ANTISENSE OLIGONUCLEOTIDES TARGETING TIA1

Abstract

The present invention relates to antisense oligonucleotides (oligomers) complementary to nucleic acids encoding mammalian T cell-restricted intracellular antigen-1 (TIA1), in particular antisense oligonucleotides targeting TIA1 pre-mRNA sequences, which are capable of inhibiting the expression of TIA1. Inhibition of TIA1expression is beneficial for a range of medical disorders including neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or Frontotemporal Dementia.

Claims

1. An antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10-30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 1, wherein the antisense oligonucleotide is capable of inhibiting the expression of human TIA1 in a cell which is expressing human TIA1; or a pharmaceutically acceptable salt thereof.

2. The antisense oligonucleotide according to claim 1, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a sequence selected from the group consisting of SEQ ID NO 4-53.

3. The antisense oligonucleotide according to any one of claims 1 to 3, wherein the contiguous nucleotide sequence is fully complementary to a region of SEQ ID NO 1, selected from the group consisting of the regions in list A.

4. The antisense oligonucleotide according to any one of claims 1 to 3, wherein the contiguous nucleotide sequence is fully complementary to a region of SEQ ID NO 1, selected from the group consisting of the regions in list B.

5. The antisense oligonucleotide according to any one of claims 1-4, wherein the antisense oligonucleotide is a gapmer oligonucleotide comprising a contiguous nucleotide sequence of formula 5′-F-G-F′-3′, where region F and F′ independently comprise 1-8 sugar modified nucleosides, and G is a region between 5 and 16 nucleosides which are capable of recruiting RNaseH.

6. The antisense oligonucleotide according to claim 5, wherein the sugar modified nucleosides of region F and F′ are independently selected from the group consisting of 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, arabino nucleic acid (ANA), 2′-fluoro-ANA and LNA nucleosides.

7. The antisense oligonucleotide according to claim 5 or 6, wherein region G comprises 5-16 contiguous DNA nucleosides.

8. The antisense oligonucleotide according to any one of claims 1-7, wherein the antisense oligonucleotide is a LNA gapmer oligonucleotide.

9. The antisense oligonucleotide according to any one of claims 5-8, wherein the LNA nucleosides are beta-D-oxy LNA nucleosides.

10. The antisense oligonucleotide according to any one of claims 1-9, wherein the internucleoside linkages between the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

11. The antisense oligonucleotide according to any one of claims 1-10, wherein the oligonucleotide comprises a contiguous nucleotide sequence selected from the group consisting of: 54-103.

12. The antisense oligonucleotide according to any one of claims 1-11, wherein the oligonucleotide comprises or consists of a contiguous nucleotide sequence, selected from the group consisting of: CCttctcatataaaaCACA (SEQ ID NO 54); CTTtactacactccCT (SEQ ID NO 55); CCACtaattcttaaaattTC (SEQ ID NO 56); CCaacaattacttcTCAA (SEQ ID NO 57); CTGatttacaacctcATC (SEQ ID NO 58); TATttttctccaaaattCC (SEQ ID NO 59); CTCAttcatccaacaaatAA (SEQ ID NO 60); CACtaaaacatcctaaaaCC (SEQ ID NO 61); TTCCattctttactctttAA (SEQ ID NO 62); ACActatattctacctaATC (SEQ ID NO 63); CCtttcccattaaaaaATTT (SEQ ID NO 64); ACCTtccatttaacattAC (SEQ ID NO 65); ATCtaccattcaacaaaCAC (SEQ ID NO 66); TGTaacttaatcttCCT (SEQ ID NO 67); CAtcctaaccttattatTAT (SEQ ID NO 68); CCctaacattcctatTTA (SEQ ID NO 69); CCttcaatctaatcTTTA (SEQ ID NO 70); ACcttgaatactccTCA (SEQ ID NO 71); TTCActacctcccaaAT (SEQ ID NO 72); ATCtcacacacaataatCAC (SEQ ID NO 73); CTCAcacacaataatcaCT (SEQ ID NO 74); ATAtattcctttacataCAA (SEQ ID NO 75); TATAttcctttacatacaAC (SEQ ID NO 76); ATattcctttacatacaACT (SEQ ID NO 77); TATTcctttacatacaacTT (SEQ ID NO 78); ATtcctttacatacaaCTTT (SEQ ID NO 79); GCCaacatttatccAC (SEQ ID NO 80); CCAacatttatccACT (SEQ ID NO 81); CTaaaactccataccTCA (SEQ ID NO 82); CCcagacattacacCA (SEQ ID NO 83); CCagacattacaccaTTC (SEQ ID NO 84); AGAcattacaccatTCA (SEQ ID NO 85); AAacagtaatcccTTCA (SEQ ID NO 86); ACAgtaatcccttcaCT (SEQ ID NO 87); CAGtaatcccttcacTT (SEQ ID NO 88); AGtaatcccttcacttTA (SEQ ID NO 89); TAatcccttcactttaTAT (SEQ ID NO 90); TATTaacacaaacacattCA (SEQ ID NO 91); ACAcaaacacattcaatCAT (SEQ ID NO 92); CACAaacacattcaatcaTA (SEQ ID NO 93); ACAaacacattcaatcaTAT (SEQ ID NO 94); CAaacacattcaatcaTATC (SEQ ID NO 95); TGAcaaatcctaaTCT (SEQ ID NO 96); TTAccttacccattaTC (SEQ ID NO 97); TAccttacccattatcTT (SEQ ID NO 98); TACccttacatccATA (SEQ ID NO 99); AAAtacccttacatccaTAA (SEQ ID NO 100); ACccttacatccaTAAT (SEQ ID NO 101); CCTtacatccataatcAT (SEQ ID NO 102); and CTTAcatccataatcatTT (SEQ ID NO 103), wherein a capital letter represents a LNA nucleoside, a lower case letter represents a DNA nucleoside.

