ANTISENSE COMPOUNDS AND USES THEREOF

Abstract

The present invention provides compounds comprising oligonucleotides complementary to a pyruvate kinase M transcript. Certain such compounds are useful for hybridizing to a pyruvate kinase M transcript, including but not limited to a pyruvate kinase M transcript in a cell. In certain embodiments, such hybridization results in modulation of splicing of the pyruvate kinase M transcript. In certain embodiments, such compounds are used to treat one or more symptoms associated with cancer.

Description

SEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0206WOSEQ.txt, created Oct. 30, 2013, which is 88 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND

[0002] The pyruvate kinase M (PK-M) gene has 12 exons. Exon 9 and exon 10 are alternatively spliced in a mutually exclusive fashion to give rise to the M1 and M2 isoforms of the PK-M gene. Inclusion of exon 9 and exclusion of exon 10 yields the PK-M1 isoform. Exclusion of exon 9 and inclusion of exon 10 yields the PK-M2 isoform. Exons 9 and 10 each encode a 56 amino acid segment that confers distinctive properties to the respective PK-M1 and PK-M2 isoforms. The PK-M2 isoform is expressed in a broad range of cancer cells, whereas PK-M1 is predominantly expressed in terminally differentiated tissues.

[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.

[0004] Certain antisense compounds have been described previously. See for example U.S. Pat. No. 7,399,845 and published International Patent Application No. WO 2008/049085, which are hereby incorporated by reference herein in their entirety.

SUMMARY

[0005] In certain embodiments, the present invention provides compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides are complementary to a pyruvate kinase M (PK-M) transcript. In certain such embodiments, oligonucleotides are complementary to a target region of the PK-M transcript comprising exon 10. In certain such embodiments, oligonucleotides are complementary to a target region of the PK-M transcript comprising an intron adjacent to exon 10. In certain such embodiments, oligonucleotides are complementary to a target region of the PK-M transcript comprising an intron adjacent to exon 10 and downstream of exon 10. In certain such embodiments, oligonucleotides are complementary to a target region of the PK-M transcript comprising an intron adjacent to exon 10 and upstream of exon 10. In certain embodiments, the PK-M transcript comprises an exonic splice enhancer for exon 10. In certain embodiments, oligonucleotides inhibit inclusion of exon 10. In certain embodiments, oligonucleotides promote skipping of exon 10. In certain embodiments, oligonucleotides promote selection of exon 9. In certain embodiments, oligonucleotides promote skipping of exon 10 and promote inclusion of exon 9. In certain such embodiments, PK-M mRNA with exon 9 mRNA is increased. In certain such embodiments, PK-M mRNA with exon 10 mRNA is decreased. In certain embodiments, the PK-M2 isoform of the PK-M protein is decreased. In certain embodiments, the PK-M1 isoform of the PK-M protein is decreased.

[0006] In certain embodiments, including, but not limited to any of the above numbered embodiments, the PK-M transcript is in a human. In certain embodiments, including, but not limited to any of the above numbered embodiments, the PK-M transcript is in a mouse.

[0007] The present disclosure provides the following non-limiting numbered embodiments:

Embodiment 1

[0008] A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region, wherein the complementary region comprises at least 8 contiguous nucleobases and is complementary to an equal-length portion within a target region of a PK-M transcript.

Embodiment 2

[0009] The compound of embodiment 1, wherein the target region of the PK-M transcript comprises exon 10 of the PK-M transcript.

Embodiment 3

[0010] The compound of embodiment 1 or 2, wherein the complementary region of the modified oligonucleotide is 100% complementary to the target region.

Embodiment 4

[0011] The compound of any of embodiments 1 to 3, wherein the complementary region of the modified oligonucleotide comprises at least 10 contiguous nucleobases.

Embodiment 5

[0012] The compound of any of embodiments 1 to 3, wherein the complementary region of the modified oligonucleotide comprises at least 15 contiguous nucleobases.

Embodiment 6

[0013] The compound of any of embodiments 1 to 3, wherein the complementary region of the modified oligonucleotide comprises at least 20 contiguous nucleobases.

Embodiment 7

[0014] The compound of any of embodiments 1-6, wherein the nucleobase sequence of the oligonucleotide is at least 80% complementary to the target region, as measured over the entire length of the oligonucleotide.

Embodiment 8

[0015] The compound of any of embodiments 1-6, wherein the nucleobase sequence of the oligonucleotide is at least 90% complementary to an equal-length region of the PK-M transcript, as measured over the entire length of the oligonucleotide.

Embodiment 9

[0016] The compound of any of embodiments 1-6, wherein the nucleobase sequence of the oligonucleotide is 100% complementary to an equal-length region of the PK-M transcript, as measured over the entire length of the oligonucleotide.

Embodiment 10

[0017] The compound of any of embodiments 1-9, wherein the target region is within exon 10 of the PK-M transcript.

Embodiment 11

[0018] The compound of any of embodiments 1-10, wherein the target region is within nucleobase 29153 and nucleobase 29281 of SEQ ID NO.: 1.

Embodiment 12

[0019] The compound of any of embodiments 1-10, wherein the target region is within nucleobase 29158 and nucleobase 29262 of SEQ ID NO.: 1.

Embodiment 13

[0020] The compound of any of embodiments 1-10, wherein the target region is within nucleobase 29164 and nucleobase 29188 of SEQ ID NO.: 1.

Embodiment 14

[0021] The compound of any of embodiments 1-10, wherein the target region is within nucleobase 29261 and nucleobase 29279 of SEQ ID NO.: 1.

Embodiment 15

[0022] The compound of any of embodiments 1-10, wherein the target region is within nucleobase 29168 and nucleobase 29183 of SEQ ID NO.: 1.

Embodiment 16

[0023] The compound of any of embodiments 1-15, wherein the antisense oligonucleotide comprises any one of SEQ ID NOs: 4 to 36.

Embodiment 17

[0024] The compound of any of embodiments 1-16, wherein the modified oligonucleotide comprises at least one modified nucleoside.

Embodiment 18

[0025] The compound of embodiment 17, wherein at least one modified nucleoside comprises a modified sugar moiety.

Embodiment 19

[0026] The compound of embodiment 18, wherein at least one modified sugar moiety is a 2'-substituted sugar moiety.

Embodiment 20

[0027] The compound of embodiment 19, wherein the 2'-substitutent of at least one 2'-substituted sugar moiety is selected from among: 2'-OMe, 2'-F, and 2'-MOE.

Embodiment 21

[0028] The compound of any of embodiments 17-20, wherein the 2'-substituent of at least one 2'-substituted sugar moiety is a 2'-MOE.

Embodiment 22

[0029] The compound of any of embodiments 1-18, wherein at least one modified sugar moiety is a bicyclic sugar moiety.

Embodiment 23

[0030] The compound of embodiment 22, wherein at least one bicyclic sugar moiety is LNA or cEt.

Embodiment 24

[0031] The compound of any of embodiments 18-23, wherein at least one sugar moiety is a sugar surrogate.

Embodiment 25

[0032] The compound of embodiment 24, wherein at least one sugar surrogate is a morpholino.

Embodiment 26

[0033] The compound of embodiment 24, wherein at least one sugar surrogate is a modified morpholino.

Embodiment 27

[0034] The compound of any of embodiment 1-26, wherein the modified oligonucleotide comprises at least 5 modified nucleosides, each independently comprising a modified sugar moiety.

Embodiment 28

[0035] The compound of embodiment 27, wherein the modified oligonucleotide comprises at least 10 modified nucleosides, each independently comprising a modified sugar moiety.

Embodiment 29

[0036] The compound of embodiment 27, wherein the modified oligonucleotide comprises at least 15 modified nucleosides, each independently comprising a modified sugar moiety.

Embodiment 30

[0037] The compound of embodiment 27, wherein each nucleoside of the modified oligonucleotide is a modified nucleoside, each independently comprising a modified sugar moiety

Embodiment 31

[0038] The compound of any of embodiments 1-30, wherein the modified oligonucleotide comprises at least two modified nucleosides comprising modified sugar moieties that are the same as one another.

Embodiment 32

[0039] The compound of any of embodiments 1-31, wherein the modified oligonucleotide comprises at least two modified nucleosides comprising modified sugar moieties that are different from one another.

Embodiment 33

[0040] The compound of any of embodiments 1-32, wherein the modified oligonucleotide comprises a modified region of at least 5 contiguous modified nucleosides.

Embodiment 34

[0041] The compound of embodiment 33, wherein the modified oligonucleotide comprises a modified region of at least 10 contiguous modified nucleosides.

Embodiment 35

[0042] The compound of embodiment 33, wherein the modified oligonucleotide comprises a modified region of at least 15 contiguous modified nucleosides.

Embodiment 36

[0043] The compound of embodiment 33, wherein the modified oligonucleotide comprises a modified region of at least 20 contiguous modified nucleosides.

Embodiment 37

[0044] The compound of any of embodiments 32-36, wherein each modified nucleoside of the modified region has a modified sugar moiety independently selected from among: 2'-F, 2'-OMe, 2'-MOE, cEt, LNA, morpholino, and modified morpholino.

Embodiment 38

[0045] The compound of any of embodiments 33-37, wherein the modified nucleosides of the modified region each comprise the same modification as one another.

Embodiment 39

[0046] The compound of embodiment 38, wherein the modified nucleosides of the modified region each comprise the same 2'-substituted sugar moiety.

Embodiment 40

[0047] The compound of embodiment 38, wherein the 2'-substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from 2'-F, 2'-OMe, and 2'-MOE.

Embodiment 41

[0048] The compound of embodiment 39, wherein the 2'-substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is 2'-MOE.

Embodiment 42

[0049] The compound of embodiment 38, wherein the modified nucleosides of the region of modified nucleosides each comprise the same bicyclic sugar moiety.

Embodiment 43

[0050] The compound of embodiment 42, wherein the bicyclic sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from LNA and cEt.

Embodiment 44

[0051] The compound of embodiment 38, wherein the modified nucleosides of the region of modified nucleosides each comprises a sugar surrogate.

Embodiment 45

[0052] The compound of embodiment 44, wherein the sugar surrogate of the modified nucleosides of the region of modified nucleosides is a morpholino.

Embodiment 46

[0053] The compound of embodiment 44, wherein the sugar surrogate of the modified nucleosides of the region of modified nucleosides is a modified morpholino.

Embodiment 47

[0054] The compound of any of embodiments 1-46, wherein the modified nucleotide comprises no more than 4 contiguous naturally occurring nucleosides.

Embodiment 48

[0055] The compound of any of embodiments 1-46, wherein each nucleoside of the modified oligonucleotide is a modified nucleoside.

Embodiment 49

[0056] The compound of embodiment 48 wherein each modified nucleoside comprises a modified sugar moiety.

Embodiment 50

[0057] The compound of embodiment 49, wherein the modified nucleosides of the modified oligonucleotide comprise the same modification as one another.

Embodiment 51

[0058] The compound of embodiment 50, wherein the modified nucleosides of the modified oligonucleotide each comprise the same 2'-substituted sugar moiety.

Embodiment 52

[0059] The compound of embodiment 51, wherein the 2'-substituted sugar moiety of the modified oligonucleotide is selected from 2'-F, 2'-OMe, and 2'-MOE.

Embodiment 53

[0060] The compound of embodiment 52, wherein the 2'-substituted sugar moiety of the modified oligonucleotide is 2'-MOE.

Embodiment 54

[0061] The compound of embodiment 50, wherein the modified nucleosides of the modified oligonucleotide each comprise the same bicyclic sugar moiety.

Embodiment 55

[0062] The compound of embodiment 54, wherein the bicyclic sugar moiety of the modified oligonucleotide is selected from LNA and cEt.

Embodiment 56

[0063] The compound of embodiment 50, wherein the modified nucleosides of the modified oligonucleotide each comprises a sugar surrogate.

Embodiment 57

[0064] The compound of embodiment 56, wherein the sugar surrogate of the modified oligonucleotide is a morpholino.

Embodiment 58

[0065] The compound of embodiment 56, wherein the sugar surrogate of the modified oligonucleotide is a modified morpholino.

Embodiment 59

[0066] The compound of any of embodiments 1-58, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.

Embodiment 60

[0067] The compound of embodiment 59, wherein each internucleoside linkage is a modified internucleoside linkage.

Embodiment 61

[0068] The compound of embodiment 59 or 60, comprising at least one phosphorothioate internucleoside linkage.

Embodiment 62

[0069] The compound of embodiment 60, wherein each internucleoside linkage is a modified internucleoside linkage and wherein each internucleoside linkage comprises the same modification.

Embodiment 63

[0070] The compound of embodiment 62, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.

Embodiment 64

[0071] The compound of any of embodiments 1-63 comprising at least one conjugate.

Embodiment 65

[0072] The compound of any of embodiments 1-64 consisting of the modified oligonucleotide.

Embodiment 66

[0073] The compound of any of embodiments 1-65, wherein the compound modulates splicing of the PK-M transcript.

Embodiment 67

[0074] The compound of any of embodiments 1-66, having a nucleobase sequence comprising any of the sequences as set forth in SEQ ID NOs. 4 to 36.

Embodiment 68

[0075] The compound of any of embodiments 1-66, having a nucleobase sequence comprising any of the sequences as set forth in SEQ ID NOs. 4 to 17.

Embodiment 69

[0076] The compound of any of embodiments 1-66, having a nucleobase sequence comprising any of the sequences as set forth in SEQ ID NOs. 18 to 28.

Embodiment 70

[0077] The compound of any of embodiments 1-66, having a nucleobase sequence comprising any of the sequences as set forth in SEQ ID NOs. 29 to 36.

Embodiment 71

[0078] The compound of any of embodiments 1-66, having a nucleobase sequence comprising SEQ ID NO. 32.

Embodiment 72

[0079] The compound of any of embodiments 1-66, having a nucleobase sequence comprising SEQ ID NO. 7.

Embodiment 73

[0080] The compound of any of embodiments 1-66, having a nucleobase sequence comprising SEQ ID NO. 24.

Embodiment 74

[0081] A pharmaceutical composition comprising a compound according to any of embodiments 1-73 and a pharmaceutically acceptable carrier or diluent.

Embodiment 75

[0082] The pharmaceutical composition of embodiment 74, wherein the pharmaceutically acceptable carrier or diluent is sterile saline.

Embodiment 76

[0083] A method of modulating splicing of a PK-M transcript in a cell comprising contacting the cell with a compound according to any of embodiments 1-75.

Embodiment 77

[0084] The method of embodiment 76, wherein the cell is in vitro.

Embodiment 78

[0085] The method of embodiment 76, wherein the cell is in an animal.

Embodiment 79

[0086] The method of any of embodiments 76-78, wherein inclusion of exon 9 is increased.

Embodiment 80

[0087] The method of any of embodiments 76-78, wherein exclusion of exon 10 is increased.

Embodiment 81

[0088] The method of any of embodiments 76-78, wherein inclusion of exon 10 is decreased.

Embodiment 82

[0089] The method of any of embodiments 76-78, wherein PK-M1 mRNA expression is increased.

Embodiment 83

[0090] The method of any of embodiments 76-78, wherein PK-M2 mRNA expression is decreased.

Embodiment 84

[0091] A method of modulating the expression of PK-M in a cell, comprising contacting the cell with a compound according to any of embodiments 1-75.

Embodiment 85

[0092] The method of embodiment 84, wherein PK-M1 expression is increased.

Embodiment 86

[0093] The method of embodiments 84 or 85, wherein PK-M2 expression is decreased.

Embodiment 87

[0094] The method of embodiment 84, wherein the cell is in vitro.

Embodiment 88

[0095] The method of embodiment 84, wherein the cell is in an animal.

Embodiment 89

[0096] A method of inducing apoptosis in a cell, comprising contacting the cell with a compound according to any of embodiments 1-75.

Embodiment 90

[0097] The method of embodiment 89, wherein the cell is in vitro.

Embodiment 91

[0098] The method of embodiment 89, wherein the cell is in an animal.

Embodiment 92

[0099] A method comprising administering the compound according to any of embodiments 1-67 or the pharmaceutical composition of embodiments 74 or 75 to an animal.

Embodiment 93

[0100] The method of embodiment 92, wherein the administration is intracerebroventricular.

Embodiment 94

[0101] The method of embodiment 92, wherein the administration is into the central nervous system.

Embodiment 95

[0102] The method of any of embodiments 92-94, wherein the animal has one or more symptoms associated with cancer.

Embodiment 96

[0103] The method embodiment 95, wherein the cancer is glioblastoma.

Embodiment 97

[0104] The method of embodiment 96, wherein the administration results in amelioration of at least one symptom of cancer.

Embodiment 98

[0105] The method of any of embodiments 91-97, wherein the animal is a mouse.

Embodiment 99

[0106] The method of any of embodiments 91-97, wherein the animal is a human.

Embodiment 100

[0107] A method of preventing or retarding the growth of a cancerous tumor, comprising administering the compound according to any of embodiments 1-73 or the pharmaceutical composition of embodiments 74 or 75 to an animal in need thereof.

Embodiment 101

[0108] The method of embodiment 100, wherein the animal is a mouse.

Embodiment 102

[0109] The method of embodiment 100, wherein the animal is a human.

Embodiment 103

[0110] The method of embodiment 100 to 102, wherein the cancerous tumor comprises glioblastoma.

Embodiment 104

[0111] Use of the compound according to any of embodiments 1-73 or the pharmaceutical composition of embodiments 74 or 75 for the preparation of a medicament for use in the treatment of cancer.

Embodiment 105

[0112] Use of the compound according to any of embodiments 1-73 or the pharmaceutical composition of embodiments 74 or 75 for the preparation of a medicament for use in the amelioration of one or more symptoms cancer.

Embodiment 106

[0113] The use of embodiment 104 or 105, wherein the cancer is glioblastoma.

BRIEF DESCRIPTION OF THE FIGURES

[0114] FIG. 1a: Diagram of the PK-M genomic region. This region comprises introns 8, 9, and 10 (represented by the lines) and portions of exon 8, intact exons 9 and 10, and portions of exon 11 (represented by boxes). Numbers above the boxes show the length in nucleotides. cDNA amplicons generated after radioactive RT-PCR are shown below and labeled accordingly. Three spliced species were observed: the shorter double-skipped species, comprising only exons 8 and 11 (D, 271 nucleotides); M1, including exon 9 (A, 398 nucleotides); and M2, including exon 10 (B, 398 nucleotides).

[0115] FIG. 1b: Initial ASO walks. ASOs were transfected at 30 nM in HEK-293 cells. Radioactive RT-PCR and restriction digest of endogenous PK-M transcripts are shown. The transfected ASO is indicated at the top. cDNA amplicons and fragments are indicated on the left. Lane numbers are indicated at the bottom.

[0116] FIG. 1c: ASO microwalks. ASOs were transfected at 60 nM in HEK-293 cells. Radioactive RT-PCR and restriction digest of endogenous PK-M transcripts are shown. The transfected ASO is indicated at the top. cDNA amplicons and fragments are indicated on the left. Lane numbers are indicated at the bottom.

[0117] FIG. 2a: Scheme of method used to duplicate the exon 10 10 W region into exon 9 in a minigene. Minigene mutant names are indicated below. The indicated exon 9 nucleotides at the top were mutated to the corresponding exon 10 sequences on the right. The ASOs that target 10 W and flanking regions are indicated below.

[0118] FIG. 2b: Mutant minigenes analyzed by transient transfection into HEK-293 cells, followed by radioactive RT-PCR and restriction digest, as in FIG. 1. Constructs from FIG. 2a are labeled at the top. The labeled bands are indicated in lower case on the left and right: uncut M1 fragment (a, 481 nucleotide); uncut M2 fragment (b, 481 nucleotides); PstI-cleaved M2 5' fragment (b2, 268 nucleotides); PstI-cleaved M2 3' fragment (b3, 213 nucleotides); a spliced mRNA that skips both exons 9 and 10 (d, 314 nucleotides); an exon 9-exon 10 doubly-included mRNA expressed from the 10B minigene (lanes 5 and 6) is indicated on the left (f, 648 nucleotides). This band is sensitive to PstI (fl, 435 nucleotides).

[0119] FIG. 2c: Wild-type minigene transcript level changes as a result of ASO co-transfection in HEK-293 cells. Labeled bands are indicated in lower case on the left.

[0120] FIG. 2d: Exon 10 duplication minigene transcript level changes as a result of ASO co-transfection in HEK-293 cells. Labeled bands are indicated in lower case on the left.

[0121] FIG. 2e: Alignment of the sequences of ISIS 461456, the complementary region in exon 10, and a homologous region in intron 9 is shown. Vertical lines show sequence identity. A diagram of the minigene mutants is shown on the right.

[0122] FIG. 2f: Minigene transcript level changes as a result of ASO co-transfection in HEK-293 cells. Labeled bands are indicated on the left: uncut M1 fragment (a, 481 nucleotide); uncut M2 fragment (b, 481 nucleotides); PstI-cleaved M2 5' fragment (b2, 268 nucleotides); PstI-cleaved M2 3' fragment (b3, 213 nucleotides); a spliced mRNA that skips both exons 9 and 10 (d, 314 nucleotides).

[0123] FIG. 3a: Effect of ISIS 549197, ISIS 461456, and ISIS 555158 on endogenous PK-M mRNAs in A172 and U87-MG glioblastoma cells.

[0124] FIG. 3b: Immunoblot analysis of PK-M protein isoform levels in A172 and U87-MG glioblastoma cells transfected with ISIS 549197, ISIS 461456, or ISIS 555158. Antibodies used are indicated on the left.

[0125] FIG. 3c: Immunofluroescence staining of glioblastoma cells with antibodies directed against PK-M2. Cell lines were stained with PK-M2 antibody and the DNA-binding fluorescent stain, DAPI.

[0126] FIG. 4: Immunoblot analysis of A172 cells transfected with ISIS 549197 or control ASO. Antibodies used are indicated on the left.

[0127] FIG. 5a: Flow cytometric analysis of A172 or U87-MG glioblastoma cells transfected with the indicated ASOs and stained with Annexin V-APC/7-AAD.

[0128] FIG. 5b: Dose-dependent apoptosis in glioblastoma cells by ASO treatment. Error bars represent s.d. (n=3).

[0129] FIG. 6a: Immunoblot analysis of A172 cells stably transduced with rtTA and doxycycline-inducible human T7-tagged PK-M1 cDNA. Antibodies used are indicated on the left.

[0130] FIG. 6b: Immunoblot analysis of A172 and U87-MG cells stably transduced with T7-tagged PK-M1 cDNA. Transduced cells and parental cell lines were transfected with ASOs. Antibodies used are indicated on the left.

[0131] FIG. 6c: Histogram analysis of cells grown as in FIG. 6a and transfected with the indicated ASOs. Doxycycline conditions are shown on the left. Error bars represent s.d. (n=3).

[0132] FIG. 6d: Histogram analysis of cells grown as in FIG. 6b and transfected with the indicated ASOs. Error bars represent s.d. (n=3).

[0133] FIG. 7: Immunoblot analysis of A172 cells independently transfected with four different PK-M2 siRNAs. Antibodies used are indicated on the left.

DETAILED DESCRIPTION

[0134] 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^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^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.

[0135] Unless otherwise indicated, the following terms have the following meanings:

[0136] 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.

[0137] As used herein, "chemical modification" means a chemical difference in a compound when compared to a naturally occurring counterpart. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications.

[0138] As used herein, "furanosyl" means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.

[0139] 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.

[0140] As used herein, "sugar moiety" means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.

[0141] As used herein, "modified sugar moiety" means a substituted sugar moiety, a bicyclic or tricyclic sugar moiety, or a sugar surrogate.

[0142] As used herein, "substituted sugar moiety" means a furanosyl comprising at least one substituent group that differs from that of a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2'-position, the 3'-position, the 5'-position and/or the 4'-position.

[0143] 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.

[0144] As used herein, "MOE" means --OCH₂CH₂OCH₃.

[0145] 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.

[0146] As used herein the term "sugar surrogate" means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside is capable of (1) incorporation into an oligonucleotide and (2) hybridization to a complementary nucleoside. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholino, modified morpholinos, cyclohexenyls and cyclohexitols.

[0147] 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).

[0148] 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.

[0149] As used herein, "heterocyclic base" or "heterocyclic nucleobase" means a nucleobase comprising a heterocyclic structure.

[0150] 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).

[0151] As used herein, "modified nucleobase" means any nucleobase that is not a naturally occurring nucleobase.

[0152] 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.

[0153] As used herein, "bicyclic nucleoside" or "BNA" means a nucleoside comprising a bicyclic sugar moiety.

[0154] As used herein, "constrained ethyl nucleoside" or "cEt" means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH₃)--O-2'bridge.

[0155] As used herein, "locked nucleic acid nucleoside" or "LNA" means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH₂--O-2'bridge.

[0156] 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.

[0157] 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).

[0158] 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.

[0159] As used herein "oligonucleoside" means an oligonucleotide in which none of the internucleoside linkages contains a phosphorus atom. As used herein, oligonucleotides include oligonucleosides.

[0160] As used herein, "modified oligonucleotide" means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.

[0161] As used herein "internucleoside linkage" means a covalent linkage between adjacent nucleosides in an oligonucleotide.

[0162] As used herein "naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage.

[0163] As used herein, "modified internucleoside linkage" means any internucleoside linkage other than a naturally occurring internucleoside linkage.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] As used herein, "conjugate linking group" means any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] As used herein, "detectable and/or measurable activity" means a statistically significant activity that is not zero.

[0172] 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.

[0173] 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.

[0174] As used herein, "target nucleic acid" means a nucleic acid molecule to which an antisense compound hybridizes.

[0175] As used herein, "mRNA" means an RNA molecule that encodes a protein.

[0176] As used herein, "pre-mRNA" means an RNA transcript that has not been fully processed into mRNA. Pre-RNA includes one or more intron.

[0177] As used herein, "transcript" means an RNA molecule transcribed from DNA. Transcripts include, but are not limited to mRNA, pre-mRNA, and partially processed RNA.

[0178] As used herein, "PK-M transcript" means a transcript transcribed from a PK-M gene. In certain embodiments, a PK-M transcript comprises SEQ ID NO: 1: the complement of GENBANK Accession No. NT_--010194.16 truncated from nucleotides 43281289 to 43314403.

[0179] As used herein, "PK-M gene" means a gene that encodes a pyruvate kinase M protein and any pyruvate kinase M protein isoforms. In certain embodiments, pyruvate kinase M protein isoforms include pyruvate kinase M1 and pyruvate kinase M2. In certain embodiments, a pyruvate kinase M gene is represented by GENBANK Accession No. NT_--010194.16 truncated from nucleotides 43281289 to 43314403, or a variant thereof. In certain embodiments, a pyruvate kinase M gene is at least 95% identical to GENBANK Accession No. NT_--010194.16 truncated from nucleotides 43281289 to 43314403. In certain embodiments, a pyruvate kinase M gene is at least 90% identical to GENBANK Accession No. NT_--010194.16 truncated from nucleotides 43281289 to 43314403.

[0180] As used herein, "PK-M1" means a pyruvate kinase M transcript that includes exon 9 but does not include exon 10.

[0181] As used herein, "PK-M1 isoform" means a pyruvate kinase M protein isoform that includes amino acids encoded from exon 9 but does not include amino acids encoded from exon 10.

[0182] As used herein, "PK-M2" means a pyruvate kinase M transcript that includes exon 10 but does not include exon 9.

[0183] As used herein, "PK-M2 isoform" means a pyruvate kinase M protein isoform that includes amino acids encoded from exon 10 but does not include amino acids encoded from exon 9.

[0184] 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.

[0185] 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.

[0186] As used herein, "non-complementary" in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] As used herein, "motif" means a pattern of chemical modifications in an oligomeric compound or a region thereof. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligomeric compound.

[0194] 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.

