U.S. patent application number 15/162337 was filed with the patent office on 2016-12-22 for therapeutic and diagnostic strategies.
The applicant listed for this patent is Children's Medical Center Corporation. Invention is credited to Dipanjan Chowdhury, Ashish Lal, Judy Lieberman.
Application Number | 20160369274 15/162337 |
Document ID | / |
Family ID | 42040163 |
Filed Date | 2016-12-22 |
United States Patent
Application |
20160369274 |
Kind Code |
A1 |
Lieberman; Judy ; et
al. |
December 22, 2016 |
THERAPEUTIC AND DIAGNOSTIC STRATEGIES
Abstract
The present invention encompasses the finding that microRNAs
(miRNAs) regulate certain key proteins involved in DNA repair. In
some embodiments, a miRNA suppresses levels and/or activity of one
or more DNA repair proteins. In some such embodiments, such
suppression renders cells hypersensitive to certain DNA damage
agents (e.g., .gamma.-irradiation and genotoxic drugs, among
others). The present invention provides various reagents and
methods associated with these findings including, among other
things, strategies for treating cell proliferative disorders,
certain diagnostic systems, etc.
Inventors: |
Lieberman; Judy; (Brookline,
MA) ; Lal; Ashish; (Brookline, MA) ;
Chowdhury; Dipanjan; (Chestnut Hill, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Children's Medical Center Corporation |
Boston |
MA |
US |
|
|
Family ID: |
42040163 |
Appl. No.: |
15/162337 |
Filed: |
May 23, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13063155 |
Oct 27, 2011 |
9347089 |
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PCT/US2009/057506 |
Sep 18, 2009 |
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15162337 |
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61098696 |
Sep 19, 2008 |
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61098707 |
Sep 19, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/111 20130101;
A61P 43/00 20180101; A61P 37/06 20180101; A61P 35/00 20180101; C12Q
1/6806 20130101; A61P 37/00 20180101; C12Q 1/6809 20130101; C12N
2310/141 20130101; C12N 15/113 20130101; C12N 2320/11 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1. A method comprising steps of: administering to an individual who
is suffering from or susceptible to a cell proliferative disorder a
composition comprising an miRNA agent in an amount sufficient to
inhibit cell proliferation.
2. A method comprising steps of: administering to an individual who
is suffering from or susceptible to a cell proliferative disorder a
composition comprising an miRNA agent in an amount sufficient to
render cells of the individual hypersensitive to DNA damage
agents.
3. The method of claim 1, wherein the amount is an amount
sufficient to suppress levels and/or activity of one or more DNA
repair proteins.
4. The method of claim 2, wherein the DNA repair proteins are
selected from the group consisting of photolyases,
methyltransferases, AP endonucleases, exonucleases (e.g. Exol),
histone variants (e.g. H2AX), transcription factors that regulate
expression of DNA repair genes (e.g. XBP1), and DNA glycosylases,
and combinations thereof.
5. The method of claim 1, wherein the step of administering
comprises administering in combination with other cell
proliferation disorder treatment methods and/or therapeutic
agents.
6. A method selected from the group consisting of: A method
comprising steps of: antagonizing an miRNA agent in a cell, such
that cell proliferation is increased; A method comprising steps of:
administering to an individual who is suffering from or susceptible
to a cell proliferative disorder a composition comprising an miR-24
agent in an amount sufficient to inhibit cell proliferation in
combination with a DNA damage agent and/or cell proliferation
disorder treatment method that produces DNA double-strand breaks,
isolating cells from the individual administered the miR-24 agent
and preparing Wright-stained metaphase chromosome spreads from the
isolated cells or staining the isolated cells from the individual
with SYBR Green and performing single cell gel electrophoresis on
the isolated cells, thereby measuring DNA damage and detecting DNA
damage repair to be reduced in the isolated cells, as compared to
control cells, thereby identifying the individual administered the
miR-24 agent to possess reduced DNA damage repair; A method
comprising steps of: administering to an individual who is
suffering from or susceptible to a cell proliferative disorder a
composition comprising an miR-24 agent in an amount sufficient to
render cells of the individual hypersensitive to a DNA damage agent
and/or cell proliferation disorder treatment method that produces
DNA double-strand breaks, isolating cells from the individual
administered the miR-24 agent and preparing Wright-stained
metaphase chromosome spreads from the isolated cells or staining
the isolated cells from the individual with SYBR Green and
performing single cell gel electrophoresis on the isolated cells,
thereby measuring DNA damage and detecting DNA damage repair to be
reduced in the isolated cells, as compared to control cells,
thereby identifying the individual administered the miR-24 agent to
possess reduced DNA damage repair; and A method comprising steps
of: administering to an individual who is suffering from or
susceptible to a cell proliferative disorder a composition
comprising an miR-24 agent in an amount sufficient to down-regulate
the expression of H2AFX, and thereby inhibit cell proliferation,
wherein the miR-24 agent is administered in combination with a DNA
damage agent and/or cell proliferation disorder treatment method
that produces DNA double-strand breaks, isolating cells from the
individual administered the miR-24 agent, preparing cDNA from the
isolated cells and performing quantitative RT-PCR on the cDNA using
primers from the H2AX coding region and SYBR Green, thereby
measuring H2AFX expression and detecting H2AFX expression to be
reduced in the isolated cells, as compared to control cells,
thereby identifying the individual administered the miR-24 agent to
possess reduced H2AFX expression.
7. The method of claim 1, wherein the miRNA agent is selected from
the group consisting of an miR-24 agent (e.g., miR-24-1; miR-24-2),
an miR-22 agent, an miR-125a agent, an miR-23 agent (e.g., miR-23a,
miR-23B), an miR-27 agent (e.g., miR-27a, miR-27b), an miR-17
agent, an miR-18 agent, an miR-19 agent, an miR-20 agent, an
miR-34a agent, an miR-92 agent, an miR-125 agent, an miR-146a
agent, an miR-155 agent, an miR-181a agent, an 200a agent, an
miR-48 agent, an miR-84 agent, and an miR-241 agent, and
combinations thereof.
8. The method of claim 1, wherein the miRNA agent comprises miR-24.
Description
RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. Ser. No.
13/063,155, filed Oct. 27, 2011, allowed, which claims the benefit
under 35 U.S.C. .sctn.371 of international application serial
number PCT/US2009/057506, filed Sep. 18, 2009, which claims
priority under 35 U.S.C. .sctn.119 to U.S. Ser. No. 61/098,696,
filed on Sep. 19, 2008, entitled "miRNA Targets", and U.S. Ser. No.
61/098,707, filed on Sep. 19, 2008, entitled "Therapeutic and
Diagnostic Strategies", the entire contents of which are
incorporated herein by reference.
BACKGROUND
[0002] DNA repair mechanisms are central to a variety of cellular
processes, including, importantly, DNA replication and cell
proliferation. Ability to influence DNA repair systems, and/or cell
cycle progression generally, can provide novel therapeutic and
diagnostic approaches to a variety of diseases, disorders, and
conditions associated with cell proliferation (including, for
example, cancer, immune-mediated disorders and/or neurodegenerative
disorders).
SUMMARY
[0003] The present invention encompasses the finding that microRNAs
(miRNAs) regulate certain key proteins involved in DNA repair
and/or cell cycle progression. In some embodiments, a miRNA
modulates levels and/or activity of one or more DNA repair and/or
cell cycle progression proteins. In some such embodiments, such
modulation renders cells hypersensitive to certain DNA damage
agents (e.g., .gamma.-irradiation and genotoxic drugs, among
others).
[0004] The present invention specifically encompasses the finding
that certain miRNAs whose expression is increased in terminally
differentiated cells regulate DNA repair and/or cell cycle
progression proteins. In some embodiments, the miRNAs show
increased expression in terminally differentiated hematopoietic
cells. In some embodiments, the miRNAs are selected from the group
consisting of miR-22, miR-125a, miR-24 (e.g., miR-24-1; miR-24-2),
miR-23 (e.g., miR-23a, miR-23B), miR-27 (e.g., miR-27a, miR-27b),
miR-17, miR-18, miR-19, miR-20, miR-34 a, miR-92, miR-125, miR-146
a, miR-155, miR-181 a, 200a, miR-48, miR-84, and miR-241. In some
embodiments, the miRNA is miR-24.
[0005] The present invention provides, among other things, systems
for modulating DNA repair and/or cell cycle progression in cells by
altering expression and/or activity of one or more such miRNAs.
[0006] The present invention provides, among other things, systems
for reducing cell proliferation and/or increasing sensitivity to
certain DNA damage agents through modulation of certain miRNAs
and/or of miRNA-based regulation of DNA repair proteins. According
to the present invention, such systems are particularly useful, for
example, in reducing undesirable cell proliferation (e.g., in the
context of cancer, transplant rejection, T cell immunity, etc.). In
some embodiments, cells whose proliferation is to be reduced
include hematopoietic cells. In some embodiments, inventive systems
for reducing cell proliferation include increasing levels and/or
activity of certain miRNAs in cells, such that DNA repair is
reduced and/or expression and/or activity of one or more DNA repair
(e.g., H2AX) or cell cycle progression proteins is altered. In some
such embodiments, cells may also be exposed to one or more DNA
damage agents (e.g., .gamma.-irradiation and genotoxic drugs, among
others). In some embodiments, the present invention provides
systems for inducing apoptosis.
[0007] The present invention provides, among other things, systems
for increasing cell proliferation and/or decreasing sensitivity to
certain DNA damage agents through modulation of certain miRNAs
and/or of miRNA-based regulation of DNA repair and/or cell cycle
progression proteins. According to the present invention, such
systems are particularly useful, for example, in cell culture
applications and/or in applications where cell proliferation is
desirably increased. In some embodiments, cells whose proliferation
is to be increased include hematopoietic cells. In some
embodiments, inventive systems for increasing cell proliferation
include decreasing levels and/or activity of certain miRNAs in
cells such that DNA repair is reduced and/or expression and/or
activity of one or more DNA repair (e.g., H2AX) and/or cell cycle
progression proteins is altered.
[0008] The present invention provides, among other things, systems
for detecting cells undergoing terminal differentiation and/or
cells whose DNA repair systems are compromised. Particularly useful
applications of such systems, according to the present invention
include, among other things, diagnostics for identifying cells
and/or individuals that are particularly susceptible to DNA damage
agents (e.g., .gamma.-irradiation and genotoxic drugs, among
others).
DESCRIPTION OF THE DRAWING
[0009] FIG. 1A to FIG. 1D show kinetics of miR-24 and H2AX
transcript levels in TPA-treated K562 and HL60 cells. TPA treatment
of K562 cells (FIGS. 1A, 1C) and HL60 cells (FIGS. 1B, 1D)
increases miR-24 levels (FIGS. 1A, 1B) with a concurrent decrease
in H2AX mRNA (FIGS. 1C, 1D).
