U.S. patent application number 16/370609 was filed with the patent office on 2020-02-20 for selective antisense compounds and uses thereof.
This patent application is currently assigned to Ionis Pharmaceuticals, Inc.. The applicant listed for this patent is Ionis Pharmaceuticals, Inc.. Invention is credited to Michael Oestergaard, Punit P. Seth, Eric E. Swayze.
Application Number | 20200056187 16/370609 |
Document ID | / |
Family ID | 51263134 |
Filed Date | 2020-02-20 |
United States Patent
Application |
20200056187 |
Kind Code |
A1 |
Oestergaard; Michael ; et
al. |
February 20, 2020 |
SELECTIVE ANTISENSE COMPOUNDS AND USES THEREOF
Abstract
The present disclosure provides oligomeric compounds. Certain
such oligomeric compounds are useful for hybridizing to a
complementary nucleic acid, including but not limited, to nucleic
acids in a cell. In certain embodiments, hybridization results in
modulation of the amount activity or expression of the target
nucleic acid in a cell. In certain embodiments, certain oligomeric
compounds selectively reduce the expression of a target nucleic
acid transcript relative to a non-target nucleic acid
transcript.
Inventors: |
Oestergaard; Michael;
(Carlsbad, CA) ; Seth; Punit P.; (Carlsbad,
CA) ; Swayze; Eric E.; (Encinitas, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ionis Pharmaceuticals, Inc. |
Carlsbad |
CA |
US |
|
|
Assignee: |
Ionis Pharmaceuticals, Inc.
Carlsbad
CA
|
Family ID: |
51263134 |
Appl. No.: |
16/370609 |
Filed: |
March 29, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14765633 |
Aug 4, 2015 |
10260069 |
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PCT/US2014/014722 |
Feb 4, 2014 |
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16370609 |
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61760610 |
Feb 4, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/322 20130101;
C12N 2310/335 20130101; C12N 2320/34 20130101; C12N 2310/351
20130101; C12N 2320/53 20130101; C12N 2310/33 20130101; C12N
2310/3231 20130101; C12N 2310/315 20130101; C12N 2310/3341
20130101; C12N 15/1138 20130101; C12N 15/113 20130101; C12N 2310/31
20130101; C07H 21/02 20130101; C12N 2310/341 20130101; C12N 2310/11
20130101; C12N 2310/321 20130101; C12N 2310/3521 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; C07H 21/02 20060101 C07H021/02 |
Claims
1.-168. (canceled)
169. A oligomeric compound comprising a modified oligonucleotide
consisting of 10 to 30 linked nucleosides, wherein the modified
oligonucleotide has a modification motif comprising: a 5'-region
consisting of 1-9 linked 5'-region nucleosides, each independently
selected from among a modified nucleoside and an unmodified
deoxynucleoside, provided that at least one 5'-region nucleoside is
a modified nucleoside and wherein the 3'-most 5'-region nucleoside
is a modified nucleoside; a 3'-region consisting of 2-10 linked
3'-region nucleosides, wherein each 3'-region nucleoside comprises
a modified nucleoside; and a central region between the 5'-region
and the 3'-region consisting of 6-10 linked central region
nucleosides, each independently selected from among: a modified
nucleoside and an unmodified deoxynucleoside, wherein the 5'-most
central region nucleoside is an unmodified deoxynucleoside and the
3'-most central region nucleoside is an unmodified deoxynucleoside;
and wherein the modified oligonucleotide has a nucleobase sequence
complementary to the nucleobase sequence of a target region of a
nucleic acid associated with a huntingtin transcript; wherein the
nucleobase sequence of the target region of the target nucleic acid
differs from the nucleobase sequence of at least one non-target
nucleic acid by a single differentiating nucleobase; and wherein
the single differentiating nucleobase is a single-nucleotide
polymorphism selected from among rs6446723 and rs363064.
170. The oligomeric compound of claim 169, wherein the
single-nucleotide polymorphism is rs6446723.
171. The oligomeric compound of claim 169, wherein the
single-nucleotide polymorphism is rs363064.
172. The oligomeric compound of claim 169, wherein the modified
oligonucleotide has a modification motif selected from:
eeekk-d7-kkeee, eekk-d8-kkeee, and ekek-d8-kekee, wherein each "e"
is a 2'MOE modified nucleoside, each "k" is a cEt modified
nucleoside, and each d is an unmodified deoxynucleoside.
173. The oligomeric compound of claim 172, wherein the modified
oligonucleotide has a eeekk-d7-kkeee modification motif, wherein
each "e" is a 2'MOE modified nucleoside, each "k" is a cEt modified
nucleoside, and each d is an unmodified deoxynucleoside.
174. The oligomeric compound of claim 172, wherein the modified
oligonucleotide has a eekk-d8-kkeee modification motif, wherein
each "e" is a 2'MOE modified nucleoside, each "k" is a cEt modified
nucleoside, and each d is an unmodified deoxynucleoside.
175. The oligomeric compound of claim 172, wherein the modified
oligonucleotide has a ekek-d8-kekee modification motif, wherein
each "e" is a 2'MOE modified nucleoside, each "k" is a cEt modified
nucleoside, and each d is an unmodified deoxynucleoside.
176. The oligomeric compound of claim 169, comprising at least one
modified internucleoside linkage.
177. The oligomeric compound of claim 176, wherein each
internucleoside linkage is a modified internucleoside linkage.
178. The oligomeric compound of claim 169, comprising at least one
phosphorothioate internucleoside linkage.
179. The oligomeric compound of claim 177, wherein each
internucleoside linkage is a phosphorothioate internucleoside
linkage.
180. The oligomeric compound of claim 176, wherein each
internucleoside linkage is a phosphorothioate internucleoside
linkage or a phosphodiester internucleoside linkage.
181. The oligomeric compound of claim 180, wherein the 5'-most
internucleoside linkage of the 5'-region is a phosphorothioate
internucleoside linkage, wherein the 3'-most internucleoside
linkage of the 3'-region is a phosphorothioate internucleoside
linkage, and wherein each internucleoside linkage of the central
region is a phosphorothioate internucleoside linkage.
182. The oligomeric compound of claim 180, wherein the 5'-most
internucleoside linkage of the 5'-region is a phosphorothioate
internucleoside linkage, wherein the 3'-most internucleoside
linkage of the 3'-region is a phosphorothioate internucleoside
linkage, wherein each internucleoside linkage of the central region
is a phosphorothioate internucleoside linkage, and wherein each
remaining internucleoside linkage is a phosphodiester
internucleoside linkage.
183. The oligomeric compound of claim 180, wherein the oligomeric
compound contains 2 phosphodiester internucleoside linkages.
184. The oligomeric compound of claim 180, wherein the oligomeric
compound contains 3 phosphodiester internucleoside linkages.
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 CORE0113USC1_ST25.txt, created Mar. 29, 2019 which is
316 Kb in size. The information in the electronic format of the
sequence listing is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] Antisense compounds have been used to modulate target
nucleic acids. Antisense compounds comprising a variety of chemical
modifications and motifs have been reported. In certain instances,
such compounds are useful as research tools, diagnostic reagents,
and as therapeutic agents. In certain instances antisense compounds
have been shown to modulate protein expression by binding to a
target messenger RNA (mRNA) encoding the protein. In certain
instances, such binding of an antisense compound to its target mRNA
results in cleavage of the mRNA. Antisense compounds that modulate
processing of a pre-mRNA have also been reported. Such antisense
compounds alter splicing, interfere with polyadenlyation or prevent
formation of the 5'-cap of a pre-mRNA.
SUMMARY OF THE INVENTION
[0003] In certain embodiments, the present invention provides
oligomeric compounds comprising oligonucleotides. In certain
embodiments, such oligonucleotides comprise a region having a
gapmer motif. In certain embodiments, such oligonucleotides consist
of a region having a gapmer motif.
[0004] The present disclosure provides the following non-limiting
numbered embodiments:
We claim:
Embodiment 1
[0005] A oligomeric compound comprising a modified oligonucleotide
consisting of 10 to 30 linked nucleosides, wherein the modified
oligonucleotide has a modification motif comprising: a 5'-region
consisting of 1-9 linked 5'-region nucleosides, each independently
selected from among a modified nucleoside and an unmodified
deoxynucleoside, provided that at least one 5'-region nucleoside is
a modified nucleoside and wherein the 3'-most 5'-region nucleoside
is a modified nucleoside; [0006] a 3'-region consisting of 2-10
linked 3'-region nucleosides, wherein each 3'-region nucleoside
comprises a modified nucleoside; and [0007] a central region
between the 5'-region and the 3'-region consisting of 6-10 linked
central region nucleosides, each independently selected from among:
a modified nucleoside and an unmodified deoxynucleoside, wherein
the 5'-most central region nucleoside is an unmodified
deoxynucleoside and the 3'-most central region nucleoside is an
unmodified deoxynucleoside; and wherein the modified
oligonucleotide has a nucleobase sequence complementary to the
nucleobase sequence of a target region of a nucleic acid associated
with a huntingtin transcript.
Embodiment 2
[0008] The oligomeric compound of embodiment 1, wherein the
nucleobase sequence of the target region of the target nucleic acid
differs from the nucleobase sequence of at least one non-target
nucleic acid by 1-3 differentiating nucleobases.
Embodiment 3
[0009] The oligomeric compound of embodiment 1, wherein the
nucleobase sequence of the target region of the target nucleic acid
differs from the nucleobase sequence of at least one non-target
nucleic acid by a single differentiating nucleobase.
Embodiment 4
[0010] The oligomeric compound of embodiment 2 or 3, wherein the
target nucleic acid and the non-target nucleic acid are transcripts
from different genes.
Embodiment 5
[0011] The oligomeric compound of any of embodiments 2 to 4,
wherein the non-target nucleic acid is bone morphogenetic protein
receptor, type IA.
Embodiment 6
[0012] The oligomeric compound of embodiment 2 or 3, wherein the
target nucleic acid and the non-target nucleic acid are alleles of
the same gene.
Embodiment 7
[0013] The oligomeric compound of embodiment 6, wherein the single
differentiating nucleobase is a single-nucleotide polymorphism.
Embodiment 8
[0014] The oligomeric compound of embodiment 7, wherein the
single-nucleotide polymorphism is associated with a disease.
Embodiment 9
[0015] The oligomeric compound of embodiment 8, wherein the disease
is Huntington's disease.
Embodiment 10
[0016] The oligomeric compound of embodiment 7, wherein the
single-nucleotide polymorphism is selected from among: rs6446723,
rs3856973, rs2285086, rs363092, rs916171, rs6844859, rs7691627,
rs4690073, rs2024115, rs11731237, rs362296, rs10015979, rs7659144,
rs363096, rs362273, rs16843804, rs362271, rs362275, rs3121419,
rs362272, rs3775061, rs34315806 rs363099, rs2298967, rs363088,
rs363064, rs363102, rs2798235, rs363080, rs363072, rs363125,
rs362303, rs362310, rs10488840, rs362325, rs35892913, rs363102,
rs363096, rs11731237, rs10015979, rs363080, rs2798235, rs1936032,
rs2276881, rs363070, rs35892913, rs12502045, rs6446723, rs7685686,
rs3733217, rs6844859, and rs362331.
Embodiment 11
[0017] The oligomeric compound of embodiment 8, wherein the
single-nucleotide polymorphism is selected from among: rs7685686,
rs362303 rs4690072, rs362273, rs2024115, rs6446723, rs363064, and
rs363088.
Embodiment 12
[0018] The oligomeric compound of embodiment 8, wherein the
single-nucleotide polymorphism is selected from among: rs7685686,
rs363088, rs362303, rs362273, rs2024115, rs6446723, and
rs363064.
Embodiment 13
[0019] The oligomeric compound of embodiment 8, wherein the
single-nucleotide polymorphism is rs7685686.
Embodiment 14
[0020] The oligomeric compound of embodiment 8, wherein the
single-nucleotide polymorphism is rs363088.
Embodiment 15
[0021] The oligomeric compound of embodiment 8, wherein the
single-nucleotide polymorphism is rs362303.
Embodiment 16
[0022] The oligomeric compound of embodiment 8, wherein the
single-nucleotide polymorphism is rs362273.
Embodiment 17
[0023] The oligomeric compound of embodiment 8, wherein the
single-nucleotide polymorphism is rs2024115.
Embodiment 18
[0024] The oligomeric compound of embodiment 8, wherein the
single-nucleotide polymorphism is rs6446723.
Embodiment 19
[0025] The oligomeric compound of embodiment 8, wherein the
single-nucleotide polymorphism is rs363064.
Embodiment 20
[0026] The oligomeric compound of any of embodiments 1-19, wherein
the 3'-most 5'-region nucleoside comprises a bicyclic sugar
moiety.
Embodiment 21
[0027] The oligomeric compound of embodiment 20, wherein the
3'-most 5'-region nucleoside comprises a cEt sugar moiety.
Embodiment 22
[0028] The oligomeric compound of embodiment 20, wherein the
3'-most 5'-region nucleoside comprises an LNA sugar moiety.
Embodiment 23
[0029] The oligomeric compound of any of embodiments 1-22, wherein
the central region consists of 6-10 linked nucleosides.
Embodiment 24
[0030] The oligomeric compound of any of embodiments 1-23, wherein
the central region consists of 6-9 linked nucleosides.
Embodiment 25
[0031] The oligomeric compound of embodiment 24, wherein the
central region consists of 6 linked nucleosides.
Embodiment 26
[0032] The oligomeric compound of embodiment 24, wherein the
central region consists of 7 linked nucleosides.
Embodiment 27
[0033] The oligomeric compound of embodiment 24, wherein the
central region consists of 8 linked nucleosides.
Embodiment 28
[0034] The oligomeric compound of embodiment 24, wherein the
central region consists of 9 linked nucleosides.
Embodiment 29
[0035] The oligomeric compound of any of embodiments 1-28, wherein
each central region nucleoside is an unmodified
deoxynucleoside.
Embodiment 30
[0036] The oligomeric compound of any of embodiments 1-28, wherein
at least one central region nucleoside is a modified
nucleoside.
Embodiment 31
[0037] The oligomeric compound of embodiment 30, wherein one
central region nucleoside is a modified nucleoside and each of the
other central region nucleosides is an unmodified
deoxynucleoside.
Embodiment 32
[0038] The oligomeric compound of any of embodiments 30-31, wherein
the modified central region nucleoside comprises a modified
nucleobase.
Embodiment 33
[0039] The oligomeric compound of embodiment 32, wherein the
modified nucleobase is selected from among a 2-thio pyrimidine and
a 5-propyne pyrimidine.
Embodiment 34
[0040] The oligomeric compound of embodiment 32, wherein the
modified nucleobase is 2-thiothymine.
Embodiment 35
[0041] The oligomeric compound of any of embodiments 30-34, wherein
the 2.sup.nd nucleoside from the 5'-end of the central region is a
modified nucleoside.
Embodiment 36
[0042] The oligomeric compound of any of embodiments 30-34, wherein
the 8.sup.th nucleoside from the 3'-end of the central region is a
modified nucleoside.
Embodiment 37
[0043] The oligomeric compound of any of embodiments 30-34, wherein
the 7.sup.th nucleoside from the 3'-end of the central region is a
modified nucleoside.
Embodiment 38
[0044] The oligomeric compound of any of embodiments 30-34, wherein
the modified nucleoside is a 2-thio pyrimidine.
Embodiment 39
[0045] The oligomeric compound of any of embodiments 30-34, wherein
the modified nucleobase is 2-thiothymine.
Embodiment 40
[0046] The oligomeric compound of any of embodiments 1-39, wherein
the central region has a nucleoside motif selected from among:
DDDDDDD, DDDDDDDD, DDDDDDDDD, DXDDDDD, DXDDDDDD, and DXDDDDDDD;
wherein [0047] each D is an unmodified deoxynucleoside; and each X
is a modified nucleoside.
Embodiment 41
[0048] The oligomeric compound of embodiment 40, wherein each X
comprises a modified nucleobase.
Embodiment 42
[0049] The oligomeric compound of embodiment 40, wherein each X
comprises 2-thio-thymidine.
Embodiment 43
[0050] The oligomeric compound of any of embodiments 1-42, wherein
the 5' region consists of 1 nucleoside.
Embodiment 44
[0051] The oligomeric compound of any of embodiments 1-42, wherein
the 5' region consists of 2 linked 5'-region nucleosides.
Embodiment 45
[0052] The oligomeric compound of any of embodiments 1-42, wherein
the 5' region consists of 3 linked 5'-region nucleosides.
Embodiment 46
[0053] The oligomeric compound of any of embodiments 1-42, wherein
the 5' region consists of 4 linked 5'-region nucleosides.
Embodiment 47
[0054] The oligomeric compound of any of embodiments 1-42, wherein
the 5' region consists of 5 linked 5'-region nucleosides.
Embodiment 48
[0055] The oligomeric compound of any of embodiments 1-42, wherein
the 5' region consists of 6 linked 5'-region nucleosides.
Embodiment 49
[0056] The oligomeric compound of any of embodiments 1-42, wherein
the 5' region consists of 7 linked 5'-region nucleosides.
Embodiment 50
[0057] The oligomeric compound of any of embodiments 1-42, wherein
the 5' region consists of 8 linked 5'-region nucleosides.
Embodiment 51
[0058] The oligomeric compound of any of embodiments 1-42, wherein
the 5' region consists of 9 linked 5'-region nucleosides.
Embodiment 52
[0059] The oligomeric compound of any of embodiments 1-51 wherein
at least one 5'-region nucleoside is an RNA-like nucleoside.
Embodiment 53
[0060] The oligomeric compound of any of embodiments 1-51 wherein
each 5'-region nucleoside is an RNA-like nucleoside.
Embodiment 54
[0061] The oligomeric compound of any of embodiments 1-53
comprising at least one modified 5'-region nucleoside comprising a
modified sugar.
Embodiment 55
[0062] The oligomeric compound of embodiment 54 comprising at least
one modified 5'-region nucleoside comprising a bicyclic sugar
moiety.
Embodiment 56
[0063] The oligomeric compound of embodiment 55 comprising at least
one modified 5'-region nucleoside comprising a cEt sugar
moiety.
Embodiment 57
[0064] The oligomeric compound of embodiment 55 comprising at least
one modified 5'-region nucleoside comprising an LNA sugar
moiety.
Embodiment 58
[0065] The oligomeric compound of any of embodiments 54-57
comprising at least one modified 5'-region nucleoside comprising a
2'-substituted sugar moiety.
Embodiment 59
[0066] The oligomeric compound of any of embodiments 54-58
comprising at least one modified 5'-region nucleoside comprising a
2'-MOE sugar moiety.
Embodiment 60
[0067] The oligomeric compound of any of embodiments 1-59, wherein
the 5'-region has a motif selected from among: eeeedk, eeeee,
eeeeedk, eeeeeeeek, eeeeeeek, eeeeek, eeeek, eeeekk, eeek, eeek,
eeekk, eek, eekk, ek, ekek, ekek, ekk, ekkdk, ekkkk, and k, wherein
each "e" is a 2'MOE modified nucleoside, each "k" is a cEt modified
nucleoside, and each "d" is an unmodified deoxynucleoside.
Embodiment 61
[0068] The oligomeric compound of any of embodiments 1-60, wherein
the 3' region consists of 2 linked 3'-region nucleosides.
Embodiment 62
[0069] The oligomeric compound of any of embodiments 1-60, wherein
the 3' region consists of 3 linked 3'-region nucleosides.
Embodiment 63
[0070] The oligomeric compound of any of embodiments 1-60, wherein
the 3' region consists of 4 linked 3'-region nucleosides.
Embodiment 64
[0071] The oligomeric compound of any of embodiments 1-60, wherein
the 3' region consists of 5 linked 3'-region nucleosides.
Embodiment 65
[0072] The oligomeric compound of any of embodiments 1-60, wherein
the 3' region consists of 6 linked 3'-region nucleosides.
Embodiment 66
[0073] The oligomeric compound of any of embodiments 1-60, wherein
the 3' region consists of 7 linked 3'-region nucleosides.
Embodiment 67
[0074] The oligomeric compound of any of embodiments 1-60, wherein
the 3' region consists of 8 linked 3'-region nucleosides.
Embodiment 68
[0075] The oligomeric compound of any of embodiments 1-60, wherein
the 3' region consists of 9 linked 3'-region nucleosides.
Embodiment 69
[0076] The oligomeric compound of any of embodiments 1-60, wherein
the 3' region consists of 10 linked 3'-region nucleosides.
Embodiment 70
[0077] The oligomeric compound of any of embodiments 1-69, wherein
each 3'-region nucleoside is a modified nucleoside.
Embodiment 71
[0078] The oligomeric compound of any of embodiments 1-69, wherein
at least one 3'-region nucleoside is an RNA-like nucleoside.
Embodiment 72
[0079] The oligomeric compound of any of embodiments 1-69, wherein
each 3'-region nucleoside is an RNA-like nucleoside.
Embodiment 73
[0080] The oligomeric compound of any of embodiments 1-72,
comprising at least one modified 3'-region nucleoside comprising a
modified sugar.
Embodiment 74
[0081] The oligomeric compound of embodiment 73, comprising at
least one modified 3'-region nucleoside comprising a bicyclic sugar
moiety.
Embodiment 75
[0082] The oligomeric compound of embodiment 74, comprising at
least one modified 3'-region nucleoside comprising a cEt sugar
moiety.
Embodiment 76
[0083] The oligomeric compound of embodiment 74, comprising at
least one modified 3'-region nucleoside comprising an LNA sugar
moiety.
Embodiment 77
[0084] The oligomeric compound of any of embodiments 1-73 wherein
each modified 3'-region nucleoside comprises a 2'-substituted sugar
moiety.
Embodiment 78
[0085] The oligomeric compound of any of embodiments 1-73 wherein
at least one modified 3'-region nucleoside comprises a
2'-substituted sugar moiety.
Embodiment 79
[0086] The oligomeric compound of embodiment 77 or 78, wherein the
modified 3'-region nucleoside is a 2'-MOE sugar moiety.
Embodiment 80
[0087] The oligomeric compound of any of embodiments 1-73, wherein
the 3'-region has a motif selected from among: eee, eeee, eeeee,
eeeeee, eeeeeee, eeeeeeee, eeeeeeeee, eeeeeeeeee, eeeekek,
eeeekeke, eeek, eeeke, eeekek, eeekeke, eeekekee, eeekk, eeke,
eekek, eekeke, eekekee, eekk, kee, keee, keeee, keeeke, keeekee,
keek, keeke, keekee, keekeee, keekk, keke, kekee, kke, kkeee,
kkeek, and kkke, wherein each "e" is a 2'MOE modified nucleoside
and each "k" is a cEt modified nucleoside.
Embodiment 81
[0088] The oligomeric compound of embodiments 1-80, wherein the
5'-region has a motif selected from among: eeeedk, eeeee, eeeeedk,
eeeeeeeek, eeeeeeek, eeeeek, eeeek, eeeekk, eeek, eeek, eeekk, eek,
eekk, ek, ekek, ekek, ekk, ekkdk, ekkkk, and k; [0089] wherein the
3'-region has a motif selected from among: eee, eeee, eeeee,
eeeeee, eeeeeee, eeeeeeee, eeeeeeeee, eeeeeeeeee, eeeekek,
eeeekeke, eeek, eeeke, eeekek, eeekeke, eeekekee, eeekk, eeke,
eekek, eekeke, eekekee, eekk, kee, keee, keeee, keeeke, keeekee,
keek, keeke, keekee, keekeee, keekk, keke, kekee, kke, kkeee,
kkeek, and kkke; [0090] wherein the central region has a nucleoside
motif selected from among: DDDDDDD, DDDDDDDD, DDDDDDDDD, DXDDDDD,
DXDDDDDD, and DXDDDDDDD; and [0091] wherein each "e" is a 2'MOE
modified nucleoside, each "k" is a cEt modified nucleoside, each
"d" is an unmodified deoxynucleoside, and each "X" is a modified
nucleoside or a modified nucleobase.
Embodiment 82
[0092] The oligomeric compound of embodiment 81, wherein each X
comprises a 2-thio-thymidine.
Embodiment 83
[0093] The oligomeric compound of any of embodiments 1-82
comprising at least one modified internucleoside linkage.
Embodiment 84
[0094] The oligomeric compound of embodiment 83, wherein each
internucleoside linkage is a modified internucleoside linkage.
Embodiment 85
[0095] The oligomeric compound of embodiment 83 or 84 comprising at
least one phosphorothioate internucleoside linkage.
Embodiment 86
[0096] The oligomeric compound of embodiment 84, wherein each
internucleoside linkage is a phosphorothioate internucleoside
linkage.
Embodiment 87
[0097] The oligomeric compound of embodiment 83, wherein the
5'-most internucleoside linkage of the 5'-region is a
phosphorothioate internucleoside linkage, wherein the 3'-most
internucleoside linkage of the 3'-region is a phosphorothioate
internucleoside linkage, and wherein each internucleoside linkage
of the central region is a phosphorothioate internucleoside
linkage.
Embodiment 88
[0098] The oligomeric compound of embodiment 83, wherein the
5'-most internucleoside linkage of the 5'-region is a
phosphorothioate internucleoside linkage, wherein the 3'-most
internucleoside linkage of the 3'-region is a phosphorothioate
internucleoside linkage, wherein each internucleoside linkage of
the central region is a phosphorothioate internucleoside linkage,
and wherein each remaining internucleoside linkage is a
phosphodiester internucleoside linkage.
Embodiment 89
[0099] The oligomeric compound of embodiment 87, wherein the
oligomeric compound contains 2 phosphodiester internucleoside
linkages.
Embodiment 90
[0100] The oligomeric compound of embodiment 87, wherein the
oligomeric compound contains 3 phosphodiester internucleoside
linkages.
Embodiment 91
[0101] The oligomeric compound of embodiment 87, wherein the
oligomeric compound contains 4 phosphodiester internucleoside
linkages.
Embodiment 92
[0102] The oligomeric compound of embodiment 87, wherein the
oligomeric compound contains 5 phosphodiester internucleoside
linkages.
Embodiment 93
[0103] The oligomeric compound of embodiment 87, wherein the
oligomeric compound contains 6 phosphodiester internucleoside
linkages.
Embodiment 94
[0104] The oligomeric compound of embodiment 87, wherein the
5'-region of the oligomeric compound contains 1 phosphodiester
internucleoside linkage.
Embodiment 95
[0105] The oligomeric compound of embodiment 87, wherein the
5'-region of the oligomeric compound contains 2 phosphodiester
internucleoside linkages.
Embodiment 96
[0106] The oligomeric compound of embodiment 87, wherein the
5'-region of the oligomeric compound contains 3 phosphodiester
internucleoside linkages.
Embodiment 97
[0107] The oligomeric compound of any of embodiments 87 or 94 to
96, wherein the 3'-region of the oligomeric compound contains 1
phosphodiester internucleoside linkage.
Embodiment 98
[0108] The oligomeric compound of any of embodiments 87 or 94 to
96, wherein the 3'-region of the oligomeric compound contains 2
phosphodiester internucleoside linkages.
Embodiment 99
[0109] The oligomeric compound of any of embodiments 87 or 94 to
96, wherein the 3'-region of the oligomeric compound contains 3
phosphodiester internucleoside linkages.
Embodiment 100
[0110] The oligomeric compound of any of embodiments 87 or 94 to
96, wherein the 3'-region of the oligomeric compound contains 4
phosphodiester internucleoside linkages.
Embodiment 101
[0111] The oligomeric compound of any of embodiments 87 or 94 to
96, wherein the 3'-region of the oligomeric compound contains 5
phosphodiester internucleoside linkages.
Embodiment 102
[0112] The oligomeric compound of any of embodiments 87 or 94 to
96, wherein the 3'-region of the oligomeric compound contains 6
phosphodiester internucleoside linkages.
Embodiment 103
[0113] The oligomeric compound of any of embodiments 87 or 94 to
96, wherein the 2nd, internucleoside bond from 3'-end of the
oligomeric compound is a phosphodiester internucleoside
linkage.
Embodiment 104
[0114] The oligomeric compound of any of embodiments 87 or 94 to
96, wherein the 3rd, internucleoside bond from 3'-end of the
oligomeric compound is a phosphodiester internucleoside
linkage.
Embodiment 105
[0115] The oligomeric compound of any of embodiments 87 or 94 to
96, wherein the 2.sup.nd, 3.sup.rd and 4.sup.th internucleoside
bonds from 3'-end of the oligomeric compound are phosphodiester
internucleoside linkages.
Embodiment 106
[0116] The oligomeric compound of any of embodiments 87 or 94 to
96, wherein the 3.sup.rd and 4.sup.th internucleoside bonds from
3'-end of the oligomeric compound are phosphodiester
internucleoside linkages.
Embodiment 107
[0117] The oligomeric compound of any of embodiments 87 or 94 to
96, wherein the 3.sup.rd, 4.sup.th, and 5.sup.th, internucleoside
bonds from 3'-end of the oligomeric compound are phosphodiester
internucleoside linkages.
Embodiment 108
[0118] The oligomeric compound of any of embodiments 87 or 94 to
96, wherein the 4.sup.th and 5.sup.th internucleoside bonds from
3'-end of the oligomeric compound are phosphodiester
internucleoside linkages.
Embodiment 109
[0119] The oligomeric compound of any of embodiments 87 or 94 to
96, wherein the 3.sup.rd, 4.sup.th, 5.sup.th, and 6.sup.th,
internucleoside bonds from 3'-end of the oligomeric compound are
phosphodiester internucleoside linkages.
Embodiment 110
[0120] The oligomeric compound of any of embodiments 87 or 94 to
96, wherein the 2.sup.nd, 3.sup.rd, 4.sup.th, 5.sup.th, and
6.sup.th, internucleoside bonds from 3'-end of the oligomeric
compound are phosphodiester internucleoside linkages.
Embodiment 111
[0121] The oligomeric compound of any of embodiments 87 or 97 to
110, wherein the 2.sup.nd internucleoside bond from 5'-end of the
oligomeric compound is a phosphodiester internucleoside
linkage.
Embodiment 112
[0122] The oligomeric compound of any of embodiments 87 or 97 to
110, wherein the 3.sup.rd internucleoside bond from 5'-end of the
oligomeric compound is a phosphodiester internucleoside
linkage.
Embodiment 113
[0123] The oligomeric compound of any of embodiments 87 or 97 to
110, wherein the 2.sup.nd and 3.sup.rd internucleoside bonds from
5'-end of the oligomeric compound are phosphodiester
internucleoside linkages.
Embodiment 114
[0124] The oligomeric compound of any of embodiments 87 or 97 to
110, wherein the 2.sup.nd 3.sup.rd, and 4.sup.th internucleoside
bonds from 5'-end of the oligomeric compound are phosphodiester
internucleoside linkages.
Embodiment 115
[0125] The oligomeric compound of any of embodiments 87 or 97 to
110, wherein the 3.sup.rd and 4.sup.th internucleoside bonds from
5'-end of the oligomeric compound are phosphodiester
internucleoside linkages.
Embodiment 116
[0126] The oligomeric compound of any of embodiments 87 or 97 to
110, wherein the 2.sup.nd, 3.sup.rd, 4.sup.th, and 5.sup.th
internucleoside bonds from 5'-end of the oligomeric compound are
phosphodiester internucleoside linkages.
Embodiment 117
[0127] The oligomeric compound of any of embodiments 87 or 97 to
110, wherein the 3.sup.rd 4.sup.th, and 5.sup.th internucleoside
bonds from 5'-end of the oligomeric compound are phosphodiester
internucleoside linkages.
Embodiment 118
[0128] The oligomeric compound of any of embodiments 87 or 97 to
110, wherein the 3.sup.rd, 4.sup.th, 5.sup.th, and 6.sup.th
internucleoside bonds from 5'-end of the oligomeric compound are
phosphodiester internucleoside linkages.
Embodiment 119
[0129] The oligomeric compound of any of embodiments 87 or 97 to
110, wherein the 3.sup.rd, 4.sup.th, 5.sup.th, 6.sup.th, and
7.sup.th internucleoside bonds from 5'-end of the oligomeric
compound are phosphodiester internucleoside linkages.
Embodiment 120
[0130] The oligomeric compound of any of embodiments 1-119
comprising at least one conjugate group.
Embodiment 121
[0131] The oligomeric compound of embodiment 1-120, wherein the
conjugate group consists of a conjugate.
Embodiment 122
[0132] The oligomeric compound of embodiment 120, wherein the
conjugate group consists of a conjugate and a conjugate linker.
Embodiment 123
[0133] The oligomeric compound of any of embodiments 1-122, wherein
the nucleobase sequence of the modified oligonucleotide is 100%
complementary to the nucleobase sequence of the target region of
the target nucleic acid.
Embodiment 124
[0134] The oligomeric compound of any of embodiments 1-122, wherein
the nucleobase sequence of the modified oligonucleotide contains
one mismatch relative to the nucleobase sequence of the target
region of the target nucleic acid.
Embodiment 125
[0135] The oligomeric compound of any of embodiments 1-122, wherein
the nucleobase sequence of the modified oligonucleotide contains
two mismatches relative to the nucleobase sequence of the target
region of the target nucleic acid.
Embodiment 126
[0136] The oligomeric compound of any of embodiments 1-122, wherein
the nucleobase sequence of the modified oligonucleotide comprises a
hybridizing region and at least one non-targeting region, wherein
the nucleobase sequence of the hybridizing region is complementary
to the nucleobase sequence of the target region of the target
nucleic acid.
Embodiment 127
[0137] The oligomeric compound of embodiment 126, wherein the
nucleobase sequence of the hybridizing region is 90% complementary
to the nucleobase sequence of the target region of the target
nucleic acid.
Embodiment 128
[0138] The oligomeric compound of embodiment 126, wherein the
nucleobase sequence of the hybridizing region is 95% complementary
to the nucleobase sequence of the target region of the target
nucleic acid.
Embodiment 129
[0139] The oligomeric compound of embodiment 126, wherein the
nucleobase sequence of the hybridizing region is 100% complementary
to the nucleobase sequence of the target region of the target
nucleic acid.
Embodiment 130
[0140] The oligomeric compound of embodiment 126, wherein the
nucleobase sequence of the hybridizing region contains one mismatch
relative to the nucleobase sequence of the target region of the
target nucleic acid.
Embodiment 131
[0141] The oligomeric compound of any of embodiments 126-129,
wherein the nucleobase sequence of at least one non-targeting
region is complementary to a portion of the hybridizing region of
the modified oligonucleotide.
Embodiment 132
[0142] The oligomeric compound of embodiment 126, wherein the
nucleobase sequence of at least one non-targeting region is 100%
complementary to a portion of the hybridizing region of the
modified oligonucleotide.
Embodiment 133
[0143] The oligomeric compound of embodiment 126 wherein the
nucleobase sequence of the modified oligonucleotide comprises two
non-targeting regions flanking a central hybridizing region.
Embodiment 134
[0144] The oligomeric compound of embodiment 133, wherein the two
non-targeting regions are complementary to one another.
Embodiment 135
[0145] The oligomeric compound of embodiment 134, wherein the two
non-targeting regions are 100% complementary to one another.
Embodiment 136
[0146] The oligomeric compound of any of embodiments 2-135, wherein
the nucleobase sequence of the modified oligonucleotide aligns with
the nucleobase sequence of the target region of the target nucleic
acid such that a distinguishing nucleobase of the target region of
the target nucleic acid aligns with a target-selective nucleoside
within the central region of the modified oligonucleotide.
Embodiment 137
[0147] The oligomeric compound of any of embodiments 3-135, wherein
the nucleobase sequence of the modified oligonucleotide aligns with
the nucleobase of the target region of the target nucleic acid such
that the single distinguishing nucleobase of the target region of
the target nucleic acid aligns with a target-selective nucleoside
within the central region of the modified oligonucleotide.
Embodiment 138
[0148] The oligomeric compound of embodiment 136 or 137, wherein
the target-selective nucleoside is the 5'-most nucleoside of the
central region.
Embodiment 139
[0149] The oligomeric compound of embodiment 136 or 137, wherein
the target-selective nucleoside is the 2.sup.nd nucleoside from the
5'-end of the central region.
Embodiment 140
[0150] The oligomeric compound of embodiment 136 or 137, wherein
the target-selective nucleoside is at the 3.sup.rd nucleoside from
the 5'-end of the central region.
Embodiment 141
[0151] The oligomeric compound of embodiment 136 or 137, wherein
the target-selective nucleoside is at the 4.sup.th nucleoside from
the 5'-end of the central region.
Embodiment 142
[0152] The oligomeric compound of embodiment 136 or 137, wherein
the target-selective nucleoside is at the 5.sup.th nucleoside from
the 5'-end of the central region.
Embodiment 143
[0153] The oligomeric compound of embodiment 136 or 137, wherein
the target-selective nucleoside is at the 6.sup.th nucleoside from
the 5'-end of the central region.
Embodiment 144
[0154] The oligomeric compound of embodiment 136 or 137, wherein
the target-selective nucleoside is at the 7.sup.th nucleoside from
the 5'-end of the central region.
Embodiment 145
[0155] The oligomeric compound of embodiment 136 or 137, wherein
the target-selective nucleoside is at the 8.sup.th nucleoside from
the 5'-end of the central region.
Embodiment 146
[0156] The oligomeric compound of embodiment 136 or 137, wherein
the target-selective nucleoside is at the 9.sup.th nucleoside from
the 5'-end of the central region.
Embodiment 147
[0157] The oligomeric compound of any of embodiments 136 or 137,
wherein the target-selective nucleoside is at the 2.sup.nd
nucleoside from the 3'-end of the central region.
Embodiment 148
[0158] The oligomeric compound of any of embodiments 136 or 137,
wherein the target-selective nucleoside is at the 3.sup.rd
nucleoside from the 3'-end of the central region.
Embodiment 149
[0159] The oligomeric compound of any of embodiments 136 or 137,
wherein the target-selective nucleoside is at the 4.sup.th
nucleoside from the 3'-end of the central region.
Embodiment 150
[0160] The oligomeric compound of any of embodiments 136 or 137,
wherein the target-selective nucleoside is at the 5.sup.th
nucleoside from the 3'-end of the central region.
Embodiment 151
[0161] The oligomeric compound of any of embodiments 136 or 137,
wherein the target-selective nucleoside is at the 6.sup.th
nucleoside from the 3'-end of the central region.
Embodiment 152
[0162] The oligomeric compound of any of embodiments 136 or 137,
wherein the target-selective nucleoside is at the 7.sup.th
nucleoside from the 3'-end of the central region.
Embodiment 153
[0163] The oligomeric compound of any of embodiments 136 or 137,
wherein the target-selective nucleoside is at the 8.sup.th
nucleoside from the 3'-end of the central region.
Embodiment 154
[0164] The oligomeric compound of any of embodiments 136 or 137,
wherein the target-selective nucleoside is at the 9.sup.th
nucleoside from the 3'-end of the central region.
Embodiment 155
[0165] The oligomeric compound of any of embodiments 1-154, wherein
the oligomeric compound has a nucleobase sequence comprising a
nucleobase sequence selected from among SEQ ID NO. 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39.
Embodiment 156
[0166] The oligomeric compound of any of embodiments 1-154, wherein
the oligomeric compound has a nucleobase sequence selected from
among SEQ ID NO. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, or 39.
Embodiment 157
[0167] The oligomeric compound of any of embodiments 1-156, wherein
target-selective nucleoside is an unmodified deoxynucleoside.
Embodiment 158
[0168] The oligomeric compound of any of embodiments 1-156, wherein
target-selective nucleoside is a modified nucleoside.
Embodiment 159
[0169] The oligomeric compound of any of embodiments 1-156, wherein
the target-selective nucleoside is a sugar modified nucleoside.
Embodiment 160
[0170] The oligomeric compound of embodiment 159, wherein the
target-selective nucleoside comprises a sugar modification selected
from among: 2'-MOE and cEt.
Embodiment 161
[0171] The oligomeric compound of any of embodiments 1-160, wherein
the target-selective nucleoside comprises a nucleobase
modification.
Embodiment 162
[0172] The oligomeric compound of embodiment 161, wherein the
modified nucleobase is selected from among: a 2-thio pyrimidine and
a 5-propyne pyrimidine.
Embodiment 163
[0173] The oligomeric compound of any of embodiments 1-162, wherein
the oligomeric compound is an antisense compound.
Embodiment 164
[0174] The oligomeric compound of embodiment 163, wherein the
oligomeric compound selectively reduces expression of the target
relative to the non-target.
Embodiment 165
[0175] The oligomeric compound of embodiment 164, wherein the
oligomeric compound reduces expression of target at least two-fold
more than it reduces expression of the non-target.
Embodiment 166
[0176] The oligomeric compound of embodiment 165, having an
EC.sub.50 for reduction of expression of target that is at least
two-fold lower than its EC.sub.50 for reduction of expression of
the non-target, when measured in cells.
Embodiment 167
[0177] The oligomeric compound of claim 165, having an ED.sub.50
for reduction of expression of target that is at least two-fold
lower than its ED.sub.50 for reduction of expression of the
non-target, when measured in an animal.
Embodiment 168
[0178] A method comprising contacting a cell with an oligomeric
compound of any of claims 1-167.
Embodiment 169
[0179] The method of claim 168, wherein the cell is in vitro.
Embodiment 170
[0180] The method of claim 168, wherein the cell is in an
animal.
Embodiment 171
[0181] The method of claim 170, wherein the animal is a human.
Embodiment 172
[0182] The method of claim 170, wherein the animal is a mouse.
Embodiment 173
[0183] A pharmaceutical composition comprising an oligomeric
compound of any of claims 1-167 and a pharmaceutically acceptable
carrier or diluent.
Embodiment 174
[0184] A method of administering a pharmaceutical composition of
claim 173 to an animal.
Embodiment 175
[0185] The method of claim 174, wherein the animal is a human.
Embodiment 176
[0186] The method of claim 174, wherein the animal is a mouse.
Embodiment 177
[0187] Use of an oligomeric compound of any of claims 1-167 for the
preparation of a medicament for the treatment or amelioration of
Huntington's disease.
Embodiment 178
[0188] A method of ameliorating a symptom of Huntington's disease,
comprising administering an oligomeric compound of any of claims
1-167 to an animal in need thereof.
Embodiment 179
[0189] The method of claim 178, wherein the animal is a human.
Embodiment 180
[0190] The method of claim 178, wherein the animal is a mouse.
[0191] In certain embodiments, including but not limited to any of
the above numbered embodiments, oligomeric compounds including
oligonucleotides described herein are capable of modulating
expression of a target RNA. In certain embodiments, the target RNA
is associated with a disease or disorder, or encodes a protein that
is associated with a disease or disorder. In certain embodiments,
the oligomeric compounds or oligonucleotides provided herein
modulate the expression of function of such RNA to alleviate one or
more symptom of the disease or disorder.
[0192] In certain embodiments, oligomeric compounds including
oligonucleotides describe herein are useful in vitro. In certain
embodiments such oligomeric compounds are used in diagnostics
and/or for target validation experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0193] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed. Herein, the use of the singular includes the plural unless
specifically stated otherwise. As used herein, the use of "or"
means "and/or" unless stated otherwise. Furthermore, the use of the
term "including" as well as other forms, such as "includes" and
"included", is not limiting. Also, terms such as "element" or
"component" encompass both elements and components comprising one
unit and elements and components that comprise more than one
subunit, unless specifically stated otherwise.
[0194] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including, but not limited to, patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference in their entirety for any purpose.
A. Definitions
[0195] Unless specific definitions are provided, the nomenclature
used in connection with, and the procedures and techniques of,
analytical chemistry, synthetic organic chemistry, and medicinal
and pharmaceutical chemistry described herein are those well known
and commonly used in the art. Standard techniques may be used for
chemical synthesis, and chemical analysis. Certain such techniques
and procedures may be found for example in "Carbohydrate
Modifications in Antisense Research" Edited by Sangvi and Cook,
American Chemical Society, Washington D.C., 1994; "Remington's
Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa.,
21.sup.st edition, 2005; and "Antisense Drug Technology,
Principles, Strategies, and Applications" Edited by Stanley T.
Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al.,
"Molecular Cloning, A laboratory Manual," 2.sup.nd Edition, Cold
Spring Harbor Laboratory Press, 1989, which are hereby incorporated
by reference for any purpose. Where permitted, all patents,
applications, published applications and other publications and
other data referred to throughout in the disclosure are
incorporated by reference herein in their entirety.
[0196] Unless otherwise indicated, the following terms have the
following meanings:
[0197] 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.
[0198] As used herein, "chemical modification" means a chemical
difference in a compound when compared to a naturally occurring
counterpart. Chemical modifications of oligonucleotides include
nucleoside modifications (including sugar moiety modifications and
nucleobase modifications) and internucleoside linkage
modifications. In reference to an oligonucleotide, chemical
modification does not include differences only in nucleobase
sequence.
[0199] As used herein, "furanosyl" means a structure comprising a
5-membered ring comprising four carbon atoms and one oxygen
atom.
[0200] 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.
[0201] As used herein, "sugar moiety" means a naturally occurring
sugar moiety or a modified sugar moiety of a nucleoside.
[0202] As used herein, "modified sugar moiety" means a substituted
sugar moiety or a sugar surrogate.
[0203] As used herein, "substituted sugar moiety" means a furanosyl
that is not a naturally occurring sugar moiety. Substituted sugar
moieties include, but are not limited to furanosyls comprising
substituents at the 2'-position, the 3'-position, the 5'-position
and/or the 4'-position. Certain substituted sugar moieties are
bicyclic sugar moieties.
[0204] 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.
[0205] As used herein, "MOE" means
--OCH.sub.2CH.sub.2OCH.sub.3.
[0206] As used herein, "2'-F nucleoside" refers to a nucleoside
comprising a sugar comprising fluoroine at the 2' position. Unless
otherwise indicated, the fluorine in a 2'-F nucleoside is in the
ribo position (replacing the OH of a natural ribose).
[0207] As used herein, "2'-(ara)-F" refers to a 2'-F substituted
nucleoside, wherein the fluoro group is in the arabino
position.
##STR00001##
[0208] As used herein the term "sugar surrogate" means a structure
that does not comprise a furanosyl and that is capable of replacing
the naturally occurring sugar moiety of a nucleoside, such that the
resulting nucleoside sub-units are capable of linking together
and/or linking to other nucleosides to form an oligomeric compound
which is capable of hybridizing to a complementary oligomeric
compound. Such structures include rings comprising a different
number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings);
replacement of the oxygen of a furanosyl with a non-oxygen atom
(e.g., carbon, sulfur, or nitrogen); or both a change in the number
of atoms and a replacement of the oxygen. Such structures may also
comprise substitutions corresponding to those described for
substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic
sugar surrogates optionally comprising additional substituents).
Sugar surrogates also include more complex sugar replacements
(e.g., the non-ring systems of peptide nucleic acid). Sugar
surrogates include without limitation morpholinos, cyclohexenyls
and cyclohexitols.
[0209] 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.
[0210] 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).
[0211] 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.
[0212] Nucleobases may be naturally occurring or may be
modified.
[0213] 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).
[0214] As used herein, "modified nucleobase" means any nucleobase
that is not a naturally occurring nucleobase.
[0215] 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.
[0216] As used herein, "bicyclic nucleoside" or "BNA" means a
nucleoside comprising a bicyclic sugar moiety.
[0217] As used herein, "constrained ethyl nucleoside" or "cEt"
means a nucleoside comprising a bicyclic sugar moiety comprising a
4'-CH(CH.sub.3)--O-2'bridge.
[0218] As used herein, "locked nucleic acid nucleoside" or "LNA"
means a nucleoside comprising a bicyclic sugar moiety comprising a
4'-CH.sub.2--O-2'bridge.
[0219] 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.
[0220] 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).
[0221] As used herein, "RNA-like nucleoside" means a modified
nucleoside that adopts a northern configuration and functions like
RNA when incorporated into an oligonucleotide. RNA-like nucleosides
include, but are not limited to 3'-endo furanosyl nucleosides and
RNA surrogates.
[0222] As used herein, "3'-endo-furanosyl nucleoside" means an
RNA-like nucleoside that comprises a substituted sugar moiety that
has a 3'-endo conformation. 3'-endo-furanosyl nucleosides include,
but are not limited to: 2'-MOE, 2'-F, 2'-OMe, LNA, ENA, and cEt
nucleosides.
[0223] As used herein, "RNA-surrogate nucleoside" means an RNA-like
nucleoside that does not comprise a furanosyl. RNA-surrogate
nucleosides include, but are not limited to hexitols and
cyclopentanes.
[0224] As used herein, "oligonucleotide" means a compound
comprising a plurality of linked nucleosides.
[0225] In certain embodiments, an oligonucleotide comprises one or
more unmodified ribonucleosides (RNA) and/or unmodified
deoxyribonucleosides (DNA) and/or one or more modified
nucleosides.
[0226] As used herein "oligonucleoside" means an oligonucleotide in
which none of the internucleoside linkages contains a phosphorus
atom. As used herein, oligonucleotides include
oligonucleosides.
[0227] As used herein, "modified oligonucleotide" means an
oligonucleotide comprising at least one modified nucleoside and/or
at least one modified internucleoside linkage.
[0228] As used herein "internucleoside linkage" means a covalent
linkage between adjacent nucleosides in an oligonucleotide.
[0229] As used herein "naturally occurring internucleoside linkage"
means a 3' to 5' phosphodiester linkage.
[0230] As used herein, "modified internucleoside linkage" means any
internucleoside linkage other than a naturally occurring
internucleoside linkage.
[0231] 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.
[0232] 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.
[0233] 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.
[0234] As used herein, "conjugate linking group" means any atom or
group of atoms used to attach a conjugate to an oligonucleotide or
oligomeric compound.
[0235] 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.
[0236] 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.
[0237] 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.
[0238] As used herein, "detectable and/or measureable activity"
means a measurable activity that is not zero.
[0239] 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.
[0240] As used herein, "expression" means the process by which a
gene ultimately results in a protein.
[0241] Expression includes, but is not limited to, transcription,
post-transcriptional modification (e.g., splicing, polyadenlyation,
addition of 5'-cap), and translation.
[0242] As used herein, "target nucleic acid" means a nucleic acid
molecule to which an antisense compound is intended to
hybridize.
[0243] As used herein, "non-target nucleic acid" means a nucleic
acid molecule to which hybridization of an antisense compound is
not intended or desired. In certain embodiments, antisense
compounds do hybridize to a non-target, due to homology between the
target (intended) and non-target (un-intended).
[0244] As used herein, "mRNA" means an RNA molecule that encodes a
protein.
[0245] As used herein, "pre-mRNA" means an RNA transcript that has
not been fully processed into mRNA.
[0246] Pre-RNA includes one or more intron.
[0247] As used herein, "object RNA" means an RNA molecule other
than a target RNA, the amount, activity, splicing, and/or function
of which is modulated, either directly or indirectly, by a target
nucleic acid.
[0248] In certain embodiments, a target nucleic acid modulates
splicing of an object RNA. In certain such embodiments, an
antisense compound modulates the amount or activity of the target
nucleic acid, resulting in a change in the splicing of an object
RNA and ultimately resulting in a change in the activity or
function of the object RNA.
[0249] As used herein, "microRNA" means a naturally occurring,
small, non-coding RNA that represses gene expression of at least
one mRNA. In certain embodiments, a microRNA represses gene
expression by binding to a target site within a 3' untranslated
region of an mRNA. In certain embodiments, a microRNA has a
nucleobase sequence as set forth in miRBase, a database of
published microRNA sequences found at
microrna.sanger.ac.uk/sequences/. In certain embodiments, a
microRNA has a nucleobase sequence as set forth in miRBase version
12.0 released September 2008, which is herein incorporated by
reference in its entirety.
[0250] As used herein, "microRNA mimic" means an oligomeric
compound having a sequence that is at least partially identical to
that of a microRNA. In certain embodiments, a microRNA mimic
comprises the microRNA seed region of a microRNA. In certain
embodiments, a microRNA mimic modulates translation of more than
one target nucleic acids. In certain embodiments, a microRNA mimic
is double-stranded.
[0251] As used herein, "differentiating nucleobase" means a
nucleobase that differs between two nucleic acids. In certain
instances, a target region of a target nucleic acid differs by 1-4
nucleobases from a non-target nucleic acid. Each of those
differences is refered to as a differentiating nucleobase. In
certain instances, a differentiating nucleobase is a
single-nucleotide polymorphism.
[0252] As used herein, "target-selective nucleoside" means a
nucleoside of an antisense compound that corresponds to a
differentiating nucleobase of a target nucleic acid.
[0253] As used herein, "allele" means one of a pair of copies of a
gene existing at a particular locus or marker on a specific
chromosome, or one member of a pair of nucleobases existing at a
particular locus or marker on a specific chromosome, or one member
of a pair of nucleobase sequences existing at a particular locus or
marker on a specific chromosome. For a diploid organism or cell or
for autosomal chromosomes, each allelic pair will normally occupy
corresponding positions (loci) on a pair of homologous chromosomes,
one inherited from the mother and one inherited from the father. If
these alleles are identical, the organism or cell is said to be
"homozygous" for that allele; if they differ, the organism or cell
is said to be "heterozygous" for that allele. "Wild-type allele"
refers to the genotype typically not associated with disease or
dysfunction of the gene product. "Mutant allele" refers to the
genotype associated with disease or dysfunction of the gene
product.
[0254] As used herein, "allelic variant" means a particular
identity of an allele, where more than one identity occurs. For
example, an allelic variant may refer to either the mutant allele
or the wild-type allele.
[0255] As used herein, "single nucleotide polymorphism" or "SNP"
means a single nucleotide variation between the genomes of
individuals of the same species. In some cases, a SNP may be a
single nucleotide deletion or insertion. In general, SNPs occur
relatively frequently in genomes and thus contribute to genetic
diversity. The location of a SNP is generally flanked by highly
conserved sequences. An individual may be homozygous or
heterozygous for an allele at each SNP site.
[0256] As used herein, "single nucleotide polymorphism site" or
"SNP site" refers to the nucleotides surrounding a SNP contained in
a target nucleic acid to which an antisense compound is
targeted.
[0257] 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.
[0258] 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.
[0259] Nucleobases comprising certain modifications may maintain
the ability to pair with a counterpart nucleobase and thus, are
still capable of nucleobase complementarity.
[0260] As used herein, "non-complementary" in reference to
nucleobases means a pair of nucleobases that do not form hydrogen
bonds with one another.
[0261] 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.
[0262] As used herein, "mismatch" means a nucleobase of a first
oligomeric compound that is not capable of pairing with a
nucleobase at a corresponding position of a second oligomeric
compound, when the first and second oligomeric compound are
aligned. Either or both of the first and second oligomeric
compounds may be oligonucleotides.
[0263] 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.
[0264] 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.
[0265] As used herein, "fully complementary" in reference to an
oligonucleotide or portion thereof means that each nucleobase of
the oligonucleotide or portion thereof is capable of pairing with a
nucleobase of a complementary nucleic acid or contiguous portion
thereof. Thus, a fully complementary region comprises no mismatches
or unhybridized nucleobases in either strand.
[0266] 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.
[0267] As used herein, "percent identity" means the number
ofnucleobases 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.
[0268] 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.
[0269] As used herein, "modification motif" means a pattern of
chemical modifications in an oligomeric compound or a region
thereof. Motifs may be defined by modifications at certain
nucleosides and/or at certain linking groups of an oligomeric
compound.
[0270] 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.
[0271] As used herein, "sugar motif" means a pattern of sugar
modifications in an oligomeric compound or a region thereof.
[0272] 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.
[0273] 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.
[0274] 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.
[0275] 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.
[0276] 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.
[0277] 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.
[0278] 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.
[0279] 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.
[0280] As used herein, "substituent" and "substituent group," means
an atom or group that replaces the atom or group of a named parent
compound. For example a substituent of a modified nucleoside is any
atom or group that differs from the atom or group found in a
naturally occurring nucleoside (e.g., a modified 2'-substuent is
any atom or group at the 2'-position of a nucleoside other than H
or OH). Substituent groups can be protected or unprotected. In
certain embodiments, compounds of the present invention have
substituents at one or at more than one position of the parent
compound. Substituents may also be further substituted with other
substituent groups and may be attached directly or via a linking
group such as an alkyl or hydrocarbyl group to a parent
compound.
[0281] Likewise, as used herein, "substituent" in reference to a
chemical functional group means an atom or group of atoms differs
from the atom or a group of atoms normally present in the named
functional group. In certain embodiments, a substituent replaces a
hydrogen atom of the functional group (e.g., in certain
embodiments, the substituent of a substituted methyl group is an
atom or group other than hydrogen which replaces one of the
hydrogen atoms of an unsubstituted methyl group). Unless otherwise
indicated, groups amenable for use as substituents include without
limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl
(--C(O)R.sub.aa), carboxyl (--C(O)O--R.sub.aa), aliphatic groups,
alicyclic groups, alkoxy, substituted oxy (--O--R.sub.aa), aryl,
aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino
(--N(R.sub.bb)(R.sub.cc)), imino(.dbd.NR.sub.bb), amido
(--C(O)N(R.sub.bb)(R.sub.cc) or --N(R.sub.bb)C(O)R.sub.aa), azido
(--N.sub.3), nitro (--NO.sub.2), cyano (--CN), carbamido
(--OC(O)N(R.sub.bb)(R.sub.cc) or --N(R.sub.bb)C(O)OR.sub.aa),
ureido (--N(R.sub.bb)C(O)N(R.sub.bb)(R.sub.cc)), thioureido
(--N(R.sub.bb)C(S)N(R.sub.bb)--(R.sub.cc)), guanidinyl
(--N(R.sub.bb)C(.dbd.NR.sub.bb)N(R.sub.bb)(R.sub.cc)), amidinyl
(--C(.dbd.NR.sub.bb)N(R.sub.bb)(R.sub.cc) or
--N(R.sub.bb)C(.dbd.NR.sub.bb)(R.sub.aa)), thiol (--SR.sub.bb),
sulfinyl (--S(O)R.sub.bb), sulfonyl (--S(O).sub.2R.sub.bb) and
sulfonamidyl (--S(O).sub.2N(R.sub.bb)(R.sub.cc) or
--N(R.sub.bb)S--(O).sub.2R.sub.bb). Wherein each R.sub.aa, R.sub.bb
and R.sub.cc is, independently, H, an optionally linked chemical
functional group or a further substituent group with a preferred
list including without limitation, alkyl, alkenyl, alkynyl,
aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic,
heterocyclic and heteroarylalkyl. Selected substituents within the
compounds described herein are present to a recursive degree.
[0282] As used herein, "alkyl," as used herein, means a saturated
straight or branched hydrocarbon radical containing up to twenty
four carbon atoms. Examples of alkyl groups include without
limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl,
octyl, decyl, dodecyl and the like. Alkyl groups typically include
from 1 to about 24 carbon atoms, more typically from 1 to about 12
carbon atoms (C.sub.1-C.sub.12 alkyl) with from 1 to about 6 carbon
atoms being more preferred.
[0283] 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.
[0284] 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.
[0285] 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.
[0286] 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.
[0287] As used herein, "aliphatic" means a straight or branched
hydrocarbon radical containing up to twenty four carbon atoms
wherein the saturation between any two carbon atoms is a single,
double or triple bond. An aliphatic group preferably contains from
1 to about 24 carbon atoms, more typically from 1 to about 12
carbon atoms with from 1 to about 6 carbon atoms being more
preferred. The straight or branched chain of an aliphatic group may
be interrupted with one or more heteroatoms that include nitrogen,
oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by
heteroatoms include without limitation, polyalkoxys, such as
polyalkylene glycols, polyamines, and polyimines. Aliphatic groups
as used herein may optionally include further substituent
groups.
[0288] 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.
[0289] As used herein, "aminoalkyl" means an amino substituted
C.sub.1-C.sub.12 alkyl radical. The alkyl portion of the radical
forms a covalent bond with a parent molecule. The amino group can
be located at any position and the aminoalkyl group can be
substituted with a further substituent group at the alkyl and/or
amino portions.
[0290] As used herein, "aralkyl" and "arylalkyl" mean an aromatic
group that is covalently linked to a C.sub.1-C.sub.12 alkyl
radical. The alkyl radical portion of the resulting aralkyl (or
arylalkyl) group forms a covalent bond with a parent molecule.
Examples include without limitation, benzyl, phenethyl and the
like. Aralkyl groups as used herein may optionally include further
substituent groups attached to the alkyl, the aryl or both groups
that form the radical group.
[0291] 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.
[0292] As used herein, "halo" and "halogen," mean an atom selected
from fluorine, chlorine, bromine and iodine.
[0293] 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.
[0294] As used herein, "huntingtin transcript" means a transcript
transcribed from a huntingtin gene.
B. Oligomeric Compounds
[0295] In certain embodiments, the present invention provides
oligomeric compounds. In certain embodiments, such oligomeric
compounds comprise oligonucleotides optionally comprising one or
more conjugate and/or terminal groups. In certain embodiments, an
oligomeric compound consists of an oligonucleotide. In certain
embodiments, oligonucleotides comprise one or more chemical
modifications. Such chemical modifications include modifications of
one or more nucleoside (including modifications to the sugar moiety
and/or the nucleobase) and/or modifications to one or more
internucleoside linkage.
[0296] a. Certain Modified Nucleosides
[0297] In certain embodiments, provided herein are oligomeric
compounds comprising or consisting of oligonucleotides comprising
at least one modified nucleoside. Such modified nucleosides
comprise a modified sugar moeity, a modified nucleobase, or both a
modified sugar moiety and a modified nucleobase.
[0298] i. Certain Modified Sugar Moieties
[0299] In certain embodiments, compounds of the invention comprise
one or more modified nucleosides comprising a modified sugar
moiety. Such compounds comprising one or more sugar-modified
nucleosides may have desirable properties, such as enhanced
nuclease stability or increased binding affinity with a target
nucleic acid relative to an oligonucleotide comprising only
nucleosides comprising naturally occurring sugar moieties. In
certain embodiments, modified sugar moieties are substituted sugar
moieties. In certain embodiments, modified sugar moieties are sugar
surrogates. Such sugar surrogates may comprise one or more
substitutions corresponding to those of substituted sugar
moieties.
[0300] In certain embodiments, modified sugar moieties are
substituted sugar moieties comprising one or more non-bridging
sugar substituent, including but not limited to substituents at the
2' and/or 5' positions. Examples of sugar substituents suitable for
the 2'-position, include, but are not limited to: 2'-F,
2'-OCH.sub.3 ("OMe" or "O-methyl"), and
2'-O(CH.sub.2).sub.2OCH.sub.3 ("MOE"). In certain embodiments,
sugar substituents at the 2' position is selected from allyl,
amino, azido, thio, O-allyl, O--C.sub.1-C.sub.10 alkyl,
O--C.sub.1-C.sub.10 substituted alkyl; OCF.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2--O--N(Rm)(Rn), and
O--CH.sub.2--C(.dbd.O)--N(Rm)(Rn), where each Rm and Rn is,
independently, H or substituted or unsubstituted C.sub.1-C.sub.10
alkyl. Examples of sugar substituents at the 5'-position, include,
but are not limited to: 5'-methyl (R or S); 5'-vinyl, and
5'-methoxy. In certain embodiments, substituted sugars comprise
more than one non-bridging sugar substituent, for example,
2'-F-5'-methyl sugar moieties (see, e.g., PCT International
Application WO 2008/101157, for additional 5', 2'-bis substituted
sugar moieties and nucleosides).
[0301] Nucleosides comprising 2'-substituted sugar moieties are
referred to as 2'-substituted nucleosides. In certain embodiments,
a 2'-substituted nucleoside comprises a 2'-substituent group
selected from halo, allyl, amino, azido, SH, CN, OCN, CF.sub.3,
OCF.sub.3, O, S, or N(R.sub.m)-alkyl; O, S, or N(R.sub.m)-alkenyl;
O, S or N(R.sub.m)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl,
aralkyl, O-alkaryl, O-aralkyl, O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n) or
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and
R.sub.n is, independently, H, an amino protecting group or
substituted or unsubstituted C.sub.1-C.sub.10 alkyl. These
2'-substituent groups can be further substituted with one or more
substituent groups independently selected from hydroxyl, amino,
alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol,
thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and
alkynyl.
[0302] In certain embodiments, a 2'-substituted nucleoside
comprises a 2'-substituent group selected from F, NH.sub.2,
N.sub.3, OCF.sub.3, O--CH.sub.3, O(CH.sub.2).sub.3NH.sub.2,
CH.sub.2--CH.dbd.CH.sub.2, O--CH.sub.2--CH.dbd.CH.sub.2,
OCH.sub.2CH.sub.2OCH.sub.3, O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n),
O(CH.sub.2).sub.2O(CH.sub.2).sub.2N(CH.sub.3).sub.2, and
N-substituted acetamide
(O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n) where each R.sub.m and
R.sub.n is, independently, H, an amino protecting group or
substituted or unsubstituted C.sub.1-C.sub.10 alkyl.
[0303] In certain embodiments, a 2'-substituted nucleoside
comprises a sugar moiety comprising a 2'-substituent group selected
from F, OCF.sub.3, O--CH.sub.3, OCH.sub.2CH.sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(CH.sub.3).sub.2,
--O(CH.sub.2).sub.2O(CH.sub.2).sub.2N(CH.sub.3).sub.2, and
O--CH.sub.2--C(.dbd.O)--N(H)CH.sub.3.
[0304] In certain embodiments, a 2'-substituted nucleoside
comprises a sugar moiety comprising a 2'-substituent group selected
from F, O--CH.sub.3, and OCH.sub.2CH.sub.2OCH.sub.3.
[0305] Certain modified sugar moieties comprise a bridging sugar
substituent that forms a second ring resulting in a bicyclic sugar
moiety. In certain such embodiments, the bicyclic sugar moiety
comprises a bridge between the 4' and the 2' furanose ring atoms.
Examples of such 4' to 2' sugar substituents, include, but are not
limited to: --[C(R.sub.a)(R.sub.b)].sub.n--,
--[C(R.sub.a)(R.sub.b)].sub.n--O--, --C(R.sub.aR.sub.b)--N(R)--O--
or, --C(R.sub.aR.sub.b)--O--N(R)--; 4'- CH.sub.2-2',
4'-(CH.sub.2).sub.2-2', 4'-(CH.sub.2).sub.3-2',4'-(CH.sub.2)--O-2'
(LNA); 4'-(CH.sub.2)--S-2'; 4'-(CH.sub.2).sub.2--O-2' (ENA);
4'-CH(CH.sub.3)--O-2' (cEt) and 4'-CH(CH.sub.2OCH.sub.3)--O-2',and
analogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul.
15, 2008); 4'-C(CH.sub.3)(CH.sub.3)--O-2' and analogs thereof,
(see, e.g., WO2009/006478, published Jan. 8, 2009);
4'-CH.sub.2--N(OCH.sub.3)-2' and analogs thereof (see, e.g.,
WO2008/150729, published Dec. 11, 2008);
4'-CH.sub.2--O--N(CH.sub.3)-2' (see, e.g., US2004/0171570,
published Sep. 2, 2004); 4'-CH.sub.2--O--N(R)-2', and
4'-CH.sub.2--N(R)-0-2'-, wherein each R is, independently, H, a
protecting group, or C.sub.1-C.sub.12 alkyl;
4'-CH.sub.2--N(R)--O-2', wherein R is H, C.sub.1-C.sub.12 alkyl, or
a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep.
23, 2008); 4'-CH.sub.2--C(H)(CH.sub.3)-2' (see, e.g.,
Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and
4'-CH.sub.2--C(.dbd.CH.sub.2)-2' and analogs thereof (see,
published PCT International Application WO 2008/154401, published
on Dec. 8, 2008).
[0306] In certain embodiments, such 4' to 2' bridges independently
comprise from 1 to 4 linked groups independently selected from
--[C(R.sub.a)(R.sub.b)].sub.n--, --C(R.sub.a).dbd.C(R.sub.b)--,
--C(R.sub.a).dbd.N--, --C(.dbd.NR.sub.a)--, --C(.dbd.O)--,
--C(.dbd.S)--, --O--, --Si(R.sub.a).sub.2--, --S(.dbd.O).sub.x--,
and --N(R.sub.a)--;
[0307] wherein:
[0308] x is 0, 1, or 2;
[0309] n is 1, 2, 3, or 4;
[0310] each R.sub.a and R.sub.b is, independently, H, a protecting
group, hydroxyl, C.sub.1-C.sub.12 alkyl, substituted
C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted
C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted
C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted
C.sub.5-C.sub.20 aryl, heterocycle radical, substituted heterocycle
radical, heteroaryl, substituted heteroaryl, C.sub.5-C.sub.7
alicyclic radical, substituted C.sub.5-C.sub.7 alicyclic radical,
halogen, OJ.sub.1, NJ.sub.1J.sub.2, SJ, N.sub.3, COOJ.sub.1, acyl
(C(.dbd.O)--H), substituted acyl, CN, sulfonyl
(S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and
[0311] each J.sub.1 and J.sub.2 is, independently, H,
C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl,
C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl,
C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, acyl
(C(.dbd.O)--H), substituted acyl, a heterocycle radical, a
substituted heterocycle radical, C.sub.1-C.sub.12 aminoalkyl,
substituted C.sub.1-C.sub.12 aminoalkyl, or a protecting group.
[0312] Nucleosides comprising bicyclic sugar moieties are referred
to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include,
but are not limited to, (A) .alpha.-L-Methyleneoxy
(4'-CH.sub.2--O-2') BNA, (B) .beta.-D-Methyleneoxy
(4'-CH.sub.2--O-2') BNA (also referred to as locked nucleic acid or
LNA), (C) Ethyleneoxy (4'-(CH.sub.2).sub.2--O-2') BNA, (D) Aminooxy
(4'-CH.sub.2--O--N(R)-2') BNA, (E) Oxyamino
(4'-CH.sub.2--N(R)--O-2') BNA, (F) Methyl(methyleneoxy)
(4'-CH(CH.sub.3)--O-2') BNA (also referred to as constrained ethyl
or cEt), (G) methylene-thio (4'-CH.sub.2--S-2') BNA, (H)
methylene-amino (4'-CH.sub.2--N(R)-2') BNA, (I) methyl carbocyclic
(4'-CH.sub.2--CH(CH.sub.3)-2') BNA, (J) propylene carbocyclic
(4'-(CH.sub.2).sub.3-2') BNA, and (K) Ethylene(methoxy)
(4'-(CH(CH.sub.2OMe)-O-2') BNA (also referred to as constrained MOE
or cMOE) as depicted below.
##STR00002## ##STR00003##
wherein Bx is a nucleobase moiety and R is, independently, H, a
protecting group, or C.sub.1-C.sub.12 alkyl.
[0313] 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.
[0314] In certain embodiments, bicyclic sugar moieties and
nucleosides incorporating such bicyclic sugar moieties are further
defined by isomeric configuration. For example, a nucleoside
comprising a 4'-2' methylene-oxy bridge, may be in the .alpha.-L
configuration or in the .beta.-D configuration. Previously,
.alpha.-L-methyleneoxy (4'-CH.sub.2--O-2') bicyclic nucleosides
have been incorporated into antisense oligonucleotides that showed
antisense activity (Frieden et al., Nucleic Acids Research, 2003,
21, 6365-6372).
[0315] 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).
[0316] In certain embodiments, modified sugar moieties are sugar
surrogates. In certain such embodiments, the oxygen atom of the
naturally occuring sugar is substituted, e.g., with a sulfer,
carbon or nitrogen atom. In certain such embodiments, such modified
sugar moiety also comprises bridging and/or non-bridging
substituents as described above. For example, certain sugar
surogates comprise a 4'-sulfer atom and a substitution at the
2'-position (see,e.g., published U.S. Patent Application
US2005/0130923, published on 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).
[0317] In certain embodiments, sugar surrogates comprise rings
having other than 5-atoms. For example, in certain embodiments, a
sugar surrogate comprises a six-membered tetrahydropyran. Such
tetrahydropyrans may be further modified or substituted.
Nucleosides comprising such modified tetrahydropyrans include, but
are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid
(ANA), manitol nucleic acid (MNA) (see Leumann, C J. Bioorg.
&Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), and those
compounds having Formula VII:
##STR00004##
wherein independently for each of said at least one tetrahydropyran
nucleoside analog of Formula VII:
[0318] Bx is a nucleobase moiety;
[0319] T.sub.3 and T.sub.4 are each, independently, an
internucleoside linking group linking the tetrahydropyran
nucleoside analog to the antisense compound or one of T.sub.3 and
T.sub.4 is an internucleoside linking group linking the
tetrahydropyran nucleoside analog to the antisense compound and the
other of T.sub.3 and T.sub.4 is H, a hydroxyl protecting group, a
linked conjugate group, or a 5' or 3'-terminal group;
q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7
are each, independently, H, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, or substituted
C.sub.2-C.sub.6 alkynyl; and
[0320] each of R.sub.1 and R.sub.2 is independently selected from
among: hydrogen, halogen, substituted or unsubstituted alkoxy,
NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, OC(.dbd.X)J.sub.1,
OC(.dbd.X)NJ.sub.1J.sub.2, NJ.sub.3C(.dbd.X)NJ.sub.1J.sub.2, and
CN, wherein X is O, S or NJ.sub.1, and each J, J.sub.2, and J.sub.3
is, independently, H or C.sub.1-C.sub.6 alkyl.
[0321] In certain embodiments, the modified THP nucleosides of
Formula VII are provided wherein q.sub.1, q.sub.2, q.sub.3,
q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each H. In certain
embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4,
q.sub.5, q.sub.6 and q.sub.7 is other than H. In certain
embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4,
q.sub.5, q.sub.6 and q.sub.7 is methyl. In certain embodiments, THP
nucleosides of Formula VII are provided wherein one of R.sub.1 and
R.sub.2 is F. In certain embodiments, R.sub.1 is fluoro and R.sub.2
is H, R.sub.1 is methoxy and R.sub.2 is H, and R.sub.1 is
methoxyethoxy and R.sub.2 is H.
[0322] Many other bicyclo and tricyclo sugar surrogate ring systems
are also known in the art that can be used to modify nucleosides
for incorporation into antisense compounds (see, e.g., review
article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002,
10, 841-854).
[0323] Combinations of modifications are also provided without
limitation, such as 2'-F-5'-methyl substituted nucleosides (see PCT
International Application WO 2008/101157 Published on Aug. 21, 2008
for other disclosed 5', 2'-bis substituted nucleosides) and
replacement of the ribosyl ring oxygen atom with S and further
substitution at the 2'-position (see published U.S. Patent
Application US2005-0130923, published on Jun. 16, 2005) or
alternatively 5'-substitution of a bicyclic nucleic acid (see PCT
International Application WO 2007/134181, published on Nov. 22,
2007 wherein a 4'-CH.sub.2--O-2' bicyclic nucleoside is further
substituted at the 5' position with a 5'-methyl or a 5'-vinyl
group). The synthesis and preparation of carbocyclic bicyclic
nucleosides along with their oligomerization and biochemical
studies have also been described (see, e.g., Srivastava et al., J.
Am. Chem. Soc. 2007, 129(26), 8362-8379).
[0324] In certain embodiments, the present invention provides
oligonucleotides comprising modified nucleosides. Those modified
nucleotides may include modified sugars, modified nucleobases,
and/or modified linkages. The specific modifications are selected
such that the resulting oligonucleotides possess desireable
characteristics. In certain embodmiments, oligonucleotides comprise
one or more RNA-like nucleosides. In certain embodiments,
oligonucleotides comprise one or more DNA-like nucleotides.
[0325] ii. Certain Modified Nucleobases
[0326] In certain embodiments, nucleosides of the present invention
comprise one or more unmodified nucleobases. In certain
embodiments, nucleosides of the present invention comprise one or
more modifed nucleobases.
[0327] In certain embodiments, modified nucleobases are selected
from: universal bases, hydrophobic bases, promiscuous bases,
size-expanded bases, and fluorinated bases as defined herein. In
certain embodiments, modified nucleobases are selected from:
5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6
substituted purines, including 2-aminopropyladenine,
5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl cytosine,
xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl
derivatives of adenine and guanine, 2-propyl and other alkyl
derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and
2-thiocytosine, 5-halouracil and cytosine, 5-propynyl
(--C.ident.C--CH.sub.3) uracil and cytosine and other alkynyl
derivatives of pyrimidine bases, 6-azo uracil, cytosine and
thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,
8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines
and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and
other 5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and
3-deazaadenine, universal bases, hydrophobic bases, promiscuous
bases, size-expanded bases, and fluorinated bases as defined
herein. Further modified nucleobases include tricyclic pyrimidines
such as phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one),
phenothiazine cytidine
(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a
substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole
cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
Modified nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia Of Polymer
Science And Engineering, Kroschwitz, J. I., Ed., John Wiley &
Sons, 1990, 858-859; those disclosed by Englisch et al., Angewandte
Chemie, International Edition, 1991, 30, 613; and those disclosed
by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications,
Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288.
[0328] 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.
[0329] b. Certain Internucleoside Linkages
[0330] In certain embodiments, nucleosides may be linked together
using any internucleoside linkage to form oligonucleotides. The two
main classes of internucleoside linking groups are defined by the
presence or absence of a phosphorus atom. Representative phosphorus
containing internucleoside linkages include, but are not limited
to, phosphodiesters (P.dbd.O), phosphotriesters,
methylphosphonates, phosphoramidate, and phosphorothioates
(P.dbd.S). Representative non-phosphorus containing internucleoside
linking groups include, but are not limited to,
methylenemethylimino (--CH.sub.2--N(CH.sub.3)--O--CH.sub.2--),
thiodiester (--O--C(O)--S--), thionocarbamate (--O--C(O)(NH)--S--);
siloxane (--O--Si(H).sub.2--O--); and N,N'-dimethylhydrazine
(--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--). Modified linkages,
compared to natural phosphodiester linkages, can be used to alter,
typically increase, nuclease resistance of the oligonucleotide. In
certain embodiments, internucleoside linkages having a chiral atom
can be prepared as a racemic mixture, or as separate enantiomers.
Representative chiral linkages include, but are not limited to,
alkylphosphonates and phosphorothioates. Methods of preparation of
phosphorous-containing and non-phosphorous-containing
internucleoside linkages are well known to those skilled in the
art.
[0331] The oligonucleotides described herein contain one or more
asymmetric centers and thus give rise to enantiomers,
diastereomers, and other stereoisomeric configurations that may be
defined, in terms of absolute stereochemistry, as (R) or (S),
.alpha. or .beta. such as for sugar anomers, or as (D) or (L) such
as for amino acids etc. Included in the antisense compounds
provided herein are all such possible isomers, as well as their
racemic and optically pure forms.
[0332] Neutral internucleoside linkages include without limitation,
phosphotriesters, methylphosphonates, MMI
(3'-CH.sub.2--N(CH.sub.3)--O-5'), amide-3
(3'-CH.sub.2--C(.dbd.O)--N(H)-5'), amide-4
(3'-CH.sub.2--N(H)--C(.dbd.O)-5'), formacetal
(3'-O--CH.sub.2--O-5'), and thioformacetal (3'-S--CH.sub.2--O-5').
Further neutral internucleoside linkages include nonionic linkages
comprising siloxane (dialkylsiloxane), carboxylate ester,
carboxamide, sulfide, sulfonate ester and amides (See for example:
Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and
P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4,
40-65). Further neutral internucleoside linkages include nonionic
linkages comprising mixed N, O, S and CH.sub.2 component parts.
[0333] i. 3'-Endo Modifications
[0334] In one aspect of the present disclosure, oligomeric
compounds include nucleosides synthetically modified to induce a
3'-endo sugar conformation. A nucleoside can incorporate synthetic
modifications of the heterocyclic base moiety, the sugar moiety or
both to induce a desired 3'-endo sugar conformation. These modified
nucleosides are used to mimic RNA like nucleosides so that
particular properties of an oligomeric compound can be enhanced
while maintaining the desirable 3'-endo conformational geometry.
There is an apparent preference for an RNA type duplex (A form
helix, predominantly 3'-endo) as a requirement of RNA interference
which is supported in part by the fact that duplexes composed of
2'-deoxy-2'-F-nucleosides appear efficient in triggering RNAi
response in the C. elegans system. Properties that are enhanced by
using more stable 3'-endo nucleosides include but aren't limited to
modulation of pharmacokinetic properties through modification of
protein binding, protein off-rate, absorption and clearance;
modulation of nuclease stability as well as chemical stability;
modulation of the binding affinity and specificity of the oligomer
(affinity and specificity for enzymes as well as for complementary
sequences); and increasing efficacy of RNA cleavage. The present
invention provides oligomeric compounds having one or more
nucleosides modified in such a way as to favor a C3'-endo type
conformation.
##STR00005##
[0335] Nucleoside conformation is influenced by various factors
including substitution at the 2', 3' or 4'-positions of the
pentofuranosyl sugar. Electronegative substituents generally prefer
the axial positions, while sterically demanding substituents
generally prefer the equatorial positions (Principles of Nucleic
Acid Structure, Wolfgang Sanger, 1984, Springer-Verlag.)
Modification of the 2' position to favor the 3'-endo conformation
can be achieved while maintaining the 2'-OH as a recognition
element, as exemplified in Example 35, below (Gallo et al.,
Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org.
Chem., (1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem.
(1999), 64, 747-754.) Alternatively, preference for the 3'-endo
conformation can be achieved by deletion of the 2'-OH as
exemplified by 2'deoxy-2'F-nucleosides (Kawasaki et al., J. Med.
Chem. (1993), 36, 831-841), which adopts the 3'-endo conformation
positioning the electronegative fluorine atom in the axial
position. Other modifications of the ribose ring, for example
substitution at the 4'-position to give 4'-F modified nucleosides
(Guillerm et al., Bioorganic and Medicinal Chemistry Letters
(1995), 5, 1455-1460 and Owen et al., J. Org. Chem. (1976), 41,
3010-3017), or for example modification to yield methanocarba
nucleoside analogs (Jacobson et al., J. Med. Chem. Lett. (2000),
43, 2196-2203 and Lee et al., Bioorganic and Medicinal Chemistry
Letters (2001), 11, 1333-1337) also induce preference for the
3'-endo conformation. Some modifications actually lock the
conformational geometry by formation of a bicyclic sugar moiety
e.g. locked nucleic acid (LNA, Singh et al, Chem. Commun. (1998),
4, 455-456), and ethylene bridged nucleic acids (ENA, Morita et al,
Bioorganic & Medicinal Chemistry Letters (2002), 12,
73-76.)
[0336] c. Certain Motifs
[0337] In certain embodiments, oligomeric compounds comprise or
consist of oligonucleotides. In certain embodiments, such
oligonucleotides comprise one or more chemical modification. In
certain embodiments, chemically modified oligonucleotides comprise
one or more modified sugars. In certain embodiments, chemically
modified oligonucleotides comprise one or more modified
nucleobases. In certain embodiments, chemically modified
oligonucleotides comprise one or more modified internucleoside
linkages. In certain embodiments, the chemical modifications (sugar
modifications, nucleobase modifications, and/or linkage
modifications) define a pattern or motif. In certain embodiments,
the patterns of chemical modifications of sugar moieties,
internucleoside linkages, and nucleobases are each independent of
one another. Thus, an oligonucleotide may be described by its sugar
modification motif, internucleoside linkage motif and/or nucleobase
modification motif (as used herein, nucleobase modification motif
describes the chemical modifications to the nucleobases independent
of the sequence of nucleobases).
[0338] i. Certain Sugar Motifs
[0339] In certain embodiments, oligonucleotides comprise one or
more type of modified sugar moieties and/or naturally occurring
sugar moieties arranged along an oligonucleotide or region thereof
in a defined pattern or sugar motif. Such sugar motifs include but
are not limited to any of the sugar modifications discussed
herein.
