U.S. patent application number 16/191774 was filed with the patent office on 2019-03-07 for inhibitors of dek protein and related methods.
The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF MICHIGAN. Invention is credited to Kristine Benford, David Engelke, Maureen Legendre, David Markovitz, Nirit Mor-Vaknin, Dave Pai.
Application Number | 20190071675 16/191774 |
Document ID | / |
Family ID | 56878916 |
Filed Date | 2019-03-07 |
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United States Patent
Application |
20190071675 |
Kind Code |
A1 |
Markovitz; David ; et
al. |
March 7, 2019 |
INHIBITORS OF DEK PROTEIN AND RELATED METHODS
Abstract
The present invention provides methods of treatment using
inhibitors of DEK protein and DEK activity. Such methods include,
but are not limited to, methods of preventing, treating, and/or
ameliorating inflammatory diseases, infections, autoimmune
diseases, malignant diseases, and other diseases or conditions in
which DEK has been implicated. Such inhibitors of DEK protein
include, but are not limited to, pharmaceutical compositions
including single stranded DNA or RNA aptamers capable of binding to
DEK. In some embodiments, such aptamers are useful for diagnosing
DEK related diseases or conditions. Related kits and compositions
are further provided.
Inventors: |
Markovitz; David; (Ann
Arbor, MI) ; Mor-Vaknin; Nirit; (Ann Arbor, MI)
; Legendre; Maureen; (Ann Arbor, MI) ; Engelke;
David; (Ann Arbor, MI) ; Benford; Kristine;
(Ann Arbor, MI) ; Pai; Dave; (Ann Arbor,
MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF MICHIGAN |
Ann Arbor |
MI |
US |
|
|
Family ID: |
56878916 |
Appl. No.: |
16/191774 |
Filed: |
November 15, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15557757 |
Sep 12, 2017 |
10138486 |
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PCT/US2016/022100 |
Mar 11, 2016 |
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16191774 |
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62132308 |
Mar 12, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/00 20130101;
A61P 37/00 20180101; G01N 2333/46 20130101; C12N 2320/31 20130101;
C12N 15/115 20130101; A61K 31/711 20130101; A61K 31/713 20130101;
C12Q 1/00 20130101; A61K 31/7115 20130101; A61K 45/06 20130101;
A61P 19/02 20180101; A61P 29/00 20180101; A61P 37/06 20180101; G01N
33/5308 20130101; A61P 43/00 20180101; A61K 31/7105 20130101; C12N
2310/16 20130101; A61K 31/713 20130101; A61K 2300/00 20130101; A61K
31/7115 20130101; A61K 2300/00 20130101; A61K 31/711 20130101; A61K
2300/00 20130101; A61K 31/7105 20130101; A61K 2300/00 20130101 |
International
Class: |
C12N 15/115 20060101
C12N015/115; A61K 45/06 20060101 A61K045/06; G01N 33/53 20060101
G01N033/53; A61K 31/7105 20060101 A61K031/7105; A61K 31/711
20060101 A61K031/711; A61K 31/7115 20060101 A61K031/7115; A61K
31/713 20060101 A61K031/713; C12Q 1/00 20060101 C12Q001/00; C12N
15/00 20060101 C12N015/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grant
number AI062248 awarded by the National Institute of Health. The
government has certain rights in the invention.
Claims
1. A method of treatment of a patient with an inflammatory
condition, comprising: a) generating a high affinity aptamer
specific for a DEK target sequence by the steps of: 1) identifying
a DEK target molecule; 2) generating a high affinity aptamer
specific for said DEK target molecule by systematic evolution of
ligands by exponential enrichment (SELEX), comprising: i) preparing
a mixture of nucleotides comprising random sequence; ii) binding
said mixture of nucleotides comprising random sequence to the said
DEK target molecule to form an affinity complex; iii) separating
said affinity complex from unbound nucleotides comprising random
sequence; iv) separating and isolating at least one bound
nucleotide comprising random sequence from said affinity complex;
v) purifying said at least one separated and isolated nucleotide
comprising random sequence thereby generating a high affinity
aptamer specific for said DEK molecule; and b) treating said
patient with said inflammatory condition with a therapeutically
effective amount said high affinity aptamer specific for said DEK
target molecule to treat said patient with said inflammatory
condition.
2. The method of claim 1, wherein said patient is a mammal, a
human, a cat, a dot, a horse, a pig, a domestic animal, a wild
animal, livestock or cattle.
3. The method of claim 1, wherein said inflammatory condition is an
acute inflammatory condition, a chronic inflammatory condition,
rheumatoid arthritis, juvenile idiopathic arthritis, systemic-onset
juvenile idiopathic arthritis, or osteoarthritis.
4. The method of claim 1, wherein said nucleotides are DNA
nucleotides.
5. The method of claim 1, wherein said nucleotides are
single-stranded nucleotides.
6. The method of claim 1, comprising one or more amplification
steps between steps i) to v).
7. The method of claim 1, wherein said high affinity aptamer
specific for said DEK target molecule is covalently bound to said
DEK target molecule.
7. The method of claim 1, wherein said high affinity aptamer
specific for said DEK target molecule comprises one or more
modified nucleotides.
8. The method of claim 7, wherein said one or more modified
nucleotides comprise one or more substitutions at the ribose and/or
phosphate base positions.
9. The method of claim 7, wherein said one more modified
nucleotides comprise at least one modified pyrimidine.
10. The method of claim 9, wherein said one more modified
nucleotides comprise at least one aromatic modified pyrimidine.
11. The method of claim 1, wherein said high affinity aptamer
specific for said DEK target molecule is administered with an
anti-inflammatory agent.
12. The method of claim 1, wherein said high affinity aptamer
specific for said DEK target molecule is administered with a
pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/557,757, filed Sep. 12, 2017, which is a
371 U.S. National Phase Entry of International Application No.
PCT/US2016/022100, filed Mar. 11, 2016, which claims priority to
U.S. Provisional Patent Application No. 62/132,308, filed Mar. 12,
2015, the contents of which are incorporated by reference in their
entireties.
FIELD OF THE INVENTION
[0003] Provided herein are methods of treatment using inhibitors of
DEK protein and DEK activity. Such methods include, but are not
limited to, methods of preventing, treating, and/or ameliorating
inflammatory diseases, infections, autoimmune diseases, malignant
diseases, and other diseases or conditions in which DEK has been
implicated. Such inhibitors of DEK protein include, but are not
limited to, pharmaceutical compositions including single stranded
DNA or RNA aptamers capable of binding to DEK. In some embodiments,
such aptamers are useful for diagnosing DEK related diseases or
conditions. Related kits and compositions are further provided.
BACKGROUND OF THE INVENTION
[0004] Tissue inflammatory responses participate in the
pathogenesis of a diversity of conditions, syndromes and disorders
including, for example, arthritis and ocular inflammation. An
important mediator of inflammation is the DEK protein, an abundant
and ubiquitous chromatin protein in multicellular organisms. DEK
comprises two DNA binding modules of which one includes a SAP box,
a sequence motif that DEK shares with other chromatin proteins. DEK
has no apparent affinity to specific DNA sequences, but
preferentially binds to super-helical and cruciform DNA, and
induces positive supercoils into closed circular DNA (Waldmann T.
et al., "The DEK protein--an abundant and ubiquitous constituent of
mammalian chromatin", Gene 343: 1-9, 2004.). DEK has recently been
found to play a significant role in inflammatory diseases like
arthritis (Mor-Vaknin N. et al., "DEK in synovium of patients with
juvenile idiopathic arthritis: characterization of DEK antibodies
and posttranslational modification of the DEK autoantigen",
Arthritis Rheum. 63: 556-567, 2011, Mor-Vanknin H. et al., "The DEK
nuclear antigen is a secreted chemotactic factor", Mol Cell Biol
26: 9484-9496, 2006.). Because of the structural motifs of DEK, DEK
is not a favorable candidate for traditional small-molecule drug
discovery.
[0005] Improved methods for treating DEK-mediated arthritis and
inflammatory disorders are needed.
SUMMARY OF THE INVENTION
[0006] Provided herein are aptamers that bind to DEK, and
compositions comprising aptamers that bind to DEK. Aptamers are
oligonucleotides that bind their targets with high affinity and
specificity. Aptamers may be selected using the SELEX (systematic
evolution of ligands by exponential enrichment) method. In some
instances, base modifications may mediate hydrophobic interactions
between the aptamer and target, leading to significant improvement
in binding affinity. The disclosed aptamers are useful as
therapeutics for preventing, treating, and/or ameliorating
inflammatory diseases, malignant diseases, infections, autoimmune
diseases, and/or other diseases or conditions in which DEK is
implicated. The aptamers also find use in research, drug screening,
and other applications. Also provided herein are pharmaceutical
compositions or formulations comprising a DEK aptamer, or a
pharmaceutically acceptable salt thereof, and at least one
pharmaceutically acceptable carrier. Such compositions can be
prepared in any suitable pharmaceutically acceptable dosage
form.
[0007] In experiments conducted during the course of developing
embodiments of the present invention, single stranded DNA aptamers
that bind and inhibit DEK with high specificity and affinity, and
that block its inflammatory effects, have been identified.
[0008] In some embodiments, provided herein are methods of
treating, ameliorating, or preventing recurrence of an inflammatory
condition in a patient, comprising administering to a patient
(e.g., a human patient) a therapeutically effective amount of a DEK
aptamer, and a pharmaceutically acceptable carrier. In certain
embodiments, the DEK aptamer is a DNA aptamer. In further
embodiments, the DNA aptamer comprises or consists of 18 to 200
nucleotides, or 18 to 150 nucleotides, or 18 to 100 nucleotides, or
18 to 75 nucleotides, or 18 to 50 nucleotides, or 20 to 150
nucleotides, or 20 to 100 nucleotides, or 20 to 75 nucleotides, or
20 to 50 nucleotides, wherein each nucleotide may, independently,
be a modified or unmodified nucleotide. In further embodiments, the
DNA aptamer comprises or is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID
NO: 6. In particular embodiments, the inflammatory condition is one
or more conditions selected from arthritis, rheumatoid arthritis,
juvenile rheumatoid arthritis, and inflammatory disease or an
autoimmune disease. In specific embodiments, the patient is a human
patient and the DEK is human DEK. Some embodiments further comprise
administering to a patient one or more anti-inflammatory agents. In
other embodiments, the anti-inflammatory agent is a steroidal
anti-inflammatory agent. In still other embodiments, the
anti-inflammatory agent is a non-steroidal anti-inflammatory
agent.
[0009] In some embodiments, provided herein are kits comprising a
pharmaceutical composition comprising a DEK aptamer, and,
optionally, instructions for administering the pharmaceutical
composition to a patient diagnosed with arthritis, rheumatoid
arthritis, juvenile rheumatoid arthritis, and uveitis. In other
embodiments, the kit comprises one or more anti-inflammatory
agents. In certain embodiments, the pharmaceutical composition is
to be administered together with one or more other
anti-inflammatory agents. In preferred embodiments, the DEK aptamer
is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2
or SEQ ID NO: 6.
[0010] In some embodiments, provided herein are methods of
inhibiting signs of inflammation, comprising exposing to a sample
comprising inflammatory cells a composition comprising an anti-DEK
aptamer, wherein said exposing results in inhibition of signs of
inflammation. In some embodiments, the DEK aptamer is SEQ ID NO: 1,
SEQ ID NO: 2 or SEQ ID NO: 6. In some embodiments, the sample is
from a human. In certain embodiments, the human is diagnosed with
arthritis, rheumatoid arthritis or juvenile rheumatoid
arthritis.
[0011] In some embodiments, provided herein are methods of
detecting DEK in a sample, comprising contacting proteins from a
sample with a DEK aptamer. In certain embodiments, the sample is a
human sample selected from the group consisting of a blood sample,
a serum sample, a plasma sample, a saliva sample, a urine sample, a
synovial fluid sample, a cartilage sample, and a tissue sample.
[0012] In some embodiments, provided herein are compositions
comprising an aptamer that specifically binds to DEK. In certain
embodiments the aptamer is selected from the group consisting of
SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 6. In further embodiments,
the aptamer comprises at least one modified pyrimidine. In still
further embodiments the modified aptamer is an aromatic modified
aptamer. In other embodiments, the aptamer comprises 2 to 6
modified pyrimidines.
[0013] In some embodiments, provided herein are pharmaceutical
compositions comprising at least one DEK aptamer, or a
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier.
[0014] Methods and pharmaceutical compositions or formulations for
preventing, treating, and/or ameliorating a disease or condition
mediated by DEK are provided. In some embodiments, a method
comprises administering a DEK aptamer, or pharmaceutical
compositions or formulations comprising a DEK aptamer, to a
subject, such as a mammal. In some embodiments, the subject is a
human.
[0015] In some embodiments, methods and pharmaceutical compositions
or formulations are provided for preventing, treating, and/or
ameliorating inflammatory diseases, malignant diseases, infections,
autoimmune diseases, and/or other diseases or conditions in which
DEK is implicated. Non-limiting exemplary inflammatory diseases
that may be treated with the DEK aptamers described herein include
rheumatoid arthritis, juvenile idiopathic arthritis, systemic-onset
juvenile idiopathic arthritis, osteoarthritis, uveitis, gout,
sepsis, asthma, interstitial lung disease, inflammatory bowel
disease, systemic sclerosis, intraocular inflammation, Grave's
disease, endometriosis, systemic sclerosis, adult-onset still
disease, amyloid A amyloidosis, polymyalgia rheumatic, remitting
seronegative symmetrical synovitis with pitting edema, Behcet's
disease, uveitis, graft-versus-host diseases, and TNFR-associated
periodic syndrome.
[0016] Malignant diseases that may be treated with the DEK aptamers
described herein include cancers and cancer-related conditions.
Non-limiting exemplary cancers include multiple myeloma, leukemia,
pancreatic cancer, breast cancer, colorectal cancer, cachexia,
melanoma, cervical cancer, ovarian cancer, lymphoma,
gastrointestinal, lung cancer, prostate cancer, renal cell
carcinoma, metastatic kidney cancer, solid tumors, non-small cell
lung carcinoma, non-Hodgkin's lymphoma, bladder cancer, oral
cancer, myeloproliferative neoplasm, B-cell lymphoproliferative
disease, and plasma cell leukemia. Non-limiting exemplary
cancer-related conditions include non-small cell lung
cancer-related fatigue and cancer related anorexia.
[0017] Non-limiting exemplary infections that may be treated with
the DEK aptamers described herein include human immunodeficiency
virus (HIV), human T-lymphotropic virus (HTLV), cerebral malaria,
urinary tract infections, and meningococcal infections.
[0018] Non-limiting exemplary autoimmune diseases that may be
treated with the DEK aptamers described herein include systemic
lupus erythromatosus, systemic sclerosis, polymyositis, vasculitis
syndrome including giant cell arteritis, takayasu aeteritis,
cryoglobulinemia, myeloperoxidase-antineutrophilcytoplasmic
antibody-associated crescentic glomerulonephritis, rheumatoid
vasculitis, Crohn's disease, relapsing polychondritis, acquired
hemophilia A, and autoimmune hemolytic anemia.
[0019] Further diseases that may be treated with the DEK aptamers
described herein include, but are not limited to, Castleman's
disease, ankylosing spondyliytis, coronary heart disease,
cardiovascular disease in rheumatoid arthritis, pulmonary arterial
hypertension, chronic obstructive pulmonary disease (COPD), atopic
dermatitis, psoriasis, sciatica, type II diabetes, obesity, giant
cell arteritis, acute graft-versus-host disease (GVHD), non-ST
elevation myocardial infarction, anti-neutrophil cytoplasmic
antibody (ANCA) associated vasculitis, neuromyelitis optica,
chronic glomerulonephritis, and Takayasu arteritis.
[0020] In some embodiments, aptamers disclosed herein have
applications ranging from biomarker discovery and diagnostics
(Ostroff, R. M., et al., PLoS One, 2010. 5(12): p. e15003; Mehan,
M., et al., PLoS One, 2012.) to histochemistry and imaging (Gupta,
S., et al., Appl Immunohistochem Mol Morphol, 2011. 19(3): p.
273-8).
[0021] In some embodiments, a therapeutic effect (e.g., treating,
preventing, and/or ameliorating inflammatory diseases, malignant
diseases, infections, autoimmune diseases, and other diseases or
conditions in which DEK has been implicated) may be achieved by
administering at least one DEK aptamer such that the aptamer is
exposed to, and can bind to, DEK. In some embodiments, such binding
occurs regardless of the method of delivery of the aptamer to the
subject being treated. In some embodiments, the therapeutic effect
may be achieved by administering at least one DEK aptamer such that
it is exposed to, and binds to, DEK and prevents or reduces the
binding of DEK to one or more cell receptors.
[0022] In some embodiments, the binding of a DEK aptamer to DEK
interferes with the binding of DEK to a DEK receptor. In some
embodiments, a DEK aptamer reduces signaling along the signal
transduction pathway of a DEK receptor.
[0023] In some embodiments, a DEK aptamer is administered with one
or more additional active agents. Such administration may be
sequential or in combination. Non-limiting exemplary additional
active agents include TNF-alpha inhibitors, IL-1 inhibitors, IL-23
inhibitors, IFN-gamma inhibitors, IL-17 inhibitors, IL-22
inhibitors, IL-4/IL-13 inhibitors, IL-13 inhibitors, IL-5
inhibitors, and JAK inhibitors.
[0024] In certain embodiments, provided herein are methods for
treating, ameliorating, or preventing recurrence of a condition
involving inflammation in a patient (e.g., a human patient)
comprising administering to the patient a therapeutically effective
amount of a DEK aptamer, including salts, esters and prodrugs
thereof, and pharmaceutically acceptable carrier. Such methods are
not limited to a particular DEK aptamer. In some embodiments, the
DEK aptamer upon administration to the patient does not induce a
cytokine response, an inflammatory response, and/or systemic
toxicity in the patient. In certain embodiments, the present
invention provides kits comprising a pharmaceutical composition
comprising a DEK aptamer and instructions for administering the
pharmaceutical composition to a patient (e.g., a human patient) an
inflammatory or autoimmune disorder. In some embodiments, the kits
further comprise one or more anti-inflammatory agents.
[0025] In some embodiments, an in vitro or in vivo diagnostic
method comprising contacting a DEK aptamer with a sample suspected
of comprising DEK is provided. In some embodiments, an in vivo
diagnostic method comprising administering a suitably labeled DEK
aptamer to an individual suspected of having a DEK-mediated disease
or disorder is provided, wherein the labeled aptamer is detected
for the purpose of diagnosing or evaluating the health status of
the individual. The label used may be selected in accordance with
the imaging modality to be used. In some embodiments, a diagnostic
kit or device comprising a DEK aptamer is provided. In some
embodiments, a DEK aptamer that specifically binds DEK is
provided.
