Selective Antisense Compounds And Uses Thereof

Abstract

Disclosed are oligomeric compounds which are useful for hybridizing to a complementary nucleic acid, including but not limited, to nucleic acids in a cell. The hybridization results in modulation of the amount activity or expression of the target nucleic acid in a cell.

Description

FIELD OF THE INVENTION

[0001] The present invention pertains generally to chemically-modified oligonucleotides for use in research, diagnostics, and/or therapeutics.

SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled CORE0099WOSEQ.txt, created Aug. 1, 2012 which is 304 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0003] Antisense compounds have been used to modulate target nucleic acids. Antisense compounds comprising a variety of chemical modifications and motifs have been reported. In certain instances, such compounds are useful as research tools, diagnostic reagents, and as therapeutic agents. In certain instances antisense compounds have been shown to modulate protein expression by binding to a target messenger RNA (mRNA) encoding the protein. In certain instances, such binding of an antisense compound to its target mRNA results in cleavage of the mRNA. Antisense compounds that modulate processing of a pre-mRNA have also been reported. Such antisense compounds alter splicing, interfere with polyadenlyation or prevent formation of the 5'-cap of a pre-mRNA.

SUMMARY OF THE INVENTION

[0004] In certain embodiments, the present invention provides oligomeric compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides comprise a region having a gapmer motif. In certain embodiments, such oligonucleotides consist of a region having a gapmer motif.

[0005] The present disclosure provides the following non-limiting numbered embodiments:

Embodiment 1

[0006] A oligomeric compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides, wherein the modified oligonucleotide has a modification motif comprising: a 5'-region consisting of 2-8 linked 5'-region nucleosides, each independently selected from among a modified nucleoside and an unmodified deoxynucleoside, provided that at least one 5'-region nucleoside is a modified nucleoside and wherein the 3'-most 5'-region nucleoside is a modified nucleoside; [0007] a 3'-region consisting of 2-8 linked 3'-region nucleosides, each independently selected from among a modified nucleoside and an unmodified deoxynucleoside, provided that at least one 3'-region nucleoside is a modified nucleoside and wherein the 5'-most 3'-region nucleoside is a modified nucleoside; and [0008] a central region between the 5'-region and the 3'-region consisting of 6-12 linked central region nucleosides, each independently selected from among: a modified nucleoside and an unmodified deoxynucleoside, wherein the 5'-most central region nucleoside is an unmodified deoxynucleoside and the 3'-most central region nucleoside is an unmodified deoxynucleoside; [0009] wherein the modified oligonucleotide has a nucleobase sequence complementary to the nucleobase sequence of a target region of a target nucleic acid.

Embodiment 2

[0009] [0010] The oligomeric compound of embodiment 1, wherein the nucleobase sequence of the target region of the target nucleic acid differs from the nucleobase sequence of at least one non-target nucleic acid by 1-3 differentiating nucleobases.

Embodiment 3

[0010] [0011] The oligomeric compound of embodiment 1, the nucleobase sequence of the target region of the target nucleic acid differs from the nucleobase sequence of at least one non-target nucleic acid by a single differentiating nucleobase.

Embodiment 4

[0011] [0012] The oligomeric compound of embodiment 2 or 3, wherein the target nucleic acid and the non-target nucleic acid are alleles of the same gene.

Embodiment 5

[0012] [0013] The oligomeric compound of embodiment 4, wherein the single differentiating nucleobase is a single-nucleotide polymorphism.

Embodiment 6

[0013] [0014] The oligomeric compound of embodiment 5, wherein the single-nucleotide polymorphism is associated with a disease.

Embodiment 7

[0014] [0015] The oligomeric compound of embodiment 6, wherein the disease is selected from among: Alzheimer's disease, Creutzfeldt-Jakob disease, fatal familial insomnia, Alexander disease, Parkinson's disease, amyotrophic lateral sclerosis, dentato-rubral and pallido-luysian atrophy DRPA, spino-cerebellar ataxia, Torsion dystonia, cardiomyopathy, chronic obstructive pulmonary disease (COPD), liver disease, hepatocellular carcinoma, systemic lupus erythematosus, hypercholesterolemia, breast cancer, asthma, Type 1 diabetes, Rheumatoid arthritis, Graves disease, SLE, spinal and bulbar muscular atrophy, Kennedy's disease, progressive childhood posterior subcapsular cataracts, cholesterol gallstone disease, arthrosclerosis, cardiovascular disease, primary hypercalciuria, alpha-thallasemia, obsessive compulsive disorder, Anxiety, comorbid depression, congenital visual defects, hypertension, metabolic syndrome, prostate cancer, congential myasthenic syndrome, peripheral arterial disease, atrial fibrillation, sporadic pheochromocytoma, congenital malformations, Machado-Joseph disease, Huntington's disease, and Autosomal Dominant Retinitis Pigmentosa disease.

Embodiment 8

[0015] [0016] The oligomeric compound of embodiment 6, wherein the single-nucleotide polymorphism is selected from among: rs6446723, rs3856973, rs2285086, rs363092, rs916171, rs6844859, rs7691627, rs4690073, rs2024115, rs11731237, rs362296, rs10015979, rs7659144, rs363096, rs362273, rs16843804, rs362271, rs362275, rs3121419, rs362272, rs3775061, rs34315806, rs363099, rs2298967, rs363088, rs363064, rs363102, rs2798235, rs363080, rs363072, rs363125, rs362303, rs362310, rs10488840, rs362325, rs35892913, rs363102, rs363096, rs11731237, rs10015979, rs363080, rs2798235, rs1936032, rs2276881, rs363070, rs35892913, rs12502045, rs6446723, rs7685686, rs3733217, rs6844859, and rs362331.

Embodiment 9

[0016] [0017] The oligomeric compound of embodiment 8, wherein the single-nucleotide polymorphism is selected from among: rs7685686, rs362303 rs4690072 and rs363088

Embodiment 10

[0017] [0018] The oligomeric compound of embodiment 2 or 3, wherein the target nucleic acid and the non-target nucleic acid are transcripts from different genes.

Embodiment 11

[0018] [0019] The oligomeric compound of any of embodiments 1-10, wherein the 3'-most 5'-region nucleoside comprises a bicyclic sugar moiety.

Embodiment 12

[0019] [0020] The oligomeric compound of embodiment 11, wherein the 3'-most 5'-region nucleoside comprises a cEt sugar moiety.

Embodiment 13

[0020] [0021] The oligomeric compound of embodiment 11, wherein the 3'-most 5'-region nucleoside comprises an LNA sugar moiety.

Embodiment 14

[0021] [0022] The oligomeric compound of any of embodiments 1-13, wherein the central region consists of 6-10 linked nucleosides.

Embodiment 15

[0022] [0023] The oligomeric compound of any of embodiments 1-14, wherein the central region consists of 6-9 linked nucleosides.

Embodiment 16

[0023] [0024] The oligomeric compound of embodiment 15, wherein the central region consists of 6 linked nucleosides.

Embodiment 17

[0024] [0025] The oligomeric compound of embodiment 15, wherein the central region consists of 7 linked nucleosides.

Embodiment 18

[0025] [0026] The oligomeric compound of embodiment 15, wherein the central region consists of 8 linked nucleosides.

Embodiment 19

[0026] [0027] The oligomeric compound of embodiment 15, wherein the central region consists of 9 linked nucleosides.

Embodiment 20

[0027] [0028] The oligomeric compound of any of embodiments 1-19, wherein each central region nucleoside is an unmodified deoxynucleoside.

Embodiment 21

[0028] [0029] The oligomeric compound of any of embodiments 1-19, wherein at least one central region nucleoside is a modified nucleoside.

Embodiment 22

[0029] [0030] The oligomeric compound of embodiment 21, wherein one central region nucleoside is a modified nucleoside and each of the other central region nucleosides is an unmodified deoxynucleoside.

Embodiment 23

[0030] [0031] The oligomeric compound of embodiment 21, wherein two central region nucleosides are modified nucleosides and each of the other central region nucleosides is an unmodified deoxynucleoside.

Embodiment 24

[0031] [0032] The oligomeric compound of any of embodiments 21-23 wherein at least one modified central region nucleoside is an RNA-like nucleoside.

Embodiment 25

[0032] [0033] The oligomeric compound of any of embodiments 21-23 comprising at least one modified central region nucleoside comprising a modified sugar moiety.

Embodiment 26

[0033] [0034] The oligomeric compound of any of embodiments 21-25 comprising at least one modified central region nucleoside comprising a 5'-methyl-2'-deoxy sugar moiety.

Embodiment 27

[0034] [0035] The oligomeric compound of any of embodiments 21-26 comprising at least one modified central region nucleoside comprising a bicyclic sugar moiety.

Embodiment 28

[0035] [0036] The oligomeric compound of any of embodiments 21-27 comprising at least one modified central region nucleoside comprising a cEt sugar moiety.

Embodiment 29

[0036] [0037] The oligomeric compound of any of embodiments 21-28 comprising at least one modified central region nucleoside comprising an LNA sugar moiety.

Embodiment 30

[0037] [0038] The oligomeric compound of any of embodiments 21-29 comprising at least one modified central region nucleoside comprising an .alpha.-LNA sugar moiety.

Embodiment 31

[0038] [0039] The oligomeric compound of any of embodiments 21-29 comprising at least one modified central region nucleoside comprising a 2'-substituted sugar moiety.

Embodiment 32

[0039] [0040] The oligomeric compound of embodiment 31 wherein at least one modified central region nucleoside comprises a 2'-substituted sugar moiety comprising a 2' substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH2)2SCH3, O--(CH2)2-O--N(Rm)(Rn) or O--CH2-C(.dbd.O)--N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C.sub.1-C.sub.10 alkyl; [0041] wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 33

[0041] [0042] The oligomeric compound of embodiment 32 wherein at least one modified central region nucleoside comprises a 2'-substituted sugar moiety comprising a 2' substituent selected from among: a halogen, OCH.sub.3, OCH.sub.2F, OCHF.sub.2, OCF.sub.3, OCH.sub.2CH.sub.3, O(CH.sub.2).sub.2F, OCH.sub.2CHF.sub.2, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--SCH.sub.3, O(CH.sub.2).sub.2--OCF.sub.3, O(CH.sub.2).sub.3--N(R.sub.1)(R.sub.2), O(CH.sub.2).sub.2--ON(R.sub.1)(R.sub.2), O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(R.sub.1)(R.sub.2), OCH.sub.2C(.dbd.O)--N(R.sub.1)(R.sub.2), OCH.sub.2C(.dbd.O)--N(R.sub.3)--(CH.sub.2).sub.2--N(R.sub.1)(R.sub.2), and O(CH.sub.2).sub.2--N(R.sub.3)--C(.dbd.NR.sub.4)[N(R.sub.1)(R.sub.2)]; wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are each, independently, H or C.sub.1-C.sub.6 alkyl.

Embodiment 34

[0042] [0043] The oligomeric compound of embodiment 33 wherein the 2' substituent is selected from among: a halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3 (MOE), O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2, OCH.sub.2C(.dbd.O)--N(H)CH.sub.3, OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2, and OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2.

Embodiment 35

[0043] [0044] The oligomeric compound of any of embodiments 21-34 comprising at least one modified central region nucleoside comprising a 2'-MOE sugar moiety.

Embodiment 36

[0044] [0045] The oligomeric compound of any of embodiments 21-35 comprising at least one modified central region nucleoside comprising a 2'-OMe sugar moiety.

Embodiment 37

[0045] [0046] The oligomeric compound of any of embodiments 21-36 comprising at least one modified central region nucleoside comprising a 2'-F sugar moiety.

Embodiment 38

[0046] [0047] The oligomeric compound of any of embodiments 21-37 comprising at least one modified central region nucleoside comprising a 2'-(ara)-F sugar moiety.

Embodiment 39

[0047] [0048] The oligomeric compound of any of embodiments 21-38 comprising at least one modified central region nucleoside comprising a sugar surrogate.

Embodiment 40

[0048] [0049] The oligomeric compound of embodiment 39 comprising at least one modified central region nucleoside comprising an F-HNA sugar moiety.

Embodiment 41

[0049] [0050] The oligomeric compound of embodiment 39 or 40 comprising at least one modified central region nucleoside comprising an HNA sugar moiety.

Embodiment 42

[0050] [0051] The oligomeric compound of any of embodiments 21-41 comprising at least one modified central region nucleoside comprising a modified nucleobase.

Embodiment 43

[0051] [0052] The oligomeric compound of embodiment 42 comprising at least one modified central region nucleoside comprising a modified nucleobase selected from a 2-thio pyrimidine and a 5-propyne pyrimidine.

Embodiment 44

[0052] [0053] The oligomeric compound of any of embodiments 21-43, wherein the 2.sup.nd nucleoside from the 5'-end of the central region is a modified nucleoside.

Embodiment 45

[0053] [0054] The oligomeric compound of any of embodiments 21-44, wherein the 3.sup.rd nucleoside from the 5'-end of the central region is a modified nucleoside.

Embodiment 46

[0054] [0055] The oligomeric compound of any of embodiments 21-45, wherein the 4.sup.th nucleoside from the 5'-end of the central region is a modified nucleoside.

Embodiment 47

[0055] [0056] The oligomeric compound of any of embodiments 21-46, wherein the 5.sup.th nucleoside from the 5'-end of the central region is a modified nucleoside.

Embodiment 48

[0056] [0057] The oligomeric compound of any of embodiments 21-47, wherein the 6.sup.th nucleoside from the 5'-end of the central region is a modified nucleoside.

Embodiment 49

[0057] [0058] The oligomeric compound of any of embodiments 21-48, wherein the 8.sup.th nucleoside from the 3'-end of the central region is a modified nucleoside.

Embodiment 50

[0058] [0059] The oligomeric compound of any of embodiments 21-49, wherein the 7.sup.th nucleoside from the 3'-end of the central region is a modified nucleoside.

Embodiment 51

[0059] [0060] The oligomeric compound of any of embodiments 21-50, wherein the 6.sup.th nucleoside from the 3'-end of the central region is a modified nucleoside.

Embodiment 52

[0060] [0061] The oligomeric compound of any of embodiments 21-51, wherein the 5.sup.th nucleoside from the 3'-end of the central region is a modified nucleoside.

Embodiment 53

[0061] [0062] The oligomeric compound of any of embodiments 21-52, wherein the 4.sup.th nucleoside from the 3'-end of the central region is a modified nucleoside.

Embodiment 54

[0062] [0063] The oligomeric compound of any of embodiments 21-53, wherein the 3.sup.rd nucleoside from the 3'-end of the central region is a modified nucleoside.

Embodiment 55

[0063] [0064] The oligomeric compound of any of embodiments 21-54, wherein the 2.sup.nd nucleoside from the 3'-end of the central region is a modified nucleoside.

Embodiment 56

[0064] [0065] The oligomeric compound of any of embodiments 21-55, wherein the modified nucleoside is a 5'-methyl-2'-deoxy sugar moiety.

Embodiment 57

[0065] [0066] The oligomeric compound of any of embodiments 21-55, wherein the modified nucleoside is a 2-thio pyrimidine.

Embodiment 58

[0066] [0067] The oligomeric compound of any of embodiments 21-55, wherein the central region comprises no region having more than 4 contiguous unmodified deoxynucleosides.

Embodiment 59

[0067] [0068] The oligomeric compound of any of embodiments 21-55, wherein the central region comprises no region having more than 5 contiguous unmodified deoxynucleosides.

Embodiment 60

[0068] [0069] The oligomeric compound of any of embodiments 21-55, wherein the central region comprises no region having more than 6 contiguous unmodified deoxynucleosides.

Embodiment 61

[0069] [0070] The oligomeric compound of any of embodiments 21-55, wherein the central region comprises no region having more than 7 contiguous unmodified deoxynucleosides.

Embodiment 62

[0070] [0071] The oligomeric compound of any of embodiments 1-14 or 21-59, wherein the central region has a nucleoside motif selected from among: DDDDDDDDDD, DDDDXDDDDD; DDDDDXDDDDD; DDDXDDDDD; DDDDXDDDDDD; DDDDXDDDD; DDXDDDDDD; DDDXDDDDDD; DXDDDDDD; DDXDDDDDDD; DDXDDDDD; DDXDDDXDDD; DDDXDDDXDDD; DXDDDXDDD; DDXDDDXDD; DDXDDDDXDDD; DDXDDDDXDD; DXDDDDXDDD; DDDDXDDD; DDDXDDD; DXDDDDDDD; DDDDXXDDD; and DXXDXXDXX; wherein [0072] each D is an unmodified deoxynucleoside; and each X is a modified nucleoside.

Embodiment 63

[0072] [0073] The oligomeric compound of any of embodiments 1-14 or 21-59, wherein the central region has a nucleoside motif selected from among: DDDDDDDDD; DXDDDDDDD; DDXDDDDDD; DDDXDDDDD; DDDDXDDDD; DDDDDXDDD; DDDDDDXDD; DDDDDDDXD; DXXDDDDDD; DDDDDDXXD; DDXXDDDDD; DDDXXDDDD; DDDDXXDDD; DDDDDXXDD; DXDDDDDXD; DXDDDDXDD; DXDDDXDDD; DXDDXDDDD; DXDXDDDDD; DDXDDDDXD; DDXDDDXDD; DDXDDXDDD; DDXDXDDDD; DDDXDDDXD; DDDXDDXDD; DDDXDXDDD; DDDDXDDXD; DDDDXDXDD; and DDDDDXDXD wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside.

Embodiment 64

[0073] [0074] The oligomeric compound of any of embodiments 1-14 or 21-59, wherein the central region has a nucleoside motif selected from among: DDDDXDDDD, DXDDDDDDD, DXXDDDDDD, DDXDDDDDD, DDDXDDDDD, DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, and DDDDDDDXD.

Embodiment 65

[0074] [0075] The oligomeric compound of any of embodiments 1-14 or 21-59, wherein the central region has a nucleoside motif selected from among: DDDDDDDD, DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDXD, DXDDDXDD, DXDDXDDD, DXDXDDDD, DXXDDDDD, DDXXDDDD, DDXDXDDD, DDXDDXDD, DXDDDDXD, DDDXXDDD, DDDXDXDD, DDDXDDXD, DDDDXXDD, DDDDXDXD, and DDDDDXXD.

Embodiment 66

[0075] [0076] The oligomeric compound of any of embodiments 1-14 or 21-59, wherein the central region has a nucleoside motif selected from among: DDDDDDD, DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD, DXDDDXD, DXDDXDD, DXDXDDD, DXXDDDD, DDXXDDD, DDXDXDD, DDXDDXD, DDDXXDD, DDDXDXD, and DDDDXXD.

Embodiment 67

[0076] [0077] The oligomeric compound of any of embodiments 1-14 or 21-59, wherein the central region has a nucleoside motif selected from among: DDDDDD, DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXXDDD, DXDXDD, DXDDXD, DDXXDD, DDXDXD, and DDDXXD.

Embodiment 68

[0077] [0078] The oligomeric compound of any of embodiments 1-14 or 21-59, wherein the central region has a nucleoside motif selected from among: DDDDDD, DDDDDDD, DDDDDDDD, DDDDDDDDD, DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD, DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDDDD; DDXDDDDDD, DDDXDDDDD, DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, DDDDDDDXD, DXDDDDDDDD, DDXDDDDDDD, DDDXDDDDDD, DDDDXDDDDD, DDDDDXDDDD, DDDDDDXDDD, DDDDDDDXDD, and DDDDDDDDXD.

Embodiment 69

[0078] [0079] The oligomeric compound of embodiments 62-68, wherein each X comprises a modified nucleobase.

Embodiment 70

[0079] [0080] The oligomeric compound of embodiments 62-68, wherein each X comprises a modified sugar moiety.

Embodiment 71

[0080] [0081] The oligomeric compound of embodiments 62-68, wherein each X comprises 2-thio-thymidine.

Embodiment 72

[0081] [0082] The oligomeric compound of embodiments 62-68, wherein each X nucleoside comprises an F-HNA sugar moiety.

Embodiment 73

[0082] [0083] The oligomeric compound of embodiments 62-68, wherein the nucleobase sequence of the target region of the target nucleic acid differs from the nucleobase sequence of at least one non-target nucleic acid by a single differentiating nucleobase, and wherein the location of the single differentiating nucleobase is represented by X.

Embodiment 74

[0083] [0084] The oligomeric compound of embodiment 73, wherein the target nucleic acid and the non-target nucleic acid are alleles of the same gene.

Embodiment 75

[0084] [0085] The oligomeric compound of embodiment 73, wherein the single differentiating nucleobase is a single-nucleotide polymorphism.

Embodiment 76

[0085] [0086] The oligomeric compound of any of embodiments 1-75, wherein the 5' region consists of 2 linked 5'-region nucleosides.

Embodiment 77

[0086] [0087] The oligomeric compound of any of embodiments 1-75, wherein the 5' region consists of 3 linked 5'-region nucleosides.

Embodiment 78

[0087] [0088] The oligomeric compound of any of embodiments 1-75, wherein the 5' region consists of 4 linked 5'-region nucleosides.

Embodiment 79

[0088] [0089] The oligomeric compound of any of embodiments 1-75, wherein the 5' region consists of 5 linked 5'-region nucleosides.

Embodiment 80

[0089] [0090] The oligomeric compound of any of embodiments 1-75, wherein the 5' region consists of 6 linked 5'-region nucleosides.

Embodiment 81

[0090] [0091] The oligomeric compound of any of embodiments 1-80, wherein at least one 5'-region nucleoside is an unmodified deoxynucleoside.

Embodiment 82

[0091] [0092] The oligomeric compound of any of embodiments 1-80, wherein each 5'-region nucleoside is a modified nucleoside.

Embodiment 83

[0092] [0093] The oligomeric compound of any of embodiments 1-80 wherein at least one 5'-region nucleoside is an RNA-like nucleoside.

Embodiment 84

[0093] [0094] The oligomeric compound of any of embodiments 1-80 wherein each 5'-region nucleoside is an RNA-like nucleoside.

Embodiment 85

[0094] [0095] The oligomeric compound of any of embodiments 1-80 comprising at least one modified 5'-region nucleoside comprising a modified sugar.

Embodiment 86

[0095] [0096] The oligomeric compound of embodiment 80 comprising at least one modified 5'-region nucleoside comprising a bicyclic sugar moiety.

Embodiment 87

[0096] [0097] The oligomeric compound of embodiment 86 comprising at least one modified 5'-region nucleoside comprising a cEt sugar moiety.

Embodiment 88

[0097] [0098] The oligomeric compound of embodiment 85 or 86 comprising at least one modified 5'-region nucleoside comprising an LNA sugar moiety.

Embodiment 89

[0098] [0099] The oligomeric compound of any of embodiments 76-80 comprising of at least one modified 5'-region nucleoside comprising a 2'-substituted sugar moiety.

Embodiment 90

[0099] [0100] The oligomeric compound of embodiment 89 wherein at least one modified central region nucleoside comprises a 2'-substituted sugar moiety comprising a 2' substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF.sub.3, OCF.sub.3, O, S, or N(R.sub.m)-alkyl; O, S, or N(R.sub.m)-alkenyl; O, S or N(R.sub.m)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH.sub.2).sub.2SCH.sub.3, O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n) or O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and R.sub.n is, independently, H, an amino protecting group or substituted or unsubstituted C.sub.1-C.sub.10 alkyl; [0101] wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 91

[0101] [0102] The oligomeric compound of embodiment 90 wherein at least one modified 5'-region nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: a halogen, OCH.sub.3, OCH.sub.2F, OCHF.sub.2, OCF.sub.3, OCH.sub.2CH.sub.3, O(CH.sub.2).sub.2F, OCH.sub.2CHF.sub.2, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3 (MOE), O(CH.sub.2).sub.2--SCH.sub.3, O(CH.sub.2).sub.2--OCF.sub.3, O(CH.sub.2).sub.3--N(R.sub.1)(R.sub.2), O(CH.sub.2).sub.2--ON(R.sub.1)(R.sub.2), O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(R.sub.1)(R.sub.2), OCH.sub.2C(.dbd.O)--N(R.sub.1)(R.sub.2), OCH.sub.2C(.dbd.O)--N(R.sub.3)--(CH.sub.2).sub.2--N(R.sub.1)(R.sub.2), and O(CH.sub.2).sub.2--N(R.sub.3)--C(.dbd.NR.sub.4)[N(R.sub.1)(R.sub.2)]; wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are each, independently, H or C.sub.1-C.sub.6 alkyl.

Embodiment 92

[0102] [0103] The oligomeric compound of embodiment 91, wherein the 2'-substituent is selected from among: a halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2, OCH.sub.2C(.dbd.O)--N(H)CH.sub.3, OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2, and OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2.

Embodiment 93

[0103] [0104] The oligomeric compound of any of embodiments 89-92 comprising at least one modified 5'-region nucleoside comprising a 2'-MOE sugar moiety.

Embodiment 94

[0104] [0105] The oligomeric compound of any of embodiments 89-92 comprising at least one modified 5'-region nucleoside comprising a 2'-OMe sugar moiety.

Embodiment 95

[0105] [0106] The oligomeric compound of any of embodiments 89-92 comprising at least one modified 5'-region nucleoside comprising a 2'-F sugar moiety.

Embodiment 96

[0106] [0107] The oligomeric compound of any of embodiments 89-92 comprising at least one modified 5'-region nucleoside comprising a 2'-(ara)-F sugar moiety.

Embodiment 97

[0107] [0108] The oligomeric compound of any of embodiments 82-96 comprising of at least one modified 5'-region nucleoside comprising a sugar surrogate.

Embodiment 98

[0108] [0109] The oligomeric compound of embodiment 97 comprising at least one modified 5'-region nucleoside comprising an F-HNA sugar moiety.

Embodiment 99

[0109] [0110] The oligomeric compound of embodiment 97 or 98 comprising at least one modified 5'-region nucleoside comprising an HNA sugar moiety.

Embodiment 100

[0110] [0111] The oligomeric compound of any of embodiments 1-99 comprising at least one modified 5'-region nucleoside comprising a modified nucleobase.

Embodiment 101

[0111] [0112] The oligomeric compound of embodiment 100, wherein the modified nucleoside comprises 2-thio-thymidine.

Embodiment 102

[0112] [0113] The oligomeric compound of any of embodiments 1-101, wherein the 5'-region has a motif selected from among: [0114] ADDA; ABDAA; ABBA; ABB; ABAA; AABAA; AAABAA; AAAABAA; AAAAABAA; AAABAA; AABAA; ABAB; ABADB; ABADDB; AAABB; AAAAA; ABBDC; ABDDC; ABBDCC; ABBDDC; ABBDCC; ABBC; AA; AAA; AAAA; AAAAB; AAAAAAA; AAAAAAAA; ABBB; AB; ABAB; AAAAB; AABBB; AAAAB; and AABBB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, each C is a modified nucleoside of a third type, and each D is an unmodified deoxynucleoside.

Embodiment 103

[0114] [0115] The oligomeric compound of any of embodiments 1-101, wherein the 5'-region has a motif selected from among: [0116] AB, ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, AAAB, ABBW, ABBWW, ABBB, ABBBB, ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW, AAAAA, BBBBAA, and AAABW wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of a third type.

Embodiment 104

[0116] [0117] The oligomeric compound of any of embodiments 1-101, wherein the 5'-region has a motif selected from among: ABB; ABAA; AABAA; AAABAA; ABAB; ABADB; AAABB; AAAAA; AA; AAA; AAAA; AAAAB; ABBB; AB; and ABAB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of a third type.

Embodiment 105

[0117] [0118] The oligomeric compound of embodiments 102-104, wherein each A nucleoside comprises a 2'-substituted sugar moiety.

Embodiment 106

[0118] [0119] The oligomeric compound of embodiment 105 wherein at least one central region nucleoside comprises a 2'-substituted sugar moiety comprising a 2' substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF.sub.3, OCF.sub.3, O, S, or N(R.sub.m)-alkyl; O, S, or N(R.sub.m)-alkenyl; O, S or N(R.sub.m)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH.sub.2).sub.2SCH.sub.3, O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n) or O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and R.sub.n is, independently, H, an amino protecting group or substituted or unsubstituted C.sub.1-C.sub.10 alkyl; wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 107

[0119] [0120] The oligomeric compound of embodiment 102-106, wherein each A nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: a halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2, OCH.sub.2C(.dbd.O)--N(H)CH.sub.3, OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2, and OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2.

Embodiment 108

[0120] [0121] The oligomeric compound of embodiment 107, wherein each A nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: F, OCH.sub.3, O(CH.sub.2).sub.2--OCH.sub.3.

Embodiment 109

[0121] [0122] The oligomeric compound of embodiments 102-106, wherein each A nucleoside comprises a bicyclic sugar moiety.

Embodiment 110

[0122] [0123] The oligomeric compound of embodiment 109, wherein each A nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, .alpha.-LNA, ENA and 2'-thio LNA.

Embodiment 111

[0123] [0124] The oligomeric compound of any of embodiments 102-110, wherein each A comprises a modified nucleobase.

Embodiment 112

[0124] [0125] The oligomeric compound of embodiment 111, wherein each A comprises a modified nucleobase selected from among a 2-thio pyrimidine and a 5-propyne pyrimidine.

Embodiment 113

[0125] [0126] The oligomeric compound of embodiment 112, wherein each A comprises 2-thio-thymidine.

Embodiment 114

[0126] [0127] The oligomeric compound of embodiment 102-106, wherein each A nucleoside comprises an unmodified 2'-deoxyfuranose sugar moiety.

Embodiment 115

[0127] [0128] The oligomeric compound of embodiment 102-106, wherein each A nucleoside comprises an F-HNA sugar moiety.

Embodiment 116

[0128] [0129] The oligomeric compound of any of embodiments 102-115, wherein each B nucleoside comprises a 2'-substituted sugar moiety.

Embodiment 117

[0129] [0130] The oligomeric compound of embodiment 116, wherein at least one central region nucleoside comprises a 2'-substituted sugar moiety comprising a 2' substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF.sub.3, OCF.sub.3, O, S, or N(R.sub.m)-alkyl; O, S, or N(R.sub.m)-alkenyl; O, S or N(R.sub.m)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH.sub.2).sub.2SCH.sub.3, O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n) or O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and R.sub.n is, independently, H, an amino protecting group or substituted or unsubstituted C.sub.1-C.sub.10 alkyl; wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 118

[0130] [0131] The oligomeric compound of embodiment 117, wherein each B nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: a halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2, OCH.sub.2C(.dbd.O)--N(H)CH.sub.3, OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2, and OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2.

Embodiment 119

[0131] [0132] The oligomeric compound of embodiment 118, wherein each B nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: F, OCH.sub.3, O(CH.sub.2).sub.2--OCH.sub.3.

Embodiment 120

[0132] [0133] The oligomeric compound of any of embodiments 102-115, wherein each B nucleoside comprises a bicyclic sugar moiety.

Embodiment 121

[0133] [0134] The oligomeric compound of embodiment 120, wherein each B nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, .alpha.-LNA, ENA and 2'-thio LNA.

Embodiment 122

[0134] [0135] The oligomeric compound of any of embodiments 102-115, wherein each B comprises a modified nucleobase.

Embodiment 123

[0135] [0136] The oligomeric compound of embodiment 122, wherein each B comprises a modified nucleobase selected from among a 2-thio pyrimidine and a 5-propyne pyrimidine.

Embodiment 124

[0136] [0137] The oligomeric compound of embodiment 123, wherein each B comprises 2-thio-thymidine.

Embodiment 125

[0137] [0138] The oligomeric compound of embodiment 102-106, wherein each B nucleoside comprises an unmodified 2'-deoxyfuranose sugar moiety.

Embodiment 126

[0138] [0139] The oligomeric compound of embodiment 102-115, wherein each B nucleoside comprises an F-HNA sugar moiety.

Embodiment 127

[0139] [0140] The oligomeric compound of any of embodiments 102-126, wherein each C nucleoside comprises a 2'-substituted sugar moiety.

Embodiment 128

[0140] [0141] The oligomeric compound of embodiment 127, wherein at least one central region nucleoside comprises a 2'-substituted sugar moiety comprising a 2' substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF.sub.3, OCF.sub.3, O, S, or N(R.sub.m)-alkyl; O, S, or N(R.sub.m)-alkenyl; O, S or N(R.sub.m)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH.sub.2).sub.2SCH.sub.3, O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n) or O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and R.sub.n is, independently, H, an amino protecting group or substituted or unsubstituted C.sub.1-C.sub.10 alkyl; wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 129

[0141] [0142] The oligomeric compound of embodiment 128, wherein each C nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: a halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2, OCH.sub.2C(.dbd.O)--N(H)CH.sub.3, OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2, and OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2.

Embodiment 130

[0142] [0143] The oligomeric compound of embodiment 129, wherein each C nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: F, OCH.sub.3, O(CH.sub.2).sub.2--OCH.sub.3.

Embodiment 131

[0143] [0144] The oligomeric compound of any of embodiments 102-126, wherein each C nucleoside comprises a bicyclic sugar moiety.

Embodiment 132

[0144] [0145] The oligomeric compound of embodiment 131, wherein each C nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, .alpha.-LNA, ENA and 2'-thio LNA.

Embodiment 133

[0145] [0146] The oligomeric compound of any of embodiments 102-126, wherein each C comprises a modified nucleobase.

Embodiment 134

[0146] [0147] The oligomeric compound of embodiment 133, wherein each C comprises a modified nucleobase selected from among a 2-thio pyrimidine and a 5-propyne pyrimidine.

Embodiment 135

[0147] [0148] The oligomeric compound of embodiment 134, wherein each C comprises 2-thio-thymidine.

Embodiment 136

[0148] [0149] The oligomeric compound of embodiment 102-126, wherein each C comprises an F--HNA sugar moiety.

Embodiment 137

[0149] [0150] The oligomeric compound of embodiment 102-126, wherein each C nucleoside comprises an unmodified 2'-deoxyfuranose sugar moiety.

Embodiment 138

[0150] [0151] The oligomeric compound of any of embodiments 102-138, wherein each W nucleoside comprises a 2'-substituted sugar moiety.

Embodiment 139

[0151] [0152] The oligomeric compound of embodiment 138, wherein at least one central region nucleoside comprises a 2'-substituted sugar moiety comprising a 2' substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF.sub.3, OCF.sub.3, O, S, or N(R.sub.m)-alkyl; O, S, or N(R.sub.m)-alkenyl; O, S or N(R.sub.m)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH.sub.2).sub.2SCH.sub.3, O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n) or O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and R.sub.n is, independently, H, an amino protecting group or substituted or unsubstituted C.sub.1-C.sub.10 alkyl; wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 140

[0152] [0153] The oligomeric compound of embodiment 139, wherein each W nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: a halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2, OCH.sub.2C(.dbd.O)--N(H)CH.sub.3, OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2, and OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2.

Embodiment 141

[0153] [0154] The oligomeric compound of embodiment 139, wherein each W nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: F, OCH.sub.3, O(CH.sub.2).sub.2--OCH.sub.3.

Embodiment 142

[0154] [0155] The oligomeric compound of any of embodiments 102-137, wherein each W nucleoside comprises a bicyclic sugar moiety.

Embodiment 143

[0155] [0156] The oligomeric compound of embodiment 142, wherein each W nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, .alpha.-LNA, ENA and 2'-thio LNA.

Embodiment 144

[0156] [0157] The oligomeric compound of any of embodiments 102-137, wherein each W comprises a modified nucleobase.

Embodiment 145

[0157] [0158] The oligomeric compound of embodiment 144, wherein each W comprises a modified nucleobase selected from among a 2-thio pyrimidine and a 5-propyne pyrimidine.

Embodiment 146

[0158] [0159] The oligomeric compound of embodiment 145, wherein each W comprises 2-thio-thymidine.

Embodiment 147

[0159] [0160] The oligomeric compound of embodiment 102-137, wherein each W comprises an F--HNA sugar moiety.

Embodiment 148

[0160] [0161] The oligomeric compound of embodiment 102-137, wherein each W nucleoside comprises an unmodified 2'-deoxyfuranose sugar moiety.

Embodiment 149

[0161] [0162] The oligomeric compound of any of embodiments 1-148, wherein the 3' region consists of 2 linked 3'-region nucleosides.

Embodiment 150

[0162] [0163] The oligomeric compound of any of embodiments 1-148, wherein the 3' region consists of 3 linked 3'-region nucleosides.

Embodiment 151

[0163] [0164] The oligomeric compound of any of embodiments 1-148, wherein the 3' region consists of 4 linked 3'-region nucleosides.

Embodiment 152

[0164] [0165] The oligomeric compound of any of embodiments 1-148, wherein the 3' region consists of 5 linked 3'-region nucleosides.

Embodiment 153

[0165] [0166] The oligomeric compound of any of embodiments 1-148, wherein the 3' region consists of 6 linked 3'-region nucleosides.

Embodiment 154

[0166] [0167] The oligomeric compound of any of embodiments 1-153, wherein at least one 3'-region nucleoside is an unmodified deoxynucleoside.

Embodiment 155

[0167] [0168] The oligomeric compound of any of embodiments 1-154, wherein each 3'-region nucleoside is a modified nucleoside.

Embodiment 156

[0168] [0169] The oligomeric compound of any of embodiments 1-153, wherein at least one 3'-region nucleoside is an RNA-like nucleoside.

Embodiment 157

[0169] [0170] The oligomeric compound of any of embodiments 1-154, wherein each 3'-region nucleoside is an RNA-like nucleoside.

Embodiment 158

[0170] [0171] The oligomeric compound of any of embodiments 1-153, comprising at least one modified 3'-region nucleoside comprising a modified sugar.

Embodiment 159

[0171] [0172] The oligomeric compound of embodiment 158, comprising at least one modified 3'-region nucleoside comprising a bicyclic sugar moiety.

Embodiment 160

[0172] [0173] The oligomeric compound of embodiment 159, comprising at least one modified 3'-region nucleoside comprising a cEt sugar moiety.

Embodiment 161

[0173] [0174] The oligomeric compound of embodiment 159, comprising at least one modified 3'-region nucleoside comprising an LNA sugar moiety.

Embodiment 162

[0174] [0175] The oligomeric compound of any of embodiments 1-162 comprising of at least one modified 3'-region nucleoside comprising a 2'-substituted sugar moiety.

Embodiment 163

[0175] [0176] The oligomeric compound of embodiment 162, wherein at least one central region nucleoside comprises a 2'-substituted sugar moiety comprising a 2' substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF.sub.3, OCF.sub.3, O, S, or N(R.sub.m)-alkyl; O, S, or N(R.sub.m)-alkenyl; O, S or N(R.sub.m)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH.sub.2).sub.2SCH.sub.3, O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n) or O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and R.sub.n is, independently, H, an amino protecting group or substituted or unsubstituted C.sub.1-C.sub.10 alkyl; wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 164

[0176] [0177] The oligomeric compound of embodiment 163 wherein at least one modified 3'-region nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: a halogen, OCH.sub.3, OCH.sub.2F, OCHF.sub.2, OCF.sub.3, OCH.sub.2CH.sub.3, O(CH.sub.2).sub.2F, OCH.sub.2CHF.sub.2, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3 (MOE), O(CH.sub.2).sub.2--SCH.sub.3, O(CH.sub.2).sub.2--OCF.sub.3, O(CH.sub.2).sub.3--N(R.sub.1)(R.sub.2), O(CH.sub.2).sub.2--ON(R.sub.1)(R.sub.2), O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(R.sub.1)(R.sub.2), OCH.sub.2C(.dbd.O)--N(R.sub.1)(R.sub.2), OCH.sub.2C(.dbd.O)--N(R.sub.3)--(CH.sub.2).sub.2--N(R.sub.1)(R.sub.2), and O(CH.sub.2).sub.2--N(R.sub.3)--C(.dbd.NR.sub.4)[N(R.sub.1)(R.sub.2)]; wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are each, independently, H or C.sub.1-C.sub.6 alkyl.

Embodiment 165

[0177] [0178] The oligomeric compound of embodiment 164, wherein the 2'-substituent is selected from among: a halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2, OCH.sub.2C(.dbd.O)--N(H)CH.sub.3, OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2, and OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2.

Embodiment 166

[0178] [0179] The oligomeric compound of any of embodiments 162-165 comprising at least one modified 3'-region nucleoside comprising a 2'-MOE sugar moiety.

Embodiment 167

[0179] [0180] The oligomeric compound of any of embodiments 162-166 comprising at least one modified 3'-region nucleoside comprising a 2'-OMe sugar moiety.

Embodiment 168

[0180] [0181] The oligomeric compound of any of embodiments 162-167 comprising at least one modified 3'-region nucleoside comprising a 2'-F sugar moiety.

Embodiment 169

[0181] [0182] The oligomeric compound of any of embodiments 162-168 comprising at least one modified 3'-region nucleoside comprising a 2'-(ara)-F sugar moiety.

Embodiment 170

[0182] [0183] The oligomeric compound of any of embodiments 162-169 comprising of at least one modified 3'-region nucleoside comprising a sugar surrogate.

Embodiment 171

[0183] [0184] The oligomeric compound of embodiment 170 comprising at least one modified 3'-region nucleoside comprising an F-HNA sugar moiety.

Embodiment 172

[0184] [0185] The oligomeric compound of embodiment 170 comprising at least one modified 3'-region nucleoside comprising an HNA sugar moiety.

Embodiment 173

[0185] [0186] The oligomeric compound of any of embodiments 1-172 comprising at least one modified 3'-region nucleoside comprising a modified nucleobase.

Embodiment 174

[0186] [0187] The oligomeric compound of any of embodiments 1-173, wherein each A comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: F, OCH.sub.3, O(CH.sub.2).sub.2--OCH.sub.3, and each B comprises a bicyclic sugar moiety selected from among: LNA and cEt.

Embodiment 175

[0187] [0188] The oligomeric compound of embodiment 174, wherein each A comprises O(CH.sub.2).sub.2--OCH.sub.3 and each B comprises cEt.

Embodiment 176

[0188] [0189] The oligomeric compound of any of embodiments 1-175, wherein the 3'-region has a motif selected from among: ABB, ABAA, AAABAA, AAAAABAA, AABAA, AAAABAA, AAABAA, ABAB, AAAAA, AAABB, AAAAAAAA, AAAAAAA, AAAAAA, AAAAB, AAAA, AAA, AA, AB, ABBB, ABAB, AABBB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type.

Embodiment 177

[0189] [0190] The oligomeric compound of embodiments 1-175, wherein the 3'-region has a motif selected from among: ABB; AAABAA; AABAA; AAAABAA; AAAAA; AAABB; AAAAAAAA; AAAAAAA; AAAAAA; AAAAB; AB; ABBB; and ABAB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type.

Embodiment 178

[0190] [0191] The oligomeric compound of embodiments 1-175, wherein the 3'-region has a motif selected from among: BBA, AAB, AAA, BBB, BBAA, AABB, WBBA, WAAB, BBBA, BBBBA, BBBB, BBBBBA, ABBBBB, BBAAA, AABBB, BBBAA, BBBBA, BBBBB, BABA, AAAAA, BBAAAA, AABBBB, BAAAA, and ABBBB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of a first type, a second type, or a third type.

Embodiment 179

[0191] [0192] The oligomeric compound of embodiments 176-178, wherein each A nucleoside comprises a 2'-substituted sugar moiety.

Embodiment 180

[0192] [0193] The oligomeric compound of embodiments 176-178, wherein at least one central region nucleoside comprises a 2'-substituted sugar moiety comprising a 2' substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF.sub.3, OCF.sub.3, O, S, or N(R.sub.m)-alkyl; O, S, or N(R.sub.m)-alkenyl; O, S or N(R.sub.m)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH.sub.2).sub.2SCH.sub.3, O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n) or O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and R.sub.n is, independently, H, an amino protecting group or substituted or unsubstituted C.sub.1-C.sub.10 alkyl; [0194] wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 181

[0194] [0195] The oligomeric compound of embodiment 180, wherein each A nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: a halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2, OCH.sub.2C(.dbd.O)--N(H)CH.sub.3, OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2, and OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2.

Embodiment 182

[0195] [0196] The oligomeric compound of embodiment 181, wherein each A nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: F, OCH.sub.3, O(CH.sub.2).sub.2--OCH.sub.3.

Embodiment 183

[0196] [0197] The oligomeric compound of embodiments 176-178, wherein each A nucleoside comprises a bicyclic sugar moiety.

Embodiment 184

[0197] [0198] The oligomeric compound of embodiment 183, wherein each A nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, .alpha.-LNA, ENA and 2'-thio LNA.

Embodiment 185

[0198] [0199] The oligomeric compound of any of embodiments 176-178, wherein each B nucleoside comprises a 2'-substituted sugar moiety.

Embodiment 186

[0199] [0200] The oligomeric compound of embodiment 185, wherein at least one modified central region nucleoside comprises a 2'-substituted sugar moiety comprising a 2' substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF.sub.3, OCF.sub.3, O, S, or N(R.sub.m)-alkyl; O, S, or N(R.sub.m)-alkenyl; O, S or N(R.sub.m)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH.sub.2).sub.2SCH.sub.3, O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n) or O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and R.sub.n is, independently, H, an amino protecting group or substituted or unsubstituted C.sub.1-C.sub.10 alkyl; [0201] wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 187

[0201] [0202] The oligomeric compound of embodiment 185, wherein each B nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: a halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2, OCH.sub.2C(.dbd.O)--N(H)CH.sub.3, OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2, and OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2.

Embodiment 188

[0202] [0203] The oligomeric compound of embodiment 187, wherein each B nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: F, OCH.sub.3, O(CH.sub.2).sub.2--OCH.sub.3.

Embodiment 189

[0203] [0204] The oligomeric compound of any of embodiments 176-178, wherein each B nucleoside comprises a bicyclic sugar moiety.

Embodiment 190

[0204] [0205] The oligomeric compound of embodiment 189, wherein each B nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, .alpha.-LNA, ENA and 2'-thio LNA.

Embodiment 191

[0205] [0206] The oligomeric compound of any of embodiments 176-190, wherein each A comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: F, OCH.sub.3, O(CH.sub.2).sub.2--OCH.sub.3, and each B comprises a bicyclic sugar moiety selected from among: LNA and cEt.

Embodiment 192

[0206] [0207] The oligomeric compound of embodiment 191, wherein each A comprises O(CH.sub.2).sub.2--OCH.sub.3 and each B comprises cEt.

Embodiment 193

[0207] [0208] The oligomeric compound of any of embodiments 176-192, wherein each W nucleoside comprises a 2'-substituted sugar moiety.

Embodiment 194

[0208] [0209] The oligomeric compound of embodiment 193, wherein at least one central region nucleoside comprises a 2'-substituted sugar moiety comprising a 2' substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF.sub.3, OCF.sub.3, O, S, or N(R.sub.m)-alkyl; O, S, or N(R.sub.m)-alkenyl; O, S or N(R.sub.m)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH.sub.2).sub.2SCH.sub.3, O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n) or O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and R.sub.n is, independently, H, an amino protecting group or substituted or unsubstituted C.sub.1-C.sub.10 alkyl; wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

Embodiment 195

[0209] [0210] The oligomeric compound of embodiment 193, wherein each W nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: a halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2, OCH.sub.2C(.dbd.O)--N(H)CH.sub.3, OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2, and OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2.

Embodiment 196

[0210] [0211] The oligomeric compound of embodiment 195, wherein each W nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: F, OCH.sub.3, O(CH.sub.2).sub.2--OCH.sub.3.

Embodiment 197

[0211] [0212] The oligomeric compound of any of embodiments 176-192, wherein each W nucleoside comprises a bicyclic sugar moiety.

Embodiment 198

[0212] [0213] The oligomeric compound of embodiment 197, wherein each W nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, .alpha.-LNA, ENA and 2'-thio LNA.

Embodiment 199

[0213] [0214] The oligomeric compound of any of embodiments 176-192, wherein each W comprises a modified nucleobase.

Embodiment 200

[0214] [0215] The oligomeric compound of embodiment 199, wherein each W comprises a modified nucleobase selected from among a 2-thio pyrimidine and a 5-propyne pyrimidine.

Embodiment 201

[0215] [0216] The oligomeric compound of embodiment 200, wherein each W comprises 2-thio-thymidine.

Embodiment 202

[0216] [0217] The oligomeric compound of embodiment 176-192, wherein each W comprises an F-HNA sugar moiety.

Embodiment 203

[0217] [0218] The oligomeric compound of embodiment 202, wherein each W nucleoside comprises an unmodified 2'-deoxyfuranose sugar moiety.

Embodiment 204

[0218] [0219] The oligomeric compound of embodiments 1-203, wherein the 5'-region has a motif selected from among: AB, ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, AAAB, ABBW, ABBWW, ABBB, ABBBB, ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW, AAAAA, and BBBBAA; [0220] wherein the 3'-region has a motif selected from among: BBA, AAB, AAA, BBB, BBAA, AABB, WBBA, WAAB, BBBA, BBBBA, BBBB, BBBBBA, ABBBBB, BBAAA, AABBB, BBBAA, BBBBA, BBBBB, BABA, AAAAA, BBAAAA, AABBBB, BAAAA, and ABBBB; [0221] wherein the central region has a nucleoside motif selected from among: DDDDDD, DDDDDDD, DDDDDDDD, DDDDDDDDD, DDDDDDDDDD, DXDDDDDDD, DDXDDDDDD, DDDXDDDDD, DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, DDDDDDDXD, DXXDDDDDD, DDDDDDXXD, DDXXDDDDD, DDDXXDDDD, DDDDXXDDD, DDDDDXXDD, DXDDDDDXD, DXDDDDXDD, DXDDDXDDD, DXDDXDDDD, DXDXDDDDD, DDXDDDDXD, DDXDDDXDD, DDXDDXDDD, DDXDXDDDD, DDDXDDDXD, DDDXDDXDD, DDDXDXDDD, DDDDXDDXD, DDDDXDXDD, and DDDDDXDXD, DDDDDDDD, DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDXD, DXDDDXDD, DXDDXDDD, DXDXDDDD, DXXDDDDD, DDXXDDDD, DDXDXDDD, DDXDDXDD, DXDDDDXD, DDDXXDDD, DDDXDXDD, DDDXDDXD, DDDDXXDD, DDDDXDXD, and DDDDDXXD, DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD, DXDDDXD, DXDDXDD, DXDXDDD, DXXDDDD, DDXXDDD, DDXDXDD, DDXDDXD, DDDXXDD, DDDXDXD, and DDDDXXD, DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXXDDD, DXDXDD, DXDDXD, DDXXDD, DDXDXD, and DDDXXD; and [0222] wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, each W is a modified nucleoside of a first type, a second type, or a third type, each D is an unmodified deoxynucleoside, and each X is a modified nucleoside or a modified nucleobase.

Embodiment 205

[0222] [0223] The oligomeric compound of embodiment 204, wherein the 5'-region has a motif selected from among: [0224] AB, ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, ABBW, ABBWW, ABBB, ABBBB, ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW, and BBBBAA; and wherein the 3'-region has a BBA motif.

Embodiment 206

[0224] [0225] The oligomeric compound of embodiment 204 or 205, wherein one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.

Embodiment 207

[0225] [0226] The oligomeric compound of embodiment 204 or 205, wherein one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises FHNA.

Embodiment 208

[0226] [0227] The oligomeric compound of embodiment 204 or 205, wherein one of A or B comprises cEt, another of A or B comprises a 2'-modified sugar moiety, and W comprises a 2-thio-thymidine nucleobase.

Embodiment 209

[0227] [0228] The oligomeric compound of embodiment 204 or 205, wherein one of A or B comprises cEt, another of A or B comprises a 2'-modified sugar moiety, and W comprises FHNA.

Embodiment 210

[0228] [0229] The oligomeric compound of embodiment 204 or 205, wherein each A comprises MOE, each B comprises cEt, and each W is selected from among cEt or FHNA.

Embodiment 211

[0229] [0230] The oligomeric compound of embodiment 204 or 205, wherein each W comprises cEt.

Embodiment 212

[0230] [0231] The oligomeric compound of embodiment 204 or 205, wherein each W comprises 2-thio-thymidine.

Embodiment 213

[0231] [0232] The oligomeric compound of embodiment 204 or 205, wherein each W comprises FHNA.

Embodiment 214

[0232] [0233] The oligomeric compound of any of embodiments 1-213 comprising at least one modified internucleoside linkage.

Embodiment 215

[0233] [0234] The oligomeric compound of embodiment 214, wherein each internucleoside linkage is a modified internucleoside linkage.

Embodiment 216

[0234] [0235] The oligomeric compound of embodiment 214 or 215 comprising at least one phosphorothioate internucleoside linkage.

Embodiment 217

[0235] [0236] The oligomeric compound of any of embodiments 214 or 215 comprising at least one methylphosphonate internucleoside linkage.

Embodiment 218

[0236] [0237] The oligomeric compound of any of embodiments 214 or 215 comprising one methylphosphonate internucleoside linkage.

Embodiment 219

[0237] [0238] The oligomeric compound of any of embodiments 214 or 215 comprising two methylphosphonate internucleoside linkages.

Embodiment 220

[0238] [0239] The oligomeric compound of embodiment 217, wherein at least one of the 3.sup.rd, 4.sup.th, 5.sup.th, 6.sup.th and/or, 7.sup.th internucleoside from the 5'-end is a methylphosphonate internucleoside linkage.

Embodiment 221

[0239] [0240] The oligomeric compound of embodiment 217, wherein at least one of the 3.sup.rd, 4.sup.th, 5.sup.th, 6.sup.th and/or 7.sup.th internucleoside from the 3'-end is a methylphosphonate internucleoside linkage.

Embodiment 222

[0240] [0241] The oligomeric compound of embodiment 217, wherein at least one of the 3.sup.rd, 4.sup.th, 5.sup.th, 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, and/or 12.sup.th internucleoside from the 5'-end is a methylphosphonate internucleoside linkage, and wherein at least one of the 3.sup.rd, 4.sup.th, 5.sup.th, 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, and/or 12.sup.th internucleoside from the 5'-end is a modified nucleoside.

Embodiment 223

[0241] [0242] The oligomeric compound of embodiment 217, wherein at least one of the 3.sup.rd, 4.sup.th, 5.sup.th, 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, and/or 12.sup.th internucleoside from the 3'-end is a methylphosphonate internucleoside linkage, and wherein at least one of the 3.sup.rd, 4.sup.th, 5.sup.th, 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th, 11.sup.th, and/or 12.sup.th internucleoside from the 3'-end is a modified nucleoside.

Embodiment 224

[0242] [0243] The oligomeric compound of any of embodiments 1-223 comprising at least one conjugate group.

Embodiment 225

[0243] [0244] The oligomeric compound of embodiment 1-223, wherein the conjugate group consists of a conjugate.

Embodiment 226

[0244] [0245] The oligomeric compound of embodiment 225, wherein the conjugate group consists of a conjugate and a conjugate linker.

Embodiment 227

[0245] [0246] The oligomeric compound of any of embodiments 1-226, wherein the nucleobase sequence of the modified oligonucleotide is 100% complementary to the nucleobase sequence of the target region of the target nucleic acid.

Embodiment 228

[0246] [0247] The oligomeric compound of any of embodiments 1-226, wherein the nucleobase sequence of the modified oligonucleotide contains one mismatch relative to the nucleobase sequence of the target region of the target nucleic acid.

Embodiment 229

[0247] [0248] The oligomeric compound of any of embodiments 1-226, wherein the nucleobase sequence of the modified oligonucleotide contains two mismatches relative to the nucleobase sequence of the target region of the target nucleic acid.

Embodiment 230

[0248] [0249] The oligomeric compound of any of embodiments 1-226, wherein the nucleobase sequence of the modified oligonucleotide comprises a hybridizing region and at least one non-targeting region, wherein the nucleobase sequence of the hybridizing region is complementary to the nucleobase sequence of the target region of the target nucleic acid.

Embodiment 231

[0249] [0250] The oligomeric compound of embodiment 230, wherein the nucleobase sequence of the hybridizing region is 100% complementary to the nucleobase sequence of the target region of the target nucleic acid.

Embodiment 232

[0250] [0251] The oligomeric compound of embodiment 230, wherein the nucleobase sequence of the hybridizing region contains one mismatched relative to the nucleobase sequence of the target region of the target nucleic acid.

Embodiment 233

[0251] [0252] The oligomeric compound of any of embodiments 230-232, wherein the nucleobase sequence of at least one non-targeting region is complementary to a portion of the hybridizing region of the modified oligonucleotide.

Embodiment 234

[0252] [0253] The oligomeric compound of embodiment 233, wherein the nucleobase sequence of at least one non-targeting region is 100% complementary to a portion of the hybridizing region of the modified oligonucleotide.

Embodiment 235

[0253] [0254] The oligomeric compound of embodiment 1-234 wherein the nucleobase sequence of the modified oligonucleotide comprises two non-targeting regions flanking a central hybridizing region.

Embodiment 236

[0254] [0255] The oligomeric compound of embodiment 235, wherein the two non-targeting regions are complementary to one another.

Embodiment 237

[0255] [0256] The oligomeric compound of embodiment 236, wherein the two non-targeting regions are 100% complementary to one another.

Embodiment 238

[0256] [0257] The oligomeric compound of any of embodiments 2-237, wherein the nucleobase sequence of the modified oligonucleotide aligns with the nucleobase of the target region of the target nucleic acid such that a distinguishing nucleobase of the target region of the target nucleic acid aligns with a target-selective nucleoside within the central region of the modified oligonucleotide.

Embodiment 239

[0257] [0258] The oligomeric compound of any of embodiments 3-237, wherein the nucleobase sequence of the modified oligonucleotide aligns with the nucleobase of the target region of the target nucleic acid such that the single distinguishing nucleobase of the target region of the target nucleic acid aligns with a target-selective nucleoside within the central region of the modified oligonucleotide.

Embodiment 240

[0258] [0259] The oligomeric compound of embodiment 238 or 239, wherein the target-selective nucleoside is the 5'-most nucleoside of the central region.

Embodiment 241

[0259] [0260] The oligomeric compound of embodiment 238 or 239, wherein the target-selective nucleoside is the 2.sup.nd nucleoside from the 5'-end of the central region.

Embodiment 242

[0260] [0261] The oligomeric compound of embodiment 238 or 239, wherein the target-selective nucleoside is at the 3.sup.rd nucleoside from the 5'-end of the central region.

Embodiment 243

[0261] [0262] The oligomeric compound of embodiment 238 or 239, wherein the target-selective nucleoside is at the 5.sup.th nucleoside from the 5'-end of the central region.

Embodiment 244

[0262] [0263] The oligomeric compound of embodiment 238 or 239, wherein the target-selective nucleoside is at the 7.sup.th nucleoside from the 5'-end of the central region.

Embodiment 245

[0263] [0264] The oligomeric compound of embodiment 238 or 239, wherein the target-selective nucleoside is at the 9.sup.th nucleoside from the 5'-end of the central region.

Embodiment 246

[0264] [0265] The oligomeric compound of any of embodiments 238 or 239, or 241-245, wherein the target-selective nucleoside is at the 2.sup.nd nucleoside from the 3'-end of the central region.

Embodiment 247

[0265] [0266] The oligomeric compound of any of embodiments 238 or 239, or 241-245, wherein the target-selective nucleoside is at the 5.sup.th nucleoside from the 3'-end of the central region.

Embodiment 248

[0266] [0267] The oligomeric compound of any of embodiments 1-247, wherein target-selective nucleoside is an unmodified deoxynucleoside.

Embodiment 249

[0267] [0268] The oligomeric compound of any of embodiments 1-247, wherein target-selective nucleoside is a modified nucleoside.

Embodiment 250

[0268] [0269] The oligomeric compound of embodiment 249, wherein the target-selective nucleoside is a sugar modified nucleoside.

Embodiment 251

[0269] [0270] The oligomeric compound of embodiment 250, wherein the target-selective nucleoside comprises a sugar modification selected from among: 2'-MOE, 2'-F, 2'-(ara)-F, HNA, FHNA, cEt, and .alpha.-L-LNA.

Embodiment 252

[0270] [0271] The oligomeric compound of any of embodiments 1-251, wherein the target-selective nucleoside comprises a nucleobase modification.

Embodiment 253

[0271] [0272] The oligomeric compound of embodiment 252, wherein the modified nucleobase is selected from among: a 2-thio pyrimidine and a 5-propyne pyrimidine.

Embodiment 254

[0272] [0273] The oligomeric compound of any of embodiments 1-253, wherein the oligomeric compound is an antisense compound.

Embodiment 255

[0273] [0274] The oligomeric compound of embodiment 254, wherein the oligomeric compound selectively reduces expression of the target relative to the non-target.

Embodiment 256

[0274] [0275] The oligomeric compound of embodiment 255, wherein the oligomeric compound reduces expression of target at least two-fold more than it reduces expression of the non-target.

Embodiment 257

[0275] [0276] The oligomeric compound of embodiment 256, having an EC.sub.50 for reduction of expression of target that is at least least two-fold lower than its EC.sub.50 for reduction of expression of the non-target, when measured in cells.

Embodiment 258

[0276] [0277] The oligomeric compound of embodiment 256, having an ED.sub.50 for reduction of expression of target that is at least least two-fold lower than its ED.sub.50 for reduction of expression of the non-target, when measured in an animal.

Embodiment 259

[0277] [0278] The oligomeric compound of embodiments 1-10, having an E-E-E-K-K-(D).sub.7-E-E-K motif, wherein each E is a 2'-MOE nucleoside and each K is a cEt nucleoside.

Embodiment 260

[0278] [0279] A method comprising contacting a cell with an oligomeric compound of any of embodiments 1-259.

Embodiment 261

[0279] [0280] The method of embodiment 260, wherein the cell is in vitro.

Embodiment 262

[0280] [0281] The method of embodiment 260, wherein the cell is in an animal.

Embodiment 263

[0281] [0282] The method of embodiment 262, wherein the animal is a human.

Embodiment 264

[0282] [0283] The method of embodiment 263, wherein the animal is a mouse.

Embodiment 265

[0283] [0284] A pharmaceutical composition comprising an oligomeric compound of any of embodiments 1-259 and a pharmaceutically acceptable carrier or diluent.

Embodiment 266

[0284] [0285] A method of administering a pharmaceutical composition of embodiment 265 to an animal.

Embodiment 267

[0285] [0286] The method of embodiment 266, wherein the animal is a human.

Embodiment 268

[0286] [0287] The method of embodiment 266, wherein the animal is a mouse.

Embodiment 269

[0287] [0288] Use of an oligomeric compound of any of embodiments 1-259 for the preparation of a medicament for the treatment or amelioration of Alzheimer's disease, Creutzfeldt-Jakob disease, fatal familial insomnia, Alexander disease, Parkinson's disease, amyotrophic lateral sclerosis, dentato-rubral and pallido-luysian atrophy DRPA, spino-cerebellar ataxia, Torsion dystonia, cardiomyopathy, chronic obstructive pulmonary disease (COPD), liver disease, hepatocellular carcinoma, systemic lupus erythematosus, hypercholesterolemia, breast cancer, asthma, Type 1 diabetes, Rheumatoid arthritis, Graves disease, SLE, spinal and bulbar muscular atrophy, Kennedy's disease, progressive childhood posterior subcapsular cataracts, cholesterol gallstone disease, arthrosclerosis, cardiovascular disease, primary hypercalciuria, alpha-thallasemia, obsessive compulsive disorder, Anxiety, comorbid depression, congenital visual defects, hypertension, metabolic syndrome, prostate cancer, congential myasthenic syndrome, peripheral arterial disease, atrial fibrillation, sporadic pheochromocytoma, congenital malformations, Machado-Joseph disease, Huntington's disease, and Autosomal Dominant Retinitis Pigmentosa disease.

Embodiment 270

[0288] [0289] A method of ameliorating a symptom of Alzheimer's disease, Creutzfeldt-Jakob disease, fatal familial insomnia, Alexander disease, Parkinson's disease, amyotrophic lateral sclerosis, dentato-rubral and pallido-luysian atrophy DRPA, spino-cerebellar ataxia, Torsion dystonia, cardiomyopathy, chronic obstructive pulmonary disease (COPD), liver disease, hepatocellular carcinoma, systemic lupus erythematosus, hypercholesterolemia, breast cancer, asthma, Type 1 diabetes, Rheumatoid arthritis, Graves disease, SLE, spinal and bulbar muscular atrophy, Kennedy's disease, progressive childhood posterior subcapsular cataracts, cholesterol gallstone disease, arthrosclerosis, cardiovascular disease, primary hypercalciuria, alpha-thallasemia, obsessive compulsive disorder, Anxiety, comorbid depression, congenital visual defects, hypertension, metabolic syndrome, prostate cancer, congential myasthenic syndrome, peripheral arterial disease, atrial fibrillation, sporadic pheochromocytoma, congenital malformations, Machado-Joseph disease, Huntington's disease, and Autosomal Dominant Retinitis Pigmentosa disease, comprising administering an oligomeric compound of any of embodiments 1-259 to an animal in need thereof.

Embodiment 271

[0289] [0290] The method of embodiment 270, wherein the animal is a human.

Embodiment 272

[0290] [0291] The method of embodiment 270, wherein the animal is a mouse.

[0292] In certain embodiments, including but not limited to any of the above numbered embodiments, oligomeric compounds including oligonucleotides described herein are capable of modulating expression of a target RNA. In certain embodiments, the target RNA is associated with a disease or disorder, or encodes a protein that is associated with a disease or disorder. In certain embodiments, the oligomeric compounds or oligonucleotides provided herein modulate the expression of function of such RNA to alleviate one or more symptom of the disease or disorder.

[0293] In certain embodiments, oligomeric compounds including oligonucleotides describe herein are useful in vitro. In certain embodiments such oligomeric compounds are used in diagnostics and/or for target validation experiments.

DETAILED DESCRIPTION OF THE INVENTION

[0294] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

[0295] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

A. DEFINITIONS

[0296] Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Certain such techniques and procedures may be found for example in "Carbohydrate Modifications in Antisense Research" Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 21.sup.st edition, 2005; and "Antisense Drug Technology, Principles, Strategies, and Applications" Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., "Molecular Cloning, A laboratory Manual," 2.sup.nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.

[0297] Unless otherwise indicated, the following terms have the following meanings:

[0298] As used herein, "nucleoside" means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.

[0299] As used herein, "chemical modification" means a chemical difference in a compound when compared to a naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence.

[0300] As used herein, "furanosyl" means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.

[0301] As used herein, "naturally occurring sugar moiety" means a ribofuranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in naturally occurring DNA.

[0302] As used herein, "sugar moiety" means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.

[0303] As used herein, "modified sugar moiety" means a substituted sugar moiety or a sugar surrogate.

[0304] As used herein, "substituted sugar moiety" means a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2'-position, the 3'-position, the 5'-position and/or the 4'-position. Certain substituted sugar moieties are bicyclic sugar moieties.

[0305] As used herein, "2'-substituted sugar moiety" means a furanosyl comprising a substituent at the 2'-position other than H or OH. Unless otherwise indicated, a 2'-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2'-substituent of a 2'-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring.

[0306] As used herein, "MOE" means --OCH.sub.2CH.sub.2OCH.sub.3.

[0307] As used herein, "2'-F nucleoside" refers to a nucleoside comprising a sugar comprising fluorine at the 2' position. Unless otherwise indicated, the fluorine in a 2'-F nucleoside is in the ribo position (replacing the OH of a natural ribose).

[0308] As used herein, "2'-(ara)-F" refers to a 2'-F substituted nucleoside, wherein the fluoro group is in the arabino position.

##STR00001##

[0309] As used herein the term "sugar surrogate" means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside sub-units are capable of linking together and/or linking to other nucleosides to form an oligomeric compound which is capable of hybridizing to a complementary oligomeric compound. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols.

[0310] As used herein, "bicyclic sugar moiety" means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2'-carbon and the 4'-carbon of the furanosyl.

[0311] As used herein, "nucleotide" means a nucleoside further comprising a phosphate linking group. As used herein, "linked nucleosides" may or may not be linked by phosphate linkages and thus includes, but is not limited to "linked nucleotides." As used herein, "linked nucleosides" are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked).

[0312] As used herein, "nucleobase" means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified.

[0313] As used herein the terms, "unmodified nucleobase" or "naturally occurring nucleobase" means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).

[0314] As used herein, "modified nucleobase" means any nucleobase that is not a naturally occurring nucleobase.

[0315] As used herein, "modified nucleoside" means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides comprise a modified sugar moiety and/or a modified nucleobase.

[0316] As used herein, "bicyclic nucleoside" or "BNA" means a nucleoside comprising a bicyclic sugar moiety.

[0317] As used herein, "constrained ethyl nucleoside" or "cEt" means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH.sub.3)--O-2' bridge.

[0318] As used herein, "locked nucleic acid nucleoside" or "LNA" means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH.sub.2--O-2' bridge.

[0319] As used herein, "2'-substituted nucleoside" means a nucleoside comprising a substituent at the 2'-position other than H or OH. Unless otherwise indicated, a 2'-substituted nucleoside is not a bicyclic nucleoside.

[0320] As used herein, "2'-deoxynucleoside" means a nucleoside comprising 2'-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2'-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).

[0321] As used herein, "RNA-like nucleoside" means a modified nucleoside that adopts a northern configuration and functions like RNA when incorporated into an oligonucleotide. RNA-like nucleosides include, but are not limited to 3'-endo furanosyl nucleosides and RNA surrogates.

[0322] As used herein, "3'-endo-furanosyl nucleoside" means an RNA-like nucleoside that comprises a substituted sugar moiety that has a 3'-endo conformation. 3'-endo-furanosyl nucleosides include, but are not limited to: 2'-MOE, 2'-F, 2'-OMe, LNA, ENA, and cEt nucleosides.

[0323] As used herein, "RNA-surrogate nucleoside" means an RNA-like nucleoside that does not comprise a furanosyl. RNA-surrogate nucleosides include, but are not limited to hexitols and cyclopentanes.

[0324] As used herein, "oligonucleotide" means a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.

[0325] As used herein "oligonucleoside" means an oligonucleotide in which none of the internucleoside linkages contains a phosphorus atom. As used herein, oligonucleotides include oligonucleosides.

[0326] As used herein, "modified oligonucleotide" means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.

[0327] As used herein "internucleoside linkage" means a covalent linkage between adjacent nucleosides in an oligonucleotide.

[0328] As used herein "naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage.

[0329] As used herein, "modified internucleoside linkage" means any internucleoside linkage other than a naturally occurring internucleoside linkage.

[0330] As used herein, "oligomeric compound" means a polymeric structure comprising two or more sub-structures. In certain embodiments, an oligomeric compound comprises an oligonucleotide. In certain embodiments, an oligomeric compound comprises one or more conjugate groups and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide.

[0331] As used herein, "terminal group" means one or more atom attached to either, or both, the 3' end or the 5' end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more terminal group nucleosides.

[0332] As used herein, "conjugate" means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.

[0333] As used herein, "conjugate linking group" means any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound.

[0334] As used herein, "antisense compound" means a compound comprising or consisting of an oligonucleotide at least a portion of which is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity.

[0335] As used herein, "antisense activity" means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid.

[0336] As used herein, "detecting" or "measuring" means that a test or assay for detecting or measuring is performed. Such detection and/or measuring may result in a value of zero. Thus, if a test for detection or measuring results in a finding of no activity (activity of zero), the step of detecting or measuring the activity has nevertheless been performed.

[0337] As used herein, "detectable and/or measurable activity" means a measurable activity that is not zero.

[0338] As used herein, "essentially unchanged" means little or no change in a particular parameter, particularly relative to another parameter which changes much more. In certain embodiments, a parameter is essentially unchanged when it changes less than 5%. In certain embodiments, a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold. For example, in certain embodiments, an antisense activity is a change in the amount of a target nucleic acid. In certain such embodiments, the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target nucleic acid does, but the change need not be zero.

[0339] As used herein, "expression" means the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenlyation, addition of 5'-cap), and translation.

[0340] As used herein, "target nucleic acid" means a nucleic acid molecule to which an antisense compound is intended to hybridize.

[0341] As used herein, "non-target nucleic acid" means a nucleic acid molecule to which hybridization of an antisense compound is not intended or desired. In certain embodiments, antisense compounds do hybridize to a non-target, due to homology between the target (intended) and non-target (un-intended).

[0342] As used herein, "mRNA" means an RNA molecule that encodes a protein.

[0343] As used herein, "pre-mRNA" means an RNA transcript that has not been fully processed into mRNA. Pre-RNA includes one or more intron.

[0344] As used herein, "object RNA" means an RNA molecule other than a target RNA, the amount, activity, splicing, and/or function of which is modulated, either directly or indirectly, by a target nucleic acid. In certain embodiments, a target nucleic acid modulates splicing of an object RNA. In certain such embodiments, an antisense compound modulates the amount or activity of the target nucleic acid, resulting in a change in the splicing of an object RNA and ultimately resulting in a change in the activity or function of the object RNA.

[0345] As used herein, "microRNA" means a naturally occurring, small, non-coding RNA that represses gene expression of at least one mRNA. In certain embodiments, a microRNA represses gene expression by binding to a target site within a 3' untranslated region of an mRNA. In certain embodiments, a microRNA has a nucleobase sequence as set forth in miRBase, a database of published microRNA sequences found at http://microrna.sanger.ac.uk/sequences/. In certain embodiments, a microRNA has a nucleobase sequence as set forth in miRBase version 12.0 released September 2008, which is herein incorporated by reference in its entirety.

[0346] As used herein, "microRNA mimic" means an oligomeric compound having a sequence that is at least partially identical to that of a microRNA. In certain embodiments, a microRNA mimic comprises the microRNA seed region of a microRNA. In certain embodiments, a microRNA mimic modulates translation of more than one target nucleic acids. In certain embodiments, a microRNA mimic is double-stranded.

[0347] As used herein, "differentiating nucleobase" means a nucleobase that differs between two nucleic acids. In certain instances, a target region of a target nucleic acid differs by 1-4 nucleobases from a non-target nucleic acid. Each of those differences is referred to as a differentiating nucleobase. In certain instances, a differentiating nucleobase is a single-nucleotide polymorphism.

[0348] As used herein, "target-selective nucleoside" means a nucleoside of an antisense compound that corresponds to a differentiating nucleobase of a target nucleic acid.

[0349] As used herein, "allele" means one of a pair of copies of a gene existing at a particular locus or marker on a specific chromosome, or one member of a pair of nucleobases existing at a particular locus or marker on a specific chromosome, or one member of a pair of nucleobase sequences existing at a particular locus or marker on a specific chromosome. For a diploid organism or cell or for autosomal chromosomes, each allelic pair will normally occupy corresponding positions (loci) on a pair of homologous chromosomes, one inherited from the mother and one inherited from the father. If these alleles are identical, the organism or cell is said to be "homozygous" for that allele; if they differ, the organism or cell is said to be "heterozygous" for that allele. "Wild-type allele" refers to the genotype typically not associated with disease or dysfunction of the gene product. "Mutant allele" refers to the genotype associated with disease or dysfunction of the gene product.

[0350] As used herein, "allelic variant" means a particular identity of an allele, where more than one identity occurs. For example, an allelic variant may refer to either the mutant allele or the wild-type allele.

[0351] As used herein, "single nucleotide polymorphism" or "SNP" means a single nucleotide variation between the genomes of individuals of the same species. In some cases, a SNP may be a single nucleotide deletion or insertion. In general, SNPs occur relatively frequently in genomes and thus contribute to genetic diversity. The location of a SNP is generally flanked by highly conserved sequences. An individual may be homozygous or heterozygous for an allele at each SNP site.

[0352] As used herein, "single nucleotide polymorphism site" or "SNP site" refers to the nucleotides surrounding a SNP contained in a target nucleic acid to which an antisense compound is targeted.

[0353] As used herein, "targeting" or "targeted to" means the association of an antisense compound to a particular target nucleic acid molecule or a particular region of a target nucleic acid molecule. An antisense compound targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions.

[0354] As used herein, "nucleobase complementarity" or "complementarity" when in reference to nucleobases means a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase means a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.

[0355] As used herein, "non-complementary" in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another.

[0356] As used herein, "complementary" in reference to oligomeric compounds (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity under stringent conditions. Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. In certain embodiments, complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary). In certain embodiments, complementary oligomeric compounds or regions are 80% complementary. In certain embodiments, complementary oligomeric compounds or regions are 90% complementary. In certain embodiments, complementary oligomeric compounds or regions are 95% complementary. In certain embodiments, complementary oligomeric compounds or regions are 100% complementary.

[0357] As used herein, "mismatch" means a nucleobase of a first oligomeric compound that is not capable of pairing with a nucleobase at a corresponding position of a second oligomeric compound, when the first and second oligomeric compound are aligned. Either or both of the first and second oligomeric compounds may be oligonucleotides.

[0358] As used herein, "hybridization" means the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.

[0359] As used herein, "specifically hybridizes" means the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site. In certain embodiments, an antisense oligonucleotide specifically hybridizes to more than one target site.

[0360] As used herein, "fully complementary" in reference to an oligonucleotide or portion thereof means that each nucleobase of the oligonucleotide or portion thereof is capable of pairing with a nucleobase of a complementary nucleic acid or contiguous portion thereof. Thus, a fully complementary region comprises no mismatches or unhybridized nucleobases in either strand.

[0361] As used herein, "percent complementarity" means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.

[0362] As used herein, "percent identity" means the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.

[0363] As used herein, "modulation" means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of a molecule, function, or activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. As a further example, modulation of expression can include a change in splice site selection of pre-mRNA processing, resulting in a change in the absolute or relative amount of a particular splice-variant compared to the amount in the absence of modulation.

[0364] As used herein, "modification motif" means a pattern of chemical modifications in an oligomeric compound or a region thereof. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligomeric compound.

[0365] As used herein, "nucleoside motif" means a pattern of nucleoside modifications in an oligomeric compound or a region thereof. The linkages of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.

[0366] As used herein, "sugar motif" means a pattern of sugar modifications in an oligomeric compound or a region thereof.

[0367] As used herein, "linkage motif" means a pattern of linkage modifications in an oligomeric compound or region thereof. The nucleosides of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.

[0368] As used herein, "nucleobase modification motif" means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence.

[0369] As used herein, "sequence motif" means a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modifications and thus may have any combination of chemical modifications, including no chemical modifications.

[0370] As used herein, "type of modification" in reference to a nucleoside or a nucleoside of a "type" means the chemical modification of a nucleoside and includes modified and unmodified nucleosides. Accordingly, unless otherwise indicated, a "nucleoside having a modification of a first type" may be an unmodified nucleoside.

[0371] As used herein, "differently modified" mean chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are "differently modified," even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are "differently modified," even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2'-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2'-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.

[0372] As used herein, "the same type of modifications" refers to modifications that are the same as one another, including absence of modifications. Thus, for example, two unmodified DNA nucleoside have "the same type of modification," even though the DNA nucleoside is unmodified. Such nucleosides having the same type modification may comprise different nucleobases.

[0373] As used herein, "pharmaceutically acceptable carrier or diluent" means any substance suitable for use in administering to an animal. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.

[0374] As used herein, "substituent" and "substituent group," means an atom or group that replaces the atom or group of a named parent compound. For example a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2'-substituent is any atom or group at the 2'-position of a nucleoside other than H or OH). Substituent groups can be protected or unprotected. In certain embodiments, compounds of the present invention have substituents at one or at more than one position of the parent compound. Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.

[0375] Likewise, as used herein, "substituent" in reference to a chemical functional group means an atom or group of atoms differs from the atom or a group of atoms normally present in the named functional group. In certain embodiments, a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group). Unless otherwise indicated, groups amenable for use as substituents include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (--C(O)R.sub.aa), carboxyl (--C(O)O--R.sub.aa), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (--O--R.sub.aa), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (--N(R.sub.bb)(R.sub.cc)), imino(.dbd.NR.sub.bb), amido (--C(O)N(R.sub.bb)(R.sub.cc) or --N(R.sub.bb)C(O)R.sub.aa), azido (--N.sub.3), nitro (--NO.sub.2), cyano (--CN), carbamido (--OC(O)N(R.sub.bb)(R.sub.cc) or --N(R.sub.bb)C(O)OR.sub.aa), ureido (--N(R.sub.bb)C(O)N(R.sub.bb)(R.sub.cc)), thioureido (--N(R.sub.bb)C(S)N(R.sub.bb)--R.sub.cc)), guanidinyl (--N(R.sub.bb)C(.dbd.NR.sub.bb)N(R.sub.bb)(R.sub.cc)), amidinyl (--C(.dbd.NR.sub.bb)N(R.sub.bb)(R.sub.cc) or --N(R.sub.bb)C(.dbd.NR.sub.bb)(R.sub.aa)), thiol (--SR.sub.bb), sulfinyl (--S(O)R.sub.bb), sulfonyl (--S(O).sub.2R.sub.bb) and sulfonamidyl (--S(O).sub.2N(R.sub.bb)(R.sub.cc) or --N(R.sub.bb)S--(O).sub.2R.sub.bb). Wherein each R.sub.aa, R.sub.bb and R.sub.cc is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.

[0376] As used herein, "alkyl," as used herein, means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C.sub.1-C.sub.12 alkyl) with from 1 to about 6 carbon atoms being more preferred.

[0377] As used herein, "alkenyl," means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.

[0378] As used herein, "alkynyl," means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.

[0379] As used herein, "acyl," means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula --C(O)--X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.

[0380] As used herein, "alicyclic" means a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substituent groups.

[0381] As used herein, "aliphatic" means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.

[0382] As used herein, "alkoxy" means a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.

[0383] As used herein, "aminoalkyl" means an amino substituted C.sub.1-C.sub.12 alkyl radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.

[0384] As used herein, "aralkyl" and "arylalkyl" mean an aromatic group that is covalently linked to a C.sub.1-C.sub.12 alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.

[0385] As used herein, "aryl" and "aromatic" mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituent groups.

[0386] As used herein, "halo" and "halogen," mean an atom selected from fluorine, chlorine, bromine and iodine.

[0387] As used herein, "heteroaryl," and "heteroaromatic," mean a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.

B. OLIGOMERIC COMPOUNDS

[0388] In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, such oligomeric compounds comprise oligonucleotides optionally comprising one or more conjugate and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide. In certain embodiments, oligonucleotides comprise one or more chemical modifications. Such chemical modifications include modifications of one or more nucleoside (including modifications to the sugar moiety and/or the nucleobase) and/or modifications to one or more internucleoside linkage.

[0389] a. Certain Modified Nucleosides

[0390] In certain embodiments, provided herein are oligomeric compounds comprising or consisting of oligonucleotides comprising at least one modified nucleoside. Such modified nucleosides comprise a modified sugar moeity, a modified nucleobase, or both a modified sugar moiety and a modified nucleobase.

[0391] i. Certain Modified Sugar Moieties

[0392] In certain embodiments, compounds of the invention comprise one or more modified nucleosides comprising a modified sugar moiety. Such compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to an oligonucleotide comprising only nucleosides comprising naturally occurring sugar moieties. In certain embodiments, modified sugar moieties are substituted sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.

[0393] In certain embodiments, modified sugar moieties are substituted sugar moieties comprising one or more non-bridging sugar substituent, including but not limited to substituents at the 2' and/or 5' positions. Examples of sugar substituents suitable for the 2'-position, include, but are not limited to: 2'-F, 2'-OCH.sub.3 ("OMe" or "O-methyl"), and 2'-O(CH.sub.2).sub.2OCH.sub.3 ("MOE"). In certain embodiments, sugar substituents at the 2' position is selected from allyl, amino, azido, thio, O-allyl, O--C.sub.1-C.sub.10 alkyl, O--C.sub.1-C.sub.10 substituted alkyl; OCF.sub.3, O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2--O--N(Rm)(Rn), and O--CH.sub.2--C(.dbd.O)--N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C.sub.1-C.sub.10 alkyl. Examples of sugar substituents at the 5'-position, include, but are not limited to: 5'-methyl (R or S); 5'-vinyl, and 5'-methoxy. In certain embodiments, substituted sugars comprise more than one non-bridging sugar substituent, for example, 2'-F-5'-methyl sugar moieties (see, e.g., PCT International Application WO 2008/101157, for additional 5',2'-bis substituted sugar moieties and nucleosides).

[0394] Nucleosides comprising 2'-substituted sugar moieties are referred to as 2'-substituted nucleosides. In certain embodiments, a 2'-substituted nucleoside comprises a 2'-substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF.sub.3, OCF.sub.3, O, S, or N(R.sub.m)-alkyl; O, S, or N(R.sub.m)-alkenyl; O, S or N(R.sub.m)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH.sub.2).sub.2SCH.sub.3, O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n) or O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and R.sub.n is, independently, H, an amino protecting group or substituted or unsubstituted C.sub.1-C.sub.10 alkyl. These 2'-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

[0395] In certain embodiments, a 2'-substituted nucleoside comprises a 2'-substituent group selected from F, NH.sub.2, N.sub.3, OCF.sub.3, O--CH.sub.3, O(CH.sub.2).sub.3NH.sub.2, CH.sub.2--CH.dbd.CH.sub.2, O--CH.sub.2--CH.dbd.CH.sub.2, OCH.sub.2CH.sub.2OCH.sub.3, O(CH.sub.2).sub.2SCH.sub.3, O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n), O(CH.sub.2).sub.2--O--(CH.sub.2).sub.2N(CH.sub.3).sub.2, and N-substituted acetamide (O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n) where each R.sub.m and R.sub.n is, independently, H, an amino protecting group or substituted or unsubstituted C.sub.1-C.sub.10 alkyl.

[0396] In certain embodiments, a 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent group selected from F, OCF.sub.3, O--CH.sub.3, OCH.sub.2CH.sub.2OCH.sub.3, O(CH.sub.2).sub.2SCH.sub.3, O--(CH.sub.2).sub.2--O--N(CH.sub.3).sub.2, --O(CH.sub.2).sub.2O(CH.sub.2).sub.2N(CH.sub.3).sub.2, and O--CH.sub.2--C(.dbd.O)--N(H)CH.sub.3.

[0397] In certain embodiments, a 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent group selected from F, O--CH.sub.3, and OCH.sub.2CH.sub.2OCH.sub.3.

[0398] Certain modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms. Examples of such 4' to 2' sugar substituents, include, but are not limited to: --[C(R.sub.a)(R.sub.b)].sub.n--, --[C(R.sub.a)(R.sub.b)].sub.n--O--, --C(R.sub.aR.sub.b)--N(R)--O-- or, --C(R.sub.aR.sub.b)--O--N(R)--; 4'-CH.sub.2-2', 4'-(CH.sub.2).sub.2-2', 4'-(CH.sub.2)--O-2' (LNA); 4'-(CH.sub.2)--S-2; 4'-(CH.sub.2).sub.2--O-2' (ENA); 4'-CH(CH.sub.3)--O-2' (cEt) and 4'-CH(CH.sub.2OCH.sub.3)--O-2', and analogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4'-C(CH.sub.3)(CH.sub.3)--O-2' and analogs thereof, (see, e.g., WO2009/006478, published Jan. 8, 2009); 4'-CH.sub.2--N(OCH.sub.3)-2' and analogs thereof (see, e.g., WO2008/150729, published Dec. 11, 2008); 4'-CH.sub.2--O--N(CH.sub.3)-2' (see, e.g., US2004/0171570, published Sep. 2, 2004); 4'-CH.sub.2--O--N(R)-2', and 4'-CH.sub.2--N(R)--O-2'-, wherein each R is, independently, H, a protecting group, or C.sub.1-C.sub.12 alkyl; 4'-CH.sub.2--N(R)--O-2', wherein R is H, C.sub.1-C.sub.12 alkyl, or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4'-CH.sub.2--C(H)(CH.sub.3)-2' (see, e.g., Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH.sub.2--C(.dbd.CH.sub.2)-2' and analogs thereof (see, published PCT International Application WO 2008/154401, published on Dec. 8, 2008).

[0399] In certain embodiments, such 4' to 2' bridges independently comprise from 1 to 4 linked groups independently selected from --[C(R.sub.a)(R.sub.b)].sub.n--, --C(R.sub.a).dbd.C(R.sub.b)--, --C(R.sub.a).dbd.N--, --C(.dbd.NR.sub.a)--, --C(.dbd.O)--, --C(.dbd.S)--, --O--, --Si(R.sub.a).sub.2--, --S(.dbd.O).sub.x--, and --N(R.sub.a)--;

[0400] wherein:

[0401] x is 0, 1, or 2;

[0402] n is 1, 2, 3, or 4;

[0403] each R.sub.a and R.sub.b is, independently, H, a protecting group, hydroxyl, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C.sub.5-C.sub.7 alicyclic radical, substituted C.sub.5-C.sub.7 alicyclic radical, halogen, OJ.sub.1, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1, acyl (C(.dbd.O)--H), substituted acyl, CN, sulfonyl (S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and

[0404] each J.sub.1 and J.sub.2 is, independently, H, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, acyl (C(.dbd.O)--H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C.sub.1-C.sub.12 aminoalkyl, substituted C.sub.1-C.sub.12 aminoalkyl, or a protecting group.

[0405] Nucleosides comprising bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to, (A) .alpha.-L-Methyleneoxy (4'-CH.sub.2--O-2') BNA, (B) .beta.-D-Methyleneoxy (4'-CH.sub.2--O-2') BNA (also referred to as locked nucleic acid or LNA), (C) Ethyleneoxy (4'-(CH.sub.2).sub.2--O-2') BNA, (D) Aminooxy (4'-CH.sub.2--O--N(R)-2') BNA, (E) Oxyamino (4'-CH.sub.2--N(R)--O-2') BNA, (F) Methyl(methyleneoxy) (4'-CH(CH.sub.3)--O-2') BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4'-CH.sub.2--S-2') BNA, (H) methylene-amino (4'-CH.sub.2--N(R)-2') BNA, (I) methyl carbocyclic (4'-CH.sub.2--CH(CH.sub.3)-2') BNA, (J) propylene carbocyclic (4'-(CH.sub.2).sub.3-2') BNA, and (K) Ethylene(methoxy) (4'-(CH(CH.sub.2OMe)-O-2') BNA (also referred to as constrained MOE or cMOE) as depicted below.

##STR00002## ##STR00003##

wherein Bx is a nucleobase moiety and R is, independently, H, a protecting group, or C.sub.1-C.sub.12 alkyl.

[0406] Additional bicyclic sugar moieties are known in the art, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO 2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; U.S. patent Ser. Nos. 12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and PCT International Applications Nos. PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.

[0407] In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, a nucleoside comprising a 4'-2' methylene-oxy bridge, may be in the .alpha.-L configuration or in the .beta.-D configuration. Previously, .alpha.-L-methyleneoxy (4'-CH.sub.2--O-2') bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0408] In certain embodiments, substituted sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5'-substituted and 4'-2' bridged sugars). (see, PCT International Application WO 2007/134181, published on Nov. 22, 2007, wherein LNA is substituted with, for example, a 5'-methyl or a 5'-vinyl group).

[0409] In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the naturally occurring sugar is substituted, e.g., with a sulfer, carbon or nitrogen atom. In certain such embodiments, such modified sugar moiety also comprises bridging and/or non-bridging substituents as described above. For example, certain sugar surrogates comprise a 4'-sulfer atom and a substitution at the 2'-position (see, e.g., published U.S. Patent Application US2005/0130923, published on Jun. 16, 2005) and/or the 5' position. By way of additional example, carbocyclic bicyclic nucleosides having a 4'-2' bridge have been described (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740).

[0410] In certain embodiments, sugar surrogates comprise rings having other than 5-atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), and those compounds having Formula VII:

##STR00004##

wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:

[0411] Bx is a nucleobase moiety;

[0412] T.sub.3 and T.sub.4 are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T.sub.3 and T.sub.4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T.sub.3 and T.sub.4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3'-terminal group;

q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each, independently, H, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, or substituted C.sub.2-C.sub.6 alkynyl; and

[0413] each of R.sub.1 and R.sub.2 is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, OC(.dbd.X)J.sub.1, OC(.dbd.X)NJ.sub.1J.sub.2, NJ.sub.3C(.dbd.X)NJ.sub.1J.sub.2, and CN, wherein X is O, S or NJ.sub.1, and each J.sub.1, J.sub.2, and J.sub.3 is, independently, H or C.sub.1-C.sub.6 alkyl.

[0414] In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each H. In certain embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 is other than H. In certain embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R.sub.1 and R.sub.2 is F. In certain embodiments, R.sub.1 is fluoro and R.sub.2 is H, R.sub.1 is methoxy and R.sub.2 is H, and R.sub.1 is methoxyethoxy and R.sub.2 is H.

[0415] Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see, e.g., review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

[0416] Combinations of modifications are also provided without limitation, such as 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5',2'-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2'-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5'-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4'-CH.sub.2--O-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

[0417] In certain embodiments, the present invention provides oligonucleotides comprising modified nucleosides. Those modified nucleotides may include modified sugars, modified nucleobases, and/or modified linkages. The specific modifications are selected such that the resulting oligonucleotides possess desirable characteristics. In certain embodiments, oligonucleotides comprise one or more RNA-like nucleosides. In certain embodiments, oligonucleotides comprise one or more DNA-like nucleotides.

[0418] ii. Certain Modified Nucleobases

[0419] In certain embodiments, nucleosides of the present invention comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present invention comprise one or more modified nucleobases.

[0420] In certain embodiments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (--C.ident.C--CH.sub.3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288.

[0421] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0422] b. Certain Internucleoside Linkages

[0423] In certain embodiments, nucleosides may be linked together using any internucleoside linkage to form oligonucleotides. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters (P.dbd.O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P.dbd.S). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (--CH.sub.2--N(CH.sub.3)--O--CH.sub.2--) thiodiester (--O--C(O)--S--), thionocarbamate (--O--C(O)(NH)--S--); siloxane (--O--Si(H).sub.2--O--); and N,N'-dimethylhydrazine (--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.

[0424] The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), .alpha. or .beta. such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.

[0425] Neutral internucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI (3'-CH.sub.2--N(CH.sub.3)--O-5'), amide-3 (3'-CH.sub.2--C(.dbd.P)--N(H)-5'), amide-4 (3'-CH.sub.2--N(H)--C(.dbd.O)-5'), formacetal (3'-O--CH.sub.2--O-5'), and thioformacetal (3'-S--CH.sub.2--O-5'). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH.sub.2 component parts.

[0426] i. 3'-Endo Modifications

[0427] In one aspect of the present disclosure, oligomeric compounds include nucleosides synthetically modified to induce a 3'-endo sugar conformation. A nucleoside can incorporate synthetic modifications of the heterocyclic base moiety, the sugar moiety or both to induce a desired 3'-endo sugar conformation. These modified nucleosides are used to mimic RNA like nucleosides so that particular properties of an oligomeric compound can be enhanced while maintaining the desirable 3'-endo conformational geometry. There is an apparent preference for an RNA type duplex (A form helix, predominantly 3'-endo) as a requirement of RNA interference which is supported in part by the fact that duplexes composed of 2'-deoxy-2'-F-nucleosides appear efficient in triggering RNAi response in the C. elegans system. Properties that are enhanced by using more stable 3'-endo nucleosides include but aren't limited to modulation of pharmacokinetic properties through modification of protein binding, protein off-rate, absorption and clearance; modulation of nuclease stability as well as chemical stability; modulation of the binding affinity and specificity of the oligomer (affinity and specificity for enzymes as well as for complementary sequences); and increasing efficacy of RNA cleavage. The present invention provides oligomeric compounds having one or more nucleosides modified in such a way as to favor a C3'-endo type conformation.

##STR00005##

[0428] Nucleoside conformation is influenced by various factors including substitution at the 2', 3' or 4'-positions of the pentofuranosyl sugar. Electronegative substituents generally prefer the axial positions, while sterically demanding substituents generally prefer the equatorial positions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984, Springer-Verlag.) Modification of the 2' position to favor the 3'-endo conformation can be achieved while maintaining the 2'-OH as a recognition element, as exemplified in Example 35, below (Gallo et al., Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem., (1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64, 747-754.) Alternatively, preference for the 3'-endo conformation can be achieved by deletion of the 2'-OH as exemplified by 2' deoxy-2'F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36, 831-841), which adopts the 3'-endo conformation positioning the electronegative fluorine atom in the axial position. Other modifications of the ribose ring, for example substitution at the 4'-position to give 4'-F modified nucleosides (Guillerm et al., Bioorganic and Medicinal Chemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem. (1976), 41, 3010-3017), or for example modification to yield methanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal Chemistry Letters (2001), 11, 1333-1337) also induce preference for the 3'-endo conformation. Some modifications actually lock the conformational geometry by formation of a bicyclic sugar moiety e.g. locked nucleic acid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), and ethylene bridged nucleic acids (ENA, Morita et al, Bioorganic & Medicinal Chemistry Letters (2002), 12, 73-76.)

[0429] c. Certain Motifs

[0430] In certain embodiments, oligomeric compounds comprise or consist of oligonucleotides. In certain embodiments, such oligonucleotides comprise one or more chemical modification. In certain embodiments, chemically modified oligonucleotides comprise one or more modified sugars. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleobases. In certain embodiments, chemically modified oligonucleotides comprise one or more modified internucleoside linkages. In certain embodiments, the chemical modifications (sugar modifications, nucleobase modifications, and/or linkage modifications) define a pattern or motif. In certain embodiments, the patterns of chemical modifications of sugar moieties, internucleoside linkages, and nucleobases are each independent of one another. Thus, an oligonucleotide may be described by its sugar modification motif, internucleoside linkage motif and/or nucleobase modification motif (as used herein, nucleobase modification motif describes the chemical modifications to the nucleobases independent of the sequence of nucleobases).

[0431] i. Certain Sugar Motifs

[0432] In certain embodiments, oligonucleotides comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar motif. Such sugar motifs include but are not limited to any of the sugar modifications discussed herein.

[0433] In certain embodiments, the oligonucleotides comprise or consist of a region having a gapmer sugar motif, which comprises two external regions or "wings" and a central or internal region or "gap." The three regions of a gapmer sugar motif (the 5'-wing, the gap, and the 3'-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3'-most nucleoside of the 5'-wing and the 5'-most nucleoside of the 3'-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap. In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two wings are the same as one another (symmetric sugar gapmer). In certain embodiments, the sugar motifs of the 5'-wing differs from the sugar motif of the 3'-wing (asymmetric sugar gapmer).

[0434] ii. Certain Nucleobase Modification Motifs

[0435] In certain embodiments, oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or nucleobases modification motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases is chemically modified.

[0436] In certain embodiments, oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3'-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 3'-end of the oligonucleotide. In certain such embodiments, the block is at the 5'-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 5'-end of the oligonucleotide.

[0437] In certain embodiments, nucleobase modifications are a function of the natural base at a particular position of an oligonucleotide. For example, in certain embodiments each purine or each pyrimidine in an oligonucleotide is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each cytosine is modified. In certain embodiments, each uracil is modified.

[0438] In certain embodiments, oligonucleotides comprise one or more nucleosides comprising a modified nucleobase. In certain embodiments, oligonucleotides having a gapmer sugar motif comprise a nucleoside comprising a modified nucleobase. In certain such embodiments, one nucleoside comprising a modified nucleobases is in the central gap of an oligonucleotide having a gapmer sugar motif. In certain embodiments, the sugar is an unmodified 2' deoxynucleoside. In certain embodiments, the modified nucleobase is selected from: a 2-thio pyrimidine and a 5-propyne pyrimidine

[0439] In certain embodiments, some, all, or none of the cytosine moieties in an oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methyl cytosine is not a "modified nucleobase." Accordingly, unless otherwise indicated, unmodified nucleobases include both cytosine residues having a 5-methyl and those lacking a 5 methyl. In certain embodiments, the methylation state of all or some cytosine nucleobases is specified.

[0440] iii. Certain Nucleoside Motifs

[0441] In certain embodiments, oligonucleotides comprise nucleosides comprising modified sugar moieties and/or nucleosides comprising modified nucleobases. Such motifs can be described by their sugar motif and their nucleobase motif separately or by their nucleoside motif, which provides positions or patterns of modified nucleosides (whether modified sugar, nucleobase, or both sugar and nucleobase) in an oligonucleotide.

[0442] In certain embodiments, the oligonucleotides comprise or consist of a region having a gapmer nucleoside motif, which comprises two external regions or "wings" and a central or internal region or "gap." The three regions of a gapmer nucleoside motif (the 5'-wing, the gap, and the 3'-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties and/or nucleobases of the nucleosides of each of the wings differ from at least some of the sugar moieties and/or nucleobase of the nucleosides of the gap. Specifically, at least the nucleosides of each wing that are closest to the gap (the 3'-most nucleoside of the 5'-wing and the 5'-most nucleoside of the 3'-wing) differ from the neighboring gap nucleosides, thus defining the boundary between the wings and the gap. In certain embodiments, the nucleosides within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside that differs from one or more other nucleosides of the gap. In certain embodiments, the nucleoside motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the nucleoside motifs of the 5'-wing differs from the nucleoside motif of the 3'-wing (asymmetric gapmer).

[0443] iv. Certain 5'-Wings

[0444] In certain embodiments, the 5'-wing of a gapmer consists of 1 to 6 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 1 to 5 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 1 to 4 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 1 or 2 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 2 or 3 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 5'-wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 3 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 5 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 6 linked nucleosides.

[0445] In certain embodiments, the 5'-wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least two bicyclic nucleosides. In certain embodiments, the 5'-wing of a gapmer comprises at least three bicyclic nucleosides. In certain embodiments, the 5'-wing of a gapmer comprises at least four bicyclic nucleosides. In certain embodiments, the 5'-wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 5'-wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 5'-wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 5'-wing of a gapmer is a LNA nucleoside.

[0446] In certain embodiments, the 5'-wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one 2'-substituted nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one 2'-MOE nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one 2'-OMe nucleoside. In certain embodiments, each nucleoside of the 5'-wing of a gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 5'-wing of a gapmer is a 2'-substituted nucleoside. In certain embodiments, each nucleoside of the 5'-wing of a gapmer is a 2'-MOE nucleoside. In certain embodiments, each nucleoside of the 5'-wing of a gapmer is a 2'-OMe nucleoside.

[0447] In certain embodiments, the 5'-wing of a gapmer comprises at least one 2'-deoxynucleoside. In certain embodiments, each nucleoside of the 5'-wing of a gapmer is a 2'-deoxynucleoside. In a certain embodiments, the 5'-wing of a gapmer comprises at least one ribonucleoside. In certain embodiments, each nucleoside of the 5'-wing of a gapmer is a ribonucleoside. In certain embodiments, one, more than one, or each of the nucleosides of the 5'-wing is an RNA-like nucleoside.

[0448] In certain embodiments, the 5'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-deoxynucleoside.

[0449] In certain embodiments, the 5'-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-deoxynucleoside.

[0450] In certain embodiments, the 5'-wing of a gapmer has a nucleoside motif selected from among the following: ADDA; ABDAA; ABBA; ABB; ABAA; AABAA; AAABAA; AAAABAA; AAAAABAA; AAABAA; AABAA; ABAB; ABADB; ABADDB; AAABB; AAAAA; ABBDC; ABDDC; ABBDCC; ABBDDC; ABBDCC; ABBC; AA; AAA; AAAA; AAAAB; AAAAAAA; AAAAAAAA; ABBB; AB; ABAB; AAAAB; AABBB; AAAAB; and AABBB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, each C is a modified nucleoside of a third type, and each D is an unmodified deoxynucleoside.

[0451] In certain embodiments, the 5'-wing of a gapmer has a nucleoside motif selected from among the following: AB, ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, AAAB, ABBW, ABBWW, ABBB, ABBBB, ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW, AAAAA, BBBBAA, and AAABW; wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of either the first type, the second type or a third type.

[0452] In certain embodiments, the 5'-wing of a gapmer has a nucleoside motif selected from among the following: ABB; ABAA; AABAA; AAABAA; ABAB; ABADB; AAABB; AAAAA; AA; AAA; AAAA; AAAAB; ABBB; AB; and ABAB; wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of either the first type, the second type or a third type.

[0453] In certain embodiments, an oligonucleotide comprises any 5'-wing motif provided herein. In certain such embodiments, the oligonucleotide is a 5'-hemimer (does not comprise a 3'-wing). In certain embodiments, such an oligonucleotide is a gapmer. In certain such embodiments, the 3'-wing of the gapmer may comprise any nucleoside motif.

[0454] In certain embodiments, the 5'-wing of a gapmer has a sugar motif selected from among those listed in the following non-limiting tables:

TABLE-US-00001 TABLE 1 Certain 5'-Wing Sugar Motifs Certain 5'-Wing Sugar Motifs AAAAA ABCBB BABCC BCBBA CBACC AAAAB ABCBC BACAA BCBBB CBBAA AAAAC ABCCA BACAB BCBBC CBBAB AAABA ABCCB BACAC BCBCA CBBAC AAABB ABCCC BACBA BCBCB CBBBA AAABC ACAAA BACBB BCBCC CBBBB AAACA ACAAB BACBC BCCAA CBBBC AAACB ACAAC BACCA BCCAB CBBCA AAACC ACABA BACCB BCCAC CBBCB AABAA ACABB BACCC BCCBA CBBCC AABAB ACABC BBAAA BCCBB CBCAA AABAC ACACA BBAAB BCCBC CBCAB AABBA ACACB BBAAC BCCCA CBCAC AABBB ACACC BBABA BCCCB CBCBA AABBC ACBAA BBABB BCCCC CBCBB AABCA ACBAB BBABC CAAAA CBCBC AABCB ACBAC BBACA CAAAB CBCCA AABCC ACBBA BBACB CAAAC CBCCB AACAA ACBBB BBACC CAABA CBCCC AACAB ACBBC BBBAA CAABB CCAAA AACAC ACBCA BBBAB CAABC CCAAB AACBA ACBCB BBBAC CAACA CCAAC AACBB ACBCC BBBBA CAACB CCABA AACBC ACCAA BBBBB CAACC CCABB AACCA ACCAB BBBBC CABAA CCABC AACCB ACCAC BBBCA CABAB CCACA AACCC ACCBA BBBCB CABAC CCACB ABAAA ACCBB BBBCC CABBA CCACC ABAAB ACCBC BBCAA CABBB CCBAA ABAAC ACCCA BBCAB CABBC CCBAB ABABA ACCCB BBCAC CABCA CCBAC ABABB ACCCC BBCBA CABCB CCBBA ABABC BAAAA BBCBB CABCC CCBBB ABACA BAAAB BBCBC CACAA CCBBC ABACB BAAAC BBCCA CACAB CCBCA ABACC BAABA BBCCB CACAC CCBCB ABBAA BAABB BBCCC CACBA CCBCC ABBAB BAABC BCAAA CACBB CCCAA ABBAC BAACA BCAAB CACBC CCCAB ABBBA BAACB BCAAC CACCA CCCAC ABBBB BAACC BCABA CACCB CCCBA ABBBC BABAA BCABB CACCC CCCBB ABBCA BABAB BCABC CBAAA CCCBC ABBCB BABAC BCACA CBAAB CCCCA ABBCC BABBA BCACB CBAAC CCCCB ABCAA BABBB BCACC CBABA CCCCC ABCAB BABBC BCBAA CBABB ABCAC BABCA BCBAB CBABC ABCBA BABCB BCBAC CBACA

TABLE-US-00002 TABLE 2 Certain 5'-Wing Sugar Motifs Certain 5'-Wing Sugar Motifs AAAAA BABC CBAB ABBB BAA AAAAB BACA CBAC BAAA BAB AAABA BACB CBBA BAAB BBA AAABB BACC CBBB BABA BBB AABAA BBAA CBBC BABB AA AABAB BBAB CBCA BBAA AB AABBA BBAC CBCB BBAB AC AABBB BBBA CBCC BBBA BA ABAAA BBBB CCAA BBBB BB ABAAB BBBC CCAB AAA BC ABABA BBCA CCAC AAB CA ABABB BBCB CCBA AAC CB ABBAA BBCC CCBB ABA CC ABBAB BCAA CCBC ABB AA ABBBA BCAB CCCA ABC AB ABBBB BCAC CCCB ACA BA BAAAA ABCB BCBA ACB BAAAB ABCC BCBB ACC BAABA ACAA BCBC BAA BAABB ACAB BCCA BAB BABAA ACAC BCCB BAC BABAB ACBA BCCC BBA BABBA ACBB CAAA BBB BABBB ACBC CAAB BBC BBAAA ACCA CAAC BCA BBAAB ACCB CABA BCB BBABA ACCC CABB BCC BBABB BAAA CABC CAA BBBAA BAAB CACA CAB BBBAB BAAC CACB CAC BBBBA BABA CACC CBA BBBBB BABB CBAA CBB AAAA AACC CCCC CBC AAAB ABAA AAAA CCA AAAC ABAB AAAB CCB AABA ABAC AABA CCC AABB ABBA AABB AAA AABC ABBB ABAA AAB AACA ABBC ABAB ABA AACB ABCA ABBA ABB

[0455] In certain embodiments, each A, each B, and each C located at the 3'-most 5'-wing nucleoside is a modified nucleoside. For example, in certain embodiments the 5'-wing motif is selected from among ABB, BBB, and CBB, wherein the underlined nucleoside represents the 3'-most 5'-wing nucleoside and wherein the underlined nucleoside is a modified nucleoside. In certain embodiments, the 3'-most 5'-wing nucleoside comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, the 3'-most 5'-wing nucleoside comprises a bicyclic sugar moiety selected from among cEt and LNA. In certain embodiments, the 3'-most 5'-wing nucleoside comprises cEt. In certain embodiments, the 3'-most 5'-wing nucleoside comprises LNA.

[0456] In certain embodiments, each A comprises an unmodified 2'-deoxyfuranose sugar moiety. In certain embodiments, each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2'-substituted sugar moiety. In certain embodiments, each A comprises a 2'-substituted sugar moiety selected from among F, ara-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certain embodiments, each A comprises an HNA. In certain embodiments, each A comprises a F-HNA. In certain embodiments, each A comprises a 5'-substituted sugar moiety selected from among 5'-Me DNA, and 5'-(R)-Me DNA.

[0457] In certain embodiments, each B comprises an unmodified 2'-deoxyfuranose sugar moiety. In certain embodiments, each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2'-substituted sugar moiety. In certain embodiments, each B comprises a 2'-substituted sugar moiety selected from among F, (ara)-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. In certain embodiments, each B comprises an HNA. In certain embodiments, each B comprises a F-HNA. In certain embodiments, each B comprises a 5'-substituted sugar moiety selected from among 5'-Me DNA, and 5'-(R)-Me DNA.

[0458] In certain embodiments, each A comprises a 2'-substituted sugar moiety selected from among F, ara-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3 and each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each A comprises O(CH.sub.2).sub.2--OCH.sub.3 and each B comprises cEt.

[0459] In certain embodiments, each C comprises an unmodified 2'-deoxyfuranose sugar moiety. In certain embodiments, each C comprises a modified sugar moiety. In certain embodiments, each C comprises a 2'-substituted sugar moiety. In certain embodiments, each C comprises a 2'-substituted sugar moiety selected from among F, (ara)-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each C comprises a 5'-substituted sugar moiety. In certain embodiments, each C comprises a 5'-substituted sugar moiety selected from among 5'-Me DNA, and 5'-(R)-Me DNA. In certain embodiments, each C comprises a bicyclic sugar moiety. In certain embodiments, each C comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each C comprises a modified nucleobase. In certain embodiments, each C comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine. In certain embodiments, each C comprises a 2-thio-thymidine nucleoside. In certain embodiments, each C comprises an HNA. In certain embodiments, each C comprises an F-HNA.

[0460] v. Certain 3'-Wings

[0461] In certain embodiments, the 3'-wing of a gapmer consists of 1 to 6 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 1 to 5 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 1 to 4 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 1 or 2 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 2 or 3 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 3'-wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 31 inked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 5 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 6 linked nucleosides.

[0462] In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 3'-wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 3'-wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 3'-wing of a gapmer is a LNA nucleoside.

[0463] In certain embodiments, the 3'-wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least two non-bicyclic modified nucleosides. In certain embodiments, the 3'-wing of a gapmer comprises at least three non-bicyclic modified nucleosides. In certain embodiments, the 3'-wing of a gapmer comprises at least four non-bicyclic modified nucleosides. In certain embodiments, the 3'-wing of a gapmer comprises at least one 2'-substituted nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one 2'-MOE nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one 2'-OMe nucleoside. In certain embodiments, each nucleoside of the 3'-wing of a gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 3'-wing of a gapmer is a 2'-substituted nucleoside. In certain embodiments, each nucleoside of the 3'-wing of a gapmer is a 2'-MOE nucleoside. In certain embodiments, each nucleoside of the 3'-wing of a gapmer is a 2'-OMe nucleoside.

[0464] In certain embodiments, the 3'-wing of a gapmer comprises at least one 2'-deoxynucleoside. In certain embodiments, each nucleoside of the 3'-wing of a gapmer is a 2'-deoxynucleoside. In a certain embodiments, the 3'-wing of a gapmer comprises at least one ribonucleoside. In certain embodiments, each nucleoside of the 3'-wing of a gapmer is a ribonucleoside. In certain embodiments, one, more than one, or each of the nucleosides of the 5'-wing is an RNA-like nucleoside.

[0465] In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-deoxynucleoside.

[0466] In certain embodiments, the 3'-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-deoxynucleoside.

[0467] In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside and at least one 2'-deoxynucleoside.

[0468] In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2'-deoxynucleoside.

[0469] In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2'-substituted nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2'-substituted nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside, at least one 2'-substituted nucleoside, and at least one 2'-deoxynucleoside.

[0470] In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2'-MOE nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2'-MOE nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside, at least one 2'-MOE nucleoside, and at least one 2'-deoxynucleoside.

[0471] In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2'-OMe nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2'-OMe nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside, at least one 2'-OMe nucleoside, and at least one 2'-deoxynucleoside.

[0472] In certain embodiments, the 3'-wing of a gapmer has a nucleoside motif selected from among the following: ABB, ABAA, AAABAA, AAAAABAA, AABAA, AAAABAA, AAABAA, ABAB, AAAAA, AAABB, AAAAAAAA, AAAAAAA, AAAAAA, AAAAB, AAAA, AAA, AA, AB, ABBB, ABAB, AABBB; wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type. In certain embodiments, an oligonucleotide comprises any 3'-wing motif provided herein. In certain such embodiments, the oligonucleotide is a 3'-hemimer (does not comprise a 5'-wing). In certain embodiments, such an oligonucleotide is a gapmer. In certain such embodiments, the 5'-wing of the gapmer may comprise any nucleoside motif.

[0473] In certain embodiments, the 3'-wing of a gapmer has a nucleoside motif selected from among the following: BBA, AAB, AAA, BBB, BBAA, AABB, WBBA, WAAB, BBBA, BBBBA, BBBB, BBBBBA, ABBBBB, BBAAA, AABBB, BBBAA, BBBBA, BBBBB, BABA, AAAAA, BBAAAA, AABBBB, BAAAA, and ABBBB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of either the first type, the second type or a third type.

[0474] In certain embodiments, the 3'-wing of a gapmer has a nucleoside motif selected from among the following: ABB; AAABAA; AABAA; AAAABAA; AAAAA; AAABB; AAAAAAAA; AAAAAAA; AAAAAA; AAAAB; AB; ABBB; and ABAB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of either the first type, the second type or a third type.

[0475] In certain embodiments, the 3'-wing of a gapmer has a sugar motif selected from among those listed in the following non-limiting tables:

TABLE-US-00003 TABLE 3 Certain 3'-Wing Sugar Motifs Certain 3'-Wing Sugar Motifs AAAAA ABCBB BABCC BCBBA CBACC AAAAB ABCBC BACAA BCBBB CBBAA AAAAC ABCCA BACAB BCBBC CBBAB AAABA ABCCB BACAC BCBCA CBBAC AAABB ABCCC BACBA BCBCB CBBBA AAABC ACAAA BACBB BCBCC CBBBB AAACA ACAAB BACBC BCCAA CBBBC AAACB ACAAC BACCA BCCAB CBBCA AAACC ACABA BACCB BCCAC CBBCB AABAA ACABB BACCC BCCBA CBBCC AABAB ACABC BBAAA BCCBB CBCAA AABAC ACACA BBAAB BCCBC CBCAB AABBA ACACB BBAAC BCCCA CBCAC AABBB ACACC BBABA BCCCB CBCBA AABBC ACBAA BBABB BCCCC CBCBB AABCA ACBAB BBABC CAAAA CBCBC AABCB ACBAC BBACA CAAAB CBCCA AABCC ACBBA BBACB CAAAC CBCCB AACAA ACBBB BBACC CAABA CBCCC AACAB ACBBC BBBAA CAABB CCAAA AACAC ACBCA BBBAB CAABC CCAAB AACBA ACBCB BBBAC CAACA CCAAC AACBB ACBCC BBBBA CAACB CCABA AACBC ACCAA BBBBB CAACC CCABB AACCA ACCAB BBBBC CABAA CCABC AACCB ACCAC BBBCA CABAB CCACA AACCC ACCBA BBBCB CABAC CCACB ABAAA ACCBB BBBCC CABBA CCACC ABAAB ACCBC BBCAA CABBB CCBAA ABAAC ACCCA BBCAB CABBC CCBAB ABABA ACCCB BBCAC CABCA CCBAC ABABB ACCCC BBCBA CABCB CCBBA ABABC BAAAA BBCBB CABCC CCBBB ABACA BAAAB BBCBC CACAA CCBBC ABACB BAAAC BBCCA CACAB CCBCA ABACC BAABA BBCCB CACAC CCBCB ABBAA BAABB BBCCC CACBA CCBCC ABBAB BAABC BCAAA CACBB CCCAA ABBAC BAACA BCAAB CACBC CCCAB ABBBA BAACB BCAAC CACCA CCCAC ABBBB BAACC BCABA CACCB CCCBA ABBBC BABAA BCABB CACCC CCCBB ABBCA BABAB BCABC CBAAA CCCBC ABBCB BABAC BCACA CBAAB CCCCA ABBCC BABBA BCACB CBAAC CCCCB ABCAA BABBB BCACC CBABA CCCCC ABCAB BABBC BCBAA CBABB ABCAC BABCA BCBAB CBABC ABCBA BABCB BCBAC CBACA

TABLE-US-00004 TABLE 4 Certain 3'-Wing Sugar Motifs Certain 3'-Wing Sugar Motifs AAAAA BABC CBAB ABBB BAA AAAAB BACA CBAC BAAA BAB AAABA BACB CBBA BAAB BBA AAABB BACC CBBB BABA BBB AABAA BBAA CBBC BABB AA AABAB BBAB CBCA BBAA AB AABBA BBAC CBCB BBAB AC AABBB BBBA CBCC BBBA BA ABAAA BBBB CCAA BBBB BB ABAAB BBBC CCAB AAA BC ABABA BBCA CCAC AAB CA ABABB BBCB CCBA AAC CB ABBAA BBCC CCBB ABA CC ABBAB BCAA CCBC ABB AA ABBBA BCAB CCCA ABC AB ABBBB BCAC CCCB ACA BA BAAAA ABCB BCBA ACB BAAAB ABCC BCBB ACC BAABA ACAA BCBC BAA BAABB ACAB BCCA BAB BABAA ACAC BCCB BAC BABAB ACBA BCCC BBA BABBA ACBB CAAA BBB BABBB ACBC CAAB BBC BBAAA ACCA CAAC BCA BBAAB ACCB CABA BCB BBABA ACCC CABB BCC BBABB BAAA CABC CAA BBBAA BAAB CACA CAB BBBAB BAAC CACB CAC BBBBA BABA CACC CBA BBBBB BABB CBAA CBB AAAA AACC CCCC CBC AAAB ABAA AAAA CCA AAAC ABAB AAAB CCB AABA ABAC AABA CCC AABB ABBA AABB AAA AABC ABBB ABAA AAB AACA ABBC ABAB ABA AACB ABCA ABBA ABB

[0476] In certain embodiments, each A, each B, and each C located at the 5'-most 3'-wing region nucleoside is a modified nucleoside. For example, in certain embodiments the 3'-wing motif is selected from among ABB, BBB, and CBB, wherein the underlined nucleoside represents the 5'-most 3'-wing region nucleoside and wherein the underlined nucleoside is a modified nucleoside.

[0477] In certain embodiments, each A comprises an unmodified 2'-deoxyfuranose sugar moiety. In certain embodiments, each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2'-substituted sugar moiety. In certain embodiments, each A comprises a 2'-substituted sugar moiety selected from among F, ara-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certain embodiments, each A comprises a 5'-substituted sugar moiety selected from among 5'-Me DNA, and 5'-(R)-Me DNA.

[0478] In certain embodiments, each B comprises an unmodified 2'-deoxyfuranose sugar moiety. In certain embodiments, each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2'-substituted sugar moiety. In certain embodiments, each B comprises a 2'-substituted sugar moiety selected from among F, (ara)-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. In certain embodiments, each B comprises an HNA. In certain embodiments, each B comprises an F-HNA. In certain embodiments, each B comprises a 5'-substituted sugar moiety selected from among 5'-Me DNA, and 5'-(R)-Me DNA.

[0479] In certain embodiments, each A comprises a 2'-substituted sugar moiety selected from among F, ara-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3 and each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each A comprises O(CH.sub.2).sub.2--OCH.sub.3 and each B comprises cEt.

[0480] In certain embodiments, each C comprises an unmodified 2'-deoxyfuranose sugar moiety. In certain embodiments, each C comprises a modified sugar moiety. In certain embodiments, each C comprises a 2'-substituted sugar moiety. In certain embodiments, each C comprises a 2'-substituted sugar moiety selected from among F, (ara)-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each C comprises a 5'-substituted sugar moiety. In certain embodiments, each C comprises a 5'-substituted sugar moiety selected from among 5'-Me, and 5'-(R)-Me. In certain embodiments, each C comprises a bicyclic sugar moiety. In certain embodiments, each C comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each C comprises a modified nucleobase. In certain embodiments, each C comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine. In certain embodiments, each C comprises a 2-thio-thymidine nucleoside. In certain embodiments, each C comprises an HNA. In certain embodiments, each C comprises an F-HNA.

[0481] vi. Certain Central Regions (Gaps)

[0482] In certain embodiments, the gap of a gapmer consists of 6 to 20 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 15 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 12 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 or 7 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 or 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 or 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 11 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 12 linked nucleosides.

[0483] In certain embodiments, each nucleoside of the gap of a gapmer is a 2'-deoxynucleoside. In certain embodiments, the gap comprises one or more modified nucleosides. In certain embodiments, each nucleoside of the gap of a gapmer is a 2'-deoxynucleoside or is a modified nucleoside that is "DNA-like." In such embodiments, "DNA-like" means that the nucleoside has similar characteristics to DNA, such that a duplex comprising the gapmer and an RNA molecule is capable of activating RNase H. For example, under certain conditions, 2'-(ara)-F have been shown to support RNase H activation, and thus is DNA-like. In certain embodiments, one or more nucleosides of the gap of a gapmer is not a 2'-deoxynucleoside and is not DNA-like. In certain such embodiments, the gapmer nonetheless supports RNase H activation (e.g., by virtue of the number or placement of the non-DNA nucleosides).

[0484] In certain embodiments, gaps comprise a stretch of unmodified 2'-deoxynucleoside interrupted by one or more modified nucleosides, thus resulting in three sub-regions (two stretches of one or more 2'-deoxynucleosides and a stretch of one or more interrupting modified nucleosides). In certain embodiments, no stretch of unmodified 2'-deoxynucleosides is longer than 5, 6, or 7 nucleosides. In certain embodiments, such short stretches is achieved by using short gap regions. In certain embodiments, short stretches are achieved by interrupting a longer gap region.

[0485] In certain embodiments, the gap comprises one or more modified nucleosides. In certain embodiments, the gap comprises one or more modified nucleosides selected from among cEt, FHNA, LNA, and 2-thio-thymidine. In certain embodiments, the gap comprises one modified nucleoside. In certain embodiments, the gap comprises a 5'-substituted sugar moiety selected from among 5'-Me, and 5'-(R)-Me. In certain embodiments, the gap comprises two modified nucleosides. In certain embodiments, the gap comprises three modified nucleosides. In certain embodiments, the gap comprises four modified nucleosides. In certain embodiments, the gap comprises two or more modified nucleosides and each modified nucleoside is the same. In certain embodiments, the gap comprises two or more modified nucleosides and each modified nucleoside is different.

[0486] In certain embodiments, the gap comprises one or more modified linkages. In certain embodiments, the gap comprises one or more methyl phosphonate linkages. In certain embodiments the gap comprises two or more modified linkages. In certain embodiments, the gap comprises one or more modified linkages and one or more modified nucleosides. In certain embodiments, the gap comprises one modified linkage and one modified nucleoside. In certain embodiments, the gap comprises two modified linkages and two or more modified nucleosides.

[0487] In certain embodiments, the gap comprises a nucleoside motif selected from among the following: DDDDXDDDDD; DDDDDXDDDDD; DDDXDDDDD; DDDDXDDDDDD; DDDDXDDDD; DDXDDDDDD; DDDXDDDDDD; DXDDDDDD; DDXDDDDDDD; DDXDDDDD; DDXDDDXDDD; DDDXDDDXDDD; DXDDDXDDD; DDXDDDXDD; DDXDDDDXDDD; DDXDDDDXDD; DXDDDDXDDD; DDDDXDDD; DDDXDDD; DXDDDDDDD; DDDDXXDDD; and DXXDXXDXX; wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.

[0488] In certain embodiments, the gap comprises a nucleoside motif selected from among the following: DDDDDDDDD; DXDDDDDDD; DDXDDDDDD; DDDXDDDDD; DDDDXDDDD; DDDDDXDDD; DDDDDDXDD; DDDDDDDXD; DXXDDDDDD; DDDDDDXXD; DDXXDDDDD; DDDXXDDDD; DDDDXXDDD; DDDDDXXDD; DXDDDDDXD; DXDDDDXDD; DXDDDXDDD; DXDDXDDDD; DXDXDDDDD; DDXDDDDXD; DDXDDDXDD; DDXDDXDDD; DDXDXDDDD; DDDXDDDXD; DDDXDDXDD; DDDXDXDDD; DDDDXDDXD; DDDDXDXDD; and DDDDDXDXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.

