U.S. patent application number 13/151053 was filed with the patent office on 2011-10-20 for modulating immune system development and function through microrna mir-146.
This patent application is currently assigned to CALIFORNIA INSTITUTE OF TECHNOLOGY. Invention is credited to David Baltimore, Mark Boldin, Konstantin Taganov.
Application Number | 20110258716 13/151053 |
Document ID | / |
Family ID | 40796135 |
Filed Date | 2011-10-20 |
United States Patent
Application |
20110258716 |
Kind Code |
A1 |
Baltimore; David ; et
al. |
October 20, 2011 |
MODULATING IMMUNE SYSTEM DEVELOPMENT AND FUNCTION THROUGH MICRORNA
MIR-146
Abstract
The present disclosure relates to the finding that microRNA-146
plays a role in modulating the development and function of the
immune system. Immune cell development and function can be
modulated by delivery of microRNA-146 (miR-146) or antisense
miR-146 to target immune cells or precursor cells. For example, in
some embodiments, activity and/or proliferation of certain immune
cells is regulated by administering miR-146 oligonucleotides or
anti-miR-146 oligonucleotides. In other embodiments,
pro-inflammatory cytokine expression in immune cells is regulated
by administering a miR-146 oligonucleotide or anti-miR-146. In
further embodiments, methods of regulating macrophage activity
using antisense miR-146 are provided. Additional methods and
compositions for regulating immune system function and development
using miR-146 are disclosed.
Inventors: |
Baltimore; David; (Pasadena,
CA) ; Boldin; Mark; (Pasadena, CA) ; Taganov;
Konstantin; (Pasadena, CA) |
Assignee: |
CALIFORNIA INSTITUTE OF
TECHNOLOGY
Pasadena
CA
|
Family ID: |
40796135 |
Appl. No.: |
13/151053 |
Filed: |
June 1, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12337525 |
Dec 17, 2008 |
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13151053 |
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61007987 |
Dec 17, 2007 |
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Current U.S.
Class: |
800/18 ; 435/354;
435/355 |
Current CPC
Class: |
C12N 2330/10 20130101;
C12N 2310/113 20130101; C12N 2310/141 20130101; C12N 2310/111
20130101; C12N 15/113 20130101 |
Class at
Publication: |
800/18 ; 435/354;
435/355 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C12N 5/10 20060101 C12N005/10 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED R&D
[0002] The U.S. Government has certain rights in this invention
pursuant to Grant No. GM039458 awarded by National Institutes of
Health.
Claims
1. A transgenic mouse, wherein the genome of the transgenic mouse
comprises a homozygous disruption of the endogenous miRNA-146
gene.
2. The transgenic mouse of claim 1, wherein the disruption
comprises a deletion of at least a portion of the endogenous
miRNA-146 gene.
3. The transgenic mouse of claim 1, wherein the disruption
comprises a replacement of at least a portion of the endogenous
miRNA-146 gene with a PGK-Neo cassette flanked by loxP sites.
4. The transgenic mouse of claim 1, wherein the disruption
comprises a replacement of at least a portion of the endogenous
miRNA-146 gene with a nucleotide sequence comprising a loxP
site.
5. The transgenic mouse of claim 1, wherein the endogenous
miRNA-146 gene is replaced with a nucleotide sequence comprising a
loxP site.
6. The transgenic mouse of claim 1, wherein the transgenic mouse
suffers from one or more autoimmune disorders.
7. The transgenic mouse of claim 6, wherein one of the one or more
autoimmune disorders is splenomegaly or lymphoadenopathy,
8. The transgenic mouse of claim 7, wherein the transgenic mouse
has an increased number of CD11b-positive cells as compared to a
wild-type mouse.
9. The transgenic mouse of claim 1, wherein the transgenic mouse
exhibits severe inflammation or tissue damage in organs selected
from the group consisting of liver, kidneys and lungs.
10. The transgenic mouse of claim 1, wherein the transgenic mouse
exhibits impaired T cell development as compared to a wild type
mouse.
11. The transgenic mouse of claim 10, wherein the impaired T cell
development is impaired negative T cell selection or an increased
number of activated T cells.
12. The transgenic mouse of claim 1, wherein the transgenic mouse
exhibits abnormal development in a cell of hematopoietic origin as
compared to the wild type mouse.
13. The transgenic mouse of claim 12, wherein the cell of
hematopoietic origin is selected from the group consisting of a B1
B cell, a B2 cell, a marginal zone B cell, a CD8+ cell, a natural
killer (NK) cell, and a CD8.alpha..alpha.+ T cell.
14. A transgenic mouse, wherein the transgenic mouse comprises a
homozygous disruption of 295 base pairs of an endogenous sequence
encoding pre-miR-146a.
15. The transgenic mouse of claim 14, wherein the 295 base pairs of
the endogenous sequence is replaced by a nucleotide sequence
comprising a selection marker flanked by two or more recombinase
recognition sites.
16. The transgenic mouse of claim 15, wherein the selection marker
is a PGK-Neo cassette.
17. The transgenic mouse of claim 15, wherein the recombinase
recognition site is a loxP site.
18. The transgenic mouse of claim 14, wherein the 295 base pairs of
the endogenous sequence is replaced by a sequence comprising a
recombination recognition site.
19. A cell from a transgenic mouse, wherein the transgenic mouse
comprises a homozygous deletion of 295 base pairs of an endogenous
sequence encoding pre-miR-146a.
20. The cell of claim 19, wherein the cell is selected from the
group consisting of a bone marrow derived macrophage (BMDM), a stem
cell, a T cell, a B1 B cell, a marginal zone B cell, a CD8+ cell, a
natural killer (NK) cell, and a CD8.alpha..alpha.+ T cell.
21. The cell of claim 20, wherein the cell is a BMDM.
22. The cell of claim 21, wherein the BMDM secrets a higher level
of a proinflammatory cytokine after lipopolysaccharide (LPS)
stimulation as compared to a BMDM in a wild type mouse.
23. The cell of claim 22, wherein the proinflammatory cytokine is
selected from the group consisting of TNF.alpha., IL-6 and
IL-1.beta..
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C.
.sctn.120 as a continuation of U.S. patent application Ser. No.
12/337,525, filed Dec. 17, 2008, which claims priority under 35
U.S.C. .sctn.119(e) to U.S. Provisional Application No. 61/007,987,
filed on Dec. 17, 2007. All of the aforementioned priority
applications are herein expressly incorporated by reference in
their entirety.
REFERENCE TO SEQUENCE LISTING
[0003] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled CALTE.045C1.TXT, created Jun. 1, 2011, which is 5.2
KB in size. The information in the electronic format of the
Sequence Listing is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention
[0005] The present application relates to modulating the activity
of the immune system using microRNA. More particularly the
application relates to modulating immune system development and
function through miR-146.
[0006] 2. Description of the Related Art
[0007] Historically, the innate and adaptive arms of the immune
response have been represented as two separate systems with
distinct properties. However, there are several types of immune
cells whose phenotypic features place them at the border of the
innate and adaptive systems and provide a bridge between the two.
This list includes B1 B cell, marginal zone (MZ) B cells, natural
killer T (NKT) cells, .gamma..delta. T cells and intestinal
epithelial lymphocytes (IEL) expressing CD8.alpha..alpha.. These
lineages are shown to be essential for several aspects of immunity
because their dysfunction or deficiency has been shown to lead to
the development of autoimmune disease and cancers. Each of the
above-mentioned cell types is characterized by a unique set of
anatomical location, self-renewing capacity, surface phenotype and
ligands.
[0008] For instance, B-1 cells are known to function as innate-like
immunity effectors and are the key players in the early humoral
response against bacteria, viruses, and certain parasites.
Montecino-Rodriguez, Trends Immunol 27, 428-433. B1 cells are
located mainly in the peritoneal and pleural cavities, and express
high levels of surface IgM and low levels of IgD, CD23, and B220.
They are thought to be the primary antibody producers in response
to T cell-independent type 2 antigens, such as capsular
polysaccharides on bacteria. Importantly, natural antibodies
produced by B1 cells also bind to self-antigens, and this property
could explain why B1 cells are often associated with autoimmune
diseases in mice and humans.
[0009] Another example of B cells that play an important role in T
cell-independent antibody response is MZ B cells. Viau and Zouali,
Clin Immunol 114, 17-26. They are located at the junction of white
and red pulp and have a surface phenotype distinct from other
spleen B cells. MZ B cells respond vigorously to blood-borne
infections, and play a role in host survival of infection by
encapsulated bacteria.
[0010] A group of unconventional T cells that possess properties of
both the innate and adaptive systems are the IEL cells. This
subtype exclusively expresses CD8.alpha..alpha.; however, they do
not express either of the well-defined CD4 or CD8.alpha..beta. TCR
coreceptors, or several molecules found on most other T
lymphocytes, such as CD2, CD28, and CD 90.
[0011] MicroRNA (miRNA) represent a newly discovered class of
endogenous .about.22-nt RNAs encoded by biological species. Owing
to their ability to post-transcriptionally regulate expression of
nearly any target gene, miRNA have been implicated in a variety of
processes in plants and animals, and have been shown to be involved
in development, apoptosis, signal transduction, fat metabolism,
insulin secretion, viral infection, and potentially many other
processes. Bartel, Cell 116, 281-297. A growing body of evidence
suggests that miRNAs play an important role in all aspects of
immune system development and function from driving differentiation
of certain cell lineages to fine-tuning of immune response to
antigen. Fontana, Nat Cell Biol 9, 775-787; Li, Cell 129,
147-161.
[0012] Inflammation is a highly complex defense reaction of the
host in response to an invading pathogen or injury, which when not
resolved swiftly can result in quite severe pathological
consequences. The importance of timely resolution of inflammatory
reaction is underscored by the number of diseases where a failure
to terminate inflammatory process is the main driving force, like
rheumatoid arthritis, septic shock, inflammatory bowel disease and
multiple sclerosis. In addition, there is mounting evidence that
sustained inflammation is linked to various human cancers.
