrimidines and modified pyrimidines.
  • nucleobase refers to unmodified or naturally occurring nucleobases which include, but are not limited to, the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U) and analogs thereof such as 5-methyl cytosine.
  • nucleobase and heterocyclic base moiety also include optional protection for any reactive functional groups such as 4-N-benzoylcytosine, 4-N-benzoyl-5-methyl- cytosine, 6-N-benzoyladenine or 2-N-isobutyrylguanine.
  • heterocyclic base moieties include without limitation modified nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- halouracil and cytosine, 5-propynyl (-C ⁇ C-CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8- amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
  • heterocyclic base moieties include without limitation tricyclic pyrimidines such as l,3-diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one and 9-(2- aminoethoxy)-l,3-diazaphenoxazine-2-one (G-clamp).
  • Heterocyclic base moieties 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.
  • Further heterocyclic base moieties include without limitation those known to the art skilled (see for example: United States Patent No.
  • Modified polycyclic heterocyclic compounds useful as heterocyclic base moieties are disclosed in 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;
  • nucleosides may be linked together using any internucleoside linkage to form oligonucleotides.
  • the two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom.
  • Representative non- phosphorus containing internucleoside linking groups include, but are not limited to,
  • internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. 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 enantiomers, diastereomers, and other stereoisomer ⁇ configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), a or ⁇ such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
  • 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,
  • oligomeric compounds comprise or consist of oligonucleotides.
  • such oligonucleotides comprise one or more chemical modifications.
  • chemically modified oligonucleotides comprise one or more modified sugars.
  • chemically modified oligonucleotides comprise one or more modified nucleobases.
  • chemically modified oligonucleotides comprise one or more modified internucleoside linkages.
  • the chemical modifications (sugar modifications, nucleobase modifications, and/or linkage modifications) define a pattern or motif.
  • the patterns of chemical modifications of sugar moieties, internucleoside linkages, and nucleobases are each independent of one another.
  • an oligonucleotide may be described by its sugar modification motif, internucleoside linkage motif and/or nucleobase modification motif (as used herein, nucleobase modification motif describes the chemical modifications to the nucleobases independent of the sequence of nucleobases).
  • oligonucleotides comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar motif.
  • sugar motifs include but are not limited to any of the sugar modifications discussed herein.
  • the oligomeric compounds provided herein comprise a gapmer sugar motif, which comprises two external regions or “wings” and a central or internal region or “gap” (also referred to as 5'-region and 3'-region).
  • the three regions of a gapmer sugar motif (the 5 '-wing, the gap, and the 3 '-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap.
  • the sugar moieties of the nucleosides of each wing that are closest to the gap differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap.
  • the sugar moieties within the gap are the same as one another.
  • the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap.
  • the sugar moieties of the two wings are the same as one another (symmetric sugar gapmer).
  • the sugar moieties of the 5 '-wing differs from the sugar moieties of the 3 '-wing (asymmetric sugar gapmer).
  • the sugar moieties in the two wings are selected from at least two different types that are different from the sugar moieties in the gap and at least one of each are in each wing.
  • the term "gapped oligomeric compound” refers to an oligomeric compound having two external regions or wings and an internal region or gap (also referred to as 5'- region and 3'-region). The three regions form a contiguous sequence of monomer subunits with the sugar moieties of the external regions (wings) being different than the sugar moieties of the internal region (gap).
  • the sugar moieties of each monomer subunit within a particular region is essentially the same.
  • the sugar moieties of each monomer subunit within each wing region is selected independently from 2 different types of modified nucleosides.
  • the sugar moieties of each monomer subunit within each wing region is selected independently from 3 different types of modified nucleosides. In certain embodiments, the sugar moieties of each monomer subunit within each wing region is selected independently from 4 different types of modified nucleosides. In certain embodiments, the sugar moiety of essentially each monomer subunit within the internal region is essentially the same. In certain embodiments, the sugar moiety of each monomer subunit within the internal region is a ⁇ - D-2'-deoxyribonucleoside, a nucleoside that is DNA-like and/or a nucleoside that supports RNaseH when in the gap region.
  • each monomer subunit within a particular region has the same sugar moiety.
  • the gapmer is a symmetric gapmer and when the sugar moiety used in the 5'-external region is different from the sugar moiety used in the 3 '-external region, the gapmer is an asymmetric gapmer.
  • the external regions are small (each independently 2, 3, 4, 5 or about 6 monomer subunits) and the monomer subunits comprise non-naturally occurring sugar moieties with the internal region comprising P-D-2'-deoxyribonucleosides.
  • the external regions each, independently, comprise from 2 to about 8 monomer subunits having non-naturally occurring sugar moieties and the internal region comprises from 6 to 14 unmodified nucleosides.
  • the internal region or the gap generally comprises P-D-2'-deoxyribonucleosides but can comprise non-naturally occurring sugar moieties.
  • the heterocyclic base and intemucleoside linkage is independently variable at each position of a gapped oligomeric compound.
  • a gapped oligomeric compound can further include one or more additional groups including but not limited to capping groups, conjugate groups and other 5' or 3'-terminal groups.
  • gapped oligomeric compounds comprise an internal region of ⁇ -D- 2'-deoxyribonucleosides with a single intemucleoside linkage having Formula I. In certain embodiments, gapped oligomeric compounds comprise an internal region of P-D-2'-deoxyribonucleosides having two intemucleoside linkages having Formula I. In certain embodiments, gapped oligomeric compounds comprise an internal region of -D-2'-deoxyribonucleosides having three
  • the 5' and 3'- wing regions of gapped oligomeric compounds comprise modified nucleosides wherein all the sugar moieties have the same type of modification such as cEt or MOE.
  • the 5' and 3'-wing regions of gapped oligomeric compounds comprise two types of modified nucleosides having sugar moieties independently selected from 2'-substituted sugar moieties and furanosyl bicyclic sugar moieties.
  • the 5' and 3'- wing regions of gapped oligomeric compounds comprise two types of modified nucleosides having sugar moieties independently selected from 2'-MOE substituted sugar moieties and furanosyl bicyclic sugar moieties each having a 4'-CH((S)-CH3)-0-2' bridge.
  • gapped oligomeric compounds are provided that are from about 10 to about 30 monomer subunits in length. In certain embodiments, gapped oligomeric compounds are provided that are from about 12 to about 20 monomer subunits in length. In certain embodiments,
  • gapped oligomeric compounds are provided that are from about 14 to about 20 monomer subunits in length. In certain embodiments, gapped oligomeric compounds are provided that are from about 14 to about 18 monomer subunits in length.
  • oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined partem or nucleobases
  • oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3 '-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 3 '-end of the oligonucleotide. In certain such embodiments, the block is at the 5 '-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 5 '-end of the oligonucleotide.
  • nucleobase modifications are a function of the natural base at a particular position of an oligonucleotide.
  • each purine or each pyrimidine in an oligonucleotide is modified.
  • each adenine is modified.
  • each guanine is modified.
  • each thymine is modified.
  • each cytosine is modified.
  • each uracil is modified.
  • oligonucleotides comprise one or more nucleosides comprising a modified nucleobase.
  • oligonucleotides having a gapmer sugar motif comprise a nucleoside comprising a modified nucleobase.
  • one nucleoside comprising a modified nucleobases is in the gap of an oligonucleotide having a gapmer sugar motif.
  • the sugar is an unmodified 2'deoxynucleoside.
  • the modified nucleobase is selected from: a 2-thio pyrimidine and a 5-propyne pyrimidine
  • cytosine moieties in an oligonucleotide are 5-methyl cytosine moieties.
  • 5-methyl cytosine is not a "modified nucleobase.”
  • unmodified nucleobases include both cytosine residues having a 5-methyl and those lacking a 5 methyl.
  • the methylation state of all or some cytosine nucleobases is specified.
  • oligomeric compounds comprise nucleosides comprising modified sugar moieties and/or nucleosides comprising modified nucleobases.
  • Such motifs can be described by their sugar motif and their nucleobase motif separately or by their nucleoside motif, which provides positions or patterns of modified nucleosides (whether modified sugar, nucleobase, or both sugar and nucleobase) in an oligonucleotide.
  • the oligomeric compounds comprise or consist of a region having a gapmer nucleoside motif, which comprises two external regions or "wings" and a central or internal region or "gap.”
  • the three regions of a gapmer nucleoside motif (the 5 '-wing, the gap, and the 3'- wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties and/or nucleobases of the nucleosides of each of the wings differ from at least some of the sugar moieties and/or nucleobase of the nucleosides of the gap.
  • the nucleosides of each wing that are closest to the gap differ from the neighboring gap nucleosides, thus defining the boundary between the wings and the gap.
  • the nucleosides within the gap are the same as one another.
  • the gap includes one or more nucleoside that differs from one or more other nucleosides of the gap.
  • the nucleoside motifs of the two wings are the same as one another (symmetric gapmer).
  • the nucleoside motifs of the 5'-wing differs from the nucleoside motif of the 3 -wing (asymmetric gapmer).
  • the 5'- wing of a gapmer consists of 2 to 8 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 3 to 5 linked nucleosides. In certain
  • the 5'- wing of a gapmer consists of 4 or 5 linked nucleosides.
  • the 5'- wing of a gapmer consists of 1 to 4 linked nucleosides.
  • the 5'- wing of a gapmer consists of 1 to 3 linked nucleosides.
  • the 5'- wing of a gapmer consists of 1 or 2 linked nucleosides.
  • the 5'- wing of a gapmer consists of 2 to 4 linked nucleosides.
  • the 5'- wing of a gapmer consists of 2 or 3 linked nucleosides.
  • the 5'- wing of a gapmer consists of 3 or 4 linked nucleosides.
  • the 5'- wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 5'- wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 3 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 5 linked nucleosides.
  • the 5'- wing of a gapmer consists of 2 to 8 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain
  • the 5'- wing of a gapmer consists of 3 to 5 linked nucleosides.
  • the 5'- wing of a gapmer consists of 4 or 5 linked nucleosides.
  • the 5'- wing of a gapmer consists of 2 to 4 linked nucleosides.
  • the 5'- wing of a gapmer consists of 2 to 3 linked nucleosides.
  • the 5'- wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 3 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 5 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 6 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 7 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 8 linked nucleosides.
  • the 5'- wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 5'- wing of a gapmer comprises at least two bicyclic nucleosides. In certain embodiments, the 5'- wing of a gapmer comprises at least three bicyclic nucleosides. In certain embodiments, the 5'- wing of a gapmer comprises at least four bicyclic nucleosides. In certain embodiments, the 5'- wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 5'- wing of a gapmer comprises at least one LNA nucleoside.
  • each nucleoside of the 5'- wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 5'- wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 5 '- wing of a gapmer is a LNA nucleoside.
  • the 5'- wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 5'- wing of a gapmer comprises at least one 2'- substituted nucleoside. In certain embodiments, the 5'- wing of a gapmer comprises at least one 2'- MOE nucleoside. In certain embodiments, the 5'- wing of a gapmer comprises at least one 2'-OMe nucleoside. In certain embodiments, each nucleoside of the 5'- wing of a gapmer is a non-bicyclic modified nucleoside.
  • each nucleoside of the 5'- wing of a gapmer is a 2'- substituted nucleoside. In certain embodiments, each nucleoside of the 5'- wing of a gapmer is a 2'- MOE nucleoside. In certain embodiments, each nucleoside of the 5'- wing of a gapmer is a 2'-OMe nucleoside.
  • the 5'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5 '-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2 '-substituted nucleoside. In certain embodiments, the 5 '-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 5 '-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-OMe nucleoside.
  • the 5 '-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5 '-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2 '-substituted nucleoside. In certain embodiments, the 5 '-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-OMe nucleoside.
  • the 5'- wing of a gapmer has a nucleoside motif selected from among the following: ABBA; ABB; ABAA; AABAA; AAABAA; AAAABAA; AAAAABAA; AAABAA; AABAA; ABAB; AAABB; AAAAA; ABBC; AA; AAA; AAAA; AAAAB;
  • each A is a modified nucleoside of a first type
  • each B is a modified nucleoside of a second type
  • each C is a modified nucleoside of a third type.
  • such an oligomeric compound is a gapmer.
  • the 3 '-wing of the gapmer may comprise any nucleoside motif.
  • the 3'- wing of a gapmer consists of 2 to 8 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 3 to 5 linked nucleosides. In certain embodiments,
  • the 3'- wing of a gapmer consists of 4 or 5 linked nucleosides.
  • the 3'- wing of a gapmer consists of 1 to 4 linked nucleosides.
  • the 3'- wing of a gapmer consists of 1 to 3 linked nucleosides.
  • the 3'- wing of a gapmer consists of 1 or 2 linked nucleosides.
  • the 3'- wing of a gapmer consists of 2 to 4 linked nucleosides.
  • the 3'- wing of a gapmer consists of 2 or 3 linked nucleosides.
  • the 3'- wing of a gapmer consists of 3 or 4 linked nucleosides.
  • the 3'- wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 3'- wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 3 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 5 linked nucleosides.
  • the 3'- wing of a gapmer consists of 2 to 8 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain
  • the 3'- wing of a gapmer consists of 3 to 5 linked nucleosides.
  • the 3'- wing of a gapmer consists of 4 or 5 linked nucleosides.
  • the 3'- wing of a gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 2 to 3 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 3 linked nucleosides.
  • the 3 '- wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 5 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 6 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 7 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 8 linked nucleosides.
  • the 3 '- wing of a gapmer comprises at least one bicyclic nucleoside.
  • the 3'- wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 3'- wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 3'- wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 3'- wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 3'- wing of a gapmer is a LNA nucleoside.
  • the 3'- wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 3'- wing of a gapmer comprises at least two non- bicyclic modified nucleosides. In certain embodiments, the 3'- wing of a gapmer comprises at least three non-bicyclic modified nucleosides. In certain embodiments, the 3'- wing of a gapmer comprises at least four non-bicyclic modified nucleosides. In certain embodiments, the 3'- wing of a gapmer comprises at least one 2 '-substituted nucleoside.
  • the 3'- wing of a gapmer comprises at least one 2'-MOE nucleoside. In certain embodiments, the 3'- wing of a gapmer comprises at least one 2'-OMe nucleoside. In certain embodiments, each nucleoside of the 3'- wing of a gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 3'- wing of a gapmer is a 2 '-substituted nucleoside. In certain embodiments, each nucleoside of the 3'- wing of a gapmer is a 2'-MOE nucleoside. In certain embodiments, each nucleoside of the 3'- wing of a gapmer is a 2'-OMe nucleoside.
  • the 3 '-wing of a gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2 '-substituted nucleoside. In certain embodiments, the 3 '-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-OMe nucleoside.
  • the 3 '-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3 '-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2 '-substituted nucleoside. In certain embodiments, the 3 '-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3 '-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-OMe nucleoside.
  • the 3 '-wing of a gapmer comprises at least one LNA nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3 '-wing of a gapmer comprises at least one LNA nucleoside and at least one 2 '-substituted nucleoside. In certain embodiments, the 3 '-wing of a gapmer comprises at least one LNA nucleoside and at least one 2'- MOE nucleoside. In certain embodiments, the 3 '-wing of a gapmer comprises at least one LNA nucleoside and at least one 2'-OMe nucleoside.
  • the 3 '- wing of a gapmer has a nucleoside motif selected from among the following: ABB; ABAA; AAABAA, AAAAABAA; AABAA; AAAABAA; AAABAA; ABAB; AAAAA; AAABB; AAAAAAAA; AAAAAAA; AAAAAA; AAAAB; AAAA; AAA; AA; AB; ABBB; ABAB; AABBB; wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type.
  • an oligonucleotide comprises any 3 '-wing motif provided herein.
  • the 5 '-wing of the gapmer may comprise any nucleoside motif.
  • the gap of a gapmer consists of 6 to 20 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 14 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 12 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 or 7 linked nucleosides.
  • the gap of a gapmer consists of 7 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 or 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 or 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 linked nucleosides.
  • the gap of a gapmer consists of 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 11 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 12 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 13 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 14 linked nucleosides.
  • each nucleoside of the gap of a gapmer is a 2'-deoxynucleoside.
  • the gap comprises one or more modified nucleosides.
  • each nucleoside of the gap of a gapmer is a 2'-deoxynucleoside or is a modified nucleoside that is "DNA-like".
  • DNA-like means that the nucleoside has similar characteristics to DNA, such that a duplex comprising the gapmer and an RNA molecule is capable of activating RNase H.
  • modified nucleosides that are DNA-like are 2'-endo. For example, under certain conditions, 2'-(ara)-F have been shown to support RNase H activation, and thus is DNA-like and further has 2 -endo conformation geometry.
  • one or more nucleosides of the gap of a gapmer is not a 2'-deoxynucleoside and is not DNA-like. In certain such embodiments, the gapmer nonetheless supports RNase H activation (e.g., by virtue of the number or placement of the non-DNA nucleosides).
  • the gap comprise a stretch of unmodified 2'-deoxynucleosides interrupted by one or more modified nucleosides, thus resulting in three sub-regions (two stretches of one or more 2'-deoxynucleosides and a stretch of one or more interrupting modified nucleosides).
  • no stretch of unmodified 2'-deoxynucleosides is longer than 5, 6, or 7 nucleosides.
  • such short stretches is achieved by using short gap regions.
  • short stretches are achieved by interrupting a longer gap region.
  • a gapmer comprises a 5 '-wing, a gap comprising at least one internucleoside linkage of Formula I, and a 3' wing, wherein the 5 '-wing, gap, and 3' wing are independently selected from among those discussed above.
  • a gapmer has a 5 '-wing, a gap, and a 3 '-wing having features selected from among those listed in the following non-limiting table: Table 1
  • each A is a modified nucleoside of a first type
  • each B is a modified nucleoside of a second type
  • each D is a p-D-2'-deoxyribonucleoside or a nucleoside that is DNA-like.
  • Each gap region includes at least one internucleoside linkage of Formula I.
  • each A comprises a modified sugar moiety.
  • each A comprises a modified nucleobase selected from among 2-thio- thymidine nucleoside and 5-propyne uridine nucleoside.
  • each B comprises a modified sugar moiety.
  • each B comprises a 2' -substituted sugar moiety.
  • each B comprises a bicyclic sugar moiety.
  • each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, a- L-LNA, EN A and 2'-thio LNA.
  • each B comprises a modified nucleobase.
  • each B comprises a modified nucleobase selected from among 2-thio- thymidine nucleoside and 5-propyne uridine nucleoside.
  • At least one of A or B comprises a bicyclic sugar moiety, and the other comprises a 2 '-substituted sugar moiety.
  • one of A or B is an LNA nucleoside and the other of A or B comprises a 2 '-substituted sugar moiety.
  • one of A or B is a cEt nucleoside and the other of A or B comprises a 2 '-substituted sugar moiety.
  • one of A or B is an a-L-LNA nucleoside and the other of A or B comprises a 2 '-substituted sugar moiety.
  • one of A or B is an LNA nucleoside and the other of A or B comprises a 2' -MOE sugar moiety.
  • one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-MOE sugar moiety.
  • one of A or B is an a -L-LNA nucleoside and the other of A or B comprises a 2'- MOE sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2'-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-F sugar moiety. In certain embodiments, one of A or B is an a-L- LNA nucleoside and the other of A or B comprises a 2'-F sugar moiety.
  • A comprises a bicyclic sugar moiety, and B comprises a 2'- substituted sugar moiety.
  • A is an LNA nucleoside and B comprises a 2'- substituted sugar moiety.
  • A is a cEt nucleoside and B comprises a 2'- substituted sugar moiety.
  • A is an a-L-LNA nucleoside and B comprises a 2 '-substituted sugar moiety.
  • A comprises a bicyclic sugar moiety, and B comprises a 2' -MOE sugar moiety.
  • A is an LNA nucleoside and B comprises a 2'-MOE sugar moiety.
  • A is a cEt nucleoside and B comprises a 2' -MOE sugar moiety.
  • A is an a-L-LNA nucleoside and B comprises a 2' -MOE sugar moiety.
  • A comprises a bicyclic sugar moiety, and B comprises a 2'-F sugar moiety.
  • A is an LNA nucleoside and B comprises a 2'-F sugar moiety.
  • A is a cEt nucleoside and B comprises a 2'-F sugar moiety.
  • A is an a-L-LNA nucleoside and B comprises a 2'-F sugar moiety.
  • B comprises a bicyclic sugar moiety, and A comprises a 2' -MOE sugar moiety.
  • B is an LNA nucleoside and A comprises a 2'-MOE sugar moiety.
  • B is a cEt nucleoside and A comprises a 2'-MOE sugar moiety.
  • B is an a-L-LNA nucleoside and A comprises a 2'-MOE sugar moiety.
  • B comprises a bicyclic sugar moiety, and A comprises a 2'-F sugar moiety.
  • B is an LNA nucleoside and A comprises a 2'-F sugar moiety.
  • B is a cEt nucleoside and A comprises a 2'-F sugar moiety.
  • B is an a-L-LNA nucleoside and A comprises a 2'-F sugar moiety.
  • each A and B is, independently, a modified nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' bridge or a modified nucleoside comprising a 2'-OCH 2 CH OCH3 (MOE) substituent group.
  • each A and B is, independently, a modified nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' bridge or a modified nucleoside comprising a 2'-OCH 2 CH OCH3 (MOE) substituent group.
  • each A and B is, independently, a modified nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' bridge or a modified nucleoside comprising a 2'-OCH 2 CH OCH3 (MOE) substituent group.
  • each A and B is,
  • each A and B is, independently, a modified nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH[(i?)-(CH3)]-0-2' bridge or a modified nucleoside comprising a 2'-OCH 2 CH 2 OCH 3 (MOE) substituent group.
  • At least one modified nucleoside comprising a 2'-OCH 2 CH 2 OCH 3 (MOE) substituent group and at least one modified nucleoside comprising a 4'-CH(CH3)-0-2' bridge is located in each of the 3' and 5 * wings.
  • at least one modified nucleoside comprising a 2'-OCH 2 CH 2 OCH3 (MOE) substituent group and at least one modified nucleoside comprising a 4'-CH[(S)-(CH 3 )]-0-2' bridge is located in each of the 3' and 5' wings.
  • At least one modified nucleoside comprising a 2'-OCH 2 CH 2 OCH3 (MOE) substituent group and at least one modified nucleoside comprising a 4'-CH[(i?)-(CH 3 )]-0-2' bridge is located in each of the 3' and 5' wings.
  • MOE 2'-OCH 2 CH 2 OCH3
  • oligomeric compounds comprise modified internucleoside linkages arranged along the oligomeric compound or region thereof in a defined pattern or modified internucleoside linkage motif provided that at least one internucleoside linkage has Formula I.
  • internucleoside linkages are arranged in a gapped motif, as described above for nucleoside motif.
  • the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region.
  • the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate.
  • the nucleoside motif is independently selected, so such oligomeric compounds having a gapped intemucleoside linkage motif may or may not have a gapped nucleoside motif and if it does have a gapped nucleoside motif, the wing and gap lengths may or may not be the same.
  • oligomeric compounds comprise a region having an alternating intemucleoside linkage motif. In certain embodiments, oligomeric compounds of the present invention comprise a region of uniformly modified intemucleoside linkages. In certain such embodiments, the oligomeric compound comprises a region that is uniformly linked by
  • each intemucleoside linkage of the oligomeric compound is selected from phosphodiester and phosphorothioate.
  • each intemucleoside linkage of the oligomeric compound is selected from
  • At least one intemucleoside linkage is phosphorothioate.
  • at least one intemucleoside linkage of the oligomeric compound is selected from other than phosphodiester and phosphorothioate.
  • the oligomeric compound comprises at least 6 phosphorothioate intemucleoside linkages. In certain embodiments, the oligomeric compound comprises at least 8 phosphorothioate intemucleoside linkages. In certain embodiments, the oligomeric compound comprises at least 10 phosphorothioate intemucleoside linkages. In certain embodiments, the oligomeric compound comprises at least one block of at least 6 consecutive phosphorothioate intemucleoside linkages. In certain embodiments, the oligomeric compound comprises at least one block of at least 8 consecutive phosphorothioate intemucleoside linkages.
  • the oligomeric compound comprises at least one block of at least 10 consecutive phosphorothioate intemucleoside linkages. In certain embodiments, the oligomeric compound comprises at least one block of at least one 12 consecutive phosphorothioate intemucleoside linkages. In certain such embodiments, at least one such block is located at the 3' end of the oligomeric compound. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3' end of the oligomeric compound. In certain embodiments, each intemucleoside linkage a phosphorothioate intemucleoside linkage.
  • Modification motifs define oligonucleotides by nucleoside motif (sugar motif and nucleobase motif) and linkage motif.
  • nucleoside motif sucrose motif and nucleobase motif
  • linkage motif For example, certain oligonucleotides have the following modification motif: AAADDDDDDDDDBBB;
  • each A is a modified nucleoside comprising a 2' -substituted sugar moiety; each D is a p-D-2'-deoxyribonucleoside or a modified nucleoside having B form conformation geometry and each B is a modified nucleoside comprising a bicyclic sugar moiety wherein at least one
  • each A is a modified nucleoside of a first type
  • each B is a modified nucleoside of a second type
  • each D is a p-D-2'-deoxyribonucleoside or a nucleoside that is DNA-like.
  • Each gap region includes at least one internucleoside linkage of Formula I.
  • each A comprises a modified sugar moiety.
  • each B comprises a modified sugar moiety.
  • At least one of A or B comprises a bicyclic sugar moiety, and the other comprises a 2 '-substituted sugar moiety.
  • one of A or B is an LNA nucleoside and the other of A or B comprises a 2 '-substituted sugar moiety.
  • one of A or B is a cEt nucleoside and the other of A or B comprises a 2 '-substituted sugar moiety.
  • one of A or B is an a-L-LNA nucleoside and the other of A or B comprises a 2 '-substituted sugar moiety.
  • one of A or B is an LNA nucleoside and the other of A or B comprises a 2' -MOE sugar moiety.
  • one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-MOE sugar moiety.
  • one of A or B is an a -L-LNA nucleoside and the other of A or B comprises a 2'- MOE sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2'-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-F sugar moiety. In certain embodiments, one of A or B is an a-L- LNA nucleoside and the other of A or B comprises a 2'-F sugar moiety.
  • A comprises a bicyclic sugar moiety, and B comprises a 2'- substituted sugar moiety.
  • A is an LNA nucleoside and B comprises a 2'- substituted sugar moiety.
  • A is a cEt nucleoside and B comprises a 2'- substituted sugar moiety.
  • A is an a-L-LNA nucleoside and B comprises a 2' -substituted sugar moiety.
  • A comprises a bicyclic sugar moiety, and B comprises a 2' -MOE sugar moiety.
  • A is an LNA nucleoside and B comprises a 2'-MOE sugar moiety.
  • A is a cEt nucleoside and B comprises a 2' -MOE sugar moiety.
  • A is an a-L-LNA nucleoside and B comprises a 2' -MOE sugar moiety.
  • A comprises a bicyclic sugar moiety, and B comprises a 2'-F sugar moiety.
  • A is an LNA nucleoside and B comprises a 2'-F sugar moiety.
  • A is a cEt nucleoside and B comprises a 2'-F sugar moiety.
  • A is an a-L-LNA nucleoside and B comprises a 2'-F sugar moiety.
  • B comprises a bicyclic sugar moiety, and A comprises a 2' -MOE sugar moiety.
  • B is an LNA nucleoside and A comprises a 2'-MOE sugar moiety.
  • B is a cEt nucleoside and A comprises a 2'-MOE sugar moiety.
  • B is an a-L-LNA nucleoside and A comprises a 2'-MOE sugar moiety.
  • B comprises a bicyclic sugar moiety, and A comprises a 2'-F sugar moiety.
  • B is an LNA nucleoside and A comprises a 2'-F sugar moiety.
  • B is a cEt nucleoside and A comprises a 2'-F sugar moiety.
  • B is an a-L-LNA nucleoside and A comprises a 2'-F sugar moiety.
  • each A and B is, independently, a modified nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' bridge or a modified nucleoside comprising a 2 -OCH2CH2OCH3 (MOE) substituent group.
  • each A and B is, independently, a modified nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' bridge or a modified nucleoside comprising a 2 -OCH2CH2OCH3 (MOE) substituent group.
  • each A and B is, independently, a modified nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' bridge or a modified nucleoside comprising a 2 -OCH2CH2OCH3 (MOE) substituent group.
  • each A and B is,
  • each A and B is, independently, a modified nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH[(i?)-(CH3)]-0-2' bridge or a modified nucleoside comprising a 2'-OCH 2 CH 2 OCH3 (MOE) substituent group.
  • At least one modified nucleoside comprising a 2'-OCH 2 CH20CH3 (MOE) substituent group and at least one modified nucleoside comprising a 4'-CH(CH3)-0-2' bridge is located in each of the 3' and 5' wings.
  • at least one modified nucleoside comprising a 2'- ⁇ 2 ⁇ 2 ⁇ 3 (MOE) substituent group and at least one modified nucleoside comprising a 4'-CH[(S)-(CH 3 )]-0-2' bridge is located in each of the 3' and 5' wings.
  • At least one modified nucleoside comprising a 2 -OCH2CH2OCH3 (MOE) substituent group and at least one modified nucleoside comprising a 4'-CH[(i?)-(CH 3 )]-0-2' bridge is located in each of the 3' and 5' wings,
  • the present invention provides oligomeric compounds including oligonucleotides of any of a variety of ranges of lengths.
  • the invention provides oligomeric compounds or 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 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 and 30; provided that X ⁇ Y.
  • the invention provides oligomeric compounds which comprise oligonucleotides consisting of 10 to 1 1 , 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, 1 1 to 13, 11 to 14, 11 to 15, 1 1 to 16, 1 1 to 17, 11 to 18, 1 1 to 19, 11 to 20, 1 1 to 21, 1 1 to 22, 11 to 23, 11 to 24, 11 to 25, 1 1 to 26, 11 to 27, 11 to 28, 1 1 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 18, 13 to 19, 13 to 20,
  • oligomeric compound or oligonucleotide may, nonetheless further comprise additional other substituents.
  • an oligonucleotide comprising 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.
  • a gapmer oligonucleotide has any of the above lengths.
  • an oligonucleotide is described by an overall length range and by regions having specified lengths, and where the sum of specified lengths of the regions is less than the upper limit of the overall length range, the oligonucleotide may have additional nucleosides, beyond those of the specified regions, provided that the total number of nucleosides does not exceed the upper limit of the overall length range.
  • oligonucleotides of the present invention are characterized by their modification motif and overall length. In certain embodiments, such parameters are each independent of one another.
  • each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications.
  • the internucleoside linkages within the wing regions of a sugar-gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region.
  • sugar-gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications.
  • oligonucleotide motifs may be combined to create a variety of oligonucleotides.
  • oligonucleotide or oligomeric compound is silent with respect to one or more parameter, such parameter is not limited.
  • an oligomeric compound described only as having a gapmer sugar motif without further description may have any length, internucleoside linkage motif, and nucleobase modification motif. Unless otherwise indicated, all chemical modifications are independent of nucleobase sequence,
  • 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.
  • a conjugate group comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (5)-(+)- 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.
  • active drug substance for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (5)-(+)- pranoprofen, carprofen, dansylsarco
  • 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-l-carboxylate
  • linking groups include, but are not limited to, substituted Ci-Qo alkyl, substituted or unsubstituted C 2 -Q 0 alkenyl or substituted or unsubstituted C 2 -Q 0 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.
  • conjugate groups are near the 3 '-end.
  • conjugates are attached at the 3 'end of an oligomeric compound, but before one or more terminal group nucleosides.
