U.S. patent application number 14/821982 was filed with the patent office on 2016-03-03 for modulation of angiopoietin-like 3 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 Rosanne M. Crooke, Kenneth W. Dobie, Mark J. Graham, Richard Lee.
Application Number | 20160060626 14/821982 |
Document ID | / |
Family ID | 44306180 |
Filed Date | 2016-03-03 |
United States Patent
Application |
20160060626 |
Kind Code |
A1 |
Crooke; Rosanne M. ; et
al. |
March 3, 2016 |
MODULATION OF ANGIOPOIETIN-LIKE 3 EXPRESSION
Abstract
Provided herein are methods, compounds, and compositions for
reducing expression of an ANGPTL3 mRNA and protein in an animal.
Also provided herein are methods, compounds, and compositions for
reducing plasma lipids, plasma glucose and atherosclerotic plaques
in an animal. Such methods, compounds, and compositions are useful
to treat, prevent, delay, or ameliorate any one or more of
cardiovascular disease or metabolic disease, or a symptom
thereof.
Inventors: |
Crooke; Rosanne M.;
(Carlsbad, CA) ; Graham; Mark J.; (San Clemente,
CA) ; Lee; Richard; (Oceanside, CA) ; Dobie;
Kenneth W.; (Solana Beach, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ISIS PHARMACEUTICALS, INC. |
Carlsbad |
CA |
US |
|
|
Assignee: |
ISIS PHARMACEUTICALS, INC.
Carlsbad
CA
|
Family ID: |
44306180 |
Appl. No.: |
14/821982 |
Filed: |
August 10, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14149715 |
Jan 7, 2014 |
9139831 |
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14821982 |
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13520997 |
Oct 4, 2012 |
8653047 |
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PCT/US2011/020606 |
Jan 7, 2011 |
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14149715 |
|
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61293604 |
Jan 8, 2010 |
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Current U.S.
Class: |
514/44A ; 435/23;
435/34; 435/6.11; 435/6.12; 435/7.1; 435/7.92; 506/9 |
Current CPC
Class: |
A61P 9/12 20180101; C12Q
2600/136 20130101; C12N 2310/321 20130101; C12N 15/1136 20130101;
C12N 2310/315 20130101; C12N 2320/30 20130101; C12N 2310/14
20130101; A61P 3/10 20180101; C12N 2310/11 20130101; C12N 2310/341
20130101; A61P 3/06 20180101; A61P 3/08 20180101; C12N 2310/3341
20130101; A61P 9/00 20180101; C12N 15/113 20130101; C12N 2310/3231
20130101; C12Q 1/6883 20130101; A61P 9/10 20180101; G01N 33/5023
20130101; A61P 3/04 20180101; A61P 1/16 20180101; C12Q 2600/158
20130101; A61P 3/00 20180101; A61P 43/00 20180101; C12N 2310/321
20130101; C12N 2310/3521 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; C12Q 1/68 20060101 C12Q001/68 |
Claims
1-13. (canceled)
14. An assay for identifying a compound that inhibits
angiopoietin-like 3 (ANGPTL3) comprising: a) contacting cells with
a compound comprising a modified oligonucleotide 10 to 30 linked
nucleosides in length targeted to ANGPTL3; b) measuring an amount
of ANGPTL3 produced by the cells contacted by the compound
targeting ANGPTL3; and c) comparing the amount of ANGPTL3 produced
from the cells contacted with the compound targeting ANGPTL3 to an
amount of ANGPTL3 produced by cells not contacted with the compound
targeting ANGPTL3; wherein a reduction in ANGPTL3 production by the
cells contacted by the compound targeting ANGPTL3 compared to the
production of ANGPTL3 produced by cells not contacted by the
compound targeting ANGPTL3 identifies the compound that inhibits
ANGPTL3.
15. A kit for decreasing angiopoietin-like 3 (ANGPTL3) wherein the
kit comprises: a) a compound comprising a modified oligonucleotide
10 to 30 linked nucleosides in length targeted to ANGPTL3, wherein
the compound reduces expression of ANGPTL3 by at least 40%, and b)
a label.
16. The kit of claim 14, wherein the kit further comprises a second
agent.
17. A method for treating cardiovascular disease in a subject,
comprising: administering a therapeutically effective amount of a
compound comprising a modified oligonucleotide 10 to 30 linked
nucleosides in length targeted to angiopoietin-like protein 3
(ANGPTL3) to a subject in need thereof.
18. The method of claim 17, comprising identifying a subject in
need of treatment of cardiovascular disease and administering the
compound to the subject.
19. The method of claim 17, wherein expression of ANGPTL3 is
reduced by at least 40% in the subject.
20. The method of claim 17, wherein the subject is a human
subject.
21. The method of claim 17, wherein the modified oligonucleotide is
targeted to the 3' UTR, 5' UTR, exons, introns, exon/intron
junctions, coding region, translation initiation region or
translation termination region of SEQ ID NO:4.
22. The method of claim 17, wherein the modified oligonucleotide
has a nucleobase sequence at least 90%, at least 95% or 100%
complementary to SEQ ID NO: 4 as measured over the entirety of said
modified oligonucleotide.
23. The method of claim 17, wherein at least one internucleoside
linkage of the modified oligonucleotide is a modified
internucleoside linkage.
24. The method of claim 23, wherein the at least one
internucleoside linkage is a phosphorothioate internucleoside
linkage.
25. The method of claim 17, wherein at least one nucleoside of the
modified oligonucleotide comprises a modified sugar.
26. The method of claim 25, wherein at least one modified sugar is
a bicyclic sugar.
27. The method of claim 25, wherein at least one modified sugar
comprises a 2'-O-methoxyethyl, a (4'-CH(CH.sub.3)--O-2') BNA, a
(4'-CH.sub.2--O-2') BNA, or a 4'-(CH.sub.2).sub.n--O-2' bridge,
wherein n is 1 or 2.
28. The method of claim 17, wherein at least one nucleoside of the
modified oligonucleotide comprises a modified nucleobase.
29. The method of claim 28, wherein the modified nucleobase is a
5-methylcytosine.
30. The method of claim 17, wherein the modified oligonucleotide
consists of 20 linked nucleosides.
31. The method of claim 30, wherein the modified oligonucleotide
comprises: a) a gap segment consisting of linked deoxynucleosides;
b) a 5' wing segment consisting of linked nucleosides; c) 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.
32. The method of claim 17, wherein the compound reduces ANGPTL3
expression in the subject and: a) reduces apoC-III expression
levels; b) reduces triglyceride levels; c) reduces cholesterol
levels; d) reduces LDL levels; e) reduces glucose levels; f)
improves insulin sensitivity; and/or g) ameliorates a metabolic or
cardiovascular disease.
33. The method of claim 17, wherein the compound is in a salt
form.
34. The method of claim 17, wherein the compound is
single-stranded.
35. The method of claim 17, wherein the compound comprises a
conjugate.
36. The method of claim 17, wherein the compound is in a
pharmaceutical composition comprising a pharmaceutically acceptable
diluent or carrier.
37. The method of claim 36, wherein the compound is in a salt
form.
38. The method of claim 17, wherein the cardiovascular disease is
atherosclerosis or hepatic steatosis.
39. The method of claim 17, wherein the cardiovascular disease is
hyperlipidemia and/or dyslipidemia.
40. The method of claim 17, wherein the administering is by
parenteral administration.
41. The method of claim 40, wherein the administering is by
injection.
Description
CROSS REFERENCED TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/149,715 filed Jan. 7, 2014 which is a divisional of U.S.
application Ser. No. 13/520,997 filed Oct. 4, 2012, issued as U.S.
Pat. No. 8,653,047 which is a U.S. National Phase filing under 35
U.S.C. 371 claiming priority to International Serial No.
PCT/US2011/020606 filed Jan. 7, 2011, which is the non-provisional
application of U.S. Provisional Application No. 61/293,604, filed
Jan. 8, 2010, 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 BIOL0120USC1SEQ.txt, created on Aug. 7, 2015 which is
76 Kb in size. The information in the electronic format of the
sequence listing is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] Provided herein are methods, compounds, and compositions for
reducing expression of angiopoietin-like 3 (ANGPTL3) mRNA and
protein in an animal. Also, provided herein are methods, compounds,
and compositions having an ANGPTL3 inhibitor for reducing ANGPTL3
related diseases or conditions in an animal. Such methods,
compounds, and compositions are useful, for example, to treat,
prevent, delay or ameliorate any one or more of cardiovascular
disease or metabolic syndrome, or a symptom thereof, in an
animal.
BACKGROUND
[0004] Diabetes and obesity (sometimes 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., 2002, 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.
[0005] Cardiovascular disease is also interrelated to obesity and
diabetes. Cardiovascular disease encompasses a wide variety of
etiologies and has an equally wide variety of causative agents and
interrelated players. Many causative agents contribute to symptoms
such as elevated plasma levels of cholesterol, including non-HDL
cholesterol, as well as other lipid-related disorders. Such
lipid-related disorders, generally referred to as dyslipidemia,
include hyperlipidemia, hypercholesterolemia and
hypertriglyceridemia among other indications. Elevated non-HDL
cholesterol is associated with atherogenesis and its sequelae,
including cardiovascular diseases such as arteriosclerosis,
coronary artery disease, myocardial infarction, ischemic stroke,
and other forms of heart disease. These rank as the most prevalent
types of illnesses in industrialized countries. Indeed, an
estimated 12 million people in the United States suffer with
coronary artery disease and about 36 million require treatment for
elevated cholesterol levels.
[0006] Epidemiological and experimental evidence has shown that
high levels of circulating triglyceride (TG) can contribute to
cardiovascular disease and a myriad of metabolic disorders
(Valdivielso et al., 2009, Atherosclerosis. 207(2):573-8; Zhang et
al., 2008, Circ Res. 1; 102(2):250-6). TG derived from either
exogenous or endogenous sources is incorporated and secreted in
chylomicrons from the intestine or in very low density lipoproteins
(VLDL) from the liver. Once in circulation, TG is hydrolyzed by
lipoprotein lipase (LpL) and the resulting free fatty acids can
then be taken up by local tissues and used as an energy source. Due
to the profound effect LpL has on plasma TG and metabolism in
general, discovering and developing compounds that affect LpL
activity are of great interest.
[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. It affects a large number of people in a clustered
fashion. In some studies, the prevalence in the USA is calculated
as being up to 25% of the population. Metabolic syndrome is known
under various other names, such as (metabolic) syndrome X, insulin
resistance syndrome, Reaven's syndrome or CHAOS. With the high
prevalence of cardiovascular disorders and metabolic disorders
there remains a need for improved approaches to treat these
conditions
[0008] The angiopoietins are a family of secreted growth factors.
Together with their respective endothelium-specific receptors, the
angiopoietins play important roles in angiogenesis. One family
member, angiopoietin-like 3 (also known as ANGPT5, ANGPTL3, or
angiopoietin 5), is predominantly expressed in the liver, and is
thought to play a role in regulating lipid metabolism (Kaplan et
al., J. Lipid Res., 2003, 44, 136-143).
[0009] The human gene for angiopoietin-like 3 was identified and
cloned as a result of searches of assembled EST databases. The
full-length human cDNA codes for a polypeptide of 460 amino acids
which has the characteristic structural features of angiopoietins:
a signal peptide, an extended helical domain, a short linker
peptide, and a globular fibrinogen homology domain (FHD). The mouse
angiopoietin-like 3 cDNA was found to encode a 455 amino acid
polypeptide with 76% identity to the human polypeptide. An
alignment of angiopoietins showed that angiopoietin-like 3, unlike
other family members, does not contain the motif of acidic residues
determining a calcium binding site. Northern blot analysis revealed
expression principally in the liver of adult tissues, with murine
embryo Northern blots showing the presence of transcripts as early
as day 15, suggesting that angiopoietin-like 3 is expressed early
during liver development and that expression is maintained in adult
liver. The mouse gene maps to chromosome 4, and the human gene was
mapped to the 1p31 region (Conklin et al., Genomics, 1999, 62,
477-482).
[0010] KK obese mice have a multigenic syndrome of moderate obesity
and a diabetic phenotype that resembles human hereditary type 2
diabetes. These mice show signs of hyperinsulinemia, hyperglycemia,
and hyperlipidemia. A strain of KK mice called KK/San has
significantly low plasma lipid levels despite signs of
hyperinsulinemia and hyperglycemia. The mutant phenotype is
inherited recessively, and the locus was named hypolipidemia
(hypl). The locus maps to the middle of chromosome 4, and the gene
was identified as angiopoietin-like 3 through positional
cloning.
[0011] Injection of recombinant adenoviruses containing the
full-length mouse or human angiopoietin-like 3 cDNA in the mutant
KK/San mice caused an increase in plasma levels of triglyceride,
total cholesterol and non-esterified fatty acids (NEFA). Similarly,
injection of recombinant angiopoietin-like 3 protein into the
mutant mice increased levels of triglycerides and non-esterified
fatty acids. (Koishi et al., Nat. Genet., 2002, 30, 151-157).
[0012] In another study focusing on the metabolic pathways of
triglycerides in KK/San mice, overexpression of angiopoietin-like 3
resulted in a marked increase of triglyceride-enriched very low
density lipoportien (VLDL). Differences in the hepatic VLDL
triglyceride secretion rate were not significant between wild-type
KK and KK/San mice. However, studies with labeled VLDL suggested
that the low plasma triglyceride levels in KK/San mice were
primarily due to enhanced lipolysis of VLDL triglycerides rather
than to enhanced whole particle uptake. The plasma apoB100 and
apoB48 levels of KK/San mice were similar to wild-type KK mice.
ApoCIII-deficient mice have a similar phenotype to KK/San mice, and
ApoCIII is thought to modulate VLDL triglyceride metabolism through
the inhibition of lipase-mediated hydrolysis of VLDL triglycerides.
In vitro analysis of recombinant protein revealed that
angiopoietin-like 3 directly inhibits lipoprotein lipase (LPL)
activity (Shimizugawa et al., J. Biol. Chem., 2002, 277,
33742-33748).
[0013] Consistent with a role in lipid metabolism,
angiopoietin-like 3 mRNA was found to be upregulated in C57BL/6J
mice fed normal chow diets with 4% cholesterol and in mice treated
with the liver X receptor (LXR) agonist T0901317. LXRs are
ligand-activated transcription factors which play a role in the
regulation of genes that govern cholesterol homeostasis in the
liver and peripheral tissues. In addition to cholesterol
metabolism, LXRs may also play a role in regulation of fatty acid
metabolism. Treatment of HepG2 cells with natural or synthetic
agents which activate LXR caused increased angiopoietin-like 3
expression. The promoter of the human angiopoietin-like 3 gene was
found to contain an LXR response element. In addition, the promoter
contained several potential binding sites for other transcription
factors including HNF-1, HNF-4, and C/EBP. (Kaplan et al., J. Lipid
Res., 2003, 44, 136-143).
[0014] Treatment of rodents with T0901317 is associated with
triglyceride accumulation in the liver and plasma. The liver
triglyceride accumulation has been explained by increased
expression of the sterol regulatory element binding protein-1c
(SREBP 1c) and fatty acid synthase (FAS), both of which are targets
of LXR. T0901317 failed to increase plasma triglyceride
concentration in angiopoietin-like 3 deficient mice, while the
stimulated accumulation of hepatic triglyceride was similar to that
observed in treated wild type mice. The rise in plasma triglyceride
in wild-type mice treated with T0901317 parallels an induction of
angiopoietin-like 3 mRNA in the liver and an increase in plasma
concentration of the protein. (Inaba et al., J. Biol. Chem., 2003,
278, 21344-21351).
[0015] Further studies addressed the mechanism of the increase in
plasma free fatty acid (FFA) levels observed in KK/Snk mice treated
with exogenous angiopoietin-like 3. Probe of fixed human tissues
with a fluorescence-labeled angiopoietin-like 3 protein
demonstrated strong binding only on adipose tissue. Furthermore,
radiolabeled protein binding was examined in 3T3-L1 adipocytes and
was found to be saturable and specific. Incubation of 3T3-L1
adipocytes with angiopoietin-like 3 led to enhanced release of FFA
and glycerol into the culture medium. (Shimamura et al., Biochem.
Biophys. Res. Commun., 2003, 301, 604-609).
[0016] In a study using streptozotocin-treated mice (STZ) to model
the insulin-deficient state and db/db mice to model the
insulin-resistant diabetic state, larger amounts of hepatic
angiopoietin-like 3 were observed in diabetic mice as compared to
control animals. Both models of diabetes showed
hypertriglyceridemia, and the hyperlipidemia observed was explained
at least partially by the increased expression of angiopoietin-like
3. These results suggested that angiopoietin-like 3 is a link
between diabetes and dyslipidemia, with elevation promoting
hyperlipidemia (Inukai et al., Biochem. Biophys. Res. Commun.,
2004, 317, 1075-1079).
[0017] A subsequent study examined the regulation of
angiopoietin-like 3 by leptin and insulin, both of which are key
players in the metabolic syndrome. Angiopoietin-like 3 expression
and plasma protein levels were increased in leptin-resistant db/db
and leptin-deficient ob/ob mice relative to controls.
Supplementation of ob/ob mice with leptin decreased
angiopoietin-like 3 levels. The alterations in expression were
associated with alterations in plasma triglyceride and free fatty
acid levels. Gene expression and plasma protein levels were also
increased in insulin-deficient STZ-treated mice. (Shimamura et al.,
Biochem Biophys Res Commun, 2004, 322, 1080-1085).
[0018] In accord with its membership in the angiopoietin family,
recombinant angiopoietin-like 3 protein was found to bind to
.alpha..sub.V.beta..sub.3 integrin and induced integrin
.alpha..sub.V.beta..sub.3-dependent haptotactic endothelial cell
adhesion and migration. It also stimulated signal transduction
pathways characteristic for integrin activation. Angiopoietin-like
3 strongly induced angiogenesis in the rat corneal angiogenesis
assay. (Camenisch et al., J. Biol. Chem., 2002, 277,
17281-17290).
[0019] Genome-wide association scans (GWAS) surveying the genome
for common variants associated with plasma concentrations of HDL,
LDL and triglyceride were undertaken by several groups. The GWAS
studies found an association between triglycerides and
single-nucleotide polymorphisms (SNPs) near ANGPTL3 (Willer et al.,
Nature Genetics, 2008, 40(2):161-169).
[0020] U.S. Pat. No. 7,267,819, application U.S. Ser. No.
12/128,545, and application U.S. Ser. No. 12/001,012 generally
describe angiopoietin-like 3 agonists and antagonists.
[0021] PCT publications WO/02101039 (EP02733390) and WO/0142499
(U.S. Ser. No. 10/164,030) disclose a nucleic acid sequence
complementary to mouse angiopoietin-like 3 (Ryuta, 2002; Ryuta,
2001).
[0022] There is a currently a lack of acceptable options for
treating cardiovascular and metabolic disorders. It is therefore an
object herein to provide compounds and methods for the treatment of
such diseases and disorder.
[0023] The potential role of angiopoietin-like 3 in lipid
metabolism makes it an attractive target for investigation.
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 angiopoietin-like
3.
SUMMARY OF THE INVENTION
[0024] Provided herein are antisense compounds useful for
modulating gene expression and associated pathways via antisense
mechanisms of action such as RNaseH, RNAi and dsRNA enzymes, as
well as other antisense mechanisms based on target degradation or
target occupancy.
[0025] Provided herein are methods, compounds, and compositions for
inhibiting expression of ANGPTL3 and treating, preventing, delaying
or ameliorating a ANGPTL3 related disease, condition or a symptom
thereof. In certain embodiments, the ANGPTL3 related disease or
condition is cardiovascular disease or metabolic disease.
[0026] In certain embodiments, the compounds or compositions of the
invention comprise a modified oligonucleotide 10 to 30 linked
nucleosides in length targeted to ANGPTL3. The ANGPTL target can
have a sequence selected from any one of SEQ ID NOs: 1-5. The
modified oligonucleotide targeting ANGPTL3 can have a nucleobase
sequence comprising at least 8 contiguous nucleobases complementary
to an equal length portion of SEQ ID NOs: 1-5. The modified
oligonucleotide targeting ANGPTL3 can have a nucleobase sequence
comprising at least 8 contiguous nucleobases of a nucleobase
sequence selected from any of SEQ ID NO: 34-182. The modified
oligonucleotide can have a nucleobase sequence comprising at least
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous
nucleobases of a nucleobase sequence selected from a sequence
recited in any one of SEQ ID NOs: 34-182. The contiguous nucleobase
portion of the modified oligonucleotide can be complementary to an
equal length portion of an ANGPTL3 region selected from any one of
SEQ ID NOs: 1-5. The ANGPTL3 region can be chosen from one or more
of the following regions: 22-52, 116-145, 637-720, 953-983,
1333-1469 and 1463-1489.
[0027] Certain embodiments provide a method of reducing ANGPTL3
expression in an animal comprising administering to the animal a
compound comprising the modified oligonucleotide targeting ANGPTL3
described herein.
[0028] Certain embodiments provide a method of reducing apoC-III
expression, triglyceride levels, cholesterol levels, low-density
lipoprotein (LDL) or glucose levels in an animal comprising
administering to the animal a compound comprising the modified
oligonucleotide targeted to ANGPTL3 described herein, wherein the
modified oligonucleotide reduces ANGPTL3 expression in the
animal.
[0029] Certain embodiments provide a method of ameliorating
cardiovascular disease or metabolic disease in an animal comprising
administering to the animal a compound comprising a modified
oligonucleotide targeted to ANGPTL3 described herein, wherein the
modified oligonucleotide reduces ANGPTL3 expression in the
animal.
[0030] Certain embodiments provide a method for treating an animal
with cardiovascular disease or metabolic disease comprising: 1)
identifying the animal with cardiovascular disease or metabolic
disease, and 2) administering to the animal a therapeutically
effective amount of a compound comprising a modified
oligonucleotide consisting of 20 linked nucleosides and having a
nucleobase sequence at least 90% complementary to SEQ ID NO: 1-5 as
measured over the entirety of said modified oligonucleotide,
thereby treating the animal with cardiovascular disease or
metabolic disease. In certain embodiments, the therapeutically
effective amount of the compound administered to the animal reduces
cardiovascular disease or metabolic disease in the animal.
[0031] Certain embodiments provide a method for decreasing one or
more of ANGPTL3 levels, LDL levels, apoC-III levels, triglyceride
levels, cholesterol levels, glucose levels, fat pad weight,
cardiovascular disease and metabolic disease in a human by
administering an ANGPTL3 inhibitor comprising a modified
oligonucleotide as described herein.
DETAILED DESCRIPTION OF THE INVENTION
[0032] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed. Herein, the use of the singular includes the plural unless
specifically stated otherwise. As used herein, the use of "or"
means "and/or" unless stated otherwise. Furthermore, the use of the
term "including" as well as other forms, such as "includes" and
"included", is not limiting. Also, terms such as "element" or
"component" encompass both elements and components comprising one
unit and elements and components that comprise more than one
subunit, unless specifically stated otherwise.
[0033] 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
[0034] 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
patents, applications, published applications and other
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.
[0035] Unless otherwise indicated, the following terms have the
following meanings:
[0036] "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.
[0037] "2'-O-methoxyethyl nucleotide" means a nucleotide comprising
a 2'-O-methoxyethyl modified sugar moiety.
[0038] "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.
[0039] "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.
[0040] "5-methylcytosine" means a cytosine modified with a methyl
group attached to the 5' position. A 5-methylcytosine is a modified
nucleobase.
[0041] "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%
[0042] "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 ANGPTL3 is an active pharmaceutical agent.
[0043] "Active target region" or "target region" means a region to
which one or more active antisense compounds is targeted.
[0044] "Active antisense compounds" means antisense compounds that
reduce target nucleic acid levels or protein levels.
[0045] "Adipogenesis" means the development of fat cells from
preadipocytes. "Lipogenesis" means the production or formation of
fat, either fatty degeneration or fatty infiltration.
[0046] "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 metabolic obesity; endocrine
obesity; familial obesity; hyperinsulinar obesity;
hyperplastic-hypertrophic obesity; hypogonadal obesity; hypothyroid
obesity; lifelong obesity; morbid obesity and exogenous
obesity.
[0047] "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.
[0048] "Administering" means providing an agent to an animal, and
includes, but is not limited to, administering by a medical
professional and self-administering.
[0049] "Agent" means an active substance that can provide a
therapeutic benefit when administered to an animal. "First Agent"
means a therapeutic compound of the invention. For example, a first
agent can be an antisense oligonucleotide targeting ANGPTL3.
"Second agent" means a second therapeutic compound of the invention
(e.g. a second antisense oligonucleotide targeting ANGPTL3) and/or
a non-ANGPTL3 therapeutic compound.
[0050] "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.
[0051] "ANGPTL3" means any nucleic acid or protein of ANGPTL3.
[0052] "ANGPTL3 expression" means the level of mRNA transcribed
from the gene encoding ANGPTL3 or the level of protein translated
from the mRNA. ANGPTL3 expression can be determined by art known
methods such as a Northern or Western blot.
[0053] "ANGPTL3 nucleic acid" means any nucleic acid encoding
ANGPTL3. For example, in certain embodiments, a ANGPTL3 nucleic
acid includes a DNA sequence encoding ANGPTL3, a RNA sequence
transcribed from DNA encoding ANGPTL3 (including genomic DNA
comprising introns and exons), and a mRNA sequence encoding
ANGPTL3. "ANGPTL3 mRNA" means a mRNA encoding an ANGPTL3
protein.
[0054] "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.
[0055] "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.
[0056] "Antisense compound" means an oligomeric compound that is
capable of undergoing hybridization to a target nucleic acid
through hydrogen bonding.
[0057] "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.
[0058] "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.
[0059] "ApoB-containing lipoprotein" means any lipoprotein that has
apolipoprotein B as its protein component, and is understood to
include LDL, VLDL, IDL, and lipoprotein(a) and can be generally
targeted by lipid lowering agent and therapies.
"ApoB-100-containing LDL" means ApoB-100 isoform containing
LDL.
[0060] "Atherosclerosis" means a hardening of the arteries
affecting large and medium-sized arteries and is characterized by
the presence of fatty deposits. The fatty deposits are called
"atheromas" or "plaques," which consist mainly of cholesterol and
other fats, calcium and scar tissue, and damage the lining of
arteries.
[0061] "Bicyclic sugar" means a furosyl ring modified by the
bridging of two non-geminal ring atoms. A bicyclic sugar is a
modified sugar.
[0062] "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.
[0063] "Cap structure" or "terminal cap moiety" means chemical
modifications, which have been incorporated at either terminus of
an antisense compound.
[0064] "Cardiovascular disease" or "cardiovascular disorder" refers
to a group of conditions related to the heart, blood vessels, or
the circulation. Examples of cardiovascular diseases or disorders
include, but are not limited to, aneurysm, angina, arrhythmia,
atherosclerosis, cerebrovascular disease (stroke), coronary heart
disease, hypertension, dyslipidemia, hyperlipidemia, and
hypercholesterolemia.
[0065] "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.
[0066] "Chimeric antisense compound" means an antisense compound
that has at least two chemically distinct regions.
[0067] "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.
[0068] "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-estrified cholesterol present
in the plasma or serum.
[0069] "Cholesterol absorption inhibitor" means an agent that
inhibits the absorption of exogenous cholesterol obtained from
diet.
[0070] "Complementarity" means the capacity for pairing between
nucleobases of a first nucleic acid and a second nucleic acid. In
certain embodiments, complementarity between the first and second
nucleic acid may be between two DNA strands, between two RNA
strands, or between a DNA and an RNA strand. In certain
embodiments, some of the nucleobases on one strand are matched to a
complementary hydrogen bonding base on the other strand. In certain
embodiments, all of the nucleobases on one strand are matched to a
complementary hydrogen bonding base on the other strand. In certain
embodiments, a first nucleic acid is an antisense compound and a
second nucleic acid is a target nucleic acid. In certain such
embodiments, an antisense oligonucleotide is a first nucleic acid
and a target nucleic acid is a second nucleic acid.
[0071] "Contiguous nucleobases" means nucleobases immediately
adjacent to each other.
[0072] "Coronary heart disease (CHD)" means a narrowing of the
small blood vessels that supply blood and oxygen to the heart,
which is often a result of atherosclerosis.
[0073] "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.
[0074] "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.
[0075] "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.
[0076] "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.
[0077] "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.
[0078] "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.
[0079] "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. Doses can be expressed as
mg/kg or g/kg.
[0080] "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.
[0081] "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.
[0082] "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."
[0083] "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.
[0084] "Glucose" is a monosaccharide used by cells as a source of
energy and metabolic intermediate. "Plasma glucose" refers to
glucose present in the plasma.
[0085] "High density lipoprotein-C(HDL-C)" means cholesterol
associated with high density lipoprotein particles. Concentration
of HDL-C in serum (or plasma) is typically quantified in mg/dL or
nmol/L. "serum HDL-C" and "plasma HDL-C" mean HDL-C in serum and
plasma, respectively.
[0086] "HMG-CoA reductase inhibitor" means an agent that acts
through the inhibition of the enzyme HMG-CoA reductase, such as
atorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin,
and simvastatin.
[0087] "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.
[0088] "Hypercholesterolemia" means a condition characterized by
elevated cholesterol or circulating (plasma) cholesterol,
LDL-cholesterol and VLDL-cholesterol, as per the guidelines of the
Expert Panel Report of the National Cholesterol Educational Program
(NCEP) of Detection, Evaluation of Treatment of high cholesterol in
adults (see, Arch. Int. Med. (1988) 148, 36-39).
[0089] "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.
[0090] "Hypertriglyceridemia" means a condition characterized by
elevated triglyceride levels.
[0091] "Identifying" or "selecting a subject having a metabolic or
cardiovascular disease" means identifying or selecting a subject
having been diagnosed with a metabolic disease, a cardiovascular
disease, or a metabolic syndrome; or, identifying or selecting a
subject having any symptom of a metabolic disease, cardiovascular
disease, or metabolic syndrome including, but not limited to,
hypercholesterolemia, hyperglycemia, hyperlipidemia,
hypertriglyceridemia, hypertension increased insulin resistance,
decreased insulin sensitivity, above normal body weight, and/or
above normal body fat content 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) cholesterol, measuring
serum or circulating (plasma) blood-glucose, measuring serum or
circulating (plasma) triglycerides, measuring blood-pressure,
measuring body fat content, measuring body weight, and the
like.
[0092] "Identifying" or "selecting a diabetic subject" means
identifying or selecting a subject having been identified as
diabetic or identifying or selecting a subject having any symptom
of diabetes (type 1 or type 2) such as, but not limited to, having
a fasting glucose of at least 110 mg/dL, glycosuria, polyuria,
polydipsia, increased insulin resistance, and/or decreased insulin
sensitivity.
[0093] "Identifying" or "selecting an obese subject" means
identifying or selecting a subject having been diagnosed as obese
or identifying or selecting a subject with a BMI over 30 and/or a
waist circumference of greater than 102 cm in men or greater than
88 cm in women.
[0094] "Identifying" or "selecting a subject having dyslipidemia"
means identifying or selecting a subject diagnosed with 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.
[0095] "Identifying" or "selecting" a subject having increased
adiposity" means identifying or selecting a subject having an
increased amount of body fat (or adiposity) that includes concern
for one or 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, computer 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.
[0096] "Improved cardiovascular outcome" means a reduction in the
occurrence of adverse cardiovascular events, or the risk thereof.
Examples of adverse cardiovascular events include, without
limitation, death, reinfarction, stroke, cardiogenic shock,
pulmonary edema, cardiac arrest, and atrial dysrhythmia.
[0097] "Immediately adjacent" means there are no intervening
elements between the immediately adjacent elements.
[0098] "Individual" or "subject" or "animal" means a human or
non-human animal selected for treatment or therapy.
[0099] "Inhibiting the expression or activity" refers to a
reduction or blockade of the expression or activity and does not
necessarily indicate a total elimination of expression or
activity.
[0100] "Insulin resistance" is defined as the condition in which
normal amounts of insulin are inadequate to produce a normal
insulin response from cells, e.g., fat, muscle and/or 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.
[0101] "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.
[0102] "Internucleoside linkage" refers to the chemical bond
between nucleosides.
[0103] "Intravenous administration" means administration into a
vein.
[0104] "Linked nucleosides" means adjacent nucleosides which are
bonded together.
[0105] "Lipid-lowering" means a reduction in one or more lipids in
a subject. Lipid-lowering can occur with one or more doses over
time.
[0106] "Lipid-lowering agent" means an agent, for example, an
ANGPTL3-specific modulator, provided to a subject to achieve a
lowering of lipids in the subject. For example, in certain
embodiments, a lipid-lowering agent is provided to a subject to
reduce one or more of apoB, apoC3, total cholesterol, LDL-C,
VLDL-C, IDL-C, non-HDL-C, triglycerides, small dense LDL particles,
and Lp(a) in a subject. "Lipid-lowering therapy" 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, apoC-III, total
cholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small
dense LDL particles, and Lp(a) in a subject.
