WO2016154096A1 - Modulation of smggds expression - Google Patents

Modulation of smggds expression Download PDF

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Publication number
WO2016154096A1
WO2016154096A1 PCT/US2016/023372 US2016023372W WO2016154096A1 WO 2016154096 A1 WO2016154096 A1 WO 2016154096A1 US 2016023372 W US2016023372 W US 2016023372W WO 2016154096 A1 WO2016154096 A1 WO 2016154096A1
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compound
modified
smggds
certain embodiments
oligonucleotide
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PCT/US2016/023372
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French (fr)
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Frank Rigo
Mark T. MCNALLY
Carol L. WILLIAMS
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Ionis Pharmaceuticals, Inc.
Medical College Of Wisconsin, Inc.
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Publication of WO2016154096A1 publication Critical patent/WO2016154096A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • Small G-protein guanine nucleotide dissociation stimulator 1 also known as Rapl, GTP- GDP dissociation stimulator 1 (RAP1GDS1)
  • Rapl GTP- GDP dissociation stimulator 1
  • RAP1GDS1 GTP- GDP dissociation stimulator 1
  • SmgGDS-558 which lacks exon 5
  • SmgGDS-558 which lacks exon 5
  • SmgGDS is over expressed in breast cancer and its expression correlates with poor prognosis.
  • 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 RNA (mRNA) 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-mRNA.
  • mRNA target messenger RNA
  • the present invention provides compounds comprising oligonucleotides.
  • such oligonucleotides are complementary to a SmgGDS transcript.
  • oligonucleotides are complementary to a target region of the SmgGDS transcript comprising exon 5.
  • oligonucleotides are complementary to a target region of the SmgGDS transcript comprising an intron adjacent to exon 5.
  • oligonucleotides are complementary to a target region of the SmgGDS transcript comprising an intron adjacent to exon 5 and downstream of exon 5.
  • oligonucleotides are complementary to a target region of the SmgGDS transcript comprising an intron adjacent to exon 5 and upstream of exon 5.
  • the SmgGDS transcript comprises an exonic splice enhancer for exon 5.
  • oligonucleotides inhibit inclusion of exon 5. In certain embodiments, oligonucleotides promote inclusion of exon 5. In certain embodiments, oligonucleotides promote skipping of exon 5. In certain embodiments, oligonucleotides inhibit skipping of exon 5. In certain embodiments, oligonucleotides promote selection of exon 5. In certain embodiments, oligonucleotides inhibit selection of exon 5. In certain embodiments, oligonucleotides increase levels of SmgGDS mRNA including exon 5 in a cell. In certain embodiments, oligonucleotides increase levels of SmgGDS mRNA lacking exon 5 in a cell. In certain embodiments, oligonucleotides increase SmgGDS-607 protein levels in a cell. In certain embodiments, oligonucleotides increase SmgGDS-558 protein levels in a cell.
  • oligonucleotides modulate the relative levels of SmgGDS mRNA including exon 5 and SmgGDS mRNA lacking exon 5 in a cell. In certain embodiments, oligonucleotides increase levels of SmgGDS mRNA including exon 5 relative to SmgGDS mRNA lacking exon 5 in a cell. In certain embodiments, oligonucleotides increase levels of SmgGDS mRNA lacking exon 5 relative to SmgGDS mRNA including exon 5 in a cell.
  • oligonucleotides modulate the relative protein levels of SmgGDS-607 and SmgGDS-558 in a cell. In certain embodiments, oligonucleotides increase protein levels of SmgGDS-607 relative to SmgGDS-558 in a cell. In certain embodiments, oligonucleotides increase protein levels of SmgGDS-558 relative to SmgGDS-607 in a cell.
  • a method comprises contacting a cancer cell with a compound or oligonucleotide provided herein, thereby (i) altering splicing of SmgGDS mRNA in the cell, (ii) increasing levels of SmgGDS-607 mRNA or protein in the cell, (iii) increasing levels of SmgGDS-558 mRNA or protein in the cell, (iv) increasing the ratio of SmgGDS-607 mRNA or protein to SmgGDS-558 mRNA or protein in the cell, (v) increasing the ratio of SmgGDS-558 mRNA or protein to SmgGDS-607 mRNA or protein in the cell, and/or (vi) inhibiting cancer cell proliferation.
  • a method comprises administering a compound or oligonucleotide provided herein to a subject having cancer, thereby (i) altering splicing of SmgGDS mRNA, (ii) increasing levels of SmgGDS-607 mRNA or protein, (iii) increasing levels of SmgGDS-558 mRNA, (iv) increasing the ratio of SmgGDS-607 mRNA or protein to SmgGDS-558 mRNA, (v) increasing the ratio of SmgGDS-558 mRNA or protein to SmgGDS-607 mRNA or protein, (vi) inhibiting cancer growth or proliferation, (vii) inhibiting metastasis, (viii) increasing survival, and/or (ix) treating cancer in the subject.
  • the present disclosure provides the following non-limiting numbered embodiments:
  • Embodiment 1 A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region, wherein the complementary region comprises at least 8 contiguous nucleobases and is complementary to an equal-length portion within a target region of a SmgGDS transcript.
  • Embodiment 2 The compound of embodiment 1, wherein the target region of the SmgGDS transcript
  • Embodiment 3 The compound of embodiment lor 2, wherein the complementary region of the modified oligonucleotide is 100% complementary to the target region.
  • Embodiment 4 The compound of any of embodiments 1 to 3, wherein the complementary region of the modified oligonucleotide comprises at least 10 contiguous nucleobases.
  • Embodiment 6 The compound of any of embodiments 1 to 3, wherein the complementary region of the modified oligonucleotide comprises at least 20 contiguous nucleobases.
  • Embodiment 7 The compound of any of embodiments 1-6, wherein the nucleobase sequence of the
  • oligonucleotide is at least 80% complementary to the target region, as measured over the entire length of the oligonucleotide.
  • Embodiment 8 The compound of any of embodiments 1-6, wherein the nucleobase sequence of the
  • oligonucleotide is at least 90% complementary to an equal-length region of the SmgGDS transcript, as measured over the entire length of the oligonucleotide.
  • Embodiment 9 The compound of any of embodiments 1-6, wherein the nucleobase sequence of the
  • oligonucleotide is 100% complementary to an equal -length region of the SmgGDS transcript, as measured over the entire length of the oligonucleotide.
  • Embodiment 10 The compound of any of embodiments 1-9, wherein the target region is within exon 5 or flanking intronic regions of the SmgGDS transcript.
  • Embodiment 11 The compound of any of embodiments 1-10, wherein the target region is within nucleobase 118642 and nucleobase 118788 of SEQ ID NO. 1.
  • Embodiment 12 The compound of any of embodiments 1-10, wherein the target region is within nucleobase 118557 and nucleobase 118641 of SEQ ID NO.: 1.
  • Embodiment 13 The compound of any of embodiments 1-10, wherein the target region is within nucleobase 118789 and nucleobase 118859 of SEQ ID NO.: 1.
  • Embodiment 14 The compound of any of embodiments 1-10, wherein the target region is within nucleobase 118557 and nucleobase 118859 of SEQ ID NO.: 1.
  • Embodiment 15 The compound of any of embodiments 1-10, wherein the target region is within nucleobase 118792 and nucleobase 118823 of SEQ ID NO.: 1.
  • Embodiment 16 The compound of any of embodiments 1-15, wherein the antisense oligonucleotide has a nucleobase sequence comprising any one of SEQ ID NOs: 2-54.
  • Embodiment 17 The compound of any of embodiments 1-16, wherein the modified oligonucleotide comprises at least one modified nucleoside.
  • Embodiment 18 The compound of embodiment 17, wherein at least one modified nucleoside
  • Embodiment 20 The compound of embodiment 19, wherein the 2'-substitutent of at least one 2'- substituted sugar moiety is selected from among: 2'-OMe, 2'-F, and 2'-MOE.
  • Embodiment 21 The compound of any of embodiments 17-20, wherein the 2'-substiuent of at least one 2 '-substituted sugar moiety is a 2'-MOE.
  • Embodiment 22 The compound of any of embodiments 1-18, wherein at least one modified sugar moiety is a bicyclic sugar moiety.
  • Embodiment 23 The compound of embodiment 22, wherein at least one bicyclic sugar moiety is LNA or cEt.
  • Embodiment 24 The compound of any of embodiments 18-23, wherein at least one sugar moiety is a sugar surrogate.
  • Embodiment 25 The compound of embodiment 24, wherein at least one sugar surrogate is a
  • Embodiment 26 The compound of embodiment 24, wherein at least one sugar surrogate is a modified morpholino.
  • Embodiment 28 The compound of embodiment 27, wherein the modified oligonucleotide comprises at least 10 modified nucleosides, each independently comprising a modified sugar moiety.
  • Embodiment 29 The compound of embodiment 27, wherein the modified oligonucleotide comprises at least 15 modified nucleosides, each independently comprising a modified sugar moiety.
  • Embodiment 30 The compound of embodiment 27, wherein each nucleoside of the modified oligonucleotide comprises at least 15 modified nucleosides, each independently comprising a modified sugar moiety.
  • Embodiment 31 The compound of any of embodiments 1-30, wherein the modified oligonucleotide comprises at least two modified nucleosides comprising modified sugar moieties that are the same as one another.
  • Embodiment 34 The compound of embodiment 33, wherein the modified oligonucleotide comprises a modified region of at least 10 contiguous modified nucleosides.
  • Embodiment 35 The compound of embodiment 33, wherein the modified oligonucleotide comprises a modified region of at least 15 contiguous modified nucleosides.
  • Embodiment 36 The compound of embodiment 33, wherein the modified oligonucleotide comprises a modified region of at least 20 contiguous modified nucleosides.
  • Embodiment 37 The compound of any of embodiments 32-36, wherein each modified nucleoside of the modified region has a modified sugar moiety independently selected from among: 2'-F, 2'-OMe, 2'- MOE, cEt, LNA, morpholino, and modified morpholino.
  • Embodiment 38 The compound of any of embodiments 33-37, wherein the modified nucleosides of the modified region each comprise the same modification as one another.
  • Embodiment 39 The compound of embodiment 38, wherein the modified nucleosides of the modified region each comprise the same 2 '-substituted sugar moiety.
  • Embodiment 40 The compound of embodiment 38, wherein the 2 '-substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from 2'-F, 2'-OMe, and 2'-MOE.
  • Embodiment 41 The compound of embodiment 39, wherein the 2 ' -substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is 2'-MOE.
  • Embodiment 43 The compound of embodiment 42, wherein the bicyclic sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from LNA and cEt.
  • Embodiment 44 The compound of embodiment 38, wherein the modified nucleosides of the region of modified nucleosides each comprises a sugar surrogate.
  • Embodiment 48 The compound of any of embodiments 1-46, wherein each nucleoside of the
  • modified oligonucleotide is a modified nucleoside.
  • Embodiment 50 The compound of embodiment 49, wherein the modified nucleosides of the modified oligonucleotide comprise the same modification as one another.
  • Embodiment 53 The compound of embodiment 52, wherein the 2 '-substituted sugar moiety of the modified oligonucleotide is 2'-MOE.
  • Embodiment 54 The compound of embodiment 50, wherein the modified nucleosides of the modified oligonucleotide each comprise the same bicyclic sugar moiety.
  • Embodiment 58 The compound of embodiment 56, wherein the sugar surrogate of the modified oligonucleotide is a modified morpholino.
  • Embodiment 59 The compound of any of embodiments 1-58, wherein the modified oligonucleotide comprises at least one modified intemucleoside linkage.
  • Embodiment 60 The compound of embodiment 59, wherein each intemucleoside linkage is a
  • Embodiment 61 The compound of embodiment 59 or 60, comprising at least one phosphorothioate intemucleoside linkage.
  • Embodiment 62 The compound of embodiment 60, wherein each intemucleoside linkage is a
  • Embodiment 64 The compound of any of embodiments 1-63 comprising at least one conjugate.
  • Embodiment 65 The compound of any of embodiments 1-64 consisting of the modified
  • Embodiment 68 The pharmaceutical composition of embodiment 67, wherein the pharmaceutically acceptable carrier or diluent is sterile saline.
  • Embodiment 69 A method of modulating splicing of a SmgGDS transcript in a cell comprising
  • Embodiment 70 The method of embodiment 69, wherein the cell is in vitro.
  • Embodiment 71 The method of embodiment 69, wherein the cell is in an animal.
  • Embodiment 72 The method of any of embodiments 69-71, wherein the cell is a cancer cell.
  • Embodiment 73 The method of any of embodiments 69-72, wherein inclusion of exon 5 is increased.
  • Embodiment 74 The method of any of embodiments 69-72, wherein exclusion of exon 5 is increased.
  • Embodiment 75 The method of any of embodiments 69-72, wherein SmgGDS-607 mRNA expression is increased.
  • Embodiment 76 The method of any of embodiments 76-72, wherein SmgGDS-558 mRNA expression is decreased.
  • Embodiment 78 The method of embodiment 77, wherein SmgGDS-607 expression is increased.
  • Embodiment 80 The method of embodiment 77, wherein the cell is in vitro.
  • Embodiment 81 The method of embodiment 77, wherein the cell is in an animal.
  • Embodiment 82 A method of inhibiting cell proliferation, comprising contacting the cell with a
  • Embodiment 83 The method of embodiment 82, wherein the cell is in vitro.
  • Embodiment 84 The method of embodiment 82, wherein the cell is in an animal.
  • Embodiment 85 The method of any of embodiments 82-84 wherein the cell is a cancer cell.
  • Embodiment 87 The method of embodiment 86, wherein the compound is administered systemically.
  • Embodiment 88 The method of embodiment 87, wherein the compound is administered via
  • Embodiment 89 The method of any of embodiments 86-88, wherein the animal has cancer.
  • Embodiment 90 The method embodiment 89, wherein the cancer is breast cancer, prostate cancer, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
  • the cancer is breast cancer, prostate cancer, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
  • Embodiment 91 The method of embodiment 89 or 90, wherein the administration results in
  • Embodiment 92 The method of any of embodiments 86-91, wherein the animal is a mouse.
  • Embodiment 93 The method of any of embodiments 86-91, wherein the animal is a human.
  • Embodiment 94 A method of treating cancer in an animal, comprising administering the compound according to any of embodiments 1-66 or composition according to embodiment 67 or 68 to an animal in need thereof.
  • Embodiment 95 The method of embodiment 94, wherein the animal is a mouse.
  • Embodiment 96 The method of embodiment 94, wherein the animal is a human.
  • Embodiment 97 The method of any of embodiments 94-96, wherein the cancer is breast cancer, prostate cancer, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer..
  • the cancer is breast cancer, prostate cancer, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
  • Embodiment 99 The compound according to any of embodiments 1-66 or composition according to embodiment 67 or 68 for use in treating cancer.
  • Embodiment 100 The use of embodiment 98 or 99, wherein the cancer is breast cancer, prostate
  • lung cancer squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
  • SCCHN squamous cell carcinoma of the head and neck
  • pancreatic cancer pancreatic cancer
  • Figure 1 Diagram of the SmgGDS genomic region.
  • the SmgGDS genomic region comprises numbered exons represented by vertical lines. Numbers under the vertical lines correspond to the exon number.
  • SmgGDS-607 includes exon 5 whereas SmgGDS-558 excludes exon 5.
  • a zoom-in of the genomic region spanning exons 4-6 and intervening introns is shown and indicates alternative splicing of exon 5 to generate either SmgGDS-607 or SmgGDS-558.
  • Figure 2 Plot of ASOs along the genomic transcript of SmgGDS. Exon 5 (147 nt) is indicated as a box and the flanking intronic regions are indicated by nucleotide position relative to the 5 ' and 3 ' ends of exon 5. Dark bars above the genomic transcript represent ASO alignment to the transcript and ASO number.
  • a zoom-in of the transcript region targeted by ISIS 704507 (CIO) and microwalk is shown.
  • FIG. 3 RT-PCR gel of ASO treated cells. ASOs were transfected in MDA-MB-231 cells. RT-PCR of endogenous SmgGDS transcripts is shown. The ASO number is indicated at the top of the gel. cDNA amplicons and fragments corresponding to SmgGDS-607 ("607") and SmgGDS-558 ("558") are labeled.