13. The antisense oligonucleotide according to any one of claims 1-12, wherein the oligonucleotide comprises or consists of a contiguous nucleotide sequence, selected from the group consisting of: CCttctcatataaaaCACA (SEQ ID NO 54); CTTtactacactccCT (SEQ ID NO 55); CCACtaattcttaaaattTC (SEQ ID NO 56); CCaacaattacttcTCAA (SEQ ID NO 57); CTGatttacaacctcATC (SEQ ID NO 58); TATttttctccaaaattCC (SEQ ID NO 59); CTCAttcatccaacaaatAA (SEQ ID NO 60); CACtaaaacatcctaaaaCC (SEQ ID NO 61); TTCCattctttactctttAA (SEQ ID NO 62); ACActatattctacctaATC (SEQ ID NO 63); CCtttcccattaaaaaATTT (SEQ ID NO 64); ACCTtccatttaacattAC (SEQ ID NO 65); ATCtaccattcaacaaaCAC (SEQ ID NO 66); TGTaacttaatcttCCT (SEQ ID NO 67); CAtcctaaccttattatTAT (SEQ ID NO 68); CCctaacattcctatTTA (SEQ ID NO 69); CCttcaatctaatcTTTA (SEQ ID NO 70); ACcttgaatactccTCA (SEQ ID NO 71); TTCActacctcccaaAT (SEQ ID NO 72); ATCtcacacacaataatCAC (SEQ ID NO 73); CTCAcacacaataatcaCT (SEQ ID NO 74); ATAtattcctttacataCAA (SEQ ID NO 75); TATAttcctttacatacaAC (SEQ ID NO 76); ATattcctttacatacaACT (SEQ ID NO 77); TATTcctttacatacaacTT (SEQ ID NO 78); ATtcctttacatacaaCTTT (SEQ ID NO 79); GCCaacatttatccAC (SEQ ID NO 80); CCAacatttatccACT (SEQ ID NO 81); CTaaaactccataccTCA (SEQ ID NO 82); CCcagacattacacCA (SEQ ID NO 83); CCagacattacaccaTTC (SEQ ID NO 84); AGAcattacaccatTCA (SEQ ID NO 85); AAacagtaatcccTTCA (SEQ ID NO 86); ACAgtaatcccttcaCT (SEQ ID NO 87); CAGtaatcccttcacTT (SEQ ID NO 88); AGtaatcccttcacttTA (SEQ ID NO 89); TAatcccttcactttaTAT (SEQ ID NO 90); TATTaacacaaacacattCA (SEQ ID NO 91); ACAcaaacacattcaatCAT (SEQ ID NO 92); CACAaacacattcaatcaTA (SEQ ID NO 93); ACAaacacattcaatcaTAT (SEQ ID NO 94); CAaacacattcaatcaTATC (SEQ ID NO 95); TGAcaaatcctaaTCT (SEQ ID NO 96); TTAccttacccattaTC (SEQ ID NO 97); TAccttacccattatcTT (SEQ ID NO 98); TACccttacatccATA (SEQ ID NO 99); AAAtacccttacatccaTAA (SEQ ID NO 100); ACccttacatccaTAAT (SEQ ID NO 101); CCTtacatccataatcAT (SEQ ID NO 102); and CTTAcatccataatcatTT (SEQ ID NO 103), wherein a capital letter represents a beta-D-oxy LNA nucleoside, a lower case letter represents a DNA nucleoside, wherein each LNA cytosine is 5-methyl cytosine, and wherein the internucleoside linkages between the nucleosides are phosphorothioate internucleoside linkages.

14. A conjugate comprising the oligonucleotide according to any one of claims 1-13, and at least one conjugate moiety covalently attached to said oligonucleotide.

15. A pharmaceutical composition comprising the oligonucleotide of claim 1-13 or the conjugate of claim 14 and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

16. An in vivo or in vitro method for modulating TIA1 expression in a target cell which is expressing TIA1, said method comprising administering an oligonucleotide of any one of claims 1-13, the conjugate according to claim 14, or the pharmaceutical composition of claim 15 in an effective amount to said cell.

17. A method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide of any one of claims 1-13 or the conjugate according to claim 14 or the pharmaceutical composition of claim 15 to a subject suffering from or susceptible to the disease.

18. The method of claim 17, wherein the disease is a neurological disorder, such as a neurological disorder selected from the group consisting of Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), tauopathy (such as primary tauopathy), frontotemporal dementia with parkinsonism (FTDP-17), frontotemporal lobar dementia (FTLD-TDP), Huntington's disease, Creutzfeld-Jacob disease, and spinomuscular atrophy, motor neuron disease, Tauopathy, Alzheimer's disease, and Welander distal myopathy.

19. The oligonucleotide of any one of claims 1-13 or the conjugate according to claim 14 or the pharmaceutical composition of claim 15 for use in medicine.

20. The oligonucleotide of any one of claims 1-13 or the conjugate according to claim 14 or the pharmaceutical composition of claim 15 for use in the treatment or prevention of a neurological disorder, such as a neurological disorder selected from the group consisting of Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), tauopathy (such as primary tauopathy), frontotemporal dementia with parkinsonism (FTDP-17), frontotemporal lobar dementia (FTLD-TDP), Huntington's disease, Creutzfeld-Jacob disease, and spinomuscular atrophy, motor neuron disease, Tauopathy, Alzheimer's disease, and Welander distal myopathy.

21. Use of the oligonucleotide of claims 1-13 or the conjugate according to claim 14 or the pharmaceutical composition of claim 15, for the preparation of a medicament for treatment or prevention of a neurological disorder, such as a neurological disorder selected from the group consisting of Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD) , frontotemporal dementia with parkinsonism (FTDP-17), frontotemporal lobar dementia (FTLD-TDP), tauopathy (such as primary tauopathy), Huntington's disease, Creutzfeld-Jacob disease, and spinomuscular atrophy, motor neuron disease, Tauopathy, Alzheimer's disease, and Welander distal myopathy.

22. The Use of method according to any one of claims 17-21 , wherein the neurological disorder is Amyotrophic Lateral Sclerosis (ALS).

23. The Use of method according to any one of claims 17-21, wherein the neurological disorder is a tauopathy, such as a primary tauopathy.

24. The Use of method according to any one of claims 17-21 , wherein the neurological disorder is frontotemporal lobar dementia (FTLD-TDP).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0221] The invention relates to oligonucleotides, such as antisense oligonucleotides, targeting TIA1 expression.

