Note: Descriptions are shown in the official language in which they were submitted.
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Oligonucleotide and use thereof
FIELD OF THE DISCLOSURE
The present invention relates to an oligonucleotide and a remedy for treating
immune-mediated disorders, using the oligonucleotide. The immune-mediated
disorder includes autoimmune disease, graft rejection, hypersensitivity,
diseases
associated with the over-stimulation of host's immune system by autoantigens,
microbes and Toll-like receptor (TLR)-mediated disease.
BACKGROUND ART
The present invention provides an oligonucleotide which has a nucleotide
sequence of 5'-cctectectectcctcctcctcct-3' (SEQ ID NO: 1) and a remedy for
treating
and/or preventing immune-mediated disorder, using the oligonuclotide.
The immune system protects human body from bacterial, parasitic, fungal, viral
infections, and from the growth of tumor cells. However, the immune response
can
sometimes be unwanted and cause immune-mediated disorder. The disorder
includes
autoimmune disease, graft rejection, hypersensitivity, diseases associated
with the
over-stimulation of host's immune system by microbes and Toll-like receptor
(TLR)-mediated disease.
The autoimmune diseases results from an adaptive immune response and innate
immune response or both against endogenous and/or exogenous antigens. Foreign
substances, derived from bacteria, parasites, fungi or viruses, may mimic self-
proteins
and stimulate the immune system to launch responses to a self-cell and tissue,
resulting in the diseases including but not limited to systemic lupus
erythematosus
SLE) and rheumatoid arthritis. The graft rejection is a consequence of organ
or
tissue transplantation caused by the immune response in the transplant
recipient (host)
to the transplanted organ/tissue. When a subject is transplanted with grafts
including
kidney, pancrea, heart, lung, bone marrow, cornea and skin, the subject can
launch an
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immune response (rejection) against the grafts. Hypersensitivity is an
inappropriate
immune response that has deleterious effects, resulting in significant tissue
damage or
even death. The hypersensitivity is divided into four types (e.g. Types I, II,
III and IV.
Disease associated with the over-stimulation of host's immune system by
microbes is
triggered by the infection of viruses such as flu viruses and other microbes.
In the case
of flu virus and Gram-negative bacterial infection, an excessive immune
response to
the invaders appears to be a fatal factor in patients. The response is
characterized by
the overproduction of cytokines. Studies of septic shock syndrome demonstrate
that
over production/aberrant production of cytokines can lead to rapid mortality
due to
cytokine-mediated lethal shock (Slifka MK, et al. J Mol Med. 2000; 78(2):74-
80).
Septic shock following gram-negative infection is a leading cause of mortality
in
critically ill patients. The exaggerated production of cytokines is known to
contribute
to sepsis characterized by cytokine-mediated lethal shock (Espat NJ, et al. J
Surg Res.
1995 Jul; 59 (1):153-8). Multiple organ dysfunction syndromes (MODS) are a
major
cause of morbidity and mortality in severe sepsis and shock. Cytokine-mediated
lethal
shock resulted from over-production of host cytokines is considered a main
mechanism leading to MODS (Wang H, et al. Am J Emerg Med. 2008 Jul; 26
(6):711-5). Toll-like receptor (TLR)-mediated disease is a disorder caused by
the
activation of Toll like receptors (TLRs). TLRs are a family of receptors that
recognize
microbe derived molecular structures (pathogen-associated molecular patterns
or
PAMPs). TLR expressing immune cells are activated upon binding of PAMPs. TLRs
recognize a range of pathogen-derived products and activated.
Lipopolysaccharide
(LPS) of bacteria recognized by TLR4, lipotechoic acid and diacylated
lipopeptides
by TLR2¨TLR6 dimmer, triacylated lipopeptides by TLR2¨TLR1 dimmer, CpG
containing oligonucleotide (CpG ODN) synthesized or derived from either
viruses or
bacteria by TLR9, bacterial flagellin by TLR5, zymosan by TLR2¨TLR6 dimmer, F
protein from respiratory syncytial virus (RSV) by TLR4, viral-derived
double-stranded RNA (dsRNA) and poly I:C, a synthetic analog of dsRNA by TLR3;
viral DNA by TLR9, single-stranded viral RNA (VSV and flu virus) by TLR7 and
TLR8 (Foo Y. Liew, et al. Nature Reviews Immunology. Vol 5, June 2005, 446-
458).
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In recent years, TLR activation has been connected to the pathogenesis of some
of
diseases including sepsis, dilated cardiomyopathy, diabetes, experimental
autoimmune
encephalomyelitis, systemic lupus erythematosus, atherosclerosis, asthma,
chronic
obstructive pulmonary disease and organ failure (Foo Y. Liew, et al. Nature
Review
Immunology, Vol 5, 2005, 446-458). Activation of TLR9 by self DNA play an
important role in the development of autoimmune diseases such as SLE
(Christensen
SR, et al. Immunity 2006; 25:417-28) and rheumatoid arthritis (Leadbetter EA,
et al.
Nature 2002; 416:603-7; Boule MW, etal. J Exp Med 2004; 199:1631-40).
Moreover,
the elevated production of interferons (IFNs) resulted from TLR-9 activation
has been
reported to contribute to the develop of systemic lupus erythematosus (Barrat
FJ, et al.
J Exp Med 2005; 202:1131-9; Wellmann U, et al. Proc Natl Acad Sci USA 2005;
102:9258-63).
SUMMARY OF INVENTION
In this invention, we disclose an oligonucleotide with a nucleotide sequence
of
5'-cctcctcctcctcctcctcctcct-3' that inhibits proliferation of human PBMC
activated by
TLR9 agonist, inhibits interferon production from human PBMC induced by TLR9
agonist, HSV-1, flu virus and serum from SLE patients, and rescues the mice
from
cytokine induced shock. Therefore, this oligonucleotide is useful as a remedy
for the
treatment immune-mediated disorders.
The oligonuleotides of the invention inhibits TLR9 activation. It has been
documented that TLR9 agonist activates both innate and adaptive immune
response
(Arthur M. Krieg. Nature Reviews Drug Discovery, Vol 5. June 2006, 471-484).
CpG
containing oligonucleotides (CpG ODN) is a TLR9 agonist [D.M. Klinman, Nat.
Rev.,
Immunol. 4 (2004) 249¨ 2581. The oligonuleotides of the invention inhibits the
proliferation and interferon production of human PBMC stimulated by CpG ODN,
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indicating that the oligonucleotide of the invention can be used as a remedy
for the
treatment of diseases related to TLR9 activation. Because TLR9 activation has
been
reported to contribute to the development of SLE (Barrat FJ, et al. J Exp Med
2005;
202:1131-9; Wellmann U, et al. Proc Nat! Acad Sci USA 2005; 102:9258-63;
Christensen SR, et al. Immunity 2006; 25:417-28) and rheumatoid arthritis
(Leadbetter EA, et al. Nature 2002; 416:603-7; Boule MW, et al. J Exp Med
2004;
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199:1631-40), the oligonucleotide of the invention can be used as a remedy for
the
treatment of SLE and rheumatoid arthritis by inhibiting the TLR9 activation.
The oligonuleotide of the invention inhibits the interferon production from
human PBMC induced by TLR9 agonist, HSV-1, flu virus and serum from SLE
patients. Because the elevated production of interferons has been reported to
contribute to the development of SLE (Barrat FJ, et al. J Exp Med 2005;
202:1131-9;
Wellmann U, et al. Proc Nat! Acad Sci USA 2005; 102:9258-63), the
oligonucleotide
of the invention can be used as a remedy for the treatment of SLE by
inhibiting IFN
production.
