U.S. patent application number 10/233121 was filed with the patent office on 2003-07-03 for agents that modulate dna-pk activity and methods of use thereof.
Invention is credited to Lois, Augusto, Raz, Eyal, Takabayashi, Kenji.
Application Number | 20030125284 10/233121 |
Document ID | / |
Family ID | 26897522 |
Filed Date | 2003-07-03 |
United States Patent
Application |
20030125284 |
Kind Code |
A1 |
Raz, Eyal ; et al. |
July 3, 2003 |
Agents that modulate DNA-PK activity and methods of use thereof
Abstract
The present invention provides methods for modulating cell death
in a eukaryotic cell, and methods for reducing DNA damage in a
eukaryotic cell. The methods generally comprise modulating a
biological activity of DNA-PK in a cell. The invention further
provides methods of treating a condition related to cell death in
an individual. The invention further provides methods of
identifying agents which modulate a biological activity of DNA-PK,
as well as agents identified by the methods. Methods of modulating
an immune response using an identified agent are also provided.
Inventors: |
Raz, Eyal; (Del Mar, CA)
; Lois, Augusto; (S. Escondido, CA) ; Takabayashi,
Kenji; (San Diego, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD RD
SUITE 200
MENLO PARK
CA
94025
US
|
Family ID: |
26897522 |
Appl. No.: |
10/233121 |
Filed: |
August 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10233121 |
Aug 30, 2002 |
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09848986 |
May 4, 2001 |
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60202274 |
May 5, 2000 |
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60262321 |
Jan 17, 2001 |
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Current U.S.
Class: |
514/44R ;
435/6.1; 435/6.18 |
Current CPC
Class: |
G01N 33/6869 20130101;
A61K 31/7088 20130101; C12Q 1/485 20130101 |
Class at
Publication: |
514/44 ;
435/6 |
International
Class: |
A61K 048/00; C12Q
001/68 |
Goverment Interests
[0002] The United States Government may have certain rights in this
application pursuant to National Institutes of Health Grant Nos. AI
40682, CA 56909, CA 31397, and CA78497.
Claims
What is claimed is:
1. A method of modulating cell death in a eukaryotic cell,
comprising contacting the cell with an agent that modulates a
biological activity of DNA-PK.
2. The method of claim 1, wherein the biological activity of DNA-PK
is a kinase activity of DNA-PKcs.
3. The method according to claim 2, wherein DNA-PKcs kinase
activity is increased in the cell, thereby decreasing cell
death.
4. The method according to claim 1, wherein the agent comprises an
immunomodulatory nucleic acid molecule.
5. The method according to claim 2, wherein DNA-PKcs kinase
activity is decreased in the cell, thereby increasing cell
death.
6. A method of reducing cell damage mediated by a hypoxic
condition, comprising contacting the cell with an agent that
modulates a biological activity of DNA-PK.
7. The method of claim 6, wherein said agent is an immunomodulatory
nucleic acid molecule.
8. A method for identifying an agent that modulates a biological
activity of DNA-PK, comprising: a) adding an agent to be tested to
a sample, the sample comprising DNA-PK and an immunomodulatory
nucleic acid molecule, under conditions which favor binding of the
immunomodulatory nucleic acid molecule to DNA-PK, thereby forming a
test sample; and b) detecting a biological activity of DNA-PK
protein in the test sample, as compared to a control sample lacking
the agent, wherein an increase or a decrease in the biological
activity of DNA-PK indicates that the agent modulates a biological
activity of DNA-PK.
9. The method of claim 8, wherein the biological activity of DNA-PK
is binding to an immunomodulatory nucleic acid molecule.
10. The method according to claim 9, wherein the method is a
cell-free method, and the immunomodulatory nucleic acid molecule is
detectably labeled.
11. The method of claim 8, wherein the biological activity of
DNA-PK is activation of DNA-PKcs kinase activity.
12. The method of claim 8, wherein the method is a cell-based
method and modulation of DNA-PK activity is detected by measuring
an amount of IL-6 or IL-12 produced by the cell.
13. A composition comprising: a) an agent identified by the method
of claim 8; and b) a pharmaceutically acceptable excipient.
14. A method for reducing DNA damage in a eukaryotic cell,
comprising contacting the cell with an agent that modulates a
biological activity of DNA-PK.
15. The method of claim 14, wherein the biological activity of
DNA-PK is a kinase activity of DNA-PKcs.
16. The method of claim 15, wherein said agent is an
immunomodulatory nucleic acid molecule.
17. A method of reducing cell death in an individual, comprising
administering to an individual an effective amount of an agent that
modulates a biological activity of DNA-PK.
18. The method of claim 17, wherein the cell death is triggered by
an ischemic condition.
19. A method of reducing cell death in an organ, comprising
contacting a cell of the organ with an effective amount of an agent
that modulates a biological activity of DNA-PK.
20. The method of claim 19, wherein the contacting is performed ex
vivo.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application serial No. 60/202,274, filed May 5, 2000, and U.S.
Provisional Application serial No. 60/262,321, filed Jan. 17, 2001,
both of which are incorporated by reference herein in their
entirety.
FIELD OF THE INVENTION
[0003] This invention is in the field of control of cellular
signaling, and in particular the use of immunomodulatory
polynucleotides to modulate cell death.
BACKGROUND OF THE INVENTION
[0004] The innate immune response to an invading pathogen involves
the effective and rapid recognition of highly conserved and
repeated foreign structures such as those found in polysaccharides,
lectins, complexed lipids (e.g., LPS) and double stranded (ds) RNA.
Medzhitov and Janeway (1997) Cell 91:295. Recently, bacterial
genomic DNA, plasmids and immunostimulatory oligodeoxynucleotides
containing CpG dinucleotides in a particular base context (ISS-ODN
or CpG motifs) have been shown to activate innate immunity. Klinman
et al. (1999) Immunity 11: 123. In contrast, mammalian DNA or
methylated bacterial DNAs are inactive. ISS stimulate
macrophages/monocytes to secrete IL-6 and IL-12, activate NK cells,
induce B-cell proliferation and polyclonal IgM production and
rescue B cells from apoptosis. Klinman et al. (1999) Immunity 11:
123. Furthermore, when ISS is co-delivered with an antigen, it
elicits cell-mediated immunity, which mimics the host immune
response against viral infection. Roman et al. (1997) Nature Med.
8:849.
[0005] Ku protein was originally discovered as an autoantigen
recognized by autoantibodies from the sera of certain patients with
systemic auto immune diseases. Mimori et al. (1981) J. Clin.
Invest. 68:611-620; and Mimori and Hardin (1986) J. Biol. Chem.
261:10375-10379; and reviewed in Reeves et al. (1992) Rhem. Dis.
Clin. North Am. 18:391-414. Ku antigen is a heterodimeric protein
consisting of two polypeptides of approximately 70 kDa and 80 kDa.
Ku antigen was subsequently shown to the regulatory component of
DNA-dependent protein kinase (DNA-PK). Dynan and Yoo (1998) Nucl.
Acids Res. 26:1551-1559.
[0006] Ku, which binds to double-stranded DNA breaks (DSB), is
believed to play a role in targeting the DNA-PK complex to DSB for
repair. Specifically, when bound to DNA, Ku interacts with and
activates the DNA-PK catalytic subunit (DNA-PKcs). DNA-PKcs is
believed to interact with and phosphorylate several DNA-binding
proteins in vitro, such as replication protein A and the tumor
suppressor protein p53, respectively, as well as other
transcription factors. Anderson and Lees-Miller (1992) Crit.
Reviews in Euk. Gene Expression 2:283; and Anderson (1993) Trends
Biochem. Sci. 18:433. DNA-PK is thought to play a role in
controlling gene regulation and cell growth. Ku has been reported
to bind to various DNA sequences, including NRE1 (negative
regulatory element 1) sequences from a viral LTR comprising repeats
of 5'-GAAAG-3' (Giffin et al. (1997) J. Biol. Chem. 272:5647-5658);
an Alu core element comprising the sequence 5'-GGAGGGC-3' (Tsuchiya
et al. (1998) J. Biochem. 123:120-127; and a mammalian DNA origin
of replication (Ruiz et al. (1999) Mol. Biol. Cell 10:567-580).
[0007] In addition to DSB repair, DNA-PK is also involved in V(D)J
recombination, isotype switching, as well as telomere length
maintenance and silencing. Weaver et al. (1996) CRC Crit. Rev.
Eukaryotic Gene Exp. 6:345-375; Chu (1997) J. Biol. Chem.
272:24097-24100; Casellas et al. (1998) EMBO J. 17:2404-2411; and
Boulton and Jackson (1998) EMBO J. 17:1819-1828. DNA-PK also
participates in the activation of NFKB by ioninizing radiation.
Basu et al. (1998) Biochem. Biophys. Res. Comm. 247:79-83.
[0008] Apoptosis, or programmed cell death (PCD) is a type of cell
death that is fundamentally distinct from degenerative death or
necrosis. It is an active process of gene-directed cellular
self-destruction which in some instances, serves a biologically
meaningful homeostatic function.
[0009] Apoptotic cell death is characterized primarily by
internucleosomal DNA cleavage and chromatin condensation, and also
by cellular shrinkage, cytoplasmic blebbing, and increased membrane
permeability. Gerschenson et al. (1992) FASEB J. 6:2450-2455; and
Cohen and Duke (1992) Ann. Rev. Immunol. 10:267-293. This can be
contrasted to necrosis, which is cell death occurring as the result
of severe injurious changes in the environment of infected cells.
Necrosis is characterized by the swelling and rupturing of cells,
the loss of membrane integrity, a random breakdown of DNA into
fragments of variable size, and the phagocytosis of cellular debris
by macrophages. The release of lysosomal enzymes damages
neighboring cells, thus, cells die in groups. This produces an
inflammatory response in tissue. Cell death by necrosis involves no
direct RNA or protein synthesis. For a general review of apoptosis,
see Tomei, L. D. and Cope, F. 0. Apoptosis: The Molecular Basis of
Cell Death (1991) Cold Spring Harbor Press, N.Y.; Tomei, L. D. and
Cope, F. O. Apoptosis II: The Molecular Basis of Apoptosis in
Disease (1994) Cold Spring Harbor Press, N.Y.; and Duvall and
Wyllie (1986) Immun. Today 7:115-119.
[0010] Apoptosis can be activated by a number of intrinsic or
extrinsic signals. These signals include the following: mild
physical signals, such as ionization radiation, ultraviolet
radiation, or hyperthermia; low to medium doses of toxic compounds,
such as azides or hydrogen peroxides; chemotherapeutic drugs, such
as etoposides and teniposides, cytokines such as tumour necrosis
factors and transforming growth factors; infection with human
immunodeficiency virus (HIV); and stimulation of T-cell receptors.
Various pathological processes, such as hormone deprivation, growth
factor deprivation, thermal stress and metabolic stress, induce
apoptosis. (Wyllie, A. H., in Bowen and Lockshin (eds.) Cell Death
in Biology and Pathology (Chapman and Hall, 1981), at 9-34).
[0011] Unregulated apoptosis can cause, or be associated with,
disease. For example, unregulated apoptosis is involved in diseases
such as cancer, heart disease, neurodegenerative disorders,
autoimmune disorders, and viral and bacterial infections. Cancer,
for example, not only triggers cells to proliferate but also blocks
apoptosis. Cancer is partly a failure of apoptosis in the sense
that the signal(s) for the cells to kill themselves by apoptosis
are blocked.
[0012] In heart disease, damage caused by trauma (e.g, resulting in
shock), and cardiac cells can be induced to undergo apoptosis. For
example, cells deprived of oxygen after a heart attack release
signals that induce apoptosis in cells in the heart. Apoptosis may
also be involved in the destruction of neurons in people afflicted
by strokes or neurodegenerative diseases such as Alzheimer's
disease, Parkinson's disease, and amyotrophic lateral sclerosis
(ALS). There is also evidence suggesting that ischemia can kill
neurons by inducing apoptosis. It has been shown that neurons that
are resistant to apoptosis are also resistant to ischemic damage,
thus, inhibition of apoptosis may be a therapeutic strategy for the
treatment of neurodegenerative or cardiovascular disorders, e.g.,
stroke.
[0013] Under normal physiological conditions, self-reactive immune
cells may be induced to undergo apoptosis, thereby removing such
self-reactive cells. A failure of the immune system to induce
apoptosis in a self-reactive immune cell can lead to autoimmune
disease. For example, autoimmune diseases such as rheumatoid
arthritis, diabetes, and multiple sclerosis, result when a small
percentage of T cells attack the body's own tissue.
[0014] There is a need in the art for methods of modulating cell
death resulting from genotoxic insults. The present invention
addresses this need, and provides related advantages as well.
SUMMARY OF THE INVENTION
[0015] The present invention provides methods for reducing DNA
damage, resulting from a genotoxic insult, in a eukaryotic cell;
and methods for modulating cell death in a eukaryotic cell. The
methods generally comprise modulating a biological activity of
DNA-PK in a cell. In some embodiments, the methods comprise
contacting a eukaryotic cell with an immunomodulatory nucleic acid
molecule that binds specifically to Ku protein, either alone or as
part of the DNA-PK complex. These methods are useful for treating
any disorder resulting from a genotoxic insult to a cell, e.g.,
necrosis, apoptosis, and disorders arising from necrosis and
apoptosis. These methods are useful in modulating cell death in an
individual, e.g., to treat various apoptosis-related and
necrosis-related disorders. Accordingly, the invention further
provides methods of treating a condition related to cell death in
an individual. In one particular embodiment, cell death triggered
by hypoxic or anoxic conditions is reduced. Accordingly, the
invention further provides methods for reducing DNA damage mediated
by hypoxic or anoxic conditions. These methods find use in treating
a variety of conditions, including, e.g., ischemic heart
disease.
[0016] The invention further provides methods of identifying agents
which modulate a biological activity of DNA-PK. Agents modulate a
biological activity of DNA-PK include DNA-PK antagonists and DNA-PK
agonists. In some embodiments, agents are those which specifically
bind the Ku polypeptide portion of the DNA-PK complex. In some
embodiments, the screening methods are cell-based methods. In other
embodiments, the screening methods are cell-free methods. In some
of these embodiments, the methods involve an assay to determine
whether a candidate agent is capable of competing with a known
immunomodulatory polynucleotide for binding to Ku polypeptide.
[0017] The invention further provides agents identified by the
screening methods of the invention, as well as compositions
comprising the agents. Agents identified may enhance, inhibit, or
mimic an activity of an immunomodulatory nucleic acid molecule. An
identified agent may be useful in modulating an immune response in
an individual. Antagonists and agonists of DNA-PK find use in a
variety of methods, including methods of reducing DNA damage
resulting from a genotoxic insult, methods of inducing apoptosis,
and methods of inhibiting apoptosis. The invention further provides
methods of modulating an immune response, generally comprising
administering an identified agent to an individual. In some
embodiments, methods of enhancing a Th1 response, and methods of
reducing a Th2 response are provided.
