WO2015023938A1 - Epigenetic regulators of frataxin - Google Patents

Epigenetic regulators of frataxin Download PDF

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Publication number
WO2015023938A1
WO2015023938A1 PCT/US2014/051258 US2014051258W WO2015023938A1 WO 2015023938 A1 WO2015023938 A1 WO 2015023938A1 US 2014051258 W US2014051258 W US 2014051258W WO 2015023938 A1 WO2015023938 A1 WO 2015023938A1
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fxn
dcs
dgs
dts
das
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PCT/US2014/051258
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French (fr)
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Fatih Ozsolak
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Rana Therapeutics, Inc.
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Priority to EP14836705.5A priority Critical patent/EP3033423A4/en
Priority to US14/911,836 priority patent/US20160201063A1/en
Publication of WO2015023938A1 publication Critical patent/WO2015023938A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===

Definitions

  • the invention relates in part to compositions and methods for modulating gene expression.
  • FRDA Friedreich's ataxia
  • Symptoms typically begin between ages of 5 and 15 years and first present as difficulty walking (gait ataxia). As the disease progresses, other symptoms develop, such as speech slurring, hearing loss, and vision loss.
  • Various forms of heart disease often accompany FRDA, including hypertrophic cardiomyopathy, myocardial fibrosis, and cardiac failure. Approximately, ten percent of those affected by FRDA develop diabetes. Symptom progression varies between individuals, but generally within 10 to 20 years from disease onset, the person is wheelchair bound and may eventually become completely incapacitated.
  • FRDA can lead to early death, often as a result of heart disease associated with FRDA.
  • Reduced expression of Frataxin (FXN) is thought to cause Friedreich's ataxia (FRDA).
  • regulatory factors have been identified that modulate expression of FXN in cells. Both negative and positive regulators of FXN expression have been discovered.
  • regulatory factors disclosed herein modulate FXN expression by modulating the epigenetic state of FXN genes.
  • inhibiting expression of a negative regulator of FXN results increased expression of FXN in cells, e.g., cells from a patient with FRDA.
  • inducing expression of a positive regulator of FXN results in increased expression of FXN in cells, e.g., cells from a patient with FRDA.
  • the invention provides methods and compositions that are useful for upregulating FXN in a cell. Accordingly, in some embodiments, methods and compositions provided herein are useful for the treatment and/or prevention (e.g., reducing the risk or delaying the onset) of FRDA.
  • aspects of the invention relate to methods for increasing FXN expression in a cell.
  • the methods involve delivering to a cell an oligonucleotide that inhibits expression or activity of a negative epigenetic regulator of FXN, thereby increasing FXN expression in the cell.
  • the cell prior to delivering the oligonucleotide, the cell has a lower level of FXN expression compared to an appropriate control level of FXN expression.
  • prior to delivering the oligonucleotide the cell has a higher level of histone H3 K27 or K9 methylation at the FXN gene compared with an appropriate control level of histone H3 K27 or K9 methylation.
  • the cell comprises an FXN gene encoding in its first intron a GAA repeat of between 10-2000 units.
  • the cell is obtained from or present in a subject having
  • the oligonucleotide in the cell results in decreased levels of mRNA of the negative epigenetic regulator of FXN .
  • the appropriate control is a level of FXN in a cell from a subject or in cells from a population of subjects that do not have Friedreich's ataxia.
  • the oligonucleotide comprises a sequence as set for in Table 4. In some embodiments, the oligonucleotide comprises a sequence as set for in Table 12. In some embodiments, the oligonucleotide is a gapmer, a mixmer, an siRNA, a single stranded
  • RNA a single stranded DNA, an aptamer, or a ribozyme.
  • the oligonucleotide comprises at least one modified nucleotide or internucleoside linkage.
  • the oligonucleotide is a single stranded oligonucleotide.
  • the single stranded oligonucleotide comprises the sequence 5'-X-Y-Z-3', wherein X comprises 1-5 modified nucleotides, Y comprises at least 6 unmodified nucleotides, and Z comprises 1-5 modified nucleotides.
  • the X comprises 1-5 LNAs
  • Y comprises at least 6 DNAs
  • Z comprises 1-5 LNAs.
  • the negative epigenetic regulator of FXN is a component of a histone H2A acetylation pathway, a NuA4 histone acetyltransferase complex, a protein amino acid acetylation pathway, a histone acetylation pathway, a protein amino acid acylation pathway, a H4/H2A histone acetyltransferase complex, a nucleotide binding pathway, a histone H4 acetylation pathway, a histone acetyltransferase complex, or an insulin receptor substrate binding pathway.
  • the component of the histone H2A acetylation pathway is MEAF6, YEATS4, ACTL6A, or DMAPl.
  • the component of the NuA4 histone acetyltransferase complex is MEAF6, YEATS4, ACTL6A, or DMAPl.
  • the component of the protein amino acid acetylation pathway is KAT2A, MEAF6, YEATS4, TAD A3, ACTL6A, or DMAPl .
  • the component of the histone acetylation pathway is KAT2A, MEAF6, YEATS4, TAD A3, ACTL6A, or DMAPl.
  • the component of the protein amino acid acylation pathway is KAT2A, MEAF6, YEATS4, TAD A3, ACTL6A, or DMAPl.
  • the component of the H4/H2A histone acetyltransferase complex is MEAF6, YEATS4, ACTL6A, or DMAPl.
  • the component of the nucleotide binding pathway is MEF2D, PRKDC, IDH1, ACTL6A, JAK2, CFTR, SPEN, or PRKCD.
  • the component of the histone H4 acetylation pathway is MEAF6, YEATS4, ACTL6A, or DMAPl.
  • the component of the histone acetyltransferase complex is KAT2A, MEAF6, YEATS4, TAD A3, ACTL6A, or DMAPl .
  • the component of the insulin receptor substrate binding pathway is JAK2 or PRKCD.
  • the negative epigenetic regulator of FXN is TNFSF9, JUND, HICl, PRKCD, JAK2, EID1, CFTR, TAD A3, MYBL2, KAT2A, IDH1, SUMOl, SPEN, PRKDC, KIR2DL4, APC, MEF2D, a component of the NuA4 Histone Acetyltransferase Complex, or a histone-lysine N-methyltransf erase.
  • the negative epigenetic regulator of FXN is a component of the NuA4 Histone Acetyltransferase Complex.
  • the component of the NuA4 Histone Acetyltransferase Complex is YEATS4, Eafl, TRRAP, P400, EPCl, DMAPl, Tip60, MRG15, MRGX, MORF4, ACTB, ACTL6A, ING1, ING2, ING3, ING4, ING5, RUVBL1, RUVBL2, AF9, ENL, or MEAF6.
  • the component of the component of the component of the NuA4 Histone Acetyltransferase Complex is YEATS4, Eafl, TRRAP, P400, EPCl, DMAPl, Tip60, MRG15, MRGX, MORF4, ACTB, ACTL6A, ING1, ING2, ING3, ING4, ING5, RUVBL1, RUVBL2, AF9, ENL, or MEAF6.
  • NuA4 Histone Acetyltransferase Complex is YEATS4, ACTL6A, DMAPl, or MEAF6.
  • the component of the NuA4 Histone Acetyltransferase Complex is YEATS4.
  • the negative epigenetic regulator of FXN is a histone-lysine N- methyltransferase. In some embodiments, the histone-lysine N-methyltransferase is
  • the histone-lysine N-methyltransferase is SUV39H1.
  • the negative epigenetic regulator of FXN is YEATS4, HICl, JUND, TNFSF9, PRKCD, KAT2A, JAK2, IDH1, EID1, or ACTL6A.
  • the negative epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change greater than 1.25.
  • the method further comprises: delivering to the cell a second oligonucleotide.
  • the second oligonucleotide inhibits expression or activity of a second negative epigenetic regulator of FXN.
  • the second negative epigenetic regulator of FXN is TNFSF9, JUND, HICl, PRKCD, JAK2, EID1, CFTR, TAD A3, MYBL2, KAT2A, IDH1, SUMOl, SPEN, PRKDC, KIR2DL4, APC, MEF2D, a component of the NuA4 Histone Acetyltransferase Complex, or a histone-lysine N-methyltransferase.
  • methods for increasing FXN expression in a cell involve delivering to a cell an expression vector that is engineered to express a positive epigenetic regulator of FXN, thereby increasing FXN expression in the cell.
  • the cell prior to delivering the expression vector, the cell has a lower level of FXN expression compared to an appropriate control level of FXN expression.
  • methods for increasing FXN expression in a cell involve expressing a exogenous positive epigenetic regulator of FXN.
  • the positive epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change less than or equal to 1.0, 0.90, 0.85, 0.80, 0.75, or 0.50.
  • oligonucleotides are provided that comprise a sequence as set forth in Table 4 or Table 12.
  • the oligonucleotide comprises at least one modified nucleotide or internucleoside linkage.
  • the oligonucleotide is 50 nucleotides or fewer in length.
  • the oligonucleotide consists of a sequence as set forth in Table 4.
  • the oligonucleotide consists of a sequence as set forth in Table 12.
  • FIG. 1 is a graph depicting epigenetic siRNA screen fold change distribution.
  • FIG. 2 is a table depicting the siRNA Screening Results.
  • FXN downregulating genes are genes for which reduced expression results in downregulation of FXN.
  • FXN upregulating genes are genes for which reduced expression results in upregulation of FXN
  • FIG. 3A is a table depicting the siRNA data related to the NuA4 Histone
  • FIG. 3B is a graph depicting that knockdown of Suv39Hl resulted in upregulation of FXN.
  • FIGs. 4A and 4B shows a screen of 80 epigenetic inhibitors from a epigenetics screening library using GM03816 FRDA diseased fibroblasts (Fig. 4A; actual data in Table 10) and GM0321 normal fibroblasts (Fig. 4B; actual data in Table 11).
  • FXN RNA levels are indicated on the y-axis and the inhibitors used at both 1 ⁇ and 5 ⁇ are shown on the x- axis.
  • FIGs. 5A-5E shows treatment of human FRDA diseased cell lines and Sarsero FXN mouse-model derived fibroblasts with a histone lysine methyltransferase inhibitor (HLMi).
  • the Sarsero mouse model was generated by inserting the diseased human FXN gene with GAA-repeated into mouse genome.
  • RQ FXN RNA quantity in compound treated cells relative to untreated cells.
  • FIG. 5A shows GM03816 cells after 2 days of treatment with the
  • FIG. 5B shows GM03816 cells after 3 days of treatment with the HLMi at the indicated concentration
  • FIG. 5C shows GM04078 cells after 3 days of treatment with the HLMi at the indicated concentration
  • FIG. 5D shows Sarsero fibroblasts after 3 days of treatment with the HLMi at the indicated concentration (mouse FXN expression)
  • FIG. 5E shows Sarsero fibroblasts 3 day treatment with the HLMi at the indicated concentration (human FXN expression).
  • FIG. 6 shows a western blot to detect FXN protein upregulation in human FRDA diseased cell lines GM03816 and GM04078 following 3 days of treatment with a HLMi at various concentrations (5 ⁇ , 2.5 ⁇ , 1.25 ⁇ ). Results from control cells treated with DMSO and without inhibitor treatment are also shown.
  • FIG. 7A and B are a series of graphs showing FXN mRNA levels in cells treated with o gapmers for human JUND, YEATS4, HICl, ACTL6A, EID1, IDH1, TNFSF9, JAK2,
  • KAT2A or PRKCD blank columns are untreated.
  • FIG. 8 is a photograph of a Western blot showing FXN protein levels in cells treated with gapmers for ACTL6A, JUND, PRKCD, and YEATS4.
  • FIG. 9 is a graph showing FXN mRNA levels in differentiated myotubes treated with 5 various gapmers for ACTL6A, EID 1 , HIC 1 , JUND, KAT2A, PRKCD, and YEATS4.
  • FIGs. 10A-D are a series of graphs showing enrichment in the FXN gene locus of H3K27me3 and H3K9me3 (10A and 10B), Tip60 (IOC), or SUV39H1 (10D) in diseased cell lines compared to normal cells.
  • FIGs. 11 A and 1 IB are a series of graphs showing showing enrichment in the FXN o gene locus of G9a (FIG. 11 A) and IgG (FIG. 1 IB) in diseased cell lines compared to normal cells.
  • regulatory factors disclosed herein modulate FXN expression by controlling the epigenetic state of FXN genes.
  • methods and compositions are provided that induce or enhance expression of FXN by decreasing expression or function of one or more negative epigenetic regulators of FXN. In some embodiments, this induced or enhanced expression of FXN is believed to result from a
  • FXN gene K9 methylation at the FXN gene.
  • methods for inducing expression of a positive regulator of FXN may be used to induce or enhance expression of FXN.
  • this induced or enhanced expression of FXN is believed to result from a change in the chromatin state of the FXN gene, e.g., a decreased level of histone H3 K27 or K9 methylation at the FXN gene.
  • FXN gene refers to a genomic region that encodes FXN 5 protein and/or controls the transcription of FXN mRNA.
  • the term encompasses coding sequences and exons as well as any non-coding elements, e.g., promoters, enhancers, silencers, introns, and 5' and 3' untranslated regions.
  • An FXN gene may include flanking sequences 5' and/or 3' to a known annotated FXN open reading frame, e.g., 1 Kb, 2Kb, 3Kb, 4Kb, 5Kb, 6Kb, 7Kb, 8Kb, 9Kb, or 10Kb or more flanking the 5' and/or 3' end of a known o annotated FXN open reading frame.
  • a FXN gene may be a human
  • FXN gene (see, e.g., NCBI Gene ID: 2395, located on chromosome 9).
  • a FXN gene may be a corresponding homolog of a FXN gene in a different species (e.g., a mouse FXN encoded by a mouse FXN gene such as NCBI Gene ID: 14297). 5 Negative Epigenetic regulators of FXN
  • a "negative epigenetic regulator” is a regulatory factor (e.g., regulatory protein) that promotes the formation or maintenance of heterochromatin, and/or that inhibits the formation or maintenance of euchromatin.
  • a negative epigenetic regulator inhibits or reduces FXN expression either directly or indirectly.
  • negative epigenetic regulators mediate reduction or silencing of FXN
  • FXN expression though an epigenetic mechanism, e.g., though heterochromatin formation at or near the FXN gene. Accordingly, in some embodiments, when the expression level of a negative epigenetic regulator of FXN is reduced (e.g., by contacting a cell with an appropriate oligonucleotide as described herein), FXN expression is upregulated.
  • heterochromatin formation at the FXN gene can be reversed, in part or in whole, by reducing the expression of one or more negative epigenetic regulators of FXN, thereby causing upregulation of FXN expression.
  • Heterochromatin formation can be measured using any method known in the art, e.g., using an immunoassay to detect methylation patterns at or near 0 the FXN gene. For example, levels of mono-, di- and tri-methylation of histone H3 at lysine
  • Negative epigenetic regulators of FXN may act directly on the FXN gene, e.g., by catalyzing methylation of a histone, or indirectly, e.g., by forming a complex with or activating other proteins that are involved in epigenetic modification of the FXN gene.
  • negative epigenetic regulators of FXN are provided in Tables 1 and 7.
  • the gene ID and transcript ID for each gene are provided, which can be used to identify any gene, mRNA transcript, and protein sequences by querying the NCBI (National Center for NCBI).
  • a negative epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change greater than 1. In some embodiments, a negative epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change greater than 1.5. In some embodiments, a negative epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change greater than 1.75. In some embodiments, a negative epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change greater than 2. In some embodiments, a negative epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change greater than 2.5.
  • one or more chromatin markers may be evaluated to assess the chromatin status of an FXN gene.
  • Histone H4 K20 trimethylation may be used as a marker to indicate heterochromatin.
  • Presence of HP1, SUV39 and/or other similar proteins may also be used to detect presence of heterochromatin at the FXN gene.
  • Other suitable markers may be used to assess chromatin status of an FXN gene.
  • PRKDC DNA-PKcs DNAPK, 5591 NM_001081640.1 19090 NM_011159.2
  • GLP GLP1, KMT1D, N M. _172545.4 bA188C12.1
  • a epigenetic regulator of FXN may be a component of the NuA4 Histone Acetyltransf erase Complex.
  • the NuA4 histone acetyltransf erase complex is a complex having histone acetylase activity on chromatin, as well as ATPase, DNA helicase 5 and structural DNA binding activities.
  • Subunits of the human complex include YEATS4, Eafl, TRRAP, P400, EPCl, DMAPl, Tip60, MRG15, MRGX, MORF4, ACTB, ACTL6A, ING1, ING2, ING3, ING4, ING5, RUVBL1, RUVBL2, AF9, ENL, and MEAF6.
  • a negative epigenetic regulator of FXN may be a histone- lysine N-methyltransferase.
  • Histone-lysine N-methyltransferases catalyze the transfer of one, o two or three methyl groups to a lysine residue of a histone protein.
  • the histone-lysine N-methyltransferase is capable of transferring one, two or three methyl groups to lysine 9 on histone H3 (H3K9me3). Methylation of lysine 9 on histone H3, especially near a gene promoter, is thought to reduce gene expression.
  • H3K9me3 histone- lysine N-methyltransferases are well-known in the art and include SUV39H1, SUV39H2, 5 SETDB 1 , PRDM2, G9 A and EHMT 1.
  • a "positive epigenetic regulator” is a regulatory factor (e.g., a regulatory protein) that inhibits the formation or maintenance of heterochromatin, and/or that o promotes the formation or maintenance of euchromatin.
  • heterochromatin formation at the FXN gene can be reversed, in part or in whole, by increasing the expression of one or more positive epigenetic regulators of FXN, thereby causing upregulation of FXN expression.
  • a positive 5 epigenetic regulator of FXN induces expression of FXN by directly or indirectly inhibiting the formation or maintenance of heterochromatin at an FXN gene, and/or promoting the formation or maintenance of euchromatin at an FXN gene. Accordingly, in some
  • a positive epigenetic regulator of FXN when the expression level of a positive epigenetic regulator of FXN is induced or increased, FXN expression may be upregulated.
  • a positive epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change less than 1.
  • a positive epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change less than 0.75.
  • a positive epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change less than 0.5.
  • a positive epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change less than 0.25.
  • a positive regulator of FXN is the product of a gene listed in Table 8.
  • an epigenetic regulator of FXN is a component of the histone H2A acetylation pathway, the NuA4 histone acetyltransferase complex, the protein amino acid acetylation pathway, the histone acetylation pathway, the protein amino acid acylation pathway, the H4/H2A histone acetyltransferase complex, the nucleotide binding pathway, the histone H4 acetylation pathway, the histone acetyltransferase complex, or the insulin receptor substrate binding pathway.
  • Components of each pathway may be identified using the Gene ontology reference ID provided for each pathway in Table 7 ("GO:######").
  • the reference ID can be entered into the search function of the Gene Ontology website, and gene product associations can be identified. These gene product associations indicate other potential epigenetic regulators of FXN.
  • negative epigenetic regulators of FXN that are components of certain pathways are provided in Table 7.
  • positive epigenetic regulators of FXN that are components of certain pathways are provided in Table 8.
  • the invention relates to methods for modulating FXN gene expression cells (e.g., cells for which FXN levels are reduced) for research purposes.
  • the invention relates to methods for modulating gene expression in cells (e.g., cells for which FXN levels are reduced) for therapeutic purposes.
  • Cells can be in vitro, ex vivo, or in vivo (e.g., in a subject who has a disease resulting from reduced expression or activity of FXN, e.g., Friedreich's ataxia.)
  • methods for modulating FXN expression in cells comprise delivering to the cells an oligonucleotide that inhibits expression or activity of a negative epigenetic regulator of FXN.
  • methods for modulating FXN expression in cells comprise delivering to the cells an inhibitor that inhibits activity of a negative epigenetic regulator of FXN. In some embodiments, methods for modulating FXN expression cells comprise delivering to the cells a cDNA engineered to express a positive epigenetic regulator of FXN.
  • any reference to uses of compounds contemplates use of the compound in preparation of a pharmaceutical composition or medicament for use in the treatment of condition or a disease (e.g., Friedreich's ataxia) associated with decreased levels or activity of FXN.
  • this aspect of the invention includes use of oligonucleotides or inhibitors in the preparation of a medicament for use in the treatment of disease, wherein the treatment involves upregulating expression of FXN.
  • this aspect of the invention includes use of expression vector (e.g., containing a coding region of a positive epigenetic regulator of FXN) in the preparation of a medicament for use in the treatment of disease, wherein the treatment involves upregulating expression of FXN.
  • expression vector e.g., containing a coding region of a positive epigenetic regulator of FXN
  • methods provided herein comprise contacting a cell having a lower level of FXN expression compared to an appropriate control level of FXN expression with a composition (e.g., oligonucleotide, expression vector, inhibitor) useful for
  • methods provided herein comprise contacting a cell having a lower level of FXN expression compared to an appropriate control level of FXN expression with an oligonucleotide specific for an mRNA of a negative epigenetic regulator of FXN as described herein, wherein the oligonucleotide reduces an expression level of the negative epigenetic regulator of FXN), thereby increasing FXN expression in the cell.
  • the cell may be contacted with more than one oligonucleotide that targets one or more negative epigenetic regulators of FXN, e.g., a first oligonucleotide that targets a first negative epigenetic regulator of FXN as described herein and a second oligonucleotide that targets a second negative epigenetic regulator of FXN as described herein.
  • provided herein are methods for inhibiting the function of a negative epigenetic regulator of FXN (e.g., by contacting a cell with an appropriate inhibitor as described herein), thereby upregulating FXN expression.
  • methods for increasing FXN expression in a cell by using one more inhibitors of histone-lysine N-methyltransferase.
  • the histone- lysine N-methyltransferase is capable of transferring one, two or three methyl groups to lysine 9 on histone H3 (H3K9me3).
  • the histone-lysine N- methyltransferase is SUV39H1.
  • the methods involve delivering to a cell an inhibitor that inhibits HLM, thereby increasing FXN expression in the cell.
  • a change in the chromatin state of the FXN gene e.g., a decreased level of histone H3 K9 methylation at the FXN gene
  • the inhibitor is a small molecule inhibitor.
  • the level of expression of FXN using a histone-lysine N- methyltransferase inhibitor is increased by at least about 1.1X-1.5X, 1.5X-2X, 2X- 2.5X, 2.5X-3X, or 3X-4X the control level of FXN expression.
  • a cell having a lower level of FXN expression compared to an appropriate control level of FXN expression has a level of FXN expression that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more lower than an appropriate control level of FXN expression.
  • a level of FXN expression may be determined using any suitable assay known in the art (see, e.g., Molecular Cloning: A Laboratory Manual, J.
  • the FXN expression level may be an mRNA level or a protein level.
  • the sequences of FXN mPvNAs and proteins are well-known in the art (see, e.g., NCBI Transcript IDs:
  • an appropriate control level of FXN expression may be, e.g., a level of FXN expression in a cell, tissue or fluid obtained from a healthy subject or population of healthy subjects.
  • a healthy subject is a subject that is apparently free of disease and has no history of disease, e.g., no history of Friedreich's ataxia.
  • an appropriate control level of is a level of FXN expression in a cell from a subject that does not have Friedreich's ataxia or a level of FXN expression in a population of cells from a population of subjects that do not have Friedreich's ataxia.
  • the subject or population of subjects that do not have Friedreich's ataxia are subjects that have a FXN gene locus that contains less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 GAA repeat units in the first intron.
  • an appropriate control level of FXN may be a level of FXN expression in a cell, tissue, or subject to which an oligonucleotide has not been delivered or to which a negative control has been delivered (e.g., a scrambled oligo, a carrier, etc.).
  • an appropriate control level of FXN expression may be a predetermined level or value, such that a control level need not be measured every time.
  • the predetermined level or value can take a variety of forms. It can be single cut-off value, such as a median or mean. It can be established based upon comparative groups, such as where one defined group is known have Friedriech's ataxia and another defined group is known to not have Friedriech' s ataxia.
  • the tested population is divided equally (or unequally) into groups, such as a group of subjects having a high number of GAA repeats in the first intron of FXN (e.g., over 1000 GAA repeats), a group of subjects having a moderate number of GAA repeats (e.g., from 20-1000 GAA repeats) and a group of subjects having a low number of GAA repeats (e.g., less than 20 GAA repeats).
  • groups such as a group of subjects having a high number of GAA repeats in the first intron of FXN (e.g., over 1000 GAA repeats), a group of subjects having a moderate number of GAA repeats (e.g., from 20-1000 GAA repeats) and a group of subjects having a low number of GAA repeats (e.g., less than 20 GAA repeats).
  • the predetermined value can depend upon the particular population selected.
  • the predetermined values selected may take into account the category in which a subject falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art.
  • a cell having a lower level of FXN expression compared to an appropriate control level of FXN expression is a cell that has a higher level of histone H3
  • An appropriate control level of histone H3 K27 or K9 methylation may be, e.g., a level of histone H3 K27 or K9 methylation in a cell, tissue or fluid obtained from a healthy subject or population of healthy subjects, such as a subject or subjects that do not have Friedreich's ataxia.
  • a level of H3 K27 or K9 methylation expression may be determined using any suitable assay known in the art. Examples of assays for detecting histone methylation levels include, but are not limited to, immunoassays such as Western blot, immunohistochemistry and ELISA assays.
  • Such assays may involve a binding partner, such as an antibody, that specifically binds to a methylated or unmethylated histone.
  • a binding partner such as an antibody
  • Antibodies that recognize specific methylation patterns on histones are known in the art and available from commercial vendors (see, e.g., AbCam and Millipore).
  • a cell having a lower level of FXN expression compared to an appropriate control level of FXN expression is a cell that comprises an FXN gene encoding in its first intron a GAA repeat of between 10-2000, 15-2000, 20-2000, 30-2000, 40-2000, 50- 2000, 100-2000, 10-1000, 15-1000, 20-1000, 30-1000, 40-1000, 50-1000, or 100-1000 units.
  • the number of GAA repeats may be determined using any method known in the art, e.g., sequencing-based assays or probe-based assays.
  • a cell having a lower level of FXN expression compared to an appropriate control level of FXN expression is a cell obtained from a subject having
  • a subject having Friedreich's ataxia can be identified, e.g., by the number of GAA repeats present in the first intron of an FXN gene of the subject and/or by other diagnostic criteria or symptoms known in the art.
  • Symptoms of Friedreich's ataxia include, but are not limited to, muscle weakness in the arms and legs, loss of coordination, vision impairment, hearing impairment, slurred speech, curvature of the spine, high plantar arches, diabetes, and/or heart disorders (e.g., cardiomegaly, atrial fibrillation, tachycardia and hypertrophic cardiomyopathy).
  • a physical examination of eye movements, deep tendon reflexes, extensor plantar responses, and cardiac sounds may aid in diagnosis of a subject suspected of having Friedreich's ataxia.
  • a genetic test e.g., a PCR-based test, may be used to identify a subject having expanded GAA triplet repeats in the first intron of FXN.
  • reducing an expression level of a negative epigenetic regulator of FXN includes reducing an expression level of the negative epigenetic regulator of FXN to 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%,
  • An appropriate control level may be, e.g., a level of the negative epigenetic regulator of FXN in a cell that has not been contacted with an oligonucleotide or inhibitor as described herein.
  • the expression level of the negative epigenetic regulator of FXN may be an mRNA level or a protein level.
  • an oligonucleotide as described herein may reduce the mRNA and/or protein level of the negative epigenetic regulator of FXN. For example, if the oligonucleotide is designed to 5 degrade the mRNA, the level of mRNA will be reduced, and subsequently the level of protein will also be reduced.
  • the level of protein will be reduced, but the level of mRNA may remain stable.
  • Assays for determining mRNA and protein levels are well-known in the art (e.g.,
  • microarrays sequencing-based assays, probe-based assays, immunoassays, mass- o spectrometry, etc . ) .
  • increasing FXN expression in a cell includes a level of FXN expression that is, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more above an appropriate control level of FXN.
  • the appropriate control level may be a level of FXN expression in a cell that has not been
  • the FXN expression may be FXN mRNA and/or protein expression. In some embodiments, increasing FXN
  • a level of FXN expression in a cell includes increasing a level of FXN expression to within 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a level of FXN expression in a cell from a healthy subject or a population of cells from a population of healthy subjects, e.g., subjects o that do not have Friedreich' s ataxia.
  • FXN expression in a cell obtained from or in a subject who is healthy is a cell obtained from or in a subject who is healthy.
  • methods comprise administering to a subject (e.g. a human) a composition as described herein (e.g., a composition comprising an oligonucleotide and/or inhibitor targeting a negative epigenetic regulator of FXN) to increase FXN protein 0 levels in the subject.
  • a composition as described herein e.g., a composition comprising an oligonucleotide and/or inhibitor targeting a negative epigenetic regulator of FXN
  • the increase in protein levels is at least 5%, 10%,
  • compositions and methods of treating a condition e.g., Friedreich's ataxia
  • An appropriate subject may be a non-human mammal, e.g. mouse, rat, guinea pig, rabbit, cat, dog, goat, cow, or horse.
  • a subject is a human.
  • Oligonucleotides and inhibitors have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Oligonucleotides and inhibitors can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.
  • Oligonucleotides for Modulating Expression of FXN e.g., Friedreich's ataxia
  • oligonucleotides are provided for modulating expression of FXN in a cell.
  • expression of FXN is upregulated or increased.
  • oligonucleotides are provided that reduce the expression level of a negative epigenetic regulator of FXN, thereby upregulating the expression of FXN.
  • the oligonucleotide is specific for an mRNA of a negative epigenetic regulator of FXN.
  • the oligonucleotide may be single stranded or double stranded. Single stranded oligonucleotides may include secondary structures, e.g., a loop or helix structure. In some embodiments, the oligonucleotide comprises at least one modified nucleotide or modified internucleoside linkage as described herein.
  • the oligonucleotide may have a sequence that does not contain guanosine nucleotide stretches (e.g., 3 or more, 4 or more, 5 or more, 6 or more consecutive guanosine
  • oligonucleotides having guanosine nucleotide stretches have increased non-specific binding and/or off-target effects, compared with oligonucleotides that do not have guanosine nucleotide stretches.
  • the oligonucleotide may have a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length, that map to a genomic position encompassing or in proximity to an off-target gene.
  • a threshold level of sequence identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity.
  • the oligonucleotide may have a sequence that is has greater than 30% G-C content, greater than 40% G-C content, greater than 50% G-C content, greater than 60% G-C content, 5 greater than 70% G-C content, or greater than 80% G-C content.
  • the oligonucleotide may have a sequence that has up to 100% G-C content, up to 95% G-C content, up to 90% G-C content, or up to 80% G-C content.
  • the oligonucleotide is 8 to 10 nucleotides in length
  • all but 1, 2, 3, 4, or 5 of the nucleotides of the complementary sequence of the mRNA of a negative epigenetic regulator of FXN are cytosine or guanosine o nucleotides.
  • the sequence of the mRNA to which the oligonucleotide is complementary comprises no more than 3 nucleotides selected from adenine and uracil.
  • the oligonucleotide may be complementary to a chromosome of a different species (e.g., a mouse, rat, rabbit, goat, monkey, etc.) at a position that encompasses or that is in proximity to that species' homolog of the negative epigenetic regulator of FXN.
  • The5 oligonucleotide may be complementary to a human genomic region encompassing or in
  • the oligonucleotide may be complementary to a sequence of a human mRNA of a negative epigenetic regulator of FXN (for example, a o human mRNA referenced in Table 1 by its NCBI accession number), and also be
  • Oligonucleotides having these characteristics may be tested in vivo or in vitro for efficacy in multiple species (e.g., human and mouse). This approach also 5 facilitates development of clinical candidates for treating human disease by selecting a
  • the region of complementarity of the oligonucleotide is complementary with at least 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 bases, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,0 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive nucleotides of an mRNA of a negative epigenetic regulator of FXN.
  • the region of complementarity is complementary with at least 8 consecutive nucleotides of an mRNA of a negative epigenetic regulator of FXN.
  • the sequence of the oligonucleotide is based on an RNA sequence that binds to an mRNA of a negative epigenetic regulator of FXN, or a portion thereof, said portion having a length of from 5 to 40 contiguous base pairs, or about 8 to 40 bases, or about 5 to 15, or about 5 to 30, or about 5 to 40 bases, or about 5 to 50 bases.
  • Complementary refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an
  • oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of an mRNA of a negative epigenetic regulator of FXN, then the oligonucleotide and the mRNA of a negative epigenetic regulator of FXN are considered to be complementary to each other at that position.
  • the oligonucleotide and the mRNA of a negative epigenetic regulator of FXN are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other through their bases.
  • complementary is a term which is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the mRNA of a negative epigenetic regulator of FXN. For example, if a base at one position of an oligonucleotide is capable of hydrogen bonding with a base at the corresponding position of an mRNA of a negative epigenetic regulator of FXN, then the bases are considered to be complementary to each other at that position. 100%
  • the oligonucleotide may be at least 80% complementary to (optionally one of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to) the consecutive nucleotides of an mRNA of a negative epigenetic regulator of FXN.
  • the oligonucleotide may contain 1, 2 or 3 base mismatches compared to the portion of the consecutive nucleotides of an mRNA of a negative epigenetic regulator of
  • the oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.
  • a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable or specific for a target molecule.
  • a complementary nucleic acid sequence for purposes of the present disclosure is specifically hybridizable or specific for the target molecule when binding of the sequence to the target molecule (e.g., mRNA) interferes with the normal function of the target (e.g., mRNA) to cause a loss of activity (e.g., inhibiting translation with consequent up-regulation of FXN gene expression) or expression (e.g., degrading the mRNA with consequent up-regulation of FXN gene expression) and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under conditions in which avoidance of non-specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under
  • the oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length. In a preferred embodiment, the oligonucleotide is 8 to 30 nucleotides in length.
  • Base pairings may include both canonical Watson-Crick base pairing and non- Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). It is understood that for complementary base pairings, adenosine-type bases (A) are
  • Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.
  • any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be replaced with any other nucleotide suitable for base pairing (e.g., via a Watson-Crick base pair) with an adenosine nucleotide.
  • any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a different pyrimidine nucleotide or vice versa.
  • any one or more thymidine (T) nucleotides (or modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing may be suitably replaced with a uridine (U) nucleotide (or a modified nucleotide thereof) or vice versa.
  • GC content of the oligonucleotide is preferably between about 30-60 %. Contiguous runs of three or more Gs or Cs may not be preferable in some embodiments. Accordingly, in some embodiments, the oligonucleotide does not comprise a stretch of three or more guanosine nucleotides.
  • oligonucleotides disclosed herein may increase expression of FXN mRNA by at least about 50% (i.e. 150% of normal or 1.5 fold), or by about 2 fold to about 5 fold. In some embodiments, expression may be increased by at least about 15 fold, 20 fold, 30 fold, 40 fold, 50 fold or 100 fold, or any range between any of o the foregoing numbers.
  • the oligonucleotide may be designed to cause degradation of an mRNA (e.g., the oligonucleotide may be a gapmer, an siRNA, a ribozyme or an aptamer that causes degradation). In some embodiments, the oligonucleotide may be designed to block
  • an oligonucleotide may be a mixmer, an siRNA or an aptamer that blocks translation.
  • an oligonucleotide may be designed to caused degradation and block translation of an mRNA.
  • the oligonucleotides described herein may be modified, e.g., comprise a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide and/or combinations thereof.
  • the oligonucleotides may exhibit one or more of the following properties: do not mediate alternative splicing; are not immune stimulatory; are nuclease resistant; have improved cell uptake compared to unmodified oligonucleotides; are not toxic 5 to cells or mammals; or have improved endosomal exit.
  • Oligonucleotides that are designed to interact with RNA to modulate gene expression are a distinct subset of base sequences from those that are designed to bind a DNA target ⁇ e.g., are complementary to the underlying genomic DNA sequence from which the RNA is transcribed).
  • oligonucleotides disclosed herein may be linked to one or more other oligonucleotides disclosed herein by a linker, e.g., a cleavable linker.
  • Oligonucleotides of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification.
  • nucleic acid sequences of the invention include a phosphorothioate at least the first, second, or third internucleoside linkage at the 5' or 3' end of the nucleotide sequence.
  • the nucleic acid sequence can include a 2'-modified nucleotide, e.g., a 2'-deoxy, 2'- deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0--N-methylacetamido (2'-0--NMA).
  • the nucleic acid sequence can include at least one 2'-0-methyl-modified o nucleotide, and in some embodiments, all of the nucleotides include a 2'-0-methyl
  • nucleic acids are "locked,” i.e., comprise nucleic acid analogues in which the ribose ring is “locked” by a methylene bridge connecting the 2'- O atom and the 4'-C atom.
  • any of the modified chemistries or formats of oligonucleotides described herein can5 be combined with each other, and that one, two, three, four, five, or more different types of modifications can be included within the same molecule.
  • an oligonucleotide may comprise one or more modified nucleotides (also referred to herein as nucleotide analogs).
  • the oligonucleotide may comprise at least one ribonucleotide, at least one deoxyribonucleotide, o and/or at least one bridged nucleotide.
  • the oligonucleotide may
  • a bridged nucleotide such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide.
  • LNA locked nucleic acid
  • cEt constrained ethyl
  • ENA ethylene bridged nucleic acid
  • the oligonucleotide comprises a nucleotide analog disclosed in one of the following United States5 Patent or Patent Application Publications: US 7,399,845, US 7,741,457, US 8,022,193, US 7,569,686, US 7,335,765, US 7,314,923, US 7,335,765, and US 7,816,333, US 20110009471, the entire contents of each of which are incorporated herein by reference for all purposes.
  • the oligonucleotide may have one or more 2' O-methyl nucleotides.
  • the oligonucleotide may consist entirely of 2' O-methyl nucleotides.
  • the oligonucleotide has one or more nucleotide analogues.
