U.S. patent application number 13/316377 was filed with the patent office on 2012-08-23 for compositions and methods for the modulation of jnk proteins.
This patent application is currently assigned to Isis Pharmaceuticals, Inc.. Invention is credited to Sanjay Bhanot, Xing-Xian Yu.
Application Number | 20120214736 13/316377 |
Document ID | / |
Family ID | 38997495 |
Filed Date | 2012-08-23 |
United States Patent
Application |
20120214736 |
Kind Code |
A1 |
Yu; Xing-Xian ; et
al. |
August 23, 2012 |
COMPOSITIONS AND METHODS FOR THE MODULATION OF JNK PROTEINS
Abstract
The invention provides compositions and methods for the
treatment and diagnosis of diseases or disorders amenable to
treatment through modulation of expression of a gene encoding a Jun
N-terminal kinase 1 (JNK1 protein.
Inventors: |
Yu; Xing-Xian; (San Diego,
CA) ; Bhanot; Sanjay; (Carlsbad, CA) |
Assignee: |
Isis Pharmaceuticals, Inc.
Carlsbad
CA
|
Family ID: |
38997495 |
Appl. No.: |
13/316377 |
Filed: |
December 9, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11834672 |
Aug 6, 2007 |
8101585 |
|
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13316377 |
|
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|
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60835822 |
Aug 4, 2006 |
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Current U.S.
Class: |
514/6.5 ;
514/44A |
Current CPC
Class: |
A61P 43/00 20180101;
C12N 2310/341 20130101; A61K 31/07 20130101; A61P 25/18 20180101;
A61P 9/10 20180101; C12N 15/1137 20130101; C12N 2310/11 20130101;
A61P 3/06 20180101; A61P 3/10 20180101; C12N 2310/346 20130101;
A61P 3/04 20180101; A61P 1/16 20180101; C12N 2310/321 20130101;
C12N 2310/3341 20130101; C12N 2310/315 20130101; A61P 3/00
20180101; A61P 9/00 20180101; C12N 2310/321 20130101; C12N
2310/3525 20130101 |
Class at
Publication: |
514/6.5 ;
514/44.A |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; A61P 3/10 20060101 A61P003/10; A61K 31/712 20060101
A61K031/712; A61K 38/28 20060101 A61K038/28 |
Claims
1-10. (canceled)
11. A method of treating diabetes in a subject comprising
administering to said subject a pharmaceutical composition
comprising a glucose-lowering agent and a therapeutically effective
amount of an antisense compound targeted to a JNK1 nucleic
acid.
12. A method of treating diabetes in a subject comprising
administering to said subject a glucose-lowering agent and a
therapeutically effective amount of an antisense compound targeted
to a JNK1 nucleic acid.
13. The method of claim 12, wherein the administering thereby
reduces glucose levels or improves insulin sensitivity or both.
14. The method of claim 12, wherein said glucose-lowering agent is
insulin or an insulin-analog, a biguanide, a meglitinide, a
thiazolidinedione, sulfonylurea or an alpha-glucosidase
inhibitor.
15. The method of claim 14, wherein the glucose lowering agent is a
biguanide.
16. The method of claim 15, wherein the biguanide is metformin.
17. The method of claim 14, wherein the glucose lowering agent is a
meglitinide.
18. The method of claim 17, wherein the meglitinide is nateglinide
or repaglinide.
19. The method of claim 14, wherein the glucose lowering agent is a
thiazolidinedione.
20. The method of claim 19, wherein the thiazolidinedione is
nateglinide pioglitazone, rosiglitazone, or troglitazone.
21. The method of claim 20, wherein the glucose lowering agent is a
rosiglitazone.
22. The method of claim 21, wherein blood glucose levels are
decreased and body weight is maintained or reduced.
23. The method of claim 14, wherein the glucose lowering agent is
an alpha-glucosidase inhibitor.
24. The method of claim 23, wherein the alpha-glucosidase inhibitor
is acarbose or miglitol.
25. The method of claim 14, wherein the glucose lowering agent is
sulfonylurea.
26-37. (canceled)
38. The method of claim 12 wherein the glucose lowering agent and
antisense compound are administered concomitantly.
39. The method of claim 12, wherein the administering comprises
parenteral administration.
40. The method of claim 39, wherein the parenteral administration
comprises subcutaneous or intravenous administration.
41. (canceled)
42. (canceled)
43. The method of claim 12, wherein the antisense compound has at
least 95% complementarity to SEQ ID NO: 87, 89, 90 or 91.
44. The method of claim 12, wherein the antisense compound has 100%
complementarity to SEQ ID NO: 87, 89, 90 or 91.
45. The method of claim 12, wherein the antisense compound is 12 to
30 nucleosides in length.
46-48. (canceled)
49. The method of claim 12, wherein the antisense compound is an
antisense oligonucleotide.
50. The method of claims 12, wherein the antisense compound
comprises at least one modified sugar moiety.
51. The method of claim 50, wherein the modified sugar moiety is a
2'-O-methoxyethyl sugar moiety.
52-58. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/834,672 filed Aug. 6, 2007, allowed Sep. 9, 2011, which
claims the benefit of priority under 35 U.S.C. 119(e) to U.S.
Provisional Application No. 60/835,822, filed Aug. 4, 2006, 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 BIOL0089USC1SEQ.txt, created on Dec. 7, 2011 which is
64 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] The present invention provides compositions and methods for
detecting and modulating levels of Jun N-terminal kinases (JNK
proteins), enzymes which are encoded by JNK genes.
BACKGROUND OF THE INVENTION
[0004] The rapid increase in the prevalence of obesity, type-2
diabetes and associated complications is a major global health
problem. About two-thirds of adults in the United States are
overweight, and almost one-third are obese, according to data from
the National Health and Nutrition Examination Survey (NHANES) 2001
to 2004. While overweight and obesity are found worldwide, the
prevalence of these conditions in the United States ranks high
among developed nations. Overweight refers to an excess of body
weight compared to set standards. The excess weight can come from
muscle, bone, fat, and/or body water. Obesity refers specifically
to having an abnormally high proportion of body fat. Individuals
who are obese have a 10- to 50-percent increased risk of death from
all causes, compared with healthy weight individuals. Most of the
increased risk is due to cardiovascular causes. Obesity is
associated with about 112,000 excess deaths per year in the U.S.
population relative to healthy weight individuals. Obesity is a
known risk factor for diabetes, coronary heart disease, high blood
cholesterol, stroke, hypertension, gallbladder disease,
osteoarthritis, sleep apnea and other breathing problems as well as
some forms of cancer (breast, colorectal, endometrial, and
kidney).
[0005] Diabetes is a disorder characterized by hyperglycemia due to
deficient insulin action that can result from reduced insulin
production or insulin resistance or both. Additionally,
glucotoxicity, which results from long-term hyperglycemia, induces
tissue-dependent insulin resistance exacerbating the disease.
Chronic hyperglycemia is also a major risk factor for
diabetes-associated complications, including heart disease,
retinopathy, nephropathy and neuropathy. Diabetes and obesity,
sometimes now collectively referred to as "diabesity") are
interrelated in that obesity is known to exacerbate the pathology
of diabetes and greater than 60% of diabetics are obese. Most human
obesity is associated with insulin resistance and leptin
resistance. Obesity can have an even greater impact on insulin
action than diabetes itself.
[0006] Effective treatments are needed for diabetes, obesity,
metabolic syndrome and other diseases and conditions associated
with glucose and/or lipid metabolism and/or the disregulation
thereof. The present invention satisfies this need and provides
related advantages as well.
SUMMARY OF THE INVENTION
[0007] The present invention provides methods of reducing glucose
levels in a subject by administering a therapeutically effective
amount of an antisense compound targeted to a JNK1 nucleic acid. In
a distinct embodiment, the invention also provides methods of
reducing lipid levels in a subject by administering a
therapeutically effective amount of an antisense compound targeted
to a JNK1 nucleic acid. In a further embodiment, methods of
treating metabolic syndrome in a subject by administering a
therapeutically effective amount of an antisense compound targeted
to a JNK1 nucleic acid also are provided by the present
invention.
[0008] In additional distinct embodiments, the invention also
provides methods of treating obesity, diabetes and metabolic
syndrome in a subject. The methods of the invention encompass
administration of a therapeutically effective amount of an
antisense compound targeted to a JNK1 nucleic acid to a subject in
need thereof.
[0009] The invention also provides methods of treating diabetes in
a subject by administering a glucose-lowering agent and a
therapeutically effective amount of an antisense compound targeted
to a JNK1 nucleic acid. In particular embodiments, the methods of
treating diabetes in a subject include administering a
pharmaceutical composition encompassing a glucose-lowering agent
and a therapeutically effective amount of an antisense compound
targeted to a JNK1 nucleic acid.
[0010] Also provided are methods of treating diabetes, diabetes
and/or metabolic syndrome in a subject by administering a lipid
lowering agent and a therapeutically effective amount of an
antisense compound targeted to a JNK1 nucleic acid. In certain
embodiments, the methods of treating diabetes in a subject include
administering a pharmaceutical composition encompassing a lipid
lowering agent and a therapeutically effective amount of an
antisense compound targeted to a JNK1 nucleic acid.
[0011] Antisense compounds useful for practicing the claimed
methods, including antisense oligonucleotides, that are
complementary to SEQ ID NOS: 87, 89, 90 and 91 also are
provided.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0012] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the invention(s) belong. 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, chemical analysis, pharmaceutical preparation,
formulation and delivery, and treatment of subjects. Certain such
techniques and procedures can be found for example in "Carbohydrate
Modifications in Antisense Research" Edited by Sangvi and Cook,
American Chemical Society, Washington D.C., 1994; and "Remington's
Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 18th
edition, 1990; and which is hereby incorporated by reference for
any purpose. Where permitted, all patents, patent applications,
published applications and publications, GENBANK sequences,
websites and other published materials referred to throughout the
entire disclosure herein, unless noted otherwise, are incorporated
by reference in their entirety. In the event that there is a
plurality of definitions for terms herein, those in this section
prevail. Where reference is made to a URL or other such identifier
or address, it is understood that such identifiers can change and
particular information on the internet can command go, but
equivalent information can be found by searching the internet.
Reference thereto evidences the availability and public
dissemination of such information.
[0013] "Obesity" is defined as an excessively high amount of body
fat or adipose tissue in relation to lean body mass. The amount of
body fat (or adiposity) includes concern for both the distribution
of fat throughout the body and the size 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.
[0014] Insulin resistance is a condition in which normal amounts of
insulin are inadequate to produce a normal insulin response from
fat, muscle and liver cells. Insulin resistance in fat cells
results in hydrolysis of stored triglycerides, which elevates free
fatty acids in the blood plasma. Insulin resistance in muscle
reduces glucose uptake whereas insulin resistance in liver reduces
glucose storage, with both effects serving to elevate blood
glucose. High plasma levels of insulin and glucose due to insulin
resistance often leads to metabolic syndrome and type 2
diabetes.
[0015] "Type 2 diabetes," (also known as diabetes mellitus type 2,
and formerly called diabetes mellitus type II,
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.
[0016] A glucose tolerance test is the administration of glucose to
determine how quickly it is cleared from the blood. The test is
usually used to test for diabetes, insulin resistance, and
sometimes reactive hypoglycemia. The glucose is most often given
orally so the common test is technically an oral glucose tolerance
test (OGTT).
[0017] "Metabolic rate" refers to the amount of energy expended.
Basal metabolic rate (also known as BMR) is the amount of energy
expended while at rest in a neutrally temperate environment, in the
post-absorptive state (meaning that the digestive system is
inactive, which requires about twelve hours of fasting in humans).
The release of energy in this state is sufficient only for the
functioning of the vital organs, such as the heart, lungs, brain
and the rest of the nervous system, liver, kidneys, sex organs,
muscles and skin. BMR decreases with age and with the loss of lean
body mass. Increased cardiovascular exercise and muscle mass can
increase BMR. Illness, previously consumed food and beverages,
environmental temperature, and stress levels can affect one's
overall energy expenditure, and can affect one's BMR as revealed by
gas analysis. It is measured when the person is at complete rest,
but awake. An accurate BMR measurement requires that the person's
sympathetic nervous system is not stimulated. Basal metabolic rate
is measured under very restrictive circumstances. A more common and
closely related measurement, used under less strict conditions, is
resting metabolic rate (RMR). "Metabolic" and "metabolism" are
terms well know 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 II), obesity, insulin
resistance and metabolic syndrome.
[0018] As used herein, the terms "treatment" and "treating" refer
to administering a composition of the invention to effect an
alteration or improvement of the disease or condition. Prevention,
amelioration, and/or treatment can require administration of
multiple doses at regular intervals, or prior to onset of the
disease or condition to alter the course of the disease or
condition. Moreover, a single agent can be used in a single
individual for each prevention, amelioration, and treatment of a
condition or disease sequentially, or concurrently.
[0019] As used herein, the term "pharmaceutical agent," including,
for example, an antisense oligonucleotide, a lipid lowering agent
or a glucose lowering agent, refers to a substance provides a
therapeutic benefit when administered to a subject. In certain
embodiments, an antisense oligonucleotide targeted to JNK1 is a
pharmaceutical agent.
[0020] "Therapeutically effective amount" means an amount of a
pharmaceutical agent that provides a therapeutic benefit to a
subject. In certain embodiments, a therapeutically effective amount
of antisense compound targeted to a JNK1 nucleic acid is an amount
that decreases LDL-C in the subject.
[0021] A "pharmaceutical composition" means a mixture of substances
suitable for administering to a subject. A pharmaceutical
composition can comprise, for example, a combination of antisense
oligonucleotides, a combination of antisense oligonucleotides and
non-antisense pharmaceutical agents as well as the presence of a
sterile aqueous solution or other standard pharmaceitcal additive
known in the art.
[0022] "Administering" means providing a pharmaceutical agent or
composition to a subject, and includes, but is not limited to
administering by a medical professional and self-administering.
Co-administration is the administration of two or more
pharmaceutical agents to an animal. The two or more pharmaceutical
agents can be in a single pharmaceutical composition, or can be in
separate pharmaceutical compositions. Each of the two or more
pharmaceutical agents can be administered through the same or
different routes of administration. Co-administration encompasses
administration in parallel, concomitant or sequentially.
[0023] As used herein, the term "subject" refers to an animal,
including, but not limited to a human, to whom a pharmaceutical
composition is administered. Animals include humans or non-human
animal, including, but not limited to, mice, rats, rabbits, dogs,
cats, pigs, and non-human primates, including, but not limited to,
monkeys and chimpanzees.
[0024] "Parenteral administration," means administration through
injection or infusion. Parenteral administration includes, but is
not limited to, subcutaneous administration, intravenous
administration, or intramuscular administration.
[0025] Duration refers to the period of time during which an
activity or event continues. In certain embodiments, the duration
of treatment is the period of time during which doses of a
pharmaceutical agent are administered.
[0026] "Subcutaneous administration" means administration just
below the skin. "Intravenous administration" means administration
into a vein.
[0027] Dose means a specified quantity of a pharmaceutical agent
provided in a single administration. In certain embodiments, a dose
can be administered in 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. In such embodiments, two
or more injections can be used to achieve the desired dose. In
certain embodiments, a dose can be administered in two or more
injections to minimize injection site reaction in a subject. Dosage
unit is the form in which a pharmaceutical agent is provided. 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.
[0028] "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).
[0029] As used herein, the term 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,
and age.
[0030] "CHD risk factors" mean CHD risk equivalents and major risk
factors.
[0031] "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.
[0032] "Reduced coronary heart disease risk" means a reduction in
the likelihood that a subject will develop coronary heart disease.
In certain embodiments, a reduction in coronary heart disease risk
is measured by an improvement in one or more CHD risk factors, for
example, a decrease in LDL-C levels.
[0033] "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.
[0034] "History of coronary heart disease" means the occurrence of
clinically evident coronary heart disease in the medical history of
a subject or a subject's family member.
[0035] "Early onset coronary heart disease" means a diagnosis of
coronary heart disease prior to age 50.
[0036] "Statin intolerant individual" means a individual who as a
result of statin therapy experiences one or more of creatine kinase
increases, liver function test abnormalities, muscle aches, or
central nervous system side effects.
[0037] "Efficacy" means the ability to produce a desired effect.
For example, efficacy of a lipid-lowering therapy can be reduction
in the concentration of one or more of LDL-C, VLDL-C, IDL-C,
non-HDL-C, ApoB, lipoprotein(a), or triglycerides.
[0038] "Acceptable safety profile" means a pattern of side effects
that is within clinically acceptable limits.
[0039] "Side effects" means physiological responses attributable to
a treatment other than desired effects. In certain embodiments,
side effects include, without limitation, injection site reactions,
liver function test abnormalities, renal function abnormalities,
liver toxicity, renal toxicity, central nervous system
abnormalities, and myopathies. 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.
[0040] "Injection site reaction" means inflammation or abnormal
redness of skin at a site of injection in a subject.
[0041] "Individual compliance" means adherence to a recommended or
prescribed therapy by a subject.
[0042] "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 provide to reduce
one or more of ApoB, total cholesterol, LDL-C, VLDL-C, IDL-C,
non-HDL-C, triglycerides, small dense LDL particles, and Lp(a) in a
subject.
[0043] "Lipid-lowering agent" means a pharmaceutical agent provided
to a subject to achieve a lowering of lipids in the individual. For
example, in certain embodiments, a lipid-lowering agent is provided
to a subject to reduce one or more of ApoB, LDL-C, total
cholesterol, and triglycerides.
[0044] "LDL-C target" means an LDL-C level that is desired
following lipid-lowering therapy.
[0045] "Comply" means the adherence with a recommended therapy by a
subject.
[0046] "Recommended therapy" means a therapeutic regimen
recommended by a medical professional for the treatment,
amelioration, or prevention of a disease.
[0047] "Low LDL-receptor activity" means LDL-receptor activity that
is not sufficiently high to maintain clinically acceptable levels
of LDL-C in the bloodstream.
[0048] "Cardiovascular outcome" means the occurrence of major
adverse cardiovascular events.
[0049] "Improved cardiovascular outcome" means a reduction in the
occurrence of major adverse cardiovascular events, or the risk
thereof. Examples of major adverse cardiovascular events include,
without limitation, death, reinfarction, stroke, cardiogenic shock,
pulmonary edema, cardiac arrest, and atrial dysrhythmia.
[0050] "Surrogate markers of cardiovascular outcome" means indirect
indicators of cardiovascular events, or the risk thereof. For
example, surrogate markers of cardiovascular outcome include
carotid intimal media thickness (CIMT). Another example of a
surrogate marker of cardiovascular outcome includes atheroma size.
Atheroma size can be determined by intravascular ultrasound
(IVUS).
[0051] "Increased HDL-C" means an increase in serum HDL-C in a
subject over time.
[0052] "Lipid-lowering" means a reduction in one or more serum
lipids in a subject over time.
[0053] "Therapeutic lifestyle change" means dietary and lifestyle
changes intended to lower cholesterol and reduce the risk of
developing heart disease, and includes recommendations for dietary
intake of total daily calories, total fat, saturated fat,
polyunsaturated fat, monounsaturated fat, carbohydrate, protein,
cholesterol, insoluble fiber, as well as recommendations for
physical activity.
[0054] "Statin" means a pharmaceutical agent that inhibits the
activity of HMG-CoA reductase.
[0055] "HMG-CoA reductase inhibitor" means a pharmaceutical agent
that acts through the inhibition of the enzyme HMG-CoA
reductase.
[0056] "Cholesterol absorption inhibitor" means a pharmaceutical
agent that inhibits the absorption of exogenous cholesterol
obtained from diet.
[0057] "LDL apheresis" means a form of apheresis by which LDL-C is
removed from blood. Typically, a subject's blood is removed from a
vein, and separated into red cells and plasma. LDL-C is filtered
out of the plasma prior to return of the plasma and red blood cells
to the individual.
[0058] "MTP inhibitor" means a pharmaceutical agent that inhibits
the enzyme microsomal triglyceride transfer protein.
[0059] "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.
[0060] "Very low density lipoprotein-cholesterol (VLDL-C)" means
cholesterol associated with very low density lipoprotein particles.
Concentration of VLDL-C in serum (or plasma) is typically
quantified in mg/dL or nmol/L. "Serum VLDL-C" and "plasma VLDL-C"
mean VLDL-C in the serum or plasma, respectively.
[0061] "Intermediate low density lipoprotein-cholesterol (IDL-C)"
means cholesterol associated with intermediate density lipoprotein.
Concentration of IDL-C in serum (or plasma) is typically quantified
in mg/mL or nmol/L. "Serum IDL-C" and "plasma IDL-C" mean IDL-C in
the serum or plasma, respectively.
[0062] "Non-high density lipoprotein-cholesterol (Non-HDL-C)" means
cholesterol associated with lipoproteins other than high density
lipoproteins, and includes, without limitation, LDL-C, VLDL-C, and
IDL-C.
[0063] "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 the serum
and plasma, respectively.
[0064] "Total cholesterol" means all types of cholesterol,
including, but not limited to, LDL-C, HDL-C, IDL-C and VLDL-C.
Concentration of total cholesterol in serum (or plasma) is
typically quantified in mg/dL or nmol/L.
[0065] "Lipoprotein(a)" or "Lp(a)" means a lipoprotein particle
that is comprised of LDL-C, an apolipoprotein(a) particle, and an
apolipoproteinB-100 particle.
[0066] "ApoA1" means apolipoprotein-A1 protein in serum.
Concentration of ApoA1 in serum is typically quantified in mg/dL or
nmol/L.
[0067] "ApoB:ApoA1 ratio" means the ratio of ApoB concentration to
ApoA1 concentration.
[0068] "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).
[0069] "Small LDL particle" means a subclass of LDL particles
characterized by a smaller, denser size compared to other LDL
particles. In certain embodiments, intermediate LDL particles are
23-27 nm in diameter. In certain embodiments, large LDL particles
are 21.2-23 nm in diameter. In certain embodiments, small LDL
particles are 18-21.2 nm in diameter. In certain embodiments,
particle size is measured by nuclear magnetic resonance
analysis.
[0070] "Small VLDL particle" means a subclass of VLDL particles
characterized by a smaller, denser size compared to other VLDL
particles. In certain embodiments, large VLDL particles are greater
than 60 nm in diameter. In certain embodiments, medium VLDL
particles are 35-60 nm in diameter. In certain embodiments, small
VLDL particles are 27-35 nm in diameter. In certain embodiments,
particle size is measured by nuclear magnetic resonance
analysis.
[0071] "Triglycerides" means lipids that are the triesters of
glycerol. "Serum triglycerides" mean triglycerides present in
serum. "Liver triglycerides" mean triglycerides present in liver
tissue.
[0072] "Serum lipids" mean cholesterol and triglycerides in the
serum.
[0073] "Elevated total cholesterol" means total cholesterol at a
concentration in a subject at which lipid-lowering therapy is
recommended, and includes, without limitation, elevated LDL-C'',
"elevated VLDL-C," "elevated IDL-C" and "elevated non-HDL-C." In
certain embodiments, total cholesterol concentrations of less than
200 mg/dL, 200-239 mg/dL, and greater than 240 mg/dL are considered
desirable, borderline high, and high, respectively. In certain
embodiments, LDL-C concentrations of 100 mg/dL, 100-129 mg/dL,
130-159 mg/dL, 160-189 mg/dL, and greater than 190 mg/dL are
considered optimal, near optimal/above optimal, borderline high,
high, and very high, respectively.
[0074] "Elevated triglyceride" means concentrations of triglyceride
in the serum or liver at which lipid-lowering therapy is
recommended, and includes "elevated serum triglyceride" and
"elevated liver triglyceride." In certain embodiments, serum
triglyceride concentration of 150-199 mg/dL, 200-499 mg/dL, and
greater than or equal to 500 mg/dL is considered borderline high,
high, and very high, respectively.
[0075] "Elevated small LDL particles" means a concentration of
small LDL particles in a subject at which lipid-lowering therapy is
recommended.
[0076] "Elevated small VLDL particles" means a concentration of
small VLDL particles in a subject at which lipid-lowering therapy
is recommended.
[0077] "Elevated lipoprotein(a)" means a concentration of
lipoprotein(a) in a subject at which lipid-lowering therapy is
recommended.
[0078] "Low HDL-C" means a concentration of HDL-C in a subject at
which lipid-lowering therapy is recommended. In certain embodiments
lipid-lowering therapy is recommended when low HDL-C is accompanied
by elevations in non-HDL-C and/or elevations in triglyceride. In
certain embodiments, HDL-C concentrations of less than 40 mg/dL are
considered low. In certain embodiments, HDL-C concentrations of
less than 50 mg/dL are considered low.
[0079] "ApoB" means apolipoprotein B-100 protein. Concentration of
ApoB in serum (or plasma) is typically quantified in mg/dL or
nmol/L. "Serum ApoB" and "plasma ApoB" mean ApoB in the serum and
plasma, respectively.
[0080] "LDL/HDL ratio" means the ratio of LDL-C to HDL-C.
[0081] "Oxidized-LDL" or "Ox-LDL-C" means LDL-C that is oxidized
following exposure to free radicals.
[0082] "Individual having elevated LDL-C levels" means a subject
who has been identified by a medical professional (e.g. a
physician) as having LDL-C levels near or above the level at which
therapeutic intervention is recommended, according to guidelines
recognized by medical professionals. Such a subject can also be
considered "in need of treatment" to decrease LDL-C levels.
[0083] "Individual having elevated apoB-100 levels" means a subject
who has been identified as having apoB-100 levels near or below the
level at which therapeutic intervention is recommended, according
to guidelines recognized by medical professionals. Such a subject
can also be considered "in need of treatment" to decrease apoB-100
levels.
[0084] "Treatment of elevated LDL-C levels" means administration of
an antisense compound targeted to a JNK1 nucleic acid to a subject
having elevated LDL-C levels.
[0085] "Treatment of atherosclerosis" means administration of an
antisense compound targeted to a JNK1 nucleic acid to a subject
who, based upon a physician's assessment, has or is likely to have
atherosclerosis. "Prevention of atherosclerosis" means
administration of an antisense compound targeted to a JNK1 nucleic
acid to a subject who, based upon a physician's assessment, is
susceptible to atherosclerosis.
[0086] As used herein, the term "modulation" refers to a
perturbation of function or activity when compared to the level of
the function or activity prior to modulation. For example,
modulation includes the change, either an increase (stimulation or
induction) or a decrease (inhibition or reduction) in gene
expression. As further example, modulation of expression can
include perturbing splice site selection of pre-mRNA
processing.
[0087] As used herein, the term "expression" refers to all the
functions and steps by which a gene's coded information is
converted into structures present and operating in a cell. Such
structures include, but are not limited to the products of
transcription and translation.
[0088] "Antisense inhibition" means reduction of a target nucleic
acid levels in the presence of an antisense compound complementary
to a target nucleic acid compared to target nucleic acid levels in
the absence of the antisense compound.
[0089] As used herein, the term "target" refers to a protein, the
modulation of which is desired.
[0090] As used herein, the term "target gene" refers to a gene
encoding a target.
[0091] "Targeting" means the process of design and selection of an
antisense compound that will specifically hybridize to a target
nucleic acid or a particular region of nucleotides within a target
nucleic acid molecule and induce a desired effect.
[0092] "Targeted" means having a nucleobase sequence that will
allow specific hybridization of an antisense compound to a target
nucleic acid or a particular region of nucleotides within a target
nucleic acid molecule to induce a desired effect. In certain
embodiments, a desired effect is reduction of a target nucleic
acid. In certain such embodiments, a desired effect is reduction of
JNK1 mRNA.
[0093] As used herein, the terms "target nucleic acid," "target
RNA," "target RNA transcript," "nucleic acid target" and "nucleic
acid molecule encoding a target" refer to any nucleic acid molecule
the expression or activity of which is capable of being modulated
by an antisense compound. Target nucleic acids include, but are not
limited to, RNA (including, but not limited to pre-mRNA and mRNA or
portions thereof) transcribed from DNA encoding a target, and also
cDNA derived from such RNA, and miRNA. For example, the target
nucleic acid can be a cellular gene (or mRNA transcribed from the
gene) whose expression is associated with a particular disorder or
disease state, or a nucleic acid molecule from an infectious
agent.
[0094] A "JNK1 nucleic acid" means any nucleic acid encoding JNK1.
For example, in certain embodiments, a JNK1 nucleic acid includes,
without limitation, a DNA sequence encoding JNK1, an RNA sequence
transcribed from DNA encoding JNK1, and an mRNA sequence encoding
JNK1. "JNK1 mRNA" means an mRNA encoding a JNK1 protein.
[0095] As used herein, the term "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.
[0096] As used herein, the term "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.
[0097] As used herein, the term "target region," refers to a
portion of a target nucleic acid to which one or more antisense
compounds is complementary.
[0098] As used herein, the term "target segment" refers to a
smaller or sub-portions of a region within a target nucleic
acid.
[0099] As used herein, the term "complementarity" refers to the
ability of a nucleobase to base pair with another nucleobase. For
example, in DNA, adenine (A) is complementary to thymine (T). For
example, in RNA, adenine (A) is complementary to uracil (U). In
certain embodiments, complementary nucleobase refers to a
nucleobase of an antisense compound that is capable of base pairing
with a nucleobase of its target nucleic acid. For example, if a
nucleobase at a certain position of an antisense compound is
capable of hydrogen bonding with a nucleobase at a certain position
of a target nucleic acid, then the position of hydrogen bonding
between the oligonucleotide and the target nucleic acid is
considered to be complementary at that nucleobase pair.
[0100] As used herein, the term "non-complementary nucleobase"
refers to a nucleobase that does not form hydrogen bonds with
another nucleobase or otherwise support hybridization.
[0101] As used herein, the term "complementary" refers to the
capacity of an oligomeric compound to hybridize to another
oligomeric compound or nucleic acid through base pairing. In
certain embodiments, an antisense compound and its target are
complementary to each other when a sufficient number of
corresponding positions in each molecule are occupied by
nucleobases that can pair with each other to allow stable
association between the antisense compound and the target. One
skilled in the art recognizes that the inclusion of mismatches is
possible without eliminating the ability of the oligomeric
compounds to remain in association. Therefore, described herein are
antisense compounds that can comprise up to about 20% nucleotides
that are mismatched (i.e., are not nucleobase complementary to the
corresponding nucleotides of the target). Preferably the antisense
compounds contain no more than about 15%, more preferably not more
than about 10%, most preferably not more than 5% or no mismatches.
The remaining nucleotides are nucleobase complementary or otherwise
do not disrupt hybridization (e.g., universal bases). One of
ordinary skill in the art would recognize the compounds provided
herein are at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
complementary to a target nucleic acid.
[0102] As used herein, the term "mismatch" refers to a
non-complementary nucleobase within a complementary oligomeric
compound.
[0103] As used herein, "hybridization" means the pairing of
complementary oligomeric compounds (e.g., an antisense compound and
its target nucleic acid). While not limited to a particular
mechanism, the most common mechanism of pairing involves hydrogen
bonding, which can be Watson-Crick, Hoogsteen or reversed Hoogsteen
hydrogen bonding, between complementary nucleoside or nucleotide
bases (nucleobases). For example, the natural base adenine is
nucleobase complementary to the natural nucleobases thymidine and
uracil which pair through the formation of hydrogen bonds. The
natural base guanine is nucleobase complementary to the natural
bases cytosine and 5-methyl cytosine. Hybridization can occur under
varying circumstances.
[0104] As used herein, the term "specifically hybridizes" refers to
the ability of an oligomeric compound to hybridize to one nucleic
acid site with greater affinity than it hybridizes to another
nucleic acid site. In certain embodiments, an antisense
oligonucleotide specifically hybridizes to more than one target
site.
[0105] As used herein, "designing" or "designed to" refer to the
process of designing an oligomeric compound that specifically
hybridizes with a selected nucleic acid molecule.
[0106] "Portion" means a defined number of contiguous (i.e. linked)
nucleobases of a target nucleic acid. In certain embodiments, a
portion is a defined number of contiguous nucleobases of a target
nucleic acid. In certain embodiments, a portion is a defined number
of contiguous nucleobases of an antisense compound.
[0107] As used herein, the term "oligomeric compound" 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.
[0108] As used herein, the term "antisense compound" refers to an
oligomeric compound that is at least partially complementary to a
target nucleic acid molecule to which it hybridizes. In certain
embodiments, an antisense compound modulates (increases or
decreases) expression of a target nucleic acid. Antisense compounds
include, but are not limited to, compounds that are
oligonucleotides, oligonucleosides, oligonucleotide analogs,
oligonucleotide mimetics, and chimeric combinations of these.
Consequently, while all antisense compounds are oligomeric
compounds, not all oligomeric compounds are antisense
compounds.
[0109] As used herein, the term "oligonucleotide" refers to an
oligomeric compound comprising a plurality of linked nucleotides.
In certain embodiment, one or more nucleotides of an
oligonucleotide is modified. In certain embodiments, an
oligonucleotide contains ribonucleic acid (RNA) or deoxyribonucleic
acid (DNA). In certain embodiments, oligonucleotides are composed
of naturally- and/or non-naturally-occurring nucleobases, sugars
and covalent internucleotide linkages, and can further include
non-nucleic acid conjugates.
[0110] "Oligonucleoside" means an oligonucleotide in which the
internucleoside linkages do not contain a phosphorus atom.
[0111] "Antisense oligonucleotide" means a single-stranded
oligonucleotide having a nucleobase sequence that will permits
hybridization to a corresponding region of a target nucleic
acid.
[0112] "Motif" means the pattern of unmodified and modified
nucleosides in an antisense compound.
[0113] "Chimeric antisense compounds" means an antisense compounds
that have at least 2 chemically distinct regions, each region
having a plurality of subunits.
[0114] As used herein, the term "gapmer" refers to a chimeric
oligomeric compound comprising a central region (a "gap") and a
region on either side of the central region (the "wings"), wherein
the gap comprises at least one modification that is different from
that of each wing. Such modifications include nucleobase, monomeric
linkage, and sugar modifications as well as the absence of
modification (unmodified). The gap region generally supports RNaseH
cleavage.
[0115] As used herein, the term "nucleoside" means a glycosylamine
comprising a nucleobase and a sugar. Nucleosides includes, but are
not limited to, naturally occurring nucleosides, abasic
nucleosides, modified nucleosides, and nucleosides having mimetic
bases and/or sugar groups.
[0116] As used herein, the term "nucleotide" refers to a
glycosomine comprising a nucleobase and a sugar having a phosphate
group covalently linked to the sugar. Nucleotides can be modified
with any of a variety of substituents.
[0117] As used herein, the term "nucleobase" refers to the base
portion of a nucleoside or nucleotide. A nucleobase can comprise
any atom or group of atoms capable of hydrogen bonding to a base of
another nucleic acid.
[0118] As used herein, the term "monomer" refers to a single unit
of an oligomer. Monomers include, but are not limited to,
nucleosides and nucleotides, whether naturally occurring or
modified.
[0119] As used herein, the term "deoxyribonucleotide" means a
nucleotide having a hydrogen at the 2' position of the sugar
portion of the nucleotide. Deoxyribonucleotides can be modified
with any of a variety of substituents.
[0120] As used herein, the term "ribonucleotide" means a nucleotide
having a hydroxy at the 2' position of the sugar portion of the
nucleotide. Ribonucleotides can be modified with any of a variety
of substituents.
[0121] "Unmodified nucleotide" means a nucleotide composed of
naturally occurring nucleobases, sugar moieties and internucleoside
linkages. In certain embodiments, an unmodified nucleotide is an
RNA nucleotide (i.e., .beta.-D-ribonucleosides) or a DNA nucleotide
(i.e., .beta.-D-deoxyribonucleoside).
[0122] "Modified nucleotide" means a nucleotide having,
independently, a modified sugar moiety, modified internucleoside
linkage, or modified nucleobase. A "modified nucleoside" means a
nucleotide having, independently, a modified sugar moiety or
modified nucleobase.
[0123] "Internucleoside linkage" means a covalent linkage between
adjacent nucleosides.
[0124] "Naturally occurring internucleoside linkage" means a 3' to
5' phosphodiester linkage.
[0125] "Modified internucleoside linkage" means substitution and/or
any change from a naturally occurring internucleoside linkage
[0126] "Modified sugar moiety" means substitution and/or any change
from a natural sugar moiety. For the purposes of this disclosure, a
"natural sugar moiety" is a sugar moiety found in DNA (2'-H) or RNA
(2'-OH).
[0127] "Modified nucleobase" means 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).
[0128] "Natural sugar moiety" means a sugar moiety found in DNA
(2'-H) or RNA (2'-OH).
[0129] "2'-O-methoxyethyl sugar moiety" means a 2'-substituted
furanosyl ring having a 2'-O(CH2)2-OCH3 (2'-O-methoxyethyl or
2'-MOE) substituent group.
[0130] "2'-O-methoxyethyl nucleotide" means a nucleotide comprising
a 2'-O-methoxyethyl modified sugar moiety.
[0131] "Bicyclic nucleic acid sugar moiety" means a furanosyl ring
modified by the bridging of two non-geminal ring atoms.
[0132] "Prodrug" means a therapeutic agent that is prepared in an
inactive form that is converted to an active form (i.e., drug)
within the body or cells thereof by the action of endogenous
enzymes or other chemicals and/or conditions.
[0133] "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.
[0134] "Cap structure" or "terminal cap moiety" means chemical
modifications, which have been incorporated at either terminus of
an antisense compound.
Overview
[0135] In the context of the invention, the terms "Jun N-terminal
kinase," "c-Jun N-terminal kinase" and "JNK1 protein" refer to
proteins actually known to phosphorylate the amino terminal
(N-terminal) portion of the Jun subunit of AP-1, as well as those
that have been tentatively identified as JNK1 proteins based on
amino acid sequence but which can in fact additionally or
alternatively bind and/or phosphorylate either other transcription
factors (e.g., ATF2) or kinase substrates that are not known to be
involved in transcription (Derijard et al., Cell, 1994, 76, 1025;
Kallunki et al., Genes & Development, 1994, 8, 2996; Gutta et
al., EMBO J., 1996, 15, 2760).
[0136] AP-1 is one member of a family of related heterodimeric
transcription factor complexes found in eukaryotic cells or viruses
(The FOR and JUN Families of Transcription Factors, Angel and
Hairlike, Eds., CBC Press, Boca Raton, Fla., 1994; Bohmann et al.,
Science, 1987, 238, 1386; Angel et al., Nature, 1988, 332, 166).
Two relatively well-characterized AP-1 subunits are c-For and
c-Jun; these two proteins are products of the c-for and c-jun
proto-oncogenes, respectively. (Rahmsdorf, Chapter 13, and Rapp et
al., Chapter 16 In: The FOS and JUN Families of Transcription
Factors, Angel and Herrlich, Eds., CBC Press, Boca Raton, Fla.,
1994)
[0137] The phosphorylation of proteins plays a key role in the
transduction of extracellular signals into the cell. Typical MAP
kinase pathways are known and recited in the literature. (See e.g.,
Cano et al., Trends Biochem. Sci., 1995, 20, 117 Cobb et al., J.
Biol. Chem., 1995, 270, 14843; Seger et al., FASEB J., 1995, 9,
726; Cano et al., Trends Biochem. Sci., 1995, 20, 117).
[0138] One of the signal transduction pathways involves the MAP
kinases Jun N-terminal kinase 1 (JNK1) and Jun N-terminal kinase 2
(JNK2) which are responsible for the phosphorylation of specific
sites (Serine 63 and Serine 73) on the amino terminal portion of
c-Jun. Phosphorylation of these sites potentiates the ability of
AP-1 to activate transcription (Binetruy et al., Nature, 1991, 351,
122; Smeal et al., Nature, 1991, 354, 494). Besides JNK1 and JNK2,
other JNK family members have been described, including JNK3 (Gutta
et al., EMBO J., 1996, 15, 2760), initially named p49.sup.3F12
kinase (Mohit et al., Neuron, 1994, 14, 67).
[0139] Recent studies have indicated that JNKs interfere with
insulin action in cultured cells and are activated by free fatty
acids and inflammatory cytokines; both implicated in the
development of type-2 diabetes. Thus, JNK can be a mediator of
obesity and insulin resistance. (Hirosumi et al., Nature, 2002,
420:333-336).
[0140] JNKs or c-Jun N-terminal kinases are a family of
serine/threonine protein kinases of the mitogen-activated protein
kinase (MAPK) group and are involved in a variety of physiological
functions. They are activated in response to different stimuli
which cause cellular stress including heat shock, irradiation,
hypoxia, chemotoxins, peroxides, and some cytokines (Bennett et
al., 2003; Bogoyevitch et al., 2004). Obesity also causes cellular
stress due to mechanical changes, excess lipid accumulation,
abnormalities in intracellular energy fluxes, and altered nutrient
availability, as well as changed plasma levels of cytokines
(Hotamisligil, 2005; Ozcan et al., 2004; Waetzig and Herdegen,
2005). JNK activity is much higher in liver, fat and muscle in both
genetically obese (ob/ob) mice and diet-induced obese (DIO) mice
than in their respective controls (Hirosumi et al., 2002; Ozcan et
al., 2004).
[0141] Obesity is considered a long-term disease. There are over
thirty serious medical concerns related to obesity. 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.
[0142] The present invention is based, in part, on the discovery of
antisense compounds that target nucleic acid encoding JNK1 and
which function to reduce JNK1 levels in a subject.
[0143] Effective treatments are needed for diabetes, obesity,
metabolic syndrome and other diseases and conditions associated
with glucose and/or lipid metabolism and/or the disregulation
thereof. Certain compounds on the market for the treatment of
diabetes are known to induce weight gain, a very undesirable side
effect to the treatment of this disease. Therefore, a compound that
has the potential to treat both diabetes and obesity would provide
a significant improvement over current treatments.
[0144] A role for JNK1 in both insulin resistance and obesity is
identified and JNK1 is presented herein as a therapeutic target for
a range of metabolic diseases and conditions, including diabetes,
obesity and metabolic syndrome. Therefore, provided herein are
compounds and compositions targeting JNK1 and methods for the
treatment of metabolic diseases and conditions. Metabolic
conditions are characterized by an alteration or disturbance in
metabolic function.
[0145] In accordance with the present invention, oligonucleotides
are provided which specifically hybridize with a nucleic acid
encoding a JNK1 protein. Certain oligonucleotides of the invention
are designed to bind either directly to mRNA transcribed from, or
to a selected DNA portion of, a JNK1 gene that encodes a JNK1
protein, thereby modulating the expression thereof and/or the
phosphorylation of one or more substrates for the JNK1 protein.
Pharmaceutical compositions comprising the oligonucleotides of the
invention, and various methods of using the oligonucleotides of the
invention, including methods of modulating one or more metastatic
events, are also herein provided.
[0146] Provided herein are methods, compounds and compositions for
modulating JNK1 expression in a subject. In certain embodiments,
methods, compounds and compositions are provided for reducing JNK1
levels, expression, and/or activity in a subject. In certain
embodiments the reduction of JNK1 expression, activity and/or
nucleic acid levels occurs in liver and fat tissues of a subject.
In certain embodiments the subject is an animal. In certain
embodiments the animal is a human.
[0147] Provided herein are methods, compounds and compositions for
the treatment, prevention and/or amelioration of diseases or
conditions associated with glucose and/or lipid metabolism and/or
the disregulation thereof. In certain embodiments, the methods,
compounds and compositions are for the treatment, prevention and/or
amelioration of diabetes, obesity and metabolic syndrome. In
certain embodiments, the methods, compounds and compositions are
for the treatment, prevention and/or amelioration of
hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk
of developing atherosclerosis, coronary heart disease, a history of
coronary heart disease, early onset coronary heart disease, one or
more risk factors for coronary heart disease, type II diabetes,
type II diabetes with dyslipidemia, dyslipidemia,
hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepatic
steatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty
liver disease. In certain embodiments, such methods, compounds and
compositions are used to treat, slow, prevent, delay or ameliorate
the sequelae of diabetes including, but not limited to,
retinopathy, neuropathy, cardiovascular complications and
nephropathy.
[0148] Provided herein are methods, compounds and compositions for
improving blood glucose control or tolerance. In certain
embodiments, the methods, compounds and compositions are for
improving insulin sensitivity. Also provided are methods, compounds
and compositions for the reduction of glucose levels. In certain
embodiments, such glucose levels can be blood, plasma and/or serum
glucose levels. In certain embodiments, such glucose levels can be
fed or fasting glucose levels
[0149] Also provided are methods, compounds and compositions for
the reduction of lipids. Also provided are methods, compounds and
compositions for the reduction of triglyceride levels in a subject.
In certain embodiments, such triglyceride levels are plasma
triglyceride levels. In certain embodiments, such triglyceride
levels are liver triglyceride levels. Also provided are methods of
improving liver steatosis. Also provided are methods, compounds and
compositions for the reduction of cholesterol levels. In certain
embodiments, such cholesterol levels are plasma cholesterol
levels.
[0150] Also provided are methods, compounds and compositions for
modulating expression of metabolic and/or lipogenic genes. In
certain embodiments, the metabolic and/or lipgenic genes listed in
Table 35 below. In certain embodiments expression levels of one or
more of ACC1, ACC2, FAS, SCD1 and DGAT1, DGAT2, RBP4, G6Pase and
PKC.epsilon. are lowered. In certain embodiments, levels are
reduced by about 30% to about 70%. Also provided are methods,
compounds and compositions for lowering lipogenesis. In certain
embodiments lipogenesis is lowered by lowering expression of such
metabolic or lipogenic genes. In certain embodiments, expression
levels of AR.beta..sub.3, UCP1, UCP2 and PPAR.alpha. are increased.
In certain embodiments, levels are increased by up to about 70%.
Such methods include administering to a subject an antisense
compound targeted to a nucleic acid encoding JNK1. In certain
embodiments, such methods include the administration of a
therapeutically effective amount of an antisense compound targeted
to a JNK1 nucleic acid. In certain embodiments, the compound is
administered in a composition. In certain embodiments the subject
is an animal. In certain embodiments the animal is a human. In
certain embodiments, the subject to which the antisense compound is
administered and in which levels are modulated has one or more of
the diseases or disorders listed above. In certain embodiments, the
subject to which the antisense compound is administered and in
which levels are lowered has obesity, hypercholesterolemia, mixed
dyslipidemia, atherosclerosis, coronary heart disease, diabetes,
type II diabetes, type II diabetes with dyslipidemia, dyslipidemia,
hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepatic
steatosis, non-alcoholic steatohepatitis (NASH), or non-alcoholic
fatty liver disease (NAFL).
[0151] In certain embodiments, the subject to which the antisense
compound is administered has elevated glucose levels, triglyceride
levels or cholesterol levels or any combination thereof. In certain
embodiments, such glucose levels can be blood, plasma and/or serum
glucose levels. In certain embodiments, such glucose levels can be
fed or fasting glucose levels. In certain embodiments, such glucose
levels are fed or fasting blood glucose levels. In certain
embodiments, such triglyceride levels are plasma triglyceride
levels. In certain embodiments, such triglyceride levels are liver
triglyceride levels. In certain embodiments, such cholesterol
levels are plasma cholesterol levels. In certain embodiments, the
administration thereby reduces glucose levels, triglyceride levels
or cholesterol levels. In certain embodiments the subject is an
animal. In certain embodiments the animal is a human.
[0152] Also provided are methods for reducing serum glucose levels,
serum triglyceride levels or plasma cholesterol levels in a subject
which include selecting a subject having elevated serum glucose
levels, serum triglyceride levels or plasma cholesterol levels, and
administering to the individual a therapeutically effective amount
of an antisense compound targeted to a JNK1 nucleic acid, and
additionally monitoring serum glucose levels, serum triglyceride
levels or plasma cholesterol levels. In certain embodiments the
individual is an animal. In certain embodiments the individual is a
human.
[0153] Further provided are methods for treating, preventing and/or
ameliorating diabetes, obesity or metabolic syndrome, or another
disease or condition associated with glucose and/or lipid
metabolism and/or the disregulation thereof, in a subject. Such
method includes selecting a subject diagnosed with diabetes,
obesity or metabolic syndrome or other disease or condition
associated with glucose and/or lipid metabolism, administering to
the individual a therapeutically effective amount of an antisense
compound targeted to a JNK1 nucleic acid, and monitoring factors
related to diabetes, obesity or metabolic syndrome or other related
disease or condition.
[0154] Further provided are methods of increasing metabolic rate.
Also provided are methods for lowering body weight gain. Also
provided are methods for lowering epididymal fat pad weight. Also
provided are methods for lowering whole body fat content. Such
methods include administering to a subject an antisense compound
targeted to a nucleic acid encoding JNK1. In certain embodiments,
such methods include the administration of a therapeutically
effective amount of an antisense compound targeted to a JNK1
nucleic acid. In certain embodiments, the compound is administered
in a composition. In certain embodiments the subject is an animal.
In certain embodiments the animal is a human. In certain
embodiments, the subject to which the antisense compound is
administered and in which metabolic rate is increased and/or weight
or fat content is lowered has one or more of the diseases or
disorders listed above. In certain embodiments, the subject to
which the antisense compound is administered and in which metabolic
rate is increased and/or weight or fat content is lowered has
obesity, diabetes or metabolic syndrome.
[0155] It is understood that the terms individual and subject are
used interchangeably herein and that any of the methods provided
herein can be useful for a subject or a subject and that subject or
individual can be an animal and particularly a human.
[0156] Any of the methods provided herein can further comprise
monitoring serum or plasma glucose levels, serum or plasma
triglyceride levels or serum or plasma cholesterol levels.
[0157] In any of the aforementioned methods, administration of the
antisense compound can comprise parenteral administration. The
parenteral administration can further comprise subcutaneous or
intravenous administration.
[0158] In any of the compounds, compositions or methods provided
herein, the antisense compound can have least 80%, at least 90%, or
at least 95% complementarity to SEQ ID NO: 87, 89, 90 or 91.
Alternatively, the antisense compound can have 100% complementarity
to SEQ ID NO: 87, 89, 90 or 91.
[0159] The antisense compounds provided herein and employed in any
of the described methods can be 8 to 80 subunits in length, 12 to
50 subunits in length, 12 to 30 subunits in length, 15 to 30
subunits in length, 18 to 24 subunits in length, 19 to 22 subunits
in length, or 20 subunits in length. Further, the antisense
compounds employed in any of the described methods can be antisense
oligonucleotides 8 to 80 nucleotides in length, 12 to 50
nucleotides in length, 12 to 30 nucleotides in length 15 to 30
nucleotides in length, 18 to 24 nucleotides in length, 19 to 22
nucleotides in length, or 20 nucleotides in length.
[0160] In any of the compounds, compositions and methods provided,
the antisense compound can be an antisense oligonucleotide.
Moreover, the antisense oligonucleotide can be chimeric. The
chimeric antisense oligonucleotide can be a gapmer antisense
oligonucleotide. The gapmer antisense oligonucleotide can comprise
a gap segment of ten 2'-deoxynucleotides positioned between wing
segments of five 2'-MOE nucleotides.
[0161] In any of the compounds, compositions and methods provided,
the antisense compounds can have at least one modified
internucleoside linkage. Additionally, each internucleoside linkage
can be a phosphorothioate internucleoside linkage. Each cytosine
can be a 5-methyl cytosine.
[0162] A compound for treatment of obesity and metabolic syndrome
can be an antisense compound 12 to 30 nucleobases targeted to a
JNK1 nucleic acid. The compound can have at least 70% to 100%
complementarity to any of SEQ ID Nos: 87, 89, 90 or 91. The
antisense oligonucleotide can be a gapmer antisense
oligonucleotide. The gapmer antisense oligonucleotide can comprise
a gap segment of ten 2'-deoxynucleotides positioned between wing
segments of five 2'-MOE nucleotides.
[0163] The antisense compounds can have at least one modified
internucleoside linkage. Additionally, each internucleoside linkage
can be a phosphorothioate internucleoside linkage. Each cytosine
can be a 5-methyl cytosine.
[0164] Both genetic and dietary mouse models of obesity were
treated with JNK1 ASO. JNK1 ASO treatment markedly and specifically
reduced the gene expression of JNK1 in both liver and fat tissues,
which resulted in a dramatic reduction of JNK1 activity in these
tissues. The treatment lowered BW (or body weight gain), fat depot
weight and whole body fat content, and increased metabolic rate
without causing liver toxicity or other side-effects as compared to
controls. The treatment markedly lowered fed and fasting plasma
glucose and insulin levels, improved glucose and insulin tolerance,
improved liver steatosis and lowered plasma cholesterol levels.
These data indicate that specific inhibition of JNK1 expression and
activity with ASO in the two major metabolic tissues improved
adiposity and related metabolic disorders in these models.
[0165] Treatment also resulted in improved feed efficiency.
Additionally, an increased metabolic rate in the ASO-treated mice
was confirmed by indirect calorimetry. Quantitative RT-PCR analysis
found increased gene expression in BAT from these mice of both
AR.beta..sub.3 and UCP1, two key genes involved in catabolism and
fuel combustion in rodents. Increased expression of PPAR.alpha.,
UCP2, and UCP3, and decreased expression of ACC2 were also found in
either liver or WAT, leading further support to the finding of an
increased metabolic rate. In addition, an extensive and profound
decrease in the expression of lipogenic genes and unchanged
expression of two key lipolytic genes, HSL and ATGL, were found in
WAT, indicating decreased lipogenesis and unchanged lipolysis with
reduction of JNK1 expression in this tissue. Marked decrease in
expression of ACC1 and FAS, two key genes involved in de novo fatty
acid synthesis, in liver was also detected. Furthermore, increased
fatty acid oxidation and decreased de novo fatty acid synthesis
were directly demonstrated in JNK1 ASO-transfected hepatocytes.
Taken together, these data demonstrate that decreased BW or BW gain
and lowered adiposity in the ASO-treated mice were attributable to
increased fuel combustion/metabolic rate and decreased
lipogenesis.
[0166] Antisense reduction of JNK1 activity lowers liver TG content
and improves hepatic steatosis. Additionally, plasma cholesterol
levels are improved. These changes were accompanied by increased
expression of hepatic UCP2 and PPAR.alpha. genes and decreased
expression of the key hepatic lipogenic genes including ACL, ACC1
and FAS. Without being bound to any theory, these changes in gene
expression indicate an increased shunting of citrate into the TCA
cycle and electron transport chain for oxidation and a reduced
breakdown of it (by ACL) to produce acetyl-CoA for cholesterol and
fatty acid synthesis. Improved hepatic steatosis and plasma
cholesterol levels in JNK1 ASO-treated mice can therefore be due to
increased hepatic substrate oxidation and decreased hepatic
lipogenesis. In vitro studies that showed decreased de novo sterol
synthesis and fatty acid synthesis and increased fatty acid
oxidation in JNK1 ASO-transfected hepatocytes provide additional
support. In addition, decreased expression of hepatic ApoB100 in
JNK1 ASO-treated mice was found. Reduction of hepatic ApoB100
expression lowers plasma cholesterol levels in different models of
hyperlipidemia due to reduced hepatic cholesterol synthesis and
export.
[0167] Specific reduction of JNK1 expression with ASO in just liver
and fat profoundly improved insulin sensitivity; normalized plasma
glucose and insulin levels and reduced glucose excursion during ITT
and GTT. The positive effects were found to be accompanied by
increased expression of hepatic GK and GS and decreased expression
of hepatic G6Pase and PKCs, and reduced expression of RBP4 in WAT.
GK is the rate-limiting enzyme for hepatic synthesis of
glucose-6-phosphate (which is further used for glycolysis or
glycogen synthesis) from glucose that is taken-up from blood,
whereas G6Pase is the final "gate" for hepatic glucose output by
breaking down glucose-6-phosphate (that is from either
gluconeogenesis or glycogenolysis) to release glucose into blood.
GS is the rate-limiting enzyme for glycogen synthesis that uses
glucose-6-phosphate as the primary substrate. These changes in gene
expression indicate that antisense reduction of JNK1 expression
improves liver and even whole body insulin sensitivity, promotes
blood glucose utilization and/or storage in liver while inhibiting
hepatic glucose output, thus, resulting in improved blood glucose
and insulin levels. An improved insulin signaling activity in JNK1
ASO treated mice showing decreased phosphorylation of
IRS1.sup.Ser302/307 and increased phosphorylation of Akt.sup.Ser473
in response to insulin further demonstrates that antisense
reduction of JNK1 expression improves insulin sensitivity.
[0168] Obesity, which is tightly associated with type 2 diabetes,
hyperlipidemia, and fatty liver diseases, has become epidemic
worldwide. JNK1 plays an important role in metabolism and energy
homeostasis and antisense reduction of its expression in liver and
fat increases metabolic rate and improves body weight and
adiposity, which is accompanied by improved liver steatosis,
hypercholesterolemia and insulin sensitivity in both genetically
leptin-deficient and diet-induced obese mice. Therefore, JNK1 is a
useful therapeutic target for the treatment of obesity and related
metabolic disorders.
[0169] The antisense compounds provided herein are therefore useful
for treating a number of metabolic conditions, including diabetes,
obesity and metabolic syndrome. Such treatments encompass a
therapeutic regimen that results in a clinically desirable outcome.