  • FIG. 4 Western Blot of ASO treated cells. ASOs were transfected in MDA-MB-231 cells. Western Blot of endogenous SmgGDS transcripts is shown. The ASO number is indicated above the gel lanes. Protein bands corresponding to SmgGDS-607 ("607") and SmgGDS-558 ("558") are labeled. Percent of SmgGDS-558 ("558") is indicated under the gel (Fig. 4a). Cells were treated with ASO for 48 hours (Fig. 4b) or 72 hours (Fig. 4c).
  • 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.
  • chemical modification means a chemical difference in a compound when compared to a naturally occurring counterpart.
  • chemical modification does not include differences only in nucleobase sequence.
  • Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications.
  • furanosyl means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.
  • sugar moiety means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.
  • modified sugar moiety means a substituted sugar moiety, a bicyclic or tricyclic sugar moiety, or a sugar surrogate.
  • substituted sugar moiety means a furanosyl comprising at least one substituent group that differs from that of 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.
  • 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 .
  • 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.
  • sugar surrogate means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside is capable of (1) incorporation into an oligonucleotide and (2) hybridization to a complementary nucleoside.
  • Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen.
  • 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).
  • heterocyclic base or “heterocyclic nucleobase” means a nucleobase comprising a heterocyclic structure.
  • unmodified nucleobase or “naturally occurring nucleobase” means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5 -methyl C), and uracil (U).
  • modified nucleobase means any nucleobase that is not a naturally occurring nucleobase.
  • modified nucleoside means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides comprise a modified sugar moiety and/or a modified nucleobase.
  • constrained ethyl nucleoside or “cEt” means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH 3 )-0-2'bridge.
  • 2 '-substituted nucleoside means a 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 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).
  • 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 internucleoside linkages contains a phosphorus atom.
  • 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.
  • oligomeric compound means a polymeric structure comprising two or more substructures.
  • an oligomeric compound comprises an oligonucleotide.
  • an oligomeric compound comprises one or more conjugate groups and/or terminal groups.
  • an oligomeric compound consists of an oligonucleotide.
  • terminal group means one or more atom attached to either, or both, the 3' end or the 5 ' end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more terminal group nucleosides.
  • conjugate means an atom or group of atoms bound to an oligonucleotide or oligomeric compound.
  • conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.
  • 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 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.
  • transcript means an RNA molecule transcribed from DNA.
  • Transcripts include, but are not limitied to mRNA, pre-mRNA, and partially processed RNA.
  • SmgGDS transcript means a transcript transcribed from a SmgGDS gene.
  • a SmgGDS transcript comprises GENBANK Accession No. NT_016354.20 truncated from nucleotides 39338995 to 39523480, which is incorporated by reference herein and designated as SEQ ID NO.: l .
  • SmgGDS gene means a gene that encodes a SmgGDS protein and any SmgGDS protein isoforms.
  • SmgGDS isoforms include SmgGDS-607 and SmgGDS-558.
  • a SmgGDS gene is represented by GENBANK Accession No. NT_016354.20 truncated from nucleotides 39338995 to 39523480, or a variant thereof.
  • a SmgGDS gene is at least 95% identical to GENBANK Accession No. NT_016354.20 truncated from nucleotides 39338995 to 39523480.
  • SmgGDS gene is at least 90% identical to GENBANK Accession No. NT_016354.20 truncated from nucleotides 39338995 to 39523480.
  • SmgGDS-607 means a SmgGDS transcript that includes exon 5.
  • SmgGDS-607 isoform means a SmgGDS protein isoform that includes amino acids encoded by exon 5.
  • SmgGDS-558 means a SmgGDS transcript that excludes exon 5.
  • SmgGDS-558 isoform means a SmgGDS protein isoform that excludes amino acids encoded by exon 5.
  • 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.
  • 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
  • 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).
  • complementary oligomeric compounds or regions are 80% complementary.
  • complementary oligomeric compounds or regions are 90% complementary.
  • complementary oligomeric compounds or regions are 95% complementary.
  • complementary oligomeric compounds or regions are 100% complementary.
  • 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.
  • 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-mRNA 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.
  • 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.
  • 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.
  • 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 R ⁇ , R ⁇ , and Rc C 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 (Ci-Ci 2 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, 1 -methyl -2 -buten-l-yl, dienes such as 1,3-butadiene and the like.
  • Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred.
  • Alkenyl groups as used herein may optionally include one or more further substituent groups.
  • alkynyl 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.
  • aminoalkyl means an amino substituted C1-C12 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.
  • 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 polycyclic 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 compounds of the invention comprise one or more modifed nucleosides comprising a modifed sugar moiety.
  • Such oligomeric 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 oligomeric compounds comprising only nucleosides comprising naturally occurring sugar moieties.
  • modified sugar moieties are substitued sugar moieties.
  • modified sugar moieties are bicyclic or tricyclic sugar moieties.
  • modified sugar moieties are sugar surrogates. Such sugar surogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.
  • 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 2008/101 157, for additional 5', 2'-bis substituted sugar moieties and 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 - 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(R a )(R b )] flesh-, -[C(R a )(R b )] admir-0-, -C(R a R b )-N(R)-0- or, -C(R a R b )-0-N(R)-; 4'-CH 2 -2', 4'-(CH 2 ) 2 -2', 4'-(CH 2 ) 3 -2',.
  • x 0, 1, or 2;
  • n 1, 2, 3, or 4;
  • Bx is a nucleobase moiety and R is, independently, H, a protecting group, or Ci-C 12 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.
  • a-L- methyleneoxy (4'-CH 2 -0-2') bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al, Nucleic Acids Research, 2003, 21, 6365-6372).
  • 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. Chem., 2006, 71, 7731-7740).
  • 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 (UNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, CJ. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), and those compounds having Formula
  • 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, q 2 , q 3 , q 4 , qs, qe and q 7 are each, independently, H, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, or substituted C 2 -C 6 alkynyl; and
  • the modified THP nucleosides of Formula VII are provided wherein q q 2 , q 3 , q , q 5 , q 6 and q 7 are each H. In certain embodiments, at least one of qi, q 2 , q 3 , q , q 5 , q 6 and q 7 is other than H. In certain embodiments, at least one of qi, q 2 , q 3 , q , q 5 , q 6 and q 7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of Ri and R 2 is F. In certain embodiments, Ri is fluoro and R 2 is H, Ri is methoxy and R 2 is H, and Ri is methoxyethoxy and R 2 is H.
  • sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom.
  • nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5,698,685; 5, 166,315; 5, 185,444; and 5,034,506).
  • morpholino means a sugar s llowing structure:
  • morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure.
  • sugar surrogates are refered to herein as "modifed morpholinos.”
  • 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.
  • modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein.
  • nucleobases include tricyclic pyrimidines such as phenoxazine cytidine( [5,4-b] [l,4]benzoxazin- 2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
  • nucleobases may 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.
  • nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J.I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al. , Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications , Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993, 273-288.
  • Non-phosphorus containing intemucleoside linking groups include, but are not limited to, methylenemethylimino (-CH 2 -N(CH 3 )-0-CH 2 -), thiodiester (-O-C(O)-S-), thionocarbamate (-0- C(0)(NH)-S-); siloxane (-0-Si(H) 2 -0-); and ⁇ , ⁇ '-dimethylhydrazine (-CH 2 -N(CH 3 )-N(CH 3 )-).
  • Modified linkages compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligomeric compound.
  • intemucleoside linkages having a chiral atom can be prepared 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 intemucleoside 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 stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), a or ⁇ such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
  • Further neutral intemucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral intemucleoside linkages include nonionic linkages comprising mixed N, O, S and CH 2 component parts.
  • the present invention provides oligomeric compounds comprising oligonucleotides.
  • such oligonucleotides comprise one or more chemical modification.
  • chemically modified oligonucleotides comprise one or more modified nucleosides.
  • chemically modified oligonucleotides comprise one or more modified nucleosides comprising modified sugars.
  • chemically modified oligonucleotides comprise one or more modified nucleosides comprising one or more modified nucleobases.
  • chemically modified oligonucleotides comprise one or more modified internucleoside linkages.
  • the chemically 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 modification motif.
  • Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.
  • the oligonucleotides comprise or consist of a region having a gapmer sugar modification motif, which comprises two external regions or "wings" and an internal region or "gap.”
  • the three regions of a gapmer 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 modification motifs of the two wings are the same as one another (symmetric gapmer).
  • the sugar modification motifs of the 5 '-wing differs from the sugar modification motif of the 3'-wing (asymmetric gapmer).
  • oligonucleotides comprise 2'-MOE modified nucleosides in the wings and 2'-F modified nucleosides in the gap.
  • oligonucleotides are fully modified. In certain such embodiments, oligonucleotides are uniformly modified. In certain embodiments, oligonucleotides are uniform 2'-MOE. In certain embodiments, oligonucleotides are uniform 2'-F. In certain embodiments, oligonucleotides are uniform morpholino. In certain embodiments, oligonucleotides are uniform BNA. In certain embodiments, oligonucleotides are uniform LNA. In certain embodiments, oligonucleotides are uniform cEt. In certain embodiments, oligonucleotides comprise a uniformly modified region and additional nucleosides that are unmodified or differently modified.
  • the uniformly modified region is at least 5, 10, 15, or 20 nucleosides in length. In certain embodiments, the uniform region is a - MOE region. In certain embodiments, the uniform region is a 2'-F region. In certain embodiments, the uniform region is a morpholino region. In certain embodiments, the uniform region is a BNA region. In certain embodiments, the uniform region is a LNA region. In certain embodiments, the uniform region is a cEt region.
  • the oligonucleotide does not comprise more than 4 contiguous unmodified 2'-deoxynucleosides.
  • antisesense oligonucleotides comprising more than 4 contiguous 2'-deoxynucleosides activate RNase H, resulting in cleavage of the target RNA.
  • such cleavage is avoided by not having more than 4 contiguous 2'-deoxynucleosides, for example, where alteration of splicing and not cleavage of a target RNA is desired.
  • oligonucleotides comprise modified intemucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified intemucleoside linkage motif.
  • intemucleoside linkages are arranged in a gapped motif, as described above for sugar modification motif.
  • the intemucleoside linkages in each of two wing regions are different from the intemucleoside linkages in the gap region.
  • the intemucleoside linkages in the wings are phosphodiester and the intemucleoside linkages in the gap are phosphorothioate.
  • the sugar modification motif is independently selected, so such oligonucleotides having a gapped intemucleoside linkage motif may or may not have a gapped sugar modification motif and if it does have a gapped sugar motif, the wing and gap lengths may or may not be the same.
  • oligonucleotides comprise a region having an alternating intemucleoside linkage motif. In certain embodiments, oligonucleotides of the present invention comprise a region of uniformly modified intemucleoside linkages. In certain such embodiments, the oligonucleotide 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 oligonucleotide is selected from phosphodiester and phosphorothioate. In certain
  • each intemucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one intemucleoside linkage is phosphorothioate.
  • the oligonucleotide comprises at least 6 phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages.
  • the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3' end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3' end of the oligonucleotide.
  • oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or nucleobases modification motif.
  • nucleobase modifications are arranged in a gapped motif.
  • nucleobase modifications are arranged in an alternating motif.
  • each nucleobase is modified.
  • none of the nucleobases is chemically modified.
  • some, all, or none of the 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.
  • the present invention provides oligomeric compounds including
  • oligonucleotides of any of a variety of ranges of lengths 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 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X ⁇ Y.
  • the 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.
  • Such additional nucleosides may be 5' of the 5 '-wing and/or 3' of the 3' wing.
  • oligonucleotides of the present invention are characterized by their sugar motif, intemucleoside linkage motif, nucleobase modification motif and overall length. In certain embodiments, such parameters are each independent of one another. Thus, each intemucleoside 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. Thus, the intemucleoside 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 intemucleoside linkages of the gap region.
  • sugar-gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications.
  • modified nucleobase independent of the gapmer pattern of the sugar modifications.
  • a description of an 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, intemucleoside 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, ( ⁇ S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • active drug substance for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, ( ⁇ S)-(+)-pranoprofen, carprofen, dansy
  • 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.
  • linking groups include, but are not limited to, substituted Ci- Cio alkyl, substituted or unsubstituted C 2 -Ci 0 alkenyl or substituted or unsubstituted C 2 -Ci 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.
  • oligomeric compounds of the present invention 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
  • 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.
  • antisense compounds and antisense oligonucleotides comprise single-strand compounds. In certain embodiments antisense compounds and antisense oligonucleotides comprise double- strand compounds.
  • Small GTPases including the Ras and Rho family members Rapl, K-Ras, RhoA, and Racl, are involved in signaling cascades that are often activated in cancer, such as breast cancer, and can lead to tumor development, progression, and escape from chemotherapy-induced apoptosis.
  • small GTPases must be prenylated at their C termini, which localizes and anchors the proteins to the plasma membrane where they interact with effector molecules.
  • SmgGDS is a master regulator of prenylation and membrane localization for the subset of GTPases that harbor a polybasic region. Consistent with its role in promoting malignancy, SmgGDS is overexpressed in cancer tissues, such as breast, prostate, and lung cancer where it promotes cell proliferation, migration, and NFkB signaling.
  • the administration of a compound or oligonucleotide provided herein to a subject having cancer or contacting a cancer cell with a compound or oligonucleotide provided herein causes a switch in the alternative splicing of the SmgGDS transcript.
  • the administration of an oligonucleotide provided herein causes increased inclusion of exon 5 mRNA of the SmgGDS transcript.
  • the administration of an oligonucleotide provided herein causes an increase in the exclusion of exon 5 mRNA of the SmgGDS transcript.
  • the administration of an oligonucleotide provided herein reduces expression of SmgGDS-558 in a cancer cell.
  • the administration of an oligonucleotide provided herein reduces expression of SmgGDS-558 in a cancer cell and inhibits cancer growth. In certain embodiments, the administration of an oligonucleotide provided herein reduces expression of SmgGDS-558 and induces apoptosis in a cancer cell.
  • the cancer cell is a breast cancer cell, prostate cancer cell, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
  • a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby reducing expression of SmgGDS-558 and inhibiting cancer cell growth or proliferation. In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby reducing expression of SmgGDS-558 and inducing apoptosis in a cancer cell.
  • the cancer cell is a breast cancer cell, prostate cancer cell, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
  • a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby reducing expression of SmgGDS-607 and inhibiting cancer cell growth or proliferation. In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby reducing expression of SmgGDS-607 and inducing apoptosis in a cancer cell.
  • the cancer cell is a breast cancer cell, prostate cancer cell, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
  • a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby increasing the relative level of SmgGDS-607 to SmgGDS-558 in a cancer cell and inhibiting cancer cell growth or proliferation.
  • a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby increasing the relative level of SmgGDS-558 to SmgGDS-607 in a cancer cell and inhibiting cancer cell growth or proliferation.
  • the cancer cell is a breast cancer cell, prostate cancer cell, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
  • a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby increasing exclusion of exon 5 of a SmgGDS transcript and inhibiting cancer growth or proliferation. In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby increasing inclusion of exon 5 of a SmgGDS transcript and inducing apoptosis in a cancer cell.
  • a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby downregulating SmgGDS-558 and leading to apoptosis of the cancer cell. In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby downregulating SmgGDS-607 and leading to apoptosis of the cancer cell.
  • the cancer cell is a breast cancer cell, prostate cancer cell, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
  • Certain embodiments are drawn to a method of treating cancer comprising administering to a subject having cancer a compound or oligonucleotide provided herein.
  • a subject having cancer a compound or oligonucleotide provided herein examples include, but are not limited to, breast cancer, prostate cancer, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
  • Certain embodiments are drawn to the use of a compound or oligonucleotide provided herein for the manufacture of a medicament for treating cancer. Certain embodiments are drawn to a compound or oligonucleotide provided herein for use in treating cancer. Examples of certain types of cancers include, but are not limited to, breast cancer, prostate cancer, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
  • Treating cancer refers to performing actions that lead to amelioration of cancer or of the symptoms accompanied therewith to a significant extent.
  • the combination of said actions is encompassed by the term treating cancer.
  • Amelioration of a cancer includes but is not limited to reducing in the number of cancer cells in an animal or reducing the number of cancer cells at a specific site in the body of an animal.
  • Said treatment as used herein also includes an entire restoration of the health with respect to the cancers referred to herein.
  • Treating cancer can be described by a number of different parameters including, but not limited to, reduction in the size of a tumor in an animal having cancer, reduction in the growth or proliferation of a tumor in an animal having cancer, preventing metastasis or reducing the extent of metastasis, and/or extending the survival of an animal having cancer.