[0018] These and other features of the invention will become
apparent to those persons skilled in the art upon reading the
details of the invention as more fully described below.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0019] FIGS. 1A-D depict various aspects of characterization of
ISS-binding Ku antigen from mouse livers. FIG. 1A depicts the
results of RT-PCR analysis to detect the presence of IL-6, IL-12,
and GAPDH transcripts in mouse livers treated with double-stranded
ISS, single-stranded ISS, or mutant ISS. FIG. 1B depicts the
results showing identification of ISS-binding protein as Ku. FIGS.
1C and 1D depict results showing DNA binding specificity of Ku
antigen for ds-ISS and ss-ISS, respectively.
[0020] FIGS. 2A-C depict results which show that Ku antigen is
required for the induction of cytokines by ISS. BMDM from
wild-type, Ku70.sup.-/- and Ku80.sup.-/- mice were treated with
LPS, ISS, or mutant ISS for 24 hours, after which IL-6 (FIG. 2A) or
IL-12 (FIG. 2B) protein levels were measured in culture
supernatants. IL-6 and IL-12 mRNA levels were also measured by
Northern blot analysis 6.5 hours after treatment of BMDM with ISS,
as shown in FIG. 2C.
[0021] FIGS. 3A-D depict results that show that DNA-PKcs is
required for the induction of IL-6 and IL-12 by ISS. BMDM from
wild-type, or DNA-PKcs.sup.-/- were treated with ISS, mutant ISS,
LPS, or were left untreated, for 24 hours, after which IL-6 (FIG.
3A) or IL-12 (FIG. 3B) protein levels were measured in culture
supernatants. IL-6 and IL-12 mRNA levels were also measured by
Northern blot analysis 6.5 hours after treatment of BMDM with ISS,
as shown in FIG. 3C. FIG. 3D shows the results of in vivo analysis
of the effect of ISS injected i.v., into wild-type or
DNA-PKcs.sup.-/- mice on IL-6 and IL-12 expression in spleen and
liver.
[0022] FIGS. 4E-H are graphs depicting results that show that
DNA-PKcs is required for the induction of IL-6 and IL-12 by ISS.
FIGS. 4E and 4F show the effect of wortmannin on IL-6 and IL-12
production, respectively. FIGS. 4G and 4H show production of IL-6
and IL-12, respectively, in BMDM from ATM.sup.-/- mice treated with
1SS, mutant ISS, LPS, or untreated.
[0023] FIGS. 5A-E depicts results that show involvement of IKK in
NF-.kappa.B activation by
[0024] FIGS. 6A-C depicts results that show the role of DNA-PKcs in
activation of IKK by ISS.
[0025] FIGS. 7A and B depict results showing that ISS activates
DNA-PK in vitro and in vivo.
[0026] FIGS. 8A and 8B depict results showing that DNA-PK activates
IKK.beta. through phosphorylation.
[0027] FIG. 9 depicts result showing induction of HSP70 by ISS.
[0028] FIG. 10 depicts results showing that induction of inducible
HSP70 by ISS is IFN.alpha./.beta.-dependent.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention is based on the following unexpected
observations: (1) nucleic acid molecules previously identified as
modulating the immune response ("immunomodulatory nucleic acid
molecule") bind to Ku antigen, resulting in activation of DNA-PKcs;
(2) immunomodulatory nucleic acid molecules activate the
anti-apoptotic PI3P-dependent kinase Akt; and (3) immunomodulatory
nucleic acid molecules induce an anti-apoptotic response in
eukaryotic cells. The present invention makes use of and extends
these observations by providing methods of reducing DNA damage in a
eukaryotic cell where the DNA damage is a result of a genotoxic
insult; and methods of increasing or decreasing cell death in a
eukaryotic cell. Such methods are useful for treating a variety of
pathological conditions relating to cell death (e.g., conditions
relating to necrosis and conditions relating to apoptosis), or lack
thereof. The invention further provides methods for identifying
agents that bind specifically to Ku antigen, and which therefore
may be useful in methods to modulate an immune response, and in
methods to modulate cell death.
[0030] Without wishing to be bound by theory, it is believed that
immunostimulatory nucleic acid molecules mimic the signal delivered
by DNA damage and activate one or more components of the molecular
machinery, e.g., DNA-PK, which are involved in repairing DNA damage
(e.g., DSB).
[0031] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0032] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention. The
upper and lower limits of these smaller ranges may independently be
included in the smaller ranges and are also encompassed within the
invention, subject to any specifically excluded limit in the stated
range. Where the stated range includes one or both of the limits,
ranges excluding either both of those included limits are also
included in the invention.
[0033] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0034] It must be noted that as used herein and in the appended
claims, the singular forms "a", "and", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "an immunomodulatory nucleic acid molecule"
includes a plurality of such nucleic acid molecules and reference
to "the agent" includes reference to one or more agents and
equivalents thereof known to those skilled in the art, and so
forth.
[0035] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
Definitions
[0036] The term "a biological activity of DNA-PK," as used herein,
refers to a biological activity of DNA-PK which, when modified,
affects the activity of DNA-PK in repairing DNA damage in a
eukaryotic cell. A biological activity of DNA-PK encompasses a
biological activity of either of its components, e.g., Ku antigen
and DNA-PKcs, separately or in complex with one another. Biological
activities of DNA-PK include, but are not limited to, binding to
double-strand breaks in DNA; Ku binding to DNA-PKcs; binding of
DNA-PK or its components to other factors, such as immunomodulatory
nucleic acid molecules, polypeptides, etc., which modulate DNA-PK
activity in repairing DNA damage; and phosphorylation of factors,
such as polypeptides, by DNA-PKcs.
[0037] The terms "Ku antigen," "Ku polypeptide," and "Ku protein,"
used interchangeably herein, refer to the heterodimeric Ku protein
(comprising the Ku70 and Ku80 chains), the isolated Ku 70 chain,
the isolated Ku 80 chain, and variants and fusion proteins of the
foregoing. Ku antigen may be derived from any organism, including,
but not limited to human, a murine, or other vertebrate. Ku antigen
may be derived from a natural source, or may be completely or
partially synthetic. The amino acid sequence of Ku antigen has been
reported for mouse and human. These sequences may be found in the
Swiss-Prot database under accession numbers P12956 and NP 001460
(human Ku 70); S25 149 (mouse Ku 70); and P13010 and A32626 (human
Ku 80). Ku antigen may have a wild-type or a variant amino acid
sequence. Variants include Ku antigen comprising one or more
truncations, internal deletions, substitutions, additions, or other
modifications such as glycosylations, phosphorylations, acylations,
etc. Variants further include fusion proteins comprising Ku antigen
and a heterologous protein, including, but not limited to, an
immunologically detectable protein, e.g., an epitope tag; a protein
which directly provides a detectable signal, e.g., a green
fluorescent protein; an enzyme which, upon action on a substrate,
can yield a detectable product, e.g., alkaline phosphatase.
Preferably, a Ku antigen or Ku antigen variant is biologically
active. As used herein, "biologically active Ku antigen" is a Ku
antigen which specifically binds an immunomodulatory nucleic acid
molecule, and/or which activates DNA-PKcs activity.
[0038] The term "immunomodulatory," as used herein in reference to
a nucleic acid molecule, refers to the ability of a nucleic acid
molecule to modulate an immune response in a vertebrate host.
"Immunomodulatory" includes "immunostimulatory" and
"immunoinhibitory." Whether an immunomodulatory nucleic acid
molecule is immunostimulatory or immunoinhibitory may be expressed
in terms of the aspect of the immune response being modulated,
e.g., relative enhancement, increase, or induction of a Th1 or a
Th2 cell-mediated immune response. Modulation of an immune response
includes, but is not limited to, enhancing or increasing a Th1
response, and/or decreasing or inhibiting a Th2 response, and/or
decreasing or inhibiting a Th1 response.
[0039] The terms "oligonucleotide," "polynucleotide," and "nucleic
acid molecule", used interchangeably herein, refer to a polymeric
forms of nucleotides of any length, either ribonucleotides or
deoxyribonucleotides. Thus, this term includes, but is not limited
to, single-, double-, or multi-stranded DNA or RNA, genomic DNA,
cDNA, DNA-RNA hybrids, or a polymer comprising purine and
pyrimidine bases or other natural, chemically or biochemically
modified, non-natural, or derivatized nucleotide bases. The
backbone of the polynucleotide can comprise sugars and phosphate
groups (as may typically be found in RNA or DNA), or modified or
substituted sugar or phosphate groups. Alternatively, the backbone
of the polynucleotide can comprise a polymer of synthetic subunits
such as phosphoramidites, and/or phosphorothioates, and thus can be
an oligodeoxynucleoside phosphoramidate or a mixed
phosphoramidate-phosphodiester oligomer. Peyrottes et al. (1996)
Nucl. Acids Res. 24:1841-1848; Chaturvedi et al. (1996) Nucl. Acids
Res. 24:2318-2323. The polynucleotide may comprise one or more
L-nucleosides. A polynucleotide may comprise modified nucleotides,
such as methylated nucleotides and nucleotide analogs, uracyl,
other sugars, and linking groups such as fluororibose and thioate,
and nucleotide branches. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after polymerization, such as by conjugation with
a labeling component. Other types of modifications included in this
definition are caps, substitution of one or more of the naturally
occurring nucleotides with an analog, and introduction of means for
attaching the polynucleotide to proteins, metal ions, labeling
components, other polynucleotides, or a solid support.
[0040] The terms "polypeptide," "peptide," and "protein", used
interchangeably herein, refer to a polymeric form of amino acids of
any length, which can include coded and non-coded amino acids,
chemically or biochemically modified or derivatized amino acids,
and polypeptides having modified peptide backbones. The term
includes polypeptide chains modified or derivatized in any manner,
including, but not limited to, glycosylation, formylation,
cyclization, acetylation, phosphorylation, and the like. The term
includes naturally-occurring peptides, synthetic peptides, and
peptides comprising one or more amino acid analogs. The term
includes fusion proteins, including, but not limited to, fusion
proteins with a heterologous amino acid sequence, fusions with
heterologous and homologous leader sequences, with or without
N-terminal methionine residues; immunologically tagged proteins;
and the like.
[0041] As used herein the term "isolated" is meant to describe a
polynucleotide, a polypeptide, an antibody, or a host cell that is
in an environment different from that in which the polynucleotide,
the polypeptide, the antibody, or the host cell naturally occurs.
As used herein, the term "substantially purified" refers to a
compound (e.g., either a polynucleotide or a polypeptide or an
antibody) that is removed from its natural environment and is at
least 60% free, preferably 75% free, and most preferably 90% free
from other components with which it is naturally associated.
[0042] As used herein, the terms "treatment," "treating," and the
like, refer to obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a disease or symptom thereof and/or may be
therapeutic in terms of a partial or complete cure for a disease
and/or adverse affect attributable to the disease. "Treatment," as
used herein, covers any treatment of a disease in a mammal,
particularly in a human, and includes: (a) preventing the disease
from occurring in a subject which may be predisposed to the disease
but has not yet been diagnosed as having it; (b) inhibiting the
disease, i.e., arresting its development; and (c) relieving the
disease, i.e., causing regression of the disease.
[0043] As used herein, the term "DNA damage" includes, but is not
limited to, single-strand breaks, double-strand breaks,
alkali-labile sites, oxidative damage, DNA cross-linking, and
incomplete excision repair sites.
[0044] As used herein, the term "cell death" refers to cell death
arising from any cause, and includes necrosis, apoptosis, and a
combination of necrosis and apoptosis.
[0045] As used herein, the terms "an cell death-related condition"
and "a condition related to cell death," and "condition caused by
cell death," are used interchangeably herein to refer to a
condition (the term "condition" being used interchangeably herein
with the terms "disease" and "disorder") associated with abnormally
high rates (e.g., higher than physiologically normal in the absence
of the condition) of cell death. It is also a condition which is
amenable to treatment by inducing cell death, e.g., to reduce
proliferation of an undesired cell.
[0046] The terms "genotoxic factor," and "genotoxic insult", used
interchangeably herein, refer to any of a variety of environmental
insults that result in DNA damage in a eukaryotic cell, e.g., may
adversely affect the structure and/or integrity of DNA in a
eukaryotic cell, and which thus may lead to cell death. Genotoxic
factors include, but are not limited to, anoxia, hypoxia, ischemia,
reperfusion injury, UV irradiation, gamma irradiation, DNA-damaging
chemicals, and anti-cancer drugs that target DNA.
[0047] "Ischemia" is defined as an insufficient supply of blood to
a specific organ or tissue. A consequence of decreased blood supply
is an inadequate supply of oxygen to the organ or tissue (hypoxia).
Prolonged hypoxia may result in injury to the affected organ or
tissue. "Anoxia" refers to a virtually complete absence of oxygen
in the organ or tissue, which, if prolonged, may result in death of
the organ or tissue.
[0048] "Hypoxia" and "anoxia" refer to a reduction of oxygen supply
to a tissue below physiological levels. A "hypoxic condition"
refers to a condition under which a particular organ or tissue
receives an inadequate supply of oxygen. An "anoxic condition"
refers to a condition under which the supply of oxygen to a
particular organ or tissue is cut off.
[0049] "Reperfusion" refers to the resumption of blood flow in a
tissue following a period of ischemia.
[0050] "Ischemic injury" refers to cellular and/or molecular damage
to an organ or tissue as a result of a period of ischemia and/or
ischemia followed by reperfusion.
[0051] The terms "preventing," "reducing," and "inhibiting" are
used interchangeably herein. In the context of modulating cell
death, these terms refer to a reduction in cell death or a
prolongation in the survival time of the cell. They also are
intended to include a diminution in the appearance or a delay in
the appearance of morphological and/or biochemical changes normally
associated with apoptosis and/or necrosis. Thus a reduction in cell
death leads to increased survival time and/or survival rate of a
cell or population of cells which, absent the use of a method to
reduce cell death, would normally be expected to die.
[0052] The term "biological sample" encompasses a variety of sample
types obtained from an organism and can be used in a diagnostic or
monitoring assay. The term encompasses blood and other liquid
samples of biological origin, solid tissue samples, such as a
biopsy specimen or tissue cultures or cells derived therefrom and
the progeny thereof. The term encompasses samples that have been
manipulated in any way after their procurement, such as by
treatment with reagents, solubilization, or enrichment for certain
components. The term encompasses a clinical sample, and also
includes cells in cell culture, cell supernatants, cell lysates,
serum, plasma, biological fluids, and tissue samples.
[0053] The terms "cancer", "neoplasm", "tumor", and "carcinoma",
are used interchangeably herein to refer to cells which exhibit
relatively autonomous growth, such that they exhibit an aberrant
growth phenotype characterized by a significant loss of control of
cell proliferation. Cancerous cells can be benign or malignant.
