U.S. patent application number 17/005213 was filed with the patent office on 2021-07-15 for compositions for modulating expression of c9orf72 antisense transcript.
This patent application is currently assigned to Ionis Pharmaceuticals, Inc.. The applicant listed for this patent is Ionis Pharmaceuticals, Inc.. Invention is credited to Frank Rigo.
Application Number | 20210214725 17/005213 |
Document ID | / |
Family ID | 1000005492739 |
Filed Date | 2021-07-15 |
United States Patent
Application |
20210214725 |
Kind Code |
A1 |
Rigo; Frank |
July 15, 2021 |
COMPOSITIONS FOR MODULATING EXPRESSION OF C9ORF72 ANTISENSE
TRANSCRIPT
Abstract
Disclosed herein are compositions and methods for reducing
expression of C9ORF72 antisense transcript in an animal with
C9ORF72 antisense transcript specific inhibitors. Such methods are
useful to treat, prevent, or ameliorate neurodegenerative diseases
in an individual in need thereof. Such C9ORF72 antisense transcript
specific inhibitors include antisense compounds.
Inventors: |
Rigo; Frank; (Carlsbad,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ionis Pharmaceuticals, Inc. |
Carlsbad |
CA |
US |
|
|
Assignee: |
Ionis Pharmaceuticals, Inc.
Carlsbad
CA
|
Family ID: |
1000005492739 |
Appl. No.: |
17/005213 |
Filed: |
August 27, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15540251 |
Jun 27, 2017 |
10793855 |
|
|
PCT/US2016/012381 |
Jan 6, 2016 |
|
|
|
17005213 |
|
|
|
|
62148657 |
Apr 16, 2015 |
|
|
|
62148621 |
Apr 16, 2015 |
|
|
|
62100439 |
Jan 6, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0085 20130101;
C12N 2310/31 20130101; C12N 15/113 20130101; A61K 45/06 20130101;
C12N 2310/315 20130101; C12N 2310/321 20130101; C12N 2310/341
20130101; C12N 2310/322 20130101; C12N 2320/31 20130101; A61K
31/7125 20130101; A61K 31/712 20130101; C12N 2310/11 20130101; C12N
2310/3231 20130101; C12N 2310/346 20130101; C12N 2310/14 20130101;
C12N 2310/351 20130101; A61K 31/713 20130101; C12N 2310/33
20130101; C12N 2310/3341 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 45/06 20060101 A61K045/06; A61K 31/7125 20060101
A61K031/7125; A61K 31/712 20060101 A61K031/712; A61K 9/00 20060101
A61K009/00; A61K 31/713 20060101 A61K031/713 |
Claims
1. A compound comprising a C9ORF72 antisense transcript specific
inhibitor, wherein the C9ORF72 antisense transcript specific
inhibitor is a modified oligonucleotide consisting of 12-30 linked
nucleosides, wherein at least one nucleoside of the antisense
oligonucleotide comprises a hypoxanthine nucleobase, wherein the
modified oligonucleotide has a nucleobase sequence that is at least
90% complementary to a C9ORF72 antisense transcript, and wherein
the modified oligonucleotide comprises at least one modified
internucleoside linkage and/or at least one modified nucleoside
comprising a modified sugar.
2. (canceled)
3. (canceled)
4. (canceled)
5. The compound of claim 1, wherein the modified oligonucleotide
consists of 16-25 linked nucleosides.
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. The compound of claim 1, wherein the modified oligonucleotide
has a nucleobase sequence that is 100% complementary to a C9ORF72
antisense transcript.
11. The compound of claim 1, wherein the C9ORF72 antisense
transcript has the nucleobase sequence of SEQ ID NO: 13.
12. The compound of claim 1, wherein the modified oligonucleotide
has a nucleobase sequence comprising at least 16 contiguous
nucleobases of a sequence selected from SEQ ID NO: 30-96, 98, or
99.
13. (canceled)
14. The compound of claim 1, wherein the modified oligonucleotide
comprises at least one modified internucleoside linkage.
15. The compound of claim 14, wherein at least one modified
internucleoside linkage is a phosphorothioate internucleoside
linkage.
16. The compound of claim 14, wherein each modified internucleoside
linkage is a phosphorothioate internucleoside linkage.
17. The compound of claim 14 wherein the modified oligonucleotide
comprises at least one phosphodiester internucleoside linkage.
18. (canceled)
19. (canceled)
20. (canceled)
21. The compound of claim 1, wherein at least one nucleoside of the
modified oligonucleotide comprises a modified sugar.
22. (canceled)
23. (canceled)
24. The compound of claim 21, wherein at least one modified sugar
comprises a 2'-O-methoxyethyl group.
25. The compound of claim 1, wherein the modified oligonucleotide
is a gapmer.
26-92. (canceled)
93. The compound of claim 1, wherein the modified oligonucleotide
is a single-stranded modified oligonucleotide.
94-145. (canceled)
146. A compound comprising a C9ORF72 antisense transcript specific
inhibitor, wherein the C9ORF72 antisense transcript specific
inhibitor is a modified oligonucleotide consisting of 12-30 linked
nucleosides, wherein the modified oligonucleotide has a nucleobase
sequence comprising at least 12 contiguous nucleobases that are
complementary to a hexanucleotide repeat expansion in a C9ORF72
antisense transcript, and wherein at least one nucleoside of the
modified oligonucleotide comprises a modified sugar.
147. The compound of claim 146, wherein the modified
oligonucleotide consists of 16-25 linked nucleosides.
148. The compound of claim 146, wherein the modified
oligonucleotide has a nucleobase sequence that is at least 90%
complementary to the C9ORF72 antisense transcript.
149. The compound of claim 146, wherein the modified
oligonucleotide has a nucleobase sequence that is 100%
complementary to the C9ORF72 antisense transcript.
150. The compound of claim 146, wherein the modified
oligonucleotide has a nucleobase sequence comprising at least 16
contiguous nucleobases of a sequence selected from SEQ ID NOs:
47-49, 85-96, 98, or 99.
151. The compound of claim 146, wherein the modified
oligonucleotide comprises at least one modified internucleoside
linkage.
152. The compound of claim 151, wherein at least one modified
internucleoside linkage is a phosphorothioate internucleoside
linkage.
153. The compound of claim 151, wherein each modified
internucleoside linkage is a phosphorothioate internucleoside
linkage.
154. The compound of claim 151, wherein the modified
oligonucleotide comprises at least one phosphodiester
internucleoside linkage.
155. The compound of claim 146, wherein at least one modified sugar
comprises a 2'-O-methoxyethyl group.
156. The compound of claim 146, wherein the modified
oligonucleotide is a gapmer.
157. The compound of claim 146, wherein the modified
oligonucleotide is a single-stranded modified oligonucleotide.
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 BIOL0262USD1SEQ_ST25.txt created Aug. 24, 2020, which
is 76 Kb in size. The information in the electronic format of the
sequence listing is incorporated herein by reference in its
entirety.
FIELD
[0002] Provided are compositions and methods for inhibiting
expression of C9ORF72 antisense transcript in an animal. Such
compositions and methods are useful to treat, prevent, or
ameliorate neurodegenerative diseases, including amyotrophic
lateral sclerosis (ALS), frontotemporal dementia (FTD),
corticobasal degeneration syndrome (CBD), atypical Parkinsonian
syndrome, and olivopontocerebellar degeneration (OPCD).
BACKGROUND
[0003] Amyotrophic lateral sclerosis (ALS) is a fatal
neurodegenerative disease characterized clinically by progressive
paralysis leading to death from respiratory failure, typically
within two to three years of symptom onset (Rowland and Shneider,
N. Engl. J. Med., 2001, 344, 1688-1700). ALS is the third most
common neurodegenerative disease in the Western world (Hirtz et
al., Neurology, 2007, 68, 326-337), and there are currently no
effective therapies. Approximately 10% of cases are familial in
nature, whereas the bulk of patients diagnosed with the disease are
classified as sporadic as they appear to occur randomly throughout
the population (Chio et al., Neurology, 2008, 70, 533-537). There
is growing recognition, based on clinical, genetic, and
epidemiological data, that ALS and frontotemporal dementia (FTD)
represent an overlapping continuum of disease, characterized
pathologically by the presence of TDP-43 positive inclusions
throughout the central nervous system (Lillo and Hodges, J. Clin.
Neurosci., 2009, 16, 1131-1135; Neumann et al., Science, 2006, 314,
130-133).
[0004] To date, a number of genes have been discovered as causative
for classical familial ALS, for example, SOD1, TARDBP, FUS, OPTN,
and VCP (Johnson et al., Neuron, 2010, 68, 857-864; Kwiatkowski et
al., Science, 2009, 323, 1205-1208; Maruyama et al., Nature, 2010,
465, 223-226; Rosen et al., Nature, 1993, 362, 59-62; Sreedharan et
al., Science, 2008, 319, 1668-1672; Vance et al., Brain, 2009, 129,
868-876). Recently, linkage analysis of kindreds involving multiple
cases of ALS, FTD, and ALS-FTD had suggested that there was an
important locus for the disease on the short arm of chromosome 9
(Boxer et al., J. Neurol. Neurosurg. Psychiatry, 2011, 82, 196-203;
Morita et al., Neurology, 2006, 66, 839-844; Pearson et al. J.
Nerol., 2011, 258, 647-655; Vance et al., Brain, 2006, 129,
868-876). This mutation has been found to be the most common
genetic cause of ALS and FTD. It is postulated that the ALS-FTD
causing mutation is a large hexanucleotide (GGGGCC) repeat
expansion in the first intron of the C9ORF72 gene (Renton et al.,
Neuron, 2011, 72, 257-268; DeJesus-Hernandez et al., Neuron, 2011,
72, 245-256). A founder haplotype, covering the C9ORF72 gene, is
present in the majority of cases linked to this region (Renton et
al., Neuron, 2011, 72, 257-268). This locus on chromosome 9p21
accounts for nearly half of familial ALS and nearly one-quarter of
all ALS cases in a cohort of 405 Finnish patients (Laaksovirta et
al, Lancet Neurol., 2010, 9, 978-985).
[0005] There are currently no effective therapies to treat such
neurodegenerative diseases. Therefore, it is an object to provide
compositions and methods for the treatment of such
neurodegenerative diseases.
Summary
[0006] Provided herein are compositions and methods for modulating
levels of C9ORF72 antisense transcript in cells, tissues, and
animals. In certain embodiments, C9ORF72 antisense transcript
specific inhibitors modulate expression of C9ORF72 antisense
transcript. In certain embodiments, C9ORF72 antisense transcript
specific inhibitors are nucleic acids, proteins, or small
molecules.
[0007] In certain embodiments, modulation can occur in a cell or
tissue. In certain embodiments, the cell or tissue is in an animal.
In certain embodiments, the animal is a human. In certain
embodiments, C9ORF72 antisense transcript levels are reduced. In
certain embodiments, C9ORF72 antisense transcript associated RAN
translation products are reduced. In certain embodiments, the
C9ORF72 antisense transcript associated RAN translation products
are poly-(proline-alanine), poly-(proline-arginine), and
poly-(proline-glycine). In certain embodiments, the C9ORF72
antisense transcript contains a hexanucleotide repeat expansion. In
certain embodiments, the hexanucleotide repeat is transcribed in
the antisense direction from the C9ORF72 gene. In certain
embodiments, the hexanucleotide repeat expansion is associated with
a C9ORF72 associated disease. In certain embodiments, the
hexanucleotide repeat expansion is associated with a C9ORF72
hexanucleotide repeat expansion associated disease. In certain
embodiments, the hexanucleotide repeat expansion comprises at least
30 GGCCCC, CCCCCC, GCCCCC, and/or CGCCCC repeats. In certain
embodiments, the hexanucleotide repeat expansion comprises more
than 30 GGCCCC, CCCCCC, GCCCCC, and/or CGCCCC repeats. In certain
embodiments, the hexanucleotide repeat expansion comprises more
than 100 GGCCCC, CCCCCC, GCCCCC, and/or CGCCCC repeats. In certain
embodiments, the hexanucleotide repeat expansion comprises more
than 500 GGCCCC, CCCCCC, GCCCCC, and/or CGCCCC repeats. In certain
embodiments, the hexanucleotide repeat expansion comprises more
than 1000 GGCCCC, CCCCCC, GCCCCC, and/or CGCCCC repeats. In certain
embodiments, the hexanucleotide repeat expansion is associated with
nuclear foci. In certain embodiments, C9ORF72 antisense transcript
associated RAN translation products are associated with nuclear
foci. In certain embodiments, the antisense transcript associated
RAN translation products are poly-(proline-alanine) and/or
poly-(proline-arginine). In certain embodiments, the compositions
and methods described herein are useful for reducing C9ORF72
antisense transcript levels, C9ORF72 antisense transcript
associated RAN translation products, and nuclear foci. Such
reduction can occur in a time-dependent manner or in a
dose-dependent manner.
[0008] Also provided are methods useful for preventing, treating,
ameliorating, and slowing progression of diseases and conditions
associated with C9ORF72. In certain embodiments, such diseases and
conditions associated with C9ORF72 are neurodegenerative diseases.
In certain embodiments, the neurodegenerative disease is
amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD),
corticobasal degeneration syndrome (CBD), atypical Parkinsonian
syndrome, and olivopontocerebellar degeneration (OPCD).
[0009] Such diseases and conditions can have one or more risk
factors, causes, or outcomes in common. Certain risk factors and
causes for development of a neurodegenerative disease, and, in
particular, ALS and FTD, include genetic predisposition and older
age.
[0010] In certain embodiments, methods of treatment include
administering a C9ORF72 antisense transcript specific inhibitor to
an individual in need thereof. In certain embodiments, the C9ORF72
antisense transcript specific inhibitor is a nucleic acid. In
certain embodiments, the nucleic acid is an antisense compound. In
certain embodiments, the antisense compound is an antisense
oligonucleotide. In certain embodiments, the antisense
oligonucleotide is complementary to a C9ORF72 antisense transcript.
In certain embodiments, the antisense oligonucleotide is a modified
antisense oligonucleotide.
DETAILED DESCRIPTION
[0011] 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. Additionally, as used
herein, the use of "and" 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.
[0012] 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 disclosure, including, but not limited to, patents, patent
applications, published patent applications, articles, books,
treatises, and GENBANK Accession Numbers and associated sequence
information obtainable through databases such as National Center
for Biotechnology Information (NCBI) and other data referred to
throughout in the disclosure herein are hereby expressly
incorporated by reference for the portions of the document
discussed herein, as well as in their entirety.
Definitions
[0013] Unless specific definitions are provided, the nomenclature
utilized 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.
[0014] Unless otherwise indicated, the following terms have the
following meanings:
[0015] "2'-O-methoxyethyl" (also 2'-MOE and
2'-OCH.sub.2CH.sub.2--OCH.sub.3 and MOE) refers to an
0-methoxy-ethyl modification of the 2' position of a furanose ring.
A 2'-O-methoxyethyl modified sugar is a modified sugar.
[0016] "2'-MOE nucleoside" (also 2'-O-methoxyethyl nucleoside)
means a nucleoside comprising a MOE modified sugar moiety.
[0017] "2'-substituted nucleoside" means a nucleoside comprising a
substituent at the 2'-position of the furanose ring other than H or
OH. In certain embodiments, 2'-substituted nucleosides include
nucleosides with bicyclic sugar modifications.
[0018] "5-methylcytosine" means a cytosine modified with a methyl
group attached to the 5' position. A 5-methylcytosine is a modified
nucleobase.
[0019] "About" means within .+-.7% of a value. For example, if it
is stated, "the compounds affected at least about 70% inhibition of
C9ORF72 antisense transcript", it is implied that the C9ORF72
antisense transcript levels are inhibited within a range of 63% and
77%.
[0020] "Administered concomitantly" refers to the co-administration
of two pharmaceutical agents in any manner in which the
pharmacological effects of both are manifest in the patient at the
same time. Concomitant administration does not require that both
pharmaceutical agents be administered in a single pharmaceutical
composition, in the same dosage form, or by the same route of
administration. The effects of both pharmaceutical agents need not
manifest themselves at the same time. The effects need only be
overlapping for a period of time and need not be coextensive.
[0021] "Administering" means providing a pharmaceutical agent to an
animal, and includes, but is not limited to administering by a
medical professional and self-administering.
[0022] "Amelioration" refers to a lessening, slowing, stopping, or
reversing of at least one indicator of the severity of a condition
or disease. The severity of indicators may be determined by
subjective or objective measures, which are known to those skilled
in the art.
[0023] "Animal" refers to a human or non-human animal, including,
but not limited to, mice, rats, rabbits, dogs, cats, pigs, and
non-human primates, including, but not limited to, monkeys and
chimpanzees.
[0024] "Antibody" refers to a molecule characterized by reacting
specifically with an antigen in some way, where the antibody and
the antigen are each defined in terms of the other. Antibody may
refer to a complete antibody molecule or any fragment or region
thereof, such as the heavy chain, the light chain, Fab region, and
Fc region.
[0025] "Antisense activity" means any detectable or measurable
activity attributable to the hybridization of an antisense compound
to its target nucleic acid. In certain embodiments, antisense
activity is a decrease in the amount or expression of a target
nucleic acid or protein product encoded by such target nucleic
acid.
[0026] "Antisense compound" means an oligomeric compound that is
capable of undergoing hybridization to a target nucleic acid
through hydrogen bonding. Examples of antisense compounds include
single-stranded and double-stranded compounds, such as, antisense
oligonucleotides, siRNAs, shRNAs, ssRNAs, and occupancy-based
compounds.
[0027] "Antisense inhibition" means reduction of target nucleic
acid levels in the presence of an antisense compound complementary
to a target nucleic acid compared to target nucleic acid levels or
in the absence of the antisense compound.
[0028] "Antisense mechanisms" are all those mechanisms involving
hybridization of a compound with a target nucleic acid, wherein the
outcome or effect of the hybridization is either target degradation
or target occupancy with concomitant stalling of the cellular
machinery involving, for example, transcription or splicing.
[0029] "Antisense oligonucleotide" means a single-stranded
oligonucleotide having a nucleobase sequence that permits
hybridization to a corresponding segment of a target nucleic
acid.
[0030] "Base complementarity" refers to the capacity for the
precise base pairing of nucleobases of an antisense oligonucleotide
with corresponding nucleobases in a target nucleic acid (i.e.,
hybridization), and is mediated by Watson-Crick, Hoogsteen or
reversed Hoogsteen hydrogen binding between corresponding
nucleobases.
[0031] "Bicyclic sugar" means a furanose ring modified by the
bridging of two atoms. A bicyclic sugar is a modified sugar.
[0032] "Bicyclic nucleoside" (also BNA) means a nucleoside having a
sugar moiety comprising a bridge connecting two carbon atoms of the
sugar ring, thereby forming a bicyclic ring system. In certain
embodiments, the bridge connects the 4'-carbon and the 2'-carbon of
the sugar ring.
[0033] "C9ORF72 antisense transcript" means transcripts produced
from the non-coding strand (also antisense strand and template
strand) of the C9ORF72 gene. The C9ORF72 antisense transcript
differs from the canonically transcribed "C9ORF72 sense
transcript", which is produced from the coding strand (also sense
strand) of the C9ORF72 gene.
[0034] "C9ORF72 antisense transcript associated RAN translation
products" means aberrant peptide or di-peptide polymers translated
through RAN translation (i.e., repeat-associated, and
non-ATG-dependent translation). In certain embodiments, the C9ORF72
antisense transcript associated RAN translation products are any of
poly-(proline-alanine), poly-(proline-arginine), and
poly-(proline-glycine).
[0035] "C9ORF72 antisense transcript specific inhibitor" refers to
any agent capable of specifically inhibiting the expression of
C9ORF72 antisense transcript and/or its expression products at the
molecular level. As used herein, "specific" means reducing or
inhibiting expression of C9ORF72 antisense transcript without
reducing non-target transcript to an appreciable degree (e.g., a
C9ORF72 antisense transcript specific inhibitor reduces expression
of C9ORF72 antisense transcript, but does not reduce expression of
C9ORF72 sense transcript to an appreciable degree). C9ORF72
specific antisense transcript inhibitors include nucleic acids
(including antisense compounds), siRNAs, aptamers, antibodies,
peptides, small molecules, and other agents capable of inhibiting
the expression of C9ORF72 antisense transcript and/or its
expression products, such as C9ORF72 antisense transcript
associated RAN translation products.
[0036] "C9ORF72 associated disease" means any disease associated
with any C9ORF72 nucleic acid or expression product thereof,
regardless of which DNA strand the C9ORF72 nucleic acid or
expression product thereof is derived from. Such diseases may
include a neurodegenerative disease. Such neurodegenerative
diseases may include ALS and FTD.
[0037] "C9ORF72 foci" means nuclear foci comprising a C9ORF72
transcript. In certain embodiments, a C9ORF72 foci comprises at
least one C9ORF72 sense transcript (herein "C9ORF72 sense foci").
In certain embodiments, C9ORF72 sense foci comprise C9ORF72 sense
transcripts comprising any of the following hexanucleotide repeats:
GGGGCC, GGGGGG, GGGGGC, and/or GGGGCG. In certain embodiments, a
C9ORF72 foci comprises at least one C9ORF72 antisense transcript
(herein "C9ORF72 antisense foci"). In certain embodiments, C9ORF72
antisense foci comprise C9ORF72 antisense transcripts comprising
any of the following hexanucleotide repeats: GGCCCC, CCCCCC,
GCCCCC, and/or CGCCCC. In certain embodiments, C9ORF72 foci
comprise both C9ORF72 sense transcripts and C9ORF72 antisense
transcripts.
[0038] "C9ORF72 hexanucleotide repeat expansion associated disease"
means any disease associated with a C9ORF72 nucleic acid containing
a hexanucleotide repeat expansion. In certain embodiments, the
hexanucleotide repeat expansion may comprise any of the following
hexanucleotide repeats: GGGGCC, GGGGGG, GGGGGC, GGGGCG, GGCCCC,
CCCCCC, GCCCCC, and/or CGCCCC. In certain embodiments, the
hexanucleotide repeat is repeated at least 30 times, more than 30
times, more than 100 times, more than 500 times, or more than 1000
times. Such diseases may include a neurodegenerative disease. Such
neurodegenerative diseases may include ALS and FTD.
[0039] "C9ORF72 nucleic acid" means any nucleic acid derived from
the C9ORF72 locus, regardless of which DNA strand the C9ORF72
nucleic acid is derived from. In certain embodiments, a C9ORF72
nucleic acid includes a DNA sequence encoding C9ORF72, an RNA
sequence transcribed from DNA encoding C9ORF72 including genomic
DNA comprising introns and exons (i.e., pre-mRNA), and an mRNA
sequence encoding C9ORF72. "C9ORF72 mRNA" means an mRNA encoding a
C9ORF72 protein. In certain embodiments, a C9ORF72 nucleic acid
includes transcripts produced from the coding strand of the C9ORF72
gene. C9ORF72 sense transcripts are examples of C9ORF72 nucleic
acids. In certain embodiments, a C9ORF72 nucleic acid includes
transcripts produced from the non-coding strand of the C9ORF72
gene. C9ORF72 antisense transcripts are examples of C9ORF72 nucleic
acids.
[0040] "C9ORF72 pathogenic associated mRNA variant" means the
C9ORF72 mRNA variant processed from a C9ORF72 pre-mRNA variant
containing the hexanucleotide repeat. A C9ORF72 pre-mRNA contains
the hexanucleotide repeat when transcription of the pre-mRNA begins
in the region from the start site of exon 1A to the start site of
exon 1B, e.g., nucleotides 1107 to 1520 of the genomic sequence
(SEQ ID NO: 2, the complement of GENBANK Accession No. NT_008413.18
truncated from nucleosides 27535000 to 27565000). In certain
embodiments, the level of a C9ORF72 pathogenic associated mRNA
variant is measured to determine the level of a C9ORF72 pre-mRNA
containing the hexanucleotide repeat in a sample.
[0041] "C9ORF72 transcript" means an RNA transcribed from C9ORF72.
In certain embodiments, a C9ORF72 transcript is a C9ORF72 sense
transcript. In certain embodiments, a C9ORF72 transcript is a
C9ORF72 antisense transcript.
[0042] "Cap structure" or "terminal cap moiety" means chemical
modifications, which have been incorporated at either terminus of
an antisense compound.
[0043] "cEt" or "constrained ethyl" means a bicyclic nucleoside
having a sugar moiety comprising a bridge connecting the 4'-carbon
and the 2'-carbon, wherein the bridge has the formula:
4'-CH(CH.sub.3)--O-2'.
[0044] "Constrained ethyl nucleoside" (also cEt nucleoside) means a
nucleoside comprising a bicyclic sugar moiety comprising a
4'-CH(CH.sub.3)--O-2' bridge.
[0045] "Chemically distinct region" refers to a region of an
antisense compound that is in some way chemically different than
another region of the same antisense compound. For example, a
region having 2'-O-methoxyethyl nucleosides is chemically distinct
from a region having nucleosides without 2'-O-methoxyethyl
modifications.
[0046] "Chimeric antisense compound" means an antisense compound
that has at least two chemically distinct regions, each position
having a plurality of subunits.
[0047] "Co-administration" means administration of two or more
pharmaceutical agents to an individual. The two or more
pharmaceutical agents may be in a single pharmaceutical
composition, or may be in separate pharmaceutical compositions.
Each of the two or more pharmaceutical agents may be administered
through the same or different routes of administration.
Co-administration encompasses parallel or sequential
administration.
[0048] "Complementarity" means the capacity for pairing between
nucleobases of a first nucleic acid and a second nucleic acid.
[0049] "Comprise," "comprises," and "comprising" will be understood
to imply the inclusion of a stated step or element or group of
steps or elements but not the exclusion of any other step or
element or group of steps or elements.
[0050] "Contiguous nucleobases" means nucleobases immediately
adjacent to each other.
[0051] "Designing" or "designed to" refer to the process of
designing an oligomeric compound that specifically hybridizes with
a selected nucleic acid molecule.
[0052] "Diluent" means an ingredient in a composition that lacks
pharmacological activity, but is pharmaceutically necessary or
desirable. For example, in drugs that are injected, the diluent may
be a liquid, e.g. saline solution.
[0053] "Dose" means a specified quantity of a pharmaceutical agent
provided in a single administration, or in a specified time period.
In certain embodiments, a dose may be administered in one, two, or
more boluses, tablets, or injections. For example, in certain
embodiments where subcutaneous administration is desired, the
desired dose requires a volume not easily accommodated by a single
injection, therefore, two or more injections may be used to achieve
the desired dose. In certain embodiments, the pharmaceutical agent
is administered by infusion over an extended period of time or
continuously. Doses may be stated as the amount of pharmaceutical
agent per hour, day, week, or month.
[0054] "Effective amount" in the context of modulating an activity
or of treating or preventing a condition means the administration
of that amount of pharmaceutical agent to a subject in need of such
modulation, treatment, or prophylaxis, either in a single dose or
as part of a series, that is effective for modulation of that
effect, or for treatment or prophylaxis or improvement of that
condition. The effective amount may vary among individuals
depending on the health and physical condition of the individual to
be treated, the taxonomic group of the individuals to be treated,
the formulation of the composition, assessment of the individual's
medical condition, and other relevant factors.
[0055] "Efficacy" means the ability to produce a desired
effect.
[0056] "Expression" includes all the functions by which a gene's
coded information, regardless of which DNA strand the coded
information is derived from, is converted into structures present
and operating in a cell. Such structures include, but are not
limited to the products of transcription and translation, including
RAN translation.
[0057] "Fully complementary" or "100% complementary" means each
nucleobase of a first nucleic acid has a complementary nucleobase
in a second nucleic acid. In certain embodiments, a first nucleic
acid is an antisense compound and a target nucleic acid is a second
nucleic acid.
[0058] "Gapmer" means a chimeric antisense compound in which an
internal region having a plurality of nucleosides that support
RNase H cleavage is positioned between external regions having one
or more nucleosides, wherein the nucleosides comprising the
internal region are chemically distinct from the nucleoside or
nucleosides comprising the external regions. The internal region
may be referred to as a "gap" and the external regions may be
referred to as the "wings."
[0059] "Gap-narrowed" means a chimeric antisense compound having a
gap segment of 9 or fewer contiguous 2'-deoxyribonucleosides
positioned between and immediately adjacent to 5' and 3' wing
segments having from 1 to 6 nucleosides.
[0060] "Gap-widened" means a chimeric antisense compound having a
gap segment of 12 or more contiguous 2'-deoxyribonucleosides
positioned between and immediately adjacent to 5' and 3' wing
segments having from 1 to 6 nucleosides.
[0061] "Hexanucleotide repeat expansion" means a series of six
bases (for example, GGGGCC, GGGGGG, GGGGGC, GGGGCG, GGCCCC, CCCCCC,
GCCCCC, and/or CGCCCC) repeated at least twice. In certain
embodiments, the hexanucleotide repeat expansion may be located in
intron 1 of a C9ORF72 nucleic acid. In certain embodiments, the
hexanucleotide repeat may be transcribed in the antisense direction
from the C9ORF72 gene. In certain embodiments, a pathogenic
hexanucleotide repeat expansion includes more than 30, more than
100, more than 500, or more than 1000 repeats of GGGGCC, GGGGGG,
GGGGGC, GGGGCG, GGCCCC, CCCCCC, GCCCCC, and/or CGCCCC in a C9ORF72
nucleic acid and is associated with disease. In certain
embodiments, the repeats are consecutive. In certain embodiments,
the repeats are interrupted by 1 or more nucleobases. In certain
embodiments, a wild-type hexanucleotide repeat expansion includes
30 or fewer repeats of GGGGCC, GGGGGG, GGGGGC, GGGGCG, GGCCCC,
CCCCCC, GCCCCC, and/or CGCCCC in a C9ORF72 nucleic acid. In certain
embodiments, the repeats are consecutive. In certain embodiments,
the repeats are interrupted by 1 or more nucleobases.
[0062] "Hybridization" means the annealing of complementary nucleic
acid molecules. In certain embodiments, complementary nucleic acid
molecules include, but are not limited to, an antisense compound
and a target nucleic acid. In certain embodiments, complementary
nucleic acid molecules include, but are not limited to, an
antisense oligonucleotide and a nucleic acid target.
[0063] "Hypoxanthine" (Hyp) also 6-Oxypurine is a purine
derivative. In certain embodiments, a hypoxanthine (Hyp) may be
used in place of a guanine (G) nucleobase to break up a series of 4
or more guanosines in a row ("G-quartet"). Hypoxanthine is a
modified nucleobase.
[0064] "Identifying an animal having a C9ORF72 associated disease"
means identifying an animal having been diagnosed with a C9ORF72
associated disease or predisposed to develop a C9ORF72 associated
disease. Individuals predisposed to develop a C9ORF72 associated
disease include those having one or more risk factors for
developing a C9ORF72 associated disease, including, having a
personal or family history or genetic predisposition of one or more
C9ORF72 associated diseases. In certain embodiments, the C9ORF72
associated disease is a C9ORF72 hexanucleotide repeat expansion
associated disease. Such identification may be accomplished by any
method including evaluating an individual's medical history and
standard clinical tests or assessments, such as genetic
testing.
[0065] "Immediately adjacent" means there are no intervening
elements between the immediately adjacent elements.
[0066] "Individual" means a human or non-human animal selected for
treatment or therapy.
[0067] "Inhibiting expression of a C9ORF72 antisense transcript"
means reducing the level or expression of a C9ORF72 antisense
transcript and/or its expression products (e.g., RAN translation
products). In certain embodiments, C9ORF72 antisense transcripts
are inhibited in the presence of an antisense compound targeting a
C9ORF72 antisense transcript, including an antisense
oligonucleotide targeting a C9ORF72 antisense transcript, as
compared to expression of C9ORF72 antisense transcript levels in
the absence of a C9ORF72 antisense compound, such as an antisense
oligonucleotide.
[0068] "Inhibiting expression of a C9ORF72 sense transcript" means
reducing the level or expression of a C9ORF72 sense transcript
and/or its expression products (e.g., a C9ORF72 mRNA and/or
protein). In certain embodiments, C9ORF72 sense transcripts are
inhibited in the presence of an antisense compound targeting a
C9ORF72 sense transcript, including an antisense oligonucleotide
targeting a C9ORF72 sense transcript, as compared to expression of
C9ORF72 sense transcript levels in the absence of a C9ORF72
antisense compound, such as an antisense oligonucleotide.
[0069] "Inhibiting the expression or activity" refers to a
reduction or blockade of the expression or activity and does not
necessarily indicate a total elimination of expression or
activity.
[0070] "Inosine" (I) or 9-.beta.-D-Ribosylhypoxanthine means a
nucleoside that contains a hypoxanthine nucleobase.
[0071] "Internucleoside linkage" refers to the chemical bond
between nucleosides.
[0072] "Linked nucleosides" means adjacent nucleosides linked
together by an internucleoside linkage.
[0073] "Locked nucleic acid" or "LNA" or "LNA nucleosides" means
nucleic acid monomers having a bridge connecting two carbon atoms
between the 4' and 2'position of the nucleoside sugar unit, thereby
forming a bicyclic sugar. Examples of such bicyclic sugar include,
but are not limited to A) .alpha.-L-Methyleneoxy
(4'-CH.sub.2--O-2') LNA, (B) .beta.-D-Methyleneoxy
(4'-CH.sub.2--O-2') LNA, (C) Ethyleneoxy
(4'-(CH.sub.2).sub.2--O-2') LNA, (D) Aminooxy
(4'-CH.sub.2--O--N(R)-2') LNA and (E) Oxyamino
(4'-CH.sub.2--N(R)--O-2') LNA, as depicted below.
##STR00001##
[0074] As used herein, LNA compounds include, but are not limited
to, compounds having at least one bridge between the 4' and the 2'
position of the sugar wherein each of the bridges independently
comprises 1 or from 2 to 4 linked groups independently selected
from --[C(R.sub.1)(R.sub.2)].sub.n--,
--C(R.sub.1).dbd.C(R.sub.2)--, --C(R.sub.1).dbd.N--,
--C(.dbd.NR.sub.1)--, --C(.dbd.O)--, --C(.dbd.S)--, --O--,
--Si(R.sub.1).sub.2--, --S(.dbd.O).sub.x-- and --N(R.sub.1)--;
wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R.sub.1 and
R.sub.2 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, a
heterocycle radical, a substituted heterocycle radical, heteroaryl,
substituted heteroaryl, C.sub.5-C.sub.7 alicyclic radical,
substituted C.sub.5-C.sub.7 alicyclic radical, halogen, OJ.sub.1,
NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1, acyl
(C(.dbd.O)--H), substituted acyl, CN, sulfonyl
(S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and
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.
[0075] Examples of 4'-2' bridging groups encompassed within the
definition of LNA include, but are not limited to one of formulae:
--[C(R.sub.1)(R.sub.2)].sub.n--,
--[C(R.sub.1)(R.sub.2)].sub.n--O--,
--C(R.sub.1R.sub.2)--N(R.sub.1)--O-- or
--C(R.sub.1R.sub.2)--O--N(R.sub.1)--. Furthermore, other bridging
groups encompassed with the definition of LNA are 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',
4'-(CH.sub.2).sub.2--O-2', 4'-CH.sub.2--O--N(R.sub.1)-2' and
4'-CH.sub.2--N(R.sub.1)--O-2'-bridges, wherein each R.sub.1 and
R.sub.2 is, independently, H, a protecting group or
C.sub.1-C.sub.12 alkyl.
[0076] Also included within the definition of LNA according to the
invention are LNAs in which the 2'-hydroxyl group of the ribosyl
sugar ring is connected to the 4' carbon atom of the sugar ring,
thereby forming a methyleneoxy (4'-CH.sub.2--O-2') bridge to form
the bicyclic sugar moiety. The bridge can also be a methylene
(--CH.sub.2--) group connecting the 2' oxygen atom and the 4'
carbon atom, for which the term methyleneoxy (4'-CH.sub.2--O-2')
LNA is used. Furthermore; in the case of the bicylic sugar moiety
having an ethylene bridging group in this position, the term
ethyleneoxy (4' CH.sub.2CH.sub.2--O-2') LNA is used.
.alpha.-L-methyleneoxy (4'-CH.sub.2--O-2'), an isomer of
methyleneoxy (4'-CH.sub.2--O-2') LNA is also encompassed within the
definition of LNA, as used herein.
[0077] "Mismatch" or "non-complementary nucleobase" refers to the
case when a nucleobase of a first nucleic acid is not capable of
pairing with the corresponding nucleobase of a second or target
nucleic acid.
[0078] "Modified internucleoside linkage" refers to a substitution
or any change from a naturally occurring internucleoside bond
(i.e., a phosphodiester internucleoside bond).
[0079] "Modified nucleobase" means any nucleobase other than
adenine, cytosine, guanine, thymidine, or uracil. Hypoxanthine
(Hyp) is a modified nucleobase. An "unmodified nucleobase" means
the purine bases adenine (A) and guanine (G); the pyrimidine bases
thymine (T), cytosine (C), and uracil (U).
[0080] "Modified nucleoside" means a nucleoside having,
independently, a modified sugar moiety and/or modified
nucleobase.
[0081] "Modified nucleotide" means a nucleotide having,
independently, a modified sugar moiety, modified internucleoside
linkage, and/or modified nucleobase.
[0082] "Modified oligonucleotide" means an oligonucleotide
comprising at least one modified internucleoside linkage, modified
sugar, and/or modified nucleobase.
[0083] "Modified sugar" means substitution and/or any change from a
natural sugar moiety.
[0084] "Monomer" means a single unit of an oligomer. Monomers
include, but are not limited to, nucleosides and nucleotides,
whether naturally occurring or modified.
[0085] "Motif" means the pattern of unmodified and modified
nucleoside in an antisense compound.
[0086] "Natural sugar moiety" means a sugar moiety found in DNA
(2'-H) or RNA (2'-OH).
[0087] "Naturally occurring internucleoside linkage" means a 3' to
5' phosphodiester linkage.
[0088] "Non-complementary nucleobase" refers to a pair of
nucleobases that do not form hydrogen bonds with one another or
otherwise support hybridization.
[0089] "Nucleic acid" refers to molecules composed of monomeric
nucleotides. A nucleic acid includes, but is not limited to,
ribonucleic acids (RNA), deoxyribonucleic acids (DNA),
single-stranded nucleic acids, double-stranded nucleic acids, small
interfering ribonucleic acids (siRNA), and microRNAs (miRNA).
[0090] "Nucleobase" means a heterocyclic moiety capable of pairing
with a base of another nucleic acid.
[0091] "Nucleobase complementarity" refers to 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). Hypoxanthine (Hyp)
binds with adenine, thymine, or cytosine with a preference for
binding with cytosine. In certain embodiments, complementary
nucleobase refers to 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.
[0092] "Nucleobase sequence" means the order of contiguous
nucleobases independent of any sugar, linkage, and/or nucleobase
modification.
[0093] "Nucleoside" means a nucleobase linked to a sugar.
[0094] "Nucleoside mimetic" includes those structures used to
replace the sugar or the sugar and the base and not necessarily the
linkage at one or more positions of an oligomeric compound such as
for example nucleoside mimetics having morpholino, cyclohexenyl,
cyclohexyl, tetrahydropyranyl, bicyclo, or tricyclo sugar mimetics,
e.g., non furanose sugar units. Nucleotide mimetic includes those
structures used to replace the nucleoside and the linkage at one or
more positions of an oligomeric compound such as for example
peptide nucleic acids or morpholinos (morpholinos linked by
--N(H)--C(.dbd.O)--O-- or other non-phosphodiester linkage). Sugar
surrogate overlaps with the slightly broader term nucleoside
mimetic but is intended to indicate replacement of the sugar unit
(furanose ring) only. The tetrahydropyranyl rings provided herein
are illustrative of an example of a sugar surrogate wherein the
furanose sugar group has been replaced with a tetrahydropyranyl
ring system. "Mimetic" refers to groups that are substituted for a
sugar, a nucleobase, and/or internucleoside linkage. Generally, a
mimetic is used in place of the sugar or sugar-internucleoside
linkage combination, and the nucleobase is maintained for
hybridization to a selected target.
[0095] "Nucleotide" means a nucleoside having a phosphate group
covalently linked to the sugar portion of the nucleoside.
[0096] "Off-target effect" refers to an unwanted or deleterious
biological effect associated with modulation of RNA or protein
expression of a gene other than the intended target nucleic
acid.
[0097] "Oligomeric compound" or "oligomer" means a polymer of
linked monomeric subunits which is capable of hybridizing to at
least a region of a nucleic acid molecule.
[0098] "Oligonucleotide" means a polymer of linked nucleosides each
of which can be modified or unmodified, independent one from
another.
[0099] "Parenteral administration" means administration through
injection (e.g., bolus injection) or infusion. Parenteral
administration includes subcutaneous administration, intravenous
administration, intramuscular administration, intraarterial
administration, intraperitoneal administration, or intracranial
administration, e.g., intrathecal or intracerebroventricular
administration.
[0100] "Peptide" means a molecule formed by linking at least two
amino acids by amide bonds. Without limitation, as used herein,
peptide refers to polypeptides and proteins.
[0101] "Pharmaceutical agent" means a substance that provides a
therapeutic benefit when administered to an individual. In certain
embodiments, an antisense oligonucleotide targeted to C9ORF72 sense
transcript is a pharmaceutical agent. In certain embodiments, an
antisense oligonucleotide targeted to C9ORF72 antisense transcript
is a pharmaceutical agent.
[0102] "Pharmaceutical composition" means a mixture of substances
suitable for administering to as subject. For example, a
pharmaceutical composition may comprise an antisense
oligonucleotide and a sterile aqueous solution.
[0103] "Pharmaceutically acceptable derivative" encompasses
pharmaceutically acceptable salts, conjugates, prodrugs or isomers
of the compounds described herein.
[0104] "Pharmaceutically acceptable salts" means physiologically
and pharmaceutically acceptable salts of antisense compounds, i.e.,
salts that retain the desired biological activity of the parent
oligonucleotide and do not impart undesired toxicological effects
thereto.
[0105] "Phosphorothioate linkage" means a linkage between
nucleosides where the phosphodiester bond is modified by replacing
one of the non-bridging oxygen atoms with a sulfur atom. A
phosphorothioate linkage is a modified internucleoside linkage.
[0106] "Portion" means a defined number of contiguous (i.e.,
linked) nucleobases of a nucleic acid. In certain embodiments, a
portion is a defined number of contiguous nucleobases of a target
nucleic acid. In certain embodiments, a portion is a defined number
of contiguous nucleobases of an antisense compound.
[0107] "Prevent" or "preventing" refers to delaying or forestalling
the onset or development of a disease, disorder, or condition for a
period of time from minutes to days, weeks to months, or
indefinitely.
[0108] "Prodrug" means a therapeutic agent that is prepared in an
inactive form that is converted to an active form within the body
or cells thereof by the action of endogenous enzymes or other
chemicals or conditions.
[0109] "Prophylactically effective amount" refers to an amount of a
pharmaceutical agent that provides a prophylactic or preventative
benefit to an animal.
[0110] "Region" is defined as a portion of the target nucleic acid
having at least one identifiable structure, function, or
characteristic.
[0111] "Ribonucleotide" means a nucleotide having a hydroxy at the
2' position of the sugar portion of the nucleotide. Ribonucleotides
may be modified with any of a variety of substituents.
[0112] "Salts" mean a physiologically and pharmaceutically
acceptable salts of antisense compounds, i.e., salts that retain
the desired biological activity of the parent oligonucleotide and
do not impart undesired toxicological effects thereto.
[0113] "Segments" are defined as smaller or sub-portions of regions
within a target nucleic acid.
[0114] "Shortened" or "truncated" versions of antisense
oligonucleotides taught herein have one, two or more nucleosides
deleted.
[0115] "Side effects" means physiological responses attributable to
a treatment other than desired effects. In certain embodiments,
side effects include, without limitation, injection site reactions,
liver function test abnormalities, renal function abnormalities,
liver toxicity, renal toxicity, central nervous system
abnormalities, and myopathies.
[0116] "Single-stranded oligonucleotide" means an oligonucleotide
which is not hybridized to a complementary strand.
[0117] "Sites," as used herein, are defined as unique nucleobase
positions within a target nucleic acid.
[0118] "Slows progression" means decrease in the development of the
disease.
[0119] "Specifically hybridizable" refers to an antisense compound
having a sufficient degree of complementarity between an antisense
oligonucleotide and a target nucleic acid to induce a desired
effect, while exhibiting minimal or no effects on non-target
nucleic acids under conditions in which specific binding is
desired, i.e., under physiological conditions in the case of in
vivo assays and therapeutic treatments.
[0120] "Stringent hybridization conditions" or "stringent
conditions" refer to conditions under which an oligomeric compound
will hybridize to its target sequence, but to a minimal number of
other sequences.