[0195] As used herein, "sugar motif" means a pattern of sugar modifications in an oligomeric compound or a region thereof.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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_aa), carboxyl (--C(O)O--R_aa), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (--O--R_aa), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (--N(R_bb)(R_cc)), imino(═NR_bb), amido (--C(O)N(R_bb)(R_cc) or --N(R_bb)C(O)R_aa), azido (--N₃), nitro (--NO₂), cyano (--CN), carbamido (--OC(O)N(R_bb)(R_cc) or --N(R_bb)C(O)OR_aa), ureido (--N(R_bb)C(O)N(R_bb)(R_cc)), thioureido (--N(R_bb)C(S)N(R_bb)--(R_cc)), guanidinyl (--N(R_bb)C(═NR_bb)N(R_bb)(R_cc)), amidinyl (--C(═NR_bb)N(R_bb)(R_cc) or --N(R_bb)C(═NR_bb)(R_aa)), thiol (--SR_bb), sulfinyl (--S(O)R_bb), sulfonyl (--S(O)₂R_bb) and sulfonamidyl (--S(O)₂N(R_bb)(R_cc) or --N(R_bb)S--(O)₂R_bb). Wherein each R_aa, R_bb and R_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.

[0205] 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 (C₁-C₁2 alkyl) with from 1 to about 6 carbon atoms being more preferred.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] As used herein, "aminoalkyl" means an amino substituted C₁-C₁2 alkyl radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.

[0214] As used herein, "aralkyl" and "arylalkyl" mean an aromatic group that is covalently linked to a C₁-C₁2 alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.

[0215] 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.

[0216] As used herein, "halo" and "halogen," mean an atom selected from fluorine, chlorine, bromine and iodine.

[0217] As used herein, "heteroaryl," and "heteroaromatic," mean a radical comprising a mono- or polycyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.

Oligomeric Compounds

[0218] In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, such oligomeric compounds comprise oligonucleotides optionally comprising one or more conjugate and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide. In certain embodiments, oligonucleotides comprise one or more chemical modifications. Such chemical modifications include modifications one or more nucleoside (including modifications to the sugar moiety and/or the nucleobase) and/or modifications to one or more internucleoside linkage.

[0219] Certain Sugar Moieties

[0220] In certain embodiments, oligomeric compounds of the invention comprise one or more modified nucleosides comprising a modified sugar moiety. Such oligomeric compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to oligomeric compounds comprising only nucleosides comprising naturally occurring sugar moieties. In certain embodiments, modified sugar moieties are substituted sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.

[0221] In certain embodiments, modified sugar moieties are substituted sugar moieties comprising one or more substituent, including but not limited to substituents at the 2' and/or 5' positions. Examples of sugar substituents suitable for the 2'-position, include, but are not limited to: 2'-F, 2'-OCH₃ ("OMe" or "O-methyl"), and 2'-O(CH₂)₂OCH₃ ("MOE"). In certain embodiments, sugar substituents at the 2' position is selected from allyl, amino, azido, thio, O-allyl, O--C₁-C₁0 alkyl, O--C₁-C₁0 substituted alkyl; O--C₁-C₁0 alkoxy; O--C₁-C₁0 substituted alkoxy, OCF₃, O(CH₂)₂SCH₃, O(CH₂)₂--O--N(Rm)(Rn), and O--CH₂--C(═O)--N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C₁-C₁0 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).

[0222] Nucleosides comprising 2'-substituted sugar moieties are referred to as 2'-substituted nucleosides. In certain embodiments, a 2'-substituted nucleoside comprises a 2'-substituent group selected from halo, allyl, amino, azido, O--C₁-C₁0 alkoxy; O--C₁-C₁0 substituted alkoxy, SH, CN, OCN, CF₃, OCF₃, O-alkyl, S-alkyl, N(R_m)-alkyl; O-alkenyl, S-alkenyl, or N(R_m)-alkenyl; O-alkynyl, S-alkynyl, N(R_m)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O--(CH₂)₂--O--N(R_m)(R_n) or O--CH₂--C(═O)--N(R_m)(R_n), where each R_m and R_n is, independently, H, an amino protecting group or substituted or unsubstituted C₁-C₁0 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 (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

[0223] In certain embodiments, a 2'-substituted nucleoside comprises a 2'-substituent group selected from F, NH₂, N₃, OCF₃, O--CH₃, O(CH₂)₃NH₂, CH₂--CH═CH₂, O--CH₂--CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O--(CH₂)₂--O--N(R_m)(R_n), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substituted acetamide (O--CH₂--C(═O)--N(R_m)(R_n) where each R_m and R_n is, independently, H, an amino protecting group or substituted or unsubstituted C₁-C₁0 alkyl.

[0224] In certain embodiments, a 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent group selected from F, OCF₃, O--CH₃, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O--(CH₂)₂--O--N(CH₃)₂, --O(CH₂)₂O(CH₂)₂N(CH₃)₂, and O--CH₂--C(═O)--N(H)CH₃.

[0225] In certain embodiments, a 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent group selected from F, O--CH₃, and OCH₂CH₂OCH₃.

[0226] 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_a)(R_b)]_n--, --[C(R_a)(R_b)]_n--O--, --C(R_aR_b)--N(R)--O-- or, --C(R_aR_b)--O--N(R)--; 4'-CH₂-2', 4'-(CH₂)₂-2', 4'-(CH₂)₃-2', 4'-(CH₂)--O-2' (LNA); 4'-(CH₂)--S-2; 4'-(CH₂)₂--O-2' (ENA); 4'-CH(CH₃)--O-2' (cEt) and 4'-CH(CH₂OCH₃)--O-2', and analogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4'-C(CH₃)(CH₃)--O-2' and analogs thereof, (see, e.g., WO2009/006478, published Jan. 8, 2009); 4'-CH₂--N(OCH₃)-2' and analogs thereof (see, e.g., WO2008/150729, published Dec. 11, 2008); 4'-CH₂--O--N(CH₃)-2' (see, e.g., US2004/0171570, published Sep. 2, 2004); 4'-CH₂--O--N(R)-2', and 4'-CH₂--N(R)--O-2'-, wherein each R is, independently, H, a protecting group, or C₁-C₁2 alkyl; 4'-CH₂--N(R)--O-2', wherein R is H, C₁-C₁2 alkyl, or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4'-CH₂--C(H)(CH₃)-2' (see, e.g., Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH₂--C(═CH₂)-2' and analogs thereof (see, published PCT International Application WO 2008/154401, published on Dec. 8, 2008).

[0227] In certain embodiments, such 4' to 2' bridges independently comprise from 1 to 4 linked groups independently selected from --[C(R_a)(R_b)]_n--, --C(R_a)═C(R_b)--, --C(R_a)═N--, --C(═NR_a)--, --C(═O)--, --C(═S)--, --O--, --Si(R_a)₂--, --S(═O)_x--, and --N(R_a)--;

[0228] wherein:

[0229] x is 0, 1, or 2;

[0230] n is 1, 2, 3, or 4;

[0231] each R_a and R_b is, independently, H, a protecting group, hydroxyl, C₁-C₁2 alkyl, substituted C₁-C₁2 alkyl, C₂-C₁2 alkenyl, substituted C₂-C₁2 alkenyl, C₂-C₁2 alkynyl, substituted C₂-C₁2 alkynyl, C₅-C₂0 aryl, substituted C₅-C₂0 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)--H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and

[0232] each J₁ and J₂ is, independently, H, C₁-C₁2 alkyl, substituted C₁-C₁2 alkyl, C₂-C₁2 alkenyl, substituted C₂-C₁2 alkenyl, C₂-C₁2 alkynyl, substituted C₂-C₁2 alkynyl, C₅-C₂0 aryl, substituted C₅-C₂0 aryl, acyl (C(═O)--H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁2 aminoalkyl, substituted C₁-C₁2 aminoalkyl, or a protecting group.

[0233] Nucleosides comprising bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to, (A) α-L-Methyleneoxy (4'-CH₂--O-2') BNA, (B) β-D-Methyleneoxy (4'-CH₂--O-2') BNA (also referred to as locked nucleic acid or LNA), (C) Ethyleneoxy (4'-(CH₂)₂--O-2') BNA, (D) Aminooxy (4'-CH₂--O--N(R)-2') BNA, (E) Oxyamino (4'-CH₂--N(R)--O-2') BNA, (F) Methyl(methyleneoxy) (4'-CH(CH₃)--O-2') BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio(4'-CH₂--S-2') BNA, (H) methylene-amino (4'-CH₂--N(R)-2') BNA, (I) methyl carbocyclic (4'-CH₂--CH(CH₃)-2') BNA, and (J) propylene carbocyclic (4'-(CH₂)₃-2') BNA as depicted below.

##STR00001## ##STR00002##

wherein Bx is a nucleobase moiety and R is, independently, H, a protecting group, or C₁-C₁2 alkyl.

[0234] 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.

[0235] 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 α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4'-CH₂--O-2') bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0236] 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).

[0237] 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 sulfur, 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'-sulfur 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).

[0238] 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:

##STR00003##

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

[0239] Bx is a nucleobase moiety;

[0240] T₃ and T₄ are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T₃ and T₄ is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3'-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, or substituted C₂-C₆ alkynyl; and each of R₁ and R₂ is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and each J₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

[0241] In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ is fluoro and R₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxy and R₂ is H.

[0242] Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used to modify nucleosides (see, e.g., review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

[0243] In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. No. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the term "morpholino" means a sugar surrogate having the following structure:

##STR00004##

In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as "modified morpholinos."

[0244] 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'-CH₂--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).

[0245] Certain Nucleobases

[0246] 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.

[0247] In certain embodiments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (--C≡C--CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][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.

[0248] 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.

[0249] Certain Internucleoside Linkages

[0250] In certain embodiments, the present invention provides oligomeric compounds comprising linked nucleosides. In such embodiments, nucleosides may be linked together using any internucleoside linkage. 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═O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P═S). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (--CH₂--N(CH₃)--O--CH₂--), thiodiester (--O--C(O)--S--), thionocarbamate (--O--C(O)(NH)--S--); siloxane (--O--Si(H)₂--O--); and N,N'-dimethylhydrazine (--CH₂--N(CH₃)--N(CH₃)--). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligomeric compound. In certain embodiments, 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.

[0251] The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), a or β such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.

[0252] Neutral internucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI (3'-CH₂--N(CH₃)--O-5'), amide-3 (3'-CH₂--C(═O)--N(H)-5'), amide-4 (3'-CH₂--N(H)--C(═O)-5'), formacetal (3'-O--CH₂--O-5'), and thioformacetal (3'-S--CH₂--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 CH₂ component parts.

[0253] Certain Motifs

[0254] In certain embodiments, the present invention provides oligomeric compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides comprise one or more chemical modification. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleosides. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleosides comprising modified sugars. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleosides comprising one or more modified nucleobases. In certain embodiments, chemically modified oligonucleotides comprise one or more modified internucleoside linkages. In certain embodiments, the chemically modifications (sugar modifications, nucleobase modifications, and/or linkage modifications) define a pattern or motif. In certain embodiments, the patterns of chemical modifications of sugar moieties, internucleoside linkages, and nucleobases are each independent of one another. Thus, an oligonucleotide may be described by its sugar modification motif, internucleoside linkage motif and/or nucleobase modification motif (as used herein, nucleobase modification motif describes the chemical modifications to the nucleobases independent of the sequence of nucleobases).

[0255] Certain Sugar Motifs

[0256] In certain embodiments, oligonucleotides comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.

[0257] In certain embodiments, the oligonucleotides comprise or consist of a region having a gapmer sugar modification motif, which comprises two external regions or "wings" and an internal region or "gap." The three regions of a gapmer motif (the 5'-wing, the gap, and the 3'-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3'-most nucleoside of the 5'-wing and the 5'-most nucleoside of the 3'-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap. In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar modification motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar modification motifs of the 5'-wing differs from the sugar modification motif of the 3'-wing (asymmetric gapmer). In certain embodiments, oligonucleotides comprise 2'-MOE modified nucleosides in the wings and 2'-F modified nucleosides in the gap.

[0258] In certain embodiments, oligonucleotides are fully modified. In certain such embodiments, oligonucleotides are uniformly modified. In certain embodiments, oligonucleotides are uniform 2'-MOE. In certain embodiments, oligonucleotides are uniform 2'-F. In certain embodiments, oligonucleotides are uniform morpholino. In certain embodiments, oligonucleotides are uniform BNA. In certain embodiments, oligonucleotides are uniform LNA. In certain embodiments, oligonucleotides are uniform cEt.

[0259] In certain embodiments, oligonucleotides comprise a uniformly modified region and additional nucleosides that are unmodified or differently modified. In certain embodiments, the uniformly modified region is at least 5, 10, 15, or 20 nucleosides in length. In certain embodiments, the uniform region is a 2'-MOE region. In certain embodiments, the uniform region is a 2'-F region. In certain embodiments, the uniform region is a morpholino region. In certain embodiments, the uniform region is a BNA region. In certain embodiments, the uniform region is a LNA region. In certain embodiments, the uniform region is a cEt region.

[0260] In certain embodiments, the oligonucleotide does not comprise more than 4 contiguous unmodified 2'-deoxynucleosides. In certain circumstances, antisesense oligonucleotides comprising more than 4 contiguous 2'-deoxynucleosides activate RNase H, resulting in cleavage of the target RNA. In certain embodiments, such cleavage is avoided by not having more than 4 contiguous 2'-deoxynucleosides, for example, where alteration of splicing and not cleavage of a target RNA is desired.

[0261] Certain Internucleoside Linkage Motifs

[0262] 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 sugar modification 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 sugar modification motif is independently selected, so such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped sugar modification motif and if it does have a gapped sugar motif, the wing and gap lengths may or may not be the same.

[0263] 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.

[0264] 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.

[0265] Certain Nucleobase Modification Motifs

[0266] In certain embodiments, oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or nucleobases modification motif. In certain such embodiments, nucleobase modifications are arranged in a gapped motif. In certain embodiments, nucleobase modifications are arranged in an alternating motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases is chemically modified.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] Certain Overall Lengths

[0271] In certain embodiments, the present invention provides oligomeric compounds including oligonucleotides of any of a variety of ranges of lengths. In certain embodiments, the invention provides oligomeric compounds or oligonucleotides consisting of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X≦Y. For example, in certain embodiments, the invention provides oligomeric compounds which comprise oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides. In embodiments where the number of nucleosides of an oligomeric compound or oligonucleotide is limited, whether to a range or to a specific number, the oligomeric compound or oligonucleotide may, nonetheless further comprise additional other substituents. For example, an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotides having 31 nucleosides, but, unless otherwise indicated, such an oligonucleotide may further comprise, for example one or more conjugates, terminal groups, or other substituents. In certain embodiments, a gapmer oligonucleotide has any of the above lengths.

[0272] One of skill in the art will appreciate that certain lengths may not be possible for certain motifs. For example: a gapmer having a 5'-wing region consisting of four nucleotides, a gap consisting of at least six nucleotides, and a 3'-wing region consisting of three nucleotides cannot have an overall length less than 13 nucleotides. Thus, one would understand that the lower length limit is 13 and that the limit of 10 in "10-20" has no effect in that embodiment.

[0273] Further, where an oligonucleotide is described by an overall length range and by regions having specified lengths, and where the sum of specified lengths of the regions is less than the upper limit of the overall length range, the oligonucleotide may have additional nucleosides, beyond those of the specified regions, provided that the total number of nucleosides does not exceed the upper limit of the overall length range. For example, an oligonucleotide consisting of 20-25 linked nucleosides comprising a 5'-wing consisting of 5 linked nucleosides; a 3'-wing consisting of 5 linked nucleosides and a central gap consisting of 10 linked nucleosides (5+5+10=20) may have up to 5 nucleosides that are not part of the 5'-wing, the 3'-wing, or the gap (before reaching the overall length limitation of 25). Such additional nucleosides may be 5' of the 5'-wing and/or 3' of the 3' wing.

[0274] Certain Oligonucleotides

[0275] In certain embodiments, oligonucleotides of the present invention are characterized by their sugar motif, internucleoside linkage motif, nucleobase modification motif and overall length. In certain embodiments, such parameters are each independent of one another. Thus, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. Thus, 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. 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.

[0276] Certain Conjugate Groups

[0277] In certain embodiments, oligomeric compounds are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligomeric compound including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligomeric compound, such as an oligonucleotide. Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 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).

[0278] 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.

[0279] 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.

[0280] 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 C₁-C₁0 alkyl, substituted or unsubstituted C₂-C₁0 alkenyl or substituted or unsubstituted C₂-C₁0 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.

[0281] Conjugate groups may be attached to either or both ends of an oligonucleotide (terminal conjugate groups) and/or at any internal position.

[0282] In certain embodiments, conjugate groups are at the 3'-end of an oligonucleotide of an oligomeric compound. In certain embodiments, conjugate groups are near the 3'-end. In certain embodiments, conjugates are attached at the 3'end of an oligomeric compound, but before one or more terminal group nucleosides. In certain embodiments, conjugate groups are placed within a terminal group.

In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, oligomeric compounds comprise an oligonucleotide. In certain embodiments, an oligomeric compound comprises an oligonucleotide and one or more conjugate and/or terminal groups. Such conjugate and/or terminal groups may be added to oligonucleotides having any of the chemical motifs discussed above. Thus, for example, an oligomeric compound comprising an oligonucleotide having region of alternating nucleosides may comprise a terminal group.

Antisense Compounds

[0283] In certain embodiments, oligomeric compounds of the present invention are antisense compounds. Such antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, antisense compounds specifically hybridize to one or more target nucleic acid. In certain embodiments, a specifically hybridizing antisense compound has a nucleobase sequence comprising a region having sufficient complementarity to a target nucleic acid to allow hybridization and result in antisense activity and insufficient complementarity to any non-target so as to avoid non-specific hybridization to any non-target nucleic acid sequences under conditions in which specific hybridization is desired (e.g., under physiological conditions for in vivo or therapeutic uses, and under conditions in which assays are performed in the case of in vitro assays).

[0284] 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.

[0285] 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.

[0286] In certain embodiments antisense compounds and antisense oligonucleotides comprise single-strand compounds. In certain embodiments antisense compounds and antisense oligonucleotides comprise double-strand compounds.

[0287] Certain Pathways and Mechanisms Associated with Cancer

[0288] Many cancer cells preferentially use the glycolytic pathway with lactate generation to produce energy, even under normal oxygen conditions. This metabolic feature of cancer is termed the Warburg effect. In certain embodiments, PK-M2 mediates the Warburg effect. In certain embodiments, expression of PK-M2 is crucial for tumor cell growth and proliferation.

[0289] In certain embodiments, reducing expression of PK-M2 inhibits cancer growth. In certain embodiments, reducing expression of PK-M2 induces apoptosis in a cell. In certain embodiments, the cell is a cancer cell. In certain embodiments, the cell is a tumor cell. In certain embodiments, the cell is a glioblastoma cell.

[0290] In certain embodiments, increasing inclusion of exon 9 of a PK-M transcript inhibits cancer growth. In certain embodiments, increasing exclusion of exon 10 of a PK-M transcript inhibits cancer growth. In certain embodiments, increasing inclusion of exon 9 of a PK-M transcript induces apoptosis in a cell. In certain embodiments, increasing exclusion of exon 10 of a PK-M transcript induces apoptosis in a cell. In certain embodiments, the cell is a cancer cell. In certain embodiments, the cell is a tumor cell. In certain embodiments, the cell is a glioblastoma cell. In certain embodiments, the downregulation of PK-M2 leads to apoptosis in certain cancer cells. In certain embodiments, the downregulation of PK-M2 leads to apoptosis in certain glioblastoma cell lines.

[0291] In certain embodiments, PK-M2 also functions as a co-activator of HIF-1 and/or β-catenin. In certain embodiments, reducing expression of PK-M2, as opposed to inhibiting its kinase function, interferes with anti-apoptotic and pro-proliferative functions associated with cancer or tumor cells. In certain embodiments, one or more antisense compounds may be used to target a PK-M2.

[0292] In certain embodiments, the administration of a modified oligonucleotide causes a switch in the alternative splicing of the PK-M transcript. In certain embodiments, the administration of a modified oligonucleotide causes increased inclusion of exon 9 mRNA of the PK-M transcript. In certain embodiments, the administration of a modified oligonucleotide causes an increase in the exclusion of exon 10 mRNA of the PK-M transcript. In certain embodiments, the administration of a modified oligonucleotide reduces expression of PK-M2 in a cell. In certain embodiments, the administration of a modified oligonucleotide reduces expression of PK-M2 in a cell and inhibits cancer growth. In certain embodiments, the administration of a modified oligonucleotide reduces expression of PK-M2 and induces apoptosis in a cell. In certain embodiments, the cell is a cancer cell. In certain embodiments, the cell is a tumor cell. In certain embodiments, the cell is a glioblastoma cell.

[0293] Certain Target Nucleic Acids and Mechanisms

[0294] 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 pre-mRNA. In certain embodiments, the target nucleic acid is a PK-M transcript. In certain embodiments, the target RNA is a PK-M pre-mRNA.

[0295] In certain embodiments, an antisense compound is complementary to a region of PK-M pre-mRNA. In certain embodiments, an antisense compound is complementary within a region of PK-M pre-mRNA comprising an exon encoding PK-M2. In certain embodiments, an antisense compound is complementary to a region of PK-M pre-mRNA comprising an intron-exon splice junction. In certain embodiments, an antisense compound is complementary to a region of PK-M pre-mRNA comprising the intron-exon splice junction adjacent to exon 10. In certain embodiments, an antisense compound is complementary within a region of PK-M pre-mRNA consisting of exon 10. In certain embodiments, an antisense compound is complementary within a region of PK-M pre-mRNA comprising an exonic splicing silencer within an exon 10. In certain embodiments, an antisense compound is complementary within a region of PK-M pre-mRNA comprising an exonic splicing enhancer within exon 10. In certain embodiments, an antisense compound is complementary within a region of PK-M pre-mRNA comprising an exonic splicing silencer within an exon 9. In certain embodiments, an antisense compound is complementary within a region of PK-M pre-mRNA comprising an exonic splicing enhancer within exon 9.

[0296] In certain embodiments, an antisense compound comprises a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a PK-M transcript. In certain embodiments, the target region is within nucleobase 29153 and nucleobase 29281 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 29158 and nucleobase 29262 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 29164 and nucleobase 29188 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 29261 and nucleobase 29279 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 29168 and nucleobase 29183 of SEQ ID NO.: 1.

[0297] In certain embodiments, an antisense oligonucleotide modulates splicing of a pre-mRNA. In certain embodiments, an antisense oligonucleotide modulates splicing a PK-M pre-mRNA. In certain embodiments, an antisense oligonucleotide increases the amount of PK-M mRNA. In certain embodiments, an antisense oligonucleotide increases the inclusion of exon 9 in PK-M mRNA. In certain embodiments, an antisense oligonucleotide decreases the inclusion of exon 10 in PK-M mRNA. In certain embodiments, an antisense oligonucleotide increases the amount of PK-M1 mRNA. In certain embodiments, an antisense oligonucleotide decreases the amount of PK-M2 mRNA.

[0298] In certain embodiments it is desirable to alter the splicing of PK-M pre-mRNA to include exon 9 and exclude exon 10. By altering the splicing of PK-M pre-mRNA to include exon 9 and exclude exon 10, expression of PK-M1 will increase and expression of PK-M2 will decrease. In certain embodiments it is desirable to alter the splicing of PK-M pre-mRNA to decrease expression of PK-M2.

[0299] Certain Pharmaceutical Compositions

[0300] 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.

[0301] 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.

[0302] 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.

[0303] 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.

[0304] 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.

[0305] 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.

[0306] 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.

[0307] 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.

[0308] In certain embodiments, a pharmaceutical composition provided herein comprises a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80® and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80®; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

[0309] In certain embodiments, a pharmaceutical composition provided herein is prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration.

[0310] In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.

[0311] In certain embodiments, a pharmaceutical composition is prepared for transmucosal administration. In certain of such embodiments penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0312] In certain embodiments, a pharmaceutical composition provided herein comprises an oligonucleotide in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

[0313] In certain embodiments, one or more modified oligonucleotide provided herein is formulated as a prodrug. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form of an oligonucleotide. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in certain instances, a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form. In certain instances, a prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, a prodrug is an ester.

[0314] In certain such embodiments, the ester is metabolically hydrolyzed to carboxylic acid upon administration. In certain instances the carboxylic acid containing compound is the corresponding active form. In certain embodiments, a prodrug comprises a short peptide (polyaminoacid) bound to an acid group. In certain of such embodiments, the peptide is cleaved upon administration to form the corresponding active form.

[0315] In certain embodiments, the present invention provides compositions and methods for reducing the amount or activity of a target nucleic acid in a cell. In certain embodiments, the cell is in an animal. In certain embodiments, the animal is a mammal. In certain embodiments, the animal is a rodent. In certain embodiments, the animal is a primate. In certain embodiments, the animal is a non-human primate. In certain embodiments, the animal is a human.

[0316] In certain embodiments, the present invention provides methods of administering a pharmaceutical composition comprising an oligomeric compound of the present invention to an animal. Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intracerebroventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous). In certain embodiments, pharmaceutical intrathecals are administered to achieve local rather than systemic exposures. For example, pharmaceutical compositions may be injected directly in the area of desired effect (e.g., into the eyes, ears).

[0317] In certain embodiments, a pharmaceutical composition is administered to an animal having at least one cancer cell. In certain embodiments, such administration results in apoptosis of at least cancer cell. In certain embodiments, a pharmaceutical composition is administered to an animal having at least one symptom associated with cancer. In certain embodiments, such administration results in amelioration of at least one symptom. In certain embodiments, administration of a pharmaceutical composition to an animal results in a decrease of PK-M2 mRNA in a cell of the animal. In certain embodiments, such administration results in an increase in PK-M1 mRNA. In certain embodiments, such administration results in a decrease in PK-M2 protein and an increase PK-M1 protein. In certain embodiments, a PK-M1 protein is preferred over a PK-M2 protein. In certain embodiments, the administration of certain antisense oligonucleotides delays the onset of cancer. In certain embodiments, the administration of certain antisense oligonucleotides slows the proliferation of cancer cells. In certain embodiments, the administration of certain antisense oligonucleotides slows the proliferation of tumor cells. In certain embodiments, the administration of certain antisense oligonucleotides prevents the growth of cancer. In certain embodiments, the administration of certain antisense oligonucleotides prevents the formation of tumors. In certain embodiments, the administration of certain antisense oligonucleotides causes tumor mass to decrease. In certain embodiments, the administration of certain antisense oligonucleotides rescues cellular phenotype.

NONLIMITING DISCLOSURE AND INCORPORATION BY REFERENCE

[0318] 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.

[0319] 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).

[0320] 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^meCGAUCG," wherein ^meC indicates a cytosine base comprising a methyl group at the 5-position.

EXAMPLES

[0321] The following examples illustrate certain embodiments of the present invention and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif And, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.

Example 1

Screening in HEK-293 Cells to Identify Antisense Oligonucleotides that Promote the Expression of the Pyruvate Kinase M1 Isoform Via Alternative Splicing

[0322] Alternative splicing of the Pyruvate kinase M (PK-M) gene involves a choice between mutually exclusive exons 9 and 10. An antisense oligonucleotide (ASO) screen was carried out to identify potent ASOs that switch the splicing of endogenous PK-M transcripts to include exon 9, thereby promoting PK-M1 isoform expression and down-regulating PK-M2 isoform expression. A diagram of the PK-M genomic region is presented in FIG. 1a.

[0323] The ASOs were designed as uniform oligonucleotides, 15 nucleotides in length, with 2'-O-methoxyethyl ribose sugar residues and a phosphorothioate backbone. All the cytosine nucleobases are 5-methylcytosines. The ASOs target exon 10 of the complement of GENBANK Accession No. NT_--010194.16 truncated from nucleotides 43281289 to 43314403 (designated herein as SEQ ID NO: 1), and cover the 167-nucleotide region of exon 10 in 5-nucleotide steps, as presented in Table 1.