  • administration or “administering” includes routes of introducing a compound or oligonucleotide provided herein to an animal to perform its intended function.
  • An example of a route of administration that can be used includes, but is not limited to parenteral administration, such as subcutaneous, intravenous, intramuscular, intraarterial, intraperitoneal, or intracranial injection or infusion.
  • 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 R A molecule.
  • the target nucleic acid is a pre-mR A.
  • the target nucleic acid is a SmgGDS transcript.
  • the target RNA is a SmgGDS pre-mRNA.
  • an antisense compound is complementary to a region of SmgGDS pre- mRNA. In certain embodiments, an antisense compound is complementary within a region of SmgGDS pre- mRNA comprising an exon encoding SmgGDS-558. In certain embodiments, an antisense compound is complementary to a region of SmgGDS pre-mRNA comprising an intron-exon splice junction. In certain embodiments, an antisense compound is complementary to a region of SmgGDS pre-mRNA comprising the intron-exon splice junction adjacent to exon 5. In certain embodiments, an antisense compound is complementary within a region of SmgGDS pre-mRNA consisting of exon 5.
  • an antisense compound is complementary within a region of SmgGDS pre-mRNA comprising an exonic splicing silencer within an exon 5. In certain embodiments, an antisense compound is complementary within a region of SmgGDS pre-mRNA comprising an exonic splicing enhancer within exon 5. In certain embodiments, an antisense compound is complementary within a region of SmgGDS pre-mRNA comprising an exonic splicing enhancer within exon 5.
  • the target region is within nucleobase 118557 and nucleobase 118859 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 118792 and nucleobase 118823 of SEQ ID NO.: 1.
  • an antisense oligonucleotide modulates splicing of a pre-mRNA. In certain embodiments, an antisense oligonucleotide modulates splicing of a SmgGDS pre-mRNA. In certain embodiments, an antisense oligonucleotide increases the amount of SmgGDS mRNA. In certain embodiments,
  • an antisense oligonucleotide increases the inclusion of exon 5 in SmgGDS mRNA. In certain embodiments, an antisense oligonucleotide decreases the inclusion of exon 5 in SmgGDS mRNA. In certain embodiments, an antisense oligonucleotide increases the amount of SmgGDS-607 mRNA. In certain embodiments, an antisense oligonucleotide decreases the amount of SmgGDS-558 mRNA.
  • SmgGDS pre-mRNA it is desirable to alter the splicing of SmgGDS pre-mRNA to include exon 5.
  • expression of SmgGDS-607 will increase and expression of SmgGDS-558 will decrease.
  • the present invention provides pharmaceutical compositions comprising one or more antisense compound.
  • 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 pharmaceutical grade PBS.
  • 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.
  • 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 polyvinylpyrrolidone.
  • 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.
  • 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 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.
  • 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 oligonucleotide.
  • 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
  • a prodrug is an ester.
  • 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 corresponding active form.
  • 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).
  • 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 eyes, ears).
  • a pharmaceutical composition is administered to an animal having at least one cancer cell. In certain embodiments, such administration results in apoptosis of at least cancer cell. In certain embodiments, a pharmaceutical composition is administered to an animal having at least one symptom associated with cancer. In certain embodiments, such administration results in amelioration of at least one symptom. In certain embodiments, administration of a pharmaceutical composition to an animal results in a decrease of SmgGDS-558 mR A in a cell of the animal. In certain embodiments, such administration results in an increase in SmgGDS-607 mRNA. In certain embodiments, such administration results in a decrease in SmgGDS-558 protein and an increase SmgGDS-607 protein.
  • a SmgGDS-607 protein is preferred over a SmgGDS-558 protein.
  • the administration of certain antisense oligonucleotides delays the onset of cancer. In certain embodiments, the administration of certain antisense oligonucleotides slows the proliferation of cancer cells. In certain embodiments, the administration of certain antisense oligonucleotides slows the proliferation of tumor cells. In certain embodiments, the administration of certain antisense oligonucleotides prevents the growth of cancer. In certain embodiments, the administration of certain antisense oligonucleotides prevents the formation of tumors. In certain embodiments, the administration of certain antisense oligonucleotides causes tumor mass to decrease. In certain embodiments, the administration of certain antisense oligonucleotides rescues cellular phenotype.
  • nucleic acid sequences provided herein are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases.
  • an oligomeric compound having the nucleobase sequence is intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases.
  • an oligomeric compound having the nucleobase sequence are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases.
  • an oligomeric compound having the nucleobase sequence is intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases.
  • ATCGATCG encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence "AUCGAUCG” and those having some DNA bases and some RNA bases such as
  • AUCGATCG and oligomeric compounds having other modified or naturally occurring bases, such as “AT ⁇ CGAUCG,” wherein “ ⁇ C indicates a cytosine base comprising a methyl group at the 5-position.
  • Alternative splicing of the SmgGDS gene involves a choice between including and excluding exon 5.
  • An antisense oligonucleotide (ASO) screen was carried out to identify potent ASOs that switch the splicing of endogenous SmgGDS transcripts to include or exclude exon 5.
  • ASOs that promote inclusion of exon 5 increase SmgGDS-607 isoform expression and SmgGDS-558 isoform downregulation.
  • ASOs that promote exclusion of exon 5 increase SmgGDS-558 isoform expression and SmgGDS-607 isoform downregulation.
  • a diagram of the SmgGDS genomic region is presented in Figure 1.
  • the ASOs were designed as uniform oligonucleotides, 18 nucleotides in length, with 2'-0- methoxyethyl ribose sugar residues and a phosphorothioate backbone. All the cytosine nucleobases are 5- methylcytosines.
  • the genomic sequence of SmgGDS is given by GENBANK Accession No. NT_016354.20 truncated from nucleotides 39338995 to 39523480 (designated herein as SEQ ID NO: 1).
  • the ASOs target the region surrounding exon 5 of SmgGDS, covering the 147-nucleotide exon 5 and the flanking intronic regions in 5-nucleotide steps, as presented in Table 1.
  • “Target Start Site” refers to the 5' position of the ASO relative to SEQ ID NO: 1.
  • Target Stop Site refers to the 3' position of the ASO relative to SEQ ID NO: 1.
  • the ASOs span nucleotides 118557 to 118859 of SEQ ID NO: 1.
  • a plot aligning the ASOs with the genomic transcript of SmgGDS is presented in Figure 2.
  • MDA-MB-231 cells were transfected with each ASO at a final concentration of 50 nM.
  • MDA- MB-231 cells were obtained from ATCC and seeded at a density of 5 x 10 5 cells per well in 6 well dishes the day before transfection in DMEM supplemented with 10% (v/v) FBS, 1% sodium pyruvate at 37°C and 5% C0 2 .
  • Transfections were performed using an ASO: LipofectAMINE2000® ratio of 25 pmoles: 1 ⁇ .
  • Splicing of the SmgGDS transcripts by radioactive RT-PCR was analyzed 72 hrs after transfection.
  • the human-specific primer sets used to amplify endogenous transcripts anneal to SmgGDS exons 4 and 6, and their sequences are: SmgGDSex4f: 5 ' -GTGCAGCTGCTAAATAGCAAAGAC -3' (designated herein as SEQ ID NO: 55) and SmgGDSex6r: 5 ' -GCATTTTGGCAGTGGATGCC-3 ' (designated herein as SEQ ID NO: 56).
  • MDA-MB-231 cells in 6-well plates were mock transfected (mock) or transfected with the indicated siRNA or ASOs.
  • Cell lysates were made 48 or 72 hours later and subjected western blotting.
  • Cells were lysed in 500 ⁇ SDS and 15 ⁇ was separated by SDS-PAGE and transferred onto polyvinyidene difluoride. Blots were blocked with 5% (w/v) milk in Tris-buffered saline with Tween-20, probed with antibodies, and visualized by enhanced chemiluminescence (MP Biomedicals).
  • the primary antibodies used were mouse antibody to ⁇ -actin (Santa Cruz Biotechnology, 1 : 20,000) and antibody to SmgGDS (BD Transduction Laboratories, 1 : 1,000). Secondary antibodies were donkey anti-mouse HRP conjugates (GE Healthcare, 1 :20,000).
  • results are presented in Figure 4.
  • SmgGDS-607 and SmgGDS-558 isoform protein levels closely mirrored their mRNA levels after ASO treatment.
  • ISIS 704507 (CIO) which most potently decreased SmgGDS-558 mRNA levels, also most potently decreased in SmgGDS-558 protein levels similar to a SmgGDS-558 isoform specific siRNA (BD).
  • ISIS 704508 (Cl l) and ISIS 704512 (D3) which had less effect at RNA level, also had less effect on protein levels.
  • ISIS 704484 (Al 1), which potently redirected splicing towards SmgGDS-558 at the RNA level, likewise increased SmgGDS-558 protein levels.
  • ISIS 704511 (D2), which had no effect on RNA levels, had no effect on protein levels.
  • Example 2 ASO microwalk centered on ISIS 704507
  • Overlapping 18-nucleotide ASOs were designed in 1-nucleotide steps.
  • the ASOs were designed as uniform oligonucleotides, 18 nucleotides in length, with 2'-0-methoxyethyl ribose sugar residues and a phosphorothioate backbone. All the cytosine nucleobases are 5-methylcytosines.
  • MDA-MB-231 cells were transfected with each ASO at a final concentration of 50 nM.
  • Cell culture, transfection and RNA analysis was conducted in a similar manner to that described in Example 1.
  • the results of the microwalks are presented in the Table below. Radioactivity in SmgGDS-607 was normalized for C content. Each product was quantified as a percentage of the total of SmgGDS-607 and -558. % SmgGDS-607 and % SmgGDS-558 are presented in the Table below.
  • Example 3 Antisense modulation of SmgGDS splicing in cancer cells
  • MDA-MB-231 human breast cancer cell line
  • MDA-MB-231 cells were obtained from ATCC and grown in DMEM supplemented with 10% (v/v) FBS, and 1% sodium pyruvate, at 37°C and 5% C0 2 .
  • ASO transfections were conducted as described above except in 96-well plates with cells plated 5 x 10 3 per well; ASOs were at 50 nM concentration with an ASO: LipofectAMINE2000® ratio of 30 pmoles: 1 ⁇ . The results are presented in the Table below.
  • Xenografts are established in the mammary fat pad of SHO mice (Charles River) by subcutaneous injection of 4 x 10 6 MDA-MB-231 luciferase-expressing cells in a 1 : 1 PBS:Matrigel solution. After 2 weeks, control or ASOs designed to shift SmgGDS splicing toward the SmgGDS-607 isoform and away from the
  • SmgGDS-558 isoform are delivered by four weekly single subcutaneous injections of 50 ⁇ g ASO per gram of body weight (6 mice / group).
  • the weekly doses of 50 ⁇ g ASO g-1 body weight is based on the very long half-life of ASOs in rodents and primates and on the observation that 1 to 4 doses was sufficient to redirect splicing, with therapeutic benefit, in mouse models of spinal muscular atrophy (Hua et al., Nature 478: 123-6, 2011; Hua et al., Genes Dev 24: 1634-44, 2010) and Usher's Syndrome (Lentz et al., Nat Med 19:345-50,
  • mice are implanted with the MDA-MB-231 cells expressing dox-inducible control shRNA (Luc- 231-TR-Scr4) or SmgGDS-558 shRNA (Luc-231-TR-BD), followed by dox-feed induction two weeks later.
  • Tumor size is measured weekly by bioluminescence imaging using a biophotonic imager (Xenogen); mice will be injected with 200 of 15 mg/mL luciferin five minutes before bioluminescent imaging.
  • ASOs The effect of ASOs on metastasis is assessed by biophotonic imaging of organs dissected from the mice at the conclusion of the experiments.
  • luciferase expressing MDA-MB-231 cells are injected in the tail vein rather than implanted in the mammary fatpads, and biophotonic imaging will be used to assess tumor burden in organs.
  • NCI- H23 and NCI-H1703 non-small cell lung carcinoma (NSCLC) cells The effect of modulating SmgGDS splicing with antisense oligonucleotides was examined in NCI- H23 and NCI-H1703 non-small cell lung carcinoma (NSCLC) cells.
  • NCI-H23 cells were seeded at a density of 5 x 10 5 cells per well in 6 well dishes in RPMI-1640 supplemented with L-glutamine (Life Technologies), 10% (v/v) heat-inactivated FBS, penicillin/streptomycin (20U/mL penicillin G sodium and 20 ⁇ g/mL streptomycin sulphate) at 37°C and 5% C0 2 .
  • transfections were performed using an ASO: LipofectAMINE2000® ratio of 25 pmoles: 1 ⁇ .
  • NCI-H23 cells were mock-transfected, or transfected with ISIS 704507 (CIO), ISIS 758698 (C10-5), ISIS 758700 (C10-7), ISIS 704484 (Al 1), or ISIS 704511 (D2) at a final concentration of 50 nM each.
  • a set of cells was transfected with the SmgGDS-558 isoform specific siRNA (BD) or scrambled siRNA.
  • the effect of treatment with antisense oligonucleotides on SmgGDS-558 protein expression was measured in MDA-MB-231 human breast cancer, NCI-H23 human NSCLC cells, NCI-H1703 human NSCLC cells, and MiaPaCa-2 human pancreatic cancer cells.
  • Cells were plated in a 24-well tissue culture plate at 2.5 x 10 4 cells per well in RPMI-1640 medium supplemented with L-glutamine, 10% heat-inactivated FBS, and antibiotics (20U/mL penicillin G sodium and 20 ⁇ g/mL streptomycin sulphate). Approximately 24 hours after plating, the cells were transfected with 50 nM of ASO using LipofectAMINE2000® or Dharmafect 1 (Dharmacon) transfection reagent, or treated with the transfection reagent in the absence of ASOs ("mock). Approximately 24 hours after transfection, the transfection media was aspirated and replaced with fresh media containing 10% FBS but no antibiotics.
  • the cells were cultured for an additional 48 hours, then harvested by aspirating the media, washing with PBS, and then adding 150 2X SDS-Loading Buffer to lyse the cells.
  • the lysates were run on a 12% SDS-PAGE gel, followed by transfer of the proteins to PVDF membranes.
  • the membranes were blocked with 5% (w/v) milk in Tris-buffered saline with Tween-20, and probed with antibodies to SmgGDS or GAPDH (as a loading control).
  • the immunoreactive proteins were detected by ECL (MP Biomedicals). Densitometry of the immunoreactive proteins were measured using ImageQuant TLv8.1 software in the ImageQuant LAS 4000 Luminescent Image Analyzer.
  • ISIS 704488 Treatment with ISIS 704488 (B3), ISIS 704492 (B7), and ISIS 704497 (B12) also increased smgGDS-588 protein expression, similar to that of ISIS 704484 (Al 1). Results are presented in the Table below.
  • Example 7 Effect of antisense inhibition of SmgGDS on cell proliferation in cancer cells
  • the cells were transfected with 50 nM of ISIS 704507 (CIO), ISIS 704484 (Al 1), ISIS 704511 (D2), ISIS 758698 (CI 0-5), ISIS 758700 (CI 0-7), or treated with the transfection reagent in the absence of ASOs ("mock). Approximately 24 hours after transfection, the transfection media was aspirated and replaced with fresh media containing 10% FBS but no antibiotics. The cells were cultured for an additional 48 hours, and then H-thymidine was added to each well to a final concentration of 0.005 ⁇ per well.
  • the cells were incubated for 3 hours, and then all but 10 of the media was removed from each well and replaced with 150 ⁇ , of 5 mM EDTA/5mM EGTA in PBS. After incubating for 30 min at 37°C and 5% C0 2 , the cells were harvested into scintillation vials with scintillation fluid, and radioactivity on the filter discs was measured by liquid scintillation counting.
  • the cells were transfected with 50 nM of ISIS 704507 (CIO), ISIS 704484 (Al l), ISIS 704511 (D2), with ISIS 704488 (B3), or ISIS 704492 (B7) using LipofectAMINE2000®, or treated with the transfection reagent in the absence of ASOs ("mock). Approximately 16-18 hours after transfection, the cells were collected using trypsin and re-plated onto 96- well plates.