The oligonuleotides of the invention inhibits the interferon production from
human PBMC induced by flu virus (PR8). Since influenza virus has been
documented
to be able to activate TLR7 and TLR8 (Wang JP, et al. Blood. 2008 Jun 10.
[Epub
ahead of print]), the oligonucleotide of the invention can be used as a remedy
for the
treatment of Toll-like receptor (TLR)-mediated disease by inhibiting TLR7 or
TLR8.
The oligonuleotides of the invention inhibits the interferon production from
human PBMC induced by HSV-1. Since HSV-1 has been documented to activate
TLR9 (Hubertus Hochrein et al. PNAS, 101, 11416-11421), the oligonucleotide of
the invention can be used as a remedy for the treatment of Toll-like receptor
(TLR)-mediated diseases including but not limited to SLE by inhibiting the
activation
of TLR9.
To study in vivo activity of the oligonucleotide of the invention, a mouse
model
of cytokine-mediated lethal shock was used. The mouse model was created by
injecting CpG ODN into the D-galactosamine (D-Gal) presensentised mice. After
being crated, the model mice died within 12 to 24 h. Analyses of plasma
cytokines
revealed over-production of tumor necrosis factor (TNF) alpha and interleukin-
12
(IL-12) and gamma interferon (IFN-gamma) (Marshall AJ, et al. Infect Immun.
1998
Apr; 66(4):1325-33; Peter M, Bode K,et al. Immunology. 2008 Jan;123(1):118-
28).
By using the model, we demonstrate that the oligonucleotide of the invention
can
rescue mice from cytokine-mediated lethal shock. Because the cytokine-mediated
lethal shock contributes to the septic shock (Slifka Wet al. J Mol Med.
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2000;78(2); Espat NJ, etal. J Surg Res. 1995 Jul;59(1):153-8) and multiple
organ dysfunction
syndromes (MODS) (Wang H, et al. Am J Emerg Med. 2008 Jul;26(6):711-5), the
oligonucleotide
of the invention can be used as a remedy for the treatment of sepsis and MUGS
by rescuing the host
from cytokine-mediated lethal shock.
Thus, in one aspect, the present invention provides an oligonucleotide
consisting of the
sequence 5'-cctectectectectectectect-3' (SEQ ID NO: 1), for use in the
treatment of an immune-
mediated disorder in a subject, the immune-mediated disorder resulting from
the activation of a
Toll-like receptor (TLR) selected from the group consisting of TLR7, TLR8 and
TLR9.
In another aspect, the present invention provides use of an oligonucleotide
consisting of the
sequence 5'-cctectectectectectectect-3' (SEQ ID NO: 1), for treating an immune-
mediated disorder
in a subject, the immune-mediated disorder resulting from the activation of a
Toll-like receptor
(TLR) selected from the group consisting of TLR7, TLR8 and TLR9.
In another aspect, the present invention provides use of an oligonucleotide
consisting of the
sequence 5'-cctectectectectectectect-3' (SEQ ID NO: 1), for preparation of a
pharmaceutical
composition for treating an immune-mediated disorder in a subject, the immune-
mediated disorder
resulting from the activation of a Toll-like receptor (TLR) selected from the
group consisting of
TLR7, TLR8 and TLR9.
In another aspect, the present invention provides a pharmaceutical comprising
the
oligonucleotide of the invention and a pharmaceutically acceptable carrier,
for use in the treatment
of an immune-mediated disorder in a subject, the immune-mediated disorder
resulting from the
activation of a Toll-like receptor (TLR) selected from the group consisting of
TLR7, TLR8 and
TLR9.
DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise noted, all terms in the invention have the same meaning as
commonly
understood by one of ordinary skill in the art to which this disclosure
belongs. The singular terms
"a," "an," and "the" include plural referents unless context indicates
otherwise. Similarly, the word
"or" is intended to include "and" unless the context indicates otherwise. In
case of conflict, the
present specification, including
CA 02638176 2014-08-19
explanations of terms, will control. In addition, the materials, methods and
examples
are illustrative only and not intended to be limiting. Treat, treating or
treatment shall
have the same meaning without concerning the grammar. Similarly, prevent,
preventing or prevention shall have the same meaning without concerning the
grammar.
"Oligonucleotide": An oligonucleotide means multiple nucleotides (i.e.
molecules comprising a sugar (e.g. deoxyribose) linked to a phosphate group
and to
an exchangeable organic base, which is either a substituted pyrimidine (Py)
(e.g.,
cytosine (C), thymine (T)) or a substituted purine (Pu) (e.g., adenine (A) or
guanine
(G)). The term oligonucleotide as used herein refers to
oligodeoxyribonucleotide
(ODN). The oligonucleotide can be obtained from existing nucleic acid sources
(e.g.,
genomic or cDNA), but are preferably synthetic. The oligonucleotide of the
invention
can be synthesized by a variety of automated nucleic acid synthesizers
available in the
market. These oligonucleotides are referred to as synthetic oligonucleotides.
"Chemical modification": The oligonucleotide disclosed in the invention can
encompass various chemical modifications, in comparison to natural DNA,
involving
a phosphodiester intemucleoside bridge, a ribose unit and/or a natural
nucleoside base
(adenine, guanine, cytosine, thymine). The modifications can occur either
during or
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after synthesis of the oligonucleotide. During the synthesis, modified bases
can be
incorporated internally or on its end. After the synthesis, the modification
can be
carried out using the active groups (via an amino modifier, via the 3' or 5'
hydroxyl
groups, or via the phosphate group). The skilled person knows examples of
chemical
modifications. An oligonucleotide according to the invention may have one or
more
modifications, wherein each modification is located at a particular
phosphodiester
intemucleoside bridge and/or at a particular ribose unit and/or at a
particular natural
nucleoside base position in comparison to an oligonucleotide of the same
sequence,
which is composed of natural DNA. The chemical modification includes "back
bone
modification" of the oligonucleotide of the invention. As used herein, the
modified
back bone of the oligonucleotide of the invention includes, but not limited to
the
"phosphorothioate backbone" that refers to a stabilized sugar phosphate
backbone of a
nucleic acid molecule in which a non-bridging phosphate oxygen is replaced by
sulfur
at least one intemucleotide linkage. In one embodiment a non-bridging
phosphate
oxygen is replaced by sulfur at each and every intemucleotide linkage. Other
back
bone modifications denote the modification with nonionic DNA analogs, such as
alkyl-and aryl-phophonates (in which the charged phosphonate oxygen is
replaced by
an alkyl or aryl group), phophodiester and alkylphosphotriesters, in which the
charged
oxygen moiety is alkylated. In other examples, the oligonucleotide can be is a
phosphorothioate/phosphodiester chimera. The chemical modification also
includes
the base substitutions of the oligonucleotide disclosed in the invention. The
substituted purines and pyrimidines can be C-5 propyne pyrimidine and
7-deaza-7-substituted purine. The substituted purines and pyrimidines include
but are
not limited to adenine, cytosine, guanine, and thymine, and other naturally
and
non-naturally occurring nucleobases. The chemical modification of the
oligonucleotide of the invention further includes the modification of the
bases of the
oligonucleotide. A modified base is any base which is chemically distinct from
the
naturally occurring bases typically found in DNA such as T, C, G and A, but
which
share basic chemical structures with these naturally occurring bases. The
oligonucleotide of the invention can be modified by using cytidine
derivatives. The
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term "cytidine derivative" refers to a cytidine-like nucleotide (excluding
cytidine) and
the term "thymidine derivative" refers to a thymidine-like nucleotide
(excluding
thymidine). In addition, the oligonucleotides of the invention can be
chemically
modified by linking a diol, such as tetraethyleneglycol or hexaethyleneglycol,
at
either or both termini of the oligonuleotide.