[0107] "Lipoprotein", such as VLDL, LDL and HDL, refers to a group
of proteins found in the serum, plasma and lymph and are important
for lipid transport. The chemical composition of each lipoprotein
differs in that the HDL has a higher proportion of protein versus
lipid, whereas the VLDL has a lower proportion of protein versus
lipid.
[0108] "Low density lipoprotein-cholesterol (LDL-C)" means
cholesterol carried in low density lipoprotein particles.
Concentration of LDL-C in serum (or plasma) is typically quantified
in mg/dL or nmol/L. "Serum LDL-C" and "plasma LDL-C" mean LDL-C in
the serum and plasma, respectively.
[0109] "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.
[0110] "Metabolic disorder" or "metabolic disease" 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 disorders include, but are not limited to, hyperglycemia,
prediabetes, diabetes (type I and type 2), obesity, insulin
resistance, metabolic syndrome and dyslipidemia due to type 2
diabetes.
[0111] "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).
[0112] "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.
[0113] "Mixed dyslipidemia" means a condition characterized by
elevated cholesterol and elevated triglycerides.
[0114] "Modified internucleoside linkage" refers to a substitution
or any change from a naturally occurring internucleoside bond (i.e.
a phosphodiester internucleoside bond).
[0115] "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).
[0116] "Modified nucleoside" means a nucleoside having,
independently, one or more modified sugar moiety or modified
nucleobase.
[0117] "Modified nucleotide" means a nucleotide having,
independently, one or more modified sugar moiety, modified
internucleoside linkage, or modified nucleobase. A "modified
nucleoside" means a nucleoside having, independently, one or more
modified sugar moiety or modified nucleobase.
[0118] "Modified oligonucleotide" means an oligonucleotide
comprising at least one modified nucleotide.
[0119] "Modified sugar" refers to a substitution or change from a
natural sugar.
[0120] "Motif" means the pattern of chemically distinct regions in
an antisense compound.
[0121] "MTP inhibitor" means an agent inhibits the enzyme
microsomal triglyceride transfer protein.
[0122] "Naturally occurring internucleoside linkage" means a 3' to
5' phosphodiester linkage.
[0123] "Natural sugar moiety" means a sugar found in DNA (2'-H) or
RNA (2'-OH).
[0124] "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 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.
[0125] "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. It has been suggested that increased
liver triglycerides lead to increased oxidative stress in
hepatocytes of animals and humans, indicating a potential
cause-and-effect relationship between hepatic triglyceride
accumulation, oxidative stress, and the progression of hepatic
steatosis to NASH (Browning and Horton, J Clin Invest, 2004, 114,
147-152). Hypertriglyceridemia and hyperfattyacidemia can cause
triglyceride accumulation in peripheral tissues (Shimamura et al.,
Biochem Biophys Res Commun, 2004, 322, 1080-1085).
[0126] "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.
[0127] "Nucleobase" means a heterocyclic moiety capable of pairing
with a base of another nucleic acid.
[0128] "Nucleobase sequence" means the order of contiguous
nucleobases independent of any sugar, linkage, or nucleobase
modification.
[0129] "Nucleoside" means a nucleobase linked to a sugar.
[0130] "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.
[0131] "Nucleotide" means a nucleoside having a phosphate group
covalently linked to the sugar portion of the nucleoside.
[0132] "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).
[0133] "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.
[0134] "Oligonucleotide" means a polymer of linked nucleosides each
of which can be modified or unmodified, independent one from
another.
[0135] "Parenteral administration" means administration by a manner
other than through the digestive tract. Parenteral administration
includes topical administration, 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.
[0136] "Peptide" means a molecule formed by linking at least two
amino acids by amide bonds. Peptide refers to polypeptides and
proteins.
[0137] "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 ANGPTL3 is pharmaceutical agent.
[0138] "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.
[0139] "Pharmaceutically acceptable carrier" means a medium or
diluent that does not interfere with the structure or function 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. Certain of such
carriers enable pharmaceutical compositions to be formulated for
injection or infusion. For example, a pharmaceutically acceptable
carrier can be a sterile aqueous solution.
[0140] "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.
[0141] "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.
[0142] "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.
[0143] "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.
[0144] "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.
[0145] "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.
[0146] "Single-stranded oligonucleotide" means an oligonucleotide
which is not hybridized to a complementary strand.
[0147] "Specifically hybridizable" refers to an antisense compound
having a sufficient degree of complementarity with 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.
[0148] "Statin" means an agent that inhibits the activity of
HMG-CoA reductase.
[0149] "Subcutaneous administration" means administration just
below the skin.
[0150] "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.
[0151] "Target nucleic acid," "target RNA," and "target RNA
transcript" all refer to a nucleic acid capable of being targeted
by antisense compounds.
[0152] "Target region" is defined as a portion of the target
nucleic acid having at least one identifiable structure, function,
or characteristic.
[0153] "Target segment" means the sequence of nucleotides of a
target nucleic acid to which one or more 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.
[0154] "Therapeutically effective amount" means an amount of an
agent that provides a therapeutic benefit to an individual.
[0155] "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 include 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.
[0156] "Triglyceride" means a lipid or neutral fat consisting of
glycerol combined with three fatty acid molecules.
[0157] "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.
[0158] "Treat" refers to administering a pharmaceutical composition
to effect an alteration or improvement of a disease, disorder, or
condition.
[0159] "Unmodified nucleotide" means a nucleotide composed of
naturally occurring nucleobases, sugar moieties, and
internucleoside linkages. In certain embodiments, an unmodified
nucleotide is a RNA nucleotide (i.e. .beta.-D-ribonucleosides) or a
DNA nucleotide (i.e. .beta.-D-deoxyribonucleoside).
Certain Embodiments
[0160] In certain embodiments, the compounds or compositions of the
invention comprise a modified oligonucleotide 10 to 30 linked
nucleosides in length targeted to ANGPTL3. The ANGPTL target can
have a sequence selected from any one of SEQ ID NOs: 1-5.
[0161] In certain embodiments, the compounds or compositions of the
invention comprise a modified oligonucleotide consisting of 10 to
30 nucleosides having a nucleobase sequence comprising at least 8
contiguous nucleobases complementary to an equal length portion of
SEQ ID NOs: 1-5.
[0162] In certain embodiments, the compounds or compositions of the
invention comprise a modified oligonucleotide consisting of 10 to
30 nucleosides having a nucleobase sequence comprising at least 8
contiguous nucleobases of a nucleobase sequence selected from any
of SEQ ID NO: 34-182.
[0163] In certain embodiments, the compounds or compositions of the
invention comprise a modified oligonucleotide consisting of 10 to
30 linked nucleosides and having a nucleobase sequence comprising
at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
contiguous nucleobases of a nucleobase sequence selected from a
sequence recited in any one of SEQ ID NOs: 34-182.
[0164] In certain embodiments, the compounds or compositions of the
invention comprise a salt of the modified oligonucleotide.
[0165] In certain embodiments, the compounds or compositions of the
invention further comprise a pharmaceutically acceptable carrier or
diluent.
[0166] In certain embodiments, the nucleobase sequence of the
modified oligonucleotide is at least 70%, 80%, 90%, 95% or 100%
complementary to any one of SEQ ID NO: 1-5 as measured over the
entirety of the modified oligonucleotide.
[0167] In certain embodiments, the compound of the invention
consists of a single-stranded modified oligonucleotide.
[0168] 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 or 30 linked nucleosides. In certain
embodiments, the modified oligonucleotide consists of 20 linked
nucleosides.
[0169] In certain embodiments, at least one internucleoside linkage
of said modified oligonucleotide is a modified internucleoside
linkage. In certain embodiments, each internucleoside linkage is a
phosphorothioate internucleoside linkage.
[0170] In certain embodiments, at least one nucleoside of the
modified oligonucleotide comprises a modified sugar. In certain
embodiments the modified oligonucleotide comprises at least one
tetrahydropyran modified nucleoside wherein a tetrahydropyran ring
replaces a furanose ring. In certain embodiments each of the
tetrahydropyran modified nucleoside has the structure:
##STR00001##
wherein Bx is an optionally protected heterocyclic base moiety. In
certain embodiments, at least one modified sugar is a bicyclic
sugar. In certain embodiments, at least one modified sugar
comprises a 2'-O-methoxyethyl or a 4'-(CH.sub.2).sub.n--O-2'
bridge, wherein n is 1 or 2.
[0171] 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.
[0172] 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. 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 sugar and each internucleoside linkage is a
phosphorothioate linkage.
[0173] In certain embodiments, the compounds or compositions of the
invention 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 SEQ ID NO: 1-5, 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
sugar, each internucleoside linkage is a phosphorothioate linkage
and each cytosine residue is a 5-methylcytosine.
[0174] In certain embodiments, the compounds or compositions of the
invention comprise a modified oligonucleotide consisting of 20
linked nucleosides having a nucleobase sequence comprising at least
8 contiguous nucleobases of a nucleobase sequence selected from any
of SEQ ID NO: 34-182, 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 sugar, each internucleoside
linkage is a phosphorothioate linkage and each cytosine residue is
a 5-methylcytosine.
[0175] Certain embodiments provide methods, compounds, and
compositions for inhibiting ANGPTL3 expression.
[0176] Certain embodiments provide a method of reducing ANGPTL3
expression in an animal comprising administering to the animal a
compound comprising a modified oligonucleotide 10 to 30 linked
nucleosides in length targeted to ANGPTL3.
[0177] Certain embodiments provide a method of reducing ApoC-III
expression in an animal comprising administering to the animal a
compound comprising a modified oligonucleotide 10 to 30 linked
nucleosides in length targeted to ANGPTL3, thereby reducing the
expression of ApoC-III in the animal.
[0178] Certain embodiments provide a method of reducing
triglyceride levels in an animal comprising administering to the
animal a compound comprising a modified oligonucleotide 10 to 30
linked nucleosides in length targeted to ANGPTL3, thereby reducing
the level of triglyceride in the animal.
[0179] Certain embodiments provide a method of reducing cholesterol
levels in an animal comprising administering to the animal a
compound comprising a modified oligonucleotide 10 to 30 linked
nucleosides in length targeted to ANGPTL3, thereby reducing the
level of cholesterol in the animal.
[0180] Certain embodiments provide a method of reducing low-density
lipoprotein (LDL) levels in an animal comprising administering to
the animal a compound comprising a modified oligonucleotide 10 to
30 linked nucleosides in length targeted to ANGPTL3, thereby
reducing the level of low-density lipoprotein (LDL) in the
animal.
[0181] Certain embodiments provide a method of reducing glucose
levels in an animal comprising administering to the animal a
compound comprising a modified oligonucleotide 10 to 30 linked
nucleosides in length targeted to ANGPTL3, thereby reducing the
level of glucose in the animal.
[0182] Certain embodiments provide a method of ameliorating
metabolic or cardiovascular disease in an animal comprising
administering to the animal a compound comprising a modified
oligonucleotide 10 to 30 linked nucleosides in length targeted to
ANGPTL3, thereby ameliorating the metabolic or cardiovascular
disease in the animal.
[0183] Certain embodiments provide a method for treating an animal
with an ANGPTL3 related disease or condition comprising: a)
identifying said animal with the ANGPTL3 related disease or
condition, and b) administering to said animal a therapeutically
effective amount of a compound comprising a modified
oligonucleotide 10 to 30 linked nucleosides in length targeted to
ANGPTL3. In certain embodiments, the therapeutically effective
amount of the compound administered to the animal reduces the
ANGPTL3 related disease or condition in the animal.
[0184] Certain embodiments provide a method for treating an animal
with metabolic or cardiovascular disease comprising: a) identifying
said animal with metabolic or cardiovascular disease, 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 90%
complementary to SEQ ID NO: 1-5 as measured over the entirety of
said modified oligonucleotide, thereby treating the animal with
metabolic or cardiovascular disease. In certain embodiments, the
therapeutically effective amount of the compound administered to
the animal reduces the metabolic or cardiovascular disease in the
animal.
[0185] Certain embodiments provide a method of decreasing one or
more of ANGPTL3 levels, LDL levels, apoC-III levels, triglyceride
levels, cholesterol levels, glucose levels, fat pad weight,
cardiovascular disease and metabolic disease in a human by
administering an ANGPTL3 inhibitor comprising a modified
oligonucleotide consisting of 20 linked nucleosides and having a
nucleobase sequence at least 90% complementary to SEQ ID NO: 1-5 as
measured over the entirety of said modified oligonucleotide.
[0186] Certain embodiments provide uses of the compounds and
compositions described herein for inhibiting ANGPTL3
expression.
[0187] Certain embodiments provide use of the compounds and
compositions described herein for reducing ANGPTL3 expression in an
animal. Certain embodiments include administering to the animal a
compound comprising a modified oligonucleotide 10 to 30 linked
nucleosides in length targeted to ANGPTL3.
[0188] Certain embodiments provide use of the compounds and
compositions described herein for reducing ApoC-III expression in
an animal. Certain embodiments include administering to the animal
a compound comprising a modified oligonucleotide 10 to 30 linked
nucleosides in length targeted to ANGPTL3, thereby reducing the
expression of ApoC-III in the animal.
[0189] Certain embodiments provide use of the compounds and
compositions described herein for reducing triglyceride levels in
an animal. Certain embodiments include administering to the animal
a compound comprising a modified oligonucleotide 10 to 30 linked
nucleosides in length targeted to ANGPTL3, thereby reducing the
level of triglyceride in the animal.
[0190] Certain embodiments provide use of the compounds and
compositions described herein for reducing cholesterol levels in an
animal. Certain embodiments include administering to the animal a
compound comprising a modified oligonucleotide 10 to 30 linked
nucleosides in length targeted to ANGPTL3, thereby reducing the
level of cholesterol in the animal.
[0191] Certain embodiments provide use of the compounds and
compositions described herein for reducing low-density lipoprotein
(LDL) levels in an animal. Certain embodiments include
administering to the animal a compound comprising a modified
oligonucleotide 10 to 30 linked nucleosides in length targeted to
ANGPTL3, thereby reducing the level of low-density lipoprotein
(LDL) in the animal.
[0192] Certain embodiments provide use of the compounds and
compositions described herein for reducing glucose levels in an
animal. Certain embodiments include administering to the animal a
compound comprising a modified oligonucleotide 10 to 30 linked
nucleosides in length targeted to ANGPTL3, thereby reducing the
level of glucose in the animal.
[0193] Certain embodiments provide use of the compounds and
compositions described herein for ameliorating metabolic or
cardiovascular disease in an animal. Certain embodiments include
administering to the animal a compound comprising a modified
oligonucleotide 10 to 30 linked nucleosides in length targeted to
ANGPTL3, thereby ameliorating the metabolic or cardiovascular
disease in the animal.
[0194] Certain embodiments provide use of the compounds and
compositions described herein for treatment. Certain embodiments
provide use of the compounds and compositions described herein for
treating an animal with an ANGPTL3 related disease or condition. In
certain embodiments, the ANGPTL3 related disease or condition is
metabolic or cardiovascular disease. Certain embodiments include:
a) identifying said animal with the ANGPTL3 related disease or
condition, and b) administering to said animal a therapeutically
effective amount of a compound comprising a modified
oligonucleotide 10 to 30 linked nucleosides in length targeted to
ANGPTL3. In certain embodiments, the therapeutically effective
amount of the compound administered to the animal reduces the
ANGPTL3 related disease or condition in the animal.
[0195] Certain embodiments provide use of the compounds and
compositions described herein for treating an animal with metabolic
or cardiovascular disease. comprising: a) identifying said animal
with metabolic or cardiovascular disease, 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 90%
complementary to SEQ ID NO: 1-5 as measured over the entirety of
said modified oligonucleotide, thereby treating the animal with
metabolic or cardiovascular disease. In certain embodiments, the
therapeutically effective amount of the compound administered to
the animal reduces the metabolic or cardiovascular disease in the
animal.
[0196] Certain embodiments provide use of the compounds and
compositions described herein for decreasing one or more of ANGPTL3
levels, LDL levels, apoC-III levels, triglyceride levels,
cholesterol levels, glucose levels, fat pad weight, cardiovascular
disease and metabolic disease in a human by administering an
ANGPTL3 inhibitor comprising a modified oligonucleotide consisting
of 20 linked nucleosides and having a nucleobase sequence at least
90% complementary to SEQ ID NO: 1-5 as measured over the entirety
of said modified oligonucleotide.
[0197] In certain embodiments, ANGPTL3 has the sequence as set
forth in GenBank Accession No. BG400407.1 (incorporated herein as
SEQ ID NO: 1). In certain embodiments, ANGPTL3 has the sequence as
set forth in GenBank Accession No. BG562555.1 (incorporated herein
as SEQ ID NO: 2). In certain embodiments, ANGPTL3 has the sequence
as set forth in GenBank Accession No. BG562798.1 (incorporated
herein as SEQ ID NO: 3). In certain embodiments, ANGPTL3 has the
sequence as set forth in GenBank Accession No. NM.sub.--014495.1
(incorporated herein as SEQ ID NO: 4). In certain embodiments,
ANGPTL3 has the sequence as set forth in GenBank Accession No. NT
032977.5 nucleotides 15511702 to Ser. No. 15/521,082 (incorporated
herein as SEQ ID NO: 5). In certain embodiments, ANGPTL3 has the
sequence as set forth in GenBank Accession No. AF162224.1
(incorporated herein as SEQ ID NO: 6). In certain embodiments,
ANGPTL3 has the sequence as set forth in GenBank Accession No.
AI195524.1 (incorporated herein as SEQ ID NO: 7). In certain
embodiments, ANGPTL3 has the sequence as set forth in GenBank
Accession No. BB717501.1 (incorporated herein as SEQ ID NO: 8).
TABLE-US-00001 TABLE 1 Gene Target Names and Sequences SEQ Target
Name Species Genbank # ID NO angiopoietin-like 3 Human BG400407.1 1
angiopoietin-like 3 Human BG562555.1 2 angiopoietin-like 3 Human
BG562798.1 3 angiopoietin-like 3 Human NM_014495.1 4
angiopoietin-like 3 Human nucleotides 15511702 to 5 15521082 of
NT_032977.5 angiopoietin-like 3 Mouse AF162224.1 6
angiopoietin-like 3 Mouse AI195524.1 7 angiopoietin-like 3 Mouse
BB717501.1 8
[0198] In certain embodiments, the animal is a human.
[0199] In certain embodiments, the compounds or compositions of the
invention are designated as a first agent and the methods or uses
of the invention further comprise administering a second agent.
[0200] In certain embodiments, the first agent and the second agent
are co-administered. In certain embodiments the first agent and the
second agent are co-administered sequentially or concomitantly.
[0201] In certain embodiments, the second agent is 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.
[0202] In certain embodiments, the second agent is a lipid-lowering
therapy. In certain embodiments the lipid lowering therapy can
include, but is not limited to, a therapeutic lifestyle change,
HMG-CoA reductase inhibitor, cholesterol absorption inhibitor, MTP
inhibitor, antisense compound targeted to ApoB or any combination
thereof. The HMG-CoA reductase inhibitor can be atorvastatin,
rosuvastatin, fluvastatin, lovastatin, pravastatin, or simvastatin.
The cholesterol absorption inhibitor can be ezetimibe.
[0203] In certain embodiments, administration comprises parenteral
administration.
[0204] In certain embodiments, the metabolic or cardiovascular
disease includes, but is not limited to, obesity, diabetes,
atherosclerosis, dyslipidemia, coronary heart disease,
non-alcoholic fatty liver disease (NAFLD), hyperfattyacidemia or
metabolic syndrome, or a combination thereof. The dyslipidemia can
be hyperlipidemia. The hyperlipidemia can be hypercholesterolemia,
hypertriglyceridemia, or both hypercholesterolemia and
hypertriglyceridemia. The NAFLD can be hepatic steatosis or
steatohepatitis. The diabetes can be type 2 diabetes or type 2
diabetes with dyslipidemia.
[0205] In certain embodiments, administering the compound of the
invention results in a reduction of lipid levels, including
triglyceride levels, cholesterol levels, insulin resistance,
glucose levels or a combination thereof. One or more of the levels
can be independently reduced by 5%, 10%, 20%, 30%, 35%, or 40%.
Administering the compound of the invention can result in improved
insulin sensitivity or hepatic insulin sensitivity. Administering
the compound of the invention can result in a reduction in
atherosclerotic plaques, obesity, glucose, lipids, glucose
resistance, cholesterol, or improvement in insulin sensitivity or
any combination thereof.
[0206] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
ameliorating, delaying or preventing one or more of a metabolic
disease or a cardiovascular disease.
[0207] Certain embodiments provide a kit for treating, preventing,
or ameliorating one or more of a metabolic disease or a
cardiovascular disease as described herein wherein the kit
comprises: a) a compound as described herein; and optionally b) an
additional agent or therapy as described herein. The kit can
further include instructions or a label for using the kit to treat,
prevent, or ameliorate one or more of a metabolic disease or a
cardiovascular disease.
Antisense Compounds
[0208] Oligomeric compounds include, but are not limited to,
oligonucleotides, oligonucleosides, oligonucleotide analogs,
oligonucleotide mimetics, antisense compounds, antisense
oligonucleotides, and siRNAs. An oligomeric compound can be
"antisense" to a target nucleic acid, meaning that is capable of
undergoing hybridization to a target nucleic acid through hydrogen
bonding.
[0209] 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.
[0210] In certain embodiments, an antisense compound targeted to
ANGPTL3 nucleic acid is 10 to 30 nucleotides in length. In other
words, antisense compounds are from 10 to 30 linked nucleobases. In
other embodiments, the antisense compound comprises a modified
oligonucleotide consisting of 8 to 80, 10-80, 12 to 50, 15 to 30,
18 to 24, 19 to 22, 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.
[0211] 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
can have two or more nucleosides deleted from the 5' end, or
alternatively can have two or more nucleosides deleted from the 3'
end. Alternatively, the deleted nucleosides can be dispersed
throughout the modified oligonucleotide, for example, in an
antisense compound having one or more nucleoside deleted from the
5' end and one or more nucleoside deleted from the 3' end.
[0212] When a single additional nucleoside is present in a
lengthened oligonucleotide, the additional nucleoside can be
located at the 5', 3' end or central portion of the
oligonucleotide. When two or more additional nucleosides are
present, the added nucleosides can 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)
or the central portion, of the oligonucleotide. Alternatively, the
added nucleoside can be dispersed throughout the antisense
compound, for example, in an oligonucleotide having one or more
nucleoside added to the 5' end, one or more nucleoside added to the
3' end, and/or one or more nucleoside added to the central
portion.
[0213] 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.
[0214] 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.
[0215] 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
[0216] In certain embodiments, antisense compounds targeted to an
ANGPTL3 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.
[0217] 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 can optionally
serve as a substrate for the cellular endonuclease RNase H, which
cleaves the RNA strand of an RNA:DNA duplex.
[0218] 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 can in some
embodiments include .beta.-D-ribonucleosides,
.beta.-D-deoxyribonucleosides, 2'-modified nucleosides (such
2'-modified nucleosides can include 2'-MOE, and 2'-O--CH.sub.3,
among others), and bicyclic sugar modified nucleosides (such
bicyclic sugar modified nucleosides can include those having a
4'-(CH2)n-O-2' bridge, where n=1 or n=2). Preferably, each distinct
region comprises uniform sugar moieties. The wing-gap-wing motif is
frequently described as "X--Y--Z", where "X" represents the length
of the 5' wing region, "Y" represents the length of the gap region,
and "Z" represents the length of the 3' wing region. As used
herein, a gapmer described as "X--Y--Z" has a configuration such
that the gap segment is positioned immediately adjacent each of the
5' wing segment and the 3' wing segment. Thus, no intervening
nucleotides exist between the 5' wing segment and gap segment, or
the gap segment and the 3' wing segment. Any of the antisense
compounds described herein can have a gapmer motif. In some
embodiments, X and Z are the same, in other embodiments they are
different. In a preferred embodiment, Y is between 8 and 15
nucleotides. X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30 or more nucleotides. Thus, gapmers include, but are not
limited to, for example 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3,
2-13-5, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 6-8-6,
5-8-5, 1-8-1, 2-6-2, 6-8-6, 5-8-5, 1-8-1, 2-6-2, 2-13-2, 1-8-2,
2-8-3, 3-10-2, 1-18-2, or 2-18-2.
[0219] In certain embodiments, the antisense compound as a
"wingmer" motif, having a wing-gap or gap-wing configuration, i.e.
an X--Y or Y--Z configuration as described above for the gapmer
configuration. Thus, wingmer configurations include, but are not
limited to, for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1,
10-3, 2-10, 1-10, 8-2, 2-13, or 5-13.
[0220] In certain embodiments, antisense compounds targeted to an
ANGPTL3 nucleic acid possess a 5-10-5 gapmer motif.
[0221] In certain embodiments, an antisense compound targeted to an
ANGPTL3 nucleic acid has a gap-widened motif.
Target Nucleic Acids, Target Regions and Nucleotide Sequences
[0222] Nucleotide sequences that encode ANGPTL3 include, without
limitation, the following: the human sequence as set forth in
GenBank Accession No. BG400407.1 (incorporated herein as SEQ ID NO:
1), GenBank Accession No. BG562555.1 (incorporated herein as SEQ ID
NO: 2), GenBank Accession No. BG562798.1 (incorporated herein as
SEQ ID NO: 3), GenBank Accession No. NM.sub.--014495.1
(incorporated herein as SEQ ID NO: 4), GenBank Accession No. NT
032977.5 nucleotides 15511702 to Ser. No. 15/521,082 (incorporated
herein as SEQ ID NO: 5), GenBank Accession No. AF162224.1
(incorporated herein as SEQ ID NO: 6), GenBank Accession No.
AI195524.1 (incorporated herein as SEQ ID NO: 7) and GenBank
Accession No. BB717501.1 (incorporated herein as SEQ ID NO: 8). 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 can 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.
[0223] In certain embodiments, a target region is a structurally
defined region of the target nucleic acid. For example, a target
region can 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 ANGPTL3 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 can 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
target region.
[0224] In certain embodiments, a "target segment" is a smaller,
sub-portion of a target region within a nucleic acid. For example,
a target segment can be the sequence of nucleotides of a target
nucleic acid to which one or more 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.
[0225] 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.
[0226] A target region can contain one or more target segments.
Multiple target segments within a target region can be overlapping.
Alternatively, they can 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
preceding 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.
[0227] Suitable target segments can 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 can
specifically exclude a certain structurally defined region such as
the start codon or stop codon.
[0228] The determination of suitable target segments can include a
comparison of the sequence of a target nucleic acid to other
sequences throughout the genome. For example, the BLAST algorithm
can be used to identify regions of similarity amongst different
nucleic acids. This comparison can prevent the selection of
antisense compound sequences that can hybridize in a non-specific
manner to sequences other than a selected target nucleic acid
(i.e., non-target or off-target sequences).
[0229] There can 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 ANGPTL3 mRNA levels are indicative of inhibition of
ANGPTL3 protein expression.
[0230] Reductions in levels of an ANGPTL3 protein are also
indicative of inhibition of target mRNA expression. Further,
phenotypic changes, such as a reduction of the level of
cholesterol, LDL, triglyceride, or glucose, can be indicative of
inhibition of ANGPTL3 mRNA and/or protein expression.
Hybridization
[0231] In some embodiments, hybridization occurs between an
antisense compound disclosed herein and a ANGPTL3 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.
[0232] 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.
[0233] Methods of determining whether a sequence is specifically
hybridizable to a target nucleic acid are well known in the art
(Sambrooke and Russell, Molecular Cloning: A Laboratory Manual,
3.sup.rd Ed., 2001). In certain embodiments, the antisense
compounds provided herein are specifically hybridizable with an
ANGPTL3 nucleic acid.
Complementarity
[0234] 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 an ANGPTL3 nucleic acid).
[0235] An antisense compound can hybridize over one or more
segments of an ANGPTL3 nucleic acid such that intervening or
adjacent segments are not involved in the hybridization event
(e.g., a loop structure, mismatch or hairpin structure).
[0236] In certain embodiments, the antisense compounds provided
herein, or a specified portion thereof, are, or are at least, 70%,
75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% complementary to an ANGPTL3 nucleic
acid, a target region, target segment, or specified portion
thereof. In certain embodiments, the antisense compounds provided
herein, or a specified portion thereof, are, or are at least, 70%,
75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% complementary to the sequence of one or
more of SEQ ID NOs: 1-5. Percent complementarity of an antisense
compound with a target nucleic acid can be determined using routine
methods.
[0237] For example, an antisense compound in which 18 of 20
nucleobases of the antisense compound are complementary to a target
region, and would therefore specifically hybridize, would represent
90 percent complementarity. In this example, the remaining
noncomplementary nucleobases can be clustered or interspersed with
complementary nucleobases and need not be contiguous to each other
or to complementary nucleobases. As such, an antisense compound
which is 18 nucleobases in length having 4 (four) noncomplementary
nucleobases which are flanked by two regions of complete
complementarity with the target nucleic acid would have 77.8%
overall complementarity with the target nucleic acid and would thus
fall within the scope of the present invention. Percent
complementarity of an antisense compound with a region of a target
nucleic acid can be determined routinely using BLAST programs
(basic local alignment search tools) and PowerBLAST programs known
in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410;
Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology,
sequence identity or complementarity, can be determined by, for
example, the Gap program (Wisconsin Sequence Analysis Package,
Version 8 for Unix, Genetics Computer Group, University Research
Park, Madison Wis.), using default settings, which uses the
algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482
489).
[0238] In certain embodiments, the antisense compounds provided
herein, or specified portions thereof, are fully complementary
(i.e. 100% complementary) to a target nucleic acid, or specified
portion thereof. For example, an antisense compound can be fully
complementary to an ANGPTL3 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 can 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.
[0239] The location of a non-complementary nucleobase can be at the
5' end or 3' end of the antisense compound. Alternatively, the
non-complementary nucleobase or nucleobases can be at an internal
position of the antisense compound. When two or more
non-complementary nucleobases are present, they can be either
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.
[0240] In certain embodiments, antisense compounds that are, or are
up to 10, 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 an ANGPTL3 nucleic acid, or specified portion
thereof.
[0241] In certain embodiments, antisense compounds that are, or are
up to 10, 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 an ANGPTL3 nucleic acid, or specified
portion thereof.
[0242] 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 10 nucleobase portion of a target segment. In certain
embodiments, the antisense compounds are complementary to at least
a 15 nucleobase portion of a target segment. Also contemplated are
antisense compounds that are complementary to at least an 8, 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
[0243] The antisense compounds provided herein can also have a
defined percent identity to a particular nucleotide sequence, SEQ
ID NO, or the sequence of a 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 can
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.
[0244] In certain embodiments, the antisense compounds, or portions
thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to one or more of the antisense compounds or
SEQ ID NOs, or a portion thereof, disclosed herein.
Modifications
[0245] 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.
[0246] 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.
[0247] Chemically modified nucleosides can 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
[0248] 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.
[0249] 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.
[0250] In certain embodiments, antisense compounds targeted to an
ANGPTL3 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
[0251] Antisense compounds can optionally contain one or more
nucleosides wherein the sugar group has been modified. Such sugar
modified nucleosides can 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
moieties. Examples of chemically modified ribofuranose rings
include without limitation, addition of substitutent groups
(including 5' and 2' substituent groups, bridging of non-geminal
ring atoms to form bicyclic nucleic acids (BNA), replacement of the
ribosyl ring oxygen atom with S, N(R), or C(R1)(R)2 (R=H, C1-C12
alkyl or a protecting group) and combinations thereof. Examples of
chemically modified sugars include 2'-F-5'-methyl substituted
nucleoside (see PCT International Application WO 2008/101157
Published on Aug. 21, 2008 for other disclosed 5',2'-bis
substituted nucleosides) or replacement of the ribosyl ring oxygen
atom with S with further substitution at the 2'-position (see
published U.S. Patent Application US2005-0130923, published on Jun.
16, 2005) or alternatively 5'-substitution of a BNA (see PCT
International Application WO 2007/134181 Published on Nov. 22, 2007
wherein LNA is substituted with for example a 5'-methyl or a
5'-vinyl group).
[0252] Examples of nucleosides having modified sugar moieties
include without limitation nucleosides comprising 5'-vinyl,
5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3 and 2'-O(CH2)2OCH3
substituent groups. The substituent at the 2' position can also be
selected from allyl, amino, azido, thio, 0-allyl, 0-C1-C10 alkyl,
OCF3, O(CH2)2SCH3, O(CH2)2-O--N(Rm)(Rn), and
O--CH2-C(.dbd.O)--N(Rm)(Rn), where each Rm and Rn is,
independently, H or substituted or unsubstituted C1-C10 alkyl.