[0013] MicroRNAs have been shown to be involved in regulation of
the innate immune response, and miRNAs that play a role in the
mammalian response to microbial infection have been identified.
Taganov, Proc Natl Acad Sci USA 103, 12481-12486.
SUMMARY OF THE INVENTION
[0014] Immune system development and function can be modulated by
means of microRNA expression or targeted delivery of said microRNA
to the immune system, and by preventing normal microRNA activity,
such as by knocking out the genes that encode said MicroRNA or by
administering antisense sequences of said microRNA.
[0015] Methods for regulating development and function of immune
cells are provided in accordance with one aspect of the present
invention. In some embodiments, a miR-146 oligonucleotide or
antisense miR-146 oligonucleotide are administered to a target
cell, such as an immune precursor cell. Proliferation of one or
more of B1 B cells, B2 B cells, Marginal Zone B cells,
CD8.alpha..alpha.+ T cells, Natural Killer cells or CD8+ T cells
can then be measured to identify an effect on immune cells. In some
embodiments, the miR-146 oligonucleotide or antisense miR-146
oligonucleotide are administered to bone marrow in a mammal.
[0016] In some embodiments, proliferation and function of immune
cells, such as B1 B cells, B2 B cells, Marginal Zone B cells,
CD8.alpha..alpha.+ T cells, Natural Killer cells and CD8+ T cells
can be regulated using miRNA-146 or antisense miRNA-146. In some
embodiments, the methods comprise administering a microRNA-146a
(miR-146a) or microRNA-146b (miR-146b) oligonucleotide to target
cells. In other embodiments, the methods comprise administering a
miR-146a or miR-146b expression vector to target cells such that a
miRNA-146 is expressed in target cells. In other embodiments, the
methods comprise administering antisense miRNA-146 or other
molecules that interfere with miR-146 expression or activity to
target cells expressing miRNA-146.
[0017] In some embodiments methods of increasing proliferation
and/or activity of immune cells, such as B1 B cells, Marginal Zone
B cells and Natural Killer cells and CD8+ T cells, are provided. In
some embodiments, the methods comprise administering a miRNA-146
oligonucleotide to a target cell or target tissue, such as bone
marrow. In other embodiments, the methods comprise administering a
miRNA-146 expression vector to target cells or a target tissue and
expressing a miRNA-146 in the target. In some embodiments, total
numbers of one or more of B1 B cells, Marginal Zone B cells and
Natural Killer cells and CD8+ T cells can be increased in a host by
these methods.
[0018] In other embodiments, methods of decreasing proliferation
and/or activity of immune cells such as B1 B cells, Marginal Zone B
cells and Natural Killer cells and CD8+ T cells are provided. In
some embodiments, the methods comprise administering antisense
miRNA-146 oligonucleotides to target cells or a target tissue, such
as bone marrow. In other embodiments, the methods comprise
administering an antisense miRNA-146 expression vector to target
cells or a target tissue such that antisense miRNA-146 is expressed
in the target. In some embodiments, total numbers of one or more of
B1 B cells, Marginal Zone B cells and Natural Killer cells and CD8+
T cells can be decreased in a host by these methods.
[0019] In other embodiments, proliferation and/or activity of
immune cells such as B2 B cells and CD8.alpha..alpha.+ T cells can
be upregulated. In some embodiments, the methods comprise
administering an antisense miRNA-146 oligonucleotide to target
cells or a target tissue, such as bone marrow. In other
embodiments, the methods comprise administering an antisense
miRNA-146 expression vector to a target tissue such that antisense
miRNA-146 is expressed in the target tissue. In some embodiments,
total numbers of B2 B cells and/or CD8.alpha..alpha.+ T cells can
be increased in a host by these methods.
[0020] In other embodiments, proliferation and/or activity of B2 B
cells and CD8.alpha..alpha.+ T cells can be downregulated. In some
embodiments, the methods comprise administering a miRNA-146
oligonucleotide to target cells or a target tissue, such as bone
marrow. In other embodiments, the methods comprise administering a
miRNA-146 expression vector to a target tissue and expressing a
miRNA-146 in the target tissue. In some embodiments, total numbers
of B2 B cells and/or CD8.alpha..alpha.+ T cells can be decreased in
a host by these methods.
[0021] Methods for modulating T cell activation are provided in
accordance with yet another aspect of the present invention. In
some embodiments, the T cell activation is downregulated by
administering a miR-146 oligonucleotide to the target T cells. In
other embodiments, the methods comprise administering a miR-146
expression vector to target T cells and expressing a miRNA-146 in
the target cells. In some embodiments, diseases or disorders
related to T cell activation, such as inflammatory bowel disease,
rheumatoid arthritis, lupus and multiple sclerosis, can be treated
by administering a miR-146 oligonucleotide or expression vector to
immune cells in a patient in need of treatment.
[0022] Methods for downregulating production of certain
pro-inflammatory cytokines by immune cells is provided in
accordance with another aspect of the present invention. In some
embodiments the production of pro-inflammatory cytokines by immune
cells, such as macrophages or T cells, is downregulated by
administering a miR-146 oligonucleotide to target immune cells, or
by expressing a miR-146 oligonucleotide in target cells. In
particular embodiments, macrophage function can be regulated by
administering miR-146 or antisense miR-146 to macrophages or
macrophage precursor cells.
[0023] In some embodiments the production of TNF.alpha. and/or IL-6
is downregulated by administering a microRNA-146 (miR-146)
oligonucleotide to target immune cells, such as macrophages or T
cells. In other embodiments, a miRNA-146 is expressed in target
immune cells by administering a miR-146 expression vector to the
target cells. If desired, levels of pro-inflammatory cytokines
TNF.alpha. and/or IL-6 can be upregulated by administering
anti-sense miR-146 to target immune cells or expressing
antisense-miR146 in target cells.
[0024] Methods for regulating activation of certain kinases in
immune cells are also provided. More specifically, in some
embodiments the activation of NF-kB and/or JNK1 can be
downregulated by administering a miRNA-146 oligonucleotide to
target immune cells. In other embodiments, a miRNA-146 expression
vector can be administered to a target cell such that a miRNA-146
is expressed in the target immune cell. In other embodiments the
activation of ERK can be downregulated in immune cells. In some
embodiments, the methods comprise administering miRNA-146
oligonucleotide to a target immune cell. In other embodiments, the
methods comprise administering an antisense miRNA-146 expression
vector to a target cell and expressing an antisense miRNA-146 in
the target cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows generation of miR-146 knock out (KO) mice. (A).
A 295 by pre-miR-146a encoding region was replaced by a PGK-Neo
cassette flanked by loxP sites, and in the next step was loxed out
with the help of Cre-deleter mice. (B). Detection of expression
levels of mature miR-146a in wild type and mutant mice by qRT-PCR
analysis of spleen, lymph nodes, and thymus. (C). Western blot
analysis of cell lysates from LPS stimulated BMDMs derived from
wild type (WT) and KO animals.
[0026] FIG. 2 shows miR-146a KO mice develop autoimmune-like
disease that is characterized by splenomegaly and lymphoadenopathy.
(A). Representative examples of large spleen and big lymph nodes
observed in KO animals (B). Increase in spleen weight in the KO
animals.
[0027] FIG. 3 shows miR-146a KO mice develop multiorgan
inflammation and follicular hyperplasia in the spleen.
Hematoxylin-eosin (H&E) staining of liver (A) and kidney (B)
sections from WT and KO mice. Arrows point to lymphocytic
infiltrates. (C). H&E staining of spleen sections at 20.times.
magnification. Note increased number and size of lymphoid
follicules and their rugged edges in the KO.
[0028] FIG. 4 shows massive myeloproliferation in the spleen of
miR-146a KO mice. Absolute cell number of B(A), T(B) and myeloid
(C) cells in the spleens of WT, HET and KO mice.
[0029] FIG. 5 shows T cell development in miR-146a KO mice. (A).
Peripheral T cells of the miR-146a KO mice display an activated
status. FACS analysis of CD4-positive T cells for the expression of
CD25, CD69, CD62L and CD69 surface markers. (B). FACS analysis of T
cell populations in the thymus of WT and KO mice.
[0030] FIG. 6 shows the effect of lack of miR-146a expression on
development of several hematopoietic cell lineages in mice. (A).
Reduction in B1 B cells numbers in the peritoneal cavity of
miR-146a KO mice. B1 B cells are CD19.sup.+B220.sup.lo cells as
determined by FACS analysis. (B). Reduction of marginal zone B cell
population (CD19.sup.+CD21.sup.+CD23.sup.-) in the spleen of
miR-146a KO mice.
[0031] FIG. 7 shows the effect of miR-146a on development of NK and
CD8.alpha..alpha. lineages. (A) FACS analysis of NK
(DX5.sup.+CD94.sup.+) cells in the peripheral blood. (B). FACS
analysis of CD8.alpha..alpha..sup.+ T cells in the peripheral lymph
nodes of miR-146a KO mice and WT control.
[0032] FIG. 8 shows miR-146a plays a negative role in the
development of CD8.alpha..alpha. T cells. FACS analysis of
CD8.alpha..alpha. population in the peripheral lymph nodes of WT
and KO animals.
[0033] FIG. 9 shows analysis of proinflammatory cytokine production
by ELISA using bone marrow-derived macrophages from WT (red bars)
and KO (blue bars) animals. (A). Mouse TNFa ELISA. (B) Mouse IL-6
ELISA. (C). Mouse IL-1b ELISA.
[0034] FIG. 10 shows that miR-146a overexpression downregulates
levels of endogenous TRAF6 and IRAK1 proteins and suppresses
production of proinflammatory cytokines in stable THP-1 cell lines.
(A). Schematic diagram of lentiviral constructs used to establish
stable THP-1 lines. (B). Northern blot analysis of miR-146a
expression in the established THP-1 cells. miR-146a marks the band
corresponding to mature exogenous miR-146a. (C). Western blot
analysis of human IRAK1 and TRAF6 protein expression in the
established THP-1 cell lines. (D). Western blot analysis of
LPS-induced activation of NF-kB, JNK and ERK pathways in the
established THP-1 lines. pJNK1- stands for phospho-JNK1;
pERK1/2-denotes phospho-ERK1/2. (D). Analysis of hTNFa, hIL-6 and
hIL-8 secretion by ELISA in the media of LPS-stimulated THP1/SCR
and THP1/146 cells. (E) Analysis of cytokine production in response
to LPS challenge.