[0054] By "individual" or "host" or "subject" or "patient" is meant
any mammalian subject for whom diagnosis, treatment, or therapy is
desired, particularly humans. Other subjects may include cattle,
dogs, cats, pigs, rabbits, rats, mice, horses, and so on.
[0055] Methods of the Invention
[0056] The present invention provides methods for modulating cell
death in a eukaryotic cell; and methods for reducing DNA damage due
to a genotoxic insult in a eukaryotic cell. The methods generally
comprise contacting the cell with an agent that modulates a
biological activity of DNA-PK. In some embodiments, the methods
provide for decreasing cell death in a eukaryotic cell by
activating DNA-PKcs in the cell. In other embodiments, the methods
provide for increasing cell death in a eukaryotic cell by
decreasing DNA-PKcs activity in the cell. The invention further
provides methods of treating a cell death-related condition in an
individual.
[0057] In some embodiments, the methods comprise contacting a cell
with an immunomodulatory nucleic acid molecule. Immunomodulatory
nucleic acid molecules suitable for use in these methods are
described in more detail below. Immunomodulatory nucleic acid
molecules can bind to Ku polypeptide and activate DNA-PKcs activity
in a cell.
[0058] In some embodiments, methods are provided for reducing DNA
damage resulting from exposure to a genotoxic factor in a
eukaryotic cell in response to a genotoxic factor. The methods
generally comprise contacting the cell with an agent that modulates
a biological activity of DNA-PK, as described above. In these
embodiments, an "effective amount" of an agent is an amount
effective to decrease the DNA damage by at least about 10%, at
least about 20%, at least about 25%, at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, or up to 100%, in the
cell, when compared to DNA damage in a cell exposed to the
genotoxic factor but not contacted with the agent.
[0059] The methods can be used in vitro (e.g., in a screening
assay), in vivo (e.g., in therapeutic methods), or ex vivo (e.g.,
in therapeutic methods such as reducing cell death in an organ or
tissue or cells to be transplanted). For in vivo use, a formulation
comprising an effective amount of an agent that modulates a
biological activity of DNA-PK in a eukaryotic cell is administered
to an individual in need thereof. An "effective amount" of an agent
that decreases a biological activity of DNA-PK activity in a
eukaryotic cell is an amount is an amount effective to decrease the
DNA-PK biological activity by at least about 10%, at least about
20%, at least about 25%, at least about 30%, at least about 40%, at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 90%, or up to 100%, in the cell. An
"effective amount" of an agent that increases a biological activity
of DNA-PK in a eukaryotic cell is an amount is an amount effective
to increase the biological activity of DNA-PK by at least about
10%, at least about 25%, at least about 50%, at least about 75%, at
least about 100% (or two-fold), at least about 5-fold, at least
about 10-fold, at least about 25-fold, or at least about 50-fold or
more, in the cell.
[0060] Any biological activity of DNA-PK can be detected,
including, but not limited to, (1) enzymatic activity of DNA-PK in
phosphorylating a substrate polypeptide; (2) the level of cell
death in a cell population as an indication of the level of DNA-PK
activity; (3) measuring Ku binding to DNA-PKcs; (4) binding of an
immunomodulatory nucleic acid molecule to Ku; (5) activity of a
polypeptide in the pathway of DNA-PK activition, e.g., Akt-1; and
(6) binding of a polypeptide (e.g., replication protein A, "RPA")
to the DNA-PK complex. "Detecting," as used herein, encompasses
determining the presence or absence of a biological activity;
determining a relative increase or decrease in a biological
activity; and measuring quantitatively the level of a biological
activity. Accordingly, "detecting" encompasses both quantitative
and qualitative determinations.
[0061] DNA-PK enzymatic activity can be detected using any method
known in the art. DNA-PK activity can be measured in a reaction
mixture comprising linear, double-stranded DNA, a suitable
polypeptide or peptide substrate, Mg.sup.2+ ions, ATP, and
gamma-labeled ATP, e.g., [.gamma..sup.32P]-label- ed ATP. Methods
for measuring DNA-PK activity have been described in the art. See,
e.g., Basu et al. (1998) Biochem. Biophys. Res. Comm. 247:79-83,
the contents of which is incorporated herein by reference for their
teaching of assays for DNA-PK activity. In general, a polypeptide
substrate for DNA-PK comprises a minimal target sequence for
phosphorylation by DNA-PK consists of a serine or threonine residue
adjacent to a glutamine (on either side) with no nearby basic amino
acids. An example of a peptide substrate specific for DNA-PK has
the following amino acid sequence:
Glu-Pro-Pro-Leu-Ser-Gln-Glu-Ala-Phe-Ala-As- p-Leu-Trp-Lys-Lys. Kits
for assaying DNA-PK activity are commercially available from, e.g.,
Promega. Reaction products are analyzed for incorporation of
labeled phosphate into the peptide or polypeptide substrate, using
standard techniques. As one example, reaction products are spotted
onto phosphocellulose paper; dried; washed to remove unincorporated
[.gamma.-.sup.32P]-ATP; dried; and spots cut out and counted in a
scintillation counter.
[0062] DNA damage can be detected using any known method,
including, but not limited to, a Comet assay (commercially
available from Trevigen, Inc.), which is based on alkaline lysis of
labile DNA at sites of damage; and immunological assays using
antibodies specific for aberrant DNA structures, e.g., 8-OHdG.
[0063] Cell death can be measured using any known method, and is
generally measured using any of a variety of known methods for
measuring cell viability. Such assays are generally based on entry
into the cell of a detectable compound (or a compound that becomes
detectable upon interacting with, or being acted on by, an
intracellular component) that would normally be excluded from a
normal, living cell by its intact cell membrane. Such compounds
include substrates for intracellular enzymes, including, but not
limited to, a fluorescent substrate for esterase; dyes that are
excluded from living cell, including, but not limited to, trypan
blue; and DNA-binding compounds, including, but not limited to, an
ethidium compound such as ethidium bromide and ethidium homodimer,
and propidium iodide.
[0064] Apoptosis can be assayed using any known method. Assays can
be conducted on cell populations or an individual cell, and include
morphological assays and biochemical assays. A non-limiting example
of a method of determining the level of apoptosis in a cell
population is TUNEL (TdT-mediated dUTP nick-end labeling) labeling
of the 3'-OH free end of DNA fragments produced during apoptosis
(Gavrieli et al. (1992) J. Cell Biol. 119:493). The TUNEL method
consists of catalytically adding a nucleotide, which has been
conjugated to a chromogen system or a to a fluorescent tag, to the
3'-OH end of the 180-bp (base pair) oligomer DNA fragments in order
to detect the fragments. The presence of a DNA ladder of 180-bp
oligomers is indicative of apoptosis. Procedures to detect cell
death based on the TUNEL method are available commercially, e.g.,
from Boehringer Mannheim (Cell Death Kit) and Oncor (Apoptag Plus).
Another marker that is currently available is annexin, sold under
the trademark APOPTEST.TM.. This marker is used in the "Apoptosis
Detection Kit," which is also commercially available, e.g., from
R&D Systems. During apoptosis, a cell membrane's phospholipid
asymmetry changes such that the phospholipids are exposed on the
outer membrane. Annexins are a homologous group of proteins that
bind phospholipids in the presence of calcium. A second reagent,
propidium iodide (PI), is a DNA binding fluorochrome. When a cell
population is exposed to both reagents, apoptotic cells stain
positive for annexin and negative for PI, necrotic cells stain
positive for both, live cells stain negative for both. Other
methods of testing for apoptosis are known in the art and can be
used, including, e.g., the method disclosed in U.S. Pat. No.
6,048,703.
[0065] Binding of Ku polypeptide to DNA-PKcs can be detected by
standard protein-protein interaction assays, e.g., immunological
assays such as co-precipitation of DNA-PKcs with an antibody to Ku
polypeptide, and the like.
[0066] In some embodiments, an immunomodulatory nucleic acid
molecule activates a kinase activity of Akt-1. Akt-1 (also known as
PKB-.alpha. and RAC-PK-.alpha.) is a member of the AKT/PKB family
of serine/threonine kinases and has been shown to be involved in a
diverse set of signaling pathways. Akt-1, like other members of the
AKT/PKB family is located in the cytosol of unstimulated cells and
translocates to the cell membrane following stimulation. Akt-1 has
been cloned and sequenced. Bellacosa et al. (1991) Science
254:274-277; Coffer and Woodgett (1991) Eur. J. Biochem.
201:475-481; Jones et al. (1991) Cell Regul. 2: 1001-1009. Akt-1 is
a phosphatidylinositol-3,4,5-trisphosphosphate (PIP3)-dependent
anti-apoptotic kinase, and plays a role in the prevention of
"programmed cell death" or apoptosis. Phosphatidylinositol 3-kinase
(PI3-K) phosphorylates Akt, which phosphorylation serves to
activate the anti-apoptotic activity of Akt. It has been
demonstrated that Akt-1 provides a survival signal to cells
protecting them from a number of agents including UV radiation,
withdrawal of IGF1 from neuronal cells, detachment from the
extracellular matrix, stress and heat shock. Dudek et al. (1997)
Science 275:661-665; and Alessi and Cohen (1998) Curr. Opin. Genet.
Dev. 8:55-62. Assays for Akt-1 kinase activity are known in the art
and have been amply described, e.g., in the above-mentioned
publications. In general, a substrate for Akt-1 and
[.gamma.-.sup.32P]-ATP are provided in a reaction mixture, and
phosphorylation of the substrate is monitored using conventional
assays.
[0067] Methods of detecting binding of DNA-PK to other polypeptidcs
such as RPA are known in the art, and any such method can be used
in conjunction with the methods of the present invention.
[0068] Immunomodulatory Nucleic Acid Molecules Suitable for Use in
the Methods of the Invention
[0069] The term "polynucleotide," as used in the context of
immunomodulatory nucleic acid molecules, is a polynucleotide as
defined above, and encompasses, inter alia, single- and
double-stranded oligonucleotides (including deoxyribonucleotides,
ribonucleotides, or both), modified oligonucleotides, and
oligonucleosides, alone or as part of a larger nucleic acid
construct, or as part of a conjugate with a non-nucleic acid
molecule such as a polypeptides. Thus immunomodufatory nucleic acid
molecules may be, for example, single-stranded DNA (ssDNA),
double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) or
double-stranded RNA (dsRNA). Immunomodulatory nucleic acid
molecules also encompasses crude, detoxified bacterial (e.g.,
mycobacterial) RNA or DNA, as well as enriched plasmids enriched
for immunomodulatory nucleic acid molecules. In some embodiments,
an "immunomodulatory nucleic acid molecules-enriched plasmid"
refers to a linear or circular plasmid that comprises or is
engineered to comprise a greater number of CpG motifs than normally
found in mammalian DNA. Exemplary immunomodulatory nucleic acid
molecules-enriched plasmids are described in, for example, Roman et
al. (1997) Nat. Med. 3(8):849-54. Modifications of oligonucleotides
include, but are not limited to, modifications of the 3'OH or 5'OH
group, modifications of the nucleotide base, modifications of the
sugar component, and modifications of the phosphate group.
[0070] An immunomodulatory nucleic acid molecule may comprise at
least one nucleoside comprising an L-sugar. The L-sugar may be
deoxyribose, ribose, pentose, deoxypentose, hexose, deoxyhexose,
glucose, galactose, arabinose, xylose, lyxose, or a sugar "analog"
cyclopentyl group. The L-sugar may be in pyranosyl or furanosyl
form.
[0071] An immunomodulatory nucleic acid molecule may comprise a
modified cytosine, e.g., as described in PCT Publication No. WO
99/62923.
[0072] Immunomodulatory nucleic acid molecules generally do not
provide for, nor is there any requirement that they provide for,
expression of any amino acid sequence encoded by the
polynucleotide, and thus the sequence of a immunomodulatory nucleic
acid molecule may be, and generally is, non-coding.
Immunomodulatory nucleic acid molecules may comprise a linear
double or single-stranded molecule, a circular molecule, or can
comprise both linear and circular segments. Immunomodulatory
nucleic acid molecules may be single-stranded, or may be completely
or partially double-stranded.
[0073] In some embodiments, an immunomodulatory nucleic acid
molecule is an oligonucleotide, e.g., consists of a sequence of
from about 6 to about 200, from about 10 to about 100, from about
12 to about 50, or from about 15 to about 25, nucleotides in
length.
[0074] In other embodiments, an immunomodulatory nucleic acid
molecule is part of a larger nucleotide construct (e.g., a plasmid
vector, a viral vector, or other such construct). A wide variety of
plasmid and viral vector are known in the art, and need not be
elaborated upon here. A large number of such vectors have been
described in various publications, including, e.g., Current
Protocols in Molecular Biology, (F. M. Ausubel, et al., Eds. 1987,
and updates). Many vectors are commercially available.
[0075] Immunomodulatory Nucleic Acid Molecules Comprising a CpG
Motif
[0076] In some embodiments, the immunomodulatory nucleic acid
molecules used in the invention comprise at least one unmethylated
CpG motif. In general, these immunomodulatory nucleic acid
molecules increase a Thl response in an individual. The relative
position of any CpG sequence in a polynucleotide having
immunomodulatory activity in certain mammalian species (e.g.,
rodents) is 5'-CG-3' (i.e., the C is in the 5' position with
respect to the G in the 3' position). Immunomodulatory nucleic acid
molecules can be conveniently obtained by substituting the cytosine
in the CpG dinucleotide with another nucleotide, particularly a
purine nucleotide. A substitution of particular interest is with a
guanine to form an immunomodulatory nucleic acid molecule
comprising a GpG dinucleotide.
[0077] Exemplary immunomodulatory nucleic acid molecules useful in
the invention include, but are not necessarily limited to, those
comprising the following core nucleotide sequences: 1) hexameric
core sequences comprising "CpG" motifs or comprising XpY motifs,
where X cannot be C if Y is G and vice-versa; 2) octameric core
sequences comprising "CpG" motifs or comprising XpY motifs, where X
cannot be C if Y is G and vice-versa; and 3) inosine and/or uracil
substitutions for nucleotides in the foregoing hexameric or
octameric sequences for use as RNA immunomodulatory nucleic acid
molecule (e.g., substituting uracil for thymine and/or substituting
inosine for a purine nucleotide). As used herein, "core sequence"
in the context of an immunomodulatory nucleic acid molecule refers
to a minimal sequence that provides for, factilitates, or confers
the immunomodulatory activity of the nucleic acid molecule.
[0078] Exemplary consensus CpG motifs of immunomodulatory nucleic
acid molecules useful in the invention include, but are not
necessarily limited to:
[0079] 5'-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3', in which
the immunomodulatory nucleic acid molecule comprises a CpG motif
flanked by at least two purine nucleotides (e.g., GG, GA, AG, AA,
II, etc.,) and at least two pyrimidine nucleotides (CC, TT, CT, TC,
UU, etc.);
[0080] 5'-Purine-TCG-Pyrimidine-Pyrimidine-3';
[0081] 5'-[TCG].sub.n-3', where n is any integer that is 1 or
greater, e.g., to provide a poly-TCG immunomodulatory nucleic acid
molecule (e.g., where n-3, the polynucleotide comprises the
sequence 5'-TCGTCGTCG-3');
[0082] 5'-Purine-Purine-CG-Pyrimidine-Pyrimidine-CG-3';
[0083] 5'-Purine-TCG-Pyrimidine-Pyrimidine-CG-3'; and
[0084] 5'-Purine-Purine-CG-Pyrimidine-Pyrimidine-CG-3'.