The clinically desired outcomes can be tied to glucose metabolism.
For example, the antisense compounds and methods provided herein
are useful for improving blood glucose control or tolerance and for
improving insulin sensitivity in a subject in need thereof. The
antisense compounds and methods provided herein are also useful for
reducing plasma resistin levels in a subject in need thereof. The
antisense compounds and methods provided herein are also useful for
reducing glucose levels in a subject in need thereof. The compounds
and methods are particularly useful for reducing blood, plasma
and/or serum glucose levels. The compounds and methods are useful
for reducing both fed and fasting glucose levels. Such clinical
outcomes are desirable in disease and disorders related to glucose
metabolism and insulin resistance including, for example, diabetes,
particularly type II diabetes, obesity and metabolic syndrome.
Therefore, the antisense compounds and methods provided herein are
useful for the treatment of such diseases and disorders.
[0170] The clinically desirable outcomes can also be tied to lipid
metabolism. For example, the antisense compounds and methods
provided herein are also useful for the reduction of lipids in a
subject in need thereof, particularly serum lipids. The reduction
in lipids can result from a lowering of lipogenisis and
particularly a lowering of lipogenic genes including, but not
limited to ACC1, ACC2, FAS, SCD1 and DGAT1. In particular, the
antisense compounds and methods are useful for reducing
triglyceride and cholesterol levels in a subject in need thereof.
The compounds and methods are particularly useful for reducing
plasma triglyceride levels and plasma cholesterol levels. The
compounds and methods are also particularly useful for reducing
liver triglyceride levels. Additionally, the antisense compounds
and methods provided herein are also useful for improving liver
steatosis. The compounds and methods are also particularly useful
for increasing metabolic rate and, in turn, lowering body weight
gain. The compounds and methods are also particularly useful for
lowering epididymal fat pad weight and whole body fat content. Such
clinical outcomes are desirable in diseases and disorders of lipid
as well as glucose metabolism and insulin resistance including, for
example, diabetes, metabolic syndrome, obesity,
hypercholesterolemia, mixed dyslipidemia, atherosclerosis, coronary
heart disease, diabetes, type II diabetes, type II diabetes with
dyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia,
hyperfattyacidemia, hepatic steatosis, non-alcoholic
steatohepatitis (NASH), or non-alcoholic fatty liver disease
(NAFLD). NAFLD is a condition characterized by fatty inflammation
of the liver that is not due to excessive alcohol use (for example,
alcohol consumption of over 20 g/day). In certain embodiments,
NAFLD is related to insulin resistance and the metabolic syndrome.
NASH is a condition characterized by inflammation and the
accumulation of fat and fibrous tissue in the liver that is not due
to excessive alcohol use. NASH is an extreme form of NAFLD.
Therefore, the antisense compounds and methods provided herein are
useful for the treatment of such diseases and disorders.
[0171] Elevated levels of blood glucose and triglycerides are
recognized as major risk factors for development of diabetes,
obesity and metabolic syndrome. Elevated blood glucose levels or
elevated triglyceride levels are also considered a risk factor in
the development and progression of atherosclerosis. Atherosclerosis
can lead to coronary heart disease, stroke, or peripheral vascular
disease. Accordingly provided herein are pharmaceutical agents for
lowering elevated levels of blood glucose and triglycerides.
[0172] Metabolic syndrome is 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). Accordingly, the compounds and
methods provided herein can be used to treat individuals exhibiting
one or more risk factors for metabolic syndrome. Particularly, the
compounds and methods provided herein can be used to reduce body
weight, thereby likely reducing waist circumference, and fasting
glucose levels.
[0173] As illustrated herein, administration of an antisense
oligonucleotide targeted to JNK1 to animals models of diabetes and
obesity which exhibit insulin resistance, hyperglycemia and
hyperlipidemia, resulted in antisense inhibition of JNK1, a
reduction in plasma glucose and triglyceride levels and reduction
of liver triglycerides. Reduction in triglycerides was also
accompanied by a reduction in lipogenic genes. Particularly,
expression of ACC1, ACC2, FAS, SCD1 and DGAT1 were reduced. Thus,
it is demonstrated that in an experimental model of hyperglycemia
and hyperlipidemia, antisense inhibition of JNK1 results in reduced
glucose and triglyceride levels and reduced lipogenesis.
Accordingly, provided herein are methods of reducing lipogenesis,
blood glucose and triglyceride levels through the administration of
an antisense compound targeted to a JNK1 nucleic acid. Blood
glucose and triglyceride levels are considered a risk factor for
development of diabetes, obesity and metabolic syndrome.
Accordingly, also provided herein are methods for the treatment,
prevention and/or amelioration of diabetes, obesity and metabolic
syndrome, and for the treatment, prevention and/or amelioration of
associated disorders. Also provided herein are methods for the
treatment of conditions characterized by elevated liver
triglycerides, such as hepatic steatosis.
Certain Indications
[0174] In certain embodiments, the invention provides methods of
treating a subject comprising administering one or more
pharmaceutical compositions of the present invention. In certain
embodiments, the individual has diabetes, obesity, metabolic
syndrome and/or associated disorders including but not limited to
hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk
of developing atherosclerosis, coronary heart disease, a history of
coronary heart disease, early onset coronary heart disease, one or
more risk factors for coronary heart disease, type II diabetes,
type II diabetes with dyslipidemia, dyslipidemia,
hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepatic
steatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty
liver disease.
[0175] Hypercholesterolemia is a condition characterized by
elevated serum cholesterol. Hyperlipidemia is a condition
characterized by elevated serum lipids. Hypertriglyceridemia is a
condition characterized by elevated triglyceride levels.
Non-familial hypercholesterolemia is a condition characterized by
elevated cholesterol that is not the result of a single gene
mutation. Is polygenic hypercholesterolemia is a condition
characterized by elevated cholesterol that results from the
influence of a variety of genetic factors. In certain embodiments,
polygenic hypercholesterolemia can be exacerbated by dietary intake
of lipids. Familial hypercholesterolemia (FH) is an autosomal
dominant metabolic disorder characterized by a mutation in the
LDL-receptor (LDL-R) gene, markedly elevated LDL-C and premature
onset of atherosclerosis. A diagnosis of familial
hypercholesterolemia is made when a individual meets one or more of
the following criteria: genetic testing confirming 2 mutated
LDL-receptor genes; genetic testing confirming one mutated
LDL-receptor gene; document history of untreated serum
LDL-cholesterol greater than 500 mg/dL; tendinous and/or cutaneous
xanthoma prior to age 10 years; or, both parents have documented
elevated serum LDL-cholesterol prior to lipid-lowering therapy
consistent with heterozygous familial hypercholesterolemia.
Homozygous familial hypercholesterolemia (HoFH) is a condition
characterized by a mutation in both maternal and paternal LDL-R
genes. Heterozygous familial hypercholesterolemia (HeFH) is a
condition characterized by a mutation in either the maternal or
paternal LDL-R gene. Mixed dyslipidemia is a condition
characterized by elevated serum cholesterol and elevated serum
triglycerides. Diabetic dyslipidemia or Type II diabetes with
dyslipidemia is a condition characterized by Type II diabetes,
reduced HDL-C, elevated serum triglycerides, and elevated small,
dense LDL particles.
[0176] In one embodiment are methods for decreasing blood glucose
levels or triglyceride levels, or alternatively methods for
treating obesity or metabolic syndrome, by administering to a
subject suffering from elevated glucose or triglyceride levels a
therapeutically effective amount of an antisense compound targeted
to a JNK1 nucleic acid. In another embodiment, a method of
decreasing blood glucose or triglyceride levels comprises selecting
a subject in need of a decrease in blood glucose or triglyceride
levels, and administering to the individual a therapeutically
effective amount of an antisense compound targeted to a JNK1
nucleic acid. In a further embodiment, a method of reducing risk of
development of obesity and metabolic syndrome includes selecting a
subject having elevated blood glucose or triglyceride levels and
one or more additional indicators risk of development of obesity or
metabolic syndrome, and administering to the individual a
therapeutically effective amount of an antisense compound targeted
to a JNK1 nucleic acid.
[0177] In one embodiment, administration of a therapeutically
effective amount of an antisense compound targeted a JNK1 nucleic
acid is accompanied by monitoring of glucose levels or triglyceride
levels in the serum of a subject, to determine a subject's response
to administration of the antisense compound. A subject's response
to administration of the antisense compound is used by a physician
to determine the amount and duration of therapeutic
intervention.
[0178] Atherosclerosis can lead to coronary heart disease, stroke,
or peripheral vascular disease. Elevated blood glucose levels or
elevated triglyceride levels are considered a risk factor in the
development and progression of atherosclerosis. Accordingly, in one
embodiment, a therapeutically effective amount of an antisense
compound targeted to a JNK1 nucleic acid is administered to a
subject having atherosclerosis. In a further embodiment a
therapeutically effective amount of antisense compound targeted to
a JNK1 nucleic acid is administered to a subject susceptible to
atherosclerosis. Atherosclerosis is assessed directly through
routine imaging techniques such as, for example, ultrasound imaging
techniques that reveal carotid intimomedial thickness. Accordingly,
treatment and/or prevention of atherosclerosis further include
monitoring atherosclerosis through routine imaging techniques. In
one embodiment, administration of an antisense compound targeted to
a JNK1 nucleic acid leads to a lessening of the severity of
atherosclerosis, as indicated by, for example, a reduction of
carotid intimomedial thickness in arteries.
[0179] Measurements of cholesterol, lipoproteins and triglycerides
are obtained using serum or plasma collected from a subject.
Methods of obtaining serum or plasma samples are routine, as are
methods of preparation of the serum samples for analysis of
cholesterol, triglycerides, and other serum markers.
[0180] A physician can determine the need for therapeutic
intervention for individuals in cases where more or less aggressive
blood glucose or triglyceride-lowering therapy is needed. The
practice of the methods herein can be applied to any altered
guidelines provided by the NCEP, or other entities that establish
guidelines for physicians used in treating any of the diseases or
conditions listed herein, for determining coronary heart disease
risk and diagnosing metabolic syndrome.
[0181] In one embodiment, administration of an antisense compound
targeted a JNK1 nucleic acid is parenteral administration.
Parenteral administration can be intravenous or subcutaneous
administration. Accordingly, in another embodiment, administration
of an antisense compound targeted to a JNK1 nucleic acid is
intravenous or subcutaneous administration. Administration can
include multiple doses of an antisense compound targeted to a JNK1
nucleic acid.
[0182] In certain embodiments a pharmaceutical composition
comprising an antisense compound targeted to JNK1 is for use in
therapy. In certain embodiments, the therapy is the reduction of
blood glucose, serum triglyceride or liver triglyceride in a
subject. In certain embodiments, the therapy is the treatment of
hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk
of developing atherosclerosis, coronary heart disease, a history of
coronary heart disease, early onset coronary heart disease, one or
more risk factors for coronary heart disease, type II diabetes,
type II diabetes with dyslipidemia, dyslipidemia,
hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepatic
steatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty
liver disease. In additional embodiments, the therapy is the
reduction of CHD risk. CHD risk equivalents refers to indicators of
clinical atherosclerotic disease that confer a high risk for
coronary heart disease, and include clinical coronary heart
disease, symptomatic carotid artery disease, peripheral arterial
disease, and/or abdominal aortic aneurysm. In certain the therapy
is prevention of atherosclerosis. In certain embodiments, the
therapy is the prevention of coronary heart disease.
[0183] In certain embodiments pharmaceutical composition comprising
an antisense compound targeted to JNK1 is used for the preparation
of a medicament for reduction of blood glucose, serum triglyceride
or liver triglyceride. In certain embodiments pharmaceutical
composition comprising an antisense compound targeted to JNK1 is
used for the preparation of a medicament for reducing coronary
heart disease risk. In certain embodiments an antisense compound
targeted to JNK1 is used for the preparation of a medicament for
the treatment of hypercholesterolemia, mixed dyslipidemia,
atherosclerosis, a risk of developing atherosclerosis, coronary
heart disease, a history of coronary heart disease, early onset
coronary heart disease, one or more risk factors for coronary heart
disease, type II diabetes, type II diabetes with dyslipidemia,
dyslipidemia, hypertriglyceridemia, hyperlipidemia,
hyperfattyacidemia, hepatic steatosis, non-alcoholic
steatohepatitis, or non-alcoholic fatty liver disease.
Certain Combination Therapies
[0184] In certain embodiments, one or more pharmaceutical
compositions of the present invention are co-administered with one
or more other pharmaceutical agents. In certain embodiments, such
one or more other pharmaceutical agents are designed to treat the
same disease or condition as the one or more pharmaceutical
compositions of the present invention. In certain embodiments, such
one or more other pharmaceutical agents are designed to treat a
different disease or condition as the one or more pharmaceutical
compositions of the present invention. In certain embodiments, such
one or more other pharmaceutical agents are designed to treat an
undesired effect of one or more pharmaceutical compositions of the
present invention. In certain embodiments, one or more
pharmaceutical compositions of the present invention are
co-administered with another pharmaceutical agent to treat an
undesired effect of that other pharmaceutical agent. In certain
embodiments, one or more pharmaceutical compositions of the present
invention and one or more other pharmaceutical agents are
administered at the same time. In certain embodiments, one or more
pharmaceutical compositions of the present invention and one or
more other pharmaceutical agents are administered at different
times. In certain embodiments, one or more pharmaceutical
compositions of the present invention and one or more other
pharmaceutical agents are prepared together in a single
formulation. In certain embodiments, one or more pharmaceutical
compositions of the present invention and one or more other
pharmaceutical agents are prepared separately.
[0185] In certain embodiments, pharmaceutical agents that can be
co-administered with a pharmaceutical composition comprising an
antisense compound targeted to a JNK1 nucleic acid include
glucose-lowering agents and therapies. In some embodiments, the
glucose-lowering agent is a PPAR agonist (gamma, dual, or pan), 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, a
meglitinide, a thiazolidinedione, or a sulfonylurea.
[0186] In some embodiments, the glucose-lowering therapeutic is a
GLP-1 analog. In some embodiments, the GLP-1 analog is exendin-4 or
liraglutide.
[0187] In other embodiments, the glucose-lowering therapeutic is a
sulfonylurea. In some embodiments, the sulfonylurea is
acetohexamide, chlorpropamide, tolbutamide, tolazamide,
glimepiride, a glipizide, a glyburide, or a gliclazide.
[0188] In some embodiments, the glucose lowering drug is a
biguanide. In some embodiments, the biguanide is metformin, and in
some embodiments, blood glucose levels are decreased without
increased lactic acidosis as compared to the lactic acidosis
observed after treatment with metformin alone.
[0189] In some embodiments, the glucose lowering drug is a
meglitinide. In some embodiments, the meglitinide is nateglinide or
repaglinide.
[0190] In some embodiments, the glucose-lowering drug is a
thiazolidinedione. In some embodiments, the thiazolidinedione is
pioglitazone, rosiglitazone, or troglitazone. In some embodiments,
blood glucose levels are decreased without greater weight gain than
observed with rosiglitazone treatment alone.
[0191] In some embodiments, the glucose-lowering drug is an
alpha-glucosidase inhibitor. In some embodiments, the
alpha-glucosidase inhibitor is acarbose or miglitol.
[0192] In a certain embodiment, a co-administered glucose-lowering
agent is ISIS 113715.
[0193] In a certain embodiment, glucose-lowering therapy is
therapeutic lifestyle change.
[0194] In certain such embodiments, the glucose-lowering agent is
administered prior to administration of a pharmaceutical
composition of the present invention. In certain such embodiments,
the glucose-lowering agent is administered following administration
of a pharmaceutical composition of the present invention. In
certain such embodiments the glucose-lowering agent is administered
at the same time as a pharmaceutical composition of the present
invention. In certain such embodiments the dose of a
co-administered glucose-lowering agent is the same as the dose that
would be administered if the glucose-lowering agent was
administered alone. In certain such embodiments the dose of a
co-administered glucose-lowering agent is lower than the dose that
would be administered if the glucose-lowering agent was
administered alone. In certain such embodiments the dose of a
co-administered glucose-lowering agent is greater than the dose
that would be administered if the glucose-lowering agent was
administered alone.
[0195] In certain embodiments, pharmaceutical agents that can be
co-administered with a pharmaceutical composition comprising an
antisense compound targeted to a JNK1 nucleic acid include
anti-obesity agents. Such anti-obesity agents therapeutics can be
administered as described above for glucose lowering agents.
[0196] Further provided is a method of administering an antisense
compound targeted to a JNK1 nucleic acid via injection and further
including administering a topical steroid at the injection
site.
[0197] In certain embodiments, pharmaceutical agents that can be
co-administered with a pharmaceutical composition of the present
invention include lipid-lowering agents. In certain such
embodiments, pharmaceutical agents that can be co-administered with
a pharmaceutical composition of the present invention include, but
are not limited to atorvastatin, simvastatin, rosuvastatin, and
ezetimibe. In certain such embodiments, the lipid-lowering agent is
administered prior to administration of a pharmaceutical
composition of the present invention. In certain such embodiments,
the lipid-lowering agent is administered following administration
of a pharmaceutical composition of the present invention. In
certain such embodiments the lipid-lowering agent is administered
at the same time as a pharmaceutical composition of the present
invention. In certain such embodiments the dose of a
co-administered lipid-lowering agent is the same as the dose that
would be administered if the lipid-lowering agent was administered
alone. In certain such embodiments the dose of a co-administered
lipid-lowering agent is lower than the dose that would be
administered if the lipid-lowering agent was administered alone. In
certain such embodiments the dose of a co-administered
lipid-lowering agent is greater than the dose that would be
administered if the lipid-lowering agent was administered
alone.
[0198] In certain embodiments, a co-administered lipid-lowering
agent is a HMG-CoA reductase inhibitor. In certain such embodiments
the HMG-CoA reductase inhibitor is a statin. In certain such
embodiments the statin is selected from atorvastatin, simvastatin,
pravastatin, fluvastatin, and rosuvastatin.
[0199] In certain embodiments, a co-administered lipid-lowering
agent is a cholesterol absorption inhibitor. In certain such
embodiments, cholesterol absorption inhibitor is ezetimibe.
[0200] In certain embodiments, a co-administered lipid-lowering
agent is a co-formulated HMG-CoA reductase inhibitor and
cholesterol absorption inhibitor. In certain such embodiments the
co-formulated lipid-lowering agent is ezetimibe/simvastatin.
[0201] In certain embodiments, a co-administered lipid-lowering
agent is a microsomal triglyceride transfer protein inhibitor (MTP
inhibitor).
[0202] In certain embodiments, a co-administered lipid-lowering
agent is an oligonucleotide targeted to ApoB.
[0203] In certain embodiments, a co-administered pharmaceutical
agent is a bile acid sequestrant. In certain such embodiments, the
bile acid sequestrant is selected from cholestyramine, colestipol,
and colesevelam.
[0204] In certain embodiments, a co-administered pharmaceutical
agent is a nicotinic acid. In certain such embodiments, the
nicotinic acid is selected from immediate release nicotinic acid,
extended release nicotinic acid, and sustained release nicotinic
acid.
[0205] In certain embodiments, a co-administered pharmaceutical
agent is a fibric acid. In certain such embodiments, a fibric acid
is selected from gemfibrozil, fenofibrate, clofibrate, bezafibrate,
and ciprofibrate.
[0206] Further examples of pharmaceutical agents that can be
co-administered with a pharmaceutical composition of the present
invention include, but are not limited to, corticosteroids,
including but not limited to prednisone; immunoglobulins,
including, but not limited to intravenous immunoglobulin (IVIg);
analgesics (e.g., acetaminophen); anti-inflammatory agents,
including, but not limited to non-steroidal anti-inflammatory drugs
(e.g., ibuprofen, COX-1 inhibitors, and COX-2, inhibitors);
salicylates; antibiotics; antivirals; antifungal agents;
antidiabetic agents (e.g., biguanides, glucosidase inhibitors,
insulins, sulfonylureas, and thiazolidenediones); adrenergic
modifiers; diuretics; hormones (e.g., anabolic steroids, androgen,
estrogen, calcitonin, progestin, somatostan, and thyroid hormones);
immunomodulators; muscle relaxants; antihistamines; osteoporosis
agents (e.g., biphosphonates, calcitonin, and estrogens);
prostaglandins, antineoplastic agents; psychotherapeutic agents;
sedatives; poison oak or poison sumac products; antibodies; and
vaccines.
[0207] In certain embodiments, the pharmaceutical compositions of
the present invention can be administered in conjunction with a
lipid-lowering therapy. In certain such embodiments, a
lipid-lowering therapy is therapeutic lifestyle change. In certain
such embodiments, a lipid-lowering therapy is LDL apheresis.
[0208] In certain embodiments obesity is drug induced. In a
particular embodiment obesity is induced by treatment with a
psychotherapeutic drug or agent. Therapeutic use of certain
psychothearapeutic agents, namely atypical antipsychotic agents can
increase the risk of metabolic abnormalities and there use is
generally associated with weight gain and impaired glucose
tolerance. The percentage of patients gaining weight following
antipsychotic therapy can reach up to 80% depending on the
antipsychotic used, with 30% or more developing obesity. Along with
associated medical complications, such metabolic abnormalities
increase the percentage of non-compliance patients and results in
an increased risk of relapse.
[0209] Due to the ability of JNK1 antisense oligonucleotides to
increase metabolic rate and insulin sensitivity and reduce
adiposity and weight gain, these compounds can be administered to
reduce metabolic abnormalities associated with treatment with
antipsychotic agents. In certain embodiments the JNK1 antisense
oligonucleotide is delivered in a method of reducing metabolic
abnormalities associated with the therapeutic use of
psychotherapeutic agents. Such weight inducing antipsychotic agents
include, but are not limited to clozapine, olanzapine,
aripiprazole, risperidone and ziprasidone.
[0210] In certain embodiments the JNK1 antisense oligonucleotide is
delivered concomitant with delivery of the psychotherapeutic agent.
Alternatively, delivery can be in the same formulation or can be
administered separately. In a certain embodiment, the JNK1
antisense oligonucleotide is administered after treatment with an
obesity inducing drug or agent is ceased. In a particular
embodiment administering of the JNK1 antisense compound results in
increased metabolic rate or decreasing adiposity or both without
affecting the CNS effects of the psychotherapeutic agent.
[0211] In certain embodiments, JNK1 antisense oligonucleotides are
administered in combination either in the same formulation or
separate formulations with other anti-obesity drugs or agents. In
certain embodiment, the anti-obesity agents are CNS based such as,
but not limited to, sibutramine or GLP-1 based such as, but not
limited to, liraglutide.
Antisense Compounds
[0212] Provided herein are antisense oligonucleotides that modulate
the JNK1, JNK2 and JNK3 proteins. Such modulation is desirable for
treating, alleviating or preventing various disorders or diseases,
such as obesity and metabolic syndrome. Such inhibition is further
desirable for preventing or modulating the development of such
diseases or disorders in an animal suspected of being, or known to
be, prone to such diseases or disorders.
[0213] Methods of modulating the expression of JNK1 proteins
comprising contacting animals with oligonucleotides specifically
hybridizable with a nucleic acid encoding a JNK1 protein are herein
provided. These methods are also useful for the diagnosis of
conditions associated with such expression and activation.
[0214] Also provided herein are methods that comprise inhibiting
JNK1-mediated activity using antisense oligonucleotides. These
methods employ the oligonucleotides of the invention and are
believed to be useful both therapeutically and as clinical research
and diagnostic tools. Provided are methods for inhibiting the
expression of JNK1 from a nucleic acid for the treatment,
prevention or amelioration of a condition comprising reducing body
weight gain, reducing epididymal fat pad weight, reducing whole
body fat content, increasing metabolic rate, reducing fed plasma
glucose, reducing fasting plasma glucose, reducing fed plasma
insulin, reducing fasted plasma insulin, improving glucose
tolerance, improving insulin tolerance, improving liver steatosis,
reducing plasma cholesterol, reducing plasma transaminase or a
combination thereof.
[0215] Oligonucleotides are used herein in antisense modulation of
the function of DNA or messenger RNA (mRNA) encoding a protein the
modulation of which is desired, and ultimately to regulate the
amount of such a protein. Hybridization of an antisense
oligonucleotide with its mRNA target interferes with the normal
role of mRNA and causes a modulation of its function in cells. The
functions of mRNA to be interfered with include all vital functions
such as translocation of the RNA to the site for protein
translation, actual translation of protein from the RNA, splicing
of the RNA to yield one or more mRNA species, and possibly even
independent catalytic activity which can be engaged in by the RNA.
The overall effect of such interference with mRNA function is
modulation of the expression of a protein, wherein modulation is
either an increase (stimulation) or a decrease (inhibition) in the
expression of the protein. In the context of the present invention,
inhibition is the preferred form of modulation of gene
expression.
[0216] It is preferred to target specific genes for antisense
attack. Targeting an oligonucleotide to the associated nucleic
acid, in the context of this invention, is a multistep process. The
process begins with the identification of a nucleic acid sequence
whose function is to be modulated. This can be, for example, a
cellular gene (or mRNA transcribed from the gene) whose expression
is associated with a particular disorder or disease state, or a
foreign nucleic acid from an infectious agent. In the present
invention, the target is a cellular gene associated with
hyperproliferative disorders. The targeting process also includes
determination of a site or sites within this gene for the
oligonucleotide interaction to occur such that the desired effect,
either detection or modulation of expression of the protein, will
result. Once the target site or sites have been identified,
oligonucleotides are chosen which are sufficiently complementary to
the target, i.e., hybridize sufficiently well and with sufficient
specificity to give the desired effect. Generally, there are five
regions of a gene that can be targeted for antisense modulation:
the 5' untranslated region (hereinafter, the "5'-UTR"), the
translation initiation codon region (hereinafter, the "tIR"), the
open reading frame (hereinafter, the "ORF"), the translation
termination codon region (hereinafter, the "tTR") and the 3'
untranslated region (hereinafter, the "3'-UTR"). As is known in the
art, these regions are arranged in a typical messenger RNA molecule
in the following order (left to right, 5' to 3'): 5'-UTR, tIR, ORF,
tTR, 3'-UTR. As is known in the art, although some eukaryotic
transcripts are directly translated, many ORFs contain one or more
sequences, known as "introns," which are excised from a transcript
before it is translated; the expressed (unexcised) portions of the
ORF are referred to as "exons" (Alberts et al., Molecular Biology
of the Cell, 1983, Garland Publishing Inc., New York, pp. 411-415).
Furthermore, because many eukaryotic ORFs are a thousand
nucleotides or more in length, it is often convenient to subdivide
the ORF into, e.g., the 5' ORF region, the central ORF region, and
the 3' ORF region. In some instances, an ORF contains one or more
sites that can be targeted due to some functional significance in
vivo. Examples of the latter types of sites include intragenic
stem-loop structures (see, e.g., U.S. Pat. No. 5,512,438) and, in
unprocessed mRNA molecules, intron/exon splice sites.
[0217] Within the context of the present invention, one preferred
intragenic site is the region encompassing the translation
initiation codon of the open reading frame (ORF) of the gene.
Because, as is known in the art, the translation initiation codon
is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the
corresponding DNA molecule), the translation initiation codon is
also referred to as the "AUG codon," the "start codon" or the "AUG
start codon." A minority of genes have a translation initiation
codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA,
5'-ACG and 5'-CUG have been shown to function in vivo. Furthermore,
5'-UUU functions as a translation initiation codon in vitro
(Brigstock et al., Growth Factors, 1990, 4, 45; Gelbert et al.,
Somat. Cell. Mol. Genet., 1990, 16, 173; Gold and Stormo, in:
Escherichia coli and Salmonella typhimurium: Cellular and Molecular
Biology, Vol. 2, 1987, Neidhardt et al., Eds., American Society for
Microbiology, Washington, D.C., p. 1303). Thus, the terms
"translation initiation codon" and "start codon" can encompass many
codon sequences, even though the initiator amino acid in each
instance is typically methionine (in eukaryotes) or
formylmethionine (prokaryotes). It is also known in the art that
eukaryotic and prokaryotic genes can have two or more alternative
start codons, any one of which can be preferentially utilized for
translation initiation in a particular cell type or tissue, or
under a particular set of conditions, in order to generate related
polypeptides having different amino terminal sequences (Markussen
et al., Development, 1995, 121, 3723; Gao et al., Cancer Res.,
1995, 55, 743; McDermott et al., Gene, 1992, 117, 193; Perri et
al., J. Biol. Chem., 1991, 266, 12536; French et al., J. Virol.,
1989, 63, 3270; Pushpa-Rekha et al., J. Biol. Chem., 1995, 270,
26993; Monaco et al., J. Biol. Chem., 1994, 269, 347; DeVirgilio et
al., Yeast, 1992, 8, 1043; Kanagasundaram et al., Biochim. Biophys.
Acta, 1992, 1171, 198; Olsen et al., Mol. Endocrinol., 1991, 5,
1246; Saul et al., Appl. Environ. Microbiol., 1990, 56, 3117;
Yaoita et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 7090; Rogers
et al., EMBO J., 1990, 9, 2273). In the context of the invention,
"start codon" and "translation initiation codon" refer to the codon
or codons that are used in vivo to initiate translation of an mRNA
molecule transcribed from a gene encoding a JNK1 protein,
regardless of the sequence(s) of such codons. It is also known in
the art that a translation termination codon (or "stop codon") of a
gene can have one of three sequences, i.e., 5'-UAA, 5'-UAG and
5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and
5'-TGA, respectively). The terms "start codon region" and
"translation initiation region" refer to a portion of such an mRNA
or gene that encompasses from about 25 to about 50 contiguous
nucleotides in either direction (i.e., 5' or 3') from a translation
initiation codon. Similarly, the terms "stop codon region" and
"translation termination region" refer to a portion of such an mRNA
or gene that encompasses from about 25 to about 50 contiguous
nucleotides in either direction (i.e., 5' or 3') from a translation
termination codon.
[0218] 1. Oligonucleotides of the Invention: The present invention
employs oligonucleotides for use in antisense modulation of one or
more JNK1 proteins. In the context of this invention, the term
oligonucleotide refers to an oligomer or polymer of ribonucleic
acid or deoxyribonucleic acid. This term includes oligonucleotides
composed of naturally-occurring nucleobases, sugars and covalent
intersugar (backbone) linkages as well as oligonucleotides having
non-naturally-occurring portions which function similarly. Such
modified or substituted oligonucleotides are often preferred over
native forms because of desirable properties such as, for example,
enhanced cellular uptake, enhanced binding to target and increased
stability in the presence of nucleases.
[0219] An oligonucleotide is a polymer of a repeating unit
generically known as a nucleotide. The oligonucleotides in
accordance with this invention preferably comprise from about 8 to
about 30 nucleotides. An unmodified (naturally occurring)
nucleotide has three components: (1) a nitrogen-containing
heterocyclic base linked by one of its nitrogen atoms to (2) a
5-pentofuranosyl sugar and (3) a phosphate esterified to one of the
5' or 3' carbon atoms of the sugar. When incorporated into an
oligonucleotide chain, the phosphate of a first nucleotide is also
esterified to an adjacent sugar of a second, adjacent nucleotide
via a 3'-5' phosphate linkage. The "backbone" of an unmodified
oligonucleotide consists of (2) and (3), that is, sugars linked
together by phosphodiester linkages between the 5' carbon of the
sugar of a first nucleotide and the 3' carbon of a second, adjacent
nucleotide. A "nucleoside" is the combination of (1) a nucleobase
and (2) a sugar in the absence of (3) a phosphate moiety (Kornberg,
A., DNA Replication, W.H. Freeman & Co., San Francisco, 1980,
pages 4-7). The backbone of an oligonucleotide positions a series
of bases in a specific order; the written representation of this
series of bases, which is conventionally written in 5' to 3' order,
is known as a nucleotide sequence.
[0220] Oligonucleotides can comprise nucleotide sequences
sufficient in identity and number to effect specific hybridization
with a particular nucleic acid. Such oligonucleotides which
specifically hybridize to a portion of the sense strand of a gene
are commonly described as antisense." In the context of the
invention, hybridization means hydrogen bonding, which can be
Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,
between complementary nucleotides. For example, adenine and thymine
are complementary nucleobases which pair through the formation of
hydrogen bonds. Complementary refers to the capacity for precise
pairing between two nucleotides. For example, if a nucleotide at a
certain position of an oligonucleotide is capable of hydrogen
bonding with a nucleotide at the same position of a DNA or RNA
molecule, then the oligonucleotide and the DNA or RNA are
considered to be complementary to each other at that position. The
oligonucleotide and the DNA or RNA are complementary to each other
when a sufficient number of corresponding positions in each
molecule are occupied by nucleotides which can hydrogen bond with
each other. An oligonucleotide is specifically hybridizable to its
target sequence due to the formation of base pairs between specific
partner nucleobases in the interior of a nucleic acid duplex. Among
the naturally occurring nucleobases, guanine (G) binds to cytosine
(C), and adenine (A) binds to thymine (T) or uracil (U). In
addition to the equivalency of U (RNA) and T (DNA) as partners for
A, other naturally occurring nucleobase equivalents are known,
including 5-methylcytosine, 5-hydroxymethylcytosine (HMC), glycosyl
HMC and gentiobiosyl HMC (C equivalents), and 5-hydroxymethyluracil
(U equivalent). Furthermore, synthetic nucleobases which retain
partner specificity are known in the art and include, for example,
7-deaza-Guanine, which retains partner specificity for C. Thus, an
oligonucleotide's capacity to specifically hybridize with its
target sequence will not be altered by any chemical modification to
a nucleobase in the nucleotide sequence of the oligonucleotide
which does not significantly effect its specificity for the partner
nucleobase in the target oligonucleotide. It is understood in the
art that an oligonucleotide need not be 100% complementary to its
target DNA sequence to be specifically hybridizable. An
oligonucleotide is specifically hybridizable when there is a
sufficient degree of complementarity to avoid non-specific binding
of the oligonucleotide to non-target sequences under conditions in
which specific binding is desired, i.e., under physiological
conditions in the case of in vivo assays or therapeutic treatment,
and in the case of in vitro assays, under conditions in which the
assays are performed.
[0221] Antisense oligonucleotides are commonly used as research
reagents, diagnostic aids, and therapeutic agents. For example,
antisense oligonucleotides, which are able to inhibit gene
expression with exquisite specificity, are often used by those of
ordinary skill to elucidate the function of particular genes, for
example to distinguish between the functions of various members of
a biological pathway. This specific inhibitory effect has,
therefore, been harnessed by those skilled in the art for research
uses. The specificity and sensitivity of oligonucleotides is also
harnessed by those of skill in the art for therapeutic uses.
[0222] Modified Linkages: Specific examples of some preferred
modified oligonucleotides envisioned for this invention include
those containing phosphorothioates, phosphotriesters, methyl
phosphonates, short chain alkyl or cycloalkyl intersugar linkages
or short chain heteroatomic or heterocyclic intersugar linkages.
Most preferred are oligonucleotides with phosphorothioates and
those with CH.sub.2--NH--O--CH.sub.2,
CH.sub.2--N(CH.sub.3)--O--CH.sub.2 [known as a
methylene(methylimino) or MMI backbone],
CH.sub.2--O--N(CH.sub.3)--CH.sub.2,
CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2 and
O--N(CH.sub.3)--CH.sub.2--CH.sub.2 backbones, wherein the native
phosphodiester backbone is represented as O--P--O--CH.sub.2). Also
preferred are oligonucleotides having morpholino backbone
structures (Summerton and Weller, U.S. Pat. No. 5,034,506). Further
preferred are oligonucleotides with NR--C(*)-CH.sub.2--CH.sub.2,
CH.sub.2--NR--C(*)-CH.sub.2, CH.sub.2--CH.sub.2--NR--C(*),
C(*)-NR--CH.sub.2--CH.sub.2 and CH.sub.2--C(*)-NR--CH.sub.2
backbones, wherein "*" represents O or S (known as amide backbones;
DeMesmaeker et al., WO 92/20823, published Nov. 26, 1992). In other
preferred embodiments, such as the peptide nucleic acid (PNA)
backbone, the phosphodiester backbone of the oligonucleotide is
replaced with a polyamide backbone, the nucleobases being bound
directly or indirectly to the aza nitrogen atoms of the polyamide
backbone (Nielsen et al., Science, 1991, 254, 1497; U.S. Pat. No.
5,539,082).
[0223] Modified Nucleobases The oligonucleotides of the invention
can additionally or alternatively include nucleobase modifications
or substitutions. As used herein, "unmodified" or "natural"
nucleobases include adenine (A), guanine (G), thymine (T), cytosine
(C) and uracil (U). Modified nucleobases include nucleobases found
only infrequently or transiently in natural nucleic acids, e.g.,
hypoxanthine, 6-methyladenine, 5-methylcytosine,
5-hydroxymethylcytosine (HMC), glycosyl HMC and gentiobiosyl HMC,
as well synthetic nucleobases, e.g., 2-aminoadenine, 2-thiouracil,
2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine,
7-deazaguanine, N.sup.6(6-aminohexyl)adenine and 2,6-diaminopurine
(Kornberg, A., DNA Replication, W.H. Freeman & Co., San
Francisco, 1980, pages-75-77; Gebeyehu, G., et al., Nucleic Acids
Res., 1987, 15, 4513).
[0224] Sugar Modifications: Modified oligonucleotides can also
contain one or more substituted sugar moieties. Preferred
oligonucleotides comprise one of the following at the 2' position:
OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or
N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and
alkynyl can be substituted or unsubstituted C.sub.1 to C.sub.10
alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly
preferred are O[(CH.sub.2).sub.nO].sub.mCH.sub.3,
O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or
O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3,
SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3,
NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. A preferred
modification includes an alkoxyalkoxy group, 2'-methoxyethoxy
(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78, 486-504). Further preferred modifications include
2'-dimethylaminooxyethoxy, i.e., a
2'-O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as
2'-DMAOE and 2'-dimethylaminoethoxyethoxy, i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2.
[0225] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (2'-F).
Similar modifications can also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Oligonucleotides can also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative United States patents that
teach the preparation of such modified sugar structures include,
but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;
5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;
5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and
5,700,920, each of which is herein incorporated by reference.
[0226] Other Modifications: Similar modifications can also be made
at other positions on the oligonucleotide, particularly the 3'
position of the sugar on the 3' terminal nucleotide and the 5'
position of 5' terminal nucleotide. The 5' and 3' termini of an
oligonucleotide can also be modified to serve as points of chemical
conjugation of, e.g., lipophilic moieties (see immediately
subsequent paragraph), intercalating agents (Kuyavin et al., WO
96/32496, published Oct. 17, 1996; Nguyen et al., U.S. Pat. No.
4,835,263, issued Can 30, 1989) or hydroxyalkyl groups (Helene et
al., WO 96/34008, published Oct. 31, 1996).
[0227] Other positions within an oligonucleotide of the invention
can be used to chemically link thereto one or more effector groups
to form an oligonucleotide conjugate. An "effector group" is a
chemical moiety that is capable of carrying out a particular
chemical or biological function. Examples of such effector groups
include, but are not limited to, an RNA cleaving group, a reporter
group, an intercalator, a group for improving the pharmacokinetic
properties of an oligonucleotide, or a group for improving the
pharmacodynamic properties of an oligonucleotide and other
substituents having similar properties. A variety of chemical
linkers can be used to conjugate an effector group to an
oligonucleotide of the invention. As an example, U.S. Pat. No.
5,578,718 to Cook et al. discloses methods of attaching an
alkylthio linker, which can be further derivatized to include
additional groups, to ribofuranosyl positions, nucleosidic base
positions, or on internucleoside linkages. Additional methods of
conjugating oligonucleotides to various effector groups are known
in the art; see, e.g., Protocols for Oligonucleotide Conjugates
(Methods in Molecular Biology, Volume 26) Agrawal, S., ed., Humana
Press, Totowa, N.J., 1994.
[0228] Another preferred additional or alternative modification of
the oligonucleotides of the invention involves chemically linking
to the oligonucleotide one or more lipophilic moieties which
enhance the cellular uptake of the oligonucleotide. Such lipophilic
moieties can be linked to an oligonucleotide at several different
positions on the oligonucleotide. Some preferred positions include
the 3' position of the sugar of the 3' terminal nucleotide, the 5'
position of the sugar of the 5' terminal nucleotide, and the 2'
position of the sugar of any nucleotide. The N.sup.6 position of a
purine nucleobase can also be utilized to link a lipophilic moiety
to an oligonucleotide of the invention (Gebeyehu, G., et al.,
Nucleic Acids Res., 1987, 15, 4513). Such lipophilic moieties
include but are not limited to a cholesteryl moiety (Letsinger et
al., Proc. Natl. Acad. Sci. U.S.A., 1989, 86, 6553), cholic acid
(Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053), a
thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y.
Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem.
Let., 1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl.
Acids Res., 1992, 20, 533), an aliphatic chain, e.g., dodecandiol
or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10,
111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al.,
Biochimie, 1993, 75, 49), a phospholipid, e.g.,
di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res.,
1990, 18, 3777), a polyamine or a polyethylene glycol chain
(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969),
or adamantane acetic acid (Manoharan et al., Tetrahedron Lett.,
1995, 36, 3651), a palmityl moiety (Mishra et al., Biochim.
Biophys. Acta, 1995, 1264, 229), or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923). Oligonucleotides comprising
lipophilic moieties, and methods for preparing such
oligonucleotides, are disclosed in U.S. Pat. Nos. 5,138,045,
5,218,105 and 5,459,255.
[0229] The present invention also includes oligonucleotides that
are substantially chirally pure with regard to particular positions
within the oligonucleotides. Examples of substantially chirally
pure oligonucleotides include, but are not limited to, those having
phosphorothioate linkages that are at least 75% Sp or Rp (Cook et
al., U.S. Pat. No. 5,587,361) and those having substantially
chirally pure (Sp or Rp) alkylphosphonate, phosphoamidate or
phosphotriester linkages (Cook, U.S. Pat. Nos. 5,212,295 and
5,521,302).
[0230] Chimeric Oligonucleotides: The present invention also
includes oligonucleotides which are chimeric oligonucleotides.
"Chimeric" oligonucleotides or "chimeras," in the context of this
invention, are oligonucleotides which contain two or more
chemically distinct regions, each made up of at least one
nucleotide. These oligonucleotides typically contain at least one
region wherein the oligonucleotide is modified so as to confer upon
the oligonucleotide increased resistance to nuclease degradation,
increased cellular uptake, and/or increased binding affinity for
the target nucleic acid. An additional region of the
oligonucleotide can serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is
a cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. Activation of RNase H, therefore, results in cleavage of
the RNA target, thereby greatly enhancing the efficiency of
antisense inhibition of gene expression. Cleavage of the RNA target
can be routinely detected by gel electrophoresis and, if necessary,
associated nucleic acid hybridization techniques known in the art.
By way of example, such "chimeras" can be "gapmers," i.e.,
oligonucleotides in which a central portion (the "gap") of the
oligonucleotide serves as a substrate for, e.g., RNase H, and the
5' and 3' portions (the "wings") are modified in such a fashion so
as to have greater affinity for the target RNA molecule but are
unable to support nuclease activity (e.g., 2'-fluoro- or
2'-methoxyethoxy-substituted). Other chimeras include "wingmers,"
that is, oligonucleotides in which the 5' portion of the
oligonucleotide serves as a substrate for, e.g., RNase H, whereas
the 3' portion is modified in such a fashion so as to have greater
affinity for the target RNA molecule but is unable to support
nuclease activity (e.g., 2'-fluoro- or
2'-methoxyethoxy-substituted), or vice-versa.
[0231] Synthesis: The oligonucleotides used in accordance with this
invention can be conveniently and routinely made through the
well-known technique of solid phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art can additionally or alternatively be
employed. It is also known to use similar techniques to prepare
other oligonucleotides such as the phosphorothioates and alkylated
derivatives.
[0232] Teachings regarding the synthesis of particular modified
oligonucleotides can be found in the following U.S. patents or
pending patent applications, each of which is commonly assigned
with this application: U.S. Pat. Nos. 5,138,045 and 5,218,105,
drawn to polyamine conjugated oligonucleotides; U.S. Pat. No.
5,212,295, drawn to monomers for the preparation of
oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos.
5,378,825 and 5,541,307, drawn to oligonucleotides having modified
backbones; U.S. Pat. No. 5,386,023, drawn to backbone modified
oligonucleotides and the preparation thereof through reductive
coupling; U.S. Pat. No. 5,457,191, drawn to modified nucleobases
based on the 3-deazapurine ring system and methods of synthesis
thereof; U.S. Pat. No. 5,459,255, drawn to modified nucleobases
based on N-2 substituted purines; U.S. Pat. No. 5,521,302, drawn to
processes for preparing oligonucleotides having chiral phosphorus
linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids;
U.S. Pat. No. 5,554,746, drawn to oligonucleotides having
.beta.-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods
and materials for the synthesis of oligonucleotides; U.S. Pat. No.
5,578,718, drawn to nucleosides having alkylthio groups, wherein
such groups can be used as linkers to other moieties attached at
any of a variety of positions of the nucleoside; U.S. Pat. Nos.
5,587,361 and 5,599,797, drawn to oligonucleotides having
phosphorothioate linkages of high chiral purity; U.S. Pat. No.
5,506,351, drawn to processes for the preparation of 2'-O-alkyl
guanosine and related compounds, including 2,6-diaminopurine
compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides
having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to
oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,223,168,
issued Jun. 29, 1993, and U.S. Pat. No. 5,608,046, both drawn to
conjugated 4'-desmethyl nucleoside analogs; U.S. Pat. No.
5,602,240, and 5,610,289, drawn to backbone modified
oligonucleotide analogs; and U.S. patent application Ser. No.
08/383,666, filed Feb. 3, 1995, and U.S. Pat. No. 5,459,255, drawn
to, inter alia, methods of synthesizing
2'-fluoro-oligonucleotides.
[0233] 5-methyl-cytosine: In 2'-methoxyethoxy-modified
oligonucleotides, 5-methyl-2'-methoxyethoxy-cytosine residues are
used and are prepared as follows.
2,2'-Anhydro[1-(.beta.-D-arabinofuranosyl)-5-methyluridine]:
5-Methyluridine (ribosylthymine, commercially available through
Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0
g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to
DMF (300 mL). The mixture was heated to reflux, with stirring,
allowing the evolved carbon dioxide gas to be released in a
controlled manner. After 1 hour, the slightly darkened solution was
concentrated under reduced pressure. The resulting syrup was poured
into diethylether (2.5 L), with stirring. The product formed a gum.
The ether was decanted and the residue was dissolved in a minimum
amount of methanol (ca. 400 mL). The solution was poured into fresh
ether (2.5 L) to yield a stiff gum. The ether was decanted and the
gum was dried in a vacuum oven (60?C at 1 mm Hg for 24 h) to give a
solid which was crushed to a light tan powder (57 g, 85% crude
yield). The material was used as is for further reactions.
2'-O-Methoxyethyl-5-methyluridine: 2,2'-Anhydro-5-methyluridine
(195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and
2-methoxyethanol (1.2 L) were added to a 2 L stainless steel
pressure vessel and placed in a pre-heated oil bath at 160?C. After
heating for 48 hours at 155-160?C, the vessel was opened and the
solution evaporated to dryness and triturated with MeOH (200 mL).
The residue was suspended in hot acetone (1 L). The insoluble salts
were filtered, washed with acetone (150 mL) and the filtrate
evaporated. The residue (280 g) was dissolved in CH.sub.3CN (600
mL) and evaporated. A silica gel column (3 kg) was packed in
CH.sub.2Cl.sub.2/acetone/MeOH (20:5:3) containing 0.5% Et.sub.3NH.
The residue was dissolved in CH.sub.2Cl.sub.2 (250 mL) and adsorbed
onto silica (150 g) prior to loading onto the column. The product
was eluted with the packing solvent to give 160 g (63%) of product.
2'-O-Methoxyethyl-5'-O -dimethoxytrityl-5-methyluridine:
2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was
co-evaporated with pyridine (250 mL) and the dried residue
dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl
chloride (94.3 g, 0.278 M) was added and the mixture stirred at
room temperature for one hour. A second aliquot of dimethoxytrityl
chloride (94.3 g, 0.278 M) was added and the reaction stirred for
an additional one hour. Methanol (170 mL) was then added to stop
the reaction. HPLC showed the presence of approximately 70%
product. The solvent was evaporated and triturated with CH.sub.3CN
(200 mL). The residue was dissolved in CHCl.sub.3 (1.5 L) and
extracted with 2.times.500 mL of saturated NaHCO.sub.3 and
2.times.500 mL of saturated NaCl. The organic phase was dried over
Na.sub.2SO.sub.4, filtered and evaporated. 275 g of residue was
obtained. The residue was purified on a 3.5 kg silica gel column,
packed and eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5%
Et.sub.3NH. The pure fractions were evaporated to give 164 g of
product. Approximately 20 g additional was obtained from the impure
fractions to give a total yield of 183 g (57%).
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine:
2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106 g,
0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562
mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL,
0.258 M) were combined and stirred at room temperature for 24
hours. The reaction was monitored by tlc by first quenching the tlc
sample with the addition of MeOH. Upon completion of the reaction,
as judged by tlc, MeOH (50 mL) was added and the mixture evaporated
at 35?C. The residue was dissolved in CHCl.sub.3 (800 mL) and
extracted with 2.times.200 mL of saturated sodium bicarbonate and
2.times.200 mL of saturated NaCl. The water layers were back
extracted with 200 mL of CHCl.sub.3. The combined organics were
dried with sodium sulfate and evaporated to give 122 g of residue
(approximately 90% product). The residue was purified on a 3.5 kg
silica gel column and eluted using EtOAc/Hexane (4:1). Pure product
fractions were evaporated to yield 96 g (84%).
3'-O-Acetyl-2'-O-methoxyethyl-5'-O
-dimethoxytrityl-5-methyl-4-triazoleuridine: A first solution was
prepared by dissolving 3'-O-acetyl-2'-O
-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M)
in CH.sub.3CN (700 mL) and set aside. Triethylamine (189 mL, 1.44
M) was added to a solution of triazole (90 g, 1.3 M) in CH.sub.3CN
(1 L), cooled to -5?C and stirred for 0.5 h using an overhead
stirrer. POCl.sub.3 was added dropwise, over a 30 minute period, to
the stirred solution maintained at 0-10?C, and the resulting
mixture stirred for an additional 2 hours. The first solution was
added dropwise, over a 45 minute period, to the later solution. The
resulting reaction mixture was stored overnight in a cold room.
Salts were filtered from the reaction mixture and the solution was
evaporated. The residue was dissolved in EtOAc (1 L) and the
insoluble solids were removed by filtration. The filtrate was
washed with 1.times.300 mL of NaHCO.sub.3 and 2.times.300 mL of
saturated NaCl, dried over sodium sulfate and evaporated. The
residue was triturated with EtOAc to give the title compound. 2'-O
-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine: A solution of
3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuri-
dine (103 g, 0.141 M) in dioxane (500 mL) and NH.sub.4OH (30 mL)
was stirred at room temperature for 2 hours. The dioxane solution
was evaporated and the residue azeotroped with MeOH (2.times.200
mL). The residue was dissolved in MeOH (300 mL) and transferred to
a 2 liter stainless steel pressure vessel. Methanol (400 mL)
saturated with NH.sub.3 gas was added and the vessel heated to
100?C for 2 hours (thin layer chromatography, tlc, showed complete
conversion). The vessel contents were evaporated to dryness and the
residue was dissolved in EtOAc (500 mL) and washed once with
saturated NaCl (200 mL). The organics were dried over sodium
sulfate and the solvent was evaporated to give 85 g (95%) of the
title compound.
N.sup.4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine:
2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85 g,
0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2
g, 0.165 M) was added with stirring. After stirring for 3 hours,
tlc showed the reaction to be approximately 95% complete. The
solvent was evaporated and the residue azeotroped with MeOH (200
mL). The residue was dissolved in CHCl.sub.3 (700 mL) and extracted
with saturated NaHCO.sub.3 (2.times.300 mL) and saturated NaCl
(2.times.300 mL), dried over MgSO.sub.4 and evaporated to give a
residue (96 g). The residue was chromatographed on a 1.5 kg silica
column using EtOAc/Hexane (1:1) containing 0.5% Et.sub.3NH as the
eluting solvent. The pure product fractions were evaporated to give
90 g (90%) of the title compound.
N.sup.4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine-3-
'-amidite: N.sup.4-Benzoyl-2'-O
-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M)
was dissolved in CH.sub.2Cl.sub.2 (1 L). Tetrazole diisopropylamine
(7.1 g) and 2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123
M) were added with stirring, under a nitrogen atmosphere. The
resulting mixture was stirred for 20 hours at room temperature (tic
showed the reaction to be 95% complete). The reaction mixture was
extracted with saturated NaHCO.sub.3 (1.times. 300 mL) and
saturated NaCl (3.times.300 mL). The aqueous washes were
back-extracted with CH.sub.2Cl.sub.2 (300 mL), and the extracts
were combined, dried over MgSO.sub.4 and concentrated. The residue
obtained was chromatographed on a 1.5 kg silica column using
EtOAc\Hexane (3:1) as the eluting solvent. The pure fractions were
combined to give 90.6 g (87%) of the title compound.
2'-O-(Aminooxyethyl) nucleoside amidites and
2'-O-(dimethylaminooxyethyl) nucleoside amidites
2'-(Dimethylaminooxyethoxy) nucleoside amidites
[0234] 2'-(Dimethylaminooxyethoxy) nucleoside amidites [also known
in the art as 2'-O-(dimethylaminooxyethyl) nucleoside amidites] are
prepared as described in the following paragraphs. Adenosine,
cytidine and guanosine nucleoside amidites are prepared similarly
to the thymidine (5-methyluridine) except the exocyclic amines are
protected with a benzoyl moiety in the case of adenosine and
cytidine and with isobutyryl in the case of guanosine.
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine
[0235] O.sup.2-2'-anhydro-5-methyluridine (Pro. Bio. Sint., Varese,
Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013
eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient
temperature under an argon atmosphere and with mechanical stirring.
tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458
mmol) was added in one portion. The reaction was stirred for 16 h
at ambient temperature. TLC (Rf 0.22, ethyl acetate) indicated a
complete reaction. The solution was concentrated under reduced
pressure to a thick oil. This was partitioned between
dichloromethane (1 L) and saturated sodium bicarbonate (2.times.1
L) and brine (1 L). The organic layer was dried over sodium sulfate
and concentrated under reduced pressure to a thick oil. The oil was
dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600
mL) and the solution was cooled to -10.degree. C. The resulting
crystalline product was collected by filtration, washed with ethyl
ether (3.times.200 mL) and dried (40.degree. C., 1 mm Hg, 24 h) to
149 g (74.8%) of white solid. TLC and NMR were consistent with pure
product.
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridi-
ne
[0236] In a 2 L stainless steel, unstirred pressure reactor was
added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the
fume hood and with manual stirring, ethylene glycol (350 mL,
excess) was added cautiously at first until the evolution of
hydrogen gas subsided.
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine
(149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were
added with manual stirring. The reactor was sealed and heated in an
oil bath until an internal temperature of 160.degree. C. was
reached and then maintained for 16 h (pressure <100 psig). The
reaction vessel was cooled to ambient and opened. TLC (Rf 0.67 for
desired product and Rf 0.82 for ara-T side product, ethyl acetate)
indicated about 70% conversion to the product. In order to avoid
additional side product formation, the reaction was stopped,
concentrated under reduced pressure (10 to 1 mm Hg) in a warm water
bath (40-100.degree. C.) with the more extreme conditions used to
remove the ethylene glycol. [Alternatively, once the low boiling
solvent is gone, the remaining solution can be partitioned between
ethyl acetate and water. The product will be in the organic phase.]
The residue was purified by column chromatography (2 kg silica gel,
ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate
fractions were combined, stripped and dried to product as a white
crisp foam (84 g, 50%), contaminated starting material (17.4 g) and
pure reusable starting material 20 g. The yield based on starting
material less pure recovered starting material was 58%. TLC and NMR
were consistent with 99% pure product.
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine
[0237]
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
(20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g,
44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was
then dried over P.sub.2O.sub.5 under high vacuum for two days at
40?C. The reaction mixture was flushed with argon and dry THF
(369.8 mL, Aldrich, sure seal bottle) was added to get a clear
solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added
dropwise to the reaction mixture. The rate of addition is
maintained such that resulting deep red coloration is just
discharged before adding the next drop. After the addition was
complete, the reaction was stirred for 4 hrs. By that time TLC
showed the completion of the reaction (ethylacetate:hexane, 60:40).
The solvent was evaporated in vacuum. Residue obtained was placed
on a flash column and eluted with ethyl acetate:hexane (60:40), to
get
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine
as white foam (21.819 g, 86%).
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methylurid-
ine
[0238]
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridi-
ne (3.1 g, 4.5 mmol) was dissolved in dry CH.sub.2Cl.sub.2 (4.5 mL)
and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at -10?C
to 0?C. After 1 h the mixture was filtered, the filtrate was washed
with ice cold CH.sub.2Cl.sub.2 and the combined organic phase was
washed with water, brine and dried over anhydrous Na.sub.2SO.sub.4.