[0035] FIG. 11 shows that expression of miR-146a in hematopoietic
system affects development of some B and T cell subsets. (A)
miR-146 expression levels as determined by quantitative RT-PCR
analysis of hematopoietic tissues of miR-146a-expressing and
control mice collected 2-3 months post transplantation. (B) FACS
analysis of GFP-positive B1 B cells from bone marrow of BMT mice.
(C) FACS analysis of GFP-positive cells from peritoneal cavity of
BMT mice. The CD19.sup.+B220.sup.lo-neg population was analyzed
further for CD5 expression. (D). B cell development in the bone
marrow. (E). Staining for CD8+ T cells (CD8+ CD3+) cells in the
spleen. (F). Staining for CD8.alpha..alpha. cells in the mesenteric
lymph nodes (MLN).
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0036] miR-146 can be used to modulate immune system development
and function. As a result, it can be used, for example, as a
therapeutic agent to treat disease states, including those
characterized by activation, particularly excessive activation of
the innate immune system. Such disease states include, for example,
sepsis and septic shock, neurodegeneration, neutrophilic
alveolitis, asthma, hepatitis, inflammatory bowel disease,
ischemia/reperfusion, septic shock, glomerulonephritis, rheumatoid
arthritis, lupus, multiple sclerosis and Crohn's disease.
[0037] When miR-146a or miRNA-146b is delivered into cells of the
innate immune system, among other things it dampens the production
of pro-inflammatory cytokines like TNF and IL-6. Such delivery can
be achieved in a variety of ways using methods well known in the
art, for example, by modification of an oligonucleotide encoding a
miR-146, such as a mature miR-146a or miR-146b, with cholesterol to
help it easily penetrate the cell membrane or by expressing the
miRNA in the cells using an appropriate expression vector. See, for
example, Krutzfeldt, J. et al., Nature 438, 685-9 (2005), herein
expressly incorporated by reference. Delivery of molecules that
inhibit miR-146 activity, such as antisense molecules, can be used
to upregulate activity of the immune system where appropriate.
These and other embodiments are discussed in more detail below.
[0038] Human miR-146a is located in the second exon of LOC285628
gene on the human chromosome 5. LOC285628 consists of two exons
separated by a long .about.16 kb long intron and is most probably a
non-coding RNA gene, since it does not contain a long, continuous
open reading frame. MiR-146b is located on human chromosome 10.
[0039] In some embodiments, immune cell function, proliferation and
numbers can be modulated by modulating levels of miR-146 in target
cells and tissues. Upregulation of miRNA-146 in target organs,
tissues and/or cells can be accomplished by, for example,
administering to the target either synthetic miR-146, such as a
miR-146 oligonucleotide, or expression vectors that express
miRNA-146. Downregulation of miRNA-146 in target cells, organs or
tissues can be accomplished by, for example, by administering to
the target an anti-miR-146, such as by administering synthetic
antisense miRNA-146, expression vectors that express antisense
miRNA-146 or administering one or more other molecules that
interfere with miR-146 expression or activity.
[0040] For example, in some embodiments B1 B cell activity and/or
proliferation can be upregulated by administering miRNA-146 to
target immune cells or precursor cells. CD8+CD3+ conventional T
cell activity and/or proliferation can be upregulated by
administering miRNA-146. B2 B cell activity and/or proliferation
can be downregulated by administering miRNA-146. CD8.alpha..alpha.+
T cell activity and/or proliferation can be downregulated by
administering miRNA-146.
[0041] CD-11b+ cell activity and/or proliferation can be
upregulated by administering anti-miR-146 to target immune cells or
precursor cells. B1 B cell activity and/or proliferation can be
downregulated by administering antisense miR-146. CD8+ T cell
activity and/or proliferation can be downregulated by administering
antisense miR-146. B2 B cell activity and/or proliferation can be
upregulated by administering antisense miRNA-146.
CD8.alpha..alpha.+ T cell activity and/or proliferation can be
upregulated by administering antisense miRNA-146.
[0042] In some embodiments, vaccination, such as cancer vaccination
can be improved by modulating miR146a levels in an animal. For
example, anti-miR-146 can be delivered to the target animal to be
vaccinated, increasing the activity and/or number of
CD8.alpha..alpha.+ T cells. Because CD.alpha..alpha.+ T cells are
considered to be precursors of memory T cells, increased activity
and/or numbers of CD.alpha..alpha.+ T cells increases the desired
immune response to a vaccine. The vaccine can be administered
before, concurrently with or after the ani-miR-146.
[0043] In some embodiments, production of pro-inflammatory
cytokines, such as TNF.alpha. and IL-6 levels, by immune cells can
be downregulated by modulating levels or activity of miRNA-146 in
target cells. TNF.alpha. and IL-6 production can be downregulated
by administering miRNA-146 to the target cells.
[0044] In some embodiments, T cell activation, for example as
indicated by the increased expression levels of surface markers,
such as CD25, CD69 and CD44, can be reduced by modulating levels of
miRNA-146 target cells. In some embodiments, T cell activation can
be downregulated by administering miRNA-146 to target cells.
Because T cell activation is related to some diseases or disorders,
such as inflammatory bowel disease, rheumatoid arthritis, lupus and
multiple sclerosis, administration of miRNA-146 to a patient in
need of treatment can be used to treat such disorders.
Definitions
[0045] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. See,
e.g. Singleton et al., Dictionary of Microbiology and Molecular
Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994);
Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold
Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). For purposes
of the present invention, the following terms are defined
below.
[0046] When used herein the terms "miR," "mir" and "miRNA" are used
to refer to microRNA, a class of small RNA molecules that are
capable of modulating RNA translation (see, Zeng and Cullen, RNA,
9(1):112-123, 2003; Kidner and Martienssen Trends Genet,
19(1):13-6, 2003; Dennis C, Nature, 420(6917):732, 2002; Couzin J,
Science 298(5602):2296-7, 2002, each of which is incorporated by
reference herein).
[0047] "MiRNA-146," "miR-146," "miR-146a/b" and "miRNA-146a/b" are
used interchangeably and, unless otherwise indicated, refer to
microRNA-146a and/or microRNA-146b, including miR-146a,
pri-miR-146a, pre-miR-146a, mature miR-146a, miR-146b,
pre-miR-146b, mature miR-146b, miRNA-146 seed sequence, sequences
comprising a miRNA-146 seed sequence, and variants thereof.
[0048] An "expression vector" is a nucleic acid construct,
generated recombinantly or synthetically, bearing a series of
specified nucleic acid elements that enable transcription of a
particular gene in a host cell. Typically, gene expression is
placed under the control of certain regulatory elements, such as
constitutive or inducible promoters.
[0049] "MiRNA nucleic acid" is defined as RNA or DNA that encodes a
miR as defined above, or is complementary to a nucleic acid
sequence encoding a miR, or hybridizes to such RNA or DNA and
remains stably bound to it under appropriate stringency conditions.
Specifically included are genomic DNA, cDNA, mRNA, miRNA and
antisense molecules, pri-miRNA, pre-miRNA, mature miRNA, miRNA seed
sequence, as well as nucleic acids based on alternative backbones
or including alternative bases. MiRNA nucleic acids can be derived
from natural sources or synthesized.
[0050] "MicroRNA seed sequence," "miRNA seed sequence," "seed
region" and "seed portion" are used to refer to nucleotides 2-7 or
2-8 of the mature miRNA sequence. The miRNA seed sequence is
typically located at the 5' end of the miRNA. A miRNA-146 seed
sequence is provided in SEQ ID NO: 14.
[0051] The term "operably linked" is used to describe the
connection between regulatory elements and a gene or its coding
region. That is, gene expression is typically placed under the
control of certain regulatory elements, for example, without
limitation, constitutive or inducible promoters, tissue-specific
regulatory elements, and enhancers. A gene or coding region is said
to be "operably linked .sup.to or "operatively linked to or
"operably associated .sup.with the regulatory elements, meaning
that the gene or coding region is controlled or influenced by the
regulatory element.
[0052] The term "mammal" is defined as an individual belonging to
the class Mammalia and includes, without limitation, humans,
domestic and farm animals, and zoo, sports, or pet animals, such as
sheep, dogs, horses, cats or cows. Preferably, the mammal herein is
human. However, in some embodiments the mammal is not a human.
[0053] As used herein, "treatment" is a clinical intervention made
in response to a disease, disorder or physiological condition
manifested by a patient or to which a patient may be susceptible.
The aim of treatment includes the alleviation or prevention of
symptoms, slowing or stopping the progression or worsening of a
disease, disorder, or condition and/or the remission of the
disease, disorder or condition. "Treatments" refer to one or both
of therapeutic treatment and prophylactic or preventative measures.
Those in need of treatment include those already affected by a
disease or disorder or undesired physiological condition as well as
those in which the disease or disorder or undesired physiological
condition is to be prevented.
[0054] The term "effective amount" refers to an amount sufficient
to effect beneficial or desirable biological and/or clinical
results.
[0055] "Pharmaceutically acceptable" carriers, excipients, or
stabilizers are ones which are nontoxic to the cell or mammal being
exposed thereto at the dosages and concentrations employed or that
have an acceptable level of toxicity as determined by the skilled
practitioner. Often the physiologically acceptable carrier is an
aqueous pH buffered solution. The physiologically acceptable
carrier may also comprise one or more of the following:
antioxidants, such as ascorbic acid, low molecular weight (less
than about 10 residues) polypeptides, proteins, such as serum
albumin, gelatin, immunoglobulins, hydrophilic polymers such as
polyvinylpyrrolidone, amino acids, carbohydrates such as glucose,
mannose, or dextrins, chelating agents such as EDTA, sugar alcohols
such as mannitol or sorbitol, salt-forming counterions such as
sodium, and nonionic surfactants such as Tween.TM.., polyethylene
glycol (PEG), and Pluronics.TM..