[0340] In certain embodiments, the oligonucleotides comprise or
consist of a region having a gapmer sugar motif, which comprises
two external regions or "wings" and a central or internal region or
"gap." The three regions of a gapmer sugar motif (the 5'-wing, the
gap, and the 3'-wing) form a contiguous sequence of nucleosides
wherein at least some of the sugar moieties of the nucleosides of
each of the wings differ from at least some of the sugar moieties
of the nucleosides of the gap. Specifically, at least the sugar
moieties of the nucleosides of each wing that are closest to the
gap (the 3'-most nucleoside of the 5'-wing and the 5'-most
nucleoside of the 3'-wing) differ from the sugar moiety of the
neighboring gap nucleosides, thus defining the boundary between the
wings and the gap. In certain embodiments, the sugar moieties
within the gap are the same as one another. In certain embodiments,
the gap includes one or more nucleoside having a sugar moiety that
differs from the sugar moiety of one or more other nucleosides of
the gap. In certain embodiments, the sugar motifs of the two wings
are the same as one another (symmetric sugar gapmer). In certain
embodiments, the sugar motifs of the 5'-wing differs from the sugar
motif of the 3'-wing (asymmetric sugar gapmer).
[0341] ii. Certain Nucleobase Modification Motifs
[0342] In certain embodiments, oligonucleotides comprise chemical
modifications to nucleobases arranged along the oligonucleotide or
region thereof in a defined pattern or nucleobases modification
motif. In certain embodiments, each nucleobase is modified. In
certain embodiments, none of the nucleobases is chemically
modified.
[0343] 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.
[0344] 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.
[0345] In certain embodiments, oligonucleotides comprise one or
more nucleosides comprising a modified nucleobase. In certain
embodiments, oligonucleotides having a gapmer sugar motif comprise
a nucleoside comprising a modified nucleobase. In certain such
embodiments, one nucleoside comprising a modified nucleobases is in
the central gap of an oligonucleotide having a gapmer sugar motif.
In certain embodiments, the sugar is an unmodified
2'deoxynucleoside. In certain embodiments, the modified nucleobase
is selected from: a 2-thio pyrimidine and a 5-propyne pyrimidine.
In certain embodiments, one nucleoside comprising a modified
nucleobases is the 5.sup.th nucleobase from the 5'-end of the
oligonucleotide. In certain embodiments, the 5.sup.th nucleobase
from the 5'-end of the oligonucleotide is 2-thiothymine.
[0346] 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.
[0347] iii. Certain Nucleoside Motifs
[0348] In certain embodiments, oligonucleotides comprise
nucleosides comprising modified sugar moieties and/or nucleosides
comprising modified nucleobases. Such motifs can be described by
their sugar motif and their nucleobase motif separately or by their
nucleoside motif, which provides positions or patterns of modified
nucleosides (whether modified sugar, nucleobase, or both sugar and
nucleobase) in an oligonucleotide.
[0349] In certain embodiments, the oligonucleotides comprise or
consist of a region having a gapmer nucleoside motif, which
comprises two external regions or "wings" and a central or internal
region or "gap." The three regions of a gapmer nucleoside motif
(the 5'-wing, the gap, and the 3'-wing) form a contiguous sequence
of nucleosides wherein at least some of the sugar moieties and/or
nucleobases of the nucleosides of each of the wings differ from at
least some of the sugar moieties and/or nucleobase of the
nucleosides of the gap. Specifically, at least the nucleosides of
each wing that are closest to the gap (the 3'-most nucleoside of
the 5'-wing and the 5'-most nucleoside of the 3'-wing) differ from
the neighboring gap nucleosides, thus defining the boundary between
the wings and the gap. In certain embodiments, the nucleosides
within the gap are the same as one another. In certain embodiments,
the gap includes one or more nucleoside that differs from one or
more other nucleosides of the gap. In certain embodiments, the
nucleoside motifs of the two wings are the same as one another
(symmetric gapmer). In certain embodiments, the nucleoside motifs
of the 5'-wing differs from the nucleoside motif of the 3'-wing
(asymmetric gapmer).
[0350] iv. Certain 5'-Wings
[0351] In certain embodiments, the 5'-wing of a gapmer consists of
1 to 9 linked nucleosides. In certain embodiments, the 5'-wing of a
gapmer consists of 1 to 8 linked nucleosides. In certain
embodiments, the 5'-wing of a gapmer consists of 1 to 7 linked
nucleosides. In certain embodiments, the 5'-wing of a gapmer
consists of 1 to 6 linked nucleosides. In certain embodiments, the
5'-wing of a gapmer consists of 1 to 5 linked nucleosides. In
certain embodiments, the 5'-wing of a gapmer consists of 2 to 5
linked nucleosides. In certain embodiments, the 5'-wing of a gapmer
consists of 3 to 5 linked nucleosides. In certain embodiments, the
5'-wing of a gapmer consists of 4 or 5 linked nucleosides. In
certain embodiments, the 5'-wing of a gapmer consists of 1 to 4
linked nucleosides. In certain embodiments, the 5'-wing of a gapmer
consists of 1 to 3 linked nucleosides. In certain embodiments, the
5'-wing of a gapmer consists of 1 or 2 linked nucleosides. In
certain embodiments, the 5'-wing of a gapmer consists of 2 to 4
linked nucleosides. In certain embodiments, the 5'-wing of a gapmer
consists of 2 or 3 linked nucleosides. In certain embodiments, the
5'-wing of a gapmer consists of 3 or 4 linked nucleosides. In
certain embodiments, the 5'-wing of a gapmer consists of 1
nucleoside. In certain embodiments, the 5'-wing of a gapmer
consists of 2 linked nucleosides. In certain embodiments, the
5'-wing of a gapmer consists of 3 linked nucleosides. In certain
embodiments, the 5'-wing of a gapmer consists of 4 linked
nucleosides. In certain embodiments, the 5'-wing of a gapmer
consists of 5 linked nucleosides. In certain embodiments, the
5'-wing of a gapmer consists of 6 linked nucleosides. In certain
embodiments, the 5'-wing of a gapmer consists of 7 linked
nucleosides. In certain embodiments, the 5'-wing of a gapmer
consists of 8 linked nucleosides. In certain embodiments, the
5'-wing of a gapmer consists of 9 linked nucleosides.
[0352] In certain embodiments, the 5'-wing of a gapmer comprises at
least one bicyclic nucleoside. In certain embodiments, the 5'-wing
of a gapmer comprises at least two bicyclic nucleosides. In certain
embodiments, the 5'-wing of a gapmer comprises at least three
bicyclic nucleosides. In certain embodiments, the 5'-wing of a
gapmer comprises at least four bicyclic nucleosides. In certain
embodiments, the 5'-wing of a gapmer comprises at least one
constrained ethyl nucleoside. In certain embodiments, the 5'-wing
of a gapmer comprises at least one LNA nucleoside. In certain
embodiments, each nucleoside of the 5'-wing of a gapmer is a
bicyclic nucleoside. In certain embodiments, each nucleoside of the
5'-wing of a gapmer is a constrained ethyl nucleoside. In certain
embodiments, each nucleoside of the 5'-wing of a gapmer is a LNA
nucleoside.
[0353] In certain embodiments, the 5'-wing of a gapmer comprises at
least one non-bicyclic modified nucleoside. In certain embodiments,
the 5'-wing of a gapmer comprises at least one 2'-substituted
nucleoside. In certain embodiments, the 5'-wing of a gapmer
comprises at least one 2'-MOE nucleoside. In certain embodiments,
the 5'-wing of a gapmer comprises at least one 2'-OMe nucleoside.
In certain embodiments, each nucleoside of the 5'-wing of a gapmer
is a non-bicyclic modified nucleoside. In certain embodiments, each
nucleoside of the 5'-wing of a gapmer is a 2'-substituted
nucleoside. In certain embodiments, each nucleoside of the 5'-wing
of a gapmer is a 2'-MOE nucleoside. In certain embodiments, each
nucleoside of the 5'-wing of a gapmer is a 2'-OMe nucleoside.
[0354] In certain embodiments, the 5'-wing of a gapmer comprises at
least one 2'-deoxynucleoside. In certain embodiments, each
nucleoside of the 5'-wing of a gapmer is a 2'-deoxynucleoside. In a
certain embodiments, the 5'-wing of a gapmer comprises at least one
ribonucleoside. In certain embodiments, each nucleoside of the
5'-wing of a gapmer is a ribonucleoside. In certain embodiments,
one, more than one, or each of the nucleosides of the 5'-wing is an
RNA-like nucleoside.
[0355] In certain embodiments, the 5'-wing of a gapmer comprises at
least one bicyclic nucleoside and at least one non-bicyclic
modified nucleoside. In certain embodiments, the 5'-wing ofa gapmer
comprises at least one bicyclic nucleoside and at least one
2'-substituted nucleoside. In certain embodiments, the 5'-wing of a
gapmer comprises at least one bicyclic nucleoside and at least one
2'-MOE nucleoside. In certain embodiments, the 5'-wing of a gapmer
comprises at least one bicyclic nucleoside and at least one 2'-OMe
nucleoside. In certain embodiments, the 5'-wing of a gapmer
comprises at least one bicyclic nucleoside and at least one
2'-deoxynucleoside.
[0356] In certain embodiments, the 5'-wing of a gapmer comprises at
least one constrained ethyl nucleoside and at least one
non-bicyclic modified nucleoside. In certain embodiments, the
5'-wing of a gapmer comprises at least one constrained ethyl
nucleoside and at least one 2'-substituted nucleoside. In certain
embodiments, the 5'-wing of a gapmer comprises at least one
constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In
certain embodiments, the 5'-wing of a gapmer comprises at least one
constrained ethyl nucleoside and at least one 2'-OMe nucleoside. In
certain embodiments, the 5'-wing of a gapmer comprises at least one
constrained ethyl nucleoside and at least one
2'-deoxynucleoside.
[0357] In certain embodiments, the 5'-wing of a gapmer has a
nucleoside motif selected from among the following: eeeedk, eeeee,
eeeeedk, eeeeeeeek, eeeeeeek, eeeeek, eeeek, eeeekk, eeek, eeekdx,
eeekk, eek, eekk, ek, ekek, ekekdx, ekk, ekkdk, ekkkk, and k,
wherein each "e" is a 2'MOE modified nucleoside, each "k" is a cEt
modified nucleoside, each "dx" is a 2-thiothymidine, and each "d"
is an unmodified deoxynucleoside.
[0358] In certain embodiments, an oligonucleotide comprises any
5'-wing motif provided herein. In certain such embodiments, the
oligonucleotide is a 5'-hemimer (does not comprise a 3'-wing). In
certain embodiments, such an oligonucleotide is a gapmer. In
certain such embodiments, the 3'-wing of the gapmer may comprise
any nucleoside motif.
[0359] v. Certain 3'-Wings
[0360] In certain embodiments, the 3'-wing of a gapmer consists of
1 to 10 linked nucleosides. In certain embodiments, the 3'-wing of
a gapmer consists of 1 to 9 linked nucleosides. In certain
embodiments, the 3'-wing of a gapmer consists of 1 to 8 linked
nucleosides. In certain embodiments, the 3'-wing of a gapmer
consists of 1 to 7 linked nucleosides. In certain embodiments, the
3'-wing of a gapmer consists of 1 to 6 linked nucleosides. In
certain embodiments, the 3'-wing of a gapmer consists of 1 to 5
linked nucleosides.
[0361] In certain embodiments, the 3'-wing of a gapmer consists of
2 to 5 linked nucleosides. In certain embodiments, the 3'-wing of a
gapmer consists of 3 to 5 linked nucleosides. In certain
embodiments, the 3'-wing of a gapmer consists of 4 or 5 linked
nucleosides. In certain embodiments, the 3'-wing of a gapmer
consists of 1 to 4 linked nucleosides. In certain embodiments, the
3'-wing of a gapmer consists of 1 to 3 linked nucleosides. In
certain embodiments, the 3'-wing of a gapmer consists of 1 or 2
linked nucleosides. In certain embodiments, the 3'-wing of a gapmer
consists of 2 to 4 linked nucleosides. In certain embodiments, the
3'-wing of a gapmer consists of 2 or 3 linked nucleosides. In
certain embodiments, the 3'-wing of a gapmer consists of 3 or 4
linked nucleosides. In certain embodiments, the 3'-wing of a gapmer
consists of 1 nucleoside. In certain embodiments, the 3'-wing of a
gapmer consists of 2 linked nucleosides. In certain embodiments,
the 3'-wing of a gapmer consists of 3 linked nucleosides. In
certain embodiments, the 3'-wing of a gapmer consists of 4 linked
nucleosides. In certain embodiments, the 3'-wing of a gapmer
consists of 5 linked nucleosides. In certain embodiments, the
3'-wing of a gapmer consists of 6 linked nucleosides. In certain
embodiments, the 3'-wing of a gapmer consists of 7 linked
nucleosides. In certain embodiments, the 3'-wing of a gapmer
consists of 8 linked nucleosides. In certain embodiments, the
3'-wing of a gapmer consists of 9 linked nucleosides. In certain
embodiments, the 3'-wing of a gapmer consists of 10 linked
nucleosides.
[0362] In certain embodiments, the 3'-wing of a gapmer comprises at
least one bicyclic nucleoside. In certain embodiments, the 3'-wing
of a gapmer comprises at least one constrained ethyl nucleoside. In
certain embodiments, the 3'-wing of a gapmer comprises at least one
LNA nucleoside. In certain embodiments, each nucleoside of the
3'-wing of a gapmer is a bicyclic nucleoside. In certain
embodiments, each nucleoside of the 3'-wing of a gapmer is a
constrained ethyl nucleoside. In certain embodiments, each
nucleoside of the 3'-wing of a gapmer is a LNA nucleoside.
[0363] In certain embodiments, the 3'-wing of a gapmer comprises at
least one non-bicyclic modified nucleoside. In certain embodiments,
the 3'-wing of a gapmer comprises at least two non-bicyclic
modified nucleosides. In certain embodiments, the 3'-wing of a
gapmer comprises at least three non-bicyclic modified nucleosides.
In certain embodiments, the 3'-wing of a gapmer comprises at least
four non-bicyclic modified nucleosides. In certain embodiments, the
3'-wing of a gapmer comprises at least one 2'-substituted
nucleoside. In certain embodiments, the 3'-wing of a gapmer
comprises at least one 2'-MOE nucleoside. In certain embodiments,
the 3'-wing of a gapmer comprises at least one 2'-OMe nucleoside.
In certain embodiments, each nucleoside of the 3'-wing of a gapmer
is a non-bicyclic modified nucleoside. In certain embodiments, each
nucleoside of the 3'-wing of a gapmer is a 2'-substituted
nucleoside. In certain embodiments, each nucleoside of the 3'-wing
of a gapmer is a 2'-MOE nucleoside. In certain embodiments, each
nucleoside of the 3'-wing of a gapmer is a 2'-OMe nucleoside.
[0364] In certain embodiments, the 3'-wing of a gapmer comprises at
least one 2'-deoxynucleoside. In certain embodiments, each
nucleoside of the 3'-wing of a gapmer is a 2'-deoxynucleoside. In a
certain embodiments, the 3'-wing of a gapmer comprises at least one
ribonucleoside. In certain embodiments, each nucleoside of the
3'-wing of a gapmer is a ribonucleoside. In certain embodiments,
one, more than one, or each of the nucleosides of the 5'-wing is an
RNA-like nucleoside.
[0365] In certain embodiments, the 3'-wing of a gapmer comprises at
least one bicyclic nucleoside and at least one non-bicyclic
modified nucleoside. In certain embodiments, the 3'-wing ofa gapmer
comprises at least one bicyclic nucleoside and at least one
2'-substituted nucleoside. In certain embodiments, the 3'-wing of a
gapmer comprises at least one bicyclic nucleoside and at least one
2'-MOE nucleoside. In certain embodiments, the 3'-wing of a gapmer
comprises at least one bicyclic nucleoside and at least one 2'-OMe
nucleoside. In certain embodiments, the 3'-wing of a gapmer
comprises at least one bicyclic nucleoside and at least one
2'-deoxynucleoside.
[0366] In certain embodiments, the 3'-wing of a gapmer comprises at
least one constrained ethyl nucleoside and at least one
non-bicyclic modified nucleoside. In certain embodiments, the
3'-wing of a gapmer comprises at least one constrained ethyl
nucleoside and at least one 2'-substituted nucleoside. In certain
embodiments, the 3'-wing of a gapmer comprises at least one
constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In
certain embodiments, the 3'-wing of a gapmer comprises at least one
constrained ethyl nucleoside and at least one 2'-OMe nucleoside. In
certain embodiments, the 3'-wing of a gapmer comprises at least one
constrained ethyl nucleoside and at least one
2'-deoxynucleoside.
[0367] In certain embodiments, the 3'-wing of a gapmer comprises at
least one LNA nucleoside and at least one non-bicyclic modified
nucleoside. In certain embodiments, the 3'-wing of a gapmer
comprises at least one LNA nucleoside and at least one
2'-substituted nucleoside. In certain embodiments, the 3'-wing of a
gapmer comprises at least one LNA nucleoside and at least one
2'-MOE nucleoside. In certain embodiments, the 3'-wing of a gapmer
comprises at least one LNA nucleoside and at least one 2'-OMe
nucleoside. In certain embodiments, the 3'-wing of a gapmer
comprises at least one LNA nucleoside and at least one
2'-deoxynucleoside.
[0368] In certain embodiments, the 3'-wing of a gapmer comprises at
least one bicyclic nucleoside, at least one non-bicyclic modified
nucleoside, and at least one 2'-deoxynucleoside. In certain
embodiments, the 3'-wing of a gapmer comprises at least one
constrained ethyl nucleoside, at least one non-bicyclic modified
nucleoside, and at least one 2'-deoxynucleoside. In certain
embodiments, the 3'-wing of a gapmer comprises at least one LNA
nucleoside, at least one non-bicyclic modified nucleoside, and at
least one 2'-deoxynucleoside.
[0369] In certain embodiments, the 3'-wing of a gapmer comprises at
least one bicyclic nucleoside, at least one 2'-substituted
nucleoside, and at least one 2'-deoxynucleoside. In certain
embodiments, the 3'-wing of a gapmer comprises at least one
constrained ethyl nucleoside, at least one 2'-substituted
nucleoside, and at least one 2'-deoxynucleoside. In certain
embodiments, the 3'-wing of a gapmer comprises at least one LNA
nucleoside, at least one 2'-substituted nucleoside, and at least
one 2'-deoxynucleoside.
[0370] In certain embodiments, the 3'-wing of a gapmer comprises at
least one bicyclic nucleoside, at least one 2'-MOE nucleoside, and
at least one 2'-deoxynucleoside. In certain embodiments, the
3'-wing of a gapmer comprises at least one constrained ethyl
nucleoside, at least one 2'-MOE nucleoside, and at least one
2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer
comprises at least one LNA nucleoside, at least one 2'-MOE
nucleoside, and at least one 2'-deoxynucleoside.
[0371] In certain embodiments, the 3'-wing of a gapmer comprises at
least one bicyclic nucleoside, at least one 2'-OMe nucleoside, and
at least one 2'-deoxynucleoside. In certain embodiments, the
3'-wing of a gapmer comprises at least one constrained ethyl
nucleoside, at least one 2'-OMe nucleoside, and at least one
2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer
comprises at least one LNA nucleoside, at least one 2'-OMe
nucleoside, and at least one 2'-deoxynucleoside.
[0372] In certain embodiments, the 3'-wing of a gapmer has a
nucleoside motif selected from among the following: eee, eeee,
eeeee, eeeeee, eeeeeee, eeeeeeee, eeeeeeeee, eeeeeeeeee, eeeekek,
eeeekeke, eeek, eeeke, eeekek, eeekeke, eeekekee, eeekk, eeke,
eekek, eekeke, eekekee, eekk, kee, keee, keeee, keeeke, keeekee,
keek, keeke, keekee, keekeee, keekk, keke, kekee, kke, kkeee,
kkeek, and kkke, wherein each "e" is a 2'MOE modified nucleoside
and each "k" is a cEt modified nucleoside.
[0373] vi. Certain Central Regions (Gaps)
[0374] In certain embodiments, the gap of a gapmer consists of 6 to
20 linked nucleosides. In certain embodiments, the gap of a gapmer
consists of 6 to 15 linked nucleosides. In certain embodiments, the
gap of a gapmer consists of 6 to 12 linked nucleosides. In certain
embodiments, the gap of a gapmer consists of 6 to 10 linked
nucleosides. In certain embodiments, the gap of a gapmer consists
of 6 to 9 linked nucleosides. In certain embodiments, the gap of a
gapmer consists of 6 to 8 linked nucleosides. In certain
embodiments, the gap of a gapmer consists of 6 or 7 linked
nucleosides. In certain embodiments, the gap of a gapmer consists
of 7 to 10 linked nucleosides. In certain embodiments, the gap of a
gapmer consists of 7 to 9 linked nucleosides. In certain
embodiments, the gap of a gapmer consists of 7 or 8 linked
nucleosides. In certain embodiments, the gap of a gapmer consists
of 8 to 10 linked nucleosides. In certain embodiments, the gap of a
gapmer consists of 8 or 9 linked nucleosides. In certain
embodiments, the gap of a gapmer consists of 6 linked nucleosides.
In certain embodiments, the gap of a gapmer consists of 7 linked
nucleosides. In certain embodiments, the gap of a gapmer consists
of 8 linked nucleosides. In certain embodiments, the gap of a
gapmer consists of 9 linked nucleosides. In certain embodiments,
the gap of a gapmer consists of 10 linked nucleosides. In certain
embodiments, the gap of a gapmer consists of 11 linked nucleosides.
In certain embodiments, the gap of a gapmer consists of 12 linked
nucleosides.
[0375] In certain embodiments, each nucleoside of the gap of a
gapmer is a 2'-deoxynucleoside. In certain embodiments, the gap
comprises one or more modified nucleosides. In certain embodiments,
each nucleoside of the gap of a gapmer is a 2'-deoxynucleoside or
is a modified nucleoside that is "DNA-like." In such embodiments,
"DNA-like" means that the nucleoside has similar characteristics to
DNA, such that a duplex comprising the gapmer and an RNA molecule
is capable of activating RNase H. For example, under certain
conditions, 2'-(ara)-F have been shown to support RNase H
activation, and thus is DNA-like. In certain embodiments, one or
more nucleosides of the gap of a gapmer is not a 2'-deoxynucleoside
and is not DNA-like. In certain such embodiments, the gapmer
nonetheless supports RNase H activation (e.g., by virtue of the
number or placement of the non-DNA nucleosides).
[0376] In certain embodiments, gaps comprise a stretch of
unmodified 2'-deoxynucleoside interrupted by one or more modified
nucleosides, thus resulting in three sub-regions (two stretches of
one or more 2'-deoxynucleosides and a stretch of one or more
interrupting modified nucleosides). In certain embodiments, no
stretch of unmodified 2'-deoxynucleosides is longer than 5, 6, or 7
nucleosides. In certain embodiments, such short stretches is
achieved by using short gap regions. In certain embodiments, short
stretches are achieved by interrupting a longer gap region.
[0377] In certain embodiments, the gap comprises one or more
modified nucleosides. In certain embodiments, the gap comprises one
or more modified nucleosides selected from among cEt, FHNA, LNA,
and 2-thio-thymidine. In certain embodiments, the gap comprises one
modified nucleoside. In certain embodiments, the gap comprises a
5'-substituted sugar moiety selected from among 5'-Me, and
5'-(R)-Me. In certain embodiments, the gap comprises two modified
nucleosides. In certain embodiments, the gap comprises three
modified nucleosides. In certain embodiments, the gap comprises
four modified nucleosides. In certain embodiments, the gap
comprises two or more modified nucleosides and each modified
nucleoside is the same. In certain embodiments, the gap comprises
two or more modified nucleosides and each modified nucleoside is
different.
[0378] In certain embodiments, the gap comprises one or more
modified linkages. In certain embodiments, the gap comprises one or
more methyl phosphonate linkages. In certain embodiments the gap
comprises two or more modified linkages. In certain embodiments,
the gap comprises one or more modified linkages and one or more
modified nucleosides. In certain embodiments, the gap comprises one
modified linkage and one modified nucleoside. In certain
embodiments, the gap comprises two modified linkages and two or
more modified nucleosides.
[0379] In certain embodiments, the gap comprises a nucleoside motif
selected from among the following: DDDDDDD, DDDDDDDD, DDDDDDDDD,
DXDDDDD, DXDDDDDD, and DXDDDDDDD, wherein each D is an unmodified
deoxynucleoside and each X is a modified nucleoside.
[0380] In certain embodiments, each X comprises an unmodified
2'-deoxyfuranose sugar moiety. In certain embodiments, each X
comprises a modified sugar moiety. In certain embodiments, each X
comprises a 2'-substituted sugar moiety. In certain embodiments,
each X comprises a 2'-substituted sugar moiety selected from among
F, (ara)-F, OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain
embodiments, each X comprises a 5'-substituted sugar moiety. In
certain embodiments, each X comprises a 2-thio-thymidine
nucleoside. In certain embodiments, each X comprises a
5'-substituted sugar moiety selected from among 5'-Me, and
5'-(R)-Me. In certain embodiments, each X comprises a bicyclic
sugar moiety. In certain embodiments, each X comprises a bicyclic
sugar moiety selected from among cEt, cMOE, LNA, .alpha.-L-LNA, ENA
and 2'-thio LNA. In certain embodiments, each X comprises a
modified nucleobase. In certain embodiments, each X comprises a
modified nucleobase selected from among 2-thio-thymidine and
5-propyne uridine. In certain embodiments, each X comprises an HNA.
In certain embodiments, each C comprises an F-HNA. In certain
embodiments, X represents the location of a single differentiating
nucleobase.
[0381] vii. Certain Gapmer Motifs
[0382] In certain embodiments, a gapmer comprises a 5'-wing, a gap,
and a 3' wing, wherein the 5'-wing, gap, and 3' wing are
independently selected from among those discussed above. For
example, in certain embodiments, a gapmer has a 5'-wing, a gap, and
a 3'-wing having features selected from among any of those listed
in the tables above and any 5'-wing may be paired with any gap and
any 3'-wing.
[0383] In certain embodiments, a gapmer has a sugar motif other
than: E-K-K-(D).sub.9-K-K-E; E-E-E-E-K-(D).sub.9-E-E-E-E-E;
E-K-K-K-(D).sub.9-K--K-K-E; K-E-E-K-(D).sub.9-K-E-E-K;
K-D-D-K-(D).sub.9-K-D-D-K; K-E-K-E-K-(D).sub.9-K-E-K-E-K;
K-D-K-D-K-(D).sub.9-K-D-K-D-K; E-K-E-K-(D).sub.9-K-E-K-E;
E-E-E-E-E-K-(D).sub.8-E-E-E-E-E; or E-K-E-K-E-(D).sub.9-E-K-E-K-E,
E-E-E-K-K-(D).sub.7-E-E-K, E-K-E-K--K-K-(D).sub.7-K-E-K-E,
E-K-E-K-E-K-(D).sub.7-K-E-K-E, wherein K is a nucleoside comprising
a cEt sugar moiety and E is a nucleoside comprising a 2'-MOE sugar
moiety.
[0384] viii. Certain Internucleoside Linkage Motifs
[0385] In certain embodiments, oligonucleotides comprise modified
internucleoside linkages arranged along the oligonucleotide or
region thereof in a defined pattern or modified internucleoside
linkage motif. In certain embodiments, internucleoside linkages are
arranged in a gapped motif, as described above for nucleoside
motif. In such embodiments, the internucleoside linkages in each of
two wing regions are different from the internucleoside linkages in
the gap region. In certain embodiments the internucleoside linkages
in the wings are phosphodiester and the internucleoside linkages in
the gap are phosphorothioate. The nucleoside motif is independently
selected, so such oligonucleotides having a gapped internucleoside
linkage motif may or may not have a gapped nucleoside motif and if
it does have a gapped nucleoside motif, the wing and gap lengths
may or may not be the same.
[0386] 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.
[0387] 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.
[0388] In certain embodiments, the oligonucleotide comprises a
mixture of phosphorothioate internucleoside linkages and
phosphodiester internucleoside linkages. In certain embodiments, a
modified oligonucleotide has one or more phosphodiester
internucleoside linkages in the 5'-wing region or 3'-wing region.
In certain embodiments, a modified oligonucleotide has one or more
phosphodiester internucleoside linkages in the 5'-wing region and
each of the remaining internucleoside linkages comprise
phosphorothioate internucleoside linkages. In certain embodiments,
a modified oligonucleotide has one or more phosphodiester
internucleoside linkages in the 3'-wing region and each of the
remaining internucleoside linkages comprise phosphorothioate
internucleoside linkages. In certain embodiments, a modified
oligonucleotide has one or more phosphodiester internucleoside
linkages in the 5'-wing region and the 3'-wing region and each of
the remaining internucleoside linkages comprise phosphorothioate
internucleoside linkages.
[0389] The following non-limiting Table further illustrates certain
internucleoside linkage motifs:
TABLE-US-00001 TABLE 1 Certain Internucleoside Linkage Motifs
5'-wing region Central gap region 3'-wing region E.sub.s E.sub.o
E.sub.o E.sub.o E.sub.o K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s K.sub.s E.sub.s E E.sub.s E.sub.o E.sub.o
E.sub.o E.sub.o K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s E.sub.s E.sub.s E E.sub.s K.sub.o E.sub.o K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s K.sub.o E.sub.s K.sub.s E E.sub.s E.sub.o E.sub.o E.sub.o
E.sub.s D.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s E.sub.o E.sub.o E.sub.s E.sub.s E E.sub.s E.sub.s
E.sub.o E.sub.o E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s E.sub.o E.sub.o E.sub.o E.sub.s E.sub.s E
E.sub.s E.sub.o E.sub.o E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s E.sub.o E.sub.o E.sub.o
E.sub.s E.sub.s E E.sub.s E.sub.o E.sub.s K.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s E.sub.o E.sub.o
E.sub.o E.sub.o E.sub.s E.sub.s E E.sub.s E.sub.o K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s E.sub.s
E.sub.o E.sub.o E.sub.o E.sub.o E.sub.s E.sub.s E E.sub.s K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s
E.sub.s E.sub.o E.sub.o E.sub.o E.sub.o E.sub.o E.sub.s E.sub.s E
E.sub.s E.sub.s E.sub.o E.sub.o E.sub.o E.sub.o E.sub.s K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.o
E.sub.s E.sub.s E E.sub.s E.sub.s E.sub.o E.sub.o E.sub.o E.sub.o
E.sub.o E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s E.sub.s E.sub.s E E.sub.s E.sub.o K.sub.o K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
K.sub.o K.sub.o E.sub.s E.sub.s E E.sub.s K.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.o
K.sub.o K.sub.s E E.sub.s E.sub.o E.sub.o E.sub.s K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.s E.sub.o
E.sub.o E.sub.s E E.sub.s E.sub.s E.sub.o K.sub.o K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.o K.sub.o
E.sub.s E.sub.s E E.sub.s E.sub.o K.sub.o K.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.o K.sub.o
E.sub.s E.sub.s E E.sub.s K.sub.o E.sub.o K.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.o E.sub.o
K.sub.s E.sub.s E E.sub.s E.sub.o K.sub.o K.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.o K.sub.o
E.sub.s E.sub.s E E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s E.sub.s E.sub.o K.sub.s K
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s E.sub.s E.sub.o K.sub.o E.sub.s K.sub.s E K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
E.sub.s E.sub.s E.sub.o K.sub.o E.sub.s K.sub.s E K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
E.sub.s E.sub.o K.sub.o E.sub.s K.sub.s E.sub.s E K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s
E.sub.s E.sub.o K.sub.o E.sub.s K.sub.s E.sub.s E K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.s
E.sub.s E.sub.o E.sub.o K.sub.s E.sub.s E K.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.s
E.sub.o E.sub.o K.sub.s E.sub.s E.sub.s E E.sub.s E.sub.s K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
E.sub.o E.sub.s E.sub.s K E.sub.s E.sub.s K.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.o
E.sub.s K.sub.s E E.sub.s E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.o E.sub.o E.sub.s
K.sub.s E E.sub.s E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s E.sub.o E.sub.o K.sub.s
E.sub.s K E.sub.s E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s K.sub.o E.sub.s E.sub.s E E.sub.s
E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s K.sub.s E.sub.o E.sub.o E.sub.s K.sub.s E E.sub.s
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
E.sub.s E.sub.s E.sub.s E.sub.o K.sub.o E.sub.s K.sub.s E E.sub.s
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s K.sub.s E.sub.s E.sub.o E.sub.o K.sub.s E.sub.s E E.sub.s
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s E.sub.s E.sub.o E.sub.o K.sub.s E.sub.s K E.sub.s E.sub.s
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s E.sub.o E.sub.o K.sub.s E.sub.s K E.sub.s K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
E.sub.s E.sub.s E.sub.o K.sub.o E.sub.s K.sub.s E K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
E.sub.s E.sub.o K.sub.o E.sub.s K.sub.s E.sub.s E E.sub.s K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
E.sub.s E.sub.s E.sub.o K.sub.o E.sub.s K.sub.s E E.sub.s K.sub.s
K.sub.s D.sub.s X.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s K.sub.s K.sub.s E E.sub.s K.sub.o E.sub.o K.sub.s
D.sub.s X.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s K.sub.o E.sub.s K.sub.s E E.sub.s K.sub.s E.sub.s K.sub.s
D.sub.s X.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s K.sub.s E.sub.s E.sub.s E E.sub.s E.sub.s E.sub.s K.sub.s
D.sub.s X.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s K.sub.s E.sub.s E.sub.s E E.sub.s K.sub.s E.sub.s K.sub.s
D.sub.s X.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s K.sub.s E.sub.s K.sub.s E E.sub.s K.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s
E.sub.o K.sub.s K E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.s E.sub.o E.sub.s K
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s E.sub.s E.sub.o K.sub.s E.sub.s K K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
K.sub.s E.sub.o E.sub.s K.sub.s K E.sub.s E.sub.s K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.o
K.sub.o E.sub.s E.sub.s K E.sub.s E.sub.s K.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.s
E.sub.o E.sub.s K.sub.s E E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s E.sub.o
K.sub.o E.sub.s K.sub.s E E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s E.sub.s E.sub.o
K.sub.o E.sub.s K.sub.s E E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.s E.sub.o
E.sub.o K.sub.s E.sub.s E E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.o E.sub.o
K.sub.s E.sub.s K E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s E.sub.s E.sub.s E.sub.o E.sub.o K.sub.s
E.sub.s K E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s E.sub.o K.sub.o E.sub.s
K.sub.s E
wherein each "E" is a 2'MOE modified nucleoside, each "K" is a cEt
modified nucleoside, each "D" is an unmodified deoxynucleoside,
each "X" comprises a 2-thiothymidine, each "s" is a
phosphorothioate internucleoside linkage, and each "o" is a
phosphodiester internucleoside linkage.
[0390] In certain embodiments, oligonucleotides comprise one or
more methylphosponate linkages. In certain embodiments,
oligonucleotides having a gapmer nucleoside motif comprise a
linkage motif comprising all phosphorothioate linkages except for
one or two methylphosponate linkages. In certain embodiments, one
methylphosponate linkage is in the central gap of an
oligonucleotide having a gapmer nucleoside motif.
[0391] i. Certain Modification Motifs
[0392] Modification motifs define oligonucleotides by nucleoside
motif (sugar motif and nucleobase motif) and linkage motif. For
example, certain oligonucleotides have the following modification
motif:
[0393]
A.sub.sA.sub.sA.sub.sD.sub.sD.sub.sD.sub.sD.sub.s(.sup.ND).sub.sD.s-
ub.sD.sub.sD.sub.sD.sub.sB.sub.sB.sub.sB;
wherein each A is a modified nucleoside comprising a 2'-substituted
sugar moiety; each D is an unmodified 2'-deoxynucleoside; each B is
a modified nucleoside comprising a bicyclic sugar moiety; .sup.ND
is a modified nucleoside comprising a modified nucleobase; and s is
a phosphorothioate internucleoside linkage. Thus, the sugar motif
is a gapmer motif. The nucleobase modification motif is a single
modified nucleobase at 8.sup.th nucleoside from the 5'-end.
Combining the sugar motif and the nucleobase modification motif,
the nucleoside motif is an interrupted gapmer where the gap of the
sugar modified gapmer is interrupted by a nucleoside comprising a
modified nucleobase. The linkage motif is uniform
phosphorothioate.
[0394] The following non-limiting Table further illustrates certain
modification motifs:
TABLE-US-00002 TABLE 2 Certain Modification Motifs 5'-wing region
Central gap region 3'-wing region A.sub.s B.sub.s B.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
B.sub.s B.sub.s A A.sub.s A.sub.s A.sub.s A.sub.s B.sub.s B.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s B.sub.s
B.sub.s A A.sub.s A.sub.s A.sub.s B.sub.s B.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s B.sub.s B.sub.s A A.sub.s
B.sub.s B.sub.s D.sub.s X.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s B.sub.s B.sub.s A A.sub.s A.sub.s A.sub.s
A.sub.s A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s A.sub.s A.sub.s A A.sub.s A.sub.s A.sub.s A.sub.s
A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s B.sub.s B.sub.s A A.sub.s A.sub.s A.sub.s A.sub.s B.sub.s
B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
B.sub.s A.sub.s A A.sub.s A.sub.s A.sub.s A.sub.s A.sub.s B.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s B.sub.s
A.sub.s A A.sub.s A.sub.o A.sub.o A.sub.o A.sub.o B.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s B.sub.s A.sub.s A
A.sub.s A.sub.o A.sub.o A.sub.o A.sub.o B.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s A.sub.s A.sub.s A A.sub.s
B.sub.o A.sub.o B.sub.s D.sub.s X.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s B.sub.o A.sub.s B.sub.s A A.sub.s
A.sub.o A.sub.o A.sub.o A.sub.s D.sub.s B.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s A.sub.o A.sub.o A.sub.s
A.sub.s A A.sub.s A.sub.s A.sub.o A.sub.o A.sub.s B.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s A.sub.o A.sub.o
A.sub.o A.sub.s A.sub.s A A.sub.s A.sub.o A.sub.o A.sub.s B.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s A.sub.s
A.sub.o A.sub.o A.sub.o A.sub.s A.sub.s A A.sub.s A.sub.o A.sub.s
B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
A.sub.s A.sub.o A.sub.o A.sub.o A.sub.o A.sub.s A.sub.s A A.sub.s
A.sub.o B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s A.sub.s A.sub.s A.sub.o A.sub.o A.sub.o A.sub.o A.sub.s
A.sub.s A A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s A.sub.s A.sub.s A.sub.o A.sub.o A.sub.o A.sub.o
A.sub.o A.sub.s A.sub.s A A.sub.s A.sub.s A.sub.o A.sub.o A.sub.o
A.sub.o A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s A.sub.o A.sub.s A.sub.s A A.sub.s A.sub.s A.sub.o
A.sub.o A.sub.o A.sub.o A.sub.o A.sub.s B.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s A.sub.s A.sub.s A A.sub.s
A.sub.o B.sub.o B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s B.sub.o B.sub.o A.sub.s A.sub.s A A.sub.s
B.sub.s A.sub.s B.sub.s D.sub.s X.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s B.sub.s A.sub.s A.sub.s A A.sub.s
A.sub.s A.sub.s B.sub.s D.sub.s X.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s B.sub.s A.sub.s A.sub.s A A.sub.s
A.sub.s A.sub.s B.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s B.sub.s B.sub.s A A.sub.s B.sub.s A.sub.s
B.sub.s D.sub.s X.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s B.sub.s A.sub.s B.sub.s A A.sub.s A.sub.s A.sub.s
A.sub.s B.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s B.sub.s B.sub.s A A.sub.s B.sub.s B.sub.s D.sub.s
B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
B.sub.s B.sub.s A A.sub.s B.sub.s B.sub.s B.sub.s B.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s B.sub.s B.sub.s A
A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s B.sub.o B.sub.o B.sub.s A A.sub.s A.sub.s
A.sub.s A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s B.sub.s A.sub.s A.sub.s A A.sub.s A.sub.s A.sub.s
A.sub.s D.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s B.sub.s A.sub.s A.sub.s A A.sub.s A.sub.o A.sub.o
A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s B.sub.s A.sub.o A.sub.o A.sub.s A A.sub.s A.sub.s A.sub.o
B.sub.o B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s B.sub.o B.sub.o A.sub.s A.sub.s A A.sub.s A.sub.o B.sub.o
B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s B.sub.o B.sub.o A.sub.s A.sub.s A A.sub.s B.sub.o A.sub.o
B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s B.sub.o A.sub.o B.sub.s A.sub.s A A.sub.s A.sub.o B.sub.o
B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s B.sub.o B.sub.o A.sub.s A.sub.s A A.sub.s B.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s A.sub.s
A.sub.s A.sub.o B.sub.s B B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s A.sub.s A.sub.o B.sub.o
A.sub.s B.sub.s A B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s A.sub.s A.sub.s A.sub.o B.sub.o A.sub.s
B.sub.s A B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s A.sub.s A.sub.o B.sub.o A.sub.s B.sub.s
A.sub.s A B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s A.sub.s A.sub.s A.sub.o B.sub.o A.sub.s B.sub.s
A.sub.s A B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s B.sub.s A.sub.s A.sub.o A.sub.o B.sub.s A.sub.s A
B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s B.sub.s A.sub.o A.sub.o B.sub.s A.sub.s A.sub.s A
A.sub.s A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s A.sub.o A.sub.s A.sub.s B A.sub.s A.sub.s
B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s A.sub.o A.sub.s B.sub.s A A.sub.s A.sub.s B.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
A.sub.o A.sub.o A.sub.s B.sub.s A A.sub.s A.sub.s B.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s A.sub.s
A.sub.o A.sub.o B.sub.s A.sub.s B A.sub.s A.sub.s B.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s B.sub.o
A.sub.s A.sub.s A A.sub.s A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s B.sub.s A.sub.o A.sub.o
A.sub.s B.sub.s A A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s A.sub.s A.sub.s A.sub.s A.sub.o B.sub.o
A.sub.s B.sub.s A A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s B.sub.s A.sub.s A.sub.o A.sub.o
B.sub.s A.sub.s A A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s A.sub.s A.sub.o A.sub.o B.sub.s
A.sub.s B A.sub.s A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s A.sub.o A.sub.o B.sub.s
A.sub.s B A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s A.sub.s A.sub.s A.sub.o B.sub.o A.sub.s
B.sub.s A B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s A.sub.s A.sub.o B.sub.o A.sub.s B.sub.s
A.sub.s A A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s A.sub.s A.sub.s A.sub.o B.sub.o A.sub.s
B.sub.s A A.sub.s A.sub.s A.sub.s A.sub.s A.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
A.sub.s A.sub.s A.sub.s A.sub.s A A.sub.s A.sub.s A.sub.s A.sub.s
A.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s A.sub.s A.sub.s A.sub.s A.sub.s A A.sub.s
B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s A.sub.s A.sub.o B.sub.s B A.sub.s B.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
B.sub.s A.sub.o A.sub.s B B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s A.sub.s A.sub.o B.sub.s
A.sub.s B B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s B.sub.s A.sub.o A.sub.s B.sub.s B A.sub.s
A.sub.s B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s B.sub.o B.sub.o A.sub.s A.sub.s B A.sub.s A.sub.s
B.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s B.sub.s A.sub.o A.sub.s B.sub.s A A.sub.s B.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s A.sub.s A.sub.o B.sub.o A.sub.s B.sub.s A A.sub.s B.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
A.sub.s A.sub.s A.sub.o B.sub.o A.sub.s B.sub.s A A.sub.s B.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s B.sub.s A.sub.o A.sub.o B.sub.s A.sub.s A A.sub.s B.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s A.sub.o A.sub.o B.sub.s A.sub.s B A.sub.s B.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s A.sub.s A.sub.s
A.sub.o A.sub.o B.sub.s A.sub.s B A.sub.s B.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s A.sub.s
A.sub.o B.sub.o A.sub.s B.sub.s A
[0395] In certain embodiments, each A comprises a modified sugar
moiety. In certain embodiments, each A comprises a 2'-substituted
sugar moiety. In certain embodiments, each A comprises a
2'-substituted sugar moiety selected from among F, (ara)-F,
OCH.sub.3 and O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments,
each A comprises a bicyclic sugar moiety. In certain embodiments,
each A comprises a bicyclic sugar moiety selected from among cEt,
cMOE, LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain
embodiments, each A comprises a modified nucleobase. In certain
embodiments, each A comprises a modified nucleobase selected from
among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside.
In certain embodiments, each B comprises a modified sugar moiety.
In certain embodiments, each B comprises a 2'-substituted sugar
moiety. In certain embodiments, each B comprises a 2'-subsituted
sugar moiety selected from among F, (ara)-F, OCH.sub.3 and
O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each B
comprises a bicyclic sugar moiety. In certain embodiments, each B
comprises a bicyclic sugar moiety selected from among cEt, cMOE,
LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments,
each B comprises a modified nucleobase. In certain embodiments,
each B comprises a modified nucleobase selected from among
2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. In
certain embodiments, each A comprises an HNA. In certain
embodiments, each A comprises an F-HNA. In certain embodiments,
each "X" comprises a 2-thiothymidine, each "s" comprises a
phosphorothioate internucleoside linkage, and each "o" comprises a
phosphodiester internucleoside linkage.
[0396] In certain embodiments, B comprises a bicyclic sugar moiety,
and A comprises a 2'-MOE sugar moiety. In certain embodiments, B is
an LNA nucleoside and A comprises a 2'-MOE sugar moiety. In certain
embodiments, B is a cEt nucleoside and A comprises a 2'-MOE sugar
moiety. In certain embodiments, B is an .alpha.-L-LNA nucleoside
and A comprises a 2'-MOE sugar moiety. In certain embodiments, B is
an LNA nucleoside and A comprises a 2'-OMe sugar moiety. In certain
embodiments, B is a cEt nucleoside and A comprises a 2'-OMe sugar
moiety. In certain embodiments, B is an .alpha.-L-LNA nucleoside
and A comprises a 2'-OMe sugar moiety.
[0397] In certain embodiments, A comprises a bicyclic sugar moiety,
and B comprises a 2'-substituted sugar moiety. In certain
embodiments, A is an LNA nucleoside and B comprises a
2'-substituted sugar moiety. In certain embodiments, A is a cEt
nucleoside and B comprises a 2'-substituted sugar moiety. In
certain embodiments, A is an .alpha.-L-LNA nucleoside and B
comprises a 2'-substituted sugar moiety. In certain embodiments, A
is a cEt nucleoside and B comprises a 2'-MOE sugar moiety.
[0398] The following non-limiting Table further illustrates certain
modification motifs:
TABLE-US-00003 TABLE 3 Certain Modification Motifs 5'-wing region
Central gap region 3'-wing region E.sub.s K.sub.s K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
K.sub.s K.sub.s E E.sub.s E.sub.s E.sub.s E.sub.s K.sub.s K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.s
K.sub.s E E.sub.s E.sub.s E.sub.s K.sub.s K.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.s K.sub.s E E.sub.s
K.sub.s K.sub.s D.sub.s X.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s K.sub.s K.sub.s E E.sub.s E.sub.s E.sub.s
E.sub.s E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s E.sub.s E.sub.s E E.sub.s E.sub.s E.sub.s E.sub.s
E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s K.sub.s K.sub.s E E.sub.s E.sub.s E.sub.s E.sub.s K.sub.s
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
K.sub.s K.sub.s E E.sub.s E.sub.s E.sub.s E.sub.s E.sub.s K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.s
K.sub.s E E.sub.s E.sub.o E.sub.o E.sub.o E.sub.o K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.s K.sub.s E
E.sub.s E.sub.o E.sub.o E.sub.o E.sub.o K.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s E.sub.s E E.sub.s
K.sub.o E.sub.o K.sub.s D.sub.s X.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s K.sub.o E.sub.s K.sub.s E E.sub.s
E.sub.o E.sub.o E.sub.o E.sub.s D.sub.s K.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.o E.sub.o E.sub.s
E.sub.s E E.sub.s E.sub.s E.sub.o E.sub.o E.sub.s K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.o E.sub.o
E.sub.o E.sub.s E.sub.s E E.sub.s E.sub.o E.sub.o E.sub.s K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s
E.sub.o E.sub.o E.sub.o E.sub.s E.sub.s E E.sub.s E.sub.o E.sub.s
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
E.sub.s E.sub.o E.sub.o E.sub.o E.sub.o E.sub.s E.sub.s E E.sub.s
E.sub.o K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s E.sub.s E.sub.s E.sub.o E.sub.o E.sub.o E.sub.o E.sub.s
E.sub.s E E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s E.sub.s E.sub.s E.sub.o E.sub.o E.sub.o E.sub.o
E.sub.o E.sub.s E.sub.s E E.sub.s E.sub.s E.sub.o E.sub.o E.sub.o
E.sub.o E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s E.sub.o E.sub.s E.sub.s E E.sub.s E.sub.s E.sub.o
E.sub.o E.sub.o E.sub.o E.sub.o E.sub.s K.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s E.sub.s E E.sub.s
E.sub.o K.sub.o K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s K.sub.o K.sub.o E.sub.s E.sub.s E E.sub.s
K.sub.s E.sub.s K.sub.s D.sub.s xD.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s K.sub.s E.sub.s E.sub.s E E.sub.s
E.sub.s E.sub.s K.sub.s D.sub.s xD.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s K.sub.s E.sub.s E.sub.s E E.sub.s
E.sub.s E.sub.s K.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s K.sub.s K.sub.s E E.sub.s K.sub.s E.sub.s
K.sub.s D.sub.s xD.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s K.sub.s E.sub.s K.sub.s E E.sub.s E.sub.s E.sub.s
E.sub.s K.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s K.sub.s K.sub.s E E.sub.s K.sub.s K.sub.s D.sub.s
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
K.sub.s K.sub.s E E.sub.s K.sub.s K.sub.s K.sub.s K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.s K.sub.s E
E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s K.sub.o K.sub.o K.sub.s E E.sub.s E.sub.s
E.sub.s E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s K.sub.s E.sub.s E.sub.s E E.sub.s E.sub.s E.sub.s
E.sub.s D.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s K.sub.s E.sub.s E.sub.s E E.sub.s E.sub.o E.sub.o
E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s K.sub.s E.sub.o E.sub.o E.sub.s E E.sub.s E.sub.s E.sub.o
K.sub.o K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s K.sub.o K.sub.o E.sub.s E.sub.s E E.sub.s E.sub.o K.sub.o
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s K.sub.o K.sub.o E.sub.s E.sub.s E E.sub.s K.sub.o E.sub.o
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s K.sub.o E.sub.o K.sub.s E.sub.s E E.sub.s E.sub.o K.sub.o
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s K.sub.o K.sub.o E.sub.s E.sub.s E E.sub.s K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s
E.sub.s E.sub.o K.sub.s K K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s E.sub.o K.sub.o
E.sub.s K.sub.s E K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s E.sub.s E.sub.s E.sub.o K.sub.o E.sub.s
K.sub.s E K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s E.sub.s E.sub.o K.sub.o E.sub.s K.sub.s
E.sub.s E K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s E.sub.s E.sub.s E.sub.o K.sub.o E.sub.s K.sub.s
E.sub.s E K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s K.sub.s E.sub.s E.sub.o E.sub.o K.sub.s E.sub.s E
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s K.sub.s E.sub.o E.sub.o K.sub.s E.sub.s E.sub.s E
E.sub.s E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s E.sub.o E.sub.s E.sub.s K E.sub.s E.sub.s
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s E.sub.o E.sub.s K.sub.s E E.sub.s E.sub.s K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
E.sub.o E.sub.o E.sub.s K.sub.s E E.sub.s E.sub.s K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s
E.sub.o E.sub.o K.sub.s E.sub.s K E.sub.s E.sub.s K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.o
E.sub.s E.sub.s E E.sub.s E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s K.sub.s E.sub.o E.sub.o
E.sub.s K.sub.s E E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s E.sub.s E.sub.s E.sub.s E.sub.o K.sub.o
E.sub.s K.sub.s E E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s K.sub.s E.sub.s E.sub.o E.sub.o
K.sub.s E.sub.s E E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s E.sub.o E.sub.o K.sub.s
E.sub.s K E.sub.s E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.o E.sub.o K.sub.s
E.sub.s K E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s E.sub.s E.sub.s E.sub.o K.sub.o E.sub.s
K.sub.s E K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s E.sub.s E.sub.o K.sub.o E.sub.s K.sub.s
E.sub.s E E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s E.sub.s E.sub.s E.sub.o K.sub.o E.sub.s
K.sub.s E E.sub.s E.sub.s E.sub.s E.sub.s E.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
E.sub.s E.sub.s E.sub.s E.sub.s E E.sub.s E.sub.s E.sub.s E.sub.s
E.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s E.sub.s E.sub.s E.sub.s E.sub.s E E.sub.s
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s E.sub.s E.sub.o K.sub.s K E.sub.s K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
K.sub.s E.sub.o E.sub.s K K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s E.sub.o K.sub.s
E.sub.s K K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s K.sub.s E.sub.o E.sub.s K.sub.s K E.sub.s
E.sub.s K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s K.sub.o K.sub.o E.sub.s E.sub.s K E.sub.s E.sub.s
K.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s K.sub.s E.sub.o E.sub.s K.sub.s E E.sub.s K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s E.sub.s E.sub.o K.sub.o E.sub.s K.sub.s E E.sub.s K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
E.sub.s E.sub.s E.sub.o K.sub.o E.sub.s K.sub.s E E.sub.s K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s K.sub.s E.sub.o E.sub.o K.sub.s E.sub.s E E.sub.s K.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s
D.sub.s E.sub.o E.sub.o K.sub.s E.sub.s K E.sub.s K.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s E.sub.s
E.sub.o E.sub.o K.sub.s E.sub.s K E.sub.s K.sub.s D.sub.s D.sub.s
D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s D.sub.s E.sub.s
E.sub.o K.sub.o E.sub.s K.sub.s E
wherein each "E" is a 2'MOE modified nucleoside, each "K" is a cEt
modified nucleoside, each "D" is an unmodified deoxynucleoside,
each "X" comprises a 2-thiothymidine, each "s" is a
phosphorothioate internucleoside linkage, and each "o" is a
phosphodiester internucleoside linkage.
[0399] d. Certain Overall Lengths
[0400] In certain embodiments, the present invention provides
oligomeric compounds including oligonucleotides of any of a variety
of ranges of lengths. In certain embodiments, the invention
provides oligomeric compounds or oligonucleotides consisting of X
to Y linked nucleosides, where X represents the fewest number of
nucleosides in the range and Y represents the largest number of
nucleosides in the range. In certain such embodiments, X and Y are
each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and
50; provided that X.ltoreq.Y. For example, in certain embodiments,
the invention provides oligomeric compounds which comprise
oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8
to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to
20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27,
8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to
14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21,
9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to
29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10
to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22,
10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to
29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11
to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23,
11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to 28, 11 to 29, 11 to
30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12
to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25,
12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to
15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13
to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28,
13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to
19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14
to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17,
15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to
24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16
to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23,
16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to
30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17
to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30,
18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to
25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19
to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29,
19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to
24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21
to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28,
21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to
27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23
to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27,
24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to
29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27
to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked
nucleosides. In embodiments where the number of nucleosides of an
oligomeric compound or oligonucleotide is limited, whether to a
range or to a specific number, the oligomeric compound or
oligonucleotide may, nonetheless further comprise additional other
substituents. For example, an oligonucleotide comprising 8-30
nucleosides excludes oligonucleotides having 31 nucleosides, but,
unless otherwise indicated, such an oligonucleotide may further
comprise, for example one or more conjugates, terminal groups, or
other substituents. In certain embodiments, a gapmer
oligonucleotide has any of the above lengths.
[0401] 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.
[0402] e. Certain Oligonucleotides
[0403] In certain embodiments, oligonucleotides of the present
invention are characterized by their modification motif and overall
length. In certain embodiments, such parameters are each
independent of one another. Thus, unless otherwise indicated, each
internucleoside linkage of an oligonucleotide having a gapmer sugar
motif may be modified or unmodified and may or may not follow the
gapmer modification pattern of the sugar modifications. For
example, the internucleoside linkages within the wing regions of a
sugar-gapmer may be the same or different from one another and may
be the same or different from the internucleoside linkages of the
gap region. Likewise, such sugar-gapmer oligonucleotides may
comprise one or more modified nucleobase independent of the gapmer
pattern of the sugar modifications. One of skill in the art will
appreciate that such motifs may be combined to create a variety of
oligonucleotides. Herein if a description of an oligonucleotide or
oligomeric compound is silent with respect to one or more
parameter, such parameter is not limited. Thus, an oligomeric
compound described only as having a gapmer sugar motif without
further description may have any length, internucleoside linkage
motif, and nucleobase modification motif. Unless otherwise
indicated, all chemical modifications are independent of nucleobase
sequence.
[0404] f. Certain Conjugate Groups
[0405] 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).
[0406] 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.
[0407] 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.
[0408] Some nonlimiting examples of conjugate linking moieties
include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO),
succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC)
and 6-aminohexanoic acid (AHEX or AHA). Other linking groups
include, but are not limited to, substituted C.sub.1-C.sub.10
alkyl, substituted or unsubstituted C.sub.2-C.sub.10 alkenyl or
substituted or unsubstituted C.sub.2-C.sub.10 alkynyl, wherein a
nonlimiting list of preferred substituent groups includes hydroxyl,
amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy,
halogen, alkyl, aryl, alkenyl and alkynyl.
[0409] Conjugate groups may be attached to either or both ends of
an oligonucleotide (terminal conjugate groups) and/or at any
internal position.
[0410] In certain embodiments, conjugate groups are at the 3'-end
of an oligonucleotide of an oligomeric compound. In certain
embodiments, conjugate groups are near the 3'-end. In certain
embodiments, conjugates are attached at the 3'end of an oligomeric
compound, but before one or more terminal group nucleosides. In
certain embodiments, conjugate groups are placed within a terminal
group. In certain embodiments, the present invention provides
oligomeric compounds. In certain embodiments, oligomeric compounds
comprise an oligonucleotide. In certain embodiments, an oligomeric
compound comprises an oligonucleotide and one or more conjugate
and/or terminal groups. Such conjugate and/or terminal groups may
be added to oligonucleotides having any of the motifs discussed
above. Thus, for example, an oligomeric compound comprising an
oligonucleotide having region of alternating nucleosides may
comprise a terminal group.
C. Antisense Compounds
[0411] In certain embodiments, oligomeric compounds provided herein
are antisense compounds. Such antisense compounds are capable of
hybridizing to a target nucleic acid, resulting in at least one
antisense activity. In certain embodiments, antisense compounds
specifically hybridize to one or more target nucleic acid. In
certain embodiments, a specifically hybridizing antisense compound
has a nucleobase sequence comprising a region having sufficient
complementarity to a target nucleic acid to allow hybridization and
result in antisense activity and insufficient complementarity to
any non-target so as to avoid non-specific hybridization to any
non-target nucleic acid sequences under conditions in which
specific hybridization is desired (e.g., under physiological
conditions for in vivo or therapeutic uses, and under conditions in
which assays are performed in the case of in vitro assays).
[0412] 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.
[0413] 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.
[0414] a. Certain Antisense Activities and Mechanisms
[0415] In certain antisense activities, hybridization of an
antisense compound results in recruitment of a protein that cleaves
of the target nucleic acid. For example, certain antisense
compounds result in RNase H mediated cleavage of target nucleic
acid. RNase H is a cellular endonuclease that cleaves the RNA
strand of an RNA:DNA duplex. The "DNA" in such an RNA:DNA duplex,
need not be unmodified DNA. In certain embodiments, the invention
provides antisense compounds that are sufficiently "DNA-like" to
elicit RNase H activity. Such DNA-like antisense compounds include,
but are not limited to gapmers having unmodified deoxyfuronose
sugar moieties in the nucleosides of the gap and modified sugar
moieties in the nucleosides of the wings.
[0416] Antisense activities may be observed directly or indirectly.
In certain embodiments, observation or detection of an antisense
activity involves observation or detection of a change in an amount
of a target nucleic acid or protein encoded by such target nucleic
acid; a change in the ratio of splice variants of a nucleic acid or
protein; and/or a phenotypic change in a cell or animal.
[0417] In certain embodiments, compounds comprising
oligonucleotides having a gapmer nucleoside motif described herein
have desirable properties compared to non-gapmer oligonucleotides
or to gapmers having other motifs. In certain circumstances, it is
desirable to identify motifs resulting in a favorable combination
of potent antisense activity and relatively low toxicity. In
certain embodiments, compounds of the present invention have a
favorable therapeutic index (measure of activity divided by measure
of toxicity).
[0418] b. Certain Selective Antisense Compounds
[0419] In certain embodiments, antisense compounds provided are
selective for a target relative to a non-target nucleic acid. In
certain embodiments, antisense compounds provided are selective for
a target relative to one or more non-target nucleic acids. In
certain embodiments, the nucleobase sequences of the target and
non-target nucleic acids differ by no more than 4 differentiating
nucleobases in the targeted region. In certain embodiments, the
nucleobase sequences of the target and non-target nucleic acids
differ by no more than 3 differentiating nucleobases in the
targeted region. In certain embodiments, the nucleobase sequences
of the target and non-target nucleic acids differ by no more than 2
differentiating nucleobases in the targeted region. In certain
embodiments, the nucleobase sequences of the target and non-target
nucleic acids differ by a single differentiating nucleobase in the
targeted region. In certain embodiments, the nucleobase sequences
of the target and non-target nucleic acids are identical in the
targeted region.
[0420] In certain embodiments, the target and non-target nucleic
acids are transcripts from different genes. In certain embodiments,
the target nucleic acid is a transcript from the huntingtin gene
and the non-target nucleic acid is a transcript from a different
gene. In certain embodiments, the non-target nucleic acid is a
transcript from a gene encoding bone morphogenetic protein
receptor, type IA and the target nucleic acid is a transcript from
a different gene. In certain embodiments, the target nucleic acid
is a transcript from the huntingtin gene and the non-target nucleic
acid is a transcript from a gene encoding bone morphogenetic
protein receptor, type IA. In certain embodiments, the target and
non-target nucleic acids are different alleles for the same gene.
In certain embodiments, the introduction of a mismatch between an
antisense compound and a non-target nucleic acid may alter the
RNase H cleavage site of a target nucleic acid compared to a
non-target nucleic acid. In certain embodiments, the target and
non-target nucleic acids are not functionally related to one
another (e.g., are transcripts from different genes). In certain
embodiments, the target and non-target nucleic acids are allelic
variants of one another. In certain embodiments, the allelic
variant contains a single nucleotide polymorphism (SNP). In certain
embodiments, a SNP is associated with a mutant allele. In certain
embodiments, a mutant SNP is associated with a disease. In certain
embodiments a mutant SNP is associated with a disease, but is not
causative of the disease. In certain embodiments, mRNA and protein
expression of a mutant allele is associated with disease.
[0421] Selectivity of antisense compounds is achieved, principally,
by nucleobase complementarity. For example, if an antisense
compound has no mismatches for a target nucleic acid and one or
more mismatches for a non-target nucleic acid, some amount of
selectivity for the target nucleic acid will result. In certain
embodiments, provided herein are antisense compounds with enhanced
selectivity (e.g. the ratio of activity for the target to the
activity for non-target is greater). For example, in certain
embodiments, a selective nucleoside comprises a particular feature
or combination of features (e.g., chemical modification, motif,
placement of selective nucleoside, and/or self-complementary
region) that increases selectivity of an antisense compound
compared to an antisense compound not having that feature or
combination of features. In certain embodiments, such feature or
combination of features increases antisense activity for the
target. In certain embodiments, such feature or combination of
features decreases activity for the target, but decreases activity
for the non-target by a greater amount, thus resulting in an
increase in selectivity.
[0422] Without being limited by mechanism, enhanced selectivity may
result from a larger difference in the affinity of an antisense
compound for its target compared to its affinity for the non-target
and/or a larger difference in RNase H activity for the resulting
duplexes. For example, in certain embodiments, a selective
antisense compound comprises a modified nucleoside at that same
position as a differentiating nucleobase (i.e., the selective
nucleoside is modified). That modification may increase the
difference in binding affinity of the antisense compound for the
target relative to the non-target. In addition or in the
alternative, the chemical modification may increase the difference
in RNAse H activity for the duplex formed by the antisense compound
and its target compared to the RNase activity for the duplex formed
by the antisense compound and the non-target. For example, the
modification may exaggerate a structure that is less compatible for
RNase H to bind, cleave and/or release the non-target.
[0423] In certain embodiments, an antisense compound binds its
intended target to form a target duplex. In certain embodiments,
RNase H cleaves the target nucleic acid of the target duplex. In
certain such embodiments, there is a primary cleavage site between
two particular nucleosides of the target nucleic acid (the primary
target cleavage site), which accounts for the largest amount of
cleavage of the target nucleic acid. In certain nembodiments, there
are one or more secondary target cleavage sites. In certain
embodiments, the same antisence compound hybridizes to a non-target
to form a non-target duplex. In certain such embodiments, the
non-target differs from the target by a single nucleobase within
the target region, and so the antisense compound hybridizes with a
single mismatch. Because of the mismatch, in certain embodiments,
RNase H cleavage of the non-target may be reduced compared to
cleavage of the target, but still occurs. In certain embodiments,
though, the primary site of that cleavage of the non-target nucleic
acid (primary non-target cleavage site) is different from that of
the target. That is, the primary site is shifted due to the
mismatch. In such a circumstance, one may use a modification placed
in the antisense compound to disrupt RNase H cleavage at the
primary non-target cleavage site. Such modification will result in
reduced cleavage of the non-target, but will result little or no
decrease in cleavage of the target. In certain embodiments, the
modification is a modified sugar, nucleobase and/or linkage.
[0424] In certain embodiments, the primary non-target cleavage site
is towards the 5'-end of the antisense compound, and the 5'-end of
an antisense compound may be modified to prevent RNaseH cleavage.
In this manner, it is thought that one having skill in the art may
modify the 5'-end of an antisense compound, or modify the
nucleosides in the gap region of the 5'-end of the antisense
compound, or modify the the 3'-most 5'-region nucleosides of the
antisense compound to selectively inhibit RNaseH cleavage of the
non-target nucleic acid duplex while retaining RNase H cleavage of
the target nucleic acid duplex. In certain embodiments, 1-3 of the
3'-most 5'-region nucleosides of the antisense compound comprises a
bicyclic sugar moiety.
[0425] For example, in certain embodiments the target nucleic acid
may have an allelic variant, e.g. a non-target nucleic acid,
containing a single nucleotide polymorphism. An antisense compound
may be designed having a single nucleobase mismatch from the
non-target nucleic acid, but which has full complementarity to the
target nucleic acid. The mismatch between the antisense compound
and the non-target nucleic acid may destabilize the antisense
compound non-target nucleic acid duplex, and consequently the
cleavage site of RNaseH may shift upstream towards the 5'-end of
the antisense compound. Modification of the 5'-end of the antisense
compound or the gap region near the 5'-end of the antisense
compound, or one or more of the 3'-most nucleosides of the 5'-wing
region, will then prevent RNaseH cleavage of the non-target nucleic
acid. Since the target nucleic acid is fully complementary to the
antisense compound, the antisense compound and the target nucleic
acid will form a more stabilized antisense compound-target nucleic
acid duplex and the cleavage site of RnaseH will be more
downstream, towards the 3' end of the antisense compound.
Accordingly, modifications at the 5'-end of the antisense compound
will prevent RNaseH cleavage of the non-target nucleic acid, but
will not substantially effect RNaseH cleavage of the target nucleic
acid, and selectivity between a target nucleic acid and its allelic
variant may be achieved. In certain embodiments, one or more of the
3'-most nucleosides of the 5'-wing region comprises a bicyclic
sugar moiety. In certain embodiments, one or more of the 3'-most
nucleosides of the 5'-wing region comprises a bicyclic sugar moiety
selected from cEt and LNA. In certain embodiments, one or more of
the 3'-most nucleosides of the 5'-wing region comprises cEt. In
certain embodiments, one or more of the 3'-most nucleosides of the
5'-wing region comprises LNA.
[0426] In certain embodiments, the introduction of a mismatch
between an antisense compound and a target nucleic acid may alter
the RNase H cleavage site of a target nucleic acid compared to a
non-target nucleic acid by shifting the RNaseH cleavage site
downstream from the mismatch site and towards the 3'-end of the
antisense compound. In certain embodiments where the cleavage site
of a target nucleic acid compared to a non-target nucleic acid has
shifted downstream towards the 3'-end of the antisense compound,
the 3'-end of an antisense compound may be modified to prevent
RNaseH cleavage. In this manner, it is thought that one having
skill in the art may modify the 3'-end of an antisense compound, or
modify the nucleosides in the gap region near the 3'-end of
antisense compound, to selectively inhibit RNaseH cleavage of the
non-target nucleic acid while retaining RNase H cleavage of the
target nucleic acid.
[0427] For example, in certain embodiments the target nucleic acid
may have an allelic variant, e.g. a non-target nucleic acid,
containing a single nucleotide polymorphism. An antisense compound
may be designed having a single nucleobase mismatch from the
non-target nucleic acid, but which has full complementarity to
target nucleic acid. The mismatch between the antisense compound
and the non-target nucleic acid may destabilize the antisense
compound-non-target nucleic acid duplex, and consequently the
cleavage site of RNaseH may shift downstream towards the 3'-end of
the antisense compound. Modification of the 3'-end of the antisense
compound, or one or more of the the 5'-most nucleosides of the
3'-wing region, or the gap region of the antisense compound near
the 3'-end will then prevent RNaseH cleavage of the non-target
nucleic acid. Since the target nucleic acid is fully complementary
to the antisense compound, the antisense compound and the target
nucleic acid will form a more stabilized antisense compound-target
nucleic acid duplex and the cleavage site of RnaseH will be more
upstream, towards the 5' end of the antisense compound.
Accordingly, modifications at the 3'-end of the antisense compound
will prevent RNaseH cleavage of the non-target nucleic acid, but
will not substantially effect RNaseH cleavage of the target nucleic
acid, and selectivity between a target nucleic acid and its allelic
variant may be achieved. In certain embodiments, one or more of the
5'-most nucleosides of the 3'-wing region comprises a bicyclic
sugar moiety. In certain embodiments, one or more of the 5'-most
nucleosides of the 3'-wing region comprises a bicyclic sugar moiety
selected from cEt and LNA. In certain embodiments, one or more of
the 5'-most nucleosides of the 3'-wing region comprises cEt. In
certain embodiments, one or more of the 5'-most nucleosides of the
3'-wing region comprises LNA.
[0428] In certain embodiments, the selectivity of antisense
compounds having certain gaps, e.g. gaps of 7 nucleosides or
longer, may be improved by the addition of one or more bicyclic
nucleosides at the 3'-most 5'-wing nucleoside. In certain
embodiments, the selectivity of antisense compounds having certain
gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the
addition of two or more bicyclic nucleosides at the 3'-most 5'-wing
nucleoside. In certain embodiments, the selectivity of antisense
compounds having certain gaps, e.g. gaps of 7 nucleosides or
longer, may be improved by the addition of one bicyclic nucleoside
at the 3'-most 5'-wing nucleoside. In certain embodiments, the
selectivity of antisense compounds having certain gaps, e.g. gaps
of 7 nucleosides or longer, may be improved by the addition of two
bicyclic nucleosides at the 3'-most 5'-wing nucleoside. In certain
embodiments, the selectivity of antisense compounds having certain
gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the
addition of three bicyclic nucleosides at the 3'-most 5'-wing
nucleoside. In certain embodiments, the selectivity of antisense
compounds having certain gaps, e.g. gaps of 7 nucleosides or
longer, may be improved by the addition of four bicyclic
nucleosides at the 3'-most 5'-wing nucleoside. In certain
embodiments, the selectivity of antisense compounds having certain
gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the
addition of five bicyclic nucleosides at the 3'-most 5'-wing
nucleoside. In certain embodiments discussed above, the bicyclic
nucleosides at the 3'-most 5'-wing nucleoside are selected from
among cEt, cMOE, LNA, .alpha.-LNA, ENA and 2'-thio LNA. In certain
embodiments discussed above, the bicyclic nucleosides at the
3'-most 5'-wing nucleoside comprise cEt. In certain embodiments
discussed above, the bicyclic nucleosides at the 3'-most 5'-wing
nucleoside comprise LNA.
[0429] In certain embodiments, the selectivity of antisense
compounds having certain gaps, e.g. gaps of 7 nucleosides or
longer, may be improved by the addition of one or more bicyclic
nucleosides at the 3'-most 5'-wing nucleoside and the addition of
one or more bicylic nucleosides at the 5'-most 3'-wing nucleoside.
In certain embodiments, the selectivity of antisense compounds
having certain gaps, e.g. gaps of 7 nucleosides or longer, may be
improved by the addition of two or more bicyclic nucleosides at the
3'-most 5'-wing nucleoside and the addition of one or more bicylic
nucleosides at the 5'-most 3'-wing nucleoside. In certain
embodiments, the selectivity of antisense compounds having certain
gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the
addition of one bicyclic nucleoside at the 3'-most 5'-wing
nucleoside and the addition of one or more bicylic nucleosides at
the 5'-most 3'-wing nucleoside. In certain embodiments, the
selectivity of antisense compounds having certain gaps, e.g. gaps
of 7 nucleosides or longer, may be improved by the addition of two
bicyclic nucleosides at the 3'-most 5'-wing nucleoside and the
addition of one or more bicylic nucleosides at the 5'-most 3'-wing
nucleoside. In certain embodiments, the selectivity of antisense
compounds having certain gaps, e.g. gaps of 7 nucleosides or
longer, may be improved by the addition of three bicyclic
nucleosides at the 3'-most 5'-wing nucleoside and the addition of
one or more bicylic nucleosides at the 5'-most 3'-wing nucleoside.
In certain embodiments, the selectivity of antisense compounds
having certain gaps, e.g. gaps of 7 nucleosides or longer, may be
improved by the addition of four bicyclic nucleosides at the
3'-most 5'-wing nucleoside and the addition of one or more bicylic
nucleosides at the 5'-most 3'-wing nucleoside. In certain
embodiments, the selectivity of antisense compounds having certain
gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the
addition of four bicyclic nucleosides at the 3'-most 5'-wing
nucleoside and the addition of one or more bicylic nucleosides at
the 5'-most 3'-wing nucleoside.
[0430] In certain embodiments, the selectivity of antisense
compounds having certain gaps, e.g. gaps of 7 nucleosides or
shorter, may be improved by the addition of one or more bicyclic
nucleosides at the 3'-most 5'-wing nucleoside. In certain
embodiments, the selectivity of antisense compounds having certain
gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the
addition of two or more bicyclic nucleosides at the 3'-most 5'-wing
nucleoside. In certain embodiments, the selectivity of antisense
compounds having certain gaps, e.g. gaps of 7 nucleosides or
shorter, may be improved by the addition of one bicyclic nucleoside
at the 3'-most 5'-wing nucleoside. In certain embodiments, the
selectivity of antisense compounds having certain gaps, e.g. gaps
of 7 nucleosides or shorter, may be improved by the addition of two
bicyclic nucleosides at the 3'-most 5'-wing nucleoside. In certain
embodiments, the selectivity of antisense compounds having certain
gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the
addition of three bicyclic nucleosides at the 3'-most 5'-wing
nucleoside. In certain embodiments, the selectivity of antisense
compounds having certain gaps, e.g. gaps of 7 nucleosides or
shorter, may be improved by the addition of four bicyclic
nucleosides at the 3'-most 5'-wing nucleoside. In certain
embodiments, the selectivity of antisense compounds having certain
gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the
addition of five bicyclic nucleosides at the 3'-most 5'-wing
nucleoside. In certain embodiments discussed above, the bicyclic
nucleosides at the 3'-most 5'-wing nucleoside are selected from
among cEt, cMOE, LNA, .alpha.-LNA, ENA and 2'-thio LNA. In certain
embodiments discussed above, the bicyclic nucleosides at the
3'-most 5'-wing nucleoside comprise cEt. In certain embodiments
discussed above, the bicyclic nucleosides at the 3'-most 5'-wing
nucleoside comprise LNA.
[0431] In certain embodiments, for example, in certain embodiments
described above, the target nucleic acid may have an allelic
variant, e.g. a non-target nucleic acid, containing a single
nucleotide polymorphism. An antisense compound may be designed
having a single nucleobase mismatch from the non-target nucleic
acid, but which has full complementarity to the target nucleic
acid, and which also has one or more mismatches towards any other
non-target nucleic acid. In this manner one having skill in the art
may design a selective antisense compound that has full
complementarity to the target nucleic acid, a single mismatch to
its allelic variant, and one or more mismatches towards any other
non-target nucleic acid.
[0432] In certain embodiments, a selective antisense compound
having a single nucleobase mismatch relative to its allelic variant
(e.g. a non-target nucleic having a single nucleobase mismatch from
the target nucleic acid) may, based on its nucleobase sequence,
have full complementarity to one or more other non-target nucleic
acids. In such certain embodiments, the sequence of the selective
antisense compound may be moved upstream or downstream, so long as
the selective antisense compound has a single nucleobase mismatch
relative to its allelic variant. For example, in certain
embodiments, the 6.sup.th nucleobase from the 5'-end of the
selective antisense compound represents the single nucleobase
mismatch relative to its allelic variant. A new selective antisense
compound may then be designed wherein the sequence of the selective
antisense compound is shifted closer to the 5'-end, and wherein the
3.sup.rd nucleobase from the 5'-end of the newly designed selective
antisense compound represents the single nucleobase mismatch
relative to its allelic variant. In this manner, the newly designed
selective antisense compound will continue to have a single
nucleobase mismatch relative to its allelic variant, but now the
newly designed selective antisense compound may have 1 or more
mismatches with other non-target nucleic acids. Since the target
nucleic acid differs from its allelic variant by only a single
nucleobase mismatch, full complementarity between the selective
antisense compound and the target nucleic acid may be maintained,
while at the same time reducing complementarity between the
selective antisense compound and other non-target nucleic acids. In
certain embodiments, such modifications will have no impact or only
a small impact on the selectivity of the selective antisense
compound relative to its allelic variant, but will reduce the
selectivity of the selective antisense compound relative to other
non-target nucleic acids.
[0433] Any of the modifications discussed herein may be used to
design a selective antisense compound that reduces the amount or
activity of a target nucleic acid, while having little to no
selectivity towards the allelic variant of the target nucleic acid
or any other non-target nucleic acids. In certain embodiments, the
target nucleic acid is a transcript from the huntingtin gene and
the non-target nucleic acid is a transcript from a gene encoding
bone morphogenetic protein receptor, type IA. In certain
embodiments, the target nucleic acid is a transcript from a mutant
huntingtin gene and the non-target nucleic acid is a transcript
from a normal huntingtin gene. In certain embodiments, the target
nucleic acid is a transcript from a mutant huntingtin gene and one
non-target nucleic acid is a transcript from a normal huntingtin
gene and another non-target nucleic acid is a nucleic acid encoding
bone morphogenetic protein receptor, type IA.
[0434] In certain embodiments, it is desirable to have a selective
antisense compound that selectively reduces the amount or activity
of a target nucleic acid and which does not significantly reduce
the amount or activity of any other non-target nucleic acid. In
certain embodiments, it is desirable to have a selective antisense
compound that selectively reduces the amount or activity of a
target nucleic acid associated with Hungtinton's Disease and which
does not significantly reduce the amount or activity of any other
non-target nucleic acid, for example BMPR1A.
[0435] In certain embodiments, a selective antisense compound that
selectively reduces the amount or activity of mutant huntingtin
allele associated with a SNP may closely align with the nucleobase
sequence of another non-target nucleic acid. For example, in
certain embodiments, an antisense compound may be complementary to
a target nucleic acid that corresponds to a mutant huntingtin
allele associated with a SNP, and this mutant huntingtin allele
associated with a SNP may have a high degree of homology with
another non-target nucleic acid. For example, in certain
embodiments, an antisense compound may be complementary to a target
nucleic acid that corresponds to a mutant huntingtin allele
associated with a SNP, and also complementary to a non-target
nucleic acid encoding bone morphogenetic protein receptor, type IA.
For example, in certain embodiments, an antisense compound may be
complementary to a target nucleic acid that corresponds to a mutant
huntingtin allele associated with a SNP, for example rs7685686, and
also complementary to a non-target nucleic acid encoding bone
morphogenetic protein receptor, type IA. In certain such
embodiments, it is desirable to design an antisemse compound that
selectively reduces the target nucleic acid associated with SNP
rs7685686 and the mutant allele associated with Huntington's
disease, but at the same time minimizes reduction between
non-target nucleic acids that have high degree of sequence homology
with the target nucleic acid nucleobase sequence surrounding SNP
rs7685686.
[0436] In certain embodiments, the nucleobase sequence surrounding
SNP rs7685686 has a high degree of homology between portions of the
non-target nucleic acid that encodes bone morphogenetic protein
receptor, type IA. In certain embodiments, the present disclosure
provides compounds having specific modification motifs and
nucleobase sequences targeted to SNP rs7685686 that selectively
reduce the amount or activity of a mutant huntintin allele of a
huntingtin transcript but do not significantly reduce the amount or
activity of a wild-type huntingtin allele and do not significantly
reduce the amount or activity of a nucleic acid that encodes bone
morphogenetic protein receptor, type IA.
[0437] Antisense compounds having certain specified motifs have
enhanced selectivity, including, but not limited to motifs
described above. In certain embodiments, enhanced selectivity is
achieved by oligonucleotides comprising any one or more of:
[0438] a modification motif comprising a long 5'-wing (longer than
5, 6, or 7 nucleosides);
[0439] a modification motif comprising a long 3'-wing (longer than
5, 6, or 7 nucleosides);
[0440] a modification motif comprising a short gap region (shorter
than 8, 7, or 6 nucleosides); and
[0441] a modification motif comprising an interrupted gap region
(having no uninterrupted stretch of unmodified 2'-deoxynucleosides
longer than 7, 6 or 5).
[0442] In certain embodiments, it is desirable to have a selective
antisense compound that selectively reduces the amount or activity
of a target nucleic acid associated with Hungtinton's Disease and
which does not significantly reduce the amount or activity of any
other non-target nucleic acid, for example BMPR1A.
[0443] In certain embodiments, a compound consists of ISIS 606561.
In certain embodiments, a compound consists of ISIS 606562. In
certain embodiments, a compound consists of ISIS 611714. In certain
embodiments, a compound consists of ISIS 611715. In certain
embodiments, a compound consists of ISIS 611717. In certain
embodiments, a compound consists of ISIS 611718. In certain
embodiments, a compound consists of ISIS 611719. In certain
embodiments, a compound consists of ISIS 611720. In certain
embodiments, a compound consists of ISIS 611721. In certain
embodiments, a compound consists of ISIS 611722. In certain
embodiments, a compound consists of ISIS 611723. In certain
embodiments, a compound consists of ISIS 613581. In certain
embodiments, a compound consists of ISIS 613582. In certain
embodiments, a compound consists of ISIS 613583. In certain
embodiments, a compound consists of ISIS 613584. In certain
embodiments, a compound consists of ISIS 613585. In certain
embodiments, a compound consists of ISIS 613586. In certain
embodiments, a compound consists of ISIS 613588.
[0444] In certain embodiments, a compound consists of ISIS 613589.
In certain embodiments, a compound consists of ISIS 617104. In
certain embodiments, a compound consists of ISIS 617105. In certain
embodiments, a compound consists of ISIS 617106. In certain
embodiments, a compound consists of ISIS 617107. In certain
embodiments, a compound consists of ISIS 617108. In certain
embodiments, a compound consists of ISIS 617109. In certain
embodiments, a compound consists of ISIS 617110. In certain
embodiments, a compound consists of ISIS 617111. In certain
embodiments, a compound consists of ISIS 617115. In certain
embodiments, a compound consists of ISIS 617116. In certain
embodiments, a compound consists of ISIS 617117. In certain
embodiments, a compound consists of ISIS 617118. In certain
embodiments, a compound consists of ISIS 617119. In certain
embodiments, a compound consists of ISIS 617425. In certain
embodiments, a compound consists of ISIS 623181. In certain
embodiments, a compound consists of ISIS 623182. In certain
embodiments, a compound consists of ISIS 623198. In certain
embodiments, a compound consists of ISIS 623199.
[0445] In certain embodiments, a compound consists of ISIS 623202.