[0085] The core structure of immunomodulatory nucleic acid
molecules useful in the invention may be flanked upstream and/or
downstream by any number or composition of nucleotides or
nucleosides. In some embodiments, the core sequence of
immunomodulatory nucleic acid molecules are at least 6 bases or 8
bases in length, and the complete immunomodulatory nucleic acid
molecules (core sequences plus flanking sequences 5', 3' or both)
are usually between 6 bases or 8 bases, and up to about 200 bases
in length to enhance uptake of the immunomodulatory nucleic acid
molecules. Those of ordinary skill in the art will be familiar
with, or can readily identify, reported nucleotide sequences of
known immunomodulatory nucleic acid molecules for reference in
preparing immunomodulatory nucleic acid molecules, see, e.g.,
Yamamoto, et al., (1992) Microbiol. Immunol., 36:983; Ballas, et
al., (1996) J. Immunol., 157:1840; Kliniman, et al., (1997) J.
Immunol., 158:3635; Sato, et al., (1996) Science, 273:352, each of
which are incorporated herein by reference. In addition,
immunomodulatory nucleic acid molecules useful in the invention
have been described in, for example, PCT publication nos. WO
98/16427, WO 98/55495, and WO 99/11275.
[0086] Exemplary DNA-based immunomodulatory nucleic acid molecules
useful in the invention include, but are not necessarily limited
to, polynucleotides comprising the following nucleotide sequences:
AACGCC, AACGCT, AACGTC, AACGTT; AGCGCC, AGCGCT, AGCGTC, AGCGTT;
GACGCC, GACGCT, GACGTC, GACGTT; GGCGCC, GGCGCT, GGCGTC, GGCGTT;
ATCGCC, ATCGCT, ATCGTC, ATCGTT; GTCGCC, GTCGCT, GTCGTC, GTCGTT; and
TCGTCG, and TCGTCGTCG.
[0087] Octameric sequences are generally the above-mentioned
hexameric sequences, with an additional 3'CG. Exemplary DNA-based
immunomodulatory nucleic acid molecules useful in the invention
include, but are not necessarily limited to, polynucleotides
comprising the following octameric nucleotide sequences:
[0088] AACGCCCG, AACGCTCG, AACGTCCG, AACGTTCG;
[0089] AGCGCCCG, AGCGCTCG, AGCGTCCG, AGCGTTCG;
[0090] GACGCCCG, GACGCTCG, GACGTCCG, GACGTTCG;
[0091] GGCGCCCG, GGCGCTCG, GGCGTCCG, GGCGTTCG;
[0092] ATCGCCCG, ATCGCTCG, ATCGTCCG, ATCGTTCG;
[0093] GTCGCCCG, GTCGCTCG, GTCGTCCG, GTCGTTCG; and
[0094] GTCGTTCG.
[0095] Immunomodulatory nucleic acid molecules useful in the
invention can comprise one or more of any of the above CpG motifs.
For example, immunomodulatory nucleic acid molecules useful in the
invention can comprise a single instance or multiple instances
(e.g., 2, 3, 5 or more) of the same CpG motif. Alternatively, the
immunomodulatory nucleic acid molecules can comprises multiple CpG
motifs (e.g., 2, 3, 5 or more) where at least two of the multiple
CpG motifs have different consensus sequences, or where all CpG
motifs in the immunomodulatory nucleic acid molecules have
different consensus sequences.
[0096] Immunomodulatory nucleic acid molecules useful in the
invention may or may not include palindromic regions. If present, a
palindrome may extend only to a CpG motif, if present, in the core
hexamer or octamer sequence, or may encompass more of the hexamer
or octamer sequence as well as flanking nucleotide sequences.
[0097] Modifications
[0098] Immunomodulatory nucleic acid molecules can be modified in a
variety of ways. For example, an immunomodulatory nucleic acid
molecules can comprise backbone phosphate group modifications
(e.g., methylphosphonate, phosphorothioate, phosphoroamidate and
phosphorodithioate internucleotide linkages), which modifications
can, for example, confer inherent anti-microbial activity on the
immunomodulatory nucleic acid molecule and enhance their stability
in vivo, making them particularly useful in therapeutic
applications. A particularly useful phosphate group modification is
the conversion to the phosphorothioate or phosphorodithioate forms
of an immunomoduiatory nucleic acid molecule. In addition to their
potentially anti-microbial properties, phosphorothioates and
phosphorodithioates are more resistant to degradation in vivo than
their unmodified oligonucleotide counterparts, increasing the
half-lives of the immunomodulatory nucleic acid molecules and
making them more available to the subject being treated.
[0099] Other modified immunomodulatory nucleic acid molecules
include immunomodulatory nucleic acid molecules having
modifications at the 5' end, the 3' end, or both the 5' and 3'
ends. For example, the 5' and/or 3' end can be covalently or
non-covalently conjugated to a molecule (either nucleic acid,
non-nucleic acid, or both) to, for example, increase the
bio-availability of the immunomodulatory nucleic acid molecules,
increase the efficiency of uptake where desirable, facilitate
delivery to cells of interest, and the like. Exemplary molecules
for conjugation to the immunomodulatory nucleic acid molecules
include, but are not necessarily limited to, cholesterol,
phospholipids, fatty acids, sterols, oligosaccharides, polypeptides
(e.g., immunoglobulins), peptides, antigens (e.g., peptides, small
molecules, etc.), linear or circular nucleic acid molecules (e.g.,
a plasmid), and the like. Additional immunomodulatory nucleic acid
conjugates, and methods for making same, are known in the art and
described in, for example, WO 98/16427 and WO 98/55495. Thus, the
term "immunomodulatory nucleic acid molecule" includes conjugates
comprising an immunomodulatory nucleic acid molecule.
[0100] Formulations
[0101] In general, immunomodulatory nucleic acid molecules are
prepared in a pharmaceutically acceptable composition for delivery
to a host. Pharmaceutically acceptable carriers preferred for use
with the immunomodulatory nucleic acid molecules in carrying out
treatment methods of the invention may include sterile aqueous of
non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol,
vegetable oils such as olive oil, and injectable organic esters
such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, and
microparticles, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers (such as those based on Ringer's
dextrose), and the like. A composition comprising a
immunomodulatory nucleic acid molecule may also be lyophilized
using means well known in the art, for subsequent reconstitution
and use according to the invention. Also contemplated are
microencapsulation carriers, such as liposomes, microspheres, and
the like.
[0102] In general, the pharmaceutical compositions can be prepared
in various forms, such as granules, tablets, pills, suppositories,
capsules, suspensions, salves, lotions and the like. Pharmaceutical
grade organic or inorganic carriers and/or diluents suitable for
oral and topical use can be used to make up compositions comprising
the therapeutically-active compounds. Diluents known to the art
include aqueous media, vegetable and animal oils and fats.
Stabilizing agents, wetting and emulsifying agents, salts for
varying the osmotic pressure or buffers for securing an adequate pH
value, and skin penetration enhancers can be used as auxiliary
agents. Preservatives and other additives may also be present such
as, for example, antimicrobials, antioxidants, chelating agents,
and inert gases and the like. In one embodiment, as discussed
above, the immunomodulatory nucleic acid molecule formulation
comprises an additional anti-mycobacterial agent.
[0103] Immunomodulatory nucleic acid molecules can be administered
in the absence of an amount of agents or compounds sufficient to
facilitate uptake by target cells (e.g., as a "naked"
polynucleotide, e.g., a polynucleotide that is not encapsulated by
a viral particle, or a nucleic acid molecule not administered with
an adjuvant). Immunomodulatory nucleic acid molecules can be
administered in microencapsulated form, e.g., in microspheres, and
the like. Immunomodulatory nucleic acid molecules can be
administered with compounds that facilitate uptake of
immunomodulatory nucleic acid molecules by target cells (e.g., by
macrophages) or otherwise enhance transport of an immunomodulatory
nucleic acid molecule to a treatment site for action. Absorption
promoters, detergents and chemical irritants (e.g., keratinolytic
agents) can enhance transmission of an immunomodulatory nucleic
acid molecule composition into a target tissue (e.g., through the
skin). For general principles regarding absorption promoters and
detergents which have been used with success in mucosal delivery of
organic and peptide-based drugs, see, e.g., Chien, Novel Drug
Delivery Systems, Ch. 4 (Marcel Dekker, 1992). Examples of suitable
nasal absorption promoters in particular are set forth at Chien,
supra at Ch. 5, Tables 2 and 3; milder agents are preferred.
Suitable agents for use in the method of this invention for
mucosal/nasal delivery are also described in Chang, et al, Nasal
Drug Delivery, "Treatise on Controlled Drug Delivery", Ch. 9 and
Tables 3-4B thereof, (Marcel Dekker, 1992). Suitable agents which
are known to enhance absorption of drugs through skin are described
in Sloan, Use of Solubility Parameters from Regular Solution Theory
to Describe Partitioning-Driven Processes, Ch. 5, "Prodrugs:
Topical and Ocular Drug Delivery" (Marcel Dekker, 1992), and at
places elsewhere in the text. All of these references are
incorporated herein for the sole purpose of illustrating the level
of knowledge and skill in the art concerning drug delivery
techniques.
[0104] A colloidal dispersion system may be used for targeted
delivery of the immunomodulatory nucleic acid molecules to specific
tissue. Colloidal dispersion systems include macromolecule
complexes, nanocapsules, microspheres, beads, and lipid-based
systems including oil-in-water emulsions, micelles, mixed micelles,
and liposomes.
[0105] Liposomes are artificial membrane vesicles which are useful
as delivery vehicles in vitro and in vivo. It has been shown that
large unilamellar vesicles (LUV), which range in size from 0.2-4.0
Fm can encapsulate a substantial percentage of an aqueous buffer
comprising large macromolecules. RNA and DNA can be encapsulated
within the aqueous interior and be delivered to cells in a
biologically active form (Fraley, et al., (1981) Trends Biochem.
Sci., 6:77). The composition of the liposome is usually a
combination of phospholipids, particularly
high-phase-transition-temperature phospholipids, usually in
combination with steroids, especially cholesterol. Other
phospholipids or other lipids may also be used. The physical
characteristics of liposomes depend on pH, ionic strength, and the
presence of divalent cations. Examples of lipids useful in liposome
production include phosphatidyl compounds, such as
phosphatidylglycerol, phosphatidylcholine, phosphatidylserine,
phosphatidylethanolamine, sphingolipids, cerebrosides, and
gangliosides. Particularly useful are diacylphosphatidylglycerols,
where the lipid moiety contains from 14-18 carbon atoms,
particularly from 16-18 carbon atoms, and is saturated.
Illustrative phospholipids include egg phosphatidylcholine,
dipalmitoylphosphatidylcholine and
distearoylphosphatidylcholine.
[0106] Where desired, targeting of liposomes can be classified
based on anatomical and mechanistic factors. Anatomical
classification is based on the level of selectivity, for example,
organ-specific, cell-specific, and organelle-specific. Mechanistic
targeting can be distinguished based upon whether it is passive or
active. Passive targeting utilizes the natural tendency of
liposomes to distribute to cells of the reticulo-endothelial system
(RES) in organs which contain sinusoidal capillaries. Active
targeting, on the other hand, involves alteration of the liposome
by coupling the liposome to a specific ligand such as a monoclonal
antibody, sugar, glycoiipid, or protein, or by changing the
composition or size of the liposome in order to achieve targeting
to organs and cell types other than the naturally occurring sites
of localization.
[0107] The surface of the targeted delivery system may be modified
in a variety of ways. In the case of a liposomal targeted delivery
system, lipid groups can be incorporated into the lipid bilayer of
the liposome in order to maintain the targeting ligand in stable
association with the liposomal bilayer. Various well known linking
groups can be used for joining the lipid chains to the targeting
ligand (see, e.g., Yanagawa, et al., (1988) Nuc. Acids Symp. Ser.,
19:189; Grabarek, et al, (1990) Anal. Biochem. 185:131; Staros et
al. (1986) Anal. Biochem. 156:220 and Boujrad, et al., (1993) Proc.
Natl. Acad. Sci. USA, 90:5728). Targeted delivery of
immunomodulatory nucleic acid molecules can also be achieved by
conjugation of the immunostimulatory nucleic acid molecules to a
the surface of viral and non-viral recombinant expression vectors,
to an antigen or other ligand, to a monoclonal antibody or to any
molecule which has the desired binding specificity.
[0108] Routes of Administration
[0109] Immunomodulatory nucleic acid molecules are administered to
an individual using any available method and route suitable for
drug delivery, including in vivo and ex vivo methods, as well as
systemic and localized routes of administration.
[0110] Conventional and pharmaceutically acceptable routes of
administration include intranasal, intramuscular, intratracheal,
intratumoral, subcutaneous, intradermal, topical application,
intravenous, rectal, nasal, oral and other parenteral routes of
administration. Routes of administration may be combined, if
desired, or adjusted depending upon the immunomodulatory nucleic
acid and/or the desired effect on the immune response. The
immunomodulatory nucleic acid composition can be administered in a
single dose or in multiple doses, and may encompass administration
of booster doses, to elicit and/or maintain the desired effect on
the immune response.
[0111] Immunomodulatory nucleic acid molecules can be administered
to a host using any available conventional methods and routes
suitable for delivery of conventional drugs, including systemic or
localized routes. In general, routes of administration contemplated
by the invention include, but are not necessarily limited to,
enteral, parenteral, or inhalational routes. Inhalational routes
may be preferred in cases of pulmonary involvement, particularly in
view of the activity of certain immunomodulatory nucleic acid
molecules as a mucosal adjuvant.
[0112] Inhalational routes of administration (e.g., intranasal,
intrapulmonary, and the like) are particularly useful in
stimulating an immune response for prevention or treatment of
infections of the respiratory tract. Such means include inhalation
of aerosol suspensions or insufflation of the polynucleotide
compositions of the invention. Nebulizer devices, metered dose
inhalers, and the like suitable for delivery of polynucleotide
compositions to the nasal mucosa, trachea and bronchioli are
well-known in the art and will therefore not be described in detail
here. For general review in regard to intranasal drug delivery,
see, e.g., Chien, Novel Drug Delivery Systems, Ch. 5 (Marcel
Dekker, 1992).
[0113] Parenteral routes of administration other than inhalation
administration include, but are not necessarily limited to,
topical, transdermal, subcutaneous, intramuscular, intraorbital,
intracapsular, intraspinal, intrasternal, and intravenous routes,
i.e., any route of administration other than through the alimentary
canal. Parenteral administration can be carried to effect systemic
or local delivery of immunomodulatory nucleic acid molecules. Where
systemic delivery is desired, administration typically involves
invasive or systemically absorbed topical or mucosal administration
of pharmaceutical preparations.