  • conjugate groups are placed within a terminal group.
  • oligomeric compounds comprise an oligonucleotide.
  • an oligomeric compound comprises an oligonucleotide and one or more conjugate and/or terminal groups.
  • conjugate and/or terminal groups may be added to oligonucleotides having any of the motifs discussed above.
  • an oligomeric compound comprising an oligonucleotide having region of alternating nucleosides may comprise a terminal group.
  • oligomeric compounds provided herein are antisense compounds. Such antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, antisense compounds specifically hybridize to one or more target nucleic acid.
  • a specifically hybridizing antisense compound has a nucleobase sequence comprising a region having sufficient complementarity to a target nucleic acid to allow hybridization and result in antisense activity and insufficient complementarity to any non-target so as to avoid non-specific hybridization to any non-target nucleic acid sequences under conditions in which specific hybridization is desired (e.g., under physiological conditions for in vivo or therapeutic uses, and under conditions in which assays are performed in the case of in vitro assays).
  • the present invention provides antisense compounds comprising oligonucleotides that are fully complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are 95% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 90% complementary to the target nucleic acid.
  • such oligonucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, an antisense compound comprises a region that is fully
  • the region of full complementarity is from 6 to 14 nucleobases in length.
  • certain antisense compounds result in RNase H mediated cleavage of target nucleic acid.
  • RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex.
  • the "DNA" in such an R A:DNA duplex need not be unmodified DNA.
  • the invention provides antisense compounds that are sufficiently "DNA-like" to elicit RNase H activity.
  • DNA-like antisense compounds include, but are not limited to gapmers having unmodified deoxyfuranose sugar moieties in the nucleosides of the gap and modified sugar moieties in the nucleosides of the wings.
  • Antisense activities may be observed directly or indirectly.
  • observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid; a change in the ratio of splice variants of a nucleic acid or protein; and/or a phenotypic change in a cell or animal.
  • compounds comprising oligonucleotides having a gapmer nucleoside motif described herein have desirable properties compared to non-gapmer
  • oligonucleotides or to gapmers having other motifs In certain circumstances, it is desirable to identify motifs resulting in a favorable combination of potent antisense activity and relatively low toxicity. In certain embodiments, compounds of the present invention have a favorable therapeutic index (measure of potency divided by measure of toxicity).
  • antisense compounds provided herein are selective for a target relative to a non-target nucleic acid.
  • the nucleobase sequences of the target and non-target nucleic acids differ by no more than 4 differentiating nucleobases in the targeted region.
  • the nucleobase sequences of the target and non-target nucleic acids differ by no more than 3 differentiating nucleobases in the targeted region.
  • the nucleobase sequences of the target and non-target nucleic acids differ by no more than 2 differentiating nucleobases in the targeted region.
  • the nucleobase sequences of the target and non-target nucleic acids differ by a single differentiating nucleobase in the targeted region.
  • the target and non-target nucleic acids are transcripts from different genes.
  • the target and non-target nucleic acids are different alleles for the same gene.
  • antisense compounds are achieved, principally, by nucleobase complementarity. For example, if an antisense compound has no mismatches for a target nucleic acid and one or more mismatches for a non-target nucleic acid, some amount of selectivity for the target nucleic acid will result. In certain embodiments, provided herein are antisense compounds with enhanced selectivity (i.e. the ratio of activity for the target to the activity for non-target is greater).
  • a selective nucleoside comprises a particular feature or combination of features (e.g., chemical modification, motif, placement of selective nucleoside, and/or self- complementary region) that increases selectivity of an antisense compound compared to an antisense compound not having that feature or combination of features.
  • a feature or combination of features increases antisense activity for the target.
  • such feature or combination of features decreases activity for the target, but decreases activity for the non-target by a greater amount, thus resulting in an increase in selectivity.
  • a selective antisense compound comprises a modified nucleoside at that same position as a differentiating nucleobase (i.e., the selective nucleoside is modified). That
  • the modification may increase the difference in binding affinity of the antisense compound for the target relative to the non-target.
  • the chemical modification may increase the difference in RNAse H activity for the duplex formed by the antisense compound and its target compared to the RNase activity for the duplex formed by the antisense compound and the non- target.
  • the modification may exaggerate a structure that is less compatible for RNase H to bind, cleave and/or release the non-target.
  • Antisense compounds having certain specified motifs have enhanced selectivity, including, but not limited to motifs described above.
  • enhanced selectivity is achieved by oligonucleotides comprising any one or more of:
  • a modification motif comprising a long 5 '-wing (longer than 5, 6, or 7 nucleosides);
  • a modification motif comprising a long 3'-wing (longer than 5, 6, or 7 nucleosides);
  • a modification motif comprising a short gap region (shorter than 8, 7, or 6 nucleosides); and a modification motif comprising an interrupted gap region (having no uninterrupted stretch of unmodified 2'-deoxynucleosides longer than 7, 6 or 5).
  • selective antisense compounds comprise nucleobase sequence elements.
  • Such nucleobase sequence elements are independent of modification motifs. Accordingly, oligonucleotides having any of the motifs (modification motifs, nucleoside motifs, sugar motifs, nucleobase modification motifs, and/or linkage motifs) may also comprise one or more of the following nucleobase sequence elements.
  • a target region and a region of a non-target nucleic acid differ by 1-
  • selective antisense compounds have a nucleobase sequence that aligns with the non-target nucleic acid with 1-4 mismatches.
  • a nucleoside of the antisense compound that corresponds to a differentiating nucleobase of the target nucleic acid is referred to herein as a target-selective nucleoside.
  • selective antisense compounds having a gapmer motif align with a non-target nucleic acid, such that a target-selective nucleoside is positioned in the gap.
  • a target-selective nucleoside is the 1 st nucleoside of the gap from the 5' end.
  • a target-selective nucleoside is the 2 nd nucleoside of the gap from the 5' end. In certain embodiments, a target-selective nucleoside is the 3 rd nucleoside of the gap from the 5 '-end. In certain embodiments, a target-selective nucleoside is the 4 th nucleoside of the gap from the 5'-end. In certain embodiments, a target-selective nucleoside is the 5 th nucleoside of the gap from the 5 '-end. In certain embodiments, a target- selective nucleoside is the 6 rd nucleoside of the gap from the 5 '-end.
  • a target-selective nucleoside is the 8 th nucleoside of the gap from the 3 '-end. In certain embodiments, a target-selective nucleoside is the 7 th nucleoside of the gap from the 3 '-end. In certain
  • a target-selective nucleoside is the 6 th nucleoside of the gap from the 3 '-end. In certain embodiments, a target-selective nucleoside is the 5 th nucleoside of the gap from the 3 '-end. In certain embodiments, a target-selective nucleoside is the 4 th nucleoside of the gap from the 3'- end. In certain embodiments, a target-selective nucleoside is the 3 rd nucleoside of the gap from the 3 '-end. In certain embodiments, a target-selective nucleoside is the 2 nd nucleoside of the gap from the 3 '-end.
  • selective antisense compounds comprise one or more mismatched nucleobases relative to the target nucleic acid.
  • antisense activity against the target is reduced by such mismatch, but activity against the non-target is reduced by a greater amount.
  • selectivity is improved.
  • Any nucleobase other than the differentiating nucleobase is suitable for a mismatch.
  • the mismatch is specifically positioned within the gap of an oligonucleotide having a gapmer motif.
  • a mismatch relative to the target nucleic acid is at positions 1, 2, 3, 4, 5, 6, 7, or 8 from the 5 '-end of the gap region.
  • a mismatch relative to the target nucleic acid is at positions 9, 8, 7, 6, 5, 4, 3, 2, 1 of the antisense compounds from the 3 '-end of the gap region. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 1 , 2, 3, or 4 of the antisense compounds from the 5'-end of the wing region. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 4, 3, 2, or 1 of the antisense compounds from the 3 '-end of the wing region.
  • selective antisense compounds comprise a region that is not complementary to the target. In certain embodiments, such region is complementary to another region of the antisense compound. Such regions are referred to herein as self-complementary regions.
  • an antisense compound has a first region at one end that is complementary to a second region at the other end. In certain embodiments, one of the first and second regions is complementary to the target nucleic acid. Unless the target nucleic acid also includes a self-complementary region, the other of the first and second region of the antisense compound will not be complementary to the target nucleic acid.
  • certain antisense compounds have the following nucleobase motif:
  • each of A, B, and C are any nucleobase; A', B', and C are the complementary bases to A, B, and C, respectively; each X is a nucleobase complementary to the target nucleic acid; and two letters in parentheses (e.g., (X C)) indicates that the nucleobase is complementary to the target nucleic acid and to the designated nucleoside within the antisense oligonucleotide.
  • such antisense compounds are expected to form self-structure, which is disrupted upon contact with a target nucleic acid.
  • a single antisense compound may include any one, two, three, or more of: self-complementary regions, a mismatch relative to the target nucleic acid, a short nucleoside gap, an interrupted gap, and specific placement of the selective nucleoside.
  • antisense compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid.
  • the target nucleic acid is an endogenous RNA molecule.
  • the target nucleic acid is a non-coding RNA.
  • the target non-coding RNA is selected from: a long-non-coding RNA, a short non-coding RNA, an intronic RNA molecule, a snoRNA, a scaRNA, a microRNA (including pre-microRNA and mature microRNA), a ribosomal RNA, and promoter directed RNA.
  • the target nucleic acid encodes a protein.
  • the target nucleic acid is selected from: an mRNA and a pre-mRNA, including intronic, exonic and untranslated regions.
  • oligomeric compounds are at least partially complementary to more than one target nucleic acid.
  • antisense compounds of the present invention may mimic microRNAs, which typically bind to multiple targets.
  • the target nucleic acid is a nucleic acid other than a mature mRNA. In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA or a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA or an intronic region of a pre-mRNA. In certain embodiments, the target nucleic acid is a long non-coding RNA. In certain embodiments, the target RNA is an mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA.
  • the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron. In certain embodiments, the target nucleic acid is selected from among non-coding RNA, including exonic regions of pre-mRNA. In certain embodiments, the target nucleic acid is a ribosomal RNA (rRNA). In certain embodiments, the target nucleic acid is a non-coding RNA associated with splicing of other pre-mRNAs. In certain embodiments, the target nucleic acid is a nuclear-retained non-coding RNA.
  • rRNA ribosomal RNA
  • antisense compounds described herein are complementary to a target nucleic acid comprising a single-nucleotide polymorphism.
  • the antisense compound is capable of modulating expression of one allele of the single-nucleotide nucleic acid to a greater or lesser extent than it modulates another allele.
  • an antisense compound hybridizes to a single-nucleotide
  • the target nucleic acid is a Huntingtin gene transcript.
  • the target nucleic acid is a single-nucleotide polymorphism-containing-target nucleic acid of a
  • the target nucleic acid is not a Huntingtin gene transcript. In certain embodiments, the target nucleic acid is a single-nucleotide polymorphism- containing-target nucleic acid of a gene transcript other than Huntingtin. In certain embodiments, the target nucleic acid is any nucleic acid other than a Huntingtin gene transcript,
  • Embodiments of the present invention provide methods, compounds, and compositions for selectively inhibiting mRNA and protein expression of an allelic variant of a particular gene or DNA sequence.
  • the allelic variant contains a single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • a SNP is associated with a mutant allele.
  • a mutant SNP is associated with a disease.
  • a mutant SNP is associated with a disease, but is not causative of the disease.
  • mRNA and protein expression of a mutant allele is associated with disease.
  • the expressed gene product of a mutant allele results in aggregation of the mutant proteins causing disease. In certain embodiments, the expressed gene product of a mutant allele results in gain of function causing disease.
  • genes with an autosomal dominant mutation resulting in a toxic gain of function of the protein are the APP gene encoding amyloid precursor protein involved in Alzheimer's disease (Gene, 371: 68, 2006); the PrP gene encoding prion protein involved in Creutzfeldt- Jakob disease and in fatal familial insomnia (Nat. Med. 1997, 3: 1009 ); GFAP gene encoding glial fibrillary acidic protein involved in
  • Alexander disease J. Neurosci. 2006, 26:111623; alpha-synuclein gene encoding alpha-synuclein protein involved in Parkinson's disease (J. Clin. Invest. 2003, 111: 145); SOD-1 gene encoding the SOD-1 protein involved in amyotrophic lateral sclerosis (Science 1998, 281: 1851); atrophin-1 gene encoding atrophin-1 protein involved in dentato-rubral and pallido-luysian atrophy (DRPA) (Trends Mol. Med. 2001, 7: 479); SCA1 gene encoding ataxin-1 protein involved in spino-cerebellar ataxia- 1 (SCA1) (Protein Sci. 2003, 12: 953); PLP gene encoding proteolipid protein involved in
  • Pelizaeus-Merzbacher disease NeuroMol Med. 2007, 4: 73
  • DYT1 gene encoding torsinA protein involved in Torsion dystonia
  • alpha-B crystalline gene encoding alpha-B crystalline protein involved in protein aggregation diseases, including cardiomyopathy (Cell 2007, 130: 427); alpha 1 -antitrypsin gene encoding alpha 1 -antitrypsin protein involved in chronic obstructive pulmonary disease (COPD), liver disease and hepatocellular carcinoma (New Engl J Med.
  • COPD chronic obstructive pulmonary disease
  • Ltk gene encoding leukocyte tyrosine kinase protein involved in systemic lupus erythematosus (Hum. Mol. Gen. 2004, 13: 171); PCSK9 gene encoding PCSK9 protein involved in hypercholesterolemia (Hum Mutat. 2009, 30: 520); prolactin receptor gene encoding prolactin receptor protein involved in breast tumors (Proc. Natl. Assoc. Sci. 2008, 105: 4533); CCL5 gene encoding the chemokine CCL5 involved in COPD and asthma (Eur. Respir. J.
  • PTPN22 gene encoding PTPN22 protein involved in Type 1 diabetes, Rheumatoid arthritis, Graves disease, and SLE (Proc. Natl. Assoc. Sci. 2007, 104: 19767); androgen receptor gene encoding the androgen receptor protein involved in spinal and bulbar muscular atrophy or Kennedy's disease (J Steroid Biochem. Mol. Biol. 2008, 108: 245); CHMP4B gene encoding chromatin modifying protein-4B involved in progressive childhood posterior subcapsular cataracts (Am. J. Hum.
  • FXR / NR1H4 gene encoding Farnesoid X receptor protein involved in cholesterol gallstone disease, arthrosclerosis and diabetes (Mol. Endocrinol. 2007, 21: 1769); ABCA1 gene encoding ABCA1 protein involved in cardiovascular disease (Transl. Res. 2007, 149: 205); CaSR gene encoding the calcium sensing receptor protein involved in primary hypercalciuria (Kidney Int. 2007, 71: 1155); alpha-globin gene encoding alpha-globin protein involved in alpha-thallasemia (Science 2006, 312: 1215); httlpr gene encoding HTTLPR protein involved in obsessive compulsive disorder (Am.
  • AChR gene encoding acetylcholine receptor involved in congential myasthenic syndrome (Neurology 2004, 62: 1090); P2Y12 gene encoding adenosine diphosphate (ADP) receptor protein involved in risk of peripheral arterial disease (Circulation 2003, 108: 2971); LQT1 gene encoding LQT1 protein involved in atrial fibrillation (Cardiology 2003, 100: 109); RET protooncogene encoding RET protein involved in sporadic pheochromocytoma (J. Clin. Endocrinol. Metab.
  • CA4 gene encoding carbonic anhydrase 4 protein, CRX gene encoding cone-rod homeobox transcription factor protein, FSCN2 gene encoding retinal fascin homolog 2 protein, IMPDH1 gene encoding inosine monophosphate dehydrogenase 1 protein, NR2E3 gene encoding nuclear receptor subfamily 2 group E3 protein, NRL gene encoding neural retina leucine zipper protein, PRPF3 (RP18) gene encoding pre-mRNA splicing factor 3 protein, PRPF8 (RP13) gene encoding pre-mRNA splicing factor 8 protein, PRPF31 (RP11) gene encoding pre-mRNA splicing factor 31 protein, RDS gene encoding peripherin 2 protein, ROM1 gene encoding rod outer membrane protein 1 protein, RHO gene encoding rhodopsin protein, RPl gene encoding RPl protein, RPGR gene encoding retinitis pigmentosa GTP
  • the mutant allele is associated with any disease from the group consisting of Alzheimer's disease, Creutzfeldt- Jakob disease, fatal familial insomnia, Alexander disease, Parkinson's disease, amyotrophic lateral sclerosis, dentato-rubral and pallido-luysian atrophy DRPA, spino-cerebellar ataxia, Torsion dystonia, cardiomyopathy, chronic obstructive pulmonary disease (COPD), liver disease, hepatocellular carcinoma, systemic lupus erythematosus, hypercholesterolemia, breast cancer, asthma, Type 1 diabetes, Rheumatoid arthritis, Graves disease, SLE, spinal and bulbar muscular atrophy, Kennedy's disease, progressive childhood posterior subcapsular cataracts, cholesterol gallstone disease, arthrosclerosis, cardiovascular disease, primary hypercalciuria, alpha-thallasemia, obsessive compulsive disorder, Anxiety, comorbid depression, congenital visual defects
  • an allelic variant of huntingtin is selectively reduced.
  • Nucleotide sequences that encode huntingtin include, without limitation, the following: GENBANK Accession No. NT_006081.18, truncated from nucleotides 1566000 to 1768000 (replaced by GENBANK Accession No. NT_006051), incorporated herein as SEQ ID NO: 8, and NM_002111.6,
  • Table 3 provides SNPs found in the GM04022, GM04281 , GM02171 , and GM02173B cell lines. Also provided are the allelic variants found at each SNP position, the genotype for each of the cell lines, and the percentage of HD patients having a particular allelic variant. For example, the two allelic variants for SNP rs6446723 are T and C.
  • the GM04022 cell line is heterozygous TC
  • the GM02171 cell line is homozygous CC
  • the GM02173 cell line is heterozygous TC
  • the GM04281 cell line is homozygous TT.
  • Fifty percent of HD patients have a T at SNP position rs6446723.
  • such pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier.
  • a pharmaceutical composition comprises a sterile saline solution and one or more antisense compound.
  • such pharmaceutical composition consists of a sterile saline solution and one or more antisense compound.
  • the sterile saline is pharmaceutical grade saline.
  • a pharmaceutical composition comprises one or more antisense compound and sterile water.
  • a pharmaceutical composition consists of one or more antisense compound and sterile water.
  • the sterile saline is pharmaceutical grade water.
  • a pharmaceutical composition comprises one or more antisense compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile phosphate-buffered saline (PBS). In certain embodiments, the sterile saline is
  • antisense 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 depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • compositions comprising antisense compounds encompass any one of
  • compositions comprising antisense 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 antisense oligomeric compound.
  • Lipid moieties 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.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
  • compositions provided herein comprise one or more modified oligonucleotides and one or more excipients.
  • excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and
  • a pharmaceutical composition provided herein comprises a delivery system.
  • delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.
  • a pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types.
  • pharmaceutical compositions include liposomes coated with a tissue-specific antibody.
  • a pharmaceutical composition provided herein comprises a co- solvent system.
  • co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • such co-solvent systems are used for hydrophobic compounds.
  • a non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80TM and 65% w/v polyethylene glycol 300.
  • the proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics.
  • co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80TM; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • a pharmaceutical composition provided herein is prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration.
  • a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.).
  • a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives).
  • injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like.
  • compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers.
  • Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Certain solvents suitable for use in pharmaceutical are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.
  • Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.
  • a pharmaceutical composition is prepared for transmucosal administration.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • a pharmaceutical composition provided herein comprises an oligonucleotide in a therapeutically effective amount.
  • the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
  • one or more modified oligonucleotide provided herein is formulated as a prodrug.
  • a prodrug upon in vivo administration, is chemically converted to the biologically, pharmaceutically or therapeutically more active form of an
  • prodrugs are useful because they are easier to administer than the corresponding active form.
  • a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form.
  • a prodrug may have improved solubility compared to the corresponding active form.
  • prodrugs are less water soluble than the corresponding active form.
  • such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility.
  • a prodrug is an ester. In certain such
  • the ester is metabolically hydrolyzed to carboxylic acid upon administration.
  • the carboxylic acid containing compound is the corresponding active form.
  • a prodrug comprises a short peptide (polyaminoacid) bound to an acid group.
  • the peptide is cleaved upon administration to form the
  • the present invention provides compositions and methods for reducing the amount or activity of a target nucleic acid in a cell.
  • the cell is in an animal.
  • the animal is a mammal.
  • the animal is a rodent.
  • the animal is a primate.
  • the animal is a non-human primate.
  • the animal is a human.
  • the present invention provides methods of administering a pharmaceutical composition comprising an oligomeric compound of the present invention to an animal.
  • Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intracerebroventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous).
  • pharmaceutical intrathecals are administered to achieve local rather than systemic exposures.
  • pharmaceutical compositions may be injected directly in the area of desired effect (e.g., into the liver).
  • RNA nucleoside comprising a 2' -OH sugar moiety and a thymine base
  • RNA having a modified sugar 2' -OH for the natural 2'-H of DNA
  • RNA having a modified base thymine (methylated uracil) for natural uracil of RNA
  • nucleic acid sequences provided herein are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases.
  • an oligomeric compound having the nucleobase sequence "ATCGATCG” encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence "AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligomeric compounds having other modified or naturally occurring bases, such as "AT me CGAUCG,” wherein me C indicates a cytosine base comprising a methyl group at the 5-position.
  • 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.
  • oligomenc 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 intemucleoside linkages can be prepared as described in U.S. Patent 5,508,270.
  • Alkyl phosphonate intemucleoside linkages can be prepared as described in U.S. Patent 4,469,863.
  • 3 '-Deoxy-3' -methylene phosphonate intemucleoside linkages can be prepared as described in U.S. Patents 5,610,289 or 5,625,050.
  • Phosphoramidite intemucleoside linkages can be prepared as described in U.S. Patent, 5,256,775 or U.S. Patent 5,366,878.
  • Alkylphosphonothioate intemucleoside linkages can be prepared as described in published
  • 3'-Deoxy-3 '-amino phosphoramidate intemucleoside linkages can be prepared as described in U.S. Patent 5,476,925.
  • Phosphotriester intemucleoside linkages can be prepared as described in U.S. Patent
  • Borano phosphate intemucleoside 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
  • the oligomeric compounds including without limitation oligonucleotides and oligonucleosides, are recovered by precipitation out of 1 M NHUOAc 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.
  • 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
  • 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).
  • 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.
  • 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-l reduced serum medium (Invitrogen Life Technologies, Carlsbad, CA) to achieve the desired concentration of the oligomeric compound(s) and a
  • transfection reagents 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, LA
  • 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, LA
  • TAMRA obtained from either PE- Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, LA
  • 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 then- 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).
  • standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed 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 ⁇ , PCR cocktail (2.5x PCR buffer minus MgCl 2 , 6.6 mM MgCl 2 , 375 ⁇ 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 ⁇ , total RNA solution (20-200 ng).
  • PCR cocktail 2.5x PCR buffer minus MgCl 2 , 6.6 mM MgCl 2 , 375 ⁇ each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM
  • 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°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).
  • RIBOGREENTM working reagent 170 ⁇ , of RIBOGREENTM working reagent (RIBOGREENTM reagent diluted 1 :350 in lOmM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 ⁇ ⁇ 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. 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.
  • 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. Louis, MO), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, CA; Amersham Biosciences, Piscataway, NJ).
  • cells determined to be appropriate for a particular phenotypic assay i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies
  • 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 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.
  • Example 10 The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.
  • Example 10 The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.
  • Poly(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 xL cold
  • 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 ⁇ , 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 ⁇ . of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl).
  • the plate is blotted on paper towels to remove excess wash buffer and then air- dried for 5 minutes.
  • 60 of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70°C, is added to each well, the plate is incubated on a 90°C 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 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 ⁇ , cold PBS. 150 ⁇ , Buffer RLT is added to each well and the plate vigorously agitated for 20 seconds. 150 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 QIAVACTM manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum is applied for 1 minute.
  • Buffer RW1 500 ⁇ , of Buffer RW1 is added to each well of the RNEASY 96TM plate and incubated for 15 minutes and the vacuum is again applied for 1 minute.
  • An additional 500 ⁇ , of Buffer RW1 is added to each well of the RNEASY 96TM plate and the vacuum is applied for 2 minutes.
  • 1 mL of Buffer RPE is then added to each well of the RNEASY 96TM 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 QIAVACTM manifold and blotted dry on paper towels.
  • RNA is then eluted by pipetting 140 ⁇ , 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).
  • Forward primer AATGGCTAAGTGAAGATGACAATCAT (SEQ ID NO: 2)
  • Reverse primer TGCACATATCATTACACCAGTTCGT (SEQ ID NO: 3)
  • FAM-TTGCAGCAATTCACTGTAAAGCTGGAAAGG-TAMRA (SEQ ID NO: 4), where FAM is the fluorescent dye and TAMRA is the quencher dye.
  • Bx is a heterocyclic base
  • Rj and R 2 are each independently H, OH, or a 2'-sugar substituent group
  • Compounds 1 and 2 are commercially available from Glen Research.
  • Compounds 3 and 4 are prepared as per the procedures well known in the art as described in the specification herein (see Seth et a/., Bioorg. Med. Chem., 2011, 21(4), 1122-1125, J Org. Chem., 2010, 75(5), 1569-1581, Nucleic Acids Symposium Series, 2008, 52(1), 553-554); and also see published PCT International Applications (WO 2011/115818, WO 2010/077578, WO2010/036698, WO2009/143369, WO 2009/006478, and WO 2007/090071), and US patent 7,569,686).
  • Compound 9 are commercially available from Sigma-Aldrich, and BOC Biosciences.
  • Bx is a heterocyclic base
  • Compound 9 are commercially available from Sigma-Aldrich, and BOC Biosciences.
  • the Grignard reagent, Compound 11 is commercially available from various sources or can be prepared using similar procedures as published in the literature (Oppolzer et al, Tet. Let., 1982, 23(38), 3901-3904).
  • Bx is a heterocyclic base
  • R 4 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl or substituted acyl; wherein the substituent groups are independently selected from halogen, oxo, hydroxyl, amino, thio, azido, cyano or carboxyl;
  • Compound 9 are commercially available from Sigma-Aldrich, and BOC Biosciences.
  • the Grignard reagent, Compound 14 is commercially available from various sources or can be prepared as per the procedures illustrated in Example 15.
  • Compound 15 used in the phosphitylation step serves only to illustrate the compounds described herein and is not intended to be limiting. Additional phosphitylating reagents known in the art as described in the specification herein can also be employed to generate various phosphoramidite analogs of Compound 16 and are used as building blocks for oligonucleotide synthesis.
  • Bx is a heterocylic base
  • R! and R 2 are each independently H, OH or a 2'-sugar substituent group
  • X is O or S
  • the UnylinkerTM 22 is commercially available.
  • Oligomeric Compound 25 comprising a modified intemucleoside linkage e.g. methyl phosphonate, phosphonoacetate or phosphonoformate
  • a modified intemucleoside linkage e.g. methyl phosphonate, phosphonoacetate or phosphonoformate
  • Phosphoramidite building blocks, Compounds 1, 2, 3, 10 and 13 are prepared as per the procedures illustrated in Examples 13 to 15.
  • reagents and solutions used for the synthesis of oligomeric compounds are purchased from commercial sources.
  • Standard phosphoramidite building blocks and solid support are used for incorporation nucleoside residues which include for example T, A, U, G, C and m C residues.
  • a 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for ⁇ - D-2'-deoxyribonucleoside, methyl phosphonate and phosphonoacetate.
  • (R)- or f$-methyl phosphonate dimeric phosphoramidite a 0.1 M solution in acetonitrile was used.
  • the oligomeric compound was synthesized on VIMAD UnyLinkerTM solid support and the appropriate amounts of solid support were packed in the column for synthesis.
  • Dichloroacetic acid (3%) in DCM was used as detritylating reagent.
  • 4,5-Dicyanoimidazole in the presence of N-methyl- imidazole or lH-tetrazole in C3 ⁇ 4CN was used as activator during the coupling step.
  • the synthesis of oligomeric compounds was performed on an ABB 94 synthesizer (Applied Biosy stems) on a 2 ⁇ scale using the procedures set forth below.
  • a solid support preloaded with the UnylinkerTM was loaded into a synthesis column after closing the column bottom outlet and CH 3 CN was added to form a slurry.
  • the swelled support- bound UnylinkerTM was treated with a detritylating reagent containing 3% dichloroacetic acid in DCM to provide the free hydroxyl groups.
  • a detritylating reagent containing 3% dichloroacetic acid in DCM to provide the free hydroxyl groups.
  • four to fourteen equivalents of phosphoramidite solutions were delivered with coupling for 6 minutes for unmodified
  • Phosphodiester linkages were introduced by oxidation with 10% t-BuOOH solution in CH 3 CN for a contact time of 10 minutes.
  • Phosphorothioate linkages were introduced by sulfurization with PADS (0.2 M) in 1 :1 pyridine/CHaCN for a contact time of 5 minutes.
  • the cyanoethyl phosphate protecting groups were deprotected using a 1 :1 (v/v) mixture of triethylamine and acetonitrile.
  • phosphonoacetate containing oligomeric compound 1.5% DBU solution in C3 ⁇ 4CN was used.
  • the solid support bound oligomeric compound was washed with acetonitrile and dried under high vacuum.
  • the solid- support bound oligomeric compound was then suspended in ammonia (28-30 wt%) at room temperature for 48 h to remove nucleobase protecting groups and to cleave from the solid support.
  • the unbound oligomeric compound was then filtered and the support was rinsed and filtered with watenethanol (1:1) followed by water. The filtrate was combined and concentrated to dryness.
  • Each nucleoside followed by a subscript "d” is a P-D-2'-deoxyribonucleoside.
  • Each nucleoside followed by a subscript “e” indicates a 2'-0-methoxyethyl (MOE) modified nucleoside.
  • Each nucleoside followed by a subscript “k” indicates a bicyclic nucleoside having a 4'-CH((5)-CH 3 )-0-2' bridge also referred to as a (S)-cEt modified nucleoside.
  • Each " m C” is a 5-methyl cytosine modified nucleoside. Nucleosides followed by subscripts "e”, or "k ; are further illustrated below.
  • hPBMC Human Peripheral Blood Mononuclear Cells
  • the hPBMC assay was performed using BD Vautainer CPT tube method.
  • a sample of whole blood from volunteered donors with informed consent at US Health Works clinic (Faraday & El Camino Real, Carlsbad) was obtained and collected in 4-15 BD Vacutainer CPT 8 ml tubes (VWR Cat.# BD362753).
  • the approximate starting total whole blood volume in the CPT tubes for each donor was recorded using the PBMC assay data sheet.
  • the blood sample was remixed immediately prior to centrifugation by gently inverting tubes 8-10 times.
  • CPT tubes were centrifuged at rt (18-25 °C) in a horizontal (swing-out) rotor for 30 min. at 1500-1800 RCF with brake off (2700 RPM Beckman Allegra 6R).
  • the cells were retrieved from the buffy coat interface (between Ficoll and polymer gel layers); transferred to a sterile 50 ml conical tube and pooled up to 5 CPT tubes/50 ml conical tube/donor.
  • the cells were then washed twice with PBS (CSL ⁇ , Mg ⁇ free; GIBCO).
  • the tubes were topped up to 50 ml and mixed by inverting several times.
  • the sample was then centrifuged at 330 x g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R) and aspirated as much supernatant as possible without disturbing pellet.
