U.S. patent application number 14/561080 was filed with the patent office on 2015-07-02 for antisense modulation of fibroblast growth factor receptor 4 expression.
This patent application is currently assigned to ISIS PHARMACEUTICALS, INC.. The applicant listed for this patent is ISIS PHARMACEUTICALS, INC.. Invention is credited to Sanjay Bhanot, Michael L. McCaleb, Xing-Xian Yu.
Application Number | 20150184164 14/561080 |
Document ID | / |
Family ID | 47357786 |
Filed Date | 2015-07-02 |
United States Patent
Application |
20150184164 |
Kind Code |
A1 |
Bhanot; Sanjay ; et
al. |
July 2, 2015 |
ANTISENSE MODULATION OF FIBROBLAST GROWTH FACTOR RECEPTOR 4
EXPRESSION
Abstract
Provided herein are methods, compounds, and compositions for
reducing expression of fibroblast growth factor receptor 4 (FGFR4)
mRNA and protein in an animal. Such methods, compounds, and
compositions are useful to treat, prevent, delay, or ameliorate a
metabolic disease, or a symptom thereof.
Inventors: |
Bhanot; Sanjay; (Carlsbad,
CA) ; Yu; Xing-Xian; (San Diego, CA) ;
McCaleb; Michael L.; (La Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ISIS PHARMACEUTICALS, INC. |
Carlsbad |
CA |
US |
|
|
Assignee: |
ISIS PHARMACEUTICALS, INC.
Carlsbad
CA
|
Family ID: |
47357786 |
Appl. No.: |
14/561080 |
Filed: |
December 4, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13631437 |
Sep 28, 2012 |
8933213 |
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14561080 |
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13525197 |
Jun 15, 2012 |
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13631437 |
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61497921 |
Jun 16, 2011 |
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Current U.S.
Class: |
514/44A ;
536/24.5 |
Current CPC
Class: |
A61P 3/00 20180101; C12N
2310/341 20130101; C12N 2310/3231 20130101; C12N 2310/315 20130101;
C12N 2310/3341 20130101; C12N 2310/11 20130101; A61P 3/10 20180101;
C12N 15/1138 20130101; A61K 31/713 20130101; C12N 2310/346
20130101; C12N 2310/323 20130101; C12N 2310/3525 20130101; A61P
3/04 20180101; C12N 2310/321 20130101; A61K 31/711 20130101; C12N
2310/321 20130101; A61K 45/06 20130101; A61P 43/00 20180101; A61P
5/50 20180101; C07H 21/04 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/713 20060101 A61K031/713 |
Claims
1. A compound comprising a modified oligonucleotide consisting of
12 to 30 linked nucleosides, wherein the linked nucleosides
comprise at least 8 contiguous nucleobases of a sequence recited in
SEQ ID NOs: 17, 45, 46, 70, 72, or 138.
2-7. (canceled)
8. The compound of claim 1 wherein the modified oligonucleotide is
a single-stranded oligonucleotide.
9. (canceled)
10. The compound of claim 1, wherein the nucleobase sequence of the
modified oligonucleotide is at least 95% complementary to SEQ ID NO
1 or 2.
11. The compound of claim 1, wherein the nucleobase sequence of the
modified oligonucleotide is 100% complementary to SEQ ID NO 1 or
2.
12. The compound of claim 1 wherein at least one internucleoside
linkage is a modified internucleoside linkage.
13. The compound of claim 12, wherein each internucleoside linkage
is a phosphorothioate internucleoside linkage.
14. The compound of claim 1, wherein at least one nucleoside of the
modified oligonucleotide comprises a modified sugar.
15. The compound of claim 14, wherein the at least one modified
sugar is a bicyclic sugar.
16. The compound of claim 15, wherein each of the at least one
bicyclic sugar comprises a 4'-CH2-N(R)--O-2' bridge wherein R is,
independently, H, C1-C12 alkyl, or a protecting group.
17. The compound of claim 15, wherein each of the at least one
bicyclic sugar comprises a 4'-CH(CH3)-O-2' bridge.
18. The compound of claim 14, wherein at least one modified sugar
comprises a 2'-O-methoxyethyl group.
19. (canceled)
20. (canceled)
21. The compound of claim 1, wherein at least one nucleoside
comprises a modified nucleobase.
22. The compound of claim 21, wherein the modified nucleobase is a
5-methylcytosine.
23. The compound of claim 1, wherein the modified oligonucleotide
comprises: a gap segment consisting of linked deoxynucleosides; a
5' wing segment consisting of linked nucleosides; and a 3' wing
segment consisting of linked nucleosides; wherein the gap segment
is positioned between the 5' wing segment and the 3' wing segment
and wherein each nucleoside of each wing segment comprises a
modified sugar.
24. The compound of claim 23, wherein the modified oligonucleotide
comprises: a gap segment consisting of ten linked deoxynucleosides;
a 5' wing segment consisting of five linked nucleosides; and a 3'
wing segment consisting of five linked nucleosides; wherein the gap
segment is positioned between the 5' wing segment and the 3' wing
segment, wherein each nucleoside of each wing segment comprises a
2'-O-methoxyethyl sugar; and wherein each internucleoside linkage
is a phosphorothioate linkage.
25. (canceled)
26. (canceled)
27. The compound of claim 1, wherein the modified oligonucleotide
consists of 20 linked nucleosides.
28. A composition comprising the compound of claim 1 or salt
thereof and at least one of a pharmaceutically acceptable carrier
or diluent.
29-46. (canceled)
47. A method for reducing or preventing a metabolic disease
comprising administering to a human a therapeutically effective
amount of a compound of claim 1, thereby reducing or preventing a
metabolic disease.
48. The method of claim 47, wherein the metabolic disease is
obesity.
49. The method of claim 47, comprising co-administering the
compound or composition and a second agent.
50-91. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/631,437, filed Sep. 28, 2012, which is a
continuation of U.S. patent application Ser. No. 13/525,197, filed
Jun. 15, 2012, which claims priority under 35 U.S.C. .sctn.119(e)
to U.S. Provisional Patent Application No. 61/497,921, filed Jun.
16, 2011, each of which is incorporated herein by reference in its
entirety.
SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled BIOL0157USC2SEQ_ST25.txt created Dec. 4, 2014, which
is 160 Kb in size. The information in the electronic format of the
sequence listing is incorporated herein by reference in its
entirety.
FIELD
[0003] Provided herein are methods, compounds, and compositions for
reducing expression of fibroblast growth factor receptor 4 (FGFR4)
mRNA and protein in an animal. Such methods, compounds, and
compositions are useful, for example, to treat, prevent, delay or
ameliorate diseases associated with metabolic disorders,
particularly disorders associated with obesity.
BACKGROUND
[0004] Obesity is considered a long-term metabolic disease. There
are several serious medical sequelae related to obesity. There are
over 1 billion overweight individuals worldwide with 100 million
clinically obese. The increasing health care costs of treating
obesity related diseases in the US alone are estimated at over $100
billion annually. Current methods for treating obesity include
behavioral modification, diet, surgery (gastroplasty),
administering pharmaceutical agents that block appetite stimulating
signals or absorption of nutrients (fat), and administering agents
that increase thermogenesis or fat metabolism. Some of these
methods have disadvantages in that they rely on patient resolve,
are invasive, or have unwanted side effects. An understanding of
the mechanisms by which obesity is regulated may provide important
therapeutic information.
[0005] Obesity is frequently associated with insulin resistance and
together constitutes risk factors for later development of type 2
diabetes and cardiovascular diseases. Insulin resistance occurs
well before development of type 2 diabetes, and insulin is
overproduced to compensate for the insulin resistance and to
maintain normal glucose levels. Type 2 diabetes ensues, as the
pancreas can no longer produce enough insulin to maintain normal
glucose levels. Early stages of type 2 diabetes are associated with
elevated levels of insulin but as the disease progresses the
pancreas may fail to produce insulin, resulting in increased blood
glucose levels. Diabetes is a significant risk factor for both
heart disease and stroke and is the leading cause of blindness and
end-stage renal failure.
[0006] Diabetes is a disorder characterized by hyperglycemia due to
deficient insulin action that may result from reduced insulin
production or insulin resistance or both. Diabetes mellitus is a
polygenic disorder affecting a significant portion of the people in
the world. It is divided into two types. In type I diabetes, or
insulin-dependent diabetes mellitus (IDDM), patients produce little
or no insulin, the hormone that regulates glucose utilization. In
type 2 diabetes, or noninsulin-dependent diabetes mellitus (NIDDM),
patients often have plasma insulin levels that are the same
compared to nondiabetic humans; however, these patients have
developed a resistance to the insulin stimulating effect of glucose
and lipid metabolism in the main insulin-sensitive tissues, i.e.,
muscle, liver and adipose tissues, and the plasma insulin levels
are insufficient to overcome the pronounced insulin resistance.
Additionally, glucotoxicity, which results from long-term
hyperglycemia, induces tissue-dependent insulin resistance (Nawano
et al., Am. J. Physiol. Endocrinol. Metab., 278, E535-543)
exacerbating the disease. Type 2 diabetes accounts for over 90% of
all diabetes cases. It is a metabolic disorder characterized by
hyperglycemia leading to secondary complications such as
neuropathy, nephropathy, retinopathy, hypertriglyceridemia,
obesity, and other cardiovascular diseases generally referred to as
metabolic syndrome.
[0007] Metabolic syndrome is a combination of medical disorders
that increase one's risk for cardiovascular disease and diabetes.
The symptoms, including high blood pressure, high triglycerides,
decreased HDL and obesity, tend to appear together in some
individuals. Metabolic syndrome is known under various other names,
such as (metabolic) syndrome X, insulin resistance syndrome or
Reaven's syndrome.
[0008] Diabetes and obesity (sometimes now collectively referred to
as "diabesity") are interrelated in that obesity is known to
exacerbate the pathology of diabetes and greater than 60% of
diabetics are obese. Most human obesity is associated with insulin
resistance and leptin resistance. In fact, it has been suggested
that obesity may have an even greater impact on insulin action than
diabetes itself (Sindelka et al., Physiol Res., 51, 85-91).
Additionally, several compounds on the market for the treatment of
diabetes are known to induce weight gain, a very undesirable side
effect to the treatment of this disease. Therefore, a compound that
has the potential to treat both diabetes and obesity would provide
a significant improvement over current treatments.
[0009] Fibroblast growth factor receptor 4 (also known as FGF
receptor-4, TKF; tyrosine kinase related to fibroblast growth
factor receptor; hydroxyaryl-protein kinase; tyrosylprotein kinase;
Fgfr4; FGFR-4; FGFR4; CD334, FGFR4_HUMAN and JTK2) has high
affinity for the acidic and/or basic fibroblast growth factors.
(Armstrong et al., Genes Chromosomes Cancer, 4, 94-98).
[0010] Although FGFRs generally have been shown to have wide
distribution throughout the body, to date, FGFR4 has only been
found in a few tissues. Among a wide variety of cells and tissues
tested, including human lymphocytes and macrophages, FGFR4 was
found to be expressed in the lung and in some tumors of lung origin
as well as in malignancies not derived from lung tissues. (Holtrich
et al., Proc. Nat. Acad. Sci., 88, 10411-10415). FGFR4 has also
been found to be expressed in the liver and in adipose tissues.
(Patel et al., JCEM, 90(2), 1226-1232). FGFR4 has also been found
to be expressed in certain carcinoma cell lines. (Bange et al.,
Cancer Res., 62, 840-847).
[0011] Additionally, FGFR4 has been shown to play a role in
systemic lipid and glucose homeostasis. FGFR4-deficient mice on a
normal diet exhibited features of metabolic syndrome that include
increase mass of insulin resistance, in addition to
hypercholesterolemia. FGFR4 deficiency was shown to alleviate
high-fat diet-induced fatty liver in a certain obese mouse model,
which is also a correlate of metabolic syndrome. Restoration of
FGFR4, specifically in hepatocytes of FGFR4 deficient mice,
decrease plasma lipid level and restored the high fat diet-induced
fatty liver but failed to restore glucose tolerance and sensitivity
to insulin. (Huang et al., Diabetes, 56, 2501-2510).
[0012] Antisense inhibition of FGFR4 provides a unique advantage
over traditional small molecule inhibitors in that antisense
inhibitors do not rely on competitive binding of the compound to
the protein and inhibit activity directly by reducing the
expression of FGFR4. A representative United States patent that
teaches FGFR4 antisense inhibitors includes US. Pat. Publication
No. US2010/0292140, of which is herein incorporated by reference in
its entirety. Antisense technology is emerging as an effective
means for reducing the expression of certain gene products and may
therefore prove to be uniquely useful in a number of therapeutic,
diagnostic, and research applications for the modulation of
FGFR4.
[0013] There is a currently a lack of acceptable options for
treating metabolic disorders. It is therefore an object herein to
provide compounds and methods for the treatment of such diseases
and disorder. This invention relates to the discovery of novel,
highly potent inhibitors of FGFR4 gene expression.
[0014] All documents, or portions of documents, cited in this
application, including, but not limited to, patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated-by-reference for the portions of the document
discussed herein, as well as in their entirety.
SUMMARY
[0015] Provided herein are methods, compounds, and compositions for
modulating expression of FGFR4 and treating, preventing, delaying
or ameliorating diseases associated with metabolic disorders,
particularly disorders associated with obesity and/or a symptom
thereof.
DETAILED DESCRIPTION
[0016] 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 described herein, as
claimed. Herein, the use of the singular includes the plural unless
specifically stated otherwise. As used herein, the use of "or"
means "and/or" unless stated otherwise. Furthermore, the use of the
term "including" as well as other forms, such as "includes" and
"included", is not limiting. Also, terms such as "element" or
"component" encompass both elements and components comprising one
unit and elements and components that comprise more than one
subunit, unless specifically stated otherwise.
[0017] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including, but not limited to, patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated-by-reference for the portions of the document
discussed herein, as well as in their entirety.
DEFINITIONS
[0018] 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 can be used for
chemical synthesis, and chemical analysis. Where permitted, all
documents, or portions of documents, cited in this application,
including, but not limited to, all patents, applications, published
applications and other journal publications, 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 incorporated by reference for the portions of the
document discussed herein, as well as in their entirety.
[0019] Unless otherwise indicated, the following terms have the
following meanings:
[0020] "2'-O-methoxyethyl" (also 2'-MOE and
2'-O(CH.sub.2).sub.2--OCH.sub.3) refers to an O-methoxy-ethyl
modification of the 2' position of a furosyl ring. A
2'-O-methoxyethyl modified sugar is a modified sugar.
[0021] "2'-O-methoxyethyl nucleotide" means a nucleotide comprising
a 2'-O-methoxyethyl modified sugar moiety.
[0022] "3' target site" refers to the nucleotide of a target
nucleic acid which is complementary to the 3'-most nucleotide of a
particular antisense compound.
[0023] "5' target site" refers to the nucleotide of a target
nucleic acid which is complementary to the 5'-most nucleotide of a
particular antisense compound.
[0024] "5-methylcytosine" means a cytosine modified with a methyl
group attached to the 5' position. A 5-methylcytosine is a modified
nucleobase.
[0025] "About" means within .+-.10% of a value. For example, if it
is stated, "a marker may be increased by about 50%", it is implied
that the marker may be increased between 45%-55%.
[0026] "Active pharmaceutical agent" means the substance or
substances in a pharmaceutical composition that provide a
therapeutic benefit when administered to an individual. For
example, in certain embodiments an antisense oligonucleotide
targeted to FGFR4 is an active pharmaceutical agent.
[0027] "Active target region" or "target region" means a region to
which one or more active antisense compounds is targeted. "Active
antisense compounds" means antisense compounds that reduce target
nucleic acid levels or protein levels.
[0028] "Adipogenesis" means the development of fat cells from
preadipocytes. "Lipogenesis" means the production or formation of
fat, either fatty degeneration or fatty infiltration.
[0029] "Adipose tissue" or "body fat" or "fat depot" is loose
connective tissue composed of adipocytes. Two types of adipose
tissue exist: white adipose tissue (WAT) and brown adipose tissue
(BAT).
[0030] "Adiposity" or "Obesity" refers to the state of being obese
or an excessively high amount of body fat or adipose tissue in
relation to lean body mass. The amount of body fat includes concern
for both the distribution of fat throughout the body and the size
and mass of the adipose tissue deposits. Body fat distribution can
be estimated by skin-fold measures, waist-to-hip circumference
ratios, or techniques such as ultrasound, computed tomography, or
magnetic resonance imaging. According to the Center for Disease
Control and Prevention, individuals with a body mass index (BMI) of
30 or more are considered obese. The term "Obesity" as used herein
includes conditions where there is an increase in body fat beyond
the physical requirement as a result of excess accumulation of
adipose tissue in the body. The term "obesity" includes, but is not
limited to, the following conditions: adult-onset obesity;
alimentary obesity; endogenous or inflammatory obesity; endocrine
obesity; familial obesity; hyperinsulinar obesity;
hyperplastic-hypertrophic obesity; hypogonadal obesity; hypothyroid
obesity; lifelong obesity; morbid obesity and exogenous
obesity.
[0031] "Administered concomitantly" refers to the co-administration
of two 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 agents be administered in
a single pharmaceutical composition, in the same dosage form, or by
the same route of administration. The effects of both 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.
[0032] "Administering" means providing an agent to an animal, and
includes, but is not limited to, administering by a medical
professional and self-administering.
[0033] "Agent" means an active substance that can provide a
therapeutic benefit when administered to an animal. "First Agent"
means a therapeutic compound provided herein. For example, a first
agent can be an antisense oligonucleotide targeting FGFR4. "Second
agent" means a second therapeutic compound described herein (e.g. a
second antisense oligonucleotide targeting FGFR4) and/or a
non-FGFR4 therapeutic compound.
[0034] "Amelioration" refers to a lessening of at least one
indicator, sign, or symptom of an associated disease, disorder, or
condition. The severity of indicators can be determined by
subjective or objective measures, which are known to those skilled
in the art.
[0035] "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.
[0036] "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 encoded by such target nucleic acid.
[0037] "Antisense compound" means an oligomeric compound that is
capable of undergoing hybridization to a target nucleic acid
through hydrogen bonding.
[0038] "Antisense inhibition" means reduction of target nucleic
acid levels or target protein levels in the presence of an
antisense compound complementary to a target nucleic acid compared
to target nucleic acid levels or target protein levels in the
absence of the antisense compound.
[0039] "Antisense oligonucleotide" means a single-stranded
oligonucleotide having a nucleobase sequence that permits
hybridization to a corresponding region or segment of a target
nucleic acid.
[0040] "Bicyclic sugar" means a furosyl ring modified by the
bridging of two non-geminal ring atoms. A bicyclic sugar is a
modified sugar.
[0041] "Bicyclic nucleic acid" or "BNA" refers to a nucleoside or
nucleotide wherein the furanose portion of the nucleoside or
nucleotide includes a bridge connecting two carbon atoms on the
furanose ring, thereby forming a bicyclic ring system.
[0042] "Biomarker" is meant to designate a gene or protein or
protein fragment which is indicative of the effect of an FGFR4
inhibitor. That means the "biomarker" is used as a detection
agent.
[0043] "Cap structure" or "terminal cap moiety" means chemical
modifications, which have been incorporated at either terminus of
an antisense compound.
[0044] "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 nucleotides is chemically distinct
from a region having nucleotides without 2'-O-methoxyethyl
modifications.
[0045] "Chimeric antisense compound" means an antisense compound
that has at least two chemically distinct regions.
[0046] "Co-administration" means administration of two or more
agents to an individual. The two or more agents can be in a single
pharmaceutical composition, or can be in separate pharmaceutical
compositions. Each of the two or more agents can be administered
through the same or different routes of administration.
Co-administration encompasses parallel or sequential
administration.
[0047] "Cholesterol" is a sterol molecule found in the cell
membranes of all animal tissues. Cholesterol must be transported in
an animal's blood plasma by lipoproteins including very low density
lipoprotein (VLDL), intermediate density lipoprotein (IDL), low
density lipoprotein (LDL), and high density lipoprotein (HDL).
"Plasma cholesterol" refers to the sum of all lipoproteins (VDL,
IDL, LDL, HDL) esterified and/or non-esterified cholesterol present
in the plasma or serum.
[0048] "Complementarity" means the capacity for pairing between
nucleobases of a first nucleic acid and a second nucleic acid.
[0049] "cEt" or "constrained ethyl" means a bicyclic 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'.
[0050] "Constrained ethyl nucleoside" (also cEt nucleoside) means a
nucleoside comprising a bicyclic sugar moiety comprising a
4'-CH(CH.sub.3)--O-2' bridge.
[0051] "Contiguous nucleobases" means nucleobases immediately
adjacent to each other.
[0052] "Deoxyribonucleotide" means a nucleotide having a hydrogen
at the 2' position of the sugar portion of the nucleotide.
Deoxyribonucleotides may be modified with any of a variety of
substituents.
[0053] "Diabetes mellitus" or "diabetes" is a syndrome
characterized by disordered metabolism and abnormally high blood
sugar (hyperglycemia) resulting from insufficient levels of insulin
or reduced insulin sensitivity. The characteristic symptoms are
excessive urine production (polyuria) due to high blood glucose
levels, excessive thirst and increased fluid intake (polydipsia)
attempting to compensate for increased urination, blurred vision
due to high blood glucose effects on the eye's optics, unexplained
weight loss, and lethargy.
[0054] "Diabetic dyslipidemia" or "type 2 diabetes with
dyslipidemia" means a condition characterized by Type 2 diabetes,
reduced HDL-C, elevated triglycerides, and elevated small, dense
LDL particles.
[0055] "Diluent" means an ingredient in a composition that lacks
pharmacological activity, but is pharmaceutically necessary or
desirable. For example, the diluent in an injected composition can
be a liquid, e.g. saline solution.
[0056] "Dyslipidemia" refers to a disorder of lipid and/or
lipoprotein metabolism, including lipid and/or lipoprotein
overproduction or deficiency. Dyslipidemias may be manifested by
elevation of lipids such as cholesterol and triglycerides as well
as lipoproteins such as low-density lipoprotein (LDL)
cholesterol.
[0057] "Dosage unit" means a form in which a pharmaceutical agent
is provided, e.g. pill, tablet, or other dosage unit known in the
art. In certain embodiments, a dosage unit is a vial containing
lyophilized antisense oligonucleotide. In certain embodiments, a
dosage unit is a vial containing reconstituted antisense
oligonucleotide.
[0058] "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 can 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 can 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 can be stated as the amount of pharmaceutical
agent per hour, day, week, or month.
[0059] "Effective amount" or "therapeutically effective amount"
means the amount of active pharmaceutical agent sufficient to
effectuate a desired physiological outcome in an individual in need
of the agent. The effective amount can 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.
[0060] "Fibroblast growth factor 4" or "FGFR4" means any nucleic
acid or protein of FGFR4.
[0061] "FGFR4 expression" means the level of mRNA transcribed from
the gene encoding FGFR4 or the level of protein translated from the
mRNA. FGFR4 expression can be determined by art known methods such
as a Northern or Western blot.
[0062] "FGFR4 nucleic acid" means any nucleic acid encoding FGFR4.
For example, in certain embodiments, a FGFR4 nucleic acid includes
a DNA sequence encoding FGFR4, a RNA sequence transcribed from DNA
encoding FGFR4 (including genomic DNA comprising introns and
exons), and a mRNA sequence encoding FGFR4. "FGFR4 mRNA" means a
mRNA encoding a FGFR4 protein.
[0063] "Fully complementary" or "100% complementary" means each
nucleobase of a nucleobase sequence of a first nucleic acid has a
complementary nucleobase in a second nucleobase sequence of 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.
[0064] "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
can be referred to as a "gap segment" and the external regions can
be referred to as "wing segments."
[0065] "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 one to six nucleosides.
[0066] "Glucose" is a monosaccharide used by cells as a source of
energy and inflammatory intermediate. "Plasma glucose" refers to
glucose present in the plasma.
[0067] "Hybridization" means the annealing of complementary nucleic
acid molecules. In certain embodiments, complementary nucleic acid
molecules include an antisense compound and a target nucleic
acid.
[0068] "Hyperlipidemia" or "hyperlipemia" is a condition
characterized by elevated serum lipids or circulating (plasma)
lipids. This condition manifests an abnormally high concentration
of fats. The lipid fractions in the circulating blood are
cholesterol, low density lipoproteins, very low density
lipoproteins and triglycerides.
[0069] "Hypertriglyceridemia" means a condition characterized by
elevated triglyceride levels.
[0070] "Identifying" or "selecting an animal with metabolic" means
identifying or selecting a subject having been diagnosed with a
metabolic disease, or a metabolic disorder; or, identifying or
selecting a subject having any symptom of a metabolic disease,
including, but not limited to, metabolic syndrome, hyperglycemia,
hypertriglyceridemia, hypertension increased insulin resistance,
decreased insulin sensitivity, above normal body weight, and/or
above normal body fat or any combination thereof. Such
identification may be accomplished by any method, including but not
limited to, standard clinical tests or assessments, such as
measuring serum or circulating (plasma) blood-glucose, measuring
serum or circulating (plasma) triglycerides, measuring
blood-pressure, measuring body fat, measuring body weight, and the
like.
[0071] "Immediately adjacent" means there are no intervening
elements between the immediately adjacent elements.
[0072] "Individual" or "subject" or "animal" means a human or
non-human animal selected for treatment or therapy.
[0073] "Inhibiting the expression or activity" refers to a
reduction or blockade of the expression or activity of a RNA or
protein and does not necessarily indicate a total elimination of
expression or activity.
[0074] "Insulin resistance" is defined as the condition in which
normal amounts of insulin are inadequate to produce a normal
insulin response from fat, muscle and liver cells. Insulin
resistance in fat cells results in hydrolysis of stored
triglycerides, which elevates free fatty acids in the blood plasma.
Insulin resistance in muscle reduces glucose uptake whereas insulin
resistance in liver reduces glucose storage, with both effects
serving to elevate blood glucose. High plasma levels of insulin and
glucose due to insulin resistance often leads to metabolic syndrome
and type 2 diabetes.
[0075] "Insulin sensitivity" is a measure of how effectively an
individual processes glucose. An individual having high insulin
sensitivity effectively processes glucose whereas an individual
with low insulin sensitivity does not effectively process
glucose.
[0076] "Internucleoside linkage" refers to the chemical bond
between nucleosides.
[0077] "Intravenous administration" means administration into a
vein.
[0078] "Linked nucleosides" means adjacent nucleosides which are
bonded together.
[0079] "Lipid-lowering therapy" or "lipid lowering agent" means a
therapeutic regimen provided to a subject to reduce one or more
lipids in a subject. In certain embodiments, a lipid-lowering
therapy is provided to reduce one or more of ApoB, total
cholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small
dense LDL particles, and Lp(a) in a subject. Examples of
lipid-lowering therapy include statins, fibrates, and MTP
inhibitors.
[0080] "Major risk factors" refers to factors that contribute to a
high risk for a particular disease or condition. In certain
embodiments, major risk factors for coronary heart disease include,
without limitation, cigarette smoking, hypertension, low HDL-C,
family history of coronary heart disease, age, and other factors
disclosed herein.
[0081] "Metabolic disease" or "metabolic disorder" refers to a
condition characterized by an alteration or disturbance in
metabolic function. "Metabolic" and "metabolism" are terms well
known in the art and generally include the whole range of
biochemical processes that occur within a living organism.
Metabolic diseases or disorders include, but are not limited to,
obesity, diabetes, hyperglycemia, prediabetes, non-alcoholic fatty
liver disease (NAFLD), metabolic syndrome, insulin resistance,
diabetic dyslipidemia, or hypertriglyceridemia or a combination
thereof.
[0082] "Metabolic syndrome" means a condition characterized by a
clustering of lipid and non-lipid cardiovascular risk factors of
metabolic origin. In certain embodiments, metabolic syndrome is
identified by the presence of any 3 of the following factors: waist
circumference of greater than 102 cm in men or greater than 88 cm
in women; serum triglyceride of at least 150 mg/dL; HDL-C less than
40 mg/dL in men or less than 50 mg/dL in women; blood pressure of
at least 130/85 mmHg; and fasting glucose of at least 110 mg/dL.
These determinants can be readily measured in clinical practice
(JAMA, 2001, 285: 2486-2497).
[0083] "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.
[0084] "Mixed dyslipidemia" means a condition characterized by
elevated cholesterol and elevated triglycerides.
[0085] "Modified internucleoside linkage" refers to a substitution
or any change from a naturally occurring internucleoside bond (i.e.
a phosphodiester internucleoside bond).
[0086] "Modified nucleobase" refers to any nucleobase other than
adenine, cytosine, guanine, thymidine, or uracil. An "unmodified
nucleobase" means the purine bases adenine (A) and guanine (G), and
the pyrimidine bases thymine (T), cytosine (C), and uracil (U).
[0087] "Modified nucleoside" means a nucleoside having,
independently, a modified sugar moiety or modified nucleobase.
[0088] "Modified nucleotide" means a nucleotide having,
independently, a modified sugar moiety, modified internucleoside
linkage, or modified nucleobase. A "modified nucleoside" means a
nucleoside having, independently, a modified sugar moiety or
modified nucleobase.
[0089] "Modified oligonucleotide" means an oligonucleotide
comprising at least one modified nucleotide.
[0090] "Modified sugar" refers to a substitution or change from a
natural sugar.
[0091] "Motif" means the pattern of chemically distinct regions in
an antisense compound.
[0092] "Naturally occurring internucleoside linkage" means a 3' to
5' phosphodiester linkage.
[0093] "Natural sugar moiety" means a sugar found in DNA (2'-H) or
RNA (2'-OH).
[0094] "Non-alcoholic fatty liver disease" or "NAFLD" means a
condition characterized by fatty inflammation of the liver that is
not due to excessive alcohol use (for example, alcohol consumption
of over 20 g/day). In certain embodiments, NAFLD is related to
insulin resistance and the metabolic syndrome. NAFLD encompasses a
disease spectrum ranging from simple triglyceride accumulation in
hepatocytes (hepatic steatosis) to hepatic steatosis with
inflammation (steatohepatitis), fibrosis, and cirrhosis.
[0095] "Nonalcoholic steatohepatitis" (NASH) occurs from
progression of NAFLD beyond deposition of triglycerides. A "second
hit" capable of inducing necrosis, inflammation, and fibrosis is
required for development of NASH. Candidates for the second-hit can
be grouped into broad categories: factors causing an increase in
oxidative stress and factors promoting expression of
proinflammatory cytokines
[0096] "Nucleic acid" refers to molecules composed of monomeric
nucleotides. A nucleic acid includes ribonucleic acids (RNA),
deoxyribonucleic acids (DNA), single-stranded nucleic acids,
double-stranded nucleic acids, small interfering ribonucleic acids
(siRNA), and microRNAs (miRNA). A nucleic acid can also comprise a
combination of these elements in a single molecule.
[0097] "Nucleobase" means a heterocyclic moiety capable of pairing
with a base of another nucleic acid.
[0098] "Nucleobase sequence" means the order of contiguous
nucleobases independent of any sugar, linkage, or nucleobase
modification.
[0099] "Nucleoside" means a nucleobase linked to a sugar.
[0100] "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.
[0101] "Nucleotide" means a nucleoside having a phosphate group
covalently linked to the sugar portion of the nucleoside.
[0102] "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).
[0103] "Oligomeric compound" or "oligomer" refers to a polymeric
structure comprising two or more sub-structures and capable of
hybridizing to a region of a nucleic acid molecule. In certain
embodiments, oligomeric compounds are oligonucleosides. In certain
embodiments, oligomeric compounds are oligonucleotides. In certain
embodiments, oligomeric compounds are antisense compounds. In
certain embodiments, oligomeric compounds are antisense
oligonucleotides. In certain embodiments, oligomeric compounds are
chimeric oligonucleotides.
[0104] "Oligonucleotide" means a polymer of linked nucleosides each
of which can be modified or unmodified, independent one from
another.
[0105] "Parenteral administration" means administration through
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.
Administration can be continuous, or chronic, or short or
intermittent.
[0106] "Peptide" means a molecule formed by linking at least two
amino acids by amide bonds. Peptide refers to polypeptides and
proteins.
[0107] "Pharmaceutical agent" means a substance that provides a
therapeutic benefit when administered to an individual. For
example, in certain embodiments, an antisense oligonucleotide
targeted to FGFR4 is pharmaceutical agent.
[0108] "Pharmaceutical composition" means a mixture of substances
suitable for administering to an individual. For example, a
pharmaceutical composition can comprise one or more active agents
and a sterile aqueous solution.
[0109] "Pharmaceutically acceptable carrier" means a medium or
diluent that does not interfere with the structure of the
oligonucleotide. Certain, of such carries enable pharmaceutical
compositions to be formulated as, for example, tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspension and
lozenges for the oral ingestion by a subject. For example, a
pharmaceutically acceptable carrier can be a sterile aqueous
solution.
[0110] "Pharmaceutically acceptable derivative" encompasses
pharmaceutically acceptable salts, conjugates, prodrugs or isomers
of the compounds described herein.
[0111] "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.
[0112] "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.
[0113] "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.
[0114] "Prevent" refers to delaying or forestalling the onset or
development of a disease, disorder, or condition for a period of
time from minutes to indefinitely. Prevent also means reducing risk
of developing a disease, disorder, or condition.
[0115] "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.
[0116] "Side effects" means physiological responses attributable to
a treatment other than the desired effects. In certain embodiments,
side effects include injection site reactions, liver function test
abnormalities, renal function abnormalities, liver toxicity, renal
toxicity, central nervous system abnormalities, myopathies, and
malaise. For example, increased aminotransferase levels in serum
can indicate liver toxicity or liver function abnormality. For
example, increased bilirubin can indicate liver toxicity or liver
function abnormality.
[0117] "Single-stranded oligonucleotide" means an oligonucleotide
which is not hybridized to a complementary strand.
[0118] "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.
[0119] "Statin" means an agent that inhibits the activity of
HMG-CoA reductase.
[0120] "Subcutaneous administration" means administration just
below the skin.
[0121] "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.
[0122] "Target nucleic acid," "target RNA," and "target RNA
transcript" all refer to a nucleic acid capable of being targeted
by antisense compounds.
[0123] "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.
[0124] "Therapeutically effective amount" means an amount of an
agent that provides a therapeutic benefit to an individual.
[0125] "Therapeutic lifestyle change" means dietary and lifestyle
changes intended to lower fat/adipose tissue mass and/or
cholesterol. Such change can reduce the risk of developing heart
disease, and may includes recommendations for dietary intake of
total daily calories, total fat, saturated fat, polyunsaturated
fat, monounsaturated fat, carbohydrate, protein, cholesterol,
insoluble fiber, as well as recommendations for physical
activity.
[0126] "Triglyceride" or "TG" means a lipid or neutral fat
consisting of glycerol combined with three fatty acid
molecules.
[0127] "Type 2 diabetes," (also known as "type 2 diabetes mellitus"
or "diabetes mellitus, type 2", and formerly called "diabetes
mellitus type 2", "non-insulin-dependent diabetes (NIDDM)",
"obesity related diabetes", or "adult-onset diabetes") is a
metabolic disorder that is primarily characterized by insulin
resistance, relative insulin deficiency, and hyperglycemia.
[0128] "Treat" refers to administering a pharmaceutical composition
to an animal to effect an alteration or improvement of a disease,
disorder, or condition.
[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).
Certain Embodiments
[0130] Certain embodiments provide methods, compounds, and
compositions for inhibiting FGFR4 expression.
[0131] Certain embodiments provide antisense compounds targeted to
a FGFR4 nucleic acid. In certain embodiments, the FGFR4 nucleic
acid is any of the sequences set forth in GENBANK Accession No.
NM.sub.--002011.3 (incorporated herein as SEQ ID NO: 1), GENBANK
Accession No: NT.sub.--023133.11 truncated from nucleosides
21323018 to 21335213 (incorporated herein as SEQ ID NO: 2); and
GENBANK Accession No. AB209631.1 (incorporated herein as SEQ ID NO:
3); and GENBANK Accession No NM.sub.--022963.2 (incorporated herein
as SEQ ID NO: 4). In certain embodiments, FGFR4 has the rhesus
monkey sequence as set forth in GENBANK Accession No.
NW.sub.--001121000.1 truncated from nucleosides 3094000 to 3109000
(SEQ ID NO: 5). In certain embodiments, FGFR4 has the murine
sequence as set forth in GENBANK Accession No. BC033313.1 (SEQ ID
NO: 6).
[0132] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
12 to 30 nucleosides having a nucleobase sequence complementary to
an equal length portion of any of SEQ ID NOs: 1-6.
[0133] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
12 to 30 nucleosides having a nucleobase sequence complementary to
an equal length portion of any of SEQ ID NOs: 1-4.
[0134] In certain embodiments, the compounds or compositions
provided herein consist of 12 to 30 linked nucleosides and have a
nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 contiguous nucleobases of any of SEQ ID
NOs: 7-322.
[0135] In certain embodiments, the compounds or compositions
provided herein can consist of 12 to 30 linked nucleosides and have
a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 contiguous nucleobases of any of SEQ ID
NOs: 16, 17, 45, 46, 70, 72, or 138.
[0136] In certain embodiments, the compound or composition provided
herein is or comprises ISIS NOs: 463588, 463589, 463690, 463691,
463835, 463837, or 464225.
[0137] In certain embodiments, the compounds or compositions
provided herein consist of 12 to 30 linked nucleosides and have a
nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 contiguous nucleobases of SEQ ID NO:
16.
[0138] In certain embodiments, the compounds or compositions
provided herein consist of 12 to 30 linked nucleosides and have a
nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 contiguous nucleobases of SEQ ID NO:
45.
[0139] In certain embodiments, the compound or composition is or
comprises ISIS NO: 463588.
[0140] In certain embodiments, the compound or composition is or
comprises ISIS NO: 463690.
[0141] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
15 to 30 nucleosides having a nucleobase sequence complementary to
an equal length portion of any of SEQ ID NOs: 1-4.
[0142] In certain embodiments, the compounds or compositions
provided herein consist of 15 to 30 linked nucleosides and have a
nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 contiguous nucleobases of any of SEQ ID
NOs: 7-322.
[0143] In certain embodiments, the compounds or compositions
provided herein consist of 15 to 30 linked nucleosides and have a
nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 contiguous nucleobases of any of SEQ ID
NOs: 16, 17, 45, 46, 70, 72, or 138.
[0144] In certain embodiments, the compound or composition provided
herein is or comprises ISIS NOs: 463588, 463589, 463690, 463691,
463835, 463837, or 464225.
[0145] In certain embodiments, the compounds or compositions
provided herein consist of 15 to 30 linked nucleosides and have a
nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 contiguous nucleobases of SEQ ID NO:
16.
[0146] In certain embodiments, the compounds or compositions
provided herein consist of 15 to 30 linked nucleosides and have a
nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 contiguous nucleobases of SEQ ID NO:
45.
[0147] In certain embodiments, the compound or composition provided
herein is or comprise ISIS NO: 463588.
[0148] In certain embodiments, the compound or composition provided
herein is or comprise ISIS NO: 463690.
[0149] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
18 to 21 nucleosides having a nucleobase sequence complementary to
an equal length portion of any of SEQ ID NOs: 1-4.
[0150] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
18 to 21 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of any of SEQ ID NOs: 7-322.
[0151] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
18 to 21 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of any of SEQ ID NOs: 16, 17, 45, 46,
70, 72, or 138
[0152] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
18 to 21 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 16.
[0153] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
18 to 21 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 45.
[0154] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 35 nucleosides having a nucleobase sequence complementary to
an equal length portion of any of SEQ ID NOs: 1-4.
[0155] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 35 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NOs: 7-322.
[0156] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 35 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NOs: 16, 17, 45, 46, 70, 72,
or 138.
[0157] In certain embodiments, the compounds or compositions
provided herein can consist of 20 to 35 linked nucleosides and have
a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 contiguous nucleobases of SEQ ID NO:
16.
[0158] In certain embodiments, the compounds or compositions
provided herein can consist of 20 to 35 linked nucleosides and have
a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 contiguous nucleobases of SEQ ID NO:
45.
[0159] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 30 nucleosides having a nucleobase sequence complementary to
an equal length portion of any of SEQ ID NOs: 1-4.
[0160] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 30 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NOs: 7-322.
[0161] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 30 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NOs: 16, 17, 45, 46, 70, 72,
or 138.
[0162] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 30 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 16.
[0163] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 30 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 45.
[0164] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 25 nucleosides having a nucleobase sequence complementary to
an equal length portion of any of SEQ ID NOs: 1-4.
[0165] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 25 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NOs: 7-322.
[0166] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 25 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 16, 17, 45, 46, 70, 72,
or 138.
[0167] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 25 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 16.
[0168] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 25 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 45.
[0169] In certain embodiments, the compounds or compositions
described herein comprise a modified oligonucleotide consisting of
20 to 24 nucleosides having a nucleobase sequence complementary to
an equal length portion of any of SEQ ID NOs: 1-4.
[0170] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 24 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NOs: 7-322.
[0171] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 24 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 16, 17, 45, 46, 70, 72,
or 138.
[0172] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 24 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 16.
[0173] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 24 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 45.
[0174] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 23 nucleosides having a nucleobase sequence complementary to
an equal length portion of any of SEQ ID NOs: 1-4.
[0175] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 23 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NOs: 7-322.
[0176] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 23 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 16, 17, 45, 46, 70, 72,
or 138.
[0177] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 23 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 16.
[0178] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 23 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 45.
[0179] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 22 nucleosides having a nucleobase sequence complementary to
an equal length portion of any of SEQ ID NOs: 1-4.
[0180] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 22 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NOs: 7-322.
[0181] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 22 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 16, 17, 45, 46, 70, 72,
or 138.
[0182] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 221 inked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 16.
[0183] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 22 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 45.
[0184] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 21 nucleosides having a nucleobase sequence complementary to
an equal length portion of any of SEQ ID NOs: 1-4.
[0185] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 21 linked nucleosides and having a nucleobase sequence
complementary to an equal length portion of any of SEQ ID NOs:
1-4.
[0186] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 21 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NOs: 7-322.
[0187] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 21 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 16, 17, 45, 46, 70, 72,
or 138.
[0188] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 21 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 16.
[0189] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 to 21 linked nucleosides and have a nucleobase sequence
comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 contiguous nucleobases of SEQ ID NO: 45.
[0190] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 nucleosides having a nucleobase sequence complementary to an
equal length portion of any of SEQ ID NOs: 1-4.
[0191] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 linked nucleosides and have a nucleobase sequence comprising at
least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
contiguous nucleobases of SEQ ID NOs: 7-322.
[0192] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 linked nucleosides and have a nucleobase sequence comprising at
least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
contiguous nucleobases of SEQ ID NO: 16, 17, 45, 46, 70, 72, or
138.
[0193] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 linked nucleosides and have a nucleobase sequence comprising at
least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
contiguous nucleobases of SEQ ID NO: 16.
[0194] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 linked nucleosides and have a nucleobase sequence comprising at
least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
contiguous nucleobases of SEQ ID NO: 45.
[0195] In certain embodiments, the compounds or compositions
provided herein comprise a salt of the modified
oligonucleotide.
[0196] In certain embodiments, the compounds or compositions
provided herein further comprise a pharmaceutically acceptable
carrier or diluent.
[0197] In certain embodiments, the nucleobase sequence of the
modified oligonucleotide is at least 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% complementary to any one of SEQ ID NOs:
1-4 as measured over the entirety of the modified
oligonucleotide.
[0198] In certain embodiments, the nucleobase sequence of the
modified oligonucleotide has at least 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 7-322
as measured over the entirety of the modified oligonucleotide.
[0199] In certain embodiments, the nucleobase sequence of the
modified oligonucleotide has at least 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 16,
17, 45, 46, 70, 72, or 138 as measured over the entirety of the
modified oligonucleotide.
[0200] In certain embodiments, the nucleobase sequence of the
modified oligonucleotide has at least 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 16 as measured
over the entirety of the modified oligonucleotide.
[0201] In certain embodiments, the nucleobase sequence of the
modified oligonucleotide has at least 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 45 as measured
over the entirety of the modified oligonucleotide.
[0202] In certain embodiments, antisense compounds or modified
oligonucleotides targets a region of a FGFR4 nucleic acid. In
certain embodiments, such compounds or oligonucleotides targeted to
a region of a FGFR4 nucleic acid have a contiguous nucleobase
portion that is complementary to an equal length nucleobase portion
of the region. For example, the portion can be at least an 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases
portion complementary to an equal length portion of a region
recited herein. In certain embodiments, such compounds or
oligonucleotide target the following nucleotide regions of SEQ ID
NO: 1: 160-179, 191-210, 191-211, 191-212, 191-213, 192-211,
192-212, 192-213, 193-212, 193-213, 194-213, 196-215, 196-216,
197-216, 200-219, 202-221, 202-222, 203-222, 290-310, 290-309,
290-311, 290-312, 290-312, 291-310, 291-311, 291-312, 292-311,
292-312, 293-312, 309-328, 332-351, 338-357, 338-358, 339-358,
347-366, 349-368, 357-376, 368-387, 368-388, 368-389, 368-390,
368-391, 369-380, 369-389, 369-390, 369-391, 370-389, 370-390,
370-391, 371-390, 371-391, 372-391, 388-407, 388-408, 389-408,
392-411, 404-423, 431-450, 431-451, 432-451, 443-462, 443-463,
444-463, 601-620, 624-643, 734-753, 757-806, 787-807, 788-807,
790-809, 790-810, 791-810, 970-989, 1024-1043, 1024-1044,
1024-1045, 1024-1046, 1024-1047, 1024-1048, 1024-1105, 1025-1044,
1025-1045, 1025-1046, 1025-1047, 1025-1048, 1026-1045, 1026-1046,
1026-1047, 1026-1048, 1027-1046, 1027-1047, 1027-1048, 1028-1047,
1028-1048, 1029-1048, 1031-1050, 1031-1051, 1032-1051, 1084-1103,
1084-1105, 1086-1105, 1097-1116, 1097-1117, 1097-1122, 1100-1119,
1100-1120, 1100-1121, 1100-1122, 1101-1120, 1101-1121, 1101-1122,
1102-1121, 1102-1122, 1103-1122, 1105-1124, 1105-1125, 1106-1125,
1110-1029, 1110-1130, 1111-1130, 1115-1134, 1185-1204, 1255-1274,
1290-1309, 1290-1310, 1291-1310, 1301-1320, 1417-1436, 1468-1487,
1468-1488, 1469-1488, 1559-1578, 1562-1581, 1564-1583, 1619-1638,
2325-2344, 2325-2345, 2326-2345, 2438-2457, 2812-2831, 2816-2835,
2816-2836, 2816-2837, 2816-2838, 2817-2836, 2817-2837, 2817-2838,
2818-2837, 2818-2838, 2819-2838, 2822-2481, 2822-2842, 2822-2843,
2822-2844, 2823-2842, 2823-2843, 2823-2844, 2824-2843, 2824-2844,
2825-2844, 2951-2970, 2951-2971, 2951-2972, 2951-2973, 2951-2974,
2951-2975, 2951-3000, 2952-2971, 2952-2972, 2952-2973, 2952-2974,
2952-2975, 2953-2972, 2953-2973, 2953-2974, 2953-2975, 2954-2973,
2954-2974, 2954-2975, 2955-2974, 2955-2975, 2956-2975.
[0203] In certain embodiments, antisense compounds or modified
oligonucleotides targets a region of a FGFR4 nucleic acid. In
certain embodiments, such compounds or oligonucleotides targeted to
a region of a FGFR4 nucleic acid have a contiguous nucleobase
portion that is complementary to an equal length nucleobase portion
of the region. For example, the portion can be at least an 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases
portion complementary to an equal length portion of a region
recited herein. In certain embodiments, such compounds or
oligonucleotide target the following nucleotide regions of SEQ ID
NO: 2: 3165-3184, 3196-3215, 3197-3216, 3196-3217, 3196-3218,
3197-3216, 3197-3217, 3197-3218, 3198-3217, 3198-3218, 3199-3218,
3201-3220, 3201-3221, 3202-3221, 3205-3224, 3207-3226, 3207-3227,
3208-3227, 3991-4011, 3991-4010, 3991-4012, 3991-4013, 3992-4011,
3992-4012, 3992-4013, 3993-4012, 3993-4013, 3994-4013, 4010-4029,
4033-4052, 4039-4058, 4039-4059, 4040-4059, 4048-4067, 4050-4069,
4058-4077, 4069-4088, 4069-4089, 4069-4091, 4069-4091, 4069-4092,
4070-4091, 4070-4090, 4070-4091, 4070-4092, 4071-4090, 4071-4091,
4071-4092, 4072-4091, 4072-4092, 4073-4092, 4089-4108, 4089-4109,
4090-4109, 4093-4112, 4105-4124, 4132-4151, 4132-4152, 4133-4152,
4144-4163, 4144-4164, 4145-4164, 4506-4522, 4528-4547, 4638-4657,
5268-5290, 5271-5291, 5272-5291, 5274-5293, 5274-5294, 5275-5294,
5966-5985, 6020-6039, 6020-6040, 6020-6041, 6020-6042, 6020-6043,
6020-6044, 6020-6235, 6021-6040, 6021-6041, 6021-6042, 6021-6043,
6021-6044, 6022-6041, 6022-6042, 6022-6043, 6022-6044, 6023-6042,
6023-6043, 6023-6044, 6024-6043, 6024-6044, 6025-6044, 6027-6046,
6027-6047, 6028-6047, 6214-6235, 6214-6233, 6214-6235, 6216-6235,
6227-6246, 6227-6247, 6227-6252, 6230-6249, 6230-6250, 6230-6251,
6230-6252, 6231-6250, 6231-6251, 6231-6252, 6232-6251, 6232-6252,
6233-6252, 6235-6254, 6235-6255, 6236-6255, 6241-6260, 6245-6264,
6315-6334, 6784-6803, 6974-6993, 7025-7044, 7025-7045, 7026-7045,
7059-7081, 7221-7240, 7223-7242, 7278-7297, 10866-10885,
10866-10866, 10867-10886, 11108-11127, 11482-11501, 11486-11505,
11486-11506, 11486-11507, 11486-11508, 11487-11506, 11487-11507,
11487-11508, 11488-11507, 11488-11508, 11489-11508, 11492-11511,
11492-11512, 11492-11513, 11492-1151, 11493-11512, 11493-11513,
11493-11514, 11494-11513, 11494-11514, 11495-11514, 11621-11640,
11621-11641, 11621-11642, 11621-11643, 11621-11644, 11621-11645,
11621-11670, 11622-11641, 11622-11642, 11622-11643, 11622-11644,
11622-11645, 11623-11642, 11623-11643, 11623-11644, 11623-11645,
11624-11643, 11624-11644, 11624-11645, 11625-11644, 11625-11645,
11626-11645.
[0204] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides wherein the
linked nucleosides comprise at least an 8 contiguous nucleobase
portion that is complementary to an equal length nucleobase portion
within the region selected from nucleotides 191-210 or 369-388 of
SEQ ID NO: 1. In certain embodiments, the modified oligonucleotide
has at least a 9, at least a 10, at least an 11, at least a 12, at
least a 13, at least a 14, at least a 15, at least a 16, at least a
17, at least an 18, at least 19 or at least a 20 contiguous
nucleobase portion of which is complementary to an equal length
portion within the region selected from nucleotides 191-210 or
369-388 of SEQ ID NO: 1. In certain embodiments, the modified
oligonucleotide is 90%, 95%, 99%, or 100% complementary to a
nucleic acid encoding human FGFR4, eg. SEQ ID No: 1.
[0205] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides wherein the
linked nucleosides comprise at least an 8 contiguous nucleobase
portion that is complementary to an equal length nucleobase portion
within the region selected from nucleotides 3196-3215 or 4070-4089
of SEQ ID NO: 2. In certain embodiments, the modified
oligonucleotide has at least a 9, at least a 10, at least an 11, at
least a 12, at least a 13, at least a 14, at least a 15, at least a
16, at least a 17, at least an 18, at least 19 or at least a 20
contiguous nucleobase portion of which is complementary to an equal
length portion within the region selected from nucleotides
3196-3215 or 4070-4089 of SEQ ID NO: 2. In certain embodiments, the
modified oligonucleotide is 90%, 95%, 99%, or 100% complementary to
a nucleic acid encoding human FGFR4, eg. SEQ ID No: 2.
[0206] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 60%
complementary within the region selected from nucleotides 191-210,
193-212, 369-388, 370-389, 788-807, 790-809 and 2954-2973 of SEQ ID
NO: 1.
[0207] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 60%
complementary within the region selected from nucleotides
3196-3215, 3198-3217, 4070-4089, 4071-4090, 5272-5291, 5274-5293,
and 11624-11643 of SEQ ID NO: 2.
[0208] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 70%
complementary within the region selected from nucleotides 191-210,
193-212, 369-388, 370-389, 188-807, 790-809 and 2954-2973 of SEQ ID
NO: 1.
[0209] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 70%
complementary within the region selected from nucleotides
3196-3215, 3198-3217, 4070-4089, 4071-4090, 5272-5291, 5274-5293,
and 11624-11643 of SEQ ID NO: 2.
[0210] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 80%
complementary within the region selected from nucleotides 191-210,
193-212, 369-388, 370-389, 188-807, 790-809 and 2954-2973 of SEQ ID
NO: 1.
[0211] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 80%
complementary within the region selected from nucleotides
3196-3215, 3198-3217, 4070-4089, 4071-4090, 5272-5291, 5274-5293,
and 11624-11643 of SEQ ID NO: 2.
[0212] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 90%
complementary within the region selected from nucleotides 191-210,
193-212, 369-388, 370-389, 188-807, 790-809 and 2954-2973 of SEQ ID
NO: 1.
[0213] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 90%
complementary within the region selected from nucleotides
3196-3215, 3198-3217, 4070-4089, 4071-4090, 5272-5291, 5274-5293,
and 11624-11643 of SEQ ID NO: 2.
[0214] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 95%
complementary within the region selected from nucleotides 191-210,
193-212, 369-388, 370-389, 188-807, 790-809 and 2954-2973 of SEQ ID
NO: 1.
[0215] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 95%
complementary within the region selected from nucleotides
3196-3215, 3198-3217, 4070-4089, 4071-4090, 5272-5291, 5274-5293,
and 11624-11643 of SEQ ID NO: 2.
[0216] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 99%
complementary within the region selected from nucleotides 191-210,
193-212, 369-388, 370-389, 188-807, 790-809 and 2954-2973 of SEQ ID
NO: 1.
[0217] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 99%
complementary within the region selected from nucleotides
3196-3215, 3198-3217, 4070-4089, 4071-4090, 5272-5291, 5274-5293,
and 11624-11643 of SEQ ID NO: 2.
[0218] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 100%
complementary within the region selected from nucleotides 191-210,
193-212, 369-388, 370-389, 188-807, 790-809 and 2954-2973 of SEQ ID
NO: 1.
[0219] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 100%
complementary within the region selected from nucleotides
3196-3215, 3198-3217, 4070-4089, 4071-4090, 5272-5291, 5274-5293,
and 11624-11643 of SEQ ID NO: 2.
[0220] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 60%
complementary within nucleotides 191-210 of SEQ ID NO: 1.
[0221] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 60%
complementary within the region selected from nucleotides 3196-3215
of SEQ ID NO: 2.
[0222] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 70%
complementary within nucleotides 191-210 of SEQ ID NO: 1.
[0223] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 70%
complementary within the region selected from nucleotides 3196-3215
of SEQ ID NO: 2.
[0224] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 80%
complementary within nucleotides 191-210 of SEQ ID NO: 1.
[0225] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 80%
complementary within the region selected from nucleotides 3196-3215
of SEQ ID NO: 2.
[0226] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 90%
complementary within nucleotides 191-210 of SEQ ID NO: 1.
[0227] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 90%
complementary within the region selected from nucleotides 3196-3215
of SEQ ID NO: 2.
[0228] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 95%
complementary within nucleotides 191-210 of SEQ ID NO: 1.
[0229] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 95%
complementary within the region selected from nucleotides 3196-3215
of SEQ ID NO: 2.
[0230] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 99%
complementary within nucleotides 191-210 of SEQ ID NO: 1.
[0231] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 99%
complementary within the region selected from nucleotides 3196-3215
of SEQ ID NO: 2.
[0232] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 100%
complementary within nucleotides 191-210 of SEQ ID NO: 1.
[0233] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 100%
complementary within the region selected from nucleotides 3196-3215
of SEQ ID NO: 2.
[0234] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 60%
complementary within nucleotides 369-388 of SEQ ID NO: 1.
[0235] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 60%
complementary within nucleotides 4070-4089 of SEQ ID NO: 2.
[0236] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 70%
complementary within nucleotides 369-388 of SEQ ID NO: 1.
[0237] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 70%
complementary within nucleotides 4070-4089 of SEQ ID NO: 2.
[0238] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 80%
complementary within nucleotides 369-388 of SEQ ID NO: 1.
[0239] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 80%
complementary within nucleotides 4070-4089 of SEQ ID NO: 2.
[0240] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 90%
complementary within nucleotides 369-388 of SEQ ID NO: 1.
[0241] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 90%
complementary within nucleotides 4070-4089 of SEQ ID NO: 2.
[0242] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 95%
complementary within nucleotides 369-388 of SEQ ID NO: 1.
[0243] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 95%
complementary within nucleotides 4070-4089 of SEQ ID NO: 2.
[0244] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 99%
complementary within nucleotides 369-388 of SEQ ID NO: 1.
[0245] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 99%
complementary within nucleotides 4070-4089 of SEQ ID NO: 2.
[0246] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 100%
complementary within nucleotides 369-388 of SEQ ID NO: 1.
[0247] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 99%
complementary within nucleotides 4070-4089 of SEQ ID NO: 2.
[0248] In certain embodiments, such compounds or oligonucleotides
targeted to a region of a FGFR4 nucleic acid have a contiguous
nucleobase portion that is complementary to an equal length
nucleobase portion of the region 191-210 or 369-388 of SEQ ID NO:
1.
[0249] In certain embodiments, such compounds or oligonucleotides
targeted to a region of a FGFR4 nucleic acid have a contiguous
nucleobase portion that is complementary to an equal length
nucleobase portion of the region 3196-3215 or 4070-4089 of SEQ ID
NO: 2.
[0250] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 1, when targeted by antisense compounds or
oligonucleotides, displays at least 65% inhibition: 160-179,
191-210, 191-211, 191-212, 191-213, 192-211, 192-212, 192-213,
193-212, 193-213, 194-213, 196-215, 196-216, 197-216, 200-219,
202-221, 202-222, 203-222, 290-210, 290-309, 290-311, 290-312,
290-312, 291-310, 291-311, 291-312, 292-311, 292-312, 293-312,
309-328, 332-351, 338-357, 338-358, 339-358, 347-366, 349-368,
357-376, 368-387, 368-388, 368-389, 368-390, 368-391, 369-380,
369-389, 369-390, 369-391, 370-389, 370-390, 370-391, 371-390,
371-391, 372-391, 388-407, 388-408, 389-408, 392-411, 404-423,
431-450, 431-451, 432-451, 443-462, 443-463, 444-463, 601-620,
624-643, 734-753, 767-806, 787-807, 788-807, 790-809, 790-810,
791-810, 970-989, 1024-1043, 1024-1044, 1024-1045, 1024-1046,
1024-1047, 1024-1048, 1024-1105, 1025-1044, 1025-1045, 1025-1046,
1025-1047, 1025-1048, 1026-1045, 1026-1046, 1026-1047, 1026-1048,
1027-1046, 1027-1047, 1027-1048, 1028-1047, 1028-1048, 1029-1048,
1031-1050, 1031-1051, 1032-1051, 1074-1051, 1084-1103, 1084-1105,
1086-1105, 1097-1116, 1097-1117, 1097-1122, 1100-1119, 1100-1119,
1100-1120, 1100-1121, 1100-1122, 1101-1120, 1101-1121, 1101-1122,
1102-1121, 1102-1122, 1103-1122, 1105-1124, 1105-1125, 1106-1125,
1110-1029, 1110-1130, 1111-1130, 1115-1134, 1185-1204, 1255-1274,
1290-1309, 1290-1310, 1291-1310, 1301-1320, 1417-1436, 1468-1487,
1468-1488, 1469-1488, 1559-1578, 1562-1581, 1564-1583, 1619-1638,
2325-2344, 2325-2345, 2326-2345, 2438-2457, 2812-2831, 2816-2835,
2816-2836, 2816-2837, 2816-2838, 2817-2836, 2817-2837, 2817-2838,
2818-2837, 2818-2838, 2819-2838, 2822-2481, 2822-2842, 2822-2843,
2822-2844, 2822-2844, 2823-2842, 2823-2843, 2823-2844, 2824-2843,
2824-2844, 2825-2844, 2951-2970, 2951-2971, 2951-2972, 2951-2973,
2951-2974, 2951-2975, 2951-2975, 2951-3000, 2952-2971, 2952-2972,
2952-2973, 2952-2974, 2952-2975, 2953-2972, 2953-2973, 2953-2974,
2953-2975, 2954-2973, 2954-2974, 2954-2975, 2955-2974, 2955-2975,
and 2956-2975.
[0251] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 2, when targeted by antisense compounds or
oligonucleotides, displays at least 65% inhibition: 3165-3184,
3196-3215, 3196-3216, 3196-3217, 3196-3218, 3197-3216, 3197-3217,
3197-3218, 3198-3217, 3198-3218, 3199-3218, 3201-3220, 3201-3221,
3202-3221, 3205-3224, 3207-3226, 3207-3227, 3208-3227, 3991-4011,
3991-4010, 3991-4012, 3991-4013, 3991-4014, 3992-4011, 3992-4012,
3992-4013, 3993-4012, 3993-4013, 3994-4013, 4010-4029, 4033-4052,
4039-4058, 4039-4059, 4040-4059, 4048-4067, 4050-4069, 4058-4077,
4069-4088, 4069-4089, 4069-4090, 4069-4091, 4069-4092, 4070-380,
4070-4090, 4070-4091, 4070-4092, 4071-4090, 4071-4091, 4071-4092,
4072-4091, 4072-4092, 4073-4092, 4089-4108, 4089-4109, 4090-4109,
4093-4112, 4105-4124, 4132-4151, 4132-4152, 4133-4152, 4144-4163,
4144-4164, 4145-4164, 4506-4522, 4528-4547, 4638-4657, 5268-5290,
5271-5291, 5272-5291, 5274-5293, 5274-5294, 5275-5294, 5966-5985,
6020-6039, 6020-6040, 6020-6041, 6020-6042, 6020-6043, 6020-6044,
6020-6045, 6021-6040, 6021-6041, 6021-6042, 6021-6043, 6021-6044,
6022-6041, 6022-6042, 6022-6043, 6022-6044, 6023-6042, 6023-6043,
6023-6047, 6024-6043, 6024-6044, 6025-6044, 6027-6046, 6027-6047,
6028-6047, 6214-6235, 6214-6233, 6214-6235, 6216-6235, 6227-6246,
6227-6247, 6227-6252, 6230-6249, 6230-6249, 6230-6250, 6230-6251,
6230-6252, 6231-6250, 6231-6251, 6231-6252, 6232-6251, 6232-6252,
6233-6252, 6235-6254, 6235-6255, 6236-6255, 6230-6260, 6241-6260,
6245-6264, 6315-6334, 6784-6803, 6974-6993, 7025-7044, 7025-7045,
7026-7045, 7221-7240, 7223-7242, 7278-7297, 10866-10885,
10866-10886, 10867-10886, 11008-11127, 11482-11501, 11486-11505,
11486-11506, 11486-11507, 11486-11508, 11487-11506, 11487-11507,
11487-11508, 11488-11507, 11488-11508, 11489-11508, 11492-11511,
11492-11512, 11492-11513, 11492-11514, 11493-11512, 11493-11513,
11493-11514, 11494-11513, 11494-11514, 11495-11514, 11621-11640,
11621-11641, 11621-11642, 11621-11643, 11621-11644, 11621-11645,
11621-11670, 11622-11641, 11622-11642, 11622-11643, 11622-11644,
11622-11645, 11623-11642, 11623-11643, 11623-11644, 11623-1645,
11624-11643, 11624-11644, 11624-11645, 11625-11644, 11625-11645,
and 11626-11645.
[0252] In certain embodiments, the nucleobase sequences recited in
the following SEQ ID NOs of SEQ ID NO: 1, when targeted by
antisense compounds or oligonucleotides, displays at least 65%
inhibition: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 59, 61, 62, 64, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, 111, 112, 113, 114, 115, and 116.
[0253] In certain embodiments, the nucleobase sequences recited in
the following SEQ ID NOs of SEQ ID NO: 1, when targeted by
antisense compounds or oligonucleotides, displays at least 70%
inhibition: 7, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 27, 28, 29,
30, 32, 33, 34, 35, 38, 39, 43, 44, 45, 46, 47, 48, 49, 50, 51, 54,
59, 61, 64, 69, 70, 72, 73, 75, 77, 78, 79, 80, 81, 82, 83, 85, 86,
87, 89, 90, 91, 92, 94, 97, 98, 103, 105, 106, 111, 112, 113, and
116.
[0254] In certain embodiments, the nucleobase sequences recited in
the following SEQ ID NOs of SEQ ID NO: 1, when targeted by
antisense compounds or oligonucleotides, displays at least 75%
inhibition: 7, 14, 16, 17, 22, 24, 28, 29, 30, 32, 33, 34, 39, 43,
44, 45, 46, 47, 49, 50, 59, 61, 69, 70, 72, 73, 75, 77, 78, 79, 80,
83, 85, 89, 90, 91, 92, 105, 106, 111, and 112.
[0255] In certain embodiments, the nucleobase sequences recited in
the following SEQ ID NOs of SEQ ID NO: 1, when targeted by
antisense compounds or oligonucleotides, displays at least 80%
inhibition: 7, 14, 16, 17, 28, 29, 33, 39, 45, 47, 49, 50, 72, 80,
90, 91, and 106.
[0256] In certain embodiments, the nucleobase sequences recited in
the following SEQ ID NOs of SEQ ID NO: 1, when targeted by
antisense compounds or oligonucleotides, displays at least 85%
inhibition 7, 14, 16, 29, 45, 50, 80, 90, and 91.
[0257] In certain embodiments, the following nucleotide regions of
SEQ ID NOs: 1 or 2, when targeted by antisense compounds or
oligonucleotides, displays at least 65% inhibition: 908-927,
992-1011, 1138-1157, 1138-1161, 1142-1161, 1345-1364, 1386-1405,
1386-1413, 1394-1413, 1461-1480, 1461-1482, 1461-1484, 1461-1486,
1461-1490, 1463-1482, 1463-1484, 1463-1486, 1463-1490, 1465-1484,
1465-1486, 1465-1490, 1467-1486, 1467-1490, 1471-1490, 1542-1561,
1941-1960, 1941-1962, 1941-1964, 1943-1962, 1943-1964, 1945-1964,
2053-2072, 2104-2123, 2104-2125, 2104-2127, 2104-2129, 2104-2131,
2104-2133, 2104-2135, 2104-2137, 2106-2125, 2106-2127, 2106-2129,
2106-2131, 2106-2133, 2106-2135, 2106-2137, 2108-2127, 2108-2129,
2108-2131, 2108-2133, 2108-2135, 2108-2137, 2110-2129, 2110-2131,
2110-2133, 2110-2135, 2110-2137, 2112-2131, 2112-2133, 2112-2135,
2112-2137, 2114-2133, 2114-2135, 2114-2137, 2116-2135, 2116-2137,
2118-2137, 2271-2290, 2838-2857, 3122-3141, 3122-3144, 3125-3144,
3165-3184, 3325-3344, 3325-3346, 3325-3348, 3325-3350, 3325-3352,
3325-3354, 3325-3356, 3325-3358, 3325-3360, 3325-3362, 3325-3362,
3327-3346, 3327-3346, 3327-3348, 3327-3350, 3327-3352, 3327-3354,
3327-3356, 3327-3358, 3327-3360, 3327-3362, 3329-3348, 3329-3348,
3329-3350, 3329-3352, 3329-3354, 3329-3356, 3329-3358, 3329-3360,
3329-3362, 3331-3350, 3331-3352, 3331-3354, 3331-3356, 3331-3358,
3331-3360, 3331-3362, 3333-3352, 3333-3354, 3333-3356, 3333-3358,
3333-3360, 3333-3362, 3335-3354, 3335-3356, 3335-3358, 3335-3360,
3335-3362, 3337-3356, 3337-3358, 3337-3360, 3337-3362, 3339-3358,
3339-3360, 3339-3362, 3341-3360, 3341-3362, 3343-3362, 3386-3405,
3386-3413, 3386-3417, 3386-3419, 3386-3423, 3386-3427, 3386-3434,
3386-3434, 3394-3413, 3394-3417, 3394-3423, 3394-3427, 3394-3434,
3398-3417, 3398-3419, 3398-3423, 3398-3427, 3398-3434, 3400-3419,
3400-3423, 3400-3427, 3400-3434, 3404-3423, 3404-3427, 3404-3434,
3408-3427, 3408-3434, 3415-3434, 3445-3464, 3445-3466, 3445-3468,
3445-3470, 3447-3466, 3447-3468, 3447-3470, 3449-3468, 3449-3470,
3451-3470, 3499-3518, 3571-3590, 3571-3592, 3571-3594, 3571-3596,
3571-3598, 3573-3592, 3573-3594, 3573-3596, 3573-3598, 3575-3594,
3575-3596, 3575-3598, 3577-3596, 3577-3598, 3579-3598, 3772-3791,
3772-3793, 3772-3795, 3772-3797, 3772-3801, 3772-3807, 3772-3817,
3774-3793, 3774-3795, 3774-3797, 3776-3795, 3776-3797, 3778-3797,
3782-3801, 3782-3817, 3788-3807, 3788-3817, 3798-3817, 3993-4012,
4799-4818, 7684-7703, 7690-7709, 7692-7711.
[0258] In certain embodiments, the nucleobase sequences recited in
the following SEQ ID NOs of SEQ ID NOs: 1 or 2, when targeted by
antisense compounds or oligonucleotides, displays at least 65%
inhibition: 29, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,
127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,
140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,
153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,
166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,
179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,
192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,
205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,
218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,
231, 232, 233, 234, and 235.
[0259] In certain embodiments, the nucleobase sequences recited in
the following SEQ ID NOs of SEQ ID NOs: 1 or 2, when targeted by
antisense compounds or oligonucleotides, displays at least 70%
inhibition: 29, 117, 119, 120, 122, 126, 127, 128, 129, 130, 131,
132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 145, 146,
147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 160, 161, 162,
163, 164, 165, 166, 167, 169, 170, 171, 174, 180, 183, 184, 185,
186, 187, 188, 189, 190, 193, 195, 198, 199, 200, 201, 202, 203,
206, 207, 208, 209, 210, 211, 213, 214, 215, 216, 217, 218, 219,
220, 221, 222, 223, 225, 226, 227, 228, 229, 231, 233, 234, and
235.
[0260] In certain embodiments, the nucleobase sequences recited in
the following SEQ ID NOs of SEQ ID NOs: 1 or 2, when targeted by
antisense compounds or oligonucleotides, displays at least 75%
inhibition: 29, 117, 120, 128, 129, 131, 132, 133, 135, 136, 137,
138, 139, 140, 141, 146, 152, 153, 154, 155, 156, 160, 161, 162,
163, 164, 165, 166, 167, 169, 174, 180, 186, 187, 188, 198, 199,
201, 202, 207, 208, 209, 213, 214, 215, 216, 217, 219, 220, 221,
223, 225, 227, 228, 229, 231, 233, and 235.
[0261] In certain embodiments, the nucleobase sequences recited in
the following SEQ ID NOs of SEQ ID NOs: 1 or 2, when targeted by
antisense compounds or oligonucleotides, displays at least 80%
inhibition: 29, 117, 131, 132, 133, 135, 136, 137, 138, 140, 141,
152, 153, 154, 155, 156, 160, 162, 163, 164, 174, 186, 187, 188,
199, 201, 202, 207, 208, 213, 214, 215, 216, 217, 219, 220, 221,
223, 227, 229, 231, and 233.
[0262] In certain embodiments, the nucleobase sequences recited in
the following SEQ ID NOs of SEQ ID NOs: 1 or 2, when targeted by
antisense compounds or oligonucleotides, displays at least 85%
inhibition: 29, 117, 132, 135, 136, 140, 141, 154, 156, 163, 164,
187, 188, 199, 201, 215, 216, 217, 219, 220, 221, 223, 227, 229,
231, and 233.
[0263] In certain embodiments, the following nucleotide regions of
SEQ ID NOs: 1 or 2 or 3, when targeted by antisense compounds or
oligonucleotides, displays at least 65% inhibition: 101-120,
101-122, 101-124, 101-125, 101-126, 101-127, 102-126, 103-122,
103-124, 103-125, 103-126, 103-127, 105-124, 105-125, 105-127,
106-125, 106-126, 106-127, 107-126, 107-127, 108-127, 1122-1141,
1165-1184, 1193-1218, 1198-1217, 1199-1218, 1323-1342, 1323-1344,
1323-1346, 1323-1347, 1323-1352, 1325-1344, 1325-1346, 1325-1347,
1327-1346, 1327-1347, 1328-1347, 1333-1352, 1333-1354, 1333-1354,
1333-1356, 1333-1358, 1333-1360, 1335-1354, 1335-1356, 1335-1358,
1335-1360, 1337-1356, 1337-1358, 1337-1360, 1339-1358, 1339-1360,
1341-1360, 1392-1411, 1392-1417, 1393-1412, 1394-1413, 1394-1415,
1394-1417, 1396-1415, 1396-1417, 1398-1417, 1413-1432, 1413-1433,
1413-1434, 1414-1433, 1414-1434, 1415-1434, 1445-1464, 1445-1466,
1445-1468, 1445-1470, 1445-1471, 1447-1466, 1447-1468, 1447-1470,
1447-1471, 1449-1468, 1449-1470, 1449-1471, 1451-1470, 1451-1471,
1452-1471, 1462-1481, 1462-1481, 1462-1482, 1462-1483, 1462-1484,
1462-1485, 1462-1487, 1463-1482, 1463-1482, 1463-1483, 1463-1484,
1463-1485, 1463-1487, 1464-1483, 1464-1484, 1464-1485, 1464-1487,
1465-1484, 1465-1485, 1465-1487, 1466-1485, 1466-1487, 1468-1487,
1501-1521, 1501-1522, 1503-1522, 1569-1588, 1569-1589, 1569-1590,
1569-1591, 1569-1592, 1569-1593, 1569-1594, 1569-1596, 1569-1598,
1570-1589, 1570-1590, 1570-1591, 1570-1592, 1570-1593, 1570-1594,
1570-1596, 1570-1598, 1571-1590, 1571-1591, 1571-1591, 1571-1592,
1571-1592, 1571-1593, 1571-1593, 1571-1594, 1571-1594, 1571-1596,
1571-1596, 1571-1598, 1571-1598, 1572-1591, 1572-1592, 1572-1593,
1572-1594, 1572-1596, 1572-1598, 1573-1592, 1573-1593, 1573-1594,
1573-1596, 1573-1598, 1574-1593, 1574-1594, 1574-1596, 1574-1598,
1575-1594, 1575-1596, 1575-1598, 1577-1596, 1577-1598, 1579-1598,
1778-1797, 1778-1799, 1778-1805, 1778-1809, 1778-1811, 1778-1821,
1780-1799, 1786-1805, 1790-1809, 1790-1811, 1790-1821, 1792-1811,
1792-1821, 1802-1821, 1944-1963, 1996-2015, 2053-2072, 2074-2093,
2418-2437, 4988-5007, 5120-5139, 5121-5140, 5121-5146, 5122-5141,
5122-5142, 5122-5143, 5122-5144, 5122-5146, 5123-5142, 5123-5143,
5123-5144, 5123-5146, 5124-5143, 5124-5144, 5124-5144, 5124-5146,
5125-5146, 5127-5146, 5150-5169, 5150-5170, 5150-5171, 5151-5170,
5151-5171, 5152-5171, 7801-7820, 7801-7822, 7803-7822.
[0264] In certain embodiments, the nucleobase sequences recited in
the following SEQ ID NOs of SEQ ID NOs: 1 or 2 or 3, when targeted
by antisense compounds or oligonucleotides, displays at least 65%
inhibition: 29, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,
249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,
262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,
275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287,
288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,
301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313,
314, 315, 316, 317, 318, 319, 320, 321, and 322.
[0265] In certain embodiments, the nucleobase sequences recited in
the following SEQ ID NOs of SEQ ID NOs: 1 or 2 or 3, when targeted
by antisense compounds or oligonucleotides, displays at least 70%
inhibition: 29, 239, 240, 241, 242, 243, 244, 245, 247, 249, 250,
251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,
264, 265, 266, 268, 269, 270, 271, 272, 274, 275, 276, 277, 278,
279, 280, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 294,
295, 296, 297, 298, 299, 300, 301, 304, 305, 306, 307, 308, 309,
310, 311, 312, 313, 314, 315, 317, 319, 320, and 322.
[0266] In certain embodiments, the nucleobase sequences recited in
the following SEQ ID NOs of SEQ ID NOs: 1 or 2 or 3, when targeted
by antisense compounds or oligonucleotides, displays at least 75%
inhibition: 29, 239, 240, 241, 242, 243, 244, 245, 247, 250, 251,
252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 264, 266,
269, 271, 272, 274, 275, 276, 277, 278, 279, 283, 284, 286, 287,
288, 291, 298, 299, 300, 305, 306, 307, 308, 309, 310, 312, 313,
315, 317, 319, and 320.
[0267] In certain embodiments, the nucleobase sequences recited in
the following SEQ ID NOs of SEQ ID NOs: 1 or 2 or 3, when targeted
by antisense compounds or oligonucleotides, displays at least 80%
inhibition: 29, 241, 242, 243, 244, 245, 247, 250, 253, 254, 255,
256, 259, 260, 261, 262, 264, 266, 271, 272, 274, 276, 278, 283,
284, 286, 287, 299, 300, 305, 306, 307, 308, 310, 312, 313, 317,
and 320.
[0268] In certain embodiments, the nucleobase sequences recited in
the following SEQ ID NOs of SEQ ID NOs: 1 or 2 or 3, when targeted
by antisense compounds or oligonucleotides, displays at least 85%
inhibition: 29, 241, 242, 243, 244, 247, 254, 256, 259, 260, 264,
272, 278, 299, 300, 305, 306, 307, 308, 310, 317, and 320.