  • MDA-MB-231 cells were plated at a concentration of 8 x 10 3 cells/well and the MiaPaCa-2 cells were plated at a concentration of 4 x 10 3 cells/well in 96-well plates. After the cells were cultured for 5-7 hours, the plates were placed in the Incucyte Live Cell Imaging System, and recordings for cell confluency were initiated. Recordings were collected for at least 70 hours.

Abstract

The present invention provides compounds comprising oligonucleotides complementary to a SmgGDS transcript. Certain such compounds are useful for hybridizing to a SmgGDS transcript, including but not limited to a SmgGDS transcript in a cell. In certain embodiments, such hybridization results in modulation of splicing of the SmgGDS transcript. In certain embodiments, such compounds are used to treat cancer.

Description

MODULATION OF SMGGDS EXPRESSION
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 BIOL0272WOSEQ_ST25.txt, created March 21, 2016, which is 248 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
BACKGROUND
Small G-protein guanine nucleotide dissociation stimulator 1 (SmgGDS), also known as Rapl, GTP- GDP dissociation stimulator 1 (RAP1GDS1), was originally identified as an exchange factor for multiple small G proteins, but recent work has uncovered additional novel activities associated with two splice isoforms of SmgGDS that established it as a positive regulator of Ras and Rho activation. Small GTPases must undergo a prenylation modification that promotes localization to the plasma membrane where interactions with various effectors occur. The 607 amino acid isoform, SmgGDS-607, which includes exon 5, binds nonprenylated small GTPases and regulates their entry into the prenylation pathway. The alternatively spliced 558 amino acid isoform, SmgGDS-558, which lacks exon 5, binds the prenylated forms and facilitates trafficking to the plasma membrane. SmgGDS is over expressed in breast cancer and its expression correlates with poor prognosis.
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 RNA (mRNA) 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-mRNA.
SUMMARY
In certain embodiments, the present invention provides compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides are complementary to a SmgGDS transcript. In certain such embodiments, oligonucleotides are complementary to a target region of the SmgGDS transcript comprising exon 5. In certain such embodiments, oligonucleotides are complementary to a target region of the SmgGDS transcript comprising an intron adjacent to exon 5. In certain such embodiments, oligonucleotides are complementary to a target region of the SmgGDS transcript comprising an intron adjacent to exon 5 and downstream of exon 5. In certain such embodiments, oligonucleotides are complementary to a target region of the SmgGDS transcript comprising an intron adjacent to exon 5 and upstream of exon 5. In certain embodiments, the SmgGDS transcript comprises an exonic splice enhancer for exon 5.
In certain embodiments, oligonucleotides inhibit inclusion of exon 5. In certain embodiments, oligonucleotides promote inclusion of exon 5. In certain embodiments, oligonucleotides promote skipping of exon 5. In certain embodiments, oligonucleotides inhibit skipping of exon 5. In certain embodiments, oligonucleotides promote selection of exon 5. In certain embodiments, oligonucleotides inhibit selection of exon 5. In certain embodiments, oligonucleotides increase levels of SmgGDS mRNA including exon 5 in a cell. In certain embodiments, oligonucleotides increase levels of SmgGDS mRNA lacking exon 5 in a cell. In certain embodiments, oligonucleotides increase SmgGDS-607 protein levels in a cell. In certain embodiments, oligonucleotides increase SmgGDS-558 protein levels in a cell.
In certain embodiments, oligonucleotides modulate the relative levels of SmgGDS mRNA including exon 5 and SmgGDS mRNA lacking exon 5 in a cell. In certain embodiments, oligonucleotides increase levels of SmgGDS mRNA including exon 5 relative to SmgGDS mRNA lacking exon 5 in a cell. In certain embodiments, oligonucleotides increase levels of SmgGDS mRNA lacking exon 5 relative to SmgGDS mRNA including exon 5 in a cell.
In certain embodiments, oligonucleotides modulate the relative protein levels of SmgGDS-607 and SmgGDS-558 in a cell. In certain embodiments, oligonucleotides increase protein levels of SmgGDS-607 relative to SmgGDS-558 in a cell. In certain embodiments, oligonucleotides increase protein levels of SmgGDS-558 relative to SmgGDS-607 in a cell.
In certain embodiments, the SmgGDS transcript is in a cell. In certain embodiments, the cell is in an animal. In certain embodiments, the animal is a mammal. In certain embodiments, the mammal is a human or rodent, such as a mouse.
In certain embodiments, the compounds and oligonucleotides provided herein are useful for inhibiting cancer cell proliferation and treating cancer. In certain embodiments, a method comprises contacting a cancer cell with a compound or oligonucleotide provided herein, thereby (i) altering splicing of SmgGDS mRNA in the cell, (ii) increasing levels of SmgGDS-607 mRNA or protein in the cell, (iii) increasing levels of SmgGDS-558 mRNA or protein in the cell, (iv) increasing the ratio of SmgGDS-607 mRNA or protein to SmgGDS-558 mRNA or protein in the cell, (v) increasing the ratio of SmgGDS-558 mRNA or protein to SmgGDS-607 mRNA or protein in the cell, and/or (vi) inhibiting cancer cell proliferation. In certain embodiments, a method comprises administering a compound or oligonucleotide provided herein to a subject having cancer, thereby (i) altering splicing of SmgGDS mRNA, (ii) increasing levels of SmgGDS-607 mRNA or protein, (iii) increasing levels of SmgGDS-558 mRNA, (iv) increasing the ratio of SmgGDS-607 mRNA or protein to SmgGDS-558 mRNA, (v) increasing the ratio of SmgGDS-558 mRNA or protein to SmgGDS-607 mRNA or protein, (vi) inhibiting cancer growth or proliferation, (vii) inhibiting metastasis, (viii) increasing survival, and/or (ix) treating cancer in the subject. The present disclosure provides the following non-limiting numbered embodiments:
Embodiment 1 : A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region, wherein the complementary region comprises at least 8 contiguous nucleobases and is complementary to an equal-length portion within a target region of a SmgGDS transcript.
Embodiment 2: The compound of embodiment 1, wherein the target region of the SmgGDS transcript
comprises exon 5 of the SmgGDS transcript. Embodiment 3: The compound of embodiment lor 2, wherein the complementary region of the modified oligonucleotide is 100% complementary to the target region.
Embodiment 4: The compound of any of embodiments 1 to 3, wherein the complementary region of the modified oligonucleotide comprises at least 10 contiguous nucleobases.
Embodiment 5: The compound of any of embodiments 1 to 3, wherein the complementary region of the modified oligonucleotide comprises at least 15 contiguous nucleobases.
Embodiment 6: The compound of any of embodiments 1 to 3, wherein the complementary region of the modified oligonucleotide comprises at least 20 contiguous nucleobases.
Embodiment 7: The compound of any of embodiments 1-6, wherein the nucleobase sequence of the
oligonucleotide is at least 80% complementary to the target region, as measured over the entire length of the oligonucleotide.
Embodiment 8: The compound of any of embodiments 1-6, wherein the nucleobase sequence of the
oligonucleotide is at least 90% complementary to an equal-length region of the SmgGDS transcript, as measured over the entire length of the oligonucleotide. Embodiment 9: The compound of any of embodiments 1-6, wherein the nucleobase sequence of the
oligonucleotide is 100% complementary to an equal -length region of the SmgGDS transcript, as measured over the entire length of the oligonucleotide.
Embodiment 10: The compound of any of embodiments 1-9, wherein the target region is within exon 5 or flanking intronic regions of the SmgGDS transcript. Embodiment 11 : The compound of any of embodiments 1-10, wherein the target region is within nucleobase 118642 and nucleobase 118788 of SEQ ID NO. 1. Embodiment 12: The compound of any of embodiments 1-10, wherein the target region is within nucleobase 118557 and nucleobase 118641 of SEQ ID NO.: 1.
Embodiment 13: The compound of any of embodiments 1-10, wherein the target region is within nucleobase 118789 and nucleobase 118859 of SEQ ID NO.: 1.
Embodiment 14: The compound of any of embodiments 1-10, wherein the target region is within nucleobase 118557 and nucleobase 118859 of SEQ ID NO.: 1.
Embodiment 15: The compound of any of embodiments 1-10, wherein the target region is within nucleobase 118792 and nucleobase 118823 of SEQ ID NO.: 1.
Embodiment 16: The compound of any of embodiments 1-15, wherein the antisense oligonucleotide has a nucleobase sequence comprising any one of SEQ ID NOs: 2-54. Embodiment 17: The compound of any of embodiments 1-16, wherein the modified oligonucleotide comprises at least one modified nucleoside.
Embodiment 18: The compound of embodiment 17, wherein at least one modified nucleoside
comprises a modified sugar moiety.
Embodiment 19: The compound of embodiment 18, wherein at least one modified sugar moiety is a
2' -substituted sugar moiety.
Embodiment 20: The compound of embodiment 19, wherein the 2'-substitutent of at least one 2'- substituted sugar moiety is selected from among: 2'-OMe, 2'-F, and 2'-MOE.
Embodiment 21 : The compound of any of embodiments 17-20, wherein the 2'-substiuent of at least one 2 '-substituted sugar moiety is a 2'-MOE. Embodiment 22: The compound of any of embodiments 1-18, wherein at least one modified sugar moiety is a bicyclic sugar moiety.
Embodiment 23 : The compound of embodiment 22, wherein at least one bicyclic sugar moiety is LNA or cEt.
Embodiment 24: The compound of any of embodiments 18-23, wherein at least one sugar moiety is a sugar surrogate. Embodiment 25 : The compound of embodiment 24, wherein at least one sugar surrogate is a
morpholino.
Embodiment 26: The compound of embodiment 24, wherein at least one sugar surrogate is a modified morpholino.
Embodiment 27: The compound of any of embodiment 1-26, wherein the modified oligonucleotide comprises at least 5 modified nucleosides, each independently comprising a modified sugar moiety.
Embodiment 28: The compound of embodiment 27, wherein the modified oligonucleotide comprises at least 10 modified nucleosides, each independently comprising a modified sugar moiety.
Embodiment 29: The compound of embodiment 27, wherein the modified oligonucleotide comprises at least 15 modified nucleosides, each independently comprising a modified sugar moiety. Embodiment 30: The compound of embodiment 27, wherein each nucleoside of the modified
oligonucleotide is a modified nucleoside, each independently comprising a modified sugar moiety
Embodiment 31 : The compound of any of embodiments 1-30, wherein the modified oligonucleotide comprises at least two modified nucleosides comprising modified sugar moieties that are the same as one another.
Embodiment 32: The compound of any of embodiments 1-31, wherein the modified oligonucleotide comprises at least two modified nucleosides comprising modified sugar moieties that are different from one another. Embodiment 33: The compound of any of embodiments 1-32, wherein the modified oligonucleotide comprises a modified region of at least 5 contiguous modified nucleosides.
Embodiment 34: The compound of embodiment 33, wherein the modified oligonucleotide comprises a modified region of at least 10 contiguous modified nucleosides.
Embodiment 35: The compound of embodiment 33, wherein the modified oligonucleotide comprises a modified region of at least 15 contiguous modified nucleosides. Embodiment 36: The compound of embodiment 33, wherein the modified oligonucleotide comprises a modified region of at least 20 contiguous modified nucleosides.
Embodiment 37: The compound of any of embodiments 32-36, wherein each modified nucleoside of the modified region has a modified sugar moiety independently selected from among: 2'-F, 2'-OMe, 2'- MOE, cEt, LNA, morpholino, and modified morpholino.
Embodiment 38: The compound of any of embodiments 33-37, wherein the modified nucleosides of the modified region each comprise the same modification as one another. Embodiment 39: The compound of embodiment 38, wherein the modified nucleosides of the modified region each comprise the same 2 '-substituted sugar moiety.
Embodiment 40: The compound of embodiment 38, wherein the 2 '-substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from 2'-F, 2'-OMe, and 2'-MOE.
Embodiment 41 : The compound of embodiment 39, wherein the 2 ' -substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is 2'-MOE.
Embodiment 42: The compound of embodiment 38, wherein the modified nucleosides of the region of modified nucleosides each comprise the same bicyclic sugar moiety.
Embodiment 43 : The compound of embodiment 42, wherein the bicyclic sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from LNA and cEt. Embodiment 44: The compound of embodiment 38, wherein the modified nucleosides of the region of modified nucleosides each comprises a sugar surrogate.
Embodiment 45 : The compound of embodiment 44, wherein the sugar surrogate of the modified nucleosides of the region of modified nucleosides is a morpholino.
Embodiment 46: The compound of embodiment 44, wherein the sugar surrogate of the modified nucleosides of the region of modified nucleosides is a modified morpholino. Embodiment 47: The compound of any of embodiments 1-46, wherein the modified nucleotide
comprises no more than 4 contiguous naturally occurring nucleosides.
Embodiment 48: The compound of any of embodiments 1-46, wherein each nucleoside of the
modified oligonucleotide is a modified nucleoside.
Embodiment 49: The compound of embodiment 48 wherein each modified nucleoside comprises a modified sugar moiety.
Embodiment 50: The compound of embodiment 49, wherein the modified nucleosides of the modified oligonucleotide comprise the same modification as one another.
Embodiment 51 : The compound of embodiment 50, wherein the modified nucleosides of the modified oligonucleotide each comprise the same 2 '-substituted sugar moiety. Embodiment 52: The compound of embodiment 51, wherein the 2 '-substituted sugar moiety of the modified oligonucleotide is selected from 2'-F, 2'-OMe, and 2'-MOE.
Embodiment 53: The compound of embodiment 52, wherein the 2 '-substituted sugar moiety of the modified oligonucleotide is 2'-MOE.
Embodiment 54: The compound of embodiment 50, wherein the modified nucleosides of the modified oligonucleotide each comprise the same bicyclic sugar moiety.
Embodiment 55: The compound of embodiment 54, wherein the bicyclic sugar moiety of the modified oligonucleotide is selected from LNA and cEt. Embodiment 56: The compound of embodiment 50, wherein the modified nucleosides of the modified oligonucleotide each comprises a sugar surrogate. Embodiment 57: The compound of embodiment 56, wherein the sugar surrogate of the modified oligonucleotide is a morpholino.
Embodiment 58: The compound of embodiment 56, wherein the sugar surrogate of the modified oligonucleotide is a modified morpholino.
Embodiment 59: The compound of any of embodiments 1-58, wherein the modified oligonucleotide comprises at least one modified intemucleoside linkage.
Embodiment 60: The compound of embodiment 59, wherein each intemucleoside linkage is a
modified intemucleoside linkage.
Embodiment 61 : The compound of embodiment 59 or 60, comprising at least one phosphorothioate intemucleoside linkage. Embodiment 62: The compound of embodiment 60, wherein each intemucleoside linkage is a
modified intemucleoside linkage and wherein each intemucleoside linkage comprises the same modification.
Embodiment 63 : The compound of embodiment 62, wherein each intemucleoside linkage is a
phosphorothioate intemucleoside linkage.
Embodiment 64: The compound of any of embodiments 1-63 comprising at least one conjugate.
Embodiment 65 : The compound of any of embodiments 1-64 consisting of the modified
oligonucleotide.
Embodiment 66: The compound of any of embodiments 1-65, wherein the compound modulates splicing of the SmgGDS transcript. Embodiment 67: A pharmaceutical composition comprising a compound according to any of embodiments 1-66 and a pharmaceutically acceptable carrier or diluent.
Embodiment 68: The pharmaceutical composition of embodiment 67, wherein the pharmaceutically acceptable carrier or diluent is sterile saline.
Embodiment 69: A method of modulating splicing of a SmgGDS transcript in a cell comprising
contacting the cell with a compound according to any of embodiments 1-66 or composition according to embodiment 67 or 68.
Embodiment 70: The method of embodiment 69, wherein the cell is in vitro.
Embodiment 71 : The method of embodiment 69, wherein the cell is in an animal.
Embodiment 72: The method of any of embodiments 69-71, wherein the cell is a cancer cell.
Embodiment 73: The method of any of embodiments 69-72, wherein inclusion of exon 5 is increased.
Embodiment 74: The method of any of embodiments 69-72, wherein exclusion of exon 5 is increased.
Embodiment 75: The method of any of embodiments 69-72, wherein SmgGDS-607 mRNA expression is increased.
Embodiment 76: The method of any of embodiments 76-72, wherein SmgGDS-558 mRNA expression is decreased.
Embodiment 77: A method of modulating expression of SmgGDS in a cell, comprising contacting the cell with a compound according to any of embodiments 1-66 or composition according to embodiment 67 or 68.