[0324] To examine the effects of antisense oligonucleotide treatment of the cells on endogenous PK-M transcripts, HEK-293 cells were transfected with each ASO at a final concentration of 30 nM. HEK-293 cells were obtained from ATCC and grown at a density of 2×10⁶ cells in 6-cm dishes in DMEM supplemented with 10% (v/v) FBS, penicillin, and streptomycin, at 37° C. and 5% CO₂. Transfections were performed using an ASO: LipofectAMINE2000® ratio of 20 pmoles: 1 μL.

[0325] Splicing of the PK-M transcripts by radioactive RT-PCR was analyzed 48 hrs after transfection. Two micrograms of total RNA was extracted from the cells using Trizol reagent (Life Technologies, Carlsbad, Calif.). Contaminating DNA was removed with DNase I (Promega). Reverse transcription was carried out using ImPromp-II reverse transcriptase (Promega). Semiquantitative PCR using Amplitaq polymerase (Applied Biosystems) was performed by including [α-³²P]-dCTP in the reactions. The human-specific primer sets used to amplify endogenous transcripts anneal to PK-M exons 8 and 11, and their sequences are: hPKMF: 5'-AGAAACAGCCAAAGGGGACT-3' (designated herein as SEQ ID NO: 2) and hPKMR: 5'-CATTCATGGCAAAGTTCACC-3' (designated herein as SEQ ID NO: 3). The primers are represented by arrows at the top portion of FIG. 1a. After 27 amplification cycles for endogenous transcripts, the reactions were divided into two aliquots for digestion with PstI (New England Biolabs) or no digestion. Pst1 digestion was carried out to distinguish between M1 and M2; only M2 has a PstI site, resulting in two cleavage products, B1 (213 nucleotides) and B2 (185 nucleotides) which are the 3' and 5' ends of M2 respectively, as shown at the bottom portion of FIG. 1a.

[0326] The products were analyzed on a 5% native polyacrylamide gel, visualized by autoradiography, and quantified on a Typhoon 9410 phosphorimager (GE Healthcare) using Multi Gauge software Version 2.3. The results are presented in FIG. 1b, and Table 1. The % M1 mRNA in endogenous transcripts was calculated using the GC-content-normalized intensities of the top undigested band (M1; depicted as A in the figure), the bottom two digested bands (M2; depicted as B1 and B2 in the figure) in the Pst1-digest lanes, and the double-skipped species (D), if detectable. Each product was quantified as a percentage of the total of M1, M2, and double-skipped species. % M1 and % M2 are presented in the Table. The first row of Table 1 denotes the numbers from the untreated control set of cells.

[0327] Some of the ASOs strongly increased the proportion of PK-M1 mRNA, with a concurrent increase in the amount of double-skipped mRNA, and a decrease in PK-M2 mRNA. The results indicate that these ASOs target functional enhancer splice elements (ESEs) in exon 10.

[0328] The two most potent ASOs were ISIS 461456 and ISIS 461472. ISIS 461472 targets the previously characterized exon 10 SRSF3 motif (Wang, Z. et al., J. Mol. Cell Biol. 4: 79-87, 2012), whereas ISIS 461456 targets a non-overlapping 15-nucleotide region in the middle of exon 10.

TABLE-US-00001 TABLE 1 RT-PCR screening of ASOs targeting exon 10 in HEK-293 cells Target Target SEQ Start Stop ID Isis No Site Site Sequence % M1 % M2 NO n/a n/a n/a n/a 2 98 n/a 461453 29153 29167 AATAATTGCAAGTGG 29 69 4 461454 29158 29172 CCTCAAATAATTGCA 26 73 5 461455 29163 29177 GAGTTCCTCAAATAA 27 70 6 461456 29168 29182 CGGCGGAGTTCCTCA 33 64 7 461457 29173 29187 CCAGGCGGCGGAGTT 25 69 8 461458 29178 29192 GGGCGCCAGGCGGCG 21 72 9 461459 29183 29197 GTAATGGGCGCCAGG 17 81 10 461460 29188 29202 CGCTGGTAATGGGCG 23 70 11 461469 29248 29262 CCCCACTGCAGCACT 13 82 12 461470 29253 29267 TATGGCCCCACTGCA 9 90 13 461471 29258 29272 ACGATTATGGCCCCA 6 94 14 461472 29263 29277 TGAGGACGATTATGG 23 74 15 461473 29268 29282 CTTGGTGAGGACGAT 4 95 16 461474 29273 29287 CCAGACTTGGTGAGG 12 88 17

Example 2

ASO Microwalk Centered on the 10 W ESE Region

[0329] An ASO microwalk was performed to find the most potent ASOs that target the exon 10 regions defined by ISIS 461456 and ISIS 461472.

[0330] Overlapping 15-nucleotide ASOs were designed in 1-nucleotide steps. The ASOs were designed as uniform oligonucleotides, 15 nucleotides in length, with 2'-O-methoxyethyl ribose sugar residues and a phosphorothioate backbone. All the cytosine nucleobases are 5-methylcytosines. The ASOs target exon 10 of SEQ ID NO: 1.

[0331] To examine the effects of antisense oligonucleotide treatment of the cells on endogenous PK-M transcripts, HEK-293 cells were transfected with each ASO at a final concentration of 60 nM. Cell culture, transfection and RNA analysis was conducted in a similar manner to that described in Example 1. The results of the microwalks are presented in FIG. 1c and Tables 2 and 3. The % M1 mRNA in endogenous transcripts was calculated using the GC-content-normalized intensities of the top undigested band (M1; depicted as A in the figure), the bottom two digested bands (M2; depicted as B1 and B2 in the figure) in the Pst1-digest lanes, and the double-skipped species (D), if detectable. Each product was quantified as a percentage of the total of M1, M2, and double-skipped species. % M1 and % M2 are presented in the Tables below. All standard deviations are ≦4% (n=3).

[0332] The results indicate that ISIS 549197 was the most potent in increasing endogenous PK-M1 mRNA and decreasing PK-M2 mRNA levels. The results also indicate that ISIS 555158 optimally abrogated the SRSF3-dependent ESE in exon 10.

TABLE-US-00002 TABLE 2 ASO microwalk around ISIS 461456 in HEK-293 cells Target Target SEQ Start Stop ID ISIS No Site Site Sequence % M1 % M2 NO n/a n/a n/a n/a 2 98 n/a 549191 29161 29175 GTTCCTCAAATAATT 11 89 18 549192 29162 29176 AGTTCCTCAAATAAT 17 83 19 549193 29164 29178 GGAGTTCCTCAAATA 28 69 20 549194 29165 29179 CGGAGTTCCTCAAAT 29 69 21 549195 29166 29180 GCGGAGTTCCTCAAA 4 95 22 549196 29167 29181 GGCGGAGTTCCTCAA 39 57 23 549197 29169 29183 GCGGCGGAGTTCCTC 41 39 24 549198 29170 29184 GGCGGCGGAGTTCCT 38 51 25 549199 29171 29185 AGGCGGCGGAGTTCC 38 53 26 549200 29172 29186 CAGGCGGCGGAGTTC 25 69 27 549201 29174 29188 GCCAGGCGGCGGAGT 25 67 28

TABLE-US-00003 TABLE 3 ASO microwalk around ISIS 461472 in HEK-293 cells Target Target SEQ Start Stop ID ISIS No Site Site Sequence % M1 % M2 NO 555155 29259 29273 GACGATTATGGCCCC 13 88 29 555156 29260 29274 GGACGATTATGGCCC 16 84 30 555157 29261 29275 AGGACGATTATGGCC 26 61 31 555158 29262 29276 GAGGACGATTATGGC 29 60 32 555159 29264 29278 GTGAGGACGATTATG 25 70 33 555160 29265 29279 GGTGAGGACGATTAT 26 68 34 555161 29266 29280 TGGTGAGGACGATTA 18 79 35 555162 29267 29281 TTGGTGAGGACGATT 14 83 36

Example 3

Characterization of the Activation Region of PK-M Exon 10

[0333] The target region of ISIS 461456 and ISIS 549197, the most potent ASOs, was characterized in detail.

[0334] To map the enhancer elements present in the target region of ISIS 461456, the high sequence identity between exons 9 and 10 was taken advantage of The PK-M2 minigene was constructed by amplifying a 6.4 kb PK-M exon 8-11 fragment from human genomic DNA (Promega), using Phusion High-Fidelity DNA polymerase and primers PKMinigeneF (5'-GGGGAAGATATCAATTCCCCATTCTGTCTTCCCATGT-3'; designated SEQ ID NO: 37) and PKMinigeneR (5'-GGGGAACTCGAGCTAGACATTCATGGCAAAGTTCACC-3'; designated SEQ ID NO: 38). The product was then digested and cloned between the BamHI and XhoI sites of pcDNA3.1+(Invitrogen). For exon-duplication and intron-deletion constructs, the upstream KpnI site 1552 nt downstream of exon 8 was removed by a 1-nt deletion, and an EcoRV restriction site was generated 90 nt upstream of exon 9 by a 2-nt insertion to create a modified wild-type minigene. To generate the 10 W, 10B7 and 10F7 constructs, modified exon 9 fragments were generated by annealing the following oligonucleotides: 10 W F (5'-CCCTAAACCTTACAGATAGCTCGTGAGGCTGAGGCAGCCATGTTCCACCGCAAGCTGTTTGAGG AACTCCGCCGAGCCTCAAGTCACTCCACAGACCTCATGGAAGCCAT-3'; designated SEQ ID NO: 39), 10F7F (5'-CCCTAAACCTTACAGATAGCTCGTGAGGCTGAGGCAGCCATGTTCCACCGCAAGCTGTTTGAGG AACTTGTGCGAGCCTCAAGTCACTCCACAGACCTCATGGAAGCCAT-3'; designated SEQ ID NO: 40), 10B7F (5'-CCCTAAACCTTACAGATAGCTCGTGAGGCTGAGGCAGCCATGTTCCACCGCAAGCTGTTTGAAG AACTCCGCCGAGCCTCAAGTCACTCCACAGACCTCATGGAAGCCAT-3'; designated SEQ ID NO: 41) with Exon 9Rev oligo (5'-CCCTTAGGGCCCTACCTGCCAGACTCCGTCAGAACTATCAAAGCTGCTGCTAAACACTTATAAG AAGCCTCCACGCTGCCCATGGCCATGGCTTCCATGAGGTCTG-3'; designated SEQ ID NO: 42) and amplifying using Ex10ADupF (5'-TTCCCCATTCTGTCTTCCCATGTGTTGTGTCTCGTTTTTTTCCTCCTCCTTCCCTCTTCCTTGCCCC CTCTTCCCCTAAACCTTACAG-3'; designated SEQ ID NO: 43) and Ex10ADupR (5'-AGTGTTACCTGCCCTTAGGGCCCTAC-3'; designated SEQ ID NO: 44). The 106-nt oligonucleotide carries mutations that duplicate specific stretches of exon 10 over the corresponding region of exon 9. Another fragment was amplified from the wild-type minigene using the following primer pairs: Ex10BF: 5'-GTAGGGCCCTAAGGGCAGGTAACAC-3' (designated SEQ ID NO: 45) and RKpnI: 5'-GGGGAAGGTACCACTGAGCAGGGCATT-3' (designated SEQ ID NO: 46). Both fragments were then gel-purified, subjected to a second overlap-extension (OE) PCR using the end primers FEcoRV (5'-GGGGAAGATATCAATTCCCCATTCTGTCTTCCCATGT-3'; designated SEQ ID NO: 47) and RKpnI (5'-GGGGAGGTACCACTGAGCAGGGCATT-3'; designated SEQ ID NO: 48).

[0335] As shown in FIG. 2a, the minigene comprises the same genomic region as indicated in FIG. 1a. The 10 W minigene duplicates the entire exon 10 10 W region into exon 9; the 1 OF minigene duplicates the first eight nucleotides of ISIS 549197; and the 10B minigene duplicates the last seven nucleotides of ISIS 549197. Due to the low baseline PK-M1 inclusion from the wild-type minigene, any strong ESEs comprised by the candidate regions was expected to lead to an increase in PK-M1 mRNAs expressed from the mini-gene.

[0336] The results are presented in FIG. 2b and Table 4. Standard deviations are 0.2%, 0.3%, and 2.6% for 10G, 10F, and 10B, respectively (n=3). The data indicate that duplication of the B7 region (10B), but not the F7 (10F) and 10 W region, lead to increased exon 9 inclusion. This result suggests that the 8-nucleotide B7 motif is a bona fide exon 10 ESE.

TABLE-US-00004 TABLE 4 Analysis of minigenes 10W, 10F and 10B Minigene % M1 10W 2 10F <1 10B 29

Example 4

Characterization of the Mechanism of Action of the ASOs

[0337] To characterize the mechanism of action of ISIS 461456 and ISIS 549197 on the inclusion of exon 9 and skipping of exon 10, these ASOs were co-transfected with the PK-M wild-type or duplicated exon 10 minigenes. The wild-type minigene comprises the flanking exons 8 and 11, and the complete genomic region between both exons, whereas the duplication construct has exon 10 replaced completely with exon 9.

[0338] HEK-293 cells were cultured, as described above. Five μg of minigene plasmid per 10-cm dish or one μg per 6-cm dish was transiently transfected using LipofectAMINE2000® (Life Technologies, Carlsbad, Calif.). ASOs were transfected, as described above, at a final concentration of 60 nM. A control ASO (5'-TCATTTGCTTCATACAGG-3', designated as SEQ ID NO: 49) was also used. The results are presented in FIGS. 2c and d, as well as in Table 5. Standard deviations for FIG. 2c are 0.6%, 4.2% and 2.9% for control, ISIS 461456 and ISIS 549197, respectively (n=3). Standard deviations for FIG. 2d are 0.8%, 0.9%, and 2.6% for control, ISIS 461456 and ISIS 549197, respectively (n=3).

[0339] As expected, both ISIS 461456 and ISIS 549197 switched the splicing of the minigene transcript by simultaneously increasing the amount of the M1 mRNA and decreasing the amount of the M2 mRNA expressed from the wild-type minigene (FIG. 2c and Table 5). However, ISIS 461456 increased exon 9 inclusion to a greater extent than ISIS 549197, although the latter decreased exon 10 inclusion to a greater extent, resulting in higher levels of double-skipped (Skp) transcripts.

[0340] Co-transfection of ISIS 461456 or ISIS 549197 with the exon 10 duplication minigene interfered with the inclusion of exon 10, leading to a large increase in double skipped species (FIG. 2d and Table 5). ISIS 461456 was especially potent, nearly converting all the mRNA to the Skp isoform.

[0341] These results suggest that both ISIS 461456 and ISIS 549197 interfere with the activation of exon 10.

TABLE-US-00005 TABLE 5 Minigene transcript level as a result of ASO co-transfection in HEK-293 cells ASO Minigene treatment % M1 % Skp % M2 Wild-type Control 1 6 93 ISIS 461456 26 63 11 ISIS 5491597 8 85 7 Exon 10 Control n/a 1 99 duplication ISIS 461456 n/a 60 40 ISIS 5491597 n/a 82 18

[0342] Alignment of the 10 W region with the PK-M exons 8-11 genomic region revealed a highly homologous region in intron 9 (FIG. 2e). To weigh the relative contributions of the exon 10 and intron 9 complementary regions for the effect of ISIS 461456 and ISIS 549197 on PK-M splicing, minigene mutation were made that eliminated the presumptive target sites in exon 10, intron 9, or both. The effect of the ASOs on splicing of the mutant minigene transcripts was then determined

[0343] Three mutants were generated (FIG. 2e). The exon 10 10 W region was mutated by duplicating the corresponding exon 9 region and termed the d10 W construct. To generate the d10 W minigene construct, a modified exon 10 fragment was constructed by annealing d10 W F (5'-ATGTTGCTCCCCTAGATTGCCCGTGAGGCAGAGGCTGCCATCTACCACTTGCAATTATTTGAAGA ACTTGTGCGCCTGGCGCCCATTACCAGCGACCCCACAGAAGCCAC-3'; designated SEQ ID NO: 50) with Exon 10 Rev (5'-CGCTGCCGCCTCCTACCTGCCAGACTTGGTGAGGACGATTATGGCCCCACTGCAGCACTTGAAG GAGGCCTCCACGGCACCCACGGCGGTGGCTTCTGTGGGGTCGCT-3'; designated SEQ ID NO: 51) and amplifying using Ex9ADupF (5'-TGGACGGATGTTGCTCCCCTAG-3'; designated SEQ ID NO: 52) and Ex9ADupR (5'-GGTACCACTGAGCAGGGCATTCCAGGGAGCCGCTGCCGCCTCCTAC-3'; designated SEQ ID NO: 53). The 108-nt oligonucleotide carries mutations that duplicate specific stretches of exon 9 over the corresponding region in exon 10. Another fragment was amplified from the wild-type minigene using the following primer pairs: FEcoRV and Ex9BR (5'-GTAGGGCCCTAAGGGCAGGTAACAC-3'; designated SEQ ID NO: 54). Both fragments were then gel-purified and subjected to a second OE PCR reaction using the FEcoRV and RKpnI primers.

[0344] A 15-nucleotide deletion was introduced in intron 9 that removed the homologous target region and this was termed the dInt9 construct. To generate the dInt9 mutant, two fragments were generated from the wild-type minigene construct, using the following primer pairs: FEcoRV and PKMdelB12R (5' TGCCCTGCCATGACCTCCCAGACGAGAAGAGGCTCTGTGCCCAG-3'; designated SEQ ID NO: 55) and PKMdelB125 (5'-ACAGAGCCTCTTCTCGTCTGGGAGGTCATGGCAGGGCAG-3'; designated SEQ ID NO: 56).

[0345] To generate the dM double mutant, the same two fragments were generated from the d10 W minigene. Both fragments were then gel-purified and subjected to a second OE PCR using FEcoRV and RKpnI. All generated fragments were then cloned between the EcoRV and KpnI sites of the modified wild-type minigene plasmid.

[0346] There was a slight decrease in baseline minigene PK-M2 mRNA expressed from the d10 W minigenes (FIG. 2f and Table 6), suggesting that the duplicated exon 9 region contains weak repressor elements. This was not the case for the dInt9 construct, suggesting that this region alone does not have a major effect in dictating M1/M2 ratios.

[0347] The loss of the 10 W binding site largely abrogated the exon 9 inclusion and exon 10 skipping promoted by ISIS 461456 and ISIS 549197 (FIG. 2f and Table 6). In contrast, removal of the intron 9 homologous region did not block the effect of ISIS 461456 and ISIS 549197 on splicing. However, when both binding sites were removed, the effect of ISIS 461456 and ISIS 549197 was completely abrogated. The results indicate that ISIS 461456 and ISIS 549197 largely mediate exon 9 inclusion through the 10 W complementary region in exon 10.

TABLE-US-00006 TABLE 6 Mutant minigene transcript level as a result of ASO co-transfection in HEK-293 cells ASO Minigene treatment % M1 % Skp % M2 D10W Control 5 9 86 ISIS 461456 11 20 68 ISIS 5491597 10 34 56 dInt9 Control 1 7 92 ISIS 461456 19 74 7 ISIS 5491597 12 80 9 dM Control 2 5 93 ISIS 461456 1 6 93 ISIS 5491597 1 5 94

Example 5

Antisense Inhibition of PK-M in Glioblastoma Cells

[0348] A characteristic splicing switch from PK-M1 to PK-M2 occurs during gliomagenesis (Clower, C. V. et al., Proc. Natl. Acad. Sci. USA 107: 1894-1899, 2010; Bluemlein, K. et al., Oncotarget. 2: 393-400, 2011). Glioblastoma cells also have a higher basal level of PK-M1 mRNA, which is expected to facilitate the ASO-mediated PK-M splicing switch (Clower, C. V. et al., Proc. Natl. Acad. Sci. USA 107: 1894-1899, 2010).

[0349] To compare the effect of ASOs targeting the 10 W region versus those targeting the SRSF3 region, side-by-side ASO transfections at final concentrations of 30 nM, 60 nM, and 90 nM in the glioblastoma cell lines A172 and U87-MG were conducted. ISIS 555158 was the ASO targeting the SRSF3 region that was chosen and was transfected at final concentrations of 60 or 90 nM. The control oligonucleotide was transfected at a final concentration of 90 nM. The experiment was run in triplicates.

[0350] U87-MG and A172 cells were obtained from ATCC and grown in DMEM supplemented with 10% (v/v) FBS, penicillin, and streptomycin, at 37° C. and 5% CO₂. ASO transfections were conducted as described above. Radioactive RT-PCR and restriction digest of endogenous PK-M transcripts were performed 36 hrs after transfection. The results are presented in FIG. 3a, as well as in Tables 7 and 8. All standard deviations are ≦4% (n=3)

[0351] As expected, there was a dose-dependent increase in exon 9 inclusion and exon 10 skipping in these cell lines, with ISIS 461456 and ISIS 549197 performing better than ISIS 555158. Consistent with the minigene experiments, treatment with ISIS 461456 resulted in greater increase in PK-M1 mRNA levels than treatment with other ASOs, whereas treatment with ISIS 549197 resulted in more double-skipped mRNA and a larger decrease in PK-M2 mRNA levels than treatment with other ASOs.

TABLE-US-00007 TABLE 7 Effect of ASO treatment on PK-M mRNA levels in A172 glioblastoma cells Treatment Dose (nM) % M1 % Skp % M2 Control 90 15 -- 85 ISIS 30 52 -- 48 461456 60 63 -- 37 90 73 -- 27 ISIS 30 49 8 42 549197 60 49 12 38 90 56 23 21 ISIS 60 39 10 51 555158 90 44 15 41

TABLE-US-00008 TABLE 8 Effect of ASO treatment on PK-M mRNA levels in U87-MG glioblastoma cells Treatment Dose (nM) % M1 % Skp % M2 Control 90 4 -- 96 ISIS 30 43 2 55 461456 60 50 3 46 90 54 5 41 ISIS 30 36 12 52 549197 60 38 17 44 90 45 22 33 ISIS 60 24 11 65 555158 90 29 14 57

[0352] To estimate the amount of PK-M1 and PK-M2 proteins in the cell lysates, isoform-specific antibodies were used. Cells were lysed in SDS, and total protein concentration was measured by the Bradford assay. Total protein of 5-30 μg was separated by SDS-PAGE and transferred onto nitrocellulose. This was followed by blocking with 5% (w/v) milk in Tris-buffered saline with Tween-20, probing with antibodies and visualization by enhanced chemiluminescence (Roche). The primary antibodies used were β-actin (Genscript mAb, 1:10,000); PK-M2 (Cell Signaling Technology, rAb, 1:2,000); and PK-M1 (ProteinTech, rAb, 1:1,000). Secondary antibodies were goat anti-mouse or anti-rabbit HRP conjugates (Bio-Rad, 1:20,000). The results are presented in FIG. 3b. A representative blot from one of three independent experiments is shown.

[0353] As expected, PK-M1 and PK-M2 protein isoform levels closely mirrored their mRNA levels. There was detectable PK-M1 protein after transfection of each of the three ASOs, but ISIS 549197 resulted in the greatest decrease in PK-M2 levels.

[0354] The data was also confirmed by immunofluorescence technique. Cells were first transfected with ASOs as described above and then plated on 4-well culture slides (BD Biosciences) 24-hrs post transfection. At 36 hrs post-transfection, the cells were washed with PBS and fixed with 3.7% formaldehyde in PBS for 20 min. Cells were then permeabilized in 0.1% Triton X-100 in PBS for 10 min after washing in PBS, and then blocked for 20 min in blocking buffer (1% goat serum in PBS). The cells were then incubated overnight with rabbit monoclonal anti-PK-M2 antibody (Cell Signaling Technology). After washing 3 times with PBS, the cells were then incubated for 1 hr in blocking buffer containing Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (Molecular Probes/Invitrogen). Cells were analyzed using a Zeiss Axiopian.Z1 upright fluorescent microscope. Downregulation of PK-M2 protein was also observed when either ISIS 461456 or ISIS 549197, but not the control ASO, was transfected into A172 or U87-MG cells (FIG. 3c).

Example 6

Effect of Antisense Inhibition of PK-M on Apoptosis in Glioblastoma Cells

[0355] The effect of treatment with ASOs targeting PK-M on apoptosis of the glioblastoma cells was studied.

[0356] Treatment with ISIS 549197 resulted in cleaved PARP as early as 24 hrs post-transfection in A172 cells, indicating that the cells were undergoing apoptosis. Cells were harvested 24 or 48 hrs after transfection, whereas the control cells were harvested after 48 hrs. The cells were lysed in SDS and total protein concentration was measured by the Bradford assay. Total protein (5-30 μg) was separated by SDS-PAGE and transferred onto nitrocellulose. The membrane was blocked with 5% (w/v) milk in Tris-buffered saline with Tween-20 and probed with PARP primary antibody (Cell Signaling Technology, rAb, 1:1,000). The bands were visualized by enhanced chemiluminescence (Roche). The results are presented in FIG. 4.

[0357] To confirm this observation, Annexin V staining assays were performed with A172 and U87-MG cells transfected with ISIS 460456, ISIS 549197, or ISIS 555158 at a final concentration of 90 nM. Cells (1×10⁶ in number) were collected 36 hrs after transfection, washed twice with PBS and resuspended in 1× Binding Buffer (10 mM HEPES, pH 7.4; 140 mM NaCl; 2.5 mM CaCl₂). The cells were then stained with 5 μl each of Annexin V-APC antibody and 7-AAD (Becton Dickinson) in the dark for 15 min, and analyzed for apoptosis for flow cytometry using an LSRII Cell Analyzer (Becton Dickinson). Both early apoptotic (7AAD.sup.-/Annexin V.sup.+) and late apoptotic (7AAD.sup.+/Annexin V.sup.+) cells were included in the quantification. The results are presented in FIG. 5a and Table 9, and are a representative of 3 biological triplicates each. Table 9 presents the percentage of Annexin V-positive cells, as indicated in the two right quadrants in each plot of the flow cytometric analysis.

TABLE-US-00009 TABLE 9 Effect of ASO treatment on apoptosis in A172 glioblastoma cells ASO: Control ISIS 555158 ISIS 461456 ISIS 549197 A172 3 23 34 48 U87-MG 4 18 32 44

[0358] To confirm this finding, cells were transfected with ISIS 461456 or ISIS 549197 at 30 nM, 60 nM, or 90 nM or with ISIS 555158 at 60 nM or 90 nM. The control ASO was transfected at 90 nM. The data is presented in FIG. 5b and Tables 10 and 11. The proportion of Annexin V-positive cells increased in an ASO dose-dependent manner, indicating that ASO-mediated switching of PK-M splicing induces apoptosis in these cell lines. ISIS 549197 was the most potent in inducing apoptosis among the three ASOs tested.

TABLE-US-00010 TABLE 10 Effect of ASO multi-dose treatment on apoptosis in A172 glioblastoma cells Treatment Dose (nM) % apoptosis Control 90 3 ISIS 30 10 461456 60 20 90 34 ISIS 30 20 549197 60 44 90 48 ISIS 60 16 555158 90 23

TABLE-US-00011 TABLE 11 Effect of ASO multi-dose treatment on apoptosis in U87-MG glioblastoma cells Treatment Dose (nM) % apoptosis Control 90 4 ISIS 30 8 461456 60 27 90 32 ISIS 30 13 549197 60 30 90 44 ISIS 60 10 555158 90 18

Example 7

Effect of Antisense Inhibition of PK-M on Apoptosis in PK-M1 Inducible Cells

[0359] To investigate the mechanism of action by which treatment with ASOs elicits apoptosis in glioblastoma cells, stable cell lines that express human PK-M1 cDNA in a doxycycline-inducible manner or PK-M2 cDNA in a constitutive manner were generated.