[0269] In certain embodiments, the following antisense compounds
target a region of SEQ ID NO: 1, a nucleic acid encoding human
FGFR4, and demonstrate at least 65% inhibition of a FGFR4 mRNA:
ISIS NOs: 299005, 299010, 299018, 299022, 299024, 299025, 299028,
299029, 299030, 463588, 463589, 463590, 463592, 463593, 463594,
463596, 463598, 463599, 463601, 463625, 463627, 463628, 463629,
463630, 463636, 463645, 463648, 463654, 463655, 463656, 463657,
463670, 463672, 463673, 463677, 463678, 463679, 463689, 463690,
463691, 463692, 463693, 463708, 463709, 463712, 463717, 463718,
463724, 463733, 463734, 463735, 463751, 463763, 463770, 463774,
463791, 463805, 463832, 463834, 463835, 463836, 463837, 463838,
463860, 463861, 463871, 463874, 463875, 463876, 463877, 463878,
463880, 463882, 463883, 463884, 463893, 463894, 463906, 463907,
463908, 463909, 463910, 463912, 463913, 463918, 463919, 463922,
463937, 463938, 463947, 463967, 463994, 464002, 464004, 464013,
464014, 464015, 464030, 464033, 464037, 464038, 464041, 464043,
464046, 464048, and 464049.
[0270] In certain embodiments, the following antisense compounds
target a region of SEQ ID NO: 1, a nucleic acid encoding human
FGFR4 and demonstrate at least 70% inhibition of a FGFR4 mRNA: ISIS
NOs: 299005, 299029, 299030, 463588, 463589, 463590, 463592,
463593, 463596, 463598, 463599, 463627, 463628, 463629, 463630,
463645, 463648, 463654, 463655, 463670, 463672, 463679, 463689,
463690, 463691, 463692, 463693, 463708, 463709, 463712, 463724,
463751, 463763, 463774, 463834, 463835, 463837, 463838, 463861,
463874, 463875, 463876, 463877, 463878, 463880, 463882, 463884,
463893, 463894, 463907, 463908, 463909, 463910, 463913, 463922,
463937, 464002, 464013, 464014, 464038, 464041, 464043, and
464049.
[0271] In certain embodiments, the following antisense compounds
target a region of SEQ ID NO: 1, a nucleic acid encoding human
FGFR4 and demonstrate at least 75% inhibition of a FGFR4 mRNA: ISIS
NOs: 299005, 299029, 463588, 463589, 463596, 463599, 463628,
463629, 463630, 463645, 463648, 463654, 463672, 463679, 463689,
463690, 463691, 463692, 463708, 463709, 463751, 463763, 463834,
463835, 463837, 463838, 463861, 463874, 463875, 463876, 463877,
463882, 463884, 463907, 463908, 463909, 463910, 464013, 464014,
464038, and 464041.
[0272] In certain embodiments, the following antisense compounds
target a region of SEQ ID NO: 1, a nucleic acid encoding human
FGFR4 and demonstrate at least 80% inhibition of a FGFR4 mRNA: ISIS
NOs: 299005, 299029, 463588, 463589, 463628, 463629, 463648,
463672, 463690, 463692, 463708, 463709, 463837, 463877, 463908,
463909, and 464014.
[0273] In certain embodiments, the following antisense compounds
target a region of SEQ ID NO: 1, a nucleic acid encoding human
FGFR4 and demonstrate at least 85% inhibition of a FGFR4 mRNA: ISIS
NOs: 299005, 299029, 463588, 463629, 463690, 463709, 463877,
463908, and 463909.
[0274] In certain embodiments, the following antisense compounds
target a region of SEQ ID NOs: 1 or 2, a nucleic acid encoding
human FGFR4, and demonstrate at least 65% inhibition of a FGFR4
mRNA: ISIS NOs: 463629, 299004, 464138, 464139, 464167, 464168,
464170, 464173, 299055, 464181, 464203, 464207, 464208, 464209,
464210, 464213, 464214, 464215, 464216, 464222, 464223, 464224,
464225, 464226, 464227, 464228, 464238, 464239, 464254, 464258,
464266, 464268, 464269, 464270, 464278, 464280, 464284, 464285,
464286, 464287, 464288, 464290, 464291, 464292, 464298, 464299,
464300, 464308, 464309, 464310, 464311, 464333, 464342, 464425,
464428, 464429, 464430, 464433, 464449, 464453, 464568, 464569,
464575, 464576, 464579, 464581, 464582, 464584, 464585, 464586,
464587, 464588, 464589, 464590, 464591, 464593, 464617, 464622,
464623, 464657, 464658, 464677, 464682, 464683, 464684, 464685,
464686, 464687, 464688, 464689, 464692, 464696, 464698, 464699,
464701, 464703, 464705, 464706, 464707, 464708, 464709, 464710,
464711, 464716, 464717, 464718, 464719, 464720, 464726, 464727,
464728, 464729, 464730, 464732, 464734, 464735, 464736, 464740,
464800, and 464801.
[0275] In certain embodiments, the following antisense compounds
target a region of SEQ ID NOs: 1 or 2, a nucleic acid encoding
human FGFR4 and demonstrate at least 70% inhibition of a FGFR4
mRNA: ISIS NOs: 463629, 299004, 464138, 464139, 464168, 464203,
464207, 464208, 464209, 464210, 464213, 464214, 464215, 464216,
464222, 464223, 464224, 464225, 464226, 464227, 464228, 464238,
464258, 464266, 464268, 464278, 464280, 464284, 464285, 464286,
464287, 464288, 464290, 464291, 464298, 464299, 464300, 464308,
464309, 464310, 464311, 464333, 464425, 464428, 464429, 464449,
464579, 464584, 464585, 464586, 464587, 464588, 464589, 464590,
464591, 464622, 464657, 464682, 464683, 464684, 464685, 464686,
464687, 464692, 464696, 464698, 464699, 464701, 464703, 464706,
464707, 464708, 464709, 464710, 464711, 464716, 464717, 464718,
464719, 464720, 464727, 464728, 464729, 464730, 464732, 464735,
464740, 464800, and 464801.
[0276] In certain embodiments, the following antisense compounds
target a region of SEQ ID NOs: 1 or 2, a nucleic acid encoding
human FGFR4 and demonstrate at least 75% inhibition of a FGFR4
mRNA: ISIS NOs: 463629, 299004, 464139, 464208, 464209, 464213,
464214, 464215, 464222, 464223, 464224, 464225, 464226, 464227,
464228, 464266, 464284, 464285, 464286, 464287, 464288, 464298,
464299, 464300, 464308, 464309, 464310, 464311, 464333, 464425,
464449, 464579, 464587, 464588, 464589, 464682, 464683, 464685,
464686, 464696, 464698, 464699, 464706, 464707, 464708, 464709,
464710, 464716, 464717, 464718, 464720, 464727, 464729, 464730,
464732, 464735, 464740, and 464801.
[0277] In certain embodiments, the following antisense compounds
target a region of SEQ ID NOs: 1 or 2, a nucleic acid encoding
human FGFR4 and demonstrate at least 80% inhibition of a FGFR4
mRNA: ISIS NOs: 463629, 299004, 464213, 464214, 464215, 464222,
464223, 464224, 464225, 464227, 464228, 464284, 464285, 464286,
464287, 464288, 464298, 464300, 464308, 464309, 464449, 464587,
464588, 464589, 464683, 464685, 464686, 464696, 464698, 464706,
464707, 464708, 464709, 464710, 464716, 464717, 464718, 464720,
464729, 464732, 464735, and 464740.
[0278] In certain embodiments, the following antisense compounds
target a region of SEQ ID NOs: 1 or 2, a nucleic acid encoding
human FGFR4 and demonstrate at least 85% inhibition of a FGFR4
mRNA: ISIS NOs: 463629, 299004, 464214, 464222, 464223, 464227,
464228, 464286, 464288, 464308, 464309, 464588, 464589, 464683,
464685, 464708, 464709, 464710, 464716, 464717, 464718, 464720,
464729, 464732, 464735, and 464740.
[0279] In certain embodiments, the following antisense compounds
target a region of SEQ ID NOs:1 or 2 or 3, a nucleic acid encoding
human FGFR4, and demonstrate at least 65% inhibition of a FGFR4
mRNA: ISIS NOs: 463629, 479530, 479532, 479533, 479534, 479535,
479536, 479537, 479538, 479539, 479540, 479541, 479542, 479543,
479544, 479545, 479546, 479547, 479548, 479549, 479550, 479551,
479552, 479553, 479554, 479555, 479556, 479557, 479558, 479560,
479561, 479562, 479564, 479565, 479566, 479567, 479568, 479569,
479570, 479572, 479573, 479574, 479576, 479577, 479582, 479583,
479584, 479585, 479594, 479596, 479597, 479608, 479613, 479614,
479622, 479625, 479626, 479641, 479682, 479689, 479690, 479691,
479692, 479693, 479694, 479696, 479697, 479698, 479699, 479703,
479704, 479705, 479706, 479716, 479721, 479722, 479725, 479731,
479732, 479736, 479737, 479738, 479739, 479740, and 479741.
[0280] In certain embodiments, the following antisense compounds
target a region of SEQ ID NOs: 1 or 2 or 3, a nucleic acid encoding
human FGFR4 and demonstrate at least 70% inhibition of a FGFR4
mRNA: ISIS NOs: 463629, 479530, 479532, 479533, 479534, 479535,
479536, 479537, 479539, 479541, 479542, 479543, 479544, 479545,
479546, 479547, 479548, 479549, 479550, 479551, 479552, 479553,
479554, 479555, 479556, 479557, 479558, 479561, 479562, 479564,
479565, 479566, 479568, 479569, 479570, 479572, 479573, 479574,
479576, 479582, 479583, 479584, 479585, 479594, 479596, 479597,
479608, 479613, 479614, 479626, 479641, 479682, 479689, 479690,
479691, 479692, 479693, 479697, 479698, 479699, 479703, 479704,
479705, 479706, 479716, 479721, 479722, 479725, 479731, 479736,
479738, 479739, and 479741.
[0281] In certain embodiments, the following antisense compounds
target a region of SEQ ID NOs: 1 or 2 or 3, a nucleic acid encoding
human FGFR4 and demonstrate at least 75% inhibition of a FGFR4
mRNA: ISIS NOs: 463629, 479530, 479532, 479533, 479534, 479535,
479536, 479537, 479539, 479542, 479543, 479544, 479545, 479546,
479547, 479548, 479549, 479550, 479551, 479552, 479553, 479554,
479556, 479558, 479562, 479565, 479566, 479568, 479569, 479570,
479572, 479573, 479574, 479583, 479584, 479594, 479596, 479597,
479614, 479690, 479691, 479692, 479698, 479699, 479703, 479704,
479705, 479706, 479721, 479722, 479731, 479736, 479738, and
479739.
[0282] In certain embodiments, the following antisense compounds
target a region of SEQ ID NOs: 1 or 2 or 3, a nucleic acid encoding
human FGFR4 and demonstrate at least 80% inhibition of a FGFR4
mRNA: ISIS NOs: 463629, 479533, 479534, 479535, 479536, 479537,
479539, 479542, 479545, 479546, 479547, 479548, 479551, 479552,
479553, 479554, 479556, 479558, 479565, 479566, 479568, 479570,
479573, 479583, 479584, 479594, 479596, 479691, 479692, 479698,
479699, 479703, 479704, 479706, 479721, 479722, 479736, and
479739.
[0283] In certain embodiments, the following antisense compounds
target a region of SEQ ID NOs: 1 or 2 or 3, a nucleic acid encoding
human FGFR4 and demonstrate at least 85% inhibition of a FGFR4
mRNA: ISIS NOs: 463629, 479533, 479534, 479535, 479536, 479539,
479546, 479548, 479551, 479552, 479556, 479566, 479573, 479691,
479692, 479698, 479699, 479703, 479704, 479706, 479736, and
479739.
[0284] In certain embodiments, the compounds provided herein have a
greater therapeutic potential than ISIS NO: 299005. In certain
embodiments, the compounds provided herein have better in vivo
inhibition over ISIS NO: 299005. In certain embodiments, the
compounds provided herein have a better tolerability profile than
ISIS NO: 299005.
[0285] In certain embodiments, the compound provided herein
consists of a single-stranded modified oligonucleotide.
[0286] In certain embodiments, the modified oligonucleotide
consists of 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 or 35 linked
nucleosides. In certain embodiments, the modified oligonucleotide
consists of 20 linked nucleosides. In certain embodiments, the
modified oligonucleotide consists of 19 linked nucleosides. In
certain embodiments, the modified oligonucleotide consists of 18
linked nucleosides. In certain embodiments, the modified
oligonucleotide consists of 17 linked nucleosides. In certain
embodiments, the modified oligonucleotide consists of 16 linked
nucleosides.
[0287] In certain embodiments, at least one internucleoside linkage
of the modified oligonucleotide is a modified internucleoside
linkage. In certain embodiments, each internucleoside linkage is a
phosphorothioate internucleoside linkage.
[0288] In certain embodiments, at least one nucleoside of said
modified oligonucleotide comprises a modified nucleobase. In
certain embodiments, the modified nucleobase is a
5-methylcytosine.
[0289] In certain embodiments, the modified oligonucleotide
comprises: a) a gap segment consisting of linked deoxynucleosides;
b) a 5' wing segment consisting of linked nucleosides; and c) a 3'
wing segment consisting of linked nucleosides. The gap segment is
positioned between the 5' wing segment and the 3' wing segment and
each nucleoside of each wing segment comprises a modified
sugar.
[0290] In certain embodiments, the modified oligonucleotide
consists of 20 linked nucleosides, the gap segment consisting of
ten linked deoxynucleosides, the 5' wing segment consisting of five
linked nucleosides, the 3' wing segment consisting of five linked
nucleosides, each nucleoside of each wing segment comprises a
2'-O-methoxyethyl modified sugar, each internucleoside linkage is a
phosphorothioate linkage and each cytosine is a
5-methylcytosine.
[0291] In certain embodiments, the modified oligonucleotide
consists of 17 linked nucleosides, the gap segment consisting of
ten linked deoxynucleosides, the 5' wing segment consisting of
three linked nucleosides, the 3' wing segment consisting of four
linked nucleosides, each nucleoside of each wing segment comprises
a 2'-O-methoxyethyl modified sugar, each internucleoside linkage is
a phosphorothioate linkage and each cytosine is a
5-methylcytosine.
[0292] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 linked nucleosides having a nucleobase sequence comprising at
least 8 contiguous nucleobases complementary to an equal length
portion of any of SEQ ID NOs: 1-4, wherein the modified
oligonucleotide comprises: a) a gap segment consisting of ten
linked deoxynucleosides; b) a 5' wing segment consisting of five
linked nucleosides; and c) a 3' wing segment consisting of five
linked nucleosides. The gap segment is positioned between the 5'
wing segment and the 3' wing segment, each nucleoside of each wing
segment comprises a 2'-O-methoxyethyl modified sugar, each
internucleoside linkage is a phosphorothioate linkage and each
cytosine residue is a 5-methylcytosine.
[0293] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 linked nucleosides having a nucleobase sequence comprising at
least 8 contiguous nucleobases complementary to an equal length
portion of any of SEQ ID NO: 1, wherein the modified
oligonucleotide comprises: a) a gap segment consisting of ten
linked deoxynucleosides; b) a 5' wing segment consisting of five
linked nucleosides; and c) a 3' wing segment consisting of five
linked nucleosides. The gap segment is positioned between the 5'
wing segment and the 3' wing segment, each nucleoside of each wing
segment comprises a 2'-O-methoxyethyl modified sugar, each
internucleoside linkage is a phosphorothioate linkage and each
cytosine residue is a 5-methylcytosine.
[0294] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 linked nucleosides having a nucleobase sequence comprising at
least 19 contiguous nucleobases of SEQ ID NOs: 7-322 wherein the
modified oligonucleotide comprises: a) a gap segment consisting of
ten linked deoxynucleosides; b) a 5' wing segment consisting of
five linked nucleosides; and c) a 3' wing segment consisting of
five linked nucleosides. The gap segment is positioned between the
5' wing segment and the 3' wing segment, each nucleoside of each
wing segment comprises a 2'-O-methoxyethyl modified sugar, each
internucleoside linkage is a phosphorothioate linkage and each
cytosine residue is a 5-methylcytosine.
[0295] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 linked nucleosides having a nucleobase sequence comprising at
least 19 contiguous nucleobases of SEQ ID NOs: 16, 17, 45, 46, 70,
72, or 138, wherein the modified oligonucleotide comprises: a) a
gap segment consisting of ten linked deoxynucleosides; b) a 5' wing
segment consisting of five linked nucleosides; and c) a 3' wing
segment consisting of five linked nucleosides. The gap segment is
positioned between the 5' wing segment and the 3' wing segment,
each nucleoside of each wing segment comprises a 2'-O-methoxyethyl
modified sugar, each internucleoside linkage is a phosphorothioate
linkage and each cytosine residue is a 5-methylcytosine.
[0296] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 linked nucleosides having a nucleobase sequence comprising at
least 19 contiguous nucleobases of SEQ ID NO: 16, wherein the
modified oligonucleotide comprises: a) a gap segment consisting of
ten linked deoxynucleosides; b) a 5' wing segment consisting of
five linked nucleosides; and c) a 3' wing segment consisting of
five linked nucleosides. The gap segment is positioned between the
5' wing segment and the 3' wing segment, each nucleoside of each
wing segment comprises a 2'-O-methoxyethyl modified sugar, each
internucleoside linkage is a phosphorothioate linkage and each
cytosine residue is a 5-methylcytosine.
[0297] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 linked nucleosides having a nucleobase sequence comprising at
least 19 contiguous nucleobases of SEQ ID NO: 45, wherein the
modified oligonucleotide comprises: a) a gap segment consisting of
ten linked deoxynucleosides; b) a 5' wing segment consisting of
five linked nucleosides; and c) a 3' wing segment consisting of
five linked nucleosides. The gap segment is positioned between the
5' wing segment and the 3' wing segment, each nucleoside of each
wing segment comprises a 2'-O-methoxyethyl modified sugar, each
internucleoside linkage is a phosphorothioate linkage and each
cytosine residue is a 5-methylcytosine.
[0298] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 linked nucleosides having a nucleobase sequence comprising at
least 20 contiguous nucleobases of SEQ ID NOs: 7-322, wherein the
modified oligonucleotide comprises: a) a gap segment consisting of
ten linked deoxynucleosides; b) a 5' wing segment consisting of
five linked nucleosides; and c) a 3' wing segment consisting of
five linked nucleosides. The gap segment is positioned between the
5' wing segment and the 3' wing segment, each nucleoside of each
wing segment comprises a 2'-O-methoxyethyl modified sugar, each
internucleoside linkage is a phosphorothioate linkage and each
cytosine residue is a 5-methylcytosine.
[0299] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 linked nucleosides having a nucleobase sequence comprising at
least 20 contiguous nucleobases of SEQ ID NOs: 16, 17, 45, 46, 70,
72, or 138, wherein the modified oligonucleotide comprises: a) a
gap segment consisting of ten linked deoxynucleosides; b) a 5' wing
segment consisting of five linked nucleosides; and c) a 3' wing
segment consisting of five linked nucleosides. The gap segment is
positioned between the 5' wing segment and the 3' wing segment,
each nucleoside of each wing segment comprises a 2'-O-methoxyethyl
modified sugar, each internucleoside linkage is a phosphorothioate
linkage and each cytosine residue is a 5-methylcytosine. In certain
embodiments, the compound or composition comprises the compound of
any of ISIS NOs: 463588, 463589, 463690, 463691, 463835, 463837, or
464225.
[0300] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 linked nucleosides having a nucleobase sequence comprising at
least 20 contiguous nucleobases of SEQ ID NO: 16, wherein the
modified oligonucleotide comprises: a) a gap segment consisting of
ten linked deoxynucleosides; b) a 5' wing segment consisting of
five linked nucleosides; and c) a 3' wing segment consisting of
five linked nucleosides. The gap segment is positioned between the
5' wing segment and the 3' wing segment, each nucleoside of each
wing segment comprises a 2'-O-methoxyethyl modified sugar, each
internucleoside linkage is a phosphorothioate linkage and each
cytosine residue is a 5-methylcytosine. In certain embodiments, the
compound or composition comprises the compound of ISIS NO:
463588.
[0301] In certain embodiments, the compounds or compositions
provided herein comprise a modified oligonucleotide consisting of
20 linked nucleosides having a nucleobase sequence comprising at
least 20 contiguous nucleobases of SEQ ID NO: 45, wherein the
modified oligonucleotide comprises: a) a gap segment consisting of
ten linked deoxynucleosides; b) a 5' wing segment consisting of
five linked nucleosides; and c) a 3' wing segment consisting of
five linked nucleosides. The gap segment is positioned between the
5' wing segment and the 3' wing segment, each nucleoside of each
wing segment comprises a 2'-O-methoxyethyl modified sugar, each
internucleoside linkage is a phosphorothioate linkage and each
cytosine residue is a 5-methylcytosine. In certain embodiments, the
compound or composition comprises the compound of ISIS NO:
463690.
[0302] Certain embodiments provide methods, compounds, and
compositions for inhibiting FGFR4 expression.
[0303] Certain embodiments provide a method of reducing FGFR4
expression in an animal comprising administering to the animal a
compound as described herein. In certain embodiments, the compound
comprises a modified oligonucleotide 12 to 30 linked nucleosides in
length targeted to FGFR4. In certain embodiments, the compound
comprises a modified oligonucleotide 15 to 30 linked nucleosides in
length targeted to FGFR4. In certain embodiments, the compound
comprises a modified oligonucleotide 18 to 21 linked nucleosides in
length targeted to FGFR4. In certain embodiments, the compound
comprises a modified oligonucleotide 20 to 35 linked nucleosides in
length targeted to FGFR4. In certain embodiments, the compound
comprises a modified oligonucleotide 20 to 25 linked nucleosides in
length targeted to FGFR4. In certain embodiments, the compound
comprises a modified oligonucleotide 20 to 24 linked nucleosides in
length targeted to FGFR4. In certain embodiments, the compound
comprises a modified oligonucleotide 20 to 23 linked nucleosides in
length targeted to FGFR4. In certain embodiments, the compound
comprises a modified oligonucleotide 20 to 22 linked nucleosides in
length targeted to FGFR4. In certain embodiments, the compound
comprises a modified oligonucleotide 20 to 21 linked nucleosides in
length targeted to FGFR4. In certain embodiments, the compound
comprises a modified oligonucleotide 20 linked nucleosides in
length targeted to FGFR4.
[0304] Certain embodiments provide a method of preventing,
ameliorating or treating a metabolic disease in an animal
comprising administering to the animal a compound as described
herein. In certain embodiments, the compound comprises a modified
oligonucleotide 12 to 30 linked nucleosides in length targeted to
FGFR4. In certain embodiments, the compound comprises a modified
oligonucleotide 20 linked nucleosides in length targeted to FGFR4.
Examples of metabolic diseases or disorders include, but are not
limited to obesity, diabetes, hyperglycemia, prediabetes,
non-alcoholic fatty liver disease (NAFLD), metabolic syndrome,
insulin resistance, diabetic dyslipidemia, or hypertriglyceridemia
or a combination thereof.
[0305] Certain embodiments provide a compound as described herein
for use in preventing, ameliorating or treating a metabolic disease
in an animal. In certain embodiments, the compound comprises a
modified oligonucleotide 12 to 30 linked nucleosides in length
targeted to FGFR4. In certain embodiments, the compound comprises a
modified oligonucleotide 20 linked nucleosides in length targeted
to FGFR4. Examples of metabolic diseases or disorders include, but
are not limited to obesity, diabetes, hyperglycemia, prediabetes,
non-alcoholic fatty liver disease (NAFLD), metabolic syndrome,
insulin resistance, diabetic dyslipidemia, or hypertriglyceridemia
or a combination thereof.
[0306] Certain embodiments provide use of a compound as described
herein in the manufacture of a medicament for preventing,
ameliorating or treating a metabolic disease in an animal. In
certain embodiments, the compound comprises a modified
oligonucleotide 12 to 30 linked nucleosides in length targeted to
FGFR4. In certain embodiments, the compound comprises a modified
oligonucleotide 20 linked nucleosides in length targeted to FGFR4.
Examples of metabolic diseases or disorders include, but are not
limited to obesity, diabetes, hyperglycemia, prediabetes,
non-alcoholic fatty liver disease (NAFLD), metabolic syndrome,
insulin resistance, diabetic dyslipidemia, or hypertriglyceridemia
or a combination thereof.
[0307] Certain embodiments provide a method of preventing,
ameliorating or treating obesity in an animal comprising
administering to the animal a compound as described herein. In
certain embodiments, the compound comprises a modified
oligonucleotide 12 to 30 linked nucleosides in length targeted to
FGFR4. In certain embodiments, the compound comprises a modified
oligonucleotide 20 linked nucleosides in length targeted to FGFR4.
In certain embodiments, the compound or composition comprises the
compound of ISIS NOs: 463588, 463589, 463690, 463691, 463835,
463837, or 464225. In certain embodiments, the compound or
composition comprises the compound of ISIS NO: 463588. In certain
embodiments, the compound or composition comprises the compound of
ISIS NO: 463690.
[0308] Certain embodiments provide a compound as described herein
for use in preventing, ameliorating or treating obesity in an
animal. In certain embodiments, the compound comprises a modified
oligonucleotide 12 to 30 linked nucleosides in length targeted to
FGFR4. In certain embodiments, the compound comprises a modified
oligonucleotide 20 linked nucleosides in length targeted to FGFR4.
In certain embodiments, the compound or composition comprises the
compound of ISIS NOs: 463588, 463589, 463690, 463691, 463835,
463837, or 464225. In certain embodiments, the compound or
composition comprises the compound of ISIS NO: 463588. In certain
embodiments, the compound or composition comprises the compound of
ISIS NO: 463690.
[0309] Certain embodiments provide use of a compound as described
herein in the manufacture of a medicament for preventing,
ameliorating or treating obesity in an animal. In certain
embodiments, the compound comprises a modified oligonucleotide 12
to 30 linked nucleosides in length targeted to FGFR4. In certain
embodiments, the compound comprises a modified oligonucleotide 20
linked nucleosides in length targeted to FGFR4. In certain
embodiments, the compound or composition comprises the compound of
ISIS NOs: 463588, 463589, 463690, 463691, 463835, 463837, or
464225. In certain embodiments, the compound or composition
comprises the compound of ISIS NO: 463588. In certain embodiments,
the compound or composition comprises the compound of ISIS NO:
463690.
[0310] Certain embodiments provide a method of reducing body weight
in an animal comprising administering to the animal a compound as
described herein. In certain embodiments, the compound comprises a
modified oligonucleotide 12 to 30 linked nucleosides in length
targeted to FGFR4. In certain embodiments, the compound comprises a
modified oligonucleotide 20 linked nucleosides in length targeted
to FGFR4. In certain embodiments, reduction of body weight in an
animal prevents, ameliorates or treats a metabolic disease. In
certain embodiments, reduction of body weight in an animal
prevents, ameliorates or treats diabetes. In certain embodiments,
reduction of body weight in an animal prevents, ameliorates or
treats obesity. In certain embodiments, reduction of body weight in
an animal prevents, ameliorates or treats metabolic syndrome. In
certain embodiments, reduction of body weight in an animal
prevents, ameliorates or treats insulin resistance. In certain
embodiments, reduction of body weight in an animal prevents,
ameliorates or treats hyperglycemia. In certain embodiments,
reduction of body weight in an animal prevents, ameliorates or
treats NAFLD. In certain embodiments, reduction of body weight in
an animal prevents, ameliorates or treats diabetic dyslipidemia. In
certain embodiments, the body weight is reduced by at least 5%,
10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or 100%.
[0311] Certain embodiments provide a compound as described herein
for use in reducing body weight in an animal. In certain
embodiments, the compound comprises a modified oligonucleotide 12
to 30 linked nucleosides in length targeted to FGFR4. In certain
embodiments, the compound comprises a modified oligonucleotide 20
linked nucleosides in length targeted to FGFR4. In certain
embodiments, reduction of body weight in an animal prevents,
ameliorates or treats a metabolic disease. In certain embodiments,
reduction of body weight in an animal prevents, ameliorates or
treats diabetes. In certain embodiments, reduction of body weight
in an animal prevents, ameliorates or treats obesity. In certain
embodiments, reduction of body weight in an animal prevents,
ameliorates or treats metabolic syndrome. In certain embodiments,
reduction of body weight in an animal prevents, ameliorates or
treats insulin resistance. In certain embodiments, reduction of
body weight in an animal prevents, ameliorates or treats
hyperglycemia. In certain embodiments, reduction of body weight in
an animal prevents, ameliorates or treats NAFLD. In certain
embodiments, reduction of body weight in an animal prevents,
ameliorates or treats diabetic dyslipidemia. In certain
embodiments, the body weight is reduced by at least 5%, 10%, 20%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95% or 100%.
[0312] Certain embodiments provide use of a compound as described
herein in the manufacture of a medicament for reducing body weight
in an animal. In certain embodiments, the compound comprises a
modified oligonucleotide 12 to 30 linked nucleosides in length
targeted to FGFR4. In certain embodiments, the compound comprises a
modified oligonucleotide 20 linked nucleosides in length targeted
to FGFR4. In certain embodiments, reduction of body weight in an
animal prevents, ameliorates or treats a metabolic disease. In
certain embodiments, reduction of body weight in an animal
prevents, ameliorates or treats diabetes. In certain embodiments,
reduction of body weight in an animal prevents, ameliorates or
treats obesity. In certain embodiments, reduction of body weight in
an animal prevents, ameliorates or treats metabolic syndrome. In
certain embodiments, reduction of body weight in an animal
prevents, ameliorates or treats insulin resistance. In certain
embodiments, reduction of body weight in an animal prevents,
ameliorates or treats hyperglycemia. In certain embodiments,
reduction of body weight in an animal prevents, ameliorates or
treats NAFLD. In certain embodiments, reduction of body weight in
an animal prevents, ameliorates or treats diabetic dyslipidemia. In
certain embodiments, the body weight is reduced by at least 5%,
10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or 100%.
[0313] Certain embodiments provide a method of reducing adipose
tissue in an animal comprising administering to the animal a
compound as described herein. In certain embodiments, the compound
comprises a modified oligonucleotide 12 to 30 linked nucleosides in
length targeted to FGFR4. In certain embodiments, the compound
comprises a modified oligonucleotide 20 linked nucleosides in
length targeted to FGFR4. In certain embodiments, reduction in
adiposity in an animal prevents, ameliorates or treats a metabolic
disease. In certain embodiments, reduction in adiposity in an
animal prevents, ameliorates or treats diabetes. In certain
embodiments, reduction in adiposity in an animal prevents,
ameliorates or treats obesity. In certain embodiments, reduction in
adiposity in an animal prevents, ameliorates or treats metabolic
syndrome. In certain embodiments, reduction in adiposity in an
animal prevents, ameliorates or treats insulin resistance. In
certain embodiments, reduction in adiposity in an animal prevents,
ameliorates or treats hyperglycemia. In certain embodiments,
reduction in adiposity in an animal prevents, ameliorates or treats
NAFLD. In certain embodiments, reduction in adiposity in an animal
prevents, ameliorates or treats diabetic dyslipidemia. In certain
embodiments, adiposity is reduced by at least 5%, 10%, 20%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or
100%.
[0314] Certain embodiments provide a compound as described herein
for use in reducing adipose tissue in an animal. In certain
embodiments, the compound comprises a modified oligonucleotide 12
to 30 linked nucleosides in length targeted to FGFR4. In certain
embodiments, the compound comprises a modified oligonucleotide 20
linked nucleosides in length targeted to FGFR4. In certain
embodiments, reduction in adiposity in an animal prevents,
ameliorates or treats a metabolic disease. In certain embodiments,
reduction in adiposity in an animal prevents, ameliorates or treats
diabetes. In certain embodiments, reduction in adiposity in an
animal prevents, ameliorates or treats obesity. In certain
embodiments, reduction in adiposity in an animal prevents,
ameliorates or treats metabolic syndrome. In certain embodiments,
reduction in adiposity in an animal prevents, ameliorates or treats
insulin resistance. In certain embodiments, reduction in adiposity
in an animal prevents, ameliorates or treats hyperglycemia. In
certain embodiments, reduction in adiposity in an animal prevents,
ameliorates or treats NAFLD. In certain embodiments, reduction in
adiposity in an animal prevents, ameliorates or treats diabetic
dyslipidemia. In certain embodiments, adiposity is reduced by at
least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95% or 100%.
[0315] Certain embodiments provide use of a compound as described
herein in the manufacture of a medicament for reducing adipose
tissue in an animal. In certain embodiments, the compound comprises
a modified oligonucleotide 12 to 30 linked nucleosides in length
targeted to FGFR4. In certain embodiments, the compound comprises a
modified oligonucleotide 20 linked nucleosides in length targeted
to FGFR4. In certain embodiments, reduction in adiposity in an
animal prevents, ameliorates or treats a metabolic disease. In
certain embodiments, reduction in adiposity in an animal prevents,
ameliorates or treats diabetes. In certain embodiments, reduction
in adiposity in an animal prevents, ameliorates or treats obesity.
In certain embodiments, reduction in adiposity in an animal
prevents, ameliorates or treats metabolic syndrome. In certain
embodiments, reduction in adiposity in an animal prevents,
ameliorates or treats insulin resistance. In certain embodiments,
reduction in adiposity in an animal prevents, ameliorates or treats
hyperglycemia. In certain embodiments, reduction in adiposity in an
animal prevents, ameliorates or treats NAFLD. In certain
embodiments, reduction in adiposity in an animal prevents,
ameliorates or treats diabetic dyslipidemia. In certain
embodiments, adiposity is reduced by at least 5%, 10%, 20%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or
100%.
[0316] Certain embodiments provide a method of increasing fatty
acid oxidation in an animal comprising administering to the animal
a compound as described herein. In certain embodiments, the
compound comprises a modified oligonucleotide 12 to 30 linked
nucleosides in length targeted to FGFR4. In certain embodiments,
the compound comprises a modified oligonucleotide 20 linked
nucleosides in length targeted to FGFR4. In certain embodiments,
increasing fatty acid oxidation in an animal prevents, ameliorates
or treats a metabolic disease. In certain embodiments, increasing
fatty acid oxidation in an animal prevents, ameliorates or treats
diabetes. In certain embodiments, increasing fatty acid oxidation
an animal prevents, ameliorates or treats obesity. In certain
embodiments, increasing fatty acid oxidation in an animal prevents,
ameliorates or treats metabolic syndrome. In certain embodiments,
increasing fatty acid oxidation in an animal prevents, ameliorates
or treats insulin resistance. In certain embodiments, increasing
fatty acid oxidation in an animal prevents, ameliorates or treats
hyperglycemia. In certain embodiments, increasing fatty acid
oxidation in an animal prevents, ameliorates or treats NAFLD. In
certain embodiments, increasing fatty acid oxidation prevents,
ameliorates or treats diabetic dyslipidemia. In certain
embodiments, the fatty acid oxidation is increased by at least 5%,
10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or 100%.
[0317] Certain embodiments provide a compound as described herein
for use in increasing fatty acid oxidation in an animal. In certain
embodiments, the compound comprises a modified oligonucleotide 12
to 30 linked nucleosides in length targeted to FGFR4. In certain
embodiments, the compound comprises a modified oligonucleotide 20
linked nucleosides in length targeted to FGFR4. In certain
embodiments, increasing fatty acid oxidation in an animal prevents,
ameliorates or treats a metabolic disease. In certain embodiments,
increasing fatty acid oxidation in an animal prevents, ameliorates
or treats diabetes. In certain embodiments, increasing fatty acid
oxidation an animal prevents, ameliorates or treats obesity. In
certain embodiments, increasing fatty acid oxidation in an animal
prevents, ameliorates or treats metabolic syndrome. In certain
embodiments, increasing fatty acid oxidation in an animal prevents,
ameliorates or treats insulin resistance. In certain embodiments,
increasing fatty acid oxidation in an animal prevents, ameliorates
or treats hyperglycemia. In certain embodiments, increasing fatty
acid oxidation in an animal prevents, ameliorates or treats NAFLD.
In certain embodiments, increasing fatty acid oxidation prevents,
ameliorates or treats diabetic dyslipidemia. In certain
embodiments, the fatty acid oxidation is increased by at least 5%,
10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or 100%.
[0318] Certain embodiments provide use of a compound as described
herein in the manufacture of a medicament for increasing fatty acid
oxidation in an animal. In certain embodiments, the compound
comprises a modified oligonucleotide 12 to 30 linked nucleosides in
length targeted to FGFR4. In certain embodiments, the compound
comprises a modified oligonucleotide 20 linked nucleosides in
length targeted to FGFR4. In certain embodiments, increasing fatty
acid oxidation in an animal prevents, ameliorates or treats a
metabolic disease. In certain embodiments, increasing fatty acid
oxidation in an animal prevents, ameliorates or treats diabetes. In
certain embodiments, increasing fatty acid oxidation an animal
prevents, ameliorates or treats obesity. In certain embodiments,
increasing fatty acid oxidation in an animal prevents, ameliorates
or treats metabolic syndrome. In certain embodiments, increasing
fatty acid oxidation in an animal prevents, ameliorates or treats
insulin resistance. In certain embodiments, increasing fatty acid
oxidation in an animal prevents, ameliorates or treats
hyperglycemia. In certain embodiments, increasing fatty acid
oxidation in an animal prevents, ameliorates or treats NAFLD. In
certain embodiments, increasing fatty acid oxidation prevents,
ameliorates or treats diabetic dyslipidemia. In certain
embodiments, the fatty acid oxidation is increased by at least 5%,
10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or 100%.
[0319] Certain embodiments provide a method of reducing glucose
levels in an animal comprising administering to the animal a
compound as described herein. In certain embodiments, the compound
comprises a modified oligonucleotide 12 to 30 linked nucleosides in
length targeted to FGFR4. In certain embodiments, the compound
comprises a modified oligonucleotide 20 linked nucleosides in
length targeted to FGFR4. In certain embodiments, reduction of
glucose levels in an animal prevents, ameliorates or treats a
metabolic disease. In certain embodiments, reduction of glucose
levels in an animal prevents, ameliorates or treats diabetes. In
certain embodiments, reduction of glucose levels in an animal
prevents, ameliorates or treats obesity. In certain embodiments,
reduction of glucose levels in an animal prevents, ameliorates or
treats metabolic syndrome. In certain embodiments, reduction of
glucose levels in an animal prevents, ameliorates or treats insulin
resistance. In certain embodiments, reduction of glucose levels in
an animal prevents, ameliorates or treats hyperglycemia. In certain
embodiments, reduction of glucose levels in an animal prevents,
ameliorates or treats NAFLD. In certain embodiments, reduction of
glucose levels in an animal prevents, ameliorates or treats
diabetic dyslipidemia. In certain embodiments, the glucose level is
reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.
[0320] Certain embodiments provide a compound as described herein
for use in reducing glucose levels in an animal. In certain
embodiments, the compound comprises a modified oligonucleotide 12
to 30 linked nucleosides in length targeted to FGFR4. In certain
embodiments, the compound comprises a modified oligonucleotide 20
linked nucleosides in length targeted to FGFR4. In certain
embodiments, reduction of glucose levels in an animal prevents,
ameliorates or treats a metabolic disease. In certain embodiments,
reduction of glucose levels in an animal prevents, ameliorates or
treats diabetes. In certain embodiments, reduction of glucose
levels in an animal prevents, ameliorates or treats obesity. In
certain embodiments, reduction of glucose levels in an animal
prevents, ameliorates or treats metabolic syndrome. In certain
embodiments, reduction of glucose levels in an animal prevents,
ameliorates or treats insulin resistance. In certain embodiments,
reduction of glucose levels in an animal prevents, ameliorates or
treats hyperglycemia. In certain embodiments, reduction of glucose
levels in an animal prevents, ameliorates or treats NAFLD. In
certain embodiments, reduction of glucose levels in an animal
prevents, ameliorates or treats diabetic dyslipidemia. In certain
embodiments, the glucose level is reduced by at least 5%, 10%, 20%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95% or 100%.
[0321] Certain embodiments provide use of a compound as described
herein in the manufacture of a medicament for reducing glucose
levels in an animal. In certain embodiments, the compound comprises
a modified oligonucleotide 12 to 30 linked nucleosides in length
targeted to FGFR4. In certain embodiments, the compound comprises a
modified oligonucleotide 20 linked nucleosides in length targeted
to FGFR4. In certain embodiments, reduction of glucose levels in an
animal prevents, ameliorates or treats a metabolic disease. In
certain embodiments, reduction of glucose levels in an animal
prevents, ameliorates or treats diabetes. In certain embodiments,
reduction of glucose levels in an animal prevents, ameliorates or
treats obesity. In certain embodiments, reduction of glucose levels
in an animal prevents, ameliorates or treats metabolic syndrome. In
certain embodiments, reduction of glucose levels in an animal
prevents, ameliorates or treats insulin resistance. In certain
embodiments, reduction of glucose levels in an animal prevents,
ameliorates or treats hyperglycemia. In certain embodiments,
reduction of glucose levels in an animal prevents, ameliorates or
treats NAFLD. In certain embodiments, reduction of glucose levels
in an animal prevents, ameliorates or treats diabetic dyslipidemia.
In certain embodiments, the glucose level is reduced by at least
5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95% or 100%.
[0322] In certain embodiments, FGFR4 has the sequence as set forth
in any of the GENBANK Accession Numbers: GENBANK Accession No.
NM.sub.--002011.3 (incorporated herein as SEQ ID NO: 1), GENBANK
Accession No: NT.sub.--023133.11 truncated from nucleosides
21323018 to 21335213 (incorporated herein as SEQ ID NO: 2); GENBANK
Accession No. AB209631.1 (incorporated herein as SEQ ID NO: 3); and
GENBANK Accession No NM.sub.--022963.2 (incorporated herein as SEQ
ID NO: 4).). In certain embodiments, FGFR4 has the human sequence
as set forth in SEQ ID NOs: 1-4. In certain embodiments, FGFR4 has
the rhesus monkey sequence as set forth in GENBANK Accession No.
NW.sub.--001121000.1 truncated from nucleosides 3094000 to 3109000
(SEQ ID NO: 5). In certain embodiments, FGFR4 has the murine
sequence as set forth in GENBANK Accession No. BC033313.1 (SEQ ID
NO: 6).
[0323] In certain embodiments, the compounds or compositions
provided herein comprise a salt thereof, and a pharmaceutically
acceptable carrier or diluent. In certain embodiments, the
composition comprises a modified oligonucleotide consisting of 20
to 35 linked nucleosides and having a nucleobase sequence
comprising at least 20 contiguous nucleobases of a nucleobase
sequence recited in SEQ ID NOs: 16, 17, 45, 46, 70, 72, or 138 or a
salt thereof and a pharmaceutically acceptable carrier or diluent.
In certain embodiments, the composition comprises a modified
oligonucleotide consisting of 20 to 25 linked nucleosides and
having a nucleobase sequence comprising at least 20 contiguous
nucleobases of a nucleobase sequence recited in SEQ ID NOs: 16, 17,
45, 46, 70, 72, or 138 or a salt thereof and a pharmaceutically
acceptable carrier or diluent. In certain embodiments, the
composition comprises a modified oligonucleotide consisting of 20
linked nucleosides and having a nucleobase sequence comprising at
least 20 contiguous nucleobases of a nucleobase sequence recited in
SEQ ID NO: 16, 17, 45, 46, 70, 72, or 138 or a salt thereof and a
pharmaceutically acceptable carrier or diluent.
[0324] In certain embodiments, the compounds or compositions
provided herein comprise a salt thereof, and a pharmaceutically
acceptable carrier or diluent. In certain embodiments, the
composition comprises a modified oligonucleotide consisting of 20
to 35 linked nucleosides and having a nucleobase sequence
comprising at least 20 contiguous nucleobases of a nucleobase
sequence recited in SEQ ID NO: 16 or a salt thereof and a
pharmaceutically acceptable carrier or diluent. In certain
embodiments, the composition comprises a modified oligonucleotide
consisting of 20 to 25 linked nucleosides and having a nucleobase
sequence comprising at least 20 contiguous nucleobases of a
nucleobase sequence recited in SEQ ID NO: 16 or a salt thereof and
a pharmaceutically acceptable carrier or diluent. In certain
embodiments, the composition comprises a modified oligonucleotide
consisting of 20 linked nucleosides and having a nucleobase
sequence comprising at least 20 contiguous nucleobases of a
nucleobase sequence recited in SEQ ID NO: 16 or a salt thereof and
a pharmaceutically acceptable carrier or diluent.
[0325] In certain embodiments, the compounds or compositions
provided herein comprise a salt thereof, and a pharmaceutically
acceptable carrier or diluent. In certain embodiments, the
composition comprises a modified oligonucleotide consisting of 20
to 35 linked nucleosides and having a nucleobase sequence
comprising at least 20 contiguous nucleobases of a nucleobase
sequence recited in SEQ ID NO: 45 or a salt thereof and a
pharmaceutically acceptable carrier or diluent. In certain
embodiments, the composition comprises a modified oligonucleotide
consisting of 20 to 25 linked nucleosides and having a nucleobase
sequence comprising at least 20 contiguous nucleobases of a
nucleobase sequence recited in SEQ ID NO: 45 or a salt thereof and
a pharmaceutically acceptable carrier or diluent. In certain
embodiments, the composition comprises a modified oligonucleotide
consisting of 20 linked nucleosides and having a nucleobase
sequence comprising at least 20 contiguous nucleobases of a
nucleobase sequence recited in SEQ ID NO: 45 or a salt thereof and
a pharmaceutically acceptable carrier or diluent.
[0326] Certain embodiments provide a method for treating an animal
with a FGFR4 related disease or condition comprising: a)
identifying said animal with the FGFR4 related disease or
condition, and b) administering to said animal a therapeutically
effective amount of a compound comprising a modified
oligonucleotide consisting of 12 to 30 linked nucleosides and
having a nucleobase sequence at least 90% complementary to any of
SEQ ID NOs: 1-4 as measured over the entirety of said modified
oligonucleotide. In certain embodiments, the therapeutically
effective amount of the compound administered to the animal treats
or reduces the FGFR4 related disease or condition, or a symptom
thereof, in the animal. In certain embodiments, the FGFR4 related
disease or condition is obesity. In certain embodiments, the FGFR4
related disease or condition is diabetes.
[0327] Certain embodiments provide a compound comprising a modified
oligonucleotide consisting of 12 to 30 linked nucleosides and
having a nucleobase sequence at least 90% complementary to any of
SEQ ID NOs: 1-4 as measured over the entirety of said modified
oligonucleotide for treating a FGFR4 related disease or condition.
In certain embodiments, the FGFR4 related disease or condition is
obesity. In certain embodiments, the FGFR4 related disease or
condition is diabetes.
[0328] Certain embodiments provide use of a compound comprising a
modified oligonucleotide consisting of 12 to 30 linked nucleosides
and having a nucleobase sequence at least 90% complementary to any
of SEQ ID NOs: 1-4 as measured over the entirety of said modified
oligonucleotide in the preparation of a medicament for treating a
FGFR4 related disease or condition. In certain embodiments, the
FGFR4 related disease or condition is obesity. In certain
embodiments, the FGFR4 related disease or condition is
diabetes.
[0329] Certain embodiments provide a method for treating an animal
with a FGFR4 related disease or condition comprising: a)
identifying said animal with the FGFR4 related disease or
condition, and b) administering to said animal a therapeutically
effective amount of a compound comprising a modified
oligonucleotide consisting of 20 linked nucleosides and having a
nucleobase sequence at least 100% complementary to any of SEQ ID
NOs: 1-4 as measured over the entirety of said modified
oligonucleotide. In certain embodiments, the therapeutically
effective amount of the compound administered to the animal treats
or reduces the FGFR4 related disease or condition, or a symptom
thereof, in the animal. In certain embodiments, the FGFR4 related
disease or condition is obesity. In certain embodiments, the FGFR4
related disease or condition is diabetes.
[0330] Certain embodiments provide a compound comprising a modified
oligonucleotide consisting of 20 linked nucleosides and having a
nucleobase sequence at least 100% complementary to any of SEQ ID
NOs: 1-4 as measured over the entirety of said modified
oligonucleotide for treating a FGFR4 related disease or condition.
In certain embodiments, the FGFR4 related disease or condition is
obesity. In certain embodiments, the FGFR4 related disease or
condition is diabetes.
[0331] Certain embodiments provide use of a compound comprising a
modified oligonucleotide consisting of 20 linked nucleosides and
having a nucleobase sequence at least 100% complementary to any of
SEQ ID NOs: 1-4 as measured over the entirety of said modified
oligonucleotide in the preparation of a medicament for treating a
FGFR4 related disease or condition. In certain embodiments, the
FGFR4 related disease or condition is obesity. In certain
embodiments, the FGFR4 related disease or condition is
diabetes.
[0332] Certain embodiments provide methods of treating, preventing,
or ameliorating a metabolic disease. In certain embodiments the
metabolic disease is obesity, diabetes, hyperglycemia, prediabetes,
non-alcoholic fatty liver disease (NAFLD), metabolic syndrome,
insulin resistance, diabetic dyslipidemia, or hypertriglyceridemia
or a combination thereof.
[0333] Certain embodiments provide compounds described herein for
treating, preventing, or ameliorating a metabolic disease. In
certain embodiments the metabolic disease is obesity, diabetes,
hyperglycemia, prediabetes, non-alcoholic fatty liver disease
(NAFLD), metabolic syndrome, insulin resistance, diabetic
dyslipidemia, or hypertriglyceridemia or a combination thereof.
[0334] Certain embodiments provide use of compounds described
herein in the preparation of a medicament for treating, preventing,
or ameliorating a metabolic disease. In certain embodiments the
metabolic disease is obesity, diabetes, hyperglycemia, prediabetes,
non-alcoholic fatty liver disease (NAFLD), metabolic syndrome,
insulin resistance, diabetic dyslipidemia, or hypertriglyceridemia
or a combination thereof.
[0335] Certain embodiments provide methods comprising administering
to an animal a compound as described herein to an animal. In
certain embodiments, the method comprises administering to an
animal a modified oligonucleotide consisting of 20 to 35 linked
nucleosides and having a nucleobase sequence comprising at least 20
contiguous nucleobases of a nucleobase sequence recited in SEQ ID
NOs: 16, 17, 45, 46, 70, 72, or 138.
[0336] Certain embodiments provide a modified oligonucleotide
consisting of 20 to 35 linked nucleosides and having a nucleobase
sequence comprising at least 20 contiguous nucleobases of a
nucleobase sequence recited in SEQ ID NOs: 16, 17, 45, 46, 70, 72,
or 138 for treating a metabolic disease, diabetes, and/or
obesity.
[0337] Certain embodiments provide use of a modified
oligonucleotide consisting of 20 to 35 linked nucleosides and
having a nucleobase sequence comprising at least 20 contiguous
nucleobases of a nucleobase sequence recited in SEQ ID NOs: 16, 17,
45, 46, 70, 72, or 138 in the preparation of a medicament for
treating metabolic disease, diabetes, and/or obesity.
[0338] Certain embodiments provide methods comprising administering
to an animal a compound as described herein to an animal. In
certain embodiments, the method comprises administering to an
animal a modified oligonucleotide consisting of 20 to 35 linked
nucleosides and having a nucleobase sequence comprising at least 20
contiguous nucleobases of a nucleobase sequence selected from among
the nucleobase sequences recited in SEQ ID NO: 16.
[0339] Certain embodiments provide a modified oligonucleotide
consisting of 20 to 35 linked nucleosides and having a nucleobase
sequence comprising at least 20 contiguous nucleobases of a
nucleobase sequence recited in SEQ ID NO: 16 for treating metabolic
disease, diabetes, and/or obesity.
[0340] Certain embodiments provide use of a modified
oligonucleotide consisting of 20 to 35 linked nucleosides and
having a nucleobase sequence comprising at least 20 contiguous
nucleobases of a nucleobase sequence recited in SEQ ID NO: 16 in
the preparation of a medicament for treating metabolic disease,
diabetes, and/or obesity.
[0341] Certain embodiments provide methods comprising administering
to an animal a compound as described herein to an animal. In
certain embodiments, the method comprises administering to an
animal a modified oligonucleotide consisting of 20 to 35 linked
nucleosides and having a nucleobase sequence comprising at least 20
contiguous nucleobases of a nucleobase sequence selected from among
the nucleobase sequences recited in SEQ ID NO: 45.
[0342] Certain embodiments provide a modified oligonucleotide
consisting of 20 to 35 linked nucleosides and having a nucleobase
sequence comprising at least 20 contiguous nucleobases of a
nucleobase sequence recited in SEQ ID NO: 45 for treating metabolic
disease, diabetes, and/or obesity.
[0343] Certain embodiments provide use of a modified
oligonucleotide consisting of 20 to 35 linked nucleosides and
having a nucleobase sequence comprising at least 20 contiguous
nucleobases of a nucleobase sequence recited in SEQ ID NO: 45 in
the preparation of a medicament for treating metabolic disease,
diabetes, and/or obesity.
[0344] Certain embodiments provide methods comprising administering
to an animal a compound as described herein to an animal. In
certain embodiments, the method comprises administering to an
animal a modified oligonucleotide consisting of 20 to 35 linked
nucleosides and having a nucleobase sequence comprising at least 20
contiguous nucleobases of a nucleobase sequence selected from among
the nucleobase sequences recited in ISIS NO: 463588.
[0345] Certain embodiments provide a modified oligonucleotide
consisting of 20 to 35 linked nucleosides and having a nucleobase
sequence comprising at least 20 contiguous nucleobases of a
nucleobase sequence of ISIS NO: 463588 for treating metabolic
disease, diabetes, and/or obesity.
[0346] Certain embodiments provide use of a modified
oligonucleotide consisting of 20 to 35 linked nucleosides and
having a nucleobase sequence comprising at least 20 contiguous
nucleobases of a nucleobase sequence of ISIS NO: 463588 in the
preparation of a medicament for treating metabolic disease,
diabetes, and/or obesity.
[0347] Certain embodiments provide methods comprising administering
to an animal a compound as described herein to an animal. In
certain embodiments, the method comprises administering to an
animal a modified oligonucleotide consisting of 20 to 35 linked
nucleosides and having a nucleobase sequence comprising at least 20
contiguous nucleobases of a nucleobase sequence selected from among
the nucleobase sequences recited in ISIS NO: 463690.
[0348] Certain embodiments provide a modified oligonucleotide
consisting of 20 to 35 linked nucleosides and having a nucleobase
sequence comprising at least 20 contiguous nucleobases of a
nucleobase sequence of ISIS NO: 463690 for treating metabolic
disease, diabetes, and/or obesity.
[0349] Certain embodiments provide use of a modified
oligonucleotide consisting of 20 to 35 linked nucleosides and
having a nucleobase sequence comprising at least 20 contiguous
nucleobases of a nucleobase sequence of ISIS NO: 463690 in the
preparation of a medicament for treating metabolic disease,
diabetes, and/or obesity.
[0350] In certain embodiments, the animal is a human.
[0351] In certain embodiments, the administering prevents, treats,
ameliorates, or slows progression of a metabolic disease as
described herein.
[0352] In certain embodiments, the administering prevents, treats,
ameliorates, or slows progression of obesity as described
herein.
[0353] In certain embodiments, the administering prevents, treats,
ameliorates, or slows progression of diabetes as described
herein.
[0354] In certain embodiments, the compound is co-administered with
a second agent.
[0355] In certain embodiments, the compound and the second agent
are administered concomitantly.
[0356] In certain embodiments, the administering is parenteral
administration.
[0357] Certain embodiments further provide a method to reduce FGFR4
mRNA or protein expression in an animal comprising administering to
the animal a compound or composition as described herein to reduce
FGFR4 mRNA or protein expression in the animal. In certain
embodiments, the animal is a human. In certain embodiments,
reducing FGFR4 mRNA or protein expression prevents, treats,
ameliorates, or slows progression of metabolic disease. In certain
embodiments, the metabolic disease or condition is diabetes. In
certain embodiments, the metabolic disease or condition is
obesity.
[0358] Certain embodiments provide a method for treating a human
with a metabolic disease comprising identifying the human with the
disease and administering to the human a therapeutically effective
amount of a compound or composition as described herein. In certain
embodiments, the treatment reduces a symptom selected from the
group consisting of metabolic syndrome, hyperglycemia,
hypertriglyceridemia, hypertension, increased glucose levels,
increased insulin resistance, decreased insulin sensitivity, above
normal body weight, and/or above normal body fat or any combination
thereof.
[0359] Certain embodiments provide a method for treating a human
with obesity comprising identifying the human with the disease and
administering to the human a therapeutically effective amount of a
compound or composition as described herein. In certain
embodiments, the treatment reduces a symptom selected from the
group consisting of metabolic syndrome, hyperglycemia,
hypertriglyceridemia, hypertension, increased glucose levels,
increased insulin resistance, decreased insulin sensitivity, above
normal body weight, and/or above normal body fat or any combination
thereof
[0360] Certain embodiments provide a method for treating a human
with diabetes comprising identifying the human with the disease and
administering to the human a therapeutically effective amount of a
compound or composition as described herein. In certain
embodiments, the treatment reduces a symptom selected from the
group consisting of metabolic syndrome, hyperglycemia,
hypertriglyceridemia, hypertension, increased glucose levels,
increased insulin resistance, decreased insulin sensitivity, above
normal body weight, and/or above normal body fat or any combination
thereof
[0361] Further provided is a method for reducing or preventing
metabolic disease comprising administering to a human a
therapeutically effective amount compound or composition as
described herein, thereby reducing or preventing metabolic
disease.
[0362] Further provided is a method for reducing or preventing
obesity comprising administering to a human a therapeutically
effective amount compound or composition as described herein,
thereby reducing or preventing diabetes.
[0363] Further provided is a method for reducing or preventing
diabetes comprising administering to a human a therapeutically
effective amount compound or composition as described herein,
thereby reducing or preventing diabetes.
[0364] Further provided is a method for ameliorating a symptom of
metabolic disease, comprising administering to a human in need
thereof a compound comprising a modified oligonucleotide consisting
of 20 to 35 linked nucleosides, wherein said modified
oligonucleotide specifically hybridizes to SEQ ID NO: 1, 2, 3, 4 or
5, thereby ameliorating a symptom of metabolic disease in the
human.
[0365] Further provided is a method for ameliorating a symptom of
obesity, comprising administering to a human in need thereof a
compound comprising a modified oligonucleotide consisting of 20 to
35 linked nucleosides, wherein said modified oligonucleotide
specifically hybridizes to SEQ ID NO: 1, 2, 3, 4 or 5, thereby
ameliorating a symptom of metabolic disease in the human.
[0366] Further provided is a method for ameliorating a symptom of
diabetes, comprising administering to a human in need thereof a
compound comprising a modified oligonucleotide consisting of 20 to
35 linked nucleosides, wherein said modified oligonucleotide
specifically hybridizes to SEQ ID NO: 1, 2, 3, 4 or 5, thereby
ameliorating a symptom of metabolic disease in the human.
[0367] Further provided is a method for ameliorating a symptom of
metabolic syndrome, comprising administering to a human in need
thereof a compound comprising a modified oligonucleotide consisting
of 12 to 30 linked nucleosides, wherein said modified
oligonucleotide specifically hybridizes to SEQ ID NO: 1, 2, 3, 4 or
5, thereby ameliorating a symptom of metabolic syndrome in the
human.
[0368] Further provided is a method for ameliorating a symptom of
metabolic disease, comprising administering to a human in need
thereof a compound comprising a modified oligonucleotide consisting
of 12 to 30 linked nucleosides, wherein said modified
oligonucleotide specifically hybridizes to SEQ ID NO: 1, 2, 3, 4 or
5, thereby ameliorating a symptom of metabolic disease in the
human.
[0369] Further provided is a method for ameliorating a symptom of
obesity, comprising administering to a human in need thereof a
compound comprising a modified oligonucleotide consisting of 12 to
30 linked nucleosides, wherein said modified oligonucleotide
specifically hybridizes to SEQ ID NO: 1, 2, 3, 4 or 5, thereby
ameliorating a symptom of obesity in the human.
[0370] Further provided is a method for ameliorating a symptom of
diabetes, comprising administering to a human in need thereof a
compound comprising a modified oligonucleotide consisting of 12 to
30 linked nucleosides, wherein said modified oligonucleotide
specifically hybridizes to SEQ ID NO: 1, 2, 3, 4 or 5, thereby
ameliorating a symptom of diabetes in the human.
[0371] Further provided is a method for ameliorating a symptom of
metabolic syndrome, comprising administering to a human in need
thereof a compound comprising a modified oligonucleotide consisting
of 12 to 30 linked nucleosides, wherein said modified
oligonucleotide specifically hybridizes to SEQ ID NO: 1, 2, 3, 4 or
5, thereby ameliorating a symptom of metabolic syndrome in the
human.
[0372] Further provided is a method for ameliorating a symptom of
metabolic disease, comprising administering to a human in need
thereof a compound comprising a modified oligonucleotide consisting
of 20 linked nucleosides, wherein said modified oligonucleotide
specifically hybridizes to SEQ ID NO: 1, 2, 3, 4 or 5, thereby
ameliorating a symptom of metabolic disease in the human.
[0373] Further provided is a method for ameliorating a symptom of
obesity, comprising administering to a human in need thereof a
compound comprising a modified oligonucleotide consisting of 20
linked nucleosides, wherein said modified oligonucleotide
specifically hybridizes to SEQ ID NO: 1, 2, 3, 4 or 5, thereby
ameliorating a symptom of obesity in the human.
[0374] Further provided is a method for ameliorating a symptom of
diabetes, comprising administering to a human in need thereof a
compound comprising a modified oligonucleotide consisting of 20
linked nucleosides, wherein said modified oligonucleotide
specifically hybridizes to SEQ ID NO: 1, 2, 3, 4 or 5, thereby
ameliorating a symptom of diabetes in the human.
[0375] Further provided is a method for ameliorating a symptom of
metabolic syndrome, comprising administering to a human in need
thereof a compound comprising a modified oligonucleotide consisting
of 20 linked nucleosides, wherein said modified oligonucleotide
specifically hybridizes to SEQ ID NO: 1, 2, 3, 4 or 5, thereby
ameliorating a symptom of metabolic syndrome in the human.
[0376] Further provided is a method for reducing the rate of
progression of a symptom associated with metabolic disease,
comprising administering to a human in need thereof a compound
comprising a modified oligonucleotide consisting of 20 to 35 linked
nucleosides, wherein said modified oligonucleotide specifically
hybridizes to SEQ ID NO: 1, 2, 3, 4 or 5, thereby reducing the rate
of progression a symptom of metabolic disease in the human.
[0377] Further provided is a method for reducing the rate of
progression of a symptom associated with obesity, comprising
administering to a human in need thereof a compound comprising a
modified oligonucleotide consisting of 20 to 35 linked nucleosides,
wherein said modified oligonucleotide specifically hybridizes to
SEQ ID NO: 1, 2, 3, 4 or 5, thereby reducing the rate of
progression a symptom of obesity in the human.
[0378] Further provided is a method for reducing the rate of
progression of a symptom associated with diabetes, comprising
administering to a human in need thereof a compound comprising a
modified oligonucleotide consisting of 20 to 35 linked nucleosides,
wherein said modified oligonucleotide specifically hybridizes to
SEQ ID NO: 1, 2, 3, 4 or 5, thereby reducing the rate of
progression a symptom of diabetes in the human.
[0379] Further provided is a method for reducing the rate of
progression of a symptom associated with metabolic syndrome,
comprising administering to a human in need thereof a compound
comprising a modified oligonucleotide consisting of 20 to 35 linked
nucleosides, wherein said modified oligonucleotide specifically
hybridizes to SEQ ID NO: 1, 2, 3, 4 or 5, thereby reducing the rate
of progression a symptom of metabolic syndrome in the human.
[0380] Further provided is a method for reducing the rate of
progression of a symptom associated with metabolic disease,
comprising administering to a human in need thereof a compound
comprising a modified oligonucleotide consisting of 12 to 30 linked
nucleosides, wherein said modified oligonucleotide specifically
hybridizes to SEQ ID NO: 1, 2, 3, 4 or 5, thereby reducing the rate
of progression a symptom of metabolic disease in the human.
[0381] Further provided is a method for reducing the rate of
progression of a symptom associated with obesity, comprising
administering to a human in need thereof a compound comprising a
modified oligonucleotide consisting of 12 to 30 linked nucleosides,
wherein said modified oligonucleotide specifically hybridizes to
SEQ ID NO: 1, 2, 3, 4 or 5, thereby reducing the rate of
progression a symptom of obesity in the human.
[0382] Further provided is a method for reducing the rate of
progression of a symptom associated with diabetes, comprising
administering to a human in need thereof a compound comprising a
modified oligonucleotide consisting of 12 to 30 linked nucleosides,
wherein said modified oligonucleotide specifically hybridizes to
SEQ ID NO: 1, 2, 3, 4 or 5, thereby reducing the rate of
progression a symptom of diabetes in the human.
[0383] Further provided is a method for reducing the rate of
progression of a symptom associated with metabolic syndrome,
comprising administering to a human in need thereof a compound
comprising a modified oligonucleotide consisting of 12 to 30 linked
nucleosides, wherein said modified oligonucleotide specifically
hybridizes to SEQ ID NO: 1, 2, 3, 4 or 5, thereby reducing the rate
of progression a symptom of metabolic syndrome in the human.
[0384] Further provided is a method for reducing the rate of
progression of a symptom associated with metabolic disease,
comprising administering to a human in need thereof a compound
comprising a modified oligonucleotide consisting of 20 linked
nucleosides, wherein said modified oligonucleotide specifically
hybridizes to SEQ ID NO: 1, 2, 3, 4 or 5, thereby reducing the rate
of progression a symptom of metabolic disease in the human.
[0385] Further provided is a method for reducing the rate of
progression of a symptom associated with obesity, comprising
administering to a human in need thereof a compound comprising a
modified oligonucleotide consisting of 20 linked nucleosides,
wherein said modified oligonucleotide specifically hybridizes to
SEQ ID NO: 1, 2, 3, 4 or 5, thereby reducing the rate of
progression a symptom of obesity in the human.
[0386] Further provided is a method for reducing the rate of
progression of a symptom associated with diabetes, comprising
administering to a human in need thereof a compound comprising a
modified oligonucleotide consisting of 20 linked nucleosides,
wherein said modified oligonucleotide specifically hybridizes to
SEQ ID NO: 1, 2, 3, 4 or 5, thereby reducing the rate of
progression a symptom of diabetes in the human.
[0387] Further provided is a method for reducing the rate of
progression of a symptom associated with metabolic syndrome,
comprising administering to a human in need thereof a compound
comprising a modified oligonucleotide consisting of 20 linked
nucleosides, wherein said modified oligonucleotide specifically
hybridizes to SEQ ID NO: 1, 2, 3, 4 or 5, thereby reducing the rate
of progression a symptom of metabolic syndrome in the human.
[0388] Also provided are methods and compounds for the preparation
of a medicament for the treatment, prevention, or amelioration of
metabolic disease.
[0389] Also provided are methods and compounds for the preparation
of a medicament for the treatment, prevention, or amelioration of
obesity.
[0390] Also provided are methods and compounds for the preparation
of a medicament for the treatment, prevention, or amelioration of
diabetes.
[0391] Also provided are methods and compounds for the preparation
of a medicament for the treatment, prevention, or amelioration of
metabolic syndrome.
[0392] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
ameliorating, or preventing metabolic disease.
[0393] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
ameliorating, or preventing obesity.
[0394] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
ameliorating, or preventing diabetes.
[0395] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
ameliorating, or preventing metabolic syndrome.
[0396] Certain embodiments provide a compound as described herein
for use in treating, preventing, or ameliorating metabolic disease
as described herein by combination therapy with an additional agent
or therapy as described herein. Agents or therapies can be
co-administered or administered concomitantly.
[0397] Certain embodiments provide a compound as described herein
for use in treating, preventing, or ameliorating diabetes as
described herein by combination therapy with an additional agent or
therapy as described herein. Agents or therapies can be
co-administered or administered concomitantly.
[0398] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
preventing, or ameliorating metabolic disease as described herein
by combination therapy with an additional agent or therapy as
described herein. Agents or therapies can be co-administered or
administered concomitantly.
[0399] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
preventing, or ameliorating obesity as described herein by
combination therapy with an additional agent or therapy as
described herein. Agents or therapies can be co-administered or
administered concomitantly.
[0400] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
preventing, or ameliorating diabetes as described herein by
combination therapy with an additional agent or therapy as
described herein. Agents or therapies can be co-administered or
administered concomitantly.
[0401] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
preventing, or ameliorating diabetes as described herein by
combination therapy with an additional agent or therapy as
described herein. Agents or therapies can be co-administered or
administered concomitantly.
[0402] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
preventing, or ameliorating metabolic disease as described herein
in a patient who is subsequently administered an additional agent
or therapy as described herein.
[0403] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
preventing, or ameliorating obesity as described herein in a
patient who is subsequently administered an additional agent or
therapy as described herein.
[0404] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
preventing, or ameliorating diabetes as described herein in a
patient who is subsequently administered an additional agent or
therapy as described herein.
[0405] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
preventing, or ameliorating metabolic syndrome as described herein
in a patient who is subsequently administered an additional agent
or therapy as described herein.
[0406] Certain embodiments provide a kit for treating, preventing,
or ameliorating metabolic disease as described herein wherein the
kit comprises:
(i) a compound as described herein; and alternatively (ii) an
additional agent or therapy as described herein.
[0407] Certain embodiments provide a kit for treating, preventing,
or ameliorating obesity as described herein wherein the kit
comprises:
(i) a compound as described herein; and alternatively (ii) an
additional agent or therapy as described herein.
[0408] Certain embodiments provide a kit for treating, preventing,
or ameliorating diabetes as described herein wherein the kit
comprises:
(i) a compound as described herein; and alternatively (ii) an
additional agent or therapy as described herein.
[0409] Certain embodiments provide a kit for treating, preventing,
or ameliorating metabolic syndrome as described herein wherein the
kit comprises:
(i) a compound as described herein; and alternatively (ii) an
additional agent or therapy as described herein.
[0410] A kit as described herein may further include instructions
for using the kit to treat, prevent, or ameliorate metabolic
disease as described herein by combination therapy as described
herein. In certain embodiments, the metabolic disease is obesity.
In certain embodiments, the metabolic disease is diabetes.
[0411] In certain embodiments, a biomarker of the anti-obesity
effect of an FGFR4 inhibitor is an increase in FGF15 and/or FGF19
protein levels. In certain embodiments, a biomarker of FGFR4
antisense oligonucleotide-caused anti-obesity effect is an increase
in FGF15 and/or FGF19 gene expression levels. In certain
embodiments, a biomarker of FGFR4 antisense oligonucleotide-caused
anti-obesity effect is an increase in FGF15 and/or FGF19 protein
levels. In certain embodiments, a biomarker of FGFR4 antisense
oligonucleotide-caused anti-obesity effect is an increase in FGF15
and/or FGF19 gene expression levels. In certain embodiments, the
FGF15 and/or FGF19 nucleic acid is any of the sequences set forth
in GENBANK Accession No. NM.sub.--008003.2 (incorporated herein as
SEQ ID NO: 345), GENBANK Accession No: XM.sub.--001100825.1
(incorporated herein as SEQ ID NO: 346); and GENBANK Accession No.
NM.sub.--005117.1 (incorporated herein as SEQ ID NO: 347).
[0412] Certain embodiments provide methods of detecting the
anti-obesity effect of a FGFR4 inhibitor in an animal by measuring
an increase in ileum FGF15 and/or ileum FGF19 gene expression and
plasma FGF15 and/or plasma FGF19 protein levels.
[0413] Certain embodiments provide methods for predicting
responsiveness of an animal to an FGFR4 inhibitor by measuring an
increase in ileum FGF15 and/or ileum FGF19 gene expression and
plasma FGF15 and/or plasma FGF19 protein levels.
[0414] Certain embodiments provide methods of detecting the
anti-obesity effect of a FGFR4 inhibitor in an animal comprising:
(a) measuring FGF15 gene expression in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF15 gene expression after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF15 gene
expression. In certain embodiments, the FGFR4 inhibitor is a
modified antisense oligonucleotide targeted to FGFR4.
[0415] Certain embodiments provide methods of detecting the
anti-obesity effect of a FGFR4 inhibitor in an animal comprising:
(a) measuring FGF19 gene expression in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF19 gene expression after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF19 gene
expression. In certain embodiments, the FGFR4 inhibitor is a
modified antisense oligonucleotide targeted to FGFR4.
[0416] Certain embodiments provide methods of detecting the
anti-obesity effect of a FGFR4 inhibitor in an animal comprising:
(a) measuring FGF15 protein levels in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF15 protein levels after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF15 protein
levels. In certain embodiments, the FGFR4 inhibitor is a modified
antisense oligonucleotide targeted to FGFR4.
[0417] Certain embodiments provide methods of detecting the
anti-obesity effect of a FGFR4 inhibitor in an animal comprising:
(a) measuring FGF19 protein levels in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF19 protein levels after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF19 protein
levels. In certain embodiments, the FGFR4 inhibitor is a modified
antisense oligonucleotide targeted to FGFR4.
[0418] Certain embodiments provide methods for predicting
responsiveness of an animal to an FGFR4 inhibitor comprising: (a)
measuring FGF15 gene expression in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF15 gene expression after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF15 gene
expression. In certain embodiments, the FGFR4 inhibitor is a
modified antisense oligonucleotide targeted to FGFR4.
[0419] Certain embodiments provide methods for predicting
responsiveness of an animal to an FGFR4 inhibitor comprising: (a)
measuring FGF19 gene expression in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF19 gene expression after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF19 gene
expression. In certain embodiments, the FGFR4 inhibitor is a
modified antisense oligonucleotide targeted to FGFR4.
[0420] Certain embodiments provide methods for predicting
responsiveness of an animal to an FGFR4 inhibitor comprising: (a)
measuring FGF15 protein levels in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF15 protein levels after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF15 protein
levels. In certain embodiments, the FGFR4 inhibitor is a modified
antisense oligonucleotide targeted to FGFR4.
[0421] Certain embodiments provide methods for predicting
responsiveness of an animal to an FGFR4 inhibitor comprising: (a)
measuring FGF19 protein levels in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF19 protein levels after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF19 protein
levels. In certain embodiments, the FGFR4 inhibitor is a modified
antisense oligonucleotide targeted to FGFR4.
[0422] Certain embodiments provide a method for treating a
metabolic disease, including obesity, diabetes, hyperglycemia,
prediabetes, non-alcoholic fatty liver disease (NAFLD), metabolic
syndrome, insulin resistance, diabetic dyslipidemia, or
hypertriglyceridemia or a combination thereof, comprising
administering a first dose of a compound or composition as
described herein to a subject having a baseline level of FGF15 or
FGF19 mRNA or protein in the blood or a tissue and administering
one or more additional doses of the compound or composition to the
subject until the level of FGF15 or FGF19 in the blood or a tissue
is not increased from the baseline level by a certain extent for a
certain amount of time.
[0423] In some aspects, one or more additional doses of the
compound or composition described herein is administered to the
subject until the level of FGF15 or FGF19 mRNA or protein in the
blood or a tissue is not increased from the baseline level for at
least about one week, two weeks, three weeks, four weeks, five
weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks,
eleven weeks, twelve weeks, thirteen weeks, fourteen weeks, fifteen
weeks, sixteen weeks, seventeen weeks, eighteen weeks, nineteen
weeks, twenty weeks, twenty-one weeks, twenty-two weeks,
twenty-three weeks, twenty-four, twenty-five, twenty-six,
twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one,
thirty-two, thirty-three, thirty-four, thirty-five, thirty-six,
thirty-seven, thirty-eight, thirty-nine, forty, forty-one,
forty-two, forty-three, forty-four, forty-five, forty-six,
forty-seven, forty-eight, forty-nine, or fifty weeks.
[0424] In certain aspects, one or more additional doses of the
compound or composition described herein is administered to the
subject until the level of FGF15 or FGF19 mRNA or protein in the
blood or a tissue is not increased from the baseline level by at
least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,
14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,
27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 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%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%
104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%,
115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%,
126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%,
137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%,
148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%,
159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%,
170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%,
181%, 182%, 183%, 184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%,
192%, 193%, 194%, 195%, 196%, 197%, 198%, 199%, 200%, 300%, 400%,
500%, 600%, 700%, 800%, 900%, 1,000%, or any value in between any
of the aforementioned percentages.
[0425] Administration of one or more additional doses of the
compound or composition described herein can continue until such
increases in the level of FGF15 or FGF19 mRNA or protein in the
blood or a tissue relative to the baseline level does not occur for
at least about one week, two weeks, three weeks, four weeks, five
weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks,
eleven weeks, twelve weeks, thirteen weeks, fourteen weeks, fifteen
weeks, sixteen weeks, seventeen weeks, eighteen weeks, nineteen
weeks, twenty weeks, twenty-one weeks, twenty-two weeks,
twenty-three weeks, twenty-four, twenty-five, twenty-six,
twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one,
thirty-two, thirty-three, thirty-four, thirty-five, thirty-six,
thirty-seven, thirty-eight, thirty-nine, forty, forty-one,
forty-two, forty-three, forty-four, forty-five, forty-six,
forty-seven, forty-eight, forty-nine, or fifty weeks. In several
aspects, each dose of compound or composition described herein can
be about 50-2000 mg, about 50-400 mg, about 50-200 mg, about 50-100
mg, about 100-200 mg, or any amount in between any of the
aforementioned ranges.