[0114] Immunomodulatory nucleic acid molecules can also be
delivered to the subject by enteral administration. Enteral routes
of administration include, but are not necessarily limited to, oral
and rectal (e.g., using a suppository) delivery.
[0115] Methods of administration of immunomodulatory nucleic acid
molecules through the skin or mucosa include, but are not
necessarily limited to, topical application of a suitable
pharmaceutical preparation, transdermal transmission, injection and
epidermal administration. For transdermal transmission, absorption
promoters or iontophoresis are suitable methods. For review
regarding such methods, those of ordinary skill in the art may wish
to consult Chien, supra at Ch. 7. Iontophoretic transmission may be
accomplished using commercially available "patches" which deliver
their product continuously via electric pulses through unbroken
skin for periods of several days or more. An exemplary patch
product for use in this method is the LECTRO PATCH.TM.
(manufactured by General Medical Company, Los Angeles, Calif.)
which electronically maintains reservoir electrodes at neutral pH
and can be adapted to provide dosages of differing concentrations,
to dose continuously and/or to dose periodically.
[0116] Epidermal administration can be accomplished by mechanically
or chemically irritating the outermost layer of the epidermis
sufficiently to provoke an immune response to the irritant. An
exemplary device for use in epidermal administration employs a
multiplicity of very narrow diameter, short tynes which can be used
to scratch immunomodulatory nucleic acid molecules coated onto the
tynes into the skin. The device included in the MONO-VACC.TM.
tuberculin test (manufactured by Pasteur Merieux, Lyon, France) is
suitable for use in epidermal administration of immunostimulatory
nucleic acid molecules.
[0117] The invention also contemplates opthalmic administration of
immunomodulatory nucleic acid molecules, which generally involves
invasive or topical application of a pharmaceutical preparation to
the eye. Eye drops, topical creams and injectable liquids are all
examples of suitable formulations for delivering drugs to the
eye.
[0118] Dosages
[0119] Although the dosage used will vary depending on the clinical
goals to be achieved, a suitable dosage range is one which provides
up to about 1 .mu.g, about 1,000 .mu.g, about 10,000 .mu.g, about
20,000 .mu.g, about 30,000 .mu.g, about 40,000 .mu.g, or about
50,000 .mu.g of immunomodulatory nucleic acid molecule can be
administered in a single dose. Alternatively, a target dosage of
immunomodulatory nucleic acid molecule can be considered to be
about 1-10 .mu.M in a sample of host blood drawn within the first
24-48 hours after administration of immunomodulatory nucleic acid
molecules. Based on current studies, immunomodulatory nucleic acid
molecules are believed to have little or no toxicity at these
dosage levels.
[0120] It should be noted that the activity of an immunomodulatory
nucleic acid molecules is generally dose-dependent. Therefore, to
increase immunomodulatory nucleic acid molecules potency by a
magnitude of two, each single dose is doubled in concentration.
Increased dosages may be needed to achieve the desired therapeutic
goal. The invention thus contemplates administration of "booster"
doses to provide and maintain a desired immune response. For
example, immunomodulatory nucleic acid molecules may be
administered at intervals ranging from at least every two weeks to
every four weeks (e.g., monthly intervals) (e.g., every four
weeks).
[0121] Cell Death-Related Disorders Which are Amenable to
Treatment
[0122] Cell death-related indications which can be treated using
the methods of the invention for reducing cell death in a
eukaryotic cell, include, but are not limited to, cell death
associated with Alzheimer's disease, Parkinson's disease,
rheumatoid arthritis, septic shock, sepsis, stroke, central nervous
system inflammation, osteoporosis, ischemia, reperfusion injury,
cell death associated with cardiovascular disease, polycystic
kidney disease, cell death of endothelial cells in cardiovascular
disease, degenerative liver disease, multiple sclerosis, amyotropic
lateral sclerosis, cerebellar degeneration, ischemic injury,
cerebral infarction, myocardial infarction, acquired
immunodeficiency syndrome (AIDS), myelodysplastic syndromes,
aplastic anemia, male pattern baldness, and head injury damage.
Also included are conditions in which DNA damage to a cell is
induced by, e.g., irradiation, radiomimetic drugs, and the like.
Also included are any hypoxic or anoxic conditions, e.g.,
conditions relating to or resulting from ischemia, myocardial
infarction, cerebral infarction, stroke, bypass heart surgery,
organ transplantation, neuronal damage, and the like.
[0123] Cell death-related indications which can be treated using
methods of the invention for activating cell death include, but are
not limited to, undesired, excessive, or uncontrolled cellular
proliferation, including, for example, neoplastic cells; as well as
any undesired cell or cell type in which induction of cell death is
desired, e.g., virus-infected cells and self-reactive immune cells.
The methods may be used to treat follicular lymphomas, carcinomas
associated with p53 mutations; autoimmune disorders, such as, for
example, systemic lupus erythematosus (SLE), immune-mediated
glomerulonephritis; hormone-dependent tumors, such as, for example,
breast cancer, prostate cancer and ovary cancer; and viral
infections, such as, for example, herpesviruses, poxviruses and
adenoviruses.
[0124] Subjects to be treated according to the methods of the
invention include any individual having any of the above-mentioned
disorders. Further included are individuals who are at risk of
developing any of the above-mentioned disorders, including, but not
limited to, an individual who has suffered a myocardial infarction,
and is therefore at risk for experiencing a subsequent myocardial
infarction; an individual who has undergone organ or tissue
transplantation; an individual who has had a stroke and is at risk
for having a subsequent stroke; and an individual at risk of
developing an autoimmune disorder due to genetic predisposition, or
due to the appearance of early symptoms of autoimmune disorder.
[0125] Methods of Identifying an Agent that Modulate a Biological
Activity of DNA-PK
[0126] The present invention provides methods of identifying agents
which modulate a biological activity of DNA-PK ("screening
methods"). In some embodiments, the screening methods are
cell-based methods. In other embodiments, the screening methods are
cell-free methods. The term "modulate" encompasses an increase or a
decrease in the detected DNA-PK biological activity when compared
to a suitable control.
[0127] The methods generally comprise:
[0128] a) contacting a substance to be tested with a sample
comprising DNA-PK, forming a test sample; and
[0129] b) detecting a biological activity of the DNA-PK in the test
sample as compared to a control sample lacking the test agent.
[0130] An increase or a decrease in the biological activity of the
DNA-PK indicates that the agent modulates a biological activity of
the DNA-PK polypeptide.
[0131] Any biological activity of DNA-PK can be detected,
including, but not limited to, (1) enzymatic activity of DNA-PK in
phosphorylating a substrate polypeptide; (2) the level of cell
death in a cell population as an indication of the level of DNA-PK
activity; (3) Ku binding to DNA-PKcs; (4) binding of an
immunomodulatory nucleic acid molecule to Ku; (5) activity of a
polypeptide in the pathway of DNA-PK activition, e.g., Akt-1; and
(6) binding of a polypeptide (e.g., replication protein A, "RPA")
to the DNA-PK complex. "Detecting," as used herein, refers to
determining the presence or absence of a biological activity;
determining a relative increase or decrease in a biological
activity; and measuring quantitatively the level of a biological
activity.
[0132] In some embodiments, a detected biological activity of a
DNA-PK is binding between the Ku polypeptide and the
immunomodulatory nucleic acid molecule, wherein an increase or a
decrease in binding activity in comparison to Ku binding activity
in a suitable control is an indication that the substance modulates
a biological activity of the Ku polypeptide.
[0133] In these embodiments, the methods comprise:
[0134] a) contacting a substance to be tested with a sample
comprising DNA-PK and an immunomodulatory nucleic acid molecule,
thereby forming a test sample; and
[0135] b) detecting the effect, if any, on a biological activity of
DNA-PK in the test sample as compared to a control sample lacking
the test agent.
[0136] In other embodiments, a detected biological activity of a
DNA-PK is activation of DNA-PKcs activity. The readout may be
direct measurement of DNA-PKcs activity (as described above);
production of IL-6 or IL-12 (as measured by a polymerase chain
reaction, e.g., for measuring IL-6 or IL-12 mRNA levels; or ELISA,
for measuring IL-6, TNF-.alpha., or IL-12 protein levels); or any
other measurement, direct or indirect, of a DNA-PK biological
activity.
[0137] In other embodiments, a detected biological activity of
DNA-PK is binding of DNA-PK with other polypeptides such as
replication protein A, heat shock factor-1, and any other
polypeptide to which DNA-PK (or a component thereof) is known to
bind. DNA-PK-interacting polypeptides include, but are not limited
to, replication protein A (RPA), a heterotrimeric single-stranded
DNA-binding protein (Zou and Stillman (2999) Mol. Cell. Biol.
20:3086-3096; and Shao et al. (1999) EMBO J. 18:1397-1406; and heat
shock factor-1 (HSF-1) (Morano and Thiele (1999) Gene Expression
7:271-282; and Nueda et al. (1999) J. Biol. Chem. 274:14988-14996).
Binding of DNA-PK to a DNA-PK-interacting polypeptide can be
measured using any method known in the art to measure
protein-protein interactions, including, but not limited to,
protein interactive trapping assays, and immunological assays
(e.g., using an antibody to one component to immunoprecipitate the
complex). Methods of measuring protein-protein interaction are
well-documented in the art in a variety of publications, including,
e.g., Current Protocols in Molecular Biology, (F. M. Ausubel, et
al., Eds. 1987, and periodic updates).
[0138] In carrying out these methods, DNA-PK complex (i.e.,
DNA-PKcs and Ku), or an isolated component of a DNA-PK complex, can
be used. The Ku polypeptide can be a full-length polypeptide (e.g.,
has an amino acid sequence of the same length as that found in its
natural environment, or "wild-type" sequence), but need not be
full-length, as long as the Ku polypeptide retains measurable
immunomodulatory nucleic acid molecule binding activity and
measurable DNA-PKcs activating activity. The Ku polypeptide used in
these assays may also contain alterations in amino acid sequence
compared to the wild-type sequence, wherein such alterations may
confer a desirable property, including, but not limited to,
enhanced stability in vitro, and the like. The Ku polypeptide may
further be a fusion protein comprising a Ku polypeptide and a
heterologous polypeptide, e.g. a non-Ku polypeptide, including, but
not limited to, a epitope to facilitate recovery of the Ku
polypeptide from the sample, and fhe like. Similary, DNA-PKcs used
in these assays may be wild-type, or may be a fragment of
wild-type; a synthetic fragment; a fusion protein comprising
DNA-PKcs or a variant thereof; may comprise one or more amino acid
substitutions, additions, insertions, and/or deletions compared to
wild-type DNA-PKcs; as long as the DNA-PKcs retains measurable
kinase activity toward one or more physiological substrates of
wild-type DNA-PKcs and/or retains measurable Ku binding and/or
retains measurable binding to other polypeptides that interact with
wild-type DNA-PK in vivo or in vitro.
[0139] Cell-free assays, i.e., assays which measure a Ku antigen
binding to an immunomodulatory nucleic acid molecule, include, but
are not limited to, protein-DNA binding assays, electrophoretic
mobility shift assays, and the like. Using these methods, one can
identify substances that bind specifically to Ku antigen and
inhibit binding of a labeled immunomodulatory nucleic acid
molecule.
[0140] The screening assay can be a binding assay, wherein one or
more of the molecules may be joined to a label, and the label
directly or indirectly provide a detectable signal. Various labels
include radioisotopes, fluorescers, chemiluminescers, enzymes,
specific binding molecules, particles, e.g. magnetic particles, and
the like. Specific binding molecules include pairs, such as biotin
and streptavidin, digoxin and antidigoxin etc. For the specific
binding members, the complementary member would normally be labeled
with a molecule that provides for detection, in accordance with
known procedures.
[0141] Where the method is a cell-free assay method, Ku polypeptide
may be, but need not be, substantially purified. In general, the Ku
polypeptide should be isolated from the source of Ku in those
instances where one or more components found in the source of Ku
interfere with binding activity or measurement of binding activity.
The sample can be a cell lysate comprising Ku, or the sample can
comprise Ku which is purified to any degree. As non-limiting
examples, the sample can be: a cell lysate of a mammalian cell line
which has been transfected with a recombinant vector ("construct")
which encodes and expresses Ku polypeptide having immunomodulatory
nucleic acid molecule binding activity; and Ku which has been
purified from a biological source.
[0142] In some embodiments, the biological activity of DNA-PK being
measured is binding of Ku to an immunomodulatory nucleic acid
molecule in the test sample. Where the biological activity of Ku
being measured is Ku-immunomodulatory nucleic acid molecule binding
activity, binding activity may be measured using any known method.
In general, the immunomodulatory nucleic acid molecule is labeled
with a detectable label, and the amount of label in a complex of Ku
and immunomodulatory nucleic acid molecule is an indication of
binding activity. Complexes formed upon binding of Ku and
immunomodulatory nucleic acid molecule can be detected using
antibody specific for Ku, or, if an epitope-tagged Ku antigen is
used, an antibody specific for the epitope tag. Immunomodulatory
nucleic acid molecules may be labeled with any of a variety of
detectable labels, including radioactive labels, biotin,
fluorescent labels, and the like. Suitable labels include
fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine,
Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein
(6-FAM), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE),
6-carboxy-X-rhodamine (ROX),
6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX),
5-carboxyfluorescein (5-FAM) or N,N,N',N'-tetramethyl-6-carboxyrho-
damine (TAMRA), radioactive labels, e.g. .sup.32P, .sup.35S,
.sup.3H; etc. The label may be a two stage system, where the
nucleic acid molecule is conjugated to biotin, haptens, etc. having
a high affinity binding partner, e.g. avidin, specific antibodies,
etc., where the binding partner is conjugated to a detectable
label.
[0143] Agents identified in a cell-free assay may be selected for
further study, and assessed for bioavailability, cellular
availability, cytotoxicity, biocompatibility, etc.
[0144] Where the method is a cell-based assay method, a test sample
comprises an intact eukaryotic cell, and an agent to be tested is
added. A biological activity of DNA-PK, as described above, can be
measured in the intact cell, or in a cell lysate made from the
cell. In some embodiments, the test sample further comprises an
immunomodulatory nucleic acid molecule.
[0145] As a non-limiting example of a cell-based method, a cell
which synthesizes DNA-PK is contacted with an immunomodulatory
nucleic acid molecule, such that the immunomodulatory nucleic acid
molecule enters the cell and binds to Ku. The cell is also
contacted with a substance to be tested. The substance to be tested
is contacted with the cell either substantially simultaneously
with, before, or after, contacting with the immunomodulatory
nucleic acid molecule. After a suitable time, Ku binding to the
immunomodulatory nucleic acid molecule is assessed, e.g., by lysing
the cells, and measuring Ku binding activity in the cell
lysates.