[0360] To generate cell lines that over-express human PK-M1 isoform in a doxycycline-dependent manner, A172 cells were first infected with MSCV-rtTA-hygro virus, and selected in hygromycin for 2 weeks. Human PK-M1 cDNA was amplified from A172 cells transfected with ISIS 549197 using the following primer pair: hPKT7cDNAF (5'-GGGGAACTCGAGATGGCTTCTAGGATGGCATCGATGACAGGTGGCCAACAGATGGGCATGTCG AAGCCCCATAGTGAAGCCG-3'; designated SEQ ID NO: 57) and hPKT7cDNAR (5'-GGGGAAGAATTCTCACGGCACAGGAACAACACGCATG-3'; designated SEQ ID NO: 58) with Phusion High-Fidelity DNA Polymerase (Finnzymes). The resulting amplicon containing the T7 tag was gel-purified and cloned between the EcoRI and XhoI sites of the retroviral TtiGP plasmid.A172-rTA cells were then infected with TtiGP-PKM1 virus. To make cells lines constitutively over-express human PK-M2, PK-M2 cDNA from A172 cells were amplified using the same primers and cloned between the EcoRI and XhoI sites of the retroviral PIG plasmid. A172 and U87-MG cells were then infected with the PIG-PKM2 virus. All infected cells were then selected with 100 μg/ml puromycin for 3 days. All plasmids were sequenced to confirm their identities.

[0361] FIG. 6a presents the immunoblot analysis of A172 cells stably transduced with rtTA and doxycycline-inducible human T7-tagged PK-M1 cDNA. FIG. 6b presents the immunoblot analysis of A172 and U87 cells stably transduced with T7-tagged human PK-M2 cDNA. Cells were grown in parallel with or without doxycycline, and harvested after 72 hrs. The cells were lysed and prepared for western blotting analysis, as described in an earlier Example. The primary antibodies used were PK-M1 (ProteinTech, rAb, 1:1,000), T7 (mAb, 1:1,000), PK-M2 (Cell Signaling Technology, rAb, 1:2,000) and β-actin (Genscript mAb, 1:10,000).

[0362] To investigate the role of PK-M1 in ASO-mediated apoptosis, doxycycline was added to the PK-M1-inducible cells for three days, after which the cells were treated with ISIS 461456, ISIS 549197, or control ASO at 60 nM final concentrations. After 36 hrs, the cells were stained for Annexin V and analyzed by flow cytometry. The results are presented in FIG. 6c and Table 10. The histograms of FIG. 6c indicate the fold increase in Annexin V-positive cells, compared to the control ASO for each condition. The data indicate that there was a similar increase in the number of Annexin V-positive cells in the cells that did or did not overexpress PK-M1, suggesting that PK-M1 induction did not cause apoptosis in these cells.

[0363] To investigate the role of PK-M2 downregulation in apoptosis, U87-MG and A172 cells overexpressing PK-M2 were treated with ISIS 461456, ISIS 549197, ISIS 555158, or control ASO at a final concentration of 90 nM. The cells were analyzed by immunoblotting as well as by Annexin V flow cytometry. The results are presented in FIGS. 6b and 6d, as well as Tables 12 and 13, and indicate that overexpression of PK-M2 in both cell lines rescued the cells from the ASO-mediated apoptosis, leading to the decrease in the number of Annexin V-positive cells to baseline levels. The histogram shown in FIG. 6d indicates the fold-increase in Annexin V-positive cells, compared to control ASO for each cell line.

TABLE-US-00012 TABLE 12 Fold change of apoptosis compared to control ASO in PK-M1-inducible cells Treatment Doxycycline Fold-change ISIS No 3.0 461456 Yes 3.3 ISIS No 8.4 549197 Yes 7.5

TABLE-US-00013 TABLE 13 Fold change of apoptosis compared to control ASO in PK-M2-overexpressing cells Cell line Treatment % apoptosis A172 ISIS 555158 7.3 ISIS 461456 10.8 ISIS 549197 15.0 0.9A172 M2 ISIS 555158 0.9 ISIS 461456 0.9 ISIS 549197 1.0 U87-MG ISIS 555158 4.6 ISIS 461456 8.2 ISIS 549197 11.3 U87-MG M2 ISIS 555158 1.0 ISIS 461456 1.4 ISIS 549197 1.1

Example 8

siRNA Knockdown of PK-M2 in A172 Cells

[0364] To confirm the effect on apoptosis in glioblastoma cells by antisense inhibition, siRNA knockdown of PK-M2 in A172 cells was employed.

[0365] Four siRNAs targeting exon 10 of human PKM2 were obtained from Sigma Genosys, and have sense-strand sequences 5'-CCAUAAUCGUCCGCACCAA-3' (M2si1; designated SEQ ID NO: 59), 5'-CAUCUACCACUUGCAAUUA-3' (M2si2; designated SEQ ID NO: 60), 5'-CCGUGGAGGCCUCCUUCAA-3' (M2si3; designated SEQ ID NO: 61) and 5'-CUUGCAAUUAUUUGAGGAA-3' (M2si4; designated SEQ ID NO: 62). A172 cells (4×10⁶) in 6-well plates were transfected with 400 pmol of siRNA duplex using LipofectAMINE2000®. Cells were harvested 48 hr later.

[0366] The results are presented in FIG. 7. Knockdown of PK-M2 also led to the appearance of cleaved PARP after 48 hrs. These observations confirm that the down-regulation of PK-M2 expression, but not the induction of PK-M1 expression, leads to apoptosis in glioblastoma cell lines.

Example 9

Effect of 2'-O-Methoxyethyl Antisense Oligonucleotides on PK-M Splicing In Vivo

[0367] The ASOs listed in Table 14 below were designed to target exon 10 of the mouse PK-M transcript comprising GENBANK Accession No. NT_--039474.8 truncated from nucleotides 5923000 to 5949000 (designated herein as SEQ ID NO: 63). (Note that the human ASOs described herein target the complement of the human genomic PK-M sequence NT_--010194.16 truncated from nucleotides 43281289 to 43314403, whereas the mouse ASOs target the mouse genomic sequence NT_--039474.8 truncated from nucleotides 5923000 to 5949000 because the mouse sequence, SEQ ID: 63, corresponds to the non-coding strand of the mouse genomic DNA. In each case, the ASOs are complementary to the RNA transcript.) Each of the ASOs in Table 14 is also complementary to the human PK-M transcript with 0-3 mismatches. Each of the ASOs is 15 nucleotides in length, with uniform 2'-O-methoxyethyl ribose sugar residues, and uniform phosphorothioate internucleoside linkages. All the cytosine nucleobases are 5-methylcytosines.

[0368] To examine the effects of antisense oligonucleotide treatment on endogenous PK-M transcripts in vivo, C57B1/6 wild-type (WT) mice were injected subcutaneously once per week for three weeks with one of the ASOs listed in Table 14 at 100 mg/kg or with PBS as a control. Each treatment group consisted of 4 animals. Four days after the administration of the last dose, the mice were sacrificed and tissues were collected.

[0369] PK-M1 and PK-M2 mRNA levels in each of the mice's livers were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. Two mouse-specific primer probe sets were used to amplify endogenous PK-M1. The first primer set anneals to PK-M exons 8 and 9 (mPKMF: 5'-TGTCTGGAGAAACAGCCAAGG-3', designated herein as SEQ ID NO: 64; mPKMR: 5'-CAAGCTCTTCAAACAGCAGACG-3', designated herein as SEQ ID NO:65; probe sequence: 5'-AGCACCTGATAGCTCGGGAGGC-3', designated herein as SEQ ID NO: 66). The second PK-M1 primer set anneals to exons 9 and 11 (mPKMF: 5'-AAGATGCCACGGTACAGATGG-3', designated herein as SEQ ID NO: 67; mPKMR 5'-CAGACCTCATGGAGGCCATG-3', designated herein as SEQ ID NO: 68; probe sequence: 5'-TGGCAGGAGTGCTCACCAAGT-3', designated herein as SEQ ID NO: 69). Two mouse-specific primer probe sets were used to amplify endogenous PK-M2. The first PK-M2 primer set anneals to PK-M exons 8 and 10 (mPKMF: 5'-GGAGTTCCTCGAATAGCTGCAAG-3', designated herein as SEQ ID NO:70; and mPKMR: 5'-AGTCCTGGATGGAGCAGACT-3', designated herein as SEQ ID NO:71; probe sequence: 5'-GCTGTTCGCATGCAGCACCT-3', designated herein as SEQ ID NO:72). The second PK-M2 primer set anneals to exons 10 and 11 (mPKMF: 5'-GCGAGCAGTCTGGGGATTTC-3', designated herein as SEQ ID NO: 73; mPKMR: 5'-ACCCCACAGAAGCTGCC-3', designated herein as SEQ ID NO:74; probe sequence: 5'-ACCAAGTCTGGCAGGAGTGCTC-3', designated herein as SEQ ID NO:75). mRNA levels were determined relative to GAPDH prior to normalization to PBS-treated controls. The results in Table 15 are presented as the average percent of PK-M1 and PK-M2 mRNA levels for each treatment group, relative to the PK-M1 and PK-M2 mRNA levels of the PBS-treated control group, respectively, and are denoted as "% PBS". The standard error for all PK-M1 results was ≦34%, and the standard error for all PK-M2 results was ≦6%. The results for each primer probe set are listed. "ND" indicates no data because the ASO targets a portion of the amplicon, thereby preventing primer binding and amplification. All of the ASOs were well tolerated, as assessed by liver weight and ALT and AST levels.

TABLE-US-00014 TABLE 14 ASOs targeting mouse PK-M exon 10 Mouse Mouse Target Target SEQ Start Stop ID Isis No. Site Site Sequence (5' to 3') NO. 606601 20994 21011 GTTCCTCGAATAGCTGCA 76 606602 20995 21012 AGTTCCTCGAATAGCTGC 77 606604 20997 21014 GGAGTTCCTCGAATAGCT 78 606651 21096 21113 TGAGCACGATAATGGCCC 79 606653 21098 21115 GGTGAGCACGATAATGGC 80 606661 21106 21123 CCAGACTTGGTGAGCACG 81

TABLE-US-00015 TABLE 15 Effect of ASOs targeting mouse PK-M exon 10 on PK-M splicing in vivo PK-M1, PK-M1, PK-M2, PK-M2, exons 8, exons 9, exons 8, exons 10, 9 primer 11 primer 10 primer 11 primer Isis probe set probe set probe set probe set No. (% PBS) (% PBS) (% PBS) (% PBS) n/a 100 100 100 100 606601 260 250 ND 50 606602 290 270 ND 50 606604 210 200 ND 60 606651 440 400 50 ND 606653 270 270 50 ND 606661 320 200 40 ND

Example 10

Effect of Deoxy, MOE, and cEt Antisense Oligonucleotides on PK-M Splicing In Vivo

[0370] The ASOs listed in Table 16 below were designed to target exon 10 of SEQ ID NO: 63. Each of the ASOs in Table 16 is also complementary to the human PK-M transcript with 0-3 mismatches. The ASOs are either 16 or 18 nucleotides in length, with deoxy sugar residues, 2'-MOE modified sugar residues, or cEt modified sugar residues, and uniform phosphorothioate internucleoside linkages. The Chemistry column presents the positions of the sugar residues; `d` signifies a deoxy sugar, `e` signifies 2'-MOE modified sugar residue, and `k` signifies a cEt modified sugar residue. All the cytosine nucleobases are 5-methylcytosines.

[0371] To examine the effects of antisense oligonucleotide treatment on endogenous PK-M transcripts in vivo, C57B1/6 WT mice were injected subcutaneously once per week for four weeks with one of the ASOs listed in Table 16 at 100 mg/kg or with PBS as a control. Each treatment group consisted of 4 animals. Two days after the administration of the last dose, the mice were sacrificed and tissues were collected.

[0372] PK-M1 and PK-M2 mRNA levels in each of the mice's livers were determined using real-time PCR according to standard protocols. Mouse-specific primer probe sets, described in Example 9, were used to amplify endogenous PK-M1 and PK-M2. mRNA levels were determined relative to GAPDH prior to normalization to PBS-treated controls. The results in Table 17 are presented as the average percent of PK-M1 and PK-M2 mRNA levels for each treatment group, relative to the PK-M1 and PK-M2 mRNA levels of the PBS-treated control group, respectively, and are denoted as "% PBS". "ND" indicates no data because the ASO targets a portion of the amplicon, thereby preventing primer binding and amplification. All of the ASOs were well tolerated, as assessed by liver weight and ALT and AST levels, except for ISIS 607034 which resulted in elevation in all three or those measures of tolerability.

TABLE-US-00016 TABLE 16 ASOs targeting mouse PK-M exon 10 Mouse Mouse Target Target SEQ ID Isis No. Start Site Stop Site Chemistry Sequence (5' to 3') NO 606989 20980 20995 kddkddkddkddkddk CAAGTGGTAGATGGCA 82 606996 21008 21023 kddkddkddkddkddk CCAGGCGGCGGAGTTC 83 607001 21044 21059 kddkddkddkddkddk CGGCGGCAGCTTCTGT 84 607003 21052 21067 kddkddkddkddkddk GGCACCCACGGCGGCA 85 607016 21104 21119 kddkddkddkddkddk ACTTGGTGAGCACGAT 86 607034 21000 21017 kkddkddkddkddkddkk GGCGGAGTTCCTCGAATA 87 607041 21044 21061 kkddkddkddkddkddkk CACGGCGGCAGCTTCTGT 88 607042 21048 21065 kkddkddkddkddkddkk CACCCACGGCGGCAGCTT 89 607055 21100 21117 kkddkddkddkddkddkk TTGGTGAGCACGATAATG 90 607057 21108 21125 kkddkddkddkddkddkk TGCCAGACTTGGTGAGCA 91 607095 21100 21115 keekeekeekeekeek GGTGAGCACGATAATG 92 607135 21100 21117 kkeekeekeekeekeeke TTGGTGAGCACGATAATG 93 607136 21104 21121 kkeekeekeekeekeeke AGACTTGGTGAGCACGAT 94

TABLE-US-00017 TABLE 17 Effect of ASOs targeting mouse PK-M exon 10 on PK-M splicing in vivo PK-M1, PK-M1, PK-M2, PK-M2, exons 8, exons 9, exons 8, exons 10, 9 primer 11 primer 10 primer 11 primer Isis probe set probe set probe set probe set No. (% PBS) (% PBS) (% PBS) (% PBS) n/a 100 100 100 100 606989 421 476 ND 86 606996 326 315 ND 25 607001 398 429 65 ND 607003 365 353 69 ND 607016 435 389 56 ND 607034 525 626 ND 148 607041 335 381 78 ND 607042 240 306 92 ND 607055 987 859 73 ND 607057 327 126 34 ND 607095 428 171 51 ND 607135 475 39 54 ND 607136 389 265 71 ND

Sequence CWU 1

1

94133115DNAHomo sapines 1acgcacaagt ctgcagctct ccccaacttt ccgttcagct cagtctccga gggtgcgcca 60gagcagacac ccggaggagt ggggagtggc agggcggggc cgggagaatg ctgccccgga 120acccataaat ctgggccctg cccaggtagg ccgggacagc tggggtggcc tgggccgaga 180gccaagaaaa gacaccccat ctggcagccc aacttggcgg caacaggtgg cccggcgccc 240gggggtctgg gaggaaagtc gctccggggg cgggccccgt tgccccgccg cgtccccatt 300ggtcatcagg tttcttaaaa tgtgactctg aatctgtgtc cttccgccgc agaatttagt 360cccaccgaaa gggcaacctg cccgcgcgtt ccgccgccgc cgccgcgctt cctcctgaag 420gtgactgcgc ccgcggggac gcagggggcg gggcccgggt cgcccggagc cgggattggg 480cagagggcgg ggcggcggag ggattgcggc ggcccgcagc gggataacct tgaggctgag 540gcagtggctc cttgcacagc agctgcacgc gccgtggctc cggatctctt cgtctttgca 600gcgtagcccg agtcggtcag cgccggaggt gagcggtgca ggaggctacg ccatcagtcc 660ccaccaaggg ccagtcgccc ggctagtgcg gaatcccggc gcgccggccg gccccgggca 720cgcaggcagg gcggcgcagg atccagggcg tctgggatgc agtggagctc agagagagga 780gaacggctcc tcacgcctgg ggcctgctct tcagaagtcc ccagcgccgt tccttccaga 840tcaggcgggt ccgccggctc ctttcccgcc ccagccctac cccctcattc tggtcccatc 900ctcttcctcc tgccccaatc ctcaatgcgc ctccatcctc gcctgccttc tctcggtccc 960tcgtgattga ttccaccctt gcttcccctt tctccgcgcc gcctgttccg tgctcgtttt 1020cccctcttcc tccttaagtc tggtccttcc accccctcct cttcaagctg tgcgtgtccc 1080ctgattctaa tgctttctgt gtaactcatt gaaactgcgt tctggttccc ctcccgcgtc 1140cattctccat tcatgcgcga ccgcccttcc cgcgccccag ttccctctcg ccgcccctcc 1200ccctgcttgc tggtcacgtc cgctcccccg catccccttc ctcgcctggc gtgtccgctc 1260cctccctccc tctctgctct ggtcgcgccc gcccacttgc tccggtctcc gagcgcggtc 1320ccacccccct tttccatcac cgcctcccag ctcccagcag gctcgggcgg tgctgaggcc 1380ccgtgtccgg ggcgggcggg cggagggctg ggctgggtgc cccgcgcggc gggcgggatg 1440cggcggcccg ggggcagctg gagacttacg taacgttggc ctgccccgct gccgggaggc 1500agggcggttg cccctgcggc gcggtgccgt cccctgtggc cgggattaga tgggcggcct 1560gcgagggcct gggaatggct cggggcccga gagctcgacc ggcccttgcc gggtggccgc 1620cagggaccac gctccatctc gccgcggccg ggctgcacgt agcggccgcg cccagggccc 1680accccgcttc accgggcgat ggccttgggc ctcgtaacgg gcgggataaa cctctgcagg 1740ctttgctggg gcctcctggc cctcgcccgt tccgcgcctc tccggcacgt ttctttcttt 1800tcctttcttt tttctttctt tttttttttt ttaattatcc ggcacatttt ttaacaaatg 1860cgtcctgatt gtggaacgcg gaggccgcgg ggtggggtgg ggatctggtt acggaggggg 1920caggaaatct gtcgcgttca ctgaacgcaa acggtgtggg tcaagggctg tttgggggtc 1980agagttagag accaggatga ctagacgagt catagcccac cgagctcaca atcttaaaat 2040gtatctcctg taatgctggg agtggggtac gagcttcctg ctgtgggagg gagggggaca 2100ggaagcctcg taaggtctca ccaggtggca agcacactgg attagaagat gcaggaaagc 2160accttccagt ctgaagatca tttaaagact cagtcatgta agggcatccc ttggcttctt 2220aatctacgta ctccagaccc agggcttgga cctttgctgg tacgggttaa agtgaggccg 2280acagtgaagg tccacgtgga gagagacatt gggaagttgt caaaaaggcg tttagaatca 2340ggtttgactc attaatttcc tgagactctg aaggagttca gcctctacac ctcagtttcc 2400cctgggaaaa ctgggagtaa cattttcctc atgatggttg aaaggattca tgcttacgtg 2460gatgtggcac actcagtaag tgtctaatct ctggcgaaat acccagaaga aaagcctgtg 2520actgaatata ttatttttga gcacaattac ttagtccaca attgagcata agggccttga 2580tccattgtct tcctactgtc cccaaacgcc tacagctaat cgtgaggaat caggctctct 2640gaataaccac gtgtctcctg gaaaccagtt ccccaaggga tctggtttaa tattgacaaa 2700gtaactgata atgtaacttt ccaactctct gcttggagca gatgcttttt ggtcatctga 2760gattgggacc tgtagacaaa ggcaggcaag gaggggctga gttaagcctt tggtgttccg 2820ctgtaagtag aatactacct accagaggaa cattccagcc cttgacttgc tctgcctgct 2880ggtgagatta ggattggagc cagggtcagt agcccccagt tggccgtctt gagggtgtgg 2940ccactcccta ggtcacagag gagatggagg tcaggactcc cctgctgcag cgttgttagc 3000aaataatttg ccctttggtg gcctgaggtg ctgtgggggc aggagtgggc tgtcttgtgg 3060ggaagggaaa tgtaacgtat caccaaatct gtgggggagt ggggctctgt ggggaagatg 3120actcaacctt tcaattattc tgcttttgag agaattatct tcgctgggtg agtggggaaa 3180ctacattgtg gttcttgcag gcttaaaaag tcccttctcc cctttcagtg agtcagataa 3240tgaagcatgc atttccccta attaaaaatg cctcaactac ggagcttgca gtgagccgag 3300atcgcgccac tgcactccag cctgggcgac agagcgagac tccttctcaa aaaaaaaaaa 3360aaaaaaagcc acaactaatt aaaaaatgat tggtccgggt cacctggttc tctgtactgc 3420cttaagagct taagtgtaga ctggcagagc ccggtgccca gctaatgccg tctgtgcagc 3480ttcattcctg gcccttttgt agcgggcaca gccgcctctc ctgggacttg attaatgatg 3540acttaaagaa ggttcttgaa ctcatttctc agttactgac ttgagccaaa atgtatggat 3600gttggtctcc tgtctagcac aactgctttc attttaagca ttgcattaga aataatgttg 3660tattcatatt ttagcaaaga gcatcattag ctctttaaga accgtacccc cagtgactta 3720gcaaatttgg tggcggtgac tctaaacagc tagcacttta tgggagttgc ttctgtgcct 3780tgaatgttgt gacactgatg tggggtcaca ggaaaaacca ttttatttcc taaagctgtt 3840tatttcttaa tattgaggca atggaagaaa tgaggaaacg gccgtgggca ggaaggtaga 3900agaaaatgca gcacttacat tacaaactca ctttacttgg gttttagttt attttgcttc 3960agttttttgt tttttttttt tttttttttt tttttttttg agatagtttt gctcttgttg 4020cccagtctgg agtgcaatgg tgtgatctcg gctcactgca acctccacct cctgggttca 4080agcaattctc ctgcctcagc ctcccgaagt agctggaatt acaggcactt gcaccacgcc 4140cagctaatat ttttgtattt ttagtagaga tggggtttca ccatgttggc caggctggtc 4200ttgaactcct gacttaggtg atccaccggc ctcggcctcc caaagtgccg ggattacggg 4260cgtgagccac tgcacctggc ctgcttcagc ttttatgaag gcaagtcagg atgtgttatc 4320ttggccaaga gctaagactg agaccggagt agaggaaaat gactcatttg acattaccct 4380tctttctgaa tcactccaaa agtccacagt caaatctcaa ttagctggta taacacaaga 4440atgcgttgac cctgtgagca gagttttatt gcatcttcaa gggtcttcgt gagattggtt 4500tatttggtat gaggtcttaa ggattttaag ttgtcccttt aagcatcagt tctgatacag 4560atgggaatag agctaaacaa ccagatatat gaagaccatt tttgatacca ccatacttgc 4620ggggagcttg cattttttta ccaaggggca gactggataa ttgagtaatt taaatacttg 4680ggactttgtt aaagaagctg ttgggggaag gtgagcctgg gtgagtggtg gagggtgatg 4740tgcagcatct gctgggcctt cctgcctctt ggtatgactg ctcagctcag gagttgggtg 4800ctttagggcc cctaccatgc agtgcggtga agccccgccc ctgcagtggt ttgtgcttgt 4860ctgcacgtag gagggaacaa gccggctgca gtaccttcgg tggcctctga gactcacctc 4920ccccacttct gtccatagac aaagctcctg gggagcccta agcctctttc ctcatgccag 4980caagcagtct tggaagcagg ctggggctgc agtgggagag atgccaagac cagttattca 5040agggcagtga ggttgccagt cctagcattt cacactgtgg accagcacag ctgctggctg 5100aatactgcag tcaagattct ctgaagggag tcctatccct tccaaacatc ttcagtttac 5160ttgcagataa tgtttcttca ttttttattt tattttattt tattttattt ttttgagacg 5220gagtctcgca ccatgaccca ggctggagtg cagtggtgca atctcagctc actgcaagct 5280ccgcctcccg ggttcatgcc attctcctgc ctcagcctcc tgagtagctg ggactacagg 5340caaccgcaac cacgcctggc tgattttttt tgtatttttt agtagagacg gggtttcact 5400gtgttagcca ggatggtctc agtctgctga cctcgtgatc cacctgtctt ggcctcccaa 5460agtgctggga ttacaggcat cagccaccgc gcctggccat gtttcttcaa ttttaaacaa 5520ctgatatctc ccttggccat gaacgaaaaa gaactgccca tcagtggagt cagtcagggc 5580acataagaca cactgtgtcc accatgccat ttcagaggag atttgattga gttaagcagg 5640gaaatagaga tgttgtaaac gttgaaacta tctgggtatc cctctttggt tattaacatt 5700agatgagcag aaaaacaaat gtcaccgatg ggcaaacatt taaaaagtct ggcagtacca 5760agtgtggaaa aaggtgtgga aggcagcaac tcagcgttca ttgatatagc cactttggag 5820agcaattcag ccttatttag taaaaataga aataaacata ctccatgacc taagacttca 5880atttctggat atatagaaac tcacacagta cagggagaca tgtactacaa gagtattgat 5940taatagaaga aacttagttt aacggggagg gagagtggcc accagtaaga cagtccataa 6000ataaaatggt ctgttgtaca gtggactagt gtaacagctt aaatgaatta gctagatcca 6060tatacccact ggatggatct taaatgtgct gctgagtgaa aaacaagtta ctcagtgata 6120tatacagtat accacttagg gcattaagaa aaccacaata ttatagggtt cagtatagac 6180atagatacac tgtacataga cttcagtgaa ctggaaggat atagagttca tgacagtgat 6240tgggtgtcaa aatgcatgat gtggctggga gcagtggctc acacctgtaa tcccagcact 6300ttgagaggct gatgcaggag gatcacttga ggccaggagg tcgagaccag cctgggcaac 6360atcgcaagat tcccatctct atttaaataa ataaaataga aaaaaaagtt taagatggag 6420gtggaaaggg ttggaggagc ttggggatgg agaaaaaatg aactgtataa aattaaaatt 6480cttgtttttg tttttagaaa gtaaagaggg ccgggcacag tggctcatgc ctgtaatccc 6540agcactttgg gaagctgagg tgggcggatc acgaggtcag gagattgaga ccatcctggc 6600taacacggtg aaaccccgtc tgtactaaaa atacaaaaaa aaaaaaaaaa aaaaattagc 6660cgggcctggt ggcgggcgcc tgtagtccca gctactcagg aggctgaggc aggagaatgg 6720cctgaacctg ggaggtggag cttgcagtga gctgagatcg cgccagtgca ctccagcctg 6780ggcaacagag cgagactcca tctcaaaaaa aaaaaaaaaa agaaaaaaga aattaaagaa 6840aatacagctc agcctttatt tgtgtttttt tttttttcct ttttttctga gacagagttt 6900ttcactctgt tgcccgggct ggagggcagt ggtgcgatct ccgttcactg cagcctccac 6960ttcctggatt caagcaattc tgtgtctcag ccacccaagt agctgggatt acaggtgcgc 7020gcctggctaa tttttgtatg tttagtagtg atggtgtttc accatattag ccaggctggt 7080ctcgaactct tagcctcaag tgatctgccc gcctcagcct ttcaaactgt tgggattaca 7140ggcgtgagcc aacacagcca gccatggctc agtgttaatg gtcagttctg ggtggtagag 7200aggcagatgt taaaactttt ttttctttaa ttcgtaacat agaagcaaac ctataaaggc 7260tgccgtagga agaccagtca tagtaactag ttcagtgctc ttggagagtt ggcactgcct 7320ttcctccttt atccccccga ctagaatgca gggcagccct tccagtaaat gttgagccag 7380tgcctcactt tgctgaggcc atcacccacc ttagttgcac ttaagaggac cctaaatcag 7440ggtcccaggt cccttgctga ttttagagtg tggatatcat acccagaaac accgccctac 7500ttttaatcct agtaaggagg caccatgtcc caggacaact aatgcttccc ccaaaccacc 7560tccttcaggc tgaaaccagt tctctgcact gagcagctgg gatggaacca ggaaatcctc 7620ggcatctgag gacattgagg ggtctctgac ttagggcttc ttcacctgaa gttgagtggt 7680ctttgaggga agtaggccca tttagcatca gctgctcttc cctattccac actctagttg 7740gaaataggac cttaggttcc tgttgacaag tcatttactt tcagccccga agaaataaaa 7800gagccaagat tttttttttt tttttaaagc cagggaattt tactagaacc tacaagtggg 7860ctcattttgt tctgtgtagc ctggtaacac catactgctt tctgctgtgg ggcctcctgg 7920ggttaaagtg tgggcttaag acccaggtct cttagctaga agatatctta tcctctgtat 7980cctgcaccca tatgcaaata cattattgtc attaccctta actatagatg aagatgaaca 8040gtgcctattc cagaccttac taggttctgc tggcccgtca cccattttga tcatgttgct 8100ggcctagttt gattagggca aatcttagaa actccatttc cattgttgag gaagagaact 8160agagagcagg ctgacctgaa tgccagcgta tcatgatgca gactttctaa cggatgcagg 8220tgttcggaag agttgtggat cgaaacgcct tcatgatggc ttggaggtgt aggtagcaaa 8280ctgacgtcac aggaaggaac acaatcttgg gtacctactg gcaacgttgg agggagaaag 8340tgagcatcag gtgccatcat tttatagttg atctatgtga tgaggttggt atcggagcat 8400aattggtaca aaggaaaaat gacttaggca gatgcagact cacgggccag gctattttat 8460tagggcagaa tgatttggtc ctttgtggaa gaattggtgg agtgaagcgt gaatctttcc 8520cagcacaacc caacaacagt cctggcccta agaagtggag catgggagtt gggtgtggtt 8580cgtgcctgta gtcccagcta cttgggatgc tgaggctgga ggatcgtttg agggtgcagt 8640gagctataat ataatataat cacaccactg cactccagcc tgggtgacag agtgaaaccc 8700tgtctgaaaa aaaaaaaaaa gaaaaaaaaa aagttggagg gaggagtgtt gggtattttt 8760tatgattttt gtctcctggt ttctgaagaa tggccaaaaa attgtgtctg acacaaagga 8820aactaatata aaaagccagg agctgtctga gatgagaaag gaaagggaga atagggcact 8880tggagctgag ctgtgattgt gcctgttcca acctgtcact ccagactaag gcctcttaga 8940gatggtgtct cctttctcac agaggagaca tggctctgag gaagatctta ctgcaggggc 9000tcgggctcaa gaataaggct cctggacctg ggcatggtgt gtgctgctat cagtggatac 9060gccaggcttc cactgctggc tggaggttgg ctctgcatgt ctgtgccttc ctaggaggag 9120gatgcaatag tgagtcagac tggcatgggt ggggccacat gtcctggcag gcactgtcca 9180ggcagctggc atgagggaga ggagtctttc ccagaagtcc gccctgagaa accaaggctg 9240ggctgctctc ctggagccag gggagtccag tgagtgtttt acatacaact ctaggtacgt 9300ggttggtggg atgggcagtt tgtgctggga agaggcttgt ggaggatttt ggagaaggca 9360ggagagctct ggccctccct gcaagggagg cttcaggtca gagcctgagg aaagacctgg 9420gcatgagata tgaggtctct ggctgggtag agagcatgaa ggacacatga gatctggagt 9480ctggatgaac ttgctgacaa gcaagctttt tttgactgct aatgcgtaaa tccactatag 9540aaattttcat ttgtcatttt tgtacttatt ggcaaaaaat tagagcttat aggcatcaga 9600tttaatttga gatagttgaa atagggtttg tgtttgaggc caatataatt ttatctgcta 9660atggcatctc gtggacttgg aggcagccct tctgtaccag aacattgtca aaagctttta 9720ctgtaaagct tgagaacaaa ctagtttgct ggatttgggc atgtaactaa caggtttgag 9780gcatgggatt atcctgtgga cttttttttt tttttttttg ctttgaggtt ctgaatgtat 9840taaggttgac cttcttaagc cgttgaaact gttctgcagt acatttgtga tgtagggcct 9900aagacttgta tcgttttttt tttttaatca caacccagtg tacagaactt gagacatgcg 9960tcttttctct gccacctttt aaaagcagat tattcttgaa gtgcatagag cagcaattga 10020ttaatggaat tggtgtcttc acatttcatt tacttcctcc caacaatttt ataggatgca 10080tataaatatt tccaaaagag gtacacataa tttcttacta taaaagtatt tttatattta 10140tatcagtaaa tttgttaata aagaggattt ttttttttct gtaatcctac caccccaatg 10200acgtcctatt aaaatttcag tatatatcct cccaggtctt ttaggtatgt ttaatttggt 10260gtcccttccc cgctcccaaa aggggaggga ccaggttctt gtatagaata gtggaatgtt 10320agtaaatcac aggtttaaag agacataaca gtggaatctc tagagcagct gtcacctgga 10380tacctggtta ttaaggtaat ttttccatta ccccaaagag ctttagttac actcagcttt 10440ttccttaatc cttgtgcagc tctccagggc acaccgtatt cagctctgag cggtctttgc 10500tagtgaggcc aaggagccac cctgagccaa aaggggagca ttatgtcacc ggaagcccaa 10560ccccagagaa ccaaaggtat gacctgatat tcagtggccc cagccaggtc tttacaggaa 10620gaccctcata tctcaggtct aagaagagcc agctgatggt ttttaaaaag agtggaatta 10680gttactccaa cccacttatt cagatcttat tttgttcaca atacagtccc tagattgtag 10740gcccattgga ggccacagca aagcctttgt gttccagttg gcctgatgtg ccatctctca 10800gtaatgttcc cttaacagcc agacttccct aagcccagct gggagctctg aaggtatgcg 10860agccctccct caaccatgag tgtagggaaa gggaccaggg gccccaggct ttcctgtcag 10920taatgcagaa gttcctcaga tttagggaag ggggagcaga ggcataactt tgattctgac 10980aaagaggcat tcagagagac tgaaaggtca tttaacaaac actggaatgc ttccacatac 11040taggtgctag gagatacaaa accatatagg tcctggaagg gaggattgat tttttcattt 11100tggtacgtag tagatattag gggcttagga aatacacatc gaaatgaaga gtgcatttgc 11160catgttgaac cgttagccgg tatcttattt ccccatttta aaagttttag aatctgtggt 11220tgaggacttg tggccatcag ttttccatag ccaacagact gttcactact gccttcagag 11280ctccttggac ctcagcgggc cttctttgga gatggcagag atggatttag atgtatactc 11340tactcgagcc acccagagag cccacaaagt cagagatgga acagggtaaa ggagtaaggg 11400tcatatgtgt gagatgcctt gatttggaac tttgagattt aggatgaggt ggggaagggc 11460taaagaggag cttgttcctg agccttgctt ggccgaagca tttaggctca agcgttttag 11520aaagagtagc ccttggtctg agaactcaag gaaacagctt tctgatgaga cgtgtagcaa 11580gcttctggtt cacatcctta cctgatagtt cttcaaacac tgcctggtct ggttcacatc 11640cttacctgat agttattcaa acactgcctg gaagcttctc ctgagttttt gtctctaatc 11700agctaactaa caggctgagt gagtttagtt gtaagtcatt aatgaagaaa gcaaaggttg 11760gggccattgt cagggttgtg acctgggcta gttaattacc tggaactgat ggtctgtgtt 11820acagagtggt ggtatacttg tcaggcttag aaaagaaatc aggatgtgta tcaaaaatca 11880tttggggaaa agatttgacc agcaacttta atttctctat gtttgcaact atcctgttaa 11940tgtagttgtg ataatttcag aattatacca gtgcccttat gttatccttg ctttgcaaat 12000tgcaaattgc tttgcgtgtc ctgacatcct tctggccaac agtagatgtg gttttaggtt 12060tagactcctg ggatggaagc ttttgcattc aggggaatga ctttgggttt gggtgaggat 12120tgtaaagagg caatatgggt gccccacgac aaagcagcta tttgtagctt tgtgacagct 12180tgacatgcag agatctaggc ttatcaaggc actaagctag gagtcagttg tttgtatcac 12240tggaagattg gttacaactt ccttcattgg aagctccttc agtgcatgtt aaatgatgtt 12300atttatagat agggtggtga gaaagctgtc taggtagatg tcagtcagcc cagtgtaaga 12360gagacctgct tactgtgggt gcttgggact atgtggagtg ggtgggaggt tttaacttgt 12420tcagtaaggt cctttccatt gttcacaatc tggtgaaccc tttttctaac atgaggagca 12480cccacataac cagatcatgt ctggcttccc tgtggcttgt gtacaaagcg tgcttattga 12540gttaatgtgt aagcaggaga cagccttctg tgctaaatgg tatattaacc acttctcagt 12600cttaccactc tctttcaatt tgtctcgacc caggacctca gcagccatgt cgaagcccca 12660tagtgaagcc gggactgcct tcattcagac ccagcagctg cacgcagcca tggctgacac 12720attcctggag cacatgtgcc gcctggacat tgattcacca cccatcacag cccggaacac 12780tggcatcatc tgtaccattg gtgagtgggt gtgccccttc ccccaaaaaa gggcttcatg 12840ggcagtgacc tttctctcct gaaaagagta actaaatgtc ctaacaaacc taggtgctac 12900atgggatact acacagattc ttatgaaagg actcaggtca taggaagttg cagtaaagaa 12960ttagtatgtg cataggatgg caaatacagt taataagaga gtattagaca tttcaaaatt 13020gctaagatgg cgaggtatgg tggctcccag cactttggga ggccaacgtg ggaggattgc 13080ctgagcctcg aaatttgaga ccagcctgag caacttagac cctgtctctc caaaaagtga 13140aaaaaaaaaa aaaaaaatta gctgggcatg gtggcatgca cctgtagttc tggctacatg 13200ggaggctgag acaaagatca cttgagtcca ggagattgaa gttgcagtga gccatgatca 13260caccactgca ctccagtcta ggcaacagag cgagatcctg tcttaagaaa aaaaaattgt 13320ccgggcgcag tggcacatgc ctgtaatcca gcacttcggg aggctgaggc aggtggatca 13380cctgaggtca ggagttcgag accagcctgg ccaacatggt gaaatcccat ctctactaaa 13440aatacaaaaa aattagccgg gggtggtggc gggtacctat aatcccagct acttgggagg 13500ctgaggcagg agaattgctt gaacctggga ggcggaggtt gcagtgagct gagatctgac 13560cattgcactc cagccttggc aacaagaacg aaactctgcc tcaaaaaaaa aaggaagaaa 13620aaagaaaaaa acatcgctaa gagtaaattt caaatgttct caccacaaaa atgttaagta 13680tttgaagtca tggatatgtt aactaacctg atttaattat tccacattgt atccaaactg 13740tatgtattgg attacataac tttgtaaccc aaattataaa ttaccagttt ataataaaaa 13800ataatttgtt gcaaaaagaa tccatatggt ttaggtttta tgctataggc aaaatttaga 13860agatgttttc cttagcaggt ctttgtagga gcaacttaaa gacctaggaa agatctttct 13920aacatgttct gtgctaccaa gattctgtgg ttggacatct ggctgggttt cagtgagggt 13980ggagaaggct ggccaagtct taacctaggc ttttctgata cagtgggagc ctgcagaact 14040tgaaggaaat ggtcgaagtg tcccagtaga tcaagaaagt aagctggcac ggtagtagcc 14100ttccatgcac tttttaaaga cttttgagct atttgggaga ggaaaagttt tcagggaaaa 14160aaattcttta aacttaagca aacttaaatg tttttccttc tttgaataat taatacttgt 14220ggctttaaaa cttttcctaa taggcccagc ttcccgatca gtggagacgt tgaaggagat 14280gattaagtct ggaatgaatg tggctcgtct gaacttctct catggaactc atgaggtgag 14340ctgtggctgg accctatggc cattgtgatg gcctgtagga aacagggagg gggtgcagtg 14400ttcgtttagc cacagtggac tagacaagga tgagtctgag tttcacagtc agtgtgaagt 14460ttgtctttac tagcccatcc ctactctcct tccctcttgt cctgacaaag caactggctg 14520agtctctttt agcaaaaagg accccctttg ttgctggctg tggttctccc acacacctct 14580cctaccctta gcttttacaa aggaagatat ggaaaggttc tactggaaaa ccctctaagc 14640cttaggtgtc ctggccacag cgcttgactc tcctgtccca gggtttctgc ttcaccttgt 14700gttgccatgg taaaccatct agcagattga ttctagctta gaaccaaaat aactgggcag 14760gtccatgaga acggtttcca ctattctaag ttttgaggga ctgagcctaa tgcataagca 14820ctatctgggg tgtaataccc cacttcctca gcactgtatt ctcagcctgt gccttcccag 14880gggttctggt acattaaaat aacaccagtt agcactcttc cccaggagcc tagtaggact 14940gtatttgtgc tgggctcttt attagctggc tttacctatg gacagaggcc ttgcccagga 15000gccaggtagc agctgttggg atggctccat tcctgcctcc