[0489] In certain embodiments, the gap comprises a nucleoside motif selected from among the following: DDDDXDDDD, DXDDDDDDD, DXXDDDDDD, DDXDDDDDD, DDDXDDDDD, DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, and DDDDDDDXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.

[0490] In certain embodiments, the gap comprises a nucleoside motif selected from among the following: DDDDDDDD, DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDXD, DXDDDXDD, DXDDXDDD, DXDXDDDD, DXXDDDDD, DDXXDDDD, DDXDXDDD, DDXDDXDD, DXDDDDXD, DDDXXDDD, DDDXDXDD, DDDXDDXD, DDDDXXDD, DDDDXDXD, and DDDDDXXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.

[0491] In certain embodiments, the gap comprises a nucleoside motif selected from among the following: DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD, DXDDDXD, DXDDXDD, DXDXDDD, DXXDDDD, DDXXDDD, DDXDXDD, DDXDDXD, DDDXXDD, DDDXDXD, and DDDDXXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.

[0492] In certain embodiments, the gap comprises a nucleoside motif selected from among the following: DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXXDDD, DXDXDD, DXDDXD, DDXXDD, DDXDXD, and DDDXXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.

[0493] In certain embodiments, the gap comprises a nucleoside motif selected from among the following: DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD, DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDDDD; DDXDDDDDD, DDDXDDDDD, DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, DDDDDDDXD, DXDDDDDDDD, DDXDDDDDDD, DDDXDDDDDD, DDDDXDDDDD, DDDDDXDDDD, DDDDDDXDDD, DDDDDDDXDD, and DDDDDDDDXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.

[0494] In certain embodiments, each X comprises an unmodified 2'-deoxyfuranose sugar moiety. In certain embodiments, each X comprises a modified sugar moiety. In certain embodiments, each X comprises a 2'-substituted sugar moiety. In certain embodiments, each X comprises a 2'-substituted sugar moiety selected from among F, (ara)-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each X comprises a 5'-substituted sugar moiety. In certain embodiments, each X comprises a 5'-substituted sugar moiety selected from among 5'-Me, and 5'-(R)-Me. In certain embodiments, each X comprises a bicyclic sugar moiety. In certain embodiments, each X comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each X comprises a modified nucleobase. In certain embodiments, each X comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine. In certain embodiments, each X comprises a 2-thio-thymidine nucleoside. In certain embodiments, each X comprises an HNA. In certain embodiments, each C comprises an F-HNA. In certain embodiments, X represents the location of a single differentiating nucleobase.

[0495] vii. Certain Gapmer Motifs

[0496] In certain embodiments, a gapmer comprises a 5'-wing, a gap, and a 3' wing, wherein the 5'-wing, gap, and 3' wing are independently selected from among those discussed above. For example, in certain embodiments, a gapmer has a 5'-wing, a gap, and a 3'-wing having features selected from among any of those listed in the tables above and any 5'-wing may be paired with any gap and any 3'-wing. For example, in certain embodiments, a 5'-wing may comprise AAABB, a 3'-wing may comprise BBA, and the gap may comprise DDDDDDD. For example, in certain embodiments, a gapmer has a 5'-wing, a gap, and a 3'-wing having features selected from among those listed in the following non-limiting table, wherein each motif is represented as (5'-wing)-(gap)-(3'-wing), wherein each number represents the number of linked nucleosides in each portion of the motif, for example, a 5-10-5 motif would have a 5'-wing comprising 5 nucleosides, a gap comprising 10 nucleosides, and a 3'-wing comprising 5 nucleosides:

TABLE-US-00005 TABLE 5 Certain Gapmer Sugar Motifs Certain Gapmer Sugar Motifs 2-10-2 3-10-2 4-10-2 5-10-2 2-10-3 3-10-3 4-10-3 5-10-3 2-10-4 3-10-4 4-10-4 5-10-4 2-10-5 3-10-5 4-10-5 5-10-5 2-9-2 3-9-2 4-9-2 5-9-2 2-9-3 3-9-3 4-9-3 5-9-3 2-9-4 3-9-4 4-9-4 5-9-4 2-9-5 3-9-5 4-9-5 5-9-5 2-11-2 3-11-2 4-11-2 5-11-2 2-11-3 3-11-3 4-11-3 5-11-3 2-11-4 3-11-4 4-11-4 5-11-4 2-11-5 3-11-5 4-11-5 5-11-5 2-8-2 3-8-2 4-8-2 5-8-2 2-8-3 3-8-3 4-8-3 5-8-3 2-8-4 3-8-4 4-8-4 5-8-4 2-8-5 3-8-5 4-8-5 5-8-5

[0497] In certain embodiments, a gapmer comprises a 5'-wing, a gap, and a 3' wing, wherein the 5'-wing, gap, and 3' wing are independently selected from among those discussed above. For example, in certain embodiments, a gapmer has a 5'-wing, a gap, and a 3'-wing having features selected from among those listed in the following non-limiting tables:

TABLE-US-00006 TABLE 6 Certain Gapmer Nucleoside Motifs 5'-wing Central gap 3'-wing region region region ADDA DDDDDD ABB ABBA DDDADDDD ABAA AAAAAAA DDDDDDDDDDD AAA AAAAABB DDDDDDDD BBAAAAA ABB DDDDADDDD ABB ABB DDDDBDDDD BBA ABB DDDDDDDDD BBA AABAA DDDDDDDDD AABAA ABB DDDDDD AABAA AAABAA DDDDDDDDD AAABAA AAABAA DDDDDDDDD AAB ABAB DDDDDDDDD ABAB AAABB DDDDDDD BBA ABADB DDDDDDD BBA ABA DBDDDDDDD BBA ABA DADDDDDDD BBA ABAB DDDDDDDD BBA AA DDDDDDDD BBBBBBBB ABB DDDDDD ABADB AAAAB DDDDDDD BAAAA ABBB DDDDDDDDD AB AB DDDDDDDDD BBBA ABBB DDDDDDDDD BBBA AB DDDDDDDD ABA ABB DDDDWDDDD BBA AAABB DDDWDDD BBAAA ABB DDDDWWDDD BBA ABADB DDDDDDD BBA ABBDC DDDDDDD BBA ABBDDC DDDDDD BBA ABBDCC DDDDDD BBA ABB DWWDWWDWW BBA ABB DWDDDDDDD BBA ABB DDWDDDDDD BBA ABB DWWDDDDDD BBA AAABB DDWDDDDDD AA BB DDWDWDDDD BBABBBB ABB DDDD(.sup.ND)DDDD BBA AAABB DDD(.sup.ND)DDD BBAAA ABB DDDD(.sup.ND)(.sup.ND)DDD BBA ABB D(.sup.ND)(.sup.ND)D(.sup.ND)(.sup.ND)D(.sup.ND)(.sup.ND) BBA ABB D(.sup.ND)DDDDDDD BBA ABB DD(.sup.ND)DDDDDD BBA ABB D(.sup.ND)(.sup.ND)DDDDDD BBA AAABB DD(.sup.ND)DDDDDD AA BB DD(.sup.ND)D(.sup.ND)DDDD BBABBBB ABAB DDDDDDDDD BABA

TABLE-US-00007 TABLE 7 Certain Gapmer Nucleoside Motifs 5'-wing Central gap 3'-wing region region region ABBW DDDDDDDD BBA ABB DWDDDDDDD BBA ABB DDWDDDDDD BBA ABB DDDWDDDDD BBA ABB DDDDWDDDD BBA ABB DDDDDWDDD BBA ABB DDDDDDWDD BBA ABB DDDDDDDWD BBA ABB DDDDDDDD WBBA ABBWW DDDDDDD BBA ABB DWWDDDDDD BBA ABB DDWWDDDDD BBA ABB DDDWWDDDD BBA ABB DDDDWWDDD BBA ABB DDDDDWWDD BBA ABB DDDDDDWWD BBA ABB DDDDDDD WWBBA ABBW DDDDDDD WBBA ABBW DDDDDDWD BBA ABBW DDDDDWDD BBA ABBW DDDDWDDD BBA ABBW DDDWDDDD BBA ABBW DDWDDDDD BBA ABBW DWDDDDDD BBA ABB DWDDDDDD WBBA ABB DWDDDDDWD BBA ABB DWDDDDWDD BBA ABB DWDDDWDDD BBA ABB DWDDWDDDD BBA ABB DWDWDDDDD BBA ABB DDWDDDDD WBBA ABB DDWDDDDWD BBA ABB DDWDDDWDD BBA ABB DDWDDWDDD BBA ABB DDWDWDDDD BBA ABB DDWWDDDDD BBA ABB DDDWDDDD WBBA ABB DDDWDDDWD BBA ABB DDDWDDWDD BBA ABB DDDWDWDDD BBA ABB DDDWWDDDD BBA ABB DDDDWDDD WBBA ABB DDDDWDDWD BBA ABB DDDDWDWDD BBA ABB DDDDWWDDD BBA ABB DDDDDWDD WBBA ABB DDDDDWDWD BBA ABB DDDDDWWDD BBA ABB DDDDDDWD WBBA

TABLE-US-00008 TABLE 8 Certain Gapmer Nucleoside Motifs 5'-wing Central gap 3'-wing region region region ABBB DDDDDDDD BBA ABB DBDDDDDDD BBA ABB DDBDDDDDD BBA ABB DDDBDDDDD BBA ABB DDDDBDDDD BBA ABB DDDDDBDDD BBA ABB DDDDDDBDD BBA ABB DDDDDDDBD BBA ABB DDDDDDDD BBBA ABBBB DDDDDDD BBA ABB DBBDDDDDD BBA ABB DDBBDDDDD BBA ABB DDDBBDDDD BBA ABB DDDDBBDDD BBA ABB DDDDDBBDD BBA ABB DDDDDDBBD BBA ABB DDDDDDD BBBBA ABBB DDDDDDD BBBA ABB DDDDDDBD BBA ABBB DDDDDBDD BBA ABBB DDDDBDDD BBA ABBB DDDBDDDD BBA ABBB DDBDDDDD BBA ABBB DBDDDDDD BBA ABB DBDDDDDD BBBA ABB DBDDDDDBD BBA ABB DBDDDDBDD BBA ABB DBDDDBDDD BBA ABB DBDDBDDDD BBA ABB DBDBDDDDD BBA ABB DDBDDDDD BBBA ABB DDBDDDDBD BBA ABB DDBDDDBDD BBA ABB DDBDDBDDD BBA ABB DDBDBDDDD BBA ABB DDBBDDDDD BBA ABB DDDBDDDD BBBA ABB DDDBDDDBD BBA ABB DDDBDDBDD BBA ABB DDDBDBDDD BBA ABB DDDBBDDDD BBA ABB DDDDBDDD BBBA ABB DDDDBDDBD BBA ABB DDDDBDBDD BBA ABB DDDDBBDDD BBA ABB DDDDDBDD BBBA ABB DDDDDBDBD BBA ABB DDDDDBBDD BBA ABB DDDDDDBD BBBA

TABLE-US-00009 TABLE 9 Certain Gapmer Nucleoside Motifs 5'-wing Central gap 3'-wing region region region ABB DDDDDDDDD BBA AB DBDDDDDDDD BBA AB DDBDDDDDDD BBA AB DDDBDDDDDD BBA AB DDDDBDDDDD BBA AB DDDDDBDDDD BBA AB DDDDDDBDDD BBA AB DDDDDDDBDD BBA AB DDDDDDDDBD BBA AB DDDDDDDDD BBBA ABBB DDDDDDDD BBA AB DBBDDDDDDD BBA AB DDBBDDDDDD BBA AB DDDBBDDDDD BBA AB DDDDBBDDDD BBA AB DDDDDBBDDD BBA AB DDDDDDBBDD BBA AB DDDDDDDBBD BBA AB DDDDDDDD BBBBA ABBBB DDDDDDD BBA AB DBBBDDDDDD BBA AB DDBBBDDDDD BBA AB DDDBBBDDDD BBA AB DDDDBBBDDD BBA AB DDDDDBBBDD BBA AB DDDDDDBBBD BBA AB DDDDDDD BBBBBA AB DDDDDDDDD BBBA AB DDDDDDDBD BBBA AB DDDDDBDD BBBA AB DDDDBDDD BBBA AB DDDBDDDD BBBA AB DDBDDDDD BBBA AB DBDDDDDD BBBA AB DDDDDBD BBBBA AB DDDDBDD BBBBA AB DDDBDDD BBBBA AB DDBDDDD BBBBA AB DBDDDDD BBBBA AB DDDDBD BBBBBA AB DDDBDD BBBBBA AB DDBDDD BBBBBA AB DBDDDD BBBBBA

TABLE-US-00010 TABLE 10 Certain Gapmer Nucleoside Motifs 5'-wing Central gap 3'-wing region region region AAAAAA DDDDDDD BABA AAAAAB DDDDDDD BABA AAAABA DDDDDDD BABA AAABAA DDDDDDD BABA AABAAA DDDDDDD BABA ABAAAA DDDDDDD BABA BAAAAA DDDDDDD BABA ABAAAB DDDDDDD BABA ABAABA DDDDDDD BABA ABABAA DDDDDDD BABA ABBAAA DDDDDDD BABA AABAAB DDDDDDD BABA AABABA DDDDDDD BABA AABBAA DDDDDDD BABA AAABAB DDDDDDD BABA AAABBA DDDDDDD BABA AAAABB DDDDDDD BABA BAAAAB DDDDDDD BABA BAAABA DDDDDDD BABA BAABAA DDDDDDD BABA BABAAA DDDDDDD BABA BBAAAA DDDDDDD BABA BBBAAA DDDDDDD BABA BBABAA DDDDDDD BABA BBAABA DDDDDDD BABA BBAAAB DDDDDDD BABA ABABAB DDDDDDD BABA BBBBAA DDDDDDD BABA BBBABA DDDDDDD BABA BBBAAB DDDDDDD BABA BBBBBA DDDDDDD BABA BBBBAB DDDDDDD BABA AAABBB DDDDDDD BABA AABABB DDDDDDD BABA ABAABB DDDDDDD BABA BAAABB DDDDDDD BABA AABBBB DDDDDDD BABA ABABBB DDDDDDD BABA BAABBB DDDDDDD BABA ABBBBB DDDDDDD BABA BABBBB DDDDDDD BABA BBBBBB DDDDDDD BABA

TABLE-US-00011 TABLE 11 Certain Gapmer Nucleoside Motifs 5'-wing Central gap 3'-wing region region region AAAAA DDDDDDD AAAAA AAAAB DDDDDDD AAAAA AAABA DDDDDDD AAAAA AAABB DDDDDDD AAAAA AABAA DDDDDDD AAAAA AABAB DDDDDDD AAAAA AABBA DDDDDDD AAAAA AABBB DDDDDDD AAAAA ABAAA DDDDDDD AAAAA ABAAB DDDDDDD AAAAA ABABA DDDDDDD AAAAA ABABB DDDDDDD AAAAA ABBAA DDDDDDD AAAAA ABBAB DDDDDDD AAAAA ABBBA DDDDDDD AAAAA ABBBB DDDDDDD AAAAA BAAAA DDDDDDD AAAAA BAAAB DDDDDDD AAAAA BAABA DDDDDDD AAAAA BAABB DDDDDDD AAAAA BABAA DDDDDDD AAAAA BABAB DDDDDDD AAAAA BABBA DDDDDDD AAAAA BABBB DDDDDDD AAAAA BBAAA DDDDDDD AAAAA BBAAB DDDDDDD AAAAA BBABA DDDDDDD AAAAA BBABB DDDDDDD AAAAA BBBAA DDDDDDD AAAAA BBBAB DDDDDDD AAAAA BBBBA DDDDDDD AAAAA BBBBB DDDDDDD AAAAA AAAAA DDDDDDD BAAAA AAAAB DDDDDDD BAAAA AAABA DDDDDDD BAAAA AAABB DDDDDDD BAAAA AABAA DDDDDDD BAAAA AABAB DDDDDDD BAAAA AABBA DDDDDDD BAAAA AABBB DDDDDDD BAAAA ABAAA DDDDDDD BAAAA ABAAB DDDDDDD BAAAA ABABA DDDDDDD BAAAA ABABB DDDDDDD BAAAA ABBAA DDDDDDD BAAAA ABBAB DDDDDDD BAAAA ABBBA DDDDDDD BAAAA ABBBB DDDDDDD BAAAA BAAAA DDDDDDD BAAAA BAAAB DDDDDDD BAAAA BAABA DDDDDDD BAAAA BAABB DDDDDDD BAAAA BABAA DDDDDDD BAAAA BABAB DDDDDDD BAAAA BABBA DDDDDDD BAAAA BABBB DDDDDDD BAAAA BBAAA DDDDDDD BAAAA BBAAB DDDDDDD BAAAA BBABA DDDDDDD BAAAA BBABB DDDDDDD BAAAA BBBAA DDDDDDD BAAAA BBBAB DDDDDDD BAAAA BBBBA DDDDDDD BAAAA BBBBB DDDDDDD BAAAA AAAAA DDDDDDD BBAAA AAAAB DDDDDDD BBAAA AAABA DDDDDDD BBAAA AAABB DDDDDDD BBAAA AABAA DDDDDDD BBAAA AABAB DDDDDDD BBAAA AABBA DDDDDDD BBAAA AABBB DDDDDDD BBAAA ABAAA DDDDDDD BBAAA ABAAB DDDDDDD BBAAA ABABA DDDDDDD BBAAA ABABB DDDDDDD BBAAA ABBAA DDDDDDD BBAAA ABBAB DDDDDDD BBAAA ABBBA DDDDDDD BBAAA ABBBB DDDDDDD BBAAA BAAAA DDDDDDD BBAAA BAAAB DDDDDDD BBAAA BAABA DDDDDDD BBAAA BAABB DDDDDDD BBAAA BABAA DDDDDDD BBAAA BABAB DDDDDDD BBAAA BABBA DDDDDDD BBAAA BABBB DDDDDDD BBAAA BBAAA DDDDDDD BBAAA BBAAB DDDDDDD BBAAA BBABA DDDDDDD BBAAA BBABB DDDDDDD BBAAA BBBAA DDDDDDD BBAAA BBBAB DDDDDDD BBAAA BBBBA DDDDDDD BBAAA BBBBB DDDDDDD BBAAA AAAAA DDDDDDD BBBAA AAAAB DDDDDDD BBBAA AAABA DDDDDDD BBBAA AAABB DDDDDDD BBBAA AABAA DDDDDDD BBBAA AABAB DDDDDDD BBBAA AABBA DDDDDDD BBBAA AABBB DDDDDDD BBBAA ABAAA DDDDDDD BBBAA ABAAB DDDDDDD BBBAA ABABA DDDDDDD BBBAA ABABB DDDDDDD BBBAA ABBAA DDDDDDD BBBAA ABBAB DDDDDDD BBBAA ABBBA DDDDDDD BBBAA ABBBB DDDDDDD BBBAA BAAAA DDDDDDD BBBAA BAAAB DDDDDDD BBBAA BAABA DDDDDDD BBBAA BAABB DDDDDDD BBBAA BABAA DDDDDDD BBBAA BABAB DDDDDDD BBBAA BABBA DDDDDDD BBBAA BABBB DDDDDDD BBBAA BBAAA DDDDDDD BBBAA BBAAB DDDDDDD BBBAA

BBABA DDDDDDD BBBAA BBABB DDDDDDD BBBAA BBBAA DDDDDDD BBBAA BBBAB DDDDDDD BBBAA BBBBA DDDDDDD BBBAA BBBBB DDDDDDD BBBAA AAAAA DDDDDDD BBBBA AAAAB DDDDDDD BBBBA AAABA DDDDDDD BBBBA AAABB DDDDDDD BBBBA AABAA DDDDDDD BBBBA AABAB DDDDDDD BBBBA AABBA DDDDDDD BBBBA AABBB DDDDDDD BBBBA ABAAA DDDDDDD BBBBA ABAAB DDDDDDD BBBBA ABABA DDDDDDD BBBBA ABABB DDDDDDD BBBBA ABBAA DDDDDDD BBBBA ABBAB DDDDDDD BBBBA ABBBA DDDDDDD BBBBA ABBBB DDDDDDD BBBBA BAAAA DDDDDDD BBBBA BAAAB DDDDDDD BBBBA BAABA DDDDDDD BBBBA BAABB DDDDDDD BBBBA BABAA DDDDDDD BBBBA BABAB DDDDDDD BBBBA BABBA DDDDDDD BBBBA BABBB DDDDDDD BBBBA BBAAA DDDDDDD BBBBA BBAAB DDDDDDD BBBBA BBABA DDDDDDD BBBBA BBABB DDDDDDD BBBBA BBBAA DDDDDDD BBBBA BBBAB DDDDDDD BBBBA BBBBA DDDDDDD BBBBA BBBBB DDDDDDD BBBBA AAAAA DDDDDDD BBBBB AAAAB DDDDDDD BBBBB AAABA DDDDDDD BBBBB AAABB DDDDDDD BBBBB AABAA DDDDDDD BBBBB AABAB DDDDDDD BBBBB AABBA DDDDDDD BBBBB AABBB DDDDDDD BBBBB ABAAA DDDDDDD BBBBB ABAAB DDDDDDD BBBBB ABABA DDDDDDD BBBBB ABABB DDDDDDD BBBBB ABBAA DDDDDDD BBBBB ABBAB DDDDDDD BBBBB ABBBA DDDDDDD BBBBB ABBBB DDDDDDD BBBBB BAAAA DDDDDDD BBBBB BAAAB DDDDDDD BBBBB BAABA DDDDDDD BBBBB BAABB DDDDDDD BBBBB BABAA DDDDDDD BBBBB BABAB DDDDDDD BBBBB BABBA DDDDDDD BBBBB BABBB DDDDDDD BBBBB BBAAA DDDDDDD BBBBB BBAAB DDDDDDD BBBBB BBABA DDDDDDD BBBBB BBABB DDDDDDD BBBBB BBBAA DDDDDDD BBBBB BBBAB DDDDDDD BBBBB BBBBA DDDDDDD BBBBB BBBBB DDDDDDD BBBBB

TABLE-US-00012 TABLE 12 Certain Gapmer Nucleoside Motifs 5'-wing Central gap 3'-wing region region region AAAW DDDDDDDD BBA AABW DDDDDDDD BBA ABAW DDDDDDDD BBA ABBW DDDDDDDD BBA BAAW DDDDDDDD BBA BABW DDDDDDDD BBA BBAW DDDDDDDD BBA BBBW DDDDDDDD BBA ABB DDDDDDDD WAAA ABB DDDDDDDD WAAB ABB DDDDDDDD WABA ABB DDDDDDDD WABB ABB DDDDDDDD WBAA ABB DDDDDDDD WBAB ABB DDDDDDDD WBBA ABB DDDDDDDD WBBB AAAWW DDDDDDD BBA AABWW DDDDDDD BBA ABAWW DDDDDDD BBA ABBWW DDDDDDD BBA BAAWW DDDDDDD BBA BABWW DDDDDDD BBA BBAWW DDDDDDD BBA BBBWW DDDDDDD BBA ABB DDDDDDD WWAAA ABB DDDDDDD WWAAB ABB DDDDDDD WWABA ABB DDDDDDD WWABB ABB DDDDDDD WWBAA ABB DDDDDDD WWBAB ABB DDDDDDD WWBBA ABB DDDDDDD WWBBB AAAAW DDDDDDD BBA AAABW DDDDDDD BBA AABAW DDDDDDD BBA AABBW DDDDDDD BBA ABAAW DDDDDDD BBA ABABW DDDDDDD BBA ABBAW DDDDDDD BBA ABBBW DDDDDDD BBA BAAAW DDDDDDD BBA BAABW DDDDDDD BBA BABAW DDDDDDD BBA BABBW DDDDDDD BBA BBAAW DDDDDDD BBA BBABW DDDDDDD BBA BBBAW DDDDDDD BBA BBBBW DDDDDDD WAAAA ABB DDDDDDD WAAAB ABB DDDDDDD WAABA ABB DDDDDDD WAABB ABB DDDDDDD WABAA ABB DDDDDDD WABAB ABB DDDDDDD WABBA ABB DDDDDDD WABBB ABB DDDDDDD WBAAA ABB DDDDDDD WBAAB ABB DDDDDDD WBABA ABB DDDDDDD WBABB ABB DDDDDDD WBBAA ABB DDDDDDD WBBAB ABB DDDDDDD WBBBA ABB DDDDDDD WBBBB

[0498] wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type and each W is a modified nucleoside or nucleobase of either the first type, the second type or a third type, each D is a nucleoside comprising an unmodified 2' deoxy sugar moiety and unmodified nucleobase, and .sup.ND is modified nucleoside comprising a modified nucleobase and an unmodified 2' deoxy sugar moiety.

[0499] In certain embodiments, each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2'-substituted sugar moiety. In certain embodiments, each A comprises a 2'-substituted sugar moiety selected from among F, ara-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certain embodiments, each A comprises an HNA. In certain embodiments, each A comprises an F-HNA. In certain embodiments, each A comprises a 5'-substituted sugar moiety selected from among 5'-Me, and 5'-(R)-Me.

[0500] In certain embodiments, each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2'-substituted sugar moiety. In certain embodiments, each B comprises a 2'-substituted sugar moiety selected from among F, (ara)-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. In certain embodiments, each B comprises an HNA. In certain embodiments, each B comprises an F-HNA. In certain embodiments, each B comprises a 5'-substituted sugar moiety selected from among 5'-Me, and 5'-(R)-Me.

[0501] In certain embodiments, each C comprises a modified sugar moiety. In certain embodiments, each C comprises a 2'-substituted sugar moiety. In certain embodiments, each C comprises a 2'-substituted sugar moiety selected from among F, (ara)-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each C comprises a 5'-substituted sugar moiety. In certain embodiments, each C comprises a 5'-substituted sugar moiety selected from among 5'-Me, and 5'-(R)-Me. In certain embodiments, each C comprises a bicyclic sugar moiety. In certain embodiments, each C comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each C comprises a modified nucleobase. In certain embodiments, each C comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine. In certain embodiments, each C comprises a 2-thio-thymidine nucleoside. In certain embodiments, each C comprises an HNA. In certain embodiments, each C comprises an F-HNA.

[0502] In certain embodiments, each W comprises a modified sugar moiety. In certain embodiments, each W comprises a 2'-substituted sugar moiety. In certain embodiments, each W comprises a 2'-substituted sugar moiety selected from among F, (ara)-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each W comprises a 5'-substituted sugar moiety. In certain embodiments, each W comprises a 5'-substituted sugar moiety selected from among 5'-Me, and 5'-(R)-Me. In certain embodiments, each W comprises a bicyclic sugar moiety. In certain embodiments, each W comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each W comprises a sugar surrogate. In certain embodiments, each W comprises a sugar surrogate selected from among HNA and F--HNA. In certain embodiments, each W comprises a 2-thio-thymidine nucleoside.

[0503] In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, and the other comprises a 2'-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2'-substituted sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-substituted sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside and the other of A or B comprises a 2'-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2'-MOE sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-MOE sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside and the other of A or B comprises a 2'-MOE sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2'-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-F sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside and the other of A or B comprises a 2'-F sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2'-(ara)-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-(ara)-F sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside and the other of A or B comprises a 2'-(ara)-F sugar moiety.

[0504] In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2'-substituted sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2'-substituted sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2'-substituted sugar moiety. In certain embodiments, A is an .alpha.-L-LNA nucleoside and B comprises a 2'-substituted sugar moiety.

[0505] In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2'-MOE sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2'-MOE sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2'-MOE sugar moiety. In certain embodiments, A is an .alpha.-L-LNA nucleoside and B comprises a 2'-MOE sugar moiety.

[0506] In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2'-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2'-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2'-F sugar moiety. In certain embodiments, A is an .alpha.-L-LNA nucleoside and B comprises a 2'-F sugar moiety.

[0507] In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2'-(ara)-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2'-(ara)-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2'-(ara)-F sugar moiety. In certain embodiments, A is an .alpha.-L-LNA nucleoside and B comprises a 2'-(ara)-F sugar moiety.

[0508] In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2'-MOE sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2'-MOE sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2'-MOE sugar moiety. In certain embodiments, B is an .alpha.-L-LNA nucleoside and A comprises a 2'-MOE sugar moiety.

[0509] In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2'-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2'-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2'-F sugar moiety. In certain embodiments, B is an .alpha.-L-LNA nucleoside and A comprises a 2'-F sugar moiety.

[0510] In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2'-(ara)-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2'-(ara)-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2'-(ara)-F sugar moiety. In certain embodiments, B is an .alpha.-L-LNA nucleoside and A comprises a 2'-(ara)-F sugar moiety.

[0511] In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-substituted sugar moiety and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-substituted sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-substituted sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-substituted sugar moiety, and W comprises a modified nucleobase.

[0512] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a modified nucleobase.

[0513] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a modified nucleobase.

[0514] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a modified nucleobase.

[0515] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase.

[0516] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.

[0517] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.

[0518] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises 2-thio-thymidine nucleobase.

[0519] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.

[0520] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.

[0521] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.

[0522] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a sugar surrogate.

[0523] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a sugar surrogate.

[0524] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises sugar surrogate.

[0525] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a HNA sugar surrogate.

[0526] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a sugar HNA surrogate.

[0527] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.

[0528] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a F-HNA sugar surrogate.

[0529] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a F-HNA sugar surrogate.

[0530] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.

[0531] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-Me DNA sugar moiety.

[0532] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety.

[0533] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety.

[0534] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety.

[0535] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety.

[0536] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety.

[0537] In certain embodiments, at least two of A, B or W comprises a 2'-substituted sugar moiety, and the other comprises a bicyclic sugar moiety. In certain embodiments, at least two of A, B or W comprises a bicyclic sugar moiety, and the other comprises a 2'-substituted sugar moiety. In certain embodiments, a gapmer has a sugar motif other than: E-K-K-(D).sub.9-K-K-E; E-E-E-E-K-(D).sub.9-E-E-E-E-E; E-K-K-K-(D).sub.9-K-K-K-E; K-E-E-K-(D).sub.9-K-E-E-K; K-D-D-K-(D).sub.9-K-D-D-K; K-E-K-E-K-(D).sub.9-K-E-K-E-K; K-D-K-D-K-(D).sub.9-K-D-K-D-K; E-K-E-K-(D).sub.9-K-E-K-E; E-E-E-E-E-K-(D).sub.8-E-E-E-E-E; or E-K-E-K-E-(D).sub.9-E-K-E-K-E, E-E-E-K-K-(D).sub.7-E-E-K, E-K-E-K-K-K-(D).sub.7-K-E-K-E, E-K-E-K-E-K-(D).sub.7-K-E-K-E, wherein K is a nucleoside comprising a cEt sugar moiety and E is a nucleoside comprising a 2'-MOE sugar moiety.

[0538] In certain embodiments a gapmer comprises a A-(D).sub.4-A-(D).sub.4-A-(D).sub.4-AA motif. In certain embodiments a gapmer comprises a B-(D).sub.4-A-(D).sub.4-A-(D).sub.4-AA motif. In certain embodiments a gapmer comprises a A-(D).sub.4-B-(D).sub.4-A-(D).sub.4-AA motif. In certain embodiments a gapmer comprises a A-(D).sub.4-A-(D).sub.4-B-(D).sub.4-AA motif. In certain embodiments a gapmer comprises a A-(D).sub.4-A-(D).sub.4-A-(D).sub.4-BA motif. In certain embodiments a gapmer comprises a A-(D).sub.4-A-(D).sub.4-A-(D).sub.4-BB motif. In certain embodiments a gapmer comprises a K-(D).sub.4-K-(D).sub.4-K-(D).sub.4-K-E motif.

[0539] viii. Certain Internucleoside Linkage Motifs

[0540] In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, internucleoside linkages are arranged in a gapped motif, as described above for nucleoside motif. In such embodiments, the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. The nucleoside motif is independently selected, so such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped nucleoside motif and if it does have a gapped nucleoside motif, the wing and gap lengths may or may not be the same.

[0541] In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides of the present invention comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

[0542] In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3' end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3' end of the oligonucleotide.

[0543] In certain embodiments, oligonucleotides comprise one or more methylphosphonate linkages. In certain embodiments, oligonucleotides having a gapmer nucleoside motif comprise a linkage motif comprising all phosphorothioate linkages except for one or two methylphosphonate linkages. In certain embodiments, one methylphosphonate linkage is in the central gap of an oligonucleotide having a gapmer nucleoside motif.

[0544] ix. Certain Modification Motifs

[0545] Modification motifs define oligonucleotides by nucleoside motif (sugar motif and nucleobase motif) and linkage motif. For example, certain oligonucleotides have the following modification motif:

[0546] A.sub.sA.sub.sA.sub.sD.sub.sD.sub.sD.sub.sD.sub.s(.sup.ND).sub.sD.s- ub.sD.sub.sD.sub.sD.sub.sB.sub.sB.sub.sB;

wherein each A is a modified nucleoside comprising a 2'-substituted sugar moiety; each D is an unmodified 2'-deoxynucleoside; each B is a modified nucleoside comprising a bicyclic sugar moiety; .sup.ND is a modified nucleoside comprising a modified nucleobase; and s is a phosphorothioate internucleoside linkage. Thus, the sugar motif is a gapmer motif. The nucleobase modification motif is a single modified nucleobase at 8.sup.th nucleoside from the 5'-end. Combining the sugar motif and the nucleobase modification motif, the nucleoside motif is an interrupted gapmer where the gap of the sugar modified gapmer is interrupted by a nucleoside comprising a modified nucleobase. The linkage motif is uniform phosphorothioate. The following non-limiting Table further illustrates certain modification motifs:

TABLE-US-00013 TABLE 13 Certain Modification Motifs 5'-wing Central gap 3'-wing region region region B.sub.sB.sub.s .sub.sD.sub.sD.sub.sD.sub.sD.sub.sD.sub.sD.sub.sD.sub.sD.sub.sD.sub.s A.sub.sA.sub.sA.sub.sA.sub.sA.sub.sA.sub.sA.sub.sA AsBsBs DsDsDsDsDsDsDsDsDs BsBsA AsBsBs DsDsDsDs(.sup.ND)sDsDsDsDs BsBsA AsBsBs DsDsDsDsAsDsDsDsDs BsBsA AsBsBs DsDsDsDsBsDsDsDsDs BsBsA AsBsBs DsDsDsDsWsDsDsDsDs BsBsA AsBsBsBs DsDsDsDsDsDsDsDsDs BsBsAsBsB AsBsBs DsDsDsDsDsDsDsDsDs BsBsAsBsB BsBsAsBsBs DsDsDsDsDsDsDsDsDs BsBsA AsBsBs DsDsDsDsDsDsDsDsDs BsBsAsBsBsBsB AsAsBsAsAs DsDsDsDsDsDsDsDsDs BsBsA AsAsAsBsAsAs DsDsDsDsDsDsDsDsDs BsBsA AsAsBsAsAs DsDsDsDsDsDsDsDsDs AsAsBsAsA AsAsAsBsAsAs DsDsDsDsDsDsDsDsDs AsAsBsAsAsA AsAsAsAsBsAsAs DsDsDsDsDsDsDsDsDs BsBsA AsBsAsBs DsDsDsDsDsDsDsDsDs BsAsBsA AsBsAsBs DsDsDsDsDsDsDsDsDs AsAsBsAsAs AsBsBs DsDsDsDsDsDsDsDsDs BsAsBsA BsBsAsBsBsBsB DsDsDsDsDsDsDsDsDs BsAsBsA AsAsAsAsAs DsDsDsDsDsDsDsDsDs AsAsAsAsA AsAsAsAsAs DsDsDsDsDsDsDs AsAsAsAsA AsAsAsAsAs DsDsDsDsDsDsDsDsDs BsBsAsBsBsBsB AsAsAsBsBs DsDsDsDsDsDsDs BsBsA AsBsAsBs DsDsDsDsDsDsDsDs BsBsA AsBsAsBs DsDsDsDsDsDsDs AsAsAsBsBs AsAsAsAsBs DsDsDsDsDsDsDs BsAsAsAsA BsBs DsDsDsDsDsDsDsDs AsA AsAs DsDsDsDsDsDsDs AsAsAsAsAsAsAsA AsAsAs DsDsDsDsDsDsDs AsAsAsAsAsAsA AsAsAs DsDsDsDsDsDsDs AsAsAsAsAsA AsBs DsDsDsDsDsDsDs BsBsBsA AsBsBsBs DsDsDsDsDsDsDsDsDs BsA AsBs DsDsDsDsDsDsDsDsDs BsBsBsA AsAsAsBsBs DsDsDs(.sup.ND)sDsDsDs BsBsAsAsA AsAsAsBsBs DsDsDsAsDsDsDs BsBsAsAsA AsAsAsBsBs DsDsDsBsDsDsDs BsBsAsAsA AsAsAsAsBs DsDsDsDsDsDsDs BsAsAsAsA AsAsBsBsBs DsDsDsDsDsDsDs BsBsBsAsA AsAsAsAsBs DsDsDsDsDsDsDs AsAsAsAsAs AsAsAsBsBs DsDsDsDsDsDsDs AsAsAsAsAs AsAsBsBsBs DsDsDsDsDsDsDs AsAsAsAsAs AsAsAsAsAs DsDsDsDsDsDsDs BsAsAsAsAs AsAsAsAsAs DsDsDsDsDsDsDs BsBsAsAsAs AsAsAsAsAs DsDsDsDsDsDsDs BsBsBsAsAs AsBsBs DsDsDsDs(.sup.ND)s(.sup.ND)sDsDsDs BsBsA AsBsBs Ds(.sup.ND)s(.sup.ND)sDs(.sup.ND)s(.sup.ND)sDs(.sup.ND)s(.sup.ND)s BsBsA AsBsBs Ds(.sup.ND)sDsDsDsDsDsDsDs BsBsA AsBsBs DsDs(.sup.ND)sDsDsDsDsDsDs BsBsA AsBsBs Ds(.sup.ND)s(.sup.ND)sDsDsDsDsDsDs BsBsA AsBsBs DsDs(D)zDsDsDsDsDsDs BsBsA AsBsBs Ds(D)zDsDsDsDsDsDsDs BsBsA AsBsBs (D)zDsDsDsDsDsDsDsDs BsBsA AsBsBs DsDsAsDsDsDsDsDsDs BsBsA AsBsBs DsDsBsDsDsDsDsDsDs BsBsA AsBsBs AsDsDsDsDsDsDsDsDs BsBsA AsBsBs BsDsDsDsDsDsDsDsDs BsBsA AsBsAsBs DsDs(D)zDsDsDsDsDsDs BsBsBsAsAs AsAsAsBsBs DsDs(.sup.ND)sDsDsDsDsDsDs AsA AsBsBsBs Ds(D)zDsDsDsDsDsDsDs AsAsAsBsBs AsBsBs DsDsDsDsDsDsDsDs(D)z BsBsA AsAsBsBsBs DsDsDsAsDsDsDs BsBsBsAsA AsAsBsBsBs DsDsDsBsDsDsDs BsBsBsAsA AsBsAsBs DsDsDsAsDsDsDs BsBsAsBsBsBsB AsBsBsBs DsDsDsDs(D)zDsDsDsDs BsA AsAsBsBsBs DsDsAsAsDsDsDs BsBsA AsBsBs DsDsDsDs(D)zDsDsDsDs BsBsBsA BsBs DsDs(.sup.ND)sDs(.sup.ND)sDsDsDsDs BsBsAsBsBsBsB

[0547] wherein each A and B are nucleosides comprising differently modified sugar moieties, each D is a nucleoside comprising an unmodified 2' deoxy sugar moiety, each W is a modified nucleoside of either the first type, the second type or a third type, each .sup.ND is a modified nucleoside comprising a modified nucleobase, s is a phosphorothioate internucleoside linkage, and z is a non-phosphorothioate internucleoside linkage.

[0548] In certain embodiments, each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2'-substituted sugar moiety. In certain embodiments, each A comprises a 2'-substituted sugar moiety selected from among F, (ara)-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certain embodiments, each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2'-substituted sugar moiety. In certain embodiments, each B comprises a 2'-substituted sugar moiety selected from among F, (ara)-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. In certain embodiments, each A comprises an HNA. In certain embodiments, each A comprises an F-HNA.

[0549] In certain embodiments, each W comprises a modified sugar moiety. In certain embodiments, each W comprises a 2'-substituted sugar moiety. In certain embodiments, each W comprises a 2'-substituted sugar moiety selected from among F, (ara)-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each W comprises a 5'-substituted sugar moiety. In certain embodiments, each W comprises a 5'-substituted sugar moiety selected from among 5'-Me, and 5'-(R)-Me. In certain embodiments, each W comprises a bicyclic sugar moiety. In certain embodiments, each W comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments, each W comprises a sugar surrogate. In certain embodiments, each W comprises a sugar surrogate selected from among HNA and F--HNA.

[0550] In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, and the other comprises a 2'-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2'-substituted sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-substituted sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside and the other of A or B comprises a 2'-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2'-MOE sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-MOE sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside and the other of A or B comprises a 2'-MOE sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2'-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-F sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside and the other of A or B comprises a 2'-F sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2'-(ara)-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2'-(ara)-F sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside and the other of A or B comprises a 2'-(ara)-F sugar moiety.

[0551] In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2'-substituted sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2'-substituted sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2'-substituted sugar moiety. In certain embodiments, A is an .alpha.-L-LNA nucleoside and B comprises a 2'-substituted sugar moiety.

[0552] In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2'-MOE sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2'-MOE sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2'-MOE sugar moiety. In certain embodiments, A is an .alpha.-L-LNA nucleoside and B comprises a 2'-MOE sugar moiety.

[0553] In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2'-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2'-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2'-F sugar moiety. In certain embodiments, A is an .alpha.-L-LNA nucleoside and B comprises a 2'-F sugar moiety.

[0554] In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2'-(ara)-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2'-(ara)-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2'-(ara)-F sugar moiety. In certain embodiments, A is an .alpha.-L-LNA nucleoside and B comprises a 2'-(ara)-F sugar moiety.

[0555] In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2'-MOE sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2'-MOE sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2'-MOE sugar moiety. In certain embodiments, B is an .alpha.-L-LNA nucleoside and A comprises a 2'-MOE sugar moiety.

[0556] In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2'-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2'-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2'-F sugar moiety. In certain embodiments, B is an .alpha.-L-LNA nucleoside and A comprises a 2'-F sugar moiety.

[0557] In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2'-(ara)-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2'-(ara)-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2'-(ara)-F sugar moiety. In certain embodiments, B is an .alpha.-L-LNA nucleoside and A comprises a 2'-(ara)-F sugar moiety.

[0558] In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-substituted sugar moiety and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-substituted sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-substituted sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-substituted sugar moiety, and W comprises a modified nucleobase.

[0559] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a modified nucleobase.

[0560] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a modified nucleobase.

[0561] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a modified nucleobase.

[0562] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase.

[0563] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.

[0564] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.

[0565] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises 2-thio-thymidine nucleobase.

[0566] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.

[0567] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.

[0568] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.

[0569] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a sugar surrogate.

[0570] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a sugar surrogate.

[0571] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises sugar surrogate.

[0572] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a HNA sugar surrogate.

[0573] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a sugar HNA surrogate.

[0574] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.

[0575] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a F--HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a F-HNA sugar surrogate.

[0576] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a F-HNA sugar surrogate.

[0577] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.

[0578] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-Me DNA sugar moiety.

[0579] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety.

[0580] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-Me DNA sugar moiety.

[0581] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety.

[0582] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety.

[0583] In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside, another of A or B comprises a 2'-(ara)-F sugar moiety, and W comprises a 5'-(R)-Me DNA sugar moiety.

[0584] In certain embodiments, at least two of A, B or W comprises a 2'-substituted sugar moiety, and the other comprises a bicyclic sugar moiety. In certain embodiments, at least two of A, B or W comprises a bicyclic sugar moiety, and the other comprises a 2'-substituted sugar moiety.

[0585] d. Certain Overall Lengths

[0586] In certain embodiments, the present invention provides oligomeric compounds including oligonucleotides of any of a variety of ranges of lengths. In certain embodiments, the invention provides oligomeric compounds or oligonucleotides consisting of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X.ltoreq.Y. For example, in certain embodiments, the invention provides oligomeric compounds which comprise oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides. In embodiments where the number of nucleosides of an oligomeric compound or oligonucleotide is limited, whether to a range or to a specific number, the oligomeric compound or oligonucleotide may, nonetheless further comprise additional other substituents. For example, an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotides having 31 nucleosides, but, unless otherwise indicated, such an oligonucleotide may further comprise, for example one or more conjugates, terminal groups, or other substituents. In certain embodiments, a gapmer oligonucleotide has any of the above lengths.

[0587] Further, where an oligonucleotide is described by an overall length range and by regions having specified lengths, and where the sum of specified lengths of the regions is less than the upper limit of the overall length range, the oligonucleotide may have additional nucleosides, beyond those of the specified regions, provided that the total number of nucleosides does not exceed the upper limit of the overall length range.

[0588] e. Certain Oligonucleotides

[0589] In certain embodiments, oligonucleotides of the present invention are characterized by their modification motif and overall length. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. For example, the internucleoside linkages within the wing regions of a sugar-gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region. Likewise, such sugar-gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. One of skill in the art will appreciate that such motifs may be combined to create a variety of oligonucleotides. Herein if a description of an oligonucleotide or oligomeric compound is silent with respect to one or more parameter, such parameter is not limited. Thus, an oligomeric compound described only as having a gapmer sugar motif without further description may have any length, internucleoside linkage motif, and nucleobase modification motif. Unless otherwise indicated, all chemical modifications are independent of nucleobase sequence.

[0590] f. Certain Conjugate Groups

[0591] In certain embodiments, oligomeric compounds are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligomeric compound including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligomeric compound, such as an oligonucleotide. Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 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 (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

[0592] In certain embodiments, a conjugate group comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

[0593] In certain embodiments, conjugate groups are directly attached to oligonucleotides in oligomeric compounds. In certain embodiments, conjugate groups are attached to oligonucleotides by a conjugate linking group. In certain such embodiments, conjugate linking groups, including, but not limited to, bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein. Conjugate linking groups are useful for attachment of conjugate groups, such as chemical stabilizing groups, functional groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound. In general a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group. In some embodiments, the conjugate linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups that are routinely used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.

[0594] Some nonlimiting examples of conjugate linking moieties include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C.sub.1-C.sub.10 alkyl, substituted or unsubstituted C.sub.2-C.sub.10 alkenyl or substituted or unsubstituted C.sub.2-C.sub.10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

[0595] Conjugate groups may be attached to either or both ends of an oligonucleotide (terminal conjugate groups) and/or at any internal position.

[0596] In certain embodiments, conjugate groups are at the 3'-end of an oligonucleotide of an oligomeric compound. In certain embodiments, conjugate groups are near the 3'-end. In certain embodiments, conjugates are attached at the 3' end of an oligomeric compound, but before one or more terminal group nucleosides. In certain embodiments, conjugate groups are placed within a terminal group. In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, oligomeric compounds comprise an oligonucleotide. In certain embodiments, an oligomeric compound comprises an oligonucleotide and one or more conjugate and/or terminal groups. Such conjugate and/or terminal groups may be added to oligonucleotides having any of the motifs discussed above. Thus, for example, an oligomeric compound comprising an oligonucleotide having region of alternating nucleosides may comprise a terminal group.

C. ANTISENSE COMPOUNDS

[0597] In certain embodiments, oligomeric compounds provided herein are antisense compounds. Such antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, antisense compounds specifically hybridize to one or more target nucleic acid. In certain embodiments, a specifically hybridizing antisense compound has a nucleobase sequence comprising a region having sufficient complementarity to a target nucleic acid to allow hybridization and result in antisense activity and insufficient complementarity to any non-target so as to avoid non-specific hybridization to any non-target nucleic acid sequences under conditions in which specific hybridization is desired (e.g., under physiological conditions for in vivo or therapeutic uses, and under conditions in which assays are performed in the case of in vitro assays).

[0598] In certain embodiments, the present invention provides antisense compounds comprising oligonucleotides that are fully complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are 95% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 90% complementary to the target nucleic acid.

[0599] In certain embodiments, such oligonucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, an antisense compound comprises a region that is fully complementary to a target nucleic acid and is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain such embodiments, the region of full complementarity is from 6 to 14 nucleobases in length.

[0600] a. Certain Antisense Activities and Mechanisms

[0601] In certain antisense activities, hybridization of an antisense compound results in recruitment of a protein that cleaves of the target nucleic acid. For example, certain antisense compounds result in RNase H mediated cleavage of target nucleic acid. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. The "DNA" in such an RNA:DNA duplex, need not be unmodified DNA. In certain embodiments, the invention provides antisense compounds that are sufficiently "DNA-like" to elicit RNase H activity. Such DNA-like antisense compounds include, but are not limited to gapmers having unmodified deoxyfuronose sugar moieties in the nucleosides of the gap and modified sugar moieties in the nucleosides of the wings.

[0602] Antisense activities may be observed directly or indirectly. In certain embodiments, observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid; a change in the ratio of splice variants of a nucleic acid or protein; and/or a phenotypic change in a cell or animal.

[0603] In certain embodiments, compounds comprising oligonucleotides having a gapmer nucleoside motif described herein have desirable properties compared to non-gapmer oligonucleotides or to gapmers having other motifs. In certain circumstances, it is desirable to identify motifs resulting in a favorable combination of potent antisense activity and relatively low toxicity. In certain embodiments, compounds of the present invention have a favorable therapeutic index (measure of activity divided by measure of toxicity).

[0604] b. Certain Selective Antisense Compounds

[0605] In certain embodiments, antisense compounds provided are selective for a target relative to a non-target nucleic acid. In certain embodiments, the nucleobase sequences of the target and non-target nucleic acids differ by no more than 4 differentiating nucleobases in the targeted region. In certain embodiments, the nucleobase sequences of the target and non-target nucleic acids differ by no more than 3 differentiating nucleobases in the targeted region. In certain embodiments, the nucleobase sequences of the target and non-target nucleic acids differ by no more than 2 differentiating nucleobases in the targeted region. In certain embodiments, the nucleobase sequences of the target and non-target nucleic acids differ by a single differentiating nucleobase in the targeted region. In certain embodiments, the target and non-target nucleic acids are transcripts from different genes. In certain embodiments, the target and non-target nucleic acids are different alleles for the same gene. In certain embodiments, the introduction of a mismatch between an antisense compound and a non-target nucleic acid may alter the RNase H cleavage site of a target nucleic acid compared to a non-target nucleic acid. In certain embodiments, the target and non-target nucleic acids are not functionally related to one another (e.g., are transcripts from different genes). In certain embodiments, the target and not-target nucleic acids are allelic variants of one another. In certain embodiments, the allelic variant contains a single nucleotide polymorphism (SNP). In certain embodiments, a SNP is associated with a mutant allele. In certain embodiments, a mutant SNP is associated with a disease. In certain embodiments a mutant SNP is associated with a disease, but is not causative of the disease. In certain embodiments, mRNA and protein expression of a mutant allele is associated with disease.

[0606] Selectivity of antisense compounds is achieved, principally, by nucleobase complementarity. For example, if an antisense compound has no mismatches for a target nucleic acid and one or more mismatches for a non-target nucleic acid, some amount of selectivity for the target nucleic acid will result. In certain embodiments, provided herein are antisense compounds with enhanced selectivity (i.e. the ratio of activity for the target to the activity for non-target is greater). For example, in certain embodiments, a selective nucleoside comprises a particular feature or combination of features (e.g., chemical modification, motif, placement of selective nucleoside, and/or self-complementary region) that increases selectivity of an antisense compound compared to an antisense compound not having that feature or combination of features. In certain embodiments, such feature or combination of features increases antisense activity for the target. In certain embodiments, such feature or combination of features decreases activity for the target, but decreases activity for the non-target by a greater amount, thus resulting in an increase in selectivity.

[0607] Without being limited by mechanism, enhanced selectivity may result from a larger difference in the affinity of an antisense compound for its target compared to its affinity for the non-target and/or a larger difference in RNase H activity for the resulting duplexes. For example, in certain embodiments, a selective antisense compound comprises a modified nucleoside at that same position as a differentiating nucleobase (i.e., the selective nucleoside is modified). That modification may increase the difference in binding affinity of the antisense compound for the target relative to the non-target. In addition or in the alternative, the chemical modification may increase the difference in RNAse H activity for the duplex formed by the antisense compound and its target compared to the RNase activity for the duplex formed by the antisense compound and the non-target. For example, the modification may exaggerate a structure that is less compatible for RNase H to bind, cleave and/or release the non-target.

[0608] In certain embodiments, an antisense compound binds its intended target to form a target duplex. In certain embodiments, RNase H cleaves the target nucleic acid of the target duplex. In certain such embodiments, there is a primary cleavage site between two particular nucleosides of the target nucleic acid (the primary target cleavage site), which accounts for the largest amount of cleavage of the target nucleic acid. In certain embodiments, there are one or more secondary target cleavage sites. In certain embodiments, the same antisense compound hybridizes to a non-target to form a non-target duplex. In certain such embodiments, the non-target differs from the target by a single nucleobase within the target region, and so the antisense compound hybridizes with a single mismatch. Because of the mismatch, in certain embodiments, RNase H cleavage of the non-target may be reduced compared to cleavage of the target, but still occurs. In certain embodiments, though, the primary site of that cleavage of the non-target nucleic acid (primary non-target cleavage site) is different from that of the target. That is; the primary site is shifted due to the mismatch. In such a circumstance, one may use a modification placed in the antisense compound to disrupt RNase H cleavage at the primary non-target cleavage site. Such modification will result in reduced cleavage of the non-target, but will result little or no decrease in cleavage of the target. In certain embodiments, the modification is a modified sugar, nucleobase and/or linkage.

[0609] In certain embodiments, the primary non-target cleavage site is towards the 5'-end of the antisense compound, and the 5'-end of an antisense compound may be modified to prevent RNaseH cleavage. In this manner, it is thought that one having skill in the art may modify the 5'-end of an antisense compound, or modify the nucleosides in the gap region of the 5'-end of the antisense compound, or modify the 3'-most 5'-region nucleosides of the antisense compound to selectively inhibit RNaseH cleavage of the non-target nucleic acid duplex while retaining RNase H cleavage of the target nucleic acid duplex. In certain embodiments, 1-3 of the 3'-most 5'-region nucleosides of the antisense compound comprises a bicyclic sugar moiety.

[0610] For example, in certain embodiments the target nucleic acid may have an allelic variant, e.g. a non-target nucleic acid, containing a single nucleotide polymorphism. An antisense compound may be designed having a single nucleobase mismatch from the non-target nucleic acid, but which has full complementarity to the target nucleic acid. The mismatch between the antisense compound and the non-target nucleic acid may destabilize the antisense compound non-target nucleic acid duplex, and consequently the cleavage site of RNaseH may shift upstream towards the 5'-end of the antisense compound. Modification of the 5'-end of the antisense compound or the gap region near the 5'-end of the antisense compound, or one or more of the 3'-most nucleosides of the 5'-wing region, will then prevent RNaseH cleavage of the non-target nucleic acid. Since the target nucleic acid is fully complementary to the antisense compound, the antisense compound and the target nucleic acid will form a more stabilized antisense compound-target nucleic acid duplex and the cleavage site of RnaseH will be more downstream, towards the 3' end of the antisense compound. Accordingly, modifications at the 5'-end of the antisense compound will prevent RNaseH cleavage of the non-target nucleic acid, but will not substantially effect RNaseH cleavage of the target nucleic acid, and selectivity between a target nucleic acid and its allelic variant may be achieved. In certain embodiments, one or more of the 3'-most nucleosides of the 5'-wing region comprises a bicyclic sugar moiety. In certain embodiments, one or more of the 3'-most nucleosides of the 5'-wing region comprises a bicyclic sugar moiety selected from cEt and LNA. In certain embodiments, one or more of the 3'-most nucleosides of the 5'-wing region comprises cEt. In certain embodiments, one or more of the 3'-most nucleosides of the 5'-wing region comprises LNA.

[0611] In certain embodiments, the introduction of a mismatch between an antisense compound and a target nucleic acid may alter the RNase H cleavage site of a target nucleic acid compared to a non-target nucleic acid by shifting the RNaseH cleavage site downstream from the mismatch site and towards the 3'-end of the antisense compound. In certain embodiments where the cleavage site of a target nucleic acid compared to a non-target nucleic acid has shifted downstream towards the 3'-end of the antisense compound, the 3'-end of an antisense compound may be modified to prevent RNaseH cleavage. In this manner, it is thought that one having skill in the art may modify the 3'-end of an antisense compound, or modify the nucleosides in the gap region near the 3'-end of antisense compound, to selectively inhibit RNaseH cleavage of the non-target nucleic acid while retaining RNase H cleavage of the target nucleic acid.

[0612] For example, in certain embodiments the target nucleic acid may have an allelic variant, e.g. a non-target nucleic acid, containing a single nucleotide polymorphism. An antisense compound may be designed having a single nucleobase mismatch from the non-target nucleic acid, but which has full complementarity to target nucleic acid. The mismatch between the antisense compound and the non-target nucleic acid may destabilize the antisense compound-non-target nucleic acid duplex, and consequently the cleavage site of RNaseH may shift downstream towards the 3'-end of the antisense compound. Modification of the 3'-end of the antisense compound, or one or more of the 5'-most nucleosides of the 3'-wing region, or the gap region of the antisense compound near the 3'-end will then prevent RNaseH cleavage of the non-target nucleic acid. Since the target nucleic acid is fully complementary to the antisense compound, the antisense compound and the target nucleic acid will form a more stabilized antisense compound-target nucleic acid duplex and the cleavage site of RnaseH will be more upstream, towards the 5' end of the antisense compound. Accordingly, modifications at the 3'-end of the antisense compound will prevent RNaseH cleavage of the non-target nucleic acid, but will not substantially effect RNaseH cleavage of the target nucleic acid, and selectivity between a target nucleic acid and its allelic variant may be achieved. In certain embodiments, one or more of the 5'-most nucleosides of the 3'-wing region comprises a bicyclic sugar moiety. In certain embodiments, one or more of the 5'-most nucleosides of the 3'-wing region comprises a bicyclic sugar moiety selected from cEt and LNA. In certain embodiments, one or more of the 5'-most nucleosides of the 3'-wing region comprises cEt. In certain embodiments, one or more of the 5'-most nucleosides of the 3'-wing region comprises LNA.

[0613] In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of one or more bicyclic nucleosides at the 3'-most 5'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of two or more bicyclic nucleosides at the 3'-most 5'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of one bicyclic nucleoside at the 3'-most 5'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of two bicyclic nucleosides at the 3'-most 5'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of three bicyclic nucleosides at the 3'-most 5'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of four bicyclic nucleosides at the 3'-most 5'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of five bicyclic nucleosides at the 3'-most 5'-wing nucleoside. In certain embodiments discussed above, the bicyclic nucleosides at the 3'-most 5'-wing nucleoside are selected from among cEt, cMOE, LNA, .alpha.-LNA, ENA and 2'-thio LNA. In certain embodiments discussed above, the bicyclic nucleosides at the 3'-most 5'-wing nucleoside comprise cEt. In certain embodiments discussed above, the bicyclic nucleosides at the 3'-most 5'-wing nucleoside comprise LNA.

[0614] In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of one or more bicyclic nucleosides at the 3'-most 5'-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5'-most 3'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of two or more bicyclic nucleosides at the 3'-most 5'-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5'-most 3'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of one bicyclic nucleoside at the 3'-most 5'-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5'-most 3'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of two bicyclic nucleosides at the 3'-most 5'-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5'-most 3'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of three bicyclic nucleosides at the 3'-most 5'-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5'-most 3'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of four bicyclic nucleosides at the 3'-most 5'-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5'-most 3'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of four bicyclic nucleosides at the 3'-most 5'-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5'-most 3'-wing nucleoside.

[0615] In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of one or more bicyclic nucleosides at the 3'-most 5'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of two or more bicyclic nucleosides at the 3'-most 5'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of one bicyclic nucleoside at the 3'-most 5'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of two bicyclic nucleosides at the 3'-most 5'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of three bicyclic nucleosides at the 3'-most 5'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of four bicyclic nucleosides at the 3'-most 5'-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of five bicyclic nucleosides at the 3'-most 5'-wing nucleoside. In certain embodiments discussed above, the bicyclic nucleosides at the 3'-most 5'-wing nucleoside are selected from among cEt, cMOE, LNA, .alpha.-LNA, ENA and 2'-thio LNA. In certain embodiments discussed above, the bicyclic nucleosides at the 3'-most 5'-wing nucleoside comprise cEt. In certain embodiments discussed above, the bicyclic nucleosides at the 3'-most 5'-wing nucleoside comprise LNA.

[0616] Antisense compounds having certain specified motifs have enhanced selectivity, including, but not limited to motifs described above. In certain embodiments, enhanced selectivity is achieved by oligonucleotides comprising any one or more of:

[0617] a modification motif comprising a long 5'-wing (longer than 5, 6, or 7 nucleosides);

[0618] a modification motif comprising a long 3'-wing (longer than 5, 6, or 7 nucleosides);

[0619] a modification motif comprising a short gap region (shorter than 8, 7, or 6 nucleosides); and

[0620] a modification motif comprising an interrupted gap region (having no uninterrupted stretch of unmodified 2'-deoxynucleosides longer than 7, 6 or 5).

[0621] i. Certain Selective Nucleobase Sequence Elements

[0622] In certain embodiments, selective antisense compounds comprise nucleobase sequence elements. Such nucleobase sequence elements are independent of modification motifs. Accordingly, oligonucleotides having any of the motifs (modification motifs, nucleoside motifs, sugar motifs, nucleobase modification motifs, and/or linkage motifs) may also comprise one or more of the following nucleobase sequence elements.

[0623] ii. Alignment of Differentiating Nucleobase/Target-Selective Nucleoside

[0624] In certain embodiments, a target region and a region of a non-target nucleic acid differ by 1-4 differentiating nucleobase. In such embodiments, selective antisense compounds have a nucleobase sequence that aligns with the non-target nucleic acid with 1-4 mismatches. A nucleoside of the antisense compound that corresponds to a differentiating nucleobase of the target nucleic acid is referred to herein as a target-selective nucleoside. In certain embodiments, selective antisense compounds having a gapmer motif align with a non-target nucleic acid, such that a target-selective nucleoside is positioned in the gap. In certain embodiments, a target-selective nucleoside is the 1.sup.st nucleoside of the gap from the 5' end. In certain embodiments, a target-selective nucleoside is the 2.sup.nd nucleoside of the gap from the 5' end. In certain embodiments, a target-selective nucleoside is the 3.sup.rd nucleoside of the gap from the 5'-end. In certain embodiments, a target-selective nucleoside is the 4.sup.th nucleoside of the gap from the 5'-end. In certain embodiments, a target-selective nucleoside is the 5.sup.th nucleoside of the gap from the 5'-end. In certain embodiments, a target-selective nucleoside is the 6.sup.rd nucleoside of the gap from the 5'-end. In certain embodiments, a target-selective nucleoside is the 8.sup.th nucleoside of the gap from the 3'-end. In certain embodiments, a target-selective nucleoside is the 7.sup.th nucleoside of the gap from the 3'-end. In certain embodiments, a target-selective nucleoside is the 6.sup.th nucleoside of the gap from the 3'-end. In certain embodiments, a target-selective nucleoside is the 5.sup.th nucleoside of the gap from the 3'-end. In certain embodiments, a target-selective nucleoside is the 4.sup.th nucleoside of the gap from the 3'-end. In certain embodiments, a target-selective nucleoside is the 3.sup.rd nucleoside of the gap from the 3'-end. In certain embodiments, a target-selective nucleoside is the 2.sup.nd nucleoside of the gap from the 3'-end.

[0625] In certain embodiments, a target-selective nucleoside comprises a modified nucleoside. In certain embodiments, a target-selective nucleoside comprises a modified sugar. In certain embodiments, a target-selective nucleoside comprises a sugar surrogate. In certain embodiments, a target-selective nucleoside comprises a sugar surrogate selected from among HNA and F-HNA. In certain embodiments, a target-selective nucleoside comprises a 2'-substituted sugar moiety. In certain embodiments, a target-selective nucleoside comprises a 2'-substituted sugar moiety selected from among MOE, F and (ara)-F. In certain embodiments, a target-selective nucleoside comprises a 5'-substituted sugar moiety. In certain embodiments, a target-selective nucleoside comprises a 5'-substituted sugar moiety selected from 5'-(R)-Me DNA. In certain embodiments, a target-selective nucleoside comprises a bicyclic sugar moiety. In certain embodiments, a target-selective nucleoside comprises a bicyclic sugar moiety selected from among cEt, and .alpha.-L-LNA. In certain embodiments, a target-selective nucleoside comprises a modified nucleobase. In certain embodiments, a target-selective nucleoside comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine.

[0626] iii. Mismatches to the Target Nucleic Acid

[0627] In certain embodiments, selective antisense compounds comprise one or more mismatched nucleobases relative to the target nucleic acid. In certain such embodiments, antisense activity against the target is reduced by such mismatch, but activity against the non-target is reduced by a greater amount. Thus, in certain embodiments selectivity is improved. Any nucleobase other than the differentiating nucleobase is suitable for a mismatch. In certain embodiments, however, the mismatch is specifically positioned within the gap of an oligonucleotide having a gapmer motif. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 1, 2, 3, 4, 5, 6, 7, or 8 from the 5'-end of the gap region. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 9, 8, 7, 6, 5, 4, 3, 2, 1 of the antisense compounds from the 3'-end of the gap region. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 1, 2, 3, or 4 of the antisense compounds from the 5'-end of the wing region. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 4, 3, 2, or 1 of the antisense compounds from the 3'-end of the wing region.

[0628] iv. Self Complementary Regions

[0629] In certain embodiments, selective antisense compounds comprise a region that is not complementary to the target. In certain embodiments, such region is complementary to another region of the antisense compound. Such regions are referred to herein as self-complementary regions. For example, in certain embodiments, an antisense compound has a first region at one end that is complementary to a second region at the other end. In certain embodiments, one of the first and second regions is complementary to the target nucleic acid. Unless the target nucleic acid also includes a self-complementary region, the other of the first and second region of the antisense compound will not be complementary to the target nucleic acid. For illustrative purposes, certain antisense compounds have the following nucleobase motif:

TABLE-US-00014 ABCXXXXXXXXXC'B'A'; ABCXXXXXXX(X/C')(X/B')(X/A'); (X/A)(X/B)(X/C)XXXXXXXXXC'B'A'

where each of A, B, and C are any nucleobase; A', B', and C' are the complementary bases to A, B, and C, respectively; each X is a nucleobase complementary to the target nucleic acid; and two letters in parentheses (e.g., (X/C')) indicates that the nucleobase is complementary to the target nucleic acid and to the designated nucleoside within the antisense oligonucleotide.

[0630] Without being bound to any mechanism, in certain embodiments, such antisense compounds are expected to form self-structure, which is disrupted upon contact with a target nucleic acid. Contact with a non-target nucleic acid is expected to disrupt the self-structure to a lesser degree, thus increasing selectivity compared to the same antisense compound lacking the self-complementary regions.

[0631] v. Combinations of Features

[0632] Though it is clear to one of skill in the art, the above motifs and other elements for increasing selectivity may be used alone or in combination. For example, a single antisense compound may include any one, two, three, or more of: self-complementary regions, a mismatch relative to the target nucleic acid, a short nucleoside gap, an interrupted gap, and specific placement of the selective nucleoside.

D. CERTAIN SHORT GAP ANTISENSE COMPOUNDS

[0633] In certain embodiments, an antisense compound of interest may modulate the expression of a target nucleic acid but possess undesirable properties. In certain embodiments, for example, an antisense compound of interest may have an undesirably high affinity for one or more non-target nucleic acids. In certain embodiments, whether as a result of such affinity for one or more non-target nucleic acid or by some other mechanism, an antisense compound of interest may produce undesirable increases in ALT and/or AST levels when administered to an animal. In certain embodiments, such an antisense compound of interest may produce undesirable increases in organ weight.

[0634] In certain such embodiments wherein an antisense compound of interest effectively modulates the expression of a target nucleic acid, but possess one or more undesirable properties, a person having skill in the art may selectively incorporate one or more modifications into the antisense compound of interest that retain some or all of the desired property of effective modulation of expression of a target nucleic acid while reducing one or more of the antisense compound's undesirable properties. In certain embodiments, the present invention provides methods of altering such an antisense compound of interest to form an improved antisense compound. In certain embodiments, altering the number of nucleosides in the 5'-region, the 3'-region, and/or the central region of such an antisense compound of interest results in improved properties. For example, in certain embodiments, one may alter the modification state of one or more nucleosides at or near the 5'-end of the central region. Having been altered, those nucleosides may then be characterized as being part of the 5'-region. Thus, in such embodiments, the overall number of nucleosides of the 5'-region is increased and the number of nucleosides in the central region is decreased. For example, an antisense compound having a modification motif of 3-10-3 could be altered to result in an improved antisense compound having a modification motif of 4-9-3 or 5-8-3. In certain embodiments, the modification state of one or more of nucleosides at or near the 3'-end of the central region may likewise be altered. In certain embodiments, the modification of one or more of the nucleosides at or near the 5'-end and the 3'-end of the central region may be altered. In such embodiments in which one or more nucleosides at or near the 5'-end and the 3'-end of the central region is altered the central region becomes shorter relative to the central region of the original antisense compound of interest. In such embodiments, the modifications to the one or more nucleosides that had been part of the central region are the same as one or more modification that had been present in the 5'-region and/or the 3'-region of the original antisense compound of interest. In certain embodiments, the improved antisense compound having a shortened central region may retain its ability to effectively modulate the expression of a target nucleic acid, but not possess some or all of the undesirable properties possessed by antisense compound of interest having a longer central region. In certain embodiments, reducing the length of the central region reduces affinity for off-target nucleic acids. In certain embodiments, reducing the length of the central region results in reduced cleavage of non-target nucleic acids by RNase H. In certain embodiments, reducing the length of the central region does not produce undesirable increases in ALT levels. In certain embodiments, reducing the length of the central region does not produce undesirable increases in AST levels. In certain embodiments, reducing the length of the central region does not produce undesirable increases organ weights.

[0635] In certain embodiments it is possible to retain the same nucleobase sequence and overall length of an antisense compound while decreasing the length of the central region. In certain embodiments retaining the same nucleobase sequence and overall length of an antisense compound while decreasing the length of the central region ameliorates one or more undesirable properties of an antisense compound. In certain embodiments retaining the same nucleobase sequence and overall length of an antisense compound while decreasing the length of the central region ameliorates one or more undesirable properties of an antisense compound but does not substantially affect the ability of the antisense compound to modulate expression of a target nucleic acid. In certain such embodiments, two or more antisense compounds would have the same overall length and nucleobase sequence, but would have a different central region length, and different properties. In certain embodiments, the length of the central region is 9 nucleobases. In certain embodiments, the length of the central region is 8 nucleobases. In certain embodiments, the length of the central region is 7 nucleobases. In certain embodiments, the central region consists of unmodified deoxynucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5'-region, the 3'-region, or both the 5'-region and the 3'-region.

[0636] In certain embodiments, the length of the central region can be decreased by increasing the length of the 5'-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5'-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5'-region with modified nucleosides comprising a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, .alpha.-LNA, ENA and 2'-thio LNA. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5'-region with a cEt substituted sugar moiety.

[0637] In certain embodiments, the length of the central region can be decreased by increasing the length of the 5'-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5'-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5'-region with modified nucleosides comprising a bicyclic sugar moiety comprising a 2' substituent selected from among: a halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3 (MOE), O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2, OCH.sub.2C(.dbd.O)--N(H)CH.sub.3, OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2, and OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5'-region with 2'-O(CH.sub.2).sub.2--OCH.sub.3 (MOE) substituted sugar moiety.

[0638] In certain embodiments, the length of the central region can be decreased by increasing the length of the 3'-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3'-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3'-region with modified nucleosides comprising a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, .alpha.-LNA, ENA and 2'-thio LNA. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3'-region with a cEt substituted sugar moiety.

[0639] In certain embodiments, the length of the central region can be decreased by increasing the length of the 3'-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3'-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3'-region with modified nucleosides comprising a bicyclic sugar moiety comprising a 2' substituent selected from among: a halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3 (MOE), O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2, OCH.sub.2C(.dbd.O)--N(H)CH.sub.3, OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2, and OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3'-region with 2'-O(CH.sub.2).sub.2--OCH.sub.3 (MOE) substituted sugar moiety.

[0640] In certain embodiments, the length of the central region can be decreased by increasing the length of the 5'-region with modified nucleosides and increasing the length of the 3'-region with modified nucleosides.

E. CERTAIN TARGET NUCLEIC ACIDS

[0641] In certain embodiments, antisense compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid is a non-coding RNA. In certain such embodiments, the target non-coding RNA is selected from: a long-non-coding RNA, a short non-coding RNA, an intronic RNA molecule, a snoRNA, a scaRNA, a microRNA (including pre-microRNA and mature microRNA), a ribosomal RNA, and promoter directed RNA. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from: an mRNA and a pre-mRNA, including intronic, exonic and untranslated regions. In certain embodiments, oligomeric compounds are at least partially complementary to more than one target nucleic acid. For example, antisense compounds of the present invention may mimic microRNAs, which typically bind to multiple targets.

[0642] In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA. In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA or a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA or an intronic region of a pre-mRNA. In certain embodiments, the target nucleic acid is a long non-coding RNA. In certain embodiments, the target RNA is an mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain such embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron. In certain embodiments, the target nucleic acid is selected from among non-coding RNA, including exonic regions of pre-mRNA. In certain embodiments, the target nucleic acid is a ribosomal RNA (rRNA). In certain embodiments, the target nucleic acid is a non-coding RNA associated with splicing of other pre-mRNAs. In certain embodiments, the target nucleic acid is a nuclear-retained non-coding RNA.

[0643] In certain embodiments, antisense compounds described herein are complementary to a target nucleic acid comprising a single-nucleotide polymorphism. In certain such embodiments, the antisense compound is capable of modulating expression of one allele of the single-nucleotide polymorphism-containing-target nucleic acid to a greater or lesser extent than it modulates another allele. In certain embodiments an antisense compound hybridizes to a single-nucleotide polymorphism-containing-target nucleic acid at the single-nucleotide polymorphism site. In certain embodiments, the target nucleic acid is a Huntingtin gene transcript. In certain embodiments, the target nucleic acid is a single-nucleotide polymorphism-containing-target nucleic acid of a Huntingtin gene transcript. In certain embodiments, the target nucleic acid is not a Huntingtin gene transcript. In certain embodiments, the target nucleic acid is a single-nucleotide polymorphism-containing-target nucleic acid of a gene transcript other than Huntingtin. In certain embodiments, the target nucleic acid is any nucleic acid other than a Huntingtin gene transcript.

[0644] a. Single-Nucleotide Polymorphism

[0645] In certain embodiments, the invention provides selective antisense compounds that have greater activity for a target nucleic acid than for a homologous or partially homologous non-target nucleic acid. In certain such embodiments, the target and non-target nucleic acids are not functionally related to one another (e.g., are transcripts from different genes). In certain embodiments, the target and not-target nucleic acids are allelic variants of one another. Certain embodiments of the present invention provide methods, compounds, and compositions for selectively inhibiting mRNA and protein expression of an allelic variant of a particular gene or DNA sequence. In certain embodiments, the allelic variant contains a single nucleotide polymorphism (SNP). In certain embodiments, a SNP is associated with a mutant allele. In certain embodiments, a mutant SNP is associated with a disease. In certain embodiments a mutant SNP is associated with a disease, but is not causative of the disease. In certain embodiments, mRNA and protein expression of a mutant allele is associated with disease.

[0646] In certain embodiments, the expressed gene product of a mutant allele results in aggregation of the mutant proteins causing disease. In certain embodiments, the expressed gene product of a mutant allele results in gain of function causing disease. In certain embodiments, genes with an autosomal dominant mutation resulting in a toxic gain of function of the protein are the APP gene encoding amyloid precursor protein involved in Alzheimer's disease (Gene, 371: 68, 2006); the PrP gene encoding prion protein involved in Creutzfeldt-Jakob disease and in fatal familial insomnia (Nat. Med. 1997, 3: 1009); GFAP gene encoding glial fibrillary acidic protein involved in Alexander disease (J. Neurosci. 2006, 26:111623); alpha-synuclein gene encoding alpha-synuclein protein involved in Parkinson's disease (J. Clin. Invest. 2003, 111: 145); SOD-1 gene encoding the SOD-1 protein involved in amyotrophic lateral sclerosis (Science 1998, 281: 1851); atrophin-1 gene encoding atrophin-1 protein involved in dentato-rubral and pallido-luysian atrophy (DRPA) (Trends Mol. Med. 2001, 7: 479); SCA1 gene encoding ataxin-1 protein involved in spino-cerebellar ataxia-1 (SCA1) (Protein Sci. 2003, 12: 953); PLP gene encoding proteolipid protein involved in Pelizaeus-Merzbacher disease (NeuroMol. Med. 2007, 4: 73); DYT1 gene encoding torsinA protein involved in Torsion dystonia (Brain Res. 2000, 877: 379); and alpha-B crystalline gene encoding alpha-B crystalline protein involved in protein aggregation diseases, including cardiomyopathy (Cell 2007, 130: 427); alpha1-antitrypsin gene encoding alpha1-antitrypsin protein involved in chronic obstructive pulmonary disease (COPD), liver disease and hepatocellular carcinoma (New Engl J. Med. 2002, 346: 45); Ltk gene encoding leukocyte tyrosine kinase protein involved in systemic lupus erythematosus (Hum. Mol. Gen. 2004, 13: 171); PCSK9 gene encoding PCSK9 protein involved in hypercholesterolemia (Hum Mutat. 2009, 30: 520); prolactin receptor gene encoding prolactin receptor protein involved in breast tumors (Proc. Natl. Assoc. Sci. 2008, 105: 4533); CCL5 gene encoding the chemokine CCL5 involved in COPD and asthma (Eur. Respir. J. 2008, 32: 327); PTPN22 gene encoding PTPN22 protein involved in Type 1 diabetes, Rheumatoid arthritis, Graves disease, and SLE (Proc. Natl. Assoc. Sci. 2007, 104: 19767); androgen receptor gene encoding the androgen receptor protein involved in spinal and bulbar muscular atrophy or Kennedy's disease (J Steroid Biochem. Mol. Biol. 2008, 108: 245); CHMP4B gene encoding chromatin modifying protein-4B involved in progressive childhood posterior subcapsular cataracts (Am. J. Hum. Genet. 2007, 81: 596); FXR/NR1H4 gene encoding Farnesoid X receptor protein involved in cholesterol gallstone disease, arthrosclerosis and diabetes (Mol. Endocrinol. 2007, 21: 1769); ABCA1 gene encoding ABCA1 protein involved in cardiovascular disease (Transl. Res. 2007, 149: 205); CaSR gene encoding the calcium sensing receptor protein involved in primary hypercalciuria (Kidney Int. 2007, 71: 1155); alpha-globin gene encoding alpha-globin protein involved in alpha-thallasemia (Science 2006, 312: 1215); httlpr gene encoding HTTLPR protein involved in obsessive compulsive disorder (Am. J. Hum. Genet. 2006, 78: 815); AVP gene encoding arginine vasopressin protein in stress-related disorders such as anxiety disorders and comorbid depression (CNS Neurol. Disord. Drug Targets 2006, 5: 167); GNAS gene encoding G proteins involved in congenital visual defects, hypertension, metabolic syndrome (Trends Pharmacol. Sci. 2006, 27: 260); APAF1 gene encoding APAF1 protein involved in a predisposition to major depression (Mol. Psychiatry. 2006, 11: 76); TGF-beta1 gene encoding TGF-beta1 protein involved in breast cancer and prostate cancer (Cancer Epidemiol. Biomarkers Prev. 2004, 13: 759); AChR gene encoding acetylcholine receptor involved in congential myasthenic syndrome (Neurology 2004, 62: 1090); P2Y12 gene encoding adenosine diphosphate (ADP) receptor protein involved in risk of peripheral arterial disease (Circulation 2003, 108: 2971); LQT1 gene encoding LQT1 protein involved in atrial fibrillation (Cardiology 2003, 100: 109); RET protooncogene encoding RET protein involved in sporadic pheochromocytoma (J. Clin. Endocrinol. Metab. 2003, 88: 4911); filamin A gene encoding filamin A protein involved in various congenital malformations (Nat. Genet. 2003, 33: 487); TARDBP gene encoding TDP-43 protein involved in amyotrophic lateral sclerosis (Hum. Mol. Gene.t 2010, 19: 671); SCA3 gene encoding ataxin-3 protein involved in Machado-Joseph disease (PLoS One 2008, 3: e3341); SCAT gene encoding ataxin-7 protein involved in spino-cerebellar ataxia-7 (PLoS One 2009, 4: e7232); and HTT gene encoding huntingtin protein involved in Huntington's disease (Neurobiol Dis. 1996, 3:183); and the CA4 gene encoding carbonic anhydrase 4 protein, CRX gene encoding cone-rod homeobox transcription factor protein, FSCN2 gene encoding retinal fascin homolog 2 protein, IMPDH1 gene encoding inosine monophosphate dehydrogenase 1 protein, NR2E3 gene encoding nuclear receptor subfamily 2 group E3 protein, NRL gene encoding neural retina leucine zipper protein, PRPF3 (RP18) gene encoding pre-mRNA splicing factor 3 protein, PRPF8 (RP13) gene encoding pre-mRNA splicing factor 8 protein, PRPF31 (RP11) gene encoding pre-mRNA splicing factor 31 protein, RDS gene encoding peripherin 2 protein, ROM1 gene encoding rod outer membrane protein 1 protein, RHO gene encoding rhodopsin protein, RP1 gene encoding RP1 protein, RPGR gene encoding retinitis pigmentosa GTPase regulator protein, all of which are involved in Autosomal Dominant Retinitis Pigmentosa disease (Adv Exp Med. Biol. 2008, 613:203)

[0647] In certain embodiments, the mutant allele is associated with any disease from the group consisting of Alzheimer's disease, Creutzfeldt-Jakob disease, fatal familial insomnia, Alexander disease, Parkinson's disease, amyotrophic lateral sclerosis, dentato-rubral and pallido-luysian atrophy DRPA, spino-cerebellar ataxia, Torsion dystonia, cardiomyopathy, chronic obstructive pulmonary disease (COPD), liver disease, hepatocellular carcinoma, systemic lupus erythematosus, hypercholesterolemia, breast cancer, asthma, Type 1 diabetes, Rheumatoid arthritis, Graves disease, SLE, spinal and bulbar muscular atrophy, Kennedy's disease, progressive childhood posterior subcapsular cataracts, cholesterol gallstone disease, arthrosclerosis, cardiovascular disease, primary hypercalciuria, alpha-thallasemia, obsessive compulsive disorder, Anxiety, comorbid depression, congenital visual defects, hypertension, metabolic syndrome, prostate cancer, congential myasthenic syndrome, peripheral arterial disease, atrial fibrillation, sporadic pheochromocytoma, congenital malformations, Machado-Joseph disease, Huntington's disease, and Autosomal Dominant Retinitis Pigmentosa disease.

[0648] i. Certain Huntingtin Targets

[0649] In certain embodiments, an allelic variant of huntingtin is selectively reduced. Nucleotide sequences that encode huntingtin include, without limitation, the following: GENBANK Accession No. NT.sub.--006081.18, truncated from nucleotides 1566000 to 1768000 (replaced by GENBANK Accession No. NT.sub.--006051), incorporated herein as SEQ ID NO: 1, and NM.sub.--002111.6, incorporated herein as SEQ ID NO: 2.

[0650] Table 14 provides SNPs found in the GM04022, GM04281, GM02171, and GM02173B cell lines. Also provided are the allelic variants found at each SNP position, the genotype for each of the cell lines, and the percentage of HD patients having a particular allelic variant. For example, the two allelic variants for SNP rs6446723 are T and C. The GM04022 cell line is heterozygous TC, the GM02171 cell line is homozygous CC, the GM02173 cell line is heterozygous TC, and the GM04281 cell line is homozygous TT. Fifty percent of HD patients have a T at SNP position rs6446723.

TABLE-US-00015 TABLE 14 Allelic Variations for SNPs Associated with HD SNP Variation GM04022 GM02171 GM02173 GM04281 TargetPOP allele rs6446723 T/C TC CC TC TT 0.50 T rs3856973 A/G AG AA AG GG 0.50 G rs2285086 A/G AG GG AG AA 0.50 A rs363092 A/C AC AA AC CC 0.49 C rs916171 C/G GC GG GC CC 0.49 C rs6844859 T/C TC CC TC TT 0.49 T rs7691627 A/G AG AA AG GG 0.49 G rs4690073 A/G AG AA AG GG 0.49 G rs2024115 A/G AG GG AG AA 0.48 A rs11731237 T/C CC CC TC TT 0.43 T rs362296 A/C CC AC AC AC 0.42 C rs10015979 A/G AA AA AG GG 0.42 G rs7659144 C/G CG CG CG CC 0.41 C rs363096 T/C CC CC TC TT 0.40 T rs362273 A/G AA AG AG AA 0.39 A rs16843804 T/C CC TC TC CC 0.38 C rs362271 A/G GG AG AG GG 0.38 G rs362275 T/C CC TC TC CC 0.38 C rs3121419 T/C CC TC TC CC 0.38 C rs362272 A/G GG -- AG GG 0.38 G rs3775061 A/G AA AG AG AA 0.38 A rs34315806 T/C CC TC TC CC 0.38 C rs363099 T/C CC TC TC CC 0.38 C rs2298967 T/C TT TC TC TT 0.38 T rs363088 A/T AA TA TA AA 0.38 A rs363064 T/C CC TC TC CC 0.35 C rs363102 A/G AG AA AA AA 0.23 G rs2798235 A/G AG GG GG GG 0.21 A rs363080 T/C TC CC CC CC 0.21 T rs363072 A/T TA TA AA AA 0.13 A rs363125 A/C AC AC CC CC 0.12 C rs362303 T/C TC TC CC CC 0.12 C rs362310 T/C TC TC CC CC 0.12 C rs10488840 A/G AG AG GG GG 0.12 G rs362325 T/C TC TC TT TT 0.11 T rs35892913 A/G GG GG GG GG 0.10 A rs363102 A/G AG AA AA AA 0.09 A rs363096 T/C CC CC TC TT 0.09 C rs11731237 T/C CC CC TC TT 0.09 C rs10015979 A/G AA AA AG GG 0.08 A rs363080 T/C TC CC CC CC 0.07 C rs2798235 A/G AG GG GG GG 0.07 G rs1936032 C/G GC CC CC CC 0.06 C rs2276881 A/G GG GG GG GG 0.06 G rs363070 A/G AA AA AA AA 0.06 A rs35892913 A/G GG GG GG GG 0.04 G rs12502045 T/C CC CC CC CC 0.04 C rs6446723 T/C TC CC TC TT 0.04 C rs7685686 A/G AG GG AG AA 0.04 G rs3733217 T/C CC CC CC CC 0.03 C rs6844859 T/C TC CC TC TT 0.03 C rs362331 T/C TC CC TC TT 0.03 C

F. CERTAIN INDICATIONS

[0651] In certain embodiments, provided herein are methods of treating an animal or individual comprising administering one or more pharmaceutical compositions as described herein. In certain embodiments, the individual or animal has Huntington's disease.

[0652] In certain embodiments, compounds targeted to huntingtin as described herein may be administered to reduce the severity of physiological symptoms of Huntington's disease. In certain embodiments, compounds targeted to huntingtin as described herein may be administered to reduce the rate of degeneration in an individual or an animal having Huntington's disease. In certain embodiments, compounds targeted to huntingtin as described herein may be administered regeneration function in an individual or an animal having Huntington's disease. In certain embodiments, symptoms of Huntingtin's disease may be reversed by treatment with a compound as described herein.

[0653] In certain embodiments, compounds targeted to huntingtin as described herein may be administered to ameliorate one or more symptoms of Huntington's disease. In certain embodiments administration of compounds targeted to huntingtin as described herein may improve the symptoms of Huntington's disease as measured by any metric known to those having skill in the art. In certain embodiments, administration of compounds targeted to huntingtin as described herein may improve a rodent's rotaraod assay performance. In certain embodiments, administration of compounds targeted to huntingtin as described herein may improve a rodent's plus maze assay. In certain embodiments, administration of compounds targeted to huntingtin as described herein may improve a rodent's open field assay performance.

[0654] Accordingly, provided herein are methods for ameliorating a symptom associated with Huntington's disease in a subject in need thereof. In certain embodiments, provided is a method for reducing the rate of onset of a symptom associated with Huntington's disease. In certain embodiments, provided is a method for reducing the severity of a symptom associated with Huntington's disease. In certain embodiments, provided is a method for regenerating neurological function as shown by an improvement of a symptom associated with Huntington's disease. In such embodiments, the methods comprise administering to an individual or animal in need thereof a therapeutically effective amount of a compound targeted to a huntingtin nucleic acid.

[0655] Huntington's disease is characterized by numerous physical, neurological, psychiatric, and/or peripheral symptoms. Any symptom known to one of skill in the art to be associated with Huntington's disease can be ameliorated or otherwise modulated as set forth above in the methods described above. In certain embodiments, the symptom is a physical symptom selected from the group consisting of restlessness, lack of coordination, unintentionally initiated motions, unintentionally uncompleted motions, unsteady gait, chorea, rigidity, writhing motions, abnormal posturing, instability, abnormal facial expressions, difficulty chewing, difficulty swallowing, difficulty speaking, seizure, and sleep disturbances. In certain embodiments, the symptom is a cognitive symptom selected from the group consisting of impaired planning, impaired flexibility, impaired abstract thinking, impaired rule acquisition, impaired initiation of appropriate actions, impaired inhibition of inappropriate actions, impaired short-term memory, impaired long-term memory, paranoia, disorientation, confusion, hallucination and dementia. In certain embodiments, the symptom is a psychiatric symptom selected from the group consisting of anxiety, depression, blunted affect, egocentrisms, aggression, compulsive behavior, irritability and suicidal ideation. In certain embodiments, the symptom is a peripheral symptom selected from the group consisting of reduced brain mass, muscle atrophy, cardiac failure, impaired glucose tolerance, weight loss, osteoporosis, and testicular atrophy.

[0656] In certain embodiments, the symptom is restlessness. In certain embodiments, the symptom is lack of coordination. In certain embodiments, the symptom is unintentionally initiated motions. In certain embodiments, the symptom is unintentionally uncompleted motions. In certain embodiments, the symptom is unsteady gait. In certain embodiments, the symptom is chorea. In certain embodiments, the symptom is rigidity. In certain embodiments, the symptom is writhing motions. In certain embodiments, the symptom is abnormal posturing. In certain embodiments, the symptom is instability. In certain embodiments, the symptom is abnormal facial expressions. In certain embodiments, the symptom is difficulty chewing. In certain embodiments, the symptom is difficulty swallowing. In certain embodiments, the symptom is difficulty speaking. In certain embodiments, the symptom is seizures. In certain embodiments, the symptom is sleep disturbances.

[0657] In certain embodiments, the symptom is impaired planning. In certain embodiments, the symptom is impaired flexibility. In certain embodiments, the symptom is impaired abstract thinking. In certain embodiments, the symptom is impaired rule acquisition. In certain embodiments, the symptom is impaired initiation of appropriate actions. In certain embodiments, the symptom is impaired inhibition of inappropriate actions. In certain embodiments, the symptom is impaired short-term memory. In certain embodiments, the symptom is impaired long-term memory. In certain embodiments, the symptom is paranoia. In certain embodiments, the symptom is disorientation. In certain embodiments, the symptom is confusion. In certain embodiments, the symptom is hallucination. In certain embodiments, the symptom is dementia.

[0658] In certain embodiments, the symptom is anxiety. In certain embodiments, the symptom is depression. In certain embodiments, the symptom is blunted affect. In certain embodiments, the symptom is egocentrism. In certain embodiments, the symptom is aggression. In certain embodiments, the symptom is compulsive behavior. In certain embodiments, the symptom is irritability. In certain embodiments, the symptom is suicidal ideation.

[0659] In certain embodiments, the symptom is reduced brain mass. In certain embodiments, the symptom is muscle atrophy. In certain embodiments, the symptom is cardiac failure. In certain embodiments, the symptom is impaired glucose tolerance. In certain embodiments, the symptom is weight loss. In certain embodiments, the symptom is osteoporosis. In certain embodiments, the symptom is testicular atrophy.

[0660] In certain embodiments, symptoms of Huntington's disease may be quantifiable. For example, osteoporosis may be measured and quantified by, for example, bone density scans. For such symptoms, in certain embodiments, the symptom may be reduced by about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.

[0661] In certain embodiments, provided are methods of treating an individual comprising administering one or more pharmaceutical compositions as described herein. In certain embodiments, the individual has Huntington's disease.

[0662] In certain embodiments, administration of an antisense compound targeted to a huntingtin nucleic acid results in reduction of huntingtin expression by at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.

[0663] In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to huntingtin are used for the preparation of a medicament for treating a patient suffering or susceptible to Huntington's disease.

G. CERTAIN PHARMACEUTICAL COMPOSITIONS

[0664] In certain embodiments, the present invention provides pharmaceutical compositions comprising one or more antisense compound. In certain embodiments, such pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more antisense compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more antisense compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and sterile water. In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile water. In certain embodiments, the sterile saline is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile phosphate-buffered saline (PBS). In certain embodiments, the sterile saline is pharmaceutical grade PBS.

[0665] In certain embodiments, antisense compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

[0666] Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising antisense compounds comprise one or more oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0667] A prodrug can include the incorporation of additional nucleosides at one or both ends of an oligomeric compound which are cleaved by endogenous nucleases within the body, to form the active antisense oligomeric compound.

[0668] Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.

[0669] In certain embodiments, pharmaceutical compositions provided herein comprise one or more modified oligonucleotides and one or more excipients. In certain such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

[0670] In certain embodiments, a pharmaceutical composition provided herein comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

[0671] In certain embodiments, a pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

[0672] In certain embodiments, a pharmaceutical composition provided herein comprises a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80.TM. and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80.TM.; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

[0673] In certain embodiments, a pharmaceutical composition provided herein is prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration.

[0674] In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain.

H. ADMINISTRATION

[0675] In certain embodiments, the compounds and compositions as described herein are administered parenterally.

[0676] In certain embodiments, parenteral administration is by infusion. Infusion can be chronic or continuous or short or intermittent. In certain embodiments, infused pharmaceutical agents are delivered with a pump. In certain embodiments, parenteral administration is by injection.

[0677] In certain embodiments, compounds and compositions are delivered to the CNS. In certain embodiments, compounds and compositions are delivered to the cerebrospinal fluid. In certain embodiments, compounds and compositions are administered to the brain parenchyma. In certain embodiments, compounds and compositions are delivered to an animal by intrathecal administration, or intracerebroventricular administration. Broad distribution of compounds and compositions, described herein, within the central nervous system may be achieved with intraparenchymal administration, intrathecal administration, or intracerebroventricular administration.

[0678] In certain embodiments, parenteral administration is by injection. The injection may be delivered with a syringe or a pump. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly to a tissue, such as striatum, caudate, cortex, hippocampus and cerebellum.

[0679] Therefore, in certain embodiments, delivery of a compound or composition described herein can affect the pharmacokinetic profile of the compound or composition. In certain embodiments, injection of a compound or composition described herein, to a targeted tissue improves the pharmacokinetic profile of the compound or composition as compared to infusion of the compound or composition. In a certain embodiment, the injection of a compound or composition improves potency compared to broad diffusion, requiring less of the compound or composition to achieve similar pharmacology. In certain embodiments, similar pharmacology refers to the amount of time that a target mRNA and/or target protein is down-regulated (e.g. duration of action). In certain embodiments, methods of specifically localizing a pharmaceutical agent, such as by bolus injection, decreases median effective concentration (EC50) by a factor of about 50 (e.g. 50 fold less concentration in tissue is required to achieve the same or similar pharmacodynamic effect). In certain embodiments, methods of specifically localizing a pharmaceutical agent, such as by bolus injection, decreases median effective concentration (EC50) by a factor of 20, 25, 30, 35, 40, 45 or 50. In certain embodiments the pharmaceutical agent in an antisense compound as further described herein. In certain embodiments, the targeted tissue is brain tissue. In certain embodiments the targeted tissue is striatal tissue. In certain embodiments, decreasing EC50 is desirable because it reduces the dose required to achieve a pharmacological result in a patient in need thereof.

[0680] In certain embodiments, an antisense oligonucleotide is delivered by injection or infusion once every month, every two months, every 90 days, every 3 months, every 6 months, twice a year or once a year.

I. CERTAIN COMBINATION THERAPIES

[0681] In certain embodiments, one or more pharmaceutical compositions are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease, disorder, or condition as the one or more pharmaceutical compositions described herein. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease, disorder, or condition as the one or more pharmaceutical compositions described herein. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired side effect of one or more pharmaceutical compositions as described herein. In certain embodiments, one or more pharmaceutical compositions are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions are co-administered with another pharmaceutical agent to produce a combinational effect. In certain embodiments, one or more pharmaceutical compositions are co-administered with another pharmaceutical agent to produce a synergistic effect.

[0682] In certain embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are prepared separately.

[0683] In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of include antipsychotic agents, such as, e.g., haloperidol, chlorpromazine, clozapine, quetiapine, and olanzapine; antidepressant agents, such as, e.g., fluoxetine, sertraline hydrochloride, venlafaxine and nortriptyline; tranquilizing agents such as, e.g., benzodiazepines, clonazepam, paroxetine, venlafaxin, and beta-blockers; mood-stabilizing agents such as, e.g., lithium, valproate, lamotrigine, and carbamazepine; paralytic agents such as, e.g., Botulinum toxin; and/or other experimental agents including, but not limited to, tetrabenazine (Xenazine), creatine, coenzyme Q10, trehalose, docosahexanoic acids, ACR16, ethyl-EPA, atomoxetine, citalopram, dimebon, memantine, sodium phenylbutyrate, ramelteon, ursodiol, zyprexa, xenasine, tiapride, riluzole, amantadine, [123I]MNI-420, atomoxetine, tetrabenazine, digoxin, detromethorphan, warfarin, alprozam, ketoconazole, omeprazole, and minocycline.

Nonlimiting Disclosure and Incorporation by Reference

[0684] While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.

[0685] Although the sequence listing accompanying this filing identifies each sequence as either "RNA" or "DNA" as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as "RNA" or "DNA" to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2'-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2'-OH for the natural 2'-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) for natural uracil of RNA).

[0686] Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligomeric compound having the nucleobase sequence "ATCGATCG" encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence "AUCGAUCG" and those having some DNA bases and some RNA bases such as "AUCGATCG" and oligomeric compounds having other modified or naturally occurring bases, such as "AT.sup.meCGAUCG," wherein .sup.meC indicates a cytosine base comprising a methyl group at the 5-position.

EXAMPLES

[0687] The following examples illustrate certain embodiments of the present invention and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.

[0688] To allow assessment of the relative effects of nucleobase sequence and chemical modification, throughout the examples, oligomeric compounds are assigned a "Sequence Code." Oligomeric compounds having the same Sequence Code have the same nucleobase sequence. Oligomeric compounds having different Sequence Codes have different nucleobase sequences.

Example 1

Modified Antisense Oligonucleotides Targeting Human Target-X

[0689] Antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 407939, which was described in an earlier publication (WO 2009/061851) was also tested.

[0690] The newly designed chimeric antisense oligonucleotides and their motifs are described in Table 15. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P.dbd.S). Nucleosides followed by "d" indicate 2'-deoxyribonucleosides. Nucleosides followed by "k" indicate 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g cEt) nucleosides. Nucleosides followed by "e" indicate 2'-O-methoxyethyl (2'-MOE) nucleosides. "N" indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).

[0691] Each gapmer listed in Table 15 is targeted to the human Target-X genomic sequence.

[0692] Activity of the newly designed gapmers was compared to a 5-10-5 2'-MOE gapmer, ISIS 407939 targeting human Target-X and is further described in USPN XXX, incorporated herein by reference. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells, and indicate that several of the newly designed antisense oligonucleotides are more potent than ISIS 407939. A total of 771 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 15. Each of the newly designed antisense oligonucleotides provided in Table 1 achieved greater than 80% inhibition and, therefore, are more active than ISIS 407939.

TABLE-US-00016 TABLE 15 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X Wing SEQ ISIS % Gap Chemistry SEQ ID Sequence (5' to 3') NO inhibition Motif Chemistry 5' 3' CODE NO NkNkNkNdNdNdNdNkNd 473359 92 3-10-3 Deoxy/ kkk eee 21 6 NdNdNdNdNeNeNe cEt NkNkNkNdNdNdNdNkNd 473360 96 3-10-3 Deoxy/ kkk eee 22 6 NdNdNdNdNeNeNe cEt NkNkNkNdNdNdNdNdNd 473168 94 3-10-3 Full kkk kkk 23 6 NdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNdNd 473317 95 3-10-3 Full kkk eee 23 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNkNd 473471 90 3-10-3 Deoxy/ kkk eee 23 6 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNdNd 473620 94 5-9-2 Full kdkdk ee 23 6 NdNdNdNdNdNeNe deoxy NkNkNdNdNdNdNdNdNd 473019 88 2-10-2 Full kk kk 24 7 NdNdNdNkNk deoxy NkNkNdNdNdNdNdNdNd 473020 93 2-10-2 Full kk kk 25 7 NdNdNdNkNk deoxy NkNkNkNdNdNdNdNdNd 473321 93 3-10-3 Full kkk eee 26 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNdNd 473322 94 3-10-3 Full kkk eee 27 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNdNd 473323 96 3-10-3 Full kkk eee 28 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNdNd 473326 94 3-10-3 Full kkk eee 29 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNkNd 473480 92 3-10-3 Deoxy/ kkk eee 29 6 NdNdNdNdNeNeNe cEt NkNkNkNdNdNdNdNdNd 473178 96 3-10-3 Full kkk kkk 30 6 NdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNdNd 473327 96 3-10-3 Full kkk eee 30 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNkNd 473481 93 3-10-3 Deoxy/ kkk eee 30 6 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNdNd 473630 89 5-9-2 Full kdkdk ee 30 6 NdNdNdNdNdNeNe deoxy NkNkNdNdNdNdNdNdNd 473029 96 2-10-2 Full kk kk 31 7 NdNdNdNkNk deoxy NkNkNdNdNdNdNdNdNd 472925 93 2-10-2 Full kk kk 32 7 NdNdNdNkNk deoxy NkNkNdNdNdNdNdNdNd 472926 85 2-10-2 Full kk kk 33 7 NdNdNdNkNk deoxy NkNkNkNdNdNdNdNdNd 473195 97 3-10-3 Full kkk kkk 34 6 NdNdNdNdNkNkNk deoxy NkNkNdNdNdNdNdNdNd 473046 90 2-10-2 Full kk kk 35 7 NdNdNdNkNk deoxy NkNkNdNdNdNdNdNdNd 472935 92 2-10-2 Full kk kk 36 7 NdNdNdNkNk deoxy NkNkNkNdNdNdNdNdNd 473089 95 3-10-3 Full kkk kkk 37 6 NdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNdNd 473350 93 3-10-3 Full kkk eee 38 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNdNd 473353 93 3-10-3 Full kkk eee 39 6 NdNdNdNdNeNeNe deoxy NkNkNdNdNdNdNdNdNd 473055 91 2-10-2 Full kk kk 40 7 NdNdNdNkNk deoxy NkNkNkNdNdNdNdNkNd 473392 95 3-10-3 Deoxy/ kkk eee 41 6 NdNdNdNdNeNeNe cEt NkNkNkNdNdNdNdNdNd 473095 100 3-10-3 Full kkk kkk 42 6 NdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNdNd 473244 99 3-10-3 Full kkk eee 42 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNkNd 473393 99 3-10-3 Deoxy/ kkk eee 42 6 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNdNd 473547 98 5-9-2 Full kdkdk ee 42 6 NdNdNdNdNdNeNe deoxy NkNkNdNdNdNdNdNdNd 472942 87 2-10-2 Full kk kk 43 7 NdNdNdNkNk deoxy NkNkNkNdNdNdNdNdNd 473098 97 3-10-3 Full kkk kkk 44 6 NdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNkNd 473408 92 3-10-3 Deoxy/ kkk eee 45 6 NdNdNdNdNeNeNe cEt NkNkNdNdNdNdNdNdNd 472958 89 2-10-2 Full kk kk 46 7 NdNdNdNkNk deoxy NkNkNdNdNdNdNdNdNd 472959 90 2-10-2 Full kk kk 47 7 NdNdNdNkNk deoxy NkNdNkNdNkNdNdNdNd 473566 94 5-9-2 Full kdkdk ee 48 6 NdNdNdNdNdNeNe deoxy NkNdNkNdNkNdNdNdNd 473567 95 5-9-2 Full kdkdk ee 49 6 NdNdNdNdNdNeNe deoxy NkNdNkNdNkNdNdNdNd 473569 92 5-9-2 Full kdkdk ee 50 6 NdNdNdNdNdNeNe deoxy NkNkNdNdNdNdNdNdNd 457851 90 2-10-2 Full kk kk 51 7 NdNdNdNkNk deoxy NkNkNdNdNdNdNdNdNd 472970 91 2-10-2 Full kk kk 32 7 NdNdNdNkNk deoxy NkNkNkNdNdNdNdNdNd 473125 90 3-10-3 Full kkk kkk 53 6 NdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNdNd 473274 98 3-10-3 Full kkk eee 53 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNkNd 473428 90 3-10-3 Deoxy/ kkk eee 53 6 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNdNd 473577 93 5-9-2 Full kdkdk ee 53 6 NdNdNdNdNdNeNe deoxy NkNkNdNdNdNdNdNdNd 472976 97 2-10-2 Full kk kk 54 7 NdNdNdNkNk deoxy NkNkNdNdNdNdNdNd 472983 94 2-10-2 Full kk kk 55 7 NdNdNdNdNkNk deoxy NkNkNdNdNdNdNdNd 472984 90 2-10-2 Full kk kk 56 7 NdNdNdNdNkNk deoxy NkNkNkNdNdNdNdNd 473135 97 3-10-3 Full kkk kkk 57 6 NdNdNdNdNdNkNkNk deoxy NkNkNdNdNdNdNdNd 472986 95 2-10-2 Full kk kk 58 7 NdNdNdNdNkNk deoxy NkNkNkNdNdNdNdNd 473137 95 3-10-3 Full kkk kkk 59 6 NdNdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNd 473286 95 3-10-3 Full kkk eee 59 6 NdNdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNkNd 473440 88 3-10-3 Deoxy/ kkk eee 59 6 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNd 473589 97 5-9-2 Full kdkdk ee 59 6 NdNdNdNdNdNdNeNe deoxy NkNkNdNdNdNdNdNd 472988 85 2-10-2 Full kk kk 60 7 NdNdNdNdNkNk deoxy NkNkNkNdNdNdNdNd 473140 96 3-10-3 Full kkk kkk 61 6 NdNdNdNdNdNkNkNk deoxy NkNkNdNdNdNdNdNd 472991 90 2-10-2 Full kk kk 62 7 NdNdNdNdNkNk deoxy NkNkNkNdNdNdNdNkNd 473444 94 3-10-3 Deoxy/ kkk eee 63 6 NdNdNdNdNeNeNe cEt NkNkNkNdNdNdNdNd 473142 96 3-10-3 Full kkk kkk 64 6 NdNdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNd 473291 95 3-10-3 Full kkk eee 64 6 NdNdNdNdNdNeNeNe deoxy NkNdNkNdNkNdNdNd 473594 95 5-9-2 Full kdkdk ee 64 6 NdNdNdNdNdNdNeNe deoxy NkNkNkNdNdNdNdNdNd 473143 97 3-10-3 Full kkk kkk 65 6 NdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNd 473292 96 3-10-3 Full kkk eee 65 6 NdNdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNkNd 473446 96 3-10-3 Deoxy/ kkk eee 65 6 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNdNd 473595 84 5-9-2 Full kdkdk ee 65 6 NdNdNdNdNdNeNe deoxy NkNkNdNdNdNdNdNdNd 472994 96 2-10-2 Full kk kk 66 7 NdNdNdNkNk deoxy NkNkNkNdNdNdNdNdNd 473144 98 3-10-3 Full kkk kkk 67 6 NdNdNdNdNkNkNk deoxy NkNkNkNdNdNdNdNdNd 473293 96 3-10-3 Full kkk eee 67 6 NdNdNdNdNeNeNe deoxy NkNkNdNdNdNdNdNdNd 472995 96 2-10-2 Full kk kk 68 7 NdNdNdNkNk deoxy NkNkNkNdNdNdNdNd 473294 91 3-10-3 Full kkk eee 69 6 NdNdNdNdNdNeNeNe deoxy NkNdNkNdNkNdNdNdNd 473597 94 5-9-2 Full kdkdk ee 69 6 NdNdNdNdNdNeNe deoxy NkNkNdNdNdNdNdNdNd 472996 94 2-10-2 Full kk kk 70 7 NdNdNdNkNk deoxy NkNkNkNdNdNdNdNd 473295 92 3-10-3 Full kkk eee 71 6 NdNdNdNdNdNeNeNe deoxy NeNeNeNeNeNdNdNdNdNd 407939 80 5-10-5 Full eeeee eeee 72 8 NdNdNdNdNdNeNeNeNeNe deoxy e NkNkNkNdNdNdNdNdNd 473296 98 3-10-3 Full kkk eee 73 6 NdNdNdNdNeNeNe deoxy NkNkNkNdNdNdNdNkNd 473450 95 3-10-3 Deoxy/ kkk eee 73 6 NdNdNdNdNeNeNe cEt NkNkNdNdNdNdNdNdNd 472998 97 2-10-2 Full kk kk 74 7 NdNdNdNkNk deoxy e = 2'MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 2

Modified Antisense Oligonucleotides Comprising 6'-(S)--CH.sub.3 Bicyclic Nucleoside (cEt) and F-HNA Modifications Targeting Human Target-X

[0693] Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 407939 was also tested.

[0694] The chimeric antisense oligonucleotides and their motifs are described in Table 16. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P.dbd.S). Nucleosides followed by "d" indicate 2'-deoxyribonucleosides. Nucleosides followed by "k" indicate 6'-(S)--CH.sub.3 bicyclic nucleosides (e.g cEt). Nucleosides followed by "e" indicate 2'-O-methoxyethyl (2'-MOE) modified nucleosides. Nucleosides followed by `g` indicate F-HNA modified nucleosides. "N" indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).

[0695] Each gapmer listed in Table 16 is targeted to the human Target-X genomic sequence.

[0696] Activity of the newly designed gapmers was compared to a 5-10-5 2'-MOE gapmer, ISIS 407939 targeting human Target-X. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells, and demonstrate that several of the newly designed gapmers are more potent than ISIS 407939. A total of 765 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 16. All but one of the newly designed antisense oligonucleotides provided in Table 16 achieved greater than 30% inhibition and, therefore, are more active than ISIS 407939.

TABLE-US-00017 TABLE 16 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X Wing SEQ ISIS % Gap Chemistry SEQ ID Sequence (5' to 3') No inhibition Motif Chemistry 5' 3' CODE NO NgNgNdNdNdNdNdNdNd 482838 81 2-10-2 Full gg gg 25 7 NdNdNdNgNg deoxy NgNgNgNdNdNdNdNdNd 482992 93 3-10-3 Full ggg ggg 28 6 NdNdNdNdNgNgNg deoxy NgNgNgNdNdNdNdNdNd 482996 97 3-10-3 Full ggg ggg 30 6 NdNdNdNdNgNgNg deoxy NgNdNgNdNgNdNdNdNd 483284 82 5-9-2 Full gdgdg ee 23 6 NdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNdNd 483289 70 5-9-2 Full gdgdg ee 27 6 NdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNdNd 483290 80 5-9-2 Full gdgdg ee 28 6 NdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNdNd 483294 69 5-9-2 Full gdgdg ee 30 6 NdNdNdNdNdNeNe deoxy NgNgNdNdNdNdNdNdNd 483438 81 2-10-4 Full gg eeee 23 6 NdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNdNd 483444 84 2-10-4 Full gg eeee 28 6 NdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNdNd 483448 77 2-10-4 Full gg eeee 30 6 NdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNdNd 482847 79 2-10-2 Full gg gg 31 7 NdNdNdNgNg deoxy NgNgNdNdNdNdNdNdNd 482747 85 2-10-2 Full gg gg 32 7 NdNdNdNgNg deoxy NgNgNdNdNdNdNdNdNd 482873 81 2-10-2 Full gg gg 40 7 NdNdNdNgNg deoxy NgNgNdNdNdNdNdNdNdNd 482874 82 2-10-2 Full gg gg 75 7 NdNdNgNg deoxy NgNgNdNdNdNdNdNd 482875 82 2-10-2 Full gg gg 76 7 NdNdNdNdNgNg deoxy NgNgNgNdNdNdNdNd 482896 95 3-10-3 Full ggg ggg 77 6 NdNdNdNdNdNgNgNg deoxy NgNgNgNdNdNdNdNdNd 483019 89 3-10-3 Full ggg ggg 38 6 NdNdNdNdNgNgNg deoxy NgNdNgNdNdNdNdNdNd 483045 92 3-10-3 Full gdg gdg 77 6 NdNdNdNdNgNdNg deoxy NgNdNgNdNgNdNdNdNd 483194 64 3-10-3 Full gdg gdg 77 6 NdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNdNd 483317 79 5-9-2 Full gdgdg ee 38 6 NdNdNdNdNdNeNe deoxy NgNgNdNdNdNdNdNdNd 483343 75 2-10-4 Full gg eeee 57 6 NdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNdNdNdN 483471 76 2-10-4 Full gg eeee 38 6 dNdNeNeNeNe deoxy NgNgNdNdNdNdNdNdNd 483478 20 2-10-4 Full gg eeee 78 6 NdNdNdNeNeNeNe deoxy NeNeNeNeNeNdNdNdNdNd 407939 30 5-10-5 Full eeeee eeeee 72 8 NdNdNdNdNdNeNeNeNeNe deoxy NgNgNdNdNdNdNdNd 482784 83 2-10-2 Full gg gg 79 7 NdNdNdNdNgNg deoxy NgNgNdNdNdNdNdNd 482794 91 2-10-2 Full gg gg 54 7 NdNdNdNdNgNg deoxy NgNgNdNdNdNdNdNd 482804 80 2-10-2 Full gg gg 58 7 NdNdNdNdNgNg deoxy NgNgNdNdNdNdNdNd 482812 81 2-10-2 Full gg gg 66 7 NdNdNdNdNgNg deoxy NgNgNdNdNdNdNdNd 482813 92 2-10-2 Full gg gg 68 7 NdNdNdNdNgNg deoxy NgNgNdNdNdNdNdNd 482814 94 2-10-2 Full gg gg 70 7 NdNdNdNdNgNg deoxy NgNgNdNdNdNdNdNd 482815 81 2-10-2 Full gg gg 80 7 NdNdNdNdNgNg deoxy NgNgNdNdNdNdNdNd 482816 71 2-10-2 Full gg gg 74 7 NdNdNdNdNgNg deoxy NgNgNgNdNdNdNdNd 482916 90 3-10-3 Full ggg ggg 44 6 NdNdNdNdNdNgNgNg deoxy NgNgNgNdNdNdNdNd 482932 89 3-10-3 Full ggg ggg 48 6 NdNdNdNdNdNgNgNg deoxy NgNgNgNdNdNdNdNd 482953 93 3-10-3 Full ggg ggg 57 6 NdNdNdNdNdNgNgNg deoxy NgNgNgNdNdNdNdNd 482962 97 3-10-3 Full ggg ggg 67 6 NdNdNdNdNdNgNgNg deoxy NgNgNgNdNdNdNdNd 482963 96 3-10-3 Full ggg ggg 69 6 NdNdNdNdNdNgNgNg deoxy NgNgNgNdNdNdNdNd 482965 89 3-10-3 Full ggg ggg 73 6 NdNdNdNdNdNgNgNg deoxy NgNdNgNdNdNdNdNd 483065 69 3-10-3 Full ggg ggg 44 6 NdNdNdNdNdNgNdNg deoxy NgNdNgNdNdNdNdNd 483092 89 3-10-3 Full gdg gdg 53 6 NdNdNdNdNdNgNdNg deoxy NgNdNgNdNgNdNdNd 483241 79 5-9-2 Full gdgdg ee 53 6 NdNdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNd 483253 76 5-9-2 Full gdgdg ee 59 6 NdNdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNd 483258 70 5-9-2 Full gdgdg ee 64 6 NdNdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNd 483260 62 5-9-2 Full gdgdg ee 67 6 NdNdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNd 483261 76 5-9-2 Full gdgdg ee 69 6 NdNdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNd 483262 75 5-9-2 Full gdgdg ee 71 6 NdNdNdNdNdNdNeNe deoxy NgNdNgNdNgNdNdNd 483263 73 5-9-2 Full gdgdg ee 73 6 NdNdNdNdNdNdNeNe deoxy NgNgNdNdNdNdNdNd 483364 78 2-10-4 Full gg eeee 81 6 NdNdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNd 483395 86 2-10-4 Full gg eeee 53 6 NdNdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNd 483413 83 2-10-4 Full gg eeee 65 6 NdNdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNd 483414 76 2-10-4 Full gg eeee 67 6 NdNdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNd 483415 85 2-10-4 Full gg eeee 69 6 NdNdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNd 483416 77 2-10-4 Full gg eeee 71 6 NdNdNdNdNeNeNeNe deoxy NgNgNdNdNdNdNdNd 483417 83 2-10-4 Full gg eeee 73 6 NdNdNdNdNeNeNeNe deoxy e = 2'-MOE, d = 2'-deoxyribonucleoside, g = F-HNA

Example 3

Modified Antisense Oligonucleotides Comprising 2'-MOE and 6'-(S)--CH.sub.3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X

[0697] Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 403052, ISIS 407594, ISIS 407606, ISIS 407939, and ISIS 416438, which were described in an earlier publication (WO 2009/061851) were also tested.

[0698] The newly designed chimeric antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 17. The chemistry column of Table 17 presents the sugar motif of each oligonucleotide, wherein "e" indicates a 2'-O-methoxyethyl (2'-MOE) nucleoside, "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g cEt) and "d" indicates a 2'-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

[0699] Each gapmer listed in Table 17 is targeted to the human Target-X genomic sequence.

[0700] Activity of the newly designed gapmers was compared to ISIS 403052, ISIS 407594, ISIS 407606, ISIS 407939, and ISIS 416438. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 380 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 17. Each of the newly designed antisense oligonucleotides provided in Table 17 achieved greater than 64% inhibition and, therefore, are more potent than each of ISIS 403052, ISIS 407594, ISIS 407606, ISIS 407939, and ISIS 416438.

TABLE-US-00018 TABLE 17 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS No Chemistry Motif % inhibition SEQ CODE 403052 eeeee-(d10)-eeeee 5-10-5 64 82 407594 eeeee-(d10)-eeeee 5-10-5 40 83 407606 eeeee-(d10)-eeeee 5-10-5 39 84 407939 eeeee-(d10)-eeeee 5-10-5 57 72 416438 eeeee-(d10)-eeeee 5-10-5 62 85 484487 kdk-(d10)-dkdk 3-10-3 91 77 484539 kdk-d(10)-kdk 3-10-3 92 53 484546 kdk-d(10)-kdk 3-10-3 92 86 484547 kdk-d(10)-kdk 3-10-3 89 87 484549 kdk-d(10)-kdk 3-10-3 91 57 484557 kdk-d(10)-kdk 3-10-3 92 65 484558 kdk-d(10)-kdk 3-10-3 94 67 484559 kdk-d(10)-kdk 3-10-3 90 69 484582 kdk-d(10)-kdk 3-10-3 88 23 484632 kk-d(10)-eeee 2-10-4 90 88 484641 kk-d(10)-eeee 2-10-4 91 77 484679 kk-d(10)-eeee 2-10-4 90 49 484693 kk-d(10)-eeee 2-10-4 93 53 484711 kk-d(10)-eeee 2-10-4 92 65 484712 kk-d(10)-eeee 2-10-4 92 67 484713 kk-d(10)-eeee 2-10-4 85 69 484714 kk-d(10)-eeee 2-10-4 83 71 484715 kk-d(10)-eeee 2-10-4 93 73 484736 kk-d(10)-eeee 2-10-4 89 23 484742 kk-d(10)-eeee 2-10-4 93 28 484746 kk-d(10)-eeee 2-10-4 88 30 484771 kk-d(10)-eeee 2-10-4 89 89 e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 4

Antisense Inhibition of Human Target-X with 5-10-5 2'-MOE Gapmers

[0701] Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested were ISIS 403094, ISIS 407641, ISIS 407643, ISIS 407662, ISIS 407900, ISIS 407910, ISIS 407935, ISIS 407936, ISIS 407939, ISIS 416446, ISIS 416449, ISIS 416455, ISIS 416472, ISIS 416477, ISIS 416507, ISIS 416508, ISIS 422086, ISIS 422087, ISIS 422140, and ISIS 422142, 5-10-5 2'-MOE gapmers targeting human Target-X, which were described in an earlier publication (WO 2009/061851), incorporated herein by reference.

[0702] The newly designed modified antisense oligonucleotides are 20 nucleotides in length and their motifs are described in Tables 18 and 19. The chemistry column of Tables 18 and 19 present the sugar motif of each oligonucleotide, wherein "e" indicates a 2'-O-methoxyethyl (2'-MOE) nucleoside and "d" indicates a 2'-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

[0703] Each gapmer listed in Table 18 is targeted to the human Target-X genomic sequence.

[0704] Activity of the newly designed gapmers was compared to ISIS 403094, ISIS 407641, ISIS 407643, ISIS 407662, ISIS 407900, ISIS 407910, ISIS 407935, ISIS 407936, ISIS 407939, ISIS 416446, ISIS 416449, ISIS 416455, ISIS 416472, ISIS 416477, ISIS 416507, ISIS 416508, ISIS 422086, ISIS 422087, ISIS 422140, and ISIS 422142. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 916 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Tables 18 and 19.

TABLE-US-00019 TABLE 18 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS No Chemistry % inhibition SEQ CODE 490275 e5-d(10)-e5 35 90 490277 e5-d(10)-e5 73 91 490278 e5-d(10)-e5 78 92 490279 e5-d(10)-e5 66 93 490323 e5-d(10)-e5 65 94 490368 e5-d(10)-e5 78 95 490396 e5-d(10)-e5 76 96 416507 e5-d(10)-e5 73 97 422140 e5-d(10)-e5 59 98 422142 e5-d(10)-e5 73 99 416508 e5-d(10)-e5 75 100 490424 e5-d(10)-e5 57 101 490803 e5-d(10)-e5 70 102 416446 e5-d(10)-e5 73 103 416449 e5-d(10)-e5 33 104 407900 e5-d(10)-e5 66 105 490103 e5-d(10)-e5 87 106 416455 e5-d(10)-e5 42 107 407910 e5-d(10)-e5 25 108 490149 e5-d(10)-e5 82 109 403094 e5-d(10)-e5 60 110 416472 e5-d(10)-e5 78 111 407641 e5-d(10)-e5 64 112 416477 e5-d(10)-e5 25 113 407643 e5-d(10)-e5 78 114 490196 e5-d(10)-e5 81 115 490197 e5-d(10)-e5 85 116 490208 e5-d(10)-e5 89 117 490209 e5-d(10)-e5 81 118 422086 e5-d(10)-e5 90 119 407935 e5-d(10)-e5 91 120 422087 e5-d(10)-e5 89 121 407936 e5-d(10)-e5 80 122 407939 e5-d(10)-e5 67 72 e = 2'-MOE, d = 2'-deoxynucleoside

TABLE-US-00020 TABLE 19 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides ISIS No Motif % inhibition SEQ CODE 407662 e5-d(10)-e5 76 123 416446 e5-d(10)-e5 73 103 e = 2'-MOE, d = 2'-deoxynucleoside

Example 5

Modified Chimeric Antisense Oligonucleotides Comprising 6'-(S)--CH.sub.3 Bicyclic Nucleoside (e.g cEt) Modifications at 5' and 3' Wing Regions Targeting Human Target-X

[0705] Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 407939, which was described in an earlier publication (WO 2009/061851) were also tested. ISIS 457851, ISIS 472925, ISIS 472926, ISIS 472935, ISIS 472942, ISIS 472958, ISIS 472959, ISIS 472970, ISIS 472976, ISIS 472983, ISIS 472984, ISIS 472988, ISIS 472991, ISIS 472994, ISIS 472995, ISIS 472996, ISIS 472998, and ISIS 473020, described in the Examples above were also included in the screen.

[0706] The newly designed chimeric antisense oligonucleotides in Table 20 were designed as 2-10-2 cEt gapmers. The newly designed gapmers are 14 nucleosides in length, wherein the central gap segment comprises of ten 2'-deoxyribonucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment comprises 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g cEt) modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

[0707] Each gapmer listed in Table 20 is targeted to the human Target-X genomic sequence.

[0708] Activity of the newly designed oligonucleotides was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 614 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 20. Many of the newly designed antisense oligonucleotides provided in Table 20 achieved greater than 72% inhibition and, therefore, are more potent than ISIS 407939.