[0010] FIG. 2A and FIG. 2B demonstrate, in FIG. 2A, that miR-328
does not target the 3'UTR of I-12AX mRNA in a luciferase reporter
assay, where HepG2 cells were transfected with control miRNA
(black) or synthetic miR-328 (white) for 48 hr and then with H2AX
3'UTR-luciferase reporter (H2AX) or vector (V) for 24 hr; and, in
FIG. 2B, that miR-328 over-expression in K562 cells has no effect
on H2AX mRNA (left), analyzed by qRT-PCR normalized to GAPDH, and
protein (right) 48 hr later.
[0011] FIG. 3A to FIG. 3C show that chromosomal aberrations after
.gamma.-irradiation are greater in K562 than HepG2 cells. HepG2
cells express less miR-24 (FIG. 3A, right panel) and more H2AX
(FIG. 3A, left panel) than K562 cells. Cells were either untreated
or irradiated and incubated for 24 h before metaphase spreads were
prepared. For each condition, at least 50 metaphase spreads were
examined. The average number of cells with chromosomal aberrations
(FIG. 3B) and chromosomal aberrations per cell (FIG. 3C) were
analyzed. In (FIG. 3B) and (FIG. 3C) white bars represent K562
cells; black bars, HepG2 cells.
[0012] FIG. 4A to FIG. 4K show that miR-24 down-regulates H2AX
expression during terminal differentiation. miR-24, analyzed by
qRT-PCR relative to U6, increases during differentiation of K562
cells (FIG. 4A) with TPA to megakaryocytes or hemin to erythrocytes
(#, p<0.001, ***, p<0.005) and during differentiation of HL60
cells (FIG. 4B) with TPA to macrophages or DMSO to granulocytes (#,
p<0.001). HL60 cells were treated with TPA for 2 days (left
panel) or DMSO for 5 days (right panel) and miR-24 levels analyzed
as described above. Under the same differentiating conditions for
K562 (FIG. 4C) and HL60 (FIG. 4D) cells, H2AX mRNA, normalized to
GAPDH mRNA, is down-regulated (**, p<0.01, K562; ***,
p<0.005, HL60). FIG. 4E shows that H2AX protein decreases after
2 d of TPA differentiation. Relative H2AX expression was quantified
by densitometry using H3 as control. FIG. 4F shows that H2AX mRNA
is selectively pulled down from K562 cell lysates with biotinylated
miR-24 (white) compared to control cel-miR-67 (black). For each
condition, pulled down RNA was first normalized to GAPDH mRNA in
the sample and then to relative input cellular RNA (***,
p<0.005). The housekeeping gene UBC was not enriched in the pull
down. FIG. 4G shows a schematic representing the location of miR-24
binding sites in the 3'UTR of H2AX mRNA. FIG. 4H shows that miR-24
targets the 3'UTR of H2AX mRNA in a luciferase reporter assay.
HepG2 cells were transfected with control miRNA (black) or
synthetic miR-24 (white) for 48 hr and then with H2AX
3'UTR-luciferase reporter (H2AX) or vector (V) for 24 hr. Mean+SD,
normalized to vector control, of 3 independent experiments are
shown (*, p<0.001). miR-24 over-expression in HepG2 cells
decreases H2AX mRNA, analyzed by qRT-PCR normalized to GAPDH (FIG.
4I; white, miR-24; black, cel-miR-67) and protein (FIG. 4J) 48 hr
later. miR-24 over-expression does not alter UBC mRNA levels. In
(FIG. 4A to FIG. 4D; FIG. 4F; and FIGS. 4H and 4I), mean.+-.SD are
shown. FIG. 4K shows suppression of the luciferase activity of a
reporter gene containing in its 3' UTR the two predicted miR-24
MRE, either wild-type (wt) or with mutated seed regions (mt). HepG2
cells were transfected with control miRNA (black) or miR-24 mimic
(white) for 48 h and then with the indicated H2AX 3' UTR-luciferase
reporters or vector (V). Luciferase activity was assayed 24 h
later. Mean.+-.s.d., normalized to vector control, of three
independent experiments is shown. In all panels, mean s.d. is
shown.
[0013] FIG. 5A to FIG. 5D show miR-24 expression impedes DSB repair
and induces chromosomal instability of .gamma.-irradiated K562
cells. FIG. 5A shows that transfection of miR-24 mimic into K562
cells reduces H2AX comparably to TPA differentiation. H2AX was
quantified relative to H3 protein by densitometry. FIG. 5B shows
representative images of metaphase chromosome spreads were prepared
from treated cells 24 h after .gamma.-irradiation. Arrows mark
chromosome breaks or fragments. FIG. 5C shows chromosome breaks
were quantified 24 hr after irradiation of K562 cells that were
either undifferentiated (white) or had been differentiated with TPA
(black, left panel) or transfected with miR-24 (black, middle
panel). In the right panel, differences in chromosome breaks that
were not present 24 hr following 0.375 Gy were significantly
different 48 hr after irradiation in miR-24 (black) vs
mock-transfected (white) cells. The mean+SD number of chromosome
breaks and fragments per cell, normalized to control is plotted.
FIG. 5D shows that overexpressing miR-24 increases unrepaired DSB
by comet assay. K562 cells, transfected with miR-24 mimic and/or
miR-24-insensitive H2AX expression plasmid, were treated or not
with bleomycin (0.5 .mu.g/ml) for 12 h and analyzed by single cell
gel electrophoresis (comet assay) 12 h later. H2AX protein is
compared to H3 level in the immunoblot. H2AX levels, reduced by the
miR-24 mimic, are rescued by the H2AX expression plasmid.
Representative images from bleomycin-treated cells are shown in the
upper panel and the mean.+-.SD comet tail moment for each condition
below (black, control mimic, expression vector; dark stippled bars,
miR-24 mimic, vector; white, control mimic, H2AX expression
plasmid; light stippled bars, miR-24 mimic, H2AX expression
plasmid). Manipulating miR-24 or H2AX levels did not affect
baseline DNA damage, but DNA damage after irradiation was
significantly increased (p<0.001) in miR-24 mimic-transfected
cells, but only in the absence of H2AX rescue.
[0014] FIG. 6A to FIG. 6D show that cells overexpressing miR-24 are
hypersensitive to DNA damage by cytotoxic drugs. FIG. 6A shows that
K562 cells overexpressing miR-24 (left) or treated with TPA (right)
are hypersensitive to bleomycin, assessed by viability relative to
mock-treated cells (.quadrature.) 2 d later. TPA treatment (u) or
transfection with miR-24 mimic (m), but not miR-328 mimic (FIG.
6A), significantly sensitizes K562 cells to DNA damage
(p<0.005). FIG. 6B shows similarly that HepG2 cells
overexpressing miR-24 (.box-solid.) are hypersensitive, compared to
mock-transfected cells (.quadrature.) to bleomycin (left) and
cisplatin (right). miR-24 over-expression significantly reduces
viability to both genotoxic agents (p<0.004). miR-24-mediated
hypersensitivity of K562 (FIG. 6C) and HepG2 (FIG. 6D) cells is
rescued by expression of miR-24-insensitive H2AX. Cells were mock
transfected (El) or transfected with miR-24 mimic (u) or H2AX cDNA
lacking the 3'UTR (FIG. 6A) or both (.cndot.). Cell viability was
assayed 2 d after exposure to DNA damage and depicted relative to
that of undamaged cells. Immunoblots demonstrate miR-24-mediated
decrease in H2AX protein and rescue by transfecting the H2AX cDNA
(no 3'UTR).
[0015] FIG. 7A to FIG. 7D shows that antagonizing miR-24 enhances
cell resistance to bleomycin. FIG. 7A shows that miR-24 knockdown
in K562 cells, treated or not with TPA, specifically decreases
miR-24 levels, assayed by qRT-PCR in cells transfected with miR-24
ASO relative to control ASO (Ctl). miR-24 expression in ASO-treated
cells, relative to U6 snRNA, is normalized to that in control
ASO-treated cells. FIG. 7B shows that miR-24 ASO enhances H2AX
transcript (left) and protein levels (right) in K562 cells treated
with TPA, but not in untreated, K562 cells. FIG. 7C shows that
relative to untreated cells (.quadrature.), TPA treatment (.cndot.)
sensitizes K562 cells to bleomycin treatment. Transfection of
miR-24 ASO significantly blocks bleomycin-induced apoptosis of
TPA-treated K562 cells (.cndot., p<0.003), but does not affect
apoptosis of untreated K562 cells (FIG. 7A). FIG. 7D shows that
transfection of miR-24 significantly enhances repair of
bleomycin-induced DNA damage, as measured by comet assay, in
TPA-treated K562 cells (*, P o 0.001). Representative images from
bleomycin-treated cells are shown on the left and the mean.+-.s.d.
comet tail moments of three independent experiments are shown on
the right.
[0016] FIG. 8A to FIG. 8C shows that inhibition of miR-24 leads to
increased cell proliferation, while miR-24 over-expression leads to
cell-cycle arrest. FIG. 8A shows that miR-24 knockdown in K562
cells specifically decreases miR-24 levels, assayed by qRT-PCR in
cells transfected with miR-24 ASO (white) relative to control ASO
(black). Expression relative to U6 snRNA is depicted normalized to
control cells. FIG. 8B shows that miR-24 knockdown with ASO
increases K562 cell proliferation measured by thymidine uptake,
both in the presence and absence of TPA. The decline in
proliferation with TPA is completely restored by antagonizing
miR-24. FIG. 8C shows that miR-24 over-expression increases the G1
compartment in HepG2 cells. HepG2 cells transfected with miR-24 or
control mimic for 48 hr were stained with propidium iodide and
analyzed by flow cytometry. Representative analysis of three
independent experiments is shown. Error bars represent standard
deviation from 3 independent experiments (FIG. 8A to FIG. 8C). *,
p<0.05; '', p<0.01; #, p<0.005.
[0017] FIG. 9A and FIG. 9B show, respectively, H2AX mRNA and
kinetics of thymidine incorporation in TPA-treated K562 cells. FIG.
9A shows H2AX mRNA analyzed by qRT-PCR using coding region primers
decreases .about.4-fold during TPA induced differentiation of K562
cells. These primers amplify both H2AX transcripts. GAPDH mRNA was
used for normalization. FIG. 9B shows kinetics of thymidine
incorporation in TPA-treated K562 cells. By 12 h there is no
thymidine incorporation, indicating that cells have stopped
dividing.