The solution was concentrated to get 2'-O-(aminooxyethyl)
thymidine, which was then dissolved in MeOH (67.5 mL). To this
formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the
resulting mixture was stirred for 1 h. Solvent was removed under
vacuum; residue chromatographed to get
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluri-
dine as white foam (1.95 g, 78%).
5'-O-tert-Butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methylurid-
ine
[0239]
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-met-
hyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M
pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium
cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at
10?C under inert atmosphere. The reaction mixture was stirred for
10 minutes at 10?C. After that the reaction vessel was removed from
the ice bath and stirred at room temperature for 2 h, the reaction
monitored by TLC (5% MeOH in CH.sub.2Cl.sub.2). Aqueous NaHCO.sub.3
solution (5%, 10 mL) was added and extracted with ethyl acetate
(2.times.20 mL). Ethyl acetate phase was dried over anhydrous
Na.sub.2SO.sub.4, evaporated to dryness. Residue was dissolved in a
solution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30
mL, 3.37 mmol) was added and the reaction mixture was stirred at
room temperature for 10 minutes. Reaction mixture cooled to 10?C in
an ice bath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added
and reaction mixture stirred at 10?C for 10 minutes. After 10
minutes, the reaction mixture was removed from the ice bath and
stirred at room temperature for 2 hrs. To the reaction mixture 5%
NaHCO.sub.3 (25 mL) solution was added and extracted with ethyl
acetate (2.times.25 mL). Ethyl acetate layer was dried over
anhydrous Na.sub.2SO.sub.4 and evaporated to dryness. The residue
obtained was purified by flash column chromatography and eluted
with 5% MeOH in CH.sub.2Cl.sub.2 to get
5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluri-
dine as a white foam (14.6 g, 80%).
2'-O-(dimethylaminooxyethyl)-5-methyluridine
[0240] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was
dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept
over KOH). This mixture of triethylamine-2HF was then added to
5-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methylurid-
ine (1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs.
Reaction was monitored by TLC (5% MeOH in CH.sub.2Cl.sub.2).
Solvent was removed under vacuum and the residue placed on a flash
column and eluted with 10% MeOH in CH.sub.2Cl.sub.2 to get 2'-O
-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%).
5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine
[0241] 2'-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17
mmol) was dried over P.sub.2O.sub.5 under high vacuum overnight at
40?C. It was then co-evaporated with anhydrous pyridine (20 mL).
The residue obtained was dissolved in pyridine (11 mL) under argon
atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol),
4,4'-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the
mixture and the reaction mixture was stirred at room temperature
until all of the starting material disappeared. Pyridine was
removed under vacuum and the residue chromatographed and eluted
with 10% MeOH in CH.sub.2Cl.sub.2 (containing a few drops of
pyridine) to get
5'-O-DMT-2'-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g,
80%).
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoet-
hyl)-N,N-diisopropylphosphoramidite]
[0242] 5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine (1.08
g, 1.67 mmol) was co-evaporated with toluene (20 mL). To the
residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was
added and dried over P.sub.2O.sub.5 under high vacuum overnight at
40?C. Then the reaction mixture was dissolved in anhydrous
acetonitrile (8.4 mL) and
2-cyanoethyl-N,N,N.sup.1,N.sup.1-tetraisopropylphosphoramidite
(2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at
ambient temperature for 4 hrs under inert atmosphere. The progress
of the reaction was monitored by TLC (hexane:ethyl acetate 1:1).
The solvent was evaporated, then the residue was dissolved in ethyl
acetate (70 mL) and washed with 5% aqueous NaHCO.sub.3 (40 mL).
Ethyl acetate layer was dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. Residue obtained was chromatographed (ethyl acetate
as eluent) to get
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoe-
thyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g,
74.9%).
2'-(Aminooxyethoxy) nucleoside amidites
[0243] 2'-(Aminooxyethoxy) nucleoside amidites [also known in the
art as 2'-O-(aminooxyethyl) nucleoside amidites] are prepared as
described in the following paragraphs. Adenosine, cytidine and
thymidine nucleoside amidites are prepared similarly.
N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimeth-
oxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]
[0244] The 2'-O-aminooxyethyl guanosine analog can be obtained by
selective 2'-O-alkylation of diaminopurine riboside. Multigram
quantities of diaminopurine riboside can be purchased from Schering
AG (Berlin) to provide 2'-O-(2-ethylacetyl) diaminopurine riboside
along with a minor amount of the 3'-O -isomer. 2'-O-(2-ethylacetyl)
diaminopurine riboside can be resolved and converted to
2'-O-(2-ethylacetyl)guanosine by treatment with adenosine
deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO
94/02501 A1 940203.) Standard protection procedures should afford
2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine and
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'--
dimethoxytrityl)guanosine which can be reduced to provide
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dime-
thoxytrityl)guanosine. As before the hydroxyl group can be
displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the
protected nucleoside can phosphitylated as usual to yield
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dime-
thoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].
[0245] Bioequivalents: The compounds of the invention encompass any
pharmaceutically acceptable salts, esters, or salts of such esters,
or any other compound which, upon administration to an animal
including a human, is capable of providing (directly or indirectly)
the biologically active metabolite or residue thereof. Accordingly,
for example, the disclosure is also drawn to "prodrugs" and
"pharmaceutically acceptable salts" of the oligonucleotides of the
invention, pharmaceutically acceptable salts of such prodrugs, and
other bioequivalents.
[0246] Oligonucleotide Prodrugs: The oligonucleotides of the
invention can additionally or alternatively be prepared to be
delivered in a "prodrug" form. The term "prodrug" indicates a
therapeutic agent that is prepared in an inactive form that is
converted to an active form (i.e., drug) within the body or cells
thereof by the action of endogenous enzymes or other chemicals
and/or conditions. In particular, prodrug versions of the
oligonucleotides of the invention are prepared as SATE
[(S-acetyl-2-thioethyl)phosphate] derivatives according to the
methods disclosed in WO 93/24510 to Gosselin et al., published Dec.
9, 1993.
[0247] Pharmaceutically Acceptable Salts: The term A
pharmaceutically acceptable salts" refers to physiologically and
pharmaceutically acceptable salts of the oligonucleotides of the
invention: i.e., salts that retain the desired biological activity
of the parent compound and do not impart undesired toxicological
effects thereto.
[0248] Pharmaceutically acceptable base addition salts are formed
with metals or amines, such as alkali and alkaline earth metals or
organic amines. Examples of metals used as cations are sodium,
potassium, magnesium, calcium, and the like. Examples of suitable
amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, dicyclohexylamine, ethylenediamine,
N-methylglucamine, and procaine (see, for example, Berge et al.,
"Pharmaceutical Salts," J. of Pharma Sci., 1977, 66, 1). The base
addition salts of said acidic compounds are prepared by contacting
the free acid form with a sufficient amount of the desired base to
produce the salt in the conventional manner. The free acid form can
be regenerated by contacting the salt form with an acid and
isolating the free acid in the conventional manner. The free acid
forms differ from their respective salt forms somewhat in certain
physical properties such as solubility in polar solvents, but
otherwise the salts are equivalent to their respective free acid
for purposes of the present invention. As used herein, a
"pharmaceutical addition salt" includes a pharmaceutically
acceptable salt of an acid form of one of the components of the
compositions of the invention. These include organic or inorganic
acid salts of the amines. Preferred acid salts are the
hydrochlorides, acetates, salicylates, nitrates and phosphates.
Other suitable pharmaceutically acceptable salts are well known to
those skilled in the art and include basic salts of a variety of
inorganic and organic acids, such as, for example, with inorganic
acids, such as for example hydrochloric acid, hydrobromic acid,
sulfuric acid or phosphoric acid; with organic carboxylic,
sulfonic, sulfo or phospho acids or N-substituted sulfamic acids,
for example acetic acid, propionic acid, glycolic acid, succinic
acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric
acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic
acid, glucaric acid, glucuronic acid, citric acid, benzoic acid,
cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic
acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid,
nicotinic acid or isonicotinic acid; and with amino acids, such as
the 20 alpha-amino acids involved in the synthesis of proteins in
nature, for example glutamic acid or aspartic acid, and also with
phenylacetic acid, methanesulfonic acid, ethanesulfonic acid,
2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,
benzenesulfonic acid, 4-methylbenzenesulonic acid,
naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or
3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid
(with the formation of cyclamates), or with other acid organic
compounds, such as ascorbic acid. Pharmaceutically acceptable salts
of compounds can also be prepared with a pharmaceutically
acceptable cation. Suitable pharmaceutically acceptable cations are
well known to those skilled in the art and include alkaline,
alkaline earth, ammonium and quaternary ammonium cations.
Carbonates or hydrogen carbonates are also possible.
[0249] For oligonucleotides, preferred examples of pharmaceutically
acceptable salts include but are not limited to (a) salts formed
with cations such as sodium, potassium, ammonium, magnesium,
calcium, polyamines such as spermine and spermidine, etc.; (b) acid
addition salts formed with inorganic acids, for example
hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric
acid, nitric acid and the like; (c) salts formed with organic acids
such as, for example, acetic acid, oxalic acid, tartaric acid,
succinic acid, maleic acid, fumaric acid, gluconic acid, citric
acid, malic acid, ascorbic acid, benzoic acid, tannic acid,
palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic
acid, methanesulfonic acid, p-toluenesulfonic acid,
naphthalenedisulfonic acid, polygalacturonic acid, and the like;
and (d) salts formed from elemental anions such as chlorine,
bromine, and iodine.
[0250] Exemplary Utilities of the Invention: The oligonucleotides
of the present invention specifically hybridize to nucleic acids
(e.g., mRNAs) encoding a JNK1 protein. The oligonucleotides of the
present invention can be utilized as therapeutic compounds, as
diagnostic tools or research reagents that can be incorporated into
kits, and in purifications and cellular product preparations, as
well as other methodologies, which are appreciated by persons of
ordinary skill in the art.
[0251] Assays and Diagnostic Applications: The oligonucleotides of
the present invention can be used to detect the presence of JNK1
protein-specific nucleic acids in a cell or tissue sample. For
example, radiolabeled oligonucleotides can be prepared by .sup.32P
labeling at the 5' end with polynucleotide kinase. (Sambrook et
al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor
Laboratory Press, 1989, Volume 2, pg. 10.59.) Radiolabeled
oligonucleotides are then contacted with cell or tissue samples
suspected of containing JNK1 protein message RNAs (and thus JNK1
proteins), and the samples are washed to remove unbound
oligonucleotide. Radioactivity remaining in the sample indicates
the presence of bound oligonucleotide, which in turn indicates the
presence of nucleic acids complementary to the oligonucleotide, and
can be quantitated using a scintillation counter or other routine
means. Expression of nucleic acids encoding these proteins is thus
detected.
[0252] Radiolabeled oligonucleotides of the present invention can
also be used to perform autoradiography of tissues to determine the
localization, distribution and quantitation of JNK1 proteins for
research, diagnostic or therapeutic purposes. In such studies,
tissue sections are treated with radiolabeled oligonucleotide and
washed as described above, then exposed to photographic emulsion
according to routine autoradiography procedures. The emulsion, when
developed, yields an image of silver grains over the regions
expressing a JNK1 protein gene. Quantitation of the silver grains
permits detection of the expression of mRNA molecules encoding
these proteins and permits targeting of oligonucleotides to these
areas.
[0253] Analogous assays for fluorescent detection of expression of
JNK1 protein nucleic acids can be developed using oligonucleotides
of the present invention which are conjugated with fluorescein or
other fluorescent tags instead of radiolabeling. Such conjugations
are routinely accomplished during solid phase synthesis using
fluorescently-labeled amidites or controlled pore glass (CPG)
columns. Fluorescein-labeled amidites and CPG are available from,
e.g., Glen Research, Sterling Va. Other means of labeling
oligonucleotides are known in the art (see, e.g., Ruth, Chapter 6
In Methods in Molecular Biology, Vol. 26: Protocols for
Oligonucleotide Conjugates, Agrawal, ed., Humana Press Inc.,
Totowa, N.J., 1994, pages 167-185).
[0254] Kits for detecting the presence or absence of expression of
a JNK1 protein can also be prepared. Such kits include an
oligonucleotide targeted to an appropriate gene, i.e., a gene
encoding a JNK1 protein. Appropriate kit and assay formats, such
as, e.g., "sandwich" assays, are known in the art and can easily be
adapted for use with the oligonucleotides of the invention.
Hybridization of the oligonucleotides of the invention with a
nucleic acid encoding a JNK1 protein can be detected by means known
in the art. Such means can include conjugation of an enzyme to the
oligonucleotide, radiolabelling of the oligonucleotide or any other
suitable detection systems.
[0255] Protein Purifications: The oligonucleotides of the invention
are also useful for the purification of specific Jun kinase
proteins from cells that normally express a set of JNK proteins
which are similar to each other in terms of their polypeptide
sequences and biochemical properties. As an example, the
purification of a JNK1 protein from cells that expresses JNK1, JNK2
and JNK3 proteins can be enhanced by first treating such cells with
oligonucleotides that inhibit the expression of JNK2 and JNK3
and/or with oligonucleotides that increase the expression of JNK1,
because such treatments will increase the relative ratio of JNK1
relative to JNK2 and JNK3. As a result, the yield of JNK1 from
subsequent purification steps will be improved as the amount of the
biochemically similar (and thus likely to contaminate) JNK2 and
JNK3 proteins in extracts prepared from cells so treated will be
diminished.
[0256] Biologically Active Oligonucleotides: The invention is also
drawn to the administration of oligonucleotides having biological
activity to cultured cells, isolated tissues and organs and
animals. By "having biological activity," it is meant that the
oligonucleotide functions to modulate the expression of one or more
genes in cultured cells, isolated tissues or organs and/or animals.
Such modulation can be achieved by an antisense oligonucleotide by
a variety of mechanisms known in the art, including but not limited
to transcriptional arrest; effects on RNA processing (capping,
polyadenylation and splicing) and transportation; enhancement of
cellular degradation of the target nucleic acid; and translational
arrest (Crooke et al., Exp. Opin. Ther. Patents, 1996 6, 855).
[0257] In an animal other than a human, the compositions and
methods of the invention can be used to study the function of one
or more genes in the animal. For example, antisense
oligonucleotides have been systemically administered to rats in
order to study the role of the N-methyl-D-aspartate receptor in
neuronal death, to mice in order to investigate the biological role
of protein kinase C-a, and to rats in order to examine the role of
the neuropeptide Y1 receptor in anxiety (Wahlestedt et al., Nature,
1993, 363, 260; Dean et al., Proc. Natl. Acad. Sci. USA., 1994, 91,
11762; and Wahlestedt et al., Science, 1993, 259, 528,
respectively). In instances where complex families of related
proteins are being investigated, "antisense knockouts" (i.e.,
inhibition of a gene by systemic administration of antisense
oligonucleotides) can represent the most accurate means for
examining a specific member of the family (see, generally, Albert
et al., Trends Pharmacol. Sci., 1994, 15, 250).
[0258] The compositions and methods of the invention also have
therapeutic uses in an animal, including a human, having (i.e.,
suffering from), or known to be or suspected of being prone to
having, a disease or disorder that is treatable in whole or in part
with one or more nucleic acids. The term "therapeutic uses" is
intended to encompass prophylactic, palliative and curative uses
wherein the oligonucleotides of the invention are contacted with
animal cells either in vivo or ex vivo. When contacted with animal
cells ex vivo, a therapeutic use includes incorporating such cells
into an animal after treatment with one or more oligonucleotides of
the invention.
[0259] For therapeutic uses, an animal suspected of having a
disease or disorder which can be treated or prevented by modulating
the expression or activity of a JNK1 protein is, for example,
treated by administering oligonucleotides in accordance with this
invention. The oligonucleotides of the invention can be utilized in
pharmaceutical compositions by adding an effective amount of an
oligonucleotide to a suitable pharmaceutically acceptable carrier
such as, e.g., a diluent. Workers in the field have identified
antisense, triplex and other oligonucleotide compositions which are
capable of modulating expression of genes implicated in viral,
fungal and metabolic diseases. Antisense oligonucleotides have been
safely administered to humans and several clinical trials are
presently underway. It is thus established that oligonucleotides
can be useful therapeutic instrumentalities that can be configured
to be useful in treatment regimes for treatment of cells, tissues
and animals, especially humans. The following U.S. patents
demonstrate palliative, therapeutic and other methods utilizing
antisense oligonucleotides. U.S. Pat. No. 5,135,917 provides
antisense oligonucleotides that inhibit human interleukin-1
receptor expression. U.S. Pat. No. 5,098,890 is directed to
antisense oligonucleotides complementary to the c-myb oncogene and
antisense oligonucleotide therapies for certain cancerous
conditions. U.S. Pat. No. 5,087,617 provides methods for treating
cancer patients with antisense oligonucleotides. U.S. Pat. No.
5,166,195 provides oligonucleotide inhibitors of Human
Immunodeficiency Virus (HIV). U.S. Pat. No. 5,004,810 provides
oligomers capable of hybridizing to herpes simplex virus Vmw65 mRNA
and inhibiting replication. U.S. Pat. No. 5,194,428 provides
antisense oligonucleotides having antiviral activity against
influenza virus. U.S. Pat. No. 5,004,810 provides antisense
oligonucleotides and methods using them to inhibit HTLV-III
replication. U.S. Pat. No. 5,286,717 provides oligonucleotides
having a complementary base sequence to a portion of an oncogene.
U.S. Pat. No. 5,276,019 and U.S. Pat. No. 5,264,423 are directed to
phosphorothioate oligonucleotide analogs used to prevent
replication of foreign nucleic acids in cells. U.S. Pat. No.
4,689,320 is directed to antisense oligonucleotides as antiviral
agents specific to cytomegalovirus (CMV). U.S. Pat. No. 5,098,890
provides oligonucleotides complementary to at least a portion of
the mRNA transcript of the human c-myb gene. U.S. Pat. No.
5,242,906 provides antisense oligonucleotides useful in the
treatment of latent Epstein-Barr virus (EBV) infections.
[0260] As used herein, the term "disease, condition or disorder"
includes any abnormal condition of an organism or part that impairs
normal physiological functioning; isuch as obesity and metabolic
syndrome. As used herein, the term "prevention" means to delay or
forestall onset or development of a condition or disease for a
period of time from hours to days, preferably weeks to months. As
used herein, the term "amelioration" means a lessening of at least
one indicator of the severity of a condition or disease. The
severity of indicators can be determined by subjective or objective
measures which are known to those skilled in the art. As used
herein, "treatment" means to administer a composition of the
invention to effect an alteration or improvement of the disease or
condition. Prevention, amelioration, and/or treatment can require
administration of multiple doses at regular intervals, or prior to
exposure to an agent (e.g., an allergen) to alter the course of the
condition or disease. Moreover, a single agent can be used in a
single individual for each prevention, amelioration, and treatment
of a condition or disease sequentially, or concurrently. The term
"a disease or disorder that is treatable in whole or in part with
one or more nucleic acids" refers to a disease or disorder, as
herein defined, the management, modulation or treatment thereof,
and/or therapeutic, curative, palliative and/or prophylactic relief
therefrom, can be provided via the administration of an antisense
oligonucleotide.
[0261] Pharmaceutical Compositions: The formulation of
pharmaceutical compositions comprising the oligonucleotides of the
invention, and their subsequent administration, are believed to be
within the skill of those in the art.
[0262] Therapeutic Considerations: In general, for therapeutic
applications, a patient (i.e., an animal, including a human, having
or predisposed to a disease or disorder) is administered one or
more oligonucleotides, in accordance with the invention in a
pharmaceutically acceptable carrier in doses ranging from 0.01 g to
100 g per kg of body weight depending on the age of the patient and
the severity of the disorder or disease state being treated.
Further, the treatment regimen can last for a period of time which
will vary depending upon the nature of the particular disease or
disorder, its severity and the overall condition of the patient,
and can extend from once daily to once every 20 years. In the
context of the invention, the term "treatment regimen" is meant to
encompass therapeutic, palliative and prophylactic modalities.
Following treatment, the patient is monitored for changes in
his/her condition and for alleviation of the symptoms of the
disorder or disease state. The dosage of the nucleic acid can
either be increased in the event the patient does not respond
significantly to current dosage levels, or the dose can be
decreased if an alleviation of the symptoms of the disorder or
disease state is observed, or if the disorder or disease state has
been ablated.
[0263] Dosing is dependent on severity and responsiveness of the
disease state to be treated, with the course of treatment lasting
from several days to several months, or until a cure is effected or
a diminution of the disease state is achieved. Optimal dosing
schedules can be calculated from measurements of drug accumulation
in the body of the patient. Persons of ordinary skill can easily
determine optimum dosages, dosing methodologies and repetition
rates. Optimum dosages can vary depending on the relative potency
of individual oligonucleotides, and can generally be estimated
based on EC.sub.50s found to be effective in in vitro and in vivo
animal models. In general, dosage is from 0.01 g to 100 g per kg of
body weight, and can be given once or more daily, weekly, monthly
or yearly, or even once every 2 to 20 years. An optimal dosing
schedule is used to deliver a therapeutically effective amount of
the oligonucleotide being administered via a particular mode of
administration.
[0264] The term "therapeutically effective amount," for the
purposes of the invention, refers to the amount of
oligonucleotide-containing pharmaceutical composition which is
effective to achieve an intended purpose without undesirable side
effects (such as toxicity, irritation or allergic response).
Although individual needs can vary, determination of optimal ranges
for effective amounts of pharmaceutical compositions is within the
skill of the art. Human doses can be extrapolated from animal
studies (Katocs et al., Chapter 27 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990).
[0265] Generally, the dosage required to provide an effective
amount of a pharmaceutical composition, which can be adjusted by
one skilled in the art, will vary depending on the age, health,
physical condition, weight, type and extent of the disease or
disorder of the recipient, frequency of treatment, the nature of
concurrent therapy (if any) and the nature and scope of the desired
effect(s) (Nies et al., Chapter 3 In: Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al.,
Eds., McGraw-Hill, New York, N.Y., 1996).
[0266] As used herein, the term "high risk individual" is meant to
refer to a subject for whom it has been determined, via, e.g.,
individual or family history or genetic testing, has a
significantly higher than normal probability of being susceptible
to the onset or recurrence of a disease or disorder. As art of
treatment regimen for a high risk individual, the individual can be
prophylactically treated to prevent the onset or recurrence of the
disease or disorder. The term "prophylactically effective amount"
is meant to refer to an amount of a pharmaceutical composition
which produces an effect observed as the prevention of the onset or
recurrence of a disease or disorder. Prophylactically effective
amounts of a pharmaceutical composition are typically determined by
the effect they have compared to the effect observed when a second
pharmaceutical composition lacking the active agent is administered
to a similarly situated individual.
[0267] Following successful treatment, it can be desirable to have
the patient undergo maintenance therapy to prevent the recurrence
of the disease state, wherein the nucleic acid is administered in
maintenance doses, ranging from 0.01 g to 100 g per kg of body
weight, once or more daily, to once every 20 years. For example, in
the case of in individual known or suspected of being prone to an
autoimmune or inflammatory condition, prophylactic effects can be
achieved by administration of preventative doses, ranging from 0.01
.mu.g to 100 g per kg of body weight, once or more daily, to once
every 20 years. In like fashion, a subject can be made less
susceptible to an inflammatory condition that is expected to occur
as a result of some medical treatment, e.g., graft versus host
disease resulting from the transplantation of cells, tissue or an
organ into the individual.
[0268] In some cases it can be more effective to treat a patient
with an oligonucleotide of the invention in conjunction with other
traditional therapeutic modalities in order to increase the
efficacy of a treatment regimen. A treatment regimen encompasses
therapeutic, palliative and prophylactic modalities. For example, a
patient can be treated with conventional chemotherapeutic agents,
particularly those used for tumor and cancer treatment. Examples of
such chemotherapeutic agents include but are not limited to
daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,
idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,
cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,
mitomycin C, actinomycin D, mithramycin, prednisone,
hydroxyprogesterone, testosterone, tamoxifen, dacarbazine,
procarbazine, hexamethylmelamine, pentamethylmelamine,
mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,
nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,
6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea,
deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil
(5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX),
colchicine, vincristine, vinblastine, etoposide, trimetrexate,
teniposide, cisplatin and diethylstilbestrol (DES). See, generally,
The Merck Manual of Diagnosis and Therapy, 15th Ed., pp. 1206-1228,
Berkow et al., Eds., Rahay, N.J., 1987). When used with the
compounds of the invention, such chemotherapeutic agents can be
used individually (e.g., 5-FU and oligonucleotide), sequentially
(e.g., 5-FU and oligonucleotide for a period of time followed by
MTX and oligonucleotide), or in combination with one or more other
such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide,
or 5-FU, radiotherapy and oligonucleotide).
[0269] In another preferred embodiment of the invention, a first
antisense oligonucleotide targeted to a first JNK1 protein is used
in combination with a second antisense oligonucleotide targeted to
a second JNK protein in order to such JNK proteins to a more
extensive degree than can be achieved when either oligonucleotide
is used individually. In various embodiments of the invention, the
first and second JNK proteins which are targeted by such
oligonucleotides are identical, are different JNK proteins or are
different isoforms of the same JNK protein.
[0270] Pharmaceutical Compositions Pharmaceutical compositions for
the non-parenteral administration of oligonucleotides can include
sterile aqueous solutions which can also contain buffers, diluents
and other suitable additives. Pharmaceutically acceptable organic
or inorganic carrier substances suitable for non-parenteral
administration which do not deleteriously react with
oligonucleotides can be used. Suitable pharmaceutically acceptable
carriers include, but are not limited to, water, salt solutions,
alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium
stearate, talc, silicic acid, viscous paraffin,
hydroxymethylcellulose, polyvinylpyrrolidone and the like. The
pharmaceutical compositions can be sterilized and, if desired,
mixed with auxiliary agents, e.g., lubricants, preservatives,
stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic pressure, buffers, colorings flavorings and/or aromatic
substances and the like which do not deleteriously react with the
oligonucleotide(s) of the pharmaceutical composition.
Pharmaceutical compositions in the form of aqueous suspensions can
contain substances which increase the viscosity of the suspension
including, for example, sodium carboxymethylcellulose, sorbitol
and/or dextran. Optionally, such suspensions can also contain
stabilizers.
[0271] In one embodiment of the invention, an oligonucleotide is
administered via the rectal mode. In particular, pharmaceutical
compositions for rectal administration include foams, solutions
(enemas) and suppositories. Rectal suppositories for adults are
usually tapered at one or both ends and typically weigh about 2 g
each, with infant rectal suppositories typically weighing about
one-half as much, when the usual base, cocoa butter, is used
(Block, Chapter 87 In: Remington's Pharmaceutical Sciences, 18th
Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990).
[0272] In a preferred embodiment of the invention, one or more
oligonucleotides are administered via oral delivery. Pharmaceutical
compositions for oral administration include powders or granules,
suspensions or solutions in water or non-aqueous media, capsules,
sachets, troches, tablets or SECs (soft elastic capsules or
"caplets"). Thickeners, flavoring agents, diluents, emulsifiers,
dispersing aids, carrier substances or binders can be desirably
added to such pharmaceutical compositions. The use of such
pharmaceutical compositions has the effect of delivering the
oligonucleotide to the alimentary canal for exposure to the mucosa
thereof. Accordingly, the pharmaceutical composition can comprise
material effective in protecting the oligonucleotide from pH
extremes of the stomach, or in releasing the oligonucleotide over
time, to optimize the delivery thereof to a particular mucosal
site. Enteric coatings for acid-resistant tablets, capsules and
caplets are known in the art and typically include acetate
phthalate, propylene glycol and sorbitan monoleate.
[0273] Various methods for producing pharmaceutical compositions
for alimentary delivery are well known in the art. See, generally,
Nairn, Chapter 83; Block, Chapter 87; Rudnic et al., Chapter 89;
Porter, Chapter 90; and Longer et al., Chapter 91 In: Remington's
Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing
Co., Easton, Pa., 1990. The oligonucleotides of the invention can
be incorporated in a known manner into customary pharmaceutical
compositions, such as tablets, coated tablets, pills, granules,
aerosols, syrups, emulsions, suspensions and solutions, using
inert, non-toxic, pharmaceutically acceptable carriers
(excipients). The therapeutically active compound should in each
case be present here in a concentration of about 0.5% to about 95%
by weight of the total mixture, i.e., in amounts which are
sufficient to achieve the stated dosage range. The pharmaceutical
compositions are prepared, for example, by diluting the active
compounds with pharmaceutically acceptable carriers, if appropriate
using emulsifying agents and/or dispersing agents, and, for
example, in the case where water is used as the diluent, organic
solvents can be used as auxiliary solvents if appropriate.
Pharmaceutical compositions can be formulated in a conventional
manner using additional pharmaceutically acceptable carriers as
appropriate. Thus, the compositions can be prepared by conventional
means with additional excipients such as binding agents (e.g.,
pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl
methylcellulose); fillers (e.g., lactose, microcrystalline
cellulose or calcium hydrogen phosphate); lubricants (e.g.,
magnesium stearate, talc or silica); disintegrates (e.g., starch or
sodium starch glycolate); or wetting agents (e.g., sodium lauryl
sulfate). Tablets can be coated by methods well known in the art.
The preparations can also contain flavoring, coloring and/or
sweetening agents as appropriate.
[0274] The pharmaceutical compositions, which can conveniently be
presented in unit dosage form, can be prepared according to
conventional techniques well known in the pharmaceutical industry.
Such techniques include the step of bringing into association the
active ingredient(s) with the pharmaceutically acceptable
carrier(s). In general the pharmaceutical compositions are prepared
by uniformly and intimately bringing into association the active
ingredient(s) with liquid excipients or finely divided solid
excipients or both, and then, if necessary, shaping the
product.
[0275] Pharmaceutical compositions of the present invention
suitable for oral administration can be presented as discrete units
such as capsules, cachets or tablets each containing predetermined
amounts of the active ingredients; as powders or granules; as
solutions or suspensions in an aqueous liquid or a non-aqueous
liquid; or as oil-in-water emulsions or water-in-oil liquid
emulsions. A tablet can be made by compression or molding,
optionally with one or more accessory ingredients. Compressed
tablets can be prepared by compressing in a suitable machine, the
active ingredients in a free-flowing form such as a powder or
granules, optionally mixed with a binder, lubricant, inert diluent,
preservative, surface active or dispersing agent. Molded tablets
can be made by molding in a suitable machine a mixture of the
powdered compound moistened with an inert liquid diluent. The
tablets can optionally be coated or scored and can be formulated so
as to provide slow or controlled release of the active ingredients
therein. Pharmaceutical compositions for parenteral, intrathecal or
intraventricular administration, or colloidal dispersion systems,
can include sterile aqueous solutions which can also contain
buffers, diluents and other suitable additives.
[0276] Penetration Enhancers: Pharmaceutical compositions
comprising the oligonucleotides of the present invention can also
include penetration enhancers in order to enhance the alimentary
delivery of the oligonucleotides. Penetration enhancers can be
classified as belonging to one of five broad categories, i.e.,
fatty acids, bile salts, chelating agents, surfactants and
non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug
Carrier Systems, 1991, 8, 91-192; Muranishi, Critical Reviews in
Therapeutic Drug Carrier Systems, 1990, 7:1).
[0277] Fatty Acids: Various fatty acids and their derivatives which
act as penetration enhancers include, for example, oleic acid,
lauric acid, capric acid, myristic acid, palmitic acid, stearic
acid, linoleic acid, linolenic acid, dicaprate, tricaprate,
recinleate, monoolein (a.k.a. 1-monooleoyl-rac-glycerol), caprylic
acid, arichidonic acid, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono-
and di-glycerides and physiologically acceptable salts thereof
(i.e., oleate, laurate, caprate, myristate, palmitate, stearate,
linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug
Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in
Therapeutic Drug Carrier Systems, 1990, 7, 1; El-Hariri et al., J.
Pharm. Pharmacol., 1992, 44, 651).
[0278] Bile Salts: The physiological roles of bile include the
facilitation of dispersion and absorption of lipids and fat-soluble
vitamins (Brunton, Chapter 38 In: Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al.,
Eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Various
natural bile salts, and their synthetic derivatives, act as
penetration enhancers. Thus, bile salts include any of the
naturally occurring components of bile as well as any of their
synthetic derivatives.
[0279] Chelating Agents: Chelating agents have the added advantage
of also serving as DNase inhibitors and include, but are not
limited to, disodium ethylenediaminetetraacetate (EDTA), citric
acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and
homovanilate), N-acyl derivatives of collagen, laureth-9 and
N-amino acyl derivatives of beta-diketones (enamines)(Lee et al.,
Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page
92; Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1; Buur et al., J. Control Rel., 1990, 14,
43).
[0280] Surfactants: Surfactants include, for example, sodium lauryl
sulfate, polyoxyethylene-9-lauryl ether and
polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, page 92); and
perfluorochemical emulsions, such as FC-43 (Takahashi et al., J.
Pharm. Phamacol., 1988, 40, 252).
[0281] Non-Surfactants: Non-surfactants include, for example,
unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone
derivatives (Lee et al., Critical Reviews in Therapeutic Drug
Carrier Systems, 1991, page 92); and non-steroidal
anti-inflammatory agents such as diclofenac sodium, indomethacin
and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987,
39, 621).
[0282] Carrier Compounds: As used herein, "carrier compound" refers
to a nucleic acid, or analog thereof, which is inert (i.e., does
not possess biological activity per se) but is recognized as a
nucleic acid by in vivo processes that reduce the bioavailability
of a nucleic acid having biological activity by, for example,
degrading the biologically active nucleic acid or promoting its
removal from circulation. The coadministration of a nucleic acid
and a carrier compound, typically with an excess of the latter
substance, can result in a substantial reduction of the amount of
nucleic acid recovered in the liver, kidney or other
extracirculatory reservoirs, presumably due to competition between
the carrier compound and the nucleic acid for a common receptor.
For example, the recovery of a partially phosphorothioated
oligonucleotide in hepatic tissue is reduced when it is
coadministered with polyinosinic acid, dextran sulfate, polycytidic
acid or 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid
(Miyao et al., Antisense Res. Dev., 1995, 5, 115; Takakura et al.,
Antisense & Nucl. Acid Drug Dev., 1996, 6, 177).
[0283] Pharmaceutically Acceptable Carriers: In contrast to a
carrier compound, a "pharmaceutically acceptable carrier"
(excipient) is a pharmaceutically acceptable solvent, suspending
agent or any other pharmacologically inert vehicle for delivering
one or more nucleic acids to an animal. The pharmaceutically
acceptable carrier can be liquid or solid and is selected with the
planned manner of administration in mind so as to provide for the
desired bulk, consistency, etc., when combined with a nucleic acid
and the other components of a given pharmaceutical composition.
Typical pharmaceutically acceptable carriers include, but are not
limited to, binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.);
fillers (e.g., lactose and other sugars, microcrystalline
cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose,
polyacrylates or calcium hydrogen phosphate, etc.); lubricants
(e.g., magnesium stearate, talc, silica, colloidal silicon dioxide,
stearic acid, metallic stearates, hydrogenated vegetable oils, corn
starch, polyethylene glycols, sodium benzoate, sodium acetate,
etc.); disintegrates (e.g., starch, sodium starch glycolate, etc.);
or wetting agents (e.g., sodium lauryl sulphate, etc.). Sustained
release oral delivery systems and/or enteric coatings for orally
administered dosage forms are described in U.S. Pat. Nos.
4,704,295; 4,556,552; 4,309,406; and 4,309,404.
[0284] Miscellaneous Additional Components: The compositions of the
present invention can additionally contain other adjunct components
conventionally found in pharmaceutical compositions, at their
art-established usage levels. Thus, for example, the compositions
can contain additional compatible pharmaceutically-active materials
such as, e.g., antipruritics, astringents, local anesthetics or
anti-inflammatory agents, or can contain additional materials
useful in physically formulating various dosage forms of the
composition of present invention, such as dyes, flavoring agents,
preservatives, antioxidants, opacifiers, thickening agents and
stabilizers. However, such materials, when added, should not unduly
interfere with the biological activities of the components of the
compositions of the invention.
[0285] Colloidal Dispersion Systems: Regardless of the method by
which the oligonucleotides of the invention are introduced into a
patient, colloidal dispersion systems can be used as delivery
vehicles to enhance the in vivo stability of the oligonucleotides
and/or to target the oligonucleotides to a particular organ, tissue
or cell type. Colloidal dispersion systems include, but are not
limited to, macromolecule complexes, nanocapsules, microspheres,
beads and lipid-based systems including oil-in-water emulsions,
micelles, mixed micelles and liposomes. A preferred colloidal
dispersion system is a plurality of liposomes, artificial membrane
vesicles which can be used as cellular delivery vehicles for
bioactive agents in vitro and in vivo (Mannino et al.,
Biotechniques, 1988, 6, 682; Blume and Cevc, Biochem. et Biophys.
Acta, 1990, 1029, 91; Lappalainen et al., Antiviral Res., 1994, 23,
119; Chonn and Cullis, Current Op. Biotech., 1995, 6, 698). It has
been shown that large unilamellar vesicles (LUV), which range in
size from 0.2-0.4 .mu.m, can encapsulate a substantial percentage
of an aqueous buffer containing large macromolecules. RNA, DNA and
intact virions can be encapsulated within the aqueous interior and
delivered to brain cells in a biologically active form (Fraley et
al., Trends Biochem. Sci., 1981, 6, 77). The composition of the
liposome is usually a combination of lipids, particularly
phospholipids, in particular, high phase transition temperature
phospholipids, usually in combination with one or more steroids,
particularly cholesterol. Examples of lipids useful in liposome
production include phosphatidyl compounds, such as
phosphatidylglycerol, phosphatidylcholine, phosphatidylserine,
sphingolipids, phosphatidylethanolamine, cerebrosides and
gangliosides. Particularly useful are diacyl phosphatidylglycerols,
where the lipid moiety contains from 14-18 carbon atoms,
particularly from 16-18 carbon atoms, and is saturated (lacking
double bonds within the 14-18 carbon atom chain). Illustrative
phospholipids include phosphatidylcholine,
dipalmitoylphosphatidylcholine and
distearoylphosphatidylcholine.
[0286] The targeting of colloidal dispersion systems, including
liposomes, can be either passive or active. Passive targeting
utilizes the natural tendency of liposomes to distribute to cells
of the reticuloendothelial system in organs that contain sinusoidal
capillaries. Active targeting, by contrast, involves modification
of the liposome by coupling thereto a specific ligand such as a
viral protein coat (Morishita et al., Proc. Natl. Acad. Sci.
(U.S.A.), 1993, 90, 8474), monoclonal antibody (or a suitable
binding portion thereof), sugar, glycolipid or protein (or a
suitable oligopeptide fragment thereof), or by changing the
composition and/or size of the liposome in order to achieve
distribution to organs and cell types other than the naturally
occurring sites of localization. The surface of the targeted
colloidal dispersion system can be modified in a variety of ways.
In the case of a liposomal targeted delivery system, lipid groups
can be incorporated into the lipid bilayer of the liposome in order
to maintain the targeting ligand in close association with the
lipid bilayer. Various linking groups can be used for joining the
lipid chains to the targeting ligand. The targeting ligand, which
binds a specific cell surface molecule found predominantly on cells
to which delivery of the oligonucleotides of the invention is
desired, can be, for example, (1) a hormone, growth factor or a
suitable oligopeptide fragment thereof which is bound by a specific
cellular receptor predominantly expressed by cells to which
delivery is desired or (2) a polyclonal or monoclonal antibody, or
a suitable fragment thereof (e.g., Fab; F(ab').sub.2) which
specifically binds an antigenic epitope found predominantly on
targeted cells. Two or more bioactive agents (e.g., an
oligonucleotide and a conventional drug; two oligonucleotides) can
be combined within, and delivered by, a single liposome. It is also
possible to add agents to colloidal dispersion systems which
enhance the intercellular stability and/or targeting of the
contents thereof.
[0287] Means of Administration: The present invention provides
compositions comprising oligonucleotides intended for
administration to an animal.
[0288] Parenteral Delivery: The administration of an
oligonucleotide of the invention to an animal in a manner other
than through the digestive canal. Means of preparing and
administering parenteral pharmaceutical compositions are known in
the art (see, e.g., Avis, Chapter 84 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 1545-1569). Parenteral means of delivery include, but
are not limited to, the following illustrative examples.
[0289] Intravitreal injection, for the direct delivery of drug to
the vitreous humor of a mammalian eye, is described in U.S. Pat.
No. 5,591,720, the contents of which are hereby incorporated by
reference. Means of preparing and administering ophthalmic
preparations are known in the art (see, e.g., Mullins et al.,
Chapter 86 In: Remington's Pharmaceutical Sciences, 18th Ed.,
Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages
1581-1595).
[0290] Intravenous administration of antisense oligonucleotides to
various non-human mammals has been described by Iversen (Chapter 26
In: Antisense Research and Applications, Crooke et al., Eds., CBC
Press, Boca Raton, Fla., 1993, pages 461-469). Systemic delivery of
oligonucleotides to non-human mammals via intraperitoneal means has
also been described (Dean et al., Proc. Natl. Acad. Sci. (U.S.A.),
1994, 91, 11766).
[0291] Intraluminal drug administration, for the direct delivery of
drug to an isolated portion of a tubular organ or tissue (e.g.,
such as an artery, vein, ureter or urethra), can be desired for the
treatment of patients with diseases or conditions afflicting the
lumen of such organs or tissues. To effect this mode of
oligonucleotide administration, a catheter or cannula is surgically
introduced by appropriate means. For example, for treatment of the
left common carotid artery, a cannula is inserted thereinto via the
external carotid artery. After isolation of a portion of the
tubular organ or tissue for which treatment is sought, a
composition comprising the oligonucleotides of the invention is
infused through the cannula or catheter into the isolated segment.
After incubation for from about 1 to about 120 minutes, during
which the oligonucleotide is taken up by cells of the interior
lumen of the vessel, the infusion cannula or catheter is removed
and flow within the tubular organ or tissue is restored by removal
of the ligatures which effected the isolation of a segment thereof
(Morishita et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 8474).
Antisense oligonucleotides can also be combined with a
biocompatible matrix, such as a hydrogel material, and applied
directly to vascular tissue in vivo (Rosenberg et al., U.S. Pat.
No. 5,593,974, issued Jan. 14, 1997).
[0292] Intraventricular drug administration, for the direct
delivery of drug to the brain of a patient, can be desired for the
treatment of patients with diseases or conditions afflicting the
brain. To effect this mode of oligonucleotide administration, a
silicon catheter is surgically introduced into a ventricle of the
brain of a human patient, and is connected to a subcutaneous
infusion pump (Medtronic Inc., Minneapolis, Minn.) that has been
surgically implanted in the abdominal region (Zimm et al., Cancer
Research, 1984, 44, 1698; Shaw, Cancer, 1993, 72(11 Suppl.), 3416).
The pump is used to inject the oligonucleotides and allows precise
dosage adjustments and variation in dosage schedules with the aid
of an external programming device. The reservoir capacity of the
pump is 18-20 mL and infusion rates can range from 0.1 mL/h to 1
mL/h. Depending on the frequency of administration, ranging from
daily to monthly, and the dose of drug to be administered, ranging
from 0.01 .mu.g to 100 g per kg of body weight, the pump reservoir
can be refilled at 3-10 week intervals. Refilling of the pump is
accomplished by percutaneous puncture of the self-sealing septum of
the pump.
[0293] Intrathecal drug administration, for the introduction of a
drug into the spinal column of a patient can be desired for the
treatment of patients with diseases of the central nervous system.
To effect this route of oligonucleotide administration, a silicon
catheter is surgically implanted into the L3-4 lumbar spinal
interspace of a human patient, and is connected to a subcutaneous
infusion pump which has been surgically implanted in the upper
abdominal region (Luer and Hatton, The Annals of Pharmacotherapy,
1993, 27, 912; Ettinger et al., Cancer, 1978, 41, 1270; Yaida et
al., Regul. Pept., 1995, 59, 193). The pump is used to inject the
oligonucleotides and allows precise dosage adjustments and
variations in dose schedules with the aid of an external
programming device. The reservoir capacity of the pump is 18-20 mL,
and infusion rates can vary from 0.1 mL/h to 1 mL/h. Depending on
the frequency of drug administration, ranging from daily to
monthly, and dosage of drug to be administered, ranging from 0.01
.mu.g to 100 g per kg of body weight, the pump reservoir can be
refilled at 3-10 week intervals. Refilling of the pump is
accomplished by a single percutaneous puncture to the self-sealing
septum of the pump. The distribution, stability and
pharmacokinetics of oligonucleotides within the central nervous
system can be followed according to known methods (Whitesell et
al., Proc. Natl. Acad. Sci. (USA), 1993, 90, 4665).
[0294] To effect delivery of oligonucleotides to areas other than
the brain or spinal column via this method, the silicon catheter is
configured to connect the subcutaneous infusion pump to, e.g., the
hepatic artery, for delivery to the liver (Kemeny et al., Cancer,
1993, 71, 1964). Infusion pumps can also be used to effect systemic
delivery of oligonucleotides (Ewel et al., Cancer Research, 1992,
52, 3005; Rubenstein et al., J. Surg. Oncol., 1996, 62, 194).
[0295] Epidermal and Transdermal Delivery, in which pharmaceutical
compositions containing drugs are applied topically, can be used to
administer drugs to be absorbed by the local dermis or for further
penetration and absorption by underlying tissues, respectively.
Means of preparing and administering medications topically are
known in the art (see, e.g., Block, Chapter 87 In: Remington's
Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing
Co., Easton, Pa., 1990, pages 1596-1609).
[0296] Vaginal Delivery provides local treatment and avoids first
pass metabolism, degradation by digestive enzymes, and potential
systemic side-effects. This mode of administration can be preferred
for antisense oligonucleotides targeted to pathogenic organisms for
which the vagina is the usual habitat, e.g., Trichomonas vaginalis.
In another embodiment, antisense oligonucleotides to genes encoding
sperm-specific antibodies can be delivered by this mode of
administration in order to increase the probability of conception
and subsequent pregnancy. Vaginal suppositories (Block, Chapter 87
In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed.,
Mack Publishing Co., Easton, Pa., 1990, pages 1609-1614) or topical
ointments can be used to effect this mode of delivery.
[0297] Intravesical Delivery provides local treatment and avoids
first pass metabolism, degradation by digestive enzymes, and
potential systemic side-effects. However, the method requires
urethral catheterization of the patient and a skilled staff.
Nevertheless, this mode of administration can be preferred for
antisense oligonucleotides targeted to pathogenic organisms, such
as T. vaginalis, which can invade the urogenital tract.
[0298] Alimentary Delivery: The administration, directly or
otherwise, to a portion of the alimentary canal of an animal. The
term "alimentary canal" refers to the tubular passage in an animal
that functions in the digestion and absorption of food and the
elimination of food residue, which runs from the mouth to the anus,
and any and all of its portions or segments, e.g., the oral cavity,
the esophagus, the stomach, the small and large intestines and the
colon, as well as compound portions thereof such as, e.g., the
gastro-intestinal tract. Thus, the term "alimentary delivery"
encompasses several routes of administration including, but not
limited to, oral, rectal, endoscopic and sublingual/buccal
administration. A common requirement for these modes of
administration is absorption over some portion or all of the
alimentary tract and a need for efficient mucosal penetration of
the nucleic acid(s) so administered.
[0299] Buccal/Sublingual Administration: Delivery of a drug via the
oral mucosa has several desirable features, including, in many
instances, a more rapid rise in plasma concentration of the drug
than via oral delivery (Harvey, Chapter 35 In: Remington's
Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing
Co., Easton, Pa., 1990, page 711). Furthermore, because venous
drainage from the mouth is to the superior vena cava, this route
also bypasses rapid first-pass metabolism by the liver. Both of
these features contribute to the sublingual route being the mode of
choice for nitroglycerin (Benet et al., Chapter 1 In: Goodman &
Gilman's The Pharmacological Basis of Therapeutics, 9th Ed.,
Hardman et al., Eds., McGraw-Hill, New York, N.Y., 1996, page
7).
[0300] Endoscopic Administration: Endoscopy can be used for drug
delivery directly to an interior portion of the alimentary tract.
For example, endoscopic retrograde cystopancreatography (ERCP)
takes advantage of extended gastroscopy and permits selective
access to the biliary tract and the pancreatic duct (Hirahata et
al., Gan To Kagaku Ryoho, 1992, 19(10 Suppl.), 1591). However, the
procedure is unpleasant for the patient, and requires a highly
skilled staff.
[0301] Rectal Administration: Drugs administered by the oral route
can often be alternatively administered by the lower enteral route,
i.e., through the anal portal into the rectum or lower intestine.
Rectal suppositories, retention enemas or rectal catheters can be
used for this purpose and can be preferred when patient compliance
might otherwise be difficult to achieve (e.g., in pediatric and
geriatric applications, or when the patient is vomiting or
unconscious). Rectal administration can result in more prompt and
higher blood levels than the oral route, but the converse can be
true as well (Harvey, Chapter 35 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, page 711). Because about 50% of the drug that is absorbed
from the rectum will bypass the liver, administration by this route
significantly reduces the potential for first-pass metabolism
(Benet et al., Chapter 1 In: Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al.,
Eds., McGraw-Hill, New York, N.Y., 1996).
[0302] Oral Administration: The preferred method of administration
is oral delivery, which is typically the most convenient route for
access to the systemic circulation. Absorption from the alimentary
canal is governed by factors that are generally applicable, e.g.,
surface area for absorption, blood flow to the site of absorption,
the physical state of the drug and its concentration at the site of
absorption (Benet et al., Chapter 1 In: Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al.,
Eds., McGraw-Hill, New York, N.Y., 1996, pages 5-7). A significant
factor which can limit the oral bioavailability of a drug is the
degree of "first pass effects." For example, some substances have
such a rapid hepatic uptake that only a fraction of the material
absorbed enters the peripheral blood (Van Berge-Henegouwen et al.,
Gastroenterology, 1977, 73, 300). The compositions and methods of
the invention circumvent, at least partially, such first pass
effects by providing improved uptake of nucleic acids and thereby,
e.g., causing the hepatic uptake system to become saturated and
allowing a significant portion of the nucleic acid so administered
to reach the peripheral circulation. Additionally or alternatively,
the hepatic uptake system is saturated with one or more inactive
carrier compounds prior to administration of the active nucleic
acid.
[0303] The following examples illustrate the invention and are not
intended to limit the same. Those skilled in the art will
recognize, or be able to ascertain through routine experimentation,
numerous equivalents to the specific substances and procedures
described herein. Such equivalents are considered to be within the
scope of the present invention.
EXAMPLES
Example 1
Synthesis of Oligonucleotides
[0304] General Synthetic Techniques: Oligonucleotides were
synthesized on an automated DNA synthesizer using standard
phosphoramidite chemistry with oxidation using iodine.
.beta.-Cyanoethyldiisopropyl phosphoramidites were purchased from
Applied Biosystems (Foster City, Calif.). For phosphorothioate
oligonucleotides, the standard oxidation bottle was replaced by a
0.2 M solution of 3H-1,2-benzodithiole-3-one-1,1-dioxide in
acetonitrile for the stepwise thiation of the phosphite
linkages.
[0305] The synthesis of 2'-O-methyl- (a.k.a. 2'-methoxy-)
phosphorothioate oligonucleotides is according to the procedures
set forth above substituting 2'-O-methyl
.beta.-cyanoethyldiisopropyl phosphoramidites (Chemgenes, Needham,
Mass.) for standard phosphoramidites and increasing the wait cycle
after the pulse delivery of tetrazole and base to 360 seconds.
[0306] Similarly, 2'-O-propyl- (a.k.a 2'-propoxy-) phosphorothioate
oligonucleotides are prepared by slight modifications of this
procedure and essentially according to procedures disclosed in U.S.
patent application Ser. No. 08/383,666, filed Feb. 3, 1995, which
is assigned to the same assignee as the instant application.
[0307] The 2'-fluoro-phosphorothioate oligonucleotides of the
invention are synthesized using
5'-dimethoxytrityl-3'-phosphoramidites and prepared as disclosed in
U.S. patent application Ser. No. 08/383,666, filed Feb. 3, 1995,
and U.S. Pat. No. 5,459,255, which issued Oct. 8, 1996, both of
which are assigned to the same assignee as the instant application.
The 2'-fluoro-oligonucleotides were prepared using phosphoramidite
chemistry and a slight modification of the standard DNA synthesis
protocol (i.e., deprotection was effected using methanolic ammonia
at room temperature).
[0308] The 2'-methoxyethoxy oligonucleotides were synthesized
essentially according to the methods of Martin et al. (Helv. Chim.
Acta, 1995, 78, 486). For ease of synthesis, the 3' nucleotide of
the 2'-methoxyethoxy oligonucleotides was a deoxynucleotide, and
2'-O--CH.sub.2CH.sub.2OCH.sub.3-cytosines were 5-methyl cytosines,
which were synthesized according to the procedures described
below.
[0309] PNA antisense analogs are prepared essentially as described
in U.S. Pat. Nos. 5,539,082 and 5,539,083, both of which (1) issued
Jul. 23, 1996, and (2) are assigned to the same assignee as the
instant application.
[0310] Purification: After cleavage from the controlled pore glass
column (Applied Biosystems) and deblocking in concentrated ammonium
hydroxide at 55?C for 18 hours, the oligonucleotides were purified
by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.
Analytical gel electrophoresis was accomplished in 20% acrylamide,
8 M urea, 45 mM Tris-borate buffer, pH 7.0. Oligodeoxynucleotides
and their phosphorothioate analogs were judged from electrophoresis
to be greater than 80% full length material.
Example 2
Assays for Oligonucleotide-Mediated Inhibition of JNK mRNA
Expression in Human Tumor Cells
[0311] In order to evaluate the activity of potential
JNK-modulating oligonucleotides, human lung carcinoma cell line
A549 (American Type Culture Collection, Rockville, Md. No. ATCC
CCL-185) cells or other cell lines as indicated in the Examples,
were grown and treated with oligonucleotides or control solutions
as detailed below. After harvesting, cellular extracts were
prepared and examined for specific JNK mRNA levels or JNK protein
levels (i.e., Northern or Western assays, respectively). In all
cases, "% expression" refers to the amount of JNK-specific signal
in an oligonucleotide-treated cell relative to an untreated cell
(or a cell treated with a control solution that lacks
oligonucleotide).
[0312] Northern Assays The mRNA expression of each JNK protein was
determined by using a nucleic acid probe specifically hybridizable
thereto. Nucleic acid probes specific for JNK1, JNK2 and JNK3 are
described in Examples 3, 4 and 5, respectively. The probes were
radiolabelled by means well known in the art (see, e.g., Short
Protocols in Molecular Biology, 2nd Ed., Ausubel et al., Eds., John
Wiley & Sons, New York, 1992, pages 3-11 to 2-3-44 and 4-17 to
4-18; Ruth, Chapter 6 In: Methods in Molecular Biology, Vol. 26:
Protocols for Oligonucleotide Conjugates, Agrawal, ed., Humana
Press Inc., Totowa, N.J., 1994, pages 167-185; and Chapter 10 In:
Molecular Cloning: A Laboratory Manual, 2nd Ed., Sambrook et al.,
Eds., pages 10.1-10.70). The blots were stripped and reprobed with
a .sup.32P-labeled glyceraldehyde 3-phosphate dehydrogenase (G3PDH)
probe (Clontech Laboratories, Inc., Palo Alto, Calif.) in order to
confirm equal loading of RNA and to allow the levels of JNK
transcripts to be normalized with regard to the G3PDH signals.
[0313] A549 cells were grown in T-75 flasks until 80-90% confluent.
At this time, the cells were washed twice with 10 mL of media
(DMEM), followed by the addition of 5 mL of DMEM containing 20
.right brkt-bot.g/mL of LIPOFECTIN.TM. (i.e., 1:1 (w/w) DOTMA/DOPE,
Life Technologies, Gaithersburg, Md.;
DOTMA=N-[1-(2,3-dioleyoxy)propyl]-N,N,N-trimethylammonium chloride;
DOPE=dioleoyl phosphatidylethanolamine). The oligonucleotides were
added from a 10 .mu.M stock solution to a final concentration of
400 nM, and the two solutions were mixed by swirling the flasks. As
a control, cells were treated with LIPOFECTIN.TM. without
oligonucleotide under the same conditions and for the same times as
the oligonucleotide-treated samples. After 4 hours at 37.degree.
C., the medium was replaced with fresh DMEM containing 10% serum.