TABLE-US-00021 TABLE 20 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS No % inhibition Motif Wing Chemistry SEQ CODE 407939 72 5-10-5 cEt 72 473020 90 2-10-2 cEt 25 492465 83 2-10-2 cEt 124 492467 74 2-10-2 cEt 125 492492 84 2-10-2 cEt 126 492494 91 2-10-2 cEt 127 492503 89 2-10-2 cEt 128 492530 91 2-10-2 cEt 129 492534 91 2-10-2 cEt 130 492536 90 2-10-2 cEt 131 492541 84 2-10-2 cEt 132 492545 89 2-10-2 cEt 133 492566 90 2-10-2 cEt 134 492571 82 2-10-2 cEt 135 492572 89 2-10-2 cEt 136 492573 90 2-10-2 cEt 137 492574 92 2-10-2 cEt 138 492575 88 2-10-2 cEt 139 492593 83 2-10-2 cEt 140 492617 91 2-10-2 cEt 141 492618 92 2-10-2 cEt 142 492619 90 2-10-2 cEt 143 492621 75 2-10-2 cEt 144 492104 89 2-10-2 cEt 145 492105 86 2-10-2 cEt 146 492189 88 2-10-2 cEt 147 492194 92 2-10-2 cEt 148 492195 90 2-10-2 cEt 149 472925 87 2-10-2 cEt 32 492196 91 2-10-2 cEt 150 472926 88 2-10-2 cEt 33 492205 92 2-10-2 cEt 151 492215 77 2-10-2 cEt 152 492221 79 2-10-2 cEt 153 472935 82 2-10-2 cEt 36 492234 86 2-10-2 cEt 154 472942 85 2-10-2 cEt 43 492276 75 2-10-2 cEt 155 492277 75 2-10-2 cEt 156 492306 85 2-10-2 cEt 157 492317 93 2-10-2 cEt 158 472958 92 2-10-2 cEt 46 472959 88 2-10-2 cEt 47 492329 88 2-10-2 cEt 159 492331 95 2-10-2 cEt 160 492333 85 2-10-2 cEt 161 492334 88 2-10-2 cEt 162 457851 89 2-10-2 cEt 51 472970 92 2-10-2 cEt 52 492365 69 2-10-2 cEt 163 472976 94 2-10-2 cEt 54 472983 76 2-10-2 cEt 55 472984 72 2-10-2 cEt 56 492377 70 2-10-2 cEt 164 492380 80 2-10-2 cEt 165 492384 61 2-10-2 cEt 166 472988 59 2-10-2 cEt 60 492388 70 2-10-2 cEt 167 492389 70 2-10-2 cEt 168 492390 89 2-10-2 cEt 169 492391 80 2-10-2 cEt 170 472991 84 2-10-2 cEt 62 492398 88 2-10-2 cEt 171 492399 94 2-10-2 cEt 172 492401 91 2-10-2 cEt 173 492403 78 2-10-2 cEt 174 472994 95 2-10-2 cEt 66 472995 91 2-10-2 cEt 68 492404 84 2-10-2 cEt 175 492405 87 2-10-2 cEt 176 472996 85 2-10-2 cEt 70 492406 43 2-10-2 cEt 177 472998 92 2-10-2 cEt 74 492440 89 2-10-2 cEt 178

Example 6

Modified Chimeric Antisense Oligonucleotides Comprising 6'-(S)--CH.sub.3 Bicyclic Nucleoside (e.g cEt) Modifications at 5' and 3' Wing Regions Targeting Human Target-X

[0709] Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X, which was described in an earlier publication (WO 2009/061851). ISIS 472998 and ISIS 473046, described in the Examples above were also included in the screen.

[0710] The newly designed chimeric antisense oligonucleotides in Table 21 were designed as 2-10-2 cEt gapmers. The newly designed gapmers are 14 nucleosides in length, wherein the central gap segment comprises of ten 2'-deoxyribonucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment comprise 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g cEt) modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

[0711] Each gapmer listed in Table 21 is targeted to the human Target-X genomic sequence.

[0712] Activity of the newly designed gapmers was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 757 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 21. Each of the newly designed antisense oligonucleotides provided in Table 21 achieved greater than 67% inhibition and, therefore, are more potent than 407939.

TABLE-US-00022 TABLE 21 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS No % inhibition Motif Wing chemistry SEQ CODE 407939 67 5-10-5 cEt 72 492651 77 2-10-2 cEt 179 492652 84 2-10-2 cEt 180 492658 87 2-10-2 cEt 181 492725 74 2-10-2 cEt 182 492730 78 2-10-2 cEt 183 492731 72 2-10-2 cEt 184 492784 72 2-10-2 cEt 185 492816 70 2-10-2 cEt 186 492818 73 2-10-2 cEt 187 492877 83 2-10-2 cEt 188 492878 79 2-10-2 cEt 189 492913 73 2-10-2 cEt 190 492914 82 2-10-2 cEt 191 492928 76 5-10-5 cEt 192 492938 80 2-10-2 cEt 193 492991 91 2-10-2 cEt 194 492992 73 2-10-2 cEt 195 493087 81 2-10-2 cEt 196 493114 80 2-10-2 cEt 197 493178 86 2-10-2 cEt 198 493179 69 2-10-2 cEt 199 493182 79 2-10-2 cEt 200 493195 71 2-10-2 cEt 201 473046 79 2-10-2 cEt 35 493201 86 2-10-2 cEt 202 493202 76 2-10-2 cEt 203 493255 80 2-10-2 cEt 204 493291 84 2-10-2 cEt 205 493292 90 2-10-2 cEt 206 493296 82 2-10-2 cEt 207 493298 77 2-10-2 cEt 208 493299 76 5-10-5 cEt 209 493304 77 2-10-2 cEt 210 493312 75 2-10-2 cEt 211 493333 76 2-10-2 cEt 212 472998 85 2-10-2 cEt 74

Example 7

Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells

[0713] Antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA, were selected and tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.67 .mu.M, 2.00 .mu.M, 1.11 .mu.M, and 6.00 .mu.M concentrations of antisense oligonucleotide, as specified in Table 22. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

[0714] The half maximal inhibitory concentration (IC.sub.50) of each oligonucleotide is also presented in Table 22. As illustrated in Table 22, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that several of the newly designed gapmers are more potent than ISIS 407939 of the previous publication.

TABLE-US-00023 TABLE 22 Dose-dependent antisense inhibition of human Target-X in Hep3B cells using electroporation 666.6667 2000.0 6000.0 IC.sub.50 ISIS No nM nM nM (.mu.M) 407939 47 68 85 0.7 457851 60 80 93 <0.6 472916 53 80 87 <0.6 472925 62 86 95 <0.6 472926 66 77 85 <0.6 472935 54 84 94 <0.6 472958 66 82 88 <0.6 472959 64 81 93 <0.6 472970 72 87 86 <0.6 472976 78 92 97 <0.6 472994 79 92 96 <0.6 472995 61 82 93 <0.6 472996 73 91 95 <0.6 472998 63 90 95 <0.6 473019 55 80 86 <0.6 473020 61 76 85 <0.6 473046 61 80 94 <0.6 473055 55 84 94 <0.6 492104 53 76 88 <0.6 492105 62 80 90 <0.6 492189 57 80 92 <0.6 492194 57 83 91 <0.6 492195 58 81 95 <0.6 492196 62 86 95 <0.6 492205 62 87 95 <0.6 492215 60 78 89 <0.6 492221 63 76 92 <0.6 492234 51 74 91 0.5 492276 50 56 95 0.8 492277 58 73 81 <0.6 492306 61 75 84 <0.6 492317 59 80 93 <0.6 492329 59 70 89 <0.6 492331 69 87 95 <0.6 492333 47 70 85 0.7 492334 57 77 90 <0.6 492390 72 88 95 <0.6 492399 68 91 96 <0.6 492401 68 89 95 <0.6 492404 65 87 94 <0.6 492405 44 81 90 0.7 492406 65 82 92 <0.6 492440 50 70 89 0.6 492465 16 80 79 1.4 492467 58 77 92 <0.6 492492 45 80 94 0.7 492494 63 82 93 <0.6 492503 55 81 93 <0.6 492530 70 86 90 <0.6 492534 67 85 91 <0.6 492536 54 81 89 <0.6 492541 54 71 85 <0.6 492545 59 78 89 <0.6 492566 59 84 85 <0.6 492571 52 81 89 <0.6 492572 67 83 90 <0.6 492573 69 83 92 <0.6 492574 65 82 91 <0.6 492575 72 83 91 <0.6 492593 61 78 90 <0.6 492617 62 80 93 <0.6 492618 47 79 94 0.6 492619 54 82 95 <0.6 492621 44 85 92 0.6 492651 53 66 91 0.6 492652 61 78 88 <0.6 492658 59 79 88 <0.6 492725 43 84 89 0.6 492730 51 87 93 0.4 492731 46 82 90 0.6 492784 56 88 96 <0.6 492816 68 89 97 <0.6 492818 64 84 96 <0.6 492877 67 91 93 <0.6 492878 80 89 93 <0.6 492913 53 87 92 <0.6 492914 75 89 96 <0.6 492928 60 83 94 <0.6 492938 70 90 92 <0.6 492991 67 93 99 <0.6 492992 0 82 95 2.1 493087 54 81 90 <0.6 493114 50 73 90 0.6 493178 71 88 96 <0.6 493179 47 82 95 0.6 493182 79 87 91 <0.6 493195 55 78 90 <0.6 493201 87 93 96 <0.6 493202 68 89 94 <0.6 493255 57 79 93 <0.6 493291 57 87 93 <0.6 493292 70 89 93 <0.6 493296 35 84 91 0.9 493298 57 84 92 <0.6 493299 65 84 93 <0.6 493304 68 86 94 <0.6 493312 53 82 91 <0.6 493333 66 84 87 <0.6

Example 8

Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells

[0715] Additional antisense oligonucleotides from the studies described above, exhibiting in vitro inhibition of Target-X mRNA, were selected and tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.67 .mu.M, 2.00 .mu.M, 1.11 .mu.M, and 6.00 .mu.M concentrations of antisense oligonucleotide, as specified in Table 23. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 23, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that several of the newly designed gapmers are more potent than ISIS 407939.

TABLE-US-00024 TABLE 23 Dose-dependent antisense inhibition of human Target-X in Hep3B cells using electroporation 0.67 2.00 6.00 IC.sub.50 ISIS No .mu.M .mu.M .mu.M (.mu.M) 407939 52 71 86 0.6 472983 49 83 97 0.5 472984 51 82 95 0.5 472991 49 82 95 0.5 472998 59 88 96 <0.6 492365 74 91 96 <0.6 492377 56 76 91 <0.6 492380 63 79 95 <0.6 492384 67 84 94 <0.6 492388 69 87 97 <0.6 492389 62 90 96 <0.6 492391 56 84 94 <0.6 492398 63 80 95 <0.6 492403 58 81 91 <0.6

Example 9

Modified Chimeric Antisense Oligonucleotides Comprising 2'-Methoxyethyl (2'-MOE) Modifications at 5' and 3' Wing Regions Targeting Human Target-X

[0716] Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested were ISIS 403052, ISIS 407939, ISIS 416446, ISIS 416472, ISIS 416507, ISIS 416508, ISIS 422087, ISIS 422096, ISIS 422130, and ISIS 422142 which were described in an earlier publication (WO 2009/061851), incorporated herein by reference. ISIS 490149, ISIS 490197, ISIS 490209, ISIS 490275, ISIS 490277, and ISIS 490424, described in the Examples above, were also included in the screen.

[0717] The newly designed chimeric antisense oligonucleotides in Table 24 were designed as 3-10-4 2'-MOE gapmers. These gapmers are 17 nucleosides in length, wherein the central gap segment comprises of ten 2'-deoxyribonucleosides and is flanked by wing segments on the 5' direction with three nucleosides and the 3' direction with four nucleosides. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has 2'-MOE modifications. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

[0718] Each gapmer listed in Table 24 is targeted to the human Target-X genomic sequence.

[0719] Activity of the newly designed oligonucleotides was compared to ISIS 403052, ISIS 407939, ISIS 416446, ISIS 416472, ISIS 416507, ISIS 416508, ISIS 422087, ISIS 422096, ISIS 422130, and ISIS 422142. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 272 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 24. Several of the newly designed antisense oligonucleotides provided in Table 24 are more potent than antisense oligonucleotides from the previous publication.

TABLE-US-00025 TABLE 24 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS No % inhibition Motif Wing Chemistry SEQ CODE 403052 51 5-10-5 2'-MOE 82 407939 78 5-10-5 2'-MOE 72 416446 70 5-10-5 2'-MOE 103 416472 79 5-10-5 2'-MOE 111 416507 84 5-10-5 2'-MOE 97 416508 80 5-10-5 2'-MOE 100 422087 89 5-10-5 2'-MOE 121 422096 78 5-10-5 2'-MOE 219 422130 81 5-10-5 2'-MOE 225 422142 84 5-10-5 2'-MOE 99 490275 77 5-10-5 2'-MOE 90 513462 79 3-10-4 2'-MOE 213 513463 81 3-10-4 2'-MOE 214 490277 74 5-10-5 2'-MOE 91 513487 83 3-10-4 2'-MOE 215 513504 81 3-10-4 2'-MOE 216 513507 86 3-10-4 2'-MOE 217 513508 85 3-10-4 2'-MOE 218 490424 69 5-10-5 2'-MOE 101 491122 87 5-10-5 2'-MOE 220 513642 79 3-10-4 2'-MOE 221 490149 71 5-10-5 2'-MOE 109 513419 90 3-10-4 2'-MOE 222 513420 89 3-10-4 2'-MOE 223 513421 88 3-10-4 2'-MOE 224 490197 77 5-10-5 2'-MOE 116 513446 89 3-10-4 2'-MOE 226 513447 83 3-10-4 2'-MOE 227 490209 79 5-10-5 2'-MOE 118 513454 84 3-10-4 2'-MOE 228 513455 92 3-10-4 2'-MOE 229 513456 89 3-10-4 2'-MOE 230 513457 83 3-10-4 2'-MOE 231

Example 10

Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells

[0720] Antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA, were selected and tested at various doses in Hep3B cells. ISIS 403052, ISIS 407643, ISIS 407935, ISIS 407936, ISIS 407939, ISIS 416446, ISIS 416459, ISIS 416472, ISIS 416507, ISIS 416508, ISIS 416549, ISIS 422086, ISIS 422087, ISIS 422130, ISIS and 422142, 5-10-5 MOE gapmers targeting human Target-X, which were described in an earlier publication (WO 2009/061851).

[0721] Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.625 .mu.M, 1.25 .mu.M, 2.50 .mu.M, 5.00 .mu.M and 10.00 .mu.M concentrations of antisense oligonucleotide, as specified in Table 25. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

[0722] The half maximal inhibitory concentration (IC.sub.50) of each oligonucleotide is also presented in Table 25. As illustrated in Table 25, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that the newly designed gapmers are potent than gapmers from the previous publication.

TABLE-US-00026 TABLE 25 Dose-dependent antisense inhibition of human Target-X in Hep3B cells using electroporation 0.625 1.25 2.50 5.00 10.00 IC.sub.50 ISIS No .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 403052 21 35 63 82 89 1.9 407643 29 46 67 83 90 1.4 407935 52 68 80 89 91 <0.6 407936 31 51 62 78 84 1.4 407939 30 61 74 83 88 1.0 416446 37 53 64 76 83 1.2 416459 51 76 83 90 92 <0.6 416472 37 52 66 78 85 1.2 416507 45 68 82 87 90 0.7 416508 33 56 74 84 89 1.1 416549 57 71 78 82 85 <0.6 422086 46 67 77 89 92 0.7 422087 50 69 74 86 91 0.6 422130 32 65 78 92 93 0.9 422142 59 73 84 86 88 <0.6 490103 52 57 66 83 88 0.9 490149 34 58 71 85 91 1.0 490196 26 59 66 79 84 1.3 490197 39 63 74 81 90 0.8 490208 44 70 76 83 88 0.6 490275 36 58 76 85 89 1.0 490277 37 63 73 87 87 0.8 490279 40 54 72 83 89 1.0 490323 49 68 79 86 90 <0.6 490368 39 62 76 86 91 0.8 490396 36 53 69 80 87 1.1 490424 45 65 69 76 82 0.6 490803 57 74 85 89 92 <0.6 513419 60 71 85 95 96 <0.6 513420 37 69 79 94 96 0.7 513421 46 64 84 95 97 0.6 513446 47 81 88 95 96 <0.6 513447 56 74 81 92 96 <0.6 513454 50 77 82 93 95 <0.6 513455 74 82 91 96 96 <0.6 513456 66 80 88 94 95 <0.6 513457 54 67 80 87 89 <0.6 513462 49 72 84 87 89 <0.6 513463 36 62 76 85 89 0.9 513487 42 56 73 87 93 0.9 513504 47 65 81 90 91 0.6 513505 39 50 78 85 92 1.0 513507 52 73 83 89 93 <0.6 513508 56 78 85 91 94 <0.6

Example 11

Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells

[0723] Additional antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA, were tested at various doses in Hep3B cells. ISIS 407935, ISIS 407939, ISIS 416446, ISIS 416472, ISIS 416507, ISIS 416549, ISIS 422086, ISIS 422087, ISIS 422096, and ISIS 422142 5-10-5 MOE gapmers targeting human Target-X, which were described in an earlier publication (WO 2009/061851).

[0724] Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.3125 .mu.M, 0.625 .mu.M, 1.25 .mu.M, 2.50 .mu.M, 5.00 .mu.M and 10.00 .mu.M concentrations of antisense oligonucleotide, as specified in Table 26. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 26, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that the newly designed gapmers are more potent than gapmers from the previous publication.

TABLE-US-00027 TABLE 26 Dose-dependent antisense inhibition of human Target-X in Hep3B cells using electroporation 0.3125 0.625 1.250 2.500 5.000 10.000 IC.sub.50 ISIS No .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 407935 30 49 75 86 91 94 0.6 407939 30 48 61 78 85 90 0.8 416446 27 52 63 75 85 90 0.7 416472 38 51 72 83 88 94 0.5 416507 58 81 76 84 89 92 <0.3 416549 52 67 75 81 88 89 0.3 422086 48 49 68 78 86 91 0.5 422087 30 56 66 83 72 92 0.6 422096 47 63 70 77 83 85 <0.3 422142 69 85 87 85 89 91 <0.3 490103 52 57 68 78 87 93 0.4 490149 33 64 62 77 86 93 0.5 490197 38 46 60 75 87 93 0.7 490208 46 62 73 83 88 91 0.4 490209 40 54 72 79 85 94 0.5 490275 52 61 67 78 85 91 0.3 490277 33 59 77 79 91 94 0.5 490323 43 61 72 69 84 87 0.4 490368 50 64 78 83 90 92 <0.3 490396 46 64 68 84 84 90 0.3 490424 24 47 58 72 76 82 1.0 490803 45 60 70 84 88 89 0.3 513419 32 53 76 88 93 95 0.5 513420 35 59 72 82 94 97 0.5 513421 46 67 78 86 94 96 <0.3 513446 26 61 77 89 91 97 0.5 513447 22 48 60 82 91 95 0.8 513454 25 59 76 86 94 96 0.5 513455 60 73 85 89 95 96 <0.3 513456 49 60 81 88 94 95 <0.3 513457 43 50 72 77 87 92 0.5 513462 25 48 58 76 83 88 0.8 513463 22 45 66 73 85 88 0.9 513487 41 56 65 79 86 90 0.4 513504 19 48 63 76 87 92 0.9 513505 11 21 54 73 85 90 1.4 513507 47 55 72 82 90 91 0.3 513508 31 59 74 85 92 93 0.5 513642 43 55 67 80 88 92 0.4

Example 12

Tolerability of 2'-MOE Gapmers Targeting Human Target-X in BALB/c Mice

[0725] BALB/c mice are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

[0726] Groups of male BALB/c mice were injected subcutaneously twice a week for 3 weeks with 50 mg/kg of ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422086, ISIS 422087, ISIS 422096, ISIS 422142, ISIS 490103, ISIS 490149, ISIS 490196, ISIS 490208, ISIS 490209, ISIS 513419, ISIS 513420, ISIS 513421, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513462, ISIS 513463, ISIS 513487, ISIS 513504, ISIS 513508, and ISIS 513642. One group of male BALB/c mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

[0727] To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).

[0728] ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422087, ISIS 422096, ISIS 490103, ISIS 490196, ISIS 490208, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513487, ISIS 513504, and ISIS 513508 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 422086, ISIS 490209, ISIS 513419, ISIS 513420, and ISIS 513463 were considered tolerable in terms of liver function.

Example 13

Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B cells

[0729] Additional antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA were selected and tested at various doses in Hep3B cells. Also tested was ISIS 407939, a 5-10-5 MOE gapmer, which was described in an earlier publication (WO 2009/061851).

[0730] Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.074 .mu.M, 0.222 .mu.M, 0.667 .mu.M, 2.000 .mu.M, and 6.000 .mu.M concentrations of antisense oligonucleotide, as specified in Table 27. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

[0731] The half maximal inhibitory concentration (IC.sub.50) of each oligonucleotide is also presented in Table 27. As illustrated in Table 27, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. Many of the newly designed antisense oligonucleotides provided in Table 27 achieved an IC.sub.50 of less than 0.9 .mu.M and, therefore, are more potent than ISIS 407939.

TABLE-US-00028 TABLE 27 Dose-dependent antisense inhibition of human Target-X in Hep3B cells using electroporation 0.074 0.222 0.667 2.000 6.000 IC.sub.50 ISIS No .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 407939 2 17 53 70 87 0.9 472970 17 47 75 92 95 0.3 472988 0 8 21 54 92 1.4 472996 18 59 74 93 95 0.2 473244 91 95 97 99 99 <0.07 473286 6 53 85 92 98 0.3 473359 2 3 20 47 67 2.6 473392 71 85 88 92 96 <0.07 473393 91 96 97 98 99 <0.07 473547 85 88 93 97 98 <0.07 473567 0 25 66 88 95 0.7 473589 8 47 79 94 99 0.3 482814 23 68 86 93 96 0.1 482815 6 48 65 90 96 0.4 482963 3 68 85 94 96 0.2 483241 14 33 44 76 93 0.6 483261 14 21 41 72 88 0.7 483290 0 1 41 69 92 1.0 483414 8 1 36 76 91 0.9 483415 0 40 52 84 94 0.6 484559 26 51 78 87 97 0.2 484713 6 5 53 64 88 0.9

Example 14

Modified Antisense Oligonucleotides Comprising 2'-O-Methoxyethyl (2'-MOE) and 6'-(S)--CH.sub.3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X

[0732] Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X, which was described in an earlier publication (WO 2009/061851). ISIS 472998, ISIS 492878, and ISIS 493201 and 493182, 2-10-2 cEt gapmers, described in the Examples above were also included in the screen.

[0733] The newly designed modified antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 28. The chemistry column of Table 28 presents the sugar motif of each oligonucleotide, wherein "e" indicates a 2'-O-methoxyethyl (2'-MOE) nucleoside, "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g cEt) and "d" indicates a 2'-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

[0734] Each gapmer listed in Table 28 is targeted to the human Target-X genomic sequence.

[0735] Activity of newly designed gapmers was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells and demonstrate that several of the newly designed gapmers are more potent than ISIS 407939. A total of 685 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 28.

TABLE-US-00029 TABLE 28 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS No % inhibition Chemistry SEQ CODE 407939 68 eeeee-d(10)-eeeee 72 492878 73 kk-d(10)-kk 493182 80 kk-d(10)-kk 493201 84 kk-d(10)-kk 472998 91 kk-d(10)-kk 515640 75 eee-d(10)-kkk 23 515637 77 eee-d(10)-kkk 232 515554 72 eee-d(10)-kkk 233 515406 80 kkk-d(10)-eee 234 515558 81 eee-d(10)-kkk 234 515407 88 kkk-d(10)-eee 235 515408 85 kkk-d(10)-eee 236 515422 86 kkk-d(10)-eee 237 515423 90 kkk-d(10)-eee 238 515575 84 eee-d(10)-kkk 238 515424 87 kkk-d(10)-eee 239 515432 78 kkk-d(10)-eee 240 515433 71 kkk-d(10)-eee 241 515434 76 kkk-d(10)-eee 242 515334 85 kkk-d(10)-eee 243 515649 61 eee-d(10)-kkk 88 515338 86 kkk-d(10)-eee 244 515438 76 kkk-d(10)-eee 245 515439 75 kkk-d(10)-eee 246 516003 87 eee-d(10)-kkk 247 515647 60 eee-d(10)-kkk 77 515639 78 eee-d(10)-kkk 34 493201 84 eee-d(10)-kkk 202 515648 36 kkk-d(10)-eee 248 515641 69 kk-d(10)-eeee 39 515650 76 kkk-d(10)-eee 44 515354 87 eee-d(10)-kkk 249 515926 87 eee-d(10)-kkk 250 515366 87 kk-d(10)-eeee 251 515642 58 kkk-d(10)-eee 252 515643 81 eee-d(10)-kkk 53 515944 84 kk-d(10)-eeee 253 515380 90 kkk-d(10)-eee 254 515532 83 kkk-d(10)-eee 254 515945 85 kk-d(10)-eeee 254 515381 82 kk-d(10)-eeee 255 515382 95 kkk-d(10)-eee 256 515948 94 eee-d(10)-kkk 256 515949 87 eee-d(10)-kkk 257 515384 89 kkk-d(10)-eee 258 515635 82 kk-d(10)-eeee 65 515638 90 kkk-d(10)-eee 67 515386 92 kk-d(10)-eeee 259 515951 84 eee-d(10)-kkk 259 515387 78 kkk-d(10)-eee 260 515952 89 kkk-d(10)-eee 260 515636 90 kkk-d(10)-eee 69 515388 84 eee-d(10)-kkk 261 e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 15

Tolerability of Modified Oligonucleotides Comprising 2'-O-Methoxyethyl (2'-MOE) and 6'-(S)--CH.sub.3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X in BALB/c Mice

[0736] BALB/c mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

[0737] Additionally, the newly designed modified antisense oligonucleotides were also added to this screen. The newly designed chimeric antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 29. The chemistry column of Table 29 presents the sugar motif of each oligonucleotide, wherein "e" indicates a 2'-O-methoxyethyl (2'-MOE) nucleoside, "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g cEt) and "d" indicates a 2'-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

[0738] Each gapmer listed in Table 29 is targeted to the human Target-X genomic sequence.

TABLE-US-00030 TABLE 29 Modified chimeric antisense oligonucleotides targeted to Target-X ISIS No Chemistry SEQ CODE 516044 eee-d(10)-kkk 21 516045 eee-d(10)-kkk 22 516058 eee-d(10)-kkk 26 516059 eee-d(10)-kkk 27 516060 eee-d(10)-kkk 28 516061 eee-d(10)-kkk 29 516062 eee-d(10)-kkk 30 516046 eee-d(10)-kkk 37 516063 eee-d(10)-kkk 38 516064 eee-d(10)-kkk 89 516065 eee-d(10)-kkk 262 516066 eee-d(10)-kkk 263 516047 eee-d(10)-kkk 41 516048 eee-d(10)-kkk 42 516049 eee-d(10)-kkk 81 516050 eee-d(10)-kkk 45 516051 eee-d(10)-kkk 48 516052 eee-d(10)-kkk 49 515652 eee-d(10)-kkk 50 508039 eee-d(10)-kkk 264 516053 eee-d(10)-kkk 265 515654 eee-d(10)-kkk 76 515656 eee-d(10)-kkk 77 516054 eee-d(10)-kkk 57 516055 eee-d(10)-kkk 59 515655 eee-d(10)-kkk 61 516056 eee-d(10)-kkk 63 516057 eee-d(10)-kkk 64 515653 eee-d(10)-kkk 71 515657 eee-d(10)-kkk 73 e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Treatment

[0739] Groups of 4-6-week old male BALB/c mice were injected subcutaneously twice a week for 3 weeks with 50 mg/kg/week of ISIS 457851, ISIS 515635, ISIS 515636, ISIS 515637, ISIS 515638, ISIS 515639, ISIS 515640, ISIS 515641, ISIS 515642, ISIS 515643, ISIS 515647, ISIS 515648, ISIS 515649, ISSI 515650, ISIS 515652, ISIS 515653, ISIS 515654, ISIS 515655, ISIS 515656, ISIS 515657, ISIS 516044, ISIS 516045, ISIS 516046, ISIS 516047, ISIS 516048, ISIS 516049, ISIS 516050, ISIS 516051, ISIS 516052, ISIS 516053, ISIS 516054, ISIS 516055, ISIS 516056, ISIS 516057, ISIS 516058, ISIS 516059, ISIS 516060, ISIS 516061, ISIS 516062, ISIS 516063, ISIS 516064, ISIS 516065, and ISIS 516066. One group of 4-6-week old male BALB/c mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

[0740] To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).

[0741] ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 515636, ISIS 515639, ISIS 515641, ISIS 515642, ISIS 515648, ISIS 515650, ISIS 515652, ISIS 515653, ISIS 515655, ISIS 515657, ISIS 516044, ISIS 516045, ISIS 516047, ISIS 516048, ISIS 516051, ISIS 516052, ISIS 516053, ISIS 516055, ISIS 516056, ISIS 516058, ISIS 516059, ISIS 516060, ISIS 516061, ISIS 516062, ISIS 516063, ISIS 516064, ISIS 516065, and ISIS 516066 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 457851, ISIS 515635, ISIS 515637, ISIS 515638, ISIS 515643, ISIS 515647, ISIS 515649, ISIS 515650, ISIS 515652, ISIS 515654, ISIS 515656, ISIS 516056, and ISIS 516057 were considered tolerable in terms of liver function.

Example 16

Efficacy of Modified Oligonucleotides Comprising 2'-O-Methoxyethyl (2'-MOE) and 6'-(S)--CH.sub.3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X in Transgenic Mice

[0742] Transgenic mice were developed at Taconic farms harboring a Target-X genomic DNA fragment. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.

Treatment

[0743] Groups of 3-4 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 20 mg/kg/week of ISIS 457851, ISIS 515636, ISIS 515639, ISIS 515653, ISIS 516053, ISIS 516065, and ISIS 516066. One group of mice was injected subcutaneously twice a week for 3 weeks with control oligonucleotide, ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, 5-10-5 MOE gapmer with no known murine target, SEQ ID NO: 9). One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

RNA Analysis

[0744] RNA was extracted from plasma for real-time PCR analysis of Target-X, using primer probe set RTS2927. The mRNA levels were normalized using RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 30, each of the antisense oligonucleotides achieved reduction of human Target-X mRNA expression over the PBS control. Treatment with the control oligonucleotide did not achieve reduction in Target-X levels, as expected.

TABLE-US-00031 TABLE 30 Percent inhibition of Target-X mRNA in transgenic mice ISIS No % inhibition 141923 0 457851 76 515636 66 515639 49 515653 78 516053 72 516065 59 516066 39

Protein Analysis

[0745] Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 31, several antisense oligonucleotides achieved reduction of human Target-X protein expression over the PBS control.

TABLE-US-00032 TABLE 31 Percent inhibition of Target-X protein levels in transgenic mice ISIS No % inhibition 141923 0 457851 64 515636 68 515639 46 515653 0 516053 19 516065 0 516066 7

Example 17

Efficacy of Modified Oligonucleotides Comprising 2'-O-Methoxyethyl (2'-MOE) and 6'-(S)--CH.sub.3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X in Transgenic Mice

[0746] Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.

Treatment

[0747] Groups of 2-4 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 10 mg/kg/week of ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422087, ISIS 422096, ISIS 473137, ISIS 473244, ISIS 473326, ISIS 473327, ISIS 473359, ISIS 473392, ISIS 473393, ISIS 473547, ISIS 473567, ISIS 473589, ISIS 473630, ISIS 484559, ISIS 484713, ISIS 490103, ISIS 490196, ISIS 490208, ISIS 513419, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513487, ISIS 513508, ISIS 515640, ISIS 515641, ISIS 515642, ISIS 515648, ISIS 515655, ISIS 515657, ISIS 516045, ISIS 516046, ISIS 516047, ISIS 516048, ISIS 516051, ISIS 516052, ISIS 516055, ISIS 516056, ISIS 516059, ISIS 516061, ISIS 516062, and ISIS 516063. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Protein Analysis

[0748] Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 32, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control.

TABLE-US-00033 TABLE 32 Percent inhibition of Target-X plasma protein levels in transgenic mice ISIS No % inhibition 407935 80 416472 49 416549 29 422087 12 422096 21 473137 57 473244 67 473326 42 473327 100 473359 0 473392 22 473393 32 473547 73 473567 77 473589 96 473630 75 484559 75 484713 56 490103 0 490196 74 490208 90 513419 90 513454 83 513455 91 513456 81 513457 12 513487 74 513508 77 515640 83 515641 87 515642 23 515648 32 515655 79 515657 81 516045 52 516046 79 516047 65 516048 79 516051 84 516052 72 516055 70 516056 0 516059 39 516061 64 516062 96 516063 24

Example 18

Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells

[0749] Antisense oligonucleotides exhibiting in vitro inhibition of Target-X mRNA were selected and tested at various doses in Hep3B cells. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X, which was described in an earlier publication (WO 2009/061851).

[0750] Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.074 .mu.M, 0.222 .mu.M, 0.667 .mu.M, 2.000 .mu.M, and 6.000 .mu.M concentrations of antisense oligonucleotide, as specified in Table 33. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

[0751] The half maximal inhibitory concentration (IC.sub.50) of each oligonucleotide is also presented in Table 33. As illustrated in Table 33, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. Many of the newly designed antisense oligonucleotides provided in Table 33 achieved an IC.sub.50 of less than 2.0 .mu.M and, therefore, are more potent than ISIS 407939.

TABLE-US-00034 TABLE 33 Dose-dependent antisense inhibition of human Target-X in Hep3B cells using electroporation 0.074 0.222 0.667 2.000 6.000 IC.sub.50 ISIS No .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 407939 0 9 21 58 76 2.0 515636 14 32 50 62 81 0.7 515639 10 24 41 61 67 1.3 515640 4 16 35 52 63 2.0 515641 0 21 27 55 66 1.9 515642 3 13 36 44 66 2.2 515648 8 10 10 5 16 >6.0 515653 9 35 26 55 71 1.5 515655 0 0 6 13 42 >6.0 515657 0 13 17 38 51 6.0 516045 0 6 15 19 40 >6.0 516046 0 7 32 48 69 2.1 516047 12 27 41 50 63 1.8 516051 9 8 34 52 66 2.0 516052 17 42 27 53 75 1.2 516053 9 7 28 63 77 1.3 516055 0 3 27 54 75 2.0 516056 0 4 14 52 66 2.6 516057 0 34 33 51 70 1.6 516058 13 12 25 47 74 2.0 516059 4 15 36 47 68 1.9 516060 0 1 39 29 63 3.2 516061 0 0 24 0 3 <6.0 516062 0 20 43 65 78 1.0 516063 0 8 10 37 61 3.8 516064 0 3 13 45 69 2.7 516065 0 14 38 63 76 1.3 516066 0 3 30 55 75 1.7

Example 19

Modified Oligonucleotides Comprising 2'-O-Methoxyethyl (2'-MOE) and 6'-(S)--CH.sub.3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X

[0752] Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 472998, ISIS 515652, ISIS 515653, ISIS 515654, ISIS 515655, ISIS 515656, and ISIS 515657, described in the Examples above were also included in the screen.

[0753] The newly designed chimeric antisense oligonucleotides are 16 or 17 nucleotides in length and their motifs are described in Table 34. The chemistry column of Table 34 presents the sugar motif of each oligonucleotide, wherein "e" indicates a 2'-O-methoxyethyl (2'-MOE) nucleoside, "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g cEt) and "d" indicates a 2'-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

[0754] Each gapmer listed in Table 34 is targeted to the human Target-X genomic sequence.

[0755] Activity of newly designed gapmers was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

TABLE-US-00035 TABLE 34 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS No % inhibition Chemistry SEQ CODE 472998 85 kk-d(10)-kk 74 515652 63 eee-d(10)-kkk 50 515653 67 eee-d(10)-kkk 71 515654 78 eee-d(10)-kkk 86 515655 41 eee-d(10)-kkk 61 515656 74 eee-d(10)-kkk 87 515657 49 eee-d(10)-kkk 73 529265 52 eek-d(10)-keke 267 529332 82 eek-d(10)-keke 268 529334 78 eek-d(10)-keke 269 529186 85 eek-d(10)-keke 213 529223 81 eek-d(10)-kkke 213 529129 75 eee-d(10)-kkk 270 529149 82 kkk-d(10)-eee 270 529177 77 eek-d(10)-keke 214 529214 78 eek-d(10)-kkke 214 529178 79 eek-d(10)-keke 271 529215 82 eek-d(10)-kkke 271 529179 71 eek-d(10)-keke 272 529216 77 eek-d(10)-kkke 272 529193 69 eek-d(10)-keke 273 529230 70 eek-d(10)-kkke 273 529136 48 eee-d(10)-kkk 274 529156 68 kkk-d(10)-eee 274 529194 44 eek-d(10)-keke 275 529231 56 eek-d(10)-kkke 275 529137 34 eee-d(10)-kkk 276 529157 79 kkk-d(10)-eee 276 529336 57 eek-d(10)-keke 277 529338 73 eek-d(10)-keke 278 529195 55 eek-d(10)-keke 279 529232 68 eek-d(10)-kkke 279 529340 65 eek-d(10)-keke 280 529342 69 eek-d(10)-keke 281 529812 69 k-d(10)-kekee 282 529831 62 k-d(10)-kdkee 282 529733 64 ke-d(10)-keke 283 529753 52 ek-d(10)-keke 283 529773 57 ke-d(10)-kdke 283 529793 36 ek-d(10)-kdke 283 529862 48 kde-d(10)-kdke 284 529882 35 edk-d(10)-kdke 284 529902 44 k-(d4)-k-(d4)-k-(d4)-ke 284 529559 71 eek-d(10)-kke 26 529584 57 kee-d(10)-kke 26 529609 58 edk-d(10)-kke 26 529634 49 kde-d(10)-kke 26 529659 52 kddk-d(9)-kke 26 529684 48 kdde-d(9)-kke 26 529709 61 eddk-d(9)-kke 26 529922 52 eeee-d(9)-kke 26 529344 50 eek-d(10)-keke 285 529138 32 eee-d(10)-kkk 286 529158 75 kkk-d(10)-eee 286 529184 75 eek-d(10)-keke 215 529221 78 eek-d(10)-kkke 215 529127 67 eee-d(10)-kkk 287 529147 79 kkk-d(10)-eee 287 529346 58 eek-d(10)-keke 288 529348 65 eek-d(10)-keke 289 529350 77 eek-d(10)-keke 290 529813 20 k-d(10)-kekee 291 529832 47 k-d(10)-kdkee 291 529734 63 ke-d(10)-keke 292 529754 58 ek-d(10)-keke 292 529774 49 ke-d(10)-kdke 292 529794 51 ek-d(10)-kdke 292 529863 64 kde-d(10)-kdke 293 529883 78 edk-d(10)-kdke 293 529903 36 k-d(4)-k-d(4)-k-d(4)-ke 293 529560 71 eek-d(10)-kke 27 529585 70 kee-d(10)-kke 27 529610 66 edk-d(10)-kke 27 529635 45 kde-d(10)-kke 27 529660 53 kddk-d(9)-kke 27 529685 42 kdde-d(9)-kke 27 529710 60 eddk-d(9)-kke 27 529923 63 eeee-d(9)-kke 27 529196 74 eek-d(10)-keke 294 529233 80 eek-d(10)-kkke 294 529139 75 eee-d(10)-kkk 295 529159 62 kkk-d(10)-eee 295 529352 74 eek-d(10)-keke 296 529354 67 eek-d(10)-keke 297 529197 43 eek-d(10)-keke 298 529234 58 eek-d(10)-kkke 298 529140 29 eee-d(10)-kkk 299 529160 59 kkk-d(10)-eee 299 529180 80 eek-d(10)-keke 216 529217 79 eek-d(10)-kkke 216 529814 51 k-d(10)-kekee 300 529833 52 k-d(10)-kdkee 300 529735 43 ke-d(10)-keke 301 529755 60 ek-d(10)-keke 301 529775 38 ke-d(10)-kdke 301 529795 58 ek-d(10)-kdke 301 529864 41 kde-d(10)-kdke 302 529884 48 edk-d(10)-kdke 302 529904 44 k-d(4)-k-(d4)-k-d(4)-ke 302 529934 61 eek-d(10)-keke 302 529356 71 eek-d(10)-keke 303 529561 75 eek-d(10)-kke 28 529586 65 kee-d(10)-kke 28 529611 54 edk-d(10)-kke 28 529636 39 kde-d(10)-kke 28 529661 67 kddk-d(9)-kke 28 529686 66 kdde-d(9)-kke 28 529711 60 eddk-d(9)-kke 28 529924 62 eeee-d(9)-kke 28 529358 82 eek-d(10)-keke 304 529181 79 eek-d(10)-keke 217 529218 73 eek-d(10)-kkke 217 529182 85 eek-d(10)-keke 218 529219 84 eek-d(10)-kkke 218 529360 84 eek-d(10)-keke 305 529362 87 eek-d(10)-keke 306 529364 81 eek-d(10)-keke 307 529366 77 eek-d(10)-keke 308 529198 28 eek-d(10)-keke 309 529235 8 eek-d(10)-kkke 309 529141 34 eee-d(10)-kkk 310 529161 66 kkk-d(10)-eee 310 529368 27 eek-d(10)-keke 311 529370 44 eek-d(10)-keke 312 529372 61 eek-d(10)-keke 313 529374 71 eek-d(10)-keke 314 529376 63 eek-d(10)-keke 315 529378 68 eek-d(10)-keke 316 529380 79 eek-d(10)-keke 317 529382 77 eek-d(10)-keke 318 529384 75 eek-d(10)-keke 319 529386 40 eek-d(10)-keke 320 529240 73 eek-d(10)-keke 321 529241 67 eek-d(10)-keke 322 529242 42 eek-d(10)-keke 323 529243 60 eek-d(10)-keke 324 529388 65 eek-d(10)-keke 325 529815 37 k-d(10)-kekee 326 529834 44 k-d(10)-kdkee 326 529736 47 ke-d(10)-keke 327 529756 78 ek-d(10)-keke 327 529776 37 ke-d(10)-kdke 327 529796 71 ek-d(10)-kdke 327 529865 70 kde-d(10)-kdke 328 529885 59 edk-d(10)-kdke 328 529905 54 k-(d4)-k-(d4)-k-(d4)-ke 328 529935 70 eek-d(10)-keke 328 529562 87 eek-d(10)-kke 29 529587 68 kee-d(10)-kke 29 529612 67 edk-d(10)-kke 29 529637 64 kde-d(10)-kke 29 529662 62 kddk-d(9)-kke 29 529687 63 kdde-d(9)-kke 29 529712 61 eddk-d(9)-kke 29 529925 61 eeee-d(9)-kke 29 529816 77 k-d(10)-kekee 329 529835 80 k-d(10)-kdkee 329 529737 82 ke-d(10)-keke 330 529757 83 ek-d(10)-keke 330 529777 68 ke-d(10)-kdke 330 529797 77 ek-d(10)-kdke 330 529866 15 kde-d(10)-kdke 331 529886 71 edk-d(10)-kdke 331 529906 63 k-(d4)-k-(d4)-k-(d4)-ke 331 529936 78 eek-d(10)-keke 331 529563 89 eek-d(10)-kke 30 529588 84 kee-d(10)-kke 30 529613 80 edk-d(10)-kke 30 529638 48 kde-d(10)-kke 30 529663 85 kddk-d(9)-kke 30 529688 42 kdde-d(9)-kke 30 529713 81 eddk-d(9)-kke 30 529926 67 eeee-d(9)-kke 30 529390 53 eek-d(10)-keke 332 529392 63 eek-d(10)-keke 333 529394 58 eek-d(10)-keke 334 529396 56 eek-d(10)-keke 335 529398 62 eek-d(10)-keke 336 529400 44 eek-d(10)-keke 337 529402 39 eek-d(10)-keke 338 529404 46 eek-d(10)-keke 339 529406 63 eek-d(10)-keke 340 529244 58 eek-d(10)-keke 341 529245 68 eek-d(10)-keke 342 529246 60 eek-d(10)-keke 343 529247 36 eek-d(10)-keke 344 529248 43 eek-d(10)-keke 345 529249 23 eek-d(10)-keke 346 529250 69 eek-d(10)-keke 347 529251 15 eek-d(10)-keke 348 529252 44 eek-d(10)-keke 349 529253 42 eek-d(10)-keke 350 529408 67 eek-d(10)-keke 351 529410 19 eek-d(10)-keke 352 529412 57 eek-d(10)-keke 353 529414 80 eek-d(10)-keke 354 529416 85 eek-d(10)-keke 355 529418 70 eek-d(10)-keke 356 529420 78 eek-d(10)-keke 357 529422 19 eek-d(10)-keke 358 529424 48 eek-d(10)-keke 359 529426 66 eek-d(10)-keke 360 529428 59 eek-d(10)-keke 361 529430 83 eek-d(10)-keke 362 529432 84 eek-d(10)-keke 363 529199 71 eek-d(10)-keke 364 529236 76 eek-d(10)-kkke 364 529142 64 eee-d(10)-kkk 365 529162 60 kkk-d(10)-eee 365 529254 46 eek-d(10)-keke 366 529255 52 eek-d(10)-keke 367 529256 57 eek-d(10)-keke 368 529257 55 eek-d(10)-keke 369 529258 3 eek-d(10)-keke 370 529259 71 eek-d(10)-keke 371 529260 72 eek-d(10)-keke 372 529261 56 eek-d(10)-keke 373 529262 56 eek-d(10)-keke 374 529263 59 eek-d(10)-keke 375 529264 49 eek-d(10)-keke 376 529434 83 eek-d(10)-keke 377 529436 80 eek-d(10)-keke 378 529438 79 eek-d(10)-keke 379 529440 87 eek-d(10)-keke 380 529442 68 eek-d(10)-keke 381 529443 72 eek-d(10)-keke 382 529444 68 eek-d(10)-keke 383 529445 85 eek-d(10)-keke 384 529446 72 eek-d(10)-keke 385 529447 60 eek-d(10)-keke 386 529448 77 eek-d(10)-keke 387 529807 78 k-d(10)-kekee 388 529826 61 k-d(10)-kdkee 388 529449 81 eek-d(10)-keke 389 529728 75 ke-d(10)-keke 390 529748 80 ek-d(10)-keke 390 529768 68 ke-d(10)-kdke 390 529788 74 ek-d(10)-kdke 390 529857 67 kde-d(10)-kdke 389 529877 77 edk-d(10)-kdke 389 529897 26 k-(d4)-k-(d4)-k-(d4)-ke 389 529200 78 eek-d(10)-keke 391 529237 84 eek-d(10)-kkke 391 529564 90 eek-d(10)-kke 34

529589 86 kee-d(10)-kke 34 529614 82 edk-d(10)-kke 34 529639 80 kde-d(10)-kke 34 529664 69 kddk-d(9)-kke 34 529689 71 kdde-d(9)-kke 34 529714 73 eddk-d(9)-kke 34 529917 73 eeee-d(9)-kke 34 529143 68 eee-d(10)-kkk 392 529163 50 kkk-d(10)-eee 392 529201 76 eek-d(10)-keke 393 529238 72 eek-d(10)-kkke 393 529144 57 eee-d(10)-kkk 394 529164 71 kkk-d(10)-eee 394 529450 91 eek-d(10)-keke 395 529451 85 eek-d(10)-keke 396 529266 63 eek-d(10)-keke 397 529806 52 k-d(10)-kekee 398 529825 44 k-d(10)-kdkee 398 529267 56 eek-d(10)-keke 399 529727 67 ke-d(10)-keke 400 529747 63 ek-d(10)-keke 400 529767 67 ke-d(10)-kdke 400 529787 68 ek-d(10)-kdke 400 529856 42 kde-d(10)-kdke 399 529876 36 edk-d(10)-kdke 399 529896 56 k-(d4)-k-(d4)-k-(d4)-ke 399 529546 65 eek-d(10)-kke 248 529571 80 kee-d(10)-kke 248 529596 43 edk-d(10)-kke 248 529621 38 kde-d(10)-kke 248 529646 68 kddk-d(9)-kke 248 529671 50 kdde-d(9)-kke 248 529696 53 eddk-d(9)-kke 248 529916 22 eeee-d(9)-kke 248 529547 86 eek-d(10)-kke 37 529572 75 kee-d(10)-kke 37 529597 58 edk-d(10)-kke 37 529622 58 kde-d(10)-kke 37 529647 18 kddk-d(9)-kke 37 529672 23 kdde-d(9)-kke 37 529697 28 eddk-d(9)-kke 37 529928 36 eeee-d(9)-kke 37 529452 63 eek-d(10)-keke 401 529453 73 eek-d(10)-keke 402 529454 82 eek-d(10)-keke 403 529455 84 eek-d(10)-keke 404 529202 61 eek-d(10)-keke 405 529239 59 eek-d(10)-kkke 405 529145 54 eee-d(10)-kkk 406 529165 77 kkk-d(10)-eee 406 529456 69 eek-d(10)-keke 407 529457 81 eek-d(10)-keke 408 529458 72 eek-d(10)-keke 409 529459 86 eek-d(10)-keke 410 529460 88 eek-d(10)-keke 411 529817 46 k-d(10)-kekee 412 529836 49 k-d(10)-kdkee 412 529738 51 ke-d(10)-keke 413 529758 53 ek-d(10)-keke 413 529778 39 ke-d(10)-kdke 413 529798 52 ek-d(10)-kdke 413 529867 56 kde-d(10)-kdke 414 529887 68 edk-d(10)-kdke 414 529907 28 k-(d4)-k-(d4)-k-(d4)-ke 414 529938 64 eek-d(10)-keke 414 529565 81 eek-d(10)-kke 38 529590 49 kee-d(10)-kke 38 529615 65 edk-d(10)-kke 38 529640 54 kde-d(10)-kke 38 529665 77 kddk-d(9)-kke 38 529690 77 kdde-d(9)-kke 38 529715 63 eddk-d(9)-kke 38 529927 62 eeee-d(9)-kke 38 529185 66 eek-d(10)-keke 221 529222 62 eek-d(10)-kkke 221 529808 75 k-d(10)-kekee 89 529827 67 k-d(10)-kdkee 89 529128 64 eee-d(10)-kkk 415 529148 78 kkk-d(10)-eee 415 529461 87 eek-d(10)-keke 416 529729 71 ke-d(10)-keke 415 529749 83 ek-d(10)-keke 415 529769 63 ke-d(10)-kdke 415 529789 10 ek-d(10)-kdke 415 529800 69 k-d(10)-kekee 415 529819 78 k-d(10)-kdkee 415 529858 60 kde-d(10)-kdke 416 529878 75 edk-d(10)-kdke 416 529898 34 k-(d4)-k-(d4)-k-(d4)-ke 416 529566 61 eek-d(10)-kke 39 529591 71 kee-d(10)-kke 39 529616 71 edk-d(10)-kke 39 529641 65 kde-d(10)-kke 39 529666 70 kddk-d(9)-kke 39 529691 67 kdde-d(9)-kke 39 529716 75 eddk-d(9)-kke 39 529721 71 ke-d(10)-keke 39 529741 81 ek-d(10)-keke 39 529761 66 ke-d(10)-kdke 39 529781 65 ek-d(10)-kdke 39 529801 71 k-d(10)-kekee 39 529820 74 k-d(10)-kdkee 39 529850 63 kde-d(10)-kdke 417 529870 72 edk-d(10)-kdke 417 529890 23 k-(d4)-k-(d4)-k-(d4)-ke 417 529918 54 eeee-d(9)-kke 39 529567 75 eek-d(10)-kke 262 529592 80 kee-d(10)-kke 262 529617 65 edk-d(10)-kke 262 529642 62 kde-d(10)-kke 262 529667 75 kddk-d(9)-kke 262 529692 53 kdde-d(9)-kke 262 529717 69 eddk-d(9)-kke 262 529722 74 ke-d(10)-keke 262 529742 81 ek-d(10)-keke 262 529762 66 ke-d(10)-kdke 262 529782 68 ek-d(10)-kdke 262 529851 68 kde-d(10)-kdke 418 529871 77 edk-d(10)-kdke 418 529891 36 k-(d4)-k-(d4)-k-(d4)-ke 418 529910 60 eeee-d(9)-kke 262 529568 79 eek-d(10)-kke 263 529593 70 kee-d(10)-kke 263 529618 77 edk-d(10)-kke 263 529643 72 kde-d(10)-kke 263 529668 73 kddk-d(9)-kke 263 529693 62 kdde-d(9)-kke 263 529718 69 eddk-d(9)-kke 263 529911 66 eeee-d(9)-kke 263 529462 76 eek-d(10)-keke 419 529268 18 eek-d(10)-keke 420 529187 46 eek-d(10)-keke 421 529224 48 eek-d(10)-kkke 421 529130 34 eee-d(10)-kkk 422 529150 51 kkk-d(10)-eee 422 529549 85 eek-d(10)-kke 42 529574 81 kee-d(10)-kke 42 529599 64 edk-d(10)-kke 42 529624 68 kde-d(10)-kke 42 529649 77 kddk-d(9)-kke 42 529674 65 kdde-d(9)-kke 42 529699 63 eddk-d(9)-kke 42 529931 59 eeee-d(9)-kke 42 529810 80 k-d(10)-kekee 423 529829 67 k-d(10)-kdkee 423 529269 65 eek-d(10)-keke 424 529731 66 ke-d(10)-keke 425 529751 76 ek-d(10)-keke 425 529771 73 ke-d(10)-kdke 425 529791 65 ek-d(10)-kdke 425 529860 73 kde-d(10)-kdke 424 529880 74 edk-d(10)-kdke 424 529900 62 k-(d4)-k-(d4)-k-(d4)-ke 424 529270 69 eek-d(10)-keke 480 529550 81 eek-d(10)-kke 44 529575 88 kee-d(10)-kke 44 529600 78 edk-d(10)-kke 44 529625 74 kde-d(10)-kke 44 529650 81 kddk-d(9)-kke 44 529675 76 kdde-d(9)-kke 44 529700 73 eddk-d(9)-kke 44 529920 67 eeee-d(9)-kke 44 529271 43 eek-d(10)-keke 427 529272 0 eek-d(10)-keke 428 529273 62 eek-d(10)-keke 429 529274 78 eek-d(10)-keke 430 529275 70 eek-d(10)-keke 431 529276 73 eek-d(10)-keke 432 529277 71 eek-d(10)-keke 433 529278 72 eek-d(10)-keke 434 529279 10 eek-d(10)-keke 435 529280 11 eek-d(10)-keke 436 529281 82 eek-d(10)-keke 437 529282 87 eek-d(10)-keke 438 529803 71 k-d(10)-kekee 250 529822 72 k-d(10)-kdkee 250 529724 76 ke-d(10)-keke 439 529744 81 ek-d(10)-keke 439 529764 65 ke-d(10)-kdke 439 529784 68 ek-d(10)-kdke 439 529853 64 kde-d(10)-kdke 440 529873 69 edk-d(10)-kdke 440 529893 45 k-(d4)-k-(d4)-k-(d4)-ke 440 529937 81 eek-d(10)-keke 440 529551 88 eek-d(10)-kke 48 529576 71 kee-d(10)-kke 48 529601 74 edk-d(10)-kke 48 529626 72 kde-d(10)-kke 48 529651 85 kddk-d(9)-kke 48 529676 67 kdde-d(9)-kke 48 529701 82 eddk-d(9)-kke 48 529913 76 eeee-d(9)-kke 48 529811 56 k-d(10)-kekee 441 529830 46 k-d(10)-kdkee 441 529732 63 ke-d(10)-keke 442 529752 72 ek-d(10)-keke 442 529772 61 ke-d(10)-kdke 442 529792 68 ek-d(10)-kdke 442 529861 54 kde-d(10)-kdke 443 529881 78 edk-d(10)-kdke 443 529901 29 k-(d4)-k-(d4)-k-(d4)-ke 443 529939 67 eek-d(10)-keke 443 529283 70 eek-d(10)-keke 444 529552 72 eek-d(10)-kke 49 529577 80 kee-d(10)-kke 49 529602 64 edk-d(10)-kke 49 529627 56 kde-d(10)-kke 49 529652 57 kddk-d(9)-kke 49 529677 43 kdde-d(9)-kke 49 529702 54 eddk-d(9)-kke 49 529921 42 eeee-d(9)-kke 49 529284 76 eek-d(10)-keke 445 529285 77 eek-d(10)-keke 446 529286 68 eek-d(10)-keke 447 529287 65 eek-d(10)-keke 448 529719 73 ke-d(10)-keke 264 529739 83 ek-d(10)-keke 264 529759 63 ke-d(10)-kdke 264 529779 70 ek-d(10)-kdke 244 529848 60 kde-d(10)-kdke 449 529868 63 edk-d(10)-kdke 449 529888 53 k-(d4)-k-(d4)-k-(d4)-ke 449 529553 81 eek-d(10)-kke 265 529578 65 kee-d(10)-kke 265 529603 60 edk-d(10)-kke 265 529628 59 kde-d(10)-kke 265 529653 76 kddk-d(9)-kke 265 529678 56 kdde-d(9)-kke 265 529703 68 eddk-d(9)-kke 265 529908 69 eeee-d(9)-kke 265 529168 64 eek-d(10)-keke 450 529205 62 eek-d(10)-kkke 450 529290 53 eek-d(10)-keke 451 529802 57 k-d(10)-kekee 452 529821 61 k-d(10)-kdkee 452 529292 74 eek-d(10)-keke 453 529723 68 ke-d(10)-keke 454 529743 84 ek-d(10)-keke 454 529763 64 ke-d(10)-kdke 454 529783 72 ek-d(10)-kdke 454 529852 66 kde-d(10)-kdke 453 529872 62 edk-d(10)-kdke 453 529892 43 k-(d4)-k-(d4)-k-(d4)-ke 453 529554 80 eek-d(10)-kke 252 529579 83 kee-d(10)-kke 252 529604 73 edk-d(10)-kke 252 529629 64 kde-d(10)-kke 252 529654 69 kddk-d(9)-kke 252 529679 52 kdde-d(9)-kke 252 529704 63 eddk-d(9)-kke 252 529912 64 eeee-d(9)-kke 252

529294 74 eek-d(10)-keke 455 529296 52 eek-d(10)-keke 456 529298 60 eek-d(10)-keke 457 529300 71 eek-d(10)-keke 458 529188 79 eek-d(10)-keke 459 529225 78 eek-d(10)-kkke 459 529131 58 eee-d(10)-kkk 460 529151 71 kkk-d(10)-eee 460 529302 74 eek-d(10)-keke 461 529189 64 eek-d(10)-keke 222 529226 50 eek-d(10)-kkke 222 529132 78 eee-d(10)-kkk 462 529152 62 kkk-d(10)-eee 462 529190 76 eek-d(10)-keke 223 529227 88 eek-d(10)-kkke 250 529133 81 eee-d(10)-kkk 463 529153 68 kkk-d(10)-eee 463 529191 78 eek-d(10)-keke 224 529228 85 eek-d(10)-kkke 224 529134 75 eee-d(10)-kkk 464 529154 61 kkk-d(10)-eee 464 529304 89 eek-d(10)-keke 465 529306 84 eek-d(10)-keke 466 529308 68 eek-d(10)-keke 467 529310 59 eek-d(10)-keke 468 529169 79 eek-d(10)-keke 469 529206 82 eek-d(10)-kkke 469 529312 68 eek-d(10)-keke 470 529314 61 eek-d(10)-keke 471 529316 62 eek-d(10)-keke 472 529555 78 eek-d(10)-kke 59 529580 73 kee-d(10)-kke 59 529605 71 edk-d(10)-kke 59 529630 64 kde-d(10)-kke 59 529655 63 kddk-d(9)-kke 59 529680 43 kdde-d(9)-kke 59 529705 63 eddk-d(9)-kke 59 529932 60 eeee-d(9)-kke 59 529318 82 eek-d(10)-keke 473 529170 85 eek-d(10)-keke 474 529207 88 eek-d(10)-kkke 474 529171 81 eek-d(10)-keke 475 529208 84 eek-d(10)-kkke 475 529805 40 k-d(10)-kekee 476 529824 32 k-d(10)-kdkee 476 529320 74 eek-d(10)-keke 477 529726 80 ke-d(10)-keke 478 529746 82 ek-d(10)-keke 478 529766 63 ke-d(10)-kdke 478 529786 69 ek-d(10)-kdke 478 529855 39 kde-d(10)-kdke 477 529875 40 edk-d(10)-kdke 477 529895 27 k-(d4)-k-(d4)-k-(d4)-ke 477 529556 72 eek-d(10)-kke 61 529581 68 kee-d(10)-kke 61 529606 54 edk-d(10)-kke 61 529631 29 kde-d(10)-kke 61 529656 74 kddk-d(9)-kke 61 529681 32 kdde-d(9)-kke 61 529706 41 eddk-d(9)-kke 61 529915 51 eeee-d(9)-kke 61 529172 88 eek-d(10)-keke 226 529209 87 eek-d(10)-kkke 226 529173 92 eek-d(10)-keke 227 529210 89 eek-d(10)-kkke 227 529183 85 eek-d(10)-keke 479 529220 92 eek-d(10)-kkke 479 529126 83 eee-d(10)-kkk 257 529146 84 kkk-d(10)-eee 257 529174 85 eek-d(10)-keke 480 529211 86 eek-d(10)-kkke 480 529322 71 eek-d(10)-keke 481 529324 79 eek-d(10)-keke 482 529326 85 eek-d(10)-keke 483 529175 92 eek-d(10)-keke 228 529212 92 eek-d(10)-kkke 228 529176 89 eek-d(10)-keke 229 529213 90 eek-d(10)-kkke 229 529804 89 k-d(10)-kekee 259 529823 89 k-d(10)-kdkee 259 529166 83 eek-d(10)-keke 230 529203 86 eek-d(10)-kkke 230 529725 92 ke-d(10)-keke 260 529745 91 ek-d(10)-keke 260 529765 88 ke-d(10)-kdke 260 529785 91 ek-d(10)-kdke 260 529799 89 k-d(10)-kekee 260 529818 88 k-d(10)-kdkee 260 529854 90 kde-d(10)-kdke 230 529874 81 edk-d(10)-kdke 230 529894 60 k-(d4)-k-(d4)-k-(d4)-ke 230 529167 71 eek-d(10)-keke 231 529204 70 eek-d(10)-kkke 231 529557 86 eek-d(10)-kke 69 529582 86 kee-d(10)-kke 69 529607 84 edk-d(10)-kke 69 529632 81 kde-d(10)-kke 69 529657 85 kddk-d(9)-kke 69 529682 78 kdde-d(9)-kke 69 529707 79 eddk-d(9)-kke 69 529720 75 ke-d(10)-keke 69 529740 70 ek-d(10)-keke 69 529760 78 ke-d(10)-kdke 69 529780 83 ek-d(10)-kdke 69 529849 80 kde-d(10)-kdke 231 529869 72 edk-d(10)-kdke 231 529889 49 k-(d4)-k-(d4)-k-(d4)-ke 231 529914 69 eeee-d(9)-kke 69 529328 68 eek-d(10)-keke 484 529558 71 eek-d(10)-kke 71 529583 81 kee-d(10)-kke 71 529608 68 edk-d(10)-kke 71 529633 73 kde-d(10)-kke 71 529658 63 kddk-d(9)-kke 71 529683 74 kdde-d(9)-kke 71 529708 70 eddk-d(9)-kke 71 529909 59 eeee-d(9)-kke 71 529192 51 eek-d(10)-keke 485 529229 69 eek-d(10)-kkke 485 529135 54 eee-d(10)-kkk 486 529155 56 kkk-d(10)-eee 486 529330 37 eek-d(10)-keke 487 e = 2' -MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 20

Design of Modified Oligonucleotides Comprising 2'-O-Methoxyethyl (2'-MOE) or 6'-(S)--CH.sub.3 Bicyclic Nucleoside (e.g cEt) Modifications

[0756] Based on the activity of the antisense oligonucleotides listed above, additional antisense oligonucleotides were designed targeting a Target-X nucleic acid targeting start positions 1147, 1154 or 12842 of Target-X.

[0757] The newly designed chimeric antisense oligonucleotides are 16 or 17 nucleotides in length and their motifs are described in Table 35. The chemistry column of Table 35 presents the sugar motif of each oligonucleotide, wherein "e" indicates a 2'-O-methoxyethyl (2'-MOE) nucleoside, "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g cEt) and "d" indicates a 2'-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosine.

[0758] Each gapmer listed in Table 35 is targeted to the human Target-X genomic sequence.

TABLE-US-00036 TABLE 35 Chimeric antisense oligonucleotides targeted to Target-X ISIS No Chemistry SEQ CODE 529544 eek-d(10)-kke 21 529569 kee-d(10)-kke 21 529594 edk-d(10)-kke 21 529619 kde-d(10)-kke 21 529644 kddk-d(9)-kke 21 529669 kdde-d(9)-kke 21 529694 eddk-d(9)-kke 21 529929 eeee-d(9)-kke 21 529809 k-d(10)-kekee 488 529828 k-d(10)-kdkee 488 529730 ke-d(10)-keke 489 529750 ek-d(10)-keke 489 529770 ke-d(10)-kdke 489 529790 ek-d(10)-kdke 489 529859 kde-d(10)-kdke 490 529879 edk-d(10)-kdke 490 529899 k-d(4)-k-d(4)-k-d(4)-ke 490 529545 eek-d(10)-kke 22 529570 kee-d(10)-kke 22 529595 edk-d(10)-kke 22 529620 kde-d(10)-kke 22 529645 kddk-d(9)-kke 22 529670 kdde-d(9)-kke 22 529695 eddk-d(9)-kke 22 529919 eeee-d(9)-kke 22 529548 eek-d(10)-kke 41 529573 kee-d(10)-kke 41 529598 edk-d(10)-kke 41 529623 kde-d(10)-kke 41 529648 kddk-d(9)-kke 41 529673 kdde-d(9)-kke 41 529698 eddk-d(9)-kke 41 529930 eeee-d(9)-kke 41 e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 21

Modified Oligonucleotides Comprising 2'-O-Methoxyethyl (2'-MOE) and 6'-(S)--CH.sub.3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X

[0759] Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 472998 and ISIS 515554, described in the Examples above were also included in the screen.

[0760] The newly designed chimeric antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 36. The chemistry column of Table 36 presents the sugar motif of each oligonucleotide, wherein "e" indicates a 2'-O-methoxyethyl (2'-MOE) nucleoside, "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g cEt) and "d" indicates a 2'-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosine.

[0761] Each gapmer listed in Table 36 is targeted to the human Target-X genomic sequence.

[0762] Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

TABLE-US-00037 TABLE 36 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS No % inhibition Chemistry SEQ CODE 472998 88 kk-d(10)-kk 74 515554 75 eee-d(10)-kkk 493 534530 92 keke-d(9)-kek 491 534563 92 kek-d(9)-ekek 491 534596 88 ekee-d(9)-kke 491 534629 89 eke-d(9)-ekke 491 534662 87 eekk-d(9)-eke 491 534695 92 eek-d(9)-keke 491 534732 90 ekek-d(8)-keke 491 534767 92 keek-d(8)-keek 491 534802 93 ekk-d(10)-kke 491 534832 83 edk-d(10)-kke 491 534862 72 kde-d(10)-kke 491 534892 82 eek-d(10)-kke 491 534922 80 kddk-d(9)-kke 491 534952 72 kdde-d(9)-kke 491 534982 77 eddk-d(9)-kke 491 535012 70 eeee-d(9)-kke 491 535045 84 eeee-d(9)-kkk 491 535078 87 eeek-d(9)-kke 491 535111 63 eeeee-d(8)-kke 491 535144 69 ededk-d(8)-kke 491 535177 68 edkde-d(8)-kke 491 534531 61 keke-d(9)-kek 492 534564 30 kek-d(9)-ekek 492 534597 67 ekee-d(9)-kke 492 534630 54 eke-d(9)-ekke 492 534663 94 eekk-d(9)-eke 492 534696 68 eek-d(9)-keke 492 534733 44 ekek-d(8)-keke 492 534768 55 keek-d(8)-keek 492 534803 73 ekk-d(10)-kke 492 534833 65 edk-d(10)-kke 492 534863 53 kde-d(10)-kke 492 534893 61 eek-d(10)-kke 492 534923 70 kddk-d(9)-kke 492 534953 54 kdde-d(9)-kke 492 534983 58 eddk-d(9)-kke 492 535013 52 eeee-d(9)-kke 492 535046 67 eeee-d(9)-kkk 492 535079 57 eeek-d(9)-kke 492 535112 42 eeeee-d(8)-kke 492 535145 41 ededk-d(8)-kke 492 535178 35 edkde-d(8)-kke 492 534565 87 kek-d(9)-ekek 493 534598 72 ekee-d(9)-kke 493 534631 70 eke-d(9)-ekke 493 534664 94 eekk-d(9)-eke 493 534697 90 eek-d(9)-keke 493 534734 74 ekek-d(8)-keke 493 534769 80 keek-d(8)-keek 493 534804 87 ekk-d(10)-kke 493 534834 76 edk-d(10)-kke 493 534864 56 kde-d(10)-kke 493 534894 67 eek-d(10)-kke 493 534924 71 kddk-d(9)-kke 493 534954 54 kdde-d(9)-kke 493 534984 48 eddk-d(9)-kke 493 535014 43 eeee-d(9)-kke 493 535047 60 eeee-d(9)-kkk 493 535080 64 eeek-d(9)-kke 493 535113 32 eeeee-d(8)-kke 493 535146 31 ededk-d(8)-kke 493 535179 28 edkde-d(8)-kke 493 534533 82 keke-d(9)-kek 494 534566 88 kek-d(9)-ekek 494 534599 65 ekee-d(9)-kke 494 534632 69 eke-d(9)-ekke 494 534665 87 eekk-d(9)-eke 494 534698 64 eek-d(9)-keke 494 534735 63 ekek-d(8)-keke 494 534770 66 keek-d(8)-keek 494 534805 87 ekk-d(10)-kke 494 534835 68 edk-d(10)-kke 494 534865 66 kde-d(10)-kke 494 534895 57 eek-d(10)-kke 494 534925 82 kddk-d(9)-kke 494 534955 76 kdde-d(9)-kke 494 534985 71 eddk-d(9)-kke 494 535015 59 eeee-d(9)-kke 494 535048 69 eeee-d(9)-kkk 494 535081 67 eeek-d(9)-kke 494 535114 37 eeeee-d(8)-kke 494 535147 32 ededk-d(8)-kke 494 535180 31 edkde-d(8)-kke 494 534534 94 keke-d(9)-kek 234 534567 92 kek-d(9)-ekek 234 534600 92 ekee-d(9)-kke 234 534633 91 eke-d(9)-ekke 234 534666 89 eekk-d(9)-eke 234 534699 91 eek-d(9)-keke 234 534736 83 ekek-d(8)-keke 234 534771 80 keek-d(8)-keek 234 534806 96 ekk-d(10)-kke 234 534836 86 edk-d(10)-kke 234 534866 82 kde-d(10)-kke 234 534896 82 eek-d(10)-kke 234 534926 89 kddk-d(9)-kke 234 534956 91 kdde-d(9)-kke 234 534986 87 eddk-d(9)-kke 234 535016 83 eeee-d(9)-kke 234 535049 87 eeee-d(9)-kkk 234 535082 87 eeek-d(9)-kke 234 535115 77 eeeee-d(8)-kke 234 535148 73 ededk-d(8)-kke 234 535181 68 edkde-d(8)-kke 234 534535 66 keke-d(9)-kek 236 534568 85 kek-d(9)-ekek 236 534601 51 ekee-d(9)-kke 236 534634 80 eke-d(9)-ekke 236 534667 90 eekk-d(9)-eke 236 534700 88 eek-d(9)-keke 236 534737 65 ekek-d(8)-keke 236 534772 77 keek-d(8)-keek 236 534807 84 ekk-d(10)-kke 236 534837 78 edk-d(10)-kke 236 534867 44 kde-d(10)-kke 236 534897 82 eek-d(10)-kke 236 534927 61 kddk-d(9)-kke 236 534957 58 kdde-d(9)-kke 236 534987 49 eddk-d(9)-kke 236 535017 38 eeee-d(9)-kke 236 535050 32 eeee-d(9)-kkk 236 535083 43 eeek-d(9)-kke 236 535116 9 eeeee-d(8)-kke 236 535149 23 ededk-d(8)-kke 236 535182 18 edkde-d(8)-kke 236 534536 89 keke-d(9)-kek 238 534569 90 kek-d(9)-ekek 238 534602 85 ekee-d(9)-kke 238 534635 87 eke-d(9)-ekke 238 534668 90 eekk-d(9)-eke 238 534701 92 eek-d(9)-keke 238 534738 81 ekek-d(8)-keke 238 534773 79 keek-d(8)-keek 238 534808 90 ekk-d(10)-kke 238 534838 88 edk-d(10)-kke 238 534868 67 kde-d(10)-kke 238 534898 89 eek-d(10)-kke 238 534928 81 kddk-d(9)-kke 238 534958 78 kdde-d(9)-kke 238 534988 66 eddk-d(9)-kke 238 535018 78 eeee-d(9)-kke 238 535051 76 eeee-d(9)-kkk 238 535084 80 eeek-d(9)-kke 238 535117 58 eeeee-d(8)-kke 238 535150 51 ededk-d(8)-kke 238 535183 53 edkde-d(8)-kke 238 534537 91 keke-d(9)-kek 239 534570 85 kek-d(9)-ekek 239 534603 79 ekee-d(9)-kke 239 534636 72 eke-d(9)-ekke 239 534669 85 eekk-d(9)-eke 239 534702 85 eek-d(9)-keke 239 534739 73 ekek-d(8)-keke 239 534774 77 keek-d(8)-keek 239 534809 91 ekk-d(10)-kke 239 534839 86 edk-d(10)-kke 239 534869 71 kde-d(10)-kke 239 534899 82 eek-d(10)-kke 239 534929 83 kddk-d(9)-kke 239 534959 80 kdde-d(9)-kke 239 534989 79 eddk-d(9)-kke 239 535019 76 eeee-d(9)-kke 239 535052 79 eeee-d(9)-kkk 239 535085 81 eeek-d(9)-kke 239 535118 58 eeeee-d(8)-kke 239 535151 65 ededk-d(8)-kke 239 535184 60 edkde-d(8)-kke 239 534516 77 keke-d(9)-kek 495 534549 80 kek-d(9)-ekek 495 534582 73 ekee-d(9)-kke 495 534615 79 eke-d(9)-ekke 495 534648 67 eekk-d(9)-eke 495 534681 87 eek-d(9)-keke 495 534718 46 ekek-d(8)-keke 495 534753 68 keek-d(8)-keek 495 534788 84 ekk-d(10)-kke 495 534818 82 edk-d(10)-kke 495 534848 75 kde-d(10)-kke 495 534878 72 eek-d(10)-kke 495 534908 81 kddk-d(9)-kke 495 534938 69 kdde-d(9)-kke 495 534968 77 eddk-d(9)-kke 495 534998 76 eeee-d(9)-kke 495 535031 76 eeee-d(9)-kkk 495 535064 70 eeek-d(9)-kke 495 535097 57 eeeee-d(8)-kke 495 535130 69 ededk-d(8)-kke 495 535163 58 edkde-d(8)-kke 495 534538 71 keke-d(9)-kek 241 534571 64 kek-d(9)-ekek 241 534604 66 ekee-d(9)-kke 241 534637 74 eke-d(9)-ekke 241 534670 87 eekk-d(9)-eke 241 534703 72 eek-d(9)-keke 241 534740 56 ekek-d(8)-keke 241 534775 53 keek-d(8)-keek 241 534810 78 ekk-d(10)-kke 241 534840 73 edk-d(10)-kke 241 534870 65 kde-d(10)-kke 241 534900 69 eek-d(10)-kke 241 534930 67 kddk-d(9)-kke 241 534960 62 kdde-d(9)-kke 241 534990 66 eddk-d(9)-kke 241 535020 61 eeee-d(9)-kke 241 535053 47 eeee-d(9)-kkk 241 535086 61 eeek-d(9)-kke 241 535119 49 eeeee-d(8)-kke 241 535152 48 ededk-d(8)-kke 241 535185 57 edkde-d(8)-kke 241 534539 70 keke-d(9)-kek 496 534572 82 kek-d(9)-ekek 496 534605 59 ekee-d(9)-kke 496 534638 69 eke-d(9)-ekke 496 534671 89 eekk-d(9)-eke 496 534704 83 eek-d(9)-keke 496 534741 47 ekek-d(8)-keke 496 534776 46 keek-d(8)-keek 496 534811 71 ekk-d(10)-kke 496 534841 61 edk-d(10)-kke 496 534871 53 kde-d(10)-kke 496 534901 55 eek-d(10)-kke 496 534931 73 kddk-d(9)-kke 496 534961 53 kdde-d(9)-kke 496 534991 56 eddk-d(9)-kke 496 535021 58 eeee-d(9)-kke 496 535054 59 eeee-d(9)-kkk 496 535087 0 eeek-d(9)-kke 496 535120 41 eeeee-d(8)-kke 496 535153 44 ededk-d(8)-kke 496 535186 35 edkde-d(8)-kke 496 534573 76 kek-d(9)-ekek 497 534606 55 ekee-d(9)-kke 497 534639 72 eke-d(9)-ekke 497 534672 89 eekk-d(9)-eke 497 534705 87 eek-d(9)-keke 497 534742 84 ekek-d(8)-keke 497 534777 79 keek-d(8)-keek 497 534812 76 ekk-d(10)-kke 497 534842 74 edk-d(10)-kke 497 534872 53 kde-d(10)-kke 497 534902 70 eek-d(10)-kke 497 534932 73 kddk-d(9)-kke 497

534962 60 kdde-d(9)-kke 497 534992 61 eddk-d(9)-kke 497 535022 38 eeee-d(9)-kke 497 535055 42 eeee-d(9)-kkk 497 535088 56 eeek-d(9)-kke 497 535121 5 eeeee-d(8)-kke 497 535154 22 ededk-d(8)-kke 497 535187 16 edkde-d(8)-kke 497 534541 86 keke-d(9)-kek 498 534574 89 kek-d(9)-ekek 498 534607 59 ekee-d(9)-kke 498 534640 76 eke-d(9)-ekke 498 534673 89 eekk-d(9)-eke 498 534706 86 eek-d(9)-keke 498 534743 79 ekek-d(8)-keke 498 534778 80 keek-d(8)-keek 498 534813 83 ekk-d(10)-kke 498 534843 82 edk-d(10)-kke 498 534873 83 kde-d(10)-kke 498 534903 78 eek-d(10)-kke 498 534933 83 kddk-d(9)-kke 498 534963 70 kdde-d(9)-kke 498 534993 78 eddk-d(9)-kke 498 535023 56 eeee-d(9)-kke 498 535056 59 eeee-d(9)-kkk 498 535089 73 eeek-d(9)-kke 498 535122 39 eeeee-d(8)-kke 498 535155 60 ededk-d(8)-kke 498 535188 41 edkde-d(8)-kke 498 534542 75 keke-d(9)-kek 499 534575 82 kek-d(9)-ekek 499 534608 72 ekee-d(9)-kke 499 534641 69 eke-d(9)-ekke 499 534674 84 eekk-d(9)-eke 499 534707 78 eek-d(9)-keke 499 534744 72 ekek-d(8)-keke 499 534779 75 keek-d(8)-keek 499 534814 81 ekk-d(10)-kke 499 534844 75 edk-d(10)-kke 499 534874 70 kde-d(10)-kke 499 534904 71 eek-d(10)-kke 499 534934 73 kddk-d(9)-kke 499 534964 72 kdde-d(9)-kke 499 534994 69 eddk-d(9)-kke 499 535024 56 eeee-d(9)-kke 499 535057 63 eeee-d(9)-kkk 499 535090 64 eeek-d(9)-kke 499 535123 40 eeeee-d(8)-kke 499 535156 47 ededk-d(8)-kke 499 535189 48 edkde-d(8)-kke 499 534515 52 keke-d(9)-kek 34 534548 85 kek-d(9)-ekek 34 534581 75 ekee-d(9)-kke 34 534614 83 eke-d(9)-ekke 34 534647 65 eekk-d(9)-eke 34 534680 88 eek-d(9)-keke 34 534717 76 ekek-d(8)-keke 34 534752 79 keek-d(8)-keek 34 534787 90 ekk-d(10)-kke 34 535030 77 eeee-d(9)-kkk 34 535063 75 eeek-d(9)-kke 34 535096 54 eeeee-d(8)-kke 34 535129 66 ededk-d(8)-kke 34 535162 49 edkde-d(8)-kke 34 534543 66 keke-d(9)-kek 500 534576 69 kek-d(9)-ekek 500 534609 77 ekee-d(9)-kke 500 534642 62 eke-d(9)-ekke 500 534675 80 eekk-d(9)-eke 500 534708 81 eek-d(9)-keke 500 534745 68 ekek-d(8)-keke 500 534780 69 keek-d(8)-keek 500 534815 85 ekk-d(10)-kke 500 534845 72 edk-d(10)-kke 500 534875 56 kde-d(10)-kke 500 534905 65 eek-d(10)-kke 500 534935 78 kddk-d(9)-kke 500 534965 48 kdde-d(9)-kke 500 534995 62 eddk-d(9)-kke 500 535025 58 eeee-d(9)-kke 500 535058 60 eeee-d(9)-kkk 500 535091 61 eeek-d(9)-kke 500 535124 51 eeeee-d(8)-kke 500 535157 55 ededk-d(8)-kke 500 535190 47 edkde-d(8)-kke 500 534517 71 keke-d(9)-kek 501 534550 80 kek-d(9)-ekek 501 534583 70 ekee-d(9)-kke 501 534616 84 eke-d(9)-ekke 501 534649 68 eekk-d(9)-eke 501 534682 87 eek-d(9)-keke 501 534719 90 ekek-d(8)-keke 501 534754 83 keek-d(8)-keek 501 534789 86 ekk-d(10)-kke 501 534819 69 edk-d(10)-kke 501 534849 62 kde-d(10)-kke 501 534879 69 eek-d(10)-kke 501 534909 73 kddk-d(9)-kke 501 534939 49 kdde-d(9)-kke 501 534969 47 eddk-d(9)-kke 501 534999 51 eeee-d(9)-kke 501 535032 51 eeee-d(9)-kkk 501 535065 64 eeek-d(9)-kke 501 535098 31 eeeee-d(8)-kke 501 535131 31 ededk-d(8)-kke 501 535164 40 edkde-d(8)-kke 501 534518 81 keke-d(9)-kek 502 534551 88 kek-d(9)-ekek 502 534584 78 ekee-d(9)-kke 502 534617 80 eke-d(9)-ekke 502 534650 83 eekk-d(9)-eke 502 534683 93 eek-d(9)-keke 502 534720 87 ekek-d(8)-keke 502 534755 82 keek-d(8)-keek 502 534790 89 ekk-d(10)-kke 502 534820 64 edk-d(10)-kke 502 534850 38 kde-d(10)-kke 502 534880 68 eek-d(10)-kke 502 534910 60 kddk-d(9)-kke 502 534940 37 kdde-d(9)-kke 502 534970 59 eddk-d(9)-kke 502 535000 30 eeee-d(9)-kke 502 535033 44 eeee-d(9)-kkk 502 535066 64 eeek-d(9)-kke 502 535099 22 eeeee-d(8)-kke 502 535132 54 ededk-d(8)-kke 502 535165 45 edkde-d(8)-kke 502 534544 80 keke-d(9)-kek 503 534577 83 kek-d(9)-ekek 503 534610 62 ekee-d(9)-kke 503 534643 66 eke-d(9)-ekke 503 534676 95 eekk-d(9)-eke 503 534709 86 eek-d(9)-keke 503 534746 73 ekek-d(8)-keke 503 534781 71 keek-d(8)-keek 503 534816 83 ekk-d(10)-kke 503 534846 73 edk-d(10)-kke 503 534876 39 kde-d(10)-kke 503 534906 67 eek-d(10)-kke 503 534936 66 kddk-d(9)-kke 503 534966 48 kdde-d(9)-kke 503 534996 56 eddk-d(9)-kke 503 535026 39 eeee-d(9)-kke 503 535059 45 eeee-d(9)-kkk 503 535092 48 eeek-d(9)-kke 503 535125 26 eeeee-d(8)-kke 503 535158 44 ededk-d(8)-kke 503 535191 34 edkde-d(8)-kke 503 534545 83 keke-d(9)-kek 504 534578 81 kek-d(9)-ekek 504 534611 78 ekee-d(9)-kke 504 534644 72 eke-d(9)-ekke 504 534677 92 eekk-d(9)-eke 504 534710 78 eek-d(9)-keke 504 534747 85 ekek-d(8)-keke 504 534782 85 keek-d(8)-keek 504 534817 88 ekk-d(10)-kke 504 534847 73 edk-d(10)-kke 504 534877 66 kde-d(10)-kke 504 534907 73 eek-d(10)-kke 504 534937 85 kddk-d(9)-kke 504 534967 80 kdde-d(9)-kke 504 534997 74 eddk-d(9)-kke 504 535027 64 eeee-d(9)-kke 504 535060 68 eeee-d(9)-kkk 504 535093 73 eeek-d(9)-kke 504 535126 42 eeeee-d(8)-kke 504 535159 49 ededk-d(8)-kke 504 535192 51 edkde-d(8)-kke 504 534519 87 keke-d(9)-kek 505 534552 85 kek-d(9)-ekek 505 534585 76 ekee-d(9)-kke 505 534618 78 eke-d(9)-ekke 505 534651 79 eekk-d(9)-eke 505 534684 87 eek-d(9)-keke 505 534721 89 ekek-d(8)-keke 505 534756 90 keek-d(8)-keek 505 534791 84 ekk-d(10)-kke 505 534821 79 edk-d(10)-kke 505 534851 64 kde-d(10)-kke 505 534881 65 eek-d(10)-kke 505 534911 85 kddk-d(9)-kke 505 534941 66 kdde-d(9)-kke 505 534971 75 eddk-d(9)-kke 505 535001 62 eeee-d(9)-kke 505 535034 65 eeee-d(9)-kkk 505 535067 76 eeek-d(9)-kke 505 535100 5 eeeee-d(8)-kke 505 535133 30 ededk-d(8)-kke 505 535166 23 edkde-d(8)-kke 505 534520 87 keke-d(9)-kek 251 534553 79 kek-d(9)-ekek 251 534586 60 ekee-d(9)-kke 251 534619 62 eke-d(9)-ekke 251 534652 84 eekk-d(9)-eke 251 534685 84 eek-d(9)-keke 251 534722 75 ekek-d(8)-keke 251 534757 81 keek-d(8)-keek 251 534792 87 ekk-d(10)-kke 251 534822 80 edk-d(10)-kke 251 534852 38 kde-d(10)-kke 251 534882 75 eek-d(10)-kke 251 534912 74 kddk-d(9)-kke 251 534942 58 kdde-d(9)-kke 251 534972 59 eddk-d(9)-kke 251 535002 50 eeee-d(9)-kke 251 535035 57 eeee-d(9)-kkk 251 535068 67 eeek-d(9)-kke 251 535101 24 eeeee-d(8)-kke 251 535134 23 ededk-d(8)-kke 251 535167 26 edkde-d(8)-kke 251 534513 90 keke-d(9)-kek 252 534546 92 kek-d(9)-ekek 252 534579 78 ekee-d(9)-kke 252 534612 82 eke-d(9)-ekke 252 534645 73 eekk-d(9)-eke 252 534678 91 eek-d(9)-keke 252 534715 87 ekek-d(8)-keke 252 534750 88 keek-d(8)-keek 252 534785 89 ekk-d(10)-kke 252 535028 52 eeee-d(9)-kkk 252 535061 73 eeek-d(9)-kke 252 535094 61 eeeee-d(8)-kke 252 535127 59 ededk-d(8)-kke 252 535160 62 edkde-d(8)-kke 252 534521 86 keke-d(9)-kek 506 534554 87 kek-d(9)-ekek 506 534587 62 ekee-d(9)-kke 506 534620 68 eke-d(9)-ekke 506 534653 77 eekk-d(9)-eke 506 534686 90 eek-d(9)-keke 506 534723 88 ekek-d(8)-keke 506 534758 79 keek-d(8)-keek 506 534793 85 ekk-d(10)-kke 506 534823 81 edk-d(10)-kke 506 534853 59 kde-d(10)-kke 506 534883 69 eek-d(10)-kke 506 534913 76 kddk-d(9)-kke 506 534943 53 kdde-d(9)-kke 506 534973 61 eddk-d(9)-kke 506 535003 53 eeee-d(9)-kke 506 535036 35 eeee-d(9)-kkk 506 535069 62 eeek-d(9)-kke 506 535102 31 eeeee-d(8)-kke 506 535135 44 ededk-d(8)-kke 506 535168 34 edkde-d(8)-kke 506 534522 83 keke-d(9)-kek 507 534555 81 kek-d(9)-ekek 507 534588 72 ekee-d(9)-kke 507 534621 74 eke-d(9)-ekke 507 534654 78 eekk-d(9)-eke 507

534687 91 eek-d(9)-keke 507 534724 84 ekek-d(8)-keke 507 534759 86 keek-d(8)-keek 507 534794 78 ekk-d(10)-kke 507 534824 75 edk-d(10)-kke 507 534854 63 kde-d(10)-kke 507 534884 60 eek-d(10)-kke 507 534914 75 kddk-d(9)-kke 507 534944 69 kdde-d(9)-kke 507 534974 66 eddk-d(9)-kke 507 535004 56 eeee-d(9)-kke 507 535037 50 eeee-d(9)-kkk 507 535070 68 eeek-d(9)-kke 507 535103 55 eeeee-d(8)-kke 507 535136 51 ededk-d(8)-kke 507 535169 54 edkde-d(8)-kke 507 534523 89 keke-d(9)-kek 253 534556 91 kek-d(9)-ekek 253 534589 88 ekee-d(9)-kke 253 534622 93 eke-d(9)-ekke 253 534655 72 eekk-d(9)-eke 253 534688 92 eek-d(9)-keke 253 534725 87 ekek-d(8)-keke 253 534760 92 keek-d(8)-keek 253 534795 93 ekk-d(10)-kke 253 534825 82 edk-d(10)-kke 253 534855 73 kde-d(10)-kke 253 534885 82 eek-d(10)-kke 253 534915 88 kddk-d(9)-kke 253 534945 82 kdde-d(9)-kke 253 534975 68 eddk-d(9)-kke 253 535005 69 eeee-d(9)-kke 253 535038 72 eeee-d(9)-kkk 253 535071 74 eeek-d(9)-kke 253 535104 61 eeeee-d(8)-kke 253 535137 67 ededk-d(8)-kke 253 535170 51 edkde-d(8)-kke 253 534524 95 keke-d(9)-kek 254 534557 98 kek-d(9)-ekek 254 534590 91 ekee-d(9)-kke 254 534623 91 eke-d(9)-ekke 254 534656 90 eekk-d(9)-eke 254 534689 92 eek-d(9)-keke 254 534726 57 ekek-d(8)-keke 254 534761 89 keek-d(8)-keek 254 534796 93 ekk-d(10)-kke 254 534826 89 edk-d(10)-kke 254 534856 87 kde-d(10)-kke 254 534886 85 eek-d(10)-kke 254 534916 87 kddk-d(9)-kke 254 534946 86 kdde-d(9)-kke 254 534976 77 eddk-d(9)-kke 254 535006 83 eeee-d(9)-kke 254 535039 86 eeee-d(9)-kkk 254 535072 87 eeek-d(9)-kke 254 535105 68 eeeee-d(8)-kke 254 535138 70 ededk-d(8)-kke 254 535171 65 edkde-d(8)-kke 254 534558 92 kek-d(9)-ekek 255 534591 91 ekee-d(9)-kke 255 534624 86 eke-d(9)-ekke 255 534657 90 eekk-d(9)-eke 255 534690 76 eek-d(9)-keke 255 534727 92 ekek-d(8)-keke 255 534762 91 keek-d(8)-keek 255 534797 94 ekk-d(10)-kke 255 534827 90 edk-d(10)-kke 255 534857 80 kde-d(10)-kke 255 534887 76 eek-d(10)-kke 255 534917 91 kddk-d(9)-kke 255 534947 91 kdde-d(9)-kke 255 534977 86 eddk-d(9)-kke 255 535007 80 eeee-d(9)-kke 255 535040 86 eeee-d(9)-kkk 255 535073 87 eeek-d(9)-kke 255 535106 70 eeeee-d(8)-kke 255 535139 73 ededk-d(8)-kke 255 535172 69 edkde-d(8)-kke 255 534514 90 keke-d(9)-kek 61 534547 92 kek-d(9)-ekek 61 534580 78 ekee-d(9)-kke 61 534613 80 eke-d(9)-ekke 61 534646 79 eekk-d(9)-eke 61 534679 93 eek-d(9)-keke 61 534716 94 ekek-d(8)-keke 61 534751 86 keek-d(8)-keek 61 534786 83 ekk-d(10)-kke 61 535029 45 eeee-d(9)-kkk 61 535062 81 eeek-d(9)-kke 61 535095 57 eeeee-d(8)-kke 61 535128 58 ededk-d(8)-kke 61 535161 49 edkde-d(8)-kke 61 534526 94 keke-d(9)-kek 256 534559 95 kek-d(9)-ekek 256 534592 93 ekee-d(9)-kke 256 534625 93 eke-d(9)-ekke 256 534658 93 eekk-d(9)-eke 256 534691 96 eek-d(9)-keke 256 534728 93 ekek-d(8)-keke 256 534763 93 keek-d(8)-keek 256 534798 97 ekk-d(10)-kke 256 534828 94 edk-d(10)-kke 256 534858 92 kde-d(10)-kke 256 534888 93 eek-d(10)-kke 256 534918 95 kddk-d(9)-kke 256 534948 93 kdde-d(9)-kke 256 534978 91 eddk-d(9)-kke 256 535008 88 eeee-d(9)-kke 256 535041 87 eeee-d(9)-kkk 256 535074 90 eeek-d(9)-kke 256 535107 78 eeeee-d(8)-kke 256 535140 81 ededk-d(8)-kke 256 535173 81 edkde-d(8)-kke 256 534527 95 keke-d(9)-kek 258 534560 96 kek-d(9)-ekek 258 534593 87 ekee-d(9)-kke 258 534626 85 eke-d(9)-ekke 258 534659 90 eekk-d(9)-eke 258 534692 91 eek-d(9)-keke 258 534729 91 ekek-d(8)-keke 258 534764 91 keek-d(8)-keek 258 534799 96 ekk-d(10)-kke 258 534829 91 edk-d(10)-kke 258 534859 87 kde-d(10)-kke 258 534889 81 eek-d(10)-kke 258 534919 92 kddk-d(9)-kke 258 534949 91 kdde-d(9)-kke 258 534979 84 eddk-d(9)-kke 258 535009 78 eeee-d(9)-kke 258 535042 76 eeee-d(9)-kkk 258 535075 83 eeek-d(9)-kke 258 535108 64 eeeee-d(8)-kke 258 535141 69 ededk-d(8)-kke 258 535174 65 edkde-d(8)-kke 258 534528 94 keke-d(9)-kek 260 534561 0 kek-d(9)-ekek 260 534594 92 ekee-d(9)-kke 260 534627 90 eke-d(9)-ekke 260 534660 92 eekk-d(9)-eke 260 534693 95 eek-d(9)-keke 260 534730 93 ekek-d(8)-keke 260 534765 92 keek-d(8)-keek 260 534800 93 ekk-d(10)-kke 260 534830 93 edk-d(10)-kke 260 534860 85 kde-d(10)-kke 260 534890 91 eek-d(10)-kke 260 534920 93 kddk-d(9)-kke 260 534950 90 kdde-d(9)-kke 260 534980 88 eddk-d(9)-kke 260 535010 88 eeee-d(9)-kke 260 535043 89 eeee-d(9)-kkk 260 535076 88 eeek-d(9)-kke 260 535109 76 eeeee-d(8)-kke 260 535142 86 ededk-d(8)-kke 260 535175 71 edkde-d(8)-kke 260 534529 70 keke-d(9)-kek 261 534562 86 kek-d(9)-ekek 261 534595 56 ekee-d(9)-kke 261 534628 73 eke-d(9)-ekke 261 534661 64 eekk-d(9)-eke 261 534694 75 eek-d(9)-keke 261 534731 47 ekek-d(8)-keke 261 534766 30 keek-d(8)-keek 261 534801 83 ekk-d(10)-kke 261 534831 84 edk-d(10)-kke 261 534861 71 kde-d(10)-kke 261 534891 73 eek-d(10)-kke 261 534921 55 kddk-d(9)-kke 261 534951 61 kdde-d(9)-kke 261 534981 48 eddk-d(9)-kke 261 535011 54 eeee-d(9)-kke 261 535044 46 eeee-d(9)-kkk 261 535077 29 eeek-d(9)-kke 261 535110 19 eeeee-d(8)-kke 261 535143 15 ededk-d(8)-kke 261 535176 37 edkde-d(8)-kke 261 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

Example 22

Modified Antisense Oligonucleotides Comprising 2'-O-Methoxyethyl (2'-MOE) and 6'-(S)--CH.sub.3Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X Targeting Intronic Repeats

[0763] Additional antisense oligonucleotides were designed targeting the intronic repeat regions of Target-X.

[0764] The newly designed chimeric antisense oligonucleotides and their motifs are described in Table 37. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P.dbd.S) and are designated as "s". Nucleosides followed by "d" indicate 2'-deoxyribonucleosides. Nucleosides followed by "k" indicate 6'-(S)--CH.sub.3 bicyclic nucleosides (e.g cEt). Nucleosides followed by "e" indicate 2'-O-methoxyethyl (2'-MOE) nucleosides. "N" indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).

[0765] Each gapmer listed in Table 37 is targeted to the intronic region of human Target-X genomic sequence, designated herein as Target-X.

[0766] Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

TABLE-US-00038 TABLE 37 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X SEQ ISIS % SEQ ID Sequence (5' to 3') No inhibition CODE NO Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds 472998 90 508 7 Nds Nks Nk Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds 473327 88 30 6 Nds Nds Nes Nes Ne Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537024 74 509 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537025 79 510 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537026 76 511 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537028 37 512 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537029 45 513 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537030 67 514 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537031 59 515 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537032 9 516 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537033 65 517 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537034 71 518 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537035 68 519 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537036 74 520 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537038 69 521 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537039 67 522 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537040 68 523 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537041 76 524 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537042 77 525 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537043 70 526 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537044 82 527 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537045 69 528 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537047 35 529 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537049 62 530 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537051 62 531 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537055 16 532 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537056 25 533 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537057 49 534 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537058 49 535 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537059 53 536 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537060 73 537 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537061 70 538 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537062 69 539 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537063 68 540 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537064 71 541 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537065 67 542 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537066 68 543 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537067 71 544 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537068 86 545 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537069 82 546 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537070 87 547 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537792 36 548 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537793 35 549 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537794 35 550 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537795 33 551 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537796 49 552 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537797 54 553 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537798 68 554 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537799 72 555 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537800 69 556 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537801 82 557 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537802 72 558 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537803 72 559 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537804 67 560 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537805 74 561 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537806 70 562 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537809 60 563 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537810 71 564 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537811 69 565 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537812 80 566 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537813 74 567 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537814 54 568 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537837 70 569 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537838 76 570 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537839 76 571 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537840 80 572 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537841 81 573 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537842 75 574 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537843 70 575 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537844 73 576 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537845 59 577 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537846 51 578 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537847 52 579 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537848 41 580 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537849 44 581 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538160 69 582 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538172 24 583 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538173 23 584 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538185 68 585 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538187 69 585 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538189 81 587 6 Nds Nds Nks Nks Nk

Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538191 66 588 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538192 59 589 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538193 16 590 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538194 10 591 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538195 15 592 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538196 3 593 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538197 36 594 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538198 49 595 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538199 47 596 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538200 57 597 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538201 71 598 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538202 60 599 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538203 55 600 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538204 62 601 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538205 68 602 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538228 63 603 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538229 26 604 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538230 75 605 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538231 75 606 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538233 52 607 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538235 26 608 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538237 28 609 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538239 54 610 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538241 73 611 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538242 68 612 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538243 61 613 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538245 75 614 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538253 37 615 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538254 45 616 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538361 56 617 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538378 70 618 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538380 68 619 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538381 57 620 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540361 71 621 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540362 73 622 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540363 78 623 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540364 89 624 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540365 83 625 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540366 84 626 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540367 65 627 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540368 55 628 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540369 82 629 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540370 86 630 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540371 74 631 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540372 82 632 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540373 81 633 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540374 87 634 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540375 78 635 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540376 69 636 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540377 88 637 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540378 85 638 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540379 77 639 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540380 84 640 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540381 85 641 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540382 69 642 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540383 85 643 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540384 88 644 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540385 87 645 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540386 86 646 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540387 77 647 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540388 86 648 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540389 86 649 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540390 85 650 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540391 83 651 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540392 43 652 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540393 88 653 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540394 68 654 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540395 87 655 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540396 87 656 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540397 59 657 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540398 36 658 6 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540399 81 659 6 Nds Nds Nks Nks Nk

Example 23

High Dose Tolerability of Modified Oligonucleotides Comprising 2'-O-Methoxyethyl (2'-MOE) and 6'-(S)--CH3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X in BALB/c Mice

[0767] BALB/c mice were treated at a high dose with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

[0768] Additionally, the newly designed antisense oligonucleotides were created with the same sequences as the antisense oligonucleotides from the study described above and were also added to this screen targeting intronic repeat regions of Target-X.

[0769] The newly designed modified antisense oligonucleotides and their motifs are described in Table 38. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P.dbd.S). Nucleosides followed by "d" indicate 2'-deoxyribonucleosides. Nucleosides followed by "k" indicate 6'-(S)--CH3 bicyclic nucleoside (e.g cEt) nucleosides. Nucleosides followed by "e" indicate 2'-O-methoxyethyl (2'-MOE) nucleosides. "N" indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).

[0770] Each gapmer listed in Table 38 is targeted to the intronic region of human Target-X genomic sequence, designated herein as Target-X. "Start site" indicates the 5'-most nucleoside to which the gapmer is targeted in the human gene sequence. "Stop site" indicates the 3'-most nucleoside to which the gapmer is targeted human gene sequence.

TABLE-US-00039 TABLE 38 Modified antisense oligonucleotides targeted to Target-X SEQ ISIS SEQ ID Sequence (5' to 3') No CODE NO Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537721 509 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537738 524 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537759 539 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537761 541 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537763 543 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537850 548 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537858 556 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537864 562 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537869 565 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537872 568 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537897 571 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540118 582 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540138 602 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540139 603 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540148 612 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540153 617 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540155 619 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540162 624 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540164 626 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540168 630 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540172 634 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540175 637 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540176 638 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540178 640 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540179 641 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540181 643 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540182 644 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540183 645 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540184 646 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540186 648 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540187 649 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540188 650 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540191 653 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540193 655 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540194 656 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544811 547 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544812 545 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544813 527 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544814 557 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544815 546 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544816 573 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544817 572 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544818 566 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544819 510 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544820 525 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544821 567 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544826 537 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544827 538 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544828 539 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544829 540 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544830 541 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545471 542 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545472 543 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545473 544 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545474 558 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545475 559 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545476 560 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545477 561 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545478 562 6 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545479 556 6 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537727 514 6

Treatment

[0771] Male BALB/c mice were injected subcutaneously with a single dose of 200 mg/kg of ISIS 422142, ISIS 457851, ISIS 473294, ISIS 473295, ISIS 473327, ISIS 484714, ISIS 515334, ISIS 515338, ISIS 515354, ISIS 515366, ISIS 515380, ISIS 515381, ISIS 515382, ISIS 515384, ISIS 515386, ISIS 515387, ISIS 515388, ISIS 515406, ISIS 515407, ISIS 515408, ISIS 515422, ISIS 515423, ISIS 515424, ISIS 515532, ISIS 515533, ISIS 515534, ISIS 515538, ISIS 515539, ISIS 515558, ISIS 515656, ISIS 515575, ISIS 515926, ISIS 515944, ISIS 515945, ISIS 515948, ISIS 515949, ISIS 515951, ISIS 515952, ISSI 516003, ISIS 516055, ISIS 516057, ISIS 516060, ISIS 516062, ISIS 529126, ISIS 529146, ISIS 529166, ISIS 529170, ISIS 529172, ISIS 529173, ISIS 529174, ISIS 529175, ISSI 529176, ISIS 529182, ISIS 529183, ISIS 529186, ISIS 529282, ISIS 529304, ISIS 529306, ISIS 529360, ISIS 529450, ISIS 529459, ISIS 529460, ISIS 529461, ISIS 529547, ISIS 529550, ISIS 529551, ISIS 529553, ISIS 529557, ISIS 529562, ISIS 529563, ISIS 529564, ISIS 529565, ISIS 529575, ISIS 529582, ISIS 529589, ISIS 529607, ISIS 529614, ISIS 529632, ISIS 529650, ISIS 529651, ISIS 529657, ISIS 529663, ISIS 529725, ISIS 529745, ISIS 529765, ISIS 529785, ISIS 529804, ISIS 529818, ISIS 529823, ISIS 529854, ISIS 534528, ISIS 534534, ISIS 534594, ISIS 534660, ISIS 534663, ISIS 534664, ISIS 534676, ISIS 534677, ISIS 537679, ISIS 537683, ISIS 534693, ISIS 534701, ISIS 534716, ISIS 534730, ISIS 534765, ISIS 534795, ISIS 534796, ISIS 534797, ISIS 534798, ISIS 534799, ISIS 534800, ISIS 534802, ISIS 534806, ISSI 534830, ISIS 534838, ISIS 534888, ISIS 534890, ISIS 534898, ISIS 534911, ISIS 534920, ISIS 534926, ISIS 534937, ISIS 534950, ISSI 534956, ISIS 534980, ISIS 534986, ISIS 535010, ISIS 535043, ISIS 535049, ISIS 535076, ISIS 535082, ISSI 535142, ISIS 537024, ISIS 537030, ISIS 537041, ISIS 537062, ISIS 537064, ISIS 537066, ISIS 537721, ISIS 537727, ISIS 537738, ISIS 537759, ISIS 537761, ISIS 537763, ISIS 537792, ISIS 537800, ISIS 537806, ISIS 537811, ISIS 537814, ISIS 537839, ISIS 537850, ISSI 537858, ISIS 537864, ISIS 537869, ISIS 537872, ISIS 537897, ISIS 538160, ISIS 538196, ISIS 538205, ISIS 538228, ISIS 538242, ISIS 538361, ISIS 538380, ISIS 540118, ISIS 540138, ISIS 540139, ISIS 540148, ISIS 540153, ISIS 540155, ISIS 540162, ISIS 540164, ISIS 540168, ISIS 540172, ISIS 540175, ISIS 540176, ISIS 540178, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540183, ISIS 540184, ISIS 540186, ISIS 540187, ISIS 540188, ISIS 540191, ISIS 540193, ISIS 540194, ISIS 544811, ISIS 544812, ISIS 544813, ISIS 544814, ISIS 544815, ISIS 544816, ISIS 544817, ISIS 544818, ISIS 544819, ISIS 544820, ISIS 544821, ISIS 544826, ISIS 544827, ISIS 544828, ISIS 544829, ISIS 544830, ISIS 545471, ISIS 545472, ISIS 545473, ISIS 545474, ISIS 545475, ISIS 545476, ISIS 545477, ISIS 545478, and ISIS 545479. One set of male BALB/c mice was injected with a single dose of PBS. Mice were euthanized 96 hours later, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

[0772] To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).

[0773] ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 529166, ISIS 529170, ISIS 529175, ISIS 529176, ISIS 529186, ISIS 529282, ISIS 529360, ISIS 529450, ISIS 529459, ISIS 529460, ISIS 529547, ISIS 529549, ISIS 529551, ISIS 529553, ISIS 529557, ISIS 529562, ISIS 529575, ISIS 529582, ISIS 529607, ISIS 529589, ISIS 529632, ISIS 529657, ISIS 529725, ISIS 529745, ISIS 529785, ISIS 529799, ISIS 529804, ISIS 529818, ISIS 529823, ISIS 534950, ISIS 534980, ISIS 535010, ISIS 537030, ISIS 537041, ISIS 537062, ISIS 537064, ISIS 537066, ISIS 537759, ISIS 537792, ISIS 537800, ISIS 537839, ISIS 538228, ISIS 473294, ISIS 473295, ISIS 484714, ISIS 515338, ISIS 515366, ISIS 515380, ISIS 515381, ISIS 515387, ISIS 515408, ISIS 515423, ISIS 515424, ISIS 515532, ISIS 515534, ISIS 515538, ISIS 515539, ISIS 515558, ISIS 515575, ISIS 515926, ISIS 515944, ISIS 515945, ISIS 515951, ISIS 515952, ISIS 529126, ISIS 529765, ISIS 534528, ISIS 534534, ISIS 534594, ISIS 534663, ISIS 534676, ISIS 534677, ISIS 534679, ISIS 534683, ISIS 534693, ISIS 534701, ISIS 534716, ISIS 534730, ISIS 534806, ISIS 534830, ISIS 534838, ISIS 534890, ISIS 534898, ISIS 534911, ISIS 534937, ISIS 534956, ISIS 534986, ISIS 535043, ISIS 535049, ISIS 535076, ISIS 535082, ISIS 535142, ISIS 538160, ISIS 538242, ISIS 538361, ISIS 538380, ISIS 534795, ISIS 534796, ISIS 534797, ISIS 540162, ISIS 540164, ISIS 540168, ISIS 540172, ISIS 540175, ISIS 540176, ISIS 540178, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540183, ISIS 540184, ISIS 540186, ISIS 540187, ISIS 540188, ISIS 540191, ISIS 540193, ISIS 540194, ISIS 544813, ISIS 544814, ISIS 544816, ISIS 544826, ISIS 544827, ISIS 544828, ISIS 544829, ISIS 545473, and ISIS 545474 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 529173, ISIS 529854, ISIS 529614, ISIS 515386, ISIS 515388, ISIS 515949, ISIS 544817, and ISIS 545479 were considered tolerable in terms of liver function.

Example 24

Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats

[0774] Sprague-Dawley rats are a multipurpose model used for safety and efficacy evaluations. The rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

[0775] Six-eight week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Teklad normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS 473286, ISIS 473547, ISIS 473567, ISIS 473589, ISIS 473630, ISIS 484559, ISIS 515636, ISIS 515640, ISIS 515641, ISIS 515655, ISIS 515657, ISIS 516046, ISIS 516048, ISIS 516051, ISIS 516052, and ISIS 516062. A group of four Sprague-Dawley rats was injected subcutaneously twice a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

[0776] To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured. Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer.

[0777] ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 473286, ISIS 473547, ISSI 473589, ISIS 473630, ISIS 484559, ISIS 515636, ISIS 515640, ISIS 515655, ISIS 516046, and ISIS 516051 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 473567, ISIS 515641, ISIS 515657, ISIS 516048, and ISIS 516051 were considered tolerable in terms of liver function.

Example 25

Tolerability of Chimeric Antisense Oligonucleotides Comprising 2'-O-Methoxyethyl (2'-MOE) Modifications Targeting Human Target-X in Sprague-Dawley Rats

[0778] Sprague-Dawley rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

[0779] Six-eight week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 407936, ISIS 416507, ISIS 416508, ISIS 490208, ISIS 490279, ISIS 490323, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513419, ISIS 513446, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513504, ISIS 513507, and ISIS 513508. A group of four Sprague-Dawley rats was injected subcutaneously twice a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

[0780] To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer.

[0781] ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 416507, ISIS 490208, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513446, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513504, and ISIS 513508 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 407936, ISIS 416508, ISIS 490279, and ISIS 513507 were considered tolerable in terms of liver function.

Example 26

Tolerability of Chimeric Antisense Oligonucleotides Comprising 2'-O-Methoxyethyl (2'-MOE) Modifications Targeting Human Target-X in CD-1 Mice

[0782] CD-1 mice are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

[0783] Groups of 3 male CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 473244, ISIS 473295, ISIS 484714, ISIS 515386, ISIS 515424, ISIS 515534, ISIS 515558, ISIS 515926, ISIS 515949, ISIS 515951, ISIS 515952, ISIS 529126, ISIS 529166, ISIS 529173, ISIS 529186, ISIS 529360, ISIS 529461, ISIS 529553, ISIS 529564, ISIS 529582, ISIS 529614, ISIS 529725, ISIS 529745, ISIS 529765, ISIS 529785, ISIS 529799, ISIS 529818, ISIS 529823, ISIS 534528, ISIS 534594, and ISIS 534664. One group of male CD-1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

[0784] To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).

[0785] ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 473295, ISIS 473714, ISIS 515558, ISIS 515926, 515951, ISIS 515952, ISIS 529126, ISIS 529166, 529564, ISIS 529582, ISIS 529614, ISIS 529725, ISIS 529765, ISIS 529799, ISIS 529823, and ISIS 534594 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 515424, ISIS 515534, ISIS 515926, ISIS 529785, and ISIS 534664 were considered tolerable in terms of liver function.

Example 27

Tolerability of Chimeric Antisense Oligonucleotides Comprising 2'-O-Methoxyethyl (2'-MOE) Modifications Targeting Human Target-X in CD-1 Mice

[0786] CD-1 mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

[0787] Groups of 3 male CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 100 mg/kg of ISIS 490208, ISIS 490279, ISIS 490323, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513419, ISIS 513446, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513504, ISIS 513507, and ISIS 513508. Groups of 3 male CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 100 mg/kg of ISIS 407936, ISIS 416507, and ISIS 416508, which are gapmers described in a previous publication. One group of male CD-1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

[0788] To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).

[0789] ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 407936, ISIS 416507, ISIS 490279, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513446, ISIS 513454, ISIS 513456, and ISIS 513504 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 490208, ISIS 513455, ISIS 513507, and ISIS 513508 were considered tolerable in terms of liver function.

Example 28

Efficacy of Modified Oligonucleotides Comprising 2'-O-Methoxyethyl (2'-MOE) and 6'-(S)--CH.sub.3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X in Transgenic Mice

[0790] Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.

Treatment

[0791] Groups of 2-3 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 5 mg/kg/week of ISIS 473244, ISIS 473295, ISIS 484714, ISIS 515926, ISIS 515951, ISIS 515952, ISIS 516062, ISIS 529126, ISIS 529553, ISIS 529745, ISIS 529799, ISIS 534664, ISIS 534826, ISIS 540168, ISIS 540175, ISIS 544826, ISIS 544827, ISIS 544828, and ISIS 544829. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Protein Analysis

[0792] Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 39, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control. `n.d.` indicates that the value for that particular oligonucleotide was not measured.

TABLE-US-00040 TABLE 39 Percent inhibition of Target-X plasma protein levels in transgenic mice ISIS No % inhibition 473244 2 473295 13 484714 19 515926 11 515951 13 515952 0 516062 62 529126 0 529553 0 529745 22 529799 26 534664 32 534826 n.d. 540168 94 540175 98 544813 0 544826 23 544827 60 544828 33 544829 53

Example 29

Efficacy of Modified Oligonucleotides Comprising 2'-Methoxyethyl (2'-MOE) and 6'-(S)--CH.sub.3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X in Transgenic Mice

[0793] Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.

Treatment

[0794] Groups of 2-3 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 1 mg/kg/week of ISIS 407936, ISIS 490197, ISIS 490275, ISIS 490278, ISIS 490279, ISIS 490323, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513446, ISIS 513447, ISIS 513504, ISIS 516062, ISIS 529166, ISIS 529173, ISIS 529360, ISIS 529725, ISIS 534557, ISIS 534594, ISIS 534664, ISIS 534688, ISIS 534689, ISIS 534915, ISIS 534916, ISIS 534917, and ISIS 534980. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Protein Analysis

[0795] Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 40, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control.

TABLE-US-00041 TABLE 40 Percent inhibition of Target-X plasm protein levels in transgenic mice ISIS No % inhibition 407936 28 490197 50 490275 21 490278 20 490279 59 490323 54 490368 22 490396 31 490803 30 491122 51 513446 29 513447 44 513504 45 516062 75 529166 37 529173 64 529360 43 529725 53 534557 76 534594 40 534664 14 534687 12 534688 48 534689 25 534915 40 534916 45 534917 66 534980 62

Example 30

Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats

[0796] Sprague-Dawley rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

[0797] Six-eight week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Teklad normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 4 weeks with ISIS 515380, ISIS 515381, ISIS 515387, ISIS 529175, ISIS 529176, ISIS 529575, ISIS 529804, and ISIS 537064. Doses 1, 5, 6, 7, and 8 were 25 mg/kg; dose 2 was 75 mg/kg; doses 3 and 4 were 50 mg/kg. One group of four Sprague-Dawley rats was injected subcutaneously twice a week for 4 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

[0798] To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured. Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer.

[0799] ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused increase in the levels within three times the upper limit of normal levels of transaminases were deemed very tolerable. ISIS oligonucleotides that caused increase in the levels of transaminases between three times and seven times the upper limit of normal levels were deemed tolerable. Based on these criteria, ISIS 515380, ISIS 515387, ISIS 529175, ISIS 529176, ISIS 529804, and ISIS 537064 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 515381 was considered tolerable in terms of liver function.

Example 31

Efficacy of Antisense Oligonucleotides Targeting Human Target-X in Transgenic Mice

[0800] Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.

Treatment

[0801] Two groups of 3 male and female transgenic mice were injected subcutaneously twice a week for 2 weeks with 0.5 mg/kg/week or 1.5 mg/kg/week of ISIS 407935 and ISIS 513455. Another group of mice was subcutaneously twice a week for 2 weeks with 0.6 mg/kg/week or 2.0 mg/kg/week of ISIS 473286. Another 16 groups of mice were subcutaneously twice a week for 2 weeks with 0.1 mg/kg/week or 0.3 mg/kg/week of ISIS 473589, ISIS 515380, ISIS 515423, ISIS 529804, ISIS 534676, ISIS 534796, ISIS 540162, ISIS 540164, ISIS 540175, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540186, ISIS 540191, ISIS 540193, ISIS 544827, or ISIS 545474. Another 3 groups of mice were injected subcutaneously twice a week for 2 weeks with 0.3 mg/kg/week of ISIS 516062, ISIS 534528 or ISIS 534693. One group of mice was injected subcutaneously twice a week for 2 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Protein Analysis

[0802] Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 41, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control.

TABLE-US-00042 TABLE 41 Percent inhibition of Target-X plasma protein levels in transgenic mice Dose % ISIS No (mg/kg/wk) inhibition 407935 1.5 65 0.5 31 513455 1.5 64 0.5 52 473286 2 67 0.6 11 473589 0.3 42 0.1 12 515380 0.3 64 0.1 32 515423 0.3 72 0.1 37 529804 0.3 36 0.1 24 534676 0.3 31 0.1 18 534796 0.3 54 0.1 43 540162 0.3 84 0.1 42 540164 0.3 25 0.1 17 540175 0.3 90 0.1 55 540179 0.3 29 0.1 24 540181 0.3 53 0.1 0 540182 0.3 78 0.1 21 540186 0.3 72 0.1 46 540191 0.3 62 0.1 35 540193 0.3 74 0.1 46 544827 0.3 28 0.1 19 545474 0.3 59 0.1 0 516062 0.3 33 534528 0.3 41 534693 0.3 34

Example 32

Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats

[0803] Sprague-Dawley rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

[0804] Five-six week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Teklad normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 4 weeks with 50 mg/kg of ISIS 515423, ISIS 515424, ISIS 515640, ISIS 534676, ISIS 534796, ISIS 534797, ISIS 540162, ISIS 540164, ISIS 540172, ISIS 540175, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540183, ISIS 540186, ISIS 540191, and ISIS 545474. A group of four Sprague-Dawley rats was injected subcutaneously twice a week for 4 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

[0805] To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured. Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer.

[0806] ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 540164, ISIS 540172, and ISIS 540175 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 534676, ISIS 534796, ISIS 534797, ISIS 540162, and ISIS 540179 were considered tolerable in terms of liver function.

Example 33

Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells

[0807] Antisense oligonucleotides selected from the studies described above were tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.05 .mu.M, 0.15 .mu.M, 0.44 .mu.M, 1.33 .mu.M, and 4.00 .mu.M concentrations of antisense oligonucleotide, as specified in Table 42. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

[0808] The half maximal inhibitory concentration (IC.sub.50) of each oligonucleotide is also presented in Table 42. As illustrated in Table 42, Target-X mRNA levels were reduced in a dose-dependent manner in several of the antisense oligonucleotide treated cells.

TABLE-US-00043 TABLE 42 Dose-dependent antisense inhibition of human Target-X in Hep3B cells using electroporation 0.05 0.15 0.44 1.33 4.00 IC.sub.50 ISIS No .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 473286 0 1 13 12 15 >4.0 457851 23 32 57 80 93 0.3 473286 3 20 43 71 88 0.5 473286 15 26 24 28 36 >4.0 473286 6 3 10 26 29 >4.0 473327 14 28 35 67 90 0.5 473589 29 53 76 89 95 0.1 515380 44 72 85 93 95 <0.05 515423 43 64 87 95 98 <0.05 515424 38 55 85 92 97 0.1 515636 21 33 74 82 93 0.2 516046 29 23 29 48 78 0.9 516048 35 24 41 67 87 0.4 516052 18 6 48 63 80 0.6 516062 24 14 21 47 68 1.6 529166 16 47 75 87 94 0.2 529173 14 49 77 91 96 0.2 529175 30 69 88 93 96 0.1 529176 34 63 85 93 96 0.1 529360 35 53 74 91 93 0.1 529725 53 69 85 92 95 <0.05 529804 37 41 71 90 94 0.1 534528 50 68 78 93 97 <0.05 534557 48 78 90 94 95 <0.05 534594 39 47 76 87 94 0.1 534676 29 20 40 64 87 0.5 534687 41 37 56 80 93 0.2 534688 16 56 88 94 96 0.1 534689 21 59 82 94 95 0.1 534693 18 58 81 93 95 0.1 534795 19 43 68 90 94 0.2 534796 25 59 80 93 96 0.1 534890 31 55 77 90 96 0.1 534898 22 61 80 94 97 0.1 534915 19 26 51 77 94 0.3 534916 20 36 66 86 93 0.2 534917 34 53 82 89 94 0.1 540162 40 64 84 90 92 <0.05 540164 34 60 83 91 92 0.1 540168 51 79 90 92 94 <0.05 540172 40 66 80 88 92 <0.05 540175 30 61 80 88 91 0.1 540176 7 17 50 75 85 0.5 540179 11 22 25 16 19 >4.0 540181 19 46 72 86 91 0.2 540182 16 66 83 86 92 0.1 540183 39 74 87 92 93 <0.05 540186 31 69 85 91 94 0.1 540191 38 54 80 88 91 0.1 540193 57 67 84 94 97 <0.05 540194 30 45 62 77 91 0.2 544827 37 42 67 82 96 0.1 544829 26 41 42 71 93 0.3 545473 28 27 49 80 97 0.3 545474 23 27 55 84 96 0.3

Example 34

Tolerability of Antisense Oligonucleotides Targeting Human Target-X in CD-1 Mice

[0809] CD-1 mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

[0810] Two groups of 4 male 6-8 week old CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 407935 and ISIS 490279. Another seven groups of 4 male 6-8 week old CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, and ISIS 540191. One group of male CD-1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

[0811] To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 43. Treatment with the newly designed antisense oligonucleotides were more tolerable compared to treatment with ISIS 407935 (disclosed in an earlier publication), which caused elevation of ALT levels greater than seven times the upper limit of normal (ULN).

TABLE-US-00044 TABLE 43 Effect of antisense oligonucleotide treatment on liver function in CD-1 mice Dose AST BUN Bilirubin Motif (mg/kg/wk) ALT(IU/L) (IU/L) (mg/dL) (mg/dL) PBS -- -- 37 47 28 0.2 407935 e5-d(10)-e5 100 373 217 24 0.2 490279 kdkdk-d(9)-ee 100 96 82 24 0.2 473589 e5-d(10)-e5 50 93 116 22 0.2 529804 k-d(10)-kekee 50 54 74 27 0.2 534796 ekk-d(10)-kke 50 60 63 27 0.2 540162 eek-d(10)-kke 50 43 55 29 0.2 540175 eek-d(10)-kke 50 113 78 24 0.3 540182 eek-d(10)-kke 50 147 95 26 0.1 540191 eek-d(10)-kke 50 79 88 28 0.2 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

Body and Organ Weights

[0812] Body weights, as well as liver, heart, lungs, spleen and kidney weights were measured at the end of the study, and are presented in Table 44. Several of the ISIS oligonucleotides did not cause any changes in organ weights outside the expected range and were therefore deemed tolerable in terms of organ weights.

TABLE-US-00045 TABLE 44 Body and organ weights (grams) of CD-1 mice Dose (mg/ Body Liv- Kid- Motif kg/wk) weight er Spleen ney PBS -- -- 42 2.2 0.12 0.64 407935 e5-d(10)-e5 100 40 2.6 0.20 0.62 490279 kdkdk-d(9)-ee 100 42 2.8 0.17 0.61 473589 e5-d(10)-e5 50 41 2.5 0.16 0.67 529804 k-d(10)-kekee 50 40 2.3 0.14 0.62 534796 ekk-d(10)-kke 50 37 2.6 0.15 0.51 540162 eek-d(10)-kke 50 42 2.4 0.15 0.60 540175 eek-d(10)-kke 50 39 2.2 0.11 0.62 540182 eek-d(10)-kke 50 41 2.6 0.16 0.61 540191 eek-d(10)-kke 50 40 2.4 0.13 0.60 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

Example 35

Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats

[0813] Sprague-Dawley rats were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

[0814] Two groups of 4 male 7-8 week old Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 407935 and ISIS 490279. Another seven groups of 4 male 6-8 week old Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, and ISIS 540191. One group of male Sprague-Dawley rats was injected subcutaneously twice a week for 6 weeks with PBS. The rats were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

[0815] To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 45. Treatment with the all antisense oligonucleotides was tolerable in terms of plasma chemistry markers in this model.