"Immune-mediated disorder": An immune-mediated disorder is a disease caused
by an unwanted immune response in a subject. The disorder includes autoimmune
disease, graft rejection, hypersensitivity, diseases associated with the over-
stimulation
of host's immune system by microbes and diseases associated with TLR
activation.
The oligonucleotide disclosed in the invention can be used as a remedy to
treat the
immune-mediated disorder.
"Immune response": A response of a cells of the immune system, such as a B
cell,
T cell, natural killer cell, dendritic cell, neutraphil and macrophage to a
stimulus. The
response includes innate immune response and adaptive (specific or acquired)
immune response. The adaptive (specific or acquired) immune response includes
humoral immune response and cellular immune response.
"Prevent or treat immune-mediated disorder": As used herein, prevent refers to
prevent the full development of an immune-mediated disorder in a subject;
treat refer
a therapeutic intervene in a subject so as to ameliorate a sign or symptom of,
halt the
progression of, or eliminate pathological condition of the immune-mediated
disorder.
"Subject": As used herein, a subject refers to a human or non-human
vertebrate.
Non-human vertebrates are non-human primates, livestock animals and companion
animals. The oligonucleotide of the invention can be administered to prevent
or/and
treat immune-mediated disorder in a subject.
"Autoimmune diseases": The term "autoimmune disease" refers to a disease
caused by a breakdown of self-tolerance such that the adaptive and innate
immune
system responds to self antigens and mediates cell and tissue damage.
Autoimmune
diseases are frequently characterized by means of their involvement of single
organ or
single cell-types or involvement of multiple organs or tissue systems.
Autoimmune
diseases have also been referred to as "collagen," or "collagen-vascular" or
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"connective tissue" diseases. Autoimmune disorders are frequently associated
with
hypersensitivity reactions. The oligonucleotides of the invention can be
useful for
treating and/or preventing various types of autoimmune diseases. Specific,
non-limiting examples of autoimmune disorders are systemic lupus
erythematosus,
insulin-dependent (type I) diabetes mellitus, inflammatory arthritis,
rheumatoid
arthritis, multiple sclerosis, autoimmune hepatitis, chronic aggressive
hepatitis,
autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmune atrophic
gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune
orchitis,
acquired hemophilia, ankylosing spondylitis, antiphospholipid syndrome,
Beh.cedilla.et's syndrome, cardiomyopathy, chronic inflammatory demyelinating
polyneuropathy, cicatricial pemphigoid, cold
agglutinin disease,
polymyositisdermatomyositis, discoid lupus, sympathetic ophthalmia, essential
mixed
cryoglobulinemia, fibromyalgia, fibromyositis, Guillain-Barr syndrome,
idiopathic
pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy,
juvenile
arthritis, systemic sclerosis, polyarteritis nodosa, polychondritis,
dermatomyositis,
primary agammaglobulinemia, primary biliary cirrhosis, hyperimmunoglobulin E,
progressive systemic sclerosis, psoriasis, Reiter's syndrome, sarcoidosis,
stiff-man
syndrome, uveitis, vasculitis, vitiligo, Hashimoto's thyroiditis, Goopasture's
disease,
pernicious anemia, Addison's disease, dermatomyositis, Sjogren's syndrome,
dermatomyositis, myasthenia gravis, Grave's disease, uveitis, allergic
encephalomyelitis, glomerulonepluitis, and the like (N Engl J Med, Vol. 345,
No. 5,
August 2, 2001, p340-350). DNA or RNA released from DNA- or RNA-containing
microbes could stimulate the production of autoantibody specific to self RNA-
or
DNA-containing complexes and consequently led to an autoimmune disease,
including but not limited to SLE.
"Hypersensitivity": A hypersensitivity is referred to the disorders wherein
tissue
injury occurs as a result of a humoral or cell-mediated response to antigens
of
endogenous or exogenous origin and has been classified into four types. Type I
hypersensitivity (frequently referred to as anaphylactic, immediate-type,
atopic,
reagenic, IgE-mediated hypersensitivity reactions or allergy) generally result
from the
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release of pharmacologically active substances such as histamine, slow-
reacting
substance of anaphylaxis (SRS-A), and eosinophilic chemotactic factor (ECF)
form
IgE-sensitized basophils and mast cells after contact with a specific
exogenous
antigen. Type I hypersensitivity includes, but not limited to, allergic
extrinsic asthma,
seasonal allergic rhinitis and systemic anaphylaxis. Type II hypersensitivity
(also
referred to as cytotoxic, cytolytic complement-dependent or cell-stimulating
hypersensitivity reaction) results when antibody reacts with antigenic
components of
cells or tissue elements or with an antigen or hapten, which has become
intimately
coupled to cells or tissue. Type II hypersensitivity includes, but not limited
to,
autoimmune hemolytic anemia, erythroblastosis fetalis and Goodpasture's
disease.
Type III hypersensitivity (also referred to as toxic complex, soluble complex,
or
immune complex hypersensitivity reactions) results from the deposition of
soluble
circulating antigen-antibody complexes in vessels or in tissues, with
accompanying
acute inflammatory reactions at the site of immune complex deposition. Type
III
hypersensitivity includes, but not limited to, Arthurs reaction, serum
sickness,
systemic lupus erythematosis, and certain types of glomerulonephritis. Type IV
hypersensitivivity (frequently called cellular, cell-mediated, delayed, or
tuberculin-type hypersensitivity reactions) are caused by sensitized T-
lymphocytes
which result from contact with a specific antigen. Type IV hypersensitivity
includes,
but not limited to, contact dermatitis and allograft rejection (Richard A. et
al.
Immunology, Fifth Edition, 2003, W.H. FREEMAN AND COMPANY).
"Diseases associated with the over-stimulation of host's immune system by
microbes": Microbe invasion, if severe, sometimes can cause systemic
inflammatory
response in a subject, leading to diseases associated with the over-
stimulation of
host's immune system by microbes. The events in the development of the
diseases,
such as in the case of influenza A (H5N1) or bacterial infection, include the
significantly elevated blood levels of tumor necrosis factor (TNF-a),
interleulcin 1
(IL-1), IL-6, IL-12, interferon a (IFN-a), interferon 13 (IFN-I3), interferon
7,
chemokines interferon-inducible protein 10, monocyte chemoattractant protein
1,
interleukin-8, interleukin-1 p, and monocyte chemoattractant protein 1. Such
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responses can result in cytokine-mediated lethal shock that is responsible in
part for
the sepsis, ARDS, and multiorgan failure observed in many patients (The
Writing
Committee of the World Health Organization (WHO) Consultation on Human
Influenza A/115. Avian Influenza A (H5N1) Infection in Humans. N Engl J Med
2005;
353:1374-85). The significantly elevated blood level of cytokines followed
microbe
infection is termed by hypercytokinemia (hypercytokinaemia) or a cytokine
storm.