[0026] In some embodiments, aptamers comprise one or more of a
linker, a modified nucleotide, an unmodified nucleotide, an
aromatic modified pyrimidine, an alkylene glycol, a polyalkylene
glycol, a substituted or unsubstituted C.sub.2-C.sub.20 linker, a
1,3-propane diol, a poly(1,3-propane diol) having from 2 to 100
1,3-propane diol units, an ethylene glycol, and a polyethylene
glycol having from 2 to 100 ethylene glycol units, In certain
embodiments, each substituted or unsubstituted C.sub.2-C.sub.20
linker is a substituted or unsubstituted C.sub.2-C.sub.8 linker, a
substituted or unsubstituted C.sub.2-C.sub.6 linker, a substituted
or unsubstituted C.sub.2-C.sub.5 linker, a substituted or
unsubstituted C.sub.2-C.sub.4 linker, or a substituted or
unsubstituted C.sub.3 linker.
[0027] In some embodiments, an aptamer comprises a G quartet motif.
In certain embodiments described herein, an aptamer may comprise at
least one modified pyrimidine.
[0028] In particular embodiments, each modified pyrimidine may be
independently selected from:
5-(N-benzylcarboxyamide)-2'-deoxyuridine (BndU),
5-(N-benzylcarboxyamide)-2'-O-methyluridine,
5-(N-benzylcarboxyamide)-2'-fluorouridine,
5-(N-phenethylcarboxyamide)-2'-deoxyuridine (PEdU),
5-(N-thiophenylmethylcarboxyamide)-2'-deoxyuridine (ThdU),
5-(N-isobutylcarboxyamide)-2'-deoxyuridine (iBudU),
5-(N-tyrosylcarboxyamide)-2'-deoxyuridine (TyrdU),
5-(N-3,4-methylenedioxybenzylcarboxyamide)-2'-deoxyuridine (MBndU),
5-(N-4-fluorobenzylcarboxyamide)-2'-deoxyuridine (FBndU),
5-(N-3-phenylpropylcarboxyamide)-2'-deoxyuridine (PPdU),
5-(N-imidizolylethylcarboxyamide)-2'-deoxyuridine (ImdU),
5-(N-isobutylcarboxyamide)-2'-O-methyluridine,
5-(N-isobutylcarboxyamide)-2'-fluorouridine,
5-(N-tryptaminocarboxyamide)-2'-deoxyuridine (TrpdU),
5-(N--R-threoninylcarboxyamide)-2'-deoxyuridine (ThrdU),
5-(N-tryptaminocarboxyamide)-2'-O-methyluridine,
5-(N-tryptaminocarboxyamide)-2'-fluorouridine,
5-(N-[1-(3-trimethylamonium)propyl]carboxyamide)-2'-deoxyuridine
chloride, 5-(N-naphthylmethylcarboxyamide)-2'-deoxyuridine (NapdU),
5-(N-naphthylmethylcarboxyamide)-2'-O-methyluridine,
5-(N-naphthylmethylcarboxyamide)-2'-fluorouridine,
5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2'-deoxyuridine),
5-(N-2-naphthylmethylcarboxyamide)-2'-deoxyuridine (2NapdU),
5-(N-2-naphthylmethylcarboxyamide)-2'-O-methyluridine,
5-(N-2-naphthylmethylcarboxyamide)-2'-fluorouridine,
5-(N-1-naphthylethylcarboxyamide)-2'-deoxyuridine (NEdU),
5-(N-1-naphthylethylcarboxyamide)-2'-O-methyluridine,
5-(N-1-naphthylethylcarboxyamide)-2'-fluorouridine,
5-(N-2-naphthylethylcarboxyamide)-2'-deoxyuridine (2NEdU),
5-(N-2-naphthylethylcarboxyamide)-2'-O-methyluridine,
5-(N-2-naphthylethylcarboxyamide)-2'-fluorouridine,
5-(N-3-benzofuranylethylcarboxyamide)-2'-deoxyuridine (BFdU),
5-(N-3-benzofuranylethylcarboxyamide)-2'-O-methyluridine,
5-(N-3-benzofuranylethylcarboxyamide)-2'-fluorouridine,
5-(N-3-benzothiophenylethylcarboxyamide)-2'-deoxyuridine (BTdU),
5-(N-3-benzothiophenylethylcarboxyamide)-2'-O-methyluridine, and
5-(N-3-benzothiophenylethylcarboxyamide)-2'-fluorouridine.
[0029] In further embodiments, aptamers comprise one or more of
5-(N-1-naphthylmethylcarboxyamide)-2'-deoxyuridine (NapdU),
5-(N-1-naphthylmethylcarboxyamide)-2'-O-methyluridine,
5-(N-1-naphthylmethylcarboxyamide)-2'-fluorouridine,
5-(N-2-naphthylmethylcarboxyamide)-2'-deoxyuridine (2NapdU),
5-(N-2-naphthylmethylcarboxyamide)-2'-O-methyluridine,
5-(N-2-naphthylmethylcarboxyamide)-2'-fluorouridine,
5-(N-1-naphthylethylcarboxyamide)-2'-deoxyuridine (NEdU),
5-(N-1-naphthylethylcarboxyamide)-2'-O-methyluridine,
5-(N-1-naphthylethylcarboxyamide)-2'-fluorouridine,
5-(N-2-naphthylethylcarboxyamide)-2'-deoxyuridine (2NEdU),
5-(N-2-naphthylethylcarboxyamide)-2'-O-methyluridine,
5-(N-2-naphthylethylcarboxyamide)-2'-fluorouridine,
5-(N-3-benzofuranylethylcarboxyamide)-2'-deoxyuridine (BFdU),
5-(N-3-benzofuranylethylcarboxyamide)-2'-O-methyluridine,
5-(N-3-benzofuranylethylcarboxyamide)-2'-fluorouridine,
5-(N-3-benzothiophenylethylcarboxyamide)-2'-deoxyuridine (BTdU),
5-(N-3-benzothiophenylethylcarboxyamide)-2'-O-methyluridine, and
5-(N-3-benzothiophenylethylcarboxyamide)-2'-fluorouridine.
[0030] In some embodiments, the aptamer comprises at least 2 to 6
modified pyrimidines and/or a 2'-OMe. In certain embodiments, an
aptamer may comprise at least one, or at least 2 to 5
phosphorothioate linkages.
[0031] In some embodiments, a pharmaceutical composition is
provided that comprises any of the aptamers described herein, or
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier. In some embodiments, the pharmaceutical
composition is for treating a disease or condition mediated by
DEK.
[0032] In some embodiments, an aptamer that binds DEK with an
affinity of less than 10 nM is provided. In some embodiments, the
aptamer binds DEK 1 with an affinity of less than 10 nM. In some
embodiments, the aptamer binds DEK with an affinity of less than 8
nM, or less than 7 nM, or less than 6 nM, or less than 5 nM, or
less than 4 nM, or less than 3 nM, or less than 2 nM, or less than
1 nM.
[0033] In embodiments described herein, the aptamer may consist of
18 to 200 nucleotides, or 18 to 150 nucleotides, or 18 to 100
nucleotides, or 18 to 75 nucleotides, or 18 to 50 nucleotides, or
20 to 150 nucleotides, or 20 to 100 nucleotides, or 20 to 75
nucleotides, or 20 to 50 nucleotides, wherein each nucleotide may,
independently, be a modified or unmodified nucleotide. In
embodiments described herein, the aptamer may comprise a detectable
label.
[0034] In some embodiments, a method of detecting DEK in a sample
is provided, comprising contacting proteins from a sample with an
aptamer described herein. In some embodiments, a method of
determining whether a sample comprises DEK, comprises contacting
proteins from the sample with an aptamer described herein. In some
embodiments, the method comprises contacting the sample with the
aptamer under stringent conditions.
[0035] In some embodiments, the sample is a sample from a human. In
some embodiments, the sample is selected from blood, serum, plasma,
saliva, urine, synovial fluid, cartilage and a tissue sample.
[0036] The foregoing and other objects, features, and advantages of
the invention will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1: Shows a DNA oligomer containing 40 central
nucleotides of sequence flanked by defined primer-binding
sites.
[0038] FIG. 2: Shows that DEK aptamers block neutrophil
extracellular trap (NET) formation by activated human
neutrophils
[0039] FIG. 3A-3D: Shows that stimulation of primary human
neutrophils from healthy donors with led to the release of DEK into
the extracellular milieu.
[0040] FIG. 4A-4B. Shows that incubation with the anti-DEK aptamer
SEQ ID NO: 6 blocked formation of PMA-induced NETs by healthy
control human peripheral blood neutrophils in a dose-dependent
manner.
[0041] FIG. 5A-5D: Shows a zyomsan-induced arthritis (ZIA) model in
which anti-DEK aptamer attenuated inflammation in WT mice.
[0042] FIG. 6: Shows the effects of DEK aptamers on zymosan
induction of joint inflammation.
[0043] FIG. 7A-7B: Shows fluorescent immunohistochemistry staining
of joint sections hours after intra-articular injection of either
control or anti-DEK aptamer.
[0044] FIG. 8A-8D: Shows reduced knee circumference and levels of
pro-inflammatory markers in DEK-KO mice.
[0045] FIG. 9: Shows that monocyte migration in response to
zymosan-induced arthritis is the same in WT and DEK-KO mice.
[0046] FIG. 10A-10E: Shows that neturophils from DEK-KO mice are
mature by flow cytometery.
[0047] FIG. 11A-11D: Shows that neutrophils from DEK-KO mice
demonstrate limited capacity to form NETs after LPS stimulation as
detected by extracellular co-localization of DAPI and anti-elastase
antibody, when compared to neutrophils from WT mice.
[0048] FIG. 12A-12B: Shows that minimal NET formation is observed
after long-term stimulation of DEK-KO neutrophils.
[0049] FIG. 13A-13B: Shows that mouse peripheral blood neutrophils
from DEK-KO mice form fewer NETs in response to stimulation than do
peripheral blood neutrophils from WT mice.
[0050] FIG. 14A-14B: Shows that peripheral and bone marrow
neutrophils from DEK-KO mice express reactive oxygen species (ROS)
to the same extent as do those from WT mice before and after PMA
stimulation.
[0051] FIG. 15A-15B: Shows no difference in expression of certain
pro-inflammatory cytokines and TRL2 in DEK-KO vs. WT mice.
[0052] FIG. 16: Shows that DEK-KO and WT cells exhibit similar
NF-.kappa.B signaling after stimulation.
[0053] FIG. 17A-17C: Shows immunostaining of non-permeabilized
sections of knees from mice treated with SEQ ID NO: 6 compared to
control aptamer, and the presence of extracellular DNA colocalizing
with MPO, in keeping with the presence of NETs in joints injected
with zymosan and treated with control aptamer control, but not with
the anti-DEK aptamer.
[0054] FIG. 18A-18C: Shows that DEK is present with NETs in the
extracellular space of DEK-KO neutrophils.
[0055] FIG. 19: Shows that bioactive DEK needed to restore NEY
formation by DEK-KO neutrophils.
DEFINITIONS AND METHODS
[0056] While the invention will be described in conjunction with
certain representative embodiments, it will be understood that the
invention is not limited to these illustrative examples. One
skilled in the art will recognize many methods and materials
similar or equivalent to those described herein may be used in the
practice of the present invention. The present invention is in no
way limited to the methods and materials described.
[0057] Unless defined otherwise, technical and scientific terms
used herein have the meaning commonly understood by one of ordinary
skill in the art to which this invention belongs. Definitions of
common terms in molecular biology may be found in Benjamin Lewin,
Genes V, published by Oxford University Press, 1994 (ISBN
0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of
Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN
0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and
Biotechnology: a Comprehensive Desk Reference, published by VCH
Publishers, Inc., 1995 (ISBN 1-56081-569-8). Although any methods,
devices, and materials similar or equivalent to those described
herein can be used in the practice of the invention, certain
methods, devices, and materials are described herein. It is further
to be understood that all base sizes or amino acid sizes, and all
molecular weight or molecular mass values, given for nucleic acids
or polypeptides are approximate, and are provided for
description.
[0058] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art(s) to which this invention belongs.
Although any methods, devices, and materials similar or equivalent
to those described herein can be used in the practice or testing of
the invention, the preferred methods, devices and materials are now
described.
[0059] As used in this disclosure, including the appended claims,
the singular forms "a," "an," and "the" include plural references,
unless the content clearly dictates otherwise, and are used
interchangeably with "at least one" and "one or more." Thus,
reference to "an aptamer" includes mixtures of aptamers, and the
like.
[0060] As used herein, the term "about" represents an insignificant
modification or variation of the numerical value such that the
basic function of the item to which the numerical value relates is
unchanged.
[0061] As used herein, the term "nucleotide" refers to a
ribonucleotide or a deoxyribonucleotide, or a modified form
thereof, as well as an analog thereof. Nucleotides include species
that include purines (e.g., adenine, hypoxanthine, guanine, and
their derivatives and analogs) as well as pyrimidines (e.g.,
cytosine, uracil, thymine, and their derivatives and analogs). When
a base is indicated as "A", "C", "G", "U", or "T", it is intended
to encompass both ribonucleotides and deoxyribonucleoties, and
modified forms and analogs thereof.
[0062] As used herein, "nucleic acid," "oligonucleotide," and
"polynucleotide" are used interchangeably to refer to a polymer of
nucleotides and include DNA, RNA, DNA/RNA hybrids and modifications
of these kinds of nucleic acids, oligonucleotides and
polynucleotides, wherein the attachment of various entities or
moieties to the nucleotide units at any position are included.
[0063] The terms "polynucleotide," "oligonucleotide," and "nucleic
acid" include double- or single-stranded molecules as well as
triple-helical molecules. Nucleic acid, oligonucleotide, and
polynucleotide are broader terms than the term aptamer and, thus,
the terms nucleic acid, oligonucleotide, and polynucleotide include
polymers of nucleotides that are aptamers but the terms nucleic
acid, oligonucleotide, and polynucleotide are not limited to
aptamers.
[0064] As used herein, the terms "modify", "modified",
"modification", and any variations thereof, when used in reference
to an oligonucleotide, means that at least one of the four
constituent nucleotide bases (i.e., A, G, T/U, and C) of the
oligonucleotide is an analog or ester of a naturally occurring
nucleotide. In some embodiments, the modified nucleotide confers
nuclease resistance to the oligonucleotide. In some embodiments,
the modified nucleotides lead to predominantly hydrophobic
interactions of aptamers with protein targets resulting in high
binding efficiency and stable co-crystal complexes. A pyrimidine
with a substitution at the C-5 position is an example of a modified
nucleotide. Modifications can include backbone modifications,
methylations, unusual base-pairing combinations such as the
isobases isocytidine and isoguanidine, and the like. Modifications
can also include 3' and 5' modifications, such as capping. Other
modifications can include substitution of one or more of the
naturally occurring nucleotides with an analog, internucleotide
modifications such as, for example, those with uncharged linkages
(e.g., methyl phosphonates, phosphotriesters, phosphoamidates,
carbamates, etc.) and those with charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), those with
intercalators (e.g., acridine, psoralen, etc.), those containing
chelators (e.g., metals, radioactive metals, boron, oxidative
metals, etc.), those containing alkylators, and those with modified
linkages (e.g., alpha anomeric nucleic acids, etc.). Further, any
of the hydroxyl groups ordinarily present on the sugar of a
nucleotide may be replaced by a phosphonate group or a phosphate
group; protected by standard protecting groups; or activated to
prepare additional linkages to additional nucleotides or to a solid
support. The 5' and 3' terminal OH groups can be phosphorylated or
substituted with amines, organic capping group moieties of from
about 1 to about 20 carbon atoms, polyethylene glycol (PEG)
polymers, in some embodiments, ranging from about 10 to about 80
kDa, PEG polymers, in some embodiments, ranging from about 20 to
about 60 kDa, or other hydrophilic or hydrophobic biological or
synthetic polymers. In one embodiment, modifications are of the C-5
position of pyrimidines. These modifications can be produced
through an amide linkage directly at the C-5 position or by other
types of linkages.
[0065] Polynucleotides can also contain analogous forms of ribose
or deoxyribose sugars that are generally known in the art,
including 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose,
carbocyclic sugar analogs, .alpha.-anomeric sugars, epimeric sugars
such as arabinose, xyloses or lyxoses, pyranose sugars, furanose
sugars, sedoheptuloses, acyclic analogs and abasic nucleoside
analogs such as methyl riboside. As noted above, one or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include embodiments
wherein phosphate is replaced by P(O)S ("thioate"), P(S)S
("dithioate"), (O)NR.sub.2 ("amidate"), P(O)R, P(O)OR', CO or
CH.sub.2 ("formacetal"), in which each R or R' is independently H
or substituted or unsubstituted alkyl (1-20 C) optionally
containing an ether (--O--) linkage, aryl, alkenyl, cycloalkyl,
cycloalkenyl or araldyl. Not all linkages in a polynucleotide need
be identical. Substitution of analogous forms of sugars, purines,
and pyrimidines can be advantageous in designing a final product,
as can alternative backbone structures like a polyamide backbone,
for example.
[0066] As used herein, the term "nuclease" refers to an enzyme
capable of cleaving the phosphodiester bond between nucleotide
subunits of an oligonucleotide. As used herein, the term
"endonuclease" refers to an enzyme that cleaves phosphodiester
bond(s) at a site internal to the oligonucleotide. As used herein,
the term "exonuclease" refers to an enzyme which cleaves
phosphodiester bond(s) linking the end nucleotides of an
oligonucleotide. Biological fluids typically contain a mixture of
both endonucleases and exonucleases.
[0067] As used herein, the terms "nuclease resistant" and "nuclease
resistance" refers to the reduced ability of an oligonucleotide to
serve as a substrate for an endo- or exonuclease, such that, when
contacted with such an enzyme, the oligonucleotide is either not
degraded (e.g., not detectably degraded) or is degraded more slowly
than an oligonucleotide composed of unmodified nucleotides.
[0068] As used herein, the term "C-5 modified pyrimidine" refers to
a pyrimidine with a modification at the C-5 position. Examples of a
C-5 modified pyrimidine include those described in U.S. Pat. Nos.
5,719,273 and 5,945,527. Examples of a C-5 modification include
substitution of deoxyuridine at the C-5 position with a substituent
independently selected from: benzylcarboxyamide (alternatively
benzylaminocarbonyl) (Bn), naphthylmethylcarboxyamide
(alternatively naphthylmethylaminocarbonyl) (Nap),
tryptaminocarboxyamide (alternatively tryptaminocarbonyl) (Trp),
phenethylcarboxyamide (alternatively phenethylamino carbonyl) (Pe),
thiophenylmethylcarboxyamide (alternatively
thiophenylmethylaminocarbonyl) (Th) and isobutylcarboxyamide
(alternatively isobutylaminocarbonyl) (iBu).