attgccagat ttagaattaa 15060cccattctga ggagcttggg gttccctgag gtaccatgac ttatttattt ttttatttta 15120taaaacaaaa ttttgctctg tcatccaggc tggactgcag tggtatgatt atggctaact 15180ggatccttga cctcccaggt tcaagtgatc ctcttgcctc agcctcccga gtagctggga 15240atacaggcat gcaccaccac acctggctaa tttaaaaatt tttttggggg aaatgaggtc 15300tcactatatt gccttggctg gtctcaaact cctgggctca agtgattctc tcaaatgttg 15360ggattacagg aatgagctac catgctcagc ctgggattgt gcctttttaa aaccttcaga 15420cttaaccata ggtttcccat agatcatggg atttcgtaat ggcattgata agaggaatta 15480cagaagaggc aaactttgca cctgtcttgg cttctgtatt tcctgttgag agtaaagaaa 15540atgctatcct gtaaggccaa ttgccttaca gaggttgccc tctggcattt ggaagttggt 15600attaagtttg gactaaaaat aaagcctcag gaaatgcaat ccaagagtga attcctcctt 15660ttgggaaaca caagactctt catcatagat tccctaacct gtgttcataa acagcctatg 15720gcctggctag tggctggccc ttaaatgtca tggggacctg accaagtcca gcagacatac 15780catgtaggtt aagacatgtc cctgtacctt ttggaaaatt ctgtagtttt ccaaaagcaa 15840ggggtcctta gcaggagtca ccgagaatta cttgttagag aattaagtgt tagcttagct 15900tagagagagc tgaagacaat gctggaggtc tgttcgctgt tgatccctgc tgctgtagtc 15960tgccatgggc tcctgcattc aggggaagga gcagaaatag atttttaaga agttgacctt 16020taagtaggct ttatggttcc ttcatccagt aaaataacac cacatagctc taacatggca 16080agggcgagtg atacctgcca cacctgctgg atgagagctg gctccgattt tggtatttta 16140aactttaaga ggcttttgga gattatctct actttcactc ctattcccag attataatta 16200agatttattt tttatttttt atctatttat tttttaaaga tgtccctctt gtgtgttcat 16260tttgaagttt tagaccaaga tgaggttgtg tgtgggctca gcttggaaac tgatctgaaa 16320ttattctaat ttatataatg taatgtaaac agtttcagcc ttaccatacg tcagggctat 16380cgtttcatgt gcacctttga ctaggggctg gggcgtactt ttccagtttc tgactatttt 16440aaatgctctt ctgagcagaa cgttgagatt actgtcttcc ctctcactct gacagaggga 16500catcaaatgt ctgcatctga tcttttaaca gctttttttt tttgagacag aatcttgctc 16560tgttgcccag gctggagtgc actggcacaa tctctgctca ccacaacctc tgcctcccag 16620gttcaagcaa ttctcatacc tcagcctcct gagtagctgg gattacagac ctgtgccacc 16680acgcccagct aattttttta tatttttagt agagacgggg tttcgccatg ttggccaggc 16740tggtcttgaa cttgtgacct caggtgatcc gactgcctcg gcctcctaaa ggcgtgagcc 16800accacgccca gccctctttt aacagctttg gcaactagtc ttcagccctc acttttggca 16860gttcacatgg gcaagatgca ttcttgctga acatgtggtt ccatatgcca tgttttccag 16920atttatttat ttatttattt atttatttag agagggagtc tcgctctgtc atccaggctg 16980gagtgcagtg gcacgatctt ggctcactgc aacctctgcc tcctgggttc aagagattct 17040tctgcctcag cctcctaagt atctgagatt acaggcacct gccaccacac ccgactaatt 17100tttgtatttt agtagagact tggtttcacc ttgttggcca ggctgatctc gaattcctga 17160cctcaagtga tccatccgcc ttggcctccc aaattgctgg gattacaggc gtgagccacc 17220acacctggcc tagaaataat gacttttaaa caacctaaat gtagagcctt ccacaggaca 17280gcattgatgg atgctttacc acataacatc ccaataaagc cacagctgaa gtggaagact 17340cagtacacct cccagagatg ctctaagaga ttatgatata tgacatagat ttgaataata 17400tacctaataa ttggtatgtt tataatatat ggttttacat ccccaagacc aaaaatgcat 17460gtttgcatga aacactcatg gttacaaaaa tatattaggc caccaaaaaa acccccacgt 17520ttcataaagt agaaattata cagacacatt ctctgataaa attttttagt ggaaattaag 17580aacaaagtca agaaaactga agtgtgctta ctttagaaag caaagatctc aaggtagatg 17640aaataaatat ttaactcaga acactagggg aggaaaaccc taaaaagggt gaagaaaata 17700attttgtaag attatagctc aatgaaatga aaataaattt gacagattaa gctaagagct 17760gattctttgt ggggaaaaaa tagtaaaata gaaagttctg agaagccagg tgaagaatgc 17820gaggatgtgc aaataagagt atgaatagaa aagagaatat ttcctacaca ttggagattt 17880taaaagtcaa gaaagactta taacttaatt cctatataga gagatgactc tggctatttt 17940caaagaaaag ataaatatcc aaaagataga aaatatgaat agaccagtgg ccataagaag 18000ttgaaaaagt gggctgggcg tgcggtggct cacacctgca atcccagcac tttgggaggc 18060caaggcggag ggatcacttg aggtcaggag ttcgagacca gcctggccaa catggtgaaa 18120ccctgtctct actaaaaata caaaaattgg ctgggcatgg tggcacatgc ctgcagtccc 18180agctactcgg gagcctgagg caggagaatc gcttgaacct gagaggtgga ggctacagtg 18240agccaagatc gcgccactgc actccagcca aaaagttgaa aaagtgatta agatctggtt 18300cacctcagaa cacttaagtc caaatgattt tagtggctga attgtctccc cttcaaagtt 18360cagttacatt gttaaactgt tccagagcct agagaaatat agaaatcttc ccactgtgtt 18420ctttgaaacc aatatacgct gatactgaga tcaaacaagg acagtaccaa aaccaggcag 18480gtactaacag ttagtgtgct agaccagtct cacttagatg cagaaaacaa ataaaattta 18540ataatccaaa tccagtagtg attgaaagga atgtcttgat ccatgaccaa gtagatttta 18600ttctaggaga acaaaattct acatcgggat taagtagagt taaggttgac attttttttt 18660ttttcttctg agacggagtc tcgctctgtc acccaggctg gaatgcagtg gcacgatctc 18720ggctcactgc aacctctgcc ttccgggttc acaccattct cttgcctcag cctcccgagt 18780agctgggact acaggcgcct gctaccacgc ccggctaatt ttgtttttgt acttttagta 18840gagacggggt ttcaccatgt tagccaggat ggtctcgatc tcctgacctt gtgatccccc 18900ctcctcggcc tcccaaagtg ctgggattac aggcgtgagc cactgcgcct ggcctgagtt 18960aaggttgact tttaaacaac ctaaatatag ctaaatatag agccttccgc aggacagcat 19020tgatgtgtgg aactcttatc cacgtgataa catcccaaca aagccacagc tgtagtggaa 19080ctcagtacac ctgagtctta tcattataag atgataatag gtaacattta ttagataatt 19140accatgtact ttgtcctaat acttcatgta ttcttttact cctcacgtca actctgaagg 19200aaaggcacca cctatcccct taaaagaaaa caactattac tattcttttt tttttttttc 19260ttttagagac ggaatctcac tctgtctgtc gcccaggctg gagtgcagtg gcacgatctc 19320ggctcactgc aacttctgcc tcctaggttc aagtggttct cttgcctcag cctcctgaat 19380agctgggact acaggtgcac gccaccacgc ccagctaatt tttgtatttt aagttgagac 19440gaggtttcac catgttggcc tggttgttgt caatctcttg atctcatgat ccacccgcct 19500tggccttcca aagtgtttag atgacaggtg tgagccaccg cgcccagcct ctattctatt 19560ctattttgtt ctatttctat tacaagccag taagcaagaa aatatcataa tttataagga 19620accctataaa aaacagacaa gccaagggtc tgtcattagg aagtatgcct gaataagaag 19680ctgaagattt ttagacacag gtttcaggca acactgtctt tagaggctag gctctggctc 19740cagctccctc cagcctcctg tgaataacag gcaggcttac ttgcaggtgc cactttcctg 19800gacagtggtg gttaaaggac aaggcccaga aagtgctgaa ttaggtgccc ttgttaccgc 19860taatgtctta ttgatgacac tatcttagag ctcttttgac atcttggctc tgcgtctttt 19920tttttttttt ttcttgagat agggtgttgc tttgttatcc aggccggagt gcagtggtgt 19980gatcatggct cactgtagcc ttgacctcct agacataacc cacctcagcc tcacaagtag 20040ctgggacccc aggcacgcac catcatgcac agttaatttt tgtgtttttt gtagagacga 20100ggtttcgcca tgttgcccag gctcatctca aactcctggc ccaaactgtc ctcccacctt 20160agcctcccaa agtgtttggt ttataggcat gagccactgt gcttagcctg agtccctctt 20220ttaaacaaac aaaatggtaa atggaaagga ggaaaggctt aagaaaaaag attgaagcca 20280ggatttgttg taagcaagga gtaataaagg gcagttcatt tagagaaagg catatgacca 20340cctttccccc tccaatcaga atctagaaag tgattgaggc cgggcgcagt ggctcacgcc 20400tgtaatccca gcactttggg aggccgaggt gggcggatca cgagatcagg agatcgagac 20460catcctggct aacatggtga aaccccgtct ctactaaaaa tcgaagaagt tagccaggcg 20520tggtggtggg cgcctgcagt cccagctact cgggaggctg agaggcagga gaatggcgtg 20580aacctgggag gtggagcttg cagtgagcct agatagtgcc actgcactcc agcctgggcg 20640acagagcaag actctgtctc aaaaaaaaaa aaaacaaagc gattgagaaa atcaggtctg 20700tgtgacctta gcaatgagtt atttagcttg ggccactgtt agcttaagtc aataacttca 20760agtttgcgtt gtagttggaa tcaatagagg aaaagctctc agcattacca catatatcag 20820aatgtgacat tgattgccag accagcctta tccaaacaca agtcctaggc tttttgccct 20880gtttatgagc tttatatgct gagggtattt gatgagtctt agggaaaaaa gaacagccct 20940ggggacacag ctgcttttat gatgagacat gtttgcaccc ataccttaat gggttttggt 21000ggcaatattc tgaaatttgc cacctacatt tcaaagattt gccctttggg tgaattagtg 21060ctgtagtaga agtgggtgga ggctgaggag gttggattaa gcaggtagag gatttctcag 21120tgcatggatc gtgctgagga tggagataga gctctaagac atccacgggc ctttcctgag 21180tgatcagctt tggctcctgg gcaggggaat tggagctgga ttctagtgtg ggagcacgct 21240tgtcatcttc cttcttttcc cccagtacca tgcggagacc atcaagaatg tgcgcacagc 21300cacggaaagc tttgcttctg accccatcct ctaccggccc gttgctgtgg ctctagacac 21360taaaggacct gagatccgaa ctgggctcat caagggcgtg agtattctgc ggagagcgag 21420gggaaggctc agtaggcaat atgccccaga gacatgtcct ccaaagcgct gggttgccat 21480gtttcttccc agtactatga aggactgcag aggagttgag gtctacaaat gaggatttat 21540tcatcactgt aaacaatgtt gatttgatct actttgctag gaaatggtac cacaaaggaa 21600cctttttttt ttaccctaaa aacctaaact ttaggctttc taacttggag aaccatctct 21660ttgtatcttt ttccccatca ttaagtagca taactgaaac atattctttt cttggattat 21720ttccgtgaag tatacagagt tagagaataa gagcaaaaaa ctgtattact tttagcagtg 21780acttgagcat tgttcccggg aggaaagagc ttttccattc cttctgaggt gatgctgcta 21840ctggtgtctc cagtttggac tcttgcttac tctcttgtcc ctagagcggc actgcagagg 21900tggagctgaa gaagggagcc actctcaaaa tcacgctgga taacgcctac atggaaaagt 21960gtgacgagaa catcctgtgg ctggactaca agaacatctg caaggtggtg gaagtgggca 22020gcaagatcta cgtggatgat gggcttattt ctctccaggt gaagcagaaa ggtacgtatg 22080ggagctggag tccagttgtc taaaacagtc ttttgtctct aaacttcctt gacacaagga 22140agatgggaag gttggttgcc tggcagtgag attgagtctg tgtgttctca ggaatccctt 22200ttataactca tttatcctca aagataggct ttaatccagc atagttacat tcttctggtt 22260ctggagaaca caggaacata catacatata tatatatata tacatatata tatatatata 22320tatatatata tatatatata tatatatttg tttcgctgtg ttttgttttg ttttcaagac 22380agagtctcgc tctgttgccc aggctggagt gcagtggcat gatcttggct cactgcaacc 22440tctgcctcca gagttctagc tattctccta cctcagcctc ctgagtagct gggattacag 22500gcacccgcca ccacacccgg ctaatttttt tgtattttta gtagagatgg cgttttgcca 22560tgttggccag gctggtctca aactcctgac ctcaggtgat ctgcctgcct tggcctccca 22620aagtcagaac agtcttaatt atccttattt atgggtgagg aaagtgaggt acagagaggt 22680taaatggctt gcccaggatt acacagtgta gtaggttttc aactctggta aaacagctcc 22740agcacccata atgcaccact tcccagctca ctgtccttgc gggaaaggtg cctgcttcct 22800gttgacctgt gccctcgtgc tctgcctccc ctacttaccc tttttcatac aggtgccgac 22860ttcctggtga cggaggtgga aaatggtggc tccttgggca gcaagaaggg tgtgaacctt 22920cctggggctg ctgtggactt gcctgctgtg tcggagaagg acatccagga tctgaagttt 22980ggggtcgagc aggatgttga tatggtgttt gcgtcattca tccgcaaggc atctgatgtc 23040catgaagtta ggaaggtcct gggagagaag ggaaagaaca tcaagattat cagcaaaatc 23100gagaatcatg agggggttcg gaggcaagtc cccgttgtcc ctgctccagt cccagcgcag 23160ctctccgaag ggcatggtcc atcctgtgaa tgtctgattc ccagccccta gcccatcaga 23220atgtagactc ccaagccagt tccaaacctg ctgaatcaga atatcttagg agagtagaag 23280gcattatgtt tttttgtttt tgtttttttg ttttttttaa aaaaaagctt cccaggtaat 23340tgagatgctg gcagcttgac attgttccct gggcctgggg accaacattt gagagaacag 23400ggtcactgct cacaggacca ggggccatga tgttctgttc ctgatcagaa acactaccag 23460tgtttgctgg aatgggggga ccagggggaa agatgacagc agacacttaa gaaagggctc 23520tttttggccc ttcctgggga gccatgtgga atttcagggc ctggtgtcca tgttaaagct 23580tatggcctcc tggtcttcac ttagaatgca gctggctcag tgatcatgct aactctggta 23640tggtccattc cactctcaga ggaagatgtg tggttcttct ccagtttcag attgccccaa 23700cttagcttac cccctcccca atgctcacaa agtagagccc agtgggcatg gccaccattt 23760ttggcatcct gctaggaata caactcagca caactaagat gctagacaca ctcttgtgga 23820ttagaagtgt gtttggggag ggtgggggag caaccctgtg cacccactgt agtggcctta 23880ctgtctgagc tttgtgtaga tatcctctgt accaggcaat ttggggtcct cccctttgcc 23940atcctgataa gccataggct agctgaactt ggccctaggc caggcaaagc cacattccct 24000cttgccttca gcaggttgga gtgggccacc tcaaagggca gtcctcaagt gtccttgact 24060agatgaggcc atgggtcttt gtggtggaag cagtcatcag gcctcaggtt ccctgtcttg 24120aagtgctgat tggaaaatgg aggccctaga gagaccccta acatgcatgg gatttggaga 24180ggagaccttg ggaatgagcc catttggatt tgccctctcc cctttcttcc gtcaatgaag 24240catccatatt ggtgttgaag cccagcaggc agaattgttg gcccactctg ggggcctaag 24300gtagctggac tgccttgcca tctgtgtgca cccatgatga tatcatggat gtctgtcctg 24360gtacaaggac atctaagtta gggaatccca gggaaacttc ttgtctactg ccatacttgt 24420ggcctctgtt ctatataacc tctctccccc caactttgtc catcaggttt gatgaaatcc 24480tggaggccag tgatgggatc atggtggctc gtggtgatct aggcattgag attcctgcag 24540agaaggtctt ccttgctcag aagatgatga ttggacggtg caaccgagct gggaagcctg 24600tcatctgtgc tactcaggca tgtgcccacc cttccccaca ttctcatgtg cacactcgca 24660tgtttgtatg ggaaagctct ggaggctgtc tgatctcttc ccatggaatt gtcgcacgta 24720acacacagat aatccccttc ccccatgtac ctacacaaag ccatactctg tgtacctact 24780cactatccag aggatcagct tgctgtcatt tgtctctgaa gacagctcaa gctacatctc 24840actaatgctc tgtcccctcc cagatgctgg agagcatgat caagaagccc cgccccactc 24900gggctgaagg cagtgatgtg gccaatgcag tcctggatgg agccgactgc atcatgctgt 24960ctggagaaac agccaaaggg gactatcctc tggaggctgt gcgcatgcag cacctggtga 25020gttctggggc ctgccccatc ccccagggct tcggactggg cctgggatgg atgcaagctc 25080tggtgcagag ctttttaggt ttctccatcc tcttatgcac agcctttcat tatcctccaa 25140gttacagcag caagagggtg ggggtggaag tggaggtggc tttttttttt ctcctgttct 25200gcattcctgc ccacaccccc acccctccat ttccttctgc tctggaggca tcctccttca 25260ttggacacca cacagtttat ttcacttctg acttcaaggt tgtgaattct tcccatggct 25320taagtcctgg gatacttctg cagtgaaagg aggtcttgta cctcttcctc agagtcagaa 25380gttctgagta cctttgccct attctgaaaa gggctagggg ctcctgctcc cagctgccct 25440cttcctttgg cttccaattc agttccctct gccccgcatc ctgcagacag gcgctcccgc 25500agggggccct tgtggacctg cactggagtc tgttgccttc actgagctgc ctgtgctggc 25560cttgcatggt gcctgtaggg ggatttgctt tgctgtgcca ttggggtaca gctgctgctc 25620ttactctaga ccaaaaagtc gggttgagtg actggtggca gggccacaga tagagacagc 25680ggggagggtg gctgaccctg gcggccctgg actgagcgtc tggaggagtc gtggaggctc 25740tttcccttct ttctcctctg agagctcgtt cttcaggctc ttccagcttg tcatgtcgag 25800tgcctggcca ctgctcaggg ttggaggctc agtccctttg ccctgtctgt tccagctctg 25860gagctaactc agggatccct gatcagggtt acataggttt ggtaaaatga gtgctggaaa 25920ttaactttct cccagtagtc ttaggtcatg ctcagtgaac ttaaacttta tccagatatg 25980gttttccttc agcctttcta ttccctttct agccagtgaa agacccgctg ccctttgacc 26040tcagcccctc caagccccca agtttaaaac gccaccccct gccaccagaa aaaacagaaa 26100aaaaaaaaaa aaaaaaaaac taaaacaccc atctggtctg ggcatcttcc tttccttttt 26160cactatgtat cctgttactg ggcttaaaca gctttcagag aagagatgtc atttctatta 26220aatgctcttt cagtagcgaa ctgagttcac acttgactaa ggatattttc cggactgtct 26280gtcatcagca tccttagtgg gtttccccat atttaaattg gtagaggcca gggatggtgg 26340ctcacacctg taatctcagt actttgggag gccaaggtag gtggattgct tgagctcaga 26400agaccagcct gggcaacctg gtgaaaccct gtctctacta aaaattcaag ttagctagct 26460gggcatggtg atgcacttct gtagtcccag ctacttggag agggggtgat gctggggcag 26520caggatcgct tgaacccagg aggttgaggt tgcagtgagc caagatggta ccagcctagg 26580tgacaaagtg acaccctgtc tcaaaaaaga aaccaaacaa acataaaaaa aaaaacaaaa 26640aaatcggtag agagtgattt ctctcccagg cccacttaat gtagactggg cctggctgac 26700acctcaccat tcgtgtgatg tgattgctgt tctgatgctt agatactctt ggcgcagtct 26760cacaattgcc accatggtag gaaggtgtcc caggagacgg tgcaccttga accagtcacc 26820actaaagtgg ctgcctttct gggtctctcc acacatcccc tctctctaat ttccctactt 26880aatcgtgtga cttcatggtc tcaaaggagg aacagaggct gatcttgact tagatatact 26940gaaccatgaa atcactgcat agaatgtggg gacttgaatg tgtctttggg caagtcattt 27000aacctcttaa gacctcatct gtaaaatgga ttagatatgt ttaattatag ccttagcatt 27060aaatattcat tgctgttatt attaagtgtc tgataagtct ctgtgtacat ggatgtaatc 27120ttcctaactc ccattacctc catttataga tgagggttat atggccaata aagcctgggt 27180ttgaatctag gtctactgcc tccaaagcca gtcttctctc ctgcaacatc atgctctgtc 27240tagcaggaga tgagaacagg tctccatttg gagcctgtca gtggggtcag agactaagat 27300tcaggctcag ggtctaaatt ccatatcctt tcttccatac cctggtgttt cctatgaaca 27360gatagatact ttagggctgc aaggtttgga ttgcatggca ctgctcagaa gataagttac 27420aggtctgggc taggctgtag ctgcccctcc aggtggctag acctttcctt tctgtgtcac 27480cagttaacac tggccaacag ttccttccat taactgttca ctgctttctc ctgtgtctaa 27540ctgatgcagt ttatgaccca taactaagag cagtaccagg tatggctctg tttcctgttc 27600atgtcccctg tcctctgggc tgcatgcatt ccgttcttac agaaagaata cctttaacct 27660agtacatcct gccacacatc tgcttctact gtgaaattga tgagggggta ttaccgattc 27720ttccctcacc catcatttac tgagatgctg gtgattgcat tataatcctc taaagcttac 27780attgtctttc tgattcttgg tcttatctga gcaagtgatc tataaataac tcagtggctt 27840tctcatgact gttttaatta ttagatttta atcaagtgtc ttattaaata tatctgcatg 27900cttccacagg catctgtctc ttcacatggc tgttcagtgt gcctctcaca agttagccca 27960cgttttctgt tctcctgctt caaactcagt tgagctgcct tgctttggct ttgatcccag 28020ctttccagcg ctgctcaatc tgttgccatg gcaggccatt ggaaaggctc agtgcatccc 28080cgtgcctgaa gccaagtgag cgctcactcc atgcatgcat ggaggctggg caggagcctg 28140cctaatcaac cagccatgtg aggagggagg gcctgttcct tcctgtaagc tatgtcatga 28200ggcagcgtgg tcaagtcctc tgccagggag tggcctgggc ccagcctggg catgttttca 28260tgccagggtg ctagagccta ctgccagatt gtctccctcc acccccaatg aaaaaatcct 28320tccagaaggg aagagccaat ttcccctgta ttggagggga agtggcagca cctcctgaag 28380cagttggact ttcatcaccc tacctctgca tctgcctgaa ggacagattt agccaattaa 28440cctaaggtta ccttcctctc tgataaattc cccattctgt cttcccatgt gttgtgtctc 28500gtttttttcc tcctccttcc ctcttccttg ccccctcttc ccctaaacct tacagatagc 28560tcgtgaggct gaggcagcca tgttccaccg caagctgttt gaagaacttg tgcgagcctc 28620aagtcactcc acagacctca tggaagccat ggccatgggc agcgtggagg cttcttataa 28680gtgtttagca gcagctttga tagttctgac ggagtctggc aggtagggcc ctaagggcag 28740gtaacactgt taggataacc agcctcttgc tccacctgct ctaggagaag acagccaggc 28800ccaacctggc atctgggcac agagcctctt ctcgtctgta ggaacaccgc cagggaggtc 28860atggcagggc aggaccaaag ggtcctgtgg ctcagtaggc acagtagatg tcacaggcac 28920ttggtgaagg actggtttct gtggagtctt gatcttggct cagctcagaa tctccagtga 28980ttgggctcct cttggccttt gttcccagga acatgttcct caccagctgt ccggtgactc 29040ttcccctccc tctccttttg tgacaaagct ctgacaaagc tctgtccccc tctcgtccct 29100ctggacggat gttgctcccc tagattgccc gtgaggcaga ggctgccatc taccacttgc 29160aattatttga ggaactccgc cgcctggcgc ccattaccag cgaccccaca gaagccaccg 29220ccgtgggtgc cgtggaggcc tccttcaagt gctgcagtgg ggccataatc gtcctcacca 29280agtctggcag gtaggaggcg gcagcggctc cctggaatgc cctgctcagt ggtacctcac 29340cttgggggtc ctgggagcag tccattgaac aatgctcagg tggcactgag ccaaggtaag 29400acccctctgc ctgccacctt gggcctgcag ggaaggattg agcagagccc cttccctggg 29460cccaaaggac tctaggtagc actcataagg aatgtcagaa catttggatc aaaagcaaat 29520ttatgctgga gatttattac ataacagtgc acaggctgac tacaaatggt tatttgatat 29580tgaaaattta gtcctctaaa attgtaaaag ataaccactt ttgcttattc cagttactat 29640gtgctcttta aaaatttcag ttgggaaatg aatttattta aatgctgttt actgtgcctc 29700catttggcac actagtccct gctgtttttg agccctaaag acaaattggg ttccagctca 29760ggagaggttg ctgtgctatc ttggctgaca ttctgtgggg cctggcagcc aggctgagga 29820ctgtgtggcc tatgctgggc ctccaacttg ggatcccttc cttggcccag gacattgagt 29880taatgtcctt cactctccta gttagggagt atgctccttg tccctgtcca cagggcagca 29940agggtttcct ggaagagggg agcaaacagg cagtgcccat gcactgagga gcagcagatg 30000ggcgtgggca gcccagagaa ccaggacaca agctctgtgc agatgccctc agcagagggc 30060tccagcctcc cactcttggc tgaacagctc caacccgtag