In certain embodiments, a compound consists of ISIS 623203. In
certain embodiments, a compound consists of ISIS 623205. In certain
embodiments, a compound consists of ISIS 623206. In certain
embodiments, a compound consists of ISIS 623208. In certain
embodiments, a compound consists of ISIS 623212. In certain
embodiments, a compound consists of ISIS 623214. In certain
embodiments, a compound consists of ISIS 623218. In certain
embodiments, a compound consists of ISIS 623220. In certain
embodiments, a compound consists of ISIS 623221. In certain
embodiments, a compound consists of ISIS 623224. In certain
embodiments, a compound consists of ISIS 623227. In certain
embodiments, a compound consists of ISIS 623230.
[0446] In certain embodiments, a compound consists of ISIS 623232.
In certain embodiments, a compound consists of ISIS 623233. In
certain embodiments, a compound consists of ISIS 623235. In certain
embodiments, a compound consists of ISIS 623236. In certain
embodiments, a compound consists of ISIS 623237. In certain
embodiments, a compound consists of ISIS 623238. In certain
embodiments, a compound consists of ISIS 623239. In certain
embodiments, a compound consists of ISIS 623241. In certain
embodiments, a compound consists of ISIS 623242. In certain
embodiments, a compound consists of ISIS 623243. In certain
embodiments, a compound consists of ISIS 623254. In certain
embodiments, a compound consists of ISIS 623262. In certain
embodiments, a compound consists of ISIS 623490. In certain
embodiments, a compound consists of ISIS 623493. In certain
embodiments, a compound consists of ISIS 623494.
[0447] i. Certain Selective Nucleobase Sequence Elements
[0448] In certain embodiments, selective antisense compounds
comprise nucleobase sequence elements.
[0449] Such nucleobase sequence elements are independent of
modification motifs. Accordingly, oligonucleotides having any of
the motifs (modification motifs, nucleoside motifs, sugar motifs,
nucleobase modification motifs, and/or linkage motifs) may also
comprise one or more of the following nucleobase sequence
elements.
[0450] ii. Alignment of Differentiating Nucleobase/Target-Selective
Nucleoside
[0451] In certain embodiments, a target region and a region of a
non-target nucleic acid differ by 1-4 differentiating nucleobase.
In such embodiments, selective antisense compounds have a
nucleobase sequence that aligns with the non-target nucleic acid
with 1-4 mismatches. A nucleoside of the antisense compound that
corresponds to a differentiating nucleobase of the target nucleic
acid is referred to herein as a target-selective nucleoside. In
certain embodiments, selective antisense compounds having a gapmer
motif align with a non-target nucleic acid, such that a
target-selective nucleoside is positioned in the gap. In certain
embodiments, a target-selective nucleoside is the 1.sup.st
nucleoside of the gap from the 5' end. In certain embodiments, a
target-selective nucleoside is the 2.sup.nd nucleoside of the gap
from the 5' end. In certain embodiments, a target-selective
nucleoside is the 3.sup.rd nucleoside of the gap from the 5'-end.
In certain embodiments, a target-selective nucleoside is the
4.sup.th nucleoside of the gap from the 5'-end. In certain
embodiments, a target-selective nucleoside is the 5.sup.th
nucleoside of the gap from the 5'-end. In certain embodiments, a
target-selective nucleoside is the 6.sup.rd nucleoside of the gap
from the 5'-end. In certain embodiments, a target-selective
nucleoside is the 8.sup.th nucleoside of the gap from the 3'-end.
In certain embodiments, a target-selective nucleoside is the
7.sup.th nucleoside of the gap from the 3'-end. In certain
embodiments, a target-selective nucleoside is the 6.sup.th
nucleoside of the gap from the 3'-end. In certain embodiments, a
target-selective nucleoside is the 5.sup.th nucleoside of the gap
from the 3'-end. In certain embodiments, a target-selective
nucleoside is the 4.sup.th nucleoside of the gap from the 3'-end.
In certain embodiments, a target-selective nucleoside is the
3.sup.rd nucleoside of the gap from the 3'-end. In certain
embodiments, a target-selective nucleoside is the 2.sup.nd
nucleoside of the gap from the 3'-end.
[0452] In certain embodiments, a target-selective nucleoside
comprises a modified nucleoside. In certain embodiments, a
target-selective nucleoside comprises a modified sugar. In certain
embodiments, a target-selective nucleoside comprises a sugar
surrogate. In certain embodiments, a target-selective nucleoside
comprises a sugar surrogate selected from among HNA and F-HNA. In
certain embodiments, a target-selective nucleoside comprises a
2'-substituted sugar moiety. In certain embodiments, a
target-selective nucleoside comprises a 2'-substituted sugar moiety
selected from among MOE, F and (ara)-F. In certain embodiments, a
target-selective nucleoside comprises a 5'-substituted sugar
moiety. In certain embodiments, a target-selective nucleoside
comprises a 5'-substituted sugar moiety selected from 5'-(R)-Me
DNA. In certain embodiments, a target-selective nucleoside
comprises a bicyclic sugar moiety. In certain embodiments, a
target-selective nucleoside comprises a bicyclic sugar moiety
selected from among cEt, and .alpha.-L-LNA. In certain embodiments,
a target-selective nucleoside comprises a modified nucleobase. In
certain embodiments, a target-selective nucleoside comprises a
modified nucleobase selected from among 2-thio-thymidine and
5-propyne uridine.
[0453] i. Alignment of Differentiating Nucleobase/Selectivity
Against One or More Non-Target Nucleic Acid Transcripts
[0454] In certain embodiments, a target region and a region of one
or more non-target nucleic acids differ by 0-4 differentiating
nucleobases. In such embodiments, selective antisense compounds
have a nucleobase sequence that aligns with one or more non-target
nucleic acids with 0-4 mismatches. In certain such embodiments,
selective antisense compounds have a nucleobase sequence that
aligns with a first non-target nucleic acid with 1-4 mismatches and
a second non-target nucleic acid with 0-4 mismatches. In certain
embodiments, selective antisense compounds have a nucleobase
sequence that aligns with a first non-target nucleic acid with 1
mismatch and a second non-target nucleic acid with 0 mismatches. In
certain embodiments, selective antisense compounds have a
nucleobase sequence that aligns with a first non-target nucleic
acid with 1 mismatch and a second non-target nucleic acid with 1
mismatch.
[0455] In certain embodiments, a selective antisense compound may
be selective against one non-target nucleic acid transcript, but
not be selective against another non-target nucleic acid
transcript. For example, in certain embodiments, a selective
antisense compound may be selective for a target relative to a
first non-target nucleic acid, but may also be selective towards a
second non-target nucleic acid relative to the first non-target
nucleic acid, wherein the first and second non-target nucleic acids
differ by 0-4 differentiating nucleobases. For example, in certain
embodiments, a selective antisense compound may be selective for a
target relative to a first non-target nucleic acid based on a
mismatch of a single differentiating nucleobase, but also be
selective for a second non-target nucleic acid relative to the
first non-target nucleic acid. In certain embodiments, it is
preferred to have a selective antisense compound be selective for a
target relative to a first non-target nucleic acid and also be
selective for a target relative to a second non-target nucleic
acid. In certain embodiments, it is preferred to have a selective
antisense compound be selective for a mutant huntingtin allele
associated with Huntington's disease, and not be selective relative
to any other non-target nucleic acids.
[0456] In certain embodiments where a selective antisense compound
is selective for a target relative to a first non-target nucleic
acid based on a single differentiating nucleobase, but wherein the
selective antisense compound is also selective relative to a second
non-target nucleic acid, it is possible to alter the position of
the target-selective nucleoside within the gap region of the
selective antisense compound to maintain selectivity relative to
the first non-target nucleic acid and to increase selectivity
relative to the second non-target nucleic acid. For example, in
certain embodiments, a target-selective nucleoside is the 3.sup.rd
nucleoside of the gap from the 5'-end of the selective antisense
compound, wherein the 5'-wing of the selective antisense compound
consists of 4 nucleosides.
[0457] In certain embodiments, a selective antisense compound may
be redesigned wherein the overall length of the selective antisense
compound remains the same, but wherein the length of the 5'-wing is
shortened to 1-4 nucleosides and/or the position of the
target-selective nucleoside is moved closer to the 5'-end of the
gap, for example the 1.sup.st or or 2.sup.nd nucleoside of the gap
from the 5'-end. In certain embodiments, a selective antisense
compound may be redesigned wherein the overall length of the
selective antisense compound remains the same, but wherein the
length of the 5'-wing is increased to 1-9 nucleosides and/or the
position of the target-selective nucleoside is moved closer to the
5'-end of the gap, for example the 1.sup.st or or 2.sup.nd
nucleoside of the gap from the 5'-end. In certain embodiments, a
selective antisense compound may be redesigned wherein the overall
length of the selective antisense compound remains the same, but
wherein the length of the 5'-wing is increased to 1-9 nucleosides
and/or the position of the target-selective nucleoside is moved
closer to the 3'-end of the gap, for example the 1.sup.St or
2.sup.nd nucleoside of the gap from the 3'-end. In certain
embodiments, a selective antisense compound may be redesigned
wherein the overall length of the selective antisense compound
remains the same, but wherein the length of the 5'-wing is
shortened to 1-4 nucleosides and/or the position of the
target-selective nucleoside is moved closer to the 3'-end of the
gap, for example the 1.sup.st or or 2.sup.nd nucleoside of the gap
from the 3'-end. In this manner, the selectivity of the selective
antisense compound relative to the first non-target nucleic acid
may be retained, while also achieving selectivity of the selective
antisense compound relative to the second non-target nucleic acid.
In certain embodiments, redesign of the selective antisense
compound will retain a single-differentiating nucleobase relative
to the first non-target nucleic acid and increase the number of
differentiating nucleobases between the selective antisense
compound and the second non-target nucleic acid. In certain
embodiments, the first non-target nucleic acid is wild-type
huntingtin and the second non-target nucleic acid is bone
morphogenetic protein receptor, type IA.
[0458] In certain embodiments, a selective antisense compound may
be redesigned wherein the overall length of the selective antisense
compound remains the same, but wherein the length of the 3'-wing is
increased to 1-10 nucleosides and/or the position of the
target-selective nucleoside is moved closer to the 5'-end of the
gap, for example the 1.sup.st or 2.sup.nd nucleoside of the gap
from the 5'-end. In certain embodiments, a selective antisense
compound may be redesigned wherein the overall length of the
selective antisense compound remains the same, but wherein the
length of the 3'-wing is increased to 1-10 nucleosides and/or the
position of the target-selective nucleoside is moved closer to the
3'-end of the gap, for example the 1.sup.th or 2.sup.nd nucleoside
of the gap from the 3'-end. In certain embodiments, a selective
antisense compound may be redesigned wherein the overall length of
the selective antisense compound remains the same, but wherein the
length of the 3'-wing is increased to 1-10 nucleosides and/or the
position of the target-selective nucleoside is moved closer to the
3'-end of the gap, for example the 1.sup.St or 2.sup.nd nucleoside
of the gap from the 3'-end. In certain embodiments, a selective
antisense compound may be redesigned wherein the overall length of
the selective antisense compound remains the same, but wherein the
length of the 3'-wing is increased to 1-10 nucleosides and/or the
position of the target-selective nucleoside is moved closer to the
5'-end of the gap, for example the 1.sup.st or 2.sup.nd nucleoside
of the gap from the 5'-end. In this manner, the selectivity of the
selective antisense compound relative to the first non-target
nucleic acid may be retained, while also increasing selectivity of
the selective antisense compound relative to the second non-target
nucleic acid. In certain embodiments, redesign of the selective
antisense compound will retain a single-differentiating nucleobase
relative to the first non-target nucleic acid and increase the
number of differentiating nucleobases between the selective
antisense compound and the second non-target nucleic acid. In
certain embodiments, the first non-target nucleic acid is wild-type
huntingtin and the second non-target nucleic acid is bone
morphogenetic protein receptor, type IA.
[0459] ii. Mismatches to the Target Nucleic Acid
[0460] In certain embodiments, selective antisense compounds
comprise one or more mismatched nucleobases relative to the target
nucleic acid. In certain such embodiments, antisense activity
against the target is reduced by such mismatch, but activity
against the non-target is reduced by a greater amount. Thus, in
certain embodiments selectivity is improved. Any nucleobase other
than the differentiating nucleobase is suitable for a mismatch. In
certain embodiments, however, the mismatch is specifically
positioned within the gap of an oligonucleotide having a gapmer
motif. In certain embodiments, a mismatch relative to the target
nucleic acid is at positions 1, 2, 3, 4, 5, 6, 7, or 8 from the
5'-end of the gap region. In certain embodiments, a mismatch
relative to the target nucleic acid is at positions 9, 8, 7, 6, 5,
4, 3, 2, 1 of the antisense compounds from the 3'-end of the gap
region. In certain embodiments, a mismatch relative to the target
nucleid acid is at positions 1, 2, 3, or 4 of the antisense
compounds from the 5'-end of the wing region. In certain
embodiments, a mismatch relative to the target nucleid acid is at
positions 4, 3, 2, or 1 of the antisense compounds from the 3'-end
of the wing region.
[0461] iii. Self Complementary Regions
[0462] In certain embodiments, selective antisense compounds
comprise a region that is not complementary to the target. In
certain embodiments, such region is complementary to another region
of the antisense compound. Such regions are referred to herein as
self-complementary regions. For example, in certain embodiments, an
antisense compound has a first region at one end that is
complementary to a second region at the other end. In certain
embodiments, one of the first and second regions is complementary
to the target nucleic acid. Unless the target nucleic acid also
includes a self-complementary region, the other of the first and
second region of the antisense compound will not be complementary
to the target nucleic acid. For illustrative purposes, certain
antisense compounds have the following nucleobase motif:
[0463] ABCXXXXXXXXXC'B'A';
[0464] ABCXXXXXXX(X/C')(X/B')(X/A');
[0465] (X/A)(X/B)(X/C)XXXXXXXXXC'B'A'
where each of A, B, and C are any nucleobase; A', B', and C' are
the complementary bases to A, B, and C, respectively; each X is a
nucleobase complementary to the target nucleic acid; and two
letters in parentheses (e.g., (X/C')) indicates that the nucleobase
is complementary to the target nucleic acid and to the designated
nucleoside within the antisense oligonucleotide.
[0466] Without being bound to any mechanism, in certain
embodiments, such antisense compounds are expected to form
self-structure, which is disrupted upon contact with a target
nucleic acid. Contact with a non-target nucleic acid is expected to
disrupt the self-structure to a lesser degree, thus increasing
selectivity compared to the same antisense compound lacking the
self-complementary regions.
[0467] iv. Combinations of Features
[0468] Though it is clear to one of skill in the art, the above
motifs and other elements for increasing selectivity may be used
alone or in combination. For example, a single antisense compound
may include any one, two, three, or more of: self-complementary
regions, a mismatch relative to the target nucleic acid, a short
nucleoside gap, an interrupted gap, and specific placement of the
selective nucleoside.
D. Certain Target Nucleic Acids
[0469] In certain embodiments, antisense compounds comprise or
consist of an oligonucleotide comprising a region that is
complementary to a target nucleic acid. In certain embodiments, the
target nucleic acid is an endogenous RNA molecule. In certain
embodiments, the target nucleic acid is a non-coding RNA. In
certain such embodiments, the target non-coding RNA is selected
from: a long-non-coding RNA, a short non-coding RNA, an intronic
RNA molecule, a snoRNA, a scaRNA, a microRNA (including
pre-microRNA and mature microRNA), a ribosomal RNA, and promoter
directed RNA. In certain embodiments, the target nucleic acid
encodes a protein. In certain such embodiments, the target nucleic
acid is selected from: an mRNA and a pre-mRNA, including intronic,
exonic and untranslated regions. In certain embodiments, oligomeric
compounds are at least partially complementary to more than one
target nucleic acid. For example, antisense compounds of the
present invention may mimic microRNAs, which typically bind to
multiple targets.
[0470] In certain embodiments, the target nucleic acid is a nucleic
acid other than a mature mRNA. In certain embodiments, the target
nucleic acid is a nucleic acid other than a mature mRNA or a
microRNA. In certain embodiments, the target nucleic acid is a
non-coding RNA other than a microRNA. In certain embodiments, the
target nucleic acid is a non-coding RNA other than a microRNA or an
intronic region of a pre-mRNA. In certain embodiments, the target
nucleic acid is a long non-coding RNA. In certain embodiments, the
target RNA is an mRNA. In certain embodiments, the target nucleic
acid is a pre-mRNA.
[0471] In certain such embodiments, the target region is entirely
within an intron. In certain embodiments, the target region spans
an intron/exon junction. In certain embodiments, the target region
is at least 50% within an intron. In certain embodiments, the
target nucleic acid is selected from among non-coding RNA,
including exonic regions of pre-mRNA. In certain embodiments, the
target nucleic acid is a ribosomal RNA (rRNA). In certain
embodiments, the target nucleic acid is a non-coding RNA associated
with splicing of other pre-mRNAs. In certain embodiments, the
target nucleic acid is a nuclear-retained non-coding RNA.
[0472] In certain embodiments, antisense compounds described herein
are complementary to a target nucleic acid comprising a
single-nucleotide polymorphism. In certain such embodiments, the
antisense compound is capable of modulating expression of one
allele of the single-nucleotide polymorphism-containing-target
nucleic acid to a greater or lesser extent than it modulates
another allele. In certain embodiments an antisense compound
hybridizes to a single-nucleotide polymorphism-containing-target
nucleic acid at the single-nucleotide polymorphism site. In certain
embodiments, the target nucleic acid is a Huntingtin gene
transcript. In certain embodiments, the target nucleic acid is a
single-nucleotide polymorphism-containing-target nucleic acid of a
Huntingtin gene transcript. In certain embodiments, the target
nucleic acid is not a Huntingtin gene transcript. In certain
embodiments, the target nucleic acid is a single-nucleotide
polymorphism-containing-target nucleic acid of a gene transcript
other than Huntingtin. In certain embodiments, the target nucleic
acid is any nucleic acid other than a Huntingtin gene
transcript.
[0473] a. Single-Nucleotide Polymorphism
[0474] In certain embodiments, the invention provides selective
antisense compounds that have greater activity for a target nucleic
acid than for a homologous or partially homologous non-target
nucleic acid. In certain such embodiments, the target and
non-target nucleic acids are not functionally related to one
another (e.g., are transcripts from different genes). In certain
embodiments, the target and not-targe nucleic acids are allelic
variants of one another. Certain embodiments of the present
invention provide methods, compounds, and compositions for
selectively inhibiting mRNA and protein expression of an allelic
variant of a particular gene or DNA sequence. In certain
embodiments, the allelic variant contains a single nucleotide
polymorphism (SNP). In certain embodiments, a SNP is associated
with a mutant allele. In certain embodiments, a mutant SNP is
associated with a disease. In certain embodiments a mutant SNP is
associated with a disease, but is not causative of the disease. In
certain embodiments, mRNA and protein expression of a mutant allele
is associated with disease.
[0475] In certain embodiments, the expressed gene product of a
mutant allele results in aggregation of the mutant proteins causing
disease. In certain embodiments, the expressed gene product of a
mutant allele results in gain of function causing disease. In
certain embodiments, genes with an autosomal dominant mutation
resulting in a toxic gain of function of the protein are the APP
gene encoding amyloid precursor protein involved in Alzheimer's
disease (Gene, 371: 68, 2006); the PrP gene encoding prion protein
involved in Creutzfeldt-Jakob disease and in fatal familial
insomnia (Nat. Med. 1997, 3: 1009); GFAP gene encoding glial
fibrillary acidic protein involved in Alexander disease (J.
Neurosci. 2006, 26:111623); alpha-synuclein gene encoding
alpha-synuclein protein involved in Parkinson's disease (J. Clin.
Invest. 2003, 111: 145); SOD-1 gene encoding the SOD-1 protein
involved in amyotrophic lateral sclerosis (Science 1998, 281:
1851); atrophin-1 gene encoding atrophin-1 protein involved in
dentato-rubral and pallido-luysian atrophy (DRPA) (Trends Mol. Med.
2001, 7: 479); SCA1 gene encoding ataxin-1 protein involved in
spino-cerebellar ataxia-1 (SCA1) (Protein Sci. 2003, 12: 953); PLP
gene encoding proteolipid protein involved in Pelizaeus-Merzbacher
disease (NeuroMol Med. 2007, 4: 73); DYT1 gene encoding torsinA
protein involved in Torsion dystonia (Brain Res. 2000, 877: 379);
and alpha-B crystalline gene encoding alpha-B crystalline protein
involved in protein aggregation diseases, including cardiomyopathy
(Cell 2007, 130: 427); alphal-antitrypsin gene encoding
alphal-antitrypsin protein involved in chronic obstructive
pulmonary disease (COPD), liver disease and hepatocellular
carcinoma (New Engl J Med. 2002, 346: 45); Ltk gene encoding
leukocyte tyrosine kinase protein involved in systemic lupus
erythematosus (Hum. Mol. Gen. 2004, 13: 171); PCSK9 gene encoding
PCSK9 protein involved in hypercholesterolemia (Hum Mutat. 2009,
30: 520); prolactin receptor gene encoding prolactin receptor
protein involved in breast tumors (Proc. Natl. Assoc. Sci. 2008,
105: 4533); CCL5 gene encoding the chemokine CCL5 involved in COPD
and asthma (Eur. Respir. J. 2008, 32: 327); PTPN22 gene encoding
PTPN22 protein involved in Type 1 diabetes, Rheumatoid arthritis,
Graves disease, and SLE (Proc. Natl. Assoc. Sci. 2007, 104: 19767);
androgen receptor gene encoding the androgen receptor protein
involved in spinal and bulbar muscular atrophy or Kennedy's disease
(J Steroid Biochem. Mol. Biol. 2008, 108: 245); CHMP4B gene
encoding chromatin modifying protein-4B involved in progressive
childhood posterior subcapsular cataracts (Am. J. Hum. Genet 2007,
81: 596); FXR/NR1H4 gene encoding Farnesoid X receptor protein
involved in cholesterol gallstone disease, arthrosclerosis and
diabetes (Mol. Endocrinol. 2007, 21: 1769); ABCA1 gene encoding
ABCA1 protein involved in cardiovascular disease (Transl. Res.
2007, 149: 205); CaSR gene encoding the calcium sensing receptor
protein involved in primary hypercalciuria (Kidney Int. 2007, 71:
1155); alpha-globin gene encoding alpha-globin protein involved in
alpha-thallasemia (Science 2006, 312: 1215); httlpr gene encoding
HTTLPR protein involved in obsessive compulsive disorder (Am. J.
Hum. Genet. 2006, 78: 815); AVP gene encoding arginine vasopressin
protein in stress-related disorders such as anxiety disorders and
comorbid depression (CNS Neurol. Disord. Drug Targets 2006, 5:
167); GNAS gene encoding G proteins involved in congenital visual
defects, hypertension, metabolic syndrome (Trends Pharmacol. Sci.
2006, 27: 260); APAF1 gene encoding APAF 1 protein involved in a
predisposition to major depression (Mol. Psychiatry 2006, 11: 76);
TGF-beta1 gene encoding TGF-beta1 protein involved in breast cancer
and prostate cancer (Cancer Epidemiol. Biomarkers Prev. 2004, 13:
759); AChR gene encoding acetylcholine receptor involved in
congential myasthenic syndrome (Neurology 2004, 62: 1090); P2Y12
gene encoding adenosine diphosphate (ADP) receptor protein involved
in risk of peripheral arterial disease (Circulation 2003, 108:
2971); LQT1 gene encoding LQT1 protein involved in atrial
fibrillation (Cardiology 2003, 100: 109); RET protooncogene
encoding RET protein involved in sporadic pheochromocytoma (J.
Clin. Endocrinol. Metab. 2003, 88: 4911); filamin A gene encoding
filamin A protein involved in various congenital malformations
(Nat. Genet. 2003, 33: 487); TARDBP gene encoding TDP-43 protein
involved in amyotrophic lateral sclerosis (Hum. Mol. Gene.t 2010,
19: 671); SCA3 gene encoding ataxin-3 protein involved in
Machado-Joseph disease (PLoS One 2008, 3: e3341); SCA7 gene
encoding ataxin-7 protein involved in spino-cerebellar ataxia-7
(PLoS One 2009, 4: e7232); and HTT gene encoding huntingtin protein
involved in Huntington's disease (Neurobiol Dis. 1996, 3:183); and
the CA4 gene encoding carbonic anhydrase 4 protein, CRX gene
encoding cone-rod homeobox transcription factor protein, FSCN2 gene
encoding retinal fascin homolog 2 protein, IMPDH1 gene encoding
inosine monophosphate dehydrogenase 1 protein, NR2E3 gene encoding
nuclear receptor subfamily 2 group E3 protein, NRL gene encoding
neural retina leucine zipper protein, PRPF3 (RP 18) gene encoding
pre-mRNA splicing factor 3 protein, PRPF8 (RP13) gene encoding
pre-mRNA splicing factor 8 protein, PRPF31 (RP11) gene encoding
pre-mRNA splicing factor 31 protein, RDS gene encoding peripherin 2
protein, ROM1 gene encoding rod outer membrane protein 1 protein,
RHO gene encoding rhodopsin protein, RP1 gene encoding RP1 protein,
RPGR gene encoding retinitis pigmentosa GTPase regulator protein,
all of which are involved in Autosomal Dominant Retinitis
Pigmentosa disease (Adv Exp Med Biol. 2008, 613:203)
[0476] In certain embodiments, the mutant allele is associated with
any disease from the group consisting of Alzheimer's disease,
Creutzfeldt-Jakob disease, fatal familial insomnia, Alexander
disease, Parkinson's disease, amyotrophic lateral sclerosis,
dentato-rubral and pallido-luysian atrophy DRPA, spino-cerebellar
ataxia, Torsion dystonia, cardiomyopathy, chronic obstructive
pulmonary disease (COPD), liver disease, hepatocellular carcinoma,
systemic lupus erythematosus, hypercholesterolemia, breast cancer,
asthma, Type 1 diabetes, Rheumatoid arthritis, Graves disease, SLE,
spinal and bulbar muscular atrophy, Kennedy's disease, progressive
childhood posterior subcapsular cataracts, cholesterol gallstone
disease, arthrosclerosis, cardiovascular disease, primary
hypercalciuria, alpha-thallasemia, obsessive compulsive disorder,
Anxiety, comorbid depression, congenital visual defects,
hypertension, metabolic syndrome, prostate cancer, congential
myasthenic syndrome, peripheral arterial disease, atrial
fibrillation, sporadic pheochromocytoma, congenital malformations,
Machado-Joseph disease, Huntington's disease, and Autosomal
Dominant Retinitis Pigmentosa disease.
[0477] i. Certain Huntingtin Targets
[0478] In certain embodiments, an allelic variant of huntingtin is
selectively reduced. Nucleotide sequences that encode huntingtin
include, without limitation, the following: GENBANK Accession No.
NT_006081.18, truncated from nucleotides 1566000 to 1768000
incorporated herein as SEQ ID NO: 1, and NM_002111.6, incorporated
herein as SEQ ID NO: 2.
[0479] Table 4 provides SNPs found in the GM04022, GM04281,
GM02171, and GM02173B cell lines. Also provided are the allelic
variants found at each SNP position, the genotype for each of the
cell lines, and the percentage of HD patients having a particular
allelic variant. For example, the two allelic variants for SNP
rs6446723 are T and C. The GM04022 cell line is heterozygous TC,
the GM02171 cell line is homozygous CC, the GM02173 cell line is
heterozygous TC, and the GM04281 cell line is homozygous TT. Fifty
percent of HD patients have a T at SNP position rs6446723.
TABLE-US-00004 TABLE 4 Allelic Variations for SNPs Associated with
HD SNP Variation GM04022 GM02171 GM02173 GM04281 TargetPOP allele
rs6446723 T/C TC CC TC TT 0.50 T rs3856973 A/G AG AA AG GG 0.50 G
rs2285086 A/G AG GG AG AA 0.50 A rs363092 A/C.sup. AC AA AC CC 0.49
C rs916171 C/G GC GG GC CC 0.49 C rs6844859 T/C TC CC TC TT 0.49 T
rs7691627 A/G AG AA AG GG 0.49 G rs4690073 A/G AG AA AG GG 0.49 G
rs2024115 A/G AG GG AG AA 0.48 A rs11731237 T/C CC CC TC TT 0.43 T
rs362296 A/C.sup. CC AC AC AC 0.42 C rs10015979 A/G AA AA AG GG
0.42 G rs7659144 C/G CG CG CG CC 0.41 C rs363096 T/C CC CC TC TT
0.40 T rs362273 A/G AA AG AG AA 0.39 A rs16843804 T/C CC TC TC CC
0.38 C rs362271 A/G GG AG AG GG 0.38 G rs362275 T/C CC TC TC CC
0.38 C rs3121419 T/C CC TC TC CC 0.38 C rs362272 A/G GG -- AG GG
0.38 G rs3775061 A/G AA AG AG AA 0.38 A rs34315806 T/C CC TC TC CC
0.38 C rs363099 T/C CC TC TC CC 0.38 C rs2298967 T/C TT TC TC TT
0.38 T rs363088 A/T.sup. AA TA TA AA 0.38 A rs363064 T/C CC TC TC
CC 0.35 C rs363102 A/G AG AA AA AA 0.23 G rs2798235 A/G AG GG GG GG
0.21 A rs363080 T/C TC CC CC CC 0.21 T rs363072 A/T.sup. TA TA AA
AA 0.13 A rs363125 A/C.sup. AC AC CC CC 0.12 C rs362303 T/C TC TC
CC CC 0.12 C rs362310 T/C TC TC CC CC 0.12 C rs10488840 A/G AG AG
GG GG 0.12 G rs362325 T/C TC TC TT TT 0.11 T rs35892913 A/G GG GG
GG GG 0.10 A rs363102 A/G AG AA AA AA 0.09 A rs363096 T/C CC CC TC
TT 0.09 C rs11731237 T/C CC CC TC TT 0.09 C rs10015979 A/G AA AA AG
GG 0.08 A rs363080 T/C TC CC CC CC 0.07 C rs2798235 A/G AG GG GG GG
0.07 G rs1936032 C/G GC CC CC CC 0.06 C rs2276881 A/G GG GG GG GG
0.06 G rs363070 A/G AA AA AA AA 0.06 A rs35892913 A/G GG GG GG GG
0.04 G rs12502045 T/C CC CC CC CC 0.04 C rs6446723 T/C TC CC TC TT
0.04 C rs7685686 A/G AG GG AG AA 0.04 G rs3733217 T/C CC CC CC CC
0.03 C rs6844859 T/C TC CC TC TT 0.03 C rs362331 T/C TC CC TC TT
0.03 C
E. Certain Indications
[0480] In certain embodiments, provided herein are methods of
treating an animal or individual comprising administering one or
more pharmaceutical compositions as described herein. In certain
embodiments, the individual or animal has Huntington's disease.
[0481] In certain embodiments, compounds targeted to huntingtin as
described herein may be administered to reduce the severity of
physiological symptoms of Huntington's disease. In certain
embodiments, compounds targeted to huntingtin as described herein
may be administered to reduce the rate of degeneration in an
individual or an animal having Huntington's disease. In certain
embodiments, compounds targeted to huntingtin as described herein
may be administered regeneration function in an individual or an
animal having Huntington's disease. In certain embodiments,
symptoms of Huntingtin's disease may be reversed by treatment with
a compound as described herein.
[0482] In certain embodiments, compounds targeted to huntingtin as
described herein may be administered to ameliorate one or more
symptoms of Huntington's disease. In certain embodiments
administration of compounds targeted to huntingtin as described
herein may improve the symptoms of Huntington's disease as measured
by any metric known to those having skill in the art. In certain
embodiments, administration of compounds targeted to huntingtin as
described herein may improve a rodent's rotaraod assay performance.
In certain embodiments, administration of compounds targeted to
huntingtin as described herein may improve a rodent's plus maze
assay. In certain embodiments, administration of compounds targeted
to huntingtin as described herein may improve a rodent's open field
assay performance.
[0483] Accordingly, provided herein are methods for ameliorating a
symptom associated with Huntington's disease in a subject in need
thereof. In certain embodiments, provided is a method for reducing
the rate of onset of a symptom associated with Huntington's
disease. In certain embodiments, provided is a method for reducing
the severity of a symptom associated with Huntington's disease. In
certain embodiments, provided is a method for regenerating
neurological function as shown by an improvement of a symptom
associated with Huntington's disease. In such embodiments, the
methods comprise administering to an individual or animal in need
thereof a therapeutically effective amount of a compound targeted
to a huntingtin nucleic acid.
[0484] Huntington's disease is characterized by numerous physical,
neurological, psychiatric, and/or peripheral symptoms. Any symptom
known to one of skill in the art to be associated with Huntington's
disease can be ameliorated or otherwise modulated as set forth
above in the methods described above. In certain embodiments, the
symptom is a physical symptom selected from the group consisting of
restlessness, lack of coordination, unintentionally initiated
motions, unintentionally uncompleted motions, unsteady gait,
chorea, rigidity, writhing motions, abnormal posturing,
instability, abnormal facial expressions, difficulty chewing,
difficulty swallowing, difficulty speaking, seizure, and sleep
disturbances. In certain embodiments, the symptom is a cognitive
symptom selected from the group consisting of impaired planning,
impaired flexibility, impaired abstract thinking, impaired rule
acquisition, impaired initiation of appropriate actions, impaired
inhibition of inappropriate actions, impaired short-term memory,
impaired long-term memory, paranoia, disorientation, confusion,
hallucination and dementia. In certain embodiments, the symptom is
a psychiatric symptom selected from the group consisting of
anxiety, depression, blunted affect, egocentrisms, aggression,
compulsive behavior, irritability and suicidal ideation. In certain
embodiments, the symptom is a peripheral symptom selected from the
group consisting of reduced brain mass, muscle atrophy, cardiac
failure, impaired glucose tolerance, weight loss, osteoporosis, and
testicular atrophy.
[0485] In certain embodiments, the symptom is restlessness. In
certain embodiments, the symptom is lack of coordination. In
certain embodiments, the symptom is unintentionally initiated
motions. In certain embodiments, the symptom is unintentionally
uncompleted motions. In certain embodiments, the symptom is
unsteady gait. In certain embodiments, the symptom is chorea. In
certain embodiments, the symptom is rigidity. In certain
embodiments, the symptom is writhing motions. In certain
embodiments, the symptom is abnormal posturing. In certain
embodiments, the symptom is instability. In certain embodiments,
the symptom is abnormal facial expressions. In certain embodiments,
the symptom is difficulty chewing. In certain embodiments, the
symptom is difficulty swallowing. In certain embodiments, the
symptom is difficulty speaking. In certain embodiments, the symptom
is seizures. In certain embodiments, the symptom is sleep
disturbances.
[0486] In certain embodiments, the symptom is impaired planning. In
certain embodiments, the symptom is impaired flexibility. In
certain embodiments, the symptom is impaired abstract thinking. In
certain embodiments, the symptom is impaired rule acquisition. In
certain embodiments, the symptom is impaired initiation of
appropriate actions. In certain embodiments, the symptom is
impaired inhibition of inappropriate actions. In certain
embodiments, the symptom is impaired short-term memory. In certain
embodiments, the symptom is impaired long-term memory. In certain
embodiments, the symptom is paranoia. In certain embodiments, the
symptom is disorientation. In certain embodiments, the symptom is
confusion. In certain embodiments, the symptom is hallucination. In
certain embodiments, the symptom is dementia.
[0487] In certain embodiments, the symptom is anxiety. In certain
embodiments, the symptom is depression. In certain embodiments, the
symptom is blunted affect. In certain embodiments, the symptom is
egocentrism. In certain embodiments, the symptom is aggression. In
certain embodiments, the symptom is compulsive behavior. In certain
embodiments, the symptom is irritability. In certain embodiments,
the symptom is suicidal ideation.
[0488] In certain embodiments, the symptom is reduced brain mass.
In certain embodiments, the symptom is muscle atrophy. In certain
embodiments, the symptom is cardiac failure. In certain
embodiments, the symptom is impaired glucose tolerance. In certain
embodiments, the symptom is weight loss. In certain embodiments,
the symptom is osteoporosis. In certain embodiments, the symptom is
testicular atrophy.
[0489] In certain embodiments, symptoms of Huntington's disease may
be quantifiable. For example, osteoporosis may be measured and
quantified by, for example, bone density scans. For such symptoms,
in certain embodiments, the symptom may be reduced by about 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%,
or a range defined by any two of these values.
[0490] In certain embodiments, provided are methods of treating an
individual comprising administering one or more pharmaceutical
compositions as described herein. In certain embodiments, the
individual has Huntington's disease.
[0491] In certain embodiments, administration of an antisense
compound targeted to a huntingtin nucleic acid results in reduction
of huntingtin expression by at least about 15, 20, 25, 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range
defined by any two of these values.
[0492] In certain embodiments, pharmaceutical compositions
comprising an antisense compound targeted to huntingtin are used
for the preparation of a medicament for treating a patient
suffering or susceptible to Huntington's disease.
F. Certain Pharmaceutical Compositions
[0493] 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.
[0494] 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.
[0495] 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.
[0496] 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.
[0497] 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.
[0498] 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.
[0499] 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.
[0500] 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.
[0501] In certain embodiments, a pharmaceutical composition
provided herein comprises a co-solvent system. Certain of such
co-solvent systems comprise, for example, benzyl alcohol, a
nonpolar surfactant, a water-miscible organic polymer, and an
aqueous phase. In certain embodiments, such co-solvent systems are
used for hydrophobic compounds. A non-limiting example of such a
co-solvent system is the VPD co-solvent system, which is a solution
of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the
nonpolar surfactant Polysorbate 80.TM. and 65% w/v polyethylene
glycol 300. The proportions of such co-solvent systems may be
varied considerably without significantly altering their solubility
and toxicity characteristics. Furthermore, the identity of
co-solvent components may be varied: for example, other surfactants
may be used instead of Polysorbate 80.TM.; the fraction size of
polyethylene glycol may be varied; other biocompatible polymers may
replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other
sugars or polysaccharides may substitute for dextrose.
[0502] In certain embodiments, a pharmaceutical composition
provided herein is prepared for oral administration. In certain
embodiments, pharmaceutical compositions are prepared for buccal
administration.
[0503] 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.
G. Administration
[0504] In certain embodiments, the compounds and compositions as
described herein are administered parenterally.
[0505] In certain embodiments, parenteral administration is by
infusion. Infusion can be chronic or continuous or short or
intermittent. In certain embodiments, infused pharmaceutical agents
are delivered with a pump. In certain embodiments, parenteral
administration is by injection.
[0506] In certain embodiments, compounds and compositions are
delivered to the CNS. In certain embodiments, compounds and
compositions are delivered to the cerebrospinal fluid. In certain
embodiments, compounds and compositions are administered to the
brain parenchyma. In certain embodiments, compounds and
compositions are delivered to an animal by intrathecal
administration, or intracerebroventricular administration. Broad
distribution of compounds and compositions, described herein,
within the central nervous system may be achieved with
intraparenchymal administration, intrathecal administration, or
intracerebroventricular administration.
[0507] In certain embodiments, parenteral administration is by
injection. The injection may be delivered with a syringe or a pump.
In certain embodiments, the injection is a bolus injection. In
certain embodiments, the injection is administered directly to a
tissue, such as striatum, caudate, cortex, hippocampus and
cerebellum.
[0508] Therefore, in certain embodiments, delivery of a compound or
composition described herein can affect the pharmacokinetic profile
of the compound or composition. In certain embodiments, injection
of a compound or composition described herein, to a targeted tissue
improves the pharmacokinetic profile of the compound or composition
as compared to infusion of the compound or composition. In a
certain embodiment, the injection of a compound or composition
improves potency compared to broad diffusion, requiring less of the
compound or composition to achieve similar pharmacology. In certain
embodiments, similar pharmacology refers to the amount of time that
a target mRNA and/or target protein is down-regulated (e.g.
duration of action). In certain embodiments, methods of
specifically localizing a pharmaceutical agent, such as by bolus
injection, decreases median effective concentration (EC50) by a
factor of about 50 (e.g. 50 fold less concentration in tissue is
required to achieve the same or similar pharmacodynamic effect). In
certain embodiments, methods of specifically localizing a
pharmaceutical agent, such as by bolus injection, decreases median
effective concentration (EC50) by a factor of 20, 25, 30, 35, 40,
45 or 50. In certain embodiments the pharmaceutical agent in an
antisense compound as further described herein. In certain
enbodiments, the targeted tissue is brain tissue. In certain
enbodiments the targeted tissue is striatal tissue. In certain
embodiments, decreasing EC50 is desirable because it reduces the
dose required to achieve a pharmacological result in a patient in
need thereof.
[0509] In certain embodiments, an antisense oligonucleotide is
delivered by injection or infusion once every month, every two
months, every 90 days, every 3 months, every 6 months, twice a year
or once a year.
H. Certain Combination Therapies
[0510] In certain embodiments, one or more pharmaceutical
compositions are co-administered with one or more other
pharmaceutical agents. In certain embodiments, such one or more
other pharmaceutical agents are designed to treat the same disease,
disorder, or condition as the one or more pharmaceutical
compositions described herein. In certain embodiments, such one or
more other pharmaceutical agents are designed to treat a different
disease, disorder, or condition as the one or more pharmaceutical
compositions described herein. In certain embodiments, such one or
more other pharmaceutical agents are designed to treat an undesired
side effect of one or more pharmaceutical compositions as described
herein. In certain embodiments, one or more pharmaceutical
compositions are co-administered with another pharmaceutical agent
to treat an undesired effect of that other pharmaceutical agent. In
certain embodiments, one or more pharmaceutical compositions are
co-administered with another pharmaceutical agent to produce a
combinational effect. In certain embodiments, one or more
pharmaceutical compositions are co-administered with another
pharmaceutical agent to produce a synergistic effect.
[0511] In certain embodiments, one or more pharmaceutical
compositions and one or more other pharmaceutical agents are
administered at the same time. In certain embodiments, one or more
pharmaceutical compositions and one or more other pharmaceutical
agents are administered at different times. In certain embodiments,
one or more pharmaceutical compositions and one or more other
pharmaceutical agents are prepared together in a single
formulation. In certain embodiments, one or more pharmaceutical
compositions and one or more other pharmaceutical agents are
prepared separately.
[0512] In certain embodiments, pharmaceutical agents that may be
co-administered with a pharmaceutical composition of include
antipsychotic agents, such as, e.g., haloperidol, chlorpromazine,
clozapine, quetapine, and olanzapine; antidepressant agents, such
as, e.g., fluoxetine, sertraline hydrochloride, venlafaxine and
nortriptyline; tranquilizing agents such as, e.g., benzodiazepines,
clonazepam, paroxetine, venlafaxin, and beta-blockers;
mood-stabilizing agents such as, e.g., lithium, valproate,
lamotrigine, and carbamazepine; paralytic agents such as, e.g.,
Botulinum toxin; and/or other experimental agents including, but
not limited to, tetrabenazine (Xenazine), creatine, conezyme Q10,
trehalose, docosahexanoic acids, ACR16, ethyl-EPA, atomoxetine,
citalopram, dimebon, memantine, sodium phenylbutyrate, ramelteon,
ursodiol, zyprexa, xenasine, tiapride, riluzole, amantadine,
[123I]MNI-420, atomoxetine, tetrabenazine, digoxin,
detromethorphan, warfarin, alprozam, ketoconazole, omeprazole, and
minocycline.
Nonlimiting Disclosure and Incorporation by Reference
[0513] 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.
[0514] 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).
[0515] Accordingly, nucleic acid sequences provided herein,
including, but not limited to those in the sequence listing, are
intended to encompass nucleic acids containing any combination of
natural or modified RNA and/or DNA, including, but not limited to
such nucleic acids having modified nucleobases. By way of further
example and without limitation, an oligomeric compound having the
nucleobase sequence "ATCGATCG" encompasses any oligomeric compounds
having such nucleobase sequence, whether modified or unmodified,
including, but not limited to, such compounds comprising RNA bases,
such as those having sequence "AUCGAUCG" and those having some DNA
bases and some RNA bases such as "AUCGATCG" and oligomeric
compounds having other modified or naturally occurring bases, such
as "AT.sup.meCGAUCG," wherein .sup.meC indicates a cytosine base
comprising a methyl group at the 5-position.
EXAMPLES
[0516] 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.
[0517] To allow assessment of the relative effects of nucleobase
sequence and chemical modification, throughout the examples,
oligomeric compounds are assigned a "Sequence Code." Oligomeric
compounds having the same Sequence Code have the same nucleobase
sequence. Oligomeric compounds having different Sequence Codes have
different nucleobase sequences.
Example 1: Single Nucleotide Polymorphisms (SNPs) in the Huntingtin
(HTT) Gene Sequence
[0518] SNP positions (identified by Hayden et al, WO/2009/135322)
associated with the HTT gene were mapped to the HTT genomic
sequence, designated herein as SEQ ID NO: 1 (NT_006081.18 truncated
from nucleotides 1566000 to 1768000). Table 5 provides SNP
positions associated with the HTT gene. Table 5 provides a
reference SNP ID number from the Entrez SNP database at the
National Center for Biotechnology Information (NCBI,
ncbi.nlm.nih.gov/sites/entrez?db=snp), incorporated herein by
reference. Table 5 furnishes further details on each SNP. The
`Reference SNP ID number` or `RS number` is the number designated
to each SNP from the Entrez SNP database at NCBI, incorporated
herein by reference. `SNP position` refers to the nucleotide
position of the SNP on SEQ ID NO: 1. `Polymorphism` indicates the
nucleotide variants at that SNP position. `Major allele` indicates
the nucleotide associated with the major allele, or the nucleotide
present in a statistically significant proportion of individuals in
the human population. `Minor allele` indicates the nucleotide
associated with the minor allele, or the nucleotide present in a
relatively small proportion of individuals in the human
population.
TABLE-US-00005 TABLE 5 Single Nuclear Polymorphisms (SNPs) and
their positions on SEQ ID NO: 1 SNP Major Minor RS No. position
Polymorphism allele allele rs2857936 1963 C/T C T rs12506200 3707
A/G G A rs762855 14449 A/G G A rs3856973 19826 G/A G A rs2285086
28912 G/A A G rs7659144 37974 C/G C G rs16843804 44043 C/T C T
rs2024115 44221 G/A A G rs10015979 49095 A/G A G rs7691627 51063
A/G G A rs2798235 54485 G/A G A rs4690072 62160 G/T.sup. T G
rs6446723 66466 C/T T C rs363081 73280 G/A G A rs363080 73564 T/C C
T rs363075 77327 G/A G A rs363064 81063 T/C C T rs3025849 83420 A/G
A G rs6855981 87929 A/G G A rs363102 88669 G/A A G rs11731237 91466
C/T C T rs4690073 99803 A/G G A rs363144 100948 .sup. T/G T G
rs3025838 101099 C/T C T rs34315806 101687 A/G G A rs363099 101709
T/C C T rs363096 119674 T/C T C rs2298967 125400 C/T T C rs2298969
125897 A/G G A rs6844859 130139 C/T T C rs363092 135682 C/A C A
rs7685686 146795 A/G A G rs363088 149983 A/T.sup. A T rs362331
155488 C/T T C rs916171 156468 G/C C G rs362322 161018 A/G A G
rs362275 164255 T/C C T rs362273 167080 A/G A G rs2276881 171314
G/A G A rs3121419 171910 T/C C T rs362272 174633 G/A G A rs362271
175171 G/A G A rs3775061 178407 C/T C T rs362310 179429 A/G G A
rs362307 181498 T/C C T rs362306 181753 G/A G A rs362303 181960 T/C
C T rs362296 186660 C/A C A rs1006798 198026 A/G A G
Example 2: Modified Oligonucleotides Targeting Huntingtin (HTT)
SNPs and Bone Morphogenetic Protein Receptor 1A (BMPR1A)
[0519] A series of modified oligonucleotides were designed to
target SNP positions associated with the HTT gene. These modified
oligonucleotides were evaluated for their ability to selectively
inhibit mutant (mut) HTT while leaving the expression of the
wild-type (wt) HTT and BMPR1A intact. In the tables, `k` subscript
indicates an (S)-cEt modification; `e` subscript indicates MOE
modification; `m` before the cytosine residue indicates a 5-methyl
cytosine; `x` before the thymine residue indicates a 2-thiothymine;
the number along with `d` indicates the number of deoxyribose
nucleosides; `o` subscript after the sugar modification subscripts
indicates a phosphodiester internucleoside linkage; `s` subscript
after the nucleoside indicates a phosphorothioate internucleoside
linkage.
[0520] As described above in Example 1, certain SNPs may have two
or more allelic variants. For example, the two allelic variants for
SNP rs7685686 are A and G. In certain embodiments, antisense
oligonucleotides can be designed that target either allelic
variant. In certain embodiments, a higher percentage of the
population may have a particular allelic variant. Modified
oligonucleotides were designed to target the G allelic variant of
rs7685686. These modified oligonucleotides are described further in
Table 6.
[0521] The modified oligonucleotides were tested in vitro.
Selective inhibition of the modified oligonucleotides targeting
BMPR1A and HTT SNP rs7685686 or rs7685686 (G) was evaluated.
Selective inhibition of modified oligonucleotides targeting SNP
rs6446723 with 4 mismatches to BMPR1A and targeting SNP rs363064
with 3 or 4 mismatches to BMPR1A were also evaluated. Human patient
fibroblasts GM04022 cell line was used. Cultured GM04022 cells at a
density of 35,000 cells per well were transfected using
electroporation at 130V with 0.0, 0.37, 1.1, 3.3 and 10 .mu.M
concentrations of modified oligonucleotides. After a treatment
period of approximately 24 hours, RNA was isolated from the cells.
Target message for HTT and BMPR1A were measured by quantitative
real-time PCR using ABI assay C_2229297_10 and RTS2623,
respectively. The target mRNA levels were adjusted according to
total RNA content, as measured by RIBOGREEN and the results are
presented in Table 7.
[0522] The IC.sub.50 of each modified oligonucleotide presented in
Table 7 was calculated by plotting the concentrations of
oligonucleotides used versus the percent inhibition of HTT or
BMPR1A mRNA expression achieved at each concentration, and noting
the concentration of oligonucleotide at which 50% inhibition of HTT
or BMPR1A mRNA expression was achieved compared to the control.
Selectivity for HTT was calculated by dividing the IC.sub.50 for
inhibition of the wild-type HTT versus the IC.sub.50 for inhibiting
expression of the mutant HTT mRNA. Selectivity for BMPR1A was
calculated by dividing the IC.sub.50 for inhibition of BMPR1A
versus the IC.sub.50 for inhibiting expression of the mutant HTT
mRNA.
[0523] ISIS 141923 and 387916 were included in the study as
negative and positive controls. ISIS 460209 or 572772 was also
included in the study for comparison.
TABLE-US-00006 TABLE 6 Modified oligonucleotides targeting
Huntingtin (HTT) SNPs and/or BMPR1A SEQ ID. Isis No. SNP Sequence
(5' to 3') Motif NO. 460209 rs7685686 T.sub.es A.sub.ks A.sub.ks
A.sub.ds T.sub.ds T.sub.ds G.sub.ds T.sub.ds mC.sub.ds A.sub.ds
T.sub.ds ekk-d9-kke 3 mC.sub.ds A.sub.ks mC.sub.ks mC.sub.e 572772
rs7685686 A.sub.es T.sub.es A.sub.es A.sub.es A.sub.ks T.sub.ks
T.sub.ds G.sub.ds T.sub.ds mC.sub.ds A.sub.ds eeeekk-d7-kke 4
T.sub.ds mC.sub.ds A.sub.ks mC.sub.ks mC.sub.e 551429 rs7685686
T.sub.es A.sub.es A.sub.es A.sub.ks T.sub.ks T.sub.ds G.sub.ds
T.sub.ds mC.sub.ds A.sub.ds T.sub.ds eeekk-d7-kke 3 mC.sub.ds
A.sub.ks mC.sub.ks mC.sub.e 556845 rs7685686 T.sub.es A.sub.ks
A.sub.ks A.sub.ds xT.sub.ds T.sub.ds G.sub.ds T.sub.ds mC.sub.ds
A.sub.ds ekk-d9-kke 3 T.sub.ds mC.sub.ds A.sub.ks mC.sub.ks
mC.sub.e 617425 rs7685686 A.sub.es T.sub.es A.sub.es A.sub.es
A.sub.es T.sub.ks T.sub.ds G.sub.ds T.sub.ds mC.sub.ds A.sub.ds
eeeeek-d7-eee 4 T.sub.ds mC.sub.ds A.sub.es mC.sub.es mC.sub.e
617115 rs7685686 A.sub.es T.sub.es A.sub.es A.sub.es A.sub.es
T.sub.ks T.sub.ds G.sub.ds T.sub.ds mC.sub.ds A.sub.ds
eeeeek-d7-kke 4 T.sub.ds mC.sub.ds A.sub.ks mC.sub.ks mC.sub.e
617116 rs7685686 A.sub.es T.sub.es A.sub.es A.sub.es A.sub.ks
T.sub.ks T.sub.ds G.sub.ds T.sub.ds mC.sub.ds A.sub.ds
eeeekk-d7-kee 4 T.sub.ds mC.sub.ds A.sub.ks mC.sub.es mC.sub.e
617117 rs7685686 A.sub.es T.sub.es A.sub.es A.sub.es A.sub.es
T.sub.ks T.sub.ds G.sub.ds T.sub.ds mC.sub.ds A.sub.ds
eeeeek-d7-kee 4 T.sub.ds mC.sub.ds A.sub.ks mC.sub.es mC.sub.e
617118 rs7685686 A.sub.es T.sub.eo A.sub.eo A.sub.eo A.sub.eo
T.sub.ks T.sub.ds G.sub.ds T.sub.ds mC.sub.ds eeeeek-d7-kee 4
A.sub.ds T.sub.ds mC.sub.ds A.sub.ks mC.sub.es mC.sub.e 617119
rs7685686 A.sub.es T.sub.eo A.sub.eo A.sub.eo A.sub.eo T.sub.ks
T.sub.ds G.sub.ds T.sub.ds mC.sub.ds eeeeek-d7-eee 4 A.sub.ds
T.sub.ds mC.sub.ds A.sub.es mC.sub.es mC.sub.e 617111 rs7685686
A.sub.es T.sub.ko A.sub.eo A.sub.ks A.sub.ds xT.sub.ds T.sub.ds
G.sub.ds T.sub.ds mC.sub.ds ekek-d9-keke 5 A.sub.ds T.sub.ds
mC.sub.ds A.sub.ko mC.sub.es mC.sub.ks Ae 613581 rs7685686 A.sub.es
A.sub.eo T.sub.eo A.sub.eo A.sub.es A.sub.ds T.sub.ks T.sub.ds
G.sub.ds T.sub.ds eeeee-d-k-d7- 6 mC.sub.ds A.sub.ds T.sub.ds
mC.sub.ds A.sub.eo mC.sub.eo mC.sub.es A.sub.es G.sub.e eeeee
613582 rs7685686 A.sub.es T.sub.es A.sub.eo A.sub.eo A.sub.es
T.sub.ks T.sub.ds G.sub.ds T.sub.ds mC.sub.ds A.sub.ds
eeeeek-d7-eeeeee 7 T.sub.ds mC.sub.ds A.sub.eo mC.sub.eo mC.sub.eo
A.sub.es G.sub.es A.sub.e 613583 rs7685686 T.sub.es A.sub.eo
A.sub.eo A.sub.es T.sub.ks T.sub.ds G.sub.ds T.sub.ds mC.sub.ds
A.sub.ds T.sub.ds eeeek-d7-eeeeeee 8 mC.sub.ds A.sub.es mC.sub.eo
mC.sub.eo A.sub.eo G.sub.es A.sub.es A.sub.e 613584 rs7685686
A.sub.es A.sub.eo A.sub.es T.sub.ks T.sub.ds G.sub.ds T.sub.ds
mC.sub.ds A.sub.ds T.sub.ds eeek-d7-eeeeeeee 9 mC.sub.ds A.sub.es
mC.sub.eo mC.sub.eo A.sub.eo G.sub.eo A.sub.es A.sub.es A.sub.e
613585 rs7685686 A.sub.es A.sub.eo T.sub.ks T.sub.ds G.sub.ds
T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds eek-d7-eeeeeeeee 10
A.sub.es mC.sub.es mC.sub.eo A.sub.eo G.sub.eo A.sub.eo A.sub.es
A.sub.es A.sub.e 613586 rs7685686 A.sub.es T.sub.ks T.sub.ds
G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.es
ek-d7-eeeeeeeeee 11 mC.sub.es mC.sub.eo A.sub.eo G.sub.eo A.sub.eo
A.sub.eo A.sub.es A.sub.es A.sub.e 613588 rs7685686 T.sub.es
A.sub.es A.sub.eo T.sub.eo A.sub.eo A.sub.eo A.sub.es T.sub.ks
T.sub.ds G.sub.ds T.sub.ds eeeeeeek-d7-eeee 12 mC.sub.ds A.sub.ds
T.sub.ds mC.sub.ds A.sub.eo mC.sub.es mC.sub.es A.sub.e 613589
rs7685686 T.sub.es T.sub.es A.sub.eo A.sub.eo T.sub.eo A.sub.eo
A.sub.eo A.sub.es T.sub.ks T.sub.ds G.sub.ds eeeeeeeek-d7-eee 13
T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.es mC.sub.es
mC.sub.e 617105 rs7685686 A.sub.es A.sub.eo A.sub.ko T.sub.ks
T.sub.ds G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds
eekk-d8-kkeee 14 mC.sub.ds A.sub.ds mC.sub.ko mC.sub.ko A.sub.es
G.sub.es A.sub.e 606561 rs7685686 A.sub.es T.sub.ks A.sub.es
A.sub.ks A.sub.ds xT.sub.ds T.sub.ds G.sub.ds T.sub.ds mC.sub.ds
A.sub.ds ekek-d9-keee 5 T.sub.ds mC.sub.ds A.sub.ks mC.sub.es
mC.sub.es A.sub.e 606562 rs7685686 A.sub.es T.sub.es A.sub.es
A.sub.ks A.sub.ds XT.sub.ds T.sub.ds G.sub.ds T.sub.ds mC.sub.ds
A.sub.ds eeek-d9-keee 5 T.sub.ds mC.sub.ds A.sub.ks mC.sub.es
mC.sub.es A.sub.e 611714 rs7685686 T.sub.es A.sub.es A.sub.es
A.sub.ks T.sub.ks T.sub.ds G.sub.ds mC.sub.ds mC.sub.ds A.sub.ds
eeekk-d7-kke 15 (G) T.sub.ds mC.sub.ds A.sub.ks mC.sub.ks mC.sub.e
611715 rs7685686 A.sub.es T.sub.ks A.sub.es A.sub.ks A.sub.ds
xT.sub.ds T.sub.ds G.sub.ds mC.sub.ds mC.sub.ds ekek-d9-keke 16 (G)
A.sub.ds T.sub.ds mC.sub.ds A.sub.ks mC.sub.es mC.sub.ks A.sub.e
611717 rs7685686 A.sub.es T.sub.es A.sub.es A.sub.es A.sub.ks
T.sub.ks T.sub.ds G.sub.ds mC.sub.ds mC.sub.ds eeeekk-d7-kke 17 (G)
A.sub.ds T.sub.ds mC.sub.ds A.sub.ks mC.sub.ks mC.sub.e 611718
rs7685686 T.sub.es A.sub.ks A.sub.ks A.sub.ds T.sub.ks T.sub.ds
G.sub.ds mC.sub.ds mC.sub.ds A.sub.ds ekk-d-k-d7-kke 15 (G)
T.sub.ds mC.sub.ds A.sub.ks mC.sub.ks mC.sub.e 611719 rs7685686
T.sub.es A.sub.ks A.sub.ks A.sub.ks T.sub.ks T.sub.ds G.sub.ds
mC.sub.ds mC.sub.ds A.sub.ds ekkkk-d7-kke 15 (G) T.sub.ds mC.sub.ds
A.sub.ks mC.sub.ks mC.sub.e 611720 rs7685686 A.sub.es T.sub.ks
T.sub.ds G.sub.ds mC.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds
A.sub.ds ek-d9-kkke 18 (G) mC.sub.ds mC.sub.ko A.sub.ko G.sub.ks
A.sub.e 611721 rs7685686 T.sub.es A.sub.es A.sub.es A.sub.es
T.sub.ks T.sub.ds G.sub.ds mC.sub.ds mC.sub.ds A.sub.ds
eeeek-d7-keee 19 (G) T.sub.ds mC.sub.ds A.sub.ks mC.sub.es
mC.sub.es A.sub.e 611722 rs7685686 A.sub.es T.sub.es A.sub.es
A.sub.es A.sub.ds T.sub.ks T.sub.ds G.sub.ds mC.sub.ds mC.sub.ds
eeee-d-k-d7-keee 16 (G) A.sub.ds T.sub.ds mC.sub.ds A.sub.ks
mC.sub.es mC.sub.es A.sub.e 611723 rs7685686 T.sub.es A.sub.eo
A.sub.eo A.sub.es T.sub.ks T.sub.ds G.sub.ds mC.sub.ds mC.sub.ds
A.sub.ds eeeek-d7-keeee 20 (G) T.sub.ds mC.sub.ds A.sub.ks
mC.sub.eo mC.sub.eo A.sub.es G.sub.e 617104 rs6446723 T.sub.es
A.sub.es A.sub.eo T.sub.ko T.sub.ks T.sub.ds T.sub.ds mC.sub.ds
T.sub.ds A.sub.ds G.sub.ds eeekk-d7-kkeee 21 A.sub.ds mC.sub.ko
T.sub.ko T.sub.es T.sub.es A.sub.e 617106 rs6446723 T.sub.es
A.sub.eo A.sub.ko T.sub.ks T.sub.ds T.sub.ds T.sub.ds mC.sub.ds
T.sub.ds A.sub.ds G.sub.ds eekk-d8-kkeee 21 A.sub.ds mC.sub.ko
T.sub.ko T.sub.es T.sub.es A.sub.e 617107 rs363064 A.sub.es
A.sub.eo T.sub.ko A.sub.ks mC.sub.ds G.sub.ds G.sub.ds G.sub.ds
T.sub.ds A.sub.ds eekk-d8-kkeee 38 A.sub.ds mC.sub.ds A.sub.ko
T.sub.ko T.sub.es T.sub.es T.sub.e 617108 rs6446723 T.sub.es
A.sub.ko A.sub.eo T.sub.ks T.sub.ds T.sub.ds T.sub.ds mC.sub.ds
T.sub.ds A.sub.ds G.sub.ds ekek-d8-kekee 21 A.sub.ds mC.sub.ko
T.sub.eo T.sub.ks T.sub.es A.sub.e 617109 rs6446723 A.sub.es
A.sub.eo T.sub.ko T.sub.ks T.sub.ds T.sub.ds mC.sub.ds T.sub.ds
A.sub.ds G.sub.ds A.sub.ds eekk-d8-kkeee 22 mC.sub.ds T.sub.ko
T.sub.ko T.sub.es A.sub.es T.sub.e 617110 rs363064 G.sub.es
A.sub.es A.sub.eo T.sub.ko A.sub.ks mC.sub.ds G.sub.ds G.sub.ds
G.sub.ds T.sub.ds eeekk-d7-kkeee 39 A.sub.ds A.sub.ds mC.sub.ko
A.sub.ko T.sub.es T.sub.es T.sub.e 623182 rs7685686 A.sub.es
T.sub.ks T.sub.ds G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds
mC.sub.ds A.sub.ds ek-d8-eeekk 23 mC.sub.es mC.sub.es A.sub.eo
G.sub.ks A.sub.k 623202 rs7685686 T.sub.ks T.sub.ds G.sub.ds
T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds mC.sub.ds
k-d9-eekeke 24 mC.sub.es A.sub.eo G.sub.ko A.sub.es A.sub.ks
A.sub.e 623203 rs7685686 T.sub.ks T.sub.ds G.sub.ds T.sub.ds
mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds mC.sub.es
k-d8-eeekeke 24 mC.sub.es A.sub.eo G.sub.ko A.sub.es A.sub.ks
A.sub.e 623205 rs7685686 T.sub.ks T.sub.ds G.sub.ds T.sub.ds
mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds mC.sub.ds
k-d9-eekekee 25 mC.sub.es A.sub.eo G.sub.ko A.sub.es A.sub.ks
A.sub.es A.sub.e 623206 rs7685686 T.sub.ks T.sub.ds G.sub.ds
T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds mC.sub.es
k-d8-eeekekee 25 mC.sub.es A.sub.eo G.sub.ko A.sub.es A.sub.ks
A.sub.es A.sub.e 623212 rs7685686 T.sub.ks T.sub.ds G.sub.ds
T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds mC.sub.ks
k-d8-keeekee 24 mC.sub.es A.sub.eo G.sub.eo A.sub.ks A.sub.es
A.sub.e 623214 rs7685686 T.sub.ks T.sub.ds G.sub.ds T.sub.ds
mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds mC.sub.ds
k-d9-keekeee 25 mC.sub.ks A.sub.eo G.sub.eo A.sub.ks A.sub.es
A.sub.es A.sub.e 623218 rs7685686 A.sub.es A.sub.es T.sub.ks
T.sub.ds G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds
eek-d8-eeek 26 A.sub.ds mC.sub.eo mC.sub.es A.sub.es G.sub.k 623220
rs7685686 A.sub.es A.sub.es T.sub.ks T.sub.ds G.sub.ds T.sub.ds
mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds eek-d9-eeke 27 A.sub.ds
mC.sub.ds mC.sub.eo A.sub.es G.sub.ks A.sub.e 623221 rs7685686
A.sub.es A.sub.es T.sub.ks T.sub.ds G.sub.ds T.sub.ds mC.sub.ds
A.sub.ds T.sub.ds mC.sub.ds eek-d8-eeeke 27 A.sub.ds mC.sub.eo
mC.sub.eo A.sub.es G.sub.ks A.sub.e 623224 rs7685686 A.sub.es
A.sub.es T.sub.ks T.sub.ds G.sub.ds T.sub.ds mC.sub.ds A.sub.ds
T.sub.ds mC.sub.ds eek-d8-eeekek 28 A.sub.ds mC.sub.es mC.sub.eo
A.sub.eo G.sub.ks A.sub.es A.sub.k 623227 rs7685686 A.sub.es
A.sub.es T.sub.ks T.sub.ds G.sub.ds T.sub.ds mC.sub.ds A.sub.ds
T.sub.ds mC.sub.ds eek-d8-keee 26 A.sub.ds mC.sub.ko mC.sub.es
A.sub.es G.sub.e 623233 rs7685686 A.sub.es A.sub.es T.sub.ks
T.sub.ds G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds
eek-d8-keeeke 28 A.sub.ds mC.sub.ks mC.sub.eo A.sub.eo G.sub.es
A.sub.ks A.sub.e 623237 rs7685686 A.sub.es T.sub.ks T.sub.ds
G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.es
ek-d7-eeeekeke 29 mC.sub.es mC.sub.es A.sub.eo G.sub.ko A.sub.es
A.sub.ks A.sub.e 623239 rs7685686 A.sub.es T.sub.ks T.sub.ds
G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds
ek-d8-keeekee 29 mC.sub.ks mC.sub.es A.sub.eo G.sub.eo A.sub.ks
A.sub.es A.sub.e 623242 rs7685686 A.sub.es T.sub.ks T.sub.ds
G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds
ek-d8-eeekek 30 mC.sub.es mC.sub.eo A.sub.eo G.sub.ks A.sub.es
A.sub.k 623254 rs7685686 A.sub.es A.sub.es T.sub.ks T.sub.ds
G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds
eek-d9-eekek 31 A.sub.ds mC.sub.ds mC.sub.eo mC.sub.eo G.sub.ks
A.sub.es A.sub.k 623262 rs7685686 A.sub.es T.sub.ks T.sub.ds
G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds
ek-d8-eeekeke 32 mC.sub.es mC.sub.es mC.sub.eo G.sub.ko A.sub.es
A.sub.ks A.sub.e 623490 rs7685686 T.sub.ks T.sub.ds G.sub.ds
T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds mC.sub.ds
k-d9-eekekee 33 mC.sub.es A.sub.eo G.sub.ko mC.sub.es A.sub.ks
A.sub.es A.sub.e 623494 rs7685686 A.sub.es T.sub.ks T.sub.ds
G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds
ek-d8-eeekeke 34 mC.sub.es mC.sub.es A.sub.eo G.sub.ko T.sub.es
A.sub.ks A.sub.e 387916 mC.sub.es mC.sub.es T.sub.es T.sub.es
mC.sub.es mC.sub.ds mC.sub.ds T.sub.ds G.sub.ds eeeee-d10-eeeee 35
(pos control) A.sub.ds A.sub.ds G.sub.ds G.sub.ds T.sub.ds T.sub.ds
mC.sub.es mC.sub.es T.sub.es mC.sub.es mC.sub.e 141923 T.sub.es
mC.sub.es T.sub.es mC.sub.es T.sub.es A.sub.ds T.sub.ds T.sub.ds
G.sub.ds mC.sub.ds eeeee-d10-eeeee 36
(neg control) A.sub.ds mC.sub.ds A.sub.ds T.sub.ds T.sub.ds
mC.sub.es mC.sub.es A.sub.es A.sub.es G.sub.e
TABLE-US-00007 TABLE 7 Selectivity of modified oligonucleotides
targeting Huntingtin (HTT) SNPs and/or BMPR1A HTT Mut BMPR1A HTT
BMPR1A SEQ ID ISIS NO. SNP IC.sub.50 (.mu.M) IC.sub.50 (.mu.M)
Selectivity Selectivity Motif NO. 460209.sup.1 rs7685686 <0.4
1.35 5.6 3.4 ekk-d9-kke 3 572772.sup.2 rs7685686 0.27 2.23 >37
8.3 eeeekk-d7-kke 4 551429 rs7685686 <0.4 1.2 >25 >3
eeekk-d7-kke 3 556845 rs7685686 <0.4 1.5 >25 >3.8
ekk-d9-kke 3 617425 rs7685686 1.3 5 >8 3.8 eeeeek-d7-eee 4
617115 rs7685686 <0.4 1.9 >25 >5 eeeeek-d7-kke 4 617116
rs7685686 <0.4 1.7 >25 >4 eeeekk-d7-kee 4 617117 rs7685686
0.7 3.1 >14 4 eeeeek-d7-kee 4 617118 rs7685686 0.4 1.8 >25 5
eeeeek-d7-kee 4 617119 rs7685686 0.8 2.9 >13 4 eeeeek-d7-eee 4
617111 rs7685686 <0.4 1.0 >25 3 ekek-d9-keke 5 613581
rs7685686 0.9 4.4 >11 5 eeeeedk-d7-eeeee 6 613582 rs7685686 0.4
5.1 >25 13 eeeeek-d7-eeeeee 7 613583 rs7685686 0.7 4.9 >14 7
eeeek-d7-eeeeeee 8 613584 rs7685686 0.4 5.6 >25 14
eeek-d7-eeeeeeee 9 613585 rs7685686 0.4 4.8 >25 11
eek-d7-eeeeeeeee 10 613586 rs7685686 0.7 >10 >14 15
ek-d7-eeeeeeeeee 11 613588 rs7685686 0.7 4.4 >14 6
eeeeeeek-d7-eeee 12 613589 rs7685686 1.2 5.6 >8 5
eeeeeeeek-d7-eee 13 617105 rs7685686 <0.4 2 15 5 eekk-d8-kkeee
14 606561 rs7685686 <4 2.2 25 25 ekek-d9-keee 5 606562 rs7685686
0.7 3.4 25 8 eeek-d9-keee 5 611714 rs7685686 (G) 0.6 9.4 8 16
eeekk-d7-kke 15 611715 rs7685686 (G) 0.6 9.0 9 14 ekek-d9-keke 16
611717 rs7685686 (G) 0.8 10 6 12 eeeekk-d7-kke 17 611718 rs7685686
(G) 0.8 >10 9 13 ekk-d-k-d7-kke 15 611719 rs7685686 (G) 0.9
>10 4 11 ekkkk-d7-kke 15 611720 rs7685686 (G) <0.4 9.4 6 23
ek-d9-kkke 18 611721 rs7685686 (G) 0.9 >10 >11 11
eeeek-d7-keee 19 611722 rs7685686 (G) 1.5 >10 >7 7
eeee-d-k-d7-keee 16 611723 rs7685686 (G) 2.7 >10 4 4
eeeek-d7-keeee 20 617104 rs6446723 <0.4 >10 17 >25
eeekk-d7-kkeee 21 617106 rs6446723 <0.4 >10 14 >25
eekk-d8-kkeee 21 617108 rs6446723 <0.4 >10 14 >25
ekek-d8-kekee 21 617109 rs6446723 <0.4 >10 9 >25
eekk-d8-kkeee 22 623182 rs7685686 0.6 >10 17 17 ek-d8-eeekk 23
623202 rs7685686 0.8 >10 4 13 k-d9-eekeke 24 623203 rs7685686
0.8 >10 12 12 k-d8-eeekeke 24 623205 rs7685686 0.6 >10 3 17
k-d9-eekekee 25 623206 rs7685686 0.9 >10 11 11 k-d8-eeekekee 25
623212 rs7685686 1.2 >10 8 8 k-d8-keeekee 24 623214 rs7685686
1.5 >10 7 7 k-d9-keekeee 25 623218 rs7685686 1.4 >10 7 7
eek-d8-eeek 26 623220 rs7685686 1.6 >10 4 6 eek-d9-eeke 27
623221 rs7685686 1.2 >10 8 8 eek-d8-eeeke 27 623224 rs7685686
0.9 >10 11 11 eek-d8-eeekek 28 623227 rs7685686 0.9 6.9 11 8
eek-d8-keee 26 623233 rs7685686 0.6 5.4 16 9 eek-d8-keeeke 28
623237 rs7685686 0.6 9.8 18 17 ek-d7-eeeekeke 29 623239 rs7685686
0.5 7.7 19 15 ek-d8-keeekee 29 623242 rs7685686 0.6 9.9 18 18
ek-d8-eeekek 30 623254 rs7685686 1.2 4.4 5 4 eek-d9-eekek 31 623262
rs7685686 1.0 4.4 10 4 ek-d8-eeekeke 32 623490 rs7685686 1.3 4.9 2
4 k-d9-eekekee 33 623494 rs7685686 0.4 6.2 25 15 ek-d8-eeekeke 34
387916 <0.4 >10 2 25 eeeee-d10-eeeee 35 (pos control) 141923
>10 >10 1 1 eeeee-d10-eeeee 36 (neg control) .sup.1IC.sub.50
measured from average of 2 independent assays .sup.2IC.sub.50
measured from average of 3 independent assays
Example 3: Modified Oligonucleotides Targeting HTT SNP rs7685686
and BMPR1A
[0524] A series of modified oligonucleotides were designed to
target SNP positions associated with the HTT gene and BMPR1A. These
modified oligonucleotides were evaluated for their ability to
selectively inhibit mutant (mut) HTT while leaving the expression
of the wild-type (wt) HTT and BMPR1A intact. In the tables, `k`
subscript indicates an (S)-cEt modification; `e` subscript
indicates MOE modification; `m` before the cytosine residue
indicates a 5-methyl cytosine; `x` before the thymine residue
indicates a 2-thiothymine; the number along with `d` indicates the
number of deoxyribose nucleosides; `o` subscript after the sugar
modification subscripts indicates a phosphodiester internucleoside
linkage; `s` subscript after the nucleoside indicates a
phosphorothioate internucleoside linkage.
[0525] The modified oligonucleotides presented in Table 8 were
tested in vitro. Selective inhibition of the modified
oligonucleotides targeting HTT SNP rs7685686 and BMPR1A was
evaluated. Human patient fibroblasts GM04022 cell line was used.
Cultured GM04022 cells at a density of 35,000 cells per well were
transfected using electroporation at 130V with 0.0, 0.12, 0.37,
1.1, 3.3 and 10 M concentrations of modified oligonucleotides.
After a treatment period of approximately 24 hours, RNA was
isolated from the cells. Target message for HTT and BMPR1A were
measured by quantitative real-time PCR using ABI assay C_2229297_10
and RTS2623, respectively. The target mRNA levels were adjusted
according to total RNA content, as measured by RIBOGREEN and the
results are presented in Table 9.
[0526] The IC.sub.50 of each modified oligonucleotide along with
HTT and BMPR1A selectivity were calculated in the same manner as
described in Example 2. Results are the average of 2 independent
assays and are presented in Table 9.
[0527] ISIS 141923 and 387916 were included in the study negative
and positive control. ISIS 460209 or 572772 was also included in
the study for comparison.
TABLE-US-00008 TABLE 8 Modified oligonucleotides targeting
Huntingtin (HTT) SNP rs7685686 and BMPR1A ISIS NO. SNP Sequence (5'
to 3') Motif SEQ ID. NO. 623181 rs7685686 A.sub.es T.sub.ks
T.sub.ds G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds
A.sub.ds ek-d9-eekk 23 mC.sub.ds mC.sub.es A.sub.eo G.sub.ks
A.sub.k 623198 rs7685686 A.sub.es T.sub.ks T.sub.ds G.sub.ds
T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds ek-d9-keek
23 mC.sub.ds mC.sub.ks A.sub.eo G.sub.es A.sub.k 623199 rs7685686
T.sub.ks T.sub.ds G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds
mC.sub.ds A.sub.ds k-d9-eekek 37 mC.sub.ds mC.sub.es A.sub.eo
G.sub.ks A.sub.es A.sub.k 623208 rs7685686 T.sub.ks T.sub.ds
G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds
k-d9-keekk 37 mC.sub.ds mC.sub.ks A.sub.eo G.sub.es A.sub.ks
A.sub.k 623230 rs7685686 A.sub.es A.sub.es T.sub.ks T.sub.ds
G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds
eek-d8-kkeek 27 A.sub.ds mC.sub.ko mC.sub.ko A.sub.es G.sub.es
A.sub.k 623232 rs7685686 A.sub.es A.sub.es T.sub.ks T.sub.ds
G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds
eek-d9-keeke 28 A.sub.ds mC.sub.ds mC.sub.ks A.sub.eo G.sub.es
A.sub.ks A.sub.e 623235 rs7685686 A.sub.es T.sub.ks T.sub.ds
G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds
ek-d9-eekeke 29 mC.sub.ds mC.sub.es A.sub.eo G.sub.ko A.sub.es
A.sub.ks A.sub.e 623236 rs7685686 A.sub.es T.sub.ks T.sub.ds
G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds
ek-d8-eeekeke 29 mC.sub.es mC.sub.es A.sub.eo G.sub.ko A.sub.es
A.sub.ks A.sub.e 623238 rs7685686 A.sub.es T.sub.ks T.sub.ds
G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds
ek-d9-keekee 29 mC.sub.ds mC.sub.ks A.sub.eo G.sub.eo A.sub.ks
A.sub.es A.sub.e 623241 rs7685686 A.sub.es T.sub.ks T.sub.ds
G.sub.ds T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds
ek-d9-eekek 30 mC.sub.ds mC.sub.eo A.sub.eo G.sub.ks A.sub.es
A.sub.k 623243 rs7685686 A.sub.es T.sub.ks T.sub.ds G.sub.ds
T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.es
ek-d7-eeeekek 30 mC.sub.es mC.sub.eo A.sub.eo G.sub.ks A.sub.es
A.sub.k 623493 rs7685686 A.sub.es T.sub.ks T.sub.ds G.sub.ds
T.sub.ds mC.sub.ds A.sub.ds T.sub.ds mC.sub.ds A.sub.ds
ek-d9-eekeke 34 mC.sub.ds mC.sub.es A.sub.eo G.sub.ko T.sub.es
A.sub.ks A.sub.e
TABLE-US-00009 TABLE 9 Selectivity of modified oligonucleotides
targeting HTT SNP rs7685686 and BMPR1A HTT Mut BMPR1A HTT BMPR1A
SEQ ID ISIS NO. SNP IC.sub.50 (.mu.M) IC.sub.50 (.mu.M) Selectivity
Selectivity Motif NO. 460209.sup.1 rs7685686 <0.4 1.35 5.6 3.4
ekk-d9-kke 3 572772.sup.2 rs7685686 0.27 2.23 >37 8.3
eeeekk-d7-kke 4 623181 rs7685686 0.35 8.9 9.3 25.4 ek-d9-keek 23
623198 rs7685686 0.4 9.25 19.5 23.1 k-d9-eekek 23 623199 rs7685686
0.35 10 9.4 28.6 eek-d8-kkeek 37 623208 rs7685686 0.3 10 33.3 33.3
eek-d9-keeke 37 623230 rs7685686 0.35 4.6 28.6 13.1 ek-d9-eekeke 27
623232 rs7685686 0.5 7.4 15.7 14.8 ek-d8-eeekeke 28 623235
rs7685686 0.4 9.75 4.1 24.4 ek-d9-keekee 29 623236 rs7685686 0.4
8.55 21.8 21.4 ek-d9-eekek 29 623238 rs7685686 0.35 6.05 21.4 17.3
ek-d7-eeeekek 29 623241 rs7685686 0.4 6.85 5.5 17.1 ek-d9-eekeke 30
623243 rs7685686 0.55 10 18.2 18.2 ek-d9-keek 30 623493 rs7685686
0.45 5.45 7.3 12.1 k-d9-eekek 34 387916 <0.4 >10 2 25
eeeee-d10-eeeee 35 (pos control) 141923 >10 >10 1 1
eeeee-d10-eeeee 36 (neg control) .sup.1IC.sub.50 measured from
average of 2 independent assays .sup.2IC.sub.50 measured from
average of 3 independent assays
Example 4: Tolerability of Modified Oligonucleotides Targeting HTT
SNPs and BMPR1A
[0528] BALB/c wild type mice were separated into groups of 4 mice.
Each mouse was administered a single 300 .mu.g ICV dose of a
modified oligonucleotide or a single ICV dose of PBS. At 3 hours
post injection, each mouse was evaluated according to 7 different
criteria. The 7 criteria are (1) the mouse was bright, alert, and
responsive; (2) the mouse was standing or hunched without stimuli;
(3) the mouse shows any movement without stimuli (4) the mouse
demonstrates forward movement after it is lifted; (5) the mouse
demonstrates any movement after it is lifted; (6) the mouse
responds to a tail pinch; (7) the mouse has a regular respiratory
rate. For each of the 7 different criteria, each mouse was given a
sub-score of 0 if it met the criteria or 1 if it did not. After
each of the 7 criteria were evaluated, the sub-scores were summed
for each mouse and then averaged for each group. For example, if a
mouse was bright, alert, and responsive 3 hours after the 300 .mu.g
ICV dose, and met all other criteria, it would get a summed score
of 0. If another mouse was not bright, alert, and responsive 3
hours after the 300 .mu.g ICV dose but met all other criteria, it
would receive a score of 1. PBS treated mice generally receive a
score of 0. A score of at the top end of the range would be
suggestive of acute toxicity.
[0529] Each mouse was then evaluated weekly by a trained observer
for 8 weeks and examined for adverse events (AEs). Adverse events
are defined as any behavior not typical in a naive matched control
animal. Animals were evaluated for adverse events including, but
not limited to: limb clasping, abnormal limb splay, abnormal gait,
tremors, abnormal respiration, paralysis, spasticity, impaired
righting reflex, hyperactivity and lethargy. For each group, the
number of animals that exhibited any adverse events during any of
the 8 weekly observations was calculated. For example, a group of
animals where no animals exhibited any adverse events is given a
score of 0.
[0530] Body weights were also measured throughout the study.
Results are presented as the average percent weight change for each
group, relative to the average pre-treatment weight for the
group.
[0531] Animals were sacrificed at 8 weeks. Brain and lumbar
sections of the spinal cords were collected from each animal, and
RT-PCR was performed. Expression levels of allograft inflammatory
factor (AIF1) were determined as a measure of imflammation.
Expression levels of glial fibrillary acidic protein (GFAP) were
also determined for some samples as a measure of glial cell
activation. Cyclophilin levels were determined as a control. After
normalization of all samples to cyclophilin, the values of AIF1
and/or GFAP for each treatment group were divided by the normalized
value for the PBS control group in order to determine the percent
AIF1 or GFAP expression relative to the PBS treated animals. Data
shown below represent the mean for each group.
[0532] The results of the tolerability studies are presented in
Tables 10, 11, and 12.