The cells were allowed to recover for 18 hours. Total cellular RNA
was then extracted in guanidinium, subject to gel electrophoresis
and transferred to a filter according to techniques known in the
art (see, e.g., Chapter 7 In: Molecular Cloning: A Laboratory
Manual, 2nd Ed., Sambrook et al., Eds., pages 7.1-7.87, and Short
Protocols in Molecular Biology, 2nd Ed., Ausubel et al., Eds., John
Wiley & Sons, New York, 1992, pages 2-24 to 2-30 and 4-14 to
4-29). Filters were typically hybridized overnight to a probe
specific for the particular JNK gene of interest in hybridization
buffer (25 mM KPO.sub.4, pH 7.4; 5.times.SSC; 5.times.Denhardt's
solution, 100 g/ml Salmon sperm DNA and 50% formamide) (Alahari et
al., Nucl. Acids Res., 1993, 21, 4079). This was followed by two
washes with 1.times.SSC, 0.1% SDS and two washes with
0.25.times.SSC, 0.1% SDS. Hybridizing bands were visualized by
exposure to X-OMAT AR film and quantitated using a
PHOSPHORIMAGER.TM. essentially according to the manufacturer's
instructions (Molecular Dynamics, Sunnyvale, Calif.).
[0314] Western Assays: A549 cells were grown and treated with
oligonucleotides as described above. Cells were lysed, and protein
extracts were electrophoresed (SDS-PAGE) and transferred to
nitrocellulose filters by means known in the art (see, e.g.,
Chapter 18 In: Molecular Cloning: A Laboratory Manual, 2nd Ed.,
Sambrook et al., Eds., pages 18.34, 18.47-18.54 and 18.60-18.75)).
The amount of each JNK protein was determined by using a primary
antibody that specifically recognizes the appropriate JNK protein.
The primary antibodies specific for each JNK protein are described
in the appropriate Examples. The primary antibodies were detected
by means well known in the art (see, e.g., Short Protocols in
Molecular Biology, 2nd Ed., Ausubel et al., Eds., John Wiley &
Sons, New York, 1992, pages 10-33 to 10-35; and Chapter 18 In:
Molecular Cloning: A Laboratory Manual, 2nd Ed., Sambrook et al.,
Eds., pages 18.1-18.75 and 18.86-18.88) and quantitated using a
PHOSPHORIMAGER.TM. essentially according to the manufacturer's
instructions (Molecular Dynamics, Sunnyvale, Calif.).
[0315] Levels of JNK proteins can also be quantitated by measuring
the level of their corresponding kinase activity. Such kinase
assays can be done in gels in situ (Hibi et al., Genes & Dev.,
1993, 7, 2135) or after immunoprecipitation from cellular extracts
(Derijard et al., Cell, 1994, 76, 1025). Substrates and/or kits for
such assays are commercially available from, for example, Upstate
Biotechnology, Inc. (Lake Placid, N.Y.), New England Biolabs, Inc.,
(Beverly, Mass.) and Calbiochem-Novabiochem Biosciences, Inc., (La
Jolla, Calif.).
Example 3
Oligonucleotide-Mediated Inhibition of JNK1 Expression
[0316] JNK1 oligonucleotide sequences: Table 1 lists the nucleotide
sequences of a set of oligonucleotides designed to specifically
hybridize to JNK1 mRNAs and their corresponding ISIS and SEQ ID
numbers. The nucleotide co-ordinates of the target gene, JNK1, and
gene target regions are also included. The nucleotide co-ordinates
are derived from GenBank accession No. L26318 (SEQ ID NO: 87),
locus name "HUMJNK1" (see also FIG. 1(A) of Derijard et al., Cell,
1994, 76, 1025). The abbreviations for gene target regions are as
follows: 5'-UTR, 5' untranslated region; tIR, translation
initiation region; ORF, open reading frame; 3'-UTR, 3' untranslated
region. The nucleotides of the oligonucleotides whose sequences are
presented in Table 1 are connected by phosphorothioate linkages and
are unmodified at the 2' position (i.e., 2'-deoxy). It should be
noted that the oligonucleotide target co-ordinate positions and
gene target regions can vary within mRNAs encoding related isoforms
of JNK1 (see subsection G, below).
[0317] In addition to hybridizing to human JNK1 mRNAs, the full
oligonucleotide sequences of ISIS Nos. 12548 (SEQ ID NO: 17) and
12551 (SEQ ID NO: 20) hybridize to the 5' ends of mRNAs from Rattus
norvegicus that encode a stress-activated protein kinase named
"p54?" (Kyriakis et al., Nature, 1994, 369, 156). Specifically,
ISIS 12548 (SEQ ID NO: 17) hybridizes to bases 498-517 of GenBank
accession No. L27129 (SEQ ID NO: 88), locus name "RATSAPKD," and
ISIS 12551 (SEQ ID NO: 20) hybridizes to bases 803-822 of the same
sequence.
[0318] JNK1-specific probes: In initial screenings of a set of
oligonucleotides derived from the JNK1 sequence (Table 2) for
biological activity, a cDNA clone of JNK1 (Derijard et al., Cell,
1994, 76, 1025) was radiolabeled and used as a JNK1-specific probe
in Northern blots. Alternatively, however, one or more of the
oligonucleotides of Table 1 is detectably labeled and used as a
JNK1-specific probe.
TABLE-US-00001 TABLE 1 Nucleotide Sequences of JNK1
Oligonucleotides TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE SEQ
NUCLEOTIDE TARGET NO. (5' -> 3') ID NO: CO-ORDINATES REGION
11978 ATT-CTT-TCC-ACT-CTT-CTA-TT 1 1062-1081 ORF 11979
CTC-CTC-CAA-GTC-CAT-AAC-TT 2 1094-1113 ORF 11980
CCC-GTA-TAA-CTC-CAT-TCT-TG 3 1119-1138 ORF 11981
CTG-TGC-TAA-AGG-AGA-GGG-CT 4 1142-1161 ORF 11982
ATG-ATG-GAT-GCT-GAG-AGC-CA 5 1178-1197 3'-UTR 11983
GTT-GAC-ATT-GAA-GAC-ACA-TC 6 1215-1234 3'-UTR 11984
CTG-TAT-CAG-AGG-CCA-AAG-TC 7 1241-1260 3'-UTR 11985
TGC-TGC-TTC-TAG-ACT-GCT-GT 8 1261-1280 3'-UTR 11986
AGT-CAT-CTA-CAG-CAG-CCC-AG 9 1290-1309 3'-UTR 11987
CCA-TCC-CTC-CCA-CCC-CCC-GA 10 1320-1339 3'-UTR 11988
ATC-AAT-GAC-TAA-CCG-ACT-CC 11 1340-1359 3'-UTR 11989
CAA-AAA-TAA-GAC-CAC-TGA-AT 12 1378-1397 3'-UTR 12463
CAC-GCT-TGC-TTC-TGC-TCA-TG 13 0018-0037 tIR 12464
CGG-CTT-AGC-TTC-TTG-ATT-GC 14 0175-0194 ORF 12538
CCC-GCT-TGG-CAT-GAG-TCT-GA 15 0207-0226 ORF 12539
CTC-TCT-GTA-GGC-CCG-CTT-GG 16 0218-0237 ORF 12548
ATT-TGC-ATC-CAT-GAG-CTC-CA 17 0341-0360 ORF 12549
CGT-TCC-TGC-AGT-CCT-GGC-CA 18 0533-0552 ORF 12550
GGA-TGA-CCT-CGG-GTG-CTC-TG 19 0591-0610 ORF 12551
CCC-ATA-ATG-CAC-CCC-ACA-GA 20 0646-0665 ORF 12552
CGG-GTG-TTG-GAG-AGC-TTC-AT 21 0956-0975 ORF 12553
TTT-GGT-GGT-GGA-GCT-TCT-GC 22 1006-1025 ORF 12554
GGC-TGC-CCC-CGT-ATA-ACT-CC 23 1126-1145 ORF 12555
TGC-TAA-AGG-AGA-GGG-CTG-CC 24 1139-1158 ORF 12556
AGG-CCA-AAG-TCG-GAT-CTG-TT 25 1232-1251 3'-UTR 12557
CCA-CCC-CCC-GAT-GGC-CCA-AG 26 1311-1330 3'-UTR
[0319] Activities of JNK1 oligonucleotides: The data from screening
a set of JNK1-specific phosphorothioate oligonucleotides (Table 2)
indicate the following results. Oligonucleotides showing activity
in this assay, as reflected by levels of inhibition of JNK1 mRNA
levels of at least 50%, include ISIS Nos. 11982, 11983, 11985,
11987, 12463, 12464, 12538, 12539, 12548, 12549, 12550, 12552,
12553, 12554, 12555, 12556 and 12557 (SEQ ID NOS: 5, 6, 8, 10, 13,
14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25 and 26, respectively).
These oligonucleotides are thus preferred embodiments of the
invention for modulating JNK1 expression. Oligonucleotides showing
levels of inhibition of JNK1 mRNAs of at least 80% in this assay,
include ISIS Nos. 11982, 12539, 12464, 12548, 12554 and 12464 (SEQ
ID NOS: 5, 14, 16, 17 and 23, respectively). These oligonucleotides
are thus more preferred embodiments of the invention for modulating
JNK1 expression.
[0320] The time course of inhibition of JNK1 mRNA expression by
ISIS 12539 (SEQ ID NO: 16) is shown in Table 3. Following the 4
hour treatment with ISIS 12539, the level of inhibition of JNK1 was
greater than about 85% (t=0 h), rose to about 95% inhibition at t=4
h, and subsequently remained at greater than or equal to about 80%
(t=12 and 48 h) or 60% (t=72 h).
TABLE-US-00002 TABLE 2 Activities of JNK1 Oligonucleotides GENE SEQ
ID TARGET ISISNo: NO: REGION % EXPRESSION: % INHIBITION: 11978 1
ORF 85% 15% 11979 2 ORF 90% 10% 11980 3 ORF 85% 15% 11981 4 ORF 62%
28% 11982 5 3'-UTR 13% 87% 11983 6 3'-UTR 40% 60% 11984 7 3'-UTR
53% 47% 11985 8 3'-UTR 47% 53% 11986 9 3'-UTR 90% 10% 11987 10
3'-UTR 47% 53% 11988 11 3'-UTR 78% 22% 11989 12 3'-UTR 60% 40%
12463 13 tIR 23% 77% 12464 14 ORF 18% 82% 12538 15 ORF 33% 67%
12539 16 ORF 9% 91% 12548 17 ORF 5% 95% 12549 18 ORF 28% 72% 12550
19 ORF 40% 60% 12551 20 ORF 52% 48% 12552 21 ORF 34% 66% 12553 22
ORF 25% 75% 12554 23 ORF 11% 89% 12555 24 ORF 27% 73% 12556 25
3'-UTR 41% 59% 12557 26 3'-UTR 29% 71%
TABLE-US-00003 TABLE 3 Time Course of Response to JNK1 Antisense
Oligonucleotides (ASOs) SEQ Normalized ID % ISIS # NO: ASO
Description Time Control % Inhibition control -- (LIPOFECTIN .TM. 0
h 100.0 0.0 only) control -- (LIPOFECTIN .TM. 4 h 100.0 0.0 only)
control -- (LIPOFECTIN .TM. 12 h 100.0 0.0 only) control --
(LIPOFECTIN .TM. 48 h 100.0 0.0 only) control -- (LIPOFECTIN .TM.
72 h 100.0 0.0 only) 12539 16 JNK1 active 0 h 14.1 85.9 12539 16 ''
4 h 5.9 94.1 12539 16 '' 12 h 11.6 88.4 12539 16 '' 48 h 21.0 79.0
12539 16 '' 272 h 41.5 58.5
[0321] Additional JNK1 oligonucleotides: The results for
JNK1-specific oligonucleotides (Table 2) indicate that one of the
most active phosphorothioate oligonucleotides for modulating JNK1
expression is ISIS 12539 (SEQ ID NO: 16). As detailed in Table 4,
additional oligonucleotides based on this oligonucleotide were
designed to confirm and extend the findings described above.
[0322] Oligonucleotides ISIS Nos. 14320 (SEQ ID NO: 27) and 14321
(SEQ ID NO: 28) are 2'-deoxy-phosphorothioate sense strand and
scrambled controls for ISIS 12539 (SEQ ID NO: 16), respectively.
ISIS Nos. 15346 and 15347 are "gapmers" corresponding to ISIS
12539; both have 2'-methoxyethoxy "wings" (having phosphorothioate
linkages in the case of ISIS 15346 and phosphodiester linkages in
the case of ISIS 15347) and a central 2'-deoxy "gap" designed to
support RNaseH activity on the target mRNA molecule. Similarly,
ISIS Nos. 15348 to 15350 are "wingmers" corresponding to ISIS 12539
and have a 5' or 3' 2'-methoxyethoxy RNaseH-refractory "wing" and a
3' or 5' (respectively) 2'-deoxy "wing" designed to support RNaseH
activity on the target JNK1 mRNA.
[0323] The chemically modified derivatives of ISIS 12539 (SEQ ID
NO: 16) were tested in the Northern assay described herein at
concentrations of 100 and 400 nM, and the data (Table 5) indicate
the following results. At 400 nM, relative to the 2'-unmodified
oligonucleotide ISIS 12539, both "gapmers" (ISIS Nos. 15346 and
15347) effected inhibition of JNK1 mRNA expression up to at least
about 88% inhibition. Similarly, the four "wingmers" (ISIS Nos.
15348 to 15351) effected inhibition of JNK1 expression of up to at
least about 60 to 70% inhibition.
TABLE-US-00004 TABLE 4 Chemically Modified JNK1 Oligonucleotides
ISIS NUCLEOTIDE SEQUENCE (5' -> 3')AND SEQID NO. CHEMICAL
MODIFICATIONS* NO: COMMENTS 12539
C.sup.ST.sup.SC.sup.ST.sup.SC.sup.ST.sup.SG.sup.ST.sup.SA.sup.SG.sup-
.SG.sup.SC.sup.SC.sup.SC.sup.SG.sup.SC.sup.ST.sup.ST.sup.SG.sup.SG
16 active 14320
C.sup.SC.sup.SA.sup.SA.sup.SG.sup.SC.sup.SG.sup.SG.sup.SG.sup.SC.sup-
.SC.sup.ST.sup.SA.sup.SC.sup.SA.sup.SG.sup.SA.sup.SG.sup.SA.sup.SG
27 12539 sense control 14321
C.sup.ST.sup.ST.sup.ST.sup.SC.sup.SC.sup.SG.sup.ST.sup.ST.sup.SG.sup-
.SG.sup.SA.sup.SC.sup.SC.sup.SC.sup.SC.sup.ST.sup.SG.sup.SG.sup.SG
28 scrambled control 15345
C.sup.ST.sup.SC.sup.ST.sup.SC.sup.ST.sup.SG.sup.ST.sup.SA.sup.SG.sup-
.SG.sup.SC.sup.SC.sup.SC.sup.SG.sup.SC.sup.ST.sup.ST.sup.SG.sup.SG
16 fully 2'- methoxyethoxy 15346
C.sup.ST.sup.SC.sup.ST.sup.SC.sup.ST.sup.SG.sup.ST.sup.SA.sup.SG.sup-
.SG.sup.SC.sup.SC.sup.SC.sup.SG.sup.SC.sup.ST.sup.ST.sup.SG.sup.SG
16 "gapmer" 15347
C.sup.OT.sup.OC.sup.OT.sup.OC.sup.ST.sup.SG.sup.ST.sup.SA.sup.SG.sup-
.SG.sup.SC.sup.SC.sup.SC.sup.SG.sup.OC.sup.OT.sup.OT.sup.OG.sup.OG
16 "gapmer" 15348
C.sup.ST.sup.SC.sup.ST.sup.SC.sup.ST.sup.SG.sup.ST.sup.SA.sup.SG.sup-
.SG.sup.SC.sup.SC.sup.SC.sup.SG.sup.SC.sup.ST.sup.ST.sup.SG.sup.SG
16 "wingmer" 15349
C.sup.ST.sup.SC.sup.ST.sup.SC.sup.ST.sup.SG.sup.ST.sup.SA.sup.SG.sup-
.SG.sup.SC.sup.SC.sup.SC.sup.SG.sup.SC.sup.ST.sup.ST.sup.SG.sup.SG
16 "wingmer" 15351
C.sup.OT.sup.OC.sup.OT.sup.OC.sup.OT.sup.OG.sup.OT.sup.OA.sup.OG.sup-
.OG.sup.SC.sup.SC.sup.SC.sup.SG.sup.SC.sup.ST.sup.ST.sup.SG.sup.SG
16 "wingmer" 15350
C.sup.ST.sup.SC.sup.ST.sup.SC.sup.ST.sup.SG.sup.ST.sup.SA.sup.SG.sup-
.OG.sup.OC.sup.OC.sup.OC.sup.OG.sup.OC.sup.OT.sup.OT.sup.OG.sup.OG
16 "wingmer" 20571
C.sup.ST.sup.SC.sup.ST.sup.SC.sup.ST.sup.SG.sup.ST.sup.SA.sup.SG.sup-
.SG.sup.SC.sup.SC.sup.SC.sup.SG.sup.SC.sup.ST.sup.ST.sup.SG.sup.SG
1 fully 5-methyl- cytosine version of ISIS 15346 *Emboldened
residues, 2'-methoxyethoxy-residues (others are 2'-deoxy-)
including "C" residues, 5-methyl-cytosines; ".sup.O",
phosphodiester linkage; ".sup.S", phosphorothioate linkage. --- "C"
residues, 2'-deoxy 5-methylcytosine residues;
TABLE-US-00005 TABLE 5 Activity of Chemically Modified JNK1
Antisense Oligonucleotides SEQ Normalized ID % ISIS # NO:
Oligonucleotide Description* Dose Control control -- No
oligonucleotide -- 100.0 (LIPOFECTIN .TM. only) 12539 16 JNK1
active, fully P.dbd.S & 100 nM 56.4 12539 16 fully 2'-deoxy 400
nM 26.7 15345 16 fully P.dbd.S & fully 2'-MOE 100 nM 95.4 15345
16 400 nM 89.1 15346 16 gapmer: P.dbd.S, 2'-MOE wings; 100 nM 22.6
15346 16 P.dbd.S, 2'-deoxy core 400 nM 11.0 15347 16 gapmer:
P.dbd.O, 2'-MOE wings; 100 nM 27.1 15347 16 P.dbd.S, 2-deoxy core
400 nM 11.7 15348 16 wingmer: fully P.dbd.S; 100 nM 30.4 15348 16
5' 2'-MOE; 3' 2-deoxy 400 nM 32.9 15349 16 wingmer: fully P.dbd.S;
100 nM 42.5 15349 16 5' 2-deoxy; 3' 2'-MOE 400 nM 35.5 15351 16
wingmer: 5' P.dbd.O & 2'-MOE; 100 nM 45.1 15351 16 3' P.dbd.S
& 2-deoxy 400 nM 39.8 15350 16 wingmer: 5' P.dbd.S & 2'-
100 nM 71.1 15350 16 deoxy; 3' P.dbd.O & 2'-MOE 400 nM 41.3
*Abbreviations: P.dbd.O, phosphodiester linkage; P.dbd.S,
hosphorothioate linkage; MOE, methoxyethoxy-.
[0324] Dose- and sequence-dependent response to JNK1
oligonucleotides: In order to demonstrate a dose-dependent response
to ISIS 12539 (SEQ ID NO: 16), different concentrations (i.e., 50,
100, 200 and 400 nM) of ISIS 12539 were tested for their effect on
JNK1 mRNA levels in A549 cells (Table 6). In addition, two control
oligonucleotides (ISIS 14320, SEQ ID NO: 27, sense control, and
ISIS 14321, SEQ ID NO: 28, scrambled control; see also Table 4)
were also applied to A549 cells in order to demonstrate the
specificity of ISIS 12539. The results (Table 6) demonstrate that
the response of A549 cells to ISIS 12539 is dependent on dose in an
approximately linear fashion. In contrast, neither of the control
oligonucleotides effect any consistent response on JNK1 mRNA
levels.
[0325] Western Assays: In order to assess the effect of
oligonucleotides targeted to JNK1 mRNAs on JNK1 protein levels,
Western assays were performed essentially as described above in
Example 2, with the following exception(s) and/or modification(s).
A primary antibody that specifically binds to JNK1 (catalog No.
sc-474-G) was purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, Calif.; other JNK1-specific antibodies are available from
StressGen Biotechnologies, Inc., Victoria, BC, Canada; and Research
Diagnostics, Inc., Flanders, N.J.). In this experiment, cells were
grown and treated with oligonucleotide at 300 nM for the initial 20
hours and then at 200 nM for 4 hours. At t=48 h, aliquots were
removed for Northern and Western analyses, and fresh media was
added to the cells. Aliquots for analysis were also taken at t=72
h. The samples from t=48 h and t=72 h were analyzed using the
Northern and Western assays described above.
TABLE-US-00006 TABLE 6 Dose-Dependent Responses to JNK1 Antisense
Oligonucleotides SEQ ID Normalized % ISIS # NO: Oligonucleotide
Description Dose Control control -- No oligonucleotide -- 100.0
(LIPOFECTIN .TM. only) 12539 16 JNK1 active 50 nM 70.3 12539 16 ''
100 nM 51.6 12539 16 '' 200 nM 22.4 12539 16 '' 400 nM 11.1 14320
27 12539 sense control 50 nM 103.6 14320 27 '' 100 nM 76.3 14320 27
'' 200 nM 98.9 14320 27 '' 400 nM 97.1 14321 28 12539 scrambled
control 50 nM 91.8 14321 28 '' 100 nM 94.1 14321 28 '' 200 nM 100.2
14321 28 '' 400 nM 79.2
[0326] The data (Table 7) indicate the following results. In this
assay, at t=48 h, oligonucleotides showing a level of mRNA %
inhibition from >about 70% to about 100% include ISIS Nos. 12539
(phosphorothioate linkages), 15346 and 15347 ("gapmers"), and 15348
and 15351 (5' "wingmers") (SEQ ID NO: 16). Oligonucleotides showing
levels of mRNA inhibition of from .gtoreq.about 90% to about 100%
of JNK1 mRNAs in this assay include ISIS Nos. 12539, 15345 AND
15346 (SEQ ID NO: 16). The oligonucleotides tested showed
approximately parallel levels of JNK1 protein inhibition; ISIS Nos.
12539, 15346-15348 and 15351 effected levels of protein inhibition
.gtoreq.about 40%, and ISIS Nos. 12539, 15346 and 15347 effected
levels of protein inhibition .gtoreq.about 55%.
[0327] At t=72 h, oligonucleotides showing a level of mRNA %
inhibition from >about 70% to about 100% include ISIS Nos. 12539
(phosphorothioate linkages), 15346 and 15347 ("gapmers"), and 15348
(5' "wingmers") (SEQ ID NO: 16). Oligonucleotides showing levels of
mRNA inhibition of from .gtoreq.about 90% to about 100% of JNK1
mRNAs at this point in the assay include ISIS Nos. 12539 and 15346
(SEQ ID NO: 16). Overall, the oligonucleotides tested showed higher
levels of JNK1 protein inhibition at this point in the assay. With
the exception of the fully 2'-methoxyethoxy-modified ISIS 15345,
all of the oligonucleotides in Table 7 effect .gtoreq.about 40%
protein inhibition. ISIS Nos. 12539, 15346-15348 and 15351 effected
levels of protein inhibition .gtoreq.about 60%, and ISIS Nos.
12539, 15346 and 15347 effected levels of protein inhibition
.gtoreq.about 70%.
TABLE-US-00007 TABLE 7 Modulation of JNK1 mRNA and JNK1 Protein
Levels by Modified JNK1 Antisense Oligonucleotides SEQ Protein ID
RNA % Protein ISIS # NO: RNA % Control % Inhibition Control %
Inhibition t = 48 h control -- 100.0 0.0 100.0 0.0 12539 16 6.7
93.3 44.3 55.7 15345 16 70.3 29.7 105.0 (0.0) 15346 16 4.3 95.7
42.7 57.3 15347 16 7.9 92.1 38.8 61.2 15348 16 24.3 75.7 58.3 41.7
15349 16 63.1 36.9 69.5 30.5 15350 16 49.2 50.8 71.7 28.3 15351 16
26.9 73.1 52.4 47.6 t = 72 h control 16 100.0 0.0 100.0 0.0 12539
16 11.7 88.3 29.2 70.8 15345 16 187.4 (0.0) 87.8 12.2 15346 16 10.6
89.4 25.7 74.3 15347 16 8.2 81.8 28.4 71.6 15348 16 28.0 72.0 41.7
58.3 15349 16 52.0 48.0 56.5 43.5 15350 16 54.4 45.6 58.4 41.6
15351 16 46.1 53.9 37.0 63.0
[0328] Oligonucleotides specific for JNK1 isoforms: Subsequent to
the initial descriptions of JNK1 (Derijard et al., Cell, 1994, 76,
1025), cDNAs encoding related isoforms of JNK1 were cloned and
their nucleotide sequences determined (Gupta et al., EMBO Journal,
1996, 15, 2760). In addition to JNK1-a1 (GenBank accession No.
L26318 (SEQ ID NO: 87), locus name "HUMJNK1"), which encodes a
polypeptide having an amino acid sequence identical to that of
JNK1, the additional isoforms include JNK1-a2 (GenBank accession
No. U34822 (SEQ ID NO: 89), locus name "U34822"), JNK1-.beta.1
(GenBank accession No. U35004 (SEQ ID NO: 90), locus name
"HSU35004") and JNK1-.beta.2 (GenBank accession No. U35005 (SEQ ID
NO: 91), locus name "HSU35005"). The four isoforms of JNK1, which
probably arise from alternative mRNA splicing, can each interact
with different transcription factors or sets of transcription
factors (Gupta et al., EMBO Journal, 1996, 15, 2760). As detailed
below, the oligonucleotides of the invention are specific for
certain members or sets of these isoforms of JNK1.
[0329] In the ORFs of mRNAs encoding JNK1/JNK1-a1 and JNK1-a2,
nucleotides (nt) 631-665 of JNK1/JNK1-a1 (Genbank accession No.
L26318 (SEQ ID NO: 87)) and nt 625-659 of JNK1-a2 (Genbank
accession No. U34822 (SEQ ID NO: 89)) have the sequence shown below
as SEQ ID NO: 63, whereas, in the ORFs of mRNAs encoding
JNK1-.beta.1 and JNK1-.beta.2, nt 631-665 of JNK1-.beta.1 (GenBank
accession No. U35004 (SEQ ID NO: 90)) and nt 626-660 of
JNK1-.beta.2 (GenBank accession No. U35005 (SEQ ID NO: 91)) have
the sequence shown below as SEQ ID NO: 64. For purposes of
illustration, SEQ ID NOS: 63 and 64 are shown aligned with each
other (vertical marks, "|," indicate bases that are identical in
both sequences):
##STR00001##
Due to this divergence between the a and b JNK1 isoforms, antisense
oligonucleotides derived from the reverse complement of SEQ ID NO:
63 (i.e., SEQ ID NO: 65, see below) can be used to modulate the
expression of JNK1/JNK1-a1 and JNK1-a2 without significantly
effecting the expression of JNK1-.beta.1 and JNK1-.beta.2. In like
fashion, antisense oligonucleotides derived from the reverse
complement of SEQ ID NO: 64 (i.e., SEQ ID NO: 66, see below) can be
selected and used to modulate the expression of JNK1-.beta.1 and
JNK1-.beta.2 without significantly effecting the expression of
JNK1/JNK1-a1 and JNK1-a2. As an example, an oligonucleotide having
a sequence derived from SEQ ID NO: 65 but not to SEQ ID NO: 66 is
specifically hybridizable to mRNAs encoding JNK1/JNK1-a1 and
JNK1-a2 but not to those encoding JNK1-.beta.1 and
JNK1-.beta.2:
##STR00002##
[0330] As a further example, in the ORFs of mRNAs encoding
JNK1/JNK1-a1 and JNK1-a2, nt 668-711 of JNK1/JNK1-a1 (Genbank
accession No. L26318 (SEQ ID NO: 87)) and nt 662-705 of JNK1-a2
(Genbank accession No. U34822 (SEQ ID NO: 89)) have the sequence
shown below as SEQ ID NO: 67, whereas, in the ORFs of mRNAs
encoding JNK1-.beta.1 and JNK1-.beta.2, nt 668-711 of JNK1-.beta.1
(GenBank accession No. U35004 (SEQ ID NO: 90)) and nt 663-706 of
JNK1-.beta.2 (GenBank accession No. U35005 (SEQ ID NO: 91)) have
the sequence shown below as SEQ ID NO: 68. For purposes of
illustration, SEQ ID NOS: 67 and 68 are shown aligned with each
other as follows:
##STR00003##
Due to this divergence between the a and b JNK1 isoforms, antisense
oligonucleotides derived from the reverse complement of SEQ ID NO:
67 (i.e., SEQ ID NO: 69, see below) are specifically hybridizable
to mRNAs encoding, and can be selected and used to modulate the
expression of, JNK1/JNK1-a1 and JNK1-a2 without significantly
effecting the expression of JNK1-.beta.1 and JNK1-.beta.2. In like
fashion, antisense oligonucleotides derived from the reverse
complement of SEQ ID NO: 68 (i.e., SEQ ID NO: 70, see below) are
specifically hybridizable to mRNAs encoding, and can be selected
and used to modulate the expression of, can be selected and used to
modulate the expression of JNK1-.beta.1 and JNK1-.beta.2 without
significantly effecting the expression of JNK/JNK1-a1 and
JNK1-a2:
##STR00004##
[0331] In the case of the carboxyl terminal portion of the JNK1
isoforms, JNK1/JNK1-a1 shares identity with JNK1-.beta.1;
similarly, JNK1-a2 and JNK1-.beta.2 have identical carboxy terminal
portions. The substantial differences in the amino acid sequences
of these isoforms (5 amino acids in JNK1/JNK1-a1 and JNK1-.beta.1
are replaced with 48 amino acids in JNK1-a2 and JNK1-.beta.2)
result from a slight difference in nucleotide sequence that shifts
the reading frame. Specifically, in the ORFs of mRNAs encoding
JNK1/JNK1-a1 and JNK1-.beta.1, nt 1144-1175 of JNK1/JNK1-a1
(Genbank accession No. L26318 (SEQ ID NO: 87)) and JNK1-.beta.1
(Genbank accession No. U35004 (SEQ ID NO: 90)) have the sequence
shown below as SEQ ID NO: 71, whereas, in the ORFs of mRNAs
encoding JNK1-a2 and JNK1-.beta.2, nt 1138-1164 of JNK1-a2 (GenBank
accession No. U34822 (SEQ ID NO: 89)) and nt 1139-1165 of
JNK1-.beta.2 (GenBank accession No. U35005 (SEQ ID NO: 91)) have
the sequence shown below as SEQ ID NO: 72. For purposes of
illustration, SEQ ID NOS: 71 and 72 are shown aligned with each
other (dashes, A-," indicate bases that are absent in the indicated
sequence, and emboldened bases indicate the stop codon for the
JNK1/JNK1-a1 and JNK1-.beta.1 ORFs):
##STR00005##
Due to this divergence between the JNK1 isoforms, antisense
oligonucleotides derived from the reverse complement of SEQ ID NO:
71 (i.e., SEQ ID NO: 73, see below) are specifically hybridizable
to mRNAs encoding, and can be selected and used to modulate the
expression of, JNK1/JNK1-a1 and JNK1-.beta.1 without significantly
effecting the expression of JNK1-a2 and JNK1-.beta.2. In like
fashion, antisense oligonucleotides derived from the reverse
complement of SEQ ID NO: 72 (i.e., SEQ ID NO: 74, see below) are
specifically hybridizable to mRNAs encoding, and can be selected
and used to modulate the expression of, JNK1-a2 and JNK1-.beta.2
without significantly effecting the expression of JNK1/JNK1-a1 and
JNK1-.beta.1:
##STR00006##
[0332] In preferred embodiments, such isoform-specific
oligonucleotides such as are described above are methoxyethoxy
"gapmers" or "wingmers" in which the RNase H-sensitive "gap" or
"wing" is positioned so as to overlap a region of nonidentity in
the above antisense sequences, i.e., SEQ ID NOS: 65, 66, 69, 70, 73
and 74.
Example 4
Oligonucleotide-Mediated Inhibition of JNK2 Expression
[0333] JNK2 oligonucleotide sequences: Table 8 lists the nucleotide
sequences of oligonucleotides designed to specifically hybridize to
JNK2 mRNAs and the corresponding ISIS and SEQ ID numbers thereof.
The target gene nucleotide co-ordinates and gene target region are
also included. The nucleotide co-ordinates are derived from GenBank
accession No. L31951 (SEQ ID NO: 92), locus name "HUMJNK2" (see
also FIG. 1(A) of Sluss et al., Mol. Cel. Biol., 1994, 14, 8376,
and Kallunki et al., Genes & Development, 1994, 8, 2996). The
abbreviations for gene target regions are as follows: 5'-UTR, 5'
untranslated region; tIR, translation initiation region; ORF, open
reading frame; 3'-UTR, 3' untranslated region. The nucleotides of
the oligonucleotides whose sequences are presented in Table 8 are
connected by phosphorothioate linkages and are unmodified at the 2'
position (i.e., 2-deoxy). It should be noted that the
oligonucleotide target co-ordinate positions and gene target
regions can vary within mRNAs encoding related isoforms of JNK2
(see subsection G, below).
[0334] In addition to hybridizing to human JNK2 mRNAs, the full
oligonucleotide sequence of ISIS No. 12562 (SEQ ID NO: 33)
hybridizes to the ORF of mRNAs from Rattus norvegicus that encode a
stress-activated protein kinase named "p54a2" (Kyriakis et al.,
Nature, 1994, 369, 156). Specifically, ISIS 12562 (SEQ ID NO: 33)
hybridizes to bases 649-668 of GenBank accession No. L27112 (SEQ ID
NO: 93), locus name "RATSAPKB." This oligonucleotide is thus a
preferred embodiment of the invention for investigating the role of
the p54a2 protein kinase in rat in vitro, i.e., in cultured cells
or tissues derived from whole animals, or in vivo.
[0335] JNK2-specific probes: In initial screenings of a set of
oligonucleotides derived from the JNK2 sequence (Table 9) for
biological activity, a cDNA clone of JNK2 (Kallunki et al., Genes
& Development, 1994, 8, 2996) was radiolabeled and used as a
JNK2-specific probe in Northern blots. Alternatively, however, one
or more of the oligonucleotides of Table 8 is detectably labeled
and used as a JNK2-specific probe.
[0336] Activities of JNK2 oligonucleotides: The data from screening
a set of JNK2-specific phosphorothioate oligonucleotides (Table 9)
indicate the following results. Oligonucleotides showing activity
in this assay, as reflected by levels of inhibition of JNK2 mRNA
levels of at least 50%, include ISIS Nos. 12558, 12559, 12560,
12563, 12564, 12565, 12566, 12567, 12568, 12569 and 12570 (SEQ ID
NOS: 29, 30, 31, 34, 35, 36, 37, 38, 39, 40 and 41, respectively).
These oligonucleotides are thus preferred embodiments of the
invention for modulating JNK2 expression. Oligonucleotides showing
levels of JNK2 mRNAs of at least 80% in this assay, include ISIS
Nos. 12558, 12560, 12565, 12567, 12568 and 12569 (SEQ ID NOS: 29,
31, 36, 38, 39 and 40, respectively). These oligonucleotides are
thus more preferred embodiments of the invention for modulating
JNK2 expression.
[0337] The time course of inhibition of JNK2 mRNA expression by
ISIS 12560 (SEQ ID NO: 31) is shown in Table 10. Following the 4
hour treatment with ISIS 12560, the level of inhibition of JNK2 was
greater than or equal to about 80% for at least about 12 hours and
greater than or equal to about 60% up to at least about t=48 h.
TABLE-US-00008 TABLE 8 Nucleotide Sequences of JNK2
Oligonucleotides TARGET GENENUCLEO- SEQID TIDECO- GENETARGET
ISISNO. NUCLEOTIDE SEQUENCE(5' -> 3') NO: ORDINATES REGION 12558
GTT-TCA-GAT-CCC-TCG-CCC-GC 29 0003-0022 5'-UTR 12559
TGC-AGC-ACA-AAC-AAT-CCC-TT 30 0168-0187 ORF 12560
GTC-CGG-GCC-AGG-CCA-AAG-TC 31 0563-0582 ORF 12561
CAG-GAT-GAC-TTC-GGG-CGC-CC 32 0633-0652 ORF 12562
GCT-CTC-CCA-TGA-TGC-AAC-CC 33 0691-0710 ORF 12563
ATG-GGT-GAC-GCA-GAG-CTT-CG 34 0997-1016 ORF 12564
CTG-CTG-CAT-CTG-AAG-GCT-GA 35 1180-1199 ORF 12565
TGA-GAA-GGA-GTG-GCG-TTG-CT 36 1205-1224 ORF 12566
TGC-TGT-CTG-TGT-CTG-AGG-CC 37 1273-1292 ORF 12567
GGT-CCC-GTC-GAG-GCA-TCA-AG 38 1295-1314 ORF 12568
CAT-TTC-AGG-CCC-ACG-GAG-GT 39 1376-1395 3'-UTR 12569
GGT-CTG-AAT-AGG-GCA-AGG-CA 40 1547-1566 3'-UTR 12570
GGG-CAA-GTC-CAA-GCA-AGC-AT 41 1669-1688 3'-UTR
TABLE-US-00009 TABLE 9 Activities of JNK2 Oligonucleotides GENE SEQ
ID TARGET ISIS NO. NO: REGION % EXPRESSION % INHIBITION 12558 29
5'-UTR 15% 85% 12559 30 ORF 28% 72% 12560 31 ORF 11% 89% 12561 32
ORF 60% 40% 12562 33 ORF 89% 11% 12563 34 ORF 22% 78% 12564 35 ORF
28% 72% 12565 36 ORF 19% 81% 12566 37 ORF 42% 58% 12567 38 ORF 18%
82% 12568 39 3'-UTR 20% 80% 12569 40 3'-UTR 13% 87% 12570 41 3'-UTR
24% 76%
TABLE-US-00010 TABLE 10 Time Course of Response to JNK2 Antisense
Oligonucleotides (ASOs) Normalized SEQ ID ASO % % ISIS # NO:
Description Time Control Inhibition control -- (LIPOFECTIN .TM. 0 h
100.0 0.0 only) control -- (LIPOFECTIN .TM. 4 h 100.0 0.0 only)
control -- (LIPOFECTIN .TM. 12 h 100.0 0.0 only) control --
(LIPOFECTIN .TM. 48 h 100.0 0.0 only) control -- (LIPOFECTIN .TM.
72 h 100.0 0.0 only) 12560 31 JNK2 active 0 h 20.2 79.8 12560 31 ''
4 h 11.1 88.9 12560 31 '' 12 h 21.8 78.2 12560 31 '' 48 h 42.7 57.3
12560 31 '' 72 h 116.8 (0.0)
[0338] Additional JNK2 oligonucleotides: The results for
JNK2-specific oligonucleotides (Table 9) indicate that one of the
most active phosphorothioate oligonucleotides for modulating JNK2
expression is ISIS 12560 (SEQ ID NO: 31). As detailed in Table 11,
additional oligonucleotides based on this oligonucleotide were
designed to confirm and extend the findings described above.
[0339] Oligonucleotides ISIS Nos. 14318 (SEQ ID NO: 42) and 14319
(SEQ ID NO: 43) are 2'-deoxy-phosphorothioate sense strand and
scrambled controls for ISIS 12560 (SEQ ID NO: 31), respectively.
ISIS Nos. 15353 and 15354 are "gapmers" corresponding to ISIS
12560; both have 2'-methoxyethoxy "wings" (having phosphorothioate
linkages in the case of ISIS 15353 and phosphodiester linkages in
the case of ISIS 15354) and a central 2'-deoxy "gap" designed to
support RNaseH activity on the target mRNA molecule. Similarly,
ISIS Nos. 15355 to 15358 are "wingmers" corresponding to ISIS 12560
and have a 5' or 3' 2'-methoxyethoxy RNaseH-refractory "wing" and a
3' or 5' (respectively) 2-deoxy "wing" designed to support RNaseH
activity on the target JNK2 mRNA.
[0340] The chemically modified derivatives of ISIS 12560 (SEQ ID
NO: 31) were tested in the Northern assay described herein at
concentrations of 100 and 400 nM, and the data (Table 12) indicate
the following results. At 400 nM, relative to the 2'-unmodified
oligonucleotide ISIS 12560, both "gapmers" (ISIS Nos. 15353 and
15354) effected approximately 80% inhibition of JNK2 mRNA
expression. Similarly, the four "wingmers" (ISIS Nos. 15355 to
15358) effected 70-90% inhibition of JNK2 expression.
[0341] Dose- and sequence-dependent response to JNK2
oligonucleotides: In order to demonstrate a dose-dependent response
to ISIS 12560 (SEQ ID NO: 31), different concentrations (i.e., 50,
100, 200 and 400 nM) of ISIS 12560 were tested for their effect on
JNK2 mRNA levels in A549 cells (Table 13). In addition, two control
oligonucleotides (ISIS 14318, SEQ ID NO: 42, sense control, and
ISIS 14319, SEQ ID NO: 43, scrambled control; see also Table 11)
were also applied to A549 cells in order to demonstrate the
specificity of ISIS 12560. The results (Table 12) demonstrate that
the response of A549 cells to ISIS 12539 is dependent on dose in an
approximately linear fashion. In contrast, neither of the control
oligonucleotides effect any consistent response on JNK2 mRNA
levels.
TABLE-US-00011 TABLE 11 Chemically Modified JNK2 Oligonucleotides
NUCLEOTIDE SEQUENCE (5' -> 3')AND SEQ ID ISISNO. CHEMICAL
MODIFICATIONS* NO: COMMENTS 12560
G.sup.ST.sup.SC.sup.SC.sup.SG.sup.SG.sup.SG.sup.SC.sup.SC.sup.SA.sup-
.SG.sup.SG.sup.SC.sup.SC.sup.SA.sup.SA.sup.SA.sup.SG.sup.ST.sup.SC
31 active 14318
G.sup.SA.sup.SC.sup.ST.sup.ST.sup.ST.sup.SG.sup.SG.sup.SC.sup.SC.sup-
.ST.sup.SG.sup.SG.sup.SC.sup.SC.sup.SC.sup.SG.sup.SG.sup.SA.sup.SC
42 12560 sense control 14319
G.sup.ST.sup.SG.sup.SC.sup.SG.sup.SC.sup.SG.sup.SC.sup.SG.sup.SA.sup-
.SG.sup.SC.sup.SC.sup.SC.sup.SG.sup.SA.sup.SA.sup.SA.sup.ST.sup.SC
43 scrambled control 15352
G.sup.ST.sup.SC.sup.SC.sup.SG.sup.SG.sup.SG.sup.SC.sup.SC.sup.SA.sup-
.SG.sup.SG.sup.SC.sup.SC.sup.SA.sup.SA.sup.SA.sup.SG.sup.ST.sup.SC
31 fully 2'- methoxyethoxy 15353
G.sup.ST.sup.SC.sup.SC.sup.SG.sup.SG.sup.SG.sup.SC.sup.SC.sup.SA.sup-
.SG.sup.SG.sup.SC.sup.SC.sup.SA.sup.SA.sup.SA.sup.SG.sup.ST.sup.SC
31 "gapmer" 15354
G.sup.OT.sup.OC.sup.OC.sup.OG.sup.SG.sup.SG.sup.SC.sup.SC.sup.SA.sup-
.SG.sup.SG.sup.SC.sup.SC.sup.SA.sup.OA.sup.OA.sup.OG.sup.OT.sup.OC
31 "gapmer" 15355
G.sup.ST.sup.SC.sup.SC.sup.SG.sup.SG.sup.SG.sup.SC.sup.SC.sup.SA.sup-
.SG.sup.SG.sup.SC.sup.SC.sup.SA.sup.SA.sup.SA.sup.SG.sup.ST.sup.SC
31 "wingmer" 15356
G.sup.ST.sup.SC.sup.SC.sup.SG.sup.SG.sup.SG.sup.SC.sup.SC.sup.SA.sup-
.SG.sup.SG.sup.SC.sup.SC.sup.SA.sup.SA.sup.SA.sup.SG.sup.ST.sup.SC
31 "wingmer" 15358
G.sup.OT.sup.OC.sup.OC.sup.OG.sup.OG.sup.OG.sup.OC.sup.OC.sup.OA.sup-
.OG.sup.SG.sup.SC.sup.SC.sup.SA.sup.SA.sup.SA.sup.SG.sup.ST.sup.SC
31 "wingmer" 15357
G.sup.ST.sup.SC.sup.SC.sup.SG.sup.SG.sup.SG.sup.SC.sup.SC.sup.SA.sup-
.OG.sup.OG.sup.OC.sup.OC.sup.OA.sup.OA.sup.OA.sup.OG.sup.OT.sup.OC
31 "wingmer" 20572
G.sup.ST.sup.SC.sup.SC.sup.SG.sup.SG.sup.SG.sup.SC.sup.SC.sup.SA.sup-
.SG.sup.SG.sup.SC.sup.SC.sup.SA.sup.SA.sup.SA.sup.SG.sup.ST.sup.SC
31 fully 5-methyl- cytosine version of ISIS 15353 *Emboldened
residues, 2'-methoxyethoxy-residues (others are 2'-deoxy-)
including "C" residues, 5-methyl-cytosines; ".sup.O",
phosphodiester linkage; ".sup.S", phosphorothioate linkage. --- "C"
residues, 2'-deoxy 5-methylcytosine residues; ---
TABLE-US-00012 TABLE 12 Activity of Chemically Modified JNK2
Antisense Oligonucleotides SEQ Normalized ID % ISIS # NO:
Oligonucleotide Description Dose Control control -- No
oligonucleotide -- 100.0 (LIPOFECTIN .TM. only) 12560 31 JNK2
active, fully P.dbd.S & 100 nM 62.1 12560 31 fully 2-deoxy 400
nM 31.4 15352 31 fully P.dbd.S & fully 2'-MOE 100 nM 132.4
15352 31 400 nM 158.4 15353 31 gapmer: P.dbd.S, 2'-MOE wings; 100
nM 56.7 15353 31 P.dbd.S, 2-deoxy core 400 nM 21.2 15354 31 gapmer:
P.dbd.O, 2'-MOE wings; 100 nM 38.3 15354 31 P.dbd.S, 2-deoxy core
400 nM 17.1 15355 31 wingmer: fully P.dbd.S; 100 nM 61.3 15355 31
5' 2'-MOE; 3' 2-deoxy 400 nM 29.1 15356 31 wingmer: fully P.dbd.S;
100 nM 38.6 15356 31 5' 2-deoxy; 3' 2'-MOE 400 nM 11.0 15358 31
wingmer: 5' P.dbd.O & 2'-MOE; 100 nM 47.4 15358 31 3' P.dbd.S
& 2-deoxy 400 nM 29.4 15357 31 wingmer: 5' P.dbd.S & 2'-
100 nM 42.8 15357 31 deoxy; 3' P.dbd.O & 2'-MOE 400 nM 13.7
TABLE-US-00013 TABLE 13 Dose-Dependent Responses to JNK2 Antisense
Oligonucleotides SEQ ID Normalized % ISIS # NO: Oligonucleotide
Description Dose Control control -- No oligonucleotide -- 100.0
(LIPOFECTIN .TM. only) 12560 31 JNK2 active 50 nM 68.1 12560 31 ''
100 nM 50.0 12560 31 '' 200 nM 25.1 12560 31 '' 400 nM 14.2 14318
42 12560 sense control 50 nM 87.1 14318 42 '' 100 nM 89.8 14318 42
'' 200 nM 92.1 14318 42 '' 400 nM 99.6 14319 43 12560 scrambled
control 50 nM 90.4 14319 43 '' 100 nM 93.7 14319 43 '' 200 nM 110.2
14319 43 '' 400 nM 100.0
[0342] Western Assays: In order to assess the effect of
oligonucleotides targeted to JNK2 mRNAs on JNK2 protein levels,
Western assays are performed essentially as described above in
Examples 2 and 3. A primary antibody that specifically binds to
JNK2 is purchased from, for example, Santa Cruz Biotechnology,
Inc., Santa Cruz, Calif.; Upstate Biotechnology, Inc., Lake Placid,
N.Y.; StressGen Biotechnologies, Inc., Victoria, BC, Canada; or
Research Diagnostics, Inc., Flanders, N.J.
[0343] Oligonucleotides specific for JNK2 isoforms: Subsequent to
the initial descriptions of JNK2 (Sluss et al., Mol. Cel. Biol.,
1994, 14, 8376; Kallunki et al., Genes & Development, 1994, 8,
2996; GenBank accession No. HSU09759 (SEQ ID NO: 94), locus name
"U09759 (SEQ ID NO: 94)"), cDNAs encoding related isoforms of JNK2
were cloned and their nucleotide sequences determined (Gupta et
al., EMBO Journal, 1996, 15, 2760). In addition to JNK2-a2 (GenBank
accession No. L31951 (SEQ ID NO: 92), locus name "HUMJNK2"), which
encodes a polypeptide having an amino acid sequence identical to
that of JNK2, the additional isoforms include JNK2-a1 (GenBank
accession No. U34821 (SEQ ID NO: 95), locus name "HSU34821"),
JNK2-.beta.1 (GenBank accession No. U35002 (SEQ ID NO: 96), locus
name "HSU35002") and JNK2-.beta.2 (GenBank accession No. U35003
(SEQ ID NO: 97), locus name "HSU35003"). The four isoforms of JNK2,
which probably arise from alternative mRNA splicing, can each
interact with different transcription factors or sets of
transcription factors (Gupta et al., EMBO Journal, 1996, 15, 2760).
As detailed below, the oligonucleotides of the invention are
specific for certain members or sets of these isoforms of JNK2.
[0344] In the ORFs of mRNAs encoding JNK2/JNK2-a2 and JNK2-a1,
nucleotides (nt) 689-748 of JNK2/JNK2-a2 (GenBank accession No.
L31951 (SEQ ID NO: 92)) and nt 675-734 of JNK2-a1 (GenBank
accession No. U34821 (SEQ ID NO: 95)) have the sequence shown below
as SEQ ID NO: 75, whereas, in the ORFs of mRNAs encoding
JNK2-.beta.1 and JNK2-.beta.2, nt 653-712 of JNK2-.beta.1 (GenBank
accession No. U35002 (SEQ ID NO: 96)) and nt 665-724 of
JNK2-.beta.2 (GenBank accession No. U35003 (SEQ ID NO: 97)) have
the sequence shown below as SEQ ID NO: 76. For purposes of
illustration, SEQ ID NOS: 75 and 76 are shown aligned with each
other (vertical marks, "|," indicate bases that are identical in
both sequences):
##STR00007##
[0345] Due to this divergence between the a and b JNK2 isoforms,
antisense oligonucleotides derived from the reverse complement of
SEQ ID NO: 75 (i.e., SEQ ID NO: 77, see below) are specifically
hybridizable to, and can be selected and used to modulate the
expression of, JNK2/JNK2-a2 and JNK2-a1 without significantly
effecting the expression of JNK1-.beta.1 and JNK1-.beta.2. In like
fashion, antisense oligonucleotides derived from the reverse
complement of SEQ ID NO: 76 (i.e., SEQ ID NO: 78, see below) are
specifically hybridizable to, and can be selected and used to
modulate the expression of, JNK2-.beta.1 and JNK2-.beta.2 without
significantly effecting the expression of JNK2/JNK2-a2 and JNK2-a1.
As an example, an oligonucleotide having a sequence derived from
SEQ ID NO: 77 but not from SEQ ID NO: 78 is specifically
hybridizable to, mRNAs encoding JNK1/JNK1-a1 and JNK1-a2 but not to
those encoding JNK2-.beta.1 and JNK2-.beta.2:
##STR00008##
[0346] In the case of the carboxyl terminal portion of the JNK2
isoforms, JNK2/JNK2-a2 shares identity with JNK1-.beta.2;
similarly, JNK2-a1 and JNK2-.beta.1 have identical carboxy terminal
portions. The substantial differences in the amino acid sequences
of these isoforms (5 amino acids in JNK2-a2 and JNK2-.beta.2 are
replaced with 47 amino acids in JNK2/JNK2-a2 and JNK2-.beta.2)
result from a slight difference in nucleotide sequence that shifts
the reading frame. Specifically, in the ORFs of mRNAs encoding
JNK2-a1 and JNK1-131, nt 1164-1198 of JNK2-a1 (GenBank accession
No. U34821 (SEQ ID NO: 95)) and nt 1142-1176 of JNK2-.beta.1
(GenBank accession No. U35002 (SEQ ID NO: 96)) have the sequence
shown below as SEQ ID NO: 79, whereas, in the ORFs of mRNAs
encoding JNK2/JNK2-a2 and JNK2-.beta.2, nt 1178-1207 of
JNK2/JNK2-a2 (GenBank accession No. L31951 (SEQ ID NO: 92)) and nt
1154-1183 of JNK2-.beta.2 (GenBank accession No. U35003 (SEQ ID NO:
97)) have the sequence shown below as SEQ ID NO: 80. For purposes
of illustration, SEQ ID NOS: 79 and 80 are shown aligned with each
other (dashes, "-," indicate bases that are absent in the indicated
sequence, and emboldened bases indicate the stop codon for the
JNK2-a1 and JNK2-.beta.1 ORFs):
##STR00009##
Due to this divergence between the JNK2 isoforms, antisense
oligonucleotides derived from the reverse complement of SEQ ID NO:
79 (i.e., SEQ ID NO: 81, see below) are specifically hybridizable
to, and can be selected and used to modulate the expression of,
mRNAs encoding JNK2-a1 and JNK2-.beta.1 without significantly
effecting the expression of JNK2/JNK2-a2 and JNK2-.beta.2. In like
fashion, antisense oligonucleotides derived from the reverse
complement of SEQ ID NO: 80 (i.e., SEQ ID NO: 82, see below) are
specifically hybridizable to, and can be selected and used to
modulate the expression of, mRNAs encoding JNK2/JNK2-a2 and
JNK2-.beta.2 without significantly effecting the expression of
JNK2-a1 and JNK2-.beta.1. As an example, ISIS 12564 (SEQ ID NO: 35)
corresponds to SEQ ID NO: 82 but not to SEQ ID NO: 81, and is thus
specifically hybridizable to, and can be used to modulate the
expression of, mRNAs encoding JNK2/JNK2-a2 and JNK2-.beta.2 but not
those encoding JNK2-a1 and JNK2-a1:
##STR00010##
[0347] In preferred embodiments, such isoform-specific
oligonucleotides such as are described above are methoxyethoxy
"gapmers" or "wingmers" in which the RNase H-sensitive "gap" or
"wing" is positioned so as to overlap a region of nonidentity in
the above antisense sequences, i.e., SEQ ID NOS: 77, 78, 81 and
82.
Example 5
Oligonucleotide-Mediated Inhibition of JNK3 Expression
[0348] A. JNK3 oligonucleotide sequences: Table 14 lists the
nucleotide sequences of oligonucleotides designed to specifically
hybridize to JNK3 mRNAs and the corresponding ISIS and SEQ ID
numbers thereof. The target gene nucleotide co-ordinates and gene
target region are also included. The nucleotide co-ordinates are
derived from GenBank accession No. U07620 (SEQ ID NO: 98), locus
name "HSU07620" see also FIG. 4(A) of Mohit et al., Neuron, 1994,
14, 67). The abbreviations for gene target regions are as follows:
5'-UTR, 5' untranslated region; tIR, translation initiation region;
ORF, open reading frame; 3'-UTR, 3' untranslated region. It should
be noted that the oligonucleotide target co-ordinate positions and
gene target regions can vary within mRNAs encoding related isoforms
of JNK3 (see subsection D, below).
[0349] The nucleotides of the oligonucleotides whose sequences are
presented in Table 14 are connected by phosphorothioate linkages
and are "gapmers." Specifically, the six nucleotides of the 3' and
5' termini are 2'-methoxyethoxy-modified and are shown emboldened
in Table 14, whereas the central eight nucleotides are unmodified
at the 2' position (i.e., 2-deoxy).
[0350] In addition to hybridizing to human JNK3 mRNAs, the full
oligonucleotide sequences of ISIS Nos. 16692, 16693, 16703, 16704,
16705, 16707, and 16708 (SEQ ID NOS: 46, 47, 56, 57, 58, 60 and 61,
respectively) specifically hybridize to mRNAs from Rattus
norvegicus that encode a stress-activated protein kinase named
"p54.beta." (Kyriakis et al., Nature, 1994, 369, 156; GenBank
accession No. L27128 (SEQ ID NO: 99), locus name "RATSAPKC."
Furthermore, the full oligonucleotide sequences of 16692, 16693,
16695, 16703, 16704, 16705, 16707 and 16708 (SEQ ID NOS: 46, 47,
49, 56, 57, 58, 60 and 61, respectively) specifically hybridize to
mRNAs from Mus musculus that encode a mitogen activated protein
(MAP) kinase stress activated protein named the "p459.sup.3F12 SAP
kinase" (Martin et al., Brain Res. Mol. Brain. Res., 1996, 35, 47;
GenBank accession No. L35236 (SEQ ID NO: 100), locus name
"MUSMAPK"). These oligonucleotides are thus preferred embodiments
of the invention for investigating the role of the p54.beta. and
p459.sup.3F12 SAP protein kinases in rat or mouse, respectively, in
vitro, i.e., in cultured cells or tissues derived from whole
animals or in vivo. The target gene nucleotide co-ordinates and
gene target regions for these oligonucleotides, as defined for
these GenBank entries, are detailed in Table 15.