  • the oligonucleotide may have at least one nucleotide analogue that results in an increase in T m of the oligonucleotide in a range of 1°C, 2 °C, 3°C, 4 °C, or 5°C compared with an oligonucleotide that does not have the at least one nucleotide analogue.
  • the oligonucleotide may have a plurality of nucleotide analogues that results in a total increase in T m of the oligonucleotide in a range of 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C or more compared with an oligonucleotide that does not have the nucleotide analogue.
  • the oligonucleotide may be of up to 50 nucleotides in length in which 2 to 10, 2 to 15 5 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or more nucleotides of the oligonucleotide are nucleotide analogues.
  • the oligonucleotide may be of 8 to 30 nucleotides in length in which 2 to 10, 2 to 15 5 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are nucleotide analogues.
  • the oligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are nucleotide analogues.
  • the oligonucleotides may have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified.
  • the oligonucleotide may consist entirely of bridged nucleotides (e.g. , LNA nucleotides, cEt nucleotides, ENA nucleotides).
  • the oligonucleotide may comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides.
  • the oligonucleotide may comprise alternating deoxyribonucleotides and 2'-0-methyl nucleotides.
  • the oligonucleotide may comprise alternating deoxyribonucleotides and ENA nucleotide analogues.
  • the oligonucleotide may comprise alternating deoxyribonucleotides and LNA nucleotides.
  • the oligonucleotide may comprise alternating LNA nucleotides and 2'-0- methyl nucleotides.
  • the oligonucleotide may have a 5' nucleotide that is a bridged nucleotide (e.g. , a LNA nucleotide, cEt nucleotide, ENA nucleotide).
  • the oligonucleotide may have a 5' nucleotide that is a deoxyribonucleotide.
  • the oligonucleotide may comprise deoxyribonucleotides flanked by at least one bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide) on each of the 5' and 3' ends of the deoxyribonucleotides.
  • the oligonucleotide may comprise
  • deoxyribonucleotides flanked by 1, 2, 3, 4, 5, 6, 7, 8 or more bridged nucleotides (e.g. , LNA nucleotides, cEt nucleotides, ENA nucleotides) on each of the 5' and 3' ends of the deoxyribonucleotides.
  • the 3' position of the oligonucleotide may have a 3' hydroxyl group.
  • the 3' position of the oligonucleotide may have a 3' thiophosphate.
  • the oligonucleotide may be conjugated with a label. For example, the
  • oligonucleotide may be conjugated with a biotin moiety, cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
  • a biotin moiety cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
  • the oligonucleotide comprises one or more modifications comprising: a modified sugar moiety, and/or a modified internucleoside linkage, and/or a modified nucleotide and/or combinations thereof. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be incorporated in a single oligonucleotide or even at within a single o nucleoside within an oligonucleotide.
  • the oligonucleotides are chimeric oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased5 uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • beneficial properties such as, for example, increased nuclease resistance, increased5 uptake into cells, increased binding affinity for the target
  • Chimeric oligonucleotides of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers.
  • the oligonucleotide comprises at least one nucleotide modified 5 at the 2' position of the sugar, preferably a 2'-0-alkyl, 2'-0-alkyl-0-alkyl or 2'-fluoro- modified nucleotide.
  • RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA.
  • modified oligonucleotides include those comprising modified backbones, for example, modified internucleoside linkages such as
  • oligonucleotides may have phosphorothioate backbones; heteroatom backbones, such as methylene(methylimino) or MMI backbones; amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones (see
  • PNA peptide nucleic acid
  • Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see US patent nos.
  • Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001 ; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216- 220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.
  • the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001 ; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).
  • PMO phosphorodiamidate morpholino oligomer
  • Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see US patent nos.
  • Modified oligonucleotides are also known that include oligonucleotides that are based on or constructed from arabinonucleotide or modified arabinonucleotide residues.
  • Arabinonucleosides are stereoisomers of ribonucleosides, differing only in the configuration at the 2'-position of the sugar ring.
  • a 2'-arabino modification is 2'-F arabino.
  • the modified oligonucleotide is 2' -fluoro-D-arabinonucleic acid (FANA) (as described in, for example, Lon et al., Biochem., 41 :3457-3467, 2002 and Min et al., Bioorg. Med. Chem. Lett., 12:2651-2654, 2002; the disclosures of which are incorporated herein by reference in their entireties). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • WO 99/67378 discloses arabinonucleic acids (ANA) oligomers and their analogues for improved sequence specific inhibition of gene expression via association to complementary messenger RNA.
  • EDAs ethylene-bridged nucleic acids
  • ENAs include, but are not limited to, 2'-0,4'-C-ethylene -bridged nucleic acids.
  • LNAs examples include compounds of the following general formula.
  • -CH CH-, where R is selected from hydrogen and Ci-4-alkyl; Z and Z* are independently selected among an intemucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety; and the asymmetric groups may be found in either orientation.
  • the LNA used in the oligonucleotides described herein comprises at least one LNA unit according any of the formulas
  • the Locked Nucleic Acid (LNA) used in the oligonucleotides described herein comprises at least one Locked Nucleic Acid (LNA) unit according any of the formulas shown in Scheme 2 of PCT/DK2006/000512.
  • the LNA used in the oligomer of the invention comprises internucleoside linkages selected from -0-P(O) 2 -O-, -0-P(0,S)-0-, -0-P(S) 2 -O-, -S-P(0) 2 -0-, -S-P(0,S)-0-, -S-P(S) 2 -0-, -0-P(0) 2 -S-, -0-P(0,S)-S-, -S-P(0) 2 -S-, -0-PO(R H )-0-, o- PO(OCH 3 )-0-, -0-PO(NR H )-0-, -0-PO(OCH 2 CH 2 S-R)-O-, -0-PO(BH 3 )-0-, -0-PO(NHR H )- 0-, -0-P(0) 2 -NR H -, -NR H -P(0) 2 -0-, -NR H -CO-0-0-
  • LNA units are shown below:
  • thio-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from S or -CH 2 -S-.
  • Thio-LNA can be in both beta-D and alpha-L-configuration.
  • amino-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from -N(H)-, N(R)-, CH 2 -N(H)-, and -CH 2 -N(R)- where R is selected from hydrogen and Ci-4-alkyl.
  • Amino-LNA can be in both beta-D and alpha- L-configuration.
  • Oxy-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above represents -O- or -CH 2 -O-. Oxy-LNA can be in both beta-D and alpha-L-configuration.
  • ena-LNA comprises a locked nucleotide in which Y in the general formula above is -CH 2 -0- (where the oxygen atom of -CH 2 -0- is attached to the 2'-position relative to the base B).
  • LNAs are described in additional detail herein.
  • One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, OCH 3 OCH 3 , OCH 3 0(CH 2 )n CH 3 , 0(CH 2 )n NH 2 or 0(CH 2 )n CH 3 where n is from 1 to about 10; CI to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF 3 ; OCF 3 ; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH 3 ; S0 2 CH 3 ; ON0 2 ; N0 2 ; N 3 ; NH2; heterocycloalkyl; heterocyclo alkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a
  • a preferred modification includes 2'-methoxyethoxy [2'-0-CH 2 CH 2 OCH , also known as 2'-0-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486).
  • Other preferred modifications include 2'- methoxy (2'-0-CH 3 ), 2'-propoxy (2'-OCH 2 CH 2 CH 3 ) and 2'-fluoro (2'-F). Similar
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
  • Oligonucleotides can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobase often referred to in the art simply as “base”
  • “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g.
  • hypoxanthine, 6-methyladenine, 5- Me pyrimidines particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2- (aminoalklyamino)adenine or other hetero substituted alkyladenines, 2-thiouracil, 2- 5 thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 5-propynyluracil, 8-azaguanine, 7- deazaguanine, N6 (6-aminohexyl)adenine, 6-aminopurine, 2-aminopurine,
  • both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for o hybridization with an appropriate nucleic acid target compound.
  • PNA peptide nucleic acid
  • Oligonucleotides can also include one or more nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • base any nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • “unmodified” or “natural” nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases comprise other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- 5 thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 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-sub
  • nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in "The Concise Encyclopedia of Polymer Science And Engineering", pages 858-859, Kroschwitz, ed. John Wiley & Sons, 1990;, those disclosed by Englisch et al., Angewandle Chemie, International Edition, 1991, 30, page 613, and those disclosed by
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5- o methylcytosine substitutions have been shown to increase nucleic acid duplex stability by
  • the oligonucleotides are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the 0 oligonucleotide.
  • one or more oligonucleotides, of the same or different types can be conjugated to each other; or oligonucleotides can be conjugated to targeting moieties with enhanced specificity for a cell type or tissue type.
  • moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci.
  • a thioether e.g., hexyl-S- tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci.,
  • a phospholipid e.g. , di-hexadecyl-rac- glycerol or triethylammonium 1,2-di-O-hexadecyl- rac-glycero-3-H-phosphonate
  • conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence- specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference.
  • Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.
  • di-hexadecyl-rac- glycerol or triethylammonium 1,2- di-O-hexadecyl-rac-glycero-3-H-phosphonate a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See, e.g. , U.S. Pat. Nos.
  • oligonucleotide modification includes modification of the 5' or 3' end of the oligonucleotide.
  • the 3' end of the oligonucleotide comprises a hydroxyl group or a thiophosphate.
  • additional molecules e.g. a biotin moiety or a fluorophor
  • the oligonucleotide comprises a biotin moiety conjugated to the 5' nucleotide.
  • the oligonucleotide comprises locked nucleic acids (LNA), ENA modified nucleotides, 2'-0-methyl nucleotides, or 2'-fluoro-deoxyribonucleotides. In some embodiments, the oligonucleotide comprises alternating deoxyribonucleotides and 2'- fluoro-deoxyribonucleotides. In some embodiments, the oligonucleotide comprises alternating deoxyribonucleotides and 2'-0-methyl nucleotides. In some embodiments, the oligonucleotide comprises alternating deoxyribonucleotides and ENA modified nucleotides.
  • LNA locked nucleic acids
  • ENA modified nucleotides 2'-0-methyl nucleotides
  • 2'-fluoro-deoxyribonucleotides In some embodiments, the oligonucleotide comprises alternating deoxyribonucleotides and 2'-fluor
  • the oligonucleotide comprises alternating deoxyribonucleotides and locked nucleic acid nucleotides. In some embodiments, the oligonucleotide comprises alternating locked nucleic acid nucleotides and 2' -O-methyl nucleotides. In some embodiments, the 5' nucleotide of the oligonucleotide is a
  • the 5' nucleotide of the oligonucleotide is a locked nucleic acid nucleotide.
  • the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one locked nucleic acid nucleotide on each 5 of the 5' and 3' ends of the deoxyribonucleotides.
  • the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group or a 3' thiophosphate.
  • the oligonucleotide comprises phosphorothioate
  • the oligonucleotide comprises
  • the oligonucleotide comprises phosphorothioate internucleoside linkages
  • oligonucleotide can have any combination of modifications as described herein.
  • an oligonucleotide described herein may be a mixmer or
  • the term 'mixmer' refers to oligonucleotides which comprise both naturally and non-naturally occurring nucleotides or comprise two different types of non-naturally occurring nucleotides.
  • Mixmers are generally known in the art to have a higher binding affinity than unmodified oligonucleotides and may be used to specifically bind a target molecule, e.g., to block a binding site on the target molecule.
  • o mixmers do not recruit an RNAse to the target molecule and thus do not promote cleavage of the target molecule.
  • the mixmer comprises or consists of a repeating pattern of nucleotide analogues and naturally occurring nucleotides, or one type of nucleotide analogue and a second type of nucleotide analogue.
  • the mixmer 5 need not comprise a repeating pattern and may instead comprise any arrangement of
  • nucleotide analogues and naturally occurring nucleotides or any arrangement of one type of nucleotide analogue and a second type of nucleotide analogue.
  • the repeating pattern may, for instance be every second or every third nucleotide is a nucleotide analogue, such as LNA, and the remaining nucleotides are naturally occurring nucleotides, such as DNA, or are a 2' 0 substituted nucleotide analogue such as 2'MOE or 2' fluoro analogues, or any other
  • nucleotide analogues described herein may be combined with nucleotide analogues at fixed positions— e.g. at the 5' or 3' termini.
  • the mixmer does not comprise a region of more than 5, more than 4, more than 3, or more than 2 consecutive naturally occurring nucleotides, such as DNA nucleotides.
  • the mixmer comprises at least a region consisting of at least two consecutive nucleotide analogues, such as at least two consecutive LNAs.
  • the mixmer comprises at least a region consisting of at least three consecutive nucleotide analogue units, such as at least three consecutive LNAs.
  • the mixmer does not comprise a region of more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2 consecutive nucleotide analogues, such as LNAs. It is to be understood that the LNA units may be replaced with other nucleotide analogues, such as those referred to herein.
  • the mixmer comprises at least one nucleotide analogue in one or more of six consecutive nucleotides.
  • the substitution pattern for the nucleotides may be selected from the group consisting of Xxxxxx, xXxxxx, xxXxxx, xxxXxx, xxxxXx and xxxxxX, wherein "X” denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA.
  • the mixmer comprises at least two nucleotide analogues in one or more of six consecutive nucleotides.
  • the substitution pattern for the nucleotides may be selected from the group consisting of XXxxxx, XxXxxx, XxxXxx, xXXxxx, xXxXxx, xXxxxX, xXxxxX, xxXXxx, xxXxXx, xxXxxX, xxxXXx, xxxXxXx, xxxXxX and xxxxXX, wherein "X” denotes a nucleotide analogue, such as an LNA, and "x” denotes a naturally occuring nucleotide, such as DNA or RNA.
  • the substitution pattern for the nucleotides may be selected from the group consisting of XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXxxxX, xxXxXx, xxXxxX and xxxXxX.
  • the substitution pattern is selected from the group consisting of xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX.
  • the substitution pattern is selected from the group consisting of xXxXxx, xXxxXx and xxXxXx.
  • the substitution pattern for the nucleotides is xXxXxx.
  • the mixmer comprises at least three nucleotide analogues in one or more of six consecutive nucleotides.
  • the substitution pattern for the nucleotides may be selected from the group consisting of XXXxxx, xXXXxx, xxXXXx, xxxXXX, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXX, XxxxXX, XxxxXX, xXxXXx, xXxxXXX, xxXXX, xXxXxX and XxXxXx, wherein "X” denotes a nucleotide analogue, such as an LNA, and "x” denotes a naturally occuring nucleotide, such as DNA or RNA.
  • the substitution pattern for the nucleotides is selected from the group consisting of XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxxxXX, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx.
  • the substitution pattern for the nucleotides is selected from the group consisting of xXXxXx, xXXxxX, xxXXxX, xXxXXx, xXxxXX, xxXxXX and xXxXxX. n some embodiments, the substitution pattern for the nucleotides is xXxXxX or XxXxXx. In some embodiments, the substitution pattern for the nucleotides is xXxXxX.
  • the mixmer comprises at least four nucleotide analogues in one or more of six consecutive nucleotides.
  • the substitution pattern for the nucleotides may be selected from the group consisting of xXXXX, xXxXXX, xXXxXX, xXXXxX, xXXXx, XxxXXX, XxXxX, XxXXxX, XxXXx, XXxxXX, XXxXxX, XXxXx, XXxxX, XXXxXx and XXXXxx, wherein "X” denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA.
  • the mixmer comprises at least five nucleotide analogues in one or more of six consecutive nucleotides.
  • the substitution pattern for the nucleotides may be selected from the group consisting of xXXXXX, XxXXXX, XXxXXX, XXXxXX,
  • XXXXxX and XXXXx wherein "X” denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA.
  • the oligonucleotide may comprise a nucleotide sequence having one or more of the following modification patterns.
  • the mixmer contains a modified nucleotide, e.g., an LNA, at the 5' end. In some embodiments, the mixmer contains a modified nucleotide, e.g., an LNA, at the first two positions, counting from the 5' end.
  • the mixmer is incapable of recruiting RNAseH.
  • Oligonucleotides that are incapable of recruiting RNAseH are well known in the literature, in example see WO2007/112754, WO2007/112753, or PCT/DK2008/000344.
  • Mixmers may be designed to comprise a mixture of affinity enhancing nucleotide analogues, such as in non- limiting example LNA nucleotides and 2'-0-methyl nucleotides.
  • the mixmer comprises modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
  • a mixmer may be produced using any method known in the art or described herein.
  • Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of mixmers include U.S. patent publication Nos. US20060128646,
  • the oligonucleotide is a gapmer.
  • a gapmer oligonucleotide generally has the formula 5'-X-Y-Z-3', with X and Z as flanking regions around a gap region
  • the Y region is a contiguous stretch of nucleotides, e.g., a region of at least 6 DNA nucleotides, which are capable of recruiting an RNAse, such as RNAseH. Without wishing to be bound by theory, it is thought that the gapmer binds to the target nucleic acid, at which point an RNAse is recruited and can then cleave the target nucleic acid.
  • the Y region is flanked both 5' and 3' by regions X and Z comprising high-affinity modified nucleotides, e.g., 1 - 6 modified nucleotides.
  • Exemplary modified oligonucleotides include, but are not limited to, 2' MOE or 2'OMe or Locked Nucleic Acid bases (LNA).
  • the flanks X and Z may be have a of length 1 - 20 nucleotides, preferably 1-8 nucleotides and even more preferred 1 - 5 nucleotides.
  • the flanks X and Z may be of similar length or of dissimilar lengths.
  • the gap-segment Y may be a nucleotide sequence of length 5 - 20 nucleotides, preferably 6-12 nucleotides and even more preferred 6 - 10 nucleotides.
  • the gap region of the gapmer oligonucleotides of the invention may contain modified nucleotides known to be acceptable for efficient RNase H action in addition to DNA nucleotides, such as C4'-substituted nucleotides, acyclic nucleotides, and arabino- configured nucleotides.
  • the gap region comprises one or more unmodified internucleosides.
  • one or both flanking regions each independently comprise one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
  • the gap region and two flanking regions each independently comprise modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
  • modified internucleoside linkages e.g., phosphorothioate internucleoside linkages or other linkages
  • a gapmer may be produced using any method known in the art or described herein.
  • Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; 5,700,922; 5,898,031; 7,432,250; and 7,683,036; U.S. patent publication Nos.
  • oligonucleotides provided herein may be in the form of small interfering RNAs (siRNA), also known as short interfering RNA or silencing RNA.
  • siRNA small interfering RNAs
  • mRNAs target nucleic acids
  • RNAi RNA interference pathway
  • Specificity of siRNA molecules may be determined by the binding of the antisense strand of the molecule to its target RNA.
  • Effective siRNA molecules are generally less than 30 to 35 base pairs in length to prevent the triggering of non-specific RNA interference pathways in the cell via the interferon response, although longer siRNA can also be effective.
  • siRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence can be designed and prepared using any method known in the art (see, e.g., PCT Publication Nos. WO08124927A1 and WO 2004/016735; and U.S. Patent
  • a number of commercial packages and services are available that are suitable for use for the preparation of siRNA molecules. These include the in vitro transcription kits available from Ambion (Austin, TX) and New England Biolabs (Beverly, MA) as described above; viral siRNA construction kits commercially available from Invitrogen (Carlsbad, CA) and Ambion (Austin, TX), and custom siRNA construction services provided by Ambion (Austin, TX), Qiagen (Valencia, CA), Dharmacon (Lafayette, CO) and Sequitur, Inc (Natick, MA).
  • a target sequence can be selected (and a siRNA sequence designed) using computer software available commercially (e.g.
  • an siRNA may be designed or obtained using the RNAi atlas (available at the RNAiAtlas website), the siRNA database (available at the Swedish Bioinformatics Website), or using DesiRM (available at the Institute of Microbial
  • the siRNA molecule can be double stranded (i.e. a dsRNA molecule comprising an antisense strand and a complementary sense strand) or single- stranded (i.e. a ssRNA molecule comprising just an antisense strand).
  • the siRNA molecules can comprise a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self- complementary sense and antisense strands.
  • Double-stranded siRNA may comprise RNA strands that are the same length or different lengths.
  • Double- stranded siRNA molecules can also be assembled from a single oligonucleotide in a stem-loop structure, wherein self-complementary sense and antisense regions of the siRNA molecule are linked by means of a nucleic acid based or non-nucleic acid-based linker(s), as well as circular single- stranded RNA having two or more loop structures and a stem comprising self-complementary sense and antisense strands, wherein the circular RNA can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.
  • Small hairpin RNA (shRNA) molecules thus are also contemplated herein. These molecules comprise a specific antisense sequence in addition to the reverse complement (sense) sequence, typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides a single- stranded RNA molecule and its reverse complement, such that they may anneal to form a dsRNA molecule (optionally with additional processing steps that may result in addition or removal of one, two, three or more 5 nucleotides from the 3' end and/or the 5' end of either or both strands).
  • shRNA Small hairpin RNA
  • a spacer can be of a sufficient length to permit the antisense and sense sequences to anneal and form a double- stranded structure (or stem) prior to cleavage of the spacer (and, optionally, subsequent processing steps that may result in addition or removal of one, two, three, four, or more nucleotides from the 3' end and/or the 5' end of either or both strands).
  • a spacer sequence is o may be an unrelated nucleotide sequence that is situated between two complementary
  • nucleotide sequence regions which, when annealed into a double- stranded nucleic acid, comprise a shRNA.
  • the overall length of the siRNA molecules can vary from about 14 to about 200 nucleotides depending on the type of siRNA molecule being designed. Generally between5 about 14 and about 50 of these nucleotides are complementary to the RNA target sequence, i.e. constitute the specific antisense sequence of the siRNA molecule. For example, when the siRNA is a double- or single- stranded siRNA, the length can vary from about 14 to about 50 nucleotides, whereas when the siRNA is a shRNA or circular molecule, the length can vary from about 40 nucleotides to about 200 nucleotides.
  • An siRNA molecule may comprise a 3' overhang at one end of the molecule, The other end may be blunt-ended or have also an overhang (5' or 3') ⁇
  • the siRNA molecule of the present invention comprises 3' overhangs of about 1 to about 3 nucleotides on both ends of the molecule.
  • an oligonucleotide may be a microRNA (miRNA).
  • MicroRNAs are small non-coding RNAs, belonging to a class of regulatory molecules that control gene expression by binding to complementary sites on a target RNA transcript.
  • miRNAs are generated from large RNA precursors (termed pri-miRNAs) that are processed in the nucleus into approximately 70 nucleotide pre- 0 miRNAs, which fold into imperfect stem-loop structures.
  • pri-miRNAs large RNA precursors
  • These pre-miRNAs typically undergo an additional processing step within the cytoplasm where mature miRNAs of 18-25 nucleotides in length are excised from one side of the pre-miRNA hairpin by an RNase III enzyme, Dicer.
  • miRNAs including pri-miRNA, pre-miRNA, mature miRNA or fragments of variants thereof that retain the biological activity of mature miRNA.
  • the size range of the miRNA can be from 21 nucleotides to 170 nucleotides, although miRNAs of up to 2000 nucleotides can be utilized. In one embodiment the size range of the miRNA is from 70 to 170 nucleotides in length. In another embodiment, mature miRNAs of from 21 to 25 nucleotides in length can be used.
  • a miRNA is expressed from a vector.
  • the vector may include a sequence encoding a mature miRNA.
  • the vector may include a sequence encoding a pre-miRNA such that the pre-miRNA is expressed and processed in a cell into a mature miRNA.
  • the vector may include a sequence encoding a pri-miRNA.
  • the primary transcript is first processed to produce the stem-loop precursor miRNA molecule.
  • the stem-loop precursor is 5 then processed to produce the mature microRNA.
  • oligonucleotides provided herein may be in the form of aptamers.
  • aptamer is any nucleic acid that binds specifically to a target, such as a small molecule, protein, nucleic acid, cell, tissue or organism.
  • the aptamer is a DNA aptamer or an RNA aptamer.
  • a nucleic acid aptamer is a o single- stranded DNA or RNA (ssDNA or ssRNA). It is to be understood that a single- stranded nucleic acid aptamer may form helices and/or loop structures.
  • the nucleic acid that forms the nucleic acid aptamer may comprise naturally occurring nucleotides, modified nucleotides, naturally occurring nucleotides with hydrocarbon linkers (e.g., an alkylene) or a polyether linker (e.g., a PEG linker) inserted between one or more nucleotides, modified 5 nucleotides with hydrocarbon or PEG linkers inserted between one or more nucleotides, or a combination of thereof.
  • hydrocarbon linkers e.g., an alkylene
  • a polyether linker e.g., a PEG linker
  • nucleic acid aptamers may be accomplished by any suitable method known in the art, including an optimized protocol for in vitro selection, known as SELEX (Systemic Evolution of Ligands by Exponential enrichment). Many factors are important for 0 successful aptamer selection. For example, the target molecule should be stable and easily reproduced for each round of SELEX, because the SELEX process involves multiple rounds of binding, selection, and amplification to enrich the nucleic acid molecules. In addition, the nucleic acids that exhibit specific binding to the target molecule have to be present in the initial library. Thus, it is advantageous to produce a highly diverse nucleic acid pool. Because the starting library is not guaranteed to contain aptamers to the target molecule, the SELEX process for a single target may need to be repeated with different starting libraries.
  • SELEX Systemic Evolution of Ligands by Exponential enrichment
  • Exemplary publications and patents describing aptamers and method of producing aptamers include, e.g., Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Pat. Nos. 5,270,163; 5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249; 5,789,157; 5,843,653; 5,864,026; 5,989,823; 6,569,630; 8,318,438 and PCT application WO 99/31275, each incorporated herein by reference.
  • oligonucleotides provided herein may be in the form of a ribozyme.
  • a ribozyme ribonucleic acid enzyme
  • Ribozymes are molecules with catalytic activities including the ability to cleave at specific phosphodiester linkages in RNA molecules to which they have hybridized, such as mRNAs, RNA-containing substrates, IncRNAs, and ribozymes, themselves.
  • Ribozymes may assume one of several physical structures, one of which is called a "hammerhead.”
  • a hammerhead ribozyme is composed of a catalytic core containing nine conserved bases, a double- stranded stem and loop structure (stem-loop II), and two regions complementary to the target RNA flanking regions the catalytic core. The flanking regions enable the ribozyme to bind to the target RNA specifically by forming double- stranded stems I and III.
  • Cleavage occurs in cis (i.e., cleavage of the same RNA molecule that contains the hammerhead motif) or in trans (cleavage of an RNA substrate other than that containing the ribozyme) next to a specific ribonucleotide triplet by a transesterification reaction from a 3', 5'-phosphate diester to a 2', 3'-cyclic phosphate diester.
  • this catalytic activity requires the presence of specific, highly conserved sequences in the catalytic region of the ribozyme.
  • Ribozyme oligonucleotides can be prepared using well known methods (see, e.g., PCT Publications W09118624; W09413688; WO9201806; and WO 92/07065; and U.S. Patents 5436143 and 5650502) or can be purchased from commercial sources (e.g., US Biochemicals) and, if desired, can incorporate nucleotide analogs to increase the resistance of o the oligonucleotide to degradation by nucleases in a cell.
  • the ribozyme may be synthesized in any known manner, e.g., by use of a commercially available synthesizer produced, e.g., by Applied Biosystems, Inc. or Milligen.
  • the ribozyme may also be produced in recombinant vectors by conventional means. See, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (Current edition).
  • the ribozyme RNA sequences maybe synth
  • RNA polymerases such as T7 or SP6.
  • Vectors include, but are not limited to, plasmids, viral vectors, other vehicles derived from viral or bacterial or other sources that have been manipulated by the insertion or incorporation of the nucleic acid sequences for expressing an RNA transcript (e.g., shRNA, miRNA, mRNA).
  • RNA transcript e.g., shRNA, miRNA, mRNA
  • expression vectors are provided that are engineered to express 5 a positive epigenetic regulator (e.g., a product of a gene as provided in Table 7).
  • expression of the positive epigenetic regulator causes upregulation of FXN.
  • an expression vector may be engineered by incorporating a cDNA comprising exons of a gene of interest into a plasmid that is suitably configured with expression elements (e.g., a promoter) for expressing the gene of interest.
  • expression elements e.g., a promoter
  • cDNA may be obtained or synthesized using a commercially available kit or any method known in the art, e.g, synthesized from mature (fully spliced) mRNA using the enzyme reverse transcriptase (see, e.g., US Patent Numbers 7470515 and 8420324, and PCT Publication Numbers WO2000052191, WO1997024455).
  • a vector may comprise one or more expression elements.
  • “Expression elements” are any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient expression of an RNA
  • the expression element may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter or a tissue specific promoter, examples of which are well known to one of ordinary skill in the art.
  • Constitutive mammalian promoters include polymerase promoters as well as the promoters for the following non-limiting genes: hypoxanthine phosphoribosyl transferase (HPTR), adenosine o deaminase, pyruvate kinase, and beta-actin.
  • HPTR hypoxanthine phosphoribosyl transferase
  • adenosine o deaminase pyruvate kinase
  • beta-actin exemplary viral promoters which function
  • constitutively in eukaryotic cells include promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus.
  • Other constitutive promoters may be5 used.
  • Inducible promoters are expressed in the presence of an inducing agent and include metal-inducible promoters and steroid-regulated promoters, for example. Other inducible promoters may be used.
  • Expression vectors may also comprise an origin of replication, a suitable promoter polyadenylation site, transcriptional termination sequences, and 5' flanking nontranscribed o sequences.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
  • Viral vectors are generally based on non-cytopathic eukaryotic viruses in which non-essential genes have 5 been replaced with the nucleic acid sequence of interest. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lines with plasmid, production of recombinant retroviruses by the packaging cell lie, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) may be used.
  • Viral and 0 retroviral vectors that may be used include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses, such as: Moloney murine leukemia virus; Murine stem cell virus, Harvey murine sarcoma virus; murine mammary tumor virus; Rous sarcoma virus; adenovirus; adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes viruses; vaccinia viruses; polio viruses; and RNA viruses such as any retrovirus.
  • retroviruses such as: Moloney murine leukemia virus; Murine stem cell virus, Harvey murine sarcoma virus; murine mammary tumor virus; Rous sarcoma virus; adenovirus; adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes viruses; vaccini
  • compositions e.g., oligonucleotides, expression vectors, inhibitors
  • a condition e.g., o Friedrich's ataxia
  • the formulations may
  • the amount of active ingredient ⁇ e.g., an oligonucleotide, expression vector, inhibitor) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of5 administration, e.g., intrathecal, intraneural, intracerebral, intramuscular, etc.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
  • compositions of this invention can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such formulations can o contain sweetening agents, flavoring agents, coloring agents and preserving agents.
  • Formulations can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture.
  • Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release
  • a formulated composition can assume a variety of states.
  • the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous ⁇ e.g., less than 80, 50, 30, 20, or 10% water).
  • the composition is in an aqueous phase, e.g., in a solution that includes water. The aqueous phase or the crystalline
  • compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle ⁇ e.g., a microparticle as can be appropriate for a crystalline composition).
  • a delivery vehicle e.g., a liposome (particularly for the aqueous phase) or a particle ⁇ e.g., a microparticle as can be appropriate for a crystalline composition.
  • the composition is formulated in a manner that is compatible with the intended method of administration.
  • the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.
  • An oligonucleotide preparation can be formulated or administered (together or separately) in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes an oligonucleotide, e.g., a protein that complexes with the oligonucleotide.
  • another agent e.g., another therapeutic agent or an agent that stabilizes an oligonucleotide, e.g., a protein that complexes with the oligonucleotide.
  • Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg 2+ ), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.
  • an oligonucleotide preparation includes another oligonucleotide, e.g., a second oligonucleotide that modulates expression of a second gene or a second oligonucleotide that modulates expression of the first gene. Still other preparation can include at least 3, 5, 10, twenty, fifty, or a hundred or more different oligonucleotide species. Such oligonucleotides can mediated gene expression with respect to a similar number of different genes.
  • the oligonucleotide preparation includes at least a second therapeutic agent (e.g., an agent other than an oligonucleotide).
  • Expression vectors expressing different positive epigenetic regulators may be similarly combined with one another. Expression vectors expressing different positive epigenetic regulators may also be combined with one or more oligonucleotides that target negative epigenetic regulators.
  • one or more oligonucleotides as provided herein is combined with the use of one or more inhibitors as described herein.
  • HLMi histone-lysine N-methyltransferase inhibitor
  • the HLMi are contacted with cells of interest, thereby inhibiting histone-lysine N-methyltransferase, decreasing the levels of histone H3 K9 methylation, and increasing FXN expression in the cell, wherein, prior to contact with the inhibitor, the cell has a lower level of FXN expression compared to an appropriate control level of FXN expression.
  • the cell is obtained from or present in a subject having Friedreich's ataxia.
  • the inhibitor is from the epipolythiodioxopiperazine class of fungal metabolites. In certain embodiments, the inhibitor is chaetocin.
  • the inhibitor comprises a quinazoline scaffold. In certain embodiments, the inhibitor comprises a 2,4-diamino-6,7-dimethoxyquinazoline scaffold. In certain embodiments the inhibitor is a compound with the following formula:
  • R is
  • R' is isopropyl, cyclohexyl, or benzyl.
  • R" is .
  • R'" is methyl, ethyl, isopropyl, benzyl, cyclohexyl, or cyclohexylmethyl.
  • the inhibitor is ⁇ 01294, UNC0224, UNC0321, UNC0638, UNC0646, UNC0631, TM2-115, UNC0642, ⁇ -01338, or E72.
  • the inhibitor comprises an indole scaffold. In certain embodiments, the inhibitor is A-366.
  • the inhibitor comprises a benzimidazole scaffold.
  • the benzimidazole scaffold is a 2-substituted benzimidazole.
  • the benzimidazole scaffold is the following:
  • the inhibitor is BRD4770.
  • the inhibitor comprises an adenosine scaffold.
  • the inhibitor comprising an adenosine scaffold is sinefungin or analogues thereof.
  • the alpha-amino acid moiety in the sinefungin analogue has been exchanged to a moiety without an amino group.
  • the inhibitor is 5'-desoxy-5'-butyladenosine.
  • the alpha-amino acid moiety in the sinefungin analogue has been exchanged to a moiety with an amino group.
  • the inhibitor is 5'-desoxy-5'-(2"-cyclohexyl-l"aminoethyl)-adenosine.
  • one or more inhibitors of HML can be used to increase FXN expression.
  • the inhibitor is one of the exemplary inhibitors listed in Table 2 or a pharmaceutically acceptable salt or solvate thereof.
  • the inhibitor includes both the neutral form and a pharmaceutically acceptable salt thereof.
  • Sinefungin analogue 5'-desoxy-5'-(2- fluorobenzyl) adeno sine
  • Sinefungin analogue 5'-desoxy-5'-(4- fluorobenzyl) adeno sine
  • Sinefungin analogue 5'-desoxy-5'-(l- thiazolylmethyl) adeno sine
  • Sinefungin analogue 5'-desoxy-5'-(3- phenylpropyl) adeno sine
  • Sinefungin analogue 5'-desoxy-5'-(2- cyanobenzyl) adeno sine
  • compositions e.g., oligonucleotides, expression vectors, inhibitors
  • routes include:
  • intrathecal intraneural, intracerebral, intramuscular, oral, intravenous, intradermal, topical, rectal, parenteral, anal, intravaginal, intranasal, pulmonary, or ocular.
  • therapeutically effective amount is the amount of active agent (e.g., oligonucleotide, expression vector, inhibitor) present in the composition that is needed to provide the desired level of FXN expression in the subject to be treated to give the anticipated physiological response.
  • physiologically effective amount is that amount delivered to a subject to give the desired palliative or curative effect.
  • pharmaceutically acceptable carrier means that the carrier can be administered to a subject with no significant adverse toxicological effects to the subject.
  • oligonucleotides, expression vectors, and inhibitors of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such oligonucleotides, expression vectors, and inhibitors of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such oligonucleotides, expression vectors, and inhibitors of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such oligonucleotides, expression vectors, and inhibitors of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such
  • compositions typically include one or more species of oligonucleotide, expression vector, or inhibitor and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or
  • the route and site of administration may be chosen to enhance targeting.
  • intramuscular injection into the muscles of interest would be a logical choice.
  • Lung cells might be targeted by administering the composition in aerosol form.
  • the vascular endothelial cells could be targeted by coating a balloon catheter with the composition and mechanically introducing the composition.
  • Targeting of neuronal cells could be accomplished by intrathecal, intraneural, intracerebral administration.
  • Topical administration refers to the delivery to a subject by contacting the formulation directly to a surface of the subject.
  • the most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g. , to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface.
  • the most common topical delivery is to the skin.
  • the term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum.
  • Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition.
  • Topical administration can also be used as a means to selectively deliver compositionsto the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.
  • Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics.
  • the dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin. Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal
  • transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or o local therapy.
  • iontophoresis transfer of ionic solutes through biological
  • phonophoresis or sonophoresis use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea
  • optimization of vehicle characteristics relative to dose position and retention at the site of administration may be useful methods for5 enhancing the transport of topically applied compositions across skin and mucosal sites.
  • oligonucleotides administered through these membranes may have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the oligonucleotides to the hostile gastrointestinal o (GI) environment. Additional advantages include easy access to the membrane sites so that the oligonucleotide can be applied, localized and removed easily.
  • GI gastrointestinal o
  • compositions can be targeted to a surface of the oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek.
  • 5 sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable
  • the sublingual mucosa is convenient, acceptable and easily accessible.
  • a pharmaceutical composition of oligonucleotide may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a
  • compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, slurries, emulsions, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches.
  • carriers that can be used include lactose, sodium citrate and salts of phosphoric acid.
  • disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets.
  • useful diluents are lactose and high molecular weight polyethylene glycols.
  • the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.
  • Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, intrathecal or intraventricular administration.
  • parental administration involves administration directly to the site of disease (e.g., neuronal tissue, neuromuscular tissue).
  • Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
  • Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir.
  • the total concentration of solutes should be controlled to render the preparation isotonic.
  • any of the oligonucleotides described herein can be administered to ocular tissue.