[0018] FIG. 10A and FIG. 10B show that miR-24 levels and H2AX
levels in primary human peripheral blood macrophages and
granulocytes are comparable to cells generated by in vitro
differentiation. FIG. 10A shows that miR-24, analyzed by qRT-PCR
relative to U6, increases during TPA-induced differentiation of
HL60 cells to macrophages, and this increased miR-24 expression is
also observed in primary human peripheral blood macrophages and
granulocytes. FIG. 10B shows that H2AX mRNA (normalized to UBC
mRNA), and protein (normalized to histone H3) is down-regulated
during differentiation of HL60 cells and is comparable to levels in
primary human peripheral blood macrophages and granulocytes.
[0019] FIG. 11 shows that representative images of metaphase
chromosome spreads were prepared from treated cells 24 h after
.gamma.-irradiation. Arrows mark chromosome breaks or
fragments.
[0020] FIG. 12 shows that miR-24-mediated hypersensitivity of K562
cells to bleomycin is not rescued by expression of
miR-24-insensitive CHEK1. Cells were mock transfected or
transfected with miR-24 mimic and/or CHEK1 cDNA lacking the 3'UTR.
Cell viability was assayed 2 d after exposure to DNA damage and
depicted relative to that of undamaged cells. Immunoblot
demonstrates miR-24-mediated decrease in CHEK1 protein and its
restoration by transfecting CHEK1 cDNA lacking the 3'UTR.
[0021] FIG. 13 shows that miR-24 is upregulated during
hematopoietic cell differentiation into multiple lineages. Heat map
for miRNA expression in HL60 and K562 cells differentiated into
four different nondividing cell lineages, showing single-linkage
hierarchical clustering, using Pearson squared as a distance
metric. miRNA expression in each lane is analyzed relative to
expression in control undifferentiated cells. The highlighted
cluster shows miRNAs with similar expression profiles. Range is
from five-fold downregulation (green) to five-fold upregulation
(red). Arrows indicate miR-24 cluster miRNAs.
[0022] FIG. 14 presents Supplemental Table 1, Primers used for
qRT-PCR, and Supplemental Table 2, Mutations introduced in miR-24
binding sites in H2AX-3'UTR.
DEFINITIONS
[0023] Cell Proliferative Disorder, Disease, or Condition: The term
"cell proliferative disease or condition" is meant to refer to any
condition characterized by aberrant cell growth, in some
embodiments abnormally increased cellular proliferation.
[0024] Combination Therapy: The term "combination therapy", as used
herein, refers to those situations in which two or more different
pharmaceutical agents are administered in overlapping regimens so
that the subject is simultaneously exposed to both agents.
[0025] DNA Damage Agents: The term "DNA damage agents", as used
herein, refer to agents that, when applied to cells, damage DNA in
the cells. In some embodiments, DNA damage agents are teratogens.
As described herein, the present invention establishes that
presence and/or activity of certain miRNAs in cells renders those
cells hypersensitive to DNA damage agents. Representative such DNA
damage agents include, but are not limited to, .gamma.-irradiation,
genotoxic drugs, etc.
[0026] Dosing Regimen: A "dosing regimen", as that term is used
herein, refers to a set of unit doses (typically more than one)
that are administered individually separated by periods of time.
The recommended set of doses (i.e., amounts, timing, route of
administration, etc.) for a particular pharmaceutical agent
constitutes its dosing regimen.
[0027] Hypersensitive: The term "hypersensitive", is used herein to
refer to cells that are more sensitive than control cells. In some
embodiments, a cell is considered to be "hypersensitive" as
compared with a relevant control if it shows at least about 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
20, 25, 30, 25, 40, 25, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 fold or more
susceptible to a particular agent or treatment.
[0028] Initiation: As used herein, the term "initiation" when
applied to a dosing regimen can be used to refer to a first
administration of a pharmaceutical agent to a subject who has not
previously received the pharmaceutical agent. Alternatively or
additionally, the term "initiation" can be used to refer to
administration of a particular unit dose of a pharmaceutical agent
during therapy of a patient.
[0029] MicroRNA Agent: A "microRNA agent" as that term is used
herein, refers to an entity whose nucleotide sequence is
substantially identical to that of a natural miRNA. As will be
appreciated by those of ordinary skill in the art,
naturally-occurring miRNAs are comprised of RNA. As will be further
appreciated by those of ordinary skill in the art, RNA is a
particularly labile chemical. Furthermore, a variety of strategies
are known for preparing molecules that are structural mimics of RNA
(and therefore have a "sequence" in the same sense as RNA) but that
may, for example, have greater stability and/or somewhat altered
hybridization characteristics. For example, in some embodiments,
such structural mimics include one or more backbone modifications
(e.g., substitution of phosphorothioate backbone structures for
phosphodiester structures found in RNA) and/or one or more base
modifications (e.g., 2'-OMe modifications). In some embodiments,
such structural mimics are encompassed within "microRNA agent" as
that term is used herein.
[0030] Pharmaceutical agent: As used herein, the phrase
"pharmaceutical agent" refers to any agent that, when administered
to a subject, has a therapeutic effect and/or elicits a desired
biological and/or pharmacological effect.
[0031] Pharmaceutically acceptable carrier or excipient: As used
herein, the term "pharmaceutically acceptable carrier or excipient"
means a non-toxic, inert solid, semi-solid or liquid filler,
diluent, encapsulating material or formulation auxiliary of any
type.
[0032] Pharmaceutically acceptable ester: As used herein, the term
"pharmaceutically acceptable ester" refers to esters which
hydrolyze in vivo and include those that break down readily in the
human body to leave the parent compound or a salt thereof. Suitable
ester groups include, for example, those derived from
pharmaceutically acceptable aliphatic carboxylic acids,
particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic
acids, in which each alkyl or alkenyl moiety advantageously has not
more than 6 carbon atoms. Examples of particular esters include,
but are not limited to, formates, acetates, propionates, butyrates,
acrylates and ethylsuccinates.
[0033] Pharmaceutically acceptable prodrug: The term
"pharmaceutically acceptable prodrugs" as used herein refers to
those prodrugs of the compounds of the present invention which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of humans and lower animals with undue
toxicity, irritation, allergic response, and the like, commensurate
with a reasonable benefit/risk ratio, and effective for their
intended use, as well as the zwitterionic forms, where possible, of
the compounds of the present invention. "Prodrug", as used herein
means a compound which is convertible in vivo by metabolic means
(e.g. by hydrolysis) to a compound of the invention. Various forms
of prodrugs are known in the art, for example, as discussed in
Bundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et
al. (ed.), Methods in Enzymology, vol. 4, Academic Press (1985);
Krogsgaard-Larsen, et al., (ed). "Design and Application of
Prodrugs, Textbook of Drug Design and Development, Chapter 5,
113-191 (1991); Bundgaard, et al., Journal of Drug Deliver Reviews,
8:1-38 (1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et
seq. (1988); Higuchi and Stella (eds.) Prodrugs as Novel Drug
Delivery Systems, American Chemical Society (1975); and Bernard
Testa & Joachim Mayer, "Hydrolysis In Drug And Prodrug
Metabolism: Chemistry, Biochemistry And Enzymology," John Wiley and
Sons, Ltd. (2002).
[0034] Pharmaceutically acceptable salt: As used herein, the term
"pharmaceutically acceptable salt" refers to those salts which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of humans and lower animals without undue
toxicity, irritation, allergic response and the like, and are
commensurate with a reasonable benefit/risk ratio. Pharmaceutically
acceptable salts are well known in the art. For example, S. M.
Berge, et al. describes pharmaceutically acceptable salts in detail
in J. Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be
prepared in situ during the final isolation and purification of the
compounds of the invention, or separately by reacting the free base
function with a suitable organic acid. Examples of pharmaceutically
acceptable include, but are not limited to, nontoxic acid addition
salts are salts of an amino group formed with inorganic acids such
as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric
acid and perchloric acid or with organic acids such as acetic acid,
maleic acid, tartaric acid, citric acid, succinic acid or malonic
acid or by using other methods used in the art such as ion
exchange. Other pharmaceutically acceptable salts include, but are
not limited to, adipate, alginate, ascorbate, aspartate,
benzenesulfonate, benzoate, bisulfate, borate, butyrate,
camphorate, camphorsulfonate, citrate, cyclopentanepropionate,
digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,
glucoheptonate, glycerophosphate, gluconate, hemisulfate,
heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate,
lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,
malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,
propionate, stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, alkyl having from 1 to 6 carbon atoms,
sulfonate and aryl sulfonate.
[0035] Substantially identical: The term "substantially identical"
is used to refer to a nucleic acid sequence that is sufficiently
duplicative of a reference sequence to share functional attributes
of the reference sequence. In particular, a sequence that is
"substantially identical" to a reference sequence hybridizes to the
complement of the reference sequence. In some embodiments, a
sequence is "substantially identical" to a reference sequence if it
shows at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or more identity to the reference sequence. In some
embodiments, a sequence is "substantially identical" to a reference
sequence if it shows 100% identity to the reference sequence (i.e.,
is identical to the reference sequence).
[0036] Susceptible to: The term "susceptible to" is used herein to
refer to an individual having higher risk (typically based on
genetic predisposition, environmental factors, personal history, or
combinations thereof) of developing a particular disease or
disorder, or symptoms thereof, than is observed in the general
population.
[0037] Prevention: The term "prevention", as used herein, refers to
a delay in onset and/or a reduction in severity of one or more
symptoms or attributes of a disease, disorder or condition, which
delay or reduction is observed when a pharmaceutical agent is
administered prior to onset of the symptom(s) or attribute(s).
[0038] Therapeutically effective amount: The term "therapeutically
effective amount" of a pharmaceutical agent or combination of
agents is intended to refer to an amount of agent(s) which confers
a therapeutic effect on the treated subject, at a reasonable
benefit/risk ratio applicable to any medical treatment. The
therapeutic effect may be objective (i.e., measurable by some test
or marker) or subjective (i.e., subject gives an indication of or
feels an effect). A therapeutically effective amount is commonly
administered in a dosing regimen that may comprise multiple unit
doses. For any particular pharmaceutical agent, a therapeutically
effective amount (and/or an appropriate unit dose within an
effective dosing regimen) may vary, for example, depending on route
of administration, on combination with other pharmaceutical agents.
Also, the specific therapeutically effective amount (and/or unit
dose) for any particular patient may depend upon a variety of
factors including the disorder being treated and the severity of
the disorder; the activity of the specific pharmaceutical agent
employed; the specific composition employed; the age, body weight,
general health, sex and diet of the patient; the time of
administration, route of administration, and/or rate of excretion
or metabolism of the specific pharmaceutical agent employed; the
duration of the treatment; and like factors as is well known in the
medical arts.