TABLE-US-00010 TABLE 10 Tolerability of modified oligonucleotides
targeting HTT SNP rs6446723, HTT SNP rs363064, or HTT SNP rs7685686
and BMPR1A Body weight AIF1 in AIF1 in Isis Score No. of mice at 8
weeks spinal cord cortex No. at 3 hrs with an AE (% change) (% PBS)
(% PBS) PBS 0.00 0 116 100 100 611714 2.25 1 101 292 153 611715
2.25 0 104 399 202 611717 3.75 0 106 353 92 611718 2.50 4 91 294
155 611719 0.50 1 105 213 148 611720 2.00 4 87 1199 127 611721 1.50
3 118 216 153 611722 1.75 3 107 271 150 611723 1.25 3 110 114 113
613581 1.50 3 105 95 131 613582 0.00 4 116 103 118 613583 0.00 4
115 132 95 613584 0.00 3 117 97 103 613585 0.00 3 113 99 103 613586
0.00 2 115 89 101 613588 0.25 2 116 81 131 613589 0.00 2 107 95 122
617104 1.00 1 112 121 160 617105 4.00 2 125 124 130 617106 0.25 3
112 110 116 617107 1.25 2 111 100 110 617108 0.25 3 115 97 99
617109 0.00 3 108 115 105 617110 1.75 3 115 103 131 617111 0.00 2
122 116 118 617115 0.75 3 125 111 141 617116 0.00 2 112 96 137
617117 1.25 4 107 105 109 617118 0.00 2 115 98 117 617119 0.00 3
120 93 116 617425 0.00 3 116 101 94
TABLE-US-00011 TABLE 11 Tolerability of modified oligonucleotides
targeting HTT SNP rs7685686 and BMPR1A Body weight AIF1 in AIF1 in
GFAP in GFAP in Score No. of mice at 8 weeks spinal cord cortex
spinal cord cortex Isis No. at 3 hrs with an AE (% change) (% PBS)
(% PBS) (% PBS) (% PBS) PBS 0.00 0 116 100 100 100 100 623202 1.25
0 122 107 100 82 104 623205 0.25 0 127 104 116 73 137 623206 0.50 0
126 72 112 68 178 623235 0.25 1 129 168 93 130 124 623241 1.50 0
128 134 116 81 120 623242 1.25 0 116 81 109 77 108
TABLE-US-00012 TABLE 12 Tolerability of modified oligonucleotides
targeting HTT SNP rs7685686 and BMPR1A Body weight AIF1 in AIF1 in
Isis Score No. of mice at 8 weeks spinal cord cortex No. at 3 hrs
with an AE (% change) (% PBS) (% PBS) PBS 0.00 0 123 100 100 623181
3.75 1 126 115 84 623182 2.50 0 128 94 103 623198 1.25 1 136 126
103 623203 0.75 0 127 116 90 623208 0.75 0 128 117 104 623214 0.50
2 121 118 76 623236 0.25 0 121 125 88 623237 0.25 0 126 161 108
623243 0.50 0 123 103 80
Sequence CWU 1
1
391202001DNAHomo sapiens 1gcccagcagg tgtcagcctc attttacccc
gcccctattc aagatgaagt tgttctggtt 60ccaacgcctc tgacatatta gctgcatcat
tttacatttc tttttttttt ttccttttaa 120atggggtctt gctctgtcac
ccaggctgga gtgctgtggt atgatctcgg ctcactgcaa 180tctccacctc
cgaggttcca gcgattctct tgcctcagcc tcccgagtag ctgggactac
240aggcacccac catcatactg ggctaatttt tgtgttttta gtagagatgg
ggtttcccca 300tgttgcccag gctgatctca aactcctggg cttaagcaat
acagccgcgt tggcctccca 360aagtgttggg attacaagca tgagctaccc
cacccagctc attttacatt tccacttgtt 420aaactgaaaa ctggcccgag
aaagcttctg tactgccatc cttgcgtcct tgcagatgaa 480tcgtaaccta
gcatagtagg taggcagact gaaaacctaa cttagcagta ggcttctgta
540acaacagctg tgtctcagcc agttcctgca gccagacttc aaccactcac
aggccgcaaa 600ctgttcaaac tgtgttcgga gaaggcgaat tcatctggct
gttaacgtgc ctcacttctg 660ctttctgtgg ccactttccc ttttctgtcc
ataaatttgc tttgaccaca cagcatccct 720agagtctccc tgaatctgct
gtgattctgg gacctgcacc atttgtgaat tgtttttttt 780ttccttgatc
agctaaactc tgttcaattc aatttgttgg aagtttttaa cataccaatg
840gtgcaccaag gttccaattt ctccacttcc tcataaataa gtcattttaa
atggcttttc 900agtattccaa tatttggaag tattaatgtt tctaccaatt
ttctattttt ggacattgag 960gttgtttcat tttttttttc tttttttgag
acagagtctc gctccgtcac ccaggctgga 1020gtgcagtggc ctgatcccgg
cccactgcaa cctccacctc cctcctcagc ctcctgagta 1080gctgggatta
caggtgcatg caccaccaca cccagctaat ttttgtattt ttagtagaga
1140tggggtttca ccatgttggt caggctggtc tcaaactcct gacctcaggt
ggtccacctg 1200ccttggcctc ccaaaatgct gggattacag gcctgagcca
ctgcgcctgg cctcatcttc 1260ttgatattaa tgttgcttta acatctttgt
ccctgtgttt tttgtttttt tttttgagac 1320ggagtctcat tcattctgtc
acccaggctg gagttcagtg gcgtgatctc agctcactgc 1380aacctctgtc
tcctgggttc cagtgattct cctgcgtcgg tctcctgagt agctgtgttc
1440ctgggtcttt cgatggttat ttaatacttc cctacagtaa tgccctgtgc
gtacatgcta 1500agtgtgatga aatggttggc acagttaaat cttttgaaag
acattgccaa gtcactcttc 1560agaaaagtga taggaggtca tagcaatttt
aagaagtcct catttctaca tttccttact 1620aatctcggtt ggtgtctctt
caatctttcc tcacactttt cttgggtttt tcctgaatca 1680tgagtctact
acatttacac attttaaagc atctttagaa acaggatctc attttgttgc
1740ccaggctaga gtttggtggc atgattatag ctcctcatac tcctgggctc
aagtgatcct 1800tccacctctg aaaccccaaa atttgagaaa ggtctcattt
aatttagaaa gtttattttg 1860ccaaggttga gggtgcacac ctgtgatgat
atacgagtta aaaagaaatt atttaggcag 1920atactgaggg taagaaagtc
ctcggtaagg ttttcttttc aatgaaaagc agcccccaag 1980cattttcttt
tctaacaaag agcagcctgt aaaatcgagc tgcagacata cacaagcaag
2040ctggaagctt gcacaggtga atgctggcag ctgtgccaat aagaaaaggc
tacctggggc 2100caggcagatc caacatggcg gctccatctt ccctttcctt
gtcaaccatg tgcacagtaa 2160ggagcaggca acatagtgtc ccccgagtag
agaccaattt gcataataaa aggtgagggt 2220agggtgggca gcttctttgc
atgctatgta aacattatgc ctggtccaac caatctttgg 2280gccctgtgta
aattagacac cacctcctca agcctgtcta taaaaccctg tccattctgc
2340cgcaggctgg aagacccact ggggcacccc tctctctcta taggagacag
ctattcattt 2400ttctctttct ttcacctatt aaagctccac tcttaacccc
actccgtgtg tatctatgtt 2460cttgatttcc ttggcatgag gcaatgaacc
ttgggtatta ccccagaacc ttgggtatta 2520tgccacttca gtgacacagc
ctcaggaaat cctgatgaca tgttcccaag atggtcgggg 2580cacagcttgg
ttttatacat tttagggaga catgagacgt caattcatat atgtaagaag
2640tacattggtt ccgtccagaa aggcggggac aacttgaggc agggagagag
cttctaggtc 2700acaggtagac aaatggttgc attcttttga atctccgata
agcctttcca aaggaggcaa 2760tcagaatatg cgtctattga ctgggcgcag
tggctcatgc ctgtaatgcc agcactttgg 2820gaggcggagg tgggtggatc
acctgaggtc aggagtttga gagcagcccg gccaacatgg 2880tgaaaccctg
tctctactaa aaatacaaaa aattagctgg gcgtggtggc gggcgcctgt
2940aatcccagct actcgggagg ctgaggcagg agaatagctt gaacccagaa
ggaagaggtt 3000gcagtgagct gagatggtgc cattgcactc cagcctgggc
aacaagagtg aaactccatc 3060tcagaaaaaa aaaaaaaagg cctgggcaaa
gtggctcacg cctgtaatcc cagcactttg 3120ggaagccgag gcgggcaggt
cacaaagtca ggagattgag accatcctgg ctaacatgat 3180gaaaccccat
ctctactaaa aaatacaaaa aactagctgg gtgtggtggc gagcacctgt
3240agtcccagct actcggcagg ctgaggcagg agaatggcgt gaaccgggga
ggcggagctt 3300gcagtgagcc gagatcacac cactgcactc cagcccggac
gacagggcaa gactctatct 3360caaattaaaa aaaaaaaaaa aaaaaaaaaa
aaagagagag agaatatgca tctatctcag 3420tgagcagaag gatgactttg
aatggaatgg gagcagttcc tagcttgaac ttccccttta 3480gcttcagtga
tttgggggct caaggtatgt tcctttcaca tacctcagcc tcccaagtag
3540ctgggaccac aagtgcatgc caccacacgt ggctaatgtt ttattttttt
tgtaggaata 3600gggtctcact atgtgtccag gctggtctaa aacccctgag
ctcaaatggt cctcccgcct 3660cagcctcccg aaatgctggg attacaggca
tgagccagca tgcccggcct agtctacatt 3720tttataaatt gctaattcaa
agttccctct ccaaaacctc atggttttcc ctgttctcat 3780cccctgcacc
ctcccttccc ctggagtact cacctggcct tggaggtctg gtgtgagccc
3840ggacttcgat tctaggcaca gcatgtgatg agcgccccca ggtcaaacac
ctcccctctg 3900cggcctgtgc ttcaccgcct tgacagtgag aaaggtctcc
cttcggctca ttctcgaagt 3960ctcaaacttc acttctcctg tgcgctgatt
ctgaattcag cccccgtcca aggtcctggc 4020ccctttctct tctgcttggc
gtgttgttca tcaccactgt gcactgctga gggtaagtgc 4080ggttctctgg
acctctgctt tatcattaga acagactctt gcggtttccc acgacattcc
4140tttcacttct cacttggaag atgagccgtg aggaaatcct gtgttgtgtg
gtatgtgggc 4200tgtgcttctg cttgacttga gggccaagca gcattgcaag
ccatggtttt aaataagaaa 4260gaacatttct aaccttcatc ttctagtaag
gaaacaagtg ggctttagag ttcttgctca 4320ggaaagacct atgtcccagt
ccaaccggac cttttactaa agagatcttc ctgatcctcc 4380tccccaggcc
aggggagggg tcctccctgg ggttggagcc tttagtaggg ggtcggagac
4440acgacgtagc cttcatgaca ttcatagtct agttacacga tccctgtaag
ggtcagttga 4500agtaagtgct acaaaggaag ggaggtgctc agtggagagg
gctctctttt atgtattata 4560tttctttcat ggggagggat atggatcagg
gatcagcaga ggtgtttcag tcccgaggga 4620aagaaagtca gcgtggcttg
ggagttggga gcagcaagac agtggctcaa gatatcttaa 4680gactagtgga
gtacaccttg catgttaaaa gccttgctca gggctgcctg gttcttgtag
4740gacgacagag atggcctagc tctgcatact gcacccccag gggctcagaa
cagtgcaaat 4800gtcagtctat ctgtcagtgg cagagccagc cttggagcag
gggtgcaagg aggtctctgc 4860actggccagg catgcagaac attctgttca
gtagcactgg acagaaggcc ccatctagat 4920gagacagagc tggtggggca
ggacaaagac tcctggcagc tcaaacggcc tggcagatgc 4980ttggagagag
ggggcttctt gagacagcac catttctggg aagagagtca cctgggaggg
5040atgaggccac gctccggctt ggaggtgaag agaggggctg ctgcaagaaa
gaattagaga 5100catgccagcc tttgctgtgt tgcccaggct ggtcatgaac
tcttggcctc aagcaatctt 5160cccacctcag cctccccaag cgctgggatt
atagacatga gcccccatgc tggccaataa 5220aagatgattt tatggagggg
atggtggtga aggttgtggg tggtatgaaa tagtaagaaa 5280tatatattgg
tctgcaccca gttcctgcca cagagctcct aaaatcctga gaacttcctg
5340ggtgagcatc ttttgttcta atgaggtgac tcttggtggc tcctggatag
gagtgaatca 5400ccagaaagat caagccagag ttagaagcag aaagtgctgg
ctataacaca ggaaagctgt 5460aacacaaata ataaagtttt tttttttttt
tttgagatgg agcctcactc tgttgcccag 5520gctggagtgc aatggtgcaa
tctcagctca ctacaagctc tgcctcccag gttcaagtga 5580ttctcctgcc
tcagcctcct gagcagttgg gactacaggt gtgtgccacc acatctggct
5640aatttttgta tttttagcag agacggggtt tcaccatatt aaccaggctg
gcctcaaact 5700ccttaccttg tgatccgcct gcctcagcct cccaaagtgc
tgggattaca ggcatgagcc 5760accgtgcctg gccaaaagac attgttctta
aaagaatcaa ctaactaacc aaataaataa 5820aaatctaacc taattaagaa
actaaaaata cacaaaaatt aatttcaagg ggagaaaaat 5880catgtaaaga
gagaaagata atgaatactt tgcagaaatt tatgaacata aacataaaac
5940ttggatgaaa tgcatttcta ggaaaacata atttatcaaa actaaccaca
agtaaaatag 6000aagcctaaat aggatatttt caagagaaga agtaaagttg
tcaaagtgct acccttcaaa 6060aaaacaccag gctcaaacaa tctgacatgg
gaatgttagc acaccttaga gagcaaataa 6120aactttgaat gggcttgaaa
tattccagac tctagaaaaa caaaacttcc caattctttt 6180tataaagcaa
gtataaattg ataccaaaat cttataaaga ccttatacaa aacttcatac
6240caatctcttt tatgaataca aaacccttaa taaagtatta ccagacagaa
cccaacaata 6300cataaaaatg tcacatcata acatagtggg gtttatttca
ataatgcatg gatggttcaa 6360tacaaggaaa ttcagtaaca caatataata
gatcatgtga atatacccaa agaaaaaata 6420gattattttc atagatgctg
taaaggcatt tgaccaaatt caacacctac tttttaggtg 6480gtcaataaaa
taaattagtt actccttctt tagcatgata aaatatattt atcagcccag
6540aaggcatcat tttacccgat aagggcacac gctggaggga ataatgttaa
aattaggaat 6600aagaggatag ctagtttctt tcttcttttt tttttttgag
acggagtctt gctctgttgc 6660caggctggag tgcagtggtg caatgttggc
tcactgcacg ccccccgcct cccaggttca 6720agcgattctc ctgcctcagc
ctcccgagta gctgggacta caggcgcgca ccaccatgcc 6780cggctaattt
ttttttgtat tttagtagag atggggtttc accatgttgg tcaggctggt
6840cttgaactcc caacctcacg tactgggatt accggtgtga gccaccacgc
cagcccaact 6900actttcaaca ttatccttaa tactgatgct tattgactta
ctatggggtt acctctagat 6960aaatccataa taagttgaaa atataagtaa
aaaatgccct taatacacct aacctaccaa 7020acatcatagc tgagcccagc
ctgccttagc tatgctcaga cactgacgtc agcctacaat 7080tggcaaaatc
acacagcagc acagtctact gcagagcatc tgctgtttgc ccttgtgact
7140gcgtggctgc ctgggagctt cccagcttca caagacagta ttacgtagca
catcactagc 7200ctggggaaag atcaaagttg aaaatttgaa gtgtggtttc
cattgaatgt gtactgcttt 7260tgcaccatca tcaagtcaaa aaattttagt
tgaaccagcc taagtttggg accatcttta 7320ttttcaggag gaacttccat
gtacattgat gacggacgat agaatccgtt tctatcatcc 7380taatgaacat
aatgaataaa tccagacaaa cataaacatt aacagagtaa gcagctttcg
7440gggctggaag ccagaagagg gtgggagcgc agagagagag gccaaacacc
agggctgctt 7500ctgctttgcg ggtatttgct gatctggaca aggtatctgg
aaggctgagc taagcctcct 7560ttttttttga ggtggcgtct cactctgttg
ccaggctgga gtgcaatggt gcgatctcag 7620ctcactgcaa cctccacctc
cctggttcaa gcgattctcc tgcctcagcc tcccgagtag 7680ctgggattac
aggctcccgc cactacaccc agctgatttt tgtaatttta gtagagacgg
7740ggtttcacca tgttggccag gatggtctcg atctcttgac gtcatgatct
gtccacctcg 7800gcctcccaaa gtgctgggat tataggcgtg acccaccgtg
ccccgtctga gctaagcctc 7860ttgagcatag gggactaaaa atgaaatcta
gcgcatgcca agtttagggt cccaggcaat 7920tcctttccac tttggggtcc
actttggggt ccaccccacc caagaagaag gatgacttgg 7980aagtaaacca
gctctgaaat atggatggtc ctctgggacc ataccaatcc cttcatatca
8040accacatcca gttcctcaaa actggaactt ggattaagat ggcctaggac
ttctagtgtc 8100ccaggagcct ggcattgcaa acaaaaatcc tctccggaag
aagataatac cttaagcttc 8160aaatgactct ctaataaatt tcaaatacaa
tgtccagcac acaaacacaa attaccagga 8220acgtgatatg aggcctgatg
gatgggaatt agcagaaact tcaggcatga gaaacatacc 8280ctcagaggcc
tagaatctat ctagtgtcta gataatggag atatgaaata cagacactta
8340aacaactatg tttcccatgt tcaaagagga aatttgcaaa acttgaaagt
gttggcagga 8400aatcagaaac tataaaatgt gacaacagca tactttagag
tcagtataaa ttacggtccc 8460gaaaactgca gaattccaga acttaatggt
aaagcaaggg tttaacagca gaatagaaat 8520agccagagag aactaggaag
taagtcagat gacactaccc agaataaggc actgagaggc 8580caaggaatgg
aaaatgcaga agaaaggata tggtgagagg atctaatata catttatttg
8640gagtaccagg gagagagaga aggagaagaa cagaagccgt gtttcaagga
cggtgactga 8700gaggcttcga aactgatgaa agccatcagt tcacaaattc
aaagcccagt gaattccaag 8760gagaaaaaaa gaaatccata ctgtgaaagc
aagtccagac aatgacaaac accatcaaca 8820atacacagga caggcataag
atgcatttaa tggggacact cagaggcaga gggttatcag 8880aaggaggcac
ttctctccca agttctcatc atcccagggc cagggacagc tggtcacacc
8940ttagggagtt cactaggaga gggatctggc ttcttgtcat tctgggtatt
tgtagggaaa 9000ttggaaggga accgagagca cctagccaat cgcatagcaa
tgggagattt caggctgtgg 9060ggaatgtctt tgctggtgaa aagaacatcc
tgaccttaga aatctttcac cgagggggat 9120ctgcgttcca gaacttctgg
agctggtata ggtaaggctt tgagctttcc tactgagcca 9180gcctgttgct
aggttaccaa aggggacctc gagggccatc tggccaacaa gcagacttgt
9240ctctccttac acccccagac gtatcactgc aaaactacag aaaaccaaag
acagagaaaa 9300tcttaaaagc agccagattt aaaaaatggc atattagttt
caaagcagca gccatgaaat 9360tgacagctga tgtctcaaca gcaagaatga
aaagtggaag acaggccagg tgtggtggct 9420caggcctgta atcccagcac
tttgggaggc cgaggcgggt ggatcacgag gtcaggagac 9480caagaccatc
ctggctaaca tggtgaaacc ccgtctctac taaaaataca aaaaaattag
9540tcgggcatgg tggtgggtgc ctgtagtccc agctactcgg gaggctgagg
caggagaatg 9600gcgtgaaccc gggaggcgga gcttgcagtg agccgagatt
gtgccactgc actccagcct 9660gggtgacaga gcaagactct gtctcaaaaa
aaaaaaaaaa aaaaaaaaaa aaagggtgac 9720gaagcttcaa tctcctgaaa
ggaagcaact gccgcctttg attcgatacc caccaaaatc 9780cgtgaagaag
gaaggcaaaa taaaaacact tcctgattga actggaaaga tttccgcaat
9840agaagaccca ctgtccaagg aattctaaag gatgctttcc aggcagaaga
aaatgacccc 9900agaggaagat cagagattca ggaaagaaat ggagagtgat
aaaaatggaa aattcggggg 9960ccaatttaaa caaaagctga ctgctctaca
actgttgtgt ctctatcttt tgtaacatat 10020atgtgtgtgt agcttttttt
tttttttttg tcaagatgga ttctcactct gtcgcccagg 10080ctacagtgaa
atggcacggt ctcggctcac tgcaacctct gccccttggg ctcaaatgat
10140tctcttgcct cagcctcctg agtagctgag attacaggtg cctggcacaa
tgcctggcta 10200atttttgtat ttttactaga gatgggattt ctccatgttg
gccaggctgg tcttgaacac 10260ctgacctcag gtgatccacc tgcctgggcc
tcccaaagtg ctaggattac aggcgcgagc 10320cactgcatct ggcctatgtg
tgtgtttata tggaattaaa acacatggca ataataccct 10380ccaaattggg
agaaaccaaa aatagcattt aaatgttgta agctccctgc ataatcaaga
10440agagaataga tttacgttag attttgatac ctggaggatg aatgttgtaa
tttctagggt 10500gaccatgaaa agaggagaca acggtgtatg tttttttttt
tttgagatgg agtctcactt 10560tgtcacccag gctggagtgt tgtggtgtga
tcttggctca ctgcaacctc ctcctcttgg 10620gttcaggcca tcctcccacc
taggcctcca gagtaggtgg gatcacaggc acctgccacc 10680acacctggct
aatttttttt tttttttaaa tatttagtag agatggggtt tcaccatgtt
10740ggccaggctg gtcttgaact cctgacctca ggcgatctgc ctacctctgc
ctctcaaagt 10800gctgggatta caggtgtgag ccatcgcgcc cggccaacag
tgatcacttt caaactaaca 10860gaggttcaaa aataaaatca gacttaacca
aaaaccaggt aacagagctg gtaggatata 10920cagaaagact gacctcacgt
atatcaacga ttacagttaa tattaatgaa ggaaatgctc 10980tagtttaaaa
acgagggttg tcaaagaccc cacataagaa gctccttacc agcggtgcac
11040ctagaaccta aggaaacagg acagatgaag gaggacgcgc ccccgccgct
gtcctgcgcc 11100tcagccatcc tatgagacgg gaaaggtttc tgtctgcagc
tgggcccgtg ctctttacca 11160gctcctggct ttcttctctg gaaggttcct
gcctgttttg ccctcacacc tgctcctctc 11220tcagccctct caggggtggg
gctggaggcc accaaagagc ctcctctgct ctccagttgc 11280tcgactgctc
ctcatttccc cctggggtct gcgtcagggt ttccttcttt tccagcccca
11340ccccgcgtgc atcccacctg gtctcgggtc ggggctgctc ccgcttactg
ccccctgccc 11400aggctggtgt gcaccccctc tggctgcttt caaggcctct
tctctcttct cggcaggaca 11460ggcacaggca ggtggccagg tgtcatgctt
agctccccgc ccagtgagat tctttcattt 11520aacaatcttc ccctgaatag
ttcatgttca ttgctgaaaa tttgaaaaat atggaaaagc 11580acaaagatta
agatataaac cgccctcaat tcccctgccc agagagagtc actgctatga
11640cttggtgact aggaacctta tttctctctc gctctttttt ttttttttga
gacagagtct 11700tgctctgtca cccaggctgg agtgcagtgg ctcgatctca
gctcactgca acctccgcct 11760cctgggttca agcgattctc ctgcctcagc
ctcttgagta gctgggatta caggcacctg 11820ccaccatgcc cggctaattt
ttgtattttt agttgagaga gggtttcatc ttgttggtca 11880ggcggacttg
aactcctgac ctcaggtgat cagcccacct cggcctccca aagtgctggg
11940attacaggtg tgagccactg cgccttcatc tctcttctgt gtatgtgtac
gctgtttttt 12000ctttagaatg ggggacgtta tcaggctcta catggtgtgt
agtcggctag catgttgtaa 12060gcctttccct gtgtcacaag tgctcatctg
gaacaggatt ctaatgactg cctgtggcta 12120tgttgggatt cctttaactc
agctccttct gcccagcatc tatctttttt ccatcttttg 12180tcctaagtgt
tgctataata aatcattgat cacacatgcc tgactgtttg cataggataa
12240attacgggaa atgtttttgc tgttcaggga ctgtgcccat ttttaggcct
cagagacacc 12300atgccagact gcccagtatt gatctttact ctttttagat
gatgccaaac ttttctgtga 12360actttaaaaa cctgtgtctt gacagtccat
ttctgtaagt ctttcacatt agatttcctg 12420tcaggatgat agtcaattct
aggcagatga tgttttctca gccatggctg aagcagttgt 12480gatttgttgt
ggccatgtaa agtcccgatg atccattgcc tccctggatg ggttggaata
12540atttggtttg ggagcatata acagaatgac ctggagtcac agcagctcag
acggaagtgt 12600atttctccct tacagatgaa agaattccag gccaggctgg
aatgacaact gcacacagtc 12660atctgggccc cctccttcca gctcccatca
ccccaggatg tggcttttat gcagatgatc 12720caaaatggct gctcaagtcc
cagccaacac atcccattcc agggagcagg aaaaaggtgt 12780gtctttccct
tcattttatg tgattccttt ctagaagtac tactcattac ttctgcttgc
12840atctccctgg ctagcactta cttagttata tggccatagc tagctgaagg
aaggacaggg 12900actgtcatac actagctaag aggcaaactg cttagataaa
aaggtctcta aagaaggtca 12960gagcggctgc tagggtgcaa ctctattact
tattgttatg ggacgaactg tgtccctcat 13020tcaggttgat gtcctaagcc
ccagaacctc agaatgggat tgtatttgga gacaggttct 13080ttaaggaggt
aaggaggcta aaatgagatc attagggtgg gccataatcc gactgatgtc
13140ttacaagaag agattaggac acggacatgc tcagagggac ggccacgtga
ggacaccaag 13200aaaggcagct gtctgcaagt caaggacagg gctcagggga
aaccaacctt gccaacacct 13260tcatctcgga cttctagcct ctaggaccat
gagaagatac atttctgttg tttaagctgc 13320ccggtctgtg gtactttgtt
atggcagccc aagtaaacaa atacagtcat ctgctgctgg 13380aacaaatcac
cccagcactg tggcttggca gcacacatgt ctagtcatag agttatatgt
13440agttacgtgt agagccatat gtatcgtcac acgttctgtg ggtcaggaat
ttggacccag 13500cttaaccagc tccacttctc gccagggttc agtcaaatac
cagctgcctc ccacctgaga 13560gctcagccgg ggaagggtcc ctttccaatc
tcacgtggtg ttggcaggat ccagttcctc 13620atggcctgct ggactgagaa
cctcagttct cactgcctgt tggccagagg ccgcctttat 13680gtcctcgcca
tgtgggcctc tccaacatgg cagctgactt catcagagca tccatgccaa
13740gaaggcaaca gagagggcca gggagactga agtcataccc ttttgcgacc
tagtcatggg 13800gtgacattcc atcacctttg cccattggtt agaagcaggc
caccaggtac agcccaagct 13860cacggggagg ggtcatacaa gggtgtcaat
accaggaggt gaggggtgct ggggccatct 13920tatgagtctg cccactgagg
taactaacaa ccttgaggcc tgacacagtg gacaaaggcc 13980cttattaaca
gcagagaact gggaacttta tttatttatt tatttttgag acagagtctc
14040actcttgtca cccaggctgg agtgcaatgg catgatcttg gctcactgca
acctccacct 14100cccaggttca agcaattctg cctcagcctc cggaatagct
gggactacag gcatgcacca 14160ctacacccgg ctaatttttg tatttttagt
agagacaggg tttcgccatg ttggccaggc 14220tggtctcgaa ctcctgacct
ctggtgatct gcctgccttg gcctcccaaa gtgctgggat 14280tacaggcgtg
agccaccgca cctcgctgga acttaatttt tttagagaca gtgtcgctct
14340atcacccaag ctggagtgca gtggtgcaat cctagctcac ttgcagcctc
aaattcctgg 14400gttcaggtga tcctcccaca tcagcctccc aagaactggg
aactaacagc tgtttctctg 14460ctgtccttct caagaaaagg gaggctactg
ctaccccact ggggacaatg ctgggtttcc 14520ctttaggaca ggctctgaga
caaggcggag gtgctgtttg tggccacaga gcaggggact 14580ctgggttgca
ggtgtggcct ggctaaagta ggctttactg ggctcctctc tgcctgcatc
14640accccccggc tgggcggttg tctctgaggc caaccttact ccctgctggg
caggctggac 14700agctgccctc tccgtttgcc cctctaccac ccaaaaggca
ggaggctctg gagaccagga 14760ccctgcccgc cacggcctgt gtcccaggcg
tgagggggtg ccccacagac ctctgctgag 14820ctgctgctga atgacgcccc
ttgggggtcc tgccggaagg tcagagcagg ggtgcactcc 14880cataaagaaa
cgcccccagg tcgggactca ttcctgtggg cggcatcttg tggccatagc
14940tgcttctcgc tgcactaatc acagtgcctc tgtgggcagc aggcgctgac
cacccaggcc 15000tgccccagac cctctcctcc cttccggggc gctgcgctgg
gaccgatggg gggcgccagg 15060cctgtggaca ccgccctgca ggggcctctc
cagctcactg ggggtggggt gggggtcaca 15120cttggggtcc tcaggtcgtg
ccgaccacgc gcattctctg cgctctgcgc aggagctcgc 15180ccaccctctc
cccgtgcaga gagccccgca gctggctccc cgcagggctg tccgggtgag
15240tatggctctg gccacgggcc agtgtggcgg gagggcaaac cccaaggcca
cctcggctca 15300gagtccacgg ccggctgtcg ccccgctcca ggcgtcggcg
ggggatcctt tccgcatggg 15360cctgcgcccg cgctcggcgc cccctccacg
gccccgcccc gtccatggcc ccgtccttca 15420tgggcgagcc cctccatggc
cctgcccctc cgcgccccac ccctccctcg ccccacctct 15480caccttcctg
ccccgccccc agcctcccca cccctcaccg gccagtcccc tcccctatcc
15540cgctccgccc ctcagccgcc ccgcccctca gccggcctgc ctaatgtccc
cgtccccagc 15600atcgccccgc cccgcccccg tctcgccccg cccctcaggc
ggcctccctg ctgtgccccg 15660ccccggcctc gccacgcccc tacctcacca
cgccccccgc atcgccacgc cccccgcatc 15720gccacgcctc ccttaccatg
cagtcccgcc ccgtcccttc ctcgtcccgc ctcgccgcga 15780cacttcacac
acagcttcgc ctcaccccat tacagtctca ccacgccccg tcccctctcc
15840gttgagcccc gcgccttcgc ccgggtgggg cgctgcgctg tcagcggcct
tgctgtgtga 15900ggcagaacct gcgggggcag gggcgggctg gttccctggc
cagccattgg cagagtccgc 15960aggctagggc tgtcaatcat gctggccggc
gtggccccgc ctccgccggc gcggccccgc 16020ctccgccggc gcagcgtctg
ggacgcaagg cgccgtgggg gctgccggga cgggtccaag 16080atggacggcc
gctcaggttc tgcttttacc tgcggcccag agccccattc attgccccgg
16140tgctgagcgg cgccgcgagt cggcccgagg cctccgggga ctgccgtgcc
gggcgggaga 16200ccgccatggc gaccctggaa aagctgatga aggccttcga
gtccctcaag tccttccagc 16260agcagcagca gcagcagcag cagcagcagc
agcagcagca gcagcagcag cagcaacagc 16320cgccaccgcc gccgccgccg
ccgccgcctc ctcagcttcc tcagccgccg ccgcaggcac 16380agccgctgct
gcctcagccg cagccgcccc cgccgccgcc cccgccgcca cccggcccgg
16440ctgtggctga ggagccgctg caccgaccgt gagtttgggc ccgctgcagc
tccctgtccc 16500ggcgggtccc aggctacggc ggggatggcg gtaaccctgc
agcctgcggg ccggcgacac 16560gaacccccgg ccccgcagag acagagtgac
ccagcaaccc agagcccatg agggacaccc 16620gccccctcct ggggcgaggc
cttcccccac ttcagccccg ctccctcact tgggtcttcc 16680cttgtcctct
cgcgagggga ggcagagcct tgttggggcc tgtcctgaat tcaccgaggg
16740gagtcacggc ctcagccctc tcgcccttcg caggatgcga agagttgggg
cgagaacttg 16800tttcttttta tttgcgagaa accagggcgg gggttctttt
aactgcgttg tgaagagaac 16860ttggaggagc cgagatttgc tcagtgccac
ttccctcttc tagtctgaga gggaagaggg 16920ctgggggcgc gggacacttc
gagaggaggc ggggtttgga gctggagaga tgtgggggca 16980gtggatgaca
taatgctttt aggacgcctc ggcgggagtg gcggggcagg gggggggcgg
17040ggagtgaggg cgcgtccaat gggagatttc ttttcctagt ggcacttaaa
acagcctgag 17100atttgaggct cttcctacat tgtcaggaca tttcatttag
ttcatgatca cggtggtagt 17160aacacgattt taagcaccac ctaagagatc
tgctcatcta agcctaagtt ggtctgcagg 17220cgtttgaatg agttgtggtt
gccaagtaaa gtggtgaact tacgtggtga ttaatgaaat 17280tatcttaaat
attaggaaga gttgattgaa gttttttgcc tatgtgtgtt gggaataaaa
17340ccaacacgtt gctgatgggg aggttaattg ccgagggatg aatgaggtgt
acattttacc 17400agtattccag tcaggcttgc cagaatacgg ggggtccgca
gactccgtgg gcatctcaga 17460tgtgccagtg aaagggtttc tgtttgcttc
attgctgaca gcttgttact ttttggaagc 17520taggggtttc tgttgcttgt
tcttggggag aatttttgaa acaggaaaag agagaccatt 17580aaaacatcta
gcggaacccc aggactttcc ctggaagtct gtgtgtcgag tgtacagtag
17640gagttaggaa gtactctggt gcagttcagg cctttctctt acctctcagt
attctatttc 17700cgatctggat gtgtcccaga tggcatttgg taagaatatc
tctgttaaga ctgattaatt 17760tttagtaata tttcttgttc tttgtttctg
ttatgatcct tgtctcgtct tcaaagttta 17820attagaaaat gattcggaga
gcagtgttag cttatttgtt ggaataaaat ttaggaataa 17880attattctaa
aggatggaaa aactttttgg atatttggag aaattttaaa acaatttggc
17940ttatctcttc agtaagtaat ttctcatcca gaaatttact gtagtgcttt
tctaggaggt 18000aggtgtcata aaagttcaca cattgcatgt atcttgtgta
aacactaaac agggctcctg 18060atgggaagga agacctttct gctgggctgc
ttcagacact tgatcattct aaaaatatgc 18120cttctctttc ttatgctgat
ttgacagaac ctgcatttgc ttatcttcaa aatatgggta 18180tcaagaaatt
tcctttgctg ccttgacaaa ggagatagat tttgtttcat tactttaagg
18240taatatatga ttaccttatt taaaaaattt aatcaggact ggcaaggtgg
cttacacctt 18300taatccgagc actttgggag gcctaggtgg acgaatcacc
tgaggtcagg agtttgagac 18360cagcctggct aacatggtga aaccctgtct
ctactaaaaa tacaaaaatt agctggtcat 18420ggtggcacgt gcctgtaatc
caagctacct gggaggctga ggcaggaaaa tcgcttgaac 18480ccgggaggca
gagtctgcag tgagttgaga tcacgccact gcactccagc ctgggtgaca
18540gagcgagact ctatctcaaa aaaaattttt tttaatgtat tatttttgca
taagtaatac 18600attgacatga tacaaattct gtaattacaa aagggcaata
attaaaatat cttccttcca 18660cccctttcct ctgagtacct aactttgtcc
ccaagaacaa gcactatttc agttcctcat 18720gtatcctgcc agatataacc
tgttcatatt gtaagataga tttaaaatgc tctaaaaaca 18780aaagtagttt
agaataatat atatctatat attttttgag atgtagtctc acattgtcac
18840ccaggctgga gtgcagtgat acaatctcgg ctcactgcag tctctgcctc
ccaggttcaa 18900atgcttctcc tgcctcagcc ttctgagtag ctgggattac
aggcgcccac caccatgtcc 18960agctaatttt tgtattttta gtagagatgg
ggtttcacca tgttggccag gctggtcttg 19020aactcctgac cttgtgatct
gtccacctcg gcctcccaaa gtgctgggat tacaggtgtg 19080agccaccatg
cctggctaga ataataactt ttaaaggttc ttagcatgct ctgaaatcaa
19140ctgcattagg tttatttata gttttatagt tattttaaat aaaatgcata
tttgtcatat 19200ttctctgtat tttgctgttg agaaaggagg tattcactaa
ttttgagtaa caaacactgc 19260tcacaaagtt tggattttgg cagttctgtt
cacgtgcttc agccaaaaaa tcctcttctc 19320aaagtaagat tgatgaaagc
aatttagaaa gtatctgttc tgtttttatg gctcttgctc 19380tttggtgtgg
aactgtggtg tcacgccatg catgggcctc agtttatgag tgtttgtgct
19440ctgctcagca tacaggatgc aggagttcct tatggggctg gctgcaggct
cagcaaatct 19500agcatgcttg ggagggtcct cacagtaatt aggaggcaat
taatacttgc ttctggcagt 19560ttcttattct ccttcagatt cctatctggt
gtttccctga ctttattcat tcatcagtaa 19620atatttacta aacatgtact
atgtgcctgg cactgttata ggtgcagggc tcagcagtga 19680gcagacaaag
ctctgccctc gtgaagcttt cattctaatg aaggacatag acagtaagca
19740agatagataa gtaaaatata cagtacgtta atacgtggag gaacttcaaa
gcagggaagg 19800ggatagggaa atgtcagggt taatcgagtg ttaacttatt
tttattttta aaaaaattgt 19860taagggcttt ccagcaaaac ccagaaagcc
tgctagacaa attccaaaag agctgtagca 19920ctaagtgttg acatttttat
tttattttgt tttgttttgt tttttttgag acagttcttg 19980ctctatcagc
caggctggag tgcactagtg tgatcttggc tcactgcaac ctctgcctct
20040tgggttcaag tgattctcat gcctcagcct cctgtttagc tgggattata
gacatgcact 20100gccatgcctg ggtaattttt tttttttccc ccgagacgga
gtcttgctct gtcgcccagg 20160ctggagtgca gtggcgcgat ctcagctcac
tgcaagctcc gcttcccgag ttcacgccat 20220tctcctgcct cagtctccca
agtagctggg actacaggcg cctgccacca cgtccagcta 20280atttttttgt
atttttaata gagacggggt ttcaccgtgt tagccaggat gatcttgatc
20340tcctgacctc gtcatccgcc gaccttgtga tccgcccacc tcggcctccc
aaagtgctgg 20400gattacaggc atgagccact gtgcccggcc acgcctgggt
aatttttgta tttttagtag 20460agatggggtt ttgccatgat gagcaggctg
gtctcgaact cccggcctca tgtgatctgc 20520ctgccttggc ctcccaaagt
gctaggatta caggcatgag ccaccatacc tggccagtgt 20580tgatatttta
aatacggtgt tcagggaagg tccactgaga agacagcttt tttttttttt
20640ttttttgggg ttggggggca aggtcttgct ctttaaccca ggctggaatg
cagtatcact 20700atcgtagctc acttcagcct tgaactcctg ggctcaagtg
atcctcccac ctcaacctca 20760caatgtgttg ggactatagg tgtgagccat
cacacctggc cagatgatgg cttttgagta 20820aagacctcaa gcgagttaag
agtctagtgt aagggtgtat gaagtagtgg tattccagat 20880ggggggaaca
ggtccaaaat cttcctgttt caggaatagc aaggatgtca ttttagttgg
20940gtgaattgag tgagggggac atttgtagta agaagtaagg tccaagaggt
caagggagtg 21000ccatatcaga ccaatactac ttgccttgta gatggaataa
agatattggc atttatgtga 21060gtgagatggg atgtcactgg aggattagag
cagaggagta gcatgatctg aatttcaatc 21120ttaagtgaac tctggctgac
aacagagtga aggggaacac cggcaaaagc agaaaccagt 21180taggaagcca
ctgcagtgct cagataagca tggtgggttc tgtcagggta ccggctgtcg
21240gctgtgggca gtgtgaggaa tgactgactg gattttgaat gcggaaccaa
ctgcacttgt 21300tgaactctgc taagtataac aatttagcag tagcttgcgt
tatcaggttt gtattcagct 21360gcaagtaaca gaaaatcctg ctgcaatagc
ttaaactggt aacaagcaag agcttatcag 21420aagacaaaaa taagtctggg
gaaattcaac aataagttaa ggaacccagg ctctttcttt 21480tttttttttt
tgaaacggag tttcgctctt gtcacccggg ctggagtgca atgatgtgat
21540ctcagctcac taaaacctct acctcctggg ttcaagtgat tcttctgcct
cagcctccca 21600agtaactggg attacaggcg tataccacca tgcccagcta
atttttgtgt ttttagtaga 21660gatggggttt caccatgttg gccaggctgg
tctcgaactt ctgacctcag gtgatccact 21720cgcctcagcc tgccaaagtg
ctgggattac aggtttgggc cactgcaccc ggtcagaacc 21780caggctcttt
cttatactta ccttgcaaac ccttgttctc attttttccc tttgtatttt
21840tattgttgaa ttgtaatagt tctttatata ttctggatac tggattctta
tcagatagat 21900gatttgtaaa aactctccct tcctttggat tgtcttttta
ctttcttgat agtgtctttt 21960gaagtgtaaa agtttttaat tttgatgaag
tcgagtttat ctattttgtc tttggttgct 22020gtgcttcaag tgtcatatct
aagaaatcat tgtctaatcc aaagtcaaaa aggtttactc 22080ctatgttttc
ttctaagaat tttagagttt tacatttaag tctgatccat tttgagttaa
22140tttttatata tggttcaggt agaagtccaa ctttattctt ttccatgtgg
ttattcagtt 22200gtcccagcac tgtttgttga agagactatt ctttccccat
ggaattatct tagtaccctt 22260gttgaaaatt aatcgtcctt aattgtataa
atttatttct agactgtcag ttctacctgt 22320tggtctttat gtcgatcctg
tgccagtacc atacagtctt gattactgaa gtttgtgtca 22380cagtttaaat
tcatgaaatg tgagttctcc aactttgttc cttttcaaga ttgatttggc
22440catgctgggt cccttgcatt tccgtacgaa ttgtaggatc agcttgtcag
tttcaacaaa 22500gaagccaagt aggattctga gagggattgt gttgaatctg
tagatcaact tggggagtat 22560tcgcatctta acaatattgt cttccaccta
tgaacatggg caaactttgt gtaaatggtc 22620agattgtaag tatttcgggc
tgtgtgggca cagtgtctct gtcacagcta cgcggctctg 22680ccattgtagc
atgaaagtag ccataagcaa tatgtatgag tgtctgtgtt ccaatagaat
22740tttattaatg acaaggaagt ttgaatttca tataattttc acctgtcatg
agatagtatt 22800tgattatttt ggtcaaccat ttaaaaatgt aaaaacattt
cttagcttgt gaactagcca 22860aaaatatgca ggttatagtt ttcccactcc
taggttaaaa tatgatagga ccacatttgg 22920aaagcatttc tttttttttt
tttttttttt tttttgagac ggagtttcac tcttgttgcc 22980caggctggag
tgcagtggcg cgatctcggc tcactgcaac ctctgcctcc caggttcaag
23040acattctcct gcacggcctc cctagtagct gggattacag gcatgcgcca
ccacacccag 23100ctaattttgt atttttagta gagacggggt ttctccatgt
tggtcaggct ggtcttgaac 23160tcctgacctc aggtgatcca cccgcctcag
cctcccaaag tgctgggatt acagggtgtg 23220agccaccaca ccctgctgga
aagcatttct tttttggctg tttttgtttt ttttttaaac 23280tagttttgaa
aattataaaa gttacacata tacattataa aaatatcttc aagcagcaca
23340gatgaaaaac aaagcccttc ttgcaagtct gtcatctttg tctaacttcc
taagaacaaa 23400agtgtttctt gtgtcttctt cccagatttt aatatgcata
tacaagcatt taaatgtgtc 23460attttttgtt tgcttgactg agatcacatt
acatatgtat ttttttactt aacaatgtgt 23520catagatatt gttccatagc
agtacctgta attcttatta attgctatgt aatattttag 23580aatttctttt
taaaagagga cttttggaga tgtaaaggca aaggtctcac atttttgtgg
23640ctgtagaatg tgctggtgac atattctctc taccttgaga agtccccatc
cccatcacct 23700ccatttcctg taaataagtc aaccacttga taaactacct
ttgaatggat ccacactcaa 23760aacatttagt cttattcaga caacaaggag
gaaaaataaa ataccttata aagcactgtt 23820taatattgta ttaaattgga
tcaatttggg ggctagaatg tatgttagag acatgatatg 23880tccataggtc
cttgctatca cagtgaggtc tcagggacag tcgtttggta tcatttggga
23940tctcataagc agactctctc tgcttgacct gacaaatcag agtctgtgtt
ttaacaggtt 24000cagtgagtga cttacatgca cattggagtt tgggaagctc
cactgtaggt gcttagacct 24060tacctttgtt gttgctaata acaatgcaag
catttgggag gaagacctgt gttgctcata 24120tgtgtccagg tgtagctgag
gtggccttgc ttatctgctg tagggccgtt gagcatttct 24180gtagctgtga
tgagtgagct gaggtgagcc tgcggagagc tcccagccat tggtagtggg
24240actcgcttag atgaactgga aggacccttt catctgagca gccactatgg
agaaaaacaa 24300ccgaatgagg ggagagacaa tgtgcaattt tatttagggc
acaaaggaga gctgtggtta 24360gaaggtgaca tttgagtgga aagggggcaa
gccatgtgta tagcgggaga agagaggtcc 24420aggcagagtt aacagaaggc
agaaatgctt tccatgtttg agaaccagta aggaggccag 24480tggctgaagt
aaggtgaagg gcagaaataa ggatgaggct gcgagagatg agaggttaga
24540gacgagcgtc ttgtgcacca agataagctt gtgtggtcaa aacaagtagt
ttaatttatg 24600tttttaaaag atcattttgg ctgggcacaa tggttcatgc
ctgtaatacc agtagtttga 24660gacggtgtgg tgggaggatt gcctgaggcc
agacgaccag catagccaac atagcagcac 24720ctataaggtc tctacaaaaa
actttaaaaa attagctggg catagtggtg tgtgcctgta 24780gtcccagcta
ctcaggaggc tgaggaggct ggaggattgc ttgagtccag gagtttgagg
24840ctgcagtgag ctatgattat gccactacac tacaacctgg gcaagagagt
gagaccctgt 24900ctctaaatat acacacacac acacacacac acacacacac
acacacacac acacacacac 24960acacacatat atatgtatat atatgcattt
agatgaaaag atcactttga caataccaca 25020tgctggtgag gatttagaaa
aactaggtca cttattgctg gtgggaatat aatatagtac 25080ggccactctg
gaaaacagtt tggcagtttg tcataaaact gaacataccg ttagtataca
25140gcccagcagc aactacaatc ctgggcatta atcctagaga aatgaaacct
taatgttcac 25200ataaaaacct atactcaagt atgcatagca gctttaccca
taatatctaa gaactggaat 25260cagctcagat gtccttcaac aggtgaatgg
ttaaactact cagtaataaa aaggaatgag 25320ctactgatag catgcaacag
tttaggtgaa gttatgctaa tgaaaaaagc caatcccaaa 25380aggttataca
tactgtatga ttctatgttt ttttgcaatg gcacagtttt agggatggag
25440aatagattag tggttgcctg gggttagaga tggggtagta gagtaggtta
gtggtggcag 25500aggagagaaa agagagggag gtgaatgtgg ttataaaagg
acaacacagg ggaatacttg 25560taatggaaat gctttgtctt tttttttttt
tttttttttt tggcgacaga gtcttgctct 25620gttgcccagg ctggagtgca
gtggcatgat cttttctcac tgcaacctct gcctcctggg 25680ttcaagtgat
acttgtgtct cagtctccca tgttcagagt gaaacaaacc agaggtaatg
25740ttcatccaaa taatccaaca cacatgacat taaaacatca agatcaggtc
ggacgtggtg 25800gctcatgcct gtaatcccag cacttttggg aggccaaggt
gggcagatca cttgaggtca 25860ggagttcgag accagccggg ccaacatgat
gaaaccccat cttgactaaa aatacaaaaa 25920ttagccgggc atggtggtgt
gcacctgtag tcccagctac ttgggaggct gaggcaagag 25980aactgcttga
acccgagggg cagaggttgc agtgagctga gagtgcgcca ttgcacttca
26040gcctgtgtga cagagtaaga ctccatctcc aaaaaaaaaa aaccaagatc
aattaaaata 26100cagcattact gggccgggtg tggtggctca cacctgtaat
cccagcactt tgggaggccg 26160agatgggcag atcacgaggt caggagatcc
agaccatccc ggctaacacg gtgaaacccc 26220gtctctacta aaaaatacaa
aaaattagcc gggtatagtg gtgggtgcct gtagtcccag 26280ctacttggga
ggctgaagca ggagaatggt gtgaacccgg gaggcagagc tggcagtgag
26340ctgagatcgc gccactgcac tccagcctgg gcgacagagc aagactccgt
ctcgggggaa 26400aaaaaaaaat aaataaatag aatgctgtag tgtccttgag
tttacatgcc cctccttacg 26460cttgtgtgcc cgtgcagatt gcttgattac
acaattagag gaggctggcg gaggattgtt 26520ttaatttttt tttttttgag
acagtctggc tctgttcccc aggctagagt gcaatggcgc 26580aatcttggtg
cactgcaacc tctgcctcct gggttcaagc agttcttctg ccgcagcctc
26640ccgagtagct gggattatag gcgcccgcca ccacgcccaa ctattttttg
tatttttagt 26700agagcagcgt ttcaccatgc tggccaggct ggtctcgaac
tcctgacctc agatgatctg 26760ctgccccagc ctcccaaagt gctgggatta
caggcgtgag ccacacctgg ccgtttgttt 26820taattttgaa ggtgaagtga
aagtgactac atttaccaaa agtgattgaa aagccaggac 26880tgttcttacc
ctgtttttcc agttcttgct cagagcaagg tggtttcttt ttcacttaat
26940caccatactt acttttcatg tagaacaagt cagtttgagt tatcagttca
tcatcttaac 27000taaattccat gggggaagga attagtttta gtttcttaaa
cttccaggtt tgcttattgg 27060acaaaatgag atagcaaggc agtgttttta
agttagattt tttatttctt tggtaataca 27120attttctcag aaacttagta
gtcttttagt ttagttgttt ttagttggtc ctatgttttg 27180gatcacccct
ctctacttta ttttgatagt gccaactgtg aagacatctg aagccatagg
27240tttggatggg aaggaggcat ctttagcctg atcatcttcg ccaggctgtt
tatctccttt 27300tgcttggctg agaagtctta ataggaggct tattcccagc
tatttgggga catagaagca 27360gttagccatt gcttatattt tactgaggtc
tgtgtggtat gttgattgta gtcagttaac 27420gattttgaga actgaaggca
gcctggtata tatagagtag gtattagact gtgtttcttc 27480taattgaatt
tcccatctct tgtaatctat gccatcatct tctgtactgc tgagaaagaa
27540agaaagtttc taatcaaact ataccactgg ttgtaagatg cagtttggct
ttagtgatgt 27600taacacatga ttcaaacgtg aaattgattg agtattggtg
aaatacagag gagatttaaa 27660gccagaagac ctgggtttaa atgctggctg
tatgacttca tatctgtgtg atcttgggca 27720tgtcatggtt ggcacttcaa
tttcttctct ctataatggg ggaagtgagg ccagtcatgg 27780tggctcatac
ctataatccc agtgctttgg gaggccaaga tgggaagatc gcttgaggcc
27840aggagtttga gcaattgggc aacatcgtga ggccccgtct ctacaaaata
ttttgaaaaa 27900attagccagg cccagtggtg cgtgcctgtg gtccgcgcca
ctcaggaggc tgagacggga 27960ggatcctttc agcctaggag tttaaggcta
aagtgagcca tgattgtgct atcgtactcc 28020agcctgggca gcagagcaag
atcctgactc taaaaaaaag taaaataaag taaaatgggg 28080gaaatgaact
gctttagtaa catcatctgt tttttctgtg agcagcgtag cttgacagcc
28140attggtgaac tcgtgccctg tgcttccctg tccagatccc cattctgccc
gcaacatgga 28200gtataacggt ttattcatag tagtcgagaa acactcactg
aatgaatgaa tgaggtgtag 28260aactaagtgg agtgggtaat tcaacacata
ttaatttcct tctttttttt atttttagaa 28320agaaagaact ttcagctacc
aagaaagacc gtgtgaatca ttgtctgaca atatgtgaaa 28380acatagtggc
acagtctgtc aggtaattgc actttgaact gtctagagaa aataagaact
28440ttgtatattt tcagtcttaa tgggctagaa tattctttgt gtcccagcta
ttttaaatgg 28500attcagaaat ccatttaaga tgaagaagga cccttttccc
atatttctgg ctatatacaa 28560ggatatccag acactgaaat gaataatgtt
ccctttttgt aatcttttat gcaaaaatta 28620aaaccattat ggtaattgaa
caacatgttt atgtttagtt aacaccctta gcaactatag 28680ttattttaaa
accatctatg gtttgatatt tttgcatttg ttgcaatagt aggaacagca
28740caagacagtt cagtttgtct ctcttatttg ctttttcttg gcagtttgct
gtcctattgt 28800acctctgctc ctagcagtgg ctggagccca ctcctctgtg
cttcgggatt agtggggatc 28860gtggggcatt gactgtaggt cagctttcct
tgcttgatct ttctcactgg gatgaactag 28920cagcaccttc ttttgtagct
gctttgcttt tgactatctt tctgaccgtt gttcctagta 28980gctgtagatg
gtaaatatat ttaggcctgt ttccaatggc tcagtaggag acatattcac
29040ctatgatatc tgaattctgt tacccacatg ggcatgcgtg aaatagttgc
cttgccttac 29100tttcccttgg aataaataat tcatgttatt ctcctggtag
aagctagaaa aagcctttat 29160agtcagtcag aaaaaaattt ttagacaaat
aatcttgatt ttagtactga caaaaacgtg 29220tggtgattct ttttttaatt
tttttttgag acggagtttc actcttgttg cccaggctgg 29280agtgcaatgg
cgtgatctcg gctcactgca acctctgcct cctgggttca agtgattctc
29340ctgcctcagc ctcccaagta gctggagtta caggcatgtg ctactgtgcc
cagctaattt 29400tgtattttta gtagagatgt tggtcaggct gatctcgaac
tcccaacctt aggtgatctg 29460cccgcctcag cctcccaaag tgctgggatt
acaggcgtga gccagggcgc ccggtgattc 29520atttgttttt tcaaaaaatt
tcctcttggc cattgctttt cacttttgtt tttttttttt 29580ttttgagacg
gagtcacgat ctgtcaccca ggctggagtg cagtggcatg atcttggctt
29640actgcaagct ctgcctccca ggttcacgcc attctcctgc ttcagcctgg
cgagtagctg 29700ggactacagg tgctcgccac cacacccggc taattttttg
tatttttagt agagatgggg 29760tttcaccgtg gtcttgatct cctgacctca
tgacccgctc aactcagcct cccaaagtgc 29820tgggattaca ggcgtgagcc
accgcgcccg gccctctctt gtctttttat tgtggtaaaa 29880tgcacataaa
attgactgtc ttaaccattt ttaggggtac agttcagtat atatattcgt
29940aatgttgtac agccatcact gccatctact tcataagttt ttcttctgtc
aaaactgaac 30000atctgtcttc attaaactcc ctatcatcca ttctttcctg
tagtcccttt ctactttctg 30060tctgtatgag tgtaactgct ctggagacct
catgtaagtg
gattcctaca ggatttgtgt 30120tttttttttg gtgatctgct tatttttaat
gcctctgtgc atttgtatta tatactttca 30180aagtgatttc acaaaaccgt
ttcattttag gttaactcat ttctgttgtt tgtgaaatac 30240tgtgtatgat
tctgttctgt ttctgtctaa tttgtggaaa tgttgtggga agaaaatgaa
30300ataacaaatg agcatatgtc ctgaaaataa aaatataaaa attctaagtt
agcatgctat 30360tgtagaatac aacgctatga taaaagtagg aaaaaaaaag
gtttgaattc tatctctgct 30420acctgtgtaa gctgggtgac tttagataag
ctgtaacgtg tttgagcctt actggctcat 30480ttttgaaatg taatccctag
ttacacagtt cttgtgggat cagatggtac atgtgaaaca 30540ctgtgaaaaa
gcaactgcat agatatgttc attagccacc tgagcgggaa gcgtatccca
30600ttgcgatgcc catcatccaa agctatatgt tatctttact tttttttttt
tgagacagag 30660tcttgctctg ttgcccaggc tagagtgcag tggtgcaatc
tcagctcact gcaagctcca 30720cctcccgggt tcacgctatt ctcctgcccc
agcctcccaa gtagctggga ctacaggcac 30780ccgccaccat gcctggctaa
atttttgtat ttttagtaga gatggggttt caccgtgtta 30840gccaggatgg
tcttgatctc ctgacctcgt gatccgcccg cctcggcctc ccaaagtgct
30900gggattacag gcgtgagcca ctgcccctgg ccatctttac tttttttgtg
aaatgacttt 30960aaatacttgg caaacatttg gtcattgttc atctgatctc
caccatccag gtctcagaga 31020acataatttc tctctgaaag cttattgacc
caggaaataa gatctctttc aatctgagtg 31080cgtcaggctt tattcttgtc
attttgtctt ttgataattt tcaaatggaa ttcatggaat 31140gttggcttat
attcatatat tagtaaagta tgttgagaca tcttaagatt gatttgtggt
31200tctatatgcc atattaaatc aaaataatag ctgttaatgg ttttcacatt
agtctgtctc 31260ttgtttttat ggagtaatgc tgagagttca ttatgcttgt
tctacagaag agcatgttaa 31320aaggagtttt tggagtcaga gaggttattc
ttggtttcat aggatacact ctatactttt 31380tagggatttc agagtatata
gctgaaggtg atattttatg taaatatgtt ttatggaaac 31440ttattgctca
tcgctgtttc ctgttaactc tcctaaaata taattaaact tttggaactt
31500ttttatagct tttgtgctag actaattttt gtctctaatg aggttatata
aatggcagct 31560tctgacgttt tcaatgtagg aagtcattta aaacttcatg
tatattgtga aaatgtagtc 31620tgctttaagc tctctaaagt ggtctaagtt
actggttcct aagtatggat gagcatcaaa 31680atcatctgga aaatttgtta
aaaatacagt aatgaaggca cctcactgtc ctttttccca 31740aacatacttc
tgcattctgt ttgagtaggt agggactaca catttttcac aagtatcctc
31800ttgggaatac ccaggaatgc ttacttgagc aacctcttac taatatgtac
cttgataagg 31860tggctaggta aacataaata tacaaaaatc catagatctc
ccatatatta gcataaatca 31920gctagaaaat ataacgttta aagatctagt
tcacagtagc accaatatat cgaactctaa 31980ggaatcgata aatatgcaaa
aactttataa aaacttctgt taatgtttct gaaagatata 32040ggtgaccact
ttctagatag gaagatttta tattactaag ttgaattttc tctaaattaa
32100cacagaaatt taaaataatc ttgatcaaaa ttctagtaga ggtatttttg
aacttgttca 32160ctgcaagaat aaatacataa ttgcaaagaa tatctcaaaa
tcatcaccag gcctggtgtg 32220gtggcccatg cctgtaatcc cagcactttg
ggaggctgag gcaggcagat cacctgaggt 32280caagagtttg agaccagctg
gaccagtgcg gtgaaacact gcctctacta aaaatacaaa 32340aattagctgg
gtgtggtggt gcatgcctgt agtcccagct acttgggagg ctgaggcagg
32400agaattgctt gaacccagga ggtacaggtt gcggtgagcc tagatcgcac
cactgcattc 32460cagcctgggc gacaagagca aaattctgtc tcaagaaaaa
agagaaaaaa gaaaaagaaa 32520tcaacactaa tatggtgaga cttaatgtat
gtgacattaa aatagtgatt ggatgttaaa 32580acaggtatag aacagaaaga
agagtgtatg tgtgtatctg tatgaattta tgatgggtgt 32640aacatatatg
tattagggaa atgagggaaa tgatacattt ctctgacttt gggagaacat
32700tatatctcta cctcatattg caaacaaaca taaagttcag attaattacc
taaatgtgaa 32760aaaatgaaat aatttcttta aaaaatgtaa tcttagtttg
aggaaggtta acattataaa 32820ggaaaaaact gttttgagtg gaatatagtt
caatatgtca aaatccacct tcaacaaaat 32880tgaaagtaaa ttgaacttgg
ggaaagtatt gacagcatat agatcaaagg ttactagcct 32940gtgtaaagag
cagttataaa tatcgttaag aaaaacactg tcgacctgtc ggcaccttgt
33000tctccgactc ccagcctcca gaactgtgac gagtaagtgc ttattgttta
aaccacccag 33060tctgtatgtg gtattttgtt atagaaactc aagctgatta
ggacactagt aatcagtaga 33120ctgaaactga aacaaaaata agaacctttt
ttacctgtca aattggcaaa cattaagaat 33180attcagattt ttgtcagagg
tgatacaacc ttctaagaag gcaatttggg aaaatataaa 33240gctttagatt
attatatgtc tgacctagca gttttacctc tagggtgctt acccctagga
33300aagtgtgtaa tgatattggt gcagtgccct tcatcccatt agaaaattaa
aaataacctt 33360aatggcctac cactaaaagg ggattgaaaa tttaagatat
atttatttat gtgtttattg 33420agatggagtc ttgcactgtc cgcctgggcc
agagtgcaat ggtgcgatct cggctcactg 33480caacctctgc ttcccgggtt
catgtgattc tcctgcctca gcctcctgag tagctgggat 33540tacaggctca
caccaccgca cccggctaat tttttgtatt tttagtagag atggggtttc
33600actgtgttgg ccagactggt ctcgaactcc tgacctcatg atccgcgccc
ctcggcctcc 33660cagtgttggg attacaggtg tgagccactg cgcctggcca
gatacattta tacaagagaa 33720tgttagttaa cattcataga tatttatatt
ttgtttactt tttattaaaa aaattttttt 33780tagagacagg atcttactct
gtcacccagg caggatgcag ttgcacaatc atagcccact 33840gcagcctgaa
ctcctgggct taagtgatcc ttctgcctca gccttttgag tacctggggg
33900actttaggca gtgctactat acctggctaa tttttaaatg ttttatagat
gagatcttgc 33960tgtattgccc aggctggtct agaattcctg ggcccaagtg
atcctcccac cttggcctcc 34020caaagcgctg agattacagg catgagccac
cacttctgac caatagatat ttatatttgt 34080gactggaaaa tatattaaca
atgtgttaaa aaattcagtt aaaaaataat gaaagatttt 34140tgcttctggc
taagatagaa taacaaggac agcatttatc ttcttgcctt gaaatagttg
34200aaaacggaag aaatatatgt aacagtggtt ttcaagttat tgggcatcag
gcaaagaaga 34260atagttatcc caggaaaatg aatgtggaga gccctacaat
ttccttacat tactgcctgg 34320tcatggcaag aggaaaaact gagaggagac
tgaggctgag ccagtggttt gctgggttga 34380ggaggcagag ctgggagtgc
agagatgcaa ggtggtgaga gcccatatgg aagaatacca 34440gggaagagag
ctgcagaggg agctccggag acctgcaccc tgccctctca gtaccctgtc
34500atgtgtgtag ctgagtactg acgagcactt gcttgtgcgg aaatgaccca
gggctggagg 34560tagagccacc tgaaaggatt agaaggaaca gttgctgaaa
gtcacacagg gccaggaaga 34620atttctaatc acaccagttg gagtggaaaa
cctcagctct catagagcag gtagggtact 34680cagaagggtt tgcccaccta
gccccagact aagtttcgtt actctgaccc tacctaatat 34740taaaaagaga
ttaattaaat tgttcgcaac aaaaataata tatttcagtg tttgtaacac
34800gtagaagtga attgtatgac aatagcataa aggctggaag agcagaaatt
gacatgtatt 34860tgcgctgggc agaataatgc tcccctcttt ccccaaaaga
tatcaagtcc taatccctgg 34920agcctgtaaa tattacttta tatggaaaat
tgttttatga tgtgattaaa ttcaggatct 34980tgagatgagg gggctatctt
ggatgatctg ggtaggcact aaatgcaatc acatatatat 35040aaaaaggagg
cagagggaga ttttacacac agagagaagg ccctgtgaag atggaacaga
35100aagatttgaa ggtgctggcc ttgaaaattg gagtgatgaa gctataagcc
aaggaatgca 35160gcagccacca aagctggaag aggcacggag cagttctcat
ttagagccta ctccagaggg 35220aatgtggtgc tgccaattcc tttttttttt
ttttttttaa gatatcattt acccctttaa 35280gttggttttt tttttttttt
ttttttttta gtatttattg atcattcttg ggtgtttctt 35340ggagaggggg
atttggcagg gtcataggac aatagtggag ggaaggtcag cagataaaca
35400tgtaaacaaa ggtctctggt tttcctaggc agagggccct gccacgttct
gcagtgtttg 35460tgtccctggg tacttgagat tagggagtgg tgatgactct
taacgagtat gctgccttca 35520agcatctgtt taacaaagca catcttgcac
cgcccttaat ccatttaacc cttagtggac 35580acagcacatg tttcagagag
cacggggttg ggggtaaggt tatagattaa cagcatccca 35640aggcagaaga
atttttctta gtacagaaca aaatggagtg tcctatgtct acttctttct
35700acgcagacac agtaacaatc tgatctctct ttcttttccc acatttcctc
cttttctatt 35760cgacaaaact gccaccgtca tcatggactg ttctcaatga
gctattgggt acacctccca 35820gatggggtgg cggccgggca gaggggctcc
tcacttccca gatggggcgg ccgggcagag 35880gcgcccccca acctcccaga
cggggcggcg gctgggcggg ggctgccccc cacctcccgg 35940acggggcggg
tggccgggcg ggggctgccc accacctccc ggacggggcg gctggccggg
36000cgggggctgc cccccacctc ccggacgggg cgggtggccg ggcgggggct
gccccccacc 36060tcccggacgg ggcggctggc cgggcggggg ctgcccccca
cctcccggac ggagcggctg 36120ccgggcggag gggctcctca cttcccggac
ggggcggctg ctgggcggag gggctcctca 36180cttctcagac ggggcggctg
gtcagagacg ctcctcacct cccagacggg gtggcagtgg 36240ggcagagaca
ttcttaagtt cccagacgga gtcacggccg ggcagaggtg ctcttcacat
36300ctcagacggg gcggcggggc agaggtgctc cccacttccc agacgatggg
cggccgggca 36360gagatgctcc tcacttccta gatgggatga cagccgggaa
gaggcgctcc tcacttccca 36420gactgggcag ccaggcagag gggctcctca
catcccagac gatgggcggc caggcagaaa 36480cgctcctcac ttcctagacg
gggtggcggc tgggcagagg ccgcaatctt ggcactttgg 36540gaggccaagg
caggcggctg ggaggtgaag gttgtagtga cccgagatca cgccactgca
36600ctccagcctg ggcaacactg agcactgagt gagcgagact ccgtctgcaa
tcccggcacc 36660tcgggaggcc gaggctggca gatcacttgc agtcaggagc
tggagaccag cccggccaac 36720acggcgaaac cccgtctcca ccaaaaaaca
cgaaaaccag tcagacatgg cggtgcgtgc 36780ctgcaatccc aggcacttgg
caggctgagg caggagaatc aggtagggag gttgcagtga 36840gtagagatgg
tggcagtaca gtccagcctt ggctcggcat cagagggaga ctgtgcgagg
36900gcgagggcga gggcgaggga attccttaat ttcagtttag tgatactaat
tttggactct 36960ggcctctaaa actgtgaaag aaaaaatttt ttgtttgttt
gtttctttta agccacatag 37020tttgtggtaa tttgttacag cagctgcagg
aaactaattt atgctgcatg tgaaatggtg 37080taataaggta gattgtgatg
aagatacata gtataaacaa ttaagcaaca actaaaagca 37140caacaaggaa
ttatagctaa tgaaccaaaa aaggagatta gaataataaa aatggtgaat
37200cccaaagaag ccagaaatag gggaagaggc aaataaagga aagaaagagc
ttgatggtag 37260atttcaacct aactatgtca aaaaggacat tacatgtaaa
aggcagcgat ttttcagatt 37320gaatggaaaa gtaagactcg gtatatgctg
ctgcctgcaa gaaacacatt ctaaatataa 37380aggcaaaaat aacctacagg
taacagaacg gaaagaagtt cactgtgctt acaagaatta 37440gatgcaagct
agactggttc tgttaatatc agacaaagtg gatttcaaag caaaggctct
37500tgcccaggat gagatggtca tttcataatg atgaagggga ttcgttcatc
agcctggcat 37560agcaagctga aatgtttatg caccggacta cagagctaaa
atacatgaag caaagcctga 37620cagaactaca agtagaaaca gacaaatcca
cagtgataga gatttcagta gccgctctca 37680atgatttgta gaacacgtag
ccataatatc tggatctaga acacttgacc aacactgtcc 37740cctgtgcaac
ctcattggca tttacaggac actccaccca gcaccagcag aagagacact
37800ctctcaagtg ctcacagaat gtttgccaag atagagcaga tgctgggcca
taaaacaagt 37860ctctaaatta aaagcattca aattattcag agtatgtttt
ctgacctcag tatcattaag 37920ttggaatata ttataggaag ataacctgga
aaagcctcag atatgtggaa aaacccattt 37980ccacatggcc catgggtcag
aagtgaagtc aaaagggaaa tttgaaagtc ttttggattg 38040actgatataa
aaacaataga tttctaaact tgtggggtgc tgttacagca tagtaaatgg
38100aaatttctag cattaaatgc ctgttttagg aaagaaagat ttcaaatcaa
tgacctcagc 38160ttctaccttt ggaaacttga aaatgacaag caaatggaat
ccagagttac cagaagggcc 38220aggtacggtg gcttatgcct gcagttctgc
cactttggga ggccgaggca ggtggattgt 38280ttgagactgg cagttgaaga
ccagcctggg cagcctaggg agaccccata tctacaaaaa 38340acaaaaaaat
tagccaggtg tggtggcatg tgcctgtagt cccagctaac caggagtcta
38400aggtgggagg attgcttgag tctgggaggt tgaggctgca gtgaactgtg
attgtgccac 38460tgtgttccat cctgggcaac agaatgagac cctgtctcaa
aaacaaaaac agttactaga 38520agaatggaca tcataaagat aggagcagaa
gtcagtaaaa tagaaaacaa aaatacatag 38580gaaatcaata aaaccaaaag
ctggttcatc aagaacatca ataaattggt aaagctgata 38640ggaaaaacag
tgaagtcaca aattagcaat atcaggaatg agggagatga cagtagtata
38700gattatatag atattaaaag gactgtatga ggcaggtgtg gtggttcacg
cctgtaatcc 38760cagcaccttg ggaggccgag gtggacagat cacctgaggt
caggagtttg ggaccagcct 38820ggccaacatg gtgaaactct gtctctacta
aaaatacaaa aattagttgg tcgtggtgct 38880gtgtgcctgt aatcccagct
acttgggagg ctgaggcagg agaattgctt gaacctggga 38940ggcggaggtt
gcagtgagct gagattgtgc cgttgcactc cagcctgggt gacagagcaa
39000gactccatct caaaacaaat aaataaataa aaaggactat atggtaatat
tatgaacaac 39060tttatgccaa taaatttgac aacttataga tgaaatggat
gagttccttg aaagacacag 39120aaactattaa agctctctca agaagatata
gataagctga ttagccctat atctatttta 39180ttgaatttaa atgtaaaaat
caatatttag ttactggaaa acttttaagt gtggttggaa 39240atggtatacg
aactttttca actgaatttt atgaagtcta atcacaggta aaggttttct
39300gatgaaaatt tagtgtctga attgagatat actgtaaaaa atgttatata
tcttaattat 39360ttcttcacat taattacatg ttgaaataat actttgggtg
tattgggtta aattaaatat 39420tatgaaaatc ttgcctgttt tctttttact
tttgatgcgt cagctaggaa atataaaagt 39480gtagctcaca ttctgtttct
gttgacagta ctgctttgga gcacagtgtt tgaatgatct 39540atcatttcaa
agacctttcc tcagttcgtt attcatggct gtctgtattc cacatagata
39600aggtctgaaa tactgctaag tggcatgttt tgttttatgc ttttataagt
ttgttgatca 39660ttactgatgt ggacttttgg tgcctcttag gctcattgct
atcttccaac cattgtttgc 39720aatttttacc tagagataaa gagaaagaga
catttggttt cagagtagtt agattgggat 39780catgaaagag caacctcatt
ttgatgcttc aaaaatagca catcccccgt attactggga 39840tttgctattc
ttgggattac ttcaagaaca tccttgtgtt actggtttgg atgcttctga
39900atgctgtgaa gtcagtttca tgtacatggc tcatcagttt agctctctct
tggctttgtt 39960tagacagttg gagcatgatg gcctaaacag cttctttcaa
ttaaacattt taaaatagtt 40020tacaaatagt aaacaaactc cagtttttgt
gactctttgt ctcgcacaac aaaaacacaa 40080tctgaccatg atcatctggc
atcttagggt gaaatatggt tatactttgg cccataccga 40140aagcaagatt
aaaaaggggc aggagagata gactgctgaa ctgattttca aggttccaag
40200aatattgtag gttaagagta aaagtaaact tttggtagaa agcagtgggt
tgtctaggat 40260tgaagtatct gaagttttta aacgaaaatt taaaaagaaa
aatgagaatt gccttacaag 40320tacaatctct tcttttttaa aaaataaact
ttattttgaa atagttttag atttatagaa 40380aaaaattaga tagggtagga
agttttcata taccctacat ccagttaccc cagttattat 40440catcctaatt
tagtgtgaga cattttcatg tttaatgaat caatattgat atgctattaa
40500cttaagtcca gactttattc agattttctt aatttctatg taatgtcctt
tttctgttcc 40560agaattccat gcaggacacc ggatacctca ttacatttca
ttgtcatgtc accttaggct 40620cctcttgaca gtttctcttc tttttttgct
tagaaattct ccagaatttc agaaacttct 40680gggcatcgct atggaacttt
ttctgctgtg cagtgatgac gcagagtcag atgtcaggat 40740ggtggctgac
gaatgcctca acaaagttat caaagtaaga accgtgtgga tgatgttctc
40800ctcagagcta tcattgttgt aggctgagag aagaagcgat cattgagtgt
tcttctgttt 40860tgagtccctg aggatgtctg cacttttttc ctttctgatg
tatggtttgg aggtgctctg 40920ttgtatggtt tggaggtgct ctgttgtatg
gtttggaggt gctctattgt atggtttgga 40980ggtgctctgt tgtatggttt
ggaggtgctc ttgtatggtt tggaggtgct cttgtatggt 41040ttggaggtgc
tctgttgtat ggtttggagg tggtcttgta tggtttgcag gtgctctatt
41100gcatggtttg caggtgctct attgtatggt ttggaagtgc tcttgtatgg
tttggaggtg 41160ctcttgtatg gtttggagat gctctattgt atggtttgca
ggtgctctat tgtatggttt 41220ggaagtgctc ttgtatggtt tggaggtgct
cttgtatggt ttggaggtgc tctgttgtat 41280ggtttggagg tgctctgttg
tatggtttgg aggtgctctt gtatggtttg gaggtgctct 41340attgtatggt
ttggagatgc tctggtatct gcctgcattg cttgccacac ctgcccggtc
41400agaaggcgct atgttgacaa ttgtgcctgc acggtgccta ggtcaatgaa
gggaaccgat 41460ggtagccact ggatgctcct gggaaaatgt cactacaggc
accagagaag ccagagctat 41520gcccaaattt ctatgagtct cagttttctt
aaccataaaa tgggatcaat gtttttgtgg 41580catgtgtatg agtgtgtgtc
tgtgtatgtg tgaggattaa attgtgtatg tgtgaggact 41640aattgccact
actggatcct caaagtggta agaagtgttc ttattaataa tgacatcctt
41700acactcttac ccagcaagat tgatgggtgt ggcactgctt ctctttttcc
atcacatggt 41760ttccatggta tccttttgcc cagggaatct ttgctttgtg
gctagcactt tgttgtttgg 41820ctaatcacgc tttctgtggt caggacgctg
gcttctctgg agccatggga ttctagctcc 41880ctgtcttgtc cctagagtgg
tcactgtctt ctctctccgc ttgcaattcc tgctttgctc 41940gcatctcact
tatgcagtga cgtatatcag tttcaccttg ttctccgtgc ctgctgatca
42000ttggcaccac ttgcatggtg ccatttaggg cctgcttcca gttaagcttg
cttctccaca 42060ggcctaaata tccttgcttg cttcttttat tctcactggc
aggaccaggg cggtctgtct 42120ttgcatgaga cagggtctcg ctcagtcacc
caggctggag tgcagtggct gatcacggct 42180cattgcagcc ttgagctacc
gggctcaagc tatcctcctg gcttggcccc ttgagtagct 42240gggactacag
gcgtgcacca ccatgcccag ctaattttta aaattatttg tagagatggg
42300atctcgccag gttgcccagg ctggtcttga acgcctgggc tcaagtgatc
ctccctcctt 42360ggtttcccaa agtgctggga tcacaggtgt gagccactgt
gcctggccct tgatgtttca 42420gttcttgata tttgatcctc agagtcagaa
aatctaaaaa gagggctatc ccaggttgcc 42480ttggttcatg gcaaatggga
cgttaagagg gcagagagaa tatgaacaga aactgttcta 42540atattggtca
tttaatgtgt aagtattgtt cttttttaaa cctccttcat tttttttcca
42600ggaattgctg gacacagtgg cttggtgtgt gtctgaggac tgtaggccat
ggccctaggt 42660tgtggtttta ggtctcaggt gctcttcctg gctgtctcct
tgcttctttc ccatgtcctc 42720ttctttgttt ccagccattt ctcccttatg
cttaagtttg gtgcagcagg gtttggctgc 42780tctcagattc ctgcttcctc
agatgctgta gttgtcaggc ccagcgggct ggcagcggga 42840tcaggatctg
gctaggtttg ctctcactgt ggcagagtag ggggaggcgt gggagagcac
42900gtgtgacccc aggccagctg tagggagcat aggcatggtc acgtagcctt
caggtcctag 42960actttgtctt ctcatgagta tggctgtgtg tgtatggtga
aaactaggtt ctacttagcc 43020caagaaaatg ggcacatttt gcatgtggtt
tctgtagaga aatgcactgg gtatctgaca 43080tagcctggca gcatgcctcc
ctcaggtagg ttagtctcag gcggtgaagc acgtgtgtcc 43140agcaagaact
tcatatgtgg cataaagtct ccgttctgtg aggtgctggc aaatcaccac
43200caccgtcaag aggctgaagt gatttttgtc tagggaggca ggaaaggctt
cctggagtca 43260gcagccagta ggtgaaagag tagattggag accttcttaa
tcatcaccgc ctcttgtctc 43320aaggggtgcc aggaagctgt ggaggctgaa
cccatcttat gctgccagag agtgggacac 43380catgagggtc aggtcaaggg
gttgtacctt gtttggtaga gaattagggg ctcttgaaga 43440ctttggatgt
ggtcagggga gtgtatcatt taggaagagt gacccggtga ggacgtgggg
43500tagaggagga caggtgggag ggagtccagg tgggagtgag tagacccagc
aggagtgcag 43560ggcctcgagc caggatggtg gcagggctgt gaggagaggc
agccacctgt gtgtctgcgg 43620aagcaggggc aagagggaag aggccagcag
cgtgctgcca tcacccagcg actggcgtag 43680attgtgagag accattccct
gctcttagga ggggctgagt tttagttttc tcttgttata 43740caataagctt
ggtatttgtt tacaaaacat ttgtaaagct aaatcaaggt ttgataaggc
43800ttctagtttt atttaagaag taatgttgaa ataaatgttt gtccaattcg
ctttgctcat 43860ttaaggactt tcagtacaaa ctgcaacaac aggattagga
tttaaacgtt tctgagatgt 43920ttttactcct cagaatttcc cagaatgtga
tctggttttg attttcaagc ttgctgaccc 43980aataggttaa cccacaagtt
ttacgaagac catctcagtc cacttacatc aactgcccat 44040gccacggtta
aagagatcat cgactgatgt ttggcacagc ttcctccctc ttgggtgggc
44100aagcatttgg aagagaaggc tcctatgggt gagagtgggg caccaaagtc
ttccctgtcc 44160catcccctag cttgagaagc ccttctctaa tgtggacttt
gtgccgttag catcgttact 44220agcttgaagt tgaccatctg gacgtacttt
ctggtttagc ctcacaagtg agcaaggagg 44280gttgagagat gtgctgtgag
gaatgtgggg ccccagctgg cagcaggctc tgggtcaggg 44340gggcagggac
cacgggcata cctgacagtg aggaggggcc acacctgcag aaaaggatgc
44400aggactccgc cttgggaagt gttctaggcc agagcgaggg tctgtggttt
ataagtacac 44460ccacagtgct cgggaccctg cagatgtcca gggtgccgtc
tgagcccgta tcatccaaca 44520gaatgttctg ctagtgaaga ttaaagattt
actccagggg ctttaggatt tattatatat 44580atataaatcc tatatatata
attttttttt tttttttttt tgagatggag tttcgctctt 44640gttgcccagg
ctggagtgca atggcgtgat cttggctcac tgcaacctcc gcctcccggg
44700ttcaaactat tctcctgcct cagcctctcg agtagctggg attacaggcg
cccaccacca 44760cacccggcta atttttgtat tttttagtag agacggagtt
tctccatgtt ggtcaggctg 44820gtcttgaact cctgacctca ggtgatctgc
ccgccttggc ctcccaaagt gctgggatta 44880caggcatgag ccaccccacc
tggccaggat ttattgtatt tgaaccatct accattttaa 44940ttttgatgtt
atgtagtatt tgatgataat gaaagttaaa ttgtttttct ttccattttt
45000ctgtttaagt gaatgacctg tatctagttt attcagtaac ttcctgcata
tatttgtttc 45060tttcattctt aatgaatata ttcttaattt agttgctatt
atgttttgct ttgccccaaa 45120attgaaatct tagtttcctt ttagctcgtt
ttagaactag
tgatgggatg tgtcttccat 45180aaatctcttg tgatttgttg taggctttga
tggattctaa tcttccaagg ttacagctcg 45240agctctataa ggaaattaaa
aaggtgggcc ttgcttttct tttttaaaaa tgttttaaat 45300tttaaatttt