[0146] Alternatively, the cells need not be lysed in order to
measure Ku binding activity. In these embodiments, Ku binding is
measured in intact cells. Ku binding may be indicated in intact
cells by assaying for secretion of a cytokine, such as IL-6 or
IL-12, which is produced by certain cells, such as macrophages,
upon Ku-immunomodulatory nucleic acid molecule binding.
[0147] As a non-limiting example, a construct comprising a
nucleotide sequence encoding Ku polypeptide is introduced into a
cell line (e.g., a macrophage cell line, or a cell line that is Ku
deficient, e.g., a BMDM cell derived or isolated from a
Ku70.sup.-/- or Ku 80.sup.-/- animal) such that Ku polypeptide is
expressed in the cells. For these assays, the Ku coding region may
be under control of an endogenous promoter, or, alternatively,
under control of an inducible promoter. Inducible promoters are
known in the art, and can be used in such a construct. Suitable
inducible promoters include, but are not limited to, a
hormone-inducible promoter. When an inducible promoter is used, the
inducer is added to the cell culture before, or simultaneously
with, the substance being tested. Controls include a culture to
which no inducer has been added, as well as a culture to which
inducer, but no substance being tested, is added. If the assay is
conducted in a cell line which is not Ku deficient, a Ku-deficient
cell line may be used as a negative control.
[0148] Assays such as those described herein are amenable to high
through-put screening assays. For example, isolated DNA-PK,
isolated Ku, or cells comprising endogenous DNA-PK, or cells
expressing a construct encoding DNA-PK complex, or cells expressing
a construct encoding Ku, each in separate well of a microtiter
plate, e.g., can be contacted with a large number of test compounds
at a time, thereby allowing automation.
[0149] The term "agent" is used interchangeably herein with the
terms "substance" and "compound". An "agent which modulates a
biological activity of DNA-PK," as used herein, describes any
molecule, e.g. protein; peptide; natural or synthetic inorganic or
organic compound, or pharmaceutical, with the capability of
altering one or more biological activities of DNA-PK, as described
herein. Generally a plurality of assay mixtures are run in parallel
with different agent concentrations to obtain a differential
response to the various concentrations. Typically, one of these
concentrations serves as a negative control, i.e. at zero
concentration or below the level of detection.
[0150] Candidate agents encompass numerous chemical classes, and
may be natural or synthetic inorganic or organic molecules, which
may be small inorganic or organic compounds having a molecular
weight of more than 50 and less than about 5000 daltons, or which
may be larger compounds (e.g., larger than 5000 daltons), such as
macromolecules (e.g., polypeptides, glycopeptides, and the like).
Candidate agents include naturally-occurring compounds, synthetic
compounds, and semi-synthethic compounds. Candidate agents may
comprise functional groups necessary for structural interaction
with proteins, particularly hydrogen bonding, and typically include
at least an amine, carbonyl, hydroxyl or carboxyl group, or at
least two of the functional chemical groups. The candidate agents
may comprise cyclical carbon or heterocyclic structures and/or
aromatic or polyaromatic structures substituted with one or more of
the above functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof.
[0151] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides and oligopeptides.
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available or
readily produced. Additionally, natural or synthetically produced
libraries and compounds are readily modified through conventional
chemical, physical and biochemical means, and may be used to
produce combinatorial libraries. Known pharmacological agents may
be subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, glycosylation,
amidification, etc. to produce structural analogs.
[0152] A variety of other reagents may be included in the screening
assay. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc., that are used to facilitate optimal
nucleic acid-protein binding, and/or protein-protein binding,
and/or reduce non-specific or background interactions. Reagents
that improve the efficiency of the assay, such as protease
inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be
used. The mixture of components are added in any order that
provides for the requisite binding. Incubations are performed at
any suitable temperature, typically between 4.degree. C. and
40.degree. C. Incubation periods are selected for optimum activity,
but may also be optimized to facilitate rapid high-throughput
screening. Typically between 0.1 hour and 1 hour will be
sufficient.
[0153] Agents Identified by the Screening Methods of the
Invention
[0154] The invention further provides an agent identified by a
screening method of the invention (an "identified agent"), and
compositions comprising an identified agent. Compositions may
comprise a single agent, or may comprise a mixture of two or more
different agents, depending on the desired effect on an immune
response or on cell viability. These compositions may comprise a
buffer, which is selected according to the desired use of the
agent(s), and may also include other substances appropriate to the
intended use. Those skilled in the art can readily select an
appropriate buffer, a wide variety of which are known in the art,
suitable for an intended use. In some instances, the composition
can comprise a pharmaceutically acceptable excipient, a variety of
which are known in the art and need not be discussed in detail
herein. Pharmaceutically acceptable excipients have been amply
described in a variety of publications, including, for example, A.
Gennaro (2000) "Remington: The Science and Practice of Pharmacy",
20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical
Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al.,
eds 7.sup.th ed., Lippincott, Williams, & Wilkins; and Handbook
of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds.,
3.sup.rd ed. Amer. Pharmaceutical Assoc.
[0155] Methods Using Identified Agents
[0156] The present invention provides methods of modulating an
immune response in an individual, generally comprising
administering an identified agent to an individual. Agents
identified by the screening methods of the invention may enhance,
mimic, or inhibit an activity of an immunomodulatory nucleic acid
molecule, and may therefore modulate an immune response in an
individual. Immunomodulatory nucleic acid molecules may increase a
Th1 or a Th2 immune response in a eukaryotic cell. Thus, an
identified agent may enhance, mimic, or inhibit a Th1 or a Th2
immune response in a eukaryotic cell. An identified agent can also
be used in methods to modulate cell death, and methods of reducing
DNA damage, which methods are described above.
[0157] In some embodiments, methods are provided for increasing a
Th1 response in an individual, comprising administering the agent
to an individual. The agent may be administered before,
simultaneously with, or after the subject is exposed to antigen.
Exposure to antigen can be via intentional introduction by a
clinician, other medical personnel, or researcher, or may be via
random, unintentional encounter with antigen. Whether a Th1
response is increased, induced, or enhanced can be determined by
measuring any parameter associated with a Th1 response, including,
but not limited to, production of cytokines normally associated
with a Th1 response, including, but not limited to, IL-2 and
IFN-.gamma.; and production of Ig2a or its equivalent, an antibody
isotype normally associated with a Th1 response. Cytokine
production can be measured by any known means, including, but not
limited to, a polymerase chain reaction (PCR), using
oligonucleotide primers specific for a cytokinc; enzyme-linked
immunosorbent assay (ELISA), using cytokine-specific antibody; and
the like.
[0158] In some embodiments, an identified agent may be used in
methods for reducing, or inhibiting, a Th2 response in a vertebrate
host, comprising administering the agent to an individual. The
agent may be administered before, simultaneously with, or after the
subject is exposed to antigen. Whether a Th2 response has been
reduced or inhibited can be determined by measuring any parameter
associated with a Th2 response, using any method known in the art,
including, but not limited to, measuring cytokine production
normally associated with a Th2 response, including, but not limited
to, IL-4, IL-6, and IL-10; and measuring production of antibody
isotypes normally associated with a Th2 response, including IgA,
and IgE, and IgG1, or their equivalents.
[0159] In the above-described methods, the identified agent is
generally administered in a formulation together with a
pharmaceutically acceptable excipient, as described above. An
identified agent is administered to an individual using any
available method and route suitable for drug delivery, including in
vivo and ex vivo methods, as well as systemic and localized routes
of administration.
[0160] Conventional and pharmaceutically acceptable routes of
administration include intranasal, intramuscular, intratracheal,
intratumoral, subcutaneous, intradermal, topical application,
intravenous, rectal, nasal, oral and other parenteral routes of
administration. Routes of administration may be combined, if
desired, or adjusted depending upon the identified agent and/or the
desired effect on the immune response. The composition comprising
an identified agent can be administered in a single dose or in
multiple doses, and may encompass administration of booster doses,
to elicit and/or maintain the desired effect on the immune
response. Although the dosage used will vary depending on the
clinical goals to be achieved, a suitable dosage range is one which
provides up to about 1 .mu.g to about 1,000 .mu.g or about 10,000
.mu.g of immunomodulatory nucleic acid molecule can be administered
in a single dose. Single or multiple doses can be administered.
Appropriate doses and regimens can be readily determined by the
clinician.
EXAMPLES
[0161] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Celsius, and pressure
is at or near atmospheric.
Example 1
Identification of an ISS-Binding Protein
[0162] The putative immunostimulatory (ISS) binding protein was
isolated by using a multi-step purification strategy. Crude mouse
liver lysates were used as the protein source after confirming
liver responsiveness to intravenous ISS injection in vivo.
[0163] Liver Cells Respond to ISS
[0164] Protocols
[0165] ISS oligodeoxynucleotide (ISS-ODN)
(5'-TGACTGTGAACGTTCGAGATGA-3'; SEQ ID NO:1) and mutated (M-ODN)
(5'-TGACTGTGAACCTTAGAGAA-3'; SEQ ID NO:2) phosphodiester or
phosphorothioate ODNs were purchased from Trilink Biotechnologies
(San Diego, Calif.). Total cellular RNA was isolated from spleen of
ISS-ODN, M-ODN or PBS injected mice using Stratagene RNA isolation
kit (San Diego, Calif.) and subjected to reverse
transcription-polymerase chain reaction (RT-PCR). First strand cDNA
preparation and PCR amplification were performed using the
SuperScript preamplification system (Gibco BRL, Gaithersburg, Md.)
and AdvanTaq Plus DNA polymerase (Clontech, San Francisco, Calif.),
respectively. The primer sequence used were as follows:
1 (SEQ ID NO:3) IL-6 sense 5'-ATGAAGTTCCTCTCTGCAAGAGACT-3' (SEQ ID
NO:4) antisense 5'-CACTAGGTTTGCCGAGTAGATC- TC-3' (SEQ ID NO:5)
IL-12p40 sense 5'-GGGACATCATCAAACCAGACC-3' (SEQ ID NO:6) antisense
5'-GCCAACCAAGCAGAAGACAGC-3' (SEQ ID NO:7) GAPDH sense
5'-ACCACAGTCCATGCCATCAC-3' (SEQ ID NO:8) antisense
5'-TCCACCACCCTGTTGCTGTA-3'
[0166] PCR were performed under the following conditions by
appropriate cycling number (94.degree. C. for 30 seconds;
65.degree. C. for 30 seconds; and 68.degree. C. for 30 seconds).
PCR products were visualized by electrophoresis on 1.5% TAE agarose
gels after being stained with ethidium bromide. BMDM isolated from
either wild type or Ku 70.sup.-/- mice were treated with ISS (10
.mu.g/ml) or LPS (10 .mu.g/ml) or left untreated for 6.5 hours.
Total RNA was isolated and 10 .mu.g of total RNA was separated on
1% agarose gel and then transferred onto a nylon membrane. The
membrane was probed with [.sup.32P]-labeled IL-6 or IL-12 or GAPDH
cDNA followed by autoradiography, as described previously. Chu et
al. (1999) Immunity 11:1.
[0167] Results
[0168] Mice were intravenously injected with 200 .mu.l (1
.mu.g/.mu.l in PBS) of single-stranded (ss) or double-stranded (ds)
ISS-ODN (5'-TGACTGTGAACGTTCGAGATGA-3'; SEQ ID NO:1), ss or ds
inactive, mutated (M), M-ODN (5'-TGACTGTGAACCTTAGAGATGA-3'; SEQ ID
NO:9), or 200 .mu.l of 1.times. PBS. After 2.5 hours, the mice were
euthanized and RNA extracted from their livers. This RNA was used
in an RT-PCR assay to detect the presence of IL-6, IL-12 and GAPDH
messages. The M-ODN did not display any immune stimulation. In
contrast, both IL-6 and IL-12 message was readily detected in mice
injected with ISS-ODN. The results are shown in FIG. 1A.
[0169] Purification of the ISS Binding Protein
[0170] Purification of the ISS binding protein was accomplished by
sequential ion exchange, heparin and ISS-ODN affinity
chromatography, as follows. Due to the relative small tissue weight
per spleen, liver lysate was used after confirming its reactivity
to in vivo ISS injection, as described above. Crude liver extracts
from 220 mice (BALB/c, Jackson Lab., Bar Harbour, Me.) or 15 New
Zealand White rabbits (Simunek, Vista, Calif.) were prepared in
homogenization buffer (20 mM HEPES, pH 7.6, 250 mM KCl, 0.1 mM
EDTA, 0.5 mM EGTA, 20% glycerol) with 250 mM KCl and protease and
phosphatase inhibitors and then centrifuged at 100,000.times.g for
1.5 hours at 4.degree. C. The supernatant (400 ml) was filtered and
loaded onto a Porous 20 QE column (17 ml, Pharmacia). Two .mu.l (10
.mu.g) of each fraction were used in a gel retardation assay to
identify the location of proteins containing DNA binding activity
using [.sup.32P]-labeled ds-ISS-ODN as a probe. Fractions
containing ISS-ODN binding activity were pooled (450 ml), buffered
exchanged to low salt (50 mM KCl) and loaded onto a Heparin column
(2 ml). The active fractions were subsequently loaded onto a 2 ml
ds-ISS-ODN affinity column [CNBr-activated Sepharose 4B (Pharmacia)
coupled to 320 .mu.g of ds-ISS-ODNs with the sequence
5'-TGACTGAACGTTCGAGATGA-3'; SEQ ID NO:21]. The column was washed
with 8 ml of homogenization buffer containing 50 mM KCl and the
bound proteins were eluted with a 20 ml linear gradient of 50 mM to
1.55 M KCl. One-ml fractions were collected and 2 .mu.l of each
were used to test for DNA binding activity. The active fractions
were concentrated, separated by SDS-PAGE and stained with Coomassie
Blue. The appearance of two bands with estimated molecular weights
of 70 KDa and 80 KDa correlated with the DNA binding activity.
Sequence analysis (Harvard Microsequencing Facility) of peptides
derived from the two bands identified Ku70 and Ku80,
respectively.
[0171] Mouse liver extracts were purified through anion (QE),
heparin and ISS-based affinity chromatography, as described above.
Two .mu.l of each fraction (10 .mu.g) were incubated with [.sup.32
P]-labeled ds-ISS-ODN at 4.degree. C. for 30 minutes. The samples
were separated on a 5% acrylamide gel and the protein-DNA complexes
were detected by autoradiography. Only three fractions from the
final affinity column displayed selective reactivity with
[.sup.32P]-labeled ds-ISS-ODN. The active fractions were separated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), stained with Coomassie blue and micro-sequenced.
[0172] Sequence analysis of peptides derived from the two bands
identified the Ku70/Ku80 heterodimer as an ISS binding protein.
