U.S. patent application number 13/294068 was filed with the patent office on 2012-05-10 for compositions and their uses directed to hepcidin.
This patent application is currently assigned to ISIS PHARMACEUTICALS, INC.. Invention is credited to C. FRANK BENNETT, WILLIAM A. GAARDE, TRISHA LOCKHART, ROBERT MCKAY, BRETT P. MONIA.
Application Number | 20120115930 13/294068 |
Document ID | / |
Family ID | 38610239 |
Filed Date | 2012-05-10 |
United States Patent
Application |
20120115930 |
Kind Code |
A1 |
MONIA; BRETT P. ; et
al. |
May 10, 2012 |
COMPOSITIONS AND THEIR USES DIRECTED TO HEPCIDIN
Abstract
Disclosed herein are compounds, compositions and methods for
modulating the expression of hepcidin in a cell, tissue or animal
or preventing, ameliorating or treating anemia. Also provided are
methods for prevention, amelioration or treatment of anemia, and
for increasing red blood cell count in an animal. Also provided are
methods for the prevention, amelioration and/or treatment of low
serum iron levels, low red blood cell count and other clinical
endpoints of anemia in an animal. These methods may be achieved by
administration of compounds or compositions including antisense
compounds targeted to a nucleic acid that expresses hepcidin
polypeptide combined with an erythropoiesis stimulating agent.
Inventors: |
MONIA; BRETT P.; (ENCINITAS,
CA) ; BENNETT; C. FRANK; (CARLSBAD, CA) ;
GAARDE; WILLIAM A.; (CARLSBAD, CA) ; LOCKHART;
TRISHA; (CARDIFF BY THE SEA, CA) ; MCKAY; ROBERT;
(POWAY, CA) |
Assignee: |
ISIS PHARMACEUTICALS, INC.
CARLSBAD
CA
|
Family ID: |
38610239 |
Appl. No.: |
13/294068 |
Filed: |
November 10, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11734562 |
Apr 12, 2007 |
8076306 |
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13294068 |
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60792004 |
Apr 12, 2006 |
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60821638 |
Aug 7, 2006 |
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60865833 |
Nov 14, 2006 |
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Current U.S.
Class: |
514/44A ;
536/24.5 |
Current CPC
Class: |
C12N 2310/346 20130101;
C12N 2310/321 20130101; C12N 2310/341 20130101; A61P 7/00 20180101;
C12N 2310/3341 20130101; C12N 15/113 20130101; C12N 2310/315
20130101; C12N 2310/321 20130101; C12N 2310/11 20130101; C12N
2310/3525 20130101; A61P 7/06 20180101 |
Class at
Publication: |
514/44.A ;
536/24.5 |
International
Class: |
A61K 31/711 20060101
A61K031/711; A61P 7/06 20060101 A61P007/06; A61K 31/7125 20060101
A61K031/7125; C07H 21/04 20060101 C07H021/04; A61K 31/712 20060101
A61K031/712 |
Claims
1. A compound comprising a modified oligonucleotide consisting of
12 to 35 linked nucleosides and having a nucleobase sequence
comprising at least 8 contiguous nucleobases complementary to an
equal length portion of a nucleobase sequence of a nucleic acid
encoding hepcidin, wherein said modified oligonucleotide
specifically hybridizes to said nucleic acid encoding hepcidin.
2. The compound of claim 1, wherein said nucleobase sequence of a
nucleic acid encoding hepcidin is selected from the group
consisting of SEQ ID NOs: 1, 2 and 3.
3. The compound of claim 2, wherein said nucleobase sequence of a
nucleic acid encoding hepcidin is SEQ ID NO: 1.
4. The compound of claim 3, wherein said modified oligonucleotide
has a nucleobase sequence comprising at least 8 contiguous
nucleobases complementary to an equal length portion of nucleotides
123-566 of SEQ ID NO: 1.
5. The compound of claim 3, wherein said modified oligonucleotide
has a nucleobase sequence comprising at least 8 contiguous
nucleobases complementary to an equal length portion of nucleotides
442-541 of SEQ ID NO: 1.
6. The compound of claim 3, wherein said compound consists of a
single-stranded modified oligonucleotide.
7. The compound of claim 1, wherein the nucleobase sequence of the
modified oligonucleotide is at least 90% complementary to at least
a 20 contiguous nucleobase portion of nucleotides 123-566 of SEQ ID
NO: 1.
8. The compound of claim 6, wherein the nucleobase sequence of the
modified oligonucleotide is 100% complementary to at least a 20
contiguous nucleobase portion of nucleotides 123-566 of SEQ ID NO:
1.
9. The compound of claim 6, wherein at least one internucleoside
linkage of said modified oligonucleotide is a modified
internucleoside linkage.
10. The compound of claim 9, wherein each internucleoside linkage
of said modified oligonucleotide is a phosphorothioate
internucleoside linkage.
11. The compound of claim 6, wherein at least one nucleoside of
said modified oligonucleotide comprises a modified sugar.
12. The compound of claim 11, wherein at least one modified sugar
is a bicyclic sugar.
13. The compound of claim 11, wherein at least one modified sugar
is selected from the group consisting of a 2'-O-(2-methoxyethyl),
and a 4'-(CH.sub.2).sub.2--O-2' bridge, wherein n is 1 or 2.
14. The compound of claim 6, wherein at least one nucleoside of
said modified oligonucleotide comprises a modified nucleobase.
15. The compound of claim 14, wherein said modified nucleobase is a
5-methylcytosine.
16. The compound of claim 3, wherein said modified oligonucleotide
comprises: a gap segment consisting of linked deoxynucleosides; a
5' wing segment consisting of linked nucleosides; and a 3' wing
segment consisting of linked nucleosides; wherein the gap segment
is positioned between the 5' wing segment and the 3' wing segment
and wherein each nucleoside of each wing segment comprises a
modified sugar.
17. The compound of claim 16, wherein said modified oligonucleotide
comprises: a gap segment consisting of ten linked deoxynucleosides;
a 5' wing segment consisting of five linked nucleosides; and a 3'
wing segment consisting of five linked nucleosides; wherein the gap
segment is positioned between the 5' wing segment and the 3' wing
segment, wherein each nucleoside of each wing segment comprises a
2'-O-methoxyethyl sugar, wherein each cytosine in said modified
oligonucleotide is a 5-methylcytosine, and wherein each
internucleoside linkage of said modified oligonucleotide is a
phosphorothioate linkage.
18. The compound of claim 17, wherein said modified oligonucleotide
consists of 20 linked nucleosides.
19. The compound of claim 6, wherein said modified oligonucleotide
consists of 20 linked nucleosides.
20. A composition comprising the compound of claim 3 or a salt
thereof and a pharmaceutically acceptable carrier or diluent.
21. The composition of claim 20, wherein said compound consists of
a single-stranded modified oligonucleotide.
22. The composition of claim 20, wherein said modified
oligonucleotide consists of 20 linked nucleosides.
23. The compound of claim 1, wherein said modified oligonucleotide
has a nucleobase sequence comprising at least 8 contiguous
nucleobases complementary to an equal length portion of a
nucleotide region selected from the group consisting of nucleotides
123-566, 123-252, 295-566, 352-405, 429-566, and 456-527 of SEQ ID
NO: 1, 204-341 and 231-302 of SEQ ID NO: 2, and 3249-4743,
3249-3454, 3398-3454, 4643-4703, and 4643-4743 of SEQ ID NO: 3.
24. The compound of claim 1, wherein said modified oligonucleotide
has a nucleobase sequence comprising at least 8 contiguous
nucleobases of a nucleobase sequence selected from the group
consisting of SEQ ID NOs: 23-37.
25. The compound of claim 1, wherein said modified oligonucleotide
has a nucleobase sequence consisting of a nucleobase sequence
selected from the group consisting of SEQ ID NOs: 23-37.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/734,562, filed Apr. 12, 2007, which claims priority under 35
U.S.C. .sctn.119(e) to U.S. Provisional Application No. 60/792,004,
filed Apr. 12, 2006, U.S. Provisional Application No. 60/821,638,
filed Aug. 7, 2006 and U.S. Provisional Application No. 60/865,833,
filed Nov. 14, 2006. Each of the above applications is herein
incorporated 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 BIOL0082USSEQ.txt, created on Nov. 9, 2011, which is
42 Kb in size. The information in the electronic format of the
sequence listing is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0003] Anemia is characterized by a lower than normal number of red
blood cells (erythrocytes) in the blood, usually measured by a
decrease in the amount of hemoglobin. The cause of anemia can
include chronic inflammation, chronic kidney disease, kidney
dialysis treatment, genetic disorders, chronic infection, acute
infection, cancer and cancer treatments. Altered iron homeostasis
and/or erythropoiesis in these conditions can result in decreased
erythrocyte (red blood cell) production. Anemia can limit the use
of chemotherapeutic agents during cancer treatment. Symptoms of
anemia include fatigue, pallor and poor exercise tolerance. Though
rarely life threatening, anemia can be severely debilitating and
difficult to treat. Clinical signs of anemia include low serum iron
(hypoferremia), low hemoglobin levels, low hematocrit levels,
decreased red blood cells, decreased reticulocytes, increased
soluble transferrin receptor and iron restricted erythropoesis.
[0004] In some cases, increasing dietary iron or intravenous iron
delivery are used to treat anemia. Erythropoietin (EPO) stimulates
erythroprogenitors and promotes red blood cell formation.
Recombinant erythropoietin and other erythropoiesis stimulating
agents (ESAs) are used for treating anemia, although certain
patients respond poorly to this treatment. EPO is well known and is
commercially available through Amgen (Thousand Oaks, Calif.).
[0005] Anemia of chronic disease (ACD) is a highly prevalent,
inflammatory-driven disorder that is poorly treated with currently
available therapies. The mechanism underlying ACD is chronic
inflammation, which results in changes in iron homeostasis and
utilization, resulting in a blunting of erythroid progenitor cell
proliferation and red cell function.
[0006] ACD is associated with increased production of inflammatory
cytokines, including, for example, tumor necrosis factor-.alpha.,
IL-1 .beta., IL-6, and interferon-.gamma. (Means (1995) Stem cells
13:32-37 and Means (1999) Int J Hematol 70:7-12). In several in
vitro and in vivo animal model systems, inflammatory cytokines
negatively affected the ability to mediate erythropoietin (EPO)
production, EPO responsiveness, and the coordinate regulation of
iron metabolism (Roodman et al. (1989) Adv Exp Med Biol
271:185-196; Fuchs et al. (1991) Eur J Hematol 46:65-70; Jelkmann
et al. (1994) Ann NY Acad Sci 718:300-311; Vannucchi et al. (1994)
Br J Hematol 87:18-23; and Oldenburg et al. (2001) Aliment
Pharmacol Ther 15:429-438). Administration of EPO failed to reverse
anemia in mice continuously exposed to TNF-.alpha. (Clibon et al.
(1990) Exp Hematol 18:438-441). Increased levels of inflammatory
cytokines, such as TNF-.alpha., IL-1 .beta., and INF-.gamma.,
contribute to defective EPO production and EPO resistance observed
in patients with anemia of chronic disease (Jelkmann et al. (1991)
Ann NY Acad Sci 718:300-311 and Macdougall and Cooper (2002) Neprol
Dial Transplant 17 (11):39-43.). Therefore, various cytokines,
e.g., inflammatory cytokines and cytokines associated with
inflammation, are involved in many aspects of the pathogenesis of
anemia of chronic disease, including inhibition of erythroid
progenitors, inhibition of EPO production, and impairment of iron
release and iron availability for erythropoiesis.
[0007] Hepcidin is an 8 kD polypeptide that is produced by
hepatocytes in response to inflammation or to rising levels of iron
in the blood. The primary role of hepcidin is to regulate blood
iron levels by facilitating a decrease in these blood iron levels.
Hepcidin binds with and down regulates ferroportin to reduce
ferroportin mediated release of iron into the blood. Hepcidin
expression is increased in conditions of acute and chronic
inflammation resulting in decreased iron availability for
erythropoeisis. Hepcidin is frequently measured in the urine or
serum as a biomarker of anemia status. (Ganz et al. (2006) Am J
Physiol Gastrointest Liver Physiol 290:G199-G203; and Ganz (2003)
Blood 102 (3):783-788). Moreover, hepcidin over expression has been
strongly linked to ACD mechanistically as a mediator of this
disorder in animal models and in humans.
SUMMARY OF THE INVENTION
[0008] Provided herein are compounds, particularly oligomeric
compounds, especially nucleic acid and nucleic acid-like oligomers,
which are targeted to a nucleic acid encoding hepcidin. Preferably,
the oligomeric compounds are antisense compounds targeted to a
nucleic acid molecule encoding hepcidin, particularly human
hepcidin (GenBank Accession Nos. BM719679.1, entered Mar. 1, 2002;
NM.sub.--021175.2, entered Jul. 23, 2004; and nucleotides 7819907
to 7825131 of NT.sub.--011196.11, entered Jan. 5, 2003; all herein
incorporated by reference and assigned SEQ ID NOS: 1, 2 and 3,
respectively), that modulate the expression of hepcidin. In a
particular embodiment, the antisense compounds are antisense
oligonucleotides. In another embodiment, the antisense compounds
are siRNAs. In one embodiment, the compounds comprise at least an 8
nucleobase portion, preferably at least a 12 nucleobase portion,
more preferably at least a 15 nucleobase portion, of the sequences
listed in Table 3. In a further embodiment, the compounds are at
least 80% identical to the sequences listed in or below Table
3.
[0009] Herein, active target segments are identified for SEQ ID
NOS: 1, 2 and 3. A series of antisense oligonucleotide compounds
was designed to target one or more of SEQ ID NOS: 1, 2, and 3.
Active target segments were then identified as being regions of the
hepcidin mRNA associated with the most active antisense
oligonucleotides. Compounds are provided that are targeted to an
active target segment of SEQ ID NO: 1, 2, or 3 and modulate the
expression of hepcidin. In one embodiment, the compounds comprise
at least an 8 nucleobase portion, preferably at least a 12
nucleobase portion, more preferably at least a 15 nucleobase
portion, that is complementary to an active target segment. In a
further embodiment, the compounds are at least 80% complementary to
an active target segments. In another embodiment, the compounds are
13 to 30 nucleobases in length. In another embodiment, the
compounds are at least about 80% complementary to at least a 20
nucleobase portion of the active target segment. In yet another
embodiment, the compounds are complementary to at least an 8
nucleobase portion of the active target segment.
[0010] In another aspect, the compounds comprise at least one
modified internucleoside linkage, modified sugar moiety, or
modified nucleobase. In another aspect, the compounds comprise a
chimeric oligonucleotide. In one aspect, the compounds comprise a
phosphorothioate linkage. In another aspect, the compounds comprise
a 2'-MOE modification. In another aspect, the compounds comprise a
5-methylcytosine modification.
[0011] Another embodiment provides for a composition comprising
erythropoietin and an antisense compound targeted to a nucleic acid
encoding hepcidin. In one aspect, the compound is an antisense
oligonucleotide targeted to a nucleic acid molecule encoding
hepcidin
[0012] A further embodiment provides methods for prevention,
amelioration or treatment of anemia in an individual comprising
administering to the individual erythropoietin and an antisense
compound targeted to an nucleic acid encoding hepcidin. In one
aspect, the compound is an antisense oligonucleotide targeted to a
nucleic acid molecule encoding hepcidin.
[0013] In one embodiment, the antisense oligonucleotide used in
this method causes an increase in serum iron levels, prevention of
iron restriction of erythropoiesis, or a combination thereof. In
yet another embodiment, the administration causes an increase in
red blood cell counts, reticulocyte counts, hemoglobin levels,
hematocrit levels, or a combination thereof.
[0014] In one aspect, the cause of anemia is chronic inflammation,
chronic kidney disease, kidney dialysis treatment, genetic
disorders, chronic infection, acute infection, cancer, or cancer
treatments. In another aspect, the anemia is associated with
inflammation. In another aspect, it is anemia of chronic disease
(ACD).
[0015] In another embodiment, the individual receiving the
administration has received or has been continuously receiving
erythropoietin prior to treatment. Another embodiment provides
methods for prevention, amelioration or treatment of anemia in an
individual who is being treated with erythropoietin comprising
administering to the individual erythropoietin and an antisense
oligonucleotide targeted to a nucleic acid molecule encoding
hepcidin.
[0016] In another embodiment, the administration comprises delivery
of the antisense oligonucleotide and erythropoietin in a single
formation. In one aspect, the delivery of the single formulation is
by injection. In another embodiment, the administration comprises
delivery of the antisense oligonucleotide and erythropoietin in
separate formulations. In one aspect delivery of separate
formulations is by injection. In another aspect, separate
formulations are delivered at distinct timepoints. In another
aspect, they are delivered simultaneously.
[0017] Another embodiment provides a method for increasing red
blood cell count in an animal that is being treated with
erythropoietin comprising administering to the animal an oligomeric
compound targeted to a nucleic acid encoding hepcidin. In one
aspect, the compound is an antisense oligonucleotide targeted to a
nucleic acid molecule encoding hepcidin.
[0018] Another embodiment provides a method for increasing red
blood cell count in an animal comprising the steps of delivering an
erythropoietin therapy and delivering an antisense oligonucleotide
therapy targeted to a nucleic acid molecule encoding hepcidin.
[0019] Another embodiment provides methods for prevention,
amelioration or treatment of anemia in an individual comprising
administering to the individual antisense compound targeted to an
nucleic acid encoding hepcidin, wherein the individual is further
receiving erythropoietin therapy. In one aspect, the erythropoietin
therapy had occurred or was continuously occurring at the time of
administration of the antisense oligonucleotide. In another aspect
the individual is therapy naive, meaning that the individual had
not before received or was not receiving erythropoietin therapy at
the initiation of the method. In one embodiment, the method further
comprises administering to the individual erythropoietin. In one
embodiment, the antisense compound and erythropoietin are
administered in a single formulation. In another embodiment, they
are administered in separate formulations. In one embodiment, the
separate formulations are administered simultaneously. In another
aspect the separate formulations are administered at distinct
times. In one aspect, the formulations are administered by
injection.