[0121] "Subject" means a human or non-human animal selected for
treatment or therapy.
[0122] "Targeting" or "targeted" means the process of design and
selection of an antisense compound that will specifically hybridize
to a target nucleic acid and induce a desired effect.
[0123] "Target nucleic acid," "target RNA," and "target RNA
transcript" and "nucleic acid target" all mean a nucleic acid
capable of being targeted by antisense compounds.
[0124] "Target region" means a portion of a target nucleic acid to
which one or more antisense compounds is targeted.
[0125] "Target segment" means the sequence of nucleotides of a
target nucleic acid to which an antisense compound is targeted. "5'
target site" refers to the 5'-most nucleotide of a target segment.
"3' target site" refers to the 3'-most nucleotide of a target
segment.
[0126] "Therapeutically effective amount" means an amount of a
pharmaceutical agent that provides a therapeutic benefit to an
individual.
[0127] "Treat" or "treating" or "treatment" means administering a
composition to effect an alteration or improvement of a disease or
condition.
[0128] "Unmodified nucleobases" means the purine bases adenine (A)
and guanine (G), and the pyrimidine bases (T), cytosine (C), and
uracil (U).
[0129] "Unmodified nucleotide" means a nucleotide composed of
naturally occurring nucleobases, sugar moieties, and
internucleoside linkages. In certain embodiments, an unmodified
nucleotide is an RNA nucleotide (i.e. .beta.-D-ribonucleosides) or
a DNA nucleotide (i.e. .beta.-D-deoxyribonucleoside).
[0130] "Wing segment" means a plurality of nucleosides modified to
impart to an oligonucleotide properties such as enhanced inhibitory
activity, increased binding affinity for a target nucleic acid, or
resistance to degradation by in vivo nucleases.
Certain Embodiments
[0131] Provided herein are compounds comprising a C9ORF72 antisense
transcript specific inhibitor.
[0132] In certain embodiments, the C9ORF72 antisense transcript
specific inhibitor is an antisense compound.
[0133] In certain embodiments, the C9ORF72 antisense transcript
specific antisense compound is an antisense oligonucleotide.
[0134] In certain embodiments, the antisense oligonucleotide
consists of 12-30 linked nucleosides.
[0135] In certain embodiments, the antisense oligonucleotide
consists of 16-25 linked nucleosides.
[0136] In certain embodiments, the antisense oligonucleotide
consists of 18-22 linked nucleosides
[0137] In certain embodiments, the antisense oligonucleotide has a
nucleobase sequence that is at least 80%, at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% complementary to a C9ORF72 antisense transcript.
[0138] In certain embodiments, the antisense oligonucleotide has a
nucleobase sequence that is at least 90% complementary to a C9ORF72
antisense transcript.
[0139] In certain embodiments, the antisense oligonucleotide has a
nucleobase sequence that is at least 95% complementary to a C9ORF72
antisense transcript.
[0140] In certain embodiments, the antisense oligonucleotide has a
nucleobase sequence that is 100% complementary to a C9ORF72
antisense transcript.
[0141] In certain embodiments, the C9ORF72 antisense transcript has
the nucleobase sequence of SEQ ID NO: 13.
[0142] In certain embodiments, the antisense oligonucleotide has a
nucleobase sequence comprising at least 8, at least 9, at least 10,
at least 11, at least 12, at least 13, at least 14, at least 15, at
least 16, at least 17, at least 18, at least 19, or 20 contiguous
nucleobases of a sequence selected from among SEQ ID NO: 30-84.
[0143] In certain embodiments, the antisense oligonucleotide is a
modified antisense oligonucleotide.
[0144] In certain embodiments, the modified antisense
oligonucleotide comprises at least one modified internucleoside
linkage.
[0145] In certain embodiments, each modified internucleoside
linkage is a phosphorothioate internucleoside linkage.
[0146] In certain embodiments, the at least one modified
internucleoside linkage is a phosphorothioate internucleoside
linkage.
[0147] In certain embodiments, the modified antisense
oligonucleotide comprises at least one phosphodiester
internucleoside linkage.
[0148] In certain embodiments, at least one modified nucleobase is
a hypoxanthine.
[0149] In certain embodiments, at least one nucleoside of the
modified antisense oligonucleotide is an inosine.
[0150] In certain embodiments, the at least one nucleoside of the
modified antisense oligonucleotide comprises a modified
nucleobase.
[0151] In certain embodiments, at least one modified nucleobase is
a 5-methylcytosine.
[0152] In certain embodiments, the at least one nucleoside of the
modified antisense oligonucleotide comprises a modified sugar.
[0153] In certain embodiments, the at least one modified sugar is a
bicyclic sugar.
[0154] In certain embodiments, the bicyclic sugar comprises a
chemical bridge between the 2' and 4' position of the sugar,
wherein the chemical bridge is selected from: 4'-CH.sub.2--O-2';
4'-CH(CH.sub.3)--O-2'; 4'-(CH.sub.2).sub.2--O-2'; and
4'-CH.sub.2--N(R)--O-2' wherein R is, independently, H,
C.sub.1-C.sub.12 alkyl, or a protecting group.
[0155] In certain embodiments, the at least one modified sugar
comprises a 2'-O-methoxyethyl group.
[0156] In certain embodiments, the antisense oligonucleotide is a
gapmer.
[0157] In certain embodiments, the compound comprises at least one
conjugate.
[0158] In certain embodiments, the C9ORF72 antisense transcript
specific antisense compound consists of an antisense
oligonucleotide.
[0159] Provided herein are pharmaceutical compositions comprising
any compound described herein and a pharmaceutically acceptable
diluent or carrier.
[0160] Provided herein are pharmaceutical compositions comprising a
C9ORF72 antisense transcript specific inhibitor.
[0161] Provided herein are pharmaceutical compositions comprising a
C9ORF72 antisense transcript specific inhibitor and a C9ORF sense
transcript specific inhibitor.
[0162] In certain embodiments, the C9ORF72 sense transcript
specific inhibitor is a C9ORF72 sense transcript specific antisense
compound.
[0163] In certain embodiments, the C9ORF72 antisense transcript
specific inhibitor is a C9ORF72 antisense transcript specific
antisense compound.
[0164] In certain embodiments, the C9ORF72 sense transcript
specific antisense compound is an antisense oligonucleotide.
[0165] In certain embodiments, the C9ORF72 antisense transcript
specific antisense compound is an antisense oligonucleotide.
[0166] In certain embodiments, the C9ORF72 antisense transcript has
the nucleobase sequence of SEQ ID NO: 13.
[0167] In certain embodiments, the C9ORF72 sense transcript has the
nucleobase sequence of SEQ ID NO: 1-10.
[0168] Provided herein are uses of any compound described herein
for the manufacture of a medicament for treating a
neurodegenerative disease.
[0169] Provided herein are methods, comprising contacting a cell
with an antisense oligonucleotide having a nucleobase sequence of
any of SEQ ID NOs: 30-84.
[0170] Provided herein are methods, comprising contacting a cell
with a C9ORF72 antisense transcript specific inhibitor.
[0171] Provided herein are methods, comprising contacting a cell
with a C9ORF72 antisense transcript specific inhibitor and a
C9ORF72 sense transcript specific inhibitor.
[0172] Provided herein are methods, comprising contacting a cell
with a C9ORF72 antisense transcript specific inhibitor; and thereby
reducing the level or expression of C9ORF72 antisense transcript in
the cell.
[0173] Provided herein are methods, comprising contacting a cell
with a C9ORF72 antisense transcript specific inhibitor and a
C9ORF72 sense transcript specific inhibitor; and thereby reducing
the level or expression of both C9ORF72 antisense transcript and
C9ORF72 sense transcript in the cell.
[0174] In certain embodiments, the C9ORF72 antisense specific
inhibitor is an antisense compound.
[0175] In certain embodiments, the C9ORF72 antisense transcript
specific inhibitor is an antisense compound.
[0176] In certain embodiments, the cell is in vitro.
[0177] In certain embodiments, the cell is in an animal.
[0178] Provided herein are methods, comprising administering to an
animal in need thereof a therapeutically effective amount of a
C9ORF72 antisense transcript specific inhibitor.
[0179] In certain embodiments the amount is effective to reduce the
level or expression of the C9ORF72 antisense transcript.
[0180] Provided herein are methods, comprising co-administering to
an animal in need thereof a therapeutically effective amount of a
C9ORF72 antisense transcript inhibitor and a therapeutically
effective amount of a C9ORF72 sense transcript inhibitor.
[0181] In certain embodiments the therapeutically effective amount
is effective to reduce the level or expression of the C9ORF72
antisense transcript and the C9ORF72 sense transcript.
[0182] In certain embodiments, wherein the C9ORF72 antisense
transcript inhibitor is a C9ORF72 antisense transcript specific
antisense compound.
[0183] In certain embodiments, the C9ORF72 sense transcript
inhibitor is a C9ORF72 sense transcript specific antisense
compound.
[0184] Provided herein are methods, comprising:
[0185] identifying an animal having a C9ORF72 associated disease;
and
[0186] administering to the animal a therapeutically effective
amount of a C9ORF72 antisense transcript specific inhibitor.
[0187] In certain embodiments the amount is effective to reduce the
level or expression of the C9OR72 antisense transcript.
[0188] Provided herein are methods, comprising:
[0189] identifying an animal having a C9ORF72 associated disease;
and
[0190] coadministering to the animal a therapeutically effective
amount of a C9ORF72 antisense transcript specific inhibitor and a
therapeutically effective amount of a C9ORF72 sense transcript
inhibitor.
[0191] In certain embodiments the amount is effective to reduce the
level or expression of the C9ORF72 antisense transcript and the
C9ORF72 sense transcript.
[0192] In certain embodiments the C9ORF72 antisense transcript
specific inhibitor is a C9ORF72 antisense transcript specific
antisense compound.
[0193] In certain embodiments the C9ORF72 sense transcript
inhibitor is a C9ORF72 sense transcript specific antisense
compound.
[0194] In certain embodiments the C9ORF72 antisense transcript
specific antisense compound is at least 80%, at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% complementary to a C9ORF72 anti sense transcript.
[0195] In certain embodiments the C9ORF72 sense transcript specific
antisense compound is at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
complementary to a C9ORF72 sense transcript.
[0196] In certain embodiments the C9ORF72 antisense transcript is
SEQ ID NO: 13.
[0197] In certain embodiments the C9ORF72 sense transcript is any
of SEQ ID NO: 1-10.
[0198] In certain embodiments the C9ORF72 associated disease is a
C9ORF72 hexanucleotide repeat expansion associated disease.
[0199] In certain embodiments the C9ORF72 associated disease or
C9ORF72 hexanucleotide repeat expansion associated disease is
amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD),
corticobasal degeneration syndrome (CBD), atypical Parkinsonian
syndrome, and olivopontocerebellar degeneration (OPCD).
[0200] In certain embodiments the amyotrophic lateral sclerosis
(ALS) is familial ALS or sporadic ALS.
[0201] In certain embodiments the contacting or administering
reduces C9ORF72 antisense transcript associated RAN translation
products.
[0202] In certain embodiments the C9ORF72 antisense transcript
associated RAN translation products are any of
poly-(proline-alanine), poly-(proline-arginine), and
poly-(proline-glycine).
[0203] In certain embodiments the administering and coadminstering
is parenteral administration.
[0204] In certain embodiments the parental administration is any of
injection or infusion.
[0205] In certain embodiments the parenteral administration is any
of intrathecal administration or intracerebroventricular
administration.
[0206] In certain embodiments at least one symptom of a C9ORF72
associated disease or a C9ORF72 hexanucleotide repeat expansion
associated disease is slowed, ameliorated, or prevented.
[0207] In certain embodiments the at least one symptom is any of
motor function, respiration, muscle weakness, fasciculation and
cramping of muscles, difficulty in projecting the voice, shortness
of breath, difficulty in breathing and swallowing, inappropriate
social behavior, lack of empathy, distractibility, changes in food
preferences, agitation, blunted emotions, neglect of personal
hygiene, repetitive or compulsive behavior, and decreased energy
and motivation.
[0208] In certain embodiments the C9ORF72 antisense transcript
specific antisense compound is an antisense oligonucleotide.
[0209] In certain embodiments the C9ORF72 sense transcript specific
antisense compound is an antisense oligonucleotide.
[0210] In certain embodiments the antisense oligonucleotide is a
modified antisense oligonucleotide.
[0211] In certain embodiments at least one internucleoside linkage
of the antisense oligonucleotide is a modified internucleoside
linkage.
[0212] In certain embodiments at least one modified internucleoside
linkage is a phosphorothioate internucleoside linkage.
[0213] In certain embodiments each modified internucleoside linkage
is a phosphorothioate internucleoside linkage.
[0214] In certain embodiments at least one nucleoside of the
modified antisense oligonucleotide comprises a modified
nucleobase.
[0215] In certain embodiments the modified nucleobase is a
5-methylcytosine.
[0216] In certain embodiments at least one nucleoside of the
modified antisense oligonucleotide comprises a modified sugar.
[0217] In certain embodiments at least one modified sugar is a
bicyclic sugar.
[0218] In certain embodiments the bicyclic sugar comprises a
chemical bridge between the 2' and 4' position of the sugar,
wherein the chemical bridge is selected from: 4'-CH.sub.2--O-2';
4'-CH(CH.sub.3)--O-2'; 4'-(CH.sub.2).sub.2--O-2'; and
4'-CH.sub.2--N(R)--O-2' wherein R is, independently, H,
C.sub.1-C.sub.12 alkyl, or a protecting group.
[0219] In certain embodiments at least one modified sugar comprises
a 2'-O-methoxyethyl group.
[0220] In certain embodiments the antisense oligonucleotide is a
gapmer.
[0221] The present disclosure provides the following non-limiting
numbered embodiments: Embodiment 1. A compound comprising a
modified oligonucleotide consisting of 12-30 linked nucleosides and
having a nucleobase sequence comprising at least 8, at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, at least 19, or at
least 20 consecutive nucleobases of any of the nucleobases
sequences of SEQ ID NOs: 30-99.
[0222] Embodiment 2. The compound of embodiment 1, wherein the
modified oligonucleotide has a nucleobase sequence that is at least
80%, at least 85%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, or 100% complementary to a C9ORF72
antisense transcript.
[0223] Embodiment 3. The compound of embodiment 2, wherein the
C9ORF72 antisense transcript has the nucleobase sequence of SEQ ID
NO: 13.
[0224] Embodiment 4. The compound of any of embodiments 1-3,
wherein the modified oligonucleotide is a single-stranded modified
oligonucleotide.
[0225] Embodiment 5. The compound of any of embodiments 1-4,
wherein the modified oligonucleotide comprises at least one
modified internucleoside linkage.
[0226] Embodiment 6. The compound of any of embodiment 5, wherein
the at least one modified internucleoside linkage is a
phosphorothioate internucleoside linkage.
[0227] Embodiment 7. The compound of embodiments 5 or 6, wherein
the modified oligonucleotide comprises at least one phosphodiester
linkage.
[0228] Embodiment 8. The compound of embodiment 6, wherein each
internucleoside linkage is a phosphorothioate internucleoside
linkage.
[0229] Embodiment 9. The compound of any of embodiments 1-8,
wherein at least one nucleoside of the modified oligonucleotide
comprises a modified nucleobase.
[0230] Embodiment 10. The compound of embodiment 9, wherein the
modified nucleobase is a 5 methylcytosine.
[0231] Embodiment 11. The compound of any of embodiments 1-10,
wherein at least one nucleoside of the modified oligonucleotide
comprises a modified sugar.
[0232] Embodiment 12. The compound of embodiment 11, wherein each
nucleoside of the modified oligonucleotide comprises a modified
sugar.
[0233] Embodiment 13. The compound of embodiments 11 or 12, wherein
the modified sugar is a bicyclic sugar.
[0234] Embodiment 14. The compound of embodiment 13, wherein the
bicyclic sugar comprises a chemical bridge between the 4' and 2'
positions of the sugar, wherein the chemical bridge is selected
from: 4'-CH(R)--O-2' and 4'-(CH.sub.2).sub.2--O-2', wherein R is
independently selected from H, C.sub.1-C.sub.6 alkyl, and
C.sub.1-C.sub.6 alkoxy.
[0235] Embodiment 15. The compound of embodiment 14, wherein the
chemical bridge is 4'-CH(R)-0-2' and wherein R is methyl.
[0236] Embodiment 16. The compound of embodiment 14, wherein the
chemical bridge is 4'-CH(R)-0-2' and wherein R is H.
[0237] Embodiment 17. The compound of embodiment 14, wherein the
chemical bridge is 4'-CH(R)-0-2' and wherein R is
--CH.sub.2--O--CH.sub.3.
[0238] Embodiment 18. The compound of embodiments 11 or 12, wherein
the modified sugar comprises a 2'-O-methoxyethyl group.
[0239] Embodiment 19. The compound of any of embodiments 1-11 and
13-18, wherein the modified oligonucleotide is a gapmer.
[0240] Embodiment 20. The compound of embodiment 19, wherein the
gapmer is selected from a 5-10-5 MOE gapmer, a 5-8-5 MOE gapmer, or
a 4-8-4 MOE gapmer.
[0241] Embodiment 21. A pharmaceutical composition comprising the
compound of any preceding embodiment or salt thereof and at least
one of a pharmaceutically acceptable carrier or diluent.
[0242] Embodiment 22. The pharmaceutical composition of embodiment
21 further comprising a C9ORF72 sense transcript specific
inhibitor.
[0243] Embodiment 23. The pharmaceutical composition of embodiment
22, wherein the C9ORF72 sense transcript specific inhibitor is any
of a nucleic acid, aptamer, antibody, peptide, or small
molecule.
[0244] Embodiment 24. The pharmaceutical composition of embodiment
23, wherein the nucleic acid is a single-stranded nucleic acids or
a double-stranded nucleic acid.
[0245] Embodiment 25. The pharmaceutical composition of embodiment
23, wherein the nucleic acid is a siRNA.
[0246] Embodiment 26. The pharmaceutical composition of embodiment
22, wherein the C9ORF72 sense transcript inhibitor is an antisense
compound.
[0247] Embodiment 27. The pharmaceutical composition of embodiment
26, wherein the antisense compound is an antisense
oligonucleotide.
[0248] Embodiment 28. The pharmaceutical composition of embodiment
26, wherein the antisense compound is a modified
oligonucleotide.
[0249] Embodiment 29. The pharmaceutical composition of embodiment
28, wherein the modified oligonucleotide is single-stranded.
[0250] Embodiment 30. The pharmaceutical composition of embodiments
28 or 29, wherein the modified oligonucleotide has a nucleobase
sequence that is at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%
complementary to a C9ORF72 sense transcript.
[0251] Embodiment 31. The pharmaceutical composition of embodiment
30 wherein the C9ORF72 sense transcript has the nucleobase sequence
of SEQ ID NO: 1-10.
[0252] Embodiment 32. Use of the compound or composition of any
preceding embodiment for the manufacture of a medicament for
treating a neurodegenerative disease.
[0253] Embodiment 33. A method comprising administering to an
animal the compound or composition of any preceding embodiment.
[0254] Embodiment 34. The method of embodiment 33, wherein the
compound prevents, treats, ameliorates, or slows progression of at
least one symptom of a C9ORF72 associated disease.
[0255] Embodiment 35. The method of embodiment 34, wherein the at
least one symptom is selected from among impaired motor function,
difficulty with respiration, muscle weakness, fasciculation and
cramping of muscles, difficulty in projecting the voice, shortness
of breath, difficulty in breathing and swallowing, inappropriate
social behavior, lack of empathy, distractibility, changes in food
preference, agitation, blunted emotions, neglect of personal
hygiene, repetitive or compulsive behavior, and decreased energy
and motivation.
[0256] Embodiment 36. The method of embodiment 34, wherein the
C9ORF72 associated disease is amyotrophic lateral sclerosis (ALS),
frontotemporal dementia (FTD), corticobasal degeneration syndrome
(CBD), atypical Parkinsonian syndrome, or olivopontocerebellar
degeneration (OPCD).
[0257] Embodiment 37. The method of embodiment 36, wherein the
amyotrophic lateral sclerosis (ALS) is familial ALS.
[0258] Embodiment 38. The method of embodiment 36, wherein the
amyotrophic lateral sclerosis (ALS) is sporadic ALS.
[0259] Embodiment 39. The method of any of embodiments 33-38,
wherein the administering reduces C9ORF72 antisense transcript
associated RAN translation products.
[0260] Embodiment 40. The method of embodiment 39, wherein the
C9ORF72 antisense transcript associated RAN translation products
are any of poly-(proline-alanine), poly-(proline-arginine), and
poly-(proline-glycine).
[0261] Embodiment 41. The method of any of embodiments 33-40,
wherein the administering reduces C9ORF72 antisense foci.
[0262] Embodiment 42. The method of any of embodiments 33-41,
wherein the administering reduces C9ORF72 sense foci.
[0263] Embodiment 43. The method of any of embodiments 33-42,
wherein the administering is parenteral administration.
[0264] Embodiment 44. The method of embodiment 43, wherein the
parenteral administration is any of injection or infusion.
[0265] Embodiment 45. The method of embodiment 43, wherein the
parenteral administration is directly into the central nervous
system (CNS).
[0266] Embodiment 46. The method of any of embodiments 43-45,
wherein the parenteral administration is any of intrathecal
administration or intracerebroventricular administration.
[0267] Embodiment 47. The compound of embodiment 11, wherein the
modified oligonucleotide comprises sugar residues in any of the
following patterns: eeedeeeeedeeeeedeeee or eeeeedeeeeedeeeeee,
wherein, [0268] e=a 2'-O-methoxyethylribose modified sugar, and
[0269] d=a 2'-deoxyribose sugar.
[0270] Embodiment 48. The compound of embodiment 5, wherein the
modified oligonucleotide comprises internucleoside linkages in any
of the following patterns: soooossssssssssooss, sooosssssssssooss,
ssososssososssososs, or ssssososssosossss, wherein, [0271] s=a
phosphorothioate linkage, and [0272] o=a phosphodiester
linkage.
Antisense Compounds
[0273] Oligomeric compounds include, but are not limited to,
oligonucleotides, oligonucleosides, oligonucleotide analogs,
oligonucleotide mimetics, antisense compounds, antisense
oligonucleotides, and siRNAs. An oligomeric compound may be
"antisense" to a target nucleic acid, meaning that is capable of
undergoing hybridization to a target nucleic acid through hydrogen
bonding.
[0274] In certain embodiments, an antisense compound has a
nucleobase sequence that, when written in the 5' to 3' direction,
comprises the reverse complement of the target segment of a target
nucleic acid to which it is targeted. In certain such embodiments,
an antisense oligonucleotide has a nucleobase sequence that, when
written in the 5' to 3' direction, comprises the reverse complement
of the target segment of a target nucleic acid to which it is
targeted.
[0275] In certain embodiments, an antisense compound targeted to a
C9ORF72 nucleic acid is 12 to 30 subunits in length. In other
words, such anti sense compounds are from 12 to 30 linked subunits.
In certain embodiments, the antisense compound is 8 to 80, 12 to
50, 15 to 30, 18 to 24, 19 to 22, or 20 linked subunits. In certain
embodiments, the antisense compounds are 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80
linked subunits in length, or a range defined by any two of the
above values. In some embodiments the antisense compound is an
antisense oligonucleotide, and the linked subunits are
nucleosides.
[0276] In certain embodiments antisense oligonucleotides targeted
to a C9ORF72 nucleic acid may be shortened or truncated. For
example, a single subunit may be deleted from the 5' end (5'
truncation), or alternatively from the 3' end (3' truncation). A
shortened or truncated antisense compound targeted to a C9ORF72
nucleic acid may have two subunits deleted from the 5' end, or
alternatively may have two subunits deleted from the 3' end, of the
antisense compound. Alternatively, the deleted nucleosides may be
dispersed throughout the antisense compound, for example, in an
antisense compound having one nucleoside deleted from the 5' end
and one nucleoside deleted from the 3' end.
[0277] When a single additional subunit is present in a lengthened
antisense compound, the additional subunit may be located at the 5'
or 3' end of the antisense compound. When two or more additional
subunits are present, the added subunits may be adjacent to each
other, for example, in an antisense compound having two subunits
added to the 5' end (5' addition), or alternatively to the 3' end
(3' addition), of the antisense compound. Alternatively, the added
subunits may be dispersed throughout the antisense compound, for
example, in an antisense compound having one subunit added to the
5' end and one subunit added to the 3' end.
[0278] It is possible to increase or decrease the length of an
antisense compound, such as an antisense oligonucleotide, and/or
introduce mismatch bases without eliminating activity. For example,
in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a
series of antisense oligonucleotides 13-25 nucleobases in length
were tested for their ability to induce cleavage of a target RNA in
an oocyte injection model. Antisense oligonucleotides 25
nucleobases in length with 8 or 11 mismatch bases near the ends of
the antisense oligonucleotides were able to direct specific
cleavage of the target mRNA, albeit to a lesser extent than the
antisense oligonucleotides that contained no mismatches. Similarly,
target specific cleavage was achieved using 13 nucleobase antisense
oligonucleotides, including those with 1 or 3 mismatches.
[0279] Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March
2001) demonstrated the ability of an oligonucleotide having 100%
complementarity to the bcl-2 mRNA and having 3 mismatches to the
bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in
vitro and in vivo. Furthermore, this oligonucleotide demonstrated
potent anti-tumor activity in vivo.
[0280] Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358,1988) tested
a series of tandem 14 nucleobase antisense oligonucleotides, and a
28 and 42 nucleobase antisense oligonucleotides comprised of the
sequence of two or three of the tandem antisense oligonucleotides,
respectively, for their ability to arrest translation of human DHFR
in a rabbit reticulocyte assay. Each of the three 14 nucleobase
antisense oligonucleotides alone was able to inhibit translation,
albeit at a more modest level than the 28 or 42 nucleobase
antisense oligonucleotides.
Antisense Compound Motifs
[0281] In certain embodiments, antisense compounds targeted to a
C9ORF72 nucleic acid have chemically modified subunits arranged in
patterns, or motifs, to confer to the antisense compounds
properties such as enhanced inhibitory activity, increased binding
affinity for a target nucleic acid, or resistance to degradation by
in vivo nucleases.
[0282] Chimeric antisense compounds typically contain at least one
region modified so as to confer increased resistance to nuclease
degradation, increased cellular uptake, increased binding affinity
for the target nucleic acid, and/or increased inhibitory activity.
A second region of a chimeric antisense compound may optionally
serve as a substrate for the cellular endonuclease RNase H, which
cleaves the RNA strand of an RNA:DNA duplex.
[0283] Antisense compounds having a gapmer motif are considered
chimeric antisense compounds. In a gapmer an internal region having
a plurality of nucleotides that supports RNaseH cleavage is
positioned between external regions having a plurality of
nucleotides that are chemically distinct from the nucleosides of
the internal region. In the case of an antisense oligonucleotide
having a gapmer motif, the gap segment generally serves as the
substrate for endonuclease cleavage, while the wing segments
comprise modified nucleosides. In certain embodiments, the regions
of a gapmer are differentiated by the types of sugar moieties
comprising each distinct region. The types of sugar moieties that
are used to differentiate the regions of a gapmer may in some
embodiments include .beta.-D-ribonucleosides,
.beta.-D-deoxyribonucleosides, 2'-modified nucleosides (such
2'-modified nucleosides may include 2'-MOE, and 2'-O--CH.sub.3,
among others), and bicyclic sugar modified nucleosides (such
bicyclic sugar modified nucleosides may include those having a
4'-(CH.sub.2)n-O-2' bridge, where n=1 or n=2 and
4'-CH.sub.2--O--CH.sub.2-2'). Preferably, each distinct region
comprises uniform sugar moieties. The wing-gap-wing motif is
frequently described as "X-Y-Z", where "X" represents the length of
the 5' wing region, "Y" represents the length of the gap region,
and "Z" represents the length of the 3' wing region. As used
herein, a gapmer described as "X-Y-Z" has a configuration such that
the gap segment is positioned immediately adjacent to each of the
5' wing segment and the 3' wing segment. Thus, no intervening
nucleotides exist between the 5' wing segment and gap segment, or
the gap segment and the 3' wing segment. Any of the antisense
compounds described herein can have a gapmer motif. In some
embodiments, X and Z are the same, in other embodiments they are
different. In a preferred embodiment, Y is between 8 and 15
nucleotides. X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides.
Thus, gapmers described herein include, but are not limited to, for
example 5-10-5, 5-10-4, 4-10-4, 4-10-3, 3-10-3, 2-10-2, 5-9-5,
5-9-4, 4-9-5, 5-8-5, 5-8-4, 4-8-5, 5-7-5, 4-7-5, 5-7-4, or
4-7-4.
[0284] In certain embodiments, the antisense compound has a
"wingmer" motif, having a wing-gap or gap-wing configuration, i.e.
an X-Y or Y-Z configuration as described above for the gapmer
configuration. Thus, wingmer configurations described herein
include, but are not limited to, for example 5-10, 8-4, 4-12, 12-4,
3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13, 5-13, 5-8, or
6-8.
[0285] In certain embodiments, an antisense compound targeted to a
C9ORF72 nucleic acid has a gap-narrowed motif. In certain
embodiments, a gap-narrowed antisense oligonucleotide targeted to a
C9ORF72 nucleic acid has a gap segment of 9, 8, 7, or 6
2'-deoxynucleotides positioned immediately adjacent to and between
wing segments of 5, 4, 3, 2, or 1 chemically modified nucleosides.
In certain embodiments, the chemical modification comprises a
bicyclic sugar. In certain embodiments, the bicyclic sugar
comprises a 4' to 2' bridge selected from among:
4'-(CH.sub.2).sub.n--O-2' bridge, wherein n is 1 or 2; and
4'-CH.sub.2--O--CH.sub.2-2'. In certain embodiments, the bicyclic
sugar is comprises a 4'-CH(CH.sub.3)--O-2' bridge. In certain
embodiments, the chemical modification comprises a non-bicyclic
2'-modified sugar moiety. In certain embodiments, the non-bicyclic
2'-modified sugar moiety comprises a 2'-O-methylethyl group or a
2'-O-methyl group.
Target Nucleic Acids, Target Regions and Nucleotide Sequences
[0286] Nucleotide sequences that encode C9ORF72 include, without
limitation, the following: the complement of GENBANK Accession No.
NM 001256054.1 (incorporated herein as SEQ ID NO: 1), GENBANK
Accession No. NT 008413.18 truncated from nucleobase 27535000 to
27565000 (incorporated herein as SEQ ID NO: 2), GENBANK Accession
No. BQ068108.1 (incorporated herein as SEQ ID NO: 3), GENBANK
Accession No. NM 018325.3 (incorporated herein as SEQ ID NO: 4),
GENBANK Accession No. DN993522.1 (incorporated herein as SEQ ID NO:
5), GENBANK Accession No. NM 145005.5 (incorporated herein as SEQ
ID NO: 6), GENBANK Accession No. DB079375.1 (incorporated herein as
SEQ ID NO: 7), GENBANK Accession No. BU194591.1 (incorporated
herein as SEQ ID NO: 8), Sequence Identifier 4141_014_A
(incorporated herein as SEQ ID NO: 9), and Sequence Identifier
4008_73_A (incorporated herein as SEQ ID NO: 10).
[0287] Nucleotide sequences that encode the C9ORF72 antisense
transcript include, without limitation, the following: SEQ ID NO:
13. The sequence of SEQ ID NO: 13 is complementary to nucleotides
1159 to 1929 of SEQ ID NO: 2 (the complement of GENBANK Accession
No. NT 008413.18 truncated from nucleotides 27535000 to 27565000)
except that SEQ ID NO: 13 has two more hexanucleotide repeats than
SEQ ID NO: 2. The sequence of the hexanucleotide repeat is GGCCCC
in SEQ ID NO: 13 and GGGGCC in SEQ ID NO: 2. Thus, SEQ ID NO: 13 is
12 nucleotides longer than nucleotides 1159 to 1929 of SEQ ID NO:
2, to which it is complementary.
[0288] It is understood that the sequence set forth in each SEQ ID
NO in the Examples contained herein is independent of any
modification to a sugar moiety, an internucleoside linkage, or a
nucleobase. As such, antisense compounds defined by a SEQ ID NO may
comprise, independently, one or more modifications to a sugar
moiety, an internucleoside linkage, or a nucleobase. Antisense
compounds described by Isis Number (Isis No) indicate a combination
of nucleobase sequence and motif.
[0289] In certain embodiments, a target region is a structurally
defined region of the target nucleic acid. For example, a target
region may encompass a 3' UTR, a 5' UTR, an exon, an intron, an
exon/intron junction, a coding region, a translation initiation
region, translation termination region, or other defined nucleic
acid region. The structurally defined regions for C9ORF72 can be
obtained by accession number from sequence databases such as NCBI
and such information is incorporated herein by reference. In
certain embodiments, a target region may encompass the sequence
from a 5' target site of one target segment within the target
region to a 3' target site of another target segment within the
same target region.
[0290] Targeting includes determination of at least one target
segment to which an antisense compound hybridizes, such that a
desired effect occurs. In certain embodiments, the desired effect
is a reduction in mRNA target nucleic acid levels. In certain
embodiments, the desired effect is reduction of levels of protein
encoded by the target nucleic acid or a phenotypic change
associated with the target nucleic acid.
[0291] A target region may contain one or more target segments.
Multiple target segments within a target region may be overlapping.
Alternatively, they may be non-overlapping. In certain embodiments,
target segments within a target region are separated by no more
than about 300 nucleotides. In certain embodiments, target segments
within a target region are separated by a number of nucleotides
that is, is about, is no more than, is no more than about, 250,
200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on
the target nucleic acid, or is a range defined by any two of the
preceeding values. In certain embodiments, target segments within a
target region are separated by no more than, or no more than about,
5 nucleotides on the target nucleic acid. In certain embodiments,
target segments are contiguous. Contemplated are target regions
defined by a range having a starting nucleic acid that is any of
the 5' target sites or 3' target sites listed herein.
[0292] Suitable target segments may be found within a 5' UTR, a
coding region, a 3' UTR, an intron, an exon, or an exon/intron
junction. Target segments containing a start codon or a stop codon
are also suitable target segments. A suitable target segment may
specifically exclude a certain structurally defined region such as
the start codon or stop codon.
[0293] The determination of suitable target segments may include a
comparison of the sequence of a target nucleic acid to other
sequences throughout the genome. For example, the BLAST algorithm
may be used to identify regions of similarity amongst different
nucleic acids. This comparison can prevent the selection of
antisense compound sequences that may hybridize in a non-specific
manner to sequences other than a selected target nucleic acid
(i.e., non-target or off-target sequences).
[0294] There may be variation in activity (e.g., as defined by
percent reduction of target nucleic acid levels) of the antisense
compounds within a target region. In certain embodiments,
reductions in C9ORF72 mRNA levels are indicative of inhibition of
C9ORF72 expression. Reductions in levels of a C9ORF72 protein are
also indicative of inhibition of target mRNA expression. Reduction
in the presence of expanded C9ORF72 RNA foci are indicative of
inhibition of C9ORF72 expression. Further, phenotypic changes are
indicative of inhibition of C9ORF72 expression. For example,
improved motor function and respiration may be indicative of
inhibition of C9ORF72 expression.
Hybridization
[0295] In some embodiments, hybridization occurs between an
antisense compound disclosed herein and a C9ORF72 nucleic acid. The
most common mechanism of hybridization involves hydrogen bonding
(e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen
bonding) between complementary nucleobases of the nucleic acid
molecules.
[0296] Hybridization can occur under varying conditions. Stringent
conditions are sequence-dependent and are determined by the nature
and composition of the nucleic acid molecules to be hybridized.
[0297] Methods of determining whether a sequence is specifically
hybridizable to a target nucleic acid are well known in the art. In
certain embodiments, the antisense compounds provided herein are
specifically hybridizable with a C9ORF72 nucleic acid.
Complementarily
[0298] An antisense compound and a target nucleic acid are
complementary to each other when a sufficient number of nucleobases
of the antisense compound can hydrogen bond with the corresponding
nucleobases of the target nucleic acid, such that a desired effect
will occur (e.g., antisense inhibition of a target nucleic acid,
such as a C9ORF72 nucleic acid).
[0299] Non-complementary nucleobases between an antisense compound
and a C9ORF72 nucleic acid may be tolerated provided that the
antisense compound remains able to specifically hybridize to a
target nucleic acid. Moreover, an antisense compound may hybridize
over one or more segments of a C9ORF72 nucleic acid such that
intervening or adjacent segments are not involved in the
hybridization event (e.g., a loop structure, mismatch or hairpin
structure).
[0300] In certain embodiments, the antisense compounds provided
herein, or a specified portion thereof, are, or are at least, 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% complementary to a C9ORF72 nucleic acid, a
target region, target segment, or specified portion thereof.
Percent complementarity of an antisense compound with a target
nucleic acid can be determined using routine methods.
[0301] For example, an antisense compound in which 18 of 20
nucleobases of the antisense compound are complementary to a target
region, and would therefore specifically hybridize, would represent
90 percent complementarity. In this example, the remaining
noncomplementary nucleobases may be clustered or interspersed with
complementary nucleobases and need not be contiguous to each other
or to complementary nucleobases. As such, an antisense compound
which is 18 nucleobases in length having 4 (four) noncomplementary
nucleobases which are flanked by two regions of complete
complementarity with the target nucleic acid would have 77.8%
overall complementarity with the target nucleic acid and would thus
fall within the scope of the present invention. Percent
complementarity of an antisense compound with a region of a target
nucleic acid can be determined routinely using BLAST programs
(basic local alignment search tools) and PowerBLAST programs known
in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410;
Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology,
sequence identity or complementarity, can be determined by, for
example, the Gap program (Wisconsin Sequence Analysis Package,
Version 8 for Unix, Genetics Computer Group, University Research
Park, Madison Wis.), using default settings, which uses the
algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482
489).
[0302] In certain embodiments, the antisense compounds provided
herein, or specified portions thereof, are fully complementary
(i.e., 100% complementary) to a target nucleic acid, or specified
portion thereof. For example, an antisense compound may be fully
complementary to a C9ORF72 nucleic acid, or a target region, or a
target segment or target sequence thereof. As used herein, "fully
complementary" means each nucleobase of an antisense compound is
capable of precise base pairing with the corresponding nucleobases
of a target nucleic acid. For example, a 20 nucleobase antisense
compound is fully complementary to a target sequence that is 400
nucleobases long, so long as there is a corresponding 20 nucleobase
portion of the target nucleic acid that is fully complementary to
the antisense compound. Fully complementary can also be used in
reference to a specified portion of the first and/or the second
nucleic acid. For example, a 20 nucleobase portion of a 30
nucleobase antisense compound can be "fully complementary" to a
target sequence that is 400 nucleobases long. The 20 nucleobase
portion of the 30 nucleobase oligonucleotide is fully complementary
to the target sequence if the target sequence has a corresponding
20 nucleobase portion wherein each nucleobase is complementary to
the 20 nucleobase portion of the antisense compound. At the same
time, the entire 30 nucleobase antisense compound may or may not be
fully complementary to the target sequence, depending on whether
the remaining 10 nucleobases of the antisense compound are also
complementary to the target sequence.
[0303] The location of a non-complementary nucleobase may be at the
5' end or 3' end of the antisense compound. Alternatively, the
non-complementary nucleobase or nucleobases may be at an internal
position of the antisense compound. When two or more
non-complementary nucleobases are present, they may be contiguous
(i.e., linked) or non-contiguous. In one embodiment, a
non-complementary nucleobase is located in the wing segment of a
gapmer antisense oligonucleotide.
[0304] In certain embodiments, antisense compounds that are, or are
up to 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length
comprise no more than 4, no more than 3, no more than 2, or no more
than 1 non-complementary nucleobase(s) relative to a target nucleic
acid, such as a C9ORF72 nucleic acid, or specified portion
thereof.
[0305] In certain embodiments, antisense compounds that are, or are
up to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, or 30 nucleobases in length comprise no more than 6, no
more than 5, no more than 4, no more than 3, no more than 2, or no
more than 1 non-complementary nucleobase(s) relative to a target
nucleic acid, such as a C9ORF72 nucleic acid, or specified portion
thereof.
[0306] The antisense compounds provided herein also include those
which are complementary to a portion of a target nucleic acid. As
used herein, "portion" refers to a defined number of contiguous
(i.e. linked) nucleobases within a region or segment of a target
nucleic acid. A "portion" can also refer to a defined number of
contiguous nucleobases of an antisense compound. In certain
embodiments, the antisense compounds, are complementary to at least
an 8 nucleobase portion of a target segment. In certain
embodiments, the antisense compounds are complementary to at least
a 9 nucleobase portion of a target segment. In certain embodiments,
the antisense compounds are complementary to at least a 10
nucleobase portion of a target segment. In certain embodiments, the
antisense compounds, are complementary to at least an 11 nucleobase
portion of a target segment. In certain embodiments, the antisense
compounds, are complementary to at least a 12 nucleobase portion of
a target segment. In certain embodiments, the antisense compounds,
are complementary to at least a 13 nucleobase portion of a target
segment. In certain embodiments, the antisense compounds, are
complementary to at least a 14 nucleobase portion of a target
segment. In certain embodiments, the antisense compounds, are
complementary to at least a 15 nucleobase portion of a target
segment. Also contemplated are antisense compounds that are
complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, or more nucleobase portion of a target segment, or a range
defined by any two of these values.
Identity
[0307] The antisense compounds provided herein may also have a
defined percent identity to a particular nucleotide sequence, SEQ
ID NO, or compound represented by a specific Isis number, or
portion thereof. As used herein, an antisense compound is identical
to the sequence disclosed herein if it has the same nucleobase
pairing ability. For example, a RNA which contains uracil in place
of thymidine in a disclosed DNA sequence would be considered
identical to the DNA sequence since both uracil and thymidine pair
with adenine. Shortened and lengthened versions of the antisense
compounds described herein as well as compounds having
non-identical bases relative to the antisense compounds provided
herein also are contemplated. The non-identical bases may be
adjacent to each other or dispersed throughout the antisense
compound. Percent identity of an antisense compound is calculated
according to the number of bases that have identical base pairing
relative to the sequence to which it is being compared.
[0308] In certain embodiments, the antisense compounds, or portions
thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to one or more of the antisense compounds or
SEQ ID NOs, or a portion thereof, disclosed herein.
[0309] In certain embodiments, a portion of the antisense compound
is compared to an equal length portion of the target nucleic acid.
In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to
an equal length portion of the target nucleic acid.
[0310] In certain embodiments, a portion of the antisense
oligonucleotide is compared to an equal length portion of the
target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase
portion is compared to an equal length portion of the target
nucleic acid.
Modifications
[0311] A nucleoside is a base-sugar combination. The nucleobase
(also known as base) portion of the nucleoside is normally a
heterocyclic base moiety. Nucleotides are nucleosides that further
include a phosphate group covalently linked to the sugar portion of
the nucleoside. For those nucleosides that include a pentofuranosyl
sugar, the phosphate group can be linked to the 2', 3' or 5'
hydroxyl moiety of the sugar. Oligonucleotides are formed through
the covalent linkage of adjacent nucleosides to one another, to
form a linear polymeric oligonucleotide. Within the oligonucleotide
structure, the phosphate groups are commonly referred to as forming
the internucleoside linkages of the oligonucleotide.
[0312] Modifications to antisense compounds encompass substitutions
or changes to internucleoside linkages, sugar moieties, or
nucleobases. Modified antisense compounds are often preferred over
native forms because of desirable properties such as, for example,
enhanced cellular uptake, enhanced affinity for nucleic acid
target, increased stability in the presence of nucleases, or
increased inhibitory activity.
[0313] Chemically modified nucleosides may also be employed to
increase the binding affinity of a shortened or truncated antisense
oligonucleotide for its target nucleic acid. Consequently,
comparable results can often be obtained with shorter antisense
compounds that have such chemically modified nucleosides.
Modified Internucleoside Linkages
[0314] The naturally occurring internucleoside linkage of RNA and
DNA is a 3' to 5' phosphodiester linkage. Antisense compounds
having one or more modified, i.e. non-naturally occurring,
internucleoside linkages are often selected over antisense
compounds having naturally occurring internucleoside linkages
because of desirable properties such as, for example, enhanced
cellular uptake, enhanced affinity for target nucleic acids, and
increased stability in the presence of nucleases.
[0315] Oligonucleotides having modified internucleoside linkages
include internucleoside linkages that retain a phosphorus atom as
well as internucleoside linkages that do not have a phosphorus
atom. Representative phosphorus containing internucleoside linkages
include, but are not limited to, phosphodiesters, phosphotriesters,
methylphosphonates, phosphoramidate, and phosphorothioates. Methods
of preparation of phosphorous-containing and
non-phosphorous-containing linkages are well known.