Fractions from the ISS-ODN affinity column were separated by
SDS-PAGE and stained with Coomassie blue. Sequence analysis of two
bands with approximate molecular sizes of 64 KDa and 97 kDa, which
correlated with the presence of DNA binding activity were
identified as Ku70 and Ku80, respectively (arrows). The results are
shown in FIG. 1B. The Ku heterodimer was identified as an ISS
binding protein in four independent experiments.
[0173] The Ku heterodimer was further confirmed as an ISS binding
protein in a supershift assay using anti-Ku Abs. Ten .mu.g of
fraction 20 from the affinity column were incubated with anti-Ku or
control (JNK) monoclonal antibodies (mAbs) for 30 minutes prior to
probing with ds [.sup.32P]-labeled ISS-ODN in an electrophoretic
mobility shift (EMSA) assay.
[0174] To verify the binding specificity of the Ku protein to
ISS-ODN, inhibition studies were performed using cold ss or ds ISS
or mutated (M, i.e., control) ODNs. The results are shown in FIGS.
1C and 1D. Ten .mu.g of fraction 20 from the affinity column were
incubated with [.sup.32P]-labeled ISS-ODN in the presence of
unlabeled (0.1, 0.2 and 0.4 .mu.g, respectively) ss-ISS-ODN (lanes
3-5), ds-ISS-ODN (lanes 7-9), ss-M-ODN (lanes 11-13) or ds-M-ODN
(lanes 15-17). As shown in FIGS. 1C and 1D, both cold ds and
ss-ISS-ODNs displaced the interaction of [.sup.32P]-labeled
ds-ISS-ODN with Ku from the eluted fraction while the ds and
ss-M-ODNs did not have any significant effect on this
interaction.
[0175] The specific interaction of Ku with ISS-ODN and the lack of
interaction of Ku with M-ODN correlates with cytokine induction by
ISS-ODN and the lack of induction by M-ODN (FIG. 1A). Following the
same methodology, the Ku protein was also isolated and identified
as a specific ISS binding protein from crude rabbit liver
extract.
Example 2
ISS Stimulate Secretion of IL-6 and IL-12 from Bone Marrow-Derived
Macrophages from Wild-Type Control Mice, but not from Ku70.sup.-/-
or Ku80.sup.-/- Mice
[0176] To evaluate the potential role of Ku in ISS induction of
IL-6 and IL-12, bone marrow derived macrophages (BMDM) were grown
from wild type (wt), Ku70.sup.-/- and Ku80.sup.-/- mice and
stimulated with ISS-ODN. Martin-Orozco et al. (1999) Int. Immunol.
11:1111.
[0177] Ku70.sup.-/- and Ku80.sup.-/- mice and their wild-type (wt)
control on the 129 genetic background were generated by Dr. G. Li
and bred at Memorial Sloan-Kettering Cancer Center (MSKCC), New
York, N.Y. BMDM from wt, Ku70.sup.-/- and Ku80.sup.-/- mice (MSKCC)
were prepared as was previously published (Martin-Orozco et al.
(1999) Int. Immunol. 11:1111), and maintained in DMEM with 10% FBS,
antibiotics and 20% L-cell medium and cultured for about 10 days to
allow them to mature. BMDM were seeded (2.5.times.10.sup.5/well in
triplicate) on 96-well plates and treated with LPS (10 .mu.g/ml),
ISS-ODN (5 .mu.g/ml) or M-ODN (5 .mu.g/ml).
[0178] The results are shown in FIGS. 2A-C. BMDM
(2.5.times.10.sup.5/well) from wt, or Ku70.sup.-/- and Ku80.sup.-/-
mice were treated with LPS (1 .mu.g/ml), ISS-ODN (5 .mu.g/ml) or
M-ODN (5 .mu.g/ml) for 24 hours. IL-6 (A) or IL-12 (B) levels in
the supernatants were determined by ELISA. Results are
means.+-.S.D, of 5 separate experiments for wt and Ku70.sup.-/- and
3 separate experiments for Ku80.sup.-/- mice. (C) BMDM were
incubated with LPS (10 .mu.g/ml) or ISS-ODN (5 .mu.g/ml) for 6.5
hours. Total RNA was isolated and analyzed for the presence of
IL-6, IL-12 and GAPDH messages by a Northern blot assay, as
described in Example 1. Results are means.+-.S.D. of 5 separate
experiments for wt and Ku70.sup.-/- and 3 separate experiments for
Ku80.sup.-/- mice.
[0179] As shown in FIGS. 2A-C, BMDM from Ku70.sup.-/- and
Ku80.sup.-/- mice secreted very low levels of IL-6 (FIG. 2A) and
IL-12 (FIG. 2B) as compared to BMDM from wt controls. The
diminished response was ISS specific since LPS stimulation resulted
in the induction of IL-6 and IL-12 from both wt and Ku70.sup.-/-
BMDM. The lack of induction of IL-6 and IL-12 by ISS in
Ku70.sup.-/- or Ku80.sup.-/- BMDM was further confirmed by Northern
blot analysis (FIG. 2C). These data further confirm that ISS binds
Ku antigen to mediate its effects.
Example 3
DNA-PKcs is Required for Innate Cytokine Induction by Bacterial DNA
and ISS-ODN
[0180] Materials and Methods
[0181] DNA-PKcs.sup.-/- and their wt control mice on the 129
genetic background were generated by Dr. GC. Li and bred at
Memorial Sloan-Kettering Cancer Center, New York, N.Y.
IKK.beta..sup.-/- Tnfr1.sup.-/- mice were generated by Drs. Z-W Li
and Karin M. ATM.sup.-/- mice on the C57BL/6 background were
generated and bred by Dr. Y. Xu (UCSD) as was previously described
(Xu et al.(1996) Genes Dev. 10: 2411-2422) while their wild-type
(wt) control were purchased from Jackson Laboratories (Bar Harbor,
Me.). BMDMs from wt, DNA-PKcs.sup.-/- mice, IKK.beta..sup.-/-
Tnfr1.sup.-/- and ATM.sup.-/- mice were prepared as was previously
published (Martin-Orozco et al. (1999) Intl. Immunol.
11:1111-1118), maintained in DMEM with 10% FBS, antibiotics and 20%
L-cell medium and cultured for 7-10 days to allow them to mature.
Prior to use BMDM were seeded (2.5.times.10.sup.5/well in
triplicate) in 96-well plates and than treated with LPS (1
.mu.g/ml), ISS-ODN (5 .mu.g/ml) or M-ODN (5 .mu.g/ml), po-ISS-ODN
(10 .mu.g/ml) or po-ds-ISS-ODN (10 .mu.g/ml), LPS-free, ultra pure
bacterial DNA (E. coli, Sigma) (15 .mu.g/ml) or methylated
bacterial DNA or LPS-free, ultra pure calf thymus DNA (Sigma) (15
.mu.g/ml). Methylation of bacterial DNA was performed by SssI
methylase (Biolab, Boston, Mass.) (15 .mu.g/ml) following
manufacturer's instruction. Where indicated the P13K inhibitor
wortmannin (Wm), at various concentrations, was added to ISS-ODN or
LPS stimulated BMDMs. After 24 hours in culture, the supernatants
were collected and assayed for IL-6 and IL-12 levels by ELISA kits
(PharMingen, San Diego Calif.).
[0182] Most of the experiments described in this study were
performed with LPS-free, single stranded (ss), 22 mer long,
phosphothioate (ps) ODNs. In some experiments, ss and double
stranded (ds) 22 mer long phosphodiester (po) ODNs were used.
(Trilink, San Diego, Calif.). The sequences of the ODNs used in
this study are as follows (where *C denotes 5-methyl C):
2 ISS-ODN (1) 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO:1) ISS-ODN
(2) 5'-TGACTGTGAACGTTAGAGATGA-3' (SEQ ID NO:10) Methylated
(.sup.5-methylC) ISS-ODN 5'-TGACTGTGAA*CGTTAGAGATGA-3' (SEQ ID
NO:11) Mutated (M)-ODN 5'-TGACTGTGAAGGTTAGAGATGA-- 3' (SEQ ID
NO:12) Control-ODN (1) 5'-TGACTGTGAACCTTAGAGATG- A-3' (SEQ ID NO:9)
Control-ODN (2) 5'-TGACTGTGTTCCTTAGAGAT- GA-3' (SEQ ID NO:13)
Control-ODN (3) 5'-TGACTGTGAATATTAGAGATGA-3' (SEQ ID NO:14)
[0183] Results
[0184] Bone marrow-derived macrophages (BMDM) respond to ISS-ODN by
secreting high levels of IL-6 and IL-12. Martin-Orozco et al.
(1999) Int'l. Immunol. 11: 1111-1118. Initially, BMDM from
DNA-PKcs-deficient mice (Kurimasa et al., 1999) were used to
explore the possible role of DNA-PKcs in induction of these innate
cytokines by ISS-ODN. Very low levels of IL-6 and IL-12 were
produced by DNA-PKcs-deficient BMDM upon ISS-ODN stimulation in
comparison to wild type (wt) BMDM, as shown in FIGS. 3A and 3B. In
contrast, DNA-PKcs.sup.-/- BMDM exhibited normal induction of IL-6
and IL-12 in response to LPS stimulation, as shown in FIGS. 3A and
3B.
[0185] Since phosphothioate (ps) ODNs differ structurally from
phosphodiester (Po) ODNs, we compared the response of ISS-ODN to
po-ISS-ODN, po-ds-ISS-ODN (unmethylated or methylated), LPS-free
bacterial DNA (E. coli,), methylated E. coli bacterial DNA or to
LPS-free calf thymus DNA. Similar activity profile was observed for
po-ds-ISS-ODN and bacterial DNA in wt BMDM (FIGS. 3C and 3D) while
po-ISS-ODN was less effective FIGS. 3C and 3D). As expected, calf
thymus DNA and methylated bacterial DNA induced a several-fold less
IL-6 and IL-12 as compared to unmethylated bacterial DNA. BMDM from
DNA-PKcs-deficient mice were also defective in induction of IL-6
and IL-12 in response to po-ISS-ODN, po-ds-ISS-ODN and bacterial
DNA (FIGS. 3C and 3D), indicating that DNA-PKcs is required for
induction of IL-6 and IL-12 by synthetic (ps) and natural forms
(po) of ISS-enriched DNAs (i.e., bacterial DNA).
[0186] We then determined whether the lack of ISS-ODN
responsiveness in DNA-PKcs-deficient BMDM was due to a defect in
mRNA induction. Little induction of IL-6 and IL-12 mRNAs in
response to ISS-ODN was observed in DNA-PKcs-deficient BMDM (FIG.
3C). In contrast, DNA-PKcs-deficient BMDM exhibited normal cytokine
mRNA induction in response to LPS stimulation.
[0187] To determine the requirement for DNA-PKcs in the induction
of IL-6 and IL-12 by ISS-ODN, po-ISS-ODN, ps-ds-ISS-ODN, bacterial
and calf thymus DNAs in vivo, we injected these DNAs to wt and
DNA-PKcs.sup.-/- mice. The levels of IL-6 and IL-12 mRNAs in liver
or spleen were examined by RT-PCR. IL-6 or IL-12 MRNA levels were
detected in the liver or the spleen of wt controls but were lacking
in the same organs in DNA-PKcs-deficient mice (FIG. 3D). Only
minute amounts of mRNAs were observed in response to calf-thymus
DNA injection into wt mice (FIG. 3D).
[0188] DNA-PKcs is a member of P13K family and its enzymatic
activity is blocked by P13K inhibitors such as wortmannin (Wm) at
high concentrations, or Ly294002 (Ly). Hartley et al.(1995) Cell
82:849-856; and Smith and Jackson (1999) Genes Dev. 13:916-934. To
further establish the role of DNA-PKcs in the induction of IL-6 and
IL-12 by ISS-ODN, we examined the effects of Wm and Ly on these
responses. High concentrations of Wm (>100 nM) significantly
inhibited the induction of IL-6 and IL-12 by ISS-ODN (Hartley et
al., 1995) (FIGS. 4E and 4F). Ly also blocked IL-6 and IL-12
induction by ISS-ODN. In contrast, both Wm and Ly did not inhibit
LPS-induced secretion of IL-6 and IL-12 FIGS. 4E and 4F).
[0189] The ATM gene product, which is also a member of the P13K
family, is functionally related to DNA-PKcs and its kinase activity
is also Wm and Ly sensitive (Hartley et al., supra; Xu et al.,
supra). We therefore examined the induction of TL-6 and IL-12 in
ATM-deficient mice. As shown in FIGS. 4G and 4H, normal induction
of IL-6 and IL-12 by ISS-ODN was observed in ATM-deficient BMDM,
excluding a role for ATM in ISS-induced activation of innate
immunity.
Example 4
IKK.beta. is Essential for ISS Activity
[0190] Materials and Methods
[0191] Oligonucleotides
[0192] Oligonucleotides were as described in Example 3, above.
[0193] Animals
[0194] Animal were as described in Example 3, above.
[0195] Kinase Assays and Immunoblotting.
[0196] Kinase assays and immunoblotting were performed according to
Li et al. ((1999) J. Exp. Med. 189:1839-1845). Briefly, BMDM were
treated with ISS-ODN (5 .mu.g/ml), M-ODN (5 .mu.g/ml) on ps and po
backbones as indicated, LPS-free bacterial DNA or methylated
bacterial DNA (5 .mu.g/ml), LPS-free calf thymus DNA (5 .mu.g/ml),
LPS (10 .mu.g/ml) or TNF.alpha. (10 ng/ml) for the indicated time
periods. Cell lysates were prepared and normalized by
immunoblotting (IB) with anti-IKK.alpha. polyclonal antibodies
(Santa Cruz, Santa Cruz Biotech Inc., CA), anti-IKKp polyclonal
antbodies (Santa Cruz) or anti-DNA-PKcs monoclonal antibodies
(NeoMarker, Calif.). IKB kinase (IKK) complex or DNA-PK complex
from 100 .mu.g of the lysates were immunoprecipitated by
anti-IKK.alpha. or by anti-DNA-PKcs antibodies. The kinase
activities (KA) were determined by a kinase assay using the
N-terminus of IKBa (for IKK) or the N-terminus of p53 (for DNA-PK)
as a substrate as previously described. Wang et al. (1992) Proc.
Natl. Acad. Sci. USA 89:4231-4235; Li et al.(1999), supra; and
Hammarsten et al. (2000) J. Biol. Chem. 275:1541-1545.
[0197] The in vitro DNA-PK phosphorylation assay was performed
according to Hammarsten et al. (2000), supra, with modification.
Briefly, affinity-purified DNA-PK (Promega, Mo.) was incubated with
various DNA preparations (described below), 0.5 .mu.g of GST-p53
(1-70) and 3.3 .mu.Ci of .gamma.-.sup.32p-ATP in a 20 .mu.l
reaction buffer (10 mM Tris-Ci, 5 mM MgCl.sub.2, 0.3 mM EDTA and 10
.mu.M ATP) at 30.degree. C. for 30 minutes. The reaction was
stopped by addition of 4.times.loading buffer. The samples were
boiled, loaded on 9% SDS-PAGE, transferred onto a PVDF membrane and
visualized by autoradiography.