TABLE-US-00046 TABLE 45 Effect of antisense oligonucleotide treatment on liver function in Sprague-Dawley rats Dose AST BUN Bilirubin Motif (mg/kg/wk) ALT(IU/L) (IU/L) (mg/dL) (mg/dL) PBS -- -- 71 83 19 0.2 407935 e5-d(10)-e5 100 74 96 22 0.2 490279 kdkdk-d(9)-ee 100 96 181 22 0.4 473589 e5-d(10)-e5 50 57 73 21 0.2 529804 k-d(10)-kekee 50 54 78 21 0.2 534796 ekk-d(10)-kke 50 68 98 22 0.2 540162 eek-d(10)-kke 50 96 82 21 0.1 540175 eek-d(10)-kke 50 55 73 18 0.2 540182 eek-d(10)-kke 50 45 87 21 0.2 540191 eek-d(10)-kke 50 77 104 21 0.2 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

Body and Organ Weights

[0816] Body weights, as well as liver, heart, lungs, spleen and kidney weights were measured at the end of the study, and are presented in Table 46. Treatment with all the antisense oligonucleotides was tolerable in terms of body and organ weights in this model.

TABLE-US-00047 TABLE 46 Body and organ weights (grams) of Sprague-Dawley rats Dose (mg/ Body Liv- Kid- Motif kg/wk) weight er Spleen ney PBS -- -- 443 16 0.8 3.5 ISIS 407935 e5-d(10)-e5 100 337 14 1.8 3.2 ISIS 490279 kdkdk-d(9)-ee 100 365 18 2.2 2.9 ISIS 473589 e5-d(10)-e5 50 432 18 1.3 3.3 ISIS 529804 k-d(10)-kekee 50 429 18 2.2 3.4 ISIS 534796 ekk-d(10)-kke 50 434 15 1.4 3.3 ISIS 540162 eek-d(10)-kke 50 446 18 1.1 3.3 ISIS 540175 eek-d(10)-kke 50 467 16 1.0 3.5 ISIS 540182 eek-d(10)-kke 50 447 22 2.5 4.5 ISIS 540191 eek-d(10)-kke 50 471 21 1.4 3.9 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

Example 36

Dose-Dependent Antisense Inhibition of Human Target-X in Cynomolgos Monkey Primary Hepatocytes

[0817] Antisense oligonucleotides selected from the studies described above were tested at various doses in cynomolgus monkey primary hepatocytes. Cells were plated at a density of 35,000 cells per well and transfected using electroporation with 0.009 .mu.M, 0.03 .mu.M, 0.08 .mu.M, 0.25 .mu.M, 0.74 .mu.M, 2.22 .mu.M, 6.67 .mu.M, and 20.00 .mu.M concentrations of antisense oligonucleotide, as specified in Table 47. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 47, Target-X mRNA levels were reduced in a dose-dependent manner with some of the antisense oligonucleotides that are cross-reactive with the rhesus monkey genomic sequence.

TABLE-US-00048 TABLE 47 Dose-dependent antisense inhibition of Target-X in cynomolgous monkey primary hepatocytes using electroporation 0.009 0.03 0.08 0.25 0.74 2.22 6.67 20.00 ISIS No .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M 407935 10 18 15 29 56 73 82 88 490279 19 12 13 0 6 18 27 22 473589 5 10 19 42 64 76 88 92 529804 10 3 23 25 57 80 86 91 534796 0 28 23 49 71 81 87 90 540162 9 14 9 6 13 13 11 31 540175 0 4 12 9 10 16 12 22 540182 0 7 0 6 36 12 10 0 540191 6 7 0 0 0 0 21 42

Example 37

Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells

[0818] Antisense oligonucleotides from the study described above were also tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.009 .mu.M, 0.03 .mu.M, 0.08 .mu.M, 0.25 .mu.M, 0.74 .mu.M, 2.22 .mu.M, 6.67 .mu.M, and 20.00 .mu.M concentrations of antisense oligonucleotide, as specified in Table 48. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM.. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 48, Target-X mRNA levels were reduced in a dose-dependent manner with several of the antisense oligonucleotides.

TABLE-US-00049 TABLE 48 Dose-dependent antisense inhibition of Target-X in Hep3B cells using electroporation 0.009 0.03 0.08 0.25 0.74 2.22 6.67 20.00 IC.sub.50 ISIS No .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 407935 3 9 11 35 64 83 87 93 4.5 473244 20 33 50 69 77 89 7 14 0.9 473589 0 14 23 44 74 88 90 94 2.7 490279 0 5 7 15 25 61 76 78 11.6 515533 0 12 21 36 63 78 88 94 3.6 515952 0 12 27 57 76 89 93 94 2.2 516066 6 0 12 26 52 70 81 86 6.0 529459 0 4 24 40 61 78 88 94 3.5 529553 9 7 17 40 58 74 87 93 4.6 529804 0 3 34 64 83 89 93 95 2.0 534796 8 18 43 67 82 89 95 96 1.4 537806 6 11 5 20 37 69 79 86 7.1 540162 18 33 63 75 87 91 91 92 0.7 540175 10 25 55 76 86 89 89 93 1.0 540182 13 36 61 75 84 88 90 93 0.7 540191 3 12 28 61 79 80 88 94 2.2

Example 38

Efficacy of Antisense Oligonucleotides Targeting Human Target-X in Transgenic Mice

[0819] Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.

Treatment

[0820] Eight groups of 3 transgenic mice each were injected subcutaneously twice a week for 3 weeks with 20 mg/kg/week, 10 mg/kg/week, 5 mg/kg/week, or 2.5 mg/kg/week of ISIS 407935 or ISIS 490279. Another 24 groups of 3 transgenic mice each were subcutaneously twice a week for 3 weeks with 5 mg/kg/week, 2.5 mg/kg/week, 1.25 mg/kg/week, or 0.625 mg/kg/week of ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, or ISIS 540191. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

RNA Analysis

[0821] RNA was extracted from plasma for real-time PCR analysis of Target-X, using primer probe set RTS2927. The mRNA levels were normalized using RIBOGREEN.RTM.. As shown in Table 49, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control. Results are presented as percent inhibition of Target-X, relative to control. Treatment with newly designed 2'-MOE gapmer, ISIS 490279, caused greater reduction in human Target-X mRNA levels than treatment with ISIS 407935, the 2'-MOE gapmer from the earlier publication. Treatment with several of the newly designed oligonucleotides also caused greater reduction in human Target-X mRNA levels than treatment with ISIS 407935.

TABLE-US-00050 TABLE 49 Percent inhibition of Target-X mRNA in transgenic mice Dose % ISIS No Motif (mg/kg/wk) inhibition 407935 e5-d(10)-e5 20.0 85 10.0 57 5.0 45 2.5 28 490279 kdkdk-d(9)-ee 20.0 88 10.0 70 5.0 51 2.5 33 473589 e5-d(10)-e5 5.00 80 2.50 62 1.25 44 0.625 25 529804 k-d(10)-kekee 5.00 55 2.50 41 1.25 0 0.625 1 534796 ekk-d(10)-kke 5.00 56 2.50 41 1.25 5 0.625 0 540162 eek-d(10)-kke 5.00 97 2.50 92 1.25 69 0.625 78 540175 eek-d(10)-kke 5.00 95 2.50 85 1.25 65 0.625 55 540182 eek-d(10)-kke 5.00 97 2.50 83 1.25 54 0.625 10 540191 eek-d(10)-kke 5.00 91 2.50 74 1.25 58 0.625 34 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

Protein Analysis

[0822] Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). As shown in Table 50, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control. Results are presented as percent inhibition of Target-X, relative to control.

TABLE-US-00051 TABLE 50 Percent inhibition of Target-X plasm protein levels in transgenic mice Dose % ISIS No Motif (mg/kg/wk) inhibition 407935 e5-d(10)-e5 20 65 10 47 5 0 2.5 3 490279 kdkdk-d(9)-ee 20 91 10 75 5 31 2.5 23 473589 e5-d(10)-e5 5 78 2.5 40 1.25 6 0.625 0 529804 k-d(10)-kekee 5 50 2.5 36 1.25 0 0.625 8 534796 ekk-d(10)-kke 5 45 2.5 26 1.25 0 0.625 8 540162 eek-d(10)-kke 5 98 2.5 96 1.25 78 0.625 74 540175 eek-d(10)-kke 5 93 2.5 83 1.25 49 0.625 24 540182 eek-d(10)-kke 5 97 2.5 71 1.25 50 0.625 0 540191 eek-d(10)-kke 5 97 2.5 74 1.25 46 0.625 25 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

Example 39

Effect of ISIS Antisense Oligonucleotides Targeting Human Target-X in Cynomolgus Monkeys

[0823] Cynomolgus monkeys were treated with ISIS antisense oligonucleotides selected from studies described above, including ISIS 407935, ISIS 490279, ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, and ISIS 540191. Antisense oligonucleotide efficacy was evaluated. ISIS 407935, from the earlier publication, was included in the study for comparison.

Treatment

[0824] Prior to the study, the monkeys were kept in quarantine for at least a 30-day period, during which the animals were observed daily for general health. Standard panels of serum chemistry and hematology, examination of fecal samples for ova and parasites, and a tuberculosis test were conducted immediately after the animals' arrival to the quarantine area. The monkeys were 2-4 years old at the start of treatment and weighed between 2 and 4 kg. Ten groups of four randomly assigned male cynomolgus monkeys each were injected subcutaneously with ISIS oligonucleotide or PBS using a stainless steel dosing needle and syringe of appropriate size into one of 4 sites on the back of the monkeys; each site used in clock-wise rotation per dose administered. Nine groups of monkeys were dosed four times a week for the first week (days 1, 3, 5, and 7) as loading doses, and subsequently once a week for weeks 2-12, with 35 mg/kg of ISIS 407935, ISIS 490279, ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, or ISIS 540191. A control group of cynomolgus monkeys was injected with PBS subcutaneously thrice four times a week for the first week (days 1, 3, 5, and 7), and subsequently once a week for weeks 2-12. The protocols described in the Example were approved by the Institutional Animal Care and Use Committee (IACUC).

Hepatic Target Reduction

RNA Analysis

[0825] On day 86, RNA was extracted from liver tissue for real-time PCR analysis of Target-X using primer probe set RTS2927. Results are presented as percent inhibition of Target-X mRNA, relative to PBS control, normalized to RIBOGREEN.RTM. or to the house keeping gene, GAPDH. As shown in Table 52, treatment with ISIS antisense oligonucleotides resulted in reduction of Target-X mRNA in comparison to the PBS control.

TABLE-US-00052 TABLE 52 Percent Inhibition of cynomolgous monkey Target-X mRNA in the cynomolgus monkey liver relative to the PBS control ISIS No Motif RTS2927/Ribogreen RTS2927/GAPDH 407935 e5-d(10)-e5 90 90 490279 kdkdk-d(9)-ee 72 66 473589 e5-d(10)-e5 96 96 529804 k-d(10)-kekee 90 87 534796 ekk-d(10)-kke 80 78 540162 eek-d(10)-kke 66 58 540175 eek-d(10)-kke 68 66 540182 eek-d(10)-kke 0 0 540191 eek-d(10)-kke 34 14 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

Protein Levels and Activity Analysis

[0826] Plasma Target-X levels were measured prior to dosing, and on day 3, day 5, day 7, day 16, day 30, day 44, day 65, and day 86 of treatment. Target-X activity was measured using Target-X deficient plasma. Approximately 1.5 mL of blood was collected from all available study animals into tubes containing 3.2% sodium citrate. The samples were placed on ice immediately after collection. Collected blood samples were processed to platelet poor plasma and the tubes were centrifuged at 3,000 rpm for 10 min at 4.degree. C. to obtain plasma.

[0827] Protein levels of Target-X were measured by a Target-X elisa kit (purchased from Hyphen BioMed). The results are presented in Table 53.

TABLE-US-00053 TABLE 53 Plasma Target-X protein levels (% reduction compared to the baseline) in the cynomolgus monkey plasma Day Day Day Day Day Day Day Day ISIS No 3 5 7 16 30 44 65 86 407935 21 62 69 82 84 85 84 90 490279 0 29 35 30 38 45 51 58 473589 12 67 85 97 98 98 98 98 529804 19 65 76 87 88 89 90 90 534796 1 46 54 64 64 67 66 70 540162 0 24 26 37 45 49 49 50 540175 0 28 36 38 47 52 55 55 540182 0 17 8 0 0 0 5 0 540191 0 12 4 0 0 4 9 10

Example 40

Single Nucleotide Polymorphisms (SNPs) in the Huntingtin (HTT) Gene Sequence

[0828] SNP positions (identified by Hayden et al, WO/2009/135322) associated with the HTT gene were mapped to the HTT genomic sequence, designated herein as SEQ ID NO: 1 (NT.sub.--006081.18 truncated from nucleotides 1566000 to 1768000). Table 56 provides SNP positions associated with the HTT gene. Table 56 provides a reference SNP ID number from the Entrez SNP database at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/sites/entrez?db=snp), incorporated herein by reference. Table 56 furnishes further details on each SNP. The `Reference SNP ID number` or `RS number` is the number designated to each SNP from the Entrez SNP database at NCBI, incorporated herein by reference. `SNP position` refers to the nucleotide position of the SNP on SEQ ID NO: 1. `Polymorphism` indicates the nucleotide variants at that SNP position. `Major allele` indicates the nucleotide associated with the major allele, or the nucleotide present in a statistically significant proportion of individuals in the human population. `Minor allele` indicates the nucleotide associated with the minor allele, or the nucleotide present in a relatively small proportion of individuals in the human population.

TABLE-US-00054 TABLE 56 Single Nuclear Polymorphisms (SNPs) and their positions on SEQ ID NO: 1 SNP Major Minor RS No. position Polymorphism allele allele rs2857936 1963 C/T C T rs12506200 3707 A/G G A rs762855 14449 A/G G A rs3856973 19826 G/A G A rs2285086 28912 G/A A G rs7659144 37974 C/G C G rs16843804 44043 C/T C T rs2024115 44221 G/A A G rs10015979 49095 A/G A G rs7691627 51063 A/G G A rs2798235 54485 G/A G A rs4690072 62160 G/T T G rs6446723 66466 C/T T C rs363081 73280 G/A G A rs363080 73564 T/C C T rs363075 77327 G/A G A rs363064 81063 T/C C T rs3025849 83420 A/G A G rs6855981 87929 A/G G A rs363102 88669 G/A A G rs11731237 91466 C/T C T rs4690073 99803 A/G G A rs363144 100948 T/G T G rs3025838 101099 C/T C T rs34315806 101687 A/G G A rs363099 101709 T/C C T rs363096 119674 T/C T C rs2298967 125400 C/T T C rs2298969 125897 A/G G A rs6844859 130139 C/T T C rs363092 135682 C/A C A rs7685686 146795 A/G A G rs363088 149983 A/T A T rs362331 155488 C/T T C rs916171 156468 G/C c G rs362322 161018 A/G A G rs362275 164255 T/C C T rs362273 167080 A/G A G rs2276881 171314 G/A G A rs3121419 171910 T/C C T rs362272 174633 G/A G A rs362271 175171 G/A G A rs3775061 178407 C/T C T rs362310 179429 A/G G A rs362307 181498 T/C C T rs362306 181753 G/A G A rs362303 181960 T/C C T rs362296 186660 C/A C A rs1006798 198026 A/G A G

Example 41

Modified Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0829] A series of modified oligonucleotides were designed based on the parent gapmer, ISIS 460209 wherein the central gap region contains nine 2'-deoxyribonucleosides. These modified oligonucleotides were designed by introducing various chemical modifications in the central gap region and were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to the parent gapmer, ISIS 460209.

[0830] The modified oligonucleotides were created with a 3-9-3 motif and are described in Table 57. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e", "k", "y", or "z" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside, a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt), a subscript "y" indicates an .alpha.-L-LNA bicyclic nucleoside and a subscript "z" indicates a F-HNA modified nucleoside. .sup.pU indicates a 5-propyne uridine nucleoside and .sup.xT indicates a 2-thio-thymidine nucleoside.

[0831] The number in parentheses indicates the position on the modified oligonucleotide opposite to the SNP position, as counted from the 5'-terminus.

Cell Culture and Transfection

[0832] The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2.times.PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented below.

Analysis of IC.sub.50's

[0833] The half maximal inhibitory concentration (IC.sub.50) of each oligonucleotide is presented in Table 58 and was calculated by plotting the concentrations of oligonucleotides used versus the percent inhibition of HTT mRNA expression achieved at each concentration, and noting the concentration of oligonucleotide at which 50% inhibition of HTT mRNA expression was achieved compared to the control. The IC.sub.50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as `mut IC.sub.50`. The IC.sub.50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as `wt IC.sub.50`. Selectivity was calculated by dividing the IC.sub.50 for inhibition of the wild-type HTT versus the IC.sub.50 for inhibiting expression of the mutant HTT mRNA.

[0834] The parent gapmer, ISIS 460209 is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which the activity and selectivity of the modified oligonucleotides targeting nucleotides overlapping the SNP position could be compared.

[0835] As illustrated in Table 58, modified oligonucleotides having chemical modifications in the central gap region at the SNP position exhibited similar activity with an increase in selectivity comparing to the parent gapmer, wherein the central gap region contains full deoxyribonucleosides.

TABLE-US-00055 TABLE 57 Modified oligonucleotides targeting HTT rs7685686 Wing SEQ ISIS Gap chemistry ID NO Sequence (5' to 3') chemistry 5' 3' NO 460209* (8) T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e Full Deoxy ekk kke 10 539560 (8) T.sub.eA.sub.kA.sub.kATTG.sup.pUCATCA.sub.kC.sub.kC.sub.e Deoxy/5-Propyne ekk kke 11 539563 (8) T.sub.eA.sub.kA.sub.kATTG.sup.xTCATCA.sub.kC.sub.kC.sub.e Deoxy/2-Thio ekk kke 10 539554 (8) T.sub.eA.sub.kA.sub.kATTGU.sub.yCATCA.sub.kC.sub.kC.sub.e Deoxy/.alpha.-L-LNA ekk kke 11 542686 (8) T.sub.eA.sub.kA.sub.kATTGT.sub.zCATCA.sub.kC.sub.kC.sub.e Deoxy/F-HNA ekk kke 10 e = 2'-MOE, k = cEt

TABLE-US-00056 TABLE 58 Comparison of inhibition of HTT mRNA levels and selectivity of modified oligonucleotides with ISIS 460209 targeted to rs7685686 in GM04022 cells ISIS Mut IC.sub.50 Wt IC.sub.50 Selectivity Wing chemistry NO (.mu.M) (.mu.M) (mut vs wt) Gap chemistry 5' 3' 460209* (8) 0.41 2.0 4.9 Full Deoxy ekk kke 539560 (8) 0.29 1.1 3.8 Deoxy/5-Propyne ekk kke 539563 (8) 0.45 3.1 6.9 Deoxy/2-Thio ekk kke 539554 (8) 3.5 >10 >3 Deoxy/.alpha.-L-LNA ekk kke 542686 (8) 0.5 3.1 6.0 Deoxy/F-HNA ekk kke

Example 42

Modified Oligonucleotides Comprising Chemical Modifications in the Gap Region Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0836] Additional modified oligonucleotides were designed in a similar manner as the antisense oligonucleotides described in Table 57. Various chemical modifications were introduced in the central gap region at the SNP position in an effort to improve selectivity while maintaining activity in reducing mutant HTT mRNA levels.

[0837] The modified oligonucleotides were created with a 3-9-3 motif and are described in Table 59. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "a", "e", "f", "h", "k", "1", "R", "w" are sugar modified nucleosides. A subscript "a" indicates a 2'-(ara)-F modified nucleoside, a subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside, a subscript "f" indicates a 2'-F modified nucleoside, a subscript "h" indicates a HNA modified nucleoside, a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt), a subscript "1" indicates a LNA modified nucleoside, a subscript "R" indicates a 5'-(R)-Me DNA, a subscript "w" indicates an unlocked nucleic acid (UNA) modified nucleoside. .sup.nT indicates an N3-ethylcyano thymidine nucleoside and .sup.bN indicates an abasic nucleoside (e.g. 2'-deoxyribonucleoside comprising a H in place of a nucleobase). Underlined nucleoside or the number in parentheses indicates the position on the modified oligonucleotide opposite to the SNP position, as counted from the 5'-terminus.

Thermal Stability Assay

[0838] The modified oligonucleotides were evaluated in thermal stability (T.sub.m) assay. The T.sub.m's were measured using the method described herein. A Cary 100 Bio spectrophotometer with the Cary Win UV Thermal program was used to measure absorbance vs. temperature. For the T.sub.m experiments, oligonucleotides were prepared at a concentration of 8 .mu.M in a buffer of 100 mM Na+, 10 mM phosphate, 0.1 mM EDTA, pH 7. Concentration of oligonucleotides were determined at 85.degree. C. The oligonucleotide concentration was 4 .mu.M with mixing of equal volumes of test oligonucleotide and mutant or wild-type RNA strand. Oligonucleotides were hybridized with the mutant or wild-type RNA strand by heating duplex to 90.degree. C. for 5 min and allowed to cool at room temperature. Using the spectrophotometer, T.sub.m measurements were taken by heating duplex solution at a rate of 0.5 C/min in cuvette starting @ 15.degree. C. and heating to 85.degree. C. T.sub.m values were determined using Vant Hoff calculations (A.sub.260 vs temperature curve) using non self-complementary sequences where the minimum absorbance which relates to the duplex and the maximum absorbance which relates to the non-duplex single strand are manually integrated into the program.

[0839] Presented in Table 60 is the T.sub.m for the modified oligonucleotides when duplexed to mutant or wild-type RNA complement. The T.sub.m of the modified oligonucleotides duplexed with mutant RNA complement is denoted as "T.sub.m (.degree. C.) mut". The T.sub.m of the modified oligonucleotides duplexed with wild-type RNA complement is denoted as "T.sub.m (.degree. C.) wt".

Cell Culture, Transfection and Selectivity Analysis

[0840] The modified oligonucleotides were also tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with a single dose at 2 .mu.M concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2.times.PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. The results in Table 60 are presented as percent of HTT mRNA expression, relative to untreated control levels and is denoted as "% UTC". Selectivity as was also evaluated and measured by dividing the percent of wild-type HTT mRNA levels vs. the percent of mutant HTT mRNA levels.

[0841] The parent gapmer, ISIS 460209 is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which the selectivity of the modified oligonucleotides targeting nucleotides overlapping the SNP position could be compared.

[0842] As illustrated in Table 60, improvement in selectivity was observed for antisense oligonucleotides comprising chemical modifications in the central gap region at the SNP site such as 5'-(R)-Me (ISIS 539558), HNA (ISIS 539559), and 2'-(ara)-F (ISIS 539565) in comparison to the parent full deoxy gapmer, ISIS 460209. Modified oligonucleotides comprising LNA (ISIS 539553) or 2'-F (ISIS 539570) showed comparable selectivity while UNA modification (ISIS 539556 or 543909) showed no selectivity. Modified oligonucleotides comprising modified nucleobase, N3-ethylcyano (ISIS 539564) or abasic nucleobase (ISIS 543525) showed little to no improvement in selectivity.

TABLE-US-00057 TABLE 59 Modified oligonucleotides comprising chemical modifications in the central gap region Wing SEQ ISIS chemistry ID NO Sequence (5' to 3') Gap chemistry 5' 3' NO. 460209* (8) T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e Full Deoxy ekk kke 10 539553 (8) T.sub.eA.sub.kA.sub.kATTGT.sub.lCATCA.sub.kC.sub.kC.sub.e Deoxy/LNA ekk kke 10 539556 (8) T.sub.eA.sub.kA.sub.kATTGU.sub.wCATCA.sub.kC.sub.kC.sub.e Deoxy/UNA ekk kke 11 539558 (8) T.sub.eA.sub.kA.sub.kATTGT.sub.RCATCA.sub.kC.sub.kC.sub.e Deoxy/5'-(R)-Me DNA ekk kke 10 539559 (8) T.sub.eA.sub.kA.sub.kATTGT.sub.hCATCA.sub.kC.sub.kC.sub.e Deoxy/HNA ekk kke 10 539564 (8) T.sub.eA.sub.kA.sub.kATTG.sup.nTCATCA.sub.kC.sub.kC.sub.e Deoxy/deoxy with N3- ekk kke 10 Ethylcyano nucleobase 539565 (8) T.sub.eA.sub.kA.sub.kATTGT.sub.aCATCA.sub.kC.sub.kC.sub.e Deoxy/2'-(ara)-F ekk kke 10 539570 (8) T.sub.eA.sub.kA.sub.kATTGT.sub.fCATCA.sub.kC.sub.kC.sub.e Deoxy/2'-F ekk kke 10 543525 (8) T.sub.eA.sub.kA.sub.kATTG.sup.bNCATCA.sub.kC.sub.kC.sub.e Deoxy/Deoxy-Abasic ekk kke 12 543909 (5) T.sub.eA.sub.kA.sub.kAU.sub.wTGTCATCA.sub.kC.sub.kC.sub.e Deoxy/UNA ekk kke 13 e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

TABLE-US-00058 TABLE 60 Comparison of selectivity in inhibition of HTT mRNA levels and Tm of modified oligonucleotides with ISIS 460209 targeted to rs7685686 in GM04022 cells Wing ISIS Tm (.degree. C.) % UTC Selectivity chemistry NO mutant wt mutant wt (wt vs mut) Gap chemistry 5' 3' 460209* (8) 53.7 52.2 23 57 2.4 Full Deoxy ekk kke 539553 (8) 57.7 55.3 54 102 1.9 Deoxy/LNA ekk kke 539556 (8) 43.7 44.1 90 105 1.2 Deoxy/UNA ekk kke 539558 (8) 51.2 49.7 25 83 3.3 Deoxy/5'-(R)-Me DNA ekk kke 539559 (8) 55.4 50.5 18 62 3.5 Deoxy/HNA ekk kke 539564 (8) 42.8 43.1 86 135 1.6 Deoxy/Deoxy N3- ekk kke ethylcyano nucleobase 539565 (8) 53.8 52.5 14 46 3.4 Deoxy/2'-(ara)-F ekk kke 539570 (8) 54.4 51.8 25 50 2.0 Deoxy/2'-F ekk kke 543525 (8) 43.1 43.8 87 97 1.1 Deoxy/Deoxy Abasic ekk kke 543909 (5) 44.7 42.1 68 79 1.2 Deoxy/UNA ekk kke e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 43

Chimeric Oligonucleotides Comprising Self-Complementary Regions Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0843] Chimeric oligonucleotides were designed based on the parent gapmer, ISIS 460209. These gapmers comprise self-complementary regions flanking the central gap region, wherein the central gap region contains nine deoxyribonucleosides and the self-complementary regions are complementary to one another. The underlined nucleosides indicate the portion of the 5'-end that is self-complement to the portion of the 3'-end.

[0844] The gapmers and their motifs are described in Table 61. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" or "k" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside and a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt).

[0845] The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with a single dose at 2 .mu.M concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2.times.PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. The results in Table 62 are presented as percent of HTT mRNA expression, relative to untreated control levels and is denoted as "% UTC". Selectivity was also evaluated and measured by dividing the percent of wild-type HTT mRNA levels vs. the percent of the mutant HTT mRNA levels.

[0846] The parent gapmer, ISIS 460209 is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which the selectivity of the modified oligonucleotides targeting nucleotides overlapping the SNP position could be compared.

[0847] As illustrated in Table 62, improvement in selectivity was observed for chimeric oligonucleotides comprising 5-9-5 (ISIS 550913), 6-9-6 (ISIS 550912), 6-9-3 (ISIS 550907) or 3-9-7 (ISIS 550904) in comparison to the parent gapmer motif, 3-9-3 (ISIS 460209). The remaining gapmers showed moderate to little improvement in selectivity.

TABLE-US-00059 TABLE 61 Chimeric oligonucleotides comprising various wing motifs targeted to HTT rs7685686 Wing SEQ ISIS chemistry ID NO Sequence (5' to 3') Motif 5' 3' NO. 460209* T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e 3-9-3 ekk kke 10 544838 T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.eA.sub.k 3-9-4 ekk kkek 14 544840 T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.eT.sub.kT.sub.kA.- sub.k 3-9-6 ekk kkekkk 15 544842 T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.eA.sub.kT.sub.kT.- sub.kT.sub.kA.sub.k 3-9-8 ekk kkekkkkk 16 550903 T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.eT.sub.kA.sub.k 3-9-5 ekk kkekk 17 550904 T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.eT.sub.kT.sub.kT.- sub.kA.sub.k 3-9-7 ekk kkekkkk 18 550905 G.sub.kT.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e 4-9-3 kekk kke 19 550906 G.sub.kG.sub.kT.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e 5-9-3 kkekk kke 20 550907 G.sub.kG.sub.kT.sub.kT.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kG.sub.kC.- sub.e 6-9-3 kkkekk kke 21 550908 G.sub.kG.sub.kT.sub.kG.sub.kT.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.- sub.kC.sub.e 7-9-3 kkkkekk kke 22 550909 G.sub.kG.sub.kT.sub.kG.sub.kA.sub.kT.sub.eA.sub.kA.sub.kATTGTCATCA.- sub.kC.sub.kC.sub.e 8-9-3 kkkkkekk kke 23 550910 G.sub.kG.sub.kC.sub.kT.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.- sub.eG.sub.kC.sub.kC.sub.k 6-9-6 kkkekk kkekkk 24 550911 G.sub.kC.sub.kT.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.eG.- sub.kC.sub.k 5-9-5 kkekk kkekk 25 550912 T.sub.kA.sub.kA.sub.kT.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.- sub.eT.sub.kT.sub.kA.sub.k 6-9-6 kkkekk kkekkk 26 550913 A.sub.kA.sub.kT.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.eT.- sub.kT.sub.k 5-9-5 kkekk kkekk 27 550914 T.sub.kC.sub.kT.sub.kT.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.- sub.eA.sub.kG.sub.kA.sub.k 6-9-6 kkkekk kkekkk 28 550915 C.sub.kT.sub.kT.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.eA.- sub.kG.sub.k 5-9-5 kkekk kkekk 29 e = 2'-MOE, k = cEt

TABLE-US-00060 TABLE 62 Comparison of selectivity in inhibition of HTT mRNA levels of chimeric oligonucleotides with ISIS 460209 targeted to rs7685686 in GM04022 cells ISIS % UTC Selectivity wing chemistry NO mut wt (wt vs. mut) Motif 5' 3' 460209* 23 57 2.4 3-9-3 ekk kke 544838 13 25 2.0 3-9-4 ekk kkek 544840 17 31 1.8 3-9-6 ekk kkekkk 544842 55 102 1.9 3-9-8 ekk kkekkkkk 550903 13 36 2.7 3-9-5 ekk kkekk 550904 23 67 3.0 3-9-7 ekk kkekkkk 550905 21 51 2.4 4-9-3 kekk kke 550906 23 67 2.9 5-9-3 kkekk kke 550907 30 93 3.1 6-9-3 kkkekk kke 550908 60 80 2.4 7-9-3 kkkkekk kke 550909 42 101 2.4 8-9-3 kkkkkekk kke 550910 57 102 1.8 6-9-6 kkkekk kkekkk 550911 18 40 2.2 5-9-5 kkekk kkekk 550912 14 51 3.6 6-9-6 kkkekk kkekkk 550913 8 36 4.5 5-9-5 kkekk kkekk 550914 29 45 1.5 6-9-6 kkkekk kkekkk 550915 13 28 2.1 5-9-5 kkekk kkekk e = 2'-MOE, k = cEt

Example 44

Chimeric Antisense Oligonucleotides Comprising Non-Self-Complementary Regions Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0848] Additional gapmers are designed based on the most selective gapmers from studies described in Tables 61 and 62 (ISIS 550912 and 550913). These gapmers are created such that they cannot form self-structure in the effort to evaluate if the increased activity simply is due to higher binding affinity. Gapmers are designed by deleting two or three nucleotides at the 3'-terminus and are created with 6-9-3 or 5-9-3 motif.

[0849] The chimeric oligonucleotides and their motifs are described in Table 63. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" or "k" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside and a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt).

[0850] The gapmers, ISIS 550912 and ISIS 550913, from which the newly designed gapmers are derived from, are marked with an asterisk (*) in the table.

TABLE-US-00061 TABLE 63 Non-self-complementary chimeric oligonucleotides targeting HTT SNP Wing SEQ ISIS chemistry ID NO Sequence (5' to 3') Motif 5' 3' NO. 550912* T.sub.kA.sub.kA.sub.kT.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC- .sub.eT.sub.kT.sub.kA.sub.k 6-9-6 kkkekk kkekkk 26 550913* A.sub.kA.sub.kT.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.eT- .sub.kT.sub.k 5-9-5 kkekk kkekk 27 556879 T.sub.kA.sub.kA.sub.kT.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.- sub.e 6-9-3 kkkekk kke 30 556880 A.sub.kA.sub.kT.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e 5-9-3 kkekk kke 31 e = 2'-MOE, k = cEt

Example 45

Chimeric Oligonucleotides Containing Mismatches Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0851] A series of chimeric antisense oligonucleotides were designed based on the parent gapmer, ISIS 460209, wherein the central gap region contains nine 2'-deoxyribonucleosides. These gapmers were designed by introducing modified nucleosides at both 5' and 3' termini. Gapmers were also created with a single mismatch shifted slightly upstream and downstream (i.e. "microwalk") within the central gap region and with the SNP position opposite position 5 of the parent gapmer, as counted from the 5'-gap terminus.

[0852] The gapmers and their motifs are described in Table 64. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" or "k" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside and a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). Underlined nucleosides indicate the mismatch position, as counted from the 5'-gap terminus.

[0853] These gapmers were evaluated for thermal stability (T.sub.m) using methods described in Example 42. Presented in Table 65 are the T.sub.m measurements for chimeric antisense oligonucleotides when duplexed to mutant or wild-type RNA complement. The T.sub.m of chimeric antisense oligonucleotides duplexed with mutant RNA complement is denoted as "T.sub.m (.degree. C.) mut". The T.sub.m of chimeric antisense oligonucleotides duplexed with wild-type RNA complement is denoted as "T.sub.m (.degree. C.) wt".

[0854] These gapmers were also tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with a single dose at 2 .mu.M concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2.times.PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. The results in Table 65 are presented as percent of HTT mRNA expression, relative to untreated control levels and is denoted as "% UTC". Selectivity was also evaluated and measured by dividing the percent of wild-type HTT mRNA levels vs. the percent of mutant HTT mRNA levels.

[0855] The parent gapmer, ISIS 460209 is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which the selectivity of the modified oligonucleotides targeting nucleotides overlapping the SNP position could be compared.

[0856] As illustrated in Table 65, improvement in selectivity was observed for gapmers comprising a 4-9-4 motif with a central deoxy gap region (ISIS 476333) or a single mismatch at position 8 within the gap region (ISIS 543531) in comparison to the parent gapmer. The remaining gapmers showed moderate to little improvement in selectivity.

TABLE-US-00062 TABLE 64 Chimeric oligonucleotides containing a single mismatch targeting mutant HTT SNP Wing SEQ ISIS Mismatch chemistry ID NO Sequence (5' to 3') position Motif 5' 3' NO. 460209* T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e -- 3-9-3 ekk kke 10 476333 A.sub.eT.sub.kA.sub.eA.sub.kATTGTCATCA.sub.kC.sub.eC.sub.kA.sub.e -- 4-9-4 ekek keke 32 543526 A.sub.eT.sub.kA.sub.eA.sub.kATTCTCATCA.sub.kC.sub.eC.sub.kA.sub.e 4 4-9-4 ekek keke 33 543527 A.sub.eT.sub.kA.sub.eA.sub.kATAGTCATCA.sub.kC.sub.eC.sub.kA.sub.e 3 4-9-4 ekek keke 34 543529 A.sub.eT.sub.kA.sub.eA.sub.kATTGTGATCA.sub.kC.sub.eC.sub.kA.sub.e 6 4-9-4 ekek keke 35 543530 A.sub.eT.sub.kA.sub.eA.sub.kATTGTCTTCA.sub.kC.sub.eC.sub.kA.sub.e 7 4-9-4 ekek keke 36 543531 A.sub.eT.sub.kA.sub.eA.sub.kATTGTCAACA.sub.kC.sub.eC.sub.kA.sub.e 8 4-9-4 ekk keke 37 543532 T.sub.eA.sub.kA.sub.kATTCTCATCA.sub.kC.sub.kC.sub.e 4 3-9-3 ekk kke 38 543534 T.sub.eA.sub.kA.sub.kAATGTCATCA.sub.kC.sub.kC.sub.e 2 3-9-3 ekk kke 39 543535 T.sub.eA.sub.kA.sub.kATTGTGATCA.sub.kC.sub.kC.sub.e 6 3-9-3 ekk kke 40 543536 T.sub.eA.sub.kA.sub.kATTGTCTTCA.sub.kC.sub.kC.sub.e 7 3-9-3 ekk kke 41 543537 T.sub.eA.sub.kA.sub.kATTGTCAACA.sub.kC.sub.kC.sub.e 8 3-9-3 ekk kke 42 e = 2'-MOE, k = cEt

TABLE-US-00063 TABLE 65 Comparison of selectivity and T.sub.m of chimeric oligonucleotides with ISIS 460209 targeted to rs7685686 in GM04022 cells ISIS Tm (.degree. C.) % UTC Selectivity Mismatch Wing chemistry NO mut wt mut wt (wt vs mut) position Motif 5' 3' 460209* 53.7 52.2 23 57 2.4 -- 3-9-3 ekk kke 476333 60.2 58.4 10 37 3.6 -- 4-9-4 ekek keke 543526 47.9 46.6 70 86 1.2 4 4-9-4 ekek keke 543527 52.6 49.9 40 103 2.6 3 4-9-4 ekek keke 543529 50.3 49.0 66 102 1.5 6 4-9-4 ekek keke 543530 52.9 50.9 67 110 1.6 7 4-9-4 ekek keke 543531 53.3 50.3 46 136 3.0 8 4-9-4 ekk keke 543532 43.6 42.8 127 151 1.2 4 3-9-3 ekk kke 543534 45.9 43.8 67 95 1.4 2 3-9-3 ekk kke 543535 44.0 43.3 96 113 1.2 6 3-9-3 ekk kke 543536 46.8 44.6 106 104 1.0 7 3-9-3 ekk kke 543537 45.9 44.3 77 81 1.1 8 3-9-3 ekk kke e = 2'-MOE, k = cEt

Example 46

Chimeric Oligonucleotides Comprising Mismatches Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0857] Additional chimeric antisense oligonucleotides are designed based on two gapmers selected from studies described in Tables 64 and 65 (ISIS 476333 and ISIS 460209) wherein the central gap region contains nine 2'-deoxyribonucleosides. These gapmers are designed by introducing a single mismatch, wherein the mismatch will be shifted throughout the antisense oligonucleotide (i.e. "microwalk"). Gapmers are also created with 4-9-4 or 3-9-3 motifs and with the SNP position opposite position 8 of the original gapmers, as counted from the 5'-terminus.

[0858] The gapmers and their motifs are described in Table 66. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" or "k" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside and a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). Underlined nucleosides indicate the mismatch position, as counted from the 5'-terminus.

[0859] The gapmers, ISIS 476333 and ISIS 460209, in which the newly designed antisense oligonucleotides are derived from, are marked with an asterisk (*) in the table.

TABLE-US-00064 TABLE 66 Chimeric oligonucleotides comprising mismatches targeting HTT SNP Wing SEQ ISIS Mismatch chemistry ID NO Sequence (5' to 3') position Motif 5' 3' NO 476333* A.sub.eT.sub.kA.sub.eA.sub.kATTGTCATCA.sub.kC.sub.eC.sub.kA.sub.e -- 4-9-4 ekek keke 32 554209 T.sub.eT.sub.kA.sub.eA.sub.kATTGTCATCA.sub.kC.sub.eC.sub.kA.sub.e 1 4-9-4 ekek keke 43 554210 A.sub.eA.sub.kA.sub.eA.sub.kATTGTCATCA.sub.kC.sub.eC.sub.kA.sub.e 2 4-9-4 ekek keke 44 554211 A.sub.eT.sub.kT.sub.eA.sub.kATTGTCATCA.sub.kC.sub.eC.sub.kA.sub.e 3 4-9-4 ekek keke 45 554212 A.sub.eT.sub.kA.sub.eT.sub.kATTGTCATCA.sub.kC.sub.eC.sub.kA.sub.e 4 4-9-4 ekek keke 46 554213 A.sub.eT.sub.kA.sub.eA.sub.kTTTGTCATCA.sub.kC.sub.eC.sub.kA.sub.e 5 4-9-4 ekek keke 47 554214 A.sub.eT.sub.kA.sub.eA.sub.kATTGTCATGA.sub.kC.sub.eC.sub.kA.sub.e 13 4-9-4 ekek keke 48 554215 A.sub.eT.sub.kA.sub.eA.sub.kATTGTCATCT.sub.kC.sub.eC.sub.kA.sub.e 14 4-9-4 ekek keke 49 554216 A.sub.eT.sub.kA.sub.eA.sub.kATTGTCATCA.sub.kG.sub.eC.sub.kA.sub.e 15 4-9-4 ekek keke 50 554217 A.sub.eT.sub.kA.sub.eA.sub.kATTGTCATCA.sub.kC.sub.eG.sub.kA.sub.e 16 4-9-4 ekek keke 51 554218 A.sub.eT.sub.kA.sub.eA.sub.kATTGTCATCA.sub.kC.sub.eC.sub.kT.sub.e 17 4-9-4 ekek keke 52 460209* T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e -- 3-9-3 ekk kke 10 562481 T.sub.eA.sub.kA.sub.kGTTGTCATCA.sub.kC.sub.kC.sub.e 4 3-9-3 ekk kke 53 554482 T.sub.eA.sub.kA.sub.kAGTGTCATCA.sub.kC.sub.kC.sub.e 5 3-9-3 ekk kke 54 554283 T.sub.eA.sub.kA.sub.kATGGTCATCA.sub.kC.sub.kC.sub.e 6 3-9-3 ekk kke 55 e = 2'-MOE, k = cEt

Example 47

Short-Gap Chimeric Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0860] Chimeric antisense oligonucleotides were designed based on the parent gapmer, ISIS 460209, wherein the central gap region contains nine 2'-deoxyribonucleosides. These gapmers were designed by shortening the central gap region to seven 2'-deoxyribonucleosides. Gapmers were also created with 5-7-5 motif and with the SNP position opposite position 8 or 9 of the parent gapmer, as counted from the 5'-terminus.

[0861] The gapmers and their motifs are described in Table 67. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" or "k" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside and a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). Underlined nucleoside or the number in parentheses indicates the position on the modified oligonucleotide opposite to the SNP position, as counted from the 5'-terminus.

[0862] The chimeric antisense oligonucleotides were tested in vitro. ISIS 141923 was included in the study as a negative control and is denoted as "neg control". A non-allele specific antisense oligonucleotide, ISIS 387916 was used as a positive control and is denoted as "pos control". ISIS 460209 was included in the study for comparison. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3, and 10 .mu.M concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2.times.PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 68.

[0863] The IC.sub.50 and selectivity were calculated using methods described previously in Example 41. As illustrated in Table 68, no improvement in potency and selectivity was observed for the chimeric antisense oligonucleotides as compared to ISIS 460209.

TABLE-US-00065 TABLE 67 Chimeric antisense oligonucleotides targeting HTT rs7685686 Wing SEQ ISIS Chemistry ID NO Sequence (5' to 3') Motif 5' 3' NO. 460209* (8) T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e 3-9-3 ekk kke 10 460085 (9) A.sub.eT.sub.eA.sub.eA.sub.eA.sub.eTTGTCATC.sub.eA.sub.eC.sub.e- C.sub.eA.sub.e 5-7-5 eeeee eeeee 32 540108 (9) A.sub.eT.sub.eA.sub.eA.sub.kA.sub.kTTGTCATC.sub.kA.sub.kC.sub.e- C.sub.eA.sub.e 5-7-5 eeekk kkeee 32 387916 T.sub.eC.sub.eT.sub.eC.sub.eT.sub.eATTGCACATTC.sub.eC.sub.eA.sub.eA- .sub.eG.sub.e 5-10-5 eeeee eeeee 56 (pos control) 141923 C.sub.eC.sub.eT.sub.eT.sub.eC.sub.eCCTGAAGGTTC.sub.eC.sub.eT.sub.eC- .sub.eC.sub.e 5-10-5 eeeee eeeee 57 (neg control) e = 2'-MOE, k = cEt

TABLE-US-00066 TABLE 68 Comparison of inhibition of HTT mRNA levels and selectivity of chimeric antisense oligonucleotides with ISIS 460209 targeted to rs7685686 in GM04022 cells Wing Mut IC.sub.50 Wt IC.sub.50 Selectivity chemistry ISIS NO (.mu.M) (.mu.M) (mut vs wt) Motif 5' 3' 460209* (8) 0.41 2.0 4.9 3-9-3 ekk kke 460085 (9) 3.5 >10 >3 5-7-5 eeeee eeeee 540108 (9) 0.41 -- -- 5-7-5 eeekk kkeee 387916 0.39 0.34 1.0 5-10-5 eeeee eeeee (pos control) 141923 >10 >10 -- 5-10-5 eeeee eeeee (neg control) e = 2'-MOE, k = cEt

Example 48

Short-Gap Chimeric Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0864] Additional chimeric antisense oligonucleotides were designed based on the parent gapmer, ISIS 460209, wherein the central gap region contains nine 2'-deoxyribonucleosides. These gapmers were designed with the central gap region shortened or interrupted by introducing various modifications either within the gap or by adding one or more modified nucleosides to the 3'-most 5'-region or to the 5'-most 3'-region. Gapmers were created with the SNP position opposite position 8 of the parent gapmer, as counted from the 5'-terminus.

[0865] The gapmers and their motifs are described in Table 69. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" or "k" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside and a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt).

[0866] The chimeric antisense oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 2 .mu.M concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2.times.PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. The results in Table 70 are presented as percent of HTT mRNA expression, relative to untreated control levels and is denoted as "% UTC". Selectivity was also evaluated and measured by dividing the percent of wild-type HTT mRNA levels vs. the percent of mutant HTT mRNA levels. ISIS 460209 marked with an asterisk (*) in the table was included in the study for comparison.

[0867] As illustrated in Table 70, modifications to the 3'-most 5'-region nucleosides that shorten the gap from 9 to 7 or 8 nucleotides (ISIS 551429 and ISIS 551426) improved selectivity and potency comparing to the parent gapmer (ISIS 460209). The remaining chimeric antisense oligonucleotides showed moderate to little improvement in selectivity.

TABLE-US-00067 TABLE 69 Short-gap antisense oligonucleotides targeting HTT rs7685686 Wing SEQ ISIS Chemistry ID NO Sequence (5' to 3') Motif 5' 3' NO. 460209* T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e 3-9-3 ekk kke 10 551426 T.sub.eA.sub.kA.sub.eA.sub.kTTGTCATCA.sub.kC.sub.kC.sub.e 4-8-3 ekek kke 10 551427 T.sub.eA.sub.kA.sub.eAT.sub.kTGTCATCA.sub.kC.sub.kC.sub.e 3-9-3 or eke or kke 10 5-7-3 ekedk 551428 T.sub.eA.sub.kA.sub.eATT.sub.kGTCATCA.sub.kC.sub.kC.sub.e 3-9-3 or eke or kke 10 6-6-3 ekeddk 551429 T.sub.eA.sub.eA.sub.eA.sub.kT.sub.kTGTCATCA.sub.kC.sub.kC.sub.e 5-7-3 eeekk kke 10 e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

TABLE-US-00068 TABLE 70 Comparison of selectivity in inhition of HTT mRNA levels of antisense oligonucleotides with ISIS 460209 targeted to rs7685686 in GM4022 cells % UTC Selectivity Wing chemistry ISIS NO mut wt (wt vs. mut) Motif 5' 3' 460209* 23 57 2.4 3-9-3 ekk kke 551426 14 66 4.8 4-8-3 ekek kke 551427 35 97 2.8 3-9-3 or eke or kke 5-7-3 ekedk 551428 61 110 1.8 3-9-3 or eke or kke 6-6-3 ekeddk 551429 19 94 5.0 5-7-3 eeekk kke e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 49

Modified Oligonucleotides Targeting HTT SNP

[0868] A series of modified antisense oligonucleotides are designed based on the parent gapmer, ISIS 460209, wherein the central gap region contains nine 2'-deoxynucleosides and is marked with an asterisk (*) in the table. These modified oligonucleotides are designed by shortening or interrupting the gap with a single mismatch or various chemical modifications within the central gap region. The modified oligonucleotides are created with the SNP position opposite position 8 of the parent gapmer, as counted from the 5'-terminus.

[0869] The gapmers and their motifs are described in Table 71. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages, except for the internucleoside linkage with a subscript "p", "pz" or "pw". Subscript "p" indicates methyl phosphonate internucleoside linkage. Subscript "pz" indicates (R)-methyl phosphonate internucleoside linkage. Subscript "pw" indicates (S)-methyl phosphonate internucleoside linkage. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. .sup.xT indicates a 2-thio thymidine nucleoside. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e", "k" or "b" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside, a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt) and a subscript "b" indicates a 5'-Me DNA modified nucleoside. Underlined nucleosides indicate the position of modification. Bold and underlined nucleosides indicate the mismatch position.

TABLE-US-00069 TABLE 71 Short-gap chimeric oligonucleotides targeting HTT SNP Wing SEQ ISIS Sequence Gap Chemistry ID NO (5' to 3') Motif Chemistry 5' 3' NO. 460209* T.sub.eA.sub.kA.sub.kATTGTC 3-9-3 -- ekk kke 10 ATCA.sub.kC.sub.kC.sub.e XXXX16 T.sub.eA.sub.kA.sub.kA.sup.xTTGT 3-9-3 Deoxy/2-thio ekk kke 10 CATCA.sub.kC.sub.kC.sub.e XXXX17 T.sub.eA.sub.kA.sub.kAT.sup.xTGT 3-9-3 Deoxy/2-thio ekk kke 10 CATCA.sub.kC.sub.kC.sub.e XXXX18 T.sub.eA.sub.kA.sub.kA.sup.xT.sup.xTGT 3-9-3 Deoxy/2-thio ekk kke 10 CATCA.sub.kC.sub.kC.sub.e XXXX19 T.sub.eA.sub.kA.sub.kATT.sub.pGT 3-9-3 Deoxy/Methyl ekk kke 10 (558257) CATCA.sub.kC.sub.kC.sub.e phosphonate XXXX20 T.sub.eA.sub.kA.sub.kAT.sub.pTGT 3-9-3 Deoxy/Methyl ekk kke 10 (558256) CATCA.sub.kC.sub.kC.sub.e phosphonate XXXX20a T.sub.eA.sub.kA.sub.kAT.sub.pzTGT 3-9-3 Deoxy/(R)- ekk kke 10 CATCA.sub.kC.sub.kC.sub.e Methyl phosphonate XXXX20b T.sub.eA.sub.kA.sub.kAT.sub.pwTG 3-9-3 Deoxy/(S)- ekk kke 10 TCATCA.sub.kC.sub.kC.sub.e Methyl phosphonate XXXX21 T.sub.eA.sub.kA.sub.kA.sub.pTTGT 3-9-3 Methyl ekk kke 10 (558255) CATCA.sub.kC.sub.kC.sub.e phosphonate XXXX22 T.sub.eA.sub.kA.sub.kATT.sub.bGT 3-9-3 5'-Me-DNA ekk kke 10 CATCA.sub.kC.sub.kC.sub.e XXXX23 T.sub.eA.sub.kA.sub.kAT.sub.bTGT 3-9-3 5'-Me-DNA ekk kke 10 CATCA.sub.kC.sub.kC.sub.e XXXX24 T.sub.eA.sub.kA.sub.kA.sub.bTTGT 3-9-3 5'-Me-DNA ekk kke 10 CATCA.sub.kC.sub.kC.sub.e XXXX25 T.sub.eA.sub.kA.sub.kGTTGTC 4-8-3 Mismatch at ekk kke 53 ATCA.sub.kC.sub.kC.sub.e position 4 XXXX26 T.sub.eA.sub.kA.sub.kAGTGT 5-7-3 Mismatch at ekk kke 54 CATCA.sub.kC.sub.kC.sub.e position 5 XXXX27 T.sub.eA.sub.kA.sub.kATGGT 6-6-3 Mismatch at ekk kke 55 CATCA.sub.kC.sub.kC.sub.e position 6 e = 2'-MOE, k = cEt

Example 50

Short-Gap Chimeric Oligonucleotides Comprising Modifications at the Wing Regions Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0870] Additional chimeric antisense oligonucleotides were designed based on the parent gapmer, ISIS 460209, wherein the central gap region contains nine 2'-deoxynucleosides. These gapmers were designed by shortening the central gap region to seven 2'-deoxynucleosides and introducing various modifications at the wing regions.

[0871] The gapmers and their motifs are described in Table 72. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" or "k" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside and a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt).

[0872] The number in parentheses indicates the position on the chimeric oligonucleotide opposite to the SNP position, as counted from the 5'-terminus.

[0873] These gapmers were evaluated for thermal stability (T.sub.m) using methods described in Example 42. Presented in Table 73 is the T.sub.m measurements for chimeric antisense oligonucleotides when duplexed to mutant or wild-type RNA complement. The T.sub.m of chimeric antisense oligonucleotides duplexed with mutant RNA complement is denoted as "T.sub.m (.degree. C.) mut". The T.sub.m of chimeric antisense oligonucleotides duplexed with wild-type RNA complement is denoted as "T.sub.m (.degree. C.) wt".

[0874] These gapmers were also tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with a single dose at 2 .mu.M concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2.times.PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. The results in Table 73 are presented as percent of HTT mRNA expression, relative to untreated control levels and is denoted as "% UTC". Selectivity was also evaluated and measured by dividing the percent of wild-type HTT mRNA levels vs. the percent of mutant HTT mRNA levels. ISIS 460209 marked with an asterisk (*) in the table was included in the study for comparison.

[0875] As illustrated in Table 73, improvement in selectivity was observed for gapmers comprising 2-7-8 or 5-7-5 motifs having cEt subunits at the wing regions in comparison to the parent gapmer, ISIS 460209. The remaining gapmers showed moderate to little improvement in selectivity.

TABLE-US-00070 TABLE 72 Short-gap chimeric oligonucleotides comprising wing modifications wing SEQ ISIS chemistry ID NO Sequence (5' to 3') Motif 5' 3' NO. 460209* (8) T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e 3-9-3 ekk kke 10 540103 (6) A.sub.kA.sub.kTTGTCATC.sub.eA.sub.eC.sub.eC.sub.eA.sub.eG.sub.e- A.sub.eA.sub.e 2-7-8 kk e8 58 540104(6) A.sub.eA.sub.eTTGTCATC.sub.eA.sub.eC.sub.eC.sub.eA.sub.eG.sub.e- A.sub.eA.sub.e 2-7-8 ee e8 59 540105 (7) A.sub.eA.sub.eA.sub.eTTGTCATC.sub.eA.sub.eC.sub.eC.sub.eA.sub.e- G.sub.eA.sub.e 3-7-7 eee e7 60 540106 (8) T.sub.eA.sub.eA.sub.eA.sub.eTTGTCATC.sub.eA.sub.eC.sub.eC.sub.e- A.sub.eG.sub.e 4-7-6 eeee e6 61 540107 (9) A.sub.eT.sub.eA.sub.eA.sub.eA.sub.kTTGTCATC.sub.kA.sub.eC.sub.e- C.sub.eA.sub.e 5-7-5 eeeek keeee 32 540109 (10) A.sub.eA.sub.eT.sub.eA.sub.eA.sub.eA.sub.eTTGTCATC.sub.eA.sub.eC.sub.eC.s- ub.e 6-7-4 e6 e4 62 540110 (11) T.sub.eA.sub.eA.sub.eT.sub.eA.sub.eA.sub.eA.sub.eTTGTCATC.sub.eA.sub.eC.s- ub.e 7-7-3 e7 eee 63 540111 (12) T.sub.eT.sub.eA.sub.eA.sub.eT.sub.eA.sub.eA.sub.eA.sub.eTTGTCATC.sub.eA.s- ub.e 8-7-2 e8 ee 64 540112 (12) T.sub.eT.sub.eA.sub.eA.sub.eT.sub.eA.sub.eA.sub.eA.sub.eTTGTCATC.sub.kA.s- ub.k 8-7-2 e8 kk 64 e = 2'-MOE (e.g. e6 = eeeeee), and k = cEt

TABLE-US-00071 TABLE 73 Comparison of selectivity in inhibition of HTT mRNA levels of antisense oligonucleotides with ISIS 460209 targeted to RS7685686 in GM04022 cells Selec- Tm tivity wing (.degree. C.) % UTC (wt vs chemistry ISIS NO mut wt mut wt mut) Motif 5' 3' 460209* (8) 53.7 52.2 23 57 2.4 3-9-3 ekk kke 540103 (6) 57.6 56.4 23 74 3.3 2-7-8 kk e8 540104 (6) 54.8 52.8 36 91 2.5 2-7-8 ee e8 540105 (7) 54.2 52.2 53 135 2.6 3-7-7 eee e7 540106 (8) 52.4 50.8 30 77 2.6 4-7-6 eeee e6 540107 (9) 56.6 54.7 19 62 3.3 5-7-5 eeeek keeee 540109 (10) 49.1 47.3 78 127 1.6 6-7-4 e6 e4 540110 (11) 42.8 41.2 89 112 1.3 7-7-3 e7 eee 540111 (12) 39.0 36.9 111 128 1.1 8-7-2 e8 ee 540112 (12) 44.2 42.4 86 102 1.2 8-7-2 e8 kk

Example 51

Chimeric Oligonucleotides with SNP Site Shifting within the Central Gap Region

[0876] Chimeric antisense oligonucleotides were designed based on the parent gapmer, ISIS 460209 wherein the SNP site aligns with position 5 of the parent gapmer, as counted from the 5'-gap terminus. These gapmers were designed by shifting the SNP site upstream or downstream (i.e. microwalk) within the central gap region of the parent gapmer.

[0877] The gapmers and their motifs are described in Table 74. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" or "k" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside and a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). Underline nucleosides indicate the position on the chimeric oligonucleotide aligns with the SNP site.

[0878] The SNP site indicates the position on the chimeric antisense oligonucleotide opposite to the SNP position, as counted from the 5'-gap terminus and is denoted as "SNP site".

[0879] The chimeric oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. ISIS 460209 marked with an asterisk (*) in the table was included in the study for comparison.

[0880] The IC.sub.50 and selectivity were calculated using the methods previously described in Example 41. As illustrated in Table 75, chimeric oligonucleotides comprising 4-9-2 (ISIS 540082) or 2-9-4 (ISIS 540095) motif with the SNP site at position 1 or 3 showed comparable activity and 2.5 fold selectivity as compared to their counterparts.

TABLE-US-00072 TABLE 74 Chimeric oligonucleotides designed by microwalk wing SEQ ISIS SNP chemistry ID NO Sequence (5' to 3') Motif site 5' 3' NO. 460209* T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e 3-9-3 5 ekk kke 10 540082 A.sub.eT.sub.kT.sub.kG.sub.kTCATCACCAG.sub.kA.sub.e 4-9-2 1 ekkk ke 65 540089 T.sub.eT.sub.kA.sub.kA.sub.kTAAATTGTCA.sub.kT.sub.e 4-9-2 8 ekkk ke 66 540095 A.sub.eT.sub.kTGTCATCACC.sub.kA.sub.kG.sub.kA.sub.e 2-9-4 3 ek kkke 65 e = 2'-MOE, and k = cEt

TABLE-US-00073 TABLE 75 Comparison of inhibition of HTT mRNA levels and selectivity of chimeric oligonucleotides with ISIS 460209 targeted to HTT SNP Mut Wing IC.sub.50 Wt IC.sub.50 Selectivity SNP chemistry ISIS NO (.mu.M) (.mu.M) (wt vs mut) Motif site 5' 3' 460209 0.41 2.0 4.9 3-9-3 5 ekk kke 540082 0.45 5.6 12 4-9-2 1 ekkk ke 540089 >10 >10 -- 4-9-2 8 ekkk ke 540095 0.69 8.4 12 2-9-4 3 ek kkke e = 2'-MOE, and k = cEt

Example 52

Chimeric Oligonucleotides with SNP Site Shifting at Various Positions

[0881] Chimeric antisense oligonucleotides were designed based on the parent gapmer, ISIS 460209 wherein the SNP site aligns with position 8 of the parent gapmer, as counted from the 5'-terminus. These gapmers were designed by shifting the SNP site upstream or downstream (i.e. microwalk) of the original oligonucleotide.

[0882] The gapmers and their motifs are described in Table 76. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" or "k" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside and a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). Underline nucleosides indicate the SNP site.

[0883] The SNP site indicates the position on the chimeric antisense oligonucleotide opposite to the SNP position, as counted from the 5'-terminus and is denoted as "SNP site".

[0884] The chimeric oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2.times.PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. The results in Table 77 are presented as percent of HTT mRNA expression, relative to untreated control levels and is denoted as "% UTC". Selectivity was also evaluated and measured by dividing the percent of wild-type HTT mRNA levels vs. the percent of mutant HTT mRNA levels.

[0885] The parent gapmer, ISIS 460209 is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which the selectivity of the modified oligonucleotides targeting nucleotides overlapping the SNP position could be compared.

[0886] As illustrated in Table 77, improvement in potency and selectivity was observed for chimeric oligonucleotides comprising 4-9-2 or 2-9-4 motif having the target SNP site at positions 3, 4, 6, 7 and 8 (ISIS540083, ISIS540084, ISIS 540085, ISIS 540094, ISIS 540096, ISIS 540097 and ISIS 540098) in comparison to position 8 of the parent gapmer (ISIS 460209). The remaining gapmers showed little to no improvement in potency or selectivity.

TABLE-US-00074 TABLE 76 Chimeric oligonucleotides designed by microwalk SEQ ISIS SNP ID NO Sequence (5' to 3') site Motif NO. 460209* T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e 8 3-9-3 10 (ekk- d9- kke) 543887 T.sub.eT.sub.kG.sub.kT.sub.kCATCACCAGA.sub.kA.sub.e 4 4-9-2 67 (ekkk- d9-ke) 540083 A.sub.eA.sub.kT.sub.kT.sub.kGTCATCACCA.sub.kG.sub.e 6 4-9-2 68 (ekkk- d9-ke) 540084 A.sub.eA.sub.kA.sub.kT.sub.kTGTCATCACC.sub.kA.sub.e 7 4-9-2 69 (ekkk- d9-ke) 540085 T.sub.eA.sub.kA.sub.kA.sub.kTTGTCATCAC.sub.kC.sub.e 8 4-9-2 10 (ekkk- d9-ke) 540087 A.sub.eA.sub.kT.sub.kA.sub.kAATTGTCATC.sub.kA.sub.e 10 4-9-2 70 (ekkk- d9-ke) 540090 A.sub.eT.sub.kT.sub.kA.sub.kATAAATTGTC.sub.kA.sub.e 13 4-9-2 71 (ekkk- d9-ke) 540091 T.sub.eA.sub.kT.sub.kT.sub.kAATAAATTGT.sub.kC.sub.e 14 4-9-2 72 (ekkk- d9-ke) 540092 G.sub.eT.sub.kCATCACCAGA.sub.kA.sub.kA.sub.kA.sub.e 2 2-9-4 73 (ek- d9- kkke) 540093 T.sub.eG.sub.kTCATCACCAG.sub.kA.sub.kA.sub.kA.sub.e 3 2-9-4 74 (ek- d9- kkke) 540094 T.sub.eT.sub.kGTCATCACCA.sub.kG.sub.kA.sub.kA.sub.e 4 2-9-4 67 (ek- d9- kkke) 540096 A.sub.eA.sub.kTTGTCATCAC.sub.kC.sub.kA.sub.kG.sub.e 6 2-9-4 68 (ek- d9- kkke) 540097 A.sub.eA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.kA.sub.e 8 2-9-4 69 (ek- d9- kkke) 540098 T.sub.eA.sub.kAATTGTCATC.sub.kA.sub.kC.sub.kC.sub.e 8 2-9-4 10 (ek- d9- kkke) 540099 A.sub.eT.sub.kAAATTGTCAT.sub.kC.sub.kA.sub.kC.sub.e 9 2-9-4 75 (ek- d9- kkke) 540100 A.sub.eA.sub.kTAAATTGTCA.sub.kT.sub.kC.sub.kA.sub.e 10 2-9-4 70 (ek- d9- kkke) 540101 T.sub.eA.sub.kATAAATTGTC.sub.kA.sub.kT.sub.kC.sub.e 11 2-9-4 76 (ek- d9- kkke) 540102 T.sub.eT.sub.kAATAAATTGT.sub.kC.sub.kA.sub.kT.sub.e 12 2-9-4 66 (ek- d9- kkke) e = 2'-MOE; k = cEt; d = 2'-deoxyribonucleoside

TABLE-US-00075 TABLE 77 Comparison of selectivity in HTT SNP inhibition of chimeric oligonucleotides with ISIS 460209 % UTC Selectivity SNP ISIS NO mut wt (wt vs. mut) site Motif 460209* 23 57 2.4 8 3-9-3 (ekk-d9-kke) 543887 18 43 2.3 4 4-9-2 (ekkk-d9-ke) 540083 18 67 3.7 6 4-9-2 (ekkk-d9-ke) 540084 10 49 4.9 7 4-9-2 (ekkk-d9-ke) 540085 21 86 4.1 8 4-9-2 (ekkk-d9-ke) 540087 60 98 1.6 10 4-9-2 (ekkk-d9-ke) 540090 129 137 1.1 13 4-9-2 (ekkk-d9-ke) 540091 93 105 1.1 14 4-9-2 (ekkk-d9-ke) 540092 28 55 2.0 2 2-9-4 (ek-d9-kkke) 540093 18 62 3.4 3 2-9-4 (ek-d9-kkke) 540094 13 45 3.4 4 2-9-4 (ek-d9-kkke) 540096 17 68 4.0 6 2-9-4 (ek-d9-kkke) 540097 8 35 4.2 8 2-9-4 (ek-d9-kkke) 540098 12 45 3.9 8 2-9-4 (ek-d9-kkke) 540099 62 91 1.5 9 2-9-4 (ek-d9-kkke) 540100 80 106 1.3 10 2-9-4 (ek-d9-kkke) 540101 154 152 1.0 11 2-9-4 (ek-d9-kkke) 540102 102 106 1.0 12 2-9-4 (ek-d9-kkke) e = 2'-MOE; k = cEt; d = 2'-deoxyribonucleoside

Example 53

Selectivity in Inhibition of HTT mRNA Levels Targeting SNP by Chimeric Oligonucleotides Designed by Microwalk

[0887] A series of modified oligonucleotides were designed based on the parent gapmer, ISIS 460209, wherein the central gap region comprises nine 2'-deoxyribonucleosides. These gapmers were created with various motifs and modifications at the wings and/or the central gap region.

[0888] The modified oligonucleotides and their motifs are described in Table 78. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e", "k", "y", or "z" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside, a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt), a subscript "y" indicates an .alpha.-L-LNA modified nucleoside, and a subscript "z" indicates a F-HNA modified nucleoside. .sup.pU indicates a 5-propyne uridine nucleoside and .sup.xT indicates a 2-thio-thymidine nucleoside. Underlined nucleosides indicate the mismatch position.

[0889] These gapmers were evaluated for thermal stability (T.sub.m) using methods described in Example 42. Presented in Table 79 are the T.sub.m measurements for chimeric antisense oligonucleotides when duplexed to mutant or wild-type RNA complement. The T.sub.m of chimeric antisense oligonucleotides duplexed with mutant RNA complement is denoted as "T.sub.m (.degree. C.) mut". The T.sub.m of chimeric antisense oligonucleotides duplexed with wild-type RNA complement is denoted as "T.sub.m (.degree. C.) wt".

[0890] These gapmers were also tested in vitro. ISIS 141923 was included in the study as a negative control and is denoted as "neg control". The non-allele specific antisense oligonucleotides, ISIS 387916 was used as a positive control and is denoted as "pos control". Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with a single dose at 2 .mu.M concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2.times.PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. ISIS 460209 marked with an asterisk (*) in the table was included in the study for comparison. The results in Table 79 are presented as percent of HTT mRNA expression, relative to untreated control levels and is denoted as "% UTC". Selectivity was also evaluated and measured by dividing the percent of wild-type HTT mRNA levels vs. the percent of mutant HTT mRNA levels.

[0891] As illustrated, several of the newly designed antisense oligonucleotides showed improvement in potency and/or selectivity in inhibiting mut HTT mRNA levels comparing to ISIS 460209.

TABLE-US-00076 TABLE 78 Modified oligonucleotides comprising various modifications targeting HTT SNP Wing SEQ ISIS Chemistry ID NO Sequence (5' to 3') Modification 5' 3' NO. 460209* T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e 3-9-3 ekk kke 10 (ekk-d9-kke) 539560 T.sub.eA.sub.kA.sub.kATTG.sup.pUCATCA.sub.kC.sub.kC.sub.e 5-propyne in ekk kke 11 gap 539563 T.sub.eA.sub.kA.sub.kATTG.sup.xTCATCA.sub.kC.sub.kC.sub.e 2-thio in gap ekk kke 10 539554 T.sub.eA.sub.kA.sub.kATTGU.sub.yCATCA.sub.kC.sub.kC.sub.e .alpha.-L-LNA in gap ekk kke 11 542686 T.sub.eA.sub.kA.sub.kATTGT.sub.zCATCA.sub.kC.sub.kC.sub.e F-HNA in gap ekk kke 10 540108 A.sub.eT.sub.eA.sub.eA.sub.kA.sub.kTTGTCATC.sub.kA.sub.kC.sub.eC.su- b.eA.sub.e 5-7-5 eeekk kkeee 23 (eeekk-d7-kkeee) 544840 T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.eT.sub.kT.sub.kA.- sub.k 3-9-6 ekk kkekkk 15 (ekk-d9-kkekkk) 550904 T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.eT.sub.kT.sub.kT.- sub.kA.sub.k 3-9-7 ekk kkekkkk 18 (ekk-d9-kkekkkk) 540082 A.sub.eT.sub.kT.sub.kG.sub.kTCATCACCAG.sub.kA.sub.e 4-9-2 ekkk ke 65 (ekkk-d9-ke) 540089 T.sub.eT.sub.kA.sub.kA.sub.kTAAATTGTCA.sub.kT.sub.e 4-9-2 ekkk ke 66 (ekkk-d9-ke) 540095 A.sub.eT.sub.kTGTCATCACC.sub.kA.sub.kG.sub.kA.sub.e 2-9-4 ek kkke 67 (ek-d9-kkke) 543528 A.sub.eT.sub.kA.sub.eA.sub.kAATGTCATCA.sub.kC.sub.eC.sub.kA.sub.e Mismatch at ekek keke 77 position 2 counting from 5' gap 543533 T.sub.eA.sub.kA.sub.kATAGTCATCA.sub.kC.sub.kC.sub.e Mismatch at ekk kke 78 position 3 counting from 5' gap 387916 T.sub.eC.sub.eT.sub.eC.sub.eT.sub.eATTGCACATTC.sub.eC.sub.eA.sub.eA- .sub.eG.sub.e 5-10-5 eeeee eeeee 56 (pos control) 141923 C.sub.eC.sub.eT.sub.eT.sub.eC.sub.eCCTGAAGGTTC.sub.eC.sub.eT.sub.eC- .sub.eC.sub.e 5-10-5 eeeee eeeee 57 (neg control) e = 2'-MOE; k = cEt; d = 2'-deoxyribonucleoside

TABLE-US-00077 TABLE 79 Comparison of selectivity in inhibition of HTT mRNA levels, and Tm of modified oligonucleotides with ISIS 460209 targeted tors7685686 in GM04022 cells Tm (.degree. C.) % UTC Selectivity Wing Chemistry ISIS NO mutant wt mut wt (wt vs mut) Modification 5' 3' 460209* 53.7 52.2 23 57 2.7 3-9-3 ekk kke (ekk-d9-kke) 539560 54.1 50.8 13 32 2.4 5-propyne in gap ekk kke 539563 53.8 49.1 13 40 3.2 2-thio in gap ekk kke 539554 56.5 54.5 54 89 1.7 .alpha.-L-LNA in gap ekk kke 542686 56.1 50.4 26 62 2.4 F-HNA in gap ekk kke 540108 60.0 57.9 27 63 2.3 5-7-5 eeekk kkeee (eeekk-d7-kkeee) 544840 -- -- 19 40 2.1 3-9-6 ekk kkekkk (ekk-d9-kkekkk) 550904 -- -- 39 65 1.7 3-9-7 ekk kkekkkk (ekk-d9- kkekkkk) 540082 -- -- 21 62 3.0 4-9-2 ekkk ke (ekkk-d9-ke) 540089 -- -- 78 86 1.1 4-9-2 ekkk ke (ekkk-d9-ke) 540095 -- -- 22 66 3.1 2-9-4 ek kkke (ek-d9-kkke) 543528 50.5 49.1 44 90 2.1 Mismatch at ekek keke position 2 counting from 5' gap 543533 47.0 44.8 83 97 1.2 Mismatch at ekk kke position 3 counting from 5' gap 387916 -- -- 21 19 0.9 5-10-5 eeeee eeeee (pos control) 141923 -- -- 95 99 1.0 5-10-5 eeeee eeeee (neg control) e = 2'-MOE; k = cEt; d = 2'-deoxyribonucleoside

Example 54

Chimeric Oligonucleotides Comprising Modifications at the SNP Site of HTT Gene

[0892] Additional gapmers are designed based on the gapmer selected from studies described in Tables 73 and 74 (ISIS 540108) and is marked with an asterisk (*). These gapmers are designed by introducing modifications at the SNP site at position 9 of the oligonucleotides, as counted from the 5'-terminus and are created with a 5-7-5 motif.

[0893] The gapmers are described in Table 80. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "a", "b", "e", or "k" are sugar modified nucleosides. A subscript "a" indicates 2'-(ara)-F modified nucleoside, a subscript "b" indicates a 5'-Me DNA modified nucleoside, a subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside, and a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). .sup.xT indicates a 2-thio-thymidine nucleoside. Underline nucleoside or the number in parentheses indicates the position on the oligonucleotides opposite to the SNP position, as counted from the 5'-terminus.

TABLE-US-00078 TABLE 80 Modified oligonucleotides targeting HTT SNP Wing SEQ ISIS Gap chemistry ID NO Sequence (5' to 3') Chemistry 5' 3' NO. 540108* (9) A.sub.eT.sub.eA.sub.eA.sub.kA.sub.kTTGTCATC.sub.kA.sub.kC.sub.eC.sub.eA.s- ub.e Deoxy eeekk kkeee 32 XXXX28 (9) A.sub.eT.sub.eA.sub.eA.sub.kA.sub.kTTG.sup.xTCATC.sub.kA.sub.kC- .sub.eC.sub.eA.sub.e Deoxy/2- eeekk kkeee 32 thio XXXX29 (9) A.sub.eT.sub.eA.sub.eA.sub.kA.sub.kTTGT.sub.aCATC.sub.kA.sub.kC- .sub.eC.sub.eA.sub.e Deoxy/2'- eeekk kkeee 32 (ara)-F XXXX30 (9) A.sub.eT.sub.eA.sub.eA.sub.kA.sub.kTTGT.sub.bCATC.sub.kA.sub.kC- .sub.eC.sub.eA.sub.e Deoxy/5'- eeekk kkeee 32 Me-DNA e = 2'-MOE, k = cEt

Example 55

Chimeric Oligonucleotides Comprising Modifications at the Wing Regions Targeting HTT SNP

[0894] Additional gapmers are designed based on the gapmer selected from studies described in Tables 89 and 21 (ISIS 540107) and is marked with an asterisk (*). These gapmers are designed by introducing bicyclic modified nucleosides at the 3' or 5' terminus and are tested to evaluate if the addition of bicyclic modified nucleosides at the wing regions improves the activity and selectivity in inhibition of mutant HTT SNP.

[0895] The gapmers comprise a 5-7-5 motif and are described in Table 81. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e", or "k" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside, and a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt).

TABLE-US-00079 TABLE 81 Modified oligonucleotides targeting HTT SNP wing SEQ ISIS chemistry ID NO Sequence (5' to 3') Motif 5' 3' NO. 540107* A.sub.eT.sub.eA.sub.eA.sub.eA.sub.kTTGTCATC.sub.kA.sub.eC.sub.eC.s- ub.eA.sub.e 5-7-5 eeeek keeee 32 (eeeek-d7-keeee) XXXX31 A.sub.eT.sub.eA.sub.kA.sub.kA.sub.kTTGTCATC.sub.kA.sub.kC.sub.kC.su- b.eA.sub.e 5-7-5 eekkk kkkee 32 (eekkk-d7-kkkee) XXXX32 A.sub.eT.sub.eA.sub.eA.sub.eA.sub.kTTGTCATC.sub.eA.sub.eC.sub.eC.su- b.eA.sub.e 5-7-5 eeeek eeeee 32 (eeeek-d7-eeeee) XXXX33 A.sub.eT.sub.eA.sub.eA.sub.kA.sub.kTTGTCATC.sub.eA.sub.eC.sub.eC.su- b.eA.sub.e 5-7-5 eeekk eeeee 32 (eeekk-d7-eeeee) XXXX34 A.sub.eT.sub.eA.sub.kA.sub.kA.sub.kTTGTCATC.sub.eA.sub.eC.sub.eC.su- b.eA.sub.e 5-7-5 eekkk eeeee 32 (eekkk-d7-eeeee) XXXX35 A.sub.eT.sub.eA.sub.eA.sub.eA.sub.eTTGTCATC.sub.kA.sub.eC.sub.eC.su- b.eA.sub.e 5-7-5 eeeee keeee 32 (eeeee-d7-keeee) XXXX36 A.sub.eT.sub.eA.sub.eA.sub.eA.sub.eTTGTCATC.sub.kA.sub.kC.sub.eC.su- b.eA.sub.e 5-7-5 eeeee kkeee 32 (eeeee-d7-kkeee) XXXX37 A.sub.eT.sub.eA.sub.eA.sub.eA.sub.eTTGTCATC.sub.kA.sub.kC.sub.kC.su- b.eA.sub.e 5-7-5 eeeee kkkee 32 (eeeee-d7-kkkee) e = 2'-MOE; k = cEt; d = 2'-deoxyribonucleoside

Example 56

Chimeric Oligonucleotides Comprising Wing and Central Gap Modifications Targeting HTT SNP

[0896] Additional gapmers are designed based on the parent gapmer, ISIS 460209, wherein the central gap region comprises nine 2'-deoxyribonucleosides and is marked with an asterisk (*) in the table. These gapmers were designed by introducing modifications at the wings or the central gap region and are created with a 3-9-3 motif.

[0897] The gapmers are described in Table 82. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e", or "k" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside, and a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). .sup.PT indicates a 5-propyne thymidine nucleoside. .sup.PC indicates a 5-propyne cytosine nucleoside. Underline nucleoside or the number in parentheses indicates the position on the oligonucleotides opposite to the SNP position, as counted from the 5'-terminus.

TABLE-US-00080 TABLE 82 Modified oligonucleotides targeting HTT SNP wing SEQ ISIS chemistry ID NO Sequence (5' to 3') Modification 5' 3' NO 460209* (8) T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e Deoxy gap ekk kke 10 (3-9-3) 552103 (8) T.sub.eA.sub.eA.sub.eATTGTCATCA.sub.kC.sub.kC.sub.k Deoxy gap eee kkk 10 (3-9-3) 552104 (8) T.sub.kA.sub.kA.sub.kATTGTCATCA.sub.eC.sub.eC.sub.e Deoxy gap kkk eee 10 (3-9-3) 552105 (8) T.sub.eA.sub.kA.sub.kATTG.sup.PT.sup.PCATCA.sub.kC.sub.kC.sub.e Deoxy/5- ekk kke 10 Propyne 552106 (8) T.sub.eA.sub.kA.sub.kA.sup.PT.sup.PTG.sup.PT.sup.PCA.sup.PT.sup- .PCA.sub.kC.sub.kC.sub.e Deoxy/5- ekk kke 10 Propyne e = 2'-MOE; k = cEt

Example 57

Modified Oligonucleotides Comprising F-HNA Modification at the Central Gap or Wing Region Targeting HTT SNP

[0898] A series of modified oligonucleotides were designed based on ISIS 460209, wherein the central gap region contains nine 2'-deoxyribonucleosides. These modified oligonucleotides were designed by incorporating one or more F-HNA(s) modification within the central gap region or on the wing regions. The F-HNA containing oligonucleotides were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.

[0899] The modified oligonucleotides and their motifs are described in Table 83. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P.dbd.S). Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicate 2'-O-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript "k" indicate 6'-(S)--CH.sub.3 bicyclic nucleosides (e.g. cEt). Nucleosides followed by a subscript "z" indicate F-HNA modified nucleosides. .sup.mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 as counted from the 5'-terminus.

[0900] The gap-interrupted antisense oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 84.

[0901] The IC.sub.50 and selectivity were calculated using methods previously described in Example 41. The IC.sub.50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as `mut IC.sub.50`. The IC.sub.50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as `wt IC.sub.50`. Selectivity was calculated by dividing the IC.sub.50 for inhibition of the wild-type HTT versus the IC.sub.50 for inhibiting expression of the mutant HTT mRNA.

[0902] The parent gapmer, 460209 is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which the activity and selectivity of antisense oligonucleotides targeting nucleotides overlapping the SNP position could be compared.

[0903] As illustrated in Table 84, oligonucleotides comprising F-HNA modification(s) showed improvement in selectivity while maintaining activity as compared to the parent gapmer, ISIS 460209.

TABLE-US-00081 TABLE 83 Gap-interrupted antisense oligonucleotides targeting HTT SNP Wing SEQ ISIS Sequence Gap chemistry ID NO. (5' to 3') Motif chemistry 5' 3' NO. 460209* T.sub.eA.sub.kA.sup.kATTGT 3-9-3 Full deoxy ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 566266 T.sub.eA.sub.kA.sub.kA.sub.zTTGT 3-9-3 or Deoxy/F- ekk or kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 4-8-3 HNA ekkz 566267 T.sub.eA.sub.kA.sub.kAT.sub.zTGT 3-9-3 or Deoxy/F- ekk or kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 5-7-3 HNA ekkdz 566268 T.sub.eA.sub.kA.sub.kATT.sub.zGT 3-9-3 or Deoxy/F- ekk or kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 6-6-3 HNA ekkddz 566269 T.sub.eA.sub.kA.sub.kATTG.sub.zT 3-9-3 or Deoxy/F- ekk or kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 7-5-3 HNA ekkdddz 567369 T.sub.eA.sub.kA.sub.kA.sub.zT.sub.zTGT 3-9-3 or Deoxy/F- ekk or kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 5-7-3 HNA ekkzz e = 2'-MOE, k = cEt, d = 2'-.beta.-deoxyribonucleoside, z = F-HNA

TABLE-US-00082 TABLE 84 Comparison of inhibition of HTT mRNA levels and selectivity of gap-interrupted antisense oligonucleotides with ISIS 460209 targeting HTT SNP IC.sub.50 (.mu.M) Selectivity Gap Wing Chemistry ISIS NO Mut Wt (wt vs mut) Motif chemistry 5' 3' 460209* 0.28 3.1 11 3-9-3 Full deoxy ekk kke 566266 0.20 >10 >50 3-9-3 or Deoxy/F- ekk or ekkz kke 4-8-3 HNA 566267 0.90 >9.9 >11 3-9-3 or Deoxy/F- ekk or ekkdz kke 5-7-3 HNA 566268 1.0 >10 >10 3-9-3 or Deoxy/F- ekk or ekkddz kke 6-6-3 HNA 566269 1.7 >10.2 >6 3-9-3 or Deoxy/F- ekk or kke 7-5-3 HNA ekkdddz 567369 0.82 >9.8 >12 3-9-3 or Deoxy/F- ekk or ekkzz kke 5-7-3 HNA e = 2'-MOE, k = cEt, d = 2'-.beta.-deoxyribonucleoside, z = F-HNA

Example 58

Modified Oligonucleotides Comprising cEt Modification(s) at the Central Gap Region Targeting HTT SNP

[0904] A series of modified oligonucleotides were designed in the same manner as described in Example 57.

[0905] These modified oligonucleotides were designed by replacing F-HNA(s) with cEt modification(s) in the central gap region while maintaining the wing configuration. The modified oligonucleotides were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.

[0906] The modified oligonucleotides and their motifs are described in Table 85. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P.dbd.S). Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicate 2'-.beta.-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript "k" indicate 6'-(S)--CH.sub.3 bicyclic nucleosides (e.g. cEt). .sup.mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 as counted from the 5'-terminus.

[0907] The gap-interrupted antisense oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented below.

[0908] The IC.sub.50 and selectivity were calculated using methods previously described in Example 41. The IC.sub.50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as `mut IC.sub.50`. The IC.sub.50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as `wt IC.sub.50`. Selectivity was calculated by dividing the IC.sub.50 for inhibition of the wild-type HTT versus the IC.sub.50 for inhibiting expression of the mutant HTT mRNA.

[0909] As illustrated in Table 86, some of the newly designed antisense oligonucleotides (ISIS 575006, 575007, and 575008) showed improvement in potency and/or selectivity in inhibiting mut HTT mRNA levels comparing to ISIS 460209.

TABLE-US-00083 TABLE 85 Gap-interrupted antisense oligonucleotides targeting HTT SNP Wing SEQ ISIS Sequence Gap chemistry ID NO. (5' to 3') Motif chemistry 5' 3' NO. 460209* T.sub.eA.sub.kA.sub.kATTGT 3-9-3 Full deoxy ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 575006 T.sub.eA.sub.kA.sub.kA.sub.kTTGT 4-8-3 Full deoxy ekkk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 575007 T.sub.eA.sub.kA.sub.kAT.sub.kTGT 3-9-3 or Full deoxy or ekk or kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 5-7-3 Deoxy/cEt ekkdk 575133 T.sub.eA.sub.kA.sub.kATT.sub.kGT 3-9-3 or Full deoxy or ekk or kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 6-6-3 Deoxy/cEt ekkddk 575134 T.sub.eA.sub.kA.sub.kATTG.sub.kT 3-9-3 or Full deoxy or ekk or kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 7-5-3 Deoxy/cEt ekkdddk 575008 T.sub.eA.sub.kA.sub.kA.sub.kT.sub.kTGT 5-7-3 Deoxy ekkkk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e e = 2'-MOE, k = cEt, d = 2'-.beta.-deoxyribonucleoside

TABLE-US-00084 TABLE 86 Comparison of inhibition of HTT mRNA levels and selectivity of gap-interrupted antisense oligonucleotides with ISIS 460209 targeting HTT SNP IC.sub.50 (.mu.M) Selectivity Gap Wing Chemistry ISIS NO Mut Wt (wt vs mut) Motif chemistry 5' 3' 460209* 0.28 3.1 11 3-9-3 Full deoxy ekk kke 575006 0.27 3.8 14 4-8-3 Full deoxy ekkk kke 575007 0.67 >10.1 >15 3-9-3 or Full deoxy or ekk or kke 5-7-3 Deoxy/cEt ekkdk 575133 3.0 >9 >3 3-9-3 or Full deoxy or ekk or kke 6-6-3 Deoxy/cEt ekkddk 575134 2.6 >10.4 >4 3-9-3 or Full deoxy or ekk or kke 7-5-3 Deoxy/cEt ekkdddk 575008 0.18 >9.9 >55 5-7-3 Full deoxy ekkkk kke e = 2'-MOE, k = cEt, d = 2'-.beta.-deoxyribonucleoside

Example 59

Modified Oligonucleotides Comprising F-HNA Modification at the 3'-End of Central Gap Region Targeting HTT SNP

[0910] A series of modified oligonucleotides were designed based on ISIS 460209, wherein the central gap region contains nine 2'-deoxyribonucleosides. These modified oligonucleotides were designed by incorporating one F-HNA modification at the 3'-end of the central gap region. The F-HNA containing oligonucleotides were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting HTT SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.

[0911] The modified oligonucleotides and their motifs are described in Table 87. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P.dbd.S). Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicate 2'-.beta.-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript "k" indicate 6'-(S)--CH.sub.3 bicyclic nucleosides (e.g. cEt). Nucleosides followed by a subscript "z" indicate F-HNA modified nucleosides. .sup.mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 as counted from the 5'-terminus.

[0912] The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 88.

[0913] The IC.sub.50 and selectivity were calculated using methods previously described in Example 41. The IC.sub.50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as `mut IC.sub.50`. The IC.sub.50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as `wt IC.sub.50`. Selectivity was calculated by dividing the IC.sub.50 for inhibition of the wild-type HTT versus the IC.sub.50 for inhibiting expression of the mutant HTT mRNA.

[0914] As illustrated in Table 88, a couple of the newly designed antisense oligonucleotides (ISIS 575833 and 575834) showed improvement in selectivity while maintaining potency as compared to ISIS 460209. ISIS 575836 showed an increase in potency without improvement in selectivity while ISIS 575835 showed comparable selectivity without improvement in potency.

TABLE-US-00085 TABLE 87 Modified oligonucleotides targeting HTT SNP Gap Wing SEQ ISIS Sequence chem- chemistry ID NO. (5' to 3') Motif istry 5' 3' NO. 460209* T.sub.eA.sub.kA.sub.kATTGT 3-9-3 Full ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e deoxy 575833 T.sub.eA.sub.kA.sub.kATTGT 3-9-3 Deoxy/ ekk kke or 10 .sup.mC.sub.zAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e or F-HNA zdddkke 3-5-7 575834 T.sub.eA.sub.kA.sub.kATTGT 3-9-3 Deoxy/ ekk kke or 10 .sup.mCA.sub.zT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e or F-HNA zddkke 3-6-6 575835 T.sub.eA.sub.kA.sub.kATTGT 3-9-3 Deoxy/ ekk kke or 10 .sup.mCAT.sub.z.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e or F-HNA zdkke 3-7-5 575836 T.sub.eA.sub.kA.sub.kATTGT 3-9-3 Deoxy/ ekk kke or 10 .sup.mCAT.sup.mC.sub.zA.sub.k.sup.mC.sub.k.sup.mC.sub.e or F-HNA zkke 3-8-4 e = 2'-MOE, k = cEt, d = 2'-.beta.-deoxyribonucleoside, z = F-HNA

TABLE-US-00086 TABLE 88 Comparison of inhibition of HTT mRNA levels and selectivity of modified oligonucleotides with ISIS 460209 targeting HTT SNP IC.sub.50 (.mu.M) Selectivity Wing Chemistry ISIS NO Mut Wt (wt vs mut) Motif Gap chemistry 5' 3' 460209* 0.28 3.1 11 3-9-3 Full deoxy ekk kke 575833 0.22 4.2 19 3-9-3 or Deoxy/F-HNA ekk kke or 3-5-7 zdddkke 575834 0.30 6.3 21 3-9-3 or Deoxy/F-HNA ekk kke or 3-6-6 zddkke 575835 0.89 9.8 11 3-9-3 or Deoxy/F-HNA ekk kke or 3-7-5 zdkke 575836 0.09 0.4 4.6 3-9-3 or Deoxy/F-HNA ekk kke or zkke 3-8-4 e = 2'-MOE, k = cEt, d = 2'-.beta.-deoxyribonucleoside, z = F-HNA

Example 60

Short-Gap Chimeric Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0915] Additional chimeric antisense oligonucleotides were designed based on ISIS 460209 and ISIS 540094 wherein the central gap region contains nine 2'-deoxynucleosides. These gapmers were designed with the central gap region shortened by introducing cEt modifications to the wing regions, or interrupted by introducing cEt modifications at the 3'-end of the central gap region. The modified oligonucleotides were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting HTT SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209 and 540094.

[0916] The gapmers and their motifs are described in Table 89. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P.dbd.S). Nucleosides without a subscript are (3-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicate 2'-O-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript "k" indicate 6'-(S)--CH.sub.3 bicyclic nucleosides (e.g. cEt). .sup.mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 4 or 8 as counted from the 5'-terminus.

[0917] The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 90.

[0918] The IC.sub.50 and selectivity were calculated using methods previously described in Example 41. The IC.sub.50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as `mut IC.sub.50`. The IC.sub.50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as `wt IC.sub.50`. Selectivity was calculated by dividing the IC.sub.50 for inhibition of the wild-type HTT versus the IC.sub.50 for inhibiting expression of the mutant HTT mRNA.

[0919] As illustrated in Table 90, the newly designed antisense oligonucleotides (ISIS 575003) showed improvement in selectivity while maintaining potency as compared to ISIS 460209.

TABLE-US-00087 TABLE 89 Short-gap antisense oligonucleotides targeting HTT SNP Wing SEQ ISIS Sequence Gap chemistry ID NO. (5' to 3') Motif chemistry 5' 3' NO. 460209* T.sub.eA.sub.kA.sub.kATTGT 3-9-3 Full ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e deoxy 540094* T.sub.eT.sub.kGT.sup.mCAT.sup.mCA 2-9-4 Full ek kkke 67 .sup.mC.sup.mCA.sub.kG.sub.kA.sub.kA.sub.e deoxy 575003 T.sub.eT.sub.kGT.sup.mCAT.sup.mCA 2-8-5 Full ek kkkke 67 .sup.mC.sup.mC.sub.kA.sub.kG.sub.kA.sub.kA.sub.e deoxy 575004 T.sub.eT.sub.kGT.sup.mCAT.sup.mCA 2-9-4 Full ek kkke 67 .sup.mC.sub.k.sup.mCA.sub.kG.sub.kA.sub.kA.sub.e or deoxy or 2-7-6 or kdkkke Deoxy/cEt 575005 T.sub.eT.sub.kGT.sup.mCAT.sup.mCA 2-7-6 Full ek kkkkke 67 .sup.mC.sub.k.sup.mC.sub.kA.sub.kG.sub.kA.sub.kA.sub.e deoxy e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

TABLE-US-00088 TABLE 90 Comparison of inhibition of HTT mRNA levels and selectivity of modified oligonucleotides with ISIS 460209 targeting HTT SNP IC.sub.50 (.mu.M) Selectivity Wing Chemistry ISIS NO Mut Wt (wt vs mut) Motif Gap chemistry 5' 3' 460209* 0.34 3.3 9.7 3-9-3 Full deoxy ekk kke 540094* 0.17 2.4 14 2-9-4 Full deoxy ek kkke 575003 0.40 10 25 2-8-5 Full deoxy ek kkkke 575004 1.2 >9.6 >8 2-9-4 or Full deoxy or ek kkke or 2-7-6 Deoxy/cEt kdkkke 575005 >10 >100 >10 2-7-6 Full deoxy ek kkkkke e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 61

Short-Gap Chimeric Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0920] Additional chimeric antisense oligonucleotides were designed based on 15-mer, ISIS 460209 and 17-mer, ISIS 476333 wherein the central gap region contains nine 2'-deoxynucleosides. These gapmers were designed with the central gap region shortened at the 5'-end of the central gap region. The gapmers were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting HTT SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the gapmers were evaluated and compared to ISIS 460209 and ISIS 476333.

[0921] The gapmers and their motifs are described in Table 91. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P.dbd.S). Nucleosides without a subscript are (3-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicate 2'-O-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript "k" indicate 6'-(S)--CH.sub.3 bicyclic nucleosides (e.g. cEt). .sup.mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 or 9 as counted from the 5'-terminus.

[0922] The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 92.

[0923] The IC.sub.50 and selectivity were calculated using methods previously described in Example 41. The IC.sub.50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as `mut IC.sub.50`. The IC.sub.50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as `wt IC.sub.50`. Selectivity was calculated by dividing the IC.sub.50 for inhibition of the wild-type HTT versus the IC.sub.50 for inhibiting expression of the mutant HTT mRNA.

[0924] As illustrated in Table 92, a couple of the newly designed antisense oligonucleotides (ISIS 571036 and 571037) showed improvement in potency and selectivity in inhibiting mut HTT mRNA levels as compared to ISIS 460209 and 476333.

TABLE-US-00089 TABLE 91 Short-gap antisense oligonucleotides targeting HTT SNP Gap Wing SEQ ISIS Sequence chem- chemistry ID NO. (5' to 3') Motif istry 5' 3' NO. 460209* T.sub.eA.sub.kA.sub.kATTGT 3-9-3 Full ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e deoxy 476333* A.sub.eT.sub.kA.sub.eA.sub.kATTGT 4-9-4 Full ekek keke 32 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.e.sup.mC.sub.kA.sub.e deoxy 571036 A.sub.eT.sub.kA.sub.eA.sub.kA.sub.eT.sub.kTGT 6-7-4 Full ekekek keke 32 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.e.sup.mC.sub.kA.sub.e deoxy 571037 A.sub.eT.sub.eA.sub.eA.sub.eA.sub.kT.sub.kTGT 6-7-4 Full eeeekk keke 32 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.e.sup.mC.sub.kA.sub.e deoxy 571038 A.sub.eT.sub.kA.sub.eA.sub.kA.sub.eT.sub.eTGT 6-7-4 Full ekekee keke 32 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.e.sup.mC.sub.kA.sub.e deoxy e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

TABLE-US-00090 TABLE 92 Comparison of inhibition of HTT mRNA levels and selectivity of modified oligonucleotides with ISIS 460209 targeting HTT SNP Selectivity Wing IC.sub.50 (.mu.M) (wt vs Gap Chemistry ISIS NO Mut Wt mut) Motif chemistry 5' 3' 460209* 0.34 3.3 9.7 3-9-3 Full deoxy ekk kke 476333* 0.32 1.5 4.7 4-9-4 Full deoxy ekek keke 571036 0.17 >10.0 >59 6-7-4 Full deoxy ekekek keke 571037 0.11 >9.9 >90 6-7-4 Full deoxy eeeekk keke 571038 1.5 >10.5 >7 6-7-4 Full deoxy ekekee keke e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 62

Short-Gap Chimeric Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0925] Additional chimeric antisense oligonucleotides were designed based on 15-mer, ISIS 460209 wherein the central gap region contains nine 2'-deoxynucleosides. These gapmers were designed by having the central gap region shortened to seven 2'-deoxynucleosides. The gapmers were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting HTT SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the gapmers were evaluated and compared to ISIS 460209.

[0926] The gapmers and their motifs are described in Table 93. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P.dbd.S). Nucleosides without a subscript are 13-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicate 2'-O-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript "k" indicate 6'-(S)--CH.sub.3 bicyclic nucleosides (e.g. cEt). .sup.mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 or 9 as counted from the 5'-terminus.

[0927] The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 94.

[0928] The IC.sub.50 and selectivity were calculated using methods previously described in Example 41. The IC.sub.50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as `mut IC.sub.50`. The IC.sub.50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as `wt IC.sub.50`. Selectivity was calculated by dividing the IC.sub.50 for inhibition of the wild-type HTT versus the IC.sub.50 for inhibiting expression of the mutant HTT mRNA.

[0929] As illustrated in Table 94, each of the newly designed antisense oligonucleotides (ISIS 540108 and 571069) showed improvement in potency and/or selectivity in inhibiting mut HTT mRNA levels as compared to ISIS 460209.

TABLE-US-00091 TABLE 93 Short-gap antisense oligonucleotides targeting HTT SNP Gap Wing SEQ ISIS Sequence Mo- chem- chemistry ID NO. (5' to 3') tif istry 5' 3' NO. 460209 T.sub.eA.sub.kA.sub.kATTGT 3-9- Full ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3 deoxy 540108 A.sub.eT.sub.eA.sub.eA.sub.kA.sub.kTTGT 5-7- Full eeekk kkeee 32 .sup.mCAT.sup.mC.sub.kA.sub.k.sup.mC.sub.e.sup.mC.sub.eA.sub.e 5 deoxy 571069 A.sub.eT.sub.eA.sub.eA.sub.eA.sub.kT.sub.kTGT 6-7- Full eeeekk kkee 32 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.eA.sub.e 4 deoxy 571173 A.sub.eT.sub.eA.sub.kA.sub.kATTGT 4-7- Full eekk kkeeee 32 .sup.mCAT.sub.k.sup.mC.sub.kA.sub.e.sup.mC.sub.e.sup.mC.sub.eA.sub.e 6 deoxy 572773 T.sub.eA.sub.eA.sub.kA.sub.kTTGT 4-7- Full eekk kkee 10 .sup.mCAT.sup.mC.sub.kA.sub.k.sup.mC.sub.e.sup.mC.sub.e 4 deoxy e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

TABLE-US-00092 TABLE 94 Comparison of inhibition of HTT mRNA levels and selectivity of modified oligonucleotides with ISIS 460209 targeting HTT SNP Selectivity ISIS IC.sub.50 (.mu.M) (wt vs Gap Wing Chemistry NO Mut Wt mut) Motif chemistry 5' 3' 460209 0.34 3.3 9.7 3-9-3 Full deoxy ekk kke 540108 0.20 >10 >50 5-7-5 Full deoxy eeekk kkeee 571069 0.29 >9.9 >34 6-7-4 Full deoxy eeeekk kkee 571173 1.0 >10 >10 4-7-6 Full deoxy eekk kkeeee 572773 0.71 >7.8 11 4-7-4 Full deoxy eekk kkee e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 63

Short-Gap Chimeric Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0930] Additional chimeric antisense oligonucleotides were designed based on 15-mer, ISIS 460209 and 17-mer, ISIS 540108 wherein the central gap region contains nine and seven 2'-deoxynucleosides, respectively. These gapmers were designed by introducing one or more cEt modification(s) at the 5'-end of the central gap region. The gapmers were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting HTT SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the gapmers were evaluated and compared to ISIS 460209 and ISIS 540108.

[0931] The gapmers and their motifs are described in Table 95. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P.dbd.S). Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicate 2'-O-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript "k" indicate 6'-(S)--CH.sub.3 bicyclic nucleosides (e.g. cEt). .sup.mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 or 9 as counted from the 5'-terminus.

[0932] The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 96.

[0933] The IC.sub.50 and selectivity were calculated using methods previously described in Example 41. The IC.sub.50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as `mut IC.sub.50`. The IC.sub.50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as `wt IC.sub.50`. Selectivity was calculated by dividing the IC.sub.50 for inhibition of the wild-type HTT versus the IC.sub.50 for inhibiting expression of the mutant HTT mRNA.

[0934] As illustrated in Table 96, most of the newly designed oligonucleotides showed improvement in selectivity while maintaining potency as compared to 460209.

TABLE-US-00093 TABLE 95 Short-gap antisense oligonucleotides targeting HTT SNP Gap Wing SEQ ISIS Sequence chem- chemistry ID NO. (5' to 3') Motif istry 5' 3' NO. 460209 T.sub.eA.sub.kA.sub.kATTGT 3-9-3 Full ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e deoxy 540108 A.sub.eT.sub.eA.sub.eA.sub.kA.sub.kTTGT 5-7-5 Full eeekk kkeee 32 .sup.mCAT.sup.mC.sub.kA.sub.k.sup.mC.sub.e.sup.mC.sub.eA.sub.e deoxy 556872 A.sub.eT.sub.eA.sub.eA.sub.eA.sub.kTTGT 5-7-5 Full eeeek eeeee 32 .sup.mCAT.sup.mC.sub.eA.sub.e.sup.mC.sub.e.sup.mC.sub.eA.sub.e deoxy 556873 A.sub.eT.sub.eA.sub.eA.sub.kA.sub.kTTGT 5-7-5 Full eeekk eeeee 32 .sup.mCAT.sup.mC.sub.eA.sub.e.sup.mC.sub.e.sup.mC.sub.eA.sub.e deoxy 556874 A.sub.eT.sub.eA.sub.kA.sub.kA.sub.kTTGT 5-7-5 Full eekkk eeeee 32 .sup.mCAT.sup.mC.sub.eA.sub.e.sup.mC.sub.e.sup.mC.sub.eA.sub.e deoxy 568877 A.sub.eT.sub.kA.sub.kA.sub.kA.sub.kTTGT 5-7-5 Full ekkkk eeeee 32 .sup.mCAT.sup.mC.sub.eA.sub.e.sup.mC.sub.e.sup.mC.sub.eA.sub.e deoxy 568878 A.sub.kT.sub.kA.sub.kA.sub.kA.sub.kTTGT 5-7-5 Full kkkkk eeeee 32 .sup.mCAT.sup.mC.sub.eA.sub.e.sup.mC.sub.e.sup.mC.sub.eA.sub.e deoxy e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

TABLE-US-00094 TABLE 96 Comparison of inhibition of HTT mRNA levels and selectivity of modified oligonucleotides with ISIS 460209 targeting HTT SNP Wing IC.sub.50 (.mu.M) Selectivity Gap Chemistry ISIS NO Mut Wt (wt vs mut) Motif chemistry 5' 3' 460209 0.45 2.3 5.1 3-9-3 Full deoxy ekk kke 540108 0.25 9.5 38 5-7-5 Full deoxy eeekk kkeee 556872 1.0 9.9 9.9 5-7-5 Full deoxy eeeek eeeee 556873 0.67 3.4 5.1 5-7-5 Full deoxy eeekk eeeee 556874 0.38 1.9 5.0 5-7-5 Full deoxy eekkk eeeee 568877 0.44 6.2 14 5-7-5 Full deoxy ekkkk eeeee 568878 0.41 8.6 21 5-7-5 Full deoxy kkkkk eeeee e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 64

Short-Gap Chimeric Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0935] Additional chimeric antisense oligonucleotides were designed based on 15-mer, ISIS 460209 and 17-mer, ISIS 540108 wherein the central gap region contains nine and seven 2'-deoxynucleosides, respectively. These gapmers were designed by introducing one or more cEt modification(s) at the 3'-end of the central gap region. The gapmers were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting HTT SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the gapmers were evaluated and compared to ISIS 460209 and ISIS 540108.

[0936] The gapmers and their motifs are described in Table 97. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P.dbd.S). Nucleosides without a subscript are (3-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicate 2'-O-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript "k" indicate 6'-(S)--CH.sub.3 bicyclic nucleosides (e.g. cEt). .sup.mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 or 9 as counted from the 5'-terminus.

[0937] The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 98.

[0938] The IC.sub.50 and selectivity were calculated using methods previously described in Example 41. The IC.sub.50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as `mut IC.sub.50`. The IC.sub.50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as `wt IC.sub.50`. Selectivity was calculated by dividing the IC.sub.50 for inhibition of the wild-type HTT versus the IC.sub.50 for inhibiting expression of the mutant HTT mRNA.

[0939] As illustrated in Table 98, each of the newly designed oligonucleotides showed improvement in selective inhibition of mutant HTT mRNA levels compared to ISIS 460209. Comparable potency was observed for ISIS 568879 and 568880 while a slight loss in potency was observed for ISIS 556875, 556876 and 556877.

TABLE-US-00095 TABLE 97 Short-gap antisense oligonucleotides targeting HTT SNP Gap Wing SEQ ISIS Sequence chem- chemistry ID NO. (5' to 3') Motif istry 5' 3' NO. 460209 T.sub.eA.sub.kA.sub.kATTGT 3-9-3 Full ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e deoxy 540108 A.sub.eT.sub.eA.sub.eA.sub.kA.sub.kTTGT 5-7-5 Full eeekk kkeee 32 .sup.mCAT.sup.mC.sub.kA.sub.k.sup.mC.sub.e.sup.mC.sub.eA.sub.e deoxy 556875 A.sub.eT.sub.eA.sub.eA.sub.eA.sub.eTTGT 5-7-5 Full eeeee keeee 32 .sup.mCAT.sup.mC.sub.kA.sub.e.sup.mC.sub.e.sup.mC.sub.eA.sub.e deoxy 556876 A.sub.eT.sub.eA.sub.eA.sub.eA.sub.eTTGT 5-7-5 Full eeeee kkeee 32 .sup.mCAT.sup.mC.sub.kA.sub.k.sup.mC.sub.e.sup.mC.sub.eA.sub.e deoxy 556877 A.sub.eT.sub.eA.sub.eA.sub.eA.sub.eTTGT 5-7-5 Full eeeee kkkee 32 .sup.mCAT.sup.mC.sub.kA.sub.k.sup.mC.sub.k.sup.mC.sub.eA.sub.e deoxy 568879 A.sub.eT.sub.eA.sub.eA.sub.eA.sub.eTTGT 5-7-5 Full eeeee kkkke 32 .sup.mCAT.sup.mC.sub.kA.sub.k.sup.mC.sub.k.sup.mC.sub.kA.sub.e deoxy 568880 A.sub.eT.sub.eA.sub.eA.sub.eA.sub.eTTGT 5-7-5 Full eeeee kkkkk 32 .sup.mCAT.sup.mC.sub.kA.sub.k.sup.mC.sub.k.sup.mC.sub.kA.sub.k deoxy e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

TABLE-US-00096 TABLE 98 Comparison of inhibition of HTT mRNA levels and selectivity of modified oligonucleotides with ISIS 460209 targeting HTT SNP Selectivity Wing IC.sub.50 (.mu.M) (wt vs Gap Chemistry ISIS NO Mut Wt mut) Motif chemistry 5' 3' 460209 0.45 2.3 5.1 3-9-3 Full deoxy ekk kke 540108 0.25 9.5 38 5-7-5 Full deoxy eeekk kkeee 556875 1.9 >9.5 >5 5-7-5 Full deoxy eeeee keeee 556876 0.99 >9.9 >10 5-7-5 Full deoxy eeeee kkeee 556877 1.0 >10 >10 5-7-5 Full deoxy eeeee kkkee 568879 0.44 >10.1 >23 5-7-5 Full deoxy eeeee kkkke 568880 0.59 >10 >17 5-7-5 Full deoxy eeeee kkkkk e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 65

Modified Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0940] A series of modified oligonucleotides were designed based on the parent gapmer, ISIS 460209 wherein the central gap region contains nine 2'-deoxyribonucleosides. These modified oligonucleotides were designed by introducing various chemical modifications in the central gap region and were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to the parent gapmer, ISIS 460209.

[0941] The modified oligonucleotides were created with a 3-9-3 motif and are described in Table 99. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages, except for the internucleoside linkage having a subscript "p" which indicates a methyl phosphonate internucleoside linkage (--O--P(CH.sub.3)(.dbd.O)--O--). Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). .sup.mC indicates a 5-methyl cytosine nucleoside. .sup.xT indicates a 2-thio-thymidine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 as counted from the 5'-terminus.

[0942] The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2.times.PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 100.

[0943] The IC.sub.50 and selectivity were calculated using methods previously described in Example 41. The IC.sub.50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as `mut IC.sub.50`. The IC.sub.50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as `wt IC.sub.50`. Selectivity was calculated by dividing the IC.sub.50 for inhibition of the wild-type HTT versus the IC.sub.50 for inhibiting expression of the mutant HTT mRNA.

[0944] As illustrated in Table 100, improvement in selectivity with a slight decrease in potency was observed for the newly designed oligonucleotides as compared to ISIS 460209.

TABLE-US-00097 TABLE 99 Short-gap antisense oligonucleotides targeting HTT SNP Wing SEQ ISIS Sequence Gap chemistry ID NO. (5' to 3') chemistry 5' 3' NO. 460209 T.sub.eA.sub.kA.sub.kATTGT Full deoxy ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 556845 T.sub.eA.sub.kA.sub.kA.sup.xTTGT Deoxy/2-Thio ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 556847 T.sub.eA.sub.kA.sub.kA.sup.xT.sup.xTGT Deoxy/2-Thio ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 558257 T.sub.eA.sub.kA.sub.kATT.sub.pGT Deoxy/Methyl ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC Phosphonate 571125 T.sub.eA.sub.kA.sub.kA.sup.xTT.sub.pGT Deoxy/2-Thio/ ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e Methyl Phosphonate e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

TABLE-US-00098 TABLE 100 Comparison of inhibition of HTT mRNA levels and selectivity of modified oligonucleotides with ISIS 460209 targeting HTT SNP Wing IC.sub.50 (.mu.M) Selectivity Chemistry ISIS NO Mut Wt (wt vs mut) Gap chemistry 5' 3' 460209 0.56 3.8 6.8 Full deoxy ekk kke 556845 0.98 >9.8 >10 Deoxy/2-Thio ekk kke 556847 1.3 >10.4 >8 Deoxy/2-Thio ekk kke 558257 1.7 >10.2 >6 Deoxy/Methyl ekk kke Phosphonate 571125 1.8 >10.8 >6 Deoxy/2- ekk kke Thio/Methyl Phosphonate e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 66

Modified Oligonucleotides Comprising Chemical Modifications in the Central Gap Region Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0945] Additional chimeric antisense oligonucleotides were designed in the same manner as the antisense oligonucleotides described in Example 65. These gapmers were designed by introducing various modifications in the central gap region and were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to the parent gapmer, ISIS 460209.

[0946] The modified oligonucleotides and their motifs are described in Table 101. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages, except for the internucleoside linkage having a subscript "p" which indicates a methyl phosphonate internucleoside linkage (--O--P(CH.sub.3)(.dbd.O)--O--). Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). .sup.mC indicates a 5-methyl cytosine nucleoside. .sup.xT indicates a 2-thio-thymidine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 as counted from the 5'-terminus.

[0947] The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2.times.PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 102.

[0948] The IC.sub.50 and selectivity were calculated using methods previously described in Example 41. The IC.sub.50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as `mut IC.sub.50`. The IC.sub.50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as `wt IC.sub.50`. Selectivity was calculated by dividing the IC.sub.50 for inhibition of the wild-type HTT versus the IC.sub.50 for inhibiting expression of the mutant HTT mRNA.

[0949] As illustrated in Table 102, some of the newly designed oligonucleotides showed improvement in selectivity while maintaining potency as compared to 460209.

TABLE-US-00099 TABLE 101 Short-gap antisense oligonucleotides targeting HTT SNP Wing SEQ ISIS Sequence Gap chemistry ID NO. (5' to 3') Motif chemistry 5' 3' NO. 460209 T.sub.eA.sub.kA.sub.kATTGT 3-9-3 Full deoxy ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 551429 T.sub.eA.sub.eA.sub.eA.sub.kT.sub.kTGT 5-7-3 Full deoxy eeekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 571122 T.sub.eA.sub.eA.sub.eA.sub.k.sup.xTTGT 4-8-3 Deoxy/2-Thio eeek kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 571123 T.sub.eA.sub.eA.sub.eA.sub.kT.sub.kT.sub.pGT 5-7-3 Deoxy/Methyl eeekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e Phosphonate 571124 T.sub.eA.sub.eA.sub.eA.sub.k.sup.xTT.sub.pGT 4-8-3 Deoxy/2- eeek kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e Thio/Methyl Phosphonate 579854 T.sub.eA.sub.eA.sub.eA.sub.kTT.sub.pGT 4-8-3 Deoxy/Methyl eeek kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e Phosphonate 566282 T.sub.eA.sub.kA.sub.kA.sub.dxT.sub.dxT.sub.dG.sub.dT.sub.d.sup.mC.s- ub.d 3-9-3 Deoxy/Methyl ekk kke 10 A.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e Phosphonate e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

TABLE-US-00100 TABLE 102 Comparison of inhibition of HTT mRNA levels and selectivity of modified oligonucleotides with ISIS 460209 targeting HTT SNP Selectivity Wing ISIS IC.sub.50 (.mu.M) (wt vs Chemistry NO Mut Wt mut) Motif Gap chemistry 5' 3' 460209 0.56 3.8 6.8 3-9-3 Full deoxy ekk kke 551429 0.50 >10 >20 5-7-3 Full deoxy eeekk kke 571122 1.8 >10.8 >6 4-8-3 Deoxy/2-Thio eeek kke 571123 0.96 >9.6 >10 5-7-3 Deoxy/Methyl eeekk kke Phosphonate 571124 2.3 >9.2 >4 4-8-3 Deoxy/2- eeek kke Thio/Methyl Phosphonate 579854 0.63 >10.1 >16 4-8-3 Deoxy/Methyl eeek kke Phosphonate 566282 0.51 6.3 12.4 3-9-3 Deoxy/Methyl ekk kke Phosphonate e = 2'-MOE, k = cEt

Example 67

Modified Oligonucleotides Comprising Chemical Modifications in the Central Gap Region Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0950] Additional chimeric antisense oligonucleotides were designed in the same manner as the antisense oligonucleotides described in Example 65. These gapmers were designed by introducing various modifications in the central gap region and were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to the parent gapmer, ISIS 460209.

[0951] The modified oligonucleotides and their motifs are described in Table 103. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages, except for the internucleoside linkage having a subscript "p" which indicates a methyl phosphonate internucleoside linkage (--O--P(CH.sub.3)(.dbd.O)--O--). Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). .sup.mC indicates a 5-methyl cytosine nucleoside. .sup.xT indicates a 2-thio-thymidine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 or 9 as counted from the 5'-terminus.

[0952] The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2.times.PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 104.

[0953] The IC.sub.50 and selectivity were calculated using methods previously described in Example 41. The IC.sub.50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as `mut IC.sub.50`. The IC.sub.50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as `wt IC.sub.50`. Selectivity was calculated by dividing the IC.sub.50 for inhibition of the wild-type HTT versus the IC.sub.50 for inhibiting expression of the mutant HTT mRNA.

[0954] As illustrated in Table 104, all but one of the newly designed oligonucleotides showed improvement in selectivity while maintaining potency as compared to ISIS 460209.

TABLE-US-00101 TABLE 103 Short-gap antisense oligonucleotides targeting HTT SNP Wing SEQ ISIS Sequence Gap chemistry ID NO. (5' to 3') Motif chemistry 5' 3' NO. 460209 T.sub.eA.sub.kA.sub.kATTGT 3-9-3 Full ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e deoxy 476333 A.sub.eT.sub.kA.sub.eA.sub.kATTGT 4-9-4 Full ekek keke 32 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.e.sup.mC.sub.kA.sub.e deoxy 571039 A.sub.eT.sub.kA.sub.eA.sub.kA.sup.xTTGT 4-9-4 Deoxy/ ekek keke 32 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.e.sup.mC.sub.kA.sub.e 2-Thio 571171 A.sub.eT.sub.kA.sub.eA.sub.kATT.sub.pGT 4-9-4 Deoxy/ ekek keke 32 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.e.sup.mC.sub.kA.sub.e Methyl Phospho- nate 571041 A.sub.eT.sub.kA.sub.eA.sub.kA.sup.xTT.sub.pGT 4-9-4 Deoxy/2- ekek keke 32 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.e.sup.mC.sub.kA.sub.e Thio/ Methyl Phospho- nate e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

TABLE-US-00102 TABLE 104 Comparison of inhibition of HTT mRNA levels and selectivity of modified oligonucleotides with ISIS 460209 targeting HTT SNP ISIS IC.sub.50 (.mu.M) Selectivity Gap Wing Chemistry NO Mut Wt (wt vs mut) chemistry 5' 3' 460209 0.56 3.8 6.8 Full deoxy ekk kke 476333 0.56 3.4 6.1 Full deoxy ekek keke 571039 0.34 >9.9 >29 Deoxy/2-Thio ekek keke 571171 0.54 >10.3 >19 Deoxy/Methyl ekek keke Phosphonate 571041 0.75 >9.8 >13 Deoxy/2- ekek keke Thio/Methyl Phosphonate e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 68

Selectivity in Inhibition of HTT mRNA Levels Targeting SNP by Gap-Interrupted Modified Oligonucleotides

[0955] Additional modified oligonucleotides were designed based on the parent gapmer, ISIS 460209 wherein the central gap region contains nine 2'-deoxyribonucleosides. These modified oligonucleotides were designed by introducing one or more modified nucleobase(s) in the central gap region and were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.

[0956] The modified oligonucleotides were created with a 3-9-3 motif and are described in Table 105. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). .sup.mC indicates a 5-methyl cytosine nucleoside. .sup.xT indicates a 2-thio-thymidine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 as counted from the 5'-terminus.

[0957] The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2.times.PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.

[0958] The IC.sub.50 and selectivity were calculated using methods previously described in Example 41. The IC.sub.50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as `mut IC.sub.50`. The IC.sub.50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as `wt IC.sub.50`. Selectivity was calculated by dividing the IC.sub.50 for inhibition of the wild-type HTT versus the IC.sub.50 for inhibiting expression of the mutant HTT mRNA.

[0959] As illustrated in Table 106, ISIS 556845 showed improvement in selectivity and potency as compared to ISIS 460209. ISIS 556847 showed improvement in selectivity with comparable potency while ISIS 556846 showed improvement in potency with comparable selectivity.

TABLE-US-00103 TABLE 105 Gap-interrupted modified oligonucleotides targeting HTT SNP Wing SEQ ISIS Sequence Gap chemistry ID NO. (5' to 3') chemistry 5' 3' NO. 460209 T.sub.eA.sub.kA.sub.kATTGT Full ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e deoxy 556845 T.sub.eA.sub.kA.sub.kA.sup.xTTGT Deoxy/ ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 2-Thio 556846 T.sub.eA.sub.kA.sub.kAT.sup.xTGT Deoxy/ ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 2-Thio 556847 T.sub.eA.sub.kA.sub.kA.sup.xT.sup.xTGT Deoxy/ ekk kke 10 .sup.mCAT.sup.mCA.sub.k.sup.mC.sub.k.sup.mC.sub.e 2-Thio e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

TABLE-US-00104 TABLE 106 Comparison of inhibition of HTT mRNA levels and selectivity of gap-interrupted modified oligonucleotides with ISIS 460209 targeting HTT SNP ISIS IC.sub.50 (.mu.M) Selectivity Gap Wing Chemistry NO Mut Wt (wt vs mut) chemistry 5' 3' 460209 0.30 0.99 3.3 Full deoxy ekk kke 556845 0.13 10.01 >77 Deoxy/2-Thio ekk kke 556846 0.19 0.48 2.5 Deoxy/2-Thio ekk kke 556847 0.45 9.9 >22 Deoxy/2-Thio ekk kke e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 69

Evaluation of Modified Oligonucleotides Targeting HTT SNP--In Vivo Study

[0960] Additional modified oligonucleotides were selected and tested for their effects on mutant and wild type HTT protein levels in vivo targeting various SNP sites as illustrated below.

[0961] The gapmers and their motifs are described in Table 107. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" or "k" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside and a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt).

[0962] The gapmer, ISIS 460209 was included in the study as a benchmark oligonucleotide against which the potency and selectivity of the modified oligonucleotides could be compared. A non-allele specific oligonucleotide, ISIS 387898, was used as a positive control.

[0963] Hu97/18 mice, the first murine model of HD that fully genetically recapitulates human HD were used in the study. They were generated in Hayden's lab by cross bred BACHD, YAC 18 and Hdh (-/-) mice.

[0964] Hu97/18 mice were treated with 300 .mu.g of modified oligonucleotides by a single unilateral intracerebroventricular (ICV) bolus injection. This treatment group consisted of 4 animals/oligonucleotide. The control group received a 10 .mu.l bolus injection of sterile PBS and consisted of 4 animals.

[0965] Animals were sacrificed at 4 weeks post-injection. The second most anterior 2 mm coronal slab for each brain hemisphere was collected using a 2 mm rodent brain matrix. The remaining portion of the brain was post-fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose and sectioned into 25 .mu.m coronal sections for immunohistochemical analysis.

[0966] The HTT protein levels were analyzed by high molecular weight western blot (modified from Invitrogen's NuPAGE Bis-Tris System Protocol). The tissue was homogenized in ice cold SDP lysis buffer. 40 .mu.g of total protein lysate was resolved on 10% low-BIS acrylamide gels (200:1 acrylamide:BIS) with tris-glycine running buffer (25 mM Tris, 190 mM Glycince, 0.1% SDS) containing 10.7 mM .beta.-mercaptoethanol added fresh. Gels were run at 90V for 40 min through the stack, then 190V for 2.5 h, or until the 75 kDa molecular weight marker band was at the bottom of the gel. Proteins were transferred to nitrocellulose at 24V for 2 h with NuPage transfer buffer (Invitrogen: 25 mM Bicine, 25 mM Bis-Tris, 1.025 mM EDTA, 5% MeOH, pH 7.2). Membranes were blocked with 5% milk in PBS, and then blotted for HTT with MAB2166 (1:1000, millipore). Anti-calnexin (Sigma C4731) immunoblotting was used as loading control. Proteins were detected with IR dye 800CW goat anti-mouse (Rockland 610-131-007) and AlexaFluor 680 goat anti-rabbit (Molecular Probes A21076)-labeled secondary antibodies, and the LiCor Odyssey Infrared Imaging system.

[0967] The results in Table 108 are presented as the average percent of HTT protein levels for each treatment group, normalized to PBS-treated control and is denoted as "% UTC". The percent of mutant HTT protein levels is denoted as "mut". The percent of wild-type HTT protein levels is denoted as "wt". Selectivity was also evaluated and measured by dividing the percent of wild-type HTT protein levels vs. the percent of the mutant HTT protein levels.

[0968] As illustrated in Table 108, treatment with the newly designed oligonucleotides, ISIS 476333 and 460085 showed improvement in potency and selectivity in inhibiting mutant HTT protein levels as compared to the parent gapmer, 460209. Comparable or a slight loss in potency and/or selectivity was observed for the remaining oligonucleotides.