tataggtaca cgtattttgt aggtacatgt aaatgtatat atttatgggg
45360tacatgagat attttgatac aggtatacaa tacataataa tcacaccatg
gaaagttgga 45420tatccatgcc ctcaagcatt tatcctttgt gttacaaaca
atccagttac atgctttact 45480tattttattt tatttttgag acagagtctt
gctttcaccc atgctagagt acagtggcat 45540gaccttggct cactgcaacc
tccgcctccc gggttcaacc gaactttggg ctggtctcaa 45600actcctgacc
tcaggtgatc cgcccgcctc ggcctcccaa agtgttggga ttacaggcgt
45660gagccactgt gccgggcctg attgtacatt ttaaaataac taaaacagtc
agggcacagt 45720ggctcatgcc tgtaatccca gcattttggg aggctgaggc
aggtgatcac ctgagatcag 45780gagttcgaga ccagcctggc caacatggag
aaaccctgtc tctactaaaa atacaaaaat 45840tagccaagtg tggtggcggg
cgcctgtaat cctggctact cgggaggctg aggtagggga 45900atcgcttgaa
cctgggggtg gaggttgcag tgagccgaga tcacgccact gcattccagc
45960ctgagcgaca gagtgagact ttgtctcaaa aaataaaaat gaaataaaat
tgggccgggt 46020gtggtggctc acaccttagt cccagcactt tgggaacctg
aggcaggtgg atgcttgaga 46080ccaggagttt gagaccagca tgggcaacat
ggcaaaacgc tgtctgtaca gaaattagct 46140gggtgtggtg gtgcacaact
atagtctcag ctacttggga gattgaggtg ggaggattaa 46200ttgagcctgg
aaggttgaat ctataggtag ctgagattgt gccactgccc ttcagcctgg
46260gcgaccaagt gagaccctgt ctcaaaagaa aaacaaaaaa acaaaaaaca
aaccactatt 46320atcgactata tattattgtc tatgatccct ctgctgtgct
gtcgaatacc aggtcttggg 46380cccttatttc catcactgag caaacttcac
tctgttaagc agcaggtgtg ggatttcatc 46440gttattcagt aattcacaat
gttagaagga aatgctgttt ggtagacgat tgctttactt 46500ttcttcaaaa
ggttactctt tattagatga gatgagaatt aaaaatggta acttacttta
46560tatctttata attgaagccc actagacctt aaagtagtta ccagatgttt
tatgcattta 46620aatggccttt tctctaaaat tagaaagtaa caaggaaaga
aaatgcttcg tttctatgca 46680accctcttgg tgactagtat gtgactctta
atgcaaccct cattgcaccc cctcagaatg 46740gtgcccctcg gagtttgcgt
gctgccctgt ggaggtttgc tgagctggct cacctggttc 46800ggcctcagaa
atgcaggtaa gttgtacact ctggatgttg gtttttgtcg ggggccagct
46860gctactgatc ctttatgtct cagctcagat gtcatttcaa aagtctgctc
tgccctctcc 46920aaattgcagt cgaccttgcc ctgtttatgt ttccctcata
gcactaatcc atgtcagaaa 46980ttgtcacgta cagtctatct gtgtgcttgt
ttattttcta tcccaccctt ccgcaagaga 47040cttatgggat gtgtgcccca
ggacagcagg ggtcttactg tcttatgctc tgttgcagcc 47100cagcagcgat
aacagtgtct gcacatagta cttgcttaaa agatacttgc caaattgttg
47160aaggttgagg taccaatttc attattgctg actataggag ttatagcaaa
atatccattt 47220gtctgttaca tgagttaaaa atatggttgt tgcactgtga
atagtttggt ttagtcaaaa 47280cagttgtatc ttaacggatt gagaaacaaa
agcaggacca cttttcatca gctccctcct 47340tctccttaac cagcaataca
tgctgatgct gatatcccat agaccctcag ctccatcctg 47400agtcactggg
aatgtggtct aaaccctcac tattaatatg aactgagttt caataagaat
47460cttatatggg tcgggcatag tggctcatac ctttgatccc agcacttcag
gaggccaagg 47520caggtggatt gcttgaccca gactaggcaa catggtgaaa
cgccgcctct acaaaaaata 47580caaaacttag ccaggcatgg tggtgcgtgc
ctgtggtcac agccactcga gaggctgagg 47640tgggaggatc acttgagcct
gggaggtgga ggtcgtgttg agccaagatc gcaccactgc 47700actccagcct
gggcaacaga gtgagacctg tctcaaaaaa accaaaatcc agaaaagaac
47760ttatatggct gcagaggtat aatcactaag gaaatttcct tttgtataat
cttttttctt 47820ttactatcat ttaaaaaaat gtgttatatt tctgaagcaa
cacatccagg ttctgcacat 47880agcagccaaa gtgaccttaa agaatataac
tgggtcttgt cattccctta tttaaactct 47940tgtacccatt tcccagtgcc
gtttagatag agattccaga ctcgtcaatg gctctgtcac 48000ctcagacacc
ctgcattgac tcattagtct gattagagtc aggtttttct tcctcctgat
48060ggtttttttt tcccccttag ttctcagcgg aacagtcact tccttaggga
ggtttcccca 48120gccaccctct gaggccgtgc ttgttgccag actctgccac
tagagggcag ggctgcacca 48180ctcctggcac ctcgcacccg gcctgccctg
tcactctgtg tgttgggtga attcctgtga 48240tctgtgactc actgctctgt
gtcctacaca ttcggctttt cttctctccc cacaacccca 48300ttttataatt
ctcctttttc aggaaagctt tattcccatt taaaaatttt tgtttttaaa
48360atggtatttt cttacactta ttttctaatt aaaaatgagt gttttaagaa
gtattatgat 48420ttactgcaaa taatttttaa acccagcctt ttagatcctc
tgtgatcata agagaaatga 48480aggatgtctc ccaacacttg agcttcatcc
acatttcatc ctcctgttct ttcagctgag 48540ttttccccat cccattaggg
actgttggaa tataaaactg gcttttccct aacagggaat 48600gaattgcttc
tgtttctcct gaaggagagc tggaagaatg acttgcgttc ttttgcatac
48660acaggcctta cctggtgaac cttctgccgt gcctgactcg aacaagcaag
agacccgaag 48720aatcagtcca ggagaccttg gctgcagctg ttcccaaaat
tatggcttct tttggcaatt 48780ttgcaaatga caatgaaatt aaggtatgat
tgttgcctca ggtcacaaac atgcgagtga 48840tgctgtgagt gagtctgtgg
agggtgaggg cttctgaaca gggagtcctg tgggagtgct 48900tcttggggta
tgttgtatgt cgtaatttag actaccatca tttgtgttat ttttgaggca
48960cctaaggact tctttccact tctcatttct tactgtgggg tgaagagttg
aattgggaga 49020tggtttctag atgcaaattg aaaaggcatt tttccagagc
agatttgttt tcggcgtact 49080agagtgactc tttaacctag ctgcgggaag
atgactgtgc caagactgca ggtaggagaa 49140agctcactga cgaggccttg
tgggtctgaa cgtcctgcag ctatcagagc ctgttggctt 49200cctgttgtgc
attccaacaa atcatcttca aacccacttt agtgttttgt ttataatgtc
49260cagaaatagt gaccctgtca catgctctac agattacagg attcttagcc
tcttcctttt 49320tggtaggtca gtcctgggtt tgagcccaag tgaccctcct
gggaggtgat gatacacact 49380gggtagagtg gaatcagatg gacttggatt
agaattctgt cctctttact agttattttc 49440ctctaggcaa actgcccaac
agctctaagc tatttccttc gtattctgaa aaataagcct 49500taatgggacc
catatagggc aactctgaga gtaaaataaa ggaatatgtg ttagagtgta
49560gcatagtcac ccacgggaag ggcttagatg ttagctgcta ctgctcttat
tagctgaatg 49620atttggaata aactgttagc ctctctcatg ttttttctct
tgagcttcga agttttcttg 49680ttaatactaa ggagatattc aaactagtca
tggggttttg gaatgacgaa gggagatgat 49740gaatctaaag aatttagtgt
aatatttctt catgctcagt aaatggtagt ttctgctgct 49800gttattttta
ttaccatctc tttggaatgg gagtaggtgc tcctttgtgg tcagaggctg
49860tgagagctcc acagcgccag tttgcccatc tgtacactgg ggtctgttga
aggcagtccc 49920ctctgtgata tctctggctg tcagagctca gatgatagat
ggtatttttg tactcttagt 49980tctcatcatt ttcatgattt cgatcaccat
ttgagtatga tgatgctaac actttgttga 50040acgtagaatc cgttaattac
ttccttcctg aacctttggc attaaaaaaa atctattctg 50100ctacctctct
gctcatttat ggttattcaa atttattatc aagagcctgg tacagtggct
50160tgtgcctata attgtagcta cttgggaggc tgaggtagga ggattgcttg
aggccaggag 50220tttgagacca gcctgggcaa gatagtgaga ccctatctct
aaaaaaactg aaaaaaaatt 50280agctggacat gatggcatgt gcctgtggtc
ctagctactc aggaggctga gacaggaggc 50340tcggttgagc ccaggagttg
gagttcgagg ctacactgag ctgtgattgt gccaccacac 50400tccagcatgg
gtggtaaaac aagatgccat ttcttaaaaa aaaaaaatat atatatatat
50460attatcaatg aaattcagta gtaccaacag gattataaac aaagatagta
gttcccttcc 50520tactttttct cttaatcctt gtgtctcaca ggcaaacata
actcttagta tttcttccaa 50580tatttacttt catgtttctt tctttctttc
tttttttttc tttgagatgg agttttgctc 50640ttgttgccaa ggctggagtg
caatgacgca atcttggctc accacaacct ctgtctcccg 50700ggttcaagcg
attctcctgc ctcagcctcc tagtagctgg gattacaggc atgcatcacc
50760acgctcggct aattttgtac ttttagtaga gatggggttt ctccgggttg
gtcaggctgg 50820tctcgaactc ctgacctcag gtgatcctcc cacctcagcc
tcccaaagtg ctgggattac 50880aggcgtgagc cactgcgccc agcaacttcc
acatttctaa ataacatgct tctactgcta 50940tttttttttt caattttaga
cattttttta ctttcactat agttctatca gaattcagtg 51000tgtacgttat
tatgcctaag taaatagtca tggttgctta cgtattatat ttctttgatt
51060gtgtttctta tttgatgaga aagctgtgtt ttttgctctg ggttgaaact
ggagagagga 51120cctggggagg aggaggagga cagatgaagt tggtgactgt
accttcatgg ccatagctgg 51180gttctcagca cccggggatc tgctgatcac
ctactcatag gccaggcccc tatcgaagtt 51240ctaggtgacc cagtgctggg
gacggggggg ccacctgcaa ggtctaatca tggaggtggg 51300ggctacagtg
ttggcttgtg ctggggccag catccttagg aaggcatctt ggaggtggag
51360gagacagccg cccacttctt gattggggcc ttcagcagca ccagcttctt
gggcaggctg 51420gtgctggctt tcatcaccat gtcgtgttca atcttcttcc
agatcctgac ttctaggttc 51480agctttcctc agaccctggt tcctttcaga
ggccattgct gctgccttgc tctttgctgg 51540cttgtgcctt gattatatgt
ctttgtacaa ctttttgttt tcctggagtt aatcttcaca 51600tctgttttct
tggagttaat cgttacctct atatcgcttg cttattattc tttggccttt
51660ttgtcttctc acaccttcca acttctttgt aatatgtgtt tagtacaatt
tttcatgaca 51720ggtagtttac tgaatcagtt tttccccagt gtggtcatcc
aacttgagtt atccagctct 51780ctgccccagt ctgggcaggt tgatcttcag
gtctgtagta cacttgtatc ctaggacttc 51840tctttgccat tagcctggaa
tttcctttgc agttctcccg ttggatgccc agttcctaga 51900tgccatatgt
ttttctatcg tctagtagct tcctgagaga agatgaatgg gagggaaatt
51960gtatgaggtt ttgcattcat aaaaatgcca ttttttttcc tgtacacttg
gctgggtatg 52020gtgttctggg gtagaaatca ttttccctca gaaatgcaaa
gtctttgccc tgttgtctta 52080aaatctccaa cgtgacccga ttccttaacc
tatgaatgta cttttctttg gaagctttcc 52140atttttgggg aggtgaagtg
ctaggtactt agtaggcctt ttaatttgga aacttacatc 52200ccttcagttc
tgggaaaatt ttcttaacat ttctctgaga agttcttgcc ttttattttc
52260tgtgttctct cctgaaattg gttagttgga tgttggtcct cctagattga
ctcacatctt 52320acctttttct tttctttttc tggtactttt tagatatcca
tctcaaactc ttctattcat 52380tgttatgttt ttaacttctt tcttttcttt
gtctcttgat ggggtcttgc cctgttgccc 52440aggttgtggt gcagtggtgc
gatcatagct cactgcagcc tcaaattcct gggctcaagc 52500agctgttctg
cctcaccctc ccaagtagtt gggactacag gtatgcacca ccacgtccag
52560ctattttctt tacttttttt tttttttttt tgagatggag tcctactctg
tcgcccaggc 52620tagagtgcgg tggtgggatt ttggctcact taagcctctg
cctcccaggt tcaagcagtt 52680ctcctgcctc agcctctcaa gtagctggga
ttacaggtgt gcaccaccat gcccggctaa 52740tttttgtatt tttagtagag
ccagagtttc accatgttgg ccaggctggt ctcgaacgcc 52800tgacctcagg
tgatccgcct gccttggcct ccgaaagtgc cgggattaca ggcgtgagcc
52860catcattaga tctttaaata ccagtatcta taagtctttt cctcttgagt
cagctagtat 52920ccctggaagg aaattactca ttttcctgct tggaggctat
aagcttggct atgtttatcc 52980tgcaaccggg gactggaagg gaggggactg
acagtgttgc tggtcagggt gccctcttac 53040tttttgtttt ctgtgtgcat
ctcacgtctg tcctcagcct atgtaaacac ctcttgagat 53100tatccctctc
aatctttgcc ggaggtgggg gaggggctgc ttcctgggct gccttggatt
53160ggagggaaga cctcaggtga gtgggtggga atttgcccaa ggagccatga
gaccagccac 53220tatttcaccc tctccatccc tccactttca gatgtatgtg
gcgcctccaa agcccgagct 53280cttcttggcg tctgtggctt caataagctt
gctttttgct ggtatccctc ctaccctccc 53340ctgtccccag caaagcttgc
atttgaactt cttcctacgg gctaacaaat cagtcagtta 53400tgtagctctt
gttacttttt agcttccgaa gttttgttga cacccgtagt ctgctaatgt
53460ccctgttctg ttctttctgt tcgtgtaaat atatgcttta tacaacttct
ttacatgatt 53520tttgtggggt ttctgggtag cagagcttca caagttcaat
ccagcgtgtt ggattagaaa 53580tctcccaccc tctggtttat tcttattctc
aaaattacct gccaaacact gatactccct 53640tgtttttcct tttcctgaca
ggaaatgtac ataccataca ggacagaaat cattagtgta 53700tcccttggtg
aataaccaca aagtgaactt aacccttgta accgccaccc aggtcaagac
53760agaatattac caagcactca gaagcctctc ccctattccc ccgtcactgc
tcctgccttc 53820ctccccaagg tcatgactgc tggcttctaa ttccagagtc
tgtttttaaa ttctgtgtac 53880atagaccatg gattaagtgt tctttttgtc
tggtttattt tggtcgacat taagttcatg 53940agagtcttct atattatcgt
gtgtattagt attcctgtag ttttaggagc ttcatagcat 54000tccattgtag
ggatatacca cagtttattc attgtattat cactgggttg tttctagttc
54060ttggctattg cgagcagtgc tactgtgacc actcttaggt gtgtcttttg
gagtacatgt 54120gcaggtttcc atcttgcaca gctagaggtg gagttgttgg
gtgatagggt gtgtgcatct 54180cagctgcagt agaaactgcc aaatagcttt
ccttgagtgc ttgtaccagc tcaccctttt 54240gccactgtgt atggggattc
caggagctct ggtcctcgct agcacttgga attgctgatg 54300cttttactct
tagccttcct gatgggtgtt ttctggaatc acattatgat tttaatttcc
54360attccttaaa gtacccttgg ctctgaagtt taatgattca tgcatctctt
cccttttgaa 54420gtactcttac aggtatgttg tgcatgtgtt gaaaagtggc
actatctatt ctaaaataca 54480gtatgcctcc tctgtgtttg aacagttgta
gcgtggcctt ggggcctcct gttagctggc 54540ttggagaagg gattcttggg
attgtagaga ttagacctga ggaggcccct tggagctctc 54600tgactaaatt
ttattcttta ttattccaaa ctatttaagc tcaccgtgtg ctgactcatc
54660ataataatga gtagctctca ttgtgcttgt ctatttggac tcatacaatg
attttttttt 54720tttctttgag acagagtctt gctctgttgc ctaggctgga
gtgcagtggc acaatctcgg 54780ctcactgcag cctccacctc ccaggttcaa
gtgattcttg tgcctcagct tctcaagtag 54840ctgagactgc aggtgcgtac
caccatgcct ggctaatgtt tgtattttta gtagagacgg 54900ggtttcacca
tgttggccag gttggtctca aactcctgac ctcaagtgat ctgccttctt
54960cagcctccca aagtgctggg attacaggtg tgagccactg agcttggcca
aagtagtttt 55020ttaagatgtt agtatctttt cttgcagcta aaaaagtttg
tcagagatga ttctactttg 55080ttctccaggt gttttctcag ggagaaattg
gaggcagtaa gccactgggg gagtcctgtg 55140gctggggggt ggggtagtcc
tgtggctcct tgtcagggag tcctgtggct ggcaaggaga 55200gaagtcctgt
ggctgggttg ggagggagtc ctgtggctgg ggtctcatcc tgtgcctaac
55260agtgtccaga ggtgccgaga ccagctcagt cggggagacc ctaacccagc
agcgctagag 55320gaattaaaga cacacacaca gaaatataga ggtgtgaagt
gggaaatcag gggtctcaca 55380gcctttagag ctgagagccc tgaacagaga
tttacccaca tatttattaa tagcaaacca 55440gtcattagca ttgtttctat
agatgttaaa ttaactaaaa gtatccctta tgggaaacga 55500ggggatgggc
cgaattaaaa gaagaggttg ggctagttaa ccgcagcagg agcatgtcct
55560taaggcacag atcgctcatg ctattgtttg tggcttaaga atgcctttaa
gcggttttcc 55620accctgggtg ggccaggtgt tccttgccct cattcctgtc
aacccacaac cttccagtgt 55680gggcattagg gccattatga acatgttaca
gtgcttcaga gattttgttt atggccagtt 55740ttggggccag tttatggcca
gattttgggg ggcctgctcc caatacagag gtctcgtgta 55800aattccctgg
gaggcgataa gcctctgaga aacagactat gctaaccacg ccatgaaaga
55860gaaacttatt tataaatcag atgccagtta ctagtttact gcttatttgc
ccaggcgtag 55920ctctgacaga gtccccgact catagtgctt gctcagtgca
tgctgaacaa tgattggaat 55980caagtcatgg ctcagagcat agttttgaat
aatgggaaat ggatgttctt aagtaacata 56040gtcaccaaga taatgcgact
agctgggtca ccccttttca attttaggat atttttatca 56100agatttaaat
ggccatcatt agagttatag cactttctcc tttggattgt cctagaggcc
56160catgagaaag tattccctaa tttcttagga gaacagtttg tgggtagtat
gcggtcatgt 56220ccagttaaat tgcagatatt tccgatcgaa gatgttccag
tcctgagaac ttcgtgacat 56280tagcaggact tctacaagcc atctcttagg
gtggggcatt tactgcagtt ggctagtact 56340cttttctcct taactttgtc
atttgttgat ttttttttaa ctgtccccaa atactgtggg 56400cagagtgtat
ctagaattga ggcctccacc attgcggaga ggacatggat gctgagcagt
56460cccctgagtg aaggttataa agaagcaaat agactacaca tgtctgtaaa
ctgctcttga 56520gtgtcccaaa tttggggtac ttcagttcag ctgtaggaaa
agcctcaaac tgtttatact 56580ttgcaagaat tggaaacttc taattcacgt
taagttttat gtaatacatg ataagcttca 56640taggagcttc atcttttatc
tacttggact tttgcttccg taggttttgt taaaggcctt 56700catagcgaac
ctgaagtcaa gctcccccac cattcggcgg acagcggctg gatcagcagt
56760gagcatctgc cagcactcaa gaaggacaca atatttctat agttggctac
taaatgtgct 56820cttaggtaag gtggaggcat atgagtggaa gagtctccag
catgtactca agatagacct 56880ttgaaataaa taaaaccaga tgatccctca
gcttctagac caggctattt ggcactggtt 56940gattgaatgt gaactgcact
ggggctgctg tgagcccgca tgggtctctg tgaccctgca 57000gatgcagccg
tgcccaggga ctgggcagtg ggtgtgggct ggtgtgagcc ctgtctgcca
57060cccagggcct ggccctctgt ctgtgtcggc catgactatg gtgagtcttg
taggcttgag 57120actgtgcctc gggttcctgc gggttctctg taggtcagtt
gacagtttct cctgttgttt 57180gggtaactgt ggaaacgaac actggcaagt
gctgaagcga gcatgtggac gtgcgatatg 57240aaataacgac ctggctttca
aaggcagtga ggctctctgg aaaggacctt gctgagctag 57300ggatgtgggt
gtgtagccat tcccagtggg cctcatggcg tactcgttca tgatcatgtt
57360tgtgccatct tgatctctca ggatctcttc ttttttaaca gattaagccg
ggaatctcca 57420aacagtgagt cagatgttaa gatgtcttgc ttccaccccc
acaggcttac tcgttcctgt 57480cgaggatgaa cactccactc tgctgattct
tggcgtgctg ctcaccctga ggtatttggt 57540gcccttgctg cagcagcagg
tcaaggacac aagcctgaaa ggcagcttcg gagtgacaag 57600gaaagaaatg
gaagtctctc cttctgcaga gcagcttgtc caggtaggag cacagggttt
57660actctaggcc ctgcatgtga atgactgaca ttcaaagaac cgattaattt
ggaagagaag 57720cggcagaacc gagagttaga ggtgtggact ctggagctgc
gctgctcgtt tccaacccta 57780ggtgctgacc tctagctgtc ttccctctgt
atgtccctgt caccgtgagt caaatgcggg 57840tgatgcctcc tcaggtgccg
tgttacctaa gcctctcaga gaccactgct accctgtttc 57900taaaaccaga
ggtcacgata tgtgttcatc cacccagtaa atactgattg agcacccact
57960gtgtgctagg ctctgggata ggggctgggt atacaatggt gagtatttca
gctgcagctt 58020ctgccccgtg gaggctgtgg cctagcacac tggtctaggc
acggtggtat atgctcactc 58080aaggagatag ggacgtggtc gtttggggtg
tcggaacaaa atgtcggaac ttctctttcc 58140aatgcagaga aaccttgcag
taattctaat gtactgtgat tggcagttga cttcagttct 58200ttgtagcacg
cttactcagg ttatttcact aactatgtaa ccatgcagcc tcattttaag
58260caattggatt ttttgaactt tacttaaaat gttatgtcag ggtttttatt
gtgcttaatg 58320tgtgccattt agctaagttt tgtaggatac gaaattgtaa
gtggcttaaa atgattctta 58380atagaatcat gaattgaaga taatgctaat
aatttaagca ctgagttagg tagtgtttgt 58440aaaatgctta gaatgcttcc
tggcacatgt taaggccatg taagtgctgc gtgttgataa 58500acagctgagc
aaaagtggac tcttaagaaa gtattggggc tgagagttct gttccaacca
58560gctgcccttt ggttattttt cagaataaaa gcagagtctc atgggatatg
acatttatat 58620ttccttcaca aaaaacactg ctgagtgttt tgttgagtaa
aaagggtgta gccatggtaa 58680taatacattt aaaatatagt ttatttcatc
tttaccttgc cttgtttttt ttttaagcta 58740gctttttatt gagaattcca
cacatacaaa agtatcaact catgaccagt tatatttcat 58800ttataatcct
acttctccct ttttttatta tttgaaagca aaccccaatt atcctcttat
58860ttcatctata agtatttcag tatctctata gatgaggact cttctttatt
tttaaaactt 58920tatttttaaa atgatggtca gatgcagtgt tcatgcctgt
aatcccagaa ctttgggagg 58980ccaagctggg cggatcactt gaacctggga
gtttgagacc agcccgggaa acatggcgaa 59040accccatgtc ttaaagaaaa
aaatcagcca agtgtggtga tgcatgcctg tagtcccagc 59100tacttgggag
gctgagatgg gagggtcaca tgagcctgga agatcaaggc tgcagtgatc
59160catgattgta ccactgcact ccatcctggg tgatggagca agattctgtc
tcaaaaaaac 59220aaaactgcaa aacaacgtca caaaacagtg ccattgttag
acctgaaaat attaaacatt 59280tcctacatca aatacccacc aactcattat
caatttttct ctctactctt ttggaatcag 59340catctaaata aaattggtcg
ataaggattg taaatctctt tgatgaactg gttcccctcc 59400atcccagttt
ttttccctta gagttcattt attgagaaac cagattgttt gtcttctaag
59460ttttcctgtg gtctgatata ctgcttccat ctccactgtg taaattaaca
cctttttctc 59520ttctctgtat ttcctgtaaa tcaataattg gaggaaaagc
cttgtcagat ttagtgtata 59580ttttatatct gagtccagta tttcttatat
aatattttaa gataagtgta ctcttttaaa 59640aagtattgaa actatatgct
caattttttt taactgatgc ttttaagaag gctgcttgat 59700cataaaagtt
tagagatcat tggtctgatg ggaaaagcaa ataattacta aaccgtttag
59760caaggttgag gtgcacatgg tggggcctgg agaagttcag tcatgagccg
tcacttatgg 59820gcacgtggaa tctgacccgg cacagagttg ggagaagaca
ggagctttat agacagaaaa 59880tgtggtcttt gctaagtccc aggagtgaaa
gggtgagaca gtgctcacag cacacgagtg 59940tgggtgcgta gacagagcaa
gggtgggtcc tgaaaaggcc tgcaggcttt ctcatagatt 60000agcaagagtg
ctggttacgg aggtttctaa catttgtgaa cagatcgaaa ctgtgttaaa
60060ttgggattgc agtaatcctg gaaggacagg gatagagggt gaaggggaaa
aaagggtatg 60120gatgtgagac ttaattgctg attttcttaa gacctttctc
caaagtaaat aaatgatgtg 60180gcacattttt gaactggcaa attctaaact
ctagatatga
ttatctctat aacatatctt 60240actccatctt cttttgacta aaaactgttc
ttaattaaat taccatgaga cgttcaattc 60300agcaaatgta gtttggctaa
ccatatttaa ttagaattta atataatcct aggcctggcc 60360aaactattaa
gcaagtgtgg gcaaaatatt gataatttta gatatgcagg aacttagttt
60420gctttccatg tgtgcttttc gaaaaaggaa taaattgaaa aatagaggaa
gccctgaaat 60480ccaagaagca aactctctca cctaggcatg cagtaaaagc
aattctagga tgattgctgt 60540ttggcgcgta gttcgtatta gaaaccattc
ttcttgaata aatagtatgt ttaagaagct 60600gggcagaggg aaggcatatg
catatattat caacaaggag ggagaaaaag gcaattagta 60660accatccata
ggagggtcag caagatttat aaaggaaatt tgtgatccaa gtatgaagca
60720aaataaggtg cagaataaat tttaagcaag taatagatta gagtaagaga
acccatttga 60780ccattaacct tgggacattc tctttcaaat gacatggagt
agtactgaaa tctttctttc 60840tttctgagtc taggttattg tgactggact
cagaaagaaa tatttcatta ttgcagtgaa 60900taacatttgt gaacattatt
gttcataaat tatgcagtga ataacattta tgaacacgtg 60960atgtgtaaga
tacatactgt ttatttttag ttaagttttt tggctcaact tctaggcaga
61020gaacattaaa tgtaaatagt gttacctagg agcatgtaaa tggaaatctc
catagtatga 61080aagcagtgct gttgctaaca gaatttagga gggggcagat
gaggtgaagg aaatgtgggt 61140gctgatttcc ttattacatt gagaggagcc
aggagattct ttgttcaaaa tggatggctt 61200aagaagtcaa agtataagct
gattacgtag agcaggtacc caaaaatgtt ttgtgtaagg 61260ggccagatag
taaatatttt cagtcttgca ggccatccca agtctgtggc agctactcaa
61320cactaccttt gtagcatgaa agcagccaca ggcagcccat aaatgtggct
ctgttccggt 61380gaaactttag gtacaaaagc aggtgcaggc cagacctgac
ctgtgcactg tggtttgctg 61440acctgggatt caggggtata gaagttacca
tcagaagagc taaaagtgag actttttact 61500ttatactctt ctacactgtc
tgattttgaa aaaaagaaac atgtatttta taatattaaa 61560gatagggttg
gcaaatagca aataaaaata cagaatacca gtgaaatttg aacttcagat
61620acattatgag taattttatg gtgtaagtat attccaaatc atgtgggaca
tacttacact 61680acaaaattat ttgttgtttg tttacagttt aaatttgagt
gccttgtatt ttatctggca 61740actgtaatta aagggaaaaa gaataaattc
attatgttca tataatgtga tatagcaggg 61800gtccccaacc cccaggctgc
agagtggtac tggtccatgg gtccccaacc cccaggctgc 61860agagcggtat
tggtccatgg cctgttagga accaggctgc ccagcaggaa gtgagcagca
61920ggtgagctgg cattcccacc tgagcaccgc ctcctgtcag atcagtggca
gcattagatt 61980cccataggag tgcaaaccct attgtgaact gcacatgtga
ggggtctagg ttgtgcgctc 62040cttatgagaa tctaatgcct gatgatctga
ggtggaacag tctcgtcttg aaaccatccc 62100ctggccctgt ggaaaaattg
tctcccatga aaccagtctc tggtgccaga aaggttgggt 62160agcactgtga
tatagtatta aaagtgctaa taaatatggc atactgcctt taaaatgtct
62220ggtagctctt tctcagtggc actcataata gtgttttttg atttttaaat
gtgtgtcaag 62280ctgactctcc cctccgtgta tgctgggctt tattttccct
ttcctagtca ccagttttgg 62340gaaatagaga tcttcattct catgctgctc
ctctagtgca agtgctccat ttatttttaa 62400ggaattaata taacaaaaaa
tcatgggaat ttagaaaaca acatggaagc taatgatcac 62460attggtggaa
gtgataggga aatatttagg gggagaagtt aaggtataaa ctttgtcaat
62520gaagtcctat taaaaacaac aaaaaagtga agcttaggat gcattttata
aactctgacc 62580agaacacctg tgtttctctg tttctaggtt tatgaactga
cgttacatca tacacagcac 62640caagaccaca atgttgtgac cggagccctg
gagctgttgc agcagctctt cagaacgcct 62700ccacccgagc ttctgcaaac
cctgaccgca gtcgggggca ttgggcagct caccgctgct 62760aaggaggagt
ctggtggccg aagccgtagt gggagtattg tggaacttat aggcaagtta
62820ttagcaaggt ctactcttac aattaacttt gcagtaatac tagttacact
ctattgatta 62880tgggcctgcc ctgtgctaag cagtctgcat tccatcttcc
ttgccaaaac ttataataca 62940aatttcatct ttattttata aataggggag
ttgggctggg tgtggtggct cacgcctgta 63000atttcagcac tttggaagga
tcgcttcagc ccaggagttt gagacaacct ggccaagtga 63060gaccctgtct
ctacaaaaaa aaaaaaaaaa aaaaaattag ctgggcatgg tggcacatgc
63120ctgtagtccc agctgctttg gaggctgagg tggtaggatt gcttaagccc
aagaggttga 63180ggctgcagtg aatcttgatg gcagctgcac tgagcctggt
gacagagcaa gatgctgtct 63240caaaataaat ttaaaaataa aataagagaa
ttaaagttta gcaggttggg tggcaaaatg 63300aggccacaca tttaaagccc
ctcctcctga ttcttttctc tgccttggct gcctcctgtg 63360gcattttagg
tgctgagaaa tgaaaacagt agggaaaata gttccaggat cctcatgtta
63420atttgccaga aatggcatct tcaagtcgtc agagggatct gagagttcct
tcctggcctg 63480acttgagaaa atccgtctgt ccccagctct gcgtctgcct
ccactgccca gtcacctcct 63540ctccatgctc ttggggctgg gccctacccc
accatgcagt gctgccctgg agcagtgagc 63600ttggtgggtc ctgtctggca
tgagagctgc ctttgggagc tggatcccag cctctaccac 63660tgggtctggt
gcctagcagg ctatggataa acttctgctg actccggcct ctcctaagcc
63720actgcaacgt ggtcggtgta gtgcacagtg tgtgtgcagc gtggccttac
tcacagcctc 63780cacattagag agaatctgac tgaagtctta ctgctgcctc
gtgtgaacat aaatgtttgc 63840cagaaccatg agcaggaaat gttaatctgc
cttgtttcct gtcctttaca cggaagaatt 63900tttttctgta tggaatgcgt
gccttacaaa taatgagtgg aaatacccat cgctaatgaa 63960aagttatact
tgactgttag tcagctaaat aatctgagat ttctaatact tttaatttgg
64020cttttacaat gcaatttatc ttagcttttt tgatttctta ggtcatatct
ttagaactat 64080atatttgaat gttaatgtaa ttttcatatt gaaattaaaa
tgttgaactg cgatgttaag 64140tgtttcctgt ggaaaaacgt tcacattttc
tctagtttta aagttgaatc aagctgtttg 64200aagattttca catttcttct
agattttatc agcttgttac tttatctgtc actttctgtg 64260atttgcagct
ggagggggtt cctcatgcag ccctgtcctt tcaagaaaac aaaaaggtga
64320ttatttcaga aatcagagtc ttgtgttgaa tcttactgat tttcttgtat
ttctgtaatg 64380taatgtatct tgtatttctt gtaatactgt attggactct
gtgtatatct cttctcagat 64440gagtgattat atgtgtgaat gttgctggaa
tctgataacc aggcctgaat agttttgtag 64500ggtggctttt aaaaattact
ttcatatcag aattgctttg tcataaattt tgaacgcatc 64560ataaatttct
aatgttcggg gtcagcagac tttttttgta aagggacaga gtgtaaacat
64620cttagcttta tgggccatat ggtctctttt gcaacattca gctctgccct
gtgacaggaa 64680tgcagttgta aagacatgag ctactggcca gctatgttcc
agtagaactt tacttacaga 64740aacagacagg ctgtagtttg ccaatacctg
ccttagggaa tgtgttgtta tattttgtga 64800gttaccttct cagtaaattt
tatttagtat tagtcaggaa tattattaag tagcttcttt 64860tccagcctgg
tcaacatagt gagacccggt ctctaccaaa acaaaacaaa acaaaaaaac
64920agccacgcat gtggcatgtg cctgtagcct cagctgctgc tcagggggct
gaggcaagag 64980gattgtttga gcccaggagt ttgaggtcac agtgagctgt
agtcatgcca ctgcactcca 65040gcctaggcaa cagaatgaga ccttgtgtct
taaaaaaaaa aagtttcctt tgttgggtta 65100ttttaatttg gacctggtta
tcatttttca gccatattta actttgtaca tatcagaatg 65160ttctgataaa
acttaacttt tattaaagtg tttgtgatat aatctgctag ttttggtaca
65220cattatcttt tgcaatgcca gttattttct tttccagtgt gggtttgcat
aggaaaagaa 65280ttgctgtcac tttctatttt gaaatcttaa aagactgatc
cttttttgtg tcatgatttg 65340agtatttaat tgagagccta atgcctaata
ttatttgcag tattaaatgg gatcttaaca 65400ggaatagcat tctagccttc
attgaattaa gtaaacattt cttaagagaa cttggaatct 65460ataatatttg
cgtcatcata gtatgagata cttaatcaag tttgagattt tagtgaaaca
65520ttgtttagaa gccaaaagga ttctaggaaa aattaatgtc tatattcttg
aattaggaga 65580gattttggga cgtgtgacta agttacgctg acacttgttt
gtttcttagt cgctttttcc 65640agtggcggtg agaacgaaga tgactgattc
acattgctca gatgagttta tcctcttctg 65700gctgggacat gggatatatc
ctgtctcttt taagcctttt tggtattttt cccccattga 65760gagctgtgtc
ttcaaactct tctgttatag ctggaaaatc ctttttaagt gaaatctgcc
65820caaattataa gacagatgaa ggtagagttg tgttggatat aggattaggg
tgaaagtagt 65880gggggtgtcc tggagcctct cttctggtgg cagcctagct
cttgtgcctt tgaggaaatt 65940accctgggga cggctctgtg gaacatattt
gcaaaccact gatttggaag atagagatgg 66000cttttgttaa gatctgaatt
cacctttttg gcattttatt tgatttctca aggtaaagaa 66060cttattttgt
aataaagttt cctattattt agtagatagg ccaagttgct gtgttaattc
66120catgtagatt ttgggtttcc tttgctcatt ttttcactct taatctcaca
tcattgtaag 66180tttatggaag ttatcatact tctgactttt tctttgaaga
gcagaaatta gaaattccca 66240ataattattt tgatagtgtc atttaatgac
actcacatgt gatgtagcca caaagattta 66300atgagttcag ttttaaatca
tattaagact gttggtttca tttgttctca ttaatgtaat 66360tctgaagatg
aacaataaaa tgtattttta gaactttcaa atgaaatatt atttcatcct
66420tccagatcat ataatgctta agttctgatt gttaatcata aagtctagaa
aattaaaaga 66480taataaaatg aaagtgactt ttaggtatta gagttttatt
ataaattctg gtgtgtcatt 66540ggagctatga catgaatatt tcaaaggcca
atagcattgg atctttacag ttataactta 66600ccatttttaa gtttaagtag
taatatagat tatttaataa tcaaaatcaa taaatattaa 66660ttattaaaat
gttttgtggt atagtttgag aatcattgct tttaactttt tccatatagg
66720tttattgact ttaatagcat tctaaacata acatctctac attctttgtg
tttaatactg 66780tggaggtata aaaatactta tatatgatga taaactatat
tagagtaaat taaatattct 66840tatgagtttc attttagagt gcatttactt
aattttgaag tccttatttt tagcaaacta 66900aaaggaatgt tggtacatta
tttactaggc aaagtgctct taggagaaga agaagccttg 66960gaggatgact
ctgaatcgag atcggatgtc agcagctctg ccttaacagg tagttctcac
67020tagttagccg ctggtgtgga ccttcactgt ctgccttcca ccccttgccc
ttcctgctcg 67080tccccctgca cctggtggac agcacgactg ggggcagcag
tggagccagg ttgcttaaat 67140ggggcatatt cgggcttctt ttataatact
tactctgaag cttgtgtgtc tgtggtgttt 67200gcatcatata tttgttgttt
tccatggttt aggctgtttt aaaattaggt ttatggcttg 67260agcatagggc
tttgtgagta ggggatggca ggtcgaaaca tctcatgagt tggatgggtt
67320atgctggggg ttgggaaatg ggatgaaaaa ttatgggatg aaaaattgcc
tatggatagt 67380ttaacttgaa agaatctgcc tttgtttaca gatagttatc
ttttttcttt tttgagatag 67440agtctcacac tgtcacccag tgcagatacc
cagtgtcact ggagtgcagt ggtgtgctct 67500tggtgcactg cagcctccgc
cttctgggtt ccagcgattc tcctgcctca gcctcccaag 67560tagctgggac
tacaggtgcc cgccaccacg cttggctaat ttttgtattt ttttgtggag
67620acgggttttt gccatgttgg tcaggctggt cttgaactcc tgacctcaag
tgatctgcct 67680gcctcagcct cccacagtgc cgggattaca ggagtgagcc
actgtgcccg gccagttaca 67740gatacttatc taatgaaatt ctctgtgtac
tttataaaag atgaggatta actgaaggta 67800ctaataactg gattatatga
gggtggtttt ggttgtataa tcctatctaa aagaatattt 67860tagctataac
tgaaagtaag acttaaatat ttagagagga aaatctgaat aattctagta
67920gtaattattt atttacaaaa taaaaataga tttttttttg attacacaaa
ttaaacaaca 67980ataaaacatc acagcaatcc ggatactata aagctcacat
gcttaccgac ccaactgccc 68040caggagtgac cactgccaac agcttcatgt
cgaccttttt gccataattt ttatatagcc 68100ttttttgttt ttaaatggta
atttagaaag tcaactagga aaatgtgtta caggtttatc 68160ttccaggaga
ataggactgg agtcgagatc ttgaatgtgg cttggaagaa ggcaagccca
68220ccccagagag atgagttgac agttgtttct gaccactgct tgcttagagg
gcctgcgtgt 68280ctgtgaccgc ctagctttgc gcccctgact aggctgcccc
ttaattacaa atgtctttat 68340atattgctcc agctaaggct tggagtagtc
ggttaagaac ttgaacttcg gtttttgcag 68400tgaaacagca tttgagaata
tcaccttctg ataagcctta ttttataagg tgggtactgt 68460agtgggaggc
agtgtgagag atgcttgaag gatgcactgc tgtcctgcat ttcagcatct
68520tcaggatgct gtgcagctga aacatttgat aacggtggaa ctgttcgtta
ttttgcaagc 68580ctgtgattcc ctattgaatg ttttctctcg ccatttgaca
aatgagtgtt tctctgtctt 68640cagcctcagt gaaggatgag atcagtggag
agctggctgc ttcttcaggg gtttccactc 68700cagggtcagc aggtcatgac
atcatcacag aacagccacg gtcacagcac acactgcagg 68760cggactcagt
ggatctggcc agctgtgact tgacaagctc tgccactgat ggggatgagg
68820aggatatctt gagccacagc tccagccagg tcagcgccgt cccatctgac
cctgccatgg 68880acctgaatga tgggacccag gcctcgtcgc ccatcagcga
cagctcccag accaccaccg 68940aagggcctga ttcagctgtt accccttcag
acagttctga aattgtaagt gggcagaggg 69000gcctgacatc ttttttttta
ttttttattt gagacagagt ctcactccat agtgcagtgg 69060aggccgggca
caggggctca tgcctgtaat cccagcactt tgggagactg aggcaggcgg
69120atcacttgag gtcaggagtt cgagaccagc ctggccaaca tggtgaaacc
ctgtctctac 69180taaaaataca aaaattagtt gggcgtggtg gcacatgtct
gtagtcccag ctgttaggga 69240ggctgaggca ggagaattgc ttgagcctgg
gaggcagagg ttgcaatgag ccgagatcgt 69300gacactgcac tccagcccgg
gcaacagagc aagactccat ttcaaaaaaa ataaaaaaat 69360aaagtgcagt
ggctcgttct cagcccactg caacttctgc ctcccaggct cgagcgattc
69420tcccgcctca gcctcctgag taggtgggat tacaggtggg caccaccaca
ctcagctaat 69480gtttgtattt tcagtagaga cagggtttca ccatgttggc
caggctggtc tcaaactcct 69540gaccttagat gatccaccca ccttggcctc
ctaaagtatt gggattatag ttgtgagcca 69600ccatgcccgg ccctgccacc
tgccatcttt tgagttcttc cctggagacc tagacctgaa 69660ccctcctgct
tgttctcttg ttatctaata cccctattga cagcgcagct tagatcatta
69720atggagagct tgacctcatc tgataccttc actgaaggaa acaacttagt
gtcttttgtg 69780ttgaacactg aggtaaaaaa ttggaatagt tgattatatg
aactctgcta aaattgagtg 69840cattttacat tttttaaggc cttgttgggc
cctggttaaa taattatttt taaaaatcct 69900taaggagcct attataaaca
gatctgtggt cttaatgaaa tgtgattaat actgtgcatt 69960attttaagaa
cttttgactt ttcaaaaaac ttttacaaca tttcccattt gatagcggca
70020taggtttaag cacttctcat ctctaagtta gtggacaaaa aaccctcatg
gatagtctaa 70080taatgtttgc tacaagtcca tgttgagttt tatactccat
tttattttca gttttaaaaa 70140ctgtggttaa atatgtgtaa cataaaattt
atgttcttaa ccattttttg cgtatacagt 70200tcgctggtat taaatacatt
taaataatgt catggaatca ttgctaccac ccatctctgt 70260aaccttttga
tcatgtaaca ctgaagctct gttcccattg aactctattc ctcctttccc
70320gccaagtccc tggcaaccac gattcttctt tctgtcttct gaatttgact
actttgggtt 70380ctcatatact ttaggagtca cacagtattt gttttactta
gcataatgtc cccaaagctc 70440atgcatgttg tagcctatgt tagaacttcc
taatgtttca ggccaaatac tattccattg 70500tatggatagg ccacattttg
cttttccatt cctctgtcca tggacacttg tattgcttca 70560tgttttagcc
attgtgaatc atgctgttat gaacgtgggt gtacagatag ctcctggaga
70620ctctgctttc catttttttg gctaaatacc cagaaatgga gttgctttta
cattccaatt 70680ttaatttaaa acattcatat cattgagtgt tttacttaat
agtatagtag ttaacaaact 70740taataaaata gtattttggt aataatttgc
tggtagtcca ttgttcagtt tttttaggta 70800aattacacag gacatttcaa
gtggacatga aacatcttgt gatgtggaat catgccccaa 70860gctgatggct
aaacatatga aataccatac cctaaattta gtagatttag tctttgcaat
70920ttaggagata acctgttata ttgttaggtt tttgtcgaaa agctttgtcc
tcatatttcc 70980aacttgctgt aaaatttgtt tgtgaagaca aatatttttg
tatgggtttt ttctttttca 71040tattaaaaag aaatgtccac attggaattt
ttttggagtt tttagagcta atagagcttt 71100tcataatgta gtgggaatga
gtgatcagta agctcttagc agtttccatg cgtgcatttc 71160tgtgccttga
aataaatgac agatgagtac atttgtgttc tgtgtgtaaa atgtgctctt
71220tcctcattgc acttccatgt tggagggctt gtctcttggt gatcacactt
caaaattctc 71280acagcccccc ttgaaccgtt taggtgttag acggtaccga
caaccagtat ttgggcctgc 71340agattggaca gccccaggat gaagatgagg
aagccacagg tattcttcct gatgaagcct 71400cggaggcctt caggaactct
tccatgggta tgtggactac aggtgatgcg ctacaaagtg 71460gtttgtattc
agacctggac atcttaatta tatctttgct tccaagaaga agtcctttga
71520tactgttttc tgagttctga atagctgatg aaaatgacca attgaggaat
aatcatactt 71580tttcttgatc taaatcttat acttttgagt tatcttagca
taaatgtata attgtatttt 71640aagtggaaat ttgtcactta atcttgattt
ctctgttttt aaagcccttc aacaggcaca 71700tttattgaaa aacatgagtc
actgcaggca gccttctgac agcagtgttg ataaatttgt 71760gttgagagat
gaagctactg aaccgggtga tcaagaaaac aaggtgaggg acataggctt
71820gagacgactt ggtgtttctg agcttgtgtg aggatttaaa atcgccctgg
ctactgtcta 71880ctttattgct ttcccatccc tgggccttta aatttcccct
ttaaatacca gctcttccca 71940ggcctgttgt tttctgcctt tccaggtact
acccacagcc ttgagaattg cctgagttct 72000gcctcctttg agagtgtgcc
ccagacaaat ctattctgta ctgaatgttt ccttgtctga 72060tttcttggat
cattcatttg atggttgcgt atggcctgca acgtttcttg ttttggttct
72120actgaactgt tctaaaagtc tctcttcata ttatcttttt acatgtaaat
gtaactgtct 72180tcacttttaa ttcctcaagg acaaggaata gcgtttcaca
gttcgtccca tcaatcagaa 72240ttatagcctt tggcatctcc ctatctacca
ggcccacttc ctcttagatt tgggcttccc 72300caggctgttg cctttcccca
agtagcttct gcttgtcctg tagaagacct ttcatgcttt 72360gcttctgcag
cagccgttcc tgaatgccta gtgtcaactg ccttcttacc acgcccaccc
72420tccctgcatg ctgcatttat cccctgccac agccctgtga ccctgtgtcc
tgctgcctct 72480gacttgtctg tttctgcttg gccatggtct ctgtgaggtc
aggtgtgcat atgggcacaa 72540accagggcat ctctttatcc ccagcacctg
gcttaagtgc tgctctggaa ctatctgttg 72600aatgaactaa tgcatgaatg
tattgttgag tatgagacaa acaagtgtca ttgtctcctt 72660tctagccttg
ccgcatcaaa ggtgacattg gacagtccac tgatgatgac tctgcacctc
72720ttgtccattg tgtccgcctt ttatctgctt cgtttttgct aacaggggga
aaaaatggtg 72780agtacaaaag gggatgtgca cagttgaagg aaataactag
gtttcagagg tcagcttggt 72840ggcctgtttt tgccttgcgt gcagcagagg
aagtagaatc tgaggatgag tttggttttc 72900actagccgag gggagggagg
aaatgatggg agcaggtagg ttattgggtc tggttttgtt 72960catttgaaaa
caatctgttg tttgaggctg aaggtggctt gggtgatttc ttggcagtgc
73020tggttccgga cagggatgtg agggtcagcg tgaaggccct ggccctcagc
tgtgtgggag 73080cagctgtggc cctccacccg gaatctttct tcagcaaact
ctataaagtt cctcttgaca 73140ccacggaata ccctggtatg ttaaaagttc
acatcttatt ttctcagatt taatcattat 73200tgtaaaaact atttcagtat
tgactatttt agttttagag cagtaagtgt tttgagttca 73260tttgggatat
ttgacctgcg ttgtagctct tcagaaaaca catgaatagt gaagttcttt
73320gtttcatggg ttccctttag atgaaaccca tagaggagaa aagtagaaac
ctcagcacgt 73380aagagccaac atatatacac atcggattta aacctaaagc
acaaattgtg cctggtcgca 73440gtggcgctga gtcgcactca gccaggccag
gcattcacac tcagggtgag tgggaaccag 73500gactggctga ggcagcagtg
gacccaagtc tccatcgcgc ccatgcttac tatggagcct 73560tctcgttctc
tctttttctt tgggtgagag ggtacacttg tgtttttgaa tttatatgag
73620gtaagtgtgt aatagggttt tttctaatct tttttaagtg gaatctggaa
ttttaatcag 73680atttattatc tgacaaccta gaattataat ccagaaagtc
tgtggtattg aggacatatt 73740ggcaatatga tgaatctcta attcttaaat
cctgaaactt tttttttttt aatcacttag 73800ggttattata gtgaagtcat
ttctgaattt ggatcttctc ttcacacctc tttttctctt 73860tcctgagaat
taagcttttg tttcgagtta gaaagttgat agtagggaat tgttccatgg
73920ctgagcaatt tatctccaca gaggaacagt atgtctcaga catcttgaac
tacatcgatc 73980atggagaccc acaggttcga ggagccactg ccattctctg
tgggaccctc atctgctcca 74040tcctcagcag gtcccgcttc cacgtgggag
attggatggg caccattaga accctcacag 74100gtaacggcca gtttttcagc
tgtgtttttt ctagttatgc ttactaaggt ttaagtttag 74160atgatgatgt
ttgttgcttg ttcttctggt taggaaatac attttctttg gcggattgca
74220ttcctttgct gcggaaaaca ctgaaggatg agtcttctgt tacttgcaag
ttagcttgta 74280cagctgtgag ggtgagcata atcttctgtg gaaccatttc
ttcacttagt ggacatttta 74340tcattgctac aattaaaatt ggagcttaat
aggaaatatt tccatgcact ctaaagctgt 74400aaccagtaat acccaccatg
tatccatctc tcagctttag aaagaaaacg ttgccagtaa 74460agttaatgct
tcataaactt cagtttaagt tctaattctc agaatatttg tttgaaatag
74520acctcttcct aaaggatata tttagaaata acctatcatt aagtgtaaag
tctgttgaat 74580atgctgggca cggtgactca cacctgtaat ctgaccactt
tgggaggcca aggtggaagg 74640attgcttgag cccaggagtt caagactatg
ggcaacatag ttgaccctgt ccctacagaa 74700aattaaaaaa aaaaaaaaaa
aaagtagctg ggtatggtgg tgcatacctg tagtctcagc 74760tactcgggaa
gctgaggtgg aggggggatt gcttgagccc cagagatcaa ggctgcagta
74820aggcgtggtt acaccactgc cctctagcct gggcaacaga gtgagactgt
ctcaaaaata 74880atagtaataa taatcagttg aattaaaaaa aaaaaaaaaa
aaaccactgt gctaggccca 74940tagtatggta agagttaaag tgagccttag
ggattattta ctcaacctct gtttctgtat 75000aaagtggaat aggctcaatt
ctttaagtga tagcatgttg aacctttcca taccaactgg 75060ctcataagtc
acaactggcc agtcaacaag agtaaaaatt aactggtaaa aatcaaagca
75120aaaaacctac aattgtcaaa tttgtgggat aactccccct tttaaaatgt
catgcctgac 75180agtaatttct ctctagtttc caggttttca gtcagttgtg
tcttttttga gcagaaggaa 75240gcatgctaag agctcaatct tgtggctagc
tgggggtctt
tgtgtcagcc atgcatgtga 75300tggtgcccct gggtgcttgg ggctgcaggg
gaggggtaca gcagtagggg cctgttctgt 75360tctctcgtgc tgtggagtac
atagtgacat agtggggtgg tccttggtgt aggtcccttg 75420ttcctacccc
tgggtctgag atttatttag aagtggtgtt ggggctgtgc ggcaggcccc
75480tctgtaactg atcaatgttt gtgaagttgc tgtttgagag ttgaaaccat
gacataagca 75540gaaatggaag gaagaaagaa ccagttatgt gaaagggaca
catttacttt taagcttgta 75600tttactgaga taaagtattc ttaatcaatg
ttcttgagag gtgtgggaaa aatgcaacat 75660cctggttgca gttaaaccca
gaacattgtg tgttgaagag tgacggttct caaaccgtca 75720agacgcgggt
actgagtggg actaacctgc tgtcctcttg ccttggacct tgtgttccag
75780aactgtgtca tgagtctctg cagcagcagc tacagtgagt taggactgca
gctgatcatc 75840gatgtgctga ctctgaggaa cagttcctat tggctggtga
ggacagagct tctggaaacc 75900cttgcagaga ttgacttcag gtaagtgagt
cacatccatt agatttcatg aactaagctc 75960aattgaaagt tctgggatca
cttgatgcaa ggaatgatgt tatcaagtac cctgtccatc 76020agaaatccga
gtggtttagg tagatgacag tgattttctc ctcccagtgg ctttttgctg
76080aactttgccc tatgcttgga attttatttt attttattat ttatttagag
acaagatctt 76140gctctgtcgc ccaggcttga atgcagtagc acaatcatag
ctcactgaag ctttgaactc 76200taggactcaa gtggtcctcc tgcctcagcc
tcccgattag ctaggagaat aggtgtgtgc 76260cgtcacactg gctaatattt
tttgtagaaa tggggtcttg ctatgttgcc caggctggtc 76320tcaaactcct
gggcttgatt gatcctccat cttggcctcc caaagtgctg ggattacagg
76380catgagccac tgtgcctggc ctagaatttt aaaatataag tagaagagta
gatttttttt 76440tttggtagtc ctcgtcattt aagtattctg gatagtggga
ataaaagagc ttagaatttt 76500tcatctttgt cttaaacttt taaaaaaatg
tagcttatat taattctgct tgtttaaaaa 76560gaatatactc ttcattatac
tgaacctagg taagacagct ggtttatatt ttgttgcaat 76620taaaaaacgt
gagctgtggt tgcagtgagc caagattgtg gccattgcac ttcagcctgg
76680caacagagtg agacttggcc tcaaaaaaaa aaaaataaca tgagctgtgt
tggcactttc 76740attttctaag agtagttttg gctggagaag ttttctttca
gtactttctt ttagaaggga 76800aattttcctt tataatttag ggtttgtttt
ttttttttcc aagccacctt ttatagagcc 76860cttgtgggtt atttcattta
atccttagaa tgtttataaa tctgggcttg ttctcggctc 76920cacccacaga
tagggacgct gagcgtgcat gagtgggcag caagatagca ggttatggag
76980ggcccagctc accccttctg tggcttgagc caattttata gggcacttac
agagtctttt 77040gaaatagtat ttattttgaa gaaaaagaaa aacagtttac
tgagtactgt cttattgagt 77100ctggaattgt gagaggaatg ccacctctat
ttatttaaag ccattggcct tttttgttgt 77160tttgagtaag tgctgcccaa
ggtccttcca gggcacctgg atgagcctgc tctggagcaa 77220gctggcggta
agtgtttact gagtaactaa atgatttcat tgttaaatgt gctcttttgt
77280taggctggtg agctttttgg aggcaaaagc agaaaactta cacagagggg
ctcatcatta 77340tacaggggta agcggtttat ttttgtgaga tgctgtttta
ccttcaagaa ggtgaaagtg 77400aggctttcct tgtggaattt ctctaaatgc
attcgtcatg ttttagatgt ttatttcaca 77460gtttatatca tgaaagttat
aatcttgtca tatggattta agtctagtaa tgttgagttc 77520tttctcacta
gctttccaaa atatcttacc taaaatttag tcaaatacaa gattatgttt
77580atttttatta tccttctctc taaagctttt aaaactgcaa gaacgagtgc
tcaataatgt 77640tgtcatccat ttgcttggag atgaagaccc cagggtgcga
catgttgccg cagcatcact 77700aattaggtat ttaccaatat tttatctctt
ttcctttttt ggttgaagta ctaaaagata 77760cgagaatgga aagagaggga
agaattcaaa ggatgtagag cagtattcct gaatctgagc 77820tcatttcagc
cattctattc ttaaactata atgaaaaaaa aatccaaaaa agtctaaaat
77880tataattaaa aaaacaacaa aatactaact gtccattgta aaaagtaatg
cactttcatt 77940gtaaaaattt tggactatag agaatagtac taagaagaaa
aaaaaaatca ccttcaattc 78000tgctgccacc tggaggtaat cactgttaat
attttgctat atactctatg agtttcttgt 78060tcaaaatcag gtcaaaatta
catgcaattt tgtaatctga caatttccac ttaatatttt 78120attagcattt
tcctgttatg aaacagtaat tttagttatg ggtcgttgtt ttgctatgcg
78180gttgggataa aattttatat actttttttg gcaattactt attatacata
aatgtttgtg 78240tatagttttc tttttctgag aattcctgga agttgagtta
ccaggcccgg ctttgaattt 78300ttttttttat tttttttttg agacagagtc
ctgctctatt gtccaggtgc tatctcggct 78360cactgcaacc tctgtctccc
tggttcaagc gattctcctg cctcagcctc ccgagtagct 78420gggattacag
gggcacacca ccacgcccaa ttaatttttg tatttttagt agagacaggg
78480tttcacgata ttggccaggc tggtctcgaa cttctgaccc cgtgatccac
ctgcattggc 78540ctcccaaagt gctgggatta caggcgtgag ccatggcgcc
tggccaggct ttaaatttaa 78600aacaaatctt ctaatagctt tatggaggtt
ataatttaca tttcttgaaa tgtactcact 78660ttgagtgtat agtaaactcc
aattttatca catttctgtc accccaaatg tatccttgtg 78720cccatttgct
gtaacctccg gttcctgccc caactcctag gcagccactc atctattttc
78780tgtcccttaa gatttgtgtt ttcgccaggc gctcatgcct gtaatcccag
cactttggga 78840ggccgaggtt ggtggatcac ttgaggtcag gagttcgaga
ccagcctggc caacatggtg 78900aaaccttgtc tctactaaaa atacaaaaat
tagtcggatg tggtggcaca cgcctgtaat 78960cccagctact cgggaggctg
aggcaggaga atcacttgaa cctgggaggc ggaggttgca 79020gtgagcagag
atcgcgccac tgccttccaa cctgggcaac agagagagac tgtctcaaaa
79080caaacaaaga tttgtatttt ctggacattt tatagtactg gggtcatagt
atagatggac 79140ttttgcattt ggcttctttt acttaattgt gagattggtt
cttgttgtag catgtatcag 79200tagtttgttc atttttattg gcgaaagtat
tctattatat gaataatacc atattttatc 79260tatccatcag atggatatta
tagagttcat gttttggcta atttatgaat tatggtactg 79320tgaacatttg
cctgcaagat tttgtgtaga catgtcttca tttctcttga gtagatcacc
79380tagaagtgga tttttaaata attttggtac ttactgtgaa actgctcttc
aaaaacatac 79440cattgttcct tccttccttc cttccttcct tccttccttc
tttccttcct cccttcctcc 79500ctcccttccc tacttccctc tccctttccc
tttcccttcc ccttttccct tccccttccc 79560gcctgcctgc ctgcctgcct
tccttccttc cttccttcgt ttctttctac atatacacat 79620ttttttaaat
ttcaatggtt tttggggtac aagtggtttt tggttacatg gctgaatttt
79680ggttacatgg tgaagtctga gattttagta cacctgtcac ccgagtagtg
taccttgtac 79740ccaatatgta gttttttgtc cctcaccttc cagccttccg
ccttgtgagt ctccaatgtc 79800cattatacca cactgtatgc ccttgcgtac
ccacagctca gctcccactt ctgagaacat 79860atagcagaaa catgccaaag
tatactccca ctaccagaat gtgattgtgc ctgattcttc 79920tcaccagtac
aaatatttca aaaaaagtta aatatgtatc agttttttgg gcagaagttg
79980atacttctct ttatttattt attttttttg agatagggtc tcattctatg
atgcccaggc 80040tggagtgtgg tggtgcgatc tcggctcact gcagtctctg
cctcccaggt tcaagtgatt 80100cccacgtcag cctcccagga agctggaatt
acaggcgagg gccaccactg ccagctaatt 80160tttgtatttt ttggtagaga
tggggtttca ccatgttggc cagactggtc tcaagctcct 80220gacctcaagt
gatccacctg ccttggcctt ccaaagtgct gggattacag gcgtgagcta
80280ccacacccgg ctgatatttc tttttaaaat aacttacctt cttttgaaag
taatacatgt 80340ttaatgaaca gaatttaagg aaaatataaa aaaacgaaat
aatctttgta atcaaactac 80400tgaaaagaaa accaaagtta cattttggtg
catattcttt ttcattttca tcattgtaat 80460ttgcatttct ttgattactt
gtgagacact cctttcattt acttaatagg tttatatgac 80520ttgcctattc
agagattttg cagctttacc attttctgca aatgatagca acttcttttt
80580gtttgtttgt ttgtggagac agagtctcgc tctgtcactc aggcaggaat
gcagtggtgg 80640aatcttggct cattgcaact attgcctcct gggttcaagc
gattttcctg cctcagcctc 80700ccaagtagct gggattacag gagtgtgcca
ccatgcccgg ctaatttttg tatctttagt 80760agagatgggg ttttgccatg
ttggccgggc tgatcttgaa ctcctggcct caagcggtcc 80820ccctgtctcg
gcctcccaaa gtgctgggat tacaggcgtg agccaccgta cccagccagt
80880agttacttct tatattctag aaaaaattct actcatgatc aagtctccat
gaggaaagag 80940actttaattg aagatcatgg ggcttgcaga ccaatatgat
aaaatagttc attgtttcta 81000aaagtattac tgagtgttga tggcagatat
gaaccctttt gtttttgtag gaaaatgtta 81060cccgtattct ccatttgaat
tcagtttaga tttgttagga atcgcagctt aagctttgcc 81120atctgggagt
gtttgggaca gttttgcaga caaaattgca aaagtgccta aggaatgcag
81180ctggcattca gacctgctct gtgctcagta ctctgtggac agacactgtt
cagcacttgt 81240tgatcagaag gtttagaaag agaactttca aagttggttt
ttaattaaag catttaatag 81300tgtaaataga aagggattaa attttatgac
agacaaaaga aagtacagca cccagctggg 81360cgtgggggct cacgcctgta
atccagcact atggggggct gaggtgggtg gatcacgagg 81420tcaggagttc
aagagttcaa gaacagcctg gccaaggtga tgaaaccctg tctctactaa
81480aactacaaaa attagccggg cgcggtggca ggcgcctgta atcccagcta
ctcaggaggc 81540tgaggcagga gaatcacttg aacctggacg gcagaggttg
cagtgagcca agattgcacc 81600attgtactcc ggcctgggcc acagagtgac
attctgtctc aaaaaaaaaa aaaaaagaaa 81660aaaagaaagt acagcaccca
gttatgtccg agtgggtgca tgagagtgac cctgagattg 81720gagacaacgc
tgtcacgtgc ttgaagaacg ccacctgaga aagggggcga gaagtggtgt
81780ccgctggtaa ccagaggtgt tggcttagcc atctgcaggg aggagggtgg
tctatcacag 81840gtgagtttca tctactttct taagcaaatt aaccttactt
ttgtgttagg cttgtcccaa 81900agctgtttta taaatgtgac caaggacaag
ctgatccagt agtggccgtg gcaagagatc 81960aaagcagtgt ttacctgaaa
cttctcatgc atgagacgca gcctccatct catttctccg 82020tcagcacaat
aaccaggtat gctgacccag tggcatcttc acattgtcgg gaaaatgccc
82080tttcctgatg cctttcttta ggctttaatt gaaaacattt tattttctag
aaaaaagctt 82140cagctcagga tgtttgagtg taggtcagtc ctttgatagg
atattatcat tttgaggatt 82200gaccacacca cctctgtatt taagctctgc
cacaatcact cagctgtgac actgtaaatc 82260tcttaatagt ttattacatt
ccatgtgctg acagttgtat ttttgtttgt gacacttacg 82320tattatctgt
taaaacattt tcactttagt tgtgttacct ttaaagagga ttgtattcta
82380tcatgcctgt tgattttttg gtgagcgggc tattaaagtc agtgttattt
agggttatcc 82440actagttcag tgatttgcga gattatcatt cacatttatt
gtggagcttt tgaatatcgt 82500gtcaaatggc cacatatatc ccattcttat
ctgcttctta ggtgagtggg acacagtgct 82560ttaatgaagc tataatcttc
agaattctag cttgcagaga agattgcaga agtgataaga 82620cttgtgcttt
ttaattttgt cttttaaatg ttattttaaa aattggcttt atatgatact
82680ctttttttct gctgagtaac agtgttttac aaaacttgga ctaaatgact
tctaagctta 82740aatgatcact tgatgctttt tttctgaatt aggaactcag
cttatcaaat atcaaagtca 82800taattcctga ataaataacg tcttttttca
tgtaaagact gctttaaaaa acacatggaa 82860ggctgggtgc ggtggctcac
gcctgtaatc ctaacacttt gggaggccca ggtgggcagg 82920tcgcttgagc
tcaggggttc aagaccaccc agggcaacat ggcaaaaccc acctctactc
82980aaatacaaaa aattagccag gcgtggtggc gggcccctgt aatcccagct
actcgggagg 83040ctgagggatg agaatcactt gagccccgga ggcagaggtt
gcagtgagcc aagattgtgc 83100cattgcactc ccagcttggg ctacagagtg
agactctgtc tcaaaaaaag acacacacac 83160aaacaaaaaa aacatggaga
catttttttg gccaccttaa tatttcccct cagataattt 83220cctttgttta
aactcagaac tggcattttc tctcttggag aagattcagg acaaatactc
83280ctttaagata agtagaagca gtgaaagagg atttgattat caggaatttg
ataagcttag 83340aataaattgt tgcttcttaa tgtcatttca gaagatgaat
atttattaat agatgccaac 83400tgagatatca ttaaaattga ttactaacta
ctacttggaa aagtctccca gttccaaact 83460tcagcaggcc tcttgacaat
tcagctgtgg tcaattgggt cttgcgtgat agatacaatg 83520accaattgtg
cagcagagtg tgctgcttag ctgcctattc tgttagcatt catgtgttaa
83580cttaaaatca taatctcctt agttttgttg agtgtctccg tggacaagac
actgtgaggg 83640atacaaaatc agattggctt tattcaaacc actggggtat
tataattcat ttataattta 83700ttttattttt tgcctttttt ccatgtgttc
taaaggaatt agagtttgta tataactata 83760atgggggata gaaattgaca
tgtgccatga agggaatgca aaaaagtgcc gtgggagatg 83820agaagtggag
aaaggaattt cttttttctt ggaagcagga ataacttcat gaagcatgta
83880tttcaactta aacagatagt aggcaacgct gtaaggggag tatggctgca
gcaaaagtgt 83940tcggggcaga ctgggaggaa gggagggaat aaattcagcc
attgttatgg aataatgatc 84000aaaatttatt ttcagcccgt ttcacttaaa
agttgagact gcttaacttt ttttaatctt 84060taatcttaaa cttttaaatg
ccatttgatc tttaaaaata tatgttttaa tagtgtattt 84120taagtctcta
tatttttgtt attagaatat atagaggcta taacctacta ccaagcataa
84180cagacgtcac tatggaaaat aacctttcaa gagttattgc agcagtttct
catgaactaa 84240tcacatcaac caccagagca ctcacagtaa gtctctttct
tgatcggtct tactgacatt 84300gtaatagttt ttggtagctt gtatggccag
ttagttgtat ggtcatctta cggtgaggtg 84360cttgtcttac agctcttact
tatccatgag gcttgctaag aaattgtgct tctgtgaaaa 84420gaatctcagc
ttactccagg aatgtaaatg actatgtttt ttctgattat taaagtaata
84480cacgcccaaa ataaaaaaat tcagccaatt taggaagaca caacaattaa
aataagccag 84540gcatggtggc tcatgcctgt aatcccagca ctttgggagg
ccaaggttgg gggctcactt 84600gaggtcagga gtcggatacc agcctggcca
acgtggtgaa accccatctc tactaaaaat 84660acaaaaatta gctgggcgtg
gtggcgggcg cctgtaatcc cagctactca ggaggctgag 84720gcaggagaat
cgcttgaacc tgggaggtag aggttgcagt gagctgaggt caagccactg
84780cactccagcc tgtgcaatag agcgagactc tgtctcaaaa aaaaaaaaaa
aaaaagaaaa 84840gaaaaaagta aactactgtc acctgcattg gtaatgtatc
agaagtttaa aatgtctaga 84900ttataattaa ctcagtgacc tggtaatata
tactaaggga aaaatattta taatttacat 84960ttttacattt ttattttttt
aattttatta tttttttttt gagacagagt tttgctcttg 85020ttgcccaggc
tggagtgcaa tggcatgatc tcagctcacc acaacctcca cctcccgggt
85080tcaagcaatt ctcctgcctc agcctcctga gtagctggga ttacaggcat
gcaccaccat 85140gcccggctaa ttttgtattt ttagtagaga cagggtttct
ccatgttggt caggctggtc 85200tcaaactccc aacctcaggt gatccgccct
cctcgacccc ccaaagtgct gggattacag 85260gtgtgagcca ccatgcctgg
ccttacattt ttataataag aatttatgtt gctgacatta 85320gaaaagaacc
ataatatcca agaatccaag aataattaaa ttatgtacat atgctagtat
85380atagtgtgat gctttggaga atttttaaca atatggagat gtataatctg
gattgtaata 85440ttgagtgaaa aaaggcagaa tacaaacctg gtgggggtat
agtcggattt cagttaagaa 85500aaataatatt tacatatata catttctcac
actggcagat aatcaccaag ataaattttg 85560ggattgtgga tgattttttt
cttctttata tttttcagat attctcaaat tttctaaaat 85620gagcaagtat
aacttttgtt atcagaaaaa aataatatac aaaagtaatg ttaatttgct
85680ggtgaccagg ttaaaccttt ttatttttat tttttgagat ggaatctcac
tctgttgccc 85740aggctagagc acagtggcat gatcttggct cactgcagcc
tccgcttcct gggttcaaat 85800gattctctgg ccccagcctc ctgagtggct
ggaattacag gcgtgtggca ccacacctgg 85860ctaatttttg tatttttagt
agaggtaggg tttcaccagg ttggtcaggc tggtctcgaa 85920ctcctgacct
cgtgatccac ccacctcggc ctcccaaagt gctgggatta caggcgtgag
85980ctactgcgcc cagccagacc tttttatttt atttgacaaa agaaatactt
ccatgttata 86040gaagactaaa tattgtttgg gctgtctgca gtatggtctt
cccttgattt gttcaaaata 86100tcgtaaactt tgcttattta tttttattgt
ggccgactgt gtcgggcact gttgtaggct 86160tgggatggaa aaacaggatt
cctgccctta gggtttctgc aggctggtca gggagacgat 86220gtggtaagct
ggagctcagc tcctaaggat gtgcaggggc agttgagagg cggaagggtg
86280ggagatcatt ccagggtgtg ggcagcacag gaacctctct tcattgggat
ataattgcca 86340ttctgataac acgtgtttga ggtgtctaaa gtaggaagtt
gtaccatggt gggacagata 86400tcctgtggtt atcatacaca gatctcagtt
ttcttctcat tgtttgtact ttttataaag 86460ggtaacagga gatataattc
aataaacctt tgtggtgttt gggtgtgatt ttattgtttc 86520tttcttctca
gtttggatgc tgtgaagctt tgtgtcttct ttccactgcc ttcccagttt
86580gcatttggag tttaggttgg cactgtgggt atgtattttc ctcagtatat
attaatagtt 86640gtctacaaca gtatgacata aacatagtta ttaggatgcc
ctttttcttt ctttttaagt 86700cttttatcaa tttggctttt tggaaaaata
tctgatggaa tacttgtttc tgctatatta 86760gctgtgtgag actagtgaca
ggagctgtgg gaaatgaatg ccaaatgttc ttaggcattg 86820atgggaattt
cagggtgtgg tcttcaagtt catttaaggg aattttcata tgctggcaaa
86880aggcttttct cattagcttg actctttcca aaattatttg ctgtgaatta
gaagtttagg 86940aacctttttt cacttaattg tgacctagca tacgaaatgg
tgatgattta ggaactactg 87000ttcttgtatt aacagctttt atttaaaaat
gattttcctc cagtagatgg ccctactagc 87060atctgggaaa taatttcaag
tcttctccag cattcaggaa taggctttca ttttgtgtat 87120caattactga
gaatgatttt ggtgactcac atcacatttg agaagtaaac ctgcagattt
87180cttgtgtgtg tcagcaaatg accaactgat atttgcttga agtggattac
attatctgct 87240ctagaatgat tgctttccca ccttcctcac atacagactg
agcagctacg gtttctaatc 87300ataggtctgg cactagactt cacttctggg
caactttggc attggagtaa aatgtattaa 87360tttaaagaaa gttaaaaatc
cgttcaagta aacatacagt tctaatactt tttacaattt 87420aaaatataga
tttaaatgat aaaataaaaa agaaaatatg ggtagacacc ataatcctcg
87480tttctgcatc tgttcacaag gggttgatat ttatgagttc tattctccat
atccattcta 87540tgttctctta atgctcagtc agcacctcag gtggttggag
ttcaatgctt ggtagtttga 87600cttacactgt cttttctagg ggattgagcc
ctgggtagtc ctgcttattt gaggttgcaa 87660tttgtctttc aataactttt
actacaagat atggcgtgtt aaaggatacc attggggaac 87720caacataata
atatcaggaa aactaaccac gtcagacctg ccccattgtg tatcaagtac
87780actatttttc catagtaata aagagttcac cccagccaat tctcttttat
tttgtgcctg 87840tttactcaat ggcattaaca tgcccaaatg tctgggtagc
tgtctcatct ccagttcagc 87900agaaccattg tcatatgccc tagtaaaagc
attccttcat tggacactta ggccccaata 87960ctttcattca gatctactac
ctgatttcat ttctcaaatg atttttatgg agctctgatt 88020tataggaaag
atgttagttg attaaaaata aaacaatttc tgagctggta taaaatgtat
88080tgtgacatgc cttcctcttg gaattgcaag agaaaggaag actgttgttt
gcttaaaaat 88140tgtctataat ttgactttgc aaatgtctgc ttccagagtg
cctccactga gtgcctcaga 88200tgagtctagg aagagctgta ccgttgggat
ggccacaatg attctgaccc tgctctcgtc 88260agcttggttc ccattggatc
tctcagccca tcaagatgct ttgattttgg ccggaaactt 88320gcttgcaggt
actggtactg agttgaaaca gggactccag gacttggatt ttgatttcct
88380tagggggaat gggggtggtg agcatatgag gggaaaatac tataaggtca
ttgccagtga 88440tggcttgtcc ctttagtcaa atttcagatg ttacctatat
gcataaacac atgcagttgg 88500cagctgttct gtgctgagta ttttaaagta
gcctcttccc aatatagccc ctcagttaac 88560tacaagtaaa ctcattttga
atttcatttt aatgggcacc atatgccagt actccctcgg 88620gcactgggat
gttaagaaag tataatgtat ggacttcatt ctcaagttag ttttagatta
88680gagggggata cacgtaaaca aaagtgcagt ggtcacacag agtggcccta
atcactctcc 88740ttgggcagat ttatgggctg gtaggaaaga gcacaacacg
gagagggtgt agcaccttgg 88800cgatgataat ggaggatgtg gccagcaagg
aagacggagt ccattgaaat tgattttggg 88860agaagttgcc aatctccatg
aaagaattgg ggcctgtgct atttgcttca gggggctata 88920ggagagtttc
gtgaaaggga ctaaaagatg agtattttaa taagatcatt catccaactt
88980gaacatgggc tggaggagaa ggtagggaga ctcaggagat taatgttgat
gctaaggcaa 89040gataatggct ttgggactgt agggaagaca ctgattgtaa
gagaatgaag gaggcagaat 89100tgccaggcct ggttcaccaa ctgaacttcg
gttgtgaaga caaagaaacc tgggatgact 89160tcacatcctg ggcaggtgtg
tggtggtgac agtcatggaa attgggaaca cagatttgtg 89220cgggaaacat
cagtttcagt ttgagtttgg cttatcagtt gaatatcagg cacagatgtc
89280tggccaactc tcaacatagg gtcttaaatg acttcagttc cccaagcaat
ttgtccttcc 89340catgctattg gggtggagag gtaatgtctg tgcccatatc
acagccagtg ctcccaaatc 89400tctgagaagt tcatgggcct ctgaagaaga
agccaaccca gcagccacca agcaagagga 89460ggtctggcca gccctggggg
accgggccct ggtgcccatg gtggagcagc tcttctctca 89520cctgctgaag
gtgattaaca tttgtgccca cgtcctggat gacgtggctc ctggacccgc
89580aataaaggta atgtcccact tgggtgctgg attcatacag ccttaatgac
tatgggtttc 89640cagactacct ttgtttagta atctgtccct tctttattct
ctttttgctt taaatgaaca 89700aaattgctca gattgtgaca ctaaatttaa
catcaaaatg tgaccatgtg gatgggtgca 89760gtggctcgtg cctgttattc
cagcactttg ggagactgag gcaagtggat cacttgaggc 89820caagagttcg
agaccagcct gggcaacatc acgaaacccc ctctctacta aaaatacaaa
89880aaattagatg ggttgggccg ggcgtggtgg ctcaagcctg taatcccagc
actttgggag 89940gccgaggtgg gcggatcacg aggtcaagag atcaagacca
tcctggctaa cacagtgaaa 90000ccccgtctct actaaaaata caaaaaaatt
atctgagcat ggtggcgggc gcctgtagtc 90060ccagctgctc gggaggctga
ggcaggagaa tggcgtgaat ccgggaggcg gagcttgcag 90120tgagccgaga
tcgtgccact gcactccagc ctgggtgaca gagcgagact ccgtctcaaa
90180aaaaaaatta gatgggcatg gtggtgcgtg cctgtaatcc cagctacttg
ggaggctgag 90240gcaagagagt tgcttgaacc tgggaggcgg agtttgcagt
aagccttgat tgtgccgctg 90300cactccagcc tgggtgacag agtcagactc
tttccaaaag
aagaaaaaaa tgtgaccatg 90360tgttttatag ctcttttagt atcatcagtc
actgttatcc ctaagaggga aatacctagc 90420tttagtttta ggtttccagc
attagccaag aaagctcaga attgatgttc ctggccaagt 90480acctcattgc
tgtctcctta aatcttggtt aatggctact gtcctggcta gcatagttat
90540ggagcatttc catggttgta gaatgttctg ccaatctcag ggacagtttt
gcttttctgt 90600gaagcaataa aatcaacttc aaaacaaatg ttaactattt
gtacaatgga tttaagatag 90660accagttcac atactttttt tttttttttt
ttttgagatg gagtttcatt cttgttgcct 90720gggctggagt gcaatggtgt
gatctcagct cactgcaact tctgcctcct gggttcaaac 90780gattcttctg
cctcagcctc tcgaggcaga ttacagctgg gattacaggc atgcaccacc
90840acacccagct aatttttttg tagttttagt agagacgggg tttcaccatg
ttggtcaggt 90900tggtctcaaa ctcctgacct gaagtgatct atccgcttcg
gcctcccaaa gtgttgggat 90960tacgggcatg agccaccacg cccagcctaa
gatagaccag ttcacttact gtttatatct 91020gattactctc tctttgcctt
gtcttctacc tttaaaaatc tccctactaa cttcccattc 91080tcctttagct
gccatcagtc ttctcccttc tctgcaaaca tctctggaga gtcccagcct
91140cagcccacag agcttcccac tgctctgagg tggaccttgt ttgcaaggct
tctttggctc 91200tcttggcctg gaccctgtct actacttcag ccatccttcc
ttaacccctg ctggtggttt 91260ctgttgccac actccatagc agcgtttccc
gcccagatca tgtctttaca tctctgggca 91320ctgctctggt cctgcctgcc
tttccctctt tgtatcctgc aggctgctac ccccatcttg 91380agtgtcctct
tcagttggct ttcagagggc ctcctgggtg ttcccttacc cacttgccac
91440tccccagtca ctgggttcag tccttcctgc ccaccagcac atgctttcta
ggctctgtcc 91500taggccgtct tctctctttg tagtctctgg gccagtgctg
ttctagagag tggcagaatt 91560ttctataacc atggcagtgc tccatagcta
tgccaggcaa gacagtagcc actaaacaca 91620tatagctgtt gagcccttga
aatgcagcta gtgtgactga agaactgaac cccgattcgg 91680tttaattttc
attaaattta aatttaaata accttatgtg ggtagtggct ccagtattgg
91740gcagggcagc ctgagagtcg gggctgttct cctgtcttca gtgtctagat
gagggacctc 91800agaggacctg tctctggagc tgcagttcaa tgtagccagc
tgccccgtga cacttacata 91860tagctgattt gtggatatgt cagacacggt
gtgatgagct cagctttctg tcctcctccc 91920cacatctgcc cctgccccat
ttaccccact ttgtgtctta tcaagctaga aacaggtcac 91980cacaagtctt
catttccact caccaagtct tttgtttccc ctactaaata ttttgcgaga
92040agaaagtgtg tacctttgta ttcacataca tgtacatgca catatacatg
cacatatgca 92100ggggtcccca acctctgtta aaaaccggac tgcaggccgt
gcgtggtggc tcacgcctgt 92160aattccagaa ctttgggagg ccgagaccag
tgcatcacaa ggtcaggaga tcgagaccat 92220tccggctcac acggtgaaac
cccgtctcta ctaaaaatac aaaaaaaaat tagccgggtg 92280tggtggcggg