[0426] In certain aspects, one or more additional doses of the
compound or composition described herein is administered to the
subject until the level of FGF15 or FGF19 protein in the blood or a
tissue is not increased from the baseline level by at least about 1
pg/mL, 5 pg/mL, 10 pg/mL, 15 pg/mL, 20 pg/mL, 25 pg/mL, 30 pg/mL,
35 pg/mL, 40 pg/mL, 45 pg/mL, 50 pg/mL, 55 pg/mL, 60 pg/mL, 65
pg/mL, 70 pg/mL, 75 pg/mL, 80 pg/mL, 85 pg/mL, 90 pg/mL, 95 pg/mL,
100 pg/mL, 105 pg/mL, 110 pg/mL, 115 pg/mL, 120 pg/mL, 125 pg/mL,
130 pg/mL, 135 pg/mL, 140 pg/mL, 145 pg/mL, 150 pg/mL, 155 pg/mL,
160 pg/mL, 165 pg/mL, 170 pg/mL, 175 pg/mL, 180 pg/mL, 185 pg/mL,
190 pg/mL, 195 pg/mL, 200 pg/mL, 205 pg/mL, 210 pg/mL, 215 pg/mL,
220 pg/mL, 225 pg/mL, 230 pg/mL, 235 pg/mL, 240 pg/mL, 245 pg/mL,
250 pg/mL, 255 pg/mL, 260 pg/mL, 265 pg/mL, 270 pg/mL, 275 pg/mL,
280 pg/mL, 290 pg/mL, 295 pg/mL, 300 pg/mL, 350 pg/mL, 400 pg/mL,
450 pg/mL, 500 pg/mL, 550 pg/mL, 600 pg/mL, 650 pg/mL, 700 pg/mL,
750 pg/mL, 800 pg/mL, 850 pg/mL, 900 pg/mL, 950 pg/mL, 1,000 pg/mL,
2,000 pg/mL, or any value in between any of the aforementioned
concentrations. Administration of one or more additional doses of
the compound or composition described herein can continue until
such increases in the level of FGF15 or FGF19 protein in the blood
or a tissue relative to the baseline level does not occur for at
least about one week, two weeks, three weeks, four weeks, five
weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks,
eleven weeks, twelve weeks, thirteen weeks, fourteen weeks, fifteen
weeks, sixteen weeks, seventeen weeks, eighteen weeks, nineteen
weeks, twenty weeks, twenty-one weeks, twenty-two weeks,
twenty-three weeks, twenty-four, twenty-five, twenty-six,
twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one,
thirty-two, thirty-three, thirty-four, thirty-five, thirty-six,
thirty-seven, thirty-eight, thirty-nine, forty, forty-one,
forty-two, forty-three, forty-four, forty-five, forty-six,
forty-seven, forty-eight, forty-nine, or fifty weeks. In several
aspects, each dose of compound or composition described herein can
be about 50-2000 mg, about 50-400 mg, about 50-200 mg, about 50-100
mg, about 100-200 mg, or any amount in between any of the
aforementioned ranges.
[0427] It will be understood that one or more doses of the compound
or composition described herein can be administered during the
aforementioned time periods. For example, a subject may have been
administered one or more doses of the compound or composition
described herein during the at least about one week, two weeks,
three weeks, four weeks, five weeks, six weeks, seven weeks, eight
weeks, nine weeks, ten weeks, eleven weeks, twelve weeks, thirteen
weeks, fourteen weeks, fifteen weeks, sixteen weeks, seventeen
weeks, eighteen weeks, nineteen weeks, twenty weeks, twenty-one
weeks, twenty-two weeks, twenty-three weeks, twenty-four,
twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine,
thirty, thirty-one, thirty-two, thirty-three, thirty-four,
thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine,
forty, forty-one, forty-two, forty-three, forty-four, forty-five,
forty-six, forty-seven, forty-eight, forty-nine, or fifty weeks. In
certain embodiments, additional doses of the compound or
composition described herein are administered to the subject until
the level of FGF15 or FGF19 mRNA or protein in the blood or a
tissue is not increased from the baseline level by at least about
1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,
16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,
29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 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%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103% 104%, 105%,
106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%,
117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%,
128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%,
139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%,
150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%,
161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%,
172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%,
183%, 184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%, 192%, 193%,
194%, 195%, 196%, 197%, 198%, 199%, 200%, 300%, 400%, 500%, 600%,
700%, 800%, 900%, 1,000%, or any value in between any of the
aforementioned percentages in the aforementioned time periods. In
several aspects, each dose of compound or composition described
herein can be about 50-2000 mg, about 50-400 mg, about 50-200 mg,
about 50-100 mg, about 100-200 mg, or any amount in between any of
the aforementioned ranges.
[0428] Various embodiments are directed to a method of treating a
metabolic disease, including obesity, diabetes, hyperglycemia,
prediabetes, non-alcoholic fatty liver disease (NAFLD), metabolic
syndrome, insulin resistance, diabetic dyslipidemia, or
hypertriglyceridemia or a combination thereof, comprising (a)
obtaining the baseline level of FGF15 or FGF19 mRNA or protein in
the blood or a tissue of a subject, (b) administering to the
subject a dose of a compound or composition described herein, (c)
obtaining the level of FGF15 or FGF19 mRNA or protein in the blood
or a tissue after the administration of the compound or composition
described herein; and (d) repeating steps (b) and (c) until the
level of FGF15 or FGF19 mRNA or protein in the blood or a tissue
does not increase by a certain extent for a certain amount of time
relative to baseline.
[0429] In several aspects, steps (b) and (c) are repeated until the
level of FGF15 or FGF19 mRNA or protein in the blood or a tissue
does not increase relative to baseline for at least about one week,
two weeks, three weeks, four weeks, five weeks, six weeks, seven
weeks, eight weeks, nine weeks, ten weeks, eleven weeks, twelve
weeks, thirteen weeks, fourteen weeks, fifteen weeks, sixteen
weeks, seventeen weeks, eighteen weeks, nineteen weeks, twenty
weeks, twenty-one weeks, twenty-two weeks, twenty-three weeks,
twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight,
twenty-nine, thirty, thirty-one, thirty-two, thirty-three,
thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight,
thirty-nine, forty, forty-one, forty-two, forty-three, forty-four,
forty-five, forty-six, forty-seven, forty-eight, forty-nine, or
fifty weeks. In several aspects, each dose of compound or
composition described herein can be about 50-2000 mg, about 50-400
mg, about 50-200 mg, about 50-100 mg, about 100-200 mg, or any
amount in between any of the aforementioned ranges.
[0430] In certain aspects, one or more additional doses of the
compound or composition described herein is administered to the
subject until the level of FGF15 or FGF19 protein in the blood or a
tissue is not increased from the baseline level by at least about 1
pg/mL, 5 pg/mL, 10 pg/mL, 15 pg/mL, 20 pg/mL, 25 pg/mL, 30 pg/mL,
35 pg/mL, 40 pg/mL, 45 pg/mL, 50 pg/mL, 55 pg/mL, 60 pg/mL, 65
pg/mL, 70 pg/mL, 75 pg/mL, 80 pg/mL, 85 pg/mL, 90 pg/mL, 95 pg/mL,
100 pg/mL, 105 pg/mL, 110 pg/mL, 115 pg/mL, 120 pg/mL, 125 pg/mL,
130 pg/mL, 135 pg/mL, 140 pg/mL, 145 pg/mL, 150 pg/mL, 155 pg/mL,
160 pg/mL, 165 pg/mL, 170 pg/mL, 175 pg/mL, 180 pg/mL, 185 pg/mL,
190 pg/mL, 195 pg/mL, 200 pg/mL, 205 pg/mL, 210 pg/mL, 215 pg/mL,
220 pg/mL, 225 pg/mL, 230 pg/mL, 235 pg/mL, 240 pg/mL, 245 pg/mL,
250 pg/mL, 255 pg/mL, 260 pg/mL, 265 pg/mL, 270 pg/mL, 275 pg/mL,
280 pg/mL, 290 pg/mL, 295 pg/mL, 300 pg/mL, 350 pg/mL, 400 pg/mL,
450 pg/mL, 500 pg/mL, 550 pg/mL, 600 pg/mL, 650 pg/mL, 700 pg/mL,
750 pg/mL, 800 pg/mL, 850 pg/mL, 900 pg/mL, 950 pg/mL, 1,000 pg/mL,
2,000 pg/mL, or any value in between any of the aforementioned
concentrations. Administration of one or more additional doses of
the compound or composition described herein can continue until
such increases in the level of FGF15 or FGF19 protein in the blood
or a tissue relative to the baseline level does not occur for at
least about one week, two weeks, three weeks, four weeks, five
weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks,
eleven weeks, twelve weeks, thirteen weeks, fourteen weeks, fifteen
weeks, sixteen weeks, seventeen weeks, eighteen weeks, nineteen
weeks, twenty weeks, twenty-one weeks, twenty-two weeks,
twenty-three weeks, twenty-four, twenty-five, twenty-six,
twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one,
thirty-two, thirty-three, thirty-four, thirty-five, thirty-six,
thirty-seven, thirty-eight, thirty-nine, forty, forty-one,
forty-two, forty-three, forty-four, forty-five, forty-six,
forty-seven, forty-eight, forty-nine, or fifty weeks. In several
aspects, each dose of compound or composition described herein can
be about 50-2000 mg, about 50-400 mg, about 50-200 mg, about 50-100
mg, about 100-200 mg, or any amount in between any of the
aforementioned ranges.
[0431] In certain embodiments, a method of treating a metabolic
disease and/or obesity comprises (a) obtaining the baseline level
of FGF15 or FGF19 mRNA or protein in the blood or a tissue of a
subject, (b) administering to the subject a dose of a compound or
composition described herein, (c) obtaining the level of FGF15 or
FGF19 mRNA or protein in the blood or a tissue after the
administration of the compound or composition described herein; and
(d) repeating steps (b) and (c) until the level of FGF15 or FGF19
mRNA or protein in the blood or a tissue does not increase by about
1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,
16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,
29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 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%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103% 104%, 105%,
106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%,
117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%,
128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%,
139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%,
150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%,
161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%,
172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%,
183%, 184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%, 192%, 193%,
194%, 195%, 196%, 197%, 198%, 199%, 200%, 300%, 400%, 500%, 600%,
700%, 800%, 900%, 1,000%, or any value in between any of the
aforementioned percentages relative to baseline for at least about
one week, two weeks, three weeks, four weeks, five weeks, six
weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven
weeks, twelve weeks, thirteen weeks, fourteen weeks, fifteen weeks,
sixteen weeks, seventeen weeks, eighteen weeks, nineteen weeks,
twenty weeks, twenty-one weeks, twenty-two weeks, twenty-three
weeks, twenty-four, twenty-five, twenty-six, twenty-seven,
twenty-eight, twenty-nine, thirty, thirty-one, thirty-two,
thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven,
thirty-eight, thirty-nine, forty, forty-one, forty-two,
forty-three, forty-four, forty-five, forty-six, forty-seven,
forty-eight, forty-nine, or fifty weeks. In several aspects, each
dose of compound or composition described herein can be about
50-2000 mg, about 50-400 mg, about 50-200 mg, about 50-100 mg,
about 100-200 mg, or any amount in between any of the
aforementioned ranges.
[0432] Certain embodiments provide a method for treating a
metabolic disease, including obesity, diabetes, hyperglycemia,
prediabetes, non-alcoholic fatty liver disease (NAFLD), metabolic
syndrome, insulin resistance, diabetic dyslipidemia, or
hypertriglyceridemia or a combination thereof, comprising
administering a first dose of a compound or composition as
described herein to a subject having a baseline level of FGF15 or
FGF19 mRNA or protein in the blood or a tissue and administering
one or more additional higher doses of the compound or composition
to the subject until the level of FGF15 or FGF19 in the blood or a
tissue is increased from the baseline level by a certain extent for
a certain amount of time. In several aspects, such method further
comprises administering additional doses of the compound or
composition to the subject to maintain FGF15 or FGF19 mRNA or
protein in the blood or a tissue at a certain level above the
baseline level. It will be understood that the one or more
additional higher doses can be relative to the first dose or the
most recently administered additional higher dose.
[0433] In some aspects, one or more additional higher doses of the
compound or composition described herein is administered to the
subject until the level of FGF15 or FGF19 mRNA or protein in the
blood or a tissue is increased from the baseline level for at least
about one week, two weeks, three weeks, four weeks, five weeks, six
weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven
weeks, twelve weeks, thirteen weeks, fourteen weeks, fifteen weeks,
sixteen weeks, seventeen weeks, eighteen weeks, nineteen weeks,
twenty weeks, twenty-one weeks, twenty-two weeks, twenty-three
weeks, twenty-four, twenty-five, twenty-six, twenty-seven,
twenty-eight, twenty-nine, thirty, thirty-one, thirty-two,
thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven,
thirty-eight, thirty-nine, forty, forty-one, forty-two,
forty-three, forty-four, forty-five, forty-six, forty-seven,
forty-eight, forty-nine, or fifty weeks.
[0434] In several aspects, one or more additional higher doses of
the compound or composition described herein is an amount at least
about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3., 3.4, 3.5,
3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,
4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,
6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,
7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0
fold greater than the first dose or most recently administered
additional higher dose. In certain aspects, each dose of compound
or composition described herein can be about 50-2000 mg, about
50-400 mg, about 50-200 mg, about 50-100 mg, about 100-200 mg, or
any amount in between any of the aforementioned ranges.
[0435] In certain aspects, one or more additional higher doses of
the compound or composition described herein is administered to the
subject until the level of FGF15 or FGF19 mRNA or protein in the
blood or a tissue is increased from the baseline level by at least
about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,
15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,
28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 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%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103% 104%,
105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%,
116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%,
127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%,
138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%,
149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%,
160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%,
171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%,
182%, 183%, 184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%, 192%,
193%, 194%, 195%, 196%, 197%, 198%, 199%, 200%, 300%, 400%, 500%,
600%, 700%, 800%, 900%, 1,000%, or any value in between any of the
aforementioned percentages.
[0436] Administration of one or more additional higher doses of the
compound or composition described herein can continue until such
increases in the level of FGF15 or FGF19 mRNA or protein in the
blood or a tissue relative to the baseline level occurs for at
least about one week, two weeks, three weeks, four weeks, five
weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks,
eleven weeks, twelve weeks, thirteen weeks, fourteen weeks, fifteen
weeks, sixteen weeks, seventeen weeks, eighteen weeks, nineteen
weeks, twenty weeks, twenty-one weeks, twenty-two weeks,
twenty-three weeks, twenty-four, twenty-five, twenty-six,
twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one,
thirty-two, thirty-three, thirty-four, thirty-five, thirty-six,
thirty-seven, thirty-eight, thirty-nine, forty, forty-one,
forty-two, forty-three, forty-four, forty-five, forty-six,
forty-seven, forty-eight, forty-nine, or fifty weeks. In certain
aspects, additional doses of the compound or composition can be
administered to the subject to maintain FGF15 or FGF19 mRNA or
protein in the blood or a tissue at a certain level above the
baseline level. In several aspects, each dose of compound or
composition described herein can be about 50-2000 mg, about 50-400
mg, about 50-200 mg, about 50-100 mg, about 100-200 mg, or any
amount in between any of the aforementioned ranges.
[0437] In certain aspects, one or more additional higher doses of
the compound or composition described herein is administered to the
subject until the level of FGF15 or FGF19 protein in the blood or a
tissue is increased from the baseline level by at least about 1
pg/mL, 5 pg/mL, 10 pg/mL, 15 pg/mL, 20 pg/mL, 25 pg/mL, 30 pg/mL,
35 pg/mL, 40 pg/mL, 45 pg/mL, 50 pg/mL, 55 pg/mL, 60 pg/mL, 65
pg/mL, 70 pg/mL, 75 pg/mL, 80 pg/mL, 85 pg/mL, 90 pg/mL, 95 pg/mL,
100 pg/mL, 105 pg/mL, 110 pg/mL, 115 pg/mL, 120 pg/mL, 125 pg/mL,
130 pg/mL, 135 pg/mL, 140 pg/mL, 145 pg/mL, 150 pg/mL, 155 pg/mL,
160 pg/mL, 165 pg/mL, 170 pg/mL, 175 pg/mL, 180 pg/mL, 185 pg/mL,
190 pg/mL, 195 pg/mL, 200 pg/mL, 205 pg/mL, 210 pg/mL, 215 pg/mL,
220 pg/mL, 225 pg/mL, 230 pg/mL, 235 pg/mL, 240 pg/mL, 245 pg/mL,
250 pg/mL, 255 pg/mL, 260 pg/mL, 265 pg/mL, 270 pg/mL, 275 pg/mL,
280 pg/mL, 290 pg/mL, 295 pg/mL, 300 pg/mL, 350 pg/mL, 400 pg/mL,
450 pg/mL, 500 pg/mL, 550 pg/mL, 600 pg/mL, 650 pg/mL, 700 pg/mL,
750 pg/mL, 800 pg/mL, 850 pg/mL, 900 pg/mL, 950 pg/mL, 1,000 pg/mL,
2,000 pg/mL, or any value in between any of the aforementioned
concentrations. Administration of one or more additional higher
doses of the compound or composition described herein can continue
until such increases in the level of FGF15 or FGF19 protein in the
blood or a tissue relative to the baseline level occurs for at
least about one week, two weeks, three weeks, four weeks, five
weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks,
eleven weeks, twelve weeks, thirteen weeks, fourteen weeks, fifteen
weeks, sixteen weeks, seventeen weeks, eighteen weeks, nineteen
weeks, twenty weeks, twenty-one weeks, twenty-two weeks,
twenty-three weeks, twenty-four, twenty-five, twenty-six,
twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one,
thirty-two, thirty-three, thirty-four, thirty-five, thirty-six,
thirty-seven, thirty-eight, thirty-nine, forty, forty-one,
forty-two, forty-three, forty-four, forty-five, forty-six,
forty-seven, forty-eight, forty-nine, or fifty weeks. In certain
aspects, additional doses of the compound or composition can be
administered to the subject to maintain FGF15 or FGF19 protein in
the blood or a tissue at any of the aforementioned concentrations
above the baseline level. In several aspects, each dose of compound
or composition described herein can be about 50-2000 mg, about
50-400 mg, about 50-200 mg, about 50-100 mg, about 100-200 mg, or
any amount in between any of the aforementioned ranges.
[0438] It will be understood that one or more higher doses of the
compound or composition described herein can be administered during
the aforementioned time periods. For example, a subject may have
been administered one or more doses of the compound or composition
described herein during the at least about one week, two weeks,
three weeks, four weeks, five weeks, six weeks, seven weeks, eight
weeks, nine weeks, ten weeks, eleven weeks, twelve weeks, thirteen
weeks, fourteen weeks, fifteen weeks, sixteen weeks, seventeen
weeks, eighteen weeks, nineteen weeks, twenty weeks, twenty-one
weeks, twenty-two weeks, twenty-three weeks, twenty-four,
twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine,
thirty, thirty-one, thirty-two, thirty-three, thirty-four,
thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine,
forty, forty-one, forty-two, forty-three, forty-four, forty-five,
forty-six, forty-seven, forty-eight, forty-nine, or fifty weeks. In
certain embodiments, additional higher doses of the compound or
composition described herein are administered to the subject until
the level of FGF15 or FGF19 mRNA or protein in the blood or a
tissue is increased from the baseline level by at least about 1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,
17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,
30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 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%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103% 104%, 105%, 106%,
107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%,
118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%,
129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%,
140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%,
151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%,
162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%,
173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%,
184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%, 192%, 193%, 194%,
195%, 196%, 197%, 198%, 199%, 200%, 300%, 400%, 500%, 600%, 700%,
800%, 900%, 1,000%, or any value in between any of the
aforementioned percentages in the aforementioned time periods. In
certain aspects, additional doses of the compound or composition
can be administered to the subject to maintain FGF15 or FGF19 mRNA
or protein in the blood or a tissue at any of the aforementioned
increased levels above the baseline level. In several aspects, each
dose of compound or composition described herein can be about
50-2000 mg, about 50-400 mg, about 50-200 mg, about 50-100 mg,
about 100-200 mg, or any amount in between any of the
aforementioned ranges.
[0439] In certain embodiments, a method of treating a metabolic
disease, including obesity, diabetes, hyperglycemia, prediabetes,
non-alcoholic fatty liver disease (NAFLD), metabolic syndrome,
insulin resistance, diabetic dyslipidemia, or hypertriglyceridemia
or a combination thereof, comprises (a) obtaining the baseline
level of FGF15 or FGF19 mRNA or protein in the blood or a tissue of
a subject, (b) administering to the subject a dose of a compound or
composition described herein, (c) obtaining the level of FGF15 or
FGF19 mRNA or protein in the blood or a tissue after the
administration of the compound or composition described herein; and
(d) repeating steps (b) and (c) until the level of FGF15 or FGF19
mRNA or protein in the blood or a tissue is increased by at least
about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,
15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,
28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 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%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103% 104%,
105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%,
116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%,
127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%,
138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%,
149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%,
160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%,
171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%,
182%, 183%, 184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%, 192%,
193%, 194%, 195%, 196%, 197%, 198%, 199%, 200%, 300%, 400%, 500%,
600%, 700%, 800%, 900%, 1,000%, or any value in between any of the
aforementioned percentages relative to the baseline level. In
certain aspects, the dose administered in step (d) can be a higher
dose than previously administered until the level of FGF15 or FGF19
mRNA or protein in the blood or a tissue is increased by any of the
aforementioned percentages. In certain aspects, such method further
comprises administering additional doses of the compound or
composition to the subject to maintain FGF15 or FGF19 mRNA or
protein in the blood or a tissue at any of the aforementioned
increased levels above the baseline level. In several aspects, each
dose of compound or composition described herein can be about
50-2000 mg, about 50-400 mg, about 50-200 mg, about 50-100 mg,
about 100-200 mg, or any amount in between any of the
aforementioned ranges.
[0440] In certain embodiments, a method of treating a metabolic
disease, including obesity, diabetes, hyperglycemia, prediabetes,
non-alcoholic fatty liver disease (NAFLD), metabolic syndrome,
insulin resistance, diabetic dyslipidemia, or hypertriglyceridemia
or a combination thereof, comprises (a) obtaining the baseline
level of FGF15 or FGF19 protein in the blood or a tissue of a
subject, (b) administering to the subject a dose of a compound or
composition described herein, (c) obtaining the level of FGF15 or
FGF19 protein in the blood or a tissue after the administration of
the compound or composition described herein; and (d) repeating
steps (b) and (c) until the level of FGF15 or FGF19 protein in the
blood or a tissue is increased by at least about 1 pg/mL, 5 pg/mL,
10 pg/mL, 15 pg/mL, 20 pg/mL, 25 pg/mL, 30 pg/mL, 35 pg/mL, 40
pg/mL, 45 pg/mL, 50 pg/mL, 55 pg/mL, 60 pg/mL, 65 pg/mL, 70 pg/mL,
75 pg/mL, 80 pg/mL, 85 pg/mL, 90 pg/mL, 95 pg/mL, 100 pg/mL, 105
pg/mL, 110 pg/mL, 115 pg/mL, 120 pg/mL, 125 pg/mL, 130 pg/mL, 135
pg/mL, 140 pg/mL, 145 pg/mL, 150 pg/mL, 155 pg/mL, 160 pg/mL, 165
pg/mL, 170 pg/mL, 175 pg/mL, 180 pg/mL, 185 pg/mL, 190 pg/mL, 195
pg/mL, 200 pg/mL, 205 pg/mL, 210 pg/mL, 215 pg/mL, 220 pg/mL, 225
pg/mL, 230 pg/mL, 235 pg/mL, 240 pg/mL, 245 pg/mL, 250 pg/mL, 255
pg/mL, 260 pg/mL, 265 pg/mL, 270 pg/mL, 275 pg/mL, 280 pg/mL, 290
pg/mL, 295 pg/mL, 300 pg/mL, 350 pg/mL, 400 pg/mL, 450 pg/mL, 500
pg/mL, 550 pg/mL, 600 pg/mL, 650 pg/mL, 700 pg/mL, 750 pg/mL, 800
pg/mL, 850 pg/mL, 900 pg/mL, 950 pg/mL, 1,000 pg/mL, 2,000 pg/mL,
or any value in between any of the aforementioned concentrations.
In certain aspects, the dose administered in step (d) can be a
higher dose than previously administered until the level of FGF15
or FGF19 mRNA or protein in the blood or a tissue is increased by
any of the aforementioned percentages. In certain aspects, such
method further comprises administering additional doses of the
compound or composition to the subject to maintain FGF15 or FGF19
protein in the blood or a tissue at any of the aforementioned
increased concentrations above the baseline level. In several
aspects, each dose of compound or composition described herein can
be about 50-2000 mg, about 50-400 mg, about 50-200 mg, about 50-100
mg, about 100-200 mg, or any amount in between any of the
aforementioned ranges.
[0441] The level of FGF15 or FGF19 mRNA or protein in a blood or a
tissue, such as liver tissue, may be obtained by several known
assays. For instance, FGF15 or FGF19 mRNA levels can be obtained by
quantitative RT-PCR. FGF15 or FGF19 protein levels can be obtained,
for example, by using any of a number of well recognized
immunological binding assays such as, but not limited to, an enzyme
linked immunosorbent assay (ELISA), which is also known as a
"sandwich assay", an enzyme immunoassay, a radioimmunoassay (RIA),
a fluoroimmunoassay (FIA), a chemiluminescent immunoassay (CLIA) a
counting immunoassay (CIA), a filter media enzyme immunoassay
(MEIA), or a fluorescence-linked immunosorbent assay (FLISA).
Several commercial antibodies against FGF15 or FGF19 mRNA or
protein are suitable for obtaining the level of FGF15 or FGF19 mRNA
or protein in a blood or a tissue. Such commercially available
antibodies can be obtained from Abcam or Santa Cruz Biotechnology,
for example. FGF15 or FGF19 protein levels can be also be obtained
by high performance liquid chromatography (HPLC), mass
spectrometry, or surface plasmon resonance.
Antisense Compounds
[0442] 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.
[0443] 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.
[0444] In certain embodiments, an antisense compound targeted to a
FGFR4 nucleic acid is 12 to 30 nucleotides in length. In other
words, antisense compounds are from 12 to 30 linked nucleobases. In
other embodiments, the antisense compound comprises a modified
oligonucleotide consisting of 8 to 80, 10 to 50, 15 to 30, 18 to
21, 20 to 80, 20 to 35, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20
to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21 or 20
linked nucleobases. In certain such embodiments, the antisense
compound comprises a modified oligonucleotide consisting of 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 nucleobases in length, or a range defined by
any two of the above values.
[0445] In certain embodiments, the antisense compound comprises a
shortened or truncated modified oligonucleotide. The shortened or
truncated modified oligonucleotide can have a single nucleoside
deleted from the 5' end (5' truncation), or alternatively from the
3' end (3' truncation). A shortened or truncated oligonucleotide
may have two nucleosides deleted from the 5' end, or alternatively
may have two subunits deleted from the 3' end. Alternatively, the
deleted nucleosides may be dispersed throughout the modified
oligonucleotide, for example, in an antisense compound having one
nucleoside deleted from the 5' end and one nucleoside deleted from
the 3' end.
[0446] When a single additional nucleoside is present in a
lengthened oligonucleotide, the additional nucleoside may be
located at the 5' or 3' end of the oligonucleotide. When two or
more additional nucleosides are present, the added nucleosides may
be adjacent to each other, for example, in an oligonucleotide
having two nucleosides added to the 5' end (5' addition), or
alternatively to the 3' end (3' addition), of the oligonucleotide.
Alternatively, the added nucleoside may be dispersed throughout the
antisense compound, for example, in an oligonucleotide having one
nucleoside added to the 5' end and one subunit added to the 3'
end.
[0447] 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.
[0448] 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.
[0449] 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
[0450] In certain embodiments, antisense compounds targeted to a
FGFR4 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.
[0451] 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.
[0452] 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
constrained ethyl). In certain embodiments, wings may include
several modified sugar moieties, including, for example 2'-MOE and
constrained ethyl. In certain embodiments, wings may include
several modified and unmodified sugar moieties. In certain
embodiments, wings may include various combinations of 2'-MOE
nucleosides, constrained ethyl nucleosides, and
2'-deoxynucleosides.
[0453] Each distinct region may comprise uniform sugar moieties,
variant, or alternating sugar moieties. The wing-gap-wing motif is
frequently described as "X-Y-Z", where "X" represents the length of
the 5'-wing, "Y" represents the length of the gap, and "Z"
represents the length of the 3'-wing. "X" and "Z" may comprise
uniform, variant, or alternating sugar moieties. In certain
embodiments, "X" and "Y" may include one or more
2'-deoxynucleosides."Y" may comprise 2'-deoxynucleosides. As used
herein, a gapmer described as "X-Y-Z" has a configuration such that
the gap is positioned immediately adjacent to each of the 5'-wing
and the 3' wing. Thus, no intervening nucleotides exist between the
5'-wing and gap, or the gap and the 3'-wing. Any of the antisense
compounds described herein can have a gapmer motif. In certain
embodiments, "X" and "Z" are the same, in other embodiments they
are different. In certain embodiments, "Y" is between 8 and 15
nucleosides. 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
nucleosides.
[0454] In certain embodiments, antisense compounds targeted to a
FGFR4 nucleic acid possess a 5-10-5 gapmer motif.
[0455] In certain embodiments, antisense compounds targeted to a
FGFR4 nucleic acid possess a 3-10-4 gapmer motif.
Target Nucleic Acids, Target Regions and Nucleotide Sequences
[0456] In certain embodiments, the FGFR4 nucleic acid is any of the
sequences set forth in GENBANK Accession No. NM.sub.--002011.3
(incorporated herein as SEQ ID NO: 1), GENBANK Accession No:
NT.sub.--023133.11 truncated from nucleosides 21323018 to 21335213
(incorporated herein as SEQ ID NO: 2); GENBANK Accession No.
AB209631.1 (incorporated herein as SEQ ID NO: 3) and GENBANK
Accession No NM.sub.--022963.2 (incorporated herein as SEQ ID NO:
4). In certain embodiments, FGFR4 has the rhesus monkey sequence as
set forth in GENBANK Accession No. NW.sub.--001121000.1 truncated
from nucleosides 3094000 to 3109000 (SEQ ID NO: 5 In certain
embodiments, FGFR4 has the murine sequence as set forth in GENBANK
Accession No. BC033313.1 (SEQ ID NO: 6).
[0457] 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.
[0458] 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 FGFR4 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.
[0459] 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.
[0460] 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.
[0461] 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.
[0462] 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).
[0463] There may be variation in activity (e.g., as defined by
percent reduction of target nucleic acid levels) of the antisense
compounds within an active target region. In certain embodiments,
reductions in FGFR4 mRNA levels are indicative of inhibition of
FGFR4 expression. Reductions in levels of a FGFR4 protein are also
indicative of inhibition of target mRNA expression. Further,
phenotypic changes are indicative of inhibition of FGFR4
expression. In certain embodiments, reduced glucose levels, reduced
lipid levels, and reduced body weight can be indicative of
inhibition of FGFR4 expression. In certain embodiments,
amelioration of symptoms associated with metabolic disease can be
indicative of inhibition of FGFR4 expression. In certain
embodiments, amelioration of symptoms associated with diabetes can
be indicative of inhibition of FGFR4 expression. In certain
embodiments, reduction of insulin resistance is indicative of
inhibition of FGFR4 expression. In certain embodiments, reduction
of diabetes biomarkers can be indicative of inhibition of FGFR4
expression. In certain embodiments, reduction of FGFR4 expression
is accompanied by an increase in FGF15 and/or FGF19 gene expression
and/or an increase in FGF15 and/or FGF19 protein levels. In certain
embodiments, reduction of FGFR4 expression is accompanied by an
increase in ileum FGF15 and/or ileum FGF19 gene expression and
plasma FGF15 and/or plasma FGF19 protein levels. Therefore, certain
embodiments provide methods of measuring reduction of FGFR
expression by measuring an increase in ileum FGF15 and/or ileum
FGF19 gene expression and plasma FGF15 and/or plasma FGF19 protein
levels. In certain embodiments, a biomarker of the anti-obesity
effect of an FGFR4 inhibitor is an increase in FGF15 and/or FGF19
gene expression levels. In certain embodiments, a biomarker of the
anti-obesity effect of an FGFR4 inhibitor is an increase in FGF15
and/or FGF19 protein levels. In certain embodiments, a biomarker of
FGFR4 antisense oligonucleotide-caused anti-obesity effect is an
increase in FGF15 and/or FGF19 gene expression levels. In certain
embodiments, a biomarker of FGFR4 antisense oligonucleotide-caused
anti-obesity effect is an increase in FGF15 and/or FGF19 protein
levels. In certain embodiments, a biomarker of FGFR4 antisense
oligonucleotide-caused anti-obesity effect is an increase in FGF15
and/or FGF19 gene expression levels.
[0464] Certain embodiments provide methods of detecting the
anti-obesity effect of a FGFR4 inhibitor in an animal by measuring
an increase in ileum FGF15 and/or ileum FGF19 gene expression and
plasma FGF15 and/or plasma FGF19 protein levels.
[0465] Certain embodiments provide methods of detecting the
anti-obesity effect of a FGFR4 inhibitor in an animal comprising:
(a) measuring FGF15 gene expression in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF15 gene expression after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF15 gene
expression. In certain embodiments, the FGFR4 inhibitor is a
modified antisense oligonucleotide targeted to FGFR4.
[0466] Certain embodiments provide methods of detecting the
anti-obesity effect of a FGFR4 inhibitor in an animal comprising:
(a) measuring FGF19 gene expression in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF19 gene expression after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF19 gene
expression. In certain embodiments, the FGFR4 inhibitor is a
modified antisense oligonucleotide targeted to FGFR4.
[0467] Certain embodiments provide methods of detecting the
anti-obesity effect of a FGFR4 inhibitor in an animal comprising:
(a) measuring FGF15 protein levels in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF15 protein levels after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF15 protein
levels. In certain embodiments, the FGFR4 inhibitor is a modified
antisense oligonucleotide targeted to FGFR4.
[0468] Certain embodiments provide methods of detecting the
anti-obesity effect of a FGFR4 inhibitor in an animal comprising:
(a) measuring FGF19 protein levels in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF19 protein levels after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF19 protein
levels. In certain embodiments, the FGFR4 inhibitor is a modified
antisense oligonucleotide targeted to FGFR4.
[0469] Certain embodiments provide methods for predicting
responsiveness of an animal to an FGFR4 inhibitor comprising: (a)
measuring FGF15 gene expression in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF15 gene expression after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF15 gene
expression. In certain embodiments, the FGFR4 inhibitor is a
modified antisense oligonucleotide targeted to FGFR4.
[0470] Certain embodiments provide methods for predicting
responsiveness of an animal to an FGFR4 inhibitor comprising: (a)
measuring FGF19 gene expression in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF19 gene expression after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF19 gene
expression. In certain embodiments, the FGFR4 inhibitor is a
modified antisense oligonucleotide targeted to FGFR4.
[0471] Certain embodiments provide methods for predicting
responsiveness of an animal to an FGFR4 inhibitor by measuring an
increase in ileum FGF15 and/or ileum FGF19 gene expression and
plasma FGF15 and/or plasma FGF19 protein levels.
[0472] Certain embodiments provide methods for predicting
responsiveness of an animal to an FGFR4 inhibitor comprising: (a)
measuring FGF15 protein levels in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF15 protein levels after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF15 protein
levels. In certain embodiments, the FGFR4 inhibitor is a modified
antisense oligonucleotide targeted to FGFR4.
[0473] Certain embodiments provide methods for predicting
responsiveness of an animal to an FGFR4 inhibitor comprising: (a)
measuring FGF19 protein levels in an individual prior to
administration of a FGFR4 inhibitor (b) administering an FGFR4
inhibitor (c) measuring FGF19 protein levels after administration
of a FGFR4 inhibitor (d) detecting an increase of FGF19 protein
levels. In certain embodiments, the FGFR4 inhibitor is a modified
antisense oligonucleotide targeted to FGFR4.
Hybridization
[0474] In some embodiments, hybridization occurs between an
antisense compound disclosed herein and a FGFR4 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.
[0475] 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.
[0476] 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 FGFR4 nucleic acid.
Complementarity
[0477] 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 FGFR4 nucleic acid).
[0478] An antisense compound may hybridize over one or more
segments of a FGFR4 nucleic acid such that intervening or adjacent
segments are not involved in the hybridization event (e.g., a loop
structure, mismatch or hairpin structure).
[0479] 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 FGFR4 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.
[0480] 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
non-complementary 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) non-complementary
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).
[0481] 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, antisense compound may be fully
complementary to a FGFR4 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.
[0482] 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.
[0483] 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 FGFR4 nucleic acid, or specified portion
thereof.
[0484] 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 FGFR4 nucleic acid, or specified portion
thereof.
[0485] 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 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. In certain
embodiments, the antisense compounds are complementary to at least
a 16 nucleobase portion of a target segment. In certain
embodiments, the antisense compounds are complementary to at least
a 17 nucleobase portion of a target segment. In certain
embodiments, the antisense compounds are complementary to at least
a 18 nucleobase portion of a target segment. In certain
embodiments, the antisense compounds are complementary to at least
a 19 nucleobase portion of a target segment. In certain
embodiments, the antisense compounds are complementary to at least
a 20 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
[0486] 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.
[0487] In certain embodiments, the antisense compounds, or portions
thereof, are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
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.
Modifications
[0488] 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.
[0489] 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.
[0490] 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
[0491] 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.
[0492] 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.
[0493] In certain embodiments, antisense compounds targeted to a
FGFR4 nucleic acid comprise one or more modified internucleoside
linkages. 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.
Modified Sugar Moieties
[0494] Antisense compounds provided herein 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 a chemically modified
ribofuranose ring moiety. 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(R1)(R)2 (R=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),
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).
[0495] 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, and
2'-O(CH.sub.2)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,
O(CH.sub.2)2SCH.sub.3, O(CH.sub.2)2-O--N(Rm)(Rn), and
O--CH.sub.2--C(.dbd.O)--N(Rm)(Rn), where each Rm and Rn is,
independently, H or substituted or unsubstituted C.sub.1-C.sub.10
alkyl.
[0496] As used herein, "bicyclic nucleosides" refer to modified
nucleosides comprising a bicyclic sugar moiety. Examples of
bicyclic nucleosides 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 bicyclic nucleosides wherein the bridge comprises a 4'
to 2' bicyclic nucleoside. Examples of such 4' to 2' bicyclic
nucleosides, include, but are not limited to, one of the formulae:
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, published
PCT International Application WO2009/006478, published Jan. 8,
2009); 4'-CH.sub.2--N(OCH.sub.3)-2', and analogs thereof (see,
published PCT International Application WO2008/150729, 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,
published PCT International Application WO 2008/154401, published
on Dec. 8, 2008). Also see, for example: Singh et al., Chem.
Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54,
3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000,
97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,
2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039;
Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379 (Jul. 4,
2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2,
558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al.,
Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos.
6,670,461, 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133,
6,525,191, 7,399,845; published PCT International applications WO
2004/106356, WO 94/14226, WO 2005/021570, and WO 2007/134181; U.S.
Patent Publication Nos. US2004/0171570, US2007/0287831, and
US2008/0039618; and U.S. patent Ser. Nos. 12/129,154, 60/989,574,
61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and
61/099,844; and PCT International Application Nos.
PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922. 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).
[0497] 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' position of the
pentofuranosyl sugar moiety wherein such bridges independently
comprises 1 or from 2 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)--;
[0498] wherein:
[0499] x is 0, 1, or 2;
[0500] n is 1, 2, 3, or 4;
[0501] 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
[0502] 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.
[0503] 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.
[0504] In certain embodiments, bicyclic nucleosides are further
defined by isomeric configuration. For example, a nucleoside
comprising a 4'-2' methylene-oxy bridge, may be in the .alpha.-L
configuration or in the .beta.-D configuration. Previously,
.alpha.-L-methyleneoxy (4'-CH.sub.2--O-2') BNA's have been
incorporated into antisense oligonucleotides that showed antisense
activity (Frieden et al., Nucleic Acids Research, 2003, 21,
6365-6372).
[0505] 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, (G) methylene-thio (4'-CH.sub.2--S-2')
BNA, (H) methylene-amino (4'-CH2-N(R)-2') BNA, (I) methyl
carbocyclic (4'-CH.sub.2--CH(CH.sub.3)-2') BNA, and (J) propylene
carbocyclic (4'-(CH.sub.2).sub.3-2') BNA as depicted below.
##STR00001## ##STR00002##
wherein Bx is the base moiety and R is, independently, H, a
protecting group or C.sub.1-C.sub.12 alkyl.
[0506] In certain embodiments, bicyclic nucleoside having Formula
I:
##STR00003##
wherein:
[0507] Bx is a heterocyclic base moiety;
[0508] -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;
[0509] R.sub.c is C.sub.1-C.sub.12 alkyl or an amino protecting
group; and
[0510] 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.
[0511] In certain embodiments, bicyclic nucleoside having Formula
II:
##STR00004##
wherein:
[0512] Bx is a heterocyclic base moiety;
[0513] 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;
[0514] 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 C.sub.2-C.sub.6
alkynyl, acyl, substituted acyl, substituted amide, thiol, or
substituted thio.
[0515] 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.e.
[0516] In certain embodiments, bicyclic nucleoside having Formula
III:
##STR00005##
wherein:
[0517] Bx is a heterocyclic base moiety;
[0518] 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;
[0519] 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)--).
[0520] In certain embodiments, bicyclic nucleoside having Formula
IV:
##STR00006##
wherein:
[0521] Bx is a heterocyclic base moiety;
[0522] 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;
[0523] 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
C.sub.2-C.sub.6 alkynyl;
[0524] 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;
[0525] In certain embodiments, bicyclic nucleoside having Formula
V:
##STR00007##
wherein:
[0526] Bx is a heterocyclic base moiety;
[0527] 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;
[0528] 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;
[0529] or q.sub.e and q.sub.f together are
.dbd.C(q.sub.g)(q.sub.h);
[0530] 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.
[0531] The synthesis and preparation of the methyleneoxy
(4'-CH.sub.2--O-2') BNA monomers adenine, cytosine, guanine,
5-methyl-cytosine, thymine, and uracil, along with their
oligomerization, and nucleic acid recognition properties have been
described (see, e.g., Koshkin et al., Tetrahedron, 1998, 54,
3607-3630). BNAs and preparation thereof are also described in WO
98/39352 and WO 99/14226.
[0532] Analogs of methyleneoxy (4'-CH.sub.2--O-2') BNA,
methyleneoxy (4'-CH.sub.2--O-2') BNA, and 2'-thio-BNAs, have also
been prepared (see, e.g., Kumar et al., Bioorg. Med. Chem. Lett.,
1998, 8, 2219-2222). Preparation of locked nucleoside analogs
comprising oligodeoxyribonucleotide duplexes as substrates for
nucleic acid polymerases has also been described (see, e.g., Wengel
et al., WO 99/14226). Furthermore, synthesis of 2'-amino-BNA, a
novel comformationally restricted high-affinity oligonucleotide
analog, has been described in the art (see, e.g., 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.
[0533] In certain embodiments, bicyclic nucleoside having Formula
VI:
##STR00008##
wherein:
[0534] Bx is a heterocyclic base moiety;
[0535] 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;
[0536] 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, 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;
and
[0537] 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.
[0538] 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 (see, e.g., Freier
et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek
et al., J. Org. Chem., 2006, 71, 7731-7740). 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).
[0539] 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 the 2' carbon atom and
the 4' carbon atom.
[0540] 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.
[0541] 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.mO].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.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; 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; and a group for
improving the pharmacodynamic properties of an antisense compound,
and other substituents having similar properties. In certain
embodiments, modifed nucleosides comprise a 2'-MOE side chain (see,
e.g., 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 (see, e.g., Martin, P., 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).
[0542] As used herein, a "modified tetrahydropyran nucleoside" or
"modified THP nucleoside" means a nucleoside having a six-membered
tetrahydropyran "sugar" substituted in for the pentofuranosyl
residue in normal nucleosides (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, C J. Bioorg. & Med.
Chem. (2002) 10:841-854), fluoro HNA (F-HNA), or those compounds
having Formula X:
[0543] Formula X:
##STR00009##
wherein independently for each of said at least one tetrahydropyran
nucleoside analog of Formula X:
[0544] Bx is a heterocyclic base moiety;
[0545] T.sub.3 and T.sub.4 are each, independently, an
internucleoside linking group linking the tetrahydropyran
nucleoside analog to the antisense compound or one of T.sub.3 and
T.sub.4 is an internucleoside linking group linking the
tetrahydropyran nucleoside analog to the antisense compound and the
other of T.sub.3 and T.sub.4 is H, a hydroxyl protecting group, a
linked conjugate group, or a 5' or 3'-terminal group;
[0546] 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
[0547] 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.
[0548] In certain embodiments, the modified THP nucleosides of
Formula X are provided wherein q.sub.m, q.sub.n, q.sub.p, q.sub.r,
q.sub.s, q.sub.t, and q.sub.u are each H. In certain embodiments,
at least one of q.sub.m, q.sub.n, q.sub.p, q.sub.r, q.sub.s,
q.sub.t, and q.sub.u is other than H. In certain embodiments, at
least one of q.sub.m, q.sub.n, q.sub.p, q.sub.r, q.sub.s, q.sub.t
and q.sub.u is methyl. In certain embodiments, THP nucleosides of
Formula X 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.
[0549] 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'-modifed nucleosides may further comprise
other modifications, for example, at other positions of the sugar
and/or at the nucleobase.
[0550] As used herein, "2'-F" refers to a sugar comprising a fluoro
group at the 2' position.
[0551] As used herein, "2'-OMe" or "2'-OCH.sub.3" or "2'-O-methyl"
each refers to a sugar comprising an --OCH.sub.3 group at the 2'
position of the sugar ring.
[0552] 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).
[0553] Many other bicyclo and tricyclo sugar surrogate ring systems
are also known in the art that can be used to modify nucleosides
for incorporation into antisense compounds (see, e.g., review
article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002,
10, 841-854).
Such ring systems can undergo various additional substitutions to
enhance activity.
[0554] Methods for the preparations of modified sugars are well
known to those skilled in the art.
[0555] 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.
[0556] In certain embodiments, antisense compounds comprise one or
more nucleotides having modified sugar moieties. In certain
embodiments, the modified sugar moiety is 2'-MOE. In certain
embodiments, the 2'-MOE modified nucleotides are arranged in a
gapmer motif. In certain embodiments, the modified sugar moiety is
a cEt. In certain embodiments, the cEt modified nucleotides are
arranged throughout the wings of a gapmer motif
Modified Nucleobases
[0557] Nucleobase (or base) modifications or substitutions are
structurally distinguishable from, yet functionally interchangeable
with, naturally occurring or synthetic unmodified nucleobases. Both
natural and modified nucleobases are capable of participating in
hydrogen bonding. Such nucleobase modifications may impart nuclease
stability, binding affinity or some other beneficial biological
property to antisense compounds. Modified nucleobases include
synthetic and natural nucleobases such as, for example,
5-methylcytosine (5-me-C). Certain nucleobase substitutions,
including 5-methylcytosine substitutions, are particularly useful
for increasing the binding affinity of an antisense compound for a
target nucleic acid. For example, 5-methylcytosine substitutions
have been shown to increase nucleic acid duplex stability by
0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B.,
eds., Antisense Research and Applications, CRC Press, Boca Raton,
1993, pp. 276-278).
[0558] Additional unmodified nucleobases include 5-hydroxymethyl
cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and
other alkyl derivatives of adenine and guanine, 2-propyl and other
alkyl derivatives of adenine and guanine, 2-thiouracil,
2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine,
5-propynyl (--C.ident.C--CH.sub.3) uracil and cytosine and other
alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and
thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,
8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines
and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and
other 5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine.
[0559] Heterocyclic base moieties may also include those in which
the purine or pyrimidine base is replaced with other heterocycles,
for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and
2-pyridone. Nucleobases that are particularly useful for increasing
the binding affinity of antisense compounds include 5-substituted
pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted
purines, including 2 aminopropyladenine, 5-propynyluracil and
5-propynylcytosine.
[0560] In certain embodiments, antisense compounds targeted to a
FGFR4 nucleic acid comprise one or more modified nucleobases. In
certain embodiments, gap-widened antisense oligonucleotides
targeted to a FGFR4 nucleic acid comprise one or more modified
nucleobases. In certain embodiments, the modified nucleobase is
5-methylcytosine. In certain embodiments, each cytosine is a
5-methylcytosine.
Compositions and Methods for Formulating Pharmaceutical
Compositions
[0561] Antisense oligonucleotides may be admixed with
pharmaceutically acceptable active or inert substance 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.
[0562] Antisense compound targeted to a FGFR4 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 FGFR4 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.
[0563] 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.
[0564] Pharmaceutically acceptable salts of the compounds described
herein may be prepared by methods well-known in the art. For a
review of pharmaceutically acceptable salts, see Stahl and Wermuth,
Handbook of Pharmaceutical Salts: Properties, Selection and Use
(Wiley-VCH, Weinheim, Germany, 2002). Sodium salts of antisense
oligonucleotides are useful and are well accepted for therapeutic
administration to humans. Accordingly, in one embodiment the
compounds described herein are in the form of a sodium salt.
[0565] 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
[0566] 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.
[0567] 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
[0568] The effects of antisense compounds on the level, activity or
expression of FGFR4 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 cells 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 and primary hepatocytes.
In Vitro Testing of Antisense Oligonucleotides
[0569] Described herein are methods for treatment of cells with
antisense oligonucleotides, which can be modified appropriately for
treatment with other antisense compounds.
[0570] In general, cells are treated with antisense
oligonucleotides when the cells reach approximately 60-80%
confluence in culture.
[0571] One reagent commonly used to introduce antisense
oligonucleotides into cultured cells includes the cationic lipid
transfection reagent LIPOFECTIN.RTM. (Invitrogen, Carlsbad,
Calif.). Antisense oligonucleotides are mixed with LIPOFECTIN.RTM.
in OPTI-MEM.RTM. 1 (Invitrogen, Carlsbad, Calif.) to achieve the
desired final concentration of antisense oligonucleotide and a
LIPOFECTIN.RTM. concentration that typically ranges 2 to 12 ug/mL
per 100 nM antisense oligonucleotide.
[0572] Another reagent used to introduce antisense oligonucleotides
into cultured cells includes LIPOFECTAMINE 2000.RTM. (Invitrogen,
Carlsbad, Calif.). Antisense oligonucleotide is mixed with
LIPOFECTAMINE 2000.RTM. in OPTI-MEM.RTM. 1 reduced serum medium
(Invitrogen, Carlsbad, Calif.) to achieve the desired concentration
of antisense oligonucleotide and a LIPOFECTAMINE.RTM. concentration
that typically ranges 2 to 12 ug/mL per 100 nM antisense
oligonucleotide.
[0573] Another reagent used to introduce antisense oligonucleotides
into cultured cells includes Cytofectin.RTM. (Invitrogen, Carlsbad,
Calif.). Antisense oligonucleotide is mixed with Cytofectin.RTM. in
OPTI-MEM.RTM. 1 reduced serum medium (Invitrogen, Carlsbad, Calif.)
to achieve the desired concentration of antisense oligonucleotide
and a Cytofectin.RTM. concentration that typically ranges 2 to 12
ug/mL per 100 nM antisense oligonucleotide.
[0574] Another technique used to introduce antisense
oligonucleotides into cultured cells includes electroporation.
[0575] 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.
[0576] 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 LIPOFECTAMINE2000.RTM., Lipofectin or Cytofectin. Antisense
oligonucleotides are used at higher concentrations ranging from 625
to 20,000 nM when transfected using electroporation.
RNA Isolation
[0577] 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.RTM. Reagent (Invitrogen, Carlsbad, Calif.)
according to the manufacturer's recommended protocols.
Analysis of Inhibition of Target Levels or Expression
[0578] Inhibition of levels or expression of a FGFR4 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.RTM. 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
[0579] Quantitation of target RNA levels may be accomplished by
quantitative real-time PCR using the ABI PRISM.RTM. 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.
[0580] 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.
[0581] 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.RTM. (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.RTM. RNA quantification
reagent (Invitrogen, Inc. Eugene, Oreg.). Methods of RNA
quantification by RIBOGREEN.RTM. are taught in Jones, L. J., et al,
(Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR.RTM.
4000 instrument (PE Applied Biosystems) is used to measure
RIBOGREEN.RTM. fluorescence.
[0582] Probes and primers are designed to hybridize to a FGFR4
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.RTM. Software (Applied Biosystems,
Foster City, Calif.).
Analysis of Protein Levels
[0583] Antisense inhibition of FGFR4 nucleic acids can be assessed
by measuring FGFR4 protein levels. Protein levels of FGFR4 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 human and rat FGFR4 are
commercially available.
In Vivo Testing of Antisense Compounds
[0584] Antisense compounds, for example, antisense
oligonucleotides, are tested in animals to assess their ability to
inhibit expression of FGFR4 and produce phenotypic changes. 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. Following a period of treatment with
antisense oligonucleotides, RNA is isolated from tissue and changes
in FGFR4 nucleic acid expression are measured. Changes in FGFR4
protein levels are also measured.
Certain Indications
[0585] In certain embodiments, provided herein are methods of
treating an individual comprising administering one or more
pharmaceutical compositions as described herein. In certain
embodiments, the individual has a metabolic disease.
[0586] As shown in the examples below, compounds targeted to FGFR4,
as described herein, have been shown to reduce the severity of
physiological symptoms of a metabolic disease, including obesity or
adiposity, metabolic syndrome, diabetes mellitus, insulin
resistance, diabetic dyslipidemia, and hypertriglyceridemia. In
certain of the experiments, the compounds reduced body weight,
e.g., the animals continued to experience symptoms, but the
symptoms were less severe compared to untreated animals. In certain
of the experiments, the compounds reduced body fat, e.g., the
animals continued to experience symptoms, but the symptoms were
less severe compared to untreated animals. In certain of the
experiments, the compounds reduced adipose tissue, e.g., the
animals continued to experience symptoms, but the symptoms were
less severe compared to untreated animals. In other of the
experiments, however, the compounds appear to reduce the symptoms
of obesity; e.g., animals treated for a longer period of time
experienced less severe symptoms than those administered the
compounds for a shorter period of time. In other of the
experiments, however, the compounds appear to reduce the symptoms
of diabetes; e.g., animals treated for a longer period of time
experienced less severe symptoms than those administered the
compounds for a shorter period of time. In other of the
experiments, however, the compounds appear to inhibit weight gain;
e.g., animals treated for a longer period of time experienced less
severe symptoms than those administered the compounds for a shorter
period of time. In other of the experiments, however, the compounds
appear to reduce glucose levels; e.g., animals treated for a longer
period of time experienced less severe symptoms than those
administered the compounds for a shorter period of time. In other
of the experiments, however, the compounds appear to increase fatty
acid oxidation; e.g., animals treated for a longer period of time
experienced less severe symptoms than those administered the
compounds for a shorter period of time. The ability of the
compounds exemplified below to restore function therefore
demonstrates that symptoms of the disease may be reversed by
treatment with a compound as described herein.
[0587] Obesity is characterized by numerous physical and
physiological symptoms. Any symptom known to one of skill in the
art to be associated with obesity can be ameliorated or otherwise
modulated as set forth above in the methods described above. In
certain embodiments, the symptom is a physical symptom selected
from the group consisting of increased adipose tissue mass or
weight, increased weight gain, increased fat pad weight, imbalance
with caloric intake and energy expenditure, increase in body fat,
increase in body mass, having a body mass index (BMI) of 30 or
higher, increase in body frame, increased sweating, sleep apnea,
difficulty in sleeping, inability to cope with sudden physical
activity, lethargy, back and joint problems, increase in
breathlessness, increase in breast region adiposity, increase in
abdomen size or fat, extreme hunger, or extreme fatigue.
[0588] In certain embodiments, the symptom is a physiological
symptom selected from the group consisting of high blood pressure,
hypertension, high cholesterol levels, type 2 diabetes, stroke,
cardiac insufficiency, heart disease, coronary artery obstruction,
breast cancer in women, gastro-oesophageal reflux disease, hip and
knee arthrosis, and reduced life expectancy.
[0589] In certain embodiments, the physical symptom is excess body
weight. In certain embodiments, the symptom is excess fat mass. In
certain embodiments, the symptom is a body mass index of 30 or
higher. In certain embodiments, the symptom is breathlessness. In
certain embodiments, the symptom is increased sweating. In certain
embodiments, the symptom is sleep apnea. In certain embodiments,
the symptom is difficulty in sleeping. In certain embodiments, the
symptom is inability to cope with sudden physical activity. In
certain embodiments, the symptom is lethargy. In certain
embodiments, the symptom is back and joint problems.
[0590] In certain embodiments, the physiological symptom is high
blood pressure. In certain embodiments, the symptom is
hypertension. In certain embodiments, the symptom is high
cholesterol levels. In certain embodiments, the symptom is type 2
diabetes. In certain embodiments, the symptom is stroke. In certain
embodiments, the symptom is cardiac insufficiency. In certain
embodiments, the symptom is heart disease. In certain embodiments,
the symptom is coronary artery obstruction. In certain embodiments,
the symptom is breast cancer in women. In certain embodiments, the
symptom is gastro-oesophageal reflux disease. In certain
embodiments, the symptom is hip and knee arthrosis. In certain
embodiments, the symptom is reduced life expectancy.
[0591] Diabetes mellitus is characterized by numerous physical and
physiological symptoms. Any symptom known to one of skill in the
art to be associated with Type 2 diabetes can be ameliorated or
otherwise modulated as set forth above in the methods described
above. In certain embodiments, the symptom is a physical symptom
selected from the group consisting of increased glucose levels,
increased weight gain, frequent urination, unusual thirst, extreme
hunger, extreme fatigue, blurred vision, frequent infections,
tingling or numbness at the extremities, dry and itchy skin, weight
loss, slow-healing sores, and swollen gums.
[0592] In certain embodiments, the symptom is a physiological
symptom selected from the group consisting of increased insulin
resistance, increased glucose levels, increased fat mass, decreased
metabolic rate, decreased glucose clearance, decreased glucose
tolerance, decreased insulin sensitivity, decreased hepatic insulin
sensitivity, increased adipose tissue size and weight, increased
body fat, and increased body weight.
[0593] In certain embodiments, the physical symptom is increased
weight gain. In certain embodiments, the symptom is frequent
urination. In certain embodiments, the symptom is unusual thirst.
In certain embodiments, the symptom is extreme hunger. In certain
embodiments, the symptom is extreme fatigue. In certain
embodiments, the symptom is blurred vision. In certain embodiments,
the symptom is frequent infections. In certain embodiments, the
symptom is tingling or numbness at the extremities. In certain
embodiments, the symptom is dry and itchy skin. In certain
embodiments, the symptom is weight loss. In certain embodiments,
the symptom is slow-healing sores. In certain embodiments, the
symptom is swollen gums. In certain embodiments, the symptom is
increased insulin resistance. In certain embodiments, the symptom
is increased fat mass. In certain embodiments, the symptom is
decreased metabolic rate. In certain embodiments, the symptom is
decreased glucose clearance. In certain embodiments, the symptom is
decreased glucose tolerance. In certain embodiments, the symptom is
decreased insulin sensitivity. In certain embodiments, the symptom
is decreased hepatic insulin sensitivity. In certain embodiments,
the symptom is increased adipose tissue size and weight. In certain
embodiments, the symptom is increased body fat. In certain
embodiments, the symptom is increased body weight.
[0594] In certain embodiments, provided are methods of treating an
individual comprising administering one or more pharmaceutical
compositions as described herein. In certain embodiments, the
individual has metabolic related disease.
[0595] In certain embodiments, administration of an antisense
compound targeted to a FGFR4 nucleic acid results in reduction of
FGFR4 expression by at least about 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by
any two of these values.
[0596] In certain embodiments, pharmaceutical compositions
comprising an antisense compound targeted to FGFR4 are used for the
preparation of a medicament for treating a patient suffering or
susceptible to a metabolic disease.
[0597] In certain embodiments, the methods described herein include
administering a compound comprising a modified oligonucleotide
having a contiguous nucleobases portion as described herein of a
sequence recited in SEQ ID NO: 16.
[0598] In certain embodiments, the methods described herein include
administering a compound comprising a modified oligonucleotide
having a contiguous nucleobases portion as described herein of a
sequence recited in SEQ ID NO: 45.
Administration
[0599] In certain embodiments, the compounds and compositions as
described herein may be administered in a number of ways depending
upon whether local or systemic treatment is desired and upon the
area to be treated. Administration may be topical, pulmonary, e.g.,
by inhalation or insufflation of powders or aerosols, including by
nebulizer; intratracheal, intranasal, epidermal and transdermal,
oral or parenteral. The compounds and compositions as described
herein can be administered directly to a tissue or organ.
[0600] In certain embodiments, the compounds and compositions as
described herein are administered parenterally. "Parenteral
administration" means administration through injection or infusion.
Parenteral administration includes subcutaneous administration,
intravenous administration, intramuscular administration,
intraarterial administration, intraperitoneal administration, or
intracranial administration, e.g. intracerebral administration,
intrathecal administration, intraventricular administration,
ventricular administration, intracerebroventricular administration,
cerebral intraventricular administration or cerebral ventricular
administration. Administration can be continuous, or chronic, or
short or intermittent.
[0601] In certain embodiments, parenteral administration is by
injection. The injection can be delivered with a syringe or a pump.
In certain embodiments, the injection is a bolus injection. In
certain embodiments, the injection is administered directly to a
tissue or organ.
[0602] In certain embodiments, the compounds and compositions as
described herein are administered parenterally.
[0603] In certain embodiments, parenteral administration is
subcutaneous.
[0604] In further embodiments, the formulation for administration
is the compounds described herein and saline.
[0605] In certain embodiments, an antisense oligonucleotide is
delivered by injection or infusion once every month, every two
months, every 90 days, every 3 months, every 6 months, twice a year
or once a year.
Certain Combination Therapies
[0606] 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 as described
herein. In certain embodiments, one or more pharmaceutical
compositions are co-administered with another pharmaceutical agent
to treat an undesired effect of that other pharmaceutical agent. In
certain embodiments, one or more pharmaceutical compositions are
co-administered with another pharmaceutical agent to produce a
combinational effect. In certain embodiments, one or more
pharmaceutical compositions are co-administered with another
pharmaceutical agent to produce a synergistic effect.
[0607] In certain embodiments, a first agent and one or more second
agents are administered at the same time. In certain embodiments,
the first agent and one or more second agents are administered at
different times. In certain embodiments, the first agent and one or
more second agents are prepared together in a single pharmaceutical
formulation. In certain embodiments, the first agent and one or
more second agents are prepared separately.
[0608] In certain embodiments, the second compound is administered
prior to administration of a pharmaceutical composition described
herein. In certain embodiments, the second compound is administered
following administration of a pharmaceutical composition described
herein. In certain embodiments, the second compound is administered
at the same time as a pharmaceutical composition described herein.
In certain embodiments, the dose of a co-administered second
compound is the same as the dose that would be administered if the
second compound was administered alone. In certain embodiments, the
dose of a co-administered second compound is lower than the dose
that would be administered if the second compound was administered
alone. In certain embodiments, the dose of a co-administered second
compound is greater than the dose that would be administered if the
second compound was administered alone.
[0609] 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 certain 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.
[0610] In certain embodiments the FGFR4 antisense oligonucleotide
is delivered concomitant with delivery of the second agent.
Alternatively, delivery can be in the same formulation or can be
administered separately. In certain embodiments, FGFR4 antisense
oligonucleotide is administered prior to the treatment with the
second agents. In a certain embodiment, the FGFR4 antisense
oligonucleotide is administered after treatment with an obesity
inducing drug or agent is ceased.
[0611] In certain embodiments, second agents include, but are not
limited to, a glucose-lowering agent. The glucose lowering agent
can include, but is not limited to, a therapeutic lifestyle change,
PPAR agonist, a dipeptidyl peptidase (IV) inhibitor, a GLP-1
analog, insulin or an insulin analog, an insulin secretagogue, a
SGLT2 inhibitor, a human amylin analog, a biguanide, an
alpha-glucosidase inhibitor, or a combination thereof. The
glucose-lowering agent can include, but is not limited to
metformin, sulfonylurea, rosiglitazone, meglitinide,
thiazolidinedione, alpha-glucosidase inhibitor or a combination
thereof. The sulfonylurea can be acetohexamide, chlorpropamide,
tolbutamide, tolazamide, glimepiride, a glipizide, a glyburide, or
a gliclazide. The meglitinide can be nateglinide or repaglinide.
The thiazolidinedione can be pioglitazone or rosiglitazone. The
alpha-glucosidase can be acarbose or miglitol.
[0612] In some embodiments, the glucose-lowering therapeutic is a
GLP-1 analog. In some embodiments, the GLP-1 analog is exendin-4 or
liraglutide.
[0613] In other embodiments, the glucose-lowering therapeutic is a
sulfonylurea. In some embodiments, the sulfonylurea is
acetohexamide, chlorpropamide, tolbutamide, tolazamide,
glimepiride, a glipizide, a glyburide, or a gliclazide.
[0614] In some embodiments, the glucose-lowering drug is a
biguanide. In some embodiments, the biguanide is metformin, and in
some embodiments, blood glucose levels are decreased without
increased lactic acidosis as compared to the lactic acidosis
observed after treatment with metformin alone.
[0615] In some embodiments, the glucose-lowering drug is a
meglitinide. In some embodiments, the meglitinide is nateglinide or
repaglinide.
[0616] In some embodiments, the glucose-lowering drug is a
thiazolidinedione. In some embodiments, the thiazolidinedione is
pioglitazone, rosiglitazone, or troglitazone. In some embodiments,
blood glucose levels are decreased without greater weight gain than
observed with rosiglitazone treatment alone.
[0617] In some embodiments, the glucose-lowering drug is an
alpha-glucosidase inhibitor. In some embodiments, the
alpha-glucosidase inhibitor is acarbose or miglitol.
[0618] In a certain embodiment, a co-administered glucose-lowering
agent is ISIS 113715.
[0619] In a certain embodiment, glucose-lowering therapy is
therapeutic lifestyle change.
[0620] In certain embodiments, second agents include, but are not
limited to, lipid-lowering agents. The lipid-lowering agent can
include, but is not limited to atorvastatin, simvastatin,
rosuvastatin, and ezetimibe. In certain such embodiments, the
lipid-lowering agent is administered prior to administration of a
pharmaceutical composition described herein. In certain such
embodiments, the lipid-lowering agent is administered following
administration of a pharmaceutical composition described herein. In
certain such embodiments the lipid-lowering agent is administered
at the same time as a pharmaceutical composition described herein.
In certain such embodiments the dose of a co-administered
lipid-lowering agent is the same as the dose that would be
administered if the lipid-lowering agent was administered alone. In
certain such embodiments the dose of a co-administered
lipid-lowering agent is lower than the dose that would be
administered if the lipid-lowering agent was administered alone. In
certain such embodiments the dose of a co-administered
lipid-lowering agent is greater than the dose that would be
administered if the lipid-lowering agent was administered
alone.
[0621] In certain embodiments, a co-administered lipid-lowering
agent is a HMG-CoA reductase inhibitor. In certain such embodiments
the HMG-CoA reductase inhibitor is a statin. In certain such
embodiments the statin is selected from atorvastatin, simvastatin,
pravastatin, fluvastatin, and rosuvastatin.
[0622] In certain embodiments, a co-administered lipid-lowering
agent is a cholesterol absorption inhibitor. In certain such
embodiments, cholesterol absorption inhibitor is ezetimibe.
[0623] In certain embodiments, a co-administered lipid-lowering
agent is a co-formulated HMG-CoA reductase inhibitor and
cholesterol absorption inhibitor. In certain such embodiments the
co-formulated lipid-lowering agent is ezetimibe/simvastatin.
[0624] In certain embodiments, a co-administered lipid-lowering
agent is a microsomal triglyceride transfer protein inhibitor (MTP
inhibitor).
[0625] In certain embodiments, a co-administered lipid-lowering
agent is an oligonucleotide targeted to ApoB.
[0626] In certain embodiments, second agents include, but are not
limited to an anti-obesity drug or agent. Such anti-obesity agents
include but are not limited to Orlistat, Sibutramine, or
Rimonabant, and may be administered as described above as adipose
or body weight lowering agents. In certain embodiments, the
antisense compound may be co-administered with appetite
suppressants. Such appetite suppressants include but are not
limited to diethylpropion tenuate, mazindol, orlistat,
phendimetrazine, phentermine, and sibutramine and may be
administered as described herein. In certain embodiment, the
anti-obesity agents are CNS based such as, but not limited to,
sibutramine or GLP-1 based such as, but not limited to,
liraglutide.
[0627] In certain embodiments, second agents include, but are not
limited to an antipsychotic drug or agent. Such antipsychotic
agents therapeutics may be administered as described above to
reduce metabolic abnormalities associated with treatment with
antipsychotic agents. In a particular embodiment administering of
the FGFR4 antisense compound results in increased metabolic rate or
decreasing adiposity or decreasing body weight or all three without
affecting the CNS effects of the psychotherapeutic agent
[0628] Due to the ability of FGFR4 antisense oligonucleotides to
increase metabolic rate and insulin sensitivity and reduce
adiposity and weight gain, these compounds can be administered to
reduce metabolic abnormalities associated with treatment with
antipsychotic agents. In certain embodiments the FGFR4 antisense
oligonucleotide is delivered in a method of reducing metabolic
abnormalities associated with the therapeutic use of
psychotherapeutic agents. Such weight inducing antipsychotic agents
include, but are not limited to clozapine, olanzapine,
aripiprazole, risperidone and ziprasidone.
[0629] Further provided is a method of administering an antisense
compound targeted to a FGFR4 nucleic acid via injection and further
including administering a topical steroid at the injection
site.
[0630] Further examples of pharmaceutical agents that may be
co-administered with a pharmaceutical composition of the present
invention include, but are not limited to, corticosteroids,
including but not limited to prednisone; immunoglobulins,
including, but not limited to intravenous immunoglobulin (IVIg);
analgesics (e.g., acetaminophen); anti-inflammatory agents,
including, but not limited to non-steroidal anti-inflammatory drugs
(e.g., ibuprofen, COX-1 inhibitors, and COX-2, inhibitors);
salicylates; antibiotics; antivirals; antifungal agents;
antidiabetic agents (e.g., biguanides, glucosidase inhibitors,
insulins, sulfonylureas, and thiazolidenediones); adrenergic
modifiers; diuretics; hormones (e.g., anabolic steroids, androgen,
estrogen, calcitonin, progestin, somatostan, and thyroid hormones);
immunomodulators; muscle relaxants; antihistamines; osteoporosis
agents (e.g., biphosphonates, calcitonin, and estrogens);
prostaglandins, antineoplastic agents; psychotherapeutic agents;
sedatives; poison oak or poison sumac products; antibodies; and
vaccines.
[0631] In certain embodiments, the pharmaceutical compositions of
the present invention may be administered in conjunction with a
lipid-lowering therapy. In certain such embodiments, a
lipid-lowering therapy is therapeutic lifestyle change. In certain
such embodiments, a lipid-lowering therapy is LDL apheresis.
Formulations
[0632] The compounds provided herein may also be admixed,
conjugated or otherwise associated with other molecules, molecule
structures or mixtures of compounds, as for example, liposomes,
receptor-targeted molecules, or other formulations, for assisting
in uptake, distribution and/or absorption. Representative United
States patents that teach the preparation of such uptake,
distribution and/or absorption-assisting formulations include, but
are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;
5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;
4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;
5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;
5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;
5,580,575; and 5,595,756, each of which is herein incorporated by
reference.
[0633] The antisense compounds provided herein encompass any
pharmaceutically acceptable salts, esters, or salts of such esters,
or any other compound which, upon administration to an animal,
including a human, is capable of providing (directly or indirectly)
the biologically active metabolite or residue thereof.
[0634] The term "pharmaceutically acceptable salts" refers to
physiologically and pharmaceutically acceptable salts of the
compounds provided herein: i.e., salts that retain the desired
biological activity of the parent compound and do not impart
undesired toxicological effects thereto. The term "pharmaceutically
acceptable salt" includes a salt prepared from pharmaceutically
acceptable non-toxic acids or bases, including inorganic or organic
acids and bases. For oligonucleotides, preferred examples of
pharmaceutically acceptable salts and their uses are further
described in U.S. Pat. No. 6,287,860, which is incorporated herein
in its entirety. Sodium salts have been shown to be suitable forms
of oligonucleotide drugs.
[0635] The term "pharmaceutically acceptable derivative"
encompasses, but is not limited to, pharmaceutically acceptable
salts, solvates, hydrates, esters, prodrugs, polymorphs, isomers,
isotopically labeled variants of the compounds described
herein.
[0636] The present invention also includes pharmaceutical
compositions and formulations which include the antisense compounds
provided herein. The pharmaceutical compositions described herein
may be administered in a number of ways depending upon whether
local or systemic treatment is desired and upon the area to be
treated. Administration may be parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion; or
intracranial, e.g., intracerebral administration, intrathecal
administration, intraventricular administration, ventricular
administration, intracerebroventricular administration, cerebral
intraventricular administration or cerebral ventricular
administration.
[0637] Parenteral administration, is preferred to target FGFR4
expression in the liver and plasma. Oligonucleotides with at least
one 2'-O-methoxyethyl modification are believed to be particularly
useful for oral administration. Pharmaceutical compositions and
formulations for topical administration may include transdermal
patches, ointments, lotions, creams, gels, drops, suppositories,
sprays, liquids and powders. Conventional pharmaceutical carriers,
aqueous, powder or oily bases, thickeners and the like may be
necessary or desirable. Coated condoms, gloves and the like may
also be useful.
[0638] The pharmaceutical formulations described herein, which may
conveniently be presented in unit dosage form, may be prepared
according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0639] The compositions described herein may be formulated into any
of many possible dosage forms such as, but not limited to, tablets,
capsules, gel capsules, liquid syrups, soft gels, suppositories,
and enemas. The compositions described herein may also be
formulated as suspensions in aqueous, non-aqueous or mixed media.
Aqueous suspensions may further contain substances which increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0640] Pharmaceutical compositions described herein include, but
are not limited to, solutions, emulsions, foams and
liposome-containing formulations. The pharmaceutical compositions
and formulations described herein may comprise one or more
penetration enhancers, carriers, excipients or other active or
inactive ingredients.
[0641] Emulsions are typically heterogenous systems of one liquid
dispersed in another in the form of droplets usually exceeding 0.1
.mu.m in diameter. Emulsions may contain additional components in
addition to the dispersed phases, and the active drug which may be
present as a solution in the aqueous phase, oily phase or itself as
a separate phase. Microemulsions are included as an embodiment
described herein. Emulsions and their uses are well known in the
art and are further described in U.S. Pat. No. 6,287,860, which is
incorporated herein in its entirety.
[0642] Formulations include liposomal formulations. As used in the
present invention, the term "liposome" means a vesicle composed of
amphiphilic lipids arranged in a spherical bilayer or bilayers.
Liposomes are unilamellar or multilamellar vesicles which have a
membrane formed from a lipophilic material and an aqueous interior
that contains the composition to be delivered. Cationic liposomes
are positively charged liposomes which are believed to interact
with negatively charged DNA molecules to form a stable complex.
Liposomes that are pH-sensitive or negatively-charged are believed
to entrap DNA rather than complex with it. Both cationic and
noncationic liposomes have been used to deliver DNA to cells.
[0643] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Liposomes and their uses are
further described in U.S. Pat. No. 6,287,860, which is incorporated
herein in its entirety.
[0644] In another embodiment, formulations include saline
formulations. In certain embodiments, a formulation consists of the
compounds described herein and saline. In certain embodiments, a
formulation consists essentially of the compounds described herein
and saline. In certain embodiments, the saline is pharmaceutically
acceptable grade saline. In certain embodiments, the saline is
buffered saline. In certain embodiments, the saline is phosphate
buffered saline (PBS).
[0645] In certain embodiments, a formulation excludes liposomes. In
certain embodiments, the formulation excludes sterically stabilized
liposomes. In certain embodiments, a formulation excludes
phospholipids. In certain embodiments, the formulation consists
essentially of the compounds described herein and saline and
excludes liposomes.
[0646] The pharmaceutical formulations and compositions may also
include surfactants. Surfactants and their uses are further
described in U.S. Pat. No. 6,287,860, which is incorporated herein
in its entirety.
[0647] In one embodiment, the present invention employs various
penetration enhancers to affect the efficient delivery of nucleic
acids, particularly oligonucleotides. Penetration enhancers and
their uses are further described in U.S. Pat. No. 6,287,860, which
is incorporated herein in its entirety.
[0648] One of skill in the art will recognize that formulations are
routinely designed according to their intended use, i.e. route of
administration.
[0649] Formulations for topical administration include those in
which the oligonucleotides provided herein are in admixture with a
topical delivery agent such as lipids, liposomes, fatty acids,
fatty acid esters, steroids, chelating agents and surfactants.
Preferred lipids and liposomes include neutral (e.g.
dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl
choline DMPC, distearolyphosphatidyl choline) negative (e.g.
dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.
dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl
ethanolamine DOTMA).
[0650] Compositions and formulations for parenteral administration,
including intravenous, intraarterial, subcutaneous,
intraperitoneal, intramuscular injection or infusion, or
intracranial may include sterile aqueous solutions which may also
contain buffers, diluents and other suitable additives such as, but
not limited to, penetration enhancers, carrier compounds and other
pharmaceutically acceptable carriers or excipients.
[0651] Certain embodiments provided herein provide pharmaceutical
compositions containing one or more oligomeric compounds and one or
more other chemotherapeutic agents which function by a
non-antisense mechanism. Examples of such chemotherapeutic agents
include but are not limited to cancer chemotherapeutic drugs such
as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,
idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,
cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,
mitomycin C, actinomycin D, mithramycin, prednisone,
hydroxyprogesterone, testosterone, tamoxifen, dacarbazine,
procarbazine, hexamethylmelamine, pentamethylmelamine,
mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,
nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea,
deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil
(5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX),
colchicine, taxol, vincristine, vinblastine, etoposide (VP-16),
trimetrexate, irinotecan, topotecan, gemcitabine, teniposide,
cisplatin and diethylstilbestrol (DES). When used with the
compounds provided herein, such chemotherapeutic agents may be used
individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,
5-FU and oligonucleotide for a period of time followed by MTX and
oligonucleotide), or in combination with one or more other such
chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or
5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs,
including but not limited to nonsteroidal anti-inflammatory drugs
and corticosteroids, and antiviral drugs, including but not limited
to ribivirin, vidarabine, acyclovir and ganciclovir, may also be
combined in compositions provided herein. Combinations of antisense
compounds and other non-antisense drugs are also within the scope
of this invention. Two or more combined compounds may be used
together or sequentially.