  • the cell pellet was dislodged by gently swirling tube and resuspended cells in RPMI+10%
  • FBS+pen/strep ( ⁇ 1 ml / 10 ml starting whole blood volume).
  • a 60 ⁇ sample was pipette into a sample vial (Beckman Coulter) with 600 ⁇ VersaLyse reagent (Beckman Coulter Cat# A09777) and was gently vortexed for 10-15 sec. The sample was allowed to incubate for 10 min. at rt and being mixed again before counting. The cell suspension was counted on Vicell XR cell viability analyzer (Beckman Coulter) using PBMC cell type (dilution factor of 1 : 11 was stored with other parameters). The live cell/ml and viability were recorded. The cell suspension was diluted to 1 x 10' live PBMC/ml in RPMI+ 10% FBS+pen/strep.
  • the cells were plated at 5 x 10 5 in 50 ⁇ /well of 96-well tissue culture plate (Falcon
  • a modified oligonucleotide was designed based on the 3/14/3 MOE gapmer, ISIS 353512. This modified oligonucleotide was created by having alternating methyl thiophosphonate
  • the hPBMCs were isolated from fresh, volunteered donors and were treated with modified oligonucleotides at 0, 0.0128, 0.064, 0.32, 1.6, 8, 40 and 200 ⁇ concentrations. After a 24 hr treatment, the cytokine levels were measured.
  • Each nucleoside followed by a subscript "d” is a ⁇ - ⁇ -2'- deoxyribonucleoside.
  • Each nucleoside followed by a subscript “e” indicates a 2'-0-methoxyethyl (MOE) modified nucleoside.
  • Each " m C” is a 5-methyl cytosine modified nucleoside. Nucleosides followed by subscripts "e” are further illustrated below.
  • SNPs Single nucleotide polymorphisms in the huntingtin (HTT) gene sequence
  • SNP positions (identified by Hayden et al, WO/2009/135322) associated with the HTT gene were mapped to the HTT genomic sequence, designated herein as SEQ ID NO: 08 (NT_006081.18 truncated from nucleotides 1566000 to 1768000).
  • NCBI National Center for Biotechnology Information
  • the 'Reference SNP ID number' or 'RS number' is the number designated to each SNP from the Entrez SNP database at NCBI, incorporated herein by reference.
  • 'SNP position' refers to the nucleotide position of the SNP on SEQ ID NO: 08.
  • 'Polymorphism' indicates the nucleotide variants at that SNP position.
  • 'Major allele' indicates the nucleotide associated with the major allele, or the nucleotide present in a statistically significant proportion of individuals in the human population.
  • 'Minor allele' indicates the nucleotide associated with the minor allele, or the nucleotide present in a relatively small proportion of individuals in the human population.
  • SNPs Single Nuclear Polymorphisms
  • rs2857936 1963 C/T C T rsl2506200 3707 A/G G A rs762855 14449 A/G G A rs3856973 19826 G/A G A rs2285086 28912 G/A A G rs7659144 37974 C/G C G rsl 6843804 44043 C/T C T rs2024115 44221 G/A A G rsl0015979 49095 A G A G rs7691627 51063 A/G G A rs2798235 54485 G/A G A rs4690072 62160 G/T T G rs6446723 66466 C/T T C rs363081 73280 G/A G A rs363080 73564 T/C C T rs363075 77327 G/A G A rs363064 81063 T/C C T rs3025849 83420 A/G
  • rs362310 179429 A/G G
  • a rs362307 181498 T/C C T rs362306 181753 G/A G
  • rs362303 181960 T/C C T rs362296 186660 C/A C A rsl 006798 198026 A/G A G.
  • modified oligonucleotides were designed based on ISIS 460209 wherein the gap region contains nine P-D-2'-deoxyribonucleosides.
  • the modified oligonucleotides were designed by introducing a methyl phosphonate internucleoside linkage within the gap region.
  • the oligonucleotides with modified phosphorus containing backbone were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact.
  • oligonucleotides opposite to the SNP position, as counted from the 5' -terminus is position 8.
  • Heterozygous fibroblast GM04022 cell line was used for the in vitro assay (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 ⁇ concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C_2229297_10 which measures at dbSNP rs362303.
  • RT-PCR method in short; A mixture was made using 2020 ⁇ , 2X PCR buffer, 101 ⁇ , primers (300 ⁇ from ABI), 1000 ⁇ , water and 40.4 ⁇ , RT MIX. To each well was added 15 nL of this mixture and 5 ⁇ . of purified RNA. The mutant and wild-type H7TmRNA levels were measured simultaneously by using two different fiuorophores, FAM for mutant allele and VIC for wild-type allele. The H rmR A levels were adjusted according to total RNA content, as measured by RIBOGREEN. Analysis of IC50 's and Selectivity
  • the half maximal inhibitory concentration (IC50) of each oligonucleotide is presented below and was calculated by plotting the concentrations of oligonucleotides used versus the percent inhibition of HT mRNA expression achieved at each concentration, and noting the concentration of oligonucleotide at which 50% inhibition of HT mR A expression was achieved compared to the control.
  • the IC50 at which each oligonucleotide inhibits the mutant H7TmRNA expression is denoted as 'mut ⁇ 1 ⁇ 2 0 '.
  • the IC50 at which each oligonucleotide inhibits the wild-type H -TmRNA expression is denoted as 'wt IC 50 '.
  • Selectivity as expressed in "fold” was calculated by dividing the IC 50 for inhibition of the wild-type HTT versus the IC 50 for inhibiting expression of the mutant HTT mRNA and the results are presented below.
  • Each nucleoside followed by a subscript "d” is a P-D-2'-deoxyribonucleoside.
  • Each nucleoside followed by a subscript "e” indicates a 2'-0-methoxyethyl (MOE) modified nucleoside.
  • Each nucleoside followed by a subscript "k” indicates a bicyclic nucleoside having a 4'- CH((S)-CH 3 )-0-2' bridge also referred to as a (5)-cEt modified nucleoside.
  • Each ,,m C" is a 5-methyl cytosine modified nucleoside. Nucleosides followed by subscripts "e” or “k” are further illustrated below.
  • Modified oligonucleotides comprising methyl phosphonate or phosphonoacetate
  • a series of modified oligonucleotides were designed based on ISIS 460209 wherein the gap region contains nine P-D-2'-deoxyribonucleosides.
  • the modified oligonucleotides were synthesized to include one or more methyl phosphonate or phosphonoacetate internucleoside linkage modifications within the gap region.
  • the oligonucleotides with modified phosphorus containing backbone were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact.
  • the potency and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.
  • the modified oligonucleotides were tested in vitro.
  • Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute).
  • Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 ⁇ concentrations of modified oligonucleotides.
  • RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C_2229297_10 which measures at dbSNP rs362303.
  • RT-PCR method in short; A mixture was made using 2020 ⁇ , 2X PCR buffer, 101 ⁇ primers (300 ⁇ from ABI), 1000 uL water and 40.4 ⁇ , RT MIX. To each well was added 15 ⁇ , of this mixture and 5 ⁇ , of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HIT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.
  • Each nucleoside followed by a subscript “d” is a P-D-2'-deoxyribonucleoside.
  • Each nucleoside followed by a subscript “e” indicates a 2'-0-methoxyethyl (MOE) modified nucleoside.
  • Each nucleoside followed by a subscript “k” indicates a bicyclic nucleoside having a 4'-CH((S)-CH 3 )-0-2' bridge also referred to as a (5)-cEt modified nucleoside.
  • Each " m C” is a 5-methyl cytosine modified nucleoside. Nucleosides followed by subscripts "e” "k” are further illustrated below.
  • a series of modified oligonucleotide was designed based on ISIS 460209 or ISIS 476333, wherein the gap region contains nine P-D-2'-deoxyribonucleosides.
  • the modified oligonucleotides were designed by introducing methyl phosphonate internucleoside linkage within the gap region at a fixed position and using different wing motifs for 3/9/3 and 4/9/4 gapmer motifs.
  • the modified oligonucleotides were tested for their ability to selectively inhibit mutant (mut) H7TmRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact.
  • the potency and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209 or ISIS 476333.
  • the position on the oligonucleotides opposite to the SNP position, as counted from the 5'- terminus is position 8 for ISIS 460209 or position 9 for ISIS 476333.
  • the modified oligonucleotides were tested in vitro.
  • Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute).
  • Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.1, 0.4, 1.1, 3.3 and 10 ⁇ concentrations of modified oligonucleotides.
  • RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C_2229297_10 which measures at dbSNP rs362303.
  • RT-PCR method in short; A mixture was made using 2020 uL 2X PCR buffer, 101 uL primers (300 ⁇ from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type H7 mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.
  • Each nucleoside followed by a subscript "d” is a p-D-2'-deoxyribonucleoside.
  • Each nucleoside followed by a subscript "e” indicates a 2'-0-methoxyethyl (MOE) modified nucleoside.
  • Each nucleoside followed by a subscript “k” indicates a bicyclic nucleoside having a 4'- CH((5)-CH 3 )-0-2 * bridge also referred to as a (S)-cEt modified nucleoside.
  • Each " m C” is a 5-methyl cytosine modified nucleoside. Nucleosides followed by subscripts "e” or “k” are further illustrated below.
  • modified oligonucleotides were designed based on ISIS 482050 and 449093 wherein the gap region contains ten P-D-2'-deoxyribonucleosides.
  • the modified oligonucleotides were designed by introducing two methyl phosphonate internucleoside linkages at the 5 '-end of the gap region with a 3/10/3 motif.
  • the oligonucleotides were evaluated for reduction in PTEN and SRB-1 mRNA expression levels in vivo.
  • the parent gapmers, ISIS 482050 and 449093 were included in the study for comparison.
  • mice Six week old BALB/C mice (purchased from Charles River) were injected subcutaneously twice a week for three weeks at dosage 10 mg/kg or 20 mg/kg with the modified oligonucleotides shown below or with saline control. Each treatment group consisted of 3 animals. The mice were sacrificed 48 hrs following last administration, and organs and plasma were harvested for further analysis. mRNA Analysis
  • Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to RIBOGREEN as described herein. The results below are listed as PTEN or SRB-1 mRNA expression for each treatment group relative to saline-treated control (% UTC). As illustrated, reduction in PTEN or SRB-1 mRNA expression levels was achieved with the oligonucleotides comprising two methyl phosphonate internucleoside linkages at the 5 '-end of the gap region, ISIS 582073 and 582074.
  • Plasma chemistry markers such as liver transaminase levels, alanine aminotranferase (ALT) in serum were measured relative to saline injected mice and the results are presented below.
  • Body weights, as well as liver, kidney and spleen weights were measured at the end of the study. The results below are presented as the average percent of body and organ weights for each treatment group relative to saline-treated control. As illustrated, treatment with ISIS 582073 resulted in a reduction in liver and spleen weights compared to treatment with the parent gapmer, ISIS 482050. The remaining oligonucleotide, ISIS 582074 did not cause any changes in body and organ weights outside the expected range as compared to ISIS 449093.
  • Each nucleoside followed by a subscript "d” is a P-D-2'-deoxyribonucleoside.
  • Each nucleoside followed by a subscript "k” indicates a bicyclic nucleoside having a 4'-CH((£)-CH 3 )-0-2' bridge also referred to as a (S)-cEt modified nucleoside.
  • Each ,,m C" is a 5-methyl cytosine modified nucleoside. Nucleosides followed by subscript "k” are further illustrated below.
  • oligonucleotides were designed in the same manner as the antisense oligonucleotides described in Example 25, wherein two methyl phosphonate internucleoside linkages are introduced at the 5 '-end of the gap region.
  • the modified oligonucleotides were designed based on ISIS 464917, 465178, 465984 and 466456 with a 3/10/3 motif.
  • oligonucleotides were evaluated for reduction in Target-Y mRNA expression levels in vivo.
  • the parent gapmers, ISIS 464917, 465178, 465984 and 466456 were included in the study for comparison.
  • mice Six week old BALB/C mice (purchased from Charles River) were injected subcutaneously twice a week for three weeks at dosage 10 mg/kg or 20 mg/kg with the modified oligonucleotides shown below or with saline control. Each treatment group consisted of 3 animals. The mice were sacrificed 48 hrs following last administration, and organs and plasma were harvested for further analysis. mRNA Analysis
  • Target-Y mRNA expression for each treatment group relative to saline-treated control (% UTC). As illustrated, reduction in Target-Y mRNA expression levels was achieved with the oligonucleotides comprising two methyl phosphonate intemucleoside linkages at the 5 '-end of the gap region, ISIS 582071, 582072, 582069 and 582070.
  • Plasma chemistry markers such as liver transaminase levels, alanine aminotranferase (ALT) in serum were measured relative to saline treated mice and the results are presented below.
  • Body weights, as well as liver, kidney and spleen weights were measured at the end of the study. The results below are presented as the average percent of body and organ weights for each treatment group relative to saline-treated control. As illustrated, treatment with ISIS 582070 resulted in a reduction in liver and spleen weights compared to treatment with the parent gapmer, ISIS 466456. An increase in body and organ weights was observed for ISIS 582071 as compared to ISIS 464917. The remaining oligonucleotides, ISIS 582072 and 582069 did not cause any changes in body and organ weights outside the expected range as compared to ISIS 465178 and 465984.
  • Each nucleoside followed by a subscript "d” is a P-D-2'-deoxyribonucleoside.
  • Each nucleoside followed by a subscript "e” indicates a 2'-0- methoxyethyl (MOE) modified nucleoside.
  • Each nucleoside followed by a subscript “k” indicates a bicyclic nucleoside having a 4'-CH((S)-CH3)-0-2' bridge also referred to as a (S)-cEt modified nucleoside.
  • Each "N” is a modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C). Nucleosides followed b subscript “e” or “k” are further illustrated below.

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Abstract

The present invention provides gapped oligomeric compounds. More particularly the gapped oligomeric compounds provided herein comprise at least one modified intemucleoside linkage in the gap region. Such gapped oligomeric compounds have one or more improved properties such as selectivity, potency, improved toxicity profile and or improved proinflammatory profile. Certain such oligomeric compounds are useful for hybridizing to a complementary nucleic acid, including but not limited, to nucleic acids in a cell. In certain embodiments, hybridization results in modulation of the amount activity or expression of the target nucleic acid in a cell.

Description

LINKAGE MODIFIED GAPPED OLIGOMERIC
COMPOUNDS AND USES THEREOF
FIELD OF THE INVENTION
The present invention pertains generally to chemically-modified oligonucleotides for use in research, diagnostics, and/or therapeutics.
SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled CHEM0084WOSEQ.txt, created July 26, 2012, which is 268 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
Antisense compounds have been used to modulate target nucleic acids. Antisense compounds comprising a variety of chemical modifications and motifs have been reported. In certain instances, such compounds are useful as research tools, diagnostic reagents, and as therapeutic agents. In certain instances antisense compounds have been shown to modulate protein expression by binding to a target messenger R A (mR A) encoding the protein. In certain instances, such binding of an antisense compound to its target mRNA results in cleavage of the mRNA. Antisense compounds that modulate processing of a pre-mRNA have also been reported. Such antisense compounds alter splicing, interfere with polyadenlyation or prevent formation of the 5 ' -cap of a pre-mRN A.
The synthesis and biochemical properties of oligonucleotides containing phosphorus- modified phosphonoacetate and thio-phosphonoacetate deoxyribonucleotides have been described in scientific journals and patent literature (see Dellinger et al, J. Am. Chem. Soc, 2003, 125, 940-950; Nucleic Acids Research, 2003, 31(14), 4109-4118^; also see published US patent applications (US 2004/0116687 and US 2002/0058802) and US patent US 6,693,187.
DNA or RNA containing oligonucleotides comprising alkylphosphonate internucleoside linkage backbone have been disclosed (see US patents 5,264,423 and 5,286,717).
The synthesis of oligodeoxyribonucleotides containing a methyl phosphonate locked nucleic acid (LNA) thymine monomer has been described. The Tm values of the duplexes with their DNA or RNA complements have also been reported {see Lauritsen et al, Bioorg. Med. Chem. Lett., 2003, 253-256).
Various dephosphono linkages (linkages without the phosphorus atom) modifications have been synthesized and studied for their antisense properties. Nonionic, achiral amide linkages (De Mesmaeker et al, Chem. Int. Ed. Engl, 1994, 33, 226-229; Just et al, Synlett, 1994, 137-139) were disclosed. A full account of the synthesis and properties of the five isomeric amide modifications was described (De Mesmaeker et al, 1994, Novel Backbone Replacements for Oligonucleotides, In Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook Eds. ACS
Symposium Series 580:24-39). Gogoi et al presented the synthesis of thioacetamido nucleic acids (TANA) backbone and thermal stability studies with complementary DNA and RNA sequences (Gogoi et al, Chem. Commun., 2006, 2373-2375).
Several nitrogen containing backbone modifications similar to the amides were evaluated as dimeric nucleosides (Sanghvi et al, Nucleosides Nucleotides, 1997, 16, pp. 907-916). Peoc'h reported the synthesis of four methylene(methylimino) (MMI) linked oligodeoxyribonucleotide dimers modified at the 2 -position with fluoro and/or methoxy groups and their incorporation into different sequences (Peoc'h et al, Nucleosides, Nucleotides & Nucleic Acids, 23, 411-438, 2004). Amino linkages have been synthesized and studied for enhanced cellular absorption (Saha et. al, Tet. Lett., 1993, 34, 6017-6020; De Mesmaeker el al, J. Bioorg. Med. Chem. Lett. 1994, 4, 395-398; Caulfield et al, Bioorg. Med. Chem. Lett. 1993, 3, 2771-2776). Other nitrogen containing backbones include oxime (Sanghvi et al, In Nucleosides and Nucleotides as Antitumor and
Antiviral Agents; C.K. Chu and D.C. Baker Eds.: Plenum Press: New York, 1993, 311-324), methyleneimino (ibid), methyleneoxy (methylimino) (MOMI) (ibid), methylene(dimethylhydrazo) (MDH) (Sanghvi et al, Collect. Czech. Chem. Commun., 1993, 58, pp. 158-162), hydroxyl
(methyliminomethylene) (HMIM) (Sanghvi et al., 11th IRT Nucleosides & Nucleotides, Leuven,
Belgium, Sept. 7-11, 1994 (poster presentation)), carbamate ( Dabkowski et al, JChem. Soc. Perkin Trans. 1, 1994, 817-829), oxyamide linkage (Burgess et al, J. Chem. Soc. Chem. Commun., 1994, 915-916), N-substituted guanidine (Vandendrissche et al, J. Chem. Soc, 1993, 1567-1575;
Pannecouque et al, Tetrahedron, 1994, 50, 7231-7246), urea (Kutterer et al, Bioorg. Med. Chem. Lett., 1994, 3, 435-438) and thiourea linkages (Vandendrissche et al, J. Chem. Soc. 1993, 1567- 1575).
Synthesis of sulfur-containing backbone modifications, such as sulfonamide (McElroy et al, Bioorg. Med. Chem. Lett., 1994, 4, 1071-1076), sulfamoyl (Dewynter et al, Acad. Set, 1992, 315, 1675-1682), sulfonate (Huang et al, Synlett, 1993, 83-84), sulfide (Wang et al, Chin. Chem. Lett, 1993, 4, 101-104; Huang et al, Synlett, 1993, 83-84; Kawai et al, Nucleic Acids Res., 1993, 21, 1473-1479; Meng et al, Angew. Chem. Int. Ed. Engl, 1993, 32, 729-731 ; Just et al. (1994), Synthesis and Hybridization Properties of DNA Oligomers Containing Sulfide-Linked
Dinucleosides. In Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook Eds. ACS Symposium Series 580; (pp. 52-65)), and sulfone linkages (Just et al. (1994), Synthesis and Hybridization Properties of DNA Oligomers Containing Sulfide-Linked Dinucleosides. In Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook Eds. ACS
Symposium Series 580; (pp. 62-65)) have been accomplished by several research groups.
Another backbone substitution is the formacetal and the related thioformacetal {Jones et al,
J. Org. Chem., 1993, 58, 2983-2991). Matteucci reported the synthesis of oligonucleotide analogs with one or more phosphodiester linkages that are replaced by a formacetal/ketal type linkage (see US Patent 5,264,562).
Oligomeric compounds have been prepared using Click chemistry wherein alkynyl phosphonate intemucleoside linkages on an oligomeric compound attached to a solid support are converted into the 1,2,3-triazolylphosphonate intemucleoside linkages and then cleaved from the solid support (Krishna et al, J. Am. Chem. Soc, DOI: 101021/ja3026714, published online May 21, 2012). SUMMARY OF THE INVENTION
Provided herein are gapped oligomeric compounds comprising at least one modified intemucleoside linkage having Formula I in the gap region. In certain embodiments, the oligomeric compounds provided herein hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA. In certain embodiments, the oligomeric compounds disclosed herein provide improved selectivity for a target RNA. In certain embodiments, the oligomeric compounds provide improved potency for a target RNA. In certain embodiments, the oligomeric compounds provided herein provide an improvement in the toxicity profile. In certain embodiments, the oligomeric compounds provided herein provide an improvement in the proinflammatory profile. In certain embodiments, the oligomeric compounds provide improved potency and selectivity for a target RNA. In certain embodiments, the oligomeric compounds provide improved potency, selectivity and an improvement in the proinflammatory profile. The variables are defined individually in further detail herein. It is to be understood that the oligomeric compounds provided herein include all combinations of the embodiments disclosed and variables defined herein.
In certain embodiments, gapped oligomeric compounds are provided comprising a contiguous sequence of linked monomer subunits having a gap region located between a 5 '-region and a 3 '-region wherein the 5' and 3'-regions each, independently, have from 2 to 8 contiguous modified nucleosides wherein essentially each modified nucleoside in the 5' and 3 '-regions are RNA-like and the gap region has from 6 to 14 contiguous monomer subunits selected from β-ϋ-2'- deoxyribonucleosides and modified nucleosides that are DNA-like and wherein at least one of the intemucleoside linking groups in the gap region or linking the gap region and the 5'-region or the 3'- region has Formula I:
Figure imgf000005_0001
I
wherein independently for each intemucleoside linking group having Formula I:
X is O or S;
Q is C C6 alkyl, substituted Ci-Ce alkyl, C2-C6 alkenyl, substituted C -C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C(=0)OH or CH2C(=0)OH;
each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ1? S and OC(=0)J!; and
each J] is, independently, H or C C6 alkyl.
In certain embodiments, gapped oligomeric compounds are provided consisting of a contiguous sequence of linked monomer subunits having a gap region located between a '-region and a 3 '-region wherein the 5' and 3 '-regions each, independently, have from 2 to 8 contiguous modified nucleosides wherein essentially each modified nucleoside in the 5' and 3 '-regions 5' and 3'- regions are RNA-like and the gap region has from 6 to 14 contiguous monomer subunits selected from p-D-2'-deoxyribonucleosides and modified nucleosides that are DNA-like and wherein at least one of the intemucleoside linking groups in the gap region or linking the gap region and the 5 - region or the 3 '-region has Formula I: X=?-Q
o
I
I
wherein independently for each internucleoside linking group having Formula I:
X is O or S;
Q is Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C(=0)OH or CH2C(=0)OH;
each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJl5 SJi and OC(=0)J!; and
each J] is, independently, H or Cj-C6 alkyl.
In certain embodiments, gapped oligomeric compounds are provided consisting of a contiguous sequence of linked monomer subunits having a gap region located between a 5 '-region and a 3'-region wherein the 5' and 3'-regions each, independently, have from 2 to 8 contiguous modified nucleosides wherein each modified nucleoside in the 5' and 3 '-regions are RNA-like and the gap region has from 6 to 14 contiguous monomer subunits selected from P-D-2'-deoxyribo- nucleosides and modified nucleosides that are DNA-like and wherein at least one of the
internucleoside linking groups in the gap region or linking the gap region and the 5'-region or the 3'- region has Formula I:
I
o
X=P-Q
o
I
I
wherein independently for each internucleoside linking group having Formula I:
X is O or S;
Q is Ct-Ce alkyl, substituted Ci-C$ alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C(=0)OH or CH2C(=0)OH;
each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJi, SJ\ and OC(=0)J!; and
each is, independently, H or Cj-C6 alkyl. In certain embodiments, gapped oligomenc compounds are provided consisting of a contiguous sequence of linked monomer subunits having a gap region located between a 5'-region and a 3 '-region wherein the 5' and 3 '-regions each, independently, have from 2 to 8 contiguous modified nucleosides wherein each modified nucleoside in the 5' and 3 '-regions are RNA-like and the gap region has from 6 to 14 contiguous monomer subunits selected from P-D-2'-deoxyribo- nucleosides and wherein at least one of the intemucleoside linking groups in the gap region or linking the gap region and the 5'-region or the 3'-region has Formula I:
I
o
X=P-Q
o
I
I
wherein independently for each intemucleoside linking group having Formula I:
X is O or S;
Q is CrC6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C(=0)OH or CH2C(=0)OH;
each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJls SJi and OC(=0)Ji; and
each Ji is, independently, H or Q-C6 alkyl.
In certain embodiments, gapped oligomenc compounds are provided having only one intemucleoside linking group of Formula I in the gap region. In certain embodiments, gapped oligomenc compounds are provided having only two intemucleoside linking groups of Formula I in the gap region. In certain embodiments, gapped oligomenc compounds are provided having only three intemucleoside linking groups of Formula I in the gap region. In certain embodiments, the intemucleoside linking groups having Formula I are contiguous. In certain embodiments, gapped oligomenc compounds are provide having at least two intemucleoside linking groups of Formula I in the gap region that are separated by at least one phosphorothioate or phosphodiester
intemucleoside linking group.
In certain embodiments, gapped oligomeric compounds are provided wherein the intemucleoside linking group linking the 5 '-region and the gap region has Formula I. In certain embodiments, gapped oligomeric compounds are provided wherein the intemucleoside linking group linking the 5'-region and the gap region and the adjacent intemucleoside linkage in the gap region has Formula I. In certain embodiments, the intemucleoside linking group linking the 3'- region and the gap region has Formula I. In certain embodiments, gapped oligomeric compounds are provided wherein the intemucleoside linking group linking the 3 -region and the gap region and the adjacent intemucleoside linkage in the gap region has Formula I. In certain embodiments, the intemucleoside linking group linking the 5 -region and the gap region has Formula I and the intemucleoside linking group linking the 3'-region and the gap region has Formula I. In certain embodiments, only one intemucleoside linking group of Formula I is located in the gap region.
In certain embodiments, each intemucleoside linking group in the 5' and 3'-regions and each intemucleoside linking group in the gap region other than intemucleoside linking groups having Formula I is a phosphodiester or a phosphorothioate intemucleoside linking group. In certain embodiments, each intemucleoside linking group in the 5' and 3 '-regions and each intemucleoside linking group in the gap region other than intemucleoside linking groups having Formula I is a phosphorothioate intemucleoside linking group. In certain embodiments, each intemucleoside linking group in the 5' and 3 '-regions and each intemucleoside linking group in the gap region other than intemucleoside linking groups having Formula I is a phosphodiester intemucleoside linking group.
In certain embodiments, gapped oligomeric compounds are provided wherein each monomer subunit comprises a heterocyclic base independently selected from an optionally protected purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, each monomer subunit comprises a heterocyclic base independently selected from uracil, thymine, cytosine, 4-N- benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine or 2-N-isobutyrylguanine.
In certain embodiments, gapped oligomeric compounds are provided wherein each Q is, independently, selected from Q-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, C(=0)OH and CH2C(=0)OH. In certain embodiments, each Q is, independently, selected from C¾, C(=0)OH, CH2C(=0)OH, (CH2)2OCH3, CH=CH2, CH2CH=CH2 and C≡CH. In certain embodiments, each Q is CH2C(=0)OH. In certain embodiments, each Q is C¾. In certain embodiments, each Q is C≡CH.
In certain embodiments, gapped oligomeric compounds are provided wherein each X is O. In certain embodiments, each X is S.
In certain embodiments, gapped oligomeric compounds are provided wherein the chirality of each intemucleoside linking group having Formula I is Rp. In certain embodiments, the chirality of each intemucleoside linking group having Formula I is Sp. In certain embodiments, each modified nucleoside in the 5' and 3'-regions provides enhanced hybridization affinity for an RNA target as compared to an unmodified nucleoside. In certain embodiments, each modified nucleoside in the 5' and 3 -regions comprises a modified sugar moiety. In certain embodiments, each modified nucleoside in the 5' and 3'-regions is, independently, a bicyclic nucleoside comprising a bicyclic furanosyl sugar moiety or a modified nucleoside comprising a furanosyl sugar moiety having at least one substituent group.
In certain embodiments, gapped oligomeric compounds are provided wherein the 5' and 3 - regions comprise one or more 2 -modified nucleosides that each have a 2'-substituent group independently selected from halogen, OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, 0(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, 0(CH2)2-OCH3, 0(CH2)2-SCH3, 0(CH2)2-OCF3, 0(CH2)3- N(R3)(R4), 0(CH2)2-ON(R3)(R4), 0(CH2)2-0(CH2)2-N(R3)(R4), OCH2C(=0)-N(R4)(R4),
OCH2C(=0)-N(R5)-(CH2)2-N(R3)(R4) and 0(CH2)2-N(R5)-C(=NR6)[N(R3)(R4)] wherein R3, R4, R5 and Re are each, independently, H and Q-C6 alkyl. In certain embodiments, each 2'-substituent group is independently selected from F, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, 0(CH2)2-OCH3, 0(CH2)2-0(CH2)2-N(CH3)2, OCH2C(=0)-N(H)CH3, OCH2C(=0)-N(H)-(CH2)2- N(CH3)2 and OCH2-N(H)-C(=NH)NH2. In certain embodiments, each 2'-substituent group is independently selected from F, OCH3, 0(CH2)2-OCH3 and OCH2C(=0)-N(H)CH3. In certain embodiments, each 2'-substituent group is 0(CH2)2-OCH3.
In certain embodiments, gapped oligomeric compounds are provided wherein the 5' and 3'- regions comprise one or more bicyclic nucleosides that each have a bridging group between the 4' and 2' carbon atoms of the furanosyl ring independently selected from 4'-(CH2)-0-2', 4'-(CH2)-S-2', 4*-(CH2)2-0-2', 4*-CH(CH3)-0-2', 4,-CH(CH20CH3)-0-2,, 4*-C(CH3)2-0-2*, 4'-CH2-N(OCH3)-2*, 4'- CH2-0-N(CH3)-2', 4'-CH2-NCH3-0-2', 4*-CH2-C(H)(CH3)-2' and 4'-CH2-C(=CH2)-2*. In certain embodiments, each of the bridging groups is independently selected from 4'-(CH2)-0-2', 4'-(CH2)2- 0-2', 4'-CH(CH3)-0-2, 5 4*-CH2-NCH3-0-2', 4'-CH2-C(H)(CH3)-2' and 4,-CH2-C(=CH2)-2*. In certain embodiments, each bridging group is 4'-CH[(S)-(CH3)]-0-2'.
In certain embodiments, gapped oligomeric compounds are provided wherein the sugar moieties of each modified nucleoside in the 5' and 3'-regions are the same. In certain embodiments, gapped oligomeric compounds are provided comprising at least two different types of modified nucleosides in the 5' and 3'-regions wherein the different types of modified nucleosides have at least different modified sugar moieties. In certain embodiments, the different types of modified nucleosides include bicyclic nucleosides comprising bicyclic furanosyl sugar moieties and modified nucleosides comprising furanosyl sugar moieties having at least one substituent group. In certain embodiments, the different types of modified nucleosides include 4'-CH[(S)-(CH3)]-0-2' bicyclic nucleosides and 2'-0(CH2)2-OCH3 substituted nucleosides. In certain embodiments, the 5' and 3'- regions include only 4'-CH[(S)-(CH3)]-0-2' bicyclic nucleosides and 2'-0(CH2)2-OCH3 substituted nucleosides.