[0020] Another embodiment provides methods for increasing red blood
cells in an individual antisense compound targeted to an nucleic
acid encoding hepcidin, wherein the individual is further receiving
erythropoietin therapy. In one aspect, the erythropoietin therapy
had occurred or was continuously occurring at the time of
administration of the antisense oligonucleotide. In another aspect
the individual is therapy naive, meaning that the individual had
not before received or was not receiving erythropoietin therapy at
the initiation of the method. In one embodiment, the method further
comprises administering to the individual erythropoietin. In one
embodiment, the antisense compound and erythropoietin are
administered in a single formulation. In another embodiment, they
are administered in separate formulations. In one embodiment, the
separate formulations are administered simultaneously. In another
aspect the separate formulations are administered at distinct
times. In one aspect, the formulations are administered by
injection.
[0021] Also provided herein are methods for providing a combination
therapy for prevention, treatment or amelioration of anemia or
increasing red blood cells in an animal comprising the step of
administering to the animal an antisense compound disclosed herein,
wherein the animal is further receiving erythropoietin therapy. In
one embodiment, the animal has been receiving erythropoietin
therapy prior to the administration of the antisense compound. In
another embodiment the animal begins erythropoietin therapy
simultaneously with the administration of the antisense compound.
In one embodiment, the combination therapy comprises receiving the
antisense compound and an erythropoietin therapy compound in a
single formulation. In another embodiment, the combination therapy
comprises receiving the antisense compound and an erythropoietin
therapy compound in separate formulations. In one embodiment,
recombinant human erythropoietin is administered as the
erythropoietin therapy.
[0022] Also provided herein is a use of an oligomeric compound
targeted to a nucleic acid encoding hepcidin for the preparation of
a medicament for the prevention, amelioration, and/or treatment of
anemia. In one aspect, the compound is an antisense oligonucleotide
targeted to a nucleic acid encoding hepcidin. Another embodiment
provides for the use of an oligomeric composition targeted to a
nucleic acid encoding hepcidin for the preparation of a medicament
for the prevention, amelioration, and/or treatment of anemia. In
one aspect the composition comprises an antisense oligonucleotide
targeted to a nucleic acid encoding hepcidin and erythropoietin. In
one aspect, the medicament is prepared for treatment of anemia in a
patient being treated with erythropoietin. In one aspect, the cause
of anemia is chronic inflammation, chronic kidney disease, kidney
dialysis treatment, genetic disorders, chronic infection, acute
infection, cancer or cancer treatments. In another embodiment, the
anemia is anemia of chronic disease (ACD). In one embodiment, the
medicament is formulated for delivery by injection.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1. Western blot of liver lysate following treatment
with antisense oligonucleotides targeting hepcidin mRNA and showing
that the hepcidin polypeptide is reduced following treatment.
Hepcidin was detected with hepcidin antibody (Alpha Diagnostics,
HEPC11-A, 0.3 .micro.g/ml).
[0024] FIG. 2. Graph illustrating that delivery of 10-75 mg/kg of
antisense oligonucleotide targeting hepcidin mRNA reduces hepcidin
expression and in turn leads to an increase in serum iron levels in
normal mice.
[0025] FIG. 3. Graph illustrating that antisense oligonucleotide
reduction of hepcidin increases serum iron levels in both basal and
LPS states in mice (N=4, SEM).
[0026] FIGS. 4a-d. Graphs illustrating responses to treatment with
a compound that reduces the expression of mRNA encoding hepcidin
for a number of clinical endpoints for anemia: a) serum levels; b)
red blood cell count; c) hemoglobin levels; and d) hematocrit
levels.
[0027] FIG. 5 (a-c). Graphs illustrating the enhanced reticulocyte
counts in turpentine-treated mice receiving erythropoietin and an
antisense oligonucleotide targeted to hepcidin.
DETAILED DESCRIPTION OF THE INVENTION
[0028] Anemia is a severely debilitating condition stemming from a
lower than normal number of red blood cells (erythrocytes) in the
blood that characterizes a number of diseases and conditions.
Therapeutic interventions for these diseases or conditions are not
completely satisfactory due to lack of efficacy and/or unwanted
side effects of the compounds. Provided herein are methods, uses,
compositions and compounds for the prevention, amelioration, and
/or treatment of anemia. 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. By way of example, some appropriate indicators include
low serum iron (hypoferremia), low hemoglobin or hematocrit levels,
decreased red blood cell or reticulocyte numbers, increased soluble
transferrin receptor, iron restricted erythropoiesis; symptoms
include fatigue, pallor and poor exercise tolerance. The severity
of indicators may be determined by subjective or objective measures
which are known to those skilled in the art. As used herein,
"treatment" or "therapy" means to administer a composition of
antisense compounds, erythropoietin (or other ESA), or of antisense
compounds combined with erythropoietin (or other ESA) to effect an
alteration or improvement of the disease or condition; the
antisense compounds being targeted to a nucleic acid encoding
hepcidin. Prevention, amelioration, and/or treatment may require
administration of multiple doses at regular intervals, or prior to
exposure to an agent (e.g., a chemotherapeutic) to alter the course
of the condition or disease. Moreover, a single agent may be used
in a single individual for each prevention, amelioration, and
treatment of a condition or disease sequentially or concurrently.
Moreover, the compounds and compositions disclosed herein may be
administered or delivered to a patient who is receiving a
particular therapy, compound, agent or composition. In this case,
the receiving can occur prior to the method of administering or
delivering, or it may occur simultaneously or following the
administration or delivery of the compound or composition.
Additionally, regardless of when the receiving begins relative to
administration or delivery, it may continue after the
administration or delivery.
[0029] Disclosed herein are antisense compounds, including
antisense oligonucleotides, for use in modulating the expression of
nucleic acid molecules encoding hepcidin. This modulation is
accomplished by providing antisense compounds that are designed to
target and hybridize with one or more target nucleic acid molecules
encoding hepcidin (SEQ ID NOS: 1, 2 or 3.). As used herein, the
terms "target nucleic acid" and "nucleic acid molecule encoding
hepcidin" have been used for convenience to encompass RNA
(including pre-mRNA and mRNA or portions thereof) transcribed from
DNA encoding hepcidin and also cDNA derived from such RNA. In a
preferred embodiment, the target nucleic acid is an mRNA encoding
hepcidin.
Target Nucleic Acids
[0030] "Targeting" refers to the design and selection of antisense
compounds capable of hybridizing to or complementary to a
particular target nucleic acid molecule. Antisense compounds
selected by this process are said to be "targeted to" the target
nucleic acid molecule. Targeting can be a multistep process that
usually begins with the identification of a target nucleic acid
whose expression is to be modulated. 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.
As disclosed herein, the target nucleic acid encodes hepcidin.
Variants
[0031] It is also known in the art that alternative RNA transcripts
can be produced from the same genomic region of DNA. These
alternative transcripts are generally known as "variants." More
specifically, "pre-mRNA variants" are transcripts produced from the
same genomic DNA that differ from other transcripts produced from
the same genomic DNA in either their start or stop position and
contain both intronic and exonic sequence. Variants can result in
mRNA variants including, but not limited to, those with alternate
splice junctions, or alternate initiation and termination codons.
Variants in genomic and mRNA sequences can result in disease.
Antisense compounds targeted to such variants are within the scope
of the oligomeric compounds as described herein.
Target Names, Synonyms, Features
[0032] Accordingly, there are provided compositions and methods for
modulating the expression of hepcidin. "Hepcidin" is also referred
to as hepcidin antimicrobial peptide; HAMP; HAMP1; HEPC; HFE2;
LEAP-1; LEAP1; and liver-expressed antimicrobial peptide. Herein
the term "hepcidin" is used. Table 1 lists the GenBank accession
numbers of sequences corresponding to nucleic acid molecules
encoding hepcidin (nt=nucleotide), the date the version of the
sequence was entered in GenBank, and the corresponding SEQ ID NO in
the instant application, when assigned. Each sequence is
incorporated herein by reference.
TABLE-US-00001 TABLE 1 Gene Targets SEQ ID Species Genbank #
Genbank Date NO Human BM719679.1 Mar. 1, 2002 1 Human NM_021175.2
Jul. 23, 2004 2 Human nucleotides 7819907 to 7825131 of Jan. 5,
2003 3 NT_011196.11 Mouse NM_032541.1 May 28, 2001 4 Mouse the
complement of nucleotides Feb. 24, 2003 5 3978217 to 3980665 of
NT_039413.1
Modulation of Target Expression
[0033] Modulation of expression of a target nucleic acid can be
achieved through alteration of any number of nucleic acid
functions. "Modulation" means a perturbation of function, for
example, either an increase (stimulation or induction) or a
decrease (inhibition or reduction) in expression. As another
example, modulation of expression can include perturbing splice
site selection of pre-mRNA processing. "Expression" includes all
the functions by which a gene's coded information is converted into
structures present and operating in a cell. These structures
include the products of transcription and translation. "Modulation
of expression" means the perturbation of such functions. The
functions of RNA to be modulated can include translocation
functions, which include, but are not limited to, translocation of
the RNA to a site of protein translation, translocation of the RNA
to sites within the cell which are distant from the site of RNA
synthesis, and translation of protein from the RNA. RNA processing
functions that can be modulated include, but are not limited to,
splicing of the RNA to yield one or more RNA species, capping of
the RNA, 3' maturation of the RNA and catalytic activity or complex
formation involving the RNA which may be engaged in or facilitated
by the RNA. Modulation of expression can result in the increased
level of one or more nucleic acid species or the decreased level of
one or more nucleic acid species, either temporally or by net
steady state level. One result of such interference with target
nucleic acid function is modulation of the expression of hepcidin.
Thus, in one embodiment modulation of expression can mean increase
or decrease in target RNA or protein levels. In another embodiment
modulation of expression can mean an increase or decrease of one or
more RNA splice products, or a change in the ratio of two or more
splice products.
[0034] The effect of antisense compounds on target nucleic acid
expression can be tested in any of a variety of cell types provided
that the target nucleic acid is present at measurable levels. The
effect of antisense compounds on target nucleic acid expression can
be routinely determined using, for example, PCR, Northern blot
analysis or measurement of hepcidin polypeptide in the urine or
serum of an animal (e.g., ELISA or Western blots). Cell lines are
derived from both normal tissues and cell types and from cells
associated with various disorders (e.g. hyperproliferative
disorders). Cell lines derived from multiple tissues and species
can be obtained from American Type Culture Collection (ATCC,
Manassas, Va.) and other public sources, and are well known to
those skilled in the art. Primary cells, or those cells which are
isolated from an animal and not subjected to continuous culture,
can be prepared according to methods known in the art, or obtained
from various commercial suppliers. Additionally, primary cells
include those obtained from donor human subjects in a clinical
setting (i.e. blood donors, surgical patients). Primary cells are
prepared by methods known in the art.
Assaying Modulation of Expression
[0035] Modulation of hepcidin expression can be assayed in a
variety of ways known in the art. Hepcidin mRNA levels can be
quantitated by, e.g., Northern blot analysis, competitive
polymerase chain reaction (PCR), or real-time PCR. RNA analysis can
be performed on total cellular RNA or poly(A)+ mRNA by methods
known in the art. Methods of RNA isolation are taught in, for
example, Ausubel, F. M. et al., Current Protocols in Molecular
Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley
& Sons, Inc., 1993.
[0036] Northern blot analysis is routine in the art and is taught
in, for example, Ausubel, F. M. et al., Current Protocols in
Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley &
Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently
accomplished using the commercially available ABI PRISM.TM. 7700
Sequence Detection System, available from PE-Applied Biosystems,
Foster City, Calif. and used according to manufacturer's
instructions. The method of analysis of modulation of RNA levels is
not a limitation of the instant description.
[0037] Levels of a protein encoded by hepcidin can be quantitated
in a variety of ways well known in the art, such as
immunoprecipitation, Western blot analysis (immunoblotting), ELISA
or fluorescence-activated cell sorting (FACS). Antibodies directed
to a protein encoded by hepcidin can be identified and obtained
from a variety of sources, such as the MSRS catalog of antibodies
(Aerie Corporation, Birmingham, Mich.), or can be prepared via
conventional antibody generation methods. Methods for preparation
of polyclonal antisera are taught in, for example, Ausubel, F. M.
et al., Current Protocols in Molecular Biology, Volume 2, pp.
11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of
monoclonal antibodies is taught in, for example, Ausubel, F. M. et
al., Current Protocols in Molecular Biology, Volume 2, pp.
11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.
[0038] Immunoprecipitation methods 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. 10.16.1-10.16.11, John Wiley
& Sons, Inc., 1998. Western blot (immunoblot) analysis is
standard in the art and can be found at, for example, Ausubel, F.
M. et al., Current Protocols in Molecular Biology, Volume 2, pp.
10.8.1-10.8.21, John Wiley & Sons, Inc., 1997.
Active Target Segments
[0039] The locations on the target nucleic acid defined by having
one or more active antisense compounds targeted thereto are
referred to as "active target segments." When an active target
segment is validated by multiple antisense compounds, the compounds
are preferably separated by no more than about 30 nucleotides on
the active target segment, more preferably no more than about 10
nucleotides on the target sequence, even more preferably the
compounds are contiguous, and most preferably the compounds are
overlapping. In a preferred embodiment, at least 50%, preferably at
least 70% of the oligonucleotides targeted to the active target
segment modulate expression of their target RNA at least 40%. There
may be substantial variation in activity (e.g., as defined by
percent reduction or activity ranking) of each antisense compounds
within an active target segment. Active antisense compounds are
those that modulate the expression of their target RNA. Active
antisense compounds reduce expression of their target RNA at least
10%, preferably at least 20%. In other embodiments at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95% or at least
99%. In a more preferred embodiment, the level of reduction
required to define an active antisense compound is based on the
results from the screen used to define the active target
segments.
Hybridization
[0040] As used herein, "hybridization" means the pairing of
complementary strands of antisense compounds to their target
sequence. While not limited to a particular mechanism, the most
common mechanism of pairing involves hydrogen bonding, which may be
Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,
between complementary nucleoside or nucleotide bases (nucleobases).
For example, the natural base adenine is complementary to the
natural nucleobases thymidine and uracil which pair through the
formation of hydrogen bonds. The natural base guanine is
complementary to the natural base 5-methylcytosine and the
artificial base known as a G-clamp. Hybridization can occur under
varying circumstances.
[0041] An antisense compound is specifically hybridizable when
there is a sufficient degree of complementarity to avoid
non-specific binding of the antisense compound to non-target
nucleic acid 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 under conditions in which
assays are performed in the case of in vitro assays.
[0042] As used herein, "stringent hybridization conditions" or
"stringent conditions" refers to conditions under which an
antisense compound will hybridize to its target sequence, but to a
minimal number of other sequences. Stringent conditions are
sequence-dependent and will be different in different
circumstances, and "stringent conditions" under which antisense
compounds hybridize to a target sequence are determined by the
nature and composition of the antisense compounds and the assays in
which they are being investigated.
Complementarity
[0043] "Complementarity," as used herein, refers to the capacity
for precise pairing between two nucleobases on either two
oligomeric compound strands or an antisense compound with 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 a complementary
position. The antisense compound and the further DNA or RNA are
complementary to each other when a sufficient number of
complementary positions in each molecule are occupied by
nucleobases which can hydrogen bond with each other. Thus,
"specifically hybridizable" and "complementary" are terms which are
used to indicate a sufficient degree of precise pairing or
complementarity over a sufficient number of nucleobases such that
stable and specific binding occurs between the antisense compound
and a target nucleic acid.
Identity
[0044] Antisense compounds, or a portion thereof, may have a
defined percent identity to a SEQ ID NO, or a compound having a
specific CMPD number. As used herein, a sequence is identical to
the sequence disclosed herein if it has the same nucleobase pairing
ability. For example, an RNA which contains uracil in place of
thymidine in the disclosed sequences would be considered identical
as they both pair with adenine. Similarly, a G-clamp modified
heterocyclic base would be considered identical to a cytosine or a
5-Me cytosine in the sequences of the instant application as it
pairs with a guanine. This identity may be over the entire length
of the oligomeric compound, or in a portion of the antisense
compound (e.g., nucleobases 1-20 of a 27-mer may be compared to a
20-mer to determine percent identity of the antisense compound to
the SEQ ID NO.) It is understood by those skilled in the art that
an antisense compound need not have an identical sequence to those
described herein to function similarly to the antisense compound
described herein. Shortened versions of antisense compound taught
herein, or non-identical versions of the antisense compound taught
herein fall within the scope of the current disclosure.
Non-identical versions are those wherein each base does not have
the same pairing activity as the antisense compounds disclosed
herein. In a preferred embodiment the antisense compounds can have
about 3 mismatched base pairs. Bases do not have the same pairing
activity by being shorter or having at least one abasic site.
Alternatively, a non-identical version can include at least one
base replaced with a different base with different pairing activity
(e.g., G can be replaced by C, A, or T). Percent identity is
calculated according to the number of bases that have identical
base pairing corresponding to the SEQ ID NO or identical bases
corresponding to the antisense compound to which it is being
compared. The non-identical bases may be adjacent to each other,
dispersed through out the oligonucleotide, or both.
[0045] For example, a 16-mer having the same sequence as
nucleobases 2-17 of a 20-mer is 80% identical to the 20-mer.
Alternatively, a 20-mer containing four nucleobases not identical
to another 20-mer is also 80% identical to that other 20-mer. A
14-mer having the same sequence as nucleobases 1-14 of an 18-mer is
78% identical to the 18-mer. Such calculations are well within the
ability of those skilled in the art.