[0652] In another related embodiment, compositions provided herein
may contain one or more antisense compounds, particularly
oligonucleotides, targeted to a first nucleic acid and one or more
additional antisense compounds targeted to a second nucleic acid
target. Alternatively, compositions provided herein may contain two
or more antisense compounds targeted to different regions of the
same nucleic acid target. Numerous examples of antisense compounds
are known in the art. Two or more combined compounds may be used
together or sequentially.
Dosing
[0653] Dosing is dependent on severity and responsiveness of the
disease state to be treated, with the course of treatment lasting
from several days to several months, or until a cure is effected or
a diminution of the disease state is achieved. Optimal dosing
schedules can be calculated from measurements of drug accumulation
in the body of the patient. Optimum dosages may vary depending on
the relative potency of individual oligonucleotides, and can
generally be estimated based on EC.sub.50s found to be effective in
in vitro and in vivo animal models. In general, dosage is from 0.01
.mu.g to 100 g per kg of body weight, and may be given once or more
daily, weekly, monthly or yearly, or at desired intervals.
Following successful treatment, it may be desirable to have the
patient undergo maintenance therapy to prevent the recurrence of
the disease state, wherein the oligonucleotide is administered in
maintenance doses, ranging from 0.01 .mu.g to 100 g per kg of body
weight, once or more daily.
[0654] While the present invention has been described with
specificity in accordance with certain of its preferred
embodiments, the following examples serve only to illustrate the
invention and are not intended to limit the same. Each of the
references, GENBANK accession numbers, and the like recited in the
present application is incorporated herein by reference in its
entirety.
Certain Compounds
[0655] About one thousand four hundred and fifty four newly
designed and previously disclosed antisense compounds of various
lengths, motifs and backbone composition were tested for their
effect on human FGFR4 mRNA in vitro in several cell types. The new
compounds were compared with nine previously designed compounds,
including ISIS 299005, ISIS 299010, ISIS 299018, ISIS 299022, ISIS
299024, ISIS 299025, ISIS 299028, ISIS 299029, and ISIS 299030
which have previously been determined to be some of the most potent
antisense compounds in vitro (see e.g., U.S. Patent Publication No.
US2010/0292140). Of the one thousand four hundred and fifty four
newly designed and the nine previously designed antisense
compounds, fifty three compounds were selected for further study
based on in vitro potency. The selected compounds were tested for
dose dependent inhibition in HepG2 (Examples 8 and 9).
[0656] Certain oligonucleotides were then tested for tolerability
in a CD1 mouse model, as well as a Sprague-Dawley rat model. The
oligonucleotides tested for tolerability include oligonucleotides,
ISIS 299005 (SEQ ID NO: 7), ISIS 463588 (SEQ ID NO: 16), ISIS
463589 (SEQ ID NO: 17), ISIS 463628 (SEQ ID NO: 28), ISIS 463690
(SEQ ID NO: 45), ISIS 463691 (SEQ ID NO: 46), ISIS 463835 (SEQ ID
NO: 70), ISIS 463837 (SEQ ID NO: 72), ISIS 464222 (SEQ ID NO: 135),
ISIS 464225 (SEQ ID NO: 138), ISIS 464228 (SEQ ID NO: 141), ISIS
464286 (SEQ ID NO: 154), ISIS 464308 (SEQ ID NO: 163), ISIS 464449
(SEQ ID NO: 174), ISIS 464587 (SEQ ID NO: 186), ISIS 464588 (SEQ ID
NO: 187), ISIS 464589 (SEQ ID NO: 188), ISIS 464718 (SEQ ID NO:
221), ISIS 479533 (SEQ ID NO: 241), ISIS 479551 (SEQ ID NO: 259),
ISIS 479691 (SEQ ID NO: 299), ISIS 479692 (SEQ ID NO: 300), ISIS
479698 (SEQ ID NO: 305), ISIS 479699 (SEQ ID NO: 306), ISIS 479703
(SEQ ID NO: 307), ISIS 479704 (SEQ ID NO: 308), ISIS 479706 (SEQ ID
NO: 310), and ISIS 479736 (SEQ ID NO: 317). By virtue of their
complementary sequence, the compounds are complementary to the
regions 192-211, 191-210, 193-212, 291-310, 369-388, 370-389,
788-807, 790-809, 2951-2970, 2954-2973, and 2981-3000 of SEQ ID NO:
1; 11621-11640, 11624-11643, 11651-11670, 1463-1482, 3325-3344,
7802-7821, 2110-2129, 2112-2131, 2114-2133, 3575-3594, 2111-2130,
3570-3589, 11623-11639, 11624-11640, 11652-11668, 11653-11669,
2113-2129, 2114-2130, 2116-2132, and 3571-3587 of SEQ ID NO: 2; and
103-122, 1569-1588, 5122-5138, 5123-5139, 5151-5167, 5152-5168,
105-121, 106-122, 108-124, and 1570-1586 of SEQ ID NO: 3.
[0657] In the in vivo models, the liver function markers, such as
alanine transaminase, aspartate transaminase and bilirubin, and
kidney function markers, such as BUN and creatinine were measured.
(Example 11).
[0658] Eight oligonucleotides having a nucleobase sequence of a
sequence recited in SEQ ID NO: 7 (ISIS 299005), 16 (ISIS 463588),
17 (ISIS 463589), 45 (ISIS 463690), 46 (ISIS 463691), 70 (ISIS
463835), 72 (ISIS 463837) and 138 (ISIS 464225) were tested. By
virtue of their complementary sequence, the compounds are
complementary to the regions 192-211, 191-210, 193-212, 369-388,
370-389, 788-807, 790-809, and 2954-2973 of SEQ ID NO: 1. In
certain embodiments, the compounds targeting the listed regions, as
further described herein, comprise a modified oligonucleotide
having some nucleobase portion of the sequence recited in the SEQ
ID NOs, as further described herein, In certain embodiments, the
compounds targeting the listed regions or having a nucleobase
portion of a sequence recited in the listed SEQ ID NOs can be of
various length, as further described herein, and can have one of
various motifs, as further described herein. In certain
embodiments, a compound targeting a region or having a nucleobase
portion of a sequence recited in the listed SEQ ID NOs has the
specific length and motif, as indicated by the ISIS NOs: 299005,
463588, 463589, 463690, 463691, 463835, 463837, and 464225.
[0659] These eight compounds, ISIS 299005 (SEQ ID NO: 7), ISIS
463588 (SEQ ID NO: 16), ISIS 463589 (SEQ ID NO: 17), ISIS 463690
(SEQ ID NO: 45), ISIS 463691 (SEQ ID NO: 46), ISIS 463835 (SEQ ID
NO: 70), ISIS 463837 (SEQ ID NO: 72), and ISIS 464225 (SEQ ID NO:
138), were assayed for long-term effects on tolerability in a
CD/1GS rat model for 13 weeks (Example 12). Body weights and organ
weights, the liver function markers, such as alanine transaminase,
aspartate transaminase and bilirubin, and kidney function markers,
such as BUN and creatinine were measured. The eight compounds were
also tested for their viscosity. (Example 14)
[0660] ISIS 463588, ISIS 463589, and ISIS 463690 which demonstrated
very good tolerability in all three in vivo models, were tested for
their half-life in CD1 mouse liver (Example 13).
[0661] These eight compounds, ISIS 299005 (SEQ ID NO: 7), ISIS
463588 (SEQ ID NO: 16), ISIS 463589 (SEQ ID NO: 17), ISIS 463690
(SEQ ID NO: 45), ISIS 463691 (SEQ ID NO: 46), ISIS 463835 (SEQ ID
NO: 70), ISIS 463837 (SEQ ID NO: 72), and ISIS 464225 (SEQ ID NO:
138), were tested for efficacy, pharmacokinetic profile and
tolerability in cynomolgus monkeys (Example 15). The inhibition
studies in these monkeys indicated that treatment with some of
these compounds caused reduction of FGFR4 mRNA in the liver
tissues. Specifically, treatment with ISIS 463588 caused
significantly greater reduction of FGFR4 mRNA in liver and kidney
tissues, respectively compared to treatment with the previously
disclosed compound, ISIS 299005. It was noted that ISIS 463588
caused the highest reduction of FGFR4 mRNA compared to the PBS
control, irrespective of the primer probe set used. Hence, in terms
of potency, treatment with ISIS 463588 was the most effective in
the monkey study. Treatment with ISIS 463690 also caused a greater
reduction of FGFR4 mRNA in liver and kidney tissues, respectively
compared to treatment with the previously disclosed compound, ISIS
299005.
[0662] FGF19 has been known to reduce adiposity and improve insulin
sensitivity in transgenic mice (Fu, L. et al., Endocrinology. 145:
2594-2603, 2004). FGF19 is also characterized as a high affinity
ligand for FGFR4 (Xie, M.-H. et al., Cytokine. 11: 729-735, 1999).
However, treating mice with FGF19 protein induces hepatocyte
proliferation consistent with the increased hepatocyte
proliferation and liver tumor formation observed in FGF19
transgenic mice (Wu, X. et al., JBC 285(8): 5165-5170, 2010).
Leptin is a hormone which has been found to be present at very high
levels in obese individuals compared to normal-weight individuals
(Considine, R. V. et al., N. Engl. J. Med. 334: 292-295, 1996).
Evaluation of FGF19 mRNA and plasma levels demonstrated the
significant increase in FGF19 mRNA and protein levels in all the
treatment groups. Specifically, monkeys treated with ISIS 463588
had the most significant increase in FGF19 levels. Evaluation of
leptin plasma levels demonstrated a significant decrease in monkeys
treated with ISIS 463588 or ISIS 463690. Tolerability studies in
cynomolgus monkeys (Example 15) were conducted after treatment with
the ISIS oligonucleotides. This included measurement plasma levels
of liver metabolites, kidney metabolites, pro-inflammatory factors,
such as C-reactive protein, complement C3 and cytokines. The
results indicated that treatment with the ISIS oligonucleotides in
Example 15 remained within acceptable levels for antisense
oligonucleotides and were therefore tolerable to the monkeys. In
particular, treatment with ISIS 463588 was very well-tolerated in
this model.
[0663] Pharmacokinetic studies of the three most well-tolerated
ISIS oligonucleotides, ISIS 463588, ISIS 463589, and ISIS 463690,
was also performed in the monkeys and indicated that the
pharmacokinetics of all three were optimal.
[0664] Hence, the in vivo studies, particularly in the cynomolgus
monkeys, indicate that ISIS 463588, ISIS 463589, and ISIS 463690,
were a more potent oligonucleotide compared to ISIS 299005 and was
also considerably more tolerable.
[0665] Accordingly, provided herein are antisense compounds with
any one or more of the improved characteristics. In a certain
embodiments, provided herein are compounds comprising a modified
oligonucleotide as further described herein targeted to or
specifically hybridizable with the region of nucleotides of SEQ ID
NO: 1.
[0666] In certain embodiments, the compounds as described herein
are efficacious by virtue of having at least one of an in vitro
IC.sub.50 of less than 1.5 .mu.M, less than 1.4 .mu.M, less than
1.3 .mu.M, less than 1.2 .mu.M, less than 1.1 .mu.M, less than 1.0
.mu.M, less than 0.9 .mu.M, less than 0.8 .mu.M, less than 0.7
.mu.M, less than 0.6 .mu.M when delivered to a HepG2 cell line
using electroporation as described in Example 8. In certain
embodiments, the compounds as described herein are efficacious in
vivo, as demonstrated by decreasing the levels of FGFR4 mRNA by
60%, 65%, 70%, 75% or 80%. In further embodiments, the compounds
are efficacious in vivo, as demonstrated by increasing the levels
of FGF15 and FGF19 mRNA and protein by 100%, 200%, 300%, 400%,
500%, 600%, 700%, 800%, 900%, or 1000%. In other embodiments, the
compounds are efficacious in vivo, as demonstrated by decreasing
plasma levels of leptin by 30%, 35%, or 40%.
[0667] In certain embodiments, the compounds as described herein
are highly tolerable, as demonstrated by having at least one of an
increase an ALT or AST value of no more than 4 fold, 3 fold, or 2
fold over saline treated animals; or an increase in liver, spleen
or kidney weight of no more than 30%, 20%, 15%, 12%, 10%, 5% or
2%.
EXAMPLES
Non-Limiting Disclosure and Incorporation by Reference
[0668] 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 Human Fibroblast Growth Factor Receptor
(FGFR4) in HepG2 Cells
[0669] Antisense oligonucleotides were designed targeting a FGFR4
nucleic acid and were tested for their effects on FGFR4 mRNA in
vitro. Cultured HepG2 cells at a density of 20,000 cells per well
were transfected using electroporation with 4,500 nM antisense
oligonucleotide. After a treatment period of approximately 24
hours, RNA was isolated from the cells and FGFR4 mRNA levels were
measured by quantitative real-time PCR. Human primer probe set
RTS3232 (forward sequence TCATCAACGGCAGCAGCTT, designated herein as
SEQ ID NO: 327; reverse sequence AGCTATTGATGTCTGCAGTCTTTAGG,
designated herein as SEQ ID NO: 328; probe sequence
AGCCGACGGTTTCCCCTATGTGCA, designated herein as SEQ ID NO: 329) was
used to measure mRNA levels. FGFR4 mRNA levels were adjusted
according to total RNA content, as measured by RIBOGREEN.RTM..
Results are presented as percent inhibition of FGFR4, relative to
untreated control cells. A total of 458 oligonucleotides were
tested. Only those oligonucleotides demonstrating greater than 65%
inhibition in vitro or which were used in subsequent assays are
shown in Table 1.
[0670] Some of the antisense oligonucleotides were also tested with
an additional primer probe set RTS1325 (forward sequence
TTGCTGTGCCGTGTCCAA, designated herein as SEQ ID NO: 330; reverse
sequence TCCAAGAAGCCGAGCAGAAC, designated herein as SEQ ID NO: 331;
probe sequence AGCTGCCGTGCCTGTGTCCTGAT, designated herein as SEQ ID
NO: 332). `n/a.` indicates that particular antisense
oligonucleotide was not tested with RTS1325.
[0671] The newly designed chimeric antisense oligonucleotides in
Table 1 were designed as 5-10-5 MOE gapmers. The gapmers are 20
nucleosides in length, wherein the central gap segment comprises of
ten 2'-deoxynucleosides and is flanked by wing segments on the 5'
and 3' directions comprising five nucleosides each. Each nucleotide
in the 5' wing segment and each nucleotide in the 3' wing segment
has a 2'-MOE modification. The internucleoside linkages throughout
each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine
residues throughout each gapmer are 5-methylcytosines. "Start site"
indicates the 5'-most nucleoside to which the gapmer is targeted in
the human gene sequence. "Stop site" indicates the 3'-most
nucleoside to which the gapmer is targeted in the human gene
sequence. Each gapmer listed in Table 1 is targeted to the human
FGFR4 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession
No. NM.sub.--002011.3).
TABLE-US-00001 TABLE 1 Inhibition of human FGFR4 mRNA levels by
chimeric antisense oligonucleotides targeted to SEQ ID NO: 1 % %
SEQ Start Stop ISIS inhibition inhibition ID Site Site Sequence No
(RTS3232) (RTS1325) NO 192 211 GGCACACTCAGCAGGACCCC 299005 85 n/a 7
304 323 AGGCTGCCCAAGGGCTACTG 299010 65 n/a 8 597 616
GTCCAGTAGGGTGCTTGCTG 299018 68 n/a 9 727 746 CCCATGAAAGGCCTGTCCAT
299022 68 n/a 10 757 776 GCGCAGCCGAATGCCTCCAA 299024 68 65 11 785
804 TCTCCATCACGAGACTCCAG 299025 65 59 12 969 988
TACACCTTGCACAGCAGCTC 299028 68 66 13 1027 1046 GCTGCTGCCGTTGATGACGA
299029 91 61 14 1032 1051 CCGAAGCTGCTGCCGTTGAT 299030 72 19 15 191
210 GCACACTCAGCAGGACCCCC 463588 87 n/a 16 193 212
AGGCACACTCAGCAGGACCC 463589 83 n/a 17 194 213 CAGGCACACTCAGCAGGACC
463590 72 n/a 18 196 215 CCCAGGCACACTCAGCAGGA 463592 73 n/a 19 197
216 GCCCAGGCACACTCAGCAGG 463593 71 n/a 20 198 217
GGCCCAGGCACACTCAGCAG 463594 69 n/a 21 200 219 GAGGCCCAGGCACACTCAGC
463596 78 n/a 22 202 221 TGGAGGCCCAGGCACACTCA 463598 72 n/a 23 203
222 CTGGAGGCCCAGGCACACTC 463599 78 n/a 24 205 224
GACTGGAGGCCCAGGCACAC 463601 69 n/a 25 287 306 CTGTCAGCTCCTGCTCTTGC
463625 65 n/a 26 290 309 CTACTGTCAGCTCCTGCTCT 463627 74 n/a 27 291
310 GCTACTGTCAGCTCCTGCTC 463628 82 n/a 28 292 311
GGCTACTGTCAGCTCCTGCT 463629 93 n/a 29 293 312 GGGCTACTGTCAGCTCCTGC
463630 75 n/a 30 299 318 GCCCAAGGGCTACTGTCAGC 463636 69 n/a 31 309
328 CGCACAGGCTGCCCAAGGGC 463645 75 n/a 32 332 351
GCTCAGCCCGCCCACAGCAC 463648 81 n/a 33 338 357 CACCACGCTCAGCCCGCCCA
463654 77 n/a 34 339 358 CCACCACGCTCAGCCCGCCC 463655 73 n/a 35 340
359 GCCACCACGCTCAGCCCGCC 463656 69 n/a 36 341 360
GGCCACCACGCTCAGCCCGC 463657 65 n/a 37 347 366 ACCAGTGGCCACCACGCTCA
463670 73 n/a 38 349 368 GTACCAGTGGCCACCACGCT 463672 81 n/a 39 350
369 TGTACCAGTGGCCACCACGC 463673 69 n/a 40 355 374
CTCCTTGTACCAGTGGCCAC 463677 67 n/a 41 356 375 CCTCCTTGTACCAGTGGCCA
463678 66 n/a 42 357 376 CCCTCCTTGTACCAGTGGCC 463679 76 n/a 43 368
387 CCAGGCGACTGCCCTCCTTG 463689 76 n/a 44 369 388
GCCAGGCGACTGCCCTCCTT 463690 85 n/a 45 370 389 TGCCAGGCGACTGCCCTCCT
463691 78 n/a 46 371 390 GTGCCAGGCGACTGCCCTCC 463692 81 n/a 47 372
391 GGTGCCAGGCGACTGCCCTC 463693 70 n/a 48 388 407
CCGTACACGGCCAGCAGGTG 463708 80 n/a 49 389 408 CCCGTACACGGCCAGCAGGT
463709 85 n/a 50 392 411 AGCCCCGTACACGGCCAGCA 463712 73 n/a 51 397
416 CCTCCAGCCCCGTACACGGC 463717 66 n/a 52 398 417
CCCTCCAGCCCCGTACACGG 463718 66 n/a 53 404 423 GGCGGCCCCTCCAGCCCCGT
463724 70 n/a 54 414 433 GCAATCTCTAGGCGGCCCCT 463733 65 n/a 55 415
434 GGCAATCTCTAGGCGGCCCC 463734 69 n/a 56 416 435
TGGCAATCTCTAGGCGGCCC 463735 67 n/a 57 431 450 CCTCAGGTAGGAAGCTGGCA
463750 56 n/a 58 432 451 TCCTCAGGTAGGAAGCTGGC 463751 76 n/a 59 443
462 AGCGGCCAGCATCCTCAGGT 463762 58 n/a 60 444 463
TAGCGGCCAGCATCCTCAGG 463763 77 n/a 61 599 618 GTGTCCAGTAGGGTGCTTGC
463770 66 n/a 62 601 620 GTGTGTCCAGTAGGGTGCTT 463771 32 n/a 63 624
643 AGTTTCTTCTCCATGCGCTG 463774 72 n/a 64 717 736
GCCTGTCCATCCTTAAGCCA 463791 68 n/a 65 732 751 TTCTCCCCATGAAAGGCCTG
463805 65 n/a 66 734 753 GGTTCTCCCCATGAAAGGCC 463807 60 n/a 67 784
803 CTCCATCACGAGACTCCAGT 463832 65 76 68 787 806
GCTCTCCATCACGAGACTCC 463834 78 59 69 788 807 CGCTCTCCATCACGAGACTC
463835 78 67 70 789 808 ACGCTCTCCATCACGAGACT 463836 69 66 71 790
809 CACGCTCTCCATCACGAGAC 463837 80 75 72 791 810
CCACGCTCTCCATCACGAGA 463838 76 67 73 968 987 ACACCTTGCACAGCAGCTCC
463860 66 67 74 970 989 GTACACCTTGCACAGCAGCT 463861 76 74 75 1021
1040 GCCGTTGATGACGATGTGCT 463871 65 46 76 1024 1043
GCTGCCGTTGATGACGATGT 463874 77 52 77 1025 1044 TGCTGCCGTTGATGACGATG
463875 78 42 78 1026 1045 CTGCTGCCGTTGATGACGAT 463876 78 10 79 1028
1047 AGCTGCTGCCGTTGATGACG 463877 90 54 80 1029 1048
AAGCTGCTGCCGTTGATGAC 463878 73 22 81 1031 1050 CGAAGCTGCTGCCGTTGATG
463880 74 3 82 1084 1103 GCTATTGATGTCTGCAGTCT 463882 76 67 83 1085
1104 AGCTATTGATGTCTGCAGTC 463883 68 56 84 1086 1105
GAGCTATTGATGTCTGCAGT 463884 75 61 85 1097 1116 CCTCCACCTCTGAGCTATTG
463893 74 73 86 1098 1117 ACCTCCACCTCTGAGCTATT 463894 71 71 87 1099
1118 GACCTCCACCTCTGAGCTAT 463906 66 55 88 1100 1119
GGACCTCCACCTCTGAGCTA 463907 77 90 89 1101 1120 AGGACCTCCACCTCTGAGCT
463908 89 47 90 1102 1121 CAGGACCTCCACCTCTGAGC 463909 89 74 91 1103
1122 ACAGGACCTCCACCTCTGAG 463910 79 55 92 1105 1124
GTACAGGACCTCCACCTCTG 463912 69 75 93 1106 1125 GGTACAGGACCTCCACCTCT
463913 71 73 94 1111 1130 CCGCAGGTACAGGACCTCCA 463918 67 72 95 1112
1131 TCCGCAGGTACAGGACCTCC 463919 65 37 96 1115 1134
CGTTCCGCAGGTACAGGACC 463922 70 72 97 1185 1204 GCAGACTGGTAGGAGAGGCC
463937 74 82 98 1186 1205 GGCAGACTGGTAGGAGAGGC 463938 66 7 99 1214
1233 GGTCCTCCTCTGGCAGCACC 463947 68 55 100 1301 1320
GCAGGAGCACAGCCAAGGCC 463967 67 77 101 1329 1348
GCCTGCCCTCGATACAGCCC 463994 68 n/a 102 1417 1436
GCCTGACTCCAGGGAGAACT 464002 73 n/a 103 1419 1438
GAGCCTGACTCCAGGGAGAA 464004 68 n/a 104 1468 1487
GGAGGAGAGACGCACGCCTC 464013 77 n/a 105 1469 1488
TGGAGGAGAGACGCACGCCT 464014 82 n/a 106 1470 1489
CTGGAGGAGAGACGCACGCC 464015 68 n/a 107 1502 1521
GACTCACGAGGCCGGCGAGC 464030 68 n/a 108 1505 1524
CTAGACTCACGAGGCCGGCG 464033 68 n/a 109 1558 1577
CCCAAGCACCAGCCTGTCCC 464037 67 n/a 110 1559 1578
TCCCAAGCACCAGCCTGTCC 464038 79 n/a 111 1562 1581
GCTTCCCAAGCACCAGCCTG 464041 75 n/a 112 1564 1583
GGGCTTCCCAAGCACCAGCC 464043 74 n/a 113 1616 1635
CCATGCCAAAGGCCTCTGCA 464046 65 n/a 114 1618 1637
GTCCATGCCAAAGGCCTCTG 464048 68 n/a 115 1619 1638
GGTCCATGCCAAAGGCCTCT 464049 73 n/a 116
Example 2
Dose-Dependent Antisense Inhibition of Human FGFR4 in HepG2
Cells
[0672] Gapmers from Example 1 exhibiting significant in vitro
inhibition of human FGFR4 were tested at various doses in HepG2
cells. Cells were plated at a density of 20,000 cells per well and
transfected using electroporation with 0.6 nM, 1.3 nM, 2.5 nM, 5.0
nM, and 10.0 .mu.M concentrations of antisense oligonucleotide, as
specified in Table 2. After a treatment period of approximately 16
hours, RNA was isolated from the cells and FGFR4 mRNA levels were
measured by quantitative real-time PCR. Human FGFR4 primer probe
set RTS3232 was used to measure mRNA levels. FGFR4 mRNA levels were
adjusted according to total RNA content, as measured by
RIBOGREEN.RTM.. Results are presented as percent inhibition of
FGFR4, relative to untreated control cells.
[0673] The half maximal inhibitory concentration (IC.sub.50) of
each oligonucleotide is also presented in Table 2 and was
calculated by plotting the concentrations of oligonucleotides used
versus the percent inhibition of FGFR4 mRNA expression achieved at
each concentration, and noting the concentration of oligonucleotide
at which 50% inhibition of FGFR4 mRNA expression was achieved
compared to the control. As illustrated in Table 2, FGFR4 mRNA
levels were significantly reduced in a dose-dependent manner in
antisense oligonucleotide treated cells.
TABLE-US-00002 TABLE 2 Dose-dependent antisense inhibition of human
FGFR4 in HepG2 cells using electroporation 0.6 1.3 2.5 5.0 10.0
IC.sub.50 ISIS No .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 299005 45
63 77 92 95 0.7 463588 43 66 86 90 97 0.6 463589 41 67 85 92 95 0.6
463628 55 68 87 94 92 0.3 463629 25 40 63 76 91 1.8 463648 36 51 71
85 96 1.1 463672 19 46 74 90 96 1.5 463690 30 66 86 94 97 0.9
463691 31 50 78 89 96 1.1 463692 33 56 75 90 94 1.1 463708 11 45 63
77 94 1.9 463709 35 50 73 86 96 1.1 463750 24 42 54 80 93 1.8
463762 57 76 90 95 98 <0.6 463771 53 44 66 83 88 1.4 463807 13
36 56 87 96 2.0 463834 32 44 68 90 97 1.3 463835 37 59 82 91 97 0.9
463837 28 61 77 92 97 1.1 463838 29 50 72 88 95 1.3 463861 44 52 78
90 97 0.8 463893 29 33 65 84 95 1.6 464013 27 34 50 75 86 2.1
464014 16 33 55 78 90 2.1 464038 37 55 74 90 96 1.0
Example 3
Antisense Inhibition of Human Fibroblast Growth Factor Receptor
(FGFR4) in HepG2 Cells
[0674] Additional antisense oligonucleotides were designed
targeting a FGFR4 nucleic acid and were tested for their effects on
FGFR4 mRNA in vitro. Some of the antisense oligonucleotides
described in Example 1 were also included in the assay for
comparison. Cultured HepG2 cells at a density of 20,000 cells per
well were transfected using electroporation with 4,500 nM antisense
oligonucleotide. After a treatment period of approximately 24
hours, RNA was isolated from the cells and FGFR4 mRNA levels were
measured by quantitative real-time PCR. Human primer probe set
RTS3232 was used to measure mRNA levels. FGFR4 mRNA levels were
adjusted according to total RNA content, as measured by
RIBOGREEN.RTM.. Results are presented as percent inhibition of
FGFR4, relative to untreated control cells. A total of 772
oligonucleotides were tested. Only those oligonucleotides
demonstrating greater than 65% inhibition are shown in Tables 3 and
4.
[0675] The newly designed chimeric antisense oligonucleotides in
Table 3 were designed as 5-10-5 MOE gapmers. The gapmers are 20
nucleosides in length, wherein the central gap segment comprises of
ten 2'-deoxynucleosides and is flanked by wing segments on the 5'
and 3' directions comprising five nucleosides each. Each nucleotide
in the 5' wing segment and each nucleotide in the 3' wing segment
has a 2'-MOE modification. The internucleoside linkages throughout
each gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine
residues throughout each gapmer are 5-methylcytosines. "Start site"
indicates the 5'-most nucleoside to which the gapmer is targeted in
the human gene sequence. "Stop site" indicates the 3'-most
nucleoside to which the gapmer is targeted in the human gene
sequence.
[0676] Each gapmer listed in Table 3 is targeted to either the
human FGFR4 mRNA, designated herein as SEQ ID NO: 1 (GENBANK
Accession No. NM.sub.--002011.3) or the human FGFR4 genomic
sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No:
NT.sub.--023133.11 truncated from nucleosides 21323018 to
21335213), or both. Some of the antisense oligonucleotides were
designed to target variant gene sequences and are listed in Table
4. Each gapmer in Table 4 is listed to either SEQ ID NO: 3 (GENBANK
Accession No. AB209631.1)
TABLE-US-00003 TABLE 3 Inhibition of human FGFR4 mRNA levels by
chimeric antisense oligonucleotides targeted to SEQ ID NO: 1 and
SEQ ID NO: 2 Start Stop Start Stop Site on Site on Site on Site on
SEQ SEQ SEQ SEQ SEQ ID ID ISIS % ID ID ID NO: 1 NO: 1 No Sequence
inhibition NO: 2 NO: 2 NO 160 179 299004 CAGCAGCCGCATCTCCTTCT 88
3165 3184 117 2497 2516 299055 GCTGAAGACAGAATCGCTGG 65 11167 11186
118 292 311 463629 GGCTACTGTCAGCTCCTGCT 87 3993 4012 29 2325 2344
464138 CAGCACTCACGCATCAGCCC 72 10866 10885 119 2326 2345 464139
CCAGCACTCACGCATCAGCC 75 10867 10886 120 2437 2456 464167
GGGTCCGAAGGTCAGGCGGA 68 11107 11126 121 2438 2457 464168
AGGGTCCGAAGGTCAGGCGG 70 11108 11127 122 2440 2459 464170
ATAGGGTCCGAAGGTCAGGC 67 11110 11129 123 2443 2462 464173
GGAATAGGGTCCGAAGGTCA 69 11113 11132 124 2582 2601 464181
GTGCCTGCACAGCCTTGAGC 66 11252 11271 125 2812 2831 464203
TCTCCAGCCAGGCTCAGCCA 72 11482 11501 126 2816 2835 464207
CAGCTCTCCAGCCAGGCTCA 72 11486 11505 127 2817 2836 464208
GCAGCTCTCCAGCCAGGCTC 78 11487 11506 128 2818 2837 464209
AGCAGCTCTCCAGCCAGGCT 79 11488 11507 129 2819 2838 464210
TAGCAGCTCTCCAGCCAGGC 70 11489 11508 130 2822 2841 464213
GCATAGCAGCTCTCCAGCCA 82 11492 11511 131 2823 2842 464214
AGCATAGCAGCTCTCCAGCC 85 11493 11512 132 2824 2843 464215
TAGCATAGCAGCTCTCCAGC 84 11494 11513 133 2825 2844 464216
TTAGCATAGCAGCTCTCCAG 72 11495 11514 134 2951 2970 464222
CCAGCTTCTCTGGGCTCAGG 88 11621 11640 135 2952 2971 464223
TCCAGCTTCTCTGGGCTCAG 86 11622 11641 136 2953 2972 464224
TTCCAGCTTCTCTGGGCTCA 81 11623 11642 137 2954 2973 464225
CTTCCAGCTTCTCTGGGCTC 82 11624 11643 138 2955 2974 464226
GCTTCCAGCTTCTCTGGGCT 79 11625 11644 139 2956 2975 464227
GGCTTCCAGCTTCTCTGGGC 87 11626 11645 140 2981 3000 464228
ACGCCATTTGCTCCTGTTTT 89 11651 11670 141 n/a n/a 464238
TGCGAATCAATGGGTCCCGA 73 908 927 142 n/a n/a 464239
GGTGCGAATCAATGGGTCCC 67 910 929 143 n/a n/a 464254
CCGCCGGCGCGAAGACAGCC 66 984 1003 144 n/a n/a 464258
CATCTCTGCCGCCGGCGCGA 71 992 1011 145 n/a n/a 464266
CTGACCGCTGACCGACCACC 76 1138 1157 146 n/a n/a 464268
GCTGCTGACCGCTGACCGAC 73 1142 1161 147 n/a n/a 464269
CTGCCCTGATATCAGAGTCC 65 1180 1199 148 n/a n/a 464270
GGCTGCCCTGATATCAGAGT 65 1182 1201 149 n/a n/a 464278
CTCAGATACTGCTGTCTCTG 71 1345 1364 150 n/a n/a 464280
TGCCCATCCCTCTGTGCCCC 72 1386 1405 151 n/a n/a 464284
TGCTCTCTTGCCCATCCCTC 82 1394 1413 152 n/a n/a 464285
CTCTTTGGTCACACCGTCTG 82 1461 1480 153 n/a n/a 464286
ATCTCTTTGGTCACACCGTC 90 1463 1482 154 n/a n/a 464287
CTATCTCTTTGGTCACACCG 82 1465 1484 155 n/a n/a 464288
GCCTATCTCTTTGGTCACAC 88 1467 1486 156 n/a n/a 464290
CGCTGCCTATCTCTTTGGTC 70 1471 1490 157 n/a n/a 464291
AGCTTGCAAGCCCTTAATGG 70 1542 1561 158 n/a n/a 464292
CCAGCTTGCAAGCCCTTAAT 69 1544 1563 159 n/a n/a 464298
ACCTTCATCTTCCAGCAGAG 80 1941 1960 160 n/a n/a 464299
CAACCTTCATCTTCCAGCAG 76 1943 1962 161 n/a n/a 464300
TTCAACCTTCATCTTCCAGC 81 1945 1964 162 n/a n/a 464308
CAGCTTTGCTCAGCCCAGCA 90 3325 3344 163 n/a n/a 464309
TCCAGCTTTGCTCAGCCCAG 87 3327 3346 164 n/a n/a 464310
TTTCCAGCTTTGCTCAGCCC 78 3329 3348 165 n/a n/a 464311
CCTTTCCAGCTTTGCTCAGC 78 3331 3350 166 n/a n/a 464333
CCAGGTCCACAGTCCAGGGC 75 4799 4818 167 n/a n/a 464342
ACTTGCCAGAGAGTAGCAGA 66 4836 4855 168 n/a n/a 464425
GCCATAGCACCTCCTCCAGG 75 7684 7703 169 n/a n/a 464428
CCCAATGCCATAGCACCTCC 73 7690 7709 170 n/a n/a 464429
GTCCCAATGCCATAGCACCT 70 7692 7711 171 n/a n/a 464430
TAGTCCCAATGCCATAGCAC 65 7694 7713 172 n/a n/a 464433
TTCTATTAGTCCCAATGCCA 69 7700 7719 173 n/a n/a 464449
GTCACTTGCCAGGGTCAGGA 81 7802 7821 174 n/a n/a 464453
GCTCAGAAGTCACTTGCCAG 68 7810 7829 175 n/a n/a 464568
GTCCATCTGGCTTCCCCTGC 68 2031 2050 176 n/a n/a 464569
CAGTCCATCTGGCTTCCCCT 68 2033 2052 177 n/a n/a 464575
CCACTCCACTTCCAGTCCAT 65 2045 2064 178 n/a n/a 464576
TGCCACTCCACTTCCAGTCC 68 2047 2066 179 n/a n/a 464579
GGTCACTGCCACTCCACTTC 78 2053 2072 180 n/a n/a 464581
CCTTGGTCACTGCCACTCCA 68 2057 2076 181 n/a n/a 464582
GGAAGCCTATCACACCTCCT 67 2080 2099 182 n/a n/a 464584
GTGTCTCTGGATCTACCCTG 71 2104 2123 183 n/a n/a 464585
TGGTGTCTCTGGATCTACCC 74 2106 2125 184 n/a n/a 464586
ACTGGTGTCTCTGGATCTAC 72 2108 2127 185 n/a n/a 464587
GCACTGGTGTCTCTGGATCT 83 2110 2129 186 n/a n/a 464588
TGGCACTGGTGTCTCTGGAT 88 2112 2131 187 n/a n/a 464589
GGTGGCACTGGTGTCTCTGG 88 2114 2133 188 n/a n/a 464590
TGGGTGGCACTGGTGTCTCT 74 2116 2135 189 n/a n/a 464591
TATGGGTGGCACTGGTGTCT 74 2118 2137 190 n/a n/a 464593
GGCCTATGGGTGGCACTGGT 68 2122 2141 191 n/a n/a 464617
GTCAGGCTGTGATGTACACA 69 2261 2280 192 n/a n/a 464622
TGCTGTTACTGTCAGGCTGT 73 2271 2290 193 n/a n/a 464623
GCCAGTCACCTCTGGTTCGG 68 2292 2311 194 n/a n/a 464657
AGCAGTTTTGGGATTCTTTT 72 2838 2857 195 n/a n/a 464658
AAAGCAGTTTTGGGATTCTT 67 2840 2859 196 n/a n/a 464677
TCCAAGTCCCTGGCCAGGCT 65 2993 3012 197 n/a n/a 464682
ATCCTTTCCAGCTTTGCTCA 78 3333 3352 198 n/a n/a 464683
GGATCCTTTCCAGCTTTGCT 90 3335 3354 199 n/a n/a 464684
AAGGATCCTTTCCAGCTTTG 72 3337 3356 200 n/a n/a 464685
GCAAGGATCCTTTCCAGCTT 88 3339 3358 201 n/a n/a 464686
GGGCAAGGATCCTTTCCAGC 82 3341 3360 202 n/a n/a 464687
CTGGGCAAGGATCCTTTCCA 71 3343 3362 203 n/a n/a 464688
GCCTGGGCAAGGATCCTTTC 69 3345 3364 204 n/a n/a 464689
GTGGTTGAGCCCTGCCCTGC 67 3380 3399 205 n/a n/a 464692
GTCTCAGTGGTTGAGCCCTG 71 3386 3405 206 n/a n/a 464696
CTGACTGAGTCTCAGTGGTT 82 3394 3413 207 n/a n/a 464698
GGCACTGACTGAGTCTCAGT 84 3398 3417 208 n/a n/a 464699
CAGGCACTGACTGAGTCTCA 79 3400 3419 209 n/a n/a 464701
AAGCCAGGCACTGACTGAGT 72 3404 3423 210 n/a n/a 464703
CTGGAAGCCAGGCACTGACT 70 3408 3427 211 n/a n/a 464705
GCTTGCTGGAAGCCAGGCAC 67 3413 3432 212 n/a n/a 464706
ATGCTTGCTGGAAGCCAGGC 80 3415 3434 213 n/a n/a 464707
GTCCTCTCTCGCAGACACAG 84 3445 3464 214 n/a n/a 464708
CAGTCCTCTCTCGCAGACAC 86 3447 3466 215 n/a n/a 464709
GCCAGTCCTCTCTCGCAGAC 86 3449 3468 216 n/a n/a 464710
AGGCCAGTCCTCTCTCGCAG 90 3451 3470 217 n/a n/a 464711
GAGCTCACCACCAGCTCTGC 70 3499 3518 218 n/a n/a 464716
GCTGCCTGGACCTCCTAGGT 90 3571 3590 219 n/a n/a 464717
ATGCTGCCTGGACCTCCTAG 85 3573 3592 220 n/a n/a 464718
ACATGCTGCCTGGACCTCCT 89 3575 3594 221 n/a n/a 464719
ACACATGCTGCCTGGACCTC 73 3577 3596 222 n/a n/a 464720
CCACACATGCTGCCTGGACC 88 3579 3598 223 n/a n/a 464726
GCAAATGCCACACTCTTGGG 67 3770 3789 224 n/a n/a 464727
GGGCAAATGCCACACTCTTG 78 3772 3791 225 n/a n/a 464728
CAGGGCAAATGCCACACTCT 71 3774 3793 226 n/a n/a 464729
CCCAGGGCAAATGCCACACT 87 3776 3795 227 n/a n/a 464730
CACCCAGGGCAAATGCCACA 78 3778 3797 228 n/a n/a 464732
GCCACACCCAGGGCAAATGC 87 3782 3801 229 n/a n/a 464734
GGATGCCACACCCAGGGCAA 66 3786 3805 230 n/a n/a 464735
GCGGATGCCACACCCAGGGC 87 3788 3807 231 n/a n/a 464736
CTGCGGATGCCACACCCAGG 67 3790 3809 232 n/a n/a 464740
GCCACATGCTGCGGATGCCA 88 3798 3817 233 n/a n/a 464800
GGACTTCCCACCAACTGCCT 71 3122 3141 234 n/a n/a 464801
GCTGGACTTCCCACCAACTG 77 3125 3144 235
TABLE-US-00004 TABLE 4 Inhibition of human FGFR4 mRNA levels by
chimeric antisense oligonucleotides targeted to SEQ ID NO: 3 Target
Start ISIS % SEQ ID Site Sequence No inhibition NO 1502
CAAGGAGCTCACCACCAGCT 464713 83 236 1504 GGCAAGGAGCTCACCACCAG 464714
76 237 1506 CAGGCAAGGAGCTCACCACC 464715 69 238
Example 4
Dose-Dependent Antisense Inhibition of Human FGFR4 in HepG2
Cells
[0677] Gapmers from Example 3 which caused significant inhibition
of FGFR4 mRNA were further tested at various doses in HepG2 cells.
Cells were plated at a density of 20,000 cells per well and
transfected using electroporation with 0.6 .mu.M, 1.3 .mu.M, 2.5
.mu.M, 5.0 .mu.M, and 10.0 .mu.M concentrations of antisense
oligonucleotide, as specified in Table 5. After a treatment period
of approximately 16 hours, RNA was isolated from the cells and
FGFR4 mRNA levels were measured by quantitative real-time PCR.
Human FGFR4 primer probe set RTS3232 was used to measure mRNA
levels. FGFR4 mRNA levels were adjusted according to total RNA
content, as measured by RIBOGREEN.RTM.. Results are presented as
percent inhibition of FGFR4, relative to untreated control
cells.
[0678] The half maximal inhibitory concentration (IC.sub.50) of
each oligonucleotide is also presented in Table 5. As illustrated
in Table 5, FGFR4 mRNA levels were significantly reduced in a
dose-dependent manner in antisense oligonucleotide treated
cells.
TABLE-US-00005 TABLE 5 Dose-dependent antisense inhibition of human
FGFR4 in HepG2 cells using electroporation 0.6 1.3 2.5 5.0 10.0
IC.sub.50 ISIS No .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 299004 20
44 73 87 96 1.5 463629 23 54 80 87 96 1.0 464138 0 32 57 84 91 2.3
464208 28 37 58 76 87 1.8 464209 22 30 64 79 80 2.0 464213 21 40 54
79 90 1.9 464214 14 31 55 84 93 2.1 464215 35 38 67 85 94 1.4
464222 29 53 73 89 93 1.2 464223 16 0 63 76 88 3.2 464225 36 43 74
85 88 1.2 464227 29 56 64 86 90 1.3 464228 52 76 82 91 92 0.3
464284 21 44 67 83 91 1.6 464285 27 36 57 81 93 1.9 464286 35 47 70
89 95 1.2 464287 26 50 68 85 90 1.4 464288 19 49 55 83 90 1.7
464300 25 34 47 75 93 2.1 464308 35 57 77 94 97 1.0 464309 4 25 65
89 95 2.1 464425 2 31 52 71 81 2.7 464449 32 59 78 88 95 1.0 464587
25 52 75 88 91 1.3 464588 26 74 84 93 93 1.0 464589 29 62 83 90 93
1.0 464683 10 35 50 71 90 2.4 464685 14 42 42 62 88 2.6 464686 12
44 66 81 95 1.8 464696 22 43 68 85 94 1.6 464698 12 10 20 44 71 5.9
464706 16 52 46 84 92 1.8 464707 26 40 69 84 93 1.5 464708 18 46 57
84 94 1.7 464709 6 14 32 58 84 3.7 464710 12 30 44 65 86 2.7 464713
9 28 47 78 92 2.4 464716 21 45 64 86 93 1.6 464717 13 37 57 86 94
2.0 464718 22 56 80 93 97 1.2 464720 15 33 49 77 92 2.2 464729 15
20 35 69 84 3.0 464732 19 55 73 85 93 1.4 464735 27 45 62 89 94 1.5
464740 10 44 65 82 89 1.9 464801 17 53 56 81 92 1.7
Example 5
Dose-Dependent Antisense Inhibition of Human FGFR4 in HepG2
Cells
[0679] Gapmers from the studies described above which caused
significant inhibition of FGFR4 mRNA were further tested at various
doses in HepG2 cells. Cells were plated at a density of 20,000
cells per well and transfected using electroporation with 0.3
.mu.M, 0.6 .mu.M, 1.3 .mu.M, 2.5 .mu.M, 5.0 .mu.M, and 10.0 .mu.M
concentrations of antisense oligonucleotide, as specified in Table
6. After a treatment period of approximately 16 hours, RNA was
isolated from the cells and FGFR4 mRNA levels were measured by
quantitative real-time PCR. Human FGFR4 primer probe set RTS3232
was used to measure mRNA levels. FGFR4 mRNA levels were adjusted
according to total RNA content, as measured by RIBOGREEN.RTM..
Results are presented as percent inhibition of FGFR4, relative to
untreated control cells.
[0680] The half maximal inhibitory concentration (IC.sub.50) of
each oligonucleotide is also presented in Table 6. As illustrated
in Table 6, FGFR4 mRNA levels were significantly reduced in a
dose-dependent manner in antisense oligonucleotide treated
cells.
TABLE-US-00006 TABLE 6 Dose-dependent antisense inhibition of human
FGFR4 in HepG2 cells using electroporation 0.3 0.6 1.3 2.5 5.0 10.0
IC.sub.50 ISIS No .mu.M .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M)
299004 0 20 14 49 70 89 2.5 299005 7 25 52 76 92 96 1.3 463588 26
22 43 84 94 98 1.3 463589 13 24 52 74 92 95 1.3 463628 27 45 57 76
94 95 0.8 463629 24 36 67 85 93 96 0.9 463648 14 21 38 54 75 90 1.9
463672 8 28 41 57 86 95 1.6 463690 22 17 59 74 91 97 1.3 463691 10
24 45 60 86 87 1.6 463692 0 10 33 56 76 92 2.2 463709 12 22 36 66
85 95 1.6 463762 0 22 16 29 0 84 >10.0 463771 0 29 38 49 66 89
2.2 463834 14 24 43 52 79 94 1.7 463835 18 35 40 58 82 94 1.4
463837 8 22 53 73 89 97 1.4 463838 12 23 44 56 77 91 1.7 463861 25
41 41 61 76 90 1.3 463907 0 25 51 68 84 95 1.6 463909 19 39 54 82
93 97 1.0 464038 8 22 36 44 72 89 2.2
Example 6
Dose-Dependent Antisense Inhibition of Human FGFR4 in HepG2
Cells
[0681] Gapmers from the study described in Example 4 exhibiting
significant in vitro inhibition of FGFR4 mRNA were selected and
tested at various doses in HepG2 cells. Cells were plated at a
density of 20,000 cells per well and transfected using
electroporation with 0.6 .mu.M, 1.3 .mu.M, 2.5 .mu.M, 5.0 .mu.M and
10.0 .mu.M concentrations of antisense oligonucleotide, as
specified in Table 7. After a treatment period of approximately 16
hours, RNA was isolated from the cells and FGFR4 mRNA levels were
measured by quantitative real-time PCR. Human FGFR4 primer probe
set RTS3232 was used to measure mRNA levels. FGFR4 mRNA levels were
adjusted according to total RNA content, as measured by
RIBOGREEN.RTM.. Results are presented as percent inhibition of
FGFR4, relative to untreated control cells.
[0682] The half maximal inhibitory concentration (IC.sub.50) of
each oligonucleotide is also presented in Table 7. As illustrated
in Table 7, FGFR4 mRNA levels were significantly reduced in a
dose-dependent manner in antisense oligonucleotide treated
cells.
TABLE-US-00007 TABLE 7 Dose-dependent antisense inhibition of human
FGFR4 in HepG2 cells using electroporation 0.6 1.3 2.5 5.0 10.0
IC.sub.50 ISIS No .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 463629 44
73 86 96 98 <0.6 464222 40 65 81 88 96 0.7 464225 47 76 84 92 86
<0.6 464228 56 80 86 92 95 <0.6 464284 24 47 62 78 90 1.6
464286 23 60 73 86 93 1.2 464287 19 62 69 89 91 1.3 464308 38 54 78
90 96 1.0 464449 27 69 80 91 94 0.9 464587 24 68 74 88 91 1.1
464588 42 75 81 88 92 <0.6 464589 36 69 78 90 92 0.8 464716 52
60 75 90 95 0.6 464718 38 61 76 91 95 0.9 464732 30 39 65 85 94
1.5
Example 7
Antisense Inhibition of Human Fibroblast Growth Factor Receptor
(FGFR4) in HepG2 Cells
[0683] Additional antisense oligonucleotides were designed
targeting a FGFR4 nucleic acid and were tested for their effects on
FGFR4 mRNA in vitro. ISIS 463629 and ISIS 463762 were also included
in the assay for comparison. Cultured HepG2 cells at a density of
20,000 cells per well were transfected using electroporation with
4,500 nM antisense oligonucleotide. After a treatment period of
approximately 24 hours, RNA was isolated from the cells and FGFR4
mRNA levels were measured by quantitative real-time PCR. Human
primer probe set RTS3232 was used to measure mRNA levels. FGFR4
mRNA levels were adjusted according to total RNA content, as
measured by RIBOGREEN.RTM.. Results are presented as percent
inhibition of FGFR4, relative to untreated control cells. A total
of 230 oligonucleotides were tested. Only those oligonucleotides
demonstrating greater than 65% inhibition are shown in Table 8.
[0684] The newly designed chimeric antisense oligonucleotides in
Table 8 were designed as 5-10-5 MOE gapmers or 3-10-4 MOE gapmers.
The 5-10-5 gapmers are 20 nucleosides in length, wherein the
central gap segment comprises of ten 2'-deoxynucleosides and is
flanked by wing segments on the 5' and 3' directions comprising
five nucleosides each. The 3-10-4 gapmers are 17 nucleosides in
length, wherein the central gap segment comprises of ten
2'-deoxynucleosides and is flanked by a wing segment in the 5'
direction comprising three nucleosides and a wing segment in the 3'
direction comprising four nucleosides. Each nucleotide in the 5'
wing segment and each nucleotide in the 3' wing segment has a
2'-MOE modification. The internucleoside linkages throughout each
gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine
residues throughout each gapmer are 5-methylcytosines. "Start site"
indicates the 5'-most nucleoside to which the gapmer is targeted in
the human gene sequence. "Stop site" indicates the 3'-most
nucleoside to which the gapmer is targeted in the human gene
sequence.
[0685] Each gapmer listed in Table 8 is targeted to either the
human FGFR4 mRNA, designated herein as SEQ ID NO: 1 (GENBANK
Accession No. NM.sub.--002011.3) or the human FGFR4 genomic
sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No:
NT.sub.--023133.11 truncated from nucleosides 21323018 to
21335213), or SEQ ID NO: 3 (GENBANK Accession No. AB209631.1), or
all three.
TABLE-US-00008 TABLE 8 Inhibition of human FGFR4 mRNA levels by
chimeric antisense oligo- nucleotides targeted to SEQ ID NO: 1, SEQ
ID NO: 2 and SEQ ID NO: 3 Start Start Start Site on Site on Site on
SEQ SEQ SEQ SEQ ID NO: ID NO: ID NO: ISIS % ID 1 2 3 Motif Sequence
No inhibition NO 292 3993 1996 5-10-5 GGCTACTGTCAGCTCCTGCT 463629
93 29 118 3123 1122 5-10-5 TGGACTTCCCACCAACTGCC 479530 77 239 n/a
2109 101 5-10-5 CACTGGTGTCTCTGGATCTA 479532 78 240 n/a 2111 103
5-10-5 GGCACTGGTGTCTCTGGATC 479533 89 241 n/a 2113 105 5-10-5
GTGGCACTGGTGTCTCTGGA 479534 92 242 n/a 2115 107 5-10-5
GGGTGGCACTGGTGTCTCTG 479535 88 243 n/a 3334 1333 5-10-5
GATCCTTTCCAGCTTTGCTC 479536 91 244 n/a 3336 1335 5-10-5
AGGATCCTTTCCAGCTTTGC 479537 84 245 n/a 3338 1337 5-10-5
CAAGGATCCTTTCCAGCTTT 479538 65 246 n/a 3340 1339 5-10-5
GGCAAGGATCCTTTCCAGCT 479539 88 247 n/a 3342 1341 5-10-5
TGGGCAAGGATCCTTTCCAG 479540 68 248 n/a 3393 1392 5-10-5
TGACTGAGTCTCAGTGGTTG 479541 71 249 n/a 3395 1394 5-10-5
ACTGACTGAGTCTCAGTGGT 479542 80 250 n/a 3397 1396 5-10-5
GCACTGACTGAGTCTCAGTG 479543 76 251 n/a 3399 1398 5-10-5
AGGCACTGACTGAGTCTCAG 479544 77 252 n/a 3414 1413 5-10-5
TGCTTGCTGGAAGCCAGGCA 479545 83 253 n/a 3446 1445 5-10-5
AGTCCTCTCTCGCAGACACA 479546 88 254 n/a 3448 1447 5-10-5
CCAGTCCTCTCTCGCAGACA 479547 80 255 n/a 3450 1449 5-10-5
GGCCAGTCCTCTCTCGCAGA 479548 92 256 n/a 3502 1501 5-10-5
AAGGAGCTCACCACCAGCTC 479549 76 257 n/a n/a 1503 5-10-5
GCAAGGAGCTCACCACCAGC 479550 79 258 n/a 3570 1569 5-10-5
CTGCCTGGACCTCCTAGGTC 479551 95 259 n/a 3572 1571 5-10-5
TGCTGCCTGGACCTCCTAGG 479552 85 260 n/a 3574 1573 5-10-5
CATGCTGCCTGGACCTCCTA 479553 80 261 n/a 3576 1575 5-10-5
CACATGCTGCCTGGACCTCC 479554 80 262 n/a 3578 1577 5-10-5
CACACATGCTGCCTGGACCT 479555 71 263 n/a 3580 1579 5-10-5
ACCACACATGCTGCCTGGAC 479556 87 264 n/a 3775 1778 5-10-5
CCAGGGCAAATGCCACACTC 479557 71 265 n/a 3777 1780 5-10-5
ACCCAGGGCAAATGCCACAC 479558 83 266 n/a 3783 1786 5-10-5
TGCCACACCCAGGGCAAATG 479560 67 267 n/a 3787 1790 5-10-5
CGGATGCCACACCCAGGGCA 479561 70 268 n/a 3789 1792 5-10-5
TGCGGATGCCACACCCAGGG 479562 78 269 n/a 3799 1802 5-10-5
AGCCACATGCTGCGGATGCC 479564 71 270 n/a 1393 n/a 5-10-5
GCTCTCTTGCCCATCCCTCT 479565 81 271 n/a 1462 n/a 5-10-5
TCTCTTTGGTCACACCGTCT 479566 90 272 n/a 1464 n/a 5-10-5
TATCTCTTTGGTCACACCGT 479567 67 273 n/a 1466 n/a 5-10-5
CCTATCTCTTTGGTCACACC 479568 83 274 n/a 1468 n/a 5-10-5
TGCCTATCTCTTTGGTCACA 479569 76 275 n/a 1944 n/a 5-10-5
TCAACCTTCATCTTCCAGCA 479570 80 276 n/a 3324 1323 5-10-5
AGCTTTGCTCAGCCCAGCAG 479572 75 277 n/a 3326 1325 5-10-5
CCAGCTTTGCTCAGCCCAGC 479573 85 278 n/a 3328 1327 5-10-5
TTCCAGCTTTGCTCAGCCCA 479574 79 279 n/a 7801 n/a 5-10-5
TCACTTGCCAGGGTCAGGAG 479576 70 280 n/a 7803 n/a 5-10-5
AGTCACTTGCCAGGGTCAGG 479577 65 281 n/a 1462 n/a 3-10-4
CTTTGGTCACACCGTCT 479582 74 282 n/a 1463 n/a 3-10-4
TCTTTGGTCACACCGTC 479583 84 283 n/a 1464 n/a 3-10-4
CTCTTTGGTCACACCGT 479584 82 284 n/a 1465 n/a 3-10-4
TCTCTTTGGTCACACCG 479585 71 285 n/a 3326 1325 3-10-4
GCTTTGCTCAGCCCAGC 479594 80 286 n/a 3328 1327 3-10-4
CAGCTTTGCTCAGCCCA 479596 81 287 n/a 3329 1328 3-10-4
CCAGCTTTGCTCAGCCC 479597 78 288 161 3166 1165 3-10-4
GCAGCCGCATCTCCTTC 479608 70 289 194 3199 1198 3-10-4
GCACACTCAGCAGGACC 479613 72 290 195 3200 1199 3-10-4
GGCACACTCAGCAGGAC 479614 78 291 349 4050 2053 3-10-4
CCAGTGGCCACCACGCT 479622 67 292 369 4070 2073 3-10-4
AGGCGACTGCCCTCCTT 479625 68 293 370 4071 2074 3-10-4
CAGGCGACTGCCCTCCT 479626 71 294 602 4506 2418 3-10-4
GTGTCCAGTAGGGTGCT 479641 70 295 2819 11489 4988 3-10-4
CAGCTCTCCAGCCAGGC 479682 71 296 2951 11621 5120 3-10-4
GCTTCTCTGGGCTCAGG 479689 72 297 2952 11622 5121 3-10-4
AGCTTCTCTGGGCTCAG 479690 78 298 2953 11623 5122 3-10-4
CAGCTTCTCTGGGCTCA 479691 87 299 2954 11624 5123 3-10-4
CCAGCTTCTCTGGGCTC 479692 87 300 2955 11625 5124 3-10-4
TCCAGCTTCTCTGGGCT 479693 71 301 2956 11626 5125 3-10-4
TTCCAGCTTCTCTGGGC 479694 67 302 2958 11628 5127 3-10-4
GCTTCCAGCTTCTCTGG 479696 65 303 2981 11651 5150 3-10-4
CCATTTGCTCCTGTTTT 479697 73 304 2982 11652 5151 3-10-4
GCCATTTGCTCCTGTTT 479698 88 305 2983 11653 5152 3-10-4
CGCCATTTGCTCCTGTT 479699 92 306 n/a 2113 105 3-10-4
GCACTGGTGTCTCTGGA 479703 88 307 n/a 2114 106 3-10-4
GGCACTGGTGTCTCTGG 479704 95 308 n/a 2115 107 3-10-4
TGGCACTGGTGTCTCTG 479705 78 309 n/a 2116 108 3-10-4
GTGGCACTGGTGTCTCT 479706 90 310 n/a 3395 1394 3-10-4
GACTGAGTCTCAGTGGT 479716 71 311 n/a 3415 1414 3-10-4
CTTGCTGGAAGCCAGGC 479721 82 312 n/a 3416 1415 3-10-4
GCTTGCTGGAAGCCAGG 479722 82 313 n/a 3446 1445 3-10-4
CCTCTCTCGCAGACACA 479725 70 314 n/a 3452 1451 3-10-4
GCCAGTCCTCTCTCGCA 479731 78 315 n/a 3453 1452 3-10-4
GGCCAGTCCTCTCTCGC 479732 69 316 n/a 3571 1570 3-10-4
GCCTGGACCTCCTAGGT 479736 97 317 n/a 3572 1571 3-10-4
TGCCTGGACCTCCTAGG 479737 69 318 n/a 3573 1572 3-10-4
CTGCCTGGACCTCCTAG 479738 76 319 n/a 3574 1573 3-10-4
GCTGCCTGGACCTCCTA 479739 88 320 n/a 3575 1574 3-10-4
TGCTGCCTGGACCTCCT 479740 66 321 n/a 3576 1575 3-10-4
ATGCTGCCTGGACCTCC 479741 72 322
Example 8
Dose-Dependent Antisense Inhibition of Human FGFR4 in HepG2
Cells
[0686] Gapmers from Examples 5, 6 and 7 exhibiting significant in
vitro inhibition of FGFR4 mRNA were further selected and tested at
various doses in HepG2 cells. Cells were plated at a density of
20,000 cells per well and transfected using electroporation with
0.6 .mu.M, 1.3 .mu.M, 2.5 .mu.M, 5.0 .mu.M, and 10.0 .mu.M
concentrations of antisense oligonucleotide, as specified in Table
9. After a treatment period of approximately 16 hours, RNA was
isolated from the cells and FGFR4 mRNA levels were measured by
quantitative real-time PCR. Human FGFR4 primer probe set RTS3232
was used to measure mRNA levels. FGFR4 mRNA levels were adjusted
according to total RNA content, as measured by RIBOGREEN.RTM..
Results are presented as percent inhibition of FGFR4, relative to
untreated control cells.
[0687] The half maximal inhibitory concentration (IC.sub.50) of
each oligonucleotide is also presented in Table 9. As illustrated
in Table 9, FGFR4 mRNA levels were significantly reduced in a
dose-dependent manner in antisense oligonucleotide treated
cells.
TABLE-US-00009 TABLE 9 Dose-dependent antisense inhibition of human
FGFR4 in HepG2 cells using electroporation 0.6 1.3 2.5 5.0 10.0
IC.sub.50 ISIS No .mu.M .mu.M .mu.M .mu.M .mu.M (.mu.M) 299005 31
50 66 89 95 1.3 463588 25 48 70 91 97 1.4 463589 33 46 69 87 96 1.3
463628 49 67 77 90 97 <0.6 463629 36 58 70 88 92 1.6 463648 34
41 49 64 84 1.9 463672 16 34 68 84 94 1.8 463690 42 58 75 88 97 0.8
463691 27 38 73 83 96 1.5 463692 3 39 57 76 94 2.2 463709 22 43 64
82 95 1.6 463762 13 29 46 74 90 2.5 463771 40 31 51 78 91 1.7
463834 23 44 55 78 93 1.8 463835 30 39 65 83 95 1.5 463837 29 43 72
87 95 1.4 463838 23 40 59 77 93 1.8 463861 9 33 61 82 97 2.1 464038
19 25 42 61 88 2.8 464222 30 56 75 87 95 1.1 464225 40 60 79 85 90
0.8 464228 50 72 86 91 94 <0.6 464284 30 52 59 84 90 1.4 464286
50 65 83 92 95 <0.6 464287 24 50 67 89 92 1.4 464308 36 56 76 90
97 1.0 464449 44 73 85 93 95 <0.6 464587 33 54 79 92 98 1.0
464588 53 76 89 95 95 <0.6 464589 30 66 80 93 95 0.9 464716 33
41 69 86 95 1.4 464718 33 56 77 93 98 1.0 464732 27 43 61 86 95 1.6
479533 68 84 89 93 95 <0.6 479534 67 74 92 95 97 <0.6 479535
54 72 81 91 95 <0.6 479536 38 68 86 96 98 0.7 479539 39 52 77 92
98 1.0 479546 32 70 78 91 98 0.9 479548 49 71 81 93 96 <0.6
479551 72 82 91 95 97 <0.6 479556 36 63 83 90 97 0.9
Example 9
Dose-Dependent Antisense Inhibition of Human FGFR4 in HepG2
Cells
[0688] Gapmers from Examples 7 and 8 exhibiting significant in
vitro inhibition of FGFR4 mRNA were further selected and tested at
various doses in HepG2 cells. Cells were plated at a density of
20,000 cells per well and transfected using electroporation with
0156 .mu.M, 0.31 .mu.M, 0.63 .mu.M, 1.25 .mu.M, 2.50 .mu.M and 5.00
.mu.M concentrations of antisense oligonucleotide, as specified in
Table 10. After a treatment period of approximately 16 hours, RNA
was isolated from the cells and FGFR4 mRNA levels were measured by
quantitative real-time PCR. Human FGFR4 primer probe set RTS3232
was used to measure mRNA levels. FGFR4 mRNA levels were adjusted
according to total RNA content, as measured by RIBOGREEN.RTM..
Results are presented as percent inhibition of FGFR4, relative to
untreated control cells.
[0689] The half maximal inhibitory concentration (IC.sub.50) of
each oligonucleotide is also presented in Table 10. As illustrated
in Table 10, FGFR4 mRNA levels were significantly reduced in a
dose-dependent manner in antisense oligonucleotide treated
cells.
TABLE-US-00010 TABLE 10 Dose-dependent antisense inhibition of
human FGFR4 in HepG2 cells using electroporation 0.156 0.31 0.63
1.25 2.50 5.00 IC.sub.50 ISIS No .mu.M .mu.M .mu.M .mu.M .mu.M
.mu.M (.mu.M) 463629 19 30 48 66 84 91 0.7 479533 18 33 63 67 84 86
0.6 479534 18 25 34 63 82 86 0.9 479535 24 28 43 62 68 81 0.9
479536 25 28 29 62 78 90 0.8 479539 8 16 36 48 75 88 1.1 479546 0
27 33 63 77 87 1.1 479548 8 39 30 62 74 85 0.9 479551 27 44 59 80
86 89 0.4 479556 16 29 32 53 71 87 1.0 479566 19 27 29 63 81 88 0.9
479584 3 22 30 58 80 88 1.0 479596 4 20 32 54 71 88 1.1 479691 18
11 50 62 80 91 0.8 479692 12 26 49 61 79 90 0.8 479698 23 40 57 73
87 92 0.5 479699 17 37 60 76 90 93 0.5 479703 18 20 41 67 82 89 0.8
479704 31 43 66 80 90 92 0.4 479706 26 18 36 58 76 90 0.9 479736 36
48 71 86 93 94 0.3
Example 10
Tolerability of Antisense Oligonucleotides Targeting Human FGFR4 in
CD1 Mice
[0690] CD1.RTM. mice (Charles River, MA) are a multipurpose mice
model, frequently utilized for safety and efficacy testing. The
mice were treated with ISIS antisense oligonucleotides selected
from studies described above and evaluated for changes in the
levels of various markers.
Treatment
[0691] Groups of five male CD1 mice were injected subcutaneously
twice a week for 6 weeks with 50 mg/kg of ISIS 299005, ISIS 463588,
ISIS 463589, ISIS 463628, ISIS 463690, ISIS 463691, ISIS 463835,
ISIS 463837, ISIS 464222, ISIS 464225, ISIS 464228, ISIS 464286,
ISIS 464308, ISIS 464449, ISIS 464587, ISIS 464588, ISIS 464589,
ISIS 464718, ISIS 479533, ISIS 479551, ISIS 479691, ISIS 479692,
ISIS 479698, ISIS 479699, ISIS 479703, ISIS 479704, ISIS 479706, or
ISIS 479736. One group of male CD1 mice was injected subcutaneously
twice a week for 6 weeks with PBS. Mice were euthanized 48 hours
after the last dose, and plasma were harvested for further
analysis. Treatment with ISIS 479691 caused death of the mice and
that ISIS oligonucleotide was therefore removed from further
study.
Plasma Chemistry Markers
[0692] To evaluate the effect of ISIS oligonucleotides on metabolic
function, plasma concentrations of transaminases, bilirubin,
albumin, creatinine, and BUN were measured using an automated
clinical chemistry analyzer (Hitachi Olympus AU400e, Melville,
N.Y.). The transaminase levels are expressed as IU/L; the
bilirubin, creatinine, and BUN levels are expressed as mg/dL; and
the albumin is expressed in g/dL. The results are presented in
Table 11. ISIS oligonucleotides that caused adverse changes in the
levels of any of the plasma chemistry markers were excluded in
further studies.
TABLE-US-00011 TABLE 11 ALT, AST, Bilirubin, BUN, Creatinine and
Albumin levels in CD1 mouse plasma at week 6 ALT AST (IU/ (IU/
Bilirubin BUN Creatinine Albumin L) L) (mg/dL) (mg/dL) (mg/dL)
(g/dL) PBS 34 59 0.2 33 0.16 3.3 ISIS 299005 50 72 0.1 27 0.12 2.8
ISIS 463588 55 72 0.1 30 0.12 3.0 ISIS 463589 64 79 0.2 28 0.10 2.7
ISIS 463628 48 83 0.1 27 0.13 3.0 ISIS 463690 71 93 0.2 29 0.13 3.0
ISIS 463691 145 134 0.2 26 0.10 3.0 ISIS 463835 159 113 0.2 26 0.11
3.0 ISIS 463837 59 78 0.1 27 0.09 2.8 ISIS 464222 559 564 0.2 23
0.09 2.7 ISIS 464225 83 88 0.1 25 0.09 2.8 ISIS 464228 58 93 0.1 29
0.10 2.8 ISIS 464286 139 154 0.1 21 0.05 2.8 ISIS 464308 2533 1673
0.2 28 0.11 3.3 ISIS 464449 748 451 0.2 24 0.08 3.0 ISIS 464587 183
159 0.1 25 0.11 3.0 ISIS 464588 256 726 0.2 21 0.03 2.0 ISIS 464589
142 126 0.2 27 0.09 2.9 ISIS 464718 789 608 0.2 19 0.03 2.7 ISIS
479533 61 76 0.1 22 0.09 2.9 ISIS 479551 81 104 0.2 26 0.13 3.0
ISIS 479692 847 1026 0.3 26 0.10 3.1 ISIS 479698 92 133 0.2 29 0.12
2.7 ISIS 479699 57 95 0.1 20 0.09 2.6 ISIS 479703 158 108 0.1 23
0.11 3.0 ISIS 479704 38 56 0.2 23 0.10 3.2 ISIS 479706 700 642 0.5
26 0.12 3.1 ISIS 479736 204 134 0.1 25 0.11 2.9
Example 11
Tolerability of Antisense Oligonucleotides Targeting Human FGFR4 in
Sprague-Dawley Rats
[0693] Sprague-Dawley rats are a multipurpose model used for safety
and efficacy evaluations. The rats were treated with ISIS antisense
oligonucleotides from the study described in Example 10 and
evaluated for changes in the levels of various plasma chemistry
markers.
Treatment
[0694] Seven week old male Sprague-Dawley rats were maintained on a
12-hour light/dark cycle and fed ad libitum with Purina normal rat
chow, diet 5001. Groups of four Sprague-Dawley rats each were
injected subcutaneously twice a week for 4 weeks with 50 mg/kg of
ISIS 299005, ISIS 463588, ISIS 463589, ISIS 463628, ISIS 463690,
ISIS 463691, ISIS 463835, ISIS 463837, ISIS 464222, ISIS 464225,
ISIS 464228, ISIS 464286, ISIS 464308, ISIS 464449, ISIS 464587,
ISIS 464718, ISIS 479533, ISIS 479551, ISIS 479691, ISIS 479692,
ISIS 479698, ISIS 479699, ISIS 479703, ISIS 479704, ISIS 479706, or
ISIS 479736. A group of rats were injected subcutaneously twice a
week for 4 weeks with PBS. Forty eight hours after the last dose,
rats were euthanized and plasmas were harvested for further
analysis.
Liver Function
[0695] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma concentrations of transaminases were measured
using an automated clinical chemistry analyzer (Hitachi Olympus
AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine
transaminase) and AST (aspartate transaminase) were measured and
the results are presented in Table 12, expressed in IU/L. Plasma
levels of bilirubin were also measured using the same clinical
chemistry analyzer and the results are also presented in Table 12,
expressed as mg/dL. ISIS oligonucleotides that caused adverse
changes were excluded in further studies.
TABLE-US-00012 TABLE 12 Effect of antisense oligonucleotide
treatment on ALT, AST, and Bilirubin in the liver of Sprague-Dawley
rats ALT AST Bilirubin (IU/L) (IU/L) (g/dL) PBS 52 206 0.15 ISIS
299005 72 387 0.16 ISIS 463588 56 305 0.13 ISIS 463589 82 553 0.15
ISIS 463628 351 351 0.13 ISIS 463690 81 367 0.14 ISIS 463691 83 368
0.13 ISIS 463835 90 345 0.13 ISIS 463837 67 301 0.11 ISIS 464222
231 322 0.19 ISIS 464225 66 241 0.11 ISIS 464228 77 359 0.57 ISIS
464286 96 207 0.11 ISIS 464308 59 295 0.12 ISIS 464449 158 509 0.15
ISIS 464587 414 373 0.29 ISIS 464588 215 278 0.40 ISIS 464589 282
482 0.32 ISIS 464718 280 577 0.43 ISIS 479533 391 457 0.29 ISIS
479551 1360 1300 0.41 ISIS 479691 383 439 0.35 ISIS 479692 674 675
0.24 ISIS 479698 354 775 0.86 ISIS 479699 145 455 0.90 ISIS 479703
779 781 0.54 ISIS 479704 790 1243 0.41 ISIS 479706 570 680 0.36
ISIS 479736 499 644 0.24
Kidney Function
[0696] To evaluate the effect of ISIS oligonucleotides on kidney
function, plasma concentrations of blood urea nitrogen (BUN) and
creatinine were measured using an automated clinical chemistry
analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are
presented in Table 13, expressed in mg/dL.
TABLE-US-00013 TABLE 13 Effect of antisense oligonucleotide
treatment on renal function markers (mg/dL) of Sprague-Dawley rats
BUN Creatinine Saline 18 0.29 ISIS 299005 20 0.33 ISIS 463588 23
0.35 ISIS 463589 19 0.32 ISIS 463628 19 0.33 ISIS 463690 19 0.35
ISIS 463691 18 0.34 ISIS 463835 19 0.34 ISIS 463837 18 0.32 ISIS
464222 21 0.37 ISIS 464225 20 0.29 ISIS 464228 22 0.32 ISIS 464286
22 0.36 ISIS 464308 18 0.32 ISIS 464449 16 0.32 ISIS 464587 23 0.38
ISIS 464588 23 0.27 ISIS 464589 24 0.35 ISIS 464718 22 0.32 ISIS
479533 28 0.31 ISIS 479551 21 0.36 ISIS 479691 29 0.36 ISIS 479692
25 0.40 ISIS 479698 30 0.34 ISIS 479699 30 0.35 ISIS 479703 28 0.31
ISIS 479704 31 0.42 ISIS 479706 26 0.38 ISIS 479736 22 0.37
Example 12
Tolerability of Antisense Oligonucleotides Targeting Human FGFR4 in
CD/IGS Rats
[0697] CD/IGS rats are a multipurpose model used for safety and
efficacy evaluations. The rats were treated with ISIS antisense
oligonucleotides selected from the study described in Examples 10
and 11 and evaluated for changes in the levels of various
markers.
Treatment
[0698] Ten-twelve week old male CD/IGS rats were maintained on a
12-hour light/dark cycle and fed ad libitum with Purina normal rat
chow, diet 5001. Groups of four CD/IGS rats each were injected
subcutaneously twice a week for 12 weeks with 30 mg/kg of ISIS
299005, ISIS 463588, ISIS 463589, ISIS 463690, ISIS 463691, ISIS
463835, ISIS 463837, or ISIS 464225. A group of 6 rats was injected
subcutaneously twice a week for 12 weeks with PBS and served as a
control group. Urine and blood samples were collected at various
time points. Forty eight hours after the last dose, body weights
were taken, rats were euthanized and organs and plasma were
harvested for further analysis.
Liver Function
[0699] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma concentrations of various liver function markers
were measured on week 8 and week 12 using an automated clinical
chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma
concentrations of ALT (alanine transaminase) and AST (aspartate
transaminase) were measured and the results are presented in Tables
14 and 15, expressed in IU/L. Plasma levels of bilirubin and BUN
were also measured using the same clinical chemistry analyzer and
the results are also presented in Tables 14 and 15, expressed as
mg/dL. ISIS oligonucleotides that caused adverse changes in the
levels of any of the markers of liver function were excluded in
further studies.
TABLE-US-00014 TABLE 14 ALT, AST, Bilirubin and BUN of CD/IGS rats
on week 8 ALT AST Bilirubin BUN (IU/L) (IU/L) (mg/dL) (mg/dL) PBS
31 71 0.16 13.6 ISIS 299005 60 121 0.15 17.4 ISIS 463588 57 103
0.19 18.6 ISIS 463589 46 136 0.14 16.8 ISIS 463690 79 91 0.24 18.1
ISIS 463691 80 93 0.18 18.8 ISIS 463835 103 118 0.18 16.6 ISIS
463837 52 101 0.14 20.7 ISIS 464225 48 253 0.14 18.9
TABLE-US-00015 TABLE 15 ALT, AST, TBIL, and BUN levels in the liver
of CD/IGS rats on week 12 ALT AST TBIL BUN (IU/L) (IU/L) (mg/dL)
(mg/dL) PBS 38 60 0.10 18.2 ISIS 299005 79 150 0.10 20.0 ISIS
463588 66 146 0.13 23.3 ISIS 463589 47 106 0.10 18.3 ISIS 463690 66
65 0.10 20.3 ISIS 463691 72 68 0.13 20.3 ISIS 463835 63 76 0.10
18.8 ISIS 463837 52 98 0.10 21.8 ISIS 464225 48 260 0.10 19.0
Example 13
Pharmacokinetic Measurement of Antisense Oligonucleotide in CD1
Mouse Liver
[0700] CD1 mice were treated with ISIS 463588, ISIS 463589, and
ISIS 463690, and the oligonucleotide half-life as well as the
elapsed time for oligonucleotide degradation and elimination from
the liver was evaluated.
Treatment
[0701] Group of ten CD1 mice each were injected subcutaneously
twice per week for 2 weeks (4 doses) with 50 mg/kg of ISIS 463588,
ISIS 463589, or ISIS 463690. Groups of five mice each from each
group were sacrificed 3 days and 56 days following the final dose.
Livers were harvested for analysis.
Measurement of Oligonucleotide Concentration
[0702] The concentration of the full-length oligonucleotide as well
as the total oligonucleotide concentration (including the degraded
form) was measured. The method used is a modification of previously
published methods (Leeds et al., 1996; Geary et al., 1999) which
consist of a phenol-chloroform (liquid-liquid) extraction followed
by a solid phase extraction. An internal standard (ISIS 355868, a
27-mer 2'-O-methoxyethyl modified phosphorothioate oligonucleotide,
GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 323)
was added prior to extraction. Tissue sample concentrations were
calculated using calibration curves, with a lower limit of
quantitation (LLOQ) of approximately 1.14 .mu.g/g. Half-lives were
then calculated using WinNonlin software (PHARSIGHT).