In certain embodiments, gapped oligomeric compounds are provided wherein one or more modified nucleosides in the 5' and 3 -regions comprise a sugar surrogate.
In certain embodiments, gapped oligomeric compounds are provided wherein each monomer subunit in the gap region is a p-D-2'-deoxyribonucleoside. In certain embodiments, at least one monomer subunit in the gap region is a modified nucleoside that is DNA-like. In certain
embodiments, each modified nucleoside that is DNA-like is a 2'-(ara)-F modified nucleoside.
In certain embodiments, gapped oligomeric compounds are provided wherein the 5' and 3'- regions each, independently, have from 2 to 8 monomer subunits. In certain embodiments, the 5' and 3 -regions each, independently, have from 3 to 6 monomer subunits. In certain embodiments, the gap region has from 6 to 14 monomer subunits. In certain embodiments, the gap region has from 8 to 10 monomer subunits. In certain embodiments, the 5' and 3'-regions each, independently, have from 3 to 6 monomer subunits and the gap region has from 8 to 14 monomer subunits. In certain embodiments, the 5' and 3'-regions each, independently, have from 3 to 6 monomer subunits and the gap region has from 6 to 10 monomer subunits. In certain embodiments, the 5' and 3'- regions each, independently, have from 3 to 6 monomer subunits and the gap region has from 6 to 8 monomer subunits. In certain embodiments, the 5' and 3'-regions each, independently, have from 4 to 5 monomer subunits and the gap region has from 7 to 8 monomer subunits.
In certain embodiments, gapped oligomeric compounds are provided wherein at least one modified nucleoside in the 5' and 3 -regions is other than a 2'-OCH3 substituted nucleoside or a 2'-0- CH2-4' bridged bicyclic nucleoside.
In certain embodiments, gapped oligomeric compounds are provided that include one 5'- terminal group. In certain embodiments, gapped oligomeric compounds are provided that include one 3 '-terminal group. In certain embodiments, gapped oligomeric compounds are provided that include at least one 5' or 3'-terminal group.
In certain embodiments, the gapped oligomeric compounds provided herein are other than the gapped oligomeric compounds listed below:
SEQ ID NO. Sequence Gap Chemistry wings 573'
ISIS # 05/558257 TeAkAkATTpGTCATCAkCkCe -(P(=0)(CH3))- ekk kke
05/558256 TeAkAkATpTGTCATCAkCkCe -(P(=0)(CH3))- ekk kke
05/558255 TeAkAkApTTGTCATCAkCkCe -(P(=0)(CH3))- ekk kke
05/571123 TeAeAeAkTkTpGTmCATmCAk mCk mCe -(P(=0)(CH3))- eeekk kke
05/571124 TeAeAeAk xTTpGTmCATmCeAk mCk mC -(P(=0)(CH3))- eeek kke
05/579854 TeAeAeAkTTpGTmCATmCAk mCk mCe -(P(=0)(CH3))- eeek kke
09/571171 AeTkAeAkATTpGTmCATmCAk mCemCkAe -(P(=0)(CH3))- ekek keke
09/571041 AsTkAeAkAxTTpGTmCATn,CAk mCe mCkAe -(P(=0)(CH3))- ekek keke
Wherein unless indicated otherwise each internucleoside linkage is a phosphorothioate. Each "XT" is a 2-thio-thymidine modified nucleoside. A subscript "p" indicates a methyl phosphonate internucleoside linkage (-(P(=0)(CH3))-). Nucleosides not followed by a subscript are β-ϋ-2'- deoxyribonucleosides. Nucleosides followed by a subscript "e" are 2'-0-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript "k" are 6'-(2> -0¾ (cEt) bicyclic modified nucleosides. Each "mC" is a 5-methyl cytosine modified nucleoside.
In certain embodiments, the gapped oligomeric compounds provided herein are other than gapped oligomeric compounds complementary to at least a region of a nucleic acid that is a
Huntingtin gene transcript. In certain embodiments, the gapped oligomeric compounds provided herein are other than gapped oligomeric compounds complementary to at least a region of a nucleic acid comprising a single-nucleotide polymorphism. In certain embodiments, the gapped oligomeric compounds provided herein are other than gapped oligomeric compounds complementary to at least a region of a nucleic acid comprising a single-nucleotide polymorphism-containing-target nucleic acid of a Huntingtin gene transcript. In certain embodiments, the gapped oligomeric compounds provided herein are other than gapped oligomeric compounds complementary to at least a region of a nucleic acid comprising a single-nucleotide polymorphism-containing-target nucleic acid of a gene transcript other than Huntingtin.
In certain embodiments, methods of inhibiting gene expression are provided comprising contacting one or more cells, a tissue or an animal with an oligomeric compound as provided herein wherein said oligomeric compound is complementary to a target RNA. In certain embodiments, the cells are in a human. In certain embodiments, the target RNA is human mRNA. In certain embodiments, the target RNA is cleaved thereby inhibiting its function.
In certain embodiments, in vitro methods of inhibiting gene expression are provided comprising contacting one or more cells or a tissue with an oligomeric compound as provided herein. In certain embodiments, oligomeric compound as provided herein are used in an in vivo method of inhibiting gene expression said method comprising contacting one or more cells, a tissue or an animal with an oligomeric compound as provided herein.
In certain embodiments, oligomeric compounds as provided herein are used in medical therapy.
DETAILED DESCRIPTION OF THE INVENTION
Provided herein are linkage modified gapped oligomeric compounds. Such gapped oligomeric compounds comprise a contiguous sequence of linked monomer subunits having a gap region located between a 5 '-region and a 3 '-region wherein the 5' and 3 '-regions each, independently, have from 2 to 8 contiguous modified nucleosides wherein essentially each modified nucleoside in the 5' and 3 '-regions are RNA-like and the gap region has from 6 to 14 contiguous monomer subunits selected from P-D-2'-deoxyribonucleosides and modified nucleosides that are DNA-like and wherein at least one of the intemucleoside linking groups in the gap region or between the gap region and the 5 '-region or the 3 '-region has Formula I:
I
O
X=P-Q
o
I
I
wherein independently for each intemucleoside linking group having Formula I:
X is O or S;
Q is C]-C6 alkyl, substituted Q-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C(=0)OH or CH2C(=0)OH;
each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJls SJi and OC(=0)J!; and
each J] is, independently, H or Ci-Ce alkyl.
The gapped oligomeric compounds provided herein have been shown to have improved properties. In certain embodiments, the activity of an otherwise unmodified gapped oligomeric compound against a target nucleic acid is enhanced by incorporation of at least one intemucleoside linking group having Formula I in the gap region. In certain embodiments, at least one
intemucleoside linking group having Formula I is located at the gap junction on the 5' side wherein the internucleoside linkage separates the gap region from the wing region. In certain embodiments, at least one internucleoside linking group having Formula I is located at the gap junction on the 3' side. As indicated in the in vitro and in vivo data provided in the example section herein, such properties include selectivity, potency, improved toxicity profile and or an improved
proinflammatory profile.
In certain embodiments, a gapped oligomeric compound of interest is identified and then a series of identical oligomeric compounds are prepared with a single internucleoside linking group having Formula I walked across the gap region. If there are 8 monomer subunits in the gap then there will be 8 oligomeric compounds prepared having the internucleoside linking group having Formula I located at a different position in each of the oligomeric compounds which are
subsequently assayed in one or more assays as illustrated herein to determine the lead from the series.
In certain embodiments, additional internucleoside linking groups having Formula I are incorporated into the gap region of the lead oligomeric compound and assayed in one or more assays as illustrated herein. In certain embodiments, the lead compound is further functionalized with one or more terminal groups such as for example a conjugate group. In certain embodiments, a gapped oligomeric compound of interest is identified and then a series of identical oligomeric compounds are prepared with blocks of at least two internucleoside linking group having Formula I walked across the gap region.
In certain embodiments, gapped oligomeric compounds are provided having a reduced proinflammatory response when compared to unmodified gapped oligomeric compounds. As shown using an in vitro hPBMC assay (Example 21), a gapped oligomeric compound having alternating methyl thiophosphonate (-P(CH3)(=S)-) internucleoside linkages in the gap reduced the
proinflammatory response compared to the an identical oligomeric compound without the modified internucleoside linkages.
In certain embodiments, gapped oligomeric compounds are provided having enhanced potency (IC50) and selectivity when compared to unmodified gapped oligomeric compounds. As shown using an in vitro HTT SNP assay (Example 23), a gapped oligomeric compound having a single methyl phosphonate (-P(CH3)(=0)-) internucleoside linkage in the gap, at either position 4 or 5 from the 5'-end, showed lower IC50 and increased selectivity when compared to the an identical oligomeric compound without the modified internucleoside linkages.
In certain embodiments, gapped oligomeric compounds are provided having enhanced selectivity with comparable potency (ICso) when compared to unmodified gapped oligomeric compounds. As shown using an in vitro HTT SNP assay (Example 24), a gapped oligomeric compound having one or two methyl phosphonate (-P(CH3)(=0)-) or one phosphonoacetate (-P(CH2C02 ")(=0)-) intemucleoside linkages in the gap typically showed enhanced selectivity with comparable or slightly lower potency.
In certain embodiments, gapped oligomeric compounds are provided having enhanced selectivity with comparable potency (IC50) when compared to an unmodified gapped oligomeric compound. As shown using an in vitro HTT SNP assay (Example 25), a gapped oligomeric compound having one or two methyl phosphonate (-P(CH3)(=0)-) intemucleoside linkages in the gap typically showed enhanced selectivity with comparable or slightly lower potency.
In certain embodiments, gapped oligomeric compounds are provided having improved hepatotoxicity profiles when compared to unmodified gapped oligomeric compounds. As shown in in vivo PTEN and SRB-1 assays (Example 26), a gapped oligomeric compound having two methyl phosphonate (-P(CH3)(=0)-) intemucleoside linkages at the 5'-end of the gap and linking the gap to the 5'-wing lowered the ALT as compared to the unmodified oligomeric compounds. The activity and organ weights are similar for the SRB-1 assay. The activity is reduced for the modified oligomeric compound in the PTEN assay. Also for the PTEN assay the liver is slightly elevated for the modified gapped oligomeric compound whereas for the unmodified oligomeric compound the liver and the spleen weights are both elevated.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.
A. Definitions
Unless specific definitions are provided, the nomenclature used 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., 21st edition, 2005; 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 are incorporated by reference herein in their entirety.
Unless otherwise indicated, the following terms have the following meanings:
As used herein, "chemical modification" means a chemical difference in a compound when compared to a naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence.
As used herein, "furanosyl" means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.
As used herein, "naturally occurring sugar moiety" means a ribofuranosyl as found in naturally occurring RNA or a 2'-deoxyribofuranosyl as found in naturally occurring DNA.
As used herein, "sugar moiety" means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.
As used herein, "modified sugar moiety" means a substituted sugar moiety or a sugar surrogate.
As used herein, "substituted sugar moiety" means a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2'-position, the 3'-position, the 5'-position and/or the 4'-position. Certain substituted sugar moieties are bicyclic sugar moieties.
As used herein, "2 '-substituted sugar moiety" means a furanosyl comprising a substituent at the 2 '-position other than H or OH. Unless otherwise indicated, a 2 '-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2 '-substituent of a 2 '-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring.
As used herein, "MOE" means -OCH2CH2OCH3.
As used herein, "2'-F nucleoside" refers to a nucleoside comprising a sugar comprising fluorine at the 2' position. Unless otherwise indicated, the fluorine in a 2'-F nucleoside is in the ribo position (replacing the OH of a natural ribose).
As used herein, "2'-(ara)-F" refers to a 2'-F substituted nucleoside, wherein the fluoro group is in the arabino position.
As used herein the term "sugar surrogate" means a structure that does not comprise a furanosyl ring and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside sub-units or monomer subunits are capable of linking together and/or linking to other nucleosides or other monomer subunits to form an oligomeric compound which is capable of hybridizing to a complementary oligomeric compound such as a nucleic acid target. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen atom of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen, wherein replacement of the oxygen atom with sulfur in furanose is generally considered a modified nucleoside as opposed to a sugar surrogate but can be considered both); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols.
As used herein, "bicyclic sugar moiety" means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2'-carbon and the 4'-carbon of the furanosyl.
As used herein, "nucleoside" means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.
As used herein, "nucleotide" means 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, but is not limited to "linked nucleotides." As used herein, "linked nucleosides" are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked).
As used herein the term "nucleobase" generally refers to the nucleobase of a nucleoside or modified nucleoside. The term "heterocyclic base moiety" is broader than the term nucleobase in that it includes any heterocyclic base that can be attached to a sugar to prepare a nucleoside or modified nucleoside. Such heterocyclic base moieties include but are not limited to naturally occurring nucleobases (adenine, guanine, thymine, cytosine and uracil) and protected forms of unmodified nucleobases (4-N-benzoylcytosine, 6-N-benzoyladenine and 2-N-isobutyrylguanine) as well as modified (5 -methyl cytosine) or non-naturally occurring heterocyclic base moieties and synthetic mimetics thereof (such as for example phenoxazines).
As used herein the term "modified nucleoside" refers to a nucleoside comprising a modified heterocyclic base and or a sugar moiety other than ribose and 2'-deoxyribose. In certain
embodiments, a modified nucleoside comprises a modified heterocyclic base moiety. In certain embodiments, a modified nucleoside comprises a sugar moiety other than ribose and 2'-deoxyribose. In certain embodiments, a modified nucleoside comprises a modified heterocyclic base moiety and a sugar moiety other than ribose and 2'-deoxyribose. The term "modified nucleoside" is intended to include all manner of modified nucleosides that can be incorporated into an oligomeric compound using standard oligomer synthesis protocols. Modified nucleosides include abasic nucleosides but in general a heterocyclic base moiety is included for hybridization to a complementary nucleic acid target.
In certain embodiments, modified nucleosides include a furanose or modified furanose sugar group such as a 4'-S analog (4'-S-modified nucleoside and 4'-S-ribonucleoside refer to replacement of the furanose oxygen atom with S). Such modified nucleosides include without limitation, substituted nucleosides (such as 2 5', and/or 4' substituted nucleosides) 4'-S-modified nucleosides, (such as 4'-S-ribonucleosides, 4'-S-2'-deoxyribonucleosides and 4'-S-2'-substituted ribonucleosides), bicyclic modified nucleosides (such as 2*-0-CH(CH3)-4*, 2'-0-CH2-4' or 2'-0-(CH2)2-4* bridged furanose analogs) and base modified nucleosides. The sugar can be modified with more than one of these modifications listed such as for example a bicyclic modified nucleoside further including a 5'- substitution or a 5' or 4' substituted nucleoside further including a 2' substituent. The term modified nucleoside also includes combinations of these modifications such as base and sugar modified nucleosides. These modifications are meant to be illustrative and not exhaustive as other modifi- cations are known in the art and are also envisioned as possible modifications for the modified nucleosides described herein.
In certain embodiments, modified nucleosides comprise a sugar surrogate wherein the furanose ring has been replaced with a mono or polycyclic ring system or a non-cyclic sugar surrogate such as that used in peptide nucleic acids. Illustrative examples of sugar moieties for such modified nucleosides includes without limitation morpholino, hexitol, cyclohexenyl, 2.2.2 and 3.2.1 cyclohexose and open non-cyclic groups.
In certain embodiments, modified nucleosides comprise a non-naturally occurring sugar moiety and a modified heterocyclic base moiety. Such modified nucleosides include without limitation modified nucleosides wherein the heterocyclic base moiety is replaced with a
phenoxazine moiety (for example the 9-(2-aminoethoxy)-l,3-diazaphenoxazine-2-one group, also referred to as a G-clamp which forms four hydrogen bonds when hybridized with a guanosine base) and further replacement of the sugar moiety with a sugar surrogate group such as for example a morpholino, a cyclohexenyl or a bicyclo[3.1.0]hexyl.
As used herein, "bicyclic nucleoside" or "BNA" means a nucleoside comprising a bicyclic sugar moiety.
As used herein, "constrained ethyl nucleoside" or "cEt" means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' bridge. In certain embodiments, the cEt comprises a comprising a 4'-CH((5)-CH3)-0-2' bridge. In certain embodiments, the cEt comprises a comprising a 4'-CH((i?)-CH3)-0-2' bridge.
As used herein, "locked nucleic acid nucleoside" or "LNA" means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH2-0-2'bridge.
As used herein, "2 '-substituted nucleoside" means a ribofuranosyl nucleoside comprising a substituent at the 2'-position other than H or OH. Unless otherwise indicated, a 2 '-substituted nucleoside is not a bicyclic nucleoside.
As used herein, "2'-deoxynucleoside" means a nucleoside comprising 2'-H(H) furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain
embodiments, a 2'-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).
As used herein, "RNA-like nucleoside" means a modified nucleoside other than a β-D-ribose nucleoside that provides an A-form (northern) duplex when incorporated into an oligomeric compound and duplexed with a complementary RNA. RNA-like nucleosides are used as replacements for RNA nucleosides in oligomeric compounds to enhance one or more properties such as, for example, nuclease resistance and or hybridization affinity. RNA-like nucleosides include, but are not limited to modified furanosyl nucleosides that adopt a 3'-endo conformational geometry when put into an oligomeric compound. RNA-like nucleosides also include RNA surrogates such as F-HNA. RNA-like nucleosides include but are not limited to modified nucleosides comprising a 2'-substituent group selected from F, 0(CH2)2OCH3 (MOE) and OCH3. RNA-like nucleosides also include but are not limited to modified nucleosides comprising bicyclic furanosyl sugar moiety comprising a 4*-CH2-0-2'5 4'-(CH2)2-0-2*, 4'-C(H)[(R)-CH3]-0-2* or 4'- C(H)[(S)-CH3]-0-2* bridging group.
As used herein, "DNA-like nucleoside" means a modified nucleoside other than a β-ϋ-2'- doxyribose nucleoside that provides a B-form (southern) duplex when incorporated into an oligomeric compound and duplexed with a complementary DNA. DNA-like nucleosides provide an intermediate duplex when incorporated into an oligomeric compound and duplexed with a complementary RNA that is between A-form and B-form. DNA -like nucleosides are used as replacements for DNA nucleosides in oligomeric compounds to enhance one or more properties. DNA-like nucleosides include, but are not limited to modified nucleosides that adopt a 2'-endo conformational geometry when put into an oligomeric compound.
As used herein, "oligonucleotide" means a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.
As used herein "oligonucleoside" means an oligonucleotide in which none of the
internucleoside linkages contains a phosphorus atom. As used herein, oligonucleotides include oligonucleosides.
As used herein, "modified oligonucleotide" means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
As used herein "internucleoside linkage" means a covalent linkage between adjacent nucleosides in an oligonucleotide.
As used herein "naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage.
As used herein, "modified internucleoside linkage" means any internucleoside linkage other than a naturally occurring internucleoside linkage.
As used herein the term "monomer subunit" is meant to include all manner of monomers that are amenable to oligomer synthesis. In general a monomer subunit includes at least a sugar moiety having at least two reactive sites that can form linkages to further monomer subunits. Essentially all monomer subunits include a heterocyclic base moiety that is hybridizable to a complementary site on a nucleic acid target. Reactive sites on monomer subunits located on the termini of an oligomeric compound can be protected or unprotected (generally OH) or can form an attachment to a terminal group (conjugate or other group). Monomer subunits include, without limitation, nucleosides and modified nucleosides. In certain embodiments, monomer subunits include nucleosides such as β-D- ribonucleosides and p-D-2'-deoxyribnucleosides and modified nucleosides including but not limited to substituted nucleosides (such as 2', 5' and bis substituted nucleosides), 4'-S-modified nucleosides (such as 4'-S-ribonucleosides, 4'-S-2'-deoxyribonucleosides and 4'-S-2'-substituted ribonucleosides), bicyclic modified nucleosides (such as bicyclic nucleosides wherein the sugar moiety has a 2'-0-
CHRa-4' bridging group, wherein Ra is H, alkyl or substituted alkyl), other modified nucleosides and nucleosides having sugar surrogates.
As used herein, "conjugate" means 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 pharmacodynamic,
pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.
As used herein, "conjugate linking group" means any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound.
As used herein, "antisense compound" means a compound comprising or consisting of an oligonucleotide or oligomeric compound wherein at least a portion of which is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity.
As used herein, "antisense activity" means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid.
As used herein, "detecting" or "measuring" means that a test or assay for detecting or measuring is performed. Such detection and/or measuring may result in a value 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.
As used herein, "detectable and/or measureable activity" means a statistically significant activity that is not zero.
As used herein, "essentially unchanged" means little or no change in a particular parameter, particularly relative to another parameter which changes much more. In certain embodiments, a parameter is essentially unchanged when it changes less than 5%. In certain embodiments, a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold. For example, in certain embodiments, an antisense activity is a change in the amount of a target nucleic acid. In certain such embodiments, the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target nucleic acid does, but the change need not be zero.
As used herein, "expression" means the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenlyation, addition of 5 '-cap), and translation.
As used herein, "target nucleic acid" means a nucleic acid molecule to which an antisense compound hybridizes.
As used herein, "mRNA" means an RNA molecule that encodes a protein.
As used herein, "pre-mRNA" means an RNA transcript that has not been fully processed into mRNA. Pre-RNA includes one or more intron.
As used herein, "object RNA" means an RNA molecule other than a target RNA, the amount, activity, splicing, and/or function of which is modulated, either directly or indirectly, by a target nucleic acid. In certain embodiments, a target nucleic acid modulates splicing of an object RNA. In certain such embodiments, an antisense compound modulates the amount or activity of the target nucleic acid, resulting in a change in the splicing of an object RNA and ultimately resulting in a change in the activity or function of the object RNA.
As used herein, "microRNA" means a naturally occurring, small, non-coding RNA that represses gene expression of at least one mRNA. In certain embodiments, a microRNA represses gene expression by binding to a target site within a 3' untranslated region of an mRNA. 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.ulc/sequences/. 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" means 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. In certain embodiments, a microRNA mimic is double-stranded.
As used herein, "differentiating nucleobase" means a nucleobase that differs between two nucleic acids. In certain instances, a target region of a target nucleic acid differs by 1-4 nucleobases from a non-target nucleic acid. Each of those differences is referred to as a differentiating nucleobase. In certain instances, a differentiating nucleobase is a single-nucleotide polymorphism.
As used herein, "target-selective nucleoside" means a nucleoside of an antisense compound that corresponds to a differentiating nucleobase of a target nucleic acid.
As used herein, "allele" means one of a pair of copies of a gene existing at a particular locus or marker on a specific chromosome, or one member of a pair of nucleobases existing at a particular locus or marker on a specific chromosome, or one member of a pair of nucleobase sequences existing at a particular locus or marker on a specific chromosome. For a diploid organism or cell or for autosomal chromosomes, each allelic pair will normally occupy corresponding positions (loci) on a pair of homologous chromosomes, one inherited from the mother and one inherited from the father. If these alleles are identical, the organism or cell is said to be "homozygous" for that allele; if they differ, the organism or cell is said to be "heterozygous" for that allele. "Wild-type allele" refers to the genotype typically not associated with disease or dysfunction of the gene product. "Mutant allele" refers to the genotype associated with disease or dysfunction of the gene product.
As used herein, "allelic variant" means a particular identity of an allele, where more than one identity occurs. For example, an allelic variant may refer to either the mutant allele or the wild-type allele.
As used herein, "single nucleotide polymorphism" or "SNP" means a single nucleotide variation between the genomes of individuals of the same species. In some cases, a SNP may be a single nucleotide deletion or insertion. In general, SNPs occur relatively frequently in genomes and thus contribute to genetic diversity. The location of a SNP is generally flanked by highly conserved sequences. An individual may be homozygous or heterozygous for an allele at each SNP site.
As used herein, "single nucleotide polymorphism site" or "SNP site" refers to the nucleotides surrounding a SNP contained in a target nucleic acid to which an antisense compound is targeted.
As used herein, "targeting" or "targeted to" means the association of an antisense compound to a particular target nucleic acid molecule or a particular region of 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, "nucleobase complementarity" or "complementarity" when in reference to nucleobases means 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 means 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 means a pair of nucleobases that do not form hydrogen bonds with one another.
As used herein, "complementary" in reference to oligomeric compounds (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity under stringent conditions. Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. In certain embodiments, complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary). In certain embodiments, complementary oligomeric compounds or regions are 80% complementary. In certain
embodiments, complementary oligomeric compounds or regions are 90% complementary. In certain embodiments, complementary oligomeric compounds or regions are 95% complementary. In certain embodiments, complementary oligomeric compounds or regions are 100%
complementary.
As used herein, "mismatch" means a nucleobase of a first oligomeric compound that is not capable of pairing with a nucleobase at a corresponding position of a second oligomeric compound, when the first and second oligomeric compound are aligned. Either or both of the first and second oligomeric compounds may be oligonucleotides.
As used herein, "hybridization" means 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 nucleobases.
As used herein, "specifically hybridizes" means 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, "fully complementary" in reference to an oligonucleotide or portion thereof means that each nucleobase of the oligonucleotide or portion thereof is capable of pairing with a nucleobase of a complementary nucleic acid or contiguous portion thereof. Thus, a fully
complementary region comprises no mismatches or unhybridized nucleobases in either strand.
As used herein, "percent complementarity" means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.
As used herein, "percent identity" means the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.
As used herein, "modulation" means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of a molecule, 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 a change in splice site selection of pre-mR A processing, resulting in a change in the absolute or relative amount of a particular splice- variant compared to the amount in the absence of modulation.
As used herein, "modification motif means a pattern of chemical 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 means 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, "sugar motif means a pattern of sugar modifications in an oligomeric compound or a region thereof.
As used herein, "linkage motif means 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, "nucleobase modification motif means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence.
As used herein, "sequence motif means a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modifications and thus may have any combination of chemical modifications, including no chemical modifications.
As used herein, "type of modification" in reference to a nucleoside or a nucleoside of a
"type" means the chemical 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, "differently modified" mean chemical modifications or chemical substituents 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 nucleobases are not differently modified. For example, a nucleoside comprising a 2'-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2'-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.
As used herein, "the same type of modifications" refers to modifications that are the same as one another, including absence of modifications. Thus, for example, two unmodified DNA nucleoside have "the same type of modification," even though the DNA nucleoside is unmodified. Such nucleosides having the same type modification may comprise different nucleobases.
As used herein, "pharmaceutically acceptable carrier or diluent" means 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, "substituent" and "substituent group," means an atom or group that replaces the atom or group of a named parent compound. For example a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2'-substuent is any atom or group at the 2'-position of a nucleoside other than H or OH). Substituent groups can be protected or unprotected. In certain embodiments, compounds of the present invention have substituents at one or at more than one position of the parent compound. Substituents 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.
Likewise, as used herein, "substituent" in reference to a chemical functional group means an atom or group of atoms differs from the atom or a group of atoms normally present in the named functional group. In certain embodiments, a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group). Unless otherwise indicated, groups amenable for use as substituents include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (-C(O)Raa), carboxyl (-C(O)O-Raa), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (-O-Raa), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (-N(Rbb)(RcC)), imino(=NRbb), amido (-C(0)N(Rbb)(Rcc) or -N(Rbb)C(0)Raa), azido (-N3), nitro (-N02), cyano (-CN), carbamido (-OC(0)N(Rbb)(Rcc) or -N(Rbb)C(0)ORaa), ureido (-N(Rbb)C(0)N(Rbb)(Rcc)), thioureido (-N(Rbb)C(S)N(Rbb)(Rcc)),
guanidinyl (-N(Rbb)C(=NRbb)N(Rbb)(Rcc)), amidinyl (-C(=NRbb)N(RbbXRcc) or
-N(Rbb)C(=NRbb)(Raa)), thiol (-SRbb), sulfmyl (-S(0)Rbb), sulfonyl (-S(0)2Rbb) and sulfonamidyl (- S(0)2N(Rbb)(Rcc) or -N(Rbb)S(0)2Rbb). Wherein each Raa, Rbb and Rcc is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, 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.
As used herein, "alkyl," as used herein, means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, 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 (C]-C12 alkyl) with from 1 to about 6 carbon atoms being more preferred.
As used herein, "alkenyl," means 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 without limitation, 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," means 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, without limitation, 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, "acyl," means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula -C(0)-X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.
As used herein, "alicyclic" means 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, "aliphatic" means 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, "alkoxy" means 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 without limitation, methoxy, ethoxy, propoxy, isopropoxy, rc-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, "aminoalkyl" means an amino substituted Ci-C alkyl radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.
As used herein, "aralkyl" and "arylalkyl" mean an aromatic group that is covalently linked to a C!-Ci2 alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, 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, "aryl" and "aromatic" mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, 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, "halo" and "halogen," mean an atom selected from fluorine, chlorine, bromine and iodine.
As used herein, "heteroaryl," and "heteroaromatic," mean a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. 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 without limitation, 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.
B. Oligomeric Compounds
As used herein, the term "oligomeric compound" refers to a contiguous sequence of linked monomer subunits. Each linked monomer subunit normally includes a heterocyclic base moiety but monomer subunits also includes those without a heterocyclic base moiety such as abasic monomer subunits. At least some and generally most if not essentially all of the heterocyclic bases in an oligomeric compound are capable of hybridizing to a nucleic acid molecule, normally a preselected RNA target. The term "oligomeric compound" therefore includes oligonucleotides, oligonucleotide analogs and oligonucleosides. It also includes polymers having one or a plurality of nucleosides having sugar surrogate groups.
In certain embodiments, oligomeric compounds comprise a plurality of monomer subunits independently selected from naturally occurring nucleosides, non-naturally occurring nucleosides, modified nucleosides and nucleosides having sugar surrogate groups. In certain embodiments, oligomeric compounds are single stranded. In certain embodiments, oligomeric compounds are double stranded comprising a double-stranded duplex. In certain embodiments, oligomeric compounds comprise one or more conjugate groups and/or terminal groups.
In certain embodiments, the oligomeric compounds as provided herein can be modified by covalent attachment of one or more terminal groups to the 5' or 3'-terminal groups. A terminal group can also be attached at any other position at one of the terminal ends of the oligomeric compound. As used herein the terms "5'-terminal group", "3'-terminal group", "terminal group" and combinations thereof are meant to include useful groups known to the art skilled that can be placed on one or both of the terminal ends, including but not limited to the 5' and 3'-ends of an oligomeric compound respectively, for various purposes such as enabling the tracking of the oligomeric compound (a fluorescent label or other reporter group), improving the pharmacokinetics or pharmacodynamics of the oligomeric compound (such as for example: uptake and/or delivery) or enhancing one or more other desirable properties of the oligomeric compound (a group for improving nuclease stability or binding affinity). In certain embodiments, 5' and 3'-terminal groups include without limitation, modified or unmodified nucleosides; two or more linked nucleosides that are independently, modified or unmodified; conjugate groups; capping groups; phosphate moieties; and protecting groups.
In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, such oligomeric compounds comprise oligonucleotides optionally comprising one or more conjugate and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide. In certain embodiments, oligonucleotides comprise one or more chemical modifications. Such chemical modifications include modifications of one or more nucleoside (including modifications to the sugar moiety and/or the nucleobase) and/or modifications to one or more internucleoside linkage.
a. Certain Modified Nucleosides
Provided herein are oligomeric compounds comprising modified nucleosides. Such modified nucleosides comprise a modified sugar moeity, a modified nucleobase, or both a modifed sugar moiety and a modified nucleobase. i. Certain Modified Sugar Moieties
In certain embodiments, compounds of the invention comprise one or more modifed nucleosides comprising a modifed sugar moiety. Such compounds comprising one or more sugar- modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to an oligonucleotide comprising only nucleosides comprising naturally occurring sugar moieties. In certain embodiments, modified sugar moieties are substitued sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.