MiR-146 Nucleic Acid Molecules
[0056] Nucleic acid molecules that encode miR-146 are used in
various embodiments of the present invention. miR-146 sequences for
mature miR-146a, pre-miR-146a, mature miR-146b and pre-miR-146b are
provided in SEQ ID NOs: 1, 2, 3 and 4, respectively and are used in
some emboidments. cDNAs encoding mature miR-146a, pre-miR-146a,
mature miR-146b and pre-miR-146b, are provided in SEQ ID NOs: 5, 6,
7 and 8, respectively. Nucleic acid molecules encoding pri-miR-146a
and pri-miR-146b sequences can also be used in accordance with some
embodiments. A miRNA sequence may comprise from about 6 to about 99
or more nucleotides. In some embodiments, a miRNA sequence
comprises about the first 6 to about the first 22 nucleotides of a
pre-miRNA-146. Isolated or purified polynucleotides having at least
6 nucleotides (i.e., a hybridizable portion) of a miR-146 coding
sequence or its complement are used in some embodiments. In other
embodiments, miR-146 polynucleotides preferably comprise at least
22 (continuous) nucleotides, or a full-length miR-146 coding
sequence.
[0057] In some embodiments, nucleic acids are used that are capable
of blocking the activity of a miRNA (anti-miRNA-146 or
anti-miR-146). Such nucleic acids include, for example, antisense
miR-146. In preferred embodiments, the anti-miR is an antisense
miR-16 nucleic acid comprising a total of about 5 to about 100 or
more, more preferably about 10 to about 60 nucleotides, and has a
sequence that is preferably complementary to at least the seed
region of miR-146. In particularly preferred embodiments, an
anti-miRNA may comprise a total of at least about 5, to about 26
nucleotides. In some embodiments, the sequence of the anti-miRNA
can comprise at least 5 nucleotides that are substantially
complementary to the 5' region of a miR-146, at least 5 nucleotides
that are substantially complementary to the 3' region of a miR-146,
at least 4-7 nucleotides that are substantially complementary to a
miR-146 seed sequence, or at least 5-12 nucleotide that are
substantially complementary to the flanking regions of a miR-146
seed sequence.
[0058] It is not intended that the methods of the present invention
be limited by the source of the miR-146 or anti-miR-146. Human and
mouse synthetic miR-146a and miR-146b are commercially available,
as are inhibitors thereof. For example, both miRNA precursors and
miRNA inhibitors for miR-146a and miR-146b can be purchased from
Ambion.RTM. It has been shown that antisense miRNAs can
specifically silence target miRNA in tissue. Krutzfeldt, J. et al.,
Nature 438, 685-9 (2005).
[0059] In some embodiments, an anti-miR-146 comprises the
complement of a sequence of a miRNA referred to in SEQ ID NOs: 1-4.
In other embodiments an anti-miR-146 comprises the complement of
the seed sequence of SEQ ID NO: 14 or is able to hybridize under
stringent conditions to the seed sequence of SEQ ID NO: 14.
Preferred molecules are those that are able to hybridize under
stringent conditions to the complement of a cDNA encoding a mature
miR-146, for example SEQ ID NO: 1 or SEQ ID NO: 3. Particular
antisense sequences for miR-146a and miR-146b are provided in SEQ
ID NOs: 9 and 10.
[0060] The miR-146 can be from a human or non-human mammal, derived
from any recombinant source, synthesized in vitro or by chemical
synthesis. The nucleotide may be DNA or RNA and may exist in a
double-stranded, single-stranded or partially double-stranded form,
depending on the particular context. miR-146 and anti-miR-146
nucleic acids may be prepared by any conventional means typically
used to prepare nucleic acids in large quantity. For example,
nucleic acids may be chemically synthesized using commercially
available reagents and synthesizers by methods that are well-known
in the art for example, the phosphotriester method of Matteucci, et
al., (J. Am. Chem. Soc. 103:3185-3191, 1981) and/or using automated
synthesis methods. (See, e.g., Gait, 1985, Oligonucleotide
Synthesis: A Practical Approach, IRL Press, Oxford, England). In
addition, larger DNA or RNA segments can readily be prepared by
well known methods, such as synthesis of a group of
oligonucleotides that define various modular segments, followed by
ligation of oligonucleotides to build the complete segment. The
scope of the current invention is not limited to naturally
occurring miR-146 sequences; mutants and variants of miR-146
sequences are also covered by the scope of the current
invention.
[0061] Nucleotide sequences that encode a mutant of a miR-146 that
is a miR-146 with one or more substitutions, additions and/or
deletions, and fragments of miR-146 as well as truncated versions
of miR-146 maybe also be useful in the methods of the present
invention.
[0062] To increase stability and/or optimize delivery of the sense
or antisense oligonucleotides, modified nucleotides or backbone
modifications can be utilized. For example, modified nucleotides
may include: linked nuclear acid (LNA), 2'-O-Me nucleotides,
2'-O-methoxyethyl, and 2' fluoro. Backbone modifications include,
for example, phosphorothioate and phosphate.
[0063] In some embodiments, a miR-146 or anti-miR-146
oligonucleotide is modified with cholesterol to enhance delivery to
target cells. The cholesterol can be linked, for example, through a
hydroxyprolinol linkage on the 3' end of the miRNA.
[0064] Nucleic acid molecules encoding miR-146 and anti-miR-146 are
used in some embodiments of the present invention, for example to
modulate function, activity and/or proliferation of immune
cells.
MiR-146 Expression Vectors
[0065] Expression vectors that contain a miR-146 or anti-miR-146
coding sequence are also useful in the present invention for
delivery of a miR-146 or anti-miR146 to target cells. Thus the
present invention also contemplates expression vectors that contain
a miR-146 sequence and/or anti-miR-146 sequence, optionally
associated with a regulatory element that directs the expression of
the coding sequence in a target cell. MiR-146 sequences are
described in detail in the previous section. The choice of vector
and/or expression control sequences to which the encoding sequence
is operably linked depends directly, as is well known in the art,
on the functional properties desired, e.g., miRNA transcription,
and the host cell to be transformed.
[0066] A vector contemplated by the present invention is preferably
capable of directing replication in an appropriate host and of
expression of a miR-146 or anti-miR-146 in a target cell. Vectors
that can be used are well known in the art and include, but are not
limited to, pUC8, pUC9, pBR322 and pBR329 available from BioRad
Laboratories, (Richmond, Calif.), pPL and pKK223 available from
Pharmacia (Piscataway, N.J.) for use in prokaryotic cells, and pSVL
and pKSV-10 (Pharmacia), pBPV-1/pML2d (International
Biotechnologies, Inc.), pCDNA and pTDT1 (ATCC, #31255), for use in
eukaryotic cells, as well as eukaryotic viral vectors such as
adenoviral or retroviral vectors.
[0067] Vectors may include a selection gene whose expression
confers a detectable marker such as a drug resistance. Typical
selection genes encode proteins that confer resistance to
antibiotics or other toxins, e.g., ampicillin, neomycin,
methotrexate, or tetracycline, complement auxotrophic deficiencies,
or supply critical nutrients withheld from the media. Such
selection systems are well known in the art. The selectable marker
can optionally be present on a separate plasmid and introduced by
co-transfection.
[0068] Expression control elements that are used for regulating the
expression of an operably linked coding sequence are known in the
art and include, but are not limited to, inducible promoters,
constitutive promoters, enhancers, and other regulatory elements.
In some embodiments an inducible promoter is used that is readily
controlled, such as being responsive to a nutrient in the target
cell's medium. In some embodiments, the promoter is the U6 promoter
or CMV promoter.
[0069] Other methods, vectors, and target cells suitable for
adaptation to the expression of miR-146 in target cells are well
known in the art and are readily adapted to the specific
circumstances.
Delivery of Oligonucleotides and Expression Vectors to a Target
Cell or Tissue
[0070] In some embodiments, a miR-146 or anti-miR-146
oligonucleotide is delivered to a target cell. In other
embodiments, an expression vector encoding a miR-146 or
anti-miR-146 is delivered to a target cell where the miR-146 or
anti-miR-146 is expressed. Methods for delivery of oligonucleotides
and expression vectors to target cells are well known in the art
and exemplary methods are described briefly below. Target cells can
be, for example, any immune cell, such as immune cells involved in
innate immunity, or precursors of immune cells. Target cells may be
present in a host, such as in a mammal, or may be in culture
outside of a host. Delivery of miR-146 or anti-miR-146 to target
cells in vivo, ex vivo and in vitro is contemplated, depending on
the particular circumstances.
[0071] In some embodiments, a miR-146 or anti-miR-146
oligonucleotide is delivered to a target organ or tissue. Target
organs and tissues may include locations where hematopoietic and/or
immune cells or precursors of such cells are known to be located
and may include, for example and without limitation, the peritoneal
cavity, spleen, lymph nodes, including mesenteric lymph nodes and
peripheral lymph nodes, thymus, and bone marrow. In some
embodiments, immune cell development, function, proliferation
and/or activity is modulated by delivering miR-146 or anti miR-146
to bone marrow. In other embodiments, the numbers and/or activity
of B1 B cells can be modulated by administering a miRNA-146 or
anti-miR-146 oligonucleotide to B1 B cells in peritoneal cavity or
to B1 B precursor cells in the bone marrow. The numbers and/or
activity of B2 B cells can be modulated by administering a
miRNA-146 or anti-miR-146 oligonucleotide to the bone marrow or to
B2 B cells or B2 B precursor cells in the bone marrow or elsewhere.