  • the compositions can be applied to the surface of the eye or nearby tissue, e.g. , the inside of the eyelid.
  • ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers.
  • Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as asorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers.
  • the oligonucleotide can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure.
  • Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably oligonucleotides, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation.
  • Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.
  • Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred.
  • Dry powder dispersion devices deliver agents that may be readily formulated as dry powders.
  • a oligonucleotide composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers.
  • the delivery of a composition for inhalation can be mediated by a dosing timing element which can include a o timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
  • binder means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device5 and subsequently inhaled by a subject so that the particles reach the lungs to permit
  • the powder is said to be “respirable.”
  • the average particle size is less than about 10 ⁇ in diameter preferably with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 ⁇ m and most preferably less than about 5.0 ⁇ m.
  • the particle size distribution is between o about 0.1 ⁇ m and about 5 ⁇ m in diameter, particularly about 0.3 ⁇ m to about 5 ⁇ m.
  • dry means that the composition has a moisture content below about 10% by weight (% w) water, usually below about 5% w and preferably less it than about 3% w.
  • a dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol.
  • the types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.
  • HSA human serum albumin
  • bulking agents such as carbohydrates, amino acids and polypeptides
  • pH adjusters or buffers such as sodium chloride
  • salts such as sodium chloride
  • Suitable pH adjusters or buffers include organic salts prepared from organic acids and 0 bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.
  • Pulmonary administration of a micellar oligonucleotide formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non- CFC and CFC propellants.
  • propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non- CFC and CFC propellants.
  • Exemplary devices include devices which are introduced into the vasculature, e.g., devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be placed in the vasculature of the lung, heart, or leg.
  • Other devices include non-vascular devices, e.g., devices implanted in the
  • the device can release a therapeutic substance in addition to an oligonucleotide.
  • unit doses or measured doses of a composition that includes oligonucleotide are dispensed by an implanted device.
  • the device can include a sensor that monitors a parameter within a subject.
  • the device can include pump, e.g., and, optionally, associated electronics.
  • Tissue e.g., cells or organs can be treated with an oligonucleotide or expression vector, ex vivo and then administered or implanted in a subject.
  • the tissue can be autologous, allogeneic, or xenogeneic tissue.
  • tissue can be treated to reduce graft v. host disease .
  • the tissue is allogeneic and the tissue is treated to treat a disorder characterized by unwanted gene expression in that tissue.
  • tissue e.g., hematopoietic cells, e.g., bone marrow hematopoietic cells, can be treated to inhibit unwanted cell proliferation.
  • Introduction of treated tissue, whether autologous or transplant can be combined with other therapies.
  • the oligonucleotide or expression vector treated cells are insulated from other cells, e.g., by a semi-permeable porous barrier that prevents the cells from leaving the implant, but enables molecules from the body to reach the cells and molecules produced by the cells to enter the body.
  • the porous barrier is formed from alginate.
  • the invention features a method of administering an oligonucleotide , expression vector, or inhibitor to a subject (e.g., a human subject).
  • a subject e.g., a human subject.
  • the unit dose is between about 10 mg and 25 mg per kg of bodyweight. In one embodiment, the unit dose is between about 1 mg and 100 mg per kg of bodyweight. In one embodiment, the unit dose is between about 0.1 mg and 500 mg per kg of bodyweight. In some embodiments, the unit dose is more than 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 25, 50 or 100 mg per kg of bodyweight.
  • the defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with a reduced level of FXN.
  • the unit dose, 5 for example, can be administered by injection (e.g. , intrathecal, intraneural, intracerebral, intravenous or intramuscular), an inhaled dose, or a topical application.
  • the unit dose is administered daily. In some embodiments, less frequently than once a day, e.g. , less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, not a regular frequency.
  • the unit dose may be administered a single time.
  • the unit dose is administered more than once a day, e.g., once an hour, two hours, four hours, eight hours, twelve hours, etc.
  • a subject is administered an initial dose and one or more maintenance doses of an oligonucleotide, expression vector, or inhibitor.
  • the maintenance5 dose or doses are generally lower than the initial dose, e.g. , one-half less of the initial dose.
  • a maintenance regimen can include treating the subject with a dose or doses ranging from 0.0001 to 100 mg/kg of body weight per day, e.g. , 100, 10, 1, 0.1, 0.01, 0.001, or 0.0001 mg per kg of bodyweight per day.
  • the maintenance doses may be administered no more than once every 1, 5, 10, or 30 days. Further, the treatment regimen may last for a period of time o which will vary depending upon the nature of the particular disease, its severity and the
  • the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g. , no more than once for every 5 or 8 days.
  • the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state.
  • the dosage may either 5 be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
  • a pharmaceutical composition includes a plurality of active species (e.g, a plurality of oligonucleotides, expression vectors and/or inhibitors).
  • an oligonucleotide species has sequences that are non-overlapping and non- adjacent to another oligonucleotide species with respect to a target sequence (e.g. , an mRNA of a negative epigenetic regulator of FXN).
  • a target sequence e.g. , an mRNA of a negative epigenetic regulator of FXN.
  • the plurality of oligonucleotide species is specific for different mRNAs of different negative epigenetic regulators of FXN.
  • the oligonucleotide is allele specific.
  • a patient is treated with an oligonucleotide, expression vector, or inhibitor in conjunction with other therapeutic modalities.
  • the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.0001 mg to 100 mg per kg of body weight.
  • the concentration of the oligonucleotide or inhibitor composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans.
  • concentration or amount of oligonucleotide or inhibitor administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary.
  • nasal formulations may tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10- 100 times in order to provide a suitable nasal formulation.
  • treatment of a subject with a therapeutically effective amount of an oligonucleotide and/or inhibitor can include a single treatment or, preferably, can include a series of treatments.
  • the effective dosage of an oligonucleotide and/or inhibitor used for treatment may increase or decrease over the course of a particular treatment.
  • the subject can be monitored after administering an oligonucleotide or inhibitor composition. Based on information from the monitoring, an additional amount of the oligonucleotide and/or inhibitor composition can be administered.
  • Optimal dosing schedules can be calculated from measurements of FXN expression levels in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models.
  • the animal models include transgenic animals that express a human FXN and/or a human negative epigenetic regulator of FXN.
  • a composition for testing in an animal model includes an
  • oligonucleotide that is complementary, at least in an internal region, to a sequence that is conserved between an mRNA of a negative epigenetic regulator of FXN in the animal model and the mRNA of the negative epigenetic regulator of FXN in a human.
  • the administration of a composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral, ocular,
  • parenteral e.g. intravenous (e.g., as a bolus or as a diffusible infusion)
  • intradermal e.g., intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral, ocular,
  • intraneuronal, intrathecal, or intracerebral administration can be provided by the subject or by another person, e.g., a health care provider.
  • the composition can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.
  • kits comprising a container housing a composition comprising an oligonucleotide, expression vector, or inhibitor.
  • the composition is a pharmaceutical composition comprising an
  • the individual components of the pharmaceutical composition may be provided in one container.
  • the kit may be packaged in a number of different configurations such as one or more containers in a single box.
  • the different components can be combined, e.g., according to instructions provided with the kit.
  • the components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition.
  • the kit can also include a delivery device.
  • RNAi based genetic screen was performed in cells from FRDA patients to identify regulators of FXN.
  • Several genes were identified as being negative regulators of FXN expression. When expression of these negative regulators is knocked down in cells, FXN expression increases in the cells.
  • Several other genes were identified as being positive regulators of FXN expression. When expression of these positive regulators is knocked down in the cells, FXN expression decreases in the cells.
  • described herein are certain regulatory factors that modulate expression of FXN in cells.
  • GM03816 cell line which is a fibroblast cell line from a patient with Friedriech's ataxia (FRDA).
  • FRDA Friedriech's ataxia
  • Cells were treated with the Human Epigenetics siGENOME® SMARTpool® siRNA Library (Dharmacon) according to the manufacturer's instructions.
  • RNA was harvested (at day 4 after treatment) and real time PCR performed to measure the level of FXN mRNA after treatment of the cells with the siRNA library.
  • Oligonucleotides were designed to target a subset of the genes identified in the siRNA screen. The sequence and structure of each oligonucleotide is shown in Table 4. Table 5 provides a description of the nucleotide analogs, modifications and intemucleoside linkages used for certain oligonucleotides described in Table 4. Oligonucleotides designed to target negative epigenetic regulators of FXN
  • CTCATCACAG CCG A EID1 human lnaCs;lnaTs;lnaCs;dAs;dTs;dCs;dAs;dCs;dAs; G dGs;dCs;dCs;lnaGs;lnaAs;lnaG-Sup
  • KAT2A- CATTGACCAGCTCCA KAT2A human lnaCs;lnaAs;lnaTs;dTs;dGs;dAs;dCs;dCs;dAs; 05 dGs;dCs;dTs;lnaCs;lnaCs;lnaA-Sup m08
  • KAT2A- GGCGATATACTCCTT KAT2A human lnaGs;lnaGs;lnaCs;dGs;dAs;dTs;dAs;dTs;dAs 06 ;dCs;dTs;dCs;lnaCs;lnaTs;lnaT-Sup m08
  • KAT2A- CCATCAGCGTCGCTC KAT2A human lnaCs;lnaCs;lnaAs;dTs;dCs;dAs;dGs;dCs;dGs 08 ;dTs;dCs;dGs;lnaCs;lnaTs;lnaC-Sup m08
  • PRKCD- TGTTGAAGCGTTCTT PRKCD human lnaTs;lnaGs;lnaTs;dTs;dGs;dAs;dAs;dGs;dCs 05 ;dGs;dTs;dTs;lnaCs;lnaTs;lnaT-Sup m08
  • PRKCD- CGATGTTGAAGCGT PRKCD human lnaCs;lnaGs;lnaAs;dTs;dGs;dTs;dTs;dGs;dAs 06 T ;dAs;dGs;dCs;lnaGs;lnaTs;lnaT-Sup m08
  • PRKCD- AAGCGGCCTTTGTCC PRKCD human lnaAs;lnaAs;lnaGs;dCs;dGs;dGs;dCs;dCs;dTs 07 ;dTs;dTs;dGs;lnaTs;lnaCs;lnaC-Sup m08
  • PRKCD- TAG AGTTCAA AG CG PRKCD human lnaTs;lnaAs;lnaGs;dAs;dGs;dTs;dTs;dCs;dAs; 08 G dAs;dAs;dGs;lnaCs;lnaGs;lnaG-Sup m08
  • TN FSF9 GTCAG AG G CGTATT TN FSF9 human lnaGs;lnaTs;lnaCs;dAs;dGs;dAs;dGs;dGs;dCs -01 C ;dGs;dTs;dAs;lnaTs;lnaTs;lnaC-Sup m08
  • TN FSF9 GACGGCGCAGGCGG TN FSF9 human lnaGs;lnaAs;lnaCs;dGs;dGs;dCs;dGs;dCs;dAs -03 C ;dGs;dGs;dCs;lnaGs;lnaGs;lnaC-Sup m08
  • TN FSF9 CTGAGCCCTCGCCG TN FSF9 human lnaCs;lnaTs;lnaGs;dAs;dGs;dCs;dCs;dCs;dTs; -04 G dCs;dGs;dCs;lnaCs;lnaGs;lnaG-Sup m08
  • TN FSF9 GGTCCACGGTCAAA TN FSF9 human lnaGs;lnaGs;lnaTs;dCs;dCs;dAs;dCs;dGs;dGs -05 G ;dTs;dCs;dAs;lnaAs;lnaAs;lnaG-Sup m08
  • TN FSF9 AGGTGCAGCAAGCG TN FSF9 human lnaAs;lnaGs;lnaGs;dTs;dGs;dCs;dAs;dGs;dCs -07 G ;dAs;dAs;dGs;lnaCs;lnaGs;lnaG-Sup m08
  • HIC1- TGACCGCGGCCTCT HICl human lnaTs;lnaGs;lnaAs;dCs;dCs;dGs;dCs;dGs;dGs 02 G ;dCs;dCs;dTs;lnaCs;lnaTs;lnaG-Sup m08
  • HIC1- ACGTACAGGTTGTC HICl human lnaAs;lnaCs;lnaGs;dTs;dAs;dCs;dAs;dGs;dGs 05 A ;dTs;dTs;dGs;lnaTs;lnaCs;lnaA-Sup m08
  • HIC1- ACACGTACAGGTTG HICl human lnaAs;lnaCs;lnaAs;dCs;dGs;dTs;dAs;dCs;dAs; 06 T dGs;dGs;dTs;lnaTs;lnaGs;lnaT-Sup m08
  • HIC1- TCTTGTCG CACG ACG H ICl human lnaTs;lnaCs;lnaTs;dTs;dGs;dTs;dCs;dGs;dCs; 07 dAs;dCs;dGs;lnaAs;lnaCs;lnaG-Sup m08
  • HIC1- TGTG CG AACTTG CC HICl human lnaTs;lnaGs;lnaTs;dGs;dCs;dGs;dAs;dAs;dCs 10 G ;dTs;dTs;dGs;lnaCs;lnaCs;lnaG-Sup m08
  • YEATS4 ATTCG G CCATTCTCT YEATS4 human lnaAs;lnaTs;lnaTs;dCs;dGs;dGs;dCs;dCs;dAs; -02 dTs;dTs;dCs;lnaTs;lnaCs;lnaT-Sup m08 YEATS4 CCCGCCGGAGTCAG YEATS4 human lnaCs;lnaCs;lnaCs;dGs;dCs;dCs;dGs;dGs;dAs -03 G ;dGs;dTs;dCs;lnaAs;lnaGs;lnaG-Sup
  • YEATS4 TTCGAATTCACCCCA YEATS4 human lnaTs;lnaTs;lnaCs;dGs;dAs;dAs;dTs;dTs;dCs; -06 dAs;dCs;dCs;lnaCs;lnaCs;lnaA-Sup
  • siRNA screen was performed in FRDA fibroblasts to identify epigenetic regulators that upregulate or downregulate FXN expression when knocked down.
  • the results of the screen are provided in Table 6 and FIG. 1 as the fold change in FXN mRNA expression compared to untreated cells.
  • Knockdown of several epigenetic regulators caused upregulation of FXN mRNA expression, indicating that FXN expression is at least partially regulated by epigenetic factors and that some of the screened epigenetic factors are negative epigenetic regulators of FXN.
  • CDY1 1.595909 0.034201 Gene Fold STDEV Change
  • CDY2A 1.436019 0.380472
  • CDK5R1 1.224767 0.449125
  • CDK2 1.009869 0.096811

Abstract

Provided herein are methods for increasing Frataxin (FXN) expression that involve targeting or expressing regulatory factors that modulate the epigenetic state of FXN genes. Also provided herein are methods for increasing FXN expression using inhibitors of a negative epigenetic regulator of FXN. Compositions and methods for treating Friedrich' s ataxia are also provided.

Description

EPIGENETIC REGULATORS OF FRATAXIN
CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/010,427, entitled "EPIGENETIC REGULATORS OF FRATAXIN", filed June 10, 2014 and of U.S. Provisional Application No. 61/866,830, entitled
"EPIGENETIC REGULATORS OF FRATAXIN", filed August 16, 2013, the contents of each of which are incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The invention relates in part to compositions and methods for modulating gene expression.
BACKGROUND OF THE INVENTION
Friedreich's ataxia (FRDA) is a rare recessive inherited disease characterized by progressive degeneration of the spinal cord and peripheral nerve tissue. Symptoms resulting from this nervous system damage include muscle weakness, loss of coordination, vision and hearing impairment, speech problems, scoliosis, diabetes, and several heart disorders.
Symptoms typically begin between ages of 5 and 15 years and first present as difficulty walking (gait ataxia). As the disease progresses, other symptoms develop, such as speech slurring, hearing loss, and vision loss. Various forms of heart disease often accompany FRDA, including hypertrophic cardiomyopathy, myocardial fibrosis, and cardiac failure. Approximately, ten percent of those affected by FRDA develop diabetes. Symptom progression varies between individuals, but generally within 10 to 20 years from disease onset, the person is wheelchair bound and may eventually become completely incapacitated. FRDA can lead to early death, often as a result of heart disease associated with FRDA. Reduced expression of Frataxin (FXN) is thought to cause Friedreich's ataxia (FRDA).
SUMMARY OF THE INVENTION
According to some aspects of the invention certain regulatory factors have been identified that modulate expression of FXN in cells. Both negative and positive regulators of FXN expression have been discovered. In some embodiments, regulatory factors disclosed herein modulate FXN expression by modulating the epigenetic state of FXN genes. In some embodiments, inhibiting expression of a negative regulator of FXN results increased expression of FXN in cells, e.g., cells from a patient with FRDA. In other embodiments, inducing expression of a positive regulator of FXN results in increased expression of FXN in cells, e.g., cells from a patient with FRDA. Thus, in certain aspects, the invention provides methods and compositions that are useful for upregulating FXN in a cell. Accordingly, in some embodiments, methods and compositions provided herein are useful for the treatment and/or prevention (e.g., reducing the risk or delaying the onset) of FRDA.
Aspects of the invention relate to methods for increasing FXN expression in a cell. In some embodiments, the methods involve delivering to a cell an oligonucleotide that inhibits expression or activity of a negative epigenetic regulator of FXN, thereby increasing FXN expression in the cell. In some embodiments, prior to delivering the oligonucleotide, the cell has a lower level of FXN expression compared to an appropriate control level of FXN expression. In some embodiments, prior to delivering the oligonucleotide, the cell has a higher level of histone H3 K27 or K9 methylation at the FXN gene compared with an appropriate control level of histone H3 K27 or K9 methylation. In some embodiments, the cell comprises an FXN gene encoding in its first intron a GAA repeat of between 10-2000 units. In some embodiments, the cell is obtained from or present in a subject having
Friedreich's ataxia. In some embodiments, presence of the oligonucleotide in the cell results in decreased levels of mRNA of the negative epigenetic regulator of FXN . In some embodiments, the appropriate control is a level of FXN in a cell from a subject or in cells from a population of subjects that do not have Friedreich's ataxia.
In some embodiments, the oligonucleotide comprises a sequence as set for in Table 4. In some embodiments, the oligonucleotide comprises a sequence as set for in Table 12. In some embodiments, the oligonucleotide is a gapmer, a mixmer, an siRNA, a single stranded
RNA, a single stranded DNA, an aptamer, or a ribozyme. In some embodiments, the oligonucleotide comprises at least one modified nucleotide or internucleoside linkage. In some embodiments, the oligonucleotide is a single stranded oligonucleotide. In some embodiments, the single stranded oligonucleotide comprises the sequence 5'-X-Y-Z-3', wherein X comprises 1-5 modified nucleotides, Y comprises at least 6 unmodified nucleotides, and Z comprises 1-5 modified nucleotides. In some embodiments, the X comprises 1-5 LNAs, Y comprises at least 6 DNAs, and Z comprises 1-5 LNAs. In some embodiments, the negative epigenetic regulator of FXN is a component of a histone H2A acetylation pathway, a NuA4 histone acetyltransferase complex, a protein amino acid acetylation pathway, a histone acetylation pathway, a protein amino acid acylation pathway, a H4/H2A histone acetyltransferase complex, a nucleotide binding pathway, a histone H4 acetylation pathway, a histone acetyltransferase complex, or an insulin receptor substrate binding pathway. In some embodiments, the component of the histone H2A acetylation pathway is MEAF6, YEATS4, ACTL6A, or DMAPl. In some embodiments, the component of the NuA4 histone acetyltransferase complex is MEAF6, YEATS4, ACTL6A, or DMAPl. In some embodiments, the component of the protein amino acid acetylation pathway is KAT2A, MEAF6, YEATS4, TAD A3, ACTL6A, or DMAPl . In some
embodiments, the component of the histone acetylation pathway is KAT2A, MEAF6, YEATS4, TAD A3, ACTL6A, or DMAPl. In some embodiments, the component of the protein amino acid acylation pathway is KAT2A, MEAF6, YEATS4, TAD A3, ACTL6A, or DMAPl. In some embodiments, the component of the H4/H2A histone acetyltransferase complex is MEAF6, YEATS4, ACTL6A, or DMAPl. In some embodiments, the component of the nucleotide binding pathway is MEF2D, PRKDC, IDH1, ACTL6A, JAK2, CFTR, SPEN, or PRKCD. In some embodiments, the component of the histone H4 acetylation pathway is MEAF6, YEATS4, ACTL6A, or DMAPl. In some embodiments, the component of the histone acetyltransferase complex is KAT2A, MEAF6, YEATS4, TAD A3, ACTL6A, or DMAPl . In some embodiments, the component of the insulin receptor substrate binding pathway is JAK2 or PRKCD.
In some embodiments, the negative epigenetic regulator of FXN is TNFSF9, JUND, HICl, PRKCD, JAK2, EID1, CFTR, TAD A3, MYBL2, KAT2A, IDH1, SUMOl, SPEN, PRKDC, KIR2DL4, APC, MEF2D, a component of the NuA4 Histone Acetyltransferase Complex, or a histone-lysine N-methyltransf erase.
In some embodiments, the negative epigenetic regulator of FXN is a component of the NuA4 Histone Acetyltransferase Complex. In some embodiments, the component of the NuA4 Histone Acetyltransferase Complex is YEATS4, Eafl, TRRAP, P400, EPCl, DMAPl, Tip60, MRG15, MRGX, MORF4, ACTB, ACTL6A, ING1, ING2, ING3, ING4, ING5, RUVBL1, RUVBL2, AF9, ENL, or MEAF6. In some embodiments, the component of the
NuA4 Histone Acetyltransferase Complex is YEATS4, ACTL6A, DMAPl, or MEAF6. In some embodiments, the component of the NuA4 Histone Acetyltransferase Complex is YEATS4.
In some embodiments, the negative epigenetic regulator of FXN is a histone-lysine N- methyltransferase. In some embodiments, the histone-lysine N-methyltransferase is
SUV39H1, SUV39H2, SETDB1, PRDM2, G9A and EHMT1. In some embodiments, the histone-lysine N-methyltransferase is SUV39H1.
In some embodiments, the negative epigenetic regulator of FXN is YEATS4, HICl, JUND, TNFSF9, PRKCD, KAT2A, JAK2, IDH1, EID1, or ACTL6A.
In some embodiments, the negative epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change greater than 1.25.
In some embodiments, the method further comprises: delivering to the cell a second oligonucleotide. In some embodiments, the second oligonucleotide inhibits expression or activity of a second negative epigenetic regulator of FXN. In some embodiments, the second negative epigenetic regulator of FXN is TNFSF9, JUND, HICl, PRKCD, JAK2, EID1, CFTR, TAD A3, MYBL2, KAT2A, IDH1, SUMOl, SPEN, PRKDC, KIR2DL4, APC, MEF2D, a component of the NuA4 Histone Acetyltransferase Complex, or a histone-lysine N-methyltransferase.
According to some aspects of the invention methods for increasing FXN expression in a cell are provided that involve delivering to a cell an expression vector that is engineered to express a positive epigenetic regulator of FXN, thereby increasing FXN expression in the cell. In some embodiments, prior to delivering the expression vector, the cell has a lower level of FXN expression compared to an appropriate control level of FXN expression.
According to some aspects of the invention methods for increasing FXN expression in a cell are provided that involve expressing a exogenous positive epigenetic regulator of FXN. In some embodiments, the positive epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change less than or equal to 1.0, 0.90, 0.85, 0.80, 0.75, or 0.50.
According to some aspects of the invention, oligonucleotides are provided that comprise a sequence as set forth in Table 4 or Table 12. In some embodiments, the oligonucleotide comprises at least one modified nucleotide or internucleoside linkage. In some embodiments, the oligonucleotide is 50 nucleotides or fewer in length. In some embodiments, the oligonucleotide consists of a sequence as set forth in Table 4. In some embodiments, the oligonucleotide consists of a sequence as set forth in Table 12.
The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appending claims.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1 is a graph depicting epigenetic siRNA screen fold change distribution.
FIG. 2 is a table depicting the siRNA Screening Results. "FXN downregulating genes" are genes for which reduced expression results in downregulation of FXN. "FXN upregulating genes" are genes for which reduced expression results in upregulation of FXN
FIG. 3A is a table depicting the siRNA data related to the NuA4 Histone
Acetyltransferase Complex.
FIG. 3B is a graph depicting that knockdown of Suv39Hl resulted in upregulation of FXN.
FIGs. 4A and 4B shows a screen of 80 epigenetic inhibitors from a epigenetics screening library using GM03816 FRDA diseased fibroblasts (Fig. 4A; actual data in Table 10) and GM0321 normal fibroblasts (Fig. 4B; actual data in Table 11). FXN RNA levels are indicated on the y-axis and the inhibitors used at both 1 μΜ and 5 μΜ are shown on the x- axis.
FIGs. 5A-5E shows treatment of human FRDA diseased cell lines and Sarsero FXN mouse-model derived fibroblasts with a histone lysine methyltransferase inhibitor (HLMi). The Sarsero mouse model was generated by inserting the diseased human FXN gene with GAA-repeated into mouse genome. RQ: FXN RNA quantity in compound treated cells relative to untreated cells. FIG. 5A shows GM03816 cells after 2 days of treatment with the
HLMi at the indicated concentration; FIG. 5B shows GM03816 cells after 3 days of treatment with the HLMi at the indicated concentration; FIG. 5C shows GM04078 cells after 3 days of treatment with the HLMi at the indicated concentration; FIG. 5D shows Sarsero fibroblasts after 3 days of treatment with the HLMi at the indicated concentration (mouse FXN expression); FIG. 5E shows Sarsero fibroblasts 3 day treatment with the HLMi at the indicated concentration (human FXN expression).
5 FIG. 6 shows a western blot to detect FXN protein upregulation in human FRDA diseased cell lines GM03816 and GM04078 following 3 days of treatment with a HLMi at various concentrations (5 μΜ, 2.5 μΜ, 1.25 μΜ). Results from control cells treated with DMSO and without inhibitor treatment are also shown.
FIG. 7A and B are a series of graphs showing FXN mRNA levels in cells treated with o gapmers for human JUND, YEATS4, HICl, ACTL6A, EID1, IDH1, TNFSF9, JAK2,
KAT2A or PRKCD; blank columns are untreated.
FIG. 8 is a photograph of a Western blot showing FXN protein levels in cells treated with gapmers for ACTL6A, JUND, PRKCD, and YEATS4.
FIG. 9 is a graph showing FXN mRNA levels in differentiated myotubes treated with 5 various gapmers for ACTL6A, EID 1 , HIC 1 , JUND, KAT2A, PRKCD, and YEATS4.
FIGs. 10A-D are a series of graphs showing enrichment in the FXN gene locus of H3K27me3 and H3K9me3 (10A and 10B), Tip60 (IOC), or SUV39H1 (10D) in diseased cell lines compared to normal cells.
FIGs. 11 A and 1 IB are a series of graphs showing showing enrichment in the FXN o gene locus of G9a (FIG. 11 A) and IgG (FIG. 1 IB) in diseased cell lines compared to normal cells.
DETAILED DESCRIPTION OF THE INVENTION
5 In some embodiments, regulatory factors disclosed herein modulate FXN expression by controlling the epigenetic state of FXN genes. In some embodiments, methods and compositions are provided that induce or enhance expression of FXN by decreasing expression or function of one or more negative epigenetic regulators of FXN. In some embodiments, this induced or enhanced expression of FXN is believed to result from a
0 change in the chromatin state of the FXN gene, e.g., a decreased level of histone H3 K27 or
K9 methylation at the FXN gene. In other aspects of the invention methods for inducing expression of a positive regulator of FXN may be used to induce or enhance expression of FXN. Here again, in some embodiments, this induced or enhanced expression of FXN is believed to result from a change in the chromatin state of the FXN gene, e.g., a decreased level of histone H3 K27 or K9 methylation at the FXN gene.
As used herein, the term "FXN gene" refers to a genomic region that encodes FXN 5 protein and/or controls the transcription of FXN mRNA. Thus, the term encompasses coding sequences and exons as well as any non-coding elements, e.g., promoters, enhancers, silencers, introns, and 5' and 3' untranslated regions. An FXN gene may include flanking sequences 5' and/or 3' to a known annotated FXN open reading frame, e.g., 1 Kb, 2Kb, 3Kb, 4Kb, 5Kb, 6Kb, 7Kb, 8Kb, 9Kb, or 10Kb or more flanking the 5' and/or 3' end of a known o annotated FXN open reading frame. In some embodiments, a FXN gene may be a human
FXN gene (see, e.g., NCBI Gene ID: 2395, located on chromosome 9). In some
embodiments, a FXN gene may be a corresponding homolog of a FXN gene in a different species (e.g., a mouse FXN encoded by a mouse FXN gene such as NCBI Gene ID: 14297). 5 Negative Epigenetic regulators of FXN
As used herein, a "negative epigenetic regulator" is a regulatory factor (e.g., regulatory protein) that promotes the formation or maintenance of heterochromatin, and/or that inhibits the formation or maintenance of euchromatin. In some embodiments, a negative epigenetic regulator inhibits or reduces FXN expression either directly or indirectly. In some o embodiments, negative epigenetic regulators mediate reduction or silencing of FXN
expression though an epigenetic mechanism, e.g., though heterochromatin formation at or near the FXN gene. Accordingly, in some embodiments, when the expression level of a negative epigenetic regulator of FXN is reduced (e.g., by contacting a cell with an appropriate oligonucleotide as described herein), FXN expression is upregulated.
5 Without wishing to be bound by theory, it is believed that in some embodiments the heterochromatin formation at the FXN gene can be reversed, in part or in whole, by reducing the expression of one or more negative epigenetic regulators of FXN, thereby causing upregulation of FXN expression. Heterochromatin formation can be measured using any method known in the art, e.g., using an immunoassay to detect methylation patterns at or near 0 the FXN gene. For example, levels of mono-, di- and tri-methylation of histone H3 at lysine
27 and/or lysine 9 may be measured at or near the FXN gene. An increase in these types of methylation may indicate the presence of heterochromatin in some embodiments. Negative epigenetic regulators of FXN may act directly on the FXN gene, e.g., by catalyzing methylation of a histone, or indirectly, e.g., by forming a complex with or activating other proteins that are involved in epigenetic modification of the FXN gene.
Examples of negative epigenetic regulators of FXN are provided in Tables 1 and 7. The gene ID and transcript ID for each gene are provided, which can be used to identify any gene, mRNA transcript, and protein sequences by querying the NCBI (National Center for
Biotechnology Information) Gene database.
In some embodiments, a negative epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change greater than 1. In some embodiments, a negative epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change greater than 1.5. In some embodiments, a negative epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change greater than 1.75. In some embodiments, a negative epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change greater than 2. In some embodiments, a negative epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change greater than 2.5.
In some embodiments, one or more chromatin markers may be evaluated to assess the chromatin status of an FXN gene. For example, Histone H4 K20 trimethylation may be used as a marker to indicate heterochromatin. Presence of HP1, SUV39 and/or other similar proteins may also be used to detect presence of heterochromatin at the FXN gene. Other suitable markers may be used to assess chromatin status of an FXN gene.
Table 1. Negative epigenetic regulators of FXN
Figure imgf000010_0001
ACTL6A ACTL6, ARPN-BETA, 86 NM_177989.2 56456 NM_019673.2 Arp4, BAF53A, NM_178042.2
INO80K NM_004301.3
JAK2 JTK10, THCYT3 3717 NM_004972.3 16452 NM_001048177.1
NM_008413.2
EID1 PNAS-22, C15orf3, 23741 NM_014335.2 58521 NM_025613.3
CRIl, EID-1, IRO45620,
PTD014, RBP21
CFTR tcag7.78, ABC35, 1080 NM_000492.3 12638 NM_021050.2
ABCC7, CF, CFTR/MRP,
MRP7, TNR-CFTR,
dJ760C5.1
TAD A3 ADA3, NGG1, STAF54, 10474 NM_001278270.1 101206 NM_133932.2
TADA3L, Hada3 NR_103488.1
NM_006354.3
NM_133480.2
MYBL2 B-MYB, BMYB 4605 NM_002466.2 17865 NM_008652.2
KAT2A GCN5, GCN5L2, PCAF- 2648 NM_021078.2 14534 NM_001038010.2 b, Hgcn5 NM_020004.5
IDH1 IDCD, IDH, IDP, IDPC, 3417 NM_005896.2 15926 NM_001111320.1
PICD NM_010497.3
SUM01 OK/SW-cl.43, DAP1, 7341 NM_001005781.1 22218 NM_009460.2
GMP1, OFC10, PICl, NM_001005782.1
SENP2, SMT3, SMT3C, NM_003352.4
SMT3H3, UBL1
SPEN RP1-134019.1, 23013 NM_015001.2 56381 NM_019763.2
HIAA0929, MINT,
RBM15C, SHARP
PRKDC DNA-PKcs, DNAPK, 5591 NM_001081640.1 19090 NM_011159.2
DNPK1, HYRC, HYRC1, NM_006904.6
XRCC7, p350
KIR2DL4 XXbac-BCX195L8.3, 3805 NM_001080770.1
CD158D, G9P, KIR103, NM_002255.5
KIR103AS
APC BTPS2, DP2, DP2.5, 324 NM_000038.5 11789 NM_007462.3
DP3, GS, PPP1R46 NM_001127510.2
NM_001127511.2
MEF2D RP11-98G7.2 4209 NM_001271629.1 17261 NM_133665.3
NM_005920.3 MEAF6 RP3-423B22.2, 64769 NM_001270875.1 70088 NM_027310.3 Clorfl49, CENP-28, NM_001270876.1
EAF6, NY-SAR-91 NM_022756.5
NR_073090.1
NR_073091.1
NR_073092.1
TRRAP PAF350/400, PAF400, 8295 NM_001244580.1 100683 NM_001081362.1
STAF40, TR-AP, Tral NM_003496.3
Eafl EAF1 85403 NM_033083.6 74427 NM_028932.4
EP400 CAGH32, P400, 57634 NM_015409.4 75560 NM_029337.2
TNRC12 NM_173066.1
EPC1 Epll 80314 NM_001272004.1 13831 NM_001276350.1
NM_001272019.1 NM_007935.2 NM_025209.3 NM_027497.3
DMAP1 RP5-891H21.2, 55929 NM_001034023.1 66233 NM_023178.2
DNMAPl, DNMTAPl, NM_019100.4
EAF2, MEAF2, SWC4 NM_001034024.1
Tip60 KAT5, ESA1, HTATIP, 10524 NM_001206833.1 81601 NM_001199247.1
HTATIP1, PLIP, TIP, NM_006388.3 NM_001199248.1 ZC2HC5, Cpla2 NM_182709.2 NM_001199249.1
NM_182710.2 NM_178637.2
NR_037603.1
MRG15 M0RF4L1, FWP006, 10933 NM_001265603.1 56397 NM_001168225.1
Eaf3, HsT17725, NM_001265604.1 NM_001168226.1 MEAF3, M0RFRG15, NM_001265605.1 NM_001168227.1 S863-6 NM_006791.3 NM_001168228.1
NM_206839.2 NM_001168229.1
NM_001168230.1 NM_019768.4 MRGX MORF4L2, MORFL2 9643 NM_001142418.1 56397 NM_001168225.1
NM_001142419.1 NM_001168226.1
NM_001142420.1 NM_001168227.1
NM_001142421.1 NM_001168228.1
NM_001142422.1 NM_001168229.1
NM_001142423.1 NM_001168230.1
NM_001142424.1 NM_019768.4
NM_001142425.1
NM_001142426.1
NM_001142427.1
NM_001142428.1
NM_001142429.1
NM_001142430.1
NM_001142431.1
NM_001142432.1
NM_012286.2
MORF4 CSR, CSRB, SEN, SENl 10934 No transcript avail 67568 NM_026242.3
ACTB BRWSl, PS1TP5BP1 60 NM_001101.3 11461 NM_007393.3
ING1 RP11-8D7.1, 3621 NM_001267728.1 26356 NM_011919.4 p24INGlc, p33, NM_198219.2
P33ingl, p33INGlb, NM_005537.4
p47, p47INGla NM_198217.2
NM_198218.2
ING2 ING1L, P33ing2 3622 NM_001564.2 69260 NM_023503.3
ING3 HSPC301, Eaf4, 54556 NM_019071.2 71777 NM_023626.4
MEAF4, p47ING3, NM_198267.1 ING4 My036, my036, 51147 NM_001127582.1 28019 NM_133345.2 p29ING4 NM_001127583.1
NM_001127584.1
NM_001127585.1
NM_001127586.1
NM_016162.3
ING5 p28ING5 84289 NM_032329.4 66262 NM_025454.2
Eaf5 Mortality factor 10934, NM_001265603.1 21761 NM_001039147.2 related genes 10933 NM_001265604.1 NM_024431.3 (MORF4, MRG15/X) NM_001265605.1
NM_006791.3
NM_206839.2
AF9 MLLT3, YEATS3 4300 NM_004529.2 70122 NM_027326.3
NM_029931.2
ENL MLLTl, LTG19, YEATS 1 4298 NM_005934.3 64144 NM_022328.2
RUVBL1 ECP54, INO80H, 8607 NM_003707.2 56505 NM_019685.2
NMP238, PONTIN,
Pontin52, RVB1, TIH1,
TIP49, TIP49A
RUVBL2 CGI-46, ECP51, 10856 NM_006666.1 20174 NM_011304.3
INO80J, REPTIN, RVB2,
TIH2, TIP48, TIP49B
SUV39H1 MG44, KMT1A, 6839 NM_003173.2 20937 NM_011514.2
SUV39H
SUV39H2 RP11-2K17.2, KMT1B 79723 NM_001193424.1 64707 NM_022724.4
NM_001193425.1
NM_001193426.1
NM_001193427.1
NM_024670.3
SETDB1 RP11-316M1.1, ESET, 9869 NM_001145415.1 84505 NM_001163641.1
H3-K9-HMTase4, NM_001243491.1 NM_001163642.1 KGIT, KMT1E, TDRD21 NM_012432.3 NM_018877.3
PRDM2 RP5-1177E19.1, 7799 NM_001007257.2 110593 NM_001081355.3
HUMHOXY1, KMT8, NM_001135610.1 NM_001256380.1 MTB-ZF, RIZ, RIZ1, NM_012231.4
RIZ2 NM_015866.4
G9A EHMT2, DAAP- 10919 NM_006709.3 110147 NM_145830.1
66K18.3, BAT8, NM_025256.5 NM_147151.1 C6orf30, G9A, GAT8,
KMT1C, NG36
EHMT1 RP11-188C12.1, 79813 NM_001145527.1 77683 NM_001012518.3 EU HMTASE1, Eu- N M_024757.4 N M. _001109686.2
HMTasel, FP13812, N M. _001109687.2
GLP, GLP1, KMT1D, N M. _172545.4 bA188C12.1
In some embodiments, a epigenetic regulator of FXN may be a component of the NuA4 Histone Acetyltransf erase Complex. The NuA4 histone acetyltransf erase complex is a complex having histone acetylase activity on chromatin, as well as ATPase, DNA helicase 5 and structural DNA binding activities. Subunits of the human complex include YEATS4, Eafl, TRRAP, P400, EPCl, DMAPl, Tip60, MRG15, MRGX, MORF4, ACTB, ACTL6A, ING1, ING2, ING3, ING4, ING5, RUVBL1, RUVBL2, AF9, ENL, and MEAF6.