[0316] In certain embodiments, antisense compounds targeted to a
C9ORF72 nucleic acid comprise one or more modified internucleoside
linkages. In certain embodiments, the modified internucleoside
linkages are interspersed throughout the antisense compound. In
certain embodiments, the modified internucleoside linkages are
phosphorothioate linkages. In certain embodiments, each
internucleoside linkage of an antisense compound is a
phosphorothioate internucleoside linkage. In certain embodiments,
the antisense compounds targeted to a C9ORF72 nucleic acid comprise
at least one phosphodiester linkage and at least one
phosphorothioate linkage.
Modified Sugar Moieties
[0317] Antisense compounds of the invention can optionally contain
one or more nucleosides wherein the sugar group has been modified.
Such sugar modified nucleosides may impart enhanced nuclease
stability, increased binding affinity, or some other beneficial
biological property to the antisense compounds. In certain
embodiments, nucleosides comprise chemically modified ribofuranose
ring moieties. Examples of chemically modified ribofuranose rings
include without limitation, addition of substitutent groups
(including 5' and 2' substituent groups, bridging of non-geminal
ring atoms to form bicyclic nucleic acids (BNA), replacement of the
ribosyl ring oxygen atom with S, N(R), or C(R.sub.1)(R.sub.2) (R,
R.sub.1 and R.sub.2 are each independently H, C.sub.1-C.sub.12
alkyl or a protecting group) and combinations thereof. Examples of
chemically modified sugars include 2'-F-5'-methyl substituted
nucleoside (see PCT International Application WO 2008/101157
Published on Aug. 21, 2008 for other disclosed 5',2'-bis
substituted nucleosides) or replacement of the ribosyl ring oxygen
atom with S with 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 BNA (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).
[0318] Examples of nucleosides having modified sugar moieties
include without limitation nucleosides comprising 5'-vinyl,
5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH.sub.3, 2'-OCH.sub.2CH.sub.3,
2'-OCH.sub.2CH.sub.2F and 2'-O(CH.sub.2).sub.2OCH.sub.3 substituent
groups. The substituent at the 2' position can also be selected
from allyl, amino, azido, thio, O-allyl, O--C.sub.1-C.sub.10 alkyl,
OCF.sub.3, OCH.sub.2F, 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--C(.dbd.O)--N(R.sub.m)(R.sub.n), and
O--CH.sub.2--C(.dbd.O)--N(R.sub.1)--(CH.sub.2).sub.2--N(R.sub.m)(R.sub.n)-
, where each R.sub.l, R.sub.m and R.sub.n is, independently, H or
substituted or unsubstituted C1-C.sub.10 alkyl.
[0319] As used herein, "bicyclic nucleosides" refer to modified
nucleosides comprising a bicyclic sugar moiety. Examples of
bicyclic nucleic acids (BNAs) include without limitation
nucleosides comprising a bridge between the 4' and the 2' ribosyl
ring atoms. In certain embodiments, antisense compounds provided
herein include one or more BNA nucleosides wherein the bridge
comprises one of the formulas: 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' and 4'-CH(CH.sub.2OCH.sub.3)--O-2' (and
analogs thereof see 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
PCT/US2008/068922 published as WO/2009/006478, published Jan. 8,
2009); 4'-CH.sub.2--N(OCH.sub.3)-2' (and analogs thereof see
PCT/US2008/064591 published as WO/2008/150729, published Dec. 11,
2008); 4'-CH.sub.2--O--N(CH.sub.3)-2' (see published U.S. Patent
Application US2004-0171570, published Sep. 2, 2004);
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 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
PCT/US2008/066154 published as WO 2008/154401, published on Dec. 8,
2008).
[0320] Further bicyclic nucleosides have been reported in published
literature (see for example: Srivastava et al., J. Am. Chem. Soc.,
2007, 129(26) 8362-8379; Frieden et al., Nucleic Acids Research,
2003, 21, 6365-6372; 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; Wahlestedt et
al., Proc. Natl. Acad. Sci. U.S.A, 2000, 97, 5633-5638; Singh et
al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron,
1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998,
8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039;
U.S. Pat. Nos. 7,399,845; 7,053,207; 7,034,133; 6,794,499;
6,770,748; 6,670,461; 6,525,191; 6,268,490; U.S. Patent Publication
Nos.: US2008-0039618; US2007-0287831; US2004-0171570; U.S. patent
application Ser. Nos. 12/129,154; 61/099,844; 61/097,787;
61/086,231; 61/056,564; 61/026,998; 61/026,995; 60/989,574;
International applications WO 2007/134181; WO 2005/021570; WO
2004/106356; and PCT International Applications Nos.:
PCT/US2008/068922; PCT/US2008/066154; and PCT/US2008/064591). Each
of the foregoing bicyclic nucleosides can be prepared having one or
more stereochemical sugar configurations including for example
.alpha.-L-ribofuranose and .beta.-D-ribofuranose (see PCT
international application PCT/DK98/00393, published on Mar. 25,
1999 as WO 99/14226).
[0321] As used herein, "monocylic nucleosides" refer to nucleosides
comprising modified sugar moieties that are not bicyclic sugar
moieties. In certain embodiments, the sugar moiety, or sugar moiety
analogue, of a nucleoside may be modified or substituted at any
position.
[0322] As used herein, "4'-2' bicyclic nucleoside" or "4' to 2'
bicyclic nucleoside" refers to a bicyclic nucleoside comprising a
furanose ring comprising a bridge connecting two carbon atoms of
the furanose ring connects the 2' carbon atom and the 4' carbon
atom of the sugar ring.
[0323] In certain embodiments, bicyclic sugar moieties of BNA
nucleosides include, but are not limited to, compounds having at
least one bridge between the 4' and the 2' carbon atoms of the
pentofuranosyl sugar moiety including without limitation, bridges
comprising 1 or 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)--;
wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R.sub.a and
R.sub.b is, independently, H, a protecting group, hydroxyl,
C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl,
C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl,
C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl,
heterocycle radical, substituted heterocycle radical, heteroaryl,
substituted heteroaryl, C.sub.5-C.sub.7 alicyclic radical,
substituted C.sub.5-C.sub.7 alicyclic radical, halogen, OJ.sub.1,
NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1, acyl
(C(.dbd.O)--H), substituted acyl, CN, sulfonyl
(S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and
[0324] 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.
[0325] In certain embodiments, the bridge of a bicyclic sugar
moiety is, --[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)--. In certain embodiments, the
bridge is 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',
4'-(CH.sub.2).sub.2--O-2', 4'-CH.sub.2--O--N(R)-2' and
4'-CH.sub.2--N(R)--O-2'- wherein each R is, independently, H, a
protecting group or C.sub.1-C.sub.12 alkyl.
[0326] In certain embodiments, bicyclic nucleosides are further
defined by isomeric configuration. For example, a nucleoside
comprising a 4'-(CH.sub.2)--O-2' bridge, may be in the .alpha.-L
configuration or in the .beta.-D configuration. Previously,
.alpha.-L-methyleneoxy (4'-CH.sub.2--O-2') BNA's have been
incorporated into antisense oligonucleotides that showed antisense
activity (Frieden et al., Nucleic Acids Research, 2003, 21,
6365-6372).
[0327] In certain embodiments, bicyclic nucleosides include those
having a 4' to 2' bridge wherein such bridges include without
limitation, .alpha.-L-4'-(CH.sub.2)--O-2',
.beta.-D-4'-CH.sub.2--O-2', 4'-(CH.sub.2).sub.2--O-2',
4'-CH.sub.2--O--N(R)-2', 4'-CH.sub.2--N(R)--O-2',
4'-CH(CH.sub.3)--O-2', 4'-CH.sub.2--S-2', 4'-CH.sub.2--N(R)-2',
4'-CH.sub.2--CH(CH.sub.3)-2', and 4'-(CH.sub.2).sub.3-2', wherein R
is H, a protecting group or C.sub.1-C.sub.12 alkyl.
[0328] In certain embodiment, bicyclic nucleosides have the
formula:
##STR00002##
wherein:
[0329] Bx is a heterocyclic base moiety;
[0330] -Q.sub.a-Q.sub.b-Q.sub.c- is
--CH.sub.2--N(R.sub.c)--CH.sub.2--,
--C(.dbd.O)--N(R.sub.c)--CH.sub.2--, --CH.sub.2--O--N(R.sub.c)--,
--CH.sub.2--N(R.sub.c)--O-- or N(R.sub.c)--O--CH.sub.2;
[0331] R.sub.c is C.sub.1-C.sub.12 alkyl or an amino protecting
group; and
[0332] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium.
[0333] In certain embodiments, bicyclic nucleosides have the
formula:
##STR00003##
wherein:
[0334] Bx is a heterocyclic base moiety;
[0335] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0336] Z.sub.a is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl,
substituted C.sub.2-C.sub.6 alkenyl, substituted C2-C6 alkynyl,
acyl, substituted acyl, substituted amide, thiol or substituted
thiol.
[0337] In one embodiment, each of the substituted groups, is,
independently, mono or poly substituted with substituent groups
independently selected from halogen, oxo, hydroxyl, OJ.sub.c,
NJ.sub.cJ.sub.d, SJ.sub.c, N.sub.3, OC(.dbd.X)J.sub.c, and
NJ.sub.eC(.dbd.X)NJ.sub.cJ.sub.d, wherein each J.sub.c, J.sub.d and
J.sub.e is, independently, H, C.sub.1-C.sub.6 alkyl, or substituted
C.sub.1-C.sub.6 alkyl and X is O or NJ.sub.c.
[0338] In certain embodiments, bicyclic nucleosides have the
formula:
##STR00004##
wherein:
[0339] Bx is a heterocyclic base moiety;
[0340] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0341] Z.sub.b is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl,
substituted C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6
alkynyl or substituted acyl (C(.dbd.O)--).
[0342] In certain embodiments, bicyclic nucleosides have the
formula:
##STR00005##
wherein: [0343] Bx is a heterocyclic base moiety; [0344] T.sub.a
and T.sub.b are each, independently H, a hydroxyl protecting group,
a conjugate group, a reactive phosphorus group, a phosphorus moiety
or a covalent attachment to a support medium; [0345] R.sub.d is
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 C2-C6 alkynyl; [0346] each
q.sub.a, q.sub.b, q.sub.c and q.sub.d is, independently, H,
halogen, 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,
C.sub.1-C.sub.6 alkoxyl, substituted C.sub.1-C.sub.6 alkoxyl, acyl,
substituted acyl, C.sub.1-C.sub.6 aminoalkyl or substituted
C.sub.1-C.sub.6 aminoalkyl;
[0347] In certain embodiments, bicyclic nucleosides have the
formula:
##STR00006##
wherein:
[0348] Bx is a heterocyclic base moiety;
[0349] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0350] q.sub.a, q.sub.b, q.sub.e and q.sub.f are each,
independently, hydrogen, halogen, 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.1-C.sub.12 alkoxy,
substituted C.sub.1-C.sub.12 alkoxy, OJ.sub.j, SJ.sub.j, SOJ.sub.j,
SO.sub.2J.sub.j, NJ.sub.jJ.sub.k, N.sub.3, CN, C(.dbd.O)OJ.sub.j,
C(.dbd.O)NJ.sub.jJ.sub.k, C(.dbd.O)J.sub.j,
O--C(.dbd.O)NJ.sub.jJ.sub.k, N(H)C(.dbd.NH)NJ.sub.jJ.sub.k,
N(H)C(.dbd.O)NJ.sub.jJ.sub.k or N(H)C(.dbd.S)NJ.sub.jJ.sub.k;
[0351] or q.sub.e and q.sub.f together are
.dbd.C(q.sub.g)(q.sub.h);
[0352] q.sub.g and q.sub.h are each, independently, H, halogen,
C.sub.1-C.sub.12 alkyl or substituted C.sub.1-C.sub.12 alkyl.
[0353] The synthesis and preparation of adenine, cytosine, guanine,
5-methyl-cytosine, thymine and uracil bicyclic nucleosides having a
4'-CH.sub.2--O-2' bridge, along with their oligomerization, and
nucleic acid recognition properties have been described (Koshkin et
al., Tetrahedron, 1998, 54, 3607-3630). The synthesis of bicyclic
nucleosides has also been described in WO 98/39352 and WO
99/14226.
[0354] Analogs of various bicyclic nucleosides that have 4' to 2'
bridging groups such as 4'-CH.sub.2--O-2' and 4'-CH.sub.2--S-2',
have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett.,
1998, 8, 2219-2222). Preparation of oligodeoxyribonucleotide
duplexes comprising bicyclic nucleosides for use as substrates for
nucleic acid polymerases has also been described (Wengel et al., WO
99/14226). Furthermore, synthesis of 2'-amino-BNA, a novel
conformationally restricted high-affinity oligonucleotide analog
has been described in the art (Singh et al., J. Org. Chem., 1998,
63, 10035-10039). In addition, 2'-amino- and 2'-methylamino-BNA's
have been prepared and the thermal stability of their duplexes with
complementary RNA and DNA strands has been previously reported.
[0355] In certain embodiments, bicyclic nucleosides have the
formula:
##STR00007##
wherein:
[0356] Bx is a heterocyclic base moiety;
[0357] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0358] each q.sub.i, q.sub.j, q.sub.k and q.sub.l is,
independently, H, halogen, 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.1-C.sub.12 alkoxyl, substituted
C.sub.1-C.sub.12 alkoxyl, OJT, SJ.sub.j, SOJ.sub.j,
SO.sub.2J.sub.j, NJ.sub.jJ.sub.k, N.sub.3, CN,
C(.dbd.O)OJ.sub.jJ.sub.k, C(.dbd.O)NJ.sub.jJ.sub.k,
C(.dbd.O)J.sub.j, O--C(.dbd.O)NJ.sub.jJ.sub.k,
N(H)C(.dbd.NH)NJ.sub.jJ.sub.k, N(H)C(.dbd.O)NJ.sub.jJ.sub.k or
N(H)C(.dbd.S)NJ.sub.jJ.sub.k; and
[0359] q.sub.i and q.sub.j or q.sub.l and q.sub.k together are
.dbd.C(q.sub.g)(q.sub.h), wherein q.sub.g and q.sub.h are each,
independently, H, halogen, C.sub.1-C.sub.12 alkyl or substituted
C.sub.1-C.sub.12 alkyl.
[0360] One carbocyclic bicyclic nucleoside having a
4'-(CH.sub.2).sub.3-2' bridge and the alkenyl analog bridge
4'-CH.dbd.CH--CH.sub.2-2' have been described (Frier et al.,
Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al.,
J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation
of carbocyclic bicyclic nucleosides along with their
oligomerization and biochemical studies have also been described
(Srivastava et al., J. Am. Chem. Soc. 2007, 129(26),
8362-8379).
[0361] In certain embodiments, 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, (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) vinyl BNA as depicted
below.
##STR00008## ##STR00009##
[0362] wherein Bx is the base moiety and R is, independently, H, a
protecting group, C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6
alkoxy.
[0363] As used herein, the term "modified tetrahydropyran
nucleoside" or "modified THP nucleoside" means a nucleoside having
a six-membered tetrahydropyran "sugar" substituted for the
pentofuranosyl residue in normal nucleosides and can be referred to
as a sugar surrogate. Modified THP nucleosides include, but are not
limited to, what is referred to in the art as hexitol nucleic acid
(HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see
Leumann, Bioorg. Med. Chem., 2002, 10, 841-854) or fluoro HNA
(F-HNA) having a tetrahydropyranyl ring system as illustrated
below.
##STR00010##
[0364] In certain embodiment, sugar surrogates are selected having
the formula:
##STR00011##
wherein:
[0365] Bx is a heterocyclic base moiety;
[0366] T.sub.3 and T.sub.4 are each, independently, an
internucleoside linking group linking the tetrahydropyran
nucleoside analog to the oligomeric compound or one of T.sub.3 and
T.sub.4 is an internucleoside linking group linking the
tetrahydropyran nucleoside analog to an oligomeric compound or
oligonucleotide 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;
[0367] 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
[0368] one of R.sub.1 and R.sub.2 is hydrogen and the other is
selected from halogen, substituted or unsubstituted alkoxy,
NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, OC(.dbd.X)J.sub.1,
OC(.dbd.X)NJ.sub.1J.sub.2, NJ.sub.3C(.dbd.X)NJ.sub.1J.sub.2 and CN,
wherein X is O, S or NJ.sub.1 and each J.sub.1, J.sub.2 and J.sub.3
is, independently, H or C.sub.1-C.sub.6 alkyl.
[0369] In certain embodiments, 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 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.
[0370] In certain embodiments, sugar surrogates comprise rings
having more than 5 atoms and more than one heteroatom. For example
nucleosides comprising morpholino sugar moieties and their use in
oligomeric compounds has been reported (see for example: Braasch et
al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos.
5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the
term "morpholino" means a sugar surrogate having the following
formula:
##STR00012##
In certain embodiments, morpholinos may be modified, for example by
adding or altering various substituent groups from the above
morpholino structure. Such sugar surrogates are referred to herein
as "modified morpholinos."
[0371] 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).
[0372] In certain embodiments, antisense compounds comprise one or
more modified cyclohexenyl nucleosides, which is a nucleoside
having a six-membered cyclohexenyl in place of the pentofuranosyl
residue in naturally occurring nucleosides. Modified cyclohexenyl
nucleosides include, but are not limited to those described in the
art (see for example commonly owned, published PCT Application WO
2010/036696, published on Apr. 10, 2010, Robeyns et al., J. Am.
Chem. Soc., 2008, 130(6), 1979-1984; Horvath et al., Tetrahedron
Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem.
Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides
& Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al.,
Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al.,
Acta Crystallographica, Section F: Structural Biology and
Crystallization Communications, 2005, F61(6), 585-586; Gu et al.,
Tetrahedron, 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides,
2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68,
4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24),
4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et
al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7),
785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published
PCT application, WO 06/047842; and Published PCT Application WO
01/049687; the text of each is incorporated by reference herein, in
their entirety). Certain modified cyclohexenyl nucleosides have
Formula X.
##STR00013##
[0373] wherein independently for each of said at least one
cyclohexenyl nucleoside analog of Formula X:
[0374] Bx is a heterocyclic base moiety;
[0375] T.sub.3 and T.sub.4 are each, independently, an
internucleoside linking group linking the cyclohexenyl nucleoside
analog to an antisense compound or one of T.sub.3 and T.sub.4 is an
internucleoside linking group linking the tetrahydropyran
nucleoside analog to an 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; and
[0376] q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6,
q.sub.7, q.sub.8 and q.sub.9 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, substituted C.sub.2-C.sub.6 alkynyl or
other sugar substituent group.
[0377] Many other monocyclic, bicyclic and tricyclic ring systems
are known in the art and are suitable as sugar surrogates that can
be used to modify nucleosides for incorporation into oligomeric
compounds as provided herein (see for example review article:
Leumann, Christian J. Bioorg. & Med. Chem., 2002, 10, 841-854).
Such ring systems can undergo various additional substitutions to
further enhance their activity.
[0378] As used herein, "2'-modified sugar" means a furanosyl sugar
modified at the 2' position. In certain embodiments, such
modifications include substituents selected from: a halide,
including, but not limited to substituted and unsubstituted alkoxy,
substituted and unsubstituted thioalkyl, substituted and
unsubstituted amino alkyl, substituted and unsubstituted alkyl,
substituted and unsubstituted allyl, and substituted and
unsubstituted alkynyl. In certain embodiments, 2' modifications are
selected from substituents including, but not limited to:
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nF,
O(CH.sub.2).sub.nONH.sub.2, OCH.sub.2C(.dbd.O)N(H)CH.sub.3, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3].sub.2, where n and m
are from 1 to about 10. Other 2'- substituent groups can also be
selected from: C.sub.1-C.sub.12 alkyl, substituted alkyl, alkenyl,
alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3,
OCN, Cl, Br, CN, F, CF.sub.3, OCF.sub.3, SOCH.sub.3,
SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving
pharmacokinetic properties, or a group for improving the
pharmacodynamic properties of an antisense compound, and other
substituents having similar properties. In certain embodiments,
modified nucleosides comprise a 2' MOE side chain (Baker et al., J.
Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitution have
been described as having improved binding affinity compared to
unmodified nucleosides and to other modified nucleosides, such as
2'-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having
the 2'-MOE substituent also have been shown to be antisense
inhibitors of gene expression with promising features for in vivo
use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al.,
Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans.,
1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides,
1997, 16, 917-926).
[0379] As used herein, "2'-modified" or "2'-substituted" refers to
a nucleoside comprising a sugar comprising a substituent at the 2'
position other than H or OH. 2'-modified nucleosides, include, but
are not limited to, bicyclic nucleosides wherein the bridge
connecting two carbon atoms of the sugar ring connects the 2'
carbon and another carbon of the sugar ring; and nucleosides with
non-bridging 2'substituents, such as allyl, amino, azido, thio,
O-allyl, O--C.sub.1-C.sub.10 alkyl, --OCF.sub.3,
O--(CH.sub.2).sub.2--O--CH.sub.3, 2'-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 or substituted or unsubstituted
C.sub.1-C.sub.10 alkyl. 2'-modified nucleosides may further
comprise other modifications, for example at other positions of the
sugar and/or at the nucleobase.
[0380] As used herein, "2'-F" refers to a nucleoside comprising a
sugar comprising a fluoro group at the 2' position of the sugar
ring.
[0381] As used herein, "2'-OMe" or "2'-OCH.sub.3", "2'-O-methyl" or
"2'-methoxy" each refers to a nucleoside comprising a sugar
comprising an --OCH.sub.3 group at the 2' position of the sugar
ring.
[0382] As used herein, "MOE" or "2'-MOE" or
"2'-OCH.sub.2CH.sub.2OCH.sub.3" or "2'-O-methoxyethyl" each refers
to a nucleoside comprising a sugar comprising a
--OCH.sub.2CH.sub.2OCH.sub.3 group at the 2' position of the sugar
ring.
[0383] Methods for the preparations of modified sugars are well
known to those skilled in the art. Some representative U.S. patents
that teach the preparation of such modified sugars include without
limitation, U.S.: 4,981,957; 5,118,800; 5,319,080; 5,359,044;
5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;
5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;
5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and
International Application PCT/US2005/019219, filed Jun. 2, 2005 and
published as WO 2005/121371 on Dec. 22, 2005, and each of which is
herein incorporated by reference in its entirety.
[0384] As used herein, "oligonucleotide" refers to a compound
comprising a plurality of linked nucleosides. In certain
embodiments, one or more of the plurality of nucleosides is
modified. In certain embodiments, an oligonucleotide comprises one
or more ribonucleosides (RNA) and/or deoxyribonucleosides
(DNA).
[0385] In nucleotides having modified sugar moieties, the
nucleobase moieties (natural, modified or a combination thereof)
are maintained for hybridization with an appropriate nucleic acid
target.
[0386] In certain embodiments, antisense compounds comprise one or
more nucleosides having modified sugar moieties. In certain
embodiments, the modified sugar moiety is 2'-MOE. In certain
embodiments, the 2'-MOE modified nucleosides are arranged in a
gapmer motif. In certain embodiments, the modified sugar moiety is
a bicyclic nucleoside having a (4'-CH(CH.sub.3)--O-2') bridging
group. In certain embodiments, the (4'-CH(CH.sub.3)--O-2') modified
nucleosides are arranged throughout the wings of a gapmer
motif.
Compositions and Methods for Formulating Pharmaceutical
Compositions
[0387] Antisense oligonucleotides may be admixed with
pharmaceutically acceptable active or inert substances for the
preparation of pharmaceutical compositions or formulations.
Compositions and methods for the formulation of pharmaceutical
compositions are dependent upon a number of criteria, including,
but not limited to, route of administration, extent of disease, or
dose to be administered.
[0388] An antisense compound targeted to a C9ORF72 nucleic acid can
be utilized in pharmaceutical compositions by combining the
antisense compound with a suitable pharmaceutically acceptable
diluent or carrier. A pharmaceutically acceptable diluent includes
phosphate-buffered saline (PBS). PBS is a diluent suitable for use
in compositions to be delivered parenterally. Accordingly, in one
embodiment, employed in the methods described herein is a
pharmaceutical composition comprising an antisense compound
targeted to a C9ORF72 nucleic acid and a pharmaceutically
acceptable diluent. In certain embodiments, the pharmaceutically
acceptable diluent is PBS. In certain embodiments, the antisense
compound is an antisense oligonucleotide.
[0389] Pharmaceutical compositions comprising antisense compounds
encompass any pharmaceutically acceptable salts, esters, or salts
of such esters, or any other 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.
[0390] A prodrug can include the incorporation of additional
nucleosides at one or both ends of an antisense compound which are
cleaved by endogenous nucleases within the body, to form the active
antisense compound.
Conjugated Antisense Compounds
[0391] Antisense compounds may be covalently linked to one or more
moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the resulting antisense
oligonucleotides. Typical conjugate groups include cholesterol
moieties and lipid moieties. Additional conjugate groups include
carbohydrates, phospholipids, biotin, phenazine, folate,
phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,
coumarins, and dyes.
[0392] Antisense compounds can also be modified to have one or more
stabilizing groups that are generally attached to one or both
termini of antisense compounds to enhance properties such as, for
example, nuclease stability. Included in stabilizing groups are cap
structures. These terminal modifications protect the antisense
compound having terminal nucleic acid from exonuclease degradation,
and can help in delivery and/or localization within a cell. The cap
can be present at the 5'-terminus (5'-cap), or at the 3'-terminus
(3'-cap), or can be present on both termini. Cap structures are
well known in the art and include, for example, inverted deoxy
abasic caps. Further 3' and 5'-stabilizing groups that can be used
to cap one or both ends of an antisense compound to impart nuclease
stability include those disclosed in WO 03/004602 published on Jan.
16, 2003.
Cell Culture and Antisense Compounds Treatment
[0393] The effects of antisense compounds on the level, activity or
expression of C9ORF72 nucleic acids can be tested in vitro in a
variety of cell types. Cell types used for such analyses are
available from commercial vendors (e.g. American Type Culture
Collection, Manassas, Va.; Zen-Bio, Inc., Research Triangle Park,
NC; Clonetics Corporation, Walkersville, Md.) and are cultured
according to the vendor's instructions using commercially available
reagents (e.g. Invitrogen Life Technologies, Carlsbad, Calif.).
Illustrative cell types include, but are not limited to, HepG2
cells, Hep3B cells, and primary hepatocytes.
In Vitro Testing of Antisense Oligonucleotides
[0394] Described herein are methods for treatment of cells with
antisense oligonucleotides, which can be modified appropriately for
treatment with other antisense compounds.
[0395] In general, cells are treated with antisense
oligonucleotides when the cells reach approximately 60-80%
confluency in culture.
[0396] One reagent commonly used to introduce antisense
oligonucleotides into cultured cells includes the cationic lipid
transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, Calif.).
Antisense oligonucleotides are mixed with LIPOFECTIN in OPTI-MEM 1
(Invitrogen, Carlsbad, Calif.) to achieve the desired final
concentration of antisense oligonucleotide and a LIPOFECTIN
concentration that typically ranges 2 to 12 ug/mL per 100 nM
antisense oligonucleotide.
[0397] Another reagent used to introduce antisense oligonucleotides
into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad,
Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE in
OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to
achieve the desired concentration of antisense oligonucleotide and
a LIPOFECTAMINE concentration that typically ranges 2 to 12 ug/mL
per 100 nM antisense oligonucleotide.
[0398] Another technique used to introduce antisense
oligonucleotides into cultured cells includes electroporation.
[0399] Cells are treated with antisense oligonucleotides by routine
methods. Cells are typically harvested 16-24 hours after antisense
oligonucleotide treatment, at which time RNA or protein levels of
target nucleic acids are measured by methods known in the art and
described herein. In general, when treatments are performed in
multiple replicates, the data are presented as the average of the
replicate treatments.
[0400] The concentration of antisense oligonucleotide used varies
from cell line to cell line. Methods to determine the optimal
antisense oligonucleotide concentration for a particular cell line
are well known in the art. Antisense oligonucleotides are typically
used at concentrations ranging from 1 nM to 300 nM when transfected
with LIPOFECTAMINE. Antisense oligonucleotides are used at higher
concentrations ranging from 625 to 20,000 nM when transfected using
electroporation.
RNA Isolation
[0401] RNA analysis can be performed on total cellular RNA or
poly(A)+ mRNA. Methods of RNA isolation are well known in the art.
RNA is prepared using methods well known in the art, for example,
using the TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according
to the manufacturer's recommended protocols.
Analysis of Inhibition of Target Levels or Expression
[0402] Inhibition of levels or expression of a C9ORF72 nucleic acid
can be assayed in a variety of ways known in the art. For example,
target nucleic acid levels can be quantitated by, e.g., Northern
blot analysis, competitive polymerase chain reaction (PCR), or
quantitative real-time PCR. RNA analysis can be performed on total
cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well
known in the art. Northern blot analysis is also routine in the
art. Quantitative real-time PCR can be conveniently accomplished
using the commercially available ABI PRISM 7600, 7700, or 7900
Sequence Detection System, available from PE-Applied Biosystems,
Foster City, Calif. and used according to manufacturer's
instructions.
Quantitative Real-Time PCR Analysis of Target RNA Levels
[0403] Quantitation of target RNA levels may be accomplished by
quantitative real-time PCR using the ABI PRISM 7600, 7700, or 7900
Sequence Detection System (PE-Applied Biosystems, Foster City,
Calif.) according to manufacturer's instructions. Methods of
quantitative real-time PCR are well known in the art.
[0404] Prior to real-time PCR, the isolated RNA is subjected to a
reverse transcriptase (RT) reaction, which produces complementary
DNA (cDNA) that is then used as the substrate for the real-time PCR
amplification. The RT and real-time PCR reactions are performed
sequentially in the same sample well. RT and real-time PCR reagents
are obtained from Invitrogen (Carlsbad, Calif.). RT real-time-PCR
reactions are carried out by methods well known to those skilled in
the art.
[0405] Gene (or RNA) target quantities obtained by real time PCR
are normalized using either the expression level of a gene whose
expression is constant, such as cyclophilin A, or by quantifying
total RNA using RIBOGREEN (Invitrogen, Inc. Carlsbad, Calif.).
Cyclophilin A expression is quantified by real time PCR, by being
run simultaneously with the target, multiplexing, or separately.
Total RNA is quantified using RIBOGREEN RNA quantification reagent
(Invetrogen, Inc. Eugene, Oreg.). Methods of RNA quantification by
RIBOGREEN are taught in Jones, L. J., et al, (Analytical
Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000 instrument (PE
Applied Biosystems) is used to measure RIBOGREEN fluorescence.
[0406] Probes and primers are designed to hybridize to a C9ORF72
nucleic acid. Methods for designing real-time PCR probes and
primers are well known in the art, and may include the use of
software such as PRIMER EXPRESS Software (Applied Biosystems,
Foster City, Calif.).
Strand Specific Semi-Quantitative PCR Analysis of Target RNA
Levels
[0407] Analysis of specific, low abundance target RNA strand levels
may be accomplished by reverse transcription, PCR, and gel
densitometry analysis using the Gel Logic 200 Imaging System and
Kodak MI software (Kodak Scientific Imaging Systems, Rochester,
N.Y., USA) according to manufacturer's instructions.
[0408] RT-PCR reactions are carried out as taught in Ladd, P. D.,
et al, (Human Molecular Genetics, 2007, 16, 3174-3187) and in
Sopher, B. L., et al, (Neuron, 2011, 70, 1071-1084) and such
methods are well known in the art.
[0409] The PCR amplification products are loaded onto gels, stained
with ethidium bromide, and subjected to densitometry analysis. Mean
intensities from regions of interest (ROI) that correspond to the
bands of interest in the gel are measured.
[0410] Gene (or RNA) target quantities obtained by PCR are
normalized using the expression level of a housekeeping gene whose
expression is constant, such as GAPDH. Expression of the
housekeeping gene (or RNA) is analyzed and measured using the same
methods as the target.
[0411] Probes and primers are designed to hybridize to a C9ORF72
nucleic acid. Methods for designing RT-PCR probes and primers are
well known in the art, and may include the use of software such as
PRIMER EXPRESS Software (Applied Biosystems, Foster City,
Calif.).
Analysis of Protein Levels
[0412] Antisense inhibition of C9ORF72 nucleic acids can be
assessed by measuring C9ORF72 protein levels. Protein levels of
C9ORF72 can be evaluated or quantitated in a variety of ways well
known in the art, such as immunoprecipitation, Western blot
analysis (immunoblotting), enzyme-linked immunosorbent assay
(ELISA), quantitative protein assays, protein activity assays (for
example, caspase activity assays), immunohistochemistry,
immunocytochemistry or fluorescence-activated cell sorting (FACS).
Antibodies directed to a target can be identified and obtained from
a variety of sources, such as the MSRS catalog of antibodies (Aerie
Corporation, Birmingham, Mich.), or can be prepared via
conventional monoclonal or polyclonal antibody generation methods
well known in the art. Antibodies useful for the detection of
mouse, rat, monkey, and human C9ORF72 are commercially
available.
In Vivo Testing of Antisense Compounds
[0413] Antisense compounds, for example, antisense
oligonucleotides, are tested in animals to assess their ability to
inhibit expression of C9ORF72 and produce phenotypic changes, such
as, improved motor function and respiration. In certain
embodiments, motor function is measured by rotarod, grip strength,
pole climb, open field performance, balance beam, hindpaw footprint
testing in the animal. In certain embodiments, respiration is
measured by whole body plethysmograph, invasive resistance, and
compliance measurements in the animal. Testing may be performed in
normal animals, or in experimental disease models. For
administration to animals, antisense oligonucleotides are
formulated in a pharmaceutically acceptable diluent, such as
phosphate-buffered saline. Administration includes parenteral
routes of administration, such as intraperitoneal, intravenous, and
subcutaneous. Calculation of antisense oligonucleotide dosage and
dosing frequency is within the abilities of those skilled in the
art, and depends upon factors such as route of administration and
animal body weight. Following a period of treatment with antisense
oligonucleotides, RNA is isolated from CNS tissue or CSF and
changes in C9ORF72 nucleic acid expression are measured.
Targeting C9ORF72
[0414] Antisense oligonucleotides described herein may hybridize to
a C9ORF72 nucleic acid derived from either DNA strand. For example,
antisense oligonucleotides described herein may hybridize to a
C9ORF72 antisense transcript or a C9ORF72 sense transcript.
Antisense oligonucleotides described herein may hybridize to a
C9ORF72 nucleic acid in any stage of RNA processing. Described
herein are antisense oligonucleotides that are complementary to a
pre-mRNA or a mature mRNA. Additionally, antisense oligonucleotides
described herein may hybridize to any element of a C9ORF72 nucleic
acid. For example, described herein are antisense oligonucleotides
that are complementary to an exon, an intron, the 5' UTR, the 3'
UTR, a repeat region, a hexanucleotide repeat expansion, a splice
junction, an exon:exon splice junction, an exonic splicing silencer
(ESS), an exonic splicing enhancer (ESE), exon 1a, exon 1b, exon
1c, exon 1d, exon 1e, exon 2, exon 3, exon 4, exon 5, exon 6, exon
7, exon 8, exon 9, exon 10, exon 11, intron 1, intron 2, intron 3,
intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, or
intron 10 of a C9ORF72 nucleic acid.
[0415] In certain embodiments, antisense oligonucleotides described
herein hybridize to all variants of C9ORF72 derived from the sense
strand. In certain embodiments, the antisense oligonucleotides
described herein selectively hybridize to certain variants of
C9ORF72 derived from the sense strand. In certain embodiments, the
antisense oligonucleotides described herein selectively hybridize
to variants of C9ORF72 derived from the sense strand containing a
hexanucleotide repeat expansion. In certain embodiments, the
antisense oligonucleotides described herein selectively hybridize
to pre-mRNA variants containing a hexanucleotide repeat. In certain
embodiments, pre-mRNA variants of C9ORF72 containing a
hexanucleotide repeat expansion include SEQ ID NO: 1-3 and 6-10. In
certain embodiments, such hexanucleotide repeat expansion comprises
at least 24 repeats of any of GGGGCC, GGGGGG, GGGGGC, or
GGGGCG.
[0416] In certain embodiments, the antisense oligonucleotides
described herein inhibit expression of all variants of C9ORF72
derived from the sense strand. In certain embodiments, the
antisense oligonucleotides described herein inhibit expression of
all variants of C9ORF72 derived from the sense strand equally. In
certain embodiments, the antisense oligonucleotides described
herein preferentially inhibit expression of one or more variants of
C9ORF72 derived from the sense strand. In certain embodiments, the
antisense oligonucleotides described herein preferentially inhibit
expression of variants of C9ORF72 derived from the sense strand
containing a hexanucleotide repeat expansion. In certain
embodiments, the antisense oligonucleotides described herein
selectively inhibit expression of pre-mRNA variants containing the
hexanucleotide repeat. In certain embodiments, the antisense
oligonucleotides described herein selectively inhibit expression of
C9ORF72 pathogenic associated mRNA variants. In certain
embodiments, pre-mRNA variants of C9ORF72 containing a
hexanucleotide repeat expansion include SEQ ID NO: 1-3 and 6-10. In
certain embodiments, such hexanucleotide repeat expansion comprises
at least 24 repeats of any of GGGGCC, GGGGGG, GGGGGC, or GGGGCG. In
certain embodiments, the hexanucleotide repeat expansion forms
C9ORF72 sense foci. In certain embodiments, antisense
oligonucleotides described herein are useful for reducing C9ORF72
sense foci. C9ORF72 sense foci may be reduced in terms of percent
of cells with foci as well as number of foci per cell.
C9OFF72 Features
[0417] Antisense oligonucleotides described herein may hybridize to
any C9ORF72 nucleic acid at any state of processing within any
element of the C9ORF72 gene. In certain embodiments, antisense
oligonucleotides described herein may target the antisense
transcript, e.g., SEQ ID NO: 13. In certain embodiments, antisense
oligonucleotides described herein may hybridize to an exon, an
intron, the 5' UTR, the 3' UTR, a repeat region, a hexanucleotide
repeat expansion, a splice junction, an exon:exon splice junction,
an exonic splicing silencer (ESS), an exonic splicing enhancer
(ESE), exon 1a, exon 1b, exon 1c, exon 1 d, exon 1e, exon 2, exon
3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon
11, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6,
intron 7, intron 8, intron 9, or intron 10. For example, antisense
oligonucleotides may target any of the exons characterized below in
Tables 1-5 described below. Antisense oligonucleotides described
herein may also target nucleic acids not characterized below and
such nucleic acid may be characterized in GENBANK. Moreover,
antisense oligonucleotides described herein may also target
elements other than exons and such elements as characterized in
GENBANK.
TABLE-US-00001 TABLE 1 Functional Segments for NM_001256054.1 (SEQ
ID NO: 1) Start site Stop site in in reference reference mRNA mRNA
to SEQ to SEQ Exon start stop ID ID Number site site NO: 2 NO: 2
exon 1C 1 158 1137 1294 exon 2 159 646 7839 8326 exon 3 647 706
9413 9472 exon 4 707 802 12527 12622 exon 5 803 867 13354 13418
exon 6 868 940 14704 14776 exon 7 941 1057 16396 16512 exon 8 1058
1293 18207 18442 exon 9 1294 1351 24296 24353 exon 10 1352 1461
26337 26446 exon 11 1462 3339 26581 28458
TABLE-US-00002 TABLE 2 Functional Segments for NM_018325.3 (SEQ ID
NO: 4) Start site Stop site in in reference reference mRNA mRNA to
SEQ to SEQ Exon start stop ID ID Number site site NO: 2 NO: 2 exon
1B 1 63 1510 1572 exon 2 64 551 7839 8326 exon 3 552 611 9413 9472
exon 4 612 707 12527 12622 exon 5 708 772 13354 13418 exon 6 773
845 14704 14776 exon 7 846 962 16396 16512 exon 8 963 1198 18207
18442 exon 9 1199 1256 24296 24353 exon 10 1257 1366 26337 26446
exon 11 1367 3244 26581 28458
TABLE-US-00003 TABLE 3 Functional Segments for NM_145005.5 (SEQ ID
NO: 6) Start site Stop site in in reference reference mRNA mRNA to
SEQ to SEQ Exon start stop ID ID Number site site NO: 2 NO: 2 exon
1A 1 80 1137 1216 exon 2 81 568 7839 8326 exon 3 569 628 9413 9472
exon 4 629 724 12527 12622 exon 5B 725 1871 13354 14500 (exon 5
into intron 5)
TABLE-US-00004 TABLE 4 Functional Segments for DB079375.1 (SEQ ID
NO: 7) Start site Stop site in in reference reference mRNA mRNA to
SEQ to SEQ Exon start stop ID ID Number site site NO: 2 NO: 2 exon
1E 1 35 1135 1169 exon 2 36 524 7839 8326 exon 3 (EST 525 562 9413
9450 ends before end of full exon)
TABLE-US-00005 TABLE 5 Functional Segments for BU194591.1 (SEQ ID
NO: 8) Start site Stop site in in reference reference mRNA mRNA to
SEQ to SEQ Exon start stop ID ID Number site site NO: 2 NO: 2 exon
1D 1 36 1241 1279 exon 2 37 524 7839 8326 exon 3 525 584 9413 9472
exon 4 585 680 12527 12622 exon 5B 681 798 13354 13465 (exon 5 into
intron 5)
Certain Indications
[0418] In certain embodiments, provided herein are methods of
treating an individual comprising administering one or more
pharmaceutical compositions described herein. In certain
embodiments, the individual has a neurodegenerative disease. In
certain embodiments, the individual is at risk for developing a
neurodegenerative disease, including, but not limited to, ALS or
FTD. In certain embodiments, the individual has been identified as
having a C9ORF72 associated disease. In certain embodiments, the
individual has been identified as having a C9ORF72 hexanucleotide
repeat expansion associated disease. In certain embodiments,
provided herein are methods for prophylactically reducing C9ORF72
expression in an individual. Certain embodiments include treating
an individual in need thereof by administering to an individual a
therapeutically effective amount of an antisense compound targeted
to a C9ORF72 nucleic acid.
[0419] In one embodiment, administration of a therapeutically
effective amount of an antisense compound targeted to a C9ORF72
nucleic acid is accompanied by monitoring of C9ORF72 levels in an
individual, to determine an individual's response to administration
of the antisense compound. An individual's response to
administration of the antisense compound may be used by a physician
to determine the amount and duration of therapeutic
intervention.
[0420] In certain embodiments, administration of an antisense
compound targeted to a C9ORF72 nucleic acid results in reduction of
C9ORF72 expression by at least 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. In certain embodiments, administration of an
antisense compound targeted to a C9ORF72 nucleic acid results in
improved motor function and respiration in an animal. In certain
embodiments, administration of a C9ORF72 antisense compound
improves motor function and respiration by at least 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.
[0421] In certain embodiments, administration of an antisense
compound targeted to a C9ORF72 antisense transcript results in
reduction of C9ORF72 antisense transcript expression by at least
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. In certain
embodiments, administration of an antisense compound targeted to a
C9ORF72 antisense transcript results in improved motor function and
respiration in an animal. In certain embodiments, administration of
a C9ORF72 antisense compound improves motor function and
respiration by at least 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. In certain embodiments, administration of a C9ORF72
antisense compound reduces the number of cells with C9ORF72
antisense foci and/or the number of C9ORF72 antisense foci per
cell.
[0422] In certain embodiments, administration of an antisense
compound targeted to a C9ORF72 sense transcript results in
reduction of a C9ORF72 sense transcript expression by at least 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. In certain
embodiments, administration of an antisense compound targeted to a
C9ORF72 sense transcript results in improved motor function and
respiration in an animal. In certain embodiments, administration of
a C9ORF72 antisense compound improves motor function and
respiration by at least 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. In certain embodiments, administration of a C9ORF72
antisense compound reduces the number of cells with C9ORF72 sense
foci and/or the number of C9ORF72 sense foci per cell.
[0423] In certain embodiments, pharmaceutical compositions
comprising an antisense compound targeted to a C9ORF72 nucleic are
used for the preparation of a medicament for treating a patient
suffering or susceptible to a neurodegenerative disease including
ALS and FTD.
Certain Combination Therapies
[0424] In certain embodiments, one or more pharmaceutical
compositions described herein 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 described
herein. In certain embodiments, one or more pharmaceutical
compositions described herein 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 described herein are co-administered
with another pharmaceutical agent to produce a combinational
effect. In certain embodiments, one or more pharmaceutical
compositions described herein are co-administered with another
pharmaceutical agent to produce a synergistic effect.
[0425] In certain embodiments, one or more pharmaceutical
compositions described herein and one or more other pharmaceutical
agents are administered at the same time. In certain embodiments,
one or more pharmaceutical compositions described herein and one or
more other pharmaceutical agents are administered at different
times. In certain embodiments, one or more pharmaceutical
compositions described herein and one or more other pharmaceutical
agents are prepared together in a single formulation. In certain
embodiments, one or more pharmaceutical compositions described
herein and one or more other pharmaceutical agents are prepared
separately.