[0046] The percent identity is based on the percent of nucleobases
in the original sequence present in a portion of the modified
sequence. Therefore, a 30 nucleobase antisense compound comprising
the full sequence of the complement of a 20 nucleobase active
target segment would have a portion of 100% identity with the
complement of the 20 nucleobase active target segment, while
further comprising an additional 10 nucleobase portion. Herein, the
complement of an active target segment may constitute a single
portion. In a preferred embodiment, the oligomeric compounds are at
least about 80%, more preferably at least about 85%, even more
preferably at least about 90%, most preferably at least 95%
identical to at least a portion of the complement of the active
target segments presented herein.
[0047] It is well known by those skilled in the art that it is
possible to increase or decrease the length of an antisense
compound and/or introduce mismatch bases without eliminating
activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA
89:7305-7309, 1992, incorporated herein by reference), a series of
ASOs 13-25 nucleobases in length were tested for their ability to
induce cleavage of a target RNA in an oocyte injection model. ASOs
25 nucleobases in length with 8 or 11 mismatch bases near the ends
of the ASOs were able to direct specific cleavage of the target
mRNA, albeit to a lesser extent than the ASOs that contained no
mismatches. Similarly, target specific cleavage was achieved using
a 13 nucleobase ASOs, including those with 1 or 3 mismatches. Maher
and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988, incorporated
herein by reference) tested a series of tandem 14 nucleobase ASOs,
and a 28 and 42 nucleobase ASOs comprised of the sequence of two or
three of the tandem ASOs, respectively, for their ability to arrest
translation of human DHFR in a rabbit reticulocyte assay. Each of
the three 14 nucleobase ASOs alone were able to reduce translation,
albeit at a more modest level than the 28 or 42 nucleobase
ASOs.
Therapeutics
[0048] The antisense compounds disclosed herein can be used to
modulate the expression of hepcidin in an animal, such as a human.
In one non-limiting embodiment, the methods comprise the step of
administering to said animal in need of therapy for a disease or
condition associated with hepcidin an effective amount of an
antisense compound that reduces expression of hepcidin. A disease
or condition associated with hepcidin includes, but is not limited
to, anemia. In one embodiment, the antisense compounds effectively
reduce the levels or function of hepcidin RNA. Because reduction in
hepcidin mRNA levels can lead to alteration in hepcidin protein
products of expression as well, such resultant alterations can also
be measured. Antisense compounds that effectively reduce the level
or function of hepcidin RNA or protein products of expression are
considered an active antisense compounds. In one embodiment, the
antisense compounds reduce the expression of hepcidin causing a
reduction of target RNA by at least about 10%, by at least about
20%, by at least about 25%, by at least about 30%, by at least
about 40%, by at least about 50%, by at least about 60%, by at
least about 70%, by at least about 75%, by at least about 80%, by
at least about 85%, by at least about 90%, by at least about 95%,
by at least about 98%, by at least about 99%, or by 100%.
[0049] For example, the reduction of the expression of hepcidin can
be measured in a bodily fluid, tissue or organ of the animal.
Methods of obtaining samples for analysis, such as body fluids
(e.g., blood, serum or urine), tissues (e.g., biopsy), or organs,
and methods of preparation of the samples to allow for analysis are
well known to those skilled in the art. Methods for analysis of RNA
and protein levels are discussed above and are well known to those
skilled in the art. The effects of treatment can be assessed by
measuring biomarkers associated with the hepcidin expression in the
aforementioned fluids, tissues or organs, collected from an animal
contacted with one or more oligomeric compounds targeted to a
nucleic acid encoding hepcidin, by routine clinical methods known
in the art. These biomarkers include but are not limited to:serum
iron levels, hemoglobin levels, transferrin levels, iron restricted
erythropoesis, liver transaminases, bilirubin, albumin, blood urea
nitrogen, creatine and other markers of kidney and liver function;
interleukins, tumor necrosis factors, chemokines, cytokines and
other markers of anemia.
[0050] The antisense compounds can be utilized in pharmaceutical
compositions by adding an effective amount of a compound to a
suitable pharmaceutically acceptable diluent or carrier. Acceptable
carriers and dilutents are well known to those skilled in the art.
Selection of a dilutent or carrier is based on a number of factors,
including, but not limited to, the solubility of the compound and
the route of administration. Such considerations are well
understood by those skilled in the art. In one aspect, the
antisense compounds reduce the expression of hepcidin. The
compounds can also be used in the manufacture of a medicament for
the treatment of diseases and conditions related to hepcidin
expression.
[0051] Methods whereby bodily fluids, organs or tissues are
contacted with an effective amount of one or more of the antisense
compounds or compositions are also contemplated. Bodily fluids,
organs or tissues can be contacted with one or more of the
antisense compounds resulting in modulation of hepcidin expression
in the cells of bodily fluids, organs or tissues. An effective
amount can be determined by monitoring the modulatory effect of the
antisense compound or compounds or compositions on target nucleic
acids or their products by methods routine to the skilled
artisan.
[0052] Thus, provided herein is the use of an isolated single- or
double-stranded antisense compound targeted to hepcidin in the
manufacture of a medicament for the treatment of a disease or
disorder by means of the method described above. In a preferred
embodiment, the antisense compound is a single stranded antisense
compound. Also provided herein is the use of a composition
comprising erythropoietin or other ESA and an antisense compound
targeted to hepcidin in the manufacture of a medicament for the
treatment of a disease or disorder by the means of the method
described above.
Kits, Research Reagents, And Diagnostics
[0053] The antisense compounds can be utilized for diagnostics, and
as research reagents and kits. Furthermore, antisense compounds,
which are able to reduce gene expression with specificity, are
often used by those of ordinary skill to elucidate the function of
particular genes or to distinguish between functions of various
members of a biological pathway.
[0054] For use in kits and diagnostics, the antisense compounds,
either alone or in combination with other compounds or
therapeutics, for example ESAs, can be used as tools in
differential and/or combinatorial analyses to elucidate expression
patterns of a portion or the entire complement of genes expressed
within cells and tissues. Methods of gene expression analysis are
well known to those skilled in the art.
Compounds
[0055] The term "oligomeric compound" refers to a polymeric
structure capable of hybridizing to a region of a nucleic acid
molecule. Generally, oligomeric compounds comprise a plurality of
monomeric subunits linked together by internucleoside linking
groups and/or internucleoside linkage mimetics. Each of the
monomeric subunits comprises a sugar, abasic sugar, modified sugar,
or a sugar mimetic, and except for the abasic sugar includes a
nucleobase, modified nucleobase or a nucleobase mimetic. Preferred
monomeric subunits comprise nucleosides and modified
nucleosides.
[0056] An "antisense compound" or "antisense oligomeric compound"
refers to an oligomeric compound that is at least partially
complementary to the region of a target nucleic acid molecule to
which it hybridizes and which modulates (increases or decreases)
its expression. This term includes oligonucleotides,
oligonucleosides, oligonucleotide analogs, oligonucleotide
mimetics, antisense compounds, antisense oligomeric compounds, and
chimeric combinations of these. Consequently, while all antisense
compounds can be said to be oligomeric compounds, not all
oligomeric compounds are antisense compounds. An "antisense
oligonucleotide" is an antisense compound that is a nucleic
acid-based oligomer. An antisense oligonucleotide can, in some
cases, include one or more chemical modifications to the sugar,
base, and/or internucleoside linkages. Nonlimiting examples of
antisense compounds include antisense compounds, antisense
oligonucleotides, external guide sequence (EGS) oligonucleotides,
alternate splicers, and siRNAs. As such, these compounds can be
introduced in the form of single-stranded, double-stranded,
circular, branched or hairpins and can contain structural elements
such as internal or terminal bulges or loops. Antisense
double-stranded compounds can be two strands hybridized to form
double-stranded compounds or a single strand with sufficient self
complementarity to allow for hybridization and formation of a fully
or partially double-stranded compound. The oligomeric compounds are
not auto-catalytic. As used herein, "auto-catalytic" means a
compound has the ability to promote cleavage of the target RNA in
the absence of accessory factors, e.g. proteins.
[0057] In one embodiment, the antisense compound comprises a single
stranded oligonucleotide. In some embodiments the antisense
compound contains chemical modifications. In a preferred
embodiment, the antisense compound is a single stranded, chimeric
oligonucleotide wherein the modifications of sugars, bases, and
internucleoside linkages are independently selected.
[0058] The antisense compounds may comprise an antisense compound
from about 12 to about 35 nucleobases (i.e. from about 12 to about
35 linked nucleosides). In other words, a single-stranded anisense
compound comprises from about 12 to about 35 nucleobases, and a
double-stranded antisense compound (such as a siRNA, for example)
comprises two strands, each of which is independently from about 12
to about 35 nucleobases. This includes oligonucleotides 15 to 35
and 16 to 35 nucleobases in length. Contained within the antisense
compounds (whether single or double stranded and on at least one
strand) are antisense portions. The "antisense portion" is that
part of the antisense compound that is designed to work by one of
the aforementioned antisense mechanisms. One of ordinary skill in
the art will appreciate that about 12 to about 35 nucleobases
includes 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases.
[0059] Antisense compounds about 12 to 35 nucleobases in length,
preferably about 15 to 35 nucleobases in length, comprising a
stretch of at least eight (8), preferably at least 12, more
preferably at least 15 consecutive nucleobases selected from within
the active target regions are considered to be suitable antisense
compounds as well.
[0060] Modifications can be made to the antisense compounds and may
include conjugate groups attached to one of the termini, selected
nucleobase positions, sugar positions or to one of the
internucleoside linkages. Possible modifications include, but are
not limited to, 2'-fluoro (2'-F), 2'-OMethyl (2'-OMe),
2'-Methoxyethoxy (2'-MOE) sugar modifications, inverted abasic
caps, deoxynucleobases, and bicyclic nucleic acids such as locked
nucleic acids (LNA) and ethylene nucleic acid (ENA).
[0061] In one embodiment, double-stranded antisense compounds
encompass short interfering RNAs (siRNAs). As used herein, the term
"siRNA" is defined as a double-stranded compound having a first and
second strand, each strand having a central portion and two
independent terminal portions. The central portion of the first
strand is complementary to the central portion of the second
strand, allowing hybridization of the strands. The terminal
portions are independently, optionally complementary to the
corresponding terminal portion of the complementary strand. The
ends of the strands may be modified by the addition of one or more
natural or modified nucleobases to form an overhang.
[0062] Each strand of the siRNA duplex may be from about 12 to
about 35 nucleobases. In a preferred embodiment, each strand of the
siRNA duplex is about 17 to about 25 nucleobases. The two strands
may be fully complementary (i.e., form a blunt ended compound), or
include a 5' or 3' overhang on one or both strands. Double-stranded
compounds can be made to include chemical modifications as
discussed herein.
Chemical Modifications
[0063] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base (sometimes referred to as a "nucleobase" or
simply a "base"). The two most common classes of such heterocyclic
bases are the purines and the pyrimidines. Nucleotides are
nucleosides that further include a phosphate group covalently
linked to the sugar portion of the nucleoside. For those
nucleosides that include a pentofuranosyl sugar, the phosphate
group can be linked to the 2', 3' or 5' hydroxyl moiety of the
sugar. In forming oligonucleotides, the phosphate groups covalently
link adjacent nucleosides to one another to form a linear polymeric
compound. Within oligonucleotides, the phosphate groups are
commonly referred to as forming the internucleoside backbone of the
oligonucleotide. The normal linkage or backbone of RNA and DNA is a
3' to 5' phosphodiester linkage. It is often preferable to include
chemical modifications in oligonucleotides to alter their activity.
Chemical modifications can alter oligonucleotide activity by, for
example: increasing affinity of an antisense oligonucleotide for
its target RNA, increasing nuclease resistance, and/or altering the
pharmacokinetics of the oligonucleotide. The use of chemistries
that increase the affinity of an oligonucleotide for its target can
allow for the use of shorter oligonucleotide compounds.
[0064] The term "nucleobase" or "heterocyclic base moiety" as used
herein, refers to the heterocyclic base portion of a nucleoside. In
general, a nucleobase is any group that contains one or more atom
or groups of atoms capable of hydrogen bonding to a base of another
nucleic acid. In addition to "unmodified" or "natural" nucleobases
such as the purine nucleobases adenine (A) and guanine (G), and the
pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U),
many modified nucleobases or nucleobase mimetics known to those
skilled in the art are amenable to the oligomeric compounds. The
terms modified nucleobase and nucleobase mimetic can overlap but
generally a modified nucleobase refers to a nucleobase that is
fairly similar in structure to the parent nucleobase, such as for
example a 7-deaza purine or a 5-methyl cytosine, whereas a
nucleobase mimetic would include more complicated structures, such
as for example a tricyclic phenoxazine nucleobase mimetic. Methods
for preparation of the above noted modified nucleobases are well
known to those skilled in the art.
[0065] Antisense compounds may also contain one or more nucleosides
having modified sugar moieties. The furanosyl sugar ring of a
nucleoside can be modified in a number of ways including, but not
limited to, addition of a substituent group bridging two
non-geminal ring atoms to form a bicyclic nucleic acid (BNA) and
substitution of an atom or group such as --S--, -N(R)-- or
--C(R.sub.1)(R.sub.2) for the ring oxygen at the 4'-position.
Modified sugar moieties are well known and can be used to alter,
typically increase, the affinity of the antisense compound for its
target and/or increase nuclease resistance. A representative list
of preferred modified sugars includes but is not limited to
bicyclic modified sugars (BNA's), including LNA and ENA
(4'-(CH.sub.2).sub.2--O-2' bridge); and substituted sugars,
especially 2'-substituted sugars having a 2'-F, 2'-OCH.sub.2 or a
2'-O(CH.sub.2).sub.2--OCH.sub.3 substituent group. Sugars can also
be replaced with sugar mimetic groups among others. Methods for the
preparations of modified sugars are well known to those skilled in
the art.
[0066] Included herein are internucleoside linking groups that link
the nucleosides or otherwise modified monomer units together
thereby forming an antisense compound. The two main classes of
internucleoside linking groups are defined by the presence or
absence of a phosphorus atom. Representative phosphorus containing
internucleoside linkages include, but are not limited to,
phosphodiesters, phosphotriesters, methylphosphonates,
phosphoramidate, and phosphorothioates. Representative
non-phosphorus containing internucleoside linking groups include,
but are not limited to, methylenemethylimino
(--CH.sub.2--N(CH.sub.3)--O--CH.sub.2--), thiodiester
(--O--C(O)--S--), thionocarbamate (--O--C(O)(NH)--S--); siloxane
(--O--Si(H)2--O--); and N,N'-dimethylhydrazine
(--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--). Antisense compounds
having non-phosphorus internucleoside linking groups are referred
to as oligonucleosides. Modified internucleoside linkages, compared
to natural phosphodiester linkages, can be used to alter, typically
increase, nuclease resistance of the antisense compound.
Internucleoside linkages having a chiral atom can be prepared
racemic, chiral, or as a mixture. Representative chiral
internucleoside linkages include, but are not limited to,
alkylphosphonates and phosphorothioates. Methods of preparation of
phosphorous-containing and non-phosphorous-containing linkages are
well known to those skilled in the art.
[0067] As used herein the term "mimetic" refers to groups that are
substituted for a sugar, a nucleobase, and/ or internucleoside
linkage. Generally, a mimetic is used in place of the sugar or
sugar-internucleoside linkage combination, and the nucleobase is
maintained for hybridization to a selected target. Representative
examples of a sugar mimetic include, but are not limited to,
cyclohexenyl or morpholino. Representative examples of a mimetic
for a sugar-internucleoside linkage combination include, but are
not limited to, peptide nucleic acids (PNA) and morpholino groups
linked by uncharged achiral linkages. In some instances a mimetic
is used in place of the nucleobase. Representative nucleobase
mimetics are well known in the art and include, but are not limited
to, tricyclic phenoxazine analogs and universal bases (Berger et
al., (2000) Nuc Acid Res., 28:2911-14, incorporated herein by
reference). Methods of synthesis of sugar, nucleoside and
nucleobase mimetics are well known to those skilled in the art.
[0068] As used herein the term "nucleoside" includes, nucleosides,
abasic nucleosides, modified nucleosides, and nucleosides having
mimetic bases and/or sugar groups.
[0069] As used herein, the term "oligonucleotide" refers to an
oligomeric compound which is an oligomer or polymer of ribonucleic
acid (RNA) or deoxyribonucleic acid (DNA). This term includes
oligonucleotides composed of naturally- and non-naturally-occurring
nucleobases, sugars and covalent internucleoside linkages, possibly
further including non-nucleic acid conjugates.
[0070] Provided herein are compounds having reactive phosphorus
groups useful for forming internucleoside linkages including for
example phosphodiester and phosphorothioate internucleoside
linkages. Methods of preparation and/or purification of precursors
or antisense compounds are not a limitation of the compositions or
methods described herein. Methods for synthesis and purification of
DNA, RNA, and the antisense compounds are well known to those
skilled in the art.
[0071] As used herein the term "chimeric antisense compound" refers
to an antisense compound, having at least one sugar, nucleobase
and/or internucleoside linkage that is differentially modified as
compared to the other sugars, nucleobases and internucleoside
linkages within the same oligomeric compound. The remainder of the
sugars, nucleobases and internucleoside linkages can be
independently modified or unmodified. In general a chimeric
oligomeric compound will have modified nucleosides that can be in
isolated positions or grouped together in regions that will define
a particular motif. Any combination of modifications and or mimetic
groups can comprise a chimeric oligomeric compound.
[0072] Chimeric oligomeric compounds typically contain at least one
region modified so as to confer increased resistance to nuclease
degradation, increased cellular uptake, and/or increased binding
affinity for the target nucleic acid. An additional region of the
oligomeric compound may 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 that 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
reduction of gene expression. Consequently, comparable results can
often be obtained with shorter oligomeric compounds when chimeras
are used, compared to for example phosphorothioate
deoxyoligonucleotides hybridizing to the same target region.