[0703] The results are presented in Table 16, expressed as .mu.g/g
liver tissue. The half-life of the ISIS oligonucleotides was
calculated from these values and is also presented in Table 17. The
half-life for each oligonucleotide was considered optimal.
TABLE-US-00016 TABLE 16 Oligonucleotide concentration of ISIS
oligonucleotides in the liver of CD1 mice ISIS ISIS ISIS 463588
463589 463690 Day 3 157 168 196 Day 56 31 17 28
TABLE-US-00017 TABLE 17 Half-life of ISIS oligonucleotides in the
liver of CD1 mice ISIS No Days 463588 22.4 463589 15.9 463690
18.7
Example 14
Measurement of Viscosity of ISIS Antisense Oligonucleotides
Targeting Human FGFR4
[0704] The viscosity of the antisense oligonucleotides selected
from in vivo studies described above was measured with the aim of
screening out antisense oligonucleotides which have a viscosity
more than 40 cP at a concentration of 165-185 mg/mL.
Oligonucleotides having a viscosity greater than 40 cP would be too
viscous to be administered to any subject.
[0705] ISIS oligonucleotides (32-35 mg) were weighed into a glass
vial, 120 .mu.L, of water was added and the antisense
oligonucleotide was dissolved into solution by heating the vial at
50.degree. C. Part of (75 .mu.L) the pre-heated sample was pipetted
to a micro-viscometer (Cambridge). The temperature of the
micro-viscometer was set to 25.degree. C. and the viscosity of the
sample was measured. Another part (20 .mu.L) of the pre-heated
sample was pipetted into 10 mL of water for UV reading at 260 nM at
85.degree. C. (Cary UV instrument). The results are presented in
Table 18 and indicate that most of the antisense oligonucleotide
solutions are optimal in their viscosity under the criterion stated
above.
TABLE-US-00018 TABLE 18 Viscosity and concentration of ISIS
antisense oligonucleotides targeting human FGFR4 ISIS Viscosity
Concentration No. (cP) (mg/mL) 299005 44 174 463588 21 189 463589
17 174 463690 12 178 463691 9 194 463835 25 174 463837 8 181 464225
21 204
Example 15
Effect of ISIS Antisense Oligonucleotides Targeting Human FGFR4 in
Cynomolgus Monkeys
[0706] Chinese cynomolgus monkeys were treated with ISIS antisense
oligonucleotides selected from studies described in Examples 11-14.
Antisense oligonucleotide efficacy and tolerability, as well as
their pharmacokinetic profile in the liver and kidney, were
evaluated. The human antisense oligonucleotides tested are also
cross-reactive with the rhesus genomic sequence GENBANK Accession
No NW.sub.--001121000.1 truncated from nucleosides 3094000 to
3109000 (SEQ ID NO: 5). The greater the complementarity between the
human oligonucleotide and the rhesus monkey sequence, the more
likely the human oligonucleotide can cross-react with the rhesus
monkey sequence. The start sites of each oligonucleotide to SEQ ID
NO: 5 is presented in Table 19. "Target start site" indicates the
5'-most nucleotide to which the gapmer is targeted in the rhesus
monkey gene sequence.
TABLE-US-00019 TABLE 19 Antisense oligonucleotides complementary to
SEQ ID NO: 5 Target SEQ ID Start Site Sequence ISIS No Motif NO
4366 GGCACACTCAGCAGGACCCC 299005 5-10-5 7 4365 GCACACTCAGCAGGACCCCC
463588 5-10-5 16 4367 AGGCACACTCAGCAGGACCC 463589 5-10-5 17 5223
GCCAGGCGACTGCCCTCCTT 463690 5-10-5 45 5224 TGCCAGGCGACTGCCCTCCT
463691 5-10-5 46 6420 CGCTCTCCATCACGAGACTC 463835 5-10-5 70 6422
CACGCTCTCCATCACGAGAC 463837 5-10-5 72 12755 CTTCCAGCTTCTCTGGGCTC
464225 5-10-5 138
Treatment
[0707] This study was conducted at Charles River Laboratories,
Nevada. Prior to the study, the monkeys were acclimated to their
designated housing for at least 13 days before the start of dosing.
The animals were confirmed to have at least one negative serum
antibody test to simian retrovirus (SRV), as well as to other
related viruses. Tuberculosis testing was also done. The animals
were housed individually in stainless steel cages, as specified in
the USDA Animal Welfare Act (9 CFR, Parts 1, 2, and 3). The monkeys
were 2.5 to 8 years old and weighed between 2.5 and 4.0 kg. Eight
groups of five randomly assigned male cynomolgus monkeys each were
injected subcutaneously with ISIS oligonucleotide using a stainless
steel dosing needle and syringe of appropriate size into any of six
dosing sites, which were used on a rotational basis. These sites
were one site each on the lateral portion of each thigh, and four
separate sites on the back. The monkeys were dosed once every other
day at a dose of 40 mg/kg for the first week (days 1, 3, and 5) as
loading doses, and subsequently twice a week at a maintenance dose
of 20 mg/kg (40 mg/kg/week) for weeks 2-13, with ISIS 299005, ISIS
463588, ISIS 463589, ISIS 463690, ISIS 463691, ISIS 463835, ISIS
463837, or ISIS 464225. A control group of 8 cynomolgus monkeys was
injected with PBS subcutaneously once every other day for the first
week (days 1, 3, and 5), and subsequently twice a week for weeks
2-13.
[0708] During the study period, the monkeys were observed twice
daily for a sign of illness or distress. Veterinary care was
available throughout the course of the study and animals were
examined by the veterinary staff, as warranted for clinical signs
or other changes. At the end of the study period, the animals were
euthanized under deep anesthesia induced by ketamine and
Beuthanasia-D.RTM., followed by exsanguination. All organs were
collected within 10 minutes of exsanguinations.
RNA analysis
[0709] Total RNA was extracted from liver and kidney tissue for
real-time PCR analysis and FGFR4 mRNA levels were measured using
human primer probe set RTS3232 and the rhesus primer probe set
rhFGFR4_LTS00467 (forward sequence TCATCAACGGCAGCAGCTT, designated
herein as SEQ ID NO: 333; reverse sequence
TGAGCTATTGATGTCTGCAGTCTTC, designated herein as SEQ ID NO: 334;
probe sequence CCGACGGCTTCCCCTATGTGCA, designated herein as SEQ ID
NO: 335). Results are presented as percent inhibition of FGFR4,
relative to PBS control, normalized to Cyclophilin expression
levels and/or directly with RIBOGREEN.RTM.. As shown in Tables 20
and 21, treatment with ISIS antisense oligonucleotides resulted in
significant reduction of FGFR4 mRNA in comparison to the PBS
control.
TABLE-US-00020 TABLE 20 % Inhibition of FGFR4 mRNA in the
cynomolgus monkey liver relative to the PBS control RTS3232/
rhFGFR4_LTS00467/ rhFGFR4_LTS00467/ ISIS No RTS3232/Ribogreen
Cyclophilm RIBOGREEN Cyclophilin 299005 42 33 41 32 463588 71 72 68
68 463589 40 38 44 43 463690 64 67 58 61 463691 47 65 41 61 463835
61 51 50 37 463837 39 34 38 29 464225 65 64 61 60
TABLE-US-00021 TABLE 21 % Inhibition of FGFR4 mRNA in the
cynomolgus monkey kidney relative to the PBS control
rhFGFR4_LTS00467/ ISIS No RTS3232/Ribogreen RIBOGREEN 299005 60 52
463588 86 85 463589 77 71 463690 76 68 463691 75 63 463835 61 52
463837 54 49 464225 87 83
FGF19 and Leptin Levels
[0710] FGF19 has been known to reduce adiposity and improve insulin
sensitivity in transgenic mice (Fu, L. et al., Endocrinology. 145:
2594-2603, 2004). FGF19 is also characterized as a high affinity
ligand for FGFR4 (Xie, M.-H. et al., Cytokine. 11: 729-735, 1999).
Leptin is a hormone which has been found to be present at very high
levels in obese individuals compared to normal-weight individuals
(Considine, R. V. et al., N. Engl. J. Med. 334: 292-295, 1996).
[0711] FGF19 mRNA levels were measured in ileum tissue samples by
RT-PCR analysis, using the primer probe set rhFGF19_LTS00681
(forward sequence CCCCATGTGGGAATTGATCT, designated herein as SEQ ID
NO: 336; reverse sequence CATGCCTGCTTCAGTCAGTTCT, designated herein
as SEQ ID NO: 337; probe sequence TTTGCCCTTCCCAAACCCCTCCA,
designated herein as SEQ ID NO: 338). The results are presented in
Table 22, expressed as percent expression over the PBS control. The
data indicates that treatment with any of the ISIS oligonucleotides
enhanced the expression of FGF19.
[0712] The plasma samples of monkeys treated with ISIS 299005, ISIS
463588, ISIS 463589, and ISIS 463690 were assessed for FGF19
levels. The plasma samples of monkeys treated with ISIS 463588 and
ISIS 463690 were assessed for leptin levels. Plasma levels of FGF19
were measured pre-dose and on days 23, 65 and 89 using an ELISA
assay kit (R&D Systems). Plasma levels of leptin measured
pre-dose and on days 58 and 93 using an ELISA assay kit (Alpco).
Results are presented in Tables 23 and 24. The data indicates that
treatment with any of the ISIS oligonucleotides increased FGF19
plasma levels and decreases leptin levels. In particular, treatment
with ISIS 463588 caused the highest increase in FGF19 plasma levels
compared to the PBS control as well as to the other experimental
plasma samples. Treatment with ISIS 463588 caused the most
significant decrease in leptin levels compared to the PBS
control.
TABLE-US-00022 TABLE 22 Ileum FGF19 mRNA levels in the cynomolgus
monkey (% expression over the PBS control) % ISIS No expression
299005 688 463588 715 463589 545 463690 1032 463691 477 463835 445
463837 384 464225 370
TABLE-US-00023 TABLE 23 Plasma FGF19 levels in the cynomolgus
monkey (pg/ml) Pre- dose Day 23 Day 65 Day 89 PBS 106 79 104 84
ISIS 299005 125 110 191 202 ISIS 463588 192 146 309 401 ISIS 463589
111 117 177 151 ISIS 463690 184 154 287 266
TABLE-US-00024 TABLE 24 Plasma leptin levels in the cynomolgus
monkey (ng/ml) Pre- dose Day 58 Day 93 PBS 0.21 0.60 0.53 ISIS
463588 0.15 0.23 0.26 ISIS 463690 0.27 0.32 0.36
Tolerability Studies
Liver Function
[0713] To evaluate the effect of ISIS oligonucleotides on hepatic
function, blood samples were collected from all the study groups.
The blood samples were collected via femoral venipuncture on day
58, 48 hrs post-dosing and processed for serum. Concentrations of
various metabolites were measured using an automated clinical
chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma
concentrations of ALT and AST were measured and the results are
presented in Table 25, expressed in IU/L. Bilirubin is also a liver
function marker, was similarly measured and is presented in Table
25, expressed in mg/dL. The results indicate that treatment with
ISIS 463588, as well as several other ISIS oligonucleotides, was
well tolerated in terms of the liver function of the monkeys.
TABLE-US-00025 TABLE 25 ALT, AST, and Bilirubin in cynomolgus
monkey plasma (on day 58) ALT AST Bilirubin (IU/L) (IU/L) (mg/dL)
PBS 47.4 40.3 0.2 ISIS 299005 45.2 37.4 0.2 ISIS 463588 77.6 73.2
0.1 ISIS 463589 33.8 29.8 0.2 ISIS 463690 103.6 47.6 0.2 ISIS
463691 76.2 72.4 1.8 ISIS 463835 116.2 42.0 0.1 ISIS 463837 121.0
43.2 0.1 ISIS 464225 81.4 40.8 0.1
Kidney Function
[0714] To evaluate the effect of ISIS oligonucleotides on kidney
function, blood samples were collected from all the study groups.
The blood samples were collected via femoral venipuncture on day
58, 48 hrs post-dosing and processed for serum. Concentrations of
BUN and creatinine were measured using an automated clinical
chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).
Results are presented in Table 26, expressed in mg/dL.
[0715] The results indicate that most of the ISIS oligonucleotides
did not have any adverse effects on the kidney function.
Specifically, treatment with ISIS 463588 was well tolerated in
terms of the kidney function of the monkeys.
TABLE-US-00026 TABLE 26 Plasma BUN and creatinine levels (mg/dL) in
cynomolgus monkeys on day 58 BUN Creatinine PBS 28.6 0.8 ISIS
299005 24.3 0.7 ISIS 463588 23.2 0.7 ISIS 463589 28.7 0.8 ISIS
463690 22.7 0.7 ISIS 463691 16.8 0.5 ISIS 463835 32.2 0.8 ISIS
463837 26.7 0.7 ISIS 464225 25.8 0.6
Analysis of Markers of Inflammation
[0716] To evaluate the effect of ISIS oligonucleotides on factors
involved in inflammation, blood was collected from all available
animals for C-reactive protein (CRP) and complement C3 analysis, as
well as for measurement of cytokine and chemokine levels. The blood
samples were collected via femoral venipuncture on day 93, 48 hrs
post-dosing and processed for separately for serum and plasma.
Serum CRP and plasma complement C3 was measured using an automated
clinical chemistry analyzer (Hitachi Olympus AU400e, Melville,
N.Y.). The data is presented in Tables 27 and 28, expressed in
mg/dL.
[0717] For cytokine level analyses, blood (1 mL each) was collected
and then centrifuged 3,000 rpm for 10 min at 2-8.degree. C. Plasma
samples of mice treated with ISIS 463588, ISIS 463589, and ISIS
463690 were sent to Aushon Biosystems Inc. (Billerica, Mass.) for
measurement of chemokine and cytokine levels. Levels of IL-6,
MIP-1.alpha., IL-8, MIP-1.beta., MCP-1, IL-1.beta., and RANTES were
measured using the respective cross-reacting human antibodies and
IFN-.gamma. and IL-1.beta. were measured using the respective
primate antibodies. Measurements were taken pre-dose and on day 93.
The results are presented in Tables 29-36.
[0718] The data indicate that most of the ISIS oligonucleotides
were not pro-inflammatory. Specifically, treatment with ISIS 463588
was well tolerated in terms of being non-pro-inflammatory in the
monkeys since there were no changes in CRP, a marker of
inflammation.
TABLE-US-00027 TABLE 27 CRP (mg/dL) in cynomolgus monkeys Pre-dose
Day 30 Day 58 Day 93 PBS 2.2 3.1 2.5 4.1 ISIS 299005 1.0 1.5 1.2
1.2 ISIS 463588 2.9 5.2 3.7 3.8 ISIS 463589 1.8 1.9 2.2 2.4 ISIS
463690 2.2 3.1 2.1 3.6 ISIS 463691 6.3 5.2 10.3 2.6 ISIS 463835 9.7
16.2 4.7 5.7 ISIS 463837 2.5 11.4 2.8 2.9 ISIS 464225 2.5 8.1 6.9
5.2
TABLE-US-00028 TABLE 28 Complement C3 (mg/dL) in cynomolgus monkeys
Pre-dose Day 30 Day 58 Day 93 PBS 114.3 109.1 112.5 113.3 ISIS
299005 108.3 92.1 99.6 91.4 ISIS 463588 106.7 91.9 94.9 95.7 ISIS
463589 116.3 102.0 105.1 100.9 ISIS 463690 113.3 89.4 85.6 78.7
ISIS 463691 123.5 89.2 70.6 97.6 ISIS 463835 105.5 66.2 66.5 69.0
ISIS 463837 107.1 91.1 88.7 86.5 ISIS 464225 104.7 91.9 92.7
80.1
TABLE-US-00029 TABLE 29 IL-6 (pg/mL) in cynomolgus monkeys Pre-dose
Day 93 PBS 1.0 1.0 ISIS 463588 0.4 0.9 ISIS 463589 0.5 2.8 ISIS
463690 1.2 10.2
TABLE-US-00030 TABLE 30 IL-8 (pg/mL) in cynomolgus monkeys Pre-dose
Day 93 PBS 544 482 ISIS 463588 1255 1159 ISIS 463589 424 636 ISIS
463690 719 1344
TABLE-US-00031 TABLE 31 MIP-1.alpha. (pg/mL) in cynomolgus monkeys
Pre-dose Day 93 PBS 7.6 8.9 ISIS 463588 8.9 10.8 ISIS 463589 7.9
11.2 ISIS 463690 13.8 18.9
TABLE-US-00032 TABLE 32 MIP-1.beta. (pg/mL) in cynomolgus monkeys
Pre-dose Day 93 PBS 249 229 ISIS 463588 219 211 ISIS 463589 175 196
ISIS 463690 362 478
TABLE-US-00033 TABLE 33 MCP-1 (pg/mL) in cynomolgus monkeys
Pre-dose Day 93 PBS 200 275 ISIS 463588 420 496 ISIS 463589 343 363
ISIS 463690 441 709
TABLE-US-00034 TABLE 34 IFN-.gamma. (pg/mL) in cynomolgus monkeys
Pre-dose Day 93 PBS 22.1 25.9 ISIS 463588 1.6 1.5 ISIS 463589 10.8
12.3 ISIS 463690 20.8 17.4
TABLE-US-00035 TABLE 35 IL-1.beta. (pg/mL) in cynomolgus monkeys
Pre-dose Day 93 PBS 0.09 0.28 ISIS 463588 0.07 0.08 ISIS 463589
0.13 0.06 ISIS 463690 0.20 0.36
TABLE-US-00036 TABLE 36 RANTES (pg/mL) in cynomolgus monkeys
Pre-dose Day 93 PBS 43339 48967 ISIS 463588 45962 51326 ISIS 463589
38382 30985 ISIS 463690 37330 29209
Hematology
[0719] To evaluate any effect of ISIS oligonucleotides in
cynomolgus monkeys on hematologic parameters, approximately 1.3 mL
of blood was collected on day 93 from each of the available study
animals in tubes containing K.sub.2-EDTA. Samples were analyzed for
red blood cell (RBC) count, white blood cells (WBC) count,
individual white blood cell counts, such as that of monocytes,
neutrophils, lymphocytes, as well as for platelet count, hemoglobin
content and hematocrit, using an ADVIA120 hematology analyzer
(Bayer, USA). The data is presented in Tables 37 and 38.
[0720] The data indicate that most of the ISIS oligonucleotides did
not have any adverse effects on the any hematologic parameters.
Specifically, treatment with ISIS 463588 was well tolerated in
terms of the hematologic parameters of the monkeys.
TABLE-US-00037 TABLE 37 Blood cells in cynomolgus monkeys RBC
Platelets WBC Neutro- Lympho- Mono- (.times.10.sup.6/
(.times.10.sup.3/ (.times.10.sup.3/ phils cytes cytes .mu.L) .mu.L)
.mu.L) (/.mu.L) (/.mu.L) (/.mu.L) PBS 6.1 426 13.8 3244 9637 483
ISIS 299005 6.2 348 13.8 3395 9378 549 ISIS 463588 6.4 331 11.7
3081 7741 387 ISIS 463589 5.7 360 12.3 3590 8037 413 ISIS 463690
6.1 430 13.1 2592 9451 571 ISIS 463691 5.3 494 17.5 7511 8534 1144
ISIS 463835 5.5 558 12.7 3129 8374 664 ISIS 463837 5.8 480 13.3
3145 9025 566 ISIS 464225 5.9 429 13.6 2994 9349 762
TABLE-US-00038 TABLE 38 Hematologic parameters in cynomolgus
monkeys Hemoglobin HCT (g/dL) (%) PBS 14.2 45.5 ISIS 299005 13.7
44.0 ISIS 463588 13.9 45.4 ISIS 463589 13.3 41.9 ISIS 463690 13.8
44.7 ISIS 463691 12.7 40.8 ISIS 463835 12.3 40.0 ISIS 463837 12.8
41.7 ISIS 464225 13.1 42.8
Pharmacokinetic Studies
Measurement of Oligonucleotide Concentration
[0721] The concentration of the full-length oligonucleotide as well
as the total oligonucleotide concentration (including the degraded
form) was measured. The method used is a modification of previously
published methods (Leeds et al., 1996; Geary et al., 1999) which
consist of a phenol-chloroform (liquid-liquid) extraction followed
by a solid phase extraction. An internal standard (ISIS 355868, a
27-mer 2'-O-methoxyethyl modified phosphorothioate oligonucleotide,
GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 323)
was added prior to extraction. Tissue sample concentrations were
calculated using calibration curves, with a lower limit of
quantitation (LLOQ) of approximately 1.14 .mu.g/g. The results are
presented in Table 39, expressed as .mu.g/g tissue. The ratio of
the concentrations in the kidney versus the liver was calculated
and presented in Table 39. Treatment with ISIS oligonucleotides did
not result in any abnormality in the ratio.
TABLE-US-00039 TABLE 39 Full-length oligonucleotide concentration
(.mu.g/g) in the liver and kidney of cynomolgus monkey ISIS No
Kidney Liver Kidney/Liver 463588 1717 1033 1.7 463589 1663 1227 1.4
463690 1395 1226 1.1
[0722] Overall, the results of the study indicate that ISIS 463588
is a potent and tolerable antisense oligonucleotide for treatment
of metabolic diseases, such as diabetes, obesity, insulin
resistance, and insulin deficiency.
Example 16
In Vivo Effect of Antisense Inhibition of Murine FGFR4 in
Diet-Induced Obesity (DIO) Mice with Caloric Restriction
[0723] DIO mice are C57BL/6 mice fed a high fat diet starting from
6 weeks of age and are a standard model used for assays related to
studying the effect of therapeutic agents on lowering adiposity and
improving insulin sensitivity. The antisense oligonucleotide, ISIS
393250, a 5-10-5 MOE gapmer, having a sequence of
5'-GCCACATTTCCTTCCAGCTG-3 (SEQ ID NO: 324), and with a target start
site of 337 on murine FGFR4 mRNA (GENBANK Accession No. BC033313.1
(SEQ ID NO: 6) was used in this assay. The effect of ISIS 393250 on
a DIO model under caloric restriction was evaluated.
Treatment
[0724] Male 6 week-old C57BL/6 mice (Jackson Laboratories) were fed
with 58 kcal % high-fat diet (Research diet D12330) ad lib for 4
months to induce obesity. The mice were divided into 4 groups based
on body weight and body fat content. The first group of mice was
treated with 25 mg/kg ISIS 393250 administered subcutaneously twice
weekly for 6 weeks. The second group of mice was treated with 25
mg/kg control oligonucleotide, ISIS 141923 (CCTTCCCTGAAGGTTCCTCC
(SEQ ID NO: 325), 5-10-5 MOE gapmer with no known murine target),
administered subcutaneously twice weekly for 6 weeks. Two control
groups of mice were treated with PBS administered subcutaneously
twice weekly for 6 weeks. After two weeks of treatment, the
oligonucleotide-treated mice and one of the PBS control group mice
were subjected to caloric restriction by providing 95% of the
amount of food consumed daily by the FGFR4 ASO-treated mice during
the first two weeks of treatment. The second PBS control group
continued to be fed ad libitum with the same amount of food as in
the first two weeks of treatment.
[0725] Weekly body weights were measured and body compositions were
monitored at different time point with an Echo MRI Body Composition
Analyzer. The mice were euthanized after 6 weeks of treatment.
RNA Analysis
[0726] RNA was extracted from the liver for RT-PCR analysis of
murine FGFR4 expression. The primer probe set mFGFR4_LTS00702
(forward sequence CCCTGAGGCCAGATACACAGATAT, designated herein as
SEQ ID NO: 339; reverse sequence ACGGATGACTTGCCGATGATA, designated
herein as SEQ ID NO: 340; probe sequence
CTCACTGGTTCTGCTTGTGCTCCTGCT, designated herein as SEQ ID NO: 341)
was used for analysis. The results indicated that treatment with
ISIS 393250 reduced murine FGFR4 levels by 76%.
Body Weight and Body Composition Analysis
[0727] Weekly body weights were measured and are presented in Table
40. Body fat content data is presented in Table 41, expressed as
percent of the corresponding body weight. Lean body mass is
presented in Table 42, expressed in grams. White adipose tissue
weight was measured after euthanizing the mice and is presented in
Table 43, expressed in grams. The data indicates calorie
restriction significantly lowered body weight and total body fat
content. Treatment with ISIS 393250 further lowered both body
weight and fat content, but had no effect on body lean mass.
Treatment with ISIS 141923 had no effect. Hence, antisense
inhibition of FGFR4 expression has a beneficial effect on body
weight and body fat content in subjects suffering from obesity.
TABLE-US-00040 TABLE 40 Weekly body weights (g) Calorie- restrict-
Pre- Week Week Week Week Week Week ed dose 1 2 3 4 5 6 PBS No 51.7
51.1 52.1 52.6 53.1 50.6 53.4 PBS Yes 50.3 50.4 51.1 50.0 47.6 46.0
45.4 ISIS 141923 Yes 49.5 50.1 50.9 49.8 48.1 46.3 45.2 ISIS 393250
Yes 50.5 50.9 50.9 48.9 46.7 44.4 42.1
TABLE-US-00041 TABLE 41 Body fat content (% body weight) Calorie-
restricted Pre-dose Week 2 Week 4 Week 6 PBS No 39.5 39.8 40 40 PBS
Yes 38.7 40.2 39.1 38.3 ISIS 141923 Yes 38.9 39.1 38.1 37.7 ISIS
393250 Yes 39.4 38.6 35.3 31.3
TABLE-US-00042 TABLE 42 Lean body mass (g) Calorie- restricted
Pre-dose Week 2 Week 4 Week 6 PBS No 27.8 28.2 28.2 28.3 PBS Yes
27.3 27.5 25.7 24.5 ISIS 141923 Yes 26.7 27.3 26 24.9 ISIS 393250
Yes 27.1 27.8 25.9 24.8
TABLE-US-00043 TABLE 43 White adipose tissue weight (g) Calorie-
restricted Epididymal Peri-renal PBS No 2.5 1.1 PBS Yes 2.2 0.9
ISIS 141923 Yes 2.2 0.9 ISIS 393250 Yes 1.8 0.7
Metabolic Rate and Locomotor Activity Analysis
[0728] The metabolic rate was assessed by measuring the oxygen
consumption and heat production of the mice. Both parameters were
measured with an indirect calorimetry system (Oxymax system,
Columbus Instruments). Locomotor activity was also assessed with
the same instrument. Metabolic rate and locomotor activity was
assessed both in darkness, when the mice are typically more active,
and in light. The results are presented in Tables 44-46. The
results indicate that calorie restriction reduced whole body oxygen
consumption. Treatment with ISIS 393250 prevented this decrease in
oxygen consumption without affecting locomotor activity. Hence,
antisense inhibition of FGFR4 expression in obese subjects with a
calorie-restricted diet would be beneficial as it would prevent any
decline in metabolic rate in the subject.
TABLE-US-00044 TABLE 44 O.sub.2 consumption (mL/kg lean tissue/hr)
Calorie- restricted dark light PBS No 4275 3327 PBS Yes 4085 3259
ISIS 141923 Yes 4094 3258 ISIS 393250 Yes 4268 3359
TABLE-US-00045 TABLE 45 Heat production (kcal/kg lean tissue/hr)
Calorie- restricted dark light PBS No 19.8 15 PBS Yes 19.1 15.1
ISIS 141923 Yes 19.1 15.1 ISIS 393250 Yes 19.7 15.7
TABLE-US-00046 TABLE 46 Locomotor activity (events/min) Calorie-
restricted dark light PBS No 16.4 1.9 PBS Yes 21.9 3.1 ISIS 141923
Yes 16.1 2.5 ISIS 393250 Yes 16.2 3
Example 17
In Vivo Effect of Antisense Inhibition of Murine FGFR4 in
Diet-Induced Obesity (DIO) Mice with Caloric Restriction
[0729] The effect of ISIS 446259 (TCCATTTCCTCAGAGGCCTC (SEQ ID NO:
326), 5-10-5 MOE gapmer, with a target start site of 407 on GENBANK
Accession No. BC033313.1 (SEQ ID NO: 6)) on DIO mice under caloric
restriction was evaluated.
Treatment
[0730] Male 6 week-old C57BL/6 mice (Jackson Laboratories) were fed
with 58 kcal % high-fat diet Research diet D12330) ad lib for 3.5
months to induce obesity. The mice were divided into 4 groups based
on body weight and body fat content. The first group of mice was
treated with 25 mg/kg ISIS 446259 administered subcutaneously twice
weekly for 8 weeks. The second group of mice was treated with 25
mg/kg control oligonucleotide, ISIS 141923 administered
subcutaneously twice weekly for 8 weeks. Two control groups of mice
were treated with PBS administered subcutaneously twice weekly for
8 weeks. After two weeks of treatment, the oligonucleotide-treated
mice and one of the PBS control group mice were subjected to
caloric restriction by providing 90% of the amount of food consumed
daily by the FGFR4 ASO-treated mice during the first two weeks of
treatment. The second PBS control group continued to be fed ad
libitum with the same amount of food as in the first two weeks of
treatment.
[0731] Weekly body weights were measured and body compositions were
monitored at different time point with an Echo MRI Body Composition
Analyzer. The mice were euthanized after 8 weeks of treatment.
RNA Analysis
[0732] RNA was extracted from the liver for RT-PCR analysis of
murine FGFR4 expression. The primer probe set mFGFR4 LTS00702 was
used to analyze mRNA levels. The results indicated that treatment
with ISIS 446259 reduced murine FGFR4 levels by 83%.
Body Weight and Body Composition Analysis
[0733] Weekly body weights were measured and are presented in Table
47. Body fat content data is presented in Table 48, expressed as
percent of the corresponding body weight. Lean body mass was
presented in Table 49, expressed in grams. The data indicates
calorie restriction significantly lowered body weight and total
body fat content. Treatment with ISIS 446259 further lowered both
body weight and fat content, but had no effect on body lean mass.
Treatment with ISIS 141923 had no further effect. Hence, antisense
inhibition of FGFR4 expression has a beneficial effect on body
weight and body fat content in subjects suffering from obesity in
addition to effects seen by caloric restriction alone.
TABLE-US-00047 TABLE 47 Weekly body weights (g) Calorie- Week Week
Week Week Week restricted 0 2 4 6 8 PBS No 48.7 49.9 51 53.4 52.5
PBS Yes 49.7 50.7 46.9 46.9 46.2 ISIS 141923 Yes 49.6 50.5 46.9
46.1 44.5 ISIS 446259 Yes 49.4 49.4 45.2 43.8 39.7
TABLE-US-00048 TABLE 48 Body fat content (% body weight) Calorie-
restricted Week 0 Week 2 Week 5 Week 8 PBS No 41 43 43 42 PBS Yes
41 43 41 39 ISIS 141923 Yes 40 40 37 35 ISIS 446259 Yes 41 41 35
30
TABLE-US-00049 TABLE 49 Lean body mass (g) Calorie- restricted Week
0 Week 2 Week 5 Week 8 PBS No 26 24 26 27 PBS Yes 26 25 25 25 ISIS
141923 Yes 27 26 25 26 ISIS 446259 Yes 26 25 25 25
Plasma Lipid Analysis
[0734] To evaluate the effect of ISIS oligonucleotides on
cholesterol and triglyceride metabolism, plasma levels of each were
measured at the end of the treatment period. The mice were
euthanized and blood was collected via cardiac puncture. The lipid
levels were measured using an automated clinical chemistry analyzer
(Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in
Table 50, expressed as mg/dL. The results indicate that treatment
with ISIS 446259 reduced both cholesterol and triglyceride levels
in the mice. Therefore, antisense inhibition of FGFR4 had a
beneficial effect on the lipid profile and may be used to reduce
adiposity in obese subjects.
TABLE-US-00050 TABLE 50 Cholesterol and lipid levels (mg/dL)
Calorie- restricted Cholesterol Triglycerides PBS No 270 137 PBS
Yes 240 128 ISIS 141923 Yes 222 113 ISIS 446259 Yes 181 84
Example 18
In Vivo Effect of Antisense Inhibition of Murine FGFR4 on FGF15
Levels in DIO Mice
[0735] The effect of ISIS 393250 and ISIS 446259 on FGF15 levels in
DIO mice was evaluated.
Treatment
[0736] Male 6 week-old C57BL/6 mice (Jackson Laboratories) were fed
with 58 kcal % high-fat diet Research diet D12330) ad lib for 3.5
months to induce obesity. A group of C57BL/6 mice were fed normal
Purina mouse chow and served as the naive control. The DIO mice
were divided into groups based on body weight and body fat content.
The first group of DIO mice was treated with 25 mg/kg ISIS 393250
administered subcutaneously twice weekly for 4 weeks. The second
group of DIO mice was treated with 25 mg/kg ISIS 446259
administered subcutaneously twice weekly for 4 weeks. The third
group of DIO mice was treated with 25 mg/kg control
oligonucleotide, ISIS 141923 administered subcutaneously twice
weekly for 4 weeks. A control group of DIO mice was treated with
PBS administered subcutaneously twice weekly for 4 weeks. The mice
were euthanized after 4 weeks of treatment.
FGF15 Levels
[0737] FGF15 is the rodent equivalent of FGF19 (Wright, T. J. et
al., Dev. Biol. 269: 264-275, 2004), and is therefore important for
the reduction of adiposity and improvement of insulin sensitivity
in mice.
[0738] RNA was extracted from liver and ileum. Liver RNA was
analyzed by RT-PCR analysis for FGFR4 mRNA levels using primer
probe set mFGFR4_LTS00702. Ileum RNA was analyzed by RT-PCR
analysis for FGF15 levels using primer probe set mFgf15_LTS00635
(forward sequence GACCAAAACGAACGAAATTTGTT, designated herein as SEQ
ID NO: 342; reverse sequence ACGTCCTTGATGGCAATCG, designated herein
as SEQ ID NO: 343; probe sequence AATTCCGCGCGGTCGCTCTG, designated
herein as SEQ ID NO: 344). The results are presented in Table 51
and demonstrate that treatment with either antisense
oligonucleotide significantly decreases FGFR4 mRNA levels and also
significantly enhances FGF15 expression levels.
[0739] Plasma samples of the mice group were also analyzed at weeks
2 and 4 for FGF15 protein levels with ELISA using an anti-FGF15
antibody (Santa Cruz Biotechnology Inc). The results are presented
in Table 52 and demonstrate that antisense inhibition of FGFR4
results in enhanced plasma levels of FGF15.
TABLE-US-00051 TABLE 51 FGFR4 and FGF15 mRNA levels relative to
control Liver Ileum FGFR4 (% FGF15 (% ISIS No inhibition)
expression) 141923 14 92 393250 96 1117 446259 94 707 C57BL/6
control 0 25
TABLE-US-00052 TABLE 52 FGF15 plasma levels at week 2 and 4 (ng/ml)
Week 2 Week 4 PBS 0.13 0.13 ISIS 141923 0.12 0.14 ISIS 393250 0.69
0.96 ISIS 446259 0.18 0.25 C57BL/6 control 0.1 0.12
Example 19
In Vivo Effect of Antisense Inhibition of Murine FGFR4 on FGF15
Levels in C57BL/6 Mice
[0740] The effect of ISIS 393250 on FGF15 levels in C57BL/6 mice
was evaluated.
Treatment
[0741] Male 6 week-old C57BL/6 mice (Jackson Laboratories) were fed
normal Purina mouse chow. The mice were randomly divided into 3
groups. The first group of mice was treated with 50 mg/kg ISIS
393250 administered subcutaneously twice weekly for 5.5 weeks. The
second group of mice was treated with 50 mg/kg control
oligonucleotide, ISIS 141923 administered subcutaneously twice
weekly for 5.5 weeks. A control group of mice was treated with PBS
administered subcutaneously twice weekly for 5.5 weeks.
FGFR4 Levels
[0742] RNA was extracted from liver and RNA was analyzed by RT-PCR
analysis for FGFR4 mRNA levels using primer probe set
mFGFR4_LTS00702. The results are presented in Table 53 and
demonstrate that treatment with ISIS 393250 significantly decreases
FGFR4 mRNA levels
TABLE-US-00053 TABLE 53 FGFR4 mRNA inhibition levels (%) ISIS No %
141923 0 393250 79
FGF15 Levels
[0743] Plasma samples of the mice group were analyzed for FGF15
protein levels using with ELISA using an anti-FGF15 antibody (Santa
Cruz Biotechnology Inc). The results are presented in Table 54 and
demonstrate that antisense inhibition of FGFR4 results in enhanced
plasma levels of FGF15.
TABLE-US-00054 TABLE 54 FGF15 plasma levels at day 16 ng/mL PBS
0.07 ISIS 141923 0.08 ISIS 393250 0.28
Example 20
In Vivo Effect of Antisense Inhibition of Murine FGFR4 on FGF15
Levels in Ob/Ob Mice
[0744] Leptin is a hormone produced by fat that regulates appetite.
Deficiency of this hormone in both humans and in non-human animals,
leads to obesity. ob/ob mice have a mutation in the leptin gene
which results in obesity and hyperglycemia. As such, these mice are
a useful model for the investigation of obesity and diabetes and
related conditions provided herein. These mice models are also
useful for testing compounds, compositions and methods designed to
treat, prevent or ameliorate such conditions.
[0745] In accordance with the present invention, the effects of
antisense inhibition of FGFR4 were investigated in the ob/ob mouse
model of obesity. Male 12 week old ob/ob
(C57Bl/6J-Lep.sup.ob/Lep.sup.ob) mice were purchased from Jackson
Laboratories (Bar Harbor, Me.) and used for the current study.
Treatment
[0746] The mice were divided into groups based on body weight and
body fat content. The first group of mice was treated with 25 mg/kg
ISIS 393250 administered subcutaneously twice weekly for 14 weeks.
The second group of mice was treated with 25 mg/kg control
oligonucleotide, ISIS 141923 administered subcutaneously twice
weekly for 14 weeks. A control group of mice was treated with PBS
administered subcutaneously twice weekly for 14 weeks.
FGFR4 Levels
[0747] RNA was extracted from liver and RNA was analyzed by RT-PCR
analysis for FGFR4 mRNA levels using primer probe set
mFGFR4_LTS00702. The results are presented in Table 55 and
demonstrate that treatment with ISIS 393250 significantly decreases
FGFR4 mRNA levels
TABLE-US-00055 TABLE 55 FGFR4 mRNA inhibition levels (%) ISIS No %
141923 0 393250 89
FGF15 Levels
[0748] Plasma samples of the mice group were analyzed for FGF15
protein levels using with ELISA using an anti-FGF15 antibody (Santa
Cruz Biotechnology Inc). The results are presented in Table 56 and
demonstrate that antisense inhibition of FGFR4 results in enhanced
plasma levels of FGF15.
TABLE-US-00056 TABLE 56 FGF15 plasma levels at week 4 and 8 (ng/mL)
Week 4 Week 8 PBS 0.5 1.2 ISIS 141923 0.8 0.5 ISIS 393250 4.2
4.2
Example 21
Effect of Antisense Inhibition of Murine FGFR4 in Monkey Primary
Hepatocytes
[0749] The effect of antisense inhibition of FGFR4 with ISIS 299004
on fatty acid oxidation in monkey hepatocytes was evaluated. AICAR
was used as a positive control.
Treatment
[0750] Primary hepatocytes purchased from APL/Lovelace In Vitro
Enterprises and cultured in William E medium. The cells were seeded
at a density of 1 million cells per 25 ml flask. After 4-5 hrs of
culture, the cells were treated with 30 nM of ISIS 299004 or 1000
.mu.M AICAR for 18 hrs. A control set of cells was treated with
PBS. FGFR4 levels were measured using the primer probe set
cynoFGFR4_MGB_LTS00689 (forward sequence GCACCAGGGATGAGCTTGAC,
designated herein as SEQ ID NO: 348; reverse sequence
CCAAGTCTCCCACTTTCCAGTT, designated herein as SEQ ID NO: 349; probe
sequence AAGAGCCTGACTCCAGT, designated herein as SEQ ID NO: 350).
Treatment with ISIS 299004 reduced FGFR4 levels by 83%.
[0751] For evaluation of fatty acid oxidation, the cells were
placed in low glucose media containing .sup.1-14cOleic acid and
BSA, and the culture flasks were capped with a rubber stopper
containing a hanging reservoir bucket. The cells were then
incubated at 37.degree. C. under 5% CO.sub.2 for 1.5 hrs. Following
incubation, 200 .mu.l of 1M hyamine hydroxide (a .sup.14CO.sub.2
trapping agent) was added to the reservoir bucket and 1 ml of 10%
perchloric acid solution was added to the cells. The flasks were
transferred to a 37.degree. C. shaking incubator for 40 min. Upon
completion of the incubation, the hanging bucket reservoir
containing the hyamine hydroxide was separated from the flask and
placed in scintillation fluid overnight, and read in the
scintillation counter the next day. Bradford-based protein
measurements were conducted on an equal number of primary monkey
hepatocytes, by using the DC.TM. Biorad protein assay kit (Bearden,
J. Biochem. Biophys. Acta. 533: 525. 1978). The values obtained
from the protein readout was used for normalization of the CO.sub.2
production counted by the scintillation counter. The results are
presented in Table 57 and indicate that antisense inhibition of
FGFR4 increased fatty acid oxidation in primary hepatocytes. Five
independent fatty acid oxidation experiments were conducted, which
demonstrated a similar trend on the results.
TABLE-US-00057 TABLE 57 CO.sub.2 production (% of the control)
CO.sub.2 ISIS 141923 +2 ISIS 299004 +48 AICAR +44
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 350 <210> SEQ ID NO 1 <211> LENGTH: 3040
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (168)..(2576) <400> SEQUENCE: 1 ctcgctcccg
gccgaggagc gctcgggctg tctgcggacc ctgccgcgtg caggggtcgc 60
ggccggctgg agctgggagt gaggcggcgg aggagccagg tgaggaggag ccaggaaggc
120 agttggtggg aagtccagct tgggtccctg agagctgtga gaaggag atg cgg ctg
176 Met Arg Leu 1 ctg ctg gcc ctg ttg ggg gtc ctg ctg agt gtg cct
ggg cct cca gtc 224 Leu Leu Ala Leu Leu Gly Val Leu Leu Ser Val Pro
Gly Pro Pro Val 5 10 15 ttg tcc ctg gag gcc tct gag gaa gtg gag ctt
gag ccc tgc ctg gct 272 Leu Ser Leu Glu Ala Ser Glu Glu Val Glu Leu
Glu Pro Cys Leu Ala 20 25 30 35 ccc agc ctg gag cag caa gag cag gag
ctg aca gta gcc ctt ggg cag 320 Pro Ser Leu Glu Gln Gln Glu Gln Glu
Leu Thr Val Ala Leu Gly Gln 40 45 50 cct gtg cgt ctg tgc tgt ggg
cgg gct gag cgt ggt ggc cac tgg tac 368 Pro Val Arg Leu Cys Cys Gly
Arg Ala Glu Arg Gly Gly His Trp Tyr 55 60 65 aag gag ggc agt cgc
ctg gca cct gct ggc cgt gta cgg ggc tgg agg 416 Lys Glu Gly Ser Arg
Leu Ala Pro Ala Gly Arg Val Arg Gly Trp Arg 70 75 80 ggc cgc cta
gag att gcc agc ttc cta cct gag gat gct ggc cgc tac 464 Gly Arg Leu
Glu Ile Ala Ser Phe Leu Pro Glu Asp Ala Gly Arg Tyr 85 90 95 ctc
tgc ctg gca cga ggc tcc atg atc gtc ctg cag aat ctc acc ttg 512 Leu
Cys Leu Ala Arg Gly Ser Met Ile Val Leu Gln Asn Leu Thr Leu 100 105
110 115 att aca ggt gac tcc ttg acc tcc agc aac gat gat gag gac ccc
aag 560 Ile Thr Gly Asp Ser Leu Thr Ser Ser Asn Asp Asp Glu Asp Pro
Lys 120 125 130 tcc cat agg gac ccc tcg aat agg cac agt tac ccc cag
caa gca ccc 608 Ser His Arg Asp Pro Ser Asn Arg His Ser Tyr Pro Gln
Gln Ala Pro 135 140 145 tac tgg aca cac ccc cag cgc atg gag aag aaa
ctg cat gca gta cct 656 Tyr Trp Thr His Pro Gln Arg Met Glu Lys Lys
Leu His Ala Val Pro 150 155 160 gcg ggg aac acc gtc aag ttc cgc tgt
cca gct gca ggc aac ccc acg 704 Ala Gly Asn Thr Val Lys Phe Arg Cys
Pro Ala Ala Gly Asn Pro Thr 165 170 175 ccc acc atc cgc tgg ctt aag
gat gga cag gcc ttt cat ggg gag aac 752 Pro Thr Ile Arg Trp Leu Lys
Asp Gly Gln Ala Phe His Gly Glu Asn 180 185 190 195 cgc att gga ggc
att cgg ctg cgc cat cag cac tgg agt ctc gtg atg 800 Arg Ile Gly Gly
Ile Arg Leu Arg His Gln His Trp Ser Leu Val Met 200 205 210 gag agc
gtg gtg ccc tcg gac cgc ggc aca tac acc tgc ctg gta gag 848 Glu Ser
Val Val Pro Ser Asp Arg Gly Thr Tyr Thr Cys Leu Val Glu 215 220 225
aac gct gtg ggc agc atc cgc tat aac tac ctg cta gat gtg ctg gag 896
Asn Ala Val Gly Ser Ile Arg Tyr Asn Tyr Leu Leu Asp Val Leu Glu 230
235 240 cgg tcc ccg cac cgg ccc atc ctg cag gcc ggg ctc ccg gcc aac
acc 944 Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn
Thr 245 250 255 aca gcc gtg gtg ggc agc gac gtg gag ctg ctg tgc aag
gtg tac agc 992 Thr Ala Val Val Gly Ser Asp Val Glu Leu Leu Cys Lys
Val Tyr Ser 260 265 270 275 gat gcc cag ccc cac atc cag tgg ctg aag
cac atc gtc atc aac ggc 1040 Asp Ala Gln Pro His Ile Gln Trp Leu
Lys His Ile Val Ile Asn Gly 280 285 290 agc agc ttc gga gcc gac ggt
ttc ccc tat gtg caa gtc cta aag act 1088 Ser Ser Phe Gly Ala Asp
Gly Phe Pro Tyr Val Gln Val Leu Lys Thr 295 300 305 gca gac atc aat
agc tca gag gtg gag gtc ctg tac ctg cgg aac gtg 1136 Ala Asp Ile
Asn Ser Ser Glu Val Glu Val Leu Tyr Leu Arg Asn Val 310 315 320 tca
gcc gag gac gca ggc gag tac acc tgc ctc gca ggc aat tcc atc 1184
Ser Ala Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly Asn Ser Ile 325
330 335 ggc ctc tcc tac cag tct gcc tgg ctc acg gtg ctg cca gag gag
gac 1232 Gly Leu Ser Tyr Gln Ser Ala Trp Leu Thr Val Leu Pro Glu
Glu Asp 340 345 350 355 ccc aca tgg acc gca gca gcg ccc gag gcc agg
tat acg gac atc atc 1280 Pro Thr Trp Thr Ala Ala Ala Pro Glu Ala
Arg Tyr Thr Asp Ile Ile 360 365 370 ctg tac gcg tcg ggc tcc ctg gcc
ttg gct gtg ctc ctg ctg ctg gcc 1328 Leu Tyr Ala Ser Gly Ser Leu
Ala Leu Ala Val Leu Leu Leu Leu Ala 375 380 385 ggg ctg tat cga ggg
cag gcg ctc cac ggc cgg cac ccc cgc ccg ccc 1376 Gly Leu Tyr Arg
Gly Gln Ala Leu His Gly Arg His Pro Arg Pro Pro 390 395 400 gcc act
gtg cag aag ctc tcc cgc ttc cct ctg gcc cga cag ttc tcc 1424 Ala
Thr Val Gln Lys Leu Ser Arg Phe Pro Leu Ala Arg Gln Phe Ser 405 410
415 ctg gag tca ggc tct tcc ggc aag tca agc tca tcc ctg gta cga ggc
1472 Leu Glu Ser Gly Ser Ser Gly Lys Ser Ser Ser Ser Leu Val Arg
Gly 420 425 430 435 gtg cgt ctc tcc tcc agc ggc ccc gcc ttg ctc gcc
ggc ctc gtg agt 1520 Val Arg Leu Ser Ser Ser Gly Pro Ala Leu Leu
Ala Gly Leu Val Ser 440 445 450 cta gat cta cct ctc gac cca cta tgg
gag ttc ccc cgg gac agg ctg 1568 Leu Asp Leu Pro Leu Asp Pro Leu
Trp Glu Phe Pro Arg Asp Arg Leu 455 460 465 gtg ctt ggg aag ccc cta
ggc gag ggc tgc ttt ggc cag gta gta cgt 1616 Val Leu Gly Lys Pro
Leu Gly Glu Gly Cys Phe Gly Gln Val Val Arg 470 475 480 gca gag gcc
ttt ggc atg gac cct gcc cgg cct gac caa gcc agc act 1664 Ala Glu
Ala Phe Gly Met Asp Pro Ala Arg Pro Asp Gln Ala Ser Thr 485 490 495
gtg gcc gtc aag atg ctc aaa gac aac gcc tct gac aag gac ctg gcc
1712 Val Ala Val Lys Met Leu Lys Asp Asn Ala Ser Asp Lys Asp Leu
Ala 500 505 510 515 gac ctg gtc tcg gag atg gag gtg atg aag ctg atc
ggc cga cac aag 1760 Asp Leu Val Ser Glu Met Glu Val Met Lys Leu
Ile Gly Arg His Lys 520 525 530 aac atc atc aac ctg ctt ggt gtc tgc
acc cag gaa ggg ccc ctg tac 1808 Asn Ile Ile Asn Leu Leu Gly Val
Cys Thr Gln Glu Gly Pro Leu Tyr 535 540 545 gtg atc gtg gag tgc gcc
gcc aag gga aac ctg cgg gag ttc ctg cgg 1856 Val Ile Val Glu Cys
Ala Ala Lys Gly Asn Leu Arg Glu Phe Leu Arg 550 555 560 gcc cgg cgc
ccc cca ggc ccc gac ctc agc ccc gac ggt cct cgg agc 1904 Ala Arg
Arg Pro Pro Gly Pro Asp Leu Ser Pro Asp Gly Pro Arg Ser 565 570 575
agt gag ggg ccg ctc tcc ttc cca gtc ctg gtc tcc tgc gcc tac cag
1952 Ser Glu Gly Pro Leu Ser Phe Pro Val Leu Val Ser Cys Ala Tyr
Gln 580 585 590 595 gtg gcc cga ggc atg cag tat ctg gag tcc cgg aag
tgt atc cac cgg 2000 Val Ala Arg Gly Met Gln Tyr Leu Glu Ser Arg
Lys Cys Ile His Arg 600 605 610 gac ctg gct gcc cgc aat gtg ctg gtg
act gag gac aat gtg atg aag 2048 Asp Leu Ala Ala Arg Asn Val Leu
Val Thr Glu Asp Asn Val Met Lys 615 620 625 att gct gac ttt ggg ctg
gcc cgc ggc gtc cac cac att gac tac tat 2096 Ile Ala Asp Phe Gly
Leu Ala Arg Gly Val His His Ile Asp Tyr Tyr 630 635 640 aag aaa acc
agc aac ggc cgc ctg cct gtg aag tgg atg gcg ccc gag 2144 Lys Lys
Thr Ser Asn Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu 645 650 655
gcc ttg ttt gac cgg gtg tac aca cac cag agt gac gtg tgg tct ttt
2192 Ala Leu Phe Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp Ser
Phe 660 665 670 675 ggg atc ctg cta tgg gag atc ttc acc ctc ggg ggc
tcc ccg tat cct 2240 Gly Ile Leu Leu Trp Glu Ile Phe Thr Leu Gly
Gly Ser Pro Tyr Pro 680 685 690 ggc atc ccg gtg gag gag ctg ttc tcg
ctg ctg cgg gag gga cat cgg 2288 Gly Ile Pro Val Glu Glu Leu Phe
Ser Leu Leu Arg Glu Gly His Arg 695 700 705 atg gac cga ccc cca cac
tgc ccc cca gag ctg tac ggg ctg atg cgt 2336 Met Asp Arg Pro Pro
His Cys Pro Pro Glu Leu Tyr Gly Leu Met Arg 710 715 720 gag tgc tgg
cac gca gcg ccc tcc cag agg cct acc ttc aag cag ctg 2384 Glu Cys
Trp His Ala Ala Pro Ser Gln Arg Pro Thr Phe Lys Gln Leu 725 730 735
gtg gag gcg ctg gac aag gtc ctg ctg gcc gtc tct gag gag tac ctc
2432 Val Glu Ala Leu Asp Lys Val Leu Leu Ala Val Ser Glu Glu Tyr
Leu 740 745 750 755 gac ctc cgc ctg acc ttc gga ccc tat tcc ccc tct
ggt ggg gac gcc 2480 Asp Leu Arg Leu Thr Phe Gly Pro Tyr Ser Pro
Ser Gly Gly Asp Ala 760 765 770 agc agc acc tgc tcc tcc agc gat tct
gtc ttc agc cac gac ccc ctg 2528 Ser Ser Thr Cys Ser Ser Ser Asp
Ser Val Phe Ser His Asp Pro Leu 775 780 785 cca ttg gga tcc agc tcc
ttc ccc ttc ggg tct ggg gtg cag aca tga 2576 Pro Leu Gly Ser Ser
Ser Phe Pro Phe Gly Ser Gly Val Gln Thr 790 795 800 gcaaggctca
aggctgtgca ggcacatagg ctggtggcct tgggccttgg ggctcagcca 2636
cagcctgaca cagtgctcga ccttgatagc atggggcccc tggcccagag ttgctgtgcc
2696 gtgtccaagg gccgtgccct tgcccttgga gctgccgtgc ctgtgtcctg
atggcccaaa 2756 tgtcagggtt ctgctcggct tcttggacct tggcgcttag
tccccatccc gggtttggct 2816 gagcctggct ggagagctgc tatgctaaac
ctcctgcctc ccaataccag caggaggttc 2876 tgggcctctg aacccccttt
ccccacacct ccccctgctg ctgctgcccc agcgtcttga 2936 cgggagcatt
ggcccctgag cccagagaag ctggaagcct gccgaaaaca ggagcaaatg 2996
gcgttttata aattattttt ttgaaataaa aaaaaaaaaa aaaa 3040 <210>
SEQ ID NO 2 <211> LENGTH: 12196 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 2
tgagggagca gaaggaaggg gttctcctat ccgctgcacg gcactgcgca gaagacaggg
60 gagccaggca ttccctgaag ggtgaaaagc aaggagtaga gctgggtagt
agactagaat 120 ttaggagcct ggcctggggc ctgggtgggg cgaaagaggc
ggagcctgaa tggggtgtgt 180 ataggggggt tgcgtgtagg ggtgtgtgta
taggctgggg cggggtcccg ggagtgggct 240 gactgggtcg ggggcggggc
tctccaggtg ggcggggatc ttggccaccc ctggccacac 300 ctctctccgg
ctcgagctgg tctaggcggg gcgggcccga gggggtgtgg caggaggtgg 360
gcgggcccgg gtgggggggg ggggggcgtg gaaggagggg cgggcccgag caggaggggg
420 cgggcccgag gggcggggtg ggacaggagg tgggccgctc gcggccacgc
cgccgtcgcg 480 ggtacattcc tcgctcccgg ccgaggagcg ctcgggctgt
ctgcggaccc tgccgcgtgc 540 aggggtcgcg gccggctgga gctgggagtg
aggcggcgga ggagccaggt gaggaggagc 600 caggtgagca ggaccctgtg
ctgggcgcgg agtcacgcag gctcgaggtg agccggaacc 660 cttgtgggcc
cgggctgcgc tcccagccgc cagggggcga gaggcggcgg ggctacgggg 720
actgcccctc ccggcgcagg ggacctgggc gtccgccggg cggcaggggg tggagggggc
780 ggtaaatcag taacccgcag tgcacacagg gccttttgtc ccgctccgtc
caaagagcac 840 cccggccgcg gagctggtta ctcattgccc accgaggcgg
gggcaggctg gccctgtgca 900 gctaccctcg ggacccattg attcgcacct
ccccccaggc tggcccggca agggtggggg 960 aggacaagcg cgcttgtccc
tgcggctgtc ttcgcgccgg cggcagagat gagggacctg 1020 aggccccgaa
aagttcagtc acttagtgcc cgggggcctc cagcgcgagt gcgggaggct 1080
gaaggagaac ccaggactgt ctgatgccta aggcaggccc tccattccca cgtggggggt
1140 ggtcggtcag cggtcagcag ccatgggtga ctcgactaag gactctgata
tcagggcagc 1200 ctggggtagg aataaactcc ccgggcctcc ccacccactc
ccagcccaag ctgtgtaccc 1260 aaagagctgc cctccctgcc aagccgagct
tggtagggag ttttaccaag gaggatccga 1320 ctggattcga gagttgaggt
gggccagaga cagcagtatc tgagtcaggt agagaagagc 1380 aatgaggggc
acagagggat gggcaagaga gcacatgtgc ccagttttga aagccaatgg 1440
cttcagcgct cctgaagggg cagacggtgt gaccaaagag ataggcagcg gcagagaggg
1500 agccctagga tgttgagctg gatcctgctg ggcacaggta gccattaagg
gcttgcaagc 1560 tggggggcat gacatggcag acttgcaggt ttttttgttt
gtttttttat tttattttat 1620 ttttttattt tgtttttttt gagacggagt
ctcactctgt cgcccaggct ggagtgcagt 1680 ggcgcgatct cggctcactg
caagctccgc ctcccgggtt cgcgccattc tcctgcctca 1740 gcctcccgag
tagctgggac tacaggcgcc cgccaccgcg cccggctaat tttttgtatt 1800
tttagtagag acggggtttc accgtgttag ccaggatgtt ctcgatctcc tgacctcgtg
1860 atccgcccac ctcggcctcc caaagtgctg ggattacagg tgtgaaccat
cgcgcccagc 1920 cgacttgtag ttttttaaaa ctctgctgga agatgaaggt
tgaagagccg agggagagga 1980 tgtttccaga ggcccatgca agagatggcc
atgacctgcc ttgagaaggg gcaggggaag 2040 ccagatggac tggaagtgga
gtggcagtga ccaaggagga ggaggtgtga taggcttccc 2100 acgcagggta
gatccagaga caccagtgcc acccataggc ccctaggact gcagtggtca 2160
ccgattcctt tgtcccagct gagactcagt tctgagtgtt ctattttggg gaacagaggc
2220 gtccttggta gcatttggaa gaggatagcc agctggggtg tgtgtacatc
acagcctgac 2280 agtaacagca tccgaaccag aggtgactgg ctaagggcag
acccagggca acaggttaac 2340 cgttctaggg ccgggcacag ggaggagaac
attccaacac tctgcgtgcc gacgcacgtt 2400 ctctctttta tcctcaaaac
agtcctatga ggatagtaag ccagagagag acagagacaa 2460 ggaattacaa
gttggtgaga gtcaggattt gaacttggct ctggcagatg gaaaattagg 2520
gtctgtattc tttacaaaac cgtgtgtgcc tcagatggag ttggtgcata acaagcagag
2580 gtatccaggg tcgcggtcct gcttgccacg gaaggggccg ccttgtcagt
tgtgaccacc 2640 cagccctgga aatgtcagta atgctgtaag gagtggggat
cggatcagat gccatccaga 2700 tgctgaagtt tgaccttgtg tcatttttca
ctttcttttt tggctcttct gcaatcaatt 2760 catttattta gcaaaaaaga
aattatgtgt gccgagagca tgcagaagat atgtctccgt 2820 tctctgcttc
cctccaaaaa agaatcccaa aactgctttc tgtgaacgtg tgccagggtc 2880
ccagcaggac tcagggagag caggaagccc agcccagacc ccttgcacaa cctaccgtgg
2940 ggaggcctta ggctctggct actacagagc tggttccagt ctgcactgcc
acagcctggc 3000 cagggacttg gacacatctg ctggccactt cctgtctcag
tttccttatc tgcaaaataa 3060 gggaaaagcc cccacaaagg tgcacgtgta
gcaggagctc ttttccctcc ctattttagg 3120 aaggcagttg gtgggaagtc
cagcttgggt ccctgagagc tgtgagaagg agatgcggct 3180 gctgctggcc
ctgttggggg tcctgctgag tgtgcctggg cctccagtct tgtccctgga 3240
ggcctctgag gaagtggagc ttggtatggc ttctgaggtg ggagagggtg gcaggggtgg
3300 gaagagtggg caccaggagg gggctgctgg gctgagcaaa gctggaaagg
atccttgccc 3360 aggccctgag aaggtggcgg cagggcaggg ctcaaccact
gagactcagt cagtgcctgg 3420 cttccagcaa gcattcatct atcactgtgt
ctgcgagaga ggactggcct tgcagggcgc 3480 agggccctaa gctgggctgc
agagctggtg gtgagctcct tacctgggtg tgtgtgcgtg 3540 tgtgtgtgtg
ttctgtgcac tgggtgtgtg acctaggagg tccaggcagc atgtgtggta 3600
taagcattat gagggtgata tgccccggtg cagcatgacc ctgtatgtgg caccaacagc
3660 atgtgccttg tgtgtgtgtg tgtccgtatg tgtgtgtgtg tatgcgtgtg
tgtgtgtgtg 3720 tgtgtcttgg ccactgtcgt gtgcactaaa tgctgtgtgt
gtgacatgcc ccaagagtgt 3780 ggcatttgcc ctgggtgtgg catccgcagc
atgtggctgt gtgggtgtca aggagtggtg 3840 gctccttcag catgcgttgc
aaagtgcttg tgccctgcat gtgcggtgtg ttctttgtac 3900 acaggaggct
gcctcagatg gggctgcggg gtctgctgac ctctgccctc tgcccacaga 3960
gccctgcctg gctcccagcc tggagcagca agagcaggag ctgacagtag cccttgggca
4020 gcctgtgcgt ctgtgctgtg ggcgggctga gcgtggtggc cactggtaca
aggagggcag 4080 tcgcctggca cctgctggcc gtgtacgggg ctggaggggc
cgcctagaga ttgccagctt 4140 cctacctgag gatgctggcc gctacctctg
cctggcacga ggctccatga tcgtcctgca 4200 gaatctcacc ttgattacag
gtggtaagag actctagcag ggagtgaagg gatgcctggg 4260 gagacagacc
tgcccctctt ggaccttaga tgcttccctc tgtccctgat gtagactcct 4320
tgacctccag caacgatgat gaggacccca agtcccatag ggacccctcg aataggcaca
4380 gttaccccca gcaaggtcag taggtctcca aggacttgtg tccccgctgc
tgctcatctg 4440 atcactgaga agaggaggcc tgtgtgggaa cacacggtca
ttctaggggc cttcccctgc 4500 cctccagcac cctactggac acacccccag
cgcatggaga agaaactgca tgcagtacct 4560 gcggggaaca ccgtcaagtt
ccgctgtcca gctgcaggca accccacgcc caccatccgc 4620 tggcttaagg
atggacaggc ctttcatggg gagaaccgca ttggaggcat tcgggtgagt 4680
ctctgggttc caagaccgtc tgctccccca ttttcattcc ttcatcagtc ccctcatacc
4740 tacaagcata cctataaatc aatcgaatga gtgaagcgat tgcggggccc
cggaaggagc 4800 cctggactgt ggacctgggc agctctggtt ccccttctgc
tactctctgg caagtgactt 4860 aacctctcag cctcagcaac tccatttgta
aagggagaag aatcactgac tggttggtct 4920 gcataagcct tagcatctca
tcgtcttgat gagaccctgc agggtcggct ccatgctgtc 4980 atgaggcaac
tgagtctcag agaaggcaag ggttggctca aagtagcaca gctagggaga 5040
gggagagcta aaattccaaa ggctcaaacc caaggctcaa gcgccctggg gagcctactc
5100 ctttgtgcca tagtccttgg cctgggcctg atgttctcag ggcctagaga
gcttgacaag 5160 agccctgtgg gcaggatgag gatctagcct cctggtcctc
tggccccctt ggtggacatg 5220 gtccggtggt cccggacact ctctctgcct
gcagctgcgc catcagcact ggagtctcgt 5280 gatggagagc gtggtgccct
cggaccgcgg cacatacacc tgcctggtag agaacgctgt 5340 gggcagcatc
cgctataact acctgctaga tgtgctgggt gagcgcgggg ctgggaacag 5400
gggaggcctg acccattttg ggctcagttg tgccctcttg gtggggtcta gtctggcagg
5460 caggatggac tcagatgagt caggcagctt ggtgagcagg tgggtcaggg
gaaagcacag 5520 gggttagtgt ggggctggag gagcagaggt ctgccaagag
gaaaaacaag aaggacatcc 5580 aggcagaggg cgcagcccga gcggagggcc
tgagtataac aaacgccctg cacttgcagg 5640 ccagcatatt cgtagggcgt
ggcgtttata tggggagcca ggtggtggag ggttttgaat 5700 gctaggctga
gatgttgtcc ttgacccgaa gcaataggga gccagggaag gtttaagcag 5760
ggtaagcagg agacagacaa gaagctgcag aaaggtccct cccttgaact tgaggaaggc
5820 tggagggagg caaacagggt gcttctatgg gtgccggtgg tcagggttga
ctgtctcgcc 5880 cggtccccag agcggtcccc gcaccggccc atcctgcagg
ccgggctccc ggccaacacc 5940 acagccgtgg tgggcagcga cgtggagctg
ctgtgcaagg tgtacagcga tgcccagccc 6000 cacatccagt ggctgaagca
catcgtcatc aacggcagca gcttcggagc cgacggtttc 6060 ccctatgtgc
aagtcctaaa ggtaaaaggt gcaccctgct gcagcctggg ccccattctt 6120
ctcccacctt gggttggggg gctccccagc ttccctgttg gccacagtgt ggccccaggc
6180 cctgctgtga ccccagagca tgtcccccac cccagactgc agacatcaat
agctcagagg 6240 tggaggtcct gtacctgcgg aacgtgtcag ccgaggacgc
aggcgagtac acctgcctcg 6300 caggcaattc catcggcctc tcctaccagt
ctgcctggct cacggtgctg ccaggtgagc 6360 acctgaaggg ccaggagatg
ctgcgagatg cccctctggg ccagcagtgg gggctgtggc 6420 ctgttgggtg
gtcagtctct gttggcctgt ggggtctggc ctggggggca gtgtgtggat 6480
ttgtgggttt gagctgtatg acagcccctc tgtgcctctc cacacgtggc cgtccatgtg
6540 accgtctgct gaggtgtggg tgcctgggac tgggcataac tacagcttcc
tccgtgtgtg 6600 tccccacata tgttgggagc tgggagggac tgagttaggg
tgcacggggc ggccagtctc 6660 accactgacc agtttgtctg tctgtgtgtg
tccatgtgcg agggcagagg aggaccccac 6720 atggaccgca gcagcgcccg
aggccaggta tacggacatc atcctgtacg cgtcgggctc 6780 cctggccttg
gctgtgctcc tgctgctggc cgggctgtat cgagggcagg cgctccacgg 6840
ccggcacccc cgcccgcccg ccactgtgca gaagctctcc cgcttccctc tggcccgaca
6900 ggtactgggc gcatccccca cctcacatgt gacagcctga ctccagcagg
cagaaccaag 6960 tctcccactt tgcagttctc cctggagtca ggctcttccg
gcaagtcaag ctcatccctg 7020 gtacgaggcg tgcgtctctc ctccagcggc
cccgccttgc tcgccggcct cgtgagtcta 7080 gatctacctc tcgacccact
atgggagttc ccccgggaca ggtgcgctga gctgtgtggg 7140 ggcagggacg
cgggcgccgg gttgcagccc gccctccgca ggagtgactc ggaggtctga 7200
ggctggactt tctccatctc caggctggtg cttgggaagc ccctaggcga gggctgcttt
7260 ggccaggtag tacgtgcaga ggcctttggc atggaccctg cccggcctga
ccaagccagc 7320 actgtggccg tcaagatgct caaaggtgag tgtggcccgg
tgtggtggct cacacctgta 7380 acgccagcac tttaggaggc tgagggtggg
aggatcgctt gaatccagga attcgaggcc 7440 agcctgggca acatggcaag
acttcatctc tacaaaaaaa aaataagaaa attagttggg 7500 tgtggtggtg
tgtgccttta gtctcagtta ctagggaggc tgaggcagga ggatcccttg 7560
aatccaggag ttggaggttg cagggagcca tgatcacgcc actgtattcc agcctgggca
7620 acacagtgag accctatctg aaaaaataaa taaataaata aaaataaaag
gtgaacgtgg 7680 cagcctggag gaggtgctat ggcattggga ctaatagaag
gggctcacgg tgccaccagg 7740 tgagccctgg agctgggaga ggctgtggga
tcccaccctt aaacctgcaa ttcacctctg 7800 ctcctgaccc tggcaagtga
cttctgagcc tcagttttcc cttgtgtcat atggggtaga 7860 taacagtccc
tactcccagc ccaaggattg tggaaagtgc ctggctcata gtcagggctc 7920
aataaatctt caccactggg gtgatgatga tgagaagaat ttggtgtgac aggcttgata
7980 tcctgtgtca gcattagtct gtgtcagctt tgacttcaca tctccttgtc
agcctcacag 8040 gccctctacc tccttcctta tggttccccc cagacacacc
ctcagcctcc cttggaccct 8100 ccctaggtct gccccccacg tccactgctg
taggaggaca gcccttctgc ttgcacccag 8160 gcccagcccc ggggtgctct
tgctgggcac tcctgcaccc cacccatcag ggcctctcct 8220 tgcagttccc
cagccccctc tgcaagaatg gcctccactg ctcttctgct cctcccctcc 8280
tctctacaca gctggggcca cctggtgctc cctgggaggc agggattgag aaatgcacat
8340 tgtgtcattg gcccagggcc acaggtcagc cccaggggct cagccagaga
agccaaagca 8400 gccttcttcc caagctcccc ggctgcaccc ggcctgccgc
cagctccctg aattcccagg 8460 ccagttggaa gccaggccct ggtcaaacag
accccagggc gccagcctgc tttccgcacc 8520 cagaagctct gaccccatgc
ggggactacc gctgacccct ccagcggcag cttccttcct 8580 tccttcctgc
tccgagctct tcccctctct cctgtgtcct gggcctgccc gctggaaggc 8640
ctgcctctta gatccttgat acagttgcat ccttgcaact gctgtgacag gcagggtgtg
8700 acccactgct ctgtttccca caagacgaac ctgaggttca gagacgctag
gagacttttt 8760 caaggccaca cagcctagca aggattcagc cctagaccta
cgtagccctg gtccagtgct 8820 gcttgtcctg cacctgcctc tgcatgctcc
ctcgtgcagt tggagggcag cctcttcacc 8880 ccgtctgctg cccttacaga
caacgcctct gacaaggacc tggccgacct ggtctcggag 8940 atggaggtga
tgaagctgat cggccgacac aagaacatca tcaacctgct tggtgtctgc 9000
acccaggaag gtggggccga ggcggggctg gctgcacggg ccgttagggt gcagagccaa
9060 agctttggca gcctctccac gctccctcca ctccctctgc agggcccctg
tacgtgatcg 9120 tggagtgcgc cgccaaggga aacctgcggg agttcctgcg
ggcccggcgc cccccaggcc 9180 ccgacctcag ccccgacggt cctcggagca
gtgaggggcc gctctccttc ccagtcctgg 9240 tctcctgcgc ctaccaggtg
gcccgaggca tgcagtatct ggagtcccgg aaggtacagg 9300 cgctagggct
ctgagcccct ctcagtctct ccagctccac tctcaggcct gtggcattca 9360
atgtcccgac ttctccctct ctgctctttt tcatgacccc acctcagtgt ccccaggcat
9420 tcacgctttc ctgcattccc cactcgttcc tcacccttcc ccagagggga
gaggggacgc 9480 aggagaaggc actccccgtt tctaaacctt gacctcctcc
tctgtaaagt gggtggaggg 9540 cccctgcccc cgggcctgct ggggggtggt
gtgtgctcaa ctccaggcca ggtgtcctga 9600 ggcacccaag cccccgctcc
ctgcagtgta tccaccggga cctggctgcc cgcaatgtgc 9660 tggtgactga
ggacaatgtg atgaagattg ctgactttgg gctggcccgc ggcgtccacc 9720
acattgacta ctataagaaa accagcaacg tgagggagat ggggcagaac tggatggggg
9780 tggaggggca ctgggcccgg ggtggcaggc acgaggacct gtgggactct
gcactgaggc 9840 cctctctccc ctccagggcc gcctgcctgt gaagtggatg
gcgcccgagg ccttgtttga 9900 ccgggtgtac acacaccaga gtgacgtgtg
agtcctgccg gcggtcactg tcctacccca 9960 caaaaagggc aaggcactgc
ccaaagtcac gtggccccag gagtcatgcg ctcgagggct 10020 ccttcagatt
tggtctggga cccgagtggg cccagactcc aggaggagcc cattccccaa 10080
cagctgtggt gggtcatgtc tgtggggtcc cccgtcctag ccccggtcgt cgggagggcg
10140 ctgagccaca ctgagccctg gccctacctc caggtggtct tttgggatcc
tgctatggga 10200 gatcttcacc ctcgggggct ccccgtatcc tggcatcccg
gtggaggagc tgttctcgct 10260 gctgcgggag ggacatcgga tggaccgacc
cccacactgc cccccagagc tgtgaggcct 10320 caccctgccc tcgaccccac
tttccagtcc tcctcctcct ctgccctgac catggcctca 10380 gggtgtgtcc
cggccagaag gacaacacta acaacaactc ctcgtcctcc tcctcctctt 10440
cctcttcctc ctcctcctct tcctcctcct cctcttcctc ctcctcttcc tcctcctcct
10500 cttcctcctc ctcctcttcc tccttctcct cctgctcctc ttcctcctcc
ttctcttcct 10560 cctcctcctc ttcctcctcc tcctcttcct cctcctcctc
ttcctccttc tcctcctgct 10620 cctcttcctc ctccttctct tcctcctcct
tctcttcctc ctcctcctcc tgctcctctt 10680 cctcctcctc ctcttcctcc
tcctcagcct agtggagtgt cctggcctgg cttctactga 10740 tgaccctcct
atccctcatc aaactcccca ccaaactcct ccccacccag agaacccccg 10800
gtcctcccct tcctcctgaa ggcctgaggc tccctgtgac cctccgcccc acctctcgca
10860 ggtacgggct gatgcgtgag tgctggcacg cagcgccctc ccagaggcct
accttcaagc 10920 agctggtgga ggcgctggac aaggtcctgc tggccgtctc
tgaggaggta cagcccctcc 10980 cacccaccac ctccctctgc ctgctcccct
ccaggcctca tctggcctga ccgcgtggac 11040 atgcgccccg tcccatcccg
ggcgctgcag aggctgacca gctccgttcc ccacagtacc 11100 tcgacctccg
cctgaccttc ggaccctatt ccccctctgg tggggacgcc agcagcacct 11160
gctcctccag cgattctgtc ttcagccacg accccctgcc attgggatcc agctccttcc
11220 ccttcgggtc tggggtgcag acatgagcaa ggctcaaggc tgtgcaggca
cataggctgg 11280 tggccttggg ccttggggct cagccacagc ctgacacagt
gctcgacctt gatagcatgg 11340 ggcccctggc ccagagttgc tgtgccgtgt
ccaagggccg tgcccttgcc cttggagctg 11400 ccgtgcctgt gtcctgatgg
cccaaatgtc agggttctgc tcggcttctt ggaccttggc 11460 gcttagtccc
catcccgggt ttggctgagc ctggctggag agctgctatg ctaaacctcc 11520
tgcctcccaa taccagcagg aggttctggg cctctgaacc ccctttcccc acacctcccc
11580 ctgctgctgc tgccccagcg tcttgacggg agcattggcc cctgagccca
gagaagctgg 11640 aagcctgccg aaaacaggag caaatggcgt tttataaatt
atttttttga aataaagctc 11700 tgtgtgcctg ggtcttccct gagcaacatg
gagtggggtg aggtggaggg atccctccag 11760 cagagttctg cctacaggac
acggactgag ggcactggac caggccatgg gctccgccac 11820 ctccactgcc
ccaggagcca gtgtgtgcct atctgggtcc gcctgtccca ccagccccat 11880
cttgtgtctg cgacagtgtg aatgagtatt aatgggctga gtccgcattg cactatacac
11940 ggtgggactc ctgtaccctc tgcacatgtg tgtgtgtgca tgtgtgccct
gcagctgtcc 12000 ccaagggagc tggcagcccc cctcccccat ctgctcagca
ttaaccaagc tgaccgttaa 12060 cacagcatga aaatctgaga gccagcctta
ggccgcggcc cgctcccacg ctctgccggc 12120 tcaggctggg ggcttgtgga
ggccatgccc gccccgccct ggccagtctc ccgggcagca 12180 gctggttgcc gcccgc
12196 <210> SEQ ID NO 3 <211> LENGTH: 5192 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
3 ccatgacctg ccttgagaag gggcagggga agccagatgg actggaagtg gagtggcagt
60 gaccaaggag gaggaggtgt gataggcttc ccacgcaggg tagatccaga
gacaccagtg 120 ccacccatag gcccctagga ctgcagtggt cacccgattc
ctttgtccca gctgagactc 180 agttctgagt gttctatttt ggggaacaga
ggcgtccttg gtagcatttg gaagaggata 240 gccagctggg gtgtgtgtac
atcacagcct gacagtaaca gcatccgaac cagaggtgac 300 tggctaaggg
cagacccagg gcaacaggtt aaccgttcta gggccgggca cagggaggag 360
aacattccaa cactctgtgt gcccagtgcc gacgcacgtt ctctctttta tcctcaaaac
420 agtcctatga ggatataagc cagagagaga cagagacaag gaattacaag
ttggtgagag 480 tcaggatttg aacttggctc tggcagatgg aaaattaggg
tctgtattct ttacaaaacc 540 gtgtgtgcct cagatggagt tggtgcataa
caagcagagg tatccagggt cgcggtcctg 600 cttgccacgg aaggggccgc
cttgtcagtt gtgaccaccc agccctggaa atgtcagtaa 660 tgctgtaagg
agtggggatc ggatcagatg ccatccagat gctgaagttt gaccttgtgt 720
catttttcac tttctttttt ggctcttctg caatcaattc atttatttag caaaaaagaa
780 attatgtgtg ccgagagcat gcagaagata tgtctccgtt ctctgcttcc
ctccaaaaaa 840 gaatcccaaa actgctttct gtgaacgtgt gccagggtcc
cagcaggact cagggagagc 900 aggaagccca gcccagaccc cttgcacaac
ctaccgtggg gaggccttag gctctggcta 960 ctacagagct ggttccagtc
tgcactgcca cagcctggcc agggacttgg acacatctgc 1020 tggccacttc
ctgtctcagt ttccttatct gcaaaataag ggaaaagccc ccacaaaggt 1080
gcacgtgtag caggagctct tttccctccc tattttagga aggcagttgg tgggaagtcc
1140 agcttgggtc cctgagagct gtgagaagga gatgcggctg ctgctggccc
tgttgggggt 1200 cctgctgagt gtgcctgggc ctccagtctt gtccctggag
gcctctgagg aagtggagct 1260 tggtatggct tctgaggtgg gagagggtgg
caggggtggg aagagtgggc accaggaggg 1320 ggctgctggg ctgagcaaag
ctggaaagga tccttgccca ggccctgaga aggtggcggc 1380 agggcagggc
tcaaccactg agactcagtc agtgcctggc ttccagcaag cattcatcta 1440
tcactgtgtc tgcgagagag gactggcctt gcagggcgca gggccctaag ctgggctgca
1500 gagctggtgg tgagctcctt gcctgggtgt gtgtgcgtgt gtgtgtgtgt
tctgtgcact 1560 gggtgtgtga cctaggaggt ccaggcagca tgtgtggtat
aagcattatg agggtgatat 1620 gccccggtgc agcatgaccc tgtatgtggc
accaacagca tgtgccttgt gtgtgtgtgt 1680 gtccgtatgt gtgtgtgtgt
atgcgtgtgt gtgtgtgtgt gtgtgtgtct tggccactgt 1740 catgtgcact
aaatgctgtg tgtgtgacat gccccaagag tgtggcattt gccctgggtg 1800
tggcatccgc agcatgtggc tgtgtgggtg tcaaggagtg gtggctcctt cagcatgcgt
1860 tgcgaagtgc ttgtgccctg catgtgcggt gtgttctctg tacacaggag
gctgcctcag 1920 atggggctgc ggggtctgct gacctctgcc ctctgcccac
agagccctgc ctggctccca 1980 gcctggagca gcaagagcag gagctgacag
tagcccttgg gcagcctgtg cggctgtgct 2040 gtgggcgggc tgagcgtggt
ggccactggt acaaggaggg cagtcgcctg gcacctgctg 2100 gccgtgtacg