[0426] In certain embodiments, pharmaceutical agents that may be
co-administered with a pharmaceutical composition described herein
include Riluzole (Rilutek), Lioresal (Lioresal), and
Dexpramipexole.
[0427] In certain embodiments, pharmaceutical agents that may be
co-administered with a C9ORF72 antisense transcript specific
inhibitor described herein include, but are not limited to, an
additional C9ORF72 inhibitor. In certain embodiments, the
co-administered pharmaceutical agent is administered prior to
administration of a pharmaceutical composition described herein. In
certain embodiments, the co-administered pharmaceutical agent is
administered following administration of a pharmaceutical
composition described herein. In certain embodiments the
co-administered pharmaceutical agent is administered at the same
time as a pharmaceutical composition described herein. In certain
embodiments the dose of a co-administered pharmaceutical agent is
the same as the dose that would be administered if the
co-administered pharmaceutical agent was administered alone. In
certain embodiments the dose of a co-administered pharmaceutical
agent is lower than the dose that would be administered if the
co-administered pharmaceutical agent was administered alone. In
certain embodiments the dose of a co-administered pharmaceutical
agent is greater than the dose that would be administered if the
co-administered pharmaceutical agent was administered alone.
[0428] In certain embodiments, the co-administration of a second
compound enhances the effect of a first compound, such that
co-administration of the compounds results in an effect that is
greater than the effect of administering the first compound alone.
In other embodiments, the co-administration results in effects that
are additive of the effects of the compounds when administered
alone. In certain embodiments, the co-administration results in
effects that are supra-additive of the effects of the compounds
when administered alone. In certain embodiments, the first compound
is an antisense compound. In certain embodiments, the second
compound is an antisense compound.
Certain Human Therapeutics
[0429] The human C9ORF72 antisense transcript specific antisense
compounds described herein are being evaluated as possible human
therapeutics. Various parameters of potency, efficacy, and/or
tolerability are being examined. Such parameters include in vitro
inhibition of C9ORF72 antisense transcript; in vitro dose response
(IC50); in vivo inhibition of C9ORF72 antisense transcript in a
transgenic animal containing a human C9ORF72 transgene in relevant
tissues (e.g., brain and/or spinal cord); and/or tolerability in
mouse, rat, dog, and/or primate. Tolerability markers that may be
measured include blood and serum chemistry parameters, CSF
chemistry parameters, body and organ weights, general observations
and/or behavioral tests, and/or biochemical markers such as GFAP
and/or AIF 1. Acute or long term tolerability may be measured.
Certain Assays for Measuring C9ORF72 Antisense Transcripts
[0430] Certain assays described herein are directed to the
reduction of C9ORF72 antisense transcript. Additional assays may be
used to measure the reduction of C9ORF72 antisense transcript.
Additional controls may be used as a baseline for measuring the
reduction of C9ORF72 transcript.
Certain Hotspot Regions
1. Nucleobases 196-280 of SEQ ID NO: 13
[0431] In certain embodiments, antisense oligonucleotides are
designed to target nucleobases 196-280 of SEQ ID NO: 13. In certain
embodiments, nucleobases 196-280 are a hotspot region. In certain
embodiments, nucleobases 196-280 are targeted by antisense
oligonucleotides. In certain embodiments, the antisense
oligonucleotides are 20 nucleobases in length. In certain
embodiments, the antisense oligonucleotides are gapmers. In certain
embodiments, the gapmers are 5-10-5 MOE gapmers.
[0432] In certain embodiments, nucleobases 196-280 are targeted by
the following ISIS numbers: 687280, 687281, 687282, 687283, 687284,
687285, 687286, 687287, 687288, 687289, 687290, 687291, 687292, and
687293.
[0433] In certain embodiments, nucleobases 196-280 are targeted by
the following SEQ ID NOs: 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, and 82.
[0434] In certain embodiments, antisense oligonucleotides targeting
nucleobases 196-280 achieve at least 45%, at least 46%, at least
47%, at least 48%, at least 49%, at least 50%, at least 51%, at
least 52%, at least 53%, at least 54%, at least 55%, at least 56%,
at least 57%, at least 58%, at least 59%, at least 60%, at least
61%, at least 62%, at least 63%, at least 64%, at least 65%, at
least 66%, at least 67%, at least 68%, at least 69%, at least 70%,
at least 71%, at least 72%, or at least 73% reduction of C9ORF72
antisense transcript in vitro.
2. Nucleobases 286-315 of SEQ ID NO: 13
[0435] In certain embodiments, antisense oligonucleotides are
designed to target nucleobases 286-315 of SEQ ID NO: 13. In certain
embodiments, nucleobases 286-315 are a hotspot region. In certain
embodiments, nucleobases 286-315 are targeted by antisense
oligonucleotides. In certain embodiments, the antisense
oligonucleotides are 20 nucleobases in length. In certain
embodiments, the antisense oligonucleotides are gapmers. In certain
embodiments, the gapmers are 5-10-5 MOE gapmers.
[0436] In certain embodiments, nucleobases 286-315 are targeted by
the following ISIS numbers: 687277, 687278, and 687279.
[0437] In certain embodiments, nucleobases 286-315 are targeted by
the following SEQ ID NOs: 66, 67, and 68.
[0438] In certain embodiments, antisense oligonucleotides targeting
nucleobases 286-315 achieve at least 6%, at least 7%, at least 8%,
at least 9%, at least 10%, at least 11%, at least 12%, at least
13%, at least 14%, at least 15%, at least 16%, at least 17%, at
least 18%, at least 19%, at least 20%, at least 21%, at least 22%,
at least 23%, at least 24%, at least 25%, at least 26%, at least
27%, at least 28%, at least 29%, at least 30%, at least 31%, at
least 32%, at least 33%, at least 34%, at least 35%, at least 36%,
at least 37%, at least 38%, at least 39%, at least 40%, at least
41%, at least 42%, at least 43%, at least 44%, at least 45%, at
least 46%, at least 47%, at least 48%, at least 49%, at least 50%,
at least 51%, at least 52%, at least 53%, at least 54%, at least
55%, at least 56%, at least 57%, at least 58%, at least 59%, at
least 60%, at least 61%, at least 62%, at least 63%, or at least
64% reduction of C9ORF72 antisense transcript in vitro.
3. Nucleobases 321-415 of SEQ ID NO: 13
[0439] In certain embodiments, antisense oligonucleotides are
designed to target nucleobases 321-415 of SEQ ID NO: 13. In certain
embodiments, nucleobases 321-415 are a hotspot region. In certain
embodiments, nucleobases 321-415 are targeted by antisense
oligonucleotides. In certain embodiments, the antisense
oligonucleotides are 20 nucleobases in length. In certain
embodiments, the antisense oligonucleotides are gapmers. In certain
embodiments, the gapmers are 5-10-5 MOE gapmers.
[0440] In certain embodiments, nucleobases 321-415 are targeted by
the following ISIS numbers: 687261, 687262, 687263, 687264, 687265,
687266, 687267, 687268, 687269, 687270, 687271, 687272, 687273,
687274, 687275, and 687276.
[0441] In certain embodiments, nucleobases 321-415 are targeted by
the following SEQ ID NOs: 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65.
[0442] In certain embodiments, antisense oligonucleotides targeting
nucleobases 321-415 achieve at least 15%, at least 16%, at least
17%, at least 18%, at least 19%, at least 20%, at least 21%, at
least 22%, at least 23%, at least 24%, at least 25%, at least 26%,
at least 27%, at least 28%, at least 29%, at least 30%, at least
31%, at least 32%, at least 33%, at least 34%, at least 35%, at
least 36%, at least 37%, at least 38%, at least 39%, at least 40%,
at least 41%, at least 42%, at least 43%, at least 44%, at least
45%, at least 46%, at least 47%, at least 48%, at least 49%, at
least 50%, at least 51%, at least 52%, at least 53%, at least 54%,
at least 55%, at least 56%, at least 57%, at least 58%, at least
59%, at least 60%, at least 61%, at least 62%, at least 63%, at
least 64%, at least 65%, at least 66%, at least 67%, at least 68%,
at least 69%, or at least 70% reduction of C9ORF72 antisense
transcript in vitro.
4. Nucleobases 451-516 of SEQ ID NO: 13
[0443] In certain embodiments, antisense oligonucleotides are
designed to target nucleobases 451-516 of SEQ ID NO: 13. In certain
embodiments, nucleobases 451-516 are a hotspot region. In certain
embodiments, nucleobases 451-516 are targeted by antisense
oligonucleotides. In certain embodiments, the antisense
oligonucleotides are 16, 18, or 20 nucleobases in length. In
certain embodiments, the antisense oligonucleotides are gapmers. In
certain embodiments, the gapmers are 5-10-5 MOE gapmers, 5-8-5 MOE
gapmers, 4-8-4 MOE gapmers. In certain embodiments, each nucleoside
of the antisense oligonucleotides is modified with a 2'-MOE
substitution. In certain embodiments, the antisense
oligonucleotides contain one or more inosine residues.
[0444] In certain embodiments, nucleobases 451-516 are targeted by
the following ISIS numbers: 687255, 687256, 687257, 687258, 687259,
687260, 730389, 730390, 730391, 730392, 730393, 730394, 730395,
730396, 730397, 730398, 730399, 730400, 730401, 730402, 730403,
730404, 730405, 730406, 730407, 730408, 730409, 730410, 730411,
730412, 737821, 742033, 742034, and 742035.
[0445] In certain embodiments, nucleobases 451-516 are targeted by
the following SEQ ID NOs: 44, 45, 46, 47, 48, 49, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 98, and 99.
[0446] In certain embodiments, antisense oligonucleotides targeting
nucleobases 451-516 achieve at least 10%, at least 11%, at least
12%, at least 13%, at least 14%, at least 15%, at least 16%, at
least 17%, at least 18%, at least 19%, at least 20%, at least 21%,
at least 22%, at least 23%, at least 24%, at least 25%, at least
26%, at least 27%, at least 28%, at least 29%, at least 30%, at
least 31%, at least 32%, at least 33%, at least 34%, at least 35%,
at least 36%, at least 37%, at least 38%, at least 39%, at least
40%, at least 41%, at least 42%, at least 43%, at least 44%, at
least 45%, at least 46%, at least 47%, at least 48%, at least 49%,
at least 50%, at least 51%, at least 52%, at least 53%, at least
54%, at least 55%, at least 56%, at least 57%, at least 58%, at
least 59%, at least 60%, at least 61%, at least 62%, at least 63%,
at least 64%, at least 65%, at least 66%, at least 67%, at least
68%, at least 69%, at least 70%, at least 71%, at least 72%, at
least 73%, at least 74%, at least 75%, at least 76%, at least 77%,
or at least 78% reduction of C9ORF72 antisense transcript in
vitro.
5. Nucleobases 527-588 of SEQ ID NO: 13
[0447] In certain embodiments, antisense oligonucleotides are
designed to target nucleobases 527-588 of SEQ ID NO: 13. In certain
embodiments, nucleobases 527-588 are a hotspot region. In certain
embodiments, nucleobases 527-588 are targeted by antisense
oligonucleotides. In certain embodiments, the antisense
oligonucleotides are 20 nucleobases in length. In certain
embodiments, the antisense oligonucleotides are gapmers. In certain
embodiments, the gapmers are 5-10-5 MOE gapmers.
[0448] In certain embodiments, nucleobases 527-588 are targeted by
the following ISIS numbers: 687247, 687248, 687249, 687250, 687251,
687252, 687253, and 687254.
[0449] In certain embodiments, nucleobases 527-588 are targeted by
the following SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, and 43.
[0450] In certain embodiments, antisense oligonucleotides targeting
nucleobases 527-588 achieve at least 21%, at least 22%, at least
23%, at least 24%, at least 25%, at least 26%, at least 27%, at
least 28%, at least 29%, at least 30%, at least 31%, at least 32%,
at least 33%, at least 34%, at least 35%, at least 36%, at least
37%, at least 38%, at least 39%, at least 40%, at least 41%, at
least 42%, at least 43%, at least 44%, at least 45%, at least 46%,
at least 47%, at least 48%, at least 49%, at least 50%, at least
51%, at least 52%, at least 53%, at least 54%, at least 55%, at
least 56%, at least 57%, at least 58%, at least 59%, at least 60%,
at least 61%, at least 62%, at least 63%, at least 64%, at least
65%, at least 66%, at least 67%, at least 68%, at least 69%, at
least 70%, at least 71%, at least 72%, or at least 73% reduction of
C9ORF72 antisense transcript in vitro.
6. Nucleobases 608-636 of SEQ ID NO: 13
[0451] In certain embodiments, antisense oligonucleotides are
designed to target nucleobases 608-636 of SEQ ID NO: 13. In certain
embodiments, nucleobases 608-636 are a hotspot region. In certain
embodiments, nucleobases 608-636 are targeted by antisense
oligonucleotides. In certain embodiments, the antisense
oligonucleotides are 20 nucleobases in length. In certain
embodiments, the antisense oligonucleotides are gapmers. In certain
embodiments, the gapmers are 5-10-5 MOE gapmers.
[0452] In certain embodiments, nucleobases 608-636 are targeted by
the following ISIS numbers: 687243, 687244, 687245, and 687246.
[0453] In certain embodiments, nucleobases 608-636 are targeted by
the following SEQ ID NOs: 32, 33, 34, and 35.
[0454] In certain embodiments, antisense oligonucleotides targeting
nucleobases 608-636 achieve at least 20%, at least 21%, at least
22%, at least 23%, at least 24%, at least 25%, at least 26%, at
least 27%, at least 28%, at least 29%, at least 30%, at least 31%,
at least 32%, at least 33%, at least 34%, at least 35%, at least
36%, at least 37%, at least 38%, at least 39%, at least 40%, at
least 41%, at least 42%, at least 43%, at least 44%, at least 45%,
at least 46%, at least 47%, at least 48%, at least 49%, at least
50%, at least 51%, at least 52%, at least 53%, at least 54%, at
least 55%, at least 56%, at least 57%, at least 58%, at least 59%,
at least 60%, at least 61%, at least 62%, at least 63%, at least
64%, at least 65%, at least 66%, at least 67%, at least 68%, at
least 69%, at least 70%, at least 71%, or at least 72% reduction of
C9ORF72 antisense transcript in vitro.
7. Nucleobases 704-726 of SEQ ID NO: 13
[0455] In certain embodiments, antisense oligonucleotides are
designed to target nucleobases 704-726 of SEQ ID NO: 13. In certain
embodiments, nucleobases 704-726 are a hotspot region. In certain
embodiments, nucleobases 704-726 are targeted by antisense
oligonucleotides. In certain embodiments, the antisense
oligonucleotides are 20 nucleobases in length. In certain
embodiments, the antisense oligonucleotides are gapmers. In certain
embodiments, the gapmers are 5-10-5 MOE gapmers.
[0456] In certain embodiments, nucleobases 704-726 are targeted by
the following ISIS numbers: 687241 and 687242.
[0457] In certain embodiments, nucleobases 704-726 are targeted by
the following SEQ ID NOs: 30 and 31.
[0458] In certain embodiments, antisense oligonucleotides targeting
nucleobases 704-726 achieve at least 19%, at least 20%, or at least
21% reduction of C9ORF72 antisense transcript in vitro.
EXAMPLES
Non-Limiting Disclosure and Incorporation by Reference
[0459] 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 recited in the present
application is incorporated herein by reference in its
entirety.
Example 1: Antisense Inhibition of C9ORF72 Antisense Transcript
with Oligonucleotides
[0460] Antisense oligonucleotides (ASOs) targeting the human
C9ORF72 antisense transcript were made and tested for inhibition of
target transcript expression in vitro. All of the ASOs in the table
below except Isis Numbers 742033 and 742035 are 5-10-5 MOE gapmers
with phosphorothioate internucleoside linkages throughout the
gapmers. They are 20 nucleosides in length, with a central gap
segment consisting of ten 2'-deoxynucleosides that is flanked by
wing segments in the 5' direction and the 3' direction consisting
of five nucleosides each. Each nucleoside in the 5' and 3' wing
segments has a 2'-MOE modification. All cytosine residues
throughout each gapmer are 5 methylcytosines. Isis No. 742033 is a
5-10-5 MOE gapmer with phosphodiester ("o") and phosphorothioate
("s") internucleoside linkages arranged in order from 5' to 3':
soooossssssssssooss. Isis No. 742035 is a 5-8-5 MOE gapmer with a
central gap segment consisting of eight 2'-deoxynucleosides and
internucleoside linkages arranged in order from 5' to 3':
sooosssssssssooss. All cytosine residues in Isis Numbers 742033 and
742035 are 5-methylcytosines. Isis Numbers 742033 and 742035 also
contain inosine residues, indicated by "I". In the table below,
"Start" indicates the 5'-most nucleoside to which the gapmer is
targeted in the target transcript sequence. "Stop" indicates the
3'-most nucleoside to which the gapmer is targeted in the target
transcript sequence. Each gapmer listed in the table below is
targeted to a putative antisense transcript sequence, designated
herein as SEQ ID NO: 13. The sequence of SEQ ID NO: 13 is
complementary to nucleotides 1159 to 1929 of SEQ ID NO: 2 (the
complement of GENBANK Accession No. NT 008413.18 truncated from
nucleotides 27535000 to 27565000) except that SEQ ID NO: 13 has two
more hexanucleotide repeats than SEQ ID NO: 2. The sequence of the
hexanucleotide repeat is GGCCCC in SEQ ID NO: 13 and GGGGCC in SEQ
ID NO: 2. Thus, SEQ ID NO: 13 is 12 nucleotides longer than
nucleotides 1159 to 1929 of SEQ ID NO: 2, to which it is
complementary. Isis No. 129700 is a negative control ASO that does
not target the C9ORF72 antisense transcript.
[0461] bEND cells were cultured in 24 well plates at 45,000-50,000
cells/well 24 hours before the first of two transfections. The
cells were first transfected with 0.2 .mu.g/well of a plasmid
expressing the C9ORF72 antisense transcript (SEQ ID NO: 13) and 0.5
.mu.L Lipofectamine 2000 (Life Technologies, Carlsbad, Calif.) in
OptiMEM medium. Four to six hours later, the media was replaced. 24
hours after the first transfection (18-20 hours after media
replacement), the bEND cells were transfected with 25 nM of an ASO
listed in the table below and 0.5 .mu.L Lipofectamine 2000 in
OptiMEM medium or with no ASO. Just prior to transfection, ASOs
comprising a GGGGCC repeat with or without guanosine to inosine
substitutions (Isis Numbers 687258, 687259, 687260, 742033, and
742035) were heated at 90.degree. C. for five minutes, then placed
on ice briefly before dilution in OptiMEM. Total RNA was isolated
from the cells 24 hours after the second transfection using TRIzol
(Life Technologies) according to the manufacturer's directions. Two
DNase reactions were performed, one on the column during RNA
purification, and one after purification using TURBO DNase (Life
Technologies).
[0462] Strand specific RT-qPCR was performed on the isolated RNA to
generate and amplify C9ORF72 antisense cDNA using one or two of
three different primer sets, LTS01222, LTS01221, and C9ATS3'-1. The
LTS01222 sequences are: RT primer:
CGACTGGAGCACGAGGACACTGAAAAGATGACGCTTGGTGTGTCA (SEQ ID NO: 14),
forward PCR primer: CCCACACCTGCTCTTGCTAGA (SEQ ID NO: 15), reverse
PCR primer: CGACTGGAGCACGAGGACACTG (SEQ ID NO: 16), and probe:
CCCAAAAGAGAAGCAACCGGGCA (SEQ ID NO: 17). The LTS01221 sequences
are: RT primer: CGACTGGAGCACGAGGACACTGACGGCTGCCGGGAAGA (SEQ ID NO:
18), forward PCR primer: AGAAATGAGAGGGAAAGTAAAAATGC (SEQ ID NO:
19), reverse PCR primer: CGACTGGAGCACGAGGACACTG (SEQ ID NO: 20),
and probe: AGGAGAGCCCCCGCTTCTACCCG (SEQ ID NO: 21). The C9ATS3'-1
sequences are: RT primer:
CGACTGGAGCACGAGGACACTGACGCTGAGGGTGAACAAGAA (SEQ ID NO: 22), forward
PCR primer: GAGTTCCAGAGCTTGCTACAG (SEQ ID NO: 23), reverse PCR
primer: CGACTGGAGCACGAGGACACTG (SEQ ID NO: 24), and probe:
CTGCGGTTGTTTCCCTCCTTGTTT (SEQ ID NO: 25). RT-qPCR was also
performed on the isolated RNA using Express One-Step Superscript
qRT-PCR Kit (Life Technologies, Carlsbad, Calif.) according to
manufacturer's instructions to generate and amplify GAPDH cDNA, as
a control, using forward PCR primer: GGCAAATTCAACGGCACAGT (SEQ ID
NO: 26), reverse PCR primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 27),
and probe: AAGGCCGAGAATGGGAAGCTTGTCATC (SEQ ID NO: 28). The
resulting C9ORF72 antisense levels were normalized to GAPDH. These
normalized values for C9ORF72 antisense transcript expression in
cells treated with an ASO were then compared to the normalized
values for C9ORF72 antisense transcript expression in control cells
that were transfected with the C9ORF72 antisense plasmid but not an
ASO. The results for each primer probe set are shown in the table
below as percent inhibition of C9ORF72 antisense transcript
expression relative to the control cells that were not transfected
with an ASO. A result of 0% inhibition indicates that the C9ORF72
antisense transcript levels were equal to that of control cells
that were not transfected with an ASO. A negative value for %
inhibition indicates that the C9ORF72 antisense transcript levels
were higher than that of control cells that were not transfected
with an ASO. A result of "n/a" indicates that the corresponding
primer probe set was not used to analyze the indicated sample. The
results show that many ASOs inhibited human C9ORF72 antisense
transcript expression. The absolute inhibition results varied
across different primer probe sets, but the relative potencies of
the ASOs were very similar across different primer probe sets.
TABLE-US-00006 TABLE 6 C9ORF72 Antisense RNA Inhibition by
Antisense Oligonucleotides SEQ Isis LTS LTS C9AT ID No. Start Stop
Sequence 01222 01221 S3'-1 No. 129700 n/a n/a TAGTGCGGACCTACCCACGA
98 92 101 29 687241 707 726 GGTGTGTCAGCCGTCCCTGC n/a 19 21 30
687242 704 723 GTGTCAGCCGTCCCTGCTGC n/a -44 -1 31 687243 617 636
TGTTTTTCCCACCCTCTCTC 70 20 n/a 32 687244 614 633
TTTTCCCACCCTCTCTCCCC 68 21 n/a 33 687245 611 630
TCCCACCCTCTCTCCCCACT 72 -5 n/a 34 687246 608 627
CACCCTCTCTCCCCACTACT 59 17 n/a 35 687247 569 588
GAGGGTGAACAAGAAAAGAC 71 51 n/a 36 687248 557 576
GAAAAGACCTGATAAAGATT 50 30 n/a 37 687249 542 561
AGATTAACCAGAAGAAAACA 54 21 n/a 38 687250 539 558
TTAACCAGAAGAAAACAAGG 63 35 n/a 39 687251 536 555
ACCAGAAGAAAACAAGGAGG 73 41 n/a 40 687252 533 552
AGAAGAAAACAAGGAGGGAA 44 33 n/a 41 687253 530 549
AGAAAACAAGGAGGGAAACA 68 47 n/a 42 687254 527 546
AAACAAGGAGGGAAACAACC 57 33 n/a 43 687255 497 516
GCAAGCTCTGGAACTCAGGA 51 n/a n/a 44 687256 494 513
AGCTCTGGAACTCAGGAGTC 57 n/a n/a 45 687257 483 502
TCAGGAGTCGCGCGCTAGGG 68 n/a n/a 46 687258 480 499
GGAGTCGCGCGCTAGGGGCC 54 n/a n/a 47 687259 477 496
GTCGCGCGCTAGGGGCCGGG 63 n/a n/a 48 687260 474 493
GCGCGCTAGGGGCCGGGGCC 44 n/a n/a 49 687261 396 415
GGGCTGCGGTTGCGGTGCCT 58 n/a 56 50 687262 391 410
GCGGTTGCGGTGCCTGCGCC 55 n/a 55 51 687263 386 405
TGCGGTGCCTGCGCCCGCGG 51 n/a 53 52 687264 381 400
TGCCTGCGCCCGCGGCGGCG 40 n/a 35 53 687265 376 395
GCGCCCGCGGCGGCGGAGGC 16 n/a 15 54 687266 371 390
CGCGGCGGCGGAGGCGCAGG 45 n/a 40 55 687267 366 385
CGGCGGAGGCGCAGGCGGTG 46 n/a 45 56 687268 361 380
GAGGCGCAGGCGGTGGCGAG 29 n/a 39 57 687269 356 375
GCAGGCGGTGGCGAGTGGGT 51 n/a 52 58 687270 351 370
CGGTGGCGAGTGGGTGAGTG 56 n/a 55 59 687271 346 365
GCGAGTGGGTGAGTGAGGAG 70 n/a 69 60 687272 341 360
TGGGTGAGTGAGGAGGCGGC 69 n/a 69 61 687273 336 355
GAGTGAGGAGGCGGCATCCT 56 n/a 51 62 687274 331 350
AGGAGGCGGCATCCTGGCGG 49 n/a 46 63 687275 326 345
GCGGCATCCTGGCGGGTGGC 54 n/a 51 64 687276 321 340
ATCCTGGCGGGTGGCTGTTT 66 n/a 64 65 687277 296 315
TCGGCTGCCGGGAAGAGGCG 62 n/a 64 66 687278 291 310
TGCCGGGAAGAGGCGCGGGT 46 n/a 41 67 687279 286 305
GGAAGAGGCGCGGGTAGAAG 6 n/a 8 68 687280 261 280 GCTCTCCTCAGAGCTCGACG
48 n/a 50 69 687281 256 275 CCTCAGAGCTCGACGCATTT 48 n/a 48 70
687282 251 270 GAGCTCGACGCATTTTTACT 57 n/a 55 71 687283 246 265
CGACGCATTTTTACTTTCCC 66 n/a 62 72 687284 241 260
CATTTTTACTTTCCCTCTCA 66 n/a 67 73 687285 236 255
TTACTTTCCCTCTCATTTCT 64 n/a 61 74 687286 231 250
TTCCCTCTCATTTCTCTGAC 60 n/a 56 75 687287 226 245
TCTCATTTCTCTGACCGAAG 64 n/a 45 76 687288 221 240
TTTCTCTGACCGAAGCTGGG 70 n/a 67 77 687289 216 235
CTGACCGAAGCTGGGTGTCG 71 n/a 66 78 687290 211 230
CGAAGCTGGGTGTCGGGCTT 64 n/a 65 79 687291 206 225
CTGGGTGTCGGGCTTTCGCC 73 n/a 68 80 687292 201 220
TGTCGGGCTTTCGCCTCTAG 66 n/a 67 81 687293 196 215
GGCTTTCGCCTCTAGCGACT 70 n/a 71 82 742033 456 475
CCGGGICCGIGGCCIGGGCC 74 36 50 83 462 481 742035 454 471
GGCCGIGGCCGGIGCCGG 37 n/a 18 84 460 477 466 483
Example 2: Dose Dependent Inhibition of C9ORF72 Antisense
Transcript with an Oligonucleotide Targeting a Hexanucleotide
Repeat
[0463] Isis No. 742033 (see Example 1) was tested for dose
dependent inhibition of C9ORF72 antisense transcript expression in
vitro. bEND cells were cultured and treated as described in Example
1. During the second transfection, cells received Isis No. 742033
at a concentration listed in the table below or they received no
ASO as a control. Total RNA was isolated and analyzed as described
in Example 1 using primer probe set C9ATS3'-1.
TABLE-US-00007 TABLE 7 C9ORF72 Antisense RNA Inhibition by Isis No.
742033 Concentration of Isis No. 742033 (nM) % Inhibition 3.125 39
6.25 65 12.5 68 25.0 72
Example 3: Antisense Inhibition of C9ORF72 Antisense Transcript
with Oligonucleotides
[0464] Antisense oligonucleotides (ASOs) targeting the human
C9ORF72 antisense transcript were made and tested for inhibition of
C9ORF72 antisense transcript expression in vitro. ASOs
730401-730406 in the table below are 5-8-5 MOE gapmers with
phosphorothioate internucleoside linkages throughout the gapmers.
ASOs 730407-730412 in the table below are 4-8-4 MOE gapmers with
phosphorothioate internucleoside linkages throughout the gapmers.
ASOs 730401-730412 all have a central gap segment consisting of
eight 2'-deoxynucleosides that is flanked by wing segments in the
5' direction and the 3' direction consisting of four or five
nucleosides each. Each nucleoside in the 5' and 3' wing segments
has a 2'-MOE modification. All cytosine residues throughout each
gapmer are 5-methylcytosines. In the table below, "Start" indicates
the 5'-most nucleoside to which the gapmer is targeted in the
target transcript sequence. "Stop" indicates the 3'-most nucleoside
to which the gapmer is targeted in the target transcript sequence.
Each gapmer listed in the table below is targeted to a putative
antisense transcript sequence, designated herein as SEQ ID NO: 13.
The sequence of SEQ ID NO: 13 is complementary to nucleotides 1159
to 1929 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT
008413.18 truncated from nucleotides 27535000 to 27565000) except
that SEQ ID NO: 13 has two more hexanucleotide repeats than SEQ ID
NO: 2. The sequence of the hexanucleotide repeat is GGCCCC in SEQ
ID NO: 13 and GGGGCC in SEQ ID NO: 2. Thus, SEQ ID NO: 13 is 12
nucleotides longer than nucleotides 1159 to 1929 of SEQ ID NO: 2,
to which it is complementary. Isis No. 129700 is a negative control
ASO that does not target the C9ORF72 antisense transcript, and
742035 was included for comparison, as it was described and tested
in Example 1.
[0465] bEND cells were cultured and treated as described in Example
1. Strand specific RT-qPCR was performed on the isolated RNA, as
described in Example 1, using the primer sets LTS01222 and
LTS01221. The resulting normalized C9ORF72 antisense levels were
then compared to the normalized values for C9ORF72 antisense
transcript expression in control cells that were transfected with
the C9ORF72 antisense plasmid but not an ASO. The results for each
primer probe set are shown in the table below as percent inhibition
of C9ORF72 antisense transcript expression relative to the control
cells that were not transfected with an ASO. The values in the
table below are the averages of two separate experiments. A result
of 0% inhibition indicates that the C9ORF72 antisense transcript
levels were equal to that of control cells that were not
transfected with an ASO. A negative value for % inhibition
indicates that the C9ORF72 antisense transcript levels were higher
than that of control cells that were not transfected with an ASO. A
result of "n/a" indicates that the corresponding primer probe set
was not used to analyze the indicated sample. The results show that
many ASOs inhibited human C9ORF72 antisense transcript expression.
The absolute inhibition results varied across different primer
probe sets, but the relative potencies of the ASOs were similar
across different primer probe sets.
TABLE-US-00008 TABLE 8 Inhibition of C9ORF72 Antisense Transcript
by Antisense Oligonucleotides SEQ Isis LTS LTS ID No. Start Stop
Sequence 01222 01221 No. 129700 n/a n/a TAGTGCGGACCTACCCACGA 11 11
29 730401 456 473 GGGGCCGGGGCCGGGGCC 71 38 85 462 479 468 485
730402 451 468 CGGGGCCGGGGCCGGGGC 67 10 86 457 474 463 480 730403
452 469 CCGGGGCCGGGGCCGGGG 64 21 87 458 475 464 481 730404 459 476
GCCGGGGCCGGGGCCGGG 65 34 88 465 482 730405 454 471
GGCCGGGGCCGGGGCCGG 49 22 89 460 477 466 483 730406 455 472
GGGCCGGGGCCGGGGCCG 41 12 90 461 478 467 484 730407 456 471
GGCCGGGGCCGGGGCC 78 44 91 462 477 468 483 730408 451 466
GGGCCGGGGCCGGGGC 68 24 92 457 472 463 478 469 484 730409 452 467
GGGGCCGGGGCCGGGG 66 38 93 458 473 464 479 470 485 730410 453 468
CGGGGCCGGGGCCGGG 67 38 94 459 474 465 480 730411 454 469
CCGGGGCCGGGGCCGG 63 16 95 460 475 466 481 730412 455 470
GCCGGGGCCGGGGCCG 73 38 96 461 476 467 482 742035 454 471
GGCCGIGGCCGGIGCCGG 68 32 84 460 477 466 483
Example 4: Antisense Inhibition of C9ORF72 Antisense Transcript
with Oligonucleotides
[0466] Antisense oligonucleotides (ASOs) described in Example 1
were tested for inhibition of C9ORF72 antisense transcript
expression in vitro using a cell line in which a CMV promoter was
installed to drive the expression of the endogenous C9ORF72
antisense gene via CRISPR/Cas9 technology.
[0467] The targeting portion of a single guide RNA (sgRNA) of the
sequence 5'-GACAAGGGTACGTAATCTGTC-3', designated herein as SEQ ID
NO: 97, was designed to target a site 1,020 base pairs downstream
of the C9ORF72 hexanucleotide repeat. An NGG PAM motif is present
at the 3' end of the target site. The targeting portion of the
sgRNA was inserted into a dual-expression plasmid to generate the
full-length sgRNA of the sequence:
5'-GACAAGGGTACGTAATCTGTCTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC
GTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT-3', designated herein as
SEQ ID NO.: 101 and Cas9 nuclease.
[0468] The donor plasmid, containing a 922 base pair 5' homology
arm, reverse CMV (CMVr), and 988 base pair 3' homology arm, was
generated in pCR4 (Life Technologies) backbone plasmid by Gibson
Assembly. The homology arm sequences were designed to center around
the CRISPR/Cas9 cleavage site and were constructed using PCR
primers: 5'-TAGTCCTGCAGGTTTAAACGAATTCGTGAGTGAGGAGGCGGCA-3', forward
primer, SEQ ID NO: 102, and
5'-AGCAGAGCTCAGATTACGTACCCTTGTTGTGAACAAC-3', reverse primer, SEQ ID
NO: 103, for the 5' arm; and
5'-CAATGTCAACGTCTGGCATTACTTCTACTTTTG-3', forward primer, SEQ ID NO:
104, and 5'-TAGGGCGAATTGAATTTAGCGGCCGCACTGGCAGGATCATAGC-3', reverse
primer, SEQ ID NO: 105, for the 3' arm. The CMVr sequence was
amplified from pCDNA3.1 using PCR primers:
5'-TACGTAATCTGAGCTCTGCTTATATAGACC-3', forward primer, SEQ ID NO:
106, and 5'-AATGCCAGACGTTGACATTGATTATTGACTAGTTATTAATAG-3', reverse
primer, SEQ ID NO: 107.
[0469] Neuroblast SH-SY5Y cells (Sigma-Aldrich) were cultured in a
1:1 mixture of MEM:F-12 (Life Technologies) supplemented with 10%
FBS, 25 mM HEPES and Antibiotic-Antimycotic. C9orf72 CRISPR/Cas9
activity was assessed by measuring indel frequency using SURVEYOR
mutation detection assay (Integrated DNA Technologies) with forward
primer: 5'-GTTAGGCTCTGGGAGAGTAGTTG-3', SEQ ID NO: 108, and reverse
primer: 5'-CCTGGAGCAGGTAAATGCTGG-3', SEQ ID NO: 109. To generate
SH-SY5Y cells expressing C9orf72 antisense transcript, SH-SY5Y
cells were transfected with a plasmid expressing C9ORF72 CRISPR
sgRNA and Cas9 and with a CMVr donor plasmid. Furthermore, the
cells were co-transfected with a plasmid expressing EGFP, then
single-cell sorted by FACS into 96-well plates. RT-qPCR was
performed to screen for increased C9ORF72 antisense RNA. Positive
clones were isolated and validated by PCR using primers inside CMVr
and outside the 5' and 3' arms, respectively. Amplicons were
further validated by sequencing to confirm on-target insertion of
CMVr. Confirmation of single or double allele targeting was
obtained by PCR with primers used in the SURVEYOR assay. Sequencing
showed that the C9ORF72 antisense transcript contains 2 full
hexanucleotide repeats.
[0470] The engineered SH-SY5Y cells were plated at 30,000 cells per
well and electroporated at 140V with 10 .mu.M ASO. 24 hours later
cells were lysed. Strand specific RT-qPCR was performed on the
isolated RNA, as described in Example 1, using the primer probe
sets LTS01221 or C9ATS3'-1. The resulting normalized C9ORF72
antisense levels were then compared to the normalized values for
C9ORF72 antisense transcript expression in control cells that were
transfected with neither the C9ORF72 antisense plasmid nor an ASO.
The results for each primer probe set are shown in the table below
as percent inhibition of C9ORF72 antisense transcript expression
relative to the control cells. A result of 0% inhibition indicates
that the C9ORF72 antisense transcript levels were equal to that of
control cells that were not transfected with an ASO. A negative
value for % inhibition indicates that the C9ORF72 antisense
transcript levels were higher than that of control cells that were
not transfected with an ASO. A result of "n/a" indicates that the
corresponding primer probe set was not used to analyze the
indicated sample. The results show that many ASOs inhibited human
C9ORF72 antisense transcript expression.
TABLE-US-00009 TABLE 9 Inhibition of C90RF72 Antisense Transcript
by Antisense Oligonucleotides Isis No. LTS 01221 C9ATS3'-1 SEQ ID
No. 129700 22 -4 29 687241 68 n/a 30 687242 60 n/a 31 687243 54 n/a
32 687244 47 n/a 33 687245 53 n/a 34 687246 52 n/a 35 687247 53 n/a
36 687248 14 n/a 37 687249 6 n/a 38 687250 12 n/a 39 687251 53 n/a
40 687252 32 n/a 41 687253 21 n/a 42 687254 20 n/a 43 687255 81 n/a
44 687256 78 n/a 45 687257 63 n/a 46 687258 54 n/a 47 687259 52 n/a
48 687260 23 n/a 49 687261 9 n/a 50 687262 41 n/a 51 687263 24 n/a
52 687264 42 n/a 53 687265 25 n/a 54 687266 51 n/a 55 687267 20 n/a
56 687268 24 n/a 57 687269 20 n/a 58 687270 36 n/a 59 687271 10 n/a
60 687272 47 n/a 61 687273 50 n/a 62 687274 66 n/a 63 687275 42 n/a
64 687276 48 n/a 65 687277 n/a 40 66 687278 n/a 38 67 687279 n/a
-24 68 687280 n/a 60 69 687281 n/a 69 70 687282 n/a 32 71 687283
n/a 69 72 687284 n/a 47 73 687285 n/a 28 74 687286 n/a 67 75 687287
n/a 71 76 687288 n/a 65 77 687289 n/a 63 78 687290 62 48 79 687291
75 71 80 687292 77 68 81 687293 76 72 82 742033 6 26 83
Example 5: Dose Dependent Inhibition of C9ORF72 Antisense
Transcript with Oligonucleotides
[0471] Isis Numbers 687241, 687255, 687256, 687280, 687281, 687283,
687286, 687287, 687288, 687289, 687291, 687292, and 687293 (see
Example 1) were tested for dose dependent inhibition of C9ORF72
antisense transcript expression in vitro. The SH-SY5Y cells
described in Example 4 were cultured at 30,000 cells per well and
electroporated at 140 V treated with an oligonucleotide at a
concentration listed in the tables below or they received no ASO as
a control. 24 hours after electroporation, cells were lysed and
total RNA was isolated and analyzed by RT-qPCR using primer probe
set LTS01221 (Tables 10 and 11) or C9ATS3'-1 (Table 12). The
results are shown below for each antisense oligonucleotide
concentration, and half maximal inhibitory concentrations were
calculated using Prism software (Graphpad).
TABLE-US-00010 TABLE 10 Dose Dependent C9ORF72 Antisense Transcript
Inhibition Concentration % IC.sub.50 Isis No. (.mu.M) Inhibition
(.mu.M) 687241 0.625 -5 3.49 1.25 17 2.5 34 5 52 10 66 20 54 687291
0.625 20 3.17 1.25 15 2.5 31 5 47 10 67 20 71
TABLE-US-00011 TABLE 11 Dose Dependent C9ORF72 Antisense Transcript
Inhibition Dose % IC.sub.50 Isis No. (nM) Inhibition (.mu.M) 687255
0.625 9 2.34 1.25 20 2.5 39 5 57 10 62 20 69 687256 0.625 15 2.78
1.25 19 2.5 37 5 46 10 56 20 68 687291 0.625 24 1.72 1.25 24 2.5 41
5 63 10 62 20 70
TABLE-US-00012 TABLE 12 Dose Dependent C9ORF72 Antisense Transcript
Inhibition Dose % IC.sub.50 Isis No. (nM) Inhibition (.mu.M) 687280
0.625 19 2.87 1.25 18 2.5 39 5 61 10 69 20 66 687281 0.625 13 5.21
1.25 12 2.5 26 5 39 10 60 20 69 687283 0.625 16 4.98 1.25 31 2.5 23
5 38 10 53 20 70 687286 0.625 -5 4.31 1.25 8 2.5 29 5 51 10 65 20
71 687287 0.625 5 3.10 1.25 24 2.5 25 5 64 10 73 20 85 687288 0.625
10 3.52 1.25 12 2.5 31 5 58 10 66 20 78 687289 0.625 14 5.43 1.25
14 2.5 23 5 43 10 61 20 60 687291 0.625 23 2.09 1.25 33 2.5 35 5 67
10 75 20 77 687292 0.625 19 3.94 1.25 11 2.5 25 5 56 10 68 20 65
687293 0.625 23 2.15 1.25 25 2.5 44 5 63 10 77 20 75
Example 6: Effect of Antisense Oligonucleotides Targeting C9ORF72
Antisense Transcript on RNA Foci
[0472] Antisense oligonucleotides described above and those
described in the tables below can be tested for their effects on
C9ORF72 antisense foci in C9ORF72 ALS/FTD patient fibroblast lines.
The antisense oligonucleotides listed in Tables 13 and 14 below
target the hexanucleotide repeat of the C9ORF72 antisense
transcript. Each nucleoside of the antisense oligonucleotides in
Table 13 below is modified with a 2'-MOE substitution. All of the
cytosines are 5-methylcytosines, and all of the internucleoside
linkages are phosphorothioate linkages. The motifs and
internucleoside linkages of the oligonucleotides in Table 14 are
shown in the table. The substitution or lack thereof at the
2'-position of each nucleoside is denoted as "d", meaning 2'-deoxy,
or "e", meaning 2'-MOE. Each internucleoside linkage is denoted as
"o", meaning phosphodiester, or "s", meaning phosphorothioate. All
of the cytosines in Table 14 are 5-methylcytosines. The
oligonucleotides in Table 14 also contain inosine residues,
indicated by "I".
TABLE-US-00013 TABLE 13 Fully modified antisense oligonucleotides
targeting the antisense transcript of C9ORF72 SEQ ID Isis No.
Sequence No. 730389 GGGGCCGGGGCCGGGGCC 85 730390 CGGGGCCGGGGCCGGGGC
86 730391 CCGGGGCCGGGGCCGGGG 87 730392 GCCGGGGCCGGGGCCGGG 88 730393
GGCCGGGGCCGGGGCCGG 89 730394 GGGCCGGGGCCGGGGCCG 90 730395
GGCCGGGGCCGGGGCC 91 730396 GGGCCGGGGCCGGGGC 92 730397
GGGGCCGGGGCCGGGG 93 730398 CGGGGCCGGGGCCGGG 94 730399
CCGGGGCCGGGGCCGG 95 730400 GCCGGGGCCGGGGCCG 96
TABLE-US-00014 TABLE 14 Antisense oligonucleotides targeting the
antisense transcript of C9ORF72 SEQ Isis Internucleoside ID No.
Sequence (5' to 3') Motif (5' to 3') linkages (5' to 3') No. 737821
CCGIGGCCGIGGCCGIGGCC eeedeeeeedeeeeedeeee ssososssososssososs 98
742034 GGCCGIGGCCGIGGCCGG eeeeedeeeeedeeeeee ssssososssosossss
99
[0473] C9ORF72 antisense foci are visualized using fluorescent in
situ hybridization with a fluorescently labeled Locked Nucleic Acid
(LNA) probe targeting the hexanucleotide repeat containing C9ORF72
antisense transcript (Exiqon, Inc. Woburn Mass.). The sequence of
the probe is presented in the table below. The probe was labeled
with fluorescent 5' TYE-563. A 5' TYE-563-labeled fluorescent probe
targeting CUG repeats is used as a negative control.