In certain embodiments, modified sugar moieties are substituted sugar moieties comprising one or more non-bridging sugar substituents, including but not limited to substituents at the 2' and/or 5' positions. Examples of sugar substituents suitable for the 2 '-position, include, but are not limited to: 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-0(CH2)2OCH3 ("MOE"). In certain embodiments, sugar substituents at the 2' position are selected from allyl, amino, azido, thio, O- allyl, O-C C10 alkyl, O-Q-Qo substituted alkyl; OCF3, 0(CH2)2SCH3, 0(CH2)2-0-N(Rm)(Rn), and 0-CH2-C(=0)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted Ci-Cto alkyl. Examples of sugar substituents at the 5'-position, include, but are not limited to:, 5'-methyl (R or S); 5 -vinyl, and 5'-methoxy. In certain embodiments, substituted sugars comprise more than one non-bridging sugar substituent, for example, 2'-F-5'-methyl sugar moieties (see,e.g., PCT International Application WO 2008/101157, for additional 5', 2'-bis substituted sugar moieties and nucleosides).
Nucleosides comprising 2' -substituted sugar moieties are referred to as 2 '-substituted nucleosides. In certain embodiments, a 2'- substituted nucleoside comprises a 2'-substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O- aralkyl, 0(CH2)2SCH3, 0-(CH2)2-0-N(Rm)(R„) or 0-CH2-C(=0)-N(Rm)(Rn), where each Rm and R„ is, independently, H, an amino protecting group or substituted or unsubstituted Q-Cio alkyl. These 2 -substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (N02), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
In certain embodiments, a 2'- substituted nucleoside comprises a 2 '-substituent group selected from F, NH2, N3, OCF3, 0-CH3, 0(CH2)3NH2, CH2-CH=CH2, 0-CH2-CH=CH2,
OCH2CH2OCH3, 0(CH2)2SCH3, 0-(CH2)2-0-N(Rm)(R„), 0(CH2)20(CH2)2N(CH3)2, and N- substituted acetamide (0-CH2-C(=0)-N(Rm)(Rn) where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted Ci-Cio alkyl.
In certain embodiments, a 2'- substituted nucleoside comprises a sugar moiety comprising a 2'-substituent group selected from F, OCF3, 0-CH3, OCH2CH2OCH3, 0(CH2)2SCH3, 0-(CH2)2-0- N(CH3)2, -0(CH2)20(CH2)2N(CH3)2, and 0-CH2-C(=0)-N(H)CH3.
In certain embodiments, a 2'- substituted nucleoside comprises a sugar moiety comprising a 2'-substituent group selected from F, 0-CH3, and OCH2CH2OCH3.
Certain modifed sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms. Examples of such 4' to 2' sugar substituents, include, but are not limited to: -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]n-0-, -C(RaRb)-N(R)-0- or, -C(RaRb)-0-N(R)-; 4,-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2, 4'-(CH2)-0-2* (LNA); 4'-(CH2)-S-2'; 4'- (CH2)2-0-2' (ENA); 4'-CH(CH3)-0-2* (cEt) and 4'-CH(CH2OCH3)-0-2,,and analogs thereof (see, e.g., U.S. Patent 7,399,845, issued on July 15, 2008); 4'-C(CH3)(CH3)-0-2'and analogs thereof, (see, e.g., WO2009/006478, published January 8, 2009); 4'-CH2-N(OCH3)-2* and analogs thereof (see, e.g., WO2008/150729, published December 11, 2008); 4,-CH2-0-N(CH3)-2' (see, e.g.,
US2004/0171570, published September 2, 2004 ); 4*-CH2-0-N(R)-2', and 4'-CH2-N(R)-0-2*-, wherein each R is, independently, H, a protecting group, or Ci-C12 alkyl; 4'-CH2-N(R)-0-2', wherein R is H, CrC12 alkyl, or a protecting group (see, U.S. Patent 7,427,672, issued on September 23, 2008); 4*-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya, et al, J. Org. Chem.,2009, 74, 118-134); and 4'-CH2-C(=CH )-2' and analogs thereof (see, published PCT International Application WO 2008/154401, published on December 8, 2008).
In certain embodiments, such 4' to 2' bridges independently comprise from 1 to 4 linked groups (generally forming a 4 to 6 membered ring with the parent sugar moiety) independently selected from -[C(Ra)(Rb)]„-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=0 , -C(=S)-, -0-, - Si(Ra)2-, -S(=0)x-, and -N(Ra)-;
wherein:
x is 0, 1, or 2;
n is 1, 2, 3, or 4;
each Ra and Rb is, independently, H, a protecting group, hydroxyl, Cj-C12 alkyl, substituted d-Ci2 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C2o aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJ!J2, SJi, N3, COOJi, acyl (C(=0)-H), substituted acyl, CN, sulfonyl (S(=O)2-J , or sulfoxyl (S(=0)-Ji); and
each i\ and J2 is, independently, H, C1-Q2 alkyl, substituted Ci-C12 alkyl, C2-C12 alkenyl, substituted C2-Q2 alkenyl, C2-Ci2 alkynyl, substituted C2-Ci2 alkynyl, C5-C2o aryl, substituted C5- C2o aryl, acyl (C(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, Q-Cn aminoalkyl, substituted CrC12 aminoalkyl, or a protecting group.
Nucleosides comprising bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to, (A) a-L-Methyleneoxy (4'-CH2-0-2') BNA , (B) β-D-Methyleneoxy (4'-CH2-0-2') BNA (also referred to as locked nucleic acid or LNA), (C) Ethyleneoxy (4'-(CH2)2-0-2') BNA , (D) Aminooxy (4'-CH2-0-N(R)-2') BNA, (E) Oxyamino (4'-CH2-N(R)-0-2') BNA, (F) Methyl(methyleneoxy) (4'-CH(CH3)-0-2') BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4'-CH2-S-2') BNA, (H) methylene-amino (4'-CH2- N(R)-2') BNA, (I) methyl carbocyclic (4'-CH2-CH(CH3)-2') BNA, (J) propylene carbocyclic (4'- (CH2)3-2') BNA, and (K) Ethylene(methoxy) (4'-(CH(CH2OMe)-0-2') BNA (also referred to as constrained MOE or cMOE) as depicted below.
Figure imgf000032_0001
Figure imgf000032_0002
wherein Bx is a nucleobase moiety and R is, independently, H, a protecting group, or C1-C12 alkyl.
Additional bicyclic sugar moieties are known in the art, 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) 8362-8379 (Jul. 4, 2007); Elayadi et al, Curr. Opinion Invens. Drugs, 2001, 2, 558- 561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al, Curr. Opinion Mol. Ther., 2001, 3, 239- 243; U.S. Patent Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO 2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; 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, and 61/099,844; and PCT International Applications Nos. PCT/US2008/064591,
PCT/US2008/066154, and PCT/US2008/068922.
In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, a nucleoside comprising a 4 '-2' methylene-oxy bridge, may be in the a-L configuration or in the β-D configuration.
Previously, a-L-methyleneoxy (4'-CH2-0-2') bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al, Nucleic Acids Research, 2003, 21, 6365-6372).
In certain embodiments, substituted sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5 '-substituted and 4'-2' bridged sugars), (see, PCT International Application WO 2007/134181, published on 11/22/07, wherein LNA is substituted with, for example, a 5'-methyl or a 5'-vinyl group).
In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the naturally occuring sugar is substituted, e.g., with a sulfer, carbon or nitrogen atom. In certain such embodiments, such modified sugar moiety also comprises bridging and or non-bridging substituents as described above. For example, certain sugar surogates comprise a 4'-sulfer atom and a substitution at the 2 -position (see,e.g., published U.S. Patent Application US2005/0130923, published on June 16, 2005) and/or the 5' position. By way of additional example, carbocyclic bicyclic nucleosides having a 4 -2' bridge have been described (see, e.g., Freier et al, Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al, J. Org.
Chem., 2006, 71, 7731-7740). In certain embodiments, sugar surrogates comprise rings having other than 5-atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) {see Leumann, CJ. Bioorg. & Med. Chem. (2002) 10:841- 854), fluoro H -HNA), and those compounds having Formula VII:
Figure imgf000034_0001
VII
wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:
Bx is a nucleobase moiety;
T3 and T4 are each, independently, an internucleoside linking group linking the
tetrahydropyran nucleoside analog to the antisense compound or one of T3 and T4 is an
internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3'-terminal group;
qi> q2, q.3, q4> q5, q6 and q7 are each, independently, H, Ci-C6 alkyl, substituted
Figure imgf000034_0002
alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and
each of Ri and R2 is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJiJ2, SJi, N3, OC(=X)J1? OC(=X)NJiJ2, Ν¾0(=Χ)ΝΙιΙ2> and CN, wherein X is O, S or NJls and each Jls J2, and J3 is, independently, H or Ci-Ce alkyl.
In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein qi> 2, q3, q4, q5, e and q7 are each H. In certain embodiments, at least one of qi, q2, q3, q4, q5, q6 and q7 is other than H. In certain embodiments, at least one of ql5 q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R\ and R2 is F.
In certain embodiments, R\ is fluoro and R2 is H, Ri is methoxy and R2 is H, and R\ is
methoxyethoxy and R2 is H. Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see, e.g., review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).
Combinations of modifications are also provided without limitation, such as 2'-F-5 -methyl substituted nucleosides (see PCT International Application WO 2008/101157 Published on 8/21 /08 for other disclosed 5', 2'-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2'-position (see published U.S. Patent Application US2005-0130923, published on June 16, 2005) or alternatively 5'-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on 11/22/07 wherein a 4'-CH2- 0-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al, J. Am. Chem. Soc. 2007, 129(26), 8362-8379).
In certain embodiments, the present invention provides oligonucleotides comprising modified nucleosides. Those modified nucleotides may include modified sugars, modified nucleobases, and or modified linkages. The specific modifications are selected such that the resulting oligonucleotides possess desirable characteristics. In certain embodiments,
oligonucleotides comprise one or more RNA-like nucleosides. In certain embodiments,
oligonucleotides comprise one or more DNA-like nucleosides.
In certain embodiments, the oligomeric compounds provided herein include RNA-like nucleosides that have been modified to influence the sugar conformation to have predominantly 3'- endo conformational geometry. In certain embodiments, such modified nucleosides include synthetic modifications of the heterocyclic base moiety, the sugar moiety or both to induce a 3'-endo sugar conformation. In certain embodiments, RNA-like nucleosides are selected from RNA surrogates such as including, but not limited to, F-HNA or cyclohexenyl nucleic acid. RNA-like nucleosides are used to replace and mimic RNA nucleosides in an oligomeric compound so that particular properties of the oligomeric compound can be enhanced. Typically RNA-like nucleosides are used in the 5' and 3 '-regions (wings) of gapped oligomeric compounds to improve stability in the presence of nucleases and also to increase the affinity for nucleic a nucleic acid target. Other properties that can also be enhanced by using RNA-like nucleosides include but aren't limited to modulation of pharmacokinetic properties through modification of protein binding, protein off-rate, absorption and clearance as well as chemical stability and specificity of the oligomeric compound (affinity and specificity for enzymes as well as for complementary sequences); and increasing efficacy of RNA cleavage.
In certain embodiments, RNA-like nucleosides include modified nucleosides comprising one or more 2 3', 4' and 5' substituent groups, bicyclic nucleosides and RNA-surrogates. In certain embodiments, RNA-like nucleosides include, but are not limited to modified nucleosides comprising Z-ribo-substituent groups selected from: F, OCH3, 0-C2-C4 alkyl, 0-CH2CH=CH2, 0-(CH2)2-0-CH3 (MOE), 0-(CH2)3-NH2, O-(CH2)2-O-N(R 2, O-CH2C(O)-N(R 2, 0-(CH2)2-0-(CH2)2-N(Ri)2, O- (CH2)3-NHRi and
Figure imgf000036_0001
wherein each Ri is, typically H, Ci-C]2 alkyl or a protecting group. RNA-like nucleosides also include but are not limited to modified nucleosides having a bicyclic furanosyl sugar moiety (bicyclic nucleosides) comprising a bridging group between the 4' and 2'-carbon atoms. Such bicyclic nucleosides include, but are not limited to bridging groups consisting of from 1 to 3 linked biradical groups selected from O, S, NRa,
C(Rb)(Rc), C=0, C(Rb)=C(Rc) and C[=C(Rb)(Rc)] wherein C(Rb)=C(Rc) counts as 2 of said biradical groups wherein each Ra, Rb and Rc is, independently, H, Ci-C6 alkyl, Ci-Ce alkoxy, C2-C6 alkenyl or C2-C6 alkynyl. In certain embodiments, the bridging groups include, but are not limited to 4'- (CH2)-0-2', 4'-(CH2)-S-2', 4'-(CH2)2-0-2', 4'-CH(CH3)-0-2', 4,-CH(CH2OCH3)-0-2', 4'-C(CH3)2-0- 2 4'-CH2-N(OCH3)-2', 4,-CH2-0-N(CH3)-2', 4,-CH2-NCH3-0-2\ 4'-CH2-C(H)(CH3)-2* and 4'-CH2- C(=CH2)-2'. In certain embodiments, the bridging groups include, but are not limited to
4'-CH2-0-2*, 4'-(CH2)2-0-2', 4'-C(H)[(R)-CH3]-0-2' and 4*-C(H)[(S)-CH3]-0-2*.
In certain embodiments, the oligomeric compounds provided herein include DNA-like nucleosides that have been modified to influence the sugar conformation to have predominantly 2'- endo conformational geometry. Such modified nucleosides can include synthetic modifications of the heterocyclic base moiety, the sugar moiety or both to induce the desired 2'-endo sugar conformation. These modified nucleosides are used to mimic RNA nucleosides so that particular properties of an oligomeric compound can be enhanced while maintaining the desirable 2'-endo conformational geometry.
In certain embodiments, DNA-like nucleosides include, but are not limited to 2 -substituted furanosyl nucleosides comprising: 2 -CH2, 2'-ara-CN, 2'-ara-F, 2'-ara-Br or 2'-ara-Cl, 2'-ara-N3, 2 - ara-OH, 2'-ara-0-CH3 or 2'-dehydro-2'-ara-CH3.
The C3'-endo and C2'-endo conformational geometries are shown below:
Figure imgf000037_0001
C2'-endo/Southern C3'-endo/Northern
ii Certain Modified Nucleobases
In certain embodiments, nucleosides of the present invention comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present invention comprise one or more modifed nucleobases (heterocyclic base moieties).
In one embodiment, a heterocyclic base moiety is any heterocyclic system that contains one or more atoms or groups of atoms capable of hydrogen bonding to a heterocyclic base of a nucleic acid. In certain embodiments, nucleobase refers to purines, modified purines, pyrimidines and modified pyrimidines. In certain embodiments, nucleobase refers to unmodified or naturally occurring nucleobases which include, but are not limited to, the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U) and analogs thereof such as 5-methyl cytosine. The terms nucleobase and heterocyclic base moiety also include optional protection for any reactive functional groups such as 4-N-benzoylcytosine, 4-N-benzoyl-5-methyl- cytosine, 6-N-benzoyladenine or 2-N-isobutyrylguanine.
In certain embodiments, heterocyclic base moieties include without limitation modified nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8- amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7- methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8- azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein.
In certain embodiments, heterocyclic base moieties include without limitation tricyclic pyrimidines such as l,3-diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one and 9-(2- aminoethoxy)-l,3-diazaphenoxazine-2-one (G-clamp). Heterocyclic base moieties 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. Further heterocyclic base moieties include without limitation those known to the art skilled (see for example: United States Patent No. 3,687,808; Swayze et al., The Medicinal Chemistry of Oligonucleotides in Antisense a Drug Technology, Chapter 6, pages 143-182, Crooke, S.T., ed., 2008); The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J.I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993, 273- 302).
Modified polycyclic heterocyclic compounds useful as heterocyclic base moieties are disclosed in 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;
5,681,941; 5,750,692; 5,763,588; 5,830,653; 6,005,096; and U.S. Patent Application Publication 20030158403, each of which is incorporated herein by reference in its entirety,
b. Certain Internucleoside Linkages
In certain embodiments, nucleosides may be linked together using any internucleoside linkage to form oligonucleotides. 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=0), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P=S). Representative non- phosphorus containing internucleoside linking groups include, but are not limited to,
methylenemethylimino (-CH2-N(CH3)-0-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-0- C(0)(NH)-S-); siloxane (-0-Si(H)2-0-); and Ν,Ν'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. 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 enantiomers, diastereomers, and other stereoisomer^ configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), a or β such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
Neutral internucleoside linkages include without limitation, phosphotriesters, methylpho- sphonates, MMI (3*-CH2-N(CH3)-0-5'), amide-3 (3,-CH2-C(=0)-N(H)-5*), amide-4 (3*-CH2-N(H)- C(=0)-5'), formacetal (3*-0-CH2-0-5'), and thioformacetal (3'-S-CH2-0-5'). 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,
c. Certain Motifs
In certain embodiments, oligomeric compounds comprise or consist of oligonucleotides. In certain embodiments, such oligonucleotides comprise one or more chemical modifications. In certain embodiments, chemically modified oligonucleotides comprise one or more modified sugars. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleobases. In certain embodiments, chemically modified oligonucleotides comprise one or more modified internucleoside linkages. In certain embodiments, the chemical modifications (sugar modifications, nucleobase modifications, and/or linkage modifications) define a pattern or motif. In certain embodiments, the patterns of chemical modifications of sugar moieties, internucleoside linkages, and nucleobases are each independent of one another. Thus, an oligonucleotide may be described by its sugar modification motif, internucleoside linkage motif and/or nucleobase modification motif (as used herein, nucleobase modification motif describes the chemical modifications to the nucleobases independent of the sequence of nucleobases).
i. Certain sugar motifs
In certain embodiments, oligonucleotides comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar motif. Such sugar motifs include but are not limited to any of the sugar modifications discussed herein.
In certain embodiments, the oligomeric compounds provided herein comprise a gapmer sugar motif, which comprises two external regions or "wings" and a central or internal region or "gap" (also referred to as 5'-region and 3'-region). The three regions of a gapmer sugar motif (the 5 '-wing, the gap, and the 3 '-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3 '-most nucleoside of the 5 '-wing and the 5 '-most nucleoside of the 3 '-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap. In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar moieties of the two wings are the same as one another (symmetric sugar gapmer). In certain embodiments, the sugar moieties of the 5 '-wing differs from the sugar moieties of the 3 '-wing (asymmetric sugar gapmer). In certain embodiments, the sugar moieties in the two wings are selected from at least two different types that are different from the sugar moieties in the gap and at least one of each are in each wing.
In certain embodiments, the term "gapped oligomeric compound" refers to an oligomeric compound having two external regions or wings and an internal region or gap (also referred to as 5'- region and 3'-region). The three regions form a contiguous sequence of monomer subunits with the sugar moieties of the external regions (wings) being different than the sugar moieties of the internal region (gap). In certain embodiments, the sugar moieties of each monomer subunit within a particular region is essentially the same. In certain embodiments, the sugar moieties of each monomer subunit within each wing region is selected independently from 2 different types of modified nucleosides. In certain embodiments, the sugar moieties of each monomer subunit within each wing region is selected independently from 3 different types of modified nucleosides. In certain embodiments, the sugar moieties of each monomer subunit within each wing region is selected independently from 4 different types of modified nucleosides. In certain embodiments, the sugar moiety of essentially each monomer subunit within the internal region is essentially the same. In certain embodiments, the sugar moiety of each monomer subunit within the internal region is a β- D-2'-deoxyribonucleoside, a nucleoside that is DNA-like and/or a nucleoside that supports RNaseH when in the gap region.
In certain embodiments, each monomer subunit within a particular region has the same sugar moiety. When the sugar moieties of the external regions are the same the gapmer is a symmetric gapmer and when the sugar moiety used in the 5'-external region is different from the sugar moiety used in the 3 '-external region, the gapmer is an asymmetric gapmer. In certain embodiments, the external regions are small (each independently 2, 3, 4, 5 or about 6 monomer subunits) and the monomer subunits comprise non-naturally occurring sugar moieties with the internal region comprising P-D-2'-deoxyribonucleosides. In certain embodiments, the external regions each, independently, comprise from 2 to about 8 monomer subunits having non-naturally occurring sugar moieties and the internal region comprises from 6 to 14 unmodified nucleosides. The internal region or the gap generally comprises P-D-2'-deoxyribonucleosides but can comprise non-naturally occurring sugar moieties. The heterocyclic base and intemucleoside linkage is independently variable at each position of a gapped oligomeric compound. A gapped oligomeric compound can further include one or more additional groups including but not limited to capping groups, conjugate groups and other 5' or 3'-terminal groups.
In certain embodiments, gapped oligomeric compounds comprise an internal region of β-D- 2'-deoxyribonucleosides with a single intemucleoside linkage having Formula I. In certain embodiments, gapped oligomeric compounds comprise an internal region of P-D-2'-deoxyribonucleosides having two intemucleoside linkages having Formula I. In certain embodiments, gapped oligomeric compounds comprise an internal region of -D-2'-deoxyribonucleosides having three
intemucleoside linkages having Formula I.
In certain embodiments, the 5' and 3'- wing regions of gapped oligomeric compounds comprise modified nucleosides wherein all the sugar moieties have the same type of modification such as cEt or MOE. In certain embodiments, the 5' and 3'-wing regions of gapped oligomeric compounds comprise two types of modified nucleosides having sugar moieties independently selected from 2'-substituted sugar moieties and furanosyl bicyclic sugar moieties. In certain embodiments, the 5' and 3'- wing regions of gapped oligomeric compounds comprise two types of modified nucleosides having sugar moieties independently selected from 2'-MOE substituted sugar moieties and furanosyl bicyclic sugar moieties each having a 4'-CH((S)-CH3)-0-2' bridge.
In certain embodiments, gapped oligomeric compounds are provided that are from about 10 to about 30 monomer subunits in length. In certain embodiments, gapped oligomeric compounds are provided that are from about 12 to about 20 monomer subunits in length. In certain
embodiments, gapped oligomeric compounds are provided that are from about 14 to about 20 monomer subunits in length. In certain embodiments, gapped oligomeric compounds are provided that are from about 14 to about 18 monomer subunits in length.
ii. Certain Nucleobase Modification Motifs
In certain embodiments, oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined partem or nucleobases
modification motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases is chemically modified. In certain embodiments, oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3 '-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 3 '-end of the oligonucleotide. In certain such embodiments, the block is at the 5 '-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 5 '-end of the oligonucleotide.
In certain embodiments, nucleobase modifications are a function of the natural base at a particular position of an oligonucleotide. For example, in certain embodiments each purine or each pyrimidine in an oligonucleotide is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each cytosine is modified. In certain embodiments, each uracil is modified.
In certain embodiments, oligonucleotides comprise one or more nucleosides comprising a modified nucleobase. In certain embodiments, oligonucleotides having a gapmer sugar motif comprise a nucleoside comprising a modified nucleobase. In certain such embodiments, one nucleoside comprising a modified nucleobases is in the gap of an oligonucleotide having a gapmer sugar motif. In certain embodiments, the sugar is an unmodified 2'deoxynucleoside. In certain embodiments, the modified nucleobase is selected from: a 2-thio pyrimidine and a 5-propyne pyrimidine
In certain embodiments, some, all, or none of the cytosine moieties in an oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methyl cytosine is not a "modified nucleobase."
Accordingly, unless otherwise indicated, unmodified nucleobases include both cytosine residues having a 5-methyl and those lacking a 5 methyl. In certain embodiments, the methylation state of all or some cytosine nucleobases is specified.
iii. Certain Nucleoside Motifs
In certain embodiments, oligomeric compounds comprise nucleosides comprising modified sugar moieties and/or nucleosides comprising modified nucleobases. Such motifs can be described by their sugar motif and their nucleobase motif separately or by their nucleoside motif, which provides positions or patterns of modified nucleosides (whether modified sugar, nucleobase, or both sugar and nucleobase) in an oligonucleotide.
In certain embodiments, the oligomeric compounds comprise or consist of a region having a gapmer nucleoside motif, which comprises two external regions or "wings" and a central or internal region or "gap." The three regions of a gapmer nucleoside motif (the 5 '-wing, the gap, and the 3'- wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties and/or nucleobases of the nucleosides of each of the wings differ from at least some of the sugar moieties and/or nucleobase of the nucleosides of the gap. Specifically, at least the nucleosides of each wing that are closest to the gap (the 3 '-most nucleoside of the 5 '-wing and the 5 '-most nucleoside of the 3 '-wing) differ from the neighboring gap nucleosides, thus defining the boundary between the wings and the gap. In certain embodiments, the nucleosides within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside that differs from one or more other nucleosides of the gap. In certain embodiments, the nucleoside motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the nucleoside motifs of the 5'-wing differs from the nucleoside motif of the 3 -wing (asymmetric gapmer).
1. Certain 5'-wings
In certain embodiments, the 5'- wing of a gapmer consists of 2 to 8 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 3 to 5 linked nucleosides. In certain
embodiments, the 5'- wing of a gapmer consists of 4 or 5 linked nucleosides. In certain
embodiments, the 5'- wing of a gapmer consists of 1 to 4 linked nucleosides. In certain
embodiments, the 5'- wing of a gapmer consists of 1 to 3 linked nucleosides. In certain
embodiments, the 5'- wing of a gapmer consists of 1 or 2 linked nucleosides. In certain
embodiments, the 5'- wing of a gapmer consists of 2 to 4 linked nucleosides. In certain
embodiments, the 5'- wing of a gapmer consists of 2 or 3 linked nucleosides. In certain
embodiments, the 5'- wing of a gapmer consists of 3 or 4 linked nucleosides. In certain
embodiments, the 5'- wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 5'- wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 3 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 5 linked nucleosides.
In certain embodiments, the 5'- wing of a gapmer consists of 2 to 8 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain
embodiments, the 5'- wing of a gapmer consists of 3 to 5 linked nucleosides. In certain
embodiments, the 5'- wing of a gapmer consists of 4 or 5 linked nucleosides. In certain
embodiments, the 5'- wing of a gapmer consists of 2 to 4 linked nucleosides. In certain
embodiments, the 5'- wing of a gapmer consists of 2 to 3 linked nucleosides. In certain
embodiments, the 5'- wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 3 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 5 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 6 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 7 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 8 linked nucleosides.
In certain embodiments, the 5'- wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 5'- wing of a gapmer comprises at least two bicyclic nucleosides. In certain embodiments, the 5'- wing of a gapmer comprises at least three bicyclic nucleosides. In certain embodiments, the 5'- wing of a gapmer comprises at least four bicyclic nucleosides. In certain embodiments, the 5'- wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 5'- wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 5'- wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 5'- wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 5 '- wing of a gapmer is a LNA nucleoside.
In certain embodiments, the 5'- wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 5'- wing of a gapmer comprises at least one 2'- substituted nucleoside. In certain embodiments, the 5'- wing of a gapmer comprises at least one 2'- MOE nucleoside. In certain embodiments, the 5'- wing of a gapmer comprises at least one 2'-OMe nucleoside. In certain embodiments, each nucleoside of the 5'- wing of a gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 5'- wing of a gapmer is a 2'- substituted nucleoside. In certain embodiments, each nucleoside of the 5'- wing of a gapmer is a 2'- MOE nucleoside. In certain embodiments, each nucleoside of the 5'- wing of a gapmer is a 2'-OMe nucleoside.
In certain embodiments, the 5'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5 '-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2 '-substituted nucleoside. In certain embodiments, the 5 '-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 5 '-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-OMe nucleoside.
In certain embodiments, the 5 '-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5 '-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2 '-substituted nucleoside. In certain embodiments, the 5 '-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-OMe nucleoside.
In certain embodiments, the 5'- wing of a gapmer has a nucleoside motif selected from among the following: ABBA; ABB; ABAA; AABAA; AAABAA; AAAABAA; AAAAABAA; AAABAA; AABAA; ABAB; AAABB; AAAAA; ABBC; AA; AAA; AAAA; AAAAB;
AAAAAAA; AAAAAAAA; ABBB; AB; ABAB; AAAAB; AABBB; AAAAB; and AABBB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type and each C is a modified nucleoside of a third type. In certain embodiments, such an oligomeric compound is a gapmer. In certain such embodiments, the 3 '-wing of the gapmer may comprise any nucleoside motif.
2. Certain 3'-wings
In certain embodiments, the 3'- wing of a gapmer consists of 2 to 8 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 3 to 5 linked nucleosides. In certain
embodiments, the 3'- wing of a gapmer consists of 4 or 5 linked nucleosides. In certain
embodiments, the 3'- wing of a gapmer consists of 1 to 4 linked nucleosides. In certain
embodiments, the 3'- wing of a gapmer consists of 1 to 3 linked nucleosides. In certain
embodiments, the 3'- wing of a gapmer consists of 1 or 2 linked nucleosides. In certain
embodiments, the 3'- wing of a gapmer consists of 2 to 4 linked nucleosides. In certain
embodiments, the 3'- wing of a gapmer consists of 2 or 3 linked nucleosides. In certain
embodiments, the 3'- wing of a gapmer consists of 3 or 4 linked nucleosides. In certain
embodiments, the 3'- wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 3'- wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 3 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 5 linked nucleosides.
In certain embodiments, the 3'- wing of a gapmer consists of 2 to 8 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 2 to 5 linked nucleosides. In certain
embodiments, the 3'- wing of a gapmer consists of 3 to 5 linked nucleosides. In certain
embodiments, the 3'- wing of a gapmer consists of 4 or 5 linked nucleosides. In certain
embodiments, the 3'- wing of a gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 2 to 3 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 3 linked nucleosides. In certain embodiments, the 3 '- wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 5 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 6 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 7 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 8 linked nucleosides.
In certain embodiments, the 3 '- wing of a gapmer comprises at least one bicyclic nucleoside.
In certain embodiments, the 3'- wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 3'- wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 3'- wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 3'- wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 3'- wing of a gapmer is a LNA nucleoside.
In certain embodiments, the 3'- wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 3'- wing of a gapmer comprises at least two non- bicyclic modified nucleosides. In certain embodiments, the 3'- wing of a gapmer comprises at least three non-bicyclic modified nucleosides. In certain embodiments, the 3'- wing of a gapmer comprises at least four non-bicyclic modified nucleosides. In certain embodiments, the 3'- wing of a gapmer comprises at least one 2 '-substituted nucleoside. In certain embodiments, the 3'- wing of a gapmer comprises at least one 2'-MOE nucleoside. In certain embodiments, the 3'- wing of a gapmer comprises at least one 2'-OMe nucleoside. In certain embodiments, each nucleoside of the 3'- wing of a gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 3'- wing of a gapmer is a 2 '-substituted nucleoside. In certain embodiments, each nucleoside of the 3'- wing of a gapmer is a 2'-MOE nucleoside. In certain embodiments, each nucleoside of the 3'- wing of a gapmer is a 2'-OMe nucleoside.
In certain embodiments, the 3 '-wing of a gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2 '-substituted nucleoside. In certain embodiments, the 3 '-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-OMe nucleoside.
In certain embodiments, the 3 '-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3 '-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2 '-substituted nucleoside. In certain embodiments, the 3 '-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3 '-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-OMe nucleoside.