Cleavage of the RNA target can be routinely detected by gel
electrophoresis and, if necessary, associated nucleic acid
hybridization techniques known in the art.
[0073] Certain chimeric as well as non-chimeric oligomeric
compounds can be further described as having a particular motif. As
used in herein the term "motif" refers to the orientation of
modified sugar moieties and/or sugar mimetic groups in an antisense
compound relative to like or differentially modified or unmodified
nucleosides. As used herein, the terms "sugars", "sugar moieties"
and "sugar mimetic groups` are used interchangeably. Such motifs
include, but are not limited to, gapped motifs, alternating motifs,
fully modified motifs, hemimer motifs, blockmer motifs, and
positionally modified motifs. The sequence and the structure of the
nucleobases and type of internucleoside linkage is not a factor in
determining the motif of an antisense compound.
[0074] As used herein the term "gapped motif" refers to an
antisense compound comprising a contiguous sequence of nucleosides
that is divided into 3 regions, an internal region (gap) flanked by
two external regions (wings). The regions are differentiated from
each other at least by having differentially modified sugar groups
that comprise the nucleosides. In some embodiments, each modified
region is uniformly modified (e.g. the modified sugar groups in a
given region are identical); however, other motifs can be applied
to regions. For example, the wings in a gapmer could have an
alternating motif. The nucleosides located in the gap of a gapped
antisense compound have sugar moieties that are different than the
modified sugar moieties in each of the wings.
[0075] As used herein the term "alternating motif" refers to an
antisense compound comprising a contiguous sequence of nucleosides
comprising two differentially sugar modified nucleosides that
alternate for essentially the entire sequence of the antisense
compound, or for essentially the entire sequence of a region of an
antisense compound.
[0076] As used herein the term "fully modified motif" refers to an
antisense compound comprising a contiguous sequence of nucleosides
wherein essentially each nucleoside is a sugar modified nucleoside
having uniform modification.
[0077] As used herein the term "hemimer motif" refers to a sequence
of nucleosides that have uniform sugar moieties (identical sugars,
modified or unmodified) and wherein one of the 5'-end or the 3'-end
has a sequence of from 2 to 12 nucleosides that are sugar modified
nucleosides that are different from the other nucleosides in the
hemimer modified antisense compound.
[0078] As used herein the term "blockmer motif" refers to a
sequence of nucleosides that have uniform sugars (identical sugars,
modified or unmodified) that is internally interrupted by a block
of sugar modified nucleosides that are uniformly modified and
wherein the modification is different from the other nucleosides.
Methods of preparation of chimeric oligonucleotide compounds are
well known to those skilled in the art.
[0079] As used herein the term "positionally modified motif"
comprises all other motifs. Methods of preparation of positionally
modified oligonucleotide compounds are well known to those skilled
in the art.
[0080] The compounds described herein contain one or more
asymmetric centers and thus give rise to enantiomers,
diastereomers, and other stereoisomeric configurations that may be
defined, in terms of absolute stereochemistry, as (R) or (S), alpha
or beta, or as (D) or (L) such as for amino acids et al. The
present description of oligomeric compounds is meant to include all
such possible isomers, as well as their racemic and optically pure
forms.
[0081] In one aspect, antisense compounds are modified by covalent
attachment of one or more conjugate groups. Conjugate groups may be
attached by reversible or irreversible attachments. Conjugate
groups may be attached directly to antisense compounds or by use of
a linker. Linkers may be mono- or bifunctional linkers. Such
attachment methods and linkers are well known to those skilled in
the art. In general, conjugate groups are attached to antisense
compounds to modify one or more properties. Such considerations are
well known to those skilled in the art.
Oligomer Synthesis
[0082] Oligomerization of modified and unmodified nucleosides can
be routinely performed according to literature procedures for DNA
(Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993),
Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-217.
Gait et al., Applications of Chemically synthesized RNA in RNA:
Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al.,
Tetrahedron (2001), 57, 5707-5713).
[0083] Antisense compounds 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 may additionally or
alternatively be employed. It is well known to use similar
techniques to prepare oligonucleotides such as the
phosphorothioates and alkylated derivatives. The current
description is not limited by the method of antisense compound
synthesis.
Oligomer Purification And Analysis
[0084] Methods of oligonucleotide purification and analysis are
known to those skilled in the art. Analysis methods include
capillary electrophoresis (CE) and electrospray-mass spectroscopy.
Such synthesis and analysis methods can be performed in multi-well
plates. The methods described herein are not limited by the method
of oligomer purification.
Salts, Prodrugs And Bioequivalents
[0085] The antisense compounds comprise any pharmaceutically
acceptable salts, esters, or salts of such esters, or any other
functional chemical equivalent 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 antisense compounds,
pharmaceutically acceptable salts of such prodrugs, and other
bioequivalents.
[0086] The term "prodrug" indicates a therapeutic agent that is
prepared in an inactive or less active form that is converted to an
active form (i.e., drug) within the body or cells thereof by the
action of endogenous enzymes, chemicals, and/or conditions. In
particular, prodrug versions of the oligonucleotides are prepared
as SATE ((S-acetyl-2-thioethyl) phosphate) derivatives according to
the methods disclosed in WO 93/24510 or WO 94/26764. Prodrugs can
also include antisense compounds wherein one or both ends comprise
nucleobases that are cleaved (e.g., phosphodiester backbone
linkages) to produce the active compound.
[0087] The term "pharmaceutically acceptable salts" refers to
physiologically and pharmaceutically acceptable salts of the
compounds: i.e., salts that retain the desired biological activity
of the parent compound and do not impart undesired toxicological
effects thereto. Sodium salts of antisense oligonucleotides are
useful and are well accepted for therapeutic administration to
humans. In another embodiment, sodium salts of dsRNA compounds are
also provided.
Combinations
[0088] Compositions can contain two or more antisense compounds. In
another related embodiment, compositions can contain one or more
antisense compounds, particularly oligonucleotides, targeted to a
first nucleic acid and one or more additional antisense compounds
targeted to a second nucleic acid target. Alternatively,
compositions can contain two or more antisense compounds targeted
to different regions of the same nucleic acid target. Compositions
can also include other non-antisense compound therapeutic agents.
For example, disclosed herein are combinations comprising an
antisense oligonucleotide targeted to hepcidin combined with
erythropoietin. Two or more combined compounds may be used together
or sequentially. Likewise, one or more compound and one or more
therapeutic agent may be used together or sequentially. For
example, disclosed herein are compounds and therapeutics and
methods of use wherein the compounds are administered
simultaneously, or at distinct timepoints. For example, an
antisense oligonucleotide targeted to a nucleic acid encoding
hepcidin can be administered either simultaneously with or at a
distinct timepoint from erythropoietin.
Nonlimiting Disclosure And Incorporation By Reference
[0089] While certain compounds, compositions and methods have been
described with specificity in accordance with certain embodiments,
the following examples serve only to illustrate the compounds and
methods and are not intended to limit the same. Each of the
references, GenBank accession numbers, and the like recited in the
present application is incorporated herein by reference in its
entirety.
EXAMPLE 1
Cell Types And Transfection Methods
[0090] The effect of oligomeric compounds on target nucleic acid
expression was tested on the following cell types.
[0091] Mouse Primary Hepatocytes: Primary mouse hepatocytes were
prepared from CD-1 mice purchased from Charles River Labs
(Wilmington, Mass.). Primary mouse hepatocytes were routinely
cultured in Hepatocyte Attachment Media supplemented with 10% fetal
bovine serum, 1% penicillin/streptomycin (both from Sigma-Aldrich,
St. Louis, Mo.), 1% antibiotic-antimitotic (Invitrogen Life
Technologies, Carlsbad, Calif.) and 10 nM bovine insulin
(Sigma-Aldrich, St. Louis, Mo.). Cells were seeded into 96-well
plates (Falcon-Primaria #3872) coated with 0.1 mg/ml collagen at a
density of approximately 10,000 cells/well for use in oligomeric
compound transfection experiments.
[0092] HepG2: The human hepatocarcinoma cell line was obtained from
the American Type Culture Collection (Manassas, Va.). HepG2 cells
were routinely cultured in minimum essential medium (Eagle) with 2
mM L-glutamine and Earle's BSS (Cambrex, East Rutherford, N.J.)
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% (sigma-Aldrich, St. Louis, Mo.) at a temperature
of 37.degrees.C. Cells were routinely passaged by trypsinization
and dilution when they reached approximately 80% confluence. Cells
were seeded into 96-well plates (Falcon-Primaria #3872) at a
density of approximately 5000 cells/well for use in oligomeric
compound transfection experiments.
[0093] When cells reached 65-75% confluency, they were treated with
oligonucleotide. Oligonucleotide was mixed with LIPOFECTIN.TM.
Invitrogen Life Technologies, Carlsbad, Calif.) in OPTI-MEM.TM.-1
reduced serum medium (Invitrogen Life Technologies, Carlsbad,
Calif.) to achieve the desired concentration of oligonucleotide and
a LIPOFECTIN.TM. concentration of 2.5 or 3 .micro.g/mL per 100 nM
oligonucleotide. This transfection mixture was incubated at room
temperature for approximately 0.5 hours. For cells grown in 96-well
plates, wells were washed once with 100 .micro.L OPTI-MEMT.TM.-1
and then treated with 130 .micro.L of the transfection mixture.
Cells were treated and data were obtained in duplicate or
triplicate. After approximately 4-7 hours of treatment at
37.degrees.C., the medium containing the transfection mixture was
replaced with fresh culture medium. Cells were harvested 16-24
hours after oligonucleotide treatment.
[0094] Control oligonucleotides were used to determine the optimal
oligomeric compound concentration for a particular cell line.
Furthermore, when oligomeric compounds were tested in oligomeric
compound screening experiments or phenotypic assays, control
oligonucleotides were tested in parallel.
[0095] The concentration of oligonucleotide used varied from cell
line to cell line. To determine the optimal oligonucleotide
concentration for a particular cell line, the cells were treated
with a positive control oligonucleotide at a range of
concentrations. The concentration of positive control
oligonucleotide that resulted in about an 80% reduction of the
target mRNA was then utilized as the screening concentration for
new oligonucleotides in subsequent experiments for that cell line.
If 80% reduction was not achieved, the lowest concentration of
positive control oligonucleotide that resulted in a 60% reduction
of the target mRNA was then utilized as the oligonucleotide
screening concentration in subsequent experiments for that cell
line. If 60% reduction was not achieved, that particular cell line
was deemed as unsuitable for oligonucleotide transfection
experiments.
EXAMPLE 2
Real-Time Quantitative PCR Analysis of Hepcidin mRNA Levels
[0096] Quantitation of hepcidin mRNA levels was accomplished by
real-time quantitative PCR using the ABI PRISM.TM. 7600, 7700, or
7900 Sequence Detection System (PE-Applied Biosystems, Foster City,
Calif.) according to manufacturer's instructions.
[0097] Prior to quantitative PCR analysis, primer-probe sets
specific to the hepcidin being measured were evaluated for their
ability to be "multiplexed" with a GAPDH amplification reaction.
After isolation the RNA was subjected to sequential reverse
transcriptase (RT) reaction and real-time PCR, both of which were
performed in the same well. RT and PCR reagents were obtained from
Invitrogen Life Technologies (Carlsbad, Calif.). RT, real-time PCR
was carried out in the same by adding 20 .micro.L PCR cocktail
(2.5.times.PCR buffer minus MgCl.sub.2, 6.6 mM MgCl.sub.2, 375
.micro.M each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward
primer and reverse primer, 125 nM of probe, 4 Units RNAse
inhibitor, 1.25 Units PLATINUM.RTM. Taq, 5 Units MuLV reverse
transcriptase, and 2.5.times.ROX dye) to 96-well plates containing
30 .micro.L total RNA solution (20-200 ng). The RT reaction was
carried out by incubation for 30 minutes at 48.degrees.C. Following
a 10 minute incubation at 95.degrees.C. to activate the
PLATINUM.RTM. Taq, 40 cycles of a two-step PCR protocol were
carried out: 95.degrees.C. for 15 seconds (denaturation) followed
by 60.degrees.C. for 1.5 minutes (annealing/extension).
[0098] Gene target quantities obtained by RT, real-time PCR were
normalized using either the expression level of GAPDH, a gene whose
expression is constant, or by quantifying total RNA using
RiboGreen.TM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH
expression was quantified by RT, real-time PCR, by being run
simultaneously with the target, multiplexing, or separately. Total
RNA was quantified using RiboGreen.TM. RNA quantification reagent
(Molecular Probes, Inc. Eugene, Oreg.).
[0099] 170 .micro.L of RiboGreen.TM. working reagent (RiboGreen.TM.
reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was
pipetted into a 96-well plate containing 30 .micro.L purified
cellular RNA. The plate was read in a CytoFluor 4000 (PE Applied
Biosystems) with excitation at 485 nm and emission at 530 nm.
[0100] The GAPDH PCR probes have JOE covalently linked to the 5'
end and TAMRA or MGB covalently linked to the 3' end, where JOE is
the fluorescent reporter dye and TAMRA or MGB is the quencher dye.
In some cell types, primers and probe designed to a GAPDH sequence
from a different species are used to measure GAPDH expression. For
example, a human GAPDH primer and probe set is used to measure
GAPDH expression in monkey-derived cells and cell lines.
[0101] Probes and primers for use in real-time PCR were designed to
hybridize to target-specific sequences. The primers and probes and
the target nucleic acid sequences to which they hybridize are
presented in Table 2. The target-specific PCR probes have FAM
covalently linked to the 5' end and TAMRA or MGB covalently linked
to the 3' end, where FAM is the fluorescent dye and TAMRA or MGB is
the quencher dye.
TABLE-US-00002 TABLE 2 Hepcidin-specific primers and probes for use
in real-time PCR Target SEQ SEQ ID Sequence ID Species NO
Description Sequence (5' to 3') NO Human 2 Forward Primer
AGGAGGCGAGACACCCACTT 6 Human 2 Reverse Primer
TCCCACACTTTGATCGATGACA 7 Human 2 Probe CATCTGCATTTTCTGCTGCGGCTG 8
Mouse 4 Forward Primer TGCAGAAGAGAAGGAAGAGAGACA 9 Mouse 4 Reverse
Primer CACACTGGGAATTGTTACAGCATT 10 Mouse 4 Probe
CAACTTCCCCATCTGCATCTTCTGCTGT 11
EXAMPLE 3
Antisense Reduction of Human Hepcidin Expression By Oligomeric
Compounds
[0102] A series of antisense oligonucleotide compounds was designed
to target different regions of human hepcidin RNA, using published
sequences or portions of published sequences as cited in Table 1.
The compounds are shown in Table 3. Methods for designing and
testing antisense oligonucleotides for reduction of mRNA target
expression are discussed herein and well known to those skilled in
the art. Although an antisense oligonucleotide is reported in Table
3 as being targeted to a particular sequence, one ordinarily
skilled in the art will know that these sequences may also target
other sequences. For example, in Table 3 SEQ ID NO: 50 is
reportedly designed to target nucleotides 261 to 280 of SEQ ID NO:
2. However, SEQ ID NO: 50 is also fully complementary to
nucleotides 486 to 505 of SEQ ID NO: 1 and nucleotides 4662 to 4681
of SEQ ID NO: 3.
[0103] The compounds were analyzed for their effect on target mRNA
levels by using quantitative real time RT-PCR as described above.
The screen identified active target segments within these regions
of the human hepcidin mRNA sequence and the genomic sequence
encoding hepcidin mRNA, specifically GenBank numbers BM719679.1;
NM.sub.--021175.2; and nucleotides 7819907 to 7825131 of
NT.sub.--011196.11 (SEQ ID NOS: 1; 2; and 3, respectively). The
activity of these antisense oligonucleotides to reduce hepcidin
mRNA expression is ranked as follows: +++++ (reduction of target by
greater than about 70%), ++++ (reduction of target by greater than
about 70% to about 74%), +++ (reduction of target by greater than
about 60% to about 69%), ++ (reduction of target by greater than
about 55% to about 60%), + (reduction of target by greater than
about 50% to about 55%)or bc (below cut-off or lesser than about
50%). Antisense compounds having an activity ranking of bc may have
shown some reduction of hepcidin expression but fell below the
cut-off level for this screening assay.