In some embodiments, a negative epigenetic regulator of FXN may be a histone- lysine N-methyltransferase. Histone-lysine N-methyltransferases catalyze the transfer of one, o two or three methyl groups to a lysine residue of a histone protein. In some embodiments, the histone-lysine N-methyltransferase is capable of transferring one, two or three methyl groups to lysine 9 on histone H3 (H3K9me3). Methylation of lysine 9 on histone H3, especially near a gene promoter, is thought to reduce gene expression. H3K9me3 histone- lysine N-methyltransferases are well-known in the art and include SUV39H1, SUV39H2, 5 SETDB 1 , PRDM2, G9 A and EHMT 1.
Positive Epigenetic regulators of FXN
As used herein, a "positive epigenetic regulator" is a regulatory factor (e.g., a regulatory protein) that inhibits the formation or maintenance of heterochromatin, and/or that o promotes the formation or maintenance of euchromatin.
Without wishing to be bound by theory, it is believed that in some embodiments the heterochromatin formation at the FXN gene can be reversed, in part or in whole, by increasing the expression of one or more positive epigenetic regulators of FXN, thereby causing upregulation of FXN expression. Accordingly, in some embodiments, a positive 5 epigenetic regulator of FXN induces expression of FXN by directly or indirectly inhibiting the formation or maintenance of heterochromatin at an FXN gene, and/or promoting the formation or maintenance of euchromatin at an FXN gene. Accordingly, in some
embodiments, when the expression level of a positive epigenetic regulator of FXN is induced or increased, FXN expression may be upregulated. In some embodiments, a positive epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change less than 1. In some embodiments, a positive epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change less than 0.75. In some embodiments, a positive epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change less than 0.5. In some embodiments, a positive epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change less than 0.25.
In some embodiments, a positive regulator of FXN is the product of a gene listed in Table 8.
FXN Epigenetic Regulatory Pathways
As described in the Examples below, several pathways were identified that are enriched for epigenetic regulators of FXN. Thus, other components of these identified pathways are also contemplated as epigenetic regulators of FXN. Accordingly, in some embodiments, an epigenetic regulator of FXN is a component of the histone H2A acetylation pathway, the NuA4 histone acetyltransferase complex, the protein amino acid acetylation pathway, the histone acetylation pathway, the protein amino acid acylation pathway, the H4/H2A histone acetyltransferase complex, the nucleotide binding pathway, the histone H4 acetylation pathway, the histone acetyltransferase complex, or the insulin receptor substrate binding pathway.
Components of each pathway may be identified using the Gene ontology reference ID provided for each pathway in Table 7 ("GO:######"). The reference ID can be entered into the search function of the Gene Ontology website, and gene product associations can be identified. These gene product associations indicate other potential epigenetic regulators of FXN. In some embodiments, negative epigenetic regulators of FXN that are components of certain pathways are provided in Table 7. In some embodiments, positive epigenetic regulators of FXN that are components of certain pathways are provided in Table 8.
Methods for Modulating FXN Gene Expression
In some aspects, the invention relates to methods for modulating FXN gene expression cells (e.g., cells for which FXN levels are reduced) for research purposes. In other aspects, the invention relates to methods for modulating gene expression in cells (e.g., cells for which FXN levels are reduced) for therapeutic purposes. Cells can be in vitro, ex vivo, or in vivo (e.g., in a subject who has a disease resulting from reduced expression or activity of FXN, e.g., Friedreich's ataxia.) In some embodiments, methods for modulating FXN expression in cells comprise delivering to the cells an oligonucleotide that inhibits expression or activity of a negative epigenetic regulator of FXN. In some embodiments, methods for modulating FXN expression in cells comprise delivering to the cells an inhibitor that inhibits activity of a negative epigenetic regulator of FXN. In some embodiments, methods for modulating FXN expression cells comprise delivering to the cells a cDNA engineered to express a positive epigenetic regulator of FXN.
It is understood that any reference to uses of compounds (e.g., oligonucleotides, expression vectors, inhibitors) throughout the description contemplates use of the compound in preparation of a pharmaceutical composition or medicament for use in the treatment of condition or a disease (e.g., Friedreich's ataxia) associated with decreased levels or activity of FXN. Thus, as one non-limiting example, this aspect of the invention includes use of oligonucleotides or inhibitors in the preparation of a medicament for use in the treatment of disease, wherein the treatment involves upregulating expression of FXN. In another non- limiting example, this aspect of the invention includes use of expression vector (e.g., containing a coding region of a positive epigenetic regulator of FXN) in the preparation of a medicament for use in the treatment of disease, wherein the treatment involves upregulating expression of FXN.
In some embodiments, methods provided herein comprise contacting a cell having a lower level of FXN expression compared to an appropriate control level of FXN expression with a composition (e.g., oligonucleotide, expression vector, inhibitor) useful for
upregulating FXN expression.
In some embodiments, methods provided herein comprise contacting a cell having a lower level of FXN expression compared to an appropriate control level of FXN expression with an oligonucleotide specific for an mRNA of a negative epigenetic regulator of FXN as described herein, wherein the oligonucleotide reduces an expression level of the negative epigenetic regulator of FXN), thereby increasing FXN expression in the cell. In some embodiments, it is contemplated that the cell may be contacted with more than one oligonucleotide that targets one or more negative epigenetic regulators of FXN, e.g., a first oligonucleotide that targets a first negative epigenetic regulator of FXN as described herein and a second oligonucleotide that targets a second negative epigenetic regulator of FXN as described herein.
In another aspect of the invention, provided herein are methods for inhibiting the function of a negative epigenetic regulator of FXN (e.g., by contacting a cell with an appropriate inhibitor as described herein), thereby upregulating FXN expression. In some embodiments, provided are methods for increasing FXN expression in a cell by using one more inhibitors of histone-lysine N-methyltransferase. In some embodiments, the histone- lysine N-methyltransferase is capable of transferring one, two or three methyl groups to lysine 9 on histone H3 (H3K9me3). In some embodiments, the histone-lysine N- methyltransferase is SUV39H1. In some embodiments, the methods involve delivering to a cell an inhibitor that inhibits HLM, thereby increasing FXN expression in the cell. In some embodiments, a change in the chromatin state of the FXN gene (e.g., a decreased level of histone H3 K9 methylation at the FXN gene) increases expression of FXN. In some embodiments, the inhibitor is a small molecule inhibitor.
In certain embodiments, the level of expression of FXN using a histone-lysine N- methyltransferase inhibitor (HLMi) is increased by at least about 1.1X-1.5X, 1.5X-2X, 2X- 2.5X, 2.5X-3X, or 3X-4X the control level of FXN expression.
In some embodiments, a cell having a lower level of FXN expression compared to an appropriate control level of FXN expression has a level of FXN expression that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more lower than an appropriate control level of FXN expression. A level of FXN expression may be determined using any suitable assay known in the art (see, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001; Current Protocols in Molecular Biology, Current Edition, John Wiley & Sons, Inc., New York; and Current Protocols in Protein Production, Purification, and Analysis, Current Edition, John Wiley & Sons, Inc., New York). The FXN expression level may be an mRNA level or a protein level. The sequences of FXN mPvNAs and proteins are well-known in the art (see, e.g., NCBI Transcript IDs:
NM_000144.4, NM_001161706.1, and NM_181425.2, and NCBI Protein IDs: NP_000135.2, NP_001155178.1, and NP_852090.1) and can be used to design suitable reagents and assays for measuring an FXN expression level. In some embodiments, an appropriate control level of FXN expression may be, e.g., a level of FXN expression in a cell, tissue or fluid obtained from a healthy subject or population of healthy subjects. As used herein, a healthy subject is a subject that is apparently free of disease and has no history of disease, e.g., no history of Friedreich's ataxia. In some embodiments, an appropriate control level of is a level of FXN expression in a cell from a subject that does not have Friedreich's ataxia or a level of FXN expression in a population of cells from a population of subjects that do not have Friedreich's ataxia. In some embodiments, the subject or population of subjects that do not have Friedreich's ataxia are subjects that have a FXN gene locus that contains less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 GAA repeat units in the first intron. In some embodiments, when a level of FXN expression is elevated or increased compared to a control level of FXN, an appropriate control level of FXN may be a level of FXN expression in a cell, tissue, or subject to which an oligonucleotide has not been delivered or to which a negative control has been delivered (e.g., a scrambled oligo, a carrier, etc.).
In some embodiments, an appropriate control level of FXN expression may be a predetermined level or value, such that a control level need not be measured every time. The predetermined level or value can take a variety of forms. It can be single cut-off value, such as a median or mean. It can be established based upon comparative groups, such as where one defined group is known have Friedriech's ataxia and another defined group is known to not have Friedriech' s ataxia. It can be a range, for example, where the tested population is divided equally (or unequally) into groups, such as a group of subjects having a high number of GAA repeats in the first intron of FXN (e.g., over 1000 GAA repeats), a group of subjects having a moderate number of GAA repeats (e.g., from 20-1000 GAA repeats) and a group of subjects having a low number of GAA repeats (e.g., less than 20 GAA repeats).
The predetermined value can depend upon the particular population selected.
Accordingly, the predetermined values selected may take into account the category in which a subject falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art.
In some embodiments, a cell having a lower level of FXN expression compared to an appropriate control level of FXN expression is a cell that has a higher level of histone H3
K27 or K9 methylation at the FXN gene compared with an appropriate control level of histone H3 K27 or K9 methylation. An appropriate control level of histone H3 K27 or K9 methylation may be, e.g., a level of histone H3 K27 or K9 methylation in a cell, tissue or fluid obtained from a healthy subject or population of healthy subjects, such as a subject or subjects that do not have Friedreich's ataxia. A level of H3 K27 or K9 methylation expression may be determined using any suitable assay known in the art. Examples of assays for detecting histone methylation levels include, but are not limited to, immunoassays such as Western blot, immunohistochemistry and ELISA assays. Such assays may involve a binding partner, such as an antibody, that specifically binds to a methylated or unmethylated histone. Antibodies that recognize specific methylation patterns on histones are known in the art and available from commercial vendors (see, e.g., AbCam and Millipore).
In some embodiments, a cell having a lower level of FXN expression compared to an appropriate control level of FXN expression is a cell that comprises an FXN gene encoding in its first intron a GAA repeat of between 10-2000, 15-2000, 20-2000, 30-2000, 40-2000, 50- 2000, 100-2000, 10-1000, 15-1000, 20-1000, 30-1000, 40-1000, 50-1000, or 100-1000 units. The number of GAA repeats may be determined using any method known in the art, e.g., sequencing-based assays or probe-based assays.
In some embodiments, a cell having a lower level of FXN expression compared to an appropriate control level of FXN expression is a cell obtained from a subject having
Friedreich's ataxia. A subject having Friedreich's ataxia can be identified, e.g., by the number of GAA repeats present in the first intron of an FXN gene of the subject and/or by other diagnostic criteria or symptoms known in the art. Symptoms of Friedreich's ataxia include, but are not limited to, muscle weakness in the arms and legs, loss of coordination, vision impairment, hearing impairment, slurred speech, curvature of the spine, high plantar arches, diabetes, and/or heart disorders (e.g., cardiomegaly, atrial fibrillation, tachycardia and hypertrophic cardiomyopathy). A physical examination of eye movements, deep tendon reflexes, extensor plantar responses, and cardiac sounds may aid in diagnosis of a subject suspected of having Friedreich's ataxia. A genetic test, e.g., a PCR-based test, may be used to identify a subject having expanded GAA triplet repeats in the first intron of FXN.
As used herein, reducing an expression level of a negative epigenetic regulator of FXN includes reducing an expression level of the negative epigenetic regulator of FXN to 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%,
400%, 500% or more lower than an appropriate control level. An appropriate control level may be, e.g., a level of the negative epigenetic regulator of FXN in a cell that has not been contacted with an oligonucleotide or inhibitor as described herein. The expression level of the negative epigenetic regulator of FXN may be an mRNA level or a protein level. Thus, an oligonucleotide as described herein may reduce the mRNA and/or protein level of the negative epigenetic regulator of FXN. For example, if the oligonucleotide is designed to 5 degrade the mRNA, the level of mRNA will be reduced, and subsequently the level of protein will also be reduced. In another example, if the oligonucleotide is designed to block translation, the level of protein will be reduced, but the level of mRNA may remain stable. Assays for determining mRNA and protein levels are well-known in the art (e.g.,
microarrays, sequencing-based assays, probe-based assays, immunoassays, mass- o spectrometry, etc . ) .
As used herein, increasing FXN expression in a cell includes a level of FXN expression that is, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more above an appropriate control level of FXN. The appropriate control level may be a level of FXN expression in a cell that has not been
5 contacted with an oligonucleotide or inhibitor as described herein. The FXN expression may be FXN mRNA and/or protein expression. In some embodiments, increasing FXN
expression in a cell includes increasing a level of FXN expression to within 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a level of FXN expression in a cell from a healthy subject or a population of cells from a population of healthy subjects, e.g., subjects o that do not have Friedreich' s ataxia. For example, it may be desirable to increase an FXN expression level in a cell obtained from or in subject having Friedreich's ataxia such that the level of FXN expression is approximately the same as the level of FXN expression in a cell obtained from or in a subject who is healthy (e.g., not having Friedreich' s ataxia). However, it is to be understood that the level of FXN expression level in a cell obtained from or in
5 subject having Friedreich's ataxia may be increased to a level that is higher than the level of
FXN expression in a cell obtained from or in a subject who is healthy.
In another aspect of the invention, methods comprise administering to a subject (e.g. a human) a composition as described herein (e.g., a composition comprising an oligonucleotide and/or inhibitor targeting a negative epigenetic regulator of FXN) to increase FXN protein 0 levels in the subject. In some embodiments, the increase in protein levels is at least 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more, higher than the amount of a protein in the subject before administering the oligonucleotide and/or inhibitor. Aspects of the invention relate to compositions and methods of treating a condition (e.g., Friedreich's ataxia) associated with decreased levels of expression of FXN in a subject. An appropriate subject may be a non-human mammal, e.g. mouse, rat, guinea pig, rabbit, cat, dog, goat, cow, or horse. In preferred embodiments, a subject is a human. Oligonucleotides and inhibitors have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Oligonucleotides and inhibitors can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans. Oligonucleotides for Modulating Expression of FXN
In one aspect of the invention, oligonucleotides are provided for modulating expression of FXN in a cell. In some embodiments, expression of FXN is upregulated or increased. In some embodiments, oligonucleotides are provided that reduce the expression level of a negative epigenetic regulator of FXN, thereby upregulating the expression of FXN. In some embodiments, the oligonucleotide is specific for an mRNA of a negative epigenetic regulator of FXN.
The oligonucleotide may be single stranded or double stranded. Single stranded oligonucleotides may include secondary structures, e.g., a loop or helix structure. In some embodiments, the oligonucleotide comprises at least one modified nucleotide or modified internucleoside linkage as described herein.
The oligonucleotide may have a sequence that does not contain guanosine nucleotide stretches (e.g., 3 or more, 4 or more, 5 or more, 6 or more consecutive guanosine
nucleotides). In some embodiments, oligonucleotides having guanosine nucleotide stretches have increased non-specific binding and/or off-target effects, compared with oligonucleotides that do not have guanosine nucleotide stretches.
The oligonucleotide may have a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length, that map to a genomic position encompassing or in proximity to an off-target gene. For example, an oligonucleotide may be designed to ensure that it does not have a sequence that maps to genomic positions encompassing or in proximity with all known genes (e.g., all known protein coding genes) other than a negative epigenetic regulator of FXN. The threshold level of sequence identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity.
The oligonucleotide may have a sequence that is has greater than 30% G-C content, greater than 40% G-C content, greater than 50% G-C content, greater than 60% G-C content, 5 greater than 70% G-C content, or greater than 80% G-C content. The oligonucleotide may have a sequence that has up to 100% G-C content, up to 95% G-C content, up to 90% G-C content, or up to 80% G-C content. In some embodiments in which the oligonucleotide is 8 to 10 nucleotides in length, all but 1, 2, 3, 4, or 5 of the nucleotides of the complementary sequence of the mRNA of a negative epigenetic regulator of FXN are cytosine or guanosine o nucleotides. In some embodiments, the sequence of the mRNA to which the oligonucleotide is complementary comprises no more than 3 nucleotides selected from adenine and uracil.
The oligonucleotide may be complementary to a chromosome of a different species (e.g., a mouse, rat, rabbit, goat, monkey, etc.) at a position that encompasses or that is in proximity to that species' homolog of the negative epigenetic regulator of FXN. The5 oligonucleotide may be complementary to a human genomic region encompassing or in
proximity to the negative epigenetic regulator of FXN and also be complementary to a mouse genomic region encompassing or in proximity to the mouse homolog of the negative epigenetic regulator of FXN. For example, the oligonucleotide may be complementary to a sequence of a human mRNA of a negative epigenetic regulator of FXN (for example, a o human mRNA referenced in Table 1 by its NCBI accession number), and also be
complementary to a sequence of the corresponding mouse mRNA of the negative epigenetic regulator of FXN (for example, a corresponding mouse mRNA referenced in Table 1 by its NCBI accession number). Oligonucleotides having these characteristics may be tested in vivo or in vitro for efficacy in multiple species (e.g., human and mouse). This approach also 5 facilitates development of clinical candidates for treating human disease by selecting a
species in which an appropriate animal exists for the disease.
In some embodiments, the region of complementarity of the oligonucleotide is complementary with at least 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 bases, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,0 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive nucleotides of an mRNA of a negative epigenetic regulator of FXN. In some embodiments, the region of complementarity is complementary with at least 8 consecutive nucleotides of an mRNA of a negative epigenetic regulator of FXN. In some embodiments the sequence of the oligonucleotide is based on an RNA sequence that binds to an mRNA of a negative epigenetic regulator of FXN, or a portion thereof, said portion having a length of from 5 to 40 contiguous base pairs, or about 8 to 40 bases, or about 5 to 15, or about 5 to 30, or about 5 to 40 bases, or about 5 to 50 bases.
Complementary, as the term is used in the art, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an
oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of an mRNA of a negative epigenetic regulator of FXN, then the oligonucleotide and the mRNA of a negative epigenetic regulator of FXN are considered to be complementary to each other at that position. The oligonucleotide and the mRNA of a negative epigenetic regulator of FXN are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other through their bases. Thus, "complementary" is a term which is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the mRNA of a negative epigenetic regulator of FXN. For example, if a base at one position of an oligonucleotide is capable of hydrogen bonding with a base at the corresponding position of an mRNA of a negative epigenetic regulator of FXN, then the bases are considered to be complementary to each other at that position. 100%
complementarity is not required.
The oligonucleotide may be at least 80% complementary to (optionally one of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to) the consecutive nucleotides of an mRNA of a negative epigenetic regulator of FXN. In some embodiments the oligonucleotide may contain 1, 2 or 3 base mismatches compared to the portion of the consecutive nucleotides of an mRNA of a negative epigenetic regulator of
FXN. In some embodiments the oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.
It is understood in the art that a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable or specific for a target molecule. In some embodiments, a complementary nucleic acid sequence for purposes of the present disclosure is specifically hybridizable or specific for the target molecule when binding of the sequence to the target molecule (e.g., mRNA) interferes with the normal function of the target (e.g., mRNA) to cause a loss of activity (e.g., inhibiting translation with consequent up-regulation of FXN gene expression) or expression (e.g., degrading the mRNA with consequent up-regulation of FXN gene expression) and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under conditions in which avoidance of non-specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency.
In some embodiments, the oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length. In a preferred embodiment, the oligonucleotide is 8 to 30 nucleotides in length.
Base pairings may include both canonical Watson-Crick base pairing and non- Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). It is understood that for complementary base pairings, adenosine-type bases (A) are
complementary to thymidine-type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guano sine-type bases (G), and that universal bases such as 3- nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T. Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.
In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be replaced with any other nucleotide suitable for base pairing (e.g., via a Watson-Crick base pair) with an adenosine nucleotide. In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a different pyrimidine nucleotide or vice versa. In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a uridine (U) nucleotide (or a modified nucleotide thereof) or vice versa. In some embodiments, GC content of the oligonucleotide is preferably between about 30-60 %. Contiguous runs of three or more Gs or Cs may not be preferable in some embodiments. Accordingly, in some embodiments, the oligonucleotide does not comprise a stretch of three or more guanosine nucleotides.
5 It is to be understood that any oligonucleotide provided herein can be excluded.
In some embodiments, it has been found that oligonucleotides disclosed herein may increase expression of FXN mRNA by at least about 50% (i.e. 150% of normal or 1.5 fold), or by about 2 fold to about 5 fold. In some embodiments, expression may be increased by at least about 15 fold, 20 fold, 30 fold, 40 fold, 50 fold or 100 fold, or any range between any of o the foregoing numbers.
Any suitable oligonucleotide for targeting an mRNA is contemplated here. In some embodiments, the oligonucleotide may be designed to cause degradation of an mRNA (e.g., the oligonucleotide may be a gapmer, an siRNA, a ribozyme or an aptamer that causes degradation). In some embodiments, the oligonucleotide may be designed to block
5 translation of an mRNA (e.g., the oligonucleotide may be a mixmer, an siRNA or an aptamer that blocks translation). In some embodiments, an oligonucleotide may be designed to caused degradation and block translation of an mRNA.
Oligonucleotide structure and modifications
o The oligonucleotides described herein may be modified, e.g., comprise a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide and/or combinations thereof. In addition, the oligonucleotides may exhibit one or more of the following properties: do not mediate alternative splicing; are not immune stimulatory; are nuclease resistant; have improved cell uptake compared to unmodified oligonucleotides; are not toxic 5 to cells or mammals; or have improved endosomal exit.
Oligonucleotides that are designed to interact with RNA to modulate gene expression are a distinct subset of base sequences from those that are designed to bind a DNA target {e.g., are complementary to the underlying genomic DNA sequence from which the RNA is transcribed).
0 Any of the oligonucleotides disclosed herein may be linked to one or more other oligonucleotides disclosed herein by a linker, e.g., a cleavable linker. Oligonucleotides of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification. For example, nucleic acid sequences of the invention include a phosphorothioate at least the first, second, or third internucleoside linkage at the 5' or 3' end of the nucleotide sequence. As another 5 example, the nucleic acid sequence can include a 2'-modified nucleotide, e.g., a 2'-deoxy, 2'- deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0--N-methylacetamido (2'-0--NMA). As another example, the nucleic acid sequence can include at least one 2'-0-methyl-modified o nucleotide, and in some embodiments, all of the nucleotides include a 2'-0-methyl
modification. In some embodiments, the nucleic acids are "locked," i.e., comprise nucleic acid analogues in which the ribose ring is "locked" by a methylene bridge connecting the 2'- O atom and the 4'-C atom.
Any of the modified chemistries or formats of oligonucleotides described herein can5 be combined with each other, and that one, two, three, four, five, or more different types of modifications can be included within the same molecule.
In some embodiments, an oligonucleotide may comprise one or more modified nucleotides (also referred to herein as nucleotide analogs). In some embodiments, the oligonucleotide may comprise at least one ribonucleotide, at least one deoxyribonucleotide, o and/or at least one bridged nucleotide. In some embodiments, the oligonucleotide may
comprise a bridged nucleotide, such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide. Examples of such nucleotides are disclosed herein and known in the art. In some embodiments, the oligonucleotide comprises a nucleotide analog disclosed in one of the following United States5 Patent or Patent Application Publications: US 7,399,845, US 7,741,457, US 8,022,193, US 7,569,686, US 7,335,765, US 7,314,923, US 7,335,765, and US 7,816,333, US 20110009471, the entire contents of each of which are incorporated herein by reference for all purposes. The oligonucleotide may have one or more 2' O-methyl nucleotides. The oligonucleotide may consist entirely of 2' O-methyl nucleotides.
0 Often the oligonucleotide has one or more nucleotide analogues. For example, the oligonucleotide may have at least one nucleotide analogue that results in an increase in Tm of the oligonucleotide in a range of 1°C, 2 °C, 3°C, 4 °C, or 5°C compared with an oligonucleotide that does not have the at least one nucleotide analogue. The oligonucleotide may have a plurality of nucleotide analogues that results in a total increase in Tm of the oligonucleotide in a range of 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C or more compared with an oligonucleotide that does not have the nucleotide analogue.
The oligonucleotide may be of up to 50 nucleotides in length in which 2 to 10, 2 to 155 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or more nucleotides of the oligonucleotide are nucleotide analogues. The oligonucleotide may be of 8 to 30 nucleotides in length in which 2 to 10, 2 to 155 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are nucleotide analogues.
The oligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are nucleotide analogues. Optionally, the oligonucleotides may have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified.
The oligonucleotide may consist entirely of bridged nucleotides (e.g. , LNA nucleotides, cEt nucleotides, ENA nucleotides). The oligonucleotide may comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides. The oligonucleotide may comprise alternating deoxyribonucleotides and 2'-0-methyl nucleotides. The oligonucleotide may comprise alternating deoxyribonucleotides and ENA nucleotide analogues. The oligonucleotide may comprise alternating deoxyribonucleotides and LNA nucleotides. The oligonucleotide may comprise alternating LNA nucleotides and 2'-0- methyl nucleotides. The oligonucleotide may have a 5' nucleotide that is a bridged nucleotide (e.g. , a LNA nucleotide, cEt nucleotide, ENA nucleotide). The oligonucleotide may have a 5' nucleotide that is a deoxyribonucleotide.
The oligonucleotide may comprise deoxyribonucleotides flanked by at least one bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide) on each of the 5' and 3' ends of the deoxyribonucleotides. The oligonucleotide may comprise
deoxyribonucleotides flanked by 1, 2, 3, 4, 5, 6, 7, 8 or more bridged nucleotides (e.g. , LNA nucleotides, cEt nucleotides, ENA nucleotides) on each of the 5' and 3' ends of the deoxyribonucleotides. The 3' position of the oligonucleotide may have a 3' hydroxyl group.
The 3' position of the oligonucleotide may have a 3' thiophosphate. The oligonucleotide may be conjugated with a label. For example, the
oligonucleotide may be conjugated with a biotin moiety, cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
5 Preferably the oligonucleotide comprises one or more modifications comprising: a modified sugar moiety, and/or a modified internucleoside linkage, and/or a modified nucleotide and/or combinations thereof. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be incorporated in a single oligonucleotide or even at within a single o nucleoside within an oligonucleotide.
In some embodiments, the oligonucleotides are chimeric oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased5 uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric oligonucleotides of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers.
o Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, US patent nos. 5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and
5,700,922, each of which is herein incorporated by reference.
In some embodiments, the oligonucleotide comprises at least one nucleotide modified 5 at the 2' position of the sugar, preferably a 2'-0-alkyl, 2'-0-alkyl-0-alkyl or 2'-fluoro- modified nucleotide. In other preferred embodiments, RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., 0 higher target binding affinity) than 2'-deoxyoligonucleotides against a given target.
A number of nucleotide modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide; these modified oligos survive intact for a longer time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, for example, modified internucleoside linkages such as
phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. In some embodiments, oligonucleotides may have phosphorothioate backbones; heteroatom backbones, such as methylene(methylimino) or MMI backbones; amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones (see
Summerton and Weller, U.S. Pat. No. 5,034,506); or peptide nucleic acid (PNA) backbones (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497). Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see US patent nos. 3,687,808; 4,469,863; 4,476,301 ; 5,023,243; 5, 177, 196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131 ; 5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925; 5,519,126; 5,536,821 ; 5,541,306; 5,550, 111 ; 5,563, 253; 5,571,799; 5,587,361 ; and 5,625,050.
Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001 ; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216- 220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001 ; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties). Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602.
Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see US patent nos. 5,034,506; 5, 166,315; 5,185,444; 5,214,134; 5,216, 141 ; 5,235,033; 5,264, 562; 5, 264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596, 086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623, 070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
Modified oligonucleotides are also known that include oligonucleotides that are based on or constructed from arabinonucleotide or modified arabinonucleotide residues.
Arabinonucleosides are stereoisomers of ribonucleosides, differing only in the configuration at the 2'-position of the sugar ring. In some embodiments, a 2'-arabino modification is 2'-F arabino. In some embodiments, the modified oligonucleotide is 2' -fluoro-D-arabinonucleic acid (FANA) (as described in, for example, Lon et al., Biochem., 41 :3457-3467, 2002 and Min et al., Bioorg. Med. Chem. Lett., 12:2651-2654, 2002; the disclosures of which are incorporated herein by reference in their entireties). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
PCT Publication No. WO 99/67378 discloses arabinonucleic acids (ANA) oligomers and their analogues for improved sequence specific inhibition of gene expression via association to complementary messenger RNA.
Other preferred modifications include ethylene-bridged nucleic acids (ENAs) (e.g. ,
International Patent Publication No. WO 2005/042777, Morita et al., Nucleic Acid Res., Suppl 1 :241-242, 2001 ; Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8: 144-149, 2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49: 171- 172, 2005; the disclosures of which are incorporated herein by reference in their entireties). Preferred ENAs include, but are not limited to, 2'-0,4'-C-ethylene -bridged nucleic acids.
Examples of LNAs are described in WO/2008/043753 and include compounds of the following general formula.
Figure imgf000032_0001
where X and Y are independently selected among the groups -0-,
-S-, -N(H)-, N(R)-, -CH2- or -CH- (if part of a double bond),
-CH2-0-, -CH2-S-, -CH2-N(H)-, -CH2-N(R)-, -CH2-CH2- or -CH2-CH- (if part of a double bond),
-CH=CH-, where R is selected from hydrogen and Ci-4-alkyl; Z and Z* are independently selected among an intemucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety; and the asymmetric groups may be found in either orientation.
In some embodiments, the LNA used in the oligonucleotides described herein comprises at least one LNA unit according any of the formulas
Figure imgf000032_0002
wherein Y is -0-, -S-, -NH-, or N(R ); Z and Z* are independently selected among an intemucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety, and RH is selected from hydrogen and Ci-4-alkyl. In some embodiments, the Locked Nucleic Acid (LNA) used in the oligonucleotides described herein comprises at least one Locked Nucleic Acid (LNA) unit according any of the formulas shown in Scheme 2 of PCT/DK2006/000512.
In some embodiments, the LNA used in the oligomer of the invention comprises internucleoside linkages selected from -0-P(O)2-O-, -0-P(0,S)-0-, -0-P(S)2-O-, -S-P(0)2-0-, -S-P(0,S)-0-, -S-P(S)2-0-, -0-P(0)2-S-, -0-P(0,S)-S-, -S-P(0)2-S-, -0-PO(RH)-0-, o- PO(OCH3)-0-, -0-PO(NRH)-0-, -0-PO(OCH2CH2S-R)-O-, -0-PO(BH3)-0-, -0-PO(NHRH)- 0-, -0-P(0)2-NRH-, -NRH-P(0)2-0-, -NRH-CO-0-, where RH is selected from hydrogen and Ci-4-alkyl.
Specifically preferred LNA units are shown below:
Figure imgf000033_0001
-D-axy-LNA
Figure imgf000033_0002
The term "thio-LNA" comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from S or -CH2-S-. Thio-LNA can be in both beta-D and alpha-L-configuration.
The term "amino-LNA" comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from -N(H)-, N(R)-, CH2-N(H)-, and -CH2-N(R)- where R is selected from hydrogen and Ci-4-alkyl. Amino-LNA can be in both beta-D and alpha- L-configuration.
The term "oxy-LNA" comprises a locked nucleotide in which at least one of X or Y in the general formula above represents -O- or -CH2-O-. Oxy-LNA can be in both beta-D and alpha-L-configuration.
The term "ena-LNA" comprises a locked nucleotide in which Y in the general formula above is -CH2-0- (where the oxygen atom of -CH2-0- is attached to the 2'-position relative to the base B).
LNAs are described in additional detail herein.
One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 0(CH2)n CH3, 0(CH2)n NH2 or 0(CH2)n CH3 where n is from 1 to about 10; CI to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF3 ; OCF3; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH3; S02 CH3; ON02; N02; N3; NH2; heterocycloalkyl; heterocyclo alkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy [2'-0-CH2CH2OCH , also known as 2'-0-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2'- methoxy (2'-0-CH3), 2'-propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F). Similar
modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
Oligonucleotides can also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g. , hypoxanthine, 6-methyladenine, 5- Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2- (aminoalklyamino)adenine or other hetero substituted alkyladenines, 2-thiouracil, 2- 5 thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 5-propynyluracil, 8-azaguanine, 7- deazaguanine, N6 (6-aminohexyl)adenine, 6-aminopurine, 2-aminopurine, 2-chloro-6- aminopurine and 2,6-diaminopurine or other diaminopurines. See, e.g. , Kornberg, "DNA Replication," W. H. Freeman & Co., San Francisco, 1980, pp75-77; and Gebeyehu, G., et al. Nucl. Acids Res., 15:4513 (1987)). A "universal" base known in the art, e.g. , inosine, can o also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex
stability by 0.6- 1.2°C. (Sanghvi, in Crooke, and Lebleu, eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and may be used as base substitutions.
It is not necessary for all positions in a given oligonucleotide to be uniformly
5 modified, and in fact more than one of the modifications described herein may be
incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.
In some embodiments, both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for o hybridization with an appropriate nucleic acid target compound. One such oligomeric
compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone. The nucleobases are retained and are bound directly or 5 indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United
States patents that teach the preparation of PNA compounds include, but are not limited to, US patent nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
0 Oligonucleotides can also include one or more nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases comprise other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- 5 thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 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- methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7- o deazaadenine and 3- deazaguanine and 3-deazaadenine.
Further, nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in "The Concise Encyclopedia of Polymer Science And Engineering", pages 858-859, Kroschwitz, ed. John Wiley & Sons, 1990;, those disclosed by Englisch et al., Angewandle Chemie, International Edition, 1991, 30, page 613, and those disclosed by
5 Sanghvi, Chapter 15, Antisense Research and Applications," pages 289- 302, Crooke, and Lebleu, eds., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5- o methylcytosine substitutions have been shown to increase nucleic acid duplex stability by
0.6-1.2<0>C (Sanghvi, et al., eds, "Antisense Research and Applications," CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos. 3,687,808, as well as 4,845,205; 5,130,302;5 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; 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of which is herein incorporated by reference.
In some embodiments, the oligonucleotides are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the 0 oligonucleotide. For example, one or more oligonucleotides, of the same or different types, can be conjugated to each other; or oligonucleotides can be conjugated to targeting moieties with enhanced specificity for a cell type or tissue type. Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci.
USA, 1989, 86, 6553-6556), 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. , dodecandiol or undecyl residues (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 triethylammonium 1,2-di-O-hexadecyl- rac-glycero-3-H-phosphonate
(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Mancharan 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-t oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). See also US patent nos. 4,828,979; 4,948,882; 5,218, 105; 5,525,465; 5,541,313; 5,545,730; 5,552, 538; 5,578,717,
5,580,731 ; 5,580,731 ; 5,591,584; 5, 109,124; 5,118,802; 5, 138,045; 5,414,077; 5,486, 603;
5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762, 779; 4,789,737;
4,824,941 ; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082, 830; 5, 112,963; 5,214, 136;
5,082,830; 5, 112,963; 5,214, 136; 5, 245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391, 723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;
5,514,785; 5, 565,552; 5,567,810; 5,574, 142; 5,585,481 ; 5,587,371 ; 5,595,726; 5,597,696;
5,599,923; 5,599, 928 and 5,688,941, each of which is herein incorporated by reference.
These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence- specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g. , di-hexadecyl-rac- glycerol or triethylammonium 1,2- di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See, e.g. , U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218, 105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731 ; 5,580,731 ; 5,591,584; 5,109, 124; 5,118,802; 5, 138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941 ; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5, 112,963; 5,214, 136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574, 142; 5,585,481 ; 5,587,371 ; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.
In some embodiments, oligonucleotide modification includes modification of the 5' or 3' end of the oligonucleotide. In some embodiments, the 3' end of the oligonucleotide comprises a hydroxyl group or a thiophosphate. It should be appreciated that additional molecules (e.g. a biotin moiety or a fluorophor) can be conjugated to the 5' or 3' end of the oligonucleotide. In some embodiments, the oligonucleotide comprises a biotin moiety conjugated to the 5' nucleotide.
