U.S. patent application number 14/082342 was filed with the patent office on 2014-07-03 for methods and products for expression of micro rnas.
This patent application is currently assigned to Whitehead Institute for Biomedical Research. The applicant listed for this patent is Whitehead Institute for Biomedical Research. Invention is credited to David P. Bartel, Chang-Zheng Chen, Harvey Lodish.
Application Number | 20140187613 14/082342 |
Document ID | / |
Family ID | 34590080 |
Filed Date | 2014-07-03 |
United States Patent
Application |
20140187613 |
Kind Code |
A1 |
Chen; Chang-Zheng ; et
al. |
July 3, 2014 |
METHODS AND PRODUCTS FOR EXPRESSION OF MICRO RNAs
Abstract
The invention relates to microRNAs, methods of producing
microRNAs and methods for using microRNAs.
Inventors: |
Chen; Chang-Zheng; (Palo
Alto, CA) ; Bartel; David P.; (Brookline, MA)
; Lodish; Harvey; (Brookline, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Whitehead Institute for Biomedical Research |
Cambridge |
MA |
US |
|
|
Assignee: |
Whitehead Institute for Biomedical
Research
Cambridge
MA
|
Family ID: |
34590080 |
Appl. No.: |
14/082342 |
Filed: |
November 18, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13326506 |
Dec 15, 2011 |
8609832 |
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14082342 |
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10913288 |
Aug 6, 2004 |
8106180 |
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13326506 |
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60493239 |
Aug 7, 2003 |
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Current U.S.
Class: |
514/44R ;
435/320.1; 435/325; 435/455; 536/24.5 |
Current CPC
Class: |
C12N 2330/30 20130101;
C12N 15/111 20130101; C12N 2310/141 20130101; C12N 2310/531
20130101; C12N 2330/51 20130101; C12N 2310/14 20130101; C07H 21/02
20130101; C12N 2310/111 20130101; C12N 15/113 20130101; C12N
2310/53 20130101; A61P 35/02 20180101; A61P 7/00 20180101 |
Class at
Publication: |
514/44.R ;
536/24.5; 435/455; 435/320.1; 435/325 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was supported, in whole or in part, by grant
number GM67031 to from National Institutes of Health. The
Government may have certain rights in the invention.
Claims
1. A precursor microRNA molecule, comprising: an isolated nucleic
acid comprising: a stem-loop structure, wherein a microRNA sequence
is incorporated into a stem of the stem-loop structure, and a
microRNA flanking sequence flanking at least one end of the
stem-loop structure.
2. The precursor microRNA molecule of claim 1, wherein the microRNA
flanking sequence is between 40 and 2,000 nucleotides in
length.
3. The precursor microRNA molecule of claim 1, wherein the microRNA
sequence and the microRNA flanking sequence are derived from the
same microRNA gene.
4-5. (canceled)
6. The precursor microRNA molecule of claim 1, wherein the
precursor microRNA molecule includes at least two stem-loop
structures.
7. The precursor microRNA molecule of claim 1, wherein the microRNA
flanking sequence is between 40 and 4,000 nucleotides in
length.
8-9. (canceled)
10. The precursor microRNA molecule of claim 1, wherein the
precursor microRNA molecule has microRNA flanking sequences
flanking each end of the stem-loop structure.
11-13. (canceled)
14. A method of altering the productive utilization of a target
mRNA, comprising: contacting a cell with a vector capable of
expressing a precursor microRNA of claim 1 wherein the precursor
microRNA includes a microRNA sequence capable of altering the
productive utilization of the target mRNA.
15. The method of claim 14, wherein the precursor microRNA is
specific for a cancer-associated RNA.
16. The method of claim 14, wherein the precursor microRNA is
specific for a viral RNA.
17. The method of claim 14, wherein the method is performed in
vivo.
18-22. (canceled)
23. A vector for producing a precursor microRNA wherein the vector
includes a sequence encoding a precursor microRNA, including a
microRNA sequence and a microRNA flanking sequence, and at least
one promoter element.
24. The vector of claim 23, wherein the vector is a viral
vector.
25. The vector of claim 24, wherein the viral vector is a
retroviral vector.
26. The vector of claim 23, wherein the at least one of the
promoter is an inducible promoter.
27-29. (canceled)
30. A host cell transfected with the vector of claim 23.
31-32. (canceled)
33. A method for modulating hematopoiesis, comprising: contacting a
hematopoietic cell with a vector capable of expressing a precursor
microRNA of claim 1 wherein the precursor microRNA includes a
microRNA sequence capable of altering accumulation of a protein
involved in hematopoiesis.
34. A method of making the vector of claim 23, the method
comprising providing a vector comprising a microRNA flanking
sequence and at least one promoter element, inserting a sequence
encoding a stem-loop structure into the vector in the context of
the microRNA flanking sequence, wherein a microRNA sequence is
incorporated into a stem of the stem-loop structure.
35. The method of claim 34, wherein the vector comprises two
microRNA flanking sequences, and wherein the sequence encoding a
stem-loop structure is flanked by the two microRNA flanking
sequences.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application filed Aug. 7, 2003, entitled "METHODS AND PRODUCTS FOR
EXPRESSION OF MICRO RNAs", having Ser. No. 60/493,239 the contents
of which are incorporated by reference herein in their
entirety.
BACKGROUND OF THE INVENTION
[0003] MicroRNAs (miRNAs) are endogenous RNAs, some of which are
known to regulate the expression of protein-coding genes at the
posttranscriptional level. During miRNA maturation in animals, the
primary transcript is first processed to a stem-loop precursor and
then the stem-loop is processed to yield a mature miRNA of about
22-nucleotides. These molecules can direct the cleavage of mRNA or
they can interfere with productive translation of the mRNA, either
of which results in reduced protein accumulation and hence the
miRNAs are able to modulate gene expression and related cellular
activities. miRNAs are important in development and
differentiation, and thus the altered expression of miRNAs could be
used to alter development and differentiation during tissue
engineering and other applications. Furthermore, miRNA-like
stem-loops can be expressed in cells as a vehicle to deliver
artificial miRNAs and short interfering RNAs (siRNAs) for the
purpose of modulating the expression of endogenous genes through
the miRNA and or RNAi pathways. This can be a useful tool for
studying gene function, human therapies, and other applications.
However, current methods for expressing miRNAs, artificial miRNAs,
and siRNAs are inefficient and are not effective for many small RNA
sequences.
SUMMARY OF THE INVENTION
[0004] The present invention relates in part to products and
methods of making and using microRNA molecules. In one aspect of
the invention a precursor microRNA molecule is provided. The
precursor microRNA molecule is an isolated nucleic acid including a
stem-loop structure wherein a microRNA sequence is incorporated
into the stem-loop structure. The precursor microRNA molecule
includes a microRNA flanking sequence on either or both sides of
the microRNA sequence.
[0005] In an embodiment of the invention the microRNA sequence and
the microRNA flanking sequence are derived from the same microRNA
gene. In another embodiment of the invention the microRNA sequence
and the microRNA flanking sequence are not derived from the same
microRNA gene.
[0006] In another aspect the invention is a precursor microRNA
molecule having a nucleic acid having a stem-loop structure,
wherein a microRNA sequence is incorporated into a stem of the
stem-loop structure, and, a microRNA flanking sequence flanking at
least one end of the stem-loop structure, wherein the microRNA
sequence and the microRNA flanking sequence are not derived from
the same microRNA gene. Optionally the microRNA sequence is an
artificial microRNA.
[0007] In one embodiment the precursor microRNA molecule includes
at least two stem-loop structures. In another embodiment the
microRNA sequences are at least 16-28 nucleotides in length. In
another embodiment the microRNA flanking sequences are 40-4,000 or
40-2,000 nucleotides in length. In yet another embodiment the
microRNA flanking sequences are at least 40 nucleotides in
length.
[0008] In yet another embodiment the precursor microRNA molecule
has the following nucleic acid sequence
##STR00001##
[0009] wherein X.sub.1, and X.sub.2 are nucleotides and wherein
N.sub.1 and N.sub.2 are nucleic acids of 16-28 nucleotides in
length and N.sub.1 and N.sub.2 have at least partial
complementarity. X.sub.1 and X.sub.2 may each be at least 40
nucleotides.
[0010] The precursor microRNA molecule in some embodiments has
microRNA flanking sequences flanking each end of the stem-loop
structure.
[0011] Another aspect of the invention provides a method of
altering the productive utilization of a target mRNA. The method
includes contacting a cell with a vector capable of expressing a
precursor microRNA wherein the microRNA sequence is capable of
altering the productive utilization of the target mRNA, either by
specifying the cleavage of the target mRNA or by altering the
accumulation of the protein of the target mRNA through another
mechanism, such as translation repression. In one embodiment the
precursor microRNA is specific for a cancer-associated RNA. In
another embodiment the precursor microRNA is specific for a viral
RNA.
[0012] In one embodiment the method of contacting a cell with a
vector capable of expressing a precursor microRNA wherein the
microRNA sequence is capable of altering the productive utilization
of a target mRNA is performed in vivo. In another embodiment the
method is performed in a subject having cancer. In yet another
embodiment the method is performed in a subject having an
infection.
[0013] In another aspect of the invention a method of altering the
productive utilization of a target mRNA including contacting a cell
with a vector capable of expressing a mature microRNA that is not
naturally expressed in the cell is provided. The mature microRNA is
expressed at a level sufficient to cause at least a 2-fold
reduction in the accumulation of a protein from the target mRNA of
the target protein. In other embodiments it is at least a 5-fold,
10-fold, 20-fold, or 30 fold reduction.
[0014] A method of altering productive utilization of a target mRNA
in primary cells is also provided. The method involves contacting a
primary cell with a vector capable of expressing a mature microRNA
that is not naturally expressed in the cell, wherein the mature
microRNA is expressed at a level sufficient to cause a reduction in
accumulation of a protein from the target mRNA in the primary cell.
In one embodiment the primary cell is in vivo.
[0015] A method for modulating hematopoiesis is also provided. The
method involves contacting a hematopoietic cell with a vector
capable of expressing a precursor microRNA, wherein the precursor
microRNA includes a microRNA sequence capable of altering
accumulation of a protein involved in hematopoiesis.
[0016] In yet another aspect of the invention a composition
including a vector for producing a precursor microRNA is provided.
The vector includes a sequence encoding a precursor microRNA,
including microRNA flanking sequences and at least one promoter
element. In one embodiment the promoter is an inducible or tissue
specific promoter.
[0017] In one embodiment the vector is a viral vector. In a second
embodiment the vector is a retroviral vector. In another embodiment
the vector includes the nucleic acid sequence of SEQ ID NO. 1 or
variants thereof.
[0018] One aspect of the invention includes a host cell transfected
with a vector capable of producing a precursor microRNA.
[0019] Another aspect of the invention encompasses a method for
detecting precursor microRNA expression. The precursor microRNA is
incorporated into a reporter system. This composition is
transfected into a host cell. The expression of a reporter gene
product is detected to detect the expression of precursor microRNA
by its effect on the to accumulation of the protein corresponding
to the target mRNA. In one embodiment the reporter system includes
a firefly luciferase reporter gene.
[0020] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The present invention may be more easily and completely
understood when taken in conjunction with the accompanying
figures.
[0022] FIG. 1. (a) Retroviral vectors for efficient expression of
microRNA hairpins. (b) Northern analysis of the expression of a
.about.70-nucleotide miR-132s hairpin from the single-copy
(SC-miR-132s) or double-copy (DC-miR-132s) constructs. (c) Northern
analysis of the expression of exact microRNA hairpin using single
copy and double-copy retroviral constructs.
[0023] FIG. 2. (a) Schematic of miR-223 genes containing the
miR-223 minimal hairpin and increasing amounts of flanking genomic
sequences. miR-223.sub.--67 is the predicted 67-nucleotide hairpin
that contains miR-223. miR-223.sub.--67 Si is a perfect complement
hairpin derived from the original bulged miR-223.sub.--67 hairpin.