[0351] JNK3-specific probes: In initial screenings of a set of
oligonucleotides derived from the JNK3 sequence for biological
activity, a cDNA clone of JNK3 (Derijard et al., Cell, 1994, 76,
1025) was radiolabeled and used as a JNK3-specific probe in
Northern blots. Alternatively, however, one or more of the
oligonucleotides of Table 14 is detectably labeled and used as a
JNK3-specific probe.
[0352] Western Assays: In order to assess the effect of
oligonucleotides targeted to JNK3 mRNAs on JNK3 protein levels,
Western assays are performed essentially as described above in
Examples 2 through 4. A primary antibody that specifically binds to
JNK3 is purchased from, for example, Upstate Biotechnology, Inc.
(Lake Placid, N.Y.), StressGen Biotechnologies Corp. (Victoria, BC,
Canada), or New England Biolabs, Inc. (Beverly, Mass.).
TABLE-US-00014 TABLE 14 Nucleotide Sequences of JNK3
Oligonucleotides TARGET GENE NUCLEOTIDE SEQID CO- GENETARGET
ISISNO. NUCLEOTIDE SEQUENCE.sup.1(5' -> 3') NO: ORDINATES REGION
16690 TTC-AAC-AGT-TTC-TTG-CAT-AA 44 0157-0176 5'-UTR 16691
CTC-ATC-TAT-AGG-AAA-CGG-GT 45 0182-0200 5'-UTR 16692
TGG-AGG-CTC-ATA-AAT-ACC-AC 46 0215-0234 tIR 16693
TAT-AAG-AAA-TGG-AGG-CTC-AT 47 0224-0243 tIR 16694
TCA-CAT-CCA-ATG-TTG-GTT-CA 48 0253-0272 ORF 16695
TTA-TCG-AAT-CCC-TGA-CAA-AA 49 0281-0300 ORF 16696
GTT-TGG-CAA-TAT-ATG-ACA-CA 50 0310-0329 ORF 16697
CTG-TCA-AGG-ACA-GCA-TCA-TA 51 0467-0486 ORF 16698
AAT-CAC-TTG-ACA-TAA-GTT-GG 52 0675-0694 ORF 16699
TAA-ATC-CCT-GTG-AAT-AAT-TC 53 0774-0793 ORF 16700
GCA-TCC-CAC-AGA-CCA-TAT-AT 54 0957-0976 ORF 16702
TGT-TCT-CTT-TCA-TCC-AAC-TG 55 1358-1377 ORF 16703
TCT-CAC-TGC-TGT-TCA-CTG-CT 56 1485-1504 tIR 16704
GGG-TCT-GGT-CGG-TGG-ACA-TG 57 1542-1561 3'-UTR 16705
AGG-CTG-CTG-TCA-GTG-TCA-GA 58 1567-1586 3'-UTR 16706
TCA-CCT-GCA-ACA-ACC-CAG-GG 59 1604-1623 3'-UTR 16707
GCG-GCT-AGT-CAC-CTG-CAA-CA 60 1612-1631 3'-UTR 16708
CGC-TGG-GTT-TCG-CAG-GCA-GG 61 1631-1650 3'-UTR 16709
ATC-ATC-TCC-TGA-AGA-ACG-CT 62 1647-1666 3'-UTR .sup.1Emboldened
residues are 2'-methoxyethoxy-modified.
TABLE-US-00015 TABLE 15 Rat and Mouse Gene Target Locations of JNK3
Oligonucleotides Rat Mouse SEQ NUCLEOTIDE GENE NUCLEOTIDE GENE ISIS
ID CO- TARGET CO- TARGET NO. NO: ORDINATES.sup.1 REGION
ORDINATES.sup.2 REGION 16692 46 0213-0232 5'-UTR 0301-0320 tIR
16693 47 0222-0241 5'-UTR 0310-0329 tIR 16695 49 -- -- 0367-0386
ORF 16703 56 1506-1525 ORF 1571-1590 tTR 16704 57 1563-1582 ORF
1628-1647 3'-UTR 16705 58 1588-1607 ORF 1653-1672 3'-UTR 16707 60
1633-1652 tTR 1698-1717 3'-UTR 16708 61 1652-1671 3'-UTR 1717-1736
3'-UTR .sup.1Co-ordinates from GenBank Accession No. L27128 (SEQ ID
NO: 99), locus name "RATSAPKC." .sup.2Co-ordinates from GenBank
Accession No. L35236 (SEQ ID NO: 100), locus name "MUSMAPK."
[0353] Oligonucleotides specific for JNK3 isoforms: Two isoforms of
JNK3 have been described. JNK3-a1 was initially cloned and named
"p49.sup.3F12 kinase" by (Mohit et al. Neuron, 1995, 14, 67).
Subsequently, two cDNAs encoding related isoforms of JNK3 were
cloned and their nucleotide sequences determined (Gupta et al.,
EMBO Journal, 1996, 15, 2760). The isoforms are named JNK3-a1
(GenBank accession No. U34820 (SEQ ID NO: 101), locus name
"HSU34820") and JNK3-a2 (GenBank accession No. U34819 (SEQ ID NO:
102), locus name "HSU34819") herein. The two isoforms of JNK3,
which probably arise from alternative mRNA splicing, can each
interact with different transcription factors or sets of
transcription factors (Gupta et al., EMBO Journal, 1996, 15, 2760).
As detailed below, certain oligonucleotides of the invention are
specific for each of these isoforms of JNK3.
[0354] JNK3-a1 and JNK-a2 differ at their carboxyl terminal
portions. The substantial differences in the amino acid sequences
of these isoforms (5 amino acids in JNK3-a1 are replaced with 47
amino acids in JNK3-a2) result from a slight difference in
nucleotide sequence that shifts the reading frame. Specifically, in
the ORF of mRNAs encoding JNK3-a1, nucleotides (nt) 1325-1362 of
JNK3-a1 (GenBank accession No. U34820 (SEQ ID NO: 101)) have the
sequence shown below as SEQ ID NO: 83, whereas, in the ORF of mRNAs
encoding JNK3-a2, nt 1301-1333 of JNK3-a2 (GenBank accession No.
U34819 (SEQ ID NO: 102)) have the sequence shown below as SEQ ID
NO: 84. For purposes of illustration, SEQ ID NOS: 83 and 202 are
shown aligned with each other (vertical marks, "|," indicate bases
that are identical in both sequences; dashes, "-," indicate bases
that are absent in the indicated sequence; and emboldened bases
indicate the stop codon for the JNK3-a1 ORF):
##STR00011##
Due to this divergence between the JNK3 isoforms, antisense
oligonucleotides derived from the reverse complement of SEQ ID NO:
83 (i.e., SEQ ID NO: 85, see below) are specifically hybridizable
to mRNAs encoding JNK3-a1, and can be selected and used to modulate
the expression of JNK3-a1 without significantly effecting the
expression of JNK3-a2. In like fashion, antisense oligonucleotides
derived from the reverse complement of SEQ ID NO: 84 (i.e., SEQ ID
NO: 86, see below) are specifically hybridizable to mRNAs encoding
JNK3-a2, and can be selected and used to modulate the expression of
JNK3-a2 without significantly effecting the expression of
JNK3-a1:
##STR00012##
[0355] In preferred embodiments, such isoform-specific
oligonucleotides such as are described above are methoxyethoxy
"gapmers" or "wingmers" in which the RNase H-sensitive "gap" or
"wing" is positioned so as to overlap a region of nonidentity in
the above antisense sequences, i.e., SEQ ID NOS: 85 and 86.
[0356] Activities of JNK3 oligonucleotides: The JNK3-specific
phosphorothioate, 2'-methoxyethoxy "gapmer" oligonucleotides (Table
14) were screened for their ability to affect JNK3 mRNA levels in
SH-SYSY cells (Biedler et al., Cancer Res., 1973, 33, 2643).
SH-SY5Y cells express a variety of mitogen-activated protein
kinases (MAPKs; see, e.g., Cheng et al., J. Biol. Chem., 1998, 273,
14560). Cells were grown in DMEM essentially as previously
described (e.g., Singleton et al., J. Biol. Chem., 1996, 271,
31791; Jalava et al., Cancer Res., 1990, 50, 3422) and treated with
oligonucleotides at a concentration of 200 nM as described in
Example 2. Control cultures were treated with an aliquot of
LIPOFECTIN.TM. that contained no oligonucleotide.
[0357] The results are shown in Table 16. Oligonucleotides showing
levels of inhibition of JNK3 mRNA levels of at least 45% include
ISIS Nos. 16692, 16693, 16694, 16695, 16696, 16697, 16702, 16703,
16704, 16705 and 16706 (SEQ ID NOS:46, 47, 48, 49, 50, 51, 55, 56,
57, 58 and 59, respectively). These oligonucleotides are preferred
embodiments of the invention for modulating JNK3 expression.
Oligonucleotides inhibiting JNK3 mRNAs by at least 60% in this
assay include ISIS Nos. 16693, 16702, 16703 and 16704 (SEQ ID NOS:
47, 55, 56 and 57, respectively). These oligonucleotides are thus
more preferred embodiments of the invention for modulating JNK3
expression.
TABLE-US-00016 TABLE 16 Activities of JNK3 Oligonucleotides SEQ ID
GENE TARGET % ISISNo: NO: REGION EXPRESSION: % INHIBITION:
control.sup.1 -- -- 100% 0% 16690 44 5'-UTR 60% 40% 16691 45 5'-UTR
66% 34% 16692 46 tIR 47% 53% 16693 47 tIR 40% 60% 16694 48 ORF 42%
58% 16695 49 ORF 44% 56% 16696 50 ORF 55% 45% 16697 51 ORF 54% 46%
16698 52 ORF 63% 37% 16699 53 ORF 61% 39% 16700 54 ORF N.D..sup.2
N.D. 16702 55 ORF 39% 61% 16703 56 tTR 30% 70% 16704 57 3'-UTR 36%
64% 16705 58 3'-UTR 42% 58% 16706 59 3'-UTR 45% 55% 16707 60 3'-UTR
73% 27% 16708 61 3'-UTR 68% 32% 16709 62 3'-UTR 66% 34% .sup.1Cells
treated with LIPOFECTIN .TM. only (no oligonucleotide). .sup.2N.D.,
not determined.
Example 6
Oligonucleotides Targeted to Genes Encoding Rat JNK Proteins
[0358] In order to study the role of JNK proteins in animal models,
oligonucleotides targeted to the genes encoding JNK1, JNK2 and JNK3
of Rattus norvegicus were prepared. These oligonucleotides are
2'-methoxyethoxy, phosphodiester/2'-hydroxyl,
phosphorothioate/2'-methoxyethoxy, phosphodiester "gapmers" in
which every cytosine residue is 5-methylcytosine (m5c). These
antisense compounds were synthesized according to the methods of
the disclosure. Certain of these oligonucleotides are additionally
specifically hybridizable to JNK genes from other species as
indicated herein. The oligonucleotides described in this Example
were tested for their ability to modulate rat JNK mRNA levels
essentially according to the methods described in the preceding
Examples, with the exceptions that the cell line used was rat A10
aortic smooth muscle cells (ATCC No. ATCC CRL-1476) and the probes
used were specific for rat JNK1, JNK2 or JNK3 (see infra). A10
cells were grown and treated with oligonucleotides essentially as
described by (Cioffi et al. Mol. Pharmacol., 1997, 51, 383).
[0359] JNK1: Table 17 describes the sequences and structures of a
set of oligonucleotides, ISIS Nos. 21857 to 21870 (SEQ ID NOS:111
to 124, respectively) that were designed to be specifically
hybridizable to nucleic acids from Rattus norvegicus that encode a
stress-activated protein kinase named "p54?" or "SAPK?" that is
homologous to the human protein JNK1 (Kyriakis et al., Nature,
1994, 369, 156; GenBank accession No. L27129 (SEQ ID NO: 88), locus
name "RATSAPKD"). In Table 17, emboldened residues are
2'-methoxyethoxy-residues (others are 2'-deoxy-); "C" residues are
2'-methoxyethoxy-5-methyl-cytosines and "C" residues are
5-methyl-cytosines; "o" indicates a phosphodiester linkage; and "s"
indicates a phosphorothioate linkage. The target gene co-ordinates
are from GenBank Accession No. L27129 (SEQ ID NO: 88), locus name
"RATSAPKD."
TABLE-US-00017 TABLE 17 Nucleotide Sequences of Rat JNK1
Oligonucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS ID CO- TARGET
NO. NUCLEOTIDE SEQUENCE(5' -> 3') NO ORDINATE REGION 21857
CoAoAoCoGsTsCsCsCsGsCsGsCsTsCsGoGoCoCoG 111 0002-0021 5'-UTR 21858
CoCoToGoCsTsCsGsCsGsGsCsTsCsCsGoCoGoToT 112 0029-0048 5'-UTR 21859
CoToCoAoTsGsAsTsGsGsCsAsAsGsCsAoAoToToA 113 0161-0180 tIR 21860
ToGoToToGsTsCsAsCsGsTsTsTsAsCsToToCoToG 114 0181-0200 ORF 21861
CoGoGoToAsGsGsCsTsCsGsCsTsTsAsGoCoAoToG 115 0371-0390 ORF 21862
CoToAoGoGsGsAsTsTsTsCsTsGsTsGsGoToGoToG 116 0451-0470 ORF 21863
CoAoGoCoAsGsAsGsTsGsAsAsGsGsTsGoCoToToG 117 0592-0611 ORF 21864
ToCoGoToTsCsCsTsGsCsAsGsTsCsCsToToGoCoC 118 0691-0710 ORF 21865
CoCoAoToTsTsCsTsCsCsCsAsTsAsAsToGoCoAoC 119 0811-0830 ORF 21866
ToGoAoAoTsTsCsAsGsGsAsCsAsAsGsGoToGoToT 120 0901-0920 ORF 21867
AoGoCoToTsCsGsTsCsTsAsCsGsGsAsGoAoToCoC 121 1101-1120 ORF 21868
CoAoCoToCsCsTsCsTsAsTsTsGsTsGsToGoCoToC 122 1211-1230 ORF 21869
GoCoToGoCsAsCsCsTsAsAsAsGsGsAsGoAoCoGoG 123 1301-1320 ORF 21870
CoCoAoGoAsGsTsCsGsGsAsTsCsTsGsToGoGoAoC 124 1381-1400 ORF
These antisense compounds were tested for their ability to modulate
levels of (JNK1) and (JNK2) mRNA in A10 cells via Northern assays.
Due to the high degree of sequence identity between the human and
rat genes, radiolabeled human JNK1 (Example 3) and JNK2 (Example 4)
cDNAs functioned as specific probes for the rat homologs.
[0360] The results are shown in Table 18. ISIS Nos. 21857 to 21870
(SEQ ID NOS:111 to 124, respectively) showed 70% to 90% inhibition
of rat JNK1 mRNA levels. These oligonucleotides are preferred
embodiments of the invention for modulating rat JNK1 expression.
Oligonucleotides showing levels of inhibition of at least 90% in
this assay include ISIS Nos. 21858, 21859, 21860, 21861, 21862,
21864, 21865, 21866 and 21867 (SEQ ID NOS:112, 113, 114, 115, 116,
118, 119, 120 and 121, respectively). These oligonucleotides are
thus more preferred embodiments of the invention for modulating rat
JNK1 expression. ISIS 21859 (SEQ ID NO:113) was chosen for use in
further studies (infra).
[0361] Two of the oligonucleotides, ISIS Nos. 21861 and 21867 (SEQ
ID NOS:115 and 121, respectively) demonstrated a capacity to
modulate both JNK1 and JNK2. Such oligonucleotides are referred to
herein as "Pan JNK" antisense compounds because the term "Pan" is
used in immunological literature to refer to an antibody that
recognizes, e.g., all isoforms of a protein or subtypes of a cell
type. The Pan JNK oligonucleotides are discussed in more detail
infra.
[0362] In addition to being specifically hybridizable to nucleic
acids encoding rat JNK1, some of the oligonucleotides described in
Table 17 are also specifically hybridizable with JNK1-encoding
nucleic acids from other species. ISIS 21859 (SEQ ID NO:113) is
complementary to bases 4 to 23 of cDNAs encoding human JNK1a1 and
JNK1.beta.1 (i.e., GenBank accession Nos. L26318 (SEQ ID NO: 87)
and U35004 (SEQ ID NO: 90), respectively). ISIS 21862 (SEQ ID
NO:116) is complementary to bases 294 to 313 of the human JNK1a1
and JNK1.beta.1 cDNAs (GenBank accession Nos. L26318 (SEQ ID NO:
87) and U35004 (SEQ ID NO: 90), respectively), bases 289 to 308 of
the human JNK1132 cDNA (GenBank accession No. U35005 (SEQ ID NO:
91)), and bases 288 to 307 of the human JNK1a2 cDNA (GenBank
accession No. U34822 (SEQ ID NO: 89)). Finally, ISIS 21865 is
complementary to bases 654 to 673 of the human JNK1a1 cDNA (GenBank
accession No. L26318 (SEQ ID NO: 87)) and to bases 648 to 667 of
the human JNK1a2 cDNA (GenBank accession No. U34822 (SEQ ID NO:
89)). These oligonucleotides are tested for their ability to
modulate mRNA levels of human JNK1 genes according to the methods
described in Example 3.
TABLE-US-00018 TABLE 18 Activities of Oligonucleotides Targeted to
Rat JNK1 SEQ ID GENE TARGET % EXPRESSION % EXPRESSION ISISNo: NO:
REGION JNK1 JNK2 control.sup.1 -- -- 100% 100% 21857 111 5'-UTR 24%
91% 21858 112 5'-UTR 8% 89% 21859 113 tIR 5% 106% 21860 114 ORF 8%
98% 21861 115 ORF 6% 13% 21862 116 ORF 6% 133% 21863 117 ORF 24%
107% 21864 118 ORF 8% 106% 21865 119 ORF 5% 50% 21866 120 ORF 8%
98% 21867 121 ORF 5% 21% 21868 122 ORF 15% 112% 21869 123 ORF 30%
93% 21870 124 ORF 11% 87% .sup.1Cells treated with LIPOFECTIN .TM.
only (no oligonucleotide).
[0363] JNK2: Table 19 describes the sequences and structures of a
set of oligonucleotides, ISIS Nos. 18254 to 18267 (SEQ ID NOS:125
to 138, respectively) that were designed to be specifically
hybridizable to nucleic acids that encode a stress-activated
protein kinase from Rattus norvegicus that encode a
stress-activated protein kinase named "p54a" or "SAPKa" (Kyriakis
et al., Nature, 1994, 369, 156). The structures of three control
oligonucleotides, ISIS Nos. 21914 to 21916 (SEQ ID NOS:139 to 141,
respectively) are also shown in the table. Two isoforms of p54a
have been described: "p54a1" (GenBank accession No. L27112 (SEQ ID
NO: 93), locus name "RATSAPKA") and "p54a2" (GenBank accession No.
L27111 (SEQ ID NO: 104), locus name "RATSAPKB"). With the exception
of ISIS 18257 (SEQ ID NO:128), the oligonucleotides described in
Table 19 are specifically hybridizable to nucleic acids encoding
either p54a1 or p54a2. ISIS 18257 is specifically hybridizable to
nucleic acids encoding p54a2 (i.e., GenBank accession No. L27112
(SEQ ID NO: 93), locus name "RATSAPKB"). In Table 19, emboldened
residues are 2'-methoxyethoxy-residues (others are 2'-deoxy-); "C"
residues are 2'-methoxyethoxy-5-methyl-cytosines and "C" residues
are 5-methyl-cytosines; "o" indicates a phosphodiester linkage; and
"s" indicates a phosphorothioate linkage. The target gene
co-ordinates are from GenBank Accession No. L27112 (SEQ ID NO: 93),
locus name "RATSAPKB."
TABLE-US-00019 TABLE 19 Nucleotide Sequences of Rat JNK2
Oligonucleotides SEQ TARGET GENE GENE ISIS ID NUCLEOTIDE TARGET NO.
NUCLEOTIDE SEQUENCE (5' -> 3') NO: CO-ORDINATES REGION 18254
ToCoAoToGsAsTsGsTsAsGsTsGsTsCs 125 0001-0020 tIR AoToAoCoA 18255
ToGoToGoGsTsGsTsGsAsAsCsAsCsAs 126 0281-0300 ORF ToToToAoA 18256
CoCoAoToAsTsGsAsAsTsAsAsCsCsTs 127 0361-0380 ORF GoAoCoAoT 18257
GoAoToAoTsCsAsAsCsAsTsTsCsTsCs 128 0621-0640 ORF CoToToGoT 18258
GoCoToToCsGsTsCsCsAsCsAsGsAsGs 129 0941-0960 ORF AoToCoCoG 18259
GoCoToCoAsGsTsGsGsAsCsAsTsGsGs 130 1201-1220 ORF AoToGoAoG 18260
AoToCoToGsCsGsAsGsGsTsTsTsCsAs 131 1281-1300 tTR ToCoGoGoC 18261
CoCoAoCoCsAsGsCsTsCsCsCsAsTsGs 132 1341-1360 3'-UTR ToGoCoToC 18262
CoAoGoToTsAsCsAsCsAsTsGsAsTsCs 133 1571-1590 3'-UTR ToGoToCoA 18263
AoAoGoAoGsGsAsTsTsAsAsGsAsGsA 134 1701-1720 3'-UTR sToToAoToT 18264
AoGoCoAoGsAsGsTsGsAsAsAsTsAsC 135 2001-2020 3'-UTR sAoAoCoToT 18265
ToGoToCoAsGsCsTsCsTsAsCsAsTsTs 136 2171-2190 3'-UTR AoGoGoCoA 18266
AoGoToAoAsGsCsCsCsGsGsTsCsTsCs 137 2371-2390 3'-UTR CoToAoAoG 18267
AoAoAoToGsGsAsAsAsAsGsGsAsCsA 138 2405-2424 3'-UTR sGoCoAoGoC 21914
GoCoToCoAsGsTsGsGsAsTsAsTsGsGs 139 18259 control -- AoToGoAoG 21915
GoCoToAoAsGsCsGsGsTsCsAsAsGsG 140 18259 control -- sToToGoAoG 21916
GoCoToCoGsGsTsGsGsAsAsAsTsGsG 141 18259 control -- sAoToCoAoG
TABLE-US-00020 TABLE 20 Activities of Oligonucleotides Targeted to
Rat JNK2 SEQ ID GENE TARGET % ISISNo: NO: REGION EXPRESSION %
INHIBITION control.sup.1 -- -- 100% 0% 18254 125 tIR 20% 80% 18255
126 ORF 21% 79% 18256 127 ORF 80% 20% 18257 128 ORF 32% 68% 18258
129 ORF 19% 81% 18259 130 ORF 15% 85% 18260 131 ORF 41% 59% 18261
132 3'-UTR 47% 53% 18262 133 3'-UTR 50% 50% 18263 134 3'-UTR 63%
37% 18264 135 3'-UTR 48% 52% 18265 136 3'-UTR 38% 62% 18266 137
3'-UTR 66% 34% 18267 138 3'-UTR 84% 16% .sup.1Cells treated with
LIPOFECTIN .TM. only (no oligonucleotide).
[0364] These antisense compounds were tested for their ability to
modulate levels of p54a (JNK2) mRNA in A10 cells using the
radiolabeled human JNK2 cDNA as a probe as described supra. The
results are shown in Table 20. Oligonucleotides showing levels of
inhibition from .gtoreq.about 60% to about 100% of rat JNK2 mRNA
levels include ISIS Nos. 18254, 18255, 18257, 18258, 18259, 18260
and 18265 (SEQ ID NOS:125, 126, 128, 129, 130, 131 and 136,
respectively). These oligonucleosides are preferred embodiments of
the invention for modulating rat JNK2 expression. Oligonucleotides
showing levels of inhibition of rat JNK1 mRNAs by at least 80% in
this assay include ISIS Nos. 18254, 18255, 18258 and 18259 (SEQ ID
NOS:125, 126, 129 and 130, respectively). These oligonucleotides
are thus more preferred embodiments of the invention for modulating
rat JNK2 expression. ISIS 18259 (SEQ ID NO:130) was chosen for use
in further studies (infra).
[0365] Dose Response: A dose response study was conducted using
oligonucleotides targeted to rat JNK1 (ISIS 21859; SEQ ID NO:113)
and JNK2 (ISIS 18259; SEQ ID NO:130) and Northern assays. The
results (Table 21) demonstrate an increasing effect as the
oligonucleotide concentration is raised and confirm that ISIS Nos.
21859 and 18259 (SEQ ID NOS:113 and 130, respectively) specifically
modulates levels of mRNA encoding JNK1 and JNK2, respectively.
TABLE-US-00021 TABLE 21 Dose-Dependent Response to Rat JNK
Antisense Oligonucleotides (ASOs) SEQ % EXPRES- % EXPRES- ID SION
SION ISIS # NO: ASODescription Dose JNK1 JNK2 21859 113 rat JNK1 0
nM 100 100 active ASO 10 nM 74 101 50 nM 25 98 100 nM 11 99 200 nM
8 101 18259 130 rat JNK2 0 nM 100 100 active ASO 10 nM 95 81 50 nM
101 35 100 nM 94 15 200 nM 89 5
[0366] JNK3: Table 22 describes the sequences and structures of a
set of oligonucleotides, ISIS Nos. 21899 to 21912 (SEQ ID NOS:142
to 155, respectively) that were designed to be specifically
hybridizable to nucleic acids from Rattus norvegicus that encode a
stress-activated protein kinase named "p54B" that is homologous to
the human protein JNK3 (Kyriakis et al., Nature, 1994, 369, 156;
GenBank accession No. L27128 (SEQ ID NO: 99), locus name
"RATSAPKC"). In Table 22, emboldened residues are
2'-methoxyethoxy-residues (others are 2'-deoxy-); "C" residues are
2'-methoxyethoxy-5-methyl-cytosines and "C" residues are
5-methyl-cytosines; "o" indicates a phosphodiester linkage; and "s"
indicates a phosphorothioate linkage. The target gene co-ordinates
are from GenBank Accession No. L27128 (SEQ ID NO: 99), locus name
"RATSAPKC." The oligonucleotides are tested for their ability to
modulate rat JNK3 mRNA levels essentially according to the methods
described in the preceding Examples.
[0367] In addition to being specifically hybridizable to nucleic
acids encoding rat JNK3, some of the oligonucleotides described in
Table 22 are also specifically hybridizable with JNK3-encoding
nucleic acids from humans and Mus musculus (mouse). Table 23 sets
out these relationships. These oligonucleotides are tested for
their ability to modulate mRNA levels of the human JNK genes
according to the methods described in Example 5.
TABLE-US-00022 TABLE 22 Nucleotide Sequences of Rat JNK3
Oligonucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE
SEQUENCE ID CO- TARGET NO. (5' -> 3') NO: ORDINATES REGION 21899
GoGoGoCoTsTsTsCsAsTsTsAs 142 0021-0040 5'-UTR GsCsCsAoCoAoToT 21900
GoGoToToGsGsTsTsCsAsCsTs 143 0241-0260 5'-UTR GsCsAsGoToAoGoT 21901
ToGoCoToCsAsTsGsTsTsGsTs 144 0351-0370 tIR AsAsTsGoToToToG 21902
GoToCoGoAsGsGsAsCsAsGsCs 145 0491-0510 ORF GsTsCsAoToAoCoG 21903
CoGoAoCoAsTsCsCsGsCsTsCs 146 0731-0750 ORF GsTsGsGoToCoCoA 21904
AoCoAoToAsCsGsGsAsGsTsCs 147 0901-0920 ORF AsTsCsAoToGoAoA 21905
GoCoAoAoTsTsTsCsTsTsCsAsT 148 1101-1120 ORF sGsAsAoToToCoT 21906
ToCoGoToAsCsCsAsAsAsCsGs 149 1321-1340 ORF TsTsGsAoToGoToA 21907
CoGoCoCoGsAsGsGsCsTsTsCs 150 1601-1620 ORF CsAsGsGoCoToGoC 21908
GoGoCoToAsGsTsCsAsCsCsTs 151 1631-1650 tTR GsCsAsAoCoAoAoC 21909
GoCoGoToGsCsGsTsGsCsGsTs 152 1771-1790 3'-UTR GsCsTsToGoCoGoT 21910
GoCoToCoAsGsCsTsGsCsGsAs 153 1891-1910 3'-UTR TsAsCsAoGoAoAoC 21911
AoGoCoGoCsGsAsCsTsAsGsAs 154 1921-1940 3'-UTR AsGsTsToAoAoGoT 21912
AoGoGoGoAsGsAsCsCsAsAsAs 155 1941-1960 3'-UTR GsTsCsGoAoGoCoG
TABLE-US-00023 TABLE 23 Cross-Hybridizations of Rat JNK3
Oligonucleotides ISIS SEQ ID Hybridizes to: NO. NO: Human
JNK3a1.sup.1 Human JNK3a2.sup.2 MouseJNK3.sup.3 21900 143 -- -- bp
329-348 21901 144 bp 193-212 bp 169-188 bp 411-430 21904 147 -- --
bp 961-980 21905 148 bp 943-962 bp 919-938 -- 21906 149 -- -- bp
1381-1400 21908 151 bp 1478-1497 bp 1449-1468 bp 1696-1715
.sup.1GenBank accession No. U34820 (SEQ ID NO: 101), locus name
"HSU34820" (see also Mohit et al., Neuron, 1995, 14, 67 and Gupta
et al., EMBO Journal, 1996, 15, 2760). .sup.2GenBank accession No.
U34819 (SEQ ID NO: 102), locus name "HSU34819" (see also Gupta et
al., EMBO Journal, 1996, 15, 2760). .sup.3Also known as
p459.sup.3F12 MAPK; GenBank accession No. L35236 (SEQ ID NO: 100),
locus name "MUSMAPK" (see also Martin et al., Brain Res. Mol. Brain
Res., 1996, 35, 47).
[0368] Pan JNK Oligonucleotides: Certain of the oligonucleotides of
the invention are capable of modulating two or more JNK proteins
and are referred to herein as "Pan JNK" oligonucleotides. For
example, ISIS Nos. 21861 and 21867 (SEQ ID NOS:115 and 121,
respectively) demonstrated a capacity to modulate both JNK1 and
JNK2 (Table 18). Such oligonucleotides are useful when the
concomitant modulation of several JNK proteins is desired.
[0369] Human Pan JNK oligonucleotides are described in Table 24.
These oligonucleotides are designed to be complementary to
sequences that are identically conserved in (i.e., SEQ ID NOS:156,
158, 159, 160 and 161), or which occur with no more than a one-base
mismatch (SEQ ID NO:157), in nucleic acids encoding human JNK1a1,
JNK1a2, JNK2a1 and JNK2a2. The oligonucleotides described in Table
24 are evaluated for their
ability to modulate JNK1 and JNK2 mRNA levels in A549 cells using
the methods and assays described in Examples 3 and 4.
[0370] In instances where such common sequences encompass one or
more base differences between the JNK genes that it is desired to
modulate, hypoxanthine (inosine) can be incorporated at the
positions of the oligonucleotide corresponding to such base
differences. ("Hypoxanthine" is the art-accepted term for the base
that corresponds to the nucleoside inosine; however, the term
"inosine" is used herein in accordance with U.S. and PCT rules
regarding nucleotide sequences.) As is known in the art, inosine
(I) is capable of hydrogen bonding with a variety of nucleobases
and thus serves as a "universal" base for hybridization purposes.
For example, an oligonucleotide having a sequence that is a
derivative of SEQ ID NO:157 having one inosine substitution
(TAGGAIATTCTTTCATGATC, SEQ ID NO:162) is predicted to bind to
nucleic acids encoding human JNK1a1, JNK1a2, JNK2a1 and JNK2a2 with
no mismatched bases. As another example, an oligonucleotide having
a sequence that is a derivative of SEQ ID NO:161 having one inosine
substitution (GGTTGCAITTTCTTCATGAA, SEQ ID NO:163) is predicted to
bind with no mismatched bases to nucleic acids encoding human
JNK3a1 and JNK3a2 in addition to JNK1a1, JNK1a2, JNK2a1 and JNK2a2.
Such oligonucleotides are evaluated for their ability to modulate
JNK1 and JNK2 mRNA levels in A549 cells, and JNK3 mRNA levels in
SH-SY5Y cells, using the methods and assays described in Examples
3, 4 and 5.
TABLE-US-00024 TABLE 24 Human Pan INK Oligonucleotides NUCLEOTIDE
SEQUENCE (5' -> 3') SEQ AND CHEMICAL MODIFICATIONS* ID NO:
A.sup.SC.sup.SA.sup.ST.sup.SC.sup.ST.sup.ST.sup.OG.sup.OA.sup.OA.sup.OA.su-
p.OT.sup.OT.sup.OC.sup.ST.sup.ST.sup.SC.sup.ST.sup.SA.sup.SG 156
T.sup.SA.sup.SG.sup.SG.sup.SA.sup.ST.sup.SA.sup.OT.sup.OT.sup.OC.sup.OT.su-
p.OT.sup.OT.sup.OC.sup.SA.sup.ST.sup.SG.sup.SA.sup.ST.sup.SC 157
A.sup.SG.sup.SA.sup.SA.sup.SG.sup.SG.sup.ST.sup.OA.sup.OG.sup.OG.sup.OA.su-
p.OC.sup.OA.sup.OT.sup.ST.sup.SC.sup.ST.sup.ST.sup.ST.sup.SC 158
T.sup.ST.sup.ST.sup.SA.sup.ST.sup.ST.sup.SC.sup.OC.sup.OA.sup.OC.sup.OT.su-
p.OG.sup.OA.sup.OT.sup.SC.sup.SA.sup.SA.sup.ST.sup.SA.sup.ST 159
T.sup.SC.sup.SA.sup.SA.sup.ST.sup.SA.sup.SA.sup.OC.sup.OT.sup.OT.sup.OT.su-
p.OA.sup.OT.sup.OT.sup.SC.sup.SC.sup.SA.sup.SC.sup.ST.sup.SG 160
G.sup.SG.sup.ST.sup.ST.sup.SG.sup.SC.sup.SA.sup.OG.sup.OT.sup.OT.sup.OT.su-
p.OC.sup.OT.sup.OT.sup.SC.sup.SA.sup.ST.sup.SG.sup.SA.sup.SA 161
*Emboldened residues, 2'-methoxyethoxy-residues (others are
2'-deoxy-); all "C" residues are 5-methyl-cytosines; ".sup.O",
phosphodiester linkage; ".sup.S", phosphorothioate linkage.
Example 7
Effect of Oligonucleotides Targeted to Human JNK1 and JNK2 on
TNFa-induced JNK Activity
[0371] Human umbilical vein endothelial cells (HUVEC, Clonetics,
San Diego Calif.) were incubated with oligonucleotide with
LipofectinJ in Opti-MEMJ for 4 hours at 37.degree. C./5% CO.sub.2.
The medium was then replaced with 1% FBS/EGM (Clonetics,
Walkersville Md.) and incubated for 24 hours at 37.degree. C./5%
CO.sub.2. Cells were treated with 5 ng/ml TNFa for 15 minutes
before lysis. JNK activity was determined by incubating lysates
(normalized for protein) with immobilized GST-c-Jun fusion protein
(e.g., New England Biolabs, Beverly, Mass.)+.sup.32P-ATP. GST-c-Jun
beads were washed and SDS-PAGE sample buffer was added. Samples
were resolved by SDS-PAGE and phosphorylated c-Jun was visualized
using a Molecular Dynamics PhosphorImager.
[0372] Compared to a control oligonucleotide, the JNK1
oligonucleotide ISIS 15346 (SEQ ID NO: 16; 100 nM concentration)
inhibited TNFa-induced JNK activity by approximately 70%. The JNK2
oligonucleotide ISIS 15353 (SEQ ID NO: 31; 100 nM) inhibited
TNFa-induced JNK activity by approximately 55%. A combination of 50
nM each oligonucleotide inhibited TNFa-induced JNK activity by
approximately 68% and a combination of 100 nM each oligonucleotide
inhibited TNFa-induced JNK activity by approximately 83%.
Example 8
Inhibition of Inflammatory Responses by Antisense Oligonucleotides
Targeting JNK Family Members
[0373] JNKs have been implicated as key mediators of a variety of
cellular responses and pathologies. JNKs can be activated by
environmental stress, such as radiation, heat shock, osmotic shock,
or growth factor withdrawal as well as by pro-inflammatory
cytokines.
[0374] Antisense oligonucleotides targeting any of the JNK family
members described in Examples 3-5 are synthesized and purified as
in Example 1 and evaluated for their activity in inhibiting
inflammatory responses. Such inhibition is evident in the reduction
of production of pro-inflammatory molecules by inflammatory cells
or upon the attenuation of proliferation of infiltrating or
inflammatory cells, the most prominent of which are lymphocytes,
neutrophils, macrophages and monocytes. Following synthesis,
oligonucleotides are tested in an appropriate model system using
optimal tissue or cell culture conditions. Inflammatory cells
including lymphocytes, neutrophils, monocytes and macrophages are
treated with the antisense oligonucleotides by the method of
electroporation. Briefly, cells (5.times.10.sup.6 cells in PBS) are
transfected with oligonucleotides by electroporation at 200V, 1000
uF using a BTX Electro Cell Manipulator 600 (Genetronics, San
Diego, Calif.). For an initial screen, cells are electroporated
with 10 uM oligonucleotide and RNA is collected 24 hours later.
Controls without oligonucleotide are subjected to the same
electroporation conditions.
[0375] Total cellular RNA is then isolated using the RNEASY7 kit
(Qiagen, Santa Clarita, Calif.). RNAse protection experiments are
conducted using RIBOQUANT.TM. kits and template sets according to
the manufacturer's instructions (Phaimingen, San Diego,
Calif.).
[0376] Adherent cells such as endothelial and A549 cells are
transfected using the LIPOFECTIN.TM. protocol described in Example
2. Reduced JNK mRNA expression is measured by Northern analysis
while protein expression is measured by Western blot analysis, both
described in Example 1. Negative control oligonucleotides with
mismatch sequences are used to establish baselines and non-specific
effects.
[0377] The degree of inflammatory response is measured by
determining the levels of inflammatory cytokine expression by
Northern or Western analysis, or cytokine secretion by
enzyme-linked immunosorbent assay (ELISA) techniques. Enzyme-linked
immunosorbent assays (ELISA) are standard in the art and can be
found at, for example, Ausubel, F. M. et al., Current Protocols in
Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley &
Sons, Inc., 1991.
[0378] The degree of inflammatory response is also determined by
measuring the expression of known immediate-early genes by the
method of Northern or Western blot analysis. Further into the
inflammatory response, levels of apoptosis are measured by flow
cytometry.
Example 9
Inhibition of Fibrosis by Antisense Oligonucleotides Targeting JNK
Family Members
[0379] Pulmonary fibrosis is characterized by inflammatory and
fibroproliferative changes in the lung and an excess accumulation
of collagen in the interstitium. There is also an increased
recruitment of immune and inflammatory cells to the lung which act
not only in the initial damage to the lung but in the progression
of the fibrotic process.
[0380] In the rodent bleomycin (BL)-induced pulmonary fibrosis
model, inhibition of fibrosis in the lung is determined by
measuring any of several markers for the condition. The BL-induced
model is widely accepted in the art and can be found at, for
example, Thrall, R. S. et al., Bleomycin In: Pulmonary Fibrosis,
pp. 777-836, Eds. Phan, S. H. and Thrall, R. S., Marcel Dekker, New
York, 1995 and Giri, S, N. et al., Miscellaneous mediator systems
in pulmonary fibrosis In: Pulmonary Fibrosis, pp. 231-292, Eds.
Phan, S. H. and Thrall, R. S., Marcel Dekker, New York, 1995.
[0381] Antisense oligonucleotides targeting any of the JNK family
members described in Examples 3-5 are synthesized and purified as
in Example 1 and evaluated for their ability to prevent or inhibit
pulmonary fibrosis. These fibrotic markers include release of
various pro-inflammatory mediators including cytokines and
chemokines such as TNFa, interleukin-8 and interleukin-6, increased
numbers of proteases and metalloproteinases, generation of reactive
oxygen species (ROS), edema, hemorrhage and cellular infiltration
predominated by neutrophils and macrophages.
[0382] Following synthesis, oligonucleotides are tested in the
rodent BL-induced pulmonary fibrosis model using optimal
conditions. Mice receive an intratracheal dose of bleomycin (0.125
U/mouse) or saline, followed by treatment with antisense
oligonucleotide (i.p.) over 2 weeks. After 2 weeks mice are
sacrificed and biochemical, histopathological and
immunohistochemical analyses are performed.
[0383] Biochemical and immunohistochemical analysis involves the
measurement of the levels of pro-inflammatory cytokine expression
by Northern or Western analysis, or cytokine secretion by
enzyme-linked immunosorbent assay (ELISA) techniques as described
in Example 8. Histopathological analyses are performed for the
presence of fibrotic lesions in the BL-treated lungs and for the
presence of and number of cells with the fibrotic phenotype by
methods which are standard in the art.
Example 10
Sensitization to Chemotherapeutic Agents by Antisense
Oligonucleotides Targeting JNK Family Members
[0384] Manipulation of cancer chemotherapeutic drug resistance can
also be accomplished using antisense oligonucleotides targeting JNK
family members. Antisense oligonucleotides targeting any of the JNK
family members described in Examples 3-5 are synthesized and
purified as in Example 1 and evaluated for their ability to
sensitize cells to the effects of chemotherapeutic agents.
Sensitization is evident in the increased number of target cells
undergoing apoptosis subsequent to treatment. Following synthesis,
oligonucleotides are tested in an appropriate model system using
optimal tissue or cell culture conditions. Cells are treated with
the compounds of the invention in conjunction with one or more
chemotherapeutic agents in a treatment regimen wherein the
chemotherapeutic agents can be used individually (e.g., 5-FU and
oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for
a period of time followed by MTX and oligonucleotide), or in
combination with one or more other such chemotherapeutic agents
(e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and
oligonucleotide).
[0385] For nonadherent cells, treatment is by the method of
electroporation. Briefly, cells (5.times.10.sup.6 cells in PBS) are
transfected with oligonucleotides by electroporation either before,
during or after treament with the chemotherapeutic agent, at 200V,
1000 uF using a BTX Electro Cell Manipulator 600 (Genetronics, San
Diego, Calif.). For an initial screen, cells are electroporated
with 10 uM oligonucleotide and RNA is collected 24 hours later.
Controls without oligonucleotide or chemotherapeutic agent are
subjected to the same electroporation conditions.
[0386] Total cellular RNA is then isolated using the RNEASY7 kit
(Qiagen, Santa Clarita, Calif.). RNAse protection experiments are
conducted using RIBOQUANT.TM. kits and template sets according to
the manufacturer's instructions (Pharmingen, San Diego,
Calif.).
[0387] Adherent cells such as endothelial and A549 cells are
transfected using the LIPOFECTIN.TM. protocol described in Example
2. Reduced JNK mRNA expression is measured by Northern analysis
while protein expression is measured by Western blot analysis, both
described in Example 1. Negative control oligonucleotides with
mismatch sequences can be used to establish baselines and
non-specific effects. The degree of apoptosis, and consequently
sensitization is measured by flow cytometry.
Example 11
Oligonucleotide-Mediated Inhibition of Human JNK2 Expression Using
a Cross-Species Oligonucleotide, ISIS 101759
[0388] In a further embodiment, chemical modifications to ISIS
18259 (SEQ ID NO: 130), designed to the rat JNK2 target were made
and the oligonucleotide was investigated for activity in human cell
lines.
[0389] The modified oligonucleotide, ISIS 101759, has identical
base and sugar compositions as ISIS 18259 and differs only in the
linker composition. ISIS 101759 contains phosphorothioate linkages
throughout. A comparison of the two oligonucleotides is shown
below.
TABLE-US-00025 "GoCoToCoAsGsTsGsGsAsCsAsTsGsGsAoToGoAoG" ISIS 18259
"GsCsTsCsAsGsTsGsGsAsCsAsTsGsGsAsTsGsAsG" ISIS 101759
[0390] Both oligonucleotides have the following base sequence
5'-GCTCAGTGGACATGGATGAG-3' and emboldened residues are
2'-methoxyethoxy-residues (others are 2'-deoxy-); "C" residues are
2'-methoxyethoxy-5-methyl-cytosines and "C" residues are
5-methyl-cytosines; "o" indicates a phosphodiester linkage; and "s"
indicates a phosphorothioate linkage.
[0391] While ISIS 18259 was designed to target gene co-ordinates
1201-1220 from GenBank Accession No. L27112 (SEQ ID NO: 93) (herein
incorporated as SEQ ID NO: 168), locus name "RATSAPKB as dileneated
in Table 19, this same sequence is also complementary over 18 of
its 20 nucleobases to coordinates 1248-1265 of human JNK2 from
GenBank accession No. L31951 (SEQ ID NO: 92) (herein incorporated
as SEQ ID NO: 167), locus name "HUMJNK2". The region of
complementarity between ISIS 18259 (and consequently 101759 since
it has the same base sequence as ISIS 18259) and the human gene is
shown here in bold, 5'-GCTCAGTGGACATGGATGAG-3'. In fact it is only
the two nucleobases at the 3' end of the oligonucleotide that are
not complementary to the human JNK2 gene.
[0392] Using three human cell lines, ISIS 101759 (SEQ ID NO: 130)
was tested for its ability to reduce human JNK2 RNA levels. The
control oligonucleotide for the three studies was ISIS 101760 (SEQ
ID NO: 166; a 7-base mismatch). The control oligonucleotide has the
same sugar and linker sequence as ISIS 101759 and the nucleobase
sequence, 5'GsCsAsCsAsTsTsGsCsAsCsGsTsGsAsAsTsTsAsC-3', where
emboldened residues are T-methoxyethoxy-residues (others are
2'-deoxy-); "C" residues are 2'-methoxyethoxy-5-methyl-cytosines
and "C" residues are 5-methyl-cytosines; and "s" indicates a
phosphorothioate linkage.
Inhibition of Human JNK2 in HuVEC Cells
HuVEC Cells:
[0393] The human umbilical vein endothilial cell line HuVEC was
obtained from Clonetics (Clonetics Corporation Walkersville, Md.).
HuVEC cells were routinely cultured in EBM (Clonetics Corporation
Walkersville, Md.) supplemented with SingleQuots supplements
(Clonetics Corporation, Walkersville, Md.). Cells were routinely
passaged by trypsinization and dilution when they reached 90%
confluence were maintained for up to 15 passages. Cells were seeded
into 100 mm dishes and incubated overnight at 37.degree. C./5%
CO.sub.2. (Falcon-Primaria #3872).
[0394] For Northern blotting or other analyses, cells can be seeded
onto 100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and oligonucleotide.
Treatment of HuVEC cells with antisense compounds:
[0395] When cells reached 70% confluency, they were treated with
oligonucleotide. For cells grown in 10 cm dishes, cells were washed
once with 5 ml PBS and then treated with 5 ml of OPTI-MEM-1
containing 3 ul LIPOFECTIN (Invitrogen Corporation, Carlsbad,
Calif.)/100 nM oligonucleotide/ml OPTI-MEM-1. For other
oligonucleotide concentrations the oligonucleotide/Lipofectin
ration was held constant. After 4-7 hours of treatment, the medium
was replaced with fresh medium. Cells were harvested 16-24 hours
after oligonucleotide treatment.
[0396] In accordance with the present invention, HuVEC cells were
treated with 100 nM ISIS 101759 or the control oligonucleotide and
mRNA levels of human JNK2 were monitored over a time-course of 0-72
hours and quantitated by Northern analysis. The data is shown in
Table 25.
TABLE-US-00026 TABLE 25 Time-course Response to Rat JNK2 Antisense
Oligonucleotides (ASOs) in HuVEC cells Percent Inhibition of human
JNK2 mRNA Expression ISIS Number 0 hr 12 hr 24 hr 48 hr 72 hr
Control 0 6 7 23 16 101759 0 93 92 88 70
[0397] From the data, it is evident that the rat JNK2
oligonucleotide was capable of reducing the expression of human
JNK2 in human HuVEC cells, and that by 72 hours the expression
began to recover.
Inhibition of Human JNK2 in HeLa Cells
HeLa Cells:
[0398] The human cervix epithelial adenocarcinoma cell line HeLa
was obtained from the American Type Culture Collection (Manassas,
Va.). HeLa cells were routinely cultured in Minimum essential
medium (Eagle) with 2 mM L-glutamine and Earle's BSS adjusted to
contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino
acids, and 1.0 mM sodium pyruvate, 90%; fetal bovine serum, 10% at
a temperature of 37.degree. C. Cells were seeded into 100 mm dishes
and incubated overnight at 37.degree. C./5% CO.sub.2
[0399] For Northern blotting or other analyses, cells can be seeded
onto 100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and
oligonucleotide.
[0400] Treatment of HeLa Cells with Antisense Compounds:
When cells reached 70% confluency, they were treated with
oligonucleotide. For cells grown in 10 cm dishes, cells were washed
once with 5 ml PBS and then treated with 5 ml of OPTI-MEM-1
containing 3 ul LIPOFECTIN (Invitrogen Corporation, Carlsbad,
Calif.)/100 nM oligonucleotide/ml OPTI-MEM-1. For other
oligonucleotide concentrations the oligonucleotide/Lipofectin
ration was held constant. After 4-7 hours of treatment, the medium
was replaced with fresh medium. Cells were harvested 16-24 hours
after oligonucleotide treatment.
[0401] In accordance with the present invention, HeLa cells were
treated with 10, 50 or 200 nM ISIS 101759 or the control
oligonucleotide and mRNA levels of human JNK2 were quantitated by
Northern analysis. The data is shown in Table 26.
TABLE-US-00027 TABLE 26 Dose Response to Rat JNK2 Antisense
Oligonucleotides (ASOs) in HeLa cells Percent Inhibition of human
JNK2 mRNA ISIS No: 10 nM 50 nM 200 nM Control 0 0 1 101759 0 90
99
[0402] From the data, it is evident that the rat JNK2
oligonucleotide was capable of reducing the expression of human
JNK2 in human HeLa cells in a dose-dependent manner. HeLa cells
were also treated with the transfection reagent, lipofectamine,
alone at 50 and 200 nM with no reduction in expression being
observed.
Inhibition of Human JNK2 in Jurkat Cells
Jurkat Cells:
[0403] The human Jurkat cell line was obtained from the American
Type Culture Collection (ATCC) (Manassas, Va.). Jurkat cells were
routinely cultured in RPMI Medium 1640 (Gibco/Life Technologies,
Gaithersburg, Md.) supplemented with 20% fetal calf serum
(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely
passaged by aspirating media that contained excess cells and
replenishing with new media.
[0404] For electroporation, cells were diluted to 28.times.10.sup.6
cells/mL and placed into 1 mm electroporation cuvettes.
Electroporation is performed by treating with 1-20 .mu.M
oligonucleotide, at 160 Volts for 6 msec. The entire electroporated
samples are then placed into 5 mL of 10% FBS/RPMI Medium 1640 in
100 mm plates. Plates are then left overnight at 37.degree. C./5%
CO.sub.2.
[0405] Each sample is then transferred to 15 mL conical tubes and
spun down at 1200 rpm for 5 minutes followed by aspiration of the
supernatant. Cells are then suspended in 5 mL PBS followed by a
second centrifugation at 1200 rpm for 5 minutes followed by
aspiration of the supernatant. Cells are then washed and lysed.
Following the lysis step, total cellular RNA is then isolated using
the RNEASY kit (Qiagen, Santa Clarita, Calif.) as described in
other examples herein.
[0406] In accordance with the present invention, Jurkat cells were
treated by electroporation with 1, 5 or 20 uM ISIS 101759 or the
control oligonucleotide and mRNA levels of human JNK2 were
quantitated by Northern analysis. The data is shown in Table
27.
TABLE-US-00028 TABLE 27 Dose Response to Rat JNK2 Antisense
Oligonucleotides (ASOs) in Jurkat cells Percent Inhibition of human
JNK2 mRNA ISIS No: 1 uM 5 uM 20 uM Control 12 18 19 101759 14 56
92
[0407] From the data, it is evident that the rat JNK2
oligonucleotide was capable of reducing the expression of human
JNK2 in human Jurkat cells in a dose-dependent manner. Jurkat cells
were also electroporated with reagents alone (no oligonucleotides)
with no reduction in expression being observed.
Targeting JNK1 for Metabolic Disorders
Example 12
In Vivo Studies in an ob/ob Model of Obesity
[0408] Leptin is a hormone produced by fat that regulates appetite.
Deficiencies in this hormone in both humans and non-human animals
leads to obesity. ob/ob mice have a mutation in the leptin gene
which results in obesity and hyperglycemia. As such, these mice are
a useful model for the investigation of obesity and metabolic
syndrome and treatments designed to treat these conditions. ob/ob
mice have higher circulating levels of insulin and are less
hyperglycemic than db/db mice, which harbor a mutation in the
leptin receptor. In accordance with the present invention, the
oligomeric compounds of the invention are tested in the ob/ob model
of obesity and diabetes.
[0409] Seven-week old male C57Bl/6J-Lep ob/ob mice (Jackson
Laboratory, Bar Harbor, Me.) were divided into a saline group
(n=6), an oligonucleotide control group (n=6) and a treatment group
(n=6). All animals were fed a diet with a fat content of 10-15% for
6 weeks. The oligonucleotide control group and the treatment group
received a 25 mg/kg subcutaneous injection of either the control
oligonucleotide or the treatment oligonucleotide twice a week for
the 6 week perion. The saline group received a saline injection on
the same injection schedule. The control oligonucleotide in this
study was a chimeric oligonucleotide that is 20 nucleosides in
length and is not targeted to a nucleic acid encoding JNK.
(CCTTCCCTGAAGGTTCCTCC, SEQ ID NO: 107, (Isis No. 141923). The
treatment oligonucleotide is also a chimeric oligonucleotide that
is 20 nucleotides in length, but it is targeted to a nucleic acid
that encodes JNK1 polypeptide. The nucleic acid encoding JNK1
polypeptide has a nucleoside sequence that is substantially similar
to GenBank Accession No.: L27129 (SEQ ID NO: 88). 1; SEQ ID NO.:
88. The oligonucleotide compound is also targeted to a nucleic acid
that encodes mouse JNK1 polypeptide with only a 1-nucleobase
mismatch. The nucleic acid preferably being substantially similar
to GenBank Accession No.: NM.sub.--016700.2; SEQ ID NO. 106. The
treatment oligo contains the nucleobase sequence of
TGTTGTCACGTTTACTTCTG, SEQ ID NO.: 114 (Isis No. 104492). The
oligonucleotide compound is also targeted to a nucleic acid that
encodes human JNK1 polypeptide with only a 2-nucleobase mismatch.
The nucleic acid preferably being substantially similar to GenBank
Accession NOs.: L26318 (SEQ ID NO: 87), U34822 (SEQ ID NO: 89),
U35004 (SEQ ID NO: 90) or U35005 (SEQ ID NO: 91); SEQ ID NOs.: 87,
89, 90, and 91 respectively.
[0410] During the treatment period, weekly food intake was
monitored, as were changes in body weight. Body composition, blood
biochemistry, metabolic rate, insulin tolerance, and oral glucose
tolerance was also measured at certain time points during the
treatment period. After the treatment period, mice were sacrificed
and target mRNA levels were evaluated in liver, brown adipose
tissue (BAT) and white adipose tissue (WAT), as were histological,
biochemical and molecular biology parameters. RNA isolation and
target mRNA expression level quantitation are performed as
described by other examples herein unless otherwise stated.
mRNA Levels
[0411] Total RNA was isolated by homogenizing tissues in RLT buffer
(Qiagen, Md.) followed by centrifugation with cesium chloride
gradient. Real-time quantitative RT-PCR analysis was then performed
to analyze the gene expression.
[0412] JNK1, but not JNK 2 mRNA levels were reduced in the liver,
white adipose tissue and brown adipiose tissue for the treatment
group compared to saline and oligonucleotide control groups(80%
reduction in liver, 80% reduction in WAT and 78% reduction in BAT).
There were no significant differences in JNK1 mRNA levels between
the saline and oligonucleotide control groups. Additionally, there
were no significant differences in JNK2 mRNA levels between
treatment, saline and oligonucleotide control groups. Thus, the
treatment compound is specific for JNK1 over JNK2 in reducing mRNA
expression.
JNK1 Activity Assay and Western Immunoblotting Analysis.
[0413] There was a corresponding reduction in JNK activity as
determined by immunoprecipitation using an antibody raised to JNK1
(Cell Signaling, Beverly, Mass.). JNK1 ASO resulted in a decrease
in JNK1 activity by greater than 95%, 80% and 65% in liver, WAT and
BAT, respectively.
Feed Efficiency, Body Weight and Fat
[0414] As compared to control groups, the treatment group had an
improved feed efficiency (change in body weight per volume of food
intake). The treatment group feed efficiency was 0.052.+-.0.0026
and the control oligonucleotide group was 0.060.+-.0.002. The
treatment group also showed a reduction in body weight gain by
about 20%, indicating an increased metabolic rate. Epididymal fat
pad weight (3.8 g v. 4.5 g) and whole body fat content (31.6% v.