gggctggagg ggccgcctag agattgccag cttcctacct gaggatgctg 2160
gccgctacct ctgcctggca cgaggctcca tgatcgtcct gcagaatctc accttgatta
2220 caggtgactc cttgacctcc agcaacgatg atgaggaccc caagtcccat
agggacctct 2280 cgaataggca cagttacccc cagcaaggtc agtaggtctc
caaggacttg tgtccccgct 2340 gctgctcatc tgatcactga gaagaggagg
cctgtgtggg aacacacggt cattctaggg 2400 gccttcccct gccctccagc
accctactgg acacaccccc agcgcatgga gaagaaactg 2460 catgcagtac
ctgcggggaa caccgtcaag ttccgctgtc cagctgcagg caaccccacg 2520
cccaccatcc gctggcttaa ggatggacag gcctttcatg gggagaaccg cattggaggc
2580 attcggctgc gccatcagca ctggagtctc gtgatggaga gcgtggtgcc
ctcggaccgc 2640 ggcacataca cctgcctggt agagaacgct gtgggcagca
tccgttataa ctacctgcta 2700 gatgtgctgg agcggtcccc gcaccggccc
atcctgcagg ccgggctccc ggccaacacc 2760 acagccgtgg tgggcagcga
cgtggagctg ctgtgcaagg tgtacagcga tgcccagccc 2820 cacatccagt
ggctgaagca catcgtcatc aacggcagca gcttcggagc cgacggtttc 2880
ccctatgtgc aagtcctaaa gactgcagac atcaatagct cagaggtgga ggtcctgtac
2940 ctgcggaacg tgtcagccga ggacgcaggc gagtacacct gcctcgcagg
caattccatc 3000 ggcctctcct accagtctgc ctggctcacg gtgctgccag
gtgagcacct gaagggccag 3060 gagatgctgc gagatgcccc tctgggccag
cagtgggggc tgtggcctgt tgggtggtca 3120 gtctctgttg gcctgtgggg
tctggcctgg ggggcagtgt gtggatttgt gggtttgagc 3180 tgtatgacag
cccctctgtg cctctccaca cgtggccgtc catgtgaccg tctgctgagg 3240
tgtgggtgcc tgggactggg cataactaca gcttcctccg tgtgtgtccc cacatatgtt
3300 gggagctggg agggactgag ttagggtgca cggggcggcc agtctcacca
ctgaccagtt 3360 tgtctgtctg tgtgtgtcca tgtgcgaggg cagaggagga
ccccacatgg accgcagcag 3420 cgcccgaggc caggtatacg gacatcatcc
tgtacgcgtc gggctccctg gccttggctg 3480 tgctcctgct gctggccagg
ctgtatcgag ggcaggcgct ccacggccgg cacccccgcc 3540 cgcccgccac
tgtgcagaag ctctcccgct tccctctggc ccgacagttc tccctggagt 3600
caggctcttc cggcaagtca agctcatccc tggtacgagg cgtgcgtctc tcctccagcg
3660 gccccgcctt gctcgccggc ctcgtgagtc tagatctacc tctcgaccca
ctatgggagt 3720 tcccccggga caggctggtg cttgggaagc ccctaggcga
gggctgcttt ggccaggtag 3780 tacgtgcaga ggcctttggc atggaccctg
cccggcctga ccaagccagc actgtggccg 3840 tcaagatgct caaagacaac
gcctctgaca aggacctggc cgacctggtc tcggagatgg 3900 aggtgatgaa
gctgatcggc cgacacaaga acatcatcaa cctgcttggt gtctgcaccc 3960
aggaagggcc cctgtacgtg atcgtggagt gcgccgccaa gggaaacctg cgggagttcc
4020 tgcgggcccg gcgcccccca ggccccgacc tcagccccga cggtcctcgg
agcagtgagg 4080 ggccgctctc cttcccagtc ctggtctcct gcgcctacca
ggtggcccga ggcatgcagt 4140 atctggagtc ccggaagtgt atccaccggg
acctggctgc ccgcaatgtg ctggtgactg 4200 aggacaatgt gatgaagatt
gctgactttg ggctggcccg cggcgtccac cacattgact 4260 actataagaa
aaccagcaac ggccgcctgc ctgtgaagtg gatggcgccc gaggccttgt 4320
ttgaccgggt gtacacacac cagagtgacg tgtggtcttt tgggatcctg ctatgggaga
4380 tcttcaccct cgggggctcc ccgtatcctg gcatcccggt ggaggagctg
ttctcgctgc 4440 tgcgggaggg acatcggatg gaccgacccc cacactgccc
cccagagctg tacgggctga 4500 tgcgtgagtg ctggcacgca gcgccctccc
agaggcctac cttcaagcag ctggtggagg 4560 cgctggacaa ggtcctgctg
gccgtctctg aggagtacct cgacctccgc ctgaccttcg 4620 gaccctattc
cccctctggt ggggacgcca gcagcacctg ctcctccagc gattctgtct 4680
tcagccacga ccccctgcca ttgggatcca gctccttccc cttcgggtct ggggtgcaga
4740 catgagcaag gctcaaggct gtgcaggcac ataggctggt ggccttgggc
cttggggctc 4800 agccacagcc tgacacagtg ctcgaccttg atagcatggg
gcccctggcc cagagttgct 4860 gtgccgtgtc caagggccgt gcccttgccc
ttggagctgc cgtgcctgtg tcctgatggc 4920 ccaaatgtca gggttctgct
cggcttcttg gaccttggcg cttagtcccc atcccgggtt 4980 tggctgagcc
tggctggaga gctgctatgc taaacctcct gcctcccaat accagcagga 5040
ggttctgggc ctctgaaccc cctttcccca cacctccccc tgctgctgct gccccagcgt
5100 cttgacggga gcattggccc ctgagcccag agaagctgga agcctgccga
aaacaggagc 5160 aaatggcgtt ttataaatta tttttttgaa at 5192
<210> SEQ ID NO 4 <211> LENGTH: 2807 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (54)..(2342)
<400> SEQUENCE: 4 gaaggcagtt ggtgggaagt ccagcttggg tccctgagag
ctgtgagaag gag atg 56 Met 1 cgg ctg ctg ctg gcc ctg ttg ggg gtc ctg
ctg agt gtg cct ggg cct 104 Arg Leu Leu Leu Ala Leu Leu Gly Val Leu
Leu Ser Val Pro Gly Pro 5 10 15 cca gtc ttg tcc ctg gag gcc tct gag
gaa gtg gag ctt gag ccc tgc 152 Pro Val Leu Ser Leu Glu Ala Ser Glu
Glu Val Glu Leu Glu Pro Cys 20 25 30 ctg gct ccc agc ctg gag cag
caa gag cag gag ctg aca gta gcc ctt 200 Leu Ala Pro Ser Leu Glu Gln
Gln Glu Gln Glu Leu Thr Val Ala Leu 35 40 45 ggg cag cct gtg cgt
ctg tgc tgt ggg cgg gct gag cgt ggt ggc cac 248 Gly Gln Pro Val Arg
Leu Cys Cys Gly Arg Ala Glu Arg Gly Gly His 50 55 60 65 tgg tac aag
gag ggc agt cgc ctg gca cct gct ggc cgt gta cgg ggc 296 Trp Tyr Lys
Glu Gly Ser Arg Leu Ala Pro Ala Gly Arg Val Arg Gly 70 75 80 tgg
agg ggc cgc cta gag att gcc agc ttc cta cct gag gat gct ggc 344 Trp
Arg Gly Arg Leu Glu Ile Ala Ser Phe Leu Pro Glu Asp Ala Gly 85 90
95 cgc tac ctc tgc ctg gca cga ggc tcc atg atc gtc ctg cag aat ctc
392 Arg Tyr Leu Cys Leu Ala Arg Gly Ser Met Ile Val Leu Gln Asn Leu
100 105 110 acc ttg att aca ggt gac tcc ttg acc tcc agc aac gat gat
gag gac 440 Thr Leu Ile Thr Gly Asp Ser Leu Thr Ser Ser Asn Asp Asp
Glu Asp 115 120 125 ccc aag tcc cat agg gac ccc tcg aat agg cac agt
tac ccc cag caa 488 Pro Lys Ser His Arg Asp Pro Ser Asn Arg His Ser
Tyr Pro Gln Gln 130 135 140 145 gca ccc tac tgg aca cac ccc cag cgc
atg gag aag aaa ctg cat gca 536 Ala Pro Tyr Trp Thr His Pro Gln Arg
Met Glu Lys Lys Leu His Ala 150 155 160 gta cct gcg ggg aac acc gtc
aag ttc cgc tgt cca gct gca ggc aac 584 Val Pro Ala Gly Asn Thr Val
Lys Phe Arg Cys Pro Ala Ala Gly Asn 165 170 175 ccc acg ccc acc atc
cgc tgg ctt aag gat gga cag gcc ttt cat ggg 632 Pro Thr Pro Thr Ile
Arg Trp Leu Lys Asp Gly Gln Ala Phe His Gly 180 185 190 gag aac cgc
att gga ggc att cgg ctg cgc cat cag cac tgg agt ctc 680 Glu Asn Arg
Ile Gly Gly Ile Arg Leu Arg His Gln His Trp Ser Leu 195 200 205 gtg
atg gag agc gtg gtg ccc tcg gac cgc ggc aca tac acc tgc ctg 728 Val
Met Glu Ser Val Val Pro Ser Asp Arg Gly Thr Tyr Thr Cys Leu 210 215
220 225 gta gag aac gct gtg ggc agc atc cgc tat aac tac ctg cta gat
gtg 776 Val Glu Asn Ala Val Gly Ser Ile Arg Tyr Asn Tyr Leu Leu Asp
Val 230 235 240 ctg gag cgg tcc ccg cac cgg ccc atc ctg cag gcc ggg
ctc ccg gcc 824 Leu Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly
Leu Pro Ala 245 250 255 aac acc aca gcc gtg gtg ggc agc gac gtg gag
ctg ctg tgc aag gtg 872 Asn Thr Thr Ala Val Val Gly Ser Asp Val Glu
Leu Leu Cys Lys Val 260 265 270 tac agc gat gcc cag ccc cac atc cag
tgg ctg aag cac atc gtc atc 920 Tyr Ser Asp Ala Gln Pro His Ile Gln
Trp Leu Lys His Ile Val Ile 275 280 285 aac ggc agc agc ttc gga gcc
gac ggt ttc ccc tat gtg caa gtc cta 968 Asn Gly Ser Ser Phe Gly Ala
Asp Gly Phe Pro Tyr Val Gln Val Leu 290 295 300 305 aag act gca gac
atc aat agc tca gag gtg gag gtc ctg tac ctg cgg 1016 Lys Thr Ala
Asp Ile Asn Ser Ser Glu Val Glu Val Leu Tyr Leu Arg 310 315 320 aac
gtg tca gcc gag gac gca ggc gag tac acc tgc ctc gca ggc aat 1064
Asn Val Ser Ala Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly Asn 325
330 335 tcc atc ggc ctc tcc tac cag tct gcc tgg ctc acg gtg ctg cca
ggt 1112 Ser Ile Gly Leu Ser Tyr Gln Ser Ala Trp Leu Thr Val Leu
Pro Gly 340 345 350 act ggg cgc atc ccc cac ctc aca tgt gac agc ctg
act cca gca ggc 1160 Thr Gly Arg Ile Pro His Leu Thr Cys Asp Ser
Leu Thr Pro Ala Gly 355 360 365 aga acc aag tct ccc act ttg cag ttc
tcc ctg gag tca ggc tct tcc 1208 Arg Thr Lys Ser Pro Thr Leu Gln
Phe Ser Leu Glu Ser Gly Ser Ser 370 375 380 385 ggc aag tca agc tca
tcc ctg gta cga ggc gtg cgt ctc tcc tcc agc 1256 Gly Lys Ser Ser
Ser Ser Leu Val Arg Gly Val Arg Leu Ser Ser Ser 390 395 400 ggc ccc
gcc ttg ctc gcc ggc ctc gtg agt cta gat cta cct ctc gac 1304 Gly
Pro Ala Leu Leu Ala Gly Leu Val Ser Leu Asp Leu Pro Leu Asp 405 410
415 cca cta tgg gag ttc ccc cgg gac agg ctg gtg ctt ggg aag ccc cta
1352 Pro Leu Trp Glu Phe Pro Arg Asp Arg Leu Val Leu Gly Lys Pro
Leu 420 425 430 ggc gag ggc tgc ttt ggc cag gta gta cgt gca gag gcc
ttt ggc atg 1400 Gly Glu Gly Cys Phe Gly Gln Val Val Arg Ala Glu
Ala Phe Gly Met 435 440 445 gac cct gcc cgg cct gac caa gcc agc act
gtg gcc gtc aag atg ctc 1448 Asp Pro Ala Arg Pro Asp Gln Ala Ser
Thr Val Ala Val Lys Met Leu 450 455 460 465 aaa gac aac gcc tct gac
aag gac ctg gcc gac ctg gtc tcg gag atg 1496 Lys Asp Asn Ala Ser
Asp Lys Asp Leu Ala Asp Leu Val Ser Glu Met 470 475 480 gag gtg atg
aag ctg atc ggc cga cac aag aac atc atc aac ctg ctt 1544 Glu Val
Met Lys Leu Ile Gly Arg His Lys Asn Ile Ile Asn Leu Leu 485 490 495
ggt gtc tgc acc cag gaa ggg ccc ctg tac gtg atc gtg gag tgc gcc
1592 Gly Val Cys Thr Gln Glu Gly Pro Leu Tyr Val Ile Val Glu Cys
Ala 500 505 510 gcc aag gga aac ctg cgg gag ttc ctg cgg gcc cgg cgc
ccc cca ggc 1640 Ala Lys Gly Asn Leu Arg Glu Phe Leu Arg Ala Arg
Arg Pro Pro Gly 515 520 525 ccc gac ctc agc ccc gac ggt cct cgg agc
agt gag ggg ccg ctc tcc 1688 Pro Asp Leu Ser Pro Asp Gly Pro Arg
Ser Ser Glu Gly Pro Leu Ser 530 535 540 545 ttc cca gtc ctg gtc tcc
tgc gcc tac cag gtg gcc cga ggc atg cag 1736 Phe Pro Val Leu Val
Ser Cys Ala Tyr Gln Val Ala Arg Gly Met Gln 550 555 560 tat ctg gag
tcc cgg aag tgt atc cac cgg gac ctg gct gcc cgc aat 1784 Tyr Leu
Glu Ser Arg Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn 565 570 575
gtg ctg gtg act gag gac aat gtg atg aag att gct gac ttt ggg ctg
1832 Val Leu Val Thr Glu Asp Asn Val Met Lys Ile Ala Asp Phe Gly
Leu 580 585 590 gcc cgc ggc gtc cac cac att gac tac tat aag aaa acc
agc aac ggc 1880 Ala Arg Gly Val His His Ile Asp Tyr Tyr Lys Lys
Thr Ser Asn Gly 595 600 605 cgc ctg cct gtg aag tgg atg gcg ccc gag
gcc ttg ttt gac cgg gtg 1928 Arg Leu Pro Val Lys Trp Met Ala Pro
Glu Ala Leu Phe Asp Arg Val 610 615 620 625 tac aca cac cag agt gac
gtg tgg tct ttt ggg atc ctg cta tgg gag 1976 Tyr Thr His Gln Ser
Asp Val Trp Ser Phe Gly Ile Leu Leu Trp Glu 630 635 640 atc ttc acc
ctc ggg ggc tcc ccg tat cct ggc atc ccg gtg gag gag 2024 Ile Phe
Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val Glu Glu 645 650 655
ctg ttc tcg ctg ctg cgg gag gga cat cgg atg gac cga ccc cca cac
2072 Leu Phe Ser Leu Leu Arg Glu Gly His Arg Met Asp Arg Pro Pro
His 660 665 670 tgc ccc cca gag ctg tac ggg ctg atg cgt gag tgc tgg
cac gca gcg 2120 Cys Pro Pro Glu Leu Tyr Gly Leu Met Arg Glu Cys
Trp His Ala Ala 675 680 685 ccc tcc cag agg cct acc ttc aag cag ctg
gtg gag gcg ctg gac aag 2168 Pro Ser Gln Arg Pro Thr Phe Lys Gln
Leu Val Glu Ala Leu Asp Lys 690 695 700 705 gtc ctg ctg gcc gtc tct
gag gag tac ctc gac ctc cgc ctg acc ttc 2216 Val Leu Leu Ala Val
Ser Glu Glu Tyr Leu Asp Leu Arg Leu Thr Phe 710 715 720 gga ccc tat
tcc ccc tct ggt ggg gac gcc agc agc acc tgc tcc tcc 2264 Gly Pro
Tyr Ser Pro Ser Gly Gly Asp Ala Ser Ser Thr Cys Ser Ser 725 730 735
agc gat tct gtc ttc agc cac gac ccc ctg cca ttg gga tcc agc tcc
2312 Ser Asp Ser Val Phe Ser His Asp Pro Leu Pro Leu Gly Ser Ser
Ser 740 745 750 ttc ccc ttc ggg tct ggg gtg cag aca tga gcaaggctca
aggctgtgca 2362 Phe Pro Phe Gly Ser Gly Val Gln Thr 755 760
ggcacatagg ctggtggcct tgggccttgg ggctcagcca cagcctgaca cagtgctcga
2422 ccttgatagc atggggcccc tggcccagag ttgctgtgcc gtgtccaagg
gccgtgccct 2482 tgcccttgga gctgccgtgc ctgtgtcctg atggcccaaa
tgtcagggtt ctgctcggct 2542 tcttggacct tggcgcttag tccccatccc
gggtttggct gagcctggct ggagagctgc 2602 tatgctaaac ctcctgcctc
ccaataccag caggaggttc tgggcctctg aacccccttt 2662 ccccacacct
ccccctgctg ctgctgcccc agcgtcttga cgggagcatt ggcccctgag 2722
cccagagaag ctggaagcct gccgaaaaca ggagcaaatg gcgttttata aattattttt
2782 ttgaaataaa aaaaaaaaaa aaaaa 2807 <210> SEQ ID NO 5
<211> LENGTH: 15001 <212> TYPE: DNA <213>
ORGANISM: Macaca mulatta <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(15001) <223> OTHER
INFORMATION: n = A,T,C or G <400> SEQUENCE: 5 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 300 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 420
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 660 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 720 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1020 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1080 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1140
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
1200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 1260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 1320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1380 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1440 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
1560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 1620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 1680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1740 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1800 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1860
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
1920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 1980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 2040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2100 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2160 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2220
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
2280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 2340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 2400 nggcgggggc aggctggccc tgtgcagcta
ccctcgggac ccattgattc gcacctcccc 2460 ccaggctggc ccggcaaggg
tgggggagga caagcttgtc cctgcggctg tcttcgcgcc 2520 ggcggcagag
atgagggagc tgaggccccg aaagtttcag tcacttagcg cccgggggcc 2580
tccaccgcga gtgcgggagg ctgtctgatg cctaaggcag gccctctgtt cccacgtgtg
2640 gggtggtcgg tcagcggtca gcagccacgg gtgacttttc cacggactct
gatatcaggg 2700 cagcctgggg taggaataaa atccccgggc ctccccagcc
actcccagcc caagctgtgt 2760 actcaaagag ctgccctccc tgccaagccg
agcttggtag ggggttttac caagggggat 2820 ccaactggat ttgagagttg
aagtggcctc agagacagca gtatctgagt caagtagaga 2880 agaggaatgg
ggggcacaga gggatgggca agagagcata tgtgaccagt tttgagagcc 2940
aatggcttca gcgttcctga agggacagac ggtgtgacca aagagatagg cagcggaaca
3000 gagggagccc taggatgctg agctgaatcc tgctgggcac aggtaaccat
taagggcttg 3060 caagctgggg gcatgacatg gcagacttgt agtcttttaa
aactctgctg gaagatgaag 3120 gttgaagagc ccagggagag gatgtttcca
aaggcccatg caagagatgg ccatgacctg 3180 ccctgagtca gggcagggga
agccagatgg actggaagtg gagtggcagt gaccaagggt 3240 aaggaggtgt
gataggcttc ccacacaggg tagatccaga gacaccagtg ccacccatag 3300
gcccctagga ctgcagtgat cacagtattc ctttgtccca gctgagactc ggttctgagt
3360 gttctgtttt ggggaacaga ggtgtccttg gtagcatttg gaagaggata
gccagctggg 3420 gtgtgtgtac atcacagcct gacagtaaca gcacccgaac
cagaggtgac tggctgaggg 3480 cagacccagg gcaacaggtg aacagttcta
gggccgggca cagggagaag aacattccaa 3540 cactctgtgt gcccagtgcc
gatgcacgtt gtctctttta tcctcaaaac agtcctatga 3600 ggatagtaac
ccagagagag acagagacaa ggaattacaa gttgatgaga gtcgggattt 3660
gaacttggct ctggtggatg gataattacg gtctgtattc tttacaaaac cgtgtgtacc
3720 tcggatggag ttggtgcatg acaagcagag gtattcaggg atgcggtcct
gcttgccacg 3780 gaaggagctg ccttgtcagt ggtgggcgcc cagctctgga
aatgtcagta atgctataag 3840 gagtggggat tggatcagat aacatccaga
tgctgaagtt tggccttgta tcatttttca 3900 ctttgttttt tggctcttcg
gcaatcaatt catttattca gcaaaaaata aattacgtgt 3960 gccaagagca
tgcagaagat agtctccgtt ctctgcttcc ctccaagaaa agaatcccaa 4020
aactgctttc tgtgaacgtg tgccagggtc ccagcaggac tcaaggagag caggaagccc
4080 agcccagacc ccttgcacaa cctactgtag gaaggcgtca ggctctggct
acagcagagc 4140 tggttccagt ctgcactgcc acagcctggc cagggacttg
gacacatcta ctggccactt 4200 cctgtctccg tttcctcatc tgcaaaataa
gggaaagccc ccgcaaaggt gcatgtggag 4260 caggagctct tttccccccc
tattctagga aggcagttgg tgggaagtcc agcctgggcc 4320 cctgagagct
gcgggaagga gatgcggctg ctgtcggccc tcttgggggt cctgctgagt 4380
gtgcctgggc ctccagtctt gtccctggag gcctcggagg aagtggagct gggtatggct
4440 tctgaggggg gagagggtgg caggggtggg aagagtgggc accaggaggg
ggctgctggg 4500 ctgagcaaag ctggaaagga tccttgccca ggccctgaga
aagtggcagc agggcagggc 4560 tcaaccactg agactcagtc agtgcctggc
ttccagcaag catccatgta tctctgtgtc 4620 tgcgagagag gactggcctc
gcagggtgca gggccctaag ctgggccgca gagctggtgg 4680 tgagctcctt
gcctgggtgt gtgcgcgcgc gtgtgtgtgt gtgtgttctg tgcactggat 4740
gtgtgaccta ggaggtccag gcagcatgtg tggtgtatgc attatgaggg tgatatgccc
4800 cagtgcagca tgaccctgta tatggcaccg atagcatgtg ccgtgtgtgt
gtgtgtccat 4860 gtgtgtgtgt gtgtgtgtct tggccagtgt catgtgcact
aaatgctgtg tgcgtgacat 4920 gccccaagag tgtggcattt gccctgggtg
tggcatccgc agcatgtggc tgtgcgggtg 4980 tcaaggagcg gtggctcctc
cagcatgcgt cgcgaggtgc ttgtgccctg catgtgtggc 5040 gtgttctctg
tacgcaggag gctgcctcag atggggctgc ggggtctgct gacctctgcc 5100
ctctgcccac agagccctgc ctggctccca gcatggagca gcaagagcag gagctgacag
5160 tagcccttgg gcagcctgtg cggctgtgct gtgggcgggc tgagcgtggt
ggccactggt 5220 acaaggaggg cagtcgcctg gcacctgctg gccgtgtacg
gggctggagg ggccgcctag 5280 agattgccag cttcctacct gaggatgctg
gccgctatct ctgcctggcc cgagcctcca 5340 tgatcgtcct gcaaaatctc
accttgacta tagatggtaa gacactctag cagttaaggg 5400 atgcctgggg
agagagacct gcccctcccg gacctcagat gcctccctct gtccttgatg 5460
tagactcctt gacctccagc aacgatgatg aggaccccca gtcccatagg gactcctcga
5520 atgggcacat ttacccccag caaggtgagt ataagtcttc aaggacttgt
gtccccgctg 5580 ctcatttaat cactgagaag aagaggtctg tgtgggaaca
cacggtcatt ctaagggcct 5640 tcctctcccc tccagcaccc tactggacac
acccccagcg catggagaag aaactgcatg 5700 cagtaccggc tgggaacacc
gtcaagttcc gctgtccggc tgcaggcaac cccacgccca 5760 ccatccgctg
gcttaaggat ggacaggcct ttcatgggga gaaccgcatt ggaggcattc 5820
gggtgagtct ctgggttcca agaccgtctg ctgcctcatt ctcattcctt cctcagtccc
5880 ctcatgccta caagcacacc tatacatcca ttgaatgagg aaagcaattg
cagggtccca 5940 gaaggagccc tggactgtgg acctgggcag ctctggttcc
tcttctgcta ctctctggca 6000 agtgacttaa cctcacagcc ttagcaactc
catttgtaaa gtgagaagaa tcacggactg 6060 gttggtctca gtaagcctta
gcatctcatc atcttgatga gaccctgcag agtcggctcc 6120 atgctgtcat
aaggcaactg agtctcagag aaggcaaggg ctggctcaga gtggcacagc 6180
cagggagagg gagagctaaa attcaaaagg ctcaaaccca aggcttaagc actctgggga
6240 gccttctcct ttgtcccata gtccttggcc tggccctgat gttctcgggg
cctagagagc 6300 ttgagaagag tcctgtgggc aggatgagga tctagccccc
tggtcctctg gcccccttgg 6360 tggacatggt ctggtggtcc tggacactcc
ctctgcctgc agctgcgcca ccagcactgg 6420 agtctcgtga tggagagcgt
ggtgccctcg gaccgcggca catacacttg cctggtggag 6480 aacgctgtgg
gcatcatccg ctataactac ctgctggatg tgctgggtga gtgcggggcc 6540
ggaaacgggg gaggcctgac ccatcctggg ctcagtcgtg ccctccggtg gggtctagtc
6600 tggcgggcag gatggactca gatgagtcaa gcagctcggt gaccaggcgg
tcaggggaaa 6660 gcacaggggt tagtgtgggg ctggaggagc agggctctgc
caagaggaaa aacaagaagg 6720 acagccaggc aaagggcgca gcctgagctg
caggcctgag tataacgaat gccgtgcact 6780 tgcaggccag cgtattcgga
gatgtggctt ttatatgggg agccaggtgg tggagggttt 6840 tgagtgctag
gctgaaacgt ccttgacccg aagcaatgag gagccaggga aggttgaaac 6900
agggtaagca ggagacggac aagaagctgc agaaaggtcc ttcccttgaa cctgaggaag
6960 gctggaggga ggcaaacagg tgcttctatg ggtgccagtg atgagggttg
actaccttgc 7020 ctggtcccca gagcggtccc cgcaccggcc catcctgcag
gctgggctcc cggccaacac 7080 cacagccgtg gtgggcagtg acgtggagct
gctgtgcaag gtgtacagcg atgcccagcc 7140 ccacatccag tggctgaagc
acatcgtcat caacggcagc agcttcgggg ccgacggctt 7200 cccctatgtg
caagtcctga aggtgcaccg tgctccagcc tgggccccac tcttctccca 7260
cccaggattg ggggctccct agcttccctg ttgatcacag tgtggcccca ggccctgctg
7320 tgaccccaga gtatgtcccc ctccccagac tgcagacatc aatagctcag
aggtggaggt 7380 cctgtacctg cggaacgtgt cagccgagga cgcaggcgag
tacacctgcc ttgcaggcaa 7440 ttccatcggc ctctcctacc agtctgcctg
gctcacggtg ctgccaggta agcacctgca 7500 gggccagaat atgctgcgag
atgcccctct gggccagcag tgggggctgt ggtctgttgg 7560 gtggtcagtc
tctgttggct tgtggggtct gccctcgggg gcagtgtgtg gatttgtgag 7620
tttgagctgt atggcagccc ctctgtgcct gtccacgtgt ggctgtccat gtgaccctct
7680 gctgagctat gggactgggc ataactacag cttcctccgt gtgtgtcccc
acatatgctg 7740 ggagctggga gggactgagt tagggcacat ggggaggcca
gtcttaccac tgaccacttg 7800 gtctgtctgt gtatgttcat gtgcgagggc
agaggaggac ctcacatgga ccgcagcaac 7860 gcccgaggcc aggtatacgg
acgtcatcct gtacgcgtcg ggctccctgg ccttggctgt 7920 gctcctgctg
ctggccgggc tgtatcgagg gcaggcgctc cacggccggc acccccgccc 7980
acccgccacc gtgcagaagc tctcccgctt ccctctggcc cgacaggtac tgggcgcatc
8040 ccccacctcg catgtggcag cctgactcca gcaggcagaa ccaagtctcc
cactttgcag 8100 ttctccctgg agtcaggctc ttccagcaag tcaagctcat
ccctggtgcg aggcgtgcgt 8160 ctctcctcca gcggccccgc cttgctcgcc
ggcctcgtga gtctagacct acctctcgac 8220 ccactgtggg agttcccccg
ggacaggtgc gctgagctgt gtgggggcag ggacacaggt 8280 gccaggtcgc
agcctgccct cagcaggagt gactcggagg tctgaggcat ggactttctc 8340
catctccagg ctggtgcttg ggaagcccct gggcgagggc tgctttggac aggtagtacg
8400 tgcagaggcc tttggcatgg accctgcccg gcctgaccaa gccagtactg
tggctgtcaa 8460 gatgctcaaa ggtgagtgtg acccagcgtg gtggctcaca
cctgtaatgc cagcactttg 8520 ggagaccgag gtgggaggat cacttgaatc
caggagttcg aggccaacct gggcaacatg 8580 gcgagacttc atctctaaaa
aaaaaaataa gaaaattagt tgggtgtggt ggtgtgtgcc 8640 tttagtccca
gttactagga aggctgaggc aggaggatcc cttgaaccca ggagttggag 8700
gttgcagtga gccatgatca cgccactgta ttccaacttg ggcaacagag tgaaacccta
8760 tctgaaaaaa aaaaaagaaa taaaagtaaa aggtgaatgt ggcagcctgg
aggaggtgct 8820 atggcattgg gactaataga aggggatcac agtgctaccg
ggtgagccct ggagctggga 8880 gaggctgtgg gatcccacct ttaaacctgc
agttcagctc tgctcctgac cctggcaagt 8940 gacttctgag cctcagtttt
ccctcgtgtc atatggggta aataacagtc cctactccca 9000 gcccagggac
tgtggaaagt gcctggctca tagtcagccc tcaacaaatc gtcaccattg 9060
gggtgacgat gatgagaagg atttggtgtg acaggcttga tatcctctgt cagcgtcagt
9120 ctgtgtcagc cttgacttca cctcttctca gcctcacagg ccctcccccg
ccttccctgt 9180 ggttcccccc agacacaccc tcagcctccc tgggaccctc
cctaggtcca tcccgcgtcc 9240 actgctctag gaggacagcc cttctgcttg
cacccagtcc cagccccgag ggtgctcttg 9300 ccaggcactc ccgtaccccg
cccatcaggt cctctcgttg cagttcccca gcgccccctg 9360 caagaatggc
ctcaactgct cttctgctcc tcccctcctc tctacacagc tggggccacc 9420
tggtgctccc tgggaggcag ggattgagaa atgtgcattg tgacattgcc gtgtgtgggt
9480 ggggagtgtt acatcactgg cccagggcca caggtcagcc ccaggggccc
agccagagaa 9540 gccagagcag ccttcttccc aagctccctg gctgcgccct
gcctgccgcc ggctccctga 9600 attcccaggc cagttggacg ccaggccctg
gtcaaacaga ccccagggtg ccagcctgct 9660 ttccgctccc agaagctctg
accccatgta gggactaccg ctaacccctc cagaggcagc 9720 ttccttcctg
ctccgagctc ttcccctctc tcctgtttcc tggacctgcc cactggaagg 9780
cctgcctctc agatccttga tatagttgca tccttgcaac tgctgtgaca ggtagggtgt
9840 gacccactgc tctgtttccc acaagacgac cctgaggttc agagacacta
agagactttt 9900 tcaaggccac acagactagc aaggaatcag ccctagacct
acgtagccct ggtccagtgc 9960 tcctcgccct gcccctgcct ctacctcgcc
ctgcccctgc ctctgcctcg ccctgcccct 10020 gccctctgcc tcgccctgcc
cctgcctctg cctgttccct catgtagttg gagagcagcc 10080 tcttcacccc
atctgctgcc cttacagaca acgcctctga caaggacctg gctgacctgg 10140
tctcggagat ggaggtgatg aagctgattg gccgacacaa gaacatcatc aacctgctgg
10200 gtgtctgcac ccaggaaggt ggggccaagg cggggccggc tgcatgggcc
gttagggtac 10260 agagccaaag ctgtggcagt ctctccgagc tccctccact
ccctctgtag ggcccctgta 10320 tgtaatcgtg gagtgcgctg ccaagggaaa
ccttcgggag ttcctgcggg cccggcgccc 10380 cccgggccct gacctcagcc
cggacggtcc tcagagcagt gaggggccac tcgccttccc 10440 agtcctggtc
tcctgcgcct accaggtggc ccgaggcatg cagtatctgg agtcccggaa 10500
ggtacaggcg gtagggctct gggcccctct cgatctctcc agctccactc tctcaggcct
10560 gtggcattaa atgtccccaa cttcttcctt tctgctcttt ttcatgaccc
cacctcagtg 10620 tccccaggca ttcatgcttt cctgccttcc ccacttgttc
ctcacacttc cctagaaggg 10680 agaggggaga ggggagaggg gagaggggac
acaggagagg gcgttcccca tttctaaacc 10740 ttgaccacct cctctgtaaa
gtgggtggag ggcccctgcc cctgggcctg ctggggggtg 10800 gtgtgtgctc
agctcaaggt caggtgtcct gatgcaccca agcccccact ccctgcagtg 10860
tatccaccgg gacctggctg cccgcaatgt gctggtgacg gaggacaatg tgatgaagat
10920 agctgacttt gggctggccc gtggcatcca ccacattgac tactataaga
aaaccagcaa 10980 cgtgagggag acggggcaga actggatggg agtggagggg
ggctgggcct ggggtggcag 11040 gcatgggaac tcgtgggact ctgcactgag
gccctccctc ccctccaggg ccgcctgcct 11100 gtcaagtgga tggcgcccga
ggccttgttt gaccgagtgt acacacacca gagtgacgtg 11160 tgagtcctgc
cagcggtcac tgtcctaccc cacaaaaagg gcaaggcact gcccaaagtc 11220
acatggcccc aggaggcctg cgctccaggg ctccttcaga tttgttctgg gatccgagtg
11280 ggaccacact ccaggaggag cccactcccc accagctggg gtgggtcatg
cctgtggggt 11340 cccccgtcct agccctggtc ttagggaggg cgctgggcca
cactgagccc tggccctgcc 11400 tccaggtggt cttttggggt cctgctgtgg
gagatcttca ccctcggggg ctccccgtat 11460 cctggcatcc cggtggagga
gctgttctca ctgctgcggg agggacatcg gatggaccga 11520 cccccacact
gccccccaga gctgtgaggc ctcatcctgc cctcgacccc cactttccag 11580
tcctcctcct ctgccctgac cttggcctca gggtttgtgc cggccagaag gacaacacta
11640 acaacaactc ctcctcctct tcctcctcct cctctcctct tcctcctcct
cctcctcctc 11700 tcctcctcct cctctttctc ctccttctcc tcctcctctt
cctcttcctg ctcctcagcc 11760 tagtggatcg tcctggcctg gcttccactg
atgaccccgc ctatccctca tcaaactccc 11820 tactcagaga acccccggtc
ctccccttcc tcctgaaggc ctgaggctcc ctatgacctt 11880 ctggcccacc
tctcccaggt acgggctgat gcgtgagtgc tggcatgcag caccctccca 11940
gaggcccacc ttcaagcagc tggtggaggc gctggacaag gtcttactgg ccgtctctga
12000 ggaggtacag ccccctccca cccaccacct ccctctgcct gctcccctcc
aggcctcgtc 12060 tggcctgacc gcgtggacat gcgccccgtc ccatcctggg
cactgcagag gctgaccggc 12120 tccgttcccc acagtacctc gacctccgcc
tgaccttcgg accctattcc cctgctggtg 12180 gggacaccag cagcacctgc
tcctccagtg actccgtctt cagccacgac cccctgccac 12240 tgggatccag
ctccttcccc tttgggtctg gggtgcagac atgagtaagg ctcaaggctg 12300
tgcaggcaca taaactagtg gccttgggcc ttggggctca gccacagcct ggcacagtgc
12360 ttgaccttgg cagcacgggg tccctggccc agagtgctgt cccaggtctc
tggttctgct 12420 ttgggtaggt cccttcttgg tcctggagtt cgtaaaggct
tcctgctctg gccttgggtt 12480 cccaacctac agctcaactc aaatctcaag
gccgtgccct tgcccttggc gctgcagtgc 12540 ctgtgtcctg atgggccaaa
cgtcagggtt ctgctcggcc cttggacctt ggcgctcagc 12600 ccccacctca
ggtttggctg agcctggctg gagagctgct atgctaaatc tcctgcctcc 12660
caataccagc agggggttca gggcctctga accccctttc cccacacctc cccctgctgc
12720 ttgccccagc gtcttgatgg gagcgtcggc ccctgagccc agagaagctg
gaagcccgcc 12780 aaaaacagga gcaaatggcg ttctataaat tatttttttg
aaataaagct ctgtgtgcct 12840 aggtcttccc tgagcaacat ggaggggagt
gggatggagg gatcccccca ggagagagtt 12900 ctgcctgcag gacacggact
gagggcgctg gaccaggccg tgggctctgc cacctcccct 12960 gccccgggag
ccagggtgtg cctgtcttgg tccgccggtc ccaccagcac catcttgtgt 13020
cggtgacagt gtgaatgagt gttaatgggc tgagtccgca ttgcaccatc tacagtggga
13080 ctcctgtgcc ctctgcacat gtgtgtgtgc gtgtgtgccc tgcagctgtc
cccgggggag 13140 caggcagccc cctcccccat ctgctcagca ttaaccaagc
tgaccgttaa cacagcatga 13200 aaatctgaaa gccagcctta ggccacggcc
tgctcccacg ctctgcctgc tcaggctggg 13260 ggcttgtggg ggccatgccc
gccccaccct ggccaatctc ccaggcagca gctggttgcc 13320 gcccgcctgg
gctgcagctg tccctgcctg cctggtcttc cactggggac ccgtcacagc 13380
cctgtaccca gagcccctca gagggagcag cttctcaggg ctctgagcct ggagccttcc
13440 ctggccccat cctggcatgt actgctactc cccagcccct gagtgggcca
tggggggcct 13500 aggcatggtg tctgacacag cgcttggcac tctcctgcct
tttcctacag gcctcaggct 13560 gggacagaac tgcccaacca ggacagcctt
tatggaggcc actgggattg cccccatctg 13620 cccccaggga gtggtggctg
aaaactctcc acatagcctc tccggctgac agtctcccac 13680 acagaactca
tcctcccccc agagcggcgc ctcctccagc gcaggctccg gggagtcacc 13740
tgaccacttc cctcaagaac ctcgttccag gccgggcacg gcggctcacg cctgtaatcc
13800 cagcagtttg gaaggccgag gaggacgcat cacctgaggt taggaattcg
agaccagcct 13860 ggacaacatg gtgaagccct gtctctacta aaaatacaaa
aattagcctg gcgtagtggg 13920 ctgcactgta gtcccagcta ctcaggaggc
tgaggcagga gaatcacttg aatccgggag 13980 gtgagggttg cagtgaaccg
agattgtgcc agcctgggct aaggagcaag gctctgtctc 14040 aaaaaaaaaa
aaaaaaagaa ccctattccc taaagtgctt ccagggtcca gccatggctc 14100
ctgaggccag cactgccacc tctctgatga tcactgtcct tgcccatgag tagggtgcgc
14160 ctgtcccgcc tgcctcctgg ggcttagggt ttaggacgaa gcgaagcaca
gtgcagatgc 14220 acataggctt gggggagggc gtaccctact tttgctccct
cccctgcagg tggctccaga 14280 ggcagcttcc aaaggcggac tgctcgcaaa
gaccccctct aactcctgaa agcccccaga 14340 ctgactttct ttttccttcc
ttcctccttc cttccctccc tccctccctt cttccctccc 14400 tcccttcctt
tctttctttc tgtcttttgt tctttcgttc tttcattcgt tcgttctttt 14460
tctttctctt tctctctctc tctttcgctc tctcttcttc tttctctttc tctctttctc
14520 ttttccttcc ttccttcctt ccttccttcc tctctttctc tctctctctt
ctttatttct 14580 ttccttcttt ctttcttttt ttttttttga gacaaggtct
tgatctattg accagattgg 14640 agtgcagtga cgccatgatc atggctctgc
tgcctcaaac tcctgggctc aagcaatcct 14700 cccacctcag cctgccaagt
gcataggact acaggtgcat gccaccacac atggataatt 14760 attttttatt
ttttgtagag atggggtttt gctatgttgt ccaagctggt ctcaaactcc 14820
tggcctcaag caatcctccc accttggcct cccaaagctc ccaaaccccg gacattctga
14880 ctgagcctca ctctacaatg tatagcctcc cattgggggt cactgtccac
tgactgtgtc 14940 cacctgtcca gccaatcaca ggagaagcag atcccttgga
cttgaccttg ggaaggagag 15000 g 15001 <210> SEQ ID NO 6
<211> LENGTH: 3323 <212> TYPE: DNA <213>
ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (350)..(2749) <400> SEQUENCE: 6
cccacgcgtc cggaacgact gagactgggc gatccagtcc caacggggag ctcccgcact
60 agggtaccgg gctgacattt gccgggtctc ggaccacgcc tctcagatca
gaagtggtcc 120 aggaggggcg gagtccgagg tgggcggggc aggagggggc
agccccccgc cacgctgcag 180 ttgcagggac attcctggct cttcggcccg
gggcggagga gctccgggcg ggtgagtgtg 240 ccagccctgc cgggatcgtg
acccgcgcgc gcgggagccg ggcggcggag gagccaggaa 300 ggtggtcagt
gggaagtctg gccctgatcc tgagatcagc tggaaggaa atg tgg ctg 358 Met Trp
Leu 1 ctc ttg gcc ctg ttg agc atc ttt cag ggg aca cca gct ttg tcc
ctt 406 Leu Leu Ala Leu Leu Ser Ile Phe Gln Gly Thr Pro Ala Leu Ser
Leu 5 10 15 gag gcc tct gag gaa atg gag cag gag ccc tgc cta gcc cca
atc ctg 454 Glu Ala Ser Glu Glu Met Glu Gln Glu Pro Cys Leu Ala Pro
Ile Leu 20 25 30 35 gag cag caa gag cag gtg ttg acg gtg gcc ctg ggg
cag cct gtg agg 502 Glu Gln Gln Glu Gln Val Leu Thr Val Ala Leu Gly
Gln Pro Val Arg 40 45 50 ctg tgc tgt ggg cgc acc gag cgt ggt cgt
cac tgg tac aaa gag ggc 550 Leu Cys Cys Gly Arg Thr Glu Arg Gly Arg
His Trp Tyr Lys Glu Gly 55 60 65 agc cgc cta gca tct gct ggg cga
gta cgg ggt tgg aga ggc cgc ctg 598 Ser Arg Leu Ala Ser Ala Gly Arg
Val Arg Gly Trp Arg Gly Arg Leu 70 75 80 gag atc gcc agc ttc ctt
cct gag gat gct ggc cga tac ctc tgc ctg 646 Glu Ile Ala Ser Phe Leu
Pro Glu Asp Ala Gly Arg Tyr Leu Cys Leu 85 90 95 gcc cgt ggc tcc
atg acc gtc gta cac aat ctt acg ttg ctt atg gat 694 Ala Arg Gly Ser
Met Thr Val Val His Asn Leu Thr Leu Leu Met Asp 100 105 110 115 gac
tcc tta acc tcc atc agt aat gat gaa gac ccc aag aca ctc agc 742 Asp
Ser Leu Thr Ser Ile Ser Asn Asp Glu Asp Pro Lys Thr Leu Ser 120 125
130 agc tcc tcg agt ggt cat gtc tac cca cag caa gca ccc tac tgg aca
790 Ser Ser Ser Ser Gly His Val Tyr Pro Gln Gln Ala Pro Tyr Trp Thr
135 140 145 cac ccc caa cgc atg gag aag aaa ctg cat gca gtg cct gcc
ggg aat 838 His Pro Gln Arg Met Glu Lys Lys Leu His Ala Val Pro Ala
Gly Asn 150 155 160 act gtc aaa ttc cgc tgt cca gct gca ggg aac ccc
atg cct acc atc 886 Thr Val Lys Phe Arg Cys Pro Ala Ala Gly Asn Pro
Met Pro Thr Ile 165 170 175 cac tgg ctc aag gat gga cag gcc ttc cac
ggg gag aat cgt att gga 934 His Trp Leu Lys Asp Gly Gln Ala Phe His
Gly Glu Asn Arg Ile Gly 180 185 190 195 ggc att cgg ctg cgc cac caa
cac tgg agc ctg gtg atg gaa agt gtg 982 Gly Ile Arg Leu Arg His Gln
His Trp Ser Leu Val Met Glu Ser Val 200 205 210 gta ccc tcg gac cgt
ggc aca tac aca tgc ctt gtg gag aac tct ctg 1030 Val Pro Ser Asp
Arg Gly Thr Tyr Thr Cys Leu Val Glu Asn Ser Leu 215 220 225 ggt agc
att cgc tac agc tat ctc ctg gat gtg ctg gag cgg tcc ccg 1078 Gly
Ser Ile Arg Tyr Ser Tyr Leu Leu Asp Val Leu Glu Arg Ser Pro 230 235
240 cac cgg ccc atc ctg cag gcg ggg ctc cca gcc aac acc aca gct gtg
1126 His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn Thr Thr Ala
Val 245 250 255 gtt ggc agc gat gtg gag cta ctc tgc aag gtg tac agc
gac gcc cag 1174 Val Gly Ser Asp Val Glu Leu Leu Cys Lys Val Tyr
Ser Asp Ala Gln 260 265 270 275 ccc cac ata cag tgg ctg aaa cac gtc
gtc atc aac ggc agc agc ttc 1222 Pro His Ile Gln Trp Leu Lys His
Val Val Ile Asn Gly Ser Ser Phe 280 285 290 ggc gcc gac ggt ttc ccc
tac gta caa gtc ctg aag aca aca gac atc 1270 Gly Ala Asp Gly Phe
Pro Tyr Val Gln Val Leu Lys Thr Thr Asp Ile 295 300 305 aat agc tcg
gag gta gag gtc ttg tat ctg agg aac gtg tcc gct gag 1318 Asn Ser
Ser Glu Val Glu Val Leu Tyr Leu Arg Asn Val Ser Ala Glu 310 315 320
gat gca gga gag tat acc tgt ctg gcg ggc aac tcc atc ggc ctt tcc
1366 Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Leu
Ser 325 330 335 tac cag tca gcg tgg ctc acg gtg ctg cca gag gaa gac
ctc acg tgg 1414 Tyr Gln Ser Ala Trp Leu Thr Val Leu Pro Glu Glu
Asp Leu Thr Trp 340 345 350 355 aca aca gca acc cct gag gcc aga tac
aca gat atc atc ctg tat gta 1462 Thr Thr Ala Thr Pro Glu Ala Arg
Tyr Thr Asp Ile Ile Leu Tyr Val 360 365 370 tca ggc tca ctg gtt ctg
ctt gtg ctc ctg ctg ctg gcc ggg gtg tat 1510 Ser Gly Ser Leu Val
Leu Leu Val Leu Leu Leu Leu Ala Gly Val Tyr 375 380 385 cat cgg caa
gtc atc cgt ggc cac tac tct cgc cag cct gtc act ata 1558 His Arg
Gln Val Ile Arg Gly His Tyr Ser Arg Gln Pro Val Thr Ile 390 395 400
caa aag ctg tcc cgt ttc cct ttg gcc cga cag ttc tct ttg gag tcg
1606 Gln Lys Leu Ser Arg Phe Pro Leu Ala Arg Gln Phe Ser Leu Glu
Ser 405 410 415 agg tcc tct ggc aag tca agt ttg tcc ctg gtg cga ggt
gtc cgt ctc 1654 Arg Ser Ser Gly Lys Ser Ser Leu Ser Leu Val Arg
Gly Val Arg Leu 420 425 430 435 tcc tcc agc ggc ccg ccc ttg ctc acg
ggc ctt gtg aat cta gac ctg 1702 Ser Ser Ser Gly Pro Pro Leu Leu
Thr Gly Leu Val Asn Leu Asp Leu 440 445 450 cct ctc gat ccg ctt tgg
gaa ttc ccc cgg gac agg ttg gtg ctc gga 1750 Pro Leu Asp Pro Leu
Trp Glu Phe Pro Arg Asp Arg Leu Val Leu Gly 455 460 465 aag ccc ctg
ggt gag ggc tgc ttt ggg caa gtg gtt cgt gca gag gcc 1798 Lys Pro
Leu Gly Glu Gly Cys Phe Gly Gln Val Val Arg Ala Glu Ala 470 475 480
ttt ggt atg gat ccc tcc cgg ccc gac caa acc agc acc gtg gct gtg
1846 Phe Gly Met Asp Pro Ser Arg Pro Asp Gln Thr Ser Thr Val Ala
Val 485 490 495 aag atg ctg aaa gac aat gcc tcc gac aag gat ttg gca
gac ctg gtc 1894 Lys Met Leu Lys Asp Asn Ala Ser Asp Lys Asp Leu
Ala Asp Leu Val 500 505 510 515 tcc gag atg gag gtg atg aag cta atc
gga aga cac aag aac atc atc 1942 Ser Glu Met Glu Val Met Lys Leu
Ile Gly Arg His Lys Asn Ile Ile 520 525 530 aac ctg ctg ggt gtc tgc
act cag gaa ggg ccc ctg tac gtg att gtg 1990 Asn Leu Leu Gly Val
Cys Thr Gln Glu Gly Pro Leu Tyr Val Ile Val 535 540 545 gaa tgt gcc
gcc aag gga aat ctt cgg gaa ttc ctc cgt gcc cgg cgc 2038 Glu Cys
Ala Ala Lys Gly Asn Leu Arg Glu Phe Leu Arg Ala Arg Arg 550 555 560
ccc cca ggc cct gat ctc agc cct gat gga cct cgg agc agc gaa gga
2086 Pro Pro Gly Pro Asp Leu Ser Pro Asp Gly Pro Arg Ser Ser Glu
Gly 565 570 575 cca ctc tcc ttc ccg gcc cta gtc tcc tgt gcc tac cag
gtg gcc cga 2134 Pro Leu Ser Phe Pro Ala Leu Val Ser Cys Ala Tyr
Gln Val Ala Arg 580 585 590 595 ggc atg cag tat ctg gag tct cgg aag
tgc atc cac cgg gac ctg gct 2182 Gly Met Gln Tyr Leu Glu Ser Arg
Lys Cys Ile His Arg Asp Leu Ala 600 605 610 gcc cga aat gtg ctg gtg
acc gag gat gat gtg atg aag atc gct gac 2230 Ala Arg Asn Val Leu
Val Thr Glu Asp Asp Val Met Lys Ile Ala Asp 615 620 625 ttt ggg ctg
gca cgt ggt gtc cac cac att gac tac tat aag aaa acc 2278 Phe Gly
Leu Ala Arg Gly Val His His Ile Asp Tyr Tyr Lys Lys Thr 630 635 640
agc aac ggc cgc ctg cca gtc aaa tgg atg gct cca gag gcg ttg ttc
2326 Ser Asn Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu
Phe 645 650 655 gac cgt gtg tac aca cac cag agt gac gtg tgg tct ttc
ggg atc ctg 2374 Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp Ser
Phe Gly Ile Leu 660 665 670 675 ctg tgg gaa atc ttc acc ctc ggg ggc
tcc cca tac cct ggc att ccg 2422 Leu Trp Glu Ile Phe Thr Leu Gly
Gly Ser Pro Tyr Pro Gly Ile Pro 680 685 690 gtg gag gag ctc ttc tca
ctg ctg cga gag ggg cac agg atg gag cgg 2470 Val Glu Glu Leu Phe
Ser Leu Leu Arg Glu Gly His Arg Met Glu Arg 695 700 705 ccc cca aac
tgc ccc tca gag ctg tat ggg cta atg agg gag tgc tgg 2518 Pro Pro
Asn Cys Pro Ser Glu Leu Tyr Gly Leu Met Arg Glu Cys Trp 710 715 720
cac gca gtc cca tct cag agg cct act ttt aag cag ctg gtg gaa gct
2566 His Ala Val Pro Ser Gln Arg Pro Thr Phe Lys Gln Leu Val Glu
Ala 725 730 735 ctg gac aag gtc ctg ctg gct gtc tct gaa gag tac ctt
gac ctc cgc 2614 Leu Asp Lys Val Leu Leu Ala Val Ser Glu Glu Tyr
Leu Asp Leu Arg 740 745 750 755 ctg acc ttt gga ccc ttt tct ccc tcc
aat ggg gat gcc agc agc acc 2662 Leu Thr Phe Gly Pro Phe Ser Pro
Ser Asn Gly Asp Ala Ser Ser Thr 760 765 770 tgc tcc tcc agt gac tcg
gtt ttc agc cac gac cct ttg ccc ctc gag 2710 Cys Ser Ser Ser Asp
Ser Val Phe Ser His Asp Pro Leu Pro Leu Glu 775 780 785 cca agc ccc
ttc cct ttc tct gac tcg cag acg aca tga gccggggagc 2759 Pro Ser Pro
Phe Pro Phe Ser Asp Ser Gln Thr Thr 790 795 agcaattttg tatgggctac
gcggcccatg gccgtgggtc tcctcgctga gctgcaacct 2819 gatgcatcga
catttaatgt tggcagtgtc aggcctctga cttgagacta ctgctgtcgc 2879
agatcctctc tctggccctg ttttggggag ggccattctt ggtcctaagg ttcatagttg
2939 aggccttctg ttccagcctt atgctcccat ctcagagttc aactctcatc
tcaagatcat 2999 ggccttgccc ttggactcat cctcagagaa gttaagcatt
aaggccttgg cacgcagcct 3059 ccgtctccgg ggctctccgg gcctagctgc
aaaacttatg ctctaaacat ttctagttcc 3119 cccaaacaac ctagaggcct
tgggacttca catcccccag cacacaagcc tcaccacccc 3179 ctgccatccc
ccctccattg cttgttccag catcttggtg aaaggggcat cagctctggt 3239
gtccctgaga gacgggaagc ctgtgggaac gacagaagaa catggcattt ttataaatta
3299 tttttttgaa aaaaaaaaaa aaaa 3323 <210> SEQ ID NO 7
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 7
ggcacactca gcaggacccc 20 <210> SEQ ID NO 8 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 8 aggctgccca
agggctactg 20 <210> SEQ ID NO 9 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 9 gtccagtagg gtgcttgctg 20
<210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 10 cccatgaaag gcctgtccat 20 <210> SEQ
ID NO 11 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
11 gcgcagccga atgcctccaa 20 <210> SEQ ID NO 12 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 12 tctccatcac
gagactccag 20 <210> SEQ ID NO 13 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 13 tacaccttgc acagcagctc 20
<210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 14 gctgctgccg ttgatgacga 20 <210> SEQ
ID NO 15 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
15 ccgaagctgc tgccgttgat 20 <210> SEQ ID NO 16 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 16 gcacactcag
caggaccccc 20 <210> SEQ ID NO 17 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 17 aggcacactc agcaggaccc 20
<210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 18 caggcacact cagcaggacc 20 <210> SEQ
ID NO 19 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
19 cccaggcaca ctcagcagga 20 <210> SEQ ID NO 20 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 20 gcccaggcac
actcagcagg 20 <210> SEQ ID NO 21 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 21 ggcccaggca cactcagcag 20
<210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 22 gaggcccagg cacactcagc 20 <210> SEQ
ID NO 23 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
23 tggaggccca ggcacactca 20 <210> SEQ ID NO 24 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 24 ctggaggccc
aggcacactc 20 <210> SEQ ID NO 25 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 25 gactggaggc ccaggcacac 20
<210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 26 ctgtcagctc ctgctcttgc 20 <210> SEQ
ID NO 27 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
27 ctactgtcag ctcctgctct 20 <210> SEQ ID NO 28 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 28 gctactgtca
gctcctgctc 20 <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 ggctactgtc agctcctgct 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 gggctactgt cagctcctgc 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 gcccaagggc tactgtcagc 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 cgcacaggct
gcccaagggc 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 gctcagcccg cccacagcac 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 caccacgctc agcccgccca 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 ccaccacgct cagcccgccc 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 gccaccacgc
tcagcccgcc 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 ggccaccacg ctcagcccgc 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 accagtggcc accacgctca 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 gtaccagtgg ccaccacgct 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 tgtaccagtg
gccaccacgc 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 ctccttgtac cagtggccac 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 cctccttgta ccagtggcca 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 ccctccttgt accagtggcc 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 ccaggcgact
gccctccttg 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 gccaggcgac tgccctcctt 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 tgccaggcga ctgccctcct 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 gtgccaggcg actgccctcc 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 ggtgccaggc
gactgccctc 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 ccgtacacgg ccagcaggtg 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 cccgtacacg gccagcaggt 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 agccccgtac acggccagca 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 cctccagccc
cgtacacggc 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 ccctccagcc ccgtacacgg 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 ggcggcccct ccagccccgt 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 gcaatctcta ggcggcccct 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 ggcaatctct
aggcggcccc 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 tggcaatctc taggcggccc 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 cctcaggtag gaagctggca 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 tcctcaggta ggaagctggc 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 agcggccagc
atcctcaggt 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 tagcggccag catcctcagg 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 gtgtccagta gggtgcttgc 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 gtgtgtccag tagggtgctt 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 agtttcttct
ccatgcgctg 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 gcctgtccat ccttaagcca 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 ttctccccat gaaaggcctg 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 ggttctcccc atgaaaggcc 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 ctccatcacg
agactccagt 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 gctctccatc acgagactcc 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 cgctctccat cacgagactc 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 acgctctcca tcacgagact 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 cacgctctcc
atcacgagac 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 ccacgctctc catcacgaga 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 acaccttgca cagcagctcc 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 gtacaccttg cacagcagct 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 gccgttgatg
acgatgtgct 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 gctgccgttg atgacgatgt 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 tgctgccgtt gatgacgatg 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 ctgctgccgt tgatgacgat 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 agctgctgcc
gttgatgacg 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 aagctgctgc cgttgatgac 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 cgaagctgct gccgttgatg 20 <210> SEQ
ID NO 83 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
83 gctattgatg tctgcagtct 20 <210> SEQ ID NO 84 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 84 agctattgat
gtctgcagtc 20 <210> SEQ ID NO 85 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 85 gagctattga tgtctgcagt 20
<210> SEQ ID NO 86 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 86 cctccacctc tgagctattg 20 <210> SEQ
ID NO 87 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
87 acctccacct ctgagctatt 20 <210> SEQ ID NO 88 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 88 gacctccacc
tctgagctat 20 <210> SEQ ID NO 89 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 89 ggacctccac ctctgagcta 20
<210> SEQ ID NO 90 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 90 aggacctcca cctctgagct 20 <210> SEQ
ID NO 91 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
91 caggacctcc acctctgagc 20 <210> SEQ ID NO 92 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 92 acaggacctc
cacctctgag 20 <210> SEQ ID NO 93 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 93 gtacaggacc tccacctctg 20
<210> SEQ ID NO 94 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 94 ggtacaggac ctccacctct 20 <210> SEQ
ID NO 95 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
95 ccgcaggtac aggacctcca 20 <210> SEQ ID NO 96 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 96 tccgcaggta
caggacctcc 20 <210> SEQ ID NO 97 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 97 cgttccgcag gtacaggacc 20
<210> SEQ ID NO 98 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 98 gcagactggt aggagaggcc 20 <210> SEQ
ID NO 99 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
99 ggcagactgg taggagaggc 20 <210> SEQ ID NO 100 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 100 ggtcctcctc
tggcagcacc 20 <210> SEQ ID NO 101 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 101 gcaggagcac agccaaggcc 20
<210> SEQ ID NO 102 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 102 gcctgccctc gatacagccc 20 <210> SEQ
ID NO 103 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
103 gcctgactcc agggagaact 20 <210> SEQ ID NO 104 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 104 gagcctgact
ccagggagaa 20 <210> SEQ ID NO 105 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 105 ggaggagaga cgcacgcctc 20
<210> SEQ ID NO 106 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 106 tggaggagag acgcacgcct 20 <210> SEQ
ID NO 107 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
107 ctggaggaga gacgcacgcc 20 <210> SEQ ID NO 108 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 108 gactcacgag
gccggcgagc 20 <210> SEQ ID NO 109 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 109 ctagactcac gaggccggcg 20
<210> SEQ ID NO 110 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 110 cccaagcacc agcctgtccc 20 <210> SEQ
ID NO 111 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
111 tcccaagcac cagcctgtcc 20 <210> SEQ ID NO 112 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 112 gcttcccaag
caccagcctg 20 <210> SEQ ID NO 113 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 113 gggcttccca agcaccagcc 20
<210> SEQ ID NO 114 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 114 ccatgccaaa ggcctctgca 20 <210> SEQ
ID NO 115 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
115 gtccatgcca aaggcctctg 20 <210> SEQ ID NO 116 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 116 ggtccatgcc
aaaggcctct 20 <210> SEQ ID NO 117 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 117 cagcagccgc atctccttct 20
<210> SEQ ID NO 118 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 118 gctgaagaca gaatcgctgg 20 <210> SEQ
ID NO 119 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
119 cagcactcac gcatcagccc 20 <210> SEQ ID NO 120 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 120 ccagcactca
cgcatcagcc 20 <210> SEQ ID NO 121 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 121 gggtccgaag gtcaggcgga 20
<210> SEQ ID NO 122 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 122 agggtccgaa ggtcaggcgg 20 <210> SEQ
ID NO 123 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
123 atagggtccg aaggtcaggc 20 <210> SEQ ID NO 124 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 124 ggaatagggt
ccgaaggtca 20 <210> SEQ ID NO 125 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 125 gtgcctgcac agccttgagc 20
<210> SEQ ID NO 126 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 126 tctccagcca ggctcagcca 20 <210> SEQ
ID NO 127 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
127 cagctctcca gccaggctca 20 <210> SEQ ID NO 128 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 128 gcagctctcc
agccaggctc 20 <210> SEQ ID NO 129 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 129 agcagctctc cagccaggct 20
<210> SEQ ID NO 130 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 130 tagcagctct ccagccaggc 20 <210> SEQ
ID NO 131 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
131 gcatagcagc tctccagcca 20 <210> SEQ ID NO 132 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 132 agcatagcag
ctctccagcc 20 <210> SEQ ID NO 133 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 133 tagcatagca gctctccagc 20
<210> SEQ ID NO 134 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 134 ttagcatagc agctctccag 20 <210> SEQ
ID NO 135 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
135 ccagcttctc tgggctcagg 20 <210> SEQ ID NO 136 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 136 tccagcttct
ctgggctcag 20 <210> SEQ ID NO 137 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 137 ttccagcttc tctgggctca 20
<210> SEQ ID NO 138 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 138 cttccagctt ctctgggctc 20 <210> SEQ
ID NO 139 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
139 gcttccagct tctctgggct 20 <210> SEQ ID NO 140 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 140 ggcttccagc
ttctctgggc 20 <210> SEQ ID NO 141 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 141 acgccatttg ctcctgtttt 20
<210> SEQ ID NO 142 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 142 tgcgaatcaa tgggtcccga 20 <210> SEQ
ID NO 143 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
143 ggtgcgaatc aatgggtccc 20 <210> SEQ ID NO 144 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 144 ccgccggcgc
gaagacagcc 20 <210> SEQ ID NO 145 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 145 catctctgcc gccggcgcga 20
<210> SEQ ID NO 146 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 146 ctgaccgctg accgaccacc 20 <210> SEQ
ID NO 147 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
147 gctgctgacc gctgaccgac 20 <210> SEQ ID NO 148 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 148 ctgccctgat
atcagagtcc 20 <210> SEQ ID NO 149 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 149 ggctgccctg atatcagagt 20
<210> SEQ ID NO 150 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 150 ctcagatact gctgtctctg 20 <210> SEQ
ID NO 151 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
151 tgcccatccc tctgtgcccc 20 <210> SEQ ID NO 152 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 152 tgctctcttg
cccatccctc 20 <210> SEQ ID NO 153 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 153 ctctttggtc acaccgtctg 20
<210> SEQ ID NO 154 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 154 atctctttgg tcacaccgtc 20 <210> SEQ
ID NO 155 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
155 ctatctcttt ggtcacaccg 20 <210> SEQ ID NO 156 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 156 gcctatctct
ttggtcacac 20 <210> SEQ ID NO 157 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 157 cgctgcctat ctctttggtc 20
<210> SEQ ID NO 158 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 158 agcttgcaag cccttaatgg 20 <210> SEQ
ID NO 159 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
159 ccagcttgca agcccttaat 20 <210> SEQ ID NO 160 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 160 accttcatct
tccagcagag 20 <210> SEQ ID NO 161 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 161 caaccttcat cttccagcag 20
<210> SEQ ID NO 162 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 162 ttcaaccttc atcttccagc 20 <210> SEQ
ID NO 163 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
163 cagctttgct cagcccagca 20 <210> SEQ ID NO 164 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 164 tccagctttg
ctcagcccag 20 <210> SEQ ID NO 165 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 165 tttccagctt tgctcagccc 20
<210> SEQ ID NO 166 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 166 cctttccagc tttgctcagc 20 <210> SEQ
ID NO 167 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
167 ccaggtccac agtccagggc 20 <210> SEQ ID NO 168 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 168 acttgccaga
gagtagcaga 20 <210> SEQ ID NO 169 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 169 gccatagcac ctcctccagg 20
<210> SEQ ID NO 170 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 170 cccaatgcca tagcacctcc 20 <210> SEQ
ID NO 171 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
171 gtcccaatgc catagcacct 20 <210> SEQ ID NO 172 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 172 tagtcccaat
gccatagcac 20 <210> SEQ ID NO 173 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 173 ttctattagt cccaatgcca 20
<210> SEQ ID NO 174 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 174 gtcacttgcc agggtcagga 20 <210> SEQ
ID NO 175 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
175 gctcagaagt cacttgccag 20 <210> SEQ ID NO 176 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 176 gtccatctgg
cttcccctgc 20 <210> SEQ ID NO 177 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 177 cagtccatct ggcttcccct 20
<210> SEQ ID NO 178 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 178 ccactccact tccagtccat 20 <210> SEQ
ID NO 179 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
179 tgccactcca cttccagtcc 20 <210> SEQ ID NO 180 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 180 ggtcactgcc
actccacttc 20 <210> SEQ ID NO 181 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 181 ccttggtcac tgccactcca 20
<210> SEQ ID NO 182 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 182 ggaagcctat cacacctcct 20 <210> SEQ
ID NO 183 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
183 gtgtctctgg atctaccctg 20 <210> SEQ ID NO 184 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 184 tggtgtctct
ggatctaccc 20 <210> SEQ ID NO 185 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 185 actggtgtct ctggatctac 20
<210> SEQ ID NO 186 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 186 gcactggtgt ctctggatct 20 <210> SEQ
ID NO 187 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
187 tggcactggt gtctctggat 20 <210> SEQ ID NO 188 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 188 ggtggcactg
gtgtctctgg 20 <210> SEQ ID NO 189 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 189 tgggtggcac tggtgtctct 20
<210> SEQ ID NO 190 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 190 tatgggtggc actggtgtct 20 <210> SEQ
ID NO 191 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
191 ggcctatggg tggcactggt 20 <210> SEQ ID NO 192 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 192 gtcaggctgt
gatgtacaca 20 <210> SEQ ID NO 193 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 193 tgctgttact gtcaggctgt 20
<210> SEQ ID NO 194 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 194 gccagtcacc tctggttcgg 20 <210> SEQ
ID NO 195 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
195 agcagttttg ggattctttt 20 <210> SEQ ID NO 196 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 196 aaagcagttt
tgggattctt 20 <210> SEQ ID NO 197 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 197 tccaagtccc tggccaggct 20
<210> SEQ ID NO 198 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 198 atcctttcca gctttgctca 20 <210> SEQ
ID NO 199 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
199 ggatcctttc cagctttgct 20 <210> SEQ ID NO 200 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 200 aaggatcctt
tccagctttg 20 <210> SEQ ID NO 201 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 201 gcaaggatcc tttccagctt 20
<210> SEQ ID NO 202 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 202 gggcaaggat cctttccagc 20 <210> SEQ
ID NO 203 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
203 ctgggcaagg atcctttcca 20 <210> SEQ ID NO 204 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 204 gcctgggcaa
ggatcctttc 20 <210> SEQ ID NO 205 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 205 gtggttgagc cctgccctgc 20
<210> SEQ ID NO 206 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 206 gtctcagtgg ttgagccctg 20 <210> SEQ
ID NO 207 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
207 ctgactgagt ctcagtggtt 20 <210> SEQ ID NO 208 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 208 ggcactgact
gagtctcagt 20 <210> SEQ ID NO 209 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 209 caggcactga ctgagtctca 20
<210> SEQ ID NO 210 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 210 aagccaggca ctgactgagt 20 <210> SEQ
ID NO 211 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
211 ctggaagcca ggcactgact 20 <210> SEQ ID NO 212 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 212 gcttgctgga
agccaggcac 20 <210> SEQ ID NO 213 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 213 atgcttgctg gaagccaggc 20
<210> SEQ ID NO 214 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 214 gtcctctctc gcagacacag 20 <210> SEQ
ID NO 215 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
215 cagtcctctc tcgcagacac 20 <210> SEQ ID NO 216 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 216 gccagtcctc
tctcgcagac 20 <210> SEQ ID NO 217 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 217 aggccagtcc tctctcgcag 20
<210> SEQ ID NO 218 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 218 gagctcacca ccagctctgc 20 <210> SEQ
ID NO 219 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
219 gctgcctgga cctcctaggt 20 <210> SEQ ID NO 220 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 220 atgctgcctg
gacctcctag 20 <210> SEQ ID NO 221 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 221 acatgctgcc tggacctcct 20
<210> SEQ ID NO 222 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 222 acacatgctg cctggacctc 20 <210> SEQ
ID NO 223 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
223 ccacacatgc tgcctggacc 20 <210> SEQ ID NO 224 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 224 gcaaatgcca
cactcttggg 20 <210> SEQ ID NO 225 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 225 gggcaaatgc cacactcttg 20
<210> SEQ ID NO 226 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 226 cagggcaaat gccacactct 20 <210> SEQ
ID NO 227 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
227 cccagggcaa atgccacact 20 <210> SEQ ID NO 228 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 228 cacccagggc
aaatgccaca 20 <210> SEQ ID NO 229 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 229 gccacaccca gggcaaatgc 20
<210> SEQ ID NO 230 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 230 ggatgccaca cccagggcaa 20 <210> SEQ
ID NO 231 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
231 gcggatgcca cacccagggc 20 <210> SEQ ID NO 232 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 232 ctgcggatgc
cacacccagg 20 <210> SEQ ID NO 233 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 233 gccacatgct gcggatgcca 20
<210> SEQ ID NO 234 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 234 ggacttccca ccaactgcct 20 <210> SEQ
ID NO 235 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
235 gctggacttc ccaccaactg 20 <210> SEQ ID NO 236 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 236 caaggagctc
accaccagct 20 <210> SEQ ID NO 237 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 237 ggcaaggagc tcaccaccag 20
<210> SEQ ID NO 238 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 238 caggcaagga gctcaccacc 20 <210> SEQ
ID NO 239 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
239 tggacttccc accaactgcc 20 <210> SEQ ID NO 240 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 240 cactggtgtc
tctggatcta 20 <210> SEQ ID NO 241 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 241 ggcactggtg tctctggatc 20
<210> SEQ ID NO 242 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 242 gtggcactgg tgtctctgga 20 <210> SEQ
ID NO 243 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
243 gggtggcact ggtgtctctg 20 <210> SEQ ID NO 244 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 244 gatcctttcc
agctttgctc 20 <210> SEQ ID NO 245 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 245 aggatccttt ccagctttgc 20
<210> SEQ ID NO 246 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 246 caaggatcct ttccagcttt 20 <210> SEQ
ID NO 247 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
247 ggcaaggatc ctttccagct 20 <210> SEQ ID NO 248 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 248 tgggcaagga
tcctttccag 20 <210> SEQ ID NO 249 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 249 tgactgagtc tcagtggttg 20
<210> SEQ ID NO 250 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 250 actgactgag tctcagtggt 20 <210> SEQ
ID NO 251 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
251 gcactgactg agtctcagtg 20 <210> SEQ ID NO 252 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 252 aggcactgac
tgagtctcag 20 <210> SEQ ID NO 253 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 253 tgcttgctgg aagccaggca 20
<210> SEQ ID NO 254 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 254 agtcctctct cgcagacaca 20 <210> SEQ
ID NO 255 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
255 ccagtcctct ctcgcagaca 20 <210> SEQ ID NO 256 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 256 ggccagtcct
ctctcgcaga 20 <210> SEQ ID NO 257 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 257 aaggagctca ccaccagctc 20
<210> SEQ ID NO 258 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 258 gcaaggagct caccaccagc 20 <210> SEQ
ID NO 259 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
259 ctgcctggac ctcctaggtc 20 <210> SEQ ID NO 260 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 260 tgctgcctgg
acctcctagg 20 <210> SEQ ID NO 261 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 261 catgctgcct ggacctccta 20
<210> SEQ ID NO 262 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 262 cacatgctgc ctggacctcc 20 <210> SEQ
ID NO 263 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
263 cacacatgct gcctggacct 20 <210> SEQ ID NO 264 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 264 accacacatg
ctgcctggac 20 <210> SEQ ID NO 265 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 265 ccagggcaaa tgccacactc 20
<210> SEQ ID NO 266 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 266 acccagggca aatgccacac 20 <210> SEQ
ID NO 267 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
267 tgccacaccc agggcaaatg 20 <210> SEQ ID NO 268 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 268 cggatgccac