[0253] Examples of bicyclic nucleic acids (BNAs) include without
limitation nucleosides comprising a bridge between the 4' and the
2' ribosyl ring atoms. In certain embodiments, antisense compounds
provided herein include one or more BNA nucleosides wherein the
bridge comprises one of the formulas: 4'-(CH2)-O-2' (LNA);
4'-(CH2)-S-2; 4'-(CH2)2-O-2' (ENA); 4'-C(CH3)2-O-2' (see
PCT/US2008/068922); 4'-CH(CH3)-O-2' and 4'-C--H(CH2OCH3)-O-2' (see
U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008);
4'-CH2-N(OCH3)-2' (see PCT/US2008/064591); 4'-CH2-O--N(CH3)-2' (see
published U.S. Patent Application US2004-0171570, published Sep. 2,
2004); 4'-CH2-N(R)--O-2' (see U.S. Pat. No. 7,427,672, issued on
Sep. 23, 2008); 4'-CH2-CH (see Chattopadhyaya et al., J. Org. Chem,
2009, 74, 118-134) (CH3)-2' and 4'-CH2-C(.dbd.CH2)-2' (see
PCT/US2008/066154); and wherein R is, independently, H, C1-C12
alkyl, or a protecting group. Each of the foregoing BNAs include
various 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). Previously, .alpha.-L-methyleneoxy
(4'-CH.sub.2--O-2') BNA's have also been incorporated into
antisense oligonucleotides that showed antisense activity (Frieden
et al., Nucleic Acids Research, 2003, 21, 6365-6372).
[0254] Further reports related to bicyclic nucleosides can be found
in published literature (see for example: Srivastava et al., J. Am.
Chem. Soc., 2007, 129, 8362-8379; U.S. Pat. Nos. 7,053,207;
6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; Elayadi
et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et
al., Chem. Biol., 2001, 8, 1-7; and Orum et al., Curr. Opinion Mol.
Ther., 2001, 3, 239-243; and U.S. Pat. No. 6,670,461; International
applications WO 2004/106356; WO 94/14226; WO 2005/021570; U.S.
Patent Publication Nos. US2004-0171570; US2007-0287831;
US2008-0039618; U.S. Pat. No. 7,399,845; 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; 61/099,844; PCT International Applications
Nos. PCT/US2008/064591; PCT/US2008/066154; PCT/US2008/068922; and
Published PCT International Applications WO 2007/134181).
[0255] 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.O)--,
--C(.dbd.NR.sub.a)--, --C(.dbd.S)--, --O--, --Si(R.sub.a).sub.2--,
--S(.dbd.O).sub.x--, and --N(R.sub.a)--;
[0256] wherein:
[0257] x is 0, 1, or 2;
[0258] n is 1, 2, 3, or 4;
[0259] 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.2J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and
[0260] 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.
[0261] 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.
[0262] 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, and (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'-CH.sub.2--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.
##STR00002## ##STR00003##
wherein Bx is the base moiety and R is independently H, a
protecting group or C.sub.1-C.sub.12 alkyl.
[0263] In certain embodiments, bicyclic nucleoside having Formula
I:
##STR00004##
wherein:
[0264] Bx is a heterocyclic base moiety;
[0265] -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;
[0266] R.sub.c is C.sub.1-C.sub.12 alkyl or an amino protecting
group; and
[0267] 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.
[0268] In certain embodiments, bicyclic nucleoside having Formula
II:
##STR00005##
wherein:
[0269] Bx is a heterocyclic base moiety;
[0270] 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;
[0271] 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.
[0272] In one embodiment, each of the substituted groups is,
independently, mono or poly substituted with substituent groups
independently selected from halogen, oxo, hydroxyl, OJ.sub.c,
NJ.sub.cJ.sub.d, SJ.sub.c, N.sub.3, OC(.dbd.X)J.sub.c, and
NJ.sub.eC(.dbd.X)NJ.sub.cJ.sub.d, wherein each J.sub.c, J.sub.d and
J.sub.e is, independently, H, C.sub.1-C.sub.6 alkyl, or substituted
C.sub.1-C.sub.6 alkyl and X is O or NJ.sub.c.
[0273] In certain embodiments, bicyclic nucleoside having Formula
III:
##STR00006##
wherein:
[0274] Bx is a heterocyclic base moiety;
[0275] 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;
[0276] 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)--).
[0277] In certain embodiments, bicyclic nucleoside having Formula
IV:
##STR00007##
wherein:
[0278] Bx is a heterocyclic base moiety;
[0279] 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;
[0280] 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;
[0281] 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;
[0282] In certain embodiments, bicyclic nucleoside having Formula
V:
##STR00008##
wherein:
[0283] Bx is a heterocyclic base moiety;
[0284] 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;
[0285] 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;
[0286] or q.sub.e and q.sub.f together are
.dbd.C(q.sub.g)(q.sub.h); [0287] 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.
[0288] 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 (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs
and preparation thereof are also described in WO 98/39352 and WO
99/14226.
[0289] Analogs of methyleneoxy (4'-CH.sub.2--O-2') BNA and
2'-thio-BNAs, have also been prepared (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 (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 (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.
[0290] In certain embodiments, bicyclic nucleoside having Formula
VI:
##STR00009##
wherein:
[0291] Bx is a heterocyclic base moiety;
[0292] 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;
[0293] 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
[0294] 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.
[0295] 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 (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
(Srivastava et al., J. Am. Chem. Soc., 2007, 129(26),
8362-8379).
[0296] In certain embodiments, nucleosides are modified by
replacement of the ribosyl ring with a sugar surrogate. Such
modification includes without limitation, replacement of the
ribosyl ring with a surrogate ring system (sometimes referred to as
DNA analogs) such as a morpholino ring, a cyclohexenyl ring, a
cyclohexyl ring or a tetrahydropyranyl ring such as one having one
of the formula:
##STR00010##
[0297] Many other bicyclo and tricyclo sugar surrogate ring systems
are also know in the art that can be used to modify nucleosides for
incorporation into antisense compounds (see for example review
article: Leumann, Christian J., Bioorganic & Medicinal
Chemistry, 2002, 10, 841-854). Such ring systems can undergo
various additional substitutions to enhance activity. See for
example compounds having Formula VII:
##STR00011##
wherein independently for each of said at least one tetrahydropyran
nucleoside analog of Formula VII:
[0298] Bx is a heterocyclic base moiety;
[0299] T.sub.a and T.sub.b are each, independently, an
internucleoside linking group linking the tetrahydropyran
nucleoside analog to the antisense compound or one of T.sub.a and
T.sub.b is an internucleoside linking group linking the
tetrahydropyran nucleoside analog to the antisense compound and the
other of T.sub.a and T.sub.b is H, a hydroxyl protecting group, a
linked conjugate group or a 5' or 3'-terminal group;
[0300] 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 each of R.sub.1 and
R.sub.2 is selected from hydrogen, hydroxyl, 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.
[0301] In certain embodiments, the modified THP nucleosides of
Formula VII are provided wherein q.sub.1, q.sub.2, q.sub.3,
q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each H (M). In certain
embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4,
q.sub.5, q.sub.6 and q.sub.7 is other than H. In certain
embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4,
q.sub.5, q.sub.6 and q.sub.7 is methyl. In certain embodiments, THP
nucleosides of Formula VII are provided wherein one of R.sub.1 and
R.sub.2 is fluoro (K). In certain embodiments, THP nucleosides of
Formula VII are provided wherein one of R.sub.1 and R.sub.2 is
methoxyethoxy. In certain embodiments, R.sub.1 is fluoro and
R.sub.2 is H; R.sub.1 is H and R.sub.2 is fluoro; R.sub.1 is
methoxy and R.sub.2 is H, and R.sub.1 is H and R.sub.2 is
methoxyethoxy. Methods for the preparations of modified sugars are
well known to those skilled in the art.
[0302] 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.
[0303] In certain embodiments, antisense compounds targeted to an
ANGPTL3 nucleic acid 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 bicyclic nucleoside having a
(4'-CH(CH.sub.3)--O-2') bridging group. In certain embodiments, the
(4'-CH(CH.sub.3)--O-2') modified nucleotides are arranged
throughout the wings of a gapmer motif.
[0304] Methods for the preparations of modified sugars are well
known to those skilled in the art.
[0305] 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.
[0306] In certain embodiments, antisense compounds targeted to an
ANGPTL3 nucleic acid 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.
Modified Nucleobases
[0307] 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 can 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).
[0308] Additional modified 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--CH3) uracil and cytosine and other alkynyl
derivatives of pyrimidine bases, 6-azo uracil, cytosine and
thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,
8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines
and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and
other 5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine.
[0309] Heterocyclic base moieties can 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.
[0310] In certain embodiments, antisense compounds targeted to an
ANGPTL3 nucleic acid comprise one or more modified nucleobases. In
certain embodiments, shortened or gap-widened antisense
oligonucleotides targeted to an ANGPTL3 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
[0311] Antisense oligonucleotides can 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.
[0312] Antisense compound targeted to an ANGPTL3 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 an ANGPTL3 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.
[0313] 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.
[0314] 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
[0315] Antisense compounds can 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.
[0316] 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 acids 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
[0317] The effects of antisense compounds on the level, activity or
expression of ANGPTL3 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,
N.C.; 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, Hep3B cells, Huh7 (hepatocellular carcinoma) cells,
primary hepatocytes, A549 cells, GM04281 fibroblasts and LLC-MK2
cells.
In Vitro Testing of Antisense Oligonucleotides
[0318] Described herein are methods for treatment of cells with
antisense oligonucleotides, which can be modified appropriately for
treatment with other antisense compounds.
[0319] In general, cells are treated with antisense
oligonucleotides when the cells reach approximately 60-80%
confluence in culture.
[0320] 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.
[0321] 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.
[0322] 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.
[0323] Another reagent used to introduce antisense oligonucleotides
into cultured cells includes Oligofectamine.TM. (Invitrogen Life
Technologies, Carlsbad, Calif.). Antisense oligonucleotide is mixed
with Oligofectamine.TM. in Opti-MEM.TM.-1 reduced serum medium
(Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the
desired concentration of oligonucleotide with an Oligofectamine.TM.
to oligonucleotide ratio of approximately 0.2 to 0.8 .mu.L per 100
nM.
[0324] Another reagent used to introduce antisense oligonucleotides
into cultured cells includes FuGENE 6 (Roche Diagnostics Corp.,
Indianapolis, Ind.). Antisense oligomeric compound was mixed with
FuGENE 6 in 1 mL of serum-free RPMI to achieve the desired
concentration of oligonucleotide with a FuGENE 6 to oligomeric
compound ratio of 1 to 4 .mu.L of FuGENE 6 per 100 nM.
[0325] Another technique used to introduce antisense
oligonucleotides into cultured cells includes electroporation
(Sambrooke and Russell, Molecular Cloning: A Laboratory Manual,
3.sup.rd Ed., 2001).
[0326] 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.
[0327] The concentration of antisense oligonucleotide used varies
from cell line to cell line.
[0328] 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
[0329] RNA analysis can be performed on total cellular RNA or
poly(A)+mRNA. Methods of RNA isolation are well known in the art
(Sambrooke and Russell, Molecular Cloning: A Laboratory Manual,
3.sup.rd Ed., 2001). 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
[0330] Inhibition of levels or expression of an ANGPTL3 nucleic
acid can be assayed in a variety of ways known in the art
(Sambrooke and Russell, Molecular Cloning: A Laboratory Manual,
3.sup.rd Ed., 2001). 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
[0331] Quantitation of target RNA levels can 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.
[0332] 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, and
real-time-PCR reactions are carried out by methods well known to
those skilled in the art.
[0333] Gene (or RNA) target quantities obtained by real time PCR
can be 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. Carlsbad, Calif.). 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.
[0334] Probes and primers are designed to hybridize to an ANGPTL3
nucleic acid. Methods for designing real-time PCR probes and
primers are well known in the art, and can include the use of
software such as PRIMER EXPRESS.RTM. Software (Applied Biosystems,
Foster City, Calif.).
[0335] Gene target quantities obtained by RT, real-time PCR can be
normalized using either the expression level of GAPDH, a gene whose
expression is constant, or by quantifying total RNA using
RiboGreen.TM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH
expression was quantified by RT, real-time PCR, by being run
simultaneously with the target, multiplexing, or separately. Total
RNA was quantified using RiboGreen.TM. RNA quantification reagent
(Molecular Probes, Inc. Eugene, Oreg.).
[0336] Presented in Table 2 are primers and probes used to measure
GAPDH expression in the cell types described herein. The GAPDH PCR
probes have JOE covalently linked to the 5' end and TAMRA or MGB
covalently linked to the 3' end, where JOE is the fluorescent
reporter dye and TAMRA or MGB is the quencher dye. In some cell
types, primers and probe designed to a GAPDH sequence from a
different species are used to measure GAPDH expression. For
example, a human GAPDH primer and probe set is used to measure
GAPDH expression in monkey-derived cells and cell lines.
TABLE-US-00002 TABLE 2 GAPDH primers and probes for use in
real-time PCR SEQ Target Sequence ID Name Species Description
Sequence (5' to 3') NO GAPDH Human Forward Primer
CAACGGATTTGGTCGTATTGG 15 GAPDH Human Reverse Primer
GGCAACAATATCCACTTTACCAGAGT 16 GAPDH Human Probe
CGCCTGGTCACCAGGGCTGCT 17 GAPDH Human Forward Primer
GAAGGTGAAGGTCGGAGTC 18 GAPDH Human Reverse Primer
GAAGATGGTGATGGGATTTC 19 GAPDH Human Probe CAAGCTTCCCGTTCTCAGCC 20
GAPDH Human Forward Primer GAAGGTGAAGGTCGGAGTC 18 GAPDH Human
Reverse Primer GAAGATGGTGATGGGATTTC 19 GAPDH Human Probe
TGGAATCATATTGGAACATG 21 GAPDH Mouse Forward Primer
GGCAAATTCAACGGCACAGT 22 GAPDH Mouse Reverse Primer
GGGTCTCGCTCCTGGAAGAT 23 GAPDH Mouse Probe
AAGGCCGAGAATGGGAAGCTTGTCATC 24 GAPDH Rat Forward Primer
TGTTCTAGAGACAGCCGCATCTT 25 GAPDH Rat Reverse Primer
CACCGACCTTCACCATCTTGT 26 GAPDH Rat Probe TTGTGCAGTGCCAGCCTCGTCTCA
27
[0337] Probes and primers for use in real-time PCR were designed to
hybridize to target-specific sequences. The primers and probes and
the target nucleic acid sequences to which they hybridize are
presented in Table 3. The target-specific PCR probes have FAM
covalently linked to the 5' end and TAMRA or MGB covalently linked
to the 3' end, where FAM is the fluorescent dye and TAMRA or MGB is
the quencher dye.
TABLE-US-00003 TABLE 3 Gene target-specific primers and probes for
use in real-time PCR Target SEQ SEQ Sequence ID Species ID NO
Description Sequence (5' to 3') NO Human 4 Forward Primer
CTTCAATGAAACGTGGGAGAACT 28 Human 4 Reverse Primer
TCTCTAGGCCCAACCAAAATTC 29 Human 4 Probe AAATATGGTTTTGGGAGGCTTGAT 30
Mouse 6 Forward Primer CAGAAGTAACATCACTCAAAAGTTTTGTAG 31 Mouse 6
Reverse Primer GACTTAATTGTTTATACTGTTCTTCCACACT 32 Mouse 6 Probe
CAGCAAGACAACAGCATAAGAGAACTCCTCCA 33
Analysis of Protein Levels
[0338] Antisense inhibition of ANGPTL3 nucleic acids can be
assessed by measuring ANGPTL3 protein levels. Protein levels of
ANGPTL3 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)
(Sambrooke and Russell, Molecular Cloning: A Laboratory Manual,
3.sup.rd Ed., 2001). 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.
In Vivo Testing of Antisense Compounds
[0339] Antisense compounds, for example, antisense
oligonucleotides, are tested in animals to assess their ability to
inhibit expression of ANGPTL3 and produce phenotypic changes.
Testing can 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 ANGPTL3 nucleic acid expression are measured.
Changes in ANGPTL3 protein levels are also measured.
Certain Indications
[0340] 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 and/or
cardiovascular disease. In certain embodiments, the individual has
atherosclerosis, hepatic steatosis or hyperlipidemia.
[0341] Accordingly, provided herein are methods for ameliorating a
symptom associated with a metabolic disease or cardiovascular
disease. Also provided herein are methods for ameliorating a
symptom associated with atherosclerosis, hepatic steatosis or
hyperlipidemia in a subject in need thereof. In certain
embodiments, provided is a method for reducing the rate of onset of
a symptom associated with a metabolic disease or cardiovascular
disease. In certain embodiments, provided is a method for reducing
the rate of onset of a symptom associated atherosclerosis, hepatic
steatosis or hyperlipidemia. In certain embodiments, provided is a
method for reducing the severity of a symptom associated with a
metabolic disease or cardiovascular disease. In certain
embodiments, provided is a method for reducing the severity of a
symptom associated with atherosclerosis, hepatic steatosis or
hyperlipidemia. In such embodiments, the methods comprise
administering to an individual in need thereof a therapeutically
effective amount of a compound targeted to an ANGPTL3 nucleic
acid.
[0342] In certain embodiments, administration of an antisense
compound targeted to an ANGPTL3 nucleic acid results in reduction
of ANGPTL3 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.
[0343] In certain embodiments, pharmaceutical compositions
comprising an antisense compound targeted to ANGPTL3 are used for
the preparation of a medicament for treating a patient suffering
from, or susceptible to, a metabolic disease or cardiovascular
disease. In certain embodiments, pharmaceutical compositions
comprising an antisense compound targeted to ANGPTL3 are used for
the preparation of a medicament for treating a patient suffering
from, or susceptible to, atherosclerosis, hepatic steatosis or
hyperlipidemia.
[0344] In certain embodiments, the methods described herein include
administering a compound comprising a modified oligonucleotide
having an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
contiguous nucleobase portion as described herein of a sequence
recited in SEQ ID NO: 34-182.
Administration
[0345] In certain embodiments, the compounds and compositions as
described herein are administered parenterally.
[0346] In certain embodiments, parenteral administration is by
infusion. Infusion can be chronic or continuous or short or
intermittent. In certain embodiments, infused pharmaceutical agents
are delivered with a pump.
[0347] 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.
Certain Combination Therapies
[0348] In certain embodiments, a first agent comprising the
modified oligonucleotide of the invention is co-administered with
one or more secondary agents. In certain embodiments, such second
agents are designed to treat the same disease, disorder or
condition as the first agent described herein. In certain
embodiments, such second agents are designed to treat a different
disease, disorder, or condition as the first agent described
herein. In certain embodiments, such second agents are designed to
treat an undesired side effect of one or more pharmaceutical
compositions as described herein. In certain embodiments, second
agents are co-administered with the first agent to treat an
undesired effect of the first agent. In certain embodiments, second
agents are co-administered with the first agent to produce a
combinational effect. In certain embodiments, second agents are
co-administered with the first agent to produce a synergistic
effect.
[0349] 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.
[0350] In certain embodiments, second agents include, but are not
limited to ascorbic acid.
EXAMPLES
Non-Limiting Disclosure and Incorporation by Reference
[0351] 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 Angiopoietin-Like 3 by Oligomeric
Compounds
[0352] A series of oligomeric compounds was designed to target
different regions of human angiopoietin-like 3, using published
sequences cited in Table 1. The compounds are shown in Table 4. All
compounds in Table 4 are chimeric oligonucleotides ("gapmers") 20
nucleotides in length, composed of a central "gap" region
consisting of 10 2'-deoxynucleotides, which is flanked on both
sides (5' and 3') by five-nucleotide "wings". The wings are
composed of 2'-O-(2-methoxyethyl) nucleotides, also known as 2'-MOE
nucleotides. The internucleoside (backbone) linkages are
phosphorothioate throughout the oligonucleotide. All cytosine
residues are 5-methylcytosines. The oligomeric compounds in Table 4
specifically hybridize to a target nucleic acid molecule encoding
angiopoietin-like 3 and are comprised of regions that increase
binding affinity, these regions being the "wings" of the oligomeric
compounds. The oligomeric compounds each comprise a region that
elicits RNase H activity, this regions being the "gap" region.
[0353] The compounds were analyzed for their effect on gene target
mRNA levels by quantitative real-time PCR as described in other
examples herein, using the target-specific primers and probe shown
in Table 3 (SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30). Data
are averages from experiments in which Huh7 cells were treated with
150 nM of the disclosed oligomeric compounds using
OLIGOFECTAMINE.TM.. Shown in Table 4 is the SEQ ID NO of the
sequence to which each oligomeric compound is targeted.
[0354] A reduction in expression is expressed as percent inhibition
in Table 4. If present, "N.D." indicates "not determined". The
target regions to which these oligomeric compounds are inhibitory
are herein referred to as "validated target segments."
TABLE-US-00004 TABLE 4 Inhibition of human angiopoietin-like 3 mRNA
levels by chimeric oligonucleotides having 2'-MOE wings and deoxy
gap Target SEQ ID Target SEQ ID ISIS # NO Site Sequence (5' to 3')
% Inhib NO 337529 1 548 ATCTGTTGTGATGTCGATAA 66 34 337527 2 336
GTATTTAGTCAAGTTTAGAG 39 35 337528 2 429 TATTACAGATTTTTACACAT 21 36
337526 3 33 CGTGGAACTGTTTTCTTCTG 63 37 337459 4 22
AGCTTAATTGTGAACATTTT 73 38 337460 4 61 ATTCTGGAGGAAATAACTAG 34 39
233675 4 116 AAATCTTGATTTTGGCTCTG 61 40 233676 4 121
ATAGCAAATCTTGATTTTGG 52 41 337461 4 126 CTAACATAGCAAATCTTGAT 53 42
337462 4 131 ATCGTCTAACATAGCAAATC 43 43 337463 4 154
AGGCCATTGGCTAAAATTTT 37 44 337464 4 171 CATGTCCCAACTGAAGGAGG 41 45
337465 4 180 CTTTAAGACCATGTCCCAAC 0 46 337466 4 203
GCCCTTCGTCTTATGGACAA 28 47 337467 4 214 TCATTAATTTGGCCCTTCGT 38 48
337468 4 223 TGAAATATGTCATTAATTTG 0 49 233690 4 247
GACTGATCAAATATGTTGAG 53 50 337469 4 271 GTTTGCAGCGATAGATCATA 56 51
337470 4 277 TCACTGGTTTGCAGCGATAG 62 52 337471 4 364
AGTTCAAGTGACATATTCTT 52 53 337472 4 496 AGTGAAGTTACTTCTGGGTG 59 54
337473 4 502 GTTTTAAGTGAAGTTACTTC 50 55 337474 4 510
CTACAAAAGTTTTAAGTGAA 25 56 337475 4 558 GGTCTTCCACGGTCTGGAGA 70 57
337476 4 624 TAGTCCTTCTGAGCTGATTT 66 58 337477 4 637
GGTTCTTGAATACTAGTCCT 80 59 337478 4 648 AAATTTCTGTGGGTTCTTGA 74 60
337479 4 665 TGGCTTGGAAGATAGAGAAA 77 61 233710 4 683
AGTAGTTCTTGGTGCTCTTG 82 62 337480 4 694 TGAAGAAAGGGAGTAGTTCT 60 63
337481 4 701 ATTCAACTGAAGAAAGGGAG 59 64 337482 4 710
TCTTATTTCATTCAACTGAA 32 65 337483 4 734 AGGAATGCCATCATGTTTTA 39 66
337484 4 762 CTCTGTTATAAATGGTGGTA 65 67 337485 4 767
TTCACCTCTGTTATAAATGG 31 68 337486 4 772 GTATGTTCACCTCTGTTATA 47 69
337487 4 777 CACTTGTATGTTCACCTCTG 75 70 337488 4 782
CATGCCACTTGTATGTTCAC 44 71 337489 4 806 AGAGTTGCTGGGTCTGATGG 52 72
337490 4 840 CTGATATAACATCACAGTAG 54 73 337491 4 850
CATGGACTACCTGATATAAC 60 74 233717 4 862 TGAATTAATGTCCATGGACT 85 75
337492 4 874 TCTATTCGATGTTGAATTAA 13 76 337493 4 909
AGTTCTCCCACGTTTCATTG 80 77 337494 4 918 CATATTTGTAGTTCTCCCAC 62 78
337495 4 923 AAAACCATATTTGTAGTTCT 25 79 337496 4 930
GCCTCCCAAAACCATATTTG 35 80 337497 4 953 GCCCAACCAAAATTCTCCAT 70 81
233721 4 959 CTCTAGGCCCAACCAAAATT 73 82 233722 4 964
ATCTTCTCTAGGCCCAACCA 91 83 337498 4 995 AACATAATTAGATTGCTTCA 26 84
337499 4 1016 GTCTTCCAACTCAATTCGTA 56 85 337500 4 1023
CTTTCCAGTCTTCCAACTCA 38 86 337501 4 1094 AACTAGATGTAGCGTATAGT 65 87
337502 4 1162 TGATCCCAAGTAGAAAACAC 33 88 337503 4 1213
CACCAGCCTCCTGAATAACC 32 89 337504 4 1245 TTAGGTTGTTTTCTCCACAC 68 90
337505 4 1301 TAATCCTCTTCTCCTCTCTG 32 91 337506 4 1315
TGAGACTTCCAAGATAATCC 63 92 337507 4 1320 CATTTTGAGACTTCCAAGAT 48 93
337508 4 1333 GAGTATAACCTTCCATTTTG 54 94 337509 4 1364
TGGATGGATCAACATTTTGG 67 95 337510 4 1385 TTCAAAGCTTTCTGAATCTG 62 96
337511 4 1397 TGCCTCAGTTCATTCAAAGC 58 97 337512 4 1410
TGCCTTTTAAATTTGCCTCA 64 98 337513 4 1443 ATTAACTTGGAATGAGGTTA 65 99
337514 4 1450 AGACCACATTAACTTGGAAT 62 100 337515 4 1458
AGATTATTAGACCACATTAA 44 101 337516 4 1463 ATACCAGATTATTAGACCAC 65
102 337517 4 1470 GGATTTAATACCAGATTATT 64 103 337518 5 1678
ACTGACTTACCTGATTTTCT 0 104 337519 5 2294 ACCTTGTAAGTCTTCATTGG 52
105 337520 5 3809 CAGTGTTATTCAGATTGTAC 56 106 337521 5 4068
AGTGTCTTACCATCATGTTT 56 107 337522 5 5100 ACAGATGTAAATAACACTTT 19
108 337523 5 5252 GTCCCCTTACCATCAAGCCT 49 109 337524 5 7150
GGGAAGATACTTTGAAGATA 50 110 337525 5 7504 CACCAGCCTCCTAAAGGAGA 27
111
Example 2
Antisense Inhibition of Mouse Angiopoietin-Like 3 by Oligomeric
Compounds
[0355] A series of oligomeric compounds was designed to target
different regions of mouse angiopoietin-like 3, using published
sequences cited in Table 1. The compounds are shown in Table 5. All
compounds in Table 5 are chimeric oligonucleotides ("gapmers") 20
nucleotides in length, composed of a central "gap" region
consisting of 10 2'-deoxynucleotides, which is flanked on both
sides (5' and 3') by five-nucleotide "wings". The wings are
composed of 2'-O-(2-methoxyethyl) nucleotides, also known as 2'-MOE
nucleotides. The internucleoside (backbone) linkages are
phosphorothioate throughout the oligonucleotide. All cytosine
residues are 5-methylcytosines. The oligomeric compounds in Table 5
specifically hybridize to a target nucleic acid molecule encoding
angiopoietin-like 3 and are comprised of regions that increase
binding affinity, these regions being the "wings" of the oligomeric
compounds. The oligomeric compounds each comprise a region that
elicits RNase H activity, this region being the "gap" region.
[0356] The compounds were analyzed for their effect on gene target
mRNA levels by quantitative real-time PCR as described in other
examples herein, using the target-specific primers and probe shown
in Table 3 (SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33). Data
are averages from experiments in which mouse primary hepatocytes
were treated with 150 nM of the disclosed oligomeric compounds
using LIPOFECTIN.TM.. The control oligomeric compounds used were
SEQ ID NOs: 9 and 10. Shown in Table 5 is the SEQ ID NO of the
sequence to which each oligomeric compound is targeted.
[0357] A reduction in expression is expressed as percent inhibition
in Table 5. If present, "N.D." indicates "not determined". The
target regions to which these oligomeric compounds are inhibitory
are herein referred to as "validated target segments". The
antisense oligonucleotides of Table 5 may also be cross reactive
with the human ANGPTL3 mRNA (GENBANK Accession NM.sub.--014495.1,
incorporated herein as SEQ ID NO: 4), depending on the number of
mismatched nucleobases the murine oligonucleotide has with the
human ANGPTL3 sequence. "Human Target Start Site" indicates the
5'-most nucleotide in the human mRNA to which the antisense
oligonucleotide is targeted. "Human Target Stop Site" indicates the
3'-most nucleotide in the human mRNA to which the antisense
oligonucleotide is targeted. `Mismatches` indicates the number of
nucleobases by which the murine oligonucleotide is mismatched with
the human gene sequence. The designation "n/a" indicates that there
was greater than 3 mismatches between the murine oligonucleotide
and the human gene sequence. The greater the complementarity
between the murine oligonucleotide and the human gene sequence, the
more likely the murine oligonucleotide can cross-react with the
human gene sequence.
TABLE-US-00005 TABLE 5 Inhibition of mouse angiopoietin-like 3 mRNA
levels by chimeric oligonucleotides having 2'-MOE wings and deoxy
gap Human Mismatches Target SEQ Target with SEQ Target % ID Start
human ISIS # ID NO Site Sequence (5' to 3') Inhib NO Site target
233671 6 19 AATAATTTAATTGTGTGCAT 13 112 n/a n/a 233672 6 56
CACTCTGGATGCAATTACTA 66 113 n/a n/a 233673 6 68
AAGGTCTGGATCCACTCTGG 74 114 n/a n/a 233674 6 100
TTTGGCTCTGAAGGTGCAGA 66 115 n/a n/a 233675 6 110
AAATCTTGATTTTGGCTCTG 80 40 116 0 233676 6 115 ATAGCAAATCTTGATTTTGG
49 41 121 0 233677 6 124 TCATCCAACATAGCAAATCT 24 116 130 2 233678 6
129 TGACATCATCCAACATAGCA 66 117 135 3 233679 6 134
AATTTTGACATCATCCAACA 76 118 140 3 233680 6 139 GCTAAAATTTTGACATCATC
72 119 145 2 233681 6 148 AGGCCATTCGCTAAAATTTT 52 120 154 1 233682
6 160 CCCAGCTGCAGGAGGCCATT 77 121 166 2 233683 6 165
CATGACCCAGCTGCAGGAGG 51 122 171 3 233684 6 172 TTAAGTCCATGACCCAGCTG
71 123 178 3 233685 6 182 GACAAAATCTTTAAGTCCAT 27 124 188 2 233686
6 187 TTATGGACAAAATCTTTAAG 29 125 193 1 233687 6 226
TTGAGCTTCTGAAATATGTC 56 126 232 2 233688 6 231 ATATGTTGAGCTTCTGAAAT
53 127 237 2 233689 6 236 ATCAAATATGTTGAGCTTCT 47 128 242 2 233690
6 241 GACTGATCAAATATGTTGAG 73 50 247 0 233691 6 266
GGTTCGAAGTGATAGGTCAT 63 129 n/a n/a 233692 6 317
TAGTGTAGATGTAGTTCTTC 44 130 323 2 233693 6 349 GACATGTTCTTCACCTCCTC
80 131 355 3 233694 6 365 TGAGTTCAGTTCTACTGACA 78 132 371 3 233695
6 373 TCAAGCTTTGAGTTCAGTTC 71 133 379 2 233696 6 394
GTCTTCTCTTCCAGCAGACT 71 134 n/a n/a 233697 6 405
GTTGAAGGGCTGTCTTCTCT 75 135 n/a n/a 233698 6 415
CTGACCTTGTGTTGAAGGGC 92 136 n/a n/a 233699 6 423
CCAAAGCCCTGACCTTGTGT 60 137 n/a n/a 233700 6 435
TTAGCTGCTCCTCCAAAGCC 68 138 n/a n/a 233701 6 451
CTTAGAATTAAGTTGGTTAG 49 139 457 3 233702 6 474 GGTGCTCCTGAGCCCCAGCT
82 140 n/a n/a 233703 6 488 TGATGTTACTTCTGGGTGCT 63 141 494 2
233704 6 511 TGCTGTTCTACAAAACTTTT 79 142 517 3 233705 6 535
AGGAGTTCTCTTATGCTGTT 86 143 n/a n/a 233706 6 540
TCTGGAGGAGTTCTCTTATG 58 144 n/a n/a 233707 6 545
CACACTCTGGAGGAGTTCTC 77 145 n/a n/a 233708 6 632
GGGTTCTTGAATACCAGTCT 58 146 638 2 233709 6 649 GAAAGAGAATTTTCTGAGGG
29 147 655 3 233710 6 677 AGTAGTTCTTGGTGCTCTTG 68 62 683 0 233711 6
730 GCAGGAAGGTCATCTTGTTC 62 148 n/a n/a 233712 6 739
GAGCAGTCGGCAGGAAGGTC 62 149 n/a n/a 233713 6 778
TACACGCCACTTGTATGTTC 55 150 784 1 233714 6 787 TTAATAGTGTACACGCCACT
62 151 n/a n/a 233715 6 816 AGACATTAAACCCTTGGGAG 47 152 n/a n/a
233716 6 838 CTGCCTGATTGGGTATCACA 76 153 n/a n/a 233717 6 856
TGAATTAATGTCCATGGACT 61 75 862 0 233718 6 871 CCATCTTTCCGGTGTTGAAT
64 154 877 3 233719 6 884 GAAGTCCTGTGAGCCATCTT 69 155 n/a n/a
233720 6 935 TTCTCCATCGAGCCTCCCAA 56 156 941 1 233721 6 953
CTCTAGGCCCAACCAAAATT 67 82 959 0 233722 6 958 ATCTTCTCTAGGCCCAACCA
63 83 964 0 233723 6 975 GTTGGACTATAGCATAGATC 70 157 n/a n/a 233724
6 988 ATGTAGTTAGACTGTTGGAC 57 158 n/a n/a 233725 6 1033
ACGTAGTGCTTGCTGTCTTT 81 159 n/a n/a 233726 6 1055
GCCCAGGTGAAAGGAGTATT 70 160 n/a n/a 233727 6 1081
TGTAGCGTGTAGTTGGTTTC 38 161 1087 1 233728 6 1086
CCACATGTAGCGTGTAGTTG 59 162 1092 3 233729 6 1091
CTCAGCCACATGTAGCGTGT 57 163 n/a n/a 233730 6 1096
GCAATCTCAGCCACATGTAG 62 164 n/a n/a 233731 6 1138
ATCAGGTCTGTGTGCTCTGG 75 165 n/a n/a 233732 6 1149
ATGTAGAAAACATCAGGTCT 66 166 n/a n/a 233733 6 1160
TCTGTGATTCCATGTAGAAA 61 167 1166 3 233734 6 1186
TCTGGACAGTAGAGCTGTCC 46 168 1192 3 233735 6 1191
AACTTTCTGGACAGTAGAGC 58 169 n/a n/a 233736 6 1209
ACCACCAGCCACCTGAGTAA 60 170 1215 2 233737 6 1229
TTCTCCACATATGTCATTCC 56 171 n/a n/a 233738 6 1236
GGTTGTTTTCTCCACATATG 70 172 n/a n/a 233739 6 1277
TGGTCTGGATTTGGTTCTGG 71 173 n/a n/a 233740 6 1283
TCTCTCTGGTCTGGATTTGG 73 174 n/a n/a 233741 6 1324
TAGAGCTTTCTGCTCTGAGG 64 175 n/a n/a 233742 6 1363
GTGGTGGGCTGGAGCATCAT 57 176 n/a n/a 233743 6 1376
TGAAGCTTCTTAGGTGGTGG 55 177 n/a n/a 233744 6 1390
TGTCTCAGTTCAGTTGAAGC 60 178 n/a n/a 233745 6 1430
TCGGGAGGACTTTAATATTT 50 179 n/a n/a 233746 7 13
GGAACTTCTCCCTCCTGTCC 49 180 n/a n/a 233747 8 202
TAACAATGAGTTTAAACCTA 17 181 n/a n/a 233748 8 210
TCTGATCTTAACAATGAGTT 0 182 n/a n/a
Example 3
Design and Screening of Duplexed Oligomeric Compounds Targeting
Angiopoietin-Like 3
[0358] In accordance with the invention, a series of duplexes,
including dsRNA and mimetics thereof, comprising oligomeric
compounds of the invention and their complements can be designed to
target angiopoietin-like 3. The nucleobase sequence of the
antisense strand of the duplex comprises at least a portion of an
oligonucleotide targeted to angiopoietin-like 3 as disclosed
herein. The ends of the strands may be modified by the addition of
one or more natural or modified nucleobases to form an overhang.