The activity and/or numbers of CD8+CD3+ conventional T cells can be
modulated by administering a miRNA-146 or anti-miR-146
oligonucleotide to these cells, for example in the spleen, or to
precursor cells in the elsewhere, such as in the bone marrow. The
numbers and/or activity of CD8.alpha..alpha.+ T cells can be
modulated by administering a miRNA-146 or anti-miR-146
oligonucleotide to these cells, for example, in mesenteric lymph
nodes, or to precursor cells in the bone marrow or elsewhere. The
numbers and/or activity of CD-11b+ cells, for example in the
spleen, can be modulated by administering a miRNA-146 or antisense
miRNA-146 to these cells or precursor cells in the spleen or
elsewhere. In some embodiments, immune cell function and/or
development is modulated by delivering miR-146 or anti-miR-146 to
the bone marrow.
[0072] Delivery of oligonucleotides and/or expression vectors to a
target cell can be achieved in a variety of ways. In some
embodiments, a transfection agent is used. A transfection agent, or
transfection reagent or delivery vehicle, is a compound or
compounds that bind(s) to or complex(es) with oligonucleotides and
polynucleotides, and enhances their entry into cells. Examples of
transfection reagents include, but are not limited to, cationic
liposomes and lipids, polyamines, calcium phosphate precipitates,
polycations, histone proteins, polyethylenimine, polylysine, and
polyampholyte complexes. Transfection reagents are well known in
the art. One transfection reagent suitable for delivery of miRNA is
siPORT.TM.. NeoFX.TM.. transfection agent (Ambion), which can be
used to transfect a variety of cell types with miRNA. miRNAs can be
readily electroporated into primary cells without inducing
significant cell death. In addition, miRNAs can be transfected at
different concentrations. The transfection efficiency of synthetic
miRNAs has been shown to be very good, and around 100% for certain
cell types (Ambion.RTM. miRNA Research Guide, page 12. See also,
www.ambion.com/miRNA).
[0073] Reagents for delivery of miRNA, anti-miRNA and expression
vectors can include, but are not limited to protein and polymer
complexes (polyplexes), lipids and liposomes (lipoplexes),
combinations of polymers and lipids (lipopolyplexes), and
multilayered and recharged particles. Transfection agents may also
condense nucleic acids. Transfection agents may also be used to
associate functional groups with a polynucleotide. Functional
groups can include cell targeting moieties, cell receptor ligands,
nuclear localization signals, compounds that enhance release of
contents from endosomes or other intracellular vesicles (such as
membrane active compounds), and other compounds that alter the
behavior or interactions of the compound or complex to which they
are attached (interaction modifiers). For delivery in vivo,
complexes made with sub-neutralizing amounts of cationic
transfection agent may be preferred.
[0074] In some embodiments, polycations are mixed with
polynucleotides for delivery to a cell. Polycations are a very
convenient linker for attaching specific receptors to DNA and as
result, DNA/polycation complexes can be targeted to specific cell
types. Here, targeting is preferably to cells involved in innate
immunity. An endocytic step in the intracellular uptake of
DNA/polycation complexes is suggested by results in which
functional DNA delivery is increased by incorporating endosome
disruptive capability into the polycation transfection vehicle.
Polycations also cause DNA condensation. The volume which one DNA
molecule occupies in complex with polycations is drastically lower
than the volume of a free DNA molecule. The size of DNA/polymer
complex may be important for gene delivery in vivo. In some
embodiments, miR-146 or anti-miR-146 nucleic acids and a
transfection reagent are delivered systematically such as by
injection. In other embodiments, they may be injected into
particular areas comprising target cells, such as particular
organs, for example the bone marrow.
[0075] Polymer reagents for delivery of miRNA, anti-miRNA and
expression vectors may incorporate compounds that increase their
utility. These groups can be incorporated into monomers prior to
polymer formation or attached to polymers after their formation. A
miRNA, anti-miRNA or expression vector transfer enhancing moiety is
typically a molecule that modifies a nucleic acid complex and can
direct it to a cell location (such as tissue cells) or location in
a cell (such as the nucleus) either in culture or in a whole
organism. By modifying the cellular or tissue location of the
complex, the desired localization and activity of the miRNA,
anti-miRNA or expression vector can be enhanced. The transfer
enhancing moiety can be, for example, a protein, peptide, lipid,
steroid, sugar, carbohydrate, nucleic acid, cell receptor ligand,
or synthetic compound. The transfer enhancing moieties can enhance
cellular binding to receptors, cytoplasmic transport to the nucleus
and nuclear entry or release from endosomes or other intracellular
vesicles.
[0076] Nuclear localizing signals can also be used to enhance the
targeting of the miRNA, anti-miRNA or expression vector into
proximity of the nucleus and/or its entry into the nucleus. Such
nuclear transport signals can be a protein or a peptide such as the
SV40 large Tag NLS or the nucleoplasmin NLS. These nuclear
localizing signals interact with a variety of nuclear transport
factors such as the NLS receptor (karyopherin alpha) which then
interacts with karyopherin beta. The nuclear transport proteins
themselves could also function as NLS's since they are targeted to
the nuclear pore and nucleus.
[0077] Compounds that enhance release from intracellular
compartments can cause DNA release from intracellular compartments
such as endosomes (early and late), lysosomes, phagosomes, vesicle,
endoplasmic reticulum, Golgi apparatus, trans Golgi network (TGN),
and sarcoplasmic reticulum and could be used to aid delivery of
miRNA-146 or anti-miR-146. Release includes movement out of an
intracellular compartment into cytoplasm or into an organelle such
as the nucleus. Such compounds include chemicals such as
chloroquine, bafilomycin or Brefeldin Al and the ER-retaining
signal (KDEL sequence), viral components such as influenza virus
hemagglutinin subunit HA-2 peptides and other types of amphipathic
peptides.
[0078] Cellular receptor moieties are any signal that enhances the
association of the miRNA, anti-miRNA or expression vector with a
cell. Enhanced cellular association can be accomplished by either
increasing the binding of the polynucleotide or polynucleotide
complex to the cell surface and/or its association with an
intracellular compartment, for example: ligands that enhance
endocytosis by enhancing binding the cell surface. Cellular
receptor moieties include agents that target to asialoglycoprotein
receptors by using asialoglycoproteins or galactose residues. Other
proteins such as insulin, EGF, or transferrin can be used for
targeting. Peptides that include the RGD sequence can also be used
to target many cells. Chemical groups that react with sulfhydryl or
disulfide groups on cells can also be used to target many types of
cells. Folate and other vitamins can also be used for targeting.
Other targeting groups include molecules that interact with
membranes such as lipids fatty acids, cholesterol, dansyl
compounds, and amphotericin derivatives. In addition viral proteins
could be used to target cells.
[0079] The skilled artisan will be able to select and use an
appropriate system for delivering miRNA-146, anti-miRNA-146 or an
expression vector to target cells in vitro or in vivo without undue
experimentation.
[0080] Modulation of Immune Cell Function and/or Proliferation
[0081] miRNA-146 can be used to modulate activity and/or
proliferation of immune cells. Development and activity of immune
cells can be modulated by administering a miR-146 oligonucleotide
or antisense miR-146 to target cells. The target cells may be in a
mammal. In some embodiments, the target cells are hematopoietic
cells. In other embodiments, miR-146 or anti miR-146 is delivered
to bone marrow.
[0082] In some embodiments, proliferation of immune cells can be
used to measure the effect of miR-146 or antisense miR-146 on
immune cells. For example, proliferation and/or activity of B1 B
cells, B2 B cells, Marginal Zone B cells, CD8.alpha..alpha.+ T
cells, Natural Killer cells and/or CD8+ T cells can be measured.
Measurements of proliferation can take place in an appropriate spot
for each cell type, such as in the bone marrow, thymus, spleen,
periphery, peritoneal cavity or lymph nodes, such as peripheral
lymph nodes or mesenteric lymph nodes. Measurement of proliferation
can be by any method known in the art, for example by FACS
analysis.
[0083] In some embodiments, miR-146 or antisense miR-146 are
administered by administering a miR-146 or anti-miR-146 expression
vector to target cells and expressing the desired miR-146 or
anti-miR-146 in the target.
[0084] In some embodiments, the activity and/or proliferation of
certain cells, such as B1 B cells, B2 B cells, Marginal Zone B
cells, CD8.alpha..alpha.+ T cells, Natural Killer cells and CD8+ T
cells is modulated using miR-146 or anti-miR-146. The activity
and/or proliferation of these cells can be either upregulated or
downregulated.
[0085] In some embodiments, activity and/or proliferation of B1 B
cells, Marginal Zone B cells, CD8+ cells and Natural Killer cells
can be upregulated by administering a miRNA-146 oligonucleotide or
a miRNA-146 expression vector to target cells, organs or tissues.
In some embodiments, the miR-146 expression vector comprises a
nucleic acid sequence encoding a miRNA-146 operably linked to a U6
promoter or a CMV promoter.
[0086] Increased numbers of B1 B cells, Marginal Zone B cells, CD8+
cells and Natural Killer cells can be detected, for example, by
FACS analysis after administering a miRNA-146 oligonucleotide or a
miRNA-146 expression vector to a target tissue.
[0087] In other embodiments, activity and/or proliferation of B1 B
cells, Marginal Zone B cells, CD8+ T cells and Natural Killer cells
is downregulated by administering an antisense miRNA-146
oligonucleotide or an anti-miR-146 expression vector to target
cells or target tissue.
[0088] Decreased numbers of B1 B cells, Marginal Zone B cells, CD8+
cells and Natural Killer cells can be detected, for example, by
FACS analysis after administering an antisense miRNA-146
oligonucleotide or an antisense miRNA-146 expression vector to a
target tissue.
[0089] In some embodiments, activity and/or proliferation of B2 B
cells and CD8.alpha..alpha.+ T cells can be upregulated by
administering an antisense miRNA-146 oligonucleotide to target
cells or target tissue. In other embodiments, the methods comprise
administering an antisense miRNA-146 expression vector to target
cells or target tissue and expressing a miRNA-146 in the target. In
some embodiments, increased numbers of B2 B cells and
CD8.alpha..alpha.+ T cells can be detected by FACS analysis after
administering an antisense miRNA-146 oligonucleotide or an
antisense miRNA-146 expression vector to a target.