cgcccatagt cccagctacc tgggaggctg atgcaggaga acggcgtgaa
92340cctgggaggc ggagcttgca gtgagccgag attgtgccat tgcactccag
cctgggcgac 92400agagcgagac tctgtctcaa aaacaaaaca aaacaaaaaa
aaaaaaaacc aggctgcaca 92460ggaagaagtg agcaagcatt accatctgag
ctctatctcc tctcaggcca gtggtggcat 92520tagattctca taggagcgtg
tatgagttcg ttctcacact tctgtaaaga catacctgag 92580acatataaag
aaaagaggtt taattggctc acagttctgc aggctgtaca ggcttctgtt
92640tctgggaagg cctcaggaaa cttgcagtca tggcagaagg tgaaggggaa
gtaggcacat 92700cttcacatgg cccacaggaa aaagagagaa ggagagagag
agagagacag agagagagag 92760agaaaaagaa agattgagag ggagagagga
gggagaaagg agagtgcctg tagggggagt 92820tgctacacaa aggagcacca
gggggatggt gctcaaccat tagaaactac ccccatgatc 92880caatcacctc
ccaccaggcc ccacctccga cactggagat tacaattcag catgagattt
92940gggtggggac acagagccaa accatatcag agcatgaacc ctattgtgaa
ctgcacattt 93000gagggatcta ggttgcatgc tccttatgag aatctaatgc
ctgatgatga tttgaggtgg 93060aacagtttca tcccgaaacc atcccccgcc
aaccctggtt tgtggaaaaa ttgtcttcca 93120cagaaccggt ccctggtgcc
aaaaagtttg gggacctctg cacatatgca tgcacctgta 93180catggacaca
taatacatgt acatatgcat actttatatt ctctgccact tctggtccag
93240actgatatac tatctcattt ggattactgc actagccttt tgttttggaa
acagcatttt 93300ttaaaaaatt taatttaatt tttttgagat agggtgtcat
tctgttgccc agcttggagt 93360gcagtgtcat gatcatagct cactgcggcc
tcgatctccc aggctcaagt gatccttctg 93420cctcagcctt ctcagtagtt
gggactacag gcatacccac catgcccagc taattttttg 93480attttttttt
ttttttgaga cagagtctca gcctgtcgcc caggctggag tgggttggcg
93540cgatctcagc tcactgcaac ttctgcctcc caggttcaag tgattctcct
gcctcagcct 93600cccgagtagt tgggattaca ggcgcctgcc accacaccca
gctaactttt tgtattttta 93660gtagagacgg ggtttcacca tgttggccag
gctggtctcg aacttgtgac ctcgtgatta 93720gcccgcctcg gcctcccaaa
gtgctgggat tacaggcgtg agctaccgct cccagccagg 93780aaacagcatt
cttgagataa ttcatataat tcacccattt aaagtatata attcattctc
93840tttagtatgc ccacagagtt gtacagccat caccagaatc agttttagaa
cccataaagg 93900aactctgtac tctttaccca aaacctccat gcctccagct
gcaggcagcc actaacctgc 93960cttctgtctc tgtgactcta cgtcttctgg
acattactgt ggatgggctc atacagtcag 94020tgagcttgtg actggtgcct
tctaccaagc agggttttca gtgtagcagc ctctctgttt 94080ttcttttttt
tttaaattgt gacggaactt ctgcctcccg ggttcaagcg attctcctgc
94140ctcagcctcc cgagtggctg ggactacagg cccatgtcac catgcctggc
taattttttt 94200tttttttttt tttagtagag atgggtttca acatgttagc
cagggtggtc tcgatctcct 94260gacttcatga tccgcctgcc tcggcctccc
aaagtgctgg gattacaggc gtgagccacc 94320atgcccggct aacctttcat
ttactgtctg catttcttcc ctgatgcctt ccagtccatg 94380cacccgattg
tagccattca tcctattatg gtttaaggtg actgtcttag tcagcatggg
94440ttgccataac aaaataccat agcctgggtg gcttcaacaa cagaatttac
ttctcacact 94500tctggaggtt gggaagtcca agatccagga ctttcgcctt
gccctcatgt ggtgaggggg 94560tgaggaagct ctgtggggcc tcttatatat
ggatgctaat ctcattcatg aggggtctgc 94620cctcatgacc cagtcacctc
ccaaaggccc cacctcctaa taccatcacc ctggtaatta 94680agtttcagtg
tataaatttg ggggactata gacattgaaa ccataacaag cacttttcta
94740agatcaggga gtgagtaagt agcagagcta ggacctcaat tccacatgtc
agtcatcttg 94800ccttcactct gctccatgat ggctgcctcc tagagcattg
ggagtctcga tgttctatat 94860gctctcatgt gttgtgtatt ggagatagtt
gaggctttat gaatacatct ggatttgttg 94920acttctagct ttgctggtaa
ccagctgtga ccttgaataa gttacttcat ctctgagcct 94980gtttcctctt
ttagaaacag gagtttaaaa tgctgctttg ggttgggcac ggtggctcat
95040gcctgtaatt ccagcacttt gggaggctga gatgggagga tcactggagc
ttggagttcg 95100agaccagcct gggcatcata gtgtgagatc ctgtctcctc
aagaaattaa aaaattagct 95160gggtgatgtg gcgtgtgcct gtggtcccat
ctactctgga ggctgaggtg ggaggattgc 95220ttgagcccag gaggttgagg
ctacaatgaa atatgattgc accccatcct gggtgacgag 95280tgagaccctg
tctcaaaaaa gaaaaaaaaa atgctgcttt gtaccccttt catgtcatgg
95340cgtcatggcc aacatagaat gccctggttg tttgctgttg gagggcatgg
gcctgggggc 95400tccctgaggg ctccttccat cttcaactca ttctctgtgc
acctgttagg aagttgtggg 95460ccagtcccta ccatgtatca ttgtgtgggt
aaaagtaaat aaaatgtgta cagtgtctga 95520actgtacata tcagggtcca
agaacaaaat gagtgacatg ggttagctct ttttaataaa 95580tggtaaaacc
aaatattcta attttcagtt ttgttatact tccatcacat gtttttgttt
95640ttttgttttt tgtttttgtt tttctatttt aggcagcctt gccttctcta
acaaaccccc 95700cttctctaag tcccatccga cgaaagggga aggagaaaga
accaggagaa caagcatctg 95760taccgttgag tcccaagaaa ggcagtgagg
ccagtgcagg taggaaacag cgtggggaag 95820ggagggacat gagtgcagca
tctgtcatgt agaaacatag gatttaagta acttggtgtt 95880ttagagaaat
aaatataata cacatcagta aagtgagaga aagtttctcc aggtgcggtt
95940caagatatta gaaactaatg actgatgtac acagaccacc ttttggtctg
aagcatttct 96000aagtgccact ggctgacatg cagcccctac agcctccagg
cttccagccc tagcatggag 96060catcactctc ctatgcttcc ctggttgcag
gtgatggctg gagaggcctc ctgattttca 96120gtaagggaag tggtgtagat
gcttaggaat agatgtagtg agtgaaaaaa ctgattctga 96180tatgtcaaaa
attctgattg gaaatggaat atttacattt ggaagagcta aaggcgagag
96240aaagtgggga taaagtcatc tgagttggag gagcttaaac cattcacaag
tttggaggac 96300ctttttttac ccatgaaaag gtcagaacag aaggggctag
gatttaggtg tgactgcagt 96360ttattgaatt cccatccata ctgctctcgg
tgggcagtgg caggggcagg agaggagcct 96420ggcaaagcat gaagtgactg
ctgctgcctc tgctatctgg gacgcctggc cacctgtctg 96480tacagtctcc
ctccagaccc attctcacgc tgtctcttgg cacccagggg ccagtgatgg
96540ttctcccatt tgttttgtgt atatagcatt tatatcaagg ctatttattt
atttatttat 96600tttatttatt tatttttttg agacagagtc tcactctgtc
acccaggctg gagtgcagtg 96660gtgcaatctc ggctcagtgc aagctctgcc
tcctgggttc aagcaattct cctgcctcag 96720cctcctgagt agctgggact
acaggtgtgc accaccacac ctggctaatt ttttgtattt 96780tttattagtg
gagacggggt ttcaccttgt tggccaggat ggtcttgatc tcctgacctc
96840gtgatccgtc cacctcagcc tctcaaagtg ctgggattac aggcatgagt
cactgtaccc 96900ggcctattta tttattttta attgacaaaa ttgtatatat
ctgtaatata caacatgatg 96960tttgaaatat gtgtacattg gccaggcgtg
gtggctcaca cctgtaatcc cagcactttg 97020ggaggctgag gtgggcggat
cacgaggtcg ggagttcaag accaaactgg ccagcatggt 97080gaaatcctgt
ctctactaaa aataccacaa aaaaaaaaaa aaaaaaaaaa agccgggcat
97140ggtggctcgc gccagtcgtc ccagctactt gggaggctga ggcaggagaa
ttgcttgaat 97200ctggcaggtg gaggttgcag tgagctgagt tcatgccact
gcactctagc ctgggcgata 97260gagcgagact ccgtctcaaa aaaaaaaaaa
aaagaagaaa tacatatgca ttgtggaatg 97320gctaattaac ctgtgcatca
cctcacgtat cattgttttg tggtgagaac acttaaaatc 97380tactctttca
gtgattttct tgcatatggt acattgctat taactgcagt caccatgcta
97440tacagtagat ctcttgaact cattcctcct gtctataaat gaaattttgt
atccttgacc 97500aacacattca aggttttttt tgagatggag tcttcttcac
ccaggctgga gtaccatggc 97560acgatctcat ctcactgcaa cctccgcctc
ccaggttcaa gcaattctcc tgcctcagcc 97620tcctgagtag ctgggattac
aggcacatgc tactgcacct ggctaatttt tgtattttta 97680gtagaagtgg
agtttcacca tgttggccag gctggtctcg aactcctgac ctcaagtgat
97740ccgcctgcct tggcctgcca aagtgctggg attacaggtg tgagccactg
cacccggcct 97800caagcgtttt aaaagatgct cttttctaag gattgactgt
agtacaggag gaagattgac 97860ctgttgaaaa gcctcagcct ttacaagtgt
aaaattatca gtatattact atcatctttc 97920tgatgaatta aataaactaa
ggactccaag tcaaaagtct tcaaactgaa gtagaatagt 97980tgtatatagt
gcttggcact ttaatattta gtatcggttt aatgataatg tttgtgcctt
98040tgccgtcttt aaaacatttt tacatcatcc ctgtttgatt acttggtgtg
ctcatgaagt 98100tgttggccac taaggaatct taggctcaga gaggttctgg
aattggccag tggtccttga 98160atcagctgct cctatgattc tctaactgat
ttctcacaaa gcaaacaagc aatcataaca 98220aaacaactgt gcacactgct
cttcttattt tgttatttaa aaagtactta ggctctactt 98280atgtttgtta
gtcaatttct cattacttct agttaatcaa aaggtcagag gaaatacttg
98340aatattttca tactagaata ctttaaaaaa tcatgatttc cagtaatctc
tttaaaactt 98400ggcaagttat tttgatctaa aagtttatct tttgtgtgca
tatttttaaa gcttctagac 98460aatctgatac ctcaggtcct gttacaacaa
gtaaatcctc atcactgggg agtttctatc 98520atcttccttc atacctcaaa
ctgcatgatg tcctgaaagc tacacacgct aactacaagg 98580tatgggcctc
tgcatctttt aaaaatatat atgcacacat acttacgtct aatggatagt
98640tgatgttttt cttatgattt gtaggatgta taagcccttt gagatatgag
ttacatttag 98700ttttttcaag tttgtttgtc tttcagcttt gtttatgata
gcttctatca tacaggtgtt 98760ttggattttc atattgtttg tactcacagc
taagattgat tacagtgaca gagctaggat 98820gtgcagccag gttatagggg
gaagtggccc tggtggagtc tggagggatc cgtgtacagg 98880cttccttccc
tcccgtgagg ctcacacaaa aatacagcaa catgctggtc ctgcaggtac
98940cctctgccta acatgagcca caattccaga ctcacagaag aaaagcaggt
gttcggcata 99000aaccatgtgt ttcaaatagt ctgggcatgg tgagccactt
gttatcagct agggaaagtt 99060tatgtcagcg taagaaactg ttcaccagat
acccccaaga gccagccttt ctgtctaggg 99120atgttttagt tttttagttc
attttttttt ttaactttaa aattttctgt tcatctgcaa 99180tttgttagat
atgaagtatg tgtctaattt aatttttgtt tttggttgtc cccaataatg
99240tttacagaag aatttttctg cactaattgg cttgagttac ttacattctc
atagttctct 99300agtttcagta gtttcattta ttattttgtt atatcaatct
atctgtctgc tcatctatta 99360gaagcatcct tgtttttttt ttttcttttt
tagacagagt cttgctctgt ccccaggttg 99420gagtgcagtg gtgcaaccat
gcctccctgc agtctcaggg ctcaagtgat cctcccacct 99480cagctcctga
gtacctggga ctaccggcat gtgccaccac acccagctaa tttttacatt
99540ttttgtagag acagggtctc cctaagttgc ctgggctggt ctcaagctcc
tggcttaagt 99600aatcctccct ccttggcctc ccaaagtgct gggattacag
gtgtgagcaa ctgcacccgg 99660ctacaagtat acttcttaat tattgtagct
taatggtatt tatgagggga tcagttcccc 99720tgttgttctt tagaattttc
tggatattct tctttattga ttttgggatg tgaacaatag 99780aatcaacttc
tacttgtaga ttgatttagg gagaacttat acctcagatg ttaagtcacc
99840ctgtccagaa tgtgggatgc tttcctattt gttcagaact ttttaaatta
cctcagaagc 99900acatgaaatt taaaggattt taaaaaaaac ttaaagatta
tttcacatag ctcttgcaca 99960tttcttgata aatgaatcct caggtattcc
tctgtttttg ttactaatag ttacttctta 100020tgggtttttt ttcccctgaa
aatcatttat caaacgtatg tggcttattt tctgaaggat 100080gtttgataat
tttggaagat atgaaagtct tcatatttta caaggtttga ggtctcttta
100140agctgcatgg ttctcatgtc agctcccaaa gcagaagacg gcatgttgaa
aaatgccgta 100200gagaagatac ttcttttcca cctgttttca actcatatca
tcttgaattt cagggcacct 100260ttccatgctc ctagtgcttg ctatctgttt
attattttcc ttcctgaata ccctgaactc 100320cagcatgttc tgctgtaatt
ctggcctccc tggcatcttg gactcctgtt tcctttgctc 100380tgtcatcccc
gcggtcagct cctgctgcgc agcttctcag ctgaagtgcg tttggagtgc
100440ctggcgtgtc ttgctggatc tttgagtatt gcctctggtt tccttggttc
cttctgctga 100500gttgctcagc gtctccactc cccatttctt gtgtggccct
tcctgcactc ctctgattcc 100560ttttgtcttc cctggtttct tgctttggtt
tcgagtctcc acagaacttt tgcagctctt 100620ctgaagacct ggaagctttt
tcatcttaat tctcatctca tgacctcttt tcccttcttt 100680gagagctaga
acttcccatg gtgaacttct ctttccagaa ttccatgcct tcttttccct
100740cccacttacc tgttgtccag gagaggtcag attgctgtgc atattggagg
agaacccttt 100800cttccctggg ctcttcatct cacatgacat caccacatca
cctcgttcct tggaccctca 100860gtggtgtcac tgctggattt ttctttcctt
tggctggcct tagggcacac ccaggttgac 100920tagcgtagtc atggtattta
gatccactca cattttcagt ttctgtgtct gtctcttgcc 100980tgcttctgac
ttcgcccaga gaaagcttct ctttcacaag ggttcttaga tttatgttca
101040ctgagcacct tcttttctga ggcagtgttt taccaatatt tattttccta
gtcagtctcg 101100ccttaccttt cttgttatgc atgtctttgg tcctgaccca
ttctctgagt ctgtaaaata 101160gaattgctgt ataatttaat tacatgaaat
cctttagaat cttaacacat cttacacctg 101220atttaatatt ttattgtatc
caaattgaac caaccctatg tgaatttgac agtgatttct 101280cccagggatc
ctagtgtata aggaatagga cttagtattt tctatttttt gatataccac
101340ataccagata ctgattatga tggacattta accctttttt ctcattatga
aagaaagtta 101400ggaattattt cttccagtag cgccagtgta acctgaaagc
ctttgaaaga gtagtttttg 101460tatagctatc tgaaaggaat ttctttccaa
aatatttttc cagtgctgac aacaaacacg 101520cagacacacc ctgcaaggtg
agtgtacggc gccgcacagt ggaggcatct gctgcagccg 101580tcgatgtttg
tgtctttggt tgtacattat gagatcgtga cagggccagt aaccgtgtgt
101640tctctccttc accttcccaa ggtcacgctg gatcttcaga acagcacgga
aaagtttgga 101700gggtttctcc gctcagcctt ggatgttctt tctcagatac
tagagctggc cacactgcag 101760gacattggga aggtttgtgt cttgtttttt
ctccttgggt tgtggctggc acacttgatg 101820tgcgtcttct gggctgagtt
catctaggat ggagcctggt tctccagggt gcctccggga 101880gactcctccc
tgccccacgt gcttgcgtca caggacccaa gtctgactct gccttagcca
101940tgaagtttag ggggaagttt ctatttgtat tctatttttg tctgttatca
tgtattagct 102000tagacccagt ttagtttgga aaatcagtgg gtttcaaaat
gtgtttgtag agtcctttat 102060ttcttaactt gaccttttca agtggaaagg
ggcaaaacag acgggtaagg gggcggggcg 102120ggaggtgtga cttgctcttt
tgtgcctgag gaagtaacag agctggggtt gacagtcata 102180ttctctgaca
cagatagtct ctgacttatc tcacagaaag tcagcggcag agcctgagtt
102240aaaagtctcg tagattttct ttttcttttt tttggtggct aatttcagtt
ttatttatat 102300ttgtttattt atttattata ctttaagttc tgggttacat
gtgcagaatg tgcagttttg 102360ttacataggt atacacgtgc catgatggtt
tgctgcaccc atcaacccat cacctacatt 102420aggtatttct cctaatgtta
tccctccccc agtcccctca ctccccatgg gccccggtgt 102480gtgatgttct
cctccctgtg cccatgtgtt ctcattgttc aatttccact tgtgagtgag
102540aacatgcggt gtttggtttt ctgatcttgt gatagtttgc tgagaatgat
ggtttccagc 102600atcatccatg tgcctgcaaa ggacatgaac tcatcctttt
ttatggctgt atagtattcc 102660atggtgtata tgtgccacat tttcttaatc
cagtctatca ttgatggaca ttcgggttgg 102720ttccaagtct ttgctattgt
gactagtgcc acaataaaca tacatgtgca tgtgtcttta 102780tcgtagaatg
atttataatc ctttgggtat atgcccagta atgggattgc tgggtcaaat
102840ggtatttcta gttctagacc tttgaggaat cgccagactg tcttccacaa
tagttgaact 102900aatttacact cccaccaaca gtgtaaaagt gttcctattt
ttccacaacc tctccagcat 102960ctgttgtttc gtgacttttt aacgatcgcc
atcctaactg gcgtgagatg gtatctcatt 103020gtgattttga tctgcatttc
tctaatgacc agtggtgatg agcatttttt cgtatgtctg 103080ttggctgcat
aaatgtcttc ttttgcgaag tgtctgttca tatcctttgt ccattttttg
103140atggggttgt ttgctttttt ttcgtaaatt tgtttaagtt ctttgtagat
tctggatgtt 103200aatcttttgt cagatgggta gattgcaaaa attttatccc
attctgtagg ttgcctgttc 103260actctgatga tagtttcttt tgctatgcag
aagctcttta gtttaattag atcccgtttg 103320tcaattttgg cttttgttgc
cattgctttt ggtgttttag acatgaagtc tttgcctatg 103380cctatgtcct
gaatgttatg gcccaggttt tcttctagga tttttatggt cctaggtctt
103440atgtttaagt ctttgatcca tcttgagttg atttttgtgt aaggtataag
gaaggggtcc 103500agtttcagtt ttctgcatgt ggctagccag ttttcccaac
accatttatt aaatagggaa 103560tcttttcccc attgcttatg tgtgtcaggt
ttgtcaaaga tcagatgatt gtagatgtgt 103620ggtggtattt ctgaggcctc
tgttctgttc cattggtcta tatatctgtt ttggtaccag 103680taccatgcag
ttttggttac tgtagtgttg tagtatagtt tgaagtcagg tagtgtgatg
103740cctccagctt tgttcttcta gcccaggatt gtcttggcta tgcaggctct
tttttggttc 103800catatgaagt ttaaaatagt tttttccaat tctgtgaaga
aagtcagtga tagcttgatg 103860gggggatagc attgaatcta taaattactt
tgggcagcaa ggccattttc acgatattga 103920ttcgtcctat ccatgaacat
ggaatgtttt tctatttgtt tgtgtcctct cttatttcct 103980tgagcagtgg
tttgtagttc tccttgaaga ggtccttcac atcccttgta agttgtcttc
104040ctaggtgttt cattccctta gtagcatttg tgaatgggag ttcactcatg
atttggctct 104100ctgtttgtct gttattggtg tataggaatg cttgtgattt
ttgcacattg attttgtatc 104160ctgagacttt gctgaagttg ctaatcagct
taaggagatt ttgagctgaa ccaatagggt 104220tttctaaata tacaatcatg
tcatctgcaa acagggacag ttttacttcc tctcttccta 104280tttgaatacc
ctttattgct ttctcttgcc tgattgcgct ggccagaact tccaatacta
104340tgttgaatag gagtggtgag agagggcatc cttgtcttgt gccggttttc
gaagggaatg 104400cttccagttt ttgcccattc agtatgatat tagctgtggg
tttgtcataa atagctctta 104460ctatgttgag atacgttcca tcgataccta
gtttattgag agtttttagc atgaaaggct 104520gttgaatttt gtcaaaggcc
ttttctgcat ctgttgagat aatcatatgg tttttgttgt 104580tggttctgtt
tatgtgatgg attacgttta ttgatttgcg tatgttgaac cagccttgca
104640ttccagggat gaagctgact tgattgtggt ggataagctt tttgatgtgc
tgctggattc 104700agtttgccag tattttattg aggattttca catcgatgtt
catcagggat attggcctaa 104760aattctcttt ttttgttgtg tctctgccag
gctttggtat caggatgatg ctggcctcat 104820aaaatgagtt agggaggatt
ctctcttttt ctattgattg gaatagtttc agaaggaatg 104880gtaccatctc
ctctttgtac ctctggtaga attcggctgt gaatccatcc tggacttttt
104940ttggttagta ggctattaac tattgcctca agtttagaac ctgttatcag
tctattcaga 105000gattcagctt ttttctggtt tagtcttggg agggtgtatg
tgtccaggaa tttatccatt 105060tcttctagat tttctagttt atttgggtag
agatgtttat agtattctct gatggtagtt 105120tgtatttctg tgggatcggt
ggtgatatcc cctttatcgt ttttattgag tctatttgat 105180tcttctctct
tttcttcttt attagtcttg ctagcggtct acctatttta ttgatctttt
105240caaaaaacca gcacctggat tcattgattt tttttggagg gttttttttc
gtgtctctat 105300ctccttcagt tctgctctga tcttagttat tttttgtctt
ctgctagctt ttgaatttgt 105360ttgctcttgc ttttctagtt cttttaattg
tgatgttagg
gtgttaattt tagatctttt 105420ctgctttctc ttgtgggcat ttagtgctat
aaatttccct ctacacactg ctttaaatgt 105480gtcccagaga ttctggtatg
ttgtgtcttc gttctcattg gtttccaaga aaatttttat 105540ttctgccttc
atttcgttat ttacccagta gtcattcaag agcaggttgt tcagtttcca
105600tgtagttgtg tggttttgag tgagattctc aatcctgagt tctaatttga
ttgcactgtg 105660gtctgacaga cagtttgttg tgatttctgt tcttttacat
ttgctgagga gtgttttact 105720tccaactatg tggtcagttt tagaataagt
gcaatgtggt gctgagaaga atgtatgttc 105780tgttgatttg gggtgcagag
ttctgtagat gtctattagg tccgcttggt ccagtgctga 105840gttcaagtcc
tggatatcct tgttaatttt ctggctcatt gatctgccta atattgacag
105900tggggtgtta aagtctccca ctattaccgg gtgggagtct ctttgtaggt
ctctaagaac 105960ttgcttcatg aatctgggtg ctcctgtatt gggggcgtgt
atatttagga tagttagctc 106020ttcttgttga attgatccct ttaccattat
gtaatggcct tctttgtctc ctttgaactt 106080tgttgattta aagtctgttt
tatcagagac taggattgca atccctgctt tttttttgct 106140ttccatttgc
ttgttagatc ttcctccatc cctttatttt gagccaatga gtgtctttgc
106200atgtgagatg ggtctcctga atacagcaca ccaatgggtc ttgactcttt
atccaatttg 106260ccagtctgtg tcttttaatt ggggcattta gcccatttac
atttaaggtt aatattgcta 106320tgtgtgaatt tgatcctgtc attatgatcc
tagttggtta ttttgcccgt taactgatgc 106380agtttcttca tagcgtcagt
agtctttaca atttggcatg tttttgcagt ggctggtact 106440ggttgttcct
ttccatgttt agtgcttcct tcaggagctc ttgtaaggca ggcctggtgg
106500tgacaaaatc tctgcatttg cttgtctgta aaggatttta tttctcgttc
acttatgaag 106560cttagtttgg ctggatatga aattctgggt tgaaaatact
ttttttaaag aatgttgaat 106620attggctccc actcttttct ggcttgtagg
atttctgcag agagatctgc tgttagtctg 106680atgggcttcc ctttgtgggt
aacccgacct ttctctctgg ctgccctttc cttcatttca 106740atcttggtgg
atctgatgat tatgtgtctt ggggttgctc ttctcgagga gtatctttgt
106800ggtgttctct gtatttcctg aatttgaatg ttggtctgcc ttgctaggtt
ggggaagttc 106860tcctggataa tatcctgaag agtgttttct aacttggttc
tattctcccc atcactttca 106920ggtacaccaa tcaaacgtag atttggtctt
ttcacatagt cccatatttc ttggaggctt 106980ggttcatttc ttttcactct
tttttctcta atcttgtctt ctcgctttat ttcattaatt 107040tgatcttcaa
tcactgatat cctttcttct gcttgattga atcggctgtc gaagcttgtg
107100tatacttcac aaaattctcg ttctgtggtt tttagctcca tcaggtcatt
taagctcttc 107160tctacactgg ttattctagc cattagtcta acattttttt
caaggttttt agcttccttg 107220tgatgggtta gaacatgctc ctttagctcg
gagaagtttg ttattaccga ccttctgaag 107280cctacttctg tcaattcatc
aaactcattc tccatccagt tttgttccct tgctggtgag 107340gagttgtgat
cctttggagg agaagaggtg ttctggtttt tggaattttc agcctttctg
107400ctatggtttc tccccatcat tgtggtttta tctacctttg gtctttgatg
ttggtgacct 107460acggatgggg ttttggtgtg ggtgtccttt ttgttgatgt
tgatgctatt cctttctgtt 107520tgttagtttt ccttctaaca gacaggcccc
tcagctgcag gtctgttgga gtttgctgga 107580ggtccactcc aggccctgtt
tgcctgggca tcaccagcag aggctgcaga acagcaaata 107640ttgctgcctg
atccttcctc tggaaacatc gtcccagagc acgaaggtgt ctgcctgtat
107700gaggtgtttg ttggccccta ctgggaggtg tctcccagtc aggctacatg
ggggtcaggg 107760acccacttga ggcagtctgt tcattatcgg agcttgaatg
ccgtaccggg agaaccactg 107820ctctcttcag agctgtcagg cacgtatgtt
taaatctgga gaagctgtct gctgcctttt 107880gttcagatgt gcccttcccc
cagaggtgga atctagagag gcagtaggcc ttgctgagct 107940gcagtgggct
ctgcccagtt cgagcttccc tgctgctttg tttacactgt gagcatagaa
108000ccacctactc tagcctcagc agtggtggac acccctcccc cagccaagct
cctgcatccc 108060aggtcgattt cagagtgctg cgctagcagt gagcaaggcc
ccatgggcgt gggacccgct 108120gagccaggca caggagagaa tctcctggtc
tgctggttgt gaagactgtg ggaaaagtgc 108180agtatttggg caggagtgta
ctgctccttc aggtacagtc actcatggct tcctttggct 108240tggaaaggga
agtcccccga ccccttgtgc ttcccaggtg aggcaacacc ccgccctgct
108300tcggcttgcc ctccgtgggc tgcacccact gtccagcaag tcccagtgag
atgaactagg 108360tacctcagtt ggaaatgcag aaatcacctg tcttctgtgt
cgatctcact gggagctgta 108420gactggagct gttcctattc ggccattttg
gaagcatccc ttgttttttg aggtggagtc 108480ttgctctgtc gcccaggctg
acgtgcatcg gcacaatctc ggcccactgc aacctttgcc 108540tcctggtttc
aagcgattct cctacctcag cctccggagt agctgggatt acaggcacct
108600gccaccatgc ctggctaatt ttttgtattt ttagtggaga tggggtttca
ccacattggc 108660caggctagtc tcgaactcct gaccttgtga tccacccacc
tcagcctcct agagtgctgg 108720gatcacaggt gtcagccacc acgcccagcc
atattttcag atctccctct ctttgcccta 108780aaccactgtg cttaataagt
agtttttagt ggccagcagt ctccatgtat aacacatttt 108840agcaaaatgg
aaaatactat atgttttaaa tttgaacgtg agattatact gaaataaaaa
108900tcatctaact gggattcttt aaatagtaag attttctttt ttgtatgtgg
gttttttttt 108960aaccttatta ttatgactgt catatataga aatggctgtt
tttcagttac agtcagtgaa 109020tgtatcaaat gctgccttat ccaaataata
aaagtaaatt attaataagt cacaatttaa 109080tgaagattga tgttagttga
tctttatatt cttgaaatca gccatatggt tgtgtgtgta 109140tgtatatatt
tttaaaggta cataaagata ataagctcat ctctgaaaat ttttacattt
109200ggcataagaa taactggata attaagcatc ttattctctg gcctgtgtct
ttacagttaa 109260aggtagattt actcacctct ccttttttgt ttttctaagt
tcatcttttt tgctgtttca 109320agacagaggc ccattttagc tttctcgcat
atccttttgt ttgtactttg gaagcctcac 109380ctgcttaatt gttgagtttt
tatccgtggt cttttagagg gggatatgta gggtagaagc 109440tttcacaggt
tcttgtttgc acttggcccc tgactgtttt gaggaatctc cctcactgac
109500tcacagcatg gcaaggtttc agatctcttt ctgccacaca gcagttctga
ggcagctgga 109560aagatatcca gatgcttaga ttgtcaggcc aggcttgaga
tatacaaact attgagcctt 109620atctgtgacc ttgcttaggt gaaggcatca
gagcccctgc accaacatgc ataggcctct 109680gcatgtgtgc ggggctgggt
gttgaggtct gagcacaagt gtagctggag aggtgagctt 109740gatgtggcga
cgggtatgag caggttttct tcagacttct gtgagtttac ctagttccag
109800gatttaaagg cacagagact ttagaattaa aatagaatca ttttcttttt
ctaaatagca 109860acactaggaa taaaaaataa taattccaca ttcttgacag
gtaatgtttt ttcttgtctt 109920ctaatcctta tttattccat actcattttt
atacataatt gaaatgtatt atgcattgga 109980tttttctttt gcattatatt
atagacgatt tttcatgtaa ctccttactg ttccatttta 110040tatgttttgt
ctggtttaag actttatctg caaaccggga aactgtctct acaaaaagaa
110100aaacaaaaat agttggccgc agtggcatgc gtctgtggtc ccagctactc
ggggctgagg 110160tgggaggatt gcttgagcct tgggaggttg aggctgcaaa
gagccatgat catgccattg 110220cactccagca tgggtgacag actttatact
gtctgttttg ggtgatttga taatgatatg 110280ccctgatgta gtttttttat
atcttgtgtt tcttgtgcct gggtttattg aggttgggtc 110340tgtggcttca
tagtattttt aaagtttgga aaattttagg ccattctttc tttctttctt
110400tctttttttt ttttttgaga cagtgtctcg ctctgtcgcc tgcgttggag
tgcagtgaca 110460ctatcttggc tcactgcaag ctctgcctcc tgggttcacg
ccattctcct gcctcagcct 110520cctgagtagc tgggactaca ggcgcctgcc
accacgcctg gctaattttt tgtattttta 110580gtagagacga ggtttcactg
tgttagccag gatggtctca atctcctgac ctcgtgatct 110640gcccgcctgg
gcctcccaaa gtgctgggat tacaggcgtg agccactgca cccagctagg
110700ccattatttc ttcaaagatt ttttttctgc cctgcctccc tccttttttc
cctctcttaa 110760aggggctgtg atttcctgaa tgattgctta gtgttgtccc
atagcttact gatgctcttt 110820tcagtgtttg attgttttat gtgttttctg
ttttgtatag tttctattat tgtgttttca 110880agttctctga tcttttcttc
tacagtgtct actctgttgt taatctgtta atctgttgtt 110940aatcctgtcc
agcgtatttt tttttttgtt tttgaaacag tctcactctg ttgcccaggc
111000tggagtttag tggtgcgata tcagctcact gcaacctcca cctcccaggc
tcaagcaatt 111060cttctgcctc agcctcccga gtagctggga ctataggcac
gtgccaccac acctggctaa 111120tttgtgtatt tttattagag atggggtttc
accatgttgg ccaaactggc cttgaactcc 111180tgacctcagg tgattcatcc
gcctcggtct cccaaagtgt tgggattata ggcatgagcc 111240accgtgtctg
gcccctgttc agtgtatatc actaattttg tttttatctc tagaagtttg
111300atttaggtct tttaaaaatg tctccctgtg tttctgttta gctttgtgaa
cacaattgta 111360ataactgttt taatatcctt ctctgctagt tctaagatct
tctaataact tcccagttct 111420tggtgtttct cattggttga ttgatactcc
tcgttttggg ttgtattttc ctgcctcttt 111480gtatggctgc caatttttta
ttggatgccc aaccttgtga attttacttt gttggatgct 111540atatattttt
gtgttcccat agatcttctt gagctttgtt ctgaggttag ttgagttaca
111600tatagatggt ttactctttt gggtcttgct ttataatttg tcagatgggt
tggagcagtg 111660cttagtttag gactaatttt ttttttggac taattattcc
tctttaggaa taattaggta 111720ccatgcttag gaggcaagac catcctgagt
actctaccta atgaaccaga aagtttgggt 111780tttccagtcc gcctgctgag
aacagtgact ttctagccct gtgtgagcgc tgagctctgc 111840tccttctaat
cctttccaat gcttctttcc ctggcctcag ggagttttct cacacacata
111900tctctgctga gtactcgaga gggaccttcc ccagatctcc agagctctct
ctgtcttgtt 111960ttctcttctc tggtgctctg tcttatgaac tgtggctgtc
ttggtctcct tagattctca 112020gcacctcttc aattcagagg gttgcctgtc
cctcctcctt gtgccacagc ctaggaactc 112080tctcaaagca gcgagttggg
gcagccatag ggctgactta gtctctcgtc tcccagggat 112140cactgtcctt
cattgctcat gtccagtgtc ttgaggactc tgggttttgt ctgttttgtt
112200ttttggtttg ctttggttgt ctcaggcagg agggtaaacc cagtccctca
ccctcattgt 112260gctcagtagt ggaagtctca ctctattaca ttagatatta
gtatttgtag cagagccctg 112320gttccctggt acttggggag ctcttgaaag
gccagaaaca gcatgctttc tcaccttttc 112380cagggcttca gtttctggtg
cacatcaagc attccataca catttgttaa agtcctttgt 112440tagacaagta
gtgattcaca ggttctattt gtaatttttt cagttaacat gtattgggta
112500tctgctggga gctagtaaaa acaaaaagtg gtgtgtgaca aattcaattc
tgacaagaac 112560aaccttaaac acttagaata tactttgagc atatcagaat
tttaaaaatg tgtggccctt 112620gagtatttga aaccaacaag aatctattgc
ttattagtag aggatatttt gttaaacaag 112680tggagagaga ggcattttca
gtctaattgg tgttggcttt tagcagctga tggaaaccag 112740ttcgtgatta
gccaggcagt ggtgaaacag gctgtgcatt ctgaatgcct aggtatctag
112800gcattcagaa tggtggcgct ctttgagtta gcatcttctt ctttcttgat
tctttttttt 112860ttttttttga gatggacttt cgctcttgtt gcccaggtaa
caactccagt gcaatggcgc 112920catctcggct cactgtaacc tctgcctccc
tggttcaagc gattctcctg cctcagcctc 112980tcaagtagct gggattacag
gtgtgcgcca ccacgcctgg ctaattttgt atttttggta 113040gagatggggt
ttcactatat tggtcaggct ggtcttgaac tcctgacctc aagtgatgca
113100cctgcctcga tctcccaaaa tgctgggatt acaggcgtga gccaccactc
ccagcccctt 113160cttgattctt gaaaaggaca ttgggtgctg tacatctcgt
tatagatgtt gataaaaatg 113220cttgtgagaa gagtaacatt aaggtagtta
tttggtcatt tttgcagatt attttaagac 113280aattctagga ctgatttgtg
gtaaatcaca cattgctgta tcatagttgt gttcactgaa 113340catattcagg
ggctctacag atgcagggct cttagctgct ttgcacactt ctgaattcct
113400gccctgcgaa caggactgga tacctaatag acaacaggta cttgataaca
gtttattgaa 113460ttaatgagtg aatgaacaga tacataaatg catgaaagaa
tggttgtaat gtatataact 113520tggatttcaa gactttttac tgactgttca
aaataagaaa ttgaaaactt tcctctgatt 113580ttcctctact atttacacaa
tttaaatgga agttatcttg taccttcaat ttctgtctag 113640gattcgtaca
ataacgggtc atctctgagt cgcttaatgt ctcacttgtc tttctacagt
113700gtgttgaaga gatcctagga tacctgaaat cctgctttag tcgagaacca
atgatggcaa 113760ctgtttgtgt tcaacaagta agagcttcat tcttttcctc
ttctgttaag acgttcgggt 113820atgacagcaa aacgctgcta ctccttaaga
ggcaggcgct gttggcataa tcagctggga 113880ggattgtggg gtccagcgca
gcactttttg gctcagtcca tgattgagcc aagaggccat 113940ccttcccttc
actccccagg aggacgaggt ctgtcactgt ggagggcaga ggacaccaga
114000agctcctctg caacctcgct agttaacttc cagtccctcg gagtttctgt
ttagaatgct 114060caatctcatt tagaattgca aggaaaccca aaacgcctat
ttaaggtaca aacagcactt 114120catacaatat ctcatgaggt attaatagtg
attcacagga agaatttcac gctgtgagtc 114180tttgctaaca tatccagtta
tttacagatg gatttgatat ttgtgtggga gattcttaaa 114240agtgttgttc
acgccacatt gttgatgcct catttttttc actgtagttg ttgaagactc
114300tctttggcac aaacttggcc tcccagtttg atggcttatc ttccaacccc
agcaagtcac 114360aaggccgagc acagcgcctt ggctcctcca gtgtgaggcc
aggcttgtac cactactgct 114420tcatggcccc gtacacccac ttcacccagg
ccctcgctga cgccagcctg aggaacatgg 114480tgcaggcgga gcaggagaac
gacacctcgg ggtaacagtt gtggcaagaa tgctgtcgtt 114540ggtggaagca
cgaaagagca agcaggaaat actttgtaaa agaataaaaa cgaaaaatgt
114600tagcgaacat cttctaatag tctgctgtat tcagagaact ctaggagata
tatatggttg 114660atgcaaagat gatttaaggc atagcccggc cttccaagaa
gtgtgtggcc agtgagtgag 114720atgggcttgg gacttacaca tctcagaggt
gggggtagag gaggaggaac actgagtggg 114780ctgagaagca gccagctctc
attgccaaag tgtgtcagca aaccagaatg cagttcataa 114840tgtccccacc
cattcaaagc acaggacctg tagagtggtg tggcatgtgt tggtggcact
114900tttcaggcct gtaacaagga tgaaagaaca gcttcatagc agcacagtag
tgctggtgtt 114960cagaggtgtg tgaaggccat agaagcatct tggatatatt
accttgtgtt ttgtcagctt 115020tatgactaga agtctctttt cacttaaatt
tgtttttttt ttttttgaga cggagtcttg 115080ctctgtcgcc caggctggag
tgcagtggtg caatctcagc tcactgcaag ctctgcatcc 115140tgggttcatg
ccattctcct gcctcagcct cccgagtagc tgggactaca ggcgcctgcc
115200atcacgcctg gctaactttt ttttgtattt ttagtagaga cggggtttca
ccatgttagc 115260caggatggtc tcgatctcct gacctcgtga tctgcccgtc
ccggcctccc aaagtgctgg 115320gattacaggc gtgagccacc gcgcccggcc
tcttttcact taaatttatg tttgtgtttt 115380taatgcctag tatacaggac
ttcttaaatt gccttaagta tgaacaggta tttgagttgc 115440taatctgtat
agtagcaata atagaatccc ttgtttttcc ttttataaat ttagcgatta
115500aatagctaca attaaaacac tagagtcagg agtcaaggaa aatacccatg
ttccaggctg 115560tatgttagtg atgtacttac tatatattgg agtttcagga
gtaagtctgt ttcaatgctt 115620tctgtaacca tttggggtat taataagcat
gtgagtgtgt gcatgtttgg gttaatttca 115680tatatgtttc ttagaaggga
tatcattgat gtaaatattt taaaggcttg tcctccaaaa 115740aaatcatgta
atttcttcta aattactgat cttttaaatg accttcacct ttctctcaaa
115800tctcacttaa gactgggctg agtagtcagt ttcctgtagc agaaaaaagc
tcagacttga 115860gtagccttct gcgagtgagg agacttgatg gctgtcaggc
agctgtaaac tctaaataga 115920gtgtcattat ctgaagaggg cgatgctgcc
acactgagtg gcctttcaag ttgtttctca 115980atctgacacg ttctgatcgt
gtgaatgtga aattggtttg agcaggagta tatctgagtg 116040cagaggagat
tatttaaaga tattctcatt ctctgcttcc cttttattcc catttggcag
116100atggtttgat gtcctccaga aagtgtctac ccagttgaag acaaacctca
cgagtgtcac 116160aaagaaccgt gcagataagg taaatggtgc cgtttgtggc
atgtgaactc aggcgtgtca 116220gtgctagaga ggaaactgga gctgagactt
tccaggtatt ttgcttgaag cttttagttg 116280aaggcttact tatggattct
ttctttcttt ttttcttttt tatagaatgc tattcataat 116340cacattcgtt
tgtttgaacc tcttgttata aaagctttaa aacagtacac gactacaaca
116400tgtgtgcagt tacagaagca ggttttagat ttgctggcgc agctggttca
gttacgggtt 116460aattactgtc ttctggattc agatcaggtt tgtcactttt
atctttcatc catcatacct 116520gttcctaatt tagtacaaat taccctaaaa
gacactgaaa tctactttaa agaaatgtgg 116580tctgcatgtt tccctcatca
gttgctgctg cttatctttt tcatgcacct agctggtgca 116640gaaggcctgg
ggcatagcca gcctcagcaa gtcagcatcc ttgccccagc tccctggact
116700caaggctaac ctggggttgg ctgttaggga tttccaaagg tttgtcccat
ccacttgcct 116760cccctccaaa ataagtttga atttaaattg tgagatacaa
ttaagattta ttgtttgggg 116820aacatttttg caaaatctag agttagttta
aacagattat caattattac cataattgat 116880catctgcagt ttcaagctat
ctaacaggtt cacttacctc tttaaaaagg aatggaattt 116940agcaggacag
taactgagac ccgtgctcct ggagtccatg tgggagctgt gtggctctgc
117000acaagcattt gcacgcttcc cctcttgact gcattacctt cctcctatag
ttgctgtggg 117060caccagattc tggctagtcc tgtcccttca tgatgcacat
tttcctcaag attcgtccca 117120gttaaatcac tgcagatgaa actgcctttt
catcgtcaaa atttaactgt catttttgag 117180ccgtgatctt gggctacttt
cttatgtggg gtaggaatat ttgtgagtta gaaatattac 117240acttctctat
ttccttctag acgtaaatct gttaatcctg tcagcactgt tactcacctg
117300aaagggtctg tttccctagg agaactgagg gcactcggtc aacactgatt
ttccacagtg 117360ggtattgggg tggtatctgc ttgttttttt tgttgttgtt
gtttgttttt ttttgttttt 117420tttttgagat ggagtctcgc tctgtcaccc
aggctggagt gcaggggtgc gatctcggct 117480cactgccagc tccgcctcag
aggttcacgc cattctcctg cctcagcctc ccgagtagct 117540gggactacag
gcacccacca ctacgccagg ctaatttttt gtatttttag tagagacgag
117600gtttcactgt gttagccagg atggtctcca tctcctgacc tcgtgatctg
cccgcctcgg 117660cctcccaaag tgctgggatg acaggcgtga gccaccgcgc
ccggcctggg gtctgctttt 117720aatgaaggag gcatcaaggg gtgggctttg
cgttggcctg atgctttcat ctttctttca 117780caaaacctgt ccgaagaaaa
tccgtctaaa tgggccattg ctctcctcag gaaatagtca 117840ttgggaactt
cttttccttt cctttgacac taggaggctg actggggaga agccctggtc
117900tatggctgtg ggcagcaggg gctgagagga gcaggctctc aggggggcac
gggtacccca 117960agggaagcca gagccctgat ttgttccatt ctagtaagaa
caaagactgc tctggtttca 118020tgtttgttct gattgccttt catcaaccgg
tcccctttct cccagttctt aagattcagt 118080acagtgacag ttttatgaac
aagaatagaa cactagaaca gacaaaccat tgaactctat 118140gctgataaag
atttattgag ctcctgctgt atgtttgcat tctgcccaga ggctctgaga
118200aaaccaggcc atatgctcca tgctttatcc atggaagctc cccgtcaggt
tgggaaagct 118260gacagctgca gggaatacag tgtgacacaa aactggctcc
catgcagccc ttacgtgtcg 118320cctctcagat ggttggggga cgaaggtcga
ctcctttggg tatcttatta ctaaaccagt 118380ttcagggaat ctgtgccacc
ctatctgcca ttaacgtgaa cagatgagtc cccaaggtgt 118440aattttgggt
attgtctgat gtctcttgga atttattatt tgtttttcca atgagatttc
118500acctcagggt atagtaaagt tgttgagggg attcctggat gtgttctgca
attatctagg 118560ctgatttcag aatagagtta tgcttatagt caaatttatc
agctgtcaag aattttattt 118620aaaatttatg cagataagca ggaggaaaag
aagcctggtt tttacatttt aatcctatta 118680ttgatgtgaa attttatttt
ccttcctgta ggtgtttatt ggctttgtat tgaaacagtt 118740tgaatacatt
gaagtgggcc agttcaggta atagcatttt attattttag atttttttct
118800tcttcttgtg tacttacatg taatttaggt tattaagtga atgtttaaac
tactgttagg 118860catttttgct gttttcttta aatggaaatc tgactaacat
actgtgcatt tttgcttctc 118920ttaaaaatta atgtatatct caagacttgt
ttggaagtag ttatgtatct gaaaattcca 118980tatgttgtca gtattcattg
cacatttcaa agcatttaat tgtgttgaca gatggtggaa 119040tgaaatcttg
tggtggagca ctagttttta aatcttctta gagaaagcag ttttatataa
119100tgttgtcttt agtaattatt atgcatttgt attctctgca gctttttctt
gctagatgtt 119160gaggttttaa tacttcttgc tagtccatta caggtttata
attattaaaa gttaaaattc 119220ttttagtacc taaaatgctt aataaacatt
gtaattagga aaatttagtg cagaaggaaa 119280gtgttcccag attccctggg
gtctggaaac atagtgttta ttctaattac atgacacctc 119340cactgtgttt
tggggcaagt tactgtttct cttttgagtt tcaatttctt caagagcaaa
119400gaggcagagg agagctagga agatcgtagc tgctgtgccc ctgtgccgtc
gggtgccttc 119460tacctgctgc ctccgaacct ttacacatgt ccctgctctg
cgcgagggca cagatgggat 119520gcactgtggc aggggtgggg ttagagtaga
tcacggacac ctgttagctt gatgtgtgct 119580tgctgtcaag gttgaatcat
gaattatttt atgttgctta tattgatatg tatcttaatt 119640ttaaaagaaa
ggtctaaatg gatgtttttg tttttaggga atcagaggca atcattccaa
119700acatcttttt cttcttggta ttactatctt atgaacgcta tcattcaaaa
cagatcattg 119760gaattcctaa aatcattcag ctctgtgatg gcatcatggc
cagtggaagg aaggctgtga 119820cacatggtaa cgggacacac ctttcactgt
cgtcttcggt gtcgtgatgt gcttggcagt 119880gttcgttttc atatacccac
tttgaacgtt gtcagtggca gccatgtgct tctcaggctc 119940tgcatgtgtg
tctgtgtatg tgaaggtact ggttagagac gtttcaaaag agaagagagc
120000atattcttta ctctcagcaa tttgtaatct tctcagggaa aaaaattcaa
gaaacagtaa 120060gataacctaa ggtacagata gattctgaat ataaagttcc
tgttcattca catgaaacgc 120120taaaagttct tcacttgatc ttagccaaaa
ggccaagaag cgatgcaaca ctaaaaattc 120180ttaaatcgaa cttgccgtga
attaaatttt gatctctcat ccagtggtat tggagatata 120240gtttgacttg
ggttcagggc tttctgtttt gcctgatgat tttgctggag cttaaataag
120300gaacccagga gatggccagc tgtgcaagcc cccagcctgt ggaaggagct
agtgtggttt 120360tatgaatgag ttgcaaatct ttctttgagc tttttgaact
gatcttccag cattgcccta 120420ttgacccctc cctgactcct ttgctggaat
ctgtaggctt
ttgaactttg acagggacac 120480atcctaagac ccttgcaaac tcccagatgt
gagaatggca ctactactta gagtcttttc 120540gactcagcgt gtgtgcagaa
gagcatcaac cgggctgtgt tgcgaggcag ggccttggct 120600gacctctcag
tgtttacata gctaagccag ttagtgtttg ccacggcctc acaagggctt
120660cagattcaca cagccaaagt atagattatt aaaggcatag gtgtttggtt
tcctggactt 120720ggagggtctt tggacagaaa atcagtaggc aaccacaccc
agtactttgt gctgggaagc 120780ttggtcatct gtgagagggt cagagagtat
acccatgcgt gcatgccacc gaagggtcag 120840tgagtattcc tgtgtgtgca
tgtctcaggg ccggagagag tatgtgtcac tgagaggtca 120900gagtgtttgt
gtgtgtgtca aagagggttg cattgtgccc ttcactgagg ggtcagaggg
120960tgcctcgcgt gtgtgtgtgt gtacgtgtgt gtgtgtcact gaggggtcag
agtgtgcctg 121020tgtgtgtgct tgtgtgtgcg tacatgtcac tgaggggtca
gagtgtgcct ctgtgtgtgt 121080gctcatgtgt gtgcatacgt gtcactgagg
ggtcagagtg tgcctctgtg tgtgctcatt 121140tgtgagcgta tgtgtcactg
agggggtcag agtgtgcctc tgtgtgtgtg ctcatgtgtg 121200agcgtatgtg
tcactgaggg ggtcagagtg tgcctctgtg tgtgtgctca tgtgtgagcg
121260tatgtgtcac tgaggggtca gtgttcctat gtgctcatga cattgagggt
cagagtgtgc 121320ctgtgtgcca atgaaaggca tttcttatat ttttttatat
gtggtcatag tagaccagtt 121380aatttatttt gactcctgtg ttagaccaaa
ataagacttg ggggaaagtc ccttatctat 121440ctaatgacag agtgagttta
cttaaaaaag cataataatc cagtggcttt gactaaatgt 121500attatgtgga
agtctttatt gtcttttcag atgaatcaag tagattattc ttgagaccag
121560gaatgttgct gttttggtta tttggaaagt tttatcattt tcaaattgac
ttttgaattt 121620gagtcacctt ttttcagaag tggtgttaaa ttataggagc
cctaggtttt ttttcttttt 121680ttagaagtca tcacaaaatg atcagtgttc
agaggaagag ctttgacctt ccacatggta 121740taatgattga taaccttaat
tcatctctta ccataaacca agtatgtgta agggttttct 121800ttatttcttg
aaagcatttt gtagatgttg agagcagttt tccaaatgta atttccatga
121860aatgcctgat aagggtaccc ttttgtcccc acagccatac cggctctgca
gcccatagtc 121920cacgacctct ttgtattaag aggaacaaat aaagctgatg
caggaaaaga gcttgaaacc 121980caaaaagagg tggtggtgtc aatgttactg
agactcatcc agtaccatca ggtaagagga 122040atgtatgttg gaactgtcgt
ggatacttta ttgacccgtg cagatggaag gaagtgccat 122100gtggtaacgc
tcactgttaa ctgtgttact ttgaaccagg tttgggcttt ctggggcctg
122160ggtagatgcc ggtgcagggg gatggggagg gaggcggggg gtgggggggt
gtggtggagt 122220tggggaggtg cagtggcagg aggtgttgtt ggtgtgtatc
cttttttttt ttttgagatg 122280gagtctctct ccgtcgccca ggctggagtg
tggtggcacg atcttggctc attgcaagct 122340ccacctcccg ggtttaagca
attctcctgc ctccacctcc cgagtagctg ggattacagg 122400catgcaccac
catgcccagc aaattttttt ttttgtattt ttagtagaga tggggtttca
122460ccatgatggc caagctgttt cgaactcctg acctcaagtg atcctcctgc
cttggcctcc 122520caaagtgcta ggattacagg cgtgagccac catgcccagc
ctggtgttta tctttaaagt 122580gggcacagcc acaggagttc acctgactcc
tggtctgaga gtcacgagat cgttcaagat 122640agtgaggccc tcttttccaa
aacgaggacc aaaaatcaat tgacagtgtt ggtcaagatg 122700gtagaaacct
taaaatgata gaaatctcaa ctctgaaata aaaactttat ttgtatattt
122760atttaccact attttgacat agggctaagg tctttttctt tgagctgatt
tctggttttg 122820ttttcttaaa gtggcataag aattcaaaga cattttgagg
aaggctgagt gcagaaatct 122880ctctttttaa atgacttctc ctttctttta
acttgcactg ttgtctagcc ctcacttatt 122940ttgtcaattc tttttagctg
tttgtctttg aatcttcata aagccatagc ttttctcata 123000agaagcagca
ctttctttgt tcattcatat tttaatgaac ccctgtagta tttaattaaa
123060tacttaatgc ctaattaaat cacataattg caatgcaaaa gtacatgtat
cataaagagg 123120tctgaaaatg agcaactggc aagcaggtgg tggcaggcag
agctgcttgg gtgggtgggt 123180gtcatggaga ggagttcatc agccacatgt
tcagtgagct ctggatatgt ctgtttagaa 123240atgatcacta ataaacttgt
gctcaaccat gtatacctct gggaagcagg tgctcttcag 123300tagattgcct
ctgcagagaa cacagaattg aagtgaatgt ccacaaaggc aatgagccac
123360ctgcagaata gtttagtcaa ggctgtgttt gaagtttgcc aaagattaat
atacatttga 123420ttttcatgtt gtgccttttc tctgattgtg aaatattaca
aattctatac aaataacaat 123480gatggcaaat cctcctgagc aaagtgtgca
ccttgtatgt gccctagagg aacttgtgtt 123540tcgttctgat tcccctacat
ttctcatgtc atagagtggg ggttgcatta gtgtccccct 123600gtcctcgctg
ggatcacatc tgtttggatc ctagagtctt ccagctgaac tgggacaagt
123660ataacagacg gacacgtagg ggtggaaagg cgtctcttgg cagcagactt
tctaattgtg 123720cacgctctta taggtgttgg agatgttcat tcttgtcctg
cagcagtgcc acaaggagaa 123780tgaagacaag tggaagcgac tgtctcgaca
gatagctgac atcatcctcc caatgttagc 123840caaacagcag gtttgtcccc
gcagccttgg cttgttgttg catagtgatg gtagcttaag 123900gtccttgtga
aaggtgggtg gctggaatca gctcttcctt cagtcctaat ctgtgccttg
123960atagcagttc tccgtgctag tcatgggaca gctgacttca tttcttctca
caatgccatc 124020tcaggttggt attgcccacc tactttacag gggggatccc
acagctccga gaggttatgg 124080aggtgatcag gcagcacaca gctttagagt
gctggggtga gggcgggcca aggctaactc 124140taaagcccga acccttacct
cctacactgc ctcctgcatt ctggtcaacc cagtgtttta 124200tttggtggtt
agatttttgt ttttgttacc ttactgcttg taatttagca gttttccttt
124260cctttccctt cctttccttt ccgacagggt ctcactctgt cacccaggct
agagtgcagt 124320cgtgtaatct cactgcaaca acctctgcct cccaggttca
accaattctc ccacctcagc 124380ctcctgagta gcaaggacca caggtgtgca
ccactacgcc tggctagttt tttgtatttt 124440tagtagagat gaggtctcgc
tgtgttgccc aggctggttt taaactcctg ggcgcaagtg 124500atccaccaac
cttggcctgc caaagtgctg gcattacagg tgtgagccac ctcgcctggc
124560ctattcatca ctaatcagaa tttctatgat caaatgacat gaatcattgt
ttccacaact 124620gcagtggaag gaaatggcct ggcagtgcca gtttcagaag
cagcctgccc ccagtcaggc 124680acaggccact gtgcccccag tgtagcagca
cctctgtagc tcacagagaa gggtggtggg 124740gacctccttg aggcagctct
gccagaaaat ctcatgagct gcctggcaca gcttgaggtt 124800gccttttaag
tggactcagc aaatacatgt ttgttcatct tgattataca caataaacaa
124860ctactctgta tagtacgagt agtccgtggt ttttggcatt tgatttaaac
ttagaggcat 124920gtgatattga tgttactgcc ttcatgactg cacccccatt
ctgatttcat aatggaatgt 124980tatcttgaga ccagttagac aacaggacag
ggatcttggc ttctggtgag attgacagca 125040gttttagtgt ggtcagggtc
tccctgccta cagatggttt tagaatggtg ccctggaagc 125100tttatcccat
tcttttctgt gcgtaatctg agtagagtgg agatcgaagg cctgaataca
125160tagtaaatac ctgacttaat atctgccgca atggaaattg tgtgatacaa
catttatgaa 125220acgcttagtg cagcacctgc caggtagctc accacaggtg
catgttgcat tcagaagtag 125280tgctagatac tatcctgtta ctggcagtgc
atacatcagt gatcaaagca gattaaagaa 125340agaccccctg ccttcttgga
gtgaagattt tgttgggatg cgggtaaggg gacagacaat 125400agaaaagcaa
gtgagtgaag tctataccat ggcggctgat caggaacacc gtacagaaga
125460atccaggagg gaagagagtt aggtggtgtc tgcggtggga gtggcattgt
tcagctggtg 125520atgagaagaa gctttggtga tctggtgaca tttgagtgaa
tttgcagaaa ggaaagatac 125580aagcctagga gatacctggg gaaggaacat
tccaggcaga gcaaatagca gtgcaaaggc 125640cctggcgggg ggcggacatg
ctgttagggt acaagcaatg agggtggagg agtggggcag 125700ccatggggag
ggaagggagt gaggcctggt ggggtgaggc cagtgtggag gagccttgag
125760agggtttgcg ctgatgtggt gtaggtttta gcaggatcat tcttattcct
gagttgagaa 125820tagccttgag ggggaggtga gggcagagca gggccaccca
tgtgagaccc ggcactggag 125880tggaatggcc caagtcagca tcccttggca
gcatgaaagc aaaaccagca aggtttgctg 125940gtggcttaga tgtggcatgt
gagagagagc agggctttgg gggtgatttc agggtgagga 126000cagggtggct
gtggacaagg tagggcagac attgggggca gcaggaggtc agagcctgtc
126060tggatgtagc agttgagacc ccataggtgc ctaatgaggt gaggccagca
tcaggtgtat 126120gagcctggag ttgtcgagag actgtggggc agggggtcag
catctgagat gtccactcac 126180agtggaccca gactggctgg agaggaggag
gagcttgaat accgagcctg ctgagtccca 126240gctccaaggt caggtaggtg
aggggagcca gtgctggggc agggggagta ggcaggtgtg 126300gggttcctaa
agccaagatt ttttttaagg cattttgtgc aggagggcga catctgctgt
126360cagcaccttg ggaacttggc ccaggtttgg cagcaccgag ggcactgatg
agtgcttttg 126420gaggagcaaa gggagccaaa ccctaatggg aatgtgttcc
tgaaaggaca ggagagagac 126480ttgggaaaag gttttacttg aagagggaac
ggagaaatag ggcagtagcc agaggaggag 126540aggagtcggc aatgggttaa
gttggcagaa atgaaggcct gtttacgcac tgagggcaga 126600agcaacaggg
aggatcagtt catgacacag gagacacaaa tcgccgttgt ggtgttcaca
126660gacatgggtt aggattggct gcatggatga cagagcactg tgggttctcc
cagagttgct 126720ggggaggagg cagagttggt gagcacaggc gagggtccag
gatgcaggaa tcctggagct 126780caagtcagtt gttcccttgt tgtaagatgt
ggccagtgtt gtgagcttca catctgtgcc 126840ttgaaaaaca ccacatctgt
ttgcagagtt gtttactatg tatacacact cagtagaaac 126900aaaaattgga
aacagtcagt gcccaccatc aataagtaat ggttgaacac actgtggtat
126960aagcttagac tattttagct tgggctattt tgcatgatta aaaatgttct
ggccaggtgt 127020ggtggctcat gcctgtaatc ccagcacttt gggaggccaa
ggcaggcaga ttgcttgagc 127080tcaggagttt gagaccagcc tgggcaacat
ggtgaaaccc tgtctctact agaaatacaa 127140aaagtagctg ggtgtggtgg
tgtgcgcctg tagtcctggc taactcagga ggctgaggtg 127200ggaggatcac
ttgagcccat tcgtgcgcca ctgcactcct ggggcacaga gtgagactct
127260gttagaaaga gagagagaga aagaagagag agggagggag gaaggaagga
aggaaataaa 127320tggaagaaat ggaagggagg aaggggaggg aggaaggaag
aaaggaagtt cagccagttg 127380ccttgggagt tctccattgc actgggttaa
gtgagaagag cagagacgtt tatgattttt 127440caaaacaact aaaacaaaac
ctctgtgggt gagggggcaa ggatatggct ataggaacat 127500ggggcagatt
aagaaaggga tatacacaca ccacttagca tttgttacaa ctgttgtggg
127560agggatggag tgcagaaaaa gaaaaaaaaa agtgcacacc atcccatgta
tgtgtataca 127620aagggacgct tggaagactg gtccccaaaa tgttggtaat
gattgtgtca gggtgctgca 127680gtgctagttg attttttttc acacttttgt
atatttgagt cttttacaga aagcatttat 127740tatttatgta ataaaaatct
aaatgacaag atttctgtta tgggaaaaat gtagctatac 127800agtgttgttg
taaaaatgtt tgcttggttc accactgaac ttaaaatgct tttaaatgag
127860ggaaggtgac gatgagatga ttatgatgat ttgcccttga gttacatagc
tggtgtacag 127920gaagctgtcg tttcttttgg cttacgtaga aatgtttgtg
gtgtctaatt ccacagatgc 127980acattgactc tcatgaagcc cttggagtgt
taaatacatt atttgagatt ttggcccctt 128040cctccctccg tccggtagac
atgcttttac ggagtatgtt cgtcactcca aacacaatgg 128100tgagtctctc
gcctggctca gcagatgaat ctggacggct tgttcaggct ctgattactg
128160ggaccacccc cagaatgtct gagtcagtca gtttgggtag ggcttcttga
gagtttgctt 128220tttttttttt tttttttttt ggtgtggggg tggtgcggaa
cagagtctca ctctgtcgcc 128280caggctggag tacagtgtca tgatctcggc
tcactgcaag ctctgccttc cagcttcaca 128340ccattctcct gcctcagcct
cccgagttgc tgggactaca agcgcccacc accacgcccg 128400gctaattttt
ttgtattttt agtagagatg gggtttcacc gtgttagcca ggatggtctt
128460gatctcctga cctcgtgacc cgcccatctc agcctcccaa agtgctggga
ttacaggcgt 128520gagccaccgc acccggcctt tttatttttt ttggagatgg
agccttgctc tgtcacccag 128580gctggagtac agtggcgcta cctcgactca
ctgcaacctc cgcctcccgg gttcaagcaa 128640ttttcctgcc tcagcctccc
gagtagctgg gactacaggt gcgtgccact gtgcccggct 128700aattttttgt
atttttagta gagacggggt ttcactgtgt tagccaggat ggtcgcgatc
128760tcctgacctt gtgatccgcc cgcctcggcc tcccaaagtg ttgggattac
aggtggctct 128820cgcaccaagc caagagtttg catttttagc aaattcccag
gtgaaactaa tgcctgcttt 128880tctgggagca cactttggga ctcagtgata
gagaggttta ttggtaggat agtaaaatag 128940gagttatttt ctttcacaaa
attggcaatt gggggaaatt taatcttcct tttttcttca 129000gctgtgactt
atgtattatg tttattttag gcgtccgtga gcactgttca actgtggata
129060tcgggaattc tggccatttt gagggttctg atttcccagt caactgaaga
tattgttctt 129120tctcgtattc aggagctctc cttctctccg tatttaatct
cctgtacagt aattaatagg 129180ttaagagatg gggacagtac ttcaacgcta
gaagaacaca gtgaagggaa acaaataaag 129240aatttgccag aagaaacatt
ttcaaggtat gctttctatc tgagcctata actaacccat 129300gccttttggg
aagtcacgtg atgtttcaca gtcagtaagt ctggaataat acctggtctt
129360gcttcacttc tgagttgggt aaagaagtct gtatcagtgt aattttctaa
tccgtcctgc 129420attatctatg gctcttggtt catacctgtc ttgaagttct
gtcatgttct gtctcttgtc 129480ctcagtagag atgctacagc agtggctcgc
ctcaggcagg gcagggcagt ggggtggctg 129540tcctgggggc aggcagtagg
ggcacgctga cgtcagggaa gttgaaaccc aagagaagcc 129600agtaaaagtg
agtctcagat tgtcaccatg tgctggcagt tttacacgct gtcagtaata
129660aaagtcttct ccctgcaggg cagcctgcct ccaataaata cgtgtagtat
caaatcctgt 129720cttccctcat aaattgtttg gaagctcccc aaggacagtg
atgaggcact cgtaagtgct 129780tgctgcctag atgggtccct ctccaccttt
gctagattct gagcattcac tgagttagag 129840ctgcttctgc aaatgtgctg
cttctgctaa gtggctgtga cttcatgcag ccttcacttg 129900gtttgtcatc
agtggagatg ccctgtgttg tcgaaggaga taagcccagt aagcctgctg
129960ggcacctttt ggtttgcagg ttcagcaggc agcccatggc tttccctgtg
tcgcattgaa 130020gcagctggct aaaattgatg atacattaaa ttcctgtgac
agatgatcag cttgtatttg 130080tgtaatggtg tacagttcac aaagcttaaa
aaaatgctac ctgccatttc atcctcagtg 130140aggaaggtga tacacagaga
gaccaagtga ctgtgtccac ggcgacggcg ctctgcattt 130200cactttagcg
gttaatgtac tctacctata tttttacttt atatttacca tatatctttt
130260catgtatact tggcgtaagt gctttatagt agtcacctaa ttcactgtca
tcttttttgt 130320ttcttggaag gtttctatta caactggttg gtattctttt
agaagacatt gttacaaaac 130380agctgaaggt ggaaatgagt gagcagcaac
atactttcta ttgccaggaa ctaggcacac 130440tgctaatgtg tctgatccac
atcttcaagt ctggtaggtg aatcacatta gtcttcctgg 130500agtgtctcgt
tccccattct gcactataca ctctcagagt gtaggagctg tgctgcccgg
130560tagaaactct gccttgccca gtgtgccagt tgaaaatatt tgttgctgta
agagtacacc 130620tgataccatg tgacccagca gttccactct tgggtatata
cccaaaagaa tggaaagcag 130680ggtggtgaaa agatatttgc atgccagcat
tcatagcagc attattcacg atagctaaaa 130740tgtggaacca actgaagtgt
ccctcgatgg atgaatggat aagcaaaatc tggtgtatat 130800ttacagtgga
atattattca gccttaaaaa aaggacattc tgacacatgc tacaacatgg
130860gtgaccctta aggacattat gctaaatgaa ataagccagt cacaaaagga
caaatactat 130920gtgattccac ttacatgagg gacctggagt agttaattca
tagatataga aagtagaatg 130980gtggttgcca ggggctgcag gggaggggag
ttatttttac aagatgaaga gagttattct 131040agaaatgaat ggtggtgatg
gttgtataac attatgaatg tacttaatgc tactgaactg 131100tacagttaaa
aatagttaag aggaccaggt gtcatggctc atgcctgaaa tccaagcact
131160ttgagaggcc aaggcaggag gattgcttga gccaaggagt ttgagaccag
cctcagcaac 131220atggtaggac cccatctgta caaacaaact agccggggat
agtggtgtgc atgtggtccc 131280agctactcag gagactgagg ctggaggatc
gcttgagccc aggaggttaa gtctctagtg 131340agatgtgttc atgccactgc
actccagcct cggctataga gtaagaccct gcctcaaaaa 131400aacaaaacaa
aacaagacaa gagccaaaaa tggttaagat gggccaatca cagtggctta
131460tgcctgtaat cccaacactt tgggaggtca aggtaaaagg atcacttgaa
gccaggagct 131520tgggaccagc ctgagcaaca tatcgagacc cctatctcta
caaagaaaat caaaaactag 131580ctagatatgg tgggcacatg cctgtagtcc
cagctacttg ggaggctgag gtgggaggat 131640ctcttgagct caggagttcg
aggctgcagg gagctattat tgcactccag cctgggctac 131700agaatgatac
cctgcctctt attaaaaaaa aatccaaaaa aaaaaaaaag taaacctgag
131760agcttcctcc tcctgtgtta aatttggagg ccaagatgtt tttgttactt
ttacaaatga 131820tcaaggacgg tgaaggttgg gcatggtagc tcacacctga
aatcccagca ctttgggagg 131880ctgaggcggg gtgatcgctt gagcttgaga
ccagcctgga caacatagca agagacccca 131940tctccacaaa aataaaaaaa
taaaaaaaaa tagccaggag tagtggcatg agcctgagcc 132000caggaggtca
agctgtagtg agccatgatc atgccactgc actccagcct gggcgagatc
132060gagaccatgt ctctagagaa agaaaatgac aaggacagtg aacccaagaa
agtcataaga 132120tgccagctgt gcagcaagca tggaaagcag ccagtccaaa
ttaggacagt gtgttttcca 132180agaagaacga tcgtttgtaa tgagaatgct
ttgctttaaa taaatgacta aatagctaga 132240agcctagttc taggggatag
gcacgtcttt cttctctcaa gaaaatagaa aggcaattct 132300aatttctagt
aacagcaaac agcattaagt catggtccaa atatgaggca aaccaaaatg
132360tggcttgatt gttcagcagt tgatctgttg gaagcccttg atattaaaaa
ggttctcctt 132420taagcggctt aggagtcacg atcaaagacc tatagaaaga
gatgccatcc ttctaggatc 132480cttggctctc ttgggaacta gattcagata
gtcataatgt aaatactgct tgagctttct 132540ttctttcttt ctttctttct
tttttttttt gagacagagt ttcactcttg ttgcccatcc 132600tggagtgcaa
tggtgccatc tcggctcacc gcaacctctg cctcccaggt tcaagcaatt
132660ctcctgcctc agcctcccga gtagctggga ttacgggcat gcaccaccac
gcctggctaa 132720ttttttgtat ttttagtaga gacagggttt ctccatgttg
aggctggtct cgaactcctg 132780acctcaggtg atccacccgc ctcggcctcc
caaagtgctg ggattacagg tgtgagccac 132840cgcacccggc ccgagctttc
atttttgaaa tcaatgtatg actgaaacac tgaagactta 132900ctgacttaat
tatggtttca gaacagaatg aaaatgtctt cggttctgat gaatataaaa
132960ggaaaactaa ccaagttaat ttggcaagta gatggtagag atagaggtgg
ggagtggaag 133020gggaactaaa atcttcacct agcattgttg ggattatatg
gttacatcat ctgaagttga 133080cagaccaaaa tatagaggct tcagaggtct
ccaaatagaa ctaaacatgt aattcagatt 133140gttaggaggt agtataaatg
agctaaatct catctttatt acggtagagt taatgggtga 133200tgtctaaagt
tgtctgaagt ctataaatca tgacaaatta tgatgtggtg attgtattca
133260acagtctttc agttgcaggg ataaaacccc agtttaaact agagtaagag
aaagaatgtg 133320ttggtttaag ctcctggaaa gtgcaggcaa gggtagttgg
taggactgca tctagtgttg 133380taattctgtg gtctgcattg tatatttatg
catctcagct ctgctttctt cttttcattt 133440atataatttt taaattttat
tttaaagata gggtctcact ttgtcgccta ggctgaagtg 133500cagtggcatg
aagtgcagtg cgaggctcac tctagcctcg aactcctggg ctctagagtt
133560cttcctgcct cagccttcta agtagctgag acaataggca tgtaccaaca
tgcctggata 133620ggttttaaaa tttttttgta gaaatggaag tcttgctgtg
ttgcccaggc gggtctttaa 133680ctcttagctt caggcgatcc tcctgcctct
gcctcccaaa atgctgaggt tataggtgtc 133740acccaccacg cccagtctca
tctctgcttc ctgtgttagt tttgttctct ggtgggctgt 133800tttcacatga
ccgaagatga cctctagcag gctgtgttct cagcccctca agtaggccta
133860tgtgattggc cttgcatgag taatatgggt gaccataaac ccctgaatgc
tctggtccac 133920atgggccaaa tgggagactg gacagcattc cattgatgag
gaggtggggc tggtctccgg 133980gagtaaggga gaggagcaca tgcagtaact
gatggtctgc tgcaagggat agcagcacag 134040cagttagaat tttggaggta
actaccagaa ctgaaaacag aaatgataac aagtagttgc 134100cttaaaaagg
gatgggagca gggtgctttt gtgatcaaag ctcctttctc ttactggatt
134160tttgtacaca ttttgcatac atatcttaga gtaaaagata gcattttcag
ccttggtcca 134220tttgaggata ctcttggcgt ggcccgcctc catgctagca
ggctctggtt gtgccaagtt 134280cagttgagca tcctggctct tgcctgcacg
gaacttccag tcagtgcgtc agtatcacaa 134340gtcttgatat ttcctatgaa
gaagaacagt agtgcagtga cagacgaaat gggtgggcag 134400gcagaggcag
gatttctgag ggagagaagt agctagcttt ttgcagagaa gagttccggc
134460acccaagaga gcagctgaga gtacaggcag gcaggcagga tgccggtagg
gcccggccgc 134520acggcgccac agaatcctgg agaaaggggc ctcttcatgg
cctctgcatt cagctgctgt 134580caccctccgc acaggccatg gccaaaattt
aattttcata gtggactcta gtttttgagc 134640cttacttgct attattgaaa
taattttctt gtttcttttt aaagatcttc ggattatgct 134700tcactgacca
ctgtaataag tttaaagttg agaaaatatg gcttgttaat gaatgatagg
134760tcaattttag tatgttggtc attttaatat tttgccacca gttggtttgg
atttgatgcc 134820aggaggagac agcctcattt ctaaggacta gtcttgcctt
tgtgggataa gggtggtgtg 134880ttctgtgtcc ttctacatgt ccgagcgatc
tctgtgcagc tcaaatgtgg tcactgtctt 134940attgcgctga tttcctctcc
ttccatctca caattgaggc aaaatattgt tactgttgaa 135000gtgttgtcca
ataggacttc cagcagagac aggatgtctg cactgtctaa tttagttgcc
135060tttagccaca tgtggtgttc tgtacctgaa atgtggctgg tctgattgga
tagcttaatt 135120tataatttta tttaatttta attaacttaa atttaaacag
ctctgtgtgg atagtggctc 135180ctgtatgaga cagtgcaggt ctgttgagaa
gcagctttac tggtgggagt ggagggcttg 135240gagagggcac gtgggtttcc
tgctggtatc ttttgacctt atttaatctg cccaacattt 135300gcaagtaagt
tgtgtgtgtg tgtatatata aatgtgtgtt tctgtcttct tgtttccttt
135360gactgcattt atttgaaaga cactaggtgg cagaattact gtatttgatt
ggtttcaaga 135420taagagttga aataattcat ctcgtgtttt tatataagta
aggtgtgttt agcatgtaaa 135480attggtaata tgtattcacg tactgcttaa
acaaaggcta
tgaattccac ccataaaccg 135540aaaatgaaga cctttaaatt tgtccatttc
aggcgtgggt acttcttaaa taatacctgg 135600ttcaggaact agtcagaatg
gcacccttga ctttttgttt cctgcttttc ctcttgttgg 135660gagaggaggg
tattcatccc aaagtggttt gcctatttca cattccatct aggataagca
135720gaatagccaa gaaagatagc tgtcctcctg tttacaacat ttggggtaac
cagcatccct 135780ctcttttggt ccaagataga ctggtttaga aacagatgat
ggcaccagag gcccaggagg 135840tggaaacatc agctttgttt gttgtccatg
tggctgaatt agagctgtct ggccttgtag 135900cctcaacacg gccttccagc
tttgctcacc gtgattttca aggacacatc ttgtgctctt 135960ccctgcctgc
catccagact atacccagtc agggtggcag gagctgctgc cccttcctcc
136020ctgagtcctg gtcgtgggtg gtggagatgt gccatgacgc tcacggaggc
atgctcaccc 136080cttcctctgt ggcagagggg atggctgcac gacagctctt
ccctgtcctt tccaaagcgt 136140ctgtggttcc actttttggg gcaaagcagg
aatactggaa gagagagaaa gtggtccttt 136200ctatagtaat aaagttgaca
ttgattcaag ttcatgcttg gggaaaggac agggctacta 136260acaattataa
tgctgggagc aatggaattt tctcatgggt atgtggtagg tttaatttta
136320attatcccag ttaattctta gaactgctct gtgaagtatt tcccgctttg
tgcttaagtt 136380ctaaaagatc ctgtgccaaa accaagaatg aaaacccaag
cattctttct tgcccatcga 136440tctttctctc atcaggccac ttcttgggtt
gatagtggtg agtgtagccg ctgccacttt 136500cagaataccc accatgggcc
ccagtcactg tgtggcgtgg agaagagatg gttctctctg 136560tgtcatagct
gaacaagccc agcccagaga ggtttctgcc ctaggagctc tcgatggtgg
136620aattgggatg cgatcccaca tcctgcctgt tttgaaaaca gcattcttta
tttccaattc 136680ctgcttccat tgttcctttt aatatttctt tgtttagctc
acaaaaacac ggcttgcgga 136740gctgctgcgt gcagctgtag ctgtttctct
gggtgcagcc tgcatccgcc ttcctgcccg 136800cctcctttcc tgcactgcca
tcgtggtctc cgggcacttg gtccctttct cttcccctga 136860gtccctttgg
ctcccctgtg ccacccttgt gatccacagg ctctgccttc tttctgtctc
136920agactgctgc tcatcactac tcgggaccct aggaagggag gttccaccga
gaagcatctt 136980ctcatctcag ccacgttctc agtgccactg ttgtctttgt
taggtaatgg tagctactgt 137040aacaaataaa ccaacatttc catggcttca
caccagagaa ggttgtttct tggttttatg 137100acaatgtatt gagggtgttc
ttggttcacg gatggttttc ctccatgtgg gaattcgggg 137160acccaggctc
ctttccttct tttggttctg ttctccaggc cttcacatcc tctgtgtctg
137220gttggggaca aggagaggga aggtaaagaa ggctttgtgg ccttggataa
gtgacaggca 137280tgcctttgct ggtgttctct cgtggtgaca ggtcacagcc
ccaccctgta aaaggggact 137340gagagacgtc gtcctgctgc ttcccagcag
cagcactgtg gtctctgatg tgttttctgt 137400gaggataaaa acaggtgatt
ccaggatgag gaaagtcagg gaaacccttg gaaggagggg 137460accaggcggg
tgtcaccatg ggattagtgg tggcttcaga atgagctgca gcgagtgcca
137520tgccttctaa agcttttgct attctgatat gcccacacca tgcccagcag
gtgtctgcct 137580tgctctccgc agagagagtg atgaatcctt ctcatgagcc
tctgtccagt tgttcctccc 137640tccacctgga agggaccctg ggttcctcat
aacatcccag cggaacaggg gaccttctat 137700cctgtcccca agttcatcct
catcctcctg ccggcttcct ggcccctctt atgtctgctt 137760cctgacgcca
catccttctg gattctctgg aattgaattt tgcctttgat gcttatttaa
137820aaatatccat tgcaggccag gtgtggtggc tcacacctgt aatcctgtgc
actttgggaa 137880gccaaggtgg gcagattgct tgagcccagg agtttgagat
tagcctgagc aacatgttga 137940aatcctgttt ctatagaaaa tacaaaaatt
agctgggcat ggtggcgcac acctatactc 138000ccagctactc aggaacctga
gacaggagga tcaattgagc cccggaggcc aaagctacag 138060tgggctgtga
tcgtgccact gtactccagt ctggtcaaac agagtgagac cctgtctgaa
138120aaaaaaaaaa aaatccattg catacttcac cgtagcgaaa catgtatgtc
ttacctttcc 138180tttcctgcct gtagctgctc ttttacactt aacagccaca
ctaagccagc cttaaatgaa 138240aaacaaacca gcacttcctg tgccctcctg
cttccttcat gaggggtccc tccctctgtg 138300tacactccat tctcattgcc
catggtggtt tgtttccctc ttgtttctca agccatggca 138360gcctgcctct
tgccctcttt actaaaaagg cctttgcaga ggctgcctgt gttctttctt
138420tctaggtctc tctcatccta ggccctccag cttgattctg tggagctgcc
ctcttgtcac 138480tcagtagctt gtggggtctt ctctgtctag ccacttaatt
gattgtgttc ctcgagttgc 138540tgtccatggt ctctcgttac tgttttctct
gtgtttctgc ctctctcctt ggccttggta 138600ggtccatccc ctttgtgacc
ttggctgttg ctctcatgga caactttctc ttgctggtcc 138660ttgtagtcct
ggcatccagc ttctcgacac gggacttgtc ctgccagtac ctcagacttg
138720cacttaaaat tgaactagca ccactgtcac tctccagggc ctcttcttgt