35.5%) were also reduced. Indirect calorimetry measurement
confirmed that the treatment group had an increased metabolic rate
as reflected in a higher VO.sub.2 as compared to the
oligonucleotide control group (VO.sub.2 increase over control group
of >7% (greater than 7%) in the dark and 23% or greater in the
light).
Glucose and Insulin Levels
[0415] Fed and fasting plasma glucose and plasma insulin levels
were improved for the treatment group over the control group. (see
Table 28). Glucose levels were completely normalized and insulin
levels were lowered by greater than 50% after 6 weeks of treatment
(Table 28) demonstrating increased insulin sensitazation. Plasma
glucose levels are measured using an Olympus Clinical Analysis
(Olympus AU400, Olympus American Inc, Melville, N.Y.) and insulin
levels are measure using an Alpco insulin-specific ELISA kit from
(Windham, N.H.).
TABLE-US-00029 TABLE 28 Plasma Glucose and Insulin Levels in ob/ob
Mice Saline Control ISIS 104492 Glucose Baseline 378.3 .+-. 33.0
374.2 .+-. 17.2 375.2 .+-. 33.7 (mg/dl) Fed 600.5 .+-. 48.7 445.5
.+-. 57.4 177.6 .+-. 12.3** Fasting 142.7 .+-. 11.8 152.3 .+-. 23.5
90.6 .+-. 10.7* Insulin Baseline 30.2 .+-. 4.1 30.6 .+-. 3.7 29.8
.+-. 1.8 (ng/ml) Fed 24.4 .+-. 3.4 26.5 .+-. 5.8 11.0 .+-. 4.8*
Fasting 17.6 .+-. 1.8 16.9 .+-. 3.4 7.9 .+-. 0.4** Data are
expressed as the mean .+-. SEM (n = 5-6). *P < 0.05 and **P <
0.01 when compared to either control group.
[0416] To confirm this ASO-caused insulin-sensitizing effect, both
Glucose (OGTT) and insulin tolerance tests (ITT) were administered
in fed and fasted mice. Mice receive intraperitoneal injections of
either glucose or insulin, and the blood glucose and insulin levels
are measured before the insulin or glucose challenge and at 15, 20
or 30 minute intervals for up to 2 hours. Blood glucose levels were
measured using a Glucometer (Abbott Laboratories, Bedford,
Mass.).
[0417] Insulin tolerance and oral glucose tolerance was improved
for the treatment group compared to the control groups. A glucose
tolerance test in medical practice is the administration of glucose
to determine how quickly it is cleared from the blood and is used
to test for diabetes, insulin resistance, and sometimes reactive
hypoglycemia. The results of an oral glucose tolerance test of the
mice of Example 12 are shown in Table 29.
TABLE-US-00030 TABLE 29 OGTT performed at 6 weeks (0.75 g/kg
Glucose) Glucose mg/dL 0 min 30 min 60 min 100 min Saline 150 425
320 375 Control 150 420 325 300 104492 100 245 210 225
[0418] In response to glucose challenge, animals treated with JNK
antisense oligonucleotide show improved glucose tolerance. Peak
plasma glucose level at the 30 minute time point was decreased by
over 40% from controls and the subsequent drop in glucose was
lessened compared to controls. The AUC for glucose excursion was
significantly lowered after treatment with JNK antisense
oligonucleotide, indicating that inhibition of JNK by antisense
improves glucose tolerance. The results indicate that glucose is
cleared much more quickly from the blood of mice treated with JNK
antisense oligonucleotide relative to the control groups. In
addition, a markedly lower level of plasma insulin was observed
during OGTT in the ASO treatment group versus controls (5 ng/ml JNK
ASO treated vs 23 ng/ml saline treated).
[0419] An insulin tolerance test was also completed. There was an
increase in rate and magnitude of glucose lowering after injecting
insulin in the animals treated with JNK antisense oligonucleotide.
AUC is reduced by about 50% by administration of JNK antisense
oligonucleotide compared to saline treated control. These data
demonstrate that reduction of JNK1 expression with JNK1 ASO
significantly improved insulin sensitivity.
[0420] These date indicate that inhibition of JNK by antisense
improves glucose tolerance and insulin sensitivity and, therefore,
JNK1 antisense oligonucleotides are useful for treating, preventing
and/or ameliorating disorders of or associated with glucose
intolerance and/or insulin resistance, such as, for example,
obesity, metabolic syndrome, diabetes, and hyperglycemia.
Liver Steatosis
[0421] To examine if the ASO treatment improved liver steatosis,
both liver TG content and histology were analyzed.
[0422] Liver TG content was found to be greater than 40% lower in
JNK ASO-treated group than in controls in ob/ob mice (120 mg/g vs
200 mg/g). Histological examinations with both H&E staining and
oil-red O staining confirmed a significant improvement in liver
steatosis in JNK1 ASO treated mice (much smaller and fewer fat
droplets than those in controls). In addition, the histological
examination did not reveal any sign of ASO-related liver damage.
Rather, improved liver steatosis was accompanied by improved liver
function, as assessed by plasma ALT and AST measurements
(133.2.+-.10.1 U/L ALT vs 311.5.+-.21.1 and 113.2.+-.8.2 U/L AST vs
187.8.+-.14.6).
[0423] In addition to Liver steatosis, plasma transaminase levels
and plasma cholesterol levels were improved for the treatment
groups over the control group.
[0424] As compared to controls, treatment with JNK1 ASO for 6 weeks
lowered plasma total cholesterol levels by 40% in ob/ob mice in the
fed state. Lipoprotein profile analysis confirmed that JNK1 ASO
treatment lowered the cholesterol content in all three major
lipoprotein fractions, namely VLDL-, LDL- and HDL-cholesterol.
[0425] Plasma triglycerides, total cholesterol, HDL-cholesterol,
LDL-cholesterol, free fatty acids and transaminases are measured by
routine clinical analyzer instruments (e.g. Olympus Clinical
Analyzer, Melville, N.Y.). Tissue triglyceride levels are measured
using a Triglyceride GPO Assay from Roche Diagnostics
(Indianapolis, Ind.). Liver triglyceride levels are used to assess
hepatic steatosis, or clearing of lipids from the liver.
[0426] Hepatic steatosis is also assessed by routine histological
analysis of frozen liver tissue sections stained with oil red O
stain, which is commonly used to visualize lipid deposits, and
counterstained with hematoxylin and eosin, to visualize nuclei and
cytoplasm, respectively. For H&E staining, liver, epididymal
WAT and intrascapular BAT samples from ob/ob mice were fixed in 10%
buffered formalin and embedded in paraffin wax. For oil-red O
staining, liver samples were collected in embedding medium.
Multiple adjacent 4-.mu.m sections were cut and mounted on glass
slides. After dehydration, the sections were stained. Images of the
histological sections were analyzed.
Metabolic Gene Expression
[0427] The ob/ob mice that received treatment were further
evaluated at the end of the treatment period for the effects of
target inhibition on the expression genes that participate in lipid
metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty
acid storage, gluconeogenesis and glucose metabolism. Briefly, mRNA
levels in liver and white and brown adipose tissue were quantitated
by real-time PCR as described in other examples herein, employing
primer-probe sets that are generated using published sequences of
each gene of interest. The observations showed 1) increased mRNA
levels of adrenoceptor .beta.3 by >2-fold and UCP1 mRNA by
>1.2-fold in BAT; 2) reduced mRNA levels of ACC1, ACC2, FAS,
SCD1, DGAT-1 and DGAT-2 by 30-60% in WAT; and 3) reduced mRNA
levels of ACC1, FAS and G6Pase by >55%, and increased mRNA
levels of both UCP2 and PPAR.alpha. by >2-fold in liver (see
Table 35).
[0428] These data indicate that specific reduction of JNK1
expression with ASOs results in increased fuel combustion and
decreased lipogenesis in this model. Thus, JNK1 appears to play an
important role in whole body metabolism and therapeutic inhibition
of JNK1 in major metabolic tissues could provide clinical benefit
for obesity and metabolic syndrome.
Example 13
In vivo Studies in a Diet-Induced Model of Obesity (DIO)
[0429] To further confirm the metabolic effects of antisense
suppression of JNK1 expression, DIO mice were also treated with
JNK1 antisense oligonucleotides.
[0430] 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 JNK1 antisense
oligonucleotides on mRNA expression in a model of diet-induced
obesity.
[0431] Male C57BL/6J mice at 6 weeks of age were fed a diet
containing 58 kcal % fat (Research Diet D12330, Research Diets
Inc., New Brunswick, N.J.) for 15 weeks to induce obesity and
insulin resistance. The animals were then divided into different
groups (n=6) and treated with JNK1 ASO or control ASO at a dose of
25 mg/kg BW or with saline twice a week for 7 weeks. Two JNK1
treatment oligonucleotides targeting two different region of JNK1
mRNA were used. The treatment oligonucleotides share the nucleobase
sequence of SEQ ID NO.: 114, Isis No. 104492, or
TGTTGTCACGTTTGCTTCTG, SEQ ID NO: 108, ISIS NO. 256463. Each were
given at the same dose. The control oligonucleotide is the same as
described above. The treatment oligonucleotides are chimeric
oligonucleotide 20 nucleotides in length, targeted to a nucleic
acid that encodes JNK1 polypeptide. The nucleic acid encoding JNK1
polypeptide has a nucleoside sequence that is substantially similar
to SEQ ID NO 87, 89, 90, 91.
[0432] During the treatment, weekly food intake and BW were
monitored, and body composition and other metabolic measurements
were conducted (see below). At the end of the studies, animals were
sacrificed. Blood samples were collected by cardiac puncture, and
tissues were dissected, weighed, and then saved for further
analysis.
mRNA Levels
[0433] Total RNA was isolated by homogenizing tissues in RLT buffer
(Qiagen, Md.) followed by centrifugation with cesium chloride
gradient. Real-time quantitative RT-PCR analysis was then performed
to analyze the gene expression. In DIO mice, treatment with 104492
reduced JNK1 mRNA by 78%, 66% and 70% in liver, WAT and BAT,
respectively. Treatment with ISIS 256463 caused similar reduction
of JNK1 expression in these tissues.
Plasma Glucose and Insulin Levels
[0434] Plasma insulin was measured with an insulin ELISA kit (ALPCO
Diagnostics, Windham, N.H.). Plasma glucose were measured with a
biochemistry analyzer (Olympus AU400, Olympus American Inc,
Melville, N.Y.).
[0435] Treatment with JNK1 ASO lowered plasma glucose and insulin
levels in both fed and fasted states when compared to controls (See
Table 30). Treatment resulted in complete normalization of glucose
levels and insulin levels in DIO mice confirming the improved
insulin sensitivity shown in the ob/ob model.
TABLE-US-00031 TABLE 30 Plasma Glucose and Insulin Levels in DIO
Mice Saline Control ISIS 104492 Glucose Baseline 207.2 .+-. 8.2
200.8 .+-. 9.7 206.3 .+-. 7.8 (mg/dl) Fed 193.5 .+-. 9.8 189.0 .+-.
6.5 174.0 .+-. 4.2* Fasting 141.0 .+-. 8.6 124.3 .+-. 13.0 76.5
.+-. 6.4** Insulin Baseline 2.23 .+-. 0.13 2.41 .+-. 0.34 2.26 .+-.
0.12 (ng/ml) Fed 2.51 .+-. 0.12 2.17 .+-. 0.39 0.75 .+-. 0.11**
Fasting 1.30 .+-. 0.50 0.92 .+-. 0.21 0.48 .+-. 0.05** Data are
expressed as the mean .+-. SEM (n = 5-6). *P < 0.05 and **P <
0.01 when compared to either control group.
Improved Hepatic Steatosis
[0436] Plasma transaminase (AST and ALT) activities were measured
with a biochemistry analyzer (Olympus AU400, Olympus American Inc,
Melville, N.Y.). Liver triglycerides (TG) was measured as
previously described (Desai et al., 2001).
[0437] JNK1 ASO treatment lowered liver TG content by greater than
40% in DIO mice without causing liver toxicity, as assessed by
plasma ALT and ASL activities.
Plasma Cholesterol Levels
[0438] Total cholesterol and FFA concentrations were measured with
a biochemistry analyzer (Olympus AU400, Olympus American Inc,
Melville, N.Y.).
[0439] As compared to controls, treatment with JNK1 ASO for 6 weeks
lowered plasma total cholesterol levels by about 35% in DIO mice in
the fasted state.
[0440] To investigate whether lowered plasma cholesterol levels
with ASO treatment in the ob/ob and DIO models were caused by
reducing hepatic synthesis and secretion, mouse primary hepatocytes
were treated with JNK1 ASO and then de novo sterol synthesis was
determined by measuring the incorporation of [14C]-acetate into
sterols. JNK1 ASO transfected hepatocytes showed reduced de novo
sterol synthesis by 13% as compared to controls. Furthermore, gene
expression analysis found that JNK1 ASO-treated mice had
significantly lower hepatic ApoB100 mRNA levels versus controls.
Reduction of ApoB100 expression has been well demonstrated to
reduce plasma cholesterol levels in rodents and several other
species. Therefore, without being bound by any theory, decreased
plasma cholesterol levels can be at least in part due to decreased
hepatic cholesterol output.
Feed Efficiency, Body Weight and Fat
[0441] In DIO mice, treatment with either of the two JNK1
oligonucleotides did not result in a change in food intake compared
to controls. Treatment with JNK1 ASOs lowered BW by greater than
10%, which resulted in significant difference from the controls.
Both JNK1 ASO-treated groups also showed greater than 35% lower
epididymal fat depot weight and greater than 20% lower percentage
body fat content with no difference on lean body mass.
[0442] Metabolic rate was measured for a 24-h period using indirect
calorimetry (Oxymax System, Columbus Instruments, Columbus, Ohio).
JNK1 ASO-treated mice had higher VO.sub.2 than controls (about 12%
or greater in the dark and about 4% or greater in the light).
Expression of Metabolic Genes
[0443] The expression of representative metabolic genes in DIO mice
was analyzed. Similar changes as seen in ob/ob mice were founding
the DIO mouse model. Additionally, about a 70% increase in the
expression of both UCP2 and UCP3 in WAT was found in JNK1 treated
DIO mice versus controls (see Table 31), further indicating that
reduction of JNK1 expression not only inhibits lipogenesis but also
increases metabolic rate.
Decreased De Novo Fatty Acid and Sterol Synthesis and Increased
Fatty Acid Oxidation
[0444] De novo fatty acid and sterol synthesis in transfected mouse
hepatocytes were determined by measuring the incorporation of
[.sup.14C]acetate into fatty acids and sterols, respectively, as
previously described (Jiang et al., 2005; Yu et al., 2005). Fatty
acid oxidation was determined by measuring the oxidation of
[.sup.14C]oleate into acid soluble products and CO.sub.2 as
described (Choi et al., 2007; Savage et al., 2006; Yu et al., 1997;
Yu et al., 2005).
[0445] To confirm that the JNK1 ASO-caused changes in gene
expression translated into functional effects, cultured mouse
primary hepatocytes were transfected with JNK1 ASO and fatty acid
oxidation and de novo fatty acid synthesis were determined.
Consistent with the changes in gene expression seen in vivo, fatty
acid oxidation rate was about 35% higher or more whereas de novo
fatty acid synthesis was about 20% lower in JNK1 ASO transfected
cells than controls.
Improved Insulin Signaling
[0446] In support of the increased insulin sensitivity seen in the
insulin and glucose tolerance tests in Example 12 above and the
fed/fasted glucose and insulin measurements in both Example 12 and
13, mechanistic insulin signaling assays were performed. The
enhanced insulin sensitivity resulting from reduction of JNK1
expression was verified by analyzing the activities of some key
insulin signaling enzymes in both WAT and liver from DIO mice
(treated with JNK1 ASO or control ASO and challenged with
insulin).
[0447] DIO mice were treated with JNK1 ASO or control ASO at a dose
of 37.5 mg/kg BW twice a week for 3 weeks. The mice were then
fasted overnight and given a bolus i.p. injection of insulin at 2
U/kg BW or vehicle. The animals were then sacrificed, and liver and
epididymal WAT were collected and quickly frozen in liquid N.sub.2
for further analysis. Equal amount of total proteins contained in
pre-cleared fat or liver homogenates were separated on gradient
SDS-PAGE gels (BioRad, Hercules, Calif.) under reduced conditions
and then transferred onto PVDF membranes. The blots were then
incubated with primary antibody against Akt,
Serine473-phosphorylated Akt (pAkt.sup.Ser437) (Cell Signaling,
Danvers, Mass.), or Ser302/307-phosphorylated IRS1
(pIRS1.sup.Ser302/307) (Biosource, Camarillo, Calif.). Signals were
then detected by using HRP-conjugated secondary antibody and ECL
detection reagents (Amershan Biosciences).
[0448] A decreased level of pIRS.sup.Ser302/307 was found not only
under basal conditions (without insulin challenge) but also after
insulin challenge in both tissues from JNK1 ASO-treated mice versus
those from control ASO-treated mice. To evaluate whether decreased
pIRS.sup.Ser302/307 caused increased downstream insulin signaling
activity, the level of pAkt.sup.Ser473 in WAT was analyzed. A much
higher level of pAkt.sup.Ser473 was found in JNK1 ASO-treated mice
versus controls after insulin challenge although its basal level
was lower in the JNK1 ASO-treated mice; the latter was probably due
to the lower plasma insulin levels in these mice. These data
indicate that reduction of JNK1 expression with ASO improved
insulin signaling activity which supports at least in part the
increased insulin sensitivity detected in the tolerance tests and
fed/fasted glucose and insulin measurements.
Statistical Analysis
[0449] Values presented represent the mean.+-.SEM of three in vitro
or 5-6 in vivo independent measures per treatment. Statistical
difference between treatment groups was determined using one-way
ANOVA with Tukey HSD multiple comparisons or two-tailed student
t-test. P<0.05 was considered to be significant.
TABLE-US-00032 TABLE 31 Metabolic Gene Expression in ob/ob and DIO
Mice Liver WAT Gene saline control ASO JNK1 ASO saline control ASO
JNK1 ASO ACL 100.0 .+-. 11.0 90.2 .+-. 7.7 54.0 .+-. 6.6** ACC1
100.0 .+-. 11.0 81.2 .+-. 12 34.3 .+-. 1.2** 100.0 .+-. 4.4 101
.+-. 9.8 39.2 .+-. 2.1** ACC2 100.0 .+-. 11 100.7 .+-. 15.sup.
121.1 .+-. 15 100.0 .+-. 9.6 77.6 .+-. 7.9 38.1 .+-. 3.7** FAS
100.0 .+-. 13.2 79.4 .+-. 13.3 44.3 .+-. 3.2** 100.0 .+-. 4.1 110.7
.+-. 9.3 44.5 .+-. 3.7** Gyk 100.0 .+-. 6.2 106.3 .+-. 5.4 107.2
.+-. 6.4 100.0 .+-. 2.7 102.5 .+-. 4.6 65.8 .+-. 2.2** SCD1 100.0
.+-. 28.4 78.3 .+-. 17.4 134.6 .+-. 12.3 100.0 .+-. 5.9 80.8 .+-.
4.3 45.3 .+-. 2.6** DGAT1 100.0 .+-. 4.0 93.4 .+-. 2.3 113.4 .+-.
6.4 100.0 .+-. 3.1 90.3 .+-. 4.1 67.2 .+-. 5.1** DGAT2 100.0 .+-.
9.8 119.6 .+-. 6.2 128 .+-. 6.7.sup. 100.0 .+-. 4.1 87.6 .+-. 3.7
67.4 .+-. 2.7** HSL 100.0 .+-. 6.8 89.2 .+-. 6.2 91.1 .+-. 3.3 ATGL
100.0 .+-. 4.2 88.1 .+-. 4.8 79.9 .+-. 11.7 PPAR.alpha. 100.0 .+-.
51.9 130.2 .+-. 28.9 212.8 .+-. 7.1** UCP2 100.0 .+-. 11.1 110.2
.+-. 21.2 209.8 .+-. 49.6** 100.0 .+-. 5.6 .sup. 93 .+-. 5.1 110.3
.+-. 6.9 AR.beta..sub.3 100.0 .+-. 7.3 152.3 .+-. 20.sup. 143.8
.+-. 20.5 GK 100.0 .+-. 7.2 97.3 .+-. 5.4 146.1 .+-. 16.6* G6Pase
100.0 .+-. 3.0 92.1 .+-. 6.7 44.6 .+-. 3.3** GS 100.0 .+-. 4.5
107.4 .+-. 7.7 185.3 .+-. 16.6** PKC.epsilon. 100.0 .+-. 8.9 84.2
.+-. 5.3 62.1 .+-. 8.3* RBP4 100.0 .+-. 13.9 118.7 .+-. 7.3 63.2
.+-. 11.2* ApoB100 100.0 .+-. 10.2 99.1 .+-. 8.9 75.1 .+-. 2.5* The
analysis was performed with quantitative RT-PCR. Total RNA was
isolated from tissues of ob/ob mice treated with JNK1 ASO or
control ASO at 25 mg/kg BW or with saline twice a week for 6 weeks.
Data are expressed as the mean .+-. SEM (n = 5-6). *P < 0.05 and
**P < 0.01 when compared to either control group.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 163 <210> SEQ ID NO 1 <211> LENGTH: 20 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 1 attctttcca ctcttctatt 20 <210> SEQ ID
NO 2 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
2 ctcctccaag tccataactt 20 <210> SEQ ID NO 3 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 3 cccgtataac
tccattcttg 20 <210> SEQ ID NO 4 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 4 ctgtgctaaa ggagagggct 20
<210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 5 atgatggatg ctgagagcca 20 <210> SEQ ID
NO 6 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
6 gttgacattg aagacacatc 20 <210> SEQ ID NO 7 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 7 ctgtatcaga
ggccaaagtc 20 <210> SEQ ID NO 8 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 8 tgctgcttct agactgctgt 20
<210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 9 agtcatctac agcagcccag 20 <210> SEQ ID
NO 10 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
10 ccatccctcc caccccccga 20 <210> SEQ ID NO 11 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 11 atcaatgact
aaccgactcc 20 <210> SEQ ID NO 12 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 12 caaaaataag accactgaat 20
<210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 13 cacgcttgct tctgctcatg 20 <210> SEQ
ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
14 cggcttagct tcttgattgc 20 <210> SEQ ID NO 15 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 15 cccgcttggc
atgagtctga 20 <210> SEQ ID NO 16 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 16 ctctctgtag gcccgcttgg 20
<210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 17 atttgcatcc atgagctcca 20 <210> SEQ
ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
18 cgttcctgca gtcctggcca 20 <210> SEQ ID NO 19 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 19 ggatgacctc
gggtgctctg 20 <210> SEQ ID NO 20 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 20 cccataatgc accccacaga 20
<210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 21 cgggtgttgg agagcttcat 20 <210> SEQ
ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
22 tttggtggtg gagcttctgc 20 <210> SEQ ID NO 23 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 23 ggctgccccc
gtataactcc 20 <210> SEQ ID NO 24 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 24 tgctaaagga gagggctgcc 20
<210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 25 aggccaaagt cggatctgtt 20 <210> SEQ
ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
26 ccaccccccg atggcccaag 20 <210> SEQ ID NO 27 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 27 ccaagcgggc
ctacagagag 20 <210> SEQ ID NO 28 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 28 ctttccgttg gacccctggg 20
<210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 29 gtttcagatc cctcgcccgc 20 <210> SEQ
ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
30 tgcagcacaa acaatccctt 20 <210> SEQ ID NO 31 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 31 gtccgggcca
ggccaaagtc 20 <210> SEQ ID NO 32 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 32 caggatgact tcgggcgccc 20
<210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 33 gctctcccat gatgcaaccc 20 <210> SEQ
ID NO 34 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
34 atgggtgacg cagagcttcg 20 <210> SEQ ID NO 35 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 35 ctgctgcatc
tgaaggctga 20 <210> SEQ ID NO 36 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 36 tgagaaggag tggcgttgct 20
<210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 37 tgctgtctgt gtctgaggcc 20 <210> SEQ
ID NO 38 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
38 ggtcccgtcg aggcatcaag 20 <210> SEQ ID NO 39 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 39 catttcaggc
ccacggaggt 20 <210> SEQ ID NO 40 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 40 ggtctgaata gggcaaggca 20
<210> SEQ ID NO 41 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 41 gggcaagtcc aagcaagcat 20 <210> SEQ
ID NO 42 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
42 gactttggcc tggcccggac 20 <210> SEQ ID NO 43 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 43 gtgcgcgcga
gcccgaaatc 20 <210> SEQ ID NO 44 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 44 ttcaacagtt tcttgcataa 20
<210> SEQ ID NO 45 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 45 ctcatctata ggaaacgggt 20 <210> SEQ
ID NO 46 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
46 tggaggctca taaataccac 20 <210> SEQ ID NO 47 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 47 tataagaaat
ggaggctcat 20 <210> SEQ ID NO 48 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 48 tcacatccaa tgttggttca 20
<210> SEQ ID NO 49 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 49 ttatcgaatc cctgacaaaa 20 <210> SEQ
ID NO 50 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
50 gtttggcaat atatgacaca 20 <210> SEQ ID NO 51 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 51 ctgtcaagga
cagcatcata 20 <210> SEQ ID NO 52 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 52 aatcacttga cataagttgg 20
<210> SEQ ID NO 53 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 53 taaatccctg tgaataattc 20 <210> SEQ
ID NO 54 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
54 gcatcccaca gaccatatat 20 <210> SEQ ID NO 55 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 55 tgttctcttt
catccaactg 20 <210> SEQ ID NO 56 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 56 tctcactgct gttcactgct 20
<210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 57 gggtctggtc ggtggacatg 20 <210> SEQ
ID NO 58 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
58 aggctgctgt cagtgtcaga 20 <210> SEQ ID NO 59 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 59 tcacctgcaa
caacccaggg 20 <210> SEQ ID NO 60 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 60 gcggctagtc acctgcaaca 20
<210> SEQ ID NO 61 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 61 cgctgggttt cgcaggcagg 20 <210> SEQ
ID NO 62 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
62 atcatctcct gaagaacgct 20 <210> SEQ ID NO 63 <211>
LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Homo sapien
<400> SEQUENCE: 63 aacgtggatt tatggtctgt ggggtgcatt atggg 35
<210> SEQ ID NO 64 <211> LENGTH: 35 <212> TYPE:
DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 64
aacgttgaca tttggtcagt tgggtgcatc atggg 35 <210> SEQ ID NO 65
<211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM:
Homo sapien <400> SEQUENCE: 65 cccataatgc accccacaga
ccataaatcc acgtt 35 <210> SEQ ID NO 66 <211> LENGTH: 35
<212> TYPE: DNA <213> ORGANISM: Homo sapien <400>
SEQUENCE: 66 cccatgatgc acccaactga ccaaatgtca acgtt 35 <210>
SEQ ID NO 67 <211> LENGTH: 44 <212> TYPE: DNA
<213> ORGANISM: Homo sapien <400> SEQUENCE: 67
aaatggtttg ccacaaaatc ctctttccag gaagggacta tatt 44 <210> SEQ
ID NO 68 <211> LENGTH: 44 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 68 aaatgatcaa
aggtggtgtt ttgttcccag gtacagatca tatt 44 <210> SEQ ID NO 69
<211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM:
Homo sapien <400> SEQUENCE: 69 aatatagtcc cttcctggaa
agaggatttt gtggcaaacc attt 44 <210> SEQ ID NO 70 <211>
LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Homo sapien
<400> SEQUENCE: 70 aatatgatct gtacctggga acaaaacacc
acctttgatc attt 44 <210> SEQ ID NO 71 <211> LENGTH: 32
<212> TYPE: DNA <213> ORGANISM: Homo sapien <400>
SEQUENCE: 71 ccctctcctt tagcacaggt gcagcagtga tc 32 <210> SEQ
ID NO 72 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 72 ccctctcctt
taggtgcagc agtgatc 27 <210> SEQ ID NO 73 <211> LENGTH:
32 <212> TYPE: DNA <213> ORGANISM: Homo sapien
<400> SEQUENCE: 73 gatcactgct gcacctgtgc taaaggagag gg 32
<210> SEQ ID NO 74 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 74
gatcactgct gcacctaaag gagaggg 27 <210> SEQ ID NO 75
<211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM:
Homo sapien <400> SEQUENCE: 75 gtgggttgca tcatgggaga
gctggtgaaa ggttgtgtga tattccaagg cactgaccat 60 <210> SEQ ID
NO 76 <211> LENGTH: 60 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 76 gtcgggtgca
tcatggcaga aatggtcctc cataaagtcc tgttcccggg aagagactat 60
<210> SEQ ID NO 77 <211> LENGTH: 60 <212> TYPE:
DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 77
atggtcagtg ccttggaata tcacacaacc tttcaccagc tctcccatga tgcaacccac
60 <210> SEQ ID NO 78 <211> LENGTH: 60 <212>
TYPE: DNA <213> ORGANISM: Homo sapien <400> SEQUENCE:
78 atagtctctt cccgggaaca ggactttatg gaggaccatt tctgccatga
tgcacccgac 60 <210> SEQ ID NO 79 <211> LENGTH: 35
<212> TYPE: DNA <213> ORGANISM: Homo sapien <400>
SEQUENCE: 79 gatcagcctt cagcacagat gcagcagtaa gtagc 35 <210>
SEQ ID NO 80 <211> LENGTH: 30 <212> TYPE: DNA
<213> ORGANISM: Homo sapien <400> SEQUENCE: 80
gatcagcctt cagatgcagc agtaagtagc 30 <210> SEQ ID NO 81
<211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM:
Homo sapien <400> SEQUENCE: 81 gctacttact gctgcatctg
tgctgaaggc tgatc 35 <210> SEQ ID NO 82 <211> LENGTH: 30
<212> TYPE: DNA <213> ORGANISM: Homo sapien <400>
SEQUENCE: 82 gctacttact gctgcatctg aaggctgatc 30 <210> SEQ ID
NO 83 <211> LENGTH: 38 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 83 ggacagcctt
ctccttcagc acaggtgcag cagtgaac 38 <210> SEQ ID NO 84
<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:
Homo sapien <400> SEQUENCE: 84 ggacagcctt ctccttcagg
tgcagcagtg aac 33 <210> SEQ ID NO 85 <211> LENGTH: 38
<212> TYPE: DNA <213> ORGANISM: Homo sapien <400>
SEQUENCE: 85 gttcactgct gcacctgtgc tgaaggagaa ggctgtcc 38
<210> SEQ ID NO 86 <211> LENGTH: 33 <212> TYPE:
DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 86
gttcactgct gcacctgaag gagaaggctg tcc 33 <210> SEQ ID NO 87
<211> LENGTH: 1418 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 87 cattaattgc
ttgccatcat gagcagaagc aagcgtgaca acaattttta tagtgtagag 60
attggagatt ctacattcac agtcctgaaa cgatatcaga atttaaaacc tataggctca
120 ggagctcaag gaatagtatg cgcagcttat gatgccattc ttgaaagaaa
tgttgcaatc 180 aagaagctaa gccgaccatt tcagaatcag actcatgcca
agcgggccta cagagagcta 240 gttcttatga aatgtgttaa tcacaaaaat
ataattggcc ttttgaatgt tttcacacca 300 cagaaatccc tagaagaatt
tcaagatgtt tacatagtca tggagctcat ggatgcaaat 360 ctttgccaag
tgattcagat ggagctagat catgaaagaa tgtcctacct tctctatcag 420
atgctgtgtg gaatcaagca ccttcattct gctggaatta ttcatcggga cttaaagccc
480 agtaatatag tagtaaaatc tgattgcact ttgaagattc ttgacttcgg
tctggccagg 540 actgcaggaa cgagttttat gatgacgcct tatgtagtga
ctcgctacta cagagcaccc 600 gaggtcatcc ttggcatggg ctacaaggaa
aacgtggatt tatggtctgt ggggtgcatt 660 atgggagaaa tggtttgcca
caaaatcctc tttccaggaa gggactatat tgatcagtgg 720 aataaagtta
ttgaacagct tggaacacca tgtcctgaat tcatgaagaa actgcaacca 780
acagtaagga cttacgttga aaacagacct aaatatgctg gatatagctt tgagaaactc
840 ttccctgatg tccttttccc agctgactca gaacacaaca aacttaaagc
cagtcaggca 900 agggatttgt tatccaaaat gctggtaata gatgcatcta
aaaggatctc tgtagatgaa 960 gctctccaac acccgtacat caatgtctgg
tatgatcctt ctgaagcaga agctccacca 1020 ccaaagatcc ctgacaagca
gttagatgaa agggaacaca caatagaaga gtggaaagaa 1080 ttgatatata
aggaagttat ggacttggag gagagaacca agaatggagt tatacggggg 1140
cagccctctc ctttagcaca ggtgcagcag tgatcaatgg ctctcagcat ccatcatcat
1200 cgtcgtctgt caatgatgtg tcttcaatgt caacagatcc gactttggcc
tctgatacag 1260 acagcagtct agaagcagca gctgggcctc tgggctgctg
tagatgacta cttgggccat 1320 cggggggtgg gagggatggg gagtcggtta
gtcattgata gaactacttt gaaaacaatt 1380 cagtggtctt atttttgggt
gatttttcaa aaaatgta 1418 <210> SEQ ID NO 88 <211>
LENGTH: 1408 <212> TYPE: DNA <213> ORGANISM: Rattus
norvegicus <400> SEQUENCE: 88 gcggccgagc gcgggacgtt
gcggccgaaa cgcggagccg cgagcaggat taagtagcgg 60 cccggccacc
ggcacggcgc cgctctccgc tactggcttc caggtctccg ttggctgcac 120
tgccggccgg ttgttgaata tttggatgaa gccattagac taattgcttg ccatcatgag
180 cagaagtaaa cgtgacaaca atttttatag tgtagagatc gcagattcta
cattcacagt 240 cctaaaacga taccagaact taaagcctat aggctcagga
gctcaaggaa tagtgtgtgc 300 agcttatgat gctattcttg aaagaaatgt
tgcaatcaag aagctcagcc ggccatttca 360 gaatcagacc catgctaagc
gagcctaccg agaactagtt cttatgaagt gtgttaatca 420 caaaaatata
attggccttt tgaatgtttt cacaccacag aaatccctag aagaatttca 480
agatgtttac atagtcatgg agctcatgga tgcaaatctt tgccaagtga ttcagatgga
540 gttagatcat gaaagaatgt cctaccttct ctatcaaatg ctgtgtggaa
tcaagcacct 600 tcactctgct ggaattattc atcgggactt aaagcctagt
aatatagtag tcaaatcaga 660 ctgcactttg aagattcttg attttggact
ggcaaggact gcaggaacga gttttatgat 720 gacgccttac gtggtaactc
gttactacag agcaccagag gtcattctcg gcatgggcta 780 caaggagaac
gtggatttat ggtctgtggg gtgcattatg ggagaaatgg tttgcctcaa 840
aatcctcttt ccaggaaggg actatattga tcagtggaat aaagttattg aacagctcgg
900 aacaccttgt cctgaattca tgaagaaact acaaccaaca gtaaggactt
acgttgaaaa 960 cagacctaag tacgctggct atagctttga gaaactgttt
cctgatgtgc ttttcccagc 1020 tgactcagaa cataacaaac ttaaagccag
tcaggcgaga gatttgttat ctaaaatgct 1080 ggtgatagat gcgtccaaaa
ggatctccgt agacgaagct ctccagcacc cgtacatcaa 1140 cgtctggtat
gatccttcag aagcagaggc cccaccacca aagatccctg acaagcagtt 1200
agatgaaagg gagcacacaa tagaggagtg gaaagaactg atatacaagg aggtcatgga
1260 tttggaggag cgaactaaga atggcgtcat aagagggcag ccgtctcctt
taggtgcagc 1320 agtgatcaat ggctctcagc atccggtctc ttcgccgtct
gtcaatgaca tgtcttcaat 1380 gtccacagat ccgactctgg cctcggat 1408
<210> SEQ ID NO 89 <211> LENGTH: 1365 <212> TYPE:
DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 89
ttgcttgcca tcatgagcag aagcaagcgt gacaacaatt tttatagtgt agagattgga
60 gattctacat tcacagtcct gaaacgatat cagaatttaa aacctatagg
ctcaggagct 120 caaggaatag tatgcgcagc ttatgatgcc attcttgaaa
gaaatgttgc aatcaagaag 180 ctaagccgac catttcagaa tcagactcat
gccaagcggg cctacagaga gctagttctt 240 atgaaatgtg ttaatcacaa
aaatataatt ggccttttga atgttttcac accacagaaa 300 tccctagaag
aatttcaaga tgtttacata gtcatggagc tcatggatgc aaatctttgc 360
caagtgattc agatggagct agatcatgaa agaatgtcct accttctcta tcagatgctg
420 tgtggaatca agcaccttca ttctgctgga attattcatc gggacttaaa
gcccagtaat 480 atagtagtaa aatctgattg cactttgaag attcttgact
tcggtctggc caggactgca 540 ggaacgagtt ttatgatgac gccttatgta
gtgactcgct actacagagc acccgaggtc 600 atccttggca tgggctacaa
ggaaaacgtg gatttatggt ctgtggggtg cattatggga 660 gaaatggttt
gccacaaaat cctctttcca ggaagggact atattgatca gtggaataaa 720
gttattgaac agcttggaac accatgtcct gaattcatga agaaactgca accaacagta
780 aggacttacg ttgaaaacag acctaaatat gctggatata gctttgagaa
actcttccct 840 gatgtccttt tcccagctga ctcagaacac aacaaactta
aagccagtca ggcaagggat 900 ttgttatcca aaatgctggt aatagatgca
tctaaaagga tctctgtaga tgaagctctc 960 caacacccgt acatcaatgt
ctggtatgat ccttctgaag cagaagctcc accaccaaag 1020 atccctgaca
agcagttaga tgaaagggaa cacacaatag aagagtggaa agaattgata 1080
tataaggaag ttatggactt ggaggagaga accaagaatg gagttatacg ggggcagccc
1140 tctcctttag gtgcagcagt gatcaatggc tctcagcatc catcatcatc
gtcgtctgtc 1200 aatgatgtgt cttcaatgtc aacagatccg actttggcct
ctgatacaga cagcagtcta 1260 gaagcagcag ctgggcctct gggctgctgt
agatgactac ttgggccatc ggggggtggg 1320 agggatgggg agtcggttag
tcattgatag aactactttg aaaac 1365 <210> SEQ ID NO 90
<211> LENGTH: 1311 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 90 cattaattgc
ttgccatcat gagcagaagc aagcgtgaca acaattttta tagtgtagag 60
attggagatt ctacattcac agtcctgaaa cgatatcaga atttaaaacc tataggctca
120 ggagctcaag gaatagtatg cgcagcttat gatgccattc ttgaaagaaa
tgttgcaatc 180 aagaagctaa gccgaccatt tcagaatcag actcatgcca
agcgggccta cagagagcta 240 gttcttatga aatgtgttaa tcacaaaaat
ataattggcc ttttgaatgt tttcacacca 300 cagaaatccc tagaagaatt
tcaagatgtt tacatagtca tggagctcat ggatgcaaat 360 ctttgccaag
tgattcagat ggagctagat catgaaagaa tgtcctacct tctctatcag 420
atgctgtgtg gaatcaagca ccttcattct gctggaatta ttcatcggga cttaaagccc
480 agtaatatag tagtaaaatc tgattgcact ttgaagattc ttgacttcgg
tctggccagg 540 actgcaggaa cgagttttat gatgacgcct tatgtagtga
ctcgctacta cagagcaccc 600 gaggtcatcc ttggcatggg ctacaaggaa
aacgttgaca tttggtcagt tgggtgcatc 660 atgggagaaa tgatcaaagg
tggtgttttg ttcccaggta cagatcatat tgatcagtgg 720 aataaagtta
ttgaacagct tggaacacca tgtcctgaat tcatgaagaa actgcaacca 780
acagtaagga cttacgttga aaacagacct aaatatgctg gatatagctt tgagaaactc
840 ttccctgatg tccttttccc agctgactca gaacacaaca aacttaaagc
cagtcaggca 900 agggatttgt tatccaaaat gctggtaata gatgcatcta
aaaggatctc tgtagatgaa 960 gctctccaac acccgtacat caatgtctgg
tatgatcctt ctgaagcaga agctccacca 1020 ccaaagatcc ctgacaagca
gttagatgaa agggaacaca caatagaaga gtggaaagaa 1080 ttgatatata
aggaagttat ggacttggag gagagaacca agaatggagt tatacggggg 1140
cagccctctc ctttagcaca ggtgcagcag tgatcaatgg ctctcagcat ccatcatcat
1200 cgtcgtctgt caatgatgtg tcttcaatgt caacagatcc gactttggcc
tctgatacag 1260 acagcagtct agaagcagca gctgggcctc tgggctgctg
tagatgacta c 1311 <210> SEQ ID NO 91 <211> LENGTH: 1349
<212> TYPE: DNA <213> ORGANISM: Homo sapien <400>
SEQUENCE: 91 attgcttgcc atcatgagca gaagcaagcg tgacaacaat ttttatagtg
tagagattgg 60 agattctaca ttcacagtcc tgaaacgata tcagaattta
aaacctatag gctcaggagc 120 tcaaggaata gtatgcgcag cttatgatgc
cattcttgaa agaaatgttg caatcaagaa 180 gctaagccga ccatttcaga
atcagactca tgccaagcgg gcctacagag agctagttct 240 tatgaaatgt
gttaatcaca aaaatataat tggccttttg aatgttttca caccacagaa 300
atccctagaa gaatttcaag atgtttacat agtcatggag ctcatggatg caaatctttg
360 ccaagtgatt cagatggagc tagatcatga aagaatgtcc taccttctct
atcagatgct 420 gtgtggaatc aagcaccttc attctgctgg aattattcat
cgggacttaa agcccagtaa 480 tatagtagta aaatctgatt gcactttgaa
gattcttgac ttcggtctgg ccaggactgc 540 aggaacgagt tttatgatga
cgccttatgt agtgactcgc tactacagag cacccgaggt 600 catccttggc
atgggctaca aggaaaacgt tgacatttgg tcagttgggt gcatcatggg 660
agaaatgatc aaaggtggtg ttttgttccc aggtacagat catattgatc agtggaataa
720 agttattgaa cagcttggaa caccatgtcc tgaattcatg aagaaactgc
aaccaacagt 780 aaggacttac gttgaaaaca gacctaaata tgctggatat
agctttgaga aactcttccc 840 tgatgtcctt ttcccagctg actcagaaca
caacaaactt aaagccagtc aggcaaggga 900 tttgttatcc aaaatgctgg
taatagatgc atctaaaagg atctctgtag atgaagctct 960 ccaacacccg
tacatcaatg tctggtatga tccttctgaa gcagaagctc caccaccaaa 1020
gatccctgac aagcagttag atgaaaggga acacacaata gaagagtgga aagaattgat
1080 atataaggaa gttatggact tggaggagag aaccaagaat ggagttatac
gggggcagcc 1140 ctctccttta ggtgcagcag tgatcaatgg ctctcagcat
ccatcatcat cgtcgtctgt 1200 caatgatgtg tcttcaatgt caacagatcc
gactttggcc tctgatacag acagcagtct 1260 agaagcagca gctgggcctc
tgggctgctg tagatgacta cttgggccat cggggggtgg 1320 gagggatggg
gagtcggtta gtcattgat 1349 <210> SEQ ID NO 92 <211>
LENGTH: 1782 <212> TYPE: DNA <213> ORGANISM: Homo
sapien <400> SEQUENCE: 92 gggcgggcga gggatctgaa acttgcccac
ccttcgggat attgcaggac gctgcatcat 60 gagcgacagt aaatgtgaca
gtcagtttta tagtgtgcaa gtggcagact caaccttcac 120 tgtcctaaaa
cgttaccagc agctgaaacc aattggctct ggggcccaag ggattgtttg 180
tgctgcattt gatacagttc ttgggataag tgttgcagtc aagaaactaa gccgtccttt
240 tcagaaccaa actcatgcaa agagagctta tcgtgaactt gtcctcttaa
aatgtgtcaa 300 tcataaaaat ataattagtt tgttaaatgt gtttacacca
caaaaaactc tagaagaatt 360 tcaagatgtg tatttggtta tggaattaat
ggatgctaac ttatgtcagg ttattcacat 420 ggagctggat catgaaagaa
tgtcctacct tctttaccag atgctttgtg gtattaaaca 480 tctgcattca
gctggtataa ttcatagaga tttgaagcct agcaacattg ttgtgaaatc 540
agactgcacc ctgaagatcc ttgactttgg cctggcccgg acagcgtgca ctaacttcat
600 gatgacccct tacgtggtga cacggtacta ccgggcgccc gaagtcatcc
tgggtatggg 660 ctacaaagag aacgttgata tctggtcagt gggttgcatc
atgggagagc tggtgaaagg 720 ttgtgtgata ttccaaggca ctgaccatat
tgatcagtgg aataaagtta ttgagcagct 780 gggaacacca tcagcagagt
tcatgaagaa acttcagcca actgtgagga attatgtcga 840 aaacagacca
aagtatcctg gaatcaaatt tgaagaactc tttccagatt ggatattccc 900
atcagaatct gagcgagaca aaataaaaac aagtcaagcc agagatctgt tatcaaaaat
960 gttagtgatt gatcctgaca agcggatctc tgtagacgaa gctctgcgtc
acccatacat 1020 cactgtttgg tatgaccccg ccgaagcaga agccccacca
cctcaaattt atgatgccca 1080 gttggaagaa agagaacatg caattgaaga
atggaaagag ctaatttaca aagaagtcat 1140 ggattgggaa gaaagaagca
agaatggtgt tgtaaaagat cagccttcag atgcagcagt 1200 aagtagcaac
gccactcctt ctcagtcttc atcgatcaat gacatttcat ccatgtccac 1260
tgagcagacg ctggcctcag acacagacag cagtcttgat gcctcgacgg gaccccttga
1320 aggctgtcga tgataggtta gaaatagcaa acctgtcagc attgaaggaa
ctctcacctc 1380 cgtgggcctg aaatgcttgg gagttgatgg aaccaaatag
aaaaactcca tgttctgcat 1440 gtaagaaaca caatgccttg ccctattcag
acctgatagg attgcctgct tagatgataa 1500 aatgaggcag aatatgtctg
aagaaaaaaa ttgcaagcca cacttctaga gattttgttc 1560 aagatcattt
caggtgagca gttagagtag gtgaatttgt ttcaaattgt actagtgaca 1620
gtttctcatc atctgtaact gttgagatgt atgtgcatgt gaccacaaat gcttgcttgg
1680 acttgcccat ctagcacttt ggaaatcagt atttaaatgc caaataatct
tccaggtagt 1740 gctgcttctg aagttatctc ttaatcctct taagtaattt gg 1782
<210> SEQ ID NO 93 <211> LENGTH: 2622 <212> TYPE:
DNA <213> ORGANISM: Rattus norvegicus <400> SEQUENCE:
93 tgtatgacac tacatcatga gtgacagtaa aagcgatggc cagttttaca
gtgtgcaagt 60 ggcagactca actttcactg ttctaaaacg ttaccagcag
ttgaaaccaa ttggctctgg 120 agcccaagga attgtttgtg ctgcttttga
tacagttctt ggaataaatg ttgctgtcaa 180 gaagttaagt cgtccttttc
agaaccaaac gcatgcaaag agagcctacc gtgaacttgt 240 cctcctaaag
tgtgtcaatc ataaaaatat aattagcttg ttaaatgtgt tcacaccaca 300
aaaaacgcta gaagaattcc aagatgtgta cttggttatg gagttaatgg acgctaactt
360 atgtcaggtt attcatatgg agctggacca tgaaagaatg tcatacctcc
tctaccagat 420 gctttgtggc attaagcacc tgcattcagc tggcataatt
catagggatt tgaagcctag 480 caacattgta gtaaaatcag actgtactct
caagatcctt gactttggcc tggcacggac 540 agcctgtacc aactttatga
tgactcccta tgtggtaact cgctactatc gggctccaga 600 agtcatcctg
ggcatgggct acaaggagaa tgttgatatc tggtcagtgg gttgcatcat 660
gggagagctg gtgaaaggtt gtgtgatatt ccaaggtact gaccatattg atcaatggaa
720 taaagttatt gaacagctag gaacaccatc cgcagagttc atgaagaaac
ttcagccaac 780 tgtaaggaat tatgtggaaa acagaccaaa gtaccctgga
atcaaatttg aagagctctt 840 tccagattgg atatttccgt cagaatccga
acgagacaaa ataaaaacaa gtcaagccag 900 agatctgtta tcgaaaatgt
tagtgattga tccggacaag cggatctctg tggacgaagc 960 cttgcgccac
ccgtatatta ctgtttggta tgaccccgct gaagcagaag cgccaccacc 1020
tcaaatttat gatgcccagt tggaagaaag agagcatgcg attgaagagt ggaaagaact
1080 aatttacaaa gaagtgatgg actgggaaga aagaagcaag aatggggtga
aagaccagcc 1140 ttcagatgca gcagtaagca gcaaggctac tccttctcag
tcgtcatcca tcaatgacat 1200 ctcatccatg tccactgagc acaccctggc
ctcagacaca gacagcagtc tcgatgcctc 1260 aaccggaccc ctggaaggct
gccgatgaaa cctcgcagat ggcgcacttg tctgtgaagg 1320 actctggctt
ccatggccct gagcacatgg gagctggtgg aacaaatcaa gaagctccat 1380
gttctgcatg taagaaacac gacgccttgc ccccactcag ttccagtagg attgcctgcg
1440 tagactgtaa catgaggcag acgatgtctg gagaaaaagt acaaaccaca
ctgttagaaa 1500 ttttgttcaa gatcattcag gtgagcaatt agaatagccg
agttcttttc aagtcgtgtg 1560 gtgtccttgg tgacagatca tgtgtaactg
tggggactcg tatgcatgtg accacaaatg 1620 cttgcttgaa cttgcccatg
tagcactttg ggaatcagta tttaaatgcc aaataatctt 1680 ccaggtagtt
ctgcttctag aataatctct taatcctctt tagtaatttg gtgtctgtcc 1740
acaaaaaaat agattatgtg tgtatgaatt ggccactatc atattatcat attttaccca
1800 cttttatggt atgatttatt ctgtcttttg tatttcagaa ggaatataat
taaatttatt 1860 taataaataa aactacagct tttcttaaat ttgtgatgtt
ttaggctgag aattaccact 1920 gctttatatc gacactctgt gtcctttaaa
ctgcccacta tgggaaactt tacgtacagc 1980 tttctgcatg acaaagttcc
aagttgtatt tcactctgct taacgactta tgtcaccttg 2040 aatcctgacc
acacatttcc tttttcttgg tcctctgaac ttggatctag aatccctcac 2100
agaacttcac cttctttatc acaaagcacc ccatctcagt agaatgaatc ggcagattcc
2160 tgagccccgc tgcctaatgt agagctgaca gggtggcttc cccagaacgg
tgggtgggtg 2220 catccttccc tgagcccacc catcctttgc tcccctctct
ttatttaagg tgaaaggtga 2280 ttgggtctca tagcctttcc ttttgtagca
ttgcctaact tgtctttctc actgacagaa 2340 gccaccacgt ccagccagag
cacatggtct cttaggagac cgggcttact taccatgcat 2400 gtttgctgct
gtccttttcc attttgtgga ggcatttcct ttttctaagg gaattcctca 2460
gatgttctag aaacattcag aagaacgcag aagaaatatt ctagagaatt gggggttcat
2520 tcttgaatat tttctgattt aaaactgctc acctgaaatt gatactttca
gatcctgatc 2580 ttgtaaatta ctcgagattt ggtaagatgc tgagttctct gt 2622
<210> SEQ ID NO 94 <211> LENGTH: 1873 <212> TYPE:
DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 94
caaactacgt gctgtacagc tgcatcagct gctcgtagac atgtccagca gctggtcgag
60 gtccacgccg cggtaggtga agttgcggaa ggtccggcga gggatctgaa