TABLE-US-00003 TABLE 3 Reduction of Human Hepcidin mRNA Levels by
Antisense Oligonucleotides Target Target SEQ CMPD SEQ ID Start
Activity ID # NO Site Sequence (5' to 3') Ranking NO 392287 1 32
AATGCACAGGCCCTGCCATC + 12 392288 1 38 CACAGAAATGCACAGGCCCT bc 13
392289 1 43 TCAAGCACAGAAATGCACAG bc 14 392290 1 48
CCCACTCAAGCACAGAAATG bc 15 392291 1 53 CAAGGCCCACTCAAGCACAG +++ 16
392292 1 58 ACTTTCAAGGCCCACTCAAG ++++ 17 392280 1 66
GCTGAACCACTTTCAAGGCC bc 18 392281 1 71 TGGTTGCTGAACCACTTTCA bc 19
392282 1 91 GAGGAATGAACACTTCTTCC bc 20 392283 1 96
TTGTCGAGGAATGAACACTT ++ 21 392284 1 101 TGTTGTTGTCGAGGAATGAA bc 22
392285 1 123 CCAAGTCACCAGAGCCCGGG +++++ 23 392286 1 128
GTCAGCCAAGTCACCAGAGC ++++ 24 392304 1 133 CCAGTGTCAGCCAAGTCACC bc
25 392305 1 138 GCCATCCAGTGTCAGCCAAG + 26 392306 1 143
CCAGGGCCATCCAGTGTCAG +++ 27 392307 1 148 TCATTCCAGGGCCATCCAGT ++++
28 392308 1 153 CTTTTTCATTCCAGGGCCAT ++ 29 392309 1 179
GGCCCTTGCACATTTTGCCT + 30 392293 1 186 CCAGATGGGCCCTTGCACAT ++++ 31
392294 1 191 TGGTTCCAGATGGGCCCTTG bc 32 392295 1 196
GGCCTTGGTTCCAGATGGGC +++ 33 392296 1 215 GTGACAGTCGCTTTTATGGG bc 34
392297 1 220 ACCGAGTGACAGTCGCTTTT bc 35 392298 1 225
CTGGGACCGAGTGACAGTCG + 36 392299 1 547 CAGGGCAGGTAGGTTCTACG +++ 37
392316 2 3 TGTCTGGGACCGAGTGACAG bc 38 392323 2 8
TCTGGTGTCTGGGACCGAGT + 39 392318 2 70 CTGGGAGCTCAGTGCCATCG +++++ 40
392319 2 75 CAGATCTGGGAGCTCAGTGC bc 41 392300 2 127
AGAGCCACTGGTCAGGCTGG +++++ 42 392320 2 132 AAAACAGAGCCACTGGTCAG bc
43 392302 2 137 GTGGGAAAACAGAGCCACTG +++ 44 392310 2 161
GCTCTGCAAGTTGTCCCGTC ++++ 45 392311 2 166 TTGCAGCTCTGCAAGTTGTC bc
46 392303 2 212 GGAACATGGGCATCCAGCTG bc 47 392313 2 217
CCTCTGGAACATGGGCATCC ++++ 48 392314 2 242 GGAAGTGGGTGTCTCGCCTC
+++++ 49 392315 2 261 CAGCAGAAAATGCAGATGGG +++++ 50 392326 2 268
GCAGCCGCAGCAGAAAATGC +++++ 51 392327 2 273 TGACAGCAGCCGCAGCAGAA +++
52 392328 2 278 ATCGATGACAGCAGCCGCAG +++++ 53 392330 2 287
CACACTTTGATCGATGACAG ++ 54 392329 2 292 CATCCCACACTTTGATCGAT ++++
55 392321 2 297 CAGCACATCCCACACTTTGA ++ 56 392322 2 302
TCTTGCAGCACATCCCACAC bc 57 392301 2 382 TTATTCCAAGACCTATGTTC ++ 58
392317 2 390 CAGCCATTTTATTCCAAGAC +++ 59 392324 2 395
AGAACCAGCCATTTTATTCC bc 60 392325 2 400 ACAAAAGAACCAGCCATTTT bc 61
392340 3 2917 CCTGACCAGGATGGAGAAAT +++ 62 392341 3 2924
CAAATTCCCTGACCAGGATG + 63 392339 3 2929 TTTAACAAATTCCCTGACCA bc 64
392338 3 2934 CAGTCTTTAACAAATTCCCT bc 65 392344 3 2942
GTTTTCATCAGTCTTTAACA +++ 66 392343 3 2963 TAGACACAATTATTTATTCA bc
67 392342 3 2968 TGTACTAGACACAATTATTT ++ 68 392332 3 2973
TAGAATGTACTAGACACAAT + 69 392333 3 2982 ATTCACGAATAGAATGTACT +++ 70
392331 3 2987 ATGAGATTCACGAATAGAAT bc 71 392337 3 3018
ATGGGTCACGGTCACTCTAC ++ 72 392336 3 3023 GGCGAATGGGTCACGGTCAC +++
73 392335 3 3028 TGTGTGGCGAATGGGTCACG bc 74 392334 3 3227
CAGCCCCTTGACCATGCCTG ++++ 75 392357 3 3260 TGTCACTTTCTCATGAAACA +++
76 392356 3 3278 TCCAAGACGAAGGTCAACTG bc 77 392355 3 3349
TTGTTCAGCCTGACCCTGGG +++++ 78 392354 3 3375 TTATCCCCAAGTCCCACCAT bc
79 392353 3 3383 CCTCAGCCTTATCCCCAAGT bc 80 392352 3 3398
TAGGGACTGCCCACCCCTCA +++++ 81 392348 3 3420 CTGCATGGTTGCCCACAAGA
++++ 82 392347 3 3425 AGTGTCTGCATGGTTGCCCA +++++ 83 392346 3 3430
AAATCAGTGTCTGCATGGTT +++++ 84 392345 3 3435 AGGAAAAATCAGTGTCTGCA
++++ 85 392351 3 3782 TCCATTTTAAAAACCTACAG bc 86 392350 3 3803
GCCAGTGAGATTGTGGTTTT bc 87 392349 3 3808 ACATGGCCAGTGAGATTGTG +++
88 392312 3 4516 GGCATCCAGCTGGCCCTGGC ++++ 89
[0104] Each active target segment was targeted by multiple, active
antisense oligonucleotides. This current example includes
identification of active target segments identified on SEQ ID NO:
1, which active target segments are referred to as Regions A-E. The
active target segments include nucleotides 456 to 527 for Region A,
nucleotides 123 to 252 for Region B, nucleotides 352 to 405 for
Region C, nucleotides 429 to 566 for Region D, and nucleotides 295
to 566 for Region E. Each of the oligonucleotides tested within
Region A reduced expression of human hepcidin at least 55%
(activity ranking ++). Four-fifths of the oligonucleotides tested
in this region reduced expression by at least 75% (activity ranking
+++++). Moreover, in Region D more than four-fifths of the
oligonucleotides tested reduced expression by at least 55%
(activity ranking ++) and more than one-half reduced expression by
at least 70% (activity ranking ++++). In Region E, three-fourths of
the oligonucleotides tested reduced expression by at least 50%
(activity ranking +) and nearly one-half reduced expression by at
least 70% (activity ranking ++++). Other active target segments on
SEQ ID NO: 1 include Region C where three-fourths of the
oligonucleotides tested reduced expression by at least 60%
(activity ranking +++), and Region B where two-thirds of the
oligonucleotides tested reduce expression by at least 50% (activity
ranking +).
[0105] Active target segments were also identified on SEQ ID NO: 2
and SEQ ID NO: 3. The active target segments of SEQ ID NO: 2 are
referred to as Regions F and G. Region F includes nucleotides 231
to 302. All of the nucleotides tested in Region F reduce expression
by at least 55% (activity ranking ++), and two-thirds reduce at
least 70% (activity ranking ++++). Region G includes nucleotides
204 to 341. More than four-fifths of the nucleotides tested in
Region G reduce expression by at least 55% (activity ranking ++)
and more than one-half reduce expression by at least 70% (activity
ranking ++++).
[0106] The active target segments of SEQ ID NO: 3 are referred to
as Regions H-K. Region H includes nucleotides 3398 to 3454 and all
of the oligonucleotides tested in this region reduce expression by
at least 70% (activity ranking ++++). Region I includes nucleotides
3249 to 3454. In Region I, three-fourths of the oligonucleotides
tested reduced expression by at least 70% (activity ranking ++++)
and one-half reduce by at least 75% (activity ranking +++++).
Region J includes nucleotides 4643 to 4703. All of the
oligonucleotides tested in this region reduce expression by at
least 60% (activity ranking +++) and four-fifths reduce expression
by at least 75% (activity ranking +++++). Region K includes
nucleotides 4643 to 4743 and at least one-half of the
oligonucleotides tested in this region reduce expression by at
least 70% (activity ranking ++++).
[0107] Thus, Applicant has discovered regions of the human hepcidin
mRNA that are highly active in response to antisense reduction.
Identification of these regions allows for the design of antisense
oligonucleotides that will modulate the expression of hepcidin.
Antisense oligonucleotides can be designed the active target
segments with at least a + activity ranking, more preferably with
at least a ++ activity ranking and most preferably with a +++
activity ranking. One skilled in the art will readily recognize
that the percent reduction is referred to herein as a relative
amount of reduction compared to other oligonucleotides tested
against SEQ ID NOS: 1, 2 or 3. Assay to assay variations in
conditions results in changes from the absolute data received,
however, the general trends and relative reduction will be
substantially the same.
EXAMPLE 4
Antisense Reduction of Mouse Hepcidin Expression By Oligomeric
Compounds
[0108] A series of antisense compounds was designed to target
different regions of mouse hepcidin RNA, using published sequences
or portions of published sequences as cited in Table 1. The
compounds are shown in Table 4. The screen identified active target
segments within these regions of the human hepcidin mRNA sequence
and the genomic sequence encoding hepcidin mRNA, specifically
GenBank numbers NM.sub.--032541.1 and the complement of nucleotides
3978217 to 3980665 of NT.sub.--039413.1 (SEQ ID NOS: 4 and 5,
respectively). The activity of these antisense oligonucleotides are
ranked based on measured percent reduction of the target from this
study. The activity of these antisense oligonucleotides to reduce
hepcidin mRNA expression is ranked as follows: +++++ (reduction of
target by greater than about 70%), ++++ (reduction of target by
greater than about 70% to about 74%), +++ (reduction of target by
greater than about 60% to about 69%), ++ (reduction of target by
greater than about 55% to about 60%), + (reduction of target by
greater than about 50% to about 55%) or bc (below cut-off or lesser
than about 50%). Antisense compounds having an activity ranking of
bc may have shown some reduction of hepcidin expression but fell
below the cut-off level for this screening assay.
TABLE-US-00004 TABLE 4 Reduction of Mouse Hepcidin mRNA Levels by
Antisense Oligonucleotides Target Target SEQ CMPD SEQ ID Start
Activity ID # NO Site Sequence (5' to 3') Ranking NO 326810 4 1
TGCTGTGCAGTCTAAGGACT +++ 90 326811 4 6 TGTTCTGCTGTGCAGTCTAA bc 91
326812 4 10 CTTCTGTTCTGCTGTGCAGT ++ 92 326813 4 15
CATGCCTTCTGTTCTGCTGT ++++ 93 326814 4 17 ATCATGCCTTCTGTTCTGCT +++
94 326815 4 22 GTGCCATCATGCCTTCTGTT +++++ 95 326816 4 24
GAGTGCCATCATGCCTTCTG ++++ 96 326817 4 27 GCTGAGTGCCATCATGCCTT +++++
97 326818 4 29 GTGCTGAGTGCCATCATGCC +++ 98 326819 4 32
CGAGTGCTGAGTGCCATCAT bc 99 326820 4 37 GGGTCCGAGTGCTGAGTGCC +++ 100
326821 4 41 GCCTGGGTCCGAGTGCTGAG +++ 101 326822 4 45
GGCAGCCTGGGTCCGAGTGC +++++ 102 326823 4 50 AGACAGGCAGCCTGGGTCCG bc
103 326824 4 55 GCAGGAGACAGGCAGCCTGG +++++ 104 326825 4 71
CTGGCAAGGAGGAGAAGCAG bc 105 326826 4 80 CTGCTCAGGCTGGCAAGGAG bc 106
326827 4 84 GGTGCTGCTCAGGCTGGCAA bc 107 326828 4 86
GTGGTGCTGCTCAGGCTGGC bc 108 326829 4 88 AGGTGGTGCTGCTCAGGCTG ++++
109 326830 4 89 TAGGTGGTGCTGCTCAGGCT +++ 110 326831 4 90
ATAGGTGGTGCTGCTCAGGC + 111 326832 4 91 GATAGGTGGTGCTGCTCAGG bc 112
326833 4 98 TGATGGAGATAGGTGGTGCT bc 113 326834 4 99
TTGATGGAGATAGGTGGTGC bc 114 326835 4 104 ATCTGTTGATGGAGATAGGT bc
115 326836 4 105 CATCTGTTGATGGAGATAGG bc 116 326837 4 112
TCTGTCTCATCTGTTGATGG bc 117 326838 4 115 TAGTCTGTCTCATCTGTTGA bc
118 326839 4 116 GTAGTCTGTCTCATCTGTTG bc 119 326840 4 121
GCTCTGTAGTCTGTCTCATC + 120 326841 4 124 GCAGCTCTGTAGTCTGTCTC +++++
121 326842 4 127 GCTGCAGCTCTGTAGTCTGT bc 122 326843 4 130
AAGGCTGCAGCTCTGTAGTC +++ 123 326844 4 135 GTGCAAAGGCTGCAGCTCTG +++
124 326845 4 141 TTCCCCGTGCAAAGGCTGCA bc 125 326846 4 145
TTTCTTCCCCGTGCAAAGGC ++ 126 326847 4 148 TGCTTTCTTCCCCGTGCAAA bc
127 326848 4 158 ATGTCTGCCCTGCTTTCTTC bc 128 326849 4 172
GCATTGGTATCGCAATGTCT + 129 326850 4 176 TTCTGCATTGGTATCGCAAT bc 130
326851 4 180 TCTCTTCTGCATTGGTATCG bc 131 326852 4 200
AAGTTGGTGTCTCTCTTCCT ++ 132 326853 4 203 GGGAAGTTGGTGTCTCTCTT +++++
133 326854 4 225 TTTACAGCAGAAGATGCAGA bc 134 326855 4 229
AGCATTTACAGCAGAAGATG ++ 135 326856 4 231 ACAGCATTTACAGCAGAAGA + 136
326857 4 233 TTACAGCATTTACAGCAGAA +++ 137 326858 4 238
AATTGTTACAGCATTTACAG bc 138 326859 4 242 TGGGAATTGTTACAGCATTT bc
139 326860 4 245 CACTGGGAATTGTTACAGCA bc 140 326861 4 252
GATACCACACTGGGAATTGT bc 141 326862 4 256 AACAGATACCACACTGGGAA bc
142 326863 4 258 GCAACAGATACCACACTGGG +++++ 143 326864 4 261
TTTGCAACAGATACCACACT +++ 144 326865 4 263 GTTTTGCAACAGATACCACA +
145 326866 4 266 TATGTTTTGCAACAGATACC bc 146 326867 4 275
GCTCTAGGCTATGTTTTGCA +++++ 147 326868 4 287 GGTCAGGATGTGGCTCTAGG
+++++ 148 326869 4 291 GAGAGGTCAGGATGTGGCTC +++++ 149 326870 4 293
TAGAGAGGTCAGGATGTGGC ++++ 150 326871 4 296 GTGTAGAGAGGTCAGGATGT
+++++ 151 326872 4 334 AGGGCAGGAATAAATAATGG bc 152 326873 4 336
GGAGGGCAGGAATAAATAAT bc 153 326874 4 355 ATTTCAAGGTCATTGGTGGG bc
154 326875 4 357 TTATTTCAAGGTCATTGGTG +++ 155 326876 4 362
CGTCTTTATTTCAAGGTCAT +++++ 156 326877 4 367 AAAATCGTCTTTATTTCAAG bc
157 326878 4 369 ATAAAATCGTCTTTATTTCA bc 158 326879 4 371
AAATAAAATCGTCTTTATTT bc 159 326880 5 512 GGGTGCTCACCTGTTGATGG bc
160 326881 5 1239 TGCTTACTGCTGACTGCCAT + 161 326882 5 1244
ATGCATGCTTACTGCTGACT + 162 326883 5 1259 GCCTATGTGTATCATATGCA ++
163 326884 5 1347 CAGCAGAACCTCTACAGCCC bc 164 326885 5 1686
TCTGTCTCATCTGTGAAAGC bc 165 326886 5 1746 ATGCTCTTACCGCAATGTCT bc
166 326887 5 1829 GCATTGGTATCTGTGTAAAG + 167
[0109] Each active target segment was targeted by multiple, active
antisense oligonucleotides. This current example includes
identification of active target segments identified on SEQ ID NO:
4, which active target segments are referred to as Regions L-0. The
active target segments include nucleotides 287 to 315 for Region L,
nucleotides 258 to 315 for Region M, nucleotides 1 to 74 for Region
N, and nucleotides 80 to 199 for Region O. Each of the
oligonucleotides tested within Region L reduced expression by at
least 70% (++++) and three-fourths of them by at least 75% (+++++).
Two-thirds of the oligonucleotides tested within Region M reduced
expression by at least 70% (++++). About one-half of the
oligonucleotides tested in Region N reduced expression by at least
70% (++++). This screen also identified regions with low activity
when targeted with an antisense oligonucleotide. In Region O more
than two-thirds of the oligonucleotides tested reduced expression
by less than 50% (bc) and more than three-fifths by less than 35%
(bc).
[0110] The active target segments for SEQ ID NO: 5 are referred to
as Regions P-R. Region P includes nucleotides 1942 to 1972 and all
of the oligonucleotides tested in this region reduce expression by
at least 70% (+++). Region Q includes nucleotides 1991 to 2047 and
three-fourths of the oligonucleotides tested in this region reduced
expression by less than 50% (bc), and thus is considered a
low-activity region. Similarly, for Region R, which includes
nucleotides 474 to 531, more than two-thirds of the
oligonucleotides tested in this region reduce expression by less
than 50% (bc).
[0111] Thus, Applicant has discovered regions of the hepcidin mRNA
that are highly responsive to antisense reduction and regions of
the hepcidin mRNA that are less responsive to antisense reduction.
Identification of these regions allows for the design of antisense
oligonucleotides that will modulate the expression of hepcidin. One
skilled in the art will readily recognize that the percent
reduction is referred to herein as a relative amount of reduction
compared to other oligonucleotides tested against SEQ ID NOS: 4 or
5. Assay to assay variations in conditions will results in changes
from the absolute data received, however, the general trends and
relative reduction will be substantially the same.
EXAMPLE 5
Antisense Oligonucleotides From About 12 To About 35
Nucleobases
[0112] As stated above, antisense oligonucleotides directed to a
target or more preferably to an active target segment can be from
about 12 to about 35 linked nucleobases. The following Table 5a
provides a non-limiting example of such antisense oligonucleotides
targeting SEQ ID NO 4.