In certain embodiments, the 3 '-wing of a gapmer comprises at least one LNA nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3 '-wing of a gapmer comprises at least one LNA nucleoside and at least one 2 '-substituted nucleoside. In certain embodiments, the 3 '-wing of a gapmer comprises at least one LNA nucleoside and at least one 2'- MOE nucleoside. In certain embodiments, the 3 '-wing of a gapmer comprises at least one LNA nucleoside and at least one 2'-OMe nucleoside.
In certain embodiments, the 3 '- wing of a gapmer has a nucleoside motif selected from among the following: ABB; ABAA; AAABAA, AAAAABAA; AABAA; AAAABAA; AAABAA; ABAB; AAAAA; AAABB; AAAAAAAA; AAAAAAA; AAAAAA; AAAAB; AAAA; AAA; AA; AB; ABBB; ABAB; AABBB; wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type. In certain embodiments, an oligonucleotide comprises any 3 '-wing motif provided herein. In certain such embodiments, the 5 '-wing of the gapmer may comprise any nucleoside motif.
3. Certain Central Regions (gap regions)
In certain embodiments, the gap of a gapmer consists of 6 to 20 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 14 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 12 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 or 7 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 or 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 or 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 11 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 12 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 13 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 14 linked nucleosides.
In certain embodiments, each nucleoside of the gap of a gapmer is a 2'-deoxynucleoside. In certain embodiments, the gap comprises one or more modified nucleosides. In certain
embodiments, each nucleoside of the gap of a gapmer is a 2'-deoxynucleoside or is a modified nucleoside that is "DNA-like". In certain embodiments, "DNA-like" means that the nucleoside has similar characteristics to DNA, such that a duplex comprising the gapmer and an RNA molecule is capable of activating RNase H. In certain embodiments, modified nucleosides that are DNA-like are 2'-endo. For example, under certain conditions, 2'-(ara)-F have been shown to support RNase H activation, and thus is DNA-like and further has 2 -endo conformation geometry. In certain embodiments, one or more nucleosides of the gap of a gapmer is not a 2'-deoxynucleoside and is not DNA-like. In certain such embodiments, the gapmer nonetheless supports RNase H activation (e.g., by virtue of the number or placement of the non-DNA nucleosides).
In certain embodiments, the gap comprise a stretch of unmodified 2'-deoxynucleosides interrupted by one or more modified nucleosides, thus resulting in three sub-regions (two stretches of one or more 2'-deoxynucleosides and a stretch of one or more interrupting modified nucleosides). In certain embodiments, no stretch of unmodified 2'-deoxynucleosides is longer than 5, 6, or 7 nucleosides. In certain embodiments, such short stretches is achieved by using short gap regions. In certain embodiments, short stretches are achieved by interrupting a longer gap region.
4. Certain Gapmer Motifs
In certain embodiments, a gapmer comprises a 5 '-wing, a gap comprising at least one internucleoside linkage of Formula I, and a 3' wing, wherein the 5 '-wing, gap, and 3' wing are independently selected from among those discussed above. For example, in certain embodiments, a gapmer has a 5 '-wing, a gap, and a 3 '-wing having features selected from among those listed in the following non-limiting table: Table 1
Certain Gapmer Nucleoside Motifs
Figure imgf000049_0001
wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type and each D is a p-D-2'-deoxyribonucleoside or a nucleoside that is DNA-like. Each gap region includes at least one internucleoside linkage of Formula I.
In certain embodiments, each A comprises a modified sugar moiety. In certain
embodiments, each A comprises a 2 '-substituted sugar moiety. In certain embodiments, each A comprises a 2 '-substituted sugar moiety selected from among F, OCH3, OCH2-C(=0)-N(H)(CH3) and 0(CH2)2-OCH3. In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, <x- L-LNA, ENA and 2'-thio LNA. In certain embodiments, each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio- thymidine nucleoside and 5-propyne uridine nucleoside. In certain embodiments, each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2' -substituted sugar moiety. In certain embodiments, each B comprises a 2'-subsituted sugar moiety selected from among F, OCH3, OCH2-C(=0)-N(H)(CH3) and 0(CH2)2-OCH3. In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, a- L-LNA, EN A and 2'-thio LNA. In certain embodiments, each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio- thymidine nucleoside and 5-propyne uridine nucleoside.
In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, and the other comprises a 2 '-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2 '-substituted sugar moiety. In certain
embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2 '-substituted sugar moiety. In certain embodiments, one of A or B is an a-L-LNA nucleoside and the other of A or B comprises a 2 '-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2' -MOE sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-MOE sugar moiety. In certain embodiments, one of A or B is an a -L-LNA nucleoside and the other of A or B comprises a 2'- MOE sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2'-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-F sugar moiety. In certain embodiments, one of A or B is an a-L- LNA nucleoside and the other of A or B comprises a 2'-F sugar moiety.
In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2'- substituted sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2'- substituted sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2'- substituted sugar moiety. In certain embodiments, A is an a-L-LNA nucleoside and B comprises a 2 '-substituted sugar moiety.
In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2' -MOE sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2'-MOE sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2' -MOE sugar moiety. In certain embodiments, A is an a-L-LNA nucleoside and B comprises a 2' -MOE sugar moiety.
In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2'-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2'-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2'-F sugar moiety. In certain embodiments, A is an a-L-LNA nucleoside and B comprises a 2'-F sugar moiety.
In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2' -MOE sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2'-MOE sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2'-MOE sugar moiety. In certain embodiments, B is an a-L-LNA nucleoside and A comprises a 2'-MOE sugar moiety.
In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2'-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2'-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2'-F sugar moiety. In certain embodiments, B is an a-L-LNA nucleoside and A comprises a 2'-F sugar moiety.
In certain embodiments, each A and B is, independently, a modified nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' bridge or a modified nucleoside comprising a 2'-OCH2CH OCH3 (MOE) substituent group. In certain embodiments, each A and B is,
independently, a modified nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH[(S)- (CH3)]-0-2' bridge or a modified nucleoside comprising a 2'-OCH2CH2OCH3 (MOE) substituent group. In certain embodiments, each A and B is, independently, a modified nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH[(i?)-(CH3)]-0-2' bridge or a modified nucleoside comprising a 2'-OCH2CH2OCH3 (MOE) substituent group. In certain embodiments, at least one modified nucleoside comprising a 2'-OCH2CH2OCH3 (MOE) substituent group and at least one modified nucleoside comprising a 4'-CH(CH3)-0-2' bridge is located in each of the 3' and 5* wings. In certain embodiments, at least one modified nucleoside comprising a 2'-OCH2CH2OCH3 (MOE) substituent group and at least one modified nucleoside comprising a 4'-CH[(S)-(CH3)]-0-2' bridge is located in each of the 3' and 5' wings. In certain embodiments, at least one modified nucleoside comprising a 2'-OCH2CH2OCH3 (MOE) substituent group and at least one modified nucleoside comprising a 4'-CH[(i?)-(CH3)]-0-2' bridge is located in each of the 3' and 5' wings.
iv. Certain Internucleoside Linkage Motifs
In certain embodiments, oligomeric compounds comprise modified internucleoside linkages arranged along the oligomeric compound or region thereof in a defined pattern or modified internucleoside linkage motif provided that at least one internucleoside linkage has Formula I. In certain embodiments, internucleoside linkages are arranged in a gapped motif, as described above for nucleoside motif. In such embodiments, the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. The nucleoside motif is independently selected, so such oligomeric compounds having a gapped intemucleoside linkage motif may or may not have a gapped nucleoside motif and if it does have a gapped nucleoside motif, the wing and gap lengths may or may not be the same.
In certain embodiments, oligomeric compounds comprise a region having an alternating intemucleoside linkage motif. In certain embodiments, oligomeric compounds of the present invention comprise a region of uniformly modified intemucleoside linkages. In certain such embodiments, the oligomeric compound comprises a region that is uniformly linked by
phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each intemucleoside linkage of the oligomeric compound is selected from phosphodiester and phosphorothioate. In certain
embodiments, each intemucleoside linkage of the oligomeric compound is selected from
phosphodiester and phosphorothioate and at least one intemucleoside linkage is phosphorothioate. In certain embodiments, at least one intemucleoside linkage of the oligomeric compound is selected from other than phosphodiester and phosphorothioate.
In certain embodiments, the oligomeric compound comprises at least 6 phosphorothioate intemucleoside linkages. In certain embodiments, the oligomeric compound comprises at least 8 phosphorothioate intemucleoside linkages. In certain embodiments, the oligomeric compound comprises at least 10 phosphorothioate intemucleoside linkages. In certain embodiments, the oligomeric compound comprises at least one block of at least 6 consecutive phosphorothioate intemucleoside linkages. In certain embodiments, the oligomeric compound comprises at least one block of at least 8 consecutive phosphorothioate intemucleoside linkages. In certain embodiments, the oligomeric compound comprises at least one block of at least 10 consecutive phosphorothioate intemucleoside linkages. In certain embodiments, the oligomeric compound comprises at least one block of at least one 12 consecutive phosphorothioate intemucleoside linkages. In certain such embodiments, at least one such block is located at the 3' end of the oligomeric compound. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3' end of the oligomeric compound. In certain embodiments, each intemucleoside linkage a phosphorothioate intemucleoside linkage.
v. Certain Modification Motifs
Modification motifs define oligonucleotides by nucleoside motif (sugar motif and nucleobase motif) and linkage motif. For example, certain oligonucleotides have the following modification motif: AAADDDDDDDDDBBB;
wherein each A is a modified nucleoside comprising a 2' -substituted sugar moiety; each D is a p-D-2'-deoxyribonucleoside or a modified nucleoside having B form conformation geometry and each B is a modified nucleoside comprising a bicyclic sugar moiety wherein at least one
intemucleoside linkage had Formula I. The following non-limiting Table further illustrates certain modification motifs:
Table 2
Certain Modification Motif
Figure imgf000053_0001
AAAAB DDDDDDD BAAAA
BB DDDDDDDD AA
AA DDDDDDD AAAAAAAA
AAA DDDDDDD AAAAAAA
AAA DDDDDDD AAAAAA
AB DDDDDDD BBBA
ABBB DDDDDDDDD BA
AB DDDDDDDDD BBBA
AAABB DDDDDDD BBAAA
AAAAB DDDDDDD BAAAA
AABBB DDDDDDD BBBAA
AAAAB DDDDDDD AAAAA
AAABB DDDDDDD AAAAA
AABBB DDDDDDD AAAAA
AAAAA DDDDDDD BAAAA
AAAAA DDDDDDD BBAAA
AAAAA DDDDDDD BBBAA wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type and each D is a p-D-2'-deoxyribonucleoside or a nucleoside that is DNA-like. Each gap region includes at least one internucleoside linkage of Formula I.
In certain embodiments, each A comprises a modified sugar moiety. In certain
embodiments, each A comprises a 2 '-substituted sugar moiety. In certain embodiments, each A comprises a 2 '-substituted sugar moiety selected from among F, OC¾, OCH2-C(=0)-N(H)(CH3) and 0(CH2)2-OCH3. In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, a- L-LNA, ENA and 2'-thio LNA. In certain embodiments, each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio- thymidine nucleoside and 5-propyne uridine nucleoside.
In certain embodiments, each B comprises a modified sugar moiety. In certain
embodiments, each B comprises a 2 '-substituted sugar moiety. In certain embodiments, each B comprises a 2'-subsituted sugar moiety selected from among F, OCH3, OCH2-C(=0)-N(H)(CH3) and 0(CH2)2-OCH3. In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, cc- L-LNA, ENA and 2'-thio LNA. In certain embodiments, each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio- thymidine nucleoside and 5-propyne urindine nucleoside.
In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, and the other comprises a 2 '-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2 '-substituted sugar moiety. In certain
embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2 '-substituted sugar moiety. In certain embodiments, one of A or B is an a-L-LNA nucleoside and the other of A or B comprises a 2 '-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2' -MOE sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-MOE sugar moiety. In certain embodiments, one of A or B is an a -L-LNA nucleoside and the other of A or B comprises a 2'- MOE sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2'-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-F sugar moiety. In certain embodiments, one of A or B is an a-L- LNA nucleoside and the other of A or B comprises a 2'-F sugar moiety.
In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2'- substituted sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2'- substituted sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2'- substituted sugar moiety. In certain embodiments, A is an a-L-LNA nucleoside and B comprises a 2' -substituted sugar moiety.
In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2' -MOE sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2'-MOE sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2' -MOE sugar moiety. In certain embodiments, A is an a-L-LNA nucleoside and B comprises a 2' -MOE sugar moiety.
In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2'-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2'-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2'-F sugar moiety. In certain embodiments, A is an a-L-LNA nucleoside and B comprises a 2'-F sugar moiety.
In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2' -MOE sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2'-MOE sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2'-MOE sugar moiety. In certain embodiments, B is an a-L-LNA nucleoside and A comprises a 2'-MOE sugar moiety.
In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2'-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2'-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2'-F sugar moiety. In certain embodiments, B is an a-L-LNA nucleoside and A comprises a 2'-F sugar moiety.
In certain embodiments, each A and B is, independently, a modified nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' bridge or a modified nucleoside comprising a 2 -OCH2CH2OCH3 (MOE) substituent group. In certain embodiments, each A and B is,
independently, a modified nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH[(S)- (CH3)]-0-2* bridge or a modified nucleoside comprising a 2'-OCH2CH2OCH3 (MOE) substituent group. In certain embodiments, each A and B is, independently, a modified nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH[(i?)-(CH3)]-0-2' bridge or a modified nucleoside comprising a 2'-OCH2CH2OCH3 (MOE) substituent group. In certain embodiments, at least one modified nucleoside comprising a 2'-OCH2CH20CH3 (MOE) substituent group and at least one modified nucleoside comprising a 4'-CH(CH3)-0-2' bridge is located in each of the 3' and 5' wings. In certain embodiments, at least one modified nucleoside comprising a 2'-ΟΟΗ2ΟΗ2θΟΗ3 (MOE) substituent group and at least one modified nucleoside comprising a 4'-CH[(S)-(CH3)]-0-2' bridge is located in each of the 3' and 5' wings. In certain embodiments, at least one modified nucleoside comprising a 2 -OCH2CH2OCH3 (MOE) substituent group and at least one modified nucleoside comprising a 4'-CH[(i?)-(CH3)]-0-2' bridge is located in each of the 3' and 5' wings,
d. Certain Overall Lengths
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 or 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 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 and 30; provided that X<Y. For example, in certain embodiments, the invention provides oligomeric compounds which comprise oligonucleotides consisting of 10 to 1 1 , 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, 1 1 to 13, 11 to 14, 11 to 15, 1 1 to 16, 1 1 to 17, 11 to 18, 1 1 to 19, 11 to 20, 1 1 to 21, 1 1 to 22, 11 to 23, 11 to 24, 11 to 25, 1 1 to 26, 11 to 27, 11 to 28, 1 1 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 comprising 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, a gapmer oligonucleotide has any of the above lengths.
Further, where an oligonucleotide is described by an overall length range and by regions having specified lengths, and where the sum of specified lengths of the regions is less than the upper limit of the overall length range, the oligonucleotide may have additional nucleosides, beyond those of the specified regions, provided that the total number of nucleosides does not exceed the upper limit of the overall length range.
e. Certain Oligonucleotides
In certain embodiments, oligonucleotides of the present invention are characterized by their modification motif and overall length. In certain embodiments, such parameters are each independent of one another. Thus, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. For example, the internucleoside linkages within the wing regions of a sugar-gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region. Likewise, such sugar-gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. One of skill in the art will appreciate that such motifs may be combined to create a variety of oligonucleotides. Herein if a description of an oligonucleotide or oligomeric compound is silent with respect to one or more parameter, such parameter is not limited. Thus, an oligomeric compound described only as having a gapmer sugar motif without further description may have any length, internucleoside linkage motif, and nucleobase modification motif. Unless otherwise indicated, all chemical modifications are independent of nucleobase sequence,
f. 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-0-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, (5)-(+)- 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.
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-l-carboxylate
(SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted Ci-Qo alkyl, substituted or unsubstituted C2-Q0 alkenyl or substituted or unsubstituted C2-Q0 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.
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 motifs discussed above. Thus, for example, an oligomeric compound comprising an oligonucleotide having region of alternating nucleosides may comprise a terminal group.
C. Antisense Compounds
In certain embodiments, oligomeric compounds provided herein are antisense compounds. Such antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, antisense compounds specifically hybridize to one or more target nucleic acid. In certain embodiments, a specifically hybridizing antisense compound has a nucleobase sequence comprising a region having sufficient complementarity to a target nucleic acid to allow hybridization and result in antisense activity and insufficient complementarity to any non-target so as to avoid non-specific hybridization to any non-target nucleic acid sequences under conditions in which specific hybridization is desired (e.g., under physiological conditions for in vivo or therapeutic uses, and under conditions in which assays are performed in the case of in vitro assays).
In certain embodiments, the present invention provides antisense compounds comprising oligonucleotides that are fully complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are 95% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 90% complementary to the target nucleic acid.
In certain embodiments, such oligonucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, an antisense compound comprises a region that is fully
complementary to a target nucleic acid and is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain such embodiments, the region of full complementarity is from 6 to 14 nucleobases in length.
a. Certain Antisense Activities and Mechanisms
In certain antisense activities, hybridization of an antisense compound results in
recruitment of a protein that cleaves a target nucleic acid. For example, certain antisense compounds result in RNase H mediated cleavage of target nucleic acid. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. The "DNA" in such an R A:DNA duplex, need not be unmodified DNA. In certain embodiments, the invention provides antisense compounds that are sufficiently "DNA-like" to elicit RNase H activity. Such DNA-like antisense compounds include, but are not limited to gapmers having unmodified deoxyfuranose sugar moieties in the nucleosides of the gap and modified sugar moieties in the nucleosides of the wings.
Antisense activities may be observed directly or indirectly. In certain embodiments, observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid; a change in the ratio of splice variants of a nucleic acid or protein; and/or a phenotypic change in a cell or animal.
In certain embodiments, compounds comprising oligonucleotides having a gapmer nucleoside motif described herein have desirable properties compared to non-gapmer
oligonucleotides or to gapmers having other motifs. In certain circumstances, it is desirable to identify motifs resulting in a favorable combination of potent antisense activity and relatively low toxicity. In certain embodiments, compounds of the present invention have a favorable therapeutic index (measure of potency divided by measure of toxicity).
b. Selective Antisense Compounds
In certain embodiments, antisense compounds provided herein are selective for a target relative to a non-target nucleic acid. In certain embodiments, the nucleobase sequences of the target and non-target nucleic acids differ by no more than 4 differentiating nucleobases in the targeted region. In certain embodiments, the nucleobase sequences of the target and non-target nucleic acids differ by no more than 3 differentiating nucleobases in the targeted region. In certain embodiments, the nucleobase sequences of the target and non-target nucleic acids differ by no more than 2 differentiating nucleobases in the targeted region. In certain embodiments, the nucleobase sequences of the target and non-target nucleic acids differ by a single differentiating nucleobase in the targeted region. In certain embodiments, the target and non-target nucleic acids are transcripts from different genes. In certain embodiments, the target and non-target nucleic acids are different alleles for the same gene.
Selectivity of antisense compounds is achieved, principally, by nucleobase complementarity. For example, if an antisense compound has no mismatches for a target nucleic acid and one or more mismatches for a non-target nucleic acid, some amount of selectivity for the target nucleic acid will result. In certain embodiments, provided herein are antisense compounds with enhanced selectivity (i.e. the ratio of activity for the target to the activity for non-target is greater). For example, in certain embodiments, a selective nucleoside comprises a particular feature or combination of features (e.g., chemical modification, motif, placement of selective nucleoside, and/or self- complementary region) that increases selectivity of an antisense compound compared to an antisense compound not having that feature or combination of features. In certain embodiments, such feature or combination of features increases antisense activity for the target. In certain embodiments, such feature or combination of features decreases activity for the target, but decreases activity for the non-target by a greater amount, thus resulting in an increase in selectivity.
Without being limited by mechanism, enhanced selectivity may result from a larger difference in the affinity of an antisense compound for its target compared to its affinity for the non- target and/or a larger difference in RNase H activity for the resulting duplexes. For example, in certain embodiments, a selective antisense compound comprises a modified nucleoside at that same position as a differentiating nucleobase (i.e., the selective nucleoside is modified). That
modification may increase the difference in binding affinity of the antisense compound for the target relative to the non-target. In addition or in the alternative, the chemical modification may increase the difference in RNAse H activity for the duplex formed by the antisense compound and its target compared to the RNase activity for the duplex formed by the antisense compound and the non- target. For example, the modification may exaggerate a structure that is less compatible for RNase H to bind, cleave and/or release the non-target.
Antisense compounds having certain specified motifs have enhanced selectivity, including, but not limited to motifs described above. In certain embodiments, enhanced selectivity is achieved by oligonucleotides comprising any one or more of:
a modification motif comprising a long 5 '-wing (longer than 5, 6, or 7 nucleosides);
a modification motif comprising a long 3'-wing (longer than 5, 6, or 7 nucleosides);
a modification motif comprising a short gap region (shorter than 8, 7, or 6 nucleosides); and a modification motif comprising an interrupted gap region (having no uninterrupted stretch of unmodified 2'-deoxynucleosides longer than 7, 6 or 5).
i. Certain Selective Nucleobase Sequence Elements In certain embodiments, selective antisense compounds comprise nucleobase sequence elements. Such nucleobase sequence elements are independent of modification motifs. Accordingly, oligonucleotides having any of the motifs (modification motifs, nucleoside motifs, sugar motifs, nucleobase modification motifs, and/or linkage motifs) may also comprise one or more of the following nucleobase sequence elements.
1. Alignment of Differentiating Nucleobase/Target-Selective Nucleoside In certain embodiments, a target region and a region of a non-target nucleic acid differ by 1-
4 differentiating nucleobase. In such embodiments, selective antisense compounds have a nucleobase sequence that aligns with the non-target nucleic acid with 1-4 mismatches. A nucleoside of the antisense compound that corresponds to a differentiating nucleobase of the target nucleic acid is referred to herein as a target-selective nucleoside. In certain embodiments, selective antisense compounds having a gapmer motif align with a non-target nucleic acid, such that a target-selective nucleoside is positioned in the gap. In certain embodiments, a target-selective nucleoside is the 1st nucleoside of the gap from the 5' end. In certain embodiments, a target-selective nucleoside is the 2nd nucleoside of the gap from the 5' end. In certain embodiments, a target-selective nucleoside is the 3rd nucleoside of the gap from the 5 '-end. In certain embodiments, a target-selective nucleoside is the 4th nucleoside of the gap from the 5'-end. In certain embodiments, a target-selective nucleoside is the 5th nucleoside of the gap from the 5 '-end. In certain embodiments, a target- selective nucleoside is the 6rd nucleoside of the gap from the 5 '-end. In certain embodiments, a target-selective nucleoside is the 8th nucleoside of the gap from the 3 '-end. In certain embodiments, a target-selective nucleoside is the 7th nucleoside of the gap from the 3 '-end. In certain
embodiments, a target-selective nucleoside is the 6th nucleoside of the gap from the 3 '-end. In certain embodiments, a target-selective nucleoside is the 5th nucleoside of the gap from the 3 '-end. In certain embodiments, a target-selective nucleoside is the 4th nucleoside of the gap from the 3'- end. In certain embodiments, a target-selective nucleoside is the 3rd nucleoside of the gap from the 3 '-end. In certain embodiments, a target-selective nucleoside is the 2nd nucleoside of the gap from the 3 '-end.
2. Mismatches to the Target Nucleic Acid
In certain embodiments, selective antisense compounds comprise one or more mismatched nucleobases relative to the target nucleic acid. In certain such embodiments, antisense activity against the target is reduced by such mismatch, but activity against the non-target is reduced by a greater amount. Thus, in certain embodiments selectivity is improved. Any nucleobase other than the differentiating nucleobase is suitable for a mismatch. In certain embodiments, however, the mismatch is specifically positioned within the gap of an oligonucleotide having a gapmer motif. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 1, 2, 3, 4, 5, 6, 7, or 8 from the 5 '-end of the gap region. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 9, 8, 7, 6, 5, 4, 3, 2, 1 of the antisense compounds from the 3 '-end of the gap region. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 1 , 2, 3, or 4 of the antisense compounds from the 5'-end of the wing region. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 4, 3, 2, or 1 of the antisense compounds from the 3 '-end of the wing region.
3. Self Complementary Regions
In certain embodiments, selective antisense compounds comprise a region that is not complementary to the target. In certain embodiments, such region is complementary to another region of the antisense compound. Such regions are referred to herein as self-complementary regions. For example, in certain embodiments, an antisense compound has a first region at one end that is complementary to a second region at the other end. In certain embodiments, one of the first and second regions is complementary to the target nucleic acid. Unless the target nucleic acid also includes a self-complementary region, the other of the first and second region of the antisense compound will not be complementary to the target nucleic acid. For illustrative purposes, certain antisense compounds have the following nucleobase motif:
ABCXXXXXXXXXC'B'A';
ABCXXXXXXX(X/C')(X/B')(X/A');
(X/A)(X/B)(X/C)XXXXXXXXXC'B'A'
where each of A, B, and C are any nucleobase; A', B', and C are the complementary bases to A, B, and C, respectively; each X is a nucleobase complementary to the target nucleic acid; and two letters in parentheses (e.g., (X C)) indicates that the nucleobase is complementary to the target nucleic acid and to the designated nucleoside within the antisense oligonucleotide.
Without being bound to any mechanism, in certain embodiments, such antisense compounds are expected to form self-structure, which is disrupted upon contact with a target nucleic acid.
Contact with a non-target nucleic acid is expected to disrupt the self-structure to a lesser degree, thus increasing selectivity compared to the same antisense compound lacking the self-complementary regions.
4. Combinations of features
Though it is clear to one of skill in the art, the above motifs and other elements for increasing selectivity may be used alone or in combination. For example, a single antisense compound may include any one, two, three, or more of: self-complementary regions, a mismatch relative to the target nucleic acid, a short nucleoside gap, an interrupted gap, and specific placement of the selective nucleoside. D. Certain Target Nucleic Acids
In certain embodiments, antisense compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid is a non-coding RNA. In certain such embodiments, the target non-coding RNA is selected from: a long-non-coding RNA, a short non-coding RNA, an intronic RNA molecule, a snoRNA, a scaRNA, a microRNA (including pre-microRNA and mature microRNA), a ribosomal RNA, and promoter directed RNA. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from: an mRNA and a pre-mRNA, including intronic, exonic and untranslated regions. In certain embodiments, oligomeric compounds are at least partially complementary to more than one target nucleic acid. For example, antisense compounds of the present invention may mimic microRNAs, which typically bind to multiple targets.
In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA. In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA or a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA or an intronic region of a pre-mRNA. In certain embodiments, the target nucleic acid is a long non-coding RNA. In certain embodiments, the target RNA is an mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain such embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron. In certain embodiments, the target nucleic acid is selected from among non-coding RNA, including exonic regions of pre-mRNA. In certain embodiments, the target nucleic acid is a ribosomal RNA (rRNA). In certain embodiments, the target nucleic acid is a non-coding RNA associated with splicing of other pre-mRNAs. In certain embodiments, the target nucleic acid is a nuclear-retained non-coding RNA.
In certain embodiments, antisense compounds described herein are complementary to a target nucleic acid comprising a single-nucleotide polymorphism. In certain such embodiments, the antisense compound is capable of modulating expression of one allele of the single-nucleotide
Figure imgf000066_0001
nucleic acid to a greater or lesser extent than it modulates another allele. In certain embodiments an antisense compound hybridizes to a single-nucleotide
polymo Wsm-containing-target nucleic acid at the single-nucleotide polymorphism site. In certain embodiments, the target nucleic acid is a Huntingtin gene transcript. In certain embodiments, the target nucleic acid is a single-nucleotide polymorphism-containing-target nucleic acid of a
Huntingtin gene transcript. In certain embodiments, the target nucleic acid is not a Huntingtin gene transcript. In certain embodiments, the target nucleic acid is a single-nucleotide polymorphism- containing-target nucleic acid of a gene transcript other than Huntingtin. In certain embodiments, the target nucleic acid is any nucleic acid other than a Huntingtin gene transcript,
a. Single-Nucleotide Polymorphism
Embodiments of the present invention provide methods, compounds, and compositions for selectively inhibiting mRNA and protein expression of an allelic variant of a particular gene or DNA sequence. In certain embodiments, the allelic variant contains a single nucleotide polymorphism (SNP). In certain embodiments, a SNP is associated with a mutant allele. In certain embodiments, a mutant SNP is associated with a disease. In certain embodiments a mutant SNP is associated with a disease, but is not causative of the disease. In certain embodiments, mRNA and protein expression of a mutant allele is associated with disease.