[0222] The oligonucleotides of the invention targeting TIA1 are capable of hybridizing to and inhibiting the expression of a TIA1 target nucleic acid in a cell which is expressing the TIA1 target nucleic acid.

[0223] The TIA1 target nucleic acid may be a mammalian TIA1 mRNA or premRNA, such as a human TIA1 mRNA or premRNA, for example a premRNA or mRNA originating from the Homo sapiens T cell-restricted intracellular antigen-1 (TIA1), RefSeqGene on Chromosome 2: 70,209,444-70,248,660 reverse strand (GRCh38:CM000664.2)—see also Ensembl ENSG00000116001 (SEQ ID NO 1).

[0224] The oligonucleotides of the invention are capable of inhibiting the expression of TIA1 target nucleic acid, such as the TIA1 mRNA, in a cell which is expressing the target nucleic acid, such as the TIA1 mRNA.

[0225] In some embodiments, the oligonucleotides of the invention are capable of inhibiting the expression of TIA1 target nucleic acid in a cell which is expressing the target nucleic acid, so to reduce the level of TIA1 target nucleic acid (e.g. the mRNA) by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% inhibition compared to the expression level of the TIA1 target nucleic acid (e.g. the mRNA) in the cell. Example 1 provides a suitable assay for evaluating the ability of the oligonucleotides of the invention to inhibit the expression of the target nucleic acid. Suitably the evaluation of a compounds ability to inhibit the expression of the target nucleic acid is performed in vitro, such a gymnotic in vitro assay, for example as according to Example 1.

[0226] An aspect of the present invention relates to an antisense oligonucleotide, such as an LNA antisense oligonucleotide gapmer which comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length with at least 90% complementarity, such as is fully complementary to SEQ ID NO 1, and/or a sequence selected from the group consisting of SEQ ID NO 4-53.

[0227] In some embodiments, the oligonucleotide comprises a contiguous sequence of 10-30 nucleotides, which is at least 90% complementary, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementary with a region of the target nucleic acid or a target sequence.

[0228] In some embodiments, the oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12-24, such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 1.

[0229] In some embodiments, the antisense oligonucleotide of the invention or the contiguous nucleotide sequence thereof is a gapmer, such as an LNA gapmer, a mixed wing gapmer, or an alternating flank gapmer.

[0230] In some embodiments, the antisense oligonucleotide according to the invention, comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides, such as at least 12 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, which is fully complementary to SEQ ID NO 1.

[0231] In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is less than 20 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12-24 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12-22 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12-20 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12-18 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12-16 nucleotides in length.

[0232] Advantageously, in some embodiments all of the internucleoside linkages between the nucleosides of the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

[0233] In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 1.

[0234] In some embodiments, the contiguous nucleotide sequence is fully complementary to a sequence selected from the group consisting of SEQ ID NO 4-53.

[0235] In some embodiments, the antisense oligonucleotide is a gapmer oligonucleotide comprising a contiguous nucleotide sequence of formula 5′-F-G-F′-3′, where region F and F′ independently comprise 1-8 sugar modified nucleosides, and G is a region between 5 and 16 nucleosides which are capable of recruiting RNaseH.

[0236] In some embodiments, the sugar modified nucleosides of region F and F′ are independently selected from the group consisting of 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, arabino nucleic acid (ANA), 2′-fluoro-ANA and LNA nucleosides.

[0237] In some embodiments, region G comprises 5-16 contiguous DNA nucleosides.

[0238] In some embodiments, wherein the antisense oligonucleotide is a gapmer oligonucleotide, such as an LNA gapmer oligonucleotide.

[0239] In some embodiments, the LNA nucleosides are beta-D-oxy LNA nucleosides.

[0240] In some embodiments, the internucleoside linkages between the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

[0241] Exemplary Sequence Motifs and Motif Sequences of the Invention are listed in the table below—see also table A in the examples.:

TABLE-US-00003 Target Target region MOTIF SEQ region sequence ID Motif Sequence SEQ ID NO 4 TGTGTTTTATATGAGAAGG SEQ ID NO 54 CCTTCTCATATAAAACACA SEQ ID NO 5 AGGGAGTGTAGTAAAG SEQ ID NO 55 CTTTACTACACTCCCT SEQ ID NO 6 GAAATTTTAAGAATTAGTGG SEQ ID NO 56 CCACTAATTCTTAAAATTTC SEQ ID NO 7 TTGAGAAGTAATTGTTGG SEQ ID NO 57 CCAACAATTACTTCTCAA SEQ ID NO 8 GATGAGGTTGTAAATCAG SEQ ID NO 58 CTGATTTACAACCTCATC SEQ ID NO 9 GGAATTTTGGAGAAAAATA SEQ ID NO 59 TATTTTTCTCCAAAATTCC SEQ ID NO 10 TTATTTGTTGGATGAATGAG SEQ ID NO 60 CTCATTCATCCAACAAATAA SEQ ID NO 11 GGTTTTAGGATGTTTTAGTG SEQ ID NO 61 CACTAAAACATCCTAAAACC SEQ ID NO 12 TTAAAGAGTAAAGAATGGAA SEQ ID NO 62 TTCCATTCTTTACTCTTTAA SEQ ID NO 13 GATTAGGTAGAATATAGTGT SEQ ID NO 63 ACACTATATTCTACCTAATC SEQ ID NO 14 AAATTTTTTAATGGGAAAGG SEQ ID NO 64 CCTTTCCCATTAAAAAATTT SEQ ID NO 15 GTAATGTTAAATGGAAGGT SEQ ID NO 65 ACCTTCCATTTAACATTAC SEQ ID NO 16 GTGTTTGTTGAATGGTAGAT SEQ ID NO 66 ATCTACCATTCAACAAACAC SEQ ID NO 17 AGGAAGATTAAGTTACA SEQ ID NO 67 TGTAACTTAATCTTCCT SEQ ID NO 18 ATAATAATAAGGTTAGGATG SEQ ID NO 68 CATCCTAACCTTATTATTAT SEQ ID NO 19 TAAATAGGAATGTTAGGG SEQ ID NO 69 CCCTAACATTCCTATTTA SEQ ID NO 20 TAAAGATTAGATTGAAGG SEQ ID NO 70 CCTTCAATCTAATCTTTA SEQ ID NO 21 TGAGGAGTATTCAAGGT SEQ ID NO 71 ACCTTGAATACTCCTCA SEQ ID NO 22 ATTTGGGAGGTAGTGAA SEQ ID NO 72 TTCACTACCTCCCAAAT SEQ ID NO 23 GTGATTATTGTGTGTGAGAT SEQ ID NO 73 ATCTCACACACAATAATCAC SEQ ID NO 24 AGTGATTATTGTGTGTGAG SEQ ID NO 74 CTCACACACAATAATCACT SEQ ID NO 25 TTGTATGTAAAGGAATATAT SEQ ID NO 75 ATATATTCCTTTACATACAA SEQ ID NO 26 GTTGTATGTAAAGGAATATA SEQ ID NO 76 TATATTCCTTTACATACAAC SEQ ID NO 27 AGTTGTATGTAAAGGAATAT SEQ ID NO 77 ATATTCCTTTACATACAACT SEQ ID NO 28 AAGTTGTATGTAAAGGAATA SEQ ID NO 78 TATTCCTTTACATACAACTT SEQ ID NO 29 AAAGTTGTATGTAAAGGAAT SEQ ID NO 79 ATTCCTTTACATACAACTTT SEQ ID NO 30 GTGGATAAATGTTGGC SEQ ID NO 80 GCCAACATTTATCCAC SEQ ID NO 31 AGTGGATAAATGTTGG SEQ ID NO 81 CCAACATTTATCCACT SEQ ID NO 32 TGAGGTATGGAGTTTTAG SEQ ID NO 82 CTAAAACTCCATACCTCA SEQ ID NO 33 TGGTGTAATGTCTGGG SEQ ID NO 83 CCCAGACATTACACCA SEQ ID NO 34 GAATGGTGTAATGTCTGG SEQ ID NO 84 CCAGACATTACACCATTC SEQ ID NO 35 TGAATGGTGTAATGTCT SEQ ID NO 85 AGACATTACACCATTCA SEQ ID NO 36 TGAAGGGATTACTGTTT SEQ ID NO 86 AAACAGTAATCCCTTCA SEQ ID NO 37 AGTGAAGGGATTACTGT SEQ ID NO 87 ACAGTAATCCCTTCACT SEQ ID NO 38 AAGTGAAGGGATTACTG SEQ ID NO 88 CAGTAATCCCTTCACTT SEQ ID NO 39 TAAAGTGAAGGGATTACT SEQ ID NO 89 AGTAATCCCTTCACTTTA SEQ ID NO 40 ATATAAAGTGAAGGGATTA SEQ ID NO 90 TAATCCCTTCACTTTATAT SEQ ID NO 41 TGAATGTGTTTGTGTTAATA SEQ ID NO 91 TATTAACACAAACACATTCA SEQ ID NO 42 ATGATTGAATGTGTTTGTGT SEQ ID NO 92 ACACAAACACATTCAATCAT SEQ ID NO 43 TATGATTGAATGTGTTTGTG SEQ ID NO 93 CACAAACACATTCAATCATA SEQ ID NO 44 ATATGATTGAATGTGTTTGT SEQ ID NO 94 ACAAACACATTCAATCATAT SEQ ID NO 45 GATATGATTGAATGTGTTTG SEQ ID NO 95 CAAACACATTCAATCATATC SEQ ID NO 46 AGATTAGGATTTGTCA SEQ ID NO 96 TGACAAATCCTAATCT SEQ ID NO 47 GATAATGGGTAAGGTAA SEQ ID NO 97 TTACCTTACCCATTATC SEQ ID NO 48 AAGATAATGGGTAAGGTA SEQ ID NO 98 TACCTTACCCATTATCTT SEQ ID NO 49 TATGGATGTAAGGGTA SEQ ID NO 99 TACCCTTACATCCATA SEQ ID NO 50 TTATGGATGTAAGGGTATTT SEQ ID NO 100 AAATACCCTTACATCCATAA SEQ ID NO 51 ATTATGGATGTAAGGGT SEQ ID NO 101 ACCCTTACATCCATAAT SEQ ID NO 52 ATGATTATGGATGTAAGG SEQ ID NO 102 CCTTACATCCATAATCAT SEQ ID NO 53 AAATGATTATGGATGTAAG SEQ ID NO 103 CTTACATCCATAATCATTT

[0242] The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12-24, such as 14-18 in length, nucleosides in length wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence comprising at least 12, such as at least 14, such as at least 15 contiguous nucleotides present in a sequence selected from the group consisting of SEQ ID NO 54-103.

[0243] The invention provides an LNA gapmer according to the invention comprising or consisting of a contiguous nucleotide sequence selected from SEQ ID NO SEQ ID NO 54-103.

[0244] The invention provides an antisense oligonucleotide selected from the group consisting of: CCttctcatataaaaCACA; CTTtactacactccCT; CCACtaattcttaaaattTC; CCaacaattacttcTCAA; CTGatttacaacctcATC; TATttttctccaaaattCC; CTCAttcatccaacaaatAA; CACtaaaacatcctaaaaCC; TTCCattctttactctttAA; ACActatattctacctaATC; CCtttcccattaaaaaATTT; ACCTtccatttaacattAC; ATCtaccattcaacaaaCAC; TGTaacttaatcttCCT; CAtcctaaccttattatTAT; CCctaacattcctatTTA; CCttcaatctaatcTTTA; ACcttgaatactccTCA; TTCActacctcccaaAT; ATCtcacacacaataatCAC; CTCAcacacaataatcaCT; ATAtattcctttacataCAA; TATAttcctttacatacaAC; ATattcctttacatacaACT; TATTcctttacatacaacTT; ATtcctttacatacaaCTTT; GCCaacatttatccAC; CCAacatttatccACT; CTaaaactccataccTCA; CCcagacattacacCA; CCagacattacaccaTTC; AGAcattacaccatTCA; AAacagtaatcccTTCA; ACAgtaatcccttcaCT; CAGtaatcccttcacTT; AGtaatcccttcacttTA; TAatcccttcactttaTAT; TATTaacacaaacacattCA; ACAcaaacacattcaatCAT; CACAaacacattcaatcaTA; ACAaacacattcaatcaTAT; CAaacacattcaatcaTATC; TGAcaaatcctaaTCT; TTAccttacccattaTC; TAccttacccattatcTT; TACccttacatccATA; AAAtacccttacatccaTAA; ACccttacatccaTAAT; CCTtacatccataatcAT; and CTTAcatccataatcatTT; wherein a capital letter is a LNA nucleoside, and a lower case letter is a DNA nucleoside. In some embodiments all internucleoside linkages in contiguous nucleoside sequence are phosphorothioate internucleoside linkages. Optionally LNA cytosine may be 5-methyl cytosine. Optionally DNA cytosine may be 5-methyl cytosine.