ggttgacctt tcttaaaagg 30120tccagttctt gctgtttggc tattttaagc tctagtcttc tggggtttca ctcagctggt 30180cctggcttca gcaattgctt ccctctgaag gccttgcata gaggccaagc gtgaagtgca 30240gggacttctc tgctgtgatg tggcttaagt ttccctgaca cctgttgagt gtcctcataa 30300cttcccttct ggtgcccctc cccagctcct gagacacagc tgcagctaca agtgtgcagt 30360gtcagtgttc aagaaagtgc ctggcagagg ggctttagaa gggtcccctg ccttccaaag 30420gagctttggc aggcagagct gctcctgcag caacactccc atttcctgtt cttgcctgct 30480gagtagcacc tagatttcta agcctcatct agatactcag atttgattct gggcctttat 30540agcccagttg ctgggactgt ttcaggagct aggggccatg tggggcaggg agagggcaca 30600aaagtagaga agcctgatgt tgattcccag ggggctggtc agctctgcta ctgctccttg 30660cagatgtcaa gagtcaggtg ctagtcacgt gctgcttggc ttgtcactgt cattggcagc 30720gagaggaatg ggtgctggtg acattgggcc agggctgcct ctctgtgtca gagttcaggg 30780tgtaggaggg gttctgccaa ccatgggctg tgtggggtaa gtgggtgagg ctgatcttgc 30840tgggtcaagg tgatcctgag cccttggcct gtggaatggg ggtagagggc aaatggtaac 30900ctagcatgct gtgggggata taggatgagg ggctgcccga gcctcgggag gggtcctagg 30960gagcagatgt tgaagaggcc agagccctca gtgagctgga tgagagggtg agctgtttga 31020acgccctgag ggtacttcct ggggcctcgt gtaatggtct cttctgtatg tcccccatcc 31080catctcaggt ctgctcacca ggtggccaga taccgcccac gtgcccccat cattgctgtg 31140acccggaatc cccagacagc tcgtcaggcc cacctgtacc gtggcatctt ccctgtgctg 31200tgcaaggacc cagtccagga ggcctgggct gaggacgtgg acctccgggt gaactttgcc 31260atgaatgttg gtacgtggct ggagcagggg ctagagccta gaggagcttg gggatgcttg 31320agcattggct tctgtgggac cccgaaagtt tggggaatag aaaggggaac acacagacct 31380tagtggggca aaaggcccag cgactgttcc tctcccttat tgggaatgtt cattctgaat 31440ctctcattct ccgaagtcct aagctgagcc aggagggaaa agggtccttt gagttgtagg 31500gctgagcaat tcagttcctc ttctcttcta gtctggggct caaagcaaaa ttgtccattt 31560tttggcatct gctcattact gagagttttt tttgtttttt gttttttttt taaataaaat 31620tggccacagc tcctgtgctg tggggtggca tacacagatt acgtactgat gtggccattg 31680tccctgtata aggtagggta tcatcagatg acaggaagca gctagctctg accctgggca 31740aggctttgca ccctctccag gatagtgaat gatgtccaaa ggtccctgcc aaccctgcca 31800tctgagtgat aaggacattt cagggccttc ctcctgtttg cctgggctgt gagtttggtg 31860ccaccttgtg gtgtgaggaa gtagtggtca gccagcctag ttcagtactc aggctatggg 31920gcagctgccc aggtgcaaac ctgcctggct tggcttttac tcaccaacct cccttctctt 31980cctccaggca aggcccgagg cttcttcaag aagggagatg tggtcattgt gctgaccgga 32040tggcgccctg gctccggctt caccaacacc atgcgtgttg ttcctgtgcc gtgatggacc 32100ccagagcccc tcctccagcc cctgtcccac ccccttcccc cagcccatcc attaggccag 32160caacgcttgt agaactcact ctgggctgta acgtggcact ggtaggttgg gacaccaggg 32220aagaagatca acgcctcact gaaacatggc tgtgtttgca gcctgctcta gtgggacagc 32280ccagagcctg gctgcccatc atgtggcccc acccaatcaa gggaagaagg aggaatgctg 32340gactggaggc ccctggagcc agatggcaag agggtgacag cttcctttcc tgtgtgtact 32400ctgtccagtt cctttagaaa aaatggatgc ccagaggact cccaaccctg gcttggggtc 32460aagaaacagc cagcaagagt taggggcctt agggcactgg gctgttgttc cattgaagcc 32520gactctggcc ctggccctta cttgcttctc tagctctcta ggcctctcca gtttgcacct 32580gtccccaccc tccactcagc tgtcctgcag caaacactcc accctccacc ttccattttc 32640ccccactact gcagcacctc caggcctgtt gctatagagc ctacctgtat gtcaataaac 32700aacagctgaa gcacctgttt cctctctttt ctgctgggga gggggaggtg gttgaaccct 32760gccctctgag caggctggga atggctgcag cctcgtgccc cgcagtggga gctatggtgg 32820tgtcacctgc catcctgccc acctcctggt gcagaggcgc tgggaaagca gtagcttact 32880atcttagggt tacaggttgc ccccttcagt gctgcgggga gactttaatg gcttacgtga 32940accgaagatg ggaaagagca gggacaaggc ctccctccca ctctggtaga taaacccaaa 33000ttgcaaagtg gccttggcca tggttcttcc actttggtct tcctgcatta gcgtatccct 33060tatgggggct gtaggaggag tcagctctgg gcgcctgaga ctgggtttgg ctccc 33115220DNAArtificial SequencePrimer 2agaaacagcc aaaggggact 20320DNAArtificial SequencePrimer 3cattcatggc aaagttcacc 20415DNAArtificial SequenceSynthetic oligonucleotide 4aataattgca agtgg 15515DNAArtificial SequenceSynthetic oligonucleotide 5cctcaaataa ttgca 15615DNAArtificial SequenceSynthetic oligonucleotide 6gagttcctca aataa 15715DNAArtificial SequenceSynthetic oligonucleotide 7cggcggagtt cctca 15815DNAArtificial SequenceSynthetic oligonucleotide 8ccaggcggcg gagtt 15915DNAArtificial SequenceSynthetic oligonucleotide 9gggcgccagg cggcg 151015DNAArtificial SequenceSynthetic oligonucleotide 10gtaatgggcg ccagg 151115DNAArtificial SequenceSynthetic oligonucleotide 11cgctggtaat gggcg 151215DNAArtificial SequenceSynthetic oligonucleotide 12ccccactgca gcact 151315DNAArtificial SequenceSynthetic oligonucleotide 13tatggcccca ctgca 151415DNAArtificial SequenceSynthetic oligonucleotide 14acgattatgg cccca 151515DNAArtificial SequenceSynthetic oligonucleotide 15tgaggacgat tatgg 151615DNAArtificial SequenceSynthetic oligonucleotide 16cttggtgagg acgat 151715DNAArtificial SequenceSynthetic oligonucleotide 17ccagacttgg tgagg 151815DNAArtificial SequenceSynthetic oligonucleotide 18gttcctcaaa taatt 151915DNAArtificial SequenceSynthetic oligonucleotide 19agttcctcaa ataat 152015DNAArtificial SequenceSynthetic oligonucleotide 20ggagttcctc aaata 152115DNAArtificial SequenceSynthetic oligonucleotide 21cggagttcct caaat 152215DNAArtificial SequenceSynthetic oligonucleotide 22gcggagttcc tcaaa 152315DNAArtificial SequenceSynthetic oligonucleotide 23ggcggagttc ctcaa 152415DNAArtificial SequenceSynthetic oligonucleotide 24gcggcggagt tcctc 152515DNAArtificial SequenceSynthetic oligonucleotide 25ggcggcggag ttcct 152615DNAArtificial SequenceSynthetic oligonucleotide 26aggcggcgga gttcc 152715DNAArtificial SequenceSynthetic oligonucleotide 27caggcggcgg agttc 152815DNAArtificial SequenceSynthetic oligonucleotide 28gccaggcggc ggagt 152915DNAArtificial SequenceSynthetic oligonucleotide 29gacgattatg gcccc 153015DNAArtificial SequenceSynthetic oligonucleotide 30ggacgattat ggccc 153115DNAArtificial SequenceSynthetic oligonucleotide 31aggacgatta tggcc 153215DNAArtificial SequenceSynthetic oligonucleotide 32gaggacgatt atggc 153315DNAArtificial SequenceSynthetic oligonucleotide 33gtgaggacga ttatg 153415DNAArtificial SequenceSynthetic oligonucleotide 34ggtgaggacg attat 153515DNAArtificial SequenceSynthetic oligonucleotide 35tggtgaggac gatta 153615DNAArtificial SequenceSynthetic oligonucleotide 36ttggtgagga cgatt 153737DNAArtificial SequencePrimer 37ggggaagata tcaattcccc attctgtctt cccatgt 373837DNAArtificial SequencePrimer 38ggggaactcg agctagacat tcatggcaaa gttcacc 3739110DNAArtificial SequenceSynthetic oligonucleotide 39ccctaaacct tacagatagc tcgtgaggct gaggcagcca tgttccaccg caagctgttt 60gaggaactcc gccgagcctc aagtcactcc acagacctca tggaagccat 11040110DNAArtificial SequenceSynthetic oligonucleotide 40ccctaaacct tacagatagc tcgtgaggct gaggcagcca tgttccaccg caagctgttt 60gaggaacttg tgcgagcctc aagtcactcc acagacctca tggaagccat 11041110DNAArtificial SequenceSynthetic oligonucleotide 41ccctaaacct tacagatagc tcgtgaggct gaggcagcca tgttccaccg caagctgttt 60gaagaactcc gccgagcctc aagtcactcc acagacctca tggaagccat 11042106DNAArtificial SequenceSynthetic oligonucleotide 42cccttagggc cctacctgcc agactccgtc agaactatca aagctgctgc taaacactta 60taagaagcct ccacgctgcc catggccatg gcttccatga ggtctg 1064388DNAArtificial SequenceSynthetic oligonucleotide 43ttccccattc tgtcttccca tgtgttgtgt ctcgtttttt tcctcctcct tccctcttcc 60ttgccccctc ttcccctaaa ccttacag 884426DNAArtificial SequenceSynthetic oligonucleotide 44agtgttacct gcccttaggg ccctac 264525DNAArtificial SequencePrimer 45gtagggccct aagggcaggt aacac 254627DNAArtificial SequencePrimer 46ggggaaggta ccactgagca gggcatt 274737DNAArtificial SequencePrimer 47ggggaagata tcaattcccc attctgtctt cccatgt 374826DNAArtificial SequencePrimer 48ggggaggtac cactgagcag ggcatt 264918DNAArtificial SequenceSynthetic oligonucleotide 49tcatttgctt catacagg 1850110DNAArtificial SequenceSynthetic oligonucleotide 50atgttgctcc cctagattgc ccgtgaggca gaggctgcca tctaccactt gcaattattt 60gaagaacttg tgcgcctggc gcccattacc agcgacccca cagaagccac 11051108DNAArtificial SequenceSynthetic oligonucleotide 51cgctgccgcc tcctacctgc cagacttggt gaggacgatt atggccccac tgcagcactt 60gaaggaggcc tccacggcac ccacggcggt ggcttctgtg gggtcgct 1085222DNAArtificial SequenceSynthetic oligonucleotide 52tggacggatg ttgctcccct ag 225346DNAArtificial SequenceSynthetic oligonucleotide 53ggtaccactg agcagggcat tccagggagc cgctgccgcc tcctac 465425DNAArtificial SequencePrimer 54gtagggccct aagggcaggt aacac 255544DNAArtificial SequencePrimer 55tgccctgcca tgacctccca gacgagaaga ggctctgtgc ccag 445639DNAArtificial SequencePrimer 56acagagcctc ttctcgtctg ggaggtcatg gcagggcag 395782DNAArtificial SequencePrimer 57ggggaactcg agatggcttc taggatggca tcgatgacag gtggccaaca gatgggcatg 60tcgaagcccc atagtgaagc cg 825837DNAArtificial SequencePrimer 58ggggaagaat tctcacggca caggaacaac acgcatg 375919RNAArtificial SequenceSynthetic oligonucleotide 59ccauaaucgu ccgcaccaa 196019RNAArtificial SequenceSynthetic oligonucleotide 60caucuaccac uugcaauua 196119RNAArtificial SequenceSynthetic oligonucleotide 61ccguggaggc cuccuucaa 196219RNAArtificial SequenceSynthetic oligonucleotide 62cuugcaauua uuugaggaa 196326001DNAMus musculus 63gtacatgtta aattataggt atagtctata gggctagtga ttaaactaga acaaagataa 60gatacaacct aactatgtac tcctctatac catgccgatt tctcagcaga agcaagatag 120taggaagtta ctgtgacaca ttctgtcatc taatgagaac acttcccata gaccccattt 180gtgtaactgt cagatcattg aagcctgcag atgaagagca attcttggct agcaccaatt 240ctccagttta atggatattc atgtcagtct gggtccatat ccagattggc ctggagttct 300gcctcagtcc ctaagtgtca aggtcacaga cttaagatca ccaagctttg tatctagatt 360tgtggtgatg gtagagttca ttaatgcatg tgtgcgtgca ttcctctgtg tgtgtgtaaa 420agaggagagg agagagaggg agggagaaaa agaatgaata tgaatctata accgaaaccc 480tgacctgcgt gggcctagat ctgtgtggct cagactgact ttggcctctt gagcgttgga 540attacaggtg tgagctactc tgcttgacgc acagtatgca cagtggacct caacatgagt 600agatagataa ctgtcaccct gttgaacact atggttcttt tgtgtgtgtg tgtccaaggc 660acaaagaggt catttcttct acccccttct cttgcagaaa ggaatgattt cctccacaga 720tataacctcc ccctccccaa cttcatgttc tttccaccat aataaccaag catttacttc 780cctaaactaa ggggaccaag gggacttata gaagaaacta aaaaacttta tttcactaca 840aattgcattt atgttaccct tccctctaat caaaactgac cgaaaatggg atgaaggcct 900gtgattcaac tacaaagaag tgtctgcttg aaatggtcac acccatcttt taccaagaaa 960aacaaaagaa caaaacaaaa ccaaaaaaaa tccaaaacct aaggtacagt cttcccattt 1020agaatgtggc aacattgtat ctgctttcaa aactctgcgg aatgaagact agtgttaaca 1080gttgggaaat tagaaaatac tttgtaggga aaaaattgtt aatgcctcca atactacaaa 1140tttaaataca tacacgcctg agtcaaaaac cagtgttgac tcctgcatag ataatggcta 1200cacgtaggtc cagttctgca ctaattacct tggattggtt tgctgcgtcc aggtccaggc 1260attttaaaag tgcaggattc gagactcaaa gtattaacat gggtccaatc tagccctcct 1320tattaggagg tcgcgctcac gttccaaaga gagacttaag ttcttcctac ccagcctcta 1380agtctggaac cctctaggag gcagcccaga ccttccacca ctctcttgct cccatcaccc 1440cagcttcccg ttccgctcag cctcagaggg tgtactgggg cgggcgccgg gagggtggag 1500agtctccggg cggggctgga ggaatgtccg tggacctata aatctgggca ccgccctggt 1560aggccagggc agatggggag acctgggcaa aaggccaaaa agggaacatt ttgtagccga 1620tctaaagcaa caggtggcgg tggcgcccgg gaacctaggg gtggtggtgg cggcggcggc 1680gctggcccgg tgggcgggcc ttgctccctc accacttccc cattggtcaa caggataacc 1740cttgagcaaa tttggtctac gatgtccttc cgccacggaa ggtagtcctc ctcaaaaggg 1800caacctgctt gtcccgccta ccctgagctt ctctcaggag gtgcgggcgc cccgttgaga 1860ggcggggcgc cgccggccgc agcccggatt gggcgagggg cggggctgcg gagggattgc 1920ggcggcccgc agctgtgata accttgaggc ccagtcggcg cagccccgca cagcagcgac 1980tcgtcttcac ttgactgacg tccgctctag gtatcgcagc aggaaccgaa gtacgcccga 2040ggtgagcggg gagaacctaa gccatctgtc cccagagccg atgcccactg gtgtgtaacg 2100caggcctgcc tgcccacgcg ggactcaggc gccaggcatc gagcatccca ctccgggttg 2160gggaggagcc acacttgagt ccaagtctta atcctccaaa ttgagggtgg ccggctagct 2220cctttcccac ggtctcattc tgctctctct tccatctcaa tctggttcca gcctcttatc 2280ttactattgc tatcctcgcc accctgttct cgccctgctc cgatgctcgt cctcctacat 2340ccactacgac ttacgacctt cctcttagct ctgtgccctc ataattcggt gccctcctct 2400taatctctac ttgaaataat tcccctactc accccacccc caccccccat tcagcctccg 2460cgttcatcct cctttcaggt cagcgttctc ttcccgcgcc agtgttcgca gctcttcgtc 2520gcccctcccc cctccgatcc ggtcacgtcc gcgccgcagc atcctttcaa cctccatatc 2580ctcgcccctc cccctcgcag cttttcccct ccccccgcat tctagtcacg tccgcggact 2640cctcgtggtc cggtgcatgg aggtaggatg cagtcactag cggtccttgg taatgcagct 2700acaagttacg taacttttgt attcccggtg gcctggcaag aaaggttggc cgctattgct 2760gcctgttttg tgtgcccgca gatttttggt ggtgggcacg ggccctcatt gctctcgcgc 2820tccatcctgc ggcggagggg cggcacgtac tgcgatgcgt ctgattaatc ggtcatgctg 2880ctcccacgtg ctggcgaggg ggagggtggg cgatcaaccc atacccgttc ccgacagaaa 2940ctggagcccg acgctaccag aacacacata gcccacagag ctcggtctaa ttagggaagc 3000agattttgtc aagtcagtgc tatgaggaag atggggctgc atgcacaagg tggccggcat 3060actggttttg aaaaagccgg gcatcttcta gtctgaaggc tataaagacc cagtaaatac 3120ccagtcatag taagataaga gctcgaccac tttttccaaa tccaggacac tggtagtttg 3180gaaaaagtga accttatagt gaaaatccag tcgtgcatgg aggcagctag gttttggaat 3240ccggtgtgat ctattaatat tcttaggttt ggcctctggt cctgtgtcac tccaggctgc 3300taaattactg gtctccttgt aagtacgggc catcctcgat gtagtgacta acctcgggtg 3360aaatatccag aagggcatgc attatatcct tttggtcaat gaatgaagta gttaccttga 3420aggctaccaa cttgattctg tctccttact atttttatgc acattagggg taagcttggg 3480gaggcaggta ggctctgaat acccagtatg cccatggaaa caaattctct ctggaatccc 3540gtcagtgttt acaaagtaat taatgttaca ttgcagctct cctgagaaag agctatctaa 3600agggagaaat ctgtgcagcc tttgtctttg gctgtgaggg tagaaacaaa gaaacaggta 3660cccagataga agccttccgg tcctctttgt tcaagtaagc cttttatgcc ttctggcttg 3720taggaagttt ggtactgttg ccccaacagt ggggtggggg ggctcagatc aagactcaag 3780gaagcattgt tagcaaataa tttgccctct gatggcctga ttatgctgta ggaaaggcag 3840tgagggtgtt ctttgggaaa gtagtgtaaa ccatcaccgg gtctgggcaa gatgactcaa 3900cctttcaact ttgcggcttt tgagaaaatt ttcttccttg ggtgagtgag gggaccacac 3960ttgttctctg aagtgttgta aggttccctc ttttttagtg agtcaaatag cgtgcatttt 4020ccctgaatta aacatgtctc actcagactg agtgtggtgg ttcagcttta atcccagtac 4080ttgggaggaa ggcctgtgag cttgagaaca tggagcgggt gataccctgt ttccagaggt 4140ggcgggtatc acctaaatga aggtgaccat aagagtggaa atgtggagtg gctgcttcta 4200gacggcatat tttcattctt gaatgattgg cacagcagct tttcatagaa caaccagtat 4260ccagattggc agtttctctc gaggcatttc tgaattattg cttaagccaa actatatgaa 4320tactaatcac ttatttagta catagctatt ttcattttaa gcattatatt ttaaattatt 4380taatttgtag ggaaatttgt taagaactgt ttgccagcac tcaggaggca gaggcatgca 4440gatctgagtt tgaggccagc ctactctaca aagtaccaag acatccagag ctacacagaa 4500accctatttt gggagtgggc tttggtggaa atgtatctca accaggtata atggcccttg 4560atccctttat tcccggtgtt ctggaggcaa actggcaaat gactttgagt ttagtccagc 4620cagtgagtga cttggcacat ttcatagctg actccaaaca gatttatggg ggctgggtat 4680gccatctgtg ttatatgaac gtgttggtca caggaaacgc tattttattt cttaaagctg 4740ttaattgctt aatattgagt ggaagggatg aggaagcggc tatggacaga aaggtagaaa 4800aagcgtttct tgccttaaac tcacattggg tttttagcat ctttagcttt tttgcagcaa 4860gtcgacacga gttgggcaag aactaagctg gagattgagg tagaggaaaa tgactcattt 4920gtctttcagt ttctcaatca tctcaaatac ccacaggcaa gttaactggt gagttttttt 4980tttttttttt ttttttactg cttctggtaa aggaccgagt catctctgta aactgcactc 5040atggagacta taataggagt agatgactgt atgacgaaga acattcttcc tgccctggaa 5100ctttcgtgcg tcttgaaatt tttaaccaag agccagactt