acttgcccct 120 tacccttcgg gatattgcag gacgctgcat catgagcgac
agtaaatgtg acagtcagtt 180 ttatagtgtc caagtggcag actcaacctt
cactgtccta aaacgttacc agcagctgaa 240 accaattggc tctggggccc
aagggattgt ttgtgctgca tttgatacag ttcttgggat 300 aaatgttgca
gtcaagaaac taagccgtcc ttttcagaac caaactcatg caaagagagc 360
ttatcgtgaa cttgtcctct taaaatgtgt caatcataaa aatataatta gtttgttaaa
420 tgtgtttaca ccacaaaaaa ctctagaaga atttcaagat gtgtatttgg
ttatggaatt 480 aatggatgct aacttatgtc aggttattca catggagctg
gatcatgaaa gaatgtccta 540 ccttctttac cagatgcttt gtggtattaa
acatctgcat tcagctggta taattcatag 600 agatttgaag cctagcaaca
ttgttgtgaa atcagactgc accctgaaga tccttgactt 660 tggcctggcc
cggacagcgt gcactaactt catgatgacc ccttacgtgg tgacacggta 720
ctaccgggcg cccgaagtca tcctgggtat gggctacaaa gagaacgttg atatctggtc
780 agtgggttgc atcatgggag agctggtgaa aggttgtgtg atattccaag
gcactgacca 840 tattgatcag tggaataaag ttattgagca gctgggaaca
ccatcagcag agttcatgaa 900 gaaacttcag ccaactgtga ggaattatgt
cgaaaacaga ccaaagtatc ctggaatcaa 960 atttgaagaa ctctttccag
attggatatt cccatcagaa tctgagcgag acaaaataaa 1020 aacaagtcaa
gccagagatc tgttatcaaa aatgttagtg attgatcctg acaagcggat 1080
ctctgtagac gaagctctgc gtcacccata catcactgtt tggtatgacc ccgccgaagc
1140 agaagcccca ccacctcaaa tttatgatgc ccagttggaa gaaagagaac
atgcaattga 1200 agaatggaaa gagctaattt acaaagaagt catggattgg
gaagaaagaa gcaagaatgg 1260 tgttgtaaaa gatcagcctc cagatgcagc
agtaagtagc aacgccactc cttctcagtc 1320 ttcatcgatc aatgacattt
catccatgtc cactgagcag acgctggcct cagacacaga 1380 cagcagtctt
gatgcctcga cgggacccct tgaaggctgt cgatgatagg ttagaaatag 1440
caaacctgtc agcattgaag gaactctcac ctccgtgggc ctgaaatgct tgggagttga
1500 tggaaccaaa tagaaaaact ccatgttctg catgtaagaa acacaatgcc
ttgccctact 1560 cagacctgat aggattgcct gcttagatga taaaatgagg
cagaatatgt ctgaagaaaa 1620 aaattgcaag ccacacttct agagattttg
ttcaagatca tttcagttga gcagttagag 1680 taggtgaatt tgtcaaattg
tactagtgac agtttctcat catctgtaac tgttgagatg 1740 attgtgcatg
tgaccacaaa tgcttgcttg gacttgccca tctagcactt tggaaatcag 1800
tatttaaatg ccaaataatc ttccaggtag tgctgcttct gaagttatct cttaatcctc
1860 ttaagtaatt tgg 1873 <210> SEQ ID NO 95 <211>
LENGTH: 1392 <212> TYPE: DNA <213> ORGANISM: Homo
sapien <400> SEQUENCE: 95 tctgaaactt gcccaccctt cgggatattg
caggacgctg catcatgagc gacagtaaat 60 gtgacagtca gttttatagt
gtgcaagtgg cagactcaac cttcactgtc ctaaaacgtt 120 accagcagct
gaaaccaatt ggctctgggg cccaagggat tgtttgtgct gcatttgata 180
cagttcttgg gataagtgtt gcagtcaaga aactaagccg tccttttcag aaccaaactc
240 atgcaaagag agcttatcgt gaacttgtcc tcttaaaatg tgtcaatcat
aaaaatataa 300 ttagtttgtt aaatgtgttt acaccacaaa aaactctaga
agaatttcaa gatgtgtatt 360 tggttatgga attaatggat gctaacttat
gtcaggttat tcacatggag ctggatcatg 420 aaagaatgtc ctaccttctt
taccagatgc tttgtggtat taaacatctg cattcagctg 480 gtataattca
tagagatttg aagcctagca acattgttgt gaaatcagac tgcaccctga 540
agatccttga ctttggcctg gcccggacag cgtgcactaa cttcatgatg accccttacg
600 tggtgacacg gtactaccgg gcgcccgaag tcatcctggg tatgggctac
aaagagaacg 660 ttgatatctg gtcagtgggt tgcatcatgg gagagctggt
gaaaggttgt gtgatattcc 720 aaggcactga ccatattgat cagtggaata
aagttattga gcagctggga acaccatcag 780 cagagttcat gaagaaactt
cagccaactg tgaggaatta tgtcgaaaac agaccaaagt 840 atcctggaat
caaatttgaa gaactctttc cagattggat attcccatca gaatctgagc 900
gagacaaaat aaaaacaagt caagccagag atctgttatc aaaaatgtta gtgattgatc
960 ctgacaagcg gatctctgta gacgaagctc tgcgtcaccc atacatcact
gtttggtatg 1020 accccgccga agcagaagcc ccaccacctc aaatttatga
tgcccagttg gaagaaagag 1080 aacatgcaat tgaagaatgg aaagagctaa
tttacaaaga agtcatggat tgggaagaaa 1140 gaagcaagaa tggtgttgta
aaagatcagc cttcagcaca gatgcagcag taagtagcaa 1200 cgccactcct
tctcagtctt catcgatcaa tgacatttca tccatgtcca ctgagcagac 1260
gctggcctca gacacagaca gcagtcttga tgcctcgacg ggaccccttg aaggctgtcg
1320 atgataggtt agaaatagca aacctgtcag cattgaagga actctcacct
ccgtgggcct 1380 gaaatgcttg gg 1392 <210> SEQ ID NO 96
<211> LENGTH: 1523 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 96 ggatattgca
ggacgctgca tcatgagcga cagtaaatgt gacagtcagt tttatagtgt 60
gcaagtggca gactcaacct tcactgtcct aaaacgttac cagcagctga aaccaattgg
120 ctctggggcc caagggattg tttgtgctgc atttgataca gttcttggga
taagtgttgc 180 agtcaagaaa ctaagccgtc cttttcagaa ccaaactcat
gcaaagagag cttatcgtga 240 acttgtcctc ttaaaatgtg tcaatcataa
aaatataatt agtttgttaa atgtgtttac 300 accacaaaaa actctagaag
aatttcaaga tgtgtatttg gttatggaat taatggatgc 360 taacttatgt
caggttattc acatggagct ggatcatgaa agaatgtcct accttcttta 420
ccagatgctt tgtggtatta aacatctgca ttcagctggt ataattcata gagatttgaa
480 gcctagcaac attgttgtga aatcagactg caccctgaag atccttgact
ttggcctggc 540 ccggacagcg tgcactaact tcatgatgac cccttacgtg
gtgacacggt actaccgggc 600 gcccgaagtc atcctgggta tgggctacaa
agagaacgtt gatatctggt cagtcgggtg 660 catcatggca gaaatggtcc
tccataaagt cctgttcccg ggaagagact atattgatca 720 gtggaataaa
gttattgagc agctgggaac accatcagca gagttcatga agaaacttca 780
gccaactgtg aggaattatg tcgaaaacag accaaagtat cctggaatca aatttgaaga
840 actctttcca gattggatat tcccatcaga atctgagcga gacaaaataa
aaacaagtca 900 agccagagat ctgttatcaa aaatgttagt gattgatcct
gacaagcgga tctctgtaga 960 cgaagctctg cgtcacccat acatcactgt
ttggtatgac cccgccgaag cagaagcccc 1020 accacctcaa atttatgatg
cccagttgga agaaagagaa catgcaattg aagaatggaa 1080 agagctaatt
tacaaagaag tcatggattg ggaagaaaga agcaagaatg gtgttgtaaa 1140
agatcagcct tcagcacaga tgcagcagta agtagcaacg ccactccttc tcagtcttca
1200 tcgatcaatg acatttcatc catgtccact gagcagacgc tggcctcaga
cacagacagc 1260 agtcttgatg cctcgacggg accccttgaa ggctgtcgat
gataggttag aaatagcaaa 1320 cctgtcagca ttgaaggaac tctcacctcc
gtgggcctga aatgcttggg agttgatgga 1380 accaaataga aaaactccat
gttctgcatg taagaaacac aatgccttgc cctattcaga 1440 cctgatagga
ttgcctgctt agatgataaa atgaggcaga atatgtctga agaaaaaaat 1500
tgcaagccac acttctagag att 1523 <210> SEQ ID NO 97 <211>
LENGTH: 1619 <212> TYPE: DNA <213> ORGANISM: Homo
sapien <400> SEQUENCE: 97 gcccaccctt cgggatattg caggacgctg
catcatgagc gacagtaaat gtgacagtca 60 gttttatagt gtgcaagtgg
cagactcaac cttcactgtc ctaaaacgtt accagcagct 120 gaaaccaatt
ggctctgggg cccaagggat tgtttgtgct gcatttgata cagttcttgg 180
gataagtgtt gcagtcaaga aactaagccg tccttttcag aaccaaactc atgcaaagag
240 agcttatcgt gaacttgtcc tcttaaaatg tgtcaatcat aaaaatataa
ttagtttgtt 300 aaatgtgttt acaccacaaa aaactctaga agaatttcaa
gatgtgtatt tggttatgga 360 attaatggat gctaacttat gtcaggttat
tcacatggag ctggatcatg aaagaatgtc 420 ctaccttctt taccagatgc
tttgtggtat taaacatctg cattcagctg gtataattca 480 tagagatttg
aagcctagca acattgttgt gaaatcagac tgcaccctga agatccttga 540
ctttggcctg gcccggacag cgtgcactaa cttcatgatg accccttacg tggtgacacg
600 gtactaccgg gcgcccgaag tcatcctggg tatgggctac aaagagaacg
ttgatatctg 660 gtcagtcggg tgcatcatgg cagaaatggt cctccataaa
gtcctgttcc cgggaagaga 720 ctatattgat cagtggaata aagttattga
gcagctggga acaccatcag cagagttcat 780 gaagaaactt cagccaactg
tgaggaatta tgtcgaaaac agaccaaagt atcctggaat 840 caaatttgaa
gaactctttc cagattggat attcccatca gaatctgagc gagacaaaat 900
aaaaacaagt caagccagag atctgttatc aaaaatgtta gtgattgatc ctgacaagcg
960 gatctctgta gacgaagctc tgcgtcaccc atacatcact gtttggtatg
accccgccga 1020 agcagaagcc ccaccacctc aaatttatga tgcccagttg
gaagaaagag aacatgcaat 1080 tgaagaatgg aaagagctaa tttacaaaga
agtcatggat tgggaagaaa gaagcaagaa 1140 tggtgttgta aaagatcagc
cttcagatgc agcagtaagt agcaacgcca ctccttctca 1200 gtcttcatcg
atcaatgaca tttcatccat gtccactgag cagacgctgg cctcagacac 1260
agacagcagt cttgatgcct cgacgggacc ccttgaaggc tgtcgatgat aggttagaaa
1320 tagcaaacct gtcagcattg aaggaactct cacctccgtg ggcctgaaat
gcttgggagt 1380 tgatggaacc aaatagaaaa actccatgtt ctgcatgtaa
gaaacacaat gccttgccct 1440 attcagacct gataggattg cctgcttaga
tgataaaatg aggcagaata tgtctgaaga 1500 aaaaaattgc aagccacact
tctagagatt ttgttcaaga tcatttcagg tgagcagtta 1560 gagtaggtga
atttgtttca aattgtacta gtgacagttt ctcatcatct gtaactgtt 1619
<210> SEQ ID NO 98 <211> LENGTH: 2372 <212> TYPE:
DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 98
gagaaatggc gtggcagggg acccagcgag cccagaggga ttttgccgct gcttcctcta
60 cccctgtatt tcacgcagct ctctaaattg actcagctcc aggctagtgt
gagaaacacc 120 aacagcaggc ccatctcaga tcttcactat ggcaacttat
gcaagaaact gttgaattag 180 acccgtttcc tatagatgag aaaccataca
agctgtggta tttatgagcc tccatttctt 240 atactactgc agtgaaccaa
cattggatgt gaaaattgcc ttttgtcagg gattcgataa 300 acaagtggat
gtgtcatata ttgccaaaca ttacaacatg agcaaaagca aagttgacaa 360
ccagttctac agtgtggaag tgggagactc aaccttcaca gttctcaagc gctaccagaa
420 tctaaagcct attggctctg gggctcaggg catagtttgt gccgcgtatg
atgctgtcct 480 tgacagaaat gtggccatta agaagctcag cagacccttt
cagaaccaaa cacatgccaa 540 gagagcgtac cgggagctgg tcctcatgaa
gtgtgtgaac cataaaaaca ttattagttt 600 attaaatgtc ttcacacccc
agaaaacgct ggaggagttc caagatgttt acttagtaat 660 ggaactgatg
gatgccaact tatgtcaagt gattcagatg gaattagacc atgagcgaat 720
gtcttacctg ctgtaccaaa tgttgtgtgg cattaagcac ctccattctg ctggaattat
780 tcacagggat ttaaaaccaa gtaacattgt agtcaagtct gattgcacat
tgaaaatcct 840 ggactttgga ctggccagga cagcaggcac aagcttcatg
atgactccat atgtggtgac 900 acgttattac agagcccctg aggtcatcct
ggggatgggc tacaaggaga acgtggatat 960 atggtctgtg ggatgcatta
tgggagaaat ggttcgccac aaaatcctct ttccaggaag 1020 ggactatatt
gaccagtgga ataaggtaat tgaacaacta ggaacaccat gtccagaatt 1080
catgaagaaa ttgcaaccca cagtaagaaa ctatgtggag aatcggccca agtatgcggg
1140 actcaccttc cccaaactct tcccagattc cctcttccca gcggactccg
agcacaataa 1200 actcaaagcc agccaagcca gggacttgtt gtcaaagatg
ctagtgattg acccagcaaa 1260 aagaatatca gtggacgacg ccttacagca
tccctacatc aacgtctggt atgacccagc 1320 cgaagtggag gcgcctccac
ctcagatata tgacaagcag ttggatgaaa gagaacacac 1380 aattgaagaa
tggaaagaac ttatctacaa ggaagtaatg aattcagaag aaaagactaa 1440
aaatggtgta gtaaaaggac agccttctcc ttcagcacag gtgcagcagt gaacagcagt
1500 gagagtctcc ctccatcctc gtctgtcaat gacatctcct ccatgtccac
cgaccagacc 1560 ctggcatctg acactgacag cagcctggaa gcctcggcag
gacccctggg ttgttgcagg 1620 tgactagccg cctgcctgcg aaacccagcg
ttcttcagga gatgatgtga tggaacacac 1680 acacacgcag acacacacac
acacacaaat gcagacacac aacatcaaga aaacagcaag 1740 ggagagaatc
caagcctaaa attaaataaa tctttcagcc tgcttcttcc ccagggttct 1800
gtattgcagc taagctcaaa tgtatattta acttctagtt gctcttgctt tggtcttctt
1860 ccaatgatgc ttactacaga aagcaaatca gacacaatta gagaagcctt
ttccataaag 1920 tgtaatttta atggctgcaa aaccggcaac ctgtaactgc
ccttttaaat ggcatgacaa 1980 ggtgtgcagt ggccccatcc agcatgtgtg
tgtctctatc ttgcatctac ctgctccttg 2040 gcctagtcag atggatgtag
atacagatcc gcatgtgtct gtattcatac agcactactt 2100 acttagagat
gctactctca gtgtcctcag ggctctacca agacataatg cactggggta 2160
ccacatggtc catttcatgt gatctattac tctgacataa acccatctgt aatatattgc
2220 cagtatataa gctgtttagt ttgttaattg attaaactgt atgtcttata
agaaaacatg 2280 taaaggggga atatattggg ggagtgagct ctctcagacc
cttgaagatg tagcttccaa 2340 atttgaatgg attaaatggc acctgtatac ca 2372
<210> SEQ ID NO 99 <211> LENGTH: 1975 <212> TYPE:
DNA <213> ORGANISM: Rattus norvegicus <400> SEQUENCE:
99 ccctccttat tccggtttgg aatgtggcta atgaaagccc agtaggagga
tttctggggc 60 aaacaggtgg accaggatcc tggttctcag gcacggaatg
gctattgtga gagcgccacc 120 agcaggacca tcgcagatct tggttatggc
tgctcacgca agaggctgtt gatgtagacc 180 ccctttcccg tagatgagaa
atcacacgag cagtggtatt tatgagcctc catttcttat 240 actactgcag
tgaaccaacc ttggatgtga aaattgcctt ttgtcaggtg tgtgttcctt 300
acaggtaaaa caaagggatt cgacaaacac gtggatgtgt cttctgttgt caaacattac
360 aacatgagca aaagcaaggt agataaccag ttctacagtg tggaagtggg
agactcaacc 420 ttcacagttc taaagcgcta ccagaacctg aagccgatcg
gctctggggc tcagggaata 480 gtttgtgctg cgtatgacgc tgtcctcgac
agaaatgtgg ccattaagaa gctcagcaga 540 cccttccaga accaaactca
tgccaagagg gcttaccggg agctggtcct catgaagtgt 600 gtgaaccata
aaaacattat tagcttatta aatgtcttta caccccagaa aacactggag 660
gagttccaag atgtttactt agtgatggaa ctgatggacg ccaacttgtg tcaggtgatt
720 cagatggagc tggaccacga gcggatgtcg tacttgctgt accagatgct
gtcggcgatc 780 aaacacctcc actccgctgg gatcatccac agggacttaa
aacccagtaa catcgtagtc 840 aagtctgatt gcacactgaa aatcctggac
tttggactgg ccaggacagc gggcacaagc 900 ttcatgatga ctccgtatgt
ggtgacgaga tattacagag cccccgaggt catcctgggc 960 atgggctaca
aggagaacgt ggacatatgg tctgtgggct gcatcatggg agaaatggtt 1020
cgtcacaaaa tcctctttcc cggaagggac tatattgacc agtggaacaa agtcatagag
1080 cagctaggaa ctccgtgtcc agaattcatg aagaaattgc agcccaccgt
cagaaactac 1140 gtggagaacc ggcccaagta tgcaggcctc accttcccca
agctctttcc agattccctc 1200 ttcccagcgg attccgagca caataaactt
aaagccagcc aagccaggga cttgttgtca 1260 aagatgttag tgattgaccc
agcgaagagg atatcggtgg atgacgcatt gcagcatccg 1320 tacatcaacg
tttggtacga ccctgctgaa gtggaggcgc ctccgcctca gatatatgac 1380
aagcaattgg atgaaaggga gcacaccatc gaagaatgga aagaactcat ctacaaggaa
1440 gtaatgaact cagaagagaa gactaagaac ggcgtagtca aaggccagcc
ctcaccttca 1500 ggtgcagcag tgaacagcag tgagagtctc cctccatcct
catctgtcaa cgacatctcc 1560 tccatgtcca ccgaccagac cctcgcatcc
gacactgaca gcagcctgga agcctcggcg 1620 ggaccgctgg gttgttgcag
gtgactagcc gcctgcctgc gaaacccagc gttcttcagg 1680 agatgacgcc
atgatagaac acagcgcaca tgcacacaca cagagcttgt acacacacac 1740
acacacacac acacacgcac gcacgcacgc acgcaagcac gcacgcacgc acaaatgcac
1800 tcacgcaatg tcaagaaaaa aaaaagtagc gagagagagc gagagagcca
acgtaaaact 1860 aagttaaatc tttctgcgtg cttctccaga gttctgtatc
gcagctgagc tgaaatgtat 1920 acttaacttc tagtcgcgct cgctcgactt
tggtctccct ccggcagtgc ttact 1975 <210> SEQ ID NO 100
<211> LENGTH: 2522 <212> TYPE: DNA <213>
ORGANISM: Mus musculus <400> SEQUENCE: 100 ggggcttgag
tgagctaaag attgggtctt cttggaaatc acctgtctgt tattattttt 60
aaacaatcgc tacacctcca aagactctgc tccttactcc ggtttggaat gtggctaatg
120 actacccagt agggaggatt tctggggcaa acagccggac caggatccta
gttctcaggc 180 acggaatggc tattgtgaga acagcaccag caggatcatc
gcagatcttg gttatggcca 240 ctcaggcaag acgctgttga gttaagaccc
ctttcccata gatgagaagc cacagaagca 300 gtggtattta tgagcctcca
tttcttatac tactgcagtg aaccaacctt ggatgtgaaa 360 attgcctttt
gtcagggatt cgataaacac gtggatgtgt catctattgc caaacattac 420
aacatgagca aaagcaaggt ggacaaccag ttctacagtg tggaagtggg ggactcaacc
480 ttcaccgttc ttaagcgcta ccagaacctg aagccaattg gctctggggc
tcagggaata 540 gtctgtgctg cgtacgacgc tgtccttgac agaaatgtgg
ccattaagaa gctcagcaga 600 cccttccaga accaaactca cgccaagagg
gcttaccggg agctggtgct catgaagtgt 660 gtgaaccata aaaacattat
tagcttatta aatgttttta caccccagaa aacgctggag 720 gagttccaag
atgtctactt agtgatggaa ctgatggacg ccaacctgtg tcaggtgatt 780
cagatggagc tggaccacga gcggatgtct tacttgctgt accagatgct gtgtggcatc
840 aagcacctcc actccgctgg gatcatccac agggacttaa aacccagtaa
cattgtagtc 900 aagtctgatt gcacactgaa aatcctcgac ttcggactgg
ccaggacagc gggtacaagc 960 ttcatgatga ctccgtatgt ggtgacgcga
tattacagag cccctgaggt catcctgggc 1020 atgggctaca aggagaacgt
ggacatatgg tctgtgggat gcatcatggg agaaatggtt 1080 cgccacaaaa
tcctctttcc cggaaggagc tatattgacc agtggaacaa agtcatcgag 1140
cagctaggaa ctccgtgtcc agagttcatg aagaaattgc agcccacagt cagaaactac
1200 gtggagaatc ggcccaagta cgcaggactc accttcccca agctctttcc
agattccctc 1260 ttcccagcgg attctgagca caataaactt aaagccagcc
aagccaggga tttgttgtct 1320 aagatgttag tgattgaccc agtgaagagg
atatcggtgg acgacgcact gcagcatccg 1380 tacatcaacg tttggtacga
cccggctgaa gtggaggcgc ctccgcctca gatatatgat 1440 aagcagctgg
atgaaaggga gcacaccatc gaagaatgga aagaacttat ctacaaggag 1500
gtaatgaact cagaagagaa gactaagaat ggcgtagtca aaagccagcc ctcgccttca
1560 gcacaggtgc agcagtgaac agcagtgaga gtctccctcc atcctcggct
gtcaacgaca 1620 tctcctccat gtccaccgac cagaccctcg catctgacac
tgacagcagc ctggaggcct 1680 cggcgggacc gttgggttgt tgcaggtgac
tagccgcctg cctgcgaaac ccagcgttct 1740 tcaggagatg acgcgataga
acacagcaca catgcacaca cacagcttgc tctcacacac 1800 actcagcttg
ctcacacaca cacacacaca tacacacaaa cacacactgt ctctctctca 1860
cacacacaca ctgtcacaac gcactcacga aaggtcaaga aaaaaataac aatagagaga
1920 tccaacataa aattaagtta aatttttctg cgtgcttctc caaagttctg
tatcacagct 1980 gagctgaaat gtatacttaa cttctagttg cgctcgcttt
ggtttccctc cagcagtgct 2040 tactacacaa gacaaatcag acacaattag
agaaaccttt ccctaaagtg taacttaagt 2100 ggctgcagaa ccagcaacct
gtaactgccc ttcaaatggc atgaggaggt gggcacgggt 2160 cccgcccagc
atgtgtgtgt ctctatctcg cgtctacctg ctcttccggc ctagtcagat 2220
ggatgtagat acagatcccg catgtgtctg tattcaaaca gcacttagag atgctcctgt
2280 cagtgtcctc caggctccac caagacacac accggggtac cacatggtcc
atttcatgtg 2340 atctattact ctgacataaa tccatctgta atatattgcc
agtatataag ctgtttagtt 2400 tgttaattgc ttaagctgta tgtcttataa
gagactatgt aaagggggaa aatggaggcg 2460 tgaactctca gacccttgaa
gatgtagctt ccgaatttga ccgttaaatg gcaccgtata 2520 cc 2522
<210> SEQ ID NO 101 <211> LENGTH: 1773 <212>
TYPE: DNA <213> ORGANISM: Homo sapien <400> SEQUENCE:
101 atctcagatc ttcactatgg caacttatgc aagaaactgt tgaattagac
ccgtttccta 60 tagatgagaa accatacaag ctgtggtatt tatgagcctc
catttcttat actactgcag 120 tgaaccaaca ttggatgtga aaattgcctt
ttgtcaggga ttcgataaac aagtggatgt 180 gtcatatatt gccaaacatt
acaacatgag caaaagcaaa gttgacaacc agttctacag 240 tgtggaagtg
ggagactcaa ccttcacagt tctcaagcgc taccagaatc taaagcctat 300
tggctctggg gctcagggca tagtttgtgc cgcgtatgat gctgtccttg acagaaatgt
360 ggccattaag aagctcagca gaccctttca gaaccaaaca catgccaaga
gagcgtaccg 420 ggagctggtc ctcatgaagt gtgtgaacca taaaaacatt
attagtttat taaatgtctt 480 cacaccccag aaaacgctgg aggagttcca
agatgtttac ttagtaatgg aactgatgga 540 tgccaactta tgtcaagtga
ttcagatgga attagaccat gagcgaatgt cttacctgct 600 gtaccaaatg
ttgtgtggca ttaagcacct ccattctgct ggaattattc acagggattt 660
aaaaccaagt aacattgtag tcaagtctga ttgcacattg aaaatcctgg actttggact
720 ggccaggaca gcaggcacaa gcttcatgat gactccatat gtggtgacac
gttattacag 780 agcccctgag gtcatcctgg ggatgggcta caaggagaac
gtggatatat ggtctgtggg 840 atgcattatg ggagaaatgg ttcgccacaa
aatcctcttt ccaggaaggg actatattga 900 ccagtggaat aaggtaattg
aacaactagg aacaccatgt ccagaattca tgaagaaatt 960 gcaacccaca
gtaagaaact atgtggagaa tcggcccaag tatgcgggac tcaccttccc 1020
caaactcttc ccagattccc tcttcccagc ggactccgag cacaataaac tcaaagccag
1080 ccaagccagg gacttgttgt caaagatgct agtgattgac ccagcaaaaa
gaatatcagt 1140 ggacgacgcc ttacagcatc cctacatcaa cgtctggtat
gacccagccg aagtggaggc 1200 gcctccacct cagatatatg acaagcagtt
ggatgaaaga gaacacacaa ttgaagaatg 1260 gaaagaactt atctacaagg
aagtaatgaa ttcagaagaa aagactaaaa atggtgtagt 1320 aaaaggacag
ccttctcctt cagcacaggt gcagcagtga acagcagtga gagtctccct 1380
ccatcctcgt ctgtcaatga catctcctcc atgtccaccg accagaccct ggcatctgac
1440 actgacagca gcctggaagc ctcggcagga cccctgggtt gttgcaggtg
actagccgcc 1500 tgcctgcgaa acccagcgtt cttcaggaga tgatgtgatg
gaacacacac acacgcagac 1560 acacacacac acacaaatgc agacacacaa
catcaagaaa acagcaaggg agagaatcca 1620 agcctaaaat taaataaatc
tttcagcctg cttcttcccc agggttctgt attgcagcta 1680 agctcaaatg
tatatttaac ttctagttgc tcttgctttg gtcttcttcc aatgatgctt 1740
actacagaaa gcaaatcaga cacaattaga gaa 1773 <210> SEQ ID NO 102
<211> LENGTH: 1505 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 102 ttatgcaaga
aactgttgaa ttagacccgt ttcctataga tgagaaacca tacaagctgt 60
ggtatttatg agcctccatt tcttatacta ctgcagtgaa ccaacattgg atgtgaaaat
120 tgccttttgt cagggattcg ataaacaagt ggatgtgtca tatattgcca
aacattacaa 180 catgagcaaa agcaaagttg acaaccagtt ctacagtgtg
gaagtgggag actcaacctt 240 cacagttctc aagcgctacc agaatctaaa
gcctattggc tctggggctc agggcatagt 300 ttgtgccgcg tatgatgctg
tccttgacag aaatgtggcc attaagaagc tcagcagacc 360 ctttcagaac
caaacacatg ccaagagagc gtaccgggag ctggtcctca tgaagtgtgt 420
gaaccataaa aacattatta gtttattaaa tgtcttcaca ccccagaaaa cgctggagga
480 gttccaagat gtttacttag taatggaact gatggatgcc aacttatgtc
aagtgattca 540 gatggaatta gaccatgagc gaatgtctta cctgctgtac
caaatgttgt gtggcattaa 600 gcacctccat tctgctggaa ttattcacag
ggatttaaaa ccaagtaaca ttgtagtcaa 660 gtctgattgc acattgaaaa
tcctggactt tggactggcc aggacagcag gcacaagctt 720 catgatgact
ccatatgtgg tgacacgtta ttacagagcc cctgaggtca tcctggggat 780
gggctacaag gagaacgtgg atatatggtc tgtgggatgc attatgggag aaatggttcg
840 ccacaaaatc ctctttccag gaagggacta tattgaccag tggaataagg
taattgaaca 900 actaggaaca ccatgtccag aattcatgaa gaaattgcaa
cccacagtaa gaaactatgt 960 ggagaatcgg cccaagtatg cgggactcac
cttccccaaa ctcttcccag attccctctt 1020 cccagcggac tccgagcaca
ataaactcaa agccagccaa gccagggact tgttgtcaaa 1080 gatgctagtg
attgacccag caaaaagaat atcagtggac gacgccttac agcatcccta 1140
catcaacgtc tggtatgacc cagccgaagt ggaggcgcct ccacctcaga tatatgacaa
1200 gcagttggat gaaagagaac acacaattga agaatggaaa gaacttatct
acaaggaagt 1260 aatgaattca gaagaaaaga ctaaaaatgg tgtagtaaaa
ggacagcctt ctccttcagg 1320 tgcagcagtg aacagcagtg agagtctccc
tccatcctcg tctgtcaatg acatctcctc 1380 catgtccacc gaccagaccc
tggcatctga cactgacagc agcctggaag cctcggcagg 1440 acccctgggt
tgttgcaggt gactagccgc ctgcctgcga aacccagcgt tcttcaggag 1500 atgat
1505 <210> SEQ ID NO 103 <400> SEQUENCE: 103 000
<210> SEQ ID NO 104 <211> LENGTH: 2622 <212>
TYPE: DNA <213> ORGANISM: Rattus norvegicus <400>
SEQUENCE: 104 tgtatgacac tacatcatga gtgacagtaa aagcgatggc
cagttttaca gtgtgcaagt 60 ggcagactca actttcactg ttctaaaacg
ttaccagcag ttgaaaccaa ttggctctgg 120 agcccaagga attgtttgtg
ctgcttttga tacagttctt ggaataaatg ttgctgtcaa 180 gaagttaagt
cgtccttttc agaaccaaac gcatgcaaag agagcctacc gtgaacttgt 240
cctcctaaag tgtgtcaatc ataaaaatat aattagcttg ttaaatgtgt tcacaccaca
300 aaaaacgcta gaagaattcc aagatgtgta cttggttatg gagttaatgg
acgctaactt 360 atgtcaggtt attcatatgg agctggacca tgaaagaatg
tcatacctcc tctaccagat 420 gctttgtggc attaagcacc tgcattcagc
tggcataatt catagggatt tgaagcctag 480 caacattgta gtaaaatcag
actgtactct caagatcctt gactttggcc tggcacggac 540 agcctgtacc
aactttatga tgactcccta tgtggtaact cgctactatc gggctccaga 600
agtcatcctg ggcatgggct acaaggagaa tgtggacatc tggtctgtcg ggtgcatcat
660 ggcagaaatg gtcctccata aatcctgttc cccaggaaga gactatattg
atcaatggaa 720 taaagttatt gaacagctag gaacaccatc cgcagagttc
atgaagaaac ttcagccaac 780 tgtaaggaat tatgtggaaa acagaccaaa
gtaccctgga atcaaatttg aagagctctt 840 tccagattgg atatttccgt
cagaatccga acgagacaaa ataaaaacaa gtcaagccag 900 agatctgtta
tcgaaaatgt tagtgattga tccggacaag cggatctctg tggacgaagc 960
cttgcgccac ccgtatatta ctgtttggta tgaccccgct gaagcagaag cgccaccacc
1020 tcaaatttat gatgcccagt tggaagaaag agagcatgcg attgaagagt
ggaaagaact 1080 aatttacaaa gaagtgatgg actgggaaga aagaagcaag
aatggggtga aagaccagcc 1140 ttcagatgca gcagtaagca gcaaggctac
tccttctcag tcgtcatcca tcaatgacat 1200 ctcatccatg tccactgagc
acaccctggc ctcagacaca gacagcagtc tcgatgcctc 1260 aaccggaccc
ctggaaggct gccgatgaaa cctcgcagat ggcgcacttg tctgtgaagg 1320
actctggctt ccatggccct gagcacatgg gagctggtgg aacaaatcaa gaagctccat
1380 gttctgcatg taagaaacac gacgccttgc ccccactcag ttccagtagg
attgcctgcg 1440 tagactgtaa catgaggcag acgatgtctg gagaaaaagt
acaaaccaca ctgttagaaa 1500 ttttgttcaa gatcattcag gtgagcaatt
agaatagccg agttcttttc aagtcgtgtg 1560 gtgtccttgg tgacagatca
tgtgtaactg tggggactcg tatgcatgtg accacaaatg 1620 cttgcttgaa
cttgcccatg tagcactttg ggaatcagta tttaaatgcc aaataatctt 1680
ccaggtagtt ctgcttctag aataatctct taatcctctt tagtaatttg gtgtctgtcc
1740 acaaaaaaat agattatgtg tgtatgaatt ggccactatc atattatcat
attttaccca 1800 cttttatggt atgatttatt ctgtcttttg tatttcagaa
ggaatataat taaatttatt 1860 taataaataa aactacagct tttcttaaat
ttgtgatgtt ttaggctgag aattaccact 1920 gctttatatc gacactctgt
gtcctttaaa ctgcccacta tgggaaactt tacgtacagc 1980 tttctgcatg
acaaagttcc aagttgtatt tcactctgct taacgactta tgtcaccttg 2040
aatcctgacc acacatttcc tttttcttgg tcctctgaac ttggatctag aatccctcac
2100 agaacttcac cttctttatc acaaagcacc ccatctcagt agaatgaatc
ggcagattcc 2160 tgagccccgc tgcctaatgt agagctgaca gggtggcttc
cccagaacgg tgggtgggtg 2220 catccttccc tgagcccacc catcctttgc
tcccctctct ttatttaagg tgaaaggtga 2280 ttgggtctca tagcctttcc
ttttgtagca ttgcctaact tgtctttctc actgacagaa 2340 gccaccacgt
ccagccagag cacatggtct cttaggagac cgggcttact taccatgcat 2400
gtttgctgct gtccttttcc attttgtgga ggcatttcct ttttctaagg gaattcctca
2460 gatgttctag aaacattcag aagaacgcag aagaaatatt ctagagaatt
gggggttcat 2520 tcttgaatat tttctgattt aaaactgctc acctgaaatt
gatactttca gatcctgatc 2580 ttgtaaatta ctcgagattt ggtaagatgc
tgagttctct gt 2622 <210> SEQ ID NO 105 <400> SEQUENCE:
105 000 <210> SEQ ID NO 106 <211> LENGTH: 1646
<212> TYPE: DNA <213> ORGANISM: Mus musculus
<400> SEQUENCE: 106 gctgttagtc agcggagcgg ccgaggccgg
acgttgcggc cgaaacgcgg agccgcgaac 60 aggattgagt agcggccgcg
gccaccgcag gacggcgccg ttctccgcta cgggcttcca 120 ggtcgccgtt
ggctgcactg ccggccttgg tgaatatttg gatgaagcca ttagactaat 180
tgcttgccat catgagcaga agcaaacgtg acaacaattt ttatagtgta gagattggag
240 attctacatt cacagtccta aaacgatacc agaatttaaa gcctataggc
tcaggagctc 300 aaggaatagt gtgtgcagct tatgatgcca ttcttgaaag
aaatgttgca atcaagaagc 360 tcagccggcc atttcagaat cagacccatg
ctaagcgcgc ctaccgagaa ctagttctta 420 tgaagtgtgt taatcacaaa
aatataattg gccttttgaa tgttttcaca ccacagaaat 480 ccctagaaga
atttcaagat gtttacatag tcatggagct catggatgca aatctttgcc 540
aagtgattca gatggagtta gatcatgaaa gaatgtccta ccttctctat caaatgctgt
600 gtggaatcaa gcaccttcac tctgctggaa ttattcatcg ggacttaaag
cctagtaata 660 tagtagtcaa atcagactgc actttgaaga ttcttgattt
tggactggcg aggactgcag 720 gaacgagttt tatgatgacg ccttatgtgg
tgactcgcta ctacagagca ccagaggtca 780 ttctcggcat gggctacaag
gagaacgtgg acttatggtc tgtggggtgc attatgggag 840 aaatggtttg
ccacaaaatc ctctttccag gaagggacta tattgatcag tggaataaag 900
ttattgaaca gctcggaaca ccttgtcctg aattcatgaa gaaactacaa ccaacagtaa
960 ggacttatgt tgaaaacagg cctaaatacg ctggatatag ctttgagaaa
ctgttccccg 1020 atgtgctttt cccagctgac tcagagcata acaaacttaa
agccagtcag gcaagagatt 1080 tgttatccaa aatgctagta atagatgcat
ccaaaaggat ctccgtagat gaagctctcc 1140 agcacccata catcaacgtc
tggtatgatc cttcagaagc agaagcccca ccaccaaaga 1200 tcccggacaa
gcagttagat gagagggagc acacaataga ggagtggaaa gaactgatat 1260
acaaggaggt aatggatttg gaggaacgaa ctaagaatgg agtcataaga gggcagccgt
1320 ctcctttagc acaggtgcag caatgatcaa tggctctcag catccatcgt
cttcgccgtc 1380 tgtcaatgac atgtcttcaa tgtccacaga tccgactttg
gcctcggata cagacagcag 1440 tctagaagca tcagctggac ctctgggctg
ctgtagatga ctacttgggc cttgggtggg 1500 tgggagggat ggggaattgg
ttagtcattg atagaactgc tttaaaaaca attcagtggt 1560 catatttttg
agtgattttt cagaaaatgt agaattcatt ttgtagtaaa gtagtttatt 1620
ttttttaatt tcaagtgttg taattc 1646 <210> SEQ ID NO 107
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 107
ccttccctga aggttcctcc 20 <210> SEQ ID NO 108 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 108 tgttgtcacg
tttgcttctg 20 <210> SEQ ID NO 109 <400> SEQUENCE: 109
000 <210> SEQ ID NO 110 <400> SEQUENCE: 110 000
<210> SEQ ID NO 111 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 111 caacgtcccg cgctcggccg 20 <210> SEQ
ID NO 112 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
112 cctgctcgcg gctccgcgtt 20 <210> SEQ ID NO 113 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 113 ctcatgatgg
caagcaatta 20 <210> SEQ ID NO 114 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 114 tgttgtcacg tttacttctg 20
<210> SEQ ID NO 115 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 115 cggtaggctc gcttagcatg 20 <210> SEQ
ID NO 116 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
116 ctagggattt ctgtggtgtg 20 <210> SEQ ID NO 117 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 117 cagcagagtg
aaggtgcttg 20 <210> SEQ ID NO 118 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 118 tcgttcctgc agtccttgcc 20
<210> SEQ ID NO 119 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 119 ccatttctcc cataatgcac 20 <210> SEQ
ID NO 120 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
120 tgaattcagg acaaggtgtt 20 <210> SEQ ID NO 121 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 121 agcttcgtct
acggagatcc 20 <210> SEQ ID NO 122 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 122 cactcctcta ttgtgtgctc 20
<210> SEQ ID NO 123 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 123 gctgcaccta aaggagacgg 20 <210> SEQ
ID NO 124 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
124 ccagagtcgg atctgtggac 20 <210> SEQ ID NO 125 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 125 tcatgatgta
gtgtcataca 20 <210> SEQ ID NO 126 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 126 tgtggtgtga acacatttaa 20
<210> SEQ ID NO 127 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 127 ccatatgaat aacctgacat 20 <210> SEQ
ID NO 128 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
128 gatatcaaca ttctccttgt 20 <210> SEQ ID NO 129 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 129 gcttcgtcca
cagagatccg 20 <210> SEQ ID NO 130 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 130 gctcagtgga catggatgag 20
<210> SEQ ID NO 131 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 131 atctgcgagg tttcatcggc 20 <210> SEQ
ID NO 132 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
132 ccaccagctc ccatgtgctc 20 <210> SEQ ID NO 133 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 133 cagttacaca
tgatctgtca 20 <210> SEQ ID NO 134 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 134 aagaggatta agagattatt 20
<210> SEQ ID NO 135 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 135 agcagagtga aatacaactt 20 <210> SEQ
ID NO 136 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
136 tgtcagctct acattaggca 20 <210> SEQ ID NO 137 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 137 agtaagcccg
gtctcctaag 20 <210> SEQ ID NO 138 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 138 aaatggaaaa ggacagcagc 20
<210> SEQ ID NO 139 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 139 gctcagtgga tatggatgag 20 <210> SEQ
ID NO 140 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
140 gctaagcggt caaggttgag 20 <210> SEQ ID NO 141 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 141 gctcggtgga
aatggatcag 20 <210> SEQ ID NO 142 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 142 gggctttcat tagccacatt 20
<210> SEQ ID NO 143 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 143 ggttggttca ctgcagtagt 20 <210> SEQ
ID NO 144 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
144 tgctcatgtt gtaatgtttg 20 <210> SEQ ID NO 145 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 145 gtcgaggaca
gcgtcatacg 20 <210> SEQ ID NO 146 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 146 cgacatccgc tcgtggtcca 20
<210> SEQ ID NO 147 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 147 acatacggag tcatcatgaa 20 <210> SEQ
ID NO 148 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
148 gcaatttctt catgaattct 20 <210> SEQ ID NO 149 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 149 tcgtaccaaa
cgttgatgta 20 <210> SEQ ID NO 150 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 150 cgccgaggct tccaggctgc 20
<210> SEQ ID NO 151 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 151 ggctagtcac ctgcaacaac 20 <210> SEQ
ID NO 152 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
152 gcgtgcgtgc gtgcttgcgt 20 <210> SEQ ID NO 153 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 153 gctcagctgc
gatacagaac 20 <210> SEQ ID NO 154 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 154 agcgcgacta gaagttaagt 20
<210> SEQ ID NO 155 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 155 agggagacca aagtcgagcg 20 <210> SEQ
ID NO 156 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
156 acatcttgaa attcttctag 20 <210> SEQ ID NO 157 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 157 taggatattc
tttcatgatc 20 <210> SEQ ID NO 158 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 158 agaaggtagg acattctttc 20
<210> SEQ ID NO 159 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 159 tttattccac tgatcaatat 20 <210> SEQ
ID NO 160 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
160 tcaataactt tattccactg 20 <210> SEQ ID NO 161 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 161 ggttgcagtt
tcttcatgaa 20 <210> SEQ ID NO 162 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: 6 <223> OTHER INFORMATION:
n = I <400> SEQUENCE: 162 tagganattc tttcatgatc 20
<210> SEQ ID NO 163 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: 8 <223> OTHER INFORMATION: n = I <400>
SEQUENCE: 163 ggttgcantt tcttcatgaa 20
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 163
<210> SEQ ID NO 1 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 1 attctttcca ctcttctatt 20 <210> SEQ ID
NO 2 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
2 ctcctccaag tccataactt 20 <210> SEQ ID NO 3 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 3 cccgtataac
tccattcttg 20 <210> SEQ ID NO 4 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 4 ctgtgctaaa ggagagggct 20
<210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 5 atgatggatg ctgagagcca 20 <210> SEQ ID
NO 6 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
6 gttgacattg aagacacatc 20 <210> SEQ ID NO 7 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 7 ctgtatcaga
ggccaaagtc 20 <210> SEQ ID NO 8 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 8 tgctgcttct agactgctgt 20
<210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 9 agtcatctac agcagcccag 20 <210> SEQ ID
NO 10 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
10 ccatccctcc caccccccga 20 <210> SEQ ID NO 11 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 11 atcaatgact
aaccgactcc 20 <210> SEQ ID NO 12 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 12 caaaaataag accactgaat 20
<210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 13 cacgcttgct tctgctcatg 20 <210> SEQ
ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
14 cggcttagct tcttgattgc 20 <210> SEQ ID NO 15 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 15 cccgcttggc
atgagtctga 20 <210> SEQ ID NO 16 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 16 ctctctgtag gcccgcttgg 20
<210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 17 atttgcatcc atgagctcca 20 <210> SEQ
ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
18 cgttcctgca gtcctggcca 20 <210> SEQ ID NO 19 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 19 ggatgacctc
gggtgctctg 20 <210> SEQ ID NO 20 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 20 cccataatgc accccacaga 20
<210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 21 cgggtgttgg agagcttcat 20
<210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 22 tttggtggtg gagcttctgc 20 <210> SEQ
ID NO 23 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
23 ggctgccccc gtataactcc 20 <210> SEQ ID NO 24 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 24 tgctaaagga
gagggctgcc 20 <210> SEQ ID NO 25 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 25 aggccaaagt cggatctgtt 20
<210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 26 ccaccccccg atggcccaag 20 <210> SEQ
ID NO 27 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
27 ccaagcgggc ctacagagag 20 <210> SEQ ID NO 28 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 28 ctttccgttg
gacccctggg 20 <210> SEQ ID NO 29 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 29 gtttcagatc cctcgcccgc 20
<210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 30 tgcagcacaa acaatccctt 20 <210> SEQ
ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
31 gtccgggcca ggccaaagtc 20 <210> SEQ ID NO 32 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 32 caggatgact
tcgggcgccc 20 <210> SEQ ID NO 33 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 33 gctctcccat gatgcaaccc 20
<210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 34 atgggtgacg cagagcttcg 20 <210> SEQ
ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
35 ctgctgcatc tgaaggctga 20 <210> SEQ ID NO 36 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 36 tgagaaggag
tggcgttgct 20 <210> SEQ ID NO 37 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 37 tgctgtctgt gtctgaggcc 20
<210> SEQ ID NO 38 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 38 ggtcccgtcg aggcatcaag 20 <210> SEQ
ID NO 39 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
39 catttcaggc ccacggaggt 20 <210> SEQ ID NO 40 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 40 ggtctgaata
gggcaaggca 20 <210> SEQ ID NO 41 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 41 gggcaagtcc aagcaagcat 20
<210> SEQ ID NO 42 <211> LENGTH: 20 <212> TYPE:
DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 42 gactttggcc tggcccggac 20 <210> SEQ
ID NO 43 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
43 gtgcgcgcga gcccgaaatc 20 <210> SEQ ID NO 44 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 44 ttcaacagtt
tcttgcataa 20 <210> SEQ ID NO 45 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 45 ctcatctata ggaaacgggt 20
<210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 46 tggaggctca taaataccac 20 <210> SEQ
ID NO 47 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
47 tataagaaat ggaggctcat 20 <210> SEQ ID NO 48 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 48 tcacatccaa
tgttggttca 20 <210> SEQ ID NO 49 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 49 ttatcgaatc cctgacaaaa 20
<210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 50 gtttggcaat atatgacaca 20 <210> SEQ
ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
51 ctgtcaagga cagcatcata 20 <210> SEQ ID NO 52 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 52 aatcacttga
cataagttgg 20 <210> SEQ ID NO 53 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 53 taaatccctg tgaataattc 20
<210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 54 gcatcccaca gaccatatat 20 <210> SEQ
ID NO 55 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
55 tgttctcttt catccaactg 20 <210> SEQ ID NO 56 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 56 tctcactgct
gttcactgct 20 <210> SEQ ID NO 57 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 57 gggtctggtc ggtggacatg 20
<210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 58 aggctgctgt cagtgtcaga 20 <210> SEQ
ID NO 59 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
59 tcacctgcaa caacccaggg 20 <210> SEQ ID NO 60 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 60 gcggctagtc
acctgcaaca 20 <210> SEQ ID NO 61 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 61 cgctgggttt cgcaggcagg 20
<210> SEQ ID NO 62 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 62 atcatctcct gaagaacgct 20 <210> SEQ
ID NO 63 <211> LENGTH: 35
<212> TYPE: DNA <213> ORGANISM: Homo sapien <400>
SEQUENCE: 63 aacgtggatt tatggtctgt ggggtgcatt atggg 35 <210>
SEQ ID NO 64 <211> LENGTH: 35 <212> TYPE: DNA
<213> ORGANISM: Homo sapien <400> SEQUENCE: 64
aacgttgaca tttggtcagt tgggtgcatc atggg 35 <210> SEQ ID NO 65
<211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM:
Homo sapien <400> SEQUENCE: 65 cccataatgc accccacaga
ccataaatcc acgtt 35 <210> SEQ ID NO 66 <211> LENGTH: 35
<212> TYPE: DNA <213> ORGANISM: Homo sapien <400>
SEQUENCE: 66 cccatgatgc acccaactga ccaaatgtca acgtt 35 <210>
SEQ ID NO 67 <211> LENGTH: 44 <212> TYPE: DNA
<213> ORGANISM: Homo sapien <400> SEQUENCE: 67
aaatggtttg ccacaaaatc ctctttccag gaagggacta tatt 44 <210> SEQ
ID NO 68 <211> LENGTH: 44 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 68 aaatgatcaa
aggtggtgtt ttgttcccag gtacagatca tatt 44 <210> SEQ ID NO 69
<211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM:
Homo sapien <400> SEQUENCE: 69 aatatagtcc cttcctggaa
agaggatttt gtggcaaacc attt 44 <210> SEQ ID NO 70 <211>
LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Homo sapien
<400> SEQUENCE: 70 aatatgatct gtacctggga acaaaacacc
acctttgatc attt 44 <210> SEQ ID NO 71 <211> LENGTH: 32
<212> TYPE: DNA <213> ORGANISM: Homo sapien <400>
SEQUENCE: 71 ccctctcctt tagcacaggt gcagcagtga tc 32 <210> SEQ
ID NO 72 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 72 ccctctcctt
taggtgcagc agtgatc 27 <210> SEQ ID NO 73 <211> LENGTH:
32 <212> TYPE: DNA <213> ORGANISM: Homo sapien
<400> SEQUENCE: 73 gatcactgct gcacctgtgc taaaggagag gg 32
<210> SEQ ID NO 74 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 74
gatcactgct gcacctaaag gagaggg 27 <210> SEQ ID NO 75
<211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM:
Homo sapien <400> SEQUENCE: 75 gtgggttgca tcatgggaga
gctggtgaaa ggttgtgtga tattccaagg cactgaccat 60 <210> SEQ ID
NO 76 <211> LENGTH: 60 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 76 gtcgggtgca
tcatggcaga aatggtcctc cataaagtcc tgttcccggg aagagactat 60
<210> SEQ ID NO 77 <211> LENGTH: 60 <212> TYPE:
DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 77
atggtcagtg ccttggaata tcacacaacc tttcaccagc tctcccatga tgcaacccac
60 <210> SEQ ID NO 78 <211> LENGTH: 60 <212>
TYPE: DNA <213> ORGANISM: Homo sapien <400> SEQUENCE:
78 atagtctctt cccgggaaca ggactttatg gaggaccatt tctgccatga
tgcacccgac 60 <210> SEQ ID NO 79 <211> LENGTH: 35
<212> TYPE: DNA <213> ORGANISM: Homo sapien <400>
SEQUENCE: 79 gatcagcctt cagcacagat gcagcagtaa gtagc 35 <210>
SEQ ID NO 80 <211> LENGTH: 30 <212> TYPE: DNA
<213> ORGANISM: Homo sapien <400> SEQUENCE: 80
gatcagcctt cagatgcagc agtaagtagc 30 <210> SEQ ID NO 81
<211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM:
Homo sapien <400> SEQUENCE: 81 gctacttact gctgcatctg
tgctgaaggc tgatc 35 <210> SEQ ID NO 82 <211> LENGTH: 30
<212> TYPE: DNA <213> ORGANISM: Homo sapien <400>
SEQUENCE: 82 gctacttact gctgcatctg aaggctgatc 30 <210> SEQ ID
NO 83 <211> LENGTH: 38 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 83 ggacagcctt
ctccttcagc acaggtgcag cagtgaac 38 <210> SEQ ID NO 84
<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:
Homo sapien <400> SEQUENCE: 84 ggacagcctt ctccttcagg
tgcagcagtg aac 33 <210> SEQ ID NO 85 <211> LENGTH: 38
<212> TYPE: DNA <213> ORGANISM: Homo sapien <400>
SEQUENCE: 85 gttcactgct gcacctgtgc tgaaggagaa ggctgtcc 38
<210> SEQ ID NO 86 <211> LENGTH: 33 <212> TYPE:
DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 86
gttcactgct gcacctgaag gagaaggctg tcc 33 <210> SEQ ID NO 87
<211> LENGTH: 1418 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 87 cattaattgc
ttgccatcat gagcagaagc aagcgtgaca acaattttta tagtgtagag 60
attggagatt ctacattcac agtcctgaaa cgatatcaga atttaaaacc tataggctca
120 ggagctcaag gaatagtatg cgcagcttat gatgccattc ttgaaagaaa
tgttgcaatc 180
aagaagctaa gccgaccatt tcagaatcag actcatgcca agcgggccta cagagagcta
240 gttcttatga aatgtgttaa tcacaaaaat ataattggcc ttttgaatgt
tttcacacca 300 cagaaatccc tagaagaatt tcaagatgtt tacatagtca
tggagctcat ggatgcaaat 360 ctttgccaag tgattcagat ggagctagat
catgaaagaa tgtcctacct tctctatcag 420 atgctgtgtg gaatcaagca
ccttcattct gctggaatta ttcatcggga cttaaagccc 480 agtaatatag
tagtaaaatc tgattgcact ttgaagattc ttgacttcgg tctggccagg 540
actgcaggaa cgagttttat gatgacgcct tatgtagtga ctcgctacta cagagcaccc
600 gaggtcatcc ttggcatggg ctacaaggaa aacgtggatt tatggtctgt
ggggtgcatt 660 atgggagaaa tggtttgcca caaaatcctc tttccaggaa
gggactatat tgatcagtgg 720 aataaagtta ttgaacagct tggaacacca
tgtcctgaat tcatgaagaa actgcaacca 780 acagtaagga cttacgttga
aaacagacct aaatatgctg gatatagctt tgagaaactc 840 ttccctgatg
tccttttccc agctgactca gaacacaaca aacttaaagc cagtcaggca 900
agggatttgt tatccaaaat gctggtaata gatgcatcta aaaggatctc tgtagatgaa
960 gctctccaac acccgtacat caatgtctgg tatgatcctt ctgaagcaga
agctccacca 1020 ccaaagatcc ctgacaagca gttagatgaa agggaacaca
caatagaaga gtggaaagaa 1080 ttgatatata aggaagttat ggacttggag
gagagaacca agaatggagt tatacggggg 1140 cagccctctc ctttagcaca
ggtgcagcag tgatcaatgg ctctcagcat ccatcatcat 1200 cgtcgtctgt
caatgatgtg tcttcaatgt caacagatcc gactttggcc tctgatacag 1260
acagcagtct agaagcagca gctgggcctc tgggctgctg tagatgacta cttgggccat
1320 cggggggtgg gagggatggg gagtcggtta gtcattgata gaactacttt
gaaaacaatt 1380 cagtggtctt atttttgggt gatttttcaa aaaatgta 1418
<210> SEQ ID NO 88 <211> LENGTH: 1408 <212> TYPE:
DNA <213> ORGANISM: Rattus norvegicus <400> SEQUENCE:
88 gcggccgagc gcgggacgtt gcggccgaaa cgcggagccg cgagcaggat
taagtagcgg 60 cccggccacc ggcacggcgc cgctctccgc tactggcttc
caggtctccg ttggctgcac 120 tgccggccgg ttgttgaata tttggatgaa
gccattagac taattgcttg ccatcatgag 180 cagaagtaaa cgtgacaaca
atttttatag tgtagagatc gcagattcta cattcacagt 240 cctaaaacga
taccagaact taaagcctat aggctcagga gctcaaggaa tagtgtgtgc 300
agcttatgat gctattcttg aaagaaatgt tgcaatcaag aagctcagcc ggccatttca