[0039] Treatment: As used herein, the term "treatment" (also
"treat" or "treating") refers to any administration of a
pharmaceutical agent that partially or completely alleviates,
ameliorates, relieves, inhibits, delays onset of, reduces severity
of and/or reduces incidence of one or more symptoms or features of
a particular disease, disorder, and/or condition. Such treatment
may be of a subject who does not exhibit signs of the relevant
disease, disorder and/or condition and/or of a subject who exhibits
only early signs of the disease, disorder, and/or condition.
Alternatively or additionally, such treatment may be of a subject
who exhibits one or more established signs of the relevant disease,
disorder and/or condition.
[0040] Unit dose: The term "unit dose", as used herein, refers to a
discrete administration of a pharmaceutical agent, typically in the
context of a dosing regiment.
DESCRIPTION OF CERTAIN EMBODIMENTS
[0041] The present invention encompasses the finding that microRNAs
(miRNAs) regulate certain key proteins involved in DNA repair
and/or cell cycle progression. In some embodiments, a miRNA
modulates levels and/or activity of one or more DNA repair and/or
cell cycle progression proteins (e.g., in some embodiments, a miRNA
suppresses levels and/or activity of one or more DNA repair and/or
cell cycle progression proteins). In some such embodiments, such
modulation renders cells hypersensitive to certain DNA damage
agents (e.g., .gamma.-irradiation and genotoxic drugs, among
others).
MicroRNAs
[0042] The present invention relates to miRNAs, and particularly to
miRNAs that regulate certain proteins involved in DNA repair and/or
cell cycle progression. In some embodiments, relevant miRNAs are
ones whose expression level increases or decreases during a
particular developmental stage of interest or in response to a
particular trigger or event of interest. In some embodiments,
relevant miRNAs are ones whose expression and/or activity levels
change during terminal differentiation of cells; in some
embodiments, relevant miRNAs are up-regulated in
terminally-differentiated cells. In some embodiments, relevant
miRNAs are up-regulated during terminal differentiation of
hematopoietic cells.
[0043] In some embodiments, relevant miRNAs are ones whose
expression changes during a particular developmental stage of
interest, or in response to a particular trigger or event of
interest, by an amount that is about 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, 60, 70, 80, 90, or 100 fold or more.
[0044] In some embodiments, relevant miRNAs are ones that regulate
cell cycle progression. In some embodiments, a relevant miRNA
suppresses the expression of cell cycle regulator genes. In some
embodiments, a relevant miRNA is characterized in that its
overexpression increases the number of cells in the G1 phase; in
some embodiments, a relevant miRNA is characterized in that its
inhibition causes differentiating cells to keep proliferating.
[0045] In some embodiments, a relevant miRNA targets genes that
initiate pathways such as synthesis of DNA building blocks; DNA
replication; DNA damage recognition; expression, transcriptional
regulation, and/or post-translational modification of cyclins,
cyclin-dependent kinases, and/or other cell cycle regulators. In
some embodiments, the miRNA targets MYC, E2F, and/or their
targets.
[0046] In some embodiments, a relevant miRNA targets genes that are
implicated in progression through the cell cycle, for example,
through G1, the G1/S checkpoint, S, and/or G2/M. In some
embodiments, a relevant miRNA targets genes that are involved in
DNA repair, including for example, genes (e.g., H2AX) that
sensitize cells to DNA damaging agents. In some embodiments, a
combination of miRNA targets is used. In some embodiments, an miRNA
targets a gene that promotes cell proliferation. In some
embodiments, an miRNA targets a gene that suppresses cell
proliferation (e.g., contributes to blocking cell cycle
progression). In some embodiments, an miRNA targets a gene that
participates in DNA repair. In some embodiments, an miRNA targets a
gene that suppresses DNA repair.
[0047] In some embodiments, a relevant miRNA is selected from the
group consisting of miR-24 and/or other miRNAs in the same cluster.
In some embodiments, a relevant miRNA is miR-22 or miR-125a. For
example, in some embodiments, a relevant miRNA is selected from the
group consisting of miR-24 (e.g., miR-24-1; miR-24-2), miR-23
(e.g., miR-23a, miR-23B), and miR-27 (e.g., miR-27a, miR-27b), etc.
In some embodiments, a relevant miRNA is a member of the let-7
family of miRNAs. In some embodiments, a relevant miRNA is selected
from the group consisting of miR-48, miR-84, and miR-241. In some
embodiments, a relevant miRNA is selected from the group consisting
of miR-17, miR-18, miR-19, miR-20, miR-34a, miR-92, miR-125,
miR-146a, miR-155, miR-181a, 200a. In some embodiments, a relevant
miRNA is one that is found on chromosome 9, or on chromosome 19. In
some embodiments, a relevant miRNA is one that is found in an
intergenic region of a chromosome (e.g., chromosome 19). In some
embodiments, a relevant miRNA is a viral miRNA. In some
embodiments, a relevant miRNA is a member of the Herpes virus
family. In some embodiments, a relevant miRNA is miR-K12-11. The
present Examples exemplify the invention with respect to
miR-24.
[0048] Among other things, the present invention provides methods
that involve increasing levels and/or activities of one or more
miRNAs, and particularly of one or more miRNAs that regulates DNA
repair and/or cell cycle progression. Some such methods involve
increasing levels of an miRNA agent, in cells. Any of a variety of
strategies may be used to increase levels of an miRNA agent in
cells, including, for example, introducing an miRNA agent into
cells (e.g., by transfection, transformation, injection, induction,
expression from a viral or other vector, etc.).
[0049] Alternatively or additionally, the present invention
provides methods that involve decreasing levels and/or activities
of one or more miRNAs, and particularly of one or more miRNAs that
regulates DNA repair and/or cell cycle progression. Some such
methods involve, for example, providing a competitor agent that
competes with a target for interaction with the miRNA. To give but
one specific example, in some embodiments, the present invention
provides methods that utilize a "miRNA sponge" that contains
multiple copies of an miRNA target sequence. In some embodiments,
the present invention provides methods that involve introducing
into a cell an agent that specifically degrades (or targets for
degradation) a particular miRNA.
DNA Repair Proteins
[0050] Cells have evolved the capacity to remove or tolerate
lesions in their DNA (Friedberg, 1985). The most direct mechanisms
for repairing DNA are those that simply reverse damage and restore
DNA to its normal structure in a single step. Cells can eliminate
three types of DNA damage by chemically reversing it. Such direct
reversal mechanisms are specific to the type of damage incurred and
do not involve breakage of the phosphodiester backbone. In some
embodiments, DNA damage can lead to formation of thymidine dimers,
methylation of guanine bases, methylation of cytosine bases, and/or
methylation of adenosine bases, or combinations thereof. Those of
ordinary skill in the art will appreciate that a variety of agents
induce DNA damage (e.g., .gamma.-irradiation and/or administration
of one or more genotoxic drugs, among others). In some embodiments,
DNA repair proteins are those that are involved in direct reversal
of DNA damage (e.g. photolyases and/or methyltransferases). In some
embodiments, DNA repair proteins involved in the direct reversal
pathway include human MGMT (Genbank Accession No. M2997 1) and
other similar proteins.
[0051] A more complex mechanism, excision repair, involves incision
of the DNA at the lesion site, removal of the damaged or
inappropriate base(s), and resynthesis of DNA using the undamaged
complementary strand as a template. This system of repair can
further be categorized into base and nucleotide excision
repair.
[0052] Base excision repair involves two major classes of repair
enzymes, namely, N-glycosylases and AP endonucleases (Wallace,
1988; Sakumi and Sekiguchi, 1990; Doetsch and Cunningham; 1990).
DNA N-glycosylases are enzymes that hydrolyze the N-glycosidic bond
between the damaged base and the deoxyribose moiety, leaving behind
an AP site on the DNA backbone. AP sites produced by the action of
N-glycosylases are acted upon by AP endonucleases, which can make
an incision either 3' to the AP site (class I AP lyase) or 5' to
the AP site (class II AP endonuclease). All those enzymes shown to
contain class I AP lyase activity possess an associated DNA
glycosylase activity; however, not all glycosylases are AP lyases.
Class II AP endonucleases are the major enzymes responsible for the
repair of AP sites in DNA.
[0053] DNA glycosylases can be defined as enzymes which recognize
specific DNA base modifications and catalyze the hydrolysis of the
N-glycosylic bond that links a base to the deoxyribose-phosphate
backbone of DNA (for review, see Sancar and Sancar, 1988; Wallace,
1988; Sakumi and Sekiguchi, 1990). This enzymatic activity results
in the generation of an AP site. To date, several DNA glycosylases
have been identified and are classified into two major families: 1)
enzymes that possess only DNA glycosylase activity and 2) enzymes
that contain both a DNA glycosylase activity and an associated
class I AP lyase activity; that is, enzymes that catalyze a
beta-elimination cleavage of the phosphodiester bond 3' to an AP
site.
[0054] In some embodiments, the present invention relates to miRNAs
that regulate certain proteins involved in DNA repair. In some
embodiments, relevant DNA repair proteins include those from the
base excision repair (BER) pathway, e.g., AP endonucleases such as
human APE (NAPE, Genbank Accession No. M80261) and related
bacterial or yeast proteins such as APN-1 (e.g., Genbank Accession
No. U33625 and M33667), exonuclease III (ExoIII, xth gene, Genbank
Accession No. M22592), exonuclease I (Exol), bacterial endonuclease
III (EndoIll, nth gene, Genbank Accession No. J02857), huEndolll
(Genbank Accession No. U79718), and endonuclease IV (EndoIV nfo
gene Genbank Accession No. M22591). In some embodiments, relevant
DNA repair proteins suitable for use in the invention include,
additional BER proteins including DNA glycosylases such as,
formamidopyrimidine-DNA glycosylase (FPG, Genbank Accession No.
X06036), human 3-alkyladenine DNA glycosylase (HAAG, also known as
human methylpurine-DNA glycosylase (hMPG, Genbank Accession No.
M74905), NTG-1 (Genbank Accession No. P31378 or 171860), SCR-1
(YAL015C), SCR-2 (Genbank Accession No. YOL043C), DNA ligase I
(Genbank Accession No. M36067), P-polymerase (Genbank Accession No.
M13140 (human)) and 8-oxoguanine DNA glycosylase (OGG1 Genbank
Accession No. U44855 (yeast); Y13479 (mouse); Y11731 (human)). In
some embodiments, relevant DNA repair proteins include histone
variants (e.g. H2AX) and transcription factors that regulate
expression of DNA repair genes (e.g. XBP1). In some embodiments,
the present invention relates to H2AX.