TABLE-US-00005 TABLE 5a Antisense Oligonucleotides from about 12 to
about 35 Nucleobases Sequence Length GGTCAGGATGTGGCTCTAGG 20
nucleobases (SEQ ID NO: 148) GGTCAGGATGTGGCT 15 nucleobases (SEQ ID
NO: 170) CAGGATGTGGCTCTA 15 nucleobases (SEQ ID NO: 171)
GTCAGGATGTGG 12 nucleobases (SEQ ID NO: 172)
GAGAGGTCAGGATGTGGCTCTAGGCTATGTTTTGCA 35 nucleobases (SEQ ID NO:
173) GTCAGGATGTGGCTCTAGGCTATGTTTT 27 nucleobases (SEQ ID NO: 174)
TGTGGCTCTAGGCTATGTTTTGC 22 nucleobases (SEQ ID NO: 175)
[0113] Antisense oligonucleotides directed to a target or more
preferably to an active target segment can also contain mismatched
nucleobases when compared to the target sequence. The following
Table 5b provides a non-limiting example of such antisense
oligonucleotides targeting nucleobases 287 to 306 of SEQ ID NO 4.
Mismatched nucleobases are underlined.
TABLE-US-00006 TABLE 5b Antisense Oligonucleotides from about 1-3
Nucleobases Mismatched to the Target Sequence Number of mismatches
to Sequence SEQ ID NO: 4 GGTCAGGATGTGGCTCTAGG None (SEQ ID NO: 148)
GGTCAGGATGTGGCTCAAGG One mismatch (SEQ ID NO: 176)
GGTCAGGAAGTGGCTCTAGG One mismatch (SEQ ID NO: 177)
GGTCAGTTTGTGGCTCTAGG Two mismatches (SEQ ID NO: 178)
GGTGAGGATGTGGCTCCAGG Two mismatches (SEQ ID NO: 179)
GGTCTGGATGTGCCTCTCGG Three mismatches (SEQ ID NO: 180)
EXAMPLE 6
Effects of Antisense Oligonucleotides Targeted To Mouse Hepcidin
mRNA: In Vivo Evaluation In Normal Mice
[0114] C57/131/6 mice were divided into treatment groups for
injection with one of the following: antisense oligonucleotides
targeting hepcidin mRNA (SEQ ID NOS: 4 and 5) at 50 mg/kg;
antisense oligonucleotides that do not target hepcidin mRNA at 50
mg/kg; or saline. The animals received an initial dose and then
were dosed twice per week for two weeks (5 doses total). The
antisense oligonucleotides used in the study were all chimeric
oligonucleotides ("gapmers") 20 nucleobases in length, composed of
a central "gap" region consisting of 10 2'-deoxynucleotides,
flanked on both the 5' and 3' ends by 5-nucleotide "wings." The
wings are composed of 2'-methoxyethoxy nucleotides (2'-MOE). The
internucleoside linkages are phosphorothioate (P=S) throughout the
oligonucleotide. All cytidine residues are 5-methylcytidines. The
sequences of the oligonucleotide are presented in Table 6.
TABLE-US-00007 TABLE 6 Antisense oligonucleotides used in normal
mouse in vivo screen assay SEQ ID Sequence NO: Targeted to hepcidin
mRNA GGTCAGGATGTGGCTCTAGG 148 CGTCTTTATTTCAAGGTCAT 156
GTGCCATCATGCCTTCTGTT 95 Not targeted to hepcidin mRNA
CCTTCCCTGAAGGTTCCTCC 168 GCTCATAGAACTGTCAGGCT 169
[0115] At the end of the treatment period animals were sacrificed
and tissues were harvested for assessment of levels of hepcidin
mRNA in the liver, levels of hepcidin polypeptide in the liver and
organ weight (specifically spleen and liver. Over the course of the
study, data collected to evaluate overt toxicity, changes in body
weight, changes in organ weight (specifically spleen and liver),
changes in food intake, blood levels of aspartate aminotransferase
(AST) and blood levels of alanine aminotransferase (ALT). There
were no signs of overt toxicity or in changes to organ or body
weight. There was an increase in levels of AST and ALT in the mice
receiving one of the antisense oligonucleotides (SEQ ID NO: 95);
however, levels for the other hepcidin targeting antisense
oligonucleotides remained comperable to the saline control. Liver
hepcidin levels were reduced by 60-70 percent for the hepcidin
targeting antisense oligonucleotides compared to controls. A
similar reduction was observed for liver hepcidin polypeptides.
Hepcidin polypeptide levels were determined by Western blot of
whole-liver lysates. Briefly, 50 .micro.g of whole-liver lysate was
electrophoresed on a 16% TG-gel. Hepcidin was detected with
hepcidin antibody (Alpha Diagnostics, HEPC11-A, 0.3 .micro.g/ml).
FIG. 1. Thus, antisense oligonucleotides targeting hepcidin mRNA
can result in a significant reduction in the levels of that mRNA
and in turn the expression thereof.
Example 7
Dose-Response of Antisense Oligonucleotide Targeting Hepcidin mRNA
In Normal Mice
[0116] C57/B1/6 mice were dosed with 10, 25, 50 or 75 mg/kg of
antisense oligonucleotide targeting hepcidin mRNA (SEQ ID NO: 145).
C57/B1/6 control mice received either saline or 10, 25, 50 or 75
mg/kg of an antisense oligonucleotide not targeted to hepcidin mRNA
(SEQ ID NO: 168) The groups were given an initial dose and then
were dosed twice per week for 2 weeks. Liver hepcidin mRNA and
serum iron levels were measured at the end of the study. All
antisense oligonucleotides used in this example were chimeric
oligonucleotides ("gapmers") 20 nucleobases in length, composed of
a central "gap" region consisting of 10 2'-deoxynucleotides,
flanked on both the 5' and 3' ends by 5-nucleotide "wings." The
wings are composed of 2'-methoxyethoxy nucleotides (2'-MOE). The
internucleoside linkages are phosphorothioate (P=S) throughout the
oligonucleotide. All cytidine residues are 5-methylcytidines.
[0117] There was a reduction in liver hepcidin mRNA for the group
receiving the antisense oligonucleotide targeting hepcidin as
compared to saline control. A reduction was not observed for the
control antisense oligonucleotide group as shown in FIG. 2. Also
observed was a corresponding increase in serum iron levels for the
treatment groups compared to saline. This same increase was not
seen in the control oligonucleotide group. These data demonstrate
that antisense oligonucleotides targeting hepcidin mRNA will cause
a decrease in liver hepcidin mRNA and a corresponding increase in
serum iron levels.
EXAMPLE 8
Effects of Antisense Reduction of Hepcidin In Normal And LPS
Stimulated Mice: Dose-Response
[0118] An ACD mouse model was prepared prior to treatment by
injecting C57B1/6 mice (Charles River Labs, Wilmington, Mass.) with
lipopolysaccaride (LPS, Sigma-Aldrich, St. Louis, Mo.) for 6 hours
(0.1 to 1 mg/kg intraperitoneal injection). (See, e.g. Ganz (2003)
Blood, 102(3):783-785, and references therein). Injection of LPS
caused an increase in cytokine production in these mice, which in
turn resulted in a three fold increase in hepcidin production and
an 80% reduction in serum iron levels.
[0119] A normal unstimulated mouse population and an LPS stimulated
mouse population were each separated into treatment groups,
antisense control groups and saline control groups. Mice in the LPS
stimulated population were prepared using a 1 mg/kg intraperitoneal
LPS injection for 6 hours. The treatment groups were treated with
an antisense oligonucleotide targeted to mouse hepcidin mRNA (SEQ
ID NOS: 4 and 5). The antisense control groups were treated with
antisense oligonucleotide not targeted to mouse hepcidin. The
saline control groups were injected with saline alone. The
antisense oligonucleotide targeted to mouse hepcidin mRNA was (SEQ
ID NO: 145). The control antisense oligonucleotide was SEQ ID NO
168. All mice were dosed at time point zero and then twice a week
for two weeks using either 10, 25, 50 or 75 mg/kg of antisense
oligonucleotide; 50 mg/kg of control oligonucleotide or using
saline. All antisense oligonucleotides used in this example were
chimeric oligonucleotides ("gapmers") 20 nucleobases in length,
composed of a central "gap" region consisting of 10
2'-deoxynucleotides, flanked on both the 5' and 3' ends by
5-nucleotide "wings." The wings are composed of 2'-methoxyethoxy
nucleotides (2'-MOE). The internucleoside linkages are
phosphorothioate (P=S) throughout the oligonucleotide. All cytidine
residues are 5-methylcytidines.
[0120] In both the normal mice and the LPS stimulated mice there
was a reduction in levels of liver hepcidin mRNA for the 25, 50 and
75 mg/kg doses as compared to saline control. This same reduction
was not seen in the antisense oligonucleotide control group.
Additionally, there was a corresponding increase in serum iron
levels for both the normal and LPS stimulated groups receiving
antisense oligonucleotide targeting hepcidin as compared to saline.
This same increase was not seen in the antisense oligonucleotide
control group. This study shows that hepcidin increases serum iron
levels in both normal and LPS stimulated mice. FIG. 3. Antisense
oligonucleotides targeting hepcidin mRNA will reduce the levels of
hepcidin mRNA in the liver, correlating with an increase in serum
iron levels. This effect of the antisense oligonucleotide is
observed in normal and LPS stimulated mice (ACD models). Thus, the
antisense oligonucleotides are useful for treating subjects having
low serum iron levels and disorders associated therewith.
EXAMPLE 9
Effects of Antisense Reduction of Hepcidin In Normal And Chronic
Anemia Mice: Turpentine Mouse Model
[0121] The effect of antisense compound treatment on acute and
chronic anemia was determined using a turpentine induced anemia
mouse model. Male C57B1/6 mice (Charles River Laboratories,
Wilmington, Mass.) weighing approximately 25 g were separated into
one of a control group, an oligonucleotide control group or a
treatment group. The control group mice received either no
treatment (n=5) or treatment with 100 microliters turpentine only
(n=5). The oligonucleotide control group mice received 100
microliters turpentine and a dosing regimen comprising either 50
mg/kg/dose of control compound (n=5) or 75 mg/kg/dose of control
compound (n=5). The control compound was an antisense compound that
is not targeted to a nucleic acid encoding hepcidin
(CCTTCCCTGAAGGTTCCTCC, SEQ ID NO: 168). The treatment group mice
received 100 microliters of turpentine and a dosing regimen
comprising either 10 mg/kg/dose of treatment compound (n=5), 25
mg/kg/dose of treatment compound (n=5), 50 mg/kg/dose of treatment
compound (n=5), or 75 mg/kg/dose of treatment compound (n=4). The
treatment oligonucleotide is Cmpd 326868 (SEQ ID NO: 148). The
turpentine injection stimulates an inflammatory response in the
mice, and subsequently produces anemia. Turpentine mouse models of
anemia are discussed in the literature. (Liuzzi, J P, Lichten, L A,
Rivera, S, Blanchard, R K, Aydemir, T B, Knutson, M D, Ganz, T, and
Cousins, R J, PNAS, v. 102, n. 19, 6843-6848 (2005); Venihaki, M,
Dikkes, P, Carrigan, A, and Karalis, K P, J. Clin. Invest.,
108:1159-1166 (2001)).
[0122] Mice in all groups were placed on a no-iron diet for two
weeks, followed by a low-iron diet. At the same time the low-iron
diet was started, the mice were subjected to the appropriate dosing
regimen as described above. Briefly, the control group received no
oligonucleotide, the oligonucleotide control group received 50 or
75 mg/kg/dose of SEQ ID NO: 168 twice a week for two weeks, and the
treatment group received 10, 25, 50 or 75 mg/kg/dose of SEQ ID NO:
148 twice a week for two weeks. After two weeks of the dosing
regimen, all mice in the oligonucleotide group and in the treatment
group received an injection comprising 100 microliters of
turpentine (Sigma-Aldrich, St. Louis, Mo.). In the control group,
only half of the mice population received an injection comprising
100 microliters of turpentine and the other half of the population
received a saline injection only. The injections were delivered
subcutaneously to the left hind limb of each mouse. To obtain acute
anemia endpoints, blood was drawn at 16 hours post turpentide
injection. For chronic anemia endpoints, the dosing regimen
described above continued for another two weeks after turpentine
injection. Blood was drawn at two weeks post-turpentine injection
and at 24 hours after the final oligonucleotide injection was
delivered. Mice were then sacrificed.
[0123] Analysis of the serum iron from acute blood draw showed that
the turpentine injection produced a significant reduction in the
serum iron levels at 16 hours post turpentine injection. The serum
iron level reduction was reversed in a dose responsive manner for
the treatment group. At two weeks post injection there was a dose
dependent improvement in the clinical endpoints of anemia compared
to the control group and the oligonucleotide control group. Table 7
and FIGS. 4a-d shows the dose dependent improvement in serum iron
levels, red blood cell count, hematocrit levels and hemoglobin
levels.
TABLE-US-00008 TABLE 7 The Effect of Hepcidin ASO in a Chronic
Turpentine Model: Serum Iron, Red Blood Cell (RBC) Count,
Hemoglobin Levels and Hematocrit Levels Oligonucleotide Turpentine
Control Group - Control Group Treatment Group (.micro.l) 0 100 100
100 100 100 100 100 CMPD none none 50 mg/kg 75 mg/kg 10 mg/kg 25
mg/kg 50 mg/kg 75 mg/kg 141923 141923 326868 326868 326868 326868
Serum Iron 190.6 170.6 186.0 208.2 182.8 260.8 266.6 325.5
(.micro.g/ (.+-.24.1) (.+-.28.1) (.+-.44.4) (.+-.40.0) (.+-.44.3)
(.+-.22.9) (.+-.46.0) (.+-.55.7) dL) (SD) RBC (10.sup.6/ 10.4 9.4
9.9 9.8 9.4 10.2 10.2 10.6 .micro.l) (SD) (.+-.0.4) (.+-.1.0)
(.+-.0.2) (.+-.0.7) (.+-.0.5) (.+-.0.3) (.+-.0.3) (.+-.0.8)
Hemoglobin 14.8 13.0 13.3 13.2 13.1 14.0 14.0 14.3 (g/dL) (SD)
(.+-.0.8) (.+-.1.2) (.+-.0.4) (.+-.0.8) (.+-.0.6) (.+-.0.5)
(.+-.0.3) (.+-.0.9) Hematocrit 53.4 47.6 49.8 47.8 47.6 51.2 51.0
51.5 (%) (SD) (.+-.2.1) (.+-.2.6) (.+-.1.1) (.+-.3.3) (.+-.1.9)
(.+-.1.9) (.+-.1.7) (.+-.3.5)
[0124] The turpentine mouse model of anemia provides data for
endpoints of chronic anemia, such as RBC count, hemoglobin levels
and hematocrit levels, as well as serum iron levels. Treating these
anemic mice with an antisense compound that targets and inhibits
expression of nucleic acids encoding hepcidin polypeptide results
in a dose dependent improvement in these endpoints.
EXAMPLE 10
Effects of Antisense Reduction of Hepcidin In Chronic Anemia
(Turpentine Mouse Model) Treated With Erythropoietin
[0125] The effect of antisense compound treatment on chronic
anemia, in combination with erythropoietin (EPO) treatment was
determined using the chronic turpentine induced anemia mouse model.
Male C57B1/6 mice (Charles River Laboratories, Wilmington, Mass.)
weighing approximately 25 g were fed a "no iron" diet (2.7 ppm
iron) for 2 weeks and subsequently switched to a "low iron" diet
(47 ppm iron) for the remainder of the study. Upon switch to the
low iron diet, two weeks prior to sacrifice, mice were treated via
subcutaneous injection with saline (vehicle) or a single 100
.micro. L dose of turpentine. Hepcidin antisense oligonucleotide
(CMPD # 326868, 50 mg/kg, which is SEQ ID NO: 148) was administered
via subcutaneous injection to the appropriate groups of animals (as
indicated in FIG. 5a-5c), two times per week, beginning 24 hours
following turpentine treatment. EPO was administered daily, for the
last four days of the study, by intraperitoneal injection to
indicated animals at indicated doses (0-25 units/day). Thus, in
this study, in indicated mice, regular treatment with antisense
oligonucleotide targeted to hepcidin began prior to regular
treatment with erythropoietin. After the first administration, each
treatment was continued at regular intervals for the remainder of
the study. At the end of the treatment period, animals were
sacrificed and tissues and blood taken for assessment of levels
hepcidin mRNA and phenotypic endpoints. Results from this study
showed that hepcidin ASO in combination with EPO was well
tolerated. Hepcidin mRNA levels in the liver, serum iron
(.micro.g/mL) and % reticulocytes (immature red blood cells) were
measured as endpoints. The results are shown in FIG. 5a-5c.
[0126] As shown in FIG. 5, in this study using the turpentine mouse
model of chronic anemia, erythropoietin treatment decreased liver
hepcidin mRNA expression and serum iron levels and increased
reticulocyte counts. Administration of the antisense
oligonucleotide targeted to hepcidin caused decreased liver
hepcidin mRNA, but an increase in serum iron. Administration of an
antisense oligonucleotide targeted to hepcidin in addition to
erythropoietin resulted in a further increase in reticulocyte
counts compared to erythropoietin treatment alone. Thus the effects
of erythropoietin and the antisense compound targeted to hepcidin
were additive. These results show that treatment with both
erythropoietin and an antisense compound targeted to hepcidin is
effective in improving endpoints of chronic anemia in a mouse model
for hypoferremia and anemia associated with inflammation.
[0127] It is contemplated herein that a further experiment,
evalutating the effectiveness of combination therapy for treating
anemia or increasing red blood cells could be performed, according
to the methods and using the mouse model described above, by
evaluating multiple experimental mice groups.