The sense strand of the nucleic acid duplex is then designed and
synthesized as the complement of the antisense strand and may also
contain modifications or additions to either terminus. The
antisense and sense strands of the duplex comprise from about 17 to
25 nucleotides, or from about 19 to 23 nucleotides. Alternatively,
the antisense and sense strands comprise 20, 21 or 22
nucleotides.
[0359] For example, in one embodiment, both strands of the dsRNA
duplex would be complementary over the central nucleobases, each
having overhangs at one or both termini.
[0360] For example, a duplex comprising an antisense strand having
the sequence CGAGAGGCGGACGGGACCG (incorporated herein as SEQ ID NO:
183) and having a two-nucleobase overhang of deoxythymidine (dT)
would have the following structure:
TABLE-US-00006 cgagaggcggacgggaccgTT Antisense Strand (SEQ ID NO:
184) ||||||||||||||||||| TTgctctccgcctgccctggc Complement (SEQ ID
NO: 185)
[0361] Overhangs can range from 2 to 6 nucleobases and these
nucleobases may or may not be complementary to the target nucleic
acid. In another embodiment, the duplexes can have an overhang on
only one terminus.
[0362] In another embodiment, a duplex comprising an antisense
strand having the same sequence, for example CGAGAGGCGGACGGGACCG
(SEQ ID NO: 183), can be prepared with blunt ends (no single
stranded overhang) as shown:
TABLE-US-00007 cgagaggcggacgggaccg Antisense Strand (SEQ ID NO:
183) ||||||||||||||||||| gctctccgcctgccctggc Complement (SEQ ID NO:
186)
[0363] The RNA duplex can be unimolecular or bimolecular; i.e, the
two strands can be part of a single molecule or may be separate
molecules.
[0364] RNA strands of the duplex can be synthesized by methods
routine to the skilled artisan or purchased from Dharmacon Research
Inc. (Lafayette, Colo.). Once synthesized, the complementary
strands are annealed. The single strands are aliquoted and diluted
to a concentration of 50 .mu.M. Once diluted, 30 .mu.L of each
strand is combined with 154, of a 5.times. solution of annealing
buffer. The final concentration of said buffer is 100 mM potassium
acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The
final volume is 75 .mu.L. This solution is incubated for 1 minute
at 90.degree. C. and then centrifuged for 15 seconds. The tube is
allowed to sit for 1 hour at 37.degree. C. at which time the dsRNA
duplexes are used in experimentation. The final concentration of
the dsRNA duplex is 20 .mu.M.
[0365] Once prepared, the duplexed compounds are evaluated for
their ability to modulate angiopoietin-like 3. When cells reached
80% confluency, they are treated with duplexed compounds of the
invention. For cells grown in 96-well plates, wells are washed once
with 200 .mu.L OPTI-MEM-1.TM. reduced-serum medium (Gibco BRL) and
then treated with 130 .mu.L of OPTI-MEM-1.TM. containing 12
.mu.g/mL LIPOFECTIN.TM. (Gibco BRL) and the desired duplex
antisense compound at a final concentration of 200 nM (a ratio of 6
.mu.g/mL LIPOFECTIN.TM. per 100 nM duplex antisense compound).
After 5 hours of treatment, the medium is replaced with fresh
medium. Cells are harvested 16 hours after treatment, at which time
RNA is isolated and target reduction measured by RT-PCR.
Example 4
Antisense Inhibition of Mouse Angiopoietin-Like 3 by Oligomeric
Compounds: Dose Response Studies
[0366] In a further embodiment of the present invention, three
oligonucleotides were selected for additional dose-response
studies. Mouse primary hepatocytes were treated with 6.25, 25, 100,
or 400 nM of ISIS 233693 (SEQ ID NO: 131), ISIS 233698 (SEQ ID NO:
136), or ISIS 233725 (SEQ ID NO: 159), or the scrambled control
oligonucleotide ISIS 113529 (5-10-5 gapmer, CTCTTACTGTGCTGTGGACA,
incorporated herein as SEQ ID NO: 11) and mRNA levels were measured
as described in other examples herein. Untreated cells served as
the control to which the data were normalized.
[0367] Results of these studies are shown in Table 6. Data are
averages from three experiments and are expressed as percent
inhibition relative to untreated control.
TABLE-US-00008 TABLE 6 Inhibition of angiopoietin-like 3 mRNA
expression in mouse primary hepatocytes % Inhibition Dose (nM)
Treatment 6.25 25 100 400 ISIS 233693 45 77 87 87 ISIS 233698 0 13
33 52 ISIS 233725 28 65 66 79 ISIS 113529 0 0 0 0
[0368] As shown in Table 6, ISIS 233693, 233698, and 233725 reduced
angiopoietin-like 3 mRNA levels in a dose-dependent manner.
Example 5
Effects of Antisense Inhibition of Angiopoietin-Like 3: In Vivo
Studies in C57BL/6 Mice
[0369] In accordance with the present invention, two
oligonucleotides targeting mouse angiopoietin-like 3 were chosen
for in vivo studies. Male C57BL/6 mice fed normal chow were
injected twice weekly with 50 mg/kg doses of either ISIS 233693
(SEQ ID NO: 131) or ISIS 233698 (SEQ ID NO: 136) for two weeks.
Each treatment group was comprised of 5 animals. A group of animals
received injections of saline twice weekly for 2 weeks. This
saline-injected group served as the control group to which the
oligonucleotide-treated groups were compared.
[0370] After the 2 week treatment period, the mice were sacrificed
and angiopoietin-like 3 mRNA levels were evaluated in liver. mRNA
expression levels were quantitated by real-time PCR as described in
other examples herein. Relative to saline-treated mice, ISIS 233693
caused a 44% decrease in angiopoietin-like 3 mRNA levels. ISIS
233698 caused a 41% decrease in angiopoietin-like 3 mRNA levels.
The data demonstrate that angiopoietin-like 3 antisense
oligonucleotide treatment can effectively inhibit target mRNA
expression in liver.
[0371] Spleen weight, body weight, and liver weight were measured
at the end of the study. Average tissue and body weights measured
(in grams) at the end of the treatment period are shown in Table 7.
As shown in Table 7, body weight, liver weight and spleen weight
were not affected by oligonucleotide treatment.
TABLE-US-00009 TABLE 7 Effect of antisense inhibition of
angiopoietin-like 3 expression on tissue and body weights in lean
mice Body Liver Spleen Treatment Weight Weight Weight Saline 24 1.1
0.1 ISIS 233693 23 1.1 0.1 ISIS 233698 22 1.0 0.1
[0372] At study termination, the animals were evaluated for serum
cholesterol, HDL, LDL, triglyceride and glucose levels by routine
analysis using an Olympus Clinical Analyzer (Olympus America Inc.,
Melville, N.Y.). The serum transaminases ALT and AST, increases in
which can indicate hepatotoxicity, were also measured. Levels of
AST or ALT associated with liver toxicity were not observed. The
levels of serum glucose (GLUC), cholesterol (CHOL), LDL, HDL, and
triglycerides (TRIG) measured are presented in Table 8 as the
average result from each treatment group in mg/dL.
TABLE-US-00010 TABLE 8 Effect of antisense inhibition of
angiopoietin-like 3 expression on serum glucose, lipids, and
transaminases in lean mice Treatment CHOL GLU HDL TG LDL Saline 80
172 58 109 14 ISIS 233693 70 251 53 72 10 ISIS 233698 87 266 67 84
12
[0373] As shown in Table 8, treatment with ISIS 233693 and ISIS
233698 reduced serum triglycerides. Treatment with ISIS 233693
reduced total cholesterol.
Example 6
Effects of Antisense Inhibition of Angiopoietin-Like 3: In Vivo
Dose-Response Studies in High-Fat Fed Mice
[0374] The C57BL/6 mouse strain is reported to be susceptible to
hyperlipidemia-induced atherosclerotic plaque formation.
Accordingly, these mice were fed a high-fat diet and used in the
following studies to evaluate the effects of angiopoietin-like 3
antisense oligonucleotides on mRNA expression.
[0375] Male C57BL/6 mice were placed on a high-fat diet containing
60% calories from fat (for example, Research Diet D12492, Research
Diets Inc., New Brunswick, N.J.). Mice receiving the high-fat diet
were divided into treatment groups. Three groups received
twice-weekly injections of ISIS 233693 (SEQ ID No: 131) at doses of
10 mg/kg, 25 mg/kg or 50 mg/kg, for 6 weeks. Three additional
groups received twice-weekly injections of ISIS 233698 (SEQ ID No:
136) at doses of 10 mg/kg, 25 mg/kg or 50 mg/kg, for 6 weeks.
[0376] A group of high-fat fed animals received injections of
saline twice weekly for 6 weeks. This saline-injected group served
as the control group to which the oligonucleotide-treated groups
were compared.
[0377] After the 6 week treatment period, the mice were sacrificed
and angiopoietin-like 3 mRNA levels were evaluated in liver. mRNA
expression levels were quantitated by real-time PCR as described in
other examples herein. Results are presented in Table 9 as the
average percentage inhibition relative to saline-treated
control.
TABLE-US-00011 TABLE 9 Antisense inhibition of angiopoietin-like 3
expression in liver from high-fat fed mice: dose-response study
Treatment % Inhibition ISIS 233693, 10 mg/kg 73 ISIS 233693, 25
mg/kg 88 ISIS 233693, 50 mg/kg 93 ISIS 233698, 10 mg/kg 17 ISIS
233698, 25 mg/kg 39 ISIS 233698, 50 mg/kg 55
[0378] These data show that antisense oligonucleotides targeted to
angiopoietin-like 3 mRNA effectively reduce target mRNA expression
in liver in a dose-dependent manner.
[0379] Body weight was monitored throughout the study. Spleen
weight, fat pad weight, and liver weight were measured at the end
of the study. Average tissue and body weights measured at the end
of the treatment period are shown in Table 10.
TABLE-US-00012 TABLE 10 Antisense inhibition of angiopoietin-like 3
expression on tissue and body weights in high-fat fed mice:
dose-response study Body Treatment Weight Liver Spleen Fat Pad
Saline 36 1.2 0.1 2.1 ISIS 233693, 10 mg/kg 37 1.5 0.1 2.0 ISIS
233693, 25 mg/kg 34 1.4 0.1 1.3 ISIS 233693, 50 mg/kg 32 1.5 0.1
1.1 ISIS 233698, 10 mg/kg 38 1.4 0.1 1.8 ISIS 233698, 25 mg/kg 33
1.2 0.1 1.5 ISIS 233698, 50 mg/kg 33 1.5 0.1 1.5
[0380] These data demonstrate that body weight, spleen weight and
liver weight were not affected. Fat pad weight was reduced in a
dose-dependent manner by treatment with ISIS 233698. Treatment with
ISIS 233698 also reduced fat pad weight.
[0381] At study termination, the animals were evaluated for serum
cholesterol, triglyceride and glucose levels by routine analysis
using an Olympus Clinical Analyzer (Olympus America Inc., Melville,
N.Y.). The serum transaminases ALT and AST, increases which can
indicate hepatotoxicity, were also measured using an Olympus
Clinical Analyzer (Olympus America Inc., Melville, N.Y.). The
levels of serum cholesterol (CHOL) and triglycerides (TRIG)
measured are presented in Table 11 as the average result from each
treatment group in mg/dL. Also shown are the average levels of HDL
and LDL as well as average glucose levels (GLUC). ALT and AST, also
shown in Table 11, are likewise shown as the average result from
each treatment group, in international units/L (IU/L).
TABLE-US-00013 TABLE 11 Effects of antisense inhibition of
angiopoietin-like 3 on serum glucose, cholesterol, triglycerides,
and liver transaminases in high-fat fed mice Treatment ALT AST CHOL
HDL LDL TRIG GLUC Saline 22 47 177 143 31 120 244 ISIS 233693, 23
60 151 127 23 81 263 10 mg/kg ISIS 233693, 21 62 125 106 18 57 254
25 mg/kg ISIS 233693, 52 79 147 125 22 44 206 50 mg/kg ISIS 233698,
28 55 151 120 27 101 285 10 mg/kg ISIS 233698, 16 53 135 110 23 89
248 25 mg/kg ISIS 233698, 192 175 158 126 31 77 215 50 mg/kg
[0382] As shown in Table 11, as compared to saline-treatment,
treatment with ISIS 233693 or ISIS 233698 resulted in decreased
cholesterol levels and dose-dependent decreases in serum
triglycerides. ISIS 233693 and ISIS 233698 also resulted in a
slight decrease in HDL which is commonly observed in mice when
hypolipidemic agents are tested due to the fact that mice, unlike
humans and other species, carry 90% of their serum cholesterol as
HDL particles. Furthermore, treatment with ISIS 233693 decreased
LDL.
Example 7
Effects of Antisense Inhibition of Angiopoietin-Like 3 Levels In
Vivo: Liver Triglycerides
[0383] Hepatic steatosis refers to the accumulation of lipids in
the liver, or "fatty liver", which is frequently caused by alcohol
consumption, diabetes and hyperlipidemia and can progress to
end-stage liver damage. Given the deleterious consequences of a
fatty liver condition, it is of use to identify compounds that
prevent or ameliorate hepatic steatosis. Hepatic steatosis may be
evaluated both by measurement of tissue triglyceride content and by
histologic examination of liver tissue.
[0384] In a further embodiment, liver tissue triglyceride content
was assessed in the animals described in Example 6. Liver tissue
triglyceride content was measured using the Triglyceride GPO assay
(Roche Diagnostics, Indianapolis, Ind.). Results for each treatment
group were normalized to saline-treated control and are presented
in Table 12.
TABLE-US-00014 TABLE 12 Effects of antisense inhibition of
angiopoietin-like 3 on liver triglycerides in high-fat fed mice
Treatment % Control ISIS 233693, 10 mg/kg 82 ISIS 233693, 25 mg/kg
54 ISIS 233693, 50 mg/kg 31 ISIS 233698, 10 mg/kg 55 ISIS 233698,
25 mg/kg 41 ISIS 233698, 50 mg/kg 47
[0385] As shown in Table 12, treatment with antisense
oligonucleotides targeted to angiopoietin-like 3 results in
dose-dependent reduction in liver triglycerides.
Example 8
Effects of Antisense Inhibition of Angiopoietin-Like 3: In Vivo
Studies with ISIS 233693 in High-Fat Fed Mice
[0386] In a study similar to that described in Example 6, male
C57BL/6 mice were placed on a high-fat diet containing 60% calories
from fat (for example, Research Diet D12492, Research Diets Inc.,
New Brunswick, N.J.). Mice receiving the high-fat diet were divided
into treatment groups. One group received twice-weekly injections
of ISIS 233693 (SEQ ID No: 131) at doses of 50 mg/kg, for 6
weeks.
[0387] Oligonucleotides were dissolved in saline for injection. A
group of high-fat fed animals received injections of saline twice
weekly for 6 weeks. This saline-injected group served as the
control group to which the oligonucleotide-treated groups were
compared.
[0388] After the 6 week treatment period, the mice were sacrificed
and angiopoietin-like 3 mRNA levels were evaluated in liver. mRNA
expression levels were quantitated by real-time PCR as described in
other examples herein. ISIS 233693 caused an 88% reduction in
target mRNA levels.
[0389] Body weight was monitored throughout the study. Spleen
weight, fat pad weight, and liver weight were measured at the end
of the study. The average body weight, liver weight, spleen weight,
and fat pad weight for animals treated with saline alone were 33 g,
1.2 g, 0.1 g, 0.7 g, respectively. The average body weight, liver
weight, spleen weight, and fat pad weight for animals treated with
ISIS 233693 were 31 g, 1.6 g, 0.2 g, and 0.2 g, respectively.
Treatment with ISIS 233693 reduced fat pad weight by 71%.
[0390] At study termination, the animals were evaluated for serum
cholesterol, triglyceride and glucose levels by routine clinical
analyses (for example at a clinical testing facility such as BTS, a
division of Lab Corp, San Diego, Calif.). The serum transaminases
ALT and AST, and serum protein, increases in which can indicate
hepatotoxicity, and levels of bilirubin, increases in which can
indicate kidney toxicity, were also measured. Toxic increases as
indicators of aberrant kidney or liver function were not observed
with ISIS 233693 treatment. ISIS 233693 caused a reduction in serum
triglyceride and glucose levels, but did not alter serum
cholesterol, LDL, or HDL levels.
[0391] Hepatic steatosis refers to the accumulation of lipids in
the liver, or "fatty liver", which is frequently caused by alcohol
consumption, diabetes and hyperlipidemia and can progress to
end-stage liver damage. Given the deleterious consequences of a
fatty liver condition, it is of use to identify compounds that
prevent or ameliorate hepatic steatosis. Hepatic steatosis may be
evaluated both by measurement of tissue triglyceride content and by
histologic examination of liver tissue.
[0392] Liver tissue triglyceride content was measured using the
Triglyceride GPO assay (Roche Diagnostics, Indianapolis, Ind.).
Average results for the ISIS 233693 treatment group were normalized
to saline-treated control. Treatment with ISIS 233693 caused a 75%
reduction in liver triglyceride levels.
[0393] Histological analysis was conducted by routine procedures.
Briefly, liver samples were procured, fixed in 10% neutral buffered
formalin and processed for H&E staining and evaluation of liver
morphology. Alternatively, liver tissue was procured, frozen,
sectioned, and subsequently stained with oil red 0 stain to
visualize lipid deposits and counterstained with eosin to mark
cytoplasm. The prepared samples were evaluated by light
microscopy.
[0394] As assessed by oil-red 0 stain and histological analysis,
livers from animals treated with ISIS 233693 presented with reduced
fat content as compared to saline-treated control livers.
[0395] Therefore, oligomeric compounds targeted to
angiopoietin-like 3 ameliorate hepatic steatosis as evaluated both
by measurement of tissue triglyceride content and by histologic
examination of liver tissue.
[0396] Taken together, the in vivo studies shown herein indicate
that antisense oligonucleotide reduction of angiopoietin-like 3
results in dose dependent reductions in liver target mRNA, as well
as reductions in serum and liver triglyceride levels. In addition,
antisense oligonucleotides targeted to angiopoietin-like 3 caused
decreases in serum cholesterol levels in both lean and high-fat fed
mice. Furthermore, reduction in fat pad weight was observed without
similar reductions in body or organ weight, indicating
target-specific reduction in fat content. Therefore, another aspect
of the invention is a method of reducing serum cholesterol, serum
triglycerides, liver triglycerides or fat pad weight for conditions
such as hyperlipidemia.
Example 9
Effects of Antisense Inhibition of Angiopoietin-Like 3 on
Atherosclerosis: Treatment with ISIS 233693 in LDLr.sup.-/-
Mice
[0397] The effect of ISIS 233693 as an anti-atherosclerotic agent
was evaluated in LDL receptor knockout mice fed on a
hypercholesterolemic diet; a model used for studying
atherosclerosis (Ishibashi et al, J Clin. Invest. 1994 May;
93:1885-93).
Treatment
[0398] C57B1/6 mice with LDL receptor gene knockout (Jackson Labs,
#2207) were fed a Harlan Tekland diet, TD 94059 (37% kCal fat, half
from cocoa butter, 1.25% cholesterol). Four weeks after the
initiation of the diet, the mice were divided into two groups
consisting of 6-8 mice each for treatment. The first group received
twice-weekly subcutaneous injections of ISIS 233693 (SEQ ID No:
131) at doses of 25 mg/kg, for 16 weeks. The second group received
twice-weekly subcutaneous injections of mismatched control
oligonucleotide, ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, incorporated
herein as SEQ ID NO: 187) at doses of 25 mg/kg, for 16 weeks.
[0399] Oligonucleotides were dissolved in saline for injection. A
group of high-fat fed animals received injections of saline twice
weekly for 16 weeks. This saline-injected group served as the
control group to which the oligonucleotide-treated groups were
compared.
[0400] Blood samples were taken every 4 weeks. At the end of the
treatment period, the mice were euthanized and liver and aorta were
collected for further analysis.
RNA Analysis
[0401] RNA was extracted from liver tissue for real-time PCR
analysis of ANGPTL3 using an ANGPTL3 primer probe set (forward
sequence CACCTGGGCAGTCACGAAA, designated herein as SEQ ID NO: 188;
reverse sequence GGAGGGCCCCAGGGATAT, designated herein as SEQ ID
NO: 189; probe sequence CACGCTACATGTGGCTGAGATTCGTGG, designated
herein as SEQ ID NO: 190). Results are presented as percent
inhibition of murine ANGPTL3, relative to PBS control. As shown in
Table 13, treatment with ISIS 233693 resulted in significant
reduction of ANGPTL3 mRNA in comparison to the PBS control.
Treatment with the control oligonucleotide, ISIS 141923 did not
result in significant reduction of ANGPTL3, as expected.
TABLE-US-00015 TABLE 13 Inhibition of ANGPTL3 mRNA in LDLr.sup.-/-
mouse liver relative to the PBS control % inhibition ISIS 141923 13
ISIS 233693 90
Cholesterol and Lipid Levels
[0402] Plasma and liver triglycerides, and cholesterol were
extracted by the method of Bligh and Dyer (Bligh, E and Dyer, W,
Can J Biochem Physiol, 37, 911-917, 1959) and measured with the use
of a commercially available triglyceride kit (DCL Triglyceride
Reagent; Diagnostic Chemicals Ltd.). The results are presented in
Tables 14-17. Table 14 demonstrates that treatment with ISIS 233693
resulted in significant decrease in cholesterol levels by 58% on
week 16 compared to the PBS control. The decrease in total
cholesterol levels was the result of significant decrease in LDL
cholesterol levels by 58% compared to the control, as presented in
Table 15. Table 16 shows a decrease in HDL that is likely mouse
model dependent, as detailed previously. Similarly, Table 17
demonstrates that treatment with ISIS 233693 decreased triglyceride
levels by 75% on week 16 compared to the PBS control.
TABLE-US-00016 TABLE 14 Effect on total cholesterol levels (mg/dL)
in LDLr.sup.-/- mice Week 0 Week 4 Week 8 Week 12 Week 16 PBS 1,313
1,618 1,398 1,083 1,629 ISIS 141923 1262 1710 1624 1102 1167 ISIS
233693 1353 1070 930 558 683
TABLE-US-00017 TABLE 15 Effect on LDL cholesterol levels (mg/dL) in
LDLr.sup.-/- mice Week 0 Week 4 Week 8 Week 12 Week 16 PBS 1,031
1,120 1,001 909 1,204 ISIS 141923 1022 1185 1116 902 843 ISIS
233693 1075 731 648 453 511
TABLE-US-00018 TABLE 16 Effect on HDL cholesterol levels (mg/dL) in
LDLr.sup.-/- mice Week 0 Week 4 Week 8 Week 12 Week 16 PBS 154 166
169 145 300 ISIS 141923 139 171 179 171 278 ISIS 233693 153 116 103
97 159
TABLE-US-00019 TABLE 17 Effect on triglyceride levels (mg/dL) in
LDLr.sup.-/- mice Week 0 Week 4 Week 8 Week 12 Week 16 PBS 230 177
134 140 268 ISIS 141923 180 154 164 156 159 ISIS 233693 191 68 61
72 67
Atherosclerotic Lesion Assessment
[0403] Atherosclerotic lesion severity was assessed in the aortae
harvested from mice after perfusion with PBS, followed by formalin
PBS solution (5% formalin in PBS). The entire mouse aorta was
dissected from the proximal ascending aorta to the bifurcation of
the iliac artery by using a dissecting microscope. Adventitial fat
was removed, and the aorta was opened longitudinally, pinned flat
onto black dissecting wax, stained with Sudan IV, and photographed
at a fixed magnification. The photographs were digitized, and total
aortic areas and lesion areas were calculated by using Adobe
Photoshop version 7.0 and NIH Scion Image software
(http://rsb.info.nih.gov/nih-image/Default.html). The results,
presented in Table 18, are reported as a percentage of the total
aortic area that contained lesions. As presented, treatment with
ISIS 233693 resulted in a significant decrease in aortic lesions
and improvement of the atherosclerotic condition.
TABLE-US-00020 TABLE 18 Effect on lesion area (% of total aortic
area) in LDLr.sup.-/- mice lesion area (%) PBS 44 ISIS 141923 40
ISIS 233693 18
Liver Function
[0404] 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 Tables 19 and 20 expressed in IU/L.
The measurements were conducted every four weeks. Week 0 is at the
start of treatment and four weeks after the initiation of the high
fat diet.
TABLE-US-00021 TABLE 19 Effect on liver ALT (IU/L) of LDLr.sup.-/-
mice Week 0 Week 4 Week 8 Week 12 Week 16 PBS 29 35 39 36 36 ISIS
141923 30 32 32 33 60 ISIS 233693 20 48 70 83 91
TABLE-US-00022 TABLE 20 Effect on liver AST (IU/L) of LDLr.sup.-/-
mice Week 0 Week 4 Week 8 Week 12 Week 16 PBS 49 63 61 82 73 ISIS
141923 57 57 61 60 81 ISIS 233693 51 60 90 95 108
Example 10
Effect of Antisense Inhibition of Angiopoietin-Like 3 on Human
apoB100 Transgenic LDLr.sup.-/- Mice
[0405] The effect of ISIS 233693 as a lipid lowering agent was
evaluated in human apoB-100 transgenic LDL receptor knockout mice
fed on a hypercholesterolemic diet. The mice used in these studies
have been described in a previous publication Sanan et al, Proc.
Natl. Acad. Sci. USA. 95: 4544-4549). In brief, this mouse strain
is a hybrid cross between the LDLr.sup.-/- mouse described
originally by Ishibashi et al. (J. Clin. Invest. 92: 883-893),
which is a hybrid of the 129sv and C57BL/6 strains, and the human
apoB-100 transgenic mouse (Linton et al, J. Clin. Invest. 92:
3029-3037), which is a hybrid of the SJL and C57BL/6B strains.
Breeding indicated that the LDLr.sup.-/- and apoB overexpression
traits were homozygous, and the mice exhibited a massive increase
in apoB-100-containing LDL.
Treatment
[0406] The mice were fed a Harlan Tekland diet, TD 88137 or
`Western diet` (21% anhydrous milkfat (butterfat), 34% sucrose, and
a total of 0.2% cholesterol). One week after the initiation of the
diet, the mice were divided into groups consisting of 5 mice each
for treatment. The first cohort received twice-weekly subcutaneous
injections of ISIS 233693 (SEQ ID No: 131) at doses of 12.5 mg/kg,
25 mg/kg or 50 mg/kg for 4 weeks. The second cohort received
twice-weekly subcutaneous injections of control oligonucleotide,
ISIS 141923 (SEQ ID NO: 187) at doses of 12.5 mg/kg or 50 mg/kg,
for 4 weeks.
[0407] Oligonucleotides were dissolved in saline for injection. A
group of high-fat fed animals received injections of saline twice
weekly for 4 weeks. This saline-injected group served as the
control group to which the oligonucleotide-treated groups were
compared.
[0408] The mice were weighed weekly. At the end of the treatment
period, the mice were euthanized and blood samples, liver, kidney,
spleen and fat pads were collected for further analysis.
RNA analysis
[0409] RNA was extracted from liver tissue for real-time PCR
analysis of ANGPTL3 using an ANGPTL3 primer probe set (forward
sequence CACCTGGGCAGTCACGAAA, designated herein as SEQ ID NO: 187;
reverse sequence GGAGGGCCCCAGGGATAT, designated herein as SEQ ID
NO: 188; probe sequence CACGCTACATGTGGCTGAGATTCGTGG, designated
herein as SEQ ID NO: 189) and of ApoCIII mRNA using an ApoCIII
primer probe set (Forward: TGCAGGGCTACATGGAACAA, incorporated
herein as SEQ ID NO: 12; Reverse: CGGACTCCTGCACGCTACTT,
incorporated herein as SEQ ID NO: 13; Probe:
CTCCAAGACGGTCCAGGATGCGC, incorporated herein as SEQ ID NO: 14).
Results are presented as percent inhibition of murine ANGPTL3 and
murine ApoCIII, relative to PBS control. As shown in Table 21,
treatment with ISIS 233693 and ISIS 233725 resulted in significant
dose-dependent reduction of ANGPTL3 mRNA in comparison to the PBS
control. Treatment with ISIS 233693 also resulted in inhibition of
ApoCIII mRNA at 50 mg/kg/week.
[0410] RNA levels of liver fatty acid binding protein (LFABP) were
also measured by real-time PCR. The results are presented in Table
21, and demonstrate that inhibition of ANGPTL3 by ISIS
oligonucleotides also influences the transport of fatty acids in
the liver, by inhibiting LFABP. Treatment with the control
oligonucleotide, ISIS 141923 did not result in significant
reduction of ANGPTL3, LFABP or apoCIII, as expected.