Cell Cycle Progression Proteins
[0055] The sequence of cell cycle events is rigorously controlled
at specific checkpoints to ensure that each discrete stage in the
cell cycle has been completed before the next is initiated. Human
diseases associated with abnormal cell proliferation, including
cancer, result when these rigorous controls on cell cycle
progression are perturbed. On the other hand, it is also sometimes
desirable to enhance proliferation of cells in a controlled manner.
For example, proliferation of cells is useful in wound healing and
where growth of tissue is desirable. Those of ordinary skill in the
art will appreciate that there may be several mechanisms for cell
cycle progression, for example the processes of mitosis and/or
meiosis.
[0056] In general, cell cycle progression is regulated by a variety
of cellular factors. For example, two relevant classes of cell
cycle progression regulatory molecules include cyclins and
cyclin-dependent kinases (CDKs). In some embodiments, cell cycle
progression proteins are selected from the group consisting of
cyclin D, cyclin dependent kinase 4 (CDK4), retinoblastoma
susceptibility protein (RB), E2F, cyclin E, cyclin A, DNA
polymerase, thymidine kinase, cyclin dependent kinase 2, cyclin B.
In some embodiments, cell cycle progression proteins prevent the
progression of the cell cycle. In some embodiments, for example,
cell cycle progression proteins are selected from the group
consisting of p21, p27, p57, p53, myc, TGFb, p16INK4a, p14arf. In
some embodiments, cell cycle progression proteins are involved in
DNA replication and repair checkpoints. In some embodiments, cell
cycle progression proteins are selected from the group consisting
of PCNA, CHEK1, BRCA1, FEN1, and UNG.
Applications
Cell Proliferative Disorders
[0057] In some embodiments, the invention provides methods and
reagents for treating cell proliferative disorders, diseases or
conditions. In general, cell proliferative disorders, diseases or
conditions encompass a variety of conditions characterized by
aberrant cell growth, preferably abnormally increased cellular
proliferation. For example, cell proliferative disorders, diseases,
or conditions include, but are not limited to, atherosclerosis,
cancer, immune-mediated responses and diseases (e.g., transplant
rejection, graft vs host disease, immune reaction to gene therapy,
autoimmune diseases, pathogen-induced immune dysregulation, etc.),
certain circulatory diseases, and certain neurodegenerative
diseases.
[0058] In certain embodiments, the invention relates to methods and
reagents for treating cancer. In general, cancer is a group of
diseases which are characterized by uncontrolled growth and spread
of abnormal cells. Examples of such diseases are carcinomas,
sarcomas, leukemias, lymphomas and the like. In certain
embodiments, the cancer is a hematological malignancy. In certain
embodiments, the cancer is a solid tumor. For example, in some
embodiments, the invention relates to treatment of rejection
following transplantation of synthetic or organic grafting
materials, cells, organs, or tissue to replace all or part of the
function of tissues, such as heart, kidney, liver, bone marrow,
skin, cornea, vessels, lung, pancreas, intestine, limb, muscle,
nerve tissue, duodenum, small-bowel, pancreatic-islet-cell,
including xeno-transplants, etc.; treatment of graft-versus-host
disease; autoimmune diseases, such as rheumatoid arthritis,
systemic lupus erythematosus, thyroiditis, Hashimoto's thyroiditis,
multiple sclerosis, myasthenia gravis, type I diabetes,
juvenile-onset or recent-onset diabetes mellitus, uveitis, Graves'
disease, psoriasis, atopic dermatitis, Crohn's disease, ulcerative
colitis, vasculitis, auto-antibody mediated diseases, aplastic
anemia, Evan's syndrome, autoimmune hemolytic anemia, and the like;
and further to treatment of infectious diseases causing aberrant
immune response and/or activation, such as traumatic or pathogen
induced immune dysregulation.
[0059] In some embodiments, the invention relates to treatment of
any of a variety of neurodegenerative diseases such as, for
example, Alzheimer's disease, Parkinson's disease, and/or
Huntington's disease.
[0060] In some embodiments, the present invention provides methods
of treating a cell proliferative disease, disorder, or condition,
by administering to an individual who is suffering from or
susceptible to the cell proliferative disease, disorder, or
condition a therapeutically effective amount of an miRNA agent. In
some embodiments, the therapeutically effective amount is an amount
sufficient to render cells of the individual hypersensitive to one
or more DNA damage agents. In some embodiments, the therapeutically
effective amount is an amount sufficient to suppress expression
and/or activity of one or more DNA repair proteins. In some
embodiments, the therapeutically effective amount is an amount
sufficient to inhibit cell proliferation. In some embodiments, the
therapeutically effective amount is an amount sufficient to induce
apoptosis.
[0061] In some embodiments, a cell is considered to be
hypersensitive to one or more DNA damage agents if it shows
increased chromosomal instability (e.g., increased numbers and/or
persistence of chromosome breaks), increased cell death rates,
and/or increased sensitivity to genotoxic stress.
Increasing Cell Division
[0062] In some embodiments, the present invention provides systems
for increasing cell division, for example by modulating (e.g.,
reducing) levels and/or activities of one or more miRNAs (e.g.,
miR-24). In some embodiments, for example, modulation of one or
more miRNA levels or activities leads to enhanced division and/or
survival as compared with control cells.
[0063] In some embodiments, miRNA levels and/or activities are
modulated through administration of an agent that modulates (e.g.,
promotes or suppresses activity of) the miRNA. To give but one
specific example, in some embodiments, level and/or activity of a
particular miRNA may be reduced by, for example, administration of
an anti-sense agent that hybridizes with the miRNA and competes
with one or more natural targets of the miRNA. In some such
embodiments, the anti-sense agent is a miRNA sponge (see
above).
[0064] In certain embodiments, systems for increasing cell division
are useful, for example, in cell culture applications. In some
embodiments, any of a variety of cell types are utilized. In some
embodiments, stem cell (e.g. embryonic stem cell, hematopoietic
stem cell, tissue stem cell, etc) proliferation is increased.
[0065] In some embodiments, said systems for increasing cell
division are useful, for example, in the preparation and/or
processing of cells or tissues for implantation. For example, in
some embodiments, cells are cultured for implantation into a
subject (e.g., for tissue replacement and/or repair
applications).
[0066] In some embodiments, cell proliferation is increased in
tissue explants.
Diagnostics
[0067] In some embodiments, the present invention provides systems
for identifying cells (and/or individuals) that are suffering from
or susceptible to one or more cell proliferative disorders, for
example by detecting unusual levels or activities of one or more
miRNAs and/or their targets (whether at the level of RNA or
protein). In some embodiments, the targets include one or more DNA
repair proteins.
[0068] In some embodiments, the present invention provides systems
for identifying cells (and/or individuals) that are hypersensitive
to DNA damage agents, for example by detecting levels or activities
of one or more miRNAs and/or their targets (whether at the level of
RNA or protein). In some embodiments, the targets include one or
more DNA repair proteins. In some embodiments, identification of
cells (and/or individuals) that are hypersensitive to DNA damage
agents allows identification of cells (and/or individuals) who are
suffering from or susceptible to one or more cell proliferative
disorders and who are likely to benefit from therapy that includes
administration of one or more DNA damaging agents or treatments
(e.g., .gamma.-irradiation and/or administration of one or more
genotoxic drugs).
Pharmaceutical Compositions
[0069] Therapeutic agents (optionally including, for example, miRNA
agents) may be administered to cells or individuals in accordance
with the present invention, in the context of a pharmaceutical
composition. In general, a pharmaceutical composition comprises at
least one therapeutically active agent and at least one
pharmaceutically acceptable carrier or excipient. Those of ordinary
skill in the art will appreciate that a therapeutically active
agent may be provided in any of a variety of forms including, for
example, in a pharmaceutically acceptable salt or ester form.
[0070] Representation pharmaceutically acceptable carriers or
excipients typically include, for example, one or more solvents,
diluents, or other liquid vehicles, dispersion or suspension aids,
surface active agents, isotonic agents, thickening or emulsifying
agents, preservatives, solid binders, lubricants, permeation
enhancers, solubilizing agents, and the like, as suited to the
particular dosage form desired. Remington's Pharmaceutical
Sciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co.,
Easton, Pa., 1975) discloses various carriers used in formulating
pharmaceutical compositions and known techniques for the
preparation thereof. Except insofar as any conventional carrier
medium is incompatible with a particular therapeutically active
agent, such as by producing any undesirable biological effect or
otherwise interacting in a deleterious manner with any other
component(s) of the pharmaceutical composition, its use is
contemplated to be within the scope of this invention.
[0071] Some examples of materials which can serve as
pharmaceutically acceptable carriers include, but are not limited
to, sugars such as lactose, glucose and sucrose; starches such as
corn starch and potato starch; cellulose and its derivatives such
as sodium carboxymethyl cellulose, hydroxymethylcellulose,
hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, and
cellulose acetate; powdered tragacanth; malt; gelatin; talc;
Cremophor (polyethoxylated caster oil); Solutol (poly-oxyethylene
esters of 12-hydroxystearic acid); excipients such as cocoa butter
and suppository waxes; oils such as peanut oil, cottonseed oil;
safflower oil; sesame oil; olive oil; corn oil and soybean oil;
glycols; such a propylene glycol; esters such as ethyl oleate and
ethyl laurate; agar; buffering agents such as magnesium hydroxide
and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic
saline; Ringer's solution; ethyl alcohol, and phosphate buffer
solutions, as well as other non-toxic compatible lubricants such as
sodium lauryl sulfate and magnesium stearate, as well as coloring
agents, releasing agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives, and antioxidants can also be
present in the composition, according to the judgment of the
formulator.
[0072] In some embodiments, a pharmaceutically acceptable carrier
is selected from the group consisting of sugars such as lactose,
glucose and sucrose; starches such as corn starch and potato
starch; cellulose and its derivatives such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; powdered
tragacanth; malt; gelatin; talc; excipients such as cocoa butter
and suppository waxes; oils such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
glycols such as propylene glycol; esters such as ethyl oleate and
ethyl laurate; agar; buffering agents such as magnesium hydroxide
and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic
saline; Ringer's solution; ethyl alcohol, and phosphate buffer
solutions; non-toxic compatible lubricants such as sodium lauryl
sulfate and magnesium stearate; coloring agents; releasing agents;
coating agents; sweetening, flavoring and perfuming agents;
preservatives and antioxidants; and combinations thereof. In some
embodiments, the pH of the ultimate pharmaceutical formulation may
be adjusted with pharmaceutically acceptable acids, bases or
buffers to enhance the stability of the formulated therapeutically
active agent or its delivery form.