[0245] The invention provides an antisense oligonucleotide selected from the group consisting of: CCttctcatataaaaCACA; CTTtactacactccCT; CCACtaattcttaaaattTC; CCaacaattacttcTCAA; CTGatttacaacctcATC; TATttttctccaaaattCC; CTCAttcatccaacaaatAA; CACtaaaacatcctaaaaCC; TTCCattctttactctttAA; ACActatattctacctaATC; CCtttcccattaaaaaATTT; ACCTtccatttaacattAC; ATCtaccattcaacaaaCAC; TGTaacttaatcttCCT; CAtcctaaccttattatTAT; CCctaacattcctatTTA; CCttcaatctaatcTTTA; ACcttgaatactccTCA; TTCActacctcccaaAT; ATCtcacacacaataatCAC; CTCAcacacaataatcaCT; ATAtattcctttacataCAA; TATAttcctttacatacaAC; ATattcctttacatacaACT; TATTcctttacatacaacTT; ATtcctttacatacaaCTTT; GCCaacatttatccAC; CCAacatttatccACT; CTaaaactccataccTCA; CCcagacattacacCA; CCagacattacaccaTTC; AGAcattacaccatTCA; AAacagtaatcccTTCA; ACAgtaatcccttcaCT; CAGtaatcccttcacTT; AGtaatcccttcacttTA; TAatcccttcactttaTAT; TATTaacacaaacacattCA; ACAcaaacacattcaatCAT; CACAaacacattcaatcaTA; ACAaacacattcaatcaTAT; CAaacacattcaatcaTATC; TGAcaaatcctaaTCT; TTAccttacccattaTC; TAccttacccattatcTT; TACccttacatccATA; AAAtacccttacatccaTAA; ACccttacatccaTAAT; CCTtacatccataatcAT; and CTTAcatccataatcatTT; wherein a capital letter is a beta-D-oxy-LNA nucleoside, and a lower case letter is a DNA nucleoside, wherein all internucleoside linkages in the oligonucleotide are phosphorothioate internucleoside linkages, and all LNA cytosines are 5-methyl cytosine.

[0246] Further Advantageous Target Site Regions

[0247] The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12-24, such as 12-18 in length, nucleosides in length wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence comprising at least 14, such as at least 15 contiguous nucleotides, which are fully complementary to a target site region selected from the group consisting of the target sequence regions in LIST A.

[0248] The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12-24, such as 12-18 in length, nucleosides in length wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence comprising at least 14, such as at least 15 contiguous nucleotides, which are fully complementary to a target site region selected from the group consisting of the target sequence regions in LIST B.

[0249] The antisense oligonucleotides according to the invention which target target sequence regions is LIST A or LIST B may be gapmer oligonucleotides, such as LNA gapmer oligonucleotides.

[0250] Method of Manufacture

[0251] In a further aspect, the invention provides methods for manufacturing the oligonucleotides of the invention comprising reacting nucleotide units and thereby forming covalently linked contiguous nucleotide units comprised in the oligonucleotide. Preferably, the method uses phophoramidite chemistry (see for example Caruthers et al, 1987, Methods in Enzymology vol. 154, pages 287-313). In a further embodiment the method further comprises reacting the contiguous nucleotide sequence with a conjugating moiety (ligand) to covalently attach the conjugate moiety to the oligonucleotide. In a further aspect a method is provided for manufacturing the composition of the invention, comprising mixing the oligonucleotide or conjugated oligonucleotide of the invention with a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

[0252] Pharmaceutical Composition

[0253] In a further aspect, the invention provides pharmaceutical compositions comprising any of the aforementioned oligonucleotides and/or oligonucleotide conjugates or salts thereof and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS) and pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments the pharmaceutically acceptable diluent is sterile phosphate buffered saline. In some embodiments the oligonucleotide is used in the pharmaceutically acceptable diluent at a concentration of 50-300 μM solution.

[0254] The compounds according to the present invention may exist in the form of their pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Acid-addition salts include for example those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethyl ammonium hydroxide. The chemical modification of a pharmaceutical compound into a salt is a technique well known to pharmaceutical chemists in order to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. It is for example described in Bastin, Organic Process Research & Development 2000, 4, 427-435 or in Ansel, In: Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995), pp. 196 and 1456-1457. For example, the pharmaceutically acceptable salt of the compounds provided herein may be a sodium salt.

[0255] Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533, 1990). WO 2007/031091 provides further suitable and preferred examples of pharmaceutically acceptable diluents, carriers and adjuvants (hereby incorporated by reference). Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in WO 2007/031091.

[0256] Oligonucleotides or oligonucleotide conjugates of the invention may be mixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

[0257] These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.

[0258] The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment. In some embodiments, the oligonucleotide or oligonucleotide conjugate of the invention is a prodrug. In particular with respect to oligonucleotide conjugates the conjugate moiety is cleaved of the oligonucleotide once the prodrug is delivered to the site of action, e.g. the target cell.

[0259] Applications

[0260] The oligonucleotides of the invention may be utilized as research reagents for, for example, diagnostics, therapeutics and prophylaxis.

[0261] In research, such oligonucleotides may be used to specifically modulate the synthesis of TIA1 protein in cells (e.g. in vitro cell cultures) and experimental animals thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention. Typically the target modulation is achieved by degrading or inhibiting the mRNA producing the protein, thereby prevent protein formation or by degrading or inhibiting a modulator of the gene or mRNA producing the protein.