TABLE-US-00105 TABLE 107 Modified oligonucleotides targeting HTT rs7685686, rs4690072 and rs363088 in Hu97/18 mice Wing SEQ ISIS Chemistry ID NO Sequence (5' to 3') Motif 5' 3' NO. 387898 C.sub.eT.sub.eC.sub.eG.sub.eA.sub.eCTAAAGCAGGA.sub.eT.sub.eT.sub.eT- .sub.eC.sub.e 5-10-5 e5 e5 79 460209 T.sub.eA.sub.kA.sub.kATTGTCATCA.sub.kC.sub.kC.sub.e 3-9-3 ekk kke 10 435879 AeA.sub.eT.sub.eA.sub.eA.sub.eATTGTCATCA.sub.eC.sub.eC.sub.eA.sub.e- G.sub.e 5-9-5 e5 e5 80 476333 A.sub.eT.sub.kA.sub.eA.sub.kATTGTCATCA.sub.kC.sub.eCkA.sub.e 4-9-4 ekek keke 32 435874 C.sub.eA.sub.eC.sub.eA.sub.eG.sub.eTGCTACCCAA.sub.eC.sub.eC.sub.eT.- sub.eT.sub.e 5-9-5 e5 e5 81 435871 T.sub.eC.sub.eA.sub.eC.sub.eA.sub.eGCTATCTTCT.sub.eC.sub.eA.sub.eT.- sub.eC.sub.e 5-9-5 e5 e5 82 460085 A.sub.eT.sub.eA.sub.eA.sub.eA.sub.eTTGTCATC.sub.eA.sub.eC.sub.eC.su- b.eA.sub.e 5-7-5 e5 e5 32 e = 2'-MOE (e.g. e5 = eeeee), k = cEt

TABLE-US-00106 TABLE 108 Effects of modified oligonucleotides on mutant and wild type HTT protein levels in Hu97/18 mice Dosage % UTC Selectivity ISIS NO SNP site (.mu.g) mut wt (wt vs mut) PBS -- 300 100 100 1 387898 -- 300 23.76 25.66 1 460209 rs7685686 300 18.16 48.99 2.7 435879 rs7685686 300 41.48 73.11 1.8 476333 rs7685686 300 6.35 22.05 3.5 460085 rs7685686 300 2.9 40.1 13.8 435874 rs4690072 300 44.18 76.63 1.7 435871 rs363088 300 33.07 89.30 2.7

Example 70

Evaluation of ISIS 435871 in Central Nervous System (CNS) Targeting HTT Rs363088--In Vivo Study

[0969] A modified oligonucleotide from Example 68, ISIS 435871 was selected and tested for its effects on mutant and wild type HTT protein levels in the CNS in vivo targeting rs363088.

[0970] Hu97/18 mouse was treated with 300 .mu.g of ISIS 435871 by a single unilateral intracerebroventricular (ICV) bolus injection. The animal was sacrificed at 4 weeks post-injection. Regional CNS structures were then micro-dissected including bilateral samples from the most anterior portion of cortex (Cortex 1), an intermediate section of cortex (Cortex 2), the most posterior section of cortex (Cortex 3), the striatum, the hippocampus, the cerebellum, and a 1 cm section of spinal cord directly below the brain stem. Tissue was homogenized and assessed for mutant and wild-type HTT levels by Western blotting using the procedures as described in Example 69. The results are presented below. As no untreated or vehicle treated control is shown, HTT intensity of each allele is expressed as a ratio of calnexin loading control intensity. The ratio of the mutant HTT to the wt HTT in the treated animal was determined and is denoted as "wt/mut". Having a ratio higher than 1 is indicative of allele-specific silencing.

[0971] As illustrated in Table 109, a single unilateral ICV bolus injection of the modified antisense oligonucleotide showed selective HTT silencing throughout the CNS except in the cerebellum, where the antisense oligonucleotide did not distribute evenly.

TABLE-US-00107 TABLE 109 Effects of ISIS 435871 on mutant and wild type HTT protein levels in CNS targeting rs363088 in Hu97/18 mice HTT intensity/calnexin intensity Tissue wt mut wt/mut Cortex 1 0.032 0.014 2.29 Cortex 2 0.027 0.009 3 Cortex 3 0.023 0.007 3.29 Striatum 0.030 0.012 2.5 Hippocampus 0.016 0.006 2.67 Cerebellum 0.023 0.019 1.21 Spinal Cord 0.014 0.007 2

Example 71

Evaluation of Modified Oligonucleotides Targeting HTT Rs7685686--In Vivo Study

[0972] Several modified oligonucleotides from Examples 43, 51, 52, 53 and 66 were selected and tested for their effects on mutant and wild type HTT protein levels in vivo targeting HTT rs7685686.

[0973] The gapmer, ISIS 460209 was included in the study as a benchmark oligonucleotide against which the potency and selectivity of the modified oligonucleotides could be compared.

[0974] Hu97/18 mice were treated with 300 .mu.g of modified oligonucleotides by a single unilateral intracerebroventricular (ICV) bolus injection. This treatment group consisted of 4 animals/oligonucleotide. The control group received a 10 .mu.l bolus injection of sterile PBS and consisted of 4 animals.

[0975] Animals were sacrificed at 4 weeks post-injection. The second most anterior 2 mm coronal slab for each brain hemisphere was collected using a 2 mm rodent brain matrix. The HTT protein levels were analyzed in the same manner as described in Example 69 and the results are presented below.

[0976] The results in Table 110 are presented as the average percent of HTT protein levels for each allele and treatment group, normalized to PBS-treated control and is denoted as "% UTC". The percent of mutant HTT protein levels is denoted as "mut". The percent of wild-type HTT protein levels is denoted as "wt".

[0977] As shown in Table 110, each of the newly designed oligonucleotides showed improvement in selective inhibition of mutant HTT protein levels as compared to ISIS 460209. ISIS 550913 and 540095 showed improvement in potency while the remaining modified oligonucleotides showed comparable or a slight decrease in potency as compared to the parent gapmer.

TABLE-US-00108 TABLE 110 Effects of modified oligonucleotides on mutant and wild type HTT protein levels targeting rs7685686 in Hu97/18 mice Wing SEQ ISIS % UTC chemistry Gap ID NO mut wt Motif 5' 3' chemistry NO PBS 100 100 -- -- -- -- -- 460209 18.16 48.99 3-9-3 ekk kke Full deoxy 10 550913 9.31 34.26 5-9-5 kkekk kkekk Full deoxy 27 540095 12.75 106.05 2-9-4 ek kkke Full deoxy 65 551429 19.07 108.31 5-7-3 eeekk kke Full deoxy 10 540094 24.68 87.56 2-9-4 ek kkke Full deoxy 67 540096 24.89 98.26 2-9-4 ek kkke Full deoxy 68 540108 28.34 85.62 5-7-5 eeekk kkeee Full deoxy 23 e = 2'-MOE, k = cEt

Example 72

Evaluation of Modified Oligonucleotides Targeting HTT Rs7685686--In Vivo Study

[0978] Several modified oligonucleotides selected from Examples 57, 58, 61 and 62 were tested and evaluated for their effects on mutant and wild type HTT protein levels in vivo targeting HTT rs7685686.

[0979] Hu97/18 mice were treated with 300 .mu.g of modified oligonucleotides by a single unilateral intracerebroventricular (ICV) bolus injection and the control group received a 10 .mu.l bolus injection of sterile PBS. Each treatment group consisted of 4 animals.

[0980] Animals were sacrificed at 4 weeks post-injection. The second most anterior 2 mm coronal slab for each brain hemisphere was collected using a 2 mm rodent brain matrix. The HTT protein levels were analyzed in the same manner as described in Example 69. The in vivo study for ISIS 575008 and 571069 marked with an asterisk (*) was performed independently and the results are presented below.

[0981] The results in Table 111 are presented as the average percent of HTT protein levels for each allele and treatment group, normalized to PBS-treated control and is denoted as "% UTC". The percent of mutant HTT protein levels is denoted as "mut". The percent of wild-type HTT protein levels is denoted as "wt".

[0982] As illustrated in Table 111, selective inhibition of mut HTT protein levels was achieved with the newly designed oligonucleotide treatment as compared to PBS treated control.

TABLE-US-00109 TABLE 111 Effects of modified oligonucleotides on mutant and wild type HTT protein levels targeting rs7685686 in Hu97/18 mice Wing SEQ ISIS % UTC chemistry Gap ID NO mut wt Motif 5' 3' chemistry NO PBS 100 100 -- -- -- -- -- 575007 26.9 104.5 3-9-3 ekk kke Deoxy/cEt 10 575008* 21.7 105.9 5-7-3 ekkkk kke Deoxy/cEt 10 566267 32.8 109.3 3-9-3 ekk kke Deoxy/F- 10 HNA 571036 30.3 103.3 6-7-4 ekekek keke Full deoxy 32 571037 32.8 111.9 6-7-4 eeeekk keke Full deoxy 32 571069* 29.4 109.8 6-7-4 eeeekk kkee Full deoxy 32 e = 2'-MOE, k = cEt

Example 73

Evaluation of Modified Oligonucleotides Targeting HTT Rs7685686--In Vivo Dose Response Study

[0983] ISIS 476333, 435871, 540108, 575007 and 551429 from previous examples were selected and evaluated at various doses for their effect on mutant and wild type HTT protein levels in vivo targeting HTT rs7685686.

[0984] Hu97/18 mice were treated with various doses of modified oligonucleotides as presented in Table 112 by a single unilateral intracerebroventricular (ICV) bolus injection. This treatment group consisted of 4 animals/oligonucleotide. The control group received a 10 .mu.l bolus injection of sterile PBS and consisted of 4 animals.

[0985] Animals were sacrificed at 4 weeks post-injection. The second most anterior 2 mm coronal slab for each brain hemisphere was collected using a 2 mm rodent brain matrix. The HTT protein levels were analyzed in the same manner as described in Example 69. The dose response study was performed independently for each modified oligonucleotide and the results are presented below.

[0986] The results in Table 112 are presented as the average percent of HTT protein levels for each allele and treatment group, normalized to PBS-treated control and is denoted as "% UTC". The percent of mutant HTT protein levels is denoted as "mut". The percent of wild-type HTT protein levels is denoted as "wt".

[0987] As illustrated in Table 112, selective inhibition of mut HTT protein levels was achieved in a dose-dependent manner for the newly designed oligonucleotides.

TABLE-US-00110 TABLE 112 Dose-dependent effect of modified oligonucleotides on mutant and wild type HTT protein levels targeting rs7685686 in Hu97/18 mice Dosage % UTC SEQ ISIS NO (.mu.g) mut wt Motif ID NO. PBS 0 100 100 -- 476333 50 48.7 115 4-9-4 32 150 23.1 53.3 (ekek-d9-keke) 300 8.8 36.7 435871 75 114 118 5-9-5 82 150 47.3 80.3 (e5-d9-e5) 300 33 89.3 500 36 97.5 540108 75 30.5 71.7 5-7-5 32 150 22 81 (eeekk-d7-kkeee) 300 8.6 59.6 575007 150 41.5 110.7 3-9-3 10 300 29 119.4 (ekk-d-k-d7-kke) (deoxy gap interrupted with cEt) 551429 75 58 101.3 5-7-3 10 150 36.2 110.4 (eeekk-d7-kke) 300 19.7 107.8 e = 2'-MOE (e.g. e5 = eeeee), k = cEt, d = 2'-deoxyribonucleoside

Example 74

Modified Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)

[0988] A series of modified oligonucleotides was designed based on a parent gapmer, ISIS 460209, wherein the central gap region contains nine .beta.-D-2'-deoxyribonucleosides. The modified oligonucleotides were designed by introducing a 5'-(R)-Me DNA modification within the central gap region. The 5'-(R)-Me DNA containing oligonucleotides were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact. The potency and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.

[0989] The position on the oligonucleotides opposite to the SNP position, as counted from the 5'-terminus is position 8.

[0990] The modified oligonucleotides were created with a 3-9-3 motif and are described in Table 113. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. Nucleosides followed by a subscript "d" are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). Nucleosides followed by a subscript "z" indicates a 5'-(R)-Me DNA. ".sup.mC" indicates a 5-methyl cytosine nucleoside.

[0991] The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with a single dose at 2 .mu.M concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2.times.PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.

[0992] The IC.sub.50s and selectivities as expressed in "fold" were measured and calculated using methods described previously in Example 41. As illustrated in Table 114, treatment with the newly designed oligonucleotides showed comparable or a slight increase in potency and/or selectivity as compared to ISIS 460209.

TABLE-US-00111 TABLE 113 Gap-interrupted oligonucleotides comprising 5'-(R)-Me DNA targeting HTT SNP Wing SEQ ISIS chemistry ID NO. Sequence (5' to 3') Gap chemistry 5' 3' NO. 460209 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e Full deoxy ekk kke 10 556848 T.sub.eA.sub.kA.sub.kA.sub.zT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e Deoxy/5'-(R)-Me DNA ekk kke 10 556849 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.zT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e Deoxy/5'-(R)-Me DNA ekk kke 10 556850 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.zG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e Deoxy/5'-(R)-Me DNA ekk kke 10 e = 2'-MOE, k = cEt

TABLE-US-00112 TABLE 114 Comparison of inhibition of HTT mRNA levels and selectivity of gap- interrupted oligonucleotides with ISIS 460209 targeting HTT SNP IC.sub.50 Wing ISIS (.mu.M) Selectivity Gap chemistry NO. Mut Wt (wt vs mut) chemistry 5' 3' 460209 0.30 0.99 3.3 Full deoxy ekk kke 556848 0.15 0.6 4.0 Deoxy/5'-(R)- ekk kke Me DNA 556849 0.16 0.46 2.9 Deoxy/5'-(R)- ekk kke Me DNA 556850 0.33 0.96 2.9 Deoxy/5'-(R)- ekk kke Me DNA e = 2'-MOE, k = cEt

Example 75

Modified Oligonucleotides Comprising 5'-(R)- or 5'-(S)-Me DNA Modification Targeting HTT SNP

[0993] A series of modified oligonucleotides was designed based on a parent gapmer, ISIS 460209, wherein the central gap region contains nine .beta.-D-2'-deoxyribonucleosides. The modified oligonucleotides were designed by introducing 5'-(S)- or 5'-(R)-Me DNA modification slightly upstream or downstream (i.e. "microwalk") within the central gap region. The gapmers were created with a 3-9-3 motif and were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression. The potency and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.

[0994] The position on the oligonucleotides opposite to the SNP position, as counted from the 5'-terminus is position 8.

[0995] The modified oligonucleotides and their motifs are described in Table 115. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. Nucleosides followed by a subscript "d" are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). Nucleosides followed by a subscript "v" indicates a 5'-(S)-Me DNA. Nucleosides followed by a subscript "z" indicates a 5'-(R)-Me DNA. ".sup.mC" indicates a 5-methyl cytosine nucleoside.

[0996] The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.1, 0.4, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2.times.PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented below.

[0997] The IC.sub.50s and selectivities as expressed in "fold" were measured and calculated using methods described previously in Example 41. The results in Table 116 demonstrated that each of the newly designed oligonucleotides comprising 5'-(S)- or 5'-(R)-Me DNA within the central gap region achieved improvement in potency and selectivity as compared to the parent gapmer, ISIS 460209.

TABLE-US-00113 TABLE 115 Gap-interrupted oligonucleotides comprising 5'-(S)- or 5'-(R)-Me DNA targeting HTT SNP Wing SEQ ISIS Gap Chemistry ID NO Sequence (5' to 3') Motif Chemistry 5' 3' NO 460209 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Full deoxy ekk kke 10 589429 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.vT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(S)- ekk kke 10 Me DNA 589430 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.vG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(S)- ekk kke 10 Me DNA 589431 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.v.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(S)- ekk kke 10 Me DNA 589432 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.v.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(S)- ekk kke 10 Me DNA 594588 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.vT.sub.vG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(S)- ekk kke 10 Me DNA 556848 T.sub.eA.sub.kA.sub.kA.sub.zT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 Me DNA 556849 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.zT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 Me DNA 556850 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.zG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 Me DNA 539558 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.z.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 Me DNA 594160 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .zA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 Me DNA 594161 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.zT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 Me DNA 589433 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.z.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 Me DNA 594162 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.zA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 Me DNA 594589 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.zT.sub.zG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 Me DNA e = 2'-MOE; k = cEt

TABLE-US-00114 TABLE 116 Comparison of inhibition of HTT mRNA levels and selectivity of gap- interrupted oligonucleotides with ISIS 460209 targeting HTT SNP ISIS IC.sub.50 (.mu.M) Selectivity Wing Chemistry NO. Mut Wt (wt vs. mut) Motif Gap Chemistry 5' 3' 460209 1.2 1.4 1.2 3-9-3 Full deoxy ekk kke 589429 0.22 3.3 15 3-9-3 Deoxy/5'-(S)-Me DNA ekk kke 589430 0.22 >10 >45.5 3-9-3 Deoxy/5'-(S)-Me DNA ekk kke 589431 0.16 1.9 11.9 3-9-3 Deoxy/5'-(S)-Me DNA ekk kke 589432 0.23 >10 >43.5 3-9-3 Deoxy/5'-(S)-Me DNA ekk kke 594588 0.81 >10 >12.3 3-9-3 Deoxy/5'-(S)-Me DNA ekk kke 556848 0.16 1.8 11.3 3-9-3 Deoxy/5'-(R)-Me DNA ekk kke 556849 0.14 1.1 7.9 3-9-3 Deoxy/5'-(R)-Me DNA ekk kke 556850 0.22 1.7 7.7 3-9-3 Deoxy/5'-(R)-Me DNA ekk kke 539558 0.38 3.8 10 3-9-3 Deoxy/5'-(R)-Me DNA ekk kke 594160 0.28 3.3 11.8 3-9-3 Deoxy/5'-(R)-Me DNA ekk kke 594161 0.28 >10 >35.7 3-9-3 Deoxy/5'-(R)-Me DNA ekk kke 589433 0.27 4.4 16.3 3-9-3 Deoxy/5'-(R)-Me DNA ekk kke 594162 0.27 3.5 13.0 3-9-3 Deoxy/5'-(R)-Me DNA ekk kke 594589 0.48 4.4 9.2 3-9-3 Deoxy/5'-(R)-Me DNA ekk kke e = 2'-MOE; k = cEt

Example 76

Inhibition of HTT mRNA Levels Targeting SNP by Modified Oligonucleotides

[0998] Additional modified oligonucleotides were designed in a similar manner as the antisense oligonucleotides described in Example 75. Various chemical modifications were introduced slightly upstream or downstream (i.e. "microwalk") within the central gap region. The gapmers were created with a 3-9-3 motif and were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression. The position on the oligonucleotides opposite to the SNP position, as counted from the 5'-terminus is position 8. The potency and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.

[0999] The modified oligonucleotides and their motifs are described in Table 117. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. Nucleosides followed by a subscript "d" are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" indicates a 2'-.beta.-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). Nucleosides followed by a subscript "b" indicates a 5'-(R)-allyl DNA. Nucleosides followed by a subscript "c" indicates a 5'-(S)-allyl DNA. Nucleosides followed by a subscript "g" indicates a 5'-(R)-hydroxyethyl DNA. Nucleosides followed by a subscript "i" indicates a 5'-(S)-hydroxyethyl DNA. ".sup.mC" indicates a 5-methyl cytosine nucleoside.

[1000] The modified oligonucleotides were tested in vitro using heterozygous fibroblast GM04022 cell line. The transfection method and analysis of HTT mRNA levels adjusted according to total RNA content, as measured by RIBOGREEN were performed in the same manner as described in Example 76. The IC.sub.50s and selectivities as expressed in "fold" were measured and calculated using methods described previously and the results are shown below. As presented in Table 118, several modified oligonucleotides achieved greater than 4.5 fold selectivity in inhibiting mutant HTT mRNA levels and, therefore, are more selective than ISIS 460209.

TABLE-US-00115 TABLE 117 Gap-interrupted oligonucleotides comprising 5'-substituted DNA targeting HTT SNP Wing SEQ ISIS Gap Chemistry Chemisty ID NO Sequence (5' to 3') Motif (mod position) 5' 3' NO 460209 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Full deoxy ekk kke 10 589414 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.bT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 allyl DNA (pos 5) 589415 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.bG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 allyl DNA (pos 6) 589416 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.b.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 allyl DNA (pos 8) 589417 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.b.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 allyl DNA (pos 11) 589418 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.cT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(S)- ekk kke 10 allyl DNA (pos 5) 589419 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.cG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(S)- ekk kke 10 allyl DNA (pos 6) 589420 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.c.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(S)- ekk kke 10 allyl DNA (pos 8) 589421 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.c.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(S)- ekk kke 10 allyl DNA (pos 11) 589422 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.gT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 hydroxyethyl DNA (pos 5) 589423 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.gG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 hydroxyethyl DNA (pos 6) 589424 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.g.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 hydroxyethyl DNA (pos 8) 589437 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.g.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(R)- ekk kke 10 hydroxyethyl DNA (pos 11) 589426 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.iT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(S)- ekk kke 10 hydroxyethyl DNA (pos 5) 589427 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.iG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(S)- ekk kke 10 hydroxyethyl DNA (pos 6) 589428 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.i.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sup.e 3-9-3 Deoxy/5'-(S)- ekk kke 10 hydroxyethyl DNA (pos 8) 589425 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.i.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/5'-(S)- ekk kke 10 hydroxyethyl DNA (pos 11) e = 2'-MOE; k = cEt

TABLE-US-00116 TABLE 118 Comparison of inhibition of HTT mRNA levels and selectivity of gap- interrupted oligonucleotides with ISIS 460209 targeting HTT SNP ISIS IC.sub.50 (.mu.M) Selectivity Gap Chemistry Wing Chemistry NO Mut Wt (wt vs. mut) (mod position) Motif 5' 3' 460209 0.47 2.1 4.5 Full deoxy 3-9-3 ekk kke 589414 1.0 7.6 7.6 Deoxy/5'-(R)-Allyl DNA 3-9-3 ekk kke (pos 5) 589415 1.4 >10 >7.1 Deoxy/5'-(R)-Allyl DNA 3-9-3 ekk kke (pos 6) 589416 2.7 >10 >3.7 Deoxy/5'-(R)-Allyl DNA 3-9-3 ekk kke (pos 8) 589417 5.4 >10 >1.9 Deoxy/5'-(R)-Allyl DNA 3-9-3 ekk kke (pos 11) 589418 1.2 >10 >8.3 Deoxy/5'-(S)-Allyl DNA 3-9-3 ekk kke (pos 5) 589419 1.1 >10 >9.1 Deoxy/5'-(S)-Allyl DNA 3-9-3 ekk kke (pos 6) 589420 3.2 >10 >3.1 Deoxy/5'-(S)-Allyl DNA 3-9-3 ekk kke (pos 8) 589421 2.0 >10 >5.0 Deoxy/5'-(S)-Allyl DNA 3-9-3 ekk kke (pos 11) 589422 0.73 3.2 4.4 Deoxy/5'-(R)- 3-9-3 ekk kke Hydroxyethyl DNA (pos 5) 589423 0.92 9.2 10 Deoxy/5'-(R)- 3-9-3 ekk kke Hydroxyethyl DNA (pos 6) 589424 0.21 4.4 21 Deoxy/5'-(R)- 3-9-3 ekk kke Hydroxyethyl DNA (pos 8) 589437 0.73 >10.2 >14 Deoxy/5'-(R)- 3-9-3 ekk kke Hydroxyethyl DNA (pos 11) 589426 0.91 5.1 5.6 Deoxy/5'-(5> 3-9-3 ekk kke Hydroxyethyl DNA (pos 5) 589427 0.91 >10 >11 Deoxy/5'-(S)- 3-9-3 ekk kke Hydroxyethyl DNA (pos 6) 589428 1.1 >11 >10 Deoxy/5'-(S)- 3-9-3 ekk kke Hydroxyethyl DNA (pos 8) 589425 1.5 >10.5 >7 Deoxy/5'-(S)- 3-9-3 ekk kke Hydroxyethyl DNA (pos 11) e = 2'-MOE; k = cEt

Example 77

Modified Oligonucleotides Comprising 5'-(R)-Me DNA(s) Targeting Human C-Reactive Protein (hCRP)

[1001] A series of modified oligonucleotides were designed based on ISIS 353512, wherein the central gap region contains fourteen .beta.-D-2'-deoxyribonucleoside. These modified oligonucleotides were designed by replacement of two or three .beta.-D-2'-deoxyribonucleoside in the 14 nucleoside gap region with 5'-(R)-Me DNA(s). The thermal stability (T.sub.m) and potency of these modified oligonucleotides targeting hCRP was evaluated. The 3-14-3 MOE gapmer, ISIS 353512 and 5-10-5 MOE gapmer, ISIS 330012 were included in the study for comparison.

[1002] The modified oligonucleotides and their motifs are described in Table 119. Each internucleoside linkage is a phosphorothioate (P.dbd.S) except for nucleosides followed by a subscript "o"which are phosphodiester internucleoside linkages (P.dbd.O). Nucleosides followed by a subscript "d" indicates a .beta.-D-2'-deoxyribonucleoside. Nucleosides followed by a subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript "z" indicates a 5'-(R)-Me DNA. ".sup.mC" indicates a 5-methyl cytosine modified nucleoside. Underlined nucleosides indicate a region comprising 5'-(R)-Me DNA modification.

Thermal Stability Assay

[1003] The modified oligonucleotides were evaluated in thermal stability (T.sub.m) assay. The T.sub.m's were measured using the method described herein. A Cary 100 Bio spectrophotometer with the Cary Win UV Thermal program was used to measure absorbance vs. temperature. For the T.sub.m experiments, oligonucleotides were prepared at a concentration of 8 .mu.M in a buffer of 100 mM Na+, 10 mM phosphate, 0.1 mM EDTA, pH 7. Concentration of oligonucleotides were determined at 85.degree. C. The oligonucleotide concentration was 4 .mu.M with mixing of equal volumes of test oligonucleotide and complimentary RNA strand. Oligonucleotides were hybridized with the complimentary RNA strand by heating duplex to 90.degree. C. for 5 min and allowed to cool at room temperature. Using the spectrophotometer, T.sub.m measurements were taken by heating duplex solution at a rate of 0.5 C/min in cuvette starting @ 15.degree. C. and heating to 85.degree. C. T.sub.m values were determined using Vant Hoff calculations (A.sub.260 vs temperature curve) using non self-complementary sequences where the minimum absorbance which relates to the duplex and the maximum absorbance which relates to the non-duplex single strand are manually integrated into the program. The results are presented below.

Cell Culture and Transfection

[1004] The modified oligonucleotides were tested in vitro. Hep3B cells were plated at a density of 40,000 cells per well and transfected using electroporation with 0.009 .mu.M, 0.027 .mu.M, 0.082 .mu.M, 0.25 .mu.M, 0.74 .mu.M, 2.2 .mu.M, 6.7 .mu.M and 20 .mu.M concentrations of antisense oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and hCRP mRNA levels were measured by quantitative real-time PCR. Human CRP primer probe set RTS1887 was used to measure mRNA levels. hCRP mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.RTM..

Analysis of IC.sub.50's

[1005] The half maximal inhibitory concentration (IC.sub.50) of each oligonucleotide is presented below and was calculated by plotting the concentrations of oligonucleotides used versus the percent inhibition of hCRP mRNA expression achieved at each concentration, and noting the concentration of oligonucleotide at which 50% inhibition of hCRP mRNA expression was achieved compared to the control.

[1006] As illustrated in Table 120, treatment with the newly designed oligonucleotides showed no improvement in potency as compared to the controls, ISIS 353512 and 330012.

TABLE-US-00117 TABLE 119 Gap-interrupted oligonucleotides comprising 5'-(R)-Me DNA targeting hCRP Wing SEQ ISIS Gap Chemistry Linkage ID NO Sequence (5' to 3') Motif Chemistry 5' 3' backbone NO 353512 T.sub.e.sup.mC.sub.e.sup.mC.sub.e.sup.mC.sub.dA.sub.dT.sub.dT.sub.d- T.sub.d.sup.mC.sub.dA.sub.d 3-14-3 Full deoxy eee eee Full PS 83 G.sub.dG.sub.dA.sub.dG.sub.dA.sub.d.sup.mC.sub.d.sup.mC.sub.dT.sub.eG.sub- .eG.sub.e 546127 T.sub.e.sup.mC.sub.e.sup.mC.sub.e.sup.mC.sub.dA.sub.dT.sub.dT.sub.d- T.sub.d.sup.mC.sub.doA.sub.zo 3-14-3 Deoxy/5'-(R)- eee eee Mixed 83 G.sub.zG.sub.dA.sub.dG.sub.dA.sub.d.sup.mC.sub.d.sup.mC.sub.dT.sub.eG.sub- .eG.sub.e Me DNA PS/PO 544810 T.sub.e.sup.mC.sub.e.sup.mC.sub.e.sup.mC.sub.dA.sub.dT.sub.dT.sub.d- T.sub.d.sup.mC.sub.dA.sub.d 3-14-3 Deoxy/5'-(R)- eee eee Mixed 83 G.sub.dG.sub.dA.sub.dG.sub.dA.sub.do.sup.mC.sub.zo.sup.mC.sub.zT.sub.eG.s- ub.eG.sub.e Me DNA PS/PO 544806 T.sub.e.sup.mC.sub.e.sup.mC.sub.eo.sup.mC.sub.zoA.sub.zoT.sub.zT.su- b.dT.sub.d.sup.mC.sub.dA.sub.d 3-14-3 Deoxy/5'-(R)- eee eee Mixed 83 G.sub.dG.sub.dA.sub.dG.sub.dA.sub.d.sup.mC.sub.d.sup.mC.sub.dT.sub.eG.sub- .eG.sub.e Me DNA PS/PO 544807 T.sub.e.sup.mC.sub.e.sup.mC.sub.e.sup.mC.sub.dA.sub.dT.sub.doT.sub.- zoT.sub.zo.sup.mC.sub.zA.sub.d 3-14-3 Deoxy/5'-(R)- eee eee Mixed 83 G.sub.dG.sub.dA.sub.dG.sub.dA.sub.d.sup.mC.sub.d.sup.mC.sub.dT.sub.eG.sub- .eG.sub.e Me DNA PS/PO 544809 T.sub.e.sup.mC.sub.e.sup.mC.sub.e.sup.mC.sub.dA.sub.dT.sub.dT.sub.d- T.sub.d.sup.mC.sub.dA.sub.d 3-14-3 Deoxy/5'-(R)- eee eee Mixed 83 G.sub.dG.sub.doA.sub.zoG.sub.zoA.sub.z.sup.mC.sub.d.sup.mC.sub.dT.sub.eG.- sub.eG.sub.e Me DNA PS/PO 330012 T.sub.e.sup.mC.sub.e.sup.mC.sub.e.sup.mC.sub.eA.sub.eT.sub.dT.sub.d- T.sub.d.sup.mC.sub.dA.sub.d 5-10-5 Full deoxy e5 e5 Full PS 83 G.sub.dG.sub.dA.sub.dG.sub.dA.sub.d.sup.mC.sub.e.sup.mC.sub.eT.sub.eG.sub- .eG.sub.e e = 2'-MOE (e.g. e5 = eeeee)

TABLE-US-00118 TABLE 120 Effect of gap-interrupted oligonucleotide treatment on Tm and hCRP inhibition Wing ISIS Tm IC.sub.50 Gap Chemistry Linkage NO (.degree. C.) (.mu.M) Motif Chemistry 5' 3' backbone 353512 66.7 1.1 3-14-3 Full deoxy eee eee Full PS 546127 65.9 2.5 3-14-3 Deoxy/5'-(R)- eee eee Mixed Me DNA PS/PO 544810 64.3 2.4 3-14-3 Deoxy/5'-(R)- eee eee Mixed Me DNA PS/PO 544806 62.8 2.8 3-14-3 Deoxy/5'-(R)- eee eee Mixed Me DNA PS/PO 544807 65.1 2.7 3-14-3 Deoxy/5'-(R)- eee eee Mixed Me DNA PS/PO 544809 64.2 5.0 3-14-3 Deoxy/5'-(R)- eee eee Mixed Me DNA PS/PO 330012 71.7 0.6 5-10-5 Full deoxy e5 e5 Full PS e = 2'-MOE (e.g. e5 = eeeee), PS/PO = phosphorothioate/phosphodiester internucleoside linkage

Example 78

Human Peripheral Blood Mononuclear Cells (hPBMC) Assay Protocol--In Vitro

[1007] The hPBMC assay was performed using BD Vacutainer CPT tube method. A sample of whole blood from volunteered donors with informed consent at US HealthWorks clinic (Faraday & El Camino Real, Carlsbad) was obtained and collected in 4-15 BD Vacutainer CPT 8 ml tubes (VWR Cat.# BD362753). The approximate starting total whole blood volume in the CPT tubes for each donor was recorded using the PBMC assay data sheet.

[1008] The blood sample was remixed immediately prior to centrifugation by gently inverting tubes 8-10 times. CPT tubes were centrifuged at rt (18-25.degree. C.) in a horizontal (swing-out) rotor for 30 min. at 1500-1800 RCF with brake off (2700 RPM Beckman Allegra 6R). The cells were retrieved from the buffy coat interface (between Ficoll and polymer gel layers); transferred to a sterile 50 ml conical tube and pooled up to 5 CPT tubes/50 ml conical tube/donor. The cells were then washed twice with PBS (Ca.sup.++, Mg.sup.++ free; GIBCO). The tubes were topped up to 50 ml and mixed by inverting several times. The sample was then centrifuged at 330.times.g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R) and aspirated as much supernatant as possible without disturbing pellet. The cell pellet was dislodged by gently swirling tube and resuspended cells in RPMI+10% FBS+pen/strep (.about.1 ml/10 ml starting whole blood volume). A 60 .mu.l sample was pipette into a sample vial (Beckman Coulter) with 600 .mu.l VersaLyse reagent (Beckman Coulter Cat# A09777) and was gently vortexed for 10-15 sec. The sample was allowed to incubate for 10 min. at rt and being mixed again before counting. The cell suspension was counted on Vicell XR cell viability analyzer (Beckman Coulter) using PBMC cell type (dilution factor of 1:11 was stored with other parameters). The live cell/ml and viability were recorded. The cell suspension was diluted to 1.times.10.sup.7 live PBMC/ml in RPMI+10% FBS+pen/strep.

[1009] The cells were plated at 5.times.10.sup.5 in 50 .mu.l/well of 96-well tissue culture plate (Falcon Microtest). 50 .mu.l/well of 2.times. concentration oligos/controls diluted in RPMI+10% FBS+pen/strep. was added according to experiment template (100 .mu.l/well total). Plates were placed on the shaker and allowed to mix for approx. 1 min. After being incubated for 24 hrs at 37.degree. C.; 5% CO.sub.2, the plates were centrifuged at 400.times.g for 10 minutes before removing the supernatant for MSD cytokine assay (i.e. human IL-6, IL-10, IL-8 and MCP-1).

Example 79

Evaluation of the Proinflammatory Effects in hPBMC Assay for 5'-(R)-Me DNA Containing Modified Oligonucleotides--In Vitro Study

[1010] The modified oligonucleotides targeting hCRP from Example 77 were tested and evaluated for the proinflammatory response in hPBMC assay using methods described previously in Example 78. The hPBMCs were isolated from fresh, volunteered donors and were treated with modified oligonucleotides at 0, 0.0128, 0.064, 0.32, 1.6, 8, 40 and 200 .mu.M concentrations using the hPBMC assay protocol described herein. After a 24 hr treatment, the cytokine levels were measured.

[1011] IL-6 was used as the primary readout. The resulting IL-6 level was compared to the positive control, ISIS 353512 and negative control, ISIS 104838. The results are presented in Table 121. As illustrated, reduction in proinflammatory response was achieved with the newly designed oligonucleotides at doses evaluated as compared to the positive control, ISIS 353512.

[1012] ISIS 104838 designated herein as SEQ ID NO: 84, is a 5-10-5 MOE gapmer with the following sequence, G.sub.e.sup.mC.sub.eT.sub.eG.sub.eA.sub.eT.sub.dT.sub.dA.sub.dG.sub.dA.su- b.dG.sub.dA.sub.dG.sub.dA.sub.dG.sub.dG.sub.eT.sub.e.sup.mC.sub.e.sup.mC.s- ub.e.sup.mC.sub.e. Each internucleoside linkage is a phosphorothioate (P.dbd.S). Each nucleoside followed by a subscript "d" is a .beta.-D-2'-deoxyribonucleoside. Each ".sup.mC" is a 5-methyl cytosine modified nucleoside and each nucleoside followed by a subscript "e" is a 2'-O-methoxyethyl(MOE) modified nucleoside.

TABLE-US-00119 TABLE 121 Effect of gap-interrupted oligonucleotide treatment on proinflammatory response in hPBMC Wing ISIS Conc. IL-6 Gap Chemistry Linkage NO (uM) (pg/mL) Motif Chemistry 5' 3' backbone 353512 0 26.9 3-14-3 Full deoxy eee eee Full PS (pos 0.0128 10.6 control) 0.064 73.3 0.32 219.8 1.6 200.1 8 287.8 40 376.9 200 181.5 546127 0 11.5 3-14-3 Deoxy/5'-(R)- eee eee Mixed 0.0128 15.1 Me DNA PS/PO 0.064 19.0 0.32 37.3 1.6 67.5 8 86.3 40 111.2 200 83.1 544810 0 11.5 3-14-3 Deoxy/5'-(R)- eee eee Mixed 0.0128 13.9 Me DNA PS/PO 0.064 15.1 0.32 24.9 1.6 34.0 8 66.2 40 96.8 200 76.5 06/544806 0 11.3 3-14-3 Deoxy/5'-(R)- eee eee Mixed 0.0128 10.8 Me DNA PS/PO 0.064 25.8 0.32 15.6 1.6 25.4 8 52.3 40 69.3 200 341.7 06/544807 0 13.3 3-14-3 Deoxy/5'-(R)- eee eee Mixed 0.0128 13.7 Me DNA PS/PO 0.064 18.4 0.32 53.3 1.6 18.4 8 164.9 40 202.7 200 606.5 06/544809 0 10.8 3-14-3 Deoxy/5'-(R)- eee eee Mixed 0.0128 13.3 Me DNA PS/PO 0.064 14.3 0.32 34.8 1.6 62.3 8 100.9 40 213.1 200 225.0 06/330012 0 10.9 5-10-5 Full deoxy e5 e5 Full PS 0.0128 12.9 0.064 10.8 0.32 25.3 1.6 44.2 8 87.5 40 80.2 200 82.3 07/104838 0 9.3 5-10-5 Full deoxy e5 e5 Full PS (neg 0.0128 10.4 control) 0.064 17.6 0.32 30.1 1.6 53.9 8 124.8 40 94.5 200 89.3 e = 2'-MOE (e.g. e5 = eeeee)

Example 80

Evaluation of the Proinflammatory Effects in hPBMC Assay for a Modified Oligonucleotide Comprising Methyl Thiophosphonate Internucleoside Linkages--In Vitro Study

[1013] A modified oligonucleotide was designed based on the 3/14/3 MOE gapmer, ISIS 353512. This modified oligonucleotide was created by having alternating methyl thiophosphonate (--P(CH.sub.3)(.dbd.S)--) internucleoside linkages throughout the gap region. The proinflammatory effect of the modified oligonucleotide targeting hCRP was evaluated in hPBMC assay using the protocol described in Example 78.

[1014] The modified oligonucleotide and its motif are described in Table 122. Each internucleoside linkage is a phosphorothioate (P.dbd.S) except for nucleosides followed by a subscript "w". Each nucleoside followed by a subscript "w" indicates a methyl thiophosphonate internucleoside linkage (--P(CH.sub.3)(.dbd.S)--). Nucleosides followed by a subscript "d" is a .beta.-D-2'-deoxyribonucleoside. Nucleosides followed by a subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside. ".sup.mC" indicates a 5-methyl cytosine modified nucleoside.

[1015] The hPBMCs were isolated from fresh, volunteered donors and were treated with modified oligonucleotides at 0, 0.0128, 0.064, 0.32, 1.6, 8, 40 and 200 .mu.M concentrations. After a 24 hr treatment, the cytokine levels were measured.

[1016] IL-6 was used as the primary readout. The resulting IL-6 level was compared to the positive control oligonucleotide, ISIS 353512 and negative control, ISIS 104838. The results from two donors denoted as "Donor 1" and "Donor 2" are presented in Table 123. As illustrated, reduction in proinflammatory response was achieved with the newly designed oligonucleotide at doses evaluated as compared to the positive control, ISIS 353512.

TABLE-US-00120 TABLE 122 Modified oligonucleotide comprising alternating methyl thiophosphonate internucleoside linkages throughout the gap region Wing SEQ ISIS Gap Chemistry ID NO Sequence (5' to 3') Motif Chemistry 5' 3' NO 353512 T.sub.e.sup.mC.sub.e.sup.mC.sub.e.sup.mC.sub.dA.sub.dT.sub.dT.sub.d- T.sub.d.sup.mC.sub.dA.sub.dG.sub.d 3-14-3 Full deoxy eee eee 83 G.sub.dA.sub.dG.sub.dA.sub.d.sup.mC.sub.d.sup.mC.sub.dT.sub.eG.sub.eG.sub- .e 560221 T.sub.e.sup.mC.sub.e.sup.mC.sub.eC.sub.dwA.sub.dT.sub.dwT.sub.dT.su- b.dw.sup.mC.sub.dA.sub.dwG.sub.dG.sub.dw 3-14-3 Deoxy/methyl eee eee 83 A.sub.dG.sub.dwA.sub.dC.sub.dw.sup.mC.sub.dT.sub.eG.sub.eG.sub.e thiophosphonate 104838 G.sub.e.sup.mC.sub.eT.sub.eG.sub.eA.sub.eT.sub.dT.sub.dA.sub.dG.sub- .dA.sub.dG.sub.dA.sub.d 5-10-5 Full deoxy e5 e5 84 G.sub.dA.sub.dG.sub.dG.sub.eT.sub.e.sup.mC.sub.e.sup.mC.sub.e.sup.mC.sub.- e e = 2'-MOE (e.g. e5 = eeeee)

TABLE-US-00121 TABLE 123 Effect of modified oligonucleotide treatment on proinflammatory response in hPBMC assay Wing ISIS Conc. IL-6 (Donor 1) IL-6 (Donor 2) Gap Chemistry NO (.mu.M) (pg/mL) (pg/mL) Motif Chemistry 5' 3' 353512 0 6.3 7.8 3-14-3 Full deoxy eee eee 0.0128 8.3 10.2 0.064 77.2 118.2 0.32 151.9 394.3 1.6 152.4 395.3 8 147.6 337.2 40 122.5 228.4 200 119.7 193.5 560221 0 5.6 7.6 3-14-3 Deoxy/methyl eee eee 0.0128 6.4 6.9 thiophosphonate 0.064 6.7 7.6 0.32 7.6 8.9 1.6 9.1 11.8 8 17.5 24.3 40 65.8 50.2 200 60.0 100.4 104838 0 5.8 7.3 5-10-5 Full deoxy e5 e5 0.0128 7.7 7.9 0.064 7.5 11.6 0.32 15.1 22.0 1.6 73.1 112.8 8 29.6 51.5 40 41.6 69.5 200 55.4 4018 e = 2'-MOE (e.g. e5 = eeeee)

Example 81

Modified Oligonucleotides Comprising Methyl Phosphonate Internucleoside Linkage Targeting HTT SNP--In Vitro Study

[1017] ISIS 558255 and 558256 from Example 49 were selected and evaluated for their effect on mutant and wild type HTT mRNA expression levels targeting rs7685686. ISIS 46020 was included in the study for comparison. The position on the oligonucleotides opposite to the SNP position, as counted from the 5'-terminus is position 8.

[1018] Heterozygous fibroblast GM04022 cell line was used for the in vitro assay (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 .mu.L 2.times.PCR buffer, 101 .mu.L primers (300 .mu.M from ABI), 1000 .mu.L water and 40.4 .mu.L RT MIX. To each well was added 15 .mu.L of this mixture and 5 .mu.L of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.

[1019] The IC.sub.50s and selectivities as expressed in "fold" were measured and calculated using methods described previously in Example 41. As illustrated in Table 124, improvement in selectivity and potency was achieved with modified oligonucleotides comprising methyl phosphonate internucleoside linkage as compared to ISIS 460209.

TABLE-US-00122 TABLE 124 Comparison of selectivity in inhition of HTT mRNA levels of antisense oligonucleotides with ISIS 460209 targeted to rs7685686 in GM4022 cells ISIS IC.sub.50 (.mu.M) Selectivity Wing Chemistry SEQ NO Mut Wt (wt vs mut) Motif Gap Chemistry 5' 3' ID NO 460209 0.30 0.99 3.3 3-9-3 Full deoxy ekk kke 10 558255 0.19 1.3 6.8 3-9-3 Deoxy/Methyl ekk kke 10 phosphonate 558256 0.20 1.3 6.5 3-9-3 Deoxy/Methyl ekk kke 10 phosphonate e = 2'-MOE (e.g. e5 = eeeee), k = cEt

Example 82

Modified Oligonucleotides Comprising Methyl Phosphonate or Phosphonoacetate Internucleoside Linkage(s) Targeting HTT SNP

[1020] A series of modified oligonucleotides were designed based on ISIS 460209 wherein the gap region contains nine .beta.-D-2'-deoxyribonucleosides. The modified oligonucleotides were synthesized to include one or more methyl phosphonate or phosphonoacetate internucleoside linkage modifications within the gap region. The oligonucleotides with modified phosphorus containing backbone were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact. The potency and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.

[1021] The position on the oligonucleotides opposite to the SNP position, as counted from the 5'-terminus is position 8.

[1022] The modified oligonucleotides and their motifs are described in Table 125. Each internucleoside linkage is a phosphorothioate (P.dbd.S) except for the internucleoside linkage having a subscript "x" or "y". Each nucleoside followed by a subscript "x" indicates a methyl phosphonate internucleoside linkage (--P(CH.sub.3)(.dbd.O)--). Each nucleoside followed by a subscript "y" indicates a phosphonoacetate internucleoside linkage (--P(CH.sub.2CO.sub.2.sup.-)(.dbd.O)--). Nucleosides followed by a subscript "d" is a .beta.-D-2'-deoxyribonucleoside. Nucleosides followed by a subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). ".sup.mC" indicates a 5-methyl cytosine modified nucleoside.

[1023] The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C.sub.--2229297.sub.--10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 .mu.L 2.times.PCR buffer, 101 .mu.L primers (300 .mu.M from ABI), 1000 uL water and 40.4 .mu.L RT MIX. To each well was added 15 .mu.L of this mixture and 5 .mu.L of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.

[1024] The IC.sub.50s and selectivities as expressed in "fold" were measured and calculated using methods described previously in Example 41. As illustrated in Table 126, most of the newly design oligonucleotides achieved improvement in selectivity while maintaining potency as compared to ISIS 460209.

TABLE-US-00123 TABLE 125 Modified oligonucleotides comprising methyl phosphonate or phosphonoacetate internucleoside linkage(s) targeting HTT SNP Wing SEQ ISIS Chemistry ID NO Sequence (5' to 3') Motif Gap Chemistry 5' 3' NO 460209 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Full deoxy ekk kke 10 566276 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dxT.sub.d.sup.mC.su- b.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/Methyl ekk kke 10 phosphonate 566277 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.dx.sup.mC.su- b.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/Methyl ekk kke 10 phosphonate 566278 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dxA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/Methyl ekk kke 10 phosphonate 566279 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dxT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/Methyl ekk kke 10 phosphonate 566280 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.dx.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/Methyl ekk kke 10 phosphonate 566283 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dxT.sub.dxG.sub.dT.sub.d.sup.mC.s- ub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/Methyl ekk kke 10 phosphonate 573815 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dyT.sub.dG.sub.dT.sub.d.sup.mC.su- b.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/ ekk kke 10 Phosphonoacetate 573816 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dyG.sub.dT.sub.d.sup.mC.su- b.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/ ekk kke 10 Phosphonoacetate 573817 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.dy.sup.mC.su- b.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/ ekk kke 10 Phosphonoacetate 573818 T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.sub- .dA.sub.dT.sub.dy.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e 3-9-3 Deoxy/ ekk kke 10 Phosphonoacetate e = 2'-MOE, k = cEt

TABLE-US-00124 TABLE 126 Comparison of selectivity in inhition of HTT mRNA levels of antisense oligonucleotides with ISIS 460209 targeted to rs7685686 in GM4022 cells ISIS Mut IC.sub.50 Selectivity Wing Chemistry SEQ NO (.mu.M)) (wt vs mut) Motif Gap Chemistry 5' 3' ID NO 460209 0.15 9.4 3-9-3 Full deoxy ekk kke 10 566276 0.76 12.8 3-9-3 Deoxy/Methyl phosphonate ekk kke 10 566277 0.20 17 3-9-3 Deoxy/Methyl phosphonate ekk kke 10 566278 0.25 8.9 3-9-3 Deoxy/Methyl phosphonate ekk kke 10 566279 0.38 -- 3-9-3 Deoxy/Methyl phosphonate ekk kke 10 566280 0.27 47 3-9-3 Deoxy/Methyl phosphonate ekk kke 10 566283 0.8 >100 3-9-3 Deoxy/Methyl phosphonate ekk kke 10 573815 0.16 18.8 3-9-3 Deoxy/Phosphonoacetate ekk kke 10 573816 0.55 18.1 3-9-3 Deoxy/Phosphonoacetate ekk kke 10 573817 0.17 22.5 3-9-3 Deoxy/Phosphonoacetate ekk kke 10 573818 0.24 13.5 3-9-3 Deoxy/Phosphonoacetate ekk kke 10 e = 2'-MOE, k = cEt

Example 83

Modified Oligonucleotides Comprising Methyl Phosphonate Internucleoside Linkages Targeting PTEN and SRB-1--In Vivo Study

[1025] Additional modified oligonucleotides were designed based on ISIS 482050 and 449093 wherein the gap region contains ten .beta.-D-2'-deoxyribonucleosides. The modified oligonucleotides were designed by introducing two methyl phosphonate internucleoside linkages at the 5'-end of the gap region with a 3/10/3 motif. The oligonucleotides were evaluated for reduction in PTEN and SRB-1 mRNA expression levels in vivo. The parent gapmers, ISIS 482050 and 449093 were included in the study for comparison.

[1026] The modified oligonucleotides and their motifs are described in Table 127. Each internucleoside linkage is a phosphorothioate (P.dbd.S) except for the internucleoside linkage having a subscript "x". Each nucleoside followed by a subscript "x" indicates a methyl phosphonate internucleoside linkage (--P(CH.sub.3)(.dbd.O)--). Nucleosides followed by a subscript "d" is a .beta.-D-2'-deoxyribonucleoside. Nucleosides followed by a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). ".sup.mC" indicates a 5-methyl cytosine modified nucleoside.

Treatment

[1027] Six week old BALB/C mice (purchased from Charles River) were injected subcutaneously twice a week for three weeks at dosage 10 mg/kg or 20 mg/kg with the modified oligonucleotides shown below or with saline control. Each treatment group consisted of 3 animals. The mice were sacrificed 48 hrs following last administration, and organs and plasma were harvested for further analysis.

mRNA Analysis

[1028] Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to RIBOGREEN as described herein. The results in Table 128 are listed as PTEN or SRB-1 mRNA expression for each treatment group relative to saline-treated control (% UTC). As illustrated, reduction in PTEN or SRB-1 mRNA expression levels was achieved with the oligonucleotides comprising two methyl phosphonate internucleoside linkages at the 5'-end of the gap region, ISIS 582073 and 582074.

Plasma Chemistry Markers

[1029] Plasma chemistry markers such as liver transaminase levels, alanine aminotranferase (ALT) in serum were measured relative to saline injected mice and the results are presented in Table 128. Treatment with the oligonucleotides resulted in reduction in ALT level compared to treatment with the parent gapmer, ISIS 482050 or 449093. The results suggest that introduction of methyl phosphonate internucleoside linkage(s) can be useful for reduction of hepatoxicity profile of otherwise unmodified parent gapmers.

Body and Organ Weights

[1030] Body weights, as well as liver, kidney and spleen weights were measured at the end of the study. The results below are presented as the average percent of body and organ weights for each treatment group relative to saline-treated control. As illustrated in Table 129, treatment with ISIS 582073 resulted in a reduction in liver and spleen weights compared to treatment with the parent gapmer, ISIS 482050. The remaining oligonucleotide, ISIS 582074 did not cause any changes in body and organ weights outside the expected range as compared to ISIS 449093.

TABLE-US-00125 TABLE 127 Modified oligonucleotides comprising methyl phosphonate internu- cleoside linkages Wing SEQ ISIS Gap Chemistry ID NO Sequence (5' to 3') Motif Chemistry 5' 3' NO. 482050 A.sub.kT.sub.k.sup.mC.sub.kA.sub.dT.sub.dG.sub.dG.sub.d.sup.mC.sub.- dT.sub.dG.sub.d.sup.mC.sub.dA.sub.dG.sub.d.sup.mC.sub.kT.sub.kT.sub.k 3-10-3 Full deoxy kkk kkk 85 582073 A.sub.kT.sub.k.sup.mC.sub.kA.sub.dxT.sub.dxG.sub.dG.sub.d.sup.mC.su- b.dT.sub.dG.sub.d.sup.mC.sub.dA.sub.dG.sub.d.sup.mC.sub.kT.sub.kT.sub.k 3-10-3 Deoxy/Methyl kkk kkk 85 phosphonate 449093 T.sub.kT.sub.k.sup.mC.sub.kA.sub.dG.sub.dT.sub.d.sup.mC.sub.dA.sub.- dT.sub.dG.sub.dA.sub.d.sup.mC.sub.dT.sub.dT.sub.k.sup.mC.sub.k.sup.mC.sub.- k 3-10-3 Full deoxy kkk kkk 86 582074 T.sub.kT.sub.k.sup.mC.sub.kA.sub.dxG.sub.dxT.sub.d.sup.mC.sub.dA.su- b.dT.sub.dG.sub.dA.sub.d.sup.mC.sub.dT.sub.dT.sub.k.sup.mC.sub.k.sup.mC.su- b.k 3-10-3 Deoxy/Methyl kkk kkk 86 phosphonate k = cEt

TABLE-US-00126 TABLE 128 Effect of modified oligonucleotide treatment on target reduction and liver function in BALB/C mice ISIS Dosage % ALT Gap Wing Chemistry SEQ NO. Target (mg/kg/wk) UTC (IU/L) Motif Chemistry 5' 3' ID NO. Saline -- 0 100 30 -- -- -- -- -- 482050 PTEN 10 50 228 3-10-3 Full deoxy kkk kkk 85 482050 20 36.1 505 582073 10 72.2 47.7 Deoxy/Methyl kkk kkk 85 582073 20 57.4 46 phosphonate 449093 SRB-1 10 48 543 3-10-3 Full deoxy kkk kkk 86 449093 20 18.5 1090 582074 10 51.3 58.3 Deoxy/Methyl kkk kkk 86 582074 20 30.3 126.3 phosphonate k = cEt

TABLE-US-00127 TABLE 129 Effect of modified oligonucleotide treatment on body and organ weights in BALB/C mice ISIS Dosage Body wt rel to Liver/Body Spleen/Body Kidney/Body SEQ NO. Target (mg/kg/wk) predose (%) Wt (%) Wt (%) Wt (%) ID NO. Saline -- 0 108.4 100 100 100 482050 PTEN 10 107.4 154.9 141.8 115.7 85 482050 20 111.3 176.7 142.3 112.5 582073 10 108.9 122.9 111.7 100.0 85 582073 20 107.9 133.8 114.6 102.9 449093 SRB-1 10 101.3 105.9 117.9 89.3 86 449093 20 95.3 118.6 129.6 93.0 582074 10 107.1 92.2 116.4 89.2 86 582074 20 103.8 95.5 128.8 91.9

Example 84

Modified Oligonucleotides Comprising Methyl Phosphonate Internucleoside Linkages Targeting Target-Y--In Vivo Study

[1031] Additional modified oligonucleotides were designed in the same manner as the antisense oligonucleotides described in Example 24, wherein two methyl phosphonate internucleoside linkages are introduced at the 5'-end of the gap region. The modified oligonucleotides were designed based on ISIS 464917, 465178, 465984 and 466456 with a 3/10/3 motif. The oligonucleotides were evaluated for reduction in Target-Y mRNA expression levels in vivo. The parent gapmers, ISIS 464917, 465178, 465984 and 466456 were included in the study for comparison.

[1032] The modified oligonucleotides and their motifs are presented in Table 130. Each internucleoside linkage is a phosphorothioate (P.dbd.S) except for the internucleoside linkage having a subscript "x". Each nucleoside followed by a subscript "x" indicates a methyl phosphonate internucleoside linkage (--P(CH.sub.3)(.dbd.O)--). Each nucleoside followed by a subscript "d" is a .beta.-D-2'-deoxyribonucleoside. Nucleosides followed by a subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt). "N" indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).

Treatment

[1033] Six week old BALB/C mice (purchased from Charles River) were injected subcutaneously twice a week for three weeks at dosage 10 mg/kg or 20 mg/kg with the modified oligonucleotides shown below or with saline control. Each treatment group consisted of 3 animals. The mice were sacrificed 48 hrs following last administration, and organs and plasma were harvested for further analysis.

mRNA Analysis

[1034] Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to RIBOGREEN as described herein. The results below are listed as Target-Y mRNA expression for each treatment group relative to saline-treated control (% UTC). As illustrated in Table 131, reduction in Target-Y mRNA expression levels was achieved with the oligonucleotides comprising two methyl phosphonate internucleoside linkages at the 5'-end of the gap region, ISIS 582071, 582072, 582069 and 582070.

Plasma Chemistry Markers

[1035] Plasma chemistry markers such as liver transaminase levels, alanine aminotranferase (ALT) in serum were measured relative to saline treated mice and the results are presented in Table 131. Treatment with the oligonucleotides resulted in reduction in ALT level compared to treatment with the parent gapmer, ISIS 464917, 465178, 465984 or 466456. The results suggest that introduction of methyl phosphonate internucleoside linkage(s) can be useful for reduction of hepatoxicity profile of otherwise unmodified parent gapmers.

Body and Organ Weights

[1036] Body weights, as well as liver, kidney and spleen weights were measured at the end of the study. The results in Table 132 are presented as the average percent of body and organ weights for each treatment group relative to saline-treated control. As illustrated, treatment with ISIS 582070 resulted in a reduction in liver and spleen weights compared to treatment with the parent gapmer, ISIS 466456. An increase in body and organ weights was observed for ISIS 582071 as compared to ISIS 464917. The remaining oligonucleotides, ISIS 582072 and 582069 did not cause any changes in body and organ weights outside the expected range as compared to ISIS 465178 and 465984.

TABLE-US-00128 TABLE 130 Modified oligonucleotides comprising methyl phosphonate internu- cleoside linkages Wing SEQ ISIS Gap Chemistry ID NO Sequence (5' to 3') Motif Chemistry 5' 3' NO. 464917 N.sub.kN.sub.kN.sub.kN.sub.dN.sub.dN.sub.dN.sub.dN.sub.dN.sub.dN.su- b.dN.sub.dN.sub.dN.sub.dN.sub.kN.sub.kN.sub.k 3-10-3 Full deoxy kkk kkk 6 582071 N.sub.kN.sub.kN.sub.kN.sub.dxN.sub.dxN.sub.dN.sub.dN.sub.dN.sub.dN.- sub.dN.sub.dN.sub.dN.sub.dN.sub.kN.sub.kN.sub.k 3-10-3 Deoxy/Methyl kkk kkk phosphonate 465178 N.sub.kN.sub.kN.sub.kN.sub.dN.sub.dN.sub.dN.sub.dN.sub.dN.sub.dN.su- b.dN.sub.dN.sub.dN.sub.dN.sub.kN.sub.kN.sub.k 3-10-3 Full deoxy kkk kkk 6 582072 N.sub.kN.sub.kN.sub.kN.sub.dxN.sub.dxN.sub.dN.sub.dN.sub.dN.sub.dN.- sub.dN.sub.dN.sub.dN.sub.dN.sub.kN.sub.kN.sub.k 3-10-3 Deoxy/Methyl kkk kkk phosphonate 465984 N.sub.kN.sub.kN.sub.kN.sub.dN.sub.dN.sub.dN.sub.dN.sub.dN.sub.dN.su- b.dN.sub.dN.sub.dN.sub.dN.sub.eN.sub.eN.sub.e 3-10-3 Full deoxy kkk eee 6 582069 N.sub.kN.sub.kN.sub.kN.sub.dxN.sub.dxN.sub.dN.sub.dN.sub.dN.sub.dN.- sub.dN.sub.dN.sub.dN.sub.dN.sub.kN.sub.kN.sub.k 3-10-3 Deoxy/Methyl kkk kkk phosphonate 466456 N.sub.kN.sub.dN.sub.kN.sub.dN.sub.kN.sub.dN.sub.dN.sub.dN.sub.dN.su- b.dN.sub.dN.sub.dN.sub.dN.sub.dN.sub.eN.sub.e 5-9-2 or Full deoxy or kdkdk ee 6 3-11-2 deoxy/cEt or kdk 582070 N.sub.kN.sub.dN.sub.kN.sub.dxN.sub.dxN.sub.dN.sub.dN.sub.dN.sub.dN.- sub.dN.sub.dN.sub.dN.sub.dN.sub.dN.sub.eN.sub.e 3-11-2 Deoxy/Methyl kdk ee phosphonate e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

TABLE-US-00129 TABLE 131 Effect of modified oligonucleotide treatment on Target-Y reduction and liver function in BALB/C mice ISIS Dosage % ALT Gap Wing Chemistry NO. (mg/kg/wk) UTC (IU/L) Motif Chemistry 5' 3' Saline 0 100 30 -- -- -- -- 464917 10 29 1244 3-10-3 Full deoxy kkk kkk 464917 20 30.1 2335 582071 20 10.2 274 3-10-3 Deoxy/Methyl kkk kkk phosphonate 465178 10 4.9 1231 3-10-3 Full deoxy kkk kkk 465178 20 10.6 6731 582072 10 36.7 44.7 3-10-3 Deoxy/Methyl kkk kkk 582072 20 23.6 43.7 phosphonate 465984 10 4.7 61 3-10-3 Full deoxy kkk eee 465984 20 0.9 57 582069 10 11.1 39.7 3-10-3 Deoxy/Methyl kkk kkk 582069 20 3.3 27.7 phosphonate 466456 10 9.5 692 5-9-2 or Full deoxy or kdkdk ee 466456 20 10.5 2209 3-11-2 deoxy/cEt or kdk 582070 10 73.9 24 3-11-2 Deoxy/Methyl kdk ee 582070 20 51.3 36.7 phosphonate e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

TABLE-US-00130 TABLE 132 Effect of modified oligonucleotide treatment on body and organ weights in BALB/C mice Body Liver/ Spleen/ Kidney/ Dosage wt rel to Body Body Body ISIS NO. (mg/kg/wk) predose (%) Wt (%) Wt (%) Wt (%) Saline 0 108 100 100 100 464917 10 92.9 125 106.2 102.3 464917 20 71.1 110.9 67.2 107.3 582071 20 104.6 135.2 142.8 89.8 465178 10 94.9 131.3 108.1 85.3 465178 20 79.5 147.5 112 95.3 582072 10 109.2 117.3 111.7 104.8 582072 20 107.1 130.1 107.2 99.8 465984 10 111.4 117.6 110.1 98.8 465984 20 111.3 122.6 134.5 96.1 582069 10 107.8 106.2 97 100.6 582069 20 105.4 115.8 106.2 100.4 466456 10 109.7 148.6 198.7 105.9 466456 20 101.2 182.3 213.7 101.9 582070 10 111.2 100.3 116.7 100.8 582070 20 111.1 108.9 115.6 95.7

Example 85

Short-Gap Chimeric Oligonucleotides Targeting Target-Y

[1037] A series of chimeric antisense oligonucleotides was designed based on ISIS 464917 or 465178, wherein the central gap region contains ten 2'-deoxyribonucleosides. These gapmers were designed by introducing 2'-MOE modified nucleoside(s) at the wing(s) and/or shortening the central gap region to nine, eight, or seven 2'-deoxyribonucleosides.

[1038] The gapmers and their motifs are described in Table 133. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" or "k" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside and a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt).

TABLE-US-00131 TABLE 133 Short-gap antisense oligonucleotides targeting Target-Y SEQ ISIS ID NO Sequence (5' to 3') Motif NO. 464917 N.sub.kN.sub.kN.sub.kNNNNNNNNNNN.sub.kN.sub.kN.sub.k 3-10-3 6 (kkk-d10-kkk) 465977 N.sub.kN.sub.kN.sub.kNNNNNNNNNNN.sub.eN.sub.eN.sub.e 3-10-3 6 (kkk-d10-eee) 573331 N.sub.eN.sub.kN.sub.kNNNNNNNNNNN.sub.kN.sub.kN.sub.e 3-10-3 6 (ekk-d10-kke) 573332 N.sub.eN.sub.eN.sub.kN.sub.kNNNNNNNNNN.sub.kN.sub.kN.sub.e 4-9-3 6 (eekk-d9-kke) 573333 N.sub.eN.sub.eN.sub.eN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.kN.sub.e 5-8-3 6 (eeekk-d8-kke) 573334 N.sub.eN.sub.eN.sub.eN.sub.eN.sub.kN.sub.kNNNNNNNN.sub.kN.sub.kN.su- b.e 6-7-3 6 (eeeekk-d7-kke) 573335 N.sub.eN.sub.kN.sub.kNNNNNNNNNN.sub.kN.sub.kN.sub.eN.sub.e 3-9-4 6 (ekk-d9-kkee) 573336 N.sub.eN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.kN.sub.eN.sub.eN.sub.e 3-8-5 6 (ekk-d8-kkeee) 573361 N.sub.eN.sub.kN.sub.kNNNNNNNN.sub.kN.sub.kN.sub.eN.sub.eN.sub.eN.su- b.e 3-7-6 6 (ekk-d7-kkeeee) 573338 N.sub.eN.sub.eN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.kN.sub.eN.sub.e 4-8-4 6 (eekk-d8-kkee) 573339 N.sub.eN.sub.eN.sub.eN.sub.kN.sub.kNNNNNNNN.sub.kN.sub.kN.sub.eN.su- b.e 5-7-4 6 (eeekk-d7-kkee) 573340 N.sub.eN.sub.eN.sub.kN.sub.kNNNNNNNN.sub.kN.sub.kN.sub.eN.sub.eN.su- b.e 4-7-5 6 (eekk-d7-kkeee) 573779 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573780 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573806 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573782 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573783 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573784 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573785 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573786 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573787 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 465178 N.sub.kN.sub.kN.sub.kNNNNNNNNNNN.sub.kN.sub.kN.sub.k 3-10-3 6 (kkk-d10-kkk) 466140 N.sub.kN.sub.kN.sub.kNNNNNNNNNNN.sub.eN.sub.eN.sub.e 3-10-3 6 (kkk-d10-eee) 573341 N.sub.eN.sub.kN.sub.kNNNNNNNNNNN.sub.kN.sub.kN.sub.e 3-10-3 6 (ekk-d10-kke) 573342 N.sub.eN.sub.eN.sub.kN.sub.kNNNNNNNNNN.sub.kN.sub.kN.sub.e 4-9-3 6 (eekk-d9-kke) 573343 N.sub.eN.sub.eN.sub.eN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.kN.sub.e 5-8-3 6 (eeekk-d8-kke) 573344 N.sub.eN.sub.eN.sub.eN.sub.eN.sub.kN.sub.kNNNNNNNN.sub.kN.sub.kN.su- b.e 6-7-3 6 (eeeekk-d7-kke) 573345 N.sub.eN.sub.kN.sub.kNNNNNNNNNN.sub.kN.sub.kN.sub.eN.sub.e 3-9-4 6 (ekk-d9-kkee) 573346 N.sub.eN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.kN.sub.eN.sub.eN.sub.e 3-8-5 6 (ekk-d8-kkeee) 573347 N.sub.eN.sub.kN.sub.kNNNNNNNN.sub.kN.sub.kN.sub.eN.sub.eN.sub.eN.su- b.e 3-7-6 6 (ekk-d7-kkeeee) 573348 N.sub.eN.sub.eN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.kN.sub.eN.sub.e 4-8-4 6 (eekk-d8-kkee) 573349 N.sub.eN.sub.eN.sub.eN.sub.kN.sub.kNNNNNNNN.sub.kN.sub.kN.sub.eN.su- b.e 5-7-4 6 (eeekk-d7-kkee) 573350 N.sub.eN.sub.eN.sub.kN.sub.kNNNNNNNN.sub.kN.sub.kN.sub.eN.sub.eN.su- b.e 4-7-5 6 (eekk-d7-kkeee) 573788 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573789 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573790 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573791 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573792 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573793 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573794 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573795 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 573796 N.sub.kN.sub.kN.sub.kNNNNNNNNN.sub.kN.sub.eN.sub.eN.sub.eN.sub.e 3-8-5 6 (kkk-d8-keeee) 141923 C.sub.eC.sub.eT.sub.eT.sub.eC.sub.eCCTGAAGGTTC.sub.eC.sub.eT.sub.eC- .sub.eC.sub.e 5-10-5 9 (neg control) (e5-d10-e5) e = 2'-MOE (e.g. e5 = eeeee), k = cEt, d = 2'-deoxyribonucleoside

Example 86

Short-Gap Chimeric Oligonucleotides Targeting Target-Y--In Vitro Study

[1039] Several short-gap chimeric oligonucleotides from Table 133 were selected and evaluated for their effects on Target-Y mRNA in vitro. The parent gapmer, ISIS 464917 and 465178 were included in the study for comparison. ISIS 141923 was used as a negative control.

[1040] The newly designed gapmers were tested in vitro. Primary mouse hepatocytes at a density of 35,000 cells per well were transfected using electroporation with 0.0625, 0.25, 1, 4 and 16 .mu.M concentrations of chimeric oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-Y mRNA levels were measured by quantitative real-time PCR. Primer probe set RTSXXXX was used to measure mRNA levels. Target-Y mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.

[1041] The half maximal inhibitory concentration (IC.sub.50) of each oligonucleotide is presented in Table 134 and was calculated by plotting the concentrations of oligonucleotides used versus the percent inhibition of Target-Y mRNA expression achieved at each concentration, and noting the concentration of oligonucleotide at which 50% inhibition of Target-Y mRNA expression was achieved compared to the control. As illustrated in Table 134 and 135, several short-gap oligonucleotides showed comparable inhibition of Target-Y mRNA levels as compared to the parent gapmers, ISIS 464917 or 465178.

TABLE-US-00132 TABLE 134 Comparison of inhibition of Target-Y mRNA levels of short-gap oligonucleotides with ISIS 464917 IC.sub.50 SEQ ID ISIS NO Motif (.mu.M) NO. 464917 3-10-3 0.5 6 (kkk-d10-kkk) 573331 3-10-3 0.5 6 (ekk-d10-kke) 573332 4-9-3 0.6 6 (eekk-d9-kke) 573333 5-8-3 0.5 6 (eeekk-d8-kke) 573335 3-9-4 0.4 6 (ekk-d9-kkee) 573336 3-8-5 0.5 6 (ekk-d8-kkeee) 573361 3-7-6 0.6 6 (ekk-d7-kkeeee) 573340 4-7-5 2.3 6 (eekk-d7-kkeee) 141923 5-10-5 >16 9 (neg control) (e5-d10-e5) e = 2'-MOE (e.g. e5 = eeeee), k = cEt, d = 2'-deoxyribonucleoside

TABLE-US-00133 TABLE 135 Comparison of inhibition of Target-Y mRNA levels of short-gap oligonucleotides with ISIS 465178 IC.sub.50 SEQ ISIS NO Motif (.mu.M) ID NO. 465178 3-10-3 0.2 6 (kkk-d10-kkk) 573341 3-10-3 0.2 6 (ekk-d10-kke) 573342 4-9-3 0.4 6 (eekk-d9-kke) 573345 3-9-4 0.2 6 (ekk-d9-kkee) 573346 3-8-5 0.4 6 573348 (ekk-d8-kkeee) 0.5 6 573350 4-8-4 0.9 6 (eekk-d8-kkee) 573806 4-7-5 0.8 6 (eekk-d7-kkeee) 573783 3-8-5 1.0 6 (kkk-d8-keeee) 573784 3-8-5 1.3 6 (kkk-d8-keeee) 573785 3-8-5 1.0 6 (kkk-8-keeee) 573792 3-8-5 0.5 6 (kkk-8-keeee) 573794 3-8-5 0.4 6 (kkk-d8-keeee) 573795 3-8-5 0.5 6 (kkk-d8-keeee) 573796 3-8-5 0.8 6 (kkk-d8-keeee) 141923 5-10-5 >16 6 (neg control) (e5-d10-e5) e = 2'-MOE (e.g. e5 = eeeee), k = cEt, d = 2'-deoxyribonucleoside

Example 87

Short-Gap Chimeric Oligonucleotides Targeting Target-Y--In Vivo Study

[1042] Several short-gap oligonucleotides described in Example 85 were selected and evaluated for efficacy in vivo and for changes in the levels of various plasma chemistry markers targeting Target-Y. The parent gapmer, ISIS 464917 was included in the study for comparison.

Treatment

[1043] Six week male BALB/C mice (purchased from Charles River) were injected subcutaneously with a single dose of antisense oligonucleotide at 10 mg/kg or 20 mg/kg or with saline control. Each treatment group consisted of 4 animals. The mice were sacrificed 96 hrs following last administration, and organs and plasma were harvested for further analysis.

mRNA Analysis

[1044] Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to Cyclophilin A as described herein. The results below are listed as Target-Y mRNA expression for each treatment group relative to saline-injected control (% UTC). As illustrated in Table 136, Target-Y mRNA expression levels were reduced in a dose-dependent manner with the newly designed oligonucleotides.

Plasma Chemistry Markers

[1045] Plasma chemistry markers such as liver transaminase levels, alanine aminotranferase (ALT) in serum were measured relative to saline treated mice and the results are presented in Table 136. Treatment with the newly designed oligonucleotides resulted in reduction in ALT levels compared to treatment with the parent gapmer, ISIS 464917. The results suggest that shortening the central gap region and introducing 2'-MOE modified nucleoside(s) at the wing(s) can be useful for the reduction of hepatoxicity profile of ISIS 464917.

Body and Organ Weights

[1046] Body weights, as well as liver, kidney and spleen weights were also measured at the end of the study. The results showed that treatment with the newly designed oligonucleotides did not cause any changes in body and organ weights outside the expected range as compared to ISIS 464917 (data not shown).

TABLE-US-00134 TABLE 136 Effect of short-gap antisense oligonucleotide treatment on Target-Y reduction and liver function in BALB/C mice Dosage % ALT SEQ ISIS NO (mg/kg/wk) UTC (IU/L) Motif ID NO. Saline 0 99 23 -- 464917 10 11.5 1834 3-10-3 6 20 5.1 8670 (kkk-d10-kkk) 573333 10 32.8 79 5-8-3 6 20 21.2 370 (eeekk-d8-kke) 573334 10 79.5 26 6-7-3 6 20 69.4 29 (eeeekk-d7-kke) 573336 10 23.2 179 3-8-5 6 20 12.0 322 (ekk-d8-kkeee) 573339 10 47.9 35 5-7-4 6 20 32.8 199 (eeekk-d7-kkee) 573340 10 81.3 63 4-7-5 6 20 66.2 33 (eekk-d7-kkeee) 573361 10 33.6 150 3-7-6 6 20 19.2 722 (ekk-d7-kkeeee) 573783 10 16.5 734 3-8-5 6 20 6.3 1774 (kkk-d8-keeee) 573785 10 20.2 61 3-8-5 6 20 14.2 40 (kkk-d8-keeee) 573806 10 19.3 346 3-8-5 6 20 15.4 1389 (kkk-d8-keeee) e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 88

Short-Gap Chimeric Oligonucleotides Targeting PTEN

[1047] A series of chimeric antisense oligonucleotides was designed based on ISIS 482050, wherein the central gap region contains ten 2'-deoxyribonucleosides. These gapmers were designed by introducing 2'-MOE modified nucleoside(s) at the wing(s) and/or shortening the central gap region to nine, or eight 2'-deoxyribonucleosides.

[1048] The gapmers and their motifs are described in Table 137. The internucleoside linkages throughout each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are .beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a subscript "e" or "k" are sugar modified nucleosides. A subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified nucleoside and a subscript "k" indicates a 6'-(S)--CH.sub.3 bicyclic nucleoside (e.g. cEt).

TABLE-US-00135 TABLE 137 Short-gap antisense oligonucleotides targeting PTEN SEQ ISIS ID NO. Sequence (5' to 3') Motif NO. 482050 A.sub.kT.sub.kC.sub.kATGGCTGCAGC.sub.kT.sub.kT.sub.k 3-10-3 85 (kkk-d10-kkk) 508033 A.sub.kT.sub.kC.sub.kATGGCTGCAGC.sub.eT.sub.eT.sub.e 3-10-3 85 (kkk-d10-eee) 573351 A.sub.eT.sub.kC.sub.kATGGCTGCAGC.sub.kT.sub.kT.sub.e 3-10-3 85 (ekk-d10-kke) 573352 A.sub.eT.sub.eC.sub.kA.sub.kTGGCTGCAGC.sub.kT.sub.kT.sub.e 4-9-3 85 (eekk-d9-kke) 573353 A.sub.eT.sub.eC.sub.eA.sub.kT.sub.kGGCTGCAGC.sub.kT.sub.kT.sub.e 5-8-3 85 (eeekk-d8-kke) 573354 A.sub.eT.sub.eC.sub.eA.sub.eT.sub.kG.sub.kGCTGCAGC.sub.kT.sub.kT.su- b.e 6-7-3 85 (eeeekk-d7-kke) 573355 A.sub.eT.sub.kC.sub.kATGGCTGCAG.sub.kC.sub.kT.sub.eT.sub.e 3-9-4 85 (ekk-d9-kkee) 573356 A.sub.eT.sub.kC.sub.kATGGCTGCA.sub.kG.sub.kC.sub.eT.sub.eT.sub.e 3-8-5 85 (ekk-d8-kkeee) 573357 A.sub.kT.sub.kC.sub.kATGGCTGC.sub.kA.sub.kG.sub.eC.sub.eT.sub.eT.su- b.e 3-7-6 85 (ekk-d7-kkeeee) 573358 A.sub.eT.sub.eC.sub.kA.sub.kTGGCTGCAG.sub.kC.sub.kT.sub.eT.sub.e 4-8-4 85 (eekk-d8-kkee) 573359 A.sub.eT.sub.eC.sub.eA.sub.kT.sub.kGGCTGCAG.sub.kC.sub.kT.sub.eT.su- b.e 5-7-4 85 (eeekk-d7-kkee) 573360 A.sub.eT.sub.eC.sub.kA.sub.kTGGCTGCA.sub.kG.sub.kC.sub.eT.sub.eT.su- b.e 4-7-5 85 (eekk-d7-kkeee) 573797 T.sub.kG.sub.kG.sub.kCTGCAGCTT.sub.kC.sub.eC.sub.eG.sub.eA.sub.e 3-8-5 87 (kkk-d8-keeee) 573798 A.sub.kT.sub.kG.sub.kGCTGCAGCT.sub.kT.sub.eC.sub.eC.sub.eG.sub.e 3-8-5 88 (kkk-d8-keeee) 573799 C.sub.kA.sub.kT.sub.kGGCTGCAGC.sub.kT.sub.eT.sub.eC.sub.eC.sub.e 3-8-5 89 (kkk-d8-keeee) 573800 T.sub.kC.sub.kA.sub.kTGGCTGCAG.sub.kC.sub.eT.sub.eT.sub.eC.sub.e 3-8-5 90 (kkk-d8-keeee) 573801 A.sub.kT.sub.kC.sub.kATGGCTGCA.sub.kG.sub.eC.sub.eT.sub.eT.sub.e 3-8-5 85 (kkk-d8-keeee) 573802 C.sub.kA.sub.kT.sub.kCATGGCTGC.sub.kA.sub.eG.sub.eC.sub.eT.sub.e 3-8-5 91 (kkk-d8-keeee) 573803 C.sub.kC.sub.kA.sub.kTCATGGCTG.sub.kC.sub.eA.sub.eG.sub.eC.sub.e 3-8-5 92 (kkk-d8-keeee) 573804 T.sub.kC.sub.kC.sub.kATCATGGCT.sub.kG.sub.eC.sub.eA.sub.eG.sub.e 3-8-5 93 (kkk-d8-keeee) 573805 T.sub.kT.sub.kC.sub.kCATCATGGC.sub.kT.sub.eG.sub.eC.sub.eA.sub.e 3-8-5 94 (kkk-d8-keeee) e = 2'-MOE, k = cEt, d = 2'-deoxyribonucleoside

Example 89

Short-Gap Chimeric Oligonucleotides Targeting PTEN--In Vitro Study

[1049] Several short-gap chimeric oligonucleotides from Table 137 were selected and evaluated for their effects on PTEN mRNA in vitro. The parent gapmer, ISIS 482050 were included in the study for comparison. ISIS 141923 was used as a negative control.

[1050] The newly designed gapmers were tested in vitro. Primary mouse hepatocytes at a density of 35,000 cells per well were transfected using electroporation with 0.0625, 0.25, 1, 4 and 16 .mu.M concentrations of chimeric oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PTEN mRNA levels were measured by quantitative real-time PCR. Primer probe set RTS186 was used to measure mRNA levels. PTEN mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.

[1051] The half maximal inhibitory concentration (IC.sub.50) of each oligonucleotide was calculated in the same manner as described previously and the results are presented in Table 138. As illustrated, most short-gap oligonucleotides showed comparable inhibition of PTEN mRNA levels as compared to ISIS 482050.

TABLE-US-00136 TABLE 138 Comparison of inhibition of PTEN mRNA levels of short-gap oligonucleotides with ISIS 482050 IC.sub.50 SEQ ISIS NO Motif (.mu.M) ID NO. 482050 3-10-3 1.9 85 (kkk-d10-kkk) 573351 3-10-3 2.8 85 573353 (ekk-d10-kke) 6.1 85 573355 3-9-4 2.6 85 (ekk-d9-kkee) 573798 3-8-5 1.6 88 (kkk-d8-keeee) 573799 3-8-5 1.9 89 (kkk-d8-keeee) 573803 3-8-5 1.4 92 (kkk-d8-keeee) 141923 5-10-5 >16 9 (neg control) (e5-d10-e5) e = 2'-MOE (e.g. e5 = eeeee), k = cEt, d = 2'-deoxyribonucleoside

Sequence CWU 1

1

941202001DNAHomo sapiens 1gcccagcagg tgtcagcctc attttacccc gcccctattc aagatgaagt tgttctggtt 60ccaacgcctc tgacatatta gctgcatcat tttacatttc tttttttttt ttccttttaa 120atggggtctt gctctgtcac ccaggctgga gtgctgtggt atgatctcgg ctcactgcaa 180tctccacctc cgaggttcca gcgattctct tgcctcagcc tcccgagtag ctgggactac 240aggcacccac catcatactg ggctaatttt tgtgttttta gtagagatgg ggtttcccca 300tgttgcccag gctgatctca aactcctggg cttaagcaat acagccgcgt tggcctccca 360aagtgttggg attacaagca tgagctaccc cacccagctc attttacatt tccacttgtt 420aaactgaaaa ctggcccgag aaagcttctg tactgccatc cttgcgtcct tgcagatgaa 480tcgtaaccta gcatagtagg taggcagact gaaaacctaa cttagcagta ggcttctgta 540acaacagctg tgtctcagcc agttcctgca gccagacttc aaccactcac aggccgcaaa 600ctgttcaaac tgtgttcgga gaaggcgaat tcatctggct gttaacgtgc ctcacttctg 660ctttctgtgg ccactttccc ttttctgtcc ataaatttgc tttgaccaca cagcatccct 720agagtctccc tgaatctgct gtgattctgg gacctgcacc atttgtgaat tgtttttttt 780ttccttgatc agctaaactc tgttcaattc aatttgttgg aagtttttaa cataccaatg 840gtgcaccaag gttccaattt ctccacttcc tcataaataa gtcattttaa atggcttttc 900agtattccaa tatttggaag tattaatgtt tctaccaatt ttctattttt ggacattgag 960gttgtttcat tttttttttc tttttttgag acagagtctc gctccgtcac ccaggctgga 1020gtgcagtggc ctgatcccgg cccactgcaa cctccacctc cctcctcagc ctcctgagta 1080gctgggatta caggtgcatg caccaccaca cccagctaat ttttgtattt ttagtagaga 1140tggggtttca ccatgttggt caggctggtc tcaaactcct gacctcaggt ggtccacctg 1200ccttggcctc ccaaaatgct gggattacag gcctgagcca ctgcgcctgg cctcatcttc 1260ttgatattaa tgttgcttta acatctttgt ccctgtgttt tttgtttttt tttttgagac 1320ggagtctcat tcattctgtc acccaggctg gagttcagtg gcgtgatctc agctcactgc 1380aacctctgtc tcctgggttc cagtgattct cctgcgtcgg tctcctgagt agctgtgttc 1440ctgggtcttt cgatggttat ttaatacttc cctacagtaa tgccctgtgc gtacatgcta 1500agtgtgatga aatggttggc acagttaaat cttttgaaag acattgccaa gtcactcttc 1560agaaaagtga taggaggtca tagcaatttt aagaagtcct catttctaca tttccttact 1620aatctcggtt ggtgtctctt caatctttcc tcacactttt cttgggtttt tcctgaatca 1680tgagtctact acatttacac attttaaagc atctttagaa acaggatctc attttgttgc 1740ccaggctaga gtttggtggc atgattatag ctcctcatac tcctgggctc aagtgatcct 1800tccacctctg aaaccccaaa atttgagaaa ggtctcattt aatttagaaa gtttattttg 1860ccaaggttga gggtgcacac ctgtgatgat atacgagtta aaaagaaatt atttaggcag 1920atactgaggg taagaaagtc ctcggtaagg ttttcttttc aatgaaaagc agcccccaag 1980cattttcttt tctaacaaag agcagcctgt aaaatcgagc tgcagacata cacaagcaag 2040ctggaagctt gcacaggtga atgctggcag ctgtgccaat aagaaaaggc tacctggggc 2100caggcagatc caacatggcg gctccatctt ccctttcctt gtcaaccatg tgcacagtaa 2160ggagcaggca acatagtgtc ccccgagtag agaccaattt gcataataaa aggtgagggt 2220agggtgggca gcttctttgc atgctatgta aacattatgc ctggtccaac caatctttgg 2280gccctgtgta aattagacac cacctcctca agcctgtcta taaaaccctg tccattctgc 2340cgcaggctgg aagacccact ggggcacccc tctctctcta taggagacag ctattcattt 2400ttctctttct ttcacctatt aaagctccac tcttaacccc actccgtgtg tatctatgtt 2460cttgatttcc ttggcatgag gcaatgaacc ttgggtatta ccccagaacc ttgggtatta 2520tgccacttca gtgacacagc ctcaggaaat cctgatgaca tgttcccaag atggtcgggg 2580cacagcttgg ttttatacat tttagggaga catgagacgt caattcatat atgtaagaag 2640tacattggtt ccgtccagaa aggcggggac aacttgaggc agggagagag cttctaggtc 2700acaggtagac aaatggttgc attcttttga atctccgata agcctttcca aaggaggcaa 2760tcagaatatg cgtctattga ctgggcgcag tggctcatgc ctgtaatgcc agcactttgg 2820gaggcggagg tgggtggatc acctgaggtc aggagtttga gagcagcccg gccaacatgg 2880tgaaaccctg tctctactaa aaatacaaaa aattagctgg gcgtggtggc gggcgcctgt 2940aatcccagct actcgggagg ctgaggcagg agaatagctt gaacccagaa ggaagaggtt 3000gcagtgagct gagatggtgc cattgcactc cagcctgggc aacaagagtg aaactccatc 3060tcagaaaaaa aaaaaaaagg cctgggcaaa gtggctcacg cctgtaatcc cagcactttg 3120ggaagccgag gcgggcaggt cacaaagtca ggagattgag accatcctgg ctaacatgat 3180gaaaccccat ctctactaaa aaatacaaaa aactagctgg gtgtggtggc gagcacctgt 3240agtcccagct actcggcagg ctgaggcagg agaatggcgt gaaccgggga ggcggagctt 3300gcagtgagcc gagatcacac cactgcactc cagcccggac gacagggcaa gactctatct 3360caaattaaaa aaaaaaaaaa aaaaaaaaaa aaagagagag agaatatgca tctatctcag 3420tgagcagaag gatgactttg aatggaatgg gagcagttcc tagcttgaac ttccccttta 3480gcttcagtga tttgggggct caaggtatgt tcctttcaca tacctcagcc tcccaagtag 3540ctgggaccac aagtgcatgc caccacacgt ggctaatgtt ttattttttt tgtaggaata 3600gggtctcact atgtgtccag gctggtctaa aacccctgag ctcaaatggt cctcccgcct 3660cagcctcccg aaatgctggg attacaggca tgagccagca tgcccggcct agtctacatt 3720tttataaatt gctaattcaa agttccctct ccaaaacctc atggttttcc ctgttctcat 3780cccctgcacc ctcccttccc ctggagtact cacctggcct tggaggtctg gtgtgagccc 3840ggacttcgat tctaggcaca gcatgtgatg agcgccccca ggtcaaacac ctcccctctg 3900cggcctgtgc ttcaccgcct tgacagtgag aaaggtctcc cttcggctca ttctcgaagt 3960ctcaaacttc acttctcctg tgcgctgatt ctgaattcag cccccgtcca aggtcctggc 4020ccctttctct tctgcttggc gtgttgttca tcaccactgt gcactgctga gggtaagtgc 4080ggttctctgg acctctgctt tatcattaga acagactctt gcggtttccc acgacattcc 4140tttcacttct cacttggaag atgagccgtg aggaaatcct gtgttgtgtg gtatgtgggc 4200tgtgcttctg cttgacttga gggccaagca gcattgcaag ccatggtttt aaataagaaa 4260gaacatttct aaccttcatc ttctagtaag gaaacaagtg ggctttagag ttcttgctca 4320ggaaagacct atgtcccagt ccaaccggac cttttactaa agagatcttc ctgatcctcc 4380tccccaggcc aggggagggg tcctccctgg ggttggagcc tttagtaggg ggtcggagac 4440acgacgtagc cttcatgaca ttcatagtct agttacacga tccctgtaag ggtcagttga 4500agtaagtgct acaaaggaag ggaggtgctc agtggagagg gctctctttt atgtattata 4560tttctttcat ggggagggat atggatcagg gatcagcaga ggtgtttcag tcccgaggga 4620aagaaagtca gcgtggcttg ggagttggga gcagcaagac agtggctcaa gatatcttaa 4680gactagtgga gtacaccttg catgttaaaa gccttgctca gggctgcctg gttcttgtag 4740gacgacagag atggcctagc tctgcatact gcacccccag gggctcagaa cagtgcaaat 4800gtcagtctat ctgtcagtgg cagagccagc cttggagcag gggtgcaagg aggtctctgc 4860actggccagg catgcagaac attctgttca gtagcactgg acagaaggcc ccatctagat 4920gagacagagc tggtggggca ggacaaagac tcctggcagc tcaaacggcc tggcagatgc 4980ttggagagag ggggcttctt gagacagcac catttctggg aagagagtca cctgggaggg 5040atgaggccac gctccggctt ggaggtgaag agaggggctg ctgcaagaaa gaattagaga 5100catgccagcc tttgctgtgt tgcccaggct ggtcatgaac tcttggcctc aagcaatctt 5160cccacctcag cctccccaag cgctgggatt atagacatga gcccccatgc tggccaataa 5220aagatgattt tatggagggg atggtggtga aggttgtggg tggtatgaaa tagtaagaaa 5280tatatattgg tctgcaccca gttcctgcca cagagctcct aaaatcctga gaacttcctg 5340ggtgagcatc ttttgttcta atgaggtgac tcttggtggc tcctggatag gagtgaatca 5400ccagaaagat caagccagag ttagaagcag aaagtgctgg ctataacaca ggaaagctgt 5460aacacaaata ataaagtttt tttttttttt tttgagatgg agcctcactc tgttgcccag 5520gctggagtgc aatggtgcaa tctcagctca ctacaagctc tgcctcccag gttcaagtga 5580ttctcctgcc tcagcctcct gagcagttgg gactacaggt gtgtgccacc acatctggct 5640aatttttgta tttttagcag agacggggtt tcaccatatt aaccaggctg gcctcaaact 5700ccttaccttg tgatccgcct gcctcagcct cccaaagtgc tgggattaca ggcatgagcc 5760accgtgcctg gccaaaagac attgttctta aaagaatcaa ctaactaacc aaataaataa 5820aaatctaacc taattaagaa actaaaaata cacaaaaatt aatttcaagg ggagaaaaat 5880catgtaaaga gagaaagata atgaatactt tgcagaaatt tatgaacata aacataaaac 5940ttggatgaaa tgcatttcta ggaaaacata atttatcaaa actaaccaca agtaaaatag 6000aagcctaaat aggatatttt caagagaaga agtaaagttg tcaaagtgct acccttcaaa 6060aaaacaccag gctcaaacaa tctgacatgg gaatgttagc acaccttaga gagcaaataa 6120aactttgaat gggcttgaaa tattccagac tctagaaaaa caaaacttcc caattctttt 6180tataaagcaa gtataaattg ataccaaaat cttataaaga ccttatacaa aacttcatac 6240caatctcttt tatgaataca aaacccttaa taaagtatta ccagacagaa cccaacaata 6300cataaaaatg tcacatcata acatagtggg gtttatttca ataatgcatg gatggttcaa 6360tacaaggaaa ttcagtaaca caatataata gatcatgtga atatacccaa agaaaaaata 6420gattattttc atagatgctg taaaggcatt tgaccaaatt caacacctac tttttaggtg 6480gtcaataaaa taaattagtt actccttctt tagcatgata aaatatattt atcagcccag 6540aaggcatcat tttacccgat aagggcacac gctggaggga ataatgttaa aattaggaat 6600aagaggatag ctagtttctt tcttcttttt tttttttgag acggagtctt gctctgttgc 6660caggctggag tgcagtggtg caatgttggc tcactgcacg ccccccgcct cccaggttca 6720agcgattctc ctgcctcagc ctcccgagta gctgggacta caggcgcgca ccaccatgcc 6780cggctaattt ttttttgtat tttagtagag atggggtttc accatgttgg tcaggctggt 6840cttgaactcc caacctcacg tactgggatt accggtgtga gccaccacgc cagcccaact 6900actttcaaca ttatccttaa tactgatgct tattgactta ctatggggtt acctctagat 6960aaatccataa taagttgaaa atataagtaa aaaatgccct taatacacct aacctaccaa 7020acatcatagc tgagcccagc ctgccttagc tatgctcaga cactgacgtc agcctacaat 7080tggcaaaatc acacagcagc acagtctact gcagagcatc tgctgtttgc ccttgtgact 7140gcgtggctgc ctgggagctt cccagcttca caagacagta ttacgtagca catcactagc 7200ctggggaaag atcaaagttg aaaatttgaa gtgtggtttc cattgaatgt gtactgcttt 7260tgcaccatca tcaagtcaaa aaattttagt tgaaccagcc taagtttggg accatcttta 7320ttttcaggag gaacttccat gtacattgat gacggacgat agaatccgtt tctatcatcc 7380taatgaacat aatgaataaa tccagacaaa cataaacatt aacagagtaa gcagctttcg 7440gggctggaag ccagaagagg gtgggagcgc agagagagag gccaaacacc agggctgctt 7500ctgctttgcg ggtatttgct gatctggaca aggtatctgg aaggctgagc taagcctcct 7560ttttttttga ggtggcgtct cactctgttg ccaggctgga gtgcaatggt gcgatctcag 7620ctcactgcaa cctccacctc cctggttcaa gcgattctcc tgcctcagcc tcccgagtag 7680ctgggattac aggctcccgc cactacaccc agctgatttt tgtaatttta gtagagacgg 7740ggtttcacca tgttggccag gatggtctcg atctcttgac gtcatgatct gtccacctcg 7800gcctcccaaa gtgctgggat tataggcgtg acccaccgtg ccccgtctga gctaagcctc 7860ttgagcatag gggactaaaa atgaaatcta gcgcatgcca agtttagggt cccaggcaat 7920tcctttccac tttggggtcc actttggggt ccaccccacc caagaagaag gatgacttgg 7980aagtaaacca gctctgaaat atggatggtc ctctgggacc ataccaatcc cttcatatca 8040accacatcca gttcctcaaa actggaactt ggattaagat ggcctaggac ttctagtgtc 8100ccaggagcct ggcattgcaa acaaaaatcc tctccggaag aagataatac cttaagcttc 8160aaatgactct ctaataaatt tcaaatacaa tgtccagcac acaaacacaa attaccagga 8220acgtgatatg aggcctgatg gatgggaatt agcagaaact tcaggcatga gaaacatacc 8280ctcagaggcc tagaatctat ctagtgtcta gataatggag atatgaaata cagacactta 8340aacaactatg tttcccatgt tcaaagagga aatttgcaaa acttgaaagt gttggcagga 8400aatcagaaac tataaaatgt gacaacagca tactttagag tcagtataaa ttacggtccc 8460gaaaactgca gaattccaga acttaatggt aaagcaaggg tttaacagca gaatagaaat 8520agccagagag aactaggaag taagtcagat gacactaccc agaataaggc actgagaggc 8580caaggaatgg aaaatgcaga agaaaggata tggtgagagg atctaatata catttatttg 8640gagtaccagg gagagagaga aggagaagaa cagaagccgt gtttcaagga cggtgactga 8700gaggcttcga aactgatgaa agccatcagt tcacaaattc aaagcccagt gaattccaag 8760gagaaaaaaa gaaatccata ctgtgaaagc aagtccagac aatgacaaac accatcaaca 8820atacacagga caggcataag atgcatttaa tggggacact cagaggcaga gggttatcag 8880aaggaggcac ttctctccca agttctcatc atcccagggc cagggacagc tggtcacacc 8940ttagggagtt cactaggaga gggatctggc ttcttgtcat tctgggtatt tgtagggaaa 9000ttggaaggga accgagagca cctagccaat cgcatagcaa tgggagattt caggctgtgg 9060ggaatgtctt tgctggtgaa aagaacatcc tgaccttaga aatctttcac cgagggggat 9120ctgcgttcca gaacttctgg agctggtata ggtaaggctt tgagctttcc tactgagcca 9180gcctgttgct aggttaccaa aggggacctc gagggccatc tggccaacaa gcagacttgt 9240ctctccttac acccccagac gtatcactgc aaaactacag aaaaccaaag acagagaaaa 9300tcttaaaagc agccagattt aaaaaatggc atattagttt caaagcagca gccatgaaat 9360tgacagctga tgtctcaaca gcaagaatga aaagtggaag acaggccagg tgtggtggct 9420caggcctgta atcccagcac tttgggaggc cgaggcgggt ggatcacgag gtcaggagac 9480caagaccatc ctggctaaca tggtgaaacc ccgtctctac taaaaataca aaaaaattag 9540tcgggcatgg tggtgggtgc ctgtagtccc agctactcgg gaggctgagg caggagaatg 9600gcgtgaaccc gggaggcgga gcttgcagtg agccgagatt gtgccactgc actccagcct 9660gggtgacaga gcaagactct gtctcaaaaa aaaaaaaaaa aaaaaaaaaa aaagggtgac 9720gaagcttcaa tctcctgaaa ggaagcaact gccgcctttg attcgatacc caccaaaatc 9780cgtgaagaag gaaggcaaaa taaaaacact tcctgattga actggaaaga tttccgcaat 9840agaagaccca ctgtccaagg aattctaaag gatgctttcc aggcagaaga aaatgacccc 9900agaggaagat cagagattca ggaaagaaat ggagagtgat aaaaatggaa aattcggggg 9960ccaatttaaa caaaagctga ctgctctaca actgttgtgt ctctatcttt tgtaacatat 10020atgtgtgtgt agcttttttt tttttttttg tcaagatgga ttctcactct gtcgcccagg 10080ctacagtgaa atggcacggt ctcggctcac tgcaacctct gccccttggg ctcaaatgat 10140tctcttgcct cagcctcctg agtagctgag attacaggtg cctggcacaa tgcctggcta 10200atttttgtat ttttactaga gatgggattt ctccatgttg gccaggctgg tcttgaacac 10260ctgacctcag gtgatccacc tgcctgggcc tcccaaagtg ctaggattac aggcgcgagc 10320cactgcatct ggcctatgtg tgtgtttata tggaattaaa acacatggca ataataccct 10380ccaaattggg agaaaccaaa aatagcattt aaatgttgta agctccctgc ataatcaaga 10440agagaataga tttacgttag attttgatac ctggaggatg aatgttgtaa tttctagggt 10500gaccatgaaa agaggagaca acggtgtatg tttttttttt tttgagatgg agtctcactt 10560tgtcacccag gctggagtgt tgtggtgtga tcttggctca ctgcaacctc ctcctcttgg 10620gttcaggcca tcctcccacc taggcctcca gagtaggtgg gatcacaggc acctgccacc 10680acacctggct aatttttttt tttttttaaa tatttagtag agatggggtt tcaccatgtt 10740ggccaggctg gtcttgaact cctgacctca ggcgatctgc ctacctctgc ctctcaaagt 10800gctgggatta caggtgtgag ccatcgcgcc cggccaacag tgatcacttt caaactaaca 10860gaggttcaaa aataaaatca gacttaacca aaaaccaggt aacagagctg gtaggatata 10920cagaaagact gacctcacgt atatcaacga ttacagttaa tattaatgaa ggaaatgctc 10980tagtttaaaa acgagggttg tcaaagaccc cacataagaa gctccttacc agcggtgcac 11040ctagaaccta aggaaacagg acagatgaag gaggacgcgc ccccgccgct gtcctgcgcc 11100tcagccatcc tatgagacgg gaaaggtttc tgtctgcagc tgggcccgtg ctctttacca 11160gctcctggct ttcttctctg gaaggttcct gcctgttttg ccctcacacc tgctcctctc 11220tcagccctct caggggtggg gctggaggcc accaaagagc ctcctctgct ctccagttgc 11280tcgactgctc ctcatttccc cctggggtct gcgtcagggt ttccttcttt tccagcccca 11340ccccgcgtgc atcccacctg gtctcgggtc ggggctgctc ccgcttactg ccccctgccc 11400aggctggtgt gcaccccctc tggctgcttt caaggcctct tctctcttct cggcaggaca 11460ggcacaggca ggtggccagg tgtcatgctt agctccccgc ccagtgagat tctttcattt 11520aacaatcttc ccctgaatag ttcatgttca ttgctgaaaa tttgaaaaat atggaaaagc 11580acaaagatta agatataaac cgccctcaat tcccctgccc agagagagtc actgctatga 11640cttggtgact aggaacctta tttctctctc gctctttttt ttttttttga gacagagtct 11700tgctctgtca cccaggctgg agtgcagtgg ctcgatctca gctcactgca acctccgcct 11760cctgggttca agcgattctc ctgcctcagc ctcttgagta gctgggatta caggcacctg 11820ccaccatgcc cggctaattt ttgtattttt agttgagaga gggtttcatc ttgttggtca 11880ggcggacttg aactcctgac ctcaggtgat cagcccacct cggcctccca aagtgctggg 11940attacaggtg tgagccactg cgccttcatc tctcttctgt gtatgtgtac gctgtttttt 12000ctttagaatg ggggacgtta tcaggctcta catggtgtgt agtcggctag catgttgtaa 12060gcctttccct gtgtcacaag tgctcatctg gaacaggatt ctaatgactg cctgtggcta 12120tgttgggatt cctttaactc agctccttct gcccagcatc tatctttttt ccatcttttg 12180tcctaagtgt tgctataata aatcattgat cacacatgcc tgactgtttg cataggataa 12240attacgggaa atgtttttgc tgttcaggga ctgtgcccat ttttaggcct cagagacacc 12300atgccagact gcccagtatt gatctttact ctttttagat gatgccaaac ttttctgtga 12360actttaaaaa cctgtgtctt gacagtccat ttctgtaagt ctttcacatt agatttcctg 12420tcaggatgat agtcaattct aggcagatga tgttttctca gccatggctg aagcagttgt 12480gatttgttgt ggccatgtaa agtcccgatg atccattgcc tccctggatg ggttggaata 12540atttggtttg ggagcatata acagaatgac ctggagtcac agcagctcag acggaagtgt 12600atttctccct tacagatgaa agaattccag gccaggctgg aatgacaact gcacacagtc 12660atctgggccc cctccttcca gctcccatca ccccaggatg tggcttttat gcagatgatc 12720caaaatggct gctcaagtcc cagccaacac atcccattcc agggagcagg aaaaaggtgt 12780gtctttccct tcattttatg tgattccttt ctagaagtac tactcattac ttctgcttgc 12840atctccctgg ctagcactta cttagttata tggccatagc tagctgaagg aaggacaggg 12900actgtcatac actagctaag aggcaaactg cttagataaa aaggtctcta aagaaggtca 12960gagcggctgc tagggtgcaa ctctattact tattgttatg ggacgaactg tgtccctcat 13020tcaggttgat gtcctaagcc ccagaacctc agaatgggat tgtatttgga gacaggttct 13080ttaaggaggt aaggaggcta aaatgagatc attagggtgg gccataatcc gactgatgtc 13140ttacaagaag agattaggac acggacatgc tcagagggac ggccacgtga ggacaccaag 13200aaaggcagct gtctgcaagt caaggacagg gctcagggga aaccaacctt gccaacacct 13260tcatctcgga cttctagcct ctaggaccat gagaagatac atttctgttg tttaagctgc 13320ccggtctgtg gtactttgtt atggcagccc aagtaaacaa atacagtcat ctgctgctgg 13380aacaaatcac cccagcactg tggcttggca gcacacatgt ctagtcatag agttatatgt 13440agttacgtgt agagccatat gtatcgtcac acgttctgtg ggtcaggaat ttggacccag 13500cttaaccagc tccacttctc gccagggttc agtcaaatac cagctgcctc ccacctgaga 13560gctcagccgg ggaagggtcc ctttccaatc tcacgtggtg ttggcaggat ccagttcctc 13620atggcctgct ggactgagaa cctcagttct cactgcctgt tggccagagg ccgcctttat 13680gtcctcgcca tgtgggcctc tccaacatgg cagctgactt catcagagca tccatgccaa 13740gaaggcaaca gagagggcca gggagactga agtcataccc ttttgcgacc tagtcatggg 13800gtgacattcc atcacctttg cccattggtt agaagcaggc caccaggtac agcccaagct 13860cacggggagg ggtcatacaa gggtgtcaat accaggaggt gaggggtgct ggggccatct 13920tatgagtctg cccactgagg taactaacaa ccttgaggcc tgacacagtg gacaaaggcc 13980cttattaaca gcagagaact gggaacttta tttatttatt tatttttgag acagagtctc 14040actcttgtca cccaggctgg agtgcaatgg catgatcttg gctcactgca acctccacct 14100cccaggttca agcaattctg cctcagcctc cggaatagct gggactacag gcatgcacca 14160ctacacccgg ctaatttttg tatttttagt agagacaggg tttcgccatg ttggccaggc 14220tggtctcgaa ctcctgacct ctggtgatct gcctgccttg gcctcccaaa gtgctgggat 14280tacaggcgtg agccaccgca cctcgctgga acttaatttt tttagagaca gtgtcgctct 14340atcacccaag ctggagtgca gtggtgcaat cctagctcac ttgcagcctc aaattcctgg 14400gttcaggtga tcctcccaca tcagcctccc aagaactggg aactaacagc tgtttctctg 14460ctgtccttct caagaaaagg gaggctactg ctaccccact ggggacaatg ctgggtttcc 14520ctttaggaca ggctctgaga caaggcggag gtgctgtttg tggccacaga gcaggggact 14580ctgggttgca ggtgtggcct ggctaaagta ggctttactg ggctcctctc tgcctgcatc 14640accccccggc tgggcggttg tctctgaggc caaccttact ccctgctggg caggctggac 14700agctgccctc tccgtttgcc cctctaccac ccaaaaggca ggaggctctg gagaccagga 14760ccctgcccgc cacggcctgt gtcccaggcg tgagggggtg ccccacagac ctctgctgag 14820ctgctgctga atgacgcccc ttgggggtcc tgccggaagg tcagagcagg ggtgcactcc 14880cataaagaaa cgcccccagg tcgggactca ttcctgtggg cggcatcttg tggccatagc 14940tgcttctcgc tgcactaatc acagtgcctc tgtgggcagc aggcgctgac cacccaggcc 15000tgccccagac cctctcctcc cttccggggc gctgcgctgg

gaccgatggg gggcgccagg 15060cctgtggaca ccgccctgca ggggcctctc cagctcactg ggggtggggt gggggtcaca 15120cttggggtcc tcaggtcgtg ccgaccacgc gcattctctg cgctctgcgc aggagctcgc 15180ccaccctctc cccgtgcaga gagccccgca gctggctccc cgcagggctg tccgggtgag 15240tatggctctg gccacgggcc agtgtggcgg gagggcaaac cccaaggcca cctcggctca 15300gagtccacgg ccggctgtcg ccccgctcca ggcgtcggcg ggggatcctt tccgcatggg 15360cctgcgcccg cgctcggcgc cccctccacg gccccgcccc gtccatggcc ccgtccttca 15420tgggcgagcc cctccatggc cctgcccctc cgcgccccac ccctccctcg ccccacctct 15480caccttcctg ccccgccccc agcctcccca cccctcaccg gccagtcccc tcccctatcc 15540cgctccgccc ctcagccgcc ccgcccctca gccggcctgc ctaatgtccc cgtccccagc 15600atcgccccgc cccgcccccg tctcgccccg cccctcaggc ggcctccctg ctgtgccccg 15660ccccggcctc gccacgcccc tacctcacca cgccccccgc atcgccacgc cccccgcatc 15720gccacgcctc ccttaccatg cagtcccgcc ccgtcccttc ctcgtcccgc ctcgccgcga 15780cacttcacac acagcttcgc ctcaccccat tacagtctca ccacgccccg tcccctctcc 15840gttgagcccc gcgccttcgc ccgggtgggg cgctgcgctg tcagcggcct tgctgtgtga 15900ggcagaacct gcgggggcag gggcgggctg gttccctggc cagccattgg cagagtccgc 15960aggctagggc tgtcaatcat gctggccggc gtggccccgc ctccgccggc gcggccccgc 16020ctccgccggc gcagcgtctg ggacgcaagg cgccgtgggg gctgccggga cgggtccaag 16080atggacggcc gctcaggttc tgcttttacc tgcggcccag agccccattc attgccccgg 16140tgctgagcgg cgccgcgagt cggcccgagg cctccgggga ctgccgtgcc gggcgggaga 16200ccgccatggc gaccctggaa aagctgatga aggccttcga gtccctcaag tccttccagc 16260agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcaacagc 16320cgccaccgcc gccgccgccg ccgccgcctc ctcagcttcc tcagccgccg ccgcaggcac 16380agccgctgct gcctcagccg cagccgcccc cgccgccgcc cccgccgcca cccggcccgg 16440ctgtggctga ggagccgctg caccgaccgt gagtttgggc ccgctgcagc tccctgtccc 16500ggcgggtccc aggctacggc ggggatggcg gtaaccctgc agcctgcggg ccggcgacac 16560gaacccccgg ccccgcagag acagagtgac ccagcaaccc agagcccatg agggacaccc 16620gccccctcct ggggcgaggc cttcccccac ttcagccccg ctccctcact tgggtcttcc 16680cttgtcctct cgcgagggga ggcagagcct tgttggggcc tgtcctgaat tcaccgaggg 16740gagtcacggc ctcagccctc tcgcccttcg caggatgcga agagttgggg cgagaacttg 16800tttcttttta tttgcgagaa accagggcgg gggttctttt aactgcgttg tgaagagaac 16860ttggaggagc cgagatttgc tcagtgccac ttccctcttc tagtctgaga gggaagaggg 16920ctgggggcgc gggacacttc gagaggaggc ggggtttgga gctggagaga tgtgggggca 16980gtggatgaca taatgctttt aggacgcctc ggcgggagtg gcggggcagg gggggggcgg 17040ggagtgaggg cgcgtccaat gggagatttc ttttcctagt ggcacttaaa acagcctgag 17100atttgaggct cttcctacat tgtcaggaca tttcatttag ttcatgatca cggtggtagt 17160aacacgattt taagcaccac ctaagagatc tgctcatcta agcctaagtt ggtctgcagg 17220cgtttgaatg agttgtggtt gccaagtaaa gtggtgaact tacgtggtga ttaatgaaat 17280tatcttaaat attaggaaga gttgattgaa gttttttgcc tatgtgtgtt gggaataaaa 17340ccaacacgtt gctgatgggg aggttaattg ccgagggatg aatgaggtgt acattttacc 17400agtattccag tcaggcttgc cagaatacgg ggggtccgca gactccgtgg gcatctcaga 17460tgtgccagtg aaagggtttc tgtttgcttc attgctgaca gcttgttact ttttggaagc 17520taggggtttc tgttgcttgt tcttggggag aatttttgaa acaggaaaag agagaccatt 17580aaaacatcta gcggaacccc aggactttcc ctggaagtct gtgtgtcgag tgtacagtag 17640gagttaggaa gtactctggt gcagttcagg cctttctctt acctctcagt attctatttc 17700cgatctggat gtgtcccaga tggcatttgg taagaatatc tctgttaaga ctgattaatt 17760tttagtaata tttcttgttc tttgtttctg ttatgatcct tgtctcgtct tcaaagttta 17820attagaaaat gattcggaga gcagtgttag cttatttgtt ggaataaaat ttaggaataa 17880attattctaa aggatggaaa aactttttgg atatttggag aaattttaaa acaatttggc 17940ttatctcttc agtaagtaat ttctcatcca gaaatttact gtagtgcttt tctaggaggt 18000aggtgtcata aaagttcaca cattgcatgt atcttgtgta aacactaaac agggctcctg 18060atgggaagga agacctttct gctgggctgc ttcagacact tgatcattct aaaaatatgc 18120cttctctttc ttatgctgat ttgacagaac ctgcatttgc ttatcttcaa aatatgggta 18180tcaagaaatt tcctttgctg ccttgacaaa ggagatagat tttgtttcat tactttaagg 18240taatatatga ttaccttatt taaaaaattt aatcaggact ggcaaggtgg cttacacctt 18300taatccgagc actttgggag gcctaggtgg acgaatcacc tgaggtcagg agtttgagac 18360cagcctggct aacatggtga aaccctgtct ctactaaaaa tacaaaaatt agctggtcat 18420ggtggcacgt gcctgtaatc caagctacct gggaggctga ggcaggaaaa tcgcttgaac 18480ccgggaggca gagtctgcag tgagttgaga tcacgccact gcactccagc ctgggtgaca 18540gagcgagact ctatctcaaa aaaaattttt tttaatgtat tatttttgca taagtaatac 18600attgacatga tacaaattct gtaattacaa aagggcaata attaaaatat cttccttcca 18660cccctttcct ctgagtacct aactttgtcc ccaagaacaa gcactatttc agttcctcat 18720gtatcctgcc agatataacc tgttcatatt gtaagataga tttaaaatgc tctaaaaaca 18780aaagtagttt agaataatat atatctatat attttttgag atgtagtctc acattgtcac 18840ccaggctgga gtgcagtgat acaatctcgg ctcactgcag tctctgcctc ccaggttcaa 18900atgcttctcc tgcctcagcc ttctgagtag ctgggattac aggcgcccac caccatgtcc 18960agctaatttt tgtattttta gtagagatgg ggtttcacca tgttggccag gctggtcttg 19020aactcctgac cttgtgatct gtccacctcg gcctcccaaa gtgctgggat tacaggtgtg 19080agccaccatg cctggctaga ataataactt ttaaaggttc ttagcatgct ctgaaatcaa 19140ctgcattagg tttatttata gttttatagt tattttaaat aaaatgcata tttgtcatat 19200ttctctgtat tttgctgttg agaaaggagg tattcactaa ttttgagtaa caaacactgc 19260tcacaaagtt tggattttgg cagttctgtt cacgtgcttc agccaaaaaa tcctcttctc 19320aaagtaagat tgatgaaagc aatttagaaa gtatctgttc tgtttttatg gctcttgctc 19380tttggtgtgg aactgtggtg tcacgccatg catgggcctc agtttatgag tgtttgtgct 19440ctgctcagca tacaggatgc aggagttcct tatggggctg gctgcaggct cagcaaatct 19500agcatgcttg ggagggtcct cacagtaatt aggaggcaat taatacttgc ttctggcagt 19560ttcttattct ccttcagatt cctatctggt gtttccctga ctttattcat tcatcagtaa 19620atatttacta aacatgtact atgtgcctgg cactgttata ggtgcagggc tcagcagtga 19680gcagacaaag ctctgccctc gtgaagcttt cattctaatg aaggacatag acagtaagca 19740agatagataa gtaaaatata cagtacgtta atacgtggag gaacttcaaa gcagggaagg 19800ggatagggaa atgtcagggt taatcgagtg ttaacttatt tttattttta aaaaaattgt 19860taagggcttt ccagcaaaac ccagaaagcc tgctagacaa attccaaaag agctgtagca 19920ctaagtgttg acatttttat tttattttgt tttgttttgt tttttttgag acagttcttg 19980ctctatcagc caggctggag tgcactagtg tgatcttggc tcactgcaac ctctgcctct 20040tgggttcaag tgattctcat gcctcagcct cctgtttagc tgggattata gacatgcact 20100gccatgcctg ggtaattttt tttttttccc ccgagacgga gtcttgctct gtcgcccagg 20160ctggagtgca gtggcgcgat ctcagctcac tgcaagctcc gcttcccgag ttcacgccat 20220tctcctgcct cagtctccca agtagctggg actacaggcg cctgccacca cgtccagcta 20280atttttttgt atttttaata gagacggggt ttcaccgtgt tagccaggat gatcttgatc 20340tcctgacctc gtcatccgcc gaccttgtga tccgcccacc tcggcctccc aaagtgctgg 20400gattacaggc atgagccact gtgcccggcc acgcctgggt aatttttgta tttttagtag 20460agatggggtt ttgccatgat gagcaggctg gtctcgaact cccggcctca tgtgatctgc 20520ctgccttggc ctcccaaagt gctaggatta caggcatgag ccaccatacc tggccagtgt 20580tgatatttta aatacggtgt tcagggaagg tccactgaga agacagcttt tttttttttt 20640ttttttgggg ttggggggca aggtcttgct ctttaaccca ggctggaatg cagtatcact 20700atcgtagctc acttcagcct tgaactcctg ggctcaagtg atcctcccac ctcaacctca 20760caatgtgttg ggactatagg tgtgagccat cacacctggc cagatgatgg cttttgagta 20820aagacctcaa gcgagttaag agtctagtgt aagggtgtat gaagtagtgg tattccagat 20880ggggggaaca ggtccaaaat cttcctgttt caggaatagc aaggatgtca ttttagttgg 20940gtgaattgag tgagggggac atttgtagta agaagtaagg tccaagaggt caagggagtg 21000ccatatcaga ccaatactac ttgccttgta gatggaataa agatattggc atttatgtga 21060gtgagatggg atgtcactgg aggattagag cagaggagta gcatgatctg aatttcaatc 21120ttaagtgaac tctggctgac aacagagtga aggggaacac cggcaaaagc agaaaccagt 21180taggaagcca ctgcagtgct cagataagca tggtgggttc tgtcagggta ccggctgtcg 21240gctgtgggca gtgtgaggaa tgactgactg gattttgaat gcggaaccaa ctgcacttgt 21300tgaactctgc taagtataac aatttagcag tagcttgcgt tatcaggttt gtattcagct 21360gcaagtaaca gaaaatcctg ctgcaatagc ttaaactggt aacaagcaag agcttatcag 21420aagacaaaaa taagtctggg gaaattcaac aataagttaa ggaacccagg ctctttcttt 21480tttttttttt tgaaacggag tttcgctctt gtcacccggg ctggagtgca atgatgtgat 21540ctcagctcac taaaacctct acctcctggg ttcaagtgat tcttctgcct cagcctccca 21600agtaactggg attacaggcg tataccacca tgcccagcta atttttgtgt ttttagtaga 21660gatggggttt caccatgttg gccaggctgg tctcgaactt ctgacctcag gtgatccact 21720cgcctcagcc tgccaaagtg ctgggattac aggtttgggc cactgcaccc ggtcagaacc 21780caggctcttt cttatactta ccttgcaaac ccttgttctc attttttccc tttgtatttt 21840tattgttgaa ttgtaatagt tctttatata ttctggatac tggattctta tcagatagat 21900gatttgtaaa aactctccct tcctttggat tgtcttttta ctttcttgat agtgtctttt 21960gaagtgtaaa agtttttaat tttgatgaag tcgagtttat ctattttgtc tttggttgct 22020gtgcttcaag tgtcatatct aagaaatcat tgtctaatcc aaagtcaaaa aggtttactc 22080ctatgttttc ttctaagaat tttagagttt tacatttaag tctgatccat tttgagttaa 22140tttttatata tggttcaggt agaagtccaa ctttattctt ttccatgtgg ttattcagtt 22200gtcccagcac tgtttgttga agagactatt ctttccccat ggaattatct tagtaccctt 22260gttgaaaatt aatcgtcctt aattgtataa atttatttct agactgtcag ttctacctgt 22320tggtctttat gtcgatcctg tgccagtacc atacagtctt gattactgaa gtttgtgtca 22380cagtttaaat tcatgaaatg tgagttctcc aactttgttc cttttcaaga ttgatttggc 22440catgctgggt cccttgcatt tccgtacgaa ttgtaggatc agcttgtcag tttcaacaaa 22500gaagccaagt aggattctga gagggattgt gttgaatctg tagatcaact tggggagtat 22560tcgcatctta acaatattgt cttccaccta tgaacatggg caaactttgt gtaaatggtc 22620agattgtaag tatttcgggc tgtgtgggca cagtgtctct gtcacagcta cgcggctctg 22680ccattgtagc atgaaagtag ccataagcaa tatgtatgag tgtctgtgtt ccaatagaat 22740tttattaatg acaaggaagt ttgaatttca tataattttc acctgtcatg agatagtatt 22800tgattatttt ggtcaaccat ttaaaaatgt aaaaacattt cttagcttgt gaactagcca 22860aaaatatgca ggttatagtt ttcccactcc taggttaaaa tatgatagga ccacatttgg 22920aaagcatttc tttttttttt tttttttttt tttttgagac ggagtttcac tcttgttgcc 22980caggctggag tgcagtggcg cgatctcggc tcactgcaac ctctgcctcc caggttcaag 23040acattctcct gcacggcctc cctagtagct gggattacag gcatgcgcca ccacacccag 23100ctaattttgt atttttagta gagacggggt ttctccatgt tggtcaggct ggtcttgaac 23160tcctgacctc aggtgatcca cccgcctcag cctcccaaag tgctgggatt acagggtgtg 23220agccaccaca ccctgctgga aagcatttct tttttggctg tttttgtttt ttttttaaac 23280tagttttgaa aattataaaa gttacacata tacattataa aaatatcttc aagcagcaca 23340gatgaaaaac aaagcccttc ttgcaagtct gtcatctttg tctaacttcc taagaacaaa 23400agtgtttctt gtgtcttctt cccagatttt aatatgcata tacaagcatt taaatgtgtc 23460attttttgtt tgcttgactg agatcacatt acatatgtat ttttttactt aacaatgtgt 23520catagatatt gttccatagc agtacctgta attcttatta attgctatgt aatattttag 23580aatttctttt taaaagagga cttttggaga tgtaaaggca aaggtctcac atttttgtgg 23640ctgtagaatg tgctggtgac atattctctc taccttgaga agtccccatc cccatcacct 23700ccatttcctg taaataagtc aaccacttga taaactacct ttgaatggat ccacactcaa 23760aacatttagt cttattcaga caacaaggag gaaaaataaa ataccttata aagcactgtt 23820taatattgta ttaaattgga tcaatttggg ggctagaatg tatgttagag acatgatatg 23880tccataggtc cttgctatca cagtgaggtc tcagggacag tcgtttggta tcatttggga 23940tctcataagc agactctctc tgcttgacct gacaaatcag agtctgtgtt ttaacaggtt 24000cagtgagtga cttacatgca cattggagtt tgggaagctc cactgtaggt gcttagacct 24060tacctttgtt gttgctaata acaatgcaag catttgggag gaagacctgt gttgctcata 24120tgtgtccagg tgtagctgag gtggccttgc ttatctgctg tagggccgtt gagcatttct 24180gtagctgtga tgagtgagct gaggtgagcc tgcggagagc tcccagccat tggtagtggg 24240actcgcttag atgaactgga aggacccttt catctgagca gccactatgg agaaaaacaa 24300ccgaatgagg ggagagacaa tgtgcaattt tatttagggc acaaaggaga gctgtggtta 24360gaaggtgaca tttgagtgga aagggggcaa gccatgtgta tagcgggaga agagaggtcc 24420aggcagagtt aacagaaggc agaaatgctt tccatgtttg agaaccagta aggaggccag 24480tggctgaagt aaggtgaagg gcagaaataa ggatgaggct gcgagagatg agaggttaga 24540gacgagcgtc ttgtgcacca agataagctt gtgtggtcaa aacaagtagt ttaatttatg 24600tttttaaaag atcattttgg ctgggcacaa tggttcatgc ctgtaatacc agtagtttga 24660gacggtgtgg tgggaggatt gcctgaggcc agacgaccag catagccaac atagcagcac 24720ctataaggtc tctacaaaaa actttaaaaa attagctggg catagtggtg tgtgcctgta 24780gtcccagcta ctcaggaggc tgaggaggct ggaggattgc ttgagtccag gagtttgagg 24840ctgcagtgag ctatgattat gccactacac tacaacctgg gcaagagagt gagaccctgt 24900ctctaaatat acacacacac acacacacac acacacacac acacacacac acacacacac 24960acacacatat atatgtatat atatgcattt agatgaaaag atcactttga caataccaca 25020tgctggtgag gatttagaaa aactaggtca cttattgctg gtgggaatat aatatagtac 25080ggccactctg gaaaacagtt tggcagtttg tcataaaact gaacataccg ttagtataca 25140gcccagcagc aactacaatc ctgggcatta atcctagaga aatgaaacct taatgttcac 25200ataaaaacct atactcaagt atgcatagca gctttaccca taatatctaa gaactggaat 25260cagctcagat gtccttcaac aggtgaatgg ttaaactact cagtaataaa aaggaatgag 25320ctactgatag catgcaacag tttaggtgaa gttatgctaa tgaaaaaagc caatcccaaa 25380aggttataca tactgtatga ttctatgttt ttttgcaatg gcacagtttt agggatggag 25440aatagattag tggttgcctg gggttagaga tggggtagta gagtaggtta gtggtggcag 25500aggagagaaa agagagggag gtgaatgtgg ttataaaagg acaacacagg ggaatacttg 25560taatggaaat gctttgtctt tttttttttt tttttttttt tggcgacaga gtcttgctct 25620gttgcccagg ctggagtgca gtggcatgat cttttctcac tgcaacctct gcctcctggg 25680ttcaagtgat acttgtgtct cagtctccca tgttcagagt gaaacaaacc agaggtaatg 25740ttcatccaaa taatccaaca cacatgacat taaaacatca agatcaggtc ggacgtggtg 25800gctcatgcct gtaatcccag cacttttggg aggccaaggt gggcagatca cttgaggtca 25860ggagttcgag accagccggg ccaacatgat gaaaccccat cttgactaaa aatacaaaaa 25920ttagccgggc atggtggtgt gcacctgtag tcccagctac ttgggaggct gaggcaagag 25980aactgcttga acccgagggg cagaggttgc agtgagctga gagtgcgcca ttgcacttca 26040gcctgtgtga cagagtaaga ctccatctcc aaaaaaaaaa aaccaagatc aattaaaata 26100cagcattact gggccgggtg tggtggctca cacctgtaat cccagcactt tgggaggccg 26160agatgggcag atcacgaggt caggagatcc agaccatccc ggctaacacg gtgaaacccc 26220gtctctacta aaaaatacaa aaaattagcc gggtatagtg gtgggtgcct gtagtcccag 26280ctacttggga ggctgaagca ggagaatggt gtgaacccgg gaggcagagc tggcagtgag 26340ctgagatcgc gccactgcac tccagcctgg gcgacagagc aagactccgt ctcgggggaa 26400aaaaaaaaat aaataaatag aatgctgtag tgtccttgag tttacatgcc cctccttacg 26460cttgtgtgcc cgtgcagatt gcttgattac acaattagag gaggctggcg gaggattgtt 26520ttaatttttt tttttttgag acagtctggc tctgttcccc aggctagagt gcaatggcgc 26580aatcttggtg cactgcaacc tctgcctcct gggttcaagc agttcttctg ccgcagcctc 26640ccgagtagct gggattatag gcgcccgcca ccacgcccaa ctattttttg tatttttagt 26700agagcagcgt ttcaccatgc tggccaggct ggtctcgaac tcctgacctc agatgatctg 26760ctgccccagc ctcccaaagt gctgggatta caggcgtgag ccacacctgg ccgtttgttt 26820taattttgaa ggtgaagtga aagtgactac atttaccaaa agtgattgaa aagccaggac 26880tgttcttacc ctgtttttcc agttcttgct cagagcaagg tggtttcttt ttcacttaat 26940caccatactt acttttcatg tagaacaagt cagtttgagt tatcagttca tcatcttaac 27000taaattccat gggggaagga attagtttta gtttcttaaa cttccaggtt tgcttattgg 27060acaaaatgag atagcaaggc agtgttttta agttagattt tttatttctt tggtaataca 27120attttctcag aaacttagta gtcttttagt ttagttgttt ttagttggtc ctatgttttg 27180gatcacccct ctctacttta ttttgatagt gccaactgtg aagacatctg aagccatagg 27240tttggatggg aaggaggcat ctttagcctg atcatcttcg ccaggctgtt tatctccttt 27300tgcttggctg agaagtctta ataggaggct tattcccagc tatttgggga catagaagca 27360gttagccatt gcttatattt tactgaggtc tgtgtggtat gttgattgta gtcagttaac 27420gattttgaga actgaaggca gcctggtata tatagagtag gtattagact gtgtttcttc 27480taattgaatt tcccatctct tgtaatctat gccatcatct tctgtactgc tgagaaagaa 27540agaaagtttc taatcaaact ataccactgg ttgtaagatg cagtttggct ttagtgatgt 27600taacacatga ttcaaacgtg aaattgattg agtattggtg aaatacagag gagatttaaa 27660gccagaagac ctgggtttaa atgctggctg tatgacttca tatctgtgtg atcttgggca 27720tgtcatggtt ggcacttcaa tttcttctct ctataatggg ggaagtgagg ccagtcatgg 27780tggctcatac ctataatccc agtgctttgg gaggccaaga tgggaagatc gcttgaggcc 27840aggagtttga gcaattgggc aacatcgtga ggccccgtct ctacaaaata ttttgaaaaa 27900attagccagg cccagtggtg cgtgcctgtg gtccgcgcca ctcaggaggc tgagacggga 27960ggatcctttc agcctaggag tttaaggcta aagtgagcca tgattgtgct atcgtactcc 28020agcctgggca gcagagcaag atcctgactc taaaaaaaag taaaataaag taaaatgggg 28080gaaatgaact gctttagtaa catcatctgt tttttctgtg agcagcgtag cttgacagcc 28140attggtgaac tcgtgccctg tgcttccctg tccagatccc cattctgccc gcaacatgga 28200gtataacggt ttattcatag tagtcgagaa acactcactg aatgaatgaa tgaggtgtag 28260aactaagtgg agtgggtaat tcaacacata ttaatttcct tctttttttt atttttagaa 28320agaaagaact ttcagctacc aagaaagacc gtgtgaatca ttgtctgaca atatgtgaaa 28380acatagtggc acagtctgtc aggtaattgc actttgaact gtctagagaa aataagaact 28440ttgtatattt tcagtcttaa tgggctagaa tattctttgt gtcccagcta ttttaaatgg 28500attcagaaat ccatttaaga tgaagaagga cccttttccc atatttctgg ctatatacaa 28560ggatatccag acactgaaat gaataatgtt ccctttttgt aatcttttat gcaaaaatta 28620aaaccattat ggtaattgaa caacatgttt atgtttagtt aacaccctta gcaactatag 28680ttattttaaa accatctatg gtttgatatt tttgcatttg ttgcaatagt aggaacagca 28740caagacagtt cagtttgtct ctcttatttg ctttttcttg gcagtttgct gtcctattgt 28800acctctgctc ctagcagtgg ctggagccca ctcctctgtg cttcgggatt agtggggatc 28860gtggggcatt gactgtaggt cagctttcct tgcttgatct ttctcactgg gatgaactag 28920cagcaccttc ttttgtagct gctttgcttt tgactatctt tctgaccgtt gttcctagta 28980gctgtagatg gtaaatatat ttaggcctgt ttccaatggc tcagtaggag acatattcac 29040ctatgatatc tgaattctgt tacccacatg ggcatgcgtg aaatagttgc cttgccttac 29100tttcccttgg aataaataat tcatgttatt ctcctggtag aagctagaaa aagcctttat 29160agtcagtcag aaaaaaattt ttagacaaat aatcttgatt ttagtactga caaaaacgtg 29220tggtgattct ttttttaatt tttttttgag acggagtttc actcttgttg cccaggctgg 29280agtgcaatgg cgtgatctcg gctcactgca acctctgcct cctgggttca agtgattctc 29340ctgcctcagc ctcccaagta gctggagtta caggcatgtg ctactgtgcc cagctaattt 29400tgtattttta gtagagatgt tggtcaggct gatctcgaac tcccaacctt aggtgatctg 29460cccgcctcag cctcccaaag tgctgggatt acaggcgtga gccagggcgc ccggtgattc 29520atttgttttt tcaaaaaatt tcctcttggc cattgctttt cacttttgtt tttttttttt 29580ttttgagacg gagtcacgat ctgtcaccca ggctggagtg cagtggcatg atcttggctt 29640actgcaagct ctgcctccca ggttcacgcc attctcctgc ttcagcctgg cgagtagctg 29700ggactacagg tgctcgccac cacacccggc taattttttg tatttttagt agagatgggg 29760tttcaccgtg gtcttgatct cctgacctca tgacccgctc aactcagcct cccaaagtgc 29820tgggattaca ggcgtgagcc accgcgcccg gccctctctt gtctttttat tgtggtaaaa 29880tgcacataaa attgactgtc ttaaccattt ttaggggtac agttcagtat atatattcgt 29940aatgttgtac agccatcact gccatctact tcataagttt ttcttctgtc aaaactgaac 30000atctgtcttc attaaactcc ctatcatcca ttctttcctg tagtcccttt ctactttctg 30060tctgtatgag tgtaactgct ctggagacct catgtaagtg