(b to d) Northern analysis of the expression of miR-223 genes with
different length of flanking sequences in 293T cells. Total
cellular RNA (b), or cytoplasmic RNA (c), or nuclear RNA (d) from
293T cells transfected with indicated miR-223 expression constructs
was analyzed.
[0024] FIG. 3. Examples of miRNA expression using longer miRNA
genes containing a miRNA hairpin and its correspondent flanking
sequences. The longer miRNA gene is about 270 nt in length, and
consists of .about.20 nt miRNA sequence and 125 nt flanking
sequence on both sides of the miRNA. Northern analysis of miR-223
(a), miR-181 (b), or miR-132s (c) expression from longer miRNA
genes of .about.270 nt in length in 293T or primary bone marrow
cells.
[0025] FIG. 4. Northern analysis of the expression and maturation
of miR-30 from a longer precursor (272 nt) and a minimal hairpin
precursor (71 nt). Northern analysis of miR-30 expression and
maturation using probes against miR-30 (a), or miR-30* (b). to
miR-30* is the small RNA processed from the opposite arm of the
miR-30 hairpin precursor.
[0026] FIG. 5. A luciferase reporter system for testing
microRNA-mediated repression in mammalian cells. (a) A schematic
diagram of retroviral reporter constructs. (b) A graph to show
repression of the target reporter gene by ectopically expressed
miR-223 in mammalian cells.
[0027] FIG. 6. (a) Schematic diagram of the predicted stem-loop
structure of the B1_C02-3 miRNA. Green letters indicate the
endogenous miRNA sequence. (b) Northern analysis of B1_C02-3
expression and maturation using probes against the miRNA or the
miRNA*.
[0028] FIG. 7. Schematic diagram of artificial-microRNA/siRNA
design.
[0029] FIG. 8 Expression of artificial-microRNA/siRNAs using a
microRNA template. (a) Northern analysis of ectopically expressed
SiLuc-1276 and SiLuc-1276*. (b) Northern analysis of ectopically
expressed SiLuc-311 and SiLuc-311*.
[0030] FIG. 9 Repression of reporter gene expression by ectopically
expressed artificial-microRNA/SiRNAs.
[0031] FIG. 10. Inducible expression of B1_C02-3 microRNA at
various concentrations of Doxycycline.
[0032] FIG. 11. Tissue and developmental expression of microRNAs
cloned from mouse bone marrow. Northern analyses were used to
determine microRNA expression in different mouse tissues, including
brain, heart, lung, liver, kidney, muscle, fetal liver, bone
marrow, spleen, and thymus.
[0033] FIG. 12. Northern blot showing microRNA expression during
hematopoietic lineage commitment.
[0034] FIG. 13. (a) Graph to show percentage Thy1.2 positive
(Thy1.2+) cells and CD-19 positive (CD-19+) cells among the
differentiating hematopoietic progenitor cells ectopically
expressing no microRNA (vector), a non-hematopoietic microRNA
(miR-30), or either of three hematopoietic microRNAs (miR-223,
miR-132, or miR-181). (b) Representative FACS analyses of Thy-1.2
(Thy-1.2 APC) and CD-19 (CD-19 PE) lineage marker expression. (c)
Graph to show percentage Mac-1 and Gr-1 negative cells (Mac-1.sup.-
Gr-1.sup.-), Mac-1 positive and Gr-1 negative to intermediate
(Mac-1+Gr-1.sup.-/low), to Mac-1 and Gr-1 positive (Mac-1+Gr-1+)
cells among the differentiating hematopoietic progenitor cells
ectopically expressing no microRNA (vector), miR-223, miR-30,
miR-132, or miR-181. The average of 12 culture replicates for each
construct is shown, with error bars indicating the standard
deviation. (d) Representative FACS analyses of Mac-1 (Mac-1 APC)
and Gr-1 (Gr-1 PE) lineage marker expression.
DETAILED DESCRIPTION
[0035] MicroRNAs (which are defined in more detail later as
including siRNAs and artificial microRNAs as well as endogenous
microRNAs) have potential for use as therapeutics as well as
research tools, e.g. analyzing gene function. Although these
molecules have potential, one limitation associated with these
molecules is the difficulty in expressing adequate quantities of
functional mature microRNA. The invention relates, in some aspects,
to methods for producing mature microRNA in sufficient quantities
for therapeutic and research applications.
[0036] The methods for efficient expression of microRNA involve the
use of a precursor microRNA molecule having a microRNA sequence in
the context of microRNA flanking sequences. The precursor microRNA
is composed of any type of nucleic acid based molecule capable of
accommodating the microRNA flanking sequences and the microRNA
sequence. Examples of precursor microRNAs and the individual
components of the precursor (flanking sequences and microRNA
sequence) are provided herein. The invention, however, is not
limited to the examples provided. The invention is based, at least
in part, on the discovery of an important component of precursor
microRNAs, that is, the microRNA flanking sequences. The nucleotide
sequence of the precursor and its components may vary widely.
[0037] In one aspect a precursor microRNA molecule is an isolated
nucleic acid including microRNA flanking sequences and having a
stem-loop structure with a microRNA sequence incorporated therein.
An "isolated molecule" is a molecule that is free of other
substances with which it is ordinarily found in nature or in vivo
systems to an extent practical and appropriate for its intended
use. In particular, the molecular species are sufficiently free
from other biological constituents of host cells or if they are
expressed in host cells they are free of the form or context in
which they are ordinarily found in nature. For instance, a nucleic
acid encoding a precursor microRNA having homologous microRNA
sequences and flanking sequences may ordinarily be found in a to
host cell in the context of the host cell genomic DNA. An isolated
nucleic acid encoding a microRNA precursor may be delivered to a
host cell, but is not found in the same context of the host genomic
DNA as the natural system. Alternatively, an isolated nucleic acid
is removed from the host cell or present in a host cell that does
not ordinarily have such a nucleic acid sequence. Because an
isolated molecular species of the invention may be admixed with a
pharmaceutically-acceptable carrier in a pharmaceutical preparation
or delivered to a host cell, the molecular species may comprise
only a small percentage by weight of the preparation or cell. The
molecular species is nonetheless isolated in that it has been
substantially separated from the substances with which it may be
associated in living systems.
[0038] An "isolated precursor microRNA molecule" is one which is
produced from a vector having a nucleic acid encoding the precursor
microRNA. Thus, the precursor microRNA produced from the vector may
be in a host cell or removed from a host cell. The isolated
precursor microRNA may be found within a host cell that is capable
of expressing the same precursor. It is nonetheless isolated in
that it is produced from a vector and, thus, is present in the cell
in a greater amount than would ordinarily be expressed in such a
cell.
[0039] The term "nucleic acid" is used to mean multiple nucleotides
(i.e. molecules comprising a sugar (e.g. ribose or deoxyribose)
linked to a phosphate group and to an exchangeable organic base,
which is either a substituted pyrimidine (e.g. cytosine (C),
thymidine (T) or uracil (U)) or a substituted purine (e.g. adenine
(A) or guanine (G)). The term shall also include polynucleosides
(i.e. a polynucleotide minus the phosphate) and any other organic
base containing polymer. Purines and pyrimidines include but are
not limited to adenine, cytosine, guanine, thymidine, inosine,
5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine,
2,6-diaminopurine, hypoxanthine, and other naturally and
non-naturally occurring nucleobases, substituted and unsubstituted
aromatic moieties. Other such modifications are well known to those
of skill in the art. Thus, the term nucleic acid also encompasses
nucleic acids with substitutions or modifications, such as in the
bases and/or sugars.
[0040] "MicroRNA flanking sequence" as used herein refers to
nucleotide sequences including microRNA processing elements.
MicroRNA processing elements are the minimal nucleic acid sequences
which contribute to the production of mature microRNA to from
precursor microRNA. Often these elements are located within a 40
nucleotide sequence that flanks a microRNA stem-loop structure. In
some instances the microRNA processing elements are found within a
stretch of nucleotide sequences of between 5 and 4,000 nucleotides
in length that flank a microRNA stem-loop structure.
[0041] Thus, in some embodiments the flanking sequences are 5-4,000
nucleotides in length. As a result, the length of the precursor
molecule may be, in some instances at least about 150 nucleotides
or 270 nucleotides in length. The total length of the precursor
molecule, however, may be greater or less than these values. In
other embodiments the minimal length of the microRNA flanking
sequence is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 and
any integer there between. In other embodiments the maximal length
of the microRNA flanking sequence is 2,000, 2,100, 2,200, 2,300,
2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200,
3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000 and any
integer there between.
[0042] The microRNA flanking sequences may be native microRNA
flanking sequences or artificial microRNA flanking sequences. A
native microRNA flanking sequence is a nucleotide sequence that is
ordinarily associated in naturally existing systems with microRNA
sequences, i.e., these sequences are found within the genomic
sequences surrounding the minimal microRNA hairpin in vivo.
Artificial microRNA flanking sequences are nucleotides sequences
that are not found to be flanking to microRNA sequences in
naturally existing systems. The artificial microRNA flanking
sequences may be flanking sequences found naturally in the context
of other microRNA sequences. Alternatively they may be composed of
minimal microRNA processing elements which are found within
naturally occurring flanking sequences and inserted into other
random nucleic acid sequences that do not naturally occur as
flanking sequences or only partially occur as natural flanking
sequences.
[0043] The microRNA flanking sequences within the precursor
microRNA molecule may flank one or both sides of the stem-loop
structure encompassing the microRNA sequence. Thus, one end (i.e.,
5') of the stem-loop structure may be adjacent to a single flanking
sequence and the other end (i.e., 3') of the stem-loop structure
may not be adjacent to a flanking sequence. Preferred structures
have flanking sequences on both ends of the stem-loop structure.
The flanking sequences may be directly adjacent to one or both ends
of the stem-loop structure or may be connected to the stem-loop
structure to through a linker, additional nucleotides or other
molecules.
[0044] A "stem-loop structure" refers to a nucleic acid having a
secondary structure that includes a region of nucleotides which are
known or predicted to form a double strand (stem portion) that is
linked on one side by a region of predominantly single-stranded
nucleotides (loop portion). The terms "hairpin" and "fold-back"
structures are also used herein to refer to stem-loop structures.
Such structures are well known in the art and the term is used
consistently with its known meaning in the art. The actual primary
sequence of nucleotides within the stem-loop structure is not
critical to the practice of the invention as long as the secondary
structure is present. As is known in the art, the secondary
structure does not require exact base-pairing. Thus, the stem may
include one or more base mismatches. Alternatively, the
base-pairing may be exact, i.e. not include any mismatches.
[0045] In some instances the precursor microRNA molecule may
include more than one stem-loop structure. The multiple stem-loop
structures may be linked to one another through a linker, such as,
for example, a nucleic acid linker or by a microRNA flanking
sequence or other molecule or some combination thereof.
[0046] An example of a precursor microRNA is the following:
##STR00002##
[0047] wherein X.sub.1, and X.sub.2 are nucleotides and wherein
N.sub.1 and N.sub.2 are nucleic acids of 16-28 nucleotides in
length. Such a structure is a single example. As described herein
the actual nucleotide sequence of the precursor molecule can vary
significantly. In general, X.sub.1, and X.sub.2 refers to the
microRNA flanking sequences and N.sub.1 and N.sub.2 refers to the
microRNA sequence and the corresponding sequence that is often
degraded and sometimes referred to as microRNA*.
[0048] N.sub.1 and N.sub.2 have at least partial complementarity.
"Partial complementarity" when used in this context refers to at
least a portion of the nucleic acid sequences that are capable of
base pairing. For instance, in some embodiments two nucleic acid
sequences that have partial complementarity have at least 10
nucleotides that are capable of base pairing. In some instances, at
least 15 nucleotides in each sequence are capable of participating
in a base paring interaction with one another. In other instances,
the two nucleic acids are perfectly complementary, and thus all
nucleotides in each sequence are capable of base pairing with a
corresponding nucleotide in the other nucleic acid sequence.
[0049] A microRNA sequence is incorporated into the stem-loop
structure of the precursor microRNA molecule. As used herein, the
term "microRNA" refers to any type of interfering RNA, including
but not limited to, endogenous microRNA and artificial microRNA.