[0069] Chemical modifications of a C-5 modified pyrimidine can also
be combined with, singly or in any combination, 2'-position sugar
modifications, modifications at exocyclic amines, and substitution
of 4-thiouridine and the like.
[0070] Representative C-5 modified pyrimidines include:
5-(N-benzylcarboxyamide)-2'-deoxyuridine (BndU),
5-(N-benzylcarboxyamide)-2'-O-methyluridine,
5-(N-benzylcarboxyamide)-2'-fluorouridine,
5-(N-isobutylcarboxyamide)-2'-deoxyuridine (iBudU),
5-(N-isobutylcarboxyamide)-2'-O-methyluridine,
5-(N-phenethylcarboxyamide)-2'-deoxyuridine (PedU),
5-(N-thiophenylmethylcarboxyamide)-2'-deoxyuridine (ThdU),
5-(N-isobutylcarboxyamide)-2'-fluorouridine,
5-(N-tryptaminocarboxyamide)-2'-deoxyuridine (TrpdU),
5-(N-tryptaminocarboxyamide)-2'-O-methyluridine,
5-(N-tryptaminocarboxyamide)-2'-fluorouridine,
5-(N-[1-(3-trimethylamonium)propyl]carboxyamide)-2'-deoxyuridine
chloride, 5-(N-naphthylmethylcarboxyamide)-2'-deoxyuridine (NapdU),
5-(N-naphthylmethylcarboxyamide)-2'-O-methyluridine,
5-(N-naphthylmethylcarboxyamide)-2'-fluorouridine or
5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2'-deoxyuridine).
[0071] Nucleotides can be modified either before or after synthesis
of an oligonucleotide. A sequence of nucleotides in an
oligonucleotide may be interrupted by one or more non-nucleotide
components. A modified oligonucleotide may be further modified
after polymerization, such as, for example, by conjugation with any
suitable labeling component. As used herein, the term "at least one
pyrimidine," when referring to modifications of a nucleic acid,
refers to one, several, or all pyrimidines in the nucleic acid,
indicating that any or all occurrences of any or all of C, T, or U
in a nucleic acid may be modified or not.
[0072] As used herein, "nucleic acid ligand," "aptamer," and
"clone" are used interchangeably to refer to a non-naturally
occurring nucleic acid that has a desirable action on a target
molecule. A desirable action includes, but is not limited to,
binding of the target, catalytically changing the target, reacting
with the target in a way that modifies or alters the target or the
functional activity of the target, covalently attaching to the
target (as in a suicide inhibitor), and facilitating the reaction
between the target and another molecule. In one embodiment, the
action is specific binding affinity for a target molecule, such
target molecule being a three dimensional chemical structure other
than a polynucleotide that binds to the nucleic acid ligand through
a mechanism which is independent of Watson/Crick base pairing or
triple helix formation, wherein the aptamer is not a nucleic acid
having the known physiological function of being bound by the
target molecule. As used herein, an "aptamer" refers to a nucleic
acid that has a specific binding affinity for a target molecule
(see e.g., Nimjee S M, Rusconi C P, Sullenger B A. "Aptamers: an
emerging class of therapeutics". Annu Rev Med. 2005; 56:555-583.
Que-Gewirth N S, Sullenger B A. "Gene therapy progress and
prospects: RNA aptamers". Gene therapy. 2007; 14(4):283-291.). It
is recognized that affinity interactions are a matter of degree.
However, in this context, the "specific binding affinity" of an
aptamer for its target means that the aptamer binds to its target
generally with a much higher degree of affinity than it binds to
other components in a test sample. An "aptamer" is a set of copies
of one type or species of nucleic acid molecule that has a
particular nucleotide sequence. An aptamer can include any suitable
number of nucleotides, including any number of chemically modified
nucleotides. "Aptamers" refers to more than one such set of
molecules. Different aptamers can have either the same or different
numbers of nucleotides. Any of the aptamer methods disclosed herein
can include the use of two or more aptamers that specifically bind
the same target molecule. An aptamer can be identified using any
known method, including the SELEX process (see below). Once
identified, an aptamer can be prepared or synthesized in accordance
with any known method, including chemical synthetic methods and
enzymatic synthetic methods. DEK aptamer, or anti-DEK aptamer, as
used herein, refers to an aptamer that specifically binds to a
mature DEK protein.
[0073] Aptamers to a given target include nucleic acids that are
identified from a candidate mixture of nucleic acids, where the
aptamer is a ligand of the target, by a method comprising: (a)
contacting the candidate mixture with the target, wherein nucleic
acids having an increased affinity to the target relative to other
nucleic acids in the candidate mixture are partitioned from the
remainder of the candidate mixture; (b) partitioning the increased
affinity nucleic acids from the remainder of the candidate mixture;
and (c) amplifying the increased affinity nucleic acids to yield a
ligand-enriched mixture of nucleic acids, whereby aptamers of the
target molecule are identified. Aptamers may be DNA or RNA and may
be single stranded, double stranded, or contain double stranded or
triple stranded regions. Aptamers can comprise chemically modified
nucleic acids and can include higher ordered structures.
[0074] As used here, a "G quartet" is a nucleotide sequence motif
that comprises four pairs of G nucleotides with at least one
nucleotide or spacer group between each pair of G nucleotides. G
quartet motifs are described, e.g., in Lane, A. N., et al., NAR,
2008. 36(17): 5482:5515.
[0075] As used herein, "protein" is used synonymously with
"peptide," "polypeptide," or "peptide fragment." A "purified"
polypeptide, protein, peptide, or peptide fragment is substantially
free of cellular material or other contaminating proteins from the
cell, tissue, or cell-free source from which the amino acid
sequence is obtained, or substantially free from chemical
precursors or other chemicals when chemically synthesized.
[0076] As used herein, "inflammatory disease" refers to a disease
or condition involving an inflammatory response. The inflammatory
response may be acute and/or chronic. In some embodiments, chronic
inflammation involves an increase in the level of DEK. Non-limiting
exemplary inflammatory diseases that may be treated with the DEK
aptamers described herein include rheumatoid arthritis, juvenile
idiopathic arthritis, systemic-onset juvenile idiopathic arthritis,
osteoarthritis, sepsis, asthma, interstitial lung disease,
inflammatory bowel disease, systemic sclerosis, intraocular
inflammation, Grave's disease, endometriosis, systemic sclerosis,
adult-onset still disease, amyloid A amyloidosis, polymyalgia
rheumatic, remitting seronegative symmetrical synovitis with
pitting edema, Behcet's disease, uveitis, graft-versus-host
diseases, and TNFR-associated periodic syndrome.
[0077] As used herein, "malignant disease" includes cancer and
cancer-related conditions.
[0078] As used herein, "cancer" means a disease or condition
involving unregulated and abnormal cell growth. Non-limiting
exemplary cancers that may be treated with the DEK aptamers
described herein include multiple myeloma, leukemia, pancreatic
cancer, breast cancer, colorectal cancer, cachexia, melanoma,
cervical cancer, ovarian cancer, lymphoma, gastrointestinal, lung
cancer, prostate cancer, renal cell carcinoma, metastatic kidney
cancer, solid tumors, non-small cell lung carcinoma, non-Hodgkin's
lymphoma, bladder cancer, oral cancer, myeloproliferative neoplasm,
B-cell lymphoproliferative disease, and plasma cell leukemia.
Non-limiting exemplary cancer-related conditions include non-small
cell lung cancer-related fatigue and cancer related anorexia.
[0079] As used herein, "infection" refers to a disease or condition
caused by a pathogen, such as a bacteria, virus, fungus, etc.
Non-limiting exemplary infections that may be treated with the DEK
aptamers described herein include human immunodeficiency virus
(HIV), human T-lymphotropic virus (HTLV), cerebral malaria, urinary
tract infections, and meningococcal infections.
[0080] As used herein, "autoimmune disease" refers to a disease or
condition arising from an inappropriate immune response against the
body's own components, such as tissues and other components. In
some embodiments, DEK levels are elevated in autoimmune disease.
Non-limiting exemplary autoimmune diseases that may be treated with
the DEK aptamers described herein include systemic lupus
erythromatosus, systemic sclerosis, polymyositis, vasculitis
syndrome including giant cell arteritis, takayasu aeteritis,
cryoglobulinemia, myeloperoxidase-antineutrophilcytoplasmic
antibody-associated crescentic glomerulonephritis, rheumatoid
vasculitis, Crohn's disease, relapsing polychondritis, acquired
hemophilia A, and autoimmune hemolytic anemia.
[0081] As used herein, a "DEK mediated disease or condition" refers
to a disease or condition in which at least some of the symptoms
and/or progression of the disease or condition is caused by
DEK-mediated signaling. Non-limiting exemplary DEK mediated
diseases or conditions include inflammatory diseases, malignant
diseases (including cancer and cancer-related conditions),
infections, and autoimmune diseases. Further non-limiting exemplary
DEK mediated diseases include, but are not limited to, Castleman's
disease, ankylosing spondyliytis, coronary heart disease,
cardiovascular disease in rheumatoid arthritis, pulmonary arterial
hypertension, chronic obstructive pulmonary disease (COPD), atopic
dermatitis, psoriasis, sciatica, type II diabetes, obesity, giant
cell arteritis, acute graft-versus-host disease (GVHD), non-ST
elevation myocardial infarction, anti-neutrophil cytoplasmic
antibody (ANCA) associated vasculitis, neuromyelitis optica,
chronic glomerulonephritis, and Takayasu arteritis.
[0082] As used herein, "modulate" means to alter, either by
increasing or decreasing, the level of a peptide or polypeptide, or
to alter, either by increasing or decreasing, the stability or
activity of a peptide or a polypeptide. The term "inhibit", as used
herein, means to prevent or reduce the expression of a peptide or a
polypeptide to an extent that the peptide or polypeptide no longer
has measurable activity or bioactivity; or to reduce the stability
and/or reduce or prevent the activity of a peptide or a polypeptide
to an extent that the peptide or polypeptide no longer has
measurable activity or bioactivity. As described herein, the
protein which is modulated or inhibited is DEK.
[0083] As used herein, the term "bioactivity" indicates an effect
on one or more cellular or extracellular process (e.g., via
binding, signaling, etc.) which can impact physiological or
pathophysiological processes. As used herein, the terms "DEK" refer
to naturally-occurring DEK, including naturally-occurring isoforms
and variants. As used herein, DEK includes all mammalian species of
DEK, including human, canine, feline, murine, primate, equine, and
bovine.
[0084] As used herein, "DEK receptor" refers to a receptor that is
bound by and activated by DEK. DEK receptors include the receptors
of any mammalian species, including, but are not limited to, human,
canine, feline, murine, equine, primate, and bovine.
[0085] A "DEK aptamer" is an aptamer that specifically binds to and
modifies the activity of DEK. In some embodiments, a DEK aptamer
inhibits at least one activity of DEK in vitro. In some
embodiments, a DEK aptamer inhibits at least one activity of DEK in
vivo. Non-limiting exemplary activities of DEK include binding to a
DEK receptor, and meditating inflammation.
[0086] As utilized herein, the term "pharmaceutically acceptable"
means approved by a regulatory agency of a federal or a state
government or listed in the U.S. Pharmacopoeia or other generally
recognized pharmacopoeia for use in animals and, more particularly,
in humans. The term "carrier" refers to a diluent, adjuvant,
excipient, or vehicle with which the therapeutic is administered
and includes, but is not limited to, such sterile liquids as water
and oils.
[0087] A "pharmaceutically acceptable salt" or "salt" of a DEK
aptamer is a product of the disclosed compound that contains an
ionic bond and is typically produced by reacting the disclosed
compound with either an acid or a base, suitable for administering
to an individual. A pharmaceutically acceptable salt can include,
but is not limited to, acid addition salts including
hydrochlorides, hydrobromides, phosphates, sulphates, hydrogen
sulphates, alkylsulphonates, arylsulphonates, arylalkylsulfonates,
acetates, benzoates, citrates, maleates, fumarates, succinates,
lactates, and tartrates; alkali metal cations such as Li, Na, K,
alkali earth metal salts such as Mg or Ca, or organic amine
salts.
[0088] A "pharmaceutical composition" is a formulation comprising a
DEK aptamer in a form suitable for administration to an individual.
A pharmaceutical composition is typically formulated to be
compatible with its intended route of administration. Examples of
routes of administration include, but are not limited to, oral and
parenteral, e.g., intravenous, intradermal, subcutaneous,
inhalation, topical, transdermal, transmucosal, intra-articular,
intra-ocular, and rectal administration.
[0089] As used herein, the term "therapeutically effective amount"
generally means the amount necessary to ameliorate at least one
symptom of a disorder or condition to be prevented, reduced, or
treated as described herein. The phrase "therapeutically effective
amount" as it relates to the DEK aptamers of the present disclosure
means the aptamer dosage that provides the specific pharmacological
response for which the aptamer is administered in a significant
number of individuals in need of such treatment. It is emphasized
that a therapeutically effective amount of an aptamer that is
administered to a particular individual in a particular instance
will not always be effective in treating the conditions/diseases
described herein, even though such dosage is deemed to be a
therapeutically effective amount by those of skill in the art.
[0090] The terms "SELEX" and "SELEX process" are used
interchangeably herein to refer generally to a combination of (1)
the selection of nucleic acids that interact with a target molecule
in a desirable manner, for example, binding with high affinity to a
protein, with (2) the amplification of those selected nucleic
acids. The SELEX process can be used to identify aptamers with high
affinity to a specific target molecule. SELEX generally includes
preparing a candidate mixture of nucleic acids, binding of the
candidate mixture to the desired target molecule to form an
affinity complex, separating the affinity complexes from the
unbound candidate nucleic acids, separating and isolating the
nucleic acid from the affinity complex, purifying the nucleic acid,
and identifying a specific aptamer sequence. The process may
include multiple rounds to further refine the affinity of the
selected aptamer. The process can include amplification steps at
one or more points in the process. (See, e.g., U.S. Pat. No.
5,475,096, entitled "Nucleic Acid Ligands.") The SELEX process can
be used to generate an aptamer that covalently binds its target as
well as an aptamer that non-covalently binds its target. (See,
e.g., U.S. Pat. No. 5,705,337 entitled "Systematic Evolution of
Nucleic Acid Ligands by Exponential Enrichment: Chemi-SELEX.")
[0091] The SELEX process can be used to identify high-affinity
aptamers containing modified nucleotides that confer improved
characteristics on the aptamer, such as, for example, improved in
vivo stability or improved delivery characteristics. Examples of
such modifications include chemical substitutions at the ribose
and/or phosphate and/or base positions. SELEX process-identified
aptamers containing modified nucleotides are described in U.S. Pat.
No. 5,660,985, entitled "High Affinity Nucleic Acid Ligands
Containing Modified Nucleotides," which describes oligonucleotides
containing nucleotide derivatives chemically modified at the C5
and/or 2'-positions of pyrimidines. U.S. Pat. No. 5,580,737, see
supra, describes highly specific aptamers containing one or more
nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F),
and/or 2'-O-methyl (2'-OMe). See also, U.S. Patent Application
Publication No. 20090098549, entitled "SELEX and PHOTOSELEX," which
describes nucleic acid libraries having expanded physical and
chemical properties and their use in SELEX and photoSELEX.
[0092] In some embodiments, provided herein are methods for
producing oligonucleotides with improved nuclease resistance. The
nuclease resistant oligonucleotides may include at least one
pyrimidine modified at the C-5 position. In certain embodiments,
the modifications include substitution of deoxyuridine at the C-5
position with a substituent independently selected from:
benzylcarboxyamide (Bn), phenethyl (Pe), thiophenylmethyl (Th),
naphthylmethylcarboxyamide (Nap), tryptaminocarboxyamide (Trp), and
isobutylcarboxyamide as illustrated above.
[0093] As used herein a "linker" is a molecular entity that
connects two or more molecular entities through covalent bond or
non-covalent interactions, and can allow spatial separation of the
molecular entities in a manner that preserves the functional
properties of one or more of the molecular entities. A linker can
also be known as a spacer. Appropriate linker sequences will be
readily ascertained by those of skill in the art based upon the
present disclosure.
[0094] As used herein, a linker can comprise one or more molecules
or sub-components, selected from the group including, but not
limited to a polynucleotide, a polypeptide, a peptide nucleic acid,
a locked nucleic acid, an oligosaccharide, a polysaccharide, an
antibody, an affybody, an antibody mimic, an aliphatic, aromatic or
heteroaromatic carbon molecule, alkylene glycol (e.g., ethylene
glycol, 1,3-propane diol), a polyalkylene glycol (e.g.,
polyethylene glycol (PEG)), a cell receptor, a ligand, a lipid, any
fragment or derivative of these structures, any combination of the
foregoing, or any other chemical structure or component.
[0095] In some embodiments, as used herein a linker or spacer may
be a backbone comprising a chain of 2 to 20 carbon atoms
(C.sub.2-C.sub.20) (saturated, unsaturated, straight chain,
branched or cyclic), 0 to 10 aryl groups, 0 to 10 heteroaryl
groups, and 0 to 10 heterocyclic groups, optionally comprising an
ether (--O--) linkage, (e.g., one or more alkylene glycol units,
including but not limited to one or more ethylene glycol units
--O--(CH.sub.2CH.sub.2O)--; one or more 1,3-propane diol
units-O--(CH.sub.2CH.sub.2CH.sub.2O)--, etc.; in some embodiments,
a linker comprises 1 to 100 units, 1 to 50 units, 1 to 40 units, 1
to 30 units, 1 to 20 units, 1 to 12 units, or 1 to 10 units); an
amine (--NH--) linkage; an amide (--NC(O)--) linkage; and a
thioether (--S--) linkage; etc.; wherein each backbone carbon atom
may be independently unsubstituted (i.e., comprising --H
substituents) or may be substituted with one or more groups
selected from a C.sub.1 to C.sub.3 alkyl, --OH, --NH.sub.2, --SH,
--O--(C.sub.1 to C.sub.6 alkyl), --S--(C.sub.1 to C.sub.6 alkyl),
halogen, --OC(O)(C.sub.1 to C.sub.6 alkyl), --NH--(C.sub.1 to
C.sub.6 alkyl), and the like. In some embodiments, a
C.sub.2-C.sub.20 linker is a C.sub.2-C.sub.8 linker, a
C.sub.2-C.sub.6 linker, a C.sub.2-C.sub.5 linker, a C.sub.2-C.sub.4
linker, or a C.sub.3 linker, wherein each carbon may be
independently substituted as described above.