TABLE-US-00023 TABLE 21 Percent inhibition of ANGPTL3 and LFABP
mRNA in mouse liver relative to the PBS control Dose ISIS No
(mg/kg) ANGPTL3 ApoCIII LFABP 233693 50 95 39 53 25 90 25 12 12.5
77 8 10 233725 50 91 27 80 25 79 27 51 12.5 62 12 11
Cholesterol and Lipid Levels
[0411] Plasma and liver triglycerides, and cholesterol were
extracted by the method of Bligh and Dyer (Bligh, E and Dyer, W,
Can J Biochem Physiol, 37, 911-917, 1959) and measured with the use
of a commercially available triglyceride kit (DCL Triglyceride
Reagent; Diagnostic Chemicals Ltd.). The results are presented in
Table 22. The study demonstrates that treatment with ISIS 233693
and ISIS 233725 decreased cholesterol levels by 63% and 37%
respectively, at 25 mg/kg/week compared to the PBS control. The
decrease in total cholesterol levels was mainly the result of
significant decreases in LDL cholesterol levels by 63% and 34%
respectively, compared to the control, as presented. The slight
decrease in HDL may be mouse model dependent as detailed previously
as HDL lowering is commonly observed in mice when hypolipidemic
agents are tested in mice. The study demonstrates that treatment
with ISIS 233693 and ISIS 233725 decreased triglyceride levels by
82% and 70% respectively, at 25 mg/kg/week compared to the PBS
control.
[0412] Therefore, treatment with ISIS oligonucleotides targeting
ANGPTL3 causes significant improvements of plasma lipid profile in
this mouse model.
TABLE-US-00024 TABLE 22 Effect on plasma lipid levels (mg/dL) Dose
Total (mg/kg) cholsterol HDL LDL Triglycerides PBS 2534 430 1591
1022 ISIS 141923 50 1730 347 998 945 12.5 2489 412 1512 1168 ISIS
233693 50 982 256 585 243 25 944 245 596 184 12.5 1480 307 990 281
ISIS 233725 50 1407 325 862 403 25 1587 312 1052 309 12.5 2060 370
1366 522
Glucose
[0413] To evaluate the effect of ISIS oligonucleotides on glucose
production, plasma glucose values were determined using a Beckman
Glucose Analyzer II (Beckman Coulter) by a glucose oxidase method.
The results are presented in Table 23 and demonstrate that
treatment with ISIS 233693 and ISIS 233725 resulted in decrease of
plasma glucose levels both by 40% at 50 mg/kg/week compared to the
PBS control.
TABLE-US-00025 TABLE 23 Effect on plasma glucose levels (mg/dL)
Dose (mg/kg) Glucose PBS 341 ISIS 141923 50 334 12.5 315 ISIS
233693 50 203 25 261 12.5 255 ISIS 233725 50 203 25 280 12.5
284
Liver Function
[0414] 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 24 expressed in IU/L.
[0415] As demonstrated by this study, treatment with ISIS 233693
did not cause any significant increase in transaminase levels and
therefore, did not result in any adverse effect on liver function.
This assay as well as the RNA analysis establishes ISIS 233693 to
be a potent and tolerable antisense oligonucleotide targeting
ANGPTL3.
TABLE-US-00026 TABLE 24 Effect on plasma transaminases (IU/L) Dose
(mg/kg) ALT AST PBS 57 80 ISIS 141923 50 61 88 12.5 43 79 ISIS
233693 50 138 152 25 43 70 12.5 45 87 ISIS 233725 50 1443 736 25
217 194 12.5 88 138
Body and Organ Weights
[0416] To evaluate the effect of ISIS oligonucleotides on organ
weight, organs were harvested and weight taken after termination of
the study. The results are presented in Table 25 and demonstrate
that treatment with ISIS oligonucleotides has no effect on liver,
spleen or kidney weights. Treatment with ISIS 233693 did decrease
fat pad weights of the mice by 77% at 50 mg/kg/week compared to the
PBS control.
TABLE-US-00027 TABLE 25 Effect on organ weights (g) Dose mg/kg/wk)
Liver Kidney Spleen Fat PBS 1.45 0.35 0.09 1.49 ISIS 141923 50 1.42
0.33 0.11 0.70 12.5 1.39 0.32 0.09 0.95 ISIS 233693 50 1.65 0.33
0.15 0.35 25 1.42 0.32 0.13 0.51 12.5 1.49 0.35 0.11 0.89
Example 11
Effects of Antisense Inhibition of ANGPTL3 Compared to Fenofibrate
Inhibition in C57BL/6 Mice
[0417] ISIS 233693 targeting mouse ANGPTL3 in comparison to
fenofibrate was evaluated in naive C57BL/6 mice. Fenofibrate is a
commercially available treatment for hypercholesterolemia and
hypertriglyceridemia in subjects and is known to reduce
cholesterol, low-density lipoprotein (LDL), very low density
lipoprotein (VLDL) and triglycerides levels, as well as increase
high-density lipoprotein (HDL) levels.
[0418] A group of five male C57BL/6 mice fed normal chow were
injected twice weekly with 50 mg/kg doses of ISIS 233693 (SEQ ID
NO: 131) for six weeks. A second group of mice was treated with
fenofibrate, administered as a daily gavage of 50 mg/kg/week. A
group of animals received injections of PBS twice weekly for 6
weeks. This PBS-injected group served as the control group to which
the oligonucleotide-treated groups were compared.
[0419] After the 6 week treatment period, the mice were sacrificed
and ANGPTL3 mRNA levels were evaluated in liver. The mRNA
expression levels were quantitated by real-time PCR, as described
in other examples herein. Relative to PBS-treated mice, ISIS 233693
caused an 85% decrease in ANGPTL3 mRNA levels. The data demonstrate
that ANGPTL3 antisense oligonucleotide treatment can effectively
inhibit target mRNA expression in liver.
Cholesterol and Lipid Levels
[0420] Plasma and liver triglycerides and cholesterol were
extracted by the method of Bligh and Dyer (Bligh, E and Dyer, W,
Can J Biochem Physiol, 37, 911-917, 1959) and measured with the use
of a commercially available triglyceride kit (DCL Triglyceride
Reagent; Diagnostic Chemicals Ltd.). The results are presented in
Table 26. The study demonstrates that treatment with ISIS 233693
decreased cholesterol levels by 23% at 50 mg/kg/week compared to
the PBS control. The study demonstrates that treatment with ISIS
233693 decreased triglyceride levels by 38% at 50 mg/kg/week
compared to the PBS control. Treatment with fenofibrate had no
effect on cholesterol or triglyceride levels.
[0421] Therefore, treatment with ISIS oligonucleotides targeting
ANGPTL3 causes significant improvements of plasma lipid profile in
this mouse model.
TABLE-US-00028 TABLE 26 Effect on plasma lipid levels (mg/dL) Total
cholesterol HDL LDL Triglycerides PBS 87 75 17 90 ISIS 233693 67 54
16 56 Fenofibrate 96 85 16 94
Glucose
[0422] To evaluate the effect of ISIS oligonucleotides on glucose
production, plasma glucose values were determined using a Beckman
Glucose Analyzer II (Beckman Coulter) by a glucose oxidase method.
The results are presented in Table 27 and demonstrate that
treatment with ISIS 233693 resulted in decrease of plasma glucose
levels by 19% at 50 mg/kg/week compared to the PBS control.
Treatment with fenofibrate had no effect on glucose levels.
TABLE-US-00029 TABLE 27 Effect on plasma glucose levels (mg/dL)
Glucose PBS 242 ISIS 233693 196 Fenofibrate 228
Liver Function
[0423] 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 28 expressed in IU/L.
[0424] As demonstrated by this study, treatment with either ISIS
2336393 or fenofibrate did not cause any significant increase in
transaminase levels and therefore, did not result in any adverse
effect on liver function.
TABLE-US-00030 TABLE 28 Effect on plasma transaminases (IU/L) ALT
AST PBS 23 83 ISIS 233693 38 70 Fenofibrate 34 70
Effect on Plasma NEFA and 3HB Levels
[0425] NEFA and 3-HB levels were assayed in the mice groups and are
shown in Table 29. NEFA and 3-HB levels, as indicators of fat
oxidation, were not significantly affected by treatment with ISIS
oligonucleotides. Treatment with fenofibrate increased fat
oxidation, as indicated by increases in the levels of both NEFA and
3HB.
TABLE-US-00031 TABLE 29 Effect on NEFA and 3HB levels NEFA 3HB PBS
0.90 393 ISIS 233693 0.98 447 Fenofibrate 1.20 641
Organ Weights
[0426] To evaluate the effect of ISIS oligonucleotides on organ
weight, organs were harvested and weight taken after termination of
the study. The results are presented in Table 30 and demonstrate
that treatment with ISIS 233693 has no effect on liver, spleen or
kidney weights. Treatment with ISIS 233693 did decrease white
adipose tissue weight of the mice by 45% at 50 mg/kg/week compared
to the PBS control.
TABLE-US-00032 TABLE 30 Effect on organ weights (g) White adipose
Liver Spleen Kidney tissue PBS 1.1 0.12 0.35 0.53 ISIS 233693 1.3
0.1 0.35 0.29 Fenofibrate 1.3 0.08 0.37 0.39
Example 12
Effects of Antisense Inhibition of ANGPTL3 in Sprague-Dawley
Rats
[0427] ISIS 360363 (GTGACATATTCTTCACCTCT; SEQ ID NO: 191) and ISIS
360382 (TTTAAGTGACGTTACCTCTG; SEQ ID NO: 192), both 5-10-5 MOE
gapmers targeting rat mRNA sequence SEQ ID NO: 193 (Genbank
Accession No. XM.sub.--233218.1) at start positions 333 and 476,
respectively, were evaluated in Sprague Dawley rats.
[0428] Two groups of Sprague Dawley rats fed normal chow were
injected weekly with 50 mg/kg doses of ISIS 360363 or ISIS 360382
for six weeks. A group of animals received injections of PBS twice
weekly for 6 weeks. This PBS-injected group served as the control
group to which the oligonucleotide-treated groups were
compared.
RNA Analysis
[0429] RNA was extracted from liver tissue for real-time PCR
analysis of ANGPTL3. Results are presented as percent inhibition of
rat ANGPTL3, relative to PBS control. As shown in Table 31,
treatment with ISIS 360363 and ISIS 360382 resulted in significant
reduction of ANGPTL3 mRNA in comparison to the PBS control.
TABLE-US-00033 TABLE 31 Inhibition of ANGPTL3 mRNA in Sprague
Dawley rat liver relative to the PBS control % ISIS No inhibition
360363 70 360382 89
Cholesterol and Lipid Levels
[0430] Plasma triglycerides and cholesterol were extracted by the
method of Bligh and Dyer (Bligh, E and Dyer, W, Can J Biochem
Physiol, 37, 911-917, 1959) and measured with the use of a
commercially available triglyceride kit (DCL Triglyceride Reagent;
Diagnostic Chemicals Ltd.). The results are presented in Table 26.
The study demonstrates that treatment with ISIS 360363 and 360382
decreased triglyceride levels by 67% and 81% respectively, compared
to the PBS control. Therefore, treatment with ISIS oligonucleotides
targeting ANGPTL3 causes significant improvements of plasma
triglyceride profile in this rat model. Treatment with ISIS
oligonucleotides in this model had no effect on total cholesterol
or LDL levels.
TABLE-US-00034 TABLE 32 Effect on plasma triglyceride levels
(mg/dL) PBS 136 ISIS 360363 45 ISIS 360382 26
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 193 <210> SEQ ID NO 1 <211> LENGTH: 833
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 1 attaatttca aaactaaaaa tcgtcagcac agagtatgtg
taaaaatctg taatacaaat 60 ttttaaactg atgcttcatt ttgctacaaa
ataatttgga gtaaatgttt gatatgattt 120 atttatgaaa cctaatgaag
cagaattaaa tactgtatta aaataagttc gctgtcttta 180 aacaaatgga
gatgactact aagtcacatt gactttaaca tgaggtatca ctatacctta 240
tttgttaaaa tatatactgt atacatttta tatattttaa cacttaatac tatgaaaaca
300 aataattgta aaggaatctt gtcagattac agtaagaatg aacatatttg
tggcatcgag 360 ttaaagttta tatttcccct aaatatgctg tgattctaat
acattcgtgt aggttttcaa 420 gtagaaataa acctcgtaac aagttactga
acgtttaaac agcctgacaa gcatgtatat 480 atgtttaaaa ttcaataaac
aaagacccag tccctaaatt atagaaattt aaattattct 540 tgcatgttta
tcgacatcac aacagatccc taaatcccta aatccctaaa gattagatac 600
aaatttttta ccacagtatc acttgtcaga atttattttt aacatatgat tttttaaaac
660 tgccagtaag aaattttaaa tttaacccat ttgttaagga tatagtgccc
aagttaattg 720 gtgacctacc tttgtcaata cttagcataa tgtatttcaa
aattatccaa tatacctgtc 780 atattaatct attatgtcca cttttttaag
atatgtatga cctatgtgaa tct 833 <210> SEQ ID NO 2 <211>
LENGTH: 795 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 2 ggtctaataa tctggtatta aatccttaag
agaaagcttg agaaatagat tttttttatc 60 ttaaagtcac tgtctattta
agattaaaca tacaatcaca taaccttaaa gaataccgtt 120 tacatttctc
aatcaaaatt cttataatac tatttgtttt aaattttgtg atgtgggaat 180
caattttaga tggtcacaat ctagattata atcaataggt gaacttatta aataactttt
240 ctaaataaaa aatttagaga cttttatttt aaaaggcatc atatgagcta
atatcacaac 300 tttcccagtt taaaaaacta gtactcttgt taaaactcta
aacttgacta aatacagagg 360 actggtaatt gtacagttct taaatgttgt
agtattaatt tcaaaactaa aaatcgtcag 420 cacagagtat gtgtaaaaat
ctgtaataca aatttttaaa ctgatgcttc attttgctac 480 aaaataattt
ggagtaaatg tttgatatga tttatttatg aaacctaatg aagcagaatt 540
aaatactgta ttaaaataag ttcgctgtct ttaaacaaat ggagatgact actaagtcac
600 attgacttta acatgaggta tcactatacc ttattgttaa acatatatac
tgtatacatt 660 ttatatattt taacacttaa tactatgaaa acaaataatt
gtaaggaatc ttgtcagatt 720 acagtaagaa tgaacatatt tgtggcatcg
agttaaagtt tatatttccc ctaaaatatg 780 cttgtgattc taata 795
<210> SEQ ID NO 3 <211> LENGTH: 743 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3
atatatagag ttaagaagtc taggtctgct tccagaagaa aacagttcca cgttgcttga
60 aattgaaaat caagataaaa atgttcacaa ttaagctcct tctttttatt
gttcctctag 120 ttatttcctc cagaattgat caagacaatt catcatttga
ttctctatct ccagagccaa 180 aatcaagatt tgctatgtta gacgatgtaa
aaattttagc caatggcctc cttcagttgg 240 gacatggtct taaagacttt
gtccataaga cgaaggggcc aaatttattg gacatatttc 300 aaaaactcaa
catatttgat cagtcttttt atgatctatc gctgcaaacc agtgaaatca 360
aagaagaaga aaaggaactg agaagaacta catataaact acaagtcaaa aatgaagagg
420 taaagaatat gtcacttgaa ctcaactcaa aacttgaaag cctcctagaa
gaaaaaattc 480 tacttcaaca aaaagtgaaa tatttagaag agcaactaac
taacttaatt caacaatcaa 540 cctgaaactc cagaacaccc agaagtaact
tcacttaaaa ctttgtagaa aaacaagata 600 atagcatcaa agaccttctc
cagaccgtgg aagaccaata taaacaatta aaccaacagc 660 aaaaaacaaa
caatcacaca cacacacaca ccaacacaac tctttttggc gccccattgc 720
cctccaaaaa aatttttaaa cac 743 <210> SEQ ID NO 4 <211>
LENGTH: 1496 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (25)..(1407) <400> SEQUENCE: 4 ggcacgagga
aaatcaagat aaaa atg ttc aca att aag ctc ctt ctt ttt 51 Met Phe Thr
Ile Lys Leu Leu Leu Phe 1 5 att gtt cct cta gtt att tcc tcc aga att
gat caa gac aat tca tca 99 Ile Val Pro Leu Val Ile Ser Ser Arg Ile
Asp Gln Asp Asn Ser Ser 10 15 20 25 ttt gat tct cta tct cca gag cca
aaa tca aga ttt gct atg tta gac 147 Phe Asp Ser Leu Ser Pro Glu Pro
Lys Ser Arg Phe Ala Met Leu Asp 30 35 40 gat gta aaa att tta gcc
aat ggc ctc ctt cag ttg gga cat ggt ctt 195 Asp Val Lys Ile Leu Ala
Asn Gly Leu Leu Gln Leu Gly His Gly Leu 45 50 55 aaa gac ttt gtc
cat aag acg aag ggc caa att aat gac ata ttt caa 243 Lys Asp Phe Val
His Lys Thr Lys Gly Gln Ile Asn Asp Ile Phe Gln 60 65 70 aaa ctc
aac ata ttt gat cag tct ttt tat gat cta tcg ctg caa acc 291 Lys Leu
Asn Ile Phe Asp Gln Ser Phe Tyr Asp Leu Ser Leu Gln Thr 75 80 85
agt gaa atc aaa gaa gaa gaa aag gaa ctg aga aga act aca tat aaa 339
Ser Glu Ile Lys Glu Glu Glu Lys Glu Leu Arg Arg Thr Thr Tyr Lys 90
95 100 105 cta caa gtc aaa aat gaa gag gta aag aat atg tca ctt gaa
ctc aac 387 Leu Gln Val Lys Asn Glu Glu Val Lys Asn Met Ser Leu Glu
Leu Asn 110 115 120 tca aaa ctt gaa agc ctc cta gaa gaa aaa att cta
ctt caa caa aaa 435 Ser Lys Leu Glu Ser Leu Leu Glu Glu Lys Ile Leu
Leu Gln Gln Lys 125 130 135 gtg aaa tat tta gaa gag caa cta act aac
tta att caa aat caa cct 483 Val Lys Tyr Leu Glu Glu Gln Leu Thr Asn
Leu Ile Gln Asn Gln Pro 140 145 150 gaa act cca gaa cac cca gaa gta
act tca ctt aaa act ttt gta gaa 531 Glu Thr Pro Glu His Pro Glu Val
Thr Ser Leu Lys Thr Phe Val Glu 155 160 165 aaa caa gat aat agc atc
aaa gac ctt ctc cag acc gtg gaa gac caa 579 Lys Gln Asp Asn Ser Ile
Lys Asp Leu Leu Gln Thr Val Glu Asp Gln 170 175 180 185 tat aaa caa
tta aac caa cag cat agt caa ata aaa gaa ata gaa aat 627 Tyr Lys Gln
Leu Asn Gln Gln His Ser Gln Ile Lys Glu Ile Glu Asn 190 195 200 cag
ctc aga agg act agt att caa gaa ccc aca gaa att tct cta tct 675 Gln
Leu Arg Arg Thr Ser Ile Gln Glu Pro Thr Glu Ile Ser Leu Ser 205 210
215 tcc aag cca aga gca cca aga act act ccc ttt ctt cag ttg aat gaa
723 Ser Lys Pro Arg Ala Pro Arg Thr Thr Pro Phe Leu Gln Leu Asn Glu
220 225 230 ata aga aat gta aaa cat gat ggc att cct gct gaa tgt acc
acc att 771 Ile Arg Asn Val Lys His Asp Gly Ile Pro Ala Glu Cys Thr
Thr Ile 235 240 245 tat aac aga ggt gaa cat aca agt ggc atg tat gcc
atc aga ccc agc 819 Tyr Asn Arg Gly Glu His Thr Ser Gly Met Tyr Ala
Ile Arg Pro Ser 250 255 260 265 aac tct caa gtt ttt cat gtc tac tgt
gat gtt ata tca ggt agt cca 867 Asn Ser Gln Val Phe His Val Tyr Cys
Asp Val Ile Ser Gly Ser Pro 270 275 280 tgg aca tta att caa cat cga
ata gat gga tca caa aac ttc aat gaa 915 Trp Thr Leu Ile Gln His Arg
Ile Asp Gly Ser Gln Asn Phe Asn Glu 285 290 295 acg tgg gag aac tac
aaa tat ggt ttt ggg agg ctt gat gga gaa ttt 963 Thr Trp Glu Asn Tyr
Lys Tyr Gly Phe Gly Arg Leu Asp Gly Glu Phe 300 305 310 tgg ttg ggc
cta gag aag ata tac tcc ata gtg aag caa tct aat tat 1011 Trp Leu
Gly Leu Glu Lys Ile Tyr Ser Ile Val Lys Gln Ser Asn Tyr 315 320 325
gtt tta cga att gag ttg gaa gac tgg aaa gac aac aaa cat tat att
1059 Val Leu Arg Ile Glu Leu Glu Asp Trp Lys Asp Asn Lys His Tyr
Ile 330 335 340 345 gaa tat tct ttt tac ttg gga aat cac gaa acc aac
tat acg cta cat 1107 Glu Tyr Ser Phe Tyr Leu Gly Asn His Glu Thr
Asn Tyr Thr Leu His 350 355 360 cta gtt gcg att act ggc aat gtc ccc
aat gca atc ccg gaa aac aaa 1155 Leu Val Ala Ile Thr Gly Asn Val
Pro Asn Ala Ile Pro Glu Asn Lys 365 370 375 gat ttg gtg ttt tct act
tgg gat cac aaa gca aaa gga cac ttc aac 1203 Asp Leu Val Phe Ser
Thr Trp Asp His Lys Ala Lys Gly His Phe Asn 380 385 390 tgt cca gag
ggt tat tca gga ggc tgg tgg tgg cat gat gag tgt gga 1251 Cys Pro
Glu Gly Tyr Ser Gly Gly Trp Trp Trp His Asp Glu Cys Gly 395 400 405
gaa aac aac cta aat ggt aaa tat aac aaa cca aga gca aaa tct aag
1299 Glu Asn Asn Leu Asn Gly Lys Tyr Asn Lys Pro Arg Ala Lys Ser
Lys 410 415 420 425 cca gag agg aga aga gga tta tct tgg aag tct caa
aat gga agg tta 1347 Pro Glu Arg Arg Arg Gly Leu Ser Trp Lys Ser
Gln Asn Gly Arg Leu 430 435 440 tac tct ata aaa tca acc aaa atg ttg
atc cat cca aca gat tca gaa 1395 Tyr Ser Ile Lys Ser Thr Lys Met
Leu Ile His Pro Thr Asp Ser Glu 445 450 455 agc ttt gaa tga
actgaggcaa atttaaaagg caataattta aacattaacc 1447 Ser Phe Glu 460
tcattccaag ttaatgtggt ctaataatct ggtattaaat ccttaagag 1496
<210> SEQ ID NO 5 <211> LENGTH: 9381 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 5
ttccaaaaga actaaagtac agtttgagaa atgcatactt aattcattac ttttttcccc
60 tcaactttaa taataaattt acccaacaaa aaagtttatt tttgacttgt
aaatctctta 120 aaatcataaa aaagtaaaat tagcttttaa aaacaggtag
tcaccatagc attgaatgtg 180 tagtttataa tacagcaaag ttaaatacaa
tttcaaatta cctattaagt tagttgctca 240 tttctttgat ttcatttagc
attgatctaa ctcaatgtgg aagaaggtta cattcgtgca 300 agttaacacg
gcttaatgat taactatgtt cacctaccaa ccttaccttt tctgggcaaa 360
tattggtata tatagagtta agaagtctag gtctgcttcc agaagaaaac agttccacgt
420 tgcttgaaat tgaaaatcaa gataaaaatg ttcacaatta agctccttct
ttttattgtt 480 cctctagtta tttcctccag aattgatcaa gacaattcat
catttgattc tctatctcca 540 gagccaaaat caagatttgc tatgttagac
gatgtaaaaa ttttagccaa tggcctcctt 600 cagttgggac atggtcttaa
agactttgtc cataagacga agggccaaat taatgacata 660 tttcaaaaac
tcaacatatt tgatcagtct ttttatgatc tatcgctgca aaccagtgaa 720
atcaaagaag aagaaaagga actgagaaga actacatata aactacaagt caaaaatgaa
780 gaggtaaaga atatgtcact tgaactcaac tcaaaacttg aaagcctcct
agaagaaaaa 840 attctacttc aacaaaaagt gaaatattta gaagagcaac
taactaactt aattcaaaat 900 caacctgaaa ctccagaaca cccagaagta
acttcactta aagtaagtag aaaataaaga 960 gggttcatgt ttatgttttc
aatgtggatc ttttaaaaaa aatatttcta aggcatgcca 1020 tttgaaatac
tttgttgcat tgttgaaata cttttttttc caagaaaaat aatctccaga 1080
aaataaaatt tcctattata atttcaagtt agttttttgt ttccctaatg ttatatatga
1140 aaacactgaa aatttgcatt ttatatgaaa attacaaatc ggttaaatta
tacaatctag 1200 aacactatgt cattacacta ttgtaaatta ctgaaggtaa
gtaaaaagtt aaaaaaaatt 1260 taaaactatt ctccagtgtt taaaacagat
taaataatac agtaaatgga aaagatttat 1320 tcatatgaaa atatgctggg
ctttttcttt taattgaagt tcagaaaatc aaattttaga 1380 gatagtacaa
tttaaataaa atgttaagga caaaaatatg tgctatttga aagaagcata 1440
caaggggaag gaattgccaa tattcatttt tcaaatccat tattagttta aaaatttaga
1500 ttatgatagt gttacaggaa attaatagaa aagaaagagg aaagcaactt
ataaccaacc 1560 tactctctat atccagactt ttgtagaaaa acaagataat
agcatcaaag accttctcca 1620 gaccgtggaa gaccaatata aacaattaaa
ccaacagcat agtcaaataa aagaaataga 1680 aaatcaggta agtcagtatt
ttaatggtat gtcccatctt tcacacaggt ctgtaaaaac 1740 actgaatcct
aaaattattt acaagcttta actggatcat gagtaaaatt atcacatcag 1800
cataactgtt aaaattgcag gctctgaagc taataaacta cctgcattta aaccatggct
1860 ctaaaacttt gtgtgacctt gaataaatta cttcacccct ttatctctca
gtttcctcac 1920 atatactaca aagataataa cagaacttat aggattattg
taagaaaaaa aattaattca 1980 tagcagccaa tgtcatctta ctaaaattca
aattagatca tgtttctctt tgctcaaaac 2040 cacacaatag ctttccattt
cactcatatt ggctctttag accaagatta cccaaccctt 2100 cgtcatctca
ctgacttcac ctcctctact ctagttattc tgaccgcttt accagtattc 2160
aaacacatca aacatactgc cacctcaaag cctttgccct tgttgtttcc tctaactgga
2220 acgctcttct gccctggtat ctacgtggcc cactctctga tttcccttag
ggtcgttatc 2280 aaacaaaaaa ttcccaatga agacttacaa ggtcacttaa
ccaaaaatca caaccgcctg 2340 gtcccatccc tgaaaacttc tacttcctta
gctacttttc tcctgcacac tcacctttat 2400 ttaacataac ataaatttta
gttatttatc tcttctattc ctgcactaaa atgtaagctc 2460 tgtgaataca
gggatttttt ccattatctt catattttcc attatttgta tatactccag 2520
aatatagaat actgtatggc acacagtagg catttctgtt gaattaataa atgtaatgtc
2580 atattcacac agaagcgtgt gctatgatta ttattacttg gattactaga
aatagtgtgc 2640 ctcataatta aaggtcaaca ttcaacaatg taattaatct
acaatgtaaa catctggtga 2700 agtgacagag ggaagcactt gtttagaaaa
aagctatgtc agaatccatg tattctaata 2760 tgcagtacaa tagtttaaaa
atattaataa tactctcaaa cagctattca agaggattca 2820 aaaaacataa
tataaactca gagaaactgg taaacaaaat cattttcaag agatataaaa 2880
caaatattat taccaatttc cactaaacaa acataatgtt agtagtgctg ctaaaaggtt
2940 ttttatcaac tacttttggt ttccatactt tccttcttat gatgttatta
ttctaaattc 3000 ttttcaatta tatcttttac tatgattaaa tgaacctgct
ccccaaagca aaatgttact 3060 atagtaatat acattgtgtc taaaaataaa
aatgtgtgaa gaaaccaaaa caatgaattt 3120 ctgagttgga agaagagtta
gatcatttaa ctttctcata tttaaattaa aaaaacaaaa 3180 ctctaaaaat
ttaagtaact ttaagatcac atagttactt agtagaaaag agtaataccc 3240
agcaagcaaa ctttacaata gatcctttta aataaggtcc taggaaatat cattcatgcc
3300 agcatcaaaa aactaacact aataatgcaa gatattatat attctgcttt
tcttactgtc 3360 aatgagaaaa actatcattc aataaattgc aaacccaaca
cacttaaata aaaataaaat 3420 gttactgcta aactaacgat aaactactga
atatatagaa agtaagcaaa caaacttgcc 3480 aacctgccaa catctacaga
tatgtttaca ggtcaaaaat tatcaaatta tcaagaaagc 3540 ctggttcaaa
ttatgtatta tgtctttatc acaggtctga agatcagtaa gacctaaaac 3600
tgaaaattat taaacttaaa atctgaacag aatatcaaat atattttatt catataaata
3660 aaagaataca ttacaatatt ctaagcaaag cagtctctac ttttggcctt
gctctgtttt 3720 ccgaccaatg tctgcttttt tgccttgctt tattttttta
tcttattaaa taatgtccct 3780 gattaaatat tttgagaaca ggtaatctgt
acaatctgaa taacactgtt tatctaaata 3840 tcaaacaccg ttataacatt
atgaactgaa agacaaactg tacttctgac atccttactc 3900 agatttcccc
taattgtata ttcagtatca ttttaaaaaa cagatttata ttcttttatc 3960
agctcagaag gactagtatt caagaaccca cagaaatttc tctatcttcc aagccaagag
4020 caccaagaac tactcccttt cttcagttga atgaaataag aaatgtaaaa
catgatggta 4080 agacactttg gtgggtttcc ttcttgaagc tattattatc
aaattcccta ttcttaggac 4140 ttgttctaga ctaaaagata gttaagagat
atccatcaaa tacaatgtat caacctaaac 4200 tggatgctgg ggttcttttt
acaccctata aaagacatac ctaagacaat cagagaaata 4260 caaatatgga
cttgattatt agataatata gaaggtttat taattttctt agatgtgatc 4320
atggtattgc agttttaaag gagaacaatc tcctgtttaa gagatacatg ctgaaatatt
4380 tacggagtta aaggtcactg gactccagac tggtgataga acaagactct
gtctctaaaa 4440 aataattaat tttttaaaag aaaatagttt ggtaagatga
ttcttacatt cttaaataac 4500 acgccatcta agaaaaatgc tttaacataa
acattactga aaaaatgcta catttgccac 4560 aacttcataa aatgtcaagt
gaaatctcaa gctccaaaga tattattcct attactaaat 4620 ctgatgtaat
aacattttat tgattctagg cattcctgct gaatgtacca ccatttataa 4680
cagaggtgaa catacaagtg gcatgtatgc catcagaccc agcaactctc aagtttttca
4740 tgtctactgt gatgttatat caggtaaaac ctgtctaagg agaatagaca
gtagttagtt 4800 caacttactc attacgtatt aggaagatta acctggttat
cattgtttta tacatatata 4860 tatgaaatat atatgagtat tcgtataaat
ataatacttt taccttgttt atgtatttac 4920 tcaatattct ccttttcctc
taaaataatc tgaagtgact attatcaata agtttactat 4980 gccaaaattc
attaattgcc tttcacttaa cttttgggac cataataaat aataaaatgt 5040
attgccataa cattaataaa ctaccttaca aaaccaccaa ttaaaatcaa acaaacaaaa
5100 aagtgttatt tacatctgtc aacataaatc tactaaaaat acatgatttc
attcattata 5160 ttcaggtagt ccatggacat taattcaaca tcgaatagat
ggatcacaaa acttcaatga 5220 aacgtgggag aactacaaat atggttttgg
gaggcttgat ggtaagggga ctacattcaa 5280 tcattcattc acttgctaat
ctacaaatat ttactgagaa cctcttatgg accaggtatt 5340 aggaaaagta
gtaacgaacg agaagcagtc tcagccttca tataatttat tatcaaacaa 5400
ttacacattt gttagtaaat tacacttatt acaactgtta ttatttgaat tatatttatc
5460 acaattacat gtctgttctt aaatatactt atcacaattt aattccacgg
cttacaatga 5520 tcataactat aattattaaa gacaattttg attaaatgtt
atgtcataag tagtaactgt 5580 tacaaataag ctgtgaaaag aaccactcct
agcattagtc actctattct ctcattaacg 5640 ttttacatat caattaattg
gaagttaaaa ggaccaggaa actcagacat acagtataca 5700 ttttaaaatt
tcaattattt aaatataata tatagaatgt atggcttata atgaattagt 5760
taactcaatg caaattattc tattttgatt acaaatagta aaataagcaa gataaaataa
5820 cagatgttta aaatccaaaa agcacataca aaaatccatg aatgatgtct
aagtactcac 5880 ttataaagta gaagacattc attattatat caaattttta
aatgctcagt actatttgac 5940 catttaaaaa ttttgtattc aaactaccag
tgaaagccct acctagaagg tatactcagt 6000 gataagtttt gtagctccaa
atcttctaat agtgagtgta accccaaaat aaaaggctga 6060 caggtaagtc
gagaatactc acttaattct ggtaagaaag caacccattt gtacttgtat 6120
ttaccagcaa tccttaaaat gaagcttcct actaactcaa tagcaataag acaatagtga
6180 atgtttaatg aaaacagtat tttataaata ctttaataaa aaggattgtg
atgaagaaca 6240 atctatttat atttgttatt tgtttttaat tccaataaaa
ataattttta aaattacaga 6300 aaaaagttat taagaaccat gcttttaaat
ttaaaatgat tttttaaatt tattcctgtc 6360 tttttctaca aagaaagcat
acattaagca aataccaaag gccaggttta catttgaaga 6420 aagtgacatt
attattactc aagtctctag gaatacttaa cacatctctt gactgtatat 6480
ggatgttaat aaatagctga cagtaaagtt tatccatata aagacttgca aatattcctc
6540 taccaatgac gagactttaa aatatctata ataatgtaac acatttcact
ggtgaaacat 6600 gtcttgtcat atgcattata gaaaggataa tcagactttc
agttatatta atatttttaa 6660 catttttgtg cacatagcta tcttcaataa
aattgtttta aaaggtatta ttttaagata 6720 cactaaaatg atcaagggat
tcaagactaa acaactcaat tagttgcacc aataaaaaac 6780 acttaaaaaa
actgtcagtg tccaacctgt acttaataac tcacagattt ttaaaacttt 6840
tcttttcagg agaattttgg ttgggcctag agaagatata ctccatagtg aagcaatcta
6900 attatgtttt acgaattgag ttggaagact ggaaagacaa caaacattat
attgaatatt 6960 ctttttactt gggaaatcac gaaaccaact atacgctaca
tctagttgcg attactggca 7020 atgtccccaa tgcaatcccg gaaaacaaag
atttggtgtt ttctacttgg gatcacaaag 7080 caaaaggaca cttcaactgt
ccagagggtt attcaggtat ctttttctga taccaatact 7140 ttattttcat
atcttcaaag tatcttccca cattattagc tattatctgc aatgacaact 7200
tttaaaaatc cgaatcccaa ataagcgttt tctctctaga cgaaaacctc ttaactataa
7260 tgaaagtgtt cattctagtt caatcaggta ttttacctct aatcttcctc
agattttcta 7320 ttttttggta gtgtatagat tatttataca gattatttaa
aattgggact tatacagatt 7380 atttaaaact gggatacatg catctaaaac