Endogenous microRNA are small RNAs naturally present in the genome
which are capable of modulating the productive utilization of mRNA.
The term artificial microRNA includes any type of RNA sequence,
other than endogenous microRNA, which is capable of modulating the
productive utilization of mRNA. For instance, it includes sequences
previously identified as siRNA, regardless of the mechanism of
down-stream processing of the RNA (i.e. although siRNAs are
believed to have a specific method of in vivo processing resulting
in the cleavage of mRNA, such sequences can be incorporated into
the vectors in the context of the flanking sequences described
herein). Thus a microRNA sequence is a nucleic acid composed of any
one or more of these sequences. MicroRNA sequences have been
described in publications such as, Lim, et al., Genes &
Development, 17, p. 991-1008 (2003), Lim et al Science 299, 1540
(2003), Lee and Ambrose Science, 294, 862 (2001), Lau et al.,
Science 294, 858-861 (2001), Lagos-Quintana et al, Current Biology,
12, 735-739 (2002), Lagos-Quintana et al, Science 294, 853-857
(2001), and Lagos-Quintana et al, RNA, 9, 175-179 (2003), which are
incorporated by reference. Multiple microRNAs may also be
incorporated into the precursor molecule.
[0050] The term "productive utilization of mRNA" refers to any
change within the cell resulting in less protein accumulating from
the mRNA. For instance a compound that interferes with translation
of mRNA, would be a compound that modulates productive utilization
of mRNA. Similarly, a compound that specifies the cleavage of an
mRNA would be a compound that modulates productive utilization of
that mRNA.
[0051] In some instances, the precursor microRNA includes a
microRNA sequence and a microRNA flanking sequence that are derived
from the same microRNA gene and in to other instances it includes a
microRNA sequence and a microRNA flanking sequence that are not
derived from the same microRNA gene. The term "that is derived from
the same microRNA gene" refers to a nucleic acid sequence that
includes both the microRNA sequence and the microRNA flanking
sequence(s) that is identical to a nucleic acid found in nature.
The term "that is not derived from the same microRNA gene" refers
to a nucleic acid sequence that includes both the microRNA sequence
and the microRNA flanking sequence(s) and that is not identical to
a nucleic acid found in nature. Thus, in some instances, the
precursor microRNA will include a flanking microRNA sequence that
is not ordinarily associated in nature with the microRNA with which
it is associated in the precursor molecule. An artificial microRNA
will always, for instance, not be derived from the same microRNA
gene as the flanking sequence with which it is associated.
Additionally, even if a microRNA sequence and a microRNA flanking
sequence are found within a common gene in nature, a precursor
microRNA molecule according to the invention is said to include a
microRNA sequence and a microRNA flanking sequence that are not
derived from the same gene, if the structure of the precursor is
modified in any way from that which is ordinarily found in nature,
i.e. a nucleotide is changed from a naturally occurring nucleotide
or an additional nucleotide (s) or linker is inserted, etc.
[0052] In some instances the precursor microRNAs described herein
do not include bantam microRNA sequences and flanking sequences. In
other instances bantam microRNA sequences and/or flanking sequences
are included within the compounds and methods of the invention.
[0053] A precursor microRNA molecule may be processed in vivo or in
vitro to produce a mature microRNA. A precursor microRNA molecule
is processed in a host cell by a ribonuclease enzyme or enzymes.
One example of a ribonuclease enzyme which processes precursor
microRNA molecules is the RNase II ribonuclease Dicer.
[0054] A mature microRNA is a functional microRNA which is capable
of modulating or altering the productive utilization of mRNA, i.e.,
regulating the expression of protein-coding genes at the
post-transcriptional level. These methods are described in more
detail below. Mature microRNAs generally have a length of between
16 and 28 nucleotides and more often between 21 and 24
nucleotides.
[0055] One advantage of the methods and products described herein
is the efficient processing of microRNAs. A related advantage is
the accuracy of processing. MicroRNAs are generally processed
asymmetrically in vivo i.e., only the functional strand is
incorporated into a ribonucleoprotein complex-miRNP. This is true
for microRNA as well as siRNAs, which are processed into
RNA-induced protein complex-RISC. This type of processing is
required for the molecule to be functional and stable. The RISC and
miRNP are similar, if not identical. Selective incorporation of the
functional strand of microRNA, artificial microRNA or siRNA into
these protein complexes will increase the efficacy, specificity,
and stability of the small RNAs. The rules for selective
incorporation of the functional strand into RISC or miRNP are not
fully known. But the methods described herein allow selective
incorporation of the functional strand into RISC or miRNP and thus
result in significantly enhanced production of functional microRNA
protein complexes.
[0056] The invention also includes vectors for producing precursor
microRNA molecules. Generally these vectors include a sequence
encoding a precursor microRNA and in vivo expression elements. The
in vivo expression elements include at least one promoter. The
vector or primary transcript is first processed to produce the
stem-loop precursor molecule. The stem-loop precursor is then
processed to produce the mature microRNA.
[0057] One example of a vector useful for expressing the precursor
microRNAs is shown in SEQ ID NO. 1. Thus the invention encompasses
the nucleotide sequence of SEQ ID NO 1 as well as variants
thereof.
[0058] In general, variants typically will share at least 40%
nucleotide identity with SEQ ID NO:1, in some instances, will share
at least 50% nucleotide identity; and in still other instances,
will share at least 60% nucleotide identity. The preferred variants
have at least 70% sequence homology to SEQ ID NO:1. More preferably
the preferred variants have at least 80% and, most preferably, at
least 90% sequence homology to SEQ ID NO:1.
[0059] Variants with high percentage sequence homology can be
identified, for example, using stringent hybridization conditions.
The term "stringent conditions", as used herein, refers to
parameters with which the art is familiar More specifically,
stringent conditions, as used herein, refer to hybridization at
65.degree. C. in hybridization buffer (3.5.times.SSC, 0.02% Ficoll,
0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin, 2.5 mM
NaH.sub.2PO.sub.4 (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodium
chloride/0.15M sodium citrate, pH 7; SDS is sodium dodecyl
sulphate; and EDTA is ethylenediaminetetraacetic acid. After
hybridization, the membrane to which the DNA is transferred is
washed at 2.times.SSC at room temperature and then at
0.1.times.SSC/0.1.times.SDS at 65.degree. C. There are other
conditions, reagents, and so forth which can be used, which result
in a similar degree of stringency.
[0060] The "in vivo expression elements" are any regulatory
nucleotide sequence, such as a promoter sequence or
promoter-enhancer combination, which facilitates the efficient
expression of the nucleic acid to produce the precursor microRNA.
The in vivo expression element may, for example, be a mammalian or
viral promoter, such as a constitutive or inducible promoter or a
tissue specific promoter. Constitutive mammalian promoters include,
but are not limited to, polymerase promoters as well as the
promoters for the following genes: hypoxanthine phosphoribosyl
transferase (HPTR), adenosine deaminase, pyruvate kinase, and
.beta.-actin. Exemplary viral promoters which function
constitutively in eukaryotic cells include, for example, promoters
from the simian virus, papilloma virus, adenovirus, human
immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus,
the long terminal repeats (LTR) of moloney leukemia virus and other
retroviruses, and the thymidine kinase promoter of herpes simplex
virus. Other constitutive promoters are known to those of ordinary
skill in the art. The promoters useful as in vivo expression
element of the invention also include inducible promoters.
Inducible promoters are expressed in the presence of an inducing
agent. For example, the metallothionein promoter is induced to
promote transcription in the presence of certain metal ions. Other
inducible promoters are known to those of ordinary skill in the
art.
[0061] In general, the in vivo expression element shall include, as
necessary, 5' non-transcribing and 5' non-translating sequences
involved with the initiation of transcription. They optionally
include enhancer sequences or upstream activator sequences as
desired.
[0062] Vectors include, but are not limited to, plasmids,
phagemids, viruses, other vehicles derived from viral or bacterial
sources that have been manipulated by the insertion or
incorporation of the nucleic acid sequences for producing the
precursor microRNA, and free nucleic acid fragments which can be
attached to these nucleic acid sequences. Viral and retroviral
vectors are a preferred type of vector and include, but are not
limited to, nucleic acid sequences from the following viruses:
retroviruses, such as: Moloney murine leukemia virus; Murine stem
cell virus, Harvey murine sarcoma virus; murine mammary tumor
virus; Rous sarcoma virus; adenovirus; adeno-associated virus;
SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma
viruses; herpes viruses; vaccinia viruses; polio viruses; and RNA
viruses such as any retrovirus. One can readily employ other
vectors not named but known in the art.
[0063] Viral vectors are generally based on non-cytopathic
eukaryotic viruses in which non-essential genes have been replaced
with the nucleic acid sequence of interest. Non-cytopathic viruses
include retroviruses, the life cycle of which involves reverse
transcription of genomic viral RNA into DNA with subsequent
proviral integration into host cellular DNA. Retroviruses have been
approved for human gene therapy trials. Genetically altered
retroviral expression vectors have general utility for the
high-efficiency transduction of nucleic acids in vivo. Standard
protocols for producing replication-deficient retroviruses
(including the steps of incorporation of exogenous genetic material
into a plasmid, transfection of a packaging cell lined with
plasmid, production of recombinant retroviruses by the packaging
cell line, collection of viral particles from tissue culture media,
and infection of the target cells with viral particles) are
provided in Kriegler, M., "Gene Transfer and Expression, A
Laboratory Manual," W.H. Freeman Co., New York (1990) and Murry, E.
J. Ed. "Methods in Molecular Biology," vol. 7, Humana Press, Inc.,
Cliffton, N.J. (1991).
[0064] The invention also encompasses host cells transfected with
these vectors. Host cells include for instance, cells and cell
lines, e.g. prokaryotic (e.g., E. coli), and eukaryotic (e.g.,
dendritic cells, CHO cells, COS cells, yeast expression systems and
recombinant baculovirus expression in insect cells).
[0065] The precursor microRNA, and subsequently the mature
functional microRNAs are useful for altering accumulation of one or
more target proteins. This may be accomplished by contacting a cell
with a vector capable of expressing a precursor microRNA as
described herein. The vector produces the microRNA transcript,
which is then processed into precursor microRNA in the cell, which
is then processed to produce the mature functional microRNA which
is capable of altering accumulation of the target protein.
Accumulation of the protein may be effected in a number of
different ways. For to instance the microRNA may directly or
indirectly affect translation or may result in cleavage of the mRNA
transcript or even effect stability of the protein being translated
from the target mRNA. MicroRNA may function through a number of
different mechanisms. The methods and products of the invention are
not limited to any one mechanism. The method may be performed in
vitro, e.g., for studying gene function, ex vivo or in vivo, e.g.
for therapeutic purposes.
[0066] An "ex vivo" method as used herein is a method which
involves isolation of a cell from a subject, manipulation of the
cell outside of the body, and reimplantation of the manipulated
cell into the subject. The ex vivo procedure may be used on
autologous or heterologous cells, but is preferably used on
autologous cells. In preferred embodiments, the ex vivo method is
performed on cells that are isolated from bodily fluids such as
peripheral blood or bone marrow, but may be isolated from any
source of cells. When returned to the subject, the manipulated cell
will be programmed for cell death or division, depending on the
treatment to which it was exposed. Ex vivo manipulation of cells
has been described in several references in the art, including
Engleman, E. G., 1997, Cytotechnology, 25:1; Van Schooten, W., et
al., 1997, Molecular Medicine Today, June, 255; Steinman, R. M.,
1996, Experimental Hematology, 24, 849; and Gluckman, J. C., 1997,
Cytokines, Cellular and Molecular Therapy, 3:187. The ex vivo
activation of cells of the invention may be performed by routine ex
vivo manipulation steps known in the art. In vivo methods are also
well known in the art. The invention thus is useful for therapeutic
purposes and also is useful for research purposes such as testing
in animal or in vitro models of medical, physiological or metabolic
pathways or conditions.
[0067] The ex vivo and in vivo methods are performed on a subject.