In certain embodiments, the expressed gene product of a mutant allele results in aggregation of the mutant proteins causing disease. In certain embodiments, the expressed gene product of a mutant allele results in gain of function causing disease. In certain embodiments, genes with an autosomal dominant mutation resulting in a toxic gain of function of the protein are the APP gene encoding amyloid precursor protein involved in Alzheimer's disease (Gene, 371: 68, 2006); the PrP gene encoding prion protein involved in Creutzfeldt- Jakob disease and in fatal familial insomnia (Nat. Med. 1997, 3: 1009 ); GFAP gene encoding glial fibrillary acidic protein involved in
Alexander disease (J. Neurosci. 2006, 26:111623); alpha-synuclein gene encoding alpha-synuclein protein involved in Parkinson's disease (J. Clin. Invest. 2003, 111: 145); SOD-1 gene encoding the SOD-1 protein involved in amyotrophic lateral sclerosis (Science 1998, 281: 1851); atrophin-1 gene encoding atrophin-1 protein involved in dentato-rubral and pallido-luysian atrophy (DRPA) (Trends Mol. Med. 2001, 7: 479); SCA1 gene encoding ataxin-1 protein involved in spino-cerebellar ataxia- 1 (SCA1) (Protein Sci. 2003, 12: 953); PLP gene encoding proteolipid protein involved in
Pelizaeus-Merzbacher disease (NeuroMol Med. 2007, 4: 73); DYT1 gene encoding torsinA protein involved in Torsion dystonia (Brain Res. 2000, 877: 379); and alpha-B crystalline gene encoding alpha-B crystalline protein involved in protein aggregation diseases, including cardiomyopathy (Cell 2007, 130: 427); alpha 1 -antitrypsin gene encoding alpha 1 -antitrypsin protein involved in chronic obstructive pulmonary disease (COPD), liver disease and hepatocellular carcinoma (New Engl J Med. 2002, 346: 45); Ltk gene encoding leukocyte tyrosine kinase protein involved in systemic lupus erythematosus (Hum. Mol. Gen. 2004, 13: 171); PCSK9 gene encoding PCSK9 protein involved in hypercholesterolemia (Hum Mutat. 2009, 30: 520); prolactin receptor gene encoding prolactin receptor protein involved in breast tumors (Proc. Natl. Assoc. Sci. 2008, 105: 4533); CCL5 gene encoding the chemokine CCL5 involved in COPD and asthma (Eur. Respir. J. 2008, 32: 327); PTPN22 gene encoding PTPN22 protein involved in Type 1 diabetes, Rheumatoid arthritis, Graves disease, and SLE (Proc. Natl. Assoc. Sci. 2007, 104: 19767); androgen receptor gene encoding the androgen receptor protein involved in spinal and bulbar muscular atrophy or Kennedy's disease (J Steroid Biochem. Mol. Biol. 2008, 108: 245); CHMP4B gene encoding chromatin modifying protein-4B involved in progressive childhood posterior subcapsular cataracts (Am. J. Hum. Genet 2007, 81 : 596); FXR / NR1H4 gene encoding Farnesoid X receptor protein involved in cholesterol gallstone disease, arthrosclerosis and diabetes (Mol. Endocrinol. 2007, 21: 1769); ABCA1 gene encoding ABCA1 protein involved in cardiovascular disease (Transl. Res. 2007, 149: 205); CaSR gene encoding the calcium sensing receptor protein involved in primary hypercalciuria (Kidney Int. 2007, 71: 1155); alpha-globin gene encoding alpha-globin protein involved in alpha-thallasemia (Science 2006, 312: 1215); httlpr gene encoding HTTLPR protein involved in obsessive compulsive disorder (Am. J. Hum. Genet. 2006, 78: 815); AVP gene encoding arginine vasopressin protein in stress-related disorders such as anxiety disorders and comorbid depression (CNS Neurol. Disord. Drug Targets 2006, 5: 167); GNAS gene encoding G proteins involved in congenital visual defects, hypertension, metabolic syndrome (Trends Pharmacol. Sci. 2006, 27: 260); APAF1 gene encoding APAFl protein involved in a predisposition to major depression (Mol. Psychiatry 2006, 11 : 76); TGF-betal gene encoding TGF-betal protein involved in breast cancer and prostate cancer (Cancer Epidemiol. Biomarkers Prev. 2004, 13: 759); AChR gene encoding acetylcholine receptor involved in congential myasthenic syndrome (Neurology 2004, 62: 1090); P2Y12 gene encoding adenosine diphosphate (ADP) receptor protein involved in risk of peripheral arterial disease (Circulation 2003, 108: 2971); LQT1 gene encoding LQT1 protein involved in atrial fibrillation (Cardiology 2003, 100: 109); RET protooncogene encoding RET protein involved in sporadic pheochromocytoma (J. Clin. Endocrinol. Metab. 2003, 88: 4911); filamin A gene encoding filamin A protein involved in various congenital malformations (Nat. Genet. 2003, 33: 487); TARDBP gene encoding TDP-43 protein involved in amyotrophic lateral sclerosis (Hum. Mol. Gene.t 2010, 19: 671); SCA3 gene encoding ataxin-3 protein involved in Machado- Joseph disease (PLoS One 2008, 3: e3341); SCA7 gene encoding ataxin-7 protein involved in spino-cerebellar ataxia-7 (PLoS One 2009, 4: e7232); and HTT gene encoding huntingtin protein involved in Huntington's disease (Neurobiol Dis. 1996, 3:183); and the CA4 gene encoding carbonic anhydrase 4 protein, CRX gene encoding cone-rod homeobox transcription factor protein, FSCN2 gene encoding retinal fascin homolog 2 protein, IMPDH1 gene encoding inosine monophosphate dehydrogenase 1 protein, NR2E3 gene encoding nuclear receptor subfamily 2 group E3 protein, NRL gene encoding neural retina leucine zipper protein, PRPF3 (RP18) gene encoding pre-mRNA splicing factor 3 protein, PRPF8 (RP13) gene encoding pre-mRNA splicing factor 8 protein, PRPF31 (RP11) gene encoding pre-mRNA splicing factor 31 protein, RDS gene encoding peripherin 2 protein, ROM1 gene encoding rod outer membrane protein 1 protein, RHO gene encoding rhodopsin protein, RPl gene encoding RPl protein, RPGR gene encoding retinitis pigmentosa GTPase regulator protein, all of which are involved in Autosomal Dominant Retinitis Pigmentosa disease (Adv Exp Med Biol. 2008, 613:203) In certain embodiments, the mutant allele is associated with any disease from the group consisting of Alzheimer's disease, Creutzfeldt- Jakob disease, fatal familial insomnia, Alexander disease, Parkinson's disease, amyotrophic lateral sclerosis, dentato-rubral and pallido-luysian atrophy DRPA, spino-cerebellar ataxia, Torsion dystonia, cardiomyopathy, chronic obstructive pulmonary disease (COPD), liver disease, hepatocellular carcinoma, systemic lupus erythematosus, hypercholesterolemia, breast cancer, asthma, Type 1 diabetes, Rheumatoid arthritis, Graves disease, SLE, spinal and bulbar muscular atrophy, Kennedy's disease, progressive childhood posterior subcapsular cataracts, cholesterol gallstone disease, arthrosclerosis, cardiovascular disease, primary hypercalciuria, alpha-thallasemia, obsessive compulsive disorder, Anxiety, comorbid depression, congenital visual defects, hypertension, metabolic syndrome, prostate cancer, congential myasthenic syndrome, peripheral arterial disease, atrial fibrillation, sporadic pheochromocytoma, congenital malformations, Machado- Joseph disease, Huntington's disease, and Autosomal Dominant Retinitis Pigmentosa disease.
i. Certain Huntingtin Targets
In certain embodiments, an allelic variant of huntingtin is selectively reduced. Nucleotide sequences that encode huntingtin include, without limitation, the following: GENBANK Accession No. NT_006081.18, truncated from nucleotides 1566000 to 1768000 (replaced by GENBANK Accession No. NT_006051), incorporated herein as SEQ ID NO: 8, and NM_002111.6,
incorporated herein as SEQ ID NO: 10.
Table 3 provides SNPs found in the GM04022, GM04281 , GM02171 , and GM02173B cell lines. Also provided are the allelic variants found at each SNP position, the genotype for each of the cell lines, and the percentage of HD patients having a particular allelic variant. For example, the two allelic variants for SNP rs6446723 are T and C. The GM04022 cell line is heterozygous TC, the GM02171 cell line is homozygous CC, the GM02173 cell line is heterozygous TC, and the GM04281 cell line is homozygous TT. Fifty percent of HD patients have a T at SNP position rs6446723.
Table 3
Allelic Variations for SNPs Associated with HD
Figure imgf000069_0001
rs2298967 T/C TT TC TC TT 0.38 T rs363088 A/T AA TA TA AA 0.38 A rs363064 T/C CC TC TC CC 0.35 C rs363102 A/G AG AA AA AA 0.23 G rs2798235 A/G AG GG GG GG 0.21 A rs363080 T/C TC CC CC CC 0.21 T rs363072 A/T TA TA AA AA 0.13 A rs363125 A/C AC AC CC CC 0.12 C rs362303 T/C TC TC CC CC 0.12 C rs362310 T/C TC TC CC CC 0.12 C rsl 0488840 A/G AG AG GG GG 0.12 G rs362325 T/C TC TC TT TT 0.11 T rs35892913 A/G GG GG GG GG 0.10 A rs363102 A/G AG AA AA AA 0.09 A rs363096 T/C CC CC TC TT 0.09 C rsl 1731237 T/C CC CC TC TT 0.09 C rsl0015979 A/G AA AA AG GG 0.08 A rs363080 T/C TC CC CC CC 0.07 C rs2798235 A/G AG GG GG GG 0.07 G rsl 936032 C/G GC CC CC CC 0.06 C rs2276881 A/G GG GG GG GG 0.06 G rs363070 A/G AA AA AA AA 0.06 A rs35892913 A/G GG GG GG GG 0.04 G rsl2502045 T/C CC CC CC CC 0.04 C rs6446723 T/C TC CC TC TT 0.04 C rs7685686 A G AG GG AG AA 0.04 G rs3733217 T/C CC CC CC CC 0.03 C rs6844859 T/C TC CC TC TT 0.03 C rs362331 T/C TC CC TC TT 0.03 C Certain Pharmaceutical Compositions In certain embodiments, the present invention provides pharmaceutical compositions comprising one or more antisense compound. In certain embodiments, such pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more antisense compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more antisense compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and sterile water. In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile water. In certain embodiments, the sterile saline is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile phosphate-buffered saline (PBS). In certain embodiments, the sterile saline is
pharmaceutical grade PBS.
In certain embodiments, antisense 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 depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
Pharmaceutical compositions comprising antisense compounds encompass any
pharmaceutically acceptable salts, esters, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising antisense 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 antisense oligomeric compound.
Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
In certain embodiments, pharmaceutical compositions provided herein comprise one or more modified oligonucleotides and one or more excipients. In certain such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and
polyvinylpyrrolidone.
In certain embodiments, a pharmaceutical composition provided herein comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.
In certain embodiments, a pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.
In certain embodiments, a pharmaceutical composition provided herein comprises a co- solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain
embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. In certain embodiments, a pharmaceutical composition provided herein is prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration.
In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical
compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical
compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.
In certain embodiments, a pharmaceutical composition is prepared for transmucosal administration. In certain of such embodiments penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
In certain embodiments, a pharmaceutical composition provided herein comprises an oligonucleotide in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
In certain embodiments, one or more modified oligonucleotide provided herein is formulated as a prodrug. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form of an
oligonucleotide. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in certain instances, a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form. In certain instances, a prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, a prodrug is an ester. In certain such
embodiments, the ester is metabolically hydrolyzed to carboxylic acid upon administration. In certain instances the carboxylic acid containing compound is the corresponding active form. In certain embodiments, a prodrug comprises a short peptide (polyaminoacid) bound to an acid group. In certain of such embodiments, the peptide is cleaved upon administration to form the
corresponding active form.
In certain embodiments, the present invention provides compositions and methods for reducing the amount or activity of a target nucleic acid in a cell. In certain embodiments, the cell is in an animal. In certain embodiments, the animal is a mammal. In certain embodiments, the animal is a rodent. In certain embodiments, the animal is a primate. In certain embodiments, the animal is a non-human primate. In certain embodiments, the animal is a human.
In certain embodiments, the present invention provides methods of administering a pharmaceutical composition comprising an oligomeric compound of the present invention to an animal. Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intracerebroventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous). In certain embodiments, pharmaceutical intrathecals are administered to achieve local rather than systemic exposures. For example, pharmaceutical compositions may be injected directly in the area of desired effect (e.g., into the liver). 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 or naturally occurring bases, such as "ATmeCGAUCG," wherein meC indicates a cytosine base comprising a methyl group at the 5-position.
Examples
The following examples illustrate certain embodiments of the present invention and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.
Example 1
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 oligomenc 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=0) 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 intemucleoside linkages (P=S) are synthesized similar to phosphodiester intemucleoside 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 intemucleoside linkages can be prepared as described in U.S. Patent 5,508,270.
Alkyl phosphonate intemucleoside linkages can be prepared as described in U.S. Patent 4,469,863.
3 '-Deoxy-3' -methylene phosphonate intemucleoside linkages can be prepared as described in U.S. Patents 5,610,289 or 5,625,050.
Phosphoramidite intemucleoside linkages can be prepared as described in U.S. Patent, 5,256,775 or U.S. Patent 5,366,878.
Alkylphosphonothioate intemucleoside 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 intemucleoside linkages can be prepared as described in U.S. Patent 5,476,925.
Phosphotriester intemucleoside linkages can be prepared as described in U.S. Patent
5,023,243.
Borano phosphate intemucleoside 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=0 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 NHUOAc 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
N¾OH at elevated temperature (55-60 °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. 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™-l 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 μΐ, ΟΡΤΙ-ΜΕΜ™-1 and then treated with 130 μΐ, 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, LA) 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, LA) 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 then- 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 μΐ, PCR cocktail (2.5x PCR buffer minus MgCl2, 6.6 mM MgCl2, 375 μΜ 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 μΐ, 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 60°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 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 μΐ, of RIBOGREEN™ working reagent (RIBOGREEN™ reagent diluted 1 :350 in lOmM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μΐ^ 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 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 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
Poly(A)+ mRNA isolation
Poly(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 xL cold
PBS. 60 vL 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 μΐ, 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 μΐ. 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 of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70°C, is added to each well, the plate is incubated on a 90°C 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 μί, cold PBS. 150 μΐ, Buffer RLT is added to each well and the plate vigorously agitated for 20 seconds. 150 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 QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum is applied for 1 minute. 500 μΐ, of Buffer RW1 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 μΐ, of Buffer RW1 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 QIAVAC™ manifold and blotted dry on paper towels. The plate is then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA is then eluted by pipetting 140 μϊ, 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 μΐ/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
General Method for the Pre aration of Phosphoramidites, Compounds 1-4
Figure imgf000085_0001
Bx is a heterocyclic base;
Rj and R2 are each independently H, OH, or a 2'-sugar substituent group
Compounds 1 and 2 are commercially available from Glen Research. Compounds 3 and 4 are prepared as per the procedures well known in the art as described in the specification herein (see Seth et a/., Bioorg. Med. Chem., 2011, 21(4), 1122-1125, J Org. Chem., 2010, 75(5), 1569-1581, Nucleic Acids Symposium Series, 2008, 52(1), 553-554); and also see published PCT International Applications (WO 2011/115818, WO 2010/077578, WO2010/036698, WO2009/143369, WO 2009/006478, and WO 2007/090071), and US patent 7,569,686).
Example 14 Preparation of Compound 10 r)2
Figure imgf000086_0001
10
Bis(diisopropylamino) chlorophosphine, Compound 5 and DMT-protected
deoxyribonucleoside, Compound 9 are commercially available from Sigma-Aldrich, and BOC Biosciences.
Compounds 7 and 10 are prepared using similar procedures as published in the literature (Yamada et al, J. Am. Chem. Soc, 2006, 128(15), 5251-5261).
Example 15
Preparation of Compound 13
C
Figure imgf000086_0002
R3 is -CH2CH3, -CH=CH2, -CH2CH=CH2, -CH2CH2OCH3, -CH2CH2OPG or -CH2CH2OCH2F; PG is a protecting group;
Bx is a heterocyclic base
Bis(diisopropylamino) chlorophosphine, Compound 5 and DTM-protected
deoxyribonucleoside, Compound 9 are commercially available from Sigma-Aldrich, and BOC Biosciences. The Grignard reagent, Compound 11 is commercially available from various sources or can be prepared using similar procedures as published in the literature (Oppolzer et al, Tet. Let., 1982, 23(38), 3901-3904).
Example 16
General Method for the Preparation of Compound 16
Figure imgf000087_0001
Bx is a heterocyclic base;
R4 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl or substituted acyl; wherein the substituent groups are independently selected from halogen, oxo, hydroxyl, amino, thio, azido, cyano or carboxyl;
Bis(diisopropylamino) chlorophosphine, Compound 5 and DTM-protected
deoxyribonucleoside, Compound 9 are commercially available from Sigma-Aldrich, and BOC Biosciences. The Grignard reagent, Compound 14 is commercially available from various sources or can be prepared as per the procedures illustrated in Example 15.
Compound 15 used in the phosphitylation step serves only to illustrate the compounds described herein and is not intended to be limiting. Additional phosphitylating reagents known in the art as described in the specification herein can also be employed to generate various phosphoramidite analogs of Compound 16 and are used as building blocks for oligonucleotide synthesis.
Example 17
Preparation of Dimeric Phosphoramidites, Compounds 20 and 21
Figure imgf000088_0001
lar)
1.
2.
Figure imgf000088_0002
Compounds 1 and 17 are commercially available from Glen Research and Chemexpress. Compounds 18 and 19 were separated by column chromatography. Although one of the
methylphosphonate intemucleoside linkages in DMT phosphoramidite dimer 20 or 21 is Rp and the other is Sp, the absolute stereochemistry at the phosphorus chiral center (P*) of the dimers has not been determined. The spectral analysis of Compounds 20 and 21 was consistent with the structures.
Example 18 General Method for the Preparation of Oligomeric Compound 25
Figure imgf000089_0001
9. NH4, rt (cleavage)
Bx is a heterocylic base;
R! and R2 are each independently H, OH or a 2'-sugar substituent group;
R5 is -CH3, -CH2(C=0)OC(CH3)2CH2CN, -(C=0)0(CH2)2Si(Ph)2CH3, -CH2CH3,
-CH=CH2, -CH2CH=CH2, -CH2CH2OCH3, -CH2CH20PG or -CH2CH2OCH2F
R6 is -CH3, -CH2(C=0)0", -(C=0)0", -CH2CH3, -CH=CH2, -CH2CH=CH2, - CH2CH2OCH3, -CH2CH2OPG or -CH2CH2OCH2F
X is O or S
= Unylinker solid support
Figure imgf000089_0002
The Unylinker™ 22 is commercially available. Oligomeric Compound 25 comprising a modified intemucleoside linkage (e.g. methyl phosphonate, phosphonoacetate or phosphonoformate) is prepared using standard procedures in automated DNA/RNA synthesis (see Dupouy et al, Angew. Chem. Int. Ed, 2006, 45, 3623-3627). Phosphoramidite building blocks, Compounds 1, 2, 3, 10 and 13 are prepared as per the procedures illustrated in Examples 13 to 15. The synthetic steps illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks which are disclosed in Examples 13 to 16a can be used in place of Compound 1, 2, 3, 10 or 13 to prepare an oligomeric compound having a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare gapped oligomeric compounds as described herein. Such gapped oligomeric compounds can have predetermined composition and base sequence as dictated by any given target. Example 19
General method for the preparation of oligomeric compounds comprising a modified phosphorus containing internucleoside linkage via solid phase techniques (preparation of 460209, 558255, 573815, 560221, A01 and A02)
Unless otherwise stated, all reagents and solutions used for the synthesis of oligomeric compounds are purchased from commercial sources. Standard phosphoramidite building blocks and solid support are used for incorporation nucleoside residues which include for example T, A, U, G, C and mC residues. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for β- D-2'-deoxyribonucleoside, methyl phosphonate and phosphonoacetate. For (R)- or f$-methyl phosphonate dimeric phosphoramidite, a 0.1 M solution in acetonitrile was used. For 2'-0-MOE phosphoramidite, a 0.2 M solution in acetonitrile was used. For constrained ethyl (cEt) BNA phosphoramidite, a 0.2 M solution in a 1 :1 (v/v) mixture of acetonitrile and toluene was used.
The oligomeric compound was synthesized on VIMAD UnyLinker™ solid support and the appropriate amounts of solid support were packed in the column for synthesis. Dichloroacetic acid (3%) in DCM was used as detritylating reagent. 4,5-Dicyanoimidazole in the presence of N-methyl- imidazole or lH-tetrazole in C¾CN was used as activator during the coupling step. The synthesis of oligomeric compounds was performed on an ABB 94 synthesizer (Applied Biosy stems) on a 2 μπιοΐ scale using the procedures set forth below.
A solid support preloaded with the Unylinker™ was loaded into a synthesis column after closing the column bottom outlet and CH3CN was added to form a slurry. The swelled support- bound Unylinker™ was treated with a detritylating reagent containing 3% dichloroacetic acid in DCM to provide the free hydroxyl groups. During the coupling step, four to fourteen equivalents of phosphoramidite solutions were delivered with coupling for 6 minutes for unmodified
deoxyribonucleoside phosphoramidites and 13 minutes for other modifications. All of the other steps followed standard protocols. Phosphodiester linkages were introduced by oxidation with 10% t-BuOOH solution in CH3CN for a contact time of 10 minutes. Phosphorothioate linkages were introduced by sulfurization with PADS (0.2 M) in 1 :1 pyridine/CHaCN for a contact time of 5 minutes.
After the desired sequence was assembled, the cyanoethyl phosphate protecting groups were deprotected using a 1 :1 (v/v) mixture of triethylamine and acetonitrile. For phosphonoacetate containing oligomeric compound, 1.5% DBU solution in C¾CN was used. The solid support bound oligomeric compound was washed with acetonitrile and dried under high vacuum. The solid- support bound oligomeric compound was then suspended in ammonia (28-30 wt%) at room temperature for 48 h to remove nucleobase protecting groups and to cleave from the solid support.
The unbound oligomeric compound was then filtered and the support was rinsed and filtered with watenethanol (1:1) followed by water. The filtrate was combined and concentrated to dryness. The residue obtained was purified by cationic ion exchange HPLC (Source 30Q resin, A - 50 mM sodium bicarbonate in CH3CN:H20 3:7 (v/v), B - 50 mM sodium bicarbonate, 1.5 M sodium bromide in CH3CN:H20 3:7 (v/v), 0-30% in 110 min, flow 6 mL/min, λ = 260 nm). Fractions containing full-length oligomeric compound were pooled together (assessed by LC/MS analysis >95%). The residue was desalted by HPLC on a reverse phase cartridge to yield the desired oligomeric compound.
SEQ ID NO. Composition (5' to 3') Gap Chemistry (motif)
/ISIS NO.
05/460209 TeAkAkAdTdTdGdTdmCdAdTdmCdAkmCkmCe Positive control (3/9/3)
05/558255 TeAkAkAd TdTdGdTdmC AdTdmCdAkmCkmCe Single -P(CH3)(=0)- (3/9/3) 05/573815 TeAkAkAdTdyTdGdTdmCdAdTdmCdAkmCkmCe Single -P(CH2C02-)(=0)- (3/9/3)
06/560221 Te mCe mCeCdwAdTdwTdTdwmCdAdwGdGdwAd Alternating -P(CH3)(=S)- GdwAdCdW mCdTeGeGe different motif (3/14/3)
05/A01 TeAkAkAdTdzTdGdTdmCdAdTdmCdAkmCkmCe Single -P((K>CH3)(=0)- (3/9/3) 05/A02 TeAkAkAdTdqTdGdTdmCdAdTd CdAk CkmCe Single -P(^-CH3)(=0)- (3/9/3)
Each intemucleoside linkage is a phosphorothioate (P=S) except for the intemucleoside linkage having a subscript "x", "y", "w", "z" or "q". Each nucleoside followed by a subscript "x" indicates a methyl phosphonate intemucleoside linkage (-P(CH3)(=0)-). Each nucleoside followed by a subscript "y" indicates a phosphonoacetate intemucleoside linkage (-P(CH2C02 ")(=0)-). Each nucleoside followed by a subscript "w" indicates a methyl thiophosphonate intemucleoside linkage (-P(CH3)(=S)-). Each nucleoside followed by a subscript "z" indicates an 7? -methyl phosphonate intemucleoside linkage (-P-(7?J-CH3)(=0)-). Each nucleoside followed by a subscript "q" indicates an (S)- methyl phosphonate intemucleoside linkage (-P-(S -CH3)(=0)-). Each nucleoside followed by a subscript "d" is a P-D-2'-deoxyribonucleoside. Each nucleoside followed by a subscript "e" indicates a 2'-0-methoxyethyl (MOE) modified nucleoside. Each nucleoside followed by a subscript "k" indicates a bicyclic nucleoside having a 4'-CH((5)-CH3)-0-2' bridge also referred to as a (S)-cEt modified nucleoside. Each "mC" is a 5-methyl cytosine modified nucleoside. Nucleosides followed by subscripts "e", or "k; are further illustrated below.
Figure imgf000092_0001
subscript e subscript k. Example 20
Human Peripheral Blood Mononuclear Cells (hPBMC) Assay Protocol
The hPBMC assay was performed using BD Vautainer CPT tube method. A sample of whole blood from volunteered donors with informed consent at US Health Works clinic (Faraday & El Camino Real, Carlsbad) was obtained and collected in 4-15 BD Vacutainer CPT 8 ml tubes (VWR Cat.# BD362753). The approximate starting total whole blood volume in the CPT tubes for each donor was recorded using the PBMC assay data sheet.
The blood sample was remixed immediately prior to centrifugation by gently inverting tubes 8-10 times. CPT tubes were centrifuged at rt (18-25 °C) in a horizontal (swing-out) rotor for 30 min. at 1500-1800 RCF with brake off (2700 RPM Beckman Allegra 6R). The cells were retrieved from the buffy coat interface (between Ficoll and polymer gel layers); transferred to a sterile 50 ml conical tube and pooled up to 5 CPT tubes/50 ml conical tube/donor. The cells were then washed twice with PBS (CSL ^, Mg ~ free; GIBCO). The tubes were topped up to 50 ml and mixed by inverting several times. The sample was then centrifuged at 330 x g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R) and aspirated as much supernatant as possible without disturbing pellet. The cell pellet was dislodged by gently swirling tube and resuspended cells in RPMI+10%
FBS+pen/strep (~1 ml / 10 ml starting whole blood volume). A 60 μΐ sample was pipette into a sample vial (Beckman Coulter) with 600 μΐ VersaLyse reagent (Beckman Coulter Cat# A09777) and was gently vortexed for 10-15 sec. The sample was allowed to incubate for 10 min. at rt and being mixed again before counting. The cell suspension was counted on Vicell XR cell viability analyzer (Beckman Coulter) using PBMC cell type (dilution factor of 1 : 11 was stored with other parameters). The live cell/ml and viability were recorded. The cell suspension was diluted to 1 x 10' live PBMC/ml in RPMI+ 10% FBS+pen/strep.
The cells were plated at 5 x 105 in 50 μΐ/well of 96-well tissue culture plate (Falcon
Microtest). 50 μΐ/well of 2x concentration oligos/controls diluted in RPMI+10% FBS+pen/strep. was added according to experiment template (100 μΐ/well total). Plates were placed on the shaker and allowed to mix for approx. 1 min. After being incubated for 24 hrs at 37 °C; 5% C02, the plates were centrifuged at 400 x g for 10 minutes before removing the supernatant for MSD cytokine assay (i.e. human IL-6, IL-10, IL-8 and MCP-1). Example 21
Evaluation of the Proinflammatory Effects in hPBMC Assay for a Modified Oligonucleotide Comprising Methyl Thiophosphonate Internucleoside Linkages - in vitro assay
A modified oligonucleotide was designed based on the 3/14/3 MOE gapmer, ISIS 353512. This modified oligonucleotide was created by having alternating methyl thiophosphonate
(-P(CH3)(=S)-) internucleoside linkages throughout the gap region. The proinflammatory effect of the modified oligonucleotide targeting hCRP was evaluated in hPBMC assay using the protocol described in Example 20.
The hPBMCs were isolated from fresh, volunteered donors and were treated with modified oligonucleotides at 0, 0.0128, 0.064, 0.32, 1.6, 8, 40 and 200 μΜ concentrations. After a 24 hr treatment, the cytokine levels were measured.
The levels of IL-6 were used as the primary readout and compared to the positive control, oligonucleotide, ISIS 353512 and negative control, ISIS 104838. The results from two donors denoted as "Donor 1" and "Donor 2" are presented below. SEQ ID NO. Composition (5' to 3')
ISIS NO.
Figure imgf000093_0001
Each internucleoside linkage is a phosphorothioate (P=S) except for nucleosides followed by a subscript "w". Each nucleoside followed by a subscript "w" indicates a methyl thiophosphonate internucleoside linkage (-P(CH3)(=S)-). Each nucleoside followed by a subscript "d" is a β-ϋ-2'- deoxyribonucleoside. Each nucleoside followed by a subscript "e" indicates a 2'-0-methoxyethyl (MOE) modified nucleoside. Each "mC" is a 5-methyl cytosine modified nucleoside. Nucleosides followed by subscripts "e" are further illustrated below.
Figure imgf000094_0001
SEQ ID NO. Cone. IL-6 (Donor 1) IL-6 (Donor 2) Gap Chemistry
ISIS NO. (uM) (pg/mL) (pg/mL) (gap motif)
06/353512 0 6.3 7.8 Positive control
0.0128 8.3 10.2 (3/14/3)
0.064 77.2 118.2
0.32 151.9 394.3
1.6 152.4 395.3
8 147.6 337.2
40 122.5 228.4
200 119.7 193.5
06/560221 0 5.6 7.6 Alternating
0.0128 6.4 6.9 P(CH3)(=S)-
0.064 6.7 7.6 (3/14/3)
0.32 7.6 8.9
1.6 9.1 11.8
8 17.5 24.3
40 65.8 50.2
200 60.0 100.4
07/104838 0 5.8 7.3 Negative control
0.0128 7.7 7.9 (5/10/5)
0.064 7.5 11.6
0.32 15.1 22.0
1.6 73.1 112.8
8 29.6 51.5
40 41.6 69.5
200 55.4 4018.
Example 22
Single nucleotide polymorphisms (SNPs) in the huntingtin (HTT) gene sequence
SNP positions (identified by Hayden et al, WO/2009/135322) associated with the HTT gene were mapped to the HTT genomic sequence, designated herein as SEQ ID NO: 08 (NT_006081.18 truncated from nucleotides 1566000 to 1768000). The chart below provides SNP positions associated with the HTT gene and a reference SNP ID number from the Entrez SNP database at the National Center for Biotechnology Information (NCBI, http ://www.ncbi .nlm.nih. gov/sites/ - entrez?db=snp , incorporated herein by reference. The chart below furnishes further details on each SNP. The 'Reference SNP ID number' or 'RS number' is the number designated to each SNP from the Entrez SNP database at NCBI, incorporated herein by reference. 'SNP position' refers to the nucleotide position of the SNP on SEQ ID NO: 08. 'Polymorphism' indicates the nucleotide variants at that SNP position. 'Major allele' indicates the nucleotide associated with the major allele, or the nucleotide present in a statistically significant proportion of individuals in the human population. 'Minor allele' indicates the nucleotide associated with the minor allele, or the nucleotide present in a relatively small proportion of individuals in the human population.
Single Nuclear Polymorphisms (SNPs) and their positions on SEQ ID NO: 08
SNP Major Minor
RS o. Polymorphism
position allele allele
rs2857936 1963 C/T C T rsl2506200 3707 A/G G A rs762855 14449 A/G G A rs3856973 19826 G/A G A rs2285086 28912 G/A A G rs7659144 37974 C/G C G rsl 6843804 44043 C/T C T rs2024115 44221 G/A A G rsl0015979 49095 A G A G rs7691627 51063 A/G G A rs2798235 54485 G/A G A rs4690072 62160 G/T T G rs6446723 66466 C/T T C rs363081 73280 G/A G A rs363080 73564 T/C C T rs363075 77327 G/A G A rs363064 81063 T/C C T rs3025849 83420 A/G A G rs6855981 87929 A/G G A rs363102 88669 G/A A G rsl 1731237 91466 C/T C T rs4690073 99803 A/G G A rs363144 100948 T/G T G rs3025838 101099 C/T C T rs34315806 101687 A/G G A rs363099 101709 T/C C T rs363096 119674 T/C T C rs2298967 125400 C/T T C rs2298969 125897 A/G G A rs6844859 130139 C/T T C rs363092 135682 C/A C A rs7685686 146795 A/G A G
rs363088 149983 A/T A T rs362331 155488 C/T T C rs916171 156468 G/C C G rs362322 161018 A/G A G rs362275 164255 T/C C T rs362273 167080 A/G A G rs2276881 171314 G/A G A
rs3121419 171910 T/C C T
rs362272 174633 G/A G A rs362271 175171 G/A G A rs3775061 178407 C/T C T
rs362310 179429 A/G G A rs362307 181498 T/C C T rs362306 181753 G/A G A rs362303 181960 T/C C T rs362296 186660 C/A C A rsl 006798 198026 A/G A G.
Example 23
Modified oligonucleotides comprising methyl phosphonate internucleoside linkage(s) targeting HTT SNP - in vitro study
A series of modified oligonucleotides were designed based on ISIS 460209 wherein the gap region contains nine P-D-2'-deoxyribonucleosides. The modified oligonucleotides were designed by introducing a methyl phosphonate internucleoside linkage within the gap region. The oligonucleotides with modified phosphorus containing backbone were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact. The potency and selectivity of the modified
oligonucleotides were evaluated and compared to ISIS 460209. The position on the
oligonucleotides opposite to the SNP position, as counted from the 5' -terminus is position 8.