Embodiment 78: The method of embodiment 77, wherein SmgGDS-607 expression is increased.
Embodiment 79: The method of embodiments 77 or 78, wherein SmgGDS-558 expression is
decreased. Embodiment 80: The method of embodiment 77, wherein the cell is in vitro.
Embodiment 81 : The method of embodiment 77, wherein the cell is in an animal. Embodiment 82: A method of inhibiting cell proliferation, comprising contacting the cell with a
compound according to any of embodiments 1-66 or composition according to embodiment 67 or 68.
Embodiment 83: The method of embodiment 82, wherein the cell is in vitro. Embodiment 84: The method of embodiment 82, wherein the cell is in an animal.
Embodiment 85: The method of any of embodiments 82-84 wherein the cell is a cancer cell.
Embodiment 86: A method comprising administering the compound according to any of embodiments 1-66 or composition according to embodiment 67 or 68 to an animal.
Embodiment 87: The method of embodiment 86, wherein the compound is administered systemically.
Embodiment 88: The method of embodiment 87, wherein the compound is administered via
subcutaneous injection or i.v. infusion.
Embodiment 89: The method of any of embodiments 86-88, wherein the animal has cancer.
Embodiment 90: The method embodiment 89, wherein the cancer is breast cancer, prostate cancer, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
Embodiment 91 : The method of embodiment 89 or 90, wherein the administration results in
amelioration of at least one symptom of cancer.
Embodiment 92: The method of any of embodiments 86-91, wherein the animal is a mouse.
Embodiment 93: The method of any of embodiments 86-91, wherein the animal is a human. Embodiment 94: A method of treating cancer in an animal, comprising administering the compound according to any of embodiments 1-66 or composition according to embodiment 67 or 68 to an animal in need thereof. Embodiment 95 : The method of embodiment 94, wherein the animal is a mouse.
Embodiment 96: The method of embodiment 94, wherein the animal is a human.
Embodiment 97: The method of any of embodiments 94-96, wherein the cancer is breast cancer, prostate cancer, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer..
Embodiment 98: Use of the compound according to any of embodiments 1-66 or composition
according to embodiment 67 or 68 for the preparation of a medicament for use in the treatment of cancer.
Embodiment 99: The compound according to any of embodiments 1-66 or composition according to embodiment 67 or 68 for use in treating cancer.
Embodiment 100: The use of embodiment 98 or 99, wherein the cancer is breast cancer, prostate
cancer, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer..
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 : Diagram of the SmgGDS genomic region. The SmgGDS genomic region comprises numbered exons represented by vertical lines. Numbers under the vertical lines correspond to the exon number.
SmgGDS-607 includes exon 5 whereas SmgGDS-558 excludes exon 5. A zoom-in of the genomic region spanning exons 4-6 and intervening introns is shown and indicates alternative splicing of exon 5 to generate either SmgGDS-607 or SmgGDS-558. Figure 2: Plot of ASOs along the genomic transcript of SmgGDS. Exon 5 (147 nt) is indicated as a box and the flanking intronic regions are indicated by nucleotide position relative to the 5 ' and 3 ' ends of exon 5. Dark bars above the genomic transcript represent ASO alignment to the transcript and ASO number. A zoom-in of the transcript region targeted by ISIS 704507 (CIO) and microwalk is shown. Figure 3: RT-PCR gel of ASO treated cells. ASOs were transfected in MDA-MB-231 cells. RT-PCR of endogenous SmgGDS transcripts is shown. The ASO number is indicated at the top of the gel. cDNA amplicons and fragments corresponding to SmgGDS-607 ("607") and SmgGDS-558 ("558") are labeled.
Figure 4: Western Blot of ASO treated cells. ASOs were transfected in MDA-MB-231 cells. Western Blot of endogenous SmgGDS transcripts is shown. The ASO number is indicated above the gel lanes. Protein bands corresponding to SmgGDS-607 ("607") and SmgGDS-558 ("558") are labeled. Percent of SmgGDS-558 ("558") is indicated under the gel (Fig. 4a). Cells were treated with ASO for 48 hours (Fig. 4b) or 72 hours (Fig. 4c).
DETAILED DESCRIPTION
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, "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, "chemical modification" means a chemical difference in a compound when compared to a naturally occurring counterpart. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications.
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 R A or a 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, a bicyclic or tricyclic sugar moiety, or a sugar surrogate.
As used herein, "substituted sugar moiety" means a furanosyl comprising at least one substituent group that differs from that of 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.
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, "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 the term "sugar surrogate" means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside is capable of (1) incorporation into an oligonucleotide and (2) hybridization to a complementary nucleoside. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); 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 morpholino, modified morpholinos, cyclohexenyls and cyclohexitols.
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, "nucleobase" means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified.
As used herein, "heterocyclic base" or "heterocyclic nucleobase" means a nucleobase comprising a heterocyclic structure.
As used herein the terms, "unmodified nucleobase" or "naturally occurring nucleobase" means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5 -methyl C), and uracil (U).
As used herein, "modified nucleobase" means any nucleobase that is not a naturally occurring nucleobase.
As used herein, "modified nucleoside" means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides comprise a modified sugar moiety and/or a modified nucleobase.
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.
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 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 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, "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, "oligomeric compound" means a polymeric structure comprising two or more substructures. In certain embodiments, an oligomeric compound comprises an oligonucleotide. In certain embodiments, an oligomeric compound comprises one or more conjugate groups and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide.
As used herein, "terminal group" means one or more atom attached to either, or both, the 3' end or the 5 ' end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more terminal group nucleosides.
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 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, "transcript" means an RNA molecule transcribed from DNA. Transcripts include, but are not limitied to mRNA, pre-mRNA, and partially processed RNA.
As used herein, "SmgGDS transcript" means a transcript transcribed from a SmgGDS gene. In certain embodiments, a SmgGDS transcript comprises GENBANK Accession No. NT_016354.20 truncated from nucleotides 39338995 to 39523480, which is incorporated by reference herein and designated as SEQ ID NO.: l .
As used herein, "SmgGDS gene" means a gene that encodes a SmgGDS protein and any SmgGDS protein isoforms. In certain embodiments, SmgGDS isoforms include SmgGDS-607 and SmgGDS-558. In certain embodiments, a SmgGDS gene is represented by GENBANK Accession No. NT_016354.20 truncated from nucleotides 39338995 to 39523480, or a variant thereof. In certain embodiments, a SmgGDS gene is at least 95% identical to GENBANK Accession No. NT_016354.20 truncated from nucleotides 39338995 to 39523480. In certain embodiments, SmgGDS gene is at least 90% identical to GENBANK Accession No. NT_016354.20 truncated from nucleotides 39338995 to 39523480.
As used herein, "SmgGDS-607" means a SmgGDS transcript that includes exon 5.
As used herein, "SmgGDS-607 isoform" means a SmgGDS protein isoform that includes amino acids encoded by exon 5.
As used herein, "SmgGDS-558" means a SmgGDS transcript that excludes exon 5.
As used herein, "SmgGDS-558 isoform" means a SmgGDS protein isoform that excludes amino acids encoded by exon 5.
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, "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, "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-mRNA 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, "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(0)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)- (Ree)), guamdinyl (-N(Rbb)C(=NRbb)N(Rbb)(Rcc)), amidinyl (-C(=NRbb)N(Rbb)(Rcc) or -N(Rbb)C(=NRbb)(Raa)), thiol (-SRbb), sulfinyl (-S(0)Rbb), sulfonyl (-S(0)2Rbb) and sulfonamidyl (-S(0)2N(Rbb)(Rcc) or -N(R„b)S- (0)2Rbb). Wherein each R^, R^, 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 (Ci-Ci2 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, 1 -methyl -2 -buten-l-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.
As used herein, "alkynyl," 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, «-butoxy, sec-butoxy, fert-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 C1-C12 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 C1-C12 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 polycyclic 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.
Oligomeric Compounds
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 one or more nucleoside (including modifications to the sugar moiety and/or the nucleobase) and/or modifications to one or more internucleoside linkage. Certain Sugar Moieties
In certain embodiments, oligomeric compounds of the invention comprise one or more modifed nucleosides comprising a modifed sugar moiety. Such oligomeric 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 oligomeric compounds 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 bicyclic or tricyclic 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 substituent, 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)20CH3 ("MOE"). In certain embodiments, sugar substituents at the 2' position is selected from allyl, amino, azido, thio, O-allyl, O-Ci-Cio alkyl, O-C l-Cio substituted alkyl; O- Ci-Cio alkoxy; O- Ci-Cio substituted alkoxy, 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-Cio 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 2008/101 157, 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, O- Ci-Ci0 alkoxy; O- Ci-Ci0 substituted alkoxy, SH, CN, OCN, CF3, OCF3, O-alkyl, S-alkyl, N(Rm)-alkyl; O- alkenyl, S- alkenyl, or -alkenyl; O- alkynyl, S- alkynyl, N(Rm)-alkynyl; O-alkylenyl- O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, 0(CH2)2SCH3, 0-(CH2)2-0-N(Rm)(Rn) or 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. 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)(Rn), 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 - 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)]„-, -[C(Ra)(Rb)]„-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 1 1, 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 Ris, independently, H, a protecting group, or C1-C12 alkyl; 4'-CH2-N(R)-0-2', wherein R is H, C1-C12 alkyl, or a protecting group (see, U.S. Patent 7,427,672, issued on September 23, 2008); 4'-CH2- C(H)(CH3)-2' (see, e.g., Chattopadhyaya, et al, J. Org. Chem., 2009, 74, 1 18-134); and 4'-CH2-C(=CH2)-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 independently selected from -[C(Ra)(Rb)]n-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=0)-, -C(=S)-, -0-, - Si(Ra)2-, -S(=0)x-, and -N(Ra)-;
wherein:
x is 0, 1, or 2;
n is 1, 2, 3, or 4;
each Ra and R¾ is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-Ci2 alkenyl, substituted C2-Ci2 alkenyl, C2-Ci2 alkynyl, substituted C2-Ci2 alkynyl, C5-C2o aryl, substituted C5-C2o aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJiJ2, SJi, N3, COOJi, acyl (C(=0)- H), substituted acyl, CN, sulfonyl (S(=0)2-Ji), or sulfoxyl (S(=0)-Ji); and
each Ji and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-Ci2 alkenyl, substituted C2-C12 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, C1-C12 aminoalkyl, substituted C1-C12 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, and (J) propylene carbocyclic (4'-(CH2)3-2') BNA as depicted below.
Figure imgf000025_0001
(A) (B) (C)
Figure imgf000025_0002
wherein Bx is a nucleobase moiety and R is, independently, H, a protecting group, or Ci-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 (UNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, CJ. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), and those compounds having Formula
Figure imgf000026_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, q3, q4, qs, qe and q7 are each, independently, H, Ci-C6 alkyl, substituted Ci-C6 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, NJ ,, SJ N3, OC(=X)Ji, OC(=X)NJ!J2, NJ3C(=X)NJ!J2, and CN, wherein X is O, S or NJi, and each Ji, J2, and J3 is, independently, H or Ci-C6 alkyl.
In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q q2, q3, q , q5, q6 and q7 are each H. In certain embodiments, at least one of qi, q2, q3, q , q5, q6 and q7 is other than H. In certain embodiments, at least one of qi, q2, q3, q , q5, q6 and q7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of Ri and R2 is F. In certain embodiments, Ri is fluoro and R2 is H, Ri is methoxy and R2 is H, and Ri is methoxyethoxy and R2 is H.
Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used to modify nucleosides (see, e.g., review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).
In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5,698,685; 5, 166,315; 5, 185,444; and 5,034,506). As used here, the term "morpholino" means a sugar s llowing structure:
Figure imgf000027_0001
In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are refered to herein as "modifed morpholinos."
Combinations of modifications are also provided without limitation, such as 2'-F-5 '-methyl substituted nucleosides (see PCT International Application WO 2008/101 157 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 1 1/22/07 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 ei /., J. Am. Chem. Soc. 2007, 129(26), 8362-8379). Certain 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.
In certain embodiments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil; 5-propynylcytosine; 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. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine( [5,4-b] [l,4]benzoxazin- 2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3- d]pyrimidin-2-one). Modified nucleobases may 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 nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J.I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al. , Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications , Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993, 273-288.
Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, U.S. 3,687,808; 4,845,205; 5, 130,302; 5, 134,066; 5,175,273; 5,367,066; 5,432,272; 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; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
Certain Intemucleoside Linkages
In certain embodiments, the present invention provides oligomeric compounds comprising linked nucleosides. In such embodiments, nucleosides may be linked together using any intemucleoside linkage. The two main classes of intemucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing intemucleoside linkages include, but are not limited to, phosphodiesters (P=0), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P=S). Representative non-phosphorus containing intemucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-0-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-0- C(0)(NH)-S-); siloxane (-0-Si(H)2-0-); and Ν,Ν'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligomeric compound. In certain embodiments, intemucleoside linkages having a chiral atom can be prepared 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 intemucleoside linkages are well known to those skilled in the art.
The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), a or β such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
Neutral intemucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI (3'-CH2-N(CH3)-0-5'), amide-3 (3'-CH2-C(=0)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=0)-5'), formacetal (3'-0-CH2-0-5'), and thioformacetal (3'-S-CH2-0-5'). Further neutral intemucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral intemucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 component parts.
Certain Motifs
In certain embodiments, the present invention provides oligomeric compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides comprise one or more chemical modification. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleosides. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleosides comprising modified sugars. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleosides comprising one or more modified nucleobases. In certain embodiments, chemically modified oligonucleotides comprise one or more modified internucleoside linkages. In certain embodiments, the chemically 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).
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 modification motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.
In certain embodiments, the oligonucleotides comprise or consist of a region having a gapmer sugar modification motif, which comprises two external regions or "wings" and an internal region or "gap." The three regions of a gapmer 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 modification motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar modification motifs of the 5 '-wing differs from the sugar modification motif of the 3'-wing (asymmetric gapmer). In certain embodiments, oligonucleotides comprise 2'-MOE modified nucleosides in the wings and 2'-F modified nucleosides in the gap.
In certain embodiments, oligonucleotides are fully modified. In certain such embodiments, oligonucleotides are uniformly modified. In certain embodiments, oligonucleotides are uniform 2'-MOE. In certain embodiments, oligonucleotides are uniform 2'-F. In certain embodiments, oligonucleotides are uniform morpholino. In certain embodiments, oligonucleotides are uniform BNA. In certain embodiments, oligonucleotides are uniform LNA. In certain embodiments, oligonucleotides are uniform cEt. In certain embodiments, oligonucleotides comprise a uniformly modified region and additional nucleosides that are unmodified or differently modified. In certain embodiments, the uniformly modified region is at least 5, 10, 15, or 20 nucleosides in length. In certain embodiments, the uniform region is a - MOE region. In certain embodiments, the uniform region is a 2'-F region. In certain embodiments, the uniform region is a morpholino region. In certain embodiments, the uniform region is a BNA region. In certain embodiments, the uniform region is a LNA region. In certain embodiments, the uniform region is a cEt region.
In certain embodiments, the oligonucleotide does not comprise more than 4 contiguous unmodified 2'-deoxynucleosides. In certain circumstances, antisesense oligonucleotides comprising more than 4 contiguous 2'-deoxynucleosides activate RNase H, resulting in cleavage of the target RNA. In certain embodiments, such cleavage is avoided by not having more than 4 contiguous 2'-deoxynucleosides, for example, where alteration of splicing and not cleavage of a target RNA is desired.
Certain Intemucleoside Linkage Motifs
In certain embodiments, oligonucleotides comprise modified intemucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified intemucleoside linkage motif. In certain embodiments, intemucleoside linkages are arranged in a gapped motif, as described above for sugar modification motif. In such embodiments, the intemucleoside linkages in each of two wing regions are different from the intemucleoside linkages in the gap region. In certain embodiments the intemucleoside linkages in the wings are phosphodiester and the intemucleoside linkages in the gap are phosphorothioate. The sugar modification motif is independently selected, so such oligonucleotides having a gapped intemucleoside linkage motif may or may not have a gapped sugar modification motif and if it does have a gapped sugar motif, the wing and gap lengths may or may not be the same.