A "subject" shall mean a human or non-human mammal, including but
not limited to, a dog, cat, horse, cow, pig, sheep, goat, primate,
rat, and mouse.
[0068] In some instances the mature microRNA is expressed at a
level sufficient to cause at least a 2-fold or in some instances a
10 fold reduction in accumulation of the target protein. The level
of accumulation of a target protein may be assessed using routine
methods known to those of skill in the art. For instance, protein
may be isolated from a target cell and quantitated using Western
blot analysis or other comparable methodologies, optionally in
comparison to a control. Protein levels may also be assessed using
reporter systems or fluorescently labeled antibodies. In other
embodiments, the mature microRNA is expressed at a level sufficient
to cause at least a 2, 5, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, or 100 fold reduction in accumulation of the target
protein. The "fold reduction" may be assessed using any parameter
for assessing a quantitative value of protein expression. For
instance, a quantitative value can be determined using a label i.e.
fluorescent, radioactive linked to an antibody. The value is a
relative value that is compared to a control or a known value.
[0069] Different microRNA sequences have different levels of
expression of mature microRNA and thus have different effects on
target mRNA and/or protein expression. For instance, in some cases
a microRNA may be expressed at a high level and may be very
efficient such that the accumulation of the target protein is
completely or near completely blocked. In other instances the
accumulation of the target protein may be only reduced slightly
over the level that would ordinarily be expressed in that cell at
that time under those conditions in the absence of the mature
microRNA. Complete inhibition of the accumulation of the target
protein is not essential for therapeutic purposes. In many cases
partial or low inhibition of accumulation may produce a preferred
phenotype. The actual amount that is useful will depend on the
particular cell type, the stage of differentiation, conditions to
which the cell is exposed, the modulation of other target proteins,
etc.
[0070] The microRNAs may be used to knock down gene expression in
vertebrate cells for gene-function studies, including
target-validation studies during the development of new
pharmaceuticals, as well as the development of human disease models
and therapies, and ultimately, human gene therapies.
[0071] The methods of the invention are useful for treating any
type of "disease", "disorder" or "condition" in which it is
desirable to reduce the expression or accumulation of a particular
target protein(s). Diseases include, for instance, but are not
limited to, cancer, infectious disease, cystic fibrosis, blood
disorders, including leukemia and lymphoma, spinal muscular
dystrophy, early-onset Parkinsonism (Waisman syndrome) and X-linked
mental retardation (MRX3).
[0072] The microRNAs are useful in research and therapeutics
related to hematopoiesis. During hematopoiesis at least eight
distinct lineages of mature blood cells are formed as the
descendents of the multipotential hematopoietic stem cells (HSCs).
Hematopoietic stem cells first arise within the extra-embryonic
yolk sac and later the aortic-gonad-mesonephros (AGM) region of the
developing embryo. Thereafter hematopoiesis normally occurs in the
fetal liver and in adult bone marrow. In the adult certain stresses
induce extramedullary hematopoiesis, especially in the spleen.
Hematopoiesis is sustained for life by self-renewal of the HSCs and
their continuous development into all blood cells types. The
extraordinary ability of HSCs to self-renew and differentiate was
demonstrated by the repopulation of the entire blood system of a
mouse by a single stem cell. Because of their ability to self-renew
and to differentiate into all blood cells, HSCs form the basis of
bone marrow transplantation for treatment of leukemias and other
cancers and several nonmalignant blood cell disorders.
[0073] In order to identify microRNAs that might play roles in
hematopoiesis, microRNAs were cloned from mouse bone marrow, the
primary adult hematopoietic organ in vertebrates. 2180 tiny RNAs
isolated from mouse bone marrow were cloned and sequenced. These
represented about 100 unique microRNA. These hematopoietic
microRNAs are listed in Table 1. Nineteen frequently cloned
microRNAs shown in Table 2 were subjected to further analysis.
These included 15 previously identified microRNAs and 4 newly
identified microRNAs. All but two of these microRNAs (LM3_A01-3 and
miR-191) were perfectly conserved in the human genome.
TABLE-US-00001 TABLE 1 Some of the microRNAs frequently cloned from
mouse bone marrow. Some microRNAs were represented by clones of
different lengths, due to heterogeneity at the microRNA 3'
terminus. The observed size range is indicated, as is the microRNA
sequence of the most abundant length. Seq. Size # of microRNA Id
microRNA sequence range clones Location begin end let-7c LM2_ (SEQ
ID NO: 7) 22-23 12 CONTIG_128829 246 267 B07-1
UGAGGUAGUAGGUUGUAUGGUU let-7g LP1_ (SEQ ID NO: 8) 20-23 11
CONTIG_265484 2018 2038 B02-8 UGAGGUAGUAGUUUGUACAGU miR-15a B1_F
(SEQ ID NO: 9) 21-22 4 CONTIG_87894 853 832 06-2
UAGCAGCACAUAAUGGUUUGUG miR-16 B1_D (SEQ ID NO: 10) 21-25 34
CONTIG_87894 713 693 01-3 UAGCAGCACGUAAAUAUUGGCG miR-19b LM1_ (SEQ
ID NO: 11) 21-23 9 CONTIG_347954 1756 1778 B07-3
UGUGCAAAUCCAUGCAAAACUGA miR-20 LM1_ (SEQ ID NO: 12) 22-24 18
Hs13_10023 1387067 1387089 B03-2 UAAAGUGCUUAUAGUGCAGGUAG miR-23a
LP1_ (SEQ ID NO: 13) 21-23 5 CONTIG_548175 437 458 B05-2
AUCACAUUGCCAGGGAUUUCCA miR-27b B2_E SEQ ID NO: 13) 20-23 7 Hs9_8633
1373759 1373779 07-1 UUCACAGUGGCUAAGUUCUGC miR-29a LM4_ (SEQ ID NO:
15) 22-22 20 Hs7_23805 55795 55774 A03-3 UAGCACCAUCUGAAAUCGGUUA
miR-30b B1_B (SEQ ID NO: 6) 22-23 12 CONTIG_572951 1505 1384 01-2
UGUAAACAUCCUACACUCAGCU miR-30c B1_C (SEQ ID NO: 16) 23-25 13
CONTIG_117246 3389 3412 07-1 UGUAAACAUCCUACACUCUCAGCU miR-104 B1_D
(SEQ ID NO: 17) 21-24 37 CONTIG_168444 1754 1732 01-7
UAGCUUAUCAGACUGAUGUUGAC miR-132s B1_G (SEQ ID NO: 5) 22-23 16
Hs17_10808 2182613 2182634 04-1 CCCAUAAAGUAGAAAGCACUAC mir-191 LM1_
(SEQ ID NO: 18) 20-24 32 CONTIG_261531 3175 3196 E02-3
CAACGGAAUCCCAAAAGCAGCU miR-223 B1_E (SEQ ID NO: 3) 20-24 65
CONTIG_202715 440 418 08-6 UGUCAGUUUGUCAAAUACCCCAA new B1_G (SEQ ID
NO: 19) 22-23 7 Hs8_8395 132054 132033 08-2 UCCUGUACUGAGCUGCCCCGAG
new LP1_ (SEQ ID NO: 20) 21-22 7 CONTIG_7440 8193 8213 B02-3
UUAUAAAGCAAUGAGACUGAU new LM3_ (SEQ ID NO: 21) 22-24 10
CONTIG_195284 13756 13734 A01-3 UGAGGUAUUAGUUUGUGCUGUUA new LM4_
(SEQ ID NO: 22) 17-23 8 Hs16_19764 637009 636987 D05-3
UACCACAGGGUAGAACCACGGAC
[0074] To identify microRNAs expressed at sites of hematopoiesis,
we probed northern blots of RNA isolated from different mouse
tissues, including brain, heart, lung, liver, kidney, muscle, fetal
liver, bone marrow, spleen, and thymus. Among these tissues, bone
marrow, spleen, and thymus represent three major adult
hematopoiesis sites. These organs play different roles in adult
hematopoiesis and comprise significantly different cell types. Bone
marrow, the primary hematopoietic organ in adult vertebrates,
provides other secondary hematopoietic organs with committed
progenitor cells. It consists of hematopoietic stem cells and
myeloid, erythroid and lymphoid cells at a variety of to
differentiation stages, although most bone marrow cells belong to
myeloid and erythroid lineages. Thymus, the primary lymphoid organ,
constitutes mainly T-lymphocytes. Spleen, the secondary lymphoid
organ, mainly comprises differentiated reticulocytes and T and B
lymphocytes. Fetal liver is the embryonic hematopoiesis site. Thus,
analysis of microRNA expression in these four tissues reveals not
only the hematopoietic-specific microRNAs but also can provide
guidance as to their differential roles during hematopoietic
development. For 17 of the 19 microRNAs, expression was detected on
the Northerns, and each of these had tissue-specific expression
patterns (Table 2). For 12 of these microRNAs, expression was
readily detected in hematopoietic tissues.
TABLE-US-00002 TABLE 3 Summary of tissue and developmental
expression of microRNAs cloned from mouse bone marrow. Northern
blots of total RNA from a variety of mouse tissues, with a focus on
hematopoietic tissues of different functions and developmental
stages, were probed for the indicated microRNA. Signal intensities
were ranked (-, negative; +, positive; +++++, most positive) after
normalization to the U6 signal to compensate for differential
sample loading. Fetal Bone microRNA Seq. id Brain Heart Lung Liver
Kidney Muscle liver marrow Spleen Thymus miR-15a B1_F06-2 - - +++ -
++ - - + + ++ miR-16 B1_D01-3 + + +++ + ++ + + +++ +++ +++ miR-20
LM1_B03-2 - - + - - - + +++ ++ ++++ miR-132s B1_G04-1 - - + - - - +
+++ +++ +++ miR-223 B1_E08-6 - - + - - - - ++++ - - let-7c
LM2_B07-1 +++ ++ ++++ - ++ + - + + + let-7g LP1_B02-8 + + +++ ++ ++
+ - + + + miR-19b LM1_B07-3 - - + - - - - - - - miR-23a LP1_B05-2 -
+ +++ - + + - - ++ - miR-27b B2_E07-1 - - ++++ - + - - - - -
miR-29a LM4_A03-3 +++++ +++ ++++ +++ +++ ++ - + ++++ ++ miR-30b
B1_B01-2 - - + - + - - - - - miR-30c B1_C07-1 - - + - + - - - - -
miR-104 B1_D01-7 - - ++++ +++ ++ + - + ++ + miR-191 LM1_E02-3 - + -
- - - - - - - new LP1_B02-3 - - + + - - - + + - new B1_G08-2 + + +
-- -- -- - -- + -- new LM3_A01-3 - - - - - - - - - - new LM4_D05-3
- - - - - - - - - -
[0075] The following five microRNAs, mir-15a, miR-16, miR-20,
miR-132s and miR-223, exhibited high and often preferential
accumulation in hematopoietic tissues and were found to be
regulated by cytokines.
[0076] miR-132s, the microRNA found at a breakpoint of a t(8:17)
translocation associated with an aggressive B-cell leukemia, is
primarily expressed in hematopoietic tissues (with the lung being
the only other tissue with appreciable expression). Significant
expression of this microRNA was seen already in E13 fetal liver.
The expression of mature miR-132s in bone marrow and spleen was
comparable and about 2-fold higher than that in thymus.
Interestingly, accumulation of the presumed miR-132 precursor was
high, and the ratio of mature and precursor RNAs varied in
different tissues, suggesting regulation at the level of Dicer
processing.
[0077] miR-20 was expressed in a similar pattern as miR-132s,
though there was substantially higher accumulation of this microRNA
in the thymus compared to the spleen.
[0078] miR-223 had the most striking tissue specificity of any of
the microRNA examined. It was very strongly expressed in bone
marrow, and was detectable in spleen but essentially negative in
thymus, E13 fetal liver and all other mouse tissues, except the
lung.
[0079] miR-16 and miR-15a are the two microRNAs that derive from
band 13q13.3 of the human chromosome 13, the site of the most
common structural aberrations in both mantle cell lymphoma and
B-cell chronic lymphocytic leukemia. Their loci are within 130 bp
of each other, suggesting that they might be transcribed and
processed from a single primary transcript. Consistent with this
idea, they have similar expression patterns, except that miR-16
appears to accumulate to much higher levels. Expression is high in
the adult hematopoietic tissues, although these microRNAs,
particularly miR-16, can be readily detected in other tissues.