[0262] If employing the oligonucleotide of the invention in research or diagnostics the target nucleic acid may be a cDNA or a synthetic nucleic acid derived from DNA or RNA.

[0263] The present invention provides an in vivo or in vitro method for modulating TIA1 expression in a target cell which is expressing TIA1, said method comprising administering an oligonucleotide of the invention in an effective amount to said cell.

[0264] In some embodiments, the target cell, is a mammalian cell in particular a human cell. The target cell may be an in vitro cell culture or an in vivo cell forming part of a tissue in a mammal. In diagnostics the oligonucleotides may be used to detect and quantitate TIA1 expression in cell and tissues by northern blotting, in-situ hybridisation or similar techniques.

[0265] For therapeutics, an animal or a human, suspected of having a disease or disorder, which can be treated by modulating the expression of TIA1

[0266] The invention provides methods for treating or preventing a disease, comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide, an oligonucleotide conjugate or a pharmaceutical composition of the invention to a subject suffering from or susceptible to the disease.

[0267] The invention also relates to an oligonucleotide, a composition or a conjugate as defined herein for use as a medicament.

[0268] The oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition according to the invention is typically administered in an effective amount.

[0269] The invention also provides for the use of the oligonucleotide or oligonucleotide conjugate of the invention as described for the manufacture of a medicament for the treatment of a disorder as referred to herein, or for a method of the treatment of as a disorder as referred to herein.

[0270] In some embodiments, the disease or disorder is a neurodegenerative disease, such as a neurodegenerative disease

[0271] In some embodiments, the disease is selected from the group consisting of Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), a tauopathy, such as a primary tauopathy, frontotemporal dementia with parkinsonism (FTDP-17), frontotemporal lobar dementia (FTLD-TDP), Huntington's disease, Creutzfeld-Jacob disease, and spinomuscular atrophy, motor neuron disease, Alzheimer's disease, and Welander distal myopathy.

[0272] In some embodiments, the disease is Amyotrophic Lateral Sclerosis.

[0273] In some embodiments, the disease is Frontotemporal Dementia (FTD).

[0274] The invention provides for the oligonucleotide, conjugate or the pharmaceutical composition of the invention for use in medicine.

[0275] The disease or disorder, as referred to herein, is associated with expression of TIA1. In some embodiments disease or disorder may be associated with a mutation in the TIA1 gene. Therefore, in some embodiments, the target nucleic acid is a mutated form of the TIA1 sequence.

[0276] The methods of the invention are preferably employed for treatment or prophylaxis against diseases caused by abnormal levels and/or activity of TIA1.

[0277] The invention further relates to use of an oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition as defined herein for the manufacture of a medicament for the treatment of abnormal levels and/or activity of TIA 1.

[0278] Administration

[0279] The oligonucleotides or pharmaceutical compositions of the present invention may be administered topical or enteral or parenteral (such as, intravenous, subcutaneous, intra-muscular, intracerebral, intracerebroventricular or intrathecal).

[0280] In a preferred embodiment the oligonucleotide or pharmaceutical compositions of the present invention are administered by a parenteral route including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g. intracerebral or intraventricular, intravitreal administration.

[0281] In some embodiments the active oligonucleotide or oligonucleotide conjugate is administered intrathecally. In some embodiments the active oligonucleotide or oligonucleotide conjugate is administered intracerebroventricularly. In some embodiments the active oligonucleotide or oligonucleotide conjugate is administered intracerebrally.

[0282] In some embodiments, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is administered ata dose of 0.1-15 mg/kg, such as from 0.2-10 mg/kg, such as from 0.25-5 mg/kg. The administration can be once a week, every 2.sup.nd week, every third week or even once a month.

[0283] Combination Therapies

[0284] In some embodiments the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is for use in a combination treatment with another therapeutic agent. The therapeutic agent can for example be the standard of care for the diseases or disorders described above.

EXAMPLES

[0285] Compounds and sequence—see Exemplary Sequence Motifs and Compounds of the Invention are listed in table A

[0286] Cell Lines

TABLE-US-00004 TABLE B Details for different cell lines used in in vitro screening of TIA1 antisense oligonucleotides. Cell lines Cell medium Hours of (All medium and incubation additives are of cells purchased from Cells/well prior to Days of Name Vendor Cat.no. Sigma Aldrich) (96 well plate) treatment treatment A431 ECACC 85090402 EMEM (Cat.no. 8000 24 3 M2279), 10% FBS (Cat.no. F7524), 2 mM Glutamine (Cat.no. G8541), 0.1 mM NEAA (Cat.no. M7145), 25 μg/ml Gentamicin (Cat.no. G1397) NCI-H23 ATCC CRL-5800 RPMI 1640 10000 24 3 (Cat.no. R2405), 10% FBS (Cat.no. F7524), 10 mM Hepes (Cat.no. H0887), 1 mM Sodium Pyruvate (Cat.no. S8636), 25 μg/ml Gentamicin (Cat.no. G1397) ARPE19 ATCC CRL-2302 DMEM/F-12 2000 0 4 HAM (Cat.no. D8437), 10% FBS (Cat.no. F7524), 25 μg/ml Gentamicin (Cat.no. G1397) U251 ECACC 9063001 EMEM (Cat.no. 2000 0 4 M2279), 10% FBS (Cat.no. F7524), 2 mM Glutamine (Cat.no. G8541), 0.1 mM NEAA (Cat.no. M7145) 1 mM Sodium Pyruvate (Cat.no. S8636), 25 μg/ml Gentamicin (Cat.no. G1397) U2O5 ATCC HTB-96 MCCoy 5A 7000 24 3 medium (Cat.no. M8403), 10% FBS (Cat.no. F7524), 1.5 mM Glutamine (Cat.no. G8541), 25 μg/ml Gentamicin (Cat.no. G1397) Compounds may also be evaluated in iPSC-derived motor neurons.