actgtaatat ttataagaaa ggaagataaa 7440 cttacgggga aatacagtaa
cagtaactac atacgagtct gtacccatta aattgcatat 7500 ctatctcctt
taggaggctg gtggtggcat gatgagtgtg gagaaaacaa cctaaatggt 7560
aaatataaca aaccaagagc aaaatctaag ccagagagga gaagaggatt atcttggaag
7620 tctcaaaatg gaaggttata ctctataaaa tcaaccaaaa tgttgatcca
tccaacagat 7680 tcagaaagct ttgaatgaac tgaggcaaat ttaaaaggca
ataatttaaa cattaacctc 7740 attccaagtt aatgtggtct aataatctgg
tattaaatcc ttaagagaaa gcttgagaaa 7800 tagatttttt ttatcttaaa
gtcactgtct atttaagatt aaacatacaa tcacataacc 7860 ttaaagaata
ccgtttacat ttctcaatca aaattcttat aatactattt gttttaaatt 7920
ttgtgatgtg ggaatcaatt ttagatggtc acaatctaga ttataatcaa taggtgaact
7980 tattaaataa cttttctaaa taaaaaattt agagactttt attttaaaag
gcatcatatg 8040 agctaatatc acaactttcc cagtttaaaa aactagtact
cttgttaaaa ctctaaactt 8100 gactaaatac agaggactgg taattgtaca
gttcttaaat gttgtagtat taatttcaaa 8160 actaaaaatc gtcagcacag
agtatgtgta aaaatctgta atacaaattt ttaaactgat 8220 gcttcatttt
gctacaaaat aatttggagt aaatgtttga tatgatttat ttatgaaacc 8280
taatgaagca gaattaaata ctgtattaaa ataagttcgc tgtctttaaa caaatggaga
8340 tgactactaa gtcacattga ctttaacatg aggtatcact ataccttatt
tgttaaaata 8400 tatactgtat acattttata tattttaaca cttaatacta
tgaaaacaaa taattgtaaa 8460 ggaatcttgt cagattacag taagaatgaa
catatttgtg gcatcgagtt aaagtttata 8520 tttcccctaa atatgctgtg
attctaatac attcgtgtag gttttcaagt agaaataaac 8580 ctcgtaacaa
gttactgaac gtttaaacag cctgacaagc atgtatatat gtttaaaatt 8640
caataaacaa agacccagtc cctaaattat agaaatttaa attattcttg catgtttatc
8700 gacatcacaa cagatcccta aatccctaaa tccctaaaga ttagatacaa
attttttacc 8760 acagtatcac ttgtcagaat ttatttttaa atatgatttt
ttaaaactgc cagtaagaaa 8820 ttttaaatta aacccatttg ttaaaggata
tagtgcccaa gttatatggt gacctacctt 8880 tgtcaatact tagcattatg
tatttcaaat tatccaatat acatgtcata tatattttta 8940 tatgtcacat
atataaaaga tatgtatgat ctatgtgaat cctaagtaaa tattttgttc 9000
cagaaaagta caaaataata aaggtaaaaa taatctataa ttttcaggac cacagactaa
9060 gctgtcgaaa ttaacgctga tttttttagg gccagaatac caaaatggct
cctctcttcc 9120 cccaaaattg gacaatttca aatgcaaaat aattcattat
ttaatatatg agttgcttcc 9180 tctatttggt ttccttaaaa aaaaaaaaaa
ctctcatagg acatgtttca ttttgttcct 9240 ttcaggagta gtaaattaga
cgttttcccc atataaagct tttttctacc agaaagatac 9300 ttctggtaga
agaagagaaa ggagctcttt atggttcaca cgactgtctc ctgtcctaac 9360
tactttgctt aaagtgctca a 9381 <210> SEQ ID NO 6 <211>
LENGTH: 1449 <212> TYPE: DNA <213> ORGANISM: Mus
musculus <220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (19)..(1386) <400> SEQUENCE: 6 aataattgag acaaaaaa
atg cac aca att aaa tta ttc ctt ttt gtt gtt 51 Met His Thr Ile Lys
Leu Phe Leu Phe Val Val 1 5 10 cct tta gta att gca tcc aga gtg gat
cca gac ctt tca tca ttt gat 99 Pro Leu Val Ile Ala Ser Arg Val Asp
Pro Asp Leu Ser Ser Phe Asp 15 20 25 tct gca cct tca gag cca aaa
tca aga ttt gct atg ttg gat gat gtc 147 Ser Ala Pro Ser Glu Pro Lys
Ser Arg Phe Ala Met Leu Asp Asp Val 30 35 40 aaa att tta gcg aat
ggc ctc ctg cag ctg ggt cat gga ctt aaa gat 195 Lys Ile Leu Ala Asn
Gly Leu Leu Gln Leu Gly His Gly Leu Lys Asp 45 50 55 ttt gtc cat
aag act aag gga caa att aac gac ata ttt cag aag ctc 243 Phe Val His
Lys Thr Lys Gly Gln Ile Asn Asp Ile Phe Gln Lys Leu 60 65 70 75 aac
ata ttt gat cag tct ttt tat gac cta tca ctt cga acc aat gaa 291 Asn
Ile Phe Asp Gln Ser Phe Tyr Asp Leu Ser Leu Arg Thr Asn Glu 80 85
90 atc aaa gaa gag gaa aag gag cta aga aga act aca tct aca cta caa
339 Ile Lys Glu Glu Glu Lys Glu Leu Arg Arg Thr Thr Ser Thr Leu Gln
95 100 105 gtt aaa aac gag gag gtg aag aac atg tca gta gaa ctg aac
tca aag 387 Val Lys Asn Glu Glu Val Lys Asn Met Ser Val Glu Leu Asn
Ser Lys 110 115 120 ctt gag agt ctg ctg gaa gag aag aca gcc ctt caa
cac aag gtc agg 435 Leu Glu Ser Leu Leu Glu Glu Lys Thr Ala Leu Gln
His Lys Val Arg 125 130 135 gct ttg gag gag cag cta acc aac tta att
cta agc cca gct ggg gct 483 Ala Leu Glu Glu Gln Leu Thr Asn Leu Ile
Leu Ser Pro Ala Gly Ala 140 145 150 155 cag gag cac cca gaa gta aca
tca ctc aaa agt ttt gta gaa cag caa 531 Gln Glu His Pro Glu Val Thr
Ser Leu Lys Ser Phe Val Glu Gln Gln 160 165 170 gac aac agc ata aga
gaa ctc ctc cag agt gtg gaa gaa cag tat aaa 579 Asp Asn Ser Ile Arg
Glu Leu Leu Gln Ser Val Glu Glu Gln Tyr Lys 175 180 185 caa tta agt
caa cag cac atg cag ata aaa gaa ata gaa aag cag ctc 627 Gln Leu Ser
Gln Gln His Met Gln Ile Lys Glu Ile Glu Lys Gln Leu 190 195 200 aga
aag act ggt att caa gaa ccc tca gaa aat tct ctt tct tct aaa 675 Arg
Lys Thr Gly Ile Gln Glu Pro Ser Glu Asn Ser Leu Ser Ser Lys 205 210
215 tca aga gca cca aga act act ccc cct ctt caa ctg aac gaa aca gaa
723 Ser Arg Ala Pro Arg Thr Thr Pro Pro Leu Gln Leu Asn Glu Thr Glu
220 225 230 235 aat aca gaa caa gat gac ctt cct gcc gac tgc tct gcc
gtt tat aac 771 Asn Thr Glu Gln Asp Asp Leu Pro Ala Asp Cys Ser Ala
Val Tyr Asn 240 245 250 aga ggc gaa cat aca agt ggc gtg tac act att
aaa cca aga aac tcc 819 Arg Gly Glu His Thr Ser Gly Val Tyr Thr Ile
Lys Pro Arg Asn Ser 255 260 265 caa ggg ttt aat gtc tac tgt gat acc
caa tca ggc agt cca tgg aca 867 Gln Gly Phe Asn Val Tyr Cys Asp Thr
Gln Ser Gly Ser Pro Trp Thr 270 275 280 tta att caa cac cgg aaa gat
ggc tca cag gac ttc aac gaa aca tgg 915 Leu Ile Gln His Arg Lys Asp
Gly Ser Gln Asp Phe Asn Glu Thr Trp 285 290 295 gaa aac tac gaa aag
ggc ttt ggg agg ctc gat gga gaa ttt tgg ttg 963 Glu Asn Tyr Glu Lys
Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu 300 305 310 315 ggc cta
gag aag atc tat gct ata gtc caa cag tct aac tac att tta 1011 Gly
Leu Glu Lys Ile Tyr Ala Ile Val Gln Gln Ser Asn Tyr Ile Leu 320 325
330 cga ctc gag cta caa gac tgg aaa gac agc aag cac tac gtt gaa tac
1059 Arg Leu Glu Leu Gln Asp Trp Lys Asp Ser Lys His Tyr Val Glu
Tyr 335 340 345 tcc ttt cac ctg ggc agt cac gaa acc aac tac acg cta
cat gtg gct 1107 Ser Phe His Leu Gly Ser His Glu Thr Asn Tyr Thr
Leu His Val Ala 350 355 360 gag att gct ggc aat atc cct ggg gcc ctc
cca gag cac aca gac ctg 1155 Glu Ile Ala Gly Asn Ile Pro Gly Ala
Leu Pro Glu His Thr Asp Leu 365 370 375 atg ttt tct aca tgg aat cac
aga gca aag gga cag ctc tac tgt cca 1203 Met Phe Ser Thr Trp Asn
His Arg Ala Lys Gly Gln Leu Tyr Cys Pro 380 385 390 395 gaa agt tac
tca ggt ggc tgg tgg tgg aat gac ata tgt gga gaa aac 1251 Glu Ser
Tyr Ser Gly Gly Trp Trp Trp Asn Asp Ile Cys Gly Glu Asn 400 405 410
aac cta aat gga aaa tac aac aaa ccc aga acc aaa tcc aga cca gag
1299 Asn Leu Asn Gly Lys Tyr Asn Lys Pro Arg Thr Lys Ser Arg Pro
Glu 415 420 425 aga aga aga ggg atc tac tgg aga cct cag agc aga aag
ctc tat gct 1347 Arg Arg Arg Gly Ile Tyr Trp Arg Pro Gln Ser Arg
Lys Leu Tyr Ala 430 435 440 atc aaa tca tcc aaa atg atg ctc cag ccc
acc acc taa gaagcttcaa 1396 Ile Lys Ser Ser Lys Met Met Leu Gln Pro
Thr Thr 445 450 455 ctgaactgag acaaaataaa agatcaataa attaaatatt
aaagtcctcc cga 1449 <210> SEQ ID NO 7 <211> LENGTH: 510
<212> TYPE: DNA <213> ORGANISM: Mus musculus
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (67)..(510) <400> SEQUENCE: 7 tgttggccta ctggacagga
gggagaagtt ccaaattgct taaaattgaa taattgagac 60 aaaaaa tgc aca caa
tta aat tat tcc ttt ttg ttg ttc ctt tag taa 108 Cys Thr Gln Leu Asn
Tyr Ser Phe Leu Leu Phe Leu 1 5 10 ttg cat cca gag tgg atc cag acc
ttt cat cat ttg att ctg cac ctt 156 Leu His Pro Glu Trp Ile Gln Thr
Phe His His Leu Ile Leu His Leu 15 20 25 cag agc caa aat caa gat
ttg cta tgt tgg atg atg tca aaa ttt tag 204 Gln Ser Gln Asn Gln Asp
Leu Leu Cys Trp Met Met Ser Lys Phe 30 35 40 cga atg gcc tcc tgc
agc tgg gtc atg gac tta aag att ttg tcc ata 252 Arg Met Ala Ser Cys
Ser Trp Val Met Asp Leu Lys Ile Leu Ser Ile 45 50 55 aga cta agg
gac aaa tta acg aca tat ttc aga agc tca aca tat ttg 300 Arg Leu Arg
Asp Lys Leu Thr Thr Tyr Phe Arg Ser Ser Thr Tyr Leu 60 65 70 75 atc
agt ctt ttt atg acc tat cac ttc gaa cca atg aaa tca aag aag 348 Ile
Ser Leu Phe Met Thr Tyr His Phe Glu Pro Met Lys Ser Lys Lys 80 85
90 agg aaa agg agc taa gaa gaa cta cat cta cac tac aag tta aaa acg
396 Arg Lys Arg Ser Glu Glu Leu His Leu His Tyr Lys Leu Lys Thr 95
100 105 agg agg tga aga aca ctt cag tag aac tga act caa agc ttg aga
gtc 444 Arg Arg Arg Thr Leu Gln Asn Thr Gln Ser Leu Arg Val 110 115
tgc tgg aag aga aga cag ccc ttc aac aca agg tca ggg ctt tgg agg 492
Cys Trp Lys Arg Arg Gln Pro Phe Asn Thr Arg Ser Gly Leu Trp Arg 120
125 130 135 agc agc taa cca act taa 510 Ser Ser Pro Thr <210>
SEQ ID NO 8 <211> LENGTH: 271 <212> TYPE: DNA
<213> ORGANISM: Mus musculus <400> SEQUENCE: 8
gagggatcta ctggagacct cagagcagaa agctctatgc tatcaaatca tccaaattga
60 tgctccaccc caccacctaa gaagcttcaa ctgaactgag acaaaataaa
agatcaataa 120 attaaatatt aaattcctcc cgatcactgt agtaatctgg
tattaaaatt ttaatggaaa 180 gcttgagaat tgaatttcaa ttaggtttaa
actcattgtt aagatcagat atcaccgaat 240 caacgtaaac aaaatttatc
tttttcaatc t 271 <210> SEQ ID NO 9 <400> SEQUENCE: 9
000 <210> SEQ ID NO 10 <400> SEQUENCE: 10 000
<210> SEQ ID NO 11 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 11 ctcttactgt gctgtggaca 20 <210> SEQ ID NO 12
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer <400> SEQUENCE: 12 tgcagggcta catggaacaa
20 <210> SEQ ID NO 13 <211> LENGTH: 20 <212>
TYPE: DNA <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Primer <400>
SEQUENCE: 13 cggactcctg cacgctactt 20 <210> SEQ ID NO 14
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Probe <400> SEQUENCE: 14 ctccaagacg gtccaggatg
cgc 23 <210> SEQ ID NO 15 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Primer <400>
SEQUENCE: 15 caacggattt ggtcgtattg g 21 <210> SEQ ID NO 16
<211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer <400> SEQUENCE: 16 ggcaacaata tccactttac
cagagt 26 <210> SEQ ID NO 17 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 17 cgcctggtca ccagggctgc t 21 <210> SEQ
ID NO 18 <211> LENGTH: 19 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 18 gaaggtgaag
gtcggagtc 19 <210> SEQ ID NO 19 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 19 gaagatggtg atgggatttc 20 <210> SEQ
ID NO 20 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Probe <400> SEQUENCE: 20 caagcttccc
gttctcagcc 20 <210> SEQ ID NO 21 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 21 tggaatcata ttggaacatg 20 <210> SEQ
ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 22 ggcaaattca
acggcacagt 20 <210> SEQ ID NO 23 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 23 gggtctcgct cctggaagat 20 <210> SEQ
ID NO 24 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Probe <400> SEQUENCE: 24 aaggccgaga
atgggaagct tgtcatc 27 <210> SEQ ID NO 25 <211> LENGTH:
23 <212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 25 tgttctagag acagccgcat ctt 23 <210>
SEQ ID NO 26 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 26
caccgacctt caccatcttg t 21 <210> SEQ ID NO 27 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 27 ttgtgcagtg ccagcctcgt ctca 24 <210>
SEQ ID NO 28 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 28
cttcaatgaa acgtgggaga act 23 <210> SEQ ID NO 29 <211>
LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 29 tctctaggcc caaccaaaat tc 22 <210>
SEQ ID NO 30 <211> LENGTH: 24 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Probe <400> SEQUENCE: 30
aaatatggtt ttgggaggct tgat 24 <210> SEQ ID NO 31 <211>
LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 31 cagaagtaac atcactcaaa agttttgtag 30
<210> SEQ ID NO 32 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 32
gacttaattg tttatactgt tcttccacac t 31 <210> SEQ ID NO 33
<211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Probe <400> SEQUENCE: 33 cagcaagaca acagcataag
agaactcctc ca 32 <210> SEQ ID NO 34 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 34 atctgttgtg atgtcgataa 20
<210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 35 gtatttagtc aagtttagag 20 <210> SEQ ID NO 36
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 36
tattacagat ttttacacat 20 <210> SEQ ID NO 37 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 37 cgtggaactg
ttttcttctg 20 <210> SEQ ID NO 38 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 38 agcttaattg tgaacatttt 20
<210> SEQ ID NO 39 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 39 attctggagg aaataactag 20 <210> SEQ ID NO 40
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 40
aaatcttgat tttggctctg 20 <210> SEQ ID NO 41 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 41 atagcaaatc
ttgattttgg 20 <210> SEQ ID NO 42 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 42 ctaacatagc aaatcttgat 20
<210> SEQ ID NO 43 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 43 atcgtctaac atagcaaatc 20 <210> SEQ ID NO 44
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 44
aggccattgg ctaaaatttt 20 <210> SEQ ID NO 45 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 45 catgtcccaa
ctgaaggagg 20 <210> SEQ ID NO 46 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 46 ctttaagacc atgtcccaac 20
<210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 47 gcccttcgtc ttatggacaa 20 <210> SEQ ID NO 48
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 48
tcattaattt ggcccttcgt 20 <210> SEQ ID NO 49 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 49 tgaaatatgt
cattaatttg 20 <210> SEQ ID NO 50 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 50 gactgatcaa atatgttgag 20
<210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 51 gtttgcagcg atagatcata 20 <210> SEQ ID NO 52
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 52
tcactggttt gcagcgatag 20 <210> SEQ ID NO 53 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 53 agttcaagtg
acatattctt 20 <210> SEQ ID NO 54 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 54 agtgaagtta cttctgggtg 20
<210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 55 gttttaagtg aagttacttc 20 <210> SEQ ID NO 56
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 56
ctacaaaagt tttaagtgaa 20 <210> SEQ ID NO 57 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 57 ggtcttccac
ggtctggaga 20 <210> SEQ ID NO 58 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 58 tagtccttct gagctgattt 20
<210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 59 ggttcttgaa tactagtcct 20 <210> SEQ ID NO 60
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 60
aaatttctgt gggttcttga 20 <210> SEQ ID NO 61 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 61 tggcttggaa
gatagagaaa 20 <210> SEQ ID NO 62 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 62 agtagttctt ggtgctcttg 20
<210> SEQ ID NO 63 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 63 tgaagaaagg gagtagttct 20 <210> SEQ ID NO 64
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 64
attcaactga agaaagggag 20 <210> SEQ ID NO 65 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 65 tcttatttca
ttcaactgaa 20 <210> SEQ ID NO 66 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 66 aggaatgcca tcatgtttta 20
<210> SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 67 ctctgttata aatggtggta 20 <210> SEQ ID NO 68
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 68
ttcacctctg ttataaatgg 20 <210> SEQ ID NO 69 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 69 gtatgttcac
ctctgttata 20 <210> SEQ ID NO 70 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 70 cacttgtatg ttcacctctg 20
<210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 71 catgccactt gtatgttcac 20 <210> SEQ ID NO 72
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 72
agagttgctg ggtctgatgg 20 <210> SEQ ID NO 73 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 73 ctgatataac
atcacagtag 20 <210> SEQ ID NO 74 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 74 catggactac ctgatataac 20
<210> SEQ ID NO 75 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 75 tgaattaatg tccatggact 20 <210> SEQ ID NO 76
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 76
tctattcgat gttgaattaa 20 <210> SEQ ID NO 77 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 77 agttctccca
cgtttcattg 20 <210> SEQ ID NO 78 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 78 catatttgta gttctcccac 20
<210> SEQ ID NO 79 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 79 aaaaccatat ttgtagttct 20 <210> SEQ ID NO 80
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 80
gcctcccaaa accatatttg 20 <210> SEQ ID NO 81 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 81 gcccaaccaa
aattctccat 20 <210> SEQ ID NO 82 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 82 ctctaggccc aaccaaaatt 20
<210> SEQ ID NO 83 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 83 atcttctcta ggcccaacca 20 <210> SEQ ID NO 84
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 84
aacataatta gattgcttca 20 <210> SEQ ID NO 85 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 85 gtcttccaac
tcaattcgta 20 <210> SEQ ID NO 86 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 86 ctttccagtc ttccaactca 20
<210> SEQ ID NO 87 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 87 aactagatgt agcgtatagt 20 <210> SEQ ID NO 88
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 88
tgatcccaag tagaaaacac 20 <210> SEQ ID NO 89 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 89 caccagcctc
ctgaataacc 20 <210> SEQ ID NO 90 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 90 ttaggttgtt ttctccacac 20
<210> SEQ ID NO 91 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 91 taatcctctt ctcctctctg 20 <210> SEQ ID NO 92
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 92
tgagacttcc aagataatcc 20 <210> SEQ ID NO 93 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 93 cattttgaga
cttccaagat 20 <210> SEQ ID NO 94 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 94 gagtataacc ttccattttg 20
<210> SEQ ID NO 95 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 95 tggatggatc aacattttgg 20 <210> SEQ ID NO 96
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 96
ttcaaagctt tctgaatctg 20 <210> SEQ ID NO 97 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 97 tgcctcagtt
cattcaaagc 20 <210> SEQ ID NO 98 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 98 tgccttttaa atttgcctca 20
<210> SEQ ID NO 99 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 99 attaacttgg aatgaggtta 20 <210> SEQ ID NO 100
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 100
agaccacatt aacttggaat 20 <210> SEQ ID NO 101 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 101 agattattag
accacattaa 20 <210> SEQ ID NO 102 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 102 ataccagatt attagaccac 20
<210> SEQ ID NO 103 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 103 ggatttaata ccagattatt 20 <210> SEQ ID NO 104
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 104
actgacttac ctgattttct 20 <210> SEQ ID NO 105 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 105 accttgtaag
tcttcattgg 20 <210> SEQ ID NO 106 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 106 cagtgttatt cagattgtac 20
<210> SEQ ID NO 107 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 107 agtgtcttac catcatgttt 20 <210> SEQ ID NO 108
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 108
acagatgtaa ataacacttt 20 <210> SEQ ID NO 109 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 109 gtccccttac
catcaagcct 20 <210> SEQ ID NO 110 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 110 gggaagatac tttgaagata 20
<210> SEQ ID NO 111 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 111 caccagcctc ctaaaggaga 20 <210> SEQ ID NO 112
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 112
aataatttaa ttgtgtgcat 20 <210> SEQ ID NO 113 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 113 cactctggat
gcaattacta 20 <210> SEQ ID NO 114 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 114 aaggtctgga tccactctgg 20
<210> SEQ ID NO 115 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 115 tttggctctg aaggtgcaga 20 <210> SEQ ID NO 116
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 116
tcatccaaca tagcaaatct 20 <210> SEQ ID NO 117 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 117 tgacatcatc
caacatagca 20 <210> SEQ ID NO 118 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 118 aattttgaca tcatccaaca 20
<210> SEQ ID NO 119 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 119 gctaaaattt tgacatcatc 20 <210> SEQ ID NO 120
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 120
aggccattcg ctaaaatttt 20 <210> SEQ ID NO 121 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 121 cccagctgca
ggaggccatt 20 <210> SEQ ID NO 122 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 122 catgacccag ctgcaggagg 20
<210> SEQ ID NO 123 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 123 ttaagtccat gacccagctg 20 <210> SEQ ID NO 124
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 124
gacaaaatct ttaagtccat 20 <210> SEQ ID NO 125 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 125 ttatggacaa
aatctttaag 20 <210> SEQ ID NO 126 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 126 ttgagcttct gaaatatgtc 20
<210> SEQ ID NO 127 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 127 atatgttgag cttctgaaat 20 <210> SEQ ID NO 128
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 128
atcaaatatg ttgagcttct 20 <210> SEQ ID NO 129 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 129 ggttcgaagt
gataggtcat 20 <210> SEQ ID NO 130 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 130 tagtgtagat gtagttcttc 20
<210> SEQ ID NO 131 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 131 gacatgttct tcacctcctc 20 <210> SEQ ID NO 132
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 132
tgagttcagt tctactgaca 20 <210> SEQ ID NO 133 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 133 tcaagctttg
agttcagttc 20 <210> SEQ ID NO 134 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 134 gtcttctctt ccagcagact 20
<210> SEQ ID NO 135 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 135 gttgaagggc tgtcttctct 20 <210> SEQ ID NO 136
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 136
ctgaccttgt gttgaagggc 20 <210> SEQ ID NO 137 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 137 ccaaagccct
gaccttgtgt 20 <210> SEQ ID NO 138 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 138 ttagctgctc ctccaaagcc 20
<210> SEQ ID NO 139 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 139 cttagaatta agttggttag 20 <210> SEQ ID NO 140
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 140
ggtgctcctg agccccagct 20 <210> SEQ ID NO 141 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 141 tgatgttact
tctgggtgct 20 <210> SEQ ID NO 142 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 142 tgctgttcta caaaactttt 20
<210> SEQ ID NO 143 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 143 aggagttctc ttatgctgtt 20 <210> SEQ ID NO 144
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 144
tctggaggag ttctcttatg 20 <210> SEQ ID NO 145 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 145 cacactctgg
aggagttctc 20 <210> SEQ ID NO 146 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 146 gggttcttga ataccagtct 20
<210> SEQ ID NO 147 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 147 gaaagagaat tttctgaggg 20 <210> SEQ ID NO 148
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 148
gcaggaaggt catcttgttc 20 <210> SEQ ID NO 149 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 149 gagcagtcgg
caggaaggtc 20 <210> SEQ ID NO 150 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 150 tacacgccac ttgtatgttc 20
<210> SEQ ID NO 151 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 151 ttaatagtgt acacgccact 20 <210> SEQ ID NO 152
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 152
agacattaaa cccttgggag 20 <210> SEQ ID NO 153 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 153 ctgcctgatt
gggtatcaca 20 <210> SEQ ID NO 154 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 154 ccatctttcc ggtgttgaat 20
<210> SEQ ID NO 155 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 155 gaagtcctgt gagccatctt 20 <210> SEQ ID NO 156
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 156
ttctccatcg agcctcccaa 20 <210> SEQ ID NO 157 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 157 gttggactat
agcatagatc 20 <210> SEQ ID NO 158 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 158 atgtagttag actgttggac 20
<210> SEQ ID NO 159 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 159 acgtagtgct tgctgtcttt 20 <210> SEQ ID NO 160
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 160
gcccaggtga aaggagtatt 20 <210> SEQ ID NO 161 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 161 tgtagcgtgt
agttggtttc 20 <210> SEQ ID NO 162 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 162 ccacatgtag cgtgtagttg 20
<210> SEQ ID NO 163 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 163 ctcagccaca tgtagcgtgt 20 <210> SEQ ID NO 164
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 164
gcaatctcag ccacatgtag 20 <210> SEQ ID NO 165 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 165 atcaggtctg
tgtgctctgg 20 <210> SEQ ID NO 166 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 166 atgtagaaaa catcaggtct 20
<210> SEQ ID NO 167 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 167 tctgtgattc catgtagaaa 20 <210> SEQ ID NO 168
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 168
tctggacagt agagctgtcc 20 <210> SEQ ID NO 169 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 169 aactttctgg
acagtagagc 20 <210> SEQ ID NO 170 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 170 accaccagcc acctgagtaa 20
<210> SEQ ID NO 171 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 171 ttctccacat atgtcattcc 20 <210> SEQ ID NO 172
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 172
ggttgttttc tccacatatg 20 <210> SEQ ID NO 173 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 173 tggtctggat
ttggttctgg 20 <210> SEQ ID NO 174 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 174 tctctctggt ctggatttgg 20
<210> SEQ ID NO 175 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 175 tagagctttc tgctctgagg 20 <210> SEQ ID NO 176
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 176
gtggtgggct ggagcatcat 20 <210> SEQ ID NO 177 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 177 tgaagcttct
taggtggtgg 20 <210> SEQ ID NO 178 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 178 tgtctcagtt cagttgaagc 20
<210> SEQ ID NO 179 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 179 tcgggaggac tttaatattt 20 <210> SEQ ID NO 180
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 180
ggaacttctc cctcctgtcc 20 <210> SEQ ID NO 181 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 181 taacaatgag
tttaaaccta 20 <210> SEQ ID NO 182 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 182 tctgatctta acaatgagtt 20