[0090] Proliferation and/or activity of B2 B cells and
CD8.alpha..alpha.+ T cells can be downregulated. In some
embodiments, the methods comprise administering a miRNA-146
oligonucleotide to a target tissue. In other embodiments, the
methods comprise administering a miRNA-146 expression vector to a
target tissue and expressing a miRNA-146 in the target tissue. In
some embodiments, decreased numbers of B2 B cells and
CD8.alpha..alpha.+ T cells can be detected by FACS analysis after
administering a miRNA-146 oligonucleotide or a miRNA-146 expression
vector to a target tissue.
[0091] Any of a variety of sequences of miRNA-146 or antisense
miRNA-146 can be used to regulate activity and proliferation of
various immune cells in the embodiments described above. In some
embodiments, the miR-146 oligonucleotide comprises mature all or a
portion of miR-146a, mature miR-146b, pre-miR-146a, pre-miR-146b,
pri-miR-146a, pri-miR-146b, or a miR-146 seed sequence. Mixtures of
various miR-146 nucleic acids can also be used. In some
embodiments, the miR-146 comprises all or a portion of a sequence
selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 14.
[0092] In some embodiments, the miR-146 expression vector comprises
a sequence encoding a miRNA-146 selected from the group consisting
of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
[0093] In some embodiments, the anti-sense miR-146 is complementary
to all or a portion of SEQ ID NOs: 1, 2, 3, 4 or 14. In some
embodiments the anti-sense miR-146 hybridizes under stringent
conditions to one or more of SEQ ID NOs: 1, 2, 3, 4 or 14. In other
embodiments the antisense miR-146 comprises a sequence selected
from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10.
[0094] In some embodiments, to regulate activity and/or
proliferation of certain immune cells, target tissues to which
miRNA-146 or antisense miRNA-146 are delivered are hematopoietic
tissues. For example, in some embodiments miR-146 oligonucleotides
or expression vectors or anti-miR-146 oligonucleotides or
expression vectors are preferably delivered to the bone marrow. In
other embodiments, targets are tissues or organs comprising immune
cells. In other embodiments the miR-146 or anti-miR-146 is
delivered directly to the immune cells to be regulated or to
precursor cells.
[0095] miRNA-146 or antisense miRNA-146 can be delivered as
described herein or as known in the art. For example, delivery can
be achieved by modification of an oligonucleotide encoding a
miR-146, such as a mature miR-146a or miR-146b, with cholesterol to
help it easily penetrate the cell membrane. Delivery can be
optimized by using modified nucleotides or utilizing backbone
modifications. Delivery can be achieved by injection into
particular areas such as hematopoietic tissue or the bone
marrow.
[0096] In other embodiments, miR-146, anti-miR-146 or expression
vectors are delivered systemically. miRNA-146 or antisense
miRNA-146 can be delivered as described herein or as known in the
art. For example, miRNA-146 or antisense miRNA-146 can be delivered
in combination with pharmaceutically acceptable carriers. In some
embodiments miRNA-146 or antisense miRNA-146 or expression vectors
can be injected intravenously.
[0097] Modulation of Production of Pro-Inflammatory Cytokines
[0098] miRNA-146 can regulate production of pro-inflammatory
cytokines in immune cells. For example, in some embodiments
production of pro-inflammatory cytokines in macrophages is
regulated using miR-146 or anti-miR-146. Pro-inflammatory cytokines
that can be regulated include, for example, TNF.alpha. and IL-6. In
some embodiments production of pro-inflammatory cytokines by immune
cells is downregulated by modulating levels of miRNA-146a in the
immune cells.
[0099] In some embodiments, the methods comprise administering a
miRNA-146 oligonucleotide to immune cells in which pro-inflammatory
cytokine production is to be reduced. In some embodiments, the
miR-146 oligonucleotide comprises all or a portion of mature
miR-146a, mature miR-146b, pre-miR-146a, pre-miR-146b,
pri-miR-146a, pri-miR-146b, or a miR-146 seed sequence. Mixtures of
various miR-146 nucleic acids can also be used. In some
embodiments, the miR-146 comprises a sequence selected from the
group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ
ID NO: 4 or SEQ ID NO: 14.
[0100] In other embodiments, the methods comprise administering a
miRNA-146 expression vector to target cells and expressing a
miRNA-146 in target cells to reduce production of pro-inflammatory
cytokines. In some embodiments, the miR-146 expression vector
comprises a nucleic acid sequence encoding a miRNA-146 operably
linked to a U6 promoter or a CMV promoter. In some embodiments, the
miR-146 expression vector comprises a sequence encoding a miRNA-146
selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6,
SEQ ID NO: 7 or SEQ ID NO: 8.
[0101] To reduce production of certain pro-inflammatory cytokines,
miRNA-146 can be delivered to immune cells that produce
pro-inflammatory cytokines, such as dendritic cells, macrophages,
Th1 helper T cells, Th2 helper T cells and regulator T cells. In
other embodiments the miR-146 is delivered to precursor cells that
develop into immune cells or tissues comprising precursor cells,
such as bone marrow. In some embodiments miR-146 is delivered to
the cells, tissues comprising the cells or systemically. miRNA-146
or antisense miRNA-146 can be delivered as described herein or as
known in the art. For example, in some embodiments the miR-146
oligonucleotide or expression vector can be administered to the
cells by transfection. In other embodiments, they may be directly
injected into bone marrow. miRNA-146 or antisense miRNA-146 can be
modified to enhance delivery. For example, these oligonucleotides
can be modified with cholesterol. In other embodiments miRNA-146 or
antisense miRNA-146 can be injected into target cells, a tissue
comprising the target cells, or injected systemically into a mammal
comprising the target cells. miRNA-146 can also be coupled to a
ligand of a target cell surface receptor and enter into target
cells through endocytosis.
[0102] In some particular embodiments, production of
pro-inflammatory cytokines by macrophages is reduced by
administering miR-146 or a miR-146 expression vector to macrophages
or macrophage precursor cells. In other embodiments, macrophage
activity is downregulated by administering miR-146 to target
tissue, such as bone marrow, or directly to macrophages.
[0103] In other embodiments, production of pro-inflammatory
cytokines by immune cells can be increased by administering
anti-miR-146 or an anti-miR-146 expression vector to the immune
cells. The anti-miR-146 can be essentially as described elsewhere
herein.
Modulation of T Cell Activation
[0104] miRNA-146 can also be used to regulate T cell activation. In
some embodiments, T cell activation is downregulated by increasing
levels of miRNA-146 in T cells. In some embodiments, the methods
comprise administering a miRNA-146 oligonucleotide to target T
cells or precursor cells. In other embodiments, the methods
comprise administering a miRNA-146 expression vector to target T
cells or precursor cells and expressing a miRNA-146 in the target
cells. In some embodiments the miR-146 oligonucleotide or
expression vector are delivered to tissues or organs comprising T
cells or precursor cells, such as bone marrow. In some embodiments,
the miR-146 expression vector comprises a nucleic acid sequence
encoding a miRNA-146 operably linked to a U6 promoter or a CMV
promoter. T cell activation can be measured by any method known in
the art, for example by measuring surface expression various
markers of activation. For example, activation can be determined by
increased surface expression of CD25, CD44 and CD69 proteins and a
decrease in CD62L expression, as discussed in the examples
below.
[0105] In other embodiments, T cell activation can be upregulated
by reducing levels or activity of miRNA-146 in target T cells. In
some embodiments, the methods comprise administering an antisense
miRNA-146 oligonucleotide to target T cells. In other embodiments,
the methods comprise administering an antisense miRNA-146
expression vector to target T cells and expressing an antisense
miRNA-146 in the target cells.
[0106] In some embodiments, the miR-146 oligonucleotide comprises
all or an effective portion of mature miR-146a, mature miR-146b,
pre-miR-146a, pre-miR-146b, pri-miR-146a, pri-miR-146b, or a
miR-146 seed sequence. Mixtures of various miR-146 nucleic acids
can also be used. In some embodiments, the miR-146 comprises all or
an effective portion of a sequence selected from the group
consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
4 or SEQ ID NO: 14. In some embodiments, the miR-146 expression
vector comprises a sequence encoding a miRNA-146 selected from the
group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ
ID NO: 8.
[0107] In some embodiments, the antisense miR-146 comprises a
sequence that is complementary to a miR-146 oligonucleotide as
discussed above, or that hybridizes under stringent conditions to a
miR-146 oligonucleotide. In some embodiments the anti-miR-146 is
selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO:
10.
[0108] Target T cells can be, for example and without limitation,
Th1 helper T cells, Th2 helper T cells and regulator T cells.
Target cells may also be precursor cells. As described above, the
target may also be tissue, such as bone marrow, that comprises T
cells or precursor cells. miRNA-146, antisense miRNA-146 and
expression vectors can be delivered as described herein or as known
in the art. For example, a miR-146 oligonucleotide or expression
vector can be administered to the cells by transfection. miRNA-146
or antisense miRNA-146 can be modified to enhance delivery. For
example, they can be modified with cholesterol. miRNA-146 or
antisense miRNA-146 or expression vectors can also be injected into
target cells. miRNA-146 or antisense miRNA-146 can also be coupled
to a ligand of a target cell surface receptor and enter into target
cells through endocytosis. In some embodiments the miR-146,
anti-miR-146 or expression vector is delivered systemically, or
injected into organs or tissues comprising target T cells. For
example, miRNA-146, anti-miR-146 or expression vectors can be
delivered to target tissues such as bone marrow, spleen or other
peripheral immune tissues.
Modulation of Certain Kinase Activation
[0109] miRNA-146 can also be used to regulate activation of certain
kinases such as NF-kB, JNK1 or ERK in immune cells. In some
embodiments the activation of these kinases can be downregulated by
modulating levels of miRNA-146 or miR-146 activity in target immune
cells or immune precursor cells.
[0110] In some embodiments, miRNA-146 can be used to downregulate
activation of NK-kB and/or JNK1 in immune cells. In some
embodiments, the methods comprise administering a miRNA-146
oligonucleotide to target immune cells, precursor cells or tissue,
such as bone marrow. In other embodiments, the methods comprise
administering a miRNA-146 expression vector to target immune cells
and expressing a miRNA-146 in the target cells.