In certain embodiments, oligonucleotides comprise a region having an alternating intemucleoside linkage motif. In certain embodiments, oligonucleotides of the present invention comprise a region of uniformly modified intemucleoside linkages. In certain such embodiments, the oligonucleotide 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 oligonucleotide is selected from phosphodiester and phosphorothioate. In certain
embodiments, each intemucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one intemucleoside linkage is phosphorothioate.
In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3' end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3' end of the oligonucleotide.
Certain Nucleobase Modification Motifs
In certain embodiments, oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or nucleobases modification motif. In certain such embodiments, nucleobase modifications are arranged in a gapped motif. In certain embodiments, nucleobase modifications are arranged in an alternating 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, 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.
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 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X<Y. For example, in certain embodiments, the invention provides oligomeric compounds which comprise oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23,
10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27,
11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to
27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to
19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30,
16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to
28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26,
17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26,
19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides. In embodiments where the number of nucleosides of an oligomeric compound or oligonucleotide is limited, whether to a range or to a specific number, the oligomeric compound or oligonucleotide may, nonetheless further comprise additional other substituents. For example, an oligonucleotide 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.
One of skill in the art will appreciate that certain lengths may not be possible for certain motifs. For example: a gapmer having a 5 '-wing region consisting of four nucleotides, a gap consisting of at least six nucleotides, and a 3'-wing region consisting of three nucleotides cannot have an overall length less than 13 nucleotides. Thus, one would understand that the lower length limit is 13 and that the limit of 10 in "10-20" has no effect in that embodiment.
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. For example, an oligonucleotide consisting of 20-25 linked nucleosides comprising a 5 '-wing consisting of 5 linked nucleosides; a 3 '-wing consisting of 5 linked nucleosides and a central gap consisting of 10 linked nucleosides (5+5+10=20) may have up to 5 nucleosides that are not part of the 5'-wing, the 3'- wing, or the gap (before reaching the overall length limitation of 25). Such additional nucleosides may be 5' of the 5 '-wing and/or 3' of the 3' wing.
Certain Oligonucleotides
In certain embodiments, oligonucleotides of the present invention are characterized by their sugar motif, intemucleoside linkage motif, nucleobase modification motif and overall length. In certain embodiments, such parameters are each independent of one another. Thus, each intemucleoside 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. Thus, the intemucleoside 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 intemucleoside 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. 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, intemucleoside linkage motif, and nucleobase modification motif. Unless otherwise indicated, all chemical modifications are independent of nucleobase sequence.
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, (<S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
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- Cio alkyl, substituted or unsubstituted C2-Ci0 alkenyl or substituted or unsubstituted C2-Ci0 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 chemical motifs discussed above. Thus, for example, an oligomeric compound comprising an oligonucleotide having region of alternating nucleosides may comprise a terminal group. Antisense Compounds
In certain embodiments, oligomeric compounds of the present invention 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.
In certain embodiments antisense compounds and antisense oligonucleotides comprise single-strand compounds. In certain embodiments antisense compounds and antisense oligonucleotides comprise double- strand compounds. Certain cancer indications, pathways, and mechanisms
Small GTPases, including the Ras and Rho family members Rapl, K-Ras, RhoA, and Racl, are involved in signaling cascades that are often activated in cancer, such as breast cancer, and can lead to tumor development, progression, and escape from chemotherapy-induced apoptosis. To be activated, small GTPases must be prenylated at their C termini, which localizes and anchors the proteins to the plasma membrane where they interact with effector molecules. SmgGDS is a master regulator of prenylation and membrane localization for the subset of GTPases that harbor a polybasic region. Consistent with its role in promoting malignancy, SmgGDS is overexpressed in cancer tissues, such as breast, prostate, and lung cancer where it promotes cell proliferation, migration, and NFkB signaling.
In certain embodiments, the administration of a compound or oligonucleotide provided herein to a subject having cancer or contacting a cancer cell with a compound or oligonucleotide provided herein causes a switch in the alternative splicing of the SmgGDS transcript. In certain embodiments, the administration of an oligonucleotide provided herein causes increased inclusion of exon 5 mRNA of the SmgGDS transcript. In certain embodiments, the administration of an oligonucleotide provided herein causes an increase in the exclusion of exon 5 mRNA of the SmgGDS transcript. In certain embodiments, the administration of an oligonucleotide provided herein reduces expression of SmgGDS-558 in a cancer cell. In certain
embodiments, the administration of an oligonucleotide provided herein reduces expression of SmgGDS-558 in a cancer cell and inhibits cancer growth. In certain embodiments, the administration of an oligonucleotide provided herein reduces expression of SmgGDS-558 and induces apoptosis in a cancer cell. In certain embodiments, the cancer cell is a breast cancer cell, prostate cancer cell, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby reducing expression of SmgGDS-558 and inhibiting cancer cell growth or proliferation. In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby reducing expression of SmgGDS-558 and inducing apoptosis in a cancer cell. In certain embodiments, the cancer cell is a breast cancer cell, prostate cancer cell, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby reducing expression of SmgGDS-607 and inhibiting cancer cell growth or proliferation. In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby reducing expression of SmgGDS-607 and inducing apoptosis in a cancer cell. In certain embodiments, the cancer cell is a breast cancer cell, prostate cancer cell, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
In certain embodiments, modulating the relative levels of SmgGDS-607 and SmgGDS-558 in a cancer cell inhibits cancer cell growth or proliferation. In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby increasing the relative level of SmgGDS-607 to SmgGDS-558 in a cancer cell and inhibiting cancer cell growth or proliferation. In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby increasing the relative level of SmgGDS-558 to SmgGDS-607 in a cancer cell and inhibiting cancer cell growth or proliferation. In certain embodiments, the cancer cell is a breast cancer cell, prostate cancer cell, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
In certain embodiments, increasing inclusion of exon 5 of a SmgGDS transcript inhibits cancer cell growth or proliferation. In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby increasing exclusion of exon 5 of a SmgGDS transcript and inhibiting cancer growth or proliferation. In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby increasing inclusion of exon 5 of a SmgGDS transcript and inducing apoptosis in a cancer cell. In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby increasing exclusion of exon 5 of a SmgGDS transcript and inducing apoptosis in a cancer cell. In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby downregulating SmgGDS-558 and leading to inhibition of cancer cell proliferation. In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby downregulating SmgGDS-607 and leading to inhibition of cancer cell proliferation. In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby downregulating SmgGDS-558 and leading to apoptosis of the cancer cell. In certain embodiments, a method comprises contacting the cancer cell with a compound or oligonucleotide provided herein, thereby downregulating SmgGDS-607 and leading to apoptosis of the cancer cell. In certain embodiments, the cancer cell is a breast cancer cell, prostate cancer cell, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer. Certain embodiments are drawn to a method of treating cancer comprising administering to a subject having cancer a compound or oligonucleotide provided herein. Examples of certain types of cancers that can be treated with the compounds and oligonucleotides described herein include, but are not limited to, breast cancer, prostate cancer, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
Certain embodiments are drawn to the use of a compound or oligonucleotide provided herein for the manufacture of a medicament for treating cancer. Certain embodiments are drawn to a compound or oligonucleotide provided herein for use in treating cancer. Examples of certain types of cancers include, but are not limited to, breast cancer, prostate cancer, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
Treating cancer refers to performing actions that lead to amelioration of cancer or of the symptoms accompanied therewith to a significant extent. The combination of said actions is encompassed by the term treating cancer. Amelioration of a cancer includes but is not limited to reducing in the number of cancer cells in an animal or reducing the number of cancer cells at a specific site in the body of an animal. Said treatment as used herein also includes an entire restoration of the health with respect to the cancers referred to herein.
Treating cancer can be described by a number of different parameters including, but not limited to, reduction in the size of a tumor in an animal having cancer, reduction in the growth or proliferation of a tumor in an animal having cancer, preventing metastasis or reducing the extent of metastasis, and/or extending the survival of an animal having cancer. The term "administration" or "administering" includes routes of introducing a compound or oligonucleotide provided herein to an animal to perform its intended function. An example of a route of administration that can be used includes, but is not limited to parenteral administration, such as subcutaneous, intravenous, intramuscular, intraarterial, intraperitoneal, or intracranial injection or infusion.
Certain Target Nucleic Acids and Mechanisms
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 R A molecule. In certain embodiments, the target nucleic acid is a pre-mR A. In certain embodiments, the target nucleic acid is a SmgGDS transcript. In certain embodiments, the target RNA is a SmgGDS pre-mRNA.
In certain embodiments, an antisense compound is complementary to a region of SmgGDS pre- mRNA. In certain embodiments, an antisense compound is complementary within a region of SmgGDS pre- mRNA comprising an exon encoding SmgGDS-558. In certain embodiments, an antisense compound is complementary to a region of SmgGDS pre-mRNA comprising an intron-exon splice junction. In certain embodiments, an antisense compound is complementary to a region of SmgGDS pre-mRNA comprising the intron-exon splice junction adjacent to exon 5. In certain embodiments, an antisense compound is complementary within a region of SmgGDS pre-mRNA consisting of exon 5. In certain embodiments, an antisense compound is complementary within a region of SmgGDS pre-mRNA comprising an exonic splicing silencer within an exon 5. In certain embodiments, an antisense compound is complementary within a region of SmgGDS pre-mRNA comprising an exonic splicing enhancer within exon 5. In certain embodiments, an antisense compound is complementary within a region of SmgGDS pre-mRNA comprising an exonic splicing silencer within an exon 5. In certain embodiments, an antisense compound is complementary within a region of SmgGDS pre-mRNA comprising an exonic splicing enhancer within exon 5.
In certain embodiments, an antisense compound comprises a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a SmgGDS transcript. In certain embodiments, the target region is within nucleobase 118642 and nucleobase 118788 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 118557 and nucleobase 118641 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 118789 and nucleobase 118859 of SEQ ID NO. : 1. In certain embodiments, the target region is within nucleobase 118557 and nucleobase 118859 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 118792 and nucleobase 118823 of SEQ ID NO.: 1.
In certain embodiments, an antisense oligonucleotide modulates splicing of a pre-mRNA. In certain embodiments, an antisense oligonucleotide modulates splicing of a SmgGDS pre-mRNA. In certain embodiments, an antisense oligonucleotide increases the amount of SmgGDS mRNA. In certain
embodiments, an antisense oligonucleotide increases the inclusion of exon 5 in SmgGDS mRNA. In certain embodiments, an antisense oligonucleotide decreases the inclusion of exon 5 in SmgGDS mRNA. In certain embodiments, an antisense oligonucleotide increases the amount of SmgGDS-607 mRNA. In certain embodiments, an antisense oligonucleotide decreases the amount of SmgGDS-558 mRNA.
In certain embodiments it is desirable to alter the splicing of SmgGDS pre-mRNA to include exon 5. By altering the splicing of SmgGDS pre-mRNA to include exon 5, expression of SmgGDS-607 will increase and expression of SmgGDS-558 will decrease. In certain embodiments it is desirable to alter the splicing of SmgGDS pre-mRNA to decrease expression of SmgGDS-558.
In certain embodiments it is desirable to alter the splicing of SmgGDS pre-mRNA to exclude exon 5.
By altering the splicing of SmgGDS pre-mRNA to exclude exon 5, expression of SmgGDS-607 will decrease and expression of SmgGDS-558 will increase. In certain embodiments it is desirable to alter the splicing of SmgGDS pre-mRNA to increase expression of SmgGDS-558. 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 eyes, ears).
In certain embodiments, a pharmaceutical composition is administered to an animal having at least one cancer cell. In certain embodiments, such administration results in apoptosis of at least cancer cell. In certain embodiments, a pharmaceutical composition is administered to an animal having at least one symptom associated with cancer. In certain embodiments, such administration results in amelioration of at least one symptom. In certain embodiments, administration of a pharmaceutical composition to an animal results in a decrease of SmgGDS-558 mR A in a cell of the animal. In certain embodiments, such administration results in an increase in SmgGDS-607 mRNA. In certain embodiments, such administration results in a decrease in SmgGDS-558 protein and an increase SmgGDS-607 protein. In certain embodiments, a SmgGDS-607 protein is preferred over a SmgGDS-558 protein. In certain embodiments, the administration of certain antisense oligonucleotides delays the onset of cancer. In certain embodiments, the administration of certain antisense oligonucleotides slows the proliferation of cancer cells. In certain embodiments, the administration of certain antisense oligonucleotides slows the proliferation of tumor cells. In certain embodiments, the administration of certain antisense oligonucleotides prevents the growth of cancer. In certain embodiments, the administration of certain antisense oligonucleotides prevents the formation of tumors. In certain embodiments, the administration of certain antisense oligonucleotides causes tumor mass to decrease. In certain embodiments, the administration of certain antisense oligonucleotides rescues cellular phenotype.
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 "AT^CGAUCG," wherein "^C 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: Screening in MDA-MB-231 cells to identify antisense oligonucleotides that modulate expression levels of SmgGDS isoforms via alternative splicing
Alternative splicing of the SmgGDS gene involves a choice between including and excluding exon 5. An antisense oligonucleotide (ASO) screen was carried out to identify potent ASOs that switch the splicing of endogenous SmgGDS transcripts to include or exclude exon 5. ASOs that promote inclusion of exon 5 increase SmgGDS-607 isoform expression and SmgGDS-558 isoform downregulation. ASOs that promote exclusion of exon 5 increase SmgGDS-558 isoform expression and SmgGDS-607 isoform downregulation. A diagram of the SmgGDS genomic region is presented in Figure 1.
The ASOs were designed as uniform oligonucleotides, 18 nucleotides in length, with 2'-0- methoxyethyl ribose sugar residues and a phosphorothioate backbone. All the cytosine nucleobases are 5- methylcytosines. The genomic sequence of SmgGDS is given by GENBANK Accession No. NT_016354.20 truncated from nucleotides 39338995 to 39523480 (designated herein as SEQ ID NO: 1). The ASOs target the region surrounding exon 5 of SmgGDS, covering the 147-nucleotide exon 5 and the flanking intronic regions in 5-nucleotide steps, as presented in Table 1. "Target Start Site" refers to the 5' position of the ASO relative to SEQ ID NO: 1. "Target Stop Site" refers to the 3' position of the ASO relative to SEQ ID NO: 1. The ASOs span nucleotides 118557 to 118859 of SEQ ID NO: 1. A plot aligning the ASOs with the genomic transcript of SmgGDS is presented in Figure 2.
To examine the effects of antisense oligonucleotide treatment of the cells on endogenous SmgGDS transcripts, MDA-MB-231 cells were transfected with each ASO at a final concentration of 50 nM. MDA- MB-231 cells were obtained from ATCC and seeded at a density of 5 x 105 cells per well in 6 well dishes the day before transfection in DMEM supplemented with 10% (v/v) FBS, 1% sodium pyruvate at 37°C and 5% C02. Transfections were performed using an ASO: LipofectAMINE2000® ratio of 25 pmoles: 1 μί.
Splicing of the SmgGDS transcripts by radioactive RT-PCR was analyzed 72 hrs after transfection.
Total RNA was extracted from cells using Qiagen RNeasy kits as per manufacturer's instructions.
Contaminating DNA was removed with DNase I on the columns as per Qiagen protocol. Reverse transcription was carried out using M-MLV reverse transcriptase (Invitrogen). Semiquantitative PCR using GoTaq Flexi DNA polymerase (Promega) was performed by including [a- 2P]-dCTP in the reactions. The human-specific primer sets used to amplify endogenous transcripts anneal to SmgGDS exons 4 and 6, and their sequences are: SmgGDSex4f: 5 ' -GTGCAGCTGCTAAATAGCAAAGAC -3' (designated herein as SEQ ID NO: 55) and SmgGDSex6r: 5 ' -GCATTTTGGCAGTGGATGCC-3 ' (designated herein as SEQ ID NO: 56).
The products were analyzed on a 5% native polyacrylamide gel, visualized and quantified on a Typhoon FLA7000 phosphorimager (GE Healthcare) using ImageQuant TLv7 software. The results are presented in Figure 3 and the Table below. Radioactivity in SmgGDS-607 was normalized for C content. Each product was quantified as a percentage of the total of SmgGDS-607 and -558. % SmgGDS-607 and % SmgGDS-558 are presented in the Table below. The first row of the Table below denotes the numbers from the mock control set of cells. Values are an average of n=3, except for the mock control (n=24), ISIS 704484 (Al l)(n=5), ISIS 704511 (D2)(n=5), ISIS 704507 (C10)(n=4), and ISIS 704489 (B4)(n=2).