TABLE-US-00015 TABLE 15 LNA probes to the C9ORF72 antisense
transcript containing the hexanucleotide repeat SEQ Description ID
Target of probe Sequence NO GGCCCC Repeat Fluorescent TYE563- 93 of
the LNA Probe GGGGCCGGGGCCGGGG Antisense Transcript CUG Repeat
Fluorescent TYE563- 100 LNA Probe CAGCAGCAGCAGCAGCAGC
[0474] All hybridization steps were performed under RNase-free
conditions. Patient fibroblast cells were plated into chamber
slides. 24 hours later, they were washed in PBS and transfected
with 25 nM of an Isis antisense oligonucleotide in the table below
or a negative control ASO that does not target any C9ORF72 RNA
using 1 .mu.l/ml Cytofectin transfection reagent (Genlantis, San
Diego, Cat #T610001). Cells were incubated for 4 hours at
37.degree. C. and 5% CO.sub.2, before the medium was replaced with
Dulbecco's modified Eagle medium (DMEM) supplemented with 20%
tetracycline-free FBS and 2% penicillin/streptomycin and 1%
amphotericin B. 24 hours after transfection, the cells were fixed
in 4% PFA, then immediately permeabilized in 0.2% Triton X-100
(Sigma Aldrich #T-8787) in PBS for 10 minutes, washed twice in PBS
for 5 minutes, dehydrated with ethanol, and air dried. The slides
were heated in 400 .mu.L hybridization buffer (50% deionized
formamide, 2.times.SCC, 50 mM Sodium Phosphate, pH 7, and 10%
dextran sulphate) at 66.degree. C. for 20-60 minutes under floating
RNase-free coverslips in a chamber humidified with hybridization
buffer. Probes were denatured at 80.degree. C. for 75 seconds and
returned immediately to ice before diluting with hybridization
buffer (40 nM final concentration). The incubating buffer was
replaced with the probe-containing mix (400 .mu.L per slide), and
slides were hybridized under floating coverslips for 12-16 hours in
a sealed, light-protected chamber.
[0475] After hybridization, floating coverslips were removed and
slides were washed at room temperature in 0.1% Tween-20/2X SCC for
5 minutes before being subjected to three 10-minute stringency
washes in 0.1.times.SCC at 65.degree. C. The slides were then
dehydrated through ethanol and air dried.
[0476] Primary visualization for quantification and imaging of foci
was performed at 100.times. magnification using a Nikon Eclipse Ti
confocal microscope system equipped with a Nikon CFI Apo TIRF 100X
Oil objective (NA 1.49). Most foci are intra-nuclear but are also
occasionally found in the cytoplasm. Treatment with RNase A, but
not DNase I, eliminated the C9ORF72 antisense foci, demonstrating
that they are comprised primarily of RNA. The foci in the
fibroblasts were counted, and the data is presented in the table
below as the number of foci per positive cell and the number of
foci per cell overall. (A positive cell is a cell that has at least
one focus.) The data in the table below show that treatment with
the antisense oligonucleotides targeting the antisense C9ORF72
transcript, listed in the table below, decreased both the number of
cells with at least one focus (foci per cell) and the number of
foci within cells that still had at least one focus (foci per
positive cell).
TABLE-US-00016 TABLE 16 Antisense C9ORF72 foci in patient
fibroblasts Foci Foci Isis No. per positive cell per cell Negative
2.99 1.50 control ASO 737821 2.73 1.00 742033 1.68 0.46 742034 1.38
0.22 742035 1.69 0.53
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 109 <210> SEQ ID NO 1 <211> LENGTH: 3339
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 1 acgtaaccta cggtgtcccg ctaggaaaga gaggtgcgtc
aaacagcgac aagttccgcc 60 cacgtaaaag atgacgcttg gtgtgtcagc
cgtccctgct gcccggttgc ttctcttttg 120 ggggcggggt ctagcaagag
caggtgtggg tttaggagat atctccggag catttggata 180 atgtgacagt
tggaatgcag tgatgtcgac tctttgccca ccgccatctc cagctgttgc 240
caagacagag attgctttaa gtggcaaatc acctttatta gcagctactt ttgcttactg
300 ggacaatatt cttggtccta gagtaaggca catttgggct ccaaagacag
aacaggtact 360 tctcagtgat ggagaaataa cttttcttgc caaccacact
ctaaatggag aaatccttcg 420 aaatgcagag agtggtgcta tagatgtaaa
gttttttgtc ttgtctgaaa agggagtgat 480 tattgtttca ttaatctttg
atggaaactg gaatggggat cgcagcacat atggactatc 540 aattatactt
ccacagacag aacttagttt ctacctccca cttcatagag tgtgtgttga 600
tagattaaca catataatcc ggaaaggaag aatatggatg cataaggaaa gacaagaaaa
660 tgtccagaag attatcttag aaggcacaga gagaatggaa gatcagggtc
agagtattat 720 tccaatgctt actggagaag tgattcctgt aatggaactg
ctttcatcta tgaaatcaca 780 cagtgttcct gaagaaatag atatagctga
tacagtactc aatgatgatg atattggtga 840 cagctgtcat gaaggctttc
ttctcaatgc catcagctca cacttgcaaa cctgtggctg 900 ttccgttgta
gtaggtagca gtgcagagaa agtaaataag atagtcagaa cattatgcct 960
ttttctgact ccagcagaga gaaaatgctc caggttatgt gaagcagaat catcatttaa
1020 atatgagtca gggctctttg tacaaggcct gctaaaggat tcaactggaa
gctttgtgct 1080 gcctttccgg caagtcatgt atgctccata tcccaccaca
cacatagatg tggatgtcaa 1140 tactgtgaag cagatgccac cctgtcatga
acatatttat aatcagcgta gatacatgag 1200 atccgagctg acagccttct
ggagagccac ttcagaagaa gacatggctc aggatacgat 1260 catctacact
gacgaaagct ttactcctga tttgaatatt tttcaagatg tcttacacag 1320
agacactcta gtgaaagcct tcctggatca ggtctttcag ctgaaacctg gcttatctct
1380 cagaagtact ttccttgcac agtttctact tgtccttcac agaaaagcct
tgacactaat 1440 aaaatatata gaagacgata cgcagaaggg aaaaaagccc
tttaaatctc ttcggaacct 1500 gaagatagac cttgatttaa cagcagaggg
cgatcttaac ataataatgg ctctggctga 1560 gaaaattaaa ccaggcctac
actcttttat ctttggaaga cctttctaca ctagtgtgca 1620 agaacgagat
gttctaatga ctttttaaat gtgtaactta ataagcctat tccatcacaa 1680
tcatgatcgc tggtaaagta gctcagtggt gtggggaaac gttcccctgg atcatactcc
1740 agaattctgc tctcagcaat tgcagttaag taagttacac tacagttctc
acaagagcct 1800 gtgaggggat gtcaggtgca tcattacatt gggtgtctct
tttcctagat ttatgctttt 1860 gggatacaga cctatgttta caatataata
aatattattg ctatctttta aagatataat 1920 aataggatgt aaacttgacc
acaactactg tttttttgaa atacatgatt catggtttac 1980 atgtgtcaag
gtgaaatctg agttggcttt tacagatagt tgactttcta tcttttggca 2040
ttctttggtg tgtagaatta ctgtaatact tctgcaatca actgaaaact agagccttta
2100 aatgatttca attccacaga aagaaagtga gcttgaacat aggatgagct
ttagaaagaa 2160 aattgatcaa gcagatgttt aattggaatt gattattaga
tcctactttg tggatttagt 2220 ccctgggatt cagtctgtag aaatgtctaa
tagttctcta tagtccttgt tcctggtgaa 2280 ccacagttag ggtgttttgt
ttattttatt gttcttgcta ttgttgatat tctatgtagt 2340 tgagctctgt
aaaaggaaat tgtattttat gttttagtaa ttgttgccaa ctttttaaat 2400
taattttcat tatttttgag ccaaattgaa atgtgcacct cctgtgcctt ttttctcctt
2460 agaaaatcta attacttgga acaagttcag atttcactgg tcagtcattt
tcatcttgtt 2520 ttcttcttgc taagtcttac catgtacctg ctttggcaat
cattgcaact ctgagattat 2580 aaaatgcctt agagaatata ctaactaata
agatcttttt ttcagaaaca gaaaatagtt 2640 ccttgagtac ttccttcttg
catttctgcc tatgtttttg aagttgttgc tgtttgcctg 2700 caataggcta
taaggaatag caggagaaat tttactgaag tgctgttttc ctaggtgcta 2760
ctttggcaga gctaagttat cttttgtttt cttaatgcgt ttggaccatt ttgctggcta
2820 taaaataact gattaatata attctaacac aatgttgaca ttgtagttac
acaaacacaa 2880 ataaatattt tatttaaaat tctggaagta atataaaagg
gaaaatatat ttataagaaa 2940 gggataaagg taatagagcc cttctgcccc
ccacccacca aatttacaca acaaaatgac 3000 atgttcgaat gtgaaaggtc
ataatagctt tcccatcatg aatcagaaag atgtggacag 3060 cttgatgttt
tagacaacca ctgaactaga tgactgttgt actgtagctc agtcatttaa 3120
aaaatatata aatactacct tgtagtgtcc catactgtgt tttttacatg gtagattctt
3180 atttaagtgc taactggtta ttttctttgg ctggtttatt gtactgttat
acagaatgta 3240 agttgtacag tgaaataagt tattaaagca tgtgtaaaca
ttgttatata tcttttctcc 3300 taaatggaga attttgaata aaatatattt
gaaattttg 3339 <210> SEQ ID NO 2 <211> LENGTH: 30001
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2 caaagaaaag ggggaggttt tgttaaaaaa gagaaatgtt
acatagtgct ctttgagaaa 60 attcattggc actattaagg atctgaggag
ctggtgagtt tcaactggtg agtgatggtg 120 gtagataaaa ttagagctgc
agcaggtcat tttagcaact attagataaa actggtctca 180 ggtcacaacg
ggcagttgca gcagctggac ttggagagaa ttacactgtg ggagcagtgt 240
catttgtcct aagtgctttt ctacccccta cccccactat tttagttggg tataaaaaga
300 atgacccaat ttgtatgatc aactttcaca aagcatagaa cagtaggaaa
agggtctgtt 360 tctgcagaag gtgtagacgt tgagagccat tttgtgtatt
tattcctccc tttcttcctc 420 ggtgaatgat taaaacgttc tgtgtgattt
ttagtgatga aaaagattaa atgctactca 480 ctgtagtaag tgccatctca
cacttgcaga tcaaaaggca cacagtttaa aaaacctttg 540 tttttttaca
catctgagtg gtgtaaatgc tactcatctg tagtaagtgg aatctataca 600
cctgcagacc aaaagacgca aggtttcaaa aatctttgtg ttttttacac atcaaacaga
660 atggtacgtt tttcaaaagt taaaaaaaaa caactcatcc acatattgca
actagcaaaa 720 atgacattcc ccagtgtgaa aatcatgctt gagagaattc
ttacatgtaa aggcaaaatt 780 gcgatgactt tgcaggggac cgtgggattc
ccgcccgcag tgccggagct gtcccctacc 840 agggtttgca gtggagtttt
gaatgcactt aacagtgtct tacggtaaaa acaaaatttc 900 atccaccaat
tatgtgttga gcgcccactg cctaccaagc acaaacaaaa ccattcaaaa 960
ccacgaaatc gtcttcactt tctccagatc cagcagcctc ccctattaag gttcgcacac
1020 gctattgcgc caacgctcct ccagagcggg tcttaagata aaagaacagg
acaagttgcc 1080 ccgccccatt tcgctagcct cgtgagaaaa cgtcatcgca
catagaaaac agacagacgt 1140 aacctacggt gtcccgctag gaaagagagg
tgcgtcaaac agcgacaagt tccgcccacg 1200 taaaagatga cgcttggtgt
gtcagccgtc cctgctgccc ggttgcttct cttttggggg 1260 cggggtctag
caagagcagg tgtgggttta ggaggtgtgt gtttttgttt ttcccaccct 1320
ctctccccac tacttgctct cacagtactc gctgagggtg aacaagaaaa gacctgataa
1380 agattaacca gaagaaaaca aggagggaaa caaccgcagc ctgtagcaag
ctctggaact 1440 caggagtcgc gcgctagggg ccggggccgg ggccggggcg
tggtcggggc gggcccgggg 1500 gcgggcccgg ggcggggctg cggttgcggt
gcctgcgccc gcggcggcgg aggcgcaggc 1560 ggtggcgagt gggtgagtga
ggaggcggca tcctggcggg tggctgtttg gggttcggct 1620 gccgggaaga
ggcgcgggta gaagcggggg ctctcctcag agctcgacgc atttttactt 1680
tccctctcat ttctctgacc gaagctgggt gtcgggcttt cgcctctagc gactggtgga
1740 attgcctgca tccgggcccc gggcttcccg gcggcggcgg cggcggcggc
ggcgcaggga 1800 caagggatgg ggatctggcc tcttccttgc tttcccgccc
tcagtacccg agctgtctcc 1860 ttcccgggga cccgctggga gcgctgccgc
tgcgggctcg agaaaaggga gcctcgggta 1920 ctgagaggcc tcgcctgggg
gaaggccgga gggtgggcgg cgcgcggctt ctgcggacca 1980 agtcggggtt
cgctaggaac ccgagacggt ccctgccggc gaggagatca tgcgggatga 2040
gatgggggtg tggagacgcc tgcacaattt cagcccaagc ttctagagag tggtgatgac
2100 ttgcatatga gggcagcaat gcaagtcggt gtgctcccca ttctgtggga
catgacctgg 2160 ttgcttcaca gctccgagat gacacagact tgcttaaagg
aagtgactat tgtgacttgg 2220 gcatcacttg actgatggta atcagttgtc
taaagaagtg cacagattac atgtccgtgt 2280 gctcattggg tctatctggc
cgcgttgaac accaccaggc tttgtattca gaaacaggag 2340 ggaggtcctg
cactttccca ggaggggtgg ccctttcaga tgcaatcgag attgttaggc 2400
tctgggagag tagttgcctg gttgtggcag ttggtaaatt tctattcaaa cagttgccat
2460 gcaccagttg ttcacaacaa gggtacgtaa tctgtctggc attacttcta
cttttgtaca 2520 aaggatcaaa aaaaaaaaag atactgttaa gatatgattt
ttctcagact ttgggaaact 2580 tttaacataa tctgtgaata tcacagaaac
aagactatca tataggggat attaataacc 2640 tggagtcaga atacttgaaa
tacggtgtca tttgacacgg gcattgttgt caccacctct 2700 gccaaggcct
gccactttag gaaaaccctg aatcagttgg aaactgctac atgctgatag 2760
tacatctgaa acaagaacga gagtaattac cacattccag attgttcact aagccagcat
2820 ttacctgctc caggaaaaaa ttacaagcac cttatgaagt tgataaaata
ttttgtttgg 2880 ctatgttggc actccacaat ttgctttcag agaaacaaag
taaaccaagg aggacttctg 2940 tttttcaagt ctgccctcgg gttctattct
acgttaatta gatagttccc aggaggacta 3000 ggttagccta cctattgtct
gagaaacttg gaactgtgag aaatggccag atagtgatat 3060 gaacttcacc
ttccagtctt ccctgatgtt gaagattgag aaagtgttgt gaactttctg 3120
gtactgtaaa cagttcactg tccttgaagt ggtcctgggc agctcctgtt gtggaaagtg
3180 gacggtttag gatcctgctt ctctttgggc tgggagaaaa taaacagcat
ggttacaagt 3240 attgagagcc aggttggaga aggtggctta cacctgtaat
gccagagctt tgggaggcgg 3300 aggcaagagg atcacttgaa gccaggagtt
caagctcaac ctgggcaacg tagaccctgt 3360 ctctacaaaa aattaaaaac
ttagccgggc gtggtgatgt gcacctgtag tcctagctac 3420 ttgggaggct
gaggcaggag ggtcatttga gcccaagagt ttgaagttac cgagagctat 3480
gatcctgcca gtgcattcca gcctggatga caaaacgaga ccctgtctct aaaaaacaag
3540 aagtgagggc tttatgattg tagaattttc actacaatag cagtggacca
accacctttc 3600 taaataccaa tcagggaaga gatggttgat tttttaacag
acgtttaaag aaaaagcaaa 3660 acctcaaact tagcactcta ctaacagttt
tagcagatgt taattaatgt aatcatgtct 3720 gcatgtatgg gattatttcc
agaaagtgta ttgggaaacc tctcatgaac cctgtgagca 3780 agccaccgtc
tcactcaatt tgaatcttgg cttccctcaa aagactggct aatgtttggt 3840
aactctctgg agtagacagc actacatgta cgtaagatag gtacataaac aactattggt
3900 tttgagctga tttttttcag ctgcatttgc atgtatggat ttttctcacc
aaagacgatg 3960 acttcaagta ttagtaaaat aattgtacag ctctcctgat
tatacttctc tgtgacattt 4020 catttcccag gctatttctt ttggtaggat
ttaaaactaa gcaattcagt atgatctttg 4080 tccttcattt tctttcttat
tctttttgtt tgtttgtttg tttgtttttt tcttgaggca 4140 gagtctctct
ctgtcgccca ggctggagtg cagtggcgcc atctcagctc attgcaacct 4200
ctgccacctc cgggttcaag agattctcct gcctcagcct cccgagtagc tgggattaca
4260 ggtgtccacc accacacccg gctaattttt tgtattttta gtagaggtgg
ggtttcacca 4320 tgttggccag gctggtcttg agctcctgac ctcaggtgat
ccacctgcct cggcctacca 4380 aagagctggg ataacaggtg tgacccacca
tgcccggccc attttttttt tcttattctg 4440 ttaggagtga gagtgtaact
agcagtataa tagttcaatt ttcacaacgt ggtaaaagtt 4500 tccctataat
tcaatcagat tttgctccag ggttcagttc tgttttagga aatactttta 4560
ttttcagttt aatgatgaaa tattagagtt gtaatattgc ctttatgatt atccaccttt
4620 ttaacctaaa agaatgaaag aaaaatatgt ttgcaatata attttatggt
tgtatgttaa 4680 cttaattcat tatgttggcc tccagtttgc tgttgttagt
tatgacagca gtagtgtcat 4740 taccatttca attcagatta cattcctata
tttgatcatt gtaaactgac tgcttacatt 4800 gtattaaaaa cagtggatat
tttaaagaag ctgtacggct tatatctagt gctgtctctt 4860 aagactatta
aattgataca acatatttaa aagtaaatat tacctaaatg aatttttgaa 4920
attacaaata cacgtgttaa aactgtcgtt gtgttcaacc atttctgtac atacttagag
4980 ttaactgttt tgccaggctc tgtatgccta ctcataatat gataaaagca
ctcatctaat 5040 gctctgtaaa tagaagtcag tgctttccat cagactgaac
tctcttgaca agatgtggat 5100 gaaattcttt aagtaaaatt gtttactttg
tcatacattt acagatcaaa tgttagctcc 5160 caaagcaatc atatggcaaa
gataggtata tcatagtttg cctattagct gctttgtatt 5220 gctattatta
taaatagact tcacagtttt agacttgctt aggtgaaatt gcaattcttt 5280
ttactttcag tcttagataa caagtcttca attatagtac aatcacacat tgcttaggaa
5340 tgcatcatta ggcgattttg tcattatgca aacatcatag agtgtactta
cacaaaccta 5400 gatagtatag cctttatgta cctaggccgt atggtatagt
ctgttgctcc taggccacaa 5460 acctgtacaa ctgttactgt actgaatact
atagacagtt gtaacacagt ggtaaatatt 5520 tatctaaata tatgcaaaca
gagaaaaggt acagtaaaag tatggtataa aagataatgg 5580 tatacctgtg
taggccactt accacgaatg gagcttgcag gactagaagt tgctctgggt 5640
gagtcagtga gtgagtggtg aattaatgtg aaggcctaga acactgtaca ccactgtaga
5700 ctataaacac agtacgctga agctacacca aatttatctt aacagttttt
cttcaataaa 5760 aaattataac tttttaactt tgtaaacttt ttaatttttt
aacttttaaa atacttagct 5820 tgaaacacaa atacattgta tagctataca
aaaatatttt ttctttgtat ccttattcta 5880 gaagcttttt tctattttct
attttaaatt ttttttttta cttgttagtc gtttttgtta 5940 aaaactaaaa
cacacacact ttcacctagg catagacagg attaggatca tcagtatcac 6000
tcccttccac ctcactgcct tccacctcca catcttgtcc cactggaagg tttttagggg
6060 caataacaca catgtagctg tcacctatga taacagtgct ttctgttgaa
tacctcctga 6120 aggacttgcc tgaggctgtt ttacatttaa cttaaaaaaa
aaaaaagtag aaggagtgca 6180 ctctaaaata acaataaaag gcatagtata
gtgaatacat aaaccagcaa tgtagtagtt 6240 tattatcaag tgttgtacac
tgtaataatt gtatgtgcta tactttaaat aacttgcaaa 6300 atagtactaa
gaccttatga tggttacagt gtcactaagg caatagcata ttttcaggtc 6360
cattgtaatc taatgggact accatcatat atgcagtcta ccattgactg aaacgttaca
6420 tggcacataa ctgtatttgc aagaatgatt tgttttacat taatatcaca
taggatgtac 6480 ctttttagag tggtatgttt atgtggatta agatgtacaa
gttgagcaag gggaccaaga 6540 gccctgggtt ctgtcttgga tgtgagcgtt
tatgttcttc tcctcatgtc tgttttctca 6600 ttaaattcaa aggcttgaac
gggccctatt tagcccttct gttttctacg tgttctaaat 6660 aactaaagct
tttaaattct agccatttag tgtagaactc tctttgcagt gatgaaatgc 6720
tgtattggtt tcttggctag catattaaat atttttatct ttgtcttgat acttcaatgt
6780 cgttttaaac atcaggatcg ggcttcagta ttctcataac cagagagttc
actgaggata 6840 caggactgtt tgcccatttt ttgttatggc tccagacttg
tggtatttcc atgtcttttt 6900 tttttttttt ttttttgacc ttttagcggc
tttaaagtat ttctgttgtt aggtgttgta 6960 ttacttttct aagattactt
aacaaagcac cacaaactga gtggctttaa acaacagcaa 7020 tttattctct
cacaattcta gaagctagaa gtccgaaatc aaagtgttga caggggcatg 7080
atcttcaaga gagaagactc tttccttgcc tcttcctggc ttctggtggt taccagcaat
7140 cctgagtgtt cctttcttgc cttgtagttt caacaatcca gtatctgcct
tttgtcttca 7200 catggctgtc taccatttgt ctctgtgtct ccaaatctct
ctccttataa acacagcagt 7260 tattggatta ggccccactc taatccagta
tgaccccatt ttaacatgat tacacttatt 7320 tctagataag gtcacattca
cgtacaccaa gggttaggaa ttgaacatat ctttttgggg 7380 gacacaattc
aacccacaag tgtcagtctc tagctgagcc tttcccttcc tgtttttctc 7440
ctttttagtt gctatgggtt aggggccaaa tctccagtca tactagaatt gcacatggac
7500 tggatatttg ggaatactgc gggtctattc tatgagcttt agtatgtaac
atttaatatc 7560 agtgtaaaga agcccttttt taagttattt ctttgaattt
ctaaatgtat gccctgaata 7620 taagtaacaa gttaccatgt cttgtaaaat
gatcatatca acaaacattt aatgtgcacc 7680 tactgtgcta gttgaatgtc
tttatcctga taggagataa caggattcca catctttgac 7740 ttaagaggac
aaaccaaata tgtctaaatc atttggggtt ttgatggata tctttaaatt 7800
gctgaaccta atcattggtt tcatatgtca ttgtttagat atctccggag catttggata
7860 atgtgacagt tggaatgcag tgatgtcgac tctttgccca ccgccatctc
cagctgttgc 7920 caagacagag attgctttaa gtggcaaatc acctttatta
gcagctactt ttgcttactg 7980 ggacaatatt cttggtccta gagtaaggca
catttgggct ccaaagacag aacaggtact 8040 tctcagtgat ggagaaataa
cttttcttgc caaccacact ctaaatggag aaatccttcg 8100 aaatgcagag
agtggtgcta tagatgtaaa gttttttgtc ttgtctgaaa agggagtgat 8160
tattgtttca ttaatctttg atggaaactg gaatggggat cgcagcacat atggactatc
8220 aattatactt ccacagacag aacttagttt ctacctccca cttcatagag
tgtgtgttga 8280 tagattaaca catataatcc ggaaaggaag aatatggatg
cataaggtaa gtgatttttc 8340 agcttattaa tcatgttaac ctatctgttg
aaagcttatt ttctggtaca tataaatctt 8400 atttttttaa ttatatgcag
tgaacatcaa acaataaatg ttatttattt tgcatttacc 8460 ctattagata
caaatacatc tggtctgata cctgtcatct tcatattaac tgtggaaggt 8520
acgaaatggt agctccacat tatagatgaa aagctaaagc ttagacaaat aaagaaactt
8580 ttagaccctg gattcttctt gggagccttt gactctaata ccttttgttt
ccctttcatt 8640 gcacaattct gtcttttgct tactactatg tgtaagtata
acagttcaaa gtaatagttt 8700 cataagctgt tggtcatgta gcctttggtc
tctttaacct ctttgccaag ttcccaggtt 8760 cataaaatga ggaggttgaa
tggaatggtt cccaagagaa ttccttttaa tcttacagaa 8820 attattgttt
tcctaaatcc tgtagttgaa tatataatgc tatttacatt tcagtatagt 8880
tttgatgtat ctaaagaaca cattgaattc tccttcctgt gttccagttt gatactaacc
8940 tgaaagtcca ttaagcatta ccagttttaa aaggcttttg cccaatagta
aggaaaaata 9000 atatctttta aaagaataat tttttactat gtttgcaggc
ttacttcctt ttttctcaca 9060 ttatgaaact cttaaaatca ggagaatctt
ttaaacaaca tcataatgtt taatttgaaa 9120 agtgcaagtc attcttttcc
tttttgaaac tatgcagatg ttacattgac tgttttctgt 9180 gaagttatct
ttttttcact gcagaataaa ggttgttttg attttatttt gtattgttta 9240
tgagaacatg catttgttgg gttaatttcc tacccctgcc cccatttttt ccctaaagta
9300 gaaagtattt ttcttgtgaa ctaaattact acacaagaac atgtctattg
aaaaataagc 9360 aagtatcaaa atgttgtggg ttgttttttt aaataaattt
tctcttgctc aggaaagaca 9420 agaaaatgtc cagaagatta tcttagaagg
cacagagaga atggaagatc aggtatatgc 9480 aaattgcata ctgtcaaatg
tttttctcac agcatgtatc tgtataaggt tgatggctac 9540 atttgtcaag
gccttggaga catacgaata agcctttaat ggagctttta tggaggtgta 9600
cagaataaac tggaggaaga tttccatatc ttaaacccaa agagttaaat cagtaaacaa
9660 aggaaaatag taattgcatc tacaaattaa tatttgctcc cttttttttt
ctgtttgccc 9720 agaataaatt ttggataact tgttcatagt aaaaataaaa
aaaattgtct ctgatatgtt 9780 ctttaaggta ctacttctcg aacctttccc
tagaagtagc tgtaacagaa ggagagcata 9840 tgtacccctg aggtatctgt
ctggggtgta ggcccaggtc cacacaatat ttcttctaag 9900 tcttatgttg
tatcgttaag actcatgcaa tttacatttt attccataac tattttagta 9960
ttaaaatttg tcagtgatat ttcttaccct ctcctctagg aaaatgtgcc atgtttatcc
10020 cttggctttg aatgcccctc aggaacagac actaagagtt tgagaagcat
ggttacaagg 10080 gtgtggcttc ccctgcggaa actaagtaca gactatttca
ctgtaaagca gagaagttct 10140 tttgaaggag aatctccagt gaagaaagag
ttcttcactt ttacttccat ttcctcttgt 10200 gggtgaccct caatgctcct
tgtaaaactc caatatttta aacatggctg ttttgccttt 10260 ctttgcttct
ttttagcatg aatgagacag atgatacttt aaaaaagtaa ttaaaaaaaa 10320
aaacttgtga aaatacatgg ccataataca gaacccaata caatgatctc ctttaccaaa
10380 ttgttatgtt tgtacttttg tagatagctt tccaattcag agacagttat
tctgtgtaaa 10440 ggtctgactt aacaagaaaa gatttccctt tacccaaaga
atcccagtcc ttatttgctg 10500 gtcaataagc agggtcccca ggaatggggt
aactttcagc accctctaac ccactagtta 10560 ttagtagact aattaagtaa
acttatcgca agttgaggaa acttagaacc aactaaaatt 10620 ctgcttttac
tgggattttg ttttttcaaa ccagaaacct ttacttaagt tgactactat 10680
taatgaattt tggtctctct tttaagtgct cttcttaaaa atgttatctt actgctgaga
10740 agttcaagtt tgggaagtac aaggaggaat agaaacttaa gagattttct
tttagagcct 10800 cttctgtatt tagccctgta ggattttttt tttttttttt
ttttttggtg ttgttgagct 10860 tcagtgaggc tattcattca cttatactga
taatgtctga gatactgtga atgaaatact 10920 atgtatgctt aaacctaaga
ggaaatattt tcccaaaatt attcttcccg aaaaggagga 10980 gttgcctttt
gattgagttc ttgcaaatct cacaacgact ttattttgaa caatactgtt 11040
tggggatgat gcattagttt gaaacaactt cagttgtagc tgtcatctga taaaattgct
11100 tcacagggaa ggaaatttaa cacggatcta gtcattattc ttgttagatt
gaatgtgtga 11160 attgtaattg taaacaggca tgataattat tactttaaaa
actaaaaaca gtgaatagtt 11220 agttgtggag gttactaaag gatggttttt
ttttaaataa aactttcagc attatgcaaa 11280 tgggcatatg gcttaggata
aaacttccag aagtagcatc acatttaaat tctcaagcaa 11340 cttaataata
tggggctctg aaaaactggt taaggttact ccaaaaatgg ccctgggtct 11400
gacaaagatt ctaacttaaa gatgcttatg aagactttga gtaaaatcat ttcataaaat
11460 aagtgaggaa aaacaactag tattaaattc atcttaaata atgtatgatt
taaaaaatat 11520 gtttagctaa aaatgcatag tcatttgaca atttcattta
tatctcaaaa aatttactta 11580 accaagttgg tcacaaaact gatgagactg
gtggtggtag tgaataaatg agggaccatc 11640 catatttgag acactttaca
tttgtgatgt gttatactga attttcagtt tgattctata 11700 gactacaaat
ttcaaaatta caatttcaag atgtaataag tagtaatatc ttgaaatagc 11760
tctaaaggga atttttctgt tttattgatt cttaaaatat atgtgctgat tttgatttgc
11820 atttgggtag attatacttt tatgagtatg gaggttaggt attgattcaa
gttttcctta 11880 cctatttggt aaggatttca aagtcttttt gtgcttggtt
ttcctcattt ttaaatatga 11940 aatatattga tgacctttaa caaatttttt
ttatctcaaa ttttaaagga gatcttttct 12000 aaaagaggca tgatgactta
atcattgcat gtaacagtaa acgataaacc aatgattcca 12060 tactctctaa
agaataaaag tgagctttag ggccgggcat ggtcagaaat ttgacaccaa 12120
cctggccaac atggcgaaac cccgtctcta ctaaaaatac aaaaatcagc cgggcatggt
12180 ggcggcacct atagtcccag ctacttggga ggatgagaca ggagagtcac
ttgaacctgg 12240 gaggagaggt tgcagtgagc tgagatcacg ccattgcact
ccagcctgag caatgaaagc 12300 aaaactccat ctcaaaaaaa aaaaaagaaa
agaaagaata aaagtgagct ttggattgca 12360 tataaatcct ttagacatgt
agtagacttg tttgatactg tgtttgaaca aattacgaag 12420 tattttcatc
aaagaatgtt attgtttgat gttattttta ttttttattg cccagcttct 12480
ctcatattac gtgattttct tcacttcatg tcactttatt gtgcagggtc agagtattat
12540 tccaatgctt actggagaag tgattcctgt aatggaactg ctttcatcta
tgaaatcaca 12600 cagtgttcct gaagaaatag atgtaagttt aaatgagagc
aattatacac tttatgagtt 12660 ttttggggtt atagtattat tatgtatatt
attaatattc taattttaat agtaaggact 12720 ttgtcataca tactattcac
atacagtatt agccacttta gcaaataagc acacacaaaa 12780 tcctggattt
tatggcaaaa cagaggcatt tttgatcagt gatgacaaaa ttaaattcat 12840
tttgtttatt tcattacttt tataattcct aaaagtggga ggatcccagc tcttatagga
12900 gcaattaata tttaatgtag tgtcttttga aacaaaactg tgtgccaaag
tagtaaccat 12960 taatggaagt ttacttgtag tcacaaattt agtttcctta
atcatttgtt gaggacgttt 13020 tgaatcacac actatgagtg ttaagagata
cctttaggaa actattcttg ttgttttctg 13080 attttgtcat ttaggttagt
ctcctgattc tgacagctca gaagaggaag ttgttcttgt 13140 aaaaattgtt
taacctgctt gaccagcttt cacatttgtt cttctgaagt ttatggtagt 13200
gcacagagat tgttttttgg ggagtcttga ttctcggaaa tgaaggcagt gtgttatatt
13260 gaatccagac ttccgaaaac ttgtatatta aaagtgttat ttcaacacta
tgttacagcc 13320 agactaattt ttttattttt tgatgcattt tagatagctg
atacagtact caatgatgat 13380 gatattggtg acagctgtca tgaaggcttt
cttctcaagt aagaattttt cttttcataa 13440 aagctggatg aagcagatac
catcttatgc tcacctatga caagatttgg aagaaagaaa 13500 ataacagact
gtctacttag attgttctag ggacattacg tatttgaact gttgcttaaa 13560
tttgtgttat ttttcactca ttatatttct atatatattt ggtgttattc catttgctat
13620 ttaaagaaac cgagtttcca tcccagacaa gaaatcatgg ccccttgctt
gattctggtt 13680 tcttgtttta cttctcatta aagctaacag aatcctttca
tattaagttg tactgtagat 13740 gaacttaagt tatttaggcg tagaacaaaa
ttattcatat ttatactgat ctttttccat 13800 ccagcagtgg agtttagtac
ttaagagttt gtgcccttaa accagactcc ctggattaat 13860 gctgtgtacc
cgtgggcaag gtgcctgaat tctctataca cctatttcct catctgtaaa 13920
atggcaataa tagtaatagt acctaatgtg tagggttgtt ataagcattg agtaagataa
13980 ataatataaa gcacttagaa cagtgcctgg aacataaaaa cacttaataa
tagctcatag 14040 ctaacatttc ctatttacat ttcttctaga aatagccagt
atttgttgag tgcctacatg 14100 ttagttcctt tactagttgc tttacatgta
ttatcttata ttctgtttta aagtttcttc 14160 acagttacag attttcatga
aattttactt ttaataaaag agaagtaaaa gtataaagta 14220 ttcactttta
tgttcacagt cttttccttt aggctcatga tggagtatca gaggcatgag 14280
tgtgtttaac ctaagagcct taatggcttg aatcagaagc actttagtcc tgtatctgtt
14340 cagtgtcagc ctttcataca tcattttaaa tcccatttga ctttaagtaa
gtcacttaat 14400 ctctctacat gtcaatttct tcagctataa aatgatggta
tttcaataaa taaatacatt 14460 aattaaatga tattatactg actaattggg
ctgttttaag gctcaataag aaaatttctg 14520 tgaaaggtct ctagaaaatg
taggttccta tacaaataaa agataacatt gtgcttatag 14580 cttcggtgtt
tatcatataa agctattctg agttatttga agagctcacc tacttttttt 14640
tgtttttagt ttgttaaatt gttttatagg caatgttttt aatctgtttt ctttaactta
14700 cagtgccatc agctcacact tgcaaacctg tggctgttcc gttgtagtag
gtagcagtgc 14760 agagaaagta aataaggtag tttattttat aatctagcaa
atgatttgac tctttaagac 14820 tgatgatata tcatggattg tcatttaaat
ggtaggttgc aattaaaatg atctagtagt 14880 ataaggaggc aatgtaatct
catcaaattg ctaagacacc ttgtggcaac agtgagtttg 14940 aaataaactg
agtaagaatc atttatcagt ttattttgat agctcggaaa taccagtgtc 15000
agtagtgtat aaatggtttt gagaatatat taaaatcaga tatataaaaa aaattactct
15060 tctatttccc aatgttatct ttaacaaatc tgaagatagt catgtacttt
tggtagtagt 15120 tccaaagaaa tgttatttgt ttattcatct tgatttcatt
gtcttcgctt tccttctaaa 15180 tctgtccctt ctagggagct attgggatta
agtggtcatt gattattata ctttattcag 15240 taatgtttct gaccctttcc
ttcagtgcta cttgagttaa ttaaggatta atgaacagtt 15300 acatttccaa
gcattagcta ataaactaaa ggattttgca cttttcttca ctgaccatta 15360
gttagaaaga gttcagagat aagtatgtgt atctttcaat ttcagcaaac ctaatttttt
15420 aaaaaaagtt ttacatagga aatatgttgg aaatgatact ttacaaagat
attcataatt 15480 tttttttgta atcagctact ttgtatattt acatgagcct
taatttatat ttctcatata 15540 accatttatg agagcttagt atacctgtgt
cattatattg catctacgaa ctagtgacct 15600 tattccttct gttacctcaa
acaggtggct ttccatctgt gatctccaaa gccttaggtt 15660 gcacagagtg
actgccgagc tgctttatga agggagaaag gctccatagt tggagtgttt 15720
tttttttttt ttttaaacat ttttcccatc ctccatcctc ttgagggaga atagcttacc
15780 ttttatcttg ttttaatttg agaaagaagt tgccaccact ctaggttgaa
aaccactcct 15840 ttaacataat aactgtggat atggtttgaa tttcaagata
gttacatgcc tttttatttt 15900 tcctaataga gctgtaggtc aaatattatt
agaatcagat ttctaaatcc cacccaatga 15960 cctgcttatt ttaaatcaaa
ttcaataatt aattctcttc tttttggagg atctggacat 16020 tctttgatat
ttcttacaac gaatttcatg tgtagaccca ctaaacagaa gctataaaag 16080
ttgcatggtc aaataagtct gagaaagtct gcagatgata taattcacct gaagagtcac
16140 agtatgtagc caaatgttaa aggttttgag atgccataca gtaaatttac
caagcatttt 16200 ctaaatttat ttgaccacag aatccctatt ttaagcaaca
actgttacat cccatggatt 16260 ccaggtgact aaagaatact tatttcttag
gatatgtttt attgataata acaattaaaa 16320 tttcagatat ctttcataag
caaatcagtg gtctttttac ttcatgtttt aatgctaaaa 16380 tattttcttt
tatagatagt cagaacatta tgcctttttc tgactccagc agagagaaaa 16440
tgctccaggt tatgtgaagc agaatcatca tttaaatatg agtcagggct ctttgtacaa
16500 ggcctgctaa aggtatagtt tctagttatc acaagtgaaa ccacttttct
aaaatcattt 16560 ttgagactct ttatagacaa atcttaaata ttagcattta
atgtatctca tattgacatg 16620 cccagagact gacttccttt acacagttct
gcacatagac tatatgtctt atggatttat 16680 agttagtatc atcagtgaaa
caccatagaa taccctttgt gttccaggtg ggtccctgtt 16740 cctacatgtc
tagcctcagg actttttttt ttttaacaca tgcttaaatc aggttgcaca 16800
tcaaaaataa gatcatttct ttttaactaa atagatttga attttattga aaaaaaattt
16860 taaacatctt taagaagctt ataggattta agcaattcct atgtatgtgt
actaaaatat 16920 atatatttct atatataata tatattagaa aaaaattgta
tttttctttt atttgagtct 16980 actgtcaagg agcaaaacag agaaatgtaa
attagcaatt atttataata cttaaaggga 17040 agaaagttgt tcaccttgtt
gaatctatta ttgttatttc aattatagtc ccaagacgtg 17100 aagaaatagc
tttcctaatg gttatgtgat tgtctcatag tgactacttt cttgaggatg 17160
tagccacggc aaaatgaaat aaaaaaattt aaaaattgtt gcaaatacaa gttatattag
17220 gcttttgtgc attttcaata atgtgctgct atgaactcag aatgatagta
tttaaatata 17280 gaaactagtt aaaggaaacg tagtttctat ttgagttata
catatctgta aattagaact 17340 tctcctgtta aaggcataat aaagtgctta
atacttttgt ttcctcagca ccctctcatt 17400 taattatata attttagttc
tgaaagggac ctataccaga tgcctagagg aaatttcaaa 17460 actatgatct
aatgaaaaaa tatttaatag ttctccatgc aaatacaaat catatagttt 17520
tccagaaaat acctttgaca ttatacaaag atgattatca cagcattata atagtaaaaa
17580 aatggaaata gcctctttct tctgttctgt tcatagcaca gtgcctcata
cgcagtaggt 17640 tattattaca tggtaactgg ctaccccaac tgattaggaa
agaagtaaat ttgttttata 17700 aaaatacata ctcattgagg tgcatagaat
aattaagaaa ttaaaagaca cttgtaattt 17760 tgaatccagt gaatacccac
tgttaatatt tggtatatct ctttctagtc tttttttccc 17820 ttttgcatgt
attttcttta agactcccac ccccactgga tcatctctgc atgttctaat 17880
ctgctttttt cacagcagat tctaagcctc tttgaatatc aacacaaact tcaacaactt
17940 catctataga tgccaaataa taaattcatt tttatttact taaccacttc
ctttggatgc 18000 ttaggtcatt ctgatgtttt gctattgaaa ccaatgctat
actgaacact tctgtcacta 18060 aaactttgca cacactcatg aatagcttct
taggataaat ttttagagat ggatttgcta 18120 aatcagagac cattttttaa
aattaaaaaa caattattca tatcgtttgg catgtaagac 18180 agtaaatttt
ccttttattt tgacaggatt caactggaag ctttgtgctg cctttccggc 18240
aagtcatgta tgctccatat cccaccacac acatagatgt ggatgtcaat actgtgaagc
18300 agatgccacc ctgtcatgaa catatttata atcagcgtag atacatgaga
tccgagctga 18360 cagccttctg gagagccact tcagaagaag acatggctca
ggatacgatc atctacactg 18420 acgaaagctt tactcctgat ttgtacgtaa
tgctctgcct gctggtactg tagtcaagca 18480 atatgaaatt gtgtctttta
cgaataaaaa caaaacagaa gttgcattta aaaagaaaga 18540 aatattacca
gcagaattat gcttgaagaa acatttaatc aagcattttt ttcttaaatg 18600
ttcttctttt tccatacaat tgtgtttacc ctaaaatagg taagattaac ccttaaagta
18660 aatatttaac tatttgttta ataaatatat attgagctcc taggcactgt
tctaggtacc 18720 gggcttaata gtggccaacc agacagcccc agccccagcc
cctacattgt gtatagtcta 18780 ttatgtaaca gttattgaat ggacttatta
acaaaaccaa agaagtaatt ctaagtcttt 18840 tttttcttga catatgaata
taaaatacag caaaactgtt aaaatatatt aatggaacat 18900 ttttttactt
tgcattttat attgttattc acttcttatt tttttttaaa aaaaaaagcc 18960
tgaacagtaa attcaaaagg aaaagtaatg ataattaatt gttgagcatg gacccaactt
19020 gaaaaaaaaa atgatgatga taaatctata atcctaaaac cctaagtaaa
cacttaaaag 19080 atgttctgaa atcaggaaaa gaattatagt atacttttgt
gtttctcttt tatcagttga 19140 aaaaaggcac agtagctcat gcctgtaaga
acagagcttt gggagtgcaa ggcaggcgga 19200 tcacttgagg ccaggagttc
cagaccagcc tgggcaacat agtgaaaccc catctctaca 19260 aaaaataaaa
aagaattatt ggaatgtgtt tctgtgtgcc tgtaatccta gctattccga 19320
aagctgaggc aggaggatct tttgagccca ggagtttgag gttacaggga gttatgatgt
19380 gccagtgtac tccagcctgg ggaacaccga gactctgtct tatttaaaaa
aaaaaaaaaa 19440 aaaatgcttg caataatgcc tggcacatag aaggtaacag
taagtgttaa ctgtaataac 19500 ccaggtctaa gtgtgtaagg caatagaaaa
attggggcaa ataagcctga cctatgtatc 19560 tacagaatca gtttgagctt
aggtaacaga cctgtggagc accagtaatt acacagtaag 19620 tgttaaccaa
aagcatagaa taggaatatc ttgttcaagg gacccccagc cttatacatc 19680
tcaaggtgca gaaagatgac ttaatatagg acccattttt tcctagttct ccagagtttt
19740 tattggttct tgagaaagta gtaggggaat gttttagaaa atgaattggt
ccaactgaaa 19800 ttacatgtca gtaagttttt atatattggt aaattttagt
agacatgtag aagttttcta 19860 attaatctgt gccttgaaac attttctttt
ttcctaaagt gcttagtatt ttttccgttt 19920 tttgattggt tacttgggag
cttttttgag gaaatttagt gaactgcaga atgggtttgc 19980 aaccatttgg
tatttttgtt ttgtttttta gaggatgtat gtgtatttta acatttctta 20040
atcattttta gccagctatg tttgttttgc tgatttgaca aactacagtt agacagctat
20100 tctcattttg ctgatcatga caaaataata tcctgaattt ttaaattttg
catccagctc 20160 taaattttct aaacataaaa ttgtccaaaa aatagtattt
tcagccacta gattgtgtgt 20220 taagtctatt gtcacagagt cattttactt
ttaagtatat gtttttacat gttaattatg 20280 tttgttattt ttaattttaa
ctttttaaaa taattccagt cactgccaat acatgaaaaa 20340 ttggtcactg
gaattttttt tttgactttt attttaggtt catgtgtaca tgtgcaggtg 20400
tgttatacag gtaaattgcg tgtcatgagg gtttggtgta caggtgattt cattacccag
20460 gtaataagca tagtacccaa taggtagttt tttgatcctc acccttctcc
caccctcaag 20520 taggccctgg tgttgctgtt tccttctttg tgtccatgta
tactcagtgt ttagctccca 20580 cttagaagtg agaacatgcg gtagttggtt
ttctgttcct ggattagttc acttaggata 20640 atgacctcta gctccatctg
gtttttatgg ctgcatagta ttccatggtg tatatgtatc 20700 acattttctt
tatccagtct accattgata ggcatttagg ttgattccct gtctttgtta 20760
tcatgaatag tgctgtgatg aacatacaca tgcatgtgtc tttatggtag aaaaatttgt
20820 attcctttag gtacatatag aataatgggg ttgctagggt gaatggtagt
tctattttca 20880 gttatttgag aaatcttcaa actgcttttc ataatagcta
aactaattta cagtcccgcc 20940 agcagtgtat aagtgttccc ttttctccac