[0080] Two of these microRNAs, mir-15a and miR-16, are within band
13q13--a region of human chromosome 13 that is thought to harbor a
lymphoid regulatory locus because it is the site of the most common
structural aberrations in both mantle cell lymphoma and B-cell
chronic lymphocytic leukemia. Another one of the hematopoietic
microRNA genes, mir-132, maps to the breakpoint junction of a
t(8;17) translocation that has been linked to an aggressive B cell
leukemia. In this translocation, a truncated MYC gene is fused to
the promoter and 5' portion of the mir-132 gene. This expression
data, together with chromosomal aberrations associated with human
leukemias, implicate microRNAs in the developmental decisions of
hematopoiesis. Thus, the microRNAs are useful in therapeutic
protocols related to hematopoeitic disorders including leukemias
and lymphomas.
[0081] Cancers include but are not limited to biliary tract cancer;
bladder cancer; breast cancer; brain cancer including glioblastomas
and medulloblastomas; cervical cancer; choriocarcinoma; colon
cancer including colorectal carcinomas; endometrial cancer;
esophageal cancer; gastric cancer; head and neck cancer;
hematological neoplasms including acute lymphocytic and myelogenous
leukemia, multiple myeloma, AIDS-associated leukemias and adult
T-cell leukemia lymphoma; intraepithelial neoplasms including
Bowen's disease and Paget's disease; liver cancer; lung cancer
including small cell lung cancer and non-small cell lung cancer;
lymphomas including Hodgkin's disease and lymphocytic lymphomas;
neuroblastomas; oral cancer including squamous cell carcinoma;
osteosarcomas; ovarian cancer including those arising from
epithelial cells, stromal cells, germ cells and mesenchymal cells;
pancreatic cancer; prostate cancer; rectal cancer; sarcomas
including leiomyosarcoma, rhabdomyosarcoma, liposarcoma,
fibrosarcoma, synovial sarcoma and osteosarcoma; skin cancer
including melanomas, Kaposi's sarcoma, basocellular cancer, and
squamous cell cancer; testicular cancer including germinal tumors
such as seminoma, non-seminoma (teratomas, choriocarcinomas),
stromal tumors, and germ cell tumors; thyroid cancer including
thyroid adenocarcinoma and medullar carcinoma; transitional cancer
and renal cancer including adenocarcinoma and Wilms tumor.
[0082] An infectious disease, as used herein, is a disease arising
from the presence of a foreign microorganism in the body. A
microbial antigen, as used herein, is an antigen of a
microorganism. Microorganisms include but are not limited to,
infectious virus, infectious bacteria, and infectious fungi.
[0083] Examples of infectious virus include but are not limited to:
Retroviridae (e.g. human immunodeficiency viruses, such as HIV-1
(also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and
other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses,
hepatitis A virus; enteroviruses, human Coxsackie viruses,
rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause
gastroenteritis); Togaviridae (e.g. equine encephalitis viruses,
rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis
viruses, yellow fever viruses); Coronoviridae (e.g. coronaviruses);
Rhabdoviradae (e.g. vesicular stomatitis viruses, rabies viruses);
Coronaviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicular
stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola
viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus,
measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.
influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga
viruses, phleboviruses and Nairo viruses); Arena viridae
(hemorrhagic fever viruses); Reoviridae (e.g. reoviruses,
orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae
(Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae
(papilloma viruses, polyoma viruses); Adenoviridae (most
adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2,
varicella zoster virus, cytomegalovirus (CMV), herpes virus;
Poxviridae (variola viruses, vaccinia viruses, pox viruses); and
Iridoviridae (e.g. African swine fever virus); and unclassified
viruses (e.g. the etiological agents of Spongiform
encephalopathies, the agent of delta hepatitis (thought to be a
defective satellite of hepatitis B virus), the agents of non-A,
non-B hepatitis (class 1=internally transmitted; class
2=parenterally transmitted (i.e. Hepatitis C); Norwalk and related
viruses, and astroviruses).
[0084] Examples of infectious bacteria include but are not limited
to: Helicobacter pyloris, Borelia burgdorferi, Legionella
pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M.
intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus,
Neisseria gonorrhoeae, Neisseria meningitidis, Listeria
monocytogenes, Streptococcus pyogenes (Group A Streptococcus),
Streptococcus agalactiae (Group B Streptococcus), Streptococcus
(viridans group), Streptococcus faecalis, Streptococcus bovis,
Streptococcus (anaerobic sps.), Streptococcus pneumoniae,
pathogenic Campylobacter sp., Enterococcus sp., Haemophilus
influenzae, Bacillus antracis, corynebacterium diphtheriae,
corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium
perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella
pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium
nucleatum, Streptobacillus moniliformis, Treponema pallidium,
Treponema pertenue, Leptospira, Rickettsia, and Actinomyces
israelli.
[0085] Examples of infectious fungi include: Cryptococcus
neoformans, Histoplasma capsulatum, Coccidioides immitis,
Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.
Other infectious organisms (i.e., protists) include: Plasmodium
such as Plasmodium falciparum, Plasmodium malariae, Plasmodium
ovale, and Plasmodium vivax and Toxoplasma gondii.
[0086] In one aspect, the invention provides a method of
administering any of the compositions described herein to a
subject. When administered, the compositions are applied in a
therapeutically effective, pharmaceutically acceptable amount as a
pharmaceutically acceptable formulation. As used herein, the term
"pharmaceutically acceptable" is given its ordinary meaning.
Pharmaceutically acceptable compounds are generally compatible with
other materials of the formulation and are not generally
deleterious to the subject. Any of the compositions of the present
invention may be to administered to the subject in a
therapeutically effective dose. A "therapeutically effective" or an
"effective" as used herein means that amount necessary to delay the
onset of, inhibit the progression of, halt altogether the onset or
progression of, diagnose a particular condition being treated, or
otherwise achieve a medically desirable result, i.e., that amount
which is capable of at least partially preventing, reversing,
reducing, decreasing, ameliorating, or otherwise suppressing the
particular condition being treated. A therapeutically effective
amount can be determined on an individual basis and will be based,
at least in part, on consideration of the species of mammal, the
mammal's age, sex, size, and health; the compound and/or
composition used, the type of delivery system used; the time of
administration relative to the severity of the disease; and whether
a single, multiple, or controlled-release dose regiment is
employed. A therapeutically effective amount can be determined by
one of ordinary skill in the art employing such factors and using
no more than routine experimentation.
[0087] The terms "treat," "treated," "treating," and the like, when
used herein, refer to administration of the systems and methods of
the invention to a subject, which may, for example, increase the
resistance of the subject to development or further development of
cancers, to administration of the composition in order to eliminate
or at least control a cancer or a infectious disease, and/or to
reduce the severity of the cancer or infectious disease. When
administered to a subject, effective amounts will depend on the
particular condition being treated and the desired outcome. A
therapeutically effective dose may be determined by those of
ordinary skill in the art, for instance, employing factors such as
those further described below and using no more than routine
experimentation.
[0088] In administering the systems and methods of the invention to
a subject, dosing amounts, dosing schedules, routes of
administration, and the like may be selected so as to affect known
activities of these systems and methods. Dosage may be adjusted
appropriately to achieve desired drug levels, local or systemic,
depending upon the mode of administration. The doses may be given
in one or several administrations per day. As one example, if daily
doses are required, daily doses may be from about 0.01 mg/kg/day to
about 1000 mg/kg/day, and in some embodiments, from about 0.1 to
about 100 mg/kg/day or from about 1 mg/kg/day to about 10
mg/kg/day. Parental administration, in some cases, may be from one
to several orders of magnitude lower dose per day, as compared to
oral doses. For example, the dosage of an active compound when
parentally administered may be between about 0.1 micrograms/kg/day
to about 10 mg/kg/day, and in some embodiments, from about 1
microgram/kg/day to about 1 mg/kg/day or from about 0.01 mg/kg/day
to about 0.1 mg/kg/day.
[0089] In some embodiments, the concentration of the active
compound(s), if administered systemically, is at a dose of about
1.0 mg to about 2000 mg for an adult of 70 kg body weight, per day.
In other embodiments, the dose is about 10 mg to about 1000 mg/70
kg/day. In yet other embodiments, the dose is about 100 mg to about
500 mg/70 kg/day. Preferably, the concentration, if applied
topically, is about 0.1 mg to about 500 mg/gm of ointment or other
base, more preferably about 1.0 mg to about 100 mg/gm of base, and
most preferably, about 30 mg to about 70 mg/gm of base. The
specific concentration partially depends upon the particular
composition used, as some are more effective than others. The
dosage concentration of the composition actually administered is
dependent at least in part upon the particular physiological
response being treated, the final concentration of composition that
is desired at the site of action, the method of administration, the
efficacy of the particular composition, the longevity of the
particular composition, and the timing of administration relative
to the severity of the disease. Preferably, the dosage form is such
that it does not substantially deleteriously effect the mammal. The
dosage can be determined by one of ordinary skill in the art
employing such factors and using no more than routine
experimentation.
[0090] In the event that the response of a particular subject is
insufficient at such doses, even higher doses (or effectively
higher doses by a different, more localized delivery route) may be
employed to the extent that subject tolerance permits. Multiple
doses per day are also contemplated in some cases to achieve
appropriate systemic levels within the subject or within the active
site of the subject. In some cases, dosing amounts, dosing
schedules, routes of administration, and the like may be selected
as described herein, whereby therapeutically effective levels for
the treatment of cancer are provided.
[0091] In certain embodiments where cancers are being treated, a
composition of the invention may be administered to a subject who
has a family history of cancer, or to a subject who has a genetic
predisposition for cancer. In other embodiments, the composition is
administered to a subject who has reached a particular age, or to a
subject more likely to get cancer. In yet other embodiments, the
compositions is administered to to subjects who exhibit symptoms of
cancer (e.g., early or advanced). In still other embodiments, the
composition may be administered to a subject as a preventive
measure. In some embodiments, the inventive composition may be
administered to a subject based on demographics or epidemiological
studies, or to a subject in a particular field or career.
[0092] Administration of a composition of the invention to a
subject may be accomplished by any medically acceptable method
which allows the composition to reach its target. The particular
mode selected will depend of course, upon factors such as those
previously described, for example, the particular composition, the
severity of the state of the subject being treated, the dosage
required for therapeutic efficacy, etc. As used herein, a
"medically acceptable" mode of treatment is a mode able to produce
effective levels of the active compound(s) of the composition
within the subject without causing clinically unacceptable adverse
effects.
[0093] Any medically acceptable method may be used to administer a
composition to the subject. The administration may be localized
(i.e., to a particular region, physiological system, tissue, organ,
or cell type) or systemic, depending on the condition being
treated. For example, the composition may be administered orally,
vaginally, rectally, buccally, pulmonary, topically, nasally,
transdermally, through parenteral injection or implantation, via
surgical administration, or any other method of administration
where suitable access to a target is achieved. Examples of
parenteral modalities that can be used with the invention include
intravenous, intradermal, subcutaneous, intracavity, intramuscular,
intraperitoneal, epidural, or intrathecal. Examples of implantation
modalities include any implantable or injectable drug delivery
system. Oral administration may be preferred in some embodiments
because of the convenience to the subject as well as the dosing
schedule. Compositions suitable for oral administration may be
presented as discrete units such as hard or soft capsules, pills,
cachettes, tablets, troches, or lozenges, each containing a
predetermined amount of the active compound. Other oral
compositions suitable for use with the invention include solutions
or suspensions in aqueous or non-aqueous liquids such as a syrup,
an elixir, or an emulsion. In another set of embodiments, the
composition may be used to fortify a food or a beverage.