<210> SEQ ID NO 183 <211> LENGTH: 19 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 183 cgagaggcgg acgggaccg 19 <210> SEQ ID NO 184
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 184
cgagaggcgg acgggaccgt t 21 <210> SEQ ID NO 185 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 185 ttgctctccg
cctgccctgg c 21 <210> SEQ ID NO 186 <211> LENGTH: 19
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 186 gctctccgcc tgccctggc 19
<210> SEQ ID NO 187 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 187 ccttccctga aggttcctcc 20 <210> SEQ ID NO 188
<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer <400> SEQUENCE: 188 cacctgggca gtcacgaaa
19 <210> SEQ ID NO 189 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Primer <400>
SEQUENCE: 189 ggagggcccc agggatat 18 <210> SEQ ID NO 190
<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Probe <400> SEQUENCE: 190 cacgctacat gtggctgaga
ttcgtgg 27 <210> SEQ ID NO 191 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 191 gtgacatatt cttcacctct 20
<210> SEQ ID NO 192 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 192 tttaagtgac gttacctctg 20 <210> SEQ ID NO 193
<211> LENGTH: 1272 <212> TYPE: DNA <213>
ORGANISM: Rattus norvegicus <220> FEATURE: <221>
NAME/KEY: CDS <222> LOCATION: (1)..(1272) <400>
SEQUENCE: 193 atg cac aca att aag ctg ctc ctt ttt gtt gtt cct cta
gta att tcg 48 Met His Thr Ile Lys Leu Leu Leu Phe Val Val Pro Leu
Val Ile Ser 1 5 10 15 tcc aga gtt gat cca gac ctt tcg cca ttt gat
tct gta ccg tca gag 96 Ser Arg Val Asp Pro Asp Leu Ser Pro Phe Asp
Ser Val Pro Ser Glu 20 25 30 cca aaa tca aga ttt gct atg ttg gat
gat gtc aaa att tta gcc aat 144 Pro Lys Ser Arg Phe Ala Met Leu Asp
Asp Val Lys Ile Leu Ala Asn 35 40 45 ggc ctc ctg cag ctg ggt cat
ggt ctt aaa gat ttt gtc cat aag aca 192 Gly Leu Leu Gln Leu Gly His
Gly Leu Lys Asp Phe Val His Lys Thr 50 55 60 aag gga caa att aat
gac ata ttt cag aag ctc aac ata ttt gat cag 240 Lys Gly Gln Ile Asn
Asp Ile Phe Gln Lys Leu Asn Ile Phe Asp Gln 65 70 75 80 tgt ttt tat
gac cta tca ctt caa acc aat gaa atc aaa gaa gag gaa 288 Cys Phe Tyr
Asp Leu Ser Leu Gln Thr Asn Glu Ile Lys Glu Glu Glu 85 90 95 aag
gag cta aga aga acc aca tct aaa cta caa gtt aaa aac gaa gag 336 Lys
Glu Leu Arg Arg Thr Thr Ser Lys Leu Gln Val Lys Asn Glu Glu 100 105
110 gtg aag aat atg tca ctt gaa ctg aac tca aag ctt gaa agt cta ctg
384 Val Lys Asn Met Ser Leu Glu Leu Asn Ser Lys Leu Glu Ser Leu Leu
115 120 125 gag gag aag atg gcg ctc caa cac aga gtc agg gct ttg gag
gaa cag 432 Glu Glu Lys Met Ala Leu Gln His Arg Val Arg Ala Leu Glu
Glu Gln 130 135 140 ctg acc agc ttg gtt cag aac ccg cct ggg gct cgg
gag cac cca gag 480 Leu Thr Ser Leu Val Gln Asn Pro Pro Gly Ala Arg
Glu His Pro Glu 145 150 155 160 gta acg tca ctt aaa agt ttt gta gaa
cag caa gat aac agc ata aga 528 Val Thr Ser Leu Lys Ser Phe Val Glu
Gln Gln Asp Asn Ser Ile Arg 165 170 175 gaa ctc ctc cag agt gtg gaa
gaa caa tat aaa caa cta agt caa cag 576 Glu Leu Leu Gln Ser Val Glu
Glu Gln Tyr Lys Gln Leu Ser Gln Gln 180 185 190 cac att cag ata aaa
gaa ata gaa aat cag ctc aga aag act ggc att 624 His Ile Gln Ile Lys
Glu Ile Glu Asn Gln Leu Arg Lys Thr Gly Ile 195 200 205 caa gaa ccc
act gaa aat tct ctt tat tct aaa cca aga gca cca aga 672 Gln Glu Pro
Thr Glu Asn Ser Leu Tyr Ser Lys Pro Arg Ala Pro Arg 210 215 220 act
act ccc cct ctt cat ctg aag gaa gca aaa aat ata gaa caa gat 720 Thr
Thr Pro Pro Leu His Leu Lys Glu Ala Lys Asn Ile Glu Gln Asp 225 230
235 240 gat ctg cct gct gac tgc tct gcc att tat aac aga ggt gaa cat
aca 768 Asp Leu Pro Ala Asp Cys Ser Ala Ile Tyr Asn Arg Gly Glu His
Thr 245 250 255 agt ggc gtg tat act att aga cca agc agc tct caa gtg
ttt aat gtc 816 Ser Gly Val Tyr Thr Ile Arg Pro Ser Ser Ser Gln Val
Phe Asn Val 260 265 270 tac tgt gac acc caa tca gga gaa ttc tgg ttg
ggc cta gag aag atc 864 Tyr Cys Asp Thr Gln Ser Gly Glu Phe Trp Leu
Gly Leu Glu Lys Ile 275 280 285 tac gct ata gtc aaa cag tct aac tac
atc tta cga ctg gag cta caa 912 Tyr Ala Ile Val Lys Gln Ser Asn Tyr
Ile Leu Arg Leu Glu Leu Gln 290 295 300 gac tgg aag gac agc aag cac
tat gct gaa tat tcc ttt cat ctg ggc 960 Asp Trp Lys Asp Ser Lys His
Tyr Ala Glu Tyr Ser Phe His Leu Gly 305 310 315 320 aat cat gaa acc
aac tac acg cta cat gtg gct gag att gct gcc aat 1008 Asn His Glu
Thr Asn Tyr Thr Leu His Val Ala Glu Ile Ala Ala Asn 325 330 335 atc
cct gag gcc cta cca gaa cac aga gac ctg atg ttt tct aca tgg 1056
Ile Pro Glu Ala Leu Pro Glu His Arg Asp Leu Met Phe Ser Thr Trp 340
345 350 gat cac aga gca aag gga cag ctc tac tgt cca gaa agt tat tca
ggt 1104 Asp His Arg Ala Lys Gly Gln Leu Tyr Cys Pro Glu Ser Tyr
Ser Gly 355 360 365 ggc tgg tgg ttc agt gac atg tgt gga gaa aac aac
cta aat ggt aaa 1152 Gly Trp Trp Phe Ser Asp Met Cys Gly Glu Asn
Asn Leu Asn Gly Lys 370 375 380 tac aac aaa ccc aga gcc aaa tcc aaa
cca gag cgg aga aga ggg atc 1200 Tyr Asn Lys Pro Arg Ala Lys Ser
Lys Pro Glu Arg Arg Arg Gly Ile 385 390 395 400 tcc tgg agg cct cgg
ggc gga aag ctc tac tct atc aaa tca tct aaa 1248 Ser Trp Arg Pro
Arg Gly Gly Lys Leu Tyr Ser Ile Lys Ser Ser Lys 405 410 415 atg atg
ctc cag ccg acc acc taa 1272 Met Met Leu Gln Pro Thr Thr 420
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 193
<210> SEQ ID NO 1 <211> LENGTH: 833 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1
attaatttca aaactaaaaa tcgtcagcac agagtatgtg taaaaatctg taatacaaat
60 ttttaaactg atgcttcatt ttgctacaaa ataatttgga gtaaatgttt
gatatgattt 120 atttatgaaa cctaatgaag cagaattaaa tactgtatta
aaataagttc gctgtcttta 180 aacaaatgga gatgactact aagtcacatt
gactttaaca tgaggtatca ctatacctta 240 tttgttaaaa tatatactgt
atacatttta tatattttaa cacttaatac tatgaaaaca 300 aataattgta
aaggaatctt gtcagattac agtaagaatg aacatatttg tggcatcgag 360
ttaaagttta tatttcccct aaatatgctg tgattctaat acattcgtgt aggttttcaa
420 gtagaaataa acctcgtaac aagttactga acgtttaaac agcctgacaa
gcatgtatat 480 atgtttaaaa ttcaataaac aaagacccag tccctaaatt
atagaaattt aaattattct 540 tgcatgttta tcgacatcac aacagatccc
taaatcccta aatccctaaa gattagatac 600 aaatttttta ccacagtatc
acttgtcaga atttattttt aacatatgat tttttaaaac 660 tgccagtaag
aaattttaaa tttaacccat ttgttaagga tatagtgccc aagttaattg 720
gtgacctacc tttgtcaata cttagcataa tgtatttcaa aattatccaa tatacctgtc
780 atattaatct attatgtcca cttttttaag atatgtatga cctatgtgaa tct 833
<210> SEQ ID NO 2 <211> LENGTH: 795 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2
ggtctaataa tctggtatta aatccttaag agaaagcttg agaaatagat tttttttatc
60 ttaaagtcac tgtctattta agattaaaca tacaatcaca taaccttaaa
gaataccgtt 120 tacatttctc aatcaaaatt cttataatac tatttgtttt
aaattttgtg atgtgggaat 180 caattttaga tggtcacaat ctagattata
atcaataggt gaacttatta aataactttt 240 ctaaataaaa aatttagaga
cttttatttt aaaaggcatc atatgagcta atatcacaac 300 tttcccagtt
taaaaaacta gtactcttgt taaaactcta aacttgacta aatacagagg 360
actggtaatt gtacagttct taaatgttgt agtattaatt tcaaaactaa aaatcgtcag
420 cacagagtat gtgtaaaaat ctgtaataca aatttttaaa ctgatgcttc
attttgctac 480 aaaataattt ggagtaaatg tttgatatga tttatttatg
aaacctaatg aagcagaatt 540 aaatactgta ttaaaataag ttcgctgtct
ttaaacaaat ggagatgact actaagtcac 600 attgacttta acatgaggta
tcactatacc ttattgttaa acatatatac tgtatacatt 660 ttatatattt
taacacttaa tactatgaaa acaaataatt gtaaggaatc ttgtcagatt 720
acagtaagaa tgaacatatt tgtggcatcg agttaaagtt tatatttccc ctaaaatatg
780 cttgtgattc taata 795 <210> SEQ ID NO 3 <211>
LENGTH: 743 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 3 atatatagag ttaagaagtc taggtctgct
tccagaagaa aacagttcca cgttgcttga 60 aattgaaaat caagataaaa
atgttcacaa ttaagctcct tctttttatt gttcctctag 120 ttatttcctc
cagaattgat caagacaatt catcatttga ttctctatct ccagagccaa 180
aatcaagatt tgctatgtta gacgatgtaa aaattttagc caatggcctc cttcagttgg
240 gacatggtct taaagacttt gtccataaga cgaaggggcc aaatttattg
gacatatttc 300 aaaaactcaa catatttgat cagtcttttt atgatctatc
gctgcaaacc agtgaaatca 360 aagaagaaga aaaggaactg agaagaacta
catataaact acaagtcaaa aatgaagagg 420 taaagaatat gtcacttgaa
ctcaactcaa aacttgaaag cctcctagaa gaaaaaattc 480 tacttcaaca
aaaagtgaaa tatttagaag agcaactaac taacttaatt caacaatcaa 540
cctgaaactc cagaacaccc agaagtaact tcacttaaaa ctttgtagaa aaacaagata
600 atagcatcaa agaccttctc cagaccgtgg aagaccaata taaacaatta
aaccaacagc 660 aaaaaacaaa caatcacaca cacacacaca ccaacacaac
tctttttggc gccccattgc 720 cctccaaaaa aatttttaaa cac 743 <210>
SEQ ID NO 4 <211> LENGTH: 1496 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: CDS <222> LOCATION: (25)..(1407) <400>
SEQUENCE: 4 ggcacgagga aaatcaagat aaaa atg ttc aca att aag ctc ctt
ctt ttt 51 Met Phe Thr Ile Lys Leu Leu Leu Phe 1 5 att gtt cct cta
gtt att tcc tcc aga att gat caa gac aat tca tca 99 Ile Val Pro Leu
Val Ile Ser Ser Arg Ile Asp Gln Asp Asn Ser Ser 10 15 20 25 ttt gat
tct cta tct cca gag cca aaa tca aga ttt gct atg tta gac 147 Phe Asp
Ser Leu Ser Pro Glu Pro Lys Ser Arg Phe Ala Met Leu Asp 30 35 40
gat gta aaa att tta gcc aat ggc ctc ctt cag ttg gga cat ggt ctt 195
Asp Val Lys Ile Leu Ala Asn Gly Leu Leu Gln Leu Gly His Gly Leu 45
50 55 aaa gac ttt gtc cat aag acg aag ggc caa att aat gac ata ttt
caa 243 Lys Asp Phe Val His Lys Thr Lys Gly Gln Ile Asn Asp Ile Phe
Gln 60 65 70 aaa ctc aac ata ttt gat cag tct ttt tat gat cta tcg
ctg caa acc 291 Lys Leu Asn Ile Phe Asp Gln Ser Phe Tyr Asp Leu Ser
Leu Gln Thr 75 80 85 agt gaa atc aaa gaa gaa gaa aag gaa ctg aga
aga act aca tat aaa 339 Ser Glu Ile Lys Glu Glu Glu Lys Glu Leu Arg
Arg Thr Thr Tyr Lys 90 95 100 105 cta caa gtc aaa aat gaa gag gta
aag aat atg tca ctt gaa ctc aac 387 Leu Gln Val Lys Asn Glu Glu Val
Lys Asn Met Ser Leu Glu Leu Asn 110 115 120 tca aaa ctt gaa agc ctc
cta gaa gaa aaa att cta ctt caa caa aaa 435 Ser Lys Leu Glu Ser Leu
Leu Glu Glu Lys Ile Leu Leu Gln Gln Lys 125 130 135 gtg aaa tat tta
gaa gag caa cta act aac tta att caa aat caa cct 483 Val Lys Tyr Leu
Glu Glu Gln Leu Thr Asn Leu Ile Gln Asn Gln Pro 140 145 150 gaa act
cca gaa cac cca gaa gta act tca ctt aaa act ttt gta gaa 531 Glu Thr
Pro Glu His Pro Glu Val Thr Ser Leu Lys Thr Phe Val Glu 155 160 165
aaa caa gat aat agc atc aaa gac ctt ctc cag acc gtg gaa gac caa 579
Lys Gln Asp Asn Ser Ile Lys Asp Leu Leu Gln Thr Val Glu Asp Gln 170
175 180 185 tat aaa caa tta aac caa cag cat agt caa ata aaa gaa ata
gaa aat 627 Tyr Lys Gln Leu Asn Gln Gln His Ser Gln Ile Lys Glu Ile
Glu Asn 190 195 200 cag ctc aga agg act agt att caa gaa ccc aca gaa
att tct cta tct 675 Gln Leu Arg Arg Thr Ser Ile Gln Glu Pro Thr Glu
Ile Ser Leu Ser 205 210 215 tcc aag cca aga gca cca aga act act ccc
ttt ctt cag ttg aat gaa 723 Ser Lys Pro Arg Ala Pro Arg Thr Thr Pro
Phe Leu Gln Leu Asn Glu 220 225 230 ata aga aat gta aaa cat gat ggc
att cct gct gaa tgt acc acc att 771 Ile Arg Asn Val Lys His Asp Gly
Ile Pro Ala Glu Cys Thr Thr Ile 235 240 245 tat aac aga ggt gaa cat
aca agt ggc atg tat gcc atc aga ccc agc 819 Tyr Asn Arg Gly Glu His
Thr Ser Gly Met Tyr Ala Ile Arg Pro Ser 250 255 260 265 aac tct caa
gtt ttt cat gtc tac tgt gat gtt ata tca ggt agt cca 867 Asn Ser Gln
Val Phe His Val Tyr Cys Asp Val Ile Ser Gly Ser Pro 270 275 280 tgg
aca tta att caa cat cga ata gat gga tca caa aac ttc aat gaa 915 Trp
Thr Leu Ile Gln His Arg Ile Asp Gly Ser Gln Asn Phe Asn Glu 285 290
295 acg tgg gag aac tac aaa tat ggt ttt ggg agg ctt gat gga gaa ttt
963 Thr Trp Glu Asn Tyr Lys Tyr Gly Phe Gly Arg Leu Asp Gly Glu Phe
300 305 310 tgg ttg ggc cta gag aag ata tac tcc ata gtg aag caa tct
aat tat 1011 Trp Leu Gly Leu Glu Lys Ile Tyr Ser Ile Val Lys Gln
Ser Asn Tyr 315 320 325 gtt tta cga att gag ttg gaa gac tgg aaa gac
aac aaa cat tat att 1059 Val Leu Arg Ile Glu Leu Glu Asp Trp Lys
Asp Asn Lys His Tyr Ile 330 335 340 345 gaa tat tct ttt tac ttg gga
aat cac gaa acc aac tat acg cta cat 1107 Glu Tyr Ser Phe Tyr Leu
Gly Asn His Glu Thr Asn Tyr Thr Leu His 350 355 360 cta gtt gcg att
act ggc aat gtc ccc aat gca atc ccg gaa aac aaa 1155 Leu Val Ala
Ile Thr Gly Asn Val Pro Asn Ala Ile Pro Glu Asn Lys 365 370 375 gat
ttg gtg ttt tct act tgg gat cac aaa gca aaa gga cac ttc aac 1203
Asp Leu Val Phe Ser Thr Trp Asp His Lys Ala Lys Gly His Phe Asn 380
385 390 tgt cca gag ggt tat tca gga ggc tgg tgg tgg cat gat gag tgt
gga 1251 Cys Pro Glu Gly Tyr Ser Gly Gly Trp Trp Trp His Asp Glu
Cys Gly 395 400 405 gaa aac aac cta aat ggt aaa tat aac aaa cca aga
gca aaa tct aag 1299 Glu Asn Asn Leu Asn Gly Lys Tyr Asn Lys Pro
Arg Ala Lys Ser Lys 410 415 420 425 cca gag agg aga aga gga tta tct
tgg aag tct caa aat gga agg tta 1347 Pro Glu Arg Arg Arg Gly Leu
Ser Trp Lys Ser Gln Asn Gly Arg Leu 430 435 440 tac tct ata aaa tca
acc aaa atg ttg atc cat cca aca gat tca gaa 1395 Tyr Ser Ile Lys
Ser Thr Lys Met Leu Ile His Pro Thr Asp Ser Glu 445 450 455 agc ttt
gaa tga actgaggcaa atttaaaagg caataattta aacattaacc 1447 Ser Phe
Glu 460 tcattccaag ttaatgtggt ctaataatct ggtattaaat ccttaagag 1496
<210> SEQ ID NO 5 <211> LENGTH: 9381 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 5 ttccaaaaga actaaagtac agtttgagaa atgcatactt
aattcattac ttttttcccc 60 tcaactttaa taataaattt acccaacaaa
aaagtttatt tttgacttgt aaatctctta 120 aaatcataaa aaagtaaaat
tagcttttaa aaacaggtag tcaccatagc attgaatgtg 180 tagtttataa
tacagcaaag ttaaatacaa tttcaaatta cctattaagt tagttgctca 240
tttctttgat ttcatttagc attgatctaa ctcaatgtgg aagaaggtta cattcgtgca
300 agttaacacg gcttaatgat taactatgtt cacctaccaa ccttaccttt
tctgggcaaa 360 tattggtata tatagagtta agaagtctag gtctgcttcc
agaagaaaac agttccacgt 420 tgcttgaaat tgaaaatcaa gataaaaatg
ttcacaatta agctccttct ttttattgtt 480 cctctagtta tttcctccag
aattgatcaa gacaattcat catttgattc tctatctcca 540 gagccaaaat
caagatttgc tatgttagac gatgtaaaaa ttttagccaa tggcctcctt 600
cagttgggac atggtcttaa agactttgtc cataagacga agggccaaat taatgacata
660 tttcaaaaac tcaacatatt tgatcagtct ttttatgatc tatcgctgca
aaccagtgaa 720 atcaaagaag aagaaaagga actgagaaga actacatata
aactacaagt caaaaatgaa 780 gaggtaaaga atatgtcact tgaactcaac
tcaaaacttg aaagcctcct agaagaaaaa 840 attctacttc aacaaaaagt
gaaatattta gaagagcaac taactaactt aattcaaaat 900 caacctgaaa
ctccagaaca cccagaagta acttcactta aagtaagtag aaaataaaga 960
gggttcatgt ttatgttttc aatgtggatc ttttaaaaaa aatatttcta aggcatgcca
1020 tttgaaatac tttgttgcat tgttgaaata cttttttttc caagaaaaat
aatctccaga 1080 aaataaaatt tcctattata atttcaagtt agttttttgt
ttccctaatg ttatatatga 1140 aaacactgaa aatttgcatt ttatatgaaa
attacaaatc ggttaaatta tacaatctag 1200 aacactatgt cattacacta
ttgtaaatta ctgaaggtaa gtaaaaagtt aaaaaaaatt 1260 taaaactatt
ctccagtgtt taaaacagat taaataatac agtaaatgga aaagatttat 1320
tcatatgaaa atatgctggg ctttttcttt taattgaagt tcagaaaatc aaattttaga
1380 gatagtacaa tttaaataaa atgttaagga caaaaatatg tgctatttga
aagaagcata 1440 caaggggaag gaattgccaa tattcatttt tcaaatccat
tattagttta aaaatttaga 1500 ttatgatagt gttacaggaa attaatagaa
aagaaagagg aaagcaactt ataaccaacc 1560 tactctctat atccagactt
ttgtagaaaa acaagataat agcatcaaag accttctcca 1620 gaccgtggaa
gaccaatata aacaattaaa ccaacagcat agtcaaataa aagaaataga 1680
aaatcaggta agtcagtatt ttaatggtat gtcccatctt tcacacaggt ctgtaaaaac
1740 actgaatcct aaaattattt acaagcttta actggatcat gagtaaaatt
atcacatcag 1800 cataactgtt aaaattgcag gctctgaagc taataaacta
cctgcattta aaccatggct 1860 ctaaaacttt gtgtgacctt gaataaatta
cttcacccct ttatctctca gtttcctcac 1920 atatactaca aagataataa
cagaacttat aggattattg taagaaaaaa aattaattca 1980 tagcagccaa
tgtcatctta ctaaaattca aattagatca tgtttctctt tgctcaaaac 2040
cacacaatag ctttccattt cactcatatt ggctctttag accaagatta cccaaccctt
2100 cgtcatctca ctgacttcac ctcctctact ctagttattc tgaccgcttt
accagtattc 2160 aaacacatca aacatactgc cacctcaaag cctttgccct
tgttgtttcc tctaactgga 2220 acgctcttct gccctggtat ctacgtggcc
cactctctga tttcccttag ggtcgttatc 2280 aaacaaaaaa ttcccaatga
agacttacaa ggtcacttaa ccaaaaatca caaccgcctg 2340 gtcccatccc
tgaaaacttc tacttcctta gctacttttc tcctgcacac tcacctttat 2400
ttaacataac ataaatttta gttatttatc tcttctattc ctgcactaaa atgtaagctc
2460 tgtgaataca gggatttttt ccattatctt catattttcc attatttgta
tatactccag 2520 aatatagaat actgtatggc acacagtagg catttctgtt
gaattaataa atgtaatgtc 2580 atattcacac agaagcgtgt gctatgatta
ttattacttg gattactaga aatagtgtgc 2640 ctcataatta aaggtcaaca
ttcaacaatg taattaatct acaatgtaaa catctggtga 2700 agtgacagag
ggaagcactt gtttagaaaa aagctatgtc agaatccatg tattctaata 2760
tgcagtacaa tagtttaaaa atattaataa tactctcaaa cagctattca agaggattca
2820 aaaaacataa tataaactca gagaaactgg taaacaaaat cattttcaag
agatataaaa 2880 caaatattat taccaatttc cactaaacaa acataatgtt
agtagtgctg ctaaaaggtt 2940 ttttatcaac tacttttggt ttccatactt
tccttcttat gatgttatta ttctaaattc 3000 ttttcaatta tatcttttac
tatgattaaa tgaacctgct ccccaaagca aaatgttact 3060 atagtaatat
acattgtgtc taaaaataaa aatgtgtgaa gaaaccaaaa caatgaattt 3120
ctgagttgga agaagagtta gatcatttaa ctttctcata tttaaattaa aaaaacaaaa
3180 ctctaaaaat ttaagtaact ttaagatcac atagttactt agtagaaaag
agtaataccc 3240 agcaagcaaa ctttacaata gatcctttta aataaggtcc
taggaaatat cattcatgcc 3300 agcatcaaaa aactaacact aataatgcaa
gatattatat attctgcttt tcttactgtc 3360 aatgagaaaa actatcattc
aataaattgc aaacccaaca cacttaaata aaaataaaat 3420 gttactgcta
aactaacgat aaactactga atatatagaa agtaagcaaa caaacttgcc 3480
aacctgccaa catctacaga tatgtttaca ggtcaaaaat tatcaaatta tcaagaaagc
3540 ctggttcaaa ttatgtatta tgtctttatc acaggtctga agatcagtaa
gacctaaaac 3600 tgaaaattat taaacttaaa atctgaacag aatatcaaat
atattttatt catataaata 3660 aaagaataca ttacaatatt ctaagcaaag
cagtctctac ttttggcctt gctctgtttt 3720 ccgaccaatg tctgcttttt
tgccttgctt tattttttta tcttattaaa taatgtccct 3780 gattaaatat
tttgagaaca ggtaatctgt acaatctgaa taacactgtt tatctaaata 3840
tcaaacaccg ttataacatt atgaactgaa agacaaactg tacttctgac atccttactc
3900 agatttcccc taattgtata ttcagtatca ttttaaaaaa cagatttata
ttcttttatc 3960 agctcagaag gactagtatt caagaaccca cagaaatttc
tctatcttcc aagccaagag 4020 caccaagaac tactcccttt cttcagttga
atgaaataag aaatgtaaaa catgatggta 4080 agacactttg gtgggtttcc
ttcttgaagc tattattatc aaattcccta ttcttaggac 4140 ttgttctaga
ctaaaagata gttaagagat atccatcaaa tacaatgtat caacctaaac 4200
tggatgctgg ggttcttttt acaccctata aaagacatac ctaagacaat cagagaaata
4260 caaatatgga cttgattatt agataatata gaaggtttat taattttctt
agatgtgatc 4320 atggtattgc agttttaaag gagaacaatc tcctgtttaa
gagatacatg ctgaaatatt 4380 tacggagtta aaggtcactg gactccagac
tggtgataga acaagactct gtctctaaaa 4440 aataattaat tttttaaaag
aaaatagttt ggtaagatga ttcttacatt cttaaataac 4500 acgccatcta
agaaaaatgc tttaacataa acattactga aaaaatgcta catttgccac 4560
aacttcataa aatgtcaagt gaaatctcaa gctccaaaga tattattcct attactaaat
4620 ctgatgtaat aacattttat tgattctagg cattcctgct gaatgtacca
ccatttataa 4680 cagaggtgaa catacaagtg gcatgtatgc catcagaccc
agcaactctc aagtttttca 4740 tgtctactgt gatgttatat caggtaaaac
ctgtctaagg agaatagaca gtagttagtt 4800 caacttactc attacgtatt
aggaagatta acctggttat cattgtttta tacatatata 4860 tatgaaatat
atatgagtat tcgtataaat ataatacttt taccttgttt atgtatttac 4920
tcaatattct ccttttcctc taaaataatc tgaagtgact attatcaata agtttactat
4980 gccaaaattc attaattgcc tttcacttaa cttttgggac cataataaat
aataaaatgt 5040 attgccataa cattaataaa ctaccttaca aaaccaccaa
ttaaaatcaa acaaacaaaa 5100 aagtgttatt tacatctgtc aacataaatc
tactaaaaat acatgatttc attcattata 5160 ttcaggtagt ccatggacat
taattcaaca tcgaatagat ggatcacaaa acttcaatga 5220 aacgtgggag
aactacaaat atggttttgg gaggcttgat ggtaagggga ctacattcaa 5280
tcattcattc acttgctaat ctacaaatat ttactgagaa cctcttatgg accaggtatt
5340 aggaaaagta gtaacgaacg agaagcagtc tcagccttca tataatttat
tatcaaacaa 5400 ttacacattt gttagtaaat tacacttatt acaactgtta
ttatttgaat tatatttatc 5460 acaattacat gtctgttctt aaatatactt
atcacaattt aattccacgg cttacaatga 5520 tcataactat aattattaaa
gacaattttg attaaatgtt atgtcataag tagtaactgt 5580 tacaaataag
ctgtgaaaag aaccactcct agcattagtc actctattct ctcattaacg 5640
ttttacatat caattaattg gaagttaaaa ggaccaggaa actcagacat acagtataca
5700 ttttaaaatt tcaattattt aaatataata tatagaatgt atggcttata
atgaattagt 5760 taactcaatg caaattattc tattttgatt acaaatagta
aaataagcaa gataaaataa 5820 cagatgttta aaatccaaaa agcacataca
aaaatccatg aatgatgtct aagtactcac 5880 ttataaagta gaagacattc
attattatat caaattttta aatgctcagt actatttgac 5940 catttaaaaa
ttttgtattc aaactaccag tgaaagccct acctagaagg tatactcagt 6000
gataagtttt gtagctccaa atcttctaat agtgagtgta accccaaaat aaaaggctga
6060 caggtaagtc gagaatactc acttaattct ggtaagaaag caacccattt
gtacttgtat 6120 ttaccagcaa tccttaaaat gaagcttcct actaactcaa
tagcaataag acaatagtga 6180 atgtttaatg aaaacagtat tttataaata
ctttaataaa aaggattgtg atgaagaaca 6240 atctatttat atttgttatt
tgtttttaat tccaataaaa ataattttta aaattacaga 6300 aaaaagttat
taagaaccat gcttttaaat ttaaaatgat tttttaaatt tattcctgtc 6360
tttttctaca aagaaagcat acattaagca aataccaaag gccaggttta catttgaaga
6420 aagtgacatt attattactc aagtctctag gaatacttaa cacatctctt
gactgtatat 6480 ggatgttaat aaatagctga cagtaaagtt tatccatata
aagacttgca aatattcctc 6540 taccaatgac gagactttaa aatatctata
ataatgtaac acatttcact ggtgaaacat 6600 gtcttgtcat atgcattata
gaaaggataa tcagactttc agttatatta atatttttaa 6660 catttttgtg
cacatagcta tcttcaataa aattgtttta aaaggtatta ttttaagata 6720
cactaaaatg atcaagggat tcaagactaa acaactcaat tagttgcacc aataaaaaac
6780 acttaaaaaa actgtcagtg tccaacctgt acttaataac tcacagattt
ttaaaacttt 6840 tcttttcagg agaattttgg ttgggcctag agaagatata
ctccatagtg aagcaatcta 6900 attatgtttt acgaattgag ttggaagact
ggaaagacaa caaacattat attgaatatt 6960 ctttttactt gggaaatcac
gaaaccaact atacgctaca tctagttgcg attactggca 7020 atgtccccaa
tgcaatcccg gaaaacaaag atttggtgtt ttctacttgg gatcacaaag 7080
caaaaggaca cttcaactgt ccagagggtt attcaggtat ctttttctga taccaatact
7140 ttattttcat atcttcaaag tatcttccca cattattagc tattatctgc
aatgacaact 7200 tttaaaaatc cgaatcccaa ataagcgttt tctctctaga
cgaaaacctc ttaactataa 7260 tgaaagtgtt cattctagtt caatcaggta
ttttacctct aatcttcctc agattttcta 7320 ttttttggta gtgtatagat
tatttataca gattatttaa aattgggact tatacagatt 7380 atttaaaact
gggatacatg catctaaaac actgtaatat ttataagaaa ggaagataaa 7440
cttacgggga aatacagtaa cagtaactac atacgagtct gtacccatta aattgcatat
7500
ctatctcctt taggaggctg gtggtggcat gatgagtgtg gagaaaacaa cctaaatggt
7560 aaatataaca aaccaagagc aaaatctaag ccagagagga gaagaggatt
atcttggaag 7620 tctcaaaatg gaaggttata ctctataaaa tcaaccaaaa
tgttgatcca tccaacagat 7680 tcagaaagct ttgaatgaac tgaggcaaat
ttaaaaggca ataatttaaa cattaacctc 7740 attccaagtt aatgtggtct
aataatctgg tattaaatcc ttaagagaaa gcttgagaaa 7800 tagatttttt
ttatcttaaa gtcactgtct atttaagatt aaacatacaa tcacataacc 7860
ttaaagaata ccgtttacat ttctcaatca aaattcttat aatactattt gttttaaatt
7920 ttgtgatgtg ggaatcaatt ttagatggtc acaatctaga ttataatcaa
taggtgaact 7980 tattaaataa cttttctaaa taaaaaattt agagactttt
attttaaaag gcatcatatg 8040 agctaatatc acaactttcc cagtttaaaa
aactagtact cttgttaaaa ctctaaactt 8100 gactaaatac agaggactgg
taattgtaca gttcttaaat gttgtagtat taatttcaaa 8160 actaaaaatc
gtcagcacag agtatgtgta aaaatctgta atacaaattt ttaaactgat 8220
gcttcatttt gctacaaaat aatttggagt aaatgtttga tatgatttat ttatgaaacc
8280 taatgaagca gaattaaata ctgtattaaa ataagttcgc tgtctttaaa
caaatggaga 8340 tgactactaa gtcacattga ctttaacatg aggtatcact
ataccttatt tgttaaaata 8400 tatactgtat acattttata tattttaaca
cttaatacta tgaaaacaaa taattgtaaa 8460 ggaatcttgt cagattacag
taagaatgaa catatttgtg gcatcgagtt aaagtttata 8520 tttcccctaa
atatgctgtg attctaatac attcgtgtag gttttcaagt agaaataaac 8580
ctcgtaacaa gttactgaac gtttaaacag cctgacaagc atgtatatat gtttaaaatt
8640 caataaacaa agacccagtc cctaaattat agaaatttaa attattcttg
catgtttatc 8700 gacatcacaa cagatcccta aatccctaaa tccctaaaga
ttagatacaa attttttacc 8760 acagtatcac ttgtcagaat ttatttttaa
atatgatttt ttaaaactgc cagtaagaaa 8820 ttttaaatta aacccatttg
ttaaaggata tagtgcccaa gttatatggt gacctacctt 8880 tgtcaatact
tagcattatg tatttcaaat tatccaatat acatgtcata tatattttta 8940
tatgtcacat atataaaaga tatgtatgat ctatgtgaat cctaagtaaa tattttgttc
9000 cagaaaagta caaaataata aaggtaaaaa taatctataa ttttcaggac
cacagactaa 9060 gctgtcgaaa ttaacgctga tttttttagg gccagaatac
caaaatggct cctctcttcc 9120 cccaaaattg gacaatttca aatgcaaaat
aattcattat ttaatatatg agttgcttcc 9180 tctatttggt ttccttaaaa
aaaaaaaaaa ctctcatagg acatgtttca ttttgttcct 9240 ttcaggagta
gtaaattaga cgttttcccc atataaagct tttttctacc agaaagatac 9300
ttctggtaga agaagagaaa ggagctcttt atggttcaca cgactgtctc ctgtcctaac
9360 tactttgctt aaagtgctca a 9381 <210> SEQ ID NO 6
<211> LENGTH: 1449 <212> TYPE: DNA <213>
ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (19)..(1386) <400> SEQUENCE: 6
aataattgag acaaaaaa atg cac aca att aaa tta ttc ctt ttt gtt gtt 51
Met His Thr Ile Lys Leu Phe Leu Phe Val Val 1 5 10 cct tta gta att
gca tcc aga gtg gat cca gac ctt tca tca ttt gat 99 Pro Leu Val Ile
Ala Ser Arg Val Asp Pro Asp Leu Ser Ser Phe Asp 15 20 25 tct gca
cct tca gag cca aaa tca aga ttt gct atg ttg gat gat gtc 147 Ser Ala
Pro Ser Glu Pro Lys Ser Arg Phe Ala Met Leu Asp Asp Val 30 35 40
aaa att tta gcg aat ggc ctc ctg cag ctg ggt cat gga ctt aaa gat 195
Lys Ile Leu Ala Asn Gly Leu Leu Gln Leu Gly His Gly Leu Lys Asp 45
50 55 ttt gtc cat aag act aag gga caa att aac gac ata ttt cag aag
ctc 243 Phe Val His Lys Thr Lys Gly Gln Ile Asn Asp Ile Phe Gln Lys
Leu 60 65 70 75 aac ata ttt gat cag tct ttt tat gac cta tca ctt cga
acc aat gaa 291 Asn Ile Phe Asp Gln Ser Phe Tyr Asp Leu Ser Leu Arg
Thr Asn Glu 80 85 90 atc aaa gaa gag gaa aag gag cta aga aga act
aca tct aca cta caa 339 Ile Lys Glu Glu Glu Lys Glu Leu Arg Arg Thr
Thr Ser Thr Leu Gln 95 100 105 gtt aaa aac gag gag gtg aag aac atg
tca gta gaa ctg aac tca aag 387 Val Lys Asn Glu Glu Val Lys Asn Met
Ser Val Glu Leu Asn Ser Lys 110 115 120 ctt gag agt ctg ctg gaa gag
aag aca gcc ctt caa cac aag gtc agg 435 Leu Glu Ser Leu Leu Glu Glu
Lys Thr Ala Leu Gln His Lys Val Arg 125 130 135 gct ttg gag gag cag
cta acc aac tta att cta agc cca gct ggg gct 483 Ala Leu Glu Glu Gln
Leu Thr Asn Leu Ile Leu Ser Pro Ala Gly Ala 140 145 150 155 cag gag
cac cca gaa gta aca tca ctc aaa agt ttt gta gaa cag caa 531 Gln Glu
His Pro Glu Val Thr Ser Leu Lys Ser Phe Val Glu Gln Gln 160 165 170
gac aac agc ata aga gaa ctc ctc cag agt gtg gaa gaa cag tat aaa 579
Asp Asn Ser Ile Arg Glu Leu Leu Gln Ser Val Glu Glu Gln Tyr Lys 175
180 185 caa tta agt caa cag cac atg cag ata aaa gaa ata gaa aag cag
ctc 627 Gln Leu Ser Gln Gln His Met Gln Ile Lys Glu Ile Glu Lys Gln
Leu 190 195 200 aga aag act ggt att caa gaa ccc tca gaa aat tct ctt
tct tct aaa 675 Arg Lys Thr Gly Ile Gln Glu Pro Ser Glu Asn Ser Leu
Ser Ser Lys 205 210 215 tca aga gca cca aga act act ccc cct ctt caa
ctg aac gaa aca gaa 723 Ser Arg Ala Pro Arg Thr Thr Pro Pro Leu Gln
Leu Asn Glu Thr Glu 220 225 230 235 aat aca gaa caa gat gac ctt cct
gcc gac tgc tct gcc gtt tat aac 771 Asn Thr Glu Gln Asp Asp Leu Pro
Ala Asp Cys Ser Ala Val Tyr Asn 240 245 250 aga ggc gaa cat aca agt
ggc gtg tac act att aaa cca aga aac tcc 819 Arg Gly Glu His Thr Ser
Gly Val Tyr Thr Ile Lys Pro Arg Asn Ser 255 260 265 caa ggg ttt aat
gtc tac tgt gat acc caa tca ggc agt cca tgg aca 867 Gln Gly Phe Asn
Val Tyr Cys Asp Thr Gln Ser Gly Ser Pro Trp Thr 270 275 280 tta att
caa cac cgg aaa gat ggc tca cag gac ttc aac gaa aca tgg 915 Leu Ile
Gln His Arg Lys Asp Gly Ser Gln Asp Phe Asn Glu Thr Trp 285 290 295