Table 1
Figure imgf000046_0001
704478 A5 118637 118654 CTTCTGCCCTCATCTGTA 39.3 60.7 6
704479 A6 118642 118659 CTGCACTTCTGCCCTCAT 39.7 60.3 7
704480 A7 118647 118664 GTCAACTGCACTTCTGCC 43.2 56.8 8
704481 A8 118652 118669 GCTTGGTCAACTGCACTT 41.1 58.9 9
704482 A9 118657 118674 CACCTGCTTGGTCAACTG 42.7 57.3 10
704483 A10 118662 118679 TGCACCACCTGCTTGGTC 23.0 77.0 11
704484 Al l 118667 118684 ATCTGTGCACCACCTGCT 12.3 87.7 12
704485 A12 118672 118689 TTACAATCTGTGCACCAC 38.7 61.4 13
704486 B l 118677 118694 GTCAATTACAATCTGTGC 22.3 77.7 14
704487 B2 118682 118699 AAATGGTCAATTACAATC 45.9 54.1 15
704488 B3 118687 118704 ACCTTAAATGGTCAATTA 30.8 69.2 16
704489 B4 118692 118709 CAGTGACCTTAAATGGTC 35.7 64.3 17
704490 B5 118697 118714 CTGCACAGTGACCTTAAA 46.2 53.8 18
704491 B6 118702 118719 TTATACTGCACAGTGACC 27.0 73.1 19
704492 B7 118707 118724 ATCTGTTATACTGCACAG 24.8 75.2 20
704493 B8 118712 118729 GCGGGATCTGTTATACTG 46.1 53.9 21
704494 B9 118717 118734 CATTGGCGGGATCTGTTA 27.2 72.8 22
704495 BIO 118722 118739 CTTCTCATTGGCGGGATC 26.3 73.7 23
704496 Bl l 118727 118744 AAGAGCTTCTCATTGGCG 27.7 72.3 24
704497 B12 118732 118749 CAGTCAAGAGCTTCTCAT 20.6 79.4 25
704498 CI 118737 118754 AAAGACAGTCAAGAGCTT 26.7 73.3 26
704499 C2 118742 118759 CCACAAAAGACAGTCAAG 20.6 79.4 27
704500 C3 118747 118764 GCATGCCACAAAAGACAG 25.4 74.6 28
704501 C4 118752 118769 CATCAGCATGCCACAAAA 22.3 77.7 29
704502 C5 118757 118774 TAGTTCATCAGCATGCCA 36.7 63.3 30
704503 C6 118762 118779 TGCTATAGTTCATCAGCA 32.0 68.0 31
704504 C7 118767 118784 CTCATTGCTATAGTTCAT 36.1 64.0 32
704505 C8 118772 118789 CCATTCTCATTGCTATAG 23.2 76.8 33
704506 C9 118777 118794 GTTTACCATTCTCATTGC 25.4 74.6 34
704507 CIO 118797 118814 TAAAGCAAGTTTTCAGTT 93.4 6.6 35
704508 Cl l 118802 118819 AGAGATAAAGCAAGTTTT 91.1 8.9 36
704509 C12 118807 118824 CCCCCAGAGATAAAGCAA 68.9 31.1 37 704510 Dl 118812 118829 TTTTTCCCCCAGAGATAA 56.6 43.4 38
704511 D2 118817 118834 TTTAATTTTTCCCCCAGA 85.1 14.9 39
704512 D3 118832 118849 ATAGTAAAGATAATTTTT 89.8 10.2 40
704513 D4 118842 118859 GTTTTGCACTATAGTAAA 79.6 20.4 41
Some of the ASOs increased the proportion of SmgGDS-607 mRNA and a decrease in SmgGDS-558 mRNA. ISIS 704507 (CIO) was the most potent at promoting exon 5 inclusion to yield SmgGDS-607. Some of the ASOs increased the proportion of SmgGDS-558 mRNA and a decrease in SmgGDS-607 mRNA. ISIS 704484 (Al 1) was the most potent at promoting exon 5 exclusion to yield SmgGDS-558.
Selected ASOs described above were evaluated for modulation of SmgGDS isoform protein expression. MDA-MB-231 cells in 6-well plates were mock transfected (mock) or transfected with the indicated siRNA or ASOs. Cell lysates were made 48 or 72 hours later and subjected western blotting. Cells were lysed in 500 μΐ SDS and 15 μΐ was separated by SDS-PAGE and transferred onto polyvinyidene difluoride. Blots were blocked with 5% (w/v) milk in Tris-buffered saline with Tween-20, probed with antibodies, and visualized by enhanced chemiluminescence (MP Biomedicals). The primary antibodies used were mouse antibody to β-actin (Santa Cruz Biotechnology, 1 : 20,000) and antibody to SmgGDS (BD Transduction Laboratories, 1 : 1,000). Secondary antibodies were donkey anti-mouse HRP conjugates (GE Healthcare, 1 :20,000).
Results are presented in Figure 4. As expected, SmgGDS-607 and SmgGDS-558 isoform protein levels closely mirrored their mRNA levels after ASO treatment. ISIS 704507 (CIO), which most potently decreased SmgGDS-558 mRNA levels, also most potently decreased in SmgGDS-558 protein levels similar to a SmgGDS-558 isoform specific siRNA (BD). Consistently, ISIS 704508 (Cl l) and ISIS 704512 (D3), which had less effect at RNA level, also had less effect on protein levels. ISIS 704484 (Al 1), which potently redirected splicing towards SmgGDS-558 at the RNA level, likewise increased SmgGDS-558 protein levels. ISIS 704511 (D2), which had no effect on RNA levels, had no effect on protein levels.
Example 2: ASO microwalk centered on ISIS 704507
An ASO microwalk centered on ISIS 704507 (CIO) was performed to find the most potent ASOs that promote inclusion of exon 5 to yield SmgGDS-607.
Overlapping 18-nucleotide ASOs were designed in 1-nucleotide steps. The ASOs were designed as uniform oligonucleotides, 18 nucleotides in length, with 2'-0-methoxyethyl ribose sugar residues and a phosphorothioate backbone. All the cytosine nucleobases are 5-methylcytosines.
To examine the effects of antisense oligonucleotide treatment of the cells on endogenous SmgGDS transcripts, MDA-MB-231 cells were transfected with each ASO at a final concentration of 50 nM. Cell culture, transfection and RNA analysis was conducted in a similar manner to that described in Example 1. The results of the microwalks are presented in the Table below. Radioactivity in SmgGDS-607 was normalized for C content. Each product was quantified as a percentage of the total of SmgGDS-607 and -558. % SmgGDS-607 and % SmgGDS-558 are presented in the Table below. The first row of the Table below denotes the numbers from the mock control set of cells. Values are an average of n=3, except for the mock control (n=9).
Table 2
ASO microwalk around ISIS 704507 in MDA-MB-231 cells
Figure imgf000049_0001
Example 3: Antisense modulation of SmgGDS splicing in cancer cells
To determine the effect of ASO modulation of SmgGDS splicing in cancer cells, selected ASOs described above were tested for effect on SmgGDS mRNA reduction and proliferation of human breast cancer cell line MDA-MB-231. MDA-MB-231 cells were obtained from ATCC and grown in DMEM supplemented with 10% (v/v) FBS, and 1% sodium pyruvate, at 37°C and 5% C02. ASO transfections were conducted as described above except in 96-well plates with cells plated 5 x 103 per well; ASOs were at 50 nM concentration with an ASO: LipofectAMINE2000® ratio of 30 pmoles: 1 μί. The results are presented in the Table below. Values are an average of n=6. ISIS 704507 (CIO) more potently inhibited cancer cell proliferation than ISIS 704512 (D3), ISIS 704508 (Cl l), and ISIS 704511 (D2). ISIS 704484 (Al l), which promotes expression of SmgGDS-558, also potently inhibited cancer cell proliferation.
Table 3
Effect of ASO treatment on proliferation in MDA-MB-231 breast cancer cells
Figure imgf000050_0002
Example 4: Antisense modulation of SmgGDS splicing in a mouse xenograft cancer model
The effect of modulating SmgGDS splicing with antisense oligonucleotides on cancer growth and metastasis in vivo is examined in a mouse xenograft breast cancer model.
Xenografts are established in the mammary fat pad of SHO mice (Charles River) by subcutaneous injection of 4 x 106 MDA-MB-231 luciferase-expressing cells in a 1 : 1 PBS:Matrigel solution. After 2 weeks, control or ASOs designed to shift SmgGDS splicing toward the SmgGDS-607 isoform and away from the
SmgGDS-558 isoform are delivered by four weekly single subcutaneous injections of 50 μg ASO per gram of body weight (6 mice / group). The weekly doses of 50 μg ASO g-1 body weight is based on the very long half-life of ASOs in rodents and primates and on the observation that 1 to 4 doses was sufficient to redirect splicing, with therapeutic benefit, in mouse models of spinal muscular atrophy (Hua et al., Nature 478: 123-6, 2011; Hua et al., Genes Dev 24: 1634-44, 2010) and Usher's Syndrome (Lentz et al., Nat Med 19:345-50,
2013) . To compare ASO administration with the shRNA-mediated knockdown of SmgGDS-558, two control groups of mice are implanted with the MDA-MB-231 cells expressing dox-inducible control shRNA (Luc- 231-TR-Scr4) or SmgGDS-558 shRNA (Luc-231-TR-BD), followed by dox-feed induction two weeks later. Tumor size is measured weekly by bioluminescence imaging using a biophotonic imager (Xenogen); mice will be injected with 200
Figure imgf000050_0001
of 15 mg/mL luciferin five minutes before bioluminescent imaging.
The effect of ASOs on metastasis is assessed by biophotonic imaging of organs dissected from the mice at the conclusion of the experiments. To assess the effects of ASOs on the early stages of metastasis, luciferase expressing MDA-MB-231 cells are injected in the tail vein rather than implanted in the mammary fatpads, and biophotonic imaging will be used to assess tumor burden in organs.
To demonstrate the efficacy of ASOs and shRNA on SmgGDS protein levels, tumors will be removed from animals and lysates subjected to western blotting. ASOs designed to shift SmgGDS splicing toward the SmgGDS-607 isoform and away from the SmgGDS-558 isoform reduce SmgGDS-558 protein levels and inhibit tumor growth.
Example 5: Effect of antisense inhibition of SmGDS in human lung cancer cells
The effect of modulating SmgGDS splicing with antisense oligonucleotides was examined in NCI- H23 and NCI-H1703 non-small cell lung carcinoma (NSCLC) cells.
NCI-H23 lung carcinoma cells
NCI-H23 cells were seeded at a density of 5 x 105 cells per well in 6 well dishes in RPMI-1640 supplemented with L-glutamine (Life Technologies), 10% (v/v) heat-inactivated FBS, penicillin/streptomycin (20U/mL penicillin G sodium and 20 μg/mL streptomycin sulphate) at 37°C and 5% C02. The following day, transfections were performed using an ASO: LipofectAMINE2000® ratio of 25 pmoles: 1 μί. NCI-H23 cells were mock-transfected, or transfected with ISIS 704507 (CIO), ISIS 758698 (C10-5), ISIS 758700 (C10-7), ISIS 704484 (Al 1), or ISIS 704511 (D2) at a final concentration of 50 nM each. As a comparison, a set of cells was transfected with the SmgGDS-558 isoform specific siRNA (BD) or scrambled siRNA.
Total RNA was extracted from cells 48-72 hours later using Qiagen RNeasy kits as per
manufacturer's instructions. Contaminating DNA was removed with DNase I on the columns as per Qiagen protocol. Reverse transcription was carried out using M-MLV reverse transcriptase (Invitrogen).
Semiquantitative PCR to detect the 607-and 588-isoforms was performed using GoTaq Flexi DNA polymerase (Promega) with the addition of [a- 2P]-dCTP in the reactions. The human-specific primer sets SmgGDSex4f and SmgGDSex6r were used to amplify endogenous transcripts anneal to SmgGDS exons 4 and 6.
The products were analyzed on a 5% native polyacrylamide gel, visualized and quantified on a Typhoon FLA7000 phosphorimager (GE Healthcare) using ImageQuant TLv7 software. The results are presented in the Table below. Radioactivity was normalized for dCTP content. % SmgGDS-558 is presented in the Table below. Similar to the results obtained in the study with MDA-MB-231 breast cancer cells, the data shows that ISIS 704507, ISIS 758698, ISIS 758700 (CIO and its derivatives) significantly decrease the SmgGDS-558 isoform, similar to the decrease by the isoform-specific siRNA. ISIS 704484 (Al l) causes significant exon skipping, increasing SmgGDS-558 expression and decreasing SmgGDS-607 expression. ISIS 704511 (D2), which had no effect on RNA levels. Table 4
% SmgGDS-558 isoform mRNA expression in NCI-H23 lung cancer cells
Figure imgf000052_0001
NCI-H1703 lung carcinoma cells
A similar experiment was conducted with conditions similar to the one described above on NCI-
H1703 human NSCLC cells. The results are presented in the Table below. Similar to the results obtained in the study with MDA-MB-231 breast cancer cells and the study above, the data shows that ISIS 704507, ISIS 758698, ISIS 758700 (CIO and its derivatives) decrease the SmgGDS-558 isoform, similar to the decrease by the siRNA. ISIS 704484 (Al l) causes significant exon skipping, increasing SmgGDS-558 expression and decreasing SmgGDS-607 expression. ISIS 704511 (D2), which had no effect on RNA levels.
Table 5
% SmgGDS-558 isoform in NCI-H1703 lung cancer cells
Figure imgf000052_0002
Example 6: Effect of antisense inhibition on SmGDS protein expression in cancer cells
The effect of treatment with antisense oligonucleotides on SmgGDS-558 protein expression was measured in MDA-MB-231 human breast cancer, NCI-H23 human NSCLC cells, NCI-H1703 human NSCLC cells, and MiaPaCa-2 human pancreatic cancer cells.
Cells were plated in a 24-well tissue culture plate at 2.5 x 104 cells per well in RPMI-1640 medium supplemented with L-glutamine, 10% heat-inactivated FBS, and antibiotics (20U/mL penicillin G sodium and 20 μg/mL streptomycin sulphate). Approximately 24 hours after plating, the cells were transfected with 50 nM of ASO using LipofectAMINE2000® or Dharmafect 1 (Dharmacon) transfection reagent, or treated with the transfection reagent in the absence of ASOs ("mock). Approximately 24 hours after transfection, the transfection media was aspirated and replaced with fresh media containing 10% FBS but no antibiotics. The cells were cultured for an additional 48 hours, then harvested by aspirating the media, washing with PBS, and then adding 150 2X SDS-Loading Buffer to lyse the cells. The lysates were run on a 12% SDS-PAGE gel, followed by transfer of the proteins to PVDF membranes. The membranes were blocked with 5% (w/v) milk in Tris-buffered saline with Tween-20, and probed with antibodies to SmgGDS or GAPDH (as a loading control). The immunoreactive proteins were detected by ECL (MP Biomedicals). Densitometry of the immunoreactive proteins were measured using ImageQuant TLv8.1 software in the ImageQuant LAS 4000 Luminescent Image Analyzer.
NCI-H23 lung carcinoma cells
Cells were transfected with ISIS 704507 (CIO), ISIS 704511 (D2), ISIS 704484 (Al 1), or treated with the transfection reagent in the absence of ASOs ("mock). Western blotting of the cells showed results were consistent with the PCR results above, demonstrating diminishing of SmgGDS-588 expression by ISIS 704507 (CIO) compared to the control, and increase in SmgGDS-588 expression and decrease in SmgGDS- 607 expression by ISIS ISIS 704484 (Al 1).
Table 6
% SmgGDS isoform protein expression in NCI-H23 lung cancer cells
Figure imgf000053_0001
NCI-H1703 lung carcinoma cells
Cells were transfected with ISIS 704507 (CIO), ISIS 704511 (Dl), ISIS 704484 (Al 1), or treated with the transfection reagent in the absence of ASOs ("mock). Western blotting of the cells showed results consistent with the PCR results above, demonstrating diminishing of SmgGDS-588 expression by ISIS 704507 (CIO) compared to the control, and increase in SmgGDS-588 expression and decrease in SmgGDS- 607 expression by ISIS ISIS 704484 (Al 1).