aaccttgcca acatctgtga ttttttgact 21000 ttttaataat agccattcct
agagaattga tttgcaattc tctattagtg atattaagca 21060 ttttttcata
tgctttttag ctgtctgtat atattcttct gaaaaatttt catgtccttt 21120
gcccagtttg tagtggggtg ggttgttttt tgcttgttaa ttagttttaa gttccttcca
21180 gattctgcat atccctttgt tggatacatg gtttgcagat atttttctcc
cattgtgtag 21240 gttgtctttt actctgttga tagtttcttt tgccatgcag
gagctcgtta ggtcccattt 21300 gtgtttgttt ttgttgcagt tgcttttggc
gtcttcatca taaaatctgt gccagggcct 21360 atgtccagaa tggtatttcc
taggttgtct tccagggttt ttacaatttt agattttacg 21420 tttatgtctt
taatccatct tgagttgatt tttgtatatg gcacaaggaa ggggtccagt 21480
ttcactccaa ttcctatggc tagcaattat cccagcacca tttattgaat acggagtcct
21540 ttccccattg cttgtttttt gtcaactttg ttgaagatca gatggttgta
agtgtgtggc 21600 tttatttctt ggctctctat tctccattgg tctatgtgtc
tgtttttata acagtaccct 21660 gctgttcagg ttcctatagc cttttagtat
aaaatcggct aatgtgatgc ctccagcttt 21720 gttctttttg cttaggattg
ctttggctat ttgggctcct ttttgggtcc atattaattt 21780 taaaacagtt
ttttctggtt ttgtgaagga tatcattggt agtttatagg aatagcattg 21840
aatctgtaga ttgctttggg cagtatggcc attttaacaa tattaattct tcctatctat
21900 gaatatggaa tgtttttcca tgtgtttgtg tcatctcttt atacctgatg
tataaagaaa 21960 agctggtatt attcctactc aatctgttcc aaaaaattga
ggaggaggaa ctcttcccta 22020 atgaggccag catcattctg ataccaaaac
ctggcagaga cacaacagaa aaaagaaaac 22080 ttcaggccaa tatccttgat
gaatatagat gcaaaaatcc tcaacaaaat actagcaaac 22140 caaatccagc
agcacatcaa aaagctgatc tactttgatc aagtaggctt tatccctggg 22200
atgcaaggtt ggttcaacat acacaaatca ataagtgtga ttcatcacat aaacagagct
22260 aaaaacaaaa accacaagat tatctcaata ggtagagaaa aggttgtcaa
taaaatttaa 22320 catcctccat gttaaaaacc ttcagtaggt caggtgtagt
gactcacacc tgtaatccca 22380 gcactttggg aggccaaggc gggcatatct
cttaagccca ggagttcaag acgagcctag 22440 gcagcatggt gaaaccccat
ctctacaaaa aaaaaaaaaa aaaaaaatta gcttggtatg 22500 gtgacatgca
cctatagtcc cagctattca ggaggttgag gtgggaggat tgtttgagcc 22560
cgggaggcag aggttggcag cgagctgaga tcatgccacc gcactccagc ctgggcaacg
22620 gagtgagacc ctgtctcaaa aaagaaaaat cacaaacaat cctaaacaaa
ctaggcattg 22680 aaggaacatg cctcaaaaaa ataagaacca tctatgacag
acccatagcc aatatcttac 22740 caaatgggca aaagctggaa gtattctcct
tgagaaccgt aacaagacaa ggatgtccac 22800 tctcaccact ccttttcagc
atagttctgg aagtcctagc cagagcaatc aggaaagaga 22860 aagaaagaaa
gacattcaga taggaagaga agaagtcaaa ctatttctgt ttgcaggcag 22920
tataattctg tacctagaaa atctcatagt ctctgcccag aaactcctaa atctgttaaa
22980 aatttcagca aagttttggc attctctata ctccaacacc ttccaaagtg
agagcaaaat 23040 caagaacaca gtcccattca caatagccgc aaaacgaata
aaatacctag gaatccagct 23100 aaccagggag gtgaaagatc tctatgagaa
ttacaaaaca ctgctgaaag aaatcagaga 23160 tgacacaaac aaatggaaat
gttctttttt aacaccttgc tttatctaat tcacttatga 23220 tgaagatact
cattcagtgg aacaggtata ataagtccac tcgattaaat ataagcctta 23280
ttctctttcc agagcccaag aaggggcact atcagtgccc agtcaataat gacgaaatgc
23340 taatattttt cccctttacg gtttctttct tctgtagtgt ggtacactcg
tttcttaaga 23400 taaggaaact tgaactacct tcctgtttgc ttctacacat
acccattctc tttttttgcc 23460 actctggtca ggtataggat gatccctacc
actttcagtt aaaaactcct cctcttacta 23520 aatgttctct taccctctgg
cctgagtaga acctagggaa aatggaagag aaaaagatga 23580 aagggaggtg
gggcctggga agggaataag tagtcctgtt tgtttgtgtg tttgctttag 23640
cacctgctat atcctaggtg ctgtgttagg cacacattat tttaagtggc cattatatta
23700 ctactactca ctctggtcgt tgccaaggta ggtagtactt tcttggatag
ttggttcatg 23760 ttacttacag atggtgggct tgttgaggca aacccagtgg
ataatcatcg gagtgtgttc 23820 tctaatctca ctcaaatttt tcttcacatt
ttttggtttg ttttggtttt tgatggtagt 23880 ggcttatttt tgttgctggt
ttgttttttg tttttttttg agatggcaag aattggtagt 23940 tttatttatt
aattgcctaa gggtctctac tttttttaaa agatgagagt agtaaaatag 24000
attgatagat acatacatac ccttactggg gactgcttat attctttaga gaaaaaatta
24060 catattagcc tgacaaacac cagtaaaatg taaatatatc cttgagtaaa
taaatgaatg 24120 tatattttgt gtctccaaat atatatatct atattcttac
aaatgtgttt atatgtaata 24180 tcaatttata agaacttaaa atgttggctc
aagtgaggga ttgtggaagg tagcattata 24240 tggccatttc aacatttgaa
cttttttctt ttcttcattt tcttcttttc ttcaggaata 24300 tttttcaaga
tgtcttacac agagacactc tagtgaaagc cttcctggat caggtaaatg 24360
ttgaacttga gattgtcaga gtgaatgata tgacatgttt tcttttttaa tatatcctac
24420 aatgcctgtt ctatatattt atattcccct ggatcatgcc ccagagttct
gctcagcaat 24480 tgcagttaag ttagttacac tacagttctc agaagagtct
gtgagggcat gtcaagtgca 24540 tcattacatt ggttgcctct tgtcctagat
ttatgcttcg ggaattcaga cctttgttta 24600 caatataata aatattattg
ctatctttta aagatataat aataagatat aaagttgacc 24660 acaactactg
ttttttgaaa catagaattc ctggtttaca tgtatcaaag tgaaatctga 24720
cttagctttt acagatataa tatatacata tatatatcct gcaatgcttg tactatatat
24780 gtagtacaag tatatatata tgtttgtgtg tgtatatata tatagtacga
gcatatatac 24840 atattaccag cattgtagga tatatatatg tttatatatt
aaaaaaaagt tataaactta 24900 aaaccctatt atgttatgta gagtatatgt
tatatatgat atgtaaaata tataacatat 24960 actctatgat agagtgtaat
atatttttta tatatatttt aacatttata aaatgataga 25020 attaagaatt
gagtcctaat ctgttttatt aggtgctttt tgtagtgtct ggtctttcta 25080
aagtgtctaa atgatttttc cttttgactt attaatgggg aagagcctgt atattaacaa
25140 ttaagagtgc agcattccat acgtcaaaca acaaacattt taattcaagc
attaacctat 25200 aacaagtaag tttttttttt ttttttgaga aagggaggtt
gtttatttgc ctgaaatgac 25260 tcaaaaatat ttttgaaaca tagtgtactt
atttaaataa catctttatt gtttcattct 25320 tttaaaaaat atctacttaa
ttacacagtt gaaggaaatc gtagattata tggaacttat 25380 ttcttaatat
attacagttt gttataataa cattctgggg atcaggccag gaaactgtgt 25440
catagataaa gctttgaaat aatgagatcc ttatgtttac tagaaatttt ggattgagat
25500 ctatgaggtc tgtgacatat tgcgaagttc aaggaaaatt cgtaggcctg
gaatttcatg 25560 cttctcaagc tgacataaaa tccctcccac tctccacctc
atcatatgca cacattctac 25620 tcctacccac ccactccacc ccctgcaaaa
gtacaggtat atgaatgtct caaaaccata 25680 ggctcatctt ctaggagctt
caatgttatt tgaagatttg ggcagaaaaa attaagtaat 25740 acgaaataac
ttatgtatga gttttaaaag tgaagtaaac atggatgtat tctgaagtag 25800
aatgcaaaat ttgaatgcat ttttaaagat aaattagaaa acttctaaaa actgtcagat
25860 tgtctgggcc tggtggctta tgcctgtaat cccagcactt tgggagtccg
aggtgggtgg 25920 atcacaaggt caggagatcg agaccatcct gccaacatgg
tgaaaccccg tctctactaa 25980 gtatacaaaa attagctggg cgtggcagcg
tgtgcctgta atcccagcta cctgggaggc 26040 tgaggcagga gaatcgcttg
aacccaggag gtgtaggttg cagtgagtca agatcgcgcc 26100 actgcacttt
agcctggtga cagagctaga ctccgtctca aaaaaaaaaa aaaatatcag 26160
attgttccta cacctagtgc ttctatacca cactcctgtt agggggcatc agtggaaatg
26220 gttaaggaga tgtttagtgt gtattgtctg ccaagcactg tcaacactgt
catagaaact 26280 tctgtacgag tagaatgtga gcaaattatg tgttgaaatg
gttcctctcc ctgcaggtct 26340 ttcagctgaa acctggctta tctctcagaa
gtactttcct tgcacagttt ctacttgtcc 26400 ttcacagaaa agccttgaca
ctaataaaat atatagaaga cgatacgtga gtaaaactcc 26460 tacacggaag
aaaaaccttt gtacattgtt tttttgtttt gtttcctttg tacattttct 26520
atatcataat ttttgcgctt cttttttttt tttttttttt tttttttcca ttatttttag
26580 gcagaaggga aaaaagccct ttaaatctct tcggaacctg aagatagacc
ttgatttaac 26640 agcagagggc gatcttaaca taataatggc tctggctgag
aaaattaaac caggcctaca 26700 ctcttttatc tttggaagac ctttctacac
tagtgtgcaa gaacgagatg ttctaatgac 26760 tttttaaatg tgtaacttaa
taagcctatt ccatcacaat catgatcgct ggtaaagtag 26820 ctcagtggtg
tggggaaacg ttcccctgga tcatactcca gaattctgct ctcagcaatt 26880
gcagttaagt aagttacact acagttctca caagagcctg tgaggggatg tcaggtgcat
26940 cattacattg ggtgtctctt ttcctagatt tatgcttttg ggatacagac
ctatgtttac 27000 aatataataa atattattgc tatcttttaa agatataata
ataggatgta aacttgacca 27060 caactactgt ttttttgaaa tacatgattc
atggtttaca tgtgtcaagg tgaaatctga 27120 gttggctttt acagatagtt
gactttctat cttttggcat tctttggtgt gtagaattac 27180 tgtaatactt
ctgcaatcaa ctgaaaacta gagcctttaa atgatttcaa ttccacagaa 27240
agaaagtgag cttgaacata ggatgagctt tagaaagaaa attgatcaag cagatgttta
27300 attggaattg attattagat cctactttgt ggatttagtc cctgggattc
agtctgtaga 27360 aatgtctaat agttctctat agtccttgtt cctggtgaac
cacagttagg gtgttttgtt 27420 tattttattg ttcttgctat tgttgatatt
ctatgtagtt gagctctgta aaaggaaatt 27480 gtattttatg ttttagtaat
tgttgccaac tttttaaatt aattttcatt atttttgagc 27540 caaattgaaa
tgtgcacctc ctgtgccttt tttctcctta gaaaatctaa ttacttggaa 27600
caagttcaga tttcactggt cagtcatttt catcttgttt tcttcttgct aagtcttacc
27660 atgtacctgc tttggcaatc attgcaactc tgagattata aaatgcctta
gagaatatac 27720 taactaataa gatctttttt tcagaaacag aaaatagttc
cttgagtact tccttcttgc 27780 atttctgcct atgtttttga agttgttgct
gtttgcctgc aataggctat aaggaatagc 27840 aggagaaatt ttactgaagt
gctgttttcc taggtgctac tttggcagag ctaagttatc 27900 ttttgttttc
ttaatgcgtt tggaccattt tgctggctat aaaataactg attaatataa 27960
ttctaacaca atgttgacat tgtagttaca caaacacaaa taaatatttt atttaaaatt
28020 ctggaagtaa tataaaaggg aaaatatatt tataagaaag ggataaaggt
aatagagccc 28080 ttctgccccc cacccaccaa atttacacaa caaaatgaca
tgttcgaatg tgaaaggtca 28140 taatagcttt cccatcatga atcagaaaga
tgtggacagc ttgatgtttt agacaaccac 28200 tgaactagat gactgttgta
ctgtagctca gtcatttaaa aaatatataa atactacctt 28260 gtagtgtccc
atactgtgtt ttttacatgg tagattctta tttaagtgct aactggttat 28320
tttctttggc tggtttattg tactgttata cagaatgtaa gttgtacagt gaaataagtt
28380 attaaagcat gtgtaaacat tgttatatat cttttctcct aaatggagaa
ttttgaataa 28440 aatatatttg aaattttgcc tctttcagtt gttcattcag
aaaaaaatac tatgatattt 28500 gaagactgat cagcttctgt tcagctgaca
gtcatgctgg atctaaactt tttttaaaat 28560 taattttgtc ttttcaaaga
aaaaatattt aaagaagctt tataatataa tcttatgtta 28620 aaaaaacttt
ctgcttaact ctctggattt cattttgatt tttcaaatta tatattaata 28680
tttcaaatgt aaaatactat ttagataaat tgtttttaaa cattcttatt attataatat
28740 taatataacc taaactgaag ttattcatcc caggtatcta atacatgtat
ccaaagtaaa 28800 aatccaagga atctgaacac tttcatctgc aaagctagga
ataggtttga cattttcact 28860 ccaagaaaaa gttttttttt gaaaatagaa
tagttgggat gagaggtttc tttaaaagaa 28920 gactaactga tcacattact
atgattctca aagaagaaac caaaacttca tataatacta 28980 taaagtaaat
ataaaatagt tccttctata gtatatttct ataatgctac agtttaaaca 29040
gatcactctt atataatact attttgattt tgatgtagaa ttgcacaaat tgatatttct
29100 cctatgatct gcagggtata gcttaaagta acaaaaacag tcaaccacct
ccatttaaca 29160 cacagtaaca ctatgggact agttttatta cttccatttt
acaaatgagg aaactaaagc 29220 ttaaagatgt gtaatacacc gcccaaggtc
acacagctgg taaaggtgga tttcatccca 29280 gacagttaca gtcattgcca
tgggcacagc tcctaactta gtaactccat gtaactggta 29340 ctcagtgtag
ctgaattgaa aggagagtaa ggaagcaggt tttacaggtc tacttgcact 29400
attcagagcc cgagtgtgaa tccctgctgt gctgcttgga gaagttactt aacctatgca
29460 aggttcattt tgtaaatatt ggaaatggag tgataatacg tacttcacca
gaggatttaa 29520 tgagacctta tacgatcctt agttcagtac ctgactagtg
cttcataaat gctttttcat 29580 ccaatctgac aatctccagc ttgtaattgg
ggcatttaga acatttaata tgattattgg 29640 catggtaggt taaagctgtc
atcttgctgt tttctatttg ttctttttgt tttctcctta 29700 cttttggatt
tttttattct actatgtctt ttctattgtc ttattaacta tactctttga 29760
tttattttag tggttgtttt agggttatac ctctttctaa tttaccagtt tataaccagt
29820 ttatatacta cttgacatat agcttaagaa acttactgtt gttgtctttt
tgctgttatg 29880 gtcttaacgt ttttatttct acaaacatta taaactccac
actttattgt tttttaattt 29940 tacttataca gtcaattatc ttttaaagat
atttaaatat aaacattcaa aacaccccaa 30000 t 30001 <210> SEQ ID
NO 3 <211> LENGTH: 1031 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 3 attcccggga
tacgtaacct acggtgtccc gctaggaaag agaggtgcgt caaacagcga 60
caagttccgc ccacgtaaaa gatgacgctt ggtgtgtcag ccgtccctgc tgcccggttg
120 cttctctttt gggggcgggg tctagcaaga gcaggtgtgg gtttaggaga
tatctccgga 180 gcatttggat aatgtgacag ttggaatgca gtgatgtcga
ctctttgccc accgccatct 240 ccagctgttg ccaagacaga gattgcttta
agtggcaaat cacctttatt agcagctact 300 tttgcttact gggacaatat
tcttggtcct agagtaaggc acatttgggc tccaaagaca 360 gaacaggtac
ttctcagtga tggagaaata acttttcttg ccaaccacac tctaaatgga 420
gaaatccttc gaaatgcaga gagtggtgct atagatgtaa agttttttgt cttgtctgaa
480 aagggagtga ttattgtttc attaatcttt gatggaaact ggaatgggga
tcgcagcaca 540 tatggactat caattatact tccacagaca gaacttagtt
tctacctccc acttcataga 600 gtgtgtgttg atagattaac acatataatc
cggaaaggaa gaatatggat gcataaggaa 660 agacaagaaa aatgtccaga
agattatctt agaaggcaca gagagaatgg aagatcaggg 720 tcagagtatt
attccaatgc ttactggaga agtgattcct gtaatggaaa ctgctttcct 780
ctatgaaatt cccccgggtt cctggaggaa atagatatag gctgatacag ttacccaatg
840 atggatgaat attgggggac cgcctggtca ttgaaaggct ttcttttctc
caggaaagaa 900 atttttttcc ttttccataa aaagcttggg aatggaagac
aacaattccc attctttttt 960 tgcgttccac ccctatgtga caacagaaat
ttttggggaa acaacaacga aaaaatttta 1020 tcccgcgcgc a 1031 <210>
SEQ ID NO 4 <211> LENGTH: 3244 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 4
gggcggggct gcggttgcgg tgcctgcgcc cgcggcggcg gaggcgcagg cggtggcgag
60 tggatatctc cggagcattt ggataatgtg acagttggaa tgcagtgatg
tcgactcttt 120 gcccaccgcc atctccagct gttgccaaga cagagattgc
tttaagtggc aaatcacctt 180 tattagcagc tacttttgct tactgggaca
atattcttgg tcctagagta aggcacattt 240 gggctccaaa gacagaacag
gtacttctca gtgatggaga aataactttt cttgccaacc 300 acactctaaa
tggagaaatc cttcgaaatg cagagagtgg tgctatagat gtaaagtttt 360
ttgtcttgtc tgaaaaggga gtgattattg tttcattaat ctttgatgga aactggaatg
420 gggatcgcag cacatatgga ctatcaatta tacttccaca gacagaactt
agtttctacc 480 tcccacttca tagagtgtgt gttgatagat taacacatat
aatccggaaa ggaagaatat 540 ggatgcataa ggaaagacaa gaaaatgtcc
agaagattat cttagaaggc acagagagaa 600 tggaagatca gggtcagagt
attattccaa tgcttactgg agaagtgatt cctgtaatgg 660 aactgctttc
atctatgaaa tcacacagtg ttcctgaaga aatagatata gctgatacag 720
tactcaatga tgatgatatt ggtgacagct gtcatgaagg ctttcttctc aatgccatca
780 gctcacactt gcaaacctgt ggctgttccg ttgtagtagg tagcagtgca
gagaaagtaa 840 ataagatagt cagaacatta tgcctttttc tgactccagc
agagagaaaa tgctccaggt 900 tatgtgaagc agaatcatca tttaaatatg
agtcagggct ctttgtacaa ggcctgctaa 960 aggattcaac tggaagcttt
gtgctgcctt tccggcaagt catgtatgct ccatatccca 1020 ccacacacat
agatgtggat gtcaatactg tgaagcagat gccaccctgt catgaacata 1080
tttataatca gcgtagatac atgagatccg agctgacagc cttctggaga gccacttcag
1140 aagaagacat ggctcaggat acgatcatct acactgacga aagctttact
cctgatttga 1200 atatttttca agatgtctta cacagagaca ctctagtgaa
agccttcctg gatcaggtct 1260 ttcagctgaa acctggctta tctctcagaa
gtactttcct tgcacagttt ctacttgtcc 1320 ttcacagaaa agccttgaca
ctaataaaat atatagaaga cgatacgcag aagggaaaaa 1380 agccctttaa
atctcttcgg aacctgaaga tagaccttga tttaacagca gagggcgatc 1440
ttaacataat aatggctctg gctgagaaaa ttaaaccagg cctacactct tttatctttg
1500 gaagaccttt ctacactagt gtgcaagaac gagatgttct aatgactttt
taaatgtgta 1560 acttaataag cctattccat cacaatcatg atcgctggta
aagtagctca gtggtgtggg 1620 gaaacgttcc cctggatcat actccagaat
tctgctctca gcaattgcag ttaagtaagt 1680 tacactacag ttctcacaag
agcctgtgag gggatgtcag gtgcatcatt acattgggtg 1740 tctcttttcc
tagatttatg cttttgggat acagacctat gtttacaata taataaatat 1800
tattgctatc ttttaaagat ataataatag gatgtaaact tgaccacaac tactgttttt
1860 ttgaaataca tgattcatgg tttacatgtg tcaaggtgaa atctgagttg
gcttttacag 1920 atagttgact ttctatcttt tggcattctt tggtgtgtag
aattactgta atacttctgc 1980 aatcaactga aaactagagc ctttaaatga
tttcaattcc acagaaagaa agtgagcttg 2040 aacataggat gagctttaga
aagaaaattg atcaagcaga tgtttaattg gaattgatta 2100 ttagatccta
ctttgtggat ttagtccctg ggattcagtc tgtagaaatg tctaatagtt 2160
ctctatagtc cttgttcctg gtgaaccaca gttagggtgt tttgtttatt ttattgttct
2220 tgctattgtt gatattctat gtagttgagc tctgtaaaag gaaattgtat
tttatgtttt 2280 agtaattgtt gccaactttt taaattaatt ttcattattt
ttgagccaaa ttgaaatgtg 2340 cacctcctgt gccttttttc tccttagaaa
atctaattac ttggaacaag ttcagatttc 2400 actggtcagt cattttcatc
ttgttttctt cttgctaagt cttaccatgt acctgctttg 2460 gcaatcattg
caactctgag attataaaat gccttagaga atatactaac taataagatc 2520
tttttttcag aaacagaaaa tagttccttg agtacttcct tcttgcattt ctgcctatgt
2580 ttttgaagtt gttgctgttt gcctgcaata ggctataagg aatagcagga
gaaattttac 2640 tgaagtgctg ttttcctagg tgctactttg gcagagctaa
gttatctttt gttttcttaa 2700 tgcgtttgga ccattttgct ggctataaaa
taactgatta atataattct aacacaatgt 2760 tgacattgta gttacacaaa
cacaaataaa tattttattt aaaattctgg aagtaatata 2820 aaagggaaaa
tatatttata agaaagggat aaaggtaata gagcccttct gccccccacc 2880
caccaaattt acacaacaaa atgacatgtt cgaatgtgaa aggtcataat agctttccca
2940 tcatgaatca gaaagatgtg gacagcttga tgttttagac aaccactgaa
ctagatgact 3000 gttgtactgt agctcagtca tttaaaaaat atataaatac
taccttgtag tgtcccatac 3060 tgtgtttttt acatggtaga ttcttattta
agtgctaact ggttattttc tttggctggt 3120 ttattgtact gttatacaga
atgtaagttg tacagtgaaa taagttatta aagcatgtgt 3180 aaacattgtt
atatatcttt tctcctaaat ggagaatttt gaataaaata tatttgaaat 3240 tttg
3244 <210> SEQ ID NO 5 <211> LENGTH: 761 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(693)..(693) <223> OTHER INFORMATION: n is a, c, g, or t
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (722)..(722) <223> OTHER INFORMATION: n is a, c, g,
or t <400> SEQUENCE: 5 cacgaggctt tgatatttct tacaacgaat
ttcatgtgta gacccactaa acagaagcta 60 taaaagttgc atggtcaaat
aagtctgaga aagtctgcag atgatataat tcacctgaag 120 agtcacagta
tgtagccaaa tgttaaaggt tttgagatgc catacagtaa atttaccaag 180
cattttctaa atttatttga ccacagaatc cctattttaa gcaacaactg ttacatccca
240 tggattccag gtgactaaag aatacttatt tcttaggata tgttttattg
ataataacaa 300 ttaaaatttc agatatcttt cataagcaaa tcagtggtct
ttttacttca tgttttaatg 360 ctaaaatatt ttcttttata gatagtcaga
acattatgcc tttttctgac tccagcagag 420 agaaaatgct ccaggttatg
tgaagcagaa tcatcattta aatatgagtc agggctcttt 480 gtacaaggcc
tgctaaagga ttcaactgga agctttgtgc tgcctttccg gcaagtcatg 540
tatgctccat atcccaccac acacatagat gtggatgtca atactgtgaa gcagatgcca
600 ccctgtcatg aacatattta taatcagcgt agatacatga gatccgagct
gacagccttc 660 tggagagcca cttcagaaga agacatggct cangatacga
tcatctacac tgacgaaagc 720 tntactcctg atttgaatat ttttcaagat
gtcttacaca g 761 <210> SEQ ID NO 6 <211> LENGTH: 1901
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 6 acgtaaccta cggtgtcccg ctaggaaaga gaggtgcgtc
aaacagcgac aagttccgcc 60 cacgtaaaag atgacgcttg atatctccgg
agcatttgga taatgtgaca gttggaatgc 120 agtgatgtcg actctttgcc
caccgccatc tccagctgtt gccaagacag agattgcttt 180 aagtggcaaa
tcacctttat tagcagctac ttttgcttac tgggacaata ttcttggtcc 240
tagagtaagg cacatttggg ctccaaagac agaacaggta cttctcagtg atggagaaat
300 aacttttctt gccaaccaca ctctaaatgg agaaatcctt cgaaatgcag
agagtggtgc 360 tatagatgta aagttttttg tcttgtctga aaagggagtg
attattgttt cattaatctt 420 tgatggaaac tggaatgggg atcgcagcac
atatggacta tcaattatac ttccacagac 480 agaacttagt ttctacctcc
cacttcatag agtgtgtgtt gatagattaa cacatataat 540 ccggaaagga
agaatatgga tgcataagga aagacaagaa aatgtccaga agattatctt 600
agaaggcaca gagagaatgg aagatcaggg tcagagtatt attccaatgc ttactggaga
660 agtgattcct gtaatggaac tgctttcatc tatgaaatca cacagtgttc
ctgaagaaat 720 agatatagct gatacagtac tcaatgatga tgatattggt
gacagctgtc atgaaggctt 780 tcttctcaag taagaatttt tcttttcata
aaagctggat gaagcagata ccatcttatg 840 ctcacctatg acaagatttg
gaagaaagaa aataacagac tgtctactta gattgttcta 900 gggacattac
gtatttgaac tgttgcttaa atttgtgtta tttttcactc attatatttc 960
tatatatatt tggtgttatt ccatttgcta tttaaagaaa ccgagtttcc atcccagaca
1020 agaaatcatg gccccttgct tgattctggt ttcttgtttt acttctcatt
aaagctaaca 1080 gaatcctttc atattaagtt gtactgtaga tgaacttaag
ttatttaggc gtagaacaaa 1140 attattcata tttatactga tctttttcca
tccagcagtg gagtttagta cttaagagtt 1200 tgtgccctta aaccagactc
cctggattaa tgctgtgtac ccgtgggcaa ggtgcctgaa 1260 ttctctatac
acctatttcc tcatctgtaa aatggcaata atagtaatag tacctaatgt 1320
gtagggttgt tataagcatt gagtaagata aataatataa agcacttaga acagtgcctg
1380 gaacataaaa acacttaata atagctcata gctaacattt cctatttaca
tttcttctag 1440 aaatagccag tatttgttga gtgcctacat gttagttcct
ttactagttg ctttacatgt 1500 attatcttat attctgtttt aaagtttctt
cacagttaca gattttcatg aaattttact 1560 tttaataaaa gagaagtaaa
agtataaagt attcactttt atgttcacag tcttttcctt 1620 taggctcatg
atggagtatc agaggcatga gtgtgtttaa cctaagagcc ttaatggctt 1680
gaatcagaag cactttagtc ctgtatctgt tcagtgtcag cctttcatac atcattttaa
1740 atcccatttg actttaagta agtcacttaa tctctctaca tgtcaatttc
ttcagctata 1800 aaatgatggt atttcaataa ataaatacat taattaaatg
atattatact gactaattgg 1860 gctgttttaa ggcaaaaaaa aaaaaaaaaa
aaaaaaaaaa a 1901 <210> SEQ ID NO 7 <211> LENGTH: 562
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (166)..(166) <223> OTHER INFORMATION: n is a, c, g,
or t <400> SEQUENCE: 7 agacgtaacc tacggtgtcc cgctaggaaa
gagagatatc tccggagcat ttggataatg 60 tgacagttgg aatgcagtga
tgtcgactct ttgcccaccg ccatctccag ctgttgccaa 120 gacagagatt
gctttaagtg gcaaatcacc tttattagca gctacntttt gcttactggg 180
acaatattct tggtcctaga gtaaggcaca tttgggctcc aaagacagaa caggtacttc
240 tcagtgatgg agaaataact tttcttgcca accacactct aaatggagaa
atccttcgaa 300 atgcagagag tggtgctata gatgtaaagt tttttgtctt
gtctgaaaag ggagtgatta 360 ttgtttcatt aatctttgat ggaaactgga
atggggatcg cagcacatat ggactatcaa 420 ttatacttcc acagacagaa
cttagtttct acctcccact tcatagagtg tgtgttgata 480 gattaacaca
tataatccgg aaaggaagaa tatggatgca taaggaaaga caagaaaatg 540
tccagaagat tatcttagaa gg 562 <210> SEQ ID NO 8 <211>
LENGTH: 798 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 8 gggctctctt ttgggggcgg ggtctagcaa
gagcagatat ctccggagca tttggataat 60 gtgacagttg gaatgcagtg
atgtcgactc tttgcccacc gccatctcca gctgttgcca 120 agacagagat
tgctttaagt ggcaaatcac ctttattagc agctactttt gcttactggg 180
acaatattct tggtcctaga gtaaggcaca tttgggctcc aaagacagaa caggtacttc
240 tcagtgatgg agaaataact tttcttgcca accacactct aaatggagaa
atccttcgaa 300 atgcagagag tggtgctata gatgtaaagt tttttgtctt
gtctgaaaag ggagtgatta 360 ttgtttcatt aatctttgat ggaaactgga
atggggatcg cagcacatat ggactatcaa 420 ttatacttcc acagacagaa
cttagtttct acctcccact tcatagagtg tgtgttgata 480 gattaacaca
tataatccgg aaaggaagaa tatggatgca taaggaaaga caagaaaatg 540
tccagaagat tatcttagaa ggcacagaga gaatggaaga tcagggtcag agtattattc
600 caatgcttac tggagaagtg attcctgtaa tgggactgct ttcatctatg
aaatcacaca 660 gtgttcctga agaaatagat atagctgata cagtactcca
tgatgatgat atttggtgac 720 agctgtcatg aaaggctttc ttctcaagta
ggaatttttt cttttcataa aagctgggat 780 gaagccagat tcccatct 798
<210> SEQ ID NO 9 <211> LENGTH: 169 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 9
aaacagcgac aagttccgcc cacgtaaaag atgatgcttg gtgtgtcagc cgtccctgct
60 gcccggttgc ttctcttttg ggggcggggt ctagcaagag cagatatctc
cggagcattt 120 ggataatgtg acagttggaa tgcggtgatg tcgactcttt
gcccaccgc 169 <210> SEQ ID NO 10 <211> LENGTH: 176
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 10 aaaacgtcat cgcacataga aaacagacag
acgtaaccta cggtgtcccg ctaggaaaga 60 gaggtgcgtc aaacagcgac
aagttccgcc cacgtaaaag atgacgcttg atatctccgg 120 agcatttgga
taatgtgaca gttggaatgc agtgatgtcg actctttgcc caccgc 176 <210>
SEQ ID NO 11 <400> SEQUENCE: 11 000 <210> SEQ ID NO 12
<400> SEQUENCE: 12 000 <210> SEQ ID NO 13 <211>
LENGTH: 783 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 13 gcctctcagt acccgaggct cccttttctc
gagcccgcag cggcagcgct cccagcgggt 60 ccccgggaag gagacagctc
gggtactgag ggcgggaaag caaggaagag gccagatccc 120 catcccttgt
ccctgcgccg ccgccgccgc cgccgccgcc gggaagcccg gggcccggat 180
gcaggcaatt ccaccagtcg ctagaggcga aagcccgaca cccagcttcg gtcagagaaa
240 tgagagggaa agtaaaaatg cgtcgagctc tgaggagagc ccccgcttct
acccgcgcct 300 cttcccggca gccgaacccc aaacagccac ccgccaggat
gccgcctcct cactcaccca 360 ctcgccaccg cctgcgcctc cgccgccgcg
ggcgcaggca ccgcaaccgc agccccgccc 420 cgggcccgcc cccgggcccg
ccccgaccac gccccggccc cggccccggc cccggccccg 480 gcccctagcg
cgcgactcct gagttccaga gcttgctaca ggctgcggtt gtttccctcc 540
ttgttttctt ctggttaatc tttatcaggt cttttcttgt tcaccctcag cgagtactgt
600 gagagcaagt agtggggaga gagggtggga aaaacaaaaa cacacacctc
ctaaacccac 660 acctgctctt gctagacccc gcccccaaaa gagaagcaac
cgggcagcag ggacggctga 720 cacaccaagc gtcatctttt acgtgggcgg
aacttgtcgc tgtttgacgc acctctcttt 780 cct 783 <210> SEQ ID NO
14 <211> LENGTH: 45 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 14 cgactggagc
acgaggacac tgaaaagatg acgcttggtg tgtca 45 <210> SEQ ID NO 15
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer <400> SEQUENCE: 15 cccacacctg ctcttgctag
a 21 <210> SEQ ID NO 16 <211> LENGTH: 22 <212>
TYPE: DNA <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Primer <400>
SEQUENCE: 16 cgactggagc acgaggacac tg 22 <210> SEQ ID NO 17
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Probe <400> SEQUENCE: 17 cccaaaagag aagcaaccgg
gca 23 <210> SEQ ID NO 18 <211> LENGTH: 38 <212>
TYPE: DNA <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Primer <400>
SEQUENCE: 18 cgactggagc acgaggacac tgacggctgc cgggaaga 38
<210> SEQ ID NO 19 <211> LENGTH: 26 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 19
agaaatgaga gggaaagtaa aaatgc 26 <210> SEQ ID NO 20
<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer <400> SEQUENCE: 20 cgactggagc acgaggacac
tg 22 <210> SEQ ID NO 21 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Probe <400> SEQUENCE:
21 aggagagccc ccgcttctac ccg 23 <210> SEQ ID NO 22
<211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer <400> SEQUENCE: 22 cgactggagc acgaggacac
tgacgctgag ggtgaacaag aa 42 <210> SEQ ID NO 23 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 23 gagttccaga gcttgctaca g 21 <210> SEQ
ID NO 24 <211> LENGTH: 22 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 24 cgactggagc
acgaggacac tg 22 <210> SEQ ID NO 25 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 25 ctgcggttgt ttccctcctt gttt 24 <210>
SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 26
ggcaaattca acggcacagt 20 <210> SEQ ID NO 27 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 27 gggtctcgct cctggaagat 20 <210> SEQ
ID NO 28 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Probe <400> SEQUENCE: 28 aaggccgaga
atgggaagct tgtcatc 27 <210> SEQ ID NO 29 <211> LENGTH:
20 <212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 29 tagtgcggac ctacccacga 20
<210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 30 ggtgtgtcag ccgtccctgc 20 <210> SEQ
ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
31 gtgtcagccg tccctgctgc 20 <210> SEQ ID NO 32 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 32 tgtttttccc
accctctctc 20 <210> SEQ ID NO 33 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 33 ttttcccacc ctctctcccc 20
<210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 34 tcccaccctc tctccccact 20 <210> SEQ
ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
35 caccctctct ccccactact 20 <210> SEQ ID NO 36 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 36 gagggtgaac
aagaaaagac 20 <210> SEQ ID NO 37 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 37 gaaaagacct gataaagatt 20
<210> SEQ ID NO 38 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 38 agattaacca gaagaaaaca 20 <210> SEQ
ID NO 39 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
39 ttaaccagaa gaaaacaagg 20 <210> SEQ ID NO 40 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 40 accagaagaa
aacaaggagg 20 <210> SEQ ID NO 41 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 41 agaagaaaac aaggagggaa 20
<210> SEQ ID NO 42 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 42 agaaaacaag gagggaaaca 20 <210> SEQ
ID NO 43 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
43 aaacaaggag ggaaacaacc 20 <210> SEQ ID NO 44 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 44 gcaagctctg
gaactcagga 20 <210> SEQ ID NO 45 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 45 agctctggaa ctcaggagtc 20
<210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 46 tcaggagtcg cgcgctaggg 20 <210> SEQ
ID NO 47 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
47 ggagtcgcgc gctaggggcc 20 <210> SEQ ID NO 48 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 48 gtcgcgcgct
aggggccggg 20 <210> SEQ ID NO 49 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 49 gcgcgctagg ggccggggcc 20
<210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 50 gggctgcggt tgcggtgcct 20 <210> SEQ
ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
51 gcggttgcgg tgcctgcgcc 20 <210> SEQ ID NO 52 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 52 tgcggtgcct
gcgcccgcgg 20 <210> SEQ ID NO 53 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 53 tgcctgcgcc cgcggcggcg 20
<210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 54 gcgcccgcgg cggcggaggc 20 <210> SEQ
ID NO 55 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
55 cgcggcggcg gaggcgcagg 20 <210> SEQ ID NO 56 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 56 cggcggaggc
gcaggcggtg 20 <210> SEQ ID NO 57 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 57 gaggcgcagg cggtggcgag 20
<210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 58 gcaggcggtg gcgagtgggt 20 <210> SEQ
ID NO 59 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
59 cggtggcgag tgggtgagtg 20 <210> SEQ ID NO 60 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 60 gcgagtgggt
gagtgaggag 20 <210> SEQ ID NO 61 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 61 tgggtgagtg aggaggcggc 20
<210> SEQ ID NO 62 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 62 gagtgaggag gcggcatcct 20 <210> SEQ
ID NO 63 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
63 aggaggcggc atcctggcgg 20 <210> SEQ ID NO 64 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 64 gcggcatcct
ggcgggtggc 20 <210> SEQ ID NO 65 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 65 atcctggcgg gtggctgttt 20
<210> SEQ ID NO 66 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 66 tcggctgccg ggaagaggcg 20 <210> SEQ
ID NO 67 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
67 tgccgggaag aggcgcgggt 20 <210> SEQ ID NO 68 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 68 ggaagaggcg
cgggtagaag 20 <210> SEQ ID NO 69 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 69 gctctcctca gagctcgacg 20
<210> SEQ ID NO 70 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 70 cctcagagct cgacgcattt 20 <210> SEQ
ID NO 71 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
71 gagctcgacg catttttact 20 <210> SEQ ID NO 72 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 72 cgacgcattt
ttactttccc 20 <210> SEQ ID NO 73 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 73 catttttact ttccctctca 20
<210> SEQ ID NO 74 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 74 ttactttccc tctcatttct 20 <210> SEQ
ID NO 75 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
75 ttccctctca tttctctgac 20 <210> SEQ ID NO 76 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 76 tctcatttct
ctgaccgaag 20 <210> SEQ ID NO 77 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 77 tttctctgac cgaagctggg 20
<210> SEQ ID NO 78 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 78 ctgaccgaag ctgggtgtcg 20 <210> SEQ
ID NO 79 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
79 cgaagctggg tgtcgggctt 20 <210> SEQ ID NO 80 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 80 ctgggtgtcg
ggctttcgcc 20 <210> SEQ ID NO 81 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 81 tgtcgggctt tcgcctctag 20
<210> SEQ ID NO 82 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 82 ggctttcgcc tctagcgact 20 <210> SEQ
ID NO 83 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION: (6)..(6)
<223> OTHER INFORMATION: I <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (10)..(10)
<223> OTHER INFORMATION: I <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (15)..(15)
<223> OTHER INFORMATION: I <400> SEQUENCE: 83
ccggggccgg ggccggggcc 20 <210> SEQ ID NO 84 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (6)..(6) <223>
OTHER INFORMATION: I <220> FEATURE: <221> NAME/KEY:
modified_base <222> LOCATION: (13)..(13) <223> OTHER
INFORMATION: I <400> SEQUENCE: 84 ggccggggcc ggggccgg 18
<210> SEQ ID NO 85 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 85 ggggccgggg ccggggcc 18 <210> SEQ ID
NO 86 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
86 cggggccggg gccggggc 18 <210> SEQ ID NO 87 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 87 ccggggccgg
ggccgggg 18 <210> SEQ ID NO 88 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 88 gccggggccg gggccggg 18
<210> SEQ ID NO 89 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 89 ggccggggcc ggggccgg 18 <210> SEQ ID
NO 90 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
90 gggccggggc cggggccg 18 <210> SEQ ID NO 91 <211>
LENGTH: 16 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 91 ggccggggcc
ggggcc 16 <210> SEQ ID NO 92 <211> LENGTH: 16
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 92 gggccggggc cggggc 16
<210> SEQ ID NO 93 <211> LENGTH: 16 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 93 ggggccgggg ccgggg 16 <210> SEQ ID NO
94 <211> LENGTH: 16 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
94 cggggccggg gccggg 16 <210> SEQ ID NO 95 <211>
LENGTH: 16 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 95 ccggggccgg
ggccgg 16 <210> SEQ ID NO 96 <211> LENGTH: 16
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 96 gccggggccg gggccg 16
<210> SEQ ID NO 97 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 97 gacaagggta cgtaatctgt c 21 <210> SEQ
ID NO 98 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION: (4)..(4)
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (10)..(10) <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (16)..(16)