In some embodiments, the oligonucleotide comprises locked nucleic acids (LNA), ENA modified nucleotides, 2'-0-methyl nucleotides, or 2'-fluoro-deoxyribonucleotides. In some embodiments, the oligonucleotide comprises alternating deoxyribonucleotides and 2'- fluoro-deoxyribonucleotides. In some embodiments, the oligonucleotide comprises alternating deoxyribonucleotides and 2'-0-methyl nucleotides. In some embodiments, the oligonucleotide comprises alternating deoxyribonucleotides and ENA modified nucleotides. In some embodiments, the oligonucleotide comprises alternating deoxyribonucleotides and locked nucleic acid nucleotides. In some embodiments, the oligonucleotide comprises alternating locked nucleic acid nucleotides and 2' -O-methyl nucleotides. In some embodiments, the 5' nucleotide of the oligonucleotide is a
deoxyribonucleotide. In some embodiments, the 5' nucleotide of the oligonucleotide is a locked nucleic acid nucleotide. In some embodiments, the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one locked nucleic acid nucleotide on each 5 of the 5' and 3' ends of the deoxyribonucleotides. In some embodiments, the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group or a 3' thiophosphate.
In some embodiments, the oligonucleotide comprises phosphorothioate
internucleoside linkages. In some embodiments, the oligonucleotide comprises
phosphorothioate internucleoside linkages between at least two nucleotides. In some o embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages
between all nucleotides.
It should be appreciated that the oligonucleotide can have any combination of modifications as described herein.
In some embodiments, an oligonucleotide described herein may be a mixmer or
5 comprise a mixmer sequence pattern. The term 'mixmer' refers to oligonucleotides which comprise both naturally and non-naturally occurring nucleotides or comprise two different types of non-naturally occurring nucleotides. Mixmers are generally known in the art to have a higher binding affinity than unmodified oligonucleotides and may be used to specifically bind a target molecule, e.g., to block a binding site on the target molecule. Generally, o mixmers do not recruit an RNAse to the target molecule and thus do not promote cleavage of the target molecule.
In some embodiments, the mixmer comprises or consists of a repeating pattern of nucleotide analogues and naturally occurring nucleotides, or one type of nucleotide analogue and a second type of nucleotide analogue. However, it is to be understood that the mixmer 5 need not comprise a repeating pattern and may instead comprise any arrangement of
nucleotide analogues and naturally occurring nucleotides or any arrangement of one type of nucleotide analogue and a second type of nucleotide analogue. The repeating pattern, may, for instance be every second or every third nucleotide is a nucleotide analogue, such as LNA, and the remaining nucleotides are naturally occurring nucleotides, such as DNA, or are a 2' 0 substituted nucleotide analogue such as 2'MOE or 2' fluoro analogues, or any other
nucleotide analogues described herein. It is recognized that the repeating pattern of nucleotide analogues, such as LNA units, may be combined with nucleotide analogues at fixed positions— e.g. at the 5' or 3' termini.
In some embodiments, the mixmer does not comprise a region of more than 5, more than 4, more than 3, or more than 2 consecutive naturally occurring nucleotides, such as DNA nucleotides. In some embodiments, the mixmer comprises at least a region consisting of at least two consecutive nucleotide analogues, such as at least two consecutive LNAs. In some embodiments, the mixmer comprises at least a region consisting of at least three consecutive nucleotide analogue units, such as at least three consecutive LNAs.
In some embodiments, the mixmer does not comprise a region of more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2 consecutive nucleotide analogues, such as LNAs. It is to be understood that the LNA units may be replaced with other nucleotide analogues, such as those referred to herein.
In some embodiments, the mixmer comprises at least one nucleotide analogue in one or more of six consecutive nucleotides. The substitution pattern for the nucleotides may be selected from the group consisting of Xxxxxx, xXxxxx, xxXxxx, xxxXxx, xxxxXx and xxxxxX, wherein "X" denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA.
In some embodiments, the mixmer comprises at least two nucleotide analogues in one or more of six consecutive nucleotides. The substitution pattern for the nucleotides may be selected from the group consisting of XXxxxx, XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXXxxx, xXxXxx, xXxxXx, xXxxxX, xxXXxx, xxXxXx, xxXxxX, xxxXXx, xxxXxX and xxxxXX, wherein "X" denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA. In some embodiments, the substitution pattern for the nucleotides may be selected from the group consisting of XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX. In some embodiments, the substitution pattern is selected from the group consisting of xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX. In some embodiments, the substitution pattern is selected from the group consisting of xXxXxx, xXxxXx and xxXxXx. In some embodiments, the substitution pattern for the nucleotides is xXxXxx.
In some embodiments, the mixmer comprises at least three nucleotide analogues in one or more of six consecutive nucleotides. The substitution pattern for the nucleotides may be selected from the group consisting of XXXxxx, xXXXxx, xxXXXx, xxxXXX, XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx, wherein "X" denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA. In some embodiments, the substitution pattern for the nucleotides is selected from the group consisting of XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx. In some embodiments, the substitution pattern for the nucleotides is selected from the group consisting of xXXxXx, xXXxxX, xxXXxX, xXxXXx, xXxxXX, xxXxXX and xXxXxX. n some embodiments, the substitution pattern for the nucleotides is xXxXxX or XxXxXx. In some embodiments, the substitution pattern for the nucleotides is xXxXxX.
In some embodiments, the mixmer comprises at least four nucleotide analogues in one or more of six consecutive nucleotides. The substitution pattern for the nucleotides may be selected from the group consisting of xXXXX, xXxXXX, xXXxXX, xXXXxX, xXXXXx, XxxXXX, XxXxXX, XxXXxX, XxXXXx, XXxxXX, XXxXxX, XXxXXx, XXXxxX, XXXxXx and XXXXxx, wherein "X" denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA.
In some embodiments, the mixmer comprises at least five nucleotide analogues in one or more of six consecutive nucleotides. The substitution pattern for the nucleotides may be selected from the group consisting of xXXXXX, XxXXXX, XXxXXX, XXXxXX,
XXXXxX and XXXXXx, wherein "X" denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA.
The oligonucleotide may comprise a nucleotide sequence having one or more of the following modification patterns.
(a) (X)Xxxxxx, (X)xXxxxx, (X)xxXxxx, (X)xxxXxx, (X)xxxxXx and (X)xxxxxX,
(b) (X)XXxxxx, (X)XxXxxx, (X)XxxXxx, (X)XxxxXx, (X)XxxxxX, (X)xXXxxx, (X)xXxXxx, (X)xXxxXx, (X)xXxxxX, (X)xxXXxx, (X)xxXxXx, (X)xxXxxX, (X)xxxXXx,
(X)xxxXxX and (X)xxxxXX,
(c) (X)XXXxxx, (X)xXXXxx, (X)xxXXXx, (X)xxxXXX, (X)XXxXxx, (X)XXxxXx, (X)XXxxxX, (X)xXXxXx, (X)xXXxxX, (X)xxXXxX, (X)XxXXxx, (X)XxxXXx
(X)XxxxXX, (X)xXxXXx, (X)xXxxXX, (X)xxXxXX, (X)xXxXxX and (X)XxXxXx,
(d) (X)xxXXX, (X)xXxXXX, (X)xXXxXX, (X)xXXXxX, (X)xXXXXx,
(X)XxxXXXX, (X)XxXxXX, (X)XxXXxX, (X)XxXXx, (X)XXxxXX, (X)XXxXxX, (X)XXxXXx, (X)XXXxxX, (X)XXXxXx, and (X)XXXXxx, (e) (X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX, (X)XXXXxX and (X)XXXXXx, and
(f) XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxXX, XXXXXxX and XXXXXXx, in which "X" denotes a nucleotide analogue, (X) denotes an optional nucleotide analogue, and "x" denotes a DNA or RNA nucleotide unit. Each of the above listed patterns may appear one or more times within an oligonucleotide, alone or in combination with any of the other disclosed modification patterns.
In some embodiments, the mixmer contains a modified nucleotide, e.g., an LNA, at the 5' end. In some embodiments, the mixmer contains a modified nucleotide, e.g., an LNA, at the first two positions, counting from the 5' end.
In some embodiments, the mixmer is incapable of recruiting RNAseH.
Oligonucleotides that are incapable of recruiting RNAseH are well known in the literature, in example see WO2007/112754, WO2007/112753, or PCT/DK2008/000344. Mixmers may be designed to comprise a mixture of affinity enhancing nucleotide analogues, such as in non- limiting example LNA nucleotides and 2'-0-methyl nucleotides. In some embodiments, the mixmer comprises modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
A mixmer may be produced using any method known in the art or described herein. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of mixmers include U.S. patent publication Nos. US20060128646,
US20090209748, US20090298916, US20110077288, and US20120322851, and U.S. patent No. 7687617.
In some embodiments, the oligonucleotide is a gapmer. A gapmer oligonucleotide generally has the formula 5'-X-Y-Z-3', with X and Z as flanking regions around a gap region
Y. In some embodiments, the Y region is a contiguous stretch of nucleotides, e.g., a region of at least 6 DNA nucleotides, which are capable of recruiting an RNAse, such as RNAseH. Without wishing to be bound by theory, it is thought that the gapmer binds to the target nucleic acid, at which point an RNAse is recruited and can then cleave the target nucleic acid. In some embodiments, the Y region is flanked both 5' and 3' by regions X and Z comprising high-affinity modified nucleotides, e.g., 1 - 6 modified nucleotides. Exemplary modified oligonucleotides include, but are not limited to, 2' MOE or 2'OMe or Locked Nucleic Acid bases (LNA). The flanks X and Z may be have a of length 1 - 20 nucleotides, preferably 1-8 nucleotides and even more preferred 1 - 5 nucleotides. The flanks X and Z may be of similar length or of dissimilar lengths. The gap-segment Y may be a nucleotide sequence of length 5 - 20 nucleotides, preferably 6-12 nucleotides and even more preferred 6 - 10 nucleotides. In some aspects, the gap region of the gapmer oligonucleotides of the invention may contain modified nucleotides known to be acceptable for efficient RNase H action in addition to DNA nucleotides, such as C4'-substituted nucleotides, acyclic nucleotides, and arabino- configured nucleotides. In some embodiments, the gap region comprises one or more unmodified internucleosides. In some embodiments, one or both flanking regions each independently comprise one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides. In some embodiments, the gap region and two flanking regions each independently comprise modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
A gapmer may be produced using any method known in the art or described herein. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; 5,700,922; 5,898,031; 7,432,250; and 7,683,036; U.S. patent publication Nos.
US20090286969, US20100197762, and US20110112170; and PCT publication Nos.
WO2008049085 and WO2009090182, each of which is herein incorporated by reference in its entirety.
In some embodiments, oligonucleotides provided herein may be in the form of small interfering RNAs (siRNA), also known as short interfering RNA or silencing RNA. SiRNA, is a class of double- stranded RNA molecules, typically about 20-25 base pairs in length that target nucleic acids (e.g., mRNAs) for degradation via the RNA interference (RNAi) pathway in cells. Specificity of siRNA molecules may be determined by the binding of the antisense strand of the molecule to its target RNA. Effective siRNA molecules are generally less than 30 to 35 base pairs in length to prevent the triggering of non-specific RNA interference pathways in the cell via the interferon response, although longer siRNA can also be effective. Following selection of an appropriate target RNA sequence, siRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence, i.e. an antisense sequence, can be designed and prepared using any method known in the art (see, e.g., PCT Publication Nos. WO08124927A1 and WO 2004/016735; and U.S. Patent
Publication Nos. 2004/0077574 and 2008/0081791). A number of commercial packages and services are available that are suitable for use for the preparation of siRNA molecules. These include the in vitro transcription kits available from Ambion (Austin, TX) and New England Biolabs (Beverly, MA) as described above; viral siRNA construction kits commercially available from Invitrogen (Carlsbad, CA) and Ambion (Austin, TX), and custom siRNA construction services provided by Ambion (Austin, TX), Qiagen (Valencia, CA), Dharmacon (Lafayette, CO) and Sequitur, Inc (Natick, MA). A target sequence can be selected (and a siRNA sequence designed) using computer software available commercially (e.g.
OligoEngine™ (Seattle, Wash.); Dharmacon, Inc. (Lafayette, Colo.); Target Finder from Ambion Inc. (Austin, Tex.) and the siRNA Design Tool from QIAGEN, Inc. (Valencia, Calif.)). In some embodiments, an siRNA may be designed or obtained using the RNAi atlas (available at the RNAiAtlas website), the siRNA database (available at the Stockholm Bioinformatics Website), or using DesiRM (available at the Institute of Microbial
Technology website).
The siRNA molecule can be double stranded (i.e. a dsRNA molecule comprising an antisense strand and a complementary sense strand) or single- stranded (i.e. a ssRNA molecule comprising just an antisense strand). The siRNA molecules can comprise a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self- complementary sense and antisense strands.
Double-stranded siRNA may comprise RNA strands that are the same length or different lengths. Double- stranded siRNA molecules can also be assembled from a single oligonucleotide in a stem-loop structure, wherein self-complementary sense and antisense regions of the siRNA molecule are linked by means of a nucleic acid based or non-nucleic acid-based linker(s), as well as circular single- stranded RNA having two or more loop structures and a stem comprising self-complementary sense and antisense strands, wherein the circular RNA can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi. Small hairpin RNA (shRNA) molecules thus are also contemplated herein. These molecules comprise a specific antisense sequence in addition to the reverse complement (sense) sequence, typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides a single- stranded RNA molecule and its reverse complement, such that they may anneal to form a dsRNA molecule (optionally with additional processing steps that may result in addition or removal of one, two, three or more 5 nucleotides from the 3' end and/or the 5' end of either or both strands). A spacer can be of a sufficient length to permit the antisense and sense sequences to anneal and form a double- stranded structure (or stem) prior to cleavage of the spacer (and, optionally, subsequent processing steps that may result in addition or removal of one, two, three, four, or more nucleotides from the 3' end and/or the 5' end of either or both strands). A spacer sequence is o may be an unrelated nucleotide sequence that is situated between two complementary
nucleotide sequence regions which, when annealed into a double- stranded nucleic acid, comprise a shRNA.
The overall length of the siRNA molecules can vary from about 14 to about 200 nucleotides depending on the type of siRNA molecule being designed. Generally between5 about 14 and about 50 of these nucleotides are complementary to the RNA target sequence, i.e. constitute the specific antisense sequence of the siRNA molecule. For example, when the siRNA is a double- or single- stranded siRNA, the length can vary from about 14 to about 50 nucleotides, whereas when the siRNA is a shRNA or circular molecule, the length can vary from about 40 nucleotides to about 200 nucleotides.
o An siRNA molecule may comprise a 3' overhang at one end of the molecule, The other end may be blunt-ended or have also an overhang (5' or 3')· When the siRNA molecule comprises an overhang at both ends of the molecule, the length of the overhangs may be the same or different. In one embodiment, the siRNA molecule of the present invention comprises 3' overhangs of about 1 to about 3 nucleotides on both ends of the molecule. 5 In some embodiments, an oligonucleotide may be a microRNA (miRNA).
MicroRNAs (referred to as "miRNAs") are small non-coding RNAs, belonging to a class of regulatory molecules that control gene expression by binding to complementary sites on a target RNA transcript. Typically, miRNAs are generated from large RNA precursors (termed pri-miRNAs) that are processed in the nucleus into approximately 70 nucleotide pre- 0 miRNAs, which fold into imperfect stem-loop structures. These pre-miRNAs typically undergo an additional processing step within the cytoplasm where mature miRNAs of 18-25 nucleotides in length are excised from one side of the pre-miRNA hairpin by an RNase III enzyme, Dicer.
As used herein, miRNAs including pri-miRNA, pre-miRNA, mature miRNA or fragments of variants thereof that retain the biological activity of mature miRNA. In one 5 embodiment, the size range of the miRNA can be from 21 nucleotides to 170 nucleotides, although miRNAs of up to 2000 nucleotides can be utilized. In one embodiment the size range of the miRNA is from 70 to 170 nucleotides in length. In another embodiment, mature miRNAs of from 21 to 25 nucleotides in length can be used.
In some embodiments, a miRNA is expressed from a vector. In some embodiments, o the vector may include a sequence encoding a mature miRNA. In some embodiments, the vector may include a sequence encoding a pre-miRNA such that the pre-miRNA is expressed and processed in a cell into a mature miRNA. In some embodiments, the vector may include a sequence encoding a pri-miRNA. In this embodiment, the primary transcript is first processed to produce the stem-loop precursor miRNA molecule. The stem-loop precursor is 5 then processed to produce the mature microRNA.
In some embodiments, oligonucleotides provided herein may be in the form of aptamers._An "aptamer" is any nucleic acid that binds specifically to a target, such as a small molecule, protein, nucleic acid, cell, tissue or organism. In some embodiments, the aptamer is a DNA aptamer or an RNA aptamer. In some embodiments, a nucleic acid aptamer is a o single- stranded DNA or RNA (ssDNA or ssRNA). It is to be understood that a single- stranded nucleic acid aptamer may form helices and/or loop structures. The nucleic acid that forms the nucleic acid aptamer may comprise naturally occurring nucleotides, modified nucleotides, naturally occurring nucleotides with hydrocarbon linkers (e.g., an alkylene) or a polyether linker (e.g., a PEG linker) inserted between one or more nucleotides, modified 5 nucleotides with hydrocarbon or PEG linkers inserted between one or more nucleotides, or a combination of thereof.
Selection of nucleic acid aptamers may be accomplished by any suitable method known in the art, including an optimized protocol for in vitro selection, known as SELEX (Systemic Evolution of Ligands by Exponential enrichment). Many factors are important for 0 successful aptamer selection. For example, the target molecule should be stable and easily reproduced for each round of SELEX, because the SELEX process involves multiple rounds of binding, selection, and amplification to enrich the nucleic acid molecules. In addition, the nucleic acids that exhibit specific binding to the target molecule have to be present in the initial library. Thus, it is advantageous to produce a highly diverse nucleic acid pool. Because the starting library is not guaranteed to contain aptamers to the target molecule, the SELEX process for a single target may need to be repeated with different starting libraries.
Exemplary publications and patents describing aptamers and method of producing aptamers include, e.g., Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Pat. Nos. 5,270,163; 5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249; 5,789,157; 5,843,653; 5,864,026; 5,989,823; 6,569,630; 8,318,438 and PCT application WO 99/31275, each incorporated herein by reference.
In some embodiments, oligonucleotides provided herein may be in the form of a ribozyme. A ribozyme (ribonucleic acid enzyme) is a molecule, typically an RNA molecule, that is capable of performing specific biochemical reactions, similar to the action of protein enzymes. Ribozymes are molecules with catalytic activities including the ability to cleave at specific phosphodiester linkages in RNA molecules to which they have hybridized, such as mRNAs, RNA-containing substrates, IncRNAs, and ribozymes, themselves.
Ribozymes may assume one of several physical structures, one of which is called a "hammerhead." A hammerhead ribozyme is composed of a catalytic core containing nine conserved bases, a double- stranded stem and loop structure (stem-loop II), and two regions complementary to the target RNA flanking regions the catalytic core. The flanking regions enable the ribozyme to bind to the target RNA specifically by forming double- stranded stems I and III. Cleavage occurs in cis (i.e., cleavage of the same RNA molecule that contains the hammerhead motif) or in trans (cleavage of an RNA substrate other than that containing the ribozyme) next to a specific ribonucleotide triplet by a transesterification reaction from a 3', 5'-phosphate diester to a 2', 3'-cyclic phosphate diester. Without wishing to be bound by theory, it is believed that this catalytic activity requires the presence of specific, highly conserved sequences in the catalytic region of the ribozyme.
Modifications in ribozyme structure have also included the substitution or
replacement of various non-core portions of the molecule with non-nucleotidic molecules. For example, Benseler et al. (J. Am. Chem. Soc. (1993) 115:8483-8484) disclosed
hammerhead-like molecules in which two of the base pairs of stem II, and all four of the nucleotides of loop II were replaced with non-nucleoside linkers based on hexaethylene glycol, propanediol, bis(triethylene glycol) phosphate, tris(propanediol)bisphosphate, or bis(propanediol) phosphate. Ma et al. (Biochem. (1993) 32: 1751-1758; Nucleic Acids Res. (1993) 21:2585-2589) replaced the six nucleotide loop of the TAR ribozyme hairpin with non-nucleotidic, ethylene glycol-related linkers. Thomson et al. (Nucleic Acids Res. (1993) 21:5600-5603) replaced loop II with linear, non-nucleotidic linkers of 13, 17, and 19 atoms in 5 length.
Ribozyme oligonucleotides can be prepared using well known methods (see, e.g., PCT Publications W09118624; W09413688; WO9201806; and WO 92/07065; and U.S. Patents 5436143 and 5650502) or can be purchased from commercial sources (e.g., US Biochemicals) and, if desired, can incorporate nucleotide analogs to increase the resistance of o the oligonucleotide to degradation by nucleases in a cell. The ribozyme may be synthesized in any known manner, e.g., by use of a commercially available synthesizer produced, e.g., by Applied Biosystems, Inc. or Milligen. The ribozyme may also be produced in recombinant vectors by conventional means. See, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (Current edition). The ribozyme RNA sequences maybe synthesized
5 conventionally, for example, by using RNA polymerases such as T7 or SP6.
Expression vectors
It is to be appreciated that use of expression vectors to deliver oligonucleotides or any other appropriate nucleic acid (e.g., a cDNA engineered to expression a positive epigenetic o regulator of FXN) is contemplated in any appropriate context. Vectors include, but are not limited to, plasmids, viral vectors, other vehicles derived from viral or bacterial or other sources that have been manipulated by the insertion or incorporation of the nucleic acid sequences for expressing an RNA transcript (e.g., shRNA, miRNA, mRNA).
In some embodiments, expression vectors are provided that are engineered to express 5 a positive epigenetic regulator (e.g., a product of a gene as provided in Table 7). In some embodiments, expression of the positive epigenetic regulator causes upregulation of FXN. In some embodiments, an expression vector may be engineered by incorporating a cDNA comprising exons of a gene of interest into a plasmid that is suitably configured with expression elements (e.g., a promoter) for expressing the gene of interest. In some
0 embodiments, cDNA may be obtained or synthesized using a commercially available kit or any method known in the art, e.g, synthesized from mature (fully spliced) mRNA using the enzyme reverse transcriptase (see, e.g., US Patent Numbers 7470515 and 8420324, and PCT Publication Numbers WO2000052191, WO1997024455).
In some embodiments, a vector may comprise one or more expression elements. "Expression elements" are any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient expression of an RNA
5 transcript (e.g., shRNA, miRNA, mRNA). The expression element may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter or a tissue specific promoter, examples of which are well known to one of ordinary skill in the art. Constitutive mammalian promoters include polymerase promoters as well as the promoters for the following non-limiting genes: hypoxanthine phosphoribosyl transferase (HPTR), adenosine o deaminase, pyruvate kinase, and beta-actin. Exemplary viral promoters which function
constitutively in eukaryotic cells include promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters may be5 used. Inducible promoters are expressed in the presence of an inducing agent and include metal-inducible promoters and steroid-regulated promoters, for example. Other inducible promoters may be used.
Expression vectors may also comprise an origin of replication, a suitable promoter polyadenylation site, transcriptional termination sequences, and 5' flanking nontranscribed o sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
One of skill in the art can readily employ other vectors known in the art. Viral vectors are generally based on non-cytopathic eukaryotic viruses in which non-essential genes have 5 been replaced with the nucleic acid sequence of interest. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lines with plasmid, production of recombinant retroviruses by the packaging cell lie, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) may be used. Viral and 0 retroviral vectors that may be used include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses, such as: Moloney murine leukemia virus; Murine stem cell virus, Harvey murine sarcoma virus; murine mammary tumor virus; Rous sarcoma virus; adenovirus; adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes viruses; vaccinia viruses; polio viruses; and RNA viruses such as any retrovirus.
Formulation, Delivery, And Dosing
The compositions (e.g., oligonucleotides, expression vectors, inhibitors) described herein can be formulated for administration to a subject for treating a condition {e.g., o Friedrich's ataxia) associated with decreased levels of FXN. The formulations may
conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient {e.g., an oligonucleotide, expression vector, inhibitor) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of5 administration, e.g., intrathecal, intraneural, intracerebral, intramuscular, etc. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
Pharmaceutical formulations of this invention can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such formulations can o contain sweetening agents, flavoring agents, coloring agents and preserving agents. A
formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release
5 formulations, tablets, pills, gels, on patches, in implants, etc.
A formulated composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous {e.g., less than 80, 50, 30, 20, or 10% water). In another example, the composition is in an aqueous phase, e.g., in a solution that includes water. The aqueous phase or the crystalline
0 compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle {e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the composition is formulated in a manner that is compatible with the intended method of administration.
In some embodiments, the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.
An oligonucleotide preparation can be formulated or administered (together or separately) in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes an oligonucleotide, e.g., a protein that complexes with the oligonucleotide. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg2+), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.
In one embodiment, an oligonucleotide preparation includes another oligonucleotide, e.g., a second oligonucleotide that modulates expression of a second gene or a second oligonucleotide that modulates expression of the first gene. Still other preparation can include at least 3, 5, 10, twenty, fifty, or a hundred or more different oligonucleotide species. Such oligonucleotides can mediated gene expression with respect to a similar number of different genes. In one embodiment, the oligonucleotide preparation includes at least a second therapeutic agent (e.g., an agent other than an oligonucleotide). Expression vectors expressing different positive epigenetic regulators may be similarly combined with one another. Expression vectors expressing different positive epigenetic regulators may also be combined with one or more oligonucleotides that target negative epigenetic regulators.
In some embodiments, one or more oligonucleotides as provided herein is combined with the use of one or more inhibitors as described herein.
Histone -lysine N-methyltransf erase Inhibitors for Modulating Expression of FXN
Provided herein are methods of increasing FXN expression (protein and/or mRNA) in a subject or cell using an inhibitor of a negative epigenetic regulator of FXN. IN some embodiments, a histone-lysine N-methyltransferase inhibitor (HLMi) is used. The HLMi are contacted with cells of interest, thereby inhibiting histone-lysine N-methyltransferase, decreasing the levels of histone H3 K9 methylation, and increasing FXN expression in the cell, wherein, prior to contact with the inhibitor, the cell has a lower level of FXN expression compared to an appropriate control level of FXN expression. The cell is obtained from or present in a subject having Friedreich's ataxia.
In certain embodiments, the inhibitor is from the epipolythiodioxopiperazine class of fungal metabolites. In certain embodiments, the inhibitor is chaetocin.
In certain embodiments, the inhibitor comprises a quinazoline scaffold. In certain embodiments, the inhibitor comprises a 2,4-diamino-6,7-dimethoxyquinazoline scaffold. In certain embodiments the inhibitor is a compound with the following formula:
Figure imgf000052_0001
pharmaceutically acceptable salts or solvates thereof. R is
Figure imgf000052_0002
R' is isopropyl, cyclohexyl, or benzyl.
R" is
Figure imgf000052_0003
. R'" is methyl, ethyl, isopropyl, benzyl, cyclohexyl, or cyclohexylmethyl. In certain embodiments, the inhibitor is ΒΓΧ01294, UNC0224, UNC0321, UNC0638, UNC0646, UNC0631, TM2-115, UNC0642, ΒΓΧ-01338, or E72.
In certain embodiments, the inhibitor comprises an indole scaffold. In certain embodiments, the inhibitor is A-366.
In certain embodiments, the inhibitor comprises a benzimidazole scaffold. In certain embodiments, the benzimidazole scaffold is a 2-substituted benzimidazole. In certain embodiments, the benzimidazole scaffold is the following:
Figure imgf000053_0001
In certain embodiments, the inhibitor is BRD4770.
In certain embodiments, the inhibitor comprises an adenosine scaffold. In certain embodiments, the inhibitor comprising an adenosine scaffold is sinefungin or analogues thereof. In certain embodiments, the alpha-amino acid moiety in the sinefungin analogue has been exchanged to a moiety without an amino group. In certain embodiments, the inhibitor is 5'-desoxy-5'-butyladenosine. In certain embodiments, the alpha-amino acid moiety in the sinefungin analogue has been exchanged to a moiety with an amino group. In certain embodiments, the inhibitor is 5'-desoxy-5'-(2"-cyclohexyl-l"aminoethyl)-adenosine.
In some embodiments, one or more inhibitors of HML can be used to increase FXN expression. In certain embodiments, the inhibitor is one of the exemplary inhibitors listed in Table 2 or a pharmaceutically acceptable salt or solvate thereof. In certain embodiments, the inhibitor includes both the neutral form and a pharmaceutically acceptable salt thereof.
Table 2. Histone-lysine N-methyltransferase Inhibitors
Figure imgf000053_0002
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Sinefungin analogue:
5'-desoxy-5'-(2- aminocarbonylethyl)adenosine
Sinefungin analogue:
5'-desoxy-5'-(2 -phenylethyl) adenosine
Sinefungin analogue:
5'-desoxy-5'-benzyladenosine
Figure imgf000058_0001
Sinefungin analogue: 5'-desoxy-5'-(2- fluorobenzyl) adeno sine
Sinefungin analogue: 5'-desoxy-5'-(4- fluorobenzyl) adeno sine
Sinefungin analogue: 5'-desoxy-5'-(l- thiazolylmethyl) adeno sine
Sinefungin analogue: 5'-desoxy-5'-(3- phenylpropyl) adeno sine
Sinefungin analogue: 5'-desoxy-5'-(2- cyanobenzyl) adeno sine
Sinefungin analogue:
5'-desoxy-5'-(3"-methyl-propyl-l")- adenosine
Sinefungin analogue:
5'-desoxy-5'-(l"-isopropyl- 1 "aminomethyl) - adeno sine
Sinefungin analogue:
5'-desoxy-5 '- (2"-cyclopentyl- 1 "aminoethyl) - adeno sine
Figure imgf000061_0001
Sinefungin analogue:
5'-desoxy-5 '- (2"-cyclohexyl- 1 "aminoethyl) - adeno sine
Sinefungin analogue:
5'-desoxy-5'-(2"-3,4- dimethoxyphenyl- 1 "aminoethyl)- adenosine
Sinefungin analogue:
5'-desoxy-5 '- (2"-4-ethylphenyl- 1 "aminoethyl) adeno sine
Figure imgf000062_0001
Sinefungin analogue:
5'-desoxy-5 '- ( 1 "-cyclohexyl- 1 "aminomethyl) - adeno sine
Route of Delivery
The compositions (e.g., oligonucleotides, expression vectors, inhibitors) described herein can be delivered to a subject by a variety of routes. Exemplary routes include:
intrathecal, intraneural, intracerebral, intramuscular, oral, intravenous, intradermal, topical, rectal, parenteral, anal, intravaginal, intranasal, pulmonary, or ocular. The term
"therapeutically effective amount" is the amount of active agent (e.g., oligonucleotide, expression vector, inhibitor) present in the composition that is needed to provide the desired level of FXN expression in the subject to be treated to give the anticipated physiological response. The term "physiologically effective amount" is that amount delivered to a subject to give the desired palliative or curative effect. The term "pharmaceutically acceptable carrier" means that the carrier can be administered to a subject with no significant adverse toxicological effects to the subject.
The oligonucleotides, expression vectors, and inhibitors of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such
compositions typically include one or more species of oligonucleotide, expression vector, or inhibitor and a pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or
intraventricular administration.
The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the composition in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the composition and mechanically introducing the composition. Targeting of neuronal cells could be accomplished by intrathecal, intraneural, intracerebral administration.
Topical administration refers to the delivery to a subject by contacting the formulation directly to a surface of the subject. The most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g. , to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface. As mentioned above, the most common topical delivery is to the skin. The term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum. Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition. Topical administration can also be used as a means to selectively deliver compositionsto the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.
Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics. The dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin. Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal
5 adsorption. Absorption via this route may be enhanced by the use of an oily vehicle
(inunction) or through the use of one or more penetration enhancers. Other effective ways to deliver a composition disclosed herein via the transdermal route include hydration of the skin and the use of controlled release topical patches. The transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or o local therapy. In addition, iontophoresis (transfer of ionic solutes through biological
membranes under the influence of an electric field), phonophoresis or sonophoresis (use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea), and optimization of vehicle characteristics relative to dose position and retention at the site of administration may be useful methods for5 enhancing the transport of topically applied compositions across skin and mucosal sites.
Both the oral and nasal membranes offer advantages over other routes of administration. For example, oligonucleotides administered through these membranes may have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the oligonucleotides to the hostile gastrointestinal o (GI) environment. Additional advantages include easy access to the membrane sites so that the oligonucleotide can be applied, localized and removed easily.
In oral delivery, compositions can be targeted to a surface of the oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek. The
5 sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable
bioavailability of many agents. Further, the sublingual mucosa is convenient, acceptable and easily accessible.
A pharmaceutical composition of oligonucleotide may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a
0 metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant. In one embodiment, the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity. Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, slurries, emulsions, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.
Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, intrathecal or intraventricular administration. In some embodiments, parental administration involves administration directly to the site of disease (e.g., neuronal tissue, neuromuscular tissue).
Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.
Any of the oligonucleotides described herein can be administered to ocular tissue. For example, the compositions can be applied to the surface of the eye or nearby tissue, e.g. , the inside of the eyelid. For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as asorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers. The oligonucleotide can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure.
Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably oligonucleotides, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs. Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is 5 that the potential for contamination is minimized because the devices are self-contained. Dry powder dispersion devices, for example, deliver agents that may be readily formulated as dry powders. A oligonucleotide composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition for inhalation can be mediated by a dosing timing element which can include a o timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
The term "powder" means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device5 and subsequently inhaled by a subject so that the particles reach the lungs to permit
penetration into the alveoli. Thus, the powder is said to be "respirable." Preferably the average particle size is less than about 10 μιη in diameter preferably with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 μ m and most preferably less than about 5.0 μ m. Usually the particle size distribution is between o about 0.1 μ m and about 5 μ m in diameter, particularly about 0.3 μ m to about 5 μ m.
The term "dry" means that the composition has a moisture content below about 10% by weight (% w) water, usually below about 5% w and preferably less it than about 3% w. A dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol.
5 The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.
Suitable pH adjusters or buffers include organic salts prepared from organic acids and 0 bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.
Pulmonary administration of a micellar oligonucleotide formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non- CFC and CFC propellants.
Exemplary devices include devices which are introduced into the vasculature, e.g., devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be placed in the vasculature of the lung, heart, or leg.
Other devices include non- vascular devices, e.g., devices implanted in the
peritoneum, or in organ or glandular tissue, e.g., artificial organs. The device can release a therapeutic substance in addition to an oligonucleotide.
In one embodiment, unit doses or measured doses of a composition that includes oligonucleotide are dispensed by an implanted device. The device can include a sensor that monitors a parameter within a subject. For example, the device can include pump, e.g., and, optionally, associated electronics.
Tissue, e.g., cells or organs can be treated with an oligonucleotide or expression vector, ex vivo and then administered or implanted in a subject. The tissue can be autologous, allogeneic, or xenogeneic tissue. E.g., tissue can be treated to reduce graft v. host disease . In other embodiments, the tissue is allogeneic and the tissue is treated to treat a disorder characterized by unwanted gene expression in that tissue. E.g., tissue, e.g., hematopoietic cells, e.g., bone marrow hematopoietic cells, can be treated to inhibit unwanted cell proliferation. Introduction of treated tissue, whether autologous or transplant, can be combined with other therapies. In some implementations, the oligonucleotide or expression vector treated cells are insulated from other cells, e.g., by a semi-permeable porous barrier that prevents the cells from leaving the implant, but enables molecules from the body to reach the cells and molecules produced by the cells to enter the body. In one embodiment, the porous barrier is formed from alginate.
Dosage
In one aspect, the invention features a method of administering an oligonucleotide , expression vector, or inhibitor to a subject (e.g., a human subject). In one embodiment, the unit dose is between about 10 mg and 25 mg per kg of bodyweight. In one embodiment, the unit dose is between about 1 mg and 100 mg per kg of bodyweight. In one embodiment, the unit dose is between about 0.1 mg and 500 mg per kg of bodyweight. In some embodiments, the unit dose is more than 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 25, 50 or 100 mg per kg of bodyweight.
The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with a reduced level of FXN. The unit dose, 5 for example, can be administered by injection (e.g. , intrathecal, intraneural, intracerebral, intravenous or intramuscular), an inhaled dose, or a topical application.
In some embodiments, the unit dose is administered daily. In some embodiments, less frequently than once a day, e.g. , less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For
o example, the unit dose may be administered a single time. In some embodiments, the unit dose is administered more than once a day, e.g., once an hour, two hours, four hours, eight hours, twelve hours, etc.
In one embodiment, a subject is administered an initial dose and one or more maintenance doses of an oligonucleotide, expression vector, or inhibitor. The maintenance5 dose or doses are generally lower than the initial dose, e.g. , one-half less of the initial dose.
A maintenance regimen can include treating the subject with a dose or doses ranging from 0.0001 to 100 mg/kg of body weight per day, e.g. , 100, 10, 1, 0.1, 0.01, 0.001, or 0.0001 mg per kg of bodyweight per day. The maintenance doses may be administered no more than once every 1, 5, 10, or 30 days. Further, the treatment regimen may last for a period of time o which will vary depending upon the nature of the particular disease, its severity and the
overall condition of the patient. In some embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g. , no more than once for every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage may either 5 be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate 0 repeated or frequent infusions, implantation of a delivery device, e.g. , a pump, semipermanent stent (e.g. , intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable. In some embodiments, a pharmaceutical composition includes a plurality of active species (e.g, a plurality of oligonucleotides, expression vectors and/or inhibitors). In some embodiment, an oligonucleotide species has sequences that are non-overlapping and non- adjacent to another oligonucleotide species with respect to a target sequence (e.g. , an mRNA of a negative epigenetic regulator of FXN). In another embodiment, the plurality of oligonucleotide species is specific for different mRNAs of different negative epigenetic regulators of FXN. In another embodiment, the oligonucleotide is allele specific.
In some cases, a patient is treated with an oligonucleotide, expression vector, or inhibitor in conjunction with other therapeutic modalities.
Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.0001 mg to 100 mg per kg of body weight.
The concentration of the oligonucleotide or inhibitor composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans. The concentration or amount of oligonucleotide or inhibitor administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary. For example, nasal formulations may tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10- 100 times in order to provide a suitable nasal formulation.
Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of an oligonucleotide and/or inhibitor can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of an oligonucleotide and/or inhibitor used for treatment may increase or decrease over the course of a particular treatment. For example, the subject can be monitored after administering an oligonucleotide or inhibitor composition. Based on information from the monitoring, an additional amount of the oligonucleotide and/or inhibitor composition can be administered. Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of FXN expression levels in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In some embodiments, the animal models include transgenic animals that express a human FXN and/or a human negative epigenetic regulator of FXN. In another embodiment, a composition for testing in an animal model includes an
oligonucleotide that is complementary, at least in an internal region, to a sequence that is conserved between an mRNA of a negative epigenetic regulator of FXN in the animal model and the mRNA of the negative epigenetic regulator of FXN in a human.
In one embodiment, the administration of a composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral, ocular,
intraneuronal, intrathecal, or intracerebral. Administration can be provided by the subject or by another person, e.g., a health care provider. The composition can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.
Kits
In certain aspects of the invention, kits are provided, comprising a container housing a composition comprising an oligonucleotide, expression vector, or inhibitor. In some embodiments, the composition is a pharmaceutical composition comprising an
oligonucleotide, expression vector, or inhibitor and a pharmaceutically acceptable carrier. In some embodiments, the individual components of the pharmaceutical composition may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical composition separately in two or more containers, e.g., one container for oligonucleotides or inhibitors, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.
The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting.
EXAMPLES
Example 1 : Knockdown of epigenetic factors and FXN expression
Introduction
An RNAi based genetic screen was performed in cells from FRDA patients to identify regulators of FXN. Several genes were identified as being negative regulators of FXN expression. When expression of these negative regulators is knocked down in cells, FXN expression increases in the cells. Several other genes were identified as being positive regulators of FXN expression. When expression of these positive regulators is knocked down in the cells, FXN expression decreases in the cells. Thus, described herein are certain regulatory factors that modulate expression of FXN in cells.
MATERIALS AND METHODS: siRNA screen
An siRNA screen was performed in the GM03816 cell line, which is a fibroblast cell line from a patient with Friedriech's ataxia (FRDA). Cells were treated with the Human Epigenetics siGENOME® SMARTpool® siRNA Library (Dharmacon) according to the manufacturer's instructions. RNA was harvested (at day 4 after treatment) and real time PCR performed to measure the level of FXN mRNA after treatment of the cells with the siRNA library.
Real Time PCR
RNA was harvested from the cells using Promega SV 96 Total RNA Isolation system or Trizol omitting the DNAse step. RNA harvested from cells was normalized so that 50ng of RNA was input to each reverse transcription reaction. For the few samples that were too dilute to reach this limit, the maximum input volume was added. Reverse transcriptase reaction was performed using the Superscript II kit and real time PCR performed on cDNA samples using icycler SYBR green chemistry (Biorad). A baseline level of mRNA expression for each target gene was determined through quantitative PCR as outlined above. Baseline levels were also determined for mRNA of various housekeeping genes which are constitutively expressed. A "control" housekeeping gene with approximately the same level of baseline expression as the target gene was chosen for comparison purposes.
Cell lines
Cells were cultured using conditions known in the art (see, e.g., Current Protocols in Cell Biology). Details of the cell lines used in the experiments described herein are provided in Table 3.
Table 3. Cell lines
Figure imgf000073_0001
Pathway enrichment analysis
Genes identified in the siRNA screen that caused greater than two-fold upregulation or downregulation of FXN mRNA were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID, available to through DAVID
Bioinformatics Resources website) to identify pathways that were enriched in the gene set. The Functional Annotation DAVID tool was used to perform the enrichment analysis.
Oligonucleotide design
Oligonucleotides were designed to target a subset of the genes identified in the siRNA screen. The sequence and structure of each oligonucleotide is shown in Table 4. Table 5 provides a description of the nucleotide analogs, modifications and intemucleoside linkages used for certain oligonucleotides described in Table 4. Oligonucleotides designed to target negative epigenetic regulators of FXN
Base Sequence Gene Organism Formatted Sequence
Name
GGCAACAAAGCGGC ACTL6A human lnaGs;lnaGs;lnaCs;dAs;dAs;dCs;dAs;dAs;dAs G ;dGs;dCs;dGs;lnaGs;lnaCs;lnaG-Sup
ATCGCCATCTATTTC ACTL6A human lnaAs;lnaTs;lnaCs;dGs;dCs;dCs;dAs;dTs;dCs;
dTs;dAs;dTs;lnaTs;lnaTs;lnaC-Sup
G AACG ACCATTAG C ACTL6A human lnaGs;lnaAs;lnaAs;dCs;dGs;dAs;dCs;dCs;dAs A ;dTs;dTs;dAs;lnaGs;lnaCs;lnaA-Sup
GCCCAGTAGAACGA ACTL6A human lnaGs;lnaCs;lnaCs;dCs;dAs;dGs;dTs;dAs;dGs C ;dAs;dAs;dCs;lnaGs;lnaAs;lnaC-Sup
CATCGTGGACTGGA ACTL6A human lnaCs;lnaAs;lnaTs;dCs;dGs;dTs;dGs;dGs;dAs A ;dCs;dTs;dGs;lnaGs;lnaAs;lnaA-Sup
TTTCA ACCG CATACT ACTL6A human lnaTs;lnaTs;lnaTs;dCs;dAs;dAs;dCs;dCs;dGs;
dCs;dAs;dTs;lnaAs;lnaCs;lnaT-Sup
GAGCTAAACCTCCGT ACTL6A human lnaGs;lnaAs;lnaGs;dCs;dTs;dAs;dAs;dAs;dCs
;dCs;dTs;dCs;lnaCs;lnaGs;lnaT-Sup
GAGCCGCCAATCCA ACTL6A human lnaGs;lnaAs;lnaGs;dCs;dCs;dGs;dCs;dCs;dAs T ;dAs;dTs;dCs;lnaCs;lnaAs;lnaT-Sup
AAATTCCTCG CCCTC EID1 human lnaAs;lnaAs;lnaAs;dTs;dTs;dCs;dCs;dTs;dCs;
dGs;dCs;dCs;lnaCs;lnaTs;lnaC-Sup
CGTAGTCGTCCTCCC EID1 human lnaCs;lnaGs;lnaTs;dAs;dGs;dTs;dCs;dGs;dTs;
dCs;dCs;dTs;lnaCs;lnaCs;lnaC-Sup
CTG AAACCCG CCATC EID1 human lnaCs;lnaTs;lnaGs;dAs;dAs;dAs;dCs;dCs;dCs;
dGs;dCs;dCs;lnaAs;lnaTs;lnaC-Sup
AGCTCTTCGATAAAA EID1 human lnaAs;lnaGs;lnaCs;dTs;dCs;dTs;dTs;dCs;dGs;
dAs;dTs;dAs;lnaAs;lnaAs;lnaA-Sup
TCGGTCAGACGATT EID1 human lnaTs;lnaCs;lnaGs;dGs;dTs;dCs;dAs;dGs;dAs G ;dCs;dGs;dAs;lnaTs;lnaTs;lnaG-Sup
CTCATCACAG CCG A EID1 human lnaCs;lnaTs;lnaCs;dAs;dTs;dCs;dAs;dCs;dAs; G dGs;dCs;dCs;lnaGs;lnaAs;lnaG-Sup
ACG ATTCTCTATG CC IDH 1 human lnaAs;lnaCs;lnaGs;dAs;dTs;dTs;dCs;dTs;dCs;
dTs;dAs;dTs;lnaGs;lnaCs;lnaC-Sup
TGGCATCACGATTCT IDH 1 human lnaTs;lnaGs;lnaGs;dCs;dAs;dTs;dCs;dAs;dCs;
dGs;dAs;dTs;lnaTs;lnaCs;lnaT-Sup I DH 1- TCAATTGACTTATCT IDH 1 human lnaTs;lnaCs;lnaAs;dAs;dTs;dTs;dGs;dAs;dCs; 03 dTs;dTs;dAs;lnaTs;lnaCs;lnaT-Sup m08
I DH 1- ACG CCCATCATATTT IDH 1 human lnaAs;lnaCs;lnaGs;dCs;dCs;dCs;dAs;dTs;dCs; 04 dAs;dTs;dAs;lnaTs;lnaTs;lnaT-Sup m08
I DH 1- TGTCTTTAAAACGCC IDH 1 human lnaTs;lnaGs;lnaTs;dCs;dTs;dTs;dTs;dAs;dAs; 05 dAs;dAs;dCs;lnaGs;lnaCs;lnaC-Sup m08
I DH 1- TTATC AAG CTTTG CT IDH 1 human lnaTs;lnaTs;lnaAs;dTs;dCs;dAs;dAs;dGs;dCs; 06 dTs;dTs;dTs;lnaGs;lnaCs;lnaT-Sup m08
JAK2- GTCATCGTA AG G CA JAK2 human lnaGs;lnaTs;lnaCs;dAs;dTs;dCs;dGs;dTs;dAs; 01 G dAs;dGs;dGs;lnaCs;lnaAs;lnaG-Sup m08
JAK2- GGATCTTTGCTCGAA JAK2 human lnaGs;lnaGs;lnaAs;dTs;dCs;dTs;dTs;dTs;dGs; 02 dCs;dTs;dCs;lnaGs;lnaAs;lnaA-Sup m08
JAK2- TAGTCTTGGATCTTT JAK2 human lnaTs;lnaAs;lnaGs;dTs;dCs;dTs;dTs;dGs;dGs; 03 dAs;dTs;dCs;lnaTs;lnaTs;lnaT-Sup m08
JAK2- TGCGAAATCTGTACC JAK2 human lnaTs;lnaGs;lnaCs;dGs;dAs;dAs;dAs;dTs;dCs 04 ;dTs;dGs;dTs;lnaAs;lnaCs;lnaC-Sup m08
JAK2- TGAATTCCACCGTTT JAK2 human lnaTs;lnaGs;lnaAs;dAs;dTs;dTs;dCs;dCs;dAs; 05 dCs;dCs;dGs;lnaTs;lnaTs;lnaT-Sup m08
JAK2- ATCG C AATATA ACTG JAK2 human lnaAs;lnaTs;lnaCs;dGs;dCs;dAs;dAs;dTs;dAs; 06 dTs;dAs;dAs;lnaCs;lnaTs;lnaG-Sup m08
JAK2- I ACA I 1 1 l U CGU JAK2 human lnaTs;lnaGs;lnaAs;dCs;dAs;dTs;dTs;dTs;dTs; 07 dCs;dTs;dCs;lnaGs;lnaCs;lnaT-Sup m08
JAK2- TCATACCGGCACATC JAK2 human lnaTs;lnaCs;lnaAs;dTs;dAs;dCs;dCs;dGs;dGs; 08 dCs;dAs;dCs;lnaAs;lnaTs;lnaC-Sup m08
JAK2- GTCTCGTAAACTTCC JAK2 human lnaGs;lnaTs;lnaCs;dTs;dCs;dGs;dTs;dAs;dAs; 09 dAs;dCs;dTs;lnaTs;lnaCs;lnaC-Sup m08
JAK2- TGATCTATCCGTTCT JAK2 human lnaTs;lnaGs;lnaAs;dTs;dCs;dTs;dAs;dTs;dCs; 10 dCs;dGs;dTs;lnaTs;lnaCs;lnaT-Sup m08
KAT2A- AGGTCGAGCCGGAT KAT2A human lnaAs;lnaGs;lnaGs;dTs;dCs;dGs;dAs;dGs;dCs 01 C ;dCs;dGs;dGs;lnaAs;lnaTs;lnaC-Sup m08
KAT2A- CCGGACTTGCGCCTT KAT2A human lnaCs;lnaCs;lnaGs;dGs;dAs;dCs;dTs;dTs;dGs 02 ;dCs;dGs;dCs;lnaCs;lnaTs;lnaT-Sup m08
KAT2A- TGAGAGCTCGAACA KAT2A human lnaTs;lnaGs;lnaAs;dGs;dAs;dGs;dCs;dTs;dCs 03 T ;dGs;dAs;dAs;lnaCs;lnaAs;lnaT-Sup m08 KAT2A- CACGGAGCCGCTTG KAT2A human lnaCs;lnaAs;lnaCs;dGs;dGs;dAs;dGs;dCs;dCs 04 G ;dGs;dCs;dTs;lnaTs;lnaGs;lnaG-Sup m08
KAT2A- CATTGACCAGCTCCA KAT2A human lnaCs;lnaAs;lnaTs;dTs;dGs;dAs;dCs;dCs;dAs; 05 dGs;dCs;dTs;lnaCs;lnaCs;lnaA-Sup m08
KAT2A- GGCGATATACTCCTT KAT2A human lnaGs;lnaGs;lnaCs;dGs;dAs;dTs;dAs;dTs;dAs 06 ;dCs;dTs;dCs;lnaCs;lnaTs;lnaT-Sup m08
KAT2A- CACCGATGACCCGC KAT2A human lnaCs;lnaAs;lnaCs;dCs;dGs;dAs;dTs;dGs;dAs 07 C ;dCs;dCs;dCs;lnaGs;lnaCs;lnaC-Sup m08
KAT2A- CCATCAGCGTCGCTC KAT2A human lnaCs;lnaCs;lnaAs;dTs;dCs;dAs;dGs;dCs;dGs 08 ;dTs;dCs;dGs;lnaCs;lnaTs;lnaC-Sup m08
KAT2A- CTGTTTGCGCTCAAT KAT2A human lnaCs;lnaTs;lnaGs;dTs;dTs;dTs;dGs;dCs;dGs; 09 dCs;dTs;dCs;lnaAs;lnaAs;lnaT-Sup m08
KAT2A- GGCGATGACCCGCT KAT2A human lnaGs;lnaGs;lnaCs;dGs;dAs;dTs;dGs;dAs;dCs 10 G ;dCs;dCs;dGs;lnaCs;lnaTs;lnaG-Sup m08
PRKCD- CATGGTCGGCTTCTT PRKCD human lnaCs;lnaAs;lnaTs;dGs;dGs;dTs;dCs;dGs;dGs 01 ;dCs;dTs;dTs;lnaCs;lnaTs;lnaT-Sup m08
PRKCD- TGCGCATAGACTGTT PRKCD human lnaTs;lnaGs;lnaCs;dGs;dCs;dAs;dTs;dAs;dGs 02 ;dAs;dCs;dTs;lnaGs;lnaTs;lnaT-Sup m08
PRKCD- GGTGGCGATAAACT PRKCD human lnaGs;lnaGs;lnaTs;dGs;dGs;dCs;dGs;dAs;dTs 03 C ;dAs;dAs;dAs;lnaCs;lnaTs;lnaC-Sup m08
PRKCD- ATCTTGTCGATGCAT PRKCD human lnaAs;lnaTs;lnaCs;dTs;dTs;dGs;dTs;dCs;dGs; 04 dAs;dTs;dGs;lnaCs;lnaAs;lnaT-Sup m08
PRKCD- TGTTGAAGCGTTCTT PRKCD human lnaTs;lnaGs;lnaTs;dTs;dGs;dAs;dAs;dGs;dCs 05 ;dGs;dTs;dTs;lnaCs;lnaTs;lnaT-Sup m08
PRKCD- CGATGTTGAAGCGT PRKCD human lnaCs;lnaGs;lnaAs;dTs;dGs;dTs;dTs;dGs;dAs 06 T ;dAs;dGs;dCs;lnaGs;lnaTs;lnaT-Sup m08
PRKCD- AAGCGGCCTTTGTCC PRKCD human lnaAs;lnaAs;lnaGs;dCs;dGs;dGs;dCs;dCs;dTs 07 ;dTs;dTs;dGs;lnaTs;lnaCs;lnaC-Sup m08
PRKCD- TAG AGTTCAA AG CG PRKCD human lnaTs;lnaAs;lnaGs;dAs;dGs;dTs;dTs;dCs;dAs; 08 G dAs;dAs;dGs;lnaCs;lnaGs;lnaG-Sup m08
PRKCD- CCCCGAAAGACCAC PRKCD human lnaCs;lnaCs;lnaCs;dCs;dGs;dAs;dAs;dAs;dGs 09 C ;dAs;dCs;dCs;lnaAs;lnaCs;lnaC-Sup m08
PRKCD- CACGGATGGACTCG PRKCD human lnaCs;lnaAs;lnaCs;dGs;dGs;dAs;dTs;dGs;dGs 10 A ;dAs;dCs;dTs;lnaCs;lnaGs;lnaA-Sup m08 PRKCD- AGTCGATGAGGTTC PRKCD human lnaAs;lnaGs;lnaTs;dCs;dGs;dAs;dTs;dGs;dAs 11 T ;dGs;dGs;dTs;lnaTs;lnaCs;lnaT-Sup m08
TN FSF9 GTCAG AG G CGTATT TN FSF9 human lnaGs;lnaTs;lnaCs;dAs;dGs;dAs;dGs;dGs;dCs -01 C ;dGs;dTs;dAs;lnaTs;lnaTs;lnaC-Sup m08
TN FSF9 AGCAGCCCCGCGAC TN FSF9 human lnaAs;lnaGs;lnaCs;dAs;dGs;dCs;dCs;dCs;dCs -02 C ;dGs;dCs;dGs;lnaAs;lnaCs;lnaC-Sup m08
TN FSF9 GACGGCGCAGGCGG TN FSF9 human lnaGs;lnaAs;lnaCs;dGs;dGs;dCs;dGs;dCs;dAs -03 C ;dGs;dGs;dCs;lnaGs;lnaGs;lnaC-Sup m08
TN FSF9 CTGAGCCCTCGCCG TN FSF9 human lnaCs;lnaTs;lnaGs;dAs;dGs;dCs;dCs;dCs;dTs; -04 G dCs;dGs;dCs;lnaCs;lnaGs;lnaG-Sup m08
TN FSF9 GGTCCACGGTCAAA TN FSF9 human lnaGs;lnaGs;lnaTs;dCs;dCs;dAs;dCs;dGs;dGs -05 G ;dTs;dCs;dAs;lnaAs;lnaAs;lnaG-Sup m08
TN FSF9 AAACCGAAGGCCGA TN FSF9 human lnaAs;lnaAs;lnaAs;dCs;dCs;dGs;dAs;dAs;dGs -06 G ;dGs;dCs;dCs;lnaGs;lnaAs;lnaG-Sup m08
TN FSF9 AGGTGCAGCAAGCG TN FSF9 human lnaAs;lnaGs;lnaGs;dTs;dGs;dCs;dAs;dGs;dCs -07 G ;dAs;dAs;dGs;lnaCs;lnaGs;lnaG-Sup m08
TN FSF9 GTCACCCGGAAGAG TN FSF9 human lnaGs;lnaTs;lnaCs;dAs;dCs;dCs;dCs;dGs;dGs -08 T ;dAs;dAs;dGs;lnaAs;lnaGs;lnaT-Sup m08
TN FSF9 AGTAGGATTCGGAC TN FSF9 human lnaAs;lnaGs;lnaTs;dAs;dGs;dGs;dAs;dTs;dTs -09 T ;dCs;dGs;dGs;lnaAs;lnaCs;lnaT-Sup m08
JU N D- CCGTAGAAGGGTGT JU N D human lnaCs;lnaCs;lnaGs;dTs;dAs;dGs;dAs;dAs;dGs 01 T ;dGs;dGs;dTs;lnaGs;lnaTs;lnaT-Sup m08
JU N D- TTCATCATGCTGCCG JUN D human lnaTs;lnaTs;lnaCs;dAs;dTs;dCs;dAs;dTs;dGs; 02 dCs;dTs;dGs;lnaCs;lnaCs;lnaG-Sup m08
JU N D- CTGTGAGCTCGTCG JU N D human lnaCs;lnaTs;lnaGs;dTs;dGs;dAs;dGs;dCs;dTs; 03 G dCs;dGs;dTs;lnaCs;lnaGs;lnaG-Sup m08
JU N D- GGAACTGTGAGCTC JU N D human lnaGs;lnaGs;lnaAs;dAs;dCs;dTs;dGs;dTs;dGs 04 G ;dAs;dGs;dCs;lnaTs;lnaCs;lnaG-Sup m08
JU N D- GCTCGTCCTTGAGCG JUN D human lnaGs;lnaCs;lnaTs;dCs;dGs;dTs;dCs;dCs;dTs; 05 dTs;dGs;dAs;lnaGs;lnaCs;lnaG-Sup m08
JU N D- TGGCTCGTCCTTGAG JUN D human lnaTs;lnaGs;lnaGs;dCs;dTs;dCs;dGs;dTs;dCs; 06 dCs;dTs;dTs;lnaGs;lnaAs;lnaG-Sup m08
JU N D- CCCGTTGGACTGGA JU N D human lnaCs;lnaCs;lnaCs;dGs;dTs;dTs;dGs;dGs;dAs 07 T ;dCs;dTs;dGs;lnaGs;lnaAs;lnaT-Sup m08 JU N D- CGCTCCGCCTTGATG JUN D human lnaCs;lnaGs;lnaCs;dTs;dCs;dCs;dGs;dCs;dCs; 08 dTs;dTs;dGs;lnaAs;lnaTs;lnaG-Sup m08
JU N D- CACCTGCTCGCGCA JU N D human lnaCs;lnaAs;lnaCs;dCs;dTs;dGs;dCs;dTs;dCs; 09 G dGs;dCs;dGs;lnaCs;lnaAs;lnaG-Sup m08
HIC1- GGCCGGTGTAGATG HICl human lnaGs;lnaGs;lnaCs;dCs;dGs;dGs;dTs;dGs;dTs 01 A ;dAs;dGs;dAs;lnaTs;lnaGs;lnaA-Sup m08
HIC1- TGACCGCGGCCTCT HICl human lnaTs;lnaGs;lnaAs;dCs;dCs;dGs;dCs;dGs;dGs 02 G ;dCs;dCs;dTs;lnaCs;lnaTs;lnaG-Sup m08
HIC1- TTGACCGCGGCCTCT H ICl human lnaTs;lnaTs;lnaGs;dAs;dCs;dCs;dGs;dCs;dGs 03 ;dGs;dCs;dCs;lnaTs;lnaCs;lnaT-Sup m08
HIC1- TACCGGTCTCCTCGC H ICl human lnaTs;lnaAs;lnaCs;dCs;dGs;dGs;dTs;dCs;dTs; 04 dCs;dCs;dTs;lnaCs;lnaGs;lnaC-Sup m08
HIC1- ACGTACAGGTTGTC HICl human lnaAs;lnaCs;lnaGs;dTs;dAs;dCs;dAs;dGs;dGs 05 A ;dTs;dTs;dGs;lnaTs;lnaCs;lnaA-Sup m08
HIC1- ACACGTACAGGTTG HICl human lnaAs;lnaCs;lnaAs;dCs;dGs;dTs;dAs;dCs;dAs; 06 T dGs;dGs;dTs;lnaTs;lnaGs;lnaT-Sup m08
HIC1- TCTTGTCG CACG ACG H ICl human lnaTs;lnaCs;lnaTs;dTs;dGs;dTs;dCs;dGs;dCs; 07 dAs;dCs;dGs;lnaAs;lnaCs;lnaG-Sup m08
HIC1- AGCTCTTGTCGCACG H ICl human lnaAs;lnaGs;lnaCs;dTs;dCs;dTs;dTs;dGs;dTs; 08 dCs;dGs;dCs;lnaAs;lnaCs;lnaG-Sup m08
HIC1- CCGCACGCGTCGCA HICl human lnaCs;lnaCs;lnaGs;dCs;dAs;dCs;dGs;dCs;dGs 09 C ;dTs;dCs;dGs;lnaCs;lnaAs;lnaC-Sup m08
HIC1- TGTG CG AACTTG CC HICl human lnaTs;lnaGs;lnaTs;dGs;dCs;dGs;dAs;dAs;dCs 10 G ;dTs;dTs;dGs;lnaCs;lnaCs;lnaG-Sup m08
HIC1- GCTGTGCG AACTTG HICl human lnaGs;lnaCs;lnaTs;dGs;dTs;dGs;dCs;dGs;dAs 11 C ;dAs;dCs;dTs;lnaTs;lnaGs;lnaC-Sup m08
HIC1- TCGAGCTTGCCCTTG H ICl human lnaTs;lnaCs;lnaGs;dAs;dGs;dCs;dTs;dTs;dGs; 12 dCs;dCs;dCs;lnaTs;lnaTs;lnaG-Sup m08
HIC1- AGAAACGGTCGATG HICl human lnaAs;lnaGs;lnaAs;dAs;dAs;dCs;dGs;dGs;dTs 13 G ;dCs;dGs;dAs;lnaTs;lnaGs;lnaG-Sup m08
YEATS4 TCG G CCATTCTCTTG YEATS4 human lnaTs;lnaCs;lnaGs;dGs;dCs;dCs;dAs;dTs;dTs; -01 dCs;dTs;dCs;lnaTs;lnaTs;lnaG-Sup m08
YEATS4 ATTCG G CCATTCTCT YEATS4 human lnaAs;lnaTs;lnaTs;dCs;dGs;dGs;dCs;dCs;dAs; -02 dTs;dTs;dCs;lnaTs;lnaCs;lnaT-Sup m08 YEATS4 CCCGCCGGAGTCAG YEATS4 human lnaCs;lnaCs;lnaCs;dGs;dCs;dCs;dGs;dGs;dAs -03 G ;dGs;dTs;dCs;lnaAs;lnaGs;lnaG-Sup
85 m08
YEATS4 ATATGGAGGTTTAG YEATS4 human lnaAs;lnaTs;lnaAs;dTs;dGs;dGs;dAs;dGs;dGs -04 T ;dTs;dTs;dTs;lnaAs;lnaGs;lnaT-Sup
86 m08
YEATS4 TCGAATTCACCCCAT YEATS4 human lnaTs;lnaCs;lnaGs;dAs;dAs;dTs;dTs;dCs;dAs; -05 dCs;dCs;dCs;lnaCs;lnaAs;lnaT-Sup
87 m08
YEATS4 TTCGAATTCACCCCA YEATS4 human lnaTs;lnaTs;lnaCs;dGs;dAs;dAs;dTs;dTs;dCs; -06 dAs;dCs;dCs;lnaCs;lnaCs;lnaA-Sup
88 m08
YEATS4 TGACGAGATGTTGT YEATS4 human lnaTs;lnaGs;lnaAs;dCs;dGs;dAs;dGs;dAs;dTs -07 C ;dGs;dTs;dTs;lnaGs;lnaTs;lnaC-Sup
89 m08
YEATS4 TAG CTG ACG AG ATG YEATS4 human lnaTs;lnaAs;lnaGs;dCs;dTs;dGs;dAs;dCs;dGs -08 T ;dAs;dGs;dAs;lnaTs;lnaGs;lnaT-Sup
90 m08
YEATS4 AGTTTCACG ACTTG C YEATS4 human lnaAs;lnaGs;lnaTs;dTs;dTs;dCs;dAs;dCs;dGs; -09 dAs;dCs;dTs;lnaTs;lnaGs;lnaC-Sup
91 m08
Table 5. Oligonucleotide Modifications
Figure imgf000079_0001
InaGs LNA w/3' thiophosphate
InaTs LNA w/3' thiophosphate
omeAs 2'-OMe w/3' thiophosphate
omeCs 2'-OMe w/3' thiophosphate
omeGs 2'-OMe w/3' thiophosphate
omeTs 2'-OMe w/3' thiophosphate
InaAs-Sup LNA w/3' thiophosphate at 3' terminus
InaCs-Sup LNA w/3' thiophosphate at 3' terminus
InaGs-Sup LNA w/3' thiophosphate at 3' terminus
InaTs-Sup LNA w/3' thiophosphate at 3' terminus
InaA-Sup LNA w/3' OH at 3' terminus
InaC-Sup LNA w/3' OH at 3' terminus
InaG-Sup LNA w/3' OH at 3' terminus
InaT-Sup LNA w/3' OH at 3' terminus
omeA-Sup 2'-OMe w/3' OH at 3' terminus
omeC-Sup 2'-OMe w/3' OH at 3' terminus
omeG-Sup 2'-OMe w/3' OH at 3' terminus
omeU-Sup 2'-OMe w/3' OH at 3' terminus
dAs-Sup DNA w/3' thiophosphate at 3' terminus
dCs-Sup DNA w/3' thiophosphate at 3' terminus
dGs-Sup DNA w/3' thiophosphate at 3' terminus
dTs-Sup DNA w/3' thiophosphate at 3' terminus
dA-Sup DNA w/3' OH at 3' terminus
dC-Sup DNA w/3' OH at 3' terminus
dG-Sup DNA w/3' OH at 3' terminus
dT-Sup DNA w/3' OH at 3' terminus
In vitro transfection of cells with oligonucleotides
Cells are seeded into each well of 24-well plates at a density of 25,000 cells per 500uL and transfections are performed with Lipofectamine and the oligonucleotides. Control wells contain Lipofectamine alone. At time points post-transfection, approximately 200 uL of cell culture supernatants is stored at -80 C for ELISA and RNA is harvested from another aliquot of cells and quantitative PCR is carried out as outlined above. The percent induction of FXN mRNA expression by each oligonucleotide is determined by normalizing mRNA levels in the presence of the oligonucleotide to the mRNA levels in the presence of control (Lipofectamine alone).
RESULTS:
An siRNA screen was performed in FRDA fibroblasts to identify epigenetic regulators that upregulate or downregulate FXN expression when knocked down. The results of the screen are provided in Table 6 and FIG. 1 as the fold change in FXN mRNA expression compared to untreated cells. Knockdown of several epigenetic regulators caused upregulation of FXN mRNA expression, indicating that FXN expression is at least partially regulated by epigenetic factors and that some of the screened epigenetic factors are negative epigenetic regulators of FXN.
Table 6. siRNA evaluation results.