taattagatc 138780attagggatg ttcagaatcc cagcatcata gtatgttcct
cctcccgcta ccccaggaac 138840cctaacctta cctcctcctc tctatctact
aggaggtggc cctcagagtc cgtctcatct 138900tccacctgaa cttccctaat
aggctccagc agctgccacc ccgggggctg agtacttcct 138960ccatgccttg
tgcagtgctg agccctttac ctgggttctc ctgtttgctc cttattacag
139020ccctgcgaac agatactgct cttaattcca tcttacacct aaggaagctg
aggccccagg 139080taaggtgcat ccaaggtcac ccaggtagta gacagtagag
ccacgatctg aaccaggcag 139140tctgattcag agcctgtgtt gacactcagc
cacctagaac acagcttgga ttgtgggttt 139200ctattacctg ttcaaaaccc
ctacatcccg ggtctgtccc tgcacgtgct ctgtggcctg 139260gctgcatctt
ccttgaaggc agtgcatgcc tcttcactca gggggcccat gcaggaacag
139320agggccccac agaaggatga ggccagtgca gaatgggctg gaggggacaa
tgctgaccag 139380gaagcaagtg tagagaaatc ccaggaaacc tggaggagcc
agagacaagg cattagaact 139440cctcgtcgtg acctggtctg cattctctga
gtgtgctgct tctgttagct cgcttccttg 139500gtctcaggtt atagtttaag
gcattgtgga gccctaaaaa gcctgtactc tgtttttacc 139560tgttttagga
ccctttcact ttggggatgt gttgattttt tttttttttt tttttttttt
139620tttgagatag agtctcgctc cattgcccag gctagagtgc agtggcacga
tcttggccac 139680tgctgcccct gcctcctggg ttcaagcaat tcttgtgctc
ccgcctccca aatacctggg 139740attacaggca cccgccacca cactcggcca
atttttgtat ttttagtgga gacagggttt 139800taccatgttg gtcaggctgg
tctcgaactc ctgacctcaa gtgatctgcc caccttggcc 139860tcccaaagtg
ctgtgattat aggcgtgagc caccacaccc ggcctgaaat ttaaatcaga
139920aataaaattt tgatcccaac agtgatgcca ggcagcccag atctggggga
gagggtggcc 139980ttggccagct gggcctttct ctgtttccca agtcttgctg
cctctccctg ctgggctttg 140040cagcctgtgc atgtctctgt gcctttgacc
ttgtttatcc aaaggagagg atagaatgaa 140100gtcatgattc ctggagccct
gagaaggatg ctgtggagaa atttgccggt agaatctagc 140160tgagtgtgtt
gctgaggtgc cagcattgtg tgtggggagg ctgaccgctt ggcctgccta
140220ggcccaggat gctccatggc cgggcacaga ggccacttgg ctgtcaggtg
tcaggagcct 140280gcagagggca cacagagcct ggaccgcagg ggggtcctgc
tttctcacct ggcctccttc 140340agcatttctg tccctcagtc cttagcaagc
ccaggagctg ttgagtttgg caggtgccga 140400gtgctgttcc tgcctgtgta
gctgtggctc agtcctgtgg gggccccgct gtggcccgag 140460tgcagtgatt
cgaggcgctg agtgttccct gactccttct ccaggagctg tgttcagact
140520ttcgcagctc ttggcttgga gctcctggag ggcttggcat tgccgaccaa
tgtggaggtc 140580gacagtgaga gaggaggaat gctagctttc ttgaccagtc
cattaaataa gtgggatatt 140640ggccaggcac ggcggctcac gccttaatcc
cagcactttg ggaggctgag gcgggtggat 140700cacgagctca ggagttcaag
accagcctgg ccaacatggt gaaaccccct ctatactaaa 140760aatacaaata
ttagctgggc gtggtggcag gcgcctgtaa tcctagctac ttgggaggct
140820gaggcaggag aacagcttga aaccggaagg tggagtttgc agtgagccaa
gattgcgcca 140880ctgcactcca acctgggcaa caagagcaaa actctatctc
aaaaaaaaaa aaaaaagtag 140940gatatctgtt tctgcttaga aaaatcagaa
ttttctaaat gccaggtgtt ctgaatacgt 141000aagtatggga gacgactcag
cctgtttcat ttttatgtaa aatcttcgcg tagccatgtg 141060gcactggacc
gagatgaaag caaagacatt tctccttaac tttgtttcta ggaatgttcc
141120ggagaatcac agcagctgcc actaggctgt tccgcagtga tggctgtggc
ggcagtttct 141180acaccctgga cagcttgaac ttgcgggctc gttccatgat
caccacccac ccggccctgg 141240tgctgctctg gtgtcagata ctgctgcttg
tcaaccacac cgactaccgc tggtgggcag 141300aagtgcagca gaccccgaag
taggttcata atgccccaca gcccagggcg ccagcccagc 141360accctgtcct
gagactccca gtaacctgag ctttggccac cgttaaagca ttttcatttt
141420ccattttttg tgagggcttg tgaaatttct gctgcatatt aatattcctt
tcatggacag 141480catattattg ggacaaacat gcggtccagc taaaggcatt
caaaatagca gttgctttct 141540aaatgcgatt ttctttggca ggttctttga
caccattgca tcttgtggga tatgcttgtc 141600atgctctgtg gctcctacta
agttctagtc cttaaattgg ttccatagcc agacatgttg 141660caatgtctta
acctcattat aaagtaaatg tggttctggt tatccttaga taatgaagta
141720acagtgtagc aaatttcaaa acctcttgga aatgttattt taccattcaa
aaaggcttac 141780taaggttctc gttatgggtg gccctctttt tgcaaaaggt
tttcaggctt aagctccatt 141840tctaggtgct ccaacactcc attatttgta
tatgtatgga aataaaagct gtgaccaccc 141900ccaaccctgg cccccgccca
gctgaatcct cagcacagta tttctggaag gctcaagatc 141960ccacgctggg
gaaaagaagt tctggagaca aaagagggca ggtgctgccg tgcctctctg
142020ctcagtatgg atactggacc ttgtgctgcc agggctccca gtagggccag
ttcatggcac 142080tcagctggaa agtccactgt tgggaggcat tcttaaccat
ccactctgtg ccgtatgtag 142140tggggtctgg tcattctgtt ggaggagaca
gaccagtgac gacatttgaa atgcttggtg 142200gatgtcttag gcctgttacg
atgactgagc actgtggggg caggagacag aaagtcagtg 142260tctcctagtt
ctgtgctgct ttaacgtgca tagaaatcag ctgcggattc agcagatcac
142320tccttttctg acagatgggc ctgcttactc tgatgttata tcagaaagct
ctgaatctgg 142380gaattgtgtc ccctgaattg gagtaacaga aatgcttaga
tgatgagtgt ttaaaagaaa 142440taaaccaaag gtaaatttag tttggaattc
agcaagcgtc ttcattcagc cctctgaggg 142500caaactacag ctttttgtaa
atgtaggtaa attctgtgac tgtttcgtga ccccctctga 142560tccagttttc
ctttataacc ttctgtattg ttccttctat tatcctgaaa taacattaat
142620agattaggct gggcgtggtg gctcatgcct ataatcccag caccttggga
agccaaggcg 142680ggcagatcac ctgaggccag gacttcgaga ccagcctggc
caacatgatg aaatgctgtc 142740tctactgaaa ataacaaaaa ttagccgagc
atggtgacag gtgcctgtag tccctgctac 142800tcagaaggct gaggcgggag
aatcgcttga acctaggagg aaaaggttgc agtgagctga 142860gatcgcgcca
ctgcactcta gcctgggtga cagagtgaga ctccatctca aaaaaaaaaa
142920aaaaaaaaaa aaattaatgg atcaatggat ttttaaccta ataattaaat
ttcaaaaaat 142980atcgttcttt aatggtaatg taaaggtaaa attaagataa
tatgtaacaa gcatgtgagt 143040gtctaaggtg tccccgtggt ggaaggaaaa
aataaatccc cataagtgtc caagatgccc 143100atagagagca gagctgttct
ggtttaaacc cctgctctta gcactgtgtt tttccagctg 143160tgggtggtgg
gggatgagta tctttttatt tccatgagat gagaaaaatg aattactaga
143220agtgtgaaat acaaaacaca gctgctcttt ttttagccat agactcagca
gccataaaat 143280tgctgtatcc agttgcagaa attcctgctg cttactcttg
accctctctc ggtttgtgtg 143340catctcctct caggctggct cccagatggg
agctggctcc aggcgacact gggtgctctg 143400ctccaggagg tccttatgtg
ggtcctgccc tagcctagcc cctctcttat ggactctgtc 143460actgtgggtt
tatgattcac tctcaatctg tcttacctct tggtgaactg ttagagtcct
143520gcctatactt tggcgcttgt gggtgtgttg tggtacacat gatgtgttgg
tcacttccca 143580gctcatcttg ttctgagtca ccctagattt gggacattca
ttcgccacca gtaccgggcg 143640gtgtatggcc tgagatttgg gggggcttgt
gctgctacaa attggggctg aatttgagtt 143700gacagtggac cttctttatg
tctactgctc atatttgaat tgcaaatact gcctcttctc 143760tttcagaggc
tcattaccct atagctgtat tattgcaaag tgcacaatta cagcttgagt
143820gtaagtcaca ctgcgctggc aggacggccc actgagaaag ggcacgtttc
ctgttcgtta 143880gttttcacat tgacacataa tttacaatac agtaaaatgt
acttttctat caactgtagt 143940cagtaacagc ccccctcccc caaccacatc
aagatataga ggagtgctgt cacttcaaac 144000agttccctct tcctctgcca
catcctgccc ctccccaggt ctaaccacca atccgtgctc 144060tgtccctctg
ttcagcccat tgcagaaggc catagaaata gaatctatag gctaggtgtg
144120gtggctcatg cctgtaatcc cagtattttg agaggctgaa gtgggaggat
gacttgaggc 144180tgggagttca agactagcct gggctgccta gcaagacccc
atctccagaa aaaaaaaatt 144240taaaaattac aatcacgtcc ctgtagttca
gctgcttggg aggctgaggc aggaggatca 144300cttgagctca ggagttagag
gttacagtga gctatgatcg tgccactgtg ctccagccta 144360ggtgacacag
caagacgttg tctctgggga aaaaagaaag aaacggaacc acgcggtgtg
144420cagccttctg agtctggccc ctttcggtga gcagtgtcta aagttctgtc
gcgtgttgcc 144480cacgcgtcgg tggctcgctc cttgcaactg ctgagcattg
tatggctagg ctgtagtttg 144540ttttcacttc accagttggg aaacagagaa
aaggcacttt ttaaaaagtt taaatctgta 144600gaattttggt ttttaccagt
tctcttctaa atcctgaggg attacaggaa aagttgttgt 144660atttcagaat
attcttagct tgatgtgacc tctgtccccg ttaaggccct ttgccgcaat
144720gggaaggacg tcgctcggtc agaccctgaa ggtcagaggg gcagtttggg
agtgtgtcaa 144780cattttaact gtatggacta gagccaagag tctcaaggtt
tataattccc acgtattcaa 144840aaagaaaaaa acaataaagt gagaagtcag
tgtagagtga aataacctgt gttagtgggg 144900aagaagtgtt tttaaacagg
atttccataa cgtataacat caacatgttt agagtggtga 144960tgtttcattg
ggaaacgaac agtaaaacat gaaagcaggg aggttttcat tctggcagtt
145020ggcaactttc acggcagatg gagaatttca aaagcaattg ctcaattatc
aaacatagcc 145080agtgtgagtt ctgaaataaa ggtgctgatt gaatgtgcag
ctttatggtg gattttgcta 145140ttcaggcaag cattttaatt ttctgcctgt
taaattctgt tttctttagt ttttcatatg 145200tggtttattg tagcttagga
atagataact gagagtatat attacacata caacattctg 145260atatggcaat
atttaaaaca acttgtctgt tttagaacta gaattaaaca taatcatctt
145320cagtattttg caaataagct cactgccatc cagaaacatt gtcaatgcat
ctgttgctcc 145380ttctagaaga cacagtctgt ccagcacaaa gttacttagt
ccccagatgt ctggagaaga 145440ggaggattct gacttggcag ccaaacttgg
aatgtgcaat agagaaatag tacgaagagg 145500ggctctcatt ctcttctgtg
attatgtcgt aagtttgaaa tgcctgtaaa cggggttgag 145560ggaggtgggg
accaggagaa catcctgtgt agatgacact tgcatggacc ctctggaacc
145620cagaccgccc ggtgtcctgc caagctccat cgaaactaaa tctagaatga
atgtttactt 145680ctgctgtgac atataattgg agaccaggcc tggccttcca
gtcactggat tctaagttgg 145740actgtgagag tttttgcagc tgactcattt
atcaaatgcc cggctattgg ctcacgccta 145800catgatgctg ggtatgtttg
ttaatttgag ggaagcaatg gaataataat aactaatgat 145860ttaaaaaaca
aagtaagtgc attgactgta gtggggttct gattttaaat ttttttaaaa
145920attaatacca ggagcagtgg cttatgccta aattccagca actcgagagg
ctgaggtagg 145980aagatcactt gagcccagga gtttgagaca agcctgggct
atggtgtgag acacccatct 146040ctaaaaaaat aaaaaataaa aaattatcca
agtgtggtgg ctcgtgcctg taatcacagc 146100tctttgagaa gctgagggcg
gaggatggct tgagcctggg agttcgagac cagcctggca 146160acacagagaa
accctgcctc taccaaaaaa agaaagagag gaagaaagaa aaattagcct
146220ggcgtggtgg tgcatgcctg tggtcccagc cacctgagag actgagaagg
gaggattgct 146280tgagcccaga agtttgaggc tgcagtgagc tgtgactgtg
tcactgcact ccggcctggg 146340tgacaaggcg agacccctgc tctaaaataa
tttttttaag ttaatttgta gaaaaggtgt 146400tagatgttct ttgtcacatt
ttatgatgga ttcctgttta aatgccgttc tctttaaaga 146460aaaaaaaata
acttgtggga gtttttaacc ataaaactag catcacatat ttaccatgga
146520gaatttacaa aaaaacaaat aaacggagga aaataaaacc tcctgtaatc
atactactca 146580gagataactt gctgttagat tttggtctag atttaatact
ttttctatat ttatattaaa 146640aatatttaaa acatatgcat ttctttgtca
caaacatggt atcttataga tactactgtc 146700acatagcaaa acagtgttaa
atattctgaa tcagaaaagg aagccgactc tccaactgaa 146760agaggtgtta
tcctagagac tttttctggt gatgacaatt tattaatagt cactttttgc
146820tttactttct ctattgaagt agtttttcta ttttgttcta cttttaagga
taatataatt 146880tataatgctg tttttcacag aaatataaga aaaaagatac
taattttata agttaataaa 146940gtttgatcat cccaaatcca aaaatctgaa
atccaaaatg ctccaaattc tgaagctttt 147000tgagtgctga cattatgttc
aaaggaaatg ttcattggaa ggtttcagat tttcggattt 147060agggagctca
acaaataagt ataatgcaca tatttcaaaa cctgaaaaaa atcctaaatt
147120cagaatactt ctgatcccaa acatttcaga taagggttat tcaacctgta
ctgtcagatg 147180atcccaaatg aaaaatatta atcgttaacc aaatatcaag
gaattgatca cattttacag 147240tttctgccta ggattatgaa tcaagatgaa
aaggctctgc atgtttaaaa atatatattt 147300ttattttctt ataaatctta
aatatctaca cttaagattt atttgatatg tgggatccat 147360tcatattttg
gattcaacag ttctgtcaaa actgtggcag tgatagggga ttcttttttt
147420cccactgaac tatcacaaaa ttggaaaaag agtaattgga gaaccccact
ggcttagccg 147480gcccgaagcc cgggagaggg caggcagtgc tgtggatggg
gtcatcccag cgcaacgctg 147540cccctgctac ctgcggatct cgctgaggcc
tgcctttgtc ctttgaccct tggccatttg 147600ttagtgtctc tgagagctgg
actgctgtac cctacttccc cagggggcct aacttcacac 147660agcctctgcc
gcagtgcgtg gttggaggtg acggccttgg taaatcgagt ttcctacctc
147720ctcaattatt tgtgctcata cactgtatat ttttagtgag gtttatattt
gggatgtgtt 147780ttctccttct taccctttct ggcctttcta tggcattaat
acctggtctc ttcttgtgta 147840cttgaaaatg aatctctcat catatttttc
cttagtgtca gaacctccat gactccgagc 147900acttaacgtg gctcattgta
aatcacattc aagatctgat cagcctttcc cacgagcctc 147960cagtacagga
cttcatcagt gccgttcatc ggaactctgc tgccagcggc ctgttcatcc
148020aggcaattca gtctcgttgt gaaaaccttt caactgtacg tcttcatcct
gccgactatt 148080gccagttgca gttttccctg ccttaaaaat ggagtattga
aatttttaac tttaatttct 148140gatttgcaaa atagtcatct tttgttcttt
tccttcttgc tgttagccaa ccatgctgaa 148200gaaaactctt cagtgcttgg
aggggatcca tctcagccag tcgggagctg tgctcacgct 148260gtatgtggac
aggcttctgt gcaccccttt ccgtgtgctg gctcgcatgg tcgacatcct
148320tgcttgtcgc cgggtagaaa tgcttctggc tgcaaattta caggtattgg
gaagagaaac 148380cctgatattg atttatattg aaaatttagc aggccaagca
aaacaggtgg ctggcttttt 148440cctccgtaag tatggtcttg acatggtcac
cgatagaaac atggaaacat ctgcaaactt 148500gccgttactc gtgtgtccga
tctgactgtt tcttgtattt ttttctagtc tgcccttact 148560aggatgaact
gtacacatca gttcatcctt tttaaatgag catgaggtta ttttgggttg
148620ttaggtgtta caaacacact aatgtgtttt tgtctattag agcagcatgg
cccagttgcc 148680aatggaagaa ctcaacagaa tccaggaata ccttcagagc
agcgggctcg ctcagaggta 148740atgctggaaa cacaggtcgt ccttgtgtta
ggacaaccca ggatataaag gatatagatt 148800tgtacgggaa taaattcaca
ggacaagaaa tcgatgtgcc ttataggtgg gtttactgca 148860gaagtgccat
aatagaacct tcctactttt aaaacaacca gatctcactt tctaaagagt
148920aaaggatgac cggcaggatc acgtctgtga cgtgagtgga ggcagtttgc
actcctggtg 148980gctgtttgag aggtagcatt tagaatgcct gtattcactg
tcctgtgatg agtgggaaaa 149040taggttatca ggtttatctt agcaaaatca
aagcatgtca tctaattgct aaacaagagt 149100tggcaaatct gagagacatt
actcaatcct tggcatgcag gacttacatc tgcatcctgt 149160tgccatttta
tgtcttcaaa gcatttaatc atttagttgt gtttgcaaag tctttgagaa
149220gcctttgtca gaaatcccta catctcctat gtgagtgtat ttccatgact
gcagaataag 149280ttaaactttt acctttttcc ttcccttgcg gggcggggtg
gggggcaggg attgtgtgtg 149340tgagagggag agagagacag cagagaagga
gaatataatt atcatgctgt gtactttgag 149400ctgaaactgc aaaaaaggaa
aaacacacaa aaattattat gcttttcagt ctttagagta 149460ccttgtctat
tatgcttttc agtctttaga gtaccttgtt gatggtgttt ttaaatggga
149520ttgggcacaa ttaggtggac agtttgggat gatttttcag tctgtagggc
caagctcttt 149580tgtaatttgc attatgaagt tgtcactctc atagcagatg
gcgggagata aactattatt 149640actttttgac cctagactta gtcttcagtc
cagatgaggg agattaaaag attataaata 149700tcttgtgcca gatgaggtga
ttttattttg aaatgaccat gaattcctat cagttgtctt 149760actgggatat
ttgatagtgg aatttgtgca tttgagtctt agatgatctg ttttacattt
149820attaagaaag cctttattag cttttatact gtgtattgcc tgttgcagtg
tttgagtata 149880aatgaaattt ctggaaaata ttaatggagt acaaactgtg
atacttaaaa gtaaactagg 149940gcctgcattt gtatcatgac ctgtttgagt
attgatgaga agatagctgt gaagaaaaag 150000gtttaaacaa gtgtattttc
ctttaagaag ccactaatag tgcatctcct tagagtgtat 150060atttctagaa
tcctagtgtg cagagtttag actaagacta aaaaaaaaaa aaaacaaatt
150120atactgtaat ttcattttta tttgtatttt agacaccaaa ggctctattc
cctgctggac 150180aggtttcgtc tctccaccat gcaagactca cttagtccct
ctcctccagt ctcttcccac 150240ccgctggacg gggatgggca cgtgtcactg
gaaacagtga gtccggacaa agtaagtgtc 150300cagcgtgtct gcatgggagg
cacagggcgc tgagtgcctc tgtcacctgt ggcagataca 150360gagagtgcag
aggaggtgcc gtggacccaa ggagttctgg cgctcggctc ggctcagtga
150420agctgtggtt agagacgtgg ggggccatca aggtctgagg gagccaagca
gtgctgatgt 150480gggacccttt tggtaggagt gtggggtgag tagttagtgg
gtgaatcaag gaatagtcgg 150540ccgtggcctg caggcccctg actgcacagg
ccttcaagca
catgtcaatg ccgttagcct 150600ccctccatct cctcatacct tctggccacc
tgtgagttgc actgccactg ccagccattc 150660tggtatgttg tcagcacctc
cactgctcat acctcatggt tagggaccac ctggagcctt 150720ggtagagcct
tggtagagcc ttggtactct actttcctgg acaaagttca gcttatgaat
150780atgaatttag atttcaaaaa ccagcagccc aagtataaga aagcgaaggt
tcagtcctgc 150840cttcttaggc tctattcgct aagcacctgc cctgccctgg
ttgctgggga gagatgagta 150900aagcagacaa cccaggagag gatggcaaag
gggccgctaa cccttagtgg tttagctata 150960tttggaaggc ctattggaag
ttcaccaggt gaagggggag gctgtgaggg tgcccaggca 151020ggtaacagaa
gtccaaaggg gaaaacctgt ggtgtggtga gccgtatagc cacagcctgc
151080cggccggcag ccctctcagc ctagtgcggt gttcccaagc actggcctag
gcctgtagct 151140ccagggatgt gaagtcccct tgaacgccgc ccatcatgtt
ccccttatcc atttttttct 151200tcccaggact ggtacgttca tcttgtcaaa
tcccagtgtt ggaccaggtc agattctgca 151260ctgctggaag gtgcagagct
ggtgaatcgg attcctgctg aagatatgaa tgccttcatg 151320atgaactcgg
tacgggggga gcagtggagg caaggaatcc tcagcttttc ttgtgacttc
151380caagtgggat ttgtctcatc atcatgtgac ccacttgttg acaacacatg
ttggggactc 151440cagtctgggc agggacggga tgtcggagag actccactct
gaatggggcc gggaagtggg 151500gaggactcca tttcagatgg ggtcgggaca
tgggggttat gctgatcgag acagaaaagc 151560acattgtttc agccacatta
gaatccacgg aggtgttgtt ttgaaatcca gctggcccca 151620aggctgggtg
tatggtttgg gatgagaact atctggcctc cactggagga acaaacacag
151680gatgttatca tctaagctcc atggccaaga cagaatggaa gtcaaggttg
cgtatttgcc 151740gtagacttca acacagtgtc gtaatgcgtg acgtcaataa
cttgtttcta gtgtcttgga 151800agttgatctt tagtcgtaaa agagaccctt
ggatgcagcg agatttcctc tactcacacc 151860tctgttagat gtagtgaggt
tcttcacccc ccaaccccag atgtcagagg gcaccctgcg 151920cagagctagg
aggccatgca aagccttggt gtccctgtcc ctcacccgtg ggcaggtcct
151980gtgagcagtg ggggggccac ctcttgggta tggtgcagcc atggcccaag
cagggcttct 152040tctcagacct actaggacgg gagaaacctc ctggtgcttt
agccctgcgt tgatatgcag 152100caaatgggag ggaagtgggc acctgggagg
acaaatgcct gtagaggccg ggagtgacgg 152160caggtgttca tgaaaagaga
ccttgtgggg agggcaacac aacagtgtgt tctgatgtac 152220tgaagagctc
aactgaaaac aacaggagaa ttagcccaaa atccatttac taaaattgtt
152280tatctttttt tttttttttg agacaaagtc tcgctgttgt cccccaggct
ggagtgcaat 152340ggcgctatct tggctcactg caacctccgc ctcctgggtt
catacgattc tcctgcctca 152400gcctcccaaa tagctggtat taacaggcat
gcaccaccac gcccggctaa tttttgtatt 152460tttagtagag acgggatttc
accatgttgg ccaggctggt ctcaaactcc tgacctcagg 152520tgatccgccc
acctcggcct cccaaagtgc tgggattata ggcctgagcc accacgcccg
152580gcctaaaatt gtttatctta agattcatgc agtgaaagct aacttactga
gtgataaatt 152640tgcttagtga tctgtttatt aggttttcca aatttgctaa
ttgggctttg aacagctgta 152700aaagttctga ctgtaaaaga aagcttcaac
ttttggcatt catgatgctt ttctgagtat 152760taaactaaga tagatgtttt
acctgaagga tcggccacca atctttaaat ggctaaacaa 152820aagggttgct
aaaacataat ccaaattgac ataagaaata ccatttttcc aaccaaaatt
152880ttggcattca tatggctact tttacgtatt tcagctgcat ttgaacatct
ttttcaaact 152940ttagggtggt tggtgtatca ctgaggtctt ggatgacact
ttagctttga ttttgttttt 153000atgaattaaa attgtcatac caaaattttt
atttcaagca aatccaagag cataaaaaat 153060taaaatatta cttaaaatac
taagagagaa cagatatata ttttactaag catatgttga 153120atgaaattgt
tcaaatattt ataacaggca tagagtagaa ttttcttaaa aatatttttg
153180atggtatacc aatttgtatt ttctcagaaa catttgcctt attctttttt
ctgttgtgtt 153240tttcttacct gattgaaagc tcataatctg ttgttattgt
ttgttaacct ttaatgctct 153300gatttcagga gttcaaccta agcctgctag
ctccatgctt aagcctaggg atgagtgaaa 153360tttctggtgg ccagaagagt
gccctttttg aagcagcccg tgaggtgact ctggcccgtg 153420tgagcggcac
cgtgcagcag ctccctgctg tccatcatgt cttccagccc gagctgcctg
153480cagagccggc ggcctactgg agcaagttga atgatctgtt tggtaattaa
aattaaaatt 153540tatcttattt ttaaaaagca ttccagggcc agtatagtac
tttgcaccaa gtaaatgtac 153600aataaaggca gtggatctaa tacattgaaa
gcgtttacag aggtagctaa agagcagcac 153660gggtgtcctc ggctcagaat
ttcttcctgt gtgtttgcca ctttgccatt cattgacatg 153720gtcatggaca
tagggctcta agcccttgag gaaggctggg ccagacctca ggggagatgc
153780agccccaaac cacgtgcagt cctgtggacg gatgtgtaga tgtgccactg
aggaacaatg 153840tcttgagctt tcatcagatt ctcagagaat tgcttgactg
cctttcgaag ttgatgcatc 153900tgtgctcacg tttgcaccca cccacgaggt
ccttctgttt caggggatgc tgcactgtat 153960cagtccctgc ccactctggc
ccgggccctg gcacagtacc tggtggtggt ctccaaactg 154020cccagtcatt
tgcaccttcc tcctgagaaa gagaaggaca ttgtgaaatt cgtggtggca
154080acccttgagg taagaggcag ctcgggagct cagtgttgct gtggggaggg
ggcatggggc 154140tgacactgaa gagggtaaag cagttttatt tgaaaagcaa
gatctctgac cagtccagtc 154200acttttccat ctcagcctgg cagtaagtct
tgtcaccgtc aagttattgt agccatcctt 154260caccctcacc tcgccactcc
tcatggtggc ctgtgaggtc agccaggtcc ccttctcatc 154320tgcacctacc
atgttaggtg gatcctaatt ttagagacat gaaaaataat catctggaag
154380tactttatgt cttaagttgg cctggacatg tcagccaagg aatacttact
tggtttgtgt 154440tagtgcttgt aattcgcccc cagaatgtgt acacgttctg
gatgcattaa agtctggcct 154500gtatccttaa agggccatcg ctgtgctgcc
tgccctcagc aaggacacac tttgcagacc 154560cacagaggct ccgcctccac
ctcacaccaa agaaagggag gagtccaaag ggcatcagtg 154620ccattactca
caaaatgata aatacaccct tattctgaac cacgtggagt catatggttt
154680gtgatccctg tccttcaggt ttcagcttag tggggaagtg ggaaagtcag
cgtgtgatca 154740cagcacaggg tgattgctgc tgattatatt atgtgcctgc
tgtatgcagg atgaaatact 154800ttatatgcgt catcttattt gactctcaca
accccctgtg agataggctc tgttactccc 154860atttgacagg tgaggaaagc
aaggcttaga gaatttcagt gacttgccca ggtcctctga 154920gctaggaagt
agccattctg gcatttgaac ccaaggcctg ctatccctag aacccacgct
154980ctcaaattca acctatgaca gaggcaagcc ctggtgctgt gggagcccca
aggaagagcc 155040tctggcctgg tggccacgta gcccaggaga gatttctaca
ggagcccaca gcgctgaagg 155100agagagaggc agcagagtaa gggggctttg
tggcagagag gggactggca ctttggggaa 155160taggtgggtc aggactgaat
gtaatggagc catgtcagag ctgtccttct ggaagggcaa 155220gggcacctgg
acgcgctgcc cctcagtgct ttggacggtt ccacaactgt gattcacacg
155280gcttccccaa acgaaggtac acgagtgggc attctgtgac tcggtacttc
cctttaggcc 155340ctgtcctggc atttgatcca tgagcagatc ccgctgagtc
tggatctcca ggcagggctg 155400gactgctgct gcctggccct gcagctgcct
ggcctctgga gcgtggtctc ctccacagag 155460tttgtgaccc acgcctgctc
cctcatctac tgtgtgcact tcatcctgga ggccggtgag 155520tccccgtcca
tgaacggtgg gttcctatca tagttcctgt ctgcttcacc atgtttttat
155580tttgtgctgc ctgtttgcca ggtactaagc taggaattgg ggatggagag
gtagataaaa 155640tatgcatcag gaagggctgg gccccatctc ttactctcca
atatattgga gtctacactg 155700gaatttaact ggaatttgct tttttagtca
ttttatttag attttgaagt ttcagctttc 155760atcaaaaata cctctaaact
ttatgtctct gtgatctttg gtcttagctg ttttatgtat 155820ttagtcttat
atgatcataa gattaataac attacattca gaagattatt tgttttctgt
155880cagagttaaa atgtttgttt ttatactgca ttgtaatatt aacgtactgt
aaaataaaag 155940tggcttgttc ttttcaagga acagtatcct caacaagggt
cattagccac aatttttaaa 156000aaattggacg tcatagttta catgttagag
ggcgttttga agctttgtat ttttaaatta 156060aatgttatag agtgatgttt
tcatgtttca taattgtttt catctgtgca tttgtagcca 156120acttgaaaac
aaagatccag ggattactac ttaaaagcca gacttcttgg aggttatagt
156180gatgattttg atagtatctt gagccgtctc ataataacct cagggtgaga
gatggccaac 156240aggagacagt cgagggactt agaaatctga atgaaatctg
aagttcaaat cttcagacat 156300ataccactaa ccaagagatt ggtacctcag
tctagtattg tctgtttgtc taaaattggt 156360tctaaggaat ctaggctagt
ctgtctatcc ctttcaactt ttgtgaggct gcacaaatgt 156420aaaatgttga
ataaaaagca ctgatggaag tgtgtagaaa ttcttctctt tgttctgttg
156480taattttagt tgcagtgcag cctggagagc agcttcttag tccagaaaga
aggacaaata 156540ccccaaaagc catcagcgag gaggaggagg aagtagatcc
aaacacacag agtaagtctc 156600aggacccatt tttttcttac atgttgttcc
tccaggactt aaaaatcatt cacagagacg 156660tgcaccgcgg tgagtgtgga
ctcctggaag cgcaccgtag ctccgctgtg tcctgctgct 156720cctccctagc
tgtcagggag gctgtagtcc attgctttgc cagctctttt gtttccgagt
156780gaacacctta tccgtacaca tgcggctgtc tctgacccta cagaccagct
gggatgccac 156840tgggggagcg ctcccttccc cccgcacttc ccacactctg
cagttattct gagatccttg 156900agggcaggga acaggtttgt cttctttgtg
ttctcagaaa ttaatgctcg gcctctggtc 156960agcaagcaac aaccttttgt
tgagtgataa tgaataaata aatgtttccc acatgagtat 157020tcagtaacct
cagtgtcagg ttcagccatc tgttttggtg gatatttaaa agaaaattcc
157080gcttttccta cagaaaaaaa aaaaaatcca aatcccagtg atttaagcca
gttatagact 157140tagacatata ctacggcttt tcatgcactt tcctcccaat
tctagagtag gtattttact 157200aggaaaatgg tggcagtgcc tgttgggagg
aagattcttt ggccaagtgt cttttgttct 157260tgccagggcc cctaggctgc
tggggtgctt cagcttcttt agcccagtgt ctggtgggga 157320atggcccctg
ttgcctgtcc cacagaggtg ggggtgcctc acctggagcc tgtccacaca
157380ttttacacag cacgcttacc tggagcatca ggcatctttt ccatgctctg
tggctcagga 157440aacacgcctt ttcaatcatg agtgcaccag tgcttttggg
ctttttctcc ccgcttttgt 157500gcaatcctgg ttgtggatgg agttttcctg
tctttagtct tctgcatagt acttttctct 157560tctggttccc ggttcaaggt
tttgtaatta gagaatgacc cagaagcaat ggcattttaa 157620tgcacagcca
aggacttctc tgaatttgta tctcaaacct ctgtgggtcc ttcaggcttc
157680agtttgtgat ttcatgattt cttgttgcta cctaaggaat atgaaaacac
ccacctccct 157740actctgcatc ttccagccga gtggcacctc aggctgtgga
tcctgtgctt ctgtggtgag 157800gataagaata gtgccaaccg tgtggattga
aatcaatcag ttaatccctc catgtaaagc 157860acctggaacg gatgacagtc
ttgttatgaa tactcaacaa atgctatcat gatttttagt 157920tagatttcca
ttgctttaaa acagttgaga catcttggcg gtttgagtta gagcaacggg
157980ccctgaagtg ggttctgttt gggtgaagat gattatgctt attccccatg
gccctcttta 158040ggcaagagtg ggaagctttc tttgtttttt taatcacctc
gataggacgt tacttcttaa 158100aggtcatcca ataaatatta ataggccggg
cgcggtggct cacgcctgta atcccagcac 158160tttgggaggc cgaggcgggc
ggatcacgag gtcaggagat cgagaccatc ccagctaaaa 158220cggtgaaacc
ccgtctctac taaaaataca aaaaattagc cgggcgtagt ggcgggcgcc
158280tgtagtccca gctacttggg aggctgaggc aggagaatgg cgtgaacccg
ggaggcggag 158340cttgcagtga gccgagatcc cgccactgca ctccagcctg
ggcgacagag caagactccg 158400tctcaaaaaa aaaaaaaaat attaataaag
ccaactcgtt agcgtggggc ttaattgctt 158460aagtccaatg agaagtcctt
ctctatccta ggaagttgcc caaactgtag aatctcgtgg 158520cctgtgggta
atagccacgt aatacacact cactgcctca acaaatcata ttttagtagg
158580tatgatattc tagactcaag acaccattct gtggatcttc ccaagggtgt
gaagtgtcca 158640cagcgtctgc cttgggagtt tccatgccca ccagaaccat
gccccaagcc cctcaagcac 158700tctgacctag gaaagccagt gaagcaagga
tgacaacatg gccctttgat actagctgag 158760ggacagacac aggtcctggg
agaccagaga aagacgaggg gcagaggagg tgtcctaaag 158820gaagtctgag
gctgaggagc cacaggatgg cttccagctg tcacaggctg ctgctggcct
158880tatcacagag agtgggccag agggctggga accaaggcca gagctcaggt
tcaggaccat 158940tccagcaatc ccagcagaaa atggggagaa ttgtatggta
taggcggata tgaaggtaga 159000atctgcaggc cttcagtggc caactcagag
tctaagtgga ttccacagtt acagcttgag 159060cagctggttg taggtcatgc
tttctacact gggcatatag gatgtgtttt ttaaaaagtc 159120ctctcttaac
cgttgcttgt ttagatccta agtatatcac tgcagcctgt gagatggtgg
159180cagaaatggt ggagtctctg cagtcggtgt tggccttggg tcataaaagg
aatagcggcg 159240tgccggcgtt tctcacgcca ttgctaagga acatcatcat
cagcctggcc cgcctgcccc 159300ttgtcaacag ctacacacgt gtgcccccac
tggtgagtct gctcgttcct tgcagaagac 159360caagtacggt gaaaggcacc
ggtaggccct gggctgggca cacgtgagag ggcgggacag 159420aatccccgca
gcccagaggc tgcctgctgt ggttctggtg cccactgtgg ttctggtgcc
159480aggctgcttt cctcaggcac cacgtgtgga ggtcgctagt agaaatactg
ggttttctaa 159540aatgaactga ggccctacat ccctaagaga ttagtgttag
acctgattct agagcaacta 159600gaccactttg cttaatagca gaccagaaac
cacaccccct cgagtgagtg agattttcct 159660ttggagataa ttcatgtttt
tctacacagt tttgcagttg tcttcagaat tggtttaaag 159720taggtgttat
tgccaggcgc agtagctcat gcctgtaatc ccagcacttt gggaagccaa
159780ggtgggcgga tcacttgagg tcaggatttc gagaccagcc tggccaacat
ggtgaaaccc 159840catctctact aaaaatataa aaattagcca ggtgtggtgg
tgtacgcctg taatcccagc 159900tactcaggag actgagacag gagaatcgct
tgaacccagg aggcgaaggt tgcagtaagc 159960cgagatcgcg ccactgcact
ctagcctggg caacagagca agactccgtc tcaaaaaaaa 160020aaaaggtagg
tgttattgat cagaaccctt gtttcagata acatgaggag cttagcttga
160080ggagagtgag ggttgatgga gggggactga cttctgccca gtgaaatggc
atcatctccc 160140accagcccgc tgaaataaga tgatggggcc tgttccttag
ggcctgcagc atcctcaggc 160200aggaaagaaa ggccgacctg gcagggtgtg
agccagcagg tgtaggtcag ggagaatgga 160260gccaggtccc agggaagagg
cttgtggctg cctgagaagg gtgcgtgcct gcctgtgtgt 160320gtgtgtgcac
gtgtgtgtat gtatgctgga gagtctaggg aggcttgctc caaggacgca
160380gtattgtttg atcctgagag ataaggattc tgccgcaggg aatgaaggta
ttccagatgg 160440cgggcttatt ccgaagaaga ggccagtgcc tggcggtgct
ggaagcagtt gcagaacagg 160500gagttgtagg ctttcctggg aagagagcag
caggggtgct ggagaagcag gccacacttg 160560ctgcatgggg ttgctctcgg
ccccactctt ggtgcacagc gagtcactgt gggttcatta 160620gcatctggtt
atgagacagt aactgctcct ttggaggggc tcgtggagac catgcaggag
160680ggcacggtct tgaggtcatg ccgtccagag cacacctgag gataggccag
gacgggctgc 160740acgctgtagg taaaattcct ccagcaagct cttcactggc
attgaggagt tccctgagtg 160800cggtcatctg gaaggcagct gtaacaggca
ctgcagtctc tccctgggtg ggtaccagag 160860aggagcatag gggagcataa
ccgatttaaa gagagggctt tcctgtggtg aggtaagaga 160920ttagctggtc
attatcatag agccccctct gcctttgtgc agatgggctg tgggaatcct
160980ggggttccgt tgggtccttt gtcacctcac tgaaggcatg taagctgagc
tggccagacc 161040gtgagctgat cctgccactt gaacagcatc aagcctgcct
ctggattctt ctgtgcatgg 161100cacttgtctg agcacctcac gcacagagaa
ctggacttca gagtttacag aaataagctg 161160tatggttcat tttcatgcct
gcttgccaat aaacatatct gagctgaacc tcattgaacg 161220cctgccttta
ttctagcaca gcacctgctg tttgtgggcg aggggtgctg tctctaactc
161280ctgcctgctt ctcccagcac tccctgagtg gggtgtgcca gcagcctcag
gatgaggaca 161340ggaagtggga gggcagagca gatttgggag ggccacttga
tggggaagga agtcccagga 161400agcagttgga gctgttttct gggggagaag
gtgccagctc tgggacagtg ttggggtagt 161460gaggagggag cccagtggag
agaagtcggg cttcctgctt cctcacagta tgtctgtcct 161520gactcaactc
ggatgatgtc acttcctttt catcttctca ggtgtggaag cttggatggt
161580cacccaaacc gggaggggat tttggcacag cattccctga gatccccgtg
gagttcctcc 161640aggaaaagga agtctttaag gagttcatct accgcatcaa
cacactaggt actcttgggg 161700cctctccttc aggtcaccat tgtcggacat
ctaccgggag gaaatccaga gcccccagta 161760ctgggatctt ctcatttgac
tccagaaaag atttaagcat gataataata caaacctatg 161820tgaatacatt
ttgcagtgtt ggcaaaactc cttttatact gagaaaatag atcccagttc
161880ctgtgttttg tggcttgaat cccagctttg tgtattccgg gcttgtttga
agtcaggaaa 161940ggttcatgtg tagtggacaa cgtgagacca aattctgcct
tagattttgc atttaggcta 162000aacagtggca gcacttgtct cagaatgttt
tcttgtgttc accagtctga tcctgttgtg 162060tctcagtggt ccattttctc
atatgggaac aagcagacgg gagcagatgg agtcaggttt 162120cttggcactc
gccttcccca gagcctagag gcagcatggg gagaaagcag gcttggggct
162180cagacagtcc tggtctgctt ccagccctcc tacctgagca gcgcagggca
agtccgtcta 162240acctctagag accctcagtt ttgtcatatg taaaatgggg
gtcgtgtcta tttcatagaa 162300ttgttgcaga tttagaaatt acatttctaa
acaaatgtta ccccttattt ctaaataagt 162360gtctaaatga ataagtcacc
acttttgccc ctatttgatg gcaagaggtg tgatcttgtg 162420gtgggactgt
aatcagtcag ttctcagtga ctgtgccctg ctgtggtgtt tcctggaatg
162480ttcctgtctt gtcctagaaa gtctggcagg ggcaccctga ctccactgtc
cagtcctctc 162540cccagtccct cgggcttctg cagatttgag gcttgtttgg
atcccagaag gttgtggcag 162600gagacacctt gcctctactt tcccctttat
aattcaatgt ccaaagagag ccctgagcag 162660gtacctcacg ccagctgcct
cacggagctc ctcctcttcc tggctgtgag gatcggtatc 162720agtggcctcc
tgctctctcc cccttgccta acacgagcac ctttgcttac ttgggtgccc
162780ttgctcttga actgcccatc ggacgtgcgt gacccaagac tgtgccgcag
tccttgcctt 162840gtctgtgctc attttctttg ttcatttttt tccctgtaac
gtaaattgtt atatttgtct 162900gtatctgtgt ctgaatcagt cctgcacgct
ctccttctct ctgtctcttg ttctttcttt 162960accccgttta tcacggggac
cccgatgtcc attgctctag ttctcctgtc ctaagcaccc 163020catcccgtct
ctctggcctt accacaagtg gcgtggctgc ctcagacatc atgatgggga
163080catgaagcac agctgtcaga aacaactgtt cgttagatac actcgaatgc
agctcatcaa 163140tagggatgga gggtctgtcg gatgtatttt cactgaatcc
ccgttcctac cttgatacac 163200tctttttaat ctattcttct agacaggtca
gaggaaccat tactttgact tttaaatttt 163260tagcagcttt attgaggtag
aattcacata ctacagattt cacccactct aagcggacag 163320cttggtggcc
attagtttta tccacagagt tgtgcagcca gctgcacagt ctcagggctg
163380gactccaggg aagattttag cccatttagt gagtggggca gaagtggccc
tggccctgca 163440cgaggttgcc tgcatgggcg tccctgccct gtccctgtgt
ctgctccact gggggttgac 163500caggctgcca gggccgactt gggcctgtgc
cacctgcctc tcatgtgtct cggacagtgc 163560agccgatgtc tatacttcgg
tttcctcaat gatgaaatgg aggggatagt gttccccgca 163620tcatagaact
gtgtgaggtt taagggactc actgcccttg gcgtggagcc ttctccaggg
163680gccgtgctgt gtcggcgtag ctgtcagctc tccgttacag gcttgagaag
ggttgacact 163740ctctcatgta acatttatat ttctaggctg gaccagtcgt
actcagtttg aagaaacttg 163800ggccaccctc cttggtgtcc tggtgacgca
gcccctcgtg atggagcagg aggagagccc 163860accagaagta aggccacacc
ctgtgctggt tggcacatgg gcagttatgg ccgcttgcag 163920gcctttggtg
gggaataaaa taaggcagca agctggtgtt ctttttttct cttaccttat
163980ttttgaaaga gtagctgaat ggtgtcttga ctgatattcc agagcaggga
caaagcctgc 164040tgaggtctgg gggctgcgat taccaatggc tggaatgcat
tttattacgg tgcattccat 164100gttaaggatc aatacgattg tgccctttct
ggaaaatatc ttttagttta tcaatattca 164160gaggagtgta ggttgaatta
aaatgaaaag gcactttata aaggccatga gtagtacctg 164220gtttcatttt
tctaatgtct tgcagagatt ttatcaggct tcttgaagtg ttcacgtaca
164280ttacgctaac acgatattaa taataactgt gctctggtac agcggagcca
gcagaatggg 164340aagttgtgga atgcaggccc ttgattctga tagaaggtgt
ggtttgaact cacagaaatg 164400acagtttgga gggtagacat atgtcacaag
tcatcaagat tgtctttaaa ttcatgcata 164460gaagctaaca gggtgtcata
agcaaggcct gtaaaatgta tgagggaatt caaagataat 164520ttattaaaaa
gtaattcatg tttggagttt tgtgcccaaa ggagtccttg atttgaaaaa
164580tgggcttttg cccatcagat tgtttcaggg cccgtgtgtg cggaggccct
gccttgtgcc 164640ccgtgagctc agcctgacag aaatcctttg gtagcactta
aggctcctct tcctcccatt 164700gaggcaggga agactctggg ttctgcaggc
agaggtggtt gtgggtgtct tgctgctctt 164760gttgacatgt gggctctcct
tccaggaaga cacagagagg acccagatca acgtcctggc 164820cgtgcaggcc
atcacctcac tggtgctcag tgcaatgact gtgcctgtgg ccggcaaccc
164880agctgtaagc tgcttggagc agcagccccg gaacaagcct ctgaaagctc
tcgacaccag 164940gtttgcttga gttcccacgt gtctctggga catagcaggt
gctggggaca gtgggttccc 165000cgctgaagcg tccagcagct tcaaccaggc
cgttttcctt cattgctaga attgaaaaca 165060ccgtccgtgt ggcctgtgca
ggagatgcag acccaaaggt ggcctcctgg tcagtgagaa 165120gctggaaacg
tgacaggaac tgacgtgggg ttattgagca tttaggggaa gacgttagca
165180gagcaggaat gagcaggcaa ctagtagaac acccacttaa gggctcacgg
acaggtgctc 165240acttaggaag tgagtttcat ttggtattac accaggttcc
tttaggcaaa gcggagggaa 165300agttctggtg tttttcactt gtaagatttt
gaaggaaaca aaacactctt tacctttttt 165360ctaaaatgta ggtttgggag
gaagctgagc attatcagag ggattgtgga gcaagagatt 165420caagcaatgg
tttcaaagag agagaatatt gccacccatc atttatatca ggcatgggat
165480cctgtccctt ctctgtctcc ggctactaca ggtacctgag ggaaagggtg
cgggggagcg 165540gttgtacttg ggctagaatg agagaagact ggcatgctca
ccacaccagt gatgcgggaa 165600gacctgagtg tggtctgagt tggaggctgt
ggtgctaaat
acgctgcccc tttcataagc 165660aggagtctta gtcaggccca gggaggaagt
aaaatctgga aatgaatgag aagcattctc 165720tcctgccagt caagaaatga
gaagcgaaag aattctcacg ggctgtaaga ccagcaggat 165780ttaaaagttg
aattagttgc ttatgttaag aactcaacca agttcatcta cacaagctga
165840atctccagct tttcctaaga aaccatgtgt ggcagtggct gcagggcagg
gcacagctgg 165900gcctgagcac cccgctccct gcacctctcc cctccctggg
ccctgcctgt cactgcccac 165960tctcccacca agccttccgg ttgtgtgcct
gccctatcac aggcatcgga gcttgtcacc 166020tggtttaaaa gaagagagtt
gtgtggggat ttgggatgca cgtttttcac tcaaaagtat 166080tttagcgtag
agctctgtga ttccgtagct atttaggagt ttaagcacct tgaaggcttt
166140aattgcagaa agttctatgt ggacgtgcaa tgtgttatac gcagtgtcta
tgagactcaa 166200atgtttatta gggcgttgaa gtaaactgag cacttggagg
gccatggatc cagccttcaa 166260ggagctcata agtcaggagg acccaggagc
aatgacctgt catagaaggc agaaaagagg 166320ggcacagagg tgggtgggag
gcatacacag gcagctcctg gagctccaag gggagcaagt 166380gcttccaggg
aagggggcgt ggaggcccct ttggaggagg caagttgatc tggggtctgg
166440cagagggtta gctggggaca tttagcggga ggctggtgcc cgggaattgg
ggggatgccc 166500agcagaaaga catgaggagg ctggcctggg gcgtgggggg
gtgtgaaagg ttaagtgggg 166560gcattatcct gctcccgctc ctgccggctg
tatctggtca gcctgggcac cgaggtgggg 166620ttctggaagg cactgttcac
caaaatgctt atctgggtcc cccagagagc ttgcctgcct 166680ggactgtcgg
ctcgcctgca actgctgact cctaagcttt tgcagctcag cccacaacca
166740gttcctattc acagaggtgg gagctgaggg gtgacaagtg actgctgcag
tcttatttgt 166800catagagaaa aagtgacaga gtccagcttg cccactggcc
ctgccagctt aactggttat 166860aaagtgacaa atccccaaga cccacagggc
tctgcacaac ctgggccctc ctgccagtgg 166920cggcgagggc aggtggctca
cggctgggtg cctgtctggg caggagctgg gctggtatgg 166980ggtgggcctg
cggccctgcc cccctgtgca gatcaagact cagggtgctg gtgttcacag
167040gtgccctcat cagccacgag aagctgctgc tacagatcaa ccccgagcgg
gagctgggga 167100gcatgagcta caaactcggc caggtcagtc tcgcgccccc
gccgcctggc ctctgtccgt 167160ttctgtcctc agactttggc gcttgacaca
cccaggagaa aagctcagtg cactttttaa 167220atgaaaggaa gttttccttt
tttttaaaaa aaaatttaat gttcattgtt tttatctgtt 167280ttattcctag
gtcccgcaag cagaggaagc attagttttg tttttattta tgttctgtat
167340tccagaaagt agttaagaga cctcacatgt agcgatagag atgtgtgtaa
gagacagtga 167400gagggcgtga cttggactta agcaaggacc gtgagacaca
aaaagggggg tgaggacaga 167460gtggagtcag ctgaaatgct caggaggaag
tagacgccat gaagggccat ggtatggggg 167520gccgcaggcg tggccgtgag
tgtccctggg gccagctctt ggggggctcc ctgagtgtcc 167580ctgtccctgt
ggccagttct gggtgggagc cccgtgtgca ggcagacagc tcggccactt
167640cctagcaggt cacattggtc tgtgcttctg tttcctcctc agataagtga
agggattcaa 167700gggtctgggt gtggtggcta acacctgtaa tctataacat
tttaggaggc tgaggcagga 167760ggcttacctg agctcaggag gttgaggctg
cagtgagcca tgattgcacc actgcactcc 167820agcctgggca acagaccagt
actctgtccc ttaaaaaaaa atgtaaacag aaacgtaggg 167880ccatttgcat
atgatggcac atggcgtgga gccctacagg tgtatgctgg gcggggcccg
167940gctgtgctgg ccgacttgca cctttccctc caccccggtg ctgtgtcttt
cgctcaccgg 168000gttcctgatt tagtgaaagc agttgtgcag gacagttctc
tttgtagctt ttgtttctgt 168060ggaaatgggt cagaatatgg tgtttagaaa
cacttatgag ctctgagagt ttcctcttct 168120gagttcctgg cctgcagcct
tcacagcaga aaccctgtga tgtcacaagc ctgtttctgt 168180tccctgctct
ctgcctgtac tgtcctgttt tgtgcctgcc ggtttcagtg acaggaagca
168240gggagctact ggaccagcct gtatttttct agacatagtt ggaaaaagaa
gtcccactct 168300tctgtccttt cacctttgac agatgtttcc accccaagat
aagtgaaaat gaccaatagg 168360atgcactgta tttttcatga aagtgtttct
gaagggcagg ctgagagtga gaggcctggg 168420gctcactggg tgcctctggc
cttgtcctgg gcccagggac actggtctgt gcccgaggta 168480ttccctatcc
ccccaacccc gctgcatttg gccacatcct tcaatgtttg cgttgtgtcc
168540agcgtccgca aaccaactgt catgggatca tactggggct gaagtacggt
cccacccctg 168600ccctgtctgg ggctgaagta cagtgccacc cctgccctgt
ctggggctga aggacagtgc 168660cacccctgcc ctgtctgggg ctgaagtaca
gtgccacccc tgccctgtct ggggctgaag 168720gacagtgcca ccccttccct
gtctggggct gaaggacagt gccacccctg ccctgtctgg 168780ggctgaagga
cagtgccacc cctgccctgt ctggggctga aggacagtgc cacccctgcc
168840ctgtctgggg ctgaaggaca gtgccacccc tgccctgtct ggggctgaag
gacagtgcca 168900cccctgccct gtctggggct gaaggacagt gccacccctg
ccctgtctgg ggctgaagga 168960cagtgccacc cctgccctgt ctggggctga
aggacagtgc cacccctgcc ctgtctgggg 169020ctgaaggaca gtgccacccc
tgccctgtct ggggctgaag gacagtgcca cccctgccct 169080gtctggggct
gaaggacagt gccacccctg ccctgtctgg ggctgaagga cagtgccacc
169140cctgccctgt ctggggctga aggacagtgc cacccctgcc ctgtctgggg
ctgaaggaca 169200gtgccacccc tgccctgtct gggatgttta gcccctagat
gccactggac tgagccgcta 169260cttgcttttg ggaaagaggg gtgggggtta
ggggtctggg cgaggggagt gcaggggctc 169320ctccttggcc tgagagctgt
tcatacagac tcctcgccca ctccctgcag ggtgctgggt 169380cccagggggg
aaatggccct tggtgccaag aacgtgagtt ggggctagtg ccagtgatga
169440tggagaacag ctttttatgg gcacacagcc cacagcactg tgccaagtgc
tcgaggcttc 169500ccgagaacca ggcagaaagg aggacagtcg aggtgtgctg
actgcgtggt ggctgcgtga 169560tctagagcgc gggtcacaaa ggcgcgaggg
agctctggcc ttgggtttac cgcaatgact 169620gccagtgcgg gagactggaa
aaggaatctc acgtattggt tccgtgtttt ggggactcca 169680ttcagatgtc
acttaggagt gaaagcatcc cttcgtagag cctctttctg tgtcaccctc
169740ctcagctgct cctggggttg actggcccct gattcatgcc tttagcatgt
gctggagctt 169800cccagcagct gtccagcccc tgccccaccc tctctgtggg
ctcccttgcc cgtaacctgg 169860ggtgtctgaa cgacccttgc taaggggcag
actgttagac ggtaggcatg tgctgagtcc 169920cagtggccac acccacccac
caggagcctg gcactgtggc cgcagcactg agcagtgccc 169980cgtttctgtg
gcaggtgtcc atacactccg tgtggctggg gaacagcatc acacccctga
170040gggaggagga atgggacgag gaagaggagg aggaggccga cgcccctgca
ccttcgtcac 170100cacccacgtc tccagtcaac tccaggtttt ccaatggcct
ttttcttttt aacagaaatt 170160tgaaatttct tatcagtcat ttgatttgtt
tgaggtgctt cttgaaatga gcctctcatc 170220tcatgtactt ggaaaatacc
catctcgcat attccacagg aaacaccggg ctggagttga 170280catccactcc
tgttcgcagt ttttgcttga gttgtacagc cgctggatcc tgccgtccag
170340ctcagccagg aggaccccgg ccatcctgat cagtgaggtg gtcagatccg
taagtgagcc 170400ttcccattcc cctcacacct gcacgtgcca cacgcaccac
acacgccaca caccccacac 170460acacacaccg cccacacaca tgccacttgc
acacacaccc ctcatgcatg caacacacac 170520acaggccaca cgcaccatag
acaccacaca cacatgccac atgcacacac atacacggca 170580tgcaccatac
acacaacaca cacagcacac atgccacaca cacacgccac accacatgca
170640ccacacacat gccacatgca cacacactcc acatgcatgc accacacaca
cacacacaca 170700ccacacacac cacatgcacc acaccacaca ggttacatgc
acacaacaca cacatgccac 170760gtgcacacac cccacacacc acatgtatgt
gccacacaca gcacacaacc acacacatgc 170820accacacaca tgccacatgt
gcatgcacca gacacatggc acacactaca cacacgccac 170880gtgcacacac
cccacacaca tgtacgcacc acacacatgc cacacacaca tgcaccacac
170940acatgccaca tgtacacaca tgtatataca caccccacac cacacacaca
ccacttgcac 171000accacgcaca cacaccacat gcgcacacac acaccacata
cgccacatgt acacaccata 171060cacacaccat acatgcacca cgtgtaccac
gcacccacac agacacagca cacgcataca 171120ccacacacac acgcacacat
gcgtcccgca cagtaatgtc tcttgggtgt aagaacacga 171180cttgccagta
gtagcgttct ggatgcgttg cctggattct aacagcgcga ttctcccctt
171240gccctcctgg ttttccacat ctccagcttc tagtggtctc agacttgttc
accgagcgca 171300accagtttga gctgatgtat gtgacgctga cagaactgcg
aagggtgcac ccttcagaag 171360acgagatcct cgctcagtac ctggtgcctg
ccacctgcaa ggcagctgcc gtccttggga 171420tggtaagtga caggtggcac
agaggtttct gtgctgaagc cacgggggcc catctgcctt 171480gggacctggt
gttggccaga ggtgccgggt gcggctgcct ccttccaaga gttgacccga
171540accggactcc acggcccacg tgagctgcag tgcttctcag atggaggggg
ttcagcgacg 171600gtcagtgcca ttcacaggtc actgtgatgt gggttgtggc
ggccaagcca tggtttgggg 171660tcccgtatcc ctgggcttat gacatcattg
tagtagccca tccccacaga accacggtgt 171720gtggtggcgc tgaggcatcg
tagatggtgg aaatgctact ggcttcccca tgctctgccc 171780tgaggcctga
ctgcctcact ccccttctca gttatgttcc aggccccccg agcttcctgg
171840ctggacagct tctctcctgg gggccgtttt gtcacagtga ccctgtgttt
ctagtcccaa 171900atctgggtgc tatagtctct ttttagcgtg gtggttgtct
tagtcttttt tggctgctac 171960cacaagttac cttagactgg gtaatttata
aacagtggaa atttacttct caccgttctg 172020ggggctggaa gttttcatgg
tcaaggtgcc agcagatttg gtgtgtgatg agggctgctc 172080tctgcttcat
agatggcatc ttctggctgg gtcctcacgg tggaaggagt gaacaagctc
172140cctcaggcct tttagaaggg ccccaatcca caagggctct cccatcatga
cctcatcacc 172200tcccaaggcc ccaccttctt gtactgtggc actgcaaatt
aggtgtcagt gtaggagttt 172260caggagggat agaaacattc agaccatccc
agcggtcaag tgttcatcct cttgagttcc 172320tccttattct gcttctggtt
tatcaggatt cagccagtgc agcatggtac ctgtattctg 172380tggcacatca
ccacatggta tttgccaagt atccatcacc tgcacacgtg aaatcattgc
172440ccgtgggtcc cgacatctgg cgaagcatat tcaaggatgg cagaactgtc
agagctggca 172500cctctggttc cttgtcatgt ggcattacct agtaatccat
tttatgatag caatggaaac 172560tcatttcttc aacaaacacc tgagtggctg
ccgtgtgcca gccgtctggg gcccttggtg 172620agaatggcat ggtggtgccc
atcagggcct gcctagcccg tgctctggac gggctcctgt 172680gtgtcaggaa
cgacaatgct gtcatgacgg tgaatgattt ttttttttgc catcactcca
172740gccgctaaca tttgcggagc tcttcctccc gcacccccac ctgacaaggc
caagggtgac 172800cttggcccca ccctaggcgg ccaaggtcag aggttagctg
gcttgtctgg gtcacacaaa 172860atgcagcaga ggttgaggtg agcacatgtc
cgtgacctgg agcctgactc cctctctgcg 172920agtcttgact gctcttgcct
agactctgtc ctccccgagc ccaaacgcca gtcatcttcc 172980cttgtgggtg
tccttcagcc tggtgccatg ctggtgactc agcagccgtc cagggagtgg
173040aaacaattga gtgtgtgggt tccctgtgtg ggcatctctc ttcacggcga
acaccctctg 173100ggtgttgccc acacgatgtc aaagcggctc ttggaagggg
tccttctcct ttgtgggaag 173160tttcagctgc tgggctaact tgaattgtaa
ctgtggtttt gtgctcaggc ccagatcccc 173220ctaggcaagt gttgtgccat
cagtaatcaa atgagaaata atcattttga aaagcagatc 173280ctaaggcagg
atggtcatgg acactcactc ccagctcttt gtgcactcat gctttctgga
173340agatggccat cctctgtgaa ggttttcagc gcgtcatgct tggtacccac
gtatccagag 173400catgtcgttt tgaggtattt gcccaccgtt gtgaaatccg
tgccacccga gagcaggtcc 173460tgatgtgggg ctttcagaag tgggacctgg
ggccgtacgc agtccttagg gaggggccgt 173520gtggcgttgt gcgtgtgagg
ggatagcaca gggtgaggtg ggggcccaag aaggaagtga 173580cccacaaaga
acagcctcct cttttggtcc ttgttcctgg gatggctggg agtggcttct
173640gtgtcgtccg gccatttccc ctgcggagag gctcctacca ctgccgagaa
cctcatcatt 173700ccacaaaaac aagaggccgc ctggccatcc agcgctccat
gggaattctg tgtccccata 173760gtcttgggct gaaggagggt gacattcctt
gctgacttct gcaggggtct cctcactgtt 173820aaagagcaga ttgaaagtga
agaacgtggg ctaagtgttt aggtcgatat ttaaccctgc 173880taggttttgg
atactaagtg aaattgaggc cattttggtt gaagttgaca gaaaccacta
173940tcagggatcc ccaagactac cccaggcttt tctagaaaga ctctcagcta
agatgtgtta 174000tggtaaaagc acacaaaaca aaatcagcaa agaaaattag
caagggcaga ggcccatggg 174060gcgatgtccc gaggacacca ggcttgagct
tccagaatcc tctcccagcg gggtcgtgca 174120ggacgcactt aactccccgc
acagtgagcc gtgacagcgc gtgtgcagtg tcgtcgccag 174180gaaagcacac
tagagactcg gtgccagggt ttttactggg ggctgggcac atgggcaccc
174240tctgcctgcc tcgtgcccag actctggact cccggaggga aggcaagttc
tcagcaccaa 174300ccctggtgcc cacacaagca gctgagcaca gggagcccct
cctcagtgag gatggtgggc 174360accgtcccaa caccagccag gggccagcct
tgcacacagg cctctcagga tggtctccgg 174420cctgctgtgt agtctcttct
gcacacaagc gtgagggcag cgcccccgcc tcggctgtgg 174480ggaggagcca
ctgggacgtg agctctggtg gcatgcagca gcttttgtct gtgtgtgcct
174540aggacaaggc cgtggcggag cctgtcagcc gcctgctgga gagcacgctc
aggagcagcc 174600acctgcccag cagggttgga gccctgcacg gcgtcctcta
tgtgctggag tgcgacctgc 174660tggacgacac tgccaagcag ctcatcccgg
tcatcagcga ctatctcctc tccaacctga 174720aagggatcgc ccagtgagtg
ggagcctggc tggggctggg gcgggggtct cagaatgagc 174780tgtgaaggaa
gcagcatcac cctctccaag tgcccaggct cctggccaga tggcaggcca
174840ggtatcagtg ggaacccagg tgggtgccat ggctgaggtc agtgagacgc
aagagcacag 174900gtgcgtccta gaggcttcct cgggcacctc cagcgagctg
gagctctcgc ctctgctgct 174960gtctcatgtg gcgcttagca cactctccca
cgtgcccatt cctgactctg ctctcgaggc 175020catcggctct cattctctgc
tcccagaacc ctgttattac ccaggctagc ctcctctctg 175080caccttcccc
gccctggccc agtacctccc tcttgtttcc actgtgattc cgacctcacc
175140ttatcttaaa gctgctggac ggcaggttct gtacacacgt gtccttgaca
aagcacggct 175200ggtgccgcaa cccctcagcg agcaagtcaa gctcttcaca
gcgatgtctt acaagcgcag 175260agggctctgt gacaccctgg tctcaccgcc
actcttccaa agtcgcagag gctttagcag 175320agatgggccc agcctctctg
agtcataggc ttctgcacac gggagctgtc tttagaggga 175380gggtggaatt
tcatcagcca cccacatggg ggagttgagg gcaagaatta ggagcaaaga
175440tgggaagggg tctgggagga atggccagtg atcccctttg acaagtgggc
aggaaacggg 175500ggctaggtca aagttgagtg gaagacctgg agggagacgg
gaaggtctct gtaggcacag 175560ttcagacagg agggaggtgt gagccagggc
acatgccggt ggccgtctgg caggatttgg 175620gacatgctgg agcagggaca
gcggctcatc aggggccatt gccctcatcc aggccagagt 175680gtcacaagcc
cgtggggagg cccttctcgc ctgtcatcct tgctgggcag tgggtgctgt
175740gctagcagga caggcggacg gctggcaact gtctctgcat ccctggagcc
tggcataggg 175800ccaagtcaca cggggcacag gcctgcaaat caggcacata
tgttggtgca gtgacgtgat 175860tttggggggc agccccagaa caggccccag
acacaggcca aagccctgcc tgtgctggtg 175920tgttgggctg ttctatggct
cttgctgtgg gcatggagga ctcagggaag gagagttgag 175980gtggtccagg
agttgcgttt gggatgcaga gagcttgtgg catccaggta gaaatggtgc
176040gtggggctga cctcagcacc atgggcagag gggccgtgtc acgtgcctcc
gaggtggagg 176100tgggaccacg tggtgacaga tatacgcatc actgggcacg
tttttgtggg tgttgggggg 176160catcgtattg gctcctctgt tcacagtggc
cactcattca gtccctggct accaggtcct 176220cactgtgcca tggggaaggc
cggcgctgtc gggggatcac agaaggcagc acgtcatgat 176280ggcatgtgcc
atgaaggaaa agcacagggc actcaggaag tagaggggac tggcctgggg
176340tgtgggaatc tagggcctcg ttgagggaca gagagaggaa gtgtgtggtg
gccagcatgg 176400aggtggccac aggggaggct gagttaggcc gagagggcag
ggcgttgggg aggtagacgg 176460gctcagccac tcagggagtg gtcaagcaga
ggctgaaggg tcaggccagg ttgcaggggc 176520ctgggggagc cactcagggt
aggcgctccc gggagcccgc ctggcccata gctctacact 176580cccgcgtggg
gccggacatg ctgtgaagcc ctctccacgt tggatggggg tggctgagcc
176640tggatgctgt ctcccgtttt cagctgcgtg aacattcaca gccagcagca
cgtactggtc 176700atgtgtgcca ctgcgtttta cctcattgag aactatcctc
tggacgtagg gccggaattt 176760tcagcatcaa taatacaggt gagtgggccc
tggctgtctt cctctgcaca cggggagtgg 176820gcttcccttc tcttttcctt
gcaggatcat accagtgggc cagttttgac ttggtcggga 176880ggaggcatga
acacctgaga ctgtgcagcg attctttgac acagaggcct ttctccctgt
176940gcagatgtgt ggggtgatgc tgtctggaag tgaggagtcc accccctcca
tcatttacca 177000ctgtgccctc agaggcctgg agcgcctcct gctctctgag
cagctctccc gcctggatgc 177060agaatcgctg gtcaagctga gtgtggacag
agtgaacgtg cacagcccgc accgggccat 177120ggcggctctg ggcctgatgc
tcacctgcat gtacacaggt gagcatgtac acggtgccca 177180taaggccagc
ccaagtcctg ttcaagggag gcaggagcat gctcactcaa gggacctcga
177240ctaggtgccc tctgatttca cacttctggt gttgccccaa gccggcccca
tcaccttgca 177300agaaaggctc tggagccccc agggctggag tacctggtca
gggttgaccg tccctgtggt 177360cactcatccc atgtggctga gctgggctgg
gtcctgggca agcaaggggc tgatatcacc 177420tgctttcaga tctccaggga
ctcactggac ccctgtgtac aaagcactgt ctacagagcc 177480tattgggttg
tatagaggta accttcgtac tgaacacttt tgttacagga aaggagaaag
177540tcagtccggg tagaacttca gaccctaatc ctgcagcccc cgacagcgag
tcagtgattg 177600ttgctatgga gcgggtatct gttctttttg ataggtaaga
agcgaagccc catccctcag 177660ccgttagctt ccctagaact ttggcctgaa
gctgtgcttt tgtgtgtgtc tgctgatccc 177720ctggcgctgt tgctggagtc
ctgccagtga ttccccacca cagcctgacc atgggctgcc 177780ttggctcagg
gttccactgg cgagctggtg gtccttggac cccagcactc aggtgtagcg
177840ttgaccagtt ccaaggttgt cccagtgcct gcccatctct cctgagggct
cagggacagt 177900acctggcagt tgggggtgtg gcagggggca ggaatgacca
gcctctggga gggtggggca 177960gaagcctgta cagtgaggag gagctggctc
agcctggctg cctatcgtga gaggggagcc 178020cacggggctg tgggaggggg
gccgtggtgc ctgtgagcag ggtgaggagc agcggcagga 178080ggatgaaggt
ggaacccaca catgcatctt tgagacccgt gtggtcagtg gcttctgccc
178140cccaccaccc cccactgctg tgcgtgcata gaattggctt ccctcacctg
ctctggaagt 178200gggttaggag cttggtaggg ctttttctca aggacaaggg
cccctgattt gctctcaggc 178260ctcagtcctg gcgacatggt ggatctggag
ccttgttgca ctgccttgcc tgtgctctcc 178320aatcagggtg gccagtgggg
agccatttgg cttttctcaa gagcatactc aggtggacct 178380tgctccactg
tttgaccaga tgaggcattc tgaacagcca agcctgtgct ggtctgtttt
178440catgttgatt tttttttttc ttttcttttt gagatggagt ttttcccttg
tcacccaggc 178500tggagtgcaa tggtgtgatc tcggctcact gcaacctccg
cctcccgggt tcaagtgatt 178560ctcctgcctc agcctcccta gtagctggga
ttacaggcac acaccaccat gcccagctaa 178620tttttgtgtt tttagtagag
acggggtttc accgtgttgg ctgggctggt ctcgaactcc 178680tgaactcaag
tgatccaccc tccttggcct cccaaagtgc tgggattgca ggcgtgagcc
178740actgcgcccg gcccccatgt cgatttttaa atgcacctct gcatcgttct
tcagtcccca 178800tatgctcact gagcaccact gcgactggca gacgggcaca
gggaggcgcc acgaccagtc 178860ctggccttca aggggcttgt ggtctagtgg
gcccaatgct aggtggcgag tgctccaaag 178920agtgtggtgc acgccttccg
cttgaccgct ctccagacgc cacagggagg cacctcgcag 178980ctgaccacag
atttctctct gtggagcagt gtcttcagag cggctgccat gccactgctg
179040ggcgagggtc tgcgggcggg tagagccagg agcacctgtg aggaagtgca
ctgccatttt 179100cgtagctgct tcccgtgtgt ctcagttaca cacggctggc
atgtgtgcac tgatgagacg 179160ggaacgtgat ggttgctttt cagcactgaa
agggatactg ctcagggggc gtgtttcagg 179220atctggttag ggaagaagca
gcgagagcac agatggggcc ctgtgtggta acaagaaaaa 179280agtcctggtt
gacaacagtg ccacgaagcg ttagaacaca tagggatgtt tgtggagcat
179340ttgcatgtgg aaagcagcaa aaacataatg ggaacgggtt cttttgttat
gatttttaaa 179400aatctctttt gtaacatcct tcccgctgcg ccgtttctgc
atattccttt atgtagcttt 179460caaactcctc ttaggagttc tggtccctac
agggcgtggg agcccaggct ttacgtagct 179520ttcaaactcc tcttaggagt
tctggtccct acagggtgtg ggagcccagg gcctgtgccg 179580agcagcctgc
ctccacgagc tagacagagg aagggctggg gttttgcctt tttagtctca
179640aaattcgtac tccagttgct taggctctga ctttccccac ttggaaagtc
cctcacggcc 179700gagggtccct cccagccctg atttcacatc ggcattttcc
ccagtattag agccaaggcc 179760ctccgcgggc aggtggggca gctgtgggag
ctggtgccag tctctgacct gcgtccctcc 179820tcccaggatc aggaaaggct
ttccttgtga agccagagtg gtggccagga tcctgcccca 179880gtttctagac
gacttcttcc caccccagga catcatgaac aaagtcatcg gagagtttct
179940gtccaaccag cagccatacc cccagttcat ggccaccgtg gtgtataagg
tgaggttgca 180000tgtgggatgg ggatggagtg ggaaagcctg gaggtggagt
tgcctccgac ttcccagcag 180060attcgccagc agagcccagc tcctccgctt
taaagcagca atgcctctgg cccccacccc 180120acccccgcca cccaggcgca
gcaggtgctt cccgtccccc cagccctgac actcaggcac 180180ctgcttgctc
cttgcaggtg tttcagactc tgcacagcac cgggcagtcg tccatggtcc
180240gggactgggt catgctgtcc ctctccaact tcacgcagag ggccccggtc
gccatggcca 180300cgtggagcct ctcctgcttc tttgtcagcg cgtccaccag
cccgtgggtc gcggcgatgt 180360atcctctctg ggtccctggt gctggccccg
tttcccttgt caacaccgag gctcatgttt 180420catgataagg ttttgaaacc
taacctttgc aaaaacccca cagatgccag ggtgacaggc 180480cctcagcccc
agggaagtaa aatgctgaca ggggtacaga aaggagcacg tccagacatt
180540tgctgaccag ggcctctcag aggggccggt gtatggcagg agggtcgcag
ctgaggggcc 180600tttctgtgga gggcctgggt gaggggagcg agggtgggcg
gtggtctctg cagacgtccc 180660gcccactcgc gggctctgtg tggctgggct
tctcctgaca
ctgcttctca ttagctttgg 180720tcattgtgcc tcgatcgccc tctcggggaa
aggcttaagt aaagatccag ttcccacccc 180780cagatgctgg ctgccaggag
tttccctttc cacagccctt ccccaagaca gaccacaaga 180840gcctccaagc
agcacagttg tcctggtgct gacagcacag ccttgcccgg cgtgcctggc
180900acggctctgc cctcactgca ttggagcagg gctagtggag gccagcggaa
gcaccggcca 180960ccagcgctgc acaggagcca ggccaggtga gtgctgccga
gtgggtgccc tgcctgcagg 181020gcatccagcc agccaagggt tgcaggaatg
gaggtggagg cgctgatgca gctggaggca 181080tccaggtggc ccttccgggg
ctctgctcgc tctccaggct ccctggaccc ctttgtagac 181140tgtttcagga
gaggaactcc caggtgagga cagggaggca gcattcccct catttgccgg
181200cctttttcct taactcctgc accagcctcc cacatgtcat cagcaggatg
ggcaagctgg 181260agcaggtgga cgtgaacctt ttctgcctgg tcgccacaga
cttctacaga caccagatag 181320aggaggagct cgaccgcagg gccttccagt
ctgtgcttga ggtggttgca gccccaggaa 181380gcccatatca ccggctgctg
acttgtttac gaaatgtcca caaggtcacc acctgctgag 181440cgccatggtg
ggagagactg tgaggcggca gctggggccg gagcctttgg aagtctgcgc
181500ccttgtgccc tgcctccacc gagccagctt ggtccctatg ggcttccgca
catgccgcgg 181560gcggccaggc aacgtgcgtg tctctgccat gtggcagaag
tgctctttgt ggcagtggcc 181620aggcagggag tgtctgcagt cctggtgggg
ctgagcctga ggccttccag aaagcaggag 181680cagctgtgct gcaccccatg
tgggtgacca ggtcctttct cctgatagtc acctgctggt 181740tgttgccagg
ttgcagctgc tcttgcatct gggccagaag tcctccctcc tgcaggctgg
181800ctgttggccc ctctgctgtc ctgcagtaga aggtgccgtg agcaggcttt
gggaacactg 181860gcctgggtct ccctggtggg gtgtgcatgc cacgccccgt
gtctggatgc acagatgcca 181920tggcctgtgc tgggccagtg gctgggggtg
ctagacaccc ggcaccattc tcccttctct 181980cttttcttct caggatttaa
aatttaatta tatcagtaaa gagattaatt ttaacgtaac 182040tctttctatg
cccgtgtaaa gtatgtgaat cgcaaggcct gtgctgcatg cgacagcgtc
182100cggggtggtg gacagggccc ccggccacgc tccctctcct gtagccactg
gcatagccct 182160cctgagcacc cgctgacatt tccgttgtac atgttcctgt
ttatgcattc acaaggtgac 182220tgggatgtag agaggcgtta gtgggcaggt
ggccacagca ggactgagga caggccccca 182280ttatcctagg ggtgcgctca
cctgcagccc ctcctcctcg ggcacagacg actgtcgttc 182340tccacccacc
agtcagggac agcagcctcc ctgtcactca gctgagaagg ccagccctcc
182400ctggctgtga gcagcctcca ctgtgtccag agacatgggc ctcccactcc
tgttccttgc 182460tagccctggg gtggcgtctg cctaggagct ggctggcagg
tgttgggacc tgctgctcca 182520tggatgcatg ccctaagagt gtcactgagc
tgtgttttgt ctgagcctct ctcggtcaac 182580agcaaagctt ggtgtcttgg
cactgttagt gacagagccc agcatccctt ctgcccccgt 182640tccagctgac
atcttgcacg gtgacccctt ttagtcagga gagtgcagat ctgtgctcat
182700cggagactgc cccacggccc tgtcagagcc gccactccta tccccaggcc
aggtccctgg 182760accagcctcc tgtttgcagg cccagaggag ccaagtcatt
aaaatggaag tggattctgg 182820atggccgggc tgctgctgat gtaggagctg
gatttgggag ctctgcttgc cgactggctg 182880tgagacgagg caggggctct
gcttcctcag ccctagaggc gagccaggca aggttggcga 182940ctgtcatgtg
gcttggtttg gtcatgcccg tcgatgtttt gggtattgaa tgtggtaagt
183000ggaggaaatg ttggaactct gtgcaggtgc tgccttgaga cccccaagct
tccacctgtc 183060cctctcctat gtggcagctg gggagcagct gagatgtgga
cttgtatgct gcccacatac 183120gtgaggggga gctgaaaggg agcccctcct
ctgagcagcc tctgccaggc ctgtatgagg 183180cttttcccac cagctcccaa
cagaggcctc ccccagccag gaccacctcg tcctcgtggc 183240ggggcagcag
gagcggtaga aaggggtccg atgtttgagg aggcccttaa gggaagctac
183300tgaattataa cacgtaagaa aatcaccatt ccgtattggt tgggggctcc
tgtttctcat 183360cctagctttt tcctggaaag cccgctagaa ggtttgggaa
cgaggggaaa gttctcagaa 183420ctgttggctg ctccccaccc gcctcccgcc
tcccccgcag gttatgtcag cagctctgag 183480acagcagtat cacaggccag
atgttgttcc tggctagatg tttacatttg taagaaataa 183540cactgtgaat
gtaaaacaga gccattccct tggaatgcat atcgctgggc tcaacataga
183600gtttgtcttc ctcttgttta cgacgtgatc taaaccagtc cttagcaagg
ggctcagaac 183660accccgctct ggcagtaggt gtcccccacc cccaaagacc
tgcctgtgtg ctccggagat 183720gaatatgagc tcattagtaa aaatgacttc
acccacgcat atacataaag tatccatgca 183780tgtgcatata gacacatcta
taattttaca cacacacctc tcaagacgga gatgcatggc 183840ctctaagagt
gcccgtgtcg gttcttcctg gaagttgact ttccttagac ccgccaggtc
183900aagttagccg cgtgacggac atccaggcgt gggacgtggt cagggcaggg
ctcattcatt 183960gcccactagg atcccactgg cgaagatggt ctccatatca
gctctctgca gaagggagga 184020agactttatc atgttcctaa aaatctgtgg
caagcaccca tcgtattatc caaattttgt 184080tgcaaatgtg attaatttgg
ttgtcaagtt ttgggggtgg gctgtgggga gattgctttt 184140gttttcctgc
tggtaatatc gggaaagatt ttaatgaaac cagggtagaa ttgtttggca
184200atgcactgaa gcgtgtttct ttcccaaaat gtgcctccct tccgctgcgg
gcccagctga 184260gtctatgtag gtgatgtttc cagctgccaa gtgctctttg
ttactgtcca ccctcatttc 184320tgccagcgca tgtgtccttt caaggggaaa
atgtgaagct gaaccccctc cagacaccca 184380gaatgtagca tctgagaagg
ccctgtgccc taaaggacac ccctcgcccc catcttcatg 184440gagggggtca
tttcagagcc ctcggagcca atgaacagct cctcctcttg gagctgagat
184500gagccccacg tggagctcgg gacggatagt agacagcaat aactcggtgt
gtggccgcct 184560ggcaggtgga acttcctccc gttgcggggt ggagtgaggt
tagttctgtg tgtctggtgg 184620gtggagtcag gcttctcttg ctacctgtga
gcatccttcc cagcagacat cctcatcggg 184680ctttgtccct cccccgcttc
ctccctctgc ggggaggacc cgggaccaca gctgctggcc 184740agggtagact
tggagctgtc ctccagaggg gtcacgtgta ggagtgagaa gaaggaagat
184800cttgagagct gctgagggac cttggagagc tcaggatggc tcagacgagg
acactcgctt 184860gccgggcctg ggcctcctgg gaaggaggga gctgctcaga
atgccgcatg acaactgaag 184920gcaacctgga aggttcaggg gccgctcttc
ccccatgtgc ctgtcacgct ctggtgcagt 184980caaaggaacg ccttcccctc
agttgtttct aagagcagag tctcccgctg caatctgggt 185040ggtaactgcc
agccttggag gatcgtggcc aacgtggacc tgcctacgga gggtgggctc