actttggcta tgtagtaatc 5160aggagaggtg agccccacgt gcatcgtggc ctgcaaaact gctggatctt aaggcctagt 5220gtagctactc aggtttggag gtggtcccag ggcaccacca gctgtgctgt gaggccacac 5280ccctactgtg gtttgtgcct gtctgcacgt aggagagagg tctgggctgc agtaccttaa 5340gtggcctcta gggagctctg agcatctcct gaggcctgcc tccaaaggaa gctgtattcc 5400ttctaaatgt gtgaataacc tttaggccat attttggcct taaagtactg gtgtctccag 5460tgtaaaagac tttacccttt gccatgccca ctttggagga gtcaaaggtt tgatttgatg 5520tagagaaggg ctgggtgtgg tggaatgtat ttttaatctc agagagacag gacagtcagg 5580atttgtagag tccttgtctc aaaaaaacaa agtagggaag gggctggcaa tgtagatctg 5640atacaaagcc atggtccatc cctactacta cataaaatgc acatagtagc acgtgtctgt 5700aatcatactg agcaaaggac agtgggattg gagaagttgt gagttggaaa aaacaaaatg 5760gactatgaac tgaataaagt cagaatgcta tttaattaag ttttggaaac tataaagata 5820aaaagtcagt gttgatgcta agttctgggt gggagaaatg gagtctttag atcccactcc 5880ttggggaaaa ggcggtcgtg caagcctgcg ggcagaagta gtaagtaggt cagtgctctt 5940ggacagctgg tgctgccttc cttccgactt taatcaccct gcccccaact gtggctccgg 6000gctgagccgg tgcctcctgt tgctgagacc attgtgcatt ctgcttacac ctggttctct 6060gggcttctga gccagggtcc agagcatggg cagcattccc ggaaacactg gccttgctaa 6120ggaagaccgc atgtgccaac cggacagaaa cttctgcaaa gtcacctcct tcctgctctg 6180gctagttctc cgctctgcac agctgagcta gaagccagga aatcccttgc tttgaggaaa 6240gtaagacctt gtcttaaaaa tcaaataagc tgggccgtga ggcaggcaga tctctcagtt 6300tgaggccagc ctggtctatt gagttccagg acagccaggg ctacacagag aaaccctgtc 6360tggaatctga ctctcaaccc ccaaaatctc tgctcaagtg atctgtaccc ttgataccgc 6420acatttaatt ttgttgtatg ctgggtactg tgctccaggc cttaccactt tctggggata 6480gaactgtcac ctgctttgac cctggtgttg gcttctcatt agggtcactg atagctggac 6540agcagccttt ctgatggatg aagtgagatg atcttgggta ctggggaaag taggcttggg 6600gtgacatcag agaaaggttg ctacaaagcc ctgttcctag gccaggctgc tttattaggg 6660cagcaatgat ttggacctac gtgacgatgt cattggtggg gtgaggcatg accttccaag 6720agtgttggct ctaagaagca gggtggcacc aggcattttt ggtttgggga tttttttttt 6780ttaatgacct ttgttcctga cacgagggag atgaatgtga agggccgagg agaactgcct 6840gagacaggaa agggcagaac caggtaatca gagccaagct gggcctgtgc atgctccaac 6900ctacagccca actgaggcct gggctggtgt ctcctttctc acagaggggc aatggctctg 6960aagaacatcc tgcttcaggg gctcaggttc aagagtaagg cttctgcccc agggcatggt 7020atctactgct gtcgtgggac acaccgggtt tctgctgccg gctggaggtt ggctgtgcat 7080gtgggtgcct cctgaggagg aggatgctgt agtgagttgg ctggcatggg tggggccgca 7140tgtcctggca gtcactgcta tctatacaag tatttggcca ataaggcaag tcctgcccag 7200gggcccacct taccctaagg aacaaggctg gagctgctgt cctgggccag aaaagacttc 7260taaaagggtg ttctatttat actatactat agataggtgg ttggctggag ttgtggggct 7320ctggaggcca tgcaatgggc agaagagctg tgggagggag ggaggggctc caggtcagag 7380ccttgggagt gaaaatagct gggttctaag gatgaggttg gtaggggact tgaaacccct 7440gtaaggtttg ttagaagtct gtttaattgt gtccatccat tggaacacat gtattttcat 7500ttgtcacttt gtatatggta gcaaggtaag tctactctgc agactttagg tttaattcta 7560gatagagaaa tgggttgtgt gtgaaatttt acctgctaaa ttgcatcctg agccctggag 7620ggcagcagtg cttgtctgtt ggaacatcct tgttgcacaa caaagttctg tcaagcctga 7680gaccggtcaa aacaggagaa gaaaattgca tggaactgat aggttgtggt catgaaattt 7740ttccatggat tagccacaca cttttgggaa ggtgactttt aaagtcagtg gattaagttg 7800cccttcctgg gtcagtgaag ctgccctgct gagcactttt gagggcatag agaacacaca 7860tctatcacta tattttagca gggtaattct gaaggacacg gctgatgtca cagtgagctt 7920tatagggtgc atagagataa atgtgtcatt ttcaaagact acttaattaa cagttttatt 7980aataaaggac acgctgggca gacagaagtg tagtaagatg ctgtctcaga agagtagggc 8040agtaacgtga tacaggctat catccaaggt ggagcagcga gggtcagtga gatagcttgg 8100aaacacacac aggagcttgc ttctgatggc tccagtgctt ctgtccccag aacctgagtg 8160gtaggagggg agagcacagt tgtggtcctc tgatccctgc aaggtgccca ccaggtaccc 8220cctaaaacta aatagtgaaa tttgtgtatt gggggaataa agtacttcaa gggatccagt 8280aaagggtcac ttttaccact gggttttttt atttatatct tctccactgt atatctgttt 8340taatgtagtt aacagcaact ttttgccttg ttattaaggt cgttaccaca aagagcttag 8400tcatattcag ctttcacctt aatccttgta gctcttcagg gcaaagcata ctccactctg 8460agcgatcttt ccaagtgagg ccaccaagcc acactgagtc aacaaaacag caatatgcca 8520tcagaagccc agacccagag aaccaaaggt atgacctgat cactgggcct cactcaggca 8580gggatttgcg agggcacact cgtgtctcag atgggaagaa cggcctggtg gctggctttt 8640tcagtagttt tcagacactg tcctggtgtc tgtcccgctc acttgtccca tctcggacac 8700agcaaggcct ttgtgtctca ttggcttggt gtaccatctg tactgtccct tagcggccac 8760ttatcctaag cctggctggg agccgtgagt ttgccagttg tgtttgtttc ggccatgagt 8820agaccaagga cctggctgcc tagggacttc tcacatttgg ggagagggaa aaccaggcag 8880aattgatctt ggcagagacc cggagctcat ttaacaaaca aggtgctggg aggcaaggag 8940ctaagactct ggagaagttg acatcttggt gtctgatgtg tcttgtagag ttaggaatga 9000acaatgttgg ccactggttt cttccttcct ttaaaggttc tcaattctgc aaagaatgta 9060gtggttgcac ccctgtaatc ccagcaccca agagctggaa catgagactg tcatcagttc 9120aagaccagcc tgtgctacat atgataactt tgtcttaaag tttttcatat ctgcatttga 9180agacttacag gtgtctggta cttggtcact agtgggtggc tgaagtctca ggttgcagag 9240acagacttag gtacatactc tccaggcgtc cagaaaagcc agagttccgg gaagagtggg 9300ggtcctcagc tgaggagaca ggggagagca aggatatgga gatactgtgg agcacaggct 9360gaagacttta tgttcaggag tttgagagga gccttgtggc ctgaaaactc agatgagaaa 9420gaatgtcagc ttcattcaag tctcagatag tctgcgagct atagcccaga gctctcccga 9480gagttctgtc ctctaatcag ctgactatcc agctcagtga gcacttagtt gtaatgaagt 9540aaaagcaaag gtcaggacag ctgttggaat tgtgacctgg gtggttaatt acctggaaca 9600gatgttcaga ctactgggtg ctatacctat cagacagaga aagaggctcc aaatagccag 9660acctcgggct ggggaagcta ggtctaatgt cagcccttgg aaagattaaa gatggtaact 9720tcaaggtcag cctaggttgc acagtggatt tcaggccagt gtagagtagc tgctgtgatt 9780ccatctccta ggttacagaa gactccttgg tgaggttata atgactttgg ggccatttca 9840atgcccttgt gcaaaatttg ctttgtatgt cttgtaggtg gtactgtgtt tttgtttttg 9900cttttgttta ttttttgcta aagaattatg gacttttgct tcctgcccag aagaactgtt 9960ttgttccctg taatttggac cttactcttg tgtctgataa aagctaaaga atacatttag 10020atagacctgg gactttggga gtaaaagaaa ggctttggga cagggtttct cttgtttgca 10080ggaccacttt aggtttgggt gaatgagaga gtgagaccat gctgagtgcc taccaaagcc 10140aatactggag attggacatc atgataggct tcccaagatg tttgtgtgct gacatactag 10200ttagaacatg cgctgggggg tgtttcttac tgtaactttg gaggtttcct tgtgtgggga 10260aagtgatgtt ctttctaggg ttgatgagaa agctcttaag acaaggtgac agcccatgtg 10320agaagcctgc ttaaactggc tcccagggtg tgtgcagtgg gtagggagtt actcttgttc 10380accaaggtct ttgctgtttg ccaatagtca caagagccct ttgcccaatt tgagtagcac 10440ccacataacc agcacgtgcc aggcttttat gctgcttgtg cacaaagcaa tcttattggg 10500ttaatgtgta agcaggagac agcttcctat gccaagtgcc tcattaagca gtgcctaatc 10560taattgcact cttcaaatct ccgggaccca ggacttcagg aaccatgccg aagccacaca 10620gtgaagcagg gactgccttc attcagaccc agcagctcca tgcagccatg gctgacacct 10680tcctggaaca catgtgccgc ctggacattg actctgcccc catcacggcc cgcaacactg 10740gcatcatttg taccattggt gagtgtggcc ctccttcctc taaatggagg cttctacctg 10800atttgaaagg catagtaacc attgcagagc tagcctaggt tctgagtgag gcacggtcca 10860catttctagg ggagtagagg tcttgggaat tggccatcaa gaagaattag tgtgcttttt 10920ctgtagttgg tgaggttcag gggtggtctt tttcatgtgc tgtcaccaac agcattcggt 10980agacagacat cttgaaggca gagaaaactg ttgccctcca cttttctggc tgaatgaggc 11040cctgcagaat ttgtagagaa tgttagtgtt cccacataat taagaaagtg actagtacaa 11100tagtcttttc tgtactttgg aaagaccttt ctcttaattt gtgaaggtaa ttaaaagcaa 11160aggatgaaga gttgtttaaa tgttgagcaa atttcagaca ttttcctacc tgagtcatga 11220ttttcttcct gtggatctaa atgtttcttg atagggcctg cttcccgatc tgtggagatg 11280ctgaaggaga tgattaagtc tggaatgaat gtggctcggc tgaatttctc tcatggaacc 11340catgaggtga gcgtcaacga gatccaggag actcagcgat tccttaacag tcgtactgca 11400ggcaggtgtg agtccagggg tcccagtgaa cggaacattg ccgtttctct cttctaactt 11460cactggaata gaaacctggc ctgctttgtc acccaccgac cagggttagc cctaccgtca 11520acctttatga aagaaggcac gtaagggttt agctggaaac cctaggccat cagatgtctt 11580ggcccccatg cttcagggtt tttacagtgg ttcctgtgtg tgaaccaaaa ggttctgagc 11640agatggatag ctggagtcat tttaagatct accttttaaa tacttctctc tccccctccc 11700tccctctcga cagggtttct ctgtatagcc ctggctgtcc tggaactcac tttgtagacc 11760aggctggcct cgaattcaga aatctacctg cctctgcctc ccgagtgctg ggattaaagg 11820cttgtgccac caccgcccag ctttttaaat actttctaac ttgactgtgg atttcctact 11880ggtattggtg aaggagggaa ggagactcct ctctgcctct tggtttctgt gtcctattta 11940gagtaaaagc attaaccctg tgctgttttg ccctctgacc tttggaagtt gtttggacta 12000aaaatagatg gagaagaatg gtccaagaag tgaaccccag aacatgagaa tcttcataga 12060ttccctaacc catattccat aaatagcttg gaggctagtg cccaaatgtc atggaacctg 12120agatagttcc tcaggcacct aagcaatatt tgacacattc ttgctgggtg tggtggtaca 12180agattgtggt tttgaggcca gaatggggac tatcctcagt aacatagtaa gatcttattg 12240caaaccagga gaggtcctta ttttccaaac tctggctttc cacaggagct cccaggaaga 12300aggtgagcct agttcagaga cagaactagg gccacttgcc atggtccttg cgagtagtgt 12360gtttagtcag gcaaaaatag atttggaggt gctgaccttt agggctcttg agtccagtaa 12420aataacacca tgcagcaggt ctaagagggc aggggacagt gagactgtcc agaccactgg 12480gcaggccagc tctgccttga tttcagacta aggcattaga gattggctgt actttgaacc 12540tttttatatc acaatataaa gcttcacaag tcagggctct tattccatgt gcaccttcag 12600tgaggctctg ggtgatggtg gtgctggtct tggtgattcc ctggagaccg tggaaaccaa 12660gctccttccc tctgacagga acatcagcca cctagctgca cctgatcttg acagctttgg 12720ctgtgtctct aattcccatc tcttgctttt cacatattca agatgtgtca ttcttgctga 12780acaggcagta ctgtactccc acactggctt ttaaacagcc taaatttaga gcctctacaa 12840ggatagcact gatggctgcc agtcttcccc atttggttac atgccagaaa aaccacagct 12900gtgataatgg atagcacgac ccagctccca gcagtgttac catgcagaga aggctaattc 12960accaccagat actccaatca caatgcagct ttatatatac gaagctgaag agtgtttatt 13020atgctcgtga atgtgctgag gtgtgtaact cagtgtgtac agccataatt cttcagtgga 13080aattaaggga gaaatccaaa cttctagagg atctctaaag caaatgaagg cagtcggtag 13140tcaatatttt gggatatctg gagctgggtt accgggtggt ggtcagcctt tgggcagctt 13200cggtccttgt ggatgaatgg tggttccagc tctgccctaa caaacaggct tgcaggtgtt 13260ttgctgtgct cagtggttca aggaccagac tcataaagtg ctgaattgaa tggtccattg 13320tcaccagtgt cacaaggata tgcactggca acaaactatt ttgctatctt ggctctgagt 13380cccagatagg aaagggaaaa ggtttgggga aactttatta caagtgaaga aagcaatggc 13440ggttgcaccg gggcaggctc ctggtcggag gaagtggtta tgaaagcagg gtctgcgtga 13500ctagagctgt ttagctcggc cattgctcac taagtcaaca gctttgagtt tgaattgcag 13560ttgggatcga tagtgaaaac agatagaggc tgccagggca gaaatcttca aacacaaatc 13620ctgggtcttt gcttgtcctg attagcatcc cttggtgagt cctagggaca ctgggacaac 13680agaagggtcc cacaggatgg gtttatagtc ttccctttaa ttgattttgg tggcagtatt 13740ctggaactgt atgagagttg gagttgatgc tgttgtgtag agggaagaat ggatattgct 13800aaggttagga gatgggtgaa ggtcaggagt cagacatact tttttttttt tatttgctaa 13860ttgatcatct ttagctccag ggtggggatt ggaaactgga cagggacacc tcacctgcca 13920atctgccttt ctttctccag taccatgcag agaccatcaa gaatgtccgt gaagccacag 13980aaagctttgc atctgatccc attctctacc gtcctgttgc ggtggctctg gatacaaagg 14040gacctgagat ccggactgga ctcatcaagg gcgtgagtat ctagaatagc ctggtagggg 14100gtcacacttt tgctatgtaa ataacctatt tagtctcact ctgggaaacg ggtattttgt 14160ttgttttatt tcttcctcaa tatacaaatt caggctttat agaaaggtga gaggtttctt 14220tggactttga gccagagttg agcgccccca tcaggggcat tggcttcttc agttcacact 14280cccatttcct gctttaatcc atagagcggc accgctgagg tggagctgaa gaagggagcc 14340actctgaaga tcaccctgga caacgcttac atggagaagt gtgacgagaa catcctgtgg 14400ctggactaca agaacatctg caaggtggtg gaggtgggca gcaagatcta cgtggacgat 14460gggctcatct cactgcaggt gaaggagaaa ggtatgtctg gtacacagtc cgtggccaat 14520gccaactcca atccccagag ctctggcaag cacagacctc gaatgtatga agatctgggt 14580ttaatctcca gaggatcaaa agtctaaggt tattgttggt ctgcgtccct gacctgtctg 14640aaatactgtc tcagaaaaaa ggcagatggg gctggagaga tggctcagta ctgactgatc 14700ttccgaagat cctgagttca aatatcccag taaccacatg gttgctcaca accatctata 14760atggttgtga tgccctcttc tggtgtctaa agagagctac agcgtgtata aaaggtcttt 14820gggccggagc aagtggggga tcctaaattc aattcccagt agccacatga tggctcacaa 14880accatctcta cagatacagt gtacagataa aacacattaa gtaaataaat aaataaataa 14940ataaatataa aaggtctttg ggccggagca agtgggggat cctaaattca attcccagta 15000gccacatgat ggctcacaaa ccatctctac agatacagtg tacagataaa atacattaag 15060taagtaaata aataaataaa taaataaata aataaatttt tttaaaaaag aaaagggcaa 15120ataacccaca aaggtccagg tacctttagt cctccgtcct agcgttcggg aatcaggaag 15180gtggagatgt ctcggtgcag catatgttag actactatta tatgccttag aatgagagtt 15240aaagttactt attctaaata ctttgtgaca gtttgagagg gtttcctata gctagccttg 15300aactcttgat tcttctgttt ccacctccca aatgctcaca ttaagaatat acaccaccag 15360ctgggcatgg tggcgcacgc ctttagtccc agtactcggg aggcagagac aggcagattt 15420ctgagttcga ggccagcctg gtctacaaag tgagttacag gacagccagg gctatacaga 15480gaaaccctgt ctcgaaaacc aaaaaagaat gtacaccacc tcgtgtggct atttatttgt 15540ttattcatta atttgaggca aggtttccct ttgtagccct gcctgacttg gaattaactg 15600tgtgtgtaga ccaggctggt cttgaactca aaggtctgtt tgcatttgcc tcttgtgcta 15660ccatacctaa aactcaaatt ttcttagcag tttgtaagta agtatttata ggtgagaaaa 15720ctgacttggc tttcctgaag tgttttgttt ggtttggttt ttgttttgtg tgtgttggag 15780tcttaattat ggctttagag tcctcctccc tctgcttctt gtaaattgag gtggtcttct 15840gtgatccctt tcacacaggc gctgacttcc tggtgacgga ggtggagaat ggtggctcct 15900tgggcagcaa gaagggcgtg aacctgccgg gcgctgctgt ggatctcccc gctgtgtcgg 15960aaaaggacat ccaggacctg aagtttgggg tggagcagga tgtggacatg gtgtttgcat 16020ctttcatccg caaggcagcc gacgtgcatg aagtcaggaa ggtgctggga gagaagggca 16080agaacatcaa gatcatcagc aaaatcgaga accatgaagg cgtccgcagg tgagtcctga 16140gacccttcca ttgcccagcc cttgagaggg gtgtggccat ggtgtgtcct ggatacctgc 16200tcagcaaaat acagcctgct gggattggtc caggcggaca tctgaatcag cattagggag 16260gccaagtatt tttagtcatc attttgggac ccggctggat actcaagggc ctcagatgtc 16320catgctaaag cttgaagcct tagaaatctt ctggtctgat aatggtgctg atgaggagtg 16380gcccattcag cttcccatag agaagcatga tgcctacgta aatggaaatt aattaaggtg 16440gcattcataa ggatgagtgg tttattgaca aatgttcata cttggctttc tccctactgc 16500ttcccctaga actgcttctg tgggttacag tgggcttggc tctgtgtcct ttgtactggg 16560caactgggag tctctttcta tcttgataag ccataggtgc tgatggcctt ggtatttggg 16620gctggggagt gggtcagctc aaagggcagc agtcagtgcc cttagctaaa tgatgatcca 16680ctttgtagaa gatccactgg cctcattctg tctttgaagt gtcgattagg gaagtacaaa 16740acggggtggg gggtgagatg cagaccaaaa cctccctgaa atatttatta tggtgtttaa 16800gaagtccagc cagtaaaatt gttgtgctgt gagtattatc atactgtgct gtggatgccc 16860cctcgcctgt gtgcccctgg gtgtgacctt tgaaagcatc tctgtcggga tcccaggtac 16920tttggttgtg cttcctgtcc tctattatct ttctcttctt taccaggttt gatgagatct 16980tggaggccag tgatgggatc atggtggctc gtggtgacct gggcattgag attcctgcag 17040agaaggtctt cctggctcag aagatgatga tcgggcgatg caaccgagct gggaagcctg 17100tcatctgtgc cacacaggca tgtgctattt cattccttct gcattctcca cctaggagac 17160ctggccttgt cctgtccttt gggcacacat agctgtgatc tgtgcacctg cacaatctta 17220agggaattat cttggcaatt atcactgaag atggcctagg atctcattta gtgatggtct 17280tttaccgaga gcccttgtct gtcccctcct agatgctgga gagcatgatc aagaagccac 17340gccccacccg tgctgaaggc agtgatgtgg ccaatgcagt cctggatgga gcagactgca 17400tcatgctgtc tggagaaaca gccaaggggg actaccctct ggaggctgtt cgcatgcagc 17460acctggtaag tcctccaagc ctaccaccaa ggcctctgca tcacccagtc ttttacctcc 17520ctccgaccac ggccagaaga gtgaggtgtg tggagcatgc tctgcttctt gattttcacg 17580ttgtgctctc gctgcctgcg ccccaccacg ttgtcctgct ctggcgatta ccttttccat 17640tacgtaggcc acatctggct aaaatattaa agtcctagga cttagtcaag ggataccttc 17700ctccctcctg aacacccaga cggcggggcg gcctctattc taaaggagcc aagagtgtgt 17760attcttggct gttcgcctgg tttggcttct aatttgatct cttgatggtc ccatgagcag 17820atgcttctct gcactgcagg ctgtagccat actaagctgc tttgagctgg ccttgcatgg 17880tgcctgtcac atgggacgtc tcttgctatg ccaaacccaa tgtagggcta gaaatagctc 17940tgggcgtggg gaatgggtgc tgaatttagc aggttctgga ctggagatta taaagacttt 18000ctctgggcaa atctatgctc tttttgacta agtcttctgg tttcagtaag atagggtctg 18060gaggtccagc attcagagcc tgaggcagga agatagcttg aggctaggct gggctagaat 18120aaggcattat cttacagaaa caacagactt ccagctgacc tgactcctgc tgactgtgat 18180gggtgaggac ccagaacttc ctgagcagag cagttagcta gggcgccagt taggaccttt 18240ccttgcctca tgaaagcatt gttggctaac tttcttggag cttttctatt cccttttcgg 18300ccagcaaaga accactgttc tttttgtgtc tccagtttcc aataagcccc caaactgaaa 18360gaaaaaaaag cccccttcag gattagacat cttaccttgc ttcatttgtg tacagctgtt 18420aagtagattc catgatctac ccatggttta tctgaattgt agctgtagcc agatgtgtgc 18480ctatattgaa acagaccagc cgttttgtag aagcttgaac tcagcttgtc tcagctggtc 18540acctcctgat ctgattggta ttgggagctg atcttcacag cttctcagta gctagcatgc 18600agacattcct tctccagcag tctgtgtgcc ttcctgatct acaccaaacc cccttctttt 18660ctagtcacct gcttagttgt cttatcacct cagagtggtc aggaacaaga ccaggtagtc 18720taaaccatgc agtcacatac atggatttat ctttgtatag ctctgggtga cttatatgac 18780cgcaagacct tgcccaaggt ggcatttgat gagattaatt ataattaatt agtcataaca 18840ttaaacaatt tactgccata atgaaatgtt agataaccct ctgggctcat tgatgtaatc 18900tttgcattct cattttcttt ttaaagggaa tcacatgtga tagtgtgttt gagcacagat 18960ttgctatcct catagggcca gccagctgtc tgtttgcacc ctgctgtagc aggtgtgggc 19020agagtaggag ttagttctca tgtcctgccc tctctcatgc cctgcccttt cctatgaaca 19080gacaccttag aacctcgagg ctgggattgc atggccctgc tcagaagatg agtcacagag 19140tccgggttag actgtggctg ccccttcagg gggtgcaagc ttctctctca tcagttaaca 19200ctcaggatag cttctcccct tcatctgttc gctgcctcct cctctgtcta actgatatag 19260ttcatgacct gtaattaaga gctagacatc ccagctatgg tcgtttcctg ttcatgtcct 19320ttgggctgca tgcattccat ttatttgtaa ctaaaagaat actttccact tgcaaatctg 19380ctaatactac caataaatgt gagttattgg ttttacatct tctctatact aaaatacttg 19440ggattgcact ctctaaaact tagattttca ttctaatgcc tggttttact tgaacagata 19500gtctatatat aacacatttg ctgttttgta acagttttaa ttgctaagtt ttaattggtg 19560tcttaaggca tgcatgcttt ctcaggcatc tgcctcttca cacggctgtc cactgtgttc 19620aagtgagcca gagttggcca ctgttctgtt tagaactggc gcaccatgta actttggctc 19680ttttgacctt tgaccccagc tttcagagct gcccagatgt ttctattata aaccaggtgc 19740aaggactcgc tcttgtatgt aggctaagct agatgtcttg taaccacaca gccgtgtgtg 19800gaggggaggc ctagttcttc ctgtaagctg tgtcatgagg cagtgtggtc aagtggaagt 19860gtggttggct ccaccttggc atctttccat gccaaggtcc tagggcctaa caatatgtcc 19920ctgtcttagc ttcaatcaaa aacaaaagaa attgatggtg cctgcctgtt atcctagcac 19980ttgggaggct gaggcaaaga aaatagtgat ttttgaggat aactgtggca agttcaaggc 20040tgataggggc tatggtaaga tcccatctca aacacatggg ggtgagtccc atctcataaa 20100cacatgggga tggggttttt tttaagaaac aaaggggaaa gtcccaaaag gataatatct 20160ttctagaacg gaaggaactt tccttgtatt tgaacagtaa