[0073] Pharmaceutical compositions may be administered in
accordance with the present invention by any appropriate means
including, for example, orally, parenterally, by inhalation spray,
topically, rectally, nasally, buccally, vaginally or via an
implanted reservoir. The term parenteral as used herein includes
subcutaneous, intracutaneous, intravenous, intramuscular,
intraarticular, intraarterial, intrasynovial, intrasternal,
intrathecal, intralesional and intracranial injection or infusion
techniques. In many embodiments, pharmaceutical compositions are
administered orally or by injection in accordance with the present
invention.
Combination Therapy
[0074] In accordance with the present invention, two or more
therapeutically active agents or regimens may be administered
simultaneously to an individual.
[0075] To give but one example, in some embodiments, it may be
desirable to administer an miRNA agent in combination with one or
more DNA damage agents or treatments (e.g., .gamma.-irradiation
and/or one or more genotoxic drugs). In some embodiments,
combinations of miRNA agents and DNA damage agents or treatments
are administered to individuals suffering from or susceptible to
one or more cell proliferation diseases, disorders, or
conditions.
EXEMPLIFICATION
Materials and Methods
Cell Culture and Differentiation
[0076] HepG2 cells were grown in DMEM supplemented with 10% FCS.
HL60 and K562 cells were grown in RPMI-1640 supplemented with 10%
FCS. K562 cells (0.5.times.10.sup.6 cells/ml) were treated with TPA
(16 nM, 2 d) or Hemin (100 .mu.M, 4 d) for differentiation into
megakaryocytes or erythrocytes, respectively. To induce macrophage
or granulocyte differentiation, HL60 cells (0.5.times.10.sup.6
cells/ml) were treated with TPA (16 nM, 2 d) or DMSO (1.25%, 5 d),
respectively. We isolated human polymorphonuclear neutrophils (PMN)
from whole blood after removing mononuclear cells and platelets by
Ficoll-Hypaque density gradient centrifugation. Erythrocytes were
lysed by treatment with ice-cold isotonic lysis buffer (0.155 M
NH4C1, pH 7.4). The remaining PMN cells were washed with Hanks'
balanced salt solution and suspended in RPMI medium containing 10%
(v/v) FCS. We isolated human macrophages from peripheral blood as
described in Song et al. 2003.
RNA Isolation and Quantitative RT-PCR
[0077] Total RNA was isolated using Trizol (Invitrogen) and reverse
transcribed using random hexamers and superscript II reverse
transcriptase (Invitrogen). qRT-PCR was performed in triplicate
samples using the SYBR Green master mix (Applied Biosystems) and
the BioRad iCycler. Primers are provided in Supplemental Table 1.
Results were normalized to GAPDH. miRNA quantitative PCR was done
in triplicate using the TaqMan MicroRNA Assay from Applied
Biosystems as per the manufacturer's instructions and normalized to
U6 SnRNA.
miRNA Microarray
[0078] We performed miRNA microarrays as described in Song et al.
2003.
miRNA Mimic and Antisense Oligonucleotide Transfection
[0079] HepG2 cells (2.5.times.10.sup.5/well) were reverse
transfected with 30 nM miRNA to control (cel-miR-67) mimics
(Dharmacon) using NeoFx (Ambion) following the manufacturer's
instructions. K562 cells were transfected with miRNA or control
mimics (100 nM) using Amaxa nucleofection following the
manufacturer's protocol. K562 cells were treated with TPA (16 nM, 2
d) and were transfected with 100 nM miR-24 ASO using lipofectamine
2000 (Invitrogen) and 36 h later these cells were exposed to
indicated concentrations of bleomycin and cell viability was
assessed 2 d later.
Biotin Pull-Down
[0080] K562 cells (1.times.10.sup.6/well) were transfected with
3'-biotinylated miR-24 (Dharmacon) or 3'-biotinylated control miRNA
(cel-miR-67) at a final concentration of 100 nM in six-well plates
in triplicate wells using Amaxa nucleofection following the
manufacturer's protocol. Twenty-four hours later, the cells were
trypsinized and pelleted at 500.times.g. After washing twice with
PBS and resuspension in 0.5 ml lysis buffer (20 mM Tris (pH 7.5),
100 mM KCl, 5 mM MgCl.sub.2, 0.3% NP-40, 50 U of RNase OUT
(Invitrogen), complete mini-protease inhibitor cocktail (Roche
Applied Science)), and incubation at 4.degree. C. for 5 min, the
cytoplasmic extract was isolated by centrifugation at
10,000.times.g for 10 min. Streptavidin-coated magnetic beads (50
.mu.l, Invitrogen) were blocked for 2 hr at 4.degree. C. in lysis
buffer containing 1 mg/ml yeast tRNA and 1 mg/ml BSA (Ambion) and
washed twice with 1 ml lysis buffer. Cytoplasmic extract was then
added to the beads and incubated for 4 h at 4.degree. C., following
which the beads were washed five times with 1 ml lysis buffer. RNA
bound to the beads (pull-down RNA) or from 10% of the extract
(input RNA), was isolated using Trizol LS reagent (Invitrogen). The
level of mRNA in the miR-24 or control pull-down was quantified by
qRT-PCR and normalized to its abundance in the input RNA.
Luciferase Assay
[0081] HepG2 cells (2.5.times.10.sup.5/well) were reverse
transfected in triplicate with 30 nM miR-24 mimic, miRNA-328 mimic
or control miRNA mimic. Two days later, cells were transfected
using Lipofectamine 2000 (Invitrogen) with psiCHECK2 (Promega)
vector (0.5 .mu.g/well) containing the 3'UTR of H2AX cloned in the
multiple cloning site of Renilla luciferase or control. After 24 hr
luciferase activities were measured using the Dual Luciferase Assay
System (Promega) and TopCount NXT microplate reader (Perkin Elmer)
per manufacturer's instructions. Data were normalized to Firefly
luciferase. To test whether H2AX mRNA is directly regulated by
miR-24, we cloned the two predicted MREs in the H2AX 3' UTR into
the multiple cloning site of psiCHECK2 and also the mutant versions
that disrupted base-pairing between the binding sites and miR-24.
HepG2 cells were cotransfected with these plasmids and miR-24 or
control mimics for 48 h using Lipofectamine 2000, before we
performed the luciferase assays as described above.
Immunoblot
[0082] K562 cells (1.times.10.sup.6) were transfected with miR-24
mimics or control miRNA mimics (cel-miR-67) as above and 48 h later
whole cell lysates were prepared using RIPA buffer (150 mM NaCl, 1%
NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0).
Protein samples were quantified using Bradford reagent (BioRad) and
resolved on 10% SDS-PAGE gels and analyzed by immunoblot probed
with antibodies to H2AX (Upstate Biotech), CHEK1 (cell Signaling),
histone H3 (Cell Signaling), tubulin (Sigma). All antibodies were
used at a dilution of 1:1000.
Chromosome Breakage Analysis
[0083] K562 and HepG2 cultures were exposed in duplicate wells to
indicated doses of .gamma.-irradiation and incubated at 37.degree.
C. for indicated times in 5% CO.sub.2. Cells were harvested and
processed for chromosomal analysis following standard methods (51).
50-75 Wright-stained metaphases for each condition were scored for
chromosomal aberrations.
Single Cell Gel Electrophoresis (Comet) Assay
[0084] Single cell comet assays were performed as per
manufacturer's instructions (Trevigen). Briefly, cells were
transfected with siRNAs and 60 h later DSBs induced with CPT (2
.mu.M, 1 h, 37.degree. C.). Treated or untreated cells were
collected, resuspended in ice cold PBS at 10.sup.5 cells/ml, mixed
with low-melt agarose (1:10 ratio) and spread on frosted glass
slides. After the agarose solidified, the slides were successively
placed in lysis and alkaline solutions (Trevigen). Slides were then
subjected to electrophoresis (1V/cm distance between electrodes)
for 10 min in 1.times.TBE buffer and cells were fixed with 70%
ethanol and stained with SYBR Green. Nuclei were visualized using
epifluorescent illumination on a Zeiss microscope and images
analyzed with the NIH Image program. DNA damage was quantified for
75 cells for each experimental condition by determining the tail
moment, a function of both the tail length and intensity of DNA in
the tail relative to the total DNA, using the software Comet Score
(TriTek). Statistical analysis was by Student's t-test.
Cell Viability Assay
[0085] microRNA-transfected K562 or HepG2 cells were seeded
(2.times.10.sup.3 cells/100 .mu.l) into octuplicate microtiter
wells, incubated overnight, and then treated with indicated
reagents or medium for 48 h. Viability was measured by CyQuant Cell
Proliferation Assay Kit as per manufacturer's instructions
(Molecular Probes). Results were expressed as OD.sub.520 relative
to that of untreated cells.
Results
[0086] Once a cell has terminally differentiated and no longer
replicates its DNA, its need to repair DNA damage is reduced.
Although ongoing DNA damage from oxidative metabolism and exogenous
agents may be similar in dividing and nondividing cells, endogenous
double stranded breaks (DSB) that occur during DNA replication and
compromise genomic integrity are radically reduced or absent and
the danger of propagating damaged chromatin in progeny cells is
minimized once a cell has stopped dividing. Nonetheless, cells that
do not divide need to maintain the integrity of the genes they
transcribe. For some long-lived and essentially irreplaceable
cells, such as neurons, DNA repair may be more essential than for
short-lived cells, such as terminally differentiated blood cells.
Dividing cells handle the risk of creating DSB during DNA
replication by expressing and activating the homologous
recombination (HR) repair machinery in a cell cycle dependent
fashion only during S phase. Moreover, during cell division, DNA
damage checkpoint proteins survey for unrepaired DNA damage to
prevent cell cycle progression at G1/S and G2/M. As a consequence
of their reduced needs for DNA repair, nondividing cells have an
attenuated response to DSB (1).
[0087] The molecular mechanisms behind the down-regulation of DNA
repair in terminally differentiated cells are generally not well
understood. In some cases, specific repair proteins are down
regulated. For instance, Chekl, the orchestrator of cell cycle
arrest in response to replication mediated DNA damage in
proliferating cells, is absent in terminally differentiated tissues
(2) Likewise, E2F1 and p53 expression are down-regulated in
terminally differentiated myotubes (3, 4). mRNA for Ku, the DNA
binding proteins of the DNA-dependent protein kinase, which plays a
central role in DSB repair by nonhomologous end joining (NHEJ),
decreases during differentiation of HL-60 cells into monocytes (5).
However, other repair pathways besides DSB repair, such as base
excision repair (BER) and transcription-coupled repair, which
repair lesions of equal importance in nondividing and dividing
cells, may be undiminished after terminal differentiation.