[0198] The ODNs (ps-ss) used include an ISS-ODN with an active CpG
motif (AACGTT), a methylated ISS-ODN at the .sup.5C of the CpG
dinucleotide (AA*CGTT) and various control ODNs. These ODNs were
incubated at concentrations of 0, 0.1, 0.3, or 1 ng/reaction. The
po-ISS-ODN was incubated at concentration of 20, 50 or 100
ng/reaction. The po-ds-ISS-ODN was incubated at concentration of 0,
0.5, 1, 2, 5 or 10 ng/reaction. The bacterial or calf thymus DNAs
were each incubated at concentration of 1, 2 or 5 ng/reaction.
Electrophoretic gel mobility shift assay (EMSA) was performed as
previously described. Chu et al. (1999) Immunity 11:721-731; and Li
et al., 1999, supra). In vitro IKK kinase assays were performed
using purified IKK derived from Sf9 insect cell lysates as was
previously described. Zandi et al. (1998) Science
281:1360-1363.
[0199] RT-PCR and Northern Blots
[0200] Total cellular RNA was isolated from spleen or liver of wt
or DNA-PKcs.sup.-/- mice injected with ISS-ODN (50 .mu.g),
po-ISS-ODN (100 .mu.g), po-ds-ISS-ODN (100 .mu.g), bacterial DNA
(100 .mu.g) or calf thymus DNA (100 .mu.g), using a RNA isolation
kit (Stratagene, San Diego, Calif.) and subjected to reverse
transcription-polymerase chain reaction (RT-PCR). First strand cDNA
preparation and PCR amplification were performed using the
SuperScript preamplification system (Gibco BRL, Gaithersburg, Md.)
and AdvanTaq Plus DNA polymerase (Clontech, San Francisco, Calif.),
respectively. The primer sequences used were as follows:
3 (SEQ ID NO:3) IL-6 sense 5'-ATGAAGTTCCTCTCTGCAAGAGACT-3' (SEQ ID
NO:4) antisense 5'-CACTAGGTTTGCCGAGTAGAT- CTC-3' (SEQ ID NO:5)
IL-12p40 sense 5'-GGGACATCATCAAACCAGACC-3' (SEQ ID NO:6) antisense
5'-GCCAACCAAGCAGAAGACAGC-3' (SEQ ID NO:7) GAPDH sense
5'-ACCACAGTCCATGCCATCAC-3' (SEQ ID NO:8) antisense
5'-TCCACCACCCTGTTGCTGTA-3'
[0201] PCR reactions were performed under the following conditions
by appropriate cycling number (94.degree. C.: 30 sec, 65.degree.
C.: 30 sec, 68.degree. C.: 30 sec). PCR products were visualized by
electrophoresis on 1.5% TAE agarose gels after being stained with
ethidium bromide. BMDM isolated from either wild type or
DNA-PKcs-deficient mice were treated with ISS-ODN (10 .mu.g/ml),
LPS (10 .mu.g/ml) or left untreated for 6.5 hrs. Total RNA was
isolated and 10 .mu.g of total RNA was separated on 1% agarose gel
and then transferred onto a nylon membrane. The membrane was probed
with .alpha.-.sub.32P-dCTP-labeled IL-6 or IL-12 or GAPDH cDNA
(generated by RT-PCR as described above) followed by
autoradiography.
[0202] Methylation and Labeling of Free Ends of Genomic DNAs
[0203] When indicated, 100 .mu.g of ultra pure bacterial DNA (E.
coli, Sigma) was incubated with or without 200 units SssI methylase
(Biolab) in a 200 .mu.l reaction buffer according to manufacture
instruction at 37.degree. C. for 3 hours and then extracted with
phenol/chloroform. The bacterial DNA was precipitated with ethanol
and dissolved in TE buffer. One .mu.g of mock and methylated
bacterial DNA was further incubated with 10 unit of BstU1 at
60.degree. C. for 4 hrs, loaded on 1% agarose gel and visualized by
ethidium bromide (EB) staining for the absence or presence of
digest products in the methylated and in the non-methylated
bacterial DNA, respectively.
[0204] For the DNA-PK assay we measure the free ends in E. coli and
calf thymus DNAs by labeling the 5' free ends of both the DNA
preparations. Thus, 0.2 .mu.g of either bacterial or calf thymus
DNAs were incubated with 15 units of T4 PNK (Stratagene, San Diego,
Calif.) and 100 .mu.Ci of .gamma.-.sup.32p-ATP in a 20 .mu.l of
reaction at 37.degree. C. for 3 hrs. To purify the labeled DNAs
from the .gamma.-.sup.32p-ATP excess, the samples were loaded onto
SephdexG50 column (Stratagene San Diego, Calif.) after the reaction
was stopped. One ill of labeled DNA was used to measure
radioactivity which yielded 4.1.times.10.sup.6.+-.2.times.10.sup.-
4 cpm/1 .mu.g for bacterial DNA and
4.2.times.10.sup.6.+-.5.2.times.10.sup- .4 cpm/1 .mu.g for calf
thymus DNA.
[0205] Results
[0206] We evaluated whether ISS-ODN activated IKK, which is
essential for NF-.kappa.B activation by pro-inflammatory stimuli.
Karin and Delhase (2000) Seminars in Immunol. 12:85-89. We observed
maximal IKK activation 30 minutes post-ISS-ODN incubation, which
lasted for about 4 hrs in wt BMDM (FIG. 5A). While bacterial DNA
and po-ISS-ODN induced IKK activation similar to ISS-ODN, little
increase in IKK activity was observed with M-ODN and or calf thymus
DNA (FIG. 5A). Optimal IKK activation was observed at an ISS-ODN
concentration of 0.65 .mu.g/ml, with little IKK activation in
response M-ODN (FIG. 5B).
[0207] We used BMDM isolated from IKK.beta..sup.-/- Tnfr1.sup.-/-
mice to determine the requirement of IKK activity, which is highly
reduced in these animals. The absence of IKK.beta. prevented IKK
and NF-.kappa.B activation by ISS-ODN as well as LPS (FIG. 5C) and
significantly reduced the induction of IL-6 and IL-12 (FIGS. 5D and
5E).
[0208] We then examined the dependence of IKK activation by ISS-ODN
on DNA-PKcs. While incubation of wt BMDM with ISS-ODN resulted in
robust IKK activation, little increase in IKK activity was observed
in similarly treated DNA-PKcs.sup.-/- BMDM (FIG. 6A). As a result,
DNA-PKcs-1-BMDM also exhibited impaired NF-.kappa.B activation upon
treatment with ISS-ODN (FIG. 6A). By contrast, DNA-PKcs-deficient
BMDM were fully responsive to LPS or TNF.alpha.. Furthermore, we
examined the dependence of IKK activation by bacterial DNA and
po-ISS-ODN on DNA-PKcs. As expected, activation of IKK by bacterial
DNA or po-ISS-ODN was largely reduced in DNA-PKcs.sup.-/- BMDM as
compared to their wt controls (FIG. 6B).
[0209] To determine whether DNA-PKes activity is required for IKK
activation we used the P13K inhibitor Wm. As found for IL-6 and
IL-12 production, only high concentrations of Wm (250 nM and above)
significantly inhibited IKK activation in wt BMDM by ISS-ODN (FIG.
6C). Even at 1000 nM Wm had no effect on IKK activation by TNFA,
while at lower concentrations (50-100 nM) Wm significantly
inhibited IKK activation by PGDF. We also compared ISS-ODN-induced
of IKK and NF-.kappa.B activation in BMDM from wt or ATM-deficient
mice (Xu et al., 1996, supra). As shown in FIG. 6D, no differences
were observed for ISS-ODN-induced IKK or NF-.kappa.B
activation-between wt and ATM-deficient BMDM, excluding a role of
ATM in this signaling.
[0210] Taken together, these results indicate that DNA-PKcs acts
upstream to IKK and is specifically required for IKK activation by
synthetic (ps) and natural forms (po) of ISS-enriched DNAs.
Example 5
ISS-ODN Directly Activates DNA-PK
[0211] Materials and Methods
[0212] Oligonucleotides and DNA-PK assays were as described in
Examples 3 and 4, above.
[0213] Results
[0214] We investigated whether ISS-ODN can directly activate DNA-PK
in vitro. The ability of an ISS-ODN containing the active CpG motif
(5'-pur-pur-CpG-pyr-pyr-3' i.e., 5'-AACGTT-3') to specifically
stimulate phosphorylation of the N-terminal portion of p53 was
compared to a battery of mutated ODNs, which include: 1) a
methylated C in the CpG dinucleotide, 2) a CpC basepair instead of
the CpG dinucleotide core, 3) a GpG basepair instead of the CpG
dinucleotide core, 4) an ApT basepair instead of the CpG
dinucleotide core, and 5) a TTCC instead of the AACG sequence of
the CpG motif (see Example 4, above). None of the mutant ODNs
induced significant secretion of IL-6 or IL-12 upon stimulation of
BMDM in vitro. Only the ISS-ODN stimulated DNA-PK activity (FIG.
7A) whereas none of the mutant ODNs, which devoid of biological
activity, led to substantial increase in DNA-PK activity.
[0215] Next, we investigated the ability of po-ISS-ODN to activate
DNA-PK in vitro. Unlike ISS-ODN (FIG. 6A), po-ISS-ODNs weakly
activated DNA-PK only at higher concentrations (50-100 ng/reaction)
(FIG. 7B). By contrast, po-ds-ISS-ODN was almost as potent as
ISS-ODN in activating this enzyme (FIG. 7C). Methylated ISS-ODN
(AA*CGTT) and methylated po-ISS-ODN (AA*CGTT) were weaker DNA-PK
activators than their unmethylated counterparts (FIGS. 7A and 7B,
respectively).
[0216] In addition, we evaluated the ability of bacterial DNA,
methylated bacterial DNA and calf thymus DNA to activate DNA-PK. To
use the same equimolar amount of the various DNA preparations in
the DNA-PK assays, we first measured the 5' free ends using T4 DNA
polynucleotide kinase in the DNA preparations. For bacterial DNA,
this labeling yielded 6.29.times.10.sup.5 cpm/0.1 .mu.g and
4.1.times.0.sup.6 cpm/1 .mu.g of DNA and for calf thymus DNA it
yielded 8.58.times.10.sup.5 cpm/0.1 .mu.g and 4.2.times.10.sup.6
cpm/1 .mu.g of DNA. Under these conditions, calf thymus DNA was a
weaker activator of DNA-PK than bacterial DNA (FIG. 7D) while
methylated bacterial DNA was a less potent DNA-PK activator than
unmethylated bacterial DNA (FIG. 7E).
[0217] To further determine whether ISS-ODN, po-ISS-ODN or
bacterial DNA activate DNA-PK in cells, we treated BMDM from either
wt or DNA-PKcs-deficient mice with ISS-ODN, po-ISS-ODN, bacterial
DNA or LPS as a control. Considerable DNA-PK activity, as measured
by immune-complex kinase assay, was found after a 30-minute
incubation with ISS-ODN, po-ISS-ODN or bacterial DNA which peaked
after 1 hour (FIG. 7F). Little or no DNA-PK activity was detected
in DNA-PKcs-deficient BMDM and LPS had no effect on DNA-PK activity
even in wt cells.
Example 6
DNA-PK Phosphorylates IKK.beta.
[0218] Materials and Methods
[0219] Oligonucleotides and assays were as described in Examples 3
and 4, above.
[0220] Results
[0221] To explore a role of ISS-activated DNA-PK in IKK activation
we tested whether affinity-purified DNA-PK can directly activate
recombinant IKK.alpha. or IKK.beta. purified from Sf9 cells (Zandi
et al., 1998, supra). Recombinant IKK.alpha. and IKK.beta. display
considerable basal kinase activity (Zandi et al., 1998, supra; Chu
et al., 1999, supra) but incubation of IKK.beta. with DNA-PK in the
presence of ISS-ODN further increased its kinase activity measured
by I.kappa.B phosphorylation (FIG. 8A). Furthermore, although
DNA-PKcs phosphorylated IKBA that activity was considerably lower
than that achieved by IKK.beta. plus DNA-PK. Only a small
enhancement of I.kappa.B kinase activity was found upon incubation
of IKK.alpha. with DNA-PK in the presence of ISS-ODN, but not
beyond the level found with DNA-PK alone (FIG. 8A, lane 6 vs. 5).
To further confirm the activation of IKK.beta. by DNA-PK, we
performed a coupled-kinase assay. Recombinant IKK.beta. was
pre-incubated with DNA-PK in the presence or absence of ISS-ODN
followed by immunoprecipitation of IKK.beta. and I.kappa.B kinase
activity was measured. Consistent with the results described above,
DNA-PK only activates IKK.beta. in the presence of ISS-ODN. We next
determined whether DNA-PK phosphorylates IKK.beta.. Recombinant
catalytically inactive IKK.beta. [IKK.beta. (KA)] purified from Sf9
cells was incubated with or without DNA-PK, in the presence or
absence of ISS-ODN. As shown in FIG. 8B, DNA-PK phosphorylated IKKP
(KA) when incubated with ISS-ODN.
Example 7
ISS Induces HSP70 Gene Transcription
[0222] A half million mouse BMDM isolated from BALB/c, IFNa
receptor knockout, or IFN.gamma. knockout mice were incubated with
ISS-ODN or M-ODN at a final concentration of 10 .mu.g/ml. After 0,
1, 2, 4, 6, and 8 hours, total RNA was prepared and analyzed by
RT-PCR (reverse transcription-polymerase chain reaction) for
induction of gene transcription of heat shock proteins. The
sequences of the primers used is as follows: hsp70 (forward) 5' GAG
ATC ATC GCC AAC GAC CA 3' (SEQ ID NO: 15) hsp70 (reverse) 5' ACA
GTC TTT CCG AGG TAT CG 3' (SEQ ID NO:16) hsc70 (forward) 5' AAT GAC
CAG GGT AAC CGC AC 3' (SEQ ID NO: 17) hsc70 (reverse) 5' ACA GTC
TTT CCG AGG TAT CG 3' (SEQ ID NO: 18) hsp90 (forward) 5' ATG AGG
GTC CTG TGG GTG TT 3' (SEQ ID NO:19) hsp90 (reverse) 5' CAC TTC AGC
TTG GAA GGC GA 3' (SEQ ID NO:20) G3PDH (forward) 5' ACC ACA GTC CAT
GCC ATC AC 3' (SEQ ID NO:7) G3PDH (reverse) 5' TCC ACC ACC CTG TTG
CTG TA 3' (SEQ ID NO:8)
[0223] The results are shown in FIGS. 9A-D and FIGS. 10A and B. The
data presented in these figures provide evidence that ISS induces
inducible HSP-70. This induction is dependent on type 1 IFN,
because stimulation of BMDM from IFN type 1 receptor knockout mice
did not result in induction of HSP-70.
[0224] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
* * * * *