[0096] SELEX can also be used to identify aptamers that have
desirable off-rate characteristics. See U.S. Pat. No. 7,947,447,
entitled "Method for Generating Aptamers with Improved Off-Rates,"
which describes improved SELEX methods for generating aptamers that
can bind to target molecules. Methods for producing aptamers having
slower rates of dissociation from their respective target molecules
are described. The methods involve contacting the candidate mixture
with the target molecule, allowing the formation of nucleic
acid-target complexes to occur, and performing a slow off-rate
enrichment process wherein nucleic acid-target complexes with fast
dissociation rates dissociate and do not reform, while complexes
with slow dissociation rates remain intact. Additionally, the
methods include the use of modified nucleotides in the production
of candidate nucleic acid mixtures to generate aptamers with
improved off-rate performance (see U.S. Patent Publication No.
2009/0098549, entitled "SELEX and PhotoSELEX"). (See also U.S. Pat.
No. 7,855,054 and U.S. Patent Publication No. 2007/0166740). Each
of these applications is incorporated herein by reference in its
entirety.
[0097] In some embodiments, methods of selecting aptamers that bind
to a target molecule including, for example, DEK are provided,
comprising: (a) preparing a candidate mixture of nucleic acids,
wherein the candidate mixture comprises modified nucleic acids in
which at least one pyrimidine in at least one, or in each, nucleic
acid of the candidate mixture is chemically modified at the
CS-position; (b) contacting the candidate mixture with a target
molecule, wherein nucleic acids having an increased affinity to the
target molecule relative to other nucleic acids in the candidate
mixture bind the target molecule, forming nucleic acid-target
molecule complexes; (c) partitioning the increased affinity nucleic
acids from the remainder of the candidate mixture; and (d)
amplifying the increased affinity nucleic acids to yield a mixture
of nucleic acids enriched in nucleic acid sequences that are
capable of binding to the target molecule with increased affinity,
whereby an aptamer to the target molecule is identified. In certain
embodiments, the method further includes performing a slow off-rate
enrichment process.
[0098] "Target" or "target molecule" or "target" refers herein to
any compound upon which a nucleic acid can act in a desirable
manner. A target molecule can be a protein, peptide, nucleic acid,
carbohydrate, lipid, polysaccharide, glycoprotein, hormone,
receptor, antigen, antibody, virus, pathogen, toxic substance,
substrate, metabolite, transition state analog, cofactor,
inhibitor, drug, dye, nutrient, growth factor, cell, tissue, any
portion or fragment of any of the foregoing, etc., without
limitation. Virtually any chemical or biological effector may be a
suitable target. Molecules of any size can serve as targets. A
target can also be modified in certain ways to enhance the
likelihood or strength of an interaction between the target and the
nucleic acid. A target can also include any minor variation of a
particular compound or molecule, such as, in the case of a protein,
for example, minor variations in amino acid sequence, disulfide
bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling component, which does not substantially
alter the identity of the molecule. A "target molecule" or "target"
is a set of copies of one type or species of molecule or
multimolecular structure that is capable of binding to an aptamer.
"Target molecules" or "targets" refer to more than one such set of
molecules. Embodiments of the SELEX process in which the target is
a peptide are described in U.S. Pat. No. 6,376,190, entitled
"Modified SELEX Processes Without Purified Proteins.
[0099] The term "second agent" refers to a therapeutic agent other
than the DEK aptamer in accordance with the present invention. In
certain instances, the second agent is an anti-inflammatory
agent.
[0100] The term "co-administration" refers to the administration of
at least two agent(s) (e.g., DEK aptamer) or therapies to a
subject. In some embodiments, the co-administration of two or more
agents/therapies is concurrent. In other embodiments, a first
agent/therapy is administered prior to a second agent/therapy.
Those of skill in the art understand that the formulations and/or
routes of administration of the various agents/therapies used may
vary. The appropriate dosage for co-administration can be readily
determined by one skilled in the art. In some embodiments, when
agents/therapies are co-administered, the respective
agents/therapies are administered at lower dosages than appropriate
for their administration alone. Thus, co-administration is
especially desirable in embodiments where the co-administration of
the agents/therapies lowers the requisite dosage of a known
potentially harmful (e.g., toxic) agent(s).
[0101] The term "combination therapy" includes the administration
of an anti-inflammatory agent (e.g., DEK aptamer) and at least a
second agent as part of a specific treatment regimen intended to
provide the beneficial effect from the co-action of these
therapeutic agents. The beneficial effect of the combination
includes, but is not limited to, pharmacokinetic or pharmacodynamic
co-action resulting from the combination of therapeutic agents.
Administration of these therapeutic agents in combination typically
is carried out over a defined time period (usually minutes, hours,
days or weeks depending upon the combination selected).
"Combination therapy" may, but generally is not, intended to
encompass the administration of two or more of these therapeutic
agents as part of separate monotherapy regimens that incidentally
and arbitrarily result in the combinations of the present
invention. "Combination therapy" is intended to embrace
administration of these therapeutic agents in a sequential manner,
that is, wherein each therapeutic agent is administered at a
different time, as well as administration of these therapeutic
agents, or at least two of the therapeutic agents, in a
substantially simultaneous manner. Substantially simultaneous
administration can be accomplished, for example, by administering
to the subject a single capsule or injection having a fixed ratio
of each therapeutic agent or in multiple, single capsules or
injections for each of the therapeutic agents. Sequential or
substantially simultaneous administration of each therapeutic agent
can be effected by any appropriate route including, but not limited
to, oral routes, intravenous routes, intramuscular routes,
intra-articular routes, corneal routes, topical routes, and direct
absorption through mucous membrane tissues. The therapeutic agents
can be administered by the same route or by different routes. For
example, a first therapeutic agent of the combination selected may
be administered by intravenous injection while the other
therapeutic agents of the combination may be administered orally.
Alternatively, for example, all therapeutic agents may be
administered orally or all therapeutic agents may be administered
by intravenous injection. The sequence in which the therapeutic
agents are administered is not narrowly critical. "Combination
therapy" also can embrace the administration of the therapeutic
agents as described above in further combination with other
biologically active ingredients and non-drug therapies (e.g.,
surgery or radiation treatment). Where the combination therapy
further comprises a non-drug treatment, the non-drug treatment may
be conducted at any suitable time so long as a beneficial effect
from the co-action of the combination of the therapeutic agents and
non-drug treatment is achieved. For example, in appropriate cases,
the beneficial effect is still achieved when the non-drug treatment
is temporally removed from the administration of the therapeutic
agents, perhaps by days or even weeks.
[0102] Sequence identity, as used herein, in the context of two or
more nucleic acid sequences is a function of the number of
identical nucleotide positions shared by the sequences (i.e., %
identity=number of identical positions/total number of
positions.times.100), taking into account the number of gaps, and
the length of each gap that needs to be introduced to optimize
alignment of two or more sequences. The comparison of sequences and
determination of percent identity between two or more sequences can
be accomplished using a mathematical algorithm, such as BLAST and
Gapped BLAST programs at their default parameters (e.g., Altschul
et al., J. Mol. Biol. 215:403, 1990; see also BLASTN at www (dot)
ncbi (dot) nlm (dot) nih (dot) gov/BLAST). For sequence
comparisons, typically one sequence acts as a reference sequence to
which test sequences are compared. When using a sequence comparison
algorithm, test and reference sequences are input into a computer,
subsequence coordinates are designated if necessary, and sequence
algorithm program parameters are designated. The sequence
comparison algorithm then calculates the percent sequence identity
for the test sequence(s) relative to the reference sequence, based
on the designated program parameters. Optimal alignment of
sequences for comparison can be conducted, e.g., by the local
homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482,
1981, by the homology alignment algorithm of Needleman and Wunsch,
J. Mol. Biol., 48:443, 1970, by the search for similarity method of
Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
visual inspection (see generally, Ausubel, F. M. et al., Current
Protocols in Molecular Biology, pub. by Greene Publishing Assoc.
and Wiley--Interscience (1987)). As used herein, when describing
the percent identity of a nucleic acid it is intended that the
nucleic acid sequence is identical to the reference sequence except
that the nucleic acid sequence may include up to five point
mutations per each 100 nucleotides of the reference nucleic acid
sequence. In other words, to obtain a desired nucleic acid
sequence, the sequence of which is at least about 95% identical to
a reference nucleic acid sequence, up to 5% of the nucleotides in
the reference sequence may be deleted or substituted with another
nucleotide, or some number of nucleotides up to 5% of the total
number of nucleotides in the reference sequence may be inserted
into the reference sequence (referred to herein as an insertion).
These mutations of the reference sequence to generate the desired
sequence may occur at the 5' or 3' terminal positions of the
reference nucleotide sequence or anywhere between those terminal
positions, interspersed either individually among nucleotides in
the reference sequence or in one or more contiguous groups within
the reference sequence.
[0103] Ranges provided herein are understood to be shorthand for
all of the values within the range. For example, a range of 1 to 50
is understood to include any number, combination of numbers, or
sub-range from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
or 50 (as well as fractions thereof unless the context clearly
dictates otherwise). Any concentration range, percentage range,
ratio range, or integer range is to be understood to include the
value of any integer within the recited range and, when
appropriate, fractions thereof (such as one tenth and one hundredth
of an integer), unless otherwise indicated. Also, any number range
recited herein relating to any physical feature, such as polymer
subunits, size or thickness, are to be understood to include any
integer within the recited range, unless otherwise indicated.
DETAILED DESCRIPTION
DEK
[0104] DEK is a nuclear protein that regulates hematopoiesis and
participates in pathways involving secretion, receptor engagement,
uptake, and subsequent modulation of heterochromatin biology, gene
expression and hematopoietic cell cycling and migration. (Broxmeyer
H E, et al. "A Role for DEK in Stem/Progenitor Cell Biology". Stem
Cells. 2013. Epub 2013/06/05.; Kappes F, et al. "The DEK
oncoprotein is a Su(var) that is essential to heterochromatin
integrity". Genes Dev. 2011; 25(7):673-678.; Mor-Vaknin N et al.
"The DEK nuclear autoantigen is a secreted chemotactic factor". Mol
Cell Biol. 2006; 26(24):9484-9496.; Kappes F, et al. "DEK is a
poly(ADP-ribose) acceptor in apoptosis and mediates resistance to
genotoxic stress". Mol Cell Biol. 2008; 28(10):3245-3257.; Saha A K
et al. "Intercellular trafficking of the nuclear oncoprotein DEK".
Proc Natl Acad Sci USA. 2013; 110(17):6847-6852.) DEK aptamers are
nucleic acid molecules, or derivatives of variants thereof, that
are selected through screening of large random libraries of nucleic
acid molecules for those that bind to a protein of interest. Hits
that emerge are put through multiple rounds of selection to
discover aptamers that bind most tightly to a protein of interest.
Using SELEX, single-stranded DNA aptamers that bind avidly and
specifically to DEK, inactivate its function, and treat arthritis
in an in vivo mouse model have been discovered. DEK aptamers of the
present invention provide utility in the treatment of, for example,
arthritis, rheumatoid arthritis (RA), juvenile rheumatoid arthritis
(JRA), juvenile idiopathic arthritis (JIA), gout, autoimmune
disorders, infectious disorders, malignant disorders and other
disorders mediated by an inflammatory response.
DEK Aptamers
[0105] In some embodiments, a DEK aptamer comprises a nucleotide
sequence shown in any one of SEQ ID NOs: 1 (5' ATA GGG AGT CGA CCG
ACC AGA AGG GGT TAA ATA
[0106] TTC [0107] CCA CAT TGC CTG CGC CAG TAC AAA TAG TAT GTG CGT
CTA CAT CTA GACT 3') (DEK aptamer 64), and SEQ ID NO: 2 (5' ATA GGG
AGT CGA CCG ACC AGA ATA CCG TGG CAT CTG GTT GTA GCA TCA CGT CTT ATG
CGG CCG TAT GTG CGT CTA CAT CTA GACT 3' (DEK aptamer 85) and SEQ ID
NO: 6 (5'-GGG GTT AAA TAT TCC CAC ATT GCC TGC GCC AGT ACA AAT
AG-3').
[0108] In some embodiments, 1 to 20, 1 to 15, 1 to 12, 1 to 8, 1 to
5, or 1 to 3 nucleotides of SEQ ID NOs: 1, 2 or 6 may be
substituted, deleted, or inserted. The number of nucleotides
substituted, deleted, or inserted is not particularly limited as
long as the aptamer specifically binds DEK with affinity (K.sub.d)
of, for example, less than 20 nM and/or has DEK antagonist activity
(IC.sub.50) of, for example, less than 10 nM (10.sup.-8 M). In some
embodiments, the DEK aptamer comprises not more than 10, and in
some embodiments, 4, 3, 2, or 1, nucleotide substitutions,
deletions, and/or insertions relative to a sequence of any one of
SEQ ID NOs: 1, 2 and 6.
[0109] In some embodiments, the present disclosure provides a DEK
aptamer that, upon binding DEK, modulates a DEK function. In some
embodiments, a DEK aptamer described herein inhibits DEK-mediated
inflammation. In various embodiments, the aptamer modulates a DEK
function in vivo, such as inhibiting inflammation. In some
embodiments, the DEK aptamer comprises at least 12, at least 13, at
least 14, at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 21, at least 22, at
least 23, at least 24, at least 25, at least 26, at least 27, at
least 28, at least 29, or at least 30 contiguous nucleotides of an
aptamer selected from SEQ ID NOs: 1, 2 and 6 wherein the aptamer
specifically binds DEK with an affinity (K.sub.d) of, for example,
less than 20 nM and/or has DEK antagonist activity (IC.sub.50) of,
for example, less than 10 nM (10.sup.-8 M).
[0110] In some embodiments, a DEK aptamer may comprise additional
nucleotides or other chemical moieties on the 5' end, the 3' end,
or both the 5' and the 3' end of the aptamer. The DEK aptamer can
contain any number of nucleotides in addition to the DEK binding
region. In various embodiments, the DEK aptamer can include up to
about 100 nucleotides, up to about 95 nucleotides, up to about 90
nucleotides, up to about 85 nucleotides, up to about 80
nucleotides, up to about 75 nucleotides, up to about 70
nucleotides, up to about 65 nucleotides, up to about 60
nucleotides, up to about 55 nucleotides, up to about 50
nucleotides, up to about 45 nucleotides, up to about 40
nucleotides, up to about 35 nucleotides, up to about 30
nucleotides, up to about 25 nucleotides, and up to about 20
nucleotides.
[0111] In some embodiments, the DEK aptamer is selected from an
aptamer that has similar binding characteristics and ability to
treat DEK associated inflammatory diseases, malignant diseases,
infections, autoimmune diseases, and other diseases or conditions
in which DEK has been implicated as an aptamer selected from SEQ ID
NOs: 1, 2 and 6. In some embodiments, a DEK aptamer is provided
that binds to the same region of DEK as an aptamer selected from
the aptamers of SEQ ID NOs: 1, 2 and 6.
[0112] In some embodiments, the DEK aptamers specifically bind
mature DEK. In some embodiments, the DEK aptamer is selected to
have any suitable dissociation constant (K.sub.d) for DEK. In some
embodiments, a DEK aptamer has a dissociation constant (K.sub.d)
for DEK of less than 30 nM, less than 25 nM, less than 20 nM, less
than 15 nM, less than 10 nM, less than 9 nM, less than 8 nM, less
than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less
than 3 nM, less than 2 nM, or less than 1 nM. Dissociation
constants may be determined with a binding assay using a
multi-point titration and fitting the equation
y=(max-min)(Protein)/(K.sub.d+Protein)+min
[0113] In some embodiments, a DEK aptamer has DEK antagonist
activity (IC.sub.50) of less than 10.sup.-8 M (<10 nM), less
than 10.sup.-9 M, less than 10.sup.-19 M, or less than 10.sup.-11
M. In various embodiments, DEK antagonist activity may be
determined using, for example, a cell inflammation assay and/or a
gene reporter assay
Methods of Detecting DEK
[0114] In some embodiments, methods of detecting DEK in a sample
are provided, comprising contacting the sample with an aptamer
described herein. In some embodiments, the method comprises
contacting the sample with a DEK aptamer described herein in the
presence of a polyanionic inhibitor. Detecting and/or quantifying
DEK bound by the DEK aptamer can be accomplished using methods in
the art and/or methods described herein. In some embodiments, the
DEK aptamer comprises a detectable label. In some embodiments, the
DEK aptamer is bound to a solid support, or comprises a member of a
binding pair that may be captured on a solid support (for example,
a biotinylated aptamer may be bound to a solid support comprising
streptavidin).
Pharmaceutical Compositions Comprising DEK Aptamers
[0115] In some embodiments, pharmaceutical compositions comprising
at least one aptamer described herein and at least one
pharmaceutically acceptable carrier are provided. Suitable carriers
are described in "Remington: The Science and Practice of Pharmacy,
Twenty-first Edition," published by Lippincott Williams &
Wilkins, which is incorporated herein by reference.
[0116] Pharmaceutical compositions that include at least one
aptamer described herein and at least one pharmaceutically
acceptable carrier may also include one or more other active
agents. The aptamers described herein can be utilized in any
pharmaceutically acceptable dosage form, including but not limited
to injectable dosage forms, liquid dispersions, gels, aerosols,
ointments, creams, lyophilized formulations, dry powders, tablets,
capsules, controlled release formulations, fast melt formulations,
delayed release formulations, extended release formulations,
pulsatile release formulations, mixed immediate release and
controlled release formulations, etc. Specifically, the aptamers
described herein can be formulated: (a) for administration selected
from any of oral, pulmonary, intravenous, intra-arterial,
intrathecal, intra-articular, rectal, ophthalmic, colonic,
parenteral, intracisternal, intravaginal, intraperitoneal, local,
buccal, nasal, and topical administration; (b) into a dosage form
selected from any of liquid dispersions, gels, aerosols, ointments,
creams, tablets, sachets and capsules; (c) into a dosage form
selected from any of lyophilized formulations, dry powders, fast
melt formulations, controlled release formulations, delayed release
formulations, extended release formulations, pulsatile release
formulations, and mixed immediate release and controlled release
formulations; or (d) any combination thereof.
[0117] Solutions or suspensions used for parenteral, intradermal,
or subcutaneous application can comprise one or more of the
following components: (1) a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents; (2)
antibacterial agents such as benzyl alcohol or methyl parabens; (3)
antioxidants such as ascorbic acid or sodium bisulfite; (4)
chelating agents such as ethylenediaminetetraacetic acid; (5)
buffers such as acetates, citrates or phosphates; and (5) agents
for the adjustment of tonicity such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric
acid or sodium hydroxide. A parenteral preparation can be enclosed
in ampoules, disposable syringes or multiple dose vials made of
glass or plastic.