The research suggested that patients who contract bird flu or SARS may need
drugs
that suppress the immune response in addition to anti-viral drugs. The
oligonucleotide
of the invention can be used to treat and/or prevent the diseases associated
with the
stimulation of host's immune system by microbes in a subject. The microbes
causing
the diseases includes, but not limited to, viruses, bacteria, fungi, parasites
and
etiological agents of Spongiforrn encephalopathies. The virus that cause the
diseases
associated with the over-stimulation of host's immune system by microbes
include:
SARS CoV, influenza viruses, avian flu virus HIV-1, polio viruses, hepatitis A
virus;
enteroviruses, human Coxsackie's viruses, rhinoviruses, echoviruses, equine
encephalitis viruses, rubella viruses, dengue viruses, encephalitis viruses,
yellow fever
viruses, corona viruses, vesicular stomatitis viruses, rabies viruses, Ebola
viruses,
parainfluenza viruses, mumps virus, measles virus, respiratory syncytial
virus,
influenza viruses, Hantan viruses, bunga viruses, phleboviruses, Nairo
viruses,
hemorrhagic fever viruses; reoviruses, orbiviurses and rotaviruses, Hepatitis
B virus,
parvoviruses, papilloma viruses, polyoma viruses, adenoviruses, herpes simplex
virus
(HSV) 1 and HSV-2, varicella zoster virus, cytomegalovinis (CMV), herpes
viruses,
variola viruses, vaccinia viruses, pox viruses, African swine fever virus, the
etiological agents of Spongiform encephalopathies, delta hepatitis virus,
Hepatitis C
virus, foot and mouth disease virus and avian flu virus. The bacteria that can
cause the
diseases associated with the over-stimulation of host's immune system by
microbes
include: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,
Mycobacteria sps (such as. M. tuberculosis, M. avium, M. E intracellulare, M.
kansaii,
M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria
meningitidis,
Listeria monocytogenes, Group A Streptococcus, Group B Streptococcus,
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Streptococcus, Streptococcusfaecalis, Streptococcus bovis, Streptococcus
(anaerobic
sps.), Streptococcus pneumoniae, pathogenic Carnpylobacter sp., Enterococcus
sp.,
Haemophilus infiuenzae, Bacillus antracis, corynebacteritun diphtheriae,
corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers,
Clostridium tetani, Enterobacter aerogeytes, Klebsiella pneumoniae, Pasturella
multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus
moniliformis,
Treponema pallidium, Treponema pertenue, Leptospira, and Actinomyces israelli.
The
fungi that can cause the diseases associated with the over-stimulation of
host's
immune system by microbes include, but not limited to, Cryptococcus
neoformans,
Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis,
Chlamydia
trachomatis, Candida albicans. The parasites that can cause the diseases
associated
with the over-stimulation of host's immune system by microbes include:
Plasmodium
falciparum and Toxoplasma gondii.
"Graft rejection": The graft rejection is an immune-mediated disorder caused
by
organ or tissue transplantation, Transplantation means the transfer of
transplants
(grafts) from a donor to a recipient. Grafts are the living cells, tissues, or
organs
transplanted from a donor to a recipient. An autograft is the a graft
transferred of one's
own tissue from one location to another; a syngeneic graft (isograft) is a
graft between
identical twins; an allogeneic graft (homograft) is a graft between
genetically
dissimilar members of the same species; and a xenogeneic graft (heterograft)
is a
transplant between members of different species. When a subject is the
recipient of an
allogeneic graft or a xenogeneic graft, the body can produce an immune
response
against the donor tissue. In this situation, there is a clear need to suppress
the immune
response, in order to avoid rejection of the graft (Richard A. et al.
Immunology, Fifth
Edition, 2003, W.H. FREEMAN AND COMPANY). The oligonucleotides of the
present invention are useful when administered for the prevention of the graft
rejection. Examples of the grafts are heart, kidney, liver, medulla ossium,
skin, cornea,
lung, pancreas, intestinum tenue, limb, muscle, nervus, duodenum, small-bowel,
pancreatic-islet-cell, and the like. In some case, the recipient may be an
animals as
defined in "subject"of the invention.
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"Toll-like receptor (TLR)-mediated diseases": A Toll-like receptor
(TLR)-mediated disease means an immune mediated disorder related to the
activation
of members of the TLR family. The disease includes, but not limited to, the
diseases
include but not limited to, sepsis associated with the activation of TLR4 by
= lipopolysaccharide (LPS), dilated cardiomyopathy associated with the
activation of
TLR2, 3, 4, 9, diabetes associated with the activation of TLR2,3,4,9,
experimental
autoimmune encephalomyelitis associated with the activation of TLR3, systemic
lupus erythematosus associated with the activation of TLR9, atherosclerosis
associated with the activation of TLR4, asthma associated with the activation
of TLR4
by LPS, chronic obstructive pulmonary disease associated with the activation
of
TLR4, EAE associated with the activation of TLR4 and organ failure associated
with
the activation of TLR4 (Foo Y. et al. Nature Review Immunology, Vol 5, 2005,
446-458). CpG-containing DNA (a TLR9 agonist) derived from a nucleic
acid-containing infectious agent could be identified from SLE serum that
induces an
efficient immune response dominated by IFN-a secretion that is thought to
contribute
the development of SLE. The oligonucleotides of the present invention can be
administered for treating and/or prevent the Toll-like receptor (TLR)-mediated
diseases including but not limited to SLE in a subject.
"CpG ODN": It has been documented that TLR9 agonist activates both innate
and adaptive immune response (Arthur M. Krieg. Nature Reviews Drug Discovery,
Vol 5. June 2006, 471-484). CpG containing oligonucleotides (CpG ODN) is a
TLR9
agonist [D.M. Klinman, Nat. Rev., Immunol. 4 (2004) 249¨ 258]. Based on the
functional characteristics, CpG ODNs are divided into three types (Tomoki Ito,
et al.
Blood, 2006, Vol 107, Num 6: 2423-2431). A-type CpG ODN activates human
plasmacytoid dendritic cells (pDCs) to produce large amount of type I
interferon
(IFN-a/13) and strongly activates natural killer cells (NK cells). B-type CpG
ODN
primarily activates B cells, resulting in their proliferation and antibody
secretion.
C-type CpG ODN shares the activities of both A- and B-type CpG ODN. As a TLR9
agonist, CpG ODN such as CpG 2216 or CpG 2006 or CpG C274 can be endocytosed
into a cellular compartment where they are exposed to and activate TLR9. In
pDC,
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TLR9 activation initiate a rapid innate immune response that is characterized
by the
secretion of pro-inflammatory cytokines [IL-6, tumor-necrosis factor-a
(TNFa)], the
secretion of type I interferon (IFN) and the secretion of secretion of IFN-
inducible
chemokines. Through both IFN-dependent and IFN-independent pathways, innate
immune cells including natural killer (NK) cells, monocytes and neutrophils
are
secondarily activated by the pDC. B cells activated through TLR9 have a
greatly
increased sensitivity to antigen stimulation and efficiently differentiate
into
antibody-secreting cells, and therefore contributing to the adaptive immune
response,
especially humoral immune response. pDC activated through TLR9 secrete IFNa,
which drives the migration and clustering of pDC to lymph nodes and other
secondary
lymphoid tissues where the pDC activates naive and memory T cells, assists the
cross-presentation of soluble protein antigens to CD8+ cytotoxic T lymphocyte
(CTL)
and promotes strong TH1 biased cellular CD4 and CD8 T-cell responses. Based on
the
above mentioned findings, it is obvious that the agents that antagonize the
activity of
CpG ODN can be used to treat or prevent the immune-mediated disorder by
inhibiting
both innate and adaptive immune response.
"Pharmaceutically acceptable carrier": A pharmaceutically acceptable carrier
denotes one or more solid or liquid filler, diluents or encapsulating
substances that are
suitable for administering the oligonucleotide of the invention to a subject.