[0111] In other embodiments, antisense miRNA-146 can be used to
downregulate activation of ERK in immune cells. In some
embodiments, the methods comprising administering an antisense
miRNA-146 oligonucleotide to target immune cells. In other
embodiments, the methods comprise administering an antisense
miRNA-146 expression vector to target immune cells and expressing
an antisense miRNA-146 in the target cells.
[0112] In some embodiments, the miR-146 or antisense miR-146
expression vector comprises a nucleic acid sequence encoding a
miRNA-146 or antisense miR-146 operably linked to a U6 promoter or
a CMV promoter.
[0113] In some embodiments, the miR-146 oligonucleotide comprises
all or an effective portion of mature miR-146a, mature miR-146b,
pre-miR-146a, pre-miR-146b, pri-miR-146a, pri-miR-146b, or a
miR-146 seed sequence. Mixtures of various miR-146 nucleic acids
can also be used. In some embodiments, the miR-146 comprises all or
an effective portion of a sequence selected from the group
consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
4 or SEQ ID NO: 14. In some embodiments, the miR-146 expression
vector comprises a sequence encoding a miRNA-146 selected from the
group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ
ID NO: 8.
[0114] In some embodiments, the antisense miR-146 comprises a
sequence that is complementary to a miR-146 oligonucleotide as
discussed above, or that hybridizes under stringent conditions to a
miR-146 oligonucleotide. In some embodiments the anti-miR-146 is
selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO:
10.
[0115] Immune target cells include, for example and without
limitation, dendritic cells, macrophages, Th1 helper T cells, Th2
helper T cells regulator T cells. Target tissue includes, for
example, bone marrow. miRNA-146 and anti-miR-146 can also be
delivered to target tissues comprising immune cells or precursor
cells. In some embodiments miR-146 is delivered to the cells,
tissues comprising the cells or systemically. In some embodiments
miR-146 or anti-miR-146 are delivered to hematopoietic tissue, such
as bone marrow.
[0116] miRNA-146, antisense miRNA-146 and expression vectors can be
delivered as described herein or as known in the art. For example,
delivery of these oligonucleotides can be enhanced by modification
with cholesterol. They can be injected to the blood surrounding the
target tissue.miRNA-146, antisense miRNA-146 and expression vectors
can be delivered as described herein or as known in the art.
Vaccination
[0117] miRNA-146 can be used to enhance vaccination in a target
mammal, such as cancer vaccination. That is, the desired effects of
vaccination can be improved by modulating levels of miRNA-146 in
target immune cells. Reducing levels of miRNA-146 or miR-146
activity can increase the number and/or activity of
CD8.alpha..alpha.+ T cells, which can in turn increase the desired
immune response to a vaccine.
[0118] In some embodiments, the methods comprise administering an
antisense miRNA-146 oligonucleotide to a mammal to be vaccinated
prior to, concurrent with, or following vaccination with an
antigen. In other embodiments, the methods comprise administering
an antisense miRNA-146 expression vector to a mammal and expressing
an antisense miRNA-146 in immune cells. In some embodiments, the
antisense miR-146 or antisense miR-146 expression vector are
delivered systemically. In other embodiments, delivery is to target
cells or tissues, such as to bone marrow. In some embodiments, the
miR-146 expression vector comprises a nucleic acid sequence
encoding a miRNA-146 operably linked to a U6 promoter or a CMV
promoter. In some embodiments, the antisense miR-146 expression
vector comprises a sequence encoding an antisense miRNA-146
selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO:
10.
[0119] In some embodiments, anti-miR-146 is delivered to target
tissues, such as tissue comprising immune cells or immune precursor
cells. In some embodiments the anti-miR-146 is delivered to
hematopoietic tissue, such as bone marrow. Anti-miR-146 or vectors
for expressing anti-miR-146 can be delivered as described herein or
as known in the art. In some embodiments they are delivered into or
in proximity of the target tissue. In other embodiments they are
delivered systemically. Anti-miR-146 or anti-miR-146 expression
vectors can be delivered in combination with pharmaceutically
acceptable carriers.
[0120] In some embodiments, the antisense miR-146 comprises a
sequence that is complementary to at least a portion of a miR-146
oligonucleotide as discussed above, or that hybridizes under
stringent conditions to a miR-146 oligonucleotide. In some
embodiments the anti-miR-146 is selected from the group consisting
of SEQ ID NO: 9 and SEQ ID NO: 10.
[0121] The following examples are offered by way of illustration
and not by way of limitation.
Knock-Out Mice
[0122] Knockout (KO) mice were produced to investigate the
biological role for miR-146a miRNA. A mir-146a KO mouse was
generated by deleting a 295 by fragment containing precursor
sequence of this miRNA (FIG. 1).
[0123] Briefly, a targeting construct was created wherein a 295 by
of genomic sequence containing mouse pre-miR-146a was replaced with
a floxed neomycin expression cassette. The assembly of the
targeting construct was done by recombineering in bacteria
following the protocol established by Copeland et al. Copeland et.
al., Nat Rev Genet 2, 769-779. The linearized targeting construct
was then electroporated into a 129/SvJ ES cell line and NeoR clones
that have undergone homologous recombination were determined by
Southern blotting. The resulting targeted ES clones were injected
into C57BL/6 blastocysts that were used to produce chimeric mice.
Gerline-competent chimeras were then used to produce heterozygous
F1 litters, which were then bred to homogeneity. Mice obtained were
analyzed in a fashion similar to what is described in the bone
marrow transfer experiments.The miR-146.sup.-/- mice were born in
Mendelian ratios, and they appeared morphologically normal and
fertile. As expected, no expression of mature miR-146a was observed
in tissues of the KO mice (FIG. 1B and FIG. 1C). In addition, bone
marrow derived macrophages (BMDMs) from KO mice have significantly
higher expression of TRAF6 protein, an observation that is
consistent with previous finding of TRAF6 as a miR-146a target gene
(Taganov et al., 2006).
[0124] Starting at about 6 month of age miR-146a KO mice developed
an autoimmune-like disorder that was characterized by splenomegaly
and lymphoadenopathy, and as a result died prematurely (FIG. 2A,
B). Histological examination of miR-146a KO mice revealed severe
inflammation and tissue damage in several peripheral organs,
including liver, kidneys and lungs (FIG. 3A, B). The enlarged
spleens of the KO mice showed signs of follicular hyperplasia with
an increase in the number and size of lymphoid follicles that
resulted in squeezing of the red pulp and the marginal zone (FIG.
3C). Flow cytometric analysis of miR-146a KO spleens revealed a
massive myeloproliferation with the number of CD11b-positive cells
increasing about 10 fold on average (FIG. 4C), while the number of
the splenic B and T cells did not change significantly. Peripheral
T cells (both CD4 from the KO animals consistently showed an
activated, effector status manifested by an increase in the cell
surface expression of CD25, CD44 and CD69 proteins and a decrease
in CD62L expression (FIG. 5A).
[0125] The activated status of peripheral T cells is a
characteristic for many autoimmune/inflammatory diseases, where T
cells cause damage to peripheral tissues in response to autoantigen
stimulation and correlates well with the histological findings of
severe inflammation in the miR-146a KO mice. Besides, T cell
development in the thymus of KO mice was found to be dysregulated:
a dramatic decrease in the number of double positive (CD4+CD8+)
thymocytes and an increase in the number of single positive (both
CD4+ and CD8+) cells (FIG. 5B) were observed, indicating that the
negative T cell selection stage is compromised and is probably a
reason for the presence of autoreactive T cells in the
periphery.
[0126] Systematic FACS analysis of cells from the KO animals
revealed that deletion of miR-146a gene affected development and
function of a number of cell lineages of hematopoietic origin. For
example, the number of B1 B cells in peritoneal cavity of miR-146a
KO animals was markedly reduced (FIG. 6A) and a decrease in the
number of marginal zone B cells in the spleen was observed (FIG.
6B). Moreover, miR-146a KO animals have fewer CD8-positive and
natural killer (NK) cells in the periphery (FIG. 7). Finally, a
dramatic increase in the number of CD8.alpha..alpha.+ T cells in
the peripheral lymph nodes of miR-146a KO mice was observed in
comparison to control (FIG. 8).
[0127] The development and function of macrophages with genetically
altered levels of miR-146a expression was also examined. Bone
marrow-derived macrophages from the KO animals secret significantly
higher levels of proinflammatory cytokines, including TNF.alpha.,
IL-6 and IL-1.beta. after LPS stimulation, indicating that miR-146a
plays a role of negative regulator of inflammation and macrophage
function (FIG. 9).
[0128] Lack of miR-146a Expression Affects Development of Several
Hematopoietic Cell Lineages in Mice
[0129] Multiple hematopoietic cell lineage cells were collected
from the miR-146 knockout mice and analyzed by FACS. The FACS
analysis was performed by using combinations of antibodies against
lineage-specific cell surface markers, and analyzed on a Becton
Dickinson FACS Balibur. The results are shown in FIG. 2. The number
of B1 B cells in peritoneal cavity of the miR-146a knockout mice
was markedly reduced (.about.4.5 folds). In contrast, the total
number of conventional B2 cells has modestly increased
(.about.25-40%). CD8.sup.+ and NK cells are also studied. A drop in
the number of these T cells was detected in miR-146a knockout mice
in thymus and peripheral lymphoid organs. Analysis of the marginal
zone B cells in spleen in the miR-146a knockout mice revealed a
severe reduction in number of the cells comparing to the wild-type
control. In addition, a dramatic increase in the number of
CD8.alpha..alpha.+ T cells in the peripheral lymph nodes of the
miR-146a knockout mice was observed.