ggggaaaagg agcagcccaa 20220aatcccacgc aaccattcca ggagcatatg ggctttgacc accctgcctc tgcatctgcc 20280tctgcatgaa gaaaagatta aacctaaacc taagggtgcc ttccttcctc tctgatgtag 20340ttccctgtct ttccatgtgt tgtctctctt gtttttgcct ttatccctct tccttatccc 20400tcctacccta aaccttacag atagctcggg aggctgaggc agccatgttc caccgtctgc 20460tgtttgaaga gcttgtgcga gcctccagtc actccacaga cctcatggag gccatggcca 20520tgggcagcgt ggaggcctct tataagtgtt tagcagcagc tttgatagtt ctcacggagt 20580ctggcaggta gggccctaag ggcaggtatc attataggat aaccagcttc tcgcgcaact 20640aggtccgcta tgtgcctgag cctaggcaca gcctctctcc ttcaggaaga cagccaaggt 20700caccataggg caggaccaaa ggattccctt gggcacagtg gaagtcacag cacctggtgc 20760aggatggttc ctgtggagtt tctaatcttg ctcagttcag aacatggagt ggctcacctt 20820ctcctggcca tttttgtgcc cagggacatg ttccttccca gttgtctgtg actcctttcc 20880tccctctcca tttgtgacaa agctctgaca aagccctgtc ccccgtcctc gtccctctgg 20940acggatgttg ctcccctaga ttgcccgaga ggcagaggct gccatctacc acttgcagct 21000attcgaggaa ctccgccgcc tggcgcccat taccagcgac cccacagaag ctgccgccgt 21060gggtgccgtg gaggcctcct tcaagtgctg cagtggggcc attatcgtgc tcaccaagtc 21120tggcaggtag gaggcggcag tggctccctg gggatgccca cgctcagttg acacctctcc 21180ttgaggatgc caagagtgag tggctctggg ccagtttaag gcccctggct gccactagag 21240gattccaggc agcactcaca gatagaccaa accaactggc tggctccagt gcacgttcaa 21300agcctgcctc acagagagct ggaacaaaca cccagtttca ccgtgattag tactgtgggg 21360ccttttgaca gaacactgcg tggcagctgg gcatggcgga gcatgcctct aatctcagca 21420cttgggccaa agaggtaggc gaatgtctgg atttgtagcc tgtctagtct acaaagtgag 21480ttccatccag gacagagccc tactcacaaa atagctcagc tggtaaagtt gcttgctaca 21540caagcttgac ccatatttgg tccccagaaa ccatggagga cggagaaggc tggccctggg 21600tatgcgcaag ggtacacact tggaggggtc ttggtggata aaggatttgc acaatcatga 21660ctatctgaat ttgaatctgg caccttaaag gttttttatt attacttttt atttttttaa 21720agtgcacaca gaggtcacca ggaaaggtgg tctggccttt aggagcactg tcagttcttt 21780cagaggccca acacctgcct atatatggca gctcacaact gtctgactcc agtcctgggg 21840aaatctaatg catgtggtca gaatacccag gcagctgtag tgaatgtacg gtggagggag 21900agagtgaggt gcccagtgtc tatctatcta tatagcacaa tagatagata aaaaataccc 21960aagtgtggtg gtgtgtgcct gtagccccag tgctcatggt gcagaggaag aaagagcaag 22020ttgtatcaca gcctagctac agaaagccaa acacatgtaa aatcagtgtg gaggactagg 22080cactggtctg tctccctaag gcagtgttca tgaactaagt agcagaaagc tacttaggcc 22140tgggctgagg atggtggcct ctgtgtaagc ttggccatga atgttggtat gtagctggaa 22200gccagggatg atggggtact gagaaatggg gacactaaaa ctatcatttt tagtcctgga 22260gttttgaaag ctctaaaata caaggtctat gctaattctg gggtttctct gaagagttcc 22320tggttccagc agctacctcc ttccttcaaa gcctatgtat gcaggctagc atgaagctct 22380gctgtggaat tcctcagtcc cccgtgccta gctaattgag taatctgata gagatagaca 22440ctatcatttg ttacaggttg agaatagggg ttccctacat tcccagggga tcttgaatgg 22500ccagatattc ctcttaccac acctgatcac cacccagatt tctttttctt tctttttttt 22560ttaaatttat ttatttatat gagtacacgt acttcagaca taccagaaga ggacattggt 22620atcggatccc attacagatg gttgctggga tcccatgtgg ttgctgggaa ttgaactcag 22680gccctctgga agagcaatca gtgctcttaa ctgctgagct atctctccag cccccagatt 22740tctttttctt tctttttttt tttaaagatt gatttattat tatatctaag tacactgtag 22800ctgtctgcag atgcaccaga agagggtgtc agatttcttt atggatagtt gtgagccacc 22860atgtggttgc cgggacctga actcaggacc ttctgaagag cagtcagtgc tcccaaccac 22920tgagctatct ctccagccct accacccaga tttctaaaac catagaaatt ctgaggtttc 22980ttttaacatt agctgctagg actcccatag gagaacagta tagtgttatg gtgaacattg 23040ttggcttcca gggcctggta actctgctgc tgttctttgc agaagaagtc aggagctagg 23100cacatggtac ttggattgta aaagttgctg gcagctacag gagtgggttc tgctgagatt 23160gggccaaagc tgcctcactg ccagatggaa gggttcattt gtgggaagaa ttctaccagc 23220catgctccta taggactgcc catactgaga gcaggataat catcttagaa aagacaggac 23280aggtctgagg ggcaggccag accttgaaac agttgtcagt gggcaaagcc tgtggtccag 23340agttgaatta gagggtatta cttttggctt aggcttactg aaagggtctt atgacatgtt 23400taacggtcag tctttccaac ctgtttccat ctcaggagtg ctcaccaagt ggccaggtac 23460cgccctcggg ctcctatcat tgccgtgact cgaaatcccc agactgctcg ccaggcccat 23520ctgtaccgtg gcatcttccc tgtgctgtgt aaggatgccg tgctgaatgc ctgggctgag 23580gatgtcgacc ttcgtgtaaa cttggccatg gatgttggta tgtagctgga aacaagggaa 23640tgatgaagtg ctaataaatg gggaccctaa aactaccact tcctgaagtc atgggctggg 23700cctatctgtt ttatcaccca gttgtaagat tagctggagc tactgtcctg agcagggtgg 23760ggttagaggg tggggaacac aagcttttgt ggccttattc ctatatagag acaaggaggc 23820agctgaccct gactctctag agtataaact gaatggtgtc caaaggtctc tgcattctga 23880gttcagcctc agcccttgtt taggctagga ggcttgcacc atcttgtggt gagaagaagt 23940aactgtcaac ctgctcccct acccagacga ggattattag ggcagttatc tgcacctgcc 24000ttgttggatt tgtgtctggg ttgggaaaag tgccactttg tgtgatcaat taggactgtg 24060gggctttgca gttttcctca agtgtatacc tctactcacc aacctccttt tccccccagg 24120caaggcccga ggcttcttca agaagggaga tgtggtcatt gtgctgaccg ggtggcgccc 24180tggctctgga ttcaccaaca ccatgcgtgt agtgcctgta ccttgatggc cctctggagc 24240ccctcttcta gcccctgtcc cttcccctcc cctatccttt ccattaggcc agcaacgctt 24300gtagtgctca ctctgggcca tagtgtggcg ctggtgggct gggacaccag ggaaaattaa 24360tgcctctaaa acatgcaata gagaccagct attattcagg gccctacctg agccaggggt 24420ggaggaggaa tgcaggactg gaaaccctga ctttatcaca gaagggcggc agcatctctg 24480ggctttgctt ctgtagaaag ttgtcagaat tcccagccct agcctggagt caggagacag 24540caaaagagta ggggctgagg gtgtggggcc cagggtccca gtgtagatga cgacttctgg 24600ccctggccct gacctgcttt cccaacagct ttggcctccc cacttcttgt gcactccact 24660tctgtcactg cagacactcc actctccacc ttgtattctg cagagtctcc aggcctgttg 24720ctatagtgcc cacctgaatg tcaataaaca gcagctgaag cacctgtgtt gtgttttgtt 24780ttgttttttt ccgggggttg gtagtggtgg tcgagccctg ccctttgagc aattgagaat 24840gacaacagcc aggaggcctg gccttgggtg ctatggatcc accagataac cttcaagcac 24900gggaccactg gagacgccgg agactgaata ctttgggagt atataagttg cctcctgcac 24960caaagtgcgg ctgtgtatcg ggaagctgat ccgttctatt tcagggcaaa tactagaaaa 25020gtctgggaca ggttcagcta gcacctagaa tggtcgaata gtgggctgat ccctgtggta 25080gaggcctgtt gagggcgctg taggatgagt ccctgattgg atacttcaca aaaccctcag 25140cctccataac agttaagcac aaagggtctc ttgttttcct gttgccctca ggaataaggt 25200acatagaaac aaggtgggac ccttgtccct ggcaactccc aacagccacc tagtcagagg 25260ctaaggcctc tgaaattgag atgttgcagc accaagagct caggttaggt aaggaggttg 25320gaaggcggag tagaatgaag agccctgagg gaacattttg atatgaagtc ccataattga 25380ggacccaggc cctggtttcc tcgacctcac aattttgaag acctacctgc agctgggacc 25440catcagggcg gtctgggagc ggggaaggct ccaccaattg agccaggctc attctgcgcg 25500agcggccaat aggcgtgtgg gggcgggcct ttcccggggt gggcggtccc cggagggcac 25560tgggtctggc gcacggcctc gggctcccgg agaaggcgct gcgatgaccg ccctgagccg 25620tagcgagcct gtcgaggcgg gcaggtaagg gaggcgtccc ggggctccag gtccaggagg 25680ctgcgacgaa gggcagggcg actctgggaa tgccgcgcct aggcgccttc tgcctcccgg 25740ctgggcactt ccaaccgcag aaatttaggc ttggaccctg cgcctccgcc cggctgtgag 25800gtgtgcgagt ctggtgcgat gtatccgatg tgtcttggtg gcttcaggcg agcggaaggc 25860acgctccctt ctggaaatca cctcgtgtcg gccaccctcg cggtccattc atttgtgttt 25920cattcatttc tcgccataac gaccccccag tcccggttgt ctccacacac agcctggcgc 25980ttccctgtgg gcctctgcaa a 260016421DNAArtificial SequencePrimer 64tgtctggaga aacagccaag g 216522DNAArtificial SequencePrimer 65caagctcttc aaacagcaga cg 226622DNAArtificial SequenceProbe 66agcacctgat agctcgggag gc 226721DNAArtificial SequencePrimer 67aagatgccac ggtacagatg g 216820DNAArtificial SequencePrimer 68cagacctcat ggaggccatg 206921DNAArtificial SequenceProbe 69tggcaggagt gctcaccaag t 217023DNAArtificial SequencePrimer 70ggagttcctc gaatagctgc aag 237120DNAArtificial SequencePrimer 71agtcctggat ggagcagact 207220DNAArtificial SequenceProbe 72gctgttcgca tgcagcacct 207320DNAArtificial SequencePrimer 73gcgagcagtc tggggatttc 207417DNAArtificial SequencePrimer 74accccacaga agctgcc 177522DNAArtificial SequenceProbe 75accaagtctg gcaggagtgc tc 227618DNAArtificial SequenceSynthetic oligonucleotide 76gttcctcgaa tagctgca 187718DNAArtificial SequenceSynthetic oligonucleotide 77agttcctcga atagctgc 187818DNAArtificial SequenceSynthetic oligonucleotide 78ggagttcctc gaatagct 187918DNAArtificial SequenceSynthetic oligonucleotide 79tgagcacgat aatggccc 188018DNAArtificial SequenceSynthetic oligonucleotide 80ggtgagcacg ataatggc 188118DNAArtificial SequenceSynthetic oligonucleotide 81ccagacttgg tgagcacg 188216DNAArtificial SequenceSynthetic oligonucleotide 82caagtggtag atggca 168316DNAArtificial SequenceSynthetic oligonucleotide 83ccaggcggcg gagttc 168416DNAArtificial SequenceSynthetic oligonucleotide 84cggcggcagc ttctgt 168516DNAArtificial SequenceSynthetic oligonucleotide 85ggcacccacg gcggca 168616DNAArtificial SequenceSynthetic oligonucleotide 86acttggtgag cacgat 168718DNAArtificial SequenceSynthetic oligonucleotide 87ggcggagttc ctcgaata 188818DNAArtificial SequenceSynthetic oligonucleotide 88cacggcggca gcttctgt 188918DNAArtificial SequenceSynthetic oligonucleotide 89cacccacggc ggcagctt 189018DNAArtificial SequenceSynthetic oligonucleotide 90ttggtgagca cgataatg 189118DNAArtificial SequenceSynthetic oligonucleotide 91tgccagactt ggtgagca 189216DNAArtificial SequenceSynthetic oligonucleotide 92ggtgagcacg ataatg 169318DNAArtificial SequenceSynthetic oligonucleotide 93ttggtgagca cgataatg 189418DNAArtificial SequenceSynthetic oligonucleotide 94agacttggtg agcacgat 18

Claims

1. A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region, wherein the complementary region comprises at least 8 contiguous nucleobases and is complementary to an equal-length portion of a target region of a PK-M transcript.

2. The compound of claim 1, wherein the target region of the PK-M transcript comprises at least a portion of exon 10 of the PK-M transcript.

3. The compound of claim 1 or 2, wherein the complementary region of the modified oligonucleotide is 100% complementary to the target region.

4. The compound of any of claims 1 to 3, wherein the complementary region of the modified oligonucleotide comprises at least 10 contiguous nucleobases.

5. The compound of any of claims 1 to 3, wherein the complementary region of the modified oligonucleotide comprises at least 15 contiguous nucleobases.

6. The compound of any of claims 1 to 3, wherein the complementary region of the modified oligonucleotide comprises at least 20 contiguous nucleobases.

7. The compound of any of claims 1-6, wherein the nucleobase sequence of the oligonucleotide is at least 80% complementary to an equal-length region of the PK-M transcript, as measured over the entire length of the oligonucleotide.

8. The compound of any of claims 1-6, wherein the nucleobase sequence of the oligonucleotide is at least 90% complementary to an equal-length region of the PK-M transcript, as measured over the entire length of the oligonucleotide.

9. The compound of any of claims 1-6, wherein the nucleobase sequence of the oligonucleotide is 100% complementary to an equal-length region of the PK-M transcript, as measured over the entire length of the oligonucleotide.

10. The compound of any of claims 1-9, wherein the target region is within exon 10 of the PK-M transcript.

11. The compound of any of claims 1-10, wherein the target region is within nucleobase 29153 and nucleobase 29281 of SEQ ID NO.: 1.

12. The compound of any of claims 1-10, wherein the target region is within nucleobase 29158 and nucleobase 29262 of SEQ ID NO.: 1.

13. The compound of any of claims 1-10, wherein the target region is within nucleobase 29164 and nucleobase 29188 of SEQ ID NO.: 1.

14. The compound of any of claims 1-10, wherein the target region is within nucleobase 29261 and nucleobase 29279 of SEQ ID NO.: 1.

15. The compound of any of claims 1-10, wherein the target region is within nucleobase 29168 and nucleobase 29183 of SEQ ID NO.: 1.

16. The compound of any of claims 1-15, wherein the nucleobase sequence of the antisense oligonucleotide comprises any one of SEQ ID NOs: 4 to 36.

17. The compound of any of claims 1-16, wherein the modified oligonucleotide comprises at least one modified nucleoside.

18. The compound of claim 17, wherein at least one modified nucleoside comprises a modified sugar moiety.

19. The compound of claim 18, wherein at least one modified sugar moiety is a 2'-substituted sugar moiety.

20. The compound of claim 19, wherein the 2'-substitutent of at least one 2'-substituted sugar moiety is selected from among: 2'-OMe, 2'-F, and 2'-MOE.

21. The compound of any of claims 17-20, wherein the 2'-substituent of at least one 2'-substituted sugar moiety is a 2'-MOE.

22. The compound of any of claims 1-18, wherein at least one modified sugar moiety is a bicyclic sugar moiety.

23. The compound of claim 22, wherein at least one bicyclic sugar moiety is LNA or cEt.

24. The compound of any of claims 18-23, wherein at least one sugar moiety is a sugar surrogate.

25. The compound of claim 24, wherein at least one sugar surrogate is a morpholino.

26. The compound of claim 24, wherein at least one sugar surrogate is a modified morpholino.

27. The compound of any of claim 1-26, wherein the modified oligonucleotide comprises at least 5 modified nucleosides, each independently comprising a modified sugar moiety.

28. The compound of claim 27, wherein the modified oligonucleotide comprises at least 10 modified nucleosides, each independently comprising a modified sugar moiety.

29. The compound of claim 27, wherein the modified oligonucleotide comprises at least 15 modified nucleosides, each independently comprising a modified sugar moiety.

30. The compound of claim 27, wherein each nucleoside of the modified oligonucleotide is a modified nucleoside, each independently comprising a modified sugar moiety

31. The compound of any of claims 1-30, wherein the modified oligonucleotide comprises at least two modified nucleosides comprising modified sugar moieties that are the same as one another.

32. The compound of any of claims 1-31, wherein the modified oligonucleotide comprises at least two modified nucleosides comprising modified sugar moieties that are different from one another.

33. The compound of any of claims 1-32, wherein the modified oligonucleotide comprises a modified region of at least 5 contiguous modified nucleosides.

34. The compound of claim 33, wherein the modified oligonucleotide comprises a modified region of at least 10 contiguous modified nucleosides.

35. The compound of claim 33, wherein the modified oligonucleotide comprises a modified region of at least 15 contiguous modified nucleosides.

36. The compound of claim 33, wherein the modified oligonucleotide comprises a modified region of at least 20 contiguous modified nucleosides.

37. The compound of any of claims 32-36, wherein each modified nucleoside of the modified region has a modified sugar moiety independently selected from among: 2'-F, 2'-OMe, 2'-MOE, cEt, LNA, morpholino, and modified morpholino.

38. The compound of any of claims 33-37, wherein the modified nucleosides of the modified region each comprise the same modification as one another.

39. The compound of claim 38, wherein the modified nucleosides of the modified region each comprise the same 2'-substituted sugar moiety.

40. The compound of claim 38, wherein the 2'-substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from 2'-F, 2'-OMe, and 2'-MOE.

41. The compound of claim 39, wherein the 2'-substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is 2'-MOE.

42. The compound of claim 38, wherein the modified nucleosides of the region of modified nucleosides each comprise the same bicyclic sugar moiety.

43. The compound of claim 42, wherein the bicyclic sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from LNA and cEt.

44. The compound of claim 38, wherein the modified nucleosides of the region of modified nucleosides each comprises a sugar surrogate.

45. The compound of claim 44, wherein the sugar surrogate of the modified nucleosides of the region of modified nucleosides is a morpholino.

46. The compound of claim 44, wherein the sugar surrogate of the modified nucleosides of the region of modified nucleosides is a modified morpholino.

47. The compound of any of claims 1-46, wherein the modified nucleotide comprises no more than 4 contiguous naturally occurring nucleosides.

48. The compound of any of claims 1-46, wherein each nucleoside of the modified oligonucleotide is a modified nucleoside.

49. The compound of claim 48 wherein each modified nucleoside comprises a modified sugar moiety.

50. The compound of claim 49, wherein the modified nucleosides of the modified oligonucleotide comprise the same modification as one another.

51. The compound of claim 50, wherein the modified nucleosides of the modified oligonucleotide each comprise the same 2'-substituted sugar moiety.

52. The compound of claim 51, wherein the 2'-substituted sugar moiety of the modified oligonucleotide is selected from 2'-F, 2'-OMe, and 2'-MOE.

53. The compound of claim 52, wherein the 2'-substituted sugar moiety of the modified oligonucleotide is 2'-MOE.

54. The compound of claim 50, wherein the modified nucleosides of the modified oligonucleotide each comprise the same bicyclic sugar moiety.

55. The compound of claim 54, wherein the bicyclic sugar moiety of the modified oligonucleotide is selected from LNA and cEt.

56. The compound of claim 50, wherein the modified nucleosides of the modified oligonucleotide each comprises a sugar surrogate.

57. The compound of claim 56, wherein the sugar surrogate of the modified oligonucleotide is a morpholino.

58. The compound of claim 56, wherein the sugar surrogate of the modified oligonucleotide is a modified morpholino.

59. The compound of any of claims 1-58, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.

60. The compound of claim 59, wherein each internucleoside linkage is a modified internucleoside linkage.

61. The compound of claim 59 or 60, comprising at least one phosphorothioate internucleoside linkage.

62. The compound of claim 60, wherein each internucleoside linkage is a modified internucleoside linkage and wherein each internucleoside linkage comprises the same modification.

63. The compound of claim 62, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.

64. The compound of any of claims 1-63 comprising at least one conjugate.

65. The compound of any of claims 1-64 consisting of the modified oligonucleotide.

66. The compound of any of claims 1-65, wherein the compound modulates splicing of the PK-M transcript.

67. The compound of any of claims 1-66, having a nucleobase sequence comprising any of the sequences as set forth in SEQ ID NOs. 4 to 36.

68. The compound of any of claims 1-66, having a nucleobase sequence comprising any of the sequences as set forth in SEQ ID NOs. 4 to 17.

69. The compound of any of claims 1-66, having a nucleobase sequence comprising any of the sequences as set forth in SEQ ID NOs. 18 to 28.

70. The compound of any of claims 1-66, having a nucleobase sequence comprising any of the sequences as set forth in SEQ ID NOs. 29 to 36.

71. The compound of any of claims 1-66, having a nucleobase sequence comprising SEQ ID NO. 32.

72. The compound of any of claims 1-66, having a nucleobase sequence comprising SEQ ID NO. 7.

73. The compound of any of claims 1-66, having a nucleobase sequence comprising SEQ ID NO. 24.

74. A pharmaceutical composition comprising a compound according to any of claims 1-73 and a pharmaceutically acceptable carrier or diluent.

75. The pharmaceutical composition of claim 74, wherein the pharmaceutically acceptable carrier or diluent is sterile saline.

76. A method of modulating splicing of a PK-M transcript in a cell comprising contacting the cell with a compound according to any of claims 1-75.

77. The method of claim 76, wherein the cell is in vitro.

78. The method of claim 76, wherein the cell is in an animal.

79. The method of any of claims 76-78, wherein inclusion of exon 9 is increased.

80. The method of any of claims 76-79, wherein exclusion of exon 10 is increased.

81. The method of any of claims 76-79, wherein inclusion of exon 10 is decreased.

82. The method of any of claims 76-81, wherein PK-M1 mRNA expression is increased.

83. The method of any of claims 76-82, wherein PK-M2 mRNA expression is decreased.

84. A method of modulating the expression of PK-M in a cell, comprising contacting the cell with a compound according to any of claims 1-75.

85. The method of claim 84, wherein PK-M1 expression is increased.

86. The method of claim 84 or 85, wherein PK-M2 expression is decreased.

87. The method of claim 84, wherein the cell is in vitro.

88. The method of claim 84, wherein the cell is in an animal.

89. A method of inducing apoptosis in a cell, comprising contacting the cell with a compound according to any of claims 1-75.

90. The method of claim 89, wherein the cell is in vitro.

91. The method of claim 89, wherein the cell is in an animal.

92. A method comprising administering the compound according to any of claims 1-67 or the pharmaceutical composition of claim 74 or 75 to an animal.

93. The method of claim 92, wherein the administration is intracerebroventricular.

94. The method of claim 92, wherein the administration is into the central nervous system.

95. The method of any of claims 92-94, wherein the animal has one or more symptoms associated with cancer.

96. The method claim 95, wherein the cancer is glioblastoma.

97. The method of claim 96, wherein the administration results in amelioration of at least one symptom of cancer.

98. The method of any of claims 91-97, wherein the animal is a mouse.

99. The method of any of claims 91-97, wherein the animal is a human.

100. A method of preventing or retarding the growth of a cancerous tumor, comprising administering the compound according to any of claims 1-73 or the pharmaceutical composition of claim 74 or 75 to an animal in need thereof.

101. The method of claim 100, wherein the animal is a mouse.

102. The method of claim 100, wherein the animal is a human.

103. The method of claims 100 to 102, wherein the cancerous tumor comprises glioblastoma.

104. Use of the compound according to any of claims 1-73 or the pharmaceutical composition of claim 74 or 75 for the preparation of a medicament for use in the treatment of cancer.

105. Use of the compound according to any of claims 1-73 or the pharmaceutical composition of claim 74 or 75 for the preparation of a medicament for use in the amelioration of one or more symptoms cancer.

106. The use of claim 104 or 105, wherein the cancer is glioblastoma.

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