185100tgacccaagt ggggcctcct tgtccaggtc tcactgcttt gcaccgtggt
cagagggact 185160gtcagctgag cttgagctcc cctggagcca gcagggctgt
gatgggcgag tcccggagcc 185220ccacccagac ctgaatgctt ctgagagcaa
agggaaggac tgacgagaga tgtatattta 185280attttttaac tgctgcaaac
attgtacatc caaattaaag gaaaaaaatg gaaaccatca 185340gttgttgctg
tgtgaggctt gctttgcttc atgagaacct agaccttgct gagctggagt
185400cttaggaagc agtctcctaa gtgcttctcc agcaggggca gaaactgtcc
caccagctaa 185460catctggcat tatggagggt cccccaggca gctgccagca
gggacaggcc ccgtgttttc 185520tgtagccagg gatgaggaag tggccccagg
gcatgggcct ggctgggtgc ttctgcaagg 185580gccttcccaa accacagtac
aggtggtctt cctgccctgc agatgggagc tgtgggagct 185640gctggagctg
ctggagcctt catggtcaag tgacatcata agcttatatg acatacacaa
185700gcctcaggac ttggcccatg gcactgaagc aggtcatcag gcccagcaca
gagactagag 185760ctgtgttctc acagggccca ccacccttcc acctccttgg
ccattgacac ctgcgtccct 185820ggcccagctg ctcccaggta acccccaaag
cagctggcac atcccacctc tggtgtggcc 185880ggggctgctg tgtgtccgca
gggcctgccc cgtctattct agcttgtttg tcctgtctga 185940accagcgcct
actccaagaa gcctctgctc agcccagcgg ggatgcttct aagctccgga
186000cgagcctctc ggaagccttg gtgattggtg gtgtagtcat cttgggatgc
agatgtctta 186060ccaacctgca agaacaaaaa ccctgtggct tcctctggtg
cagggtattt agtcaatgtt 186120tgctgaggtc ccgtctggtt ctggctaatt
ggcaggggtc gtccacccat tctttccctg 186180ctctgctgtc tgtgccagga
gagacggggg ccagtcggcc aaggggccag ctcctgctgc 186240ctgctcctct
tgggcacgtg cgggggcccc ctttctctga gcagggatag ggatcagtct
186300gccggaggga tgtggtggac aggcctaaag catttggggc ggggcatgcc
acttgagctc 186360cctaaatctg tctcctcata ggtgacaccg ctccagggcc
ccccagtggc ctctcctttc 186420agagctacct aaattctggt cacttcagag
aaatggagca cccccttctc cctggtccag 186480gtgtggacag cctggcacac
tgagcacacc tggcatggct ggtaatttca gaaagaagag 186540gggccggggt
ccagtgggaa gcagcggtga acccctcgtg agtgggcttt gcagtccctc
186600cccatgccac ggcagagctg ccctcaacac agccttcctc ttcctcatcg
gagagcacac 186660cctgtcccct tgccgagctg tgccctgtgc cttcggtggt
atttgatttt ggctgctact 186720ggctttgttg ggatctggaa gtcgcttccc
ctgcgtggtg cgtggagcac tgtaagtcag 186780atgagggaag tagccagggt
gaggtgagta ccgggtggag ccgccactga agggactggg 186840taggggggcc
ttgcctctac atgatgtgac acagccaacc gaggacagag gaagccccgt
186900tcctgggggt gtggggtgca cccctcaggg aagcctgcag tggggcctga
ggaaaggcat 186960cctccgcgag cccacgagtc tggtccatga gcaccgtgac
agtgtctgtg ggtagaggtg 187020gacccggcct tgtgtcatca ccaggacctc
ttttgggaaa ccatgtggac atcgcttgcg 187080ggtcccccag gctctgcagc
cccagcagcc tggctgcctt ttgggcaagt ggcttgagcc 187140acagaggacc
cagtcctgtt gcagccacat cctctggggg ggcccgccag tgtggccggc
187200tttctccacc ctacaccagg cctccaggtg tcctggtcgg gggtgtctgg
gccctgggtg 187260ggccctgtgg acctgtgagg tcagggtcag ggcatcactg
gaggcagagg gctgaagttg 187320tgggtctggg ttccccttgt gtgcacaggc
ccctgccctc catgcttggt caggcagcta 187380cccccaaaac tgctaggaca
ggctggtcct gaggtggatc ctggcccctg taccctctgg 187440acagcccacc
cgcccaacct tctaccctgc cccagcggcg gcagtgttgg ccacatcctt
187500cccctcctgg ccccaattgc tctggggaag tccaggctcc ggagcctgcc
caggggcccc 187560ccgtgatttg ggcccaggac tccacgtggt tctctgcctt
cacccaagcc ctgaactcct 187620cagctgccaa atccccaccc atctgcacag
gctgtgctca ccactgctgc tcctggaagg 187680tgcccctcag tgggacgccc
acctcctctc tgggcttctg tgtttgggag ccctgctgcc 187740cccacccttg
gtcagtcccc atgtcctgct ggcctgtcag gcagggcaga aaatccaccc
187800agaaatgctg agcaggatga gagtctagtt gggcccagcc tcattattta
gaagggatgg 187860aggcctaggg agcatgcttc tagcctgagc ccagcagggc
cccgcccatg tcccaggtct 187920gcaccaggga cagctcctgc cgaggcctga
cctgcccctt ctccctcagg tgctgctggt 187980tgaccagcct ctggccctag
gagaccccgt agcgactgag ggtcccagca ggccatgcag 188040ctttgccaag
gtacgagccc ctccccagca ggggacagat gtggggaccc tcccaggcag
188100gagcagctgg gtgcctggtg ctgccatctg ctgcctgcct ggttcttgtc
ctcacattgg 188160aggtcagtgt gagggctctg cctcgggaaa ggccatggag
cttgccctgt ccagggcctc 188220ccatgtgcac tgagcctggg aagagagggt
tggagttgag ccttttaccc tgggaatgct 188280gcctggagga tggtgcgggt
gtggggtggc accctgccag gcagggccct gcctccctgc 188340gcccactgga
actcgggcag gcaggggtgt aggtgcctcc tctagagccg tccggtgggg
188400gcccccggca gtggtggtgg tgtccactgg ccagcagctg ccccttcagc
caggacagta 188460ggcctgacgc tgtccccagc agctccaagg tggatttgtg
gaagggggta gagggcacgt 188520agaggcccca tgacctcccc agggttctgg
gagggctgtg cccccttagc cagcaccatg 188580ctgggtgata tagtcagatc
ctgttacccc tgttgtggag gtgaggaaac aggttagtgg 188640ggaggacatg
actaaggtcc atgctgagtc gctagagctg cacccagaac cactgctggg
188700accccatgcc tttctgctta ccccttgtgc cgggagatgc caagagatgc
tgggagccag 188760ccccacctct gcccttggag tcatggctac ggaaagggca
ttcggaccgg tccctgacct 188820caccggggag ggccgaaccc tgttcctgag
gagccagggc ttcctagagg aggtaggcct 188880tctagtcact ccttcatctg
caggcactcc acagagctct ctgtgccagc ccccagcacg 188940gagggctgac
cttagtcgag tggagatgcc ccagtgccag gcagtaggga tgatgtctcc
189000tgaggcccag atggaaggga ctggactagt ctcatggggc tgatggtggg
gccaggcctt 189060gaccagggac ccagtgtagg gggtgcagag acccctctga
gttcctcaca catccctggg 189120gccctcccca tacacttcct atcctgactg
cgggcaagag ggagccccag ttcgccttcc 189180ctatgctggg cacccacagt
ggggctgggc acccccgcca tgcccctgcc ctgtccttcc 189240cctgagagcc
tcggtcccac ctccaaggtg cctcagagga cagcaggggc agcgggcaga
189300ggccgagatg cctcctcatt ccaggctcag ctgcccttct tggggcagcc
cacacctgag 189360agtctcctgc agttggtcag gcctgaggag ggcagggggg
tgcctgctgt ccctctgctg 189420accacagtgg catttagcct gggcaccgcg
cccagcacag tccatgctgc acaggtgccg 189480tgggctccac agagccctgc
ctgacatgca tgtgttacgt ttcgggtgcc gatgcccttg 189540ggcggcactt
ctccgggcag aacccccagg ccaccgctcc ggttccggtt ccgctgcatc
189600tggggctctc ggcaggctgt ggtcctccgg ccagcctggg ggcatctcag
tccctcagcc 189660ccacaggggc ctgccccgca gcctgggcct cgagccccgt
ctccgcacgc tgtgccgaat 189720ctggctgccc atcagctccc tgcgtaccca
gactgtgccc tgccatgccc gtggctcttc 189780ccaggagtgc cctgtggcct
ccccctggct tgctgggctg attccctcct gtgtctcaaa 189840cagagctcac
ctttgccatc actgctgtcc tcaccggccg gtgccagagg cccgtgtctg
189900tgtaccctgt gtctgcacct ctgggcaggg cctggctctg accaacccgg
gcttccagtg 189960tccacagacc taaggcccag ggcgcctggg ggctggagca
agagaagcaa aaggagccaa 190020gggtgggggt ttggggttct tgtgagggcc
cagccccagg accccaggac caggacaccc 190080aggagcccca gggcccagcc
ccagttcaga aggcaggggc cttctgaggg agcttaaggg 190140tcccacagcc
caggaccccc accagggcca gtggccagcg ttgggggact cagcctcctc
190200gtcgctcgtc ctctctgttt ctcccacctt ttgccccctt tctccttgcc
tgttcccacc 190260cgaggccccc tcttggcctg cgtgagccgg ggcggcactg
aactgggggc cgatccgcct 190320gggcggcggt gagaggcagg gccgggagcc
gggccgctgg gtttgggcct ggcccgctcg 190380ccgcaatatt gatggcccgt
cagtgcagcc ctgattcctg tgctttcagt taaaaggttt 190440ctgttgttgt
agcttatgca gttgctctgt tgctatggaa acgtgacatc aaaatgacgt
190500ttcccgttta aaagctttta actaaattcc tgcctgtcag atgtaggccc
cattttgagc 190560gtggagctgc cttcgagcga gcgtgagcgg cgcctcccgc
ccatggtgcg tggggccggg 190620ccggggccct cgctgagcgc gctctctcac
cccacaggcg cctccggcat ggcggcggcc 190680gaggggcccg gctacctcgt
gtctccccag gcggagaagc accggcgggc ccgcaactgg 190740acggacgccg
agatgcgcgg cctcatgctg gtctgggagg agttcttcga cgagctcaag
190800cagaccaagc gcaacgccaa ggtgtacgag aagatggcca gcaagctctt
cgagatgacc 190860ggcgagcgca ggctgggcga ggagatcaag atcaagatca
ccaacatgac cttccagtac 190920aggtgggcga gcgggcagtg tgggccccac
caggacgggc gggcccgggc gtggcgggcc 190980gctcctgact ttcttggagc
tctgagtcgg gacgatgtgt gggtcgtggc ctgcctgtcg 191040gtctcctctg
gccgggtatg ggcagaaccc cacggggtga gacggggccc acggaaaccg
191100tgtgtgcagc cttccattgg ggaagtgggg aaactgaggc ccagcaaggg
caggaaacca 191160gtctaagagc tgaggggtag caggggtggg gctggtgctg
ggcagaggcc aggatggctc 191220ccaggacgta tgggcggtct gggcactgtc
cctcggaggc agcaacactc atggtggtgc 191280ccactgacct cacaccctgc
tcccccatag ggaggcggcg gctgccagtg ccctccccac 191340caccaagctc
ccaagctcag caggggtttc aggggcctac tgcgtcattg gggaaattga
191400gactgcaagt gagaaggagg ctcagtgctc tgcgacttgg agcatccact
gagcctctgc 191460catgagccgg tgagccccac tggggctggc cctagggtca
cggtggggta tttccagaaa 191520tcaccaggtg aggtgcagga ccagccagcg
catgggtggg gcttacggtg cgaagaagaa 191580agaggtggag gcctgccctg
gcccaggact cccagcgtgg gggctcccgg cctggcccca 191640cctctgctcc
tgctacatgg caggtgggcc cttcctgccc tggcaacctg cagggaaggc
191700cggaggggac cacccagcca gggagatgtt ggcgtctagg aggggacagg
tgtggtccca 191760cacacccagc atcttaaagt gcgtgggtcc ccagcccatt
aggacagggt cccgggtggg 191820caggggtcat ggtggggtga aggtctcagg
cacaggcaag gtcacaggtg cggtgagggt 191880cttgcagggt gtgaaggtca
taggtgtgcg gtgaaggtca caggtgtggg gtgatggttt 191940tgggtgtggg
gagggtcttg cacggagcga gggtggcagc aagagctgga agctgcaggg
192000ggagaatggc agcagagagc acccggccct gtgggcggcc tggacagggc
tgggcctggg 192060gctgccggag agcctgtcag cttccaggat gggagtggcc
tcactcagct gctccacctc 192120cgggtcaggc aggtgagcct ggggcagaga
ggctgagagc acctgagcca cttgtgggag 192180aggccacccc cactgccccc
ctcaggcgag gagccggcct ccagcacagc agaagggaac 192240ccccagtccc
cagccctagt gggagtgggg aagaggccca gcaaggcccc ggacagaccg
192300ccagcctgtg aggtctccgc tttcagttgc gttgatttga ttttttctga
gccttgaagg 192360aggggtccgg ggcctggccc tgcccaaagg cccctaggca
ggccccaaag ccgggaccta 192420gggtgctgag catgacggat gttgggtttg
agcggctggc ttgcgacgtg agggctgagg 192480tgtgagcctg ggtatcttca
gaggttcggt ggacacaggc agctgcccgc ggccccactg 192540ttcccgtggc
ctcctagtcc tgctcaggca cctggtgagg aagggacgca gagggcagtg
192600ggaggtggcc acgactgttc cagcaggctc ccctctgact caggaattca
cgggcaccac 192660ctccctggct ggctctggtt ggtgtctggc caggttattc
attatttatg ctgaaagcct 192720cttcagagtc ccaggggagg gtttctgtct
ccattcctgg aggctgagag atgagggtgc 192780agcagagtgg gggcctccac
tccagaccct gcagtctggg ctggccaagg gctgcaccgg 192840tgcactgcac
gtcatggctg atgaagcact tccacaccgc agcccctcag agctgccaca
192900gtcagcctta gttcaccgag ggggaagctg aggcccagag catgagaggg
acttgcccag 192960ggccacatag tccttagcag aggaagctgt ggctgggtga
ctcgatcttt gtcctttttc 193020tttatacccg cagtctcccc atagcagagg
cttttctttt ttttttcttt ttcttttttt 193080tttttttaca agaactcttt
atatattaag gctgttgggc tgaagaagcc tgagagggtg 193140gctggttctg
tggagcatgg tttgttgaag tacagtttgg gggcctccta cactgagaat
193200aggccttttc tcgtttctcc aaagagtggg ctggctcaag tagggcagag
agagaagcct 193260ggggcagagg ttagggatgg gcacccagcg cctgccctca
cacgctctgt gctggtgtct 193320tcacagccac gtgccaccct gggcagcatc
ccctgctcac catctggctg tgcctgtttg 193380ctgggggcac ctcattcaga
atccagctta ttgtttccaa cggccaatgg ccacaccctg 193440gcaggtagca
agagtaggag agaggagaca cccactccga gcacaggttg ggtttggagc
193500ccggccttgg ggcactctgt cactcaaagg cagagtgggg agtgggcact
gggccttagg 193560aggtactggg tccagtgagg cagagatgcc cctgccccac
ccccaccttg tggcttcttc 193620cctggcctgg ccagagctgt ctggccgcca
tggggccctg tgtctcctgc cttgacctcc 193680cagagggcag ccgaggccca
ggggaggcct ggggacttag cctctcaggg caggacctgt 193740ctgcaggagt
aggtgggtgc tgggggtccc agtggtaatg aggcatcagg cagtgtggga
193800aggggcccat ccggcccacc ccagggcctc tgggcaggtt gcaggttgta
gcgctggatc 193860taggctcctg cccagactgt aggttcaacc aagaatggca
tgggagccca gcctgctgtt 193920tgctttatta aatctgccct gtagctgggg
gaggggctta ctttgatcat cactatgtca 193980ttgatataaa aatagaggct
cagagaggtg aatgaacctg cccaaagtca cacagcaaag 194040tgtggagatg
agatactgac tcagggctgt ggacactgaa gcctgtgctc taacgccagt
194100ggctgtcgct ccctgaggca ttctctcccg aacaacacag ttattatatt
acaaaatatt 194160atcactatat ttatatatct tataatacct tattattaca
ataaaacctt attactctac 194220ctttcaaaat gaattattta aaaagcagta
tttgctcatt gcagagagtc tagaaactat 194280agaaaagcaa gggaaaagca
ataggaccag ccccaaggtc ccagcatgca cagataacct 194340tagtaatact
gggacgtgtg cttccttttt aacatctgag cccgtgtagg tcctgaagcc
194400cagcttcttt ctaagtccat tgtcatcttg accctggagc ctggccgatt
ttgctgggga 194460ggcccttgcc agccgagagc ggctcctgcc tgtgccggcg
tggcgcgccc ctctgctgag 194520gctgggcagg acaggggctg ggccagctct
gtttctcacc cttggctctt gtgtctctcg 194580tttcaggaaa ttaaaatgca
tgacagatag cgagtccgcc ccgcccgact ggccctatta 194640cctagccatt
gatgggattc tggccaaggt ccccgagtcc tgtgatggca aactgccgga
194700cagccagccg ccggggccct ccacgtccca gaccgaggcg tccctgtcgc
cgcccgctaa 194760gtccacccct ctgtacttcc cgtataacca gtgctcctac
gaaggccgct tcgaggatga 194820tcgctccgac agctcctcca gcttactgtc
ccttaagttc aggtagtgtg tctgcttgtc 194880cttcccctgc cctggggtat
ctcagccccc accatttaga gaaagggact gggagtggca 194940aggccggcgg
cggcggccac agtggttgca gaggccgtgg ctgcgggcag cgcctccagg
195000gacaggcggc ctcagaccag ggagggcttt agtgtccaca ggcagaccga
gtttgtctcc 195060cagctccatc acttttgagc tgcacggaaa gttccttgac
ttctctggcc tcagtctccc 195120tcctataaaa tgggggtaaa tcagtacctt
tctcagaggg tggctgggag catcacagga 195180gagaagacgc agcatggggc
ccggcacacg gagggagacc aagccccaga ccccagaatg 195240cgccccctgg
cctcccttag cccacacaga ccccaccctc acaggctagc tgccctctca
195300gcactgggga gggtgtcggg ctgcacctca tcacgtgttg ccgtgggcat
gacccgtccc 195360ctctgccatc catcccacac ctcagacccg tcccgtgctg
gccacgtgac tgtgcctgca 195420agatgctcac agggcagccg ggagccaggc
agcatgcagg acagacacct gcggggtggg 195480cctggggagc ccagagaagg
tgcttttgag gaggggacat ttggggtggg ctttcaaggt 195540aaaatagaag
ttggccattt ggaggcaaga acaggaagat tgtggatttg agtcacagct
195600tctcccctgc cctggtcttc aagtctttct gacaggaggt gtcagaaaag
tatctttagt 195660agagaaggcg tctccgagga gggtccctct catgccgggg
gccgctgctt gactcaggat 195720ttctcattga agacctgaga caaaaacgct
tttgctggca
gctagaagga accagcagga 195780ggcctgagat ttgtggctgt tgttcccgtg
gactgagccc agttctcaga ctcagctgcc 195840tggggccttg cacaggactg
gggcgtgggg gctgccctcc ctgatcaggc ccaaagcgcg 195900gatctcacgc
ccctgaggtt ggctgtaccc tctcagctca gagcagagtg tgggccaggg
195960atgagcaggc actggagcag ggccctgggg tctgtgggtt ttggcagctc
cctgcccttc 196020agggaggtct gctgagacca cgggtggccc ctaccccagc
agcagagctc tcaggaggcg 196080cccacagggc tggactgcct ttactcacca
cctctaccag agctctgagg tcctggggag 196140agagcccagg cctcttgtgg
gccccacacc ctctaggtgc ctgtccttct gcctctctac 196200caaggtgtgc
cggccccatt tctaggccgc cgggagataa gggggctcac atctcaggcc
196260cttccttctg ggacctcagt ttccccatct gcctaaggcc gggtggggct
ggtggtcttg 196320gcttccctac aggggtcctg agtactctgc actacccagc
accccccacc cctgccttca 196380tctctccctg ggggtggtct ctccacccct
ggcccccaac tggggctgag cccccacctg 196440cccagtttgg tgggtgaagg
gtgctccctg gcaggatatg cccctctgca gcccagaaca 196500tcccaccctt
tccagaccga aggggtgtgg attgtcctgg gaccctggtc attggggtca
196560tccgctagtc gcaaaggacg gcaatgcctg tggcctctct ttctttcttt
ttcttttttt 196620ttttttttga gacggagtct cgctcttgtg cagagagcag
tggcgcgatc ttggctcact 196680gcaacctccg cctcgtgggt tcaagcgatt
ctcctgcctc agcctcccga gtagctggga 196740ttacaggcac ccgccacaac
gcctggctaa tttttgtatt tttagtagag atggggtttc 196800accatgttgg
ccaggctggt cttgaactcc tgacctcagg tgatccacct gcctctgcct
196860cccaaagtgc tgggattaca ggcataagcc tccacacccg gccacccctg
ttactttctg 196920tcaaaggcgg tgggttctgg cccctccttt gcacatggaa
tatgagaccc tgagtaagtg 196980acctgactcc ctggggcctc agtttcccca
tttgcccagt aggattgtcg ggagggtccg 197040gtgaggcccc tggtgtgccc
aggctctgtg gccagcacgt ccacagccgg cactgtcctt 197100ccaggtcgga
ggagcggccg gtgaagaagc gcaaggtgca gagctgccac ctgcagaaga
197160agcagctgcg gctgctggag gccatggtgg aggagcagcg ccggctgagc
cgcgccgtgg 197220aggagacctg ccgcgaggtg cgccgcgtgc tggaccagca
gcacatcctg caggtgcaga 197280gcctgcagct gcaggagcgc atgatgagtc
tgctggagag gatcatcacc aagtccagcg 197340tctaggccag caggcggcgg
cggcggcggg gccgggcggc tggtggtact gctcaggcca 197400cccagggcag
gccactcagg ccaggcgggc aagggggccg ccccgcgagc ggagaccgcc
197460ttccacctgg cctctggcag gatgtccctt ctgaggggta ttttgaggaa
cccccaggcc 197520ctggggaccg tgaggctcca gtctccagca tgaatgccct
tcctcggaca caggccaggg 197580cctctggggt tcactccgag taagaacgtc
ctagagccac tctccagtgt cgttactatc 197640aatgatactt gacgtggctt
tgatattaaa cgtatacttt ttcattcttg cctggaacgc 197700acagtttgct
gttgctggct tggtgaggat gccctgattg atggatcccg aaaatgaaag
197760cagatggaaa cgggttgggg caggctggag ctgggggagc tctctcctga
agggaaccct 197820gtgtcctccc tcaccaggac ctctgcgtct ctccttaaat
ggcctctgac gcctgatgaa 197880aaccccagcg accttccagg aggcttttat
tcagctctgt ttggagcatc aggtgtttcc 197940actgcctcct tagcaatgac
actaataaaa gtcgtaacac ctgttcacat gcacagccct 198000gttgagtgtt
ctgggtgctg gagatatcat ggtggatgac acaaaggccc tggcctcttg
198060gagcttatgc tcccatgcgg ggaagacaca tgggtcagta gagaaatggt
tgcaggttgt 198120gataagtgct ggaagggagg ggttggcctg aggacacgga
ggcagacata cgtggagctg 198180ggaacagtgg ccacacaggg aacggccagt
gcgaaggccc agaggcagag gacactggag 198240caagcccagg agcagctagg
aggctggtgg ccagcagcca ggccacggaa gcccgtgcag 198300cccgtgggga
ggagtgttca tgcttttcaa gcttagtggg agtcttttgg ccagtgcagc
198360tctgggtctg acatcggtgg gggacagagg ggtggtggag cggccacagc
tgcaagctca 198420cctcactgcc ggcccttcca ccagtttcaa actctttcta
gaagctccag ctttcccaaa 198480gctgaattct ctatgagcct ccttggccgg
gactcgggcg tctggttgcc ctggctgcaa 198540aggaggctgg ggccaggtgt
gtttgagtca cctcctggaa ttaggcaagt tgctgcccaa 198600atagaaggtt
gttggcaggt gggtcagcag gtgaacagca tggtttgact cagggttcag
198660aaaaatctcc ctctggctgc caagcgagca ggccgtggag acaggtgcag
aggcaggtgt 198720ggcagcaggc atcctgccag gcagtgctgc agtcatcctg
cgacaagcag cagcagctca 198780tcctaccctc tagggggtct tgaggtcagc
caggcaagag agcagcttgg actccactgg 198840gtgtgggacc agcctgtgga
ccatggtggt gtggagggtg ccctcggcct gcctgtgtga 198900aggagaggcc
ggcgtgttct gtggagccca aaggggagct gggcaagcag gattcacttc
198960actctgaggg tcctggagct cccaccctcc tcagccatct ccccagagcc
tgtgtgccga 199020ggactcggcc catgttgctg tgggatgaga ggcagagtgt
cgtgagggtg taaggagcgg 199080cggcagtggt gggaggaggg agcagcagcc
agcgctacgg tgccagtttc cagctgccag 199140atgacgccgc tgaccctgtg
gttgagaaga gatgcacaga gccagctctt gcaagccagt 199200gtggctgcca
tagcacctgc cgagaagcag aaggaagggt ggccccagga ggacagagga
199260tgcgggcaca tctgatgcgg gcctgagttt tgggagcttt tgctctagcc
agtttccagc 199320tccgggaccc acccgcctcg taggcaagac accacccaag
aaatcatttg cttaacaaac 199380acactgggct ccaactggac acctgtgcca
ccctagatgc tgggaaccca gccatgacac 199440aggcacctgc ccccagctgc
tgaccactga ggctggctag cagctcccat ggggccagtg 199500tggggttccc
cagcctccta acagggagcc agtcacaagc cctcgagagg gaagggtgcc
199560cgcggccctg gcaggaaggt taggctggac gctcccacaa gacataacag
atggaggttc 199620taaatgatgt agcaacttct tcaccctgaa actgctgtag
agtcagccat gacgcaccgg 199680tacttcagta actgccaggc atccgggaca
gcacaccgcg agtcgctgct gtgcttgggt 199740tagaagtggt ttggtctgtt
ttcttctcgc cctctctaat cagagtcagt gattcatgcc 199800cttccatcac
cttagagaag gggcaggcgc tgcccgacct tctccaggct ggagcagcat
199860cgcctcatgt cagcagaact cagctgtaga atatcgtggg gttggtgcct
ttcatcagca 199920gcatgtcctt aacaactttc tgatttcttc cttagttgtt
ggtccattaa ggagaaaaaa 199980aatgatctca gccattgcta aaatatttga
taagattcag caaagcagca tgttaacatt 200040gaaaactaga atcaggagcc
aggcagatgt gcttgctttt cacctgtagt atttcatgtt 200100gttttgacgt
ttttagctaa tgcattaaga taaataaaca aaagccgggc acggtggttc
200160acgcctgtaa tcccagcact ttgggaggct gaggcgggag gatcctctga
ggtcaggagt 200220tcaagaccag cctgaccaac atggagaaac ctcgtcatta
ctaaaaatac aaaattagct 200280gggcgtggtg gtgcatgcct gtaatcccag
ctacttggga ggctgaggca ggagaatcgc 200340ttgaacccgg gaggcggagg
ttgcagtgag ctgagattgc accactgcac tccagcctgg 200400gtgacagtga
aactcggtct caaaaaaaaa aaaaaattaa aaaaagataa ataaaataag
200460caggataaga aatgaagaaa gtagagttac ctttgttttc agatttcatt
tttgtatacc 200520cagaaagcca aatgtacaaa agactgggag ctctttaaac
cagcttaaac ttgttgaaaa 200580tgaggatgaa gaaatatccc attcagagtt
ggaatgaatt taacccagaa ggaacaggac 200640ctctactgaa gagaactatg
cagtcttact gaaaaatcta aataatacct gagcgctgga 200700gaaacttcgc
acactcctga aagctccaaa gtcaatgtca tcattttatt aatgtcattc
200760caaacatagt ctcaataata tcacttcttg gttttgacat ggacgcgatg
atgtttaaat 200820tcatatgaaa aaagaacggg gccaaaagtc caaggccagt
cagcgtgaga agaccgctcg 200880gcctccctcg gagtcgggga gttggaaccg
cagactgaga tcatgtggct gctggaggcc 200940aggacgaacg tcgggaaatg
gagactcctg cgttgctggt gggatgtggt gcagccgctt 201000ccaggagcaa
tttggtgtcc cgtcctaaag ctgaagaaac gcatttcctc tggtcagtgc
201060cactcctaga caggccaccc tgcggcagcc gtcctcaaac tggtctgagg
acccctcaac 201120gctcttaaaa atcattaaaa gtgggccagg tgcggtggct
cacacctgta atcccagcac 201180tttgggaggc caagacaggc ggatcacgag
gtcaggacat tgagatcatc ctggctaaca 201240cggtgaaacc ccgtctctac
taaaaataca aaaaattagc cgggcgtggt ggcgggcgcc 201300tgtagtccca
gctacttggg aggctgagcc aggagaatgg cgtgaaccca ggaggtggag
201360cttgcagtga gctgagatca ctccactgca ctccagcctg ggcagcagag
cgagactctg 201420tctcaaaaaa aaataataaa taaataaata aaaataaaat
aaaataaaat tcattaaaag 201480tgccaaagaa cttttgctta tgtgagttct
aatgaccaat attaatacac attagaatat 201540cttattagaa attaaacctg
agacctttag aaaacatgta ttcatttcaa aatagcaata 201600aacccatgac
atattaacat aaataacaat tgtatgaaaa atatattttc caaaacaaaa
201660agttttcggg agaagtgtgg catagtttta catggtcgta aatctctggc
ttaagagaag 201720cccactggcc tctcagcagg ctctgggtcc gtccactttg
ggggtgtttt ggttgtgaag 201780tataggagtg aatggagaag ctcattctta
cccagatgtg tatttgaaaa gaaaaggaac 201840attttaataa cctttgcaaa
taatcggtat attcttccgt gatcctattc caacactgga 201900caggtggtgg
tttgtttttt ttttttggag acggagtccc gctctgtcac tcaggctgga
201960gtgcagtggc gcgatttcag ctcactgcaa gctccgcctc c
202001213481DNAHomo sapiensCDS(146)..(9580) 2gctgccggga cgggtccaag
atggacggcc gctcaggttc tgcttttacc tgcggcccag 60agccccattc attgccccgg
tgctgagcgg cgccgcgagt cggcccgagg cctccgggga 120ctgccgtgcc
gggcgggaga ccgcc atg gcg acc ctg gaa aag ctg atg aag 172 Met Ala
Thr Leu Glu Lys Leu Met Lys 1 5gcc ttc gag tcc ctc aag tcc ttc cag
cag cag cag cag cag cag cag 220Ala Phe Glu Ser Leu Lys Ser Phe Gln
Gln Gln Gln Gln Gln Gln Gln10 15 20 25cag cag cag cag cag cag cag
cag cag cag cag cag cag caa cag ccg 268Gln Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gln Gln Gln Gln Gln Pro 30 35 40cca ccg ccg ccg ccg ccg
ccg ccg cct cct cag ctt cct cag ccg ccg 316Pro Pro Pro Pro Pro Pro
Pro Pro Pro Pro Gln Leu Pro Gln Pro Pro 45 50 55ccg cag gca cag ccg
ctg ctg cct cag ccg cag ccg ccc ccg ccg ccg 364Pro Gln Ala Gln Pro
Leu Leu Pro Gln Pro Gln Pro Pro Pro Pro Pro 60 65 70ccc ccg ccg cca
ccc ggc ccg gct gtg gct gag gag ccg ctg cac cga 412Pro Pro Pro Pro
Pro Gly Pro Ala Val Ala Glu Glu Pro Leu His Arg 75 80 85cca aag aaa
gaa ctt tca gct acc aag aaa gac cgt gtg aat cat tgt 460Pro Lys Lys
Glu Leu Ser Ala Thr Lys Lys Asp Arg Val Asn His Cys90 95 100 105ctg
aca ata tgt gaa aac ata gtg gca cag tct gtc aga aat tct cca 508Leu
Thr Ile Cys Glu Asn Ile Val Ala Gln Ser Val Arg Asn Ser Pro 110 115
120gaa ttt cag aaa ctt ctg ggc atc gct atg gaa ctt ttt ctg ctg tgc
556Glu Phe Gln Lys Leu Leu Gly Ile Ala Met Glu Leu Phe Leu Leu Cys
125 130 135agt gat gac gca gag tca gat gtc agg atg gtg gct gac gaa
tgc ctc 604Ser Asp Asp Ala Glu Ser Asp Val Arg Met Val Ala Asp Glu
Cys Leu 140 145 150aac aaa gtt atc aaa gct ttg atg gat tct aat ctt
cca agg tta cag 652Asn Lys Val Ile Lys Ala Leu Met Asp Ser Asn Leu
Pro Arg Leu Gln 155 160 165ctc gag ctc tat aag gaa att aaa aag aat
ggt gcc cct cgg agt ttg 700Leu Glu Leu Tyr Lys Glu Ile Lys Lys Asn
Gly Ala Pro Arg Ser Leu170 175 180 185cgt gct gcc ctg tgg agg ttt
gct gag ctg gct cac ctg gtt cgg cct 748Arg Ala Ala Leu Trp Arg Phe
Ala Glu Leu Ala His Leu Val Arg Pro 190 195 200cag aaa tgc agg cct
tac ctg gtg aac ctt ctg ccg tgc ctg act cga 796Gln Lys Cys Arg Pro
Tyr Leu Val Asn Leu Leu Pro Cys Leu Thr Arg 205 210 215aca agc aag
aga ccc gaa gaa tca gtc cag gag acc ttg gct gca gct 844Thr Ser Lys
Arg Pro Glu Glu Ser Val Gln Glu Thr Leu Ala Ala Ala 220 225 230gtt
ccc aaa att atg gct tct ttt ggc aat ttt gca aat gac aat gaa 892Val
Pro Lys Ile Met Ala Ser Phe Gly Asn Phe Ala Asn Asp Asn Glu 235 240
245att aag gtt ttg tta aag gcc ttc ata gcg aac ctg aag tca agc tcc
940Ile Lys Val Leu Leu Lys Ala Phe Ile Ala Asn Leu Lys Ser Ser
Ser250 255 260 265ccc acc att cgg cgg aca gcg gct gga tca gca gtg
agc atc tgc cag 988Pro Thr Ile Arg Arg Thr Ala Ala Gly Ser Ala Val
Ser Ile Cys Gln 270 275 280cac tca aga agg aca caa tat ttc tat agt
tgg cta cta aat gtg ctc 1036His Ser Arg Arg Thr Gln Tyr Phe Tyr Ser
Trp Leu Leu Asn Val Leu 285 290 295tta ggc tta ctc gtt cct gtc gag
gat gaa cac tcc act ctg ctg att 1084Leu Gly Leu Leu Val Pro Val Glu
Asp Glu His Ser Thr Leu Leu Ile 300 305 310ctt ggc gtg ctg ctc acc
ctg agg tat ttg gtg ccc ttg ctg cag cag 1132Leu Gly Val Leu Leu Thr
Leu Arg Tyr Leu Val Pro Leu Leu Gln Gln 315 320 325cag gtc aag gac
aca agc ctg aaa ggc agc ttc gga gtg aca agg aaa 1180Gln Val Lys Asp
Thr Ser Leu Lys Gly Ser Phe Gly Val Thr Arg Lys330 335 340 345gaa
atg gaa gtc tct cct tct gca gag cag ctt gtc cag gtt tat gaa 1228Glu
Met Glu Val Ser Pro Ser Ala Glu Gln Leu Val Gln Val Tyr Glu 350 355
360ctg acg tta cat cat aca cag cac caa gac cac aat gtt gtg acc gga
1276Leu Thr Leu His His Thr Gln His Gln Asp His Asn Val Val Thr Gly
365 370 375gcc ctg gag ctg ttg cag cag ctc ttc aga acg cct cca ccc
gag ctt 1324Ala Leu Glu Leu Leu Gln Gln Leu Phe Arg Thr Pro Pro Pro
Glu Leu 380 385 390ctg caa acc ctg acc gca gtc ggg ggc att ggg cag
ctc acc gct gct 1372Leu Gln Thr Leu Thr Ala Val Gly Gly Ile Gly Gln
Leu Thr Ala Ala 395 400 405aag gag gag tct ggt ggc cga agc cgt agt
ggg agt att gtg gaa ctt 1420Lys Glu Glu Ser Gly Gly Arg Ser Arg Ser
Gly Ser Ile Val Glu Leu410 415 420 425ata gct gga ggg ggt tcc tca
tgc agc cct gtc ctt tca aga aaa caa 1468Ile Ala Gly Gly Gly Ser Ser
Cys Ser Pro Val Leu Ser Arg Lys Gln 430 435 440aaa ggc aaa gtg ctc
tta gga gaa gaa gaa gcc ttg gag gat gac tct 1516Lys Gly Lys Val Leu
Leu Gly Glu Glu Glu Ala Leu Glu Asp Asp Ser 445 450 455gaa tcg aga
tcg gat gtc agc agc tct gcc tta aca gcc tca gtg aag 1564Glu Ser Arg
Ser Asp Val Ser Ser Ser Ala Leu Thr Ala Ser Val Lys 460 465 470gat
gag atc agt gga gag ctg gct gct tct tca ggg gtt tcc act cca 1612Asp
Glu Ile Ser Gly Glu Leu Ala Ala Ser Ser Gly Val Ser Thr Pro 475 480
485ggg tca gca ggt cat gac atc atc aca gaa cag cca cgg tca cag cac
1660Gly Ser Ala Gly His Asp Ile Ile Thr Glu Gln Pro Arg Ser Gln
His490 495 500 505aca ctg cag gcg gac tca gtg gat ctg gcc agc tgt
gac ttg aca agc 1708Thr Leu Gln Ala Asp Ser Val Asp Leu Ala Ser Cys
Asp Leu Thr Ser 510 515 520tct gcc act gat ggg gat gag gag gat atc
ttg agc cac agc tcc agc 1756Ser Ala Thr Asp Gly Asp Glu Glu Asp Ile
Leu Ser His Ser Ser Ser 525 530 535cag gtc agc gcc gtc cca tct gac
cct gcc atg gac ctg aat gat ggg 1804Gln Val Ser Ala Val Pro Ser Asp
Pro Ala Met Asp Leu Asn Asp Gly 540 545 550acc cag gcc tcg tcg ccc
atc agc gac agc tcc cag acc acc acc gaa 1852Thr Gln Ala Ser Ser Pro
Ile Ser Asp Ser Ser Gln Thr Thr Thr Glu 555 560 565ggg cct gat tca
gct gtt acc cct tca gac agt tct gaa att gtg tta 1900Gly Pro Asp Ser
Ala Val Thr Pro Ser Asp Ser Ser Glu Ile Val Leu570 575 580 585gac
ggt acc gac aac cag tat ttg ggc ctg cag att gga cag ccc cag 1948Asp
Gly Thr Asp Asn Gln Tyr Leu Gly Leu Gln Ile Gly Gln Pro Gln 590 595
600gat gaa gat gag gaa gcc aca ggt att ctt cct gat gaa gcc tcg gag
1996Asp Glu Asp Glu Glu Ala Thr Gly Ile Leu Pro Asp Glu Ala Ser Glu
605 610 615gcc ttc agg aac tct tcc atg gcc ctt caa cag gca cat tta
ttg aaa 2044Ala Phe Arg Asn Ser Ser Met Ala Leu Gln Gln Ala His Leu
Leu Lys 620 625 630aac atg agt cac tgc agg cag cct tct gac agc agt
gtt gat aaa ttt 2092Asn Met Ser His Cys Arg Gln Pro Ser Asp Ser Ser
Val Asp Lys Phe 635 640 645gtg ttg aga gat gaa gct act gaa ccg ggt
gat caa gaa aac aag cct 2140Val Leu Arg Asp Glu Ala Thr Glu Pro Gly
Asp Gln Glu Asn Lys Pro650 655 660 665tgc cgc atc aaa ggt gac att
gga cag tcc act gat gat gac tct gca 2188Cys Arg Ile Lys Gly Asp Ile
Gly Gln Ser Thr Asp Asp Asp Ser Ala 670 675 680cct ctt gtc cat tgt
gtc cgc ctt tta tct gct tcg ttt ttg cta aca 2236Pro Leu Val His Cys
Val Arg Leu Leu Ser Ala Ser Phe Leu Leu Thr 685 690 695ggg gga aaa
aat gtg ctg gtt ccg gac agg gat gtg agg gtc agc gtg 2284Gly Gly Lys
Asn Val Leu Val Pro Asp Arg Asp Val Arg Val Ser Val 700 705 710aag
gcc ctg gcc ctc agc tgt gtg gga gca gct gtg gcc ctc cac ccg 2332Lys
Ala Leu Ala Leu Ser Cys Val Gly Ala Ala Val Ala Leu His Pro 715 720
725gaa tct ttc ttc agc aaa ctc tat aaa gtt cct ctt gac acc acg gaa
2380Glu Ser Phe Phe Ser Lys Leu Tyr Lys Val Pro Leu Asp Thr Thr
Glu730 735 740 745tac cct gag gaa cag tat gtc tca gac atc ttg aac
tac atc gat cat 2428Tyr Pro Glu Glu Gln Tyr Val Ser Asp Ile Leu Asn
Tyr Ile Asp His 750 755 760gga gac cca cag gtt cga gga gcc act gcc
att ctc tgt ggg acc ctc 2476Gly Asp Pro Gln Val Arg Gly Ala Thr Ala
Ile Leu Cys Gly Thr Leu 765 770 775atc tgc tcc atc ctc agc agg tcc
cgc ttc cac gtg gga gat tgg atg 2524Ile Cys Ser Ile Leu Ser Arg Ser
Arg Phe His Val Gly Asp Trp Met 780 785 790ggc acc att aga acc ctc
aca gga aat aca ttt tct ttg gcg gat tgc 2572Gly Thr Ile Arg Thr Leu
Thr Gly Asn Thr Phe Ser Leu Ala Asp Cys 795 800 805att cct ttg ctg
cgg aaa aca ctg aag gat gag tct tct gtt act tgc 2620Ile Pro Leu Leu
Arg Lys Thr Leu Lys Asp Glu Ser Ser Val Thr Cys810 815 820 825aag
tta gct tgt aca gct gtg agg aac tgt gtc atg agt ctc tgc agc 2668Lys
Leu Ala Cys Thr Ala Val Arg Asn Cys Val Met Ser Leu Cys Ser 830 835
840agc agc tac agt gag tta gga ctg cag ctg atc atc gat gtg ctg act
2716Ser Ser Tyr Ser Glu Leu Gly Leu Gln Leu Ile Ile Asp Val Leu Thr
845 850 855ctg agg aac agt tcc tat tgg ctg gtg agg aca gag ctt ctg
gaa acc 2764Leu Arg Asn Ser Ser Tyr Trp Leu Val Arg Thr Glu Leu Leu
Glu Thr 860 865 870ctt gca gag att gac ttc agg ctg gtg agc ttt ttg
gag gca
aaa gca 2812Leu Ala Glu Ile Asp Phe Arg Leu Val Ser Phe Leu Glu Ala
Lys Ala 875 880 885gaa aac tta cac aga ggg gct cat cat tat aca ggg
ctt tta aaa ctg 2860Glu Asn Leu His Arg Gly Ala His His Tyr Thr Gly
Leu Leu Lys Leu890 895 900 905caa gaa cga gtg ctc aat aat gtt gtc
atc cat ttg ctt gga gat gaa 2908Gln Glu Arg Val Leu Asn Asn Val Val
Ile His Leu Leu Gly Asp Glu 910 915 920gac ccc agg gtg cga cat gtt
gcc gca gca tca cta att agg ctt gtc 2956Asp Pro Arg Val Arg His Val
Ala Ala Ala Ser Leu Ile Arg Leu Val 925 930 935cca aag ctg ttt tat
aaa tgt gac caa gga caa gct gat cca gta gtg 3004Pro Lys Leu Phe Tyr
Lys Cys Asp Gln Gly Gln Ala Asp Pro Val Val 940 945 950gcc gtg gca
aga gat caa agc agt gtt tac ctg aaa ctt ctc atg cat 3052Ala Val Ala
Arg Asp Gln Ser Ser Val Tyr Leu Lys Leu Leu Met His 955 960 965gag
acg cag cct cca tct cat ttc tcc gtc agc aca ata acc aga ata 3100Glu
Thr Gln Pro Pro Ser His Phe Ser Val Ser Thr Ile Thr Arg Ile970 975
980 985tat aga ggc tat aac cta cta cca agc ata aca gac gtc act atg
gaa 3148Tyr Arg Gly Tyr Asn Leu Leu Pro Ser Ile Thr Asp Val Thr Met
Glu 990 995 1000aat aac ctt tca aga gtt att gca gca gtt tct cat gaa
cta atc 3193Asn Asn Leu Ser Arg Val Ile Ala Ala Val Ser His Glu Leu
Ile 1005 1010 1015aca tca acc acc aga gca ctc aca ttt gga tgc tgt
gaa gct ttg 3238Thr Ser Thr Thr Arg Ala Leu Thr Phe Gly Cys Cys Glu
Ala Leu 1020 1025 1030tgt ctt ctt tcc act gcc ttc cca gtt tgc att
tgg agt tta ggt 3283Cys Leu Leu Ser Thr Ala Phe Pro Val Cys Ile Trp
Ser Leu Gly 1035 1040 1045tgg cac tgt gga gtg cct cca ctg agt gcc
tca gat gag tct agg 3328Trp His Cys Gly Val Pro Pro Leu Ser Ala Ser
Asp Glu Ser Arg 1050 1055 1060aag agc tgt acc gtt ggg atg gcc aca
atg att ctg acc ctg ctc 3373Lys Ser Cys Thr Val Gly Met Ala Thr Met
Ile Leu Thr Leu Leu 1065 1070 1075tcg tca gct tgg ttc cca ttg gat
ctc tca gcc cat caa gat gct 3418Ser Ser Ala Trp Phe Pro Leu Asp Leu
Ser Ala His Gln Asp Ala 1080 1085 1090ttg att ttg gcc gga aac ttg
ctt gca gcc agt gct ccc aaa tct 3463Leu Ile Leu Ala Gly Asn Leu Leu
Ala Ala Ser Ala Pro Lys Ser 1095 1100 1105ctg aga agt tca tgg gcc
tct gaa gaa gaa gcc aac cca gca gcc 3508Leu Arg Ser Ser Trp Ala Ser
Glu Glu Glu Ala Asn Pro Ala Ala 1110 1115 1120acc aag caa gag gag
gtc tgg cca gcc ctg ggg gac cgg gcc ctg 3553Thr Lys Gln Glu Glu Val
Trp Pro Ala Leu Gly Asp Arg Ala Leu 1125 1130 1135gtg ccc atg gtg
gag cag ctc ttc tct cac ctg ctg aag gtg att 3598Val Pro Met Val Glu
Gln Leu Phe Ser His Leu Leu Lys Val Ile 1140 1145 1150aac att tgt
gcc cac gtc ctg gat gac gtg gct cct gga ccc gca 3643Asn Ile Cys Ala
His Val Leu Asp Asp Val Ala Pro Gly Pro Ala 1155 1160 1165ata aag
gca gcc ttg cct tct cta aca aac ccc cct tct cta agt 3688Ile Lys Ala
Ala Leu Pro Ser Leu Thr Asn Pro Pro Ser Leu Ser 1170 1175 1180ccc
atc cga cga aag ggg aag gag aaa gaa cca gga gaa caa gca 3733Pro Ile
Arg Arg Lys Gly Lys Glu Lys Glu Pro Gly Glu Gln Ala 1185 1190
1195tct gta ccg ttg agt ccc aag aaa ggc agt gag gcc agt gca gct
3778Ser Val Pro Leu Ser Pro Lys Lys Gly Ser Glu Ala Ser Ala Ala
1200 1205 1210tct aga caa tct gat acc tca ggt cct gtt aca aca agt
aaa tcc 3823Ser Arg Gln Ser Asp Thr Ser Gly Pro Val Thr Thr Ser Lys
Ser 1215 1220 1225tca tca ctg ggg agt ttc tat cat ctt cct tca tac
ctc aaa ctg 3868Ser Ser Leu Gly Ser Phe Tyr His Leu Pro Ser Tyr Leu
Lys Leu 1230 1235 1240cat gat gtc ctg aaa gct aca cac gct aac tac
aag gtc acg ctg 3913His Asp Val Leu Lys Ala Thr His Ala Asn Tyr Lys
Val Thr Leu 1245 1250 1255gat ctt cag aac agc acg gaa aag ttt gga
ggg ttt ctc cgc tca 3958Asp Leu Gln Asn Ser Thr Glu Lys Phe Gly Gly
Phe Leu Arg Ser 1260 1265 1270gcc ttg gat gtt ctt tct cag ata cta
gag ctg gcc aca ctg cag 4003Ala Leu Asp Val Leu Ser Gln Ile Leu Glu
Leu Ala Thr Leu Gln 1275 1280 1285gac att ggg aag tgt gtt gaa gag
atc cta gga tac ctg aaa tcc 4048Asp Ile Gly Lys Cys Val Glu Glu Ile
Leu Gly Tyr Leu Lys Ser 1290 1295 1300tgc ttt agt cga gaa cca atg
atg gca act gtt tgt gtt caa caa 4093Cys Phe Ser Arg Glu Pro Met Met
Ala Thr Val Cys Val Gln Gln 1305 1310 1315ttg ttg aag act ctc ttt
ggc aca aac ttg gcc tcc cag ttt gat 4138Leu Leu Lys Thr Leu Phe Gly
Thr Asn Leu Ala Ser Gln Phe Asp 1320 1325 1330ggc tta tct tcc aac
ccc agc aag tca caa ggc cga gca cag cgc 4183Gly Leu Ser Ser Asn Pro
Ser Lys Ser Gln Gly Arg Ala Gln Arg 1335 1340 1345ctt ggc tcc tcc
agt gtg agg cca ggc ttg tac cac tac tgc ttc 4228Leu Gly Ser Ser Ser
Val Arg Pro Gly Leu Tyr His Tyr Cys Phe 1350 1355 1360atg gcc ccg
tac acc cac ttc acc cag gcc ctc gct gac gcc agc 4273Met Ala Pro Tyr
Thr His Phe Thr Gln Ala Leu Ala Asp Ala Ser 1365 1370 1375ctg agg
aac atg gtg cag gcg gag cag gag aac gac acc tcg gga 4318Leu Arg Asn
Met Val Gln Ala Glu Gln Glu Asn Asp Thr Ser Gly 1380 1385 1390tgg
ttt gat gtc ctc cag aaa gtg tct acc cag ttg aag aca aac 4363Trp Phe
Asp Val Leu Gln Lys Val Ser Thr Gln Leu Lys Thr Asn 1395 1400
1405ctc acg agt gtc aca aag aac cgt gca gat aag aat gct att cat
4408Leu Thr Ser Val Thr Lys Asn Arg Ala Asp Lys Asn Ala Ile His
1410 1415 1420aat cac att cgt ttg ttt gaa cct ctt gtt ata aaa gct
tta aaa 4453Asn His Ile Arg Leu Phe Glu Pro Leu Val Ile Lys Ala Leu
Lys 1425 1430 1435cag tac acg act aca aca tgt gtg cag tta cag aag
cag gtt tta 4498Gln Tyr Thr Thr Thr Thr Cys Val Gln Leu Gln Lys Gln
Val Leu 1440 1445 1450gat ttg ctg gcg cag ctg gtt cag tta cgg gtt
aat tac tgt ctt 4543Asp Leu Leu Ala Gln Leu Val Gln Leu Arg Val Asn
Tyr Cys Leu 1455 1460 1465ctg gat tca gat cag gtg ttt att ggc ttt
gta ttg aaa cag ttt 4588Leu Asp Ser Asp Gln Val Phe Ile Gly Phe Val
Leu Lys Gln Phe 1470 1475 1480gaa tac att gaa gtg ggc cag ttc agg
gaa tca gag gca atc att 4633Glu Tyr Ile Glu Val Gly Gln Phe Arg Glu
Ser Glu Ala Ile Ile 1485 1490 1495cca aac atc ttt ttc ttc ttg gta
tta cta tct tat gaa cgc tat 4678Pro Asn Ile Phe Phe Phe Leu Val Leu
Leu Ser Tyr Glu Arg Tyr 1500 1505 1510cat tca aaa cag atc att gga
att cct aaa atc att cag ctc tgt 4723His Ser Lys Gln Ile Ile Gly Ile
Pro Lys Ile Ile Gln Leu Cys 1515 1520 1525gat ggc atc atg gcc agt
gga agg aag gct gtg aca cat gcc ata 4768Asp Gly Ile Met Ala Ser Gly
Arg Lys Ala Val Thr His Ala Ile 1530 1535 1540ccg gct ctg cag ccc
ata gtc cac gac ctc ttt gta tta aga gga 4813Pro Ala Leu Gln Pro Ile
Val His Asp Leu Phe Val Leu Arg Gly 1545 1550 1555aca aat aaa gct
gat gca gga aaa gag ctt gaa acc caa aaa gag 4858Thr Asn Lys Ala Asp
Ala Gly Lys Glu Leu Glu Thr Gln Lys Glu 1560 1565 1570gtg gtg gtg
tca atg tta ctg aga ctc atc cag tac cat cag gtg 4903Val Val Val Ser
Met Leu Leu Arg Leu Ile Gln Tyr His Gln Val 1575 1580 1585ttg gag
atg ttc att ctt gtc ctg cag cag tgc cac aag gag aat 4948Leu Glu Met
Phe Ile Leu Val Leu Gln Gln Cys His Lys Glu Asn 1590 1595 1600gaa
gac aag tgg aag cga ctg tct cga cag ata gct gac atc atc 4993Glu Asp
Lys Trp Lys Arg Leu Ser Arg Gln Ile Ala Asp Ile Ile 1605 1610
1615ctc cca atg tta gcc aaa cag cag atg cac att gac tct cat gaa
5038Leu Pro Met Leu Ala Lys Gln Gln Met His Ile Asp Ser His Glu
1620 1625 1630gcc ctt gga gtg tta aat aca tta ttt gag att ttg gcc
cct tcc 5083Ala Leu Gly Val Leu Asn Thr Leu Phe Glu Ile Leu Ala Pro
Ser 1635 1640 1645tcc ctc cgt ccg gta gac atg ctt tta cgg agt atg
ttc gtc act 5128Ser Leu Arg Pro Val Asp Met Leu Leu Arg Ser Met Phe
Val Thr 1650 1655 1660cca aac aca atg gcg tcc gtg agc act gtt caa
ctg tgg ata tcg 5173Pro Asn Thr Met Ala Ser Val Ser Thr Val Gln Leu
Trp Ile Ser 1665 1670 1675gga att ctg gcc att ttg agg gtt ctg att
tcc cag tca act gaa 5218Gly Ile Leu Ala Ile Leu Arg Val Leu Ile Ser
Gln Ser Thr Glu 1680 1685 1690gat att gtt ctt tct cgt att cag gag
ctc tcc ttc tct ccg tat 5263Asp Ile Val Leu Ser Arg Ile Gln Glu Leu
Ser Phe Ser Pro Tyr 1695 1700 1705tta atc tcc tgt aca gta att aat
agg tta aga gat ggg gac agt 5308Leu Ile Ser Cys Thr Val Ile Asn Arg
Leu Arg Asp Gly Asp Ser 1710 1715 1720act tca acg cta gaa gaa cac
agt gaa ggg aaa caa ata aag aat 5353Thr Ser Thr Leu Glu Glu His Ser
Glu Gly Lys Gln Ile Lys Asn 1725 1730 1735ttg cca gaa gaa aca ttt
tca agg ttt cta tta caa ctg gtt ggt 5398Leu Pro Glu Glu Thr Phe Ser
Arg Phe Leu Leu Gln Leu Val Gly 1740 1745 1750att ctt tta gaa gac
att gtt aca aaa cag ctg aag gtg gaa atg 5443Ile Leu Leu Glu Asp Ile
Val Thr Lys Gln Leu Lys Val Glu Met 1755 1760 1765agt gag cag caa
cat act ttc tat tgc cag gaa cta ggc aca ctg 5488Ser Glu Gln Gln His
Thr Phe Tyr Cys Gln Glu Leu Gly Thr Leu 1770 1775 1780cta atg tgt
ctg atc cac atc ttc aag tct gga atg ttc cgg aga 5533Leu Met Cys Leu
Ile His Ile Phe Lys Ser Gly Met Phe Arg Arg 1785 1790 1795atc aca
gca gct gcc act agg ctg ttc cgc agt gat ggc tgt ggc 5578Ile Thr Ala
Ala Ala Thr Arg Leu Phe Arg Ser Asp Gly Cys Gly 1800 1805 1810ggc
agt ttc tac acc ctg gac agc ttg aac ttg cgg gct cgt tcc 5623Gly Ser
Phe Tyr Thr Leu Asp Ser Leu Asn Leu Arg Ala Arg Ser 1815 1820
1825atg atc acc acc cac ccg gcc ctg gtg ctg ctc tgg tgt cag ata
5668Met Ile Thr Thr His Pro Ala Leu Val Leu Leu Trp Cys Gln Ile
1830 1835 1840ctg ctg ctt gtc aac cac acc gac tac cgc tgg tgg gca
gaa gtg 5713Leu Leu Leu Val Asn His Thr Asp Tyr Arg Trp Trp Ala Glu
Val 1845 1850 1855cag cag acc ccg aaa aga cac agt ctg tcc agc aca
aag tta ctt 5758Gln Gln Thr Pro Lys Arg His Ser Leu Ser Ser Thr Lys
Leu Leu 1860 1865 1870agt ccc cag atg tct gga gaa gag gag gat tct
gac ttg gca gcc 5803Ser Pro Gln Met Ser Gly Glu Glu Glu Asp Ser Asp
Leu Ala Ala 1875 1880 1885aaa ctt gga atg tgc aat aga gaa ata gta
cga aga ggg gct ctc 5848Lys Leu Gly Met Cys Asn Arg Glu Ile Val Arg
Arg Gly Ala Leu 1890 1895 1900att ctc ttc tgt gat tat gtc tgt cag
aac ctc cat gac tcc gag 5893Ile Leu Phe Cys Asp Tyr Val Cys Gln Asn
Leu His Asp Ser Glu 1905 1910 1915cac tta acg tgg ctc att gta aat
cac att caa gat ctg atc agc 5938His Leu Thr Trp Leu Ile Val Asn His
Ile Gln Asp Leu Ile Ser 1920 1925 1930ctt tcc cac gag cct cca gta
cag gac ttc atc agt gcc gtt cat 5983Leu Ser His Glu Pro Pro Val Gln
Asp Phe Ile Ser Ala Val His 1935 1940 1945cgg aac tct gct gcc agc
ggc ctg ttc atc cag gca att cag tct 6028Arg Asn Ser Ala Ala Ser Gly
Leu Phe Ile Gln Ala Ile Gln Ser 1950 1955 1960cgt tgt gaa aac ctt
tca act cca acc atg ctg aag aaa act ctt 6073Arg Cys Glu Asn Leu Ser
Thr Pro Thr Met Leu Lys Lys Thr Leu 1965 1970 1975cag tgc ttg gag
ggg atc cat ctc agc cag tcg gga gct gtg ctc 6118Gln Cys Leu Glu Gly
Ile His Leu Ser Gln Ser Gly Ala Val Leu 1980 1985 1990acg ctg tat
gtg gac agg ctt ctg tgc acc cct ttc cgt gtg ctg 6163Thr Leu Tyr Val
Asp Arg Leu Leu Cys Thr Pro Phe Arg Val Leu 1995 2000 2005gct cgc
atg gtc gac atc ctt gct tgt cgc cgg gta gaa atg ctt 6208Ala Arg Met
Val Asp Ile Leu Ala Cys Arg Arg Val Glu Met Leu 2010 2015 2020ctg
gct gca aat tta cag agc agc atg gcc cag ttg cca atg gaa 6253Leu Ala
Ala Asn Leu Gln Ser Ser Met Ala Gln Leu Pro Met Glu 2025 2030
2035gaa ctc aac aga atc cag gaa tac ctt cag agc agc ggg ctc gct
6298Glu Leu Asn Arg Ile Gln Glu Tyr Leu Gln Ser Ser Gly Leu Ala
2040 2045 2050cag aga cac caa agg ctc tat tcc ctg ctg gac agg ttt
cgt ctc 6343Gln Arg His Gln Arg Leu Tyr Ser Leu Leu Asp Arg Phe Arg
Leu 2055 2060 2065tcc acc atg caa gac tca ctt agt ccc tct cct cca
gtc tct tcc 6388Ser Thr Met Gln Asp Ser Leu Ser Pro Ser Pro Pro Val
Ser Ser 2070 2075 2080cac ccg ctg gac ggg gat ggg cac gtg tca ctg
gaa aca gtg agt 6433His Pro Leu Asp Gly Asp Gly His Val Ser Leu Glu
Thr Val Ser 2085 2090 2095ccg gac aaa gac tgg tac gtt cat ctt gtc
aaa tcc cag tgt tgg 6478Pro Asp Lys Asp Trp Tyr Val His Leu Val Lys
Ser Gln Cys Trp 2100 2105 2110acc agg tca gat tct gca ctg ctg gaa
ggt gca gag ctg gtg aat 6523Thr Arg Ser Asp Ser Ala Leu Leu Glu Gly
Ala Glu Leu Val Asn 2115 2120 2125cgg att cct gct gaa gat atg aat
gcc ttc atg atg aac tcg gag 6568Arg Ile Pro Ala Glu Asp Met Asn Ala
Phe Met Met Asn Ser Glu 2130 2135 2140ttc aac cta agc ctg cta gct
cca tgc tta agc cta ggg atg agt 6613Phe Asn Leu Ser Leu Leu Ala Pro
Cys Leu Ser Leu Gly Met Ser 2145 2150 2155gaa att tct ggt ggc cag
aag agt gcc ctt ttt gaa gca gcc cgt 6658Glu Ile Ser Gly Gly Gln Lys
Ser Ala Leu Phe Glu Ala Ala Arg 2160 2165 2170gag gtg act ctg gcc
cgt gtg agc ggc acc gtg cag cag ctc cct 6703Glu Val Thr Leu Ala Arg
Val Ser Gly Thr Val Gln Gln Leu Pro 2175 2180 2185gct gtc cat cat
gtc ttc cag ccc gag ctg cct gca gag ccg gcg 6748Ala Val His His Val
Phe Gln Pro Glu Leu Pro Ala Glu Pro Ala 2190 2195 2200gcc tac tgg
agc aag ttg aat gat ctg ttt ggg gat gct gca ctg 6793Ala Tyr Trp Ser
Lys Leu Asn Asp Leu Phe Gly Asp Ala Ala Leu 2205 2210 2215tat cag
tcc ctg ccc act ctg gcc cgg gcc ctg gca cag tac ctg 6838Tyr Gln Ser
Leu Pro Thr Leu Ala Arg Ala Leu Ala Gln Tyr Leu 2220 2225 2230gtg
gtg gtc tcc aaa ctg ccc agt cat ttg cac ctt cct cct gag 6883Val Val
Val Ser Lys Leu Pro Ser His Leu His Leu Pro Pro Glu 2235 2240
2245aaa gag aag gac att gtg aaa ttc gtg gtg gca acc ctt gag gcc
6928Lys Glu Lys Asp Ile Val Lys Phe Val Val Ala Thr Leu Glu Ala
2250 2255 2260ctg tcc tgg cat ttg atc cat gag cag atc ccg ctg agt
ctg gat 6973Leu Ser Trp His Leu Ile His Glu Gln Ile Pro Leu Ser Leu
Asp 2265 2270 2275ctc cag gca ggg ctg gac tgc tgc tgc ctg gcc ctg
cag ctg cct 7018Leu Gln Ala Gly Leu Asp Cys Cys Cys Leu Ala Leu Gln
Leu Pro 2280 2285 2290ggc ctc tgg agc gtg gtc tcc tcc aca gag ttt
gtg acc cac gcc 7063Gly Leu Trp Ser Val Val Ser Ser Thr Glu Phe
Val Thr His Ala 2295 2300 2305tgc tcc ctc atc tac tgt gtg cac ttc
atc ctg gag gcc gtt gca 7108Cys Ser Leu Ile Tyr Cys Val His Phe Ile
Leu Glu Ala Val Ala 2310 2315 2320gtg cag cct gga gag cag ctt ctt
agt cca gaa aga agg aca aat 7153Val Gln Pro Gly Glu Gln Leu Leu Ser
Pro Glu Arg Arg Thr Asn 2325 2330 2335acc cca aaa gcc atc agc gag
gag gag gag gaa gta gat cca aac 7198Thr Pro Lys Ala Ile Ser Glu Glu
Glu Glu Glu Val Asp Pro Asn 2340 2345 2350aca cag aat cct aag tat
atc act gca gcc tgt gag atg gtg gca 7243Thr Gln Asn Pro Lys Tyr Ile
Thr Ala Ala Cys Glu Met Val Ala 2355 2360 2365gaa atg gtg gag tct
ctg cag tcg gtg ttg gcc ttg ggt cat aaa 7288Glu Met Val Glu Ser Leu
Gln Ser Val Leu Ala Leu Gly His Lys 2370 2375 2380agg aat agc ggc
gtg ccg gcg ttt ctc acg cca ttg cta agg aac 7333Arg Asn Ser Gly Val
Pro Ala Phe Leu Thr Pro Leu Leu Arg Asn 2385 2390 2395atc atc atc
agc ctg gcc cgc ctg ccc ctt gtc aac agc tac aca 7378Ile Ile Ile Ser
Leu Ala Arg Leu Pro Leu Val Asn Ser Tyr Thr 2400 2405 2410cgt gtg
ccc cca ctg gtg tgg aag ctt gga tgg tca ccc aaa ccg 7423Arg Val Pro
Pro Leu Val Trp Lys Leu Gly Trp Ser Pro Lys Pro 2415 2420 2425gga
ggg gat ttt ggc aca gca ttc cct gag atc ccc gtg gag ttc 7468Gly Gly
Asp Phe Gly Thr Ala Phe Pro Glu Ile Pro Val Glu Phe 2430 2435
2440ctc cag gaa aag gaa gtc ttt aag gag ttc atc tac cgc atc aac
7513Leu Gln Glu Lys Glu Val Phe Lys Glu Phe Ile Tyr Arg Ile Asn
2445 2450 2455aca cta ggc tgg acc agt cgt act cag ttt gaa gaa act
tgg gcc 7558Thr Leu Gly Trp Thr Ser Arg Thr Gln Phe Glu Glu Thr Trp
Ala 2460 2465 2470acc ctc ctt ggt gtc ctg gtg acg cag ccc ctc gtg
atg gag cag 7603Thr Leu Leu Gly Val Leu Val Thr Gln Pro Leu Val Met
Glu Gln 2475 2480 2485gag gag agc cca cca gaa gaa gac aca gag agg
acc cag atc aac 7648Glu Glu Ser Pro Pro Glu Glu Asp Thr Glu Arg Thr
Gln Ile Asn 2490 2495 2500gtc ctg gcc gtg cag gcc atc acc tca ctg
gtg ctc agt gca atg 7693Val Leu Ala Val Gln Ala Ile Thr Ser Leu Val
Leu Ser Ala Met 2505 2510 2515act gtg cct gtg gcc ggc aac cca gct
gta agc tgc ttg gag cag 7738Thr Val Pro Val Ala Gly Asn Pro Ala Val
Ser Cys Leu Glu Gln 2520 2525 2530cag ccc cgg aac aag cct ctg aaa
gct ctc gac acc agg ttt ggg 7783Gln Pro Arg Asn Lys Pro Leu Lys Ala
Leu Asp Thr Arg Phe Gly 2535 2540 2545agg aag ctg agc att atc aga
ggg att gtg gag caa gag att caa 7828Arg Lys Leu Ser Ile Ile Arg Gly
Ile Val Glu Gln Glu Ile Gln 2550 2555 2560gca atg gtt tca aag aga
gag aat att gcc acc cat cat tta tat 7873Ala Met Val Ser Lys Arg Glu
Asn Ile Ala Thr His His Leu Tyr 2565 2570 2575cag gca tgg gat cct
gtc cct tct ctg tct ccg gct act aca ggt 7918Gln Ala Trp Asp Pro Val
Pro Ser Leu Ser Pro Ala Thr Thr Gly 2580 2585 2590gcc ctc atc agc
cac gag aag ctg ctg cta cag atc aac ccc gag 7963Ala Leu Ile Ser His
Glu Lys Leu Leu Leu Gln Ile Asn Pro Glu 2595 2600 2605cgg gag ctg
ggg agc atg agc tac aaa ctc ggc cag gtg tcc ata 8008Arg Glu Leu Gly
Ser Met Ser Tyr Lys Leu Gly Gln Val Ser Ile 2610 2615 2620cac tcc
gtg tgg ctg ggg aac agc atc aca ccc ctg agg gag gag 8053His Ser Val
Trp Leu Gly Asn Ser Ile Thr Pro Leu Arg Glu Glu 2625 2630 2635gaa
tgg gac gag gaa gag gag gag gag gcc gac gcc cct gca cct 8098Glu Trp
Asp Glu Glu Glu Glu Glu Glu Ala Asp Ala Pro Ala Pro 2640 2645
2650tcg tca cca ccc acg tct cca gtc aac tcc agg aaa cac cgg gct
8143Ser Ser Pro Pro Thr Ser Pro Val Asn Ser Arg Lys His Arg Ala
2655 2660 2665gga gtt gac atc cac tcc tgt tcg cag ttt ttg ctt gag
ttg tac 8188Gly Val Asp Ile His Ser Cys Ser Gln Phe Leu Leu Glu Leu
Tyr 2670 2675 2680agc cgc tgg atc ctg ccg tcc agc tca gcc agg agg
acc ccg gcc 8233Ser Arg Trp Ile Leu Pro Ser Ser Ser Ala Arg Arg Thr
Pro Ala 2685 2690 2695atc ctg atc agt gag gtg gtc aga tcc ctt cta
gtg gtc tca gac 8278Ile Leu Ile Ser Glu Val Val Arg Ser Leu Leu Val
Val Ser Asp 2700 2705 2710ttg ttc acc gag cgc aac cag ttt gag ctg
atg tat gtg acg ctg 8323Leu Phe Thr Glu Arg Asn Gln Phe Glu Leu Met
Tyr Val Thr Leu 2715 2720 2725aca gaa ctg cga agg gtg cac cct tca
gaa gac gag atc ctc gct 8368Thr Glu Leu Arg Arg Val His Pro Ser Glu
Asp Glu Ile Leu Ala 2730 2735 2740cag tac ctg gtg cct gcc acc tgc
aag gca gct gcc gtc ctt ggg 8413Gln Tyr Leu Val Pro Ala Thr Cys Lys
Ala Ala Ala Val Leu Gly 2745 2750 2755atg gac aag gcc gtg gcg gag
cct gtc agc cgc ctg ctg gag agc 8458Met Asp Lys Ala Val Ala Glu Pro
Val Ser Arg Leu Leu Glu Ser 2760 2765 2770acg ctc agg agc agc cac
ctg ccc agc agg gtt gga gcc ctg cac 8503Thr Leu Arg Ser Ser His Leu
Pro Ser Arg Val Gly Ala Leu His 2775 2780 2785ggc gtc ctc tat gtg
ctg gag tgc gac ctg ctg gac gac act gcc 8548Gly Val Leu Tyr Val Leu
Glu Cys Asp Leu Leu Asp Asp Thr Ala 2790 2795 2800aag cag ctc atc
ccg gtc atc agc gac tat ctc ctc tcc aac ctg 8593Lys Gln Leu Ile Pro
Val Ile Ser Asp Tyr Leu Leu Ser Asn Leu 2805 2810 2815aaa ggg atc
gcc cac tgc gtg aac att cac agc cag cag cac gta 8638Lys Gly Ile Ala
His Cys Val Asn Ile His Ser Gln Gln His Val 2820 2825 2830ctg gtc
atg tgt gcc act gcg ttt tac ctc att gag aac tat cct 8683Leu Val Met
Cys Ala Thr Ala Phe Tyr Leu Ile Glu Asn Tyr Pro 2835 2840 2845ctg
gac gta ggg ccg gaa ttt tca gca tca ata ata cag atg tgt 8728Leu Asp
Val Gly Pro Glu Phe Ser Ala Ser Ile Ile Gln Met Cys 2850 2855
2860ggg gtg atg ctg tct gga agt gag gag tcc acc ccc tcc atc att
8773Gly Val Met Leu Ser Gly Ser Glu Glu Ser Thr Pro Ser Ile Ile
2865 2870 2875tac cac tgt gcc ctc aga ggc ctg gag cgc ctc ctg ctc
tct gag 8818Tyr His Cys Ala Leu Arg Gly Leu Glu Arg Leu Leu Leu Ser
Glu 2880 2885 2890cag ctc tcc cgc ctg gat gca gaa tcg ctg gtc aag
ctg agt gtg 8863Gln Leu Ser Arg Leu Asp Ala Glu Ser Leu Val Lys Leu
Ser Val 2895 2900 2905gac aga gtg aac gtg cac agc ccg cac cgg gcc
atg gcg gct ctg 8908Asp Arg Val Asn Val His Ser Pro His Arg Ala Met
Ala Ala Leu 2910 2915 2920ggc ctg atg ctc acc tgc atg tac aca gga
aag gag aaa gtc agt 8953Gly Leu Met Leu Thr Cys Met Tyr Thr Gly Lys
Glu Lys Val Ser 2925 2930 2935ccg ggt aga act tca gac cct aat cct
gca gcc ccc gac agc gag 8998Pro Gly Arg Thr Ser Asp Pro Asn Pro Ala
Ala Pro Asp Ser Glu 2940 2945 2950tca gtg att gtt gct atg gag cgg
gta tct gtt ctt ttt gat agg 9043Ser Val Ile Val Ala Met Glu Arg Val
Ser Val Leu Phe Asp Arg 2955 2960 2965atc agg aaa ggc ttt cct tgt
gaa gcc aga gtg gtg gcc agg atc 9088Ile Arg Lys Gly Phe Pro Cys Glu
Ala Arg Val Val Ala Arg Ile 2970 2975 2980ctg ccc cag ttt cta gac
gac ttc ttc cca ccc cag gac atc atg 9133Leu Pro Gln Phe Leu Asp Asp
Phe Phe Pro Pro Gln Asp Ile Met 2985 2990 2995aac aaa gtc atc gga
gag ttt ctg tcc aac cag cag cca tac ccc 9178Asn Lys Val Ile Gly Glu
Phe Leu Ser Asn Gln Gln Pro Tyr Pro 3000 3005 3010cag ttc atg gcc
acc gtg gtg tat aag gtg ttt cag act ctg cac 9223Gln Phe Met Ala Thr
Val Val Tyr Lys Val Phe Gln Thr Leu His 3015 3020 3025agc acc ggg
cag tcg tcc atg gtc cgg gac tgg gtc atg ctg tcc 9268Ser Thr Gly Gln
Ser Ser Met Val Arg Asp Trp Val Met Leu Ser 3030 3035 3040ctc tcc
aac ttc acg cag agg gcc ccg gtc gcc atg gcc acg tgg 9313Leu Ser Asn
Phe Thr Gln Arg Ala Pro Val Ala Met Ala Thr Trp 3045 3050 3055agc
ctc tcc tgc ttc ttt gtc agc gcg tcc acc agc ccg tgg gtc 9358Ser Leu
Ser Cys Phe Phe Val Ser Ala Ser Thr Ser Pro Trp Val 3060 3065
3070gcg gcg atc ctc cca cat gtc atc agc agg atg ggc aag ctg gag
9403Ala Ala Ile Leu Pro His Val Ile Ser Arg Met Gly Lys Leu Glu
3075 3080 3085cag gtg gac gtg aac ctt ttc tgc ctg gtc gcc aca gac
ttc tac 9448Gln Val Asp Val Asn Leu Phe Cys Leu Val Ala Thr Asp Phe
Tyr 3090 3095 3100aga cac cag ata gag gag gag ctc gac cgc agg gcc
ttc cag tct 9493Arg His Gln Ile Glu Glu Glu Leu Asp Arg Arg Ala Phe
Gln Ser 3105 3110 3115gtg ctt gag gtg gtt gca gcc cca gga agc cca
tat cac cgg ctg 9538Val Leu Glu Val Val Ala Ala Pro Gly Ser Pro Tyr
His Arg Leu 3120 3125 3130ctg act tgt tta cga aat gtc cac aag gtc
acc acc tgc tga 9580Leu Thr Cys Leu Arg Asn Val His Lys Val Thr Thr
Cys 3135 3140gcgccatggt gggagagact gtgaggcggc agctggggcc ggagcctttg
gaagtctgcg 9640cccttgtgcc ctgcctccac cgagccagct tggtccctat
gggcttccgc acatgccgcg 9700ggcggccagg caacgtgcgt gtctctgcca
tgtggcagaa gtgctctttg tggcagtggc 9760caggcaggga gtgtctgcag
tcctggtggg gctgagcctg aggccttcca gaaagcagga 9820gcagctgtgc
tgcaccccat gtgggtgacc aggtcctttc tcctgatagt cacctgctgg
9880ttgttgccag gttgcagctg ctcttgcatc tgggccagaa gtcctccctc
ctgcaggctg 9940gctgttggcc cctctgctgt cctgcagtag aaggtgccgt
gagcaggctt tgggaacact 10000ggcctgggtc tccctggtgg ggtgtgcatg
ccacgccccg tgtctggatg cacagatgcc 10060atggcctgtg ctgggccagt
ggctgggggt gctagacacc cggcaccatt ctcccttctc 10120tcttttcttc
tcaggattta aaatttaatt atatcagtaa agagattaat tttaacgtaa
10180ctctttctat gcccgtgtaa agtatgtgaa tcgcaaggcc tgtgctgcat
gcgacagcgt 10240ccggggtggt ggacagggcc cccggccacg ctccctctcc
tgtagccact ggcatagccc 10300tcctgagcac ccgctgacat ttccgttgta
catgttcctg tttatgcatt cacaaggtga 10360ctgggatgta gagaggcgtt
agtgggcagg tggccacagc aggactgagg acaggccccc 10420attatcctag
gggtgcgctc acctgcagcc cctcctcctc gggcacagac gactgtcgtt
10480ctccacccac cagtcaggga cagcagcctc cctgtcactc agctgagaag
gccagccctc 10540cctggctgtg agcagcctcc actgtgtcca gagacatggg
cctcccactc ctgttccttg 10600ctagccctgg ggtggcgtct gcctaggagc
tggctggcag gtgttgggac ctgctgctcc 10660atggatgcat gccctaagag
tgtcactgag ctgtgttttg tctgagcctc tctcggtcaa 10720cagcaaagct
tggtgtcttg gcactgttag tgacagagcc cagcatccct tctgcccccg
10780ttccagctga catcttgcac ggtgacccct tttagtcagg agagtgcaga
tctgtgctca 10840tcggagactg ccccacggcc ctgtcagagc cgccactcct
atccccaggc caggtccctg 10900gaccagcctc ctgtttgcag gcccagagga
gccaagtcat taaaatggaa gtggattctg 10960gatggccggg ctgctgctga
tgtaggagct ggatttggga gctctgcttg ccgactggct 11020gtgagacgag
gcaggggctc tgcttcctca gccctagagg cgagccaggc aaggttggcg
11080actgtcatgt ggcttggttt ggtcatgccc gtcgatgttt tgggtattga
atgtggtaag 11140tggaggaaat gttggaactc tgtgcaggtg ctgccttgag
acccccaagc ttccacctgt 11200ccctctccta tgtggcagct ggggagcagc
tgagatgtgg acttgtatgc tgcccacata 11260cgtgaggggg agctgaaagg
gagcccctcc tctgagcagc ctctgccagg cctgtatgag 11320gcttttccca
ccagctccca acagaggcct cccccagcca ggaccacctc gtcctcgtgg
11380cggggcagca ggagcggtag aaaggggtcc gatgtttgag gaggccctta
agggaagcta 11440ctgaattata acacgtaaga aaatcaccat tccgtattgg
ttgggggctc ctgtttctca 11500tcctagcttt ttcctggaaa gcccgctaga
aggtttggga acgaggggaa agttctcaga 11560actgttggct gctccccacc
cgcctcccgc ctcccccgca ggttatgtca gcagctctga 11620gacagcagta
tcacaggcca gatgttgttc ctggctagat gtttacattt gtaagaaata
11680acactgtgaa tgtaaaacag agccattccc ttggaatgca tatcgctggg
ctcaacatag 11740agtttgtctt cctcttgttt acgacgtgat ctaaaccagt
ccttagcaag gggctcagaa 11800caccccgctc tggcagtagg tgtcccccac
ccccaaagac ctgcctgtgt gctccggaga 11860tgaatatgag ctcattagta
aaaatgactt cacccacgca tatacataaa gtatccatgc 11920atgtgcatat
agacacatct ataattttac acacacacct ctcaagacgg agatgcatgg
11980cctctaagag tgcccgtgtc ggttcttcct ggaagttgac tttccttaga
cccgccaggt 12040caagttagcc gcgtgacgga catccaggcg tgggacgtgg
tcagggcagg gctcattcat 12100tgcccactag gatcccactg gcgaagatgg
tctccatatc agctctctgc agaagggagg 12160aagactttat catgttccta
aaaatctgtg gcaagcaccc atcgtattat ccaaattttg 12220ttgcaaatgt
gattaatttg gttgtcaagt tttgggggtg ggctgtgggg agattgcttt
12280tgttttcctg ctggtaatat cgggaaagat tttaatgaaa ccagggtaga
attgtttggc 12340aatgcactga agcgtgtttc tttcccaaaa tgtgcctccc
ttccgctgcg ggcccagctg 12400agtctatgta ggtgatgttt ccagctgcca
agtgctcttt gttactgtcc accctcattt 12460ctgccagcgc atgtgtcctt
tcaaggggaa aatgtgaagc tgaaccccct ccagacaccc 12520agaatgtagc
atctgagaag gccctgtgcc ctaaaggaca cccctcgccc ccatcttcat
12580ggagggggtc atttcagagc cctcggagcc aatgaacagc tcctcctctt
ggagctgaga 12640tgagccccac gtggagctcg ggacggatag tagacagcaa
taactcggtg tgtggccgcc 12700tggcaggtgg aacttcctcc cgttgcgggg
tggagtgagg ttagttctgt gtgtctggtg 12760ggtggagtca ggcttctctt
gctacctgtg agcatccttc ccagcagaca tcctcatcgg 12820gctttgtccc
tcccccgctt cctccctctg cggggaggac ccgggaccac agctgctggc
12880cagggtagac ttggagctgt cctccagagg ggtcacgtgt aggagtgaga
agaaggaaga 12940tcttgagagc tgctgaggga ccttggagag ctcaggatgg
ctcagacgag gacactcgct 13000tgccgggcct gggcctcctg ggaaggaggg
agctgctcag aatgccgcat gacaactgaa 13060ggcaacctgg aaggttcagg
ggccgctctt cccccatgtg cctgtcacgc tctggtgcag 13120tcaaaggaac
gccttcccct cagttgtttc taagagcaga gtctcccgct gcaatctggg
13180tggtaactgc cagccttgga ggatcgtggc caacgtggac ctgcctacgg
agggtgggct 13240ctgacccaag tggggcctcc ttgtccaggt ctcactgctt
tgcaccgtgg tcagagggac 13300tgtcagctga gcttgagctc ccctggagcc
agcagggctg tgatgggcga gtcccggagc 13360cccacccaga cctgaatgct
tctgagagca aagggaagga ctgacgagag atgtatattt 13420aattttttaa
ctgctgcaaa cattgtacat ccaaattaaa ggaaaaaaat ggaaaccatc 13480a
13481315DNAArtificial sequenceSynthetic oligonucleotide 3taaattgtca
tcacc 15416DNAArtificial sequenceSynthetic oligonucleotide
4ataaattgtc atcacc 16517DNAArtificial sequenceSynthetic
oligonucleotide 5ataaattgtc atcacca 17619DNAArtificial
sequenceSynthetic oligonucleotide 6aataaattgt catcaccag
19719DNAArtificial sequenceSynthetic oligonucleotide 7ataaattgtc
atcaccaga 19819DNAArtificial sequenceSynthetic oligonucleotide
8taaattgtca tcaccagaa 19919DNAArtificial sequenceSynthetic
oligonucleotide 9aaattgtcat caccagaaa 191019DNAArtificial
sequenceSynthetic oligonucleotide 10aattgtcatc accagaaaa
191119DNAArtificial sequenceSynthetic oligonucleotide 11attgtcatca
ccagaaaaa 191219DNAArtificial sequenceSynthetic oligonucleotide
12taataaattg tcatcacca 191319DNAArtificial sequenceSynthetic
oligonucleotide 13ttaataaatt gtcatcacc 191417DNAArtificial
sequenceSynthetic oligonucleotide 14aaattgtcat caccaga
171515DNAArtificial sequenceSynthetic oligonucleotide 15taaattgcca
tcacc 151617DNAArtificial sequenceSynthetic oligonucleotide
16ataaattgcc atcacca 171716DNAArtificial sequenceSynthetic
oligonucleotide 17ataaattgcc atcacc 161815DNAArtificial
sequenceSynthetic oligonucleotide 18attgccatca ccaga
151916DNAArtificial sequenceSynthetic oligonucleotide 19taaattgcca
tcacca 162017DNAArtificial sequenceSynthetic oligonucleotide
20taaattgcca tcaccag 172117DNAArtificial sequenceSynthetic
oligonucleotide 21taattttcta gacttta
172217DNAArtificial sequenceSynthetic oligonucleotide 22aattttctag
actttat 172315DNAArtificial sequenceSynthetic oligonucleotide
23attgtcatca ccaga 152416DNAArtificial sequenceSynthetic
oligonucleotide 24ttgtcatcac cagaaa 162517DNAArtificial
sequenceSynthetic oligonucleotide 25ttgtcatcac cagaaaa
172615DNAArtificial sequenceSynthetic oligonucleotide 26aattgtcatc
a