360 gaatcagacc catgctaagc gagcctaccg agaactagtt cttatgaagt
gtgttaatca 420 caaaaatata attggccttt tgaatgtttt cacaccacag
aaatccctag aagaatttca 480 agatgtttac atagtcatgg agctcatgga
tgcaaatctt tgccaagtga ttcagatgga 540 gttagatcat gaaagaatgt
cctaccttct ctatcaaatg ctgtgtggaa tcaagcacct 600 tcactctgct
ggaattattc atcgggactt aaagcctagt aatatagtag tcaaatcaga 660
ctgcactttg aagattcttg attttggact ggcaaggact gcaggaacga gttttatgat
720 gacgccttac gtggtaactc gttactacag agcaccagag gtcattctcg
gcatgggcta 780 caaggagaac gtggatttat ggtctgtggg gtgcattatg
ggagaaatgg tttgcctcaa 840 aatcctcttt ccaggaaggg actatattga
tcagtggaat aaagttattg aacagctcgg 900 aacaccttgt cctgaattca
tgaagaaact acaaccaaca gtaaggactt acgttgaaaa 960 cagacctaag
tacgctggct atagctttga gaaactgttt cctgatgtgc ttttcccagc 1020
tgactcagaa cataacaaac ttaaagccag tcaggcgaga gatttgttat ctaaaatgct
1080 ggtgatagat gcgtccaaaa ggatctccgt agacgaagct ctccagcacc
cgtacatcaa 1140 cgtctggtat gatccttcag aagcagaggc cccaccacca
aagatccctg acaagcagtt 1200 agatgaaagg gagcacacaa tagaggagtg
gaaagaactg atatacaagg aggtcatgga 1260 tttggaggag cgaactaaga
atggcgtcat aagagggcag ccgtctcctt taggtgcagc 1320 agtgatcaat
ggctctcagc atccggtctc ttcgccgtct gtcaatgaca tgtcttcaat 1380
gtccacagat ccgactctgg cctcggat 1408 <210> SEQ ID NO 89
<211> LENGTH: 1365 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 89 ttgcttgcca
tcatgagcag aagcaagcgt gacaacaatt tttatagtgt agagattgga 60
gattctacat tcacagtcct gaaacgatat cagaatttaa aacctatagg ctcaggagct
120 caaggaatag tatgcgcagc ttatgatgcc attcttgaaa gaaatgttgc
aatcaagaag 180 ctaagccgac catttcagaa tcagactcat gccaagcggg
cctacagaga gctagttctt 240 atgaaatgtg ttaatcacaa aaatataatt
ggccttttga atgttttcac accacagaaa 300 tccctagaag aatttcaaga
tgtttacata gtcatggagc tcatggatgc aaatctttgc 360 caagtgattc
agatggagct agatcatgaa agaatgtcct accttctcta tcagatgctg 420
tgtggaatca agcaccttca ttctgctgga attattcatc gggacttaaa gcccagtaat
480 atagtagtaa aatctgattg cactttgaag attcttgact tcggtctggc
caggactgca 540 ggaacgagtt ttatgatgac gccttatgta gtgactcgct
actacagagc acccgaggtc 600 atccttggca tgggctacaa ggaaaacgtg
gatttatggt ctgtggggtg cattatggga 660 gaaatggttt gccacaaaat
cctctttcca ggaagggact atattgatca gtggaataaa 720 gttattgaac
agcttggaac accatgtcct gaattcatga agaaactgca accaacagta 780
aggacttacg ttgaaaacag acctaaatat gctggatata gctttgagaa actcttccct
840 gatgtccttt tcccagctga ctcagaacac aacaaactta aagccagtca
ggcaagggat 900 ttgttatcca aaatgctggt aatagatgca tctaaaagga
tctctgtaga tgaagctctc 960 caacacccgt acatcaatgt ctggtatgat
ccttctgaag cagaagctcc accaccaaag 1020 atccctgaca agcagttaga
tgaaagggaa cacacaatag aagagtggaa agaattgata 1080 tataaggaag
ttatggactt ggaggagaga accaagaatg gagttatacg ggggcagccc 1140
tctcctttag gtgcagcagt gatcaatggc tctcagcatc catcatcatc gtcgtctgtc
1200 aatgatgtgt cttcaatgtc aacagatccg actttggcct ctgatacaga
cagcagtcta 1260 gaagcagcag ctgggcctct gggctgctgt agatgactac
ttgggccatc ggggggtggg 1320 agggatgggg agtcggttag tcattgatag
aactactttg aaaac 1365 <210> SEQ ID NO 90 <211> LENGTH:
1311 <212> TYPE: DNA <213> ORGANISM: Homo sapien
<400> SEQUENCE: 90 cattaattgc ttgccatcat gagcagaagc
aagcgtgaca acaattttta tagtgtagag 60 attggagatt ctacattcac
agtcctgaaa cgatatcaga atttaaaacc tataggctca 120 ggagctcaag
gaatagtatg cgcagcttat gatgccattc ttgaaagaaa tgttgcaatc 180
aagaagctaa gccgaccatt tcagaatcag actcatgcca agcgggccta cagagagcta
240 gttcttatga aatgtgttaa tcacaaaaat ataattggcc ttttgaatgt
tttcacacca 300 cagaaatccc tagaagaatt tcaagatgtt tacatagtca
tggagctcat ggatgcaaat 360 ctttgccaag tgattcagat ggagctagat
catgaaagaa tgtcctacct tctctatcag 420 atgctgtgtg gaatcaagca
ccttcattct gctggaatta ttcatcggga cttaaagccc 480 agtaatatag
tagtaaaatc tgattgcact ttgaagattc ttgacttcgg tctggccagg 540
actgcaggaa cgagttttat gatgacgcct tatgtagtga ctcgctacta cagagcaccc
600 gaggtcatcc ttggcatggg ctacaaggaa aacgttgaca tttggtcagt
tgggtgcatc 660 atgggagaaa tgatcaaagg tggtgttttg ttcccaggta
cagatcatat tgatcagtgg 720 aataaagtta ttgaacagct tggaacacca
tgtcctgaat tcatgaagaa actgcaacca 780 acagtaagga cttacgttga
aaacagacct aaatatgctg gatatagctt tgagaaactc 840 ttccctgatg
tccttttccc agctgactca gaacacaaca aacttaaagc cagtcaggca 900
agggatttgt tatccaaaat gctggtaata gatgcatcta aaaggatctc tgtagatgaa
960 gctctccaac acccgtacat caatgtctgg tatgatcctt ctgaagcaga
agctccacca 1020 ccaaagatcc ctgacaagca gttagatgaa agggaacaca
caatagaaga gtggaaagaa 1080 ttgatatata aggaagttat ggacttggag
gagagaacca agaatggagt tatacggggg 1140 cagccctctc ctttagcaca
ggtgcagcag tgatcaatgg ctctcagcat ccatcatcat 1200 cgtcgtctgt
caatgatgtg tcttcaatgt caacagatcc gactttggcc tctgatacag 1260
acagcagtct agaagcagca gctgggcctc tgggctgctg tagatgacta c 1311
<210> SEQ ID NO 91 <211> LENGTH: 1349 <212> TYPE:
DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 91
attgcttgcc atcatgagca gaagcaagcg tgacaacaat ttttatagtg tagagattgg
60 agattctaca ttcacagtcc tgaaacgata tcagaattta aaacctatag
gctcaggagc 120 tcaaggaata gtatgcgcag cttatgatgc cattcttgaa
agaaatgttg caatcaagaa 180 gctaagccga ccatttcaga atcagactca
tgccaagcgg gcctacagag agctagttct 240 tatgaaatgt gttaatcaca
aaaatataat tggccttttg aatgttttca caccacagaa 300 atccctagaa
gaatttcaag atgtttacat agtcatggag ctcatggatg caaatctttg 360
ccaagtgatt cagatggagc tagatcatga aagaatgtcc taccttctct atcagatgct
420 gtgtggaatc aagcaccttc attctgctgg aattattcat cgggacttaa
agcccagtaa 480 tatagtagta aaatctgatt gcactttgaa gattcttgac
ttcggtctgg ccaggactgc 540 aggaacgagt tttatgatga cgccttatgt
agtgactcgc tactacagag cacccgaggt 600 catccttggc atgggctaca
aggaaaacgt tgacatttgg tcagttgggt gcatcatggg 660 agaaatgatc
aaaggtggtg ttttgttccc aggtacagat catattgatc agtggaataa 720
agttattgaa cagcttggaa caccatgtcc tgaattcatg aagaaactgc aaccaacagt
780 aaggacttac gttgaaaaca gacctaaata tgctggatat agctttgaga
aactcttccc 840 tgatgtcctt ttcccagctg actcagaaca caacaaactt
aaagccagtc aggcaaggga 900 tttgttatcc aaaatgctgg taatagatgc
atctaaaagg atctctgtag atgaagctct 960 ccaacacccg tacatcaatg
tctggtatga tccttctgaa gcagaagctc caccaccaaa 1020 gatccctgac
aagcagttag atgaaaggga acacacaata gaagagtgga aagaattgat 1080
atataaggaa gttatggact tggaggagag aaccaagaat ggagttatac gggggcagcc
1140 ctctccttta ggtgcagcag tgatcaatgg ctctcagcat ccatcatcat
cgtcgtctgt 1200
caatgatgtg tcttcaatgt caacagatcc gactttggcc tctgatacag acagcagtct
1260 agaagcagca gctgggcctc tgggctgctg tagatgacta cttgggccat
cggggggtgg 1320 gagggatggg gagtcggtta gtcattgat 1349 <210>
SEQ ID NO 92 <211> LENGTH: 1782 <212> TYPE: DNA
<213> ORGANISM: Homo sapien <400> SEQUENCE: 92
gggcgggcga gggatctgaa acttgcccac ccttcgggat attgcaggac gctgcatcat
60 gagcgacagt aaatgtgaca gtcagtttta tagtgtgcaa gtggcagact
caaccttcac 120 tgtcctaaaa cgttaccagc agctgaaacc aattggctct
ggggcccaag ggattgtttg 180 tgctgcattt gatacagttc ttgggataag
tgttgcagtc aagaaactaa gccgtccttt 240 tcagaaccaa actcatgcaa
agagagctta tcgtgaactt gtcctcttaa aatgtgtcaa 300 tcataaaaat
ataattagtt tgttaaatgt gtttacacca caaaaaactc tagaagaatt 360
tcaagatgtg tatttggtta tggaattaat ggatgctaac ttatgtcagg ttattcacat
420 ggagctggat catgaaagaa tgtcctacct tctttaccag atgctttgtg
gtattaaaca 480 tctgcattca gctggtataa ttcatagaga tttgaagcct
agcaacattg ttgtgaaatc 540 agactgcacc ctgaagatcc ttgactttgg
cctggcccgg acagcgtgca ctaacttcat 600 gatgacccct tacgtggtga
cacggtacta ccgggcgccc gaagtcatcc tgggtatggg 660 ctacaaagag
aacgttgata tctggtcagt gggttgcatc atgggagagc tggtgaaagg 720
ttgtgtgata ttccaaggca ctgaccatat tgatcagtgg aataaagtta ttgagcagct
780 gggaacacca tcagcagagt tcatgaagaa acttcagcca actgtgagga
attatgtcga 840 aaacagacca aagtatcctg gaatcaaatt tgaagaactc
tttccagatt ggatattccc 900 atcagaatct gagcgagaca aaataaaaac
aagtcaagcc agagatctgt tatcaaaaat 960 gttagtgatt gatcctgaca
agcggatctc tgtagacgaa gctctgcgtc acccatacat 1020 cactgtttgg
tatgaccccg ccgaagcaga agccccacca cctcaaattt atgatgccca 1080
gttggaagaa agagaacatg caattgaaga atggaaagag ctaatttaca aagaagtcat
1140 ggattgggaa gaaagaagca agaatggtgt tgtaaaagat cagccttcag
atgcagcagt 1200 aagtagcaac gccactcctt ctcagtcttc atcgatcaat
gacatttcat ccatgtccac 1260 tgagcagacg ctggcctcag acacagacag
cagtcttgat gcctcgacgg gaccccttga 1320 aggctgtcga tgataggtta
gaaatagcaa acctgtcagc attgaaggaa ctctcacctc 1380 cgtgggcctg
aaatgcttgg gagttgatgg aaccaaatag aaaaactcca tgttctgcat 1440
gtaagaaaca caatgccttg ccctattcag acctgatagg attgcctgct tagatgataa
1500 aatgaggcag aatatgtctg aagaaaaaaa ttgcaagcca cacttctaga
gattttgttc 1560 aagatcattt caggtgagca gttagagtag gtgaatttgt
ttcaaattgt actagtgaca 1620 gtttctcatc atctgtaact gttgagatgt
atgtgcatgt gaccacaaat gcttgcttgg 1680 acttgcccat ctagcacttt
ggaaatcagt atttaaatgc caaataatct tccaggtagt 1740 gctgcttctg
aagttatctc ttaatcctct taagtaattt gg 1782 <210> SEQ ID NO 93
<211> LENGTH: 2622 <212> TYPE: DNA <213>
ORGANISM: Rattus norvegicus <400> SEQUENCE: 93 tgtatgacac
tacatcatga gtgacagtaa aagcgatggc cagttttaca gtgtgcaagt 60
ggcagactca actttcactg ttctaaaacg ttaccagcag ttgaaaccaa ttggctctgg
120 agcccaagga attgtttgtg ctgcttttga tacagttctt ggaataaatg
ttgctgtcaa 180 gaagttaagt cgtccttttc agaaccaaac gcatgcaaag
agagcctacc gtgaacttgt 240 cctcctaaag tgtgtcaatc ataaaaatat
aattagcttg ttaaatgtgt tcacaccaca 300 aaaaacgcta gaagaattcc
aagatgtgta cttggttatg gagttaatgg acgctaactt 360 atgtcaggtt
attcatatgg agctggacca tgaaagaatg tcatacctcc tctaccagat 420
gctttgtggc attaagcacc tgcattcagc tggcataatt catagggatt tgaagcctag
480 caacattgta gtaaaatcag actgtactct caagatcctt gactttggcc
tggcacggac 540 agcctgtacc aactttatga tgactcccta tgtggtaact
cgctactatc gggctccaga 600 agtcatcctg ggcatgggct acaaggagaa
tgttgatatc tggtcagtgg gttgcatcat 660 gggagagctg gtgaaaggtt
gtgtgatatt ccaaggtact gaccatattg atcaatggaa 720 taaagttatt
gaacagctag gaacaccatc cgcagagttc atgaagaaac ttcagccaac 780
tgtaaggaat tatgtggaaa acagaccaaa gtaccctgga atcaaatttg aagagctctt
840 tccagattgg atatttccgt cagaatccga acgagacaaa ataaaaacaa
gtcaagccag 900 agatctgtta tcgaaaatgt tagtgattga tccggacaag
cggatctctg tggacgaagc 960 cttgcgccac ccgtatatta ctgtttggta
tgaccccgct gaagcagaag cgccaccacc 1020 tcaaatttat gatgcccagt
tggaagaaag agagcatgcg attgaagagt ggaaagaact 1080 aatttacaaa
gaagtgatgg actgggaaga aagaagcaag aatggggtga aagaccagcc 1140
ttcagatgca gcagtaagca gcaaggctac tccttctcag tcgtcatcca tcaatgacat
1200 ctcatccatg tccactgagc acaccctggc ctcagacaca gacagcagtc
tcgatgcctc 1260 aaccggaccc ctggaaggct gccgatgaaa cctcgcagat
ggcgcacttg tctgtgaagg 1320 actctggctt ccatggccct gagcacatgg
gagctggtgg aacaaatcaa gaagctccat 1380 gttctgcatg taagaaacac
gacgccttgc ccccactcag ttccagtagg attgcctgcg 1440 tagactgtaa
catgaggcag acgatgtctg gagaaaaagt acaaaccaca ctgttagaaa 1500
ttttgttcaa gatcattcag gtgagcaatt agaatagccg agttcttttc aagtcgtgtg
1560 gtgtccttgg tgacagatca tgtgtaactg tggggactcg tatgcatgtg
accacaaatg 1620 cttgcttgaa cttgcccatg tagcactttg ggaatcagta
tttaaatgcc aaataatctt 1680 ccaggtagtt ctgcttctag aataatctct
taatcctctt tagtaatttg gtgtctgtcc 1740 acaaaaaaat agattatgtg
tgtatgaatt ggccactatc atattatcat attttaccca 1800 cttttatggt
atgatttatt ctgtcttttg tatttcagaa ggaatataat taaatttatt 1860
taataaataa aactacagct tttcttaaat ttgtgatgtt ttaggctgag aattaccact
1920 gctttatatc gacactctgt gtcctttaaa ctgcccacta tgggaaactt
tacgtacagc 1980 tttctgcatg acaaagttcc aagttgtatt tcactctgct
taacgactta tgtcaccttg 2040 aatcctgacc acacatttcc tttttcttgg
tcctctgaac ttggatctag aatccctcac 2100 agaacttcac cttctttatc
acaaagcacc ccatctcagt agaatgaatc ggcagattcc 2160 tgagccccgc
tgcctaatgt agagctgaca gggtggcttc cccagaacgg tgggtgggtg 2220
catccttccc tgagcccacc catcctttgc tcccctctct ttatttaagg tgaaaggtga
2280 ttgggtctca tagcctttcc ttttgtagca ttgcctaact tgtctttctc
actgacagaa 2340 gccaccacgt ccagccagag cacatggtct cttaggagac
cgggcttact taccatgcat 2400 gtttgctgct gtccttttcc attttgtgga
ggcatttcct ttttctaagg gaattcctca 2460 gatgttctag aaacattcag
aagaacgcag aagaaatatt ctagagaatt gggggttcat 2520 tcttgaatat
tttctgattt aaaactgctc acctgaaatt gatactttca gatcctgatc 2580
ttgtaaatta ctcgagattt ggtaagatgc tgagttctct gt 2622 <210> SEQ
ID NO 94 <211> LENGTH: 1873 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 94 caaactacgt
gctgtacagc tgcatcagct gctcgtagac atgtccagca gctggtcgag 60
gtccacgccg cggtaggtga agttgcggaa ggtccggcga gggatctgaa acttgcccct
120 tacccttcgg gatattgcag gacgctgcat catgagcgac agtaaatgtg
acagtcagtt 180 ttatagtgtc caagtggcag actcaacctt cactgtccta
aaacgttacc agcagctgaa 240 accaattggc tctggggccc aagggattgt
ttgtgctgca tttgatacag ttcttgggat 300 aaatgttgca gtcaagaaac
taagccgtcc ttttcagaac caaactcatg caaagagagc 360 ttatcgtgaa
cttgtcctct taaaatgtgt caatcataaa aatataatta gtttgttaaa 420
tgtgtttaca ccacaaaaaa ctctagaaga atttcaagat gtgtatttgg ttatggaatt
480 aatggatgct aacttatgtc aggttattca catggagctg gatcatgaaa
gaatgtccta 540 ccttctttac cagatgcttt gtggtattaa acatctgcat
tcagctggta taattcatag 600 agatttgaag cctagcaaca ttgttgtgaa
atcagactgc accctgaaga tccttgactt 660 tggcctggcc cggacagcgt
gcactaactt catgatgacc ccttacgtgg tgacacggta 720 ctaccgggcg
cccgaagtca tcctgggtat gggctacaaa gagaacgttg atatctggtc 780
agtgggttgc atcatgggag agctggtgaa aggttgtgtg atattccaag gcactgacca
840 tattgatcag tggaataaag ttattgagca gctgggaaca ccatcagcag
agttcatgaa 900 gaaacttcag ccaactgtga ggaattatgt cgaaaacaga
ccaaagtatc ctggaatcaa 960 atttgaagaa ctctttccag attggatatt
cccatcagaa tctgagcgag acaaaataaa 1020 aacaagtcaa gccagagatc
tgttatcaaa aatgttagtg attgatcctg acaagcggat 1080 ctctgtagac
gaagctctgc gtcacccata catcactgtt tggtatgacc ccgccgaagc 1140
agaagcccca ccacctcaaa tttatgatgc ccagttggaa gaaagagaac atgcaattga
1200 agaatggaaa gagctaattt acaaagaagt catggattgg gaagaaagaa
gcaagaatgg 1260 tgttgtaaaa gatcagcctc cagatgcagc agtaagtagc
aacgccactc cttctcagtc 1320 ttcatcgatc aatgacattt catccatgtc
cactgagcag acgctggcct cagacacaga 1380 cagcagtctt gatgcctcga
cgggacccct tgaaggctgt cgatgatagg ttagaaatag 1440 caaacctgtc
agcattgaag gaactctcac ctccgtgggc ctgaaatgct tgggagttga 1500
tggaaccaaa tagaaaaact ccatgttctg catgtaagaa acacaatgcc ttgccctact
1560 cagacctgat aggattgcct gcttagatga taaaatgagg cagaatatgt
ctgaagaaaa 1620 aaattgcaag ccacacttct agagattttg ttcaagatca
tttcagttga gcagttagag 1680 taggtgaatt tgtcaaattg tactagtgac
agtttctcat catctgtaac tgttgagatg 1740 attgtgcatg tgaccacaaa
tgcttgcttg gacttgccca tctagcactt tggaaatcag 1800 tatttaaatg
ccaaataatc ttccaggtag tgctgcttct gaagttatct cttaatcctc 1860
ttaagtaatt tgg 1873 <210> SEQ ID NO 95 <211> LENGTH:
1392 <212> TYPE: DNA <213> ORGANISM: Homo sapien
<400> SEQUENCE: 95
tctgaaactt gcccaccctt cgggatattg caggacgctg catcatgagc gacagtaaat
60 gtgacagtca gttttatagt gtgcaagtgg cagactcaac cttcactgtc
ctaaaacgtt 120 accagcagct gaaaccaatt ggctctgggg cccaagggat
tgtttgtgct gcatttgata 180 cagttcttgg gataagtgtt gcagtcaaga
aactaagccg tccttttcag aaccaaactc 240 atgcaaagag agcttatcgt
gaacttgtcc tcttaaaatg tgtcaatcat aaaaatataa 300 ttagtttgtt
aaatgtgttt acaccacaaa aaactctaga agaatttcaa gatgtgtatt 360
tggttatgga attaatggat gctaacttat gtcaggttat tcacatggag ctggatcatg
420 aaagaatgtc ctaccttctt taccagatgc tttgtggtat taaacatctg
cattcagctg 480 gtataattca tagagatttg aagcctagca acattgttgt
gaaatcagac tgcaccctga 540 agatccttga ctttggcctg gcccggacag
cgtgcactaa cttcatgatg accccttacg 600 tggtgacacg gtactaccgg
gcgcccgaag tcatcctggg tatgggctac aaagagaacg 660 ttgatatctg
gtcagtgggt tgcatcatgg gagagctggt gaaaggttgt gtgatattcc 720
aaggcactga ccatattgat cagtggaata aagttattga gcagctggga acaccatcag
780 cagagttcat gaagaaactt cagccaactg tgaggaatta tgtcgaaaac
agaccaaagt 840 atcctggaat caaatttgaa gaactctttc cagattggat
attcccatca gaatctgagc 900 gagacaaaat aaaaacaagt caagccagag
atctgttatc aaaaatgtta gtgattgatc 960 ctgacaagcg gatctctgta
gacgaagctc tgcgtcaccc atacatcact gtttggtatg 1020 accccgccga
agcagaagcc ccaccacctc aaatttatga tgcccagttg gaagaaagag 1080
aacatgcaat tgaagaatgg aaagagctaa tttacaaaga agtcatggat tgggaagaaa
1140 gaagcaagaa tggtgttgta aaagatcagc cttcagcaca gatgcagcag
taagtagcaa 1200 cgccactcct tctcagtctt catcgatcaa tgacatttca
tccatgtcca ctgagcagac 1260 gctggcctca gacacagaca gcagtcttga
tgcctcgacg ggaccccttg aaggctgtcg 1320 atgataggtt agaaatagca
aacctgtcag cattgaagga actctcacct ccgtgggcct 1380 gaaatgcttg gg 1392
<210> SEQ ID NO 96 <211> LENGTH: 1523 <212> TYPE:
DNA <213> ORGANISM: Homo sapien <400> SEQUENCE: 96
ggatattgca ggacgctgca tcatgagcga cagtaaatgt gacagtcagt tttatagtgt
60 gcaagtggca gactcaacct tcactgtcct aaaacgttac cagcagctga
aaccaattgg 120 ctctggggcc caagggattg tttgtgctgc atttgataca
gttcttggga taagtgttgc 180 agtcaagaaa ctaagccgtc cttttcagaa
ccaaactcat gcaaagagag cttatcgtga 240 acttgtcctc ttaaaatgtg
tcaatcataa aaatataatt agtttgttaa atgtgtttac 300 accacaaaaa
actctagaag aatttcaaga tgtgtatttg gttatggaat taatggatgc 360
taacttatgt caggttattc acatggagct ggatcatgaa agaatgtcct accttcttta
420 ccagatgctt tgtggtatta aacatctgca ttcagctggt ataattcata
gagatttgaa 480 gcctagcaac attgttgtga aatcagactg caccctgaag
atccttgact ttggcctggc 540 ccggacagcg tgcactaact tcatgatgac
cccttacgtg gtgacacggt actaccgggc 600 gcccgaagtc atcctgggta
tgggctacaa agagaacgtt gatatctggt cagtcgggtg 660 catcatggca
gaaatggtcc tccataaagt cctgttcccg ggaagagact atattgatca 720
gtggaataaa gttattgagc agctgggaac accatcagca gagttcatga agaaacttca
780 gccaactgtg aggaattatg tcgaaaacag accaaagtat cctggaatca
aatttgaaga 840 actctttcca gattggatat tcccatcaga atctgagcga
gacaaaataa aaacaagtca 900 agccagagat ctgttatcaa aaatgttagt
gattgatcct gacaagcgga tctctgtaga 960 cgaagctctg cgtcacccat
acatcactgt ttggtatgac cccgccgaag cagaagcccc 1020 accacctcaa
atttatgatg cccagttgga agaaagagaa catgcaattg aagaatggaa 1080
agagctaatt tacaaagaag tcatggattg ggaagaaaga agcaagaatg gtgttgtaaa
1140 agatcagcct tcagcacaga tgcagcagta agtagcaacg ccactccttc
tcagtcttca 1200 tcgatcaatg acatttcatc catgtccact gagcagacgc
tggcctcaga cacagacagc 1260 agtcttgatg cctcgacggg accccttgaa
ggctgtcgat gataggttag aaatagcaaa 1320 cctgtcagca ttgaaggaac
tctcacctcc gtgggcctga aatgcttggg agttgatgga 1380 accaaataga
aaaactccat gttctgcatg taagaaacac aatgccttgc cctattcaga 1440
cctgatagga ttgcctgctt agatgataaa atgaggcaga atatgtctga agaaaaaaat
1500 tgcaagccac acttctagag att 1523 <210> SEQ ID NO 97
<211> LENGTH: 1619 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 97 gcccaccctt
cgggatattg caggacgctg catcatgagc gacagtaaat gtgacagtca 60
gttttatagt gtgcaagtgg cagactcaac cttcactgtc ctaaaacgtt accagcagct
120 gaaaccaatt ggctctgggg cccaagggat tgtttgtgct gcatttgata
cagttcttgg 180 gataagtgtt gcagtcaaga aactaagccg tccttttcag
aaccaaactc atgcaaagag 240 agcttatcgt gaacttgtcc tcttaaaatg
tgtcaatcat aaaaatataa ttagtttgtt 300 aaatgtgttt acaccacaaa
aaactctaga agaatttcaa gatgtgtatt tggttatgga 360 attaatggat
gctaacttat gtcaggttat tcacatggag ctggatcatg aaagaatgtc 420
ctaccttctt taccagatgc tttgtggtat taaacatctg cattcagctg gtataattca
480 tagagatttg aagcctagca acattgttgt gaaatcagac tgcaccctga
agatccttga 540 ctttggcctg gcccggacag cgtgcactaa cttcatgatg
accccttacg tggtgacacg 600 gtactaccgg gcgcccgaag tcatcctggg
tatgggctac aaagagaacg ttgatatctg 660 gtcagtcggg tgcatcatgg
cagaaatggt cctccataaa gtcctgttcc cgggaagaga 720 ctatattgat
cagtggaata aagttattga gcagctggga acaccatcag cagagttcat 780
gaagaaactt cagccaactg tgaggaatta tgtcgaaaac agaccaaagt atcctggaat
840 caaatttgaa gaactctttc cagattggat attcccatca gaatctgagc
gagacaaaat 900 aaaaacaagt caagccagag atctgttatc aaaaatgtta
gtgattgatc ctgacaagcg 960 gatctctgta gacgaagctc tgcgtcaccc
atacatcact gtttggtatg accccgccga 1020 agcagaagcc ccaccacctc
aaatttatga tgcccagttg gaagaaagag aacatgcaat 1080 tgaagaatgg
aaagagctaa tttacaaaga agtcatggat tgggaagaaa gaagcaagaa 1140
tggtgttgta aaagatcagc cttcagatgc agcagtaagt agcaacgcca ctccttctca
1200 gtcttcatcg atcaatgaca tttcatccat gtccactgag cagacgctgg
cctcagacac 1260 agacagcagt cttgatgcct cgacgggacc ccttgaaggc
tgtcgatgat aggttagaaa 1320 tagcaaacct gtcagcattg aaggaactct
cacctccgtg ggcctgaaat gcttgggagt 1380 tgatggaacc aaatagaaaa
actccatgtt ctgcatgtaa gaaacacaat gccttgccct 1440 attcagacct
gataggattg cctgcttaga tgataaaatg aggcagaata tgtctgaaga 1500
aaaaaattgc aagccacact tctagagatt ttgttcaaga tcatttcagg tgagcagtta
1560 gagtaggtga atttgtttca aattgtacta gtgacagttt ctcatcatct
gtaactgtt 1619 <210> SEQ ID NO 98 <211> LENGTH: 2372
<212> TYPE: DNA <213> ORGANISM: Homo sapien <400>
SEQUENCE: 98 gagaaatggc gtggcagggg acccagcgag cccagaggga ttttgccgct
gcttcctcta 60 cccctgtatt tcacgcagct ctctaaattg actcagctcc
aggctagtgt gagaaacacc 120 aacagcaggc ccatctcaga tcttcactat
ggcaacttat gcaagaaact gttgaattag 180 acccgtttcc tatagatgag
aaaccataca agctgtggta tttatgagcc tccatttctt 240 atactactgc
agtgaaccaa cattggatgt gaaaattgcc ttttgtcagg gattcgataa 300
acaagtggat gtgtcatata ttgccaaaca ttacaacatg agcaaaagca aagttgacaa
360 ccagttctac agtgtggaag tgggagactc aaccttcaca gttctcaagc
gctaccagaa 420 tctaaagcct attggctctg gggctcaggg catagtttgt
gccgcgtatg atgctgtcct 480 tgacagaaat gtggccatta agaagctcag
cagacccttt cagaaccaaa cacatgccaa 540 gagagcgtac cgggagctgg
tcctcatgaa gtgtgtgaac cataaaaaca ttattagttt 600 attaaatgtc
ttcacacccc agaaaacgct ggaggagttc caagatgttt acttagtaat 660
ggaactgatg gatgccaact tatgtcaagt gattcagatg gaattagacc atgagcgaat
720 gtcttacctg ctgtaccaaa tgttgtgtgg cattaagcac ctccattctg
ctggaattat 780 tcacagggat ttaaaaccaa gtaacattgt agtcaagtct
gattgcacat tgaaaatcct 840 ggactttgga ctggccagga cagcaggcac
aagcttcatg atgactccat atgtggtgac 900 acgttattac agagcccctg
aggtcatcct ggggatgggc tacaaggaga acgtggatat 960 atggtctgtg
ggatgcatta tgggagaaat ggttcgccac aaaatcctct ttccaggaag 1020
ggactatatt gaccagtgga ataaggtaat tgaacaacta ggaacaccat gtccagaatt
1080 catgaagaaa ttgcaaccca cagtaagaaa ctatgtggag aatcggccca
agtatgcggg 1140 actcaccttc cccaaactct tcccagattc cctcttccca
gcggactccg agcacaataa 1200 actcaaagcc agccaagcca gggacttgtt
gtcaaagatg ctagtgattg acccagcaaa 1260 aagaatatca gtggacgacg
ccttacagca tccctacatc aacgtctggt atgacccagc 1320 cgaagtggag
gcgcctccac ctcagatata tgacaagcag ttggatgaaa gagaacacac 1380
aattgaagaa tggaaagaac ttatctacaa ggaagtaatg aattcagaag aaaagactaa
1440 aaatggtgta gtaaaaggac agccttctcc ttcagcacag gtgcagcagt
gaacagcagt 1500 gagagtctcc ctccatcctc gtctgtcaat gacatctcct
ccatgtccac cgaccagacc 1560 ctggcatctg acactgacag cagcctggaa
gcctcggcag gacccctggg ttgttgcagg 1620 tgactagccg cctgcctgcg
aaacccagcg ttcttcagga gatgatgtga tggaacacac 1680 acacacgcag
acacacacac acacacaaat gcagacacac aacatcaaga aaacagcaag 1740
ggagagaatc caagcctaaa attaaataaa tctttcagcc tgcttcttcc ccagggttct
1800 gtattgcagc taagctcaaa tgtatattta acttctagtt gctcttgctt
tggtcttctt 1860 ccaatgatgc ttactacaga aagcaaatca gacacaatta
gagaagcctt ttccataaag 1920 tgtaatttta atggctgcaa aaccggcaac
ctgtaactgc ccttttaaat ggcatgacaa 1980 ggtgtgcagt ggccccatcc
agcatgtgtg tgtctctatc ttgcatctac ctgctccttg 2040 gcctagtcag
atggatgtag atacagatcc gcatgtgtct gtattcatac agcactactt 2100
acttagagat gctactctca gtgtcctcag ggctctacca agacataatg cactggggta
2160 ccacatggtc catttcatgt gatctattac tctgacataa acccatctgt
aatatattgc 2220
cagtatataa gctgtttagt ttgttaattg attaaactgt atgtcttata agaaaacatg
2280 taaaggggga atatattggg ggagtgagct ctctcagacc cttgaagatg
tagcttccaa 2340 atttgaatgg attaaatggc acctgtatac ca 2372
<210> SEQ ID NO 99 <211> LENGTH: 1975 <212> TYPE:
DNA <213> ORGANISM: Rattus norvegicus <400> SEQUENCE:
99 ccctccttat tccggtttgg aatgtggcta atgaaagccc agtaggagga
tttctggggc 60 aaacaggtgg accaggatcc tggttctcag gcacggaatg
gctattgtga gagcgccacc 120 agcaggacca tcgcagatct tggttatggc
tgctcacgca agaggctgtt gatgtagacc 180 ccctttcccg tagatgagaa
atcacacgag cagtggtatt tatgagcctc catttcttat 240 actactgcag
tgaaccaacc ttggatgtga aaattgcctt ttgtcaggtg tgtgttcctt 300
acaggtaaaa caaagggatt cgacaaacac gtggatgtgt cttctgttgt caaacattac
360 aacatgagca aaagcaaggt agataaccag ttctacagtg tggaagtggg
agactcaacc 420 ttcacagttc taaagcgcta ccagaacctg aagccgatcg
gctctggggc tcagggaata 480 gtttgtgctg cgtatgacgc tgtcctcgac
agaaatgtgg ccattaagaa gctcagcaga 540 cccttccaga accaaactca
tgccaagagg gcttaccggg agctggtcct catgaagtgt 600 gtgaaccata
aaaacattat tagcttatta aatgtcttta caccccagaa aacactggag 660
gagttccaag atgtttactt agtgatggaa ctgatggacg ccaacttgtg tcaggtgatt
720 cagatggagc tggaccacga gcggatgtcg tacttgctgt accagatgct
gtcggcgatc 780 aaacacctcc actccgctgg gatcatccac agggacttaa
aacccagtaa catcgtagtc 840 aagtctgatt gcacactgaa aatcctggac
tttggactgg ccaggacagc gggcacaagc 900 ttcatgatga ctccgtatgt
ggtgacgaga tattacagag cccccgaggt catcctgggc 960 atgggctaca
aggagaacgt ggacatatgg tctgtgggct gcatcatggg agaaatggtt 1020
cgtcacaaaa tcctctttcc cggaagggac tatattgacc agtggaacaa agtcatagag
1080 cagctaggaa ctccgtgtcc agaattcatg aagaaattgc agcccaccgt
cagaaactac 1140 gtggagaacc ggcccaagta tgcaggcctc accttcccca
agctctttcc agattccctc 1200 ttcccagcgg attccgagca caataaactt
aaagccagcc aagccaggga cttgttgtca 1260 aagatgttag tgattgaccc
agcgaagagg atatcggtgg atgacgcatt gcagcatccg 1320 tacatcaacg
tttggtacga ccctgctgaa gtggaggcgc ctccgcctca gatatatgac 1380
aagcaattgg atgaaaggga gcacaccatc gaagaatgga aagaactcat ctacaaggaa
1440 gtaatgaact cagaagagaa gactaagaac ggcgtagtca aaggccagcc
ctcaccttca 1500 ggtgcagcag tgaacagcag tgagagtctc cctccatcct
catctgtcaa cgacatctcc 1560 tccatgtcca ccgaccagac cctcgcatcc
gacactgaca gcagcctgga agcctcggcg 1620 ggaccgctgg gttgttgcag
gtgactagcc gcctgcctgc gaaacccagc gttcttcagg 1680 agatgacgcc
atgatagaac acagcgcaca tgcacacaca cagagcttgt acacacacac 1740
acacacacac acacacgcac gcacgcacgc acgcaagcac gcacgcacgc acaaatgcac
1800 tcacgcaatg tcaagaaaaa aaaaagtagc gagagagagc gagagagcca
acgtaaaact 1860 aagttaaatc tttctgcgtg cttctccaga gttctgtatc
gcagctgagc tgaaatgtat 1920 acttaacttc tagtcgcgct cgctcgactt
tggtctccct ccggcagtgc ttact 1975 <210> SEQ ID NO 100
<211> LENGTH: 2522 <212> TYPE: DNA <213>
ORGANISM: Mus musculus <400> SEQUENCE: 100 ggggcttgag
tgagctaaag attgggtctt cttggaaatc acctgtctgt tattattttt 60
aaacaatcgc tacacctcca aagactctgc tccttactcc ggtttggaat gtggctaatg
120 actacccagt agggaggatt tctggggcaa acagccggac caggatccta
gttctcaggc 180 acggaatggc tattgtgaga acagcaccag caggatcatc
gcagatcttg gttatggcca 240 ctcaggcaag acgctgttga gttaagaccc
ctttcccata gatgagaagc cacagaagca 300 gtggtattta tgagcctcca
tttcttatac tactgcagtg aaccaacctt ggatgtgaaa 360 attgcctttt
gtcagggatt cgataaacac gtggatgtgt catctattgc caaacattac 420
aacatgagca aaagcaaggt ggacaaccag ttctacagtg tggaagtggg ggactcaacc
480 ttcaccgttc ttaagcgcta ccagaacctg aagccaattg gctctggggc
tcagggaata 540 gtctgtgctg cgtacgacgc tgtccttgac agaaatgtgg
ccattaagaa gctcagcaga 600 cccttccaga accaaactca cgccaagagg
gcttaccggg agctggtgct catgaagtgt 660 gtgaaccata aaaacattat
tagcttatta aatgttttta caccccagaa aacgctggag 720 gagttccaag
atgtctactt agtgatggaa ctgatggacg ccaacctgtg tcaggtgatt 780
cagatggagc tggaccacga gcggatgtct tacttgctgt accagatgct gtgtggcatc
840 aagcacctcc actccgctgg gatcatccac agggacttaa aacccagtaa
cattgtagtc 900 aagtctgatt gcacactgaa aatcctcgac ttcggactgg
ccaggacagc gggtacaagc 960 ttcatgatga ctccgtatgt ggtgacgcga
tattacagag cccctgaggt catcctgggc 1020 atgggctaca aggagaacgt
ggacatatgg tctgtgggat gcatcatggg agaaatggtt 1080 cgccacaaaa
tcctctttcc cggaaggagc tatattgacc agtggaacaa agtcatcgag 1140
cagctaggaa ctccgtgtcc agagttcatg aagaaattgc agcccacagt cagaaactac
1200 gtggagaatc ggcccaagta cgcaggactc accttcccca agctctttcc
agattccctc 1260 ttcccagcgg attctgagca caataaactt aaagccagcc
aagccaggga tttgttgtct 1320 aagatgttag tgattgaccc agtgaagagg
atatcggtgg acgacgcact gcagcatccg 1380 tacatcaacg tttggtacga
cccggctgaa gtggaggcgc ctccgcctca gatatatgat 1440 aagcagctgg
atgaaaggga gcacaccatc gaagaatgga aagaacttat ctacaaggag 1500
gtaatgaact cagaagagaa gactaagaat ggcgtagtca aaagccagcc ctcgccttca
1560 gcacaggtgc agcagtgaac agcagtgaga gtctccctcc atcctcggct
gtcaacgaca 1620 tctcctccat gtccaccgac cagaccctcg catctgacac
tgacagcagc ctggaggcct 1680 cggcgggacc gttgggttgt tgcaggtgac
tagccgcctg cctgcgaaac ccagcgttct 1740 tcaggagatg acgcgataga
acacagcaca catgcacaca cacagcttgc tctcacacac 1800 actcagcttg
ctcacacaca cacacacaca tacacacaaa cacacactgt ctctctctca 1860
cacacacaca ctgtcacaac gcactcacga aaggtcaaga aaaaaataac aatagagaga
1920 tccaacataa aattaagtta aatttttctg cgtgcttctc caaagttctg
tatcacagct 1980 gagctgaaat gtatacttaa cttctagttg cgctcgcttt
ggtttccctc cagcagtgct 2040 tactacacaa gacaaatcag acacaattag
agaaaccttt ccctaaagtg taacttaagt 2100 ggctgcagaa ccagcaacct
gtaactgccc ttcaaatggc atgaggaggt gggcacgggt 2160 cccgcccagc
atgtgtgtgt ctctatctcg cgtctacctg ctcttccggc ctagtcagat 2220
ggatgtagat acagatcccg catgtgtctg tattcaaaca gcacttagag atgctcctgt
2280 cagtgtcctc caggctccac caagacacac accggggtac cacatggtcc
atttcatgtg 2340 atctattact ctgacataaa tccatctgta atatattgcc
agtatataag ctgtttagtt 2400 tgttaattgc ttaagctgta tgtcttataa
gagactatgt aaagggggaa aatggaggcg 2460 tgaactctca gacccttgaa
gatgtagctt ccgaatttga ccgttaaatg gcaccgtata 2520 cc 2522
<210> SEQ ID NO 101 <211> LENGTH: 1773 <212>
TYPE: DNA <213> ORGANISM: Homo sapien <400> SEQUENCE:
101 atctcagatc ttcactatgg caacttatgc aagaaactgt tgaattagac
ccgtttccta 60 tagatgagaa accatacaag ctgtggtatt tatgagcctc
catttcttat actactgcag 120 tgaaccaaca ttggatgtga aaattgcctt
ttgtcaggga ttcgataaac aagtggatgt 180 gtcatatatt gccaaacatt
acaacatgag caaaagcaaa gttgacaacc agttctacag 240 tgtggaagtg
ggagactcaa ccttcacagt tctcaagcgc taccagaatc taaagcctat 300
tggctctggg gctcagggca tagtttgtgc cgcgtatgat gctgtccttg acagaaatgt
360 ggccattaag aagctcagca gaccctttca gaaccaaaca catgccaaga
gagcgtaccg 420 ggagctggtc ctcatgaagt gtgtgaacca taaaaacatt
attagtttat taaatgtctt 480 cacaccccag aaaacgctgg aggagttcca
agatgtttac ttagtaatgg aactgatgga 540 tgccaactta tgtcaagtga
ttcagatgga attagaccat gagcgaatgt cttacctgct 600 gtaccaaatg
ttgtgtggca ttaagcacct ccattctgct ggaattattc acagggattt 660
aaaaccaagt aacattgtag tcaagtctga ttgcacattg aaaatcctgg actttggact
720 ggccaggaca gcaggcacaa gcttcatgat gactccatat gtggtgacac
gttattacag 780 agcccctgag gtcatcctgg ggatgggcta caaggagaac
gtggatatat ggtctgtggg 840 atgcattatg ggagaaatgg ttcgccacaa
aatcctcttt ccaggaaggg actatattga 900 ccagtggaat aaggtaattg
aacaactagg aacaccatgt ccagaattca tgaagaaatt 960 gcaacccaca
gtaagaaact atgtggagaa tcggcccaag tatgcgggac tcaccttccc 1020
caaactcttc ccagattccc tcttcccagc ggactccgag cacaataaac tcaaagccag
1080 ccaagccagg gacttgttgt caaagatgct agtgattgac ccagcaaaaa
gaatatcagt 1140 ggacgacgcc ttacagcatc cctacatcaa cgtctggtat
gacccagccg aagtggaggc 1200 gcctccacct cagatatatg acaagcagtt
ggatgaaaga gaacacacaa ttgaagaatg 1260 gaaagaactt atctacaagg
aagtaatgaa ttcagaagaa aagactaaaa atggtgtagt 1320 aaaaggacag
ccttctcctt cagcacaggt gcagcagtga acagcagtga gagtctccct 1380
ccatcctcgt ctgtcaatga catctcctcc atgtccaccg accagaccct ggcatctgac
1440 actgacagca gcctggaagc ctcggcagga cccctgggtt gttgcaggtg
actagccgcc 1500 tgcctgcgaa acccagcgtt cttcaggaga tgatgtgatg
gaacacacac acacgcagac 1560 acacacacac acacaaatgc agacacacaa
catcaagaaa acagcaaggg agagaatcca 1620 agcctaaaat taaataaatc
tttcagcctg cttcttcccc agggttctgt attgcagcta 1680 agctcaaatg
tatatttaac ttctagttgc tcttgctttg gtcttcttcc aatgatgctt 1740
actacagaaa gcaaatcaga cacaattaga gaa 1773 <210> SEQ ID NO 102
<211> LENGTH: 1505 <212> TYPE: DNA <213>
ORGANISM: Homo sapien <400> SEQUENCE: 102
ttatgcaaga aactgttgaa ttagacccgt ttcctataga tgagaaacca tacaagctgt
60 ggtatttatg agcctccatt tcttatacta ctgcagtgaa ccaacattgg
atgtgaaaat 120 tgccttttgt cagggattcg ataaacaagt ggatgtgtca
tatattgcca aacattacaa 180 catgagcaaa agcaaagttg acaaccagtt
ctacagtgtg gaagtgggag actcaacctt 240 cacagttctc aagcgctacc
agaatctaaa gcctattggc tctggggctc agggcatagt 300 ttgtgccgcg
tatgatgctg tccttgacag aaatgtggcc attaagaagc tcagcagacc 360
ctttcagaac caaacacatg ccaagagagc gtaccgggag ctggtcctca tgaagtgtgt
420 gaaccataaa aacattatta gtttattaaa tgtcttcaca ccccagaaaa
cgctggagga 480 gttccaagat gtttacttag taatggaact gatggatgcc
aacttatgtc aagtgattca 540 gatggaatta gaccatgagc gaatgtctta
cctgctgtac caaatgttgt gtggcattaa 600 gcacctccat tctgctggaa
ttattcacag ggatttaaaa ccaagtaaca ttgtagtcaa 660 gtctgattgc
acattgaaaa tcctggactt tggactggcc aggacagcag gcacaagctt 720
catgatgact ccatatgtgg tgacacgtta ttacagagcc cctgaggtca tcctggggat
780 gggctacaag gagaacgtgg atatatggtc tgtgggatgc attatgggag
aaatggttcg 840 ccacaaaatc ctctttccag gaagggacta tattgaccag
tggaataagg taattgaaca 900 actaggaaca ccatgtccag aattcatgaa
gaaattgcaa cccacagtaa gaaactatgt 960 ggagaatcgg cccaagtatg
cgggactcac cttccccaaa ctcttcccag attccctctt 1020 cccagcggac
tccgagcaca ataaactcaa agccagccaa gccagggact tgttgtcaaa 1080
gatgctagtg attgacccag caaaaagaat atcagtggac gacgccttac agcatcccta
1140 catcaacgtc tggtatgacc cagccgaagt ggaggcgcct ccacctcaga
tatatgacaa 1200 gcagttggat gaaagagaac acacaattga agaatggaaa
gaacttatct acaaggaagt 1260 aatgaattca gaagaaaaga ctaaaaatgg
tgtagtaaaa ggacagcctt ctccttcagg 1320 tgcagcagtg aacagcagtg
agagtctccc tccatcctcg tctgtcaatg acatctcctc 1380 catgtccacc
gaccagaccc tggcatctga cactgacagc agcctggaag cctcggcagg 1440
acccctgggt tgttgcaggt gactagccgc ctgcctgcga aacccagcgt tcttcaggag
1500 atgat 1505 <210> SEQ ID NO 103 <400> SEQUENCE: 103
000 <210> SEQ ID NO 104 <211> LENGTH: 2622 <212>
TYPE: DNA <213> ORGANISM: Rattus norvegicus <400>
SEQUENCE: 104 tgtatgacac tacatcatga gtgacagtaa aagcgatggc
cagttttaca gtgtgcaagt 60 ggcagactca actttcactg ttctaaaacg
ttaccagcag ttgaaaccaa ttggctctgg 120 agcccaagga attgtttgtg
ctgcttttga tacagttctt ggaataaatg ttgctgtcaa 180 gaagttaagt
cgtccttttc agaaccaaac gcatgcaaag agagcctacc gtgaacttgt 240
cctcctaaag tgtgtcaatc ataaaaatat aattagcttg ttaaatgtgt tcacaccaca
300 aaaaacgcta gaagaattcc aagatgtgta cttggttatg gagttaatgg
acgctaactt 360 atgtcaggtt attcatatgg agctggacca tgaaagaatg
tcatacctcc tctaccagat 420 gctttgtggc attaagcacc tgcattcagc
tggcataatt catagggatt tgaagcctag 480 caacattgta gtaaaatcag
actgtactct caagatcctt gactttggcc tggcacggac 540 agcctgtacc
aactttatga tgactcccta tgtggtaact cgctactatc gggctccaga 600
agtcatcctg ggcatgggct acaaggagaa tgtggacatc tggtctgtcg ggtgcatcat
660 ggcagaaatg gtcctccata aatcctgttc cccaggaaga gactatattg
atcaatggaa 720 taaagttatt gaacagctag gaacaccatc cgcagagttc
atgaagaaac ttcagccaac 780 tgtaaggaat tatgtggaaa acagaccaaa
gtaccctgga atcaaatttg aagagctctt 840 tccagattgg atatttccgt
cagaatccga acgagacaaa ataaaaacaa gtcaagccag 900 agatctgtta
tcgaaaatgt tagtgattga tccggacaag cggatctctg tggacgaagc 960
cttgcgccac ccgtatatta ctgtttggta tgaccccgct gaagcagaag cgccaccacc
1020 tcaaatttat gatgcccagt tggaagaaag agagcatgcg attgaagagt
ggaaagaact 1080 aatttacaaa gaagtgatgg actgggaaga aagaagcaag
aatggggtga aagaccagcc 1140 ttcagatgca gcagtaagca gcaaggctac
tccttctcag tcgtcatcca tcaatgacat 1200 ctcatccatg tccactgagc
acaccctggc ctcagacaca gacagcagtc tcgatgcctc 1260 aaccggaccc
ctggaaggct gccgatgaaa cctcgcagat ggcgcacttg tctgtgaagg 1320
actctggctt ccatggccct gagcacatgg gagctggtgg aacaaatcaa gaagctccat
1380 gttctgcatg taagaaacac gacgccttgc ccccactcag ttccagtagg
attgcctgcg 1440 tagactgtaa catgaggcag acgatgtctg gagaaaaagt
acaaaccaca ctgttagaaa 1500 ttttgttcaa gatcattcag gtgagcaatt
agaatagccg agttcttttc aagtcgtgtg 1560 gtgtccttgg tgacagatca
tgtgtaactg tggggactcg tatgcatgtg accacaaatg 1620 cttgcttgaa
cttgcccatg tagcactttg ggaatcagta tttaaatgcc aaataatctt 1680
ccaggtagtt ctgcttctag aataatctct taatcctctt tagtaatttg gtgtctgtcc
1740 acaaaaaaat agattatgtg tgtatgaatt ggccactatc atattatcat
attttaccca 1800 cttttatggt atgatttatt ctgtcttttg tatttcagaa
ggaatataat taaatttatt 1860 taataaataa aactacagct tttcttaaat
ttgtgatgtt ttaggctgag aattaccact 1920 gctttatatc gacactctgt
gtcctttaaa ctgcccacta tgggaaactt tacgtacagc 1980 tttctgcatg
acaaagttcc aagttgtatt tcactctgct taacgactta tgtcaccttg 2040
aatcctgacc acacatttcc tttttcttgg tcctctgaac ttggatctag aatccctcac
2100 agaacttcac cttctttatc acaaagcacc ccatctcagt agaatgaatc
ggcagattcc 2160 tgagccccgc tgcctaatgt agagctgaca gggtggcttc
cccagaacgg tgggtgggtg 2220 catccttccc tgagcccacc catcctttgc
tcccctctct ttatttaagg tgaaaggtga 2280 ttgggtctca tagcctttcc
ttttgtagca ttgcctaact tgtctttctc actgacagaa 2340 gccaccacgt
ccagccagag cacatggtct cttaggagac cgggcttact taccatgcat 2400
gtttgctgct gtccttttcc attttgtgga ggcatttcct ttttctaagg gaattcctca
2460 gatgttctag aaacattcag aagaacgcag aagaaatatt ctagagaatt
gggggttcat 2520 tcttgaatat tttctgattt aaaactgctc acctgaaatt
gatactttca gatcctgatc 2580 ttgtaaatta ctcgagattt ggtaagatgc
tgagttctct gt 2622 <210> SEQ ID NO 105 <400> SEQUENCE:
105 000 <210> SEQ ID NO 106 <211> LENGTH: 1646
<212> TYPE: DNA <213> ORGANISM: Mus musculus
<400> SEQUENCE: 106 gctgttagtc agcggagcgg ccgaggccgg
acgttgcggc cgaaacgcgg agccgcgaac 60 aggattgagt agcggccgcg
gccaccgcag gacggcgccg ttctccgcta cgggcttcca 120 ggtcgccgtt
ggctgcactg ccggccttgg tgaatatttg gatgaagcca ttagactaat 180
tgcttgccat catgagcaga agcaaacgtg acaacaattt ttatagtgta gagattggag
240 attctacatt cacagtccta aaacgatacc agaatttaaa gcctataggc
tcaggagctc 300 aaggaatagt gtgtgcagct tatgatgcca ttcttgaaag
aaatgttgca atcaagaagc 360 tcagccggcc atttcagaat cagacccatg
ctaagcgcgc ctaccgagaa ctagttctta 420 tgaagtgtgt taatcacaaa
aatataattg gccttttgaa tgttttcaca ccacagaaat 480 ccctagaaga
atttcaagat gtttacatag tcatggagct catggatgca aatctttgcc 540
aagtgattca gatggagtta gatcatgaaa gaatgtccta ccttctctat caaatgctgt
600 gtggaatcaa gcaccttcac tctgctggaa ttattcatcg ggacttaaag
cctagtaata 660 tagtagtcaa atcagactgc actttgaaga ttcttgattt
tggactggcg aggactgcag 720 gaacgagttt tatgatgacg ccttatgtgg
tgactcgcta ctacagagca ccagaggtca 780 ttctcggcat gggctacaag
gagaacgtgg acttatggtc tgtggggtgc attatgggag 840 aaatggtttg
ccacaaaatc ctctttccag gaagggacta tattgatcag tggaataaag 900
ttattgaaca gctcggaaca ccttgtcctg aattcatgaa gaaactacaa ccaacagtaa
960 ggacttatgt tgaaaacagg cctaaatacg ctggatatag ctttgagaaa
ctgttccccg 1020 atgtgctttt cccagctgac tcagagcata acaaacttaa
agccagtcag gcaagagatt 1080 tgttatccaa aatgctagta atagatgcat
ccaaaaggat ctccgtagat gaagctctcc 1140 agcacccata catcaacgtc
tggtatgatc cttcagaagc agaagcccca ccaccaaaga 1200 tcccggacaa
gcagttagat gagagggagc acacaataga ggagtggaaa gaactgatat 1260
acaaggaggt aatggatttg gaggaacgaa ctaagaatgg agtcataaga gggcagccgt
1320 ctcctttagc acaggtgcag caatgatcaa tggctctcag catccatcgt
cttcgccgtc 1380 tgtcaatgac atgtcttcaa tgtccacaga tccgactttg
gcctcggata cagacagcag 1440 tctagaagca tcagctggac ctctgggctg
ctgtagatga ctacttgggc cttgggtggg 1500 tgggagggat ggggaattgg
ttagtcattg atagaactgc tttaaaaaca attcagtggt 1560 catatttttg
agtgattttt cagaaaatgt agaattcatt ttgtagtaaa gtagtttatt 1620
ttttttaatt tcaagtgttg taattc 1646 <210> SEQ ID NO 107
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 107
ccttccctga aggttcctcc 20 <210> SEQ ID NO 108 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 108 tgttgtcacg
tttgcttctg 20
<210> SEQ ID NO 109 <400> SEQUENCE: 109 000 <210>
SEQ ID NO 110 <400> SEQUENCE: 110 000 <210> SEQ ID NO
111 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
111 caacgtcccg cgctcggccg 20 <210> SEQ ID NO 112 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 112 cctgctcgcg
gctccgcgtt 20 <210> SEQ ID NO 113 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 113 ctcatgatgg caagcaatta 20
<210> SEQ ID NO 114 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 114 tgttgtcacg tttacttctg 20 <210> SEQ
ID NO 115 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
115 cggtaggctc gcttagcatg 20 <210> SEQ ID NO 116 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 116 ctagggattt
ctgtggtgtg 20 <210> SEQ ID NO 117 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 117 cagcagagtg aaggtgcttg 20
<210> SEQ ID NO 118 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 118 tcgttcctgc agtccttgcc 20 <210> SEQ
ID NO 119 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
119 ccatttctcc cataatgcac 20 <210> SEQ ID NO 120 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 120 tgaattcagg
acaaggtgtt 20 <210> SEQ ID NO 121 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 121 agcttcgtct acggagatcc 20
<210> SEQ ID NO 122 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 122 cactcctcta ttgtgtgctc 20 <210> SEQ
ID NO 123 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
123 gctgcaccta aaggagacgg 20 <210> SEQ ID NO 124 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 124 ccagagtcgg
atctgtggac 20 <210> SEQ ID NO 125 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 125 tcatgatgta gtgtcataca 20
<210> SEQ ID NO 126 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 126 tgtggtgtga acacatttaa 20 <210> SEQ
ID NO 127 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
127 ccatatgaat aacctgacat 20 <210> SEQ ID NO 128 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 128 gatatcaaca
ttctccttgt 20 <210> SEQ ID NO 129 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 129 gcttcgtcca cagagatccg 20
<210> SEQ ID NO 130 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 130
gctcagtgga catggatgag 20 <210> SEQ ID NO 131 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 131 atctgcgagg
tttcatcggc 20 <210> SEQ ID NO 132 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 132 ccaccagctc ccatgtgctc 20
<210> SEQ ID NO 133 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 133 cagttacaca tgatctgtca 20 <210> SEQ
ID NO 134 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
134 aagaggatta agagattatt 20 <210> SEQ ID NO 135 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 135 agcagagtga
aatacaactt 20 <210> SEQ ID NO 136 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 136 tgtcagctct acattaggca 20
<210> SEQ ID NO 137 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 137 agtaagcccg gtctcctaag 20 <210> SEQ
ID NO 138 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
138 aaatggaaaa ggacagcagc 20 <210> SEQ ID NO 139 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 139 gctcagtgga
tatggatgag 20 <210> SEQ ID NO 140 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 140 gctaagcggt caaggttgag 20
<210> SEQ ID NO 141 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 141 gctcggtgga aatggatcag 20 <210> SEQ
ID NO 142 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
142 gggctttcat tagccacatt 20 <210> SEQ ID NO 143 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 143 ggttggttca
ctgcagtagt 20 <210> SEQ ID NO 144 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 144 tgctcatgtt gtaatgtttg 20
<210> SEQ ID NO 145 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 145 gtcgaggaca gcgtcatacg 20 <210> SEQ
ID NO 146 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
146 cgacatccgc tcgtggtcca 20 <210> SEQ ID NO 147 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 147 acatacggag
tcatcatgaa 20 <210> SEQ ID NO 148 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 148 gcaatttctt catgaattct 20
<210> SEQ ID NO 149 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 149 tcgtaccaaa cgttgatgta 20 <210> SEQ
ID NO 150 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
150 cgccgaggct tccaggctgc 20 <210> SEQ ID NO 151 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 151
ggctagtcac ctgcaacaac 20 <210> SEQ ID NO 152 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 152 gcgtgcgtgc
gtgcttgcgt 20 <210> SEQ ID NO 153 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 153 gctcagctgc gatacagaac 20
<210> SEQ ID NO 154 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 154 agcgcgacta gaagttaagt 20 <210> SEQ
ID NO 155 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
155 agggagacca aagtcgagcg 20 <210> SEQ ID NO 156 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 156 acatcttgaa
attcttctag 20 <210> SEQ ID NO 157 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 157 taggatattc tttcatgatc 20
<210> SEQ ID NO 158 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 158 agaaggtagg acattctttc 20 <210> SEQ
ID NO 159 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
159 tttattccac tgatcaatat 20 <210> SEQ ID NO 160 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 160 tcaataactt
tattccactg 20 <210> SEQ ID NO 161 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 161 ggttgcagtt tcttcatgaa 20
<210> SEQ ID NO 162 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: 6 <223> OTHER INFORMATION: n = I <400>
SEQUENCE: 162 tagganattc tttcatgatc 20 <210> SEQ ID NO 163
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: 8
<223> OTHER INFORMATION: n = I <400> SEQUENCE: 163
ggttgcantt tcttcatgaa 20
* * * * *