[0128] In such an experiment, a control group would receive only
saline. Experimental groups would all receive turpentine. A first
experimental group would receive only turpentine (no treatment or
therapy). A second experimental group would receive erythropoietin
treatment continuously, meaning regularly, for over a week, prior
to receiving antisense compound targeted to hepcidin, and would
continue to receive erythropoietin treatment throughout the
treatment period with antisense compound. Thus, this second
experimental group will have been receiving erythropoietin therapy
prior to administration of the antisense compound. Erythropoietin
therapy treatment regimens are well known in the art. Thus, in the
second experimental group, the antisense compound and the
erythropoietin would be administered in separate formulations, at
distinct timepoints. A third experimental group would have received
erythropoietin therapy at some time prior to receiving antisense
compound. A fourth experimental group would receive erythropoietin
therapy and antisense compound treatment simultaneously. Thus, in
this group, the group would begin erythropoietin therapy
simultaneously with the administration of the antisense compound.
In this case, the two treatments could be administered in separate
formulations or in single formulations. A fifth experimental group
would receive antisense compound prior to erythropoietin therapy.
Thus, the second through fifth experimental groups would be
receiving a combination therapy, wherein they would receive
antisense compound and be further receiving erythropoietin. A sixth
experimental group would receive only erythropoietin. A seventh
experimental group would receive only antisense compound.
[0129] It is further contemplated that a combination therapy method
could be used for preventing, treating or ameliorating anemia or
for increasing red blood cell counts in a patient. It is
contemplated that this combination method could include
administration of an antisense compound targeted to hepcidin and an
ESA. It is contemplated that the ESA used in such a therapy could
be erythropoietin, recombinant human erythropoietin, EPOGEN or
PROCRIT. It is further contemplated that such a combination therapy
method could comprise administration of the antisense compound and
erythropoietin simultaneously. Alternatively, administration of the
erythropoietin and antisense compound could be done at distinct
timepoints.
[0130] If administered simultaneously, the antisense compound and
erythropoietin could be given in a single formulation, or in
separate formulations. Also, this combination therapy could be used
to treat a patient who is receiving erythropoietin. The patient
could be receiving erythropoietin prior to initiation of the
combination therapy, or the patient could begin receiving the
erythropoietin at the same time the combination therapy is
initiated.
[0131] It is contemplated that an antisense compound used in this
therapy could be an antisense oligonucleotide targeted to a nucleic
acid molecule encoding hepcidin. Alternatively, the antisense
compound could be double stranded. It is contemplated that the
double stranded antisense compound could be an siRNA.
Sequence CWU 1
1
1801588DNAHomo sapiens 1gagcctccca cgtggtgtgg atgaggaggc agatggcagg
gcctgtgcat ttctgtgctt 60gagtgggcct tgaaagtggt tcagcaacca ggaagaagtg
ttcattcctc gacaacaaca 120tccccgggct ctggtgactt ggctgacact
ggatggccct ggaatgaaaa aggcaaagag 180gcaaaatgtg caagggccca
tctggaacca aggccccata aaagcgactg tcactcggtc 240ccagacacca
gagcaagctc aagacccagc agtgggacag ccagacagac ggcacgatgg
300cactgagctc ccagatctgg gccgcttgcc tcctgctcct cctcctcctc
gccagcctga 360ccagtggctc tgttttccca caacagacgg gacaacttgc
agagctgcaa ccccaggaca 420gagctggagc cagggccagc tggatgccca
tgttccagag gcgaaggagg cgagacaccc 480acttccccat ctgcattttc
tgctgcggct gctgtcatcg atcaaagtgt gggatgtgct 540gcaagacgta
gaacctacct gccctgcccc cgtcccctcc cttcctta 5882430DNAHomo sapiens
2gactgtcact cggtcccaga caccagagca agctcaagac ccagcagtgg gacagccaga
60cagacggcac gatggcactg agctcccaga tctgggccgc ttgcctcctg ctcctcctcc
120tcctcgccag cctgaccagt ggctctgttt tcccacaaca gacgggacaa
cttgcagagc 180tgcaacccca ggacagagct ggagccaggg ccagctggat
gcccatgttc cagaggcgaa 240ggaggcgaga cacccacttc cccatctgca
ttttctgctg cggctgctgt catcgatcaa 300agtgtgggat gtgctgcaag
acgtagaacc tacctgccct gcccccgtcc cctcccttcc 360ttatttattc
ctgctgcccc agaacatagg tcttggaata aaatggctgg ttcttttgtt
420ttccaaaaaa 43035225DNAHomo sapiens 3atgcctggta gggctggggg
ctgctcctgt gtctccccag gtgagcacac ccctattcac 60tgggccctgc ttcagcctgc
agcacccttc aactcccagg agctgggctt gccactctgc 120tcaccttgtg
gagctccatc tgcctttcct ccccaattcc cccactccct gcactcgtct
180cttcccacaa gagccctgtc tccttttcct agctattccc atctgaggcc
atctttattc 240atttagtttt tagagacagg gtttcactct cacccaggct
ggggtgcagt ggcacacaat 300cacggctcac tgcagccttg accaactaca
ggtgcgtagc accacagcca agtttttgta 360tagatggggt ctcgctttgt
tacccaggct gtgacaagag gagcctccca cgtggtgtgg 420atgaggaggc
agatggcagg gcctgtgcat ttctgtgctt gagtgggcct tgaaagtggt
480tcagcaacca ggaagaagtg ttcattcctc gacaacaaca tccccgggct
ctggtgactt 540ggctgacact ggatggccct ggaatgaaaa aggcaaagag
gcaaaatgtg caagggccca 600tctggaacca aggtttgttg atcccctggg
ccgtgtgcac cctgagctgg gcctggtagt 660ggaaaggaat gaaggcactg
cagtcaggca gcctgggttc atcccccagc tagtggtgtc 720ctaaggaacc
ggctccccaa aaacatccct ggcttgtagt gcttgccaat ttctgggtgt
780caagactccc actgctgctg atttcaggat accagcatga tgccactgaa
tgcagagttt 840cgagatgtgc atggtctgct atgttgagcc aggtctagca
taccgctgtg ccctgctgtg 900ttttagggga gatggggaaa cctggtgggt
aagagcaaaa gccctggagt caggctgtcc 960aggctagaat ctcagctctg
cctctggctg agcaagcttg ggccatgccc tgatctctgc 1020cttcagtgcc
ttttctgtaa agtgaaggaa atgagtgtcc gacggggagg aggttcctaa
1080aagggagcag ggtctgggga gcccaggcct ctggggttgg gtgactgaga
aggcagcccc 1140tgaatacaga gcagagctga aggtggggca gtaagtgctg
ctgggagaac aggcagcaca 1200ggctgagttg gtgcagaagt gagtcaacat
atgtgccatc gtataaaatg tactcatcgg 1260actgtagatg ttagctatta
ctattactgc tattttatgt tttatagaca gggtctcact 1320ctgtcaccca
ggctggagtg cagtcacaca atcatagctc actgcaacct cagcctcctg
1380ggcttaagcg atctgcctca gcctcccaag tagctgggac tacagatgtg
tgccaccacg 1440cctggctaaa tttgtttaaa attttttttg tagagatggg
gtctccctat gttgcccagg 1500ctagtcttga acttctgggc tcaagcgacc
ctcctgcctt ggcctcccaa attgctggga 1560ttacaggcat aagccactgt
gctgggccat attactgctg tcatttatgg ccaaaagttt 1620gctcaaacat
tttccagtta ccagagccac atctcaaggg tctgacactg ggaaaacacc
1680acgtgcggat cgggcacacg ctgatgcttg ccctgctcag ggctatctag
tgttccctgc 1740cagaacctat gcacgtgtgg tgagagctta aagcaatgga
tgcttccccc aacatgccag 1800acactcctga ggagcctggc ggctgctggc
catgccccgt gtgcatgtag gcgatgggga 1860agtgagtgga ggagagcgga
accttgattc tgctcatcaa actgcttaac cgctgaagca 1920aaagggggaa
cttttttccc gatcagcaga atgacatcgt gatggggaaa gggctcccca
1980gatggctggt gagcagtgtg tgtctgtgac cccgtctgcc ccaccccctg
aacacacctc 2040tgccggctga gggtgacaca accctgttcc ctgtcgctct
gttcccgctt atctctcccg 2100ccttttcggc gccaccacct tcttggaaat
gagacagagc aaaggggagg gggctcagac 2160caccgcctcc cctggcaggc
cccataaaag cgactgtcac tcggtcccag acaccagagc 2220aagctcaaga
cccagcagtg ggacagccag acagacggca cgatggcact gagctcccag
2280atctgggccg cttgcctcct gctcctcctc ctcctcgcca gcctgaccag
tggctctgtt 2340ttcccacaac aggtgagagc ccagtggcct gggtccttag
cagggcagca gggatgggag 2400agccaggcct cagcctaggg cactggagac
acccgagcac tgagcagagc tcaggacgtc 2460tcaggagtac tggcagctga
acaggaacca ggacaggcac ggtggctcat gcctgtaatc 2520ccagcacttt
gggaggttga ggcaggcagc ccacttgagg tcagtttgag accagcctgg
2580ccaacatggt aaaaccccgt ctctactaaa aatacaaaag ttagccaggc
ttggtggcag 2640gtgcctgtaa tcccagctac tcgggagact gaggcaggag
aattgcttga acccgcaagg 2700tggaggttgc acagtgagct gagattgcac
cactgcactc cagcctggca acagagcaag 2760actccatctc caaaaaagaa
cagaaatcaa tgaagcaccg agtgacaggg actggaaggt 2820cctaattcca
tgggtattta cggaacccct acgccgtgtg gagtcttatt ctagacagtg
2880gggacgaggc catgaacaag gtagatgaga gaggagattt ctccatcctg
gtcagggaat 2940ttgttaaaga ctgatgaaaa catgaataaa taattgtgtc
tagtacattc tattcgtgaa 3000tctcataaca gacagtggta gagtgaccgt
gacccattcg ccacacagta gagtcacttt 3060tttggtttgt tttttagaga
cagggtcttc ctctgttgct gaggctggag tgcagtggtg 3120cagtcatagt
tcactgcagc ctcaacctcc tgtgctcaag caatcctccc acctcagcgt
3180cccaagtagc tgggacagca ggcacatgcc acgggttggg ggaccacagg
catggtcaag 3240gggctggcag tcaagcaagt gtttcatgag aaagtgacag
ttgaccttcg tcttggaggg 3300tgagagatgg aggcagcaaa gacctaagga
gaggacaagc cagcatagcc cagggtcagg 3360ctgaacaaga ggagatggtg
ggacttgggg ataaggctga ggggtgggca gtccctaagt 3420cttgtgggca
accatgcaga cactgatttt tccttggaat aaagaggaag cccccataag
3480cttttttttt tttttctgag atagggtctc gctctgtcgt tcaggctggt
gtgcagtggc 3540atcatctggg ctcactgcaa cctccgcctc ccgggttcaa
gcaattctcc tgcctcagct 3600tcccgagcag ctgggattac aggcggctgc
caccacgccc ggctaatttt tgttttttta 3660gtagagacag ggtttcacca
tgttggccag actggtcttg aactcctgac ctcaggtgat 3720tctcccacct
cggcttccca aagtgctggg attacaggcg tgagccactg cgcccagcct
3780cctgtaggtt tttaaaatgg agaaaaccac aatctcactg gccatgtttt
aaaaaactta 3840atctgccagt caggcaccat ggctcacacc tgtaatccca
gagttttggg aggccaaggt 3900aggaagatca gttgagccca ggagttcaag
accagcttgg gcaacacaac cagaccccac 3960ctctacaaaa aattaaaaaa
ttagccgggt gtggtggcgt gcacctgctg tcccagctac 4020tcgggaagct
gaggcgggag catcgcttga gcacaggagg tcaaggctgc agggagctat
4080gactgtgcca ctgcactctg gcctgggcaa cagaggaaga ctctgtctaa
aaaacaaaca 4140aaaaaagtga ctctgctgtg tggcaaatgg attgaggggc
aagaatgcag ggaggtgtgt 4200taggaggctg gcactggcat ccaggcaggg
gaaggtgata tcccaaagaa gagtagcagc 4260tgtggaaaga ggaggaggcg
gatctgggag gttttttttt ttaggaaaag ccgcccatgg 4320gaaggtgagc
agaagcaaga aagcaaggcc cctcctaaga gtccatttga gctctgggtt
4380taaaccactt ggagaggagc aggttgccgg gagccagtct cagaggtcca
ctgggccccc 4440tgccatcctc tgcaccccct tctgctttca cagacgggac
aacttgcaga gctgcaaccc 4500caggacagag ctggagccag ggccagctgg
atggtgagcg caacagtgat gcctttccta 4560gccccctgct ccctccccat
gctaaggccg gttccctgct cacattccct tccttcccac 4620agcccatgtt
ccagaggcga aggaggcgag acacccactt ccccatctgc attttctgct
4680gcggctgctg tcatcgatca aagtgtggga tgtgctgcaa gacgtagaac
ctacctgccc 4740tgcccccgtc ccctcccttc cttatttatt cctgctgccc
cagaacatag gtcttggaat 4800aaaatggctg gttcttttgt tttccaaacc
agagtgtctg ttgtcctttc tctctgccga 4860gtgtctgtgc taagagcttg
tcctgaccct gccttgcaag caccagtgct tggtgggtca 4920tgtggggctg
gtgtgtcctg gaggttgcca ggaaagttgg tgaagaaaat ttgtttctgt
4980tctccccctt catgttgcaa taatagggga tgaaagttaa tgtttcctct
ccttgagatc 5040ttcctaaaac agctgtagaa atcagtgcct gtaaggcaag
cttgtccaac ctggaggcca 5100catgcagccc tggatggctt tgaatgcacc
caacacaaat ttgtagtttc ttaaggcatt 5160atgagatttt tccgcaattt
ttttttttct catcagctgt cattagtgtt agtgtgtttt 5220atgtg
52254410DNAMus musculus 4agtccttaga ctgcacagca gaacagaagg
catgatggca ctcagcactc ggacccaggc 60tgcctgtctc ctgcttctcc tccttgccag
cctgagcagc accacctatc tccatcaaca 120gatgagacag actacagagc
tgcagccttt gcacggggaa gaaagcaggg cagacattgc 180gataccaatg
cagaagagaa ggaagagaga caccaacttc cccatctgca tcttctgctg
240taaatgctgt aacaattccc agtgtggtat ctgttgcaaa acatagccta
gagccacatc 300ctgacctctc tacacccctg cagcccctca accccattat
ttattcctgc cctccccacc 360aatgaccttg aaataaagac gattttattt
tcaaaaaaaa aaaaaaaaaa 41052449DNAMus musculus 5gacattgctg
ggtccttctg agaaacccag tgagtaacag ccatactgaa ggcactgata 60ggggtaaagg
tacagttctt ccactcacca atccaatcac tgtttagggg aaagaagggg
120aatttttctg agagccacag tgtgacatca caggtggctg gctgcaggct
tgtgtccctg 180gttctgtctg ccccaccctc tggatgcacc tctgctggct
gtaggtgaca caaccctgtc 240ccctgtcact gttcccgctt atctctcccg
cctgtttggc gccactattt tcttggaaat 300gagtcagagc aaaatggggg
tgggtgaggc gcaggtgacc ctcccctacc actagtccca 360taaaaaggac
tgggactggc tcctagacag ccaccacaca agtccttaga ctgcacagca
420gaacagaagg catgatggca ctcagcactc ggacccaggc tgcctgtctc
ctgcttctcc 480tccttgccag cctgagcagc accacctatc tccatcaaca
ggtgagcacc ccaggcccat 540tgtggtggga gagccaggtc ccaggcaggc
aggagctgct caccactgag tagttagaat 600ggctcaggag tgatggcagc
tgctgacaag gaagagggtg gtccttagtg ggagctggga 660agctgcacag
gtgtccttga atagctactc tgttgtccta ctgtggaaaa tgaagcatgg
720tgggagccaa acaaaagtgt tccttggctg tcccaccccg tcagggcatt
ctttaagcag 780cctttacatg agtattttat aaagaattac tgtggatagt
acaaaagaca atgggcagaa 840aaactctaat gaggaaggac cagaggtggg
gctaagaggc tgacagccag gcaaagtatt 900ctatgagaaa atgatacaga
agtcgggcag tggtggcaca tgcctttaat cccagcattt 960gggaggcaga
ggcaggtgga tttctgagtt tgaatccagc ctggtctaca aagtgagttt
1020caagacagcc agggctacac agagaaatcc tgtctgaaaa aaaaaaaaaa
acaaaaaaag 1080aaaaaaaaaa tgatacagaa gggtctggag agatggctta
gctgttagga acatttgatg 1140cttgtgcata ggacctagag tcagttccca
gcacccatgt ggtggatcac aaccatcctg 1200aactctactt ccagggtacc
tgatgccttc tgccctagat ggcagtcagc agtaagcatg 1260catatgatac
acataggcac tcaaggcaat cacaagaccc ttggggactg tagggtctga
1320taagtgaagc cagtgttggc aataaagggc tgtagaggtt ctgctgtgcc
gagctttgtg 1380gacagctgtg cagatgatga tctgtcctgg aaagccacaa
tccagatgaa tgtgctataa 1440gcctttgtgc tatggggtga cctggttata
agagataaga tgcagggaaa actgtccgga 1500gtgtgcaaaa gcaaagaaaa
gtgggtgctt ttaggagcat ccaaggaatg gtgaggggac 1560acagggcagt
aggagcccct tctagaaatt ctgtctaagc acagtcccta aatctctggg
1620gagaagctgg cagagaaaag tcaggaagct atgccgggta ctccacaaga
ttcaatacct 1680cttctgcttt cacagatgag acagactaca gagctgcagc
ctttgcacgg ggaagaaagc 1740agggcagaca ttgcggtaag agcatctggg
actccctccc tgatccccag cctctcccat 1800gcccaagcta ggctgcttac
ctctctttct ttacacagat accaatgcag aagagaagga 1860agagagacac
caacttcccc atctgcatct tctgctgtaa atgctgtaac aattcccagt
1920gtggtatctg ttgcaaaaca tagcctagag ccacatcctg acctctctac