[0094] Injections can be e.g., intravenous, intradermal,
subcutaneous, intramuscular, or interperitoneal. The composition
can be injected interdermally for treatment or to prevention of
infectious disease, for example. In some embodiments, the
injections can be given at multiple locations. Implantation
includes inserting implantable drug delivery systems, e.g.,
microspheres, hydrogels, polymeric reservoirs, cholesterol
matrixes, polymeric systems, e.g., matrix erosion and/or diffusion
systems and non-polymeric systems, e.g., compressed, fused, or
partially-fused pellets. Inhalation includes administering the
composition with an aerosol in an inhaler, either alone or attached
to a carrier that can be absorbed. For systemic administration, it
may be preferred that the composition is encapsulated in
liposomes.
[0095] In general, the compositions of the invention may be
delivered using a bioerodible implant by way of diffusion, or more
preferably, by degradation of the polymeric matrix. Exemplary
synthetic polymers which can be used to form the biodegradable
delivery system include: polyamides, polycarbonates, polyalkylenes,
polyalkylene glycols, polyalkylene oxides, polyalkylene
terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl
esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides,
polysiloxanes, polyurethanes and co-polymers thereof, alkyl
cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose
esters, nitro celluloses, polymers of acrylic and methacrylic
esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,
hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose,
cellulose acetate, cellulose propionate, cellulose acetate
butyrate, cellulose acetate phthalate, carboxylethyl cellulose,
cellulose triacetate, cellulose sulphate sodium salt, poly(methyl
methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),
poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene, poly(ethylene glycol), poly(ethylene oxide),
poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl
acetate, poly vinyl chloride, polystyrene, polyvinylpyrrolidone,
and polymers of lactic acid and glycolic acid, polyanhydrides,
poly(ortho)esters, poly(butic acid), poly(valeric acid), and
poly(lactide-cocaprolactone), and natural polymers such as alginate
and other polysaccharides including dextran and cellulose,
collagen, chemical derivatives thereof (substitutions, additions of
chemical groups, for example, alkyl, alkylene, hydroxylations,
oxidations, and other modifications routinely made by those skilled
in the art), albumin and other hydrophilic proteins, zein and other
prolamines and to hydrophobic proteins, copolymers and mixtures
thereof. In general, these materials degrade either by enzymatic
hydrolysis or exposure to water in vivo, by surface or bulk
erosion. Examples of non-biodegradable polymers include ethylene
vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and
mixtures thereof.
[0096] Bioadhesive polymers of particular interest include
bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and
J. A. Hubell in Macromolecules, (1993) 26:581-587, the teachings of
which are incorporated herein, polyhyaluronic acids, casein,
gelatin, glutin, polyanhydrides, polyacrylic acid, alginate,
chitosan, poly(methyl methacrylates), poly(ethyl methacrylates),
poly(butylmethacrylate), poly(isobutyl methacrylate),
poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl
methacrylate), poly(phenyl methacrylate), poly(methyl acrylate),
poly(isopropyl acrylate), poly(isobutyl acrylate), and
poly(octadecyl acrylate).
[0097] In certain embodiments of the invention, the administration
of the composition of the invention may be designed so as to result
in sequential exposures to the composition over a certain time
period, for example, hours, days, weeks, months or years. This may
be accomplished, for example, by repeated administrations of a
composition of the invention by one of the methods described above,
or by a sustained or controlled release delivery system in which
the composition is delivered over a prolonged period without
repeated administrations. Administration of the composition using
such a delivery system may be, for example, by oral dosage forms,
bolus injections, transdermal patches or subcutaneous implants.
Maintaining a substantially constant concentration of the
composition may be preferred in some cases.
[0098] Other delivery systems suitable for use with the present
invention include time-release, delayed release, sustained release,
or controlled release delivery systems. Such systems may avoid
repeated administrations in many cases, increasing convenience to
the subject and the physician. Many types of release delivery
systems are available and known to those of ordinary skill in the
art. They include, for example, polymer-based systems such as
polylactic and/or polyglycolic acids, polyanhydrides,
polycaprolactones, copolyoxalates, polyesteramides,
polyorthoesters, polyhydroxybutyric acid, and/or combinations of
these. Microcapsules of the foregoing polymers containing drugs are
described in, for example, U.S. Pat. No. 5,075,109. Other examples
include nonpolymer systems that are lipid-based including sterols
such as cholesterol, cholesterol esters, and fatty acids or neutral
fats such as mono-, di- and triglycerides; hydrogel release
systems; liposome-based systems; phospholipid based-systems;
silastic systems; peptide based systems; wax coatings; compressed
tablets using conventional binders and excipients; or partially
fused implants. Specific examples include, but are not limited to,
erosional systems in which the composition is contained in a form
within a matrix (for example, as described in U.S. Pat. Nos.
4,452,775, 4,675,189, 5,736,152, 4,667,013, 4,748,034 and
5,239,660), or diffusional systems in which an active component
controls the release rate (for example, as described in U.S. Pat.
Nos. 3,832,253, 3,854,480, 5,133,974 and 5,407,686). The
formulation may be as, for example, microspheres, hydrogels,
polymeric reservoirs, cholesterol matrices, or polymeric systems.
In some embodiments, the system may allow sustained or controlled
release of the composition to occur, for example, through control
of the diffusion or erosion/degradation rate of the formulation
containing the composition. In addition, a pump-based hardware
delivery system may be used to deliver one or more embodiments of
the invention.
[0099] Examples of systems in which release occurs in bursts
includes, e.g., systems in which the composition is entrapped in
liposomes which are encapsulated in a polymer matrix, the liposomes
being sensitive to specific stimuli, e.g., temperature, pH, light
or a degrading enzyme and systems in which the composition is
encapsulated by an ionically-coated microcapsule with a
microcapsule core degrading enzyme. Examples of systems in which
release of the inhibitor is gradual and continuous include, e.g.,
erosional systems in which the composition is contained in a form
within a matrix and effusional systems in which the composition
permeates at a controlled rate, e.g., through a polymer. Such
sustained release systems can be e.g., in the form of pellets, or
capsules.
[0100] Use of a long-term release implant may be particularly
suitable in some embodiments of the invention. "Long-term release,"
as used herein, means that the implant containing the composition
is constructed and arranged to deliver therapeutically effective
levels of the composition for at least 30 or 45 days, and
preferably at least 60 or 90 days, or even longer in some cases.
Long-term release implants are well known to those of ordinary
skill in the art, and include some of the release systems described
above.
[0101] In some embodiments, the compositions of the invention may
include pharmaceutically acceptable carriers with formulation
ingredients such as salts, carriers, buffering agents, emulsifiers,
diluents, excipients, chelating agents, fillers, drying agents,
antioxidants, antimicrobials, preservatives, binding agents,
bulking agents, silicas, solubilizers, or stabilizers that may be
used with the active compound. For example, if the formulation is a
liquid, the carrier may be a solvent, partial solvent, or
non-solvent, and may be aqueous or organically based. Examples of
suitable formulation ingredients include diluents such as calcium
carbonate, sodium carbonate, lactose, kaolin, calcium phosphate, or
sodium phosphate; granulating and disintegrating agents such as
corn starch or algenic acid; binding agents such as starch, gelatin
or acacia; lubricating agents such as magnesium stearate, stearic
acid, or talc; time-delay materials such as glycerol monostearate
or glycerol distearate; suspending agents such as sodium
carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone; dispersing or wetting agents such as lecithin
or other naturally-occurring phosphatides; thickening agents such
as cetyl alcohol or beeswax; buffering agents such as acetic acid
and salts thereof, citric acid and salts thereof, boric acid and
salts thereof, or phosphoric acid and salts thereof; or
preservatives such as benzalkonium chloride, chlorobutanol,
parabens, or thimerosal. Suitable carrier concentrations can be
determined by those of ordinary skill in the art, using no more
than routine experimentation. The compositions of the invention may
be formulated into preparations in solid, semi-solid, liquid or
gaseous forms such as tablets, capsules, elixirs, powders,
granules, ointments, solutions, depositories, inhalants or
injectables. Those of ordinary skill in the art will know of other
suitable formulation ingredients, or will be able to ascertain
such, using only routine experimentation.
[0102] Preparations include sterile aqueous or nonaqueous
solutions, suspensions and emulsions, which can be isotonic with
the blood of the subject in certain embodiments. Examples of
nonaqueous solvents are polypropylene glycol, polyethylene glycol,
vegetable oil such as olive oil, sesame oil, coconut oil, arachis
oil, peanut oil, mineral oil, injectable organic esters such as
ethyl oleate, or fixed oils including synthetic mono or
di-glycerides. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media. Parenteral vehicles include sodium to chloride solution,
1,3-butandiol, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's or fixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers (such as those
based on Ringer's dextrose), and the like. Preservatives and other
additives may also be present such as, for example, antimicrobials,
antioxidants, chelating agents and inert gases and the like. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono- or di-glycerides. In
addition, fatty acids such as oleic acid may be used in the
preparation of injectables. Carrier formulation suitable for oral,
subcutaneous, intravenous, intramuscular, etc. administrations can
be found in Remington's Pharmaceutical Sciences, Mack Publishing
Co., Easton, Pa. Those of skill in the art can readily determine
the various parameters for preparing and formulating the
compositions of the invention without resort to undue
experimentation.
[0103] In some embodiments, the present invention includes the step
of forming a composition of the invention by bringing an active
compound into association or contact with a suitable carrier, which
may constitute one or more accessory ingredients. The final
compositions may be prepared by any suitable technique, for
example, by uniformly and intimately bringing the composition into
association with a liquid carrier, a finely divided solid carrier
or both, optionally with one or more formulation ingredients as
previously described, and then, if necessary, shaping the
product.
[0104] In some embodiments, the compositions of the present
invention may be present as pharmaceutically acceptable salts. The
term "pharmaceutically acceptable salts" includes salts of the
composition, prepared in combination with, for example, acids or
bases, depending on the particular compounds found within the
composition and the treatment modality desired. Pharmaceutically
acceptable salts can be prepared as alkaline metal salts, such as
lithium, sodium, or potassium salts; or as alkaline earth salts,
such as beryllium, magnesium or calcium salts. Examples of suitable
bases that may be used to form salts include ammonium, or mineral
bases such as sodium hydroxide, lithium hydroxide, potassium
hydroxide, calcium hydroxide, magnesium hydroxide, and the like.
Examples of suitable acids that may be used to form salts include
inorganic or mineral acids such as hydrochloric, hydrobromic,
hydroiodic, hydrofluoric, nitric, carbonic, monohydrogencarbonic,
phosphoric, monohydrogenphosphoric, to dihydrogenphosphoric,
sulfuric, monohydrogensulfuric, phosphorous acids and the like.
Other suitable acids include organic acids, for example, acetic,
propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic,
fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic,
citric, tartaric, methanesulfonic, glucuronic, galacturonic,
salicylic, formic, naphthalene-2-sulfonic, and the like. Still
other suitable acids include amino acids such as arginate,
aspartate, glutamate, and the like.
[0105] The invention also includes methods for quantitating a level
of precursor microRNA expression. The method involves incorporating
a precursor microRNA into a reporter system, transfecting a host
cell with the reporter system, and detecting expression of a
reporter gene product to quantitate the level of precursor microRNA
expression. In some embodiments the reporter system includes a
firefly luciferase reporter gene.
[0106] The present invention is further illustrated by the
following Examples, which in no way should be construed as further
limiting. The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by
reference.
EXAMPLES
Methods
[0107] Retroviral Constructs for microRNA Expression.
[0108] We have developed a retroviral vector using the murine stem
cell virus (Clonetech) backbone (FIG. 1a). A pol III expression
cassette, which consists of the human H1 promoter (P.sub.H1), the
microRNA hairpin and a polyT termination sequence (T5), was placed
after the viral 5'LTR resulting in a single copy (SC) of the
microRNA, or in the viral 3'LTR whereby viral replication resulted
in a double copy (DC) of the microRNA gene (FIG. 1a).