Bone Marrow Transfer (BMT) Experiments
[0130] 5-fluorouracil (5-FU) enriched bone marrow-derived
hematopoietic stem cells (HSC) from C57BL/6 mouse were infected
with a retrovirus carrying a miR-146a expression cassette (or
control virus) and transplanted into lethally .gamma.-irradiated
C57BL/6 recipient mice. Eight mice were transplanted and analyzed
for each miRNA construct or control construct. Secondary bone
marrow transfer experiments were carried in a similar fashion,
except infection of HSCs was not applied.
[0131] The transduced HSCs and their progenitors were traced by
expression of green fluorescent protein (GFP), which was inserted
into the retrovirus upstream of the miR-146a cassette under the
control of mouse stem cell virus (MSCV) LTR. Hematopoietic organs
and tissues from miR-146a-expressing and control mice were
collected 2-3 months post transplantation and subjected to a
quantitative RT-PCR analysis to determine the level of miR-146a
expression as well as FACS analysis using combinations of
antibodies against lineage-specific cell surface markers. To
perform the RT-PCR analysis, total RNA was isolated using mirVana
miRNA Isolation kit (Ambion). miRNA expression was measured with
MirVana qRT-PCR miRNA Detectin kit (Ambion) according to vendor
protocol and normalized by 5S rRNA levels.
[0132] A high level of repopulation of the primary and secondary
lymphoid organs by GFP-positive cells were found in both control
and miR-146a mice and a strong expression of mature miR-146A was
found in the bone marrow (-50 fold increase over control) and in
the periphery (-20 fold increase over control in spleen) of
miR-146a-overexpressing animals (FIG. 11A). Analysis of multiple
hematopoietic cell lineages in miR-146a-overexpressing mice
revealed a dramatic increase (.about.10 fold) in numbers of B-1 B
precursor cells (B220.sup.lo-negCD19.sup.+ IgM.sup.high) in the
bone marrow compartment, which was mirrored by a significant
elevation in numbers of mature B-1 B cells in the peritoneal cavity
(FIG. 11B). Analysis of the transplanted animals over a period of
one month revealed an inverse correlation over time between the
number of B-1 B precursor cells in the bone marrow compartment and
the number of mature B cells in the periphery. In addition,
analysis of miR-146a-expressing animals revealed a significant drop
in the number of B2 cells in bone marrow. A reduction in the number
of CD8.alpha..alpha. T cells in the mesenteric lymph nodes was also
observed while the number of CD8.sup.+CD3.sup.+ conventional T
cells in spleen was increased (FIG. 11 E, F).
[0133] Overexpression of miR-146a in Human Monocytic THP-1 Cell
Line
[0134] Enforced expression of miR-146a in human monocytic THP-1
cell line confirmed that miR-146a could modulate cytokine
production in response to LPS challenge (FIG. 10E). A
miR-146a-overexpressing THP-1 line (THP/146) was established that
produced approximately 8 fold more mature miR-146 than THP/SCR
control cells (FIG. 10B). Enforced expression of miR-146a in human
monocytic THP-1 cell line could indeed modulate response to LPS
challenge. The increase in miR-146 amounts in THP/146 cells
correlated with a significant drop in protein levels of IRAK1 and a
somewhat smaller effect (.about.30-40%) on TRAF6 protein (FIG.
10C). The observed change in the levels of these two adapter
proteins had a strong functional consequence--miR-146a expressing
cells (THP/146) secreted much less proinflammatory cytokines in
comparison to control (THP/SCR) after LPS treatment. The effects of
miR-146a overexpression on TLR4 signaling in these two lines were
also examined by assessing the activation of three major kinase
cascades downstream of TLRs: the NF-kB, JNK and ERK activation
pathways. Constitutive miR-146a overexpression resulted in
attenuated NF-kB activation as can be seen from changed kinetics of
IkBa degradation and resynthesis (FIG. 10D). JNK1 kinase activation
was also negatively affected in THP/146 cells, while activation of
ERK1 pathway, in contrast, was upregulated. These results suggest
that miR-146a plays an important role in fine-tuning TLR signaling
by affecting expression of TRAF6 and IRAK1 at the protein
level.
[0135] Although the foregoing invention has been described in terms
of certain embodiments, other embodiments will be apparent to those
of ordinary skill in the art. Additionally, other combinations,
omissions, substitutions and modification will be apparent to the
skilled artisan, in view of the disclosure herein. Accordingly, the
present invention is not intended to be limited by the recitation
of the preferred embodiments, but is instead to be defined by
reference to the appended claims. All references cited herein are
incorporated by reference in their entirety.
Sequence CWU 1
1
14122RNAHuman 1ugagaacuga auuccauggg uu 22299RNAHuman 2ccgaugugua
uccucagcuu ugagaacuga auuccauggg uugugucagu gucagaccuc 60ugaaauucag
uucuucagcu gggauaucuc ugucaucgu 99322RNAHuman 3ugagaacuga
auuccauagg cu 22473RNAHuman 4ccuggcacug agaacugaau uccauaggcu
gugagcucua gcaaugcccu guggacucag 60uucuggugcc cgg 73522DNAHuman
5tgagaactga attccatggg tt 22699DNAHuman 6ccgatgtgta tcctcagctt
tgagaactga attccatggg ttgtgtcagt gtcagacctc 60tgaaattcag ttcttcagct
gggatatctc tgtcatcgt 99722DNAHuman 7tgagaactga attccatagg ct
22873DNAHuman 8cctggcactg agaactgaat tccataggct gtgagctcta
gcaatgccct gtggactcag 60ttctggtgcc cgg 73922RNAArtificial
SequenceAnti-sense miR-146a 9aacccaugga auucaguucu ca
221022RNAArtificial SequenceAnti-sense miR-146b 10agccuaugga
auucaguucu ca 221122DNAArtificial SequenceAnti-sense miR-146a
11aacccatgga attcagttct ca 221222DNAArtificial SequenceAnti-sense
miR-146b 12agcctatgga attcagttct ca 22132337DNAHuman 13tctccaagac
gcttgaccgc tcttcctttc ctggatggca ccagcagggc cgattggagt 60ggtaaaccct
gggccggaag gcatgccaaa gggtggacag gatggacagg agacagtagc
120acaacgagga gggggagaac agtggctgaa ttggaaatga taaaataaaa
tgaaatttta 180ggagctcgct ggctgggaca ggcctggact gcaaggaggg
gtctttgcac catctctgaa 240aagccgatgt gtatcctcag ctttgagaac
tgaattccat gggttgtgtc agtgtcagac 300ctgtgaaatt cagttcttca
gctgggatat ctctgtcatc gtgggcttga ggacctggag 360agagtagatc
ctgaagaact ttttcagtct gctgaagagc ttggaagact ggagacagaa
420ggcagagtct caggctctga aggtataagg agtgtgagtt cctgtgagaa
acactcattt 480gattgtgaaa agacttgaat tctatgctaa gcagggttcc
aagtagctaa atgaatgatc 540tcagcaagtc tctcttgctg ctgctgctac
tcgtttacat ttattgatta cttacgatga 600ttcaggtact gttgtaagtg
ctttacatgc tgttatacga gactcttggg agaaatcact 660ttaatgaagc
ttgagacaca tggcattgcc atgcaatgat ttttcccccc tcttcacggg
720atcagaggga actaatagaa tgtgacaatg attctttagc agggactgct
gaggcttctg 780gttccttttt aagatctgca gtgaaagaag atgagaaaca
tggatatgcc cttcttttgg 840tccccctctt cctttatttg atctctactt
ccttctataa atatattagg gctacattgt 900ccctttgtat ttcaaacaag
gcaaaaagag gttgtaatta cactttactg caatcctcag 960tttctccagg
gaacaggaat gcaaaggctt tgaaggcctc tctatttgct gacatggtca
1020gctgggtgcc atgggccaag tccttctgtt gccctcctct gtcaccaagt
aagctaggtc 1080ctttctgagg ctcaggtttg ctgtgatgat gatcactttt
aggcagaagg ttagaggcct 1140catgagtgct atatggactt tattaggctt
tagatttgat ggggaataag ggatgtgatt 1200tgtcttttgg gaactcatct
ttgattcatc attgtctctt ggtatcttgg aatttccatg 1260tcattacagt
ctacagaatg aaagagtaac ctgtcccaga ggagaggcag gtgaaagact
1320ccacagcatg ctcattctca ttctgtcttc tcagtgacac cgaggtttac
tgagtgccca 1380ctatgtgcca agcactgtgc tcagggcttt ctttgtatgc
atgatctcag tgaatctcac 1440caagcctcat ctggaaaacg gggacaaatt
aacaacagga tggcaaattg aaaaacacgt 1500aaccatgttc tacagatgga
aaggggtgct tggttattat gaaggccccc tcgcaagcgt 1560gtgggacatg
ggtgtgttct ctgggttgta ctgatcagat caaggacctc ccccaccctt
1620ctcacactct gcccacttcc gccctttgct tatcagaccc ttagccagtg
actcattcca 1680gaaccagaac cttggtgaaa tctcaaccga caccagagat
cggtgtcttc agtcctagac 1740tgatggagaa aatccagaat atatactaga
agctccaaat gctctgggtt tcagctcctc 1800tgtgctgtgg acactgactt
tggctcagaa ctccgattta gtacaaaagg ctcattttta 1860tttcaggggc
actcttccta aagcaaacct aataaatgaa atatggaatt cacagataca
1920cacacacatt aaaaaattaa cctagtgtat ctgtgaggag taggcagaaa
ttcactgtat 1980aaaagaatgc ttcatttcat agagaatttg tgttaagatt
ccattagata gtacatttct 2040caaagatttt tgaggttgta tttgctttac
caaaacttgg tttatgtaag tggaaaaagc 2100atgttgcaaa ataacttggt
gtctatgatt cagtttatgt aaaataataa atgtatgtag 2160gaatacgtgt
gttgaaagat gtacatcaat ttgctaacaa tggttatctc tgacgtggtg
2220ggatttgaga tgtgtttttc tttttggttg tatttttctc tattgtttga
cttaacacag 2280aacatgcttg gttacaacaa taaagttatt gaagacaaaa
aaaaaaaaaa aaaaaaa 2337146RNAHuman 14gagaac 6
* * * * *
References