[0118] Pharmaceutical compositions suitable for injectable use may
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition
should be sterile and should be fluid to the extent that easy
syringability exists. The pharmaceutical composition should be
stable under the conditions of manufacture and storage and should
be preserved against the contaminating action of microorganisms
such as bacteria and fungi. The term "stable", as used herein,
means remaining in a state or condition that is suitable for
administration to a subject.
[0119] The carrier can be a solvent or dispersion medium,
including, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol or sorbitol, and inorganic salts such
as sodium chloride in the composition. Prolonged absorption of the
injectable compositions can be brought about by including in the
composition an agent which delays absorption, for example, aluminum
monostearate and gelatin.
[0120] Sterile injectable solutions can be prepared by
incorporating the active reagent (e.g., a DEK aptamer) in an
appropriate amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as desired, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating at least one DEK aptamer into a sterile vehicle that
contains a basic dispersion medium and any other desired
ingredient. In the case of sterile powders for the preparation of
sterile injectable solutions, exemplary methods of preparation
include vacuum drying and freeze-drying, both of which will yield a
powder of a DEK aptamer plus any additional desired ingredient from
a previously sterile-filtered solution thereof. Oral compositions
generally include an inert diluent or an edible carrier. They can
be enclosed, for example, in gelatin capsules or compressed into
tablets. For the purpose of oral therapeutic administration, the
DEK aptamer can be incorporated with excipients and used in the
form of tablets, troches, or capsules. Oral compositions can also
be prepared using a fluid carrier for use as a mouthwash, wherein
the compound in the fluid carrier is applied orally and swished and
expectorated or swallowed. Pharmaceutically compatible binding
agents, and/or adjuvant materials can be included as part of the
composition.
[0121] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from a pressured
container or dispenser that contains a suitable propellant, e.g., a
gas such as carbon dioxide, a nebulized liquid, or a dry powder
from a suitable device. For transmucosal or transdermal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art, and include, for example, for
transmucosal administration, detergents, bile salts, and fusidic
acid derivatives. Transmucosal administration can be accomplished
through the use of nasal sprays or suppositories. For transdermal
administration, the active reagents are formulated into ointments,
salves, gels, or creams as generally known in the art. The reagents
can also be prepared in the form of suppositories (e.g., with
conventional suppository bases such as cocoa butter and other
glycerides) or retention enemas for rectal delivery.
[0122] In some embodiments, a DEK aptamer is prepared with a
carrier that protects against rapid elimination from the body. For
example, a controlled release formulation can be used, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc.
[0123] Liposomal suspensions (including liposomes targeted to
infected cells with monoclonal antibodies to viral antigens) can
also be used as pharmaceutically acceptable carriers. These can be
prepared according to methods known to those skilled in the art,
for example, as described in U.S. Pat. No. 4,522,811.
[0124] Additionally, suspensions of a DEK aptamer may be prepared
as appropriate oily injection suspensions. Suitable lipophilic
solvents or vehicles include fatty oils, such as sesame oil, or
synthetic fatty acid esters, such as ethyl oleate, triglycerides,
or liposomes. Non-lipid polycationic amino polymers may also be
used for delivery. Optionally, the suspension may also include
suitable stabilizers or agents to increase the solubility of the
compounds and allow for the preparation of highly concentrated
solutions.
[0125] In some embodiments, it is especially advantageous to
formulate oral or parenteral compositions in dosage unit form for
ease of administration and uniformity of dosage. Dosage unit form
as used herein refers to physically discrete units suited as
unitary dosages for the subject to be treated; each unit containing
a predetermined quantity of a DEK aptamer calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of DEK aptamers described herein are dictated by and directly
dependent on the characteristics of the particular DEK aptamer and
the particular therapeutic effect to be achieved, and the
limitations inherent in the art of compounding such an active agent
for the treatment of individuals.
[0126] Pharmaceutical compositions comprising at least one DEK
aptamer can include one or more pharmaceutical excipients. Examples
of such excipients include, but are not limited to, binding agents,
filling agents, lubricating agents, suspending agents, sweeteners,
flavoring agents, preservatives, buffers, wetting agents,
disintegrants, effervescent agents, and other excipients. Such
excipients are known in the art. Exemplary excipients include: (1)
binding agents which include various celluloses and cross-linked
polyvinylpyrrolidone, microcrystalline cellulose, such as AVICEL.
PH101 and AVICEL. PH102, silicified microcrystalline cellulose
(ProSolv SMCC), gum tragacanth and gelatin; (2) filling agents such
as various starches, lactose, lactose monohydrate, and lactose
anhydrous; (3) disintegrating agents such as alginic acid,
Primogel, corn starch, lightly crosslinked polyvinyl pyrrolidone,
potato starch, maize starch, and modified starches, croscarmellose
sodium, cross-povidone, sodium starch glycolate, and mixtures
thereof; (4) lubricants, including agents that act on the
flowability of a powder to be compressed, include magnesium
stearate, colloidal silicon dioxide, such as AEROSIL 200, talc,
stearic acid, calcium stearate, and silica gel; (5) glidants such
as colloidal silicon dioxide; (6) preservatives, such as potassium
sorbate, methylparaben, propylparaben, benzoic acid and its salts,
other esters of parahydroxybenzoic acid such as butylparaben,
alcohols such as ethyl or benzyl alcohol, phenolic compounds such
as phenol, or quaternary compounds such as benzalkonium chloride;
(7) diluents such as pharmaceutically acceptable inert fillers,
such as microcrystalline cellulose, lactose, dibasic calcium
phosphate, saccharides, and/or mixtures of any of the foregoing;
examples of diluents include microcrystalline cellulose, such as
AVICEL PH101 and AVICEL. PH102; lactose such as lactose
monohydrate, lactose anhydrous, and PHARMATOSE. DCL21; dibasic
calcium phosphate such as EMCOMPRESS; mannitol; starch; sorbitol;
sucrose; and glucose; (8) sweetening agents, including any natural
or artificial sweetener, such as sucrose, saccharin sucrose,
xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame;
(9) flavoring agents, such as peppermint, methyl salicylate, orange
flavoring, MAGNASWEET (trademark of MAFCO), bubble gum flavor,
fruit flavors, and the like; and (10) effervescent agents,
including effervescent couples such as an organic acid and a
carbonate or bicarbonate. Suitable organic acids include, for
example, citric, tartaric, malic, fumaric, adipic, succinic, and
alginic acids and anhydrides and acid salts. Suitable carbonates
and bicarbonates include, for example, sodium carbonate, sodium
bicarbonate, potassium carbonate, potassium bicarbonate, magnesium
carbonate, sodium glycine carbonate, L-lysine carbonate, and
arginine carbonate. Alternatively, only the sodium bicarbonate
component of the effervescent couple may be present.
[0127] In various embodiments, the formulations described herein
are substantially pure. As used herein, "substantially pure" means
the active ingredient (e.g., a DEK aptamer) is the predominant
species present (i.e., on a molar basis it is more abundant than
any other individual species in the composition). In one
embodiment, a substantially purified fraction is a composition
wherein the active ingredient comprises at least about 50 percent
(on a molar basis) of all macromolecular species present.
Generally, a substantially pure composition will include more than
about 80% of all macromolecular species present in the composition.
In various embodiments, a substantially pure composition will
include at least about 85%, at least about 90%, at least about 95%,
or at least about 99% of all macromolecular species present in the
composition. In various embodiments, the active ingredient is
purified to homogeneity (contaminant species cannot be detected in
the composition by conventional detection methods) wherein the
composition consists essentially of a single macromolecular
species.
Kits Comprising DEK Aptamer Compositions
[0128] The present disclosure provides kits comprising any of the
DEK aptamers described herein. Such kits can comprise, for example,
(1) at least one DEK aptamer; and (2) at least one pharmaceutically
acceptable carrier, such as a solvent or solution. Additional kit
components can optionally include, for example: (1) any of the
pharmaceutically acceptable excipients identified herein, such as
stabilizers, buffers, etc., (2) at least one container, vial or
similar apparatus for holding and/or mixing the kit components; and
(3) delivery apparatus.
[0129] In certain embodiments, the present invention provides
instructions for administering said inhibitors of inflammation
(e.g., DEK aptamer) to a subject. In certain embodiments, the
present invention provides instructions for using the compositions
contained in a kit for the treatment of conditions characterized by
inflammation in a cell or tissue (e.g., providing dosing, route of
administration, decision trees for treating physicians for
correlating patient-specific characteristics with therapeutic
courses of action). In certain embodiments, the present invention
provides instructions for using the compositions contained in the
kit to treat a variety of medical conditions associated with
inflammation (e.g., arthritis, rheumatoid arthritis, juvenile
rheumatoid arthritis). In certain embodiments, the present
invention provides instructions for using the compositions
contained in the kit to treat a variety of medical conditions
associated with inflammation, and/or autoimmune conditions.
Methods of Treatment
[0130] In some embodiments, provided herein are methods of
preventing or treating (e.g., alleviating one or more symptoms of)
medical conditions through the use of a DEK aptamer. The methods
comprise administering a therapeutically effective amount of a DEK
aptamer to a subject in need thereof. The described aptamers can
also be used for prophylactic therapy. In some embodiments, the DEK
aptamer is administered intra-articularly. In some embodiments the
DEK aptamer is administered intra-ocularly. The DEK aptamer used in
methods of treatment can be: (1) a DEK aptamer described herein, or
a pharmaceutically acceptable salt thereof, or a prodrug thereof.
The individual or subject can be any animal (domestic, livestock or
wild), including, but not limited to, cats, dogs, horses, pigs and
cattle, and preferably human subjects. As used herein, the terms
patient, individual, and subject may be used interchangeably.
[0131] As used herein, "treating" describes the management and care
of a patient for the purpose of treating a disease, condition, or
disorder and includes the administration of a DEK aptamer to
prevent the onset of the symptoms or complications of a disease,
condition or disorder; to alleviate symptoms or complications of
the disease, condition, or disorder; or to eliminate the presence
of the disease, condition or disorder in the patient. More
specifically, "treating" includes reversing, attenuating,
alleviating, minimizing, suppressing or halting at least one
deleterious symptom or effect of a disease (disorder) state,
disease progression, disease causative agent or other abnormal
condition. Treatment is generally continued as long as symptoms
and/or pathology ameliorate.
[0132] In some embodiments, compositions and methods of the present
invention are used to prevent, treat, and/or ameliorate
inflammatory diseases, malignant diseases, infections, autoimmune
diseases, and/or other diseases or conditions in which DEK is
implicated. Non-limiting exemplary inflammatory diseases that may
be treated with the DEK aptamers described herein include
rheumatoid arthritis, juvenile idiopathic arthritis, systemic-onset
juvenile idiopathic arthritis, gout, osteoarthritis, sepsis,
asthma, interstitial lung disease, inflammatory bowel disease,
systemic sclerosis, intraocular inflammation, Grave's disease,
endometriosis, systemic sclerosis, adult-onset still disease,
amyloid A amyloidosis, polymyalgia rheumatic, remitting
seronegative symmetrical synovitis with pitting edema, Behcet's
disease, uveitis, graft-versus-host diseases, and TNFR-associated
periodic syndrome. Malignant diseases that may be treated with the
DEK aptamers described herein include cancers and cancer-related
conditions. Non-limiting exemplary cancers include multiple
myeloma, leukemia, pancreatic cancer, breast cancer, colorectal
cancer, cachexia, melanoma, cervical cancer, ovarian cancer,
lymphoma, gastrointestinal, lung cancer, prostate cancer, renal
cell carcinoma, metastatic kidney cancer, solid tumors, non-small
cell lung carcinoma, non-Hodgkin's lymphoma, bladder cancer, oral
cancer, myeloproliferative neoplasm, B-cell lymphoproliferative
disease, and plasma cell leukemia. Non-limiting exemplary
cancer-related conditions include non-small cell lung
cancer-related fatigue and cancer related anorexia. Non-limiting
exemplary infections that may be treated with the DEK aptamers
described herein include human immunodeficiency virus (HIV), human
T-lymphotropic virus (HTLV), cerebral malaria, urinary tract
infections, and meningococcal infections. Non-limiting exemplary
autoimmune diseases that may be treated with the DEK aptamers
described herein include systemic lupus erythromatosus, systemic
sclerosis, polymyositis, vasculitis syndrome including giant cell
arteritis, takayasu aeteritis, cryoglobulinemia,
myeloperoxidase-antineutrophilcytoplasmic antibody-associated
crescentic glomerulonephritis, rheumatoid vasculitis, Crohn's
disease, relapsing polychondritis, acquired hemophilia A, and
autoimmune hemolytic anemia. Further diseases that may be treated
with the DEK aptamers described herein include, but are not limited
to, Castleman's disease, ankylosing spondyliytis, coronary heart
disease, cardiovascular disease in rheumatoid arthritis, pulmonary
arterial hypertension, chronic obstructive pulmonary disease
(COPD), atopic dermatitis, psoriasis, sciatica, type II diabetes,
obesity, giant cell arteritis, acute graft-versus-host disease
(GVHD), non-ST elevation myocardial infarction, anti-neutrophil
cytoplasmic antibody (ANCA) associated vasculitis, neuromyelitis
optica, chronic glomerulonephritis, and Takayasu arteritis.
[0133] In some embodiments, the disclosed compounds or
pharmaceutically acceptable salts thereof, or prodrugs, can be
administered in combination with other active agents. Compositions
including the disclosed DEK aptamers may contain, for example, more
than one aptamer. In some embodiments, a composition containing one
or more DEK aptamers is administered in combination with one or
more additional agents for preventing, treating, and/or
ameliorating inflammatory diseases, malignant diseases, infections,
autoimmune diseases, and/or other diseases or conditions in which
DEK is implicated.
[0134] The dosage regimen utilizing the DEK aptamers is selected in
accordance with a variety of factors, including, for example, type,
species, age, weight, sex and medical condition of the subject; the
severity of the condition to be treated; the route of
administration; the renal and hepatic function of the subject; and
the particular aptamer or salts thereof employed. An ordinarily
skilled physician or veterinarian can readily determine and
prescribe the effective amount of the composition required to
prevent, counter or arrest the progress of the condition. In
general, the dosage, i.e., the therapeutically effective amount,
ranges from about 1 ng/kg to about 1 g/kg body weight, in some
embodiments about 1 ug/kg to about 1 g/kg body weight, in some
embodiments about 1 ug/kg to about 100 mg/kg body weight, in some
embodiments about 1 ug/kg to about 10 mg/kg body weight of the
subject being treated, per day.
Methods for Diagnosing and Detecting
[0135] Aptamers that bind DEK, described herein, find use as
diagnostic reagents, either in vitro or in vivo. The DEK aptamers
identified herein can be used in any diagnostic, detection,
imaging, high throughput screening or target validation techniques
or procedures or assays for which aptamers, oligonucleotides,
antibodies and ligands, without limitation can be used. For
example, DEK aptamers identified herein can be used according to
the methods described in detail in U.S. Pat. No. 7,855,054,
entitled "Multiplexed Analyses of Test Samples", which is
incorporated by reference herein in its entirety.
[0136] Aptamers capable of binding DEK, described herein, find use
in a variety of assays including, assays that use planar arrays,
beads, and other types of solid supports. The assays may be used in
a variety of contexts including in life science research
applications, clinical diagnostic applications, (e.g., a diagnostic
test for a disease, or a "wellness" test for preventative
healthcare); ALONA and UPS assays, and in vivo imaging
applications. For some applications, multiplexed assays employing
the described DEK aptamers and may be used.
[0137] In some embodiments, the DEK aptamers are used as sensitive
and specific reagents for incorporation into a variety of in vitro
diagnostic methods or kits. In some embodiments, the DEK aptamers
are used as substitutes for antibodies in a number of infectious,
or other type of, disease detection methods where the aptamer to
DEK includes either or both a detectable material and an
immobilization or capture component. In these embodiments, after
the aptamer from the kit is mixed with a clinical specimen, a
variety of assay formats may be utilized. In one embodiment, the
aptamer also includes a detectable label, such as a fluorophore. In
other embodiments, the assay format may include fluorescence
quenching, hybridization methods, flow cytometry, mass
spectroscopy, inhibition or competition methods, enzyme linked
oligonucleotide assays, SPR, evanescent wave methods, etc. In some
embodiments, the aptamer is provided in the kit in solution. In
other embodiments, the aptamer in the kit is immobilized onto a
solid support used in conjunction with the assay for testing the
specimen. In various embodiments, the solid support is designed for
the detection of one or more targets of interest. In other
embodiments, the kit may further include reagents to extract the
target of interest, reagents for amplifying the aptamer, reagents
for performing washing, detection reagents, etc.
[0138] Diagnostic or assay devices, e.g. columns, test strips or
biochips, having one or more DEK aptamers adhered to a solid
surface of the device are also provided. The aptamer(s) may be
positioned so as to be capable of binding DEK molecules that are
contacted with the solid surface to form aptamer-target complexes
that remain adhered to the surface of the device, thereby capturing
the target and enabling detection and optionally quantitation of
the target. An array of aptamers (which may be the same or
different) may be provided on such a device.
[0139] In one embodiment for detecting DEK, an aptamer affinity
complex or aptamer covalent complex is contacted with a labeling
agent that includes a binding partner that is specific for DEK. The
specific binding partner may be any suitable moiety, including an
antibody, an antibody fragment, a synthetic antibody mimetic, a
biomimetic, an aptamer, a molecular imprinted ligand, and the like.
The specific binding partner is conjugated or linked to another
labeling agent component, usually, a detectable moiety or label. In
one embodiment for detecting DEK, an aptamer affinity complex or
aptamer covalent complex is contacted with a labeling agent that is
capable of labeling DEK, without a binding partner, and comprises a
detectable moiety or label.
[0140] The detectable moiety or label is capable of being detected
directly or indirectly. In general, any reporter molecule that is
detectable can be a label. Labels include, for example, (i)
reporter molecules that can be detected directly by virtue of
generating a signal, (ii) specific binding pair members that may be
detected indirectly by subsequent binding to a cognate that
contains a reporter molecule, (iii) mass tags detectable by mass
spectrometry, (iv) oligonucleotide primers that can provide a
template for amplification or ligation, and (v) a specific
polynucleotide sequence or recognition sequence that can act as a
ligand, such as, for example, a repressor protein, wherein in the
latter two instances the oligonucleotide primer or repressor
protein will have, or be capable of having, a reporter molecule,
and so forth. The reporter molecule can be a catalyst, such as an
enzyme, a polynucleotide coding for a catalyst, promoter, dye,
fluorescent molecule, quantum dot, chemiluminescent molecule,
coenzyme, enzyme substrate, radioactive group, a small organic
molecule, amplifiable polynucleotide sequence, a particle such as
latex or carbon particle, metal sol, crystallite, liposome, cell,
etc., which may or may not be further labeled with a dye, catalyst
or other detectable group, a mass tag that alters the weight of the
molecule to which it is conjugated for mass spectrometry purposes,
and the like. The label can be selected from electromagnetic or
electrochemical materials. In one embodiment, the detectable label
is a fluorescent dye. Other labels and labeling schemes will be
evident to one skilled in the art based on the disclosure
herein.