gattcctaca ggatttgtgt 30120tttttttttg gtgatctgct tatttttaat gcctctgtgc atttgtatta tatactttca 30180aagtgatttc acaaaaccgt ttcattttag gttaactcat ttctgttgtt tgtgaaatac 30240tgtgtatgat tctgttctgt ttctgtctaa tttgtggaaa tgttgtggga agaaaatgaa 30300ataacaaatg agcatatgtc ctgaaaataa aaatataaaa attctaagtt agcatgctat 30360tgtagaatac aacgctatga taaaagtagg aaaaaaaaag gtttgaattc tatctctgct 30420acctgtgtaa gctgggtgac tttagataag ctgtaacgtg tttgagcctt actggctcat 30480ttttgaaatg taatccctag ttacacagtt cttgtgggat cagatggtac atgtgaaaca 30540ctgtgaaaaa gcaactgcat agatatgttc attagccacc tgagcgggaa gcgtatccca 30600ttgcgatgcc catcatccaa agctatatgt tatctttact tttttttttt tgagacagag 30660tcttgctctg ttgcccaggc tagagtgcag tggtgcaatc tcagctcact gcaagctcca 30720cctcccgggt tcacgctatt ctcctgcccc agcctcccaa gtagctggga ctacaggcac 30780ccgccaccat gcctggctaa atttttgtat ttttagtaga gatggggttt caccgtgtta 30840gccaggatgg tcttgatctc ctgacctcgt gatccgcccg cctcggcctc ccaaagtgct 30900gggattacag gcgtgagcca ctgcccctgg ccatctttac tttttttgtg aaatgacttt 30960aaatacttgg caaacatttg gtcattgttc atctgatctc caccatccag gtctcagaga 31020acataatttc tctctgaaag cttattgacc caggaaataa gatctctttc aatctgagtg 31080cgtcaggctt tattcttgtc attttgtctt ttgataattt tcaaatggaa ttcatggaat 31140gttggcttat attcatatat tagtaaagta tgttgagaca tcttaagatt gatttgtggt 31200tctatatgcc atattaaatc aaaataatag ctgttaatgg ttttcacatt agtctgtctc 31260ttgtttttat ggagtaatgc tgagagttca ttatgcttgt tctacagaag agcatgttaa 31320aaggagtttt tggagtcaga gaggttattc ttggtttcat aggatacact ctatactttt 31380tagggatttc agagtatata gctgaaggtg atattttatg taaatatgtt ttatggaaac 31440ttattgctca tcgctgtttc ctgttaactc tcctaaaata taattaaact tttggaactt 31500ttttatagct tttgtgctag actaattttt gtctctaatg aggttatata aatggcagct 31560tctgacgttt tcaatgtagg aagtcattta aaacttcatg tatattgtga aaatgtagtc 31620tgctttaagc tctctaaagt ggtctaagtt actggttcct aagtatggat gagcatcaaa 31680atcatctgga aaatttgtta aaaatacagt aatgaaggca cctcactgtc ctttttccca 31740aacatacttc tgcattctgt ttgagtaggt agggactaca catttttcac aagtatcctc 31800ttgggaatac ccaggaatgc ttacttgagc aacctcttac taatatgtac cttgataagg 31860tggctaggta aacataaata tacaaaaatc catagatctc ccatatatta gcataaatca 31920gctagaaaat ataacgttta aagatctagt tcacagtagc accaatatat cgaactctaa 31980ggaatcgata aatatgcaaa aactttataa aaacttctgt taatgtttct gaaagatata 32040ggtgaccact ttctagatag gaagatttta tattactaag ttgaattttc tctaaattaa 32100cacagaaatt taaaataatc ttgatcaaaa ttctagtaga ggtatttttg aacttgttca 32160ctgcaagaat aaatacataa ttgcaaagaa tatctcaaaa tcatcaccag gcctggtgtg 32220gtggcccatg cctgtaatcc cagcactttg ggaggctgag gcaggcagat cacctgaggt 32280caagagtttg agaccagctg gaccagtgcg gtgaaacact gcctctacta aaaatacaaa 32340aattagctgg gtgtggtggt gcatgcctgt agtcccagct acttgggagg ctgaggcagg 32400agaattgctt gaacccagga ggtacaggtt gcggtgagcc tagatcgcac cactgcattc 32460cagcctgggc gacaagagca aaattctgtc tcaagaaaaa agagaaaaaa gaaaaagaaa 32520tcaacactaa tatggtgaga cttaatgtat gtgacattaa aatagtgatt ggatgttaaa 32580acaggtatag aacagaaaga agagtgtatg tgtgtatctg tatgaattta tgatgggtgt 32640aacatatatg tattagggaa atgagggaaa tgatacattt ctctgacttt gggagaacat 32700tatatctcta cctcatattg caaacaaaca taaagttcag attaattacc taaatgtgaa 32760aaaatgaaat aatttcttta aaaaatgtaa tcttagtttg aggaaggtta acattataaa 32820ggaaaaaact gttttgagtg gaatatagtt caatatgtca aaatccacct tcaacaaaat 32880tgaaagtaaa ttgaacttgg ggaaagtatt gacagcatat agatcaaagg ttactagcct 32940gtgtaaagag cagttataaa tatcgttaag aaaaacactg tcgacctgtc ggcaccttgt 33000tctccgactc ccagcctcca gaactgtgac gagtaagtgc ttattgttta aaccacccag 33060tctgtatgtg gtattttgtt atagaaactc aagctgatta ggacactagt aatcagtaga 33120ctgaaactga aacaaaaata agaacctttt ttacctgtca aattggcaaa cattaagaat 33180attcagattt ttgtcagagg tgatacaacc ttctaagaag gcaatttggg aaaatataaa 33240gctttagatt attatatgtc tgacctagca gttttacctc tagggtgctt acccctagga 33300aagtgtgtaa tgatattggt gcagtgccct tcatcccatt agaaaattaa aaataacctt 33360aatggcctac cactaaaagg ggattgaaaa tttaagatat atttatttat gtgtttattg 33420agatggagtc ttgcactgtc cgcctgggcc agagtgcaat ggtgcgatct cggctcactg 33480caacctctgc ttcccgggtt catgtgattc tcctgcctca gcctcctgag tagctgggat 33540tacaggctca caccaccgca cccggctaat tttttgtatt tttagtagag atggggtttc 33600actgtgttgg ccagactggt ctcgaactcc tgacctcatg atccgcgccc ctcggcctcc 33660cagtgttggg attacaggtg tgagccactg cgcctggcca gatacattta tacaagagaa 33720tgttagttaa cattcataga tatttatatt ttgtttactt tttattaaaa aaattttttt 33780tagagacagg atcttactct gtcacccagg caggatgcag ttgcacaatc atagcccact 33840gcagcctgaa ctcctgggct taagtgatcc ttctgcctca gccttttgag tacctggggg 33900actttaggca gtgctactat acctggctaa tttttaaatg ttttatagat gagatcttgc 33960tgtattgccc aggctggtct agaattcctg ggcccaagtg atcctcccac cttggcctcc 34020caaagcgctg agattacagg catgagccac cacttctgac caatagatat ttatatttgt 34080gactggaaaa tatattaaca atgtgttaaa aaattcagtt aaaaaataat gaaagatttt 34140tgcttctggc taagatagaa taacaaggac agcatttatc ttcttgcctt gaaatagttg 34200aaaacggaag aaatatatgt aacagtggtt ttcaagttat tgggcatcag gcaaagaaga 34260atagttatcc caggaaaatg aatgtggaga gccctacaat ttccttacat tactgcctgg 34320tcatggcaag aggaaaaact gagaggagac tgaggctgag ccagtggttt gctgggttga 34380ggaggcagag ctgggagtgc agagatgcaa ggtggtgaga gcccatatgg aagaatacca 34440gggaagagag ctgcagaggg agctccggag acctgcaccc tgccctctca gtaccctgtc 34500atgtgtgtag ctgagtactg acgagcactt gcttgtgcgg aaatgaccca gggctggagg 34560tagagccacc tgaaaggatt agaaggaaca gttgctgaaa gtcacacagg gccaggaaga 34620atttctaatc acaccagttg gagtggaaaa cctcagctct catagagcag gtagggtact 34680cagaagggtt tgcccaccta gccccagact aagtttcgtt actctgaccc tacctaatat 34740taaaaagaga ttaattaaat tgttcgcaac aaaaataata tatttcagtg tttgtaacac 34800gtagaagtga attgtatgac aatagcataa aggctggaag agcagaaatt gacatgtatt 34860tgcgctgggc agaataatgc tcccctcttt ccccaaaaga tatcaagtcc taatccctgg 34920agcctgtaaa tattacttta tatggaaaat tgttttatga tgtgattaaa ttcaggatct 34980tgagatgagg gggctatctt ggatgatctg ggtaggcact aaatgcaatc acatatatat 35040aaaaaggagg cagagggaga ttttacacac agagagaagg ccctgtgaag atggaacaga 35100aagatttgaa ggtgctggcc ttgaaaattg gagtgatgaa gctataagcc aaggaatgca 35160gcagccacca aagctggaag aggcacggag cagttctcat ttagagccta ctccagaggg 35220aatgtggtgc tgccaattcc tttttttttt ttttttttaa gatatcattt acccctttaa 35280gttggttttt tttttttttt ttttttttta gtatttattg atcattcttg ggtgtttctt 35340ggagaggggg atttggcagg gtcataggac aatagtggag ggaaggtcag cagataaaca 35400tgtaaacaaa ggtctctggt tttcctaggc agagggccct gccacgttct gcagtgtttg 35460tgtccctggg tacttgagat tagggagtgg tgatgactct taacgagtat gctgccttca 35520agcatctgtt taacaaagca catcttgcac cgcccttaat ccatttaacc cttagtggac 35580acagcacatg tttcagagag cacggggttg ggggtaaggt tatagattaa cagcatccca 35640aggcagaaga atttttctta gtacagaaca aaatggagtg tcctatgtct acttctttct 35700acgcagacac agtaacaatc tgatctctct ttcttttccc acatttcctc cttttctatt 35760cgacaaaact gccaccgtca tcatggactg ttctcaatga gctattgggt acacctccca 35820gatggggtgg cggccgggca gaggggctcc tcacttccca gatggggcgg ccgggcagag 35880gcgcccccca acctcccaga cggggcggcg gctgggcggg ggctgccccc cacctcccgg 35940acggggcggg tggccgggcg ggggctgccc accacctccc ggacggggcg gctggccggg 36000cgggggctgc cccccacctc ccggacgggg cgggtggccg ggcgggggct gccccccacc 36060tcccggacgg ggcggctggc cgggcggggg ctgcccccca cctcccggac ggagcggctg 36120ccgggcggag gggctcctca cttcccggac ggggcggctg ctgggcggag gggctcctca 36180cttctcagac ggggcggctg gtcagagacg ctcctcacct cccagacggg gtggcagtgg 36240ggcagagaca ttcttaagtt cccagacgga gtcacggccg ggcagaggtg ctcttcacat 36300ctcagacggg gcggcggggc agaggtgctc cccacttccc agacgatggg cggccgggca 36360gagatgctcc tcacttccta gatgggatga cagccgggaa gaggcgctcc tcacttccca 36420gactgggcag ccaggcagag gggctcctca catcccagac gatgggcggc caggcagaaa 36480cgctcctcac ttcctagacg gggtggcggc tgggcagagg ccgcaatctt ggcactttgg 36540gaggccaagg caggcggctg ggaggtgaag gttgtagtga cccgagatca cgccactgca 36600ctccagcctg ggcaacactg agcactgagt gagcgagact ccgtctgcaa tcccggcacc 36660tcgggaggcc gaggctggca gatcacttgc agtcaggagc tggagaccag cccggccaac 36720acggcgaaac cccgtctcca ccaaaaaaca cgaaaaccag tcagacatgg cggtgcgtgc 36780ctgcaatccc aggcacttgg caggctgagg caggagaatc aggtagggag gttgcagtga 36840gtagagatgg tggcagtaca gtccagcctt ggctcggcat cagagggaga ctgtgcgagg 36900gcgagggcga gggcgaggga attccttaat ttcagtttag tgatactaat tttggactct 36960ggcctctaaa actgtgaaag aaaaaatttt ttgtttgttt gtttctttta agccacatag 37020tttgtggtaa tttgttacag cagctgcagg aaactaattt atgctgcatg tgaaatggtg 37080taataaggta gattgtgatg aagatacata gtataaacaa ttaagcaaca actaaaagca 37140caacaaggaa ttatagctaa tgaaccaaaa aaggagatta gaataataaa aatggtgaat 37200cccaaagaag ccagaaatag gggaagaggc aaataaagga aagaaagagc ttgatggtag 37260atttcaacct aactatgtca aaaaggacat tacatgtaaa aggcagcgat ttttcagatt 37320gaatggaaaa gtaagactcg gtatatgctg ctgcctgcaa gaaacacatt ctaaatataa 37380aggcaaaaat aacctacagg taacagaacg gaaagaagtt cactgtgctt acaagaatta 37440gatgcaagct agactggttc tgttaatatc agacaaagtg gatttcaaag caaaggctct 37500tgcccaggat gagatggtca tttcataatg atgaagggga ttcgttcatc agcctggcat 37560agcaagctga aatgtttatg caccggacta cagagctaaa atacatgaag caaagcctga 37620cagaactaca agtagaaaca gacaaatcca cagtgataga gatttcagta gccgctctca 37680atgatttgta gaacacgtag ccataatatc tggatctaga acacttgacc aacactgtcc 37740cctgtgcaac ctcattggca tttacaggac actccaccca gcaccagcag aagagacact 37800ctctcaagtg ctcacagaat gtttgccaag atagagcaga tgctgggcca taaaacaagt 37860ctctaaatta aaagcattca aattattcag agtatgtttt ctgacctcag tatcattaag 37920ttggaatata ttataggaag ataacctgga aaagcctcag atatgtggaa aaacccattt 37980ccacatggcc catgggtcag aagtgaagtc aaaagggaaa tttgaaagtc ttttggattg 38040actgatataa aaacaataga tttctaaact tgtggggtgc tgttacagca tagtaaatgg 38100aaatttctag cattaaatgc ctgttttagg aaagaaagat ttcaaatcaa tgacctcagc 38160ttctaccttt ggaaacttga aaatgacaag caaatggaat ccagagttac cagaagggcc 38220aggtacggtg gcttatgcct gcagttctgc cactttggga ggccgaggca ggtggattgt 38280ttgagactgg cagttgaaga ccagcctggg cagcctaggg agaccccata tctacaaaaa 38340acaaaaaaat tagccaggtg tggtggcatg tgcctgtagt cccagctaac caggagtcta 38400aggtgggagg attgcttgag tctgggaggt tgaggctgca gtgaactgtg attgtgccac 38460tgtgttccat cctgggcaac agaatgagac cctgtctcaa aaacaaaaac agttactaga 38520agaatggaca tcataaagat aggagcagaa gtcagtaaaa tagaaaacaa aaatacatag 38580gaaatcaata aaaccaaaag ctggttcatc aagaacatca ataaattggt aaagctgata 38640ggaaaaacag tgaagtcaca aattagcaat atcaggaatg agggagatga cagtagtata 38700gattatatag atattaaaag gactgtatga ggcaggtgtg gtggttcacg cctgtaatcc 38760cagcaccttg ggaggccgag gtggacagat cacctgaggt caggagtttg ggaccagcct 38820ggccaacatg gtgaaactct gtctctacta aaaatacaaa aattagttgg tcgtggtgct 38880gtgtgcctgt aatcccagct acttgggagg ctgaggcagg agaattgctt gaacctggga 38940ggcggaggtt gcagtgagct gagattgtgc cgttgcactc cagcctgggt gacagagcaa 39000gactccatct caaaacaaat aaataaataa aaaggactat atggtaatat tatgaacaac 39060tttatgccaa taaatttgac aacttataga tgaaatggat gagttccttg aaagacacag 39120aaactattaa agctctctca agaagatata gataagctga ttagccctat atctatttta 39180ttgaatttaa atgtaaaaat caatatttag ttactggaaa acttttaagt gtggttggaa 39240atggtatacg aactttttca actgaatttt atgaagtcta atcacaggta aaggttttct 39300gatgaaaatt tagtgtctga attgagatat actgtaaaaa atgttatata tcttaattat 39360ttcttcacat taattacatg ttgaaataat actttgggtg tattgggtta aattaaatat 39420tatgaaaatc ttgcctgttt tctttttact tttgatgcgt cagctaggaa atataaaagt 39480gtagctcaca ttctgtttct gttgacagta ctgctttgga gcacagtgtt tgaatgatct 39540atcatttcaa agacctttcc tcagttcgtt attcatggct gtctgtattc cacatagata 39600aggtctgaaa tactgctaag tggcatgttt tgttttatgc ttttataagt ttgttgatca 39660ttactgatgt ggacttttgg tgcctcttag gctcattgct atcttccaac cattgtttgc 39720aatttttacc tagagataaa gagaaagaga catttggttt cagagtagtt agattgggat 39780catgaaagag caacctcatt ttgatgcttc aaaaatagca catcccccgt attactggga 39840tttgctattc ttgggattac ttcaagaaca tccttgtgtt actggtttgg atgcttctga 39900atgctgtgaa gtcagtttca tgtacatggc tcatcagttt agctctctct tggctttgtt 39960tagacagttg gagcatgatg gcctaaacag cttctttcaa ttaaacattt taaaatagtt 40020tacaaatagt aaacaaactc cagtttttgt gactctttgt ctcgcacaac aaaaacacaa 40080tctgaccatg atcatctggc atcttagggt gaaatatggt tatactttgg cccataccga 40140aagcaagatt aaaaaggggc aggagagata gactgctgaa ctgattttca aggttccaag 40200aatattgtag gttaagagta aaagtaaact tttggtagaa agcagtgggt tgtctaggat 40260tgaagtatct gaagttttta aacgaaaatt taaaaagaaa aatgagaatt gccttacaag 40320tacaatctct tcttttttaa aaaataaact ttattttgaa atagttttag atttatagaa 40380aaaaattaga tagggtagga agttttcata taccctacat ccagttaccc cagttattat 40440catcctaatt tagtgtgaga cattttcatg tttaatgaat caatattgat atgctattaa 40500cttaagtcca gactttattc agattttctt aatttctatg taatgtcctt tttctgttcc 40560agaattccat gcaggacacc ggatacctca ttacatttca ttgtcatgtc accttaggct 40620cctcttgaca gtttctcttc tttttttgct tagaaattct ccagaatttc agaaacttct 40680gggcatcgct atggaacttt ttctgctgtg cagtgatgac gcagagtcag atgtcaggat 40740ggtggctgac gaatgcctca acaaagttat caaagtaaga accgtgtgga tgatgttctc 40800ctcagagcta tcattgttgt aggctgagag aagaagcgat cattgagtgt tcttctgttt 40860tgagtccctg aggatgtctg cacttttttc ctttctgatg tatggtttgg aggtgctctg 40920ttgtatggtt tggaggtgct ctgttgtatg gtttggaggt gctctattgt atggtttgga 40980ggtgctctgt tgtatggttt ggaggtgctc ttgtatggtt tggaggtgct cttgtatggt 41040ttggaggtgc tctgttgtat ggtttggagg tggtcttgta tggtttgcag gtgctctatt 41100gcatggtttg caggtgctct attgtatggt ttggaagtgc tcttgtatgg tttggaggtg 41160ctcttgtatg gtttggagat gctctattgt atggtttgca ggtgctctat tgtatggttt 41220ggaagtgctc ttgtatggtt tggaggtgct cttgtatggt ttggaggtgc tctgttgtat 41280ggtttggagg tgctctgttg tatggtttgg aggtgctctt gtatggtttg gaggtgctct 41340attgtatggt ttggagatgc tctggtatct gcctgcattg cttgccacac ctgcccggtc 41400agaaggcgct atgttgacaa ttgtgcctgc acggtgccta ggtcaatgaa gggaaccgat 41460ggtagccact ggatgctcct gggaaaatgt cactacaggc accagagaag ccagagctat 41520gcccaaattt ctatgagtct cagttttctt aaccataaaa tgggatcaat gtttttgtgg 41580catgtgtatg agtgtgtgtc tgtgtatgtg tgaggattaa attgtgtatg tgtgaggact 41640aattgccact actggatcct caaagtggta agaagtgttc ttattaataa tgacatcctt 41700acactcttac ccagcaagat tgatgggtgt ggcactgctt ctctttttcc atcacatggt 41760ttccatggta tccttttgcc cagggaatct ttgctttgtg gctagcactt tgttgtttgg 41820ctaatcacgc tttctgtggt caggacgctg gcttctctgg agccatggga ttctagctcc 41880ctgtcttgtc cctagagtgg tcactgtctt ctctctccgc ttgcaattcc tgctttgctc 41940gcatctcact tatgcagtga cgtatatcag tttcaccttg ttctccgtgc ctgctgatca 42000ttggcaccac ttgcatggtg ccatttaggg cctgcttcca gttaagcttg cttctccaca 42060ggcctaaata tccttgcttg cttcttttat tctcactggc aggaccaggg cggtctgtct 42120ttgcatgaga cagggtctcg ctcagtcacc caggctggag tgcagtggct gatcacggct 42180cattgcagcc ttgagctacc gggctcaagc tatcctcctg gcttggcccc ttgagtagct 42240gggactacag gcgtgcacca ccatgcccag ctaattttta aaattatttg tagagatggg 42300atctcgccag gttgcccagg ctggtcttga acgcctgggc tcaagtgatc ctccctcctt 42360ggtttcccaa agtgctggga tcacaggtgt gagccactgt gcctggccct tgatgtttca 42420gttcttgata tttgatcctc agagtcagaa aatctaaaaa gagggctatc ccaggttgcc 42480ttggttcatg gcaaatggga cgttaagagg gcagagagaa tatgaacaga aactgttcta 42540atattggtca tttaatgtgt aagtattgtt cttttttaaa cctccttcat tttttttcca 42600ggaattgctg gacacagtgg cttggtgtgt gtctgaggac tgtaggccat ggccctaggt 42660tgtggtttta ggtctcaggt gctcttcctg gctgtctcct tgcttctttc ccatgtcctc 42720ttctttgttt ccagccattt ctcccttatg cttaagtttg gtgcagcagg gtttggctgc 42780tctcagattc ctgcttcctc agatgctgta gttgtcaggc ccagcgggct ggcagcggga 42840tcaggatctg gctaggtttg ctctcactgt ggcagagtag ggggaggcgt gggagagcac 42900gtgtgacccc aggccagctg tagggagcat aggcatggtc acgtagcctt caggtcctag 42960actttgtctt ctcatgagta tggctgtgtg tgtatggtga aaactaggtt ctacttagcc 43020caagaaaatg ggcacatttt gcatgtggtt tctgtagaga aatgcactgg gtatctgaca 43080tagcctggca gcatgcctcc ctcaggtagg ttagtctcag gcggtgaagc acgtgtgtcc 43140agcaagaact tcatatgtgg cataaagtct ccgttctgtg aggtgctggc aaatcaccac 43200caccgtcaag aggctgaagt gatttttgtc tagggaggca ggaaaggctt cctggagtca 43260gcagccagta ggtgaaagag tagattggag accttcttaa tcatcaccgc ctcttgtctc 43320aaggggtgcc aggaagctgt ggaggctgaa cccatcttat gctgccagag agtgggacac 43380catgagggtc aggtcaaggg gttgtacctt gtttggtaga gaattagggg ctcttgaaga 43440ctttggatgt ggtcagggga gtgtatcatt taggaagagt gacccggtga ggacgtgggg 43500tagaggagga caggtgggag ggagtccagg tgggagtgag tagacccagc aggagtgcag 43560ggcctcgagc caggatggtg gcagggctgt gaggagaggc agccacctgt gtgtctgcgg 43620aagcaggggc aagagggaag aggccagcag cgtgctgcca tcacccagcg actggcgtag 43680attgtgagag accattccct gctcttagga ggggctgagt tttagttttc tcttgttata 43740caataagctt ggtatttgtt tacaaaacat ttgtaaagct aaatcaaggt ttgataaggc 43800ttctagtttt atttaagaag taatgttgaa ataaatgttt gtccaattcg ctttgctcat 43860ttaaggactt tcagtacaaa ctgcaacaac aggattagga tttaaacgtt tctgagatgt 43920ttttactcct cagaatttcc cagaatgtga tctggttttg attttcaagc ttgctgaccc 43980aataggttaa cccacaagtt ttacgaagac catctcagtc cacttacatc aactgcccat 44040gccacggtta aagagatcat cgactgatgt ttggcacagc ttcctccctc ttgggtgggc 44100aagcatttgg aagagaaggc tcctatgggt gagagtgggg caccaaagtc ttccctgtcc 44160catcccctag cttgagaagc ccttctctaa tgtggacttt gtgccgttag catcgttact 44220agcttgaagt tgaccatctg gacgtacttt ctggtttagc ctcacaagtg agcaaggagg 44280gttgagagat gtgctgtgag gaatgtgggg ccccagctgg cagcaggctc tgggtcaggg 44340gggcagggac cacgggcata cctgacagtg aggaggggcc acacctgcag aaaaggatgc 44400aggactccgc cttgggaagt gttctaggcc agagcgaggg tctgtggttt ataagtacac 44460ccacagtgct cgggaccctg cagatgtcca gggtgccgtc tgagcccgta tcatccaaca 44520gaatgttctg ctagtgaaga ttaaagattt actccagggg ctttaggatt tattatatat 44580atataaatcc tatatatata attttttttt tttttttttt tgagatggag tttcgctctt 44640gttgcccagg ctggagtgca atggcgtgat cttggctcac tgcaacctcc gcctcccggg 44700ttcaaactat tctcctgcct cagcctctcg agtagctggg attacaggcg cccaccacca 44760cacccggcta atttttgtat tttttagtag agacggagtt tctccatgtt ggtcaggctg 44820gtcttgaact cctgacctca ggtgatctgc ccgccttggc ctcccaaagt gctgggatta 44880caggcatgag ccaccccacc tggccaggat ttattgtatt tgaaccatct accattttaa 44940ttttgatgtt atgtagtatt tgatgataat gaaagttaaa ttgtttttct ttccattttt 45000ctgtttaagt gaatgacctg tatctagttt attcagtaac ttcctgcata tatttgtttc 45060tttcattctt aatgaatata ttcttaattt agttgctatt atgttttgct ttgccccaaa 45120attgaaatct tagtttcctt ttagctcgtt ttagaactag

tgatgggatg tgtcttccat 45180aaatctcttg tgatttgttg taggctttga tggattctaa tcttccaagg ttacagctcg 45240agctctataa ggaaattaaa aaggtgggcc ttgcttttct tttttaaaaa tgttttaaat 45300tttaaatttt tataggtaca cgtattttgt aggtacatgt aaatgtatat atttatgggg 45360tacatgagat attttgatac aggtatacaa tacataataa tcacaccatg gaaagttgga 45420tatccatgcc ctcaagcatt tatcctttgt gttacaaaca atccagttac atgctttact 45480tattttattt tatttttgag acagagtctt gctttcaccc atgctagagt acagtggcat 45540gaccttggct cactgcaacc tccgcctccc gggttcaacc gaactttggg ctggtctcaa 45600actcctgacc tcaggtgatc cgcccgcctc ggcctcccaa agtgttggga ttacaggcgt 45660gagccactgt gccgggcctg attgtacatt ttaaaataac taaaacagtc agggcacagt 45720ggctcatgcc tgtaatccca gcattttggg aggctgaggc aggtgatcac ctgagatcag 45780gagttcgaga ccagcctggc caacatggag aaaccctgtc tctactaaaa atacaaaaat 45840tagccaagtg tggtggcggg cgcctgtaat cctggctact cgggaggctg aggtagggga 45900atcgcttgaa cctgggggtg gaggttgcag tgagccgaga tcacgccact gcattccagc 45960ctgagcgaca gagtgagact ttgtctcaaa aaataaaaat gaaataaaat tgggccgggt 46020gtggtggctc acaccttagt cccagcactt tgggaacctg aggcaggtgg atgcttgaga 46080ccaggagttt gagaccagca tgggcaacat ggcaaaacgc tgtctgtaca gaaattagct 46140gggtgtggtg gtgcacaact atagtctcag ctacttggga gattgaggtg ggaggattaa 46200ttgagcctgg aaggttgaat ctataggtag ctgagattgt gccactgccc ttcagcctgg 46260gcgaccaagt gagaccctgt ctcaaaagaa aaacaaaaaa acaaaaaaca aaccactatt 46320atcgactata tattattgtc tatgatccct ctgctgtgct gtcgaatacc aggtcttggg 46380cccttatttc catcactgag caaacttcac tctgttaagc agcaggtgtg ggatttcatc 46440gttattcagt aattcacaat gttagaagga aatgctgttt ggtagacgat tgctttactt 46500ttcttcaaaa ggttactctt tattagatga gatgagaatt aaaaatggta acttacttta 46560tatctttata attgaagccc actagacctt aaagtagtta ccagatgttt tatgcattta 46620aatggccttt tctctaaaat tagaaagtaa caaggaaaga aaatgcttcg tttctatgca 46680accctcttgg tgactagtat gtgactctta atgcaaccct cattgcaccc cctcagaatg 46740gtgcccctcg gagtttgcgt gctgccctgt ggaggtttgc tgagctggct cacctggttc 46800ggcctcagaa atgcaggtaa gttgtacact ctggatgttg gtttttgtcg ggggccagct 46860gctactgatc ctttatgtct cagctcagat gtcatttcaa aagtctgctc tgccctctcc 46920aaattgcagt cgaccttgcc ctgtttatgt ttccctcata gcactaatcc atgtcagaaa 46980ttgtcacgta cagtctatct gtgtgcttgt ttattttcta tcccaccctt ccgcaagaga 47040cttatgggat gtgtgcccca ggacagcagg ggtcttactg tcttatgctc tgttgcagcc 47100cagcagcgat aacagtgtct gcacatagta cttgcttaaa agatacttgc caaattgttg 47160aaggttgagg taccaatttc attattgctg actataggag ttatagcaaa atatccattt 47220gtctgttaca tgagttaaaa atatggttgt tgcactgtga atagtttggt ttagtcaaaa 47280cagttgtatc ttaacggatt gagaaacaaa agcaggacca cttttcatca gctccctcct 47340tctccttaac cagcaataca tgctgatgct gatatcccat agaccctcag ctccatcctg 47400agtcactggg aatgtggtct aaaccctcac tattaatatg aactgagttt caataagaat 47460cttatatggg tcgggcatag tggctcatac ctttgatccc agcacttcag gaggccaagg 47520caggtggatt gcttgaccca gactaggcaa catggtgaaa cgccgcctct acaaaaaata 47580caaaacttag ccaggcatgg tggtgcgtgc ctgtggtcac agccactcga gaggctgagg 47640tgggaggatc acttgagcct gggaggtgga ggtcgtgttg agccaagatc gcaccactgc 47700actccagcct gggcaacaga gtgagacctg tctcaaaaaa accaaaatcc agaaaagaac 47760ttatatggct gcagaggtat aatcactaag gaaatttcct tttgtataat cttttttctt 47820ttactatcat ttaaaaaaat gtgttatatt tctgaagcaa cacatccagg ttctgcacat 47880agcagccaaa gtgaccttaa agaatataac tgggtcttgt cattccctta tttaaactct 47940tgtacccatt tcccagtgcc gtttagatag agattccaga ctcgtcaatg gctctgtcac 48000ctcagacacc ctgcattgac tcattagtct gattagagtc aggtttttct tcctcctgat 48060ggtttttttt tcccccttag ttctcagcgg aacagtcact tccttaggga ggtttcccca 48120gccaccctct gaggccgtgc ttgttgccag actctgccac tagagggcag ggctgcacca 48180ctcctggcac ctcgcacccg gcctgccctg tcactctgtg tgttgggtga attcctgtga 48240tctgtgactc actgctctgt gtcctacaca ttcggctttt cttctctccc cacaacccca 48300ttttataatt ctcctttttc aggaaagctt tattcccatt taaaaatttt tgtttttaaa 48360atggtatttt cttacactta ttttctaatt aaaaatgagt gttttaagaa gtattatgat 48420ttactgcaaa taatttttaa acccagcctt ttagatcctc tgtgatcata agagaaatga 48480aggatgtctc ccaacacttg agcttcatcc acatttcatc ctcctgttct ttcagctgag 48540ttttccccat cccattaggg actgttggaa tataaaactg gcttttccct aacagggaat 48600gaattgcttc tgtttctcct gaaggagagc tggaagaatg acttgcgttc ttttgcatac 48660acaggcctta cctggtgaac cttctgccgt gcctgactcg aacaagcaag agacccgaag 48720aatcagtcca ggagaccttg gctgcagctg ttcccaaaat tatggcttct tttggcaatt 48780ttgcaaatga caatgaaatt aaggtatgat tgttgcctca ggtcacaaac atgcgagtga 48840tgctgtgagt gagtctgtgg agggtgaggg cttctgaaca gggagtcctg tgggagtgct 48900tcttggggta tgttgtatgt cgtaatttag actaccatca tttgtgttat ttttgaggca 48960cctaaggact tctttccact tctcatttct tactgtgggg tgaagagttg aattgggaga 49020tggtttctag atgcaaattg aaaaggcatt tttccagagc agatttgttt tcggcgtact 49080agagtgactc tttaacctag ctgcgggaag atgactgtgc caagactgca ggtaggagaa 49140agctcactga cgaggccttg tgggtctgaa cgtcctgcag ctatcagagc ctgttggctt 49200cctgttgtgc attccaacaa atcatcttca aacccacttt agtgttttgt ttataatgtc 49260cagaaatagt gaccctgtca catgctctac agattacagg attcttagcc tcttcctttt 49320tggtaggtca gtcctgggtt tgagcccaag tgaccctcct gggaggtgat gatacacact 49380gggtagagtg gaatcagatg gacttggatt agaattctgt cctctttact agttattttc 49440ctctaggcaa actgcccaac agctctaagc tatttccttc gtattctgaa aaataagcct 49500taatgggacc catatagggc aactctgaga gtaaaataaa ggaatatgtg ttagagtgta 49560gcatagtcac ccacgggaag ggcttagatg ttagctgcta ctgctcttat tagctgaatg 49620atttggaata aactgttagc ctctctcatg ttttttctct tgagcttcga agttttcttg 49680ttaatactaa ggagatattc aaactagtca tggggttttg gaatgacgaa gggagatgat 49740gaatctaaag aatttagtgt aatatttctt catgctcagt aaatggtagt ttctgctgct 49800gttattttta ttaccatctc tttggaatgg gagtaggtgc tcctttgtgg tcagaggctg 49860tgagagctcc acagcgccag tttgcccatc tgtacactgg ggtctgttga aggcagtccc 49920ctctgtgata tctctggctg tcagagctca gatgatagat ggtatttttg tactcttagt 49980tctcatcatt ttcatgattt cgatcaccat ttgagtatga tgatgctaac actttgttga 50040acgtagaatc cgttaattac ttccttcctg aacctttggc attaaaaaaa atctattctg 50100ctacctctct gctcatttat ggttattcaa atttattatc aagagcctgg tacagtggct 50160tgtgcctata attgtagcta cttgggaggc tgaggtagga ggattgcttg aggccaggag 50220tttgagacca gcctgggcaa gatagtgaga ccctatctct aaaaaaactg aaaaaaaatt 50280agctggacat gatggcatgt gcctgtggtc ctagctactc aggaggctga gacaggaggc 50340tcggttgagc ccaggagttg gagttcgagg ctacactgag ctgtgattgt gccaccacac 50400tccagcatgg gtggtaaaac aagatgccat ttcttaaaaa aaaaaaatat atatatatat 50460attatcaatg aaattcagta gtaccaacag gattataaac aaagatagta gttcccttcc 50520tactttttct cttaatcctt gtgtctcaca ggcaaacata actcttagta tttcttccaa 50580tatttacttt catgtttctt tctttctttc tttttttttc tttgagatgg agttttgctc 50640ttgttgccaa ggctggagtg caatgacgca atcttggctc accacaacct ctgtctcccg 50700ggttcaagcg attctcctgc ctcagcctcc tagtagctgg gattacaggc atgcatcacc 50760acgctcggct aattttgtac ttttagtaga gatggggttt ctccgggttg gtcaggctgg 50820tctcgaactc ctgacctcag gtgatcctcc cacctcagcc tcccaaagtg ctgggattac 50880aggcgtgagc cactgcgccc agcaacttcc acatttctaa ataacatgct tctactgcta 50940tttttttttt caattttaga cattttttta ctttcactat agttctatca gaattcagtg 51000tgtacgttat tatgcctaag taaatagtca tggttgctta cgtattatat ttctttgatt 51060gtgtttctta tttgatgaga aagctgtgtt ttttgctctg ggttgaaact ggagagagga 51120cctggggagg aggaggagga cagatgaagt tggtgactgt accttcatgg ccatagctgg 51180gttctcagca cccggggatc tgctgatcac ctactcatag gccaggcccc tatcgaagtt 51240ctaggtgacc cagtgctggg gacggggggg ccacctgcaa ggtctaatca tggaggtggg 51300ggctacagtg ttggcttgtg ctggggccag catccttagg aaggcatctt ggaggtggag 51360gagacagccg cccacttctt gattggggcc ttcagcagca ccagcttctt gggcaggctg 51420gtgctggctt tcatcaccat gtcgtgttca atcttcttcc agatcctgac ttctaggttc 51480agctttcctc agaccctggt tcctttcaga ggccattgct gctgccttgc tctttgctgg 51540cttgtgcctt gattatatgt ctttgtacaa ctttttgttt tcctggagtt aatcttcaca 51600tctgttttct tggagttaat cgttacctct atatcgcttg cttattattc tttggccttt 51660ttgtcttctc acaccttcca acttctttgt aatatgtgtt tagtacaatt tttcatgaca 51720ggtagtttac tgaatcagtt tttccccagt gtggtcatcc aacttgagtt atccagctct 51780ctgccccagt ctgggcaggt tgatcttcag gtctgtagta cacttgtatc ctaggacttc 51840tctttgccat tagcctggaa tttcctttgc agttctcccg ttggatgccc agttcctaga 51900tgccatatgt ttttctatcg tctagtagct tcctgagaga agatgaatgg gagggaaatt 51960gtatgaggtt ttgcattcat aaaaatgcca ttttttttcc tgtacacttg gctgggtatg 52020gtgttctggg gtagaaatca ttttccctca gaaatgcaaa gtctttgccc tgttgtctta 52080aaatctccaa cgtgacccga ttccttaacc tatgaatgta cttttctttg gaagctttcc 52140atttttgggg aggtgaagtg ctaggtactt agtaggcctt ttaatttgga aacttacatc 52200ccttcagttc tgggaaaatt ttcttaacat ttctctgaga agttcttgcc ttttattttc 52260tgtgttctct cctgaaattg gttagttgga tgttggtcct cctagattga ctcacatctt 52320acctttttct tttctttttc tggtactttt tagatatcca tctcaaactc ttctattcat 52380tgttatgttt ttaacttctt tcttttcttt gtctcttgat ggggtcttgc cctgttgccc 52440aggttgtggt gcagtggtgc gatcatagct cactgcagcc tcaaattcct gggctcaagc 52500agctgttctg cctcaccctc ccaagtagtt gggactacag gtatgcacca ccacgtccag 52560ctattttctt tacttttttt tttttttttt tgagatggag tcctactctg tcgcccaggc 52620tagagtgcgg tggtgggatt ttggctcact taagcctctg cctcccaggt tcaagcagtt 52680ctcctgcctc agcctctcaa gtagctggga ttacaggtgt gcaccaccat gcccggctaa 52740tttttgtatt tttagtagag ccagagtttc accatgttgg ccaggctggt ctcgaacgcc 52800tgacctcagg tgatccgcct gccttggcct ccgaaagtgc cgggattaca ggcgtgagcc 52860catcattaga tctttaaata ccagtatcta taagtctttt cctcttgagt cagctagtat 52920ccctggaagg aaattactca ttttcctgct tggaggctat aagcttggct atgtttatcc 52980tgcaaccggg gactggaagg gaggggactg acagtgttgc tggtcagggt gccctcttac 53040tttttgtttt ctgtgtgcat ctcacgtctg tcctcagcct atgtaaacac ctcttgagat 53100tatccctctc aatctttgcc ggaggtgggg gaggggctgc ttcctgggct gccttggatt 53160ggagggaaga cctcaggtga gtgggtggga atttgcccaa ggagccatga gaccagccac 53220tatttcaccc tctccatccc tccactttca gatgtatgtg gcgcctccaa agcccgagct 53280cttcttggcg tctgtggctt caataagctt gctttttgct ggtatccctc ctaccctccc 53340ctgtccccag caaagcttgc atttgaactt cttcctacgg gctaacaaat cagtcagtta 53400tgtagctctt gttacttttt agcttccgaa gttttgttga cacccgtagt ctgctaatgt 53460ccctgttctg ttctttctgt tcgtgtaaat atatgcttta tacaacttct ttacatgatt 53520tttgtggggt ttctgggtag cagagcttca caagttcaat ccagcgtgtt ggattagaaa 53580tctcccaccc tctggtttat tcttattctc aaaattacct gccaaacact gatactccct 53640tgtttttcct tttcctgaca ggaaatgtac ataccataca ggacagaaat cattagtgta 53700tcccttggtg aataaccaca aagtgaactt aacccttgta accgccaccc aggtcaagac 53760agaatattac caagcactca gaagcctctc ccctattccc ccgtcactgc tcctgccttc 53820ctccccaagg tcatgactgc tggcttctaa ttccagagtc tgtttttaaa ttctgtgtac 53880atagaccatg gattaagtgt tctttttgtc tggtttattt tggtcgacat taagttcatg 53940agagtcttct atattatcgt gtgtattagt attcctgtag ttttaggagc ttcatagcat 54000tccattgtag ggatatacca cagtttattc attgtattat cactgggttg tttctagttc 54060ttggctattg cgagcagtgc tactgtgacc actcttaggt gtgtcttttg gagtacatgt 54120gcaggtttcc atcttgcaca gctagaggtg gagttgttgg gtgatagggt gtgtgcatct 54180cagctgcagt agaaactgcc aaatagcttt ccttgagtgc ttgtaccagc tcaccctttt 54240gccactgtgt atggggattc caggagctct ggtcctcgct agcacttgga attgctgatg 54300cttttactct tagccttcct gatgggtgtt ttctggaatc acattatgat tttaatttcc 54360attccttaaa gtacccttgg ctctgaagtt taatgattca tgcatctctt cccttttgaa 54420gtactcttac aggtatgttg tgcatgtgtt gaaaagtggc actatctatt ctaaaataca 54480gtatgcctcc tctgtgtttg aacagttgta gcgtggcctt ggggcctcct gttagctggc 54540ttggagaagg gattcttggg attgtagaga ttagacctga ggaggcccct tggagctctc 54600tgactaaatt ttattcttta ttattccaaa ctatttaagc tcaccgtgtg ctgactcatc 54660ataataatga gtagctctca ttgtgcttgt ctatttggac tcatacaatg attttttttt 54720tttctttgag acagagtctt gctctgttgc ctaggctgga gtgcagtggc acaatctcgg 54780ctcactgcag cctccacctc ccaggttcaa gtgattcttg tgcctcagct tctcaagtag 54840ctgagactgc aggtgcgtac caccatgcct ggctaatgtt tgtattttta gtagagacgg 54900ggtttcacca tgttggccag gttggtctca aactcctgac ctcaagtgat ctgccttctt 54960cagcctccca aagtgctggg attacaggtg tgagccactg agcttggcca aagtagtttt 55020ttaagatgtt agtatctttt cttgcagcta aaaaagtttg tcagagatga ttctactttg 55080ttctccaggt gttttctcag ggagaaattg gaggcagtaa gccactgggg gagtcctgtg 55140gctggggggt ggggtagtcc tgtggctcct tgtcagggag tcctgtggct ggcaaggaga 55200gaagtcctgt ggctgggttg ggagggagtc ctgtggctgg ggtctcatcc tgtgcctaac 55260agtgtccaga ggtgccgaga ccagctcagt cggggagacc ctaacccagc agcgctagag 55320gaattaaaga cacacacaca gaaatataga ggtgtgaagt gggaaatcag gggtctcaca 55380gcctttagag ctgagagccc tgaacagaga tttacccaca tatttattaa tagcaaacca 55440gtcattagca ttgtttctat agatgttaaa ttaactaaaa gtatccctta tgggaaacga 55500ggggatgggc cgaattaaaa gaagaggttg ggctagttaa ccgcagcagg agcatgtcct 55560taaggcacag atcgctcatg ctattgtttg tggcttaaga atgcctttaa gcggttttcc 55620accctgggtg ggccaggtgt tccttgccct cattcctgtc aacccacaac cttccagtgt 55680gggcattagg gccattatga acatgttaca gtgcttcaga gattttgttt atggccagtt 55740ttggggccag tttatggcca gattttgggg ggcctgctcc caatacagag gtctcgtgta 55800aattccctgg gaggcgataa gcctctgaga aacagactat gctaaccacg ccatgaaaga 55860gaaacttatt tataaatcag atgccagtta ctagtttact gcttatttgc ccaggcgtag 55920ctctgacaga gtccccgact catagtgctt gctcagtgca tgctgaacaa tgattggaat 55980caagtcatgg ctcagagcat agttttgaat aatgggaaat ggatgttctt aagtaacata 56040gtcaccaaga taatgcgact agctgggtca ccccttttca attttaggat atttttatca 56100agatttaaat ggccatcatt agagttatag cactttctcc tttggattgt cctagaggcc 56160catgagaaag tattccctaa tttcttagga gaacagtttg tgggtagtat gcggtcatgt 56220ccagttaaat tgcagatatt tccgatcgaa gatgttccag tcctgagaac ttcgtgacat 56280tagcaggact tctacaagcc atctcttagg gtggggcatt tactgcagtt ggctagtact 56340cttttctcct taactttgtc atttgttgat ttttttttaa ctgtccccaa atactgtggg 56400cagagtgtat ctagaattga ggcctccacc attgcggaga ggacatggat gctgagcagt 56460cccctgagtg aaggttataa agaagcaaat agactacaca tgtctgtaaa ctgctcttga 56520gtgtcccaaa tttggggtac ttcagttcag ctgtaggaaa agcctcaaac tgtttatact 56580ttgcaagaat tggaaacttc taattcacgt taagttttat gtaatacatg ataagcttca 56640taggagcttc atcttttatc tacttggact tttgcttccg taggttttgt taaaggcctt 56700catagcgaac ctgaagtcaa gctcccccac cattcggcgg acagcggctg gatcagcagt 56760gagcatctgc cagcactcaa gaaggacaca atatttctat agttggctac taaatgtgct 56820cttaggtaag gtggaggcat atgagtggaa gagtctccag catgtactca agatagacct 56880ttgaaataaa taaaaccaga tgatccctca gcttctagac caggctattt ggcactggtt 56940gattgaatgt gaactgcact ggggctgctg tgagcccgca tgggtctctg tgaccctgca 57000gatgcagccg tgcccaggga ctgggcagtg ggtgtgggct ggtgtgagcc ctgtctgcca 57060cccagggcct ggccctctgt ctgtgtcggc catgactatg gtgagtcttg taggcttgag 57120actgtgcctc gggttcctgc gggttctctg taggtcagtt gacagtttct cctgttgttt 57180gggtaactgt ggaaacgaac actggcaagt gctgaagcga gcatgtggac gtgcgatatg 57240aaataacgac ctggctttca aaggcagtga ggctctctgg aaaggacctt gctgagctag 57300ggatgtgggt gtgtagccat tcccagtggg cctcatggcg tactcgttca tgatcatgtt 57360tgtgccatct tgatctctca ggatctcttc ttttttaaca gattaagccg ggaatctcca 57420aacagtgagt cagatgttaa gatgtcttgc ttccaccccc acaggcttac tcgttcctgt 57480cgaggatgaa cactccactc tgctgattct tggcgtgctg ctcaccctga ggtatttggt 57540gcccttgctg cagcagcagg tcaaggacac aagcctgaaa ggcagcttcg gagtgacaag 57600gaaagaaatg gaagtctctc cttctgcaga gcagcttgtc caggtaggag cacagggttt 57660actctaggcc ctgcatgtga atgactgaca ttcaaagaac cgattaattt ggaagagaag 57720cggcagaacc gagagttaga ggtgtggact ctggagctgc gctgctcgtt tccaacccta 57780ggtgctgacc tctagctgtc ttccctctgt atgtccctgt caccgtgagt caaatgcggg 57840tgatgcctcc tcaggtgccg tgttacctaa gcctctcaga gaccactgct accctgtttc 57900taaaaccaga ggtcacgata tgtgttcatc cacccagtaa atactgattg agcacccact 57960gtgtgctagg ctctgggata ggggctgggt atacaatggt gagtatttca gctgcagctt 58020ctgccccgtg gaggctgtgg cctagcacac tggtctaggc acggtggtat atgctcactc 58080aaggagatag ggacgtggtc gtttggggtg tcggaacaaa atgtcggaac ttctctttcc 58140aatgcagaga aaccttgcag taattctaat gtactgtgat tggcagttga cttcagttct 58200ttgtagcacg cttactcagg ttatttcact aactatgtaa ccatgcagcc tcattttaag 58260caattggatt ttttgaactt tacttaaaat gttatgtcag ggtttttatt gtgcttaatg 58320tgtgccattt agctaagttt tgtaggatac gaaattgtaa gtggcttaaa atgattctta 58380atagaatcat gaattgaaga taatgctaat aatttaagca ctgagttagg tagtgtttgt 58440aaaatgctta gaatgcttcc tggcacatgt taaggccatg taagtgctgc gtgttgataa 58500acagctgagc aaaagtggac tcttaagaaa gtattggggc tgagagttct gttccaacca 58560gctgcccttt ggttattttt cagaataaaa gcagagtctc atgggatatg acatttatat 58620ttccttcaca aaaaacactg ctgagtgttt tgttgagtaa aaagggtgta gccatggtaa 58680taatacattt aaaatatagt ttatttcatc tttaccttgc cttgtttttt ttttaagcta 58740gctttttatt gagaattcca cacatacaaa agtatcaact catgaccagt tatatttcat 58800ttataatcct acttctccct ttttttatta tttgaaagca aaccccaatt atcctcttat 58860ttcatctata agtatttcag tatctctata gatgaggact cttctttatt tttaaaactt 58920tatttttaaa atgatggtca gatgcagtgt tcatgcctgt aatcccagaa ctttgggagg 58980ccaagctggg cggatcactt gaacctggga gtttgagacc agcccgggaa acatggcgaa 59040accccatgtc ttaaagaaaa aaatcagcca agtgtggtga tgcatgcctg tagtcccagc 59100tacttgggag gctgagatgg gagggtcaca tgagcctgga agatcaaggc tgcagtgatc 59160catgattgta ccactgcact ccatcctggg tgatggagca agattctgtc tcaaaaaaac 59220aaaactgcaa aacaacgtca caaaacagtg ccattgttag acctgaaaat attaaacatt 59280tcctacatca aatacccacc aactcattat caatttttct ctctactctt ttggaatcag 59340catctaaata aaattggtcg ataaggattg taaatctctt tgatgaactg gttcccctcc 59400atcccagttt ttttccctta gagttcattt attgagaaac cagattgttt gtcttctaag 59460ttttcctgtg gtctgatata ctgcttccat ctccactgtg taaattaaca cctttttctc 59520ttctctgtat ttcctgtaaa tcaataattg gaggaaaagc cttgtcagat ttagtgtata 59580ttttatatct gagtccagta tttcttatat aatattttaa gataagtgta ctcttttaaa 59640aagtattgaa actatatgct caattttttt taactgatgc ttttaagaag gctgcttgat 59700cataaaagtt tagagatcat tggtctgatg ggaaaagcaa ataattacta aaccgtttag 59760caaggttgag gtgcacatgg tggggcctgg agaagttcag tcatgagccg tcacttatgg 59820gcacgtggaa tctgacccgg cacagagttg ggagaagaca ggagctttat agacagaaaa 59880tgtggtcttt gctaagtccc aggagtgaaa gggtgagaca gtgctcacag cacacgagtg 59940tgggtgcgta gacagagcaa gggtgggtcc tgaaaaggcc tgcaggcttt ctcatagatt 60000agcaagagtg ctggttacgg aggtttctaa catttgtgaa cagatcgaaa ctgtgttaaa 60060ttgggattgc agtaatcctg gaaggacagg gatagagggt gaaggggaaa aaagggtatg 60120gatgtgagac ttaattgctg attttcttaa gacctttctc caaagtaaat aaatgatgtg 60180gcacattttt gaactggcaa attctaaact ctagatatga

ttatctctat aacatatctt 60240actccatctt cttttgacta aaaactgttc ttaattaaat taccatgaga cgttcaattc 60300agcaaatgta gtttggctaa ccatatttaa ttagaattta atataatcct aggcctggcc 60360aaactattaa gcaagtgtgg gcaaaatatt gataatttta gatatgcagg aacttagttt 60420gctttccatg tgtgcttttc gaaaaaggaa taaattgaaa aatagaggaa gccctgaaat 60480ccaagaagca aactctctca cctaggcatg cagtaaaagc aattctagga tgattgctgt 60540ttggcgcgta gttcgtatta gaaaccattc ttcttgaata aatagtatgt ttaagaagct 60600gggcagaggg aaggcatatg catatattat caacaaggag ggagaaaaag gcaattagta 60660accatccata ggagggtcag caagatttat aaaggaaatt tgtgatccaa gtatgaagca 60720aaataaggtg cagaataaat tttaagcaag taatagatta gagtaagaga acccatttga 60780ccattaacct tgggacattc tctttcaaat gacatggagt agtactgaaa tctttctttc 60840tttctgagtc taggttattg tgactggact cagaaagaaa tatttcatta ttgcagtgaa 60900taacatttgt gaacattatt gttcataaat tatgcagtga ataacattta tgaacacgtg 60960atgtgtaaga tacatactgt ttatttttag ttaagttttt tggctcaact tctaggcaga 61020gaacattaaa tgtaaatagt gttacctagg agcatgtaaa tggaaatctc catagtatga 61080aagcagtgct gttgctaaca gaatttagga gggggcagat gaggtgaagg aaatgtgggt 61140gctgatttcc ttattacatt gagaggagcc aggagattct ttgttcaaaa tggatggctt 61200aagaagtcaa agtataagct gattacgtag agcaggtacc caaaaatgtt ttgtgtaagg 61260ggccagatag taaatatttt cagtcttgca ggccatccca agtctgtggc agctactcaa 61320cactaccttt gtagcatgaa agcagccaca ggcagcccat aaatgtggct ctgttccggt 61380gaaactttag gtacaaaagc aggtgcaggc cagacctgac ctgtgcactg tggtttgctg 61440acctgggatt caggggtata gaagttacca tcagaagagc taaaagtgag actttttact 61500ttatactctt ctacactgtc tgattttgaa aaaaagaaac atgtatttta taatattaaa 61560gatagggttg gcaaatagca aataaaaata cagaatacca gtgaaatttg aacttcagat 61620acattatgag taattttatg gtgtaagtat attccaaatc atgtgggaca tacttacact 61680acaaaattat ttgttgtttg tttacagttt aaatttgagt gccttgtatt ttatctggca 61740actgtaatta aagggaaaaa gaataaattc attatgttca tataatgtga tatagcaggg 61800gtccccaacc cccaggctgc agagtggtac tggtccatgg gtccccaacc cccaggctgc 61860agagcggtat tggtccatgg cctgttagga accaggctgc ccagcaggaa gtgagcagca 61920ggtgagctgg cattcccacc tgagcaccgc ctcctgtcag atcagtggca gcattagatt 61980cccataggag tgcaaaccct attgtgaact gcacatgtga ggggtctagg ttgtgcgctc 62040cttatgagaa tctaatgcct gatgatctga ggtggaacag tctcgtcttg aaaccatccc 62100ctggccctgt ggaaaaattg tctcccatga aaccagtctc tggtgccaga aaggttgggt 62160agcactgtga tatagtatta aaagtgctaa taaatatggc atactgcctt taaaatgtct 62220ggtagctctt tctcagtggc actcataata gtgttttttg atttttaaat gtgtgtcaag 62280ctgactctcc cctccgtgta tgctgggctt tattttccct ttcctagtca ccagttttgg 62340gaaatagaga tcttcattct catgctgctc ctctagtgca agtgctccat ttatttttaa 62400ggaattaata taacaaaaaa tcatgggaat ttagaaaaca acatggaagc taatgatcac 62460attggtggaa gtgataggga aatatttagg gggagaagtt aaggtataaa ctttgtcaat 62520gaagtcctat taaaaacaac aaaaaagtga agcttaggat gcattttata aactctgacc 62580agaacacctg tgtttctctg tttctaggtt tatgaactga cgttacatca tacacagcac 62640caagaccaca atgttgtgac cggagccctg gagctgttgc agcagctctt cagaacgcct 62700ccacccgagc ttctgcaaac cctgaccgca gtcgggggca ttgggcagct caccgctgct 62760aaggaggagt ctggtggccg aagccgtagt gggagtattg tggaacttat aggcaagtta 62820ttagcaaggt ctactcttac aattaacttt gcagtaatac tagttacact ctattgatta 62880tgggcctgcc ctgtgctaag cagtctgcat tccatcttcc ttgccaaaac ttataataca 62940aatttcatct ttattttata aataggggag ttgggctggg tgtggtggct cacgcctgta 63000atttcagcac tttggaagga tcgcttcagc ccaggagttt gagacaacct ggccaagtga 63060gaccctgtct ctacaaaaaa aaaaaaaaaa aaaaaattag ctgggcatgg tggcacatgc 63120ctgtagtccc agctgctttg gaggctgagg tggtaggatt gcttaagccc aagaggttga 63180ggctgcagtg aatcttgatg gcagctgcac tgagcctggt gacagagcaa gatgctgtct 63240caaaataaat ttaaaaataa aataagagaa ttaaagttta gcaggttggg tggcaaaatg 63300aggccacaca tttaaagccc ctcctcctga ttcttttctc tgccttggct gcctcctgtg 63360gcattttagg tgctgagaaa tgaaaacagt agggaaaata gttccaggat cctcatgtta 63420atttgccaga aatggcatct tcaagtcgtc agagggatct gagagttcct tcctggcctg 63480acttgagaaa atccgtctgt ccccagctct gcgtctgcct ccactgccca gtcacctcct 63540ctccatgctc ttggggctgg gccctacccc accatgcagt gctgccctgg agcagtgagc 63600ttggtgggtc ctgtctggca tgagagctgc ctttgggagc tggatcccag cctctaccac 63660tgggtctggt gcctagcagg ctatggataa acttctgctg actccggcct ctcctaagcc 63720actgcaacgt ggtcggtgta gtgcacagtg tgtgtgcagc gtggccttac tcacagcctc 63780cacattagag agaatctgac tgaagtctta ctgctgcctc gtgtgaacat aaatgtttgc 63840cagaaccatg agcaggaaat gttaatctgc cttgtttcct gtcctttaca cggaagaatt 63900tttttctgta tggaatgcgt gccttacaaa taatgagtgg aaatacccat cgctaatgaa 63960aagttatact tgactgttag tcagctaaat aatctgagat ttctaatact tttaatttgg 64020cttttacaat gcaatttatc ttagcttttt tgatttctta ggtcatatct ttagaactat 64080atatttgaat gttaatgtaa ttttcatatt gaaattaaaa tgttgaactg cgatgttaag 64140tgtttcctgt ggaaaaacgt tcacattttc tctagtttta aagttgaatc aagctgtttg 64200aagattttca catttcttct agattttatc agcttgttac tttatctgtc actttctgtg 64260atttgcagct ggagggggtt cctcatgcag ccctgtcctt tcaagaaaac aaaaaggtga 64320ttatttcaga aatcagagtc ttgtgttgaa tcttactgat tttcttgtat ttctgtaatg 64380taatgtatct tgtatttctt gtaatactgt attggactct gtgtatatct cttctcagat 64440gagtgattat atgtgtgaat gttgctggaa tctgataacc aggcctgaat agttttgtag 64500ggtggctttt aaaaattact ttcatatcag aattgctttg tcataaattt tgaacgcatc 64560ataaatttct aatgttcggg gtcagcagac tttttttgta aagggacaga gtgtaaacat 64620cttagcttta tgggccatat ggtctctttt gcaacattca gctctgccct gtgacaggaa 64680tgcagttgta aagacatgag ctactggcca gctatgttcc agtagaactt tacttacaga 64740aacagacagg ctgtagtttg ccaatacctg ccttagggaa tgtgttgtta tattttgtga 64800gttaccttct cagtaaattt tatttagtat tagtcaggaa tattattaag tagcttcttt 64860tccagcctgg tcaacatagt gagacccggt ctctaccaaa acaaaacaaa acaaaaaaac 64920agccacgcat gtggcatgtg cctgtagcct cagctgctgc tcagggggct gaggcaagag 64980gattgtttga gcccaggagt ttgaggtcac agtgagctgt agtcatgcca ctgcactcca 65040gcctaggcaa cagaatgaga ccttgtgtct taaaaaaaaa aagtttcctt tgttgggtta 65100ttttaatttg gacctggtta tcatttttca gccatattta actttgtaca tatcagaatg 65160ttctgataaa acttaacttt tattaaagtg tttgtgatat aatctgctag ttttggtaca 65220cattatcttt tgcaatgcca gttattttct tttccagtgt gggtttgcat aggaaaagaa 65280ttgctgtcac tttctatttt gaaatcttaa aagactgatc cttttttgtg tcatgatttg 65340agtatttaat tgagagccta atgcctaata ttatttgcag tattaaatgg gatcttaaca 65400ggaatagcat tctagccttc attgaattaa gtaaacattt cttaagagaa cttggaatct 65460ataatatttg cgtcatcata gtatgagata cttaatcaag tttgagattt tagtgaaaca 65520ttgtttagaa gccaaaagga ttctaggaaa aattaatgtc tatattcttg aattaggaga 65580gattttggga cgtgtgacta agttacgctg acacttgttt gtttcttagt cgctttttcc 65640agtggcggtg agaacgaaga tgactgattc acattgctca gatgagttta tcctcttctg 65700gctgggacat gggatatatc ctgtctcttt taagcctttt tggtattttt cccccattga 65760gagctgtgtc ttcaaactct tctgttatag ctggaaaatc ctttttaagt gaaatctgcc 65820caaattataa gacagatgaa ggtagagttg tgttggatat aggattaggg tgaaagtagt 65880gggggtgtcc tggagcctct cttctggtgg cagcctagct cttgtgcctt tgaggaaatt 65940accctgggga cggctctgtg gaacatattt gcaaaccact gatttggaag atagagatgg 66000cttttgttaa gatctgaatt cacctttttg gcattttatt tgatttctca aggtaaagaa 66060cttattttgt aataaagttt cctattattt agtagatagg ccaagttgct gtgttaattc 66120catgtagatt ttgggtttcc tttgctcatt ttttcactct taatctcaca tcattgtaag 66180tttatggaag ttatcatact tctgactttt tctttgaaga gcagaaatta gaaattccca 66240ataattattt tgatagtgtc atttaatgac actcacatgt gatgtagcca caaagattta 66300atgagttcag ttttaaatca tattaagact gttggtttca tttgttctca ttaatgtaat 66360tctgaagatg aacaataaaa tgtattttta gaactttcaa atgaaatatt atttcatcct 66420tccagatcat ataatgctta agttctgatt gttaatcata aagtctagaa aattaaaaga 66480taataaaatg aaagtgactt ttaggtatta gagttttatt ataaattctg gtgtgtcatt 66540ggagctatga catgaatatt tcaaaggcca atagcattgg atctttacag ttataactta 66600ccatttttaa gtttaagtag taatatagat tatttaataa tcaaaatcaa taaatattaa 66660ttattaaaat gttttgtggt atagtttgag aatcattgct tttaactttt tccatatagg 66720tttattgact ttaatagcat tctaaacata acatctctac attctttgtg tttaatactg 66780tggaggtata aaaatactta tatatgatga taaactatat tagagtaaat taaatattct 66840tatgagtttc attttagagt gcatttactt aattttgaag tccttatttt tagcaaacta 66900aaaggaatgt tggtacatta tttactaggc aaagtgctct taggagaaga agaagccttg 66960gaggatgact ctgaatcgag atcggatgtc agcagctctg ccttaacagg tagttctcac 67020tagttagccg ctggtgtgga ccttcactgt ctgccttcca ccccttgccc ttcctgctcg 67080tccccctgca cctggtggac agcacgactg ggggcagcag tggagccagg ttgcttaaat 67140ggggcatatt cgggcttctt ttataatact tactctgaag cttgtgtgtc tgtggtgttt 67200gcatcatata tttgttgttt tccatggttt aggctgtttt aaaattaggt ttatggcttg 67260agcatagggc tttgtgagta ggggatggca ggtcgaaaca tctcatgagt tggatgggtt 67320atgctggggg ttgggaaatg ggatgaaaaa ttatgggatg aaaaattgcc tatggatagt 67380ttaacttgaa agaatctgcc tttgtttaca gatagttatc ttttttcttt tttgagatag 67440agtctcacac tgtcacccag tgcagatacc cagtgtcact ggagtgcagt ggtgtgctct 67500tggtgcactg cagcctccgc cttctgggtt ccagcgattc tcctgcctca gcctcccaag 67560tagctgggac tacaggtgcc cgccaccacg cttggctaat ttttgtattt ttttgtggag 67620acgggttttt gccatgttgg tcaggctggt cttgaactcc tgacctcaag tgatctgcct 67680gcctcagcct cccacagtgc cgggattaca ggagtgagcc actgtgcccg gccagttaca 67740gatacttatc taatgaaatt ctctgtgtac tttataaaag atgaggatta actgaaggta 67800ctaataactg gattatatga gggtggtttt ggttgtataa tcctatctaa aagaatattt 67860tagctataac tgaaagtaag acttaaatat ttagagagga aaatctgaat aattctagta 67920gtaattattt atttacaaaa taaaaataga tttttttttg attacacaaa ttaaacaaca 67980ataaaacatc acagcaatcc ggatactata aagctcacat gcttaccgac ccaactgccc 68040caggagtgac cactgccaac agcttcatgt cgaccttttt gccataattt ttatatagcc 68100ttttttgttt ttaaatggta atttagaaag tcaactagga aaatgtgtta caggtttatc 68160ttccaggaga ataggactgg agtcgagatc ttgaatgtgg cttggaagaa ggcaagccca 68220ccccagagag atgagttgac agttgtttct gaccactgct tgcttagagg gcctgcgtgt 68280ctgtgaccgc ctagctttgc gcccctgact aggctgcccc ttaattacaa atgtctttat 68340atattgctcc agctaaggct tggagtagtc ggttaagaac ttgaacttcg gtttttgcag 68400tgaaacagca tttgagaata tcaccttctg ataagcctta ttttataagg tgggtactgt 68460agtgggaggc agtgtgagag atgcttgaag gatgcactgc tgtcctgcat ttcagcatct 68520tcaggatgct gtgcagctga aacatttgat aacggtggaa ctgttcgtta ttttgcaagc 68580ctgtgattcc ctattgaatg ttttctctcg ccatttgaca aatgagtgtt tctctgtctt 68640cagcctcagt gaaggatgag atcagtggag agctggctgc ttcttcaggg gtttccactc 68700cagggtcagc aggtcatgac atcatcacag aacagccacg gtcacagcac acactgcagg 68760cggactcagt ggatctggcc agctgtgact tgacaagctc tgccactgat ggggatgagg 68820aggatatctt gagccacagc tccagccagg tcagcgccgt cccatctgac cctgccatgg 68880acctgaatga tgggacccag gcctcgtcgc ccatcagcga cagctcccag accaccaccg 68940aagggcctga ttcagctgtt accccttcag acagttctga aattgtaagt gggcagaggg 69000gcctgacatc ttttttttta ttttttattt gagacagagt ctcactccat agtgcagtgg 69060aggccgggca caggggctca tgcctgtaat cccagcactt tgggagactg aggcaggcgg 69120atcacttgag gtcaggagtt cgagaccagc ctggccaaca tggtgaaacc ctgtctctac 69180taaaaataca aaaattagtt gggcgtggtg gcacatgtct gtagtcccag ctgttaggga 69240ggctgaggca ggagaattgc ttgagcctgg gaggcagagg ttgcaatgag ccgagatcgt 69300gacactgcac tccagcccgg gcaacagagc aagactccat ttcaaaaaaa ataaaaaaat 69360aaagtgcagt ggctcgttct cagcccactg caacttctgc ctcccaggct cgagcgattc 69420tcccgcctca gcctcctgag taggtgggat tacaggtggg caccaccaca ctcagctaat 69480gtttgtattt tcagtagaga cagggtttca ccatgttggc caggctggtc tcaaactcct 69540gaccttagat gatccaccca ccttggcctc ctaaagtatt gggattatag ttgtgagcca 69600ccatgcccgg ccctgccacc tgccatcttt tgagttcttc cctggagacc tagacctgaa 69660ccctcctgct tgttctcttg ttatctaata cccctattga cagcgcagct tagatcatta 69720atggagagct tgacctcatc tgataccttc actgaaggaa acaacttagt gtcttttgtg 69780ttgaacactg aggtaaaaaa ttggaatagt tgattatatg aactctgcta aaattgagtg 69840cattttacat tttttaaggc cttgttgggc cctggttaaa taattatttt taaaaatcct 69900taaggagcct attataaaca gatctgtggt cttaatgaaa tgtgattaat actgtgcatt 69960attttaagaa cttttgactt ttcaaaaaac ttttacaaca tttcccattt gatagcggca 70020taggtttaag cacttctcat ctctaagtta gtggacaaaa aaccctcatg gatagtctaa 70080taatgtttgc tacaagtcca tgttgagttt tatactccat tttattttca gttttaaaaa 70140ctgtggttaa atatgtgtaa cataaaattt atgttcttaa ccattttttg cgtatacagt 70200tcgctggtat taaatacatt taaataatgt catggaatca ttgctaccac ccatctctgt 70260aaccttttga tcatgtaaca ctgaagctct gttcccattg aactctattc ctcctttccc 70320gccaagtccc tggcaaccac gattcttctt tctgtcttct gaatttgact actttgggtt 70380ctcatatact ttaggagtca cacagtattt gttttactta gcataatgtc cccaaagctc 70440atgcatgttg tagcctatgt tagaacttcc taatgtttca ggccaaatac tattccattg 70500tatggatagg ccacattttg cttttccatt cctctgtcca tggacacttg tattgcttca 70560tgttttagcc attgtgaatc atgctgttat gaacgtgggt gtacagatag ctcctggaga 70620ctctgctttc catttttttg gctaaatacc cagaaatgga gttgctttta cattccaatt 70680ttaatttaaa acattcatat cattgagtgt tttacttaat agtatagtag ttaacaaact 70740taataaaata gtattttggt aataatttgc tggtagtcca ttgttcagtt tttttaggta 70800aattacacag gacatttcaa gtggacatga aacatcttgt gatgtggaat catgccccaa 70860gctgatggct aaacatatga aataccatac cctaaattta gtagatttag tctttgcaat 70920ttaggagata acctgttata ttgttaggtt tttgtcgaaa agctttgtcc tcatatttcc 70980aacttgctgt aaaatttgtt tgtgaagaca aatatttttg tatgggtttt ttctttttca 71040tattaaaaag aaatgtccac attggaattt ttttggagtt tttagagcta atagagcttt 71100tcataatgta gtgggaatga gtgatcagta agctcttagc agtttccatg cgtgcatttc 71160tgtgccttga aataaatgac agatgagtac atttgtgttc tgtgtgtaaa atgtgctctt 71220tcctcattgc acttccatgt tggagggctt gtctcttggt gatcacactt caaaattctc 71280acagcccccc ttgaaccgtt taggtgttag acggtaccga caaccagtat ttgggcctgc 71340agattggaca gccccaggat gaagatgagg aagccacagg tattcttcct gatgaagcct 71400cggaggcctt caggaactct tccatgggta tgtggactac aggtgatgcg ctacaaagtg 71460gtttgtattc agacctggac atcttaatta tatctttgct tccaagaaga agtcctttga 71520tactgttttc tgagttctga atagctgatg aaaatgacca attgaggaat aatcatactt 71580tttcttgatc taaatcttat acttttgagt tatcttagca taaatgtata attgtatttt 71640aagtggaaat ttgtcactta atcttgattt ctctgttttt aaagcccttc aacaggcaca 71700tttattgaaa aacatgagtc actgcaggca gccttctgac agcagtgttg ataaatttgt 71760gttgagagat gaagctactg aaccgggtga tcaagaaaac aaggtgaggg acataggctt 71820gagacgactt ggtgtttctg agcttgtgtg aggatttaaa atcgccctgg ctactgtcta 71880ctttattgct ttcccatccc tgggccttta aatttcccct ttaaatacca gctcttccca 71940ggcctgttgt tttctgcctt tccaggtact acccacagcc ttgagaattg cctgagttct 72000gcctcctttg agagtgtgcc ccagacaaat ctattctgta ctgaatgttt ccttgtctga 72060tttcttggat cattcatttg atggttgcgt atggcctgca acgtttcttg ttttggttct 72120actgaactgt tctaaaagtc tctcttcata ttatcttttt acatgtaaat gtaactgtct 72180tcacttttaa ttcctcaagg acaaggaata gcgtttcaca gttcgtccca tcaatcagaa 72240ttatagcctt tggcatctcc ctatctacca ggcccacttc ctcttagatt tgggcttccc 72300caggctgttg cctttcccca agtagcttct gcttgtcctg tagaagacct ttcatgcttt 72360gcttctgcag cagccgttcc tgaatgccta gtgtcaactg ccttcttacc acgcccaccc 72420tccctgcatg ctgcatttat cccctgccac agccctgtga ccctgtgtcc tgctgcctct 72480gacttgtctg tttctgcttg gccatggtct ctgtgaggtc aggtgtgcat atgggcacaa 72540accagggcat ctctttatcc ccagcacctg gcttaagtgc tgctctggaa ctatctgttg 72600aatgaactaa tgcatgaatg tattgttgag tatgagacaa acaagtgtca ttgtctcctt 72660tctagccttg ccgcatcaaa ggtgacattg gacagtccac tgatgatgac tctgcacctc 72720ttgtccattg tgtccgcctt ttatctgctt cgtttttgct aacaggggga aaaaatggtg 72780agtacaaaag gggatgtgca cagttgaagg aaataactag gtttcagagg tcagcttggt 72840ggcctgtttt tgccttgcgt gcagcagagg aagtagaatc tgaggatgag tttggttttc 72900actagccgag gggagggagg aaatgatggg agcaggtagg ttattgggtc tggttttgtt 72960catttgaaaa caatctgttg tttgaggctg aaggtggctt gggtgatttc ttggcagtgc 73020tggttccgga cagggatgtg agggtcagcg tgaaggccct ggccctcagc tgtgtgggag 73080cagctgtggc cctccacccg gaatctttct tcagcaaact ctataaagtt cctcttgaca 73140ccacggaata ccctggtatg ttaaaagttc acatcttatt ttctcagatt taatcattat 73200tgtaaaaact atttcagtat tgactatttt agttttagag cagtaagtgt tttgagttca 73260tttgggatat ttgacctgcg ttgtagctct tcagaaaaca catgaatagt gaagttcttt 73320gtttcatggg ttccctttag atgaaaccca tagaggagaa aagtagaaac ctcagcacgt 73380aagagccaac atatatacac atcggattta aacctaaagc acaaattgtg cctggtcgca 73440gtggcgctga gtcgcactca gccaggccag gcattcacac tcagggtgag tgggaaccag 73500gactggctga ggcagcagtg gacccaagtc tccatcgcgc ccatgcttac tatggagcct 73560tctcgttctc tctttttctt tgggtgagag ggtacacttg tgtttttgaa tttatatgag 73620gtaagtgtgt aatagggttt tttctaatct tttttaagtg gaatctggaa ttttaatcag 73680atttattatc tgacaaccta gaattataat ccagaaagtc tgtggtattg aggacatatt 73740ggcaatatga tgaatctcta attcttaaat cctgaaactt tttttttttt aatcacttag 73800ggttattata gtgaagtcat ttctgaattt ggatcttctc ttcacacctc tttttctctt 73860tcctgagaat taagcttttg tttcgagtta gaaagttgat agtagggaat tgttccatgg 73920ctgagcaatt tatctccaca gaggaacagt atgtctcaga catcttgaac tacatcgatc 73980atggagaccc acaggttcga ggagccactg ccattctctg tgggaccctc atctgctcca 74040tcctcagcag gtcccgcttc cacgtgggag attggatggg caccattaga accctcacag 74100gtaacggcca gtttttcagc tgtgtttttt ctagttatgc ttactaaggt ttaagtttag 74160atgatgatgt ttgttgcttg ttcttctggt taggaaatac attttctttg gcggattgca 74220ttcctttgct gcggaaaaca ctgaaggatg agtcttctgt tacttgcaag ttagcttgta 74280cagctgtgag ggtgagcata atcttctgtg gaaccatttc ttcacttagt ggacatttta 74340tcattgctac aattaaaatt ggagcttaat aggaaatatt tccatgcact ctaaagctgt 74400aaccagtaat acccaccatg tatccatctc tcagctttag aaagaaaacg ttgccagtaa 74460agttaatgct tcataaactt cagtttaagt tctaattctc agaatatttg tttgaaatag 74520acctcttcct aaaggatata tttagaaata acctatcatt aagtgtaaag tctgttgaat 74580atgctgggca cggtgactca cacctgtaat ctgaccactt tgggaggcca aggtggaagg 74640attgcttgag cccaggagtt caagactatg ggcaacatag ttgaccctgt ccctacagaa 74700aattaaaaaa aaaaaaaaaa aaagtagctg ggtatggtgg tgcatacctg tagtctcagc 74760tactcgggaa gctgaggtgg aggggggatt gcttgagccc cagagatcaa ggctgcagta 74820aggcgtggtt acaccactgc cctctagcct gggcaacaga gtgagactgt ctcaaaaata 74880atagtaataa taatcagttg aattaaaaaa aaaaaaaaaa aaaccactgt gctaggccca 74940tagtatggta agagttaaag tgagccttag ggattattta ctcaacctct gtttctgtat 75000aaagtggaat aggctcaatt ctttaagtga tagcatgttg aacctttcca taccaactgg 75060ctcataagtc acaactggcc agtcaacaag agtaaaaatt aactggtaaa aatcaaagca 75120aaaaacctac aattgtcaaa tttgtgggat aactccccct tttaaaatgt catgcctgac 75180agtaatttct ctctagtttc caggttttca gtcagttgtg tcttttttga gcagaaggaa 75240gcatgctaag agctcaatct tgtggctagc tgggggtctt

tgtgtcagcc atgcatgtga 75300tggtgcccct gggtgcttgg ggctgcaggg gaggggtaca gcagtagggg cctgttctgt 75360tctctcgtgc tgtggagtac atagtgacat agtggggtgg tccttggtgt aggtcccttg 75420ttcctacccc tgggtctgag atttatttag aagtggtgtt ggggctgtgc ggcaggcccc 75480tctgtaactg atcaatgttt gtgaagttgc tgtttgagag ttgaaaccat gacataagca 75540gaaatggaag gaagaaagaa ccagttatgt gaaagggaca catttacttt taagcttgta 75600tttactgaga taaagtattc ttaatcaatg ttcttgagag gtgtgggaaa aatgcaacat 75660cctggttgca gttaaaccca gaacattgtg tgttgaagag tgacggttct caaaccgtca 75720agacgcgggt actgagtggg actaacctgc tgtcctcttg ccttggacct tgtgttccag 75780aactgtgtca tgagtctctg cagcagcagc tacagtgagt taggactgca gctgatcatc 75840gatgtgctga ctctgaggaa cagttcctat tggctggtga ggacagagct tctggaaacc 75900cttgcagaga ttgacttcag gtaagtgagt cacatccatt agatttcatg aactaagctc 75960aattgaaagt tctgggatca cttgatgcaa ggaatgatgt tatcaagtac cctgtccatc 76020agaaatccga gtggtttagg tagatgacag tgattttctc ctcccagtgg ctttttgctg 76080aactttgccc tatgcttgga attttatttt attttattat ttatttagag acaagatctt 76140gctctgtcgc ccaggcttga atgcagtagc acaatcatag ctcactgaag ctttgaactc 76200taggactcaa gtggtcctcc tgcctcagcc tcccgattag ctaggagaat aggtgtgtgc 76260cgtcacactg gctaatattt tttgtagaaa tggggtcttg ctatgttgcc caggctggtc 76320tcaaactcct gggcttgatt gatcctccat cttggcctcc caaagtgctg ggattacagg 76380catgagccac tgtgcctggc ctagaatttt aaaatataag tagaagagta gatttttttt 76440tttggtagtc ctcgtcattt aagtattctg gatagtggga ataaaagagc ttagaatttt 76500tcatctttgt cttaaacttt taaaaaaatg tagcttatat taattctgct tgtttaaaaa 76560gaatatactc ttcattatac tgaacctagg taagacagct ggtttatatt ttgttgcaat 76620taaaaaacgt gagctgtggt tgcagtgagc caagattgtg gccattgcac ttcagcctgg 76680caacagagtg agacttggcc tcaaaaaaaa aaaaataaca tgagctgtgt tggcactttc 76740attttctaag agtagttttg gctggagaag ttttctttca gtactttctt ttagaaggga 76800aattttcctt tataatttag ggtttgtttt ttttttttcc aagccacctt ttatagagcc 76860cttgtgggtt atttcattta atccttagaa tgtttataaa tctgggcttg ttctcggctc 76920cacccacaga tagggacgct gagcgtgcat gagtgggcag caagatagca ggttatggag 76980ggcccagctc accccttctg tggcttgagc caattttata gggcacttac agagtctttt 77040gaaatagtat ttattttgaa gaaaaagaaa aacagtttac tgagtactgt cttattgagt 77100ctggaattgt gagaggaatg ccacctctat ttatttaaag ccattggcct tttttgttgt 77160tttgagtaag tgctgcccaa ggtccttcca gggcacctgg atgagcctgc tctggagcaa 77220gctggcggta agtgtttact gagtaactaa atgatttcat tgttaaatgt gctcttttgt 77280taggctggtg agctttttgg aggcaaaagc agaaaactta cacagagggg ctcatcatta 77340tacaggggta agcggtttat ttttgtgaga tgctgtttta ccttcaagaa ggtgaaagtg 77400aggctttcct tgtggaattt ctctaaatgc attcgtcatg ttttagatgt ttatttcaca 77460gtttatatca tgaaagttat aatcttgtca tatggattta agtctagtaa tgttgagttc 77520tttctcacta gctttccaaa atatcttacc taaaatttag tcaaatacaa gattatgttt 77580atttttatta tccttctctc taaagctttt aaaactgcaa gaacgagtgc tcaataatgt 77640tgtcatccat ttgcttggag atgaagaccc cagggtgcga catgttgccg cagcatcact 77700aattaggtat ttaccaatat tttatctctt ttcctttttt ggttgaagta ctaaaagata 77760cgagaatgga aagagaggga agaattcaaa ggatgtagag cagtattcct gaatctgagc 77820tcatttcagc cattctattc ttaaactata atgaaaaaaa aatccaaaaa agtctaaaat 77880tataattaaa aaaacaacaa aatactaact gtccattgta aaaagtaatg cactttcatt 77940gtaaaaattt tggactatag agaatagtac taagaagaaa aaaaaaatca ccttcaattc 78000tgctgccacc tggaggtaat cactgttaat attttgctat atactctatg agtttcttgt 78060tcaaaatcag gtcaaaatta catgcaattt tgtaatctga caatttccac ttaatatttt 78120attagcattt tcctgttatg aaacagtaat tttagttatg ggtcgttgtt ttgctatgcg 78180gttgggataa aattttatat actttttttg gcaattactt attatacata aatgtttgtg 78240tatagttttc tttttctgag aattcctgga agttgagtta ccaggcccgg ctttgaattt 78300ttttttttat tttttttttg agacagagtc ctgctctatt gtccaggtgc tatctcggct 78360cactgcaacc tctgtctccc tggttcaagc gattctcctg cctcagcctc ccgagtagct 78420gggattacag gggcacacca ccacgcccaa ttaatttttg tatttttagt agagacaggg 78480tttcacgata ttggccaggc tggtctcgaa cttctgaccc cgtgatccac ctgcattggc 78540ctcccaaagt gctgggatta caggcgtgag ccatggcgcc tggccaggct ttaaatttaa 78600aacaaatctt ctaatagctt tatggaggtt ataatttaca tttcttgaaa tgtactcact 78660ttgagtgtat agtaaactcc aattttatca catttctgtc accccaaatg tatccttgtg 78720cccatttgct gtaacctccg gttcctgccc caactcctag gcagccactc atctattttc 78780tgtcccttaa gatttgtgtt ttcgccaggc gctcatgcct gtaatcccag cactttggga 78840ggccgaggtt ggtggatcac ttgaggtcag gagttcgaga ccagcctggc caacatggtg 78900aaaccttgtc tctactaaaa atacaaaaat tagtcggatg tggtggcaca cgcctgtaat 78960cccagctact cgggaggctg aggcaggaga atcacttgaa cctgggaggc ggaggttgca 79020gtgagcagag atcgcgccac tgccttccaa cctgggcaac agagagagac tgtctcaaaa 79080caaacaaaga tttgtatttt ctggacattt tatagtactg gggtcatagt atagatggac 79140ttttgcattt ggcttctttt acttaattgt gagattggtt cttgttgtag catgtatcag 79200tagtttgttc atttttattg gcgaaagtat tctattatat gaataatacc atattttatc 79260tatccatcag atggatatta tagagttcat gttttggcta atttatgaat tatggtactg 79320tgaacatttg cctgcaagat tttgtgtaga catgtcttca tttctcttga gtagatcacc 79380tagaagtgga tttttaaata attttggtac ttactgtgaa actgctcttc aaaaacatac 79440cattgttcct tccttccttc cttccttcct tccttccttc tttccttcct cccttcctcc 79500ctcccttccc tacttccctc tccctttccc tttcccttcc ccttttccct tccccttccc 79560gcctgcctgc ctgcctgcct tccttccttc cttccttcgt ttctttctac atatacacat 79620ttttttaaat ttcaatggtt tttggggtac aagtggtttt tggttacatg gctgaatttt 79680ggttacatgg tgaagtctga gattttagta cacctgtcac ccgagtagtg taccttgtac 79740ccaatatgta gttttttgtc cctcaccttc cagccttccg ccttgtgagt ctccaatgtc 79800cattatacca cactgtatgc ccttgcgtac ccacagctca gctcccactt ctgagaacat 79860atagcagaaa catgccaaag tatactccca ctaccagaat gtgattgtgc ctgattcttc 79920tcaccagtac aaatatttca aaaaaagtta aatatgtatc agttttttgg gcagaagttg 79980atacttctct ttatttattt attttttttg agatagggtc tcattctatg atgcccaggc 80040tggagtgtgg tggtgcgatc tcggctcact gcagtctctg cctcccaggt tcaagtgatt 80100cccacgtcag cctcccagga agctggaatt acaggcgagg gccaccactg ccagctaatt 80160tttgtatttt ttggtagaga tggggtttca ccatgttggc cagactggtc tcaagctcct 80220gacctcaagt gatccacctg ccttggcctt ccaaagtgct gggattacag gcgtgagcta 80280ccacacccgg ctgatatttc tttttaaaat aacttacctt cttttgaaag taatacatgt 80340ttaatgaaca gaatttaagg aaaatataaa aaaacgaaat aatctttgta atcaaactac 80400tgaaaagaaa accaaagtta cattttggtg catattcttt ttcattttca tcattgtaat 80460ttgcatttct ttgattactt gtgagacact cctttcattt acttaatagg tttatatgac 80520ttgcctattc agagattttg cagctttacc attttctgca aatgatagca acttcttttt 80580gtttgtttgt ttgtggagac agagtctcgc tctgtcactc aggcaggaat gcagtggtgg 80640aatcttggct cattgcaact attgcctcct gggttcaagc gattttcctg cctcagcctc 80700ccaagtagct gggattacag gagtgtgcca ccatgcccgg ctaatttttg tatctttagt 80760agagatgggg ttttgccatg ttggccgggc tgatcttgaa ctcctggcct caagcggtcc 80820ccctgtctcg gcctcccaaa gtgctgggat tacaggcgtg agccaccgta cccagccagt 80880agttacttct tatattctag aaaaaattct actcatgatc aagtctccat gaggaaagag 80940actttaattg aagatcatgg ggcttgcaga ccaatatgat aaaatagttc attgtttcta 81000aaagtattac tgagtgttga tggcagatat gaaccctttt gtttttgtag gaaaatgtta 81060cccgtattct ccatttgaat tcagtttaga tttgttagga atcgcagctt aagctttgcc 81120atctgggagt gtttgggaca gttttgcaga caaaattgca aaagtgccta aggaatgcag 81180ctggcattca gacctgctct gtgctcagta ctctgtggac agacactgtt cagcacttgt 81240tgatcagaag gtttagaaag agaactttca aagttggttt ttaattaaag catttaatag 81300tgtaaataga aagggattaa attttatgac agacaaaaga aagtacagca cccagctggg 81360cgtgggggct cacgcctgta atccagcact atggggggct gaggtgggtg gatcacgagg 81420tcaggagttc aagagttcaa gaacagcctg gccaaggtga tgaaaccctg tctctactaa 81480aactacaaaa attagccggg cgcggtggca ggcgcctgta atcccagcta ctcaggaggc 81540tgaggcagga gaatcacttg aacctggacg gcagaggttg cagtgagcca agattgcacc 81600attgtactcc ggcctgggcc acagagtgac attctgtctc aaaaaaaaaa aaaaaagaaa 81660aaaagaaagt acagcaccca gttatgtccg agtgggtgca tgagagtgac cctgagattg 81720gagacaacgc tgtcacgtgc ttgaagaacg ccacctgaga aagggggcga gaagtggtgt 81780ccgctggtaa ccagaggtgt tggcttagcc atctgcaggg aggagggtgg tctatcacag 81840gtgagtttca tctactttct taagcaaatt aaccttactt ttgtgttagg cttgtcccaa 81900agctgtttta taaatgtgac caaggacaag ctgatccagt agtggccgtg gcaagagatc 81960aaagcagtgt ttacctgaaa cttctcatgc atgagacgca gcctccatct catttctccg 82020tcagcacaat aaccaggtat gctgacccag tggcatcttc acattgtcgg gaaaatgccc 82080tttcctgatg cctttcttta ggctttaatt gaaaacattt tattttctag aaaaaagctt 82140cagctcagga tgtttgagtg taggtcagtc ctttgatagg atattatcat tttgaggatt 82200gaccacacca cctctgtatt taagctctgc cacaatcact cagctgtgac actgtaaatc 82260tcttaatagt ttattacatt ccatgtgctg acagttgtat ttttgtttgt gacacttacg 82320tattatctgt taaaacattt tcactttagt tgtgttacct ttaaagagga ttgtattcta 82380tcatgcctgt tgattttttg gtgagcgggc tattaaagtc agtgttattt agggttatcc 82440actagttcag tgatttgcga gattatcatt cacatttatt gtggagcttt tgaatatcgt 82500gtcaaatggc cacatatatc ccattcttat ctgcttctta ggtgagtggg acacagtgct 82560ttaatgaagc tataatcttc agaattctag cttgcagaga agattgcaga agtgataaga 82620cttgtgcttt ttaattttgt cttttaaatg ttattttaaa aattggcttt atatgatact 82680ctttttttct gctgagtaac agtgttttac aaaacttgga ctaaatgact tctaagctta 82740aatgatcact tgatgctttt tttctgaatt aggaactcag cttatcaaat atcaaagtca 82800taattcctga ataaataacg tcttttttca tgtaaagact gctttaaaaa acacatggaa 82860ggctgggtgc ggtggctcac gcctgtaatc ctaacacttt gggaggccca ggtgggcagg 82920tcgcttgagc tcaggggttc aagaccaccc agggcaacat ggcaaaaccc acctctactc 82980aaatacaaaa aattagccag gcgtggtggc gggcccctgt aatcccagct actcgggagg 83040ctgagggatg agaatcactt gagccccgga ggcagaggtt gcagtgagcc aagattgtgc 83100cattgcactc ccagcttggg ctacagagtg agactctgtc tcaaaaaaag acacacacac 83160aaacaaaaaa aacatggaga catttttttg gccaccttaa tatttcccct cagataattt 83220cctttgttta aactcagaac tggcattttc tctcttggag aagattcagg acaaatactc 83280ctttaagata agtagaagca gtgaaagagg atttgattat caggaatttg ataagcttag 83340aataaattgt tgcttcttaa tgtcatttca gaagatgaat atttattaat agatgccaac 83400tgagatatca ttaaaattga ttactaacta ctacttggaa aagtctccca gttccaaact 83460tcagcaggcc tcttgacaat tcagctgtgg tcaattgggt cttgcgtgat agatacaatg 83520accaattgtg cagcagagtg tgctgcttag ctgcctattc tgttagcatt catgtgttaa 83580cttaaaatca taatctcctt agttttgttg agtgtctccg tggacaagac actgtgaggg 83640atacaaaatc agattggctt tattcaaacc actggggtat tataattcat ttataattta 83700ttttattttt tgcctttttt ccatgtgttc taaaggaatt agagtttgta tataactata 83760atgggggata gaaattgaca tgtgccatga agggaatgca aaaaagtgcc gtgggagatg 83820agaagtggag aaaggaattt cttttttctt ggaagcagga ataacttcat gaagcatgta 83880tttcaactta aacagatagt aggcaacgct gtaaggggag tatggctgca gcaaaagtgt 83940tcggggcaga ctgggaggaa gggagggaat aaattcagcc attgttatgg aataatgatc 84000aaaatttatt ttcagcccgt ttcacttaaa agttgagact gcttaacttt ttttaatctt 84060taatcttaaa cttttaaatg ccatttgatc tttaaaaata tatgttttaa tagtgtattt 84120taagtctcta tatttttgtt attagaatat atagaggcta taacctacta ccaagcataa 84180cagacgtcac tatggaaaat aacctttcaa gagttattgc agcagtttct catgaactaa 84240tcacatcaac caccagagca ctcacagtaa gtctctttct tgatcggtct tactgacatt 84300gtaatagttt ttggtagctt gtatggccag ttagttgtat ggtcatctta cggtgaggtg 84360cttgtcttac agctcttact tatccatgag gcttgctaag aaattgtgct tctgtgaaaa 84420gaatctcagc ttactccagg aatgtaaatg actatgtttt ttctgattat taaagtaata 84480cacgcccaaa ataaaaaaat tcagccaatt taggaagaca caacaattaa aataagccag 84540gcatggtggc tcatgcctgt aatcccagca ctttgggagg ccaaggttgg gggctcactt 84600gaggtcagga gtcggatacc agcctggcca acgtggtgaa accccatctc tactaaaaat 84660acaaaaatta gctgggcgtg gtggcgggcg cctgtaatcc cagctactca ggaggctgag 84720gcaggagaat cgcttgaacc tgggaggtag aggttgcagt gagctgaggt caagccactg 84780cactccagcc tgtgcaatag agcgagactc tgtctcaaaa aaaaaaaaaa aaaaagaaaa 84840gaaaaaagta aactactgtc acctgcattg gtaatgtatc agaagtttaa aatgtctaga 84900ttataattaa ctcagtgacc tggtaatata tactaaggga aaaatattta taatttacat 84960ttttacattt ttattttttt aattttatta tttttttttt gagacagagt tttgctcttg 85020ttgcccaggc tggagtgcaa tggcatgatc tcagctcacc acaacctcca cctcccgggt 85080tcaagcaatt ctcctgcctc agcctcctga gtagctggga ttacaggcat gcaccaccat 85140gcccggctaa ttttgtattt ttagtagaga cagggtttct ccatgttggt caggctggtc 85200tcaaactccc aacctcaggt gatccgccct cctcgacccc ccaaagtgct gggattacag 85260gtgtgagcca ccatgcctgg ccttacattt ttataataag aatttatgtt gctgacatta 85320gaaaagaacc ataatatcca agaatccaag aataattaaa ttatgtacat atgctagtat 85380atagtgtgat gctttggaga atttttaaca atatggagat gtataatctg gattgtaata 85440ttgagtgaaa aaaggcagaa tacaaacctg gtgggggtat agtcggattt cagttaagaa 85500aaataatatt tacatatata catttctcac actggcagat aatcaccaag ataaattttg 85560ggattgtgga tgattttttt cttctttata tttttcagat attctcaaat tttctaaaat 85620gagcaagtat aacttttgtt atcagaaaaa aataatatac aaaagtaatg ttaatttgct 85680ggtgaccagg ttaaaccttt ttatttttat tttttgagat ggaatctcac tctgttgccc 85740aggctagagc acagtggcat gatcttggct cactgcagcc tccgcttcct gggttcaaat 85800gattctctgg ccccagcctc ctgagtggct ggaattacag gcgtgtggca ccacacctgg 85860ctaatttttg tatttttagt agaggtaggg tttcaccagg ttggtcaggc tggtctcgaa 85920ctcctgacct cgtgatccac ccacctcggc ctcccaaagt gctgggatta caggcgtgag 85980ctactgcgcc cagccagacc tttttatttt atttgacaaa agaaatactt ccatgttata 86040gaagactaaa tattgtttgg gctgtctgca gtatggtctt cccttgattt gttcaaaata 86100tcgtaaactt tgcttattta tttttattgt ggccgactgt gtcgggcact gttgtaggct 86160tgggatggaa aaacaggatt cctgccctta gggtttctgc aggctggtca gggagacgat 86220gtggtaagct ggagctcagc tcctaaggat gtgcaggggc agttgagagg cggaagggtg 86280ggagatcatt ccagggtgtg ggcagcacag gaacctctct tcattgggat ataattgcca 86340ttctgataac acgtgtttga ggtgtctaaa gtaggaagtt gtaccatggt gggacagata 86400tcctgtggtt atcatacaca gatctcagtt ttcttctcat tgtttgtact ttttataaag 86460ggtaacagga gatataattc aataaacctt tgtggtgttt gggtgtgatt ttattgtttc 86520tttcttctca gtttggatgc tgtgaagctt tgtgtcttct ttccactgcc ttcccagttt 86580gcatttggag tttaggttgg cactgtgggt atgtattttc ctcagtatat attaatagtt 86640gtctacaaca gtatgacata aacatagtta ttaggatgcc ctttttcttt ctttttaagt 86700cttttatcaa tttggctttt tggaaaaata tctgatggaa tacttgtttc tgctatatta 86760gctgtgtgag actagtgaca ggagctgtgg gaaatgaatg ccaaatgttc ttaggcattg 86820atgggaattt cagggtgtgg tcttcaagtt catttaaggg aattttcata tgctggcaaa 86880aggcttttct cattagcttg actctttcca aaattatttg ctgtgaatta gaagtttagg 86940aacctttttt cacttaattg tgacctagca tacgaaatgg tgatgattta ggaactactg 87000ttcttgtatt aacagctttt atttaaaaat gattttcctc cagtagatgg ccctactagc 87060atctgggaaa taatttcaag tcttctccag cattcaggaa taggctttca ttttgtgtat 87120caattactga gaatgatttt ggtgactcac atcacatttg agaagtaaac ctgcagattt 87180cttgtgtgtg tcagcaaatg accaactgat atttgcttga agtggattac attatctgct 87240ctagaatgat tgctttccca ccttcctcac atacagactg agcagctacg gtttctaatc 87300ataggtctgg cactagactt cacttctggg caactttggc attggagtaa aatgtattaa 87360tttaaagaaa gttaaaaatc cgttcaagta aacatacagt tctaatactt tttacaattt 87420aaaatataga tttaaatgat aaaataaaaa agaaaatatg ggtagacacc ataatcctcg 87480tttctgcatc tgttcacaag gggttgatat ttatgagttc tattctccat atccattcta 87540tgttctctta atgctcagtc agcacctcag gtggttggag ttcaatgctt ggtagtttga 87600cttacactgt cttttctagg ggattgagcc ctgggtagtc ctgcttattt gaggttgcaa 87660tttgtctttc aataactttt actacaagat atggcgtgtt aaaggatacc attggggaac 87720caacataata atatcaggaa aactaaccac gtcagacctg ccccattgtg tatcaagtac 87780actatttttc catagtaata aagagttcac cccagccaat tctcttttat tttgtgcctg 87840tttactcaat ggcattaaca tgcccaaatg tctgggtagc tgtctcatct ccagttcagc 87900agaaccattg tcatatgccc tagtaaaagc attccttcat tggacactta ggccccaata 87960ctttcattca gatctactac ctgatttcat ttctcaaatg atttttatgg agctctgatt 88020tataggaaag atgttagttg attaaaaata aaacaatttc tgagctggta taaaatgtat 88080tgtgacatgc cttcctcttg gaattgcaag agaaaggaag actgttgttt gcttaaaaat 88140tgtctataat ttgactttgc aaatgtctgc ttccagagtg cctccactga gtgcctcaga 88200tgagtctagg aagagctgta ccgttgggat ggccacaatg attctgaccc tgctctcgtc 88260agcttggttc ccattggatc tctcagccca tcaagatgct ttgattttgg ccggaaactt 88320gcttgcaggt actggtactg agttgaaaca gggactccag gacttggatt ttgatttcct 88380tagggggaat gggggtggtg agcatatgag gggaaaatac tataaggtca ttgccagtga 88440tggcttgtcc ctttagtcaa atttcagatg ttacctatat gcataaacac atgcagttgg 88500cagctgttct gtgctgagta ttttaaagta gcctcttccc aatatagccc ctcagttaac 88560tacaagtaaa ctcattttga atttcatttt aatgggcacc atatgccagt actccctcgg 88620gcactgggat gttaagaaag tataatgtat ggacttcatt ctcaagttag ttttagatta 88680gagggggata cacgtaaaca aaagtgcagt ggtcacacag agtggcccta atcactctcc 88740ttgggcagat ttatgggctg gtaggaaaga gcacaacacg gagagggtgt agcaccttgg 88800cgatgataat ggaggatgtg gccagcaagg aagacggagt ccattgaaat tgattttggg 88860agaagttgcc aatctccatg aaagaattgg ggcctgtgct atttgcttca gggggctata 88920ggagagtttc gtgaaaggga ctaaaagatg agtattttaa taagatcatt catccaactt 88980gaacatgggc tggaggagaa ggtagggaga ctcaggagat taatgttgat gctaaggcaa 89040gataatggct ttgggactgt agggaagaca ctgattgtaa gagaatgaag gaggcagaat 89100tgccaggcct ggttcaccaa ctgaacttcg gttgtgaaga caaagaaacc tgggatgact 89160tcacatcctg ggcaggtgtg tggtggtgac agtcatggaa attgggaaca cagatttgtg 89220cgggaaacat cagtttcagt ttgagtttgg cttatcagtt gaatatcagg cacagatgtc 89280tggccaactc tcaacatagg gtcttaaatg acttcagttc cccaagcaat ttgtccttcc 89340catgctattg gggtggagag gtaatgtctg tgcccatatc acagccagtg ctcccaaatc 89400tctgagaagt tcatgggcct ctgaagaaga agccaaccca gcagccacca agcaagagga 89460ggtctggcca gccctggggg accgggccct ggtgcccatg gtggagcagc tcttctctca 89520cctgctgaag gtgattaaca tttgtgccca cgtcctggat gacgtggctc ctggacccgc 89580aataaaggta atgtcccact tgggtgctgg attcatacag ccttaatgac tatgggtttc 89640cagactacct ttgtttagta atctgtccct tctttattct ctttttgctt taaatgaaca 89700aaattgctca gattgtgaca ctaaatttaa catcaaaatg tgaccatgtg gatgggtgca 89760gtggctcgtg cctgttattc cagcactttg ggagactgag gcaagtggat cacttgaggc 89820caagagttcg agaccagcct gggcaacatc acgaaacccc ctctctacta aaaatacaaa 89880aaattagatg ggttgggccg ggcgtggtgg ctcaagcctg taatcccagc actttgggag 89940gccgaggtgg gcggatcacg aggtcaagag atcaagacca tcctggctaa cacagtgaaa 90000ccccgtctct actaaaaata caaaaaaatt atctgagcat ggtggcgggc gcctgtagtc 90060ccagctgctc gggaggctga ggcaggagaa tggcgtgaat ccgggaggcg gagcttgcag 90120tgagccgaga tcgtgccact gcactccagc ctgggtgaca gagcgagact ccgtctcaaa 90180aaaaaaatta gatgggcatg gtggtgcgtg cctgtaatcc cagctacttg ggaggctgag 90240gcaagagagt tgcttgaacc tgggaggcgg agtttgcagt aagccttgat tgtgccgctg 90300cactccagcc tgggtgacag agtcagactc tttccaaaag