Heterozygous fibroblast GM04022 cell line was used for the in vitro assay (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μΜ concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C_2229297_10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 μΐ, 2X PCR buffer, 101 μΐ, primers (300 μΜ from ABI), 1000 μΐ, water and 40.4 μΐ, RT MIX. To each well was added 15 nL of this mixture and 5 μΐ. of purified RNA. The mutant and wild-type H7TmRNA levels were measured simultaneously by using two different fiuorophores, FAM for mutant allele and VIC for wild-type allele. The H rmR A levels were adjusted according to total RNA content, as measured by RIBOGREEN. Analysis of IC50 's and Selectivity
The half maximal inhibitory concentration (IC50) of each oligonucleotide is presented below and was calculated by plotting the concentrations of oligonucleotides used versus the percent inhibition of HT mRNA expression achieved at each concentration, and noting the concentration of oligonucleotide at which 50% inhibition of HT mR A expression was achieved compared to the control. The IC50 at which each oligonucleotide inhibits the mutant H7TmRNA expression is denoted as 'mut Κ½0'. The IC50 at which each oligonucleotide inhibits the wild-type H -TmRNA expression is denoted as 'wt IC50'. Selectivity as expressed in "fold" was calculated by dividing the IC50 for inhibition of the wild-type HTT versus the IC50 for inhibiting expression of the mutant HTT mRNA and the results are presented below.
SEQ ID NO. Composition (5' to 3')
ISIS NO.
05/460209 TeAkAkAdTdTdGdTdmCdAdTdmCdAkmCkmCe
05/558255 TeAkAkAdxTdTdGdTdmCdAdTdmCdAkmCkmCe
05/558256 TeAkAkAdTdXTdGdTdmCdAdTdmCdA mCkmCe
Each internucleoside linkage is a phosphorothioate (P=S) except for the internucleoside linkage having a subscript "x" which indicates a methyl phosphonate internucleoside linkage (-P(CH3)(=0)-). Each nucleoside followed by a subscript "d" is a P-D-2'-deoxyribonucleoside. Each nucleoside followed by a subscript "e" indicates a 2'-0-methoxyethyl (MOE) modified nucleoside. Each nucleoside followed by a subscript "k" indicates a bicyclic nucleoside having a 4'- CH((S)-CH3)-0-2' bridge also referred to as a (5)-cEt modified nucleoside. Each ,,mC" is a 5-methyl cytosine modified nucleoside. Nucleosides followed by subscripts "e" or "k" are further illustrated below.
Figure imgf000097_0001
subscript e subscript k. SEQ ID NO. ICso Fold Selectivity Gap Chemistry
/ISIS NO. (μΜ) (mut vs. wt)
05/460209 0.30 3.3 Positive control (3/9/3)
05/558255 0.19 6.8 Single -P(CH3)(=0)- (3/9/3)
05/558256 0.20 6.5 Single -P(CH3)(=0)- (3/9/3)
Example 24
Modified oligonucleotides comprising methyl phosphonate or phosphonoacetate
internucleoside linkage(s) targeting HTT SNJ* - in vitro study
A series of modified oligonucleotides were designed based on ISIS 460209 wherein the gap region contains nine P-D-2'-deoxyribonucleosides. The modified oligonucleotides were synthesized to include one or more methyl phosphonate or phosphonoacetate internucleoside linkage modifications within the gap region. The oligonucleotides with modified phosphorus containing backbone were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact. The potency and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.
The position on the oligonucleotides opposite to the SNP position, as counted from the 5'- terminus is position 8.
The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μΜ concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C_2229297_10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 μί, 2X PCR buffer, 101 μΐ primers (300 μΜ from ABI), 1000 uL water and 40.4 μΐ, RT MIX. To each well was added 15 μΐ, of this mixture and 5 μΐ, of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HIT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.
The IC50s and selectivities as expressed in "fold" were measured and calculated using methods described previously in Example 23. The results are presented below. SEQ ID NO. Composition (5' to 3')
ISIS NO.
05/460209 TeAkAkAdTdTdGdTdmCdAdTdmCdAkmCkmCe
05/566276 TeAkAkAdTdTdGdxTdmCdAdTdmCdAkmCkmCe
05/566277 TeAkAkAdTdTdGdTdxmCdAdTdmCdAkmCkmCe
05/566278 TeAkAkAdTdTdGdTdmCdxAdTdmCdAkmCkmCe
05/566279 TeAkAkAdTdTdGdTdmCdAdxTdmCdAkmCkmCe
05/566280 TeAkAkAdTdTdGdTdmCdAdTdxmCdAkmCkmCe
05/566283 TeAkAkAdTdxTdxGdTdmCdAdTdmCdAkmCkmCl e
05/573815 TeAkAkAdTdyTdGdTdmCdAdTdmCdAkmCkmCe
05/573816 TeAkAkAdTdTdyGdTdmCdAdTdmCdAkmCkmCe
05/573817 TeAkAkAdTdTdGdTdy mCdAdTdmCdAkmCkmCe
05/573818 TeAkAkAdTdTdGdTdmCdAdTdymCdAkmCkmCe Each intemucleoside linkage is a phosphorothioate (P=S) except for the intemucleoside linkage having a subscript "x" or "y". Each nucleoside followed by a subscript "x" indicates a methyl phosphonate intemucleoside linkage (-P(CH3)(=0)-). Each nucleoside followed by a subscript "y" indicates a phosphonoacetate intemucleoside linkage (-P(CH2C02 ~)(=0)-). Each nucleoside followed by a subscript "d" is a P-D-2'-deoxyribonucleoside. Each nucleoside followed by a subscript "e" indicates a 2'-0-methoxyethyl (MOE) modified nucleoside. Each nucleoside followed by a subscript "k" indicates a bicyclic nucleoside having a 4'-CH((S)-CH3)-0-2' bridge also referred to as a (5)-cEt modified nucleoside. Each "mC" is a 5-methyl cytosine modified nucleoside. Nucleosides followed by subscripts "e" "k" are further illustrated below.
Figure imgf000099_0001
subscript e subscript k.
SEQ ID NO. IC50 Fold Selectivity Gap Chemistry
/ISIS NO. (μΜ) (mut vs. wt)
05/460209 0.15 9.4 Positive control (3/9/3)
05/566276 0.76 12.8 Single -P(CH3)(=0)- (3/9/3)
05/566277 0.20 17 Single -P(CH3)(=0)- (3/9/3)
05/566278 0.25 8.9 Single -P(CH3)(=0)- (3/9/3)
05/566279 0.38 ~ Single -P(CH3)(=0)- (3/9/3
05/566280 0.27 47 Single -P(CH3)(=0)- (3/9/3
05/566283 0.8 >100 Two -P(CH3)(=0)- (3/9/3) 05/573815 0.16 18.8 Single -P(CH2C02 ")(=0)- (3/9/3) 05/573816 0.55 18.1 Single -P(CH2C02 ")(=0)- (3/9/3) 05/573817 0.17 22.5 Single -P(CH2C02 ")(=0)- (3/9/3) 05/573818 0.24 13.5 Single -P(CH2C02 ")(=0)- (3/9/3).
Example 25
Modified oligonucleotides comprising methyl phosphonate internucleoside linkage(s) targeting HTT SNP - in vitro study
A series of modified oligonucleotide was designed based on ISIS 460209 or ISIS 476333, wherein the gap region contains nine P-D-2'-deoxyribonucleosides. The modified oligonucleotides were designed by introducing methyl phosphonate internucleoside linkage within the gap region at a fixed position and using different wing motifs for 3/9/3 and 4/9/4 gapmer motifs. The modified oligonucleotides were tested for their ability to selectively inhibit mutant (mut) H7TmRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact. The potency and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209 or ISIS 476333.
The position on the oligonucleotides opposite to the SNP position, as counted from the 5'- terminus is position 8 for ISIS 460209 or position 9 for ISIS 476333.
The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.1, 0.4, 1.1, 3.3 and 10 μΜ concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C_2229297_10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2X PCR buffer, 101 uL primers (300 μΜ from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type H7 mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.
The IC50s and selectivities as expressed in "fold" were measured and calculated using methods described previously in Example 23. The results are presented below.
SEQ ID NO. Composition (5' to 3') /ISIS NO.
05/460209 TeAkAkAdTdTdGdTdmCdAdTdmCdAic mCkmCe
05/558257 TeAkAkAdTdTdxGdTdmCdAdTdmCdAicmCkmCe
05/571123 TeAeAeAk kTdxGdTdmCdAdTdmCdAkmCkmCe
05/579854 TeAeAeAkTdTdxGdTdmCdAdTdmCdAkmCkmCe
05/566282 TeAkAkAdxTdxTdGdTdmCdAdTdmCdAkmCkmCe
09/476333 AeTkAeAkAdTdTdGdTdmCdAdTdmCdAkmCemCkA e(
09/571171 AeTkAeAkAdTdTdxGdTdmCdAdTdmCdA mCe mCkAe
Each internucleoside linkage is a phosphorothioate (P=S) except for the internucleoside linkage having a subscript "x" which indicates a methyl phosphonate internucleoside linkage (-P(CH3)(=0)-). Each nucleoside followed by a subscript "d" is a p-D-2'-deoxyribonucleoside. Each nucleoside followed by a subscript "e" indicates a 2'-0-methoxyethyl (MOE) modified nucleoside. Each nucleoside followed by a subscript "k" indicates a bicyclic nucleoside having a 4'- CH((5)-CH3)-0-2* bridge also referred to as a (S)-cEt modified nucleoside. Each "mC" is a 5-methyl cytosine modified nucleoside. Nucleosides followed by subscripts "e" or "k" are further illustrated below.
Figure imgf000101_0001
subscript e subscript k.
SEQ ID NO. Mut IC50 Wt IC50 Fold Selectivity Gap Chemistry (motif)
/ISIS NO. (μΜ) (μΜ) (mut vs. wt)
05/460209 0.56 3.8 6.8 Positive control (3/9/3)
05/558257 1.7 >10 > 5.9 Single -P(CH3)(=0)- (3/9/3)
05/571123 0.96 >10 > 10.4 Single -P(CH3)(=0)- (5/7/3)
05/579854 0.63 >10 > 15.9 Single -P(CH3)(=0)- (4/8/3)
05/566282 0.51 6.3 12.4 Two -P(CH3)(=0)- (3/9/3)
09/476333 0.56 3.4 6.1 Positive control (4/9/4)
09/571171 0.54 >10 > 18.5 Single -P(CH3)(=0)- (4/9/4).
Example 26
Modified oligonucleotides comprising methyl phosphonate internucleoside linkages targeting PTEN and SRB-1 - in vivo study
Additional modified oligonucleotides were designed based on ISIS 482050 and 449093 wherein the gap region contains ten P-D-2'-deoxyribonucleosides. The modified oligonucleotides were designed by introducing two methyl phosphonate internucleoside linkages at the 5 '-end of the gap region with a 3/10/3 motif. The oligonucleotides were evaluated for reduction in PTEN and SRB-1 mRNA expression levels in vivo. The parent gapmers, ISIS 482050 and 449093 were included in the study for comparison.
Treatment
Six week old BALB/C mice (purchased from Charles River) were injected subcutaneously twice a week for three weeks at dosage 10 mg/kg or 20 mg/kg with the modified oligonucleotides shown below or with saline control. Each treatment group consisted of 3 animals. The mice were sacrificed 48 hrs following last administration, and organs and plasma were harvested for further analysis. mRNA Analysis
Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to RIBOGREEN as described herein. The results below are listed as PTEN or SRB-1 mRNA expression for each treatment group relative to saline-treated control (% UTC). As illustrated, reduction in PTEN or SRB-1 mRNA expression levels was achieved with the oligonucleotides comprising two methyl phosphonate internucleoside linkages at the 5 '-end of the gap region, ISIS 582073 and 582074.
Plasma chemistry markers
Plasma chemistry markers such as liver transaminase levels, alanine aminotranferase (ALT) in serum were measured relative to saline injected mice and the results are presented below.
Treatment with the oligonucleotides resulted in a reduction in ALT level compared to treatment with the parent gapmer, ISIS 482050 or 449093. The results suggest that introduction of methyl phosphonate internucleoside linkage(s) can be useful for reduction of hepatotoxicity profile of otherwise unmodified parent gapmers.
Body and organ weights
Body weights, as well as liver, kidney and spleen weights were measured at the end of the study. The results below are presented as the average percent of body and organ weights for each treatment group relative to saline-treated control. As illustrated, treatment with ISIS 582073 resulted in a reduction in liver and spleen weights compared to treatment with the parent gapmer, ISIS 482050. The remaining oligonucleotide, ISIS 582074 did not cause any changes in body and organ weights outside the expected range as compared to ISIS 449093.
SEQ ID NO. Composition (5' to 3')
/ISIS NO.
11/482050 AicTic mCkAdTdGdGdmCdT(1GdmCdAdGdmCkTicTk
11/582073 AkTkmCkAdxTdxGdGdmCdTdGdmCdAdGdmCkTkTk
12/449093 l k * k ^kAdlJd l d ^d^d l d^dAd ^d d l k <-k ^k
12/582074 I k t k ^k dx<Jd l d dAd l dtJdAd -d l d t k <-k <-k
Each intemucleoside linkage is a phosphorothioate (P=S) except for the intemucleoside linkage having a subscript "x". Each nucleoside followed by a subscript "x" indicates a methyl phosphonate intemucleoside linkage (-P(CH3)(=0)-). Each nucleoside followed by a subscript "d" is a P-D-2'-deoxyribonucleoside. Each nucleoside followed by a subscript "k" indicates a bicyclic nucleoside having a 4'-CH((£)-CH3)-0-2' bridge also referred to as a (S)-cEt modified nucleoside. Each ,,mC" is a 5-methyl cytosine modified nucleoside. Nucleosides followed by subscript "k" are further illustrated below.
Figure imgf000103_0001
subscript k.
SEQ ID NO. Target Dosage % UTC ALT Chemistry
/ISIS NO. (mg/kg/wk) (IU/L)
Saline ~ 0 100 30 ~
11/482050 PTEN 10 50 228 Full (P=S)
11/482050 20 36.1 505
11/582073 10 72.2 47.7 Two -P(CH3)(=0)
11/582073 20 57.4 46
12/449093 SRB-1 10 48 543 Full (P=S)
12/449093 20 18.5 1090
12/582074 10 51.3 58.3 Two -P(CH3)(=0)
12/582074 20 30.3 126.3 SEQ ID NO. Target Dosage Body wt rel to Liver/Body Spleen/Body Kidney/Body ISIS NO. (mg kg/wk) predose (%) Wt (%) Wt (%) Wt (%)
Saline — 0 108.4 100 100 100
11/482050 PTEN 10 107.4 154.9 141.8 115.7
11/482050 20 111.3 176.7 142.3 112.5
11/582073 10 108.9 122.9 111.7 100.0
11/582073 20 107.9 133.8 114.6 102.9
12/449093 SRB-1 10 101.3 105.9 117.9 89.3
12/449093 20 95.3 118.6 129.6 93.0
12/582074 10 107.1 92.2 116.4 89.2
12/582074 20 103.8 95.5 128.8 91.9.
Example 27
Modifled oligonucleotides comprising methyl phosphonate internucleoside linkages targeting Target-Y - in vivo study
Additional modifled oligonucleotides were designed in the same manner as the antisense oligonucleotides described in Example 25, wherein two methyl phosphonate internucleoside linkages are introduced at the 5 '-end of the gap region. The modified oligonucleotides were designed based on ISIS 464917, 465178, 465984 and 466456 with a 3/10/3 motif. The
oligonucleotides were evaluated for reduction in Target-Y mRNA expression levels in vivo. The parent gapmers, ISIS 464917, 465178, 465984 and 466456 were included in the study for comparison. Treatment
Six week old BALB/C mice (purchased from Charles River) were injected subcutaneously twice a week for three weeks at dosage 10 mg/kg or 20 mg/kg with the modified oligonucleotides shown below or with saline control. Each treatment group consisted of 3 animals. The mice were sacrificed 48 hrs following last administration, and organs and plasma were harvested for further analysis. mRNA Analysis
Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to RIBOGREEN as described herein. The results below are listed as Target-Y mRNA expression for each treatment group relative to saline-treated control (% UTC). As illustrated, reduction in Target-Y mRNA expression levels was achieved with the oligonucleotides comprising two methyl phosphonate intemucleoside linkages at the 5 '-end of the gap region, ISIS 582071, 582072, 582069 and 582070.
Plasma chemistry markers
Plasma chemistry markers such as liver transaminase levels, alanine aminotranferase (ALT) in serum were measured relative to saline treated mice and the results are presented below.
Treatment with the oligonucleotides resulted in a reduction in ALT level compared to treatment with the parent gapmer, ISIS 464917, 465178, 465984 or 466456. The results suggest that introduction of methyl phosphonate intemucleoside linkage(s) can be useful for reducing the hepatotoxicity profile of otherwise unmodified parent gapmers.
Body and organ weights
Body weights, as well as liver, kidney and spleen weights were measured at the end of the study. The results below are presented as the average percent of body and organ weights for each treatment group relative to saline-treated control. As illustrated, treatment with ISIS 582070 resulted in a reduction in liver and spleen weights compared to treatment with the parent gapmer, ISIS 466456. An increase in body and organ weights was observed for ISIS 582071 as compared to ISIS 464917. The remaining oligonucleotides, ISIS 582072 and 582069 did not cause any changes in body and organ weights outside the expected range as compared to ISIS 465178 and 465984.
SEQ ID NO. Composition (5' to 3')
/ISIS NO.
13/464917 NkNkNkNdNdNdNdNdNdNdNdNdNdNkNkNk
13/582071 NkNkNkNdxNcuNdNdNdNdNdNdNdNdNkNkNk
14/465178 NkNkNkNdNdNdNdNdNdNdNdNdNdNkNkNk
14/582072 NkNkNkNdxNdxNdNdNdNdNdNdNdNdNkNkNk
15/465984 NkNkNkNdNdNdNdNdNdNdNdNdNdNeNeNe
15/582069 NkNkNkNdxNdxNdNdNdNdNcNdNdNdNkNfcNk
16/466456 NkNdNkNdNkNdNdNdNdNdNdNdNdNdNeNe
16/582070 NkNdNkNdxNdxNdNdNdNdNdNdNdNdNdNeNe
Each intemucleoside linkage is a phosphorothioate (P=S) except for the intemucleoside linkage having a subscript "x". Each nucleoside followed by a subscript "x" indicates a methyl phosphonate intemucleoside linkage (-P(CH3)(=0)-). Each nucleoside followed by a subscript "d" is a P-D-2'-deoxyribonucleoside. Each nucleoside followed by a subscript "e" indicates a 2'-0- methoxyethyl (MOE) modified nucleoside. Each nucleoside followed by a subscript "k" indicates a bicyclic nucleoside having a 4'-CH((S)-CH3)-0-2' bridge also referred to as a (S)-cEt modified nucleoside. Each "N" is a modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C). Nucleosides followed b subscript "e" or "k" are further illustrated below.
Figure imgf000106_0001
subscript e subscript k.
SEQ ID NO. Dosage % UTC ALT Chemistry
/ISIS NO. (mg kg/wk) (IU L)
Saline 0 100 30 —
13/464917 10 29 1244 Full (P=S)
13/464917 20 30.1 2335
13/582071 20 10.2 274 Two -P(CH3)(=0)-
14/465178 10 4.9 1231 Full (P=S)
14/465178 20 10.6 6731
14/582072 10 36.7 44.7 Two -P(CH3)(=0)-
14/582072 20 23.6 43.7
15/465984 10 4.7 61 Full (P=S)
15/465984 20 0.9 57
15/582069 10 1 1.1 39.7 Two -P(CH3)(=0)-
15/582069 20 3.3 27.7
16/466456 10 9.5 692 Full (P=S)
16/466456 20 10.5 2209
16/582070 10 73.9 24 Two -P(CH3)(=0)-
16/582070 20 51.3 36.7 SEQ ID NO. Dosage Body wt rel to Liver/Body Spleen/Body Kidney/Body
/ISIS NO. (mg/kg/wk) predose (%) Wt (%) Wt (%) Wt (%)
Saline 0 108 100 100 100
13/464917 10 92.9 125 106.2 102.3
13/464917 20 71.1 110.9 67.2 107.3
13/582071 20 104.6 135.2 142.8 89.8
14/465178 10 94.9 131.3 108.1 85.3
14/465178 20 79.5 147.5 112 95.3
14/582072 10 109.2 1 17.3 1 1 1.7 104.8 14/582072 20 107.1 130.1 107.2 99.8
15/465984 10 111.4 117.6 110.1 98.8
15/465984 20 111.3 122.6 134.5 96.1 15/582069 10 107.8 106.2 97 100.6
15/582069 20 105.4 115.8 106.2 100.4
16/466456 10 109.7 148.6 198.7 105.9
16/466456 20 101.2 182.3 213.7 101.9 16/582070 10 111.2 100.3 116.7 100.8
16/582070 20 111.1 108.9 115.6 95.7.

Claims

Claims:
1. A gapped oligomeric compound comprising a contiguous sequence of linked monomer subunits having a gap region located between a 5'-region and a 3'-region wherein the 5' and 3'- regions each, independently, have from 2 to 8 contiguous modified nucleosides wherein essentially each modified nucleoside in the 5' and 3'-regions are RNA-like and the gap region has from 6 to 14 contiguous monomer subunits selected from p-D-2'-deoxyribonucleosides and modified nucleosides that are DNA like and wherein at least one of the intemucleoside linking groups in the gap region or linking the gap region and the 5 -region or the 3'-region has Formula I:
I
o
X=P-Q
o
I
I
wherein independently for each intemucleoside linking group having Formula I:
X is O or S;
Q is C C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C(=0)OH or CH2C(=0)OH;
each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJls SJi and OC(=0)J!; and
each Ji is, independently, H or Ci-C6 alkyl.
2. The gapped oligomeric compound of claim 1 having only two intemucleoside linking groups of Formula I in the gap region.
3. The gapped oligomeric compound of claim 1 having only three intemucleoside linking groups of Formula I in the gap region.
4. The gapped oligomeric compound of any of claims 2 or 3 wherein the intemucleoside linking groups having Formula I are contiguous.
5. The gapped oligomeric compound of claim 1 having at least two intemucleoside linking groups of Formula I in the gap region that are separated by at least one phosphorothioate or phosphodiester intemucleoside linking group.
6. The gapped oligomeric compound of any of claims 1 to 5 wherein the intemucleoside linking group linking the 5'-region and the gap region has Formula I.
7. The gapped oligomeric compound of any of claims 1 to 6 wherein the intemucleoside linking group linking the 3'-region and the gap region has Formula I.
8. The gapped oligomeric compound of any of claims 1 to 6 wherein the intemucleoside linking group linking the 5 -region and the gap region has Formula I and the intemucleoside linking group linking the 3 '-region and the gap region has Formula I.
9. The gapped oligomeric compound of claim 1 having only one intemucleoside linking group of Formula I in the gap region.
10. The gapped oligomeric compound of any of claims 1 to 9 wherein each intemucleoside linking group in the 5' and 3 '-regions and each intemucleoside linking group in the gap region other than intemucleoside linking groups having Formula I is a phosphodiester or a phosphorothioate intemucleoside linking group.
11. The gapped oligomeric compound of any of claims 1 to 9 wherein each intemucleoside linking group in the 5' and 3 '-regions and each intemucleoside linking group in the gap region other than intemucleoside linking groups having Formula I is a phosphorothioate intemucleoside linking group.
12. The gapped oligomeric compound of any of claims 1 to 11 wherein each monomer subunit comprises a heterocyclic base independently selected from an optionally protected purine, substituted purine, pyrimidine or substituted pyrimidine.
13. The gapped oligomeric compound of any of claims 1 to 12 wherein each monomer subunit comprises a heterocyclic base independently selected from uracil, thymine, cytosine, 4-N- benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine or 2-N-isobutyrylguanine.
14. The gapped oligomeric compound of any of claims 1 to 13 wherein each Q is,
independently, selected from Q-C6 alkyl, substituted C!-C6 alkyl, C2-C6 alkenyl, C(=0)OH and CH2C(=0)OH.
15. The gapped oligomeric compound of any of claims 1 to 14 wherein each Q is, independently, selected from CH3, C(=0)OH, CH2C(=0)OH, (CH2)2OCH3, CH=CH2, CH2CH=CH2 and C≡CH.
16. The gapped oligomeric compound of any of claims 1 to 15 wherein each Q is CH2C(=0)OH.
17. The gapped oligomeric compound of any of claims 1 to 15 wherein each Q is CH3.
18. The gapped oligomeric compound of any of claims 1 to 17 wherein each X is O.
19. The gapped oligomeric compound of any of claims 1 to 17 wherein each X is S.
20. The gapped oligomeric compound of any of claims 1 to 19 wherein the chirality of each internucleoside linking group having Formula I is Rp.
21. The gapped oligomeric compound of any of claims 1 to 19 wherein the chirality of each internucleoside linking group having Formula I is Sp.
22. The gapped oligomeric compound of any of claims 1 to 21 wherein each modified nucleoside in the 5' and 3'-regions provides enhanced hybridization affinity for an RNA target as compared to an unmodified nucleoside.
23. The gapped oligomeric compound of any of claims 1 to 22 wherein each modified nucleoside in the 5' and 3'-regions comprises a modified sugar moiety.
24. The gapped oligomeric compound of claim 23 wherein each modified nucleoside in the 5' and 3 '-regions is, independently, a bicyclic nucleoside comprising a bicyclic furanosyl sugar moiety or a modified nucleoside comprising a furanosyl sugar moiety having at least one substituent group.
25. The gapped oligomeric compound of claim 24 comprising one or more 2'-modified nucleosides that each have a 2'-substituent group independently selected from halogen, OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, 0(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, 0(CH2)2- OCH3, 0(C¾)2-SCH3, 0(CH2)2-OCF3, 0(CH2)3-N(R3)(R4), 0(CH2)2-ON(R3)(R4), 0(CH2)2- 0(CH2)2-N(R3)(R4), OCH2C(=0)-N(R4)(R4), OCH2C(=0)-N(R5)-(CH2)2-N(R3)(R4) and 0(CH2)2- N(R5)-C(=NR6)[N(R3)(R4)] wherein R3, R4, R5 and ¾ are each, independently, H and C C6 alkyl.
26. The gapped oligomeric compound of claim 25 wherein each 2'-substituent group is independently selected from F, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, 0(CH2)2- OCH3, 0(CH2)2-0(CH2)2-N(CH3)2, OCH2C(=0)-N(H)CH3, OCH2C(=0)-N(H)-(CH2)2-N(CH3)2 and OCH2-N(H)-C(=NH)NH2.
27. The gapped oligomeric compound of claim 26 wherein each 2'-substituent group is independently selected from F, OCH3, 0(CH2)2-OCH3 and OCH2C(=0)-N(H)CH3.
28. The gapped oligomeric compound of claim 27 wherein each 2'-substituent group is 0(CH2)2- OCH3.
29. The gapped oligomeric compound of claim 24 comprising one or more bicyclic nucleosides that each have a bridging group between the 4' and 2' carbon atoms of the furanosyl ring
independently selected from 4,-(CH2)-0-2*, 4'-(CH2)-S-2, 5 4*-(CH2)2-0-2', 4'-CH(CH3)-0-2', 4'-C- H(CH2OCH3)-0-2', 4,-C(CH3)2-0-2', 4'-CH2-N(OCH3)-2', 4'-CH2-0-N(CH3)-2', 4'-CH2-NCH3-0-2*, 4'-CH2-C(H)(CH3)-2* and 4'-CH2-C(=CH2)-2'.
30. The gapped oligomeric compound of claim 29 wherein each of the bridging groups is independently selected from 4'-(CH2)-0-2', 4'-(CH2)2-0-2', 4'-CH(CH3)-0-2*, 4*-CH2-NCH3-0-2*, 4'- CH2-C(H)(CH3)-2' and 4'-CH2-C(=CH2)-2'.
31. The gapped oligomenc compound of claim 30 wherein each bridging group is 4'-CH[(_S)- (CH3)]-0-2'.
32. The gapped oligomeric compound of any one of claims 1 to 31 wherein the sugar moieties of each modified nucleoside in the 5' and 3 '-regions are the same.
33. The gapped oligomeric compound of any of claims 1 to 31 comprising at least two different types of modified nucleosides in the 5' and 3 '-regions wherein the different types of modified nucleosides have at least different modified sugar moieties.
34. The gapped oligomeric compound of claim 33 wherein the different types of modified nucleosides include bicyclic nucleosides comprising bicyclic furanosyl sugar moieties and modified nucleosides comprising furanosyl sugar moieties having at least one substituent group.
35. The gapped oligomeric compound of claim 34 wherein the different types of modified nucleosides include 4'-CH[(S)-(CH3)]-0-2' bicyclic nucleosides and 2'-0(CH2)2-OCH3 substituted nucleosides.
36. The gapped oligomeric compound of claim 35 wherein the 5' and 3'-regions include only 4'- CH[(5)-(CH3)]-0-2' bicyclic nucleosides and 2'-0(CH2)2-OCH3 substituted nucleosides.
37. The gapped oligomeric compound of any of claims 1 to 35 wherein one or more modified nucleosides in the 5' and 3'-regions comprise a sugar surrogate.
38. The gapped oligomeric compound of any of claims 1 to 37 wherein each monomer subunit in the gap region is a P-D-2'-deoxyribonucleoside.
39. The gapped oligomeric compound of any of claims 1 to 37 wherein at least one monomer subunit in the gap region is a modified nucleoside that is DNA-like.
40. The gapped oligomeric compound of claim 39 wherein each modified nucleoside that is DNA-like in the gap region is a 2'-(ara)-F modified nucleoside.
I l l
41. The gapped oligomeric compound of any of claims 1 to 40 wherein the 5' and 3 '-regions each, independently, have from 2 to 8 monomer subunits.
42. The gapped oligomeric compound of any of claims 1 to 40 wherein the 5' and 3 -regions each, independently, have from 3 to 6 monomer subunits.
43. The gapped oligomeric compound of any of claims 1 to 42 wherein the gap region has from 6 to 14 monomer subunits.
44. The gapped oligomeric compound of any of claims 1 to 42 wherein the gap region has from 8 to 10 monomer subunits.
45. The gapped oligomeric compound of any of claims 1 to 40 wherein the 5' and 3 -regions each, independently, have from 3 to 6 monomer subunits and the gap region has from 6 to 10 monomer subunits.
46. The gapped oligomeric compound of any of claims 1 to 40 wherein the 5' and 3'-regions each, independently, have from 3 to 6 monomer subunits and the gap region has from 6 to 8 monomer subunits.
47. The gapped oligomeric compound of any of claims 1 to 40 wherein the 5' and 3 -regions each, independently, have from 4 to 5 monomer subunits and the gap region has from 7 to 8 monomer subunits.
48. The gapped oligomeric compound of claim 1 wherein at least one modified nucleoside in the 5' and 3'-regions is other than a 2'-OCH3 substituted nucleoside or a 2'-0-CH2-4' bridged bicyclic nucleoside.
49. The gapped oligomeric compound of any of claims 1 to 48 further comprising at least one 5' or 3 '-terminal group.
50. A method of inhibiting gene expression comprising contacting one or more cells, a tissue or an animal with an oligomeric compound of any of claims 1 to 49 wherein said oligomeric compound is complementary to a target RNA.
51. The method of claim 50 wherein said cells are in a human.
52. The method of claim 50 wherein said target RNA is human mRNA.
53. The method of claim 50 wherein said target RNA is cleaved thereby inhibiting its function.
54. An in vitro method of inhibiting gene expression comprising contacting one or more cells or a tissue with an oligomeric compound of any one of claims 1 to 49.
55. An oligomeric compound of any one of claims 1 to 49 for use in an in vivo method of inhibiting gene expression said method comprising contacting one or more cells, a tissue or an animal with an oligomeric compound of any of claims 1 to 49.
56. An oligomeric compound of any one of claims 1 to 49 for use in medical therapy.
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