Example 1: Testing In Vitro Efficacy of LNA Oligonucleotides of the Compounds Listed in Table A in U2OS Cell Line at 25 and 5 μM

[0287] An oligonucleotide screen was done in the human cell line, U2OS, using the 50 LNA oligonucleotides listed in table X. The U2OS cell line was purchased from ATCC (cat. no.: HTB-96) and maintained as recommended by the supplier in a humidified incubator at 37° C. with 5% CO.sub.2. For the screening assays, cells were seeded in 96 multi well plates in media recommended by the supplier (MCCoy 5A medium [Cat.no. M8403], 10% FBS [Cat.no. F7524], 1.5mM Glutamine [Cat.no. G8541], 25 μg/ml Gentamicin [Cat.no. G1397]). The number of cells/well has been optimized to 7000 cells/well in a 96 well format

[0288] Cells were incubated 24 hours before addition of the oligonucleotide in concentrations of 5 or 25 μM (dissolved in PBS). 3 days after addition of the oligonucleotide, the cells were harvested. RNA was extracted using the Qiagen RNeasy 96 kit (74182), according to the manufacturer's instructions). cDNA synthesis and qPCR was performed using qScript XLT one-step RT-qPCR ToughMix Low ROX, 95134-100 (Quanta Biosciences). Target transcript levels were quantified using FAM labeled TaqMan assays from Thermo Fisher Scientific in a multiplex reaction with a VIC labelled GAPDH control. TaqMan primer assays for the target transcript of interest TIA1 (Hs00234977_m1 (FAM-MGB)), and a house keeping gene GAPDH (4326317E VIC-MGB probe). A technical duplex set up was used, n=2 biological independent replicates.

[0289] The relative TIA1 mRNA expression levels are shown in Table X as % of control (PBS-treated cells) i.e. the lower the value the larger the inhibition.

TABLE-US-00005 TABLE C in vitro efficacy of anti-TIA1 compounds (average of 2 biological independent with stdev). TIA1 mRNA levels are normalized experiments to GAPDH and shown as % of control (PBS treated cells). U2OS screening 3 days gymnosis TIA1/GAPDH RICC-18-5324 n = 2, 7000 cells/well Compound ID # 25 μM 5 μM COMP# 54 17 25 COMP# 55 9 20 COMP# 56 104 95 COMP# 57 36 57 COMP# 58 0.8 3 COMP# 59 57 72 COMP# 60 32 50 COMP# 61 99 90 COMP# 62 11 20 COMP# 63 27 45 COMP# 64 78 85 COMP# 65 32 43 COMP# 66 71 82 COMP# 67 26 47 COMP# 68 113 94 COMP# 69 101 103 COMP# 70 73 88 COMP# 71 4 11 COMP# 72 20 47 COMP# 73 60 95 COMP# 74 48 67 COMP# 75 69 75 COMP# 76 48 74 COMP# 77 41 68 COMP# 78 15 30 COMP# 79 25 38 COMP# 80 11 25 COMP# 81 4 12 COMP# 82 15 36 COMP# 83 2 8 COMP# 84 0.6 2 COMP# 85 0.3 0.8 COMP# 86 6 19 COMP# 87 10 24 COMP# 88 20 38 COMP# 89 57 72 COMP# 90 77 82 COMP# 91 42 69 COMP# 92 57 76 COMP# 93 41 60 COMP# 94 84 87 COMP# 95 79 94 COMP# 96 90 92 COMP# 97 81 93 COMP# 98 88 93 COMP# 99 82 89 COMP# 100 95 96 COMP# 101 89 95 COMP# 102 96 91 COMP# 103 96 105

[0290] Exemplary Sequence Motifs and Compounds of the Invention are listed in table A:

TABLE-US-00006 TABLE A Target Target region Compound region sequence MOTIF SEQ ID Motif Sequence ID NO # Compound SEQ ID NO 4 TGTGTTTTATATGAGAAGG SEQ ID NO 54 CCTTCTCATATAAAACACA #54 CCttctcatataaaaCACA SEQ ID NO 5 AGGGAGTGTAGTAAAG SEQ ID NO 55 CTTTACTACACTCCCT #55 CTTtactacactccCT SEQ ID NO 6 GAAATTTTAAGAATTAGTGG SEQ ID NO 56 CCACTAATTCTTAAAATTTC #56 CCACtaattcttaaaattTC SEQ ID NO 7 TTGAGAAGTAATTGTTGG SEQ ID NO 57 CCAACAATTACTTCTCAA #57 CCaacaattacttcTCAA SEQ ID NO 8 GATGAGGTTGTAAATCAG SEQ ID NO 58 CTGATTTACAACCTCATC #58 CTGatttacaacctcATC SEQ ID NO 9 GGAATTTTGGAGAAAAATA SEQ ID NO 59 TATTTTTCTCCAAAATTCC #59 TATffitctccaaaattCC SEQ ID NO 10 TTATTTGTTGGATGAATGAG SEQ ID NO 60 CTCATTCATCCAACAAATAA #60 CTCAttcatccaacaaatAA SEQ ID NO 11 GGTTTTAGGATGTTTTAGTG SEQ ID NO 61 CACTAAAACATCCTAAAACC #61 CACtaaaacatcctaaaaCC SEQ ID NO 12 TTAAAGAGTAAAGAATGGAA SEQ ID NO 62 TTCCATTCTTTACTCTTTAA #62 TTCCattctttactctttAA SEQ ID NO 13 GATTAGGTAGAATATAGTGT SEQ ID NO 63 ACACTATATTCTACCTAATC #63 ACActatattctacctaATC SEQ ID NO 14 AAATTTTTTAATGGGAAAGG SEQ ID NO 64 CCTTTCCCATTAAAAAATTT #64 CCtttcccattaaaaaATTT SEQ ID NO 15 GTAATGTTAAATGGAAGGT SEQ ID NO 65 ACCTTCCATTTAACATTAC #65 ACCTtccatttaacattAC SEQ ID NO 16 GTGTTTGTTGAATGGTAGAT SEQ ID NO 66 ATCTACCATTCAACAAACAC #66 ATCtaccattcaacaaaCAC SEQ ID NO 17 AGGAAGATTAAGTTACA SEQ ID NO 67 TGTAACTTAATCTTCCT #67 TGTaacttaatcttCCT SEQ ID NO 18 ATAATAATAAGGTTAGGATG SEQ ID NO 68 CATCCTAACCTTATTATTAT #68 CAtcctaaccttattatTAT SEQ ID NO 19 TAAATAGGAATGTTAGGG SEQ ID NO 69 CCCTAACATTCCTATTTA #69 CCctaacattcctatTTA SEQ ID NO 20 TAAAGATTAGATTGAAGG SEQ ID NO 70 CCTTCAATCTAATCTTTA #70 CCttcaatctaatcTTTA SEQ ID NO 21 TGAGGAGTATTCAAGGT SEQ ID NO 71 ACCTTGAATACTCCTCA #71 ACcttgaatactccTCA SEQ ID NO 22 ATTTGGGAGGTAGTGAA SEQ ID NO 72 TTCACTACCTCCCAAAT #72 TTCActacctcccaaAT SEQ ID NO 23 GTGATTATTGTGTGTGAGAT SEQ ID NO 73 ATCTCACACACAATAATCAC #73 ATCtcacacacaataatCAC SEQ ID NO 24 AGTGATTATTGTGTGTGAG SEQ ID NO 74 CTCACACACAATAATCACT #74 CTCAcacacaataatcaCT SEQ ID NO 25 TTGTATGTAAAGGAATATAT SEQ ID NO 75 ATATATTCCTTTACATACAA #75 ATAtattcctttacataCAA SEQ ID NO 26 GTTGTATGTAAAGGAATATA SEQ ID NO 76 TATATTCCTTTACATACAAC #76 TATAttcctttacatacaAC SEQ ID NO 27 AGTTGTATGTAAAGGAATAT SEQ ID NO 77 ATATTCCTTTACATACAACT #77 ATattcctttacatacaACT SEQ ID NO 28 AAGTTGTATGTAAAGGAATA SEQ ID NO 78 TATTCCTTTACATACAACTT #78 TATTcctttacatacaacTT SEQ ID NO 29 AAAGTTGTATGTAAAGGAAT SEQ ID NO 79 ATTCCTTTACATACAACTTT #79 ATtcctttacatacaaCTTT SEQ ID NO 30 GTGGATAAATGTTGGC SEQ ID NO 80 GCCAACATTTATCCAC #80 GCCaacatttatccAC SEQ ID NO 31 AGTGGATAAATGTTGG SEQ ID NO 81 CCAACATTTATCCACT #81 CCAacatttatccACT SEQ ID NO 32 TGAGGTATGGAGTTTTAG SEQ ID NO 82 CTAAAACTCCATACCTCA #82 CTaaaactccataccTCA SEQ ID NO 33 TGGTGTAATGTCTGGG SEQ ID NO 83 CCCAGACATTACACCA #83 CCcagacattacacCA SEQ ID NO 34 GAATGGTGTAATGTCTGG SEQ ID NO 84 CCAGACATTACACCATTC #84 CCagacattacaccaTTC SEQ ID NO 35 TGAATGGTGTAATGTCT SEQ ID NO 85 AGACATTACACCATTCA #85 AGAcattacaccatTCA SEQ ID NO 36 TGAAGGGATTACTGTTT SEQ ID NO 86 AAACAGTAATCCCTTCA #86 AAacagtaatcccTTCA SEQ ID NO 37 AGTGAAGGGATTACTGT SEQ ID NO 87 ACAGTAATCCCTTCACT #87 ACAgtaatcccttcaCT SEQ ID NO 38 AAGTGAAGGGATTACTG SEQ ID NO 88 CAGTAATCCCTTCACTT #88 CAGtaatcccttcacTT SEQ ID NO 39 TAAAGTGAAGGGATTACT SEQ ID NO 89 AGTAATCCCTTCACTTTA #89 AGtaatcccttcacttTA SEQ ID NO 40 ATATAAAGTGAAGGGATTA SEQ ID NO 90 TAATCCCTTCACTTTATAT #90 TAatcccttcactttaTAT SEQ ID NO 41 TGAATGTGTTTGTGTTAATA SEQ ID NO 91 TATTAACACAAACACATTCA #91 TATTaacacaaacacattCA SEQ ID NO 42 ATGATTGAATGTGTTTGTGT SEQ ID NO 92 ACACAAACACATTCAATCAT #92 ACAcaaacacattcaatCAT SEQ ID NO 43 TATGATTGAATGTGTTTGTG SEQ ID NO 93 CACAAACACATTCAATCATA #93 CACAaacacattcaatcaTA SEQ ID NO 44 ATATGATTGAATGTGTTTGT SEQ ID NO 94 ACAAACACATTCAATCATAT #94 ACAaacacattcaatcaTAT SEQ ID NO 45 GATATGATTGAATGTGTTTG SEQ ID NO 95 CAAACACATTCAATCATATC #95 CAaacacattcaatcaTATC SEQ ID NO 46 AGATTAGGATTTGTCA SEQ ID NO 96 TGACAAATCCTAATCT #96 TGAcaaatcctaaTCT SEQ ID NO 47 GATAATGGGTAAGGTAA SEQ ID NO 97 TTACCTTACCCATTATC #97 TTAccttacccattaTC SEQ ID NO 48 AAGATAATGGGTAAGGTA SEQ ID NO 98 TACCTTACCCATTATCTT #98 TAccttacccattatcTT SEQ ID NO 49 TATGGATGTAAGGGTA SEQ ID NO 99 TACCCTTACATCCATA #99 TACccttacatccATA SEQ ID NO 50 TTATGGATGTAAGGGTATTT SEQ ID NO 100 AAATACCCTTACATCCATAA #100  AAAtacccttacatccaTAA SEQ ID NO 51 ATTATGGATGTAAGGGT SEQ ID NO 101 ACCCTTACATCCATAAT #101  ACccttacatccaTAAT SEQ ID NO 52 ATGATTATGGATGTAAGG SEQ ID NO 102 CCTTACATCCATAATCAT #102  CCTtacatccataatcAT SEQ ID NO 53 AAATGATTATGGATGTAAG SEQ ID NO 103 CTTACATCCATAATCATTT #103  CTTAcatccataatcatTT In the compound column, capital letters are beta-D-oxy LNA nucleosides, and LNA C are all 5-methyl C, lower case letters are DNA nucleosides, and optionally a superscript m before a lower case c represent a 5-methyl cytosine DNA nucleoside, and all internucleoside linkages are phosphorothioate internucleoside linkages.