The carrier
can be organic, inorganic, natural or synthetic. The carrier includes any and
all
solutions, diluents, solvents, dispersion media, liposome, emulsions,
coatings,
antibacterial and anti-fungal agents, isotonic and absorption delaying agents,
and any
other carrier suitable for administering the oligonucleotide of the invention
and their
use is well known in the art. The pharmaceutically acceptable carriers are
selected
depending on the particular mode of administration of the oligonucleotide. The
parenteral formulations usually comprise injectable fluids that include
pharmaceutically and physiologically acceptable fluids such as water,
physiological
saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a
vehicle. For
solid compositions (e. g., powder, pill, tablet, or capsule forms),
conventional
non-toxic solid carriers can include, for example, pharmaceutical grades of
mannitol,
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lactose, starch, or magnesium stearate. In addition to biologically-neutral
carriers,
pharmaceutical compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying agents,
preservatives,
and pH buffering agents and the like, for example sodium acetate or sorbitan
monolaurate.
"Therapeutically effective amount" : In order to treat or prevent an
immune-mediated disorder, a therapeutically effective amount of an
oligonucleotide
of the invention is administered to a subject. The "therapeutically effective
amount" of
one of the oligonucleotides means a sufficient amount of the oligonucleotide
used to
achieve a desired result of treating or preventing an immune-mediated disorder
in a
subject. The oligonucleotides of the present invention may be employed in pure
form
or in pharmaceutically acceptable carriers. Alternatively, the
oligonucleotides may be
administered as pharmaceutical compositions. The "amount" in the invention
shall
refer to a dose. The dose can be determined by standard techniques well known
to
those skilled in the art and can vary depending the factors including, but not
limited to
the size or/and overall health of the subject or the severity of the disease.
Introduction
of the oligonucleotide of the invention can be carried out as a single
treatment or over
a series of treatments. Subject doses of the oligonucleotide of the invention
for the
administration range from about 1 tig to 100 mg per administration. However,
doses
for the treatment of immune-mediated disorder may be used in a range of 10 to
1,000
times higher than the doses described above. The more preferable doses can be
adjusted to provide the optimum therapeutic effect by those skilled in the
art, for
example, by the attending physician within the scope of sound medical
judgment.
"Route of administration": For clinical use, the oligonucleotides of the
invention
can be administered alone or formulated in a pharmaceutical composition via
any
suitable route of administration that is effective to achieve the desired
therapeutic
result. The "route" of administering the oligonucleotide of the invention
shall mean
the enteral, parenteral and topical administration or inhalation. The enteral
routes of
administration of the oligonucleotide of the invention include oral, gastric,
intestinal,
and rectal. The parenteral route includes intravenous, intraperitoneal,
intramuscular,
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intrathecal, subcutaneous, local injection, vaginal, topical, nasal, mucosal,
and
pulmonary administration. The topical route of administration of the
oligonucleotide
of the invention denotes the application of the oligonucleotide externally to
the
epidermis, to the buccal cavity and into the ear, eye and nose.
"Pharmaceutical composition": A pharmaceutical composition shall mean the
composition comprising an therapeutically effective amount of the
oligonucleotide of
the invention with or without a pharmaceutically acceptable carrier. The
pharmaceutical compositions can comprise one or more oligonucleotides of the
invention. The composition includes but not limited to aqueous or saline
solutions,
particles, aerosols, pellets, granules, powders, tablets, coated tablets,
(micro) capsules,
suppositories, syrups, emulsions, suspensions, creams, drops and other
pharmaceutical compositions suitable for use in a variety of drug delivery
systems.
The compositions may be administered parenterally, orally, rectally,
intravaginally,
intraperitoneally, topically (in a dosage form as powders, ointments, gels,
drops or
transdermal patch), bucally, or as an oral or nasal spray. In all cases, the
composition
must be sterile and stable under the conditions of manufacture and storage and
preserved against the microbial contamination. Pharmaceutical compositions of
this
invention for parenteral injection comprise pharmaceutically-acceptable
sterile
aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as
well as
sterile powders for reconstitution into sterile injectable solutions or
dispersions just
prior to use. The oligonucleotide of the invention can be suspended in an
aqueous
carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to
about 8.0,
preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5Ø
The buffer
solution includes sodium citrate-citric acid and sodium phosphate-phosphoric
acid,
and sodium acetate-acetic acid buffers. For oral administration, the
composition will
be formulated with edible carriers to form powders tablets, pills, dragees,
capsules,
liquids, gels, syrups, slurries, suspensions and the like. For solid
compositions,
conventional non-toxic solid carriers can include pharmaceutical grades of
mannitol,
lactose, starch, or magnesium stearate. For buccal administration, the
composition
will be tablets or lozenges in conventional manner. For inhalation, the
composition
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will be an aerosol spray from pressurized packs or a nebulizer or a dry powder
and
can be selected by one of skill in the art. In some cases, in order to prolong
the effect
of the oligonucleotide of the invention, the oligonucleotides of the invention
are also
suitably administered by sustained-release systems. The oligonucleotide of the
invention can be used in a liquid suspension of crystalline or amorphous
material with
poor water solubility to slow the releasing of the oligonucleotide.
Alternatively,
delayed releasing of a parenterally administered drug form of the
oligonucleotide is
accomplished by dissolving or suspending the oligonucleotide in hydrophobic
materials (such as an acceptable oil vehicle). Injectable depot forms are made
by
entrapping the oligonucleotide in liposomes or microemulsions or other
biodegradable
semi-permeable polymer matrices such as polylactide-polyglycolide, poly
(orthoesters)
and poly (anhydrides).
"Active ingredients": The oligonucleotides of the invention can be used alone,
in
combination with themselves, in a pharmaceutically acceptable carrier, in
combination with one or more additional active ingredients. The administration
of the
oligonucleotide of the invention and additional active ingredients can be
sequential or
simultaneous. The active ingredients include non-steroidal anti-inflammatory
agents,
steroids, nonspecific immunosuppressive agent, biological response modifier,
chemical compound, small molecule, nucleic acid molecule and TLR antagonists.
The
active ingredients also denote the agents that suppress the immune activation
by
antagonizing chemochines, by inducing the generation of regulatory T cells
(CD4+CD25+ T cells), by inhibiting a complement, matrix metalloproteases and
nitric oxide synthase, by blocking costimulatory factors and by inhibiting the
signaling cascades in the immune cells. The non-steroidal anti-inflammatory
agents
include, but unlimited to, diclofenac, diflunisal, etodolac, flurbiprofen,
ibuprofen,
indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin,
piroxicam,
sulindac, tohnetin, celecoxib and rofecoxib. The steroids include, but
unlimited to,
cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone,
prednisone, and triamcinolone. A nonspecific immunosuppressive agent means the
agent used to prevent the development of immune-mediated disorder. The
nonspecific
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immunosuppressive agents include but not limited to cyclophosphamide,
cyclosporine,
methotrexate, steroids, FK506, tacrolimus, mycophenolic acid and sirolimus.
The
biological response modifier includes a recombinant interleukin-l¨receptor
antagonist
(Kineret or anakima), a soluble p75 TNF-a receptor-IgG1 fusion protein
(etanercept
or Enbrel), or a monoclonal antibody against TNF-a (infliximab or RemicadeX).
The
agents also include Interferon beta-1a, interleukin-10 and TGF13.