Table 7
% SmgGDS isoform protein expression in NCI-H1703 lung cancer cells
Figure imgf000054_0001
MDA-MB-231 breast cancer cells
Cells were transfected with ISIS 704488 (B3), ISIS 704492 (B7), ISIS 704497 (B12), ISIS 704511 (D2), ISIS 704484 (Al 1), or treated with the transfection reagent in the absence of ASOs ("mock). The results indicate that ISIS 704511 (D2) did not alter SmgGDS protein expression compared to mock- transfected cells. In contrast, ISIS 704484 (Al 1) increased the expression of SmgGDS protein in MDA-MB- 231 cells. Treatment with ISIS 704488 (B3), ISIS 704492 (B7), and ISIS 704497 (B12) also increased smgGDS-588 protein expression, similar to that of ISIS 704484 (Al 1). Results are presented in the Table below.
Table 8
SmgGDS isoform protein expression (%) in MDA-MB-231 human breast cancer cells
Figure imgf000054_0002
MiaPaCa-2 human pancreatic cancer cells
Cells were transfected with ISIS 704507 (CIO), ISIS 704484 (Al 1), ISIS 704511 (D2), ISIS 758698 (C10-5), ISIS 758700 (C10-7), or treated with the transfection reagent in the absence of ASOs ("mock). The results indicate that tranfection with ISIS 704507 (CIO), ISIS 758698 (C10-5), or ISIS 758700 (C10-7) diminished SmgGDS-588 expression, whereas transfection with ISIS 704484 (Al 1) diminished SmgGDS- 607 expression. Results are presented in the Table below. Table 9
SmgGDS isoform protein expression (%) in MiaPaCa-2 human pancreatic cancer cells
Figure imgf000055_0001
In another experiment, cells were transfected with ISIS ISIS 704507 (CIO), ISIS 704484 (Al 1), ISIS 704511 (D2), or treated with the transfection reagent in the absence of ASOs ("mock). The results indicate that tranfection with ISIS 704507 (CIO) diminished SmgGDS-588 expression, whereas transfection with ISIS 704484 (Al 1) diminished SmgGDS-607 expression. Results are presented in the Table below.
Table 10
SmgGDS isoform protein expression (%) in MiaPaCa-2 human pancreatic cancer cells
Figure imgf000055_0002
Example 7: Effect of antisense inhibition of SmgGDS on cell proliferation in cancer cells
The effect of treatment with antisense oligonucleotides on cell proliferation as measured by H- thymidine uptake, as well as by the Incucyte Live Cell Imaging System, was measured in MDA-MB-231 human breast cancer and pancreatic cancer cells.
H-thymidine uptake
MiaPaCa2 pancreatic cancer cells were plated in a 96-well tissue culture plate at 7 x 103 cells per well in RPMI-1640 medium supplemented with L-glutamine, 10% heat-inactivated FBS, and antibiotics
(20U/mL penicillin G sodium and 20 μg/mL streptomycin sulphate). Approximately 24 hours after plating, the cells were transfected with 50 nM of ISIS 704507 (CIO), ISIS 704484 (Al 1), ISIS 704511 (D2), ISIS 758698 (CI 0-5), ISIS 758700 (CI 0-7), or treated with the transfection reagent in the absence of ASOs ("mock). Approximately 24 hours after transfection, the transfection media was aspirated and replaced with fresh media containing 10% FBS but no antibiotics. The cells were cultured for an additional 48 hours, and then H-thymidine was added to each well to a final concentration of 0.005 μθ per well. The cells were incubated for 3 hours, and then all but 10 of the media was removed from each well and replaced with 150 μΐ, of 5 mM EDTA/5mM EGTA in PBS. After incubating for 30 min at 37°C and 5% C02, the cells were harvested into scintillation vials with scintillation fluid, and radioactivity on the filter discs was measured by liquid scintillation counting.
The results are presented in the Table below as a representative of 4 independent experiments done with similar results and demonstrate that several ISIS oligonucleotides inhibit cell proliferation in these cancer cells.
Table 11
Cell proliferation (%) in MiaPaCa-2 human pancreatic cancer cells
Figure imgf000056_0001
Incucyte Live Cell Imaging System
Cells were plated at 3 x 106 cells per dish in 10 cm dishes in RPMI-1640 medium supplemented with
L-glutamine, 10% heat-inactivated FBS, and antibiotics (20U/mL penicillin G sodium and 20 μg/mL streptomycin sulphate). Approximately 24-30 hours after plating, the cells were transfected with 50 nM of ISIS 704507 (CIO), ISIS 704484 (Al l), ISIS 704511 (D2), with ISIS 704488 (B3), or ISIS 704492 (B7) using LipofectAMINE2000®, or treated with the transfection reagent in the absence of ASOs ("mock). Approximately 16-18 hours after transfection, the cells were collected using trypsin and re-plated onto 96- well plates. MDA-MB-231 cells were plated at a concentration of 8 x 103 cells/well and the MiaPaCa-2 cells were plated at a concentration of 4 x 103 cells/well in 96-well plates. After the cells were cultured for 5-7 hours, the plates were placed in the Incucyte Live Cell Imaging System, and recordings for cell confluency were initiated. Recordings were collected for at least 70 hours.
The results are presented in the Tables below, showing a representative experiment using MDA-MB-
231 cells (representative of two independent experiments, each conducted using five technical replicates) and MiaPaCa2 cells (representative of three independent experiments, each conducted using ten technical replicates). Proliferation is expressed as the percent of confluency of the cells. The data demonstrate that ISIS 704484 (Al 1) and ISIS 704492 (B7) have the most inhibitory effect on cell proliferation in both these types of cancer cells.
Table 12
Confluency (%) in MDA-MB-231 breast cancer cells
Figure imgf000057_0001
Table 13
Confluency (%) in MiaPaCa-2 pancreatic cancer cells
Figure imgf000057_0002

Claims

Claims:
1. A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region, wherein the complementary region comprises at least 8 contiguous nucleobases and is complementary to an equal-length portion of a target region of a SmgGDS transcript.
2. The compound of claim 1, wherein the target region of the SmgGDS transcript comprises at least a portion of exon 5 of the SmgGDS transcript.
3. The compound of claim 1 or 2, wherein the complementary region of the modified oligonucleotide is 100% complementary to the target region.
4. The compound of any of claims 1 to 3, wherein the complementary region of the modified
oligonucleotide comprises at least 10 contiguous nucleobases.
5. The compound of any of claims 1 to 3, wherein the complementary region of the modified
oligonucleotide comprises at least 15 contiguous nucleobases.
6. The compound of any of claims 1 to 3, wherein the complementary region of the modified
oligonucleotide comprises at least 20 contiguous nucleobases.
7. The compound of any of claims 1-6, wherein the nucleobase sequence of the oligonucleotide is at least 80% complementary to an equal-length region of the SmgGDS transcript, as measured over the entire length of the oligonucleotide.
8. The compound of any of claims 1-6, wherein the nucleobase sequence of the oligonucleotide is at least 90% complementary to an equal-length region of the SmgGDS transcript, as measured over the entire length of the oligonucleotide.
9. The compound of any of claims 1-6, wherein the nucleobase sequence of the oligonucleotide is 100% complementary to an equal-length region of the SmgGDS transcript, as measured over the entire length of the oligonucleotide.
10. The compound of any of claims 1-9, wherein the target region is within exon 5 or flanking intronic regions of the SmgGDS transcript.
11. The compound of any of claims 1-10, wherein the target region is within nucleobase 118642 and nucleobase 118788 of SEQ ID NO.: 1.
12. The compound of any of claims 1-10, wherein the target region is within nucleobase 118557 and nucleobase 118641 of SEQ ID NO.: 1.
13. The compound of any of claims 1-10, wherein the target region is within nucleobase 118789 and nucleobase 118859 of SEQ ID NO.: 1.
14. The compound of any of claims 1-10, wherein the target region is within nucleobase 118557 and nucleobase 118859 of SEQ ID NO.: 1.
15. The compound of any of claims 1-10, wherein the target region is within nucleobase 118792 and nucleobase 118823 of SEQ ID NO.: 1.
16. The compound of any of claims 1-15, wherein the nucleobase sequence of the antisense
oligonucleotide comprises any one of SEQ ID NOs: 2-54.
17. The compound of any of claims 1-16, wherein the modified oligonucleotide comprises at least one modified nucleoside.
18. The compound of claim 17, wherein at least one modified nucleoside comprises a modified sugar moiety.
19. The compound of claim 18, wherein at least one modified sugar moiety is a 2' -substituted sugar moiety.
20. The compound of claim 19, wherein the 2' -substitutent of at least one 2 '-substituted sugar moiety is selected from among: 2'-OMe, 2'-F, and 2'-MOE.
21. The compound of any of claims 17-20, wherein the 2'-substiuent of at least one 2 '-substituted sugar moiety is a 2'-MOE.
22. The compound of any of claims 1-18, wherein at least one modified sugar moiety is a bicyclic sugar moiety.
23. The compound of claim 22, wherein at least one bicyclic sugar moiety is LNA or cEt.
24. The compound of any of claims 18-23, wherein at least one sugar moiety is a sugar surrogate.
25. The compound of claim 24, wherein at least one sugar surrogate is a morpholino.
26. The compound of claim 24, wherein at least one sugar surrogate is a modified morpholino.
27. The compound of any of claim 1-26, wherein the modified oligonucleotide comprises at least 5 modified nucleosides, each independently comprising a modified sugar moiety.
28. The compound of claim 27, wherein the modified oligonucleotide comprises at least 10 modified nucleosides, each independently comprising a modified sugar moiety.
29. The compound of claim 27, wherein the modified oligonucleotide comprises at least 15 modified nucleosides, each independently comprising a modified sugar moiety.
30. The compound of claim 27, wherein each nucleoside of the modified oligonucleotide is a modified nucleoside, each independently comprising a modified sugar moiety
31. The compound of any of claims 1-30, wherein the modified oligonucleotide comprises at least two modified nucleosides comprising modified sugar moieties that are the same as one another.
32. The compound of any of claims 1-31, wherein the modified oligonucleotide comprises at least two modified nucleosides comprising modified sugar moieties that are different from one another.
33. The compound of any of claims 1-32, wherein the modified oligonucleotide comprises a modified region of at least 5 contiguous modified nucleosides.
34. The compound of claim 33, wherein the modified oligonucleotide comprises a modified region of at least 10 contiguous modified nucleosides.
35. The compound of claim 33, wherein the modified oligonucleotide comprises a modified region of at least 15 contiguous modified nucleosides.
36. The compound of claim 33, wherein the modified oligonucleotide comprises a modified region of at least 20 contiguous modified nucleosides.
37. The compound of any of claims 32-36, wherein each modified nucleoside of the modified region has a modified sugar moiety independently selected from among: 2'-F, 2'-OMe, 2'-MOE, cEt, LNA, morpholino, and modified morpholino.
38. The compound of any of claims 33-37, wherein the modified nucleosides of the modified region each comprise the same modification as one another.
39. The compound of claim 38, wherein the modified nucleosides of the modified region each comprise the same 2 '-substituted sugar moiety.
40. The compound of claim 38, wherein the 2' -substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from 2'-F, 2'-OMe, and 2'-MOE.
41. The compound of claim 39, wherein the 2' -substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is 2'-MOE.
42. The compound of claim 38, wherein the modified nucleosides of the region of modified nucleosides each comprise the same bicyclic sugar moiety.
43. The compound of claim 42, wherein the bicyclic sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from LNA and cEt.
44. The compound of claim 38, wherein the modified nucleosides of the region of modified nucleosides each comprises a sugar surrogate.
45. The compound of claim 44, wherein the sugar surrogate of the modified nucleosides of the region of modified nucleosides is a morpholino.
46. The compound of claim 44, wherein the sugar surrogate of the modified nucleosides of the region of modified nucleosides is a modified morpholino.
47. The compound of any of claims 1-46, wherein the modified nucleotide comprises no more than 4 contiguous naturally occurring nucleosides.
48. The compound of any of claims 1-46, wherein each nucleoside of the modified oligonucleotide is a modified nucleoside.
49. The compound of claim 48 wherein each modified nucleoside comprises a modified sugar moiety.
50. The compound of claim 49, wherein the modified nucleosides of the modified oligonucleotide comprise the same modification as one another.
51. The compound of claim 50, wherein the modified nucleosides of the modified oligonucleotide each comprise the same 2 '-substituted sugar moiety.
52. The compound of claim 51, wherein the 2' -substituted sugar moiety of the modified oligonucleotide is selected from 2'-F, 2'-OMe, and 2'-MOE.
53. The compound of claim 52, wherein the 2' -substituted sugar moiety of the modified oligonucleotide is 2'-MOE.
54. The compound of claim 50, wherein the modified nucleosides of the modified oligonucleotide each comprise the same bicyclic sugar moiety.
55. The compound of claim 54, wherein the bicyclic sugar moiety of the modified oligonucleotide is selected from LNA and cEt.
56. The compound of claim 50, wherein the modified nucleosides of the modified oligonucleotide each comprises a sugar surrogate.
57. The compound of claim 56, wherein the sugar surrogate of the modified oligonucleotide is a
morpholino.
58. The compound of claim 56, wherein the sugar surrogate of the modified oligonucleotide is a modified morpholino.
59. The compound of any of claims 1-58, wherein the modified oligonucleotide comprises at least one modified intemucleoside linkage.
60. The compound of claim 59, wherein each intemucleoside linkage is a modified intemucleoside
linkage.
61. The compound of claim 59 or 60, comprising at least one phosphorothioate intemucleoside linkage.
62. The compound of claim 60, wherein each intemucleoside linkage is a modified intemucleoside
linkage and wherein each intemucleoside linkage comprises the same modification.
63. The compound of claim 62, wherein each intemucleoside linkage is a phosphorothioate
intemucleoside linkage.
64. The compound of any of claims 1-63 comprising at least one conjugate.
65. The compound of any of claims 1-64 consisting of the modified oligonucleotide.
66. The compound of any of claims 1-65, wherein the compound modulates splicing of the SmgGDS transcript.
67. A pharmaceutical composition comprising a compound according to any of claims 1-66 and a
pharmaceutically acceptable carrier or diluent.
68. The pharmaceutical composition of claim 67, wherein the pharmaceutically acceptable carrier or diluent is sterile saline.
69. A method of modulating splicing of a SmgGDS transcript in a cancer cell comprising contacting the cancer cell with the compound according to any of claims 1 -66 or composition according to claim 67 or 68.
70. The method of claim 69, wherein the cell is in vitro.
71. The method of claim 69, wherein the cell is in an animal.
72. The method of any of claims 69-71, wherein inclusion of exon 5 is increased.
73. The method of any of claims 69-72, wherein exclusion of exon 5 is increased.
74. The method of any of claims 69-73, wherein inclusion of exon 5 is decreased.
75. The method of any of claims 69-74, wherein SmgGDS-607 mR A expression is increased.
76. The method of any of claims 69-75, wherein SmgGDS-558 mRNA expression is decreased.
77. A method of modulating the expression of SmgGDS in a cancer cell, comprising contacting the cancer cell with the compound according to any of claims 1 -66 or composition according to claim 67 or 68.
78. The method of claim 77, wherein SmgGDS-607 expression is increased.
79. The method of claims 77 or 78, wherein SmgGDS-558 expression is decreased.
80. The method of claim 79, wherein the cell is in vitro.
81. The method of claim 79, wherein the cell is in an animal.
82. A method of inhibiting cancer cell growth or proliferation, comprising contacting the cancer cell with the compound according to any of claims 1-66 or composition according to claim 67 or 68.
83. The method of claim 82, wherein the cell is in vitro.
84. The method of claim 82, wherein the cell is in an animal.
85. A method of treating cancer in a subject comprising administering to the subject the compound
according to any of claims 1-66 or composition according to claim 67 or 68.
86. The method of claim 85, wherein the subject is a human.
87. The method of claim 85 or 86, wherein administering the compound to the subject inhibits cancer growth, inhibits metastasis, and/or increases survival of the subject.
88. The method of any one of claims 69-87, wherein the cancer is breast cancer, prostate cancer, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
89. Use of the compound according to any of claims 1-66 or composition according to claim 67 or 68 for the preparation of a medicament for use in the treatment of cancer.
90. A compound according to any of claims 1-66 or composition according to claim 67 or 68 for use in treating cancer.
91. The use of claim 89 or 90, wherein the cancer is breast cancer, prostate cancer, lung cancer, squamous cell carcinoma of the head and neck (SCCHN), colon cancer, and pancreatic cancer.
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