Figure imgf000081_0001
Gene Fold STDEV Change
KI 2DL4 2.022482 0.168389
APC 2.020118 0.014562
MEF2D 2.018638 0.205228
TRRAP 1.985487 0.034685
BAZ2A 1.963241 0.057413
SUV39H1 1.958921 0.343539
GTF3C4 1.956398 0.011402
PADI4 1.936776 0.110304
ZNF148 1.926952 0.101175
NC0A2 1.914404 0.13113
BAZ2B 1.912741 0.213506
KAT5 1.899023 0.042116
GSTP1 1.872775 0.103465
C20orf20 1.841951 0.132104
SMAD3 1.835045 0.000715
NC0A1 1.822696 0.033774
CDY1B 1.811376 0.66951
PHF16 1.791378 0.032315
MLH1 1.774602 0.041801
NR2C1 1.763209 0.143539
CDKN1A 1.75747 0.009267
GABPA 1.745357 0.26862
GMNN 1.744971 0.106884
GSG2 1.743216 0.094324
ATAD2 1.737997 0.014856
IFI16 1.731342 0.037746
CCND1 1.728521 0.03016
PLA2G16 1.710823 0.064799
TWIST1 1.70272 0.360724
MBD2 1.693068 0.20412
IFNB1 1.686817 0.117278
CHD3 1.685103 0.025845
SPI1 1.680621 0.19609
HIF1A 1.669651 0.088802
RCOR1 1.667394 0.113233
RUNXITI 1.66331 0.22652
CFLAR 1.663004 0.100039
CDY2B 1.632484 0.246241
TFCP2 1.620896 0.066323
ZMYM2 1.600876 0.048111
CDY1 1.595909 0.034201 Gene Fold STDEV Change
MAPK8 1.595626 0.105843
E2F1 1.581609 0.317282
IDH2 1.571751 0.224545
CCNB1 1.558334 0.149589
CLGN 1.534736 0.19762
TP53 1.528972 0.061943
TARDBP 1.527738 0.074554
KAT2B 1.522754 0.055204
RERE 1.517957 0.036769
HDAC5 1.514749 0.052821
TGFB1 1.512247 0.009211
MKI67 1.509564 0.279975
SP2 1.507101 0.26521
WDR82 1.50316 0.068023
H2AFZ 1.493633 0.029669
F0XP3 1.487303 0.358275
KRT23 1.484133 0.092025
MICB 1.476221 0.083149
CCDC101 1.471987 0.192941
RFC1 1.470751 0.107359
RAPIGAP 1.47004 0.055147
EGFR 1.466418 0.160559
HDAC8 1.459179 0.070388
NIPBL 1.458736 0.019252
LATS1 1.45133 0.02422
MLL 1.446532 0.161901
TGFBR1 1.446509 0.2189
HNRNPU 1.444872 0.195762
PKN2 1.443438 0.312938
BCAS3 1.442768 0.115198
TERT 1.441849 0.060187
BRMS1 1.437768 0.019876
CDY2A 1.436019 0.380472
NC0R2 1.430601 0.027334
SATB2 1.428661 0.052337
SALL1 1.428215 0.042382
SETD1B 1.418378 0.352216
RARA 1.409808 0.511328
MEF2C 1.408539 0.342254
CDK1 1.406172 0.049133
BCL11B 1.396615 0.106804 Gene Fold STDEV Change
YEATS2 1.392736 0.000645
CHD1 1.385509 0.054394
ELP4 1.381654 0.012584
NOC2L 1.381621 0.005973
MLL3 1.381313 0.069742
BRPF3 1.378898 0.167998
NFKB1 1.360332 0.069843
CASP8 1.359734 0.021175
PEA15 1.3581 0.077961
CD4 1.357769 0.123564
CBX5 1.356671 0.093802
USF1 1.356343 0.423359
MAP2K4 1.356072 0.033336
NFKB2 1.352113 0.117321
EP300 1.351336 0.008618
HDAC2 1.34946 0.244825
IFNG 1.340469 0.214563
RUVBL2 1.337488 0.148837
RHOB 1.336061 0.186296
MEF2A 1.325135 0.058873
CTR9 1.324755 0.120487
NCOA3 1.316417 0.09717
IL5 1.31449 0.074414
BRCA2 1.307471 0.146177
HNF4A 1.303522 0.083443
DDX53 1.300401 0.058196
EED 1.294184 0.290409
IL24 1.284432 0.336421
PROM1 1.281521 0.135175
VEGFA 1.275085 0.578396
SUPT3H 1.273981 0.109976
ATXN7 1.266507 0.320093
DNMT1 1.265959 0.02695
SDC1 1.263003 0.079211
TAF7 1.261491 0.130196
KDM4A 1.259146 0.613633
AES 1.256752 0.192033
NUDT21 1.249718 0.058988
NFYB 1.249438 0.130802
UHRFIBPI 1.246025 0.121585
BRD1 1.236461 0.058755 Gene Fold STDEV Change UVBL1 1.235495 0.124098
SMAD7 1.232964 0.12095
BAZ1B 1.232312 0.045874
RBPJ 1.231345 0.033005
MTA2 1.230373 0.045221
SUPT7L 1.230167 0.060147
ZMYM3 1.228456 0.113153
MAGEA2 1.227242 0.030533
CDK5R1 1.224767 0.449125
MLL2 1.223347 0.207863
RBI 1.220636 0.027249
PHF21A 1.219026 0.041777
TAF6L 1.216089 0.071181
SMYD2 1.213695 0.057812
CLOCK 1.208267 0.065789
SETDB2 1.206353 0.029933
PML 1.200961 0.071679
PRDM1 1.199998 0.031529
DPY30 1.19371 0.059896
MGMT 1.189418 0.050988
MYC 1.185714 0.215821
SALL3 1.185291 0.085353
IL13 1.168584 0.141312
CXXC1 1.162825 0.100761
TOP2B 1.160575 0.164227
ZNF217 1.156789 0.113157
KLF14 1.156194 0.006712
NKX3-1 1.153988 0.02949
TRIM29 1.152696 0.001042
KDM1A 1.148962 0.210084
RECK 1.145797 0.037568
SETD3 1.145261 0.285076
CSRP2BP 1.138262 0.100684
SETD1A 1.136995 0.134904
USP22 1.136202 0.114927
ING2 1.134242 0.182222
KAT6A 1.13154 0.402474
C16orf53 1.128591 0.149928
TADA1 1.126079 0.364332
SUZ12 1.119057 1.223702
N6AMT1 1.1181 0.146334 Gene Fold STDEV Change
KPNA2 1.117656 0.217184
PEX14 1.117068 0.255198
A ID1A 1.115998 0.078403
MBD3 1.113038 0.105622
SF3B3 1.108815 0.08127
SIN3B 1.105758 0.054678
NKAP 1.10296 0.071532
CAMTA2 1.099637 0.111942
F3 1.094147 0.094744
CIRl 1.088826 0.085504
CECR2 1.077054 0.064017
ANKRD1 1.076987 0.168455
NFE2L2 1.075071 0.228188
NR3C1 1.07376 0.223865
TUBA8 1.073504 0.01955
MTF1 1.070018 0.222578
EHMT2 1.068657 0.004415
FGFR3 1.066121 0.004651
MECOM 1.062529 0.019563
CREBBP 1.057707 0.164042
BRCA1 1.055937 0.097398
DAXX 1.052737 0.001142
ABCB1 1.052273 0.146848
NSD1 1.047448 0.125504
MIER1 1.043516 0.036453
BHLHE41 1.042951 0.164137
CDC73 1.039137 0.181696
TNFRSF9 1.039129 0.058632
PAX6 1.037401 0.468393
KAT7 1.037004 0.123777
VDR 1.033722 0.060818
PRKAB1 1.033714 0.117138
DCC 1.03253 0.178887
PRDX1 1.030073 0.010926
CITED2 1.029938 0.315317
NC0R1 1.027946 0.163666
ESR1 1.027744 0.25989
KRAS 1.027201 0.003579
HDAC6 1.024242 0.234
AURKB 1.023532 0.074672
UHRF1 1.021851 0.190562 Gene Fold STDEV Change
MSH2 1.016581 0.039809
TADA2B 1.016425 0.044812
MLL5 1.015729 0.047391
CDK2 1.009869 0.096811
MORF4L1 1.008464 0.213184
KAT6B 1.008352 0.020562
BIRC5 1.003109 0.047286
FES 1.002961 0.06576
CDYL 1.00149 0.132779
H AS 1.000696 0.154032
CIITA 0.999334 0.045764
SENP1 0.99401 0.04888
PRDM14 0.992507 0.00532
RBBP4 0.990023 0.041874
SUN1 0.988567 0.067196
PKN1 0.985086 0.122444
RELN 0.985057 0.134355
BRPF1 0.981796 0.080468
TAF10 0.97957 0.192907
CSNK2A1 0.978534 0.101121
TAF5L 0.975694 0.257281
SUV39H2 0.971945 0.072917
TRAF6 0.971874 0.036465
SPOP 0.971624 0.221885
MYB 0.970653 0.020559
MMP9 0.97026 0.032324
TUBA1B 0.961907 0.158951
SETD8 0.961421 0.090324
NAT10 0.953699 0.085111
DIP2B 0.949158 0.117463
HMG20B 0.946187 0.038692
EZH1 0.94278 0.059411
PRMT7 0.942367 0.092663
BRMS1L 0.941621 0.00168
TOP2A 0.941452 0.029488
MED24 0.939427 0.064759
HNF1A 0.939188 0.042303
TUBA3C 0.934268 0.074515
PRMT1 0.932275 0.139063
HPGD 0.927544 0.047463
MYOCD 0.926614 0.130867 Gene Fold STDEV Change
P MT2 0.925298 0.066321
SAP18 0.92247 0.099177
JARID2 0.919535 0.202206
SUV420H1 0.918594 0.173778
CXADR 0.916546 0.053083
PRF1 0.904105 0.011029
SLC2A4 0.904059 0.009424
D0T1L 0.902864 0.16045
TNFSF10 0.901029 0.18135
EDF1 0.900237 0.014248
SUV420H2 0.897968 0.172116
LEOl 0.896723 0.050959
PTGS2 0.894102 0.497395
MIF 0.893397 0.194554
AR 0.886335 0.078388
H2AFX 0.883926 0.422759
NQOl 0.883865 0.111466
NFYA 0.883787 0.137681
RUNX2 0.883346 0.174298
SETDB1 0.882963 0.062165
PHF20 0.88212 0.121558
HINFP 0.881388 0.051187
JUN 0.8802 0.059006
EPAS1 0.879998 0.033154
ARRB1 0.878782 0.009635
TGFBR2 0.875804 0.019383
PDK4 0.874145 0.012672
YWHAB 0.873961 0.082014
RUNX1 0.873116 0.011271
ABCC1 0.87122 0.09479
CDK11A 0.870977 0.003716
SIRT2 0.859159 0.02776
PHB 0.857689 0.119633
DNMT3L 0.856446 0.116672
SUDS3 0.856007 0.031787
ING4 0.854957 0.14643
PRMT6 0.85259 0.015261
TCF21 0.849189 0.095611
RBBP7 0.848903 0.079747
CXCL12 0.84876 0.079965
TUBA4A 0.844379 0.279532 Gene Fold STDEV Change
HIC2 0.844321 0.025511
HPSE 0.843136 0.160091
WHSC1 0.842313 0.122163
CXC 4 0.840796 0.058992
P KCA 0.839601 0.066119
E2F6 0.838928 0.065776
CHRNE 0.836508 0.038432
STAT3 0.835259 0.017378
HEY2 0.825149 0.023005
NFYC 0.822831 0.015783
EZH2 0.821495 0.085455
TAF12 0.820841 0.053659
TPPP 0.820309 0.008001
BCOR 0.818979 0.006866
SP1 0.818448 0.09332
PAXIP1 0.818335 0.047976
ULBP2 0.813605 0.099239
MLL4 0.813084 0.148387
MAGEA1 0.811527 0.118212
TBL1XR1 0.810227 0.193963
RELA 0.80782 0.08096
DEK 0.806225 0.088955
AKT1 0.805037 0.496321
WNK4 0.802826 0.060557
ETV6 0.797355 0.050518
BMI1 0.795599 0.236267
NRIP1 0.79462 0.179617
ANKRA2 0.791872 0.004883
SOD2 0.791698 0.164835
GAT A3 0.790259 0.1663
TALI 0.787454 0.141897
N6AMT2 0.786784 0.106426
PHF17 0.785948 0.206761
NFATC1 0.781722 0.116544
TFAP4 0.780982 0.038572
GADD45A 0.780126 0.145771
TUBA1A 0.777987 0.070961
HIPK2 0.777929 0.270004
ELP3 0.771886 0.025216
HAT1 0.768189 0.056701
ING5 0.76759 0.133088 Gene Fold STDEV Change
TH 0.765825 0.161472
POU2F1 0.765584 0.056692
H 0.764332 0.055399
L0XL1 0.763227 0.091752
BAZ1A 0.763213 0.083356
CYP7A1 0.760439 0.093737
HDAC10 0.759193 0.076176
DUSP4 0.757087 0.10024
SNAI1 0.752073 0.159888
GPS2 0.751587 0.070912
BRD8 0.750801 0.03568
SRCAP 0.750639 0.032659
CD70 0.747283 0.029157
GCM1 0.742235 0.04318
BANP 0.741337 0.004149
GLI3 0.739888 0.021878
ASH2L 0.738826 0.107893
RUNX3 0.738624 0.028648
PAF1 0.738335 0.119545
AMD1 0.733483 0.175237
CYBB 0.728539 0.045344
CSK 0.727852 0.044279
HDAC1 0.726997 0.062123
SIRT1 0.725727 0.112278
HDAC7 0.724565 0.147498
SETD2 0.721119 0.024466
TUBA1C 0.717963 0.198771
HDAC3 0.717661 0.038437
MCRS1 0.713804 0.140658
EP400 0.713145 0.070764
PAEP 0.710022 0.066282
PYG02 0.706929 0.028742
CD7 0.706233 0.045954
PPARG 0.702346 0.05323
OGT 0.692176 0.039326
TAF5 0.690125 0.067591
PTPN6 0.68906 0.066074
DNMT3B 0.683883 0.044812
HDAC9 0.68266 0.047391
VIM 0.682407 0.096811
ATM 0.679982 0.013184 Gene Fold STDEV Change
P MT5 0.675565 0.020562
DSG1 0.67532 0.047286
ING3 0.672389 0.06576
CASP2 0.669253 0.132779
PWWP2A 0.669005 0.154032
EHMT1 0.666011 0.045764
YWHAE 0.663513 0.04888
ZBTB7A 0.659107 0.00532
C10orf90 0.65765 0.041874
TADA2A 0.648606 0.067196
SAP30 0.646996 0.122444
AU KA 0.643001 0.134355
BPTF 0.63983 0.080468
ZBTB16 0.634699 0.192907
PHF15 0.63377 0.101121
HDAC4 0.632405 0.257281
NSUN5 0.631909 0.072917
RAD9A 0.631834 0.036465
DNMT3A 0.628056 0.221885
0RC1 0.62 0.020559
SP3 0.617935 0.032324
EPC1 0.61629 0.158951
SMYD3 0.612742 0.090324
MEN1 0.596165 0.085111
PCNA 0.595327 0.117463
CARM1 0.594609 0.038692
MBD5 0.59023 0.059411
CTNNB1 0.589965 0.092663
TAF1 0.588343 0.00168
HDAC11 0.588213 0.029488
EGR1 0.588046 0.064759
CDKN2A 0.586904 0.042303
HES1 0.58637 0.074515
RFXANK 0.58438 0.139063
HDLBP 0.582397 0.047463
ARID4B 0.582345 0.130867
HBB 0.573392 0.066321
TAF1L 0.571685 0.099177
TRIM68 0.569824 0.202206
SKI 0.55455 0.173778
MTA1 0.547317 0.053083 Gene Fold STDEV
Change
HMGA2 0.545919 0.011029
ATG7 0.545569 0.009424
ACTB 0.540832 0.16045
MAPT 0.537573 0.18135
HOXA10 0.535626 0.014248
EFCAB6 0.533825 0.172116
HIST4H4 0.526152 0.050959
STAT1 0.525319 0.497395
WD 5 0.52448 0.194554
CTAG1B 0.512581 0.078388
PWWP2B 0.510513 0.422759
PRKCB 0.509845 0.111466
MGEA5 0.500946 0.137681
CTBP1 0.496068 0.174298
TRDMT1 0.491156 0.062165
KIAA1310 0.482535 0.121558
CBX4 0.469116 0.051187
KIAA1267 0.461575 0.059006
CBFA2T2 0.460239 0.033154
ARID4A 0.419073 0.009635
CHEK1 0.416027 0.019383
SIN3A 0.404453 0.012672
YY1 0.394037 0.082014
SIRT6 0.389797 0.011271
SAP130 0.345884 0.09479
SETD7 0.345217 0.003716
SAP30L 0.332915 0.02776
ATF2 0.315087 0.119633
KAT8 0.290235 0.116672
RBBP5 0.287091 0.031787
C12orf41 0.286897 0.14643
HCFC1 0.274921 0.015261
TBL1X 0.236665 0.095611
CTCF 0.219316 0.079747
Gene: Target of an siRNA, Fold change: Fold change in FXN mRNA expression compared to control, Stdev: standard deviation
FIG. 2 depicts a list of the genes that upon knockdown downregulated or upregulated FXN mRNA at least two-fold. These genes were analyzed using the Database for
Annotation, Visualization and Integrated Discovery (DAVID) Functional annotation tool to identify pathways that were enriched in the FXN upregulating and downregulating gene sets. Tables 7 and 8 show the pathways identified.
Table 7. Pathways identified using the FXN upregulating gene set
Figure imgf000093_0001
5 Category: Database/resource where the terms orient; Term: Enriched terms associated with the gene set; Count: Number of genes involved in the term; %: Percentage of the genes involved out of the total gene set; Pvalue: Modified Fisher Exact P- value, EASE Score; Genes: Genes involved in the term l o Table 8. Pathways identified using the FXN downregulating gene set
Figure imgf000093_0002
CBFA2T2, ATF2
GOTERM. _M F GO:0016564~transcriptio 8 36.363636 0.018617 CTBP1, SIN3A, ARI D4A, YY1,
_FAT n repressor activity 36 445 CBX4, CTCF, TBL1X,
CBFA2T2
GOTERM. .BP_ GO:0031327~negative 9 40.909090 0.046413 CTBP1, SIN3A, ARI D4A,
FAT regulation of cellular 91 359 MGEA5, CBX4, SI RT6, CTCF, biosynthetic process TBL1X, CBFA2T2
GOTERM. .BP_ GO:0009890~negative 9 40.909090 0.046413 CTBP1, SIN3A, ARI D4A,
FAT regulation of biosynthetic 91 359 MGEA5, CBX4, SI RT6, CTCF, process TBL1X, CBFA2T2
GOTERM. .BP_ GO:0010558~negative 9 40.909090 0.046413 CTBP1, SIN3A, ARI D4A,
FAT regulation of 91 359 MGEA5, CBX4, SI RT6, CTCF, macromolecule TBL1X, CBFA2T2 biosynthetic process
GOTERM. .BP_ GO:0051253~negative 8 36.363636 0.047407 CTBP1, SIN3A, ARI D4A,
FAT regulation of RNA 36 616 CBX4, SI RT6, CTCF, TBL1X, metabolic process CBFA2T2
GOTERM. .BP_ GO:0045892~negative 8 36.363636 0.047407 CTBP1, SIN3A, ARI D4A,
FAT regulation of 36 616 CBX4, SI RT6, CTCF, TBL1X, transcription, DNA- CBFA2T2
dependent
GOTERM. .cc_ GO:0005694~chromosom 7 31.818181 0.060583 SI N3A, ARID4A, CBX4,
FAT e 82 982 SETD7, SI RT6, CH EK1, CTCF
GOTERM. .BP_ GO:0010605~negative 9 40.909090 0.074797 CTBP1, SIN3A, ARI D4A,
FAT regulation of 91 395 MGEA5, CBX4, SI RT6, CTCF, macromolecule metabolic TBL1X, CBFA2T2 process
SP_PIR_KEYW phosphoprotein 19 86.363636 0.097277 CTBP1, RBBP5, ARI D4A, YY1,
ORDS 36 404 CBX4, HCFC1, SIRT6, CH EK1,
CTCF, CBFA2T2, C120RF41, ATF2, PRKCB, KIAA1310, SI N3A, KIAA1267, SAP130, MGEA5, SAP30L
GOTERM. .BP_ GO:0016481~negative 8 36.363636 0.099303 CTBP1, SIN3A, ARI D4A,
FAT regulation of 36 629 CBX4, SI RT6, CTCF, TBL1X, transcription CBFA2T2
Knockdown of the YEATS gene was found to upregulate FXN to the greatest extent under the conditions evaluated. YEATS is known to be a component of the NuA4 Histone Acetyltransferase complex, which was identified as an enriched pathway by DAVID analysis. The siRNA results for other components of the NuA4 Histone Acetyltransferase complex were examined to see if knockdown of other NuA4 Histone Acetyltransferase complex components also resulted in upregulation of FXN mRNA. FIG. 3A shows that knockdown of several of the components of the NuA4 Histone Acetyltransferase complex caused
upregulation of FXN mRNA. These results suggest that the NuA4 Histone Acetyltransferase complex is involved in downregulating FXN expression, and that knockdown of one or more components of the complex may result in FXN mRNA upregulation.
Knockdown of the histone-lysine N-methyltransferase SUV39Hlwas also found to upregulate FXN mRNA levels (Table 6, FIG. 3B). These results suggest that histone-lysine N-methyltransferases, such as SUV39H1, are also involved in downregulating FXN expression and that knockdown of one or more histone-lysine N-methyltransferases may result in FXN mRNA upregulation.
Example 2: Validation of siRNA hits in a second cell line
The same siRNA pool was tested in a second FRDA cell line (GM04078) using the same methods as described in Example 1. The summary of the data is provided in Table 9. The correlation of fold change of FXN mRNA for each siRNA target between the first and second cell lines was very high (0.85) and all the top upregulating/downregulating responders for FXN mRNA were 100% reproducible in both lines. These results indicate that the negative epigenetic regulators of FXN identified in Example 1 are capable of regulating FXN levels in multiple cell lines.
Table 9. siRNA evaluation results in GM04078 FRDA cell line
Figure imgf000095_0001
Gene Fold STDEV Change
IFNB1 2.572815 0.434231
RAPIGAP 2.555712 0.063877
MEF2C 2.547859 0.546463
SMAD3 2.534138 0.312204
CDK1 2.51694 0.108525
HNF1A 2.48174 0.706655
HNRNPU 2.477997 0.34863
SALL1 2.445191 0.611896
NFKB1 2.414303 0.449406
SATB2 2.387531 0.017553
MAPK8 2.358503 0.443562
IDH1 2.314138 0.171112
USF1 2.281319 0.203232
F0XP3 2.251071 0.577201
RERE 2.235115 0.108411
BCL11B 2.228773 0.433105
SUV39H1 2.213635 0.703845
C20orf20 2.18294 0.173146
NIPBL 2.164491 0.019096
CCND1 2.163809 0.030755
SPEN 2.158165 0.190154
RARA 2.132107 0.245035
ATXN7 2.111207 0.028972
CIRl 2.107452 0.001033
BAZ2A 2.105362 0.083562
CHD1 2.104623 0.177214
PEA15 2.104046 0.456311
TADA1 2.079549 1.081316
IFNG 2.068108 0.504758
YWHAE 2.055071 0.920124
NC0A2 2.045579 0.132251
GMNN 2.035796 0.188315
ZMYM3 2.025216 0.282962
H2AFZ 2.021116 0.241134
SPI1 2.014361 0.244248
NC0A1 2.000822 0.18508
CREBBP 1.987823 0.666173
RC0R1 1.986894 0.345896
MEAF6 1.984914 0.31389
CDY1 1.975774 0.300964
KIR2DL4 1.972702 0.134292 Gene Fold STDEV Change
SMYD2 1.972411 0.218997
RBI 1.971005 0.177512
TCF21 1.970225 0.547094
KRAS 1.96191 0.414211
GSTP1 1.960572 0.171787
TERT 1.945413 0.339613
GSG2 1.94361 0.222426
IFI16 1.936336 0.155478
TRRAP 1.935236 0.125117
CDY1B 1.928019 0.504413
KAT5 1.924108 0.207072
UHRFIBPI 1.898266 0.163547
MBD2 1.896313 0.788364
TAF7 1.894265 0.331599
EP300 1.887058 0.33762
NKAP 1.880505 0.718167
USP22 1.878313 0.404658
MLL3 1.877268 0.289596
PLA2G16 1.866196 0.031098
SRCAP 1.864067 0.435432
CTR9 1.862841 0.006391
RUVBL2 1.860471 0.242777
MLL2 1.858536 0.403066
SETDB2 1.846362 0.105822
CDY2B 1.840375 3.3E-15
E2F1 1.834249 0.197402
NFYB 1.833774 0.542814
ATAD2 1.8298 0.104873
RFC1 1.828699 0.187891
JAK2 1.827241 0.378771
CDY2A 1.823435 0.170451
SP2 1.820392 0.082948
HNF4A 1.77444 0.780363
HIF1A 1.771572 0.549254
MEF2A 1.76059 0.022435
ZNF217 1.758519 0.203813
BAZ1B 1.75718 0.958769
PRKAB1 1.743587 0.022219
TAF6L 1.743031 0.473319
BIRC5 1.729221 0.224809
EED 1.728013 0.184284 Gene Fold STDEV Change
IL24 1.724212 0.087851
PADI4 1.724158 0.435553
ELP4 1.718547 0.18495
TAF12 1.716845 0.216524
NCOR2 1.713332 0.339531
MICB 1.706158 0.485012
PRDM1 1.697253 0.277432
RBPJ 1.696552 0.093915
EPC2 1.685061 0.134478
TFCP2 1.68223 0.103822
SETD1A 1.681001 0.119366
BRMS1 1.673389 0.616959
BCAS3 1.671848 0.137507
KLF14 1.670757 0.004094
M0RF4L1 1.669224 0.458002
IDH2 1.665195 0.038356
CITED2 1.664975 0.062006
MTA2 1.659185 0.122683
ZMYM2 1.657127 0.349069
PRMT2 1.652586 0.402557
NR2C1 1.649787 0.189604
DNMT1 1.642562 0.411042
RUVBL1 1.642233 0.153513
CDKN1A 1.635584 0.14411
CDYL 1.631171 0.276091
NSD1 1.629025 0.007984
EID1 1.626959 0.446406
HPGD 1.626224 0.249293
BRCA2 1.623737 0.266985
SETD1B 1.622057 0.230569
BRCA1 1.616865 0.038035
KAT2B 1.615289 0.154147
KRT23 1.593617 0.298943
TARDBP 1.593323 0.168367
WDR82 1.590596 0.086493
MKI67 1.587659 0.079339
MLL 1.572763 0.251758
HDAC2 1.568287 0.168779
FGFR3 1.564608 0.045238
GTF3C4 1.559982 0.083301
MLH1 1.558135 0.179829 Gene Fold STDEV Change
SALL3 1.557784 0.208577
PHF16 1.554078 0.019804
EGF 1.549861 0.277291
BRPF3 1.549197 0.319648
RECK 1.546451 0.356814
MSH2 1.541732 0.256483
PTGS2 1.541399 0.320257
BAZ2B 1.541001 0.492843
CD4 1.539739 0.088248
HDAC8 1.534718 0.126978
KAT6B 1.524366 0.154392
T0P2B 1.519738 0.04543
NC0A3 1.515915 0.234586
ZNF148 1.515718 0.002972
EHMT2 1.507618 0.10558
CYP7A1 1.501413 0.106614
SUV420H2 1.499707 0.218269
PKN1 1.495558 0.315035
CLGN 1.489705 0.214646
DCC 1.489164 0.003649
TAF5L 1.484746 0.036382
TNFRSF9 1.473452 0.034662
HDAC5 1.470134 0.196127
NAT10 1.462026 0.694932
N0C2L 1.451464 0.09384
MAGEA2 1.448303 0.320333
CBX5 1.43789 0.249626
NUDT21 1.434775 0.203947
PML 1.425367 0.043307
TFAP4 1.418233 0.454898
GABPA 1.416678 0.533298
ING2 1.413519 0.040177
SUV420H1 1.411008 0.082251
PR0M1 1.408753 0.071088
DOT1L 1.407563 0.033112
NR3C1 1.407268 0.087541
CASP8 1.405206 0.039253
PRKCA 1.404985 0.055076
ING5 1.404806 0.283788
SLC2A4 1.394821 0.214496
C16orf53 1.392853 0.18717 Gene Fold STDEV Change
CLOCK 1.387177 0.1655 UNXITI 1.383064 0.123211
CSK 1.382241 0.006775
CIITA 1.381362 0.116989
MYC 1.3759 0.084916
RELA 1.374278 0.138521
MGMT 1.371708 0.071233
DNMT3L 1.370523 0.417233
TUBA1B 1.369124 0.036232
CCDC101 1.359121 0.136993
MBD3 1.357939 0.068524
SETD3 1.357248 0.2859
CHRNE 1.356685 0.276021
GLI3 1.355936 0.081031
NQOl 1.350978 0.255378
AKT1 1.350557 0.016548
SENP1 1.349455 0.043645
DSG1 1.341291 0.456941
SF3B3 1.33678 0.036031
ARID1A 1.335636 0.100065
RHOB 1.335009 0.169666
SMAD7 1.332608 0.035266
TGFB1 1.328402 0.030598
KAT7 1.325775 0.355004
TUBA8 1.321374 0.163435
MECOM 1.319809 0.105974
HR 1.31965 0.056254
AES 1.317987 0.11677
PRF1 1.308304 0.089703
ANKRA2 1.305973 0.328053
CXXC1 1.304489 0.16769
KDM1A 1.301986 0.327669
TRAF6 1.299649 0.168962
SDC1 1.294594 0.000635
YEATS2 1.288982 0.188225
SP1 1.288461 0.087082
CDK2 1.288352 0.336452
CD7 1.284259 0.044682
CECR2 1.283142 0.18675
RUNX1 1.282277 0.030793
MIER1 1.281159 0.045202 Gene Fold STDEV Change
B D1 1.280489 0.153395
SOD2 1.276576 0.146712
MAGEA1 1.27447 0.278228
TALI 1.273042 0.146306
TUBA4A 1.272406 0.271077
TADA2B 1.268166 0.555467
EP400 1.265391 0.157126
HPSE 1.26532 0.003101
EDF1 1.264755 0.102171
PRDX1 1.262873 0.270256
APC 1.261094 0.027812
DEK 1.259664 0.133109
PKN2 1.257164 1.030992
RAD9A 1.252458 0.033759
NKX3-1 1.249019 0.088081
RFXANK 1.246019 0.119515
HIC2 1.242777 0.067579
PHF21A 1.241645 0.032859
BRD8 1.241255 0.250153
MTF1 1.239474 0.133996
VDR 1.237271 0.095719
SETDB1 1.236989 0.305437
IL5 1.235128 0.313773
CHD3 1.229314 0.075268
CSRP2BP 1.223999 0.12218
EZH2 1.221456 0.081359
ABCC1 1.220525 0.002991
MYB 1.213922 0.026772
LATS1 1.207816 0.040248
EHMT1 1.206821 0.356664
UHRF1 1.2045 0.199216
BANP 1.199461 0.097482
DIP2B 1.195987 0.195495
BMI1 1.194471 0.085986
SUPT7L 1.189915 0.191049
ZBTB16 1.189615 0.159281
F3 1.188948 0.068143
NC0R1 1.18696 0.083124
N6AMT2 1.186816 0.048267
DNMT3B 1.186363 0.176691
HIPK2 1.173761 0.02301 Gene Fold STDEV Change
MYOCD 1.17372 0.018408
ES 1 1.171536 0.155722
RBBP4 1.169651 0.077336
BCOR 1.164514 0.125892
PAX6 1.159433 0.072691
MMP9 1.159397 0.071555
FES 1.156963 0.126767
ANKRD1 1.155667 0.028885
SUV39H2 1.155644 0.079236
LEOl 1.152133 0.152024
CSNK2A1 1.146438 0.351157
YWHAB 1.141826 0.125109
E2F6 1.141423 0.241531
WNK4 1.140949 0.029076
PHF17 1.13958 0.094842
CYBB 1.136424 0.119533
EZH1 1.135539 0.036727
PHF15 1.132499 0.073218
CDK11A 1.131643 0.057105
JUN 1.129327 0.071908
DPY30 1.126064 0.067294
TRIM29 1.116065 0.345501
CXADR 1.112672 0.009816
ARRB1 1.112455 0.077363
SUN1 1.111646 0.076219
MAP2K4 1.110548 0.030478
PYG02 1.110056 0.176079
PAXIP1 1.109915 0.03916
TUBA1A 1.101101 0.275545
KAT6A 1.100018 0.042044
CXCR4 1.09997 0.070039
TH 1.097428 0.045707
ASH2L 1.096056 0.07783
EFCAB6 1.089647 0.234229
SP3 1.085129 0.034565
SAP30 1.082599 0.120729
PRMT1 1.081514 0.237139
DDX53 1.079228 0.001058
DAXX 1.079173 0.154447
TGFBR1 1.072997 0.021561
VIM 1.072353 0.129505 Gene Fold STDEV Change
CDC73 1.072147 0.002627
CCNB1 1.067277 0.278213
VEGFA 1.063288 0.1051
CBX4 1.060953 0.122965
PTPN6 1.059981 0.118723
CD70 1.059567 0.241441
A ID4B 1.059367 0.248975
HDAC11 1.058263 0.346318
ETV6 1.055826 0.076005
PRKDC 1.055754 0.201604
SUZ12 1.055297 0.071324
NRIP1 1.046621 0.119775
CXCL12 1.044301 0.010749
JARID2 1.041966 0.485277
NSUN5 1.041342 0.121705
NFE2L2 1.041249 0.049485
HMGA2 1.038839 0.158746
TUBA3C 1.038519 0.095052
AURKB 1.034848 0.06488
TAF10 1.033087 0.216131
ABCB1 1.028707 0.254008
RBBP7 1.025944 0.242586
PRDM14 1.023945 0.020073
PWWP2B 1.02175 0.245001
MGEA5 1.021146 0.342213
BRMS1L 1.020885 0.117824
NFKB2 1.020123 0.088886
TBL1XR1 1.019141 0.105209
EPAS1 1.019089 0.11712
HDAC3 1.015263 0.134457
IL13 1.010273 0.161229
PEX14 1.007146 0.079884
SIRT2 1.006013 0.067993
RELN 1.005239 0.010346
CASP2 1.004814 0.09785
HIST4H4 1.003723 0.116332
GADD45A 1.003043 0.049633
SIRT1 0.993451 0.156621
PAF1 0.992577 0.252626
KPNA2 0.992185 0.144404
PRMT5 0.99125 0.070388 Gene Fold STDEV Change
HDAC9 0.988737 0.29638
MC S1 0.988526 0.27915
CDK5R1 0.984716 0.180926
WHSC1 0.984353 0.057862
BHLHE41 0.980776 0.089288
ULBP2 0.980401 0.080637
SAP18 0.977365 0.336118
TPPP 0.977044 0.120371
KDM4A 0.975707 0.216738
P MT7 0.974304 0.080135
0RC1 0.972961 0.227276
TUBA1C 0.958293 0.009863
HDAC1 0.954666 0.037891
SUPT3H 0.954629 0.005615
SIN3B 0.954596 0.034148
AR 0.954527 0.153256
SMYD3 0.953981 0.062143
HRAS 0.952721 0.03968
DNMT3A 0.949286 0.077614
PHB 0.944421 0.0786
POU2F1 0.943371 0.205946
TOP2A 0.941112 0.177447
MED24 0.939721 0.118972
HAT1 0.938107 0.133348
MBD5 0.937029 0.053246
PAEP 0.93251 0.087624
TGFBR2 0.931022 0.164776
HOXA10 0.92795 0.242772
EPC1 0.924411 0.272768
PRMT6 0.918015 0.123808
BPTF 0.917084 0.051215
BRPF1 0.916583 0.081207
AMD1 0.914359 0.043902
TADA2A 0.913515 0.001343
N6AMT1 0.912432 0.153556
H2AFX 0.909985 0.074401
TNFSF10 0.90863 0.041848
PHF20 0.907036 0.132451
CARM1 0.906589 0.164824
C10orf90 0.904815 0.108391
GCM1 0.895003 0.093703 Gene Fold STDEV Change
GATA3 0.889519 0.132912
MIF 0.887288 0.202184
MLL4 0.884682 0.18084
ATG7 0.884404 0.257644 UNX2 0.878571 0.179591
CDKN2A 0.876777 0.04081
TRDMT1 0.874763 0.316913
PRKCB 0.865363 0.027988
ELP3 0.852046 0.002506
SNAI1 0.848042 0.47434
NFYA 0.846109 0.069178
SKI 0.840185 0.317046
STAT3 0.837697 0.000411
TAF1L 0.828767 0.092826
EGR1 0.825164 0.319625
PCNA 0.822489 0.032645
HDAC7 0.81853 0.062524
ATM 0.818324 0.130596
PWWP2A 0.817376 0.200661
GPS2 0.814992 0.130853
SIRT6 0.81375 0.163215
MLL5 0.812817 0.00239
HDAC6 0.810317 0.172632
OGT 0.807573 0.272133
HDAC10 0.801948 0.060865
HDLBP 0.79396 0.150848
HINFP 0.792256 0.097991
MEN1 0.791006 0.103989
PDK4 0.789981 0.081937
CBFA2T2 0.787079 0.059737
TBL1X 0.780768 0.370097
MTA1 0.775538 0.133515
LOXL1 0.773899 0.034506
DUSP4 0.772888 0.046944
SPOP 0.769197 0.026385
CAMTA2 0.768023 0.053034
CTNNB1 0.765634 0.082771
HEY2 0.752609 0.039083
SUDS3 0.752453 0.075844
CTAG1B 0.747344 0.023073
PPARG 0.736318 0.024536 Gene Fold STDEV
Change
ING4 0.73434 0.005399
KIAA1310 0.728351 0.138733
SETD7 0.724616 0.168574
NFYC 0.722623 0.236815
SETD2 0.719231 0.02749
NFATC1 0.714875 0.19817
KAT8 0.713947 0.023441
HDAC4 0.704703 0.141005
BAZ1A 0.704298 0.066868
HBB 0.687953 0.233731
ACTB 0.67941 0.190259
T IM68 0.659491 0.022622
CHEK1 0.647456 0.039642
HMG20B 0.6231 0.130657
RBBP5 0.609692 0.01255
TAF5 0.602427 0.048666
ZBTB7A 0.598118 0.001466
YY1 0.585861 0.275667
KIAA1267 0.584697 0.076867
ATF2 0.573167 0.005057
ING3 0.567088 0.011395
MAPT 0.559047 0.103793
AU KA 0.558069 0.097153
SETD8 0.554762 0.035052
ARID4A 0.55208 0.040552
STAT1 0.54645 0.042275
C12orf41 0.538001 0.078301
TAF1 0.523895 0.025411
HES1 0.506581 0.206968
CTBP1 0.473647 0.187538
WDR5 0.462155 0.016306
SAP30L 0.457775 0.119981
HCFC1 0.410236 0.062887
SAP130 0.331476 0.085623
SIN3A 0.309358 0.015005
CTCF 0.164847 0.071473
RUNX3 No data No data
Gene: Target of an siRNA, Fold change: Fold change in FXN mRNA expression compared to control, Stdev: standard deviation Example 3: Upregulation of FXN expression in cells treated with a histone lysine methyltransferase inhibitor
Epigenetic inhibitors
A screen of a library of eighty epigenetic inhibitors (Cayman Chemical) was
5 performed in GM03816 FRDA fibroblasts to identify epigenetic regulators that upregulate FXN expression. The results of the screen are provided in FIG. 4 and Tables 10 and 11. The data shows FXN mRNA fold changes in response to 1 μΜ and 5 μΜ inhibitor treatment following 3 days of treatment.
FXN RNA measurements in cells treated with histone lysine methyltransferase inhibitors o GM03816 and GM04078 cells were plated at 4000 cells/well. Sarsero mouse model derived fibroblasts were plated at 6000/well. Sarsero mouse model (B6.Cg-Tg(FXN)lSars FxntmlMkn/J; see catalog from The Jackson Laboratory atjaxmice.jax.org/strain/008586.html) was generated by inserting a human BAC containing FXN genomic region with repeat expansion into mouse genome. The resulting Sarsero mouse model and cell lines derived5 from it expressed mouse FXN and human FXN mRNAs. The histone lysine methyltranferase inhibitor, 2-(Hexahydro-4-methyl-lH-l,4-diazepin-l-yl)-6,7-dimethoxy-N-(l- (phenylmethyl)-4-piperidinyl)-4-quinazolinamine, was dissolved in DMSO and cells were treated at various concentrations and times shown in FIG. 5A-5E. Cells-to-Ct (Life
Technologies) procedure was used to analyze RNA levels of FXN following manufacturer's o protocol. The Taqman probes used were from Life Technologies are: FXN Hs00175940_ml,
Actin Hs01060665_gl, Gapdh Hs02758991_gl, Gusb Hs00939627_ml, PPIB
Hs00168719_ml, HPRT1 Hs01003267_ml.
FXN protein measurements in cells treated with histone lysine methyltransferase inhibitors5 Human FRDA diseased cell lines GM03816 and GM04078 were plated at 150000 cells/well. The cells were treated at various concentrations and times with a histone lysine methyltransferase inhibitor dissolved in DMSO. The antibody used for detection of FXM protein was Abeam human FXN antibody (ab48281). FIG. 6 shows that 3 days of HLMi treatment of human FRDA diseased cell lines GM03816 and GM04078 result in FXN human 0 protein upregulation. Table 10: Data from screen using GM03816 FRDA diseased fibroblasts
Figure imgf000108_0001
Agent 3816 5uM stdev 3816 luM stdev
Chidamide 0.967882215 0.165489129 0.924187158 0.032437385
Tricho statin A 0.692090238 0.209259791 0.93376589 0.17184603
2-PCPA (hydrochloride) 0.878612395 0.086658859 1.057774688 0.02611407
Sirtinol 0.919798649 0.078151446 1.055841853 0.015847805
(-)-Neplanocin A 0.94296969 0.002108911 1.067286134 0.071807707
Zebularine 1.092659413 0.043133835 1.067746106 0.235460396
AG-014699 0.892105436 0.114867995 1.06102006 0.219749097
GSK-J4 (hydrochloride) 0.876192868 0.06583111 0.927049179 0.057838291
CAY 10603 0.070044821 0.054894784 0.520549919 0.085497828
Pimelic Diphenylamide 106 0.485794097 0.090529435 0.745731428 0.072483905
3-Deazaneplanocin A 0.210248073 0.083585131 0.151359548 0.027764356
CAY10398 0.233878822 0.156585213 0.692101588 0.031409382
1 -Naphthoic Acid 0.868571662 0.46279875 1.054960967 0.052008788
C646 0.579816754 0.035944263 0.871311642 0.044661075
Cl-Amidine 0.902809074 0.175193443 1.152685863 0.264103724
Delphinidin chloride 0.884641057 0.063871379 0.953502066 0.035334428
IOX1 0.872237598 0.00494326 1.271688367 0.350500763
GSK-J5 (hydrochloride) 0.945088335 0.089468976 1.183072281 0.212452207
Chaetocin 0.777759373 0.132934472 0.930191108 0.065426579
(S)-HDAC-42 0.189368291 0.037760292 0.241864641 0.095041055
N- Oxalylglycine 1.034288774 0.313210364 0.707956911 0.167271222
2,4-Pyridinedicarboxylic
Acid 0.887726645 0.17653368 0.884632145 0.027040586
Nicotinamide 0.775449369 0.068537188 0.852389108 0.039413998
Tubastatin A 0.919227107 0.022271163 0.927598152 0.042551002
F-Amidine 0.648423217 0.056320466 0.781768651 0.001388982
PFI-1 0.922957467 0.205213618 0.917510237 0.217110895
MI-2 0.873563465 0.146408285 0.966216849 0.135292559
Valproic acid 1.00366769 0.000706899 0.900512098 0.153127208
Splitomicin 0.938571429 0.081980311 0.957073894 0.205902804
MS-275 0.233294275 0.039550745 0.226635876 0.14653073
AMI-1 0.565963811 0.000433569 0.55791848 0.119762235
CAY 10433 0.882676256 0.151293133 0.830938143 0.000967259
Sinefungin 0.861913953 0.176780362 0.894909785 0.001918967
Garcinol 1.094467111 0.056066205 1.008756637 0.0273752
JGB1741 1.169911294 0.048402996 1.006483881 0.124181838
5-azacytidine 1.110725939 0.126874492 1.13359584 0.062404931
Ml-nc 0.924552089 0.077595364 1.002692191 0.207187812 tenovin- 1 UD UD UD UD
CBHA 0.132678455 0.062288615 0.467814763 0.061188269 Agent 3816 5uM stdev 3816 luM stdev
RG-108 0.89656673 0.069990726 1.001076646 0.02581795
UNC1215 0.874283351 0.043449042 0.691060773 0.220171187
Picetannol 0.543519173 0.137963482 0.786661105 0.001686848
Suramin 0.835317044 0.142772218 0.954129468 0.033488309
UD=undetermined
Table 11: Data from screen using GM0321 normal fibroblasts
Figure imgf000110_0001
Agent 321B 5uM stdev 321B luM stdev
4-iodo-SAHA 0.590087532 0.242476585 0.667981929 0.152571564
UNC0321 (trifluoroacetate
0.605016162 0.034453671 0.751678309 0.046343556 salt)
CAY 10669 0.30939022 0.394654889 0.779613873 0.005299845
BSI-201 0.985335599 0.139941948 1.027793404 0.046394853
GSK-J2 (sodium salt) UD UD 1.062992432 0.187553234
AGK2 1.052526568 0.242975783 1.115129842 0.217839055
Mirin 1.305145804 1.197784414 1.078594045 0.148150954
Chidamide 0.79851937 0.0065555 0.835394192 0.203443672
Tricho statin A 0.772139968 0.41928721 1.022984586 0.084573946
2-PCPA (hydrochloride) 0.788826909 0.039516071 0.714929222 0.053957012
Sirtinol 1.024038665 0.229799104 0.809656129 0.256257926
(-)-Neplanocin A 1.104954517 0.228649677 0.873369703 0.062499549
Zebularine 1.030528935 0.005429716 1.070144275 0.125660328
AG-014699 1.000307564 0.112404334 0.89512822 0.04347404
GSK-J4 (hydrochloride) 0.874380992 0.161812285 1.001650686 0.323202006
CAY 10603 UD UD 0.359151916 0.124031472
Pimelic Diphenylamide 106 0.209357829 0.103834077 0.694010453 0.178324186
3-Deazaneplanocin A 0.126529782 0.075077401 0.264148931 0.061938623
CAY10398 0.210602925 0.157813128 0.504871673 0.169165631
1 -Naphthoic Acid 0.719662274 0.150818584 0.819305969 0.017213854
C646 1.194906621 0.00951685 0.9473841 0.173551083
Cl-Amidine 0.993094863 0.112319322 0.943203062 0.291605431
Delphinidin chloride 1.036030794 0.2792543 1.087397481 0.100124886
IOX1 0.718470223 0.119958931 0.947879144 0.033846977
GSK-J5 (hydrochloride) 0.974105226 0.452288393 0.859683667 0.325237732
Chaetocin 0.995203166 0.21904671 0.908355762 0.005729835
(S)-HDAC-42 0.69412966 0.551769336 UD UD
N- Oxalylglycine 0.680352211 0.307987658 UD UD
2,4-Pyridinedicarboxylic
0.645136143 0.061643985 0.458469394 0.112579758 Acid
Nicotinamide 0.701881607 0.206529425 0.686626406 0.171214858
Tubastatin A 1.037805434 0.111309254 0.899268368 0.344604097
F-Amidine 0.969736884 0.117230202 0.915167018 0.054748715
PFI-1 0.907082488 0.100382273 0.957719401 0.126896814
MI-2 0.796570274 0.20585434 0.880135212 0.347344616
Valproic acid 0.846928872 0.426766495 1.263618913 0.42865371
Splitomicin 1.005944436 0.545406716 1.082820732 0.174970499
MS-275 0.271301857 0.096951699 UD UD
AMI-1 0.407259491 0.094404401 UD UD Agent 321B 5uM stdev 321B luM stdev
CAY 10433 0.778212312 0.187992917 1.03043719 0.643456098
Sinefungin 0.821339027 0.302536905 0.550738646 0.507012577
Garcinol 1.327373705 0.403942211 0.629526279 0.057578097
JGB1741 0.722420283 0.164874607 0.996271558 0.197368868
5-azacytidine 0.753502134 0.516661665 1.045672178 0.124313534
Ml-nc 1.043599944 0.17903364 0.613264168 0.386029314 tenovin- 1 UD UD 1.444268369 0.10811216
CBHA 0.48745022 0.185880937 0.720833131 0.299071952
RG-108 0.622247164 0.398108985 UD UD
UNC1215 UD UD UD UD
Picetannol 0.537994883 0.205766438 0.500760875 0.09776034
Suramin 0.772191365 0.146805196 UD UD
UD=undetermined
Example 4. Data for gapmers to JunD, YEATS4, HICl, ACTL6A, EIDl, IDHl, TNFSF9,
JAK2, KAT2A and PRKCD
Gapmers for human JunD, YEATS4, HICl, ACTL6A, EIDl, IDHl, TNFSF9, JAK2, KAT2A and PRKCD were designed against the genes identified within the epigenetic siRNA screen, whose knockdown was hypothesized to lead to FXN mRNA upregulation. The oligo sequences are shown in Table 12.