<400> SEQUENCE: 98 ccggggccgg ggccggggcc 20 <210> SEQ
ID NO 99 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION: (6)..(6)
<223> OTHER INFORMATION: I <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (12)..(12)
<223> OTHER INFORMATION: I <400> SEQUENCE: 99
ggccggggcc ggggccgg 18 <210> SEQ ID NO 100 <211>
LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 100 cagcagcagc
agcagcagc 19 <210> SEQ ID NO 101 <211> LENGTH: 99
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 101 gacaagggta cgtaatctgt
ctagagctag aaatagcaag ttaaaataag gctagtccgt 60 tatcaacttg
aaaaagtggc accgagtcgg tgctttttt 99 <210> SEQ ID NO 102
<211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer <400> SEQUENCE: 102 tagtcctgca ggtttaaacg
aattcgtgag tgaggaggcg gca 43 <210> SEQ ID NO 103 <211>
LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 103 agcagagctc agattacgta cccttgttgt gaacaac
37 <210> SEQ ID NO 104 <211> LENGTH: 33 <212>
TYPE: DNA <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Primer <400>
SEQUENCE: 104 caatgtcaac gtctggcatt acttctactt ttg 33 <210>
SEQ ID NO 105 <211> LENGTH: 43 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 105
tagggcgaat tgaatttagc ggccgcactg gcaggatcat agc 43 <210> SEQ
ID NO 106 <211> LENGTH: 30 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 106 tacgtaatct
gagctctgct tatatagacc 30 <210> SEQ ID NO 107 <211>
LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 107 aatgccagac gttgacattg attattgact
agttattaat ag 42 <210> SEQ ID NO 108 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 108 gttaggctct gggagagtag ttg 23 <210>
SEQ ID NO 109 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 109
cctggagcag gtaaatgctg g 21
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 109
<210> SEQ ID NO 1 <211> LENGTH: 3339 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1
acgtaaccta cggtgtcccg ctaggaaaga gaggtgcgtc aaacagcgac aagttccgcc
60 cacgtaaaag atgacgcttg gtgtgtcagc cgtccctgct gcccggttgc
ttctcttttg 120 ggggcggggt ctagcaagag caggtgtggg tttaggagat
atctccggag catttggata 180 atgtgacagt tggaatgcag tgatgtcgac
tctttgccca ccgccatctc cagctgttgc 240 caagacagag attgctttaa
gtggcaaatc acctttatta gcagctactt ttgcttactg 300 ggacaatatt
cttggtccta gagtaaggca catttgggct ccaaagacag aacaggtact 360
tctcagtgat ggagaaataa cttttcttgc caaccacact ctaaatggag aaatccttcg
420 aaatgcagag agtggtgcta tagatgtaaa gttttttgtc ttgtctgaaa
agggagtgat 480 tattgtttca ttaatctttg atggaaactg gaatggggat
cgcagcacat atggactatc 540 aattatactt ccacagacag aacttagttt
ctacctccca cttcatagag tgtgtgttga 600 tagattaaca catataatcc
ggaaaggaag aatatggatg cataaggaaa gacaagaaaa 660 tgtccagaag
attatcttag aaggcacaga gagaatggaa gatcagggtc agagtattat 720
tccaatgctt actggagaag tgattcctgt aatggaactg ctttcatcta tgaaatcaca
780 cagtgttcct gaagaaatag atatagctga tacagtactc aatgatgatg
atattggtga 840 cagctgtcat gaaggctttc ttctcaatgc catcagctca
cacttgcaaa cctgtggctg 900 ttccgttgta gtaggtagca gtgcagagaa
agtaaataag atagtcagaa cattatgcct 960 ttttctgact ccagcagaga
gaaaatgctc caggttatgt gaagcagaat catcatttaa 1020 atatgagtca
gggctctttg tacaaggcct gctaaaggat tcaactggaa gctttgtgct 1080
gcctttccgg caagtcatgt atgctccata tcccaccaca cacatagatg tggatgtcaa
1140 tactgtgaag cagatgccac cctgtcatga acatatttat aatcagcgta
gatacatgag 1200 atccgagctg acagccttct ggagagccac ttcagaagaa
gacatggctc aggatacgat 1260 catctacact gacgaaagct ttactcctga
tttgaatatt tttcaagatg tcttacacag 1320 agacactcta gtgaaagcct
tcctggatca ggtctttcag ctgaaacctg gcttatctct 1380 cagaagtact
ttccttgcac agtttctact tgtccttcac agaaaagcct tgacactaat 1440
aaaatatata gaagacgata cgcagaaggg aaaaaagccc tttaaatctc ttcggaacct
1500 gaagatagac cttgatttaa cagcagaggg cgatcttaac ataataatgg
ctctggctga 1560 gaaaattaaa ccaggcctac actcttttat ctttggaaga
cctttctaca ctagtgtgca 1620 agaacgagat gttctaatga ctttttaaat
gtgtaactta ataagcctat tccatcacaa 1680 tcatgatcgc tggtaaagta
gctcagtggt gtggggaaac gttcccctgg atcatactcc 1740 agaattctgc
tctcagcaat tgcagttaag taagttacac tacagttctc acaagagcct 1800
gtgaggggat gtcaggtgca tcattacatt gggtgtctct tttcctagat ttatgctttt
1860 gggatacaga cctatgttta caatataata aatattattg ctatctttta
aagatataat 1920 aataggatgt aaacttgacc acaactactg tttttttgaa
atacatgatt catggtttac 1980 atgtgtcaag gtgaaatctg agttggcttt
tacagatagt tgactttcta tcttttggca 2040 ttctttggtg tgtagaatta
ctgtaatact tctgcaatca actgaaaact agagccttta 2100 aatgatttca
attccacaga aagaaagtga gcttgaacat aggatgagct ttagaaagaa 2160
aattgatcaa gcagatgttt aattggaatt gattattaga tcctactttg tggatttagt
2220 ccctgggatt cagtctgtag aaatgtctaa tagttctcta tagtccttgt
tcctggtgaa 2280 ccacagttag ggtgttttgt ttattttatt gttcttgcta
ttgttgatat tctatgtagt 2340 tgagctctgt aaaaggaaat tgtattttat
gttttagtaa ttgttgccaa ctttttaaat 2400 taattttcat tatttttgag
ccaaattgaa atgtgcacct cctgtgcctt ttttctcctt 2460 agaaaatcta
attacttgga acaagttcag atttcactgg tcagtcattt tcatcttgtt 2520
ttcttcttgc taagtcttac catgtacctg ctttggcaat cattgcaact ctgagattat
2580 aaaatgcctt agagaatata ctaactaata agatcttttt ttcagaaaca
gaaaatagtt 2640 ccttgagtac ttccttcttg catttctgcc tatgtttttg
aagttgttgc tgtttgcctg 2700 caataggcta taaggaatag caggagaaat
tttactgaag tgctgttttc ctaggtgcta 2760 ctttggcaga gctaagttat
cttttgtttt cttaatgcgt ttggaccatt ttgctggcta 2820 taaaataact
gattaatata attctaacac aatgttgaca ttgtagttac acaaacacaa 2880
ataaatattt tatttaaaat tctggaagta atataaaagg gaaaatatat ttataagaaa
2940 gggataaagg taatagagcc cttctgcccc ccacccacca aatttacaca
acaaaatgac 3000 atgttcgaat gtgaaaggtc ataatagctt tcccatcatg
aatcagaaag atgtggacag 3060 cttgatgttt tagacaacca ctgaactaga
tgactgttgt actgtagctc agtcatttaa 3120 aaaatatata aatactacct
tgtagtgtcc catactgtgt tttttacatg gtagattctt 3180 atttaagtgc
taactggtta ttttctttgg ctggtttatt gtactgttat acagaatgta 3240
agttgtacag tgaaataagt tattaaagca tgtgtaaaca ttgttatata tcttttctcc
3300 taaatggaga attttgaata aaatatattt gaaattttg 3339 <210>
SEQ ID NO 2 <211> LENGTH: 30001 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 2
caaagaaaag ggggaggttt tgttaaaaaa gagaaatgtt acatagtgct ctttgagaaa
60 attcattggc actattaagg atctgaggag ctggtgagtt tcaactggtg
agtgatggtg 120 gtagataaaa ttagagctgc agcaggtcat tttagcaact
attagataaa actggtctca 180 ggtcacaacg ggcagttgca gcagctggac
ttggagagaa ttacactgtg ggagcagtgt 240 catttgtcct aagtgctttt
ctacccccta cccccactat tttagttggg tataaaaaga 300 atgacccaat
ttgtatgatc aactttcaca aagcatagaa cagtaggaaa agggtctgtt 360
tctgcagaag gtgtagacgt tgagagccat tttgtgtatt tattcctccc tttcttcctc
420 ggtgaatgat taaaacgttc tgtgtgattt ttagtgatga aaaagattaa
atgctactca 480 ctgtagtaag tgccatctca cacttgcaga tcaaaaggca
cacagtttaa aaaacctttg 540 tttttttaca catctgagtg gtgtaaatgc
tactcatctg tagtaagtgg aatctataca 600 cctgcagacc aaaagacgca
aggtttcaaa aatctttgtg ttttttacac atcaaacaga 660 atggtacgtt
tttcaaaagt taaaaaaaaa caactcatcc acatattgca actagcaaaa 720
atgacattcc ccagtgtgaa aatcatgctt gagagaattc ttacatgtaa aggcaaaatt
780 gcgatgactt tgcaggggac cgtgggattc ccgcccgcag tgccggagct
gtcccctacc 840 agggtttgca gtggagtttt gaatgcactt aacagtgtct
tacggtaaaa acaaaatttc 900 atccaccaat tatgtgttga gcgcccactg
cctaccaagc acaaacaaaa ccattcaaaa 960 ccacgaaatc gtcttcactt
tctccagatc cagcagcctc ccctattaag gttcgcacac 1020 gctattgcgc
caacgctcct ccagagcggg tcttaagata aaagaacagg acaagttgcc 1080
ccgccccatt tcgctagcct cgtgagaaaa cgtcatcgca catagaaaac agacagacgt
1140 aacctacggt gtcccgctag gaaagagagg tgcgtcaaac agcgacaagt
tccgcccacg 1200 taaaagatga cgcttggtgt gtcagccgtc cctgctgccc
ggttgcttct cttttggggg 1260 cggggtctag caagagcagg tgtgggttta
ggaggtgtgt gtttttgttt ttcccaccct 1320 ctctccccac tacttgctct
cacagtactc gctgagggtg aacaagaaaa gacctgataa 1380 agattaacca
gaagaaaaca aggagggaaa caaccgcagc ctgtagcaag ctctggaact 1440
caggagtcgc gcgctagggg ccggggccgg ggccggggcg tggtcggggc gggcccgggg
1500 gcgggcccgg ggcggggctg cggttgcggt gcctgcgccc gcggcggcgg
aggcgcaggc 1560 ggtggcgagt gggtgagtga ggaggcggca tcctggcggg
tggctgtttg gggttcggct 1620 gccgggaaga ggcgcgggta gaagcggggg
ctctcctcag agctcgacgc atttttactt 1680 tccctctcat ttctctgacc
gaagctgggt gtcgggcttt cgcctctagc gactggtgga 1740 attgcctgca
tccgggcccc gggcttcccg gcggcggcgg cggcggcggc ggcgcaggga 1800
caagggatgg ggatctggcc tcttccttgc tttcccgccc tcagtacccg agctgtctcc
1860 ttcccgggga cccgctggga gcgctgccgc tgcgggctcg agaaaaggga
gcctcgggta 1920 ctgagaggcc tcgcctgggg gaaggccgga gggtgggcgg
cgcgcggctt ctgcggacca 1980 agtcggggtt cgctaggaac ccgagacggt
ccctgccggc gaggagatca tgcgggatga 2040 gatgggggtg tggagacgcc
tgcacaattt cagcccaagc ttctagagag tggtgatgac 2100 ttgcatatga
gggcagcaat gcaagtcggt gtgctcccca ttctgtggga catgacctgg 2160
ttgcttcaca gctccgagat gacacagact tgcttaaagg aagtgactat tgtgacttgg
2220 gcatcacttg actgatggta atcagttgtc taaagaagtg cacagattac
atgtccgtgt 2280 gctcattggg tctatctggc cgcgttgaac accaccaggc
tttgtattca gaaacaggag 2340 ggaggtcctg cactttccca ggaggggtgg
ccctttcaga tgcaatcgag attgttaggc 2400 tctgggagag tagttgcctg
gttgtggcag ttggtaaatt tctattcaaa cagttgccat 2460 gcaccagttg
ttcacaacaa gggtacgtaa tctgtctggc attacttcta cttttgtaca 2520
aaggatcaaa aaaaaaaaag atactgttaa gatatgattt ttctcagact ttgggaaact
2580 tttaacataa tctgtgaata tcacagaaac aagactatca tataggggat
attaataacc 2640 tggagtcaga atacttgaaa tacggtgtca tttgacacgg
gcattgttgt caccacctct 2700 gccaaggcct gccactttag gaaaaccctg
aatcagttgg aaactgctac atgctgatag 2760 tacatctgaa acaagaacga
gagtaattac cacattccag attgttcact aagccagcat 2820 ttacctgctc
caggaaaaaa ttacaagcac cttatgaagt tgataaaata ttttgtttgg 2880
ctatgttggc actccacaat ttgctttcag agaaacaaag taaaccaagg aggacttctg
2940 tttttcaagt ctgccctcgg gttctattct acgttaatta gatagttccc
aggaggacta 3000 ggttagccta cctattgtct gagaaacttg gaactgtgag
aaatggccag atagtgatat 3060 gaacttcacc ttccagtctt ccctgatgtt
gaagattgag aaagtgttgt gaactttctg 3120 gtactgtaaa cagttcactg
tccttgaagt ggtcctgggc agctcctgtt gtggaaagtg 3180 gacggtttag
gatcctgctt ctctttgggc tgggagaaaa taaacagcat ggttacaagt 3240
attgagagcc aggttggaga aggtggctta cacctgtaat gccagagctt tgggaggcgg
3300 aggcaagagg atcacttgaa gccaggagtt caagctcaac ctgggcaacg
tagaccctgt 3360 ctctacaaaa aattaaaaac ttagccgggc gtggtgatgt
gcacctgtag tcctagctac 3420 ttgggaggct gaggcaggag ggtcatttga
gcccaagagt ttgaagttac cgagagctat 3480 gatcctgcca gtgcattcca
gcctggatga caaaacgaga ccctgtctct aaaaaacaag 3540
aagtgagggc tttatgattg tagaattttc actacaatag cagtggacca accacctttc
3600 taaataccaa tcagggaaga gatggttgat tttttaacag acgtttaaag
aaaaagcaaa 3660 acctcaaact tagcactcta ctaacagttt tagcagatgt
taattaatgt aatcatgtct 3720 gcatgtatgg gattatttcc agaaagtgta
ttgggaaacc tctcatgaac cctgtgagca 3780 agccaccgtc tcactcaatt
tgaatcttgg cttccctcaa aagactggct aatgtttggt 3840 aactctctgg
agtagacagc actacatgta cgtaagatag gtacataaac aactattggt 3900
tttgagctga tttttttcag ctgcatttgc atgtatggat ttttctcacc aaagacgatg
3960 acttcaagta ttagtaaaat aattgtacag ctctcctgat tatacttctc
tgtgacattt 4020 catttcccag gctatttctt ttggtaggat ttaaaactaa
gcaattcagt atgatctttg 4080 tccttcattt tctttcttat tctttttgtt
tgtttgtttg tttgtttttt tcttgaggca 4140 gagtctctct ctgtcgccca
ggctggagtg cagtggcgcc atctcagctc attgcaacct 4200 ctgccacctc
cgggttcaag agattctcct gcctcagcct cccgagtagc tgggattaca 4260
ggtgtccacc accacacccg gctaattttt tgtattttta gtagaggtgg ggtttcacca
4320 tgttggccag gctggtcttg agctcctgac ctcaggtgat ccacctgcct
cggcctacca 4380 aagagctggg ataacaggtg tgacccacca tgcccggccc
attttttttt tcttattctg 4440 ttaggagtga gagtgtaact agcagtataa
tagttcaatt ttcacaacgt ggtaaaagtt 4500 tccctataat tcaatcagat
tttgctccag ggttcagttc tgttttagga aatactttta 4560 ttttcagttt
aatgatgaaa tattagagtt gtaatattgc ctttatgatt atccaccttt 4620
ttaacctaaa agaatgaaag aaaaatatgt ttgcaatata attttatggt tgtatgttaa
4680 cttaattcat tatgttggcc tccagtttgc tgttgttagt tatgacagca
gtagtgtcat 4740 taccatttca attcagatta cattcctata tttgatcatt
gtaaactgac tgcttacatt 4800 gtattaaaaa cagtggatat tttaaagaag
ctgtacggct tatatctagt gctgtctctt 4860 aagactatta aattgataca
acatatttaa aagtaaatat tacctaaatg aatttttgaa 4920 attacaaata
cacgtgttaa aactgtcgtt gtgttcaacc atttctgtac atacttagag 4980
ttaactgttt tgccaggctc tgtatgccta ctcataatat gataaaagca ctcatctaat
5040 gctctgtaaa tagaagtcag tgctttccat cagactgaac tctcttgaca
agatgtggat 5100 gaaattcttt aagtaaaatt gtttactttg tcatacattt
acagatcaaa tgttagctcc 5160 caaagcaatc atatggcaaa gataggtata
tcatagtttg cctattagct gctttgtatt 5220 gctattatta taaatagact
tcacagtttt agacttgctt aggtgaaatt gcaattcttt 5280 ttactttcag
tcttagataa caagtcttca attatagtac aatcacacat tgcttaggaa 5340
tgcatcatta ggcgattttg tcattatgca aacatcatag agtgtactta cacaaaccta
5400 gatagtatag cctttatgta cctaggccgt atggtatagt ctgttgctcc
taggccacaa 5460 acctgtacaa ctgttactgt actgaatact atagacagtt
gtaacacagt ggtaaatatt 5520 tatctaaata tatgcaaaca gagaaaaggt
acagtaaaag tatggtataa aagataatgg 5580 tatacctgtg taggccactt
accacgaatg gagcttgcag gactagaagt tgctctgggt 5640 gagtcagtga
gtgagtggtg aattaatgtg aaggcctaga acactgtaca ccactgtaga 5700
ctataaacac agtacgctga agctacacca aatttatctt aacagttttt cttcaataaa
5760 aaattataac tttttaactt tgtaaacttt ttaatttttt aacttttaaa
atacttagct 5820 tgaaacacaa atacattgta tagctataca aaaatatttt
ttctttgtat ccttattcta 5880 gaagcttttt tctattttct attttaaatt
ttttttttta cttgttagtc gtttttgtta 5940 aaaactaaaa cacacacact
ttcacctagg catagacagg attaggatca tcagtatcac 6000 tcccttccac
ctcactgcct tccacctcca catcttgtcc cactggaagg tttttagggg 6060
caataacaca catgtagctg tcacctatga taacagtgct ttctgttgaa tacctcctga
6120 aggacttgcc tgaggctgtt ttacatttaa cttaaaaaaa aaaaaagtag
aaggagtgca 6180 ctctaaaata acaataaaag gcatagtata gtgaatacat
aaaccagcaa tgtagtagtt 6240 tattatcaag tgttgtacac tgtaataatt
gtatgtgcta tactttaaat aacttgcaaa 6300 atagtactaa gaccttatga
tggttacagt gtcactaagg caatagcata ttttcaggtc 6360 cattgtaatc
taatgggact accatcatat atgcagtcta ccattgactg aaacgttaca 6420
tggcacataa ctgtatttgc aagaatgatt tgttttacat taatatcaca taggatgtac
6480 ctttttagag tggtatgttt atgtggatta agatgtacaa gttgagcaag
gggaccaaga 6540 gccctgggtt ctgtcttgga tgtgagcgtt tatgttcttc
tcctcatgtc tgttttctca 6600 ttaaattcaa aggcttgaac gggccctatt
tagcccttct gttttctacg tgttctaaat 6660 aactaaagct tttaaattct
agccatttag tgtagaactc tctttgcagt gatgaaatgc 6720 tgtattggtt
tcttggctag catattaaat atttttatct ttgtcttgat acttcaatgt 6780
cgttttaaac atcaggatcg ggcttcagta ttctcataac cagagagttc actgaggata
6840 caggactgtt tgcccatttt ttgttatggc tccagacttg tggtatttcc
atgtcttttt 6900 tttttttttt ttttttgacc ttttagcggc tttaaagtat
ttctgttgtt aggtgttgta 6960 ttacttttct aagattactt aacaaagcac
cacaaactga gtggctttaa acaacagcaa 7020 tttattctct cacaattcta
gaagctagaa gtccgaaatc aaagtgttga caggggcatg 7080 atcttcaaga
gagaagactc tttccttgcc tcttcctggc ttctggtggt taccagcaat 7140
cctgagtgtt cctttcttgc cttgtagttt caacaatcca gtatctgcct tttgtcttca
7200 catggctgtc taccatttgt ctctgtgtct ccaaatctct ctccttataa
acacagcagt 7260 tattggatta ggccccactc taatccagta tgaccccatt
ttaacatgat tacacttatt 7320 tctagataag gtcacattca cgtacaccaa
gggttaggaa ttgaacatat ctttttgggg 7380 gacacaattc aacccacaag
tgtcagtctc tagctgagcc tttcccttcc tgtttttctc 7440 ctttttagtt
gctatgggtt aggggccaaa tctccagtca tactagaatt gcacatggac 7500
tggatatttg ggaatactgc gggtctattc tatgagcttt agtatgtaac atttaatatc
7560 agtgtaaaga agcccttttt taagttattt ctttgaattt ctaaatgtat
gccctgaata 7620 taagtaacaa gttaccatgt cttgtaaaat gatcatatca
acaaacattt aatgtgcacc 7680 tactgtgcta gttgaatgtc tttatcctga
taggagataa caggattcca catctttgac 7740 ttaagaggac aaaccaaata
tgtctaaatc atttggggtt ttgatggata tctttaaatt 7800 gctgaaccta
atcattggtt tcatatgtca ttgtttagat atctccggag catttggata 7860
atgtgacagt tggaatgcag tgatgtcgac tctttgccca ccgccatctc cagctgttgc
7920 caagacagag attgctttaa gtggcaaatc acctttatta gcagctactt
ttgcttactg 7980 ggacaatatt cttggtccta gagtaaggca catttgggct
ccaaagacag aacaggtact 8040 tctcagtgat ggagaaataa cttttcttgc
caaccacact ctaaatggag aaatccttcg 8100 aaatgcagag agtggtgcta
tagatgtaaa gttttttgtc ttgtctgaaa agggagtgat 8160 tattgtttca
ttaatctttg atggaaactg gaatggggat cgcagcacat atggactatc 8220
aattatactt ccacagacag aacttagttt ctacctccca cttcatagag tgtgtgttga
8280 tagattaaca catataatcc ggaaaggaag aatatggatg cataaggtaa
gtgatttttc 8340 agcttattaa tcatgttaac ctatctgttg aaagcttatt
ttctggtaca tataaatctt 8400 atttttttaa ttatatgcag tgaacatcaa
acaataaatg ttatttattt tgcatttacc 8460 ctattagata caaatacatc
tggtctgata cctgtcatct tcatattaac tgtggaaggt 8520 acgaaatggt
agctccacat tatagatgaa aagctaaagc ttagacaaat aaagaaactt 8580
ttagaccctg gattcttctt gggagccttt gactctaata ccttttgttt ccctttcatt
8640 gcacaattct gtcttttgct tactactatg tgtaagtata acagttcaaa
gtaatagttt 8700 cataagctgt tggtcatgta gcctttggtc tctttaacct
ctttgccaag ttcccaggtt 8760 cataaaatga ggaggttgaa tggaatggtt
cccaagagaa ttccttttaa tcttacagaa 8820 attattgttt tcctaaatcc
tgtagttgaa tatataatgc tatttacatt tcagtatagt 8880 tttgatgtat
ctaaagaaca cattgaattc tccttcctgt gttccagttt gatactaacc 8940
tgaaagtcca ttaagcatta ccagttttaa aaggcttttg cccaatagta aggaaaaata
9000 atatctttta aaagaataat tttttactat gtttgcaggc ttacttcctt
ttttctcaca 9060 ttatgaaact cttaaaatca ggagaatctt ttaaacaaca
tcataatgtt taatttgaaa 9120 agtgcaagtc attcttttcc tttttgaaac
tatgcagatg ttacattgac tgttttctgt 9180 gaagttatct ttttttcact
gcagaataaa ggttgttttg attttatttt gtattgttta 9240 tgagaacatg
catttgttgg gttaatttcc tacccctgcc cccatttttt ccctaaagta 9300
gaaagtattt ttcttgtgaa ctaaattact acacaagaac atgtctattg aaaaataagc
9360 aagtatcaaa atgttgtggg ttgttttttt aaataaattt tctcttgctc
aggaaagaca 9420 agaaaatgtc cagaagatta tcttagaagg cacagagaga
atggaagatc aggtatatgc 9480 aaattgcata ctgtcaaatg tttttctcac
agcatgtatc tgtataaggt tgatggctac 9540 atttgtcaag gccttggaga
catacgaata agcctttaat ggagctttta tggaggtgta 9600 cagaataaac
tggaggaaga tttccatatc ttaaacccaa agagttaaat cagtaaacaa 9660
aggaaaatag taattgcatc tacaaattaa tatttgctcc cttttttttt ctgtttgccc
9720 agaataaatt ttggataact tgttcatagt aaaaataaaa aaaattgtct
ctgatatgtt 9780 ctttaaggta ctacttctcg aacctttccc tagaagtagc
tgtaacagaa ggagagcata 9840 tgtacccctg aggtatctgt ctggggtgta
ggcccaggtc cacacaatat ttcttctaag 9900 tcttatgttg tatcgttaag
actcatgcaa tttacatttt attccataac tattttagta 9960 ttaaaatttg
tcagtgatat ttcttaccct ctcctctagg aaaatgtgcc atgtttatcc 10020
cttggctttg aatgcccctc aggaacagac actaagagtt tgagaagcat ggttacaagg
10080 gtgtggcttc ccctgcggaa actaagtaca gactatttca ctgtaaagca
gagaagttct 10140 tttgaaggag aatctccagt gaagaaagag ttcttcactt
ttacttccat ttcctcttgt 10200 gggtgaccct caatgctcct tgtaaaactc
caatatttta aacatggctg ttttgccttt 10260 ctttgcttct ttttagcatg
aatgagacag atgatacttt aaaaaagtaa ttaaaaaaaa 10320 aaacttgtga
aaatacatgg ccataataca gaacccaata caatgatctc ctttaccaaa 10380
ttgttatgtt tgtacttttg tagatagctt tccaattcag agacagttat tctgtgtaaa
10440 ggtctgactt aacaagaaaa gatttccctt tacccaaaga atcccagtcc
ttatttgctg 10500 gtcaataagc agggtcccca ggaatggggt aactttcagc
accctctaac ccactagtta 10560 ttagtagact aattaagtaa acttatcgca
agttgaggaa acttagaacc aactaaaatt 10620 ctgcttttac tgggattttg
ttttttcaaa ccagaaacct ttacttaagt tgactactat 10680 taatgaattt
tggtctctct tttaagtgct cttcttaaaa atgttatctt actgctgaga 10740
agttcaagtt tgggaagtac aaggaggaat agaaacttaa gagattttct tttagagcct
10800 cttctgtatt tagccctgta ggattttttt tttttttttt ttttttggtg
ttgttgagct 10860 tcagtgaggc tattcattca cttatactga taatgtctga
gatactgtga atgaaatact 10920 atgtatgctt aaacctaaga ggaaatattt
tcccaaaatt attcttcccg aaaaggagga 10980 gttgcctttt gattgagttc
ttgcaaatct cacaacgact ttattttgaa caatactgtt 11040
tggggatgat gcattagttt gaaacaactt cagttgtagc tgtcatctga taaaattgct
11100 tcacagggaa ggaaatttaa cacggatcta gtcattattc ttgttagatt
gaatgtgtga 11160 attgtaattg taaacaggca tgataattat tactttaaaa
actaaaaaca gtgaatagtt 11220 agttgtggag gttactaaag gatggttttt
ttttaaataa aactttcagc attatgcaaa 11280 tgggcatatg gcttaggata
aaacttccag aagtagcatc acatttaaat tctcaagcaa 11340 cttaataata
tggggctctg aaaaactggt taaggttact ccaaaaatgg ccctgggtct 11400
gacaaagatt ctaacttaaa gatgcttatg aagactttga gtaaaatcat ttcataaaat
11460 aagtgaggaa aaacaactag tattaaattc atcttaaata atgtatgatt
taaaaaatat 11520 gtttagctaa aaatgcatag tcatttgaca atttcattta
tatctcaaaa aatttactta 11580 accaagttgg tcacaaaact gatgagactg
gtggtggtag tgaataaatg agggaccatc 11640 catatttgag acactttaca
tttgtgatgt gttatactga attttcagtt tgattctata 11700 gactacaaat
ttcaaaatta caatttcaag atgtaataag tagtaatatc ttgaaatagc 11760
tctaaaggga atttttctgt tttattgatt cttaaaatat atgtgctgat tttgatttgc
11820 atttgggtag attatacttt tatgagtatg gaggttaggt attgattcaa
gttttcctta 11880 cctatttggt aaggatttca aagtcttttt gtgcttggtt
ttcctcattt ttaaatatga 11940 aatatattga tgacctttaa caaatttttt
ttatctcaaa ttttaaagga gatcttttct 12000 aaaagaggca tgatgactta
atcattgcat gtaacagtaa acgataaacc aatgattcca 12060 tactctctaa
agaataaaag tgagctttag ggccgggcat ggtcagaaat ttgacaccaa 12120
cctggccaac atggcgaaac cccgtctcta ctaaaaatac aaaaatcagc cgggcatggt
12180 ggcggcacct atagtcccag ctacttggga ggatgagaca ggagagtcac
ttgaacctgg 12240 gaggagaggt tgcagtgagc tgagatcacg ccattgcact
ccagcctgag caatgaaagc 12300 aaaactccat ctcaaaaaaa aaaaaagaaa
agaaagaata aaagtgagct ttggattgca 12360 tataaatcct ttagacatgt
agtagacttg tttgatactg tgtttgaaca aattacgaag 12420 tattttcatc
aaagaatgtt attgtttgat gttattttta ttttttattg cccagcttct 12480
ctcatattac gtgattttct tcacttcatg tcactttatt gtgcagggtc agagtattat
12540 tccaatgctt actggagaag tgattcctgt aatggaactg ctttcatcta
tgaaatcaca 12600 cagtgttcct gaagaaatag atgtaagttt aaatgagagc
aattatacac tttatgagtt 12660 ttttggggtt atagtattat tatgtatatt
attaatattc taattttaat agtaaggact 12720 ttgtcataca tactattcac
atacagtatt agccacttta gcaaataagc acacacaaaa 12780 tcctggattt
tatggcaaaa cagaggcatt tttgatcagt gatgacaaaa ttaaattcat 12840
tttgtttatt tcattacttt tataattcct aaaagtggga ggatcccagc tcttatagga
12900 gcaattaata tttaatgtag tgtcttttga aacaaaactg tgtgccaaag
tagtaaccat 12960 taatggaagt ttacttgtag tcacaaattt agtttcctta
atcatttgtt gaggacgttt 13020 tgaatcacac actatgagtg ttaagagata
cctttaggaa actattcttg ttgttttctg 13080 attttgtcat ttaggttagt
ctcctgattc tgacagctca gaagaggaag ttgttcttgt 13140 aaaaattgtt
taacctgctt gaccagcttt cacatttgtt cttctgaagt ttatggtagt 13200
gcacagagat tgttttttgg ggagtcttga ttctcggaaa tgaaggcagt gtgttatatt
13260 gaatccagac ttccgaaaac ttgtatatta aaagtgttat ttcaacacta
tgttacagcc 13320 agactaattt ttttattttt tgatgcattt tagatagctg
atacagtact caatgatgat 13380 gatattggtg acagctgtca tgaaggcttt
cttctcaagt aagaattttt cttttcataa 13440 aagctggatg aagcagatac
catcttatgc tcacctatga caagatttgg aagaaagaaa 13500 ataacagact
gtctacttag attgttctag ggacattacg tatttgaact gttgcttaaa 13560
tttgtgttat ttttcactca ttatatttct atatatattt ggtgttattc catttgctat
13620 ttaaagaaac cgagtttcca tcccagacaa gaaatcatgg ccccttgctt
gattctggtt 13680 tcttgtttta cttctcatta aagctaacag aatcctttca
tattaagttg tactgtagat 13740 gaacttaagt tatttaggcg tagaacaaaa
ttattcatat ttatactgat ctttttccat 13800 ccagcagtgg agtttagtac
ttaagagttt gtgcccttaa accagactcc ctggattaat 13860 gctgtgtacc
cgtgggcaag gtgcctgaat tctctataca cctatttcct catctgtaaa 13920
atggcaataa tagtaatagt acctaatgtg tagggttgtt ataagcattg agtaagataa
13980 ataatataaa gcacttagaa cagtgcctgg aacataaaaa cacttaataa
tagctcatag 14040 ctaacatttc ctatttacat ttcttctaga aatagccagt
atttgttgag tgcctacatg 14100 ttagttcctt tactagttgc tttacatgta
ttatcttata ttctgtttta aagtttcttc 14160 acagttacag attttcatga
aattttactt ttaataaaag agaagtaaaa gtataaagta 14220 ttcactttta
tgttcacagt cttttccttt aggctcatga tggagtatca gaggcatgag 14280
tgtgtttaac ctaagagcct taatggcttg aatcagaagc actttagtcc tgtatctgtt
14340 cagtgtcagc ctttcataca tcattttaaa tcccatttga ctttaagtaa
gtcacttaat 14400 ctctctacat gtcaatttct tcagctataa aatgatggta
tttcaataaa taaatacatt 14460 aattaaatga tattatactg actaattggg
ctgttttaag gctcaataag aaaatttctg 14520 tgaaaggtct ctagaaaatg
taggttccta tacaaataaa agataacatt gtgcttatag 14580 cttcggtgtt
tatcatataa agctattctg agttatttga agagctcacc tacttttttt 14640
tgtttttagt ttgttaaatt gttttatagg caatgttttt aatctgtttt ctttaactta
14700 cagtgccatc agctcacact tgcaaacctg tggctgttcc gttgtagtag
gtagcagtgc 14760 agagaaagta aataaggtag tttattttat aatctagcaa
atgatttgac tctttaagac 14820 tgatgatata tcatggattg tcatttaaat
ggtaggttgc aattaaaatg atctagtagt 14880 ataaggaggc aatgtaatct
catcaaattg ctaagacacc ttgtggcaac agtgagtttg 14940 aaataaactg
agtaagaatc atttatcagt ttattttgat agctcggaaa taccagtgtc 15000
agtagtgtat aaatggtttt gagaatatat taaaatcaga tatataaaaa aaattactct
15060 tctatttccc aatgttatct ttaacaaatc tgaagatagt catgtacttt
tggtagtagt 15120 tccaaagaaa tgttatttgt ttattcatct tgatttcatt
gtcttcgctt tccttctaaa 15180 tctgtccctt ctagggagct attgggatta
agtggtcatt gattattata ctttattcag 15240 taatgtttct gaccctttcc
ttcagtgcta cttgagttaa ttaaggatta atgaacagtt 15300 acatttccaa
gcattagcta ataaactaaa ggattttgca cttttcttca ctgaccatta 15360
gttagaaaga gttcagagat aagtatgtgt atctttcaat ttcagcaaac ctaatttttt
15420 aaaaaaagtt ttacatagga aatatgttgg aaatgatact ttacaaagat
attcataatt 15480 tttttttgta atcagctact ttgtatattt acatgagcct
taatttatat ttctcatata 15540 accatttatg agagcttagt atacctgtgt
cattatattg catctacgaa ctagtgacct 15600 tattccttct gttacctcaa
acaggtggct ttccatctgt gatctccaaa gccttaggtt 15660 gcacagagtg
actgccgagc tgctttatga agggagaaag gctccatagt tggagtgttt 15720
tttttttttt ttttaaacat ttttcccatc ctccatcctc ttgagggaga atagcttacc
15780 ttttatcttg ttttaatttg agaaagaagt tgccaccact ctaggttgaa
aaccactcct 15840 ttaacataat aactgtggat atggtttgaa tttcaagata
gttacatgcc tttttatttt 15900 tcctaataga gctgtaggtc aaatattatt
agaatcagat ttctaaatcc cacccaatga 15960 cctgcttatt ttaaatcaaa
ttcaataatt aattctcttc tttttggagg atctggacat 16020 tctttgatat
ttcttacaac gaatttcatg tgtagaccca ctaaacagaa gctataaaag 16080
ttgcatggtc aaataagtct gagaaagtct gcagatgata taattcacct gaagagtcac
16140 agtatgtagc caaatgttaa aggttttgag atgccataca gtaaatttac
caagcatttt 16200 ctaaatttat ttgaccacag aatccctatt ttaagcaaca
actgttacat cccatggatt 16260 ccaggtgact aaagaatact tatttcttag
gatatgtttt attgataata acaattaaaa 16320 tttcagatat ctttcataag
caaatcagtg gtctttttac ttcatgtttt aatgctaaaa 16380 tattttcttt
tatagatagt cagaacatta tgcctttttc tgactccagc agagagaaaa 16440
tgctccaggt tatgtgaagc agaatcatca tttaaatatg agtcagggct ctttgtacaa
16500 ggcctgctaa aggtatagtt tctagttatc acaagtgaaa ccacttttct
aaaatcattt 16560 ttgagactct ttatagacaa atcttaaata ttagcattta
atgtatctca tattgacatg 16620 cccagagact gacttccttt acacagttct
gcacatagac tatatgtctt atggatttat 16680 agttagtatc atcagtgaaa
caccatagaa taccctttgt gttccaggtg ggtccctgtt 16740 cctacatgtc
tagcctcagg actttttttt ttttaacaca tgcttaaatc aggttgcaca 16800
tcaaaaataa gatcatttct ttttaactaa atagatttga attttattga aaaaaaattt
16860 taaacatctt taagaagctt ataggattta agcaattcct atgtatgtgt
actaaaatat 16920 atatatttct atatataata tatattagaa aaaaattgta
tttttctttt atttgagtct 16980 actgtcaagg agcaaaacag agaaatgtaa
attagcaatt atttataata cttaaaggga 17040 agaaagttgt tcaccttgtt
gaatctatta ttgttatttc aattatagtc ccaagacgtg 17100 aagaaatagc
tttcctaatg gttatgtgat tgtctcatag tgactacttt cttgaggatg 17160
tagccacggc aaaatgaaat aaaaaaattt aaaaattgtt gcaaatacaa gttatattag
17220 gcttttgtgc attttcaata atgtgctgct atgaactcag aatgatagta
tttaaatata 17280 gaaactagtt aaaggaaacg tagtttctat ttgagttata
catatctgta aattagaact 17340 tctcctgtta aaggcataat aaagtgctta
atacttttgt ttcctcagca ccctctcatt 17400 taattatata attttagttc
tgaaagggac ctataccaga tgcctagagg aaatttcaaa 17460 actatgatct
aatgaaaaaa tatttaatag ttctccatgc aaatacaaat catatagttt 17520
tccagaaaat acctttgaca ttatacaaag atgattatca cagcattata atagtaaaaa
17580 aatggaaata gcctctttct tctgttctgt tcatagcaca gtgcctcata
cgcagtaggt 17640 tattattaca tggtaactgg ctaccccaac tgattaggaa
agaagtaaat ttgttttata 17700 aaaatacata ctcattgagg tgcatagaat
aattaagaaa ttaaaagaca cttgtaattt 17760 tgaatccagt gaatacccac
tgttaatatt tggtatatct ctttctagtc tttttttccc 17820 ttttgcatgt
attttcttta agactcccac ccccactgga tcatctctgc atgttctaat 17880
ctgctttttt cacagcagat tctaagcctc tttgaatatc aacacaaact tcaacaactt
17940 catctataga tgccaaataa taaattcatt tttatttact taaccacttc
ctttggatgc 18000 ttaggtcatt ctgatgtttt gctattgaaa ccaatgctat
actgaacact tctgtcacta 18060 aaactttgca cacactcatg aatagcttct
taggataaat ttttagagat ggatttgcta 18120 aatcagagac cattttttaa
aattaaaaaa caattattca tatcgtttgg catgtaagac 18180 agtaaatttt
ccttttattt tgacaggatt caactggaag ctttgtgctg cctttccggc 18240
aagtcatgta tgctccatat cccaccacac acatagatgt ggatgtcaat actgtgaagc
18300 agatgccacc ctgtcatgaa catatttata atcagcgtag atacatgaga
tccgagctga 18360 cagccttctg gagagccact tcagaagaag acatggctca
ggatacgatc atctacactg 18420 acgaaagctt tactcctgat ttgtacgtaa
tgctctgcct gctggtactg tagtcaagca 18480 atatgaaatt gtgtctttta
cgaataaaaa caaaacagaa gttgcattta aaaagaaaga 18540 aatattacca
gcagaattat gcttgaagaa acatttaatc aagcattttt ttcttaaatg 18600
ttcttctttt tccatacaat tgtgtttacc ctaaaatagg taagattaac ccttaaagta
18660 aatatttaac tatttgttta ataaatatat attgagctcc taggcactgt
tctaggtacc 18720 gggcttaata gtggccaacc agacagcccc agccccagcc
cctacattgt gtatagtcta 18780 ttatgtaaca gttattgaat ggacttatta
acaaaaccaa agaagtaatt ctaagtcttt 18840 tttttcttga catatgaata
taaaatacag caaaactgtt aaaatatatt aatggaacat 18900 ttttttactt
tgcattttat attgttattc acttcttatt tttttttaaa aaaaaaagcc 18960
tgaacagtaa attcaaaagg aaaagtaatg ataattaatt gttgagcatg gacccaactt
19020 gaaaaaaaaa atgatgatga taaatctata atcctaaaac cctaagtaaa
cacttaaaag 19080 atgttctgaa atcaggaaaa gaattatagt atacttttgt
gtttctcttt tatcagttga 19140 aaaaaggcac agtagctcat gcctgtaaga
acagagcttt gggagtgcaa ggcaggcgga 19200 tcacttgagg ccaggagttc
cagaccagcc tgggcaacat agtgaaaccc catctctaca 19260 aaaaataaaa
aagaattatt ggaatgtgtt tctgtgtgcc tgtaatccta gctattccga 19320
aagctgaggc aggaggatct tttgagccca ggagtttgag gttacaggga gttatgatgt
19380 gccagtgtac tccagcctgg ggaacaccga gactctgtct tatttaaaaa
aaaaaaaaaa 19440 aaaatgcttg caataatgcc tggcacatag aaggtaacag
taagtgttaa ctgtaataac 19500 ccaggtctaa gtgtgtaagg caatagaaaa
attggggcaa ataagcctga cctatgtatc 19560 tacagaatca gtttgagctt
aggtaacaga cctgtggagc accagtaatt acacagtaag 19620 tgttaaccaa
aagcatagaa taggaatatc ttgttcaagg gacccccagc cttatacatc 19680
tcaaggtgca gaaagatgac ttaatatagg acccattttt tcctagttct ccagagtttt
19740 tattggttct tgagaaagta gtaggggaat gttttagaaa atgaattggt
ccaactgaaa 19800 ttacatgtca gtaagttttt atatattggt aaattttagt
agacatgtag aagttttcta 19860 attaatctgt gccttgaaac attttctttt
ttcctaaagt gcttagtatt ttttccgttt 19920 tttgattggt tacttgggag
cttttttgag gaaatttagt gaactgcaga atgggtttgc 19980 aaccatttgg
tatttttgtt ttgtttttta gaggatgtat gtgtatttta acatttctta 20040
atcattttta gccagctatg tttgttttgc tgatttgaca aactacagtt agacagctat
20100 tctcattttg ctgatcatga caaaataata tcctgaattt ttaaattttg
catccagctc 20160 taaattttct aaacataaaa ttgtccaaaa aatagtattt
tcagccacta gattgtgtgt 20220 taagtctatt gtcacagagt cattttactt
ttaagtatat gtttttacat gttaattatg 20280 tttgttattt ttaattttaa
ctttttaaaa taattccagt cactgccaat acatgaaaaa 20340 ttggtcactg
gaattttttt tttgactttt attttaggtt catgtgtaca tgtgcaggtg 20400
tgttatacag gtaaattgcg tgtcatgagg gtttggtgta caggtgattt cattacccag
20460 gtaataagca tagtacccaa taggtagttt tttgatcctc acccttctcc
caccctcaag 20520 taggccctgg tgttgctgtt tccttctttg tgtccatgta
tactcagtgt ttagctccca 20580 cttagaagtg agaacatgcg gtagttggtt
ttctgttcct ggattagttc acttaggata 20640 atgacctcta gctccatctg
gtttttatgg ctgcatagta ttccatggtg tatatgtatc 20700 acattttctt
tatccagtct accattgata ggcatttagg ttgattccct gtctttgtta 20760
tcatgaatag tgctgtgatg aacatacaca tgcatgtgtc tttatggtag aaaaatttgt
20820 attcctttag gtacatatag aataatgggg ttgctagggt gaatggtagt
tctattttca 20880 gttatttgag aaatcttcaa actgcttttc ataatagcta
aactaattta cagtcccgcc 20940 agcagtgtat aagtgttccc ttttctccac
aaccttgcca acatctgtga ttttttgact 21000 ttttaataat agccattcct
agagaattga tttgcaattc tctattagtg atattaagca 21060 ttttttcata
tgctttttag ctgtctgtat atattcttct gaaaaatttt catgtccttt 21120
gcccagtttg tagtggggtg ggttgttttt tgcttgttaa ttagttttaa gttccttcca
21180 gattctgcat atccctttgt tggatacatg gtttgcagat atttttctcc
cattgtgtag 21240 gttgtctttt actctgttga tagtttcttt tgccatgcag
gagctcgtta ggtcccattt 21300 gtgtttgttt ttgttgcagt tgcttttggc
gtcttcatca taaaatctgt gccagggcct 21360 atgtccagaa tggtatttcc
taggttgtct tccagggttt ttacaatttt agattttacg 21420 tttatgtctt
taatccatct tgagttgatt tttgtatatg gcacaaggaa ggggtccagt 21480
ttcactccaa ttcctatggc tagcaattat cccagcacca tttattgaat acggagtcct
21540 ttccccattg cttgtttttt gtcaactttg ttgaagatca gatggttgta
agtgtgtggc 21600 tttatttctt ggctctctat tctccattgg tctatgtgtc
tgtttttata acagtaccct 21660 gctgttcagg ttcctatagc cttttagtat
aaaatcggct aatgtgatgc ctccagcttt 21720 gttctttttg cttaggattg
ctttggctat ttgggctcct ttttgggtcc atattaattt 21780 taaaacagtt
ttttctggtt ttgtgaagga tatcattggt agtttatagg aatagcattg 21840
aatctgtaga ttgctttggg cagtatggcc attttaacaa tattaattct tcctatctat
21900 gaatatggaa tgtttttcca tgtgtttgtg tcatctcttt atacctgatg
tataaagaaa 21960 agctggtatt attcctactc aatctgttcc aaaaaattga
ggaggaggaa ctcttcccta 22020 atgaggccag catcattctg ataccaaaac
ctggcagaga cacaacagaa aaaagaaaac 22080 ttcaggccaa tatccttgat
gaatatagat gcaaaaatcc tcaacaaaat actagcaaac 22140 caaatccagc
agcacatcaa aaagctgatc tactttgatc aagtaggctt tatccctggg 22200
atgcaaggtt ggttcaacat acacaaatca ataagtgtga ttcatcacat aaacagagct
22260 aaaaacaaaa accacaagat tatctcaata ggtagagaaa aggttgtcaa
taaaatttaa 22320 catcctccat gttaaaaacc ttcagtaggt caggtgtagt