"Delivery vehicle": The oligonucleotides of the invention can be administered
in/with a delivery vehicle or in a form linked with a vehicle. The vehicle
includes, but
not limited to, sterol (e.g., cholesterol), cochleates, emulsomes, ISCOMs; a
lipid (e.g.,
a cationic lipid, anionic lipid), liposomes; ethylene glycol (PEG); live
bacterial
vectors (e.g., Salmonella, Escherichia coli, bacillus Calmette-Gurin,
Shigella,
Lactobacillus), live viral vectors (e.g., Vaccinia, adenovirus, Herpes
simplex),
virosomes, virus-like particles, microspheres, nucleic acid vaccines, polymers
(e.g.,
carboxymethylcellulose, chitosan), polymer rings and a targeting agent that
recognizes target cell by specific receptors.
"Pegylation": Pegylation is the process of covalent attachment of poly
(ethylene
glycol) polymer chains to another molecule, normally a drug or therapeutic
protein.
Pegylation is routinely achieved by incubation of a reactive derivative of PEG
with
the target agent. The pegylated agent can "mask" the agent from the host's
immune
system, increase the hydrodynamic size of the agent which prolongs its
circulatory
time. The oligonucleotides of the invention can be pegylated.
BRIEF SUMMARY OF SPECIFIC EMBODIMENTS
In the first embodiment, the present invention provides an oligonucleotide
with a
nucleotide sequence of 5'-cctectectectectectectect-3' (SEQ ID NO: 1) and the
oligonucleotides that fit the formula of (5' CCT 3') n.
In the second embodiment, the present invention provides a remedy for treating
immune-mediated disorder using the oligonucleotide of the invention. The
immune-mediated disorder includes autoimmune disease, graft rejection,
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hypersensitivity, diseases associated with the over-stimulation by of host's
immune
system by autoantigens, microbes and Toll-like receptor (TLR)-mediated
disease.
In the third embodiment, the present invention provides a remedy for treating
immune-mediated disorder using the oligonucleotides of the invention by
inhibiting
the TLR activation and IFN production induced by DNA virus, RNA virus, the
serum
from SLE patients, and by rescuing a subject from cytokine-mediated lethal
shock.
In the fourth embodiment, the present invention provides a remedy for treating
SLE, sepsis and multiple organ dysfunction syndromes in a subject using the
oligonucleotides of the invention.
In the fifth embodiment, the present invention provides a remedy for treating
immune-mediated disorder by administering the oligonucleotide of the invention
alone or with a pharmaceutically acceptable carrier to a subject through the
route of
enteral, parenteral and topical administration or inhalation.
In the sixth embodiment, the present invention provides a composition
comprising therapeutically effective amount of the oligonucleotides of the
invention
for the treatment of immune-mediated disorder.
In another embodiment, the present invention provides a remedy for the
treatment of immune-mediated disorder by administering the oligonucleotides of
the
invention alone or in combination with additional active ingredients.
In another embodiment, the present invention provides a remedy for the
treatment of immune-mediated disorder by administering the oligonucleotide of
the
invention in delivery vehicles.
Thereby, in a aspect, the present invention provides the oligonucleotide
includes a
sequence that fits the formula of (5' CCT 3') n, wherein 5' CCT 3' is a repeat
unit and
n is an integer from 2 to 50, preferably, it is 5'-cctectectectcctcctcctect-3'
(SEQ ID NO:
I).
In a preferable embodiment, the phosphate backbone of the oligonucleotide can
be
partly or completely phosphorothioate-modified, or unmodified.
In another preferable embodiment, the oligonucleotide can be developed into
their
derivatives by adding in one or several nucleotides to each end of the
oligonucleotide
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and by changing one or several bases in the oligonucleotide.
In another preferable embodiment, the oligonucleotide constitutes a part of
other
DNA molecules, plasmid or viral vectors.
In an even more preferable embodiment, the oligonucleotide can undergo
chemical modification.
In another aspect, the present invention provides use of above oligonuleotide
for
preparing a remedy for treating an immune-mediated disorder in a subject.
Preferably,
the subject is a human or non-human vertebrate.
In a preferable embodiment, the immune-mediated disorder is autoimmtme
disease, or hypersensitivity, or graft rejection, or disease associated with
the
over-stimulation of host's immune system by microbes or Toll-like receptor
(TLR)-mediated disease.
In a more preferable embodiment, the treatment of immune-mediated disorder is
carried out by a mechanism selected from a group comprising inhibiting the
proliferation of immune cells activated with Toll like receptor 9 agonist,
inhibiting the
activation of Toll like receptor 9, inhibiting interferon production and
rescuing a
subject from cytokine-mediated lethal shock.
Preferably, the immune-mediated disorder is systemic lupus erythematosus (SLE)
which is treated by inhibiting TLR9 activation and interferon production
induced by
TLR9 agonists, viruses and the serum of SLE patient, the immune-mediated
disorder
is sepsis which is treated by rescuing a subject from cytokine-mediated lethal
shock,
or the immune-mediated disorder is multiple organ dysfunction syndromes which
is
treated by rescuing a subject from cytokine-mediated lethal shock.
In another aspect, the present invention provides a remedy for administrating
to a
subject having or at risk of developing the immune-mediated disorder
comprising
above oligonucleotide.
Preferably, the remedy further comprises a pharmaceutically acceptable carrier
and/or additional active ingredients. And more preferably, the remedy is in a
form for
administrating through the route including the enteral, parenteral and topical
administration or inhalation.
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In a preferable embodiment, the oligonucleotide can be pegylated.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows graphs depicting the inhibition of SATO5f on the proliferation
of
human PBMC stimulated by CpG 2006 and CpG C274.
Figure 2 shows a graph depicting the inhibition of SATO5f on interferon
production from human PBMC stimulated by CpG C274.
Figure 3 shows graphs depicting the inhibition of SATO5f on interferon
production
from human PBMCs stimulated with herpes simplex virus-type 1 virus and flu
virus
(A) Comparison of the anti-viral activity between the interferon (IFN) induced
by
inactivated HSV-1 and recombinant human interferon (IFN)-a. 'IU' represents
the
international unit. (B) Inhibitory effect of SATO5F on IFN production from
human
PBMC (hPBMC) stimulated by inactivated HSV-1. (C) The dose effect of SATO5F
on IFN production from human PBMCs stimulated by HSV-1. (D) Comparison of the
anti-viral activity between the interferon (IFN) induced by inactivated PR8
and
recombinant human interferon (IFN)-a. (E) Inhibitory effect of SATO5F on IFN
production from hPBMC stimulated with inactivated PR8. (F) The dose effect of
SATO5F on IFN production from hPBMC stimulated with inactivated PR8.
Data from one representative experiment of three are shown. HSV-1 represents
herpes simplex virus-type 1 virus. PR8 represents flu virus (H1N1/PR8).
Figure 4 is a graph showing that SATO5F inhibits interferon production of from
hPBMCs stimulated with serum of SLE patients.
Figure 5 is a graph showing that SATO5F rescues mice from cytokine-mediated
lethal shock.
EXAMPLES
The invention will now be described in more detail in the following Examples.
But the invention is not limited to these Examples. In these Examples, herein,
experiments using commercially available kits and reagents were done according
to
attached protocols, unless otherwise stated. The skilled artisan will
appreciate that the
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oligonucleotides of the present invention can easily be applied to treat an
immune-mediated disorder. The present invention will now be demonstrated by
the
following non-limiting examples.