Table 12. Gapmers targeting various genes identified with the epigenetic siRNA screen.
Figure imgf000112_0001
JUND-04 GGAACTGTGAG JUND human lnaGs;lnaGs;lnaAs;dAs;dCs;dTs;dGs;d m08 CTCG Ts;dGs;dAs;dGs;dCs;lnaTs;lnaCs;lnaG
95 -Sup
JUND-05 GCTCGTCCTTGA JUND human lnaGs;lnaCs;lnaTs;dCs;dGs;dTs;dCs;d m08 GCG Cs;dTs;dTs;dGs;dAs;lnaGs;lnaCs;lnaG
96 -Sup
JUND-06 TGGCTCGTCCTT JUND human lnaTs;lnaGs;lnaGs;dCs;dTs;dCs;dGs;d m08 GAG Ts;dCs;dCs;dTs;dTs;lnaGs;lnaAs;lnaG-
97 Sup
JUND-07 CCCGTTGGACT JUND human lnaCs;lnaCs;lnaCs;dGs;dTs;dTs;dGs;d m08 GGAT Gs;dAs;dCs;dTs;dGs;lnaGs;lnaAs;lnaT
98 -Sup
JUND-08 CGCTCCGCCTTG JUND human lnaCs;lnaGs;lnaCs;dTs;dCs;dCs;dGs;d m08 ATG Cs;dCs;dTs;dTs;dGs;lnaAs;lnaTs;lnaG-
99 Sup
JUND-09 CACCTGCTCGC JUND human lnaCs;lnaAs;lnaCs;dCs;dTs;dGs;dCs;d m08 GCAG Ts;dCs;dGs;dCs;dGs;lnaCs;lnaAs;lnaG
100 -Sup
HICl-01 GGCCGGTGTAG HICl human lnaGs;lnaGs;lnaCs;dCs;dGs;dGs;dTs;d m08 ATG A Gs;dTs;dAs;dGs;dAs;lnaTs;lnaGs;lnaA
101 -Sup
HICl-02 TGACCGCGGCC HICl human lnaTs;lnaGs;lnaAs;dCs;dCs;dGs;dCs;d m08 TCTG Gs;dGs;dCs;dCs;dTs;lnaCs;lnaTs;lnaG
102 -Sup
HICl-03 TTGACCGCGGC HICl human lnaTs;lnaTs;lnaGs;dAs;dCs;dCs;dGs;d m08 CTCT Cs;dGs;dGs;dCs;dCs;lnaTs;lnaCs;lnaT-
103 Sup
HICl-04 TACCGGTCTCCT HICl human lnaTs;lnaAs;lnaCs;dCs;dGs;dGs;dTs;d m08 CGC Cs;dTs;dCs;dCs;dTs;lnaCs;lnaGs;lnaC-
104 Sup
HICl-05 ACGTACAGGTT HICl human lnaAs;lnaCs;lnaGs;dTs;dAs;dCs;dAs;d m08 GTCA Gs;dGs;dTs;dTs;dGs;lnaTs;lnaCs;lnaA
105 -Sup
- Ill - HICl-06 ACACGTACAGG HICl human lnaAs;lnaCs;lnaAs;dCs;dGs;dTs;dAs;d m08 TTGT Cs;dAs;dGs;dGs;dTs;lnaTs;lnaGs;lnaT
106 -Sup
HICl-07 TCTTGTCGCACG HICl human lnaTs;lnaCs;lnaTs;dTs;dGs;dTs;dCs;d m08 ACG Gs;dCs;dAs;dCs;dGs;lnaAs;lnaCs;lnaG
107 -Sup
HICl-08 AGCTCTTGTCGC HICl human lnaAs;lnaGs;lnaCs;dTs;dCs;dTs;dTs;d m08 ACG Gs;dTs;dCs;dGs;dCs;lnaAs;lnaCs;lnaG
108 -Sup
HICl-09 CCGCACGCGTC HICl human lnaCs;lnaCs;lnaGs;dCs;dAs;dCs;dGs;d m08 GCAC Cs;dGs;dTs;dCs;dGs;lnaCs;lnaAs;lnaC
109 -Sup
HICl-10 TGTGCGAACTT HICl human lnaTs;lnaGs;lnaTs;dGs;dCs;dGs;dAs;d m08 GCCG As;dCs;dTs;dTs;dGs;lnaCs;lnaCs;lnaG
110 -Sup
HICl-11 GCTGTGCGAAC HICl human lnaGs;lnaCs;lnaTs;dGs;dTs;dGs;dCs;d m08 TTGC Gs;dAs;dAs;dCs;dTs;lnaTs;lnaGs;lnaC
111 -Sup
HICl-12 TCGAGCTTGCC HICl human lnaTs;lnaCs;lnaGs;dAs;dGs;dCs;dTs;d m08 CTTG Ts;dGs;dCs;dCs;dCs;lnaTs;lnaTs;lnaG-
112 Sup
HICl-13 AGAAACGGTCG HICl human lnaAs;lnaGs;lnaAs;dAs;dAs;dCs;dGs;d m08 ATGG Gs;dTs;dCs;dGs;dAs;lnaTs;lnaGs;lnaG
113 -Sup
YEATS4- TCGGCCATTCTC YEATS4 human lnaTs;lnaCs;lnaGs;dGs;dCs;dCs;dAs;d 01 m08 TTG Ts;dTs;dCs;dTs;dCs;lnaTs;lnaTs;lnaG-
114 Sup
YEATS4- ATTCGGCCATTC YEATS4 human lnaAs;lnaTs;lnaTs;dCs;dGs;dGs;dCs;d 02 m08 TCT Cs;dAs;dTs;dTs;dCs;lnaTs;lnaCs;lnaT-
115 Sup
YEATS4- CCCGCCGGAGT YEATS4 human lnaCs;lnaCs;lnaCs;dGs;dCs;dCs;dGs;d 03 m08 CAGG Gs;dAs;dGs;dTs;dCs;lnaAs;lnaGs;lnaG
116 -Sup YEATS4- ATATGGAGGTT YEATS4 human lnaAs;lnaTs;lnaAs;dTs;dGs;dGs;dAs;d 04 m08 TAGT Gs;dGs;dTs;dTs;dTs;lnaAs;lnaGs;lnaT
-Sup
YEATS4- TCGAATTCACCC YEATS4 human lnaTs;lnaCs;lnaGs;dAs;dAs;dTs;dTs;d 05 m08 CAT Cs;dAs;dCs;dCs;dCs;lnaCs;lnaAs;lnaT-
Sup
YEATS4- TTCGAATTCACC YEATS4 human lnaTs;lnaTs;lnaCs;dGs;dAs;dAs;dTs;d 06 m08 CCA Ts;dCs;dAs;dCs;dCs;lnaCs;lnaCs;lnaA-
Sup
YEATS4- TGACGAGATGT YEATS4 human lnaTs;lnaGs;lnaAs;dCs;dGs;dAs;dGs;d 07 m08 TGTC As;dTs;dGs;dTs;dTs;lnaGs;lnaTs;lnaC- Sup
YEATS4- TAGCTGACGAG YEATS4 human lnaTs;lnaAs;lnaGs;dCs;dTs;dGs;dAs;d 08 m08 ATGT Cs;dGs;dAs;dGs;dAs;lnaTs;lnaGs;lnaT
-Sup
YEATS4- AGTTTCACGACT YEATS4 human lnaAs;lnaGs;lnaTs;dTs;dTs;dCs;dAs;d 09 m08 TGC Cs;dGs;dAs;dCs;dTs;lnaTs;lnaGs;lnaC
-Sup
ACTL6A- GGCAACAAAGC ACTL6A human lnaGs;lnaGs;lnaCs;dAs;dAs;dCs;dAs;d 01 m08 GGCG As;dAs;dGs;dCs;dGs;lnaGs;lnaCs;lna
G-Sup
ACTL6A- ATCGCCATCTAT ACTL6A human lnaAs;lnaTs;lnaCs;dGs;dCs;dCs;dAs;d 02 m08 TTC Ts;dCs;dTs;dAs;dTs;lnaTs;lnaTs;lnaC- Sup
ACTL6A- GAACGACCATT ACTL6A human lnaGs;lnaAs;lnaAs;dCs;dGs;dAs;dCs;d 03 m08 AGCA Cs;dAs;dTs;dTs;dAs;lnaGs;lnaCs;lnaA
-Sup
ACTL6A- GCCCAGTAGAA ACTL6A human lnaGs;lnaCs;lnaCs;dCs;dAs;dGs;dTs;d 04 m08 CGAC As;dGs;dAs;dAs;dCs;lnaGs;lnaAs;lnaC
-Sup
ACTL6A- CATCGTGGACT ACTL6A human lnaCs;lnaAs;lnaTs;dCs;dGs;dTs;dGs;d 05 m08 GGAA Gs;dAs;dCs;dTs;dGs;lnaGs;lnaAs;lnaA
-Sup ACTL6A- TTTCAACCGCAT ACTL6A human lnaTs;lnaTs;lnaTs;dCs;dAs;dAs;dCs;d 06 m08 ACT Cs;dGs;dCs;dAs;dTs;lnaAs;lnaCs;lnaT-
Sup
ACTL6A- GAGCTAAACCT ACTL6A human lnaGs;lnaAs;lnaGs;dCs;dTs;dAs;dAs;d 07 m08 CCGT As;dCs;dCs;dTs;dCs;lnaCs;lnaGs;lnaT- Sup
ACTL6A- GAGCCGCCAAT ACTL6A human lnaGs;lnaAs;lnaGs;dCs;dCs;dGs;dCs;d 08 m08 CCAT Cs;dAs;dAs;dTs;dCs;lnaCs;lnaAs;lnaT- Sup
EIDl-01 AAATTCCTCGCC EID1 human lnaAs;lnaAs;lnaAs;dTs;dTs;dCs;dCs;d m08 CTC Ts;dCs;dGs;dCs;dCs;lnaCs;lnaTs;lnaC-
Sup
EID1-02 CGTAGTCGTCCT EID1 human lnaCs;lnaGs;lnaTs;dAs;dGs;dTs;dCs;d m08 CCC Gs;dTs;dCs;dCs;dTs;lnaCs;lnaCs;lnaC- Sup
EID1-03 CTGAAACCCGC EID1 human lnaCs;lnaTs;lnaGs;dAs;dAs;dAs;dCs;d m08 CATC Cs;dCs;dGs;dCs;dCs;lnaAs;lnaTs;lnaC- Sup
EID1-04 AGCTCTTCGATA EID1 human lnaAs;lnaGs;lnaCs;dTs;dCs;dTs;dTs;d m08 AAA Cs;dGs;dAs;dTs;dAs;lnaAs;lnaAs;lnaA
-Sup
EID1-05 TCGGTCAGACG EID1 human lnaTs;lnaCs;lnaGs;dGs;dTs;dCs;dAs;d m08 ATTG Gs;dAs;dCs;dGs;dAs;lnaTs;lnaTs;lnaG
-Sup
EID1-06 CTCATCACAGCC EID1 human lnaCs;lnaTs;lnaCs;dAs;dTs;dCs;dAs;d m08 GAG Cs;dAs;dGs;dCs;dCs;lnaGs;lnaAs;lnaG
-Sup
I DH l-01 ACGATTCTCTAT I DH 1 human lnaAs;lnaCs;lnaGs;dAs;dTs;dTs;dCs;d m08 GCC Ts;dCs;dTs;dAs;dTs;lnaGs;lnaCs;lnaC- Sup
I DH 1-02 TGGCATCACGA I DH 1 human lnaTs;lnaGs;lnaGs;dCs;dAs;dTs;dCs;d m08 TTCT As;dCs;dGs;dAs;dTs;lnaTs;lnaCs;lnaT- Sup I DH 1-03 TCAATTG ACTTA I DH 1 human lnaTs;lnaCs;lnaAs;dAs;dTs;dTs;dGs;d m08 TCT As;dCs;dTs;dTs;dAs;lnaTs;lnaCs;lnaT-
139 Sup
I DH 1-04 ACGCCCATCAT I DH 1 human lnaAs;lnaCs;lnaGs;dCs;dCs;dCs;dAs;d m08 ATTT Ts;dCs;dAs;dTs;dAs;lnaTs;lnaTs;lnaT-
140 Sup
I DH 1-05 TGTCTTTAAAAC I DH 1 human lnaTs;lnaGs;lnaTs;dCs;dTs;dTs;dTs;dA m08 GCC s;dAs;dAs;dAs;dCs;lnaGs;lnaCs;lnaC-
141 Sup
I DH 1-06 TTATCAAGCTTT I DH 1 human lnaTs;lnaTs;lnaAs;dTs;dCs;dAs;dAs;d m08 GCT Gs;dCs;dTs;dTs;dTs;lnaGs;lnaCs;lnaT-
142 Sup
JAK2-01 GTCATCGTAAG JAK2 human lnaGs;lnaTs;lnaCs;dAs;dTs;dCs;dGs;d m08 GCAG Ts;dAs;dAs;dGs;dGs;lnaCs;lnaAs;lnaG
143 -Sup
JAK2-02 GGATCTTTGCTC JAK2 human lnaGs;lnaGs;lnaAs;dTs;dCs;dTs;dTs;d m08 GAA Ts;dGs;dCs;dTs;dCs;lnaGs;lnaAs;lnaA
144 -Sup
JAK2-03 TAGTCTTGGATC JAK2 human lnaTs;lnaAs;lnaGs;dTs;dCs;dTs;dTs;d m08 TTT Gs;dGs;dAs;dTs;dCs;lnaTs;lnaTs;lnaT-
145 Sup
JAK2-04 TGCGAAATCTG JAK2 human lnaTs;lnaGs;lnaCs;dGs;dAs;dAs;dAs;d m08 TACC Ts;dCs;dTs;dGs;dTs;lnaAs;lnaCs;lnaC-
146 Sup
JAK2-05 TGAATTCCACC JAK2 human lnaTs;lnaGs;lnaAs;dAs;dTs;dTs;dCs;d m08 GTTT Cs;dAs;dCs;dCs;dGs;lnaTs;lnaTs;lnaT-
147 Sup
JAK2-06 ATCGCAATATA JAK2 human lnaAs;lnaTs;lnaCs;dGs;dCs;dAs;dAs;d m08 ACTG Ts;dAs;dTs;dAs;dAs;lnaCs;lnaTs;lnaG-
148 Sup
JAK2-07 TGACATTTTCTC JAK2 human lnaTs;lnaGs;lnaAs;dCs;dAs;dTs;dTs;d m08 GCT Ts;dTs;dCs;dTs;dCs;lnaGs;lnaCs;lnaT-
149 Sup JAK2-08 TCATACCGGCA JAK2 human lnaTs;lnaCs;lnaAs;dTs;dAs;dCs;dCs;d m08 CATC Gs;dGs;dCs;dAs;dCs;lnaAs;lnaTs;lnaC
-Sup
JAK2-09 GTCTCGTAAACT JAK2 human lnaGs;lnaTs;lnaCs;dTs;dCs;dGs;dTs;d m08 TCC As;dAs;dAs;dCs;dTs;lnaTs;lnaCs;lnaC-
Sup
JAK2-10 TGATCTATCCGT JAK2 human lnaTs;lnaGs;lnaAs;dTs;dCs;dTs;dAs;d m08 TCT Ts;dCs;dCs;dGs;dTs;lnaTs;lnaCs;lnaT- Sup
KAT2A-01 AGGTCGAGCCG KAT2A human lnaAs;lnaGs;lnaGs;dTs;dCs;dGs;dAs;d m08 GATC Gs;dCs;dCs;dGs;dGs;lnaAs;lnaTs;lnaC
-Sup
KAT2A-02 CCGGACTTGCG KAT2A human lnaCs;lnaCs;lnaGs;dGs;dAs;dCs;dTs;d m08 CCTT Ts;dGs;dCs;dGs;dCs;lnaCs;lnaTs;lnaT- Sup
KAT2A-03 TGAGAGCTCGA KAT2A human lnaTs;lnaGs;lnaAs;dGs;dAs;dGs;dCs;d m08 ACAT Ts;dCs;dGs;dAs;dAs;lnaCs;lnaAs;lnaT
-Sup
KAT2A-04 CACGGAGCCGC KAT2A human lnaCs;lnaAs;lnaCs;dGs;dGs;dAs;dGs;d m08 TTGG Cs;dCs;dGs;dCs;dTs;lnaTs;lnaGs;lnaG
-Sup
KAT2A-05 CATTGACCAGC KAT2A human lnaCs;lnaAs;lnaTs;dTs;dGs;dAs;dCs;d m08 TCCA Cs;dAs;dGs;dCs;dTs;lnaCs;lnaCs;lnaA
-Sup
KAT2A-06 GGCGATATACT KAT2A human lnaGs;lnaGs;lnaCs;dGs;dAs;dTs;dAs;d m08 CCTT Ts;dAs;dCs;dTs;dCs;lnaCs;lnaTs;lnaT- Sup
KAT2A-07 CACCGATGACC KAT2A human lnaCs;lnaAs;lnaCs;dCs;dGs;dAs;dTs;d m08 CGCC Gs;dAs;dCs;dCs;dCs;lnaGs;lnaCs;lnaC
-Sup
KAT2A-08 CCATCAGCGTC KAT2A human lnaCs;lnaCs;lnaAs;dTs;dCs;dAs;dGs;d m08 GCTC Cs;dGs;dTs;dCs;dGs;lnaCs;lnaTs;lnaC-
Sup KAT2A-09 CTGTTTGCGCTC KAT2A human lnaCs;lnaTs;lnaGs;dTs;dTs;dTs;dGs;d m08 AAT Cs;dGs;dCs;dTs;dCs;lnaAs;lnaAs;lnaT-
Sup
KAT2A-10 GGCGATGACCC KAT2A human lnaGs;lnaGs;lnaCs;dGs;dAs;dTs;dGs;d m08 GCTG As;dCs;dCs;dCs;dGs;lnaCs;lnaTs;lnaG
-Sup
PRKCD-01 CATGGTCGGCT PRKCD human lnaCs;lnaAs;lnaTs;dGs;dGs;dTs;dCs;d m08 TCTT Gs;dGs;dCs;dTs;dTs;lnaCs;lnaTs;lnaT- Sup
PRKCD-02 TGCGCATAGAC PRKCD human lnaTs;lnaGs;lnaCs;dGs;dCs;dAs;dTs;d m08 TGTT As;dGs;dAs;dCs;dTs;lnaGs;lnaTs;lnaT
-Sup
PRKCD-03 GGTGGCGATAA PRKCD human lnaGs;lnaGs;lnaTs;dGs;dGs;dCs;dGs;d m08 ACTC As;dTs;dAs;dAs;dAs;lnaCs;lnaTs;lnaC- Sup
PRKCD-04 ATCTTGTCGATG PRKCD human lnaAs;lnaTs;lnaCs;dTs;dTs;dGs;dTs;d m08 CAT Cs;dGs;dAs;dTs;dGs;lnaCs;lnaAs;lnaT
-Sup
PRKCD-05 TGTTGAAGCGT PRKCD human lnaTs;lnaGs;lnaTs;dTs;dGs;dAs;dAs;d m08 TCTT Gs;dCs;dGs;dTs;dTs;lnaCs;lnaTs;lnaT- Sup
PRKCD-06 CGATGTTGAAG PRKCD human lnaCs;lnaGs;lnaAs;dTs;dGs;dTs;dTs;d m08 CGTT Gs;dAs;dAs;dGs;dCs;lnaGs;lnaTs;lnaT
-Sup
PRKCD-07 AAGCGGCCTTT PRKCD human lnaAs;lnaAs;lnaGs;dCs;dGs;dGs;dCs;d m08 GTCC Cs;dTs;dTs;dTs;dGs;lnaTs;lnaCs;lnaC- Sup
PRKCD-08 TAGAGTTCAAA PRKCD human lnaTs;lnaAs;lnaGs;dAs;dGs;dTs;dTs;d m08 GCGG Cs;dAs;dAs;dAs;dGs;lnaCs;lnaGs;lnaG
-Sup
PRKCD-09 CCCCGAAAGAC PRKCD human lnaCs;lnaCs;lnaCs;dCs;dGs;dAs;dAs;d m08 CACC As;dGs;dAs;dCs;dCs;lnaAs;lnaCs;lnaC
-Sup PRKCD-10 CACGGATGGAC PRKCD human lnaCs;lnaAs;lnaCs;dGs;dGs;dAs;dTs;d m08 TCGA Gs;dGs;dAs;dCs;dTs;lnaCs;lnaGs;lnaA
-Sup
PRKCD-11 AGTCGATGAGG PRKCD human lnaAs;lnaGs;lnaTs;dCs;dGs;dAs;dTs;d m08 TTCT Gs;dAs;dGs;dGs;dTs;lnaTs;lnaCs;lnaT
-Sup
TN FSF9- GTCAGAGGCGT TN FSF9 human lnaGs;lnaTs;lnaCs;dAs;dGs;dAs;dGs;d 01 m08 ATTC Gs;dCs;dGs;dTs;dAs;lnaTs;lnaTs;lnaC- Sup
TN FSF9- AGCAGCCCCGC TN FSF9 human lnaAs;lnaGs;lnaCs;dAs;dGs;dCs;dCs;d 02 m08 GACC Cs;dCs;dGs;dCs;dGs;lnaAs;lnaCs;lnaC
-Sup
TN FSF9- GACGGCGCAGG TN FSF9 human lnaGs;lnaAs;lnaCs;dGs;dGs;dCs;dGs;d 03 m08 CGGC Cs;dAs;dGs;dGs;dCs;lnaGs;lnaGs;lnaC
-Sup
TN FSF9- CTGAGCCCTCG TN FSF9 human lnaCs;lnaTs;lnaGs;dAs;dGs;dCs;dCs;d 04 m08 CCGG Cs;dTs;dCs;dGs;dCs;lnaCs;lnaGs;lnaG
-Sup
TN FSF9- GGTCCACGGTC TN FSF9 human lnaGs;lnaGs;lnaTs;dCs;dCs;dAs;dCs;d 05 m08 AAAG Gs;dGs;dTs;dCs;dAs;lnaAs;lnaAs;lnaG
-Sup
TN FSF9- AAACCGAAGGC TN FSF9 human lnaAs;lnaAs;lnaAs;dCs;dCs;dGs;dAs;d 06 m08 CGAG As;dGs;dGs;dCs;dCs;lnaGs;lnaAs;lna
G-Sup
TN FSF9- AGGTGCAGCAA TN FSF9 human lnaAs;lnaGs;lnaGs;dTs;dGs;dCs;dAs;d 07 m08 GCGG Gs;dCs;dAs;dAs;dGs;lnaCs;lnaGs;lna
G-Sup
TN FSF9- GTCACCCGGAA TN FSF9 human lnaGs;lnaTs;lnaCs;dAs;dCs;dCs;dCs;d 08 m08 GAGT Gs;dGs;dAs;dAs;dGs;lnaAs;lnaGs;lna
T-Sup
TN FSF9- AGTAGGATTCG TN FSF9 human lnaAs;lnaGs;lnaTs;dAs;dGs;dGs;dAs;d 09 m08 GACT Ts;dTs;dCs;dGs;dGs;lnaAs;lnaCs;lnaT- Sup The gapmers were screened in GM03816 cells via lipofection at 60nM concentration. In general, at least one gapmer from each gene caused upregulation of FXN mRNA (FIG. 7A and B).
Next, a Western blot with the Abeam ab48281 FXN and Abeam ab 125267 tubulin antibodies were run using treated and untreated GM03816 lysates. Several strong
upregulation oligos were identified, including ACTL6A-3, JUND-1, and PRKCD-2 (FIG. 8).
Subsequently, various oligos targeting ACTL6A, EID1, HICl, JUND, KAT2A, PRKCD, and YEATS4 were screened in differentiated myotubes for FXN mRNA levels. Measurements were taken 4 days after transfection. Several of the oligos showed upregulation of FXN mRNA, including ACTL6A-02, 03, 04, EID1-04, HICl-1, JUND-1, JUND-6, KAT2A-05, KAT2A-06, PRKCD-2, YEATS4-5, and YEATS4-9 (FIG. 9).
These results demonstrate that oligos targeting epigenetic silencers of FXN can be used to upregulate FXN levels.
Example 5. Data for SUV39H1 and Tip60
SUV39H1 and Tip60, as well as HDAC1, HDAC2, HDAC3, and G9a, were tested as potential drivers of FRDA epigenetic silencing. ChIP for candidate chromatin modifying enzymes that may be responsible for GAA-repeat associated silencing was done in diseased (GM03816 fibroblasts, GM16209 lymphoblasts) and normal (GM15851 lymphoblasts) cells. The antibodies used were HDAC1 ab46985, HDAC2 ab51832, HDAC3 ab96005, G9a ab40542, SUV39H1 abl2405, Tip60 ab23886, H3K27me3 ab6002, and H3K9me3 ab8898.
Enrichment obtained in each diseased line was normalized to the normal line levels. H3K27me3 and H3K9me3 enrichment patterns in disease tissue was at least partly mirrored by Tip60 and SUV39H1 patterns (FIG. 10A-D). Enrichment patterns for G9a (an H3K9 methyltranserase) were also measured (FIGs. 11A). Enrichment of IgG was used as a control (FIG. 1 IB). These data indicate that Tip60 and SUV39H1 may be involved in the FRDA epigenetic silencing.
Without further elaboration, it is believed that one skilled in the art can, based on the description provided herein, utilize the present invention to its fullest extent. The specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated
5 otherwise, each feature disclosed is only an example of a generic series of equivalent or
similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various o usages and conditions. Thus, other embodiments are also within the claims.
While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications5 is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will o recognize, or be able to ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is 5 directed to each individual feature, system, article, material, and/or method described herein.
In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present invention.
The indefinite articles "a" and "an," as used herein in the specification and in the 0 claims, unless clearly indicated to the contrary, should be understood to mean "at least one."
The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and/or B," when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another
embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and 5 the like are to be understood to be open-ended, i.e., to mean including but not limited to.
Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a l o claim element does not by itself connote any priority, precedence, or order of one claim
element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

Claims

What is claimed is: L A method for increasing FXN expression in a cell, the method comprising: delivering to a cell an oligonucleotide that inhibits expression or activity of a negative epigenetic regulator of FXN, thereby increasing FXN expression in the cell, wherein, prior to delivering, the cell has a lower level of FXN expression compared to an appropriate control level of FXN expression.
2. The method of claim 1, wherein, prior to delivering, the cell has a higher level of histone H3 K27 or K9 methylation at the FXN gene compared with an appropriate control level of histone H3 K27 or K9 methylation.
3. The method of claim 1, wherein the cell comprises an FXN gene encoding in its first intron a GAA repeat of between 10-2000 units.
4. The method of claim 1, wherein the cell is obtained from or present in a subject having Friedreich's ataxia.
5. The method of any one the preceding claims, wherein the negative epigenetic regulator of FXN is a component of a histone H2A acetylation pathway, a NuA4 histone acetyltransferase complex, a protein amino acid acetylation pathway, a histone acetylation pathway, a protein amino acid acylation pathway, a H4/H2A histone acetyltransferase complex, a nucleotide binding pathway, a histone H4 acetylation pathway, a histone acetyltransferase complex, or an insulin receptor substrate binding pathway.
6. The method of claim 5, wherein
(i) the component of the histone H2A acetylation pathway is MEAF6, YEATS4, ACTL6 A, or DM AP 1 ; or
(ii) the component of the NuA4 histone acetyltransferase complex is MEAF6, YEATS4, ACTL6A, or DMAP1; or (iii) the component of the protein amino acid acetylation pathway is KAT2A, MEAF6, YEATS4, TAD A3, ACTL6A, or DMAPl; or
(iv) the component of the histone acetylation pathway is KAT2A, MEAF6, YEATS4, TAD A3, ACTL6A, or DMAPl; or
(v) the component of the protein amino acid acylation pathway is KAT2A, MEAF6, YEATS4, TAD A3, ACTL6A, or DMAPl; or
(vi) the component of the H4/H2A histone acetyltransferase complex is MEAF6, YEATS4, ACTL6A, or DMAPl; or
(vii) the component of the nucleotide binding pathway is MEF2D, PRKDC, IDHl, ACTL6A, JAK2, CFTR, SPEN, or PRKCD; or
(viii) the component of the histone H4 acetylation pathway is MEAF6, YEATS4, ACTL6A, or DMAPl; or
(ix) the component of the histone acetyltransferase complex is KAT2A, MEAF6, YEATS4, TAD A3, ACTL6A, or DMAPl; or
(x) the component of the insulin receptor substrate binding pathway is JAK2 or PRKCD.
7. The method of any one of claims 1 to 4, wherein the negative epigenetic regulator of FXN is TNFSF9, JUND, HICl, PRKCD, JAK2, EID1, CFTR, TAD A3, o MYBL2, KAT2A, IDHl, SUMOl, SPEN, PRKDC, KIR2DL4, APC, MEF2D, a component of the NuA4 Histone Acetyltransferase Complex, or a histone-lysine N-methyltransferase.
8. The method of any one of the claims 1 to 4, wherein the negative epigenetic regulator of FXN is a component of the NuA4 Histone Acetyltransferase Complex.
5
9. The method of claim 8, wherein the component of the NuA4 Histone
Acetyltransferase Complex is YEATS4, Eafl, TRRAP, P400, EPCl, DMAPl, Tip60, MRG15, MRGX, MORF4, ACTB, ACTL6A, INGl, ING2, ING3, ING4, ING5, RUVBLl, RUVBL2, AF9, ENL, or MEAF6.
0
10. The method of claim 9, wherein the component of the NuA4 Histone
Acetyltransferase Complex is YEATS4, ACTL6A, DMAPl, or MEAF6.
11. The method of claim 9, wherein the component of the NuA4 Histone
Acetyltransferase Complex is YEATS4.
12. The method of any one of claims 1 or 4 or 7, wherein the negative epigenetic regulator of FXN is a histone-lysine N-methyltransferase.
13. The method of claim 12, wherein the histone-lysine N-methyltransferase is SUV39H1, SUV39H2, SETDB1, PRDM2, G9A and EHMT1.
14. The method of claim 12 or 13, wherein the histone-lysine N-methyltransferase is SUV39H1.
15. The method of any one of claims 1 to 4, wherein the negative epigenetic regulator of FXN is YEATS4, HICl, JUND, TNFSF9, PRKCD, KAT2A, JAK2, IDHl,
EID1, or ACTL6A.
16. The method of claim 15, wherein the oligonucleotide comprises a sequence as set for in Table 4.
17. The method of claim 15, wherein the oligonucleotide comprises a sequence as set for in Table 12.
18. The method of claims 1 to 4, wherein the negative epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change greater than
1.25.
19. The method of any one of the preceding claims, wherein presence of the oligonucleotide in the cell results in decreased levels of mRNA of the negative epigenetic regulator of FXN .
20. The method of any one of the preceding claims, wherein the appropriate control is a level of FXN in a cell from a subject or in cells from a population of subjects that do not have Friedreich's ataxia.
21. The method of any one of the preceding claims, wherein the oligonucleotide is a gapmer, a mixmer, an siRNA, a single stranded RNA, a single stranded DNA, an aptamer, or a ribozyme.
22. The method of any one of the preceding claims, wherein the oligonucleotide comprises at least one modified nucleotide or internucleoside linkage.
23. The method of claim 22, wherein the oligonucleotide is a single stranded oligonucleotide.
24. The method of claim 23, wherein the single stranded oligonucleotide comprises the sequence 5'-X-Y-Z-3', wherein X comprises 1-5 modified nucleotides, Y comprises at least 6 unmodified nucleotides, and Z comprises 1-5 modified nucleotides.
25. The method of claim 24, wherein the X comprises 1-5 LNAs, Y comprises at least 6 DNAs, and Z comprises 1-5 LNAs.
26. The method of any one of the preceding claims, wherein the method further comprises:
delivering to the cell a second oligonucleotide that inhibits expression or activity of a second negative epigenetic regulator of FXN.
27. The method of claim 26, wherein the second negative epigenetic regulator of FXN is TNFSF9, JUND, HICl, PRKCD, JAK2, EIDl, CFTR, TAD A3, MYBL2, KAT2A, IDH1, SUMOl, SPEN, PRKDC, KIR2DL4, APC, MEF2D, a component of the NuA4 Histone Acetyltransferase Complex, or a histone-lysine N-methyltransferase.
28. An oligonucleotide comprising a sequence as set forth in Table 4.
29. An oligonucleotide comprising a sequence as set forth in Table 12.
30. The oligonucleotide of claims 28 or 29, wherein the oligonucleotide comprises at least one modified nucleotide or internucleoside linkage.
31. The oligonucleotide of any one of claims 28 to 30, wherein the
5 oligonucleotide is 50 nucleotides or fewer in length.
32. The oligonucleotide of claim 31, wherein the oligonucleotide consists of a sequence as set forth in Table 4.
33. The oligonucleotide of claim 31, wherein the oligonucleotide consists of a sequence as set forth in Table 12. 0
34. A method for increasing FXN expression in a cell, the method comprising: delivering to a cell an expression vector that is engineered to express a positive epigenetic regulator of FXN, thereby increasing FXN expression in the cell, wherein, prior to delivering, the cell has a lower level of FXN expression compared to an appropriate control level of FXN expression. 5
35. The method of claims 34, wherein positive epigenetic regulator of FXN is the product of a gene listed in Table 6 and/or 9 that has a fold change less than or equal to 0.75.
36. A method for modulating FXN expression in a cell, the method comprising delivering to a cell an effective amount of a histone-lysine N-methyltransferase inhibitor.
37. The method of claim 36, wherein the inhibitor is listed in Table 2 or otherwise o disclosed herein.
38. A method for modulating FXN expression in a cell, the method comprising delivering to a cell an effective amount of an agent listed in Table 10 or 11 that modulates FXN expression.
39. The method of claim 38, wherein delivery of the agent results in an increase in FXN expression in the cell.
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