acccagggca 20 <210> SEQ ID NO 269 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 269 tgcggatgcc acacccaggg 20
<210> SEQ ID NO 270 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 270 agccacatgc tgcggatgcc 20 <210> SEQ
ID NO 271 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
271 gctctcttgc ccatccctct 20 <210> SEQ ID NO 272 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 272 tctctttggt
cacaccgtct 20 <210> SEQ ID NO 273 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 273 tatctctttg gtcacaccgt 20
<210> SEQ ID NO 274 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 274 cctatctctt tggtcacacc 20 <210> SEQ
ID NO 275 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
275 tgcctatctc tttggtcaca 20 <210> SEQ ID NO 276 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 276 tcaaccttca
tcttccagca 20 <210> SEQ ID NO 277 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 277 agctttgctc agcccagcag 20
<210> SEQ ID NO 278 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 278 ccagctttgc tcagcccagc 20 <210> SEQ
ID NO 279 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
279 ttccagcttt gctcagccca 20 <210> SEQ ID NO 280 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 280 tcacttgcca
gggtcaggag 20 <210> SEQ ID NO 281 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 281 agtcacttgc cagggtcagg 20
<210> SEQ ID NO 282 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 282 ctttggtcac accgtct 17 <210> SEQ ID
NO 283 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
283 tctttggtca caccgtc 17 <210> SEQ ID NO 284 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 284 ctctttggtc
acaccgt 17 <210> SEQ ID NO 285 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 285 tctctttggt cacaccg 17
<210> SEQ ID NO 286 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 286 gctttgctca gcccagc 17 <210> SEQ ID
NO 287 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
287 cagctttgct cagccca 17 <210> SEQ ID NO 288 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 288 ccagctttgc
tcagccc 17 <210> SEQ ID NO 289 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 289 gcagccgcat ctccttc 17
<210> SEQ ID NO 290 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 290 gcacactcag caggacc 17 <210> SEQ ID
NO 291 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
291 ggcacactca gcaggac 17 <210> SEQ ID NO 292 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 292 ccagtggcca
ccacgct 17 <210> SEQ ID NO 293 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 293 aggcgactgc cctcctt 17
<210> SEQ ID NO 294 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 294 caggcgactg ccctcct 17 <210> SEQ ID
NO 295 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
295 gtgtccagta gggtgct 17 <210> SEQ ID NO 296 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 296 cagctctcca
gccaggc 17 <210> SEQ ID NO 297 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 297 gcttctctgg gctcagg 17
<210> SEQ ID NO 298 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 298 agcttctctg ggctcag 17 <210> SEQ ID
NO 299 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
299 cagcttctct gggctca 17 <210> SEQ ID NO 300 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 300 ccagcttctc
tgggctc 17 <210> SEQ ID NO 301 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 301 tccagcttct ctgggct 17
<210> SEQ ID NO 302 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 302 ttccagcttc tctgggc 17 <210> SEQ ID
NO 303 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
303 gcttccagct tctctgg 17 <210> SEQ ID NO 304 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 304 ccatttgctc
ctgtttt 17 <210> SEQ ID NO 305 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 305 gccatttgct cctgttt 17
<210> SEQ ID NO 306 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 306 cgccatttgc tcctgtt 17 <210> SEQ ID
NO 307 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
307 gcactggtgt ctctgga 17 <210> SEQ ID NO 308 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 308 ggcactggtg
tctctgg 17 <210> SEQ ID NO 309 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 309 tggcactggt gtctctg 17
<210> SEQ ID NO 310 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 310 gtggcactgg tgtctct 17 <210> SEQ ID
NO 311 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
311 gactgagtct cagtggt 17 <210> SEQ ID NO 312 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 312 cttgctggaa
gccaggc 17 <210> SEQ ID NO 313 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 313 gcttgctgga agccagg 17
<210> SEQ ID NO 314 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 314 cctctctcgc agacaca 17 <210> SEQ ID
NO 315 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
315 gccagtcctc tctcgca 17 <210> SEQ ID NO 316 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 316 ggccagtcct
ctctcgc 17 <210> SEQ ID NO 317 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 317 gcctggacct cctaggt 17
<210> SEQ ID NO 318 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 318 tgcctggacc tcctagg 17 <210> SEQ ID
NO 319 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
319 ctgcctggac ctcctag 17 <210> SEQ ID NO 320 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 320 gctgcctgga
cctccta 17 <210> SEQ ID NO 321 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 321 tgctgcctgg acctcct 17
<210> SEQ ID NO 322 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 322 atgctgcctg gacctcc 17 <210> SEQ ID
NO 323 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
323 gcgtttgctc ttcttcttgc gtttttt 27 <210> SEQ ID NO 324
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 324
gccacatttc cttccagctg 20 <210> SEQ ID NO 325 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 325 ccttccctga
aggttcctcc 20 <210> SEQ ID NO 326 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 326 tccatttcct cagaggcctc 20
<210> SEQ ID NO 327 <211> LENGTH: 19 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 327
tcatcaacgg cagcagctt 19 <210> SEQ ID NO 328 <211>
LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 328 agctattgat gtctgcagtc tttagg 26
<210> SEQ ID NO 329 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Probe <400> SEQUENCE: 329
agccgacggt ttcccctatg tgca 24 <210> SEQ ID NO 330 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 330 ttgctgtgcc gtgtccaa 18 <210> SEQ ID
NO 331 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 331 tccaagaagc
cgagcagaac 20 <210> SEQ ID NO 332 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 332 agctgccgtg cctgtgtcct gat 23 <210>
SEQ ID NO 333 <211> LENGTH: 19 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 333
tcatcaacgg cagcagctt 19 <210> SEQ ID NO 334 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 334 tgagctattg atgtctgcag tcttc 25
<210> SEQ ID NO 335 <211> LENGTH: 22 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Probe <400> SEQUENCE: 335
ccgacggctt cccctatgtg ca 22 <210> SEQ ID NO 336 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 336 ccccatgtgg gaattgatct 20 <210> SEQ
ID NO 337 <211> LENGTH: 22 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 337 catgcctgct
tcagtcagtt ct 22 <210> SEQ ID NO 338 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 338 tttgcccttc ccaaacccct cca 23 <210>
SEQ ID NO 339 <211> LENGTH: 24 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 339
ccctgaggcc agatacacag atat 24 <210> SEQ ID NO 340 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 340 acggatgact tgccgatgat a 21 <210>
SEQ ID NO 341 <211> LENGTH: 27 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Probe <400> SEQUENCE: 341
ctcactggtt ctgcttgtgc tcctgct 27 <210> SEQ ID NO 342
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer <400> SEQUENCE: 342 gaccaaaacg aacgaaattt
gtt 23 <210> SEQ ID NO 343 <211> LENGTH: 19 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Primer <400>
SEQUENCE: 343 acgtccttga tggcaatcg 19 <210> SEQ ID NO 344
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Probe <400> SEQUENCE: 344 aattccgcgc ggtcgctctg
20 <210> SEQ ID NO 345 <211> LENGTH: 1809 <212>
TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (148)..(804)
<400> SEQUENCE: 345 ctgtcggagc agtaactctg tgcgccccac
gccacaagcg cccagttgct ttgtgggttg 60 tgcctgccct gcgcctgcaa
cttgagtccc cgccgcatcg cagtctccgc gccacctttg 120 taacggcctt
caggaccccg aggtgtc atg gcg aga aag tgg aac ggg cgt gcg 174 Met Ala
Arg Lys Trp Asn Gly Arg Ala 1 5 gtg gcc cga gcc ctg gtc ctg gcc act
ctg tgg ctg gct gtg tct ggg 222 Val Ala Arg Ala Leu Val Leu Ala Thr
Leu Trp Leu Ala Val Ser Gly 10 15 20 25 cgt ccc ctg gct cag caa tcc
cag tct gtg tca gat gaa gat cca ctc 270 Arg Pro Leu Ala Gln Gln Ser
Gln Ser Val Ser Asp Glu Asp Pro Leu 30 35 40 ttt ctc tac ggc tgg
ggc aag att acc cgc ctg cag tac ctg tac tcc 318 Phe Leu Tyr Gly Trp
Gly Lys Ile Thr Arg Leu Gln Tyr Leu Tyr Ser 45 50 55 gct ggt ccc
tat gtc tcc aac tgc ttc ctc cga atc cgg agc gac ggc 366 Ala Gly Pro
Tyr Val Ser Asn Cys Phe Leu Arg Ile Arg Ser Asp Gly 60 65 70 tct
gtg gac tgc gag gag gac caa aac gaa cga aat ttg ttg gaa ttc 414 Ser
Val Asp Cys Glu Glu Asp Gln Asn Glu Arg Asn Leu Leu Glu Phe 75 80
85 cgc gcg gtc gct ctg aag acg att gcc atc aag gac gtc agc agc gtg
462 Arg Ala Val Ala Leu Lys Thr Ile Ala Ile Lys Asp Val Ser Ser Val
90 95 100 105 cgg tac ctc tgc atg agc gcg gac ggc aag ata tac ggg
ctg att cgc 510 Arg Tyr Leu Cys Met Ser Ala Asp Gly Lys Ile Tyr Gly
Leu Ile Arg 110 115 120 tac tcg gag gaa gac tgt acc ttc agg gag gaa
atg gac tgt tta ggc 558 Tyr Ser Glu Glu Asp Cys Thr Phe Arg Glu Glu
Met Asp Cys Leu Gly 125 130 135 tac aac cag tac aga tcc atg aag cac
cat ctc cat atc atc ttc atc 606 Tyr Asn Gln Tyr Arg Ser Met Lys His
His Leu His Ile Ile Phe Ile 140 145 150 cag gcc aag ccc aga gaa cag
ctc cag gac cag aaa ccc tca aac ttt 654 Gln Ala Lys Pro Arg Glu Gln
Leu Gln Asp Gln Lys Pro Ser Asn Phe 155 160 165 atc ccc gtg ttt cac
cgc tcc ttc ttt gaa acc ggg gac cag ctg agg 702 Ile Pro Val Phe His
Arg Ser Phe Phe Glu Thr Gly Asp Gln Leu Arg 170 175 180 185 tct aaa
atg ttc tcc ctg ccc ctg gag agt gac agc atg gat ccg ttc 750 Ser Lys
Met Phe Ser Leu Pro Leu Glu Ser Asp Ser Met Asp Pro Phe 190 195 200
agg atg gtg gag gat gta gac cac cta gtg aag agt ccc agc ttc cag 798
Arg Met Val Glu Asp Val Asp His Leu Val Lys Ser Pro Ser Phe Gln 205
210 215 aaa tga caggattccg acaggatgga gaaaacccca aggtcccgtg
aacttccccc 854 Lys ttaggaagct gtacatattc taagtctcac atggaccctg
ttgtgttagt ggctagactt 914 gatcatgaac ttaagttgac aacctgcctg
gctgccatcg gagccccact gactttggag 974 gctgctgata tgtgcctaag
ttactccagt tctgtttgaa tacctccact aatagggaac 1034 ttactcctgt
gaaacattct tagttttgag ccaaatctgt gacttggatg gttttagcga 1094
ggaagccaga aggtatgaag tcaaatgata aaattcatgt atagaaagtg ggctctaaaa
1154 tatatattcc ctatatggat ctcatgggat cttagcttgc cccccaaatg
tctcctggcc 1214 agaactaact ggggttacaa acttggaaca aaggacagcc
tagaaaactt tgggagcctt 1274 gaaggatggt cttaggatta cgaattccag
ctgactacgt agcttccccc ttttccactt 1334 ataaatgtca gatggaagtg
acccttagct gagtgcatag ccaagctgcc acttaggccc 1394 caggagcttg
tctctgtccc atgaccccag atttccagga cctggatctt ctcctctgac 1454
ctttcccaga gttcacctgg gctctccaac cccagagcag gtagcttatg agccatccag
1514 ttgtgtcccc agctcctggc tctcagttct ggtcaccaaa cattgtgaat
caacgtgtct 1574 gctgcctgtg tcaacctgga cccctcattt acaaacaaga
ttaggaagcc ccaaattctc 1634 cagtggcagc tggggaactg tggagtcctt
tccccggcac ttacgtggca gcatgatatt 1694 tataagtaat ttattgtgtg
tgtgtcttct attttcttac tttatttatg ccccagatca 1754 tatttatgta
catgacttgt tttctacatt aaaaaggagt tggtttgtat caaaa 1809 <210>
SEQ ID NO 346 <211> LENGTH: 2442 <212> TYPE: DNA
<213> ORGANISM: Macaca mulatta <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (1)..(651)
<400> SEQUENCE: 346 tgg gag aac ctg aga cgg tcg gaa ctg cgg
ggg gaa att cta ttg gac 48 Trp Glu Asn Leu Arg Arg Ser Glu Leu Arg
Gly Glu Ile Leu Leu Asp 1 5 10 15 ggc ttt gac gtc aga tgt gct ggg
ctg gga aag tcg ggg gag gga gtg 96 Gly Phe Asp Val Arg Cys Ala Gly
Leu Gly Lys Ser Gly Glu Gly Val 20 25 30 cga gtg gcc ttt taa ggg
gaa gga ccc taa ggc cga ggt gag gct ttt 144 Arg Val Ala Phe Gly Glu
Gly Pro Gly Arg Gly Glu Ala Phe 35 40 45 acc gac tag agg gtg aag
gag gca aaa ctc ggt gcc ccc aaa cct ctg 192 Thr Asp Arg Val Lys Glu
Ala Lys Leu Gly Ala Pro Lys Pro Leu 50 55 60 acc ccg ggg ttc ctg
acc ccg ccc ctg ctg gtg ccc cat gcc gag cgc 240 Thr Pro Gly Phe Leu
Thr Pro Pro Leu Leu Val Pro His Ala Glu Arg 65 70 75 atc cac tgg
gag ccc gac gcc tgg ggg agg ggc ccc agt tgt cga ttg 288 Ile His Trp
Glu Pro Asp Ala Trp Gly Arg Gly Pro Ser Cys Arg Leu 80 85 90 ctt
tgc aaa atc aaa ctc tcc cag cca aga acc tcg ggg ccg ctg cgc 336 Leu
Cys Lys Ile Lys Leu Ser Gln Pro Arg Thr Ser Gly Pro Leu Arg 95 100
105 ggt ggg gag gag ttc ccc gaa acc cgg ccg cta aac gag gcc tcc tcc
384 Gly Gly Glu Glu Phe Pro Glu Thr Arg Pro Leu Asn Glu Ala Ser Ser
110 115 120 125 tcc cgc aga tcc gaa cgg cct ggg cgg ggt cac ccc ggc
tgg gac aag 432 Ser Arg Arg Ser Glu Arg Pro Gly Arg Gly His Pro Gly
Trp Asp Lys 130 135 140 aag ccg ccg cct gcc tgc ccg ggc ccg ggg agg
ggg ctg ggg ccg gag 480 Lys Pro Pro Pro Ala Cys Pro Gly Pro Gly Arg
Gly Leu Gly Pro Glu 145 150 155 gcg ggg tgt gag tgg gtg tgt gcg ggg
ggc gga ggc ttg atg caa tcc 528 Ala Gly Cys Glu Trp Val Cys Ala Gly
Gly Gly Gly Leu Met Gln Ser 160 165 170 cga taa gaa atg ctc ggg tgt
ctt ggg cac cta ccc gcg ggg ccc gta 576 Arg Glu Met Leu Gly Cys Leu
Gly His Leu Pro Ala Gly Pro Val 175 180 185 agg cgc tac tat ata agg
ttg ccg gcc cgg agc cgc cgc gcc gtc gga 624 Arg Arg Tyr Tyr Ile Arg
Leu Pro Ala Arg Ser Arg Arg Ala Val Gly 190 195 200 gca gga gcg ctg
cgt cca gga tcg agg gccacggcca tcccaatccg 671 Ala Gly Ala Leu Arg
Pro Gly Ser Arg 205 210 gcactcacag ccccgcagcg catcccggtc gccgcccagc
ctcccgcacc cccatcgccg 731 gaactgcgcc gagagcccca gggaggtgcc
atgaggagcg ggtgtgtggt ggtccacgcc 791 tggatcctgg ccagcctctg
gctggccgtg gccgggcgtc ccctcgcctt ctcggacgcg 851 gggccccacg
tgcactacgg ctggggcgac cccatccgcc tgcggcacct gtacacctcc 911
ggcccccatg ggctctccag ctgcttcctg cgcatccgca ccgacggcgt cgtggactgc
971 gcgcggggcc aaagcgcgca cagtttgctg gagatcaagg cagtagctct
gcggaccgtg 1031 gccatcaagg gcgtgcacag cgtgcggtac ctctgcatgg
gcgccgacgg caagatgcag 1091 gggctgcttc agtactcaga ggaagactgt
gctttcgagg aggagatccg ccctgatggc 1151 tacaatgtat accgatccga
gaagcaccgc ctcccggtct ctctgagcag tgccaaacag 1211 aggcagctgt
acaagaacag aggctttctt ccgctctctc atttcctacc catgctgccc 1271
atggccccag aggagcctga ggacctcagg ggccacttgg aatctgacat gttctcttcg
1331 cccctggaga ctgacagcat ggacccattt gggcttgtca ccggactgga
ggcggtgagg 1391 agtcccagct ttgagaaata actgaggcca tgcccgggcc
tcttcactgc tgccaggggc 1451 tgtggtacct gcagagtgga ggccgtgctt
ctacaagagc agtcccgagt ccacgttctg 1511 tttagcttta ggaagaaaca
tctagaagtt gtacatattc agagttttcc attggccgtg 1571 ccagtttcta
gccaatagac ttgtctgatc ataacattgt aagcttgtag cttgcccagc 1631
tgctgcccgg gcccccattc tgctccctcg aggttgctgg acaagctgct gtgctgtctc
1691 agtcctgctt gaatacctcc actgatgggg aactcacttc ctttggaaaa
attcttatgt 1751 caagctgaaa ttctctaatt tttttttctc atcacttccc
caggagcagc cggaagatag 1811 gcagtggttt aaatttcagg aacaggtgat
ccactctgta aaacagcacg tacatttcac 1871 tcaaccccat gtgggaattg
atctatatct acttccaggg accgtttgcc cttcccaaac 1931 ccctccaggc
cagaactgac tgaagcaggc atggcccaag aggcttcagg agtaggggaa 1991
gcccagagcc ccactccagc cctgggacat cttgagaatt ccccctgagg ccagttctgt
2051 catggatgct gtcctgagaa taacttgctg tccccggtgt cacctgcttc
caccccccag 2111 cccaccagcc ctctgcccac ctcacatgtc tccccatgga
ttggggcttc ccaggccccc 2171 catcttatgt caacctgcac ttctcgttca
aaaatcagga aaagaaaaga tttgaagacc 2231 ccaagtcttg tcaataactt
gcggtgtgga agcagcgggg gaagacttag aaccctttcc 2291 ccagcactta
gttttccaac atgatattta tgagtaattt attttgatat gtacatctct 2351
tattttctta cattatttat gcccccaaat tatatttatg tatgtaagtg aggtttgttt
2411 tgtacattaa aatggagttt gtttgtatga a 2442 <210> SEQ ID NO
347 <211> LENGTH: 651 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens 220> <221> NAME/KEY: CDS
<222> LOCATION: (1)..(651) <400> SEQUENCE: 347 atg cgg
agc ggg tgt gtg gtg gtc cac gta tgg atc ctg gcc ggc ctc 48 Met Arg
Ser Gly Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15
tgg ctg gcc gtg gcc ggg cgc ccc ctc gcc ttc tcg gac gcg ggg ccc 96
Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20
25 30 cac gtg cac tac ggc tgg ggc gac ccc atc cgc ctg cgg cac ctg
tac 144 His Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu
Tyr 35 40 45 acc tcc ggc ccc cac ggg ctc tcc agc tgc ttc ctg cgc
atc cgt gcc 192 Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg
Ile Arg Ala 50 55 60 gac ggc gtc gtg gac tgc gcg cgg ggc cag agc
gcg cac agt ttg ctg 240 Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser
Ala His Ser Leu Leu 65 70 75 80 gag atc aag gca gtc gct ctg cgg acc
gtg gcc atc aag ggc gtg cac 288 Glu Ile Lys Ala Val Ala Leu Arg Thr
Val Ala Ile Lys Gly Val His 85 90 95 agc gtg cgg tac ctc tgc atg
ggc gcc gac ggc aag atg cag ggg ctg 336 Ser Val Arg Tyr Leu Cys Met
Gly Ala Asp Gly Lys Met Gln Gly Leu 100 105 110 ctt cag tac tcg gag
gaa gac tgt gct ttc gag gag gag atc cgc cca 384 Leu Gln Tyr Ser Glu
Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro 115 120 125 gat ggc tac
aat gtg tac cga tcc gag aag cac cgc ctc ccg gtc tcc 432 Asp Gly Tyr
Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser 130 135 140 ctg
agc agt gcc aaa cag cgg cag ctg tac aag aac aga ggc ttt ctt 480 Leu
Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu 145 150
155 160 cca ctc tct cat ttc ctg ccc atg ctg ccc atg gtc cca gag gag
cct 528 Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu
Pro 165 170 175 gag gac ctc agg ggc cac ttg gaa tct gac atg ttc tct
tcg ccc ctg 576 Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser
Ser Pro Leu 180 185 190 gag acc gac agc atg gac cca ttt ggg ctt gtc
acc gga ctg gag gcc 624 Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val
Thr Gly Leu Glu Ala 195 200 205 gtg agg agt ccc agc ttt gag aag taa
651 Val Arg Ser Pro Ser Phe Glu Lys 210 215 <210> SEQ ID NO
348 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 348 gcaccaggga
tgagcttgac 20 <210> SEQ ID NO 349 <211> LENGTH: 22
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 349 ccaagtctcc cactttccag tt 22 <210>
SEQ ID NO 350 <211> LENGTH: 17 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Probe <400> SEQUENCE: 350
aagagcctga ctccagt 17
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 350
<210> SEQ ID NO 1 <211> LENGTH: 3040 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (168)..(2576)
<400> SEQUENCE: 1 ctcgctcccg gccgaggagc gctcgggctg tctgcggacc
ctgccgcgtg caggggtcgc 60 ggccggctgg agctgggagt gaggcggcgg
aggagccagg tgaggaggag ccaggaaggc 120 agttggtggg aagtccagct
tgggtccctg agagctgtga gaaggag atg cgg ctg 176 Met Arg Leu 1 ctg ctg
gcc ctg ttg ggg gtc ctg ctg agt gtg cct ggg cct cca gtc 224 Leu Leu
Ala Leu Leu Gly Val Leu Leu Ser Val Pro Gly Pro Pro Val 5 10 15 ttg
tcc ctg gag gcc tct gag gaa gtg gag ctt gag ccc tgc ctg gct 272 Leu
Ser Leu Glu Ala Ser Glu Glu Val Glu Leu Glu Pro Cys Leu Ala 20 25
30 35 ccc agc ctg gag cag caa gag cag gag ctg aca gta gcc ctt ggg
cag 320 Pro Ser Leu Glu Gln Gln Glu Gln Glu Leu Thr Val Ala Leu Gly
Gln 40 45 50 cct gtg cgt ctg tgc tgt ggg cgg gct gag cgt ggt ggc
cac tgg tac 368 Pro Val Arg Leu Cys Cys Gly Arg Ala Glu Arg Gly Gly
His Trp Tyr 55 60 65 aag gag ggc agt cgc ctg gca cct gct ggc cgt
gta cgg ggc tgg agg 416 Lys Glu Gly Ser Arg Leu Ala Pro Ala Gly Arg
Val Arg Gly Trp Arg 70 75 80 ggc cgc cta gag att gcc agc ttc cta
cct gag gat gct ggc cgc tac 464 Gly Arg Leu Glu Ile Ala Ser Phe Leu
Pro Glu Asp Ala Gly Arg Tyr 85 90 95 ctc tgc ctg gca cga ggc tcc
atg atc gtc ctg cag aat ctc acc ttg 512 Leu Cys Leu Ala Arg Gly Ser
Met Ile Val Leu Gln Asn Leu Thr Leu 100 105 110 115 att aca ggt gac
tcc ttg acc tcc agc aac gat gat gag gac ccc aag 560 Ile Thr Gly Asp
Ser Leu Thr Ser Ser Asn Asp Asp Glu Asp Pro Lys 120 125 130 tcc cat
agg gac ccc tcg aat agg cac agt tac ccc cag caa gca ccc 608 Ser His
Arg Asp Pro Ser Asn Arg His Ser Tyr Pro Gln Gln Ala Pro 135 140 145
tac tgg aca cac ccc cag cgc atg gag aag aaa ctg cat gca gta cct 656
Tyr Trp Thr His Pro Gln Arg Met Glu Lys Lys Leu His Ala Val Pro 150
155 160 gcg ggg aac acc gtc aag ttc cgc tgt cca gct gca ggc aac ccc
acg 704 Ala Gly Asn Thr Val Lys Phe Arg Cys Pro Ala Ala Gly Asn Pro
Thr 165 170 175 ccc acc atc cgc tgg ctt aag gat gga cag gcc ttt cat
ggg gag aac 752 Pro Thr Ile Arg Trp Leu Lys Asp Gly Gln Ala Phe His
Gly Glu Asn 180 185 190 195 cgc att gga ggc att cgg ctg cgc cat cag
cac tgg agt ctc gtg atg 800 Arg Ile Gly Gly Ile Arg Leu Arg His Gln
His Trp Ser Leu Val Met 200 205 210 gag agc gtg gtg ccc tcg gac cgc
ggc aca tac acc tgc ctg gta gag 848 Glu Ser Val Val Pro Ser Asp Arg
Gly Thr Tyr Thr Cys Leu Val Glu 215 220 225 aac gct gtg ggc agc atc
cgc tat aac tac ctg cta gat gtg ctg gag 896 Asn Ala Val Gly Ser Ile
Arg Tyr Asn Tyr Leu Leu Asp Val Leu Glu 230 235 240 cgg tcc ccg cac
cgg ccc atc ctg cag gcc ggg ctc ccg gcc aac acc 944 Arg Ser Pro His
Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn Thr 245 250 255 aca gcc
gtg gtg ggc agc gac gtg gag ctg ctg tgc aag gtg tac agc 992 Thr Ala
Val Val Gly Ser Asp Val Glu Leu Leu Cys Lys Val Tyr Ser 260 265 270
275 gat gcc cag ccc cac atc cag tgg ctg aag cac atc gtc atc aac ggc
1040 Asp Ala Gln Pro His Ile Gln Trp Leu Lys His Ile Val Ile Asn
Gly 280 285 290 agc agc ttc gga gcc gac ggt ttc ccc tat gtg caa gtc
cta aag act 1088 Ser Ser Phe Gly Ala Asp Gly Phe Pro Tyr Val Gln
Val Leu Lys Thr 295 300 305 gca gac atc aat agc tca gag gtg gag gtc
ctg tac ctg cgg aac gtg 1136 Ala Asp Ile Asn Ser Ser Glu Val Glu
Val Leu Tyr Leu Arg Asn Val 310 315 320 tca gcc gag gac gca ggc gag
tac acc tgc ctc gca ggc aat tcc atc 1184 Ser Ala Glu Asp Ala Gly
Glu Tyr Thr Cys Leu Ala Gly Asn Ser Ile 325 330 335 ggc ctc tcc tac
cag tct gcc tgg ctc acg gtg ctg cca gag gag gac 1232 Gly Leu Ser
Tyr Gln Ser Ala Trp Leu Thr Val Leu Pro Glu Glu Asp 340 345 350 355
ccc aca tgg acc gca gca gcg ccc gag gcc agg tat acg gac atc atc
1280 Pro Thr Trp Thr Ala Ala Ala Pro Glu Ala Arg Tyr Thr Asp Ile
Ile 360 365 370 ctg tac gcg tcg ggc tcc ctg gcc ttg gct gtg ctc ctg
ctg ctg gcc 1328 Leu Tyr Ala Ser Gly Ser Leu Ala Leu Ala Val Leu
Leu Leu Leu Ala 375 380 385 ggg ctg tat cga ggg cag gcg ctc cac ggc
cgg cac ccc cgc ccg ccc 1376 Gly Leu Tyr Arg Gly Gln Ala Leu His
Gly Arg His Pro Arg Pro Pro 390 395 400 gcc act gtg cag aag ctc tcc
cgc ttc cct ctg gcc cga cag ttc tcc 1424 Ala Thr Val Gln Lys Leu
Ser Arg Phe Pro Leu Ala Arg Gln Phe Ser 405 410 415 ctg gag tca ggc
tct tcc ggc aag tca agc tca tcc ctg gta cga ggc 1472 Leu Glu Ser
Gly Ser Ser Gly Lys Ser Ser Ser Ser Leu Val Arg Gly 420 425 430 435
gtg cgt ctc tcc tcc agc ggc ccc gcc ttg ctc gcc ggc ctc gtg agt
1520 Val Arg Leu Ser Ser Ser Gly Pro Ala Leu Leu Ala Gly Leu Val
Ser 440 445 450 cta gat cta cct ctc gac cca cta tgg gag ttc ccc cgg
gac agg ctg 1568 Leu Asp Leu Pro Leu Asp Pro Leu Trp Glu Phe Pro
Arg Asp Arg Leu 455 460 465 gtg ctt ggg aag ccc cta ggc gag ggc tgc
ttt ggc cag gta gta cgt 1616 Val Leu Gly Lys Pro Leu Gly Glu Gly
Cys Phe Gly Gln Val Val Arg 470 475 480 gca gag gcc ttt ggc atg gac
cct gcc cgg cct gac caa gcc agc act 1664 Ala Glu Ala Phe Gly Met
Asp Pro Ala Arg Pro Asp Gln Ala Ser Thr 485 490 495 gtg gcc gtc aag
atg ctc aaa gac aac gcc tct gac aag gac ctg gcc 1712 Val Ala Val
Lys Met Leu Lys Asp Asn Ala Ser Asp Lys Asp Leu Ala 500 505 510 515
gac ctg gtc tcg gag atg gag gtg atg aag ctg atc ggc cga cac aag
1760 Asp Leu Val Ser Glu Met Glu Val Met Lys Leu Ile Gly Arg His
Lys 520 525 530 aac atc atc aac ctg ctt ggt gtc tgc acc cag gaa ggg
ccc ctg tac 1808 Asn Ile Ile Asn Leu Leu Gly Val Cys Thr Gln Glu
Gly Pro Leu Tyr 535 540 545 gtg atc gtg gag tgc gcc gcc aag gga aac
ctg cgg gag ttc ctg cgg 1856 Val Ile Val Glu Cys Ala Ala Lys Gly
Asn Leu Arg Glu Phe Leu Arg 550 555 560 gcc cgg cgc ccc cca ggc ccc
gac ctc agc ccc gac ggt cct cgg agc 1904 Ala Arg Arg Pro Pro Gly
Pro Asp Leu Ser Pro Asp Gly Pro Arg Ser 565 570 575 agt gag ggg ccg
ctc tcc ttc cca gtc ctg gtc tcc tgc gcc tac cag 1952 Ser Glu Gly
Pro Leu Ser Phe Pro Val Leu Val Ser Cys Ala Tyr Gln 580 585 590 595
gtg gcc cga ggc atg cag tat ctg gag tcc cgg aag tgt atc cac cgg
2000 Val Ala Arg Gly Met Gln Tyr Leu Glu Ser Arg Lys Cys Ile His
Arg 600 605 610 gac ctg gct gcc cgc aat gtg ctg gtg act gag gac aat
gtg atg aag 2048 Asp Leu Ala Ala Arg Asn Val Leu Val Thr Glu Asp
Asn Val Met Lys 615 620 625 att gct gac ttt ggg ctg gcc cgc ggc gtc
cac cac att gac tac tat 2096 Ile Ala Asp Phe Gly Leu Ala Arg Gly
Val His His Ile Asp Tyr Tyr 630 635 640 aag aaa acc agc aac ggc cgc
ctg cct gtg aag tgg atg gcg ccc gag 2144 Lys Lys Thr Ser Asn Gly
Arg Leu Pro Val Lys Trp Met Ala Pro Glu 645 650 655 gcc ttg ttt gac
cgg gtg tac aca cac cag agt gac gtg tgg tct ttt 2192 Ala Leu Phe
Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp Ser Phe 660 665 670 675
ggg atc ctg cta tgg gag atc ttc acc ctc ggg ggc tcc ccg tat cct
2240 Gly Ile Leu Leu Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro Tyr
Pro 680 685 690 ggc atc ccg gtg gag gag ctg ttc tcg ctg ctg cgg gag
gga cat cgg 2288 Gly Ile Pro Val Glu Glu Leu Phe Ser Leu Leu Arg
Glu Gly His Arg 695 700 705 atg gac cga ccc cca cac tgc ccc cca gag
ctg tac ggg ctg atg cgt 2336 Met Asp Arg Pro Pro His Cys Pro Pro
Glu Leu Tyr Gly Leu Met Arg 710 715 720 gag tgc tgg cac gca gcg ccc
tcc cag agg cct acc ttc aag cag ctg 2384 Glu Cys Trp His Ala Ala
Pro Ser Gln Arg Pro Thr Phe Lys Gln Leu 725 730 735 gtg gag gcg ctg
gac aag gtc ctg ctg gcc gtc tct gag gag tac ctc 2432 Val Glu Ala
Leu Asp Lys Val Leu Leu Ala Val Ser Glu Glu Tyr Leu 740 745 750 755
gac ctc cgc ctg acc ttc gga ccc tat tcc ccc tct ggt ggg gac gcc
2480 Asp Leu Arg Leu Thr Phe Gly Pro Tyr Ser Pro Ser Gly Gly Asp
Ala 760 765 770 agc agc acc tgc tcc tcc agc gat tct gtc ttc agc cac
gac ccc ctg 2528 Ser Ser Thr Cys Ser Ser Ser Asp Ser Val Phe Ser
His Asp Pro Leu 775 780 785 cca ttg gga tcc agc tcc ttc ccc ttc ggg
tct ggg gtg cag aca tga 2576 Pro Leu Gly Ser Ser Ser Phe Pro Phe
Gly Ser Gly Val Gln Thr 790 795 800 gcaaggctca aggctgtgca
ggcacatagg ctggtggcct tgggccttgg ggctcagcca 2636 cagcctgaca
cagtgctcga ccttgatagc atggggcccc tggcccagag ttgctgtgcc 2696
gtgtccaagg gccgtgccct tgcccttgga gctgccgtgc ctgtgtcctg atggcccaaa
2756 tgtcagggtt ctgctcggct tcttggacct tggcgcttag tccccatccc
gggtttggct 2816 gagcctggct ggagagctgc tatgctaaac ctcctgcctc
ccaataccag caggaggttc 2876 tgggcctctg aacccccttt ccccacacct
ccccctgctg ctgctgcccc agcgtcttga 2936 cgggagcatt ggcccctgag
cccagagaag ctggaagcct gccgaaaaca ggagcaaatg 2996 gcgttttata
aattattttt ttgaaataaa aaaaaaaaaa aaaa 3040 <210> SEQ ID NO 2
<211> LENGTH: 12196 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 2 tgagggagca
gaaggaaggg gttctcctat ccgctgcacg gcactgcgca gaagacaggg 60
gagccaggca ttccctgaag ggtgaaaagc aaggagtaga gctgggtagt agactagaat
120 ttaggagcct ggcctggggc ctgggtgggg cgaaagaggc ggagcctgaa
tggggtgtgt 180 ataggggggt tgcgtgtagg ggtgtgtgta taggctgggg
cggggtcccg ggagtgggct 240 gactgggtcg ggggcggggc tctccaggtg
ggcggggatc ttggccaccc ctggccacac 300 ctctctccgg ctcgagctgg
tctaggcggg gcgggcccga gggggtgtgg caggaggtgg 360 gcgggcccgg
gtgggggggg ggggggcgtg gaaggagggg cgggcccgag caggaggggg 420
cgggcccgag gggcggggtg ggacaggagg tgggccgctc gcggccacgc cgccgtcgcg
480 ggtacattcc tcgctcccgg ccgaggagcg ctcgggctgt ctgcggaccc
tgccgcgtgc 540 aggggtcgcg gccggctgga gctgggagtg aggcggcgga
ggagccaggt gaggaggagc 600 caggtgagca ggaccctgtg ctgggcgcgg
agtcacgcag gctcgaggtg agccggaacc 660 cttgtgggcc cgggctgcgc
tcccagccgc cagggggcga gaggcggcgg ggctacgggg 720 actgcccctc
ccggcgcagg ggacctgggc gtccgccggg cggcaggggg tggagggggc 780
ggtaaatcag taacccgcag tgcacacagg gccttttgtc ccgctccgtc caaagagcac
840 cccggccgcg gagctggtta ctcattgccc accgaggcgg gggcaggctg
gccctgtgca 900 gctaccctcg ggacccattg attcgcacct ccccccaggc
tggcccggca agggtggggg 960 aggacaagcg cgcttgtccc tgcggctgtc
ttcgcgccgg cggcagagat gagggacctg 1020 aggccccgaa aagttcagtc
acttagtgcc cgggggcctc cagcgcgagt gcgggaggct 1080 gaaggagaac
ccaggactgt ctgatgccta aggcaggccc tccattccca cgtggggggt 1140
ggtcggtcag cggtcagcag ccatgggtga ctcgactaag gactctgata tcagggcagc
1200 ctggggtagg aataaactcc ccgggcctcc ccacccactc ccagcccaag
ctgtgtaccc 1260 aaagagctgc cctccctgcc aagccgagct tggtagggag
ttttaccaag gaggatccga 1320 ctggattcga gagttgaggt gggccagaga
cagcagtatc tgagtcaggt agagaagagc 1380 aatgaggggc acagagggat
gggcaagaga gcacatgtgc ccagttttga aagccaatgg 1440 cttcagcgct
cctgaagggg cagacggtgt gaccaaagag ataggcagcg gcagagaggg 1500
agccctagga tgttgagctg gatcctgctg ggcacaggta gccattaagg gcttgcaagc
1560 tggggggcat gacatggcag acttgcaggt ttttttgttt gtttttttat
tttattttat 1620 ttttttattt tgtttttttt gagacggagt ctcactctgt
cgcccaggct ggagtgcagt 1680 ggcgcgatct cggctcactg caagctccgc
ctcccgggtt cgcgccattc tcctgcctca 1740 gcctcccgag tagctgggac
tacaggcgcc cgccaccgcg cccggctaat tttttgtatt 1800 tttagtagag
acggggtttc accgtgttag ccaggatgtt ctcgatctcc tgacctcgtg 1860
atccgcccac ctcggcctcc caaagtgctg ggattacagg tgtgaaccat cgcgcccagc
1920 cgacttgtag ttttttaaaa ctctgctgga agatgaaggt tgaagagccg
agggagagga 1980 tgtttccaga ggcccatgca agagatggcc atgacctgcc
ttgagaaggg gcaggggaag 2040 ccagatggac tggaagtgga gtggcagtga
ccaaggagga ggaggtgtga taggcttccc 2100 acgcagggta gatccagaga
caccagtgcc acccataggc ccctaggact gcagtggtca 2160 ccgattcctt
tgtcccagct gagactcagt tctgagtgtt ctattttggg gaacagaggc 2220
gtccttggta gcatttggaa gaggatagcc agctggggtg tgtgtacatc acagcctgac
2280 agtaacagca tccgaaccag aggtgactgg ctaagggcag acccagggca
acaggttaac 2340 cgttctaggg ccgggcacag ggaggagaac attccaacac
tctgcgtgcc gacgcacgtt 2400 ctctctttta tcctcaaaac agtcctatga
ggatagtaag ccagagagag acagagacaa 2460 ggaattacaa gttggtgaga
gtcaggattt gaacttggct ctggcagatg gaaaattagg 2520 gtctgtattc
tttacaaaac cgtgtgtgcc tcagatggag ttggtgcata acaagcagag 2580
gtatccaggg tcgcggtcct gcttgccacg gaaggggccg ccttgtcagt tgtgaccacc
2640 cagccctgga aatgtcagta atgctgtaag gagtggggat cggatcagat
gccatccaga 2700 tgctgaagtt tgaccttgtg tcatttttca ctttcttttt
tggctcttct gcaatcaatt 2760 catttattta gcaaaaaaga aattatgtgt
gccgagagca tgcagaagat atgtctccgt 2820 tctctgcttc cctccaaaaa
agaatcccaa aactgctttc tgtgaacgtg tgccagggtc 2880 ccagcaggac
tcagggagag caggaagccc agcccagacc ccttgcacaa cctaccgtgg 2940
ggaggcctta ggctctggct actacagagc tggttccagt ctgcactgcc acagcctggc
3000 cagggacttg gacacatctg ctggccactt cctgtctcag tttccttatc
tgcaaaataa 3060 gggaaaagcc cccacaaagg tgcacgtgta gcaggagctc
ttttccctcc ctattttagg 3120 aaggcagttg gtgggaagtc cagcttgggt
ccctgagagc tgtgagaagg agatgcggct 3180 gctgctggcc ctgttggggg
tcctgctgag tgtgcctggg cctccagtct tgtccctgga 3240 ggcctctgag
gaagtggagc ttggtatggc ttctgaggtg ggagagggtg gcaggggtgg 3300
gaagagtggg caccaggagg gggctgctgg gctgagcaaa gctggaaagg atccttgccc
3360 aggccctgag aaggtggcgg cagggcaggg ctcaaccact gagactcagt
cagtgcctgg 3420 cttccagcaa gcattcatct atcactgtgt ctgcgagaga
ggactggcct tgcagggcgc 3480 agggccctaa gctgggctgc agagctggtg
gtgagctcct tacctgggtg tgtgtgcgtg 3540 tgtgtgtgtg ttctgtgcac
tgggtgtgtg acctaggagg tccaggcagc atgtgtggta 3600 taagcattat
gagggtgata tgccccggtg cagcatgacc ctgtatgtgg caccaacagc 3660
atgtgccttg tgtgtgtgtg tgtccgtatg tgtgtgtgtg tatgcgtgtg tgtgtgtgtg
3720 tgtgtcttgg ccactgtcgt gtgcactaaa tgctgtgtgt gtgacatgcc
ccaagagtgt 3780 ggcatttgcc ctgggtgtgg catccgcagc atgtggctgt
gtgggtgtca aggagtggtg 3840 gctccttcag catgcgttgc aaagtgcttg
tgccctgcat gtgcggtgtg ttctttgtac 3900 acaggaggct gcctcagatg
gggctgcggg gtctgctgac ctctgccctc tgcccacaga 3960 gccctgcctg
gctcccagcc tggagcagca agagcaggag ctgacagtag cccttgggca 4020
gcctgtgcgt ctgtgctgtg ggcgggctga gcgtggtggc cactggtaca aggagggcag
4080 tcgcctggca cctgctggcc gtgtacgggg ctggaggggc cgcctagaga
ttgccagctt 4140 cctacctgag gatgctggcc gctacctctg cctggcacga
ggctccatga tcgtcctgca 4200 gaatctcacc ttgattacag gtggtaagag
actctagcag ggagtgaagg gatgcctggg 4260 gagacagacc tgcccctctt
ggaccttaga tgcttccctc tgtccctgat gtagactcct 4320 tgacctccag
caacgatgat gaggacccca agtcccatag ggacccctcg aataggcaca 4380
gttaccccca gcaaggtcag taggtctcca aggacttgtg tccccgctgc tgctcatctg
4440 atcactgaga agaggaggcc tgtgtgggaa cacacggtca ttctaggggc
cttcccctgc 4500 cctccagcac cctactggac acacccccag cgcatggaga
agaaactgca tgcagtacct 4560 gcggggaaca ccgtcaagtt ccgctgtcca
gctgcaggca accccacgcc caccatccgc 4620 tggcttaagg atggacaggc
ctttcatggg gagaaccgca ttggaggcat tcgggtgagt 4680 ctctgggttc
caagaccgtc tgctccccca ttttcattcc ttcatcagtc ccctcatacc 4740
tacaagcata cctataaatc aatcgaatga gtgaagcgat tgcggggccc cggaaggagc
4800 cctggactgt ggacctgggc agctctggtt ccccttctgc tactctctgg
caagtgactt 4860 aacctctcag cctcagcaac tccatttgta aagggagaag
aatcactgac tggttggtct 4920 gcataagcct tagcatctca tcgtcttgat
gagaccctgc agggtcggct ccatgctgtc 4980 atgaggcaac tgagtctcag
agaaggcaag ggttggctca aagtagcaca gctagggaga 5040 gggagagcta
aaattccaaa ggctcaaacc caaggctcaa gcgccctggg gagcctactc 5100
ctttgtgcca tagtccttgg cctgggcctg atgttctcag ggcctagaga gcttgacaag
5160 agccctgtgg gcaggatgag gatctagcct cctggtcctc tggccccctt
ggtggacatg 5220 gtccggtggt cccggacact ctctctgcct gcagctgcgc
catcagcact ggagtctcgt 5280 gatggagagc gtggtgccct cggaccgcgg
cacatacacc tgcctggtag agaacgctgt 5340 gggcagcatc cgctataact
acctgctaga tgtgctgggt gagcgcgggg ctgggaacag 5400 gggaggcctg
acccattttg ggctcagttg tgccctcttg gtggggtcta gtctggcagg 5460
caggatggac tcagatgagt caggcagctt ggtgagcagg tgggtcaggg gaaagcacag
5520 gggttagtgt ggggctggag gagcagaggt ctgccaagag gaaaaacaag
aaggacatcc 5580 aggcagaggg cgcagcccga gcggagggcc tgagtataac
aaacgccctg cacttgcagg 5640 ccagcatatt cgtagggcgt ggcgtttata
tggggagcca ggtggtggag ggttttgaat 5700 gctaggctga gatgttgtcc
ttgacccgaa gcaataggga gccagggaag gtttaagcag 5760 ggtaagcagg
agacagacaa gaagctgcag aaaggtccct cccttgaact tgaggaaggc 5820
tggagggagg caaacagggt gcttctatgg gtgccggtgg tcagggttga ctgtctcgcc
5880 cggtccccag agcggtcccc gcaccggccc atcctgcagg ccgggctccc
ggccaacacc 5940 acagccgtgg tgggcagcga cgtggagctg ctgtgcaagg
tgtacagcga tgcccagccc 6000 cacatccagt ggctgaagca catcgtcatc
aacggcagca gcttcggagc cgacggtttc 6060 ccctatgtgc aagtcctaaa
ggtaaaaggt gcaccctgct gcagcctggg ccccattctt 6120 ctcccacctt
gggttggggg gctccccagc ttccctgttg gccacagtgt ggccccaggc 6180
cctgctgtga ccccagagca tgtcccccac cccagactgc agacatcaat agctcagagg
6240 tggaggtcct gtacctgcgg aacgtgtcag ccgaggacgc aggcgagtac
acctgcctcg 6300 caggcaattc catcggcctc tcctaccagt ctgcctggct
cacggtgctg ccaggtgagc 6360 acctgaaggg ccaggagatg ctgcgagatg
cccctctggg ccagcagtgg gggctgtggc 6420 ctgttgggtg gtcagtctct
gttggcctgt ggggtctggc ctggggggca gtgtgtggat 6480 ttgtgggttt
gagctgtatg acagcccctc tgtgcctctc cacacgtggc cgtccatgtg 6540
accgtctgct gaggtgtggg tgcctgggac tgggcataac tacagcttcc tccgtgtgtg
6600 tccccacata tgttgggagc tgggagggac tgagttaggg tgcacggggc
ggccagtctc 6660 accactgacc agtttgtctg tctgtgtgtg tccatgtgcg
agggcagagg aggaccccac 6720 atggaccgca gcagcgcccg aggccaggta
tacggacatc atcctgtacg cgtcgggctc 6780 cctggccttg gctgtgctcc
tgctgctggc cgggctgtat cgagggcagg cgctccacgg 6840 ccggcacccc
cgcccgcccg ccactgtgca gaagctctcc cgcttccctc tggcccgaca 6900
ggtactgggc gcatccccca cctcacatgt gacagcctga ctccagcagg cagaaccaag
6960 tctcccactt tgcagttctc cctggagtca ggctcttccg gcaagtcaag
ctcatccctg 7020 gtacgaggcg tgcgtctctc ctccagcggc cccgccttgc
tcgccggcct cgtgagtcta 7080 gatctacctc tcgacccact atgggagttc
ccccgggaca ggtgcgctga gctgtgtggg 7140 ggcagggacg cgggcgccgg
gttgcagccc gccctccgca ggagtgactc ggaggtctga 7200 ggctggactt
tctccatctc caggctggtg cttgggaagc ccctaggcga gggctgcttt 7260
ggccaggtag tacgtgcaga ggcctttggc atggaccctg cccggcctga ccaagccagc
7320 actgtggccg tcaagatgct caaaggtgag tgtggcccgg tgtggtggct
cacacctgta 7380 acgccagcac tttaggaggc tgagggtggg aggatcgctt
gaatccagga attcgaggcc 7440 agcctgggca acatggcaag acttcatctc
tacaaaaaaa aaataagaaa attagttggg 7500 tgtggtggtg tgtgccttta
gtctcagtta ctagggaggc tgaggcagga ggatcccttg 7560 aatccaggag
ttggaggttg cagggagcca tgatcacgcc actgtattcc agcctgggca 7620
acacagtgag accctatctg aaaaaataaa taaataaata aaaataaaag gtgaacgtgg
7680 cagcctggag gaggtgctat ggcattggga ctaatagaag gggctcacgg
tgccaccagg 7740 tgagccctgg agctgggaga ggctgtggga tcccaccctt
aaacctgcaa ttcacctctg 7800 ctcctgaccc tggcaagtga cttctgagcc
tcagttttcc cttgtgtcat atggggtaga 7860 taacagtccc tactcccagc
ccaaggattg tggaaagtgc ctggctcata gtcagggctc 7920 aataaatctt
caccactggg gtgatgatga tgagaagaat ttggtgtgac aggcttgata 7980
tcctgtgtca gcattagtct gtgtcagctt tgacttcaca tctccttgtc agcctcacag
8040 gccctctacc tccttcctta tggttccccc cagacacacc ctcagcctcc
cttggaccct 8100 ccctaggtct gccccccacg tccactgctg taggaggaca
gcccttctgc ttgcacccag 8160 gcccagcccc ggggtgctct tgctgggcac
tcctgcaccc cacccatcag ggcctctcct 8220 tgcagttccc cagccccctc
tgcaagaatg gcctccactg ctcttctgct cctcccctcc 8280 tctctacaca
gctggggcca cctggtgctc cctgggaggc agggattgag aaatgcacat 8340
tgtgtcattg gcccagggcc acaggtcagc cccaggggct cagccagaga agccaaagca
8400 gccttcttcc caagctcccc ggctgcaccc ggcctgccgc cagctccctg
aattcccagg 8460 ccagttggaa gccaggccct ggtcaaacag accccagggc
gccagcctgc tttccgcacc 8520 cagaagctct gaccccatgc ggggactacc
gctgacccct ccagcggcag cttccttcct 8580 tccttcctgc tccgagctct
tcccctctct cctgtgtcct gggcctgccc gctggaaggc 8640 ctgcctctta
gatccttgat acagttgcat ccttgcaact gctgtgacag gcagggtgtg 8700
acccactgct ctgtttccca caagacgaac ctgaggttca gagacgctag gagacttttt
8760 caaggccaca cagcctagca aggattcagc cctagaccta cgtagccctg
gtccagtgct 8820 gcttgtcctg cacctgcctc tgcatgctcc ctcgtgcagt
tggagggcag cctcttcacc 8880 ccgtctgctg cccttacaga caacgcctct
gacaaggacc tggccgacct ggtctcggag 8940 atggaggtga tgaagctgat
cggccgacac aagaacatca tcaacctgct tggtgtctgc 9000 acccaggaag
gtggggccga ggcggggctg gctgcacggg ccgttagggt gcagagccaa 9060
agctttggca gcctctccac gctccctcca ctccctctgc agggcccctg tacgtgatcg
9120 tggagtgcgc cgccaaggga aacctgcggg agttcctgcg ggcccggcgc
cccccaggcc 9180 ccgacctcag ccccgacggt cctcggagca gtgaggggcc
gctctccttc ccagtcctgg 9240 tctcctgcgc ctaccaggtg gcccgaggca
tgcagtatct ggagtcccgg aaggtacagg 9300 cgctagggct ctgagcccct
ctcagtctct ccagctccac tctcaggcct gtggcattca 9360 atgtcccgac
ttctccctct ctgctctttt tcatgacccc acctcagtgt ccccaggcat 9420
tcacgctttc ctgcattccc cactcgttcc tcacccttcc ccagagggga gaggggacgc
9480 aggagaaggc actccccgtt tctaaacctt gacctcctcc tctgtaaagt
gggtggaggg 9540 cccctgcccc cgggcctgct ggggggtggt gtgtgctcaa
ctccaggcca ggtgtcctga 9600 ggcacccaag cccccgctcc ctgcagtgta
tccaccggga cctggctgcc cgcaatgtgc 9660 tggtgactga ggacaatgtg
atgaagattg ctgactttgg gctggcccgc ggcgtccacc 9720 acattgacta
ctataagaaa accagcaacg tgagggagat ggggcagaac tggatggggg 9780
tggaggggca ctgggcccgg ggtggcaggc acgaggacct gtgggactct gcactgaggc
9840 cctctctccc ctccagggcc gcctgcctgt gaagtggatg gcgcccgagg
ccttgtttga 9900 ccgggtgtac acacaccaga gtgacgtgtg agtcctgccg
gcggtcactg tcctacccca 9960 caaaaagggc aaggcactgc ccaaagtcac
gtggccccag gagtcatgcg ctcgagggct 10020 ccttcagatt tggtctggga
cccgagtggg cccagactcc aggaggagcc cattccccaa 10080 cagctgtggt
gggtcatgtc tgtggggtcc cccgtcctag ccccggtcgt cgggagggcg 10140
ctgagccaca ctgagccctg gccctacctc caggtggtct tttgggatcc tgctatggga
10200 gatcttcacc ctcgggggct ccccgtatcc tggcatcccg gtggaggagc
tgttctcgct 10260 gctgcgggag ggacatcgga tggaccgacc cccacactgc
cccccagagc tgtgaggcct 10320 caccctgccc tcgaccccac tttccagtcc
tcctcctcct ctgccctgac catggcctca 10380 gggtgtgtcc cggccagaag
gacaacacta acaacaactc ctcgtcctcc tcctcctctt 10440 cctcttcctc
ctcctcctct tcctcctcct cctcttcctc ctcctcttcc tcctcctcct 10500
cttcctcctc ctcctcttcc tccttctcct cctgctcctc ttcctcctcc ttctcttcct
10560 cctcctcctc ttcctcctcc tcctcttcct cctcctcctc ttcctccttc
tcctcctgct 10620 cctcttcctc ctccttctct tcctcctcct tctcttcctc
ctcctcctcc tgctcctctt 10680 cctcctcctc ctcttcctcc tcctcagcct
agtggagtgt cctggcctgg cttctactga 10740 tgaccctcct atccctcatc
aaactcccca ccaaactcct ccccacccag agaacccccg 10800 gtcctcccct
tcctcctgaa ggcctgaggc tccctgtgac cctccgcccc acctctcgca 10860
ggtacgggct gatgcgtgag tgctggcacg cagcgccctc ccagaggcct accttcaagc
10920 agctggtgga ggcgctggac aaggtcctgc tggccgtctc tgaggaggta
cagcccctcc 10980 cacccaccac ctccctctgc ctgctcccct ccaggcctca
tctggcctga ccgcgtggac 11040 atgcgccccg tcccatcccg ggcgctgcag
aggctgacca gctccgttcc ccacagtacc 11100 tcgacctccg cctgaccttc
ggaccctatt ccccctctgg tggggacgcc agcagcacct 11160 gctcctccag
cgattctgtc ttcagccacg accccctgcc attgggatcc agctccttcc 11220
ccttcgggtc tggggtgcag acatgagcaa ggctcaaggc tgtgcaggca cataggctgg
11280 tggccttggg ccttggggct cagccacagc ctgacacagt gctcgacctt
gatagcatgg 11340 ggcccctggc ccagagttgc tgtgccgtgt ccaagggccg
tgcccttgcc cttggagctg 11400 ccgtgcctgt gtcctgatgg cccaaatgtc
agggttctgc tcggcttctt ggaccttggc 11460 gcttagtccc catcccgggt
ttggctgagc ctggctggag agctgctatg ctaaacctcc 11520 tgcctcccaa
taccagcagg aggttctggg cctctgaacc ccctttcccc acacctcccc 11580
ctgctgctgc tgccccagcg tcttgacggg agcattggcc cctgagccca gagaagctgg
11640 aagcctgccg aaaacaggag caaatggcgt tttataaatt atttttttga
aataaagctc 11700 tgtgtgcctg ggtcttccct gagcaacatg gagtggggtg
aggtggaggg atccctccag 11760 cagagttctg cctacaggac acggactgag
ggcactggac caggccatgg gctccgccac 11820 ctccactgcc ccaggagcca
gtgtgtgcct atctgggtcc gcctgtccca ccagccccat 11880 cttgtgtctg
cgacagtgtg aatgagtatt aatgggctga gtccgcattg cactatacac 11940
ggtgggactc ctgtaccctc tgcacatgtg tgtgtgtgca tgtgtgccct gcagctgtcc
12000 ccaagggagc tggcagcccc cctcccccat ctgctcagca ttaaccaagc
tgaccgttaa 12060 cacagcatga aaatctgaga gccagcctta ggccgcggcc
cgctcccacg ctctgccggc 12120 tcaggctggg ggcttgtgga ggccatgccc
gccccgccct ggccagtctc ccgggcagca 12180 gctggttgcc gcccgc 12196
<210> SEQ ID NO 3 <211> LENGTH: 5192 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3
ccatgacctg ccttgagaag gggcagggga agccagatgg actggaagtg gagtggcagt
60 gaccaaggag gaggaggtgt gataggcttc ccacgcaggg tagatccaga
gacaccagtg 120 ccacccatag gcccctagga ctgcagtggt cacccgattc
ctttgtccca gctgagactc 180 agttctgagt gttctatttt ggggaacaga
ggcgtccttg gtagcatttg gaagaggata 240 gccagctggg gtgtgtgtac
atcacagcct gacagtaaca gcatccgaac cagaggtgac 300 tggctaaggg
cagacccagg gcaacaggtt aaccgttcta gggccgggca cagggaggag 360
aacattccaa cactctgtgt gcccagtgcc gacgcacgtt ctctctttta tcctcaaaac
420 agtcctatga ggatataagc cagagagaga cagagacaag gaattacaag
ttggtgagag 480 tcaggatttg aacttggctc tggcagatgg aaaattaggg
tctgtattct ttacaaaacc 540 gtgtgtgcct cagatggagt tggtgcataa
caagcagagg tatccagggt cgcggtcctg 600 cttgccacgg aaggggccgc
cttgtcagtt gtgaccaccc agccctggaa atgtcagtaa 660 tgctgtaagg
agtggggatc ggatcagatg ccatccagat gctgaagttt gaccttgtgt 720
catttttcac tttctttttt ggctcttctg caatcaattc atttatttag caaaaaagaa
780 attatgtgtg ccgagagcat gcagaagata tgtctccgtt ctctgcttcc
ctccaaaaaa 840 gaatcccaaa actgctttct gtgaacgtgt gccagggtcc
cagcaggact cagggagagc 900 aggaagccca gcccagaccc cttgcacaac
ctaccgtggg gaggccttag gctctggcta 960 ctacagagct ggttccagtc
tgcactgcca cagcctggcc agggacttgg acacatctgc 1020 tggccacttc
ctgtctcagt ttccttatct gcaaaataag ggaaaagccc ccacaaaggt 1080
gcacgtgtag caggagctct tttccctccc tattttagga aggcagttgg tgggaagtcc
1140 agcttgggtc cctgagagct gtgagaagga gatgcggctg ctgctggccc
tgttgggggt 1200 cctgctgagt gtgcctgggc ctccagtctt gtccctggag
gcctctgagg aagtggagct 1260 tggtatggct tctgaggtgg gagagggtgg
caggggtggg aagagtgggc accaggaggg 1320 ggctgctggg ctgagcaaag
ctggaaagga tccttgccca ggccctgaga aggtggcggc 1380 agggcagggc
tcaaccactg agactcagtc agtgcctggc ttccagcaag cattcatcta 1440
tcactgtgtc tgcgagagag gactggcctt gcagggcgca gggccctaag ctgggctgca
1500 gagctggtgg tgagctcctt gcctgggtgt gtgtgcgtgt gtgtgtgtgt
tctgtgcact 1560 gggtgtgtga cctaggaggt ccaggcagca tgtgtggtat
aagcattatg agggtgatat 1620 gccccggtgc agcatgaccc tgtatgtggc
accaacagca tgtgccttgt gtgtgtgtgt 1680 gtccgtatgt gtgtgtgtgt
atgcgtgtgt gtgtgtgtgt gtgtgtgtct tggccactgt 1740 catgtgcact
aaatgctgtg tgtgtgacat gccccaagag tgtggcattt gccctgggtg 1800
tggcatccgc agcatgtggc tgtgtgggtg tcaaggagtg gtggctcctt cagcatgcgt
1860 tgcgaagtgc ttgtgccctg catgtgcggt gtgttctctg tacacaggag
gctgcctcag 1920 atggggctgc ggggtctgct gacctctgcc ctctgcccac
agagccctgc ctggctccca 1980 gcctggagca gcaagagcag gagctgacag
tagcccttgg gcagcctgtg cggctgtgct 2040 gtgggcgggc tgagcgtggt
ggccactggt acaaggaggg cagtcgcctg gcacctgctg 2100 gccgtgtacg
gggctggagg ggccgcctag agattgccag cttcctacct gaggatgctg 2160
gccgctacct ctgcctggca cgaggctcca tgatcgtcct gcagaatctc accttgatta
2220 caggtgactc cttgacctcc agcaacgatg atgaggaccc caagtcccat
agggacctct 2280 cgaataggca cagttacccc cagcaaggtc agtaggtctc
caaggacttg tgtccccgct 2340 gctgctcatc tgatcactga gaagaggagg
cctgtgtggg aacacacggt cattctaggg 2400 gccttcccct gccctccagc
accctactgg acacaccccc agcgcatgga gaagaaactg 2460 catgcagtac
ctgcggggaa caccgtcaag ttccgctgtc cagctgcagg caaccccacg 2520
cccaccatcc gctggcttaa ggatggacag gcctttcatg gggagaaccg cattggaggc
2580 attcggctgc gccatcagca ctggagtctc gtgatggaga gcgtggtgcc
ctcggaccgc 2640
ggcacataca cctgcctggt agagaacgct gtgggcagca tccgttataa ctacctgcta
2700 gatgtgctgg agcggtcccc gcaccggccc atcctgcagg ccgggctccc
ggccaacacc 2760 acagccgtgg tgggcagcga cgtggagctg ctgtgcaagg
tgtacagcga tgcccagccc 2820 cacatccagt ggctgaagca catcgtcatc
aacggcagca gcttcggagc cgacggtttc 2880 ccctatgtgc aagtcctaaa
gactgcagac atcaatagct cagaggtgga ggtcctgtac 2940 ctgcggaacg
tgtcagccga ggacgcaggc gagtacacct gcctcgcagg caattccatc 3000
ggcctctcct accagtctgc ctggctcacg gtgctgccag gtgagcacct gaagggccag
3060 gagatgctgc gagatgcccc tctgggccag cagtgggggc tgtggcctgt
tgggtggtca 3120 gtctctgttg gcctgtgggg tctggcctgg ggggcagtgt
gtggatttgt gggtttgagc 3180 tgtatgacag cccctctgtg cctctccaca
cgtggccgtc catgtgaccg tctgctgagg 3240 tgtgggtgcc tgggactggg
cataactaca gcttcctccg tgtgtgtccc cacatatgtt 3300 gggagctggg
agggactgag ttagggtgca cggggcggcc agtctcacca ctgaccagtt 3360
tgtctgtctg tgtgtgtcca tgtgcgaggg cagaggagga ccccacatgg accgcagcag
3420 cgcccgaggc caggtatacg gacatcatcc tgtacgcgtc gggctccctg
gccttggctg 3480 tgctcctgct gctggccagg ctgtatcgag ggcaggcgct
ccacggccgg cacccccgcc 3540 cgcccgccac tgtgcagaag ctctcccgct
tccctctggc ccgacagttc tccctggagt 3600 caggctcttc cggcaagtca
agctcatccc tggtacgagg cgtgcgtctc tcctccagcg 3660 gccccgcctt
gctcgccggc ctcgtgagtc tagatctacc tctcgaccca ctatgggagt 3720
tcccccggga caggctggtg cttgggaagc ccctaggcga gggctgcttt ggccaggtag
3780 tacgtgcaga ggcctttggc atggaccctg cccggcctga ccaagccagc
actgtggccg 3840 tcaagatgct caaagacaac gcctctgaca aggacctggc
cgacctggtc tcggagatgg 3900 aggtgatgaa gctgatcggc cgacacaaga
acatcatcaa cctgcttggt gtctgcaccc 3960 aggaagggcc cctgtacgtg
atcgtggagt gcgccgccaa gggaaacctg cgggagttcc 4020 tgcgggcccg
gcgcccccca ggccccgacc tcagccccga cggtcctcgg agcagtgagg 4080
ggccgctctc cttcccagtc ctggtctcct gcgcctacca ggtggcccga ggcatgcagt
4140 atctggagtc ccggaagtgt atccaccggg acctggctgc ccgcaatgtg
ctggtgactg 4200 aggacaatgt gatgaagatt gctgactttg ggctggcccg
cggcgtccac cacattgact 4260 actataagaa aaccagcaac ggccgcctgc
ctgtgaagtg gatggcgccc gaggccttgt 4320 ttgaccgggt gtacacacac
cagagtgacg tgtggtcttt tgggatcctg ctatgggaga 4380 tcttcaccct
cgggggctcc ccgtatcctg gcatcccggt ggaggagctg ttctcgctgc 4440
tgcgggaggg acatcggatg gaccgacccc cacactgccc cccagagctg tacgggctga
4500 tgcgtgagtg ctggcacgca gcgccctccc agaggcctac cttcaagcag
ctggtggagg 4560 cgctggacaa ggtcctgctg gccgtctctg aggagtacct
cgacctccgc ctgaccttcg 4620 gaccctattc cccctctggt ggggacgcca
gcagcacctg ctcctccagc gattctgtct 4680 tcagccacga ccccctgcca
ttgggatcca gctccttccc cttcgggtct ggggtgcaga 4740 catgagcaag
gctcaaggct gtgcaggcac ataggctggt ggccttgggc cttggggctc 4800
agccacagcc tgacacagtg ctcgaccttg atagcatggg gcccctggcc cagagttgct
4860 gtgccgtgtc caagggccgt gcccttgccc ttggagctgc cgtgcctgtg
tcctgatggc 4920 ccaaatgtca gggttctgct cggcttcttg gaccttggcg
cttagtcccc atcccgggtt 4980 tggctgagcc tggctggaga gctgctatgc
taaacctcct gcctcccaat accagcagga 5040 ggttctgggc ctctgaaccc
cctttcccca cacctccccc tgctgctgct gccccagcgt 5100 cttgacggga
gcattggccc ctgagcccag agaagctgga agcctgccga aaacaggagc 5160
aaatggcgtt ttataaatta tttttttgaa at 5192 <210> SEQ ID NO 4
<211> LENGTH: 2807 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (54)..(2342) <400> SEQUENCE: 4
gaaggcagtt ggtgggaagt ccagcttggg tccctgagag ctgtgagaag gag atg 56
Met 1 cgg ctg ctg ctg gcc ctg ttg ggg gtc ctg ctg agt gtg cct ggg
cct 104 Arg Leu Leu Leu Ala Leu Leu Gly Val Leu Leu Ser Val Pro Gly
Pro 5 10 15 cca gtc ttg tcc ctg gag gcc tct gag gaa gtg gag ctt gag
ccc tgc 152 Pro Val Leu Ser Leu Glu Ala Ser Glu Glu Val Glu Leu Glu
Pro Cys 20 25 30 ctg gct ccc agc ctg gag cag caa gag cag gag ctg
aca gta gcc ctt 200 Leu Ala Pro Ser Leu Glu Gln Gln Glu Gln Glu Leu
Thr Val Ala Leu 35 40 45 ggg cag cct gtg cgt ctg tgc tgt ggg cgg
gct gag cgt ggt ggc cac 248 Gly Gln Pro Val Arg Leu Cys Cys Gly Arg
Ala Glu Arg Gly Gly His 50 55 60 65 tgg tac aag gag ggc agt cgc ctg
gca cct gct ggc cgt gta cgg ggc 296 Trp Tyr Lys Glu Gly Ser Arg Leu
Ala Pro Ala Gly Arg Val Arg Gly 70 75 80 tgg agg ggc cgc cta gag
att gcc agc ttc cta cct gag gat gct ggc 344 Trp Arg Gly Arg Leu Glu
Ile Ala Ser Phe Leu Pro Glu Asp Ala Gly 85 90 95 cgc tac ctc tgc
ctg gca cga ggc tcc atg atc gtc ctg cag aat ctc 392 Arg Tyr Leu Cys
Leu Ala Arg Gly Ser Met Ile Val Leu Gln Asn Leu 100 105 110 acc ttg
att aca ggt gac tcc ttg acc tcc agc aac gat gat gag gac 440 Thr Leu
Ile Thr Gly Asp Ser Leu Thr Ser Ser Asn Asp Asp Glu Asp 115 120 125
ccc aag tcc cat agg gac ccc tcg aat agg cac agt tac ccc cag caa 488
Pro Lys Ser His Arg Asp Pro Ser Asn Arg His Ser Tyr Pro Gln Gln 130
135 140 145 gca ccc tac tgg aca cac ccc cag cgc atg gag aag aaa ctg
cat gca 536 Ala Pro Tyr Trp Thr His Pro Gln Arg Met Glu Lys Lys Leu
His Ala 150 155 160 gta cct gcg ggg aac acc gtc aag ttc cgc tgt cca
gct gca ggc aac 584 Val Pro Ala Gly Asn Thr Val Lys Phe Arg Cys Pro
Ala Ala Gly Asn 165 170 175 ccc acg ccc acc atc cgc tgg ctt aag gat
gga cag gcc ttt cat ggg 632 Pro Thr Pro Thr Ile Arg Trp Leu Lys Asp
Gly Gln Ala Phe His Gly 180 185 190 gag aac cgc att gga ggc att cgg
ctg cgc cat cag cac tgg agt ctc 680 Glu Asn Arg Ile Gly Gly Ile Arg
Leu Arg His Gln His Trp Ser Leu 195 200 205 gtg atg gag agc gtg gtg
ccc tcg gac cgc ggc aca tac acc tgc ctg 728 Val Met Glu Ser Val Val
Pro Ser Asp Arg Gly Thr Tyr Thr Cys Leu 210 215 220 225 gta gag aac
gct gtg ggc agc atc cgc tat aac tac ctg cta gat gtg 776 Val Glu Asn
Ala Val Gly Ser Ile Arg Tyr Asn Tyr Leu Leu Asp Val 230 235 240 ctg
gag cgg tcc ccg cac cgg ccc atc ctg cag gcc ggg ctc ccg gcc 824 Leu
Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala 245 250
255 aac acc aca gcc gtg gtg ggc agc gac gtg gag ctg ctg tgc aag gtg
872 Asn Thr Thr Ala Val Val Gly Ser Asp Val Glu Leu Leu Cys Lys Val
260 265 270 tac agc gat gcc cag ccc cac atc cag tgg ctg aag cac atc
gtc atc 920 Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Leu Lys His Ile
Val Ile 275 280 285 aac ggc agc agc ttc gga gcc gac ggt ttc ccc tat
gtg caa gtc cta 968 Asn Gly Ser Ser Phe Gly Ala Asp Gly Phe Pro Tyr
Val Gln Val Leu 290 295 300 305 aag act gca gac atc aat agc tca gag
gtg gag gtc ctg tac ctg cgg 1016 Lys Thr Ala Asp Ile Asn Ser Ser
Glu Val Glu Val Leu Tyr Leu Arg 310 315 320 aac gtg tca gcc gag gac
gca ggc gag tac acc tgc ctc gca ggc aat 1064 Asn Val Ser Ala Glu
Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly Asn 325 330 335 tcc atc ggc
ctc tcc tac cag tct gcc tgg ctc acg gtg ctg cca ggt 1112 Ser Ile
Gly Leu Ser Tyr Gln Ser Ala Trp Leu Thr Val Leu Pro Gly 340 345 350
act ggg cgc atc ccc cac ctc aca tgt gac agc ctg act cca gca ggc
1160 Thr Gly Arg Ile Pro His Leu Thr Cys Asp Ser Leu Thr Pro Ala
Gly 355 360 365 aga acc aag tct ccc act ttg cag ttc tcc ctg gag tca
ggc tct tcc 1208 Arg Thr Lys Ser Pro Thr Leu Gln Phe Ser Leu Glu
Ser Gly Ser Ser 370 375 380 385 ggc aag tca agc tca tcc ctg gta cga
ggc gtg cgt ctc tcc tcc agc 1256 Gly Lys Ser Ser Ser Ser Leu Val
Arg Gly Val Arg Leu Ser Ser Ser 390 395 400 ggc ccc gcc ttg ctc gcc
ggc ctc gtg agt cta gat cta cct ctc gac 1304 Gly Pro Ala Leu Leu
Ala Gly Leu Val Ser Leu Asp Leu Pro Leu Asp 405 410 415 cca cta tgg
gag ttc ccc cgg gac agg ctg gtg ctt ggg aag ccc cta 1352 Pro Leu
Trp Glu Phe Pro Arg Asp Arg Leu Val Leu Gly Lys Pro Leu 420 425 430
ggc gag ggc tgc ttt ggc cag gta gta cgt gca gag gcc ttt ggc atg
1400 Gly Glu Gly Cys Phe Gly Gln Val Val Arg Ala Glu Ala Phe Gly
Met 435 440 445 gac cct gcc cgg cct gac caa gcc agc act gtg gcc gtc
aag atg ctc 1448 Asp Pro Ala Arg Pro Asp Gln Ala Ser Thr Val Ala
Val Lys Met Leu 450 455 460 465 aaa gac aac gcc tct gac aag gac ctg
gcc gac ctg gtc tcg gag atg 1496 Lys Asp Asn Ala Ser Asp Lys Asp
Leu Ala Asp Leu Val Ser Glu Met 470 475 480 gag gtg atg aag ctg atc
ggc cga cac aag aac atc atc aac ctg ctt 1544 Glu Val Met Lys Leu
Ile Gly Arg His Lys Asn Ile Ile Asn Leu Leu 485 490 495 ggt gtc tgc
acc cag gaa ggg ccc ctg tac gtg atc gtg gag tgc gcc 1592 Gly Val
Cys Thr Gln Glu Gly Pro Leu Tyr Val Ile Val Glu Cys Ala 500 505 510
gcc aag gga aac ctg cgg gag ttc ctg cgg gcc cgg cgc ccc cca ggc
1640 Ala Lys Gly Asn Leu Arg Glu Phe Leu Arg Ala Arg Arg Pro Pro
Gly 515 520 525 ccc gac ctc agc ccc gac ggt cct cgg agc agt gag ggg
ccg ctc tcc 1688 Pro Asp Leu Ser Pro Asp Gly Pro Arg Ser Ser Glu
Gly Pro Leu Ser 530 535 540 545 ttc cca gtc ctg gtc tcc tgc gcc tac
cag gtg gcc cga ggc atg cag 1736 Phe Pro Val Leu Val Ser Cys Ala
Tyr Gln Val Ala Arg Gly Met Gln 550 555 560 tat ctg gag tcc cgg aag
tgt atc cac cgg gac ctg gct gcc cgc aat 1784 Tyr Leu Glu Ser Arg
Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn 565 570 575 gtg ctg gtg
act gag gac aat gtg atg aag att gct gac ttt ggg ctg 1832 Val Leu
Val Thr Glu Asp Asn Val Met Lys Ile Ala Asp Phe Gly Leu 580 585 590
gcc cgc ggc gtc cac cac att gac tac tat aag aaa acc agc aac ggc
1880 Ala Arg Gly Val His His Ile Asp Tyr Tyr Lys Lys Thr Ser Asn
Gly
595 600 605 cgc ctg cct gtg aag tgg atg gcg ccc gag gcc ttg ttt gac
cgg gtg 1928 Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe
Asp Arg Val 610 615 620 625 tac aca cac cag agt gac gtg tgg tct ttt
ggg atc ctg cta tgg gag 1976 Tyr Thr His Gln Ser Asp Val Trp Ser
Phe Gly Ile Leu Leu Trp Glu 630 635 640 atc ttc acc ctc ggg ggc tcc
ccg tat cct ggc atc ccg gtg gag gag 2024 Ile Phe Thr Leu Gly Gly
Ser Pro Tyr Pro Gly Ile Pro Val Glu Glu 645 650 655 ctg ttc tcg ctg
ctg cgg gag gga cat cgg atg gac cga ccc cca cac 2072 Leu Phe Ser
Leu Leu Arg Glu Gly His Arg Met Asp Arg Pro Pro His 660 665 670 tgc
ccc cca gag ctg tac ggg ctg atg cgt gag tgc tgg cac gca gcg 2120
Cys Pro Pro Glu Leu Tyr Gly Leu Met Arg Glu Cys Trp His Ala Ala 675
680 685 ccc tcc cag agg cct acc ttc aag cag ctg gtg gag gcg ctg gac
aag 2168 Pro Ser Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Ala Leu
Asp Lys 690 695 700 705 gtc ctg ctg gcc gtc tct gag gag tac ctc gac
ctc cgc ctg acc ttc 2216 Val Leu Leu Ala Val Ser Glu Glu Tyr Leu
Asp Leu Arg Leu Thr Phe 710 715 720 gga ccc tat tcc ccc tct ggt ggg
gac gcc agc agc acc tgc tcc tcc 2264 Gly Pro Tyr Ser Pro Ser Gly
Gly Asp Ala Ser Ser Thr Cys Ser Ser 725 730 735 agc gat tct gtc ttc
agc cac gac ccc ctg cca ttg gga tcc agc tcc 2312 Ser Asp Ser Val
Phe Ser His Asp Pro Leu Pro Leu Gly Ser Ser Ser 740 745 750 ttc ccc
ttc ggg tct ggg gtg cag aca tga gcaaggctca aggctgtgca 2362 Phe Pro
Phe Gly Ser Gly Val Gln Thr 755 760 ggcacatagg ctggtggcct
tgggccttgg ggctcagcca cagcctgaca cagtgctcga 2422 ccttgatagc
atggggcccc tggcccagag ttgctgtgcc gtgtccaagg gccgtgccct 2482
tgcccttgga gctgccgtgc ctgtgtcctg atggcccaaa tgtcagggtt ctgctcggct
2542 tcttggacct tggcgcttag tccccatccc gggtttggct gagcctggct
ggagagctgc 2602 tatgctaaac ctcctgcctc ccaataccag caggaggttc
tgggcctctg aacccccttt 2662 ccccacacct ccccctgctg ctgctgcccc
agcgtcttga cgggagcatt ggcccctgag 2722 cccagagaag ctggaagcct
gccgaaaaca ggagcaaatg gcgttttata aattattttt 2782 ttgaaataaa
aaaaaaaaaa aaaaa 2807 <210> SEQ ID NO 5 <211> LENGTH:
15001 <212> TYPE: DNA <213> ORGANISM: Macaca mulatta
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(15001) <223> OTHER INFORMATION: n = A,T,C or
G <400> SEQUENCE: 5 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 120 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 420 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 480 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 540
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 840 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 900
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 1020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 1080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1140 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1200 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1260
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
1320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 1380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 1440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1620
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
1680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 1740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 1800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1860 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1920 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1980
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
2040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 2100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 2160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2220 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2280 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2340
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
2400 nggcgggggc aggctggccc tgtgcagcta ccctcgggac ccattgattc
gcacctcccc 2460 ccaggctggc ccggcaaggg tgggggagga caagcttgtc
cctgcggctg tcttcgcgcc 2520 ggcggcagag atgagggagc tgaggccccg
aaagtttcag tcacttagcg cccgggggcc 2580 tccaccgcga gtgcgggagg
ctgtctgatg cctaaggcag gccctctgtt cccacgtgtg 2640 gggtggtcgg
tcagcggtca gcagccacgg gtgacttttc cacggactct gatatcaggg 2700
cagcctgggg taggaataaa atccccgggc ctccccagcc actcccagcc caagctgtgt
2760 actcaaagag ctgccctccc tgccaagccg agcttggtag ggggttttac
caagggggat 2820 ccaactggat ttgagagttg aagtggcctc agagacagca
gtatctgagt caagtagaga 2880 agaggaatgg ggggcacaga gggatgggca
agagagcata tgtgaccagt tttgagagcc 2940 aatggcttca gcgttcctga
agggacagac ggtgtgacca aagagatagg cagcggaaca 3000 gagggagccc
taggatgctg agctgaatcc tgctgggcac aggtaaccat taagggcttg 3060
caagctgggg gcatgacatg gcagacttgt agtcttttaa aactctgctg gaagatgaag
3120 gttgaagagc ccagggagag gatgtttcca aaggcccatg caagagatgg
ccatgacctg 3180 ccctgagtca gggcagggga agccagatgg actggaagtg
gagtggcagt gaccaagggt 3240 aaggaggtgt gataggcttc ccacacaggg
tagatccaga gacaccagtg ccacccatag 3300 gcccctagga ctgcagtgat
cacagtattc ctttgtccca gctgagactc ggttctgagt 3360 gttctgtttt
ggggaacaga ggtgtccttg gtagcatttg gaagaggata gccagctggg 3420
gtgtgtgtac atcacagcct gacagtaaca gcacccgaac cagaggtgac tggctgaggg
3480 cagacccagg gcaacaggtg aacagttcta gggccgggca cagggagaag
aacattccaa 3540 cactctgtgt gcccagtgcc gatgcacgtt gtctctttta
tcctcaaaac agtcctatga 3600 ggatagtaac ccagagagag acagagacaa
ggaattacaa gttgatgaga gtcgggattt 3660 gaacttggct ctggtggatg
gataattacg gtctgtattc tttacaaaac cgtgtgtacc 3720 tcggatggag
ttggtgcatg acaagcagag gtattcaggg atgcggtcct gcttgccacg 3780
gaaggagctg ccttgtcagt ggtgggcgcc cagctctgga aatgtcagta atgctataag
3840 gagtggggat tggatcagat aacatccaga tgctgaagtt tggccttgta
tcatttttca 3900 ctttgttttt tggctcttcg gcaatcaatt catttattca
gcaaaaaata aattacgtgt 3960 gccaagagca tgcagaagat agtctccgtt
ctctgcttcc ctccaagaaa agaatcccaa 4020 aactgctttc tgtgaacgtg
tgccagggtc ccagcaggac tcaaggagag caggaagccc 4080 agcccagacc
ccttgcacaa cctactgtag gaaggcgtca ggctctggct acagcagagc 4140
tggttccagt ctgcactgcc acagcctggc cagggacttg gacacatcta ctggccactt
4200 cctgtctccg tttcctcatc tgcaaaataa gggaaagccc ccgcaaaggt
gcatgtggag 4260 caggagctct tttccccccc tattctagga aggcagttgg
tgggaagtcc agcctgggcc 4320 cctgagagct gcgggaagga gatgcggctg
ctgtcggccc tcttgggggt cctgctgagt 4380 gtgcctgggc ctccagtctt
gtccctggag gcctcggagg aagtggagct gggtatggct 4440 tctgaggggg
gagagggtgg caggggtggg aagagtgggc accaggaggg ggctgctggg 4500
ctgagcaaag ctggaaagga tccttgccca ggccctgaga aagtggcagc agggcagggc
4560 tcaaccactg agactcagtc agtgcctggc ttccagcaag catccatgta
tctctgtgtc 4620 tgcgagagag gactggcctc gcagggtgca gggccctaag
ctgggccgca gagctggtgg 4680 tgagctcctt gcctgggtgt gtgcgcgcgc
gtgtgtgtgt gtgtgttctg tgcactggat 4740 gtgtgaccta ggaggtccag
gcagcatgtg tggtgtatgc attatgaggg tgatatgccc 4800 cagtgcagca
tgaccctgta tatggcaccg atagcatgtg ccgtgtgtgt gtgtgtccat 4860
gtgtgtgtgt gtgtgtgtct tggccagtgt catgtgcact aaatgctgtg tgcgtgacat
4920 gccccaagag tgtggcattt gccctgggtg tggcatccgc agcatgtggc
tgtgcgggtg 4980 tcaaggagcg gtggctcctc cagcatgcgt cgcgaggtgc
ttgtgccctg catgtgtggc 5040 gtgttctctg tacgcaggag gctgcctcag
atggggctgc ggggtctgct gacctctgcc 5100 ctctgcccac agagccctgc
ctggctccca gcatggagca gcaagagcag gagctgacag 5160 tagcccttgg
gcagcctgtg cggctgtgct gtgggcgggc tgagcgtggt ggccactggt 5220
acaaggaggg cagtcgcctg gcacctgctg gccgtgtacg gggctggagg ggccgcctag
5280 agattgccag cttcctacct gaggatgctg gccgctatct ctgcctggcc
cgagcctcca 5340 tgatcgtcct gcaaaatctc accttgacta tagatggtaa
gacactctag cagttaaggg 5400 atgcctgggg agagagacct gcccctcccg
gacctcagat gcctccctct gtccttgatg 5460
tagactcctt gacctccagc aacgatgatg aggaccccca gtcccatagg gactcctcga
5520 atgggcacat ttacccccag caaggtgagt ataagtcttc aaggacttgt
gtccccgctg 5580 ctcatttaat cactgagaag aagaggtctg tgtgggaaca
cacggtcatt ctaagggcct 5640 tcctctcccc tccagcaccc tactggacac
acccccagcg catggagaag aaactgcatg 5700 cagtaccggc tgggaacacc
gtcaagttcc gctgtccggc tgcaggcaac cccacgccca 5760 ccatccgctg
gcttaaggat ggacaggcct ttcatgggga gaaccgcatt ggaggcattc 5820
gggtgagtct ctgggttcca agaccgtctg ctgcctcatt ctcattcctt cctcagtccc
5880 ctcatgccta caagcacacc tatacatcca ttgaatgagg aaagcaattg
cagggtccca 5940 gaaggagccc tggactgtgg acctgggcag ctctggttcc
tcttctgcta ctctctggca 6000 agtgacttaa cctcacagcc ttagcaactc
catttgtaaa gtgagaagaa tcacggactg 6060 gttggtctca gtaagcctta
gcatctcatc atcttgatga gaccctgcag agtcggctcc 6120 atgctgtcat
aaggcaactg agtctcagag aaggcaaggg ctggctcaga gtggcacagc 6180
cagggagagg gagagctaaa attcaaaagg ctcaaaccca aggcttaagc actctgggga
6240 gccttctcct ttgtcccata gtccttggcc tggccctgat gttctcgggg
cctagagagc 6300 ttgagaagag tcctgtgggc aggatgagga tctagccccc
tggtcctctg gcccccttgg 6360 tggacatggt ctggtggtcc tggacactcc
ctctgcctgc agctgcgcca ccagcactgg 6420 agtctcgtga tggagagcgt
ggtgccctcg gaccgcggca catacacttg cctggtggag 6480 aacgctgtgg
gcatcatccg ctataactac ctgctggatg tgctgggtga gtgcggggcc 6540
ggaaacgggg gaggcctgac ccatcctggg ctcagtcgtg ccctccggtg gggtctagtc
6600 tggcgggcag gatggactca gatgagtcaa gcagctcggt gaccaggcgg
tcaggggaaa 6660 gcacaggggt tagtgtgggg ctggaggagc agggctctgc
caagaggaaa aacaagaagg 6720 acagccaggc aaagggcgca gcctgagctg
caggcctgag tataacgaat gccgtgcact 6780 tgcaggccag cgtattcgga