gactcacacc tgtaatccca 22380 gcactttggg aggccaaggc gggcatatct
cttaagccca ggagttcaag acgagcctag 22440 gcagcatggt gaaaccccat
ctctacaaaa aaaaaaaaaa aaaaaaatta gcttggtatg 22500 gtgacatgca
cctatagtcc cagctattca ggaggttgag gtgggaggat tgtttgagcc 22560
cgggaggcag aggttggcag cgagctgaga tcatgccacc gcactccagc ctgggcaacg
22620 gagtgagacc ctgtctcaaa aaagaaaaat cacaaacaat cctaaacaaa
ctaggcattg 22680 aaggaacatg cctcaaaaaa ataagaacca tctatgacag
acccatagcc aatatcttac 22740 caaatgggca aaagctggaa gtattctcct
tgagaaccgt aacaagacaa ggatgtccac 22800 tctcaccact ccttttcagc
atagttctgg aagtcctagc cagagcaatc aggaaagaga 22860 aagaaagaaa
gacattcaga taggaagaga agaagtcaaa ctatttctgt ttgcaggcag 22920
tataattctg tacctagaaa atctcatagt ctctgcccag aaactcctaa atctgttaaa
22980 aatttcagca aagttttggc attctctata ctccaacacc ttccaaagtg
agagcaaaat 23040 caagaacaca gtcccattca caatagccgc aaaacgaata
aaatacctag gaatccagct 23100 aaccagggag gtgaaagatc tctatgagaa
ttacaaaaca ctgctgaaag aaatcagaga 23160 tgacacaaac aaatggaaat
gttctttttt aacaccttgc tttatctaat tcacttatga 23220 tgaagatact
cattcagtgg aacaggtata ataagtccac tcgattaaat ataagcctta 23280
ttctctttcc agagcccaag aaggggcact atcagtgccc agtcaataat gacgaaatgc
23340 taatattttt cccctttacg gtttctttct tctgtagtgt ggtacactcg
tttcttaaga 23400 taaggaaact tgaactacct tcctgtttgc ttctacacat
acccattctc tttttttgcc 23460 actctggtca ggtataggat gatccctacc
actttcagtt aaaaactcct cctcttacta 23520 aatgttctct taccctctgg
cctgagtaga acctagggaa aatggaagag aaaaagatga 23580 aagggaggtg
gggcctggga agggaataag tagtcctgtt tgtttgtgtg tttgctttag 23640
cacctgctat atcctaggtg ctgtgttagg cacacattat tttaagtggc cattatatta
23700 ctactactca ctctggtcgt tgccaaggta ggtagtactt tcttggatag
ttggttcatg 23760 ttacttacag atggtgggct tgttgaggca aacccagtgg
ataatcatcg gagtgtgttc 23820 tctaatctca ctcaaatttt tcttcacatt
ttttggtttg ttttggtttt tgatggtagt 23880 ggcttatttt tgttgctggt
ttgttttttg tttttttttg agatggcaag aattggtagt 23940 tttatttatt
aattgcctaa gggtctctac tttttttaaa agatgagagt agtaaaatag 24000
attgatagat acatacatac ccttactggg gactgcttat attctttaga gaaaaaatta
24060 catattagcc tgacaaacac cagtaaaatg taaatatatc cttgagtaaa
taaatgaatg 24120 tatattttgt gtctccaaat atatatatct atattcttac
aaatgtgttt atatgtaata 24180 tcaatttata agaacttaaa atgttggctc
aagtgaggga ttgtggaagg tagcattata 24240 tggccatttc aacatttgaa
cttttttctt ttcttcattt tcttcttttc ttcaggaata 24300 tttttcaaga
tgtcttacac agagacactc tagtgaaagc cttcctggat caggtaaatg 24360
ttgaacttga gattgtcaga gtgaatgata tgacatgttt tcttttttaa tatatcctac
24420 aatgcctgtt ctatatattt atattcccct ggatcatgcc ccagagttct
gctcagcaat 24480 tgcagttaag ttagttacac tacagttctc agaagagtct
gtgagggcat gtcaagtgca 24540 tcattacatt ggttgcctct tgtcctagat
ttatgcttcg ggaattcaga cctttgttta 24600 caatataata aatattattg
ctatctttta aagatataat aataagatat aaagttgacc 24660 acaactactg
ttttttgaaa catagaattc ctggtttaca tgtatcaaag tgaaatctga 24720
cttagctttt acagatataa tatatacata tatatatcct gcaatgcttg tactatatat
24780 gtagtacaag tatatatata tgtttgtgtg tgtatatata tatagtacga
gcatatatac 24840 atattaccag cattgtagga tatatatatg tttatatatt
aaaaaaaagt tataaactta 24900 aaaccctatt atgttatgta gagtatatgt
tatatatgat atgtaaaata tataacatat 24960 actctatgat agagtgtaat
atatttttta tatatatttt aacatttata aaatgataga 25020 attaagaatt
gagtcctaat ctgttttatt aggtgctttt tgtagtgtct ggtctttcta 25080
aagtgtctaa atgatttttc cttttgactt attaatgggg aagagcctgt atattaacaa
25140 ttaagagtgc agcattccat acgtcaaaca acaaacattt taattcaagc
attaacctat 25200 aacaagtaag tttttttttt ttttttgaga aagggaggtt
gtttatttgc ctgaaatgac 25260 tcaaaaatat ttttgaaaca tagtgtactt
atttaaataa catctttatt gtttcattct 25320 tttaaaaaat atctacttaa
ttacacagtt gaaggaaatc gtagattata tggaacttat 25380 ttcttaatat
attacagttt gttataataa cattctgggg atcaggccag gaaactgtgt 25440
catagataaa gctttgaaat aatgagatcc ttatgtttac tagaaatttt ggattgagat
25500 ctatgaggtc tgtgacatat tgcgaagttc aaggaaaatt cgtaggcctg
gaatttcatg 25560 cttctcaagc tgacataaaa tccctcccac tctccacctc
atcatatgca cacattctac 25620 tcctacccac ccactccacc ccctgcaaaa
gtacaggtat atgaatgtct caaaaccata 25680 ggctcatctt ctaggagctt
caatgttatt tgaagatttg ggcagaaaaa attaagtaat 25740 acgaaataac
ttatgtatga gttttaaaag tgaagtaaac atggatgtat tctgaagtag 25800
aatgcaaaat ttgaatgcat ttttaaagat aaattagaaa acttctaaaa actgtcagat
25860 tgtctgggcc tggtggctta tgcctgtaat cccagcactt tgggagtccg
aggtgggtgg 25920 atcacaaggt caggagatcg agaccatcct gccaacatgg
tgaaaccccg tctctactaa 25980 gtatacaaaa attagctggg cgtggcagcg
tgtgcctgta atcccagcta cctgggaggc 26040 tgaggcagga gaatcgcttg
aacccaggag gtgtaggttg cagtgagtca agatcgcgcc 26100
actgcacttt agcctggtga cagagctaga ctccgtctca aaaaaaaaaa aaaatatcag
26160 attgttccta cacctagtgc ttctatacca cactcctgtt agggggcatc
agtggaaatg 26220 gttaaggaga tgtttagtgt gtattgtctg ccaagcactg
tcaacactgt catagaaact 26280 tctgtacgag tagaatgtga gcaaattatg
tgttgaaatg gttcctctcc ctgcaggtct 26340 ttcagctgaa acctggctta
tctctcagaa gtactttcct tgcacagttt ctacttgtcc 26400 ttcacagaaa
agccttgaca ctaataaaat atatagaaga cgatacgtga gtaaaactcc 26460
tacacggaag aaaaaccttt gtacattgtt tttttgtttt gtttcctttg tacattttct
26520 atatcataat ttttgcgctt cttttttttt tttttttttt tttttttcca
ttatttttag 26580 gcagaaggga aaaaagccct ttaaatctct tcggaacctg
aagatagacc ttgatttaac 26640 agcagagggc gatcttaaca taataatggc
tctggctgag aaaattaaac caggcctaca 26700 ctcttttatc tttggaagac
ctttctacac tagtgtgcaa gaacgagatg ttctaatgac 26760 tttttaaatg
tgtaacttaa taagcctatt ccatcacaat catgatcgct ggtaaagtag 26820
ctcagtggtg tggggaaacg ttcccctgga tcatactcca gaattctgct ctcagcaatt
26880 gcagttaagt aagttacact acagttctca caagagcctg tgaggggatg
tcaggtgcat 26940 cattacattg ggtgtctctt ttcctagatt tatgcttttg
ggatacagac ctatgtttac 27000 aatataataa atattattgc tatcttttaa
agatataata ataggatgta aacttgacca 27060 caactactgt ttttttgaaa
tacatgattc atggtttaca tgtgtcaagg tgaaatctga 27120 gttggctttt
acagatagtt gactttctat cttttggcat tctttggtgt gtagaattac 27180
tgtaatactt ctgcaatcaa ctgaaaacta gagcctttaa atgatttcaa ttccacagaa
27240 agaaagtgag cttgaacata ggatgagctt tagaaagaaa attgatcaag
cagatgttta 27300 attggaattg attattagat cctactttgt ggatttagtc
cctgggattc agtctgtaga 27360 aatgtctaat agttctctat agtccttgtt
cctggtgaac cacagttagg gtgttttgtt 27420 tattttattg ttcttgctat
tgttgatatt ctatgtagtt gagctctgta aaaggaaatt 27480 gtattttatg
ttttagtaat tgttgccaac tttttaaatt aattttcatt atttttgagc 27540
caaattgaaa tgtgcacctc ctgtgccttt tttctcctta gaaaatctaa ttacttggaa
27600 caagttcaga tttcactggt cagtcatttt catcttgttt tcttcttgct
aagtcttacc 27660 atgtacctgc tttggcaatc attgcaactc tgagattata
aaatgcctta gagaatatac 27720 taactaataa gatctttttt tcagaaacag
aaaatagttc cttgagtact tccttcttgc 27780 atttctgcct atgtttttga
agttgttgct gtttgcctgc aataggctat aaggaatagc 27840 aggagaaatt
ttactgaagt gctgttttcc taggtgctac tttggcagag ctaagttatc 27900
ttttgttttc ttaatgcgtt tggaccattt tgctggctat aaaataactg attaatataa
27960 ttctaacaca atgttgacat tgtagttaca caaacacaaa taaatatttt
atttaaaatt 28020 ctggaagtaa tataaaaggg aaaatatatt tataagaaag
ggataaaggt aatagagccc 28080 ttctgccccc cacccaccaa atttacacaa
caaaatgaca tgttcgaatg tgaaaggtca 28140 taatagcttt cccatcatga
atcagaaaga tgtggacagc ttgatgtttt agacaaccac 28200 tgaactagat
gactgttgta ctgtagctca gtcatttaaa aaatatataa atactacctt 28260
gtagtgtccc atactgtgtt ttttacatgg tagattctta tttaagtgct aactggttat
28320 tttctttggc tggtttattg tactgttata cagaatgtaa gttgtacagt
gaaataagtt 28380 attaaagcat gtgtaaacat tgttatatat cttttctcct
aaatggagaa ttttgaataa 28440 aatatatttg aaattttgcc tctttcagtt
gttcattcag aaaaaaatac tatgatattt 28500 gaagactgat cagcttctgt
tcagctgaca gtcatgctgg atctaaactt tttttaaaat 28560 taattttgtc
ttttcaaaga aaaaatattt aaagaagctt tataatataa tcttatgtta 28620
aaaaaacttt ctgcttaact ctctggattt cattttgatt tttcaaatta tatattaata
28680 tttcaaatgt aaaatactat ttagataaat tgtttttaaa cattcttatt
attataatat 28740 taatataacc taaactgaag ttattcatcc caggtatcta
atacatgtat ccaaagtaaa 28800 aatccaagga atctgaacac tttcatctgc
aaagctagga ataggtttga cattttcact 28860 ccaagaaaaa gttttttttt
gaaaatagaa tagttgggat gagaggtttc tttaaaagaa 28920 gactaactga
tcacattact atgattctca aagaagaaac caaaacttca tataatacta 28980
taaagtaaat ataaaatagt tccttctata gtatatttct ataatgctac agtttaaaca
29040 gatcactctt atataatact attttgattt tgatgtagaa ttgcacaaat
tgatatttct 29100 cctatgatct gcagggtata gcttaaagta acaaaaacag
tcaaccacct ccatttaaca 29160 cacagtaaca ctatgggact agttttatta
cttccatttt acaaatgagg aaactaaagc 29220 ttaaagatgt gtaatacacc
gcccaaggtc acacagctgg taaaggtgga tttcatccca 29280 gacagttaca
gtcattgcca tgggcacagc tcctaactta gtaactccat gtaactggta 29340
ctcagtgtag ctgaattgaa aggagagtaa ggaagcaggt tttacaggtc tacttgcact
29400 attcagagcc cgagtgtgaa tccctgctgt gctgcttgga gaagttactt
aacctatgca 29460 aggttcattt tgtaaatatt ggaaatggag tgataatacg
tacttcacca gaggatttaa 29520 tgagacctta tacgatcctt agttcagtac
ctgactagtg cttcataaat gctttttcat 29580 ccaatctgac aatctccagc
ttgtaattgg ggcatttaga acatttaata tgattattgg 29640 catggtaggt
taaagctgtc atcttgctgt tttctatttg ttctttttgt tttctcctta 29700
cttttggatt tttttattct actatgtctt ttctattgtc ttattaacta tactctttga
29760 tttattttag tggttgtttt agggttatac ctctttctaa tttaccagtt
tataaccagt 29820 ttatatacta cttgacatat agcttaagaa acttactgtt
gttgtctttt tgctgttatg 29880 gtcttaacgt ttttatttct acaaacatta
taaactccac actttattgt tttttaattt 29940 tacttataca gtcaattatc
ttttaaagat atttaaatat aaacattcaa aacaccccaa 30000 t 30001
<210> SEQ ID NO 3 <211> LENGTH: 1031 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3
attcccggga tacgtaacct acggtgtccc gctaggaaag agaggtgcgt caaacagcga
60 caagttccgc ccacgtaaaa gatgacgctt ggtgtgtcag ccgtccctgc
tgcccggttg 120 cttctctttt gggggcgggg tctagcaaga gcaggtgtgg
gtttaggaga tatctccgga 180 gcatttggat aatgtgacag ttggaatgca
gtgatgtcga ctctttgccc accgccatct 240 ccagctgttg ccaagacaga
gattgcttta agtggcaaat cacctttatt agcagctact 300 tttgcttact
gggacaatat tcttggtcct agagtaaggc acatttgggc tccaaagaca 360
gaacaggtac ttctcagtga tggagaaata acttttcttg ccaaccacac tctaaatgga
420 gaaatccttc gaaatgcaga gagtggtgct atagatgtaa agttttttgt
cttgtctgaa 480 aagggagtga ttattgtttc attaatcttt gatggaaact
ggaatgggga tcgcagcaca 540 tatggactat caattatact tccacagaca
gaacttagtt tctacctccc acttcataga 600 gtgtgtgttg atagattaac
acatataatc cggaaaggaa gaatatggat gcataaggaa 660 agacaagaaa
aatgtccaga agattatctt agaaggcaca gagagaatgg aagatcaggg 720
tcagagtatt attccaatgc ttactggaga agtgattcct gtaatggaaa ctgctttcct
780 ctatgaaatt cccccgggtt cctggaggaa atagatatag gctgatacag
ttacccaatg 840 atggatgaat attgggggac cgcctggtca ttgaaaggct
ttcttttctc caggaaagaa 900 atttttttcc ttttccataa aaagcttggg
aatggaagac aacaattccc attctttttt 960 tgcgttccac ccctatgtga
caacagaaat ttttggggaa acaacaacga aaaaatttta 1020 tcccgcgcgc a 1031
<210> SEQ ID NO 4 <211> LENGTH: 3244 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4
gggcggggct gcggttgcgg tgcctgcgcc cgcggcggcg gaggcgcagg cggtggcgag
60 tggatatctc cggagcattt ggataatgtg acagttggaa tgcagtgatg
tcgactcttt 120 gcccaccgcc atctccagct gttgccaaga cagagattgc
tttaagtggc aaatcacctt 180 tattagcagc tacttttgct tactgggaca
atattcttgg tcctagagta aggcacattt 240 gggctccaaa gacagaacag
gtacttctca gtgatggaga aataactttt cttgccaacc 300 acactctaaa
tggagaaatc cttcgaaatg cagagagtgg tgctatagat gtaaagtttt 360
ttgtcttgtc tgaaaaggga gtgattattg tttcattaat ctttgatgga aactggaatg
420 gggatcgcag cacatatgga ctatcaatta tacttccaca gacagaactt
agtttctacc 480 tcccacttca tagagtgtgt gttgatagat taacacatat
aatccggaaa ggaagaatat 540 ggatgcataa ggaaagacaa gaaaatgtcc
agaagattat cttagaaggc acagagagaa 600 tggaagatca gggtcagagt
attattccaa tgcttactgg agaagtgatt cctgtaatgg 660 aactgctttc
atctatgaaa tcacacagtg ttcctgaaga aatagatata gctgatacag 720
tactcaatga tgatgatatt ggtgacagct gtcatgaagg ctttcttctc aatgccatca
780 gctcacactt gcaaacctgt ggctgttccg ttgtagtagg tagcagtgca
gagaaagtaa 840 ataagatagt cagaacatta tgcctttttc tgactccagc
agagagaaaa tgctccaggt 900 tatgtgaagc agaatcatca tttaaatatg
agtcagggct ctttgtacaa ggcctgctaa 960 aggattcaac tggaagcttt
gtgctgcctt tccggcaagt catgtatgct ccatatccca 1020 ccacacacat
agatgtggat gtcaatactg tgaagcagat gccaccctgt catgaacata 1080
tttataatca gcgtagatac atgagatccg agctgacagc cttctggaga gccacttcag
1140 aagaagacat ggctcaggat acgatcatct acactgacga aagctttact
cctgatttga 1200 atatttttca agatgtctta cacagagaca ctctagtgaa
agccttcctg gatcaggtct 1260 ttcagctgaa acctggctta tctctcagaa
gtactttcct tgcacagttt ctacttgtcc 1320 ttcacagaaa agccttgaca
ctaataaaat atatagaaga cgatacgcag aagggaaaaa 1380 agccctttaa
atctcttcgg aacctgaaga tagaccttga tttaacagca gagggcgatc 1440
ttaacataat aatggctctg gctgagaaaa ttaaaccagg cctacactct tttatctttg
1500 gaagaccttt ctacactagt gtgcaagaac gagatgttct aatgactttt
taaatgtgta 1560 acttaataag cctattccat cacaatcatg atcgctggta
aagtagctca gtggtgtggg 1620 gaaacgttcc cctggatcat actccagaat
tctgctctca gcaattgcag ttaagtaagt 1680 tacactacag ttctcacaag
agcctgtgag gggatgtcag gtgcatcatt acattgggtg 1740 tctcttttcc
tagatttatg cttttgggat acagacctat gtttacaata taataaatat 1800
tattgctatc ttttaaagat ataataatag gatgtaaact tgaccacaac tactgttttt
1860 ttgaaataca tgattcatgg tttacatgtg tcaaggtgaa atctgagttg
gcttttacag 1920 atagttgact ttctatcttt tggcattctt tggtgtgtag
aattactgta atacttctgc 1980 aatcaactga aaactagagc ctttaaatga
tttcaattcc acagaaagaa agtgagcttg 2040
aacataggat gagctttaga aagaaaattg atcaagcaga tgtttaattg gaattgatta
2100 ttagatccta ctttgtggat ttagtccctg ggattcagtc tgtagaaatg
tctaatagtt 2160 ctctatagtc cttgttcctg gtgaaccaca gttagggtgt
tttgtttatt ttattgttct 2220 tgctattgtt gatattctat gtagttgagc
tctgtaaaag gaaattgtat tttatgtttt 2280 agtaattgtt gccaactttt
taaattaatt ttcattattt ttgagccaaa ttgaaatgtg 2340 cacctcctgt
gccttttttc tccttagaaa atctaattac ttggaacaag ttcagatttc 2400
actggtcagt cattttcatc ttgttttctt cttgctaagt cttaccatgt acctgctttg
2460 gcaatcattg caactctgag attataaaat gccttagaga atatactaac
taataagatc 2520 tttttttcag aaacagaaaa tagttccttg agtacttcct
tcttgcattt ctgcctatgt 2580 ttttgaagtt gttgctgttt gcctgcaata
ggctataagg aatagcagga gaaattttac 2640 tgaagtgctg ttttcctagg
tgctactttg gcagagctaa gttatctttt gttttcttaa 2700 tgcgtttgga
ccattttgct ggctataaaa taactgatta atataattct aacacaatgt 2760
tgacattgta gttacacaaa cacaaataaa tattttattt aaaattctgg aagtaatata
2820 aaagggaaaa tatatttata agaaagggat aaaggtaata gagcccttct
gccccccacc 2880 caccaaattt acacaacaaa atgacatgtt cgaatgtgaa
aggtcataat agctttccca 2940 tcatgaatca gaaagatgtg gacagcttga
tgttttagac aaccactgaa ctagatgact 3000 gttgtactgt agctcagtca
tttaaaaaat atataaatac taccttgtag tgtcccatac 3060 tgtgtttttt
acatggtaga ttcttattta agtgctaact ggttattttc tttggctggt 3120
ttattgtact gttatacaga atgtaagttg tacagtgaaa taagttatta aagcatgtgt
3180 aaacattgtt atatatcttt tctcctaaat ggagaatttt gaataaaata
tatttgaaat 3240 tttg 3244 <210> SEQ ID NO 5 <211>
LENGTH: 761 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (693)..(693) <223> OTHER INFORMATION: n
is a, c, g, or t <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (722)..(722) <223> OTHER
INFORMATION: n is a, c, g, or t <400> SEQUENCE: 5 cacgaggctt
tgatatttct tacaacgaat ttcatgtgta gacccactaa acagaagcta 60
taaaagttgc atggtcaaat aagtctgaga aagtctgcag atgatataat tcacctgaag
120 agtcacagta tgtagccaaa tgttaaaggt tttgagatgc catacagtaa
atttaccaag 180 cattttctaa atttatttga ccacagaatc cctattttaa
gcaacaactg ttacatccca 240 tggattccag gtgactaaag aatacttatt
tcttaggata tgttttattg ataataacaa 300 ttaaaatttc agatatcttt
cataagcaaa tcagtggtct ttttacttca tgttttaatg 360 ctaaaatatt
ttcttttata gatagtcaga acattatgcc tttttctgac tccagcagag 420
agaaaatgct ccaggttatg tgaagcagaa tcatcattta aatatgagtc agggctcttt
480 gtacaaggcc tgctaaagga ttcaactgga agctttgtgc tgcctttccg
gcaagtcatg 540 tatgctccat atcccaccac acacatagat gtggatgtca
atactgtgaa gcagatgcca 600 ccctgtcatg aacatattta taatcagcgt
agatacatga gatccgagct gacagccttc 660 tggagagcca cttcagaaga
agacatggct cangatacga tcatctacac tgacgaaagc 720 tntactcctg
atttgaatat ttttcaagat gtcttacaca g 761 <210> SEQ ID NO 6
<211> LENGTH: 1901 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 6 acgtaaccta
cggtgtcccg ctaggaaaga gaggtgcgtc aaacagcgac aagttccgcc 60
cacgtaaaag atgacgcttg atatctccgg agcatttgga taatgtgaca gttggaatgc
120 agtgatgtcg actctttgcc caccgccatc tccagctgtt gccaagacag
agattgcttt 180 aagtggcaaa tcacctttat tagcagctac ttttgcttac
tgggacaata ttcttggtcc 240 tagagtaagg cacatttggg ctccaaagac
agaacaggta cttctcagtg atggagaaat 300 aacttttctt gccaaccaca
ctctaaatgg agaaatcctt cgaaatgcag agagtggtgc 360 tatagatgta
aagttttttg tcttgtctga aaagggagtg attattgttt cattaatctt 420
tgatggaaac tggaatgggg atcgcagcac atatggacta tcaattatac ttccacagac
480 agaacttagt ttctacctcc cacttcatag agtgtgtgtt gatagattaa
cacatataat 540 ccggaaagga agaatatgga tgcataagga aagacaagaa
aatgtccaga agattatctt 600 agaaggcaca gagagaatgg aagatcaggg
tcagagtatt attccaatgc ttactggaga 660 agtgattcct gtaatggaac
tgctttcatc tatgaaatca cacagtgttc ctgaagaaat 720 agatatagct
gatacagtac tcaatgatga tgatattggt gacagctgtc atgaaggctt 780
tcttctcaag taagaatttt tcttttcata aaagctggat gaagcagata ccatcttatg
840 ctcacctatg acaagatttg gaagaaagaa aataacagac tgtctactta
gattgttcta 900 gggacattac gtatttgaac tgttgcttaa atttgtgtta
tttttcactc attatatttc 960 tatatatatt tggtgttatt ccatttgcta
tttaaagaaa ccgagtttcc atcccagaca 1020 agaaatcatg gccccttgct
tgattctggt ttcttgtttt acttctcatt aaagctaaca 1080 gaatcctttc
atattaagtt gtactgtaga tgaacttaag ttatttaggc gtagaacaaa 1140
attattcata tttatactga tctttttcca tccagcagtg gagtttagta cttaagagtt
1200 tgtgccctta aaccagactc cctggattaa tgctgtgtac ccgtgggcaa
ggtgcctgaa 1260 ttctctatac acctatttcc tcatctgtaa aatggcaata
atagtaatag tacctaatgt 1320 gtagggttgt tataagcatt gagtaagata
aataatataa agcacttaga acagtgcctg 1380 gaacataaaa acacttaata
atagctcata gctaacattt cctatttaca tttcttctag 1440 aaatagccag
tatttgttga gtgcctacat gttagttcct ttactagttg ctttacatgt 1500
attatcttat attctgtttt aaagtttctt cacagttaca gattttcatg aaattttact
1560 tttaataaaa gagaagtaaa agtataaagt attcactttt atgttcacag
tcttttcctt 1620 taggctcatg atggagtatc agaggcatga gtgtgtttaa
cctaagagcc ttaatggctt 1680 gaatcagaag cactttagtc ctgtatctgt
tcagtgtcag cctttcatac atcattttaa 1740 atcccatttg actttaagta
agtcacttaa tctctctaca tgtcaatttc ttcagctata 1800 aaatgatggt
atttcaataa ataaatacat taattaaatg atattatact gactaattgg 1860
gctgttttaa ggcaaaaaaa aaaaaaaaaa aaaaaaaaaa a 1901 <210> SEQ
ID NO 7 <211> LENGTH: 562 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (166)..(166) <223> OTHER
INFORMATION: n is a, c, g, or t <400> SEQUENCE: 7 agacgtaacc
tacggtgtcc cgctaggaaa gagagatatc tccggagcat ttggataatg 60
tgacagttgg aatgcagtga tgtcgactct ttgcccaccg ccatctccag ctgttgccaa
120 gacagagatt gctttaagtg gcaaatcacc tttattagca gctacntttt
gcttactggg 180 acaatattct tggtcctaga gtaaggcaca tttgggctcc
aaagacagaa caggtacttc 240 tcagtgatgg agaaataact tttcttgcca
accacactct aaatggagaa atccttcgaa 300 atgcagagag tggtgctata
gatgtaaagt tttttgtctt gtctgaaaag ggagtgatta 360 ttgtttcatt
aatctttgat ggaaactgga atggggatcg cagcacatat ggactatcaa 420
ttatacttcc acagacagaa cttagtttct acctcccact tcatagagtg tgtgttgata
480 gattaacaca tataatccgg aaaggaagaa tatggatgca taaggaaaga
caagaaaatg 540 tccagaagat tatcttagaa gg 562 <210> SEQ ID NO 8
<211> LENGTH: 798 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 8 gggctctctt ttgggggcgg
ggtctagcaa gagcagatat ctccggagca tttggataat 60 gtgacagttg
gaatgcagtg atgtcgactc tttgcccacc gccatctcca gctgttgcca 120
agacagagat tgctttaagt ggcaaatcac ctttattagc agctactttt gcttactggg
180 acaatattct tggtcctaga gtaaggcaca tttgggctcc aaagacagaa
caggtacttc 240 tcagtgatgg agaaataact tttcttgcca accacactct
aaatggagaa atccttcgaa 300 atgcagagag tggtgctata gatgtaaagt
tttttgtctt gtctgaaaag ggagtgatta 360 ttgtttcatt aatctttgat
ggaaactgga atggggatcg cagcacatat ggactatcaa 420 ttatacttcc
acagacagaa cttagtttct acctcccact tcatagagtg tgtgttgata 480
gattaacaca tataatccgg aaaggaagaa tatggatgca taaggaaaga caagaaaatg
540 tccagaagat tatcttagaa ggcacagaga gaatggaaga tcagggtcag
agtattattc 600 caatgcttac tggagaagtg attcctgtaa tgggactgct
ttcatctatg aaatcacaca 660 gtgttcctga agaaatagat atagctgata
cagtactcca tgatgatgat atttggtgac 720 agctgtcatg aaaggctttc
ttctcaagta ggaatttttt cttttcataa aagctgggat 780 gaagccagat tcccatct
798 <210> SEQ ID NO 9 <211> LENGTH: 169 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
9 aaacagcgac aagttccgcc cacgtaaaag atgatgcttg gtgtgtcagc cgtccctgct
60 gcccggttgc ttctcttttg ggggcggggt ctagcaagag cagatatctc
cggagcattt 120 ggataatgtg acagttggaa tgcggtgatg tcgactcttt
gcccaccgc 169 <210> SEQ ID NO 10 <211> LENGTH: 176
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 10 aaaacgtcat cgcacataga aaacagacag
acgtaaccta cggtgtcccg ctaggaaaga 60 gaggtgcgtc aaacagcgac
aagttccgcc cacgtaaaag atgacgcttg atatctccgg 120
agcatttgga taatgtgaca gttggaatgc agtgatgtcg actctttgcc caccgc 176
<210> SEQ ID NO 11 <400> SEQUENCE: 11 000 <210>
SEQ ID NO 12 <400> SEQUENCE: 12 000 <210> SEQ ID NO 13
<211> LENGTH: 783 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 13 gcctctcagt acccgaggct
cccttttctc gagcccgcag cggcagcgct cccagcgggt 60 ccccgggaag
gagacagctc gggtactgag ggcgggaaag caaggaagag gccagatccc 120
catcccttgt ccctgcgccg ccgccgccgc cgccgccgcc gggaagcccg gggcccggat
180 gcaggcaatt ccaccagtcg ctagaggcga aagcccgaca cccagcttcg
gtcagagaaa 240 tgagagggaa agtaaaaatg cgtcgagctc tgaggagagc
ccccgcttct acccgcgcct 300 cttcccggca gccgaacccc aaacagccac
ccgccaggat gccgcctcct cactcaccca 360 ctcgccaccg cctgcgcctc
cgccgccgcg ggcgcaggca ccgcaaccgc agccccgccc 420 cgggcccgcc
cccgggcccg ccccgaccac gccccggccc cggccccggc cccggccccg 480
gcccctagcg cgcgactcct gagttccaga gcttgctaca ggctgcggtt gtttccctcc
540 ttgttttctt ctggttaatc tttatcaggt cttttcttgt tcaccctcag
cgagtactgt 600 gagagcaagt agtggggaga gagggtggga aaaacaaaaa
cacacacctc ctaaacccac 660 acctgctctt gctagacccc gcccccaaaa
gagaagcaac cgggcagcag ggacggctga 720 cacaccaagc gtcatctttt
acgtgggcgg aacttgtcgc tgtttgacgc acctctcttt 780 cct 783 <210>
SEQ ID NO 14 <211> LENGTH: 45 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 14
cgactggagc acgaggacac tgaaaagatg acgcttggtg tgtca 45 <210>
SEQ ID NO 15 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 15
cccacacctg ctcttgctag a 21 <210> SEQ ID NO 16 <211>
LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 16 cgactggagc acgaggacac tg 22 <210>
SEQ ID NO 17 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Probe <400> SEQUENCE: 17
cccaaaagag aagcaaccgg gca 23 <210> SEQ ID NO 18 <211>
LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 18 cgactggagc acgaggacac tgacggctgc cgggaaga
38 <210> SEQ ID NO 19 <211> LENGTH: 26 <212>
TYPE: DNA <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Primer <400>
SEQUENCE: 19 agaaatgaga gggaaagtaa aaatgc 26 <210> SEQ ID NO
20 <211> LENGTH: 22 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 20 cgactggagc
acgaggacac tg 22 <210> SEQ ID NO 21 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 21 aggagagccc ccgcttctac ccg 23 <210>
SEQ ID NO 22 <211> LENGTH: 42 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 22
cgactggagc acgaggacac tgacgctgag ggtgaacaag aa 42 <210> SEQ
ID NO 23 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 23 gagttccaga
gcttgctaca g 21 <210> SEQ ID NO 24 <211> LENGTH: 22
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 24 cgactggagc acgaggacac tg 22 <210>
SEQ ID NO 25 <211> LENGTH: 24 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Probe <400> SEQUENCE: 25
ctgcggttgt ttccctcctt gttt 24 <210> SEQ ID NO 26 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 26 ggcaaattca acggcacagt 20 <210> SEQ
ID NO 27 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 27 gggtctcgct
cctggaagat 20 <210> SEQ ID NO 28 <211> LENGTH: 27
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 28 aaggccgaga atgggaagct tgtcatc 27
<210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 29 tagtgcggac ctacccacga 20 <210> SEQ
ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 30 ggtgtgtcag ccgtccctgc 20 <210> SEQ
ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
31 gtgtcagccg tccctgctgc 20 <210> SEQ ID NO 32 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 32 tgtttttccc
accctctctc 20 <210> SEQ ID NO 33 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 33 ttttcccacc ctctctcccc 20
<210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 34 tcccaccctc tctccccact 20 <210> SEQ
ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
35 caccctctct ccccactact 20 <210> SEQ ID NO 36 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 36 gagggtgaac
aagaaaagac 20 <210> SEQ ID NO 37 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 37 gaaaagacct gataaagatt 20
<210> SEQ ID NO 38 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 38 agattaacca gaagaaaaca 20 <210> SEQ
ID NO 39 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
39 ttaaccagaa gaaaacaagg 20 <210> SEQ ID NO 40 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 40 accagaagaa
aacaaggagg 20 <210> SEQ ID NO 41 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 41 agaagaaaac aaggagggaa 20
<210> SEQ ID NO 42 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 42 agaaaacaag gagggaaaca 20 <210> SEQ
ID NO 43 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
43 aaacaaggag ggaaacaacc 20 <210> SEQ ID NO 44 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 44 gcaagctctg
gaactcagga 20 <210> SEQ ID NO 45 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 45 agctctggaa ctcaggagtc 20
<210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 46 tcaggagtcg cgcgctaggg 20 <210> SEQ
ID NO 47 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
47 ggagtcgcgc gctaggggcc 20 <210> SEQ ID NO 48 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 48 gtcgcgcgct
aggggccggg 20 <210> SEQ ID NO 49 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 49 gcgcgctagg ggccggggcc 20
<210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 50 gggctgcggt tgcggtgcct 20 <210> SEQ
ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 51 gcggttgcgg tgcctgcgcc 20 <210> SEQ
ID NO 52 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
52 tgcggtgcct gcgcccgcgg 20 <210> SEQ ID NO 53 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 53 tgcctgcgcc
cgcggcggcg 20 <210> SEQ ID NO 54 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 54 gcgcccgcgg cggcggaggc 20
<210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 55 cgcggcggcg gaggcgcagg 20 <210> SEQ
ID NO 56 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
56 cggcggaggc gcaggcggtg 20 <210> SEQ ID NO 57 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 57 gaggcgcagg
cggtggcgag 20 <210> SEQ ID NO 58 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 58 gcaggcggtg gcgagtgggt 20
<210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 59 cggtggcgag tgggtgagtg 20 <210> SEQ
ID NO 60 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
60 gcgagtgggt gagtgaggag 20 <210> SEQ ID NO 61 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 61 tgggtgagtg
aggaggcggc 20 <210> SEQ ID NO 62 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 62 gagtgaggag gcggcatcct 20
<210> SEQ ID NO 63 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 63 aggaggcggc atcctggcgg 20 <210> SEQ
ID NO 64 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
64 gcggcatcct ggcgggtggc 20 <210> SEQ ID NO 65 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 65 atcctggcgg
gtggctgttt 20 <210> SEQ ID NO 66 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 66 tcggctgccg ggaagaggcg 20
<210> SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 67 tgccgggaag aggcgcgggt 20 <210> SEQ
ID NO 68 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
68 ggaagaggcg cgggtagaag 20 <210> SEQ ID NO 69 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 69 gctctcctca
gagctcgacg 20 <210> SEQ ID NO 70 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 70 cctcagagct cgacgcattt 20
<210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 71 gagctcgacg catttttact 20 <210> SEQ
ID NO 72 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 72 cgacgcattt ttactttccc 20
<210> SEQ ID NO 73 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 73 catttttact ttccctctca 20 <210> SEQ
ID NO 74 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
74 ttactttccc tctcatttct 20 <210> SEQ ID NO 75 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 75 ttccctctca
tttctctgac 20 <210> SEQ ID NO 76 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 76 tctcatttct ctgaccgaag 20
<210> SEQ ID NO 77 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 77 tttctctgac cgaagctggg 20 <210> SEQ
ID NO 78 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
78 ctgaccgaag ctgggtgtcg 20 <210> SEQ ID NO 79 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 79 cgaagctggg
tgtcgggctt 20 <210> SEQ ID NO 80 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 80 ctgggtgtcg ggctttcgcc 20
<210> SEQ ID NO 81 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 81 tgtcgggctt tcgcctctag 20 <210> SEQ
ID NO 82 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
82 ggctttcgcc tctagcgact 20 <210> SEQ ID NO 83 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (6)..(6) <223>
OTHER INFORMATION: I <220> FEATURE: <221> NAME/KEY:
modified_base <222> LOCATION: (10)..(10) <223> OTHER
INFORMATION: I <220> FEATURE: <221> NAME/KEY:
modified_base <222> LOCATION: (15)..(15) <223> OTHER
INFORMATION: I <400> SEQUENCE: 83 ccggggccgg ggccggggcc 20
<210> SEQ ID NO 84 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (6)..(6) <223> OTHER INFORMATION: I
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (13)..(13) <223> OTHER INFORMATION: I
<400> SEQUENCE: 84 ggccggggcc ggggccgg 18 <210> SEQ ID
NO 85 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
85 ggggccgggg ccggggcc 18 <210> SEQ ID NO 86 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 86 cggggccggg
gccggggc 18 <210> SEQ ID NO 87 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 87 ccggggccgg ggccgggg 18
<210> SEQ ID NO 88 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 88 gccggggccg gggccggg 18 <210> SEQ ID
NO 89 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
89 ggccggggcc ggggccgg 18 <210> SEQ ID NO 90 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 90 gggccggggc
cggggccg 18 <210> SEQ ID NO 91 <211> LENGTH: 16
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide
<400> SEQUENCE: 91 ggccggggcc ggggcc 16 <210> SEQ ID NO
92 <211> LENGTH: 16 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
92 gggccggggc cggggc 16 <210> SEQ ID NO 93 <211>
LENGTH: 16 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 93 ggggccgggg
ccgggg 16 <210> SEQ ID NO 94 <211> LENGTH: 16
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 94 cggggccggg gccggg 16
<210> SEQ ID NO 95 <211> LENGTH: 16 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 95 ccggggccgg ggccgg 16 <210> SEQ ID NO
96 <211> LENGTH: 16 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
96 gccggggccg gggccg 16 <210> SEQ ID NO 97 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 97 gacaagggta
cgtaatctgt c 21 <210> SEQ ID NO 98 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
modified_base <222> LOCATION: (4)..(4) <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION:
(10)..(10) <220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (16)..(16) <400> SEQUENCE: 98
ccggggccgg ggccggggcc 20 <210> SEQ ID NO 99 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (6)..(6) <223>
OTHER INFORMATION: I <220> FEATURE: <221> NAME/KEY:
modified_base <222> LOCATION: (12)..(12) <223> OTHER
INFORMATION: I <400> SEQUENCE: 99 ggccggggcc ggggccgg 18
<210> SEQ ID NO 100 <211> LENGTH: 19 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 100 cagcagcagc agcagcagc 19 <210> SEQ
ID NO 101 <211> LENGTH: 99 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
101 gacaagggta cgtaatctgt ctagagctag aaatagcaag ttaaaataag
gctagtccgt 60 tatcaacttg aaaaagtggc accgagtcgg tgctttttt 99
<210> SEQ ID NO 102 <211> LENGTH: 43 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 102
tagtcctgca ggtttaaacg aattcgtgag tgaggaggcg gca 43 <210> SEQ
ID NO 103 <211> LENGTH: 37 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 103 agcagagctc
agattacgta cccttgttgt gaacaac 37 <210> SEQ ID NO 104
<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer <400> SEQUENCE: 104 caatgtcaac gtctggcatt
acttctactt ttg 33 <210> SEQ ID NO 105 <211> LENGTH: 43
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 105 tagggcgaat tgaatttagc ggccgcactg
gcaggatcat agc 43 <210> SEQ ID NO 106 <211> LENGTH: 30
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 106 tacgtaatct gagctctgct tatatagacc 30
<210> SEQ ID NO 107 <211> LENGTH: 42 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 107
aatgccagac gttgacattg attattgact agttattaat ag 42 <210> SEQ
ID NO 108 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 108 gttaggctct
gggagagtag ttg 23 <210> SEQ ID NO 109 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 109 cctggagcag gtaaatgctg g 21
* * * * *