gaa aac tac gaa aag ggc ttt ggg agg ctc gat gga gaa ttt tgg ttg 963
Glu Asn Tyr Glu Lys Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu 300
305 310 315 ggc cta gag aag atc tat gct ata gtc caa cag tct aac tac
att tta 1011 Gly Leu Glu Lys Ile Tyr Ala Ile Val Gln Gln Ser Asn
Tyr Ile Leu 320 325 330 cga ctc gag cta caa gac tgg aaa gac agc aag
cac tac gtt gaa tac 1059 Arg Leu Glu Leu Gln Asp Trp Lys Asp Ser
Lys His Tyr Val Glu Tyr 335 340 345 tcc ttt cac ctg ggc agt cac gaa
acc aac tac acg cta cat gtg gct 1107 Ser Phe His Leu Gly Ser His
Glu Thr Asn Tyr Thr Leu His Val Ala 350 355 360 gag att gct ggc aat
atc cct ggg gcc ctc cca gag cac aca gac ctg 1155 Glu Ile Ala Gly
Asn Ile Pro Gly Ala Leu Pro Glu His Thr Asp Leu 365 370 375 atg ttt
tct aca tgg aat cac aga gca aag gga cag ctc tac tgt cca 1203 Met
Phe Ser Thr Trp Asn His Arg Ala Lys Gly Gln Leu Tyr Cys Pro 380 385
390 395 gaa agt tac tca ggt ggc tgg tgg tgg aat gac ata tgt gga gaa
aac 1251 Glu Ser Tyr Ser Gly Gly Trp Trp Trp Asn Asp Ile Cys Gly
Glu Asn 400 405 410 aac cta aat gga aaa tac aac aaa ccc aga acc aaa
tcc aga cca gag 1299 Asn Leu Asn Gly Lys Tyr Asn Lys Pro Arg Thr
Lys Ser Arg Pro Glu 415 420 425 aga aga aga ggg atc tac tgg aga cct
cag agc aga aag ctc tat gct 1347 Arg Arg Arg Gly Ile Tyr Trp Arg
Pro Gln Ser Arg Lys Leu Tyr Ala 430 435 440 atc aaa tca tcc aaa atg
atg ctc cag ccc acc acc taa gaagcttcaa 1396 Ile Lys Ser Ser Lys Met
Met Leu Gln Pro Thr Thr 445 450 455 ctgaactgag acaaaataaa
agatcaataa attaaatatt aaagtcctcc cga 1449 <210> SEQ ID NO 7
<211> LENGTH: 510 <212> TYPE: DNA <213> ORGANISM:
Mus musculus <220> FEATURE: <221> NAME/KEY: CDS
<222> LOCATION: (67)..(510) <400> SEQUENCE: 7
tgttggccta ctggacagga gggagaagtt ccaaattgct taaaattgaa taattgagac
60 aaaaaa tgc aca caa tta aat tat tcc ttt ttg ttg ttc ctt tag taa
108 Cys Thr Gln Leu Asn Tyr Ser Phe Leu Leu Phe Leu 1 5 10 ttg cat
cca gag tgg atc cag acc ttt cat cat ttg att ctg cac ctt 156 Leu His
Pro Glu Trp Ile Gln Thr Phe His His Leu Ile Leu His Leu 15 20 25
cag agc caa aat caa gat ttg cta tgt tgg atg atg tca aaa ttt tag 204
Gln Ser Gln Asn Gln Asp Leu Leu Cys Trp Met Met Ser Lys Phe 30 35
40 cga atg gcc tcc tgc agc tgg gtc atg gac tta aag att ttg tcc ata
252 Arg Met Ala Ser Cys Ser Trp Val Met Asp Leu Lys Ile Leu Ser Ile
45 50 55 aga cta agg gac aaa tta acg aca tat ttc aga agc tca aca
tat ttg 300 Arg Leu Arg Asp Lys Leu Thr Thr Tyr Phe Arg Ser Ser Thr
Tyr Leu 60 65 70 75 atc agt ctt ttt atg acc tat cac ttc gaa cca atg
aaa tca aag aag 348 Ile Ser Leu Phe Met Thr Tyr His Phe Glu Pro Met
Lys Ser Lys Lys 80 85 90 agg aaa agg agc taa gaa gaa cta cat cta
cac tac aag tta aaa acg 396 Arg Lys Arg Ser Glu Glu Leu His Leu His
Tyr Lys Leu Lys Thr 95 100 105 agg agg tga aga aca ctt cag tag aac
tga act caa agc ttg aga gtc 444 Arg Arg Arg Thr Leu Gln Asn Thr Gln
Ser Leu Arg Val 110 115 tgc tgg aag aga aga cag ccc ttc aac aca agg
tca ggg ctt tgg agg 492 Cys Trp Lys Arg Arg Gln Pro Phe Asn Thr Arg
Ser Gly Leu Trp Arg 120 125 130 135 agc agc taa cca act taa 510 Ser
Ser Pro Thr <210> SEQ ID NO 8 <211> LENGTH: 271
<212> TYPE: DNA <213> ORGANISM: Mus musculus
<400> SEQUENCE: 8 gagggatcta ctggagacct cagagcagaa agctctatgc
tatcaaatca tccaaattga 60 tgctccaccc caccacctaa gaagcttcaa
ctgaactgag acaaaataaa agatcaataa 120 attaaatatt aaattcctcc
cgatcactgt agtaatctgg tattaaaatt ttaatggaaa 180 gcttgagaat
tgaatttcaa ttaggtttaa actcattgtt aagatcagat atcaccgaat 240
caacgtaaac aaaatttatc tttttcaatc t 271 <210> SEQ ID NO 9
<400> SEQUENCE: 9 000 <210> SEQ ID NO 10 <400>
SEQUENCE: 10 000 <210> SEQ ID NO 11 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 11 ctcttactgt gctgtggaca 20
<210> SEQ ID NO 12 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 12
tgcagggcta catggaacaa 20 <210> SEQ ID NO 13 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 13 cggactcctg cacgctactt 20 <210> SEQ
ID NO 14 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Probe <400> SEQUENCE: 14 ctccaagacg
gtccaggatg cgc 23 <210> SEQ ID NO 15 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 15 caacggattt ggtcgtattg g 21 <210> SEQ
ID NO 16 <211> LENGTH: 26 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 16 ggcaacaata
tccactttac cagagt 26 <210> SEQ ID NO 17 <211> LENGTH:
21 <212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 17 cgcctggtca ccagggctgc t 21 <210> SEQ
ID NO 18 <211> LENGTH: 19 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 18 gaaggtgaag
gtcggagtc 19 <210> SEQ ID NO 19 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 19 gaagatggtg atgggatttc 20 <210> SEQ
ID NO 20 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Probe <400> SEQUENCE: 20 caagcttccc
gttctcagcc 20 <210> SEQ ID NO 21 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 21 tggaatcata ttggaacatg 20 <210> SEQ
ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 22 ggcaaattca
acggcacagt 20 <210> SEQ ID NO 23 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 23 gggtctcgct cctggaagat 20 <210> SEQ
ID NO 24 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Probe <400> SEQUENCE: 24 aaggccgaga
atgggaagct tgtcatc 27 <210> SEQ ID NO 25 <211> LENGTH:
23 <212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 25 tgttctagag acagccgcat ctt 23 <210>
SEQ ID NO 26 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 26
caccgacctt caccatcttg t 21 <210> SEQ ID NO 27 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 27 ttgtgcagtg ccagcctcgt ctca 24 <210>
SEQ ID NO 28 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 28
cttcaatgaa acgtgggaga act 23 <210> SEQ ID NO 29 <211>
LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Primer
<400> SEQUENCE: 29 tctctaggcc caaccaaaat tc 22 <210>
SEQ ID NO 30 <211> LENGTH: 24 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Probe <400> SEQUENCE: 30
aaatatggtt ttgggaggct tgat 24 <210> SEQ ID NO 31 <211>
LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 31 cagaagtaac atcactcaaa agttttgtag 30
<210> SEQ ID NO 32 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 32
gacttaattg tttatactgt tcttccacac t 31 <210> SEQ ID NO 33
<211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Probe <400> SEQUENCE: 33 cagcaagaca acagcataag
agaactcctc ca 32 <210> SEQ ID NO 34 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 34 atctgttgtg atgtcgataa 20
<210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 35 gtatttagtc aagtttagag 20 <210> SEQ ID NO 36
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 36
tattacagat ttttacacat 20 <210> SEQ ID NO 37 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 37 cgtggaactg
ttttcttctg 20 <210> SEQ ID NO 38 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 38 agcttaattg tgaacatttt 20
<210> SEQ ID NO 39 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 39 attctggagg aaataactag 20 <210> SEQ ID NO 40
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 40
aaatcttgat tttggctctg 20 <210> SEQ ID NO 41 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 41 atagcaaatc
ttgattttgg 20 <210> SEQ ID NO 42 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 42 ctaacatagc aaatcttgat 20
<210> SEQ ID NO 43 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 43 atcgtctaac atagcaaatc 20 <210> SEQ ID NO 44
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 44
aggccattgg ctaaaatttt 20 <210> SEQ ID NO 45 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 45 catgtcccaa
ctgaaggagg 20 <210> SEQ ID NO 46 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 46 ctttaagacc atgtcccaac 20
<210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 47 gcccttcgtc ttatggacaa 20 <210> SEQ ID NO 48
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 48
tcattaattt ggcccttcgt 20 <210> SEQ ID NO 49 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 49 tgaaatatgt
cattaatttg 20 <210> SEQ ID NO 50 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide
<400> SEQUENCE: 50 gactgatcaa atatgttgag 20 <210> SEQ
ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonuclotide <400> SEQUENCE:
51 gtttgcagcg atagatcata 20 <210> SEQ ID NO 52 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 52 tcactggttt
gcagcgatag 20 <210> SEQ ID NO 53 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 53 agttcaagtg acatattctt 20
<210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 54 agtgaagtta cttctgggtg 20 <210> SEQ ID NO 55
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 55
gttttaagtg aagttacttc 20 <210> SEQ ID NO 56 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 56 ctacaaaagt
tttaagtgaa 20 <210> SEQ ID NO 57 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 57 ggtcttccac ggtctggaga 20
<210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 58 tagtccttct gagctgattt 20 <210> SEQ ID NO 59
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 59
ggttcttgaa tactagtcct 20 <210> SEQ ID NO 60 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 60 aaatttctgt
gggttcttga 20 <210> SEQ ID NO 61 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 61 tggcttggaa gatagagaaa 20
<210> SEQ ID NO 62 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 62 agtagttctt ggtgctcttg 20 <210> SEQ ID NO 63
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 63
tgaagaaagg gagtagttct 20 <210> SEQ ID NO 64 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 64 attcaactga
agaaagggag 20 <210> SEQ ID NO 65 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 65 tcttatttca ttcaactgaa 20
<210> SEQ ID NO 66 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 66 aggaatgcca tcatgtttta 20 <210> SEQ ID NO 67
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 67
ctctgttata aatggtggta 20 <210> SEQ ID NO 68 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 68 ttcacctctg
ttataaatgg 20 <210> SEQ ID NO 69 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 69 gtatgttcac ctctgttata 20
<210> SEQ ID NO 70 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 70 cacttgtatg ttcacctctg 20 <210> SEQ ID NO 71
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 71 catgccactt gtatgttcac 20 <210> SEQ ID NO 72
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 72
agagttgctg ggtctgatgg 20 <210> SEQ ID NO 73 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 73 ctgatataac
atcacagtag 20 <210> SEQ ID NO 74 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 74 catggactac ctgatataac 20
<210> SEQ ID NO 75 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 75 tgaattaatg tccatggact 20 <210> SEQ ID NO 76
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 76
tctattcgat gttgaattaa 20 <210> SEQ ID NO 77 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 77 agttctccca
cgtttcattg 20 <210> SEQ ID NO 78 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 78 catatttgta gttctcccac 20
<210> SEQ ID NO 79 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 79 aaaaccatat ttgtagttct 20 <210> SEQ ID NO 80
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 80
gcctcccaaa accatatttg 20 <210> SEQ ID NO 81 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 81 gcccaaccaa
aattctccat 20 <210> SEQ ID NO 82 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 82 ctctaggccc aaccaaaatt 20
<210> SEQ ID NO 83 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 83 atcttctcta ggcccaacca 20 <210> SEQ ID NO 84
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 84
aacataatta gattgcttca 20 <210> SEQ ID NO 85 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 85 gtcttccaac
tcaattcgta 20 <210> SEQ ID NO 86 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 86 ctttccagtc ttccaactca 20
<210> SEQ ID NO 87 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 87 aactagatgt agcgtatagt 20 <210> SEQ ID NO 88
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 88
tgatcccaag tagaaaacac 20 <210> SEQ ID NO 89 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 89 caccagcctc
ctgaataacc 20 <210> SEQ ID NO 90 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 90 ttaggttgtt ttctccacac 20
<210> SEQ ID NO 91 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 91 taatcctctt ctcctctctg 20 <210> SEQ ID NO 92
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 92 tgagacttcc aagataatcc 20
<210> SEQ ID NO 93 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 93 cattttgaga cttccaagat 20 <210> SEQ ID NO 94
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 94
gagtataacc ttccattttg 20 <210> SEQ ID NO 95 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 95 tggatggatc
aacattttgg 20 <210> SEQ ID NO 96 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 96 ttcaaagctt tctgaatctg 20
<210> SEQ ID NO 97 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 97 tgcctcagtt cattcaaagc 20 <210> SEQ ID NO 98
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 98
tgccttttaa atttgcctca 20 <210> SEQ ID NO 99 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 99 attaacttgg
aatgaggtta 20 <210> SEQ ID NO 100 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 100 agaccacatt aacttggaat 20
<210> SEQ ID NO 101 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 101 agattattag accacattaa 20 <210> SEQ ID NO 102
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 102
ataccagatt attagaccac 20 <210> SEQ ID NO 103 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 103 ggatttaata
ccagattatt 20 <210> SEQ ID NO 104 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 104 actgacttac ctgattttct 20
<210> SEQ ID NO 105 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 105 accttgtaag tcttcattgg 20 <210> SEQ ID NO 106
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 106
cagtgttatt cagattgtac 20 <210> SEQ ID NO 107 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 107 agtgtcttac
catcatgttt 20 <210> SEQ ID NO 108 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 108 acagatgtaa ataacacttt 20
<210> SEQ ID NO 109 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 109 gtccccttac catcaagcct 20 <210> SEQ ID NO 110
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 110
gggaagatac tttgaagata 20 <210> SEQ ID NO 111 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 111 caccagcctc
ctaaaggaga 20 <210> SEQ ID NO 112 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 112 aataatttaa ttgtgtgcat 20
<210> SEQ ID NO 113 <211> LENGTH: 20 <212> TYPE:
DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 113 cactctggat gcaattacta 20 <210> SEQ ID NO 114
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 114
aaggtctgga tccactctgg 20 <210> SEQ ID NO 115 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 115 tttggctctg
aaggtgcaga 20 <210> SEQ ID NO 116 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 116 tcatccaaca tagcaaatct 20
<210> SEQ ID NO 117 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 117 tgacatcatc caacatagca 20 <210> SEQ ID NO 118
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 118
aattttgaca tcatccaaca 20 <210> SEQ ID NO 119 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 119 gctaaaattt
tgacatcatc 20 <210> SEQ ID NO 120 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 120 aggccattcg ctaaaatttt 20
<210> SEQ ID NO 121 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 121 cccagctgca ggaggccatt 20 <210> SEQ ID NO 122
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 122
catgacccag ctgcaggagg 20 <210> SEQ ID NO 123 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 123 ttaagtccat
gacccagctg 20 <210> SEQ ID NO 124 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 124 gacaaaatct ttaagtccat 20
<210> SEQ ID NO 125 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 125 ttatggacaa aatctttaag 20 <210> SEQ ID NO 126
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 126
ttgagcttct gaaatatgtc 20 <210> SEQ ID NO 127 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 127 atatgttgag
cttctgaaat 20 <210> SEQ ID NO 128 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 128 atcaaatatg ttgagcttct 20
<210> SEQ ID NO 129 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 129 ggttcgaagt gataggtcat 20 <210> SEQ ID NO 130
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 130
tagtgtagat gtagttcttc 20 <210> SEQ ID NO 131 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 131 gacatgttct
tcacctcctc 20 <210> SEQ ID NO 132 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 132 tgagttcagt tctactgaca 20
<210> SEQ ID NO 133 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 133 tcaagctttg agttcagttc 20 <210> SEQ ID NO 134
<211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 134 gtcttctctt ccagcagact 20
<210> SEQ ID NO 135 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 135 gttgaagggc tgtcttctct 20 <210> SEQ ID NO 136
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 136
ctgaccttgt gttgaagggc 20 <210> SEQ ID NO 137 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 137 ccaaagccct
gaccttgtgt 20 <210> SEQ ID NO 138 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 138 ttagctgctc ctccaaagcc 20
<210> SEQ ID NO 139 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 139 cttagaatta agttggttag 20 <210> SEQ ID NO 140
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 140
ggtgctcctg agccccagct 20 <210> SEQ ID NO 141 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 141 tgatgttact
tctgggtgct 20 <210> SEQ ID NO 142 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 142 tgctgttcta caaaactttt 20
<210> SEQ ID NO 143 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 143 aggagttctc ttatgctgtt 20 <210> SEQ ID NO 144
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 144
tctggaggag ttctcttatg 20 <210> SEQ ID NO 145 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 145 cacactctgg
aggagttctc 20 <210> SEQ ID NO 146 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 146 gggttcttga ataccagtct 20
<210> SEQ ID NO 147 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 147 gaaagagaat tttctgaggg 20 <210> SEQ ID NO 148
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 148
gcaggaaggt catcttgttc 20 <210> SEQ ID NO 149 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 149 gagcagtcgg
caggaaggtc 20 <210> SEQ ID NO 150 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 150 tacacgccac ttgtatgttc 20
<210> SEQ ID NO 151 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 151 ttaatagtgt acacgccact 20 <210> SEQ ID NO 152
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 152
agacattaaa cccttgggag 20 <210> SEQ ID NO 153 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 153 ctgcctgatt
gggtatcaca 20 <210> SEQ ID NO 154 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 154 ccatctttcc ggtgttgaat 20
<210> SEQ ID NO 155
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 155
gaagtcctgt gagccatctt 20 <210> SEQ ID NO 156 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 156 ttctccatcg
agcctcccaa 20 <210> SEQ ID NO 157 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 157 gttggactat agcatagatc 20
<210> SEQ ID NO 158 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 158 atgtagttag actgttggac 20 <210> SEQ ID NO 159
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 159
acgtagtgct tgctgtcttt 20 <210> SEQ ID NO 160 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 160 gcccaggtga
aaggagtatt 20 <210> SEQ ID NO 161 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 161 tgtagcgtgt agttggtttc 20
<210> SEQ ID NO 162 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 162 ccacatgtag cgtgtagttg 20 <210> SEQ ID NO 163
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 163
ctcagccaca tgtagcgtgt 20 <210> SEQ ID NO 164 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 164 gcaatctcag
ccacatgtag 20 <210> SEQ ID NO 165 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 165 atcaggtctg tgtgctctgg 20
<210> SEQ ID NO 166 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 166 atgtagaaaa catcaggtct 20 <210> SEQ ID NO 167
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 167
tctgtgattc catgtagaaa 20 <210> SEQ ID NO 168 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 168 tctggacagt
agagctgtcc 20 <210> SEQ ID NO 169 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 169 aactttctgg acagtagagc 20
<210> SEQ ID NO 170 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 170 accaccagcc acctgagtaa 20 <210> SEQ ID NO 171
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 171
ttctccacat atgtcattcc 20 <210> SEQ ID NO 172 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 172 ggttgttttc
tccacatatg 20 <210> SEQ ID NO 173 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 173 tggtctggat ttggttctgg 20
<210> SEQ ID NO 174 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 174 tctctctggt ctggatttgg 20 <210> SEQ ID NO 175
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 175
tagagctttc tgctctgagg 20
<210> SEQ ID NO 176 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 176 gtggtgggct ggagcatcat 20 <210> SEQ ID NO 177
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 177
tgaagcttct taggtggtgg 20 <210> SEQ ID NO 178 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 178 tgtctcagtt
cagttgaagc 20 <210> SEQ ID NO 179 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 179 tcgggaggac tttaatattt 20
<210> SEQ ID NO 180 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 180 ggaacttctc cctcctgtcc 20 <210> SEQ ID NO 181
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 181
taacaatgag tttaaaccta 20 <210> SEQ ID NO 182 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 182 tctgatctta
acaatgagtt 20 <210> SEQ ID NO 183 <211> LENGTH: 19
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 183 cgagaggcgg acgggaccg 19
<210> SEQ ID NO 184 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 184 cgagaggcgg acgggaccgt t 21 <210> SEQ ID NO 185
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonuclotide <400> SEQUENCE: 185
ttgctctccg cctgccctgg c 21 <210> SEQ ID NO 186 <211>
LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonuclotide <400> SEQUENCE: 186 gctctccgcc
tgccctggc 19 <210> SEQ ID NO 187 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 187 ccttccctga aggttcctcc 20
<210> SEQ ID NO 188 <211> LENGTH: 19 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 188
cacctgggca gtcacgaaa 19 <210> SEQ ID NO 189 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 189 ggagggcccc agggatat 18 <210> SEQ ID
NO 190 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Probe <400> SEQUENCE: 190 cacgctacat
gtggctgaga ttcgtgg 27 <210> SEQ ID NO 191 <211> LENGTH:
20 <212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonuclotide <400> SEQUENCE: 191 gtgacatatt cttcacctct 20
<210> SEQ ID NO 192 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonuclotide <400>
SEQUENCE: 192 tttaagtgac gttacctctg 20 <210> SEQ ID NO 193
<211> LENGTH: 1272 <212> TYPE: DNA <213>
ORGANISM: Rattus norvegicus <220> FEATURE: <221>
NAME/KEY: CDS <222> LOCATION: (1)..(1272) <400>
SEQUENCE: 193 atg cac aca att aag ctg ctc ctt ttt gtt gtt cct cta
gta att tcg 48 Met His Thr Ile Lys Leu Leu Leu Phe Val Val Pro Leu
Val Ile Ser 1 5 10 15 tcc aga gtt gat cca gac ctt tcg cca ttt gat
tct gta ccg tca gag 96 Ser Arg Val Asp Pro Asp Leu Ser Pro Phe Asp
Ser Val Pro Ser Glu 20 25 30 cca aaa tca aga ttt gct atg ttg gat
gat gtc aaa att tta gcc aat 144 Pro Lys Ser Arg Phe Ala Met Leu Asp
Asp Val Lys Ile Leu Ala Asn 35 40 45 ggc ctc ctg cag ctg ggt cat
ggt ctt aaa gat ttt gtc cat aag aca 192 Gly Leu Leu Gln Leu Gly His
Gly Leu Lys Asp Phe Val His Lys Thr 50 55 60 aag gga caa att aat
gac ata ttt cag aag ctc aac ata ttt gat cag 240 Lys Gly Gln Ile Asn
Asp Ile Phe Gln Lys Leu Asn Ile Phe Asp Gln 65 70 75 80 tgt ttt tat
gac cta tca ctt caa acc aat gaa atc aaa gaa gag gaa 288 Cys Phe Tyr
Asp Leu Ser Leu Gln Thr Asn Glu Ile Lys Glu Glu Glu 85 90 95 aag
gag cta aga aga acc aca tct aaa cta caa gtt aaa aac gaa gag 336 Lys
Glu Leu Arg Arg Thr Thr Ser Lys Leu Gln Val Lys Asn Glu Glu 100 105
110 gtg aag aat atg tca ctt gaa ctg aac tca aag ctt gaa agt cta ctg
384 Val Lys Asn Met Ser Leu Glu Leu Asn Ser Lys Leu Glu Ser Leu Leu
115 120 125 gag gag aag atg gcg ctc caa cac aga gtc agg gct ttg gag
gaa cag 432 Glu Glu Lys Met Ala Leu Gln His Arg Val Arg Ala Leu Glu
Glu Gln 130 135 140 ctg acc agc ttg gtt cag aac ccg cct ggg gct cgg
gag cac cca gag 480
Leu Thr Ser Leu Val Gln Asn Pro Pro Gly Ala Arg Glu His Pro Glu 145
150 155 160 gta acg tca ctt aaa agt ttt gta gaa cag caa gat aac agc
ata aga 528 Val Thr Ser Leu Lys Ser Phe Val Glu Gln Gln Asp Asn Ser
Ile Arg 165 170 175 gaa ctc ctc cag agt gtg gaa gaa caa tat aaa caa
cta agt caa cag 576 Glu Leu Leu Gln Ser Val Glu Glu Gln Tyr Lys Gln
Leu Ser Gln Gln 180 185 190 cac att cag ata aaa gaa ata gaa aat cag
ctc aga aag act ggc att 624 His Ile Gln Ile Lys Glu Ile Glu Asn Gln
Leu Arg Lys Thr Gly Ile 195 200 205 caa gaa ccc act gaa aat tct ctt
tat tct aaa cca aga gca cca aga 672 Gln Glu Pro Thr Glu Asn Ser Leu
Tyr Ser Lys Pro Arg Ala Pro Arg 210 215 220 act act ccc cct ctt cat
ctg aag gaa gca aaa aat ata gaa caa gat 720 Thr Thr Pro Pro Leu His
Leu Lys Glu Ala Lys Asn Ile Glu Gln Asp 225 230 235 240 gat ctg cct
gct gac tgc tct gcc att tat aac aga ggt gaa cat aca 768 Asp Leu Pro
Ala Asp Cys Ser Ala Ile Tyr Asn Arg Gly Glu His Thr 245 250 255 agt
ggc gtg tat act att aga cca agc agc tct caa gtg ttt aat gtc 816 Ser
Gly Val Tyr Thr Ile Arg Pro Ser Ser Ser Gln Val Phe Asn Val 260 265
270 tac tgt gac acc caa tca gga gaa ttc tgg ttg ggc cta gag aag atc
864 Tyr Cys Asp Thr Gln Ser Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile
275 280 285 tac gct ata gtc aaa cag tct aac tac atc tta cga ctg gag
cta caa 912 Tyr Ala Ile Val Lys Gln Ser Asn Tyr Ile Leu Arg Leu Glu
Leu Gln 290 295 300 gac tgg aag gac agc aag cac tat gct gaa tat tcc
ttt cat ctg ggc 960 Asp Trp Lys Asp Ser Lys His Tyr Ala Glu Tyr Ser
Phe His Leu Gly 305 310 315 320 aat cat gaa acc aac tac acg cta cat
gtg gct gag att gct gcc aat 1008 Asn His Glu Thr Asn Tyr Thr Leu
His Val Ala Glu Ile Ala Ala Asn 325 330 335 atc cct gag gcc cta cca
gaa cac aga gac ctg atg ttt tct aca tgg 1056 Ile Pro Glu Ala Leu
Pro Glu His Arg Asp Leu Met Phe Ser Thr Trp 340 345 350 gat cac aga
gca aag gga cag ctc tac tgt cca gaa agt tat tca ggt 1104 Asp His
Arg Ala Lys Gly Gln Leu Tyr Cys Pro Glu Ser Tyr Ser Gly 355 360 365
ggc tgg tgg ttc agt gac atg tgt gga gaa aac aac cta aat ggt aaa
1152 Gly Trp Trp Phe Ser Asp Met Cys Gly Glu Asn Asn Leu Asn Gly
Lys 370 375 380 tac aac aaa ccc aga gcc aaa tcc aaa cca gag cgg aga
aga ggg atc 1200 Tyr Asn Lys Pro Arg Ala Lys Ser Lys Pro Glu Arg
Arg Arg Gly Ile 385 390 395 400 tcc tgg agg cct cgg ggc gga aag ctc
tac tct atc aaa tca tct aaa 1248 Ser Trp Arg Pro Arg Gly Gly Lys
Leu Tyr Ser Ile Lys Ser Ser Lys 405 410 415 atg atg ctc cag ccg acc
acc taa 1272 Met Met Leu Gln Pro Thr Thr 420
* * * * *
References