TABLE-US-00003 Table of miRNA sequences miR-223 B1_E08-6
UGUCAGUUUGUCAAAUACCCCAA (SEQ ID NO: 3) miR-181 LM3_A05-1
AACAUUCAACGCUGUCGGUGA (SEQ ID NO: 4) miR-132s B1_G04-1
CCCAUAAAGUAGAAAGCACUAC (SEQ ID NO: 5) miR-30 B1_B01-2
UGUAAACAUCCUACACUCAGCU (SEQ ID NO: 6)
Construction of Precursor microRNA with Flanking Sequences.
[0109] We amplified miR-223 gene segments from mouse genomic DNA
with lengths indicated. Each fragment was designed to give rise to
a transcript containing the miR-223 minimal hairpin (67 nt) and
either 0-, 10-, 20-, 40-, 60-, 80-nucleotide flanking sequences on
both sides of the hairpin. microRNA constructs miR-223, miR-181 and
miR-132, each designed to to produce transcripts of 271-273
nucleotides in length, were also made. These constructs were each
cloned into the H1 expression cassette of the double-copy
retroviral construct (FIG. 1a). The constructs were transiently
transfected into 293T cells and expressed.
Ectopic Expression of microRNAs.
[0110] MicroRNAs were transiently expressed by transfecting
double-copy miRNA constructs into a mammalian cell line, i.e. 293T
cells. Or, double-copy miRNA constructs were transfected into a
retroviral packaging cell line to generate retrovirus, and miRNAs
were stably expressed by tranducing miRNA virus into mammalian
cells, such as, NIH3T3 or hematopoietic stem/progenitor cells.
Expression of candidate microRNA loci was examined using Northern
blots and radiolabeled DNA probes. To maintain hybridization
specificity without varying hybridization or washing conditions,
the length of probes for different sequences was adjusted so that
the predicted melting temperatures of the microRNA-probe duplexes
did not exceed 60.degree. C. Probes not corresponding to the entire
microRNA sequence were designed to hybridize to the 3' region of
the microRNA, which is most divergent among related microRNA
sequences. The radiolabeled probes against the miRNA, or the miRNA*
(the small RNA processed from the opposite arm of the miRNA
stem-loop structure) were used to detect corresponding mature RNAs
and unprocessed precursors. Ethidium staining of 5S RNA served as a
loading control.
Luciferase Reporter System for Detection of miRNA-Mediated
Translational Repression.
[0111] The firefly luciferase gene (Promega) was fused to a mutated
C. elegans lin-41 3'UTR (lin-41 3'UTR-delta, Genebank accession no,
AF195610) in which an 80-nt segment containing both let-7
complementary sites (green) was deleted, and replaced with a
miR-223 perfect complementary site (FIG. 5a). In a control
reporter, the Renilla luciferase gene (Promega) was fused to lin-41
3'UTR-delta. All luciferase genes were under the to control of the
viral LTR promotor. To detect translational repression of the
luciferase gene, virus was produced by transfecting the constructs
into the BOSC23 viral packaging cell line (Pear et al., 1993) and
titred by infecting NIH3T3 cells, analyzing CD4 expression using
FACS. NIH3T3 cells stably expressing miR-223 or other miRNAs were
infected with equal titer of fLuc-lin41UTRdelta+miR-223 target or
fLuc-lin41UTRdelta-miR-223 target virus along with control virus
rLuc-lin41UTRdelta. Four days after infection, luciferase activity
was measured using the luciferase assay (Promega). Firefly
luciferase activity was normalized using Renilla luciferase
activity.
Construction and Expression of Artificial microRNA/SiRNA
[0112] Artificial microRNA (A_miRNAs or siRNAs) were designed to
target 21-nucleotide unique sequences in the firefly luciferase
gene. These constructs were cloned into the B1_C02-3 microRNA
template in a retroviral construct as indicated in FIG. 7.
Expression of siRNAs was first tested in 293T cells by transient
transfection. Radiolabeled probes against SiLuc311 or SiLuc1276,
and their complement strands, SiLuc311* or SiLuc1276* respectively,
were used to detect corresponding mature A_miRNAs/siRNAs and
unprocessed precursors on Northern blots. Ethidium staining of 5S
RNA served as a loading control. Stable cell lines expressing
individual A_miRNA/siRNAs were generated by viral infection and
FACS enrichment of cells expressing green fluorescent protein
(GFP), a reporter for viral infection (Liu et al., 2000).
A_miRNA/siRNAs expressing cell lines were then infected with
firefly Luciferase virus along with control Renilla Luciferase
virus. Four days after infection, luciferase activity was measured
using the luciferase assay. Firefly luciferase activity was
normalized using Renilla luciferase activity.
Inducible Expression of B1_C02-3 microRNA from a Polymerase II
Promotor.
[0113] A 500 base pair B1_C02-3 microRNA gene, with predicted
B1_C02-3 microRNA sequence in the center, was amplified from mouse
genomic DNA. This was cloned into the TetOn inducible expression
vector (Clontech). A tetracycline-inducible cell line was generated
using this vector, and induced at various doxycycline
concentrations. A radiolabeled probe against B1_C02-3 was used to
detect the mature microRNA and unprocessed precursor, using
Northern blot analysis. Ethidium staining of 5S RNA served as a
loading control.
Expression of microRNAs in Developmental Hematopoietic Organs.
[0114] To determine whether microRNAs are components of the
molecular machinery that regulate mouse hematopoiesis, we cloned
over 100 microRNAs from mouse bone marrow and uncovered three
microRNAs: miR-181, miR-223 and miR-132, that are differentially or
preferentially expressed in hematopoietic tissues and are thus
considered hematopoietic-specific.
Expression of microRNAs in During Hematopoietic Lineage
Commitment.
[0115] To test whether microRNAs can regulate hematopoietic lineage
differentiation, the effect of microRNA expression in bone marrow
progenitor cells was examined. Lineage-negative bone marrow cells
were isolated and infected with control retroviral construct
(vector), or retroviral constructs expressing hematopoietic
microRNAs, miR-30, miR-132s, or miR-181 and miR-223. All constructs
contained a GFP reporter to indicate virally infected cells.
Infected hematopoietic progenitor cells were seeded onto S 17
stromal cells, and cultured in medium containing IL-3, IL-6, IL-7,
and stem cell factor. For each infection, twelve culture replicates
were conducted. Cells were fed with fresh growth medium every five
days. After 10 days of culture, both suspended and adherent cells
were harvested and stained with the indicated lineage markers.
Virally infected cells (GFP-positive cells) were analyzed for the
lineage profiles using FACS. In all cases, more than 50% of the
cells were GFP positive at the time of analysis. Hematopoietic
precursor cells were infected with vectors that express a control
vector (no microRNA), miR-30, or the hematopoietic microRNAs,
miR-181, miR-132s or miR-223. miR-30 which was cloned from bone
marrow but only detectable in lung and kidney on Northern was used
as negative control.
Results
[0116] 1. Expression of Endogenous microRNAs Ectopic Expression of
microRNAs Using Short miRNA Hairpins.
[0117] The expression of .about.70 nt-microRNA hairpins (miR-132s)
using the H1 expression cassette of the "double-copy" retroviral
constructs compared with the "single-copy" constructs was
determined using Northern blot analysis. This "double-copy" (DC)
configuration, provided robust and consistent expression of the
microRNA hairpin precursors. Although the transcribed microRNA
hairpin precursors (band depicted with arrow in FIG. 1b) were in
abundance, mature microRNA products were not detected in 293T,
NIH3T3 or bone marrow cells (FIG. 1b). The reduction of the
hairpins to the minimal-length miR-223 did not detectably improve
the efficiency of microRNA processing (FIG. 1c). The substitution
of a perfect match microRNA complementary strand miR-223-Si and
miR-132s-Si also did not significantly improve the efficiency of
microRNA processing (FIG. 1c). These results demonstrated that
short miRNA hairpins, while effectively transcribed in variety cell
types using the double-copy expression constructs, cannot be
effectively processed into mature miRNAs of endogenous forms.
Changing the miRNA hairpin into a siRNA-like stem-loop (i.e., from
the normally non-perfectly paired stem-loop to a perfectly paired
stem-loop), did not significantly improve the miRNA processing
efficiency. The resulting small RNA products, when detected are
typically different from the endogenous miRNAs in their size and
heterogeneity pattern (FIG. 1c). All these results suggest that
additional elements, beside the hairpin itself, are required for
proper miRNA expression and processing, and that additional
elements might be located within a larger miRNA genes that contains
the miRNA hairpin.
Expression of microRNA+Flanking Sequences.
[0118] We hypothesized that elements required for miRNA maturation
might be contained in the genomic sequences flanking a predicted
miRNA hairpin. To test this, we amplified miR-223 genes of
different length, which contained the miR-223 minimal hairpin and
the indicated length of genomic sequences flanking the predicted
miRNA-223 hairpin (FIG. 2a). These gene segments were cloned into
the H1 expression cassette of the double-copy MSCV constructs (FIG.
1a). Northern analysis of these miR-223 expression revealed that
miR-223 genes at least 137 nucleotides long could be effectively
processed into the hairpin precursor (FIG. 2b, arrow, P) and mature
miR-223 (FIG. 2b, arrow, miR). The matured miR-223 RNAs have the
sizes and heterogeneity pattern similar to the endogenous bone
marrow miR-223 RNAs. Interestingly, the smaller miR-223 genes
resulted accumulation of miR-223 gene transcripts that were not
processed into the hairpin precursor and mature miR-223 to (FIG.
2b).
[0119] Further analysis of the cytoplasmic (FIG. 2c) and nuclear
(FIG. 2d) localization of miR-223 transcripts and processed
products revealed that miRNA processing was not limited by nuclear
export of miR-223 transcripts. Abundant miR-223 transcripts
(miR-223-67Si to miR-223.sub.--107) were present in the cytoplasm
but were not processed into miR-223 hairpin precursor and mature
miRNA (FIG. 2c). This result demonstrates that the Dicer enzyme (a
cytoplasmic enzyme that is partly responsible for miRNA processing)
cannot directly process these longer hairpins, despite their
presence in the cytoplasm. When the longer miR-223 genes were used
(miR-223.sub.--137 to miR-223.sub.--500), mature miR-223 can be
readily detected on the northern blot (FIG. 2b). Interestingly, a
band of the size of the predicted hairpin precursor of miR-223 was
also observed in these lanes (FIG. 2b, P). Furthermore, mature
miR-223 and its hairpin precursor can only be seen in cytoplasm
fraction, but not in the nuclear fraction. Take together, these
results suggest that a pre-Dicer processing step is be required to
generate the miRNA hairpin precursor that can be recognized by
Dicer. The flanking sequence of miRNA hairpin gene is essential for
this non-Dicer processing step.
[0120] Based on the example of miR-223 gene expression (FIG. 3), 40
nt or longer flanking sequence on one or both sides of the miR-223
hairpin precursor is required for miR-223 expression and
maturation. The lower limit of the length of the flanking sequences
may vary from one miRNA gene to another. Thus, to ensure the
flanking sequences contain proper processing signal, we choose to
express miRNA genes with 125 nt flanking sequences amplified from
predicted miRNA genomic loci. Amplified miRNA genes were place into
the H1 expression cassette of the double-copy retroviral construct.
We were able to express miR-223, miR-181 and miR-132s (FIG. 3a-c),
as well as 10 other miRNA tested (data not shown). Over-expression
of a hematopoietic miRNA can also be achieved in hematopoietic
progenitor cells, where the endogeneous miRNA is also present (FIG.
3c).
[0121] We also compared the expression of miR-30 when expressed
from a shorter hairpin (71 nt) or a longer (272 nt) miRNA gene. The
longer miR-30 gene can be efficiently expressed and processed into
the hairpin precursor and mature miR-30 (FIG. 4a). While trace
amount of mature miR-30 is processed from a shorter hairpin (71
nt), its processing is very inefficient. Moreover, we noted that
using miRNA flanking-sequence to facilitate miRNA expression not
only increased miRNA processing efficient but also helped to
maintain asymmetric miRNA expression. For example, only the miR-30
strand but not the miR-30* strand can be detected when miR-30 was
expressed from the longer (272 nt) precursor (FIG. 4). In contrast,
others have shown that both miR-30 and miR-30* strand were detected
when miR-30 was expressed from a shorter hairpin precursor placed
in the context of heterogenous mRNA transcripts (Zeng et al. Mol.