EXAMPLES
[0141] The invention, now being generally described, will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention, and are not intended to
limit the invention. Data are presented as means.+-.SEM. The
difference between means was analyzed using the unpaired Student's
t-test. A p value <0.05 was considered significant. All
protocols for animal and human studies were approved by the
University of Michigan's Committee on Use and Care of Animal or the
Institutional Review Board.
Example 1
[0142] This example describes a selection protocol for DEK
aptamers.
[0143] SELEX technology, a method that selects for either
single-stranded DNAs or RNAs that bind tightly to the protein of
interest and can potentially inactivate its function, was used to
generate anti-DEK apamters. (Nimjee, S. M., Rusconi, C. P. &
Sullenger, B. A. Aptamers: an emerging class of therapeutics. Annu
Rev Med 56, 555-583, doi:10.1146/annurev.med.56.062904.144915
(2005), Que-Gewirth, N. S. & Sullenger, B. A. Gene therapy
progress and prospects: RNA aptamers. Gene therapy 14, 283-291,
doi:10.1038/sj.gt.3302900 (2007).) SELEX is performed using
multiple rounds of selection
[0144] and [0145] involves screening large numbers of random DNA or
RNA sequences to identify a sequence of [0146] interest. A 40
nucleotide, single-stranded DNA 5'-GGG GTT AAA TAT TCC CAC ATT GCC
TGC GCC AGT ACA AAT AG-3' (SEQ ID NO: 6), with high affinity for
recombinant DEK protein produced in a baculovirus system which
binds tightly to DEK.
[0147] A pool of 86-nucleotide DNA oligomer containing 40 central
nucleotides of random sequence flanked by defined primer-binding
sites (FIG. 1) was synthesized to order by Integrated DNA
Technologies (Coralville, Iowa). This resulted in an initial pool
with estimated complexity of 10.sup.14-10.sup.16 different
sequences: (5'-ATAGGAGTC-GACCGACCAGAA [N]40
TATGTGCGTCTACATCTA-GACTCAT-3') (SEQ ID NO: 3). Short DNA
oligonucleotides for amplifying selected sequences were: 5'-primer,
5'-ATAGGAGTCGACCGACCAGA A (SEQ ID NO: 4); 3'-primer,
5'-ATGAGTCTAGATGTAGACGCACATA (SEQ ID NO: 5). FIG. 1 shows
recombinant DEK protein conjugated to nickel agarose beads
incubated with a library including a pool of 10.sup.14-10.sup.16
random single-stranded DNA sequences. Each sequence includes 40
nucleotides (nt) flanked by 22 known nt on each side that serve as
primers for amplification by PCR. After extensive washes, the bound
nt are eluted and amplified by PCR reaction. The steps were
repeated up to 6 times to achieve specificity and to eliminate
nonspecific binding of the sequences to the naked beads.
Round 1
[0148] For the first round of selection, approximately 1 mg of
randomized single-stranded DNA library (Integrated DNA
Technologies, Corralville, Iowa) was incubated with 1.5 mL bed
volume of nickel-nitrilotriacetic acid (Ni-NTA) agarose resin
(Qiagen, Germantown, Md.) conjugated to histidine-tagged DEK
protein (at .about.1 mg DEK per mL of resin) in 5 mL of binding
buffer (20 mM Tris pH 7.6; 100 mM NaCl; 5 mM MgCl.sub.2). To obtain
the DEK protein, the open reading frame of human DEK was cloned
into the multiple cloning site of pBlueBacHis2A (Invitrogen). SF-9
cells (Invitrogen) were co-transfected with linearized Autographa
californica nuclear polyhedrosis virus DNA with the Bac-N-Blue DNA
transfection kit (Invitrogen) in accordance with the manufacturer's
protocol. Five days after cotransfection, the supernatant was
harvested and subjected to plaque assays. Single plaques were
picked, and a high-titer virus stock was raised. Three days
post-infection with a high-titer virus stock, HighFive cells were
harvested and washed three times with phosphate-buffered saline
prior to lysis with 2 ml of lysis buffer per 175-cm.sup.2 flask
(100 mM Tris-Cl [pH 7.5], 150 mM NaCl, 5 mM KCl, 0.5 mM MgCl.sub.2,
1% NP-40.5 mM imidazole). To disrupt DNA-protein and
protein-protein interactions, the lysed cells were further
incubated in the presence of 1.3 M NaCl for 20 min at room
temperature. The lysate was cleared (100,000.times.g, 10 min),
adjusted to 10% glycerol, diluted with lysis buffer to a final
concentration of 700 mM NaCl, and incubated with 10 ul of
equilibrated 50% Ni-nitrilotriacetic acid (NTA)-agarose (Qiagen,
Germantown, Md.) per 2 ml of lysate. After binding for 1 h at
4.degree. C., the beads were washed three times with 10 volumes of
buffer 1 [(50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 50 mM imidazole)],
three times with 10 volumes of buffer 2 [(50 mM Tris-Cl [pH 7.5],
300 mM NaCl, and 50 mM imidazole)], and again with 10 volumes of
buffer 1. Elution was performed with 50 mM Tris-Cl [pH 7.5]-150 mM
NaCl-500 mM imidazole. Aliquots of recombinant protein were stored
at -70.degree. C. in elution buffer. (See, for example, Kappes et
al., Phosphorylation or Protein Kinase CK2 Changes the DNA Binding
Properties of Human Chromatin Protein DEK. Molecular and Cellular
Biology. 24: 6011-6020, 2004.) The library was allowed to bind to
the resin-conjugated DEK for one hour at room temperature on a
rotating wheel. The resin was spun down gently in a swinging-bucket
centrifuge and the supernatant was removed. In fresh binding
buffer, the resin was transferred to a clean 50-mL conical tube and
washed 4 times with 25 mL binding buffer, 15 minutes each wash,
transferring to a clean conical tube after the second wash. To
elute, the resin was transferred to a clean 15-mL conical tube and
incubated with 1.5 mL elution buffer (20 mM Tris pH 7.6; 5 mM
MgCl.sub.2; 1 M NaCl; 7 M urea) for 1 hour on a rotating wheel at
room temperature. The resin slurry in elution buffer was applied to
0.45 .mu.m spin columns (Millipore, Darmstadt, Germany), and spun
in a microcentrifuge to separate the eluate from the beads. An
additional 500 .mu.L of elution buffer was applied to the 15-mL
conical tube to recover any additional beads that had stuck to the
sides during the elution step; this too was spun through the spin
columns. Thus, the total recovery volume was .about.2 mL. This
volume was then extracted twice, with equal volume
phenol:chloroform:isoamyl alcohol (25:24:1), extracted once with
equal volume chloroform, then precipitated with 2.5 volumes of
ethanol and resuspended in 20 mM Tris pH 7.6.
[0149] To amplify aptamers that bind to DEK, asymmetric PCR
amplification was performed with DNA oligonucleotide primers
complementary to end sequences (5:1 ratio of 5' primer to 3'
primer) (5'-primer, 5'-ATAGGAGTCGACCGACCAGA A (SEQ ID NO. 4);
3'-primer, 5'-ATGAGTCTAGATGTAGACGCACATA SEQ ID No. 5; Integrated
DNA Technologies, Coralville, Iowa). The amplified DNA pool was
then gel purified through denaturing polyacrylamide gel
electrophoresis (PAGE), and soaking elution/ethanol precipitation
to proceed to the next round.
Round 2
[0150] One-fourth of the resin from the Round 1 step (i.e., approx.
375 .mu.L bed volume) was used for Round 2 and subsequent rounds.
The resin was washed in binding buffer and transferred to a
pre-lubricated Eppendorf tube (Sorenson BioScience, #11700, Salt
Lake City, Utah). To this resin was added the amplified and
gel-purified DNA pool from Round 1. This mixture was allowed to
bind for one hour at room temperature on a rotating wheel. The
mixture was then transferred to a 0.45 .mu.m spin column and the
unbound fraction was spun out. The resin was washed 4 times, each
in 500 .mu.L binding buffer, keeping the resin in the spin column
during the washes, inverting vigorously several times each, and
spinning out. To elute, with the resin still in the column, 200
.mu.L of elution buffer was added, and eluted for 15 minutes on a
room temperature rotating wheel. The eluate was spun through in a
microcentrifuge to separate away from the resin. In order to dilute
the salt and the urea, 200 uL of water was added to the eluate
prior to proceeding with ethanol precipitation.
[0151] Asymmetric PCR amplification was performed with DNA
oligonucleotide primers complementary to end sequences (5:1 ratio
of 5' primer to 3' primer) as above. The amplified DNA pool was
then gel purified through denaturing polyacrylamide gel
electrophoresis, and soaking elution/ethanol precipitation before
proceeding to the next round.
Rounds 3-5
[0152] Prior to proceeding to Round 3, a subtraction was performed
to remove nonspecific binders. 100 .mu.L of Ni-NTA slurry (with no
DEK conjugated) was washed once in binding buffer and added to the
Round 2 DNA pool in fresh binding buffer. This mixture was
incubated for 30 minutes on a rotating wheel at room temperature to
allow nonspecific binders to separate. To continue with Round 3,
the supernatant was spun out and added to the same DEK-conjugated
resin used for Round 2. Round 3 proceeded exactly as Round 2.
[0153] After PCR and gel purification, Round 4 was performed
without subtraction. Between Rounds 4 and 5, a second subtraction
step was performed as above. Round 5 selection was done as with
other rounds. Asymmetric PCR amplification was performed with DNA
oligonucleotide primers complementary to end sequences (5:1 ratio
of 5' primer to 3' primer) as above. The amplified DNA pool was
then gel purified through denaturing polyacrylamide gel
electrophoresis and soaking elution/ethanol precipitation to
proceed to the next round.
Round 6
[0154] The gel-purified DNA pool from Round 5 was radiolabeled to
provide visualization via native PAGE gel shift. Approximately 1
million counts (<1 microgram) of labeled DNA was incubated with
titrated amounts of soluble (i.e., not conjugated to [his.times.6])
DEK protein (starting at 128 ng, with two-fold dilutions down to 1
ng) in 10 microliters. Bound DEK-DNA complex was separated from
free DNA on a 6% native gel supplemented with 5 mM MgCl.sub.2 and
5% glycerol. From the lane that gave the greatest % shift of DNA in
a single band, the bound complex was excised and eluted in 20 mM
Tris pH 7.6.
[0155] The Round 6 DEK pool was amplified via asymmetric PCR (5:1
(250 uM: 50 uM) ratio of 5' primer to 3' primer) and radiolabeled
following the T4-polynucleotide kinase (PNK) protocol (New England
Bioscience, Ipswich, Mass.): 1 ul T4 PNK, 1 ul .sup.32P ATP (3,000
Ci/mmol, 5 mCi/ml), 2 ul 10.times. T4 PNK buffer, 1 ug DNA, and
H.sub.2O up to 20 uL). The radiolabeled DNA was separated from the
unincorporated .sup.32P via a 6% denaturing gel. The bands
corresponding to the correct size were excised and soaked in 400 uL
of water overnight. The DNA was then precipitated and re-suspended
in water. The amount of .sup.32P labeled DNA in each tube was
determined using a scintillation counter (Beckman LS6500, Beckman
Coulter, Indianapolis, Ind.).
DEK Round 6 Binding
[0156] To test binding of the radiolabeled pools to the DEK
protein, a dot blot was performed. Glass fiber filter paper A
(Whatman/GE Healthcare, Pittsburgh, Pa.) and DEAE filters
(Whatman/GE Healthcare, Pittsburgh, Pa.) were soaked in binding
buffer for one-hour prior to use. 100 ng of DEK protein was
incubated in binding buffer and 100 ug/ml of salmon sperm DNA for
15 min. 5,000 CPM of the radiolabeled DNA pool was incubated with
the DEK and salmon sperm solution on a rotator for one hour. The
dot blot vacuum filter apparatus was set up with the DEAE paper on
the bottom and the GFC/A paper on top. Each well was washed with
100 uL of cold binding buffer (see Round 1 above). 100 uL of the
radiolabeled pool/DEK protein solution was placed into each well 8
samples at a time, and immediately vacuumed until the solution
passed through the membrane. The wells were then immediately washed
with 100 uL of cold binding buffer. When all of the samples were
loaded, all of the wells were washed 3.times. with 200 uL of
binding buffer. The membrane was then dried, and signal determined
using a Typhoon phosporimager (GE Healtcare, Pittsburgh, Pa.) for
1-2 hours to determine individual aptamers that had signal above
background of the same reaction without DEK protein.
Example 2
[0157] This example describes cloning via pGEM-T kit to obtain
"monoclonal" aptamers with individual sequences and properties.
[0158] Cloning was performed following the protocol in the Promega
pGEM-T kit (Model A3600, Madison, Wis.) using a 3:1 molar ratio of
final aptamer pool made double-stranded by PCR to pGEM plasmid, and
the electroporation transformation (BioRad Gene Pulser, according
to manufacturer's instructions (Hercules, Calif.). After plating on
LB/Amp media and incubating overnight, white colonies were randomly
selected and placed into a symmetric (1:1 (125 uM: 125 nM) 5'
primer to 3' primer PCR reaction. (Symmetric PCR conditions:
94.degree. C. 5' [94.degree. C. 30 s, 55.degree. C. 30 s,
72.degree. C. 30 s].times.20 rounds, 72.degree. C. C 10', 4.degree.
C. hold) using the 5' and 3' primers as above.
Example 3
[0159] This example describes PCR amplification with
radiolabeling.
[0160] After the symmetric PCR was complete to create
double-stranded DNA corresponding to the DEK-binding aptamers, the
DNA was gel-isolated through 6% denaturing polyacrylamide gels,
soaked out into water, and ethanol precipitated with 2.5 volumes of
ethanol. This DNA was used with an approximately 5.times. molar
excess of only the 5' primer (sequence same as above) that had been
radiolabeled to approximately 10,000,000 counts per microgram
labeling with T4 polynucleotide kinase and gamma-.sup.32P-ATP as
above. The asymmetric PCR (repeated unidirectional primer
extension) components were as follows:
TABLE-US-00001 Reagents Amount per reaction (uL) DNA from Symmetric
PCR 10 Taq polymerase 1 standard unit Taq Buffer 10x (no gelatin)
10 dNTPs (8 mM) 2.5 5' LIB SEL No T7 (non-radiolabeled) 0.25
.sup.32p 5' 1,000,000 CPM Taq DNA pol (1:20 dilution of stock) 5
H.sub.2O Up to 100 uL
[0161] The asymmetric PCR conditions were: 94.degree. C. 5'',
[94.degree. C. 30'', 59.degree. C. 0'', 72.degree. C.
30''].times.15 rounds, 72.degree. C. 7'', 4.degree. hold.
Example 4
[0162] This example describes DEK binding.
[0163] ssDNA aptamers were amplified from the 96 colonies, with
single-stranded asymmetric PCR products radiolabeled as above, and
tested to determine their binding ability. Dot blots were performed
as in DEK Round 6 binding above. Dot blots of aptamers giving the
highest binding signal were repeated in quadruplicate to verify
reproducibility.
Example 5
[0164] This example describes sequencing of plasmid DNA comprising
aptamer sequences.
[0165] The corresponding plasmid DNA of high affinity aptamer
positive clones were purified using QIAprep spin miniprep kits
(Qiagen) and sequenced by the University of Michigan DNA Sequencing
Core.
Example 6
[0166] This example describes Southwestern blotting to determine
aptamer binding to denatured DEK.
[0167] After determining the sequences of individual aptamer
clones, the corresponding DNA oligonucleotides with and without 5'
and 3' priming sequences were ordered from IDT (Coralville, Iowa),
and radiolabeled with .sup.32P according to the NEB T4 Kinase kit
protocol as above. Binding was then tested using a southwestern
blot of purified DEK protein. A conventional western blot was
performed using PVDF Immobillon P (EMDMillipore, Darmstadt,
Germany). After the transfer, the membrane was soaked in PreHyb
buffer [(10 mM Hepes (pH 7.9), 100 ug/mL salmon sperm DNA
(Sigma-Aldrich, St. Louis, Mo.), 5% bovine serum albumin
(Sigma-Aldrich, St. Louis, Mo.)] for 30 min on the shaker at room
temperature to block the membrane. The PreHyb buffer was then
removed and 5 mL of Hyb Buffer [(10 mM Hepes (pH 7.9), 50 mM NaCl,
1 mM EDTA, 1 mM DTT, 0.25% BSA, 2 ug/ul salmon sperm DNA, 50
million CPM aptamer)] was added and incubated on the shaker at room
temperature for one hour. The Hyb Buffer was removed and the
membrane was washed 3.times. on the shaker at room temperature for
20 min with wash buffer [(10 mM Hepes (pH 7.9), 300 mM NaCl, 1 mM
EDTA, 1 mM DTT, 0.25% BSA)]. The membranes were dried and exposed
on a Typhoon Phosphor imager (GE Healthcare, Pittsburgh, Pa.)
cassette overnight to visualize the radiolabeled DNA bound to the
bands corresponding to DEK protein. SEQ ID NOs 1 and 2 provide
positive DNA aptamer sequences comprising 5' and 3' priming
sequences. No quantitation was determined.
Example 7
[0168] This example describes DEK aptamers that block neutrophil
extracellular trap (NET) formation by activated human neutrophils.
[0169] DEK protein participates in the formation of NETs i.e.,
chromatin-containing structures that are vital to the innate immune
response (Kessenbrock K. et al. "Netting neutrophils in autoimmune
small-vessel vasculitis". Nat Med. 2009; 15(6):623-625, Kahlenberg
J M et al., "Neutrophil extracellular trap-associated protein
activation of the NLRP3 inflammasome is enhanced in lupus
macrophages". J Immunol. 2013; 190(3):1217-1226.). NETs are
extracellular structures composed of chromatin, DNA, histones and
antimicrobial factors such as neutrophil elastase and
myeloperoxidase. DEK is found in the extracellular space and is
important for chromatin architecture, and is linked to the
pathogenesis of autoimmunity. (Kaplan, M. J. & Radic, M.
Neutrophil extracellular traps: double-edged swords of innate
immunity. J Immunol 189, 2689-2695, doi:10.4049/jimmunol.1201719
189/6/2689 [pii] (2012).)