acccctgcag 1980cccctcaacc ccattattta ttcctgccct ccccaccaat
gaccttgaaa taaagacgat 2040tttattttca aacctaggtg tctaggtttt
ttaccctttt tttttcttgc caaagaaatg 2100gactttttgc aagccctgct
gaggacaggc actgagctgg gtttacaggt ctatgtacaa 2160tgaggctgct
ggaacgctgg ggaacagcat ttcttgtctg ttcttcactt tgcttcttat
2220ttcaaaagat ctggagttta tgtagcagcc tggaaggaac ctgaaaagac
tttccttagt 2280ctttttctgt ctttcattct tcttaaaagt tttttacaaa
tgtgtgtatg catgtgtgtg 2340tgtgtgtgtg tgtgtgtgtg agagagagag
cacatggaag agccagagta gccccagctg 2400atatgaaact tgctttgtca
aactcagaga tcatttgcct ttgccttct 2449620DNAArtificial
SequenceOligomeric Compound 6aggaggcgag acacccactt
20722DNAArtificial SequenceOligomeric Compound 7tcccacactt
tgatcgatga ca 22824DNAArtificial SequenceOligomeric Compound
8catctgcatt ttctgctgcg gctg 24924DNAArtificial SequenceOligomeric
Compound 9tgcagaagag aaggaagaga gaca 241024DNAArtificial
SequenceOligomeric Compound 10cacactggga attgttacag catt
241128DNAArtificial SequenceOligomeric Compound 11caacttcccc
atctgcatct tctgctgt 281220DNAArtificial SequenceOligomeric Compound
12aatgcacagg ccctgccatc 201320DNAArtificial SequenceOligomeric
Compound 13cacagaaatg cacaggccct 201420DNAArtificial
SequenceOligomeric Compound 14tcaagcacag aaatgcacag
201520DNAArtificial SequenceOligomeric Compound 15cccactcaag
cacagaaatg 201620DNAArtificial SequenceOligomeric Compound
16caaggcccac tcaagcacag 201720DNAArtificial SequenceOligomeric
Compound 17actttcaagg cccactcaag 201820DNAArtificial
SequenceOligomeric Compound 18gctgaaccac tttcaaggcc
201920DNAArtificial SequenceOligomeric Compound 19tggttgctga
accactttca 202020DNAArtificial SequenceOligomeric Compound
20gaggaatgaa cacttcttcc 202120DNAArtificial SequenceOligomeric
Compound 21ttgtcgagga atgaacactt 202220DNAArtificial
SequenceOligomeric Compound 22tgttgttgtc gaggaatgaa
202320DNAArtificial SequenceOligomeric Compound 23ccaagtcacc
agagcccggg 202420DNAArtificial SequenceOligomeric Compound
24gtcagccaag tcaccagagc 202520DNAArtificial SequenceOligomeric
Compound 25ccagtgtcag ccaagtcacc 202620DNAArtificial
SequenceOligomeric Compound 26gccatccagt gtcagccaag
202720DNAArtificial SequenceOligomeric Compound 27ccagggccat
ccagtgtcag 202820DNAArtificial SequenceOligomeric Compound
28tcattccagg gccatccagt 202920DNAArtificial SequenceOligomeric
Compound 29ctttttcatt ccagggccat 203020DNAArtificial
SequenceOligomeric Compound 30ggcccttgca cattttgcct
203120DNAArtificial SequenceOligomeric Compound 31ccagatgggc
ccttgcacat 203220DNAArtificial SequenceOligomeric Compound
32tggttccaga tgggcccttg 203320DNAArtificial SequenceOligomeric
Compound 33ggccttggtt ccagatgggc 203420DNAArtificial
SequenceOligomeric Compound 34gtgacagtcg cttttatggg
203520DNAArtificial SequenceOligomeric Compound 35accgagtgac
agtcgctttt 203620DNAArtificial SequenceOligomeric Compound
36ctgggaccga gtgacagtcg 203720DNAArtificial SequenceOligomeric
Compound 37cagggcaggt aggttctacg 203820DNAArtificial
SequenceOligomeric Compound 38tgtctgggac cgagtgacag
203920DNAArtificial SequenceOligomeric Compound 39tctggtgtct
gggaccgagt 204020DNAArtificial SequenceOligomeric Compound
40ctgggagctc agtgccatcg 204120DNAArtificial SequenceOligomeric
Compound 41cagatctggg agctcagtgc 204220DNAArtificial
SequenceOligomeric Compound 42agagccactg gtcaggctgg
204320DNAArtificial SequenceOligomeric Compound 43aaaacagagc
cactggtcag 204420DNAArtificial SequenceOligomeric Compound
44gtgggaaaac agagccactg 204520DNAArtificial SequenceOligomeric
Compound 45gctctgcaag ttgtcccgtc 204620DNAArtificial
SequenceOligomeric Compound 46ttgcagctct gcaagttgtc
204720DNAArtificial SequenceOligomeric Compound 47ggaacatggg
catccagctg 204820DNAArtificial SequenceOligomeric Compound
48cctctggaac atgggcatcc 204920DNAArtificial SequenceOligomeric
Compound 49ggaagtgggt gtctcgcctc 205020DNAArtificial
SequenceOligomeric Compound 50cagcagaaaa tgcagatggg
205120DNAArtificial SequenceOligomeric Compound 51gcagccgcag
cagaaaatgc 205220DNAArtificial SequenceOligomeric Compound
52tgacagcagc cgcagcagaa 205320DNAArtificial SequenceOligomeric
Compound 53atcgatgaca gcagccgcag 205420DNAArtificial
SequenceOligomeric Compound 54cacactttga tcgatgacag
205520DNAArtificial SequenceOligomeric Compound 55catcccacac
tttgatcgat 205620DNAArtificial SequenceOligomeric Compound
56cagcacatcc cacactttga 205720DNAArtificial SequenceOligomeric
Compound 57tcttgcagca catcccacac 205820DNAArtificial
SequenceOligomeric Compound 58ttattccaag acctatgttc
205920DNAArtificial SequenceOligomeric Compound 59cagccatttt
attccaagac 206020DNAArtificial SequenceOligomeric Compound
60agaaccagcc attttattcc 206120DNAArtificial SequenceOligomeric
Compound 61acaaaagaac cagccatttt 206220DNAArtificial
SequenceOligomeric Compound 62cctgaccagg atggagaaat
206320DNAArtificial
SequenceOligomeric Compound 63caaattccct gaccaggatg
206420DNAArtificial SequenceOligomeric Compound 64tttaacaaat
tccctgacca 206520DNAArtificial SequenceOligomeric Compound
65cagtctttaa caaattccct 206620DNAArtificial SequenceOligomeric
Compound 66gttttcatca gtctttaaca 206720DNAArtificial
SequenceOligomeric Compound 67tagacacaat tatttattca
206820DNAArtificial SequenceOligomeric Compound 68tgtactagac
acaattattt 206920DNAArtificial SequenceOligomeric Compound
69tagaatgtac tagacacaat 207020DNAArtificial SequenceOligomeric
Compound 70attcacgaat agaatgtact 207120DNAArtificial
SequenceOligomeric Compound 71atgagattca cgaatagaat
207220DNAArtificial SequenceOligomeric Compound 72atgggtcacg
gtcactctac 207320DNAArtificial SequenceOligomeric Compound
73ggcgaatggg tcacggtcac 207420DNAArtificial SequenceOligomeric
Compound 74tgtgtggcga atgggtcacg 207520DNAArtificial
SequenceOligomeric Compound 75cagccccttg accatgcctg
207620DNAArtificial SequenceOligomeric Compound 76tgtcactttc
tcatgaaaca 207720DNAArtificial SequenceOligomeric Compound
77tccaagacga aggtcaactg 207820DNAArtificial SequenceOligomeric
Compound 78ttgttcagcc tgaccctggg 207920DNAArtificial
SequenceOligomeric Compound 79ttatccccaa gtcccaccat
208020DNAArtificial SequenceOligomeric Compound 80cctcagcctt
atccccaagt 208120DNAArtificial SequenceOligomeric Compound
81tagggactgc ccacccctca 208220DNAArtificial SequenceOligomeric
Compound 82ctgcatggtt gcccacaaga 208320DNAArtificial
SequenceOligomeric Compound 83agtgtctgca tggttgccca
208420DNAArtificial SequenceOligomeric Compound 84aaatcagtgt
ctgcatggtt 208520DNAArtificial SequenceOligomeric Compound
85aggaaaaatc agtgtctgca 208620DNAArtificial SequenceOligomeric
Compound 86tccattttaa aaacctacag 208720DNAArtificial
SequenceOligomeric Compound 87gccagtgaga ttgtggtttt
208820DNAArtificial SequenceOligomeric Compound 88acatggccag
tgagattgtg 208920DNAArtificial SequenceOligomeric Compound
89ggcatccagc tggccctggc 209020DNAArtificial SequenceOligomeric
Compound 90tgctgtgcag tctaaggact 209120DNAArtificial
SequenceOligomeric Compound 91tgttctgctg tgcagtctaa
209220DNAArtificial SequenceOligomeric Compound 92cttctgttct
gctgtgcagt 209320DNAArtificial SequenceOligomeric Compound
93catgccttct gttctgctgt 209420DNAArtificial SequenceOligomeric
Compound 94atcatgcctt ctgttctgct 209520DNAArtificial
SequenceOligomeric Compound 95gtgccatcat gccttctgtt
209620DNAArtificial SequenceOligomeric Compound 96gagtgccatc
atgccttctg 209720DNAArtificial SequenceOligomeric Compound
97gctgagtgcc atcatgcctt 209820DNAArtificial SequenceOligomeric
Compound 98gtgctgagtg ccatcatgcc 209920DNAArtificial
SequenceOligomeric Compound 99cgagtgctga gtgccatcat
2010020DNAArtificial SequenceOligomeric Compound 100gggtccgagt
gctgagtgcc 2010120DNAArtificial SequenceOligomeric Compound
101gcctgggtcc gagtgctgag 2010220DNAArtificial SequenceOligomeric
Compound 102ggcagcctgg gtccgagtgc 2010320DNAArtificial
SequenceOligomeric Compound 103agacaggcag cctgggtccg
2010420DNAArtificial SequenceOligomeric Compound 104gcaggagaca
ggcagcctgg 2010520DNAArtificial SequenceOligomeric Compound
105ctggcaagga ggagaagcag 2010620DNAArtificial SequenceOligomeric
Compound 106ctgctcaggc tggcaaggag 2010720DNAArtificial
SequenceOligomeric Compound 107ggtgctgctc aggctggcaa
2010820DNAArtificial SequenceOligomeric Compound 108gtggtgctgc
tcaggctggc 2010920DNAArtificial SequenceOligomeric Compound
109aggtggtgct gctcaggctg 2011020DNAArtificial SequenceOligomeric
Compound 110taggtggtgc tgctcaggct 2011120DNAArtificial
SequenceOligomeric Compound 111ataggtggtg ctgctcaggc
2011220DNAArtificial SequenceOligomeric Compound 112gataggtggt
gctgctcagg 2011320DNAArtificial SequenceOligomeric Compound
113tgatggagat aggtggtgct 2011420DNAArtificial SequenceOligomeric
Compound 114ttgatggaga taggtggtgc 2011520DNAArtificial
SequenceOligomeric Compound 115atctgttgat ggagataggt
2011620DNAArtificial SequenceOligomeric Compound 116catctgttga
tggagatagg 2011720DNAArtificial SequenceOligomeric Compound
117tctgtctcat ctgttgatgg 2011820DNAArtificial SequenceOligomeric
Compound 118tagtctgtct catctgttga 2011920DNAArtificial
SequenceOligomeric Compound 119gtagtctgtc tcatctgttg
2012020DNAArtificial SequenceOligomeric Compound 120gctctgtagt
ctgtctcatc 2012120DNAArtificial SequenceOligomeric Compound
121gcagctctgt agtctgtctc 2012220DNAArtificial SequenceOligomeric
Compound 122gctgcagctc tgtagtctgt 2012320DNAArtificial
SequenceOligomeric Compound 123aaggctgcag ctctgtagtc
2012420DNAArtificial SequenceOligomeric Compound 124gtgcaaaggc
tgcagctctg 2012520DNAArtificial SequenceOligomeric Compound
125ttccccgtgc aaaggctgca 2012620DNAArtificial SequenceOligomeric
Compound 126tttcttcccc gtgcaaaggc 2012720DNAArtificial
SequenceOligomeric Compound 127tgctttcttc cccgtgcaaa
2012820DNAArtificial SequenceOligomeric Compound 128atgtctgccc
tgctttcttc 2012920DNAArtificial SequenceOligomeric Compound
129gcattggtat cgcaatgtct 2013020DNAArtificial SequenceOligomeric
Compound 130ttctgcattg gtatcgcaat 2013120DNAArtificial
SequenceOligomeric Compound 131tctcttctgc attggtatcg
2013220DNAArtificial SequenceOligomeric Compound 132aagttggtgt
ctctcttcct 2013320DNAArtificial SequenceOligomeric Compound
133gggaagttgg tgtctctctt 2013420DNAArtificial SequenceOligomeric
Compound 134tttacagcag aagatgcaga 2013520DNAArtificial
SequenceOligomeric Compound 135agcatttaca gcagaagatg
2013620DNAArtificial SequenceOligomeric Compound 136acagcattta
cagcagaaga 2013720DNAArtificial SequenceOligomeric Compound
137ttacagcatt tacagcagaa 2013820DNAArtificial SequenceOligomeric
Compound 138aattgttaca gcatttacag 2013920DNAArtificial
SequenceOligomeric Compound 139tgggaattgt tacagcattt
2014020DNAArtificial SequenceOligomeric Compound 140cactgggaat
tgttacagca 2014120DNAArtificial SequenceOligomeric Compound
141gataccacac tgggaattgt 2014220DNAArtificial SequenceOligomeric
Compound 142aacagatacc acactgggaa 2014320DNAArtificial
SequenceOligomeric Compound 143gcaacagata ccacactggg
2014420DNAArtificial SequenceOligomeric Compound 144tttgcaacag
ataccacact 2014520DNAArtificial SequenceOligomeric Compound
145gttttgcaac agataccaca 2014620DNAArtificial SequenceOligomeric
Compound 146tatgttttgc aacagatacc 2014720DNAArtificial
SequenceOligomeric Compound 147gctctaggct atgttttgca
2014820DNAArtificial SequenceOligomeric Compound 148ggtcaggatg
tggctctagg 2014920DNAArtificial SequenceOligomeric Compound
149gagaggtcag gatgtggctc 2015020DNAArtificial SequenceOligomeric
Compound 150tagagaggtc aggatgtggc 2015120DNAArtificial
SequenceOligomeric Compound 151gtgtagagag gtcaggatgt
2015220DNAArtificial SequenceOligomeric Compound 152agggcaggaa
taaataatgg 2015320DNAArtificial SequenceOligomeric Compound
153ggagggcagg aataaataat 2015420DNAArtificial SequenceOligomeric
Compound 154atttcaaggt cattggtggg 2015520DNAArtificial
SequenceOligomeric Compound 155ttatttcaag gtcattggtg
2015620DNAArtificial SequenceOligomeric Compound 156cgtctttatt
tcaaggtcat 2015720DNAArtificial SequenceOligomeric Compound
157aaaatcgtct ttatttcaag 2015820DNAArtificial SequenceOligomeric
Compound 158ataaaatcgt ctttatttca 2015920DNAArtificial
SequenceOligomeric Compound 159aaataaaatc gtctttattt
2016020DNAArtificial SequenceOligomeric Compound 160gggtgctcac
ctgttgatgg 2016120DNAArtificial SequenceOligomeric Compound
161tgcttactgc tgactgccat 2016220DNAArtificial SequenceOligomeric
Compound 162atgcatgctt actgctgact 2016320DNAArtificial
SequenceOligomeric Compound 163gcctatgtgt atcatatgca
2016420DNAArtificial SequenceOligomeric Compound 164cagcagaacc
tctacagccc 2016520DNAArtificial SequenceOligomeric Compound
165tctgtctcat ctgtgaaagc 2016620DNAArtificial SequenceOligomeric
Compound 166atgctcttac cgcaatgtct 2016720DNAArtificial
SequenceOligomeric Compound 167gcattggtat ctgtgtaaag
2016820DNAArtificial SequenceOligomeric Compound 168ccttccctga
aggttcctcc 2016920DNAArtificial SequenceOligomeric Compound
169gctcatagaa ctgtcaggct 2017015DNAArtificial SequenceOligomeric
Compound 170ggtcaggatg tggct 1517115DNAArtificial
SequenceOligomeric Compound 171caggatgtgg ctcta
1517212DNAArtificial SequenceOligomeric Compound 172gtcaggatgt gg
1217336DNAArtificial SequenceOligomeric Compound 173gagaggtcag
gatgtggctc taggctatgt tttgca 3617428DNAArtificial
SequenceOligomeric Compound 174gtcaggatgt ggctctaggc tatgtttt
2817523DNAArtificial SequenceOligomeric Compound 175tgtggctcta
ggctatgttt tgc 2317620DNAArtificial SequenceOligomeric Compound
176ggtcaggatg tggctcaagg 2017720DNAArtificial SequenceOligomeric
Compound 177ggtcaggaag tggctctagg 2017820DNAArtificial
SequenceOligomeric Compound 178ggtcagtttg tggctctagg
2017920DNAArtificial SequenceOligomeric Compound 179ggtgaggatg
tggctccagg 2018020DNAArtificial SequenceOligomeric Compound
180ggtctggatg tgcctctcgg 20
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