[0088] The microRNA (miRNA) miR-24 is uniformly up-regulated during
terminal differentiation of 2 hematopoietic leukemia cell lines,
HL60 and K562, into multiple cell lineages as well as in CD8 T cell
and muscle cell differentiation (accompanying manuscript, (6, 7))
(FIG. 4A and FIG. 4B). In the accompanying patent application, U.S.
Ser. No. 61/098,696, filed on Sep. 19, 2008, we developed a
biochemical approach to identify the genes regulated by a miRNA by
isolating mRNAs that bind to a transfected biotinylated mimic of
the processed active miRNA transcript. 269 mRNAs were significantly
enriched in the miR-24 pull-down. Genes involved in DNA repair and
regulating cell cycle progression were highly significantly
enriched in the pull down.
[0089] In particular, of the top 15 enriched gene ontology (GO)
processes, 11 were involved in various aspects of cell cycle
regulation and 2 were in the response to DNA damage and DNA repair.
miR-24 pulled down 31 of 401 genes identified as participating in
response to DNA damage (7.7%, p=7E-13) and 26 of 313 genes
associated with DNA repair (8.3%, p=1E-11). Some of the key nodes
in a bioinformatic analysis of the network of known direct
interactions of the pulled-down gene products included PCNA, which
localizes to DNA replication forks and is required to repair and
resolve stalled replication forks (8); CHEK1, the checkpoint
protein that is activated by ATR and induces cell cycle arrest at
G2/M in response to unresolved DNA damage; BRCA1, which
participates in a complex that activates DSB repair (9); FEN1, a
flap endonuclease which removes the 5'ends of Okazaki fragments
during lagging-strand DNA synthesis and participates in BER (10).
In addition to these nodes many of the miR-24-bound genes are key
players in DNA repair, including UNG, the major cellular uracil DNA
glycosylase (11), EXO1, a 5'-3' exonuclease that has a key role in
multiple DNA repair and replication pathways (12); H2AX, a histone
variant that gets phosphorylated at DSB where it serves to
stabilize checkpoint and repair factors (13); and XBP1, a
transcription factor that upregulates DNA repair genes (including
FEN 1 and H2AX) (14). The top 3 over-represented GO processes
identified in another data set--the overlap of genes whose mRNA
expression was significantly reduced by miR-24 overexpression with
the set of miR-24 predicted targets by TargetScan 4.2--were DNA
damage checkpoint (4 of 44 genes, 9.1%, p=0.0001), DSB repair via
HR (3 of 17 genes, 17.6%, p=0.0001) and recombinational repair (3
of 17 genes, 17.6%, p=0.0001). These analyses strongly suggested
that miR-24 might inhibit DNA repair, with perhaps a special
emphasis on genes involved in repairing lesions that occur during
DNA replication.
[0090] To identify miRNAs regulating DNA repair during terminal
hematopoietic cell differentiation, we analyzed miRNA expression by
microarray in two human leukemia cell lines--K562 cells
differentiated to megakaryocytes using
12-0-tetradecanoylphorbol-13-acetate (TPA) or to erythrocytes with
hemin, and HL60 cells differentiated to macrophages using TPA or to
monocytes using vitamin D3 (FIG. 4). Only a few miRNAs were
consistently upregulated (by at least 40%) in all four systems of
terminal differentiation: miR-22, miR-125a and members of the two
miR-24 clusters--miR-24, miR-23a, miR-23b and miR-27a. miR-24 stood
out as the most upregulated miRNA. The only member of the two
miR-24 clusters that was not consistently upregulated was miR-27b,
whose hybridization signal was substantially lower for all
conditions than the other cluster members, suggesting that
hybridization to that probe was inefficient. We therefore focused
our study on miR-24, which we hypothesized might regulate terminal
differentiation in nondividing cells across multiple cell
lineages.
[0091] We verified the microarray results by quantitative reverse
transcription PCR (qRT-PCR). miR-24 was consistently upregulated
during terminal differentiation of HL60 and K562 cells (FIG. 4A and
FIG. 4B) and in differentiation of CD8 T cells, muscle cells and
embryonic stem cells 15-17. One of the biggest challenges in
studying miRNAs is to identify target genes and correlate their
downregulation with cellular properties. Computational algorithms
have been developed to predict putative miRNA targets based on
complementarity to the 3' untranslated region (UTR) of the target
message, particularly of miRNA nucleotides 2-8 (the `seed`
region)(18). These tools (TargetScan, PicTar, ma22, miRanda)
predict overlapping, but distinct, miR-24 target gene sets (18).
One strategy to counter this problem is to pursue targets predicted
by multiple algorithms, and with a high prediction score. The DSB
repair gene predicted by all algorithms with a high recognition
score was H2AFX, encoding histone variant H2AX.
[0092] One of the earliest events in the DSB response is
phosphorylation of H2AX at Ser139 by members of the
phosphatidylinositol-3 kinase-like family of kinases (13).
Phosphorylated H2AX (termed .gamma.-H2AX) participates in DNA
repair, replication, and recombination and cell cycle regulation
(13). The large domains of .gamma.-H2AX generated at each DSB can
be visualized by immunostaining as nuclear foci. .gamma.-H2AX foci
bind and retain an array of cell cycle and DNA repair factors
(cohesins, MDC1, Mrel 1, BRCA1, 53BP1, etc.) at the break site (15,
16). Importantly, loss of a single H2AX allele compromises genomic
integrity and enhances cancer susceptibility in mice (17, 18). This
observation has both clinical and mechanistic implications. The
H2AX dosage effect may reflect its structural role in chromatin.
H2AX comprises -15% of cellular H2A, and there are two H2A
molecules per nucleosome. Thus, H2AX should be present, on average,
in about one of three nucleosomes, and this density likely is
reduced in cells with less H2AX, which may interfere with H2AX
function. Therefore, a subtle change in cellular H2AX, as might
occur with miRNA targeting, may significantly impact DSB repair.
Because of the critical role of H2AX in DNA repair and the known
consequences of haploinsufficiency, we focused on validating and
studying the effect of miR-24 on H2AX.
[0093] H2AX mRNA and protein declined during K562 and HL60 cell
differentiation (FIG. 4C to FIG. 4E). The H2AX transcript can be
processed alternatively to a B1.6-kb replication-independent
transcript with a poly(A) tail or a B0.6-kb transcript found only
in dividing cells, which has a short 3' UTR and lacks a poly(A)
tail (19). The shorter transcript, whose sequence is not annotated,
might lack miR-24 recognition sites, because the H2AX transcript
without the 3' UTR is 505 bases long, leaving only about 100 by for
the 3' UTR. This H2AX transcript containing a shorter 3' UTR and
expressed only in dividing cells could be an example of the
recently described principle of preferential miRNA regulation of
longer transcripts in nondividing cells.
[0094] To investigate whether miR-24 regulates H2AX expression, we
first quantified H2AX mRNA in streptavidin pull-downs from K562
cells transfected for 24 hr with 3'-biotinylated miR-24 or control
biotinylated miRNA (cel-miR-67) (FIG. 4F) (accompanying patent
application U.S. Ser. No. 61/098,696, filed on Sep. 19, 2008).
Capture of H2AX mRNA in the miR-24 pull-down, analyzed by qRT-PCR
normalized to GAPDH, was enhanced by more than 3-fold compared to
pull-down with the control miRNA. Pull-down of another housekeeping
gene (UBC) did not differ from background. Unlike most of the mRNAs
pulled-down by miR-24, H2AX is a predicted target of miR-24 by both
TargetScan 4.2 and PicTar. Its 3'UTR, which is 1086 nucleotides
long, encodes 2 evolutionarily conserved 7-mer exact matches to the
miR-24 seed at positions 88-94 and 971-977 and each site has
additional pairings to the 3'-region of miR-24 (FIG. 4G). PicTar
predicts another conserved miRNA interaction (miR-328) with the
H2AX 3'UTR.
[0095] Next, by qRT-PCR, using primers from the H2AX coding region
that measure both transcripts, we found a four-fold reduction in
H2AX mRNA in TPA-treated K562 cells (data not shown). Using primers
specific for the longer transcript, H2AX mRNA declined by two-fold
when K562 cells were differentiated by TPA to megakaryocytes or by
hemin to erythrocytes, and when HL60 cells were differentiated by
TPA to macrophages or by DMSO to granulocytes (FIG. 4C and FIG.
4D). The level of H2AX protein, measured after TPA induction,
dropped by 14-fold in K562 cells and 4-fold in HL60 cells (FIG.
4E). The strong decrease in H2AX protein levels (relative to the
modest decrease in H2AX mRNA level) during differentiation may be
attributed to miR-24--mediated translational inhibition of the
residual H2AX transcripts. We first detected increased miR-24 and
reduced H2AX mRNA levels in TPA-differentiated K562 and HL60 cells
12 h after adding TPA at which time the cells had stopped dividing
(FIG. 1A to FIG. 1D, and FIG. 9B). The relatively high miR-24 and
low H2AX mRNA and proteins levels in vitro differentiated cells
were comparable to levels in primary human peripheral blood
monocytes and granulocytes (data not shown). The reduction in H2AX
mRNA coincident with increased miR-24 in differentiated cell lines
and primary blood cells could be due to miR-24 inhibition of H2AX
mRNA expression and/or stability.
[0096] We next tested the effect of miR-24 on luciferase expression
from control or H2AX 3'UTR-containing reporter genes in HepG2
cells. Luciferase activity was unchanged from control reporters,
but was reduced more than 2-fold by miR-24 expression (FIG. 4H).
miR-24 over-expression in HepG2 cells decreased H2AX mRNA by
2-fold, while protein expression was reduced even more
(.about.8-fold) (FIG. 4I and FIG. 4J) Overexpressing miR-328
predicted (by PicTar) to target the 3'UTR of H2AX had no effect on
luciferase activity or H2AX mRNA or protein levels, further
underlining the specificity of the miR-24/H2AX interaction (FIG. 2A
and FIG. 2B). Collectively, these results demonstrate that miR-24
binds to the 3'UTR of H2AX mRNA and down-regulates its expression
likely by promoting both mRNA decay and inhibiting translation.
[0097] H2AX is a predicted miR-24 target by both TargetScan 4.2 and
PicTar. Its 3' UTR, which is 1,086 nucleotides long, encodes two
evolutionarily conserved heptamer exact matches to the miR-24 seed,
at positions 88-94 and 971-977, and each site has additional
pairings to the 3' region of miR-24 (FIG. 4G). PicTar predicts
another conserved miRNA interaction (miR-328) with the H2AX 3' UTR.
To identify the miR-24 miRNA recognition elements (MRE) in the H2AX
3' UTR, we inserted each of the predicted miR-24 MREs, as well as
MREs with a mutated seed region, into the 3' UTR of luciferase
reporter genes. Luciferase activity was reduced approximately
four-fold when either of the wild-type miR-24 MREs was inserted,
but the mutated MREs (Supplementary Table 2 of FIG. 14) had little
effect (FIG. 4K). Therefore, miR-24 regulates H2AX expression by
binding to the two sites predicted by TargetScan and PicTar.