aagaaaaaaa tgtgaccatg 90360tgttttatag ctcttttagt atcatcagtc actgttatcc ctaagaggga aatacctagc 90420tttagtttta ggtttccagc attagccaag aaagctcaga attgatgttc ctggccaagt 90480acctcattgc tgtctcctta aatcttggtt aatggctact gtcctggcta gcatagttat 90540ggagcatttc catggttgta gaatgttctg ccaatctcag ggacagtttt gcttttctgt 90600gaagcaataa aatcaacttc aaaacaaatg ttaactattt gtacaatgga tttaagatag 90660accagttcac atactttttt tttttttttt ttttgagatg gagtttcatt cttgttgcct 90720gggctggagt gcaatggtgt gatctcagct cactgcaact tctgcctcct gggttcaaac 90780gattcttctg cctcagcctc tcgaggcaga ttacagctgg gattacaggc atgcaccacc 90840acacccagct aatttttttg tagttttagt agagacgggg tttcaccatg ttggtcaggt 90900tggtctcaaa ctcctgacct gaagtgatct atccgcttcg gcctcccaaa gtgttgggat 90960tacgggcatg agccaccacg cccagcctaa gatagaccag ttcacttact gtttatatct 91020gattactctc tctttgcctt gtcttctacc tttaaaaatc tccctactaa cttcccattc 91080tcctttagct gccatcagtc ttctcccttc tctgcaaaca tctctggaga gtcccagcct 91140cagcccacag agcttcccac tgctctgagg tggaccttgt ttgcaaggct tctttggctc 91200tcttggcctg gaccctgtct actacttcag ccatccttcc ttaacccctg ctggtggttt 91260ctgttgccac actccatagc agcgtttccc gcccagatca tgtctttaca tctctgggca 91320ctgctctggt cctgcctgcc tttccctctt tgtatcctgc aggctgctac ccccatcttg 91380agtgtcctct tcagttggct ttcagagggc ctcctgggtg ttcccttacc cacttgccac 91440tccccagtca ctgggttcag tccttcctgc ccaccagcac atgctttcta ggctctgtcc 91500taggccgtct tctctctttg tagtctctgg gccagtgctg ttctagagag tggcagaatt 91560ttctataacc atggcagtgc tccatagcta tgccaggcaa gacagtagcc actaaacaca 91620tatagctgtt gagcccttga aatgcagcta gtgtgactga agaactgaac cccgattcgg 91680tttaattttc attaaattta aatttaaata accttatgtg ggtagtggct ccagtattgg 91740gcagggcagc ctgagagtcg gggctgttct cctgtcttca gtgtctagat gagggacctc 91800agaggacctg tctctggagc tgcagttcaa tgtagccagc tgccccgtga cacttacata 91860tagctgattt gtggatatgt cagacacggt gtgatgagct cagctttctg tcctcctccc 91920cacatctgcc cctgccccat ttaccccact ttgtgtctta tcaagctaga aacaggtcac 91980cacaagtctt catttccact caccaagtct tttgtttccc ctactaaata ttttgcgaga 92040agaaagtgtg tacctttgta ttcacataca tgtacatgca catatacatg cacatatgca 92100ggggtcccca acctctgtta aaaaccggac tgcaggccgt gcgtggtggc tcacgcctgt 92160aattccagaa ctttgggagg ccgagaccag tgcatcacaa ggtcaggaga tcgagaccat 92220tccggctcac acggtgaaac cccgtctcta ctaaaaatac aaaaaaaaat tagccgggtg 92280tggtggcggg cgcccatagt cccagctacc tgggaggctg atgcaggaga acggcgtgaa 92340cctgggaggc ggagcttgca gtgagccgag attgtgccat tgcactccag cctgggcgac 92400agagcgagac tctgtctcaa aaacaaaaca aaacaaaaaa aaaaaaaacc aggctgcaca 92460ggaagaagtg agcaagcatt accatctgag ctctatctcc tctcaggcca gtggtggcat 92520tagattctca taggagcgtg tatgagttcg ttctcacact tctgtaaaga catacctgag 92580acatataaag aaaagaggtt taattggctc acagttctgc aggctgtaca ggcttctgtt 92640tctgggaagg cctcaggaaa cttgcagtca tggcagaagg tgaaggggaa gtaggcacat 92700cttcacatgg cccacaggaa aaagagagaa ggagagagag agagagacag agagagagag 92760agaaaaagaa agattgagag ggagagagga gggagaaagg agagtgcctg tagggggagt 92820tgctacacaa aggagcacca gggggatggt gctcaaccat tagaaactac ccccatgatc 92880caatcacctc ccaccaggcc ccacctccga cactggagat tacaattcag catgagattt 92940gggtggggac acagagccaa accatatcag agcatgaacc ctattgtgaa ctgcacattt 93000gagggatcta ggttgcatgc tccttatgag aatctaatgc ctgatgatga tttgaggtgg 93060aacagtttca tcccgaaacc atcccccgcc aaccctggtt tgtggaaaaa ttgtcttcca 93120cagaaccggt ccctggtgcc aaaaagtttg gggacctctg cacatatgca tgcacctgta 93180catggacaca taatacatgt acatatgcat actttatatt ctctgccact tctggtccag 93240actgatatac tatctcattt ggattactgc actagccttt tgttttggaa acagcatttt 93300ttaaaaaatt taatttaatt tttttgagat agggtgtcat tctgttgccc agcttggagt 93360gcagtgtcat gatcatagct cactgcggcc tcgatctccc aggctcaagt gatccttctg 93420cctcagcctt ctcagtagtt gggactacag gcatacccac catgcccagc taattttttg 93480attttttttt ttttttgaga cagagtctca gcctgtcgcc caggctggag tgggttggcg 93540cgatctcagc tcactgcaac ttctgcctcc caggttcaag tgattctcct gcctcagcct 93600cccgagtagt tgggattaca ggcgcctgcc accacaccca gctaactttt tgtattttta 93660gtagagacgg ggtttcacca tgttggccag gctggtctcg aacttgtgac ctcgtgatta 93720gcccgcctcg gcctcccaaa gtgctgggat tacaggcgtg agctaccgct cccagccagg 93780aaacagcatt cttgagataa ttcatataat tcacccattt aaagtatata attcattctc 93840tttagtatgc ccacagagtt gtacagccat caccagaatc agttttagaa cccataaagg 93900aactctgtac tctttaccca aaacctccat gcctccagct gcaggcagcc actaacctgc 93960cttctgtctc tgtgactcta cgtcttctgg acattactgt ggatgggctc atacagtcag 94020tgagcttgtg actggtgcct tctaccaagc agggttttca gtgtagcagc ctctctgttt 94080ttcttttttt tttaaattgt gacggaactt ctgcctcccg ggttcaagcg attctcctgc 94140ctcagcctcc cgagtggctg ggactacagg cccatgtcac catgcctggc taattttttt 94200tttttttttt tttagtagag atgggtttca acatgttagc cagggtggtc tcgatctcct 94260gacttcatga tccgcctgcc tcggcctccc aaagtgctgg gattacaggc gtgagccacc 94320atgcccggct aacctttcat ttactgtctg catttcttcc ctgatgcctt ccagtccatg 94380cacccgattg tagccattca tcctattatg gtttaaggtg actgtcttag tcagcatggg 94440ttgccataac aaaataccat agcctgggtg gcttcaacaa cagaatttac ttctcacact 94500tctggaggtt gggaagtcca agatccagga ctttcgcctt gccctcatgt ggtgaggggg 94560tgaggaagct ctgtggggcc tcttatatat ggatgctaat ctcattcatg aggggtctgc 94620cctcatgacc cagtcacctc ccaaaggccc cacctcctaa taccatcacc ctggtaatta 94680agtttcagtg tataaatttg ggggactata gacattgaaa ccataacaag cacttttcta 94740agatcaggga gtgagtaagt agcagagcta ggacctcaat tccacatgtc agtcatcttg 94800ccttcactct gctccatgat ggctgcctcc tagagcattg ggagtctcga tgttctatat 94860gctctcatgt gttgtgtatt ggagatagtt gaggctttat gaatacatct ggatttgttg 94920acttctagct ttgctggtaa ccagctgtga ccttgaataa gttacttcat ctctgagcct 94980gtttcctctt ttagaaacag gagtttaaaa tgctgctttg ggttgggcac ggtggctcat 95040gcctgtaatt ccagcacttt gggaggctga gatgggagga tcactggagc ttggagttcg 95100agaccagcct gggcatcata gtgtgagatc ctgtctcctc aagaaattaa aaaattagct 95160gggtgatgtg gcgtgtgcct gtggtcccat ctactctgga ggctgaggtg ggaggattgc 95220ttgagcccag gaggttgagg ctacaatgaa atatgattgc accccatcct gggtgacgag 95280tgagaccctg tctcaaaaaa gaaaaaaaaa atgctgcttt gtaccccttt catgtcatgg 95340cgtcatggcc aacatagaat gccctggttg tttgctgttg gagggcatgg gcctgggggc 95400tccctgaggg ctccttccat cttcaactca ttctctgtgc acctgttagg aagttgtggg 95460ccagtcccta ccatgtatca ttgtgtgggt aaaagtaaat aaaatgtgta cagtgtctga 95520actgtacata tcagggtcca agaacaaaat gagtgacatg ggttagctct ttttaataaa 95580tggtaaaacc aaatattcta attttcagtt ttgttatact tccatcacat gtttttgttt 95640ttttgttttt tgtttttgtt tttctatttt aggcagcctt gccttctcta acaaaccccc 95700cttctctaag tcccatccga cgaaagggga aggagaaaga accaggagaa caagcatctg 95760taccgttgag tcccaagaaa ggcagtgagg ccagtgcagg taggaaacag cgtggggaag 95820ggagggacat gagtgcagca tctgtcatgt agaaacatag gatttaagta acttggtgtt 95880ttagagaaat aaatataata cacatcagta aagtgagaga aagtttctcc aggtgcggtt 95940caagatatta gaaactaatg actgatgtac acagaccacc ttttggtctg aagcatttct 96000aagtgccact ggctgacatg cagcccctac agcctccagg cttccagccc tagcatggag 96060catcactctc ctatgcttcc ctggttgcag gtgatggctg gagaggcctc ctgattttca 96120gtaagggaag tggtgtagat gcttaggaat agatgtagtg agtgaaaaaa ctgattctga 96180tatgtcaaaa attctgattg gaaatggaat atttacattt ggaagagcta aaggcgagag 96240aaagtgggga taaagtcatc tgagttggag gagcttaaac cattcacaag tttggaggac 96300ctttttttac ccatgaaaag gtcagaacag aaggggctag gatttaggtg tgactgcagt 96360ttattgaatt cccatccata ctgctctcgg tgggcagtgg caggggcagg agaggagcct 96420ggcaaagcat gaagtgactg ctgctgcctc tgctatctgg gacgcctggc cacctgtctg 96480tacagtctcc ctccagaccc attctcacgc tgtctcttgg cacccagggg ccagtgatgg 96540ttctcccatt tgttttgtgt atatagcatt tatatcaagg ctatttattt atttatttat 96600tttatttatt tatttttttg agacagagtc tcactctgtc acccaggctg gagtgcagtg 96660gtgcaatctc ggctcagtgc aagctctgcc tcctgggttc aagcaattct cctgcctcag 96720cctcctgagt agctgggact acaggtgtgc accaccacac ctggctaatt ttttgtattt 96780tttattagtg gagacggggt ttcaccttgt tggccaggat ggtcttgatc tcctgacctc 96840gtgatccgtc cacctcagcc tctcaaagtg ctgggattac aggcatgagt cactgtaccc 96900ggcctattta tttattttta attgacaaaa ttgtatatat ctgtaatata caacatgatg 96960tttgaaatat gtgtacattg gccaggcgtg gtggctcaca cctgtaatcc cagcactttg 97020ggaggctgag gtgggcggat cacgaggtcg ggagttcaag accaaactgg ccagcatggt 97080gaaatcctgt ctctactaaa aataccacaa aaaaaaaaaa aaaaaaaaaa agccgggcat 97140ggtggctcgc gccagtcgtc ccagctactt gggaggctga ggcaggagaa ttgcttgaat 97200ctggcaggtg gaggttgcag tgagctgagt tcatgccact gcactctagc ctgggcgata 97260gagcgagact ccgtctcaaa aaaaaaaaaa aaagaagaaa tacatatgca ttgtggaatg 97320gctaattaac ctgtgcatca cctcacgtat cattgttttg tggtgagaac acttaaaatc 97380tactctttca gtgattttct tgcatatggt acattgctat taactgcagt caccatgcta 97440tacagtagat ctcttgaact cattcctcct gtctataaat gaaattttgt atccttgacc 97500aacacattca aggttttttt tgagatggag tcttcttcac ccaggctgga gtaccatggc 97560acgatctcat ctcactgcaa cctccgcctc ccaggttcaa gcaattctcc tgcctcagcc 97620tcctgagtag ctgggattac aggcacatgc tactgcacct ggctaatttt tgtattttta 97680gtagaagtgg agtttcacca tgttggccag gctggtctcg aactcctgac ctcaagtgat 97740ccgcctgcct tggcctgcca aagtgctggg attacaggtg tgagccactg cacccggcct 97800caagcgtttt aaaagatgct cttttctaag gattgactgt agtacaggag gaagattgac 97860ctgttgaaaa gcctcagcct ttacaagtgt aaaattatca gtatattact atcatctttc 97920tgatgaatta aataaactaa ggactccaag tcaaaagtct tcaaactgaa gtagaatagt 97980tgtatatagt gcttggcact ttaatattta gtatcggttt aatgataatg tttgtgcctt 98040tgccgtcttt aaaacatttt tacatcatcc ctgtttgatt acttggtgtg ctcatgaagt 98100tgttggccac taaggaatct taggctcaga gaggttctgg aattggccag tggtccttga 98160atcagctgct cctatgattc tctaactgat ttctcacaaa gcaaacaagc aatcataaca 98220aaacaactgt gcacactgct cttcttattt tgttatttaa aaagtactta ggctctactt 98280atgtttgtta gtcaatttct cattacttct agttaatcaa aaggtcagag gaaatacttg 98340aatattttca tactagaata ctttaaaaaa tcatgatttc cagtaatctc tttaaaactt 98400ggcaagttat tttgatctaa aagtttatct tttgtgtgca tatttttaaa gcttctagac 98460aatctgatac ctcaggtcct gttacaacaa gtaaatcctc atcactgggg agtttctatc 98520atcttccttc atacctcaaa ctgcatgatg tcctgaaagc tacacacgct aactacaagg 98580tatgggcctc tgcatctttt aaaaatatat atgcacacat acttacgtct aatggatagt 98640tgatgttttt cttatgattt gtaggatgta taagcccttt gagatatgag ttacatttag 98700ttttttcaag tttgtttgtc tttcagcttt gtttatgata gcttctatca tacaggtgtt 98760ttggattttc atattgtttg tactcacagc taagattgat tacagtgaca gagctaggat 98820gtgcagccag gttatagggg gaagtggccc tggtggagtc tggagggatc cgtgtacagg 98880cttccttccc tcccgtgagg ctcacacaaa aatacagcaa catgctggtc ctgcaggtac 98940cctctgccta acatgagcca caattccaga ctcacagaag aaaagcaggt gttcggcata 99000aaccatgtgt ttcaaatagt ctgggcatgg tgagccactt gttatcagct agggaaagtt 99060tatgtcagcg taagaaactg ttcaccagat acccccaaga gccagccttt ctgtctaggg 99120atgttttagt tttttagttc attttttttt ttaactttaa aattttctgt tcatctgcaa 99180tttgttagat atgaagtatg tgtctaattt aatttttgtt tttggttgtc cccaataatg 99240tttacagaag aatttttctg cactaattgg cttgagttac ttacattctc atagttctct 99300agtttcagta gtttcattta ttattttgtt atatcaatct atctgtctgc tcatctatta 99360gaagcatcct tgtttttttt ttttcttttt tagacagagt cttgctctgt ccccaggttg 99420gagtgcagtg gtgcaaccat gcctccctgc agtctcaggg ctcaagtgat cctcccacct 99480cagctcctga gtacctggga ctaccggcat gtgccaccac acccagctaa tttttacatt 99540ttttgtagag acagggtctc cctaagttgc ctgggctggt ctcaagctcc tggcttaagt 99600aatcctccct ccttggcctc ccaaagtgct gggattacag gtgtgagcaa ctgcacccgg 99660ctacaagtat acttcttaat tattgtagct taatggtatt tatgagggga tcagttcccc 99720tgttgttctt tagaattttc tggatattct tctttattga ttttgggatg tgaacaatag 99780aatcaacttc tacttgtaga ttgatttagg gagaacttat acctcagatg ttaagtcacc 99840ctgtccagaa tgtgggatgc tttcctattt gttcagaact ttttaaatta cctcagaagc 99900acatgaaatt taaaggattt taaaaaaaac ttaaagatta tttcacatag ctcttgcaca 99960tttcttgata aatgaatcct caggtattcc tctgtttttg ttactaatag ttacttctta 100020tgggtttttt ttcccctgaa aatcatttat caaacgtatg tggcttattt tctgaaggat 100080gtttgataat tttggaagat atgaaagtct tcatatttta caaggtttga ggtctcttta 100140agctgcatgg ttctcatgtc agctcccaaa gcagaagacg gcatgttgaa aaatgccgta 100200gagaagatac ttcttttcca cctgttttca actcatatca tcttgaattt cagggcacct 100260ttccatgctc ctagtgcttg ctatctgttt attattttcc ttcctgaata ccctgaactc 100320cagcatgttc tgctgtaatt ctggcctccc tggcatcttg gactcctgtt tcctttgctc 100380tgtcatcccc gcggtcagct cctgctgcgc agcttctcag ctgaagtgcg tttggagtgc 100440ctggcgtgtc ttgctggatc tttgagtatt gcctctggtt tccttggttc cttctgctga 100500gttgctcagc gtctccactc cccatttctt gtgtggccct tcctgcactc ctctgattcc 100560ttttgtcttc cctggtttct tgctttggtt tcgagtctcc acagaacttt tgcagctctt 100620ctgaagacct ggaagctttt tcatcttaat tctcatctca tgacctcttt tcccttcttt 100680gagagctaga acttcccatg gtgaacttct ctttccagaa ttccatgcct tcttttccct 100740cccacttacc tgttgtccag gagaggtcag attgctgtgc atattggagg agaacccttt 100800cttccctggg ctcttcatct cacatgacat caccacatca cctcgttcct tggaccctca 100860gtggtgtcac tgctggattt ttctttcctt tggctggcct tagggcacac ccaggttgac 100920tagcgtagtc atggtattta gatccactca cattttcagt ttctgtgtct gtctcttgcc 100980tgcttctgac ttcgcccaga gaaagcttct ctttcacaag ggttcttaga tttatgttca 101040ctgagcacct tcttttctga ggcagtgttt taccaatatt tattttccta gtcagtctcg 101100ccttaccttt cttgttatgc atgtctttgg tcctgaccca ttctctgagt ctgtaaaata 101160gaattgctgt ataatttaat tacatgaaat cctttagaat cttaacacat cttacacctg 101220atttaatatt ttattgtatc caaattgaac caaccctatg tgaatttgac agtgatttct 101280cccagggatc ctagtgtata aggaatagga cttagtattt tctatttttt gatataccac 101340ataccagata ctgattatga tggacattta accctttttt ctcattatga aagaaagtta 101400ggaattattt cttccagtag cgccagtgta acctgaaagc ctttgaaaga gtagtttttg 101460tatagctatc tgaaaggaat ttctttccaa aatatttttc cagtgctgac aacaaacacg 101520cagacacacc ctgcaaggtg agtgtacggc gccgcacagt ggaggcatct gctgcagccg 101580tcgatgtttg tgtctttggt tgtacattat gagatcgtga cagggccagt aaccgtgtgt 101640tctctccttc accttcccaa ggtcacgctg gatcttcaga acagcacgga aaagtttgga 101700gggtttctcc gctcagcctt ggatgttctt tctcagatac tagagctggc cacactgcag 101760gacattggga aggtttgtgt cttgtttttt ctccttgggt tgtggctggc acacttgatg 101820tgcgtcttct gggctgagtt catctaggat ggagcctggt tctccagggt gcctccggga 101880gactcctccc tgccccacgt gcttgcgtca caggacccaa gtctgactct gccttagcca 101940tgaagtttag ggggaagttt ctatttgtat tctatttttg tctgttatca tgtattagct 102000tagacccagt ttagtttgga aaatcagtgg gtttcaaaat gtgtttgtag agtcctttat 102060ttcttaactt gaccttttca agtggaaagg ggcaaaacag acgggtaagg gggcggggcg 102120ggaggtgtga cttgctcttt tgtgcctgag gaagtaacag agctggggtt gacagtcata 102180ttctctgaca cagatagtct ctgacttatc tcacagaaag tcagcggcag agcctgagtt 102240aaaagtctcg tagattttct ttttcttttt tttggtggct aatttcagtt ttatttatat 102300ttgtttattt atttattata ctttaagttc tgggttacat gtgcagaatg tgcagttttg 102360ttacataggt atacacgtgc catgatggtt tgctgcaccc atcaacccat cacctacatt 102420aggtatttct cctaatgtta tccctccccc agtcccctca ctccccatgg gccccggtgt 102480gtgatgttct cctccctgtg cccatgtgtt ctcattgttc aatttccact tgtgagtgag 102540aacatgcggt gtttggtttt ctgatcttgt gatagtttgc tgagaatgat ggtttccagc 102600atcatccatg tgcctgcaaa ggacatgaac tcatcctttt ttatggctgt atagtattcc 102660atggtgtata tgtgccacat tttcttaatc cagtctatca ttgatggaca ttcgggttgg 102720ttccaagtct ttgctattgt gactagtgcc acaataaaca tacatgtgca tgtgtcttta 102780tcgtagaatg atttataatc ctttgggtat atgcccagta atgggattgc tgggtcaaat 102840ggtatttcta gttctagacc tttgaggaat cgccagactg tcttccacaa tagttgaact 102900aatttacact cccaccaaca gtgtaaaagt gttcctattt ttccacaacc tctccagcat 102960ctgttgtttc gtgacttttt aacgatcgcc atcctaactg gcgtgagatg gtatctcatt 103020gtgattttga tctgcatttc tctaatgacc agtggtgatg agcatttttt cgtatgtctg 103080ttggctgcat aaatgtcttc ttttgcgaag tgtctgttca tatcctttgt ccattttttg 103140atggggttgt ttgctttttt ttcgtaaatt tgtttaagtt ctttgtagat tctggatgtt 103200aatcttttgt cagatgggta gattgcaaaa attttatccc attctgtagg ttgcctgttc 103260actctgatga tagtttcttt tgctatgcag aagctcttta gtttaattag atcccgtttg 103320tcaattttgg cttttgttgc cattgctttt ggtgttttag acatgaagtc tttgcctatg 103380cctatgtcct gaatgttatg gcccaggttt tcttctagga tttttatggt cctaggtctt 103440atgtttaagt ctttgatcca tcttgagttg atttttgtgt aaggtataag gaaggggtcc 103500agtttcagtt ttctgcatgt ggctagccag ttttcccaac accatttatt aaatagggaa 103560tcttttcccc attgcttatg tgtgtcaggt ttgtcaaaga tcagatgatt gtagatgtgt 103620ggtggtattt ctgaggcctc tgttctgttc cattggtcta tatatctgtt ttggtaccag 103680taccatgcag ttttggttac tgtagtgttg tagtatagtt tgaagtcagg tagtgtgatg 103740cctccagctt tgttcttcta gcccaggatt gtcttggcta tgcaggctct tttttggttc 103800catatgaagt ttaaaatagt tttttccaat tctgtgaaga aagtcagtga tagcttgatg 103860gggggatagc attgaatcta taaattactt tgggcagcaa ggccattttc acgatattga 103920ttcgtcctat ccatgaacat ggaatgtttt tctatttgtt tgtgtcctct cttatttcct 103980tgagcagtgg tttgtagttc tccttgaaga ggtccttcac atcccttgta agttgtcttc 104040ctaggtgttt cattccctta gtagcatttg tgaatgggag ttcactcatg atttggctct 104100ctgtttgtct gttattggtg tataggaatg cttgtgattt ttgcacattg attttgtatc 104160ctgagacttt gctgaagttg ctaatcagct taaggagatt ttgagctgaa ccaatagggt 104220tttctaaata tacaatcatg tcatctgcaa acagggacag ttttacttcc tctcttccta 104280tttgaatacc ctttattgct ttctcttgcc tgattgcgct ggccagaact tccaatacta 104340tgttgaatag gagtggtgag agagggcatc cttgtcttgt gccggttttc gaagggaatg 104400cttccagttt ttgcccattc agtatgatat tagctgtggg tttgtcataa atagctctta 104460ctatgttgag atacgttcca tcgataccta gtttattgag agtttttagc atgaaaggct 104520gttgaatttt gtcaaaggcc ttttctgcat ctgttgagat aatcatatgg tttttgttgt 104580tggttctgtt tatgtgatgg attacgttta ttgatttgcg tatgttgaac cagccttgca 104640ttccagggat gaagctgact tgattgtggt ggataagctt tttgatgtgc tgctggattc 104700agtttgccag tattttattg aggattttca catcgatgtt catcagggat attggcctaa 104760aattctcttt ttttgttgtg tctctgccag gctttggtat caggatgatg ctggcctcat 104820aaaatgagtt agggaggatt ctctcttttt ctattgattg gaatagtttc agaaggaatg 104880gtaccatctc ctctttgtac ctctggtaga attcggctgt gaatccatcc tggacttttt 104940ttggttagta ggctattaac tattgcctca agtttagaac ctgttatcag tctattcaga 105000gattcagctt ttttctggtt tagtcttggg agggtgtatg tgtccaggaa tttatccatt 105060tcttctagat tttctagttt atttgggtag agatgtttat agtattctct gatggtagtt 105120tgtatttctg tgggatcggt ggtgatatcc cctttatcgt ttttattgag tctatttgat 105180tcttctctct tttcttcttt attagtcttg ctagcggtct acctatttta ttgatctttt 105240caaaaaacca gcacctggat tcattgattt tttttggagg gttttttttc gtgtctctat 105300ctccttcagt tctgctctga tcttagttat tttttgtctt ctgctagctt ttgaatttgt 105360ttgctcttgc ttttctagtt cttttaattg tgatgttagg

gtgttaattt tagatctttt 105420ctgctttctc ttgtgggcat ttagtgctat aaatttccct ctacacactg ctttaaatgt 105480gtcccagaga ttctggtatg ttgtgtcttc gttctcattg gtttccaaga aaatttttat 105540ttctgccttc atttcgttat ttacccagta gtcattcaag agcaggttgt tcagtttcca 105600tgtagttgtg tggttttgag tgagattctc aatcctgagt tctaatttga ttgcactgtg 105660gtctgacaga cagtttgttg tgatttctgt tcttttacat ttgctgagga gtgttttact 105720tccaactatg tggtcagttt tagaataagt gcaatgtggt gctgagaaga atgtatgttc 105780tgttgatttg gggtgcagag ttctgtagat gtctattagg tccgcttggt ccagtgctga 105840gttcaagtcc tggatatcct tgttaatttt ctggctcatt gatctgccta atattgacag 105900tggggtgtta aagtctccca ctattaccgg gtgggagtct ctttgtaggt ctctaagaac 105960ttgcttcatg aatctgggtg ctcctgtatt gggggcgtgt atatttagga tagttagctc 106020ttcttgttga attgatccct ttaccattat gtaatggcct tctttgtctc ctttgaactt 106080tgttgattta aagtctgttt tatcagagac taggattgca atccctgctt tttttttgct 106140ttccatttgc ttgttagatc ttcctccatc cctttatttt gagccaatga gtgtctttgc 106200atgtgagatg ggtctcctga atacagcaca ccaatgggtc ttgactcttt atccaatttg 106260ccagtctgtg tcttttaatt ggggcattta gcccatttac atttaaggtt aatattgcta 106320tgtgtgaatt tgatcctgtc attatgatcc tagttggtta ttttgcccgt taactgatgc 106380agtttcttca tagcgtcagt agtctttaca atttggcatg tttttgcagt ggctggtact 106440ggttgttcct ttccatgttt agtgcttcct tcaggagctc ttgtaaggca ggcctggtgg 106500tgacaaaatc tctgcatttg cttgtctgta aaggatttta tttctcgttc acttatgaag 106560cttagtttgg ctggatatga aattctgggt tgaaaatact ttttttaaag aatgttgaat 106620attggctccc actcttttct ggcttgtagg atttctgcag agagatctgc tgttagtctg 106680atgggcttcc ctttgtgggt aacccgacct ttctctctgg ctgccctttc cttcatttca 106740atcttggtgg atctgatgat tatgtgtctt ggggttgctc ttctcgagga gtatctttgt 106800ggtgttctct gtatttcctg aatttgaatg ttggtctgcc ttgctaggtt ggggaagttc 106860tcctggataa tatcctgaag agtgttttct aacttggttc tattctcccc atcactttca 106920ggtacaccaa tcaaacgtag atttggtctt ttcacatagt cccatatttc ttggaggctt 106980ggttcatttc ttttcactct tttttctcta atcttgtctt ctcgctttat ttcattaatt 107040tgatcttcaa tcactgatat cctttcttct gcttgattga atcggctgtc gaagcttgtg 107100tatacttcac aaaattctcg ttctgtggtt tttagctcca tcaggtcatt taagctcttc 107160tctacactgg ttattctagc cattagtcta acattttttt caaggttttt agcttccttg 107220tgatgggtta gaacatgctc ctttagctcg gagaagtttg ttattaccga ccttctgaag 107280cctacttctg tcaattcatc aaactcattc tccatccagt tttgttccct tgctggtgag 107340gagttgtgat cctttggagg agaagaggtg ttctggtttt tggaattttc agcctttctg 107400ctatggtttc tccccatcat tgtggtttta tctacctttg gtctttgatg ttggtgacct 107460acggatgggg ttttggtgtg ggtgtccttt ttgttgatgt tgatgctatt cctttctgtt 107520tgttagtttt ccttctaaca gacaggcccc tcagctgcag gtctgttgga gtttgctgga 107580ggtccactcc aggccctgtt tgcctgggca tcaccagcag aggctgcaga acagcaaata 107640ttgctgcctg atccttcctc tggaaacatc gtcccagagc acgaaggtgt ctgcctgtat 107700gaggtgtttg ttggccccta ctgggaggtg tctcccagtc aggctacatg ggggtcaggg 107760acccacttga ggcagtctgt tcattatcgg agcttgaatg ccgtaccggg agaaccactg 107820ctctcttcag agctgtcagg cacgtatgtt taaatctgga gaagctgtct gctgcctttt 107880gttcagatgt gcccttcccc cagaggtgga atctagagag gcagtaggcc ttgctgagct 107940gcagtgggct ctgcccagtt cgagcttccc tgctgctttg tttacactgt gagcatagaa 108000ccacctactc tagcctcagc agtggtggac acccctcccc cagccaagct cctgcatccc 108060aggtcgattt cagagtgctg cgctagcagt gagcaaggcc ccatgggcgt gggacccgct 108120gagccaggca caggagagaa tctcctggtc tgctggttgt gaagactgtg ggaaaagtgc 108180agtatttggg caggagtgta ctgctccttc aggtacagtc actcatggct tcctttggct 108240tggaaaggga agtcccccga ccccttgtgc ttcccaggtg aggcaacacc ccgccctgct 108300tcggcttgcc ctccgtgggc tgcacccact gtccagcaag tcccagtgag atgaactagg 108360tacctcagtt ggaaatgcag aaatcacctg tcttctgtgt cgatctcact gggagctgta 108420gactggagct gttcctattc ggccattttg gaagcatccc ttgttttttg aggtggagtc 108480ttgctctgtc gcccaggctg acgtgcatcg gcacaatctc ggcccactgc aacctttgcc 108540tcctggtttc aagcgattct cctacctcag cctccggagt agctgggatt acaggcacct 108600gccaccatgc ctggctaatt ttttgtattt ttagtggaga tggggtttca ccacattggc 108660caggctagtc tcgaactcct gaccttgtga tccacccacc tcagcctcct agagtgctgg 108720gatcacaggt gtcagccacc acgcccagcc atattttcag atctccctct ctttgcccta 108780aaccactgtg cttaataagt agtttttagt ggccagcagt ctccatgtat aacacatttt 108840agcaaaatgg aaaatactat atgttttaaa tttgaacgtg agattatact gaaataaaaa 108900tcatctaact gggattcttt aaatagtaag attttctttt ttgtatgtgg gttttttttt 108960aaccttatta ttatgactgt catatataga aatggctgtt tttcagttac agtcagtgaa 109020tgtatcaaat gctgccttat ccaaataata aaagtaaatt attaataagt cacaatttaa 109080tgaagattga tgttagttga tctttatatt cttgaaatca gccatatggt tgtgtgtgta 109140tgtatatatt tttaaaggta cataaagata ataagctcat ctctgaaaat ttttacattt 109200ggcataagaa taactggata attaagcatc ttattctctg gcctgtgtct ttacagttaa 109260aggtagattt actcacctct ccttttttgt ttttctaagt tcatcttttt tgctgtttca 109320agacagaggc ccattttagc tttctcgcat atccttttgt ttgtactttg gaagcctcac 109380ctgcttaatt gttgagtttt tatccgtggt cttttagagg gggatatgta gggtagaagc 109440tttcacaggt tcttgtttgc acttggcccc tgactgtttt gaggaatctc cctcactgac 109500tcacagcatg gcaaggtttc agatctcttt ctgccacaca gcagttctga ggcagctgga 109560aagatatcca gatgcttaga ttgtcaggcc aggcttgaga tatacaaact attgagcctt 109620atctgtgacc ttgcttaggt gaaggcatca gagcccctgc accaacatgc ataggcctct 109680gcatgtgtgc ggggctgggt gttgaggtct gagcacaagt gtagctggag aggtgagctt 109740gatgtggcga cgggtatgag caggttttct tcagacttct gtgagtttac ctagttccag 109800gatttaaagg cacagagact ttagaattaa aatagaatca ttttcttttt ctaaatagca 109860acactaggaa taaaaaataa taattccaca ttcttgacag gtaatgtttt ttcttgtctt 109920ctaatcctta tttattccat actcattttt atacataatt gaaatgtatt atgcattgga 109980tttttctttt gcattatatt atagacgatt tttcatgtaa ctccttactg ttccatttta 110040tatgttttgt ctggtttaag actttatctg caaaccggga aactgtctct acaaaaagaa 110100aaacaaaaat agttggccgc agtggcatgc gtctgtggtc ccagctactc ggggctgagg 110160tgggaggatt gcttgagcct tgggaggttg aggctgcaaa gagccatgat catgccattg 110220cactccagca tgggtgacag actttatact gtctgttttg ggtgatttga taatgatatg 110280ccctgatgta gtttttttat atcttgtgtt tcttgtgcct gggtttattg aggttgggtc 110340tgtggcttca tagtattttt aaagtttgga aaattttagg ccattctttc tttctttctt 110400tctttttttt ttttttgaga cagtgtctcg ctctgtcgcc tgcgttggag tgcagtgaca 110460ctatcttggc tcactgcaag ctctgcctcc tgggttcacg ccattctcct gcctcagcct 110520cctgagtagc tgggactaca ggcgcctgcc accacgcctg gctaattttt tgtattttta 110580gtagagacga ggtttcactg tgttagccag gatggtctca atctcctgac ctcgtgatct 110640gcccgcctgg gcctcccaaa gtgctgggat tacaggcgtg agccactgca cccagctagg 110700ccattatttc ttcaaagatt ttttttctgc cctgcctccc tccttttttc cctctcttaa 110760aggggctgtg atttcctgaa tgattgctta gtgttgtccc atagcttact gatgctcttt 110820tcagtgtttg attgttttat gtgttttctg ttttgtatag tttctattat tgtgttttca 110880agttctctga tcttttcttc tacagtgtct actctgttgt taatctgtta atctgttgtt 110940aatcctgtcc agcgtatttt tttttttgtt tttgaaacag tctcactctg ttgcccaggc 111000tggagtttag tggtgcgata tcagctcact gcaacctcca cctcccaggc tcaagcaatt 111060cttctgcctc agcctcccga gtagctggga ctataggcac gtgccaccac acctggctaa 111120tttgtgtatt tttattagag atggggtttc accatgttgg ccaaactggc cttgaactcc 111180tgacctcagg tgattcatcc gcctcggtct cccaaagtgt tgggattata ggcatgagcc 111240accgtgtctg gcccctgttc agtgtatatc actaattttg tttttatctc tagaagtttg 111300atttaggtct tttaaaaatg tctccctgtg tttctgttta gctttgtgaa cacaattgta 111360ataactgttt taatatcctt ctctgctagt tctaagatct tctaataact tcccagttct 111420tggtgtttct cattggttga ttgatactcc tcgttttggg ttgtattttc ctgcctcttt 111480gtatggctgc caatttttta ttggatgccc aaccttgtga attttacttt gttggatgct 111540atatattttt gtgttcccat agatcttctt gagctttgtt ctgaggttag ttgagttaca 111600tatagatggt ttactctttt gggtcttgct ttataatttg tcagatgggt tggagcagtg 111660cttagtttag gactaatttt ttttttggac taattattcc tctttaggaa taattaggta 111720ccatgcttag gaggcaagac catcctgagt actctaccta atgaaccaga aagtttgggt 111780tttccagtcc gcctgctgag aacagtgact ttctagccct gtgtgagcgc tgagctctgc 111840tccttctaat cctttccaat gcttctttcc ctggcctcag ggagttttct cacacacata 111900tctctgctga gtactcgaga gggaccttcc ccagatctcc agagctctct ctgtcttgtt 111960ttctcttctc tggtgctctg tcttatgaac tgtggctgtc ttggtctcct tagattctca 112020gcacctcttc aattcagagg gttgcctgtc cctcctcctt gtgccacagc ctaggaactc 112080tctcaaagca gcgagttggg gcagccatag ggctgactta gtctctcgtc tcccagggat 112140cactgtcctt cattgctcat gtccagtgtc ttgaggactc tgggttttgt ctgttttgtt 112200ttttggtttg ctttggttgt ctcaggcagg agggtaaacc cagtccctca ccctcattgt 112260gctcagtagt ggaagtctca ctctattaca ttagatatta gtatttgtag cagagccctg 112320gttccctggt acttggggag ctcttgaaag gccagaaaca gcatgctttc tcaccttttc 112380cagggcttca gtttctggtg cacatcaagc attccataca catttgttaa agtcctttgt 112440tagacaagta gtgattcaca ggttctattt gtaatttttt cagttaacat gtattgggta 112500tctgctggga gctagtaaaa acaaaaagtg gtgtgtgaca aattcaattc tgacaagaac 112560aaccttaaac acttagaata tactttgagc atatcagaat tttaaaaatg tgtggccctt 112620gagtatttga aaccaacaag aatctattgc ttattagtag aggatatttt gttaaacaag 112680tggagagaga ggcattttca gtctaattgg tgttggcttt tagcagctga tggaaaccag 112740ttcgtgatta gccaggcagt ggtgaaacag gctgtgcatt ctgaatgcct aggtatctag 112800gcattcagaa tggtggcgct ctttgagtta gcatcttctt ctttcttgat tctttttttt 112860ttttttttga gatggacttt cgctcttgtt gcccaggtaa caactccagt gcaatggcgc 112920catctcggct cactgtaacc tctgcctccc tggttcaagc gattctcctg cctcagcctc 112980tcaagtagct gggattacag gtgtgcgcca ccacgcctgg ctaattttgt atttttggta 113040gagatggggt ttcactatat tggtcaggct ggtcttgaac tcctgacctc aagtgatgca 113100cctgcctcga tctcccaaaa tgctgggatt acaggcgtga gccaccactc ccagcccctt 113160cttgattctt gaaaaggaca ttgggtgctg tacatctcgt tatagatgtt gataaaaatg 113220cttgtgagaa gagtaacatt aaggtagtta tttggtcatt tttgcagatt attttaagac 113280aattctagga ctgatttgtg gtaaatcaca cattgctgta tcatagttgt gttcactgaa 113340catattcagg ggctctacag atgcagggct cttagctgct ttgcacactt ctgaattcct 113400gccctgcgaa caggactgga tacctaatag acaacaggta cttgataaca gtttattgaa 113460ttaatgagtg aatgaacaga tacataaatg catgaaagaa tggttgtaat gtatataact 113520tggatttcaa gactttttac tgactgttca aaataagaaa ttgaaaactt tcctctgatt 113580ttcctctact atttacacaa tttaaatgga agttatcttg taccttcaat ttctgtctag 113640gattcgtaca ataacgggtc atctctgagt cgcttaatgt ctcacttgtc tttctacagt 113700gtgttgaaga gatcctagga tacctgaaat cctgctttag tcgagaacca atgatggcaa 113760ctgtttgtgt tcaacaagta agagcttcat tcttttcctc ttctgttaag acgttcgggt 113820atgacagcaa aacgctgcta ctccttaaga ggcaggcgct gttggcataa tcagctggga 113880ggattgtggg gtccagcgca gcactttttg gctcagtcca tgattgagcc aagaggccat 113940ccttcccttc actccccagg aggacgaggt ctgtcactgt ggagggcaga ggacaccaga 114000agctcctctg caacctcgct agttaacttc cagtccctcg gagtttctgt ttagaatgct 114060caatctcatt tagaattgca aggaaaccca aaacgcctat ttaaggtaca aacagcactt 114120catacaatat ctcatgaggt attaatagtg attcacagga agaatttcac gctgtgagtc 114180tttgctaaca tatccagtta tttacagatg gatttgatat ttgtgtggga gattcttaaa 114240agtgttgttc acgccacatt gttgatgcct catttttttc actgtagttg ttgaagactc 114300tctttggcac aaacttggcc tcccagtttg atggcttatc ttccaacccc agcaagtcac 114360aaggccgagc acagcgcctt ggctcctcca gtgtgaggcc aggcttgtac cactactgct 114420tcatggcccc gtacacccac ttcacccagg ccctcgctga cgccagcctg aggaacatgg 114480tgcaggcgga gcaggagaac gacacctcgg ggtaacagtt gtggcaagaa tgctgtcgtt 114540ggtggaagca cgaaagagca agcaggaaat actttgtaaa agaataaaaa cgaaaaatgt 114600tagcgaacat cttctaatag tctgctgtat tcagagaact ctaggagata tatatggttg 114660atgcaaagat gatttaaggc atagcccggc cttccaagaa gtgtgtggcc agtgagtgag 114720atgggcttgg gacttacaca tctcagaggt gggggtagag gaggaggaac actgagtggg 114780ctgagaagca gccagctctc attgccaaag tgtgtcagca aaccagaatg cagttcataa 114840tgtccccacc cattcaaagc acaggacctg tagagtggtg tggcatgtgt tggtggcact 114900tttcaggcct gtaacaagga tgaaagaaca gcttcatagc agcacagtag tgctggtgtt 114960cagaggtgtg tgaaggccat agaagcatct tggatatatt accttgtgtt ttgtcagctt 115020tatgactaga agtctctttt cacttaaatt tgtttttttt ttttttgaga cggagtcttg 115080ctctgtcgcc caggctggag tgcagtggtg caatctcagc tcactgcaag ctctgcatcc 115140tgggttcatg ccattctcct gcctcagcct cccgagtagc tgggactaca ggcgcctgcc 115200atcacgcctg gctaactttt ttttgtattt ttagtagaga cggggtttca ccatgttagc 115260caggatggtc tcgatctcct gacctcgtga tctgcccgtc ccggcctccc aaagtgctgg 115320gattacaggc gtgagccacc gcgcccggcc tcttttcact taaatttatg tttgtgtttt 115380taatgcctag tatacaggac ttcttaaatt gccttaagta tgaacaggta tttgagttgc 115440taatctgtat agtagcaata atagaatccc ttgtttttcc ttttataaat ttagcgatta 115500aatagctaca attaaaacac tagagtcagg agtcaaggaa aatacccatg ttccaggctg 115560tatgttagtg atgtacttac tatatattgg agtttcagga gtaagtctgt ttcaatgctt 115620tctgtaacca tttggggtat taataagcat gtgagtgtgt gcatgtttgg gttaatttca 115680tatatgtttc ttagaaggga tatcattgat gtaaatattt taaaggcttg tcctccaaaa 115740aaatcatgta atttcttcta aattactgat cttttaaatg accttcacct ttctctcaaa 115800tctcacttaa gactgggctg agtagtcagt ttcctgtagc agaaaaaagc tcagacttga 115860gtagccttct gcgagtgagg agacttgatg gctgtcaggc agctgtaaac tctaaataga 115920gtgtcattat ctgaagaggg cgatgctgcc acactgagtg gcctttcaag ttgtttctca 115980atctgacacg ttctgatcgt gtgaatgtga aattggtttg agcaggagta tatctgagtg 116040cagaggagat tatttaaaga tattctcatt ctctgcttcc cttttattcc catttggcag 116100atggtttgat gtcctccaga aagtgtctac ccagttgaag acaaacctca cgagtgtcac 116160aaagaaccgt gcagataagg taaatggtgc cgtttgtggc atgtgaactc aggcgtgtca 116220gtgctagaga ggaaactgga gctgagactt tccaggtatt ttgcttgaag cttttagttg 116280aaggcttact tatggattct ttctttcttt ttttcttttt tatagaatgc tattcataat 116340cacattcgtt tgtttgaacc tcttgttata aaagctttaa aacagtacac gactacaaca 116400tgtgtgcagt tacagaagca ggttttagat ttgctggcgc agctggttca gttacgggtt 116460aattactgtc ttctggattc agatcaggtt tgtcactttt atctttcatc catcatacct 116520gttcctaatt tagtacaaat taccctaaaa gacactgaaa tctactttaa agaaatgtgg 116580tctgcatgtt tccctcatca gttgctgctg cttatctttt tcatgcacct agctggtgca 116640gaaggcctgg ggcatagcca gcctcagcaa gtcagcatcc ttgccccagc tccctggact 116700caaggctaac ctggggttgg ctgttaggga tttccaaagg tttgtcccat ccacttgcct 116760cccctccaaa ataagtttga atttaaattg tgagatacaa ttaagattta ttgtttgggg 116820aacatttttg caaaatctag agttagttta aacagattat caattattac cataattgat 116880catctgcagt ttcaagctat ctaacaggtt cacttacctc tttaaaaagg aatggaattt 116940agcaggacag taactgagac ccgtgctcct ggagtccatg tgggagctgt gtggctctgc 117000acaagcattt gcacgcttcc cctcttgact gcattacctt cctcctatag ttgctgtggg 117060caccagattc tggctagtcc tgtcccttca tgatgcacat tttcctcaag attcgtccca 117120gttaaatcac tgcagatgaa actgcctttt catcgtcaaa atttaactgt catttttgag 117180ccgtgatctt gggctacttt cttatgtggg gtaggaatat ttgtgagtta gaaatattac 117240acttctctat ttccttctag acgtaaatct gttaatcctg tcagcactgt tactcacctg 117300aaagggtctg tttccctagg agaactgagg gcactcggtc aacactgatt ttccacagtg 117360ggtattgggg tggtatctgc ttgttttttt tgttgttgtt gtttgttttt ttttgttttt 117420tttttgagat ggagtctcgc tctgtcaccc aggctggagt gcaggggtgc gatctcggct 117480cactgccagc tccgcctcag aggttcacgc cattctcctg cctcagcctc ccgagtagct 117540gggactacag gcacccacca ctacgccagg ctaatttttt gtatttttag tagagacgag 117600gtttcactgt gttagccagg atggtctcca tctcctgacc tcgtgatctg cccgcctcgg 117660cctcccaaag tgctgggatg acaggcgtga gccaccgcgc ccggcctggg gtctgctttt 117720aatgaaggag gcatcaaggg gtgggctttg cgttggcctg atgctttcat ctttctttca 117780caaaacctgt ccgaagaaaa tccgtctaaa tgggccattg ctctcctcag gaaatagtca 117840ttgggaactt cttttccttt cctttgacac taggaggctg actggggaga agccctggtc 117900tatggctgtg ggcagcaggg gctgagagga gcaggctctc aggggggcac gggtacccca 117960agggaagcca gagccctgat ttgttccatt ctagtaagaa caaagactgc tctggtttca 118020tgtttgttct gattgccttt catcaaccgg tcccctttct cccagttctt aagattcagt 118080acagtgacag ttttatgaac aagaatagaa cactagaaca gacaaaccat tgaactctat 118140gctgataaag atttattgag ctcctgctgt atgtttgcat tctgcccaga ggctctgaga 118200aaaccaggcc atatgctcca tgctttatcc atggaagctc cccgtcaggt tgggaaagct 118260gacagctgca gggaatacag tgtgacacaa aactggctcc catgcagccc ttacgtgtcg 118320cctctcagat ggttggggga cgaaggtcga ctcctttggg tatcttatta ctaaaccagt 118380ttcagggaat ctgtgccacc ctatctgcca ttaacgtgaa cagatgagtc cccaaggtgt 118440aattttgggt attgtctgat gtctcttgga atttattatt tgtttttcca atgagatttc 118500acctcagggt atagtaaagt tgttgagggg attcctggat gtgttctgca attatctagg 118560ctgatttcag aatagagtta tgcttatagt caaatttatc agctgtcaag aattttattt 118620aaaatttatg cagataagca ggaggaaaag aagcctggtt tttacatttt aatcctatta 118680ttgatgtgaa attttatttt ccttcctgta ggtgtttatt ggctttgtat tgaaacagtt 118740tgaatacatt gaagtgggcc agttcaggta atagcatttt attattttag atttttttct 118800tcttcttgtg tacttacatg taatttaggt tattaagtga atgtttaaac tactgttagg 118860catttttgct gttttcttta aatggaaatc tgactaacat actgtgcatt tttgcttctc 118920ttaaaaatta atgtatatct caagacttgt ttggaagtag ttatgtatct gaaaattcca 118980tatgttgtca gtattcattg cacatttcaa agcatttaat tgtgttgaca gatggtggaa 119040tgaaatcttg tggtggagca ctagttttta aatcttctta gagaaagcag ttttatataa 119100tgttgtcttt agtaattatt atgcatttgt attctctgca gctttttctt gctagatgtt 119160gaggttttaa tacttcttgc tagtccatta caggtttata attattaaaa gttaaaattc 119220ttttagtacc taaaatgctt aataaacatt gtaattagga aaatttagtg cagaaggaaa 119280gtgttcccag attccctggg gtctggaaac atagtgttta ttctaattac atgacacctc 119340cactgtgttt tggggcaagt tactgtttct cttttgagtt tcaatttctt caagagcaaa 119400gaggcagagg agagctagga agatcgtagc tgctgtgccc ctgtgccgtc gggtgccttc 119460tacctgctgc ctccgaacct ttacacatgt ccctgctctg cgcgagggca cagatgggat 119520gcactgtggc aggggtgggg ttagagtaga tcacggacac ctgttagctt gatgtgtgct 119580tgctgtcaag gttgaatcat gaattatttt atgttgctta tattgatatg tatcttaatt 119640ttaaaagaaa ggtctaaatg gatgtttttg tttttaggga atcagaggca atcattccaa 119700acatcttttt cttcttggta ttactatctt atgaacgcta tcattcaaaa cagatcattg 119760gaattcctaa aatcattcag ctctgtgatg gcatcatggc cagtggaagg aaggctgtga 119820cacatggtaa cgggacacac ctttcactgt cgtcttcggt gtcgtgatgt gcttggcagt 119880gttcgttttc atatacccac tttgaacgtt gtcagtggca gccatgtgct tctcaggctc 119940tgcatgtgtg tctgtgtatg tgaaggtact ggttagagac gtttcaaaag agaagagagc 120000atattcttta ctctcagcaa tttgtaatct tctcagggaa aaaaattcaa gaaacagtaa 120060gataacctaa ggtacagata gattctgaat ataaagttcc tgttcattca catgaaacgc 120120taaaagttct tcacttgatc ttagccaaaa ggccaagaag cgatgcaaca ctaaaaattc 120180ttaaatcgaa cttgccgtga attaaatttt gatctctcat ccagtggtat tggagatata 120240gtttgacttg ggttcagggc tttctgtttt gcctgatgat tttgctggag cttaaataag 120300gaacccagga gatggccagc tgtgcaagcc cccagcctgt ggaaggagct agtgtggttt 120360tatgaatgag ttgcaaatct ttctttgagc tttttgaact gatcttccag cattgcccta 120420ttgacccctc cctgactcct ttgctggaat ctgtaggctt

ttgaactttg acagggacac 120480atcctaagac ccttgcaaac tcccagatgt gagaatggca ctactactta gagtcttttc 120540gactcagcgt gtgtgcagaa gagcatcaac cgggctgtgt tgcgaggcag ggccttggct 120600gacctctcag tgtttacata gctaagccag ttagtgtttg ccacggcctc acaagggctt 120660cagattcaca cagccaaagt atagattatt aaaggcatag gtgtttggtt tcctggactt 120720ggagggtctt tggacagaaa atcagtaggc aaccacaccc agtactttgt gctgggaagc 120780ttggtcatct gtgagagggt cagagagtat acccatgcgt gcatgccacc gaagggtcag 120840tgagtattcc tgtgtgtgca tgtctcaggg ccggagagag tatgtgtcac tgagaggtca 120900gagtgtttgt gtgtgtgtca aagagggttg cattgtgccc ttcactgagg ggtcagaggg 120960tgcctcgcgt gtgtgtgtgt gtacgtgtgt gtgtgtcact gaggggtcag agtgtgcctg 121020tgtgtgtgct tgtgtgtgcg tacatgtcac tgaggggtca gagtgtgcct ctgtgtgtgt 121080gctcatgtgt gtgcatacgt gtcactgagg ggtcagagtg tgcctctgtg tgtgctcatt 121140tgtgagcgta tgtgtcactg agggggtcag agtgtgcctc tgtgtgtgtg ctcatgtgtg 121200agcgtatgtg tcactgaggg ggtcagagtg tgcctctgtg tgtgtgctca tgtgtgagcg 121260tatgtgtcac tgaggggtca gtgttcctat gtgctcatga cattgagggt cagagtgtgc 121320ctgtgtgcca atgaaaggca tttcttatat ttttttatat gtggtcatag tagaccagtt 121380aatttatttt gactcctgtg ttagaccaaa ataagacttg ggggaaagtc ccttatctat 121440ctaatgacag agtgagttta cttaaaaaag cataataatc cagtggcttt gactaaatgt 121500attatgtgga agtctttatt gtcttttcag atgaatcaag tagattattc ttgagaccag 121560gaatgttgct gttttggtta tttggaaagt tttatcattt tcaaattgac ttttgaattt 121620gagtcacctt ttttcagaag tggtgttaaa ttataggagc cctaggtttt ttttcttttt 121680ttagaagtca tcacaaaatg atcagtgttc agaggaagag ctttgacctt ccacatggta 121740taatgattga taaccttaat tcatctctta ccataaacca agtatgtgta agggttttct 121800ttatttcttg aaagcatttt gtagatgttg agagcagttt tccaaatgta atttccatga 121860aatgcctgat aagggtaccc ttttgtcccc acagccatac cggctctgca gcccatagtc 121920cacgacctct ttgtattaag aggaacaaat aaagctgatg caggaaaaga gcttgaaacc 121980caaaaagagg tggtggtgtc aatgttactg agactcatcc agtaccatca ggtaagagga 122040atgtatgttg gaactgtcgt ggatacttta ttgacccgtg cagatggaag gaagtgccat 122100gtggtaacgc tcactgttaa ctgtgttact ttgaaccagg tttgggcttt ctggggcctg 122160ggtagatgcc ggtgcagggg gatggggagg gaggcggggg gtgggggggt gtggtggagt 122220tggggaggtg cagtggcagg aggtgttgtt ggtgtgtatc cttttttttt ttttgagatg 122280gagtctctct ccgtcgccca ggctggagtg tggtggcacg atcttggctc attgcaagct 122340ccacctcccg ggtttaagca attctcctgc ctccacctcc cgagtagctg ggattacagg 122400catgcaccac catgcccagc aaattttttt ttttgtattt ttagtagaga tggggtttca 122460ccatgatggc caagctgttt cgaactcctg acctcaagtg atcctcctgc cttggcctcc 122520caaagtgcta ggattacagg cgtgagccac catgcccagc ctggtgttta tctttaaagt 122580gggcacagcc acaggagttc acctgactcc tggtctgaga gtcacgagat cgttcaagat 122640agtgaggccc tcttttccaa aacgaggacc aaaaatcaat tgacagtgtt ggtcaagatg 122700gtagaaacct taaaatgata gaaatctcaa ctctgaaata aaaactttat ttgtatattt 122760atttaccact attttgacat agggctaagg tctttttctt tgagctgatt tctggttttg 122820ttttcttaaa gtggcataag aattcaaaga cattttgagg aaggctgagt gcagaaatct 122880ctctttttaa atgacttctc ctttctttta acttgcactg ttgtctagcc ctcacttatt 122940ttgtcaattc tttttagctg tttgtctttg aatcttcata aagccatagc ttttctcata 123000agaagcagca ctttctttgt tcattcatat tttaatgaac ccctgtagta tttaattaaa 123060tacttaatgc ctaattaaat cacataattg caatgcaaaa gtacatgtat cataaagagg 123120tctgaaaatg agcaactggc aagcaggtgg tggcaggcag agctgcttgg gtgggtgggt 123180gtcatggaga ggagttcatc agccacatgt tcagtgagct ctggatatgt ctgtttagaa 123240atgatcacta ataaacttgt gctcaaccat gtatacctct gggaagcagg tgctcttcag 123300tagattgcct ctgcagagaa cacagaattg aagtgaatgt ccacaaaggc aatgagccac 123360ctgcagaata gtttagtcaa ggctgtgttt gaagtttgcc aaagattaat atacatttga 123420ttttcatgtt gtgccttttc tctgattgtg aaatattaca aattctatac aaataacaat 123480gatggcaaat cctcctgagc aaagtgtgca ccttgtatgt gccctagagg aacttgtgtt 123540tcgttctgat tcccctacat ttctcatgtc atagagtggg ggttgcatta gtgtccccct 123600gtcctcgctg ggatcacatc tgtttggatc ctagagtctt ccagctgaac tgggacaagt 123660ataacagacg gacacgtagg ggtggaaagg cgtctcttgg cagcagactt tctaattgtg 123720cacgctctta taggtgttgg agatgttcat tcttgtcctg cagcagtgcc acaaggagaa 123780tgaagacaag tggaagcgac tgtctcgaca gatagctgac atcatcctcc caatgttagc 123840caaacagcag gtttgtcccc gcagccttgg cttgttgttg catagtgatg gtagcttaag 123900gtccttgtga aaggtgggtg gctggaatca gctcttcctt cagtcctaat ctgtgccttg 123960atagcagttc tccgtgctag tcatgggaca gctgacttca tttcttctca caatgccatc 124020tcaggttggt attgcccacc tactttacag gggggatccc acagctccga gaggttatgg 124080aggtgatcag gcagcacaca gctttagagt gctggggtga gggcgggcca aggctaactc 124140taaagcccga acccttacct cctacactgc ctcctgcatt ctggtcaacc cagtgtttta 124200tttggtggtt agatttttgt ttttgttacc ttactgcttg taatttagca gttttccttt 124260cctttccctt cctttccttt ccgacagggt ctcactctgt cacccaggct agagtgcagt 124320cgtgtaatct cactgcaaca acctctgcct cccaggttca accaattctc ccacctcagc 124380ctcctgagta gcaaggacca caggtgtgca ccactacgcc tggctagttt tttgtatttt 124440tagtagagat gaggtctcgc tgtgttgccc aggctggttt taaactcctg ggcgcaagtg 124500atccaccaac cttggcctgc caaagtgctg gcattacagg tgtgagccac ctcgcctggc 124560ctattcatca ctaatcagaa tttctatgat caaatgacat gaatcattgt ttccacaact 124620gcagtggaag gaaatggcct ggcagtgcca gtttcagaag cagcctgccc ccagtcaggc 124680acaggccact gtgcccccag tgtagcagca cctctgtagc tcacagagaa gggtggtggg 124740gacctccttg aggcagctct gccagaaaat ctcatgagct gcctggcaca gcttgaggtt 124800gccttttaag tggactcagc aaatacatgt ttgttcatct tgattataca caataaacaa 124860ctactctgta tagtacgagt agtccgtggt ttttggcatt tgatttaaac ttagaggcat 124920gtgatattga tgttactgcc ttcatgactg cacccccatt ctgatttcat aatggaatgt 124980tatcttgaga ccagttagac aacaggacag ggatcttggc ttctggtgag attgacagca 125040gttttagtgt ggtcagggtc tccctgccta cagatggttt tagaatggtg ccctggaagc 125100tttatcccat tcttttctgt gcgtaatctg agtagagtgg agatcgaagg cctgaataca 125160tagtaaatac ctgacttaat atctgccgca atggaaattg tgtgatacaa catttatgaa 125220acgcttagtg cagcacctgc caggtagctc accacaggtg catgttgcat tcagaagtag 125280tgctagatac tatcctgtta ctggcagtgc atacatcagt gatcaaagca gattaaagaa 125340agaccccctg ccttcttgga gtgaagattt tgttgggatg cgggtaaggg gacagacaat 125400agaaaagcaa gtgagtgaag tctataccat ggcggctgat caggaacacc gtacagaaga 125460atccaggagg gaagagagtt aggtggtgtc tgcggtggga gtggcattgt tcagctggtg 125520atgagaagaa gctttggtga tctggtgaca tttgagtgaa tttgcagaaa ggaaagatac 125580aagcctagga gatacctggg gaaggaacat tccaggcaga gcaaatagca gtgcaaaggc 125640cctggcgggg ggcggacatg ctgttagggt acaagcaatg agggtggagg agtggggcag 125700ccatggggag ggaagggagt gaggcctggt ggggtgaggc cagtgtggag gagccttgag 125760agggtttgcg ctgatgtggt gtaggtttta gcaggatcat tcttattcct gagttgagaa 125820tagccttgag ggggaggtga gggcagagca gggccaccca tgtgagaccc ggcactggag 125880tggaatggcc caagtcagca tcccttggca gcatgaaagc aaaaccagca aggtttgctg 125940gtggcttaga tgtggcatgt gagagagagc agggctttgg gggtgatttc agggtgagga 126000cagggtggct gtggacaagg tagggcagac attgggggca gcaggaggtc agagcctgtc 126060tggatgtagc agttgagacc ccataggtgc ctaatgaggt gaggccagca tcaggtgtat 126120gagcctggag ttgtcgagag actgtggggc agggggtcag catctgagat gtccactcac 126180agtggaccca gactggctgg agaggaggag gagcttgaat accgagcctg ctgagtccca 126240gctccaaggt caggtaggtg aggggagcca gtgctggggc agggggagta ggcaggtgtg 126300gggttcctaa agccaagatt ttttttaagg cattttgtgc aggagggcga catctgctgt 126360cagcaccttg ggaacttggc ccaggtttgg cagcaccgag ggcactgatg agtgcttttg 126420gaggagcaaa gggagccaaa ccctaatggg aatgtgttcc tgaaaggaca ggagagagac 126480ttgggaaaag gttttacttg aagagggaac ggagaaatag ggcagtagcc agaggaggag 126540aggagtcggc aatgggttaa gttggcagaa atgaaggcct gtttacgcac tgagggcaga 126600agcaacaggg aggatcagtt catgacacag gagacacaaa tcgccgttgt ggtgttcaca 126660gacatgggtt aggattggct gcatggatga cagagcactg tgggttctcc cagagttgct 126720ggggaggagg cagagttggt gagcacaggc gagggtccag gatgcaggaa tcctggagct 126780caagtcagtt gttcccttgt tgtaagatgt ggccagtgtt gtgagcttca catctgtgcc 126840ttgaaaaaca ccacatctgt ttgcagagtt gtttactatg tatacacact cagtagaaac 126900aaaaattgga aacagtcagt gcccaccatc aataagtaat ggttgaacac actgtggtat 126960aagcttagac tattttagct tgggctattt tgcatgatta aaaatgttct ggccaggtgt 127020ggtggctcat gcctgtaatc ccagcacttt gggaggccaa ggcaggcaga ttgcttgagc 127080tcaggagttt gagaccagcc tgggcaacat ggtgaaaccc tgtctctact agaaatacaa 127140aaagtagctg ggtgtggtgg tgtgcgcctg tagtcctggc taactcagga ggctgaggtg 127200ggaggatcac ttgagcccat tcgtgcgcca ctgcactcct ggggcacaga gtgagactct 127260gttagaaaga gagagagaga aagaagagag agggagggag gaaggaagga aggaaataaa 127320tggaagaaat ggaagggagg aaggggaggg aggaaggaag aaaggaagtt cagccagttg 127380ccttgggagt tctccattgc actgggttaa gtgagaagag cagagacgtt tatgattttt 127440caaaacaact aaaacaaaac ctctgtgggt gagggggcaa ggatatggct ataggaacat 127500ggggcagatt aagaaaggga tatacacaca ccacttagca tttgttacaa ctgttgtggg 127560agggatggag tgcagaaaaa gaaaaaaaaa agtgcacacc atcccatgta tgtgtataca 127620aagggacgct tggaagactg gtccccaaaa tgttggtaat gattgtgtca gggtgctgca 127680gtgctagttg attttttttc acacttttgt atatttgagt cttttacaga aagcatttat 127740tatttatgta ataaaaatct aaatgacaag atttctgtta tgggaaaaat gtagctatac 127800agtgttgttg taaaaatgtt tgcttggttc accactgaac ttaaaatgct tttaaatgag 127860ggaaggtgac gatgagatga ttatgatgat ttgcccttga gttacatagc tggtgtacag 127920gaagctgtcg tttcttttgg cttacgtaga aatgtttgtg gtgtctaatt ccacagatgc 127980acattgactc tcatgaagcc cttggagtgt taaatacatt atttgagatt ttggcccctt 128040cctccctccg tccggtagac atgcttttac ggagtatgtt cgtcactcca aacacaatgg 128100tgagtctctc gcctggctca gcagatgaat ctggacggct tgttcaggct ctgattactg 128160ggaccacccc cagaatgtct gagtcagtca gtttgggtag ggcttcttga gagtttgctt 128220tttttttttt tttttttttt ggtgtggggg tggtgcggaa cagagtctca ctctgtcgcc 128280caggctggag tacagtgtca tgatctcggc tcactgcaag ctctgccttc cagcttcaca 128340ccattctcct gcctcagcct cccgagttgc tgggactaca agcgcccacc accacgcccg 128400gctaattttt ttgtattttt agtagagatg gggtttcacc gtgttagcca ggatggtctt 128460gatctcctga cctcgtgacc cgcccatctc agcctcccaa agtgctggga ttacaggcgt 128520gagccaccgc acccggcctt tttatttttt ttggagatgg agccttgctc tgtcacccag 128580gctggagtac agtggcgcta cctcgactca ctgcaacctc cgcctcccgg gttcaagcaa 128640ttttcctgcc tcagcctccc gagtagctgg gactacaggt gcgtgccact gtgcccggct 128700aattttttgt atttttagta gagacggggt ttcactgtgt tagccaggat ggtcgcgatc 128760tcctgacctt gtgatccgcc cgcctcggcc tcccaaagtg ttgggattac aggtggctct 128820cgcaccaagc caagagtttg catttttagc aaattcccag gtgaaactaa tgcctgcttt 128880tctgggagca cactttggga ctcagtgata gagaggttta ttggtaggat agtaaaatag 128940gagttatttt ctttcacaaa attggcaatt gggggaaatt taatcttcct tttttcttca 129000gctgtgactt atgtattatg tttattttag gcgtccgtga gcactgttca actgtggata 129060tcgggaattc tggccatttt gagggttctg atttcccagt caactgaaga tattgttctt 129120tctcgtattc aggagctctc cttctctccg tatttaatct cctgtacagt aattaatagg 129180ttaagagatg gggacagtac ttcaacgcta gaagaacaca gtgaagggaa acaaataaag 129240aatttgccag aagaaacatt ttcaaggtat gctttctatc tgagcctata actaacccat 129300gccttttggg aagtcacgtg atgtttcaca gtcagtaagt ctggaataat acctggtctt 129360gcttcacttc tgagttgggt aaagaagtct gtatcagtgt aattttctaa tccgtcctgc 129420attatctatg gctcttggtt catacctgtc ttgaagttct gtcatgttct gtctcttgtc 129480ctcagtagag atgctacagc agtggctcgc ctcaggcagg gcagggcagt ggggtggctg 129540tcctgggggc aggcagtagg ggcacgctga cgtcagggaa gttgaaaccc aagagaagcc 129600agtaaaagtg agtctcagat tgtcaccatg tgctggcagt tttacacgct gtcagtaata 129660aaagtcttct ccctgcaggg cagcctgcct ccaataaata cgtgtagtat caaatcctgt 129720cttccctcat aaattgtttg gaagctcccc aaggacagtg atgaggcact cgtaagtgct 129780tgctgcctag atgggtccct ctccaccttt gctagattct gagcattcac tgagttagag 129840ctgcttctgc aaatgtgctg cttctgctaa gtggctgtga cttcatgcag ccttcacttg 129900gtttgtcatc agtggagatg ccctgtgttg tcgaaggaga taagcccagt aagcctgctg 129960ggcacctttt ggtttgcagg ttcagcaggc agcccatggc tttccctgtg tcgcattgaa 130020gcagctggct aaaattgatg atacattaaa ttcctgtgac agatgatcag cttgtatttg 130080tgtaatggtg tacagttcac aaagcttaaa aaaatgctac ctgccatttc atcctcagtg 130140aggaaggtga tacacagaga gaccaagtga ctgtgtccac ggcgacggcg ctctgcattt 130200cactttagcg gttaatgtac tctacctata tttttacttt atatttacca tatatctttt 130260catgtatact tggcgtaagt gctttatagt agtcacctaa ttcactgtca tcttttttgt 130320ttcttggaag gtttctatta caactggttg gtattctttt agaagacatt gttacaaaac 130380agctgaaggt ggaaatgagt gagcagcaac atactttcta ttgccaggaa ctaggcacac 130440tgctaatgtg tctgatccac atcttcaagt ctggtaggtg aatcacatta gtcttcctgg 130500agtgtctcgt tccccattct gcactataca ctctcagagt gtaggagctg tgctgcccgg 130560tagaaactct gccttgccca gtgtgccagt tgaaaatatt tgttgctgta agagtacacc 130620tgataccatg tgacccagca gttccactct tgggtatata cccaaaagaa tggaaagcag 130680ggtggtgaaa agatatttgc atgccagcat tcatagcagc attattcacg atagctaaaa 130740tgtggaacca actgaagtgt ccctcgatgg atgaatggat aagcaaaatc tggtgtatat 130800ttacagtgga atattattca gccttaaaaa aaggacattc tgacacatgc tacaacatgg 130860gtgaccctta aggacattat gctaaatgaa ataagccagt cacaaaagga caaatactat 130920gtgattccac ttacatgagg gacctggagt agttaattca tagatataga aagtagaatg 130980gtggttgcca ggggctgcag gggaggggag ttatttttac aagatgaaga gagttattct 131040agaaatgaat ggtggtgatg gttgtataac attatgaatg tacttaatgc tactgaactg 131100tacagttaaa aatagttaag aggaccaggt gtcatggctc atgcctgaaa tccaagcact 131160ttgagaggcc aaggcaggag gattgcttga gccaaggagt ttgagaccag cctcagcaac 131220atggtaggac cccatctgta caaacaaact agccggggat agtggtgtgc atgtggtccc 131280agctactcag gagactgagg ctggaggatc gcttgagccc aggaggttaa gtctctagtg 131340agatgtgttc atgccactgc actccagcct cggctataga gtaagaccct gcctcaaaaa 131400aacaaaacaa aacaagacaa gagccaaaaa tggttaagat gggccaatca cagtggctta 131460tgcctgtaat cccaacactt tgggaggtca aggtaaaagg atcacttgaa gccaggagct 131520tgggaccagc ctgagcaaca tatcgagacc cctatctcta caaagaaaat caaaaactag 131580ctagatatgg tgggcacatg cctgtagtcc cagctacttg ggaggctgag gtgggaggat 131640ctcttgagct caggagttcg aggctgcagg gagctattat tgcactccag cctgggctac 131700agaatgatac cctgcctctt attaaaaaaa aatccaaaaa aaaaaaaaag taaacctgag 131760agcttcctcc tcctgtgtta aatttggagg ccaagatgtt tttgttactt ttacaaatga 131820tcaaggacgg tgaaggttgg gcatggtagc tcacacctga aatcccagca ctttgggagg 131880ctgaggcggg gtgatcgctt gagcttgaga ccagcctgga caacatagca agagacccca 131940tctccacaaa aataaaaaaa taaaaaaaaa tagccaggag tagtggcatg agcctgagcc 132000caggaggtca agctgtagtg agccatgatc atgccactgc actccagcct gggcgagatc 132060gagaccatgt ctctagagaa agaaaatgac aaggacagtg aacccaagaa agtcataaga 132120tgccagctgt gcagcaagca tggaaagcag ccagtccaaa ttaggacagt gtgttttcca 132180agaagaacga tcgtttgtaa tgagaatgct ttgctttaaa taaatgacta aatagctaga 132240agcctagttc taggggatag gcacgtcttt cttctctcaa gaaaatagaa aggcaattct 132300aatttctagt aacagcaaac agcattaagt catggtccaa atatgaggca aaccaaaatg 132360tggcttgatt gttcagcagt tgatctgttg gaagcccttg atattaaaaa ggttctcctt 132420taagcggctt aggagtcacg atcaaagacc tatagaaaga gatgccatcc ttctaggatc 132480cttggctctc ttgggaacta gattcagata gtcataatgt aaatactgct tgagctttct 132540ttctttcttt ctttctttct tttttttttt gagacagagt ttcactcttg ttgcccatcc 132600tggagtgcaa tggtgccatc tcggctcacc gcaacctctg cctcccaggt tcaagcaatt 132660ctcctgcctc agcctcccga gtagctggga ttacgggcat gcaccaccac gcctggctaa 132720ttttttgtat ttttagtaga gacagggttt ctccatgttg aggctggtct cgaactcctg 132780acctcaggtg atccacccgc ctcggcctcc caaagtgctg ggattacagg tgtgagccac 132840cgcacccggc ccgagctttc atttttgaaa tcaatgtatg actgaaacac tgaagactta 132900ctgacttaat tatggtttca gaacagaatg aaaatgtctt cggttctgat gaatataaaa 132960ggaaaactaa ccaagttaat ttggcaagta gatggtagag atagaggtgg ggagtggaag 133020gggaactaaa atcttcacct agcattgttg ggattatatg gttacatcat ctgaagttga 133080cagaccaaaa tatagaggct tcagaggtct ccaaatagaa ctaaacatgt aattcagatt 133140gttaggaggt agtataaatg agctaaatct catctttatt acggtagagt taatgggtga 133200tgtctaaagt tgtctgaagt ctataaatca tgacaaatta tgatgtggtg attgtattca 133260acagtctttc agttgcaggg ataaaacccc agtttaaact agagtaagag aaagaatgtg 133320ttggtttaag ctcctggaaa gtgcaggcaa gggtagttgg taggactgca tctagtgttg 133380taattctgtg gtctgcattg tatatttatg catctcagct ctgctttctt cttttcattt 133440atataatttt taaattttat tttaaagata gggtctcact ttgtcgccta ggctgaagtg 133500cagtggcatg aagtgcagtg cgaggctcac tctagcctcg aactcctggg ctctagagtt 133560cttcctgcct cagccttcta agtagctgag acaataggca tgtaccaaca tgcctggata 133620ggttttaaaa tttttttgta gaaatggaag tcttgctgtg ttgcccaggc gggtctttaa 133680ctcttagctt caggcgatcc tcctgcctct gcctcccaaa atgctgaggt tataggtgtc 133740acccaccacg cccagtctca tctctgcttc ctgtgttagt tttgttctct ggtgggctgt 133800tttcacatga ccgaagatga cctctagcag gctgtgttct cagcccctca agtaggccta 133860tgtgattggc cttgcatgag taatatgggt gaccataaac ccctgaatgc tctggtccac 133920atgggccaaa tgggagactg gacagcattc cattgatgag gaggtggggc tggtctccgg 133980gagtaaggga gaggagcaca tgcagtaact gatggtctgc tgcaagggat agcagcacag 134040cagttagaat tttggaggta actaccagaa ctgaaaacag aaatgataac aagtagttgc 134100cttaaaaagg gatgggagca gggtgctttt gtgatcaaag ctcctttctc ttactggatt 134160tttgtacaca ttttgcatac atatcttaga gtaaaagata gcattttcag ccttggtcca 134220tttgaggata ctcttggcgt ggcccgcctc catgctagca ggctctggtt gtgccaagtt 134280cagttgagca tcctggctct tgcctgcacg gaacttccag tcagtgcgtc agtatcacaa 134340gtcttgatat ttcctatgaa gaagaacagt agtgcagtga cagacgaaat gggtgggcag 134400gcagaggcag gatttctgag ggagagaagt agctagcttt ttgcagagaa gagttccggc 134460acccaagaga gcagctgaga gtacaggcag gcaggcagga tgccggtagg gcccggccgc 134520acggcgccac agaatcctgg agaaaggggc ctcttcatgg cctctgcatt cagctgctgt 134580caccctccgc acaggccatg gccaaaattt aattttcata gtggactcta gtttttgagc 134640cttacttgct attattgaaa taattttctt gtttcttttt aaagatcttc ggattatgct 134700tcactgacca ctgtaataag tttaaagttg agaaaatatg gcttgttaat gaatgatagg 134760tcaattttag tatgttggtc attttaatat tttgccacca gttggtttgg atttgatgcc 134820aggaggagac agcctcattt ctaaggacta gtcttgcctt tgtgggataa gggtggtgtg 134880ttctgtgtcc ttctacatgt ccgagcgatc tctgtgcagc tcaaatgtgg tcactgtctt 134940attgcgctga tttcctctcc ttccatctca caattgaggc aaaatattgt tactgttgaa 135000gtgttgtcca ataggacttc cagcagagac aggatgtctg cactgtctaa tttagttgcc 135060tttagccaca tgtggtgttc tgtacctgaa atgtggctgg tctgattgga tagcttaatt 135120tataatttta tttaatttta attaacttaa atttaaacag ctctgtgtgg atagtggctc 135180ctgtatgaga cagtgcaggt ctgttgagaa gcagctttac tggtgggagt ggagggcttg 135240gagagggcac gtgggtttcc tgctggtatc ttttgacctt atttaatctg cccaacattt 135300gcaagtaagt tgtgtgtgtg tgtatatata aatgtgtgtt tctgtcttct tgtttccttt 135360gactgcattt atttgaaaga cactaggtgg cagaattact gtatttgatt ggtttcaaga 135420taagagttga aataattcat ctcgtgtttt tatataagta aggtgtgttt agcatgtaaa 135480attggtaata tgtattcacg tactgcttaa acaaaggcta

tgaattccac ccataaaccg 135540aaaatgaaga cctttaaatt tgtccatttc aggcgtgggt acttcttaaa taatacctgg 135600ttcaggaact agtcagaatg gcacccttga ctttttgttt cctgcttttc ctcttgttgg 135660gagaggaggg tattcatccc aaagtggttt gcctatttca cattccatct aggataagca 135720gaatagccaa gaaagatagc tgtcctcctg tttacaacat ttggggtaac cagcatccct 135780ctcttttggt ccaagataga ctggtttaga aacagatgat ggcaccagag gcccaggagg 135840tggaaacatc agctttgttt gttgtccatg tggctgaatt agagctgtct ggccttgtag 135900cctcaacacg gccttccagc tttgctcacc gtgattttca aggacacatc ttgtgctctt 135960ccctgcctgc catccagact atacccagtc agggtggcag gagctgctgc cccttcctcc 136020ctgagtcctg gtcgtgggtg gtggagatgt gccatgacgc tcacggaggc atgctcaccc 136080cttcctctgt ggcagagggg atggctgcac gacagctctt ccctgtcctt tccaaagcgt 136140ctgtggttcc actttttggg gcaaagcagg aatactggaa gagagagaaa gtggtccttt 136200ctatagtaat aaagttgaca ttgattcaag ttcatgcttg gggaaaggac agggctacta 136260acaattataa tgctgggagc aatggaattt tctcatgggt atgtggtagg tttaatttta 136320attatcccag ttaattctta gaactgctct gtgaagtatt tcccgctttg tgcttaagtt 136380ctaaaagatc ctgtgccaaa accaagaatg aaaacccaag cattctttct tgcccatcga 136440tctttctctc atcaggccac ttcttgggtt gatagtggtg agtgtagccg ctgccacttt 136500cagaataccc accatgggcc ccagtcactg tgtggcgtgg agaagagatg gttctctctg 136560tgtcatagct gaacaagccc agcccagaga ggtttctgcc ctaggagctc tcgatggtgg 136620aattgggatg cgatcccaca tcctgcctgt tttgaaaaca gcattcttta tttccaattc 136680ctgcttccat tgttcctttt aatatttctt tgtttagctc acaaaaacac ggcttgcgga 136740gctgctgcgt gcagctgtag ctgtttctct gggtgcagcc tgcatccgcc ttcctgcccg 136800cctcctttcc tgcactgcca tcgtggtctc cgggcacttg gtccctttct cttcccctga 136860gtccctttgg ctcccctgtg ccacccttgt gatccacagg ctctgccttc tttctgtctc 136920agactgctgc tcatcactac tcgggaccct aggaagggag gttccaccga gaagcatctt 136980ctcatctcag ccacgttctc agtgccactg ttgtctttgt taggtaatgg tagctactgt 137040aacaaataaa ccaacatttc catggcttca caccagagaa ggttgtttct tggttttatg 137100acaatgtatt gagggtgttc ttggttcacg gatggttttc ctccatgtgg gaattcgggg 137160acccaggctc ctttccttct tttggttctg ttctccaggc cttcacatcc tctgtgtctg 137220gttggggaca aggagaggga aggtaaagaa ggctttgtgg ccttggataa gtgacaggca 137280tgcctttgct ggtgttctct cgtggtgaca ggtcacagcc ccaccctgta aaaggggact 137340gagagacgtc gtcctgctgc ttcccagcag cagcactgtg gtctctgatg tgttttctgt 137400gaggataaaa acaggtgatt ccaggatgag gaaagtcagg gaaacccttg gaaggagggg 137460accaggcggg tgtcaccatg ggattagtgg tggcttcaga atgagctgca gcgagtgcca 137520tgccttctaa agcttttgct attctgatat gcccacacca tgcccagcag gtgtctgcct 137580tgctctccgc agagagagtg atgaatcctt ctcatgagcc tctgtccagt tgttcctccc 137640tccacctgga agggaccctg ggttcctcat aacatcccag cggaacaggg gaccttctat 137700cctgtcccca agttcatcct catcctcctg ccggcttcct ggcccctctt atgtctgctt 137760cctgacgcca catccttctg gattctctgg aattgaattt tgcctttgat gcttatttaa 137820aaatatccat tgcaggccag gtgtggtggc tcacacctgt aatcctgtgc actttgggaa 137880gccaaggtgg gcagattgct tgagcccagg agtttgagat tagcctgagc aacatgttga 137940aatcctgttt ctatagaaaa tacaaaaatt agctgggcat ggtggcgcac acctatactc 138000ccagctactc aggaacctga gacaggagga tcaattgagc cccggaggcc aaagctacag 138060tgggctgtga tcgtgccact gtactccagt ctggtcaaac agagtgagac cctgtctgaa 138120aaaaaaaaaa aaatccattg catacttcac cgtagcgaaa catgtatgtc ttacctttcc 138180tttcctgcct gtagctgctc ttttacactt aacagccaca ctaagccagc cttaaatgaa 138240aaacaaacca gcacttcctg tgccctcctg cttccttcat gaggggtccc tccctctgtg 138300tacactccat tctcattgcc catggtggtt tgtttccctc ttgtttctca agccatggca 138360gcctgcctct tgccctcttt actaaaaagg cctttgcaga ggctgcctgt gttctttctt 138420tctaggtctc tctcatccta ggccctccag cttgattctg tggagctgcc ctcttgtcac 138480tcagtagctt gtggggtctt ctctgtctag ccacttaatt gattgtgttc ctcgagttgc 138540tgtccatggt ctctcgttac tgttttctct gtgtttctgc ctctctcctt ggccttggta 138600ggtccatccc ctttgtgacc ttggctgttg ctctcatgga caactttctc ttgctggtcc 138660ttgtagtcct ggcatccagc ttctcgacac gggacttgtc ctgccagtac ctcagacttg 138720cacttaaaat tgaactagca ccactgtcac tctccagggc ctcttcttgt taattagatc 138780attagggatg ttcagaatcc cagcatcata gtatgttcct cctcccgcta ccccaggaac 138840cctaacctta cctcctcctc tctatctact aggaggtggc cctcagagtc cgtctcatct 138900tccacctgaa cttccctaat aggctccagc agctgccacc ccgggggctg agtacttcct 138960ccatgccttg tgcagtgctg agccctttac ctgggttctc ctgtttgctc cttattacag 139020ccctgcgaac agatactgct cttaattcca tcttacacct aaggaagctg aggccccagg 139080taaggtgcat ccaaggtcac ccaggtagta gacagtagag ccacgatctg aaccaggcag 139140tctgattcag agcctgtgtt gacactcagc cacctagaac acagcttgga ttgtgggttt 139200ctattacctg ttcaaaaccc ctacatcccg ggtctgtccc tgcacgtgct ctgtggcctg 139260gctgcatctt ccttgaaggc agtgcatgcc tcttcactca gggggcccat gcaggaacag 139320agggccccac agaaggatga ggccagtgca gaatgggctg gaggggacaa tgctgaccag 139380gaagcaagtg tagagaaatc ccaggaaacc tggaggagcc agagacaagg cattagaact 139440cctcgtcgtg acctggtctg cattctctga gtgtgctgct tctgttagct cgcttccttg 139500gtctcaggtt atagtttaag gcattgtgga gccctaaaaa gcctgtactc tgtttttacc 139560tgttttagga ccctttcact ttggggatgt gttgattttt tttttttttt tttttttttt 139620tttgagatag agtctcgctc cattgcccag gctagagtgc agtggcacga tcttggccac 139680tgctgcccct gcctcctggg ttcaagcaat tcttgtgctc ccgcctccca aatacctggg 139740attacaggca cccgccacca cactcggcca atttttgtat ttttagtgga gacagggttt 139800taccatgttg gtcaggctgg tctcgaactc ctgacctcaa gtgatctgcc caccttggcc 139860tcccaaagtg ctgtgattat aggcgtgagc caccacaccc ggcctgaaat ttaaatcaga 139920aataaaattt tgatcccaac agtgatgcca ggcagcccag atctggggga gagggtggcc 139980ttggccagct gggcctttct ctgtttccca agtcttgctg cctctccctg ctgggctttg 140040cagcctgtgc atgtctctgt gcctttgacc ttgtttatcc aaaggagagg atagaatgaa 140100gtcatgattc ctggagccct gagaaggatg ctgtggagaa atttgccggt agaatctagc 140160tgagtgtgtt gctgaggtgc cagcattgtg tgtggggagg ctgaccgctt ggcctgccta 140220ggcccaggat gctccatggc cgggcacaga ggccacttgg ctgtcaggtg tcaggagcct 140280gcagagggca cacagagcct ggaccgcagg ggggtcctgc tttctcacct ggcctccttc 140340agcatttctg tccctcagtc cttagcaagc ccaggagctg ttgagtttgg caggtgccga 140400gtgctgttcc tgcctgtgta gctgtggctc agtcctgtgg gggccccgct gtggcccgag 140460tgcagtgatt cgaggcgctg agtgttccct gactccttct ccaggagctg tgttcagact 140520ttcgcagctc ttggcttgga gctcctggag ggcttggcat tgccgaccaa tgtggaggtc 140580gacagtgaga gaggaggaat gctagctttc ttgaccagtc cattaaataa gtgggatatt 140640ggccaggcac ggcggctcac gccttaatcc cagcactttg ggaggctgag gcgggtggat 140700cacgagctca ggagttcaag accagcctgg ccaacatggt gaaaccccct ctatactaaa 140760aatacaaata ttagctgggc gtggtggcag gcgcctgtaa tcctagctac ttgggaggct 140820gaggcaggag aacagcttga aaccggaagg tggagtttgc agtgagccaa gattgcgcca 140880ctgcactcca acctgggcaa caagagcaaa actctatctc aaaaaaaaaa aaaaaagtag 140940gatatctgtt tctgcttaga aaaatcagaa ttttctaaat gccaggtgtt ctgaatacgt 141000aagtatggga gacgactcag cctgtttcat ttttatgtaa aatcttcgcg tagccatgtg 141060gcactggacc gagatgaaag caaagacatt tctccttaac tttgtttcta ggaatgttcc 141120ggagaatcac agcagctgcc actaggctgt tccgcagtga tggctgtggc ggcagtttct 141180acaccctgga cagcttgaac ttgcgggctc gttccatgat caccacccac ccggccctgg 141240tgctgctctg gtgtcagata ctgctgcttg tcaaccacac cgactaccgc tggtgggcag 141300aagtgcagca gaccccgaag taggttcata atgccccaca gcccagggcg ccagcccagc 141360accctgtcct gagactccca gtaacctgag ctttggccac cgttaaagca ttttcatttt 141420ccattttttg tgagggcttg tgaaatttct gctgcatatt aatattcctt tcatggacag 141480catattattg ggacaaacat gcggtccagc taaaggcatt caaaatagca gttgctttct 141540aaatgcgatt ttctttggca ggttctttga caccattgca tcttgtggga tatgcttgtc 141600atgctctgtg gctcctacta agttctagtc cttaaattgg ttccatagcc agacatgttg 141660caatgtctta acctcattat aaagtaaatg tggttctggt tatccttaga taatgaagta 141720acagtgtagc aaatttcaaa acctcttgga aatgttattt taccattcaa aaaggcttac 141780taaggttctc gttatgggtg gccctctttt tgcaaaaggt tttcaggctt aagctccatt 141840tctaggtgct ccaacactcc attatttgta tatgtatgga aataaaagct gtgaccaccc 141900ccaaccctgg cccccgccca gctgaatcct cagcacagta tttctggaag gctcaagatc 141960ccacgctggg gaaaagaagt tctggagaca aaagagggca ggtgctgccg tgcctctctg 142020ctcagtatgg atactggacc ttgtgctgcc agggctccca gtagggccag ttcatggcac 142080tcagctggaa agtccactgt tgggaggcat tcttaaccat ccactctgtg ccgtatgtag 142140tggggtctgg tcattctgtt ggaggagaca gaccagtgac gacatttgaa atgcttggtg 142200gatgtcttag gcctgttacg atgactgagc actgtggggg caggagacag aaagtcagtg 142260tctcctagtt ctgtgctgct ttaacgtgca tagaaatcag ctgcggattc agcagatcac 142320tccttttctg acagatgggc ctgcttactc tgatgttata tcagaaagct ctgaatctgg 142380gaattgtgtc ccctgaattg gagtaacaga aatgcttaga tgatgagtgt ttaaaagaaa 142440taaaccaaag gtaaatttag tttggaattc agcaagcgtc ttcattcagc cctctgaggg 142500caaactacag ctttttgtaa atgtaggtaa attctgtgac tgtttcgtga ccccctctga 142560tccagttttc ctttataacc ttctgtattg ttccttctat tatcctgaaa taacattaat 142620agattaggct gggcgtggtg gctcatgcct ataatcccag caccttggga agccaaggcg 142680ggcagatcac ctgaggccag gacttcgaga ccagcctggc caacatgatg aaatgctgtc 142740tctactgaaa ataacaaaaa ttagccgagc atggtgacag gtgcctgtag tccctgctac 142800tcagaaggct gaggcgggag aatcgcttga acctaggagg aaaaggttgc agtgagctga 142860gatcgcgcca ctgcactcta gcctgggtga cagagtgaga ctccatctca aaaaaaaaaa 142920aaaaaaaaaa aaattaatgg atcaatggat ttttaaccta ataattaaat ttcaaaaaat 142980atcgttcttt aatggtaatg taaaggtaaa attaagataa tatgtaacaa gcatgtgagt 143040gtctaaggtg tccccgtggt ggaaggaaaa aataaatccc cataagtgtc caagatgccc 143100atagagagca gagctgttct ggtttaaacc cctgctctta gcactgtgtt tttccagctg 143160tgggtggtgg gggatgagta tctttttatt tccatgagat gagaaaaatg aattactaga 143220agtgtgaaat acaaaacaca gctgctcttt ttttagccat agactcagca gccataaaat 143280tgctgtatcc agttgcagaa attcctgctg cttactcttg accctctctc ggtttgtgtg 143340catctcctct caggctggct cccagatggg agctggctcc aggcgacact gggtgctctg 143400ctccaggagg tccttatgtg ggtcctgccc tagcctagcc cctctcttat ggactctgtc 143460actgtgggtt tatgattcac tctcaatctg tcttacctct tggtgaactg ttagagtcct 143520gcctatactt tggcgcttgt gggtgtgttg tggtacacat gatgtgttgg tcacttccca 143580gctcatcttg ttctgagtca ccctagattt gggacattca ttcgccacca gtaccgggcg 143640gtgtatggcc tgagatttgg gggggcttgt gctgctacaa attggggctg aatttgagtt 143700gacagtggac cttctttatg tctactgctc atatttgaat tgcaaatact gcctcttctc 143760tttcagaggc tcattaccct atagctgtat tattgcaaag tgcacaatta cagcttgagt 143820gtaagtcaca ctgcgctggc aggacggccc actgagaaag ggcacgtttc ctgttcgtta 143880gttttcacat tgacacataa tttacaatac agtaaaatgt acttttctat caactgtagt 143940cagtaacagc ccccctcccc caaccacatc aagatataga ggagtgctgt cacttcaaac 144000agttccctct tcctctgcca catcctgccc ctccccaggt ctaaccacca atccgtgctc 144060tgtccctctg ttcagcccat tgcagaaggc catagaaata gaatctatag gctaggtgtg 144120gtggctcatg cctgtaatcc cagtattttg agaggctgaa gtgggaggat gacttgaggc 144180tgggagttca agactagcct gggctgccta gcaagacccc atctccagaa aaaaaaaatt 144240taaaaattac aatcacgtcc ctgtagttca gctgcttggg aggctgaggc aggaggatca 144300cttgagctca ggagttagag gttacagtga gctatgatcg tgccactgtg ctccagccta 144360ggtgacacag caagacgttg tctctgggga aaaaagaaag aaacggaacc acgcggtgtg 144420cagccttctg agtctggccc ctttcggtga gcagtgtcta aagttctgtc gcgtgttgcc 144480cacgcgtcgg tggctcgctc cttgcaactg ctgagcattg tatggctagg ctgtagtttg 144540ttttcacttc accagttggg aaacagagaa aaggcacttt ttaaaaagtt taaatctgta 144600gaattttggt ttttaccagt tctcttctaa atcctgaggg attacaggaa aagttgttgt 144660atttcagaat attcttagct tgatgtgacc tctgtccccg ttaaggccct ttgccgcaat 144720gggaaggacg tcgctcggtc agaccctgaa ggtcagaggg gcagtttggg agtgtgtcaa 144780cattttaact gtatggacta gagccaagag tctcaaggtt tataattccc acgtattcaa 144840aaagaaaaaa acaataaagt gagaagtcag tgtagagtga aataacctgt gttagtgggg 144900aagaagtgtt tttaaacagg atttccataa cgtataacat caacatgttt agagtggtga 144960tgtttcattg ggaaacgaac agtaaaacat gaaagcaggg aggttttcat tctggcagtt 145020ggcaactttc acggcagatg gagaatttca aaagcaattg ctcaattatc aaacatagcc 145080agtgtgagtt ctgaaataaa ggtgctgatt gaatgtgcag ctttatggtg gattttgcta 145140ttcaggcaag cattttaatt ttctgcctgt taaattctgt tttctttagt ttttcatatg 145200tggtttattg tagcttagga atagataact gagagtatat attacacata caacattctg 145260atatggcaat atttaaaaca acttgtctgt tttagaacta gaattaaaca taatcatctt 145320cagtattttg caaataagct cactgccatc cagaaacatt gtcaatgcat ctgttgctcc 145380ttctagaaga cacagtctgt ccagcacaaa gttacttagt ccccagatgt ctggagaaga 145440ggaggattct gacttggcag ccaaacttgg aatgtgcaat agagaaatag tacgaagagg 145500ggctctcatt ctcttctgtg attatgtcgt aagtttgaaa tgcctgtaaa cggggttgag 145560ggaggtgggg accaggagaa catcctgtgt agatgacact tgcatggacc ctctggaacc 145620cagaccgccc ggtgtcctgc caagctccat cgaaactaaa tctagaatga atgtttactt 145680ctgctgtgac atataattgg agaccaggcc tggccttcca gtcactggat tctaagttgg 145740actgtgagag tttttgcagc tgactcattt atcaaatgcc cggctattgg ctcacgccta 145800catgatgctg ggtatgtttg ttaatttgag ggaagcaatg gaataataat aactaatgat 145860ttaaaaaaca aagtaagtgc attgactgta gtggggttct gattttaaat ttttttaaaa 145920attaatacca ggagcagtgg cttatgccta aattccagca actcgagagg ctgaggtagg 145980aagatcactt gagcccagga gtttgagaca agcctgggct atggtgtgag acacccatct 146040ctaaaaaaat aaaaaataaa aaattatcca agtgtggtgg ctcgtgcctg taatcacagc 146100tctttgagaa gctgagggcg gaggatggct tgagcctggg agttcgagac cagcctggca 146160acacagagaa accctgcctc taccaaaaaa agaaagagag gaagaaagaa aaattagcct 146220ggcgtggtgg tgcatgcctg tggtcccagc cacctgagag actgagaagg gaggattgct 146280tgagcccaga agtttgaggc tgcagtgagc tgtgactgtg tcactgcact ccggcctggg 146340tgacaaggcg agacccctgc tctaaaataa tttttttaag ttaatttgta gaaaaggtgt 146400tagatgttct ttgtcacatt ttatgatgga ttcctgttta aatgccgttc tctttaaaga 146460aaaaaaaata acttgtggga gtttttaacc ataaaactag catcacatat ttaccatgga 146520gaatttacaa aaaaacaaat aaacggagga aaataaaacc tcctgtaatc atactactca 146580gagataactt gctgttagat tttggtctag atttaatact ttttctatat ttatattaaa 146640aatatttaaa acatatgcat ttctttgtca caaacatggt atcttataga tactactgtc 146700acatagcaaa acagtgttaa atattctgaa tcagaaaagg aagccgactc tccaactgaa 146760agaggtgtta tcctagagac tttttctggt gatgacaatt tattaatagt cactttttgc 146820tttactttct ctattgaagt agtttttcta ttttgttcta cttttaagga taatataatt 146880tataatgctg tttttcacag aaatataaga aaaaagatac taattttata agttaataaa 146940gtttgatcat cccaaatcca aaaatctgaa atccaaaatg ctccaaattc tgaagctttt 147000tgagtgctga cattatgttc aaaggaaatg ttcattggaa ggtttcagat tttcggattt 147060agggagctca acaaataagt ataatgcaca tatttcaaaa cctgaaaaaa atcctaaatt 147120cagaatactt ctgatcccaa acatttcaga taagggttat tcaacctgta ctgtcagatg 147180atcccaaatg aaaaatatta atcgttaacc aaatatcaag gaattgatca cattttacag 147240tttctgccta ggattatgaa tcaagatgaa aaggctctgc atgtttaaaa atatatattt 147300ttattttctt ataaatctta aatatctaca cttaagattt atttgatatg tgggatccat 147360tcatattttg gattcaacag ttctgtcaaa actgtggcag tgatagggga ttcttttttt 147420cccactgaac tatcacaaaa ttggaaaaag agtaattgga gaaccccact ggcttagccg 147480gcccgaagcc cgggagaggg caggcagtgc tgtggatggg gtcatcccag cgcaacgctg 147540cccctgctac ctgcggatct cgctgaggcc tgcctttgtc ctttgaccct tggccatttg 147600ttagtgtctc tgagagctgg actgctgtac cctacttccc cagggggcct aacttcacac 147660agcctctgcc gcagtgcgtg gttggaggtg acggccttgg taaatcgagt ttcctacctc 147720ctcaattatt tgtgctcata cactgtatat ttttagtgag gtttatattt gggatgtgtt 147780ttctccttct taccctttct ggcctttcta tggcattaat acctggtctc ttcttgtgta 147840cttgaaaatg aatctctcat catatttttc cttagtgtca gaacctccat gactccgagc 147900acttaacgtg gctcattgta aatcacattc aagatctgat cagcctttcc cacgagcctc 147960cagtacagga cttcatcagt gccgttcatc ggaactctgc tgccagcggc ctgttcatcc 148020aggcaattca gtctcgttgt gaaaaccttt caactgtacg tcttcatcct gccgactatt 148080gccagttgca gttttccctg ccttaaaaat ggagtattga aatttttaac tttaatttct 148140gatttgcaaa atagtcatct tttgttcttt tccttcttgc tgttagccaa ccatgctgaa 148200gaaaactctt cagtgcttgg aggggatcca tctcagccag tcgggagctg tgctcacgct 148260gtatgtggac aggcttctgt gcaccccttt ccgtgtgctg gctcgcatgg tcgacatcct 148320tgcttgtcgc cgggtagaaa tgcttctggc tgcaaattta caggtattgg gaagagaaac 148380cctgatattg atttatattg aaaatttagc aggccaagca aaacaggtgg ctggcttttt 148440cctccgtaag tatggtcttg acatggtcac cgatagaaac atggaaacat ctgcaaactt 148500gccgttactc gtgtgtccga tctgactgtt tcttgtattt ttttctagtc tgcccttact 148560aggatgaact gtacacatca gttcatcctt tttaaatgag catgaggtta ttttgggttg 148620ttaggtgtta caaacacact aatgtgtttt tgtctattag agcagcatgg cccagttgcc 148680aatggaagaa ctcaacagaa tccaggaata ccttcagagc agcgggctcg ctcagaggta 148740atgctggaaa cacaggtcgt ccttgtgtta ggacaaccca ggatataaag gatatagatt 148800tgtacgggaa taaattcaca ggacaagaaa tcgatgtgcc ttataggtgg gtttactgca 148860gaagtgccat aatagaacct tcctactttt aaaacaacca gatctcactt tctaaagagt 148920aaaggatgac cggcaggatc acgtctgtga cgtgagtgga ggcagtttgc actcctggtg 148980gctgtttgag aggtagcatt tagaatgcct gtattcactg tcctgtgatg agtgggaaaa 149040taggttatca ggtttatctt agcaaaatca aagcatgtca tctaattgct aaacaagagt 149100tggcaaatct gagagacatt actcaatcct tggcatgcag gacttacatc tgcatcctgt 149160tgccatttta tgtcttcaaa gcatttaatc atttagttgt gtttgcaaag tctttgagaa 149220gcctttgtca gaaatcccta catctcctat gtgagtgtat ttccatgact gcagaataag 149280ttaaactttt acctttttcc ttcccttgcg gggcggggtg gggggcaggg attgtgtgtg 149340tgagagggag agagagacag cagagaagga gaatataatt atcatgctgt gtactttgag 149400ctgaaactgc aaaaaaggaa aaacacacaa aaattattat gcttttcagt ctttagagta 149460ccttgtctat tatgcttttc agtctttaga gtaccttgtt gatggtgttt ttaaatggga 149520ttgggcacaa ttaggtggac agtttgggat gatttttcag tctgtagggc caagctcttt 149580tgtaatttgc attatgaagt tgtcactctc atagcagatg gcgggagata aactattatt 149640actttttgac cctagactta gtcttcagtc cagatgaggg agattaaaag attataaata 149700tcttgtgcca gatgaggtga ttttattttg aaatgaccat gaattcctat cagttgtctt 149760actgggatat ttgatagtgg aatttgtgca tttgagtctt agatgatctg ttttacattt 149820attaagaaag cctttattag cttttatact gtgtattgcc tgttgcagtg tttgagtata 149880aatgaaattt ctggaaaata ttaatggagt acaaactgtg atacttaaaa gtaaactagg 149940gcctgcattt gtatcatgac ctgtttgagt attgatgaga agatagctgt gaagaaaaag 150000gtttaaacaa gtgtattttc ctttaagaag ccactaatag tgcatctcct tagagtgtat 150060atttctagaa tcctagtgtg cagagtttag actaagacta aaaaaaaaaa aaaacaaatt 150120atactgtaat ttcattttta tttgtatttt agacaccaaa ggctctattc cctgctggac 150180aggtttcgtc tctccaccat gcaagactca cttagtccct ctcctccagt ctcttcccac 150240ccgctggacg gggatgggca cgtgtcactg gaaacagtga gtccggacaa agtaagtgtc 150300cagcgtgtct gcatgggagg cacagggcgc tgagtgcctc tgtcacctgt ggcagataca 150360gagagtgcag aggaggtgcc gtggacccaa ggagttctgg cgctcggctc ggctcagtga 150420agctgtggtt agagacgtgg ggggccatca aggtctgagg gagccaagca gtgctgatgt 150480gggacccttt tggtaggagt gtggggtgag tagttagtgg gtgaatcaag gaatagtcgg 150540ccgtggcctg caggcccctg actgcacagg ccttcaagca

catgtcaatg ccgttagcct 150600ccctccatct cctcatacct tctggccacc tgtgagttgc actgccactg ccagccattc 150660tggtatgttg tcagcacctc cactgctcat acctcatggt tagggaccac ctggagcctt 150720ggtagagcct tggtagagcc ttggtactct actttcctgg acaaagttca gcttatgaat 150780atgaatttag atttcaaaaa ccagcagccc aagtataaga aagcgaaggt tcagtcctgc 150840cttcttaggc tctattcgct aagcacctgc cctgccctgg ttgctgggga gagatgagta 150900aagcagacaa cccaggagag gatggcaaag gggccgctaa cccttagtgg tttagctata 150960tttggaaggc ctattggaag ttcaccaggt gaagggggag gctgtgaggg tgcccaggca 151020ggtaacagaa gtccaaaggg gaaaacctgt ggtgtggtga gccgtatagc cacagcctgc 151080cggccggcag ccctctcagc ctagtgcggt gttcccaagc actggcctag gcctgtagct 151140ccagggatgt gaagtcccct tgaacgccgc ccatcatgtt ccccttatcc atttttttct 151200tcccaggact ggtacgttca tcttgtcaaa tcccagtgtt ggaccaggtc agattctgca 151260ctgctggaag gtgcagagct ggtgaatcgg attcctgctg aagatatgaa tgccttcatg 151320atgaactcgg tacgggggga gcagtggagg caaggaatcc tcagcttttc ttgtgacttc 151380caagtgggat ttgtctcatc atcatgtgac ccacttgttg acaacacatg ttggggactc 151440cagtctgggc agggacggga tgtcggagag actccactct gaatggggcc gggaagtggg 151500gaggactcca tttcagatgg ggtcgggaca tgggggttat gctgatcgag acagaaaagc 151560acattgtttc agccacatta gaatccacgg aggtgttgtt ttgaaatcca gctggcccca 151620aggctgggtg tatggtttgg gatgagaact atctggcctc cactggagga acaaacacag 151680gatgttatca tctaagctcc atggccaaga cagaatggaa gtcaaggttg cgtatttgcc 151740gtagacttca acacagtgtc gtaatgcgtg acgtcaataa cttgtttcta gtgtcttgga 151800agttgatctt tagtcgtaaa agagaccctt ggatgcagcg agatttcctc tactcacacc 151860tctgttagat gtagtgaggt tcttcacccc ccaaccccag atgtcagagg gcaccctgcg 151920cagagctagg aggccatgca aagccttggt gtccctgtcc ctcacccgtg ggcaggtcct 151980gtgagcagtg ggggggccac ctcttgggta tggtgcagcc atggcccaag cagggcttct 152040tctcagacct actaggacgg gagaaacctc ctggtgcttt agccctgcgt tgatatgcag 152100caaatgggag ggaagtgggc acctgggagg acaaatgcct gtagaggccg ggagtgacgg 152160caggtgttca tgaaaagaga ccttgtgggg agggcaacac aacagtgtgt tctgatgtac 152220tgaagagctc aactgaaaac aacaggagaa ttagcccaaa atccatttac taaaattgtt 152280tatctttttt tttttttttg agacaaagtc tcgctgttgt cccccaggct ggagtgcaat 152340ggcgctatct tggctcactg caacctccgc ctcctgggtt catacgattc tcctgcctca 152400gcctcccaaa tagctggtat taacaggcat gcaccaccac gcccggctaa tttttgtatt 152460tttagtagag acgggatttc accatgttgg ccaggctggt ctcaaactcc tgacctcagg 152520tgatccgccc acctcggcct cccaaagtgc tgggattata ggcctgagcc accacgcccg 152580gcctaaaatt gtttatctta agattcatgc agtgaaagct aacttactga gtgataaatt 152640tgcttagtga tctgtttatt aggttttcca aatttgctaa ttgggctttg aacagctgta 152700aaagttctga ctgtaaaaga aagcttcaac ttttggcatt catgatgctt ttctgagtat 152760taaactaaga tagatgtttt acctgaagga tcggccacca atctttaaat ggctaaacaa 152820aagggttgct aaaacataat ccaaattgac ataagaaata ccatttttcc aaccaaaatt 152880ttggcattca tatggctact tttacgtatt tcagctgcat ttgaacatct ttttcaaact 152940ttagggtggt tggtgtatca ctgaggtctt ggatgacact ttagctttga ttttgttttt 153000atgaattaaa attgtcatac caaaattttt atttcaagca aatccaagag cataaaaaat 153060taaaatatta cttaaaatac taagagagaa cagatatata ttttactaag catatgttga 153120atgaaattgt tcaaatattt ataacaggca tagagtagaa ttttcttaaa aatatttttg 153180atggtatacc aatttgtatt ttctcagaaa catttgcctt attctttttt ctgttgtgtt 153240tttcttacct gattgaaagc tcataatctg ttgttattgt ttgttaacct ttaatgctct 153300gatttcagga gttcaaccta agcctgctag ctccatgctt aagcctaggg atgagtgaaa 153360tttctggtgg ccagaagagt gccctttttg aagcagcccg tgaggtgact ctggcccgtg 153420tgagcggcac cgtgcagcag ctccctgctg tccatcatgt cttccagccc gagctgcctg 153480cagagccggc ggcctactgg agcaagttga atgatctgtt tggtaattaa aattaaaatt 153540tatcttattt ttaaaaagca ttccagggcc agtatagtac tttgcaccaa gtaaatgtac 153600aataaaggca gtggatctaa tacattgaaa gcgtttacag aggtagctaa agagcagcac 153660gggtgtcctc ggctcagaat ttcttcctgt gtgtttgcca ctttgccatt cattgacatg 153720gtcatggaca tagggctcta agcccttgag gaaggctggg ccagacctca ggggagatgc 153780agccccaaac cacgtgcagt cctgtggacg gatgtgtaga tgtgccactg aggaacaatg 153840tcttgagctt tcatcagatt ctcagagaat tgcttgactg cctttcgaag ttgatgcatc 153900tgtgctcacg tttgcaccca cccacgaggt ccttctgttt caggggatgc tgcactgtat 153960cagtccctgc ccactctggc ccgggccctg gcacagtacc tggtggtggt ctccaaactg 154020cccagtcatt tgcaccttcc tcctgagaaa gagaaggaca ttgtgaaatt cgtggtggca 154080acccttgagg taagaggcag ctcgggagct cagtgttgct gtggggaggg ggcatggggc 154140tgacactgaa gagggtaaag cagttttatt tgaaaagcaa gatctctgac cagtccagtc 154200acttttccat ctcagcctgg cagtaagtct tgtcaccgtc aagttattgt agccatcctt 154260caccctcacc tcgccactcc tcatggtggc ctgtgaggtc agccaggtcc ccttctcatc 154320tgcacctacc atgttaggtg gatcctaatt ttagagacat gaaaaataat catctggaag 154380tactttatgt cttaagttgg cctggacatg tcagccaagg aatacttact tggtttgtgt 154440tagtgcttgt aattcgcccc cagaatgtgt acacgttctg gatgcattaa agtctggcct 154500gtatccttaa agggccatcg ctgtgctgcc tgccctcagc aaggacacac tttgcagacc 154560cacagaggct ccgcctccac ctcacaccaa agaaagggag gagtccaaag ggcatcagtg 154620ccattactca caaaatgata aatacaccct tattctgaac cacgtggagt catatggttt 154680gtgatccctg tccttcaggt ttcagcttag tggggaagtg ggaaagtcag cgtgtgatca 154740cagcacaggg tgattgctgc tgattatatt atgtgcctgc tgtatgcagg atgaaatact 154800ttatatgcgt catcttattt gactctcaca accccctgtg agataggctc tgttactccc 154860atttgacagg tgaggaaagc aaggcttaga gaatttcagt gacttgccca ggtcctctga 154920gctaggaagt agccattctg gcatttgaac ccaaggcctg ctatccctag aacccacgct 154980ctcaaattca acctatgaca gaggcaagcc ctggtgctgt gggagcccca aggaagagcc 155040tctggcctgg tggccacgta gcccaggaga gatttctaca ggagcccaca gcgctgaagg 155100agagagaggc agcagagtaa gggggctttg tggcagagag gggactggca ctttggggaa 155160taggtgggtc aggactgaat gtaatggagc catgtcagag ctgtccttct ggaagggcaa 155220gggcacctgg acgcgctgcc cctcagtgct ttggacggtt ccacaactgt gattcacacg 155280gcttccccaa acgaaggtac acgagtgggc attctgtgac tcggtacttc cctttaggcc 155340ctgtcctggc atttgatcca tgagcagatc ccgctgagtc tggatctcca ggcagggctg 155400gactgctgct gcctggccct gcagctgcct ggcctctgga gcgtggtctc ctccacagag 155460tttgtgaccc acgcctgctc cctcatctac tgtgtgcact tcatcctgga ggccggtgag 155520tccccgtcca tgaacggtgg gttcctatca tagttcctgt ctgcttcacc atgtttttat 155580tttgtgctgc ctgtttgcca ggtactaagc taggaattgg ggatggagag gtagataaaa 155640tatgcatcag gaagggctgg gccccatctc ttactctcca atatattgga gtctacactg 155700gaatttaact ggaatttgct tttttagtca ttttatttag attttgaagt ttcagctttc 155760atcaaaaata cctctaaact ttatgtctct gtgatctttg gtcttagctg ttttatgtat 155820ttagtcttat atgatcataa gattaataac attacattca gaagattatt tgttttctgt 155880cagagttaaa atgtttgttt ttatactgca ttgtaatatt aacgtactgt aaaataaaag 155940tggcttgttc ttttcaagga acagtatcct caacaagggt cattagccac aatttttaaa 156000aaattggacg tcatagttta catgttagag ggcgttttga agctttgtat ttttaaatta 156060aatgttatag agtgatgttt tcatgtttca taattgtttt catctgtgca tttgtagcca 156120acttgaaaac aaagatccag ggattactac ttaaaagcca gacttcttgg aggttatagt 156180gatgattttg atagtatctt gagccgtctc ataataacct cagggtgaga gatggccaac 156240aggagacagt cgagggactt agaaatctga atgaaatctg aagttcaaat cttcagacat 156300ataccactaa ccaagagatt ggtacctcag tctagtattg tctgtttgtc taaaattggt 156360tctaaggaat ctaggctagt ctgtctatcc ctttcaactt ttgtgaggct gcacaaatgt 156420aaaatgttga ataaaaagca ctgatggaag tgtgtagaaa ttcttctctt tgttctgttg 156480taattttagt tgcagtgcag cctggagagc agcttcttag tccagaaaga aggacaaata 156540ccccaaaagc catcagcgag gaggaggagg aagtagatcc aaacacacag agtaagtctc 156600aggacccatt tttttcttac atgttgttcc tccaggactt aaaaatcatt cacagagacg 156660tgcaccgcgg tgagtgtgga ctcctggaag cgcaccgtag ctccgctgtg tcctgctgct 156720cctccctagc tgtcagggag gctgtagtcc attgctttgc cagctctttt gtttccgagt 156780gaacacctta tccgtacaca tgcggctgtc tctgacccta cagaccagct gggatgccac 156840tgggggagcg ctcccttccc cccgcacttc ccacactctg cagttattct gagatccttg 156900agggcaggga acaggtttgt cttctttgtg ttctcagaaa ttaatgctcg gcctctggtc 156960agcaagcaac aaccttttgt tgagtgataa tgaataaata aatgtttccc acatgagtat 157020tcagtaacct cagtgtcagg ttcagccatc tgttttggtg gatatttaaa agaaaattcc 157080gcttttccta cagaaaaaaa aaaaaatcca aatcccagtg atttaagcca gttatagact 157140tagacatata ctacggcttt tcatgcactt tcctcccaat tctagagtag gtattttact 157200aggaaaatgg tggcagtgcc tgttgggagg aagattcttt ggccaagtgt cttttgttct 157260tgccagggcc cctaggctgc tggggtgctt cagcttcttt agcccagtgt ctggtgggga 157320atggcccctg ttgcctgtcc cacagaggtg ggggtgcctc acctggagcc tgtccacaca 157380ttttacacag cacgcttacc tggagcatca ggcatctttt ccatgctctg tggctcagga 157440aacacgcctt ttcaatcatg agtgcaccag tgcttttggg ctttttctcc ccgcttttgt 157500gcaatcctgg ttgtggatgg agttttcctg tctttagtct tctgcatagt acttttctct 157560tctggttccc ggttcaaggt tttgtaatta gagaatgacc cagaagcaat ggcattttaa 157620tgcacagcca aggacttctc tgaatttgta tctcaaacct ctgtgggtcc ttcaggcttc 157680agtttgtgat ttcatgattt cttgttgcta cctaaggaat atgaaaacac ccacctccct 157740actctgcatc ttccagccga gtggcacctc aggctgtgga tcctgtgctt ctgtggtgag 157800gataagaata gtgccaaccg tgtggattga aatcaatcag ttaatccctc catgtaaagc 157860acctggaacg gatgacagtc ttgttatgaa tactcaacaa atgctatcat gatttttagt 157920tagatttcca ttgctttaaa acagttgaga catcttggcg gtttgagtta gagcaacggg 157980ccctgaagtg ggttctgttt gggtgaagat gattatgctt attccccatg gccctcttta 158040ggcaagagtg ggaagctttc tttgtttttt taatcacctc gataggacgt tacttcttaa 158100aggtcatcca ataaatatta ataggccggg cgcggtggct cacgcctgta atcccagcac 158160tttgggaggc cgaggcgggc ggatcacgag gtcaggagat cgagaccatc ccagctaaaa 158220cggtgaaacc ccgtctctac taaaaataca aaaaattagc cgggcgtagt ggcgggcgcc 158280tgtagtccca gctacttggg aggctgaggc aggagaatgg cgtgaacccg ggaggcggag 158340cttgcagtga gccgagatcc cgccactgca ctccagcctg ggcgacagag caagactccg 158400tctcaaaaaa aaaaaaaaat attaataaag ccaactcgtt agcgtggggc ttaattgctt 158460aagtccaatg agaagtcctt ctctatccta ggaagttgcc caaactgtag aatctcgtgg 158520cctgtgggta atagccacgt aatacacact cactgcctca acaaatcata ttttagtagg 158580tatgatattc tagactcaag acaccattct gtggatcttc ccaagggtgt gaagtgtcca 158640cagcgtctgc cttgggagtt tccatgccca ccagaaccat gccccaagcc cctcaagcac 158700tctgacctag gaaagccagt gaagcaagga tgacaacatg gccctttgat actagctgag 158760ggacagacac aggtcctggg agaccagaga aagacgaggg gcagaggagg tgtcctaaag 158820gaagtctgag gctgaggagc cacaggatgg cttccagctg tcacaggctg ctgctggcct 158880tatcacagag agtgggccag agggctggga accaaggcca gagctcaggt tcaggaccat 158940tccagcaatc ccagcagaaa atggggagaa ttgtatggta taggcggata tgaaggtaga 159000atctgcaggc cttcagtggc caactcagag tctaagtgga ttccacagtt acagcttgag 159060cagctggttg taggtcatgc tttctacact gggcatatag gatgtgtttt ttaaaaagtc 159120ctctcttaac cgttgcttgt ttagatccta agtatatcac tgcagcctgt gagatggtgg 159180cagaaatggt ggagtctctg cagtcggtgt tggccttggg tcataaaagg aatagcggcg 159240tgccggcgtt tctcacgcca ttgctaagga acatcatcat cagcctggcc cgcctgcccc 159300ttgtcaacag ctacacacgt gtgcccccac tggtgagtct gctcgttcct tgcagaagac 159360caagtacggt gaaaggcacc ggtaggccct gggctgggca cacgtgagag ggcgggacag 159420aatccccgca gcccagaggc tgcctgctgt ggttctggtg cccactgtgg ttctggtgcc 159480aggctgcttt cctcaggcac cacgtgtgga ggtcgctagt agaaatactg ggttttctaa 159540aatgaactga ggccctacat ccctaagaga ttagtgttag acctgattct agagcaacta 159600gaccactttg cttaatagca gaccagaaac cacaccccct cgagtgagtg agattttcct 159660ttggagataa ttcatgtttt tctacacagt tttgcagttg tcttcagaat tggtttaaag 159720taggtgttat tgccaggcgc agtagctcat gcctgtaatc ccagcacttt gggaagccaa 159780ggtgggcgga tcacttgagg tcaggatttc gagaccagcc tggccaacat ggtgaaaccc 159840catctctact aaaaatataa aaattagcca ggtgtggtgg tgtacgcctg taatcccagc 159900tactcaggag actgagacag gagaatcgct tgaacccagg aggcgaaggt tgcagtaagc 159960cgagatcgcg ccactgcact ctagcctggg caacagagca agactccgtc tcaaaaaaaa 160020aaaaggtagg tgttattgat cagaaccctt gtttcagata acatgaggag cttagcttga 160080ggagagtgag ggttgatgga gggggactga cttctgccca gtgaaatggc atcatctccc 160140accagcccgc tgaaataaga tgatggggcc tgttccttag ggcctgcagc atcctcaggc 160200aggaaagaaa ggccgacctg gcagggtgtg agccagcagg tgtaggtcag ggagaatgga 160260gccaggtccc agggaagagg cttgtggctg cctgagaagg gtgcgtgcct gcctgtgtgt 160320gtgtgtgcac gtgtgtgtat gtatgctgga gagtctaggg aggcttgctc caaggacgca 160380gtattgtttg atcctgagag ataaggattc tgccgcaggg aatgaaggta ttccagatgg 160440cgggcttatt ccgaagaaga ggccagtgcc tggcggtgct ggaagcagtt gcagaacagg 160500gagttgtagg ctttcctggg aagagagcag caggggtgct ggagaagcag gccacacttg 160560ctgcatgggg ttgctctcgg ccccactctt ggtgcacagc gagtcactgt gggttcatta 160620gcatctggtt atgagacagt aactgctcct ttggaggggc tcgtggagac catgcaggag 160680ggcacggtct tgaggtcatg ccgtccagag cacacctgag gataggccag gacgggctgc 160740acgctgtagg taaaattcct ccagcaagct cttcactggc attgaggagt tccctgagtg 160800cggtcatctg gaaggcagct gtaacaggca ctgcagtctc tccctgggtg ggtaccagag 160860aggagcatag gggagcataa ccgatttaaa gagagggctt tcctgtggtg aggtaagaga 160920ttagctggtc attatcatag agccccctct gcctttgtgc agatgggctg tgggaatcct 160980ggggttccgt tgggtccttt gtcacctcac tgaaggcatg taagctgagc tggccagacc 161040gtgagctgat cctgccactt gaacagcatc aagcctgcct ctggattctt ctgtgcatgg 161100cacttgtctg agcacctcac gcacagagaa ctggacttca gagtttacag aaataagctg 161160tatggttcat tttcatgcct gcttgccaat aaacatatct gagctgaacc tcattgaacg 161220cctgccttta ttctagcaca gcacctgctg tttgtgggcg aggggtgctg tctctaactc 161280ctgcctgctt ctcccagcac tccctgagtg gggtgtgcca gcagcctcag gatgaggaca 161340ggaagtggga gggcagagca gatttgggag ggccacttga tggggaagga agtcccagga 161400agcagttgga gctgttttct gggggagaag gtgccagctc tgggacagtg ttggggtagt 161460gaggagggag cccagtggag agaagtcggg cttcctgctt cctcacagta tgtctgtcct 161520gactcaactc ggatgatgtc acttcctttt catcttctca ggtgtggaag cttggatggt 161580cacccaaacc gggaggggat tttggcacag cattccctga gatccccgtg gagttcctcc 161640aggaaaagga agtctttaag gagttcatct accgcatcaa cacactaggt actcttgggg 161700cctctccttc aggtcaccat tgtcggacat ctaccgggag gaaatccaga gcccccagta 161760ctgggatctt ctcatttgac tccagaaaag atttaagcat gataataata caaacctatg 161820tgaatacatt ttgcagtgtt ggcaaaactc cttttatact gagaaaatag atcccagttc 161880ctgtgttttg tggcttgaat cccagctttg tgtattccgg gcttgtttga agtcaggaaa 161940ggttcatgtg tagtggacaa cgtgagacca aattctgcct tagattttgc atttaggcta 162000aacagtggca gcacttgtct cagaatgttt tcttgtgttc accagtctga tcctgttgtg 162060tctcagtggt ccattttctc atatgggaac aagcagacgg gagcagatgg agtcaggttt 162120cttggcactc gccttcccca gagcctagag gcagcatggg gagaaagcag gcttggggct 162180cagacagtcc tggtctgctt ccagccctcc tacctgagca gcgcagggca agtccgtcta 162240acctctagag accctcagtt ttgtcatatg taaaatgggg gtcgtgtcta tttcatagaa 162300ttgttgcaga tttagaaatt acatttctaa acaaatgtta ccccttattt ctaaataagt 162360gtctaaatga ataagtcacc acttttgccc ctatttgatg gcaagaggtg tgatcttgtg 162420gtgggactgt aatcagtcag ttctcagtga ctgtgccctg ctgtggtgtt tcctggaatg 162480ttcctgtctt gtcctagaaa gtctggcagg ggcaccctga ctccactgtc cagtcctctc 162540cccagtccct cgggcttctg cagatttgag gcttgtttgg atcccagaag gttgtggcag 162600gagacacctt gcctctactt tcccctttat aattcaatgt ccaaagagag ccctgagcag 162660gtacctcacg ccagctgcct cacggagctc ctcctcttcc tggctgtgag gatcggtatc 162720agtggcctcc tgctctctcc cccttgccta acacgagcac ctttgcttac ttgggtgccc 162780ttgctcttga actgcccatc ggacgtgcgt gacccaagac tgtgccgcag tccttgcctt 162840gtctgtgctc attttctttg ttcatttttt tccctgtaac gtaaattgtt atatttgtct 162900gtatctgtgt ctgaatcagt cctgcacgct ctccttctct ctgtctcttg ttctttcttt 162960accccgttta tcacggggac cccgatgtcc attgctctag ttctcctgtc ctaagcaccc 163020catcccgtct ctctggcctt accacaagtg gcgtggctgc ctcagacatc atgatgggga 163080catgaagcac agctgtcaga aacaactgtt cgttagatac actcgaatgc agctcatcaa 163140tagggatgga gggtctgtcg gatgtatttt cactgaatcc ccgttcctac cttgatacac 163200tctttttaat ctattcttct agacaggtca gaggaaccat tactttgact tttaaatttt 163260tagcagcttt attgaggtag aattcacata ctacagattt cacccactct aagcggacag 163320cttggtggcc attagtttta tccacagagt tgtgcagcca gctgcacagt ctcagggctg 163380gactccaggg aagattttag cccatttagt gagtggggca gaagtggccc tggccctgca 163440cgaggttgcc tgcatgggcg tccctgccct gtccctgtgt ctgctccact gggggttgac 163500caggctgcca gggccgactt gggcctgtgc cacctgcctc tcatgtgtct cggacagtgc 163560agccgatgtc tatacttcgg tttcctcaat gatgaaatgg aggggatagt gttccccgca 163620tcatagaact gtgtgaggtt taagggactc actgcccttg gcgtggagcc ttctccaggg 163680gccgtgctgt gtcggcgtag ctgtcagctc tccgttacag gcttgagaag ggttgacact 163740ctctcatgta acatttatat ttctaggctg gaccagtcgt actcagtttg aagaaacttg 163800ggccaccctc cttggtgtcc tggtgacgca gcccctcgtg atggagcagg aggagagccc 163860accagaagta aggccacacc ctgtgctggt tggcacatgg gcagttatgg ccgcttgcag 163920gcctttggtg gggaataaaa taaggcagca agctggtgtt ctttttttct cttaccttat 163980ttttgaaaga gtagctgaat ggtgtcttga ctgatattcc agagcaggga caaagcctgc 164040tgaggtctgg gggctgcgat taccaatggc tggaatgcat tttattacgg tgcattccat 164100gttaaggatc aatacgattg tgccctttct ggaaaatatc ttttagttta tcaatattca 164160gaggagtgta ggttgaatta aaatgaaaag gcactttata aaggccatga gtagtacctg 164220gtttcatttt tctaatgtct tgcagagatt ttatcaggct tcttgaagtg ttcacgtaca 164280ttacgctaac acgatattaa taataactgt gctctggtac agcggagcca gcagaatggg 164340aagttgtgga atgcaggccc ttgattctga tagaaggtgt ggtttgaact cacagaaatg 164400acagtttgga gggtagacat atgtcacaag tcatcaagat tgtctttaaa ttcatgcata 164460gaagctaaca gggtgtcata agcaaggcct gtaaaatgta tgagggaatt caaagataat 164520ttattaaaaa gtaattcatg tttggagttt tgtgcccaaa ggagtccttg atttgaaaaa 164580tgggcttttg cccatcagat tgtttcaggg cccgtgtgtg cggaggccct gccttgtgcc 164640ccgtgagctc agcctgacag aaatcctttg gtagcactta aggctcctct tcctcccatt 164700gaggcaggga agactctggg ttctgcaggc agaggtggtt gtgggtgtct tgctgctctt 164760gttgacatgt gggctctcct tccaggaaga cacagagagg acccagatca acgtcctggc 164820cgtgcaggcc atcacctcac tggtgctcag tgcaatgact gtgcctgtgg ccggcaaccc 164880agctgtaagc tgcttggagc agcagccccg gaacaagcct ctgaaagctc tcgacaccag 164940gtttgcttga gttcccacgt gtctctggga catagcaggt gctggggaca gtgggttccc 165000cgctgaagcg tccagcagct tcaaccaggc cgttttcctt cattgctaga attgaaaaca 165060ccgtccgtgt ggcctgtgca ggagatgcag acccaaaggt ggcctcctgg tcagtgagaa 165120gctggaaacg tgacaggaac tgacgtgggg ttattgagca tttaggggaa gacgttagca 165180gagcaggaat gagcaggcaa ctagtagaac acccacttaa gggctcacgg acaggtgctc 165240acttaggaag tgagtttcat ttggtattac accaggttcc tttaggcaaa gcggagggaa 165300agttctggtg tttttcactt gtaagatttt gaaggaaaca aaacactctt tacctttttt 165360ctaaaatgta ggtttgggag gaagctgagc attatcagag ggattgtgga gcaagagatt 165420caagcaatgg tttcaaagag agagaatatt gccacccatc atttatatca ggcatgggat 165480cctgtccctt ctctgtctcc ggctactaca ggtacctgag ggaaagggtg cgggggagcg 165540gttgtacttg ggctagaatg agagaagact ggcatgctca ccacaccagt gatgcgggaa 165600gacctgagtg tggtctgagt tggaggctgt ggtgctaaat