[0170] Forty milliliters of venous blood was collected from each
healthy volunteer into a 60 ml sterile syringe containing 7 ml 0.25
M Citrate (0.17 M sodium citrate and 0.083 M citric acid) and 6%
Dextran in PBS buffer (without calcium or magnesium). The blood was
incubated for 30 min at room temperature prior to collecting the
upper phase by Histopaque-1077 (Sigma, St Louis, Mo., USA) and
centrifugation for 30 min at 700.times.g. The neutrophil fraction
was resuspended in 10 ml HBSS buffer, after which it was layered on
Histopaque-1119 (Sigma, St Louis, Mo., USA) for an additional 30
minute separation by centrifugation at 700.times.g. The neutrophil
fraction was collected, washed once in HBSS, and resuspended to a
concentration of 500,000 cells/ml/coverslip in RPMI supplemented
with 2% BSA. Cells were mounted on 22.times.22 mm, 2.5 .mu.m glass
coverslips treated with 0.001% poly-L-Lysine (Sigma, St Louis, Mo.,
USA). A one-hour treatment with LPS (1 .mu.g/ml) or PMA (10 ng/ml)
(Sigma, St Louis, Mo., USA) was used to induce NET formation.
Aptamers to DEK were added at 1-50 ng/ml (0.78 nM-3.94 nM) to the
neutrophil culture prior to phorbol myristate acetate (PMA)
stimulation (10 ng/ml) (Sigma, St Louis, Mo., USA) used to induce
NETs formation. Cells were fixed in 4% paraformaldehyde/PBS, pH=7,
prior to immune-histochemical examination.
[0171] Neutrophils were incubated for 1 hour at 37.degree. C., then
fixed and stained for NETs with anti-myeloperoxidase (MPO)
antibodies and 4, 6-diamidino-2-phenylindole (DAPI) for DNA.
(40.times. magnification) at room temperature for 1 hour followed
by incubation with AlexaFluor 488 goat anti-rabbit or AlexaFluor
594 goat anti-mouse antibody (Invitrogen). Other cells were stained
with rabbit anti-DEK antibody (1:100) or mouse anti-DEK antibody
(1:50 or 1:500, BD Bioscience, 610948), mouse monoclonal
anti-elastase (1:500, Abcam, Cambridge, Mass. ab78187), rabbit
anti-elastase (1:1000, Abcam, ab 21595), mouse anti-LL-37 (1:100,
Abcam, ab64892), or rabbit anti-MPO (1:500, Dako A0398) Nuclei and
NETs were visualized by DAPI--Prolong gold antifade (Invitrogen
P-36931) or stained with Hoechst. Slides were analyzed using a
fluorescence microscope (BX; Olympus) or confocal microscope
(Nikon) including Z-stacks of 80 0.3 micron optical sections
(60.times.). Ten high power (40.times.) images were captured.
Images were loaded onto Adobe Photoshop (Adobe System) and NETs
were counted manually and shown as a percentage of total
neutrophils per field. NETs were counted by at least three
independent observers and identified based on overlap of DAPI
staining with the NET markers elastase and MPO. For NET
quantification Metamorph 7.7 was used compare NET to neutrophil
ratios by the Center for Live Cell Imaging (CLCI) at the University
of Michigan. The program takes two 24 bit (color) images, a DAPI
and a FITC stained image. The RGB channels are split and then the
two green channels are added together as well as the two blue
channels. The minimum value is set to the average+standard
deviation/2 of all the pixels in the image. This threshold is then
turned into a binary, where it passes through image filters that
connect some of the finer image structures, showing the presence of
NETs. Regions are automatically created around the NETs and
information is pulled into an excel sheet indicating image number,
plane number, region number, area, integrated green signal, nucleus
count, green nets, and nets/nuclei.
[0172] Two DEK aptamers (SEQ ID NO: 1 (aptamer 64) and SEQ ID NO: 2
(aptamer 85) impeded formation of NETs in human primary neutrophils
from healthy volunteers (FIG. 2). Stimulation of primary human
neutrophils from healthy donors with Escherichia coli (E. coli)
(FIG. 3A) or 10 ng/ml PMA (FIG. 3B) led to the release of DEK into
the extracellular milieu. The banding patterns demonstrated by
immunoblot analysis are consistent with previously reported
findings of numerous DEK isoforms in primary cells. (Mor-Vaknin, N.
et al. DEK in the synovium of patients with juvenile idiopathic
arthritis: characterization of DEK antibodies and posttranslational
modification of the DEK autoantigen. Arthritis Rheum 63, 556-567,
doi:10.1002/art.30138 (2011)., Sierakowska, H., Williams, K. R.,
Szer, I. S. & Szer, W. The putative oncoprotein DEK, part of a
chimera protein associated with acute myeloid leukaemia, is an
autoantigen in juvenile rheumatoid arthritis. Clin Exp Immunol 94,
435-439 (1993).) Exposure of fresh neutrophils to E. coli or PMA
for two hours primarily induced the release of the 35 kDa and 45
kDa forms of DEK, suggesting that DEK is modified as a result of
neutrophil activation by E. coli or PMA. A 60 kDa form of DEK is
detected in the supernatant and in cell extracts of the
unstimulated cells. Human peripheral blood neutrophils subjected to
LPS or PMA treatment to induce NET formation demonostrated
co-localization of DEK with the NET markers LL-37 and neutrophil
elastase. (Brinkmann, V. & Zychlinsky, A. Neutrophil
extracellular traps: is immunity the second function of chromatin?
J Cell Biol 198, 773-783, doi:10.1083/jcb.201203170 jcb.201203170
[pii] (2012).) (FIG. 3C) Incubation with the anti-DEK aptamer SEQ
ID NO: 6 blocked formation of PMA-induced NETs by healthy control
human peripheral blood neutrophils in a dose-dependent manner (FIG.
4A and FIG. 4B).
[0173] These results indicate that DEK is present in human NETs,
and underscore the important role of DEK in NET formation and
inflammation as a target in the treatment of human inflammatory
diseases. Treatment of activated human neurophils with anti-DEK
apatamer resulted in loss of their ability to generate NETs which
are extracellular containing structures.
Example 8
[0174] This example describes treatment of arthritis in a mouse
model with a DEK aptamer.
[0175] Zymosan is a polysaccharide composed primarily of glucan and
mannan residues from the cell wall of Saccharomyces cervesiae (Di
Carlo F J, Fiore J V. "On the composition of zymosan". Science.
1958; 127(3301):756-757.) Intra-articular (i.a.) injection of
zymosan is used to induce inflammatory arthritis in mice because
zymosan is stimulates an innate immune response (Frasnelli M E,
Tarussio D, Chobaz-Peclat V, Busso N, So A. "TLR2 modulates
inflammation in zymosan-induced arthritis in mice". Arthritis Res
Ther. 2005; 7(2):R370-3799.). Monocytes, macrophages, and
neutrophils are cell types that recognize and ingest zymosan,
thereby leading to their activation.
[0176] Zymosan A from Saccharomyces cerevisiae (Sigma, St Louis,
Mo., USA) (30 mg) was resuspended in 2 ml of endotoxin-free saline,
and was subsequently boiled and homogenized by sonic
emulsification. Arthritis was induced by intra-articular (i.a.)
injection of 300 .mu.g (20 .mu.l) of zymosan through the
suprapatellar ligament into the joint space. The contralateral knee
was injected with an equal volume of sterile saline (20 .mu.l) as a
control. Injection with PBS alone and zymosan alone provided
further negative and positive controls. Twelve to 13-week-old
female mice (WT) were injected i.a. with zymosan into the knees of
both hind legs on day 0. Knee circumference was measured by 2
different investigators in a blind fashion before injection on day
0 and at 24 and 48 hours after injection. Knee circumference
determination was achieved by measuring two perpendicular diameters
of the joint with calipers. Knee circumference was determined using
the following geometric formula: circumference=2.pi.(
(a.sup.2+b.sup.2/2)), where a is the latero-lateral diameter, and b
is the antero-posterior diameter, as previously described. (Woods,
J. M. et al. IL-4 adenoviral gene therapy reduces inflammation,
proinflammatory cytokines, vascularization, and bony destruction in
rat adjuvant-induced arthritis. J Immunol 166, 1214-1222
(2001).
[0177] Aptamers to DEK and a control from the library (e.g.,
scrambled/random sequence) were diluted in PBS to a concentration
of 5-500 ng/20 ul volume for i.a. injection. Anti-DEK aptamers were
selected using SELEX technology and a single stranded DNA with a 40
nucleotide core flanked by 22 pre-determined bases on each side
with high affinity for recombinant DEK protein (FIG. 1) (SEQ ID NO;
6) was tested. To measure its anti-inflammatory capabilities in
vivo, it was injected into the knee joint 30 minutes before
administration of zymosan; the contralateral knee of each mouse was
injected with and equal volume (20 .mu.l) of sterile saline, or
with control aptamer (5 or 50 ng/knee) vs. DEK aptamer (5 or 50
ng/contralateral knee). As illustrated in FIG. 5A the contralateral
knee was injected with an equal volume (20 .mu.l) of sterile
saline. The 12-13 week old mice (129/SVEV on B6) obtained from
Jackson laboratory at age of 10 weeks were injected with aptamers
30-60 min before zymosan injection. Circumferences of the knees
were measured 24 and 48 hours after injection as described above.
Mice were sacrificed 48 hours after injection and knees were
harvested for histology and pathological assessment. FIG. 6 shows
mean values of increased knee circumference at 48 hours after
injection. Measurements of mouse knee circumference at 48 hours
demonstrated that administration of 5 ng of DEK aptamer plus
zymosan led to almost no inflammation (0 mm.sup.3.+-.0.588) (*
p=0.00074) (Student's T-test) compared to administration of 5 ng of
library control aptamer (2.94 mm.sup.3.+-.0.8). Fifty ng of DEK
aptamer reduced the inflammation in the knee to 0.58 mm.sup.3
(.+-.1.12) following zymosan injection (* p=0.0183). The
DEK-targeting aptamer SEQ ID NO: 6 ("DTA 64") significantly reduced
joint inflammation at 5 ng/knee (p=0.004), 50 ng/knee (p=0.0006)
and 100 ng/knee (p=0.032) compared to control aptamers, as measured
by knee circumference 48 hours after injection of aptamers and
zymosan (FIG. 5B). Similar results were observed 24 hours after
injection. Histopathological assessment of DEK-targeted vs. control
aptamer-treated joints revealed an overall significant reduction in
inflammatory cell migration in the presence of SEQ ID NO: 6 vs.
control aptamer as determined by pathological assessment of H&E
sections (FIG. 5C and FIG. 5D). Fluorescent immunohistochemistry
staining revealed fewer Ly6G-positive cells in DTA64-treated knees
(FIG. 7), similar to the phenotype of DEK-KO mice (see below). FIG.
7A shows joint sections analyzed for neutrophils by
immunohistochemistry 48 hours after intra-articular injection of
either control or anti-DEK aptamer using the murine neutrophil
surface marker LY6-G. Cell nuclei are stained with DAPI. The
magnification is 40.times.. FIG. 7B shows that joints injected with
anti-DEK aptamers (i.e., "DTA 64") exhibit significantly lower
numbers of Ly6G positive cells as compared to joints injected with
control aptamer. Results shown reflect the percentage of Ly6G
positive cells from 5 different fields from each joint section from
3 different mice. (*p=0.031 as determined by Students t-test.) In
contrast, there was no significant difference in the infiltration
of CD11b-positive cells. Accordingly, DEK-targeting aptamers
neutralize neutrophil recruitment and the inflammatory response in
the ZIA murine model, and protect mice from developing
zymosan-induced knee inflammation. DEK-specific aptamer inhibits
the development of inflammation in mouse knees induced by zymosan,
compared to those mice treated with nonspecific control aptamers
(i.e., random single strand DNA molecules of length comparable to
DEK aptamers).
[0178] These results demonstrate that single-stranded anti-DEK DNA
aptamers attenuate inflammation in WT mice subjected to ZIA.
Example 9
[0179] This example describes joint inflammation in wild-type (WT)
mice compared to DEK knockout (KO) mice in the zymosan-induced
arthritis (ZIA) model.
[0180] DEK knockout (KO) mice (129/SVEV on B6), were employed.
Control mice were generated by back-breeding DEK-KO mice with the
B6 WT strain for a minimum of 10 generations, bred as
heterozygotes. Mice were housed in specific pathogen-free
conditions at the Animal Maintenance Facility of the University of
Michigan Medical Center, until they were used for experiments at
10-13 weeks of age.
[0181] WT mouse knees were at least 2-fold larger (3.794
mm.sup.3.+-.0.412) than DEK-KO mouse knees (1.689
mm.sup.3.+-.0.282) 24 hours following zymosan injection (FIG. 8A;
p=0.0006). Histopathologic analysis of total inflammatory cell
infiltration by hematoxylin and eosin (H&E) staining of knee
sections 24 hours post-injection demonstrated differences between
DEK-KO and WT mice. Immunostaining for the myeloid marker CD11b
showed no differences (FIG. 9). FIG. 9 shows that monocyte
migration in response to zymosan-induced arthritis is the same in
WT and DEK-KO mice. Joint sections from WT and DEK-KO
zymosan-injected knees were analyzed for monocytes by
immunohistochemistry 24 hours after intra-articular injection using
the murine leukocyte/monocytic surface marker CD11b. Examples of
positive cells are marked by arrows. Sections were also stained for
cell nuclei with DAPI. The magnification is 40.times.. DEK-KO
injected joints exhibit the same number of CD11b positive cells as
do WT injected joints.
[0182] Together, these results demonstrate that genetic depletion
of DEK confers protection against arthritis in a murine model of
inflammatory arthritis.
Example 10
[0183] This example describes neutrophil extracellular trap (NET)
induction in mouse bone marrow neutrophils.
[0184] To purify neutrophils from mouse bone marrow, harvested bone
marrow was rinsed with 50 mL PBS prior to centrifugation at 500
g.times.5 min. The pellet was resuspended in 5 mL PBS and cells
were layered on a discontinuous gradient of 1 mL Histopaque-1119
and 5 mL Histopaque-1083. Tubes were centrifuged at room
temperature (without brake) at 700 g.times.30 min. All but the last
1.5-2 mL was removed, after which cells were rinsed with 50 mL PBS
prior to repeat centrifugation at 500 g.times.5 min. Cells were
resuspended in 5 mL PBS prior to being counted and prepared for
immunohistochemistry as described above and below, with the
exception that rather than a 1 hour incubation as for human
neutrophils, the mouse neutrophils were incubated for of 2 hours
with 1 .mu.g/ml LPS to induce NETs.
[0185] Purified and stimulated neutrophils from the bone marrow of
DEK-KO and WT mice were tested for their capacity to generate NETs
in vitro. Purity of bone marrow neutrophils from both WT and DEK-KO
mice was confirmed by CD11b and Ly6G staining (FIG. 10A and FIG.
10B). Staining for the neutrophil marker Ly6G revealed a
significantly reduced neutrophil infiltration in DEK-KO mice as
compared to WT mice (FIG. 10A and FIG. 10B). FIG. 10 shows flow
cytometry analysis of neutrophils purified from bone marrow of WT
(A) and DEK-KO (B) mice using Ly6G-FITC (neutrophils) and
CD11b-ECy5 (found on neutrophils and monocytes). No difference in
expression of Ly6G in DEK-KO neutrophils was observed when compared
to WT as shown by dot plots and histograms. Moreover, no
differences in neutrophil nuclear morphology or spontaneous NET
formation were observed in unstimulated neutrophils from WT vs.
DEK-KO mice (FIG. 11A). However, neutrophils from DEK-KO mice
demonstrated limited capacity to form NETs after LPS stimulation,
as detected by extracellular co-localization of DAPI and
anti-elastase antibody, when compared to neutrophils from WT mice
(FIG. 11B and FIG. 11 D; p=0.00019). Parallel observations were
noted after phorbol myristate acetate (PMA) activation of
neutrophils from DEK-KO and WT mice was extended for up to 8 hours
(FIG. 12). FIG. 12 shows that minimal NET formation is seen even
after long-term stimulation of DEK-KO neutrophils. FIG. 12A shows
neutrophils purified from the bone marrow of WT and DEK-KO mice.
Neutrophils were stimulated with PMA for 8 hours and then fixed and
stained with MPO (1:500, Dako) and DAPI. No formation of
fully-developed NETs was detected from the stimulated DEK-KO cells.
WT neutrophils readily generated NETs. FIG. 12B shows the
percentage of PMA-stimulated neutrophils with NETs seen in 10
different fields of WT and DEK-KO cells as counted by two
independent individuals. Although altered neutrophil development
may contribute to the lessened neutrophil-specific response
exhibited by DEK-KO mice, bone marrow and peripheral blood
neutrophil counts (see below) by Ly6G staining displayed no
differences between DEK-KO and WT mice (FIG. 10).
[0186] These results demonstrate that neutrophils from DEK-KO mice
have reduced capacity to form NETs after short-term and long-term
stimulation with PMA.
Example 11
[0187] This example describes neutrophil extracellular trap (NET)
induction in mouse peripheral blood neutrophils.
[0188] Blood was obtained from WT and DEK-KO mice by cardiac
puncture under terminal anesthesia. Blood was collected into
heparinized tubes (500 U/1 ml blood). Cells were isolated by
Histopaque 1083 in 1:1 ratio in 15 ml tubes. Neutrophils were
recovered from the red blood cell (RBC) fraction in the bottom of
the tube by 20% dextran solution (half the volume of the RBC),
mixed, and RBCs allowed to sediment for 10 min at room temperature.
Leukocyte-rich supernatant was collected from the top fraction and
washed twice by 8 ml 0.2% BSA in PBS by centrifugation (1500 RPM).
Red blood cells were lysed with 2 ml RBC lysis buffer (BioLedgened)
for 4 min on ice followed by 10 ml PBS wash. 1-2.times.10.sup.6
cells were plated on coverslips (as described above for isolation
of neutrophils from human blood) in RPMI with 2% BSA and stimulated
with 1 ng/ml PMA for 2 hours. FIG. 10C shows a representative flow
cytometry histogram of whole blood cells obtained from WT and
DEK-KO mice using Ly6G-FITC. No difference in expression of Ly6G
was detected in DEK-KO v.s WT peripheral blood. FIG. 10D. and FIG.