Although MRE2 would be found only in the longer H2AX transcript,
MRE1 could potentially be present in both transcripts. The shorter
transcript will need to be cloned to determine whether this is the
case. Overexpressing miR-328, which is predicted (by PicTar) to
target the 3' UTR of H2AX, had no effect on luciferase activity or
H2AX mRNA or protein levels, further underlining the specificity of
the miR-24--H2AX interaction (FIG. 2A and FIG. 2B). Collectively,
these results strongly suggest that miR-24 binds to the 3' UTR of
H2AX mRNA and downregulates its expression, probably by promoting
both mRNA decay and inhibiting translation.
[0098] To determine whether miR-24-mediated H2AX down-regulation
affects DSB repair, we first evaluated the most serious consequence
of unrepaired DSB, chromosomal instability, in K562 cells that were
transfected with miR-24 or mock transfected. The transfection
conditions were chosen to achieve a level of H2AX knockdown similar
to what is observed during TPA differentiation (FIG. 5A). Metaphase
spreads were prepared 24 hr after low dose .gamma.-irradiation
(FIG. 5B). K562 cells over-expressing miR-24 had twice as many
chromosome breaks and fragments as control cells after exposure to
0.75 Gy (p<0.001; FIG. 5C, left). Similarly TPA-differentiated
K562 cells were significantly more sensitive to 0.75 Gy radiation
than undifferentiated cells (p<0.003; FIG. 5C, middle). Although
there were not significantly more breaks 24 hr after exposure to a
lower dose of radiation (0.38 Gy), more chromosomal instability was
seen at this dose the next day in miR-24 transfected cells (FIG.
5C, right). Undifferentiated and untransfected K562 cells, which
have significantly higher endogenous expression of miR-24 and
4-fold less relative H2AX mRNA than HepG2 cells, also show more
chromosomal aberrations after irradiation than HepG2 cells (FIG.
3).
[0099] As another indicator of unrepaired DNA damage, the
persistence of DSB was measured by single cell gel electrophoresis
(comet assay) after low dose bleomycin treatment (FIG. 5D). The
comet moment quantifies the extent of unrepaired DNA damage.
Although the basal comet moment was not significantly changed by
miR-24 transfection, the comet tails were 5-fold higher
(p<0.001) in miR-24 transfected cells compared to control
miRNA-transfected cells after bleomycin treatment. To determine
whether the effect of miR-24 on DSB repair was mediated via its
effect on H2AX, K562 cells were co-transfected with miR-24 and a
miR-24-insensitive H2AX expression plasmid without the H2AX 3'UTR.
The expression plasmid fully rescued the cells; cells
over-expressing miR-24 and H2AX lacking the 3'UTR had no
significant increase in comet moment after bleomycin compared to
cells transfected with the miRNA control and expression vector.
This result strongly suggests that miR-24 regulates DSB repair by
controlling H2AX.
[0100] Because of impaired DNA damage repair, H2AX deficiency also
leads to increased cell death after exposure to genotoxic drugs. We
compared cell viability of K562 cells over-expressing miR-24,
miR-328 or mock transfected after treatment with bleomycin (FIG.
6A, left). Consistent with the chromosomal breakage and comet assay
analysis, cells over-expressing miR-24 were significantly
hypersensitive to DNA damage as were TPA-differentiated cells
relative to undifferentiated cells (FIG. 6A, right). miR-328
over-expression, however, had no effect, suggesting that miR-328 is
not a physiologically relevant regulator of H2AX. We also found
that unlike miR-24, transfection of miR-328 mimics does not alter
H2AX protein levels in K562 cells (FIG. 2B). The effect of miR-24
on DNA damage sensitivity was further confirmed by treating miR-24
mimic-transfected HepG2 cells with bleomycin (FIG. 6B, left) and
cisplatin (FIG. 6B, right). miR-24 significantly enhanced
cytotoxicity caused by both drugs. The effect of miR-24 on survival
was fully rescued by over-expressing miR-24-insensitive H2AX in
both K562 (FIG. 6C) and HepG2 (FIG. 6D) cells. Together these
results suggest that miR-24-mediated down-regulation of H2AX
inhibits the DNA damage response in terminally differentiated
cells.
[0101] We next tested the effect of inhibiting miR-24 on
sensitivity to genotoxic stress. When K562 cells were transfected
with miR-24 antisense oligonucleotides (ASO), miR-24 expression was
reduced even during TPA differentiation (FIG. 7A). The reduction in
miR-24, which correlated with enhanced H2AX mRNA and protein (FIG.
7B), had no effect on undifferentiated K562 cells, but
significantly reduced sensitivity to bleomycin in differentiated
cells (FIG. 7C).
[0102] Why is there a mechanism to dampen DSB repair in terminally
differentiated cells? One explanation is that most DSBs are
generated during DNA replication and this mode of regulation allows
differentiated cells to economize and conserve cellular resources
under stress-free conditions. Another possibility is that
suppression of repair triggers apoptosis, and this may be preferred
to error prone repair via NHEJ (the primary mode of DSB repair in
these cells), which would result in viable, but malfunctioning,
cells. Although this solution makes sense for regenerating cells,
such as hematopoietic cells and myocytes, it might not be a good
solution for long-lived terminally differentiated cells, like
neurons, with poor regenerative capacity. It will be worthwhile to
determine whether miR-24 is up-regulated during terminal
differentiation of all cell types or only in lineages that are
continuously renewing. It is noteworthy that at least one miR-24
cluster has been reported to be deleted in some poor prognosis
cases of CLL (19), a leukemia known to dysregulate key
anti-apoptotic genes. Based on our findings here, inappropriate
under-expression of miR-24 would be predicted to enhance DNA repair
and thereby enhance resistance to cytotoxic cancer therapies.
[0103] This study focused on the effect of miR-24 on H2AX and DSB
repair. Both H2AX mRNA and protein are reduced by miR-24
expression. miR-24 is likely operating predominantly by inhibiting
translation since the effect on protein levels is much greater than
on mRNA. Other proteins recruited to DSB and required for their
repair are BRCA1, PCNA and CHEK1, whose transcripts both
precipitate with miR-24 and show protein down-regulation upon
miR-24 over-expression (accompanying patent application U.S. Ser.
No. 61/098,696, filed on Sep. 19, 2008). BRCA1 and PCNA are
important in repairing DNA replication-mediated breaks by HR and
CHEK1 arrests dividing cells in response to DNA damage. However,
H2AX is required for DSB repair without bias for dividing or
non-dividing cells--it is important for both HR (active only in
dividing cells) and NHEJ (throughout the cell cycle) (20). The
observation that DSB repair was completely restored by
over-expressing H2AX in differentiating cells, or cells
over-expressing exogenous miR-24, suggests that the key target of
miR-24 in DSB repair is H2AX.
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EQUIVALENTS
[0136] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention, described
herein. The scope of the present invention is not intended to be
limited to the above Description, but rather is as set forth in the
appended claims.
[0137] In the claims articles such as "a," "an," and "the" may mean
one or more than one unless indicated to the contrary or otherwise
evident from the context. Claims or descriptions that include "or"
between one or more members of a group are considered satisfied if
one, more than one, or all of the group members are present in,
employed in, or otherwise relevant to a given product or process
unless indicated to the contrary or otherwise evident from the
context. The invention includes embodiments in which exactly one
member of the group is present in, employed in, or otherwise
relevant to a given product or process. The invention includes
embodiments in which more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process. Furthermore, it is to be understood that the invention
encompasses all variations, combinations, and permutations in which
one or more limitations, elements, clauses, descriptive terms,
etc., from one or more of the listed claims is introduced into
another claim. For example, any claim that is dependent on another
claim can be modified to include one or more limitations found in
any other claim that is dependent on the same base claim.
[0138] Where elements are presented as lists, e.g., in Markush
group format, it is to be understood that each subgroup of the
elements is also disclosed, and any element(s) can be removed from
the group. It should it be understood that, in general, where the
invention, or aspects of the invention, is/are referred to as
comprising particular elements, features, etc., certain embodiments
of the invention or aspects of the invention consist, or consist
essentially of, such elements, features, etc. For purposes of
simplicity those embodiments have not been specifically set forth
in haec verba herein. It is noted that the term "comprising" is
intended to be open and permits the inclusion of additional
elements or steps.
[0139] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0140] In addition, it is to be understood that any particular
embodiment of the present invention that falls within the prior art
may be explicitly excluded from any one or more of the claims.
Since such embodiments are deemed to be known to one of ordinary
skill in the art, they may be excluded even if the exclusion is not
set forth explicitly herein. Any particular embodiment of the
compositions of the invention (e.g., any targeting moiety, any
disease, disorder, and/or condition, any linking agent, any method
of administration, any therapeutic application, etc.) can be
excluded from any one or more claims, for any reason, whether or
not related to the existence of prior art.
[0141] Publications discussed above and throughout the text 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 inventors are not entitled to antedate such
disclosure by virtue of prior disclosure.
Sequence CWU 1
1
15122RNAArtificial SequenceSynthetic oligonucleotide 1uggcucaguu
cagcaggaac ag 22221RNAArtificial SequenceSynthetic oligonucleotide
2cucauggaaa gagcugagcc g 21319RNAArtificial SequenceSynthetic
oligonucleotide 3cccugucugg acugagccu 19420DNAArtificial
SequenceSynthetic primer 4tgcaccacca actgcttagc 20521DNAArtificial
SequenceSynthetic primer 5ggcatggact gtggtcatga g
21619DNAArtificial SequenceSynthetic primer 6atttgggtcg cggttcttg
19721DNAArtificial SequenceSynthetic primer 7tgccttgaca ttctcgatgg
t 21820DNAArtificial SequenceSynthetic primer 8agcaaactca
actcggcaat 20920DNAArtificial SequenceSynthetic primer 9actccccaat
gcctaaggtt 201018DNAArtificial SequenceSynthetic primer
10ggcctccagt tcccagtg 181120DNAArtificial SequenceSynthetic primer
11tcagcggtga ggtactccag 201219RNAArtificial SequenceSynthetic
oligonucleotide 12ucauggaaag agcugagcc 191319RNAArtificial
SequenceSynthetic oligonucleotide 13ucauggaaag aggauuagg
191417RNAArtificial SequenceSynthetic oligonucleotide 14ccugucugga
cugagcc 171517RNAArtificial SequenceSynthetic oligonucleotide
15ccugucugga gauuagg 17
* * * * *