acgctgcccc tttcataagc 165660aggagtctta gtcaggccca gggaggaagt aaaatctgga aatgaatgag aagcattctc 165720tcctgccagt caagaaatga gaagcgaaag aattctcacg ggctgtaaga ccagcaggat 165780ttaaaagttg aattagttgc ttatgttaag aactcaacca agttcatcta cacaagctga 165840atctccagct tttcctaaga aaccatgtgt ggcagtggct gcagggcagg gcacagctgg 165900gcctgagcac cccgctccct gcacctctcc cctccctggg ccctgcctgt cactgcccac 165960tctcccacca agccttccgg ttgtgtgcct gccctatcac aggcatcgga gcttgtcacc 166020tggtttaaaa gaagagagtt gtgtggggat ttgggatgca cgtttttcac tcaaaagtat 166080tttagcgtag agctctgtga ttccgtagct atttaggagt ttaagcacct tgaaggcttt 166140aattgcagaa agttctatgt ggacgtgcaa tgtgttatac gcagtgtcta tgagactcaa 166200atgtttatta gggcgttgaa gtaaactgag cacttggagg gccatggatc cagccttcaa 166260ggagctcata agtcaggagg acccaggagc aatgacctgt catagaaggc agaaaagagg 166320ggcacagagg tgggtgggag gcatacacag gcagctcctg gagctccaag gggagcaagt 166380gcttccaggg aagggggcgt ggaggcccct ttggaggagg caagttgatc tggggtctgg 166440cagagggtta gctggggaca tttagcggga ggctggtgcc cgggaattgg ggggatgccc 166500agcagaaaga catgaggagg ctggcctggg gcgtgggggg gtgtgaaagg ttaagtgggg 166560gcattatcct gctcccgctc ctgccggctg tatctggtca gcctgggcac cgaggtgggg 166620ttctggaagg cactgttcac caaaatgctt atctgggtcc cccagagagc ttgcctgcct 166680ggactgtcgg ctcgcctgca actgctgact cctaagcttt tgcagctcag cccacaacca 166740gttcctattc acagaggtgg gagctgaggg gtgacaagtg actgctgcag tcttatttgt 166800catagagaaa aagtgacaga gtccagcttg cccactggcc ctgccagctt aactggttat 166860aaagtgacaa atccccaaga cccacagggc tctgcacaac ctgggccctc ctgccagtgg 166920cggcgagggc aggtggctca cggctgggtg cctgtctggg caggagctgg gctggtatgg 166980ggtgggcctg cggccctgcc cccctgtgca gatcaagact cagggtgctg gtgttcacag 167040gtgccctcat cagccacgag aagctgctgc tacagatcaa ccccgagcgg gagctgggga 167100gcatgagcta caaactcggc caggtcagtc tcgcgccccc gccgcctggc ctctgtccgt 167160ttctgtcctc agactttggc gcttgacaca cccaggagaa aagctcagtg cactttttaa 167220atgaaaggaa gttttccttt tttttaaaaa aaaatttaat gttcattgtt tttatctgtt 167280ttattcctag gtcccgcaag cagaggaagc attagttttg tttttattta tgttctgtat 167340tccagaaagt agttaagaga cctcacatgt agcgatagag atgtgtgtaa gagacagtga 167400gagggcgtga cttggactta agcaaggacc gtgagacaca aaaagggggg tgaggacaga 167460gtggagtcag ctgaaatgct caggaggaag tagacgccat gaagggccat ggtatggggg 167520gccgcaggcg tggccgtgag tgtccctggg gccagctctt ggggggctcc ctgagtgtcc 167580ctgtccctgt ggccagttct gggtgggagc cccgtgtgca ggcagacagc tcggccactt 167640cctagcaggt cacattggtc tgtgcttctg tttcctcctc agataagtga agggattcaa 167700gggtctgggt gtggtggcta acacctgtaa tctataacat tttaggaggc tgaggcagga 167760ggcttacctg agctcaggag gttgaggctg cagtgagcca tgattgcacc actgcactcc 167820agcctgggca acagaccagt actctgtccc ttaaaaaaaa atgtaaacag aaacgtaggg 167880ccatttgcat atgatggcac atggcgtgga gccctacagg tgtatgctgg gcggggcccg 167940gctgtgctgg ccgacttgca cctttccctc caccccggtg ctgtgtcttt cgctcaccgg 168000gttcctgatt tagtgaaagc agttgtgcag gacagttctc tttgtagctt ttgtttctgt 168060ggaaatgggt cagaatatgg tgtttagaaa cacttatgag ctctgagagt ttcctcttct 168120gagttcctgg cctgcagcct tcacagcaga aaccctgtga tgtcacaagc ctgtttctgt 168180tccctgctct ctgcctgtac tgtcctgttt tgtgcctgcc ggtttcagtg acaggaagca 168240gggagctact ggaccagcct gtatttttct agacatagtt ggaaaaagaa gtcccactct 168300tctgtccttt cacctttgac agatgtttcc accccaagat aagtgaaaat gaccaatagg 168360atgcactgta tttttcatga aagtgtttct gaagggcagg ctgagagtga gaggcctggg 168420gctcactggg tgcctctggc cttgtcctgg gcccagggac actggtctgt gcccgaggta 168480ttccctatcc ccccaacccc gctgcatttg gccacatcct tcaatgtttg cgttgtgtcc 168540agcgtccgca aaccaactgt catgggatca tactggggct gaagtacggt cccacccctg 168600ccctgtctgg ggctgaagta cagtgccacc cctgccctgt ctggggctga aggacagtgc 168660cacccctgcc ctgtctgggg ctgaagtaca gtgccacccc tgccctgtct ggggctgaag 168720gacagtgcca ccccttccct gtctggggct gaaggacagt gccacccctg ccctgtctgg 168780ggctgaagga cagtgccacc cctgccctgt ctggggctga aggacagtgc cacccctgcc 168840ctgtctgggg ctgaaggaca gtgccacccc tgccctgtct ggggctgaag gacagtgcca 168900cccctgccct gtctggggct gaaggacagt gccacccctg ccctgtctgg ggctgaagga 168960cagtgccacc cctgccctgt ctggggctga aggacagtgc cacccctgcc ctgtctgggg 169020ctgaaggaca gtgccacccc tgccctgtct ggggctgaag gacagtgcca cccctgccct 169080gtctggggct gaaggacagt gccacccctg ccctgtctgg ggctgaagga cagtgccacc 169140cctgccctgt ctggggctga aggacagtgc cacccctgcc ctgtctgggg ctgaaggaca 169200gtgccacccc tgccctgtct gggatgttta gcccctagat gccactggac tgagccgcta 169260cttgcttttg ggaaagaggg gtgggggtta ggggtctggg cgaggggagt gcaggggctc 169320ctccttggcc tgagagctgt tcatacagac tcctcgccca ctccctgcag ggtgctgggt 169380cccagggggg aaatggccct tggtgccaag aacgtgagtt ggggctagtg ccagtgatga 169440tggagaacag ctttttatgg gcacacagcc cacagcactg tgccaagtgc tcgaggcttc 169500ccgagaacca ggcagaaagg aggacagtcg aggtgtgctg actgcgtggt ggctgcgtga 169560tctagagcgc gggtcacaaa ggcgcgaggg agctctggcc ttgggtttac cgcaatgact 169620gccagtgcgg gagactggaa aaggaatctc acgtattggt tccgtgtttt ggggactcca 169680ttcagatgtc acttaggagt gaaagcatcc cttcgtagag cctctttctg tgtcaccctc 169740ctcagctgct cctggggttg actggcccct gattcatgcc tttagcatgt gctggagctt 169800cccagcagct gtccagcccc tgccccaccc tctctgtggg ctcccttgcc cgtaacctgg 169860ggtgtctgaa cgacccttgc taaggggcag actgttagac ggtaggcatg tgctgagtcc 169920cagtggccac acccacccac caggagcctg gcactgtggc cgcagcactg agcagtgccc 169980cgtttctgtg gcaggtgtcc atacactccg tgtggctggg gaacagcatc acacccctga 170040gggaggagga atgggacgag gaagaggagg aggaggccga cgcccctgca ccttcgtcac 170100cacccacgtc tccagtcaac tccaggtttt ccaatggcct ttttcttttt aacagaaatt 170160tgaaatttct tatcagtcat ttgatttgtt tgaggtgctt cttgaaatga gcctctcatc 170220tcatgtactt ggaaaatacc catctcgcat attccacagg aaacaccggg ctggagttga 170280catccactcc tgttcgcagt ttttgcttga gttgtacagc cgctggatcc tgccgtccag 170340ctcagccagg aggaccccgg ccatcctgat cagtgaggtg gtcagatccg taagtgagcc 170400ttcccattcc cctcacacct gcacgtgcca cacgcaccac acacgccaca caccccacac 170460acacacaccg cccacacaca tgccacttgc acacacaccc ctcatgcatg caacacacac 170520acaggccaca cgcaccatag acaccacaca cacatgccac atgcacacac atacacggca 170580tgcaccatac acacaacaca cacagcacac atgccacaca cacacgccac accacatgca 170640ccacacacat gccacatgca cacacactcc acatgcatgc accacacaca cacacacaca 170700ccacacacac cacatgcacc acaccacaca ggttacatgc acacaacaca cacatgccac 170760gtgcacacac cccacacacc acatgtatgt gccacacaca gcacacaacc acacacatgc 170820accacacaca tgccacatgt gcatgcacca gacacatggc acacactaca cacacgccac 170880gtgcacacac cccacacaca tgtacgcacc acacacatgc cacacacaca tgcaccacac 170940acatgccaca tgtacacaca tgtatataca caccccacac cacacacaca ccacttgcac 171000accacgcaca cacaccacat gcgcacacac acaccacata cgccacatgt acacaccata 171060cacacaccat acatgcacca cgtgtaccac gcacccacac agacacagca cacgcataca 171120ccacacacac acgcacacat gcgtcccgca cagtaatgtc tcttgggtgt aagaacacga 171180cttgccagta gtagcgttct ggatgcgttg cctggattct aacagcgcga ttctcccctt 171240gccctcctgg ttttccacat ctccagcttc tagtggtctc agacttgttc accgagcgca 171300accagtttga gctgatgtat gtgacgctga cagaactgcg aagggtgcac ccttcagaag 171360acgagatcct cgctcagtac ctggtgcctg ccacctgcaa ggcagctgcc gtccttggga 171420tggtaagtga caggtggcac agaggtttct gtgctgaagc cacgggggcc catctgcctt 171480gggacctggt gttggccaga ggtgccgggt gcggctgcct ccttccaaga gttgacccga 171540accggactcc acggcccacg tgagctgcag tgcttctcag atggaggggg ttcagcgacg 171600gtcagtgcca ttcacaggtc actgtgatgt gggttgtggc ggccaagcca tggtttgggg 171660tcccgtatcc ctgggcttat gacatcattg tagtagccca tccccacaga accacggtgt 171720gtggtggcgc tgaggcatcg tagatggtgg aaatgctact ggcttcccca tgctctgccc 171780tgaggcctga ctgcctcact ccccttctca gttatgttcc aggccccccg agcttcctgg 171840ctggacagct tctctcctgg gggccgtttt gtcacagtga ccctgtgttt ctagtcccaa 171900atctgggtgc tatagtctct ttttagcgtg gtggttgtct tagtcttttt tggctgctac 171960cacaagttac cttagactgg gtaatttata aacagtggaa atttacttct caccgttctg 172020ggggctggaa gttttcatgg tcaaggtgcc agcagatttg gtgtgtgatg agggctgctc 172080tctgcttcat agatggcatc ttctggctgg gtcctcacgg tggaaggagt gaacaagctc 172140cctcaggcct tttagaaggg ccccaatcca caagggctct cccatcatga cctcatcacc 172200tcccaaggcc ccaccttctt gtactgtggc actgcaaatt aggtgtcagt gtaggagttt 172260caggagggat agaaacattc agaccatccc agcggtcaag tgttcatcct cttgagttcc 172320tccttattct gcttctggtt tatcaggatt cagccagtgc agcatggtac ctgtattctg 172380tggcacatca ccacatggta tttgccaagt atccatcacc tgcacacgtg aaatcattgc 172440ccgtgggtcc cgacatctgg cgaagcatat tcaaggatgg cagaactgtc agagctggca 172500cctctggttc cttgtcatgt ggcattacct agtaatccat tttatgatag caatggaaac 172560tcatttcttc aacaaacacc tgagtggctg ccgtgtgcca gccgtctggg gcccttggtg 172620agaatggcat ggtggtgccc atcagggcct gcctagcccg tgctctggac gggctcctgt 172680gtgtcaggaa cgacaatgct gtcatgacgg tgaatgattt ttttttttgc catcactcca 172740gccgctaaca tttgcggagc tcttcctccc gcacccccac ctgacaaggc caagggtgac 172800cttggcccca ccctaggcgg ccaaggtcag aggttagctg gcttgtctgg gtcacacaaa 172860atgcagcaga ggttgaggtg agcacatgtc cgtgacctgg agcctgactc cctctctgcg 172920agtcttgact gctcttgcct agactctgtc ctccccgagc ccaaacgcca gtcatcttcc 172980cttgtgggtg tccttcagcc tggtgccatg ctggtgactc agcagccgtc cagggagtgg 173040aaacaattga gtgtgtgggt tccctgtgtg ggcatctctc ttcacggcga acaccctctg 173100ggtgttgccc acacgatgtc aaagcggctc ttggaagggg tccttctcct ttgtgggaag 173160tttcagctgc tgggctaact tgaattgtaa ctgtggtttt gtgctcaggc ccagatcccc 173220ctaggcaagt gttgtgccat cagtaatcaa atgagaaata atcattttga aaagcagatc 173280ctaaggcagg atggtcatgg acactcactc ccagctcttt gtgcactcat gctttctgga 173340agatggccat cctctgtgaa ggttttcagc gcgtcatgct tggtacccac gtatccagag 173400catgtcgttt tgaggtattt gcccaccgtt gtgaaatccg tgccacccga gagcaggtcc 173460tgatgtgggg ctttcagaag tgggacctgg ggccgtacgc agtccttagg gaggggccgt 173520gtggcgttgt gcgtgtgagg ggatagcaca gggtgaggtg ggggcccaag aaggaagtga 173580cccacaaaga acagcctcct cttttggtcc ttgttcctgg gatggctggg agtggcttct 173640gtgtcgtccg gccatttccc ctgcggagag gctcctacca ctgccgagaa cctcatcatt 173700ccacaaaaac aagaggccgc ctggccatcc agcgctccat gggaattctg tgtccccata 173760gtcttgggct gaaggagggt gacattcctt gctgacttct gcaggggtct cctcactgtt 173820aaagagcaga ttgaaagtga agaacgtggg ctaagtgttt aggtcgatat ttaaccctgc 173880taggttttgg atactaagtg aaattgaggc cattttggtt gaagttgaca gaaaccacta 173940tcagggatcc ccaagactac cccaggcttt tctagaaaga ctctcagcta agatgtgtta 174000tggtaaaagc acacaaaaca aaatcagcaa agaaaattag caagggcaga ggcccatggg 174060gcgatgtccc gaggacacca ggcttgagct tccagaatcc tctcccagcg gggtcgtgca 174120ggacgcactt aactccccgc acagtgagcc gtgacagcgc gtgtgcagtg tcgtcgccag 174180gaaagcacac tagagactcg gtgccagggt ttttactggg ggctgggcac atgggcaccc 174240tctgcctgcc tcgtgcccag actctggact cccggaggga aggcaagttc tcagcaccaa 174300ccctggtgcc cacacaagca gctgagcaca gggagcccct cctcagtgag gatggtgggc 174360accgtcccaa caccagccag gggccagcct tgcacacagg cctctcagga tggtctccgg 174420cctgctgtgt agtctcttct gcacacaagc gtgagggcag cgcccccgcc tcggctgtgg 174480ggaggagcca ctgggacgtg agctctggtg gcatgcagca gcttttgtct gtgtgtgcct 174540aggacaaggc cgtggcggag cctgtcagcc gcctgctgga gagcacgctc aggagcagcc 174600acctgcccag cagggttgga gccctgcacg gcgtcctcta tgtgctggag tgcgacctgc 174660tggacgacac tgccaagcag ctcatcccgg tcatcagcga ctatctcctc tccaacctga 174720aagggatcgc ccagtgagtg ggagcctggc tggggctggg gcgggggtct cagaatgagc 174780tgtgaaggaa gcagcatcac cctctccaag tgcccaggct cctggccaga tggcaggcca 174840ggtatcagtg ggaacccagg tgggtgccat ggctgaggtc agtgagacgc aagagcacag 174900gtgcgtccta gaggcttcct cgggcacctc cagcgagctg gagctctcgc ctctgctgct 174960gtctcatgtg gcgcttagca cactctccca cgtgcccatt cctgactctg ctctcgaggc 175020catcggctct cattctctgc tcccagaacc ctgttattac ccaggctagc ctcctctctg 175080caccttcccc gccctggccc agtacctccc tcttgtttcc actgtgattc cgacctcacc 175140ttatcttaaa gctgctggac ggcaggttct gtacacacgt gtccttgaca aagcacggct 175200ggtgccgcaa cccctcagcg agcaagtcaa gctcttcaca gcgatgtctt acaagcgcag 175260agggctctgt gacaccctgg tctcaccgcc actcttccaa agtcgcagag gctttagcag 175320agatgggccc agcctctctg agtcataggc ttctgcacac gggagctgtc tttagaggga 175380gggtggaatt tcatcagcca cccacatggg ggagttgagg gcaagaatta ggagcaaaga 175440tgggaagggg tctgggagga atggccagtg atcccctttg acaagtgggc aggaaacggg 175500ggctaggtca aagttgagtg gaagacctgg agggagacgg gaaggtctct gtaggcacag 175560ttcagacagg agggaggtgt gagccagggc acatgccggt ggccgtctgg caggatttgg 175620gacatgctgg agcagggaca gcggctcatc aggggccatt gccctcatcc aggccagagt 175680gtcacaagcc cgtggggagg cccttctcgc ctgtcatcct tgctgggcag tgggtgctgt 175740gctagcagga caggcggacg gctggcaact gtctctgcat ccctggagcc tggcataggg 175800ccaagtcaca cggggcacag gcctgcaaat caggcacata tgttggtgca gtgacgtgat 175860tttggggggc agccccagaa caggccccag acacaggcca aagccctgcc tgtgctggtg 175920tgttgggctg ttctatggct cttgctgtgg gcatggagga ctcagggaag gagagttgag 175980gtggtccagg agttgcgttt gggatgcaga gagcttgtgg catccaggta gaaatggtgc 176040gtggggctga cctcagcacc atgggcagag gggccgtgtc acgtgcctcc gaggtggagg 176100tgggaccacg tggtgacaga tatacgcatc actgggcacg tttttgtggg tgttgggggg 176160catcgtattg gctcctctgt tcacagtggc cactcattca gtccctggct accaggtcct 176220cactgtgcca tggggaaggc cggcgctgtc gggggatcac agaaggcagc acgtcatgat 176280ggcatgtgcc atgaaggaaa agcacagggc actcaggaag tagaggggac tggcctgggg 176340tgtgggaatc tagggcctcg ttgagggaca gagagaggaa gtgtgtggtg gccagcatgg 176400aggtggccac aggggaggct gagttaggcc gagagggcag ggcgttgggg aggtagacgg 176460gctcagccac tcagggagtg gtcaagcaga ggctgaaggg tcaggccagg ttgcaggggc 176520ctgggggagc cactcagggt aggcgctccc gggagcccgc ctggcccata gctctacact 176580cccgcgtggg gccggacatg ctgtgaagcc ctctccacgt tggatggggg tggctgagcc 176640tggatgctgt ctcccgtttt cagctgcgtg aacattcaca gccagcagca cgtactggtc 176700atgtgtgcca ctgcgtttta cctcattgag aactatcctc tggacgtagg gccggaattt 176760tcagcatcaa taatacaggt gagtgggccc tggctgtctt cctctgcaca cggggagtgg 176820gcttcccttc tcttttcctt gcaggatcat accagtgggc cagttttgac ttggtcggga 176880ggaggcatga acacctgaga ctgtgcagcg attctttgac acagaggcct ttctccctgt 176940gcagatgtgt ggggtgatgc tgtctggaag tgaggagtcc accccctcca tcatttacca 177000ctgtgccctc agaggcctgg agcgcctcct gctctctgag cagctctccc gcctggatgc 177060agaatcgctg gtcaagctga gtgtggacag agtgaacgtg cacagcccgc accgggccat 177120ggcggctctg ggcctgatgc tcacctgcat gtacacaggt gagcatgtac acggtgccca 177180taaggccagc ccaagtcctg ttcaagggag gcaggagcat gctcactcaa gggacctcga 177240ctaggtgccc tctgatttca cacttctggt gttgccccaa gccggcccca tcaccttgca 177300agaaaggctc tggagccccc agggctggag tacctggtca gggttgaccg tccctgtggt 177360cactcatccc atgtggctga gctgggctgg gtcctgggca agcaaggggc tgatatcacc 177420tgctttcaga tctccaggga ctcactggac ccctgtgtac aaagcactgt ctacagagcc 177480tattgggttg tatagaggta accttcgtac tgaacacttt tgttacagga aaggagaaag 177540tcagtccggg tagaacttca gaccctaatc ctgcagcccc cgacagcgag tcagtgattg 177600ttgctatgga gcgggtatct gttctttttg ataggtaaga agcgaagccc catccctcag 177660ccgttagctt ccctagaact ttggcctgaa gctgtgcttt tgtgtgtgtc tgctgatccc 177720ctggcgctgt tgctggagtc ctgccagtga ttccccacca cagcctgacc atgggctgcc 177780ttggctcagg gttccactgg cgagctggtg gtccttggac cccagcactc aggtgtagcg 177840ttgaccagtt ccaaggttgt cccagtgcct gcccatctct cctgagggct cagggacagt 177900acctggcagt tgggggtgtg gcagggggca ggaatgacca gcctctggga gggtggggca 177960gaagcctgta cagtgaggag gagctggctc agcctggctg cctatcgtga gaggggagcc 178020cacggggctg tgggaggggg gccgtggtgc ctgtgagcag ggtgaggagc agcggcagga 178080ggatgaaggt ggaacccaca catgcatctt tgagacccgt gtggtcagtg gcttctgccc 178140cccaccaccc cccactgctg tgcgtgcata gaattggctt ccctcacctg ctctggaagt 178200gggttaggag cttggtaggg ctttttctca aggacaaggg cccctgattt gctctcaggc 178260ctcagtcctg gcgacatggt ggatctggag ccttgttgca ctgccttgcc tgtgctctcc 178320aatcagggtg gccagtgggg agccatttgg cttttctcaa gagcatactc aggtggacct 178380tgctccactg tttgaccaga tgaggcattc tgaacagcca agcctgtgct ggtctgtttt 178440catgttgatt tttttttttc ttttcttttt gagatggagt ttttcccttg tcacccaggc 178500tggagtgcaa tggtgtgatc tcggctcact gcaacctccg cctcccgggt tcaagtgatt 178560ctcctgcctc agcctcccta gtagctggga ttacaggcac acaccaccat gcccagctaa 178620tttttgtgtt tttagtagag acggggtttc accgtgttgg ctgggctggt ctcgaactcc 178680tgaactcaag tgatccaccc tccttggcct cccaaagtgc tgggattgca ggcgtgagcc 178740actgcgcccg gcccccatgt cgatttttaa atgcacctct gcatcgttct tcagtcccca 178800tatgctcact gagcaccact gcgactggca gacgggcaca gggaggcgcc acgaccagtc 178860ctggccttca aggggcttgt ggtctagtgg gcccaatgct aggtggcgag tgctccaaag 178920agtgtggtgc acgccttccg cttgaccgct ctccagacgc cacagggagg cacctcgcag 178980ctgaccacag atttctctct gtggagcagt gtcttcagag cggctgccat gccactgctg 179040ggcgagggtc tgcgggcggg tagagccagg agcacctgtg aggaagtgca ctgccatttt 179100cgtagctgct tcccgtgtgt ctcagttaca cacggctggc atgtgtgcac tgatgagacg 179160ggaacgtgat ggttgctttt cagcactgaa agggatactg ctcagggggc gtgtttcagg 179220atctggttag ggaagaagca gcgagagcac agatggggcc ctgtgtggta acaagaaaaa 179280agtcctggtt gacaacagtg ccacgaagcg ttagaacaca tagggatgtt tgtggagcat 179340ttgcatgtgg aaagcagcaa aaacataatg ggaacgggtt cttttgttat gatttttaaa 179400aatctctttt gtaacatcct tcccgctgcg ccgtttctgc atattccttt atgtagcttt 179460caaactcctc ttaggagttc tggtccctac agggcgtggg agcccaggct ttacgtagct 179520ttcaaactcc tcttaggagt tctggtccct acagggtgtg ggagcccagg gcctgtgccg 179580agcagcctgc ctccacgagc tagacagagg aagggctggg gttttgcctt tttagtctca 179640aaattcgtac tccagttgct taggctctga ctttccccac ttggaaagtc cctcacggcc 179700gagggtccct cccagccctg atttcacatc ggcattttcc ccagtattag agccaaggcc 179760ctccgcgggc aggtggggca gctgtgggag ctggtgccag tctctgacct gcgtccctcc 179820tcccaggatc aggaaaggct ttccttgtga agccagagtg gtggccagga tcctgcccca 179880gtttctagac gacttcttcc caccccagga catcatgaac aaagtcatcg gagagtttct 179940gtccaaccag cagccatacc cccagttcat ggccaccgtg gtgtataagg tgaggttgca 180000tgtgggatgg ggatggagtg ggaaagcctg gaggtggagt tgcctccgac ttcccagcag 180060attcgccagc agagcccagc tcctccgctt taaagcagca atgcctctgg cccccacccc 180120acccccgcca cccaggcgca gcaggtgctt cccgtccccc cagccctgac actcaggcac 180180ctgcttgctc cttgcaggtg tttcagactc tgcacagcac cgggcagtcg tccatggtcc 180240gggactgggt catgctgtcc ctctccaact tcacgcagag ggccccggtc gccatggcca 180300cgtggagcct ctcctgcttc tttgtcagcg cgtccaccag cccgtgggtc gcggcgatgt 180360atcctctctg ggtccctggt gctggccccg tttcccttgt caacaccgag gctcatgttt 180420catgataagg ttttgaaacc taacctttgc aaaaacccca cagatgccag ggtgacaggc 180480cctcagcccc agggaagtaa aatgctgaca ggggtacaga aaggagcacg tccagacatt 180540tgctgaccag ggcctctcag aggggccggt gtatggcagg agggtcgcag ctgaggggcc 180600tttctgtgga gggcctgggt gaggggagcg agggtgggcg gtggtctctg cagacgtccc 180660gcccactcgc gggctctgtg tggctgggct tctcctgaca

ctgcttctca ttagctttgg 180720tcattgtgcc tcgatcgccc tctcggggaa aggcttaagt aaagatccag ttcccacccc 180780cagatgctgg ctgccaggag tttccctttc cacagccctt ccccaagaca gaccacaaga 180840gcctccaagc agcacagttg tcctggtgct gacagcacag ccttgcccgg cgtgcctggc 180900acggctctgc cctcactgca ttggagcagg gctagtggag gccagcggaa gcaccggcca 180960ccagcgctgc acaggagcca ggccaggtga gtgctgccga gtgggtgccc tgcctgcagg 181020gcatccagcc agccaagggt tgcaggaatg gaggtggagg cgctgatgca gctggaggca 181080tccaggtggc ccttccgggg ctctgctcgc tctccaggct ccctggaccc ctttgtagac 181140tgtttcagga gaggaactcc caggtgagga cagggaggca gcattcccct catttgccgg 181200cctttttcct taactcctgc accagcctcc cacatgtcat cagcaggatg ggcaagctgg 181260agcaggtgga cgtgaacctt ttctgcctgg tcgccacaga cttctacaga caccagatag 181320aggaggagct cgaccgcagg gccttccagt ctgtgcttga ggtggttgca gccccaggaa 181380gcccatatca ccggctgctg acttgtttac gaaatgtcca caaggtcacc acctgctgag 181440cgccatggtg ggagagactg tgaggcggca gctggggccg gagcctttgg aagtctgcgc 181500ccttgtgccc tgcctccacc gagccagctt ggtccctatg ggcttccgca catgccgcgg 181560gcggccaggc aacgtgcgtg tctctgccat gtggcagaag tgctctttgt ggcagtggcc 181620aggcagggag tgtctgcagt cctggtgggg ctgagcctga ggccttccag aaagcaggag 181680cagctgtgct gcaccccatg tgggtgacca ggtcctttct cctgatagtc acctgctggt 181740tgttgccagg ttgcagctgc tcttgcatct gggccagaag tcctccctcc tgcaggctgg 181800ctgttggccc ctctgctgtc ctgcagtaga aggtgccgtg agcaggcttt gggaacactg 181860gcctgggtct ccctggtggg gtgtgcatgc cacgccccgt gtctggatgc acagatgcca 181920tggcctgtgc tgggccagtg gctgggggtg ctagacaccc ggcaccattc tcccttctct 181980cttttcttct caggatttaa aatttaatta tatcagtaaa gagattaatt ttaacgtaac 182040tctttctatg cccgtgtaaa gtatgtgaat cgcaaggcct gtgctgcatg cgacagcgtc 182100cggggtggtg gacagggccc ccggccacgc tccctctcct gtagccactg gcatagccct 182160cctgagcacc cgctgacatt tccgttgtac atgttcctgt ttatgcattc acaaggtgac 182220tgggatgtag agaggcgtta gtgggcaggt ggccacagca ggactgagga caggccccca 182280ttatcctagg ggtgcgctca cctgcagccc ctcctcctcg ggcacagacg actgtcgttc 182340tccacccacc agtcagggac agcagcctcc ctgtcactca gctgagaagg ccagccctcc 182400ctggctgtga gcagcctcca ctgtgtccag agacatgggc ctcccactcc tgttccttgc 182460tagccctggg gtggcgtctg cctaggagct ggctggcagg tgttgggacc tgctgctcca 182520tggatgcatg ccctaagagt gtcactgagc tgtgttttgt ctgagcctct ctcggtcaac 182580agcaaagctt ggtgtcttgg cactgttagt gacagagccc agcatccctt ctgcccccgt 182640tccagctgac atcttgcacg gtgacccctt ttagtcagga gagtgcagat ctgtgctcat 182700cggagactgc cccacggccc tgtcagagcc gccactccta tccccaggcc aggtccctgg 182760accagcctcc tgtttgcagg cccagaggag ccaagtcatt aaaatggaag tggattctgg 182820atggccgggc tgctgctgat gtaggagctg gatttgggag ctctgcttgc cgactggctg 182880tgagacgagg caggggctct gcttcctcag ccctagaggc gagccaggca aggttggcga 182940ctgtcatgtg gcttggtttg gtcatgcccg tcgatgtttt gggtattgaa tgtggtaagt 183000ggaggaaatg ttggaactct gtgcaggtgc tgccttgaga cccccaagct tccacctgtc 183060cctctcctat gtggcagctg gggagcagct gagatgtgga cttgtatgct gcccacatac 183120gtgaggggga gctgaaaggg agcccctcct ctgagcagcc tctgccaggc ctgtatgagg 183180cttttcccac cagctcccaa cagaggcctc ccccagccag gaccacctcg tcctcgtggc 183240ggggcagcag gagcggtaga aaggggtccg atgtttgagg aggcccttaa gggaagctac 183300tgaattataa cacgtaagaa aatcaccatt ccgtattggt tgggggctcc tgtttctcat 183360cctagctttt tcctggaaag cccgctagaa ggtttgggaa cgaggggaaa gttctcagaa 183420ctgttggctg ctccccaccc gcctcccgcc tcccccgcag gttatgtcag cagctctgag 183480acagcagtat cacaggccag atgttgttcc tggctagatg tttacatttg taagaaataa 183540cactgtgaat gtaaaacaga gccattccct tggaatgcat atcgctgggc tcaacataga 183600gtttgtcttc ctcttgttta cgacgtgatc taaaccagtc cttagcaagg ggctcagaac 183660accccgctct ggcagtaggt gtcccccacc cccaaagacc tgcctgtgtg ctccggagat 183720gaatatgagc tcattagtaa aaatgacttc acccacgcat atacataaag tatccatgca 183780tgtgcatata gacacatcta taattttaca cacacacctc tcaagacgga gatgcatggc 183840ctctaagagt gcccgtgtcg gttcttcctg gaagttgact ttccttagac ccgccaggtc 183900aagttagccg cgtgacggac atccaggcgt gggacgtggt cagggcaggg ctcattcatt 183960gcccactagg atcccactgg cgaagatggt ctccatatca gctctctgca gaagggagga 184020agactttatc atgttcctaa aaatctgtgg caagcaccca tcgtattatc caaattttgt 184080tgcaaatgtg attaatttgg ttgtcaagtt ttgggggtgg gctgtgggga gattgctttt 184140gttttcctgc tggtaatatc gggaaagatt ttaatgaaac cagggtagaa ttgtttggca 184200atgcactgaa gcgtgtttct ttcccaaaat gtgcctccct tccgctgcgg gcccagctga 184260gtctatgtag gtgatgtttc cagctgccaa gtgctctttg ttactgtcca ccctcatttc 184320tgccagcgca tgtgtccttt caaggggaaa atgtgaagct gaaccccctc cagacaccca 184380gaatgtagca tctgagaagg ccctgtgccc taaaggacac ccctcgcccc catcttcatg 184440gagggggtca tttcagagcc ctcggagcca atgaacagct cctcctcttg gagctgagat 184500gagccccacg tggagctcgg gacggatagt agacagcaat aactcggtgt gtggccgcct 184560ggcaggtgga acttcctccc gttgcggggt ggagtgaggt tagttctgtg tgtctggtgg 184620gtggagtcag gcttctcttg ctacctgtga gcatccttcc cagcagacat cctcatcggg 184680ctttgtccct cccccgcttc ctccctctgc ggggaggacc cgggaccaca gctgctggcc 184740agggtagact tggagctgtc ctccagaggg gtcacgtgta ggagtgagaa gaaggaagat 184800cttgagagct gctgagggac cttggagagc tcaggatggc tcagacgagg acactcgctt 184860gccgggcctg ggcctcctgg gaaggaggga gctgctcaga atgccgcatg acaactgaag 184920gcaacctgga aggttcaggg gccgctcttc ccccatgtgc ctgtcacgct ctggtgcagt 184980caaaggaacg ccttcccctc agttgtttct aagagcagag tctcccgctg caatctgggt 185040ggtaactgcc agccttggag gatcgtggcc aacgtggacc tgcctacgga gggtgggctc 185100tgacccaagt ggggcctcct tgtccaggtc tcactgcttt gcaccgtggt cagagggact 185160gtcagctgag cttgagctcc cctggagcca gcagggctgt gatgggcgag tcccggagcc 185220ccacccagac ctgaatgctt ctgagagcaa agggaaggac tgacgagaga tgtatattta 185280attttttaac tgctgcaaac attgtacatc caaattaaag gaaaaaaatg gaaaccatca 185340gttgttgctg tgtgaggctt gctttgcttc atgagaacct agaccttgct gagctggagt 185400cttaggaagc agtctcctaa gtgcttctcc agcaggggca gaaactgtcc caccagctaa 185460catctggcat tatggagggt cccccaggca gctgccagca gggacaggcc ccgtgttttc 185520tgtagccagg gatgaggaag tggccccagg gcatgggcct ggctgggtgc ttctgcaagg 185580gccttcccaa accacagtac aggtggtctt cctgccctgc agatgggagc tgtgggagct 185640gctggagctg ctggagcctt catggtcaag tgacatcata agcttatatg acatacacaa 185700gcctcaggac ttggcccatg gcactgaagc aggtcatcag gcccagcaca gagactagag 185760ctgtgttctc acagggccca ccacccttcc acctccttgg ccattgacac ctgcgtccct 185820ggcccagctg ctcccaggta acccccaaag cagctggcac atcccacctc tggtgtggcc 185880ggggctgctg tgtgtccgca gggcctgccc cgtctattct agcttgtttg tcctgtctga 185940accagcgcct actccaagaa gcctctgctc agcccagcgg ggatgcttct aagctccgga 186000cgagcctctc ggaagccttg gtgattggtg gtgtagtcat cttgggatgc agatgtctta 186060ccaacctgca agaacaaaaa ccctgtggct tcctctggtg cagggtattt agtcaatgtt 186120tgctgaggtc ccgtctggtt ctggctaatt ggcaggggtc gtccacccat tctttccctg 186180ctctgctgtc tgtgccagga gagacggggg ccagtcggcc aaggggccag ctcctgctgc 186240ctgctcctct tgggcacgtg cgggggcccc ctttctctga gcagggatag ggatcagtct 186300gccggaggga tgtggtggac aggcctaaag catttggggc ggggcatgcc acttgagctc 186360cctaaatctg tctcctcata ggtgacaccg ctccagggcc ccccagtggc ctctcctttc 186420agagctacct aaattctggt cacttcagag aaatggagca cccccttctc cctggtccag 186480gtgtggacag cctggcacac tgagcacacc tggcatggct ggtaatttca gaaagaagag 186540gggccggggt ccagtgggaa gcagcggtga acccctcgtg agtgggcttt gcagtccctc 186600cccatgccac ggcagagctg ccctcaacac agccttcctc ttcctcatcg gagagcacac 186660cctgtcccct tgccgagctg tgccctgtgc cttcggtggt atttgatttt ggctgctact 186720ggctttgttg ggatctggaa gtcgcttccc ctgcgtggtg cgtggagcac tgtaagtcag 186780atgagggaag tagccagggt gaggtgagta ccgggtggag ccgccactga agggactggg 186840taggggggcc ttgcctctac atgatgtgac acagccaacc gaggacagag gaagccccgt 186900tcctgggggt gtggggtgca cccctcaggg aagcctgcag tggggcctga ggaaaggcat 186960cctccgcgag cccacgagtc tggtccatga gcaccgtgac agtgtctgtg ggtagaggtg 187020gacccggcct tgtgtcatca ccaggacctc ttttgggaaa ccatgtggac atcgcttgcg 187080ggtcccccag gctctgcagc cccagcagcc tggctgcctt ttgggcaagt ggcttgagcc 187140acagaggacc cagtcctgtt gcagccacat cctctggggg ggcccgccag tgtggccggc 187200tttctccacc ctacaccagg cctccaggtg tcctggtcgg gggtgtctgg gccctgggtg 187260ggccctgtgg acctgtgagg tcagggtcag ggcatcactg gaggcagagg gctgaagttg 187320tgggtctggg ttccccttgt gtgcacaggc ccctgccctc catgcttggt caggcagcta 187380cccccaaaac tgctaggaca ggctggtcct gaggtggatc ctggcccctg taccctctgg 187440acagcccacc cgcccaacct tctaccctgc cccagcggcg gcagtgttgg ccacatcctt 187500cccctcctgg ccccaattgc tctggggaag tccaggctcc ggagcctgcc caggggcccc 187560ccgtgatttg ggcccaggac tccacgtggt tctctgcctt cacccaagcc ctgaactcct 187620cagctgccaa atccccaccc atctgcacag gctgtgctca ccactgctgc tcctggaagg 187680tgcccctcag tgggacgccc acctcctctc tgggcttctg tgtttgggag ccctgctgcc 187740cccacccttg gtcagtcccc atgtcctgct ggcctgtcag gcagggcaga aaatccaccc 187800agaaatgctg agcaggatga gagtctagtt gggcccagcc tcattattta gaagggatgg 187860aggcctaggg agcatgcttc tagcctgagc ccagcagggc cccgcccatg tcccaggtct 187920gcaccaggga cagctcctgc cgaggcctga cctgcccctt ctccctcagg tgctgctggt 187980tgaccagcct ctggccctag gagaccccgt agcgactgag ggtcccagca ggccatgcag 188040ctttgccaag gtacgagccc ctccccagca ggggacagat gtggggaccc tcccaggcag 188100gagcagctgg gtgcctggtg ctgccatctg ctgcctgcct ggttcttgtc ctcacattgg 188160aggtcagtgt gagggctctg cctcgggaaa ggccatggag cttgccctgt ccagggcctc 188220ccatgtgcac tgagcctggg aagagagggt tggagttgag ccttttaccc tgggaatgct 188280gcctggagga tggtgcgggt gtggggtggc accctgccag gcagggccct gcctccctgc 188340gcccactgga actcgggcag gcaggggtgt aggtgcctcc tctagagccg tccggtgggg 188400gcccccggca gtggtggtgg tgtccactgg ccagcagctg ccccttcagc caggacagta 188460ggcctgacgc tgtccccagc agctccaagg tggatttgtg gaagggggta gagggcacgt 188520agaggcccca tgacctcccc agggttctgg gagggctgtg cccccttagc cagcaccatg 188580ctgggtgata tagtcagatc ctgttacccc tgttgtggag gtgaggaaac aggttagtgg 188640ggaggacatg actaaggtcc atgctgagtc gctagagctg cacccagaac cactgctggg 188700accccatgcc tttctgctta ccccttgtgc cgggagatgc caagagatgc tgggagccag 188760ccccacctct gcccttggag tcatggctac ggaaagggca ttcggaccgg tccctgacct 188820caccggggag ggccgaaccc tgttcctgag gagccagggc ttcctagagg aggtaggcct 188880tctagtcact ccttcatctg caggcactcc acagagctct ctgtgccagc ccccagcacg 188940gagggctgac cttagtcgag tggagatgcc ccagtgccag gcagtaggga tgatgtctcc 189000tgaggcccag atggaaggga ctggactagt ctcatggggc tgatggtggg gccaggcctt 189060gaccagggac ccagtgtagg gggtgcagag acccctctga gttcctcaca catccctggg 189120gccctcccca tacacttcct atcctgactg cgggcaagag ggagccccag ttcgccttcc 189180ctatgctggg cacccacagt ggggctgggc acccccgcca tgcccctgcc ctgtccttcc 189240cctgagagcc tcggtcccac ctccaaggtg cctcagagga cagcaggggc agcgggcaga 189300ggccgagatg cctcctcatt ccaggctcag ctgcccttct tggggcagcc cacacctgag 189360agtctcctgc agttggtcag gcctgaggag ggcagggggg tgcctgctgt ccctctgctg 189420accacagtgg catttagcct gggcaccgcg cccagcacag tccatgctgc acaggtgccg 189480tgggctccac agagccctgc ctgacatgca tgtgttacgt ttcgggtgcc gatgcccttg 189540ggcggcactt ctccgggcag aacccccagg ccaccgctcc ggttccggtt ccgctgcatc 189600tggggctctc ggcaggctgt ggtcctccgg ccagcctggg ggcatctcag tccctcagcc 189660ccacaggggc ctgccccgca gcctgggcct cgagccccgt ctccgcacgc tgtgccgaat 189720ctggctgccc atcagctccc tgcgtaccca gactgtgccc tgccatgccc gtggctcttc 189780ccaggagtgc cctgtggcct ccccctggct tgctgggctg attccctcct gtgtctcaaa 189840cagagctcac ctttgccatc actgctgtcc tcaccggccg gtgccagagg cccgtgtctg 189900tgtaccctgt gtctgcacct ctgggcaggg cctggctctg accaacccgg gcttccagtg 189960tccacagacc taaggcccag ggcgcctggg ggctggagca agagaagcaa aaggagccaa 190020gggtgggggt ttggggttct tgtgagggcc cagccccagg accccaggac caggacaccc 190080aggagcccca gggcccagcc ccagttcaga aggcaggggc cttctgaggg agcttaaggg 190140tcccacagcc caggaccccc accagggcca gtggccagcg ttgggggact cagcctcctc 190200gtcgctcgtc ctctctgttt ctcccacctt ttgccccctt tctccttgcc tgttcccacc 190260cgaggccccc tcttggcctg cgtgagccgg ggcggcactg aactgggggc cgatccgcct 190320gggcggcggt gagaggcagg gccgggagcc gggccgctgg gtttgggcct ggcccgctcg 190380ccgcaatatt gatggcccgt cagtgcagcc ctgattcctg tgctttcagt taaaaggttt 190440ctgttgttgt agcttatgca gttgctctgt tgctatggaa acgtgacatc aaaatgacgt 190500ttcccgttta aaagctttta actaaattcc tgcctgtcag atgtaggccc cattttgagc 190560gtggagctgc cttcgagcga gcgtgagcgg cgcctcccgc ccatggtgcg tggggccggg 190620ccggggccct cgctgagcgc gctctctcac cccacaggcg cctccggcat ggcggcggcc 190680gaggggcccg gctacctcgt gtctccccag gcggagaagc accggcgggc ccgcaactgg 190740acggacgccg agatgcgcgg cctcatgctg gtctgggagg agttcttcga cgagctcaag 190800cagaccaagc gcaacgccaa ggtgtacgag aagatggcca gcaagctctt cgagatgacc 190860ggcgagcgca ggctgggcga ggagatcaag atcaagatca ccaacatgac cttccagtac 190920aggtgggcga gcgggcagtg tgggccccac caggacgggc gggcccgggc gtggcgggcc 190980gctcctgact ttcttggagc tctgagtcgg gacgatgtgt gggtcgtggc ctgcctgtcg 191040gtctcctctg gccgggtatg ggcagaaccc cacggggtga gacggggccc acggaaaccg 191100tgtgtgcagc cttccattgg ggaagtgggg aaactgaggc ccagcaaggg caggaaacca 191160gtctaagagc tgaggggtag caggggtggg gctggtgctg ggcagaggcc aggatggctc 191220ccaggacgta tgggcggtct gggcactgtc cctcggaggc agcaacactc atggtggtgc 191280ccactgacct cacaccctgc tcccccatag ggaggcggcg gctgccagtg ccctccccac 191340caccaagctc ccaagctcag caggggtttc aggggcctac tgcgtcattg gggaaattga 191400gactgcaagt gagaaggagg ctcagtgctc tgcgacttgg agcatccact gagcctctgc 191460catgagccgg tgagccccac tggggctggc cctagggtca cggtggggta tttccagaaa 191520tcaccaggtg aggtgcagga ccagccagcg catgggtggg gcttacggtg cgaagaagaa 191580agaggtggag gcctgccctg gcccaggact cccagcgtgg gggctcccgg cctggcccca 191640cctctgctcc tgctacatgg caggtgggcc cttcctgccc tggcaacctg cagggaaggc 191700cggaggggac cacccagcca gggagatgtt ggcgtctagg aggggacagg tgtggtccca 191760cacacccagc atcttaaagt gcgtgggtcc ccagcccatt aggacagggt cccgggtggg 191820caggggtcat ggtggggtga aggtctcagg cacaggcaag gtcacaggtg cggtgagggt 191880cttgcagggt gtgaaggtca taggtgtgcg gtgaaggtca caggtgtggg gtgatggttt 191940tgggtgtggg gagggtcttg cacggagcga gggtggcagc aagagctgga agctgcaggg 192000ggagaatggc agcagagagc acccggccct gtgggcggcc tggacagggc tgggcctggg 192060gctgccggag agcctgtcag cttccaggat gggagtggcc tcactcagct gctccacctc 192120cgggtcaggc aggtgagcct ggggcagaga ggctgagagc acctgagcca cttgtgggag 192180aggccacccc cactgccccc ctcaggcgag gagccggcct ccagcacagc agaagggaac 192240ccccagtccc cagccctagt gggagtgggg aagaggccca gcaaggcccc ggacagaccg 192300ccagcctgtg aggtctccgc tttcagttgc gttgatttga ttttttctga gccttgaagg 192360aggggtccgg ggcctggccc tgcccaaagg cccctaggca ggccccaaag ccgggaccta 192420gggtgctgag catgacggat gttgggtttg agcggctggc ttgcgacgtg agggctgagg 192480tgtgagcctg ggtatcttca gaggttcggt ggacacaggc agctgcccgc ggccccactg 192540ttcccgtggc ctcctagtcc tgctcaggca cctggtgagg aagggacgca gagggcagtg 192600ggaggtggcc acgactgttc cagcaggctc ccctctgact caggaattca cgggcaccac 192660ctccctggct ggctctggtt ggtgtctggc caggttattc attatttatg ctgaaagcct 192720cttcagagtc ccaggggagg gtttctgtct ccattcctgg aggctgagag atgagggtgc 192780agcagagtgg gggcctccac tccagaccct gcagtctggg ctggccaagg gctgcaccgg 192840tgcactgcac gtcatggctg atgaagcact tccacaccgc agcccctcag agctgccaca 192900gtcagcctta gttcaccgag ggggaagctg aggcccagag catgagaggg acttgcccag 192960ggccacatag tccttagcag aggaagctgt ggctgggtga ctcgatcttt gtcctttttc 193020tttatacccg cagtctcccc atagcagagg cttttctttt ttttttcttt ttcttttttt 193080tttttttaca agaactcttt atatattaag gctgttgggc tgaagaagcc tgagagggtg 193140gctggttctg tggagcatgg tttgttgaag tacagtttgg gggcctccta cactgagaat 193200aggccttttc tcgtttctcc aaagagtggg ctggctcaag tagggcagag agagaagcct 193260ggggcagagg ttagggatgg gcacccagcg cctgccctca cacgctctgt gctggtgtct 193320tcacagccac gtgccaccct gggcagcatc ccctgctcac catctggctg tgcctgtttg 193380ctgggggcac ctcattcaga atccagctta ttgtttccaa cggccaatgg ccacaccctg 193440gcaggtagca agagtaggag agaggagaca cccactccga gcacaggttg ggtttggagc 193500ccggccttgg ggcactctgt cactcaaagg cagagtgggg agtgggcact gggccttagg 193560aggtactggg tccagtgagg cagagatgcc cctgccccac ccccaccttg tggcttcttc 193620cctggcctgg ccagagctgt ctggccgcca tggggccctg tgtctcctgc cttgacctcc 193680cagagggcag ccgaggccca ggggaggcct ggggacttag cctctcaggg caggacctgt 193740ctgcaggagt aggtgggtgc tgggggtccc agtggtaatg aggcatcagg cagtgtggga 193800aggggcccat ccggcccacc ccagggcctc tgggcaggtt gcaggttgta gcgctggatc 193860taggctcctg cccagactgt aggttcaacc aagaatggca tgggagccca gcctgctgtt 193920tgctttatta aatctgccct gtagctgggg gaggggctta ctttgatcat cactatgtca 193980ttgatataaa aatagaggct cagagaggtg aatgaacctg cccaaagtca cacagcaaag 194040tgtggagatg agatactgac tcagggctgt ggacactgaa gcctgtgctc taacgccagt 194100ggctgtcgct ccctgaggca ttctctcccg aacaacacag ttattatatt acaaaatatt 194160atcactatat ttatatatct tataatacct tattattaca ataaaacctt attactctac 194220ctttcaaaat gaattattta aaaagcagta tttgctcatt gcagagagtc tagaaactat 194280agaaaagcaa gggaaaagca ataggaccag ccccaaggtc ccagcatgca cagataacct 194340tagtaatact gggacgtgtg cttccttttt aacatctgag cccgtgtagg tcctgaagcc 194400cagcttcttt ctaagtccat tgtcatcttg accctggagc ctggccgatt ttgctgggga 194460ggcccttgcc agccgagagc ggctcctgcc tgtgccggcg tggcgcgccc ctctgctgag 194520gctgggcagg acaggggctg ggccagctct gtttctcacc cttggctctt gtgtctctcg 194580tttcaggaaa ttaaaatgca tgacagatag cgagtccgcc ccgcccgact ggccctatta 194640cctagccatt gatgggattc tggccaaggt ccccgagtcc tgtgatggca aactgccgga 194700cagccagccg ccggggccct ccacgtccca gaccgaggcg tccctgtcgc cgcccgctaa 194760gtccacccct ctgtacttcc cgtataacca gtgctcctac gaaggccgct tcgaggatga 194820tcgctccgac agctcctcca gcttactgtc ccttaagttc aggtagtgtg tctgcttgtc 194880cttcccctgc cctggggtat ctcagccccc accatttaga gaaagggact gggagtggca 194940aggccggcgg cggcggccac agtggttgca gaggccgtgg ctgcgggcag cgcctccagg 195000gacaggcggc ctcagaccag ggagggcttt agtgtccaca ggcagaccga gtttgtctcc 195060cagctccatc acttttgagc tgcacggaaa gttccttgac ttctctggcc tcagtctccc 195120tcctataaaa tgggggtaaa tcagtacctt tctcagaggg tggctgggag catcacagga 195180gagaagacgc agcatggggc ccggcacacg gagggagacc aagccccaga ccccagaatg 195240cgccccctgg cctcccttag cccacacaga ccccaccctc acaggctagc tgccctctca 195300gcactgggga gggtgtcggg ctgcacctca tcacgtgttg ccgtgggcat gacccgtccc 195360ctctgccatc catcccacac ctcagacccg tcccgtgctg gccacgtgac tgtgcctgca 195420agatgctcac agggcagccg ggagccaggc agcatgcagg acagacacct gcggggtggg 195480cctggggagc ccagagaagg tgcttttgag gaggggacat ttggggtggg ctttcaaggt 195540aaaatagaag ttggccattt ggaggcaaga acaggaagat tgtggatttg agtcacagct 195600tctcccctgc cctggtcttc aagtctttct gacaggaggt gtcagaaaag tatctttagt 195660agagaaggcg tctccgagga gggtccctct catgccgggg gccgctgctt gactcaggat 195720ttctcattga agacctgaga caaaaacgct tttgctggca

gctagaagga accagcagga 195780ggcctgagat ttgtggctgt tgttcccgtg gactgagccc agttctcaga ctcagctgcc 195840tggggccttg cacaggactg gggcgtgggg gctgccctcc ctgatcaggc ccaaagcgcg 195900gatctcacgc ccctgaggtt ggctgtaccc tctcagctca gagcagagtg tgggccaggg 195960atgagcaggc actggagcag ggccctgggg tctgtgggtt ttggcagctc cctgcccttc 196020agggaggtct gctgagacca cgggtggccc ctaccccagc agcagagctc tcaggaggcg 196080cccacagggc tggactgcct ttactcacca cctctaccag agctctgagg tcctggggag 196140agagcccagg cctcttgtgg gccccacacc ctctaggtgc ctgtccttct gcctctctac 196200caaggtgtgc cggccccatt tctaggccgc cgggagataa gggggctcac atctcaggcc 196260cttccttctg ggacctcagt ttccccatct gcctaaggcc gggtggggct ggtggtcttg 196320gcttccctac aggggtcctg agtactctgc actacccagc accccccacc cctgccttca 196380tctctccctg ggggtggtct ctccacccct ggcccccaac tggggctgag cccccacctg 196440cccagtttgg tgggtgaagg gtgctccctg gcaggatatg cccctctgca gcccagaaca 196500tcccaccctt tccagaccga aggggtgtgg attgtcctgg gaccctggtc attggggtca 196560tccgctagtc gcaaaggacg gcaatgcctg tggcctctct ttctttcttt ttcttttttt 196620ttttttttga gacggagtct cgctcttgtg cagagagcag tggcgcgatc ttggctcact 196680gcaacctccg cctcgtgggt tcaagcgatt ctcctgcctc agcctcccga gtagctggga 196740ttacaggcac ccgccacaac gcctggctaa tttttgtatt tttagtagag atggggtttc 196800accatgttgg ccaggctggt cttgaactcc tgacctcagg tgatccacct gcctctgcct 196860cccaaagtgc tgggattaca ggcataagcc tccacacccg gccacccctg ttactttctg 196920tcaaaggcgg tgggttctgg cccctccttt gcacatggaa tatgagaccc tgagtaagtg 196980acctgactcc ctggggcctc agtttcccca tttgcccagt aggattgtcg ggagggtccg 197040gtgaggcccc tggtgtgccc aggctctgtg gccagcacgt ccacagccgg cactgtcctt 197100ccaggtcgga ggagcggccg gtgaagaagc gcaaggtgca gagctgccac ctgcagaaga 197160agcagctgcg gctgctggag gccatggtgg aggagcagcg ccggctgagc cgcgccgtgg 197220aggagacctg ccgcgaggtg cgccgcgtgc tggaccagca gcacatcctg caggtgcaga 197280gcctgcagct gcaggagcgc atgatgagtc tgctggagag gatcatcacc aagtccagcg 197340tctaggccag caggcggcgg cggcggcggg gccgggcggc tggtggtact gctcaggcca 197400cccagggcag gccactcagg ccaggcgggc aagggggccg ccccgcgagc ggagaccgcc 197460ttccacctgg cctctggcag gatgtccctt ctgaggggta ttttgaggaa cccccaggcc 197520ctggggaccg tgaggctcca gtctccagca tgaatgccct tcctcggaca caggccaggg 197580cctctggggt tcactccgag taagaacgtc ctagagccac tctccagtgt cgttactatc 197640aatgatactt gacgtggctt tgatattaaa cgtatacttt ttcattcttg cctggaacgc 197700acagtttgct gttgctggct tggtgaggat gccctgattg atggatcccg aaaatgaaag 197760cagatggaaa cgggttgggg caggctggag ctgggggagc tctctcctga agggaaccct 197820gtgtcctccc tcaccaggac ctctgcgtct ctccttaaat ggcctctgac gcctgatgaa 197880aaccccagcg accttccagg aggcttttat tcagctctgt ttggagcatc aggtgtttcc 197940actgcctcct tagcaatgac actaataaaa gtcgtaacac ctgttcacat gcacagccct 198000gttgagtgtt ctgggtgctg gagatatcat ggtggatgac acaaaggccc tggcctcttg 198060gagcttatgc tcccatgcgg ggaagacaca tgggtcagta gagaaatggt tgcaggttgt 198120gataagtgct ggaagggagg ggttggcctg aggacacgga ggcagacata cgtggagctg 198180ggaacagtgg ccacacaggg aacggccagt gcgaaggccc agaggcagag gacactggag 198240caagcccagg agcagctagg aggctggtgg ccagcagcca ggccacggaa gcccgtgcag 198300cccgtgggga ggagtgttca tgcttttcaa gcttagtggg agtcttttgg ccagtgcagc 198360tctgggtctg acatcggtgg gggacagagg ggtggtggag cggccacagc tgcaagctca 198420cctcactgcc ggcccttcca ccagtttcaa actctttcta gaagctccag ctttcccaaa 198480gctgaattct ctatgagcct ccttggccgg gactcgggcg tctggttgcc ctggctgcaa 198540aggaggctgg ggccaggtgt gtttgagtca cctcctggaa ttaggcaagt tgctgcccaa 198600atagaaggtt gttggcaggt gggtcagcag gtgaacagca tggtttgact cagggttcag 198660aaaaatctcc ctctggctgc caagcgagca ggccgtggag acaggtgcag aggcaggtgt 198720ggcagcaggc atcctgccag gcagtgctgc agtcatcctg cgacaagcag cagcagctca 198780tcctaccctc tagggggtct tgaggtcagc caggcaagag agcagcttgg actccactgg 198840gtgtgggacc agcctgtgga ccatggtggt gtggagggtg ccctcggcct gcctgtgtga 198900aggagaggcc ggcgtgttct gtggagccca aaggggagct gggcaagcag gattcacttc 198960actctgaggg tcctggagct cccaccctcc tcagccatct ccccagagcc tgtgtgccga 199020ggactcggcc catgttgctg tgggatgaga ggcagagtgt cgtgagggtg taaggagcgg 199080cggcagtggt gggaggaggg agcagcagcc agcgctacgg tgccagtttc cagctgccag 199140atgacgccgc tgaccctgtg gttgagaaga gatgcacaga gccagctctt gcaagccagt 199200gtggctgcca tagcacctgc cgagaagcag aaggaagggt ggccccagga ggacagagga 199260tgcgggcaca tctgatgcgg gcctgagttt tgggagcttt tgctctagcc agtttccagc 199320tccgggaccc acccgcctcg taggcaagac accacccaag aaatcatttg cttaacaaac 199380acactgggct ccaactggac acctgtgcca ccctagatgc tgggaaccca gccatgacac 199440aggcacctgc ccccagctgc tgaccactga ggctggctag cagctcccat ggggccagtg 199500tggggttccc cagcctccta acagggagcc agtcacaagc cctcgagagg gaagggtgcc 199560cgcggccctg gcaggaaggt taggctggac gctcccacaa gacataacag atggaggttc 199620taaatgatgt agcaacttct tcaccctgaa actgctgtag agtcagccat gacgcaccgg 199680tacttcagta actgccaggc atccgggaca gcacaccgcg agtcgctgct gtgcttgggt 199740tagaagtggt ttggtctgtt ttcttctcgc cctctctaat cagagtcagt gattcatgcc 199800cttccatcac cttagagaag gggcaggcgc tgcccgacct tctccaggct ggagcagcat 199860cgcctcatgt cagcagaact cagctgtaga atatcgtggg gttggtgcct ttcatcagca 199920gcatgtcctt aacaactttc tgatttcttc cttagttgtt ggtccattaa ggagaaaaaa 199980aatgatctca gccattgcta aaatatttga taagattcag caaagcagca tgttaacatt 200040gaaaactaga atcaggagcc aggcagatgt gcttgctttt cacctgtagt atttcatgtt 200100gttttgacgt ttttagctaa tgcattaaga taaataaaca aaagccgggc acggtggttc 200160acgcctgtaa tcccagcact ttgggaggct gaggcgggag gatcctctga ggtcaggagt 200220tcaagaccag cctgaccaac atggagaaac ctcgtcatta ctaaaaatac aaaattagct 200280gggcgtggtg gtgcatgcct gtaatcccag ctacttggga ggctgaggca ggagaatcgc 200340ttgaacccgg gaggcggagg ttgcagtgag ctgagattgc accactgcac tccagcctgg 200400gtgacagtga aactcggtct caaaaaaaaa aaaaaattaa aaaaagataa ataaaataag 200460caggataaga aatgaagaaa gtagagttac ctttgttttc agatttcatt tttgtatacc 200520cagaaagcca aatgtacaaa agactgggag ctctttaaac cagcttaaac ttgttgaaaa 200580tgaggatgaa gaaatatccc attcagagtt ggaatgaatt taacccagaa ggaacaggac 200640ctctactgaa gagaactatg cagtcttact gaaaaatcta aataatacct gagcgctgga 200700gaaacttcgc acactcctga aagctccaaa gtcaatgtca tcattttatt aatgtcattc 200760caaacatagt ctcaataata tcacttcttg gttttgacat ggacgcgatg atgtttaaat 200820tcatatgaaa aaagaacggg gccaaaagtc caaggccagt cagcgtgaga agaccgctcg 200880gcctccctcg gagtcgggga gttggaaccg cagactgaga tcatgtggct gctggaggcc 200940aggacgaacg tcgggaaatg gagactcctg cgttgctggt gggatgtggt gcagccgctt 201000ccaggagcaa tttggtgtcc cgtcctaaag ctgaagaaac gcatttcctc tggtcagtgc 201060cactcctaga caggccaccc tgcggcagcc gtcctcaaac tggtctgagg acccctcaac 201120gctcttaaaa atcattaaaa gtgggccagg tgcggtggct cacacctgta atcccagcac 201180tttgggaggc caagacaggc ggatcacgag gtcaggacat tgagatcatc ctggctaaca 201240cggtgaaacc ccgtctctac taaaaataca aaaaattagc cgggcgtggt ggcgggcgcc 201300tgtagtccca gctacttggg aggctgagcc aggagaatgg cgtgaaccca ggaggtggag 201360cttgcagtga gctgagatca ctccactgca ctccagcctg ggcagcagag cgagactctg 201420tctcaaaaaa aaataataaa taaataaata aaaataaaat aaaataaaat tcattaaaag 201480tgccaaagaa cttttgctta tgtgagttct aatgaccaat attaatacac attagaatat 201540cttattagaa attaaacctg agacctttag aaaacatgta ttcatttcaa aatagcaata 201600aacccatgac atattaacat aaataacaat tgtatgaaaa atatattttc caaaacaaaa 201660agttttcggg agaagtgtgg catagtttta catggtcgta aatctctggc ttaagagaag 201720cccactggcc tctcagcagg ctctgggtcc gtccactttg ggggtgtttt ggttgtgaag 201780tataggagtg aatggagaag ctcattctta cccagatgtg tatttgaaaa gaaaaggaac 201840attttaataa cctttgcaaa taatcggtat attcttccgt gatcctattc caacactgga 201900caggtggtgg tttgtttttt ttttttggag acggagtccc gctctgtcac tcaggctgga 201960gtgcagtggc gcgatttcag ctcactgcaa gctccgcctc c 202001213481DNAHomo sapiensCDS(146)..(9580) 2gctgccggga cgggtccaag atggacggcc gctcaggttc tgcttttacc tgcggcccag 60agccccattc attgccccgg tgctgagcgg cgccgcgagt cggcccgagg cctccgggga 120ctgccgtgcc gggcgggaga ccgcc atg gcg acc ctg gaa aag ctg atg aag 172 Met Ala Thr Leu Glu Lys Leu Met Lys 1 5 gcc ttc gag tcc ctc aag tcc ttc cag cag cag cag cag cag cag cag 220Ala Phe Glu Ser Leu Lys Ser Phe Gln Gln Gln Gln Gln Gln Gln Gln 10 15 20 25 cag cag cag cag cag cag cag cag cag cag cag cag cag caa cag ccg 268Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Pro 30 35 40 cca ccg ccg ccg ccg ccg ccg ccg cct cct cag ctt cct cag ccg ccg 316Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Gln Leu Pro Gln Pro Pro 45 50 55 ccg cag gca cag ccg ctg ctg cct cag ccg cag ccg ccc ccg ccg ccg 364Pro Gln Ala Gln Pro Leu Leu Pro Gln Pro Gln Pro Pro Pro Pro Pro 60 65 70 ccc ccg ccg cca ccc ggc ccg gct gtg gct gag gag ccg ctg cac cga 412Pro Pro Pro Pro Pro Gly Pro Ala Val Ala Glu Glu Pro Leu His Arg 75 80 85 cca aag aaa gaa ctt tca gct acc aag aaa gac cgt gtg aat cat tgt 460Pro Lys Lys Glu Leu Ser Ala Thr Lys Lys Asp Arg Val Asn His Cys 90 95 100 105 ctg aca ata tgt gaa aac ata gtg gca cag tct gtc aga aat tct cca 508Leu Thr Ile Cys Glu Asn Ile Val Ala Gln Ser Val Arg Asn Ser Pro 110 115 120 gaa ttt cag aaa ctt ctg ggc atc gct atg gaa ctt ttt ctg ctg tgc 556Glu Phe Gln Lys Leu Leu Gly Ile Ala Met Glu Leu Phe Leu Leu Cys 125 130 135 agt gat gac gca gag tca gat gtc agg atg gtg gct gac gaa tgc ctc 604Ser Asp Asp Ala Glu Ser Asp Val Arg Met Val Ala Asp Glu Cys Leu 140 145 150 aac aaa gtt atc aaa gct ttg atg gat tct aat ctt cca agg tta cag 652Asn Lys Val Ile Lys Ala Leu Met Asp Ser Asn Leu Pro Arg Leu Gln 155 160 165 ctc gag ctc tat aag gaa att aaa aag aat ggt gcc cct cgg agt ttg 700Leu Glu Leu Tyr Lys Glu Ile Lys Lys Asn Gly Ala Pro Arg Ser Leu 170 175 180 185 cgt gct gcc ctg tgg agg ttt gct gag ctg gct cac ctg gtt cgg cct 748Arg Ala Ala Leu Trp Arg Phe Ala Glu Leu Ala His Leu Val Arg Pro 190 195 200 cag aaa tgc agg cct tac ctg gtg aac ctt ctg ccg tgc ctg act cga 796Gln Lys Cys Arg Pro Tyr Leu Val Asn Leu Leu Pro Cys Leu Thr Arg 205 210 215 aca agc aag aga ccc gaa gaa tca gtc cag gag acc ttg gct gca gct 844Thr Ser Lys Arg Pro Glu Glu Ser Val Gln Glu Thr Leu Ala Ala Ala 220 225 230 gtt ccc aaa att atg gct tct ttt ggc aat ttt gca aat gac aat gaa 892Val Pro Lys Ile Met Ala Ser Phe Gly Asn Phe Ala Asn Asp Asn Glu 235 240 245 att aag gtt ttg tta aag gcc ttc ata gcg aac ctg aag tca agc tcc 940Ile Lys Val Leu Leu Lys Ala Phe Ile Ala Asn Leu Lys Ser Ser Ser 250 255 260 265 ccc acc att cgg cgg aca gcg gct gga tca gca gtg agc atc tgc cag 988Pro Thr Ile Arg Arg Thr Ala Ala Gly Ser Ala Val Ser Ile Cys Gln 270 275 280 cac tca aga agg aca caa tat ttc tat agt tgg cta cta aat gtg ctc 1036His Ser Arg Arg Thr Gln Tyr Phe Tyr Ser Trp Leu Leu Asn Val Leu 285 290 295 tta ggc tta ctc gtt cct gtc gag gat gaa cac tcc act ctg ctg att 1084Leu Gly Leu Leu Val Pro Val Glu Asp Glu His Ser Thr Leu Leu Ile 300 305 310 ctt ggc gtg ctg ctc acc ctg agg tat ttg gtg ccc ttg ctg cag cag 1132Leu Gly Val Leu Leu Thr Leu Arg Tyr Leu Val Pro Leu Leu Gln Gln 315 320 325 cag gtc aag gac aca agc ctg aaa ggc agc ttc gga gtg aca agg aaa 1180Gln Val Lys Asp Thr Ser Leu Lys Gly Ser Phe Gly Val Thr Arg Lys 330 335 340 345 gaa atg gaa gtc tct cct tct gca gag cag ctt gtc cag gtt tat gaa 1228Glu Met Glu Val Ser Pro Ser Ala Glu Gln Leu Val Gln Val Tyr Glu 350 355 360 ctg acg tta cat cat aca cag cac caa gac cac aat gtt gtg acc gga 1276Leu Thr Leu His His Thr Gln His Gln Asp His Asn Val Val Thr Gly 365 370 375 gcc ctg gag ctg ttg cag cag ctc ttc aga acg cct cca ccc gag ctt 1324Ala Leu Glu Leu Leu Gln Gln Leu Phe Arg Thr Pro Pro Pro Glu Leu 380 385 390 ctg caa acc ctg acc gca gtc ggg ggc att ggg cag ctc acc gct gct 1372Leu Gln Thr Leu Thr Ala Val Gly Gly Ile Gly Gln Leu Thr Ala Ala 395 400 405 aag gag gag tct ggt ggc cga agc cgt agt ggg agt att gtg gaa ctt 1420Lys Glu Glu Ser Gly Gly Arg Ser Arg Ser Gly Ser Ile Val Glu Leu 410 415 420 425 ata gct gga ggg ggt tcc tca tgc agc cct gtc ctt tca aga aaa caa 1468Ile Ala Gly Gly Gly Ser Ser Cys Ser Pro Val Leu Ser Arg Lys Gln 430 435 440 aaa ggc aaa gtg ctc tta gga gaa gaa gaa gcc ttg gag gat gac tct 1516Lys Gly Lys Val Leu Leu Gly Glu Glu Glu Ala Leu Glu Asp Asp Ser 445 450 455 gaa tcg aga tcg gat gtc agc agc tct gcc tta aca gcc tca gtg aag 1564Glu Ser Arg Ser Asp Val Ser Ser Ser Ala Leu Thr Ala Ser Val Lys 460 465 470 gat gag atc agt gga gag ctg gct gct tct tca ggg gtt tcc act cca 1612Asp Glu Ile Ser Gly Glu Leu Ala Ala Ser Ser Gly Val Ser Thr Pro 475 480 485 ggg tca gca ggt cat gac atc atc aca gaa cag cca cgg tca cag cac 1660Gly Ser Ala Gly His Asp Ile Ile Thr Glu Gln Pro Arg Ser Gln His 490 495 500 505 aca ctg cag gcg gac tca gtg gat ctg gcc agc tgt gac ttg aca agc 1708Thr Leu Gln Ala Asp Ser Val Asp Leu Ala Ser Cys Asp Leu Thr Ser 510 515 520 tct gcc act gat ggg gat gag gag gat atc ttg agc cac agc tcc agc 1756Ser Ala Thr Asp Gly Asp Glu Glu Asp Ile Leu Ser His Ser Ser Ser 525 530 535 cag gtc agc gcc gtc cca tct gac cct gcc atg gac ctg aat gat ggg 1804Gln Val Ser Ala Val Pro Ser Asp Pro Ala Met Asp Leu Asn Asp Gly 540 545 550 acc cag gcc tcg tcg ccc atc agc gac agc tcc cag acc acc acc gaa 1852Thr Gln Ala Ser Ser Pro Ile Ser Asp Ser Ser Gln Thr Thr Thr Glu 555 560 565 ggg cct gat tca gct gtt acc cct tca gac agt tct gaa att gtg tta 1900Gly Pro Asp Ser Ala Val Thr Pro Ser Asp Ser Ser Glu Ile Val Leu 570 575 580 585 gac ggt acc gac aac cag tat ttg ggc ctg cag att gga cag ccc cag 1948Asp Gly Thr Asp Asn Gln Tyr Leu Gly Leu Gln Ile Gly Gln Pro Gln 590 595 600 gat gaa gat gag gaa gcc aca ggt att ctt cct gat gaa gcc tcg gag 1996Asp Glu Asp Glu Glu Ala Thr Gly Ile Leu Pro Asp Glu Ala Ser Glu 605 610 615 gcc ttc agg aac tct tcc atg gcc ctt caa cag gca cat tta ttg aaa 2044Ala Phe Arg Asn Ser Ser Met Ala Leu Gln Gln Ala His Leu Leu Lys 620 625 630 aac atg agt cac tgc agg cag cct tct gac agc agt gtt gat aaa ttt 2092Asn Met Ser His Cys Arg Gln Pro Ser Asp Ser Ser Val Asp Lys Phe 635 640 645 gtg ttg aga gat gaa gct act gaa ccg ggt gat caa gaa aac aag cct 2140Val Leu Arg Asp Glu Ala Thr Glu Pro Gly Asp Gln Glu Asn Lys Pro 650 655 660 665 tgc cgc atc aaa ggt gac att gga cag tcc act gat gat gac tct gca 2188Cys Arg Ile Lys Gly Asp Ile Gly Gln Ser Thr Asp Asp Asp Ser Ala 670 675 680 cct ctt gtc cat tgt gtc cgc ctt tta tct gct tcg ttt ttg cta aca 2236Pro Leu Val His Cys Val Arg Leu Leu Ser Ala Ser Phe Leu Leu Thr 685 690 695 ggg gga aaa aat gtg ctg gtt ccg gac agg gat gtg agg gtc agc gtg 2284Gly Gly Lys Asn Val Leu Val Pro Asp Arg Asp Val Arg Val Ser Val 700 705 710 aag gcc ctg gcc ctc agc tgt gtg gga gca gct gtg gcc ctc cac ccg 2332Lys Ala Leu Ala Leu Ser Cys Val Gly Ala Ala Val Ala Leu His Pro 715 720 725 gaa tct ttc ttc agc aaa ctc tat aaa gtt cct ctt gac acc acg gaa 2380Glu Ser Phe Phe Ser Lys Leu Tyr Lys Val Pro Leu Asp Thr Thr Glu 730 735 740 745 tac cct gag gaa cag tat gtc tca gac atc ttg aac tac atc gat cat 2428Tyr Pro Glu Glu Gln Tyr Val Ser Asp Ile Leu Asn Tyr Ile Asp His 750 755 760

gga gac cca cag gtt cga gga gcc act gcc att ctc tgt ggg acc ctc 2476Gly Asp Pro Gln Val Arg Gly Ala Thr Ala Ile Leu Cys Gly Thr Leu 765 770 775 atc tgc tcc atc ctc agc agg tcc cgc ttc cac gtg gga gat tgg atg 2524Ile Cys Ser Ile Leu Ser Arg Ser Arg Phe His Val Gly Asp Trp Met 780 785 790 ggc acc att aga acc ctc aca gga aat aca ttt tct ttg gcg gat tgc 2572Gly Thr Ile Arg Thr Leu Thr Gly Asn Thr Phe Ser Leu Ala Asp Cys 795 800 805 att cct ttg ctg cgg aaa aca ctg aag gat gag tct tct gtt act tgc 2620Ile Pro Leu Leu Arg Lys Thr Leu Lys Asp Glu Ser Ser Val Thr Cys 810 815 820 825 aag tta gct tgt aca gct gtg agg aac tgt gtc atg agt ctc tgc agc 2668Lys Leu Ala Cys Thr Ala Val Arg Asn Cys Val Met Ser Leu Cys Ser 830 835 840 agc agc tac agt gag tta gga ctg cag ctg atc atc gat gtg ctg act 2716Ser Ser Tyr Ser Glu Leu Gly Leu Gln Leu Ile Ile Asp Val Leu Thr 845 850 855 ctg agg aac agt tcc tat tgg ctg gtg agg aca gag ctt ctg gaa acc 2764Leu Arg Asn Ser Ser Tyr Trp Leu Val Arg Thr Glu Leu Leu Glu Thr 860 865 870 ctt gca gag att gac ttc agg ctg gtg agc ttt ttg gag gca aaa gca 2812Leu Ala Glu Ile Asp Phe Arg Leu Val Ser Phe Leu Glu Ala Lys Ala 875 880 885 gaa aac tta cac aga ggg gct cat cat tat aca ggg ctt tta aaa ctg 2860Glu Asn Leu His Arg Gly Ala His His Tyr Thr Gly Leu Leu Lys Leu 890 895 900 905 caa gaa cga gtg ctc aat aat gtt gtc atc cat ttg ctt gga gat gaa 2908Gln Glu Arg Val Leu Asn Asn Val Val Ile His Leu Leu Gly Asp Glu 910 915 920 gac ccc agg gtg cga cat gtt gcc gca gca tca cta att agg ctt gtc 2956Asp Pro Arg Val Arg His Val Ala Ala Ala Ser Leu Ile Arg Leu Val 925 930 935 cca aag ctg ttt tat aaa tgt gac caa gga caa gct gat cca gta gtg 3004Pro Lys Leu Phe Tyr Lys Cys Asp Gln Gly Gln Ala Asp Pro Val Val 940 945 950 gcc gtg gca aga gat caa agc agt gtt tac ctg aaa ctt ctc atg cat 3052Ala Val Ala Arg Asp Gln Ser Ser Val Tyr Leu Lys Leu Leu Met His 955 960 965 gag acg cag cct cca tct cat ttc tcc gtc agc aca ata acc aga ata 3100Glu Thr Gln Pro Pro Ser His Phe Ser Val Ser Thr Ile Thr Arg Ile 970 975 980 985 tat aga ggc tat aac cta cta cca agc ata aca gac gtc act atg gaa 3148Tyr Arg Gly Tyr Asn Leu Leu Pro Ser Ile Thr Asp Val Thr Met Glu 990 995 1000 aat aac ctt tca aga gtt att gca gca gtt tct cat gaa cta atc 3193Asn Asn Leu Ser Arg Val Ile Ala Ala Val Ser His Glu Leu Ile 1005 1010 1015 aca tca acc acc aga gca ctc aca ttt gga tgc tgt gaa gct ttg 3238Thr Ser Thr Thr Arg Ala Leu Thr Phe Gly Cys Cys Glu Ala Leu 1020 1025 1030 tgt ctt ctt tcc act gcc ttc cca gtt tgc att tgg agt tta ggt 3283Cys Leu Leu Ser Thr Ala Phe Pro Val Cys Ile Trp Ser Leu Gly 1035 1040 1045 tgg cac tgt gga gtg cct cca ctg agt gcc tca gat gag tct agg 3328Trp His Cys Gly Val Pro Pro Leu Ser Ala Ser Asp Glu Ser Arg 1050 1055 1060 aag agc tgt acc gtt ggg atg gcc aca atg att ctg acc ctg ctc 3373Lys Ser Cys Thr Val Gly Met Ala Thr Met Ile Leu Thr Leu Leu 1065 1070 1075 tcg tca gct tgg ttc cca ttg gat ctc tca gcc cat caa gat gct 3418Ser Ser Ala Trp Phe Pro Leu Asp Leu Ser Ala His Gln Asp Ala 1080 1085 1090 ttg att ttg gcc gga aac ttg ctt gca gcc agt gct ccc aaa tct 3463Leu Ile Leu Ala Gly Asn Leu Leu Ala Ala Ser Ala Pro Lys Ser 1095 1100 1105 ctg aga agt tca tgg gcc tct gaa gaa gaa gcc aac cca gca gcc 3508Leu Arg Ser Ser Trp Ala Ser Glu Glu Glu Ala Asn Pro Ala Ala 1110 1115 1120 acc aag caa gag gag gtc tgg cca gcc ctg ggg gac cgg gcc ctg 3553Thr Lys Gln Glu Glu Val Trp Pro Ala Leu Gly Asp Arg Ala Leu 1125 1130 1135 gtg ccc atg gtg gag cag ctc ttc tct cac ctg ctg aag gtg att 3598Val Pro Met Val Glu Gln Leu Phe Ser His Leu Leu Lys Val Ile 1140 1145 1150 aac att tgt gcc cac gtc ctg gat gac gtg gct cct gga ccc gca 3643Asn Ile Cys Ala His Val Leu Asp Asp Val Ala Pro Gly Pro Ala 1155 1160 1165 ata aag gca gcc ttg cct tct cta aca aac ccc cct tct cta agt 3688Ile Lys Ala Ala Leu Pro Ser Leu Thr Asn Pro Pro Ser Leu Ser 1170 1175 1180 ccc atc cga cga aag ggg aag gag aaa gaa cca gga gaa caa gca 3733Pro Ile Arg Arg Lys Gly Lys Glu Lys Glu Pro Gly Glu Gln Ala 1185 1190 1195 tct gta ccg ttg agt ccc aag aaa ggc agt gag gcc agt gca gct 3778Ser Val Pro Leu Ser Pro Lys Lys Gly Ser Glu Ala Ser Ala Ala 1200 1205 1210 tct aga caa tct gat acc tca ggt cct gtt aca aca agt aaa tcc 3823Ser Arg Gln Ser Asp Thr Ser Gly Pro Val Thr Thr Ser Lys Ser 1215 1220 1225 tca tca ctg ggg agt ttc tat cat ctt cct tca tac ctc aaa ctg 3868Ser Ser Leu Gly Ser Phe Tyr His Leu Pro Ser Tyr Leu Lys Leu 1230 1235 1240 cat gat gtc ctg aaa gct aca cac gct aac tac aag gtc acg ctg 3913His Asp Val Leu Lys Ala Thr His Ala Asn Tyr Lys Val Thr Leu 1245 1250 1255 gat ctt cag aac agc acg gaa aag ttt gga ggg ttt ctc cgc tca 3958Asp Leu Gln Asn Ser Thr Glu Lys Phe Gly Gly Phe Leu Arg Ser 1260 1265 1270 gcc ttg gat gtt ctt tct cag ata cta gag ctg gcc aca ctg cag 4003Ala Leu Asp Val Leu Ser Gln Ile Leu Glu Leu Ala Thr Leu Gln 1275 1280 1285 gac att ggg aag tgt gtt gaa gag atc cta gga tac ctg aaa tcc 4048Asp Ile Gly Lys Cys Val Glu Glu Ile Leu Gly Tyr Leu Lys Ser 1290 1295 1300 tgc ttt agt cga gaa cca atg atg gca act gtt tgt gtt caa caa 4093Cys Phe Ser Arg Glu Pro Met Met Ala Thr Val Cys Val Gln Gln 1305 1310 1315 ttg ttg aag act ctc ttt ggc aca aac ttg gcc tcc cag ttt gat 4138Leu Leu Lys Thr Leu Phe Gly Thr Asn Leu Ala Ser Gln Phe Asp 1320 1325 1330 ggc tta tct tcc aac ccc agc aag tca caa ggc cga gca cag cgc 4183Gly Leu Ser Ser Asn Pro Ser Lys Ser Gln Gly Arg Ala Gln Arg 1335 1340 1345 ctt ggc tcc tcc agt gtg agg cca ggc ttg tac cac tac tgc ttc 4228Leu Gly Ser Ser Ser Val Arg Pro Gly Leu Tyr His Tyr Cys Phe 1350 1355 1360 atg gcc ccg tac acc cac ttc acc cag gcc ctc gct gac gcc agc 4273Met Ala Pro Tyr Thr His Phe Thr Gln Ala Leu Ala Asp Ala Ser 1365 1370 1375 ctg agg aac atg gtg cag gcg gag cag gag aac gac acc tcg gga 4318Leu Arg Asn Met Val Gln Ala Glu Gln Glu Asn Asp Thr Ser Gly 1380 1385 1390 tgg ttt gat gtc ctc cag aaa gtg tct acc cag ttg aag aca aac 4363Trp Phe Asp Val Leu Gln Lys Val Ser Thr Gln Leu Lys Thr Asn 1395 1400 1405 ctc acg agt gtc aca aag aac cgt gca gat aag aat gct att cat 4408Leu Thr Ser Val Thr Lys Asn Arg Ala Asp Lys Asn Ala Ile His 1410 1415 1420 aat cac att cgt ttg ttt gaa cct ctt gtt ata aaa gct tta aaa 4453Asn His Ile Arg Leu Phe Glu Pro Leu Val Ile Lys Ala Leu Lys 1425 1430 1435 cag tac acg act aca aca tgt gtg cag tta cag aag cag gtt tta 4498Gln Tyr Thr Thr Thr Thr Cys Val Gln Leu Gln Lys Gln Val Leu 1440 1445 1450 gat ttg ctg gcg cag ctg gtt cag tta cgg gtt aat tac tgt ctt 4543Asp Leu Leu Ala Gln Leu Val Gln Leu Arg Val Asn Tyr Cys Leu 1455 1460 1465 ctg gat tca gat cag gtg ttt att ggc ttt gta ttg aaa cag ttt 4588Leu Asp Ser Asp Gln Val Phe Ile Gly Phe Val Leu Lys Gln Phe 1470 1475 1480 gaa tac att gaa gtg ggc cag ttc agg gaa tca gag gca atc att 4633Glu Tyr Ile Glu Val Gly Gln Phe Arg Glu Ser Glu Ala Ile Ile 1485 1490 1495 cca aac atc ttt ttc ttc ttg gta tta cta tct tat gaa cgc tat 4678Pro Asn Ile Phe Phe Phe Leu Val Leu Leu Ser Tyr Glu Arg Tyr 1500 1505 1510 cat tca aaa cag atc att gga att cct aaa atc att cag ctc tgt 4723His Ser Lys Gln Ile Ile Gly Ile Pro Lys Ile Ile Gln Leu Cys 1515 1520 1525 gat ggc atc atg gcc agt gga agg aag gct gtg aca cat gcc ata 4768Asp Gly Ile Met Ala Ser Gly Arg Lys Ala Val Thr His Ala Ile 1530 1535 1540 ccg gct ctg cag ccc ata gtc cac gac ctc ttt gta tta aga gga 4813Pro Ala Leu Gln Pro Ile Val His Asp Leu Phe Val Leu Arg Gly 1545 1550 1555 aca aat aaa gct gat gca gga aaa gag ctt gaa acc caa aaa gag 4858Thr Asn Lys Ala Asp Ala Gly Lys Glu Leu Glu Thr Gln Lys Glu 1560 1565 1570 gtg gtg gtg tca atg tta ctg aga ctc atc cag tac cat cag gtg 4903Val Val Val Ser Met Leu Leu Arg Leu Ile Gln Tyr His Gln Val 1575 1580 1585 ttg gag atg ttc att ctt gtc ctg cag cag tgc cac aag gag aat 4948Leu Glu Met Phe Ile Leu Val Leu Gln Gln Cys His Lys Glu Asn 1590 1595 1600 gaa gac aag tgg aag cga ctg tct cga cag ata gct gac atc atc 4993Glu Asp Lys Trp Lys Arg Leu Ser Arg Gln Ile Ala Asp Ile Ile 1605 1610 1615 ctc cca atg tta gcc aaa cag cag atg cac att gac tct cat gaa 5038Leu Pro Met Leu Ala Lys Gln Gln Met His Ile Asp Ser His Glu 1620 1625 1630 gcc ctt gga gtg tta aat aca tta ttt gag att ttg gcc cct tcc 5083Ala Leu Gly Val Leu Asn Thr Leu Phe Glu Ile Leu Ala Pro Ser 1635 1640 1645 tcc ctc cgt ccg gta gac atg ctt tta cgg agt atg ttc gtc act 5128Ser Leu Arg Pro Val Asp Met Leu Leu Arg Ser Met Phe Val Thr 1650 1655 1660 cca aac aca atg gcg tcc gtg agc act gtt caa ctg tgg ata tcg 5173Pro Asn Thr Met Ala Ser Val Ser Thr Val Gln Leu Trp Ile Ser 1665 1670 1675 gga att ctg gcc att ttg agg gtt ctg att tcc cag tca act gaa 5218Gly Ile Leu Ala Ile Leu Arg Val Leu Ile Ser Gln Ser Thr Glu 1680 1685 1690 gat att gtt ctt tct cgt att cag gag ctc tcc ttc tct ccg tat 5263Asp Ile Val Leu Ser Arg Ile Gln Glu Leu Ser Phe Ser Pro Tyr 1695 1700 1705 tta atc tcc tgt aca gta att aat agg tta aga gat ggg gac agt 5308Leu Ile Ser Cys Thr Val Ile Asn Arg Leu Arg Asp Gly Asp Ser 1710 1715 1720 act tca acg cta gaa gaa cac agt gaa ggg aaa caa ata aag aat 5353Thr Ser Thr Leu Glu Glu His Ser Glu Gly Lys Gln Ile Lys Asn 1725 1730 1735 ttg cca gaa gaa aca ttt tca agg ttt cta tta caa ctg gtt ggt 5398Leu Pro Glu Glu Thr Phe Ser Arg Phe Leu Leu Gln Leu Val Gly 1740 1745 1750 att ctt tta gaa gac att gtt aca aaa cag ctg aag gtg gaa atg 5443Ile Leu Leu Glu Asp Ile Val Thr Lys Gln Leu Lys Val Glu Met 1755 1760 1765 agt gag cag caa cat act ttc tat tgc cag gaa cta ggc aca ctg 5488Ser Glu Gln Gln His Thr Phe Tyr Cys Gln Glu Leu Gly Thr Leu 1770 1775 1780 cta atg tgt ctg atc cac atc ttc aag tct gga atg ttc cgg aga 5533Leu Met Cys Leu Ile His Ile Phe Lys Ser Gly Met Phe Arg Arg 1785 1790 1795 atc aca gca gct gcc act agg ctg ttc cgc agt gat ggc tgt ggc 5578Ile Thr Ala Ala Ala Thr Arg Leu Phe Arg Ser Asp Gly Cys Gly 1800 1805 1810 ggc agt ttc tac acc ctg gac agc ttg aac ttg cgg gct cgt tcc 5623Gly Ser Phe Tyr Thr Leu Asp Ser Leu Asn Leu Arg Ala Arg Ser 1815 1820 1825 atg atc acc acc cac ccg gcc ctg gtg ctg ctc tgg tgt cag ata 5668Met Ile Thr Thr His Pro Ala Leu Val Leu Leu Trp Cys Gln Ile 1830 1835 1840 ctg ctg ctt gtc aac cac acc gac tac cgc tgg tgg gca gaa gtg 5713Leu Leu Leu Val Asn His Thr Asp Tyr Arg Trp Trp Ala Glu Val 1845 1850 1855 cag cag acc ccg aaa aga cac agt ctg tcc agc aca aag tta ctt 5758Gln Gln Thr Pro Lys Arg His Ser Leu Ser Ser Thr Lys Leu Leu 1860 1865 1870 agt ccc cag atg tct gga gaa gag gag gat tct gac ttg gca gcc 5803Ser Pro Gln Met Ser Gly Glu Glu Glu Asp Ser Asp Leu Ala Ala 1875 1880 1885 aaa ctt gga atg tgc aat aga gaa ata gta cga aga ggg gct ctc 5848Lys Leu Gly Met Cys Asn Arg Glu Ile Val Arg Arg Gly Ala Leu 1890 1895 1900 att ctc ttc tgt gat tat gtc tgt cag aac ctc cat gac tcc gag 5893Ile Leu Phe Cys Asp Tyr Val Cys Gln Asn Leu His Asp Ser Glu 1905 1910 1915 cac tta acg tgg ctc att gta aat cac att caa gat ctg atc agc 5938His Leu Thr Trp Leu Ile Val Asn His Ile Gln Asp Leu Ile Ser 1920 1925 1930 ctt tcc cac gag cct cca gta cag gac ttc atc agt gcc gtt cat 5983Leu Ser His Glu Pro Pro Val Gln Asp Phe Ile Ser Ala Val His 1935 1940 1945 cgg aac tct gct gcc agc ggc ctg ttc atc cag gca att cag tct 6028Arg Asn Ser Ala Ala Ser Gly Leu Phe Ile Gln Ala Ile Gln Ser 1950 1955 1960 cgt tgt gaa aac ctt tca act cca acc atg ctg aag aaa act ctt 6073Arg Cys Glu Asn Leu Ser Thr Pro Thr Met Leu Lys Lys Thr Leu 1965 1970 1975 cag tgc ttg gag ggg atc cat ctc agc cag tcg gga gct gtg ctc 6118Gln Cys Leu Glu Gly Ile His Leu Ser Gln Ser Gly Ala Val Leu 1980 1985 1990 acg ctg tat gtg gac agg ctt ctg tgc acc cct ttc cgt gtg ctg 6163Thr Leu Tyr Val Asp Arg Leu Leu Cys Thr Pro Phe Arg Val Leu 1995 2000 2005 gct cgc atg gtc gac atc ctt gct tgt cgc cgg gta gaa atg ctt 6208Ala Arg Met Val Asp Ile Leu Ala Cys Arg Arg Val Glu Met Leu 2010 2015 2020 ctg gct gca aat tta cag agc agc atg gcc cag ttg cca atg gaa 6253Leu Ala Ala Asn Leu Gln Ser Ser Met Ala Gln Leu Pro Met Glu

2025 2030 2035 gaa ctc aac aga atc cag gaa tac ctt cag agc agc ggg ctc gct 6298Glu Leu Asn Arg Ile Gln Glu Tyr Leu Gln Ser Ser Gly Leu Ala 2040 2045 2050 cag aga cac caa agg ctc tat tcc ctg ctg gac agg ttt cgt ctc 6343Gln Arg His Gln Arg Leu Tyr Ser Leu Leu Asp Arg Phe Arg Leu 2055 2060 2065 tcc acc atg caa gac tca ctt agt ccc tct cct cca gtc tct tcc 6388Ser Thr Met Gln Asp Ser Leu Ser Pro Ser Pro Pro Val Ser Ser 2070 2075 2080 cac ccg ctg gac ggg gat ggg cac gtg tca ctg gaa aca gtg agt 6433His Pro Leu Asp Gly Asp Gly His Val Ser Leu Glu Thr Val Ser 2085 2090 2095 ccg gac aaa gac tgg tac gtt cat ctt gtc aaa tcc cag tgt tgg 6478Pro Asp Lys Asp Trp Tyr Val His Leu Val Lys Ser Gln Cys Trp 2100 2105 2110 acc agg tca gat tct gca ctg ctg gaa ggt gca gag ctg gtg aat 6523Thr Arg Ser Asp Ser Ala Leu Leu Glu Gly Ala Glu Leu Val Asn 2115 2120 2125 cgg att cct gct gaa gat atg aat gcc ttc atg atg aac tcg gag 6568Arg Ile Pro Ala Glu Asp Met Asn Ala Phe Met Met Asn Ser Glu 2130 2135 2140 ttc aac cta agc ctg cta gct cca tgc tta agc cta ggg atg agt 6613Phe Asn Leu Ser Leu Leu Ala Pro Cys Leu Ser Leu Gly Met Ser 2145 2150 2155 gaa att tct ggt ggc cag aag agt gcc ctt ttt gaa gca gcc cgt 6658Glu Ile Ser Gly Gly Gln Lys Ser Ala Leu Phe Glu Ala Ala Arg 2160 2165 2170 gag gtg act ctg gcc cgt gtg agc ggc acc gtg cag cag ctc cct 6703Glu Val Thr Leu Ala Arg Val Ser Gly Thr Val Gln Gln Leu Pro 2175 2180 2185 gct gtc cat cat gtc ttc cag ccc gag ctg cct gca gag ccg gcg 6748Ala Val His His Val Phe Gln Pro Glu Leu Pro Ala Glu Pro Ala 2190 2195 2200 gcc tac tgg agc aag ttg aat gat ctg ttt ggg gat gct gca ctg 6793Ala Tyr Trp Ser Lys Leu Asn Asp Leu Phe Gly Asp Ala Ala Leu 2205 2210 2215 tat cag tcc ctg ccc act ctg gcc cgg gcc ctg gca cag tac ctg 6838Tyr Gln Ser Leu Pro Thr Leu Ala Arg Ala Leu Ala Gln Tyr Leu 2220 2225 2230 gtg gtg gtc tcc aaa ctg ccc agt cat ttg cac ctt cct cct gag 6883Val Val Val Ser Lys Leu Pro Ser His Leu His Leu Pro Pro Glu 2235 2240 2245 aaa gag aag gac att gtg aaa ttc gtg gtg gca acc ctt gag gcc 6928Lys Glu Lys Asp Ile Val Lys Phe Val Val Ala Thr Leu Glu Ala 2250 2255 2260 ctg tcc tgg cat ttg atc cat gag cag atc ccg ctg agt ctg gat 6973Leu Ser Trp His Leu Ile His Glu Gln Ile Pro Leu Ser Leu Asp 2265 2270 2275 ctc cag gca ggg ctg gac tgc tgc tgc ctg gcc ctg cag ctg cct 7018Leu Gln Ala Gly Leu Asp Cys Cys Cys Leu Ala Leu Gln Leu Pro 2280 2285 2290 ggc ctc tgg agc gtg gtc tcc tcc aca gag ttt gtg acc cac gcc 7063Gly Leu Trp Ser Val Val Ser Ser Thr Glu Phe Val Thr His Ala 2295 2300 2305 tgc tcc ctc atc tac tgt gtg cac ttc atc ctg gag gcc gtt gca 7108Cys Ser Leu Ile Tyr Cys Val His Phe Ile Leu Glu Ala Val Ala 2310 2315 2320 gtg cag cct gga gag cag ctt ctt agt cca gaa aga agg aca aat 7153Val Gln Pro Gly Glu Gln Leu Leu Ser Pro Glu Arg Arg Thr Asn 2325 2330 2335 acc cca aaa gcc atc agc gag gag gag gag gaa gta gat cca aac 7198Thr Pro Lys Ala Ile Ser Glu Glu Glu Glu Glu Val Asp Pro Asn 2340 2345 2350 aca cag aat cct aag tat atc act gca gcc tgt gag atg gtg gca 7243Thr Gln Asn Pro Lys Tyr Ile Thr Ala Ala Cys Glu Met Val Ala 2355 2360 2365 gaa atg gtg gag tct ctg cag tcg gtg ttg gcc ttg ggt cat aaa 7288Glu Met Val Glu Ser Leu Gln Ser Val Leu Ala Leu Gly His Lys 2370 2375 2380 agg aat agc ggc gtg ccg gcg ttt ctc acg cca ttg cta agg aac 7333Arg Asn Ser Gly Val Pro Ala Phe Leu Thr Pro Leu Leu Arg Asn 2385 2390 2395 atc atc atc agc ctg gcc cgc ctg ccc ctt gtc aac agc tac aca 7378Ile Ile Ile Ser Leu Ala Arg Leu Pro Leu Val Asn Ser Tyr Thr 2400 2405 2410 cgt gtg ccc cca ctg gtg tgg aag ctt gga tgg tca ccc aaa ccg 7423Arg Val Pro Pro Leu Val Trp Lys Leu Gly Trp Ser Pro Lys Pro 2415 2420 2425 gga ggg gat ttt ggc aca gca ttc cct gag atc ccc gtg gag ttc 7468Gly Gly Asp Phe Gly Thr Ala Phe Pro Glu Ile Pro Val Glu Phe 2430 2435 2440 ctc cag gaa aag gaa gtc ttt aag gag ttc atc tac cgc atc aac 7513Leu Gln Glu Lys Glu Val Phe Lys Glu Phe Ile Tyr Arg Ile Asn 2445 2450 2455 aca cta ggc tgg acc agt cgt act cag ttt gaa gaa act tgg gcc 7558Thr Leu Gly Trp Thr Ser Arg Thr Gln Phe Glu Glu Thr Trp Ala 2460 2465 2470 acc ctc ctt ggt gtc ctg gtg acg cag ccc ctc gtg atg gag cag 7603Thr Leu Leu Gly Val Leu Val Thr Gln Pro Leu Val Met Glu Gln 2475 2480 2485 gag gag agc cca cca gaa gaa gac aca gag agg acc cag atc aac 7648Glu Glu Ser Pro Pro Glu Glu Asp Thr Glu Arg Thr Gln Ile Asn 2490 2495 2500 gtc ctg gcc gtg cag gcc atc acc tca ctg gtg ctc agt gca atg 7693Val Leu Ala Val Gln Ala Ile Thr Ser Leu Val Leu Ser Ala Met 2505 2510 2515 act gtg cct gtg gcc ggc aac cca gct gta agc tgc ttg gag cag 7738Thr Val Pro Val Ala Gly Asn Pro Ala Val Ser Cys Leu Glu Gln 2520 2525 2530 cag ccc cgg aac aag cct ctg aaa gct ctc gac acc agg ttt ggg 7783Gln Pro Arg Asn Lys Pro Leu Lys Ala Leu Asp Thr Arg Phe Gly 2535 2540 2545 agg aag ctg agc att atc aga ggg att gtg gag caa gag att caa 7828Arg Lys Leu Ser Ile Ile Arg Gly Ile Val Glu Gln Glu Ile Gln 2550 2555 2560 gca atg gtt tca aag aga gag aat att gcc acc cat cat tta tat 7873Ala Met Val Ser Lys Arg Glu Asn Ile Ala Thr His His Leu Tyr 2565 2570 2575 cag gca tgg gat cct gtc cct tct ctg tct ccg gct act aca ggt 7918Gln Ala Trp Asp Pro Val Pro Ser Leu Ser Pro Ala Thr Thr Gly 2580 2585 2590 gcc ctc atc agc cac gag aag ctg ctg cta cag atc aac ccc gag 7963Ala Leu Ile Ser His Glu Lys Leu Leu Leu Gln Ile Asn Pro Glu 2595 2600 2605 cgg gag ctg ggg agc atg agc tac aaa ctc ggc cag gtg tcc ata 8008Arg Glu Leu Gly Ser Met Ser Tyr Lys Leu Gly Gln Val Ser Ile 2610 2615 2620 cac tcc gtg tgg ctg ggg aac agc atc aca ccc ctg agg gag gag 8053His Ser Val Trp Leu Gly Asn Ser Ile Thr Pro Leu Arg Glu Glu 2625 2630 2635 gaa tgg gac gag gaa gag gag gag gag gcc gac gcc cct gca cct 8098Glu Trp Asp Glu Glu Glu Glu Glu Glu Ala Asp Ala Pro Ala Pro 2640 2645 2650 tcg tca cca ccc acg tct cca gtc aac tcc agg aaa cac cgg gct 8143Ser Ser Pro Pro Thr Ser Pro Val Asn Ser Arg Lys His Arg Ala 2655 2660 2665 gga gtt gac atc cac tcc tgt tcg cag ttt ttg ctt gag ttg tac 8188Gly Val Asp Ile His Ser Cys Ser Gln Phe Leu Leu Glu Leu Tyr 2670 2675 2680 agc cgc tgg atc ctg ccg tcc agc tca gcc agg agg acc ccg gcc 8233Ser Arg Trp Ile Leu Pro Ser Ser Ser Ala Arg Arg Thr Pro Ala 2685 2690 2695 atc ctg atc agt gag gtg gtc aga tcc ctt cta gtg gtc tca gac 8278Ile Leu Ile Ser Glu Val Val Arg Ser Leu Leu Val Val Ser Asp 2700 2705 2710 ttg ttc acc gag cgc aac cag ttt gag ctg atg tat gtg acg ctg 8323Leu Phe Thr Glu Arg Asn Gln Phe Glu Leu Met Tyr Val Thr Leu 2715 2720 2725 aca gaa ctg cga agg gtg cac cct tca gaa gac gag atc ctc gct 8368Thr Glu Leu Arg Arg Val His Pro Ser Glu Asp Glu Ile Leu Ala 2730 2735 2740 cag tac ctg gtg cct gcc acc tgc aag gca gct gcc gtc ctt ggg 8413Gln Tyr Leu Val Pro Ala Thr Cys Lys Ala Ala Ala Val Leu Gly 2745 2750 2755 atg gac aag gcc gtg gcg gag cct gtc agc cgc ctg ctg gag agc 8458Met Asp Lys Ala Val Ala Glu Pro Val Ser Arg Leu Leu Glu Ser 2760 2765 2770 acg ctc agg agc agc cac ctg ccc agc agg gtt gga gcc ctg cac 8503Thr Leu Arg Ser Ser His Leu Pro Ser Arg Val Gly Ala Leu His 2775 2780 2785 ggc gtc ctc tat gtg ctg gag tgc gac ctg ctg gac gac act gcc 8548Gly Val Leu Tyr Val Leu Glu Cys Asp Leu Leu Asp Asp Thr Ala 2790 2795 2800 aag cag ctc atc ccg gtc atc agc gac tat ctc ctc tcc aac ctg 8593Lys Gln Leu Ile Pro Val Ile Ser Asp Tyr Leu Leu Ser Asn Leu 2805 2810 2815 aaa ggg atc gcc cac tgc gtg aac att cac agc cag cag cac gta 8638Lys Gly Ile Ala His Cys Val Asn Ile His Ser Gln Gln His Val 2820 2825 2830 ctg gtc atg tgt gcc act gcg ttt tac ctc att gag aac tat cct 8683Leu Val Met Cys Ala Thr Ala Phe Tyr Leu Ile Glu Asn Tyr Pro 2835 2840 2845 ctg gac gta ggg ccg gaa ttt tca gca tca ata ata cag atg tgt 8728Leu Asp Val Gly Pro Glu Phe Ser Ala Ser Ile Ile Gln Met Cys 2850 2855 2860 ggg gtg atg ctg tct gga agt gag gag tcc acc ccc tcc atc att 8773Gly Val Met Leu Ser Gly Ser Glu Glu Ser Thr Pro Ser Ile Ile 2865 2870 2875 tac cac tgt gcc ctc aga ggc ctg gag cgc ctc ctg ctc tct gag 8818Tyr His Cys Ala Leu Arg Gly Leu Glu Arg Leu Leu Leu Ser Glu 2880 2885 2890 cag ctc tcc cgc ctg gat gca gaa tcg ctg gtc aag ctg agt gtg 8863Gln Leu Ser Arg Leu Asp Ala Glu Ser Leu Val Lys Leu Ser Val 2895 2900 2905 gac aga gtg aac gtg cac agc ccg cac cgg gcc atg gcg gct ctg 8908Asp Arg Val Asn Val His Ser Pro His Arg Ala Met Ala Ala Leu 2910 2915 2920 ggc ctg atg ctc acc tgc atg tac aca gga aag gag aaa gtc agt 8953Gly Leu Met Leu Thr Cys Met Tyr Thr Gly Lys Glu Lys Val Ser 2925 2930 2935 ccg ggt aga act tca gac cct aat cct gca gcc ccc gac agc gag 8998Pro Gly Arg Thr Ser Asp Pro Asn Pro Ala Ala Pro Asp Ser Glu 2940 2945 2950 tca gtg att gtt gct atg gag cgg gta tct gtt ctt ttt gat agg 9043Ser Val Ile Val Ala Met Glu Arg Val Ser Val Leu Phe Asp Arg 2955 2960 2965 atc agg aaa ggc ttt cct tgt gaa gcc aga gtg gtg gcc agg atc 9088Ile Arg Lys Gly Phe Pro Cys Glu Ala Arg Val Val Ala Arg Ile 2970 2975 2980 ctg ccc cag ttt cta gac gac ttc ttc cca ccc cag gac atc atg 9133Leu Pro Gln Phe Leu Asp Asp Phe Phe Pro Pro Gln Asp Ile Met 2985 2990 2995 aac aaa gtc atc gga gag ttt ctg tcc aac cag cag cca tac ccc 9178Asn Lys Val Ile Gly Glu Phe Leu Ser Asn Gln Gln Pro Tyr Pro 3000 3005 3010 cag ttc atg gcc acc gtg gtg tat aag gtg ttt cag act ctg cac 9223Gln Phe Met Ala Thr Val Val Tyr Lys Val Phe Gln Thr Leu His 3015 3020 3025 agc acc ggg cag tcg tcc atg gtc cgg gac tgg gtc atg ctg tcc 9268Ser Thr Gly Gln Ser Ser Met Val Arg Asp Trp Val Met Leu Ser 3030 3035 3040 ctc tcc aac ttc acg cag agg gcc ccg gtc gcc atg gcc acg tgg 9313Leu Ser Asn Phe Thr Gln Arg Ala Pro Val Ala Met Ala Thr Trp 3045 3050 3055 agc ctc tcc tgc ttc ttt gtc agc gcg tcc acc agc ccg tgg gtc 9358Ser Leu Ser Cys Phe Phe Val Ser Ala Ser Thr Ser Pro Trp Val 3060 3065 3070 gcg gcg atc ctc cca cat gtc atc agc agg atg ggc aag ctg gag 9403Ala Ala Ile Leu Pro His Val Ile Ser Arg Met Gly Lys Leu Glu 3075 3080 3085 cag gtg gac gtg aac ctt ttc tgc ctg gtc gcc aca gac ttc tac 9448Gln Val Asp Val Asn Leu Phe Cys Leu Val Ala Thr Asp Phe Tyr 3090 3095 3100 aga cac cag ata gag gag gag ctc gac cgc agg gcc ttc cag tct 9493Arg His Gln Ile Glu Glu Glu Leu Asp Arg Arg Ala Phe Gln Ser 3105 3110 3115 gtg ctt gag gtg gtt gca gcc cca gga agc cca tat cac cgg ctg 9538Val Leu Glu Val Val Ala Ala Pro Gly Ser Pro Tyr His Arg Leu 3120 3125 3130 ctg act tgt tta cga aat gtc cac aag gtc acc acc tgc tga 9580Leu Thr Cys Leu Arg Asn Val His Lys Val Thr Thr Cys 3135 3140 gcgccatggt gggagagact gtgaggcggc agctggggcc ggagcctttg gaagtctgcg 9640cccttgtgcc ctgcctccac cgagccagct tggtccctat gggcttccgc acatgccgcg 9700ggcggccagg caacgtgcgt gtctctgcca tgtggcagaa gtgctctttg tggcagtggc 9760caggcaggga gtgtctgcag tcctggtggg gctgagcctg aggccttcca gaaagcagga 9820gcagctgtgc tgcaccccat gtgggtgacc aggtcctttc tcctgatagt cacctgctgg 9880ttgttgccag gttgcagctg ctcttgcatc tgggccagaa gtcctccctc ctgcaggctg 9940gctgttggcc cctctgctgt cctgcagtag aaggtgccgt gagcaggctt tgggaacact 10000ggcctgggtc tccctggtgg ggtgtgcatg ccacgccccg tgtctggatg cacagatgcc 10060atggcctgtg ctgggccagt ggctgggggt gctagacacc cggcaccatt ctcccttctc 10120tcttttcttc tcaggattta aaatttaatt atatcagtaa agagattaat tttaacgtaa 10180ctctttctat gcccgtgtaa agtatgtgaa tcgcaaggcc tgtgctgcat gcgacagcgt 10240ccggggtggt ggacagggcc cccggccacg ctccctctcc tgtagccact ggcatagccc 10300tcctgagcac ccgctgacat ttccgttgta catgttcctg tttatgcatt cacaaggtga 10360ctgggatgta gagaggcgtt agtgggcagg tggccacagc aggactgagg acaggccccc 10420attatcctag gggtgcgctc acctgcagcc cctcctcctc gggcacagac gactgtcgtt 10480ctccacccac cagtcaggga cagcagcctc cctgtcactc agctgagaag gccagccctc 10540cctggctgtg agcagcctcc actgtgtcca gagacatggg cctcccactc ctgttccttg 10600ctagccctgg ggtggcgtct gcctaggagc tggctggcag gtgttgggac ctgctgctcc 10660atggatgcat gccctaagag tgtcactgag ctgtgttttg tctgagcctc tctcggtcaa 10720cagcaaagct tggtgtcttg gcactgttag tgacagagcc cagcatccct tctgcccccg 10780ttccagctga catcttgcac ggtgacccct tttagtcagg agagtgcaga tctgtgctca 10840tcggagactg ccccacggcc ctgtcagagc cgccactcct atccccaggc caggtccctg 10900gaccagcctc ctgtttgcag gcccagagga gccaagtcat taaaatggaa gtggattctg 10960gatggccggg ctgctgctga tgtaggagct ggatttggga gctctgcttg ccgactggct 11020gtgagacgag gcaggggctc tgcttcctca gccctagagg cgagccaggc aaggttggcg 11080actgtcatgt ggcttggttt ggtcatgccc gtcgatgttt tgggtattga atgtggtaag 11140tggaggaaat gttggaactc tgtgcaggtg ctgccttgag acccccaagc ttccacctgt 11200ccctctccta tgtggcagct ggggagcagc tgagatgtgg acttgtatgc tgcccacata

11260cgtgaggggg agctgaaagg gagcccctcc tctgagcagc ctctgccagg cctgtatgag 11320gcttttccca ccagctccca acagaggcct cccccagcca ggaccacctc gtcctcgtgg 11380cggggcagca ggagcggtag aaaggggtcc gatgtttgag gaggccctta agggaagcta 11440ctgaattata acacgtaaga aaatcaccat tccgtattgg ttgggggctc ctgtttctca 11500tcctagcttt ttcctggaaa gcccgctaga aggtttggga acgaggggaa agttctcaga 11560actgttggct gctccccacc cgcctcccgc ctcccccgca ggttatgtca gcagctctga 11620gacagcagta tcacaggcca gatgttgttc ctggctagat gtttacattt gtaagaaata 11680acactgtgaa tgtaaaacag agccattccc ttggaatgca tatcgctggg ctcaacatag 11740agtttgtctt cctcttgttt acgacgtgat ctaaaccagt ccttagcaag gggctcagaa 11800caccccgctc tggcagtagg tgtcccccac ccccaaagac ctgcctgtgt gctccggaga 11860tgaatatgag ctcattagta aaaatgactt cacccacgca tatacataaa gtatccatgc 11920atgtgcatat agacacatct ataattttac acacacacct ctcaagacgg agatgcatgg 11980cctctaagag tgcccgtgtc ggttcttcct ggaagttgac tttccttaga cccgccaggt 12040caagttagcc gcgtgacgga catccaggcg tgggacgtgg tcagggcagg gctcattcat 12100tgcccactag gatcccactg gcgaagatgg tctccatatc agctctctgc agaagggagg 12160aagactttat catgttccta aaaatctgtg gcaagcaccc atcgtattat ccaaattttg 12220ttgcaaatgt gattaatttg gttgtcaagt tttgggggtg ggctgtgggg agattgcttt 12280tgttttcctg ctggtaatat cgggaaagat tttaatgaaa ccagggtaga attgtttggc 12340aatgcactga agcgtgtttc tttcccaaaa tgtgcctccc ttccgctgcg ggcccagctg 12400agtctatgta ggtgatgttt ccagctgcca agtgctcttt gttactgtcc accctcattt 12460ctgccagcgc atgtgtcctt tcaaggggaa aatgtgaagc tgaaccccct ccagacaccc 12520agaatgtagc atctgagaag gccctgtgcc ctaaaggaca cccctcgccc ccatcttcat 12580ggagggggtc atttcagagc cctcggagcc aatgaacagc tcctcctctt ggagctgaga 12640tgagccccac gtggagctcg ggacggatag tagacagcaa taactcggtg tgtggccgcc 12700tggcaggtgg aacttcctcc cgttgcgggg tggagtgagg ttagttctgt gtgtctggtg 12760ggtggagtca ggcttctctt gctacctgtg agcatccttc ccagcagaca tcctcatcgg 12820gctttgtccc tcccccgctt cctccctctg cggggaggac ccgggaccac agctgctggc 12880cagggtagac ttggagctgt cctccagagg ggtcacgtgt aggagtgaga agaaggaaga 12940tcttgagagc tgctgaggga ccttggagag ctcaggatgg ctcagacgag gacactcgct 13000tgccgggcct gggcctcctg ggaaggaggg agctgctcag aatgccgcat gacaactgaa 13060ggcaacctgg aaggttcagg ggccgctctt cccccatgtg cctgtcacgc tctggtgcag 13120tcaaaggaac gccttcccct cagttgtttc taagagcaga gtctcccgct gcaatctggg 13180tggtaactgc cagccttgga ggatcgtggc caacgtggac ctgcctacgg agggtgggct 13240ctgacccaag tggggcctcc ttgtccaggt ctcactgctt tgcaccgtgg tcagagggac 13300tgtcagctga gcttgagctc ccctggagcc agcagggctg tgatgggcga gtcccggagc 13360cccacccaga cctgaatgct tctgagagca aagggaagga ctgacgagag atgtatattt 13420aattttttaa ctgctgcaaa cattgtacat ccaaattaaa ggaaaaaaat ggaaaccatc 13480a 13481320DNAArtificial sequencePrimer 3gggaccctga tcaacaccat 20424DNAArtificial sequencePrimer 4ccagttcttg attttgtcga aaca 24518DNAArtificial sequenceProbe 5tgggtggtct ccgcggcc 18616DNAArtificial sequenceSynthetic oligonucleotide 6nnnnnnnnnn nnnnnn 16714DNAArtificial sequenceSynthetic oligonucleotide 7nnnnnnnnnn nnnn 14820DNAArtificial sequenceSynthetic oligonucleotide 8nnnnnnnnnn nnnnnnnnnn 20920DNAArtificial sequenceSynthetic oligonucleotide 9ccttccctga aggttcctcc 201015DNAArtificial sequenceSynthetic oligonucleotide 10taaattgtca tcacc 151115DNAArtificial sequenceSynthetic oligonucleotide 11taaattguca tcacc 151215DNAArtificial sequenceSynthetic oligonucleotide 12taaattgnca tcacc 151315DNAArtificial sequenceSynthetic oligonucleotide 13taaautgtca tcacc 151416DNAArtificial sequenceSynthetic oligonucleotide 14taaattgtca tcacca 161518DNAArtificial sequenceSynthetic oligonucleotide 15taaattgtca tcacctta 181620DNAArtificial sequenceSynthetic oligonucleotide 16taaattgtca tcaccattta 201717DNAArtificial sequenceSynthetic oligonucleotide 17taaattgtca tcaccta 171819DNAArtificial sequenceSynthetic oligonucleotide 18taaattgtca tcaccttta 191916DNAArtificial sequenceSynthetic oligonucleotide 19gtaaattgtc atcacc 162017DNAArtificial sequenceSynthetic oligonucleotide 20ggtaaattgt catcacc 172118DNAArtificial sequenceSynthetic oligonucleotide 21ggttaaattg tcatcacc 182219DNAArtificial sequenceSynthetic oligonucleotide 22ggtgtaaatt gtcatcacc 192320DNAArtificial sequenceSynthetic oligonucleotide 23ggtgataaat tgtcatcacc 202421DNAArtificial sequenceSynthetic oligonucleotide 24ggctaaattg tcatcaccgc c 212519DNAArtificial sequenceSynthetic oligonucleotide 25gctaaattgt catcaccgc 192621DNAArtificial sequenceSynthetic oligonucleotide 26taataaattg tcatcacctt a 212719DNAArtificial sequenceSynthetic oligonucleotide 27aataaattgt catcacctt 192821DNAArtificial sequenceSynthetic oligonucleotide 28tcttaaattg tcatcaccag a 212919DNAArtificial sequenceSynthetic oligonucleotide 29cttaaattgt catcaccag 193018DNAArtificial sequenceSynthetic oligonucleotide 30taataaattg tcatcacc 183117DNAArtificial sequenceSynthetic oligonucleotide 31aataaattgt catcacc 173217DNAArtificial sequenceSynthetic oligonucleotide 32ataaattgtc atcacca 173317DNAArtificial sequenceSynthetic oligonucleotide 33ataaattctc atcacca 173417DNAArtificial sequenceSynthetic oligonucleotide 34ataaatagtc atcacca 173517DNAArtificial sequenceSynthetic oligonucleotide 35ataaattgtg atcacca 173617DNAArtificial sequenceSynthetic oligonucleotide 36ataaattgtc ttcacca 173717DNAArtificial sequenceSynthetic oligonucleotide 37ataaattgtc aacacca 173815DNAArtificial sequenceSynthetic oligonucleotide 38taaattctca tcacc 153915DNAArtificial sequenceSynthetic oligonucleotide 39taaaatgtca tcacc 154015DNAArtificial sequenceSynthetic oligonucleotide 40taaattgtga tcacc 154115DNAArtificial sequenceSynthetic oligonucleotide 41taaattgtct tcacc 154215DNAArtificial sequenceSynthetic oligonucleotide 42taaattgtca acacc 154317DNAArtificial sequenceSynthetic oligonucleotide 43ttaaattgtc atcacca 174417DNAArtificial sequenceSynthetic oligonucleotide 44aaaaattgtc atcacca 174517DNAArtificial sequenceSynthetic oligonucleotide 45attaattgtc atcacca 174617DNAArtificial sequenceSynthetic oligonucleotide 46atatattgtc atcacca 174717DNAArtificial sequenceSynthetic oligonucleotide 47ataatttgtc atcacca 174817DNAArtificial sequenceSynthetic oligonucleotide 48ataaattgtc atgacca 174917DNAArtificial sequenceSynthetic oligonucleotide 49ataaattgtc atctcca 175017DNAArtificial sequenceSynthetic oligonucleotide 50ataaattgtc atcagca 175117DNAArtificial sequenceSynthetic oligonucleotide 51ataaattgtc atcacga 175217DNAArtificial sequenceSynthetic oligonucleotide 52ataaattgtc atcacct 175315DNAArtificial sequenceSynthetic oligonucleotide 53taagttgtca tcacc 155415DNAArtificial sequenceSynthetic oligonucleotide 54taaagtgtca tcacc 155515DNAArtificial sequenceSynthetic oligonucleotide 55taaatggtca tcacc 155620DNAArtificial sequenceSynthetic oligonucleotide 56tctctattgc acattccaag 205720DNAArtificial sequenceSynthetic oligonucleotide 57ccttccctga aggttcctcc 205817DNAArtificial sequenceSynthetic oligonucleotide 58aattgtcatc accagaa 175917DNAArtificial sequenceSynthetic oligonucleotide 59aattgtcatc accagaa 176017DNAArtificial sequenceSynthetic oligonucleotide 60aaattgtcat caccaga 176117DNAArtificial sequenceSynthetic oligonucleotide 61taaattgtca tcaccag 176217DNAArtificial sequenceSynthetic oligonucleotide 62aataaattgt catcacc 176317DNAArtificial sequenceSynthetic oligonucleotide 63taataaattg tcatcac 176417DNAArtificial sequenceSynthetic oligonucleotide 64ttaataaatt gtcatca 176515DNAArtificial sequenceSynthetic oligonucleotide 65attgtcatca ccaga 156615DNAArtificial sequenceSynthetic oligonucleotide 66ttaataaatt gtcat 156715DNAArtificial sequenceSynthetic oligonucleotide 67ttgtcatcac cagaa 156815DNAArtificial sequenceSynthetic oligonucleotide 68aattgtcatc accag 156915DNAArtificial sequenceSynthetic oligonucleotide 69aaattgtcat cacca 157015DNAArtificial sequenceSynthetic oligonucleotide 70aataaattgt catca 157115DNAArtificial sequenceSynthetic oligonucleotide 71attaataaat tgtca 157215DNAArtificial sequenceSynthetic oligonucleotide 72tattaataaa ttgtc 157315DNAArtificial sequenceSynthetic oligonucleotide 73gtcatcacca gaaaa 157415DNAArtificial sequenceSynthetic oligonucleotide 74tgtcatcacc agaaa 157515DNAArtificial sequenceSynthetic oligonucleotide 75ataaattgtc atcac 157615DNAArtificial sequenceSynthetic oligonucleotide 76taataaattg tcatc 157717DNAArtificial sequenceSynthetic oligonucleotide 77ataaaatgtc atcacca 177815DNAArtificial sequenceSynthetic oligonucleotide 78taaatagtca tcacc 157920DNAArtificial sequenceSynthetic oligonucleotide 79ctcgactaaa gcaggatttc 208019DNAArtificial sequenceSynthetic oligonucleotide 80aataaattgt catcaccag 198119DNAArtificial sequenceSynthetic oligonucleotide 81cacagtgcta cccaacctt 198219DNAArtificial sequenceSynthetic oligonucleotide 82tcacagctat cttctcatc 198320DNAArtificial sequenceSynthetic oligonucleotide 83tcccatttca ggagacctgg 208420DNAArtificial sequenceSynthetic oligonucleotide 84gctgattaga gagaggtccc 208516DNAArtificial sequenceSynthetic oligonucleotide 85atcatggctg cagctt 168616DNAArtificial sequenceSynthetic oligonucleotide 86ttcagtcatg acttcc 168716DNAArtificial sequenceSynthetic oligonucleotide 87tggctgcagc ttccga 168816DNAArtificial sequenceSynthetic oligonucleotide 88atggctgcag cttccg 168916DNAArtificial sequenceSynthetic oligonucleotide 89catggctgca gcttcc 169016DNAArtificial sequenceSynthetic oligonucleotide 90tcatggctgc agcttc 169116DNAArtificial sequenceSynthetic oligonucleotide 91catcatggct gcagct 169216DNAArtificial sequenceSynthetic oligonucleotide 92ccatcatggc tgcagc 169316DNAArtificial sequenceSynthetic oligonucleotide 93tccatcatgg ctgcag 169416DNAArtificial sequenceSynthetic oligonucleotide 94ttccatcatg gctgca 16

Claims

1.-272. (canceled)

273. A oligomeric compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides, wherein the modified oligonucleotide has a modification motif comprising: a 5'-region consisting of 2-8 linked 5'-region nucleosides, each independently selected from among a modified nucleoside and an unmodified deoxynucleoside, provided that at least one 5'-region nucleoside is a modified nucleoside and wherein the 3'-most 5'-region nucleoside is a modified nucleoside; a 3'-region consisting of 2-8 linked 3'-region nucleosides, each independently selected from among a modified nucleoside and an unmodified deoxynucleoside, provided that at least one 3'-region nucleoside is a modified nucleoside and wherein the 5'-most 3'-region nucleoside is a modified nucleoside; and a central region between the 5'-region and the 3'-region consisting of 6-12 linked central region nucleosides, each independently selected from among: a modified nucleoside and an unmodified deoxynucleoside, wherein the 5'-most central region nucleoside is an unmodified deoxynucleoside and the 3'-most central region nucleoside is an unmodified deoxynucleoside; wherein the modified oligonucleotide has a nucleobase sequence complementary to the nucleobase sequence of a target region of a target nucleic acid.

274. The oligomeric compound of claim 273, wherein the 5'-region has a motif selected from among: AB, ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, AAAB, ABBW, ABBWW, ABBB, ABBBB, ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW, AAAAA, and BBBBAA; wherein the 3'-region has a motif selected from among: BBA, AAB, AAA, BBB, BBAA, AABB, WBBA, WAAB, BBBA, BBBBA, BBBB, BBBBBA, ABBBBB, BBAAA, AABBB, BBBAA, BBBBA, BBBBB, BABA, AAAAA, BBAAAA, AABBBB, BAAAA, and ABBBB; wherein the central region has a nucleoside motif selected from among: DDDDDD, DDDDDDD, DDDDDDDD, DDDDDDDDD, DDDDDDDDDD, DDDDDDDDD, DXDDDDDDD, DDXDDDDDD, DDDXDDDDD, DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, DDDDDDDXD, DXXDDDDDD, DDDDDDXXD, DDXXDDDDD, DDDXXDDDD, DDDDXXDDD, DDDDDXXDD, DXDDDDDXD, DXDDDDXDD, DXDDDXDDD, DXDDXDDDD, DXDXDDDDD, DDXDDDDXD, DDXDDDXDD, DDXDDXDDD, DDXDXDDDD, DDDXDDDXD, DDDXDDXDD, DDDXDXDDD, DDDDXDDXD, DDDDXDXDD, and DDDDDXDXD, DDDDDDDD, DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDXD, DXDDDXDD, DXDDXDDD, DXDXDDDD, DXXDDDDD, DDXXDDDD, DDXDXDDD, DDXDDXDD, DXDDDDXD, DDDXXDDD, DDDXDXDD, DDDXDDXD, DDDDXXDD, DDDDXDXD, and DDDDDXXD, DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD, DXDDDXD, DXDDXDD, DXDXDDD, DXXDDDD, DDXXDDD, DDXDXDD, DDXDDXD, DDDXXDD, DDDXDXD, and DDDDXXD, DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXXDDD, DXDXDD, DXDDXD, DDXXDD, DDXDXD, and DDDXXD; and wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, each W is a modified nucleoside of a first type, a second type, or a third type, each D is an unmodified deoxynucleoside, and each X is a modified nucleoside or a modified nucleobase.

275. The oligomeric compound of claim 274, wherein the 5'-region has a motif selected from among: AB, ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, ABBW, ABBWW, ABBB, ABBBB, ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW, and BBBBAA; and wherein the 3'-region has a BBA motif.

276. The oligomeric compound of claim 274, wherein each A nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, .alpha.-LNA, ENA and 2'-thio LNA.

277. The oligomeric compound of claim 274, wherein each A nucleoside comprises a cEt.

278. The oligomeric compound of claim 276, wherein each B nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: a halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2, OCH.sub.2C(.dbd.O)--N(H)CH.sub.3, OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2, and OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2.

279. The oligomeric compound of claim 278, wherein each B nucleoside comprises a 2'-substituted sugar moiety comprising a 2'-substituent selected from among: F, OCH.sub.3, O(CH.sub.2).sub.2--OCH.sub.3.

280. The oligomeric compound of claim 274, wherein one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.

281. The oligomeric compound of claim 274, wherein one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2'-MOE sugar moiety, and W comprises FHNA.

282. The oligomeric compound of claim 274, wherein one of A or B comprises cEt, another of A or B comprises a 2'-modified sugar moiety, and W comprises a 2-thio-thymidine nucleobase.

283. The oligomeric compound of claim 274, wherein one of A or B comprises cEt, another of A or B comprises a 2'-modified sugar moiety, and W comprises FHNA.

284. The oligomeric compound of claim 274, wherein each A comprises MOE, each B comprises cEt, and each W is selected from among cEt or FHNA.

285. The oligomeric compound of claim 284, wherein each W comprises cEt.

286. The oligomeric compound of claim 284, wherein each W comprises 2-thio-thymidine.

287. The oligomeric compound of claim 284, wherein each W comprises FHNA.

288. The oligomeric compound of claim 274 comprising at least one modified internucleoside linkage.

289. The oligomeric compound of claim 288, wherein each internucleoside linkage is a modified internucleoside linkage.

290. The oligomeric compound of claim 289 comprising at least one phosphorothioate internucleoside linkage.

291. The oligomeric compound of claim 288 comprising at least one methylphosphonate internucleoside linkage.

292. The oligomeric compound of claim 275, wherein each A nucleoside comprises a bicyclic sugar moiety selected from among: cEt and LNA and each B nucleoside comprises a 2'-MOE.