10E show the percentage of Ly6G positive cells in peripheral blood
of WT and DEK-KKO mice 24 hours after the received intraarticular
injections with (FIG. 10D) PBS control or (FIG. 10E) zymosan from 3
different WT or KED-KO mice. Reports demonstrate that aberrant
granulocyte differentiation results from DEK knockdown in CD34+
human bone marrow cells (Koleva, R. I. et al. C/EBPalpha and DEK
coordinately regulate myeloid differentiation. Blood 119,
4878-4888, doi:10.1182/blood-2011-10-383083 blood-2011-10-383083
[pii] (2012). Findings in peripheral blood neutrophils were similar
to those from bone marrow neutrophils from DEK-KO mice (FIG. 13)
i.e., DEK-KO neutrophils are unable to form NETs both in vivo and
in vitro. FIG. 13 shows that mouse peripheral blood neutrophils
from DEK-KO mice form fewer NETs in response to stimulation than do
peripheral blood neutrophils from WT mice. FIG. 13A shows
mmuno-staining of peripheral blood neutrophils purified from DEK-KO
and WT mice after 2 hour stimulation with 1 ng/ml PMA. Neutrophils
were fixed and stained by MPO and DAPI. The magnification is
40.times.. WT neutrophils show NET formation as expected after PMA
stimulation, but minimal NET formation is detected in the
stimulated DEK-KO neutrophils as indicated by MPO and DAPI. FIG.
13B shows the percentage of neutrophils that formed NETs after PMA
stimulation as calculated from 5 different fields of WT and DEK-KO
peripheral blood neutrophils. Reconstitution with recombinant DEK
protein one hour prior to activation with LPS rescued the ability
of DEK-KO neutrophils to generate NETs (FIG. 11C and FIG. 11D).
These results demonstrate that addition of recombinant DEK allows
DEK-KO neutrophils to create fully-formed NETs without entry of DEK
into the cytoplasm or the nucleus.
Example 12
[0189] This example describes H.sub.2O.sub.2 generation in mouse
bone marrow derived macrophages.
[0190] Bone marrow macrophages (BMM) from WT and DEK-KO were plated
on 96-well plates. Secretion of H.sub.2O.sub.2 was determined
colorimetrically using Amplex Red reagent (Molecular Probes,
Eugene, Oreg.) according to the instructions of the manufacturer
and as previously described (Serezani, C. H., Aronoff, D. M.,
Jancar, S., Mancuso, P., and Peters-Golden, M. (2005). Leukotrienes
enhance the bactericidal activity of alveolar macrophages against
Klebsiella pneumoniae through the activation of NADPH oxidase.
Blood 106, 1067-1075.). Briefly, a solution containing 50
.upsilon.M Amplex Red reagent and 10 U/mL HRP was prepared in PBS,
and 0.1 mL of the suspension was added to BMM cultures
(5.times.10.sup.5 per well). Cells were incubated at 37.degree. C.
for 60 min. H.sub.2O.sub.2 concentrations in the culture media were
determined using a standard curve generated with known
H.sub.2O.sub.2 concentrations with a detection limit of 0.625 nM.
Samples were measured at A560 nM wavelength using a Tecan GENios
plate reader (Phenix, Australia).
[0191] The observed defect in NET formation in the absence of DEK
was not explained by a reactive oxygen species (ROS) driven
mechanism; no difference in H.sub.2O.sub.2 generation was noted
between DEK-KO and WT mice (FIG. 14A and FIG. 14B). FIG. 14 shows
that peripheral and bone marrow neutrophils from DEK-KO mice
express ROS to the same extent as do those from WT mice before and
after PMA stimulation. FIG. 14A bone marrow and FIG. 14B peripheral
blood neutrophils obtained from DEK-KO and WT mice were incubated
with or without PMA (1 ng/ml) prior to determining H.sub.2O.sub.2
concentration in the supernatants. Data shown represent 2
independent experiments performed in triplicate.
Example 13
[0192] This example describes inflammatory cytokines within mouse
knee homogenates.
[0193] Knees were skinned prior to freezing at -80.degree. C.
Frozen knees were homogenized in 0.5 ml of cold PBS, and then
centrifuged at 14,000.times.g for 10 min at 4.degree. C.
Supernatants were collected and analyzed for protein concentration
and for levels of mouse IL-1.alpha., IL-1.beta., TNF-.alpha.,
IL-12p70, IL-12 p40, IL-23, RANTES (Regulated on Activation Normal
T cell Expressed and Secreted), MIP-2, IL-10, MCP-1, IFN.gamma.,
and TGF.beta. using ELISA. Splenocytes were purified as previously
described (Zhang, M., Berndt, B. E., Chen, J. J., and Kao, J. Y.
Expression of a soluble TGF-beta receptor by tumor cells enhances
dendritic cell/tumor fusion vaccine efficacy. J Immunol 181,
3690-3697, 2008.), and TLR2 was detected by monoclonal
anti-TLR2-FITC (Imegenex, IMG-6320C). Ficoll-purified bone marrow
of WT control mice and DEK-KO mice or whole blood samples were
resuspended in 1% BSA and 1% horse serum in PBS. Samples were spun
at 1600 rpm for 5 min at 4.degree. C. Cell pellets were resuspended
with anti-Ly6G-FITC and or anti-CD11b--PE-Cy5 (BD Pharmingen,
#553312) antibodies and incubated on ice for 30 min.
Isotype-matched IgGs were used as negative control antibodies.
Samples were centrifuged at 1600 rpm for 5 min at 4.degree. C. and
fixed with 2% paraformaldehyde. Cell surface markers were analyzed
by FACS. cDNA was prepared from WT and DEK-KO knee joint tissue
that was collected 24 hours after zymosan injection. TLR2 levels
were assessed as part of a cytokine PCR array (SABiosciences
QIAGEN, # MCA, CA).
[0194] In DEK-KO mice, levels of interleukin-1.alpha.
(IL-1.alpha.), tumor necrosis factor-.alpha. (TNF-.alpha.),
IL-12p40, IL-12p70, IL-23, and regulated on activation normal T
cell expressed and secreted (RANTES) were all significantly reduced
in knee homogenates from DEK-KO mice 24 hours post-injection as
compared to WT counterparts (FIG. 8D). In contrast, other
inflammatory cytokines did not display significant differences
(FIG. 15A). FIG. 15 shows that no significant differences in
expression of certain pro-inflammatory cytokines and TLR2 between
DEK-KO vs. WT mice were observed. FIG. 15A shows no significant
difference in IL-6, IL-1.beta., MIP-2, IL-10, IFN-.gamma.,
TGF-.beta. and MCP-1 levels in knee homogenates of WT and DEK-KO
mice after zymosan injection. Cytokine levels were analyzed by
ELISA and normalized by protein concentration.
[0195] To test if the difference in inflammatory responses between
WT and DEK-KO mice is due to differences in cell signaling, the
expression levels of the cell surface receptor for zymosan,
toll-like receptor 2 (TLR2) was analyzed. TLR2 mRNA and protein
levels in knee homogenates and cells purified from WT spleens did
not differ from expression detected in DEK-KO mice (FIG. 15B). FIG.
15B shows that TLR2 levels of expression are similar in DEK-KO and
WT mice. cDNA was prepared from WT or DEK-KO zymosan-injected knees
and qPCR was used to determine TLR2 RNA levels, which showed no
significant differences (left panel). TLR2 levels were also
measured by flow cytometry of cells isolated from naive WT and
DEK-KO spleens (right panel). Low levels of TLR2 were detected, but
no difference in TLR2 expression was observed consistent with the
RNA data shown in the left panel. Moreover, NF-.kappa.B, a
regulator of inflammatory cytokines, was not differentially
regulated in DEK-KO vs. WT bone marrow macrophages and neutrophils
upon in vitro stimulation with LPS or zymosan (FIG. 16). FIG. 16
shows that bone marrow-derived macrophages collected from WT and
DEK-KO mice were stimulated with 1 .mu.g/ml LPS or Zymosan for the
indicated times. Levels of phosphorylated I.kappa.B (p I.kappa.B)
were analyzed in the cytosolic fraction. DEK-KO mice develop
significantly less inflammation compared to WT mice in the setting
of ZIA as measured by marked changes in joint circumference and
cytokine profile, and a decrease in neutrophil migration. These
effects are not due to differences in neutrophil development or
cell signaling events.
[0196] These results demonstrate that homogenates of
zymosan-injected joints from DEK-KO vs. WT mice had significantly
lower levels of inflammatory cytokines such as IL-1.alpha.,
TNF-.alpha., and RANTES, the latter of which can be produced by
T-cells in response to TNF-.alpha. and IL-1-.alpha.. IL-1.alpha.
and TNF-.alpha., inflammatory cytokines that have been targeted
successfully in the treatment of JIA, rheumatoid arthritis, and
other autoimmune diseases (Nash, P. T. & Florin, T. H. Tumour
necrosis factor inhibitors. Med J Aust 183, 205-208,
doi:nas10250_fm [pii] (2005), Lovell, D. J., Bowyer, S. L. &
Solinger, A. M. Interleukin-1 blockade by anakinra improves
clinical symptoms in patients with neonatal-onset multisystem
inflammatory disease. Arthritis Rheum 52, 1283-1286,
doi:10.1002/art.20953 (2005) are produced in greater abundance in
WT as compared to DEK-KO mice. The lower levels of IL-12p40,
IL-12p70, and IL-23 in the joints of DEK-KO vs. DEK WT mice
injected with zymosan indicates that DEK has an effect on T-cell
response. [0197] These results demonstrate that DEK regulates the
production of both inflammatory cytokines and NETs. (Keshari, R. S.
et al. Cytokines induced neutrophil extracellular traps formation:
implication for the inflammatory disease condition. PLoS One 7,
e48111, doi:10.1371/journal.pone.0048111 (2012).)
[0198] These results demonstrate that depletion of DEK confers
protection against arthrities in a murine model of inflammatory
arthrities with reduced levels of pro-inflammatory cytokines in the
knees of DEK-KO mice.
Example 14
[0199] This example describes immunohistochemistry in sections of
mouse joints.
[0200] Frozen sections of mouse joints were thawed rapidly and then
fixed in 2% paraformaldehyde/PBS (pH=7.4) for 12 min at room
temperature. Sections were washed twice for 2 min in PBS, and then
permeabilized in 0.05% Triton X-100 in PBS for 10 min at room
temperature. Sections were washed with PBS 3 times for 5 min each,
followed by blocking with 10% normal goat serum overnight at
4.degree. C. Sections were probed with rabbit anti-MPO (Dako
Denmark A0398) at dilution of 1:500, rat anti-Ly6C/Ly6G antibody
(BD Pharmingen 550327) at a dilution of 1:20 for 3 hours at room
temperature, or with custom-made rabbit anti-DEK antibody diluted
in 10% normal goat serum at a dilutions of 1:50, 1:100, or 1:200
overnight at 4.degree. C. After washing in PBS for 5 min.times.3,
sections were incubated with secondary antibody; goat anti-rat
IgG-AlexaFluor 594 (Invitrogen A-11007), at 1:200 for Ly6C/Ly6G or
goat anti-rabbit IgG-AlexaFluor 594 (Invitrogen A-11037) at 1:200
for DEK. Antibodies were diluted in 10% normal goat serum; sections
were incubated for 45 min at room temperature.
[0201] Immunostaining of non-permeabilized sections of the knees
from mice treated with SEQ ID NO: 6 compared to control aptamer for
myeloperoxidase (MPO), a known marker of NETs, revealed a reduction
in staining (FIG. 17A and FIG. 17B). Co-localization of MPO and
extracellular DAPI staining further indicates that the reduction in
MPO staining reflects a reduction in extracellular NETs in the SEQ
ID NO: 6-treated mice. Combining all the Z-stacks to a 3-D imaging
of the knee section further demonstrates the presence of
extracellular DNA colocalizing with MPO, supporting the presence of
NETs in joints injected with zymosan and treated with control
aptamer control, but not with the anti-DEK aptamer (FIG. 17C).
Example 15
[0202] This example describes DEK protein in synovial fluid (SF) of
patients with juvenile idiopathic arthritis (JIA).
[0203] SFs were obtained from JIA patients during therapeutic
arthrocentesis by medical staff of the Pediatric Rheumatology
division at the University of Michigan. SFs were diluted 1:1 with
PBS followed by separation on Histopaque-1077. Neutrophils were
plated on cover slips as described above without additional
stimulation. Neutrophils were purified from synovial fluids (SFs)
and subjected to immunohistochemical analysis using monoclonal
anti-DEK antibody (FIG. 5C, bottom).
[0204] Neutrophils purified from SFs of JIA patients are
significantly activated, such that they form NETs without
stimulation. The NETs demonstrated positive DEK staining, and
significant co-localization of DEK with elastase, LL-37, and DNA
(stained by Hoechst). Affinity-purified DEK autoantibodies isolated
from the SFs of JIA patients that show specific recognition of DEK
(Ng, E. W. et al. Pegaptanib, a targeted anti-VEGF aptamer for
ocular vascular disease. Nat Rev Drug Discov 5, 123-132,
doi:10.1038/nrd1955 (2006.) recognized NETs formed by synovial
neutrophils (FIG. 5D).
Example 16
[0205] This example describes purification of DEK antibodies from
human synovial fluid (SF).
[0206] SulfoLink Coupling Gel (Pierce Biotechnology, Rockford,
Ill., USA) was used to couple the recombinant human DEK protein and
purify DEK antibodies. 100 .mu.g of the DEK protein was dissolved
in 500 .mu.l of coupling buffer [(1.times. NET buffer: 50 mM Tris,
150 mM NaCl, 5 mM EDTA, pH 8.5)] coupled to 500 .mu.l of washed
SulfoLink gel (in a 10 ml chromatography column) by mixing with the
resin for 20 min followed by 40 min incubation. Non-specific
binding sites were blocked with 50 mM cysteine by mixing for 15 min
followed by 30 min incubation. The column was washed with 16.times.
column volume of 1 M NaCl followed by another wash with distilled
water. The column was equilibrated with 1.times. NET buffer prior
to the affinity purification step. All steps were performed at room
temperature.
[0207] Human synovial fluids were adjusted to 10 mM Tris, pH 8.0
and centrifuged to remove any precipitates. A total of 3 to 5 ml of
synovial fluids was used to purify DEK antibodies on the prepared
column. The column was mixed and then rotated for at least 4 h (up
to 16 hours) at room temperature. The column was washed with
10.times. column volume of 1.times. NET buffer+0.5 M NaCl+0.5%
NP-40 followed by a wash with 1.times. NET buffer+0.5% NP-40,
another wash of 1.times. NET buffer and a final wash of 0.1.times.
NET buffer. Antibody was eluted with 0.1 M glycine (pH 3.0) and
neutralized with 1M Tris (pH 8.0). The antibodies are highly
specific for recombinant DEK (Ng, E. W. et al. Pegaptanib, a
targeted anti-VEGF aptamer for ocular vascular disease. Nat Rev
Drug Discov 5, 123-132, doi:10.1038/nrd1955 (2006.).
[0208] These results demonstrate that NETs are formed spontaneously
bye JIA synovial neutrophils.
Example 17
[0209] This example describes extracellular and intracellular
DEK.
[0210] DEK participates in global heterochromatin integrity within
the nucleus (Kappes, F. et al. The DEK oncoprotein is a Su(var)
that is essential to heterochromatin integrity. Genes Dev 25,
673-678, doi:10.1101/gad.2036411 25/7/673 [pii] (2011).) Since NETs
are chromatin-containing structures, DEK may affect chromatin
structure and hence NET formation by one of two basic mechanisms:
(1) DEK is known to modulate intranuclear chromatin structure, and
in a number of different cell types recombinant DEK is taken up by
cells and can go directly to the nucleus and affect chromatin
structure and cell function (Saha, A. K. et al. Intercellular
trafficking of the nuclear oncoprotein DEK. Proc Natl Acad Sci USA
110, 6847-6852, doi:10.1073/pnas.1220751110 1220751110 [pii]
(2013).), in this capacity, DEK may participate in the early events
of NET formation; (2) DEK may affect NET formation in the cytoplasm
or extracellular space. To test if extracellular rDEK enters the
nucleus of DEK-KO neutrophils to restore NET formation, recombinant
DEK was added and the nuclear envelope was stained with Lamin B. As
shown in FIG. 11E, recombinant DEK does not enter the nucleus of
the neutrophil, but associates with the NET structures. In
addition, the cell was stained with wheat germ agglutinin (WGA), a
cytoplasm marker, and again recombinant DEK added to neutrophils
does not enter the cell, and is found primarily mainly in the
extracellular space (FIG. 18). FIG. 18 shows DEK-KO neutrophils
treated with recombinant DEK prior to PMA stimulation. Cells were
fixed at FIG. 18A 2 hours, FIG. 18B 3 hours and FIG. 18C 4 hours of
PMA treatment, and then permeabilized and stained for DEK and wheat
germ agglutinin (WGA), a cytoplasm marker. DEK is detected outside
of the cell in the NETs. Accordingly, DEK does not promote NET
formation from within the cell, but acts as a component of NET
architecture in the extracellular space. Denaturation of
recombinant DEK protein prior to addition to DEK-KO neutrophils
prevented restoration of NET formation (FIG. 19). FIG. 19 shows
that bioactive DEK is needed to restore NET formation by DEK-KO
neutrophils. DEK-KO neutrophils were treated with boiled
recombinant DEK (middle panel) or native recombinant DEK (lower
panel) prior to PMA stimulation. Cells were fixed and stained for
DEK and Lamin B. NETs were observed only with the native
recombinant DEK, further confirming the specific role of
biologically competent DEK in NET biology. In aggregate, these
findings indicate that bioactive DEK plays a central role in NET
formation through its effects on the extracellular chromatin
component of these structures. These results indicate that
neutrophils release DEK into the extracellular space, and that DEK
participates in extracellular structures that regulate innate
immunity and generation of autoantibodies.
EQUIVALENTS
[0211] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes that come within the meaning and range of equivalency of
the claims are intended to be embraced therein.
INCORPORATION BY REFERENCE
[0212] All publications, published patent documents, and patent
applications cited herein are hereby incorporated by reference to
the same extent as though each individual publication, published
patent document, or patent application was specifically and
individually indicated as being incorporated by reference.
Sequence CWU 1
1
6185DNAArtificial sequenceSynthetic 1atagggagtc gaccgaccag
aaggggttaa atattcccac attgcctgcg ccagtacaaa 60tagtatgtgc gtctacatct
agact 85285DNAArtificial sequenceSynthetic 2atagggagtc gaccgaccag
aataccgtgg catctggttg tagcatcacg tcttatgcgg 60ccgtatgtgc gtctacatct
agact 85386DNAArtificial sequenceSyntheticmisc_feature(22)..(22)n
is a, c, g, or tmisc_feature(23)..(77)n is a, c, g, t or u
3ataggagtcg accgaccaga annnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
60ntatgtgcgt ctacatctag actcat 86421DNAArtificial sequenceSynthetic
4ataggagtcg accgaccaga a 21525DNAArtificial sequenceSynthetic
5atgagtctag atgtagacgc acata 25641DNAArtificial sequenceSynthetic
6ggggttaaat attcccacat tgcctgcgcc agtacaaata g 41
* * * * *