All reagents used to manipulate the oligonucleotides (ODNs) in the following
examples were pyrogen-free. The endotoxin in the ODN preparations was tested
by
using the Limulus amebocyte lysate assay (Associates of Cape Cod, Inc).Human
PBMCs (hPBMC), used in the following samples, were isolated from buffy coats
(The Blood Center of Jilin Province, China) by Ficoll¨Hypaque (Pharmacia)
density
gradient centrifugation (P. M. Daftarian et al., (1996): Journal of
Immunology, 157,
12-20). The cells were cultured in IMDM supplemented with 10% (v/v)
heat-inactivated fetal bovine serum (FBS; GIBCO) and antibiotics (100 IU of
penicillin/ml and 100 IU of streptomycin/m1) at 37 C in a 5% CO2 humidified
incubator. The viability of the cells was 95-99% as determined by trypan blue
exclusion.
Example 1
Effect of SATO5f on CpGODN induced proliferation of human PBMC
The oligonucleotides (ODNs) used in the example were synthesized in Sangon
Biotech Company (Shanghai, China) and were CpG2006
(5'tcgtcgttttgtegttttgtcgtt-3'), CpG C274 (5'-tcgtegaacgttcgagatgat 3'), A151
(51-ttagggttagggttagggttaggg-3') (Hidekazu Shirota, etal. The Journal of
Immunology,
2005, 174: 4579-4583), SATO5f (5'-cctectectectcctectectect-3') and Control ODN
(5'-gttagagattaggca-3` ).
Cp02006 (Dominique De Wit, et al. Blood, 2004, Vol 103, Num 3:1030-103) is
a prototype of B-type CpG ODN. CpG C274 (Omar Duramad, et al. The Journal of
Immunology, 2005, 174: 5193-5200) is a prototype of C type CpG ODN.
CH] thymidine incorporation assay was used to test whether SATO5f inhibited
the proliferation of hPBMC stimulated by CpG2006 or CpG C274. Briefly, hPBMCs
x 105/well) were plated in 96-well U-bottomed plates (Costar) and cultured
with
CpG2006 (1 gimp or CpG C274 (1 ptg/m1) in the presence of SATO5f or A151 or
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Control ODN for 48 h, followed by pulsing with [3H] thymidine (New England
Nuclear, Boston, MA) for 16 h. The cells were harvested on glass fiber filters
and
detected in a scintillation counter. The cell proliferation in triplet wells
was expressed
as mean cpm (counts per minute) SD.
As shown in Figure 1, SATO5f inhibits the proliferation of hPBMCs stimulated
with CpG ODN 2006 or CpG ODN C274. The inhibitory effect is stronger than that
induced by A151. Control ODN can not induce the inhibition. Since CpG ODN
including CpG2006 or CpG C274 is a TLR9 agonist [D.M. Klinman, Nat. Rev.,
Immunol. 4 (2004) 249¨ 258], the data indicate that SATO5f inhibits TLR9
activation
and can be used to treat the diseases related to TLR9 activation and other
Toll-like
receptor (TLR)-mediated diseases.
Example 2
Effect of SATO5f on CpGODN-induced interferon production from human PBMC
<Experimental Method>
Vero E6 cells (African green monkey kidney cell line, American Type Culture
Collection) were cultured at 37 C in a 5% CO2 humidified incubator and
maintained
in IMDM supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS;
GIBCO) and antibiotics (100 IU of penicillin/ml and 100 IU of
streptomycin/m1).
An interferon (IFN) bioassay using Vero E6 cells and VSV was performed to test
whether SATO5f inhibited the IFN production from hPBMC stimulated by CpG C274.
The ODNs including CpG C274, A151, SATO5f and Control ODN were synthesized
in Sangon Biotech Company (Shanghai, China). The sequences of CpG C274, A151,
SATO5f and Control ODN are indicated as in example 1. CpG C274 is a prototype
of
C type CpG ODN and shares the activities of both A type CpG ODN and B type CpG
ODN. A-type CpG ODN and capable of activating human plasmacytoid dendritic
cells (pDCs) to produce large amount of type I interferon. hPBMCs (5
x105/well) were
plated in 96-well U-bottomed plates (Costar) and cultured with CpG C274 (1
1.tg/m1)
in the presence of SATO5f or A151 or Control ODN for 48 h and the supernatants
were then collected for assaying their IFN activity. Vero E6 cells
(3X104/well) were
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seeded into 96-well flat-bottomed plates and cultured for 24 h to confluence.
The cells
were then incubated with 100 ill of the supernatants for 18 h and then
challenged with
10X TCID50 (50% tissue culture infectious doses) of VSV for another 48 h.
Vesicular
stomatitis virus (VSV) was grown in Vero E6 cells. After titration, the virus
was
stored in aliquots at -70oC until use. After staining with 0.5% crystal
violet, the
cytopathic effect of virus was examined using Multi-well Microtiter Plate
Reader at
A578 nm and expressed as OD values.
<Experimental Results>
As shown in Figure 2, SATO5f inhibits IFN production from hPBMC stimulated
with CpG C274. Since elevated production of IFNs resulted from TLR-9
activation
has been reported to contribute to the developing systemic lupus erythematosus
(SLE)
(Barrat FJ, et al. J Exp Med 2005; 202:1131-9; Wellmann U, et al. Proc Natl
Acad Sci
USA 2005; 102:9258-63), the data indicate that SATO5f can be used as a remedy
to
treat SLE and other Toll-like receptor (TLR)-mediated diseases by inhibiting
elevated
IFN production.
Example 3. Inhibitory effect of SATO5F on interferon production from human
PBMCs
stimulated with herpes simplex virus-type 1 and flu virus
<Experimental Method>
Vero E6 cells were cultured as described in example 2. HSV-1(herpes simplex
virus-type 1) and PR8 (H1N1/PR8, a lab used flu virus) were originally
obtained from
Department of Immunology, Medical College of Norman Bethune, Jilin University,
Changchun. HSV-1 (M0I=200) was propagated on Vero E6 cells and PR8 (MOI=3)
was propagated on MDCK cells (Madin-Darby Canine Kidney Cells, ATCC). The
MDCK cells were maintained in IMDM supplemented with 10% (v/v)
heat-inactivated fetal bovine serum (FBS; GIBCO) and antibiotics (100 IU of
penicillin/ml and 100 IU of streptomycin/m1). HSV-1 in IMDM supplemented with
2% (v/v) FBS was inactivated by heating at 70 C in a water bath for 10 min and
PR8
in IMDM supplemented with 2% (v/v) FBS was inactivated by heating at 56 C in a
water bath for 30 min.
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The ODNs including SATO5F and CTRLODN (indicated as CTRL in the Figure
3) with nucleotide sequence of 5'-aaaaataaaaataaaataaaat-3' were synthesized
by
Takara Co (Dalian, China). The sequence of SATO5F is indicated as in the
example 1.
hPBMCs (5x106/m1) were cultured in 96 well plate with inactivated HSV-1 or
inactivated PR8 in the absence or presence of SATO5F or control ODN (CTRL ODN)
at 37 C for 48 h. The supernatants were then collected for assaying. The Vero
E6 cells
were seeded into 96-well flat-bottomed plates (3X104/well) and cultured for 24
h to
confluence. The cells were incubated with 100 1.1.1 of the diluted
supernatants (1:20
diluted for the HSV-1 induced and 1:80 diluted for the PR8 induced) for 16 h
and then
challenged with 10xTCID50 (50% tissue culture infectious doses) of VSV for
another
48 h. After staining with 0.5% crystal violet, the cytopathic effects were
examined
using Multi-well Microtiter Plate Reader at A570 nm and expressed as mean OD
value SD. Data from one representative experiment of three are shown.