gatgtggctt ttatatgggg agccaggtgg tggagggttt 6840 tgagtgctag
gctgaaacgt ccttgacccg aagcaatgag gagccaggga aggttgaaac 6900
agggtaagca ggagacggac aagaagctgc agaaaggtcc ttcccttgaa cctgaggaag
6960 gctggaggga ggcaaacagg tgcttctatg ggtgccagtg atgagggttg
actaccttgc 7020 ctggtcccca gagcggtccc cgcaccggcc catcctgcag
gctgggctcc cggccaacac 7080 cacagccgtg gtgggcagtg acgtggagct
gctgtgcaag gtgtacagcg atgcccagcc 7140 ccacatccag tggctgaagc
acatcgtcat caacggcagc agcttcgggg ccgacggctt 7200 cccctatgtg
caagtcctga aggtgcaccg tgctccagcc tgggccccac tcttctccca 7260
cccaggattg ggggctccct agcttccctg ttgatcacag tgtggcccca ggccctgctg
7320 tgaccccaga gtatgtcccc ctccccagac tgcagacatc aatagctcag
aggtggaggt 7380 cctgtacctg cggaacgtgt cagccgagga cgcaggcgag
tacacctgcc ttgcaggcaa 7440 ttccatcggc ctctcctacc agtctgcctg
gctcacggtg ctgccaggta agcacctgca 7500 gggccagaat atgctgcgag
atgcccctct gggccagcag tgggggctgt ggtctgttgg 7560 gtggtcagtc
tctgttggct tgtggggtct gccctcgggg gcagtgtgtg gatttgtgag 7620
tttgagctgt atggcagccc ctctgtgcct gtccacgtgt ggctgtccat gtgaccctct
7680 gctgagctat gggactgggc ataactacag cttcctccgt gtgtgtcccc
acatatgctg 7740 ggagctggga gggactgagt tagggcacat ggggaggcca
gtcttaccac tgaccacttg 7800 gtctgtctgt gtatgttcat gtgcgagggc
agaggaggac ctcacatgga ccgcagcaac 7860 gcccgaggcc aggtatacgg
acgtcatcct gtacgcgtcg ggctccctgg ccttggctgt 7920 gctcctgctg
ctggccgggc tgtatcgagg gcaggcgctc cacggccggc acccccgccc 7980
acccgccacc gtgcagaagc tctcccgctt ccctctggcc cgacaggtac tgggcgcatc
8040 ccccacctcg catgtggcag cctgactcca gcaggcagaa ccaagtctcc
cactttgcag 8100 ttctccctgg agtcaggctc ttccagcaag tcaagctcat
ccctggtgcg aggcgtgcgt 8160 ctctcctcca gcggccccgc cttgctcgcc
ggcctcgtga gtctagacct acctctcgac 8220 ccactgtggg agttcccccg
ggacaggtgc gctgagctgt gtgggggcag ggacacaggt 8280 gccaggtcgc
agcctgccct cagcaggagt gactcggagg tctgaggcat ggactttctc 8340
catctccagg ctggtgcttg ggaagcccct gggcgagggc tgctttggac aggtagtacg
8400 tgcagaggcc tttggcatgg accctgcccg gcctgaccaa gccagtactg
tggctgtcaa 8460 gatgctcaaa ggtgagtgtg acccagcgtg gtggctcaca
cctgtaatgc cagcactttg 8520 ggagaccgag gtgggaggat cacttgaatc
caggagttcg aggccaacct gggcaacatg 8580 gcgagacttc atctctaaaa
aaaaaaataa gaaaattagt tgggtgtggt ggtgtgtgcc 8640 tttagtccca
gttactagga aggctgaggc aggaggatcc cttgaaccca ggagttggag 8700
gttgcagtga gccatgatca cgccactgta ttccaacttg ggcaacagag tgaaacccta
8760 tctgaaaaaa aaaaaagaaa taaaagtaaa aggtgaatgt ggcagcctgg
aggaggtgct 8820 atggcattgg gactaataga aggggatcac agtgctaccg
ggtgagccct ggagctggga 8880 gaggctgtgg gatcccacct ttaaacctgc
agttcagctc tgctcctgac cctggcaagt 8940 gacttctgag cctcagtttt
ccctcgtgtc atatggggta aataacagtc cctactccca 9000 gcccagggac
tgtggaaagt gcctggctca tagtcagccc tcaacaaatc gtcaccattg 9060
gggtgacgat gatgagaagg atttggtgtg acaggcttga tatcctctgt cagcgtcagt
9120 ctgtgtcagc cttgacttca cctcttctca gcctcacagg ccctcccccg
ccttccctgt 9180 ggttcccccc agacacaccc tcagcctccc tgggaccctc
cctaggtcca tcccgcgtcc 9240 actgctctag gaggacagcc cttctgcttg
cacccagtcc cagccccgag ggtgctcttg 9300 ccaggcactc ccgtaccccg
cccatcaggt cctctcgttg cagttcccca gcgccccctg 9360 caagaatggc
ctcaactgct cttctgctcc tcccctcctc tctacacagc tggggccacc 9420
tggtgctccc tgggaggcag ggattgagaa atgtgcattg tgacattgcc gtgtgtgggt
9480 ggggagtgtt acatcactgg cccagggcca caggtcagcc ccaggggccc
agccagagaa 9540 gccagagcag ccttcttccc aagctccctg gctgcgccct
gcctgccgcc ggctccctga 9600 attcccaggc cagttggacg ccaggccctg
gtcaaacaga ccccagggtg ccagcctgct 9660 ttccgctccc agaagctctg
accccatgta gggactaccg ctaacccctc cagaggcagc 9720 ttccttcctg
ctccgagctc ttcccctctc tcctgtttcc tggacctgcc cactggaagg 9780
cctgcctctc agatccttga tatagttgca tccttgcaac tgctgtgaca ggtagggtgt
9840 gacccactgc tctgtttccc acaagacgac cctgaggttc agagacacta
agagactttt 9900 tcaaggccac acagactagc aaggaatcag ccctagacct
acgtagccct ggtccagtgc 9960 tcctcgccct gcccctgcct ctacctcgcc
ctgcccctgc ctctgcctcg ccctgcccct 10020 gccctctgcc tcgccctgcc
cctgcctctg cctgttccct catgtagttg gagagcagcc 10080 tcttcacccc
atctgctgcc cttacagaca acgcctctga caaggacctg gctgacctgg 10140
tctcggagat ggaggtgatg aagctgattg gccgacacaa gaacatcatc aacctgctgg
10200 gtgtctgcac ccaggaaggt ggggccaagg cggggccggc tgcatgggcc
gttagggtac 10260 agagccaaag ctgtggcagt ctctccgagc tccctccact
ccctctgtag ggcccctgta 10320 tgtaatcgtg gagtgcgctg ccaagggaaa
ccttcgggag ttcctgcggg cccggcgccc 10380 cccgggccct gacctcagcc
cggacggtcc tcagagcagt gaggggccac tcgccttccc 10440 agtcctggtc
tcctgcgcct accaggtggc ccgaggcatg cagtatctgg agtcccggaa 10500
ggtacaggcg gtagggctct gggcccctct cgatctctcc agctccactc tctcaggcct
10560 gtggcattaa atgtccccaa cttcttcctt tctgctcttt ttcatgaccc
cacctcagtg 10620 tccccaggca ttcatgcttt cctgccttcc ccacttgttc
ctcacacttc cctagaaggg 10680 agaggggaga ggggagaggg gagaggggac
acaggagagg gcgttcccca tttctaaacc 10740 ttgaccacct cctctgtaaa
gtgggtggag ggcccctgcc cctgggcctg ctggggggtg 10800 gtgtgtgctc
agctcaaggt caggtgtcct gatgcaccca agcccccact ccctgcagtg 10860
tatccaccgg gacctggctg cccgcaatgt gctggtgacg gaggacaatg tgatgaagat
10920 agctgacttt gggctggccc gtggcatcca ccacattgac tactataaga
aaaccagcaa 10980 cgtgagggag acggggcaga actggatggg agtggagggg
ggctgggcct ggggtggcag 11040 gcatgggaac tcgtgggact ctgcactgag
gccctccctc ccctccaggg ccgcctgcct 11100 gtcaagtgga tggcgcccga
ggccttgttt gaccgagtgt acacacacca gagtgacgtg 11160 tgagtcctgc
cagcggtcac tgtcctaccc cacaaaaagg gcaaggcact gcccaaagtc 11220
acatggcccc aggaggcctg cgctccaggg ctccttcaga tttgttctgg gatccgagtg
11280 ggaccacact ccaggaggag cccactcccc accagctggg gtgggtcatg
cctgtggggt 11340 cccccgtcct agccctggtc ttagggaggg cgctgggcca
cactgagccc tggccctgcc 11400 tccaggtggt cttttggggt cctgctgtgg
gagatcttca ccctcggggg ctccccgtat 11460 cctggcatcc cggtggagga
gctgttctca ctgctgcggg agggacatcg gatggaccga 11520 cccccacact
gccccccaga gctgtgaggc ctcatcctgc cctcgacccc cactttccag 11580
tcctcctcct ctgccctgac cttggcctca gggtttgtgc cggccagaag gacaacacta
11640 acaacaactc ctcctcctct tcctcctcct cctctcctct tcctcctcct
cctcctcctc 11700 tcctcctcct cctctttctc ctccttctcc tcctcctctt
cctcttcctg ctcctcagcc 11760 tagtggatcg tcctggcctg gcttccactg
atgaccccgc ctatccctca tcaaactccc 11820 tactcagaga acccccggtc
ctccccttcc tcctgaaggc ctgaggctcc ctatgacctt 11880 ctggcccacc
tctcccaggt acgggctgat gcgtgagtgc tggcatgcag caccctccca 11940
gaggcccacc ttcaagcagc tggtggaggc gctggacaag gtcttactgg ccgtctctga
12000 ggaggtacag ccccctccca cccaccacct ccctctgcct gctcccctcc
aggcctcgtc 12060 tggcctgacc gcgtggacat gcgccccgtc ccatcctggg
cactgcagag gctgaccggc 12120 tccgttcccc acagtacctc gacctccgcc
tgaccttcgg accctattcc cctgctggtg 12180 gggacaccag cagcacctgc
tcctccagtg actccgtctt cagccacgac cccctgccac 12240 tgggatccag
ctccttcccc tttgggtctg gggtgcagac atgagtaagg ctcaaggctg 12300
tgcaggcaca taaactagtg gccttgggcc ttggggctca gccacagcct ggcacagtgc
12360 ttgaccttgg cagcacgggg tccctggccc agagtgctgt cccaggtctc
tggttctgct 12420 ttgggtaggt cccttcttgg tcctggagtt cgtaaaggct
tcctgctctg gccttgggtt 12480 cccaacctac agctcaactc aaatctcaag
gccgtgccct tgcccttggc gctgcagtgc 12540 ctgtgtcctg atgggccaaa
cgtcagggtt ctgctcggcc cttggacctt ggcgctcagc 12600 ccccacctca
ggtttggctg agcctggctg gagagctgct atgctaaatc tcctgcctcc 12660
caataccagc agggggttca gggcctctga accccctttc cccacacctc cccctgctgc
12720 ttgccccagc gtcttgatgg gagcgtcggc ccctgagccc agagaagctg
gaagcccgcc 12780 aaaaacagga gcaaatggcg ttctataaat tatttttttg
aaataaagct ctgtgtgcct 12840 aggtcttccc tgagcaacat ggaggggagt
gggatggagg gatcccccca ggagagagtt 12900 ctgcctgcag gacacggact
gagggcgctg gaccaggccg tgggctctgc cacctcccct 12960
gccccgggag ccagggtgtg cctgtcttgg tccgccggtc ccaccagcac catcttgtgt
13020 cggtgacagt gtgaatgagt gttaatgggc tgagtccgca ttgcaccatc
tacagtggga 13080 ctcctgtgcc ctctgcacat gtgtgtgtgc gtgtgtgccc
tgcagctgtc cccgggggag 13140 caggcagccc cctcccccat ctgctcagca
ttaaccaagc tgaccgttaa cacagcatga 13200 aaatctgaaa gccagcctta
ggccacggcc tgctcccacg ctctgcctgc tcaggctggg 13260 ggcttgtggg
ggccatgccc gccccaccct ggccaatctc ccaggcagca gctggttgcc 13320
gcccgcctgg gctgcagctg tccctgcctg cctggtcttc cactggggac ccgtcacagc
13380 cctgtaccca gagcccctca gagggagcag cttctcaggg ctctgagcct
ggagccttcc 13440 ctggccccat cctggcatgt actgctactc cccagcccct
gagtgggcca tggggggcct 13500 aggcatggtg tctgacacag cgcttggcac
tctcctgcct tttcctacag gcctcaggct 13560 gggacagaac tgcccaacca
ggacagcctt tatggaggcc actgggattg cccccatctg 13620 cccccaggga
gtggtggctg aaaactctcc acatagcctc tccggctgac agtctcccac 13680
acagaactca tcctcccccc agagcggcgc ctcctccagc gcaggctccg gggagtcacc
13740 tgaccacttc cctcaagaac ctcgttccag gccgggcacg gcggctcacg
cctgtaatcc 13800 cagcagtttg gaaggccgag gaggacgcat cacctgaggt
taggaattcg agaccagcct 13860 ggacaacatg gtgaagccct gtctctacta
aaaatacaaa aattagcctg gcgtagtggg 13920 ctgcactgta gtcccagcta
ctcaggaggc tgaggcagga gaatcacttg aatccgggag 13980 gtgagggttg
cagtgaaccg agattgtgcc agcctgggct aaggagcaag gctctgtctc 14040
aaaaaaaaaa aaaaaaagaa ccctattccc taaagtgctt ccagggtcca gccatggctc
14100 ctgaggccag cactgccacc tctctgatga tcactgtcct tgcccatgag
tagggtgcgc 14160 ctgtcccgcc tgcctcctgg ggcttagggt ttaggacgaa
gcgaagcaca gtgcagatgc 14220 acataggctt gggggagggc gtaccctact
tttgctccct cccctgcagg tggctccaga 14280 ggcagcttcc aaaggcggac
tgctcgcaaa gaccccctct aactcctgaa agcccccaga 14340 ctgactttct
ttttccttcc ttcctccttc cttccctccc tccctccctt cttccctccc 14400
tcccttcctt tctttctttc tgtcttttgt tctttcgttc tttcattcgt tcgttctttt
14460 tctttctctt tctctctctc tctttcgctc tctcttcttc tttctctttc
tctctttctc 14520 ttttccttcc ttccttcctt ccttccttcc tctctttctc
tctctctctt ctttatttct 14580 ttccttcttt ctttcttttt ttttttttga
gacaaggtct tgatctattg accagattgg 14640 agtgcagtga cgccatgatc
atggctctgc tgcctcaaac tcctgggctc aagcaatcct 14700 cccacctcag
cctgccaagt gcataggact acaggtgcat gccaccacac atggataatt 14760
attttttatt ttttgtagag atggggtttt gctatgttgt ccaagctggt ctcaaactcc
14820 tggcctcaag caatcctccc accttggcct cccaaagctc ccaaaccccg
gacattctga 14880 ctgagcctca ctctacaatg tatagcctcc cattgggggt
cactgtccac tgactgtgtc 14940 cacctgtcca gccaatcaca ggagaagcag
atcccttgga cttgaccttg ggaaggagag 15000 g 15001 <210> SEQ ID
NO 6 <211> LENGTH: 3323 <212> TYPE: DNA <213>
ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (350)..(2749) <400> SEQUENCE: 6
cccacgcgtc cggaacgact gagactgggc gatccagtcc caacggggag ctcccgcact
60 agggtaccgg gctgacattt gccgggtctc ggaccacgcc tctcagatca
gaagtggtcc 120 aggaggggcg gagtccgagg tgggcggggc aggagggggc
agccccccgc cacgctgcag 180 ttgcagggac attcctggct cttcggcccg
gggcggagga gctccgggcg ggtgagtgtg 240 ccagccctgc cgggatcgtg
acccgcgcgc gcgggagccg ggcggcggag gagccaggaa 300 ggtggtcagt
gggaagtctg gccctgatcc tgagatcagc tggaaggaa atg tgg ctg 358 Met Trp
Leu 1 ctc ttg gcc ctg ttg agc atc ttt cag ggg aca cca gct ttg tcc
ctt 406 Leu Leu Ala Leu Leu Ser Ile Phe Gln Gly Thr Pro Ala Leu Ser
Leu 5 10 15 gag gcc tct gag gaa atg gag cag gag ccc tgc cta gcc cca
atc ctg 454 Glu Ala Ser Glu Glu Met Glu Gln Glu Pro Cys Leu Ala Pro
Ile Leu 20 25 30 35 gag cag caa gag cag gtg ttg acg gtg gcc ctg ggg
cag cct gtg agg 502 Glu Gln Gln Glu Gln Val Leu Thr Val Ala Leu Gly
Gln Pro Val Arg 40 45 50 ctg tgc tgt ggg cgc acc gag cgt ggt cgt
cac tgg tac aaa gag ggc 550 Leu Cys Cys Gly Arg Thr Glu Arg Gly Arg
His Trp Tyr Lys Glu Gly 55 60 65 agc cgc cta gca tct gct ggg cga
gta cgg ggt tgg aga ggc cgc ctg 598 Ser Arg Leu Ala Ser Ala Gly Arg
Val Arg Gly Trp Arg Gly Arg Leu 70 75 80 gag atc gcc agc ttc ctt
cct gag gat gct ggc cga tac ctc tgc ctg 646 Glu Ile Ala Ser Phe Leu
Pro Glu Asp Ala Gly Arg Tyr Leu Cys Leu 85 90 95 gcc cgt ggc tcc
atg acc gtc gta cac aat ctt acg ttg ctt atg gat 694 Ala Arg Gly Ser
Met Thr Val Val His Asn Leu Thr Leu Leu Met Asp 100 105 110 115 gac
tcc tta acc tcc atc agt aat gat gaa gac ccc aag aca ctc agc 742 Asp
Ser Leu Thr Ser Ile Ser Asn Asp Glu Asp Pro Lys Thr Leu Ser 120 125
130 agc tcc tcg agt ggt cat gtc tac cca cag caa gca ccc tac tgg aca
790 Ser Ser Ser Ser Gly His Val Tyr Pro Gln Gln Ala Pro Tyr Trp Thr
135 140 145 cac ccc caa cgc atg gag aag aaa ctg cat gca gtg cct gcc
ggg aat 838 His Pro Gln Arg Met Glu Lys Lys Leu His Ala Val Pro Ala
Gly Asn 150 155 160 act gtc aaa ttc cgc tgt cca gct gca ggg aac ccc
atg cct acc atc 886 Thr Val Lys Phe Arg Cys Pro Ala Ala Gly Asn Pro
Met Pro Thr Ile 165 170 175 cac tgg ctc aag gat gga cag gcc ttc cac
ggg gag aat cgt att gga 934 His Trp Leu Lys Asp Gly Gln Ala Phe His
Gly Glu Asn Arg Ile Gly 180 185 190 195 ggc att cgg ctg cgc cac caa
cac tgg agc ctg gtg atg gaa agt gtg 982 Gly Ile Arg Leu Arg His Gln
His Trp Ser Leu Val Met Glu Ser Val 200 205 210 gta ccc tcg gac cgt
ggc aca tac aca tgc ctt gtg gag aac tct ctg 1030 Val Pro Ser Asp
Arg Gly Thr Tyr Thr Cys Leu Val Glu Asn Ser Leu 215 220 225 ggt agc
att cgc tac agc tat ctc ctg gat gtg ctg gag cgg tcc ccg 1078 Gly
Ser Ile Arg Tyr Ser Tyr Leu Leu Asp Val Leu Glu Arg Ser Pro 230 235
240 cac cgg ccc atc ctg cag gcg ggg ctc cca gcc aac acc aca gct gtg
1126 His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn Thr Thr Ala
Val 245 250 255 gtt ggc agc gat gtg gag cta ctc tgc aag gtg tac agc
gac gcc cag 1174 Val Gly Ser Asp Val Glu Leu Leu Cys Lys Val Tyr
Ser Asp Ala Gln 260 265 270 275 ccc cac ata cag tgg ctg aaa cac gtc
gtc atc aac ggc agc agc ttc 1222 Pro His Ile Gln Trp Leu Lys His
Val Val Ile Asn Gly Ser Ser Phe 280 285 290 ggc gcc gac ggt ttc ccc
tac gta caa gtc ctg aag aca aca gac atc 1270 Gly Ala Asp Gly Phe
Pro Tyr Val Gln Val Leu Lys Thr Thr Asp Ile 295 300 305 aat agc tcg
gag gta gag gtc ttg tat ctg agg aac gtg tcc gct gag 1318 Asn Ser
Ser Glu Val Glu Val Leu Tyr Leu Arg Asn Val Ser Ala Glu 310 315 320
gat gca gga gag tat acc tgt ctg gcg ggc aac tcc atc ggc ctt tcc
1366 Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Leu
Ser 325 330 335 tac cag tca gcg tgg ctc acg gtg ctg cca gag gaa gac
ctc acg tgg 1414 Tyr Gln Ser Ala Trp Leu Thr Val Leu Pro Glu Glu
Asp Leu Thr Trp 340 345 350 355 aca aca gca acc cct gag gcc aga tac
aca gat atc atc ctg tat gta 1462 Thr Thr Ala Thr Pro Glu Ala Arg
Tyr Thr Asp Ile Ile Leu Tyr Val 360 365 370 tca ggc tca ctg gtt ctg
ctt gtg ctc ctg ctg ctg gcc ggg gtg tat 1510 Ser Gly Ser Leu Val
Leu Leu Val Leu Leu Leu Leu Ala Gly Val Tyr 375 380 385 cat cgg caa
gtc atc cgt ggc cac tac tct cgc cag cct gtc act ata 1558 His Arg
Gln Val Ile Arg Gly His Tyr Ser Arg Gln Pro Val Thr Ile 390 395 400
caa aag ctg tcc cgt ttc cct ttg gcc cga cag ttc tct ttg gag tcg
1606 Gln Lys Leu Ser Arg Phe Pro Leu Ala Arg Gln Phe Ser Leu Glu
Ser 405 410 415 agg tcc tct ggc aag tca agt ttg tcc ctg gtg cga ggt
gtc cgt ctc 1654 Arg Ser Ser Gly Lys Ser Ser Leu Ser Leu Val Arg
Gly Val Arg Leu 420 425 430 435 tcc tcc agc ggc ccg ccc ttg ctc acg
ggc ctt gtg aat cta gac ctg 1702 Ser Ser Ser Gly Pro Pro Leu Leu
Thr Gly Leu Val Asn Leu Asp Leu 440 445 450 cct ctc gat ccg ctt tgg
gaa ttc ccc cgg gac agg ttg gtg ctc gga 1750 Pro Leu Asp Pro Leu
Trp Glu Phe Pro Arg Asp Arg Leu Val Leu Gly 455 460 465 aag ccc ctg
ggt gag ggc tgc ttt ggg caa gtg gtt cgt gca gag gcc 1798 Lys Pro
Leu Gly Glu Gly Cys Phe Gly Gln Val Val Arg Ala Glu Ala 470 475 480
ttt ggt atg gat ccc tcc cgg ccc gac caa acc agc acc gtg gct gtg
1846 Phe Gly Met Asp Pro Ser Arg Pro Asp Gln Thr Ser Thr Val Ala
Val 485 490 495 aag atg ctg aaa gac aat gcc tcc gac aag gat ttg gca
gac ctg gtc 1894 Lys Met Leu Lys Asp Asn Ala Ser Asp Lys Asp Leu
Ala Asp Leu Val 500 505 510 515 tcc gag atg gag gtg atg aag cta atc
gga aga cac aag aac atc atc 1942 Ser Glu Met Glu Val Met Lys Leu
Ile Gly Arg His Lys Asn Ile Ile 520 525 530 aac ctg ctg ggt gtc tgc
act cag gaa ggg ccc ctg tac gtg att gtg 1990 Asn Leu Leu Gly Val
Cys Thr Gln Glu Gly Pro Leu Tyr Val Ile Val 535 540 545 gaa tgt gcc
gcc aag gga aat ctt cgg gaa ttc ctc cgt gcc cgg cgc 2038 Glu Cys
Ala Ala Lys Gly Asn Leu Arg Glu Phe Leu Arg Ala Arg Arg 550 555 560
ccc cca ggc cct gat ctc agc cct gat gga cct cgg agc agc gaa gga
2086 Pro Pro Gly Pro Asp Leu Ser Pro Asp Gly Pro Arg Ser Ser Glu
Gly 565 570 575 cca ctc tcc ttc ccg gcc cta gtc tcc tgt gcc tac cag
gtg gcc cga 2134 Pro Leu Ser Phe Pro Ala Leu Val Ser Cys Ala Tyr
Gln Val Ala Arg 580 585 590 595 ggc atg cag tat ctg gag tct cgg aag
tgc atc cac cgg gac ctg gct 2182 Gly Met Gln Tyr Leu Glu Ser Arg
Lys Cys Ile His Arg Asp Leu Ala 600 605 610 gcc cga aat gtg ctg gtg
acc gag gat gat gtg atg aag atc gct gac 2230 Ala Arg Asn Val Leu
Val Thr Glu Asp Asp Val Met Lys Ile Ala Asp 615 620 625
ttt ggg ctg gca cgt ggt gtc cac cac att gac tac tat aag aaa acc
2278 Phe Gly Leu Ala Arg Gly Val His His Ile Asp Tyr Tyr Lys Lys
Thr 630 635 640 agc aac ggc cgc ctg cca gtc aaa tgg atg gct cca gag
gcg ttg ttc 2326 Ser Asn Gly Arg Leu Pro Val Lys Trp Met Ala Pro
Glu Ala Leu Phe 645 650 655 gac cgt gtg tac aca cac cag agt gac gtg
tgg tct ttc ggg atc ctg 2374 Asp Arg Val Tyr Thr His Gln Ser Asp
Val Trp Ser Phe Gly Ile Leu 660 665 670 675 ctg tgg gaa atc ttc acc
ctc ggg ggc tcc cca tac cct ggc att ccg 2422 Leu Trp Glu Ile Phe
Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro 680 685 690 gtg gag gag
ctc ttc tca ctg ctg cga gag ggg cac agg atg gag cgg 2470 Val Glu
Glu Leu Phe Ser Leu Leu Arg Glu Gly His Arg Met Glu Arg 695 700 705
ccc cca aac tgc ccc tca gag ctg tat ggg cta atg agg gag tgc tgg
2518 Pro Pro Asn Cys Pro Ser Glu Leu Tyr Gly Leu Met Arg Glu Cys
Trp 710 715 720 cac gca gtc cca tct cag agg cct act ttt aag cag ctg
gtg gaa gct 2566 His Ala Val Pro Ser Gln Arg Pro Thr Phe Lys Gln
Leu Val Glu Ala 725 730 735 ctg gac aag gtc ctg ctg gct gtc tct gaa
gag tac ctt gac ctc cgc 2614 Leu Asp Lys Val Leu Leu Ala Val Ser
Glu Glu Tyr Leu Asp Leu Arg 740 745 750 755 ctg acc ttt gga ccc ttt
tct ccc tcc aat ggg gat gcc agc agc acc 2662 Leu Thr Phe Gly Pro
Phe Ser Pro Ser Asn Gly Asp Ala Ser Ser Thr 760 765 770 tgc tcc tcc
agt gac tcg gtt ttc agc cac gac cct ttg ccc ctc gag 2710 Cys Ser
Ser Ser Asp Ser Val Phe Ser His Asp Pro Leu Pro Leu Glu 775 780 785
cca agc ccc ttc cct ttc tct gac tcg cag acg aca tga gccggggagc 2759
Pro Ser Pro Phe Pro Phe Ser Asp Ser Gln Thr Thr 790 795 agcaattttg
tatgggctac gcggcccatg gccgtgggtc tcctcgctga gctgcaacct 2819
gatgcatcga catttaatgt tggcagtgtc aggcctctga cttgagacta ctgctgtcgc
2879 agatcctctc tctggccctg ttttggggag ggccattctt ggtcctaagg
ttcatagttg 2939 aggccttctg ttccagcctt atgctcccat ctcagagttc
aactctcatc tcaagatcat 2999 ggccttgccc ttggactcat cctcagagaa
gttaagcatt aaggccttgg cacgcagcct 3059 ccgtctccgg ggctctccgg
gcctagctgc aaaacttatg ctctaaacat ttctagttcc 3119 cccaaacaac
ctagaggcct tgggacttca catcccccag cacacaagcc tcaccacccc 3179
ctgccatccc ccctccattg cttgttccag catcttggtg aaaggggcat cagctctggt
3239 gtccctgaga gacgggaagc ctgtgggaac gacagaagaa catggcattt
ttataaatta 3299 tttttttgaa aaaaaaaaaa aaaa 3323 <210> SEQ ID
NO 7 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
7 ggcacactca gcaggacccc 20 <210> SEQ ID NO 8 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 8 aggctgccca
agggctactg 20 <210> SEQ ID NO 9 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 9 gtccagtagg gtgcttgctg 20
<210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 10 cccatgaaag gcctgtccat 20 <210> SEQ
ID NO 11 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
11 gcgcagccga atgcctccaa 20 <210> SEQ ID NO 12 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 12 tctccatcac
gagactccag 20 <210> SEQ ID NO 13 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 13 tacaccttgc acagcagctc 20
<210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 14 gctgctgccg ttgatgacga 20 <210> SEQ
ID NO 15 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
15 ccgaagctgc tgccgttgat 20 <210> SEQ ID NO 16 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 16 gcacactcag
caggaccccc 20 <210> SEQ ID NO 17 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 17 aggcacactc agcaggaccc 20
<210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 18 caggcacact cagcaggacc 20 <210> SEQ
ID NO 19 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
19 cccaggcaca ctcagcagga 20 <210> SEQ ID NO 20 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 20 gcccaggcac
actcagcagg 20 <210> SEQ ID NO 21 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 21 ggcccaggca cactcagcag 20
<210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 22 gaggcccagg cacactcagc 20 <210> SEQ
ID NO 23 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
23 tggaggccca ggcacactca 20 <210> SEQ ID NO 24 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 24 ctggaggccc
aggcacactc 20 <210> SEQ ID NO 25 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 25 gactggaggc ccaggcacac 20
<210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 26 ctgtcagctc ctgctcttgc 20 <210> SEQ
ID NO 27 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
27 ctactgtcag ctcctgctct 20 <210> SEQ ID NO 28 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 28 gctactgtca
gctcctgctc 20 <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 ggctactgtc agctcctgct 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 gggctactgt cagctcctgc 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 gcccaagggc tactgtcagc 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 cgcacaggct
gcccaagggc 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 gctcagcccg cccacagcac 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 caccacgctc agcccgccca 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 ccaccacgct cagcccgccc 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 gccaccacgc
tcagcccgcc 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 ggccaccacg ctcagcccgc 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 accagtggcc accacgctca 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 gtaccagtgg ccaccacgct 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 tgtaccagtg
gccaccacgc 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 ctccttgtac cagtggccac 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 cctccttgta ccagtggcca 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 ccctccttgt accagtggcc 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 ccaggcgact gccctccttg 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 gccaggcgac
tgccctcctt 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 tgccaggcga ctgccctcct 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 gtgccaggcg actgccctcc 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 ggtgccaggc gactgccctc 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 ccgtacacgg
ccagcaggtg 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 cccgtacacg gccagcaggt 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 agccccgtac acggccagca 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 cctccagccc cgtacacggc 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 ccctccagcc
ccgtacacgg 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 ggcggcccct ccagccccgt 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 gcaatctcta ggcggcccct 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 ggcaatctct aggcggcccc 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 tggcaatctc
taggcggccc 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 cctcaggtag gaagctggca 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 tcctcaggta ggaagctggc 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 agcggccagc atcctcaggt 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 tagcggccag
catcctcagg 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 gtgtccagta gggtgcttgc 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 gtgtgtccag tagggtgctt 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 agtttcttct ccatgcgctg 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 gcctgtccat ccttaagcca 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 ttctccccat gaaaggcctg 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 ggttctcccc
atgaaaggcc 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 ctccatcacg agactccagt 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 gctctccatc acgagactcc 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 cgctctccat cacgagactc 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 acgctctcca
tcacgagact 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 cacgctctcc atcacgagac 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 ccacgctctc catcacgaga 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 acaccttgca cagcagctcc 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 gtacaccttg
cacagcagct 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 gccgttgatg acgatgtgct 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 gctgccgttg atgacgatgt 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 tgctgccgtt gatgacgatg 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 ctgctgccgt
tgatgacgat 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 agctgctgcc gttgatgacg 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 aagctgctgc cgttgatgac 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 cgaagctgct gccgttgatg 20 <210> SEQ ID NO 83 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 83 gctattgatg
tctgcagtct 20 <210> SEQ ID NO 84 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 84 agctattgat gtctgcagtc 20
<210> SEQ ID NO 85 <211> LENGTH: 20 <212> TYPE:
DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 85 gagctattga tgtctgcagt 20 <210> SEQ
ID NO 86 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
86 cctccacctc tgagctattg 20 <210> SEQ ID NO 87 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 87 acctccacct
ctgagctatt 20 <210> SEQ ID NO 88 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 88 gacctccacc tctgagctat 20
<210> SEQ ID NO 89 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 89 ggacctccac ctctgagcta 20 <210> SEQ
ID NO 90 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
90 aggacctcca cctctgagct 20 <210> SEQ ID NO 91 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 91 caggacctcc
acctctgagc 20 <210> SEQ ID NO 92 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 92 acaggacctc cacctctgag 20
<210> SEQ ID NO 93 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 93 gtacaggacc tccacctctg 20 <210> SEQ
ID NO 94 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
94 ggtacaggac ctccacctct 20 <210> SEQ ID NO 95 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 95 ccgcaggtac
aggacctcca 20 <210> SEQ ID NO 96 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 96 tccgcaggta caggacctcc 20
<210> SEQ ID NO 97 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 97 cgttccgcag gtacaggacc 20 <210> SEQ
ID NO 98 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
98 gcagactggt aggagaggcc 20 <210> SEQ ID NO 99 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 99 ggcagactgg
taggagaggc 20 <210> SEQ ID NO 100 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 100 ggtcctcctc tggcagcacc 20
<210> SEQ ID NO 101 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 101 gcaggagcac agccaaggcc 20 <210> SEQ
ID NO 102 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
102 gcctgccctc gatacagccc 20 <210> SEQ ID NO 103 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 103 gcctgactcc
agggagaact 20 <210> SEQ ID NO 104 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 104 gagcctgact ccagggagaa 20
<210> SEQ ID NO 105 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 105 ggaggagaga cgcacgcctc 20 <210> SEQ
ID NO 106 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 106 tggaggagag acgcacgcct 20
<210> SEQ ID NO 107 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 107 ctggaggaga gacgcacgcc 20 <210> SEQ
ID NO 108 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
108 gactcacgag gccggcgagc 20 <210> SEQ ID NO 109 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 109 ctagactcac
gaggccggcg 20 <210> SEQ ID NO 110 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 110 cccaagcacc agcctgtccc 20
<210> SEQ ID NO 111 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 111 tcccaagcac cagcctgtcc 20 <210> SEQ
ID NO 112 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
112 gcttcccaag caccagcctg 20 <210> SEQ ID NO 113 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 113 gggcttccca
agcaccagcc 20 <210> SEQ ID NO 114 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 114 ccatgccaaa ggcctctgca 20
<210> SEQ ID NO 115 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 115 gtccatgcca aaggcctctg 20 <210> SEQ
ID NO 116 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
116 ggtccatgcc aaaggcctct 20 <210> SEQ ID NO 117 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 117 cagcagccgc
atctccttct 20 <210> SEQ ID NO 118 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 118 gctgaagaca gaatcgctgg 20
<210> SEQ ID NO 119 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 119 cagcactcac gcatcagccc 20 <210> SEQ
ID NO 120 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
120 ccagcactca cgcatcagcc 20 <210> SEQ ID NO 121 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 121 gggtccgaag
gtcaggcgga 20 <210> SEQ ID NO 122 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 122 agggtccgaa ggtcaggcgg 20
<210> SEQ ID NO 123 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 123 atagggtccg aaggtcaggc 20 <210> SEQ
ID NO 124 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
124 ggaatagggt ccgaaggtca 20 <210> SEQ ID NO 125 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 125 gtgcctgcac
agccttgagc 20 <210> SEQ ID NO 126 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 126 tctccagcca ggctcagcca 20
<210> SEQ ID NO 127
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 127
cagctctcca gccaggctca 20 <210> SEQ ID NO 128 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 128 gcagctctcc
agccaggctc 20 <210> SEQ ID NO 129 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 129 agcagctctc cagccaggct 20
<210> SEQ ID NO 130 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 130 tagcagctct ccagccaggc 20 <210> SEQ
ID NO 131 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
131 gcatagcagc tctccagcca 20 <210> SEQ ID NO 132 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 132 agcatagcag
ctctccagcc 20 <210> SEQ ID NO 133 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 133 tagcatagca gctctccagc 20
<210> SEQ ID NO 134 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 134 ttagcatagc agctctccag 20 <210> SEQ
ID NO 135 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
135 ccagcttctc tgggctcagg 20 <210> SEQ ID NO 136 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 136 tccagcttct
ctgggctcag 20 <210> SEQ ID NO 137 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 137 ttccagcttc tctgggctca 20
<210> SEQ ID NO 138 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 138 cttccagctt ctctgggctc 20 <210> SEQ
ID NO 139 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
139 gcttccagct tctctgggct 20 <210> SEQ ID NO 140 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 140 ggcttccagc
ttctctgggc 20 <210> SEQ ID NO 141 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 141 acgccatttg ctcctgtttt 20
<210> SEQ ID NO 142 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 142 tgcgaatcaa tgggtcccga 20 <210> SEQ
ID NO 143 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
143 ggtgcgaatc aatgggtccc 20 <210> SEQ ID NO 144 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 144 ccgccggcgc
gaagacagcc 20 <210> SEQ ID NO 145 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 145 catctctgcc gccggcgcga 20
<210> SEQ ID NO 146 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 146 ctgaccgctg accgaccacc 20 <210> SEQ
ID NO 147 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
147 gctgctgacc gctgaccgac 20
<210> SEQ ID NO 148 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 148 ctgccctgat atcagagtcc 20 <210> SEQ
ID NO 149 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
149 ggctgccctg atatcagagt 20 <210> SEQ ID NO 150 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 150 ctcagatact
gctgtctctg 20 <210> SEQ ID NO 151 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 151 tgcccatccc tctgtgcccc 20
<210> SEQ ID NO 152 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 152 tgctctcttg cccatccctc 20 <210> SEQ
ID NO 153 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
153 ctctttggtc acaccgtctg 20 <210> SEQ ID NO 154 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 154 atctctttgg
tcacaccgtc 20 <210> SEQ ID NO 155 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 155 ctatctcttt ggtcacaccg 20
<210> SEQ ID NO 156 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 156 gcctatctct ttggtcacac 20 <210> SEQ
ID NO 157 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
157 cgctgcctat ctctttggtc 20 <210> SEQ ID NO 158 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 158 agcttgcaag
cccttaatgg 20 <210> SEQ ID NO 159 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 159 ccagcttgca agcccttaat 20
<210> SEQ ID NO 160 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 160 accttcatct tccagcagag 20 <210> SEQ
ID NO 161 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
161 caaccttcat cttccagcag 20 <210> SEQ ID NO 162 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 162 ttcaaccttc
atcttccagc 20 <210> SEQ ID NO 163 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 163 cagctttgct cagcccagca 20
<210> SEQ ID NO 164 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 164 tccagctttg ctcagcccag 20 <210> SEQ
ID NO 165 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
165 tttccagctt tgctcagccc 20 <210> SEQ ID NO 166 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 166 cctttccagc
tttgctcagc 20 <210> SEQ ID NO 167 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 167 ccaggtccac agtccagggc 20
<210> SEQ ID NO 168 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 168 acttgccaga gagtagcaga 20
<210> SEQ ID NO 169 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 169 gccatagcac ctcctccagg 20 <210> SEQ
ID NO 170 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
170 cccaatgcca tagcacctcc 20 <210> SEQ ID NO 171 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 171 gtcccaatgc
catagcacct 20 <210> SEQ ID NO 172 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 172 tagtcccaat gccatagcac 20
<210> SEQ ID NO 173 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 173 ttctattagt cccaatgcca 20 <210> SEQ
ID NO 174 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
174 gtcacttgcc agggtcagga 20 <210> SEQ ID NO 175 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 175 gctcagaagt
cacttgccag 20 <210> SEQ ID NO 176 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 176 gtccatctgg cttcccctgc 20
<210> SEQ ID NO 177 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 177 cagtccatct ggcttcccct 20 <210> SEQ
ID NO 178 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
178 ccactccact tccagtccat 20 <210> SEQ ID NO 179 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 179 tgccactcca
cttccagtcc 20 <210> SEQ ID NO 180 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 180 ggtcactgcc actccacttc 20
<210> SEQ ID NO 181 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 181 ccttggtcac tgccactcca 20 <210> SEQ
ID NO 182 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
182 ggaagcctat cacacctcct 20 <210> SEQ ID NO 183 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 183 gtgtctctgg
atctaccctg 20 <210> SEQ ID NO 184 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 184 tggtgtctct ggatctaccc 20
<210> SEQ ID NO 185 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 185 actggtgtct ctggatctac 20 <210> SEQ
ID NO 186 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
186 gcactggtgt ctctggatct 20 <210> SEQ ID NO 187 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 187 tggcactggt
gtctctggat 20 <210> SEQ ID NO 188 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 188 ggtggcactg gtgtctctgg 20
<210> SEQ ID NO 189 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 189 tgggtggcac tggtgtctct 20
<210> SEQ ID NO 190 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 190 tatgggtggc actggtgtct 20 <210> SEQ
ID NO 191 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
191 ggcctatggg tggcactggt 20 <210> SEQ ID NO 192 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 192 gtcaggctgt
gatgtacaca 20 <210> SEQ ID NO 193 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 193 tgctgttact gtcaggctgt 20
<210> SEQ ID NO 194 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 194 gccagtcacc tctggttcgg 20 <210> SEQ
ID NO 195 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
195 agcagttttg ggattctttt 20 <210> SEQ ID NO 196 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 196 aaagcagttt
tgggattctt 20 <210> SEQ ID NO 197 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 197 tccaagtccc tggccaggct 20
<210> SEQ ID NO 198 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 198 atcctttcca gctttgctca 20 <210> SEQ
ID NO 199 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
199 ggatcctttc cagctttgct 20 <210> SEQ ID NO 200 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 200 aaggatcctt
tccagctttg 20 <210> SEQ ID NO 201 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 201 gcaaggatcc tttccagctt 20
<210> SEQ ID NO 202 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 202 gggcaaggat cctttccagc 20 <210> SEQ
ID NO 203 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
203 ctgggcaagg atcctttcca 20 <210> SEQ ID NO 204 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 204 gcctgggcaa
ggatcctttc 20 <210> SEQ ID NO 205 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 205 gtggttgagc cctgccctgc 20
<210> SEQ ID NO 206 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 206 gtctcagtgg ttgagccctg 20 <210> SEQ
ID NO 207 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
207 ctgactgagt ctcagtggtt 20 <210> SEQ ID NO 208 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 208 ggcactgact
gagtctcagt 20 <210> SEQ ID NO 209 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 209 caggcactga ctgagtctca 20
<210> SEQ ID NO 210 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 210
aagccaggca ctgactgagt 20 <210> SEQ ID NO 211 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 211 ctggaagcca
ggcactgact 20 <210> SEQ ID NO 212 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 212 gcttgctgga agccaggcac 20
<210> SEQ ID NO 213 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 213 atgcttgctg gaagccaggc 20 <210> SEQ
ID NO 214 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
214 gtcctctctc gcagacacag 20 <210> SEQ ID NO 215 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 215 cagtcctctc
tcgcagacac 20 <210> SEQ ID NO 216 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 216 gccagtcctc tctcgcagac 20
<210> SEQ ID NO 217 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 217 aggccagtcc tctctcgcag 20 <210> SEQ
ID NO 218 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
218 gagctcacca ccagctctgc 20 <210> SEQ ID NO 219 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 219 gctgcctgga
cctcctaggt 20 <210> SEQ ID NO 220 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 220 atgctgcctg gacctcctag 20
<210> SEQ ID NO 221 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 221 acatgctgcc tggacctcct 20 <210> SEQ
ID NO 222 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
222 acacatgctg cctggacctc 20 <210> SEQ ID NO 223 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 223 ccacacatgc
tgcctggacc 20 <210> SEQ ID NO 224 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 224 gcaaatgcca cactcttggg 20
<210> SEQ ID NO 225 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 225 gggcaaatgc cacactcttg 20 <210> SEQ
ID NO 226 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
226 cagggcaaat gccacactct 20 <210> SEQ ID NO 227 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 227 cccagggcaa
atgccacact 20 <210> SEQ ID NO 228 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 228 cacccagggc aaatgccaca 20
<210> SEQ ID NO 229 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 229 gccacaccca gggcaaatgc 20 <210> SEQ
ID NO 230 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
230 ggatgccaca cccagggcaa 20 <210> SEQ ID NO 231 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 231
gcggatgcca cacccagggc 20 <210> SEQ ID NO 232 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 232 ctgcggatgc
cacacccagg 20 <210> SEQ ID NO 233 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 233 gccacatgct gcggatgcca 20
<210> SEQ ID NO 234 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 234 ggacttccca ccaactgcct 20 <210> SEQ
ID NO 235 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
235 gctggacttc ccaccaactg 20 <210> SEQ ID NO 236 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 236 caaggagctc
accaccagct 20 <210> SEQ ID NO 237 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 237 ggcaaggagc tcaccaccag 20
<210> SEQ ID NO 238 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 238 caggcaagga gctcaccacc 20 <210> SEQ
ID NO 239 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
239 tggacttccc accaactgcc 20 <210> SEQ ID NO 240 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 240 cactggtgtc
tctggatcta 20 <210> SEQ ID NO 241 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 241 ggcactggtg tctctggatc 20
<210> SEQ ID NO 242 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 242 gtggcactgg tgtctctgga 20 <210> SEQ
ID NO 243 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
243 gggtggcact ggtgtctctg 20 <210> SEQ ID NO 244 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 244 gatcctttcc
agctttgctc 20 <210> SEQ ID NO 245 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 245 aggatccttt ccagctttgc 20
<210> SEQ ID NO 246 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 246 caaggatcct ttccagcttt 20 <210> SEQ
ID NO 247 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
247 ggcaaggatc ctttccagct 20 <210> SEQ ID NO 248 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 248 tgggcaagga
tcctttccag 20 <210> SEQ ID NO 249 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 249 tgactgagtc tcagtggttg 20
<210> SEQ ID NO 250 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 250 actgactgag tctcagtggt 20 <210> SEQ
ID NO 251 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
251 gcactgactg agtctcagtg 20 <210> SEQ ID NO 252 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide
<400> SEQUENCE: 252 aggcactgac tgagtctcag 20 <210> SEQ
ID NO 253 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
253 tgcttgctgg aagccaggca 20 <210> SEQ ID NO 254 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 254 agtcctctct
cgcagacaca 20 <210> SEQ ID NO 255 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 255 ccagtcctct ctcgcagaca 20
<210> SEQ ID NO 256 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 256 ggccagtcct ctctcgcaga 20 <210> SEQ
ID NO 257 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
257 aaggagctca ccaccagctc 20 <210> SEQ ID NO 258 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 258 gcaaggagct
caccaccagc 20 <210> SEQ ID NO 259 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 259 ctgcctggac ctcctaggtc 20
<210> SEQ ID NO 260 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 260 tgctgcctgg acctcctagg 20 <210> SEQ
ID NO 261 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
261 catgctgcct ggacctccta 20 <210> SEQ ID NO 262 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 262 cacatgctgc
ctggacctcc 20 <210> SEQ ID NO 263 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 263 cacacatgct gcctggacct 20
<210> SEQ ID NO 264 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 264 accacacatg ctgcctggac 20 <210> SEQ
ID NO 265 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
265 ccagggcaaa tgccacactc 20 <210> SEQ ID NO 266 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 266 acccagggca
aatgccacac 20 <210> SEQ ID NO 267 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 267 tgccacaccc agggcaaatg 20
<210> SEQ ID NO 268 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 268 cggatgccac acccagggca 20 <210> SEQ
ID NO 269 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
269 tgcggatgcc acacccaggg 20 <210> SEQ ID NO 270 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 270 agccacatgc
tgcggatgcc 20 <210> SEQ ID NO 271 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 271 gctctcttgc ccatccctct 20
<210> SEQ ID NO 272 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 272 tctctttggt cacaccgtct 20 <210> SEQ
ID NO 273 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 273 tatctctttg gtcacaccgt 20 <210> SEQ
ID NO 274 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
274 cctatctctt tggtcacacc 20 <210> SEQ ID NO 275 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 275 tgcctatctc
tttggtcaca 20 <210> SEQ ID NO 276 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 276 tcaaccttca tcttccagca 20
<210> SEQ ID NO 277 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 277 agctttgctc agcccagcag 20 <210> SEQ
ID NO 278 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
278 ccagctttgc tcagcccagc 20 <210> SEQ ID NO 279 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 279 ttccagcttt
gctcagccca 20 <210> SEQ ID NO 280 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 280 tcacttgcca gggtcaggag 20
<210> SEQ ID NO 281 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 281 agtcacttgc cagggtcagg 20 <210> SEQ
ID NO 282 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
282 ctttggtcac accgtct 17 <210> SEQ ID NO 283 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 283 tctttggtca
caccgtc 17 <210> SEQ ID NO 284 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 284 ctctttggtc acaccgt 17
<210> SEQ ID NO 285 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 285 tctctttggt cacaccg 17 <210> SEQ ID
NO 286 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
286 gctttgctca gcccagc 17 <210> SEQ ID NO 287 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 287 cagctttgct
cagccca 17 <210> SEQ ID NO 288 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 288 ccagctttgc tcagccc 17
<210> SEQ ID NO 289 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 289 gcagccgcat ctccttc 17 <210> SEQ ID
NO 290 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
290 gcacactcag caggacc 17 <210> SEQ ID NO 291 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 291 ggcacactca
gcaggac 17 <210> SEQ ID NO 292 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 292 ccagtggcca ccacgct 17
<210> SEQ ID NO 293 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 293 aggcgactgc cctcctt 17 <210> SEQ ID
NO 294 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 294 caggcgactg ccctcct 17 <210> SEQ ID
NO 295 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
295 gtgtccagta gggtgct 17 <210> SEQ ID NO 296 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 296 cagctctcca
gccaggc 17 <210> SEQ ID NO 297 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 297 gcttctctgg gctcagg 17
<210> SEQ ID NO 298 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 298 agcttctctg ggctcag 17 <210> SEQ ID
NO 299 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
299 cagcttctct gggctca 17 <210> SEQ ID NO 300 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 300 ccagcttctc
tgggctc 17 <210> SEQ ID NO 301 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 301 tccagcttct ctgggct 17
<210> SEQ ID NO 302 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 302 ttccagcttc tctgggc 17 <210> SEQ ID
NO 303 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
303 gcttccagct tctctgg 17 <210> SEQ ID NO 304 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 304 ccatttgctc
ctgtttt 17 <210> SEQ ID NO 305 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 305 gccatttgct cctgttt 17
<210> SEQ ID NO 306 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 306 cgccatttgc tcctgtt 17 <210> SEQ ID
NO 307 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
307 gcactggtgt ctctgga 17 <210> SEQ ID NO 308 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 308 ggcactggtg
tctctgg 17 <210> SEQ ID NO 309 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 309 tggcactggt gtctctg 17
<210> SEQ ID NO 310 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 310 gtggcactgg tgtctct 17 <210> SEQ ID
NO 311 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
311 gactgagtct cagtggt 17 <210> SEQ ID NO 312 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 312 cttgctggaa
gccaggc 17 <210> SEQ ID NO 313 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 313 gcttgctgga agccagg 17
<210> SEQ ID NO 314 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 314 cctctctcgc agacaca 17 <210> SEQ ID
NO 315 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 315 gccagtcctc tctcgca 17
<210> SEQ ID NO 316 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 316 ggccagtcct ctctcgc 17 <210> SEQ ID
NO 317 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
317 gcctggacct cctaggt 17 <210> SEQ ID NO 318 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 318 tgcctggacc
tcctagg 17 <210> SEQ ID NO 319 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 319 ctgcctggac ctcctag 17
<210> SEQ ID NO 320 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 320 gctgcctgga cctccta 17 <210> SEQ ID
NO 321 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
321 tgctgcctgg acctcct 17 <210> SEQ ID NO 322 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 322 atgctgcctg
gacctcc 17 <210> SEQ ID NO 323 <211> LENGTH: 27
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 323 gcgtttgctc ttcttcttgc
gtttttt 27 <210> SEQ ID NO 324 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 324 gccacatttc cttccagctg 20
<210> SEQ ID NO 325 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 325 ccttccctga aggttcctcc 20 <210> SEQ
ID NO 326 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
326 tccatttcct cagaggcctc 20 <210> SEQ ID NO 327 <211>
LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 327 tcatcaacgg cagcagctt 19 <210> SEQ
ID NO 328 <211> LENGTH: 26 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 328 agctattgat
gtctgcagtc tttagg 26 <210> SEQ ID NO 329 <211> LENGTH:
24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 329 agccgacggt ttcccctatg tgca 24 <210>
SEQ ID NO 330 <211> LENGTH: 18 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 330
ttgctgtgcc gtgtccaa 18 <210> SEQ ID NO 331 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 331 tccaagaagc cgagcagaac 20 <210> SEQ
ID NO 332 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Probe <400> SEQUENCE: 332 agctgccgtg
cctgtgtcct gat 23 <210> SEQ ID NO 333 <211> LENGTH: 19
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 333 tcatcaacgg cagcagctt 19 <210> SEQ
ID NO 334 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 334 tgagctattg
atgtctgcag tcttc 25 <210> SEQ ID NO 335 <211> LENGTH:
22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 335 ccgacggctt cccctatgtg ca 22 <210>
SEQ ID NO 336 <211> LENGTH: 20 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 336
ccccatgtgg gaattgatct 20 <210> SEQ ID NO 337 <211>
LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 337 catgcctgct tcagtcagtt ct 22 <210>
SEQ ID NO 338 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Probe <400> SEQUENCE: 338
tttgcccttc ccaaacccct cca 23 <210> SEQ ID NO 339 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 339 ccctgaggcc agatacacag atat 24 <210>
SEQ ID NO 340 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 340
acggatgact tgccgatgat a 21 <210> SEQ ID NO 341 <211>
LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 341 ctcactggtt ctgcttgtgc tcctgct 27
<210> SEQ ID NO 342 <211> LENGTH: 23 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 342
gaccaaaacg aacgaaattt gtt 23 <210> SEQ ID NO 343 <211>
LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 343 acgtccttga tggcaatcg 19 <210> SEQ
ID NO 344 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Probe <400> SEQUENCE: 344 aattccgcgc
ggtcgctctg 20 <210> SEQ ID NO 345 <211> LENGTH: 1809
<212> TYPE: DNA <213> ORGANISM: Mus musculus
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (148)..(804) <400> SEQUENCE: 345 ctgtcggagc
agtaactctg tgcgccccac gccacaagcg cccagttgct ttgtgggttg 60
tgcctgccct gcgcctgcaa cttgagtccc cgccgcatcg cagtctccgc gccacctttg
120 taacggcctt caggaccccg aggtgtc atg gcg aga aag tgg aac ggg cgt
gcg 174 Met Ala Arg Lys Trp Asn Gly Arg Ala 1 5 gtg gcc cga gcc ctg
gtc ctg gcc act ctg tgg ctg gct gtg tct ggg 222 Val Ala Arg Ala Leu
Val Leu Ala Thr Leu Trp Leu Ala Val Ser Gly 10 15 20 25 cgt ccc ctg
gct cag caa tcc cag tct gtg tca gat gaa gat cca ctc 270 Arg Pro Leu
Ala Gln Gln Ser Gln Ser Val Ser Asp Glu Asp Pro Leu 30 35 40 ttt
ctc tac ggc tgg ggc aag att acc cgc ctg cag tac ctg tac tcc 318 Phe
Leu Tyr Gly Trp Gly Lys Ile Thr Arg Leu Gln Tyr Leu Tyr Ser 45 50
55 gct ggt ccc tat gtc tcc aac tgc ttc ctc cga atc cgg agc gac ggc
366 Ala Gly Pro Tyr Val Ser Asn Cys Phe Leu Arg Ile Arg Ser Asp Gly
60 65 70 tct gtg gac tgc gag gag gac caa aac gaa cga aat ttg ttg
gaa ttc 414 Ser Val Asp Cys Glu Glu Asp Gln Asn Glu Arg Asn Leu Leu
Glu Phe 75 80 85 cgc gcg gtc gct ctg aag acg att gcc atc aag gac
gtc agc agc gtg 462 Arg Ala Val Ala Leu Lys Thr Ile Ala Ile Lys Asp
Val Ser Ser Val 90 95 100 105 cgg tac ctc tgc atg agc gcg gac ggc
aag ata tac ggg ctg att cgc 510 Arg Tyr Leu Cys Met Ser Ala Asp Gly
Lys Ile Tyr Gly Leu Ile Arg 110 115 120 tac tcg gag gaa gac tgt acc
ttc agg gag gaa atg gac tgt tta ggc 558 Tyr Ser Glu Glu Asp Cys Thr
Phe Arg Glu Glu Met Asp Cys Leu Gly 125 130 135 tac aac cag tac aga
tcc atg aag cac cat ctc cat atc atc ttc atc 606 Tyr Asn Gln Tyr Arg
Ser Met Lys His His Leu His Ile Ile Phe Ile 140 145 150 cag gcc aag
ccc aga gaa cag ctc cag gac cag aaa ccc tca aac ttt 654 Gln Ala Lys
Pro Arg Glu Gln Leu Gln Asp Gln Lys Pro Ser Asn Phe 155 160 165 atc
ccc gtg ttt cac cgc tcc ttc ttt gaa acc ggg gac cag ctg agg 702 Ile
Pro Val Phe His Arg Ser Phe Phe Glu Thr Gly Asp Gln Leu Arg 170 175
180 185 tct aaa atg ttc tcc ctg ccc ctg gag agt gac agc atg gat ccg
ttc 750 Ser Lys Met Phe Ser Leu Pro Leu Glu Ser Asp Ser Met Asp Pro
Phe 190 195 200 agg atg gtg gag gat gta gac cac cta gtg aag agt ccc
agc ttc cag 798 Arg Met Val Glu Asp Val Asp His Leu Val Lys Ser Pro
Ser Phe Gln 205 210 215 aaa tga caggattccg acaggatgga gaaaacccca
aggtcccgtg aacttccccc 854 Lys ttaggaagct gtacatattc taagtctcac
atggaccctg ttgtgttagt ggctagactt 914 gatcatgaac ttaagttgac
aacctgcctg gctgccatcg gagccccact gactttggag 974 gctgctgata
tgtgcctaag ttactccagt tctgtttgaa tacctccact aatagggaac 1034
ttactcctgt gaaacattct tagttttgag ccaaatctgt gacttggatg gttttagcga
1094 ggaagccaga aggtatgaag tcaaatgata aaattcatgt atagaaagtg
ggctctaaaa 1154 tatatattcc ctatatggat ctcatgggat cttagcttgc
cccccaaatg tctcctggcc 1214 agaactaact ggggttacaa acttggaaca
aaggacagcc tagaaaactt tgggagcctt 1274 gaaggatggt cttaggatta
cgaattccag ctgactacgt agcttccccc ttttccactt 1334 ataaatgtca
gatggaagtg acccttagct gagtgcatag ccaagctgcc acttaggccc 1394
caggagcttg tctctgtccc atgaccccag atttccagga cctggatctt ctcctctgac
1454 ctttcccaga gttcacctgg gctctccaac cccagagcag gtagcttatg
agccatccag 1514 ttgtgtcccc agctcctggc tctcagttct ggtcaccaaa
cattgtgaat caacgtgtct 1574 gctgcctgtg tcaacctgga cccctcattt
acaaacaaga ttaggaagcc ccaaattctc 1634 cagtggcagc tggggaactg
tggagtcctt tccccggcac ttacgtggca gcatgatatt 1694 tataagtaat
ttattgtgtg tgtgtcttct attttcttac tttatttatg ccccagatca 1754
tatttatgta catgacttgt tttctacatt aaaaaggagt tggtttgtat caaaa 1809
<210> SEQ ID NO 346 <211> LENGTH: 2442 <212>
TYPE: DNA <213> ORGANISM: Macaca mulatta <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (1)..(651)
<400> SEQUENCE: 346 tgg gag aac ctg aga cgg tcg gaa ctg cgg
ggg gaa att cta ttg gac 48 Trp Glu Asn Leu Arg Arg Ser Glu Leu Arg
Gly Glu Ile Leu Leu Asp 1 5 10 15 ggc ttt gac gtc aga tgt gct ggg
ctg gga aag tcg ggg gag gga gtg 96 Gly Phe Asp Val Arg Cys Ala Gly
Leu Gly Lys Ser Gly Glu Gly Val 20 25 30 cga gtg gcc ttt taa ggg
gaa gga ccc taa ggc cga ggt gag gct ttt 144 Arg Val Ala Phe Gly Glu
Gly Pro Gly Arg Gly Glu Ala Phe 35 40 45 acc gac tag agg gtg aag
gag gca aaa ctc ggt gcc ccc aaa cct ctg 192 Thr Asp Arg Val Lys Glu
Ala Lys Leu Gly Ala Pro Lys Pro Leu 50 55 60 acc ccg ggg ttc ctg
acc ccg ccc ctg ctg gtg ccc cat gcc gag cgc 240 Thr Pro Gly Phe Leu
Thr Pro Pro Leu Leu Val Pro His Ala Glu Arg 65 70 75 atc cac tgg
gag ccc gac gcc tgg ggg agg ggc ccc agt tgt cga ttg 288 Ile His Trp
Glu Pro Asp Ala Trp Gly Arg Gly Pro Ser Cys Arg Leu 80 85 90 ctt
tgc aaa atc aaa ctc tcc cag cca aga acc tcg ggg ccg ctg cgc 336 Leu
Cys Lys Ile Lys Leu Ser Gln Pro Arg Thr Ser Gly Pro Leu Arg 95 100
105 ggt ggg gag gag ttc ccc gaa acc cgg ccg cta aac gag gcc tcc tcc
384 Gly Gly Glu Glu Phe Pro Glu Thr Arg Pro Leu Asn Glu Ala Ser Ser
110 115 120 125
tcc cgc aga tcc gaa cgg cct ggg cgg ggt cac ccc ggc tgg gac aag 432
Ser Arg Arg Ser Glu Arg Pro Gly Arg Gly His Pro Gly Trp Asp Lys 130
135 140 aag ccg ccg cct gcc tgc ccg ggc ccg ggg agg ggg ctg ggg ccg
gag 480 Lys Pro Pro Pro Ala Cys Pro Gly Pro Gly Arg Gly Leu Gly Pro
Glu 145 150 155 gcg ggg tgt gag tgg gtg tgt gcg ggg ggc gga ggc ttg
atg caa tcc 528 Ala Gly Cys Glu Trp Val Cys Ala Gly Gly Gly Gly Leu
Met Gln Ser 160 165 170 cga taa gaa atg ctc ggg tgt ctt ggg cac cta
ccc gcg ggg ccc gta 576 Arg Glu Met Leu Gly Cys Leu Gly His Leu Pro
Ala Gly Pro Val 175 180 185 agg cgc tac tat ata agg ttg ccg gcc cgg
agc cgc cgc gcc gtc gga 624 Arg Arg Tyr Tyr Ile Arg Leu Pro Ala Arg
Ser Arg Arg Ala Val Gly 190 195 200 gca gga gcg ctg cgt cca gga tcg
agg gccacggcca tcccaatccg 671 Ala Gly Ala Leu Arg Pro Gly Ser Arg
205 210 gcactcacag ccccgcagcg catcccggtc gccgcccagc ctcccgcacc
cccatcgccg 731 gaactgcgcc gagagcccca gggaggtgcc atgaggagcg
ggtgtgtggt ggtccacgcc 791 tggatcctgg ccagcctctg gctggccgtg
gccgggcgtc ccctcgcctt ctcggacgcg 851 gggccccacg tgcactacgg
ctggggcgac cccatccgcc tgcggcacct gtacacctcc 911 ggcccccatg
ggctctccag ctgcttcctg cgcatccgca ccgacggcgt cgtggactgc 971
gcgcggggcc aaagcgcgca cagtttgctg gagatcaagg cagtagctct gcggaccgtg
1031 gccatcaagg gcgtgcacag cgtgcggtac ctctgcatgg gcgccgacgg
caagatgcag 1091 gggctgcttc agtactcaga ggaagactgt gctttcgagg
aggagatccg ccctgatggc 1151 tacaatgtat accgatccga gaagcaccgc
ctcccggtct ctctgagcag tgccaaacag 1211 aggcagctgt acaagaacag
aggctttctt ccgctctctc atttcctacc catgctgccc 1271 atggccccag
aggagcctga ggacctcagg ggccacttgg aatctgacat gttctcttcg 1331
cccctggaga ctgacagcat ggacccattt gggcttgtca ccggactgga ggcggtgagg
1391 agtcccagct ttgagaaata actgaggcca tgcccgggcc tcttcactgc
tgccaggggc 1451 tgtggtacct gcagagtgga ggccgtgctt ctacaagagc
agtcccgagt ccacgttctg 1511 tttagcttta ggaagaaaca tctagaagtt
gtacatattc agagttttcc attggccgtg 1571 ccagtttcta gccaatagac
ttgtctgatc ataacattgt aagcttgtag cttgcccagc 1631 tgctgcccgg
gcccccattc tgctccctcg aggttgctgg acaagctgct gtgctgtctc 1691
agtcctgctt gaatacctcc actgatgggg aactcacttc ctttggaaaa attcttatgt
1751 caagctgaaa ttctctaatt tttttttctc atcacttccc caggagcagc
cggaagatag 1811 gcagtggttt aaatttcagg aacaggtgat ccactctgta
aaacagcacg tacatttcac 1871 tcaaccccat gtgggaattg atctatatct
acttccaggg accgtttgcc cttcccaaac 1931 ccctccaggc cagaactgac
tgaagcaggc atggcccaag aggcttcagg agtaggggaa 1991 gcccagagcc
ccactccagc cctgggacat cttgagaatt ccccctgagg ccagttctgt 2051
catggatgct gtcctgagaa taacttgctg tccccggtgt cacctgcttc caccccccag
2111 cccaccagcc ctctgcccac ctcacatgtc tccccatgga ttggggcttc
ccaggccccc 2171 catcttatgt caacctgcac ttctcgttca aaaatcagga
aaagaaaaga tttgaagacc 2231 ccaagtcttg tcaataactt gcggtgtgga
agcagcgggg gaagacttag aaccctttcc 2291 ccagcactta gttttccaac
atgatattta tgagtaattt attttgatat gtacatctct 2351 tattttctta
cattatttat gcccccaaat tatatttatg tatgtaagtg aggtttgttt 2411
tgtacattaa aatggagttt gtttgtatga a 2442 <210> SEQ ID NO 347
<211> LENGTH: 651 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens 220> <221> NAME/KEY: CDS <222>
LOCATION: (1)..(651) <400> SEQUENCE: 347 atg cgg agc ggg tgt
gtg gtg gtc cac gta tgg atc ctg gcc ggc ctc 48 Met Arg Ser Gly Cys
Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 tgg ctg gcc
gtg gcc ggg cgc ccc ctc gcc ttc tcg gac gcg ggg ccc 96 Trp Leu Ala
Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 cac
gtg cac tac ggc tgg ggc gac ccc atc cgc ctg cgg cac ctg tac 144 His
Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr 35 40
45 acc tcc ggc ccc cac ggg ctc tcc agc tgc ttc ctg cgc atc cgt gcc
192 Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala
50 55 60 gac ggc gtc gtg gac tgc gcg cgg ggc cag agc gcg cac agt
ttg ctg 240 Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser
Leu Leu 65 70 75 80 gag atc aag gca gtc gct ctg cgg acc gtg gcc atc
aag ggc gtg cac 288 Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile
Lys Gly Val His 85 90 95 agc gtg cgg tac ctc tgc atg ggc gcc gac
ggc aag atg cag ggg ctg 336 Ser Val Arg Tyr Leu Cys Met Gly Ala Asp
Gly Lys Met Gln Gly Leu 100 105 110 ctt cag tac tcg gag gaa gac tgt
gct ttc gag gag gag atc cgc cca 384 Leu Gln Tyr Ser Glu Glu Asp Cys
Ala Phe Glu Glu Glu Ile Arg Pro 115 120 125 gat ggc tac aat gtg tac
cga tcc gag aag cac cgc ctc ccg gtc tcc 432 Asp Gly Tyr Asn Val Tyr
Arg Ser Glu Lys His Arg Leu Pro Val Ser 130 135 140 ctg agc agt gcc
aaa cag cgg cag ctg tac aag aac aga ggc ttt ctt 480 Leu Ser Ser Ala
Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu 145 150 155 160 cca
ctc tct cat ttc ctg ccc atg ctg ccc atg gtc cca gag gag cct 528 Pro
Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro 165 170
175 gag gac ctc agg ggc cac ttg gaa tct gac atg ttc tct tcg ccc ctg
576 Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu
180 185 190 gag acc gac agc atg gac cca ttt ggg ctt gtc acc gga ctg
gag gcc 624 Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu
Glu Ala 195 200 205 gtg agg agt ccc agc ttt gag aag taa 651 Val Arg
Ser Pro Ser Phe Glu Lys 210 215 <210> SEQ ID NO 348
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer <400> SEQUENCE: 348 gcaccaggga tgagcttgac
20 <210> SEQ ID NO 349 <211> LENGTH: 22 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Primer <400>
SEQUENCE: 349 ccaagtctcc cactttccag tt 22 <210> SEQ ID NO 350
<211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Probe <400> SEQUENCE: 350 aagagcctga ctccagt
17
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