<Experimental Results>
As shown in Figure 3-A, inactivated HSV-1 virus could induced IFN that
protected the Vero E6 cells from VSV attacking as recombinant human interferon
IFN-adid. As shown in Figure 3-B, SATO5F inhibited IFN production from human
PBMC (hPBMC) stimulated by inactivated HSV-1 virus. Dose effect analysis
revealed that SATO5F could significant IFN production from human PBMC
stimulated with inactivated HSV-1 in a dose dependent way. SATO5F at 111g/m1
is
efficient to mediate the inhibition (Figure 3-C). As shown in Figure 3-D,
inactivated
flu virus (PR8) could induced IFN efficient to protect the Vero E6 cells from
VSV
attacking as recombinant human interferon (IFN)-adid. As shown in Figure 3-E,
SATO5F inhibited interferon (IFN) production from human PBMC (hPBMC)
stimulated by inactivated flu virus (PR8). Dose effect analysis revealed that
SATO5F
at 2p,g/m1 was efficient to inhibit the IFN production from hPBMC stimulated
with
inactivated PR8 and the inhibition reaches the maximum when SATO5F wass used
at
41.1g/m1 (Figure 3-F).
It has been demonstrated that flu virus recognizes and activates TLR7 (Wang
JP,
et al. Blood. 2008 Jun 10. [Epub ahead of print]), and that HSV-1 recognizes
and
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activates TLR9 (Hubertus Hochrein et al. PNAS, 101, 11416-11421), stimulating
the
production of IFN. Together with the results of the example, the
oligonuleotides of the
invention can be used as a remedy for the treatment of Toll-like receptor
(TLR)-mediated disease such as SLE by inhibiting TLR7 or TLR9 activation and
IFN
production induced by virus.
Example 4.
The inhibitory effect of SATO5F on interferon production of from hPBMCs
stimulated
with serum of SLE patients.
<Experimental Method>
Anti-dsDNA¨positive sera of SLE patients were obtained from Department of
Rheumatology, China-Japan Union Hospital, Jilin University. ODNs including
SATO5F and CTRL-ODN ( indicated as CTRL in the Figure 4) with the sequence of
5'-AAAAATAAAAATAAAATAAAAT-3' were synthesized by Takara Co (Dalian,
China). The sequence of SATO5F is indicated as in example 1.
hPBMCs (5x 106/m1) were cultured in 96 well plate with 1:1 diluted
anti-dsDNA¨positive serum of SLE patient in the absence or presence of SATO5F
or
CTRL ODN for 48 h. hPBMCs incubated with medium alone or with
anti-dsDNA¨negative serum of healthy blood donor were set as controls. The
supernatants were collected for assaying their IFN activity. The Vero cells
were
incubated with the collected supernatant (1:5 diluted) for 16 h. and then
attacked by
10TCID50 of VSV for 48 h. After staining with 0.5% crystal violet, the
cytopathic
effects were examined using a multi-well microtiter plate reader at A570 nm.
<Experimental Results>
As indicated in Figure 4, the serum from SLE patient stimulates IFN production
from hPBMC, and SATO5F inhibits the IFN production from human PBMC
stimulated with the serum from SLE patient. Data from one representative
experiment
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of three are shown. It is well established that elevated production of IFNs
contributes to the development of SLE (Barrat FJ, et al. J Exp Med 2005;
202:1131-9;
Wellmann U, et al. Proc Nat! Acad Sci USA 2005; 102:9258-63). It has been
demonstrated that endogenous IFN inducing factors has been reported existed in
the
serum of SLE patient (Kwok SK, et al. Arthritis Res Ther. 2008;10(2):R29), SLE
patients have a circulating inducer of IFN production, sera from SLE patient
frequently induce the production of IFN in cultures of PBMC from healthy blood
donors (Vallin H, et al Clin Exp Immunol. 1999 Jan;115(1):196-202), and that
anti-double-stranded DNA antibodies or DNA-anti-DNA Ab complexes functions as
endogenous IFN-alpha inducers in SLE patient and contribute to the
pathogenesis of
SLE (Vallin H, et al. J Immtmol. 1999 Dec 1;163(11):6306-13). Together, the
data
indicate that SATO5F can be used for the treatment of SLE patients by
inhibiting IFN
production,
Example 5
The effect of SATO5F on rescuing mice from cytokine-mediated lethal shock
<Experimental Method>
In order to elucidate the in vivo functions of SATO5F, a model of
cytokine-mediated lethal shock was induced with the reference of a general
method
(Peter M, et al. Immunology. 2008 Jan;123(1):118-28; Marshall AJ, et al.
Infect
Immun. 1998 Apr; 66(4):1325-33)..
Female BALB/C mice (20 1g weight) obtained from the Experimental Animal
Center, Medical College of Norman Bethune, Jilin University) were given free
access
to food and water during the experiment. The experiments were in accordance
with
local legislation.
Oligonucleotides including SATO5F with the sequence of
'-cctcctcctcctcctcctcctcct-3 CTRL-ODN with the
sequence of
5'-aaaaataaaaataaaataaaat-3' and CpG-ODN 1826 (1826) with the sequence of
5'-tccatgacgttcctgacgtt-31Sanjai Kumar, et al. Infection and Immunity,
February 2004,
26
CA 02638176 2008-08-01
F1080083
p. 949-957, Vol. 72, No. 2] were synthesized by Takara Co (Dalian, China).
D-galactosamin (D-(+)-Galactosamine HCL, D-GALN) was from DeBioChem,
Nanjing, China.
The BALB/C mice, five in each group, were divided into groups of
D-GALN+1826, D-GALN+1826+SATO5F, D-GALN+1826+CTRL-ODN. The mice
were injected intraperitoneally (i.p.) with 500111 of D-galactosamin (32 mg/ml
in PBS).
1.5h later, the mice were intraperitoneally (i.p.) injected with 1826 (10p,g/
per mouse
in PBS) and subsequently injected (i.p.) with 50 g of SATO5F (in PBS) (in
D-GALN+1826+SATO5F group) or CTRL-ODN (in D-GALN+1826+C'TRL-ODN
group). In D-GALN+1826 group, the mice were injected with D-galactosamin and
1826 only. The mice were monitored and lethality was recorded.
<Experimental Results>
As indicated in Figure 5 , in the D-GALN+1826 group or
D-GALN+1826+CTRL-ODN group of the model, all of the five mice were deprived
of life in 24 after the D-galactosamin injection, Comparatively, in 168 hours
after the
D-galactosamin injection, all of five mice in D-GALN+1826+SATO5F group
survived,
demonstrating that SATO5F can rescue the mice received D-galactosamin and
1826.
It was documented that D-galactosamin presensitized mice could be created into
cytokine-mediated lethal shock animal models by injecting CpG OD (Peter M, et
al.
Immunology. 2008 Jan;123(1):118-28). As apparently shown in these results, the
data
indicate that SATO5F is suppressive in vivo and inhibits cytokine-mediated
lethal
shock. Data from one representative experiment of two are shown.
27
CA 02638176 2008-08-01
SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with section 111(1) of the Patent Rules, this description
contains a sequence
listing in electronic form in ASCII text format (file: 93865-3 SL 08-08-01
vl.txt).
A copy of the sequence listing in electronic form is available from the
Canadian Intellectual
Property Office.
The sequences in the sequence listing in electronic form are reproduced in the
following
table.
SEQUENCE TABLE
<110> Changchun Huapu Biotechnology Co., Ltd.
<120> OLIGONUCLEOTIDE AND USE THEREOF
<130> 93865-3
<160> 1
<170> Patentln version 3.3
<210> 1
<211> 24
<212> DNA
<213> artificial
<220>
<223> oligonucleotide
<400> 1
cctcctcctc ctcctcctcc tcct 24
28