Cell, 9:1327-1333). Maintaining asymmetrical miRNA processing may
be critical for ensuring the specificity of miRNA regulation in
vivo.
Ectopically Expressed miRNAs Repress Reporter Gene Expression.
[0122] A luciferase reporter system was generated for testing
microRNA-mediated repression in mammalian cells (FIG. 5a). The
firefly luciferase gene was fused to a mutated C. elegans lin-41
3'UTR (lin-41 3'UTR-delta) in which an 80-nt segment containing
both let-7 complementary sites (green) was deleted, and replaced
with a miR-223 perfect complementary site. In a control reporter,
the Renilla luciferase gene was fused to the lin-41 3'UTR-delta.
All luciferase genes are under the control of the viral LTR
promotor. Virus was produced by transfecting the constructs into
the BOSC23 viral packaging cell line and titered by infecting
NIH3T3 cells. NIH3T3 cells stably expressing miR-223 or vector were
infected with equal titer of fLuc-lin41UTRdelta+miR-223 target or
fLuc-lin41UTRdelta-miR-223 target virus along with control virus
rLuc-lin41UTRdelta. Four days after infection, luciferase activity
was measured and firefly luciferase activity was normalized using
Renilla reporter. The ectopically expressed miR-223 reduced by
4-fold expression of a luciferase reporter gene that had a miR-223
complementary site within its 3' UTR, confirming that the miRNA was
incorporated into a miRNA ribonucleoprotein complex (miRNP) and was
capable of gene silencing (FIG. 5b).
2. Expression of Artificial-microRNA/siRNAs Utilizing
miRNA-Processing Signals
[0123] DNA vectors that express perfect complementary short
hairpins RNAs are commonly used to generate functional siRNAs.
However, the efficacy of gene silencing mediated by different
short-hairpin derived siRNAs is inconsistent, and a substantial
number of short-hairpin siRNA expression vectors can trigger an
anti-viral interferon response (Nature Genetics, 2003, 34:263).
Moreover, siRNA short-hairpins are typically processed
symmetrically, in that both the functional siRNA strand and its
complement strand are incorporated into the RISC complex. Entry of
both strands into the RISC can decrease the efficiency of the
desired regulation and increase the number of off-target mRNAs that
are influenced. In comparison, endogenous miRNA processing and
maturation is a fairly efficient process that is not expected to
trigger an anti-viral interferon response. This process involves
sequential steps that are specified by the information contained in
miRNA hairpin and its flanking sequences (FIG. 2). Thus, we
designed a novel strategy to express artificial-microRNA/siRNAs
utilizing miRNA-processing signals.
[0124] To this end, we selected the B1_C02-3 miRNA as a template
for artificial-microRNA/siRNA expression (FIG. 6). Shown in FIG. 6a
are the predicted stem-loop structure of B1_C02-3 miRNA and the
actual sequence of B1_C02-3 miRNA (Green letters). Based on cloning
analysis, all B1_C02-3 clones are 23 nt in length, and no miR*
strand was found in cloning, while B1_C-02-3 was cloned over 18
times. Consistent with cloning analysis, northern analysis of
ectopically expressed B1_C02-3 revealed that the expressed miRNA
was asymmetrically processed into a defined length (FIG. 6b). In
comparison, many miRNAs are processed into different lengths with
heterogeneity mostly seen in the 3' end; a few miRNAs are also not
asymmetrically processed in that both miR and miR* strands can be
found at similar frequency. Thus, we hypothesized that the
information for specific B1_C02-3 processing is defined by its
stem-loop and flanking sequences, and this information can be
utilized to express artificial-microRNA/SiRNA with defined length
and asymmetrical strand accumulation.
[0125] Based on this hypothesis we designed the following strategy
to express artificial-microRNA/siRNAs utilizing B1_C02-3 templates
(FIG. 7). In brief, a B1_C02-3 gene of about 273 nt in length
containing the miRNA and 125 nt flanking sequences on each side of
the miRNA was cloned into the double-copy retroviral expression
constructs. This construct can effectively express B1_C02-3 (FIG.
6b). A XhoI restriction enzyme site was introduced into the
B1_C02-3 hairpin flanking sequence. This allowed us to easily
replace the B1_C02-3 miRNA hairpin with an
artificial-microRNA/siRNA hairpin. Some features of the B1_C02-3
miRNA hairpin structure, the loop and buldges (indicated with
arrows in FIG. 7) surrounding the miRNA stem, were preserved in all
artificial-microRNA/siRNA hairpins. These features might be
important to determine the boundary of miRNA processing. A stem
formed by an artificial-microRNA/siRNA sequence (with a length of
19 to 23 nt) and its perfect complement strand replaced the
B1_C02-3 miRNA stem. Expression of the artificial-microRNA/siRNA
and B1_C02-3 chimeric gene should produce an asymmetric
artificial-microRNA/SiRNA sequence with "CA" on its 5'end (FIG. 7).
Because the expression and processing of artificial-microRNA/SiRNA
utilizes the miRNA processing machinery, the final
artificial-microRNA/SiRNA products should be effectively
incorporated into the functional miRNP/RISC.
[0126] To test this design, artificial-miRNAs/SiRNAs were designed
to target 21-nucleotide unique sequences in the firefly luciferase
gene. Northern blot analyses (FIG. 8), using probes against sense
and antisense strands of the artificial-miRNAs/siRNAs, were used to
determine the expression and processing of SiLuc-1276 and
SiLuc-311. In both cases, we observed asymmetrically processed
SiLuc-1276 and SiLuc-311 products of .about.23 nt in length. These
results demonstrate that our design properly preserved processing
information for B1_C02-3, and can be used to express virtually any
desired artificial-miRNAs/siRNAs.
[0127] We analyzed the function of ectopically expressed
artificial-miRNAs/siRNAs in their ability to mediate gene
silencing. A series of artificial-miRNAs/SiRNAs expression
constructs were designed to target a luciferase reporter gene.
NIH3T3 cells stably expressing artificial-miRNAs/siRNAs or vector
were infected with the firefly Luciferase reporter along with a
control Renilla reporter. Four days after infection, luciferase
activity was measured and firefly luciferase activity was
normalized using the Renilla reporter. The ectopically expressed
artificial-miRNAs/siRNAs against Luciferase can specifically reduce
firefly luciferase activity up to 90%. Four out of five constructs
could reduce reporter expression by at least 60%. These results
confirm that the expressed artificial-miRNAs/SiRNAs was
incorporated into a miRNA ribonucleoprotein complex (miRNP/RISC)
and was capable of gene silencing (FIG. 9).
3. Expression of miRNAs Using Different Promoters Expression of
microRNAs from Pol II and Pol III Promoters.
[0128] One strategy to express the miRNA is to include a
sufficiently large DNA fragment such that the miRNA is expressed
under the control of its native promoter. A second strategy is to
use heterologous promoters. We have demonstrated that a microRNA
can be effectively expressed and processed from a microRNA
transcript containing the microRNA and corresponding genomic
flanking sequences using a pol III expression cassette. In FIG. 10
we also show that pol II heterologous promoters can be used.
Inducible Expression of microRNA Using the TetOn System.
[0129] (FIG. 10) One strategy to express the miRNA is to include a
sufficiently large DNA fragment such that the miRNA is expressed
under the control of its native promoter. A second strategy is to
use heterologous promoters. We have demonstrated that a microRNA
can be effectively expressed and processed from a microRNA
transcript containing the microRNA and corresponding genomic
flanking sequences using a pol III expression cassette. In FIG. 10
we also show that pol II heterologous promoters can be used. Such
promoters include those that are used in available mammalian
expression systems including tissue-specific promoters and
inducible promoters. TetOn system is a commercial inducible
expression system from Clontech Inc. This is of particular interest
because current siRNA expression systems utilize pol III promoters,
which are difficult to adapt for inducible expression. In FIG. 10,
we showed the expression of miRNA (B1_C02-3) using the Clontech
TetOn expression system. A B1_C02-3 gene with 500 nt flanking
sequence was cloned into the pRev-TRE vector. This construct was
then packaged into retrovirus and used to infect a Tet-On cell line
expressing the reverse tetracycline-controlled transactivator
(rtTA). B1_C02-3 is inducibly expressed in response to varying
concentrations of the teratcycline derivate doxycycline (Dox).
Similarly, this system can be used for expression of artificial
microRNAs.
4. miRNAs Modulate Hematopoietic Lineage Differentiation Expression
of microRNAs in Hematopoietic Tissues.
[0130] To investigate whether microRNAs play a role in mammalian
development and in particular might regulate mammalian
hematopoiesis, we cloned more than 70 unique microRNAs from mouse
bone marrow. miR-132s, the microRNA found at a breakpoint of a
t(8:17) translocation associated with an aggressive B-cell leukemia
(Gauwerky et al., 1989, Proc. Natl. Acad. Sci. USA, 86:8867-8871),
was expressed in all four hematopoietic tissues tested: fetal
liver, and adult bone marrow, spleen, and thymus, with little or no
expression in the non-hematopoietic tissues. The lung was the only
other tissue with appreciable miR-132s expression (FIG. 11).
Expression in E13 fetal liver suggested that miR-132s might
function in early hematopoietic development. Expression of mature
miR-132s was highest in thymus, the primary lymphoid organ that
mainly contains T-lymphocytes. Expression was much lower in bone
marrow, which consists of hematopoietic stem cells and myeloid,
erythroid and lymphoid cells at a variety of differentiation
stages, and spleen. Interestingly, accumulation of the presumed
miR-132 precursor (.about.60 nt band, FIG. 11) was high and the
ratio of mature to precursor 21 nt RNAs varied in different
tissues, suggesting post transcriptional regulation of microRNA
expression at the level of precursor processing or RNA
stability.
[0131] miR-223 was very strongly expressed in bone marrow and was
detectable in spleen but essentially absent in E13 fetal liver,
thymus, and all other adult mouse tissues tested (FIG. 11). miR-181
was very strongly expressed in thymus and was detectable in bone
marrow and spleen but essentially negative in E13 fetal liver and
all other mouse tissues tested except for high expression in brain
and lung. miR-181 and miR-223 expression in fetal liver was barely
detectable, suggesting that they only function in adult
hematopoiesis. Thus miR-181, miR-223 and miR-132s are
differentially expressed in hematopoietic tissues and their
expression is regulated during development.
microRNAs are Regulated in Hematopoietic Lineage Commitment.
[0132] Because bone marrow, spleen, and thymus, each have
specialized functions in adult hematopoiesis and comprise
significantly different cell types, the differential expression of
the miRNAs in these complex tissues suggested that individual
hematopoietic cell types might differentially express the miRNAs.
Indeed, when cells within bone marrow were sorted based on lineage
markers, they were found to to differentially express the
hematopoietic miRNAs (FIG. 12). In contrast, expression of miR-16,
an miRNA seen in a broad range of tissues, was more constant.
[0133] Mature miR-181 expression in the bone marrow cells was
detectable in undifferentiated progenitor cells (Lin.sup.-) and
up-regulated in the differentiated B-lymphocytes, marked by the
B220 surface antigen. In other differentiated lineages, miR-181
expression did not increase over that seen in Lin.sup.- cells. Note
that sorted lineage cell populations are about 85% pure, thus some
residual miRNA signal in the other lineages might be caused by
contamination of B220.sup.+ cells. Expression of the miR-181
precursor was at similar levels in all lineages, suggesting that
the differential accumulation of the mature miR-181 during
hematopoietic lineage commitment might be regulated at the level of
miRNA processing or the rate of turnover.
[0134] miR-223 expression was confined to myeloid lineages
(Gr-1.sup.+ and Mac-1.sup.+), with barely detectable expression in
T- and B-lymphoid and erythroid lineages (CD3e.sup.+, B220.sup.+,
and Ter119.sup.+, respectively; FIG. 12). This observation is
consistent with miR-223 expression in bone marrow but not spleen
and thymus (FIG. 11). miR-132s expression was lowest in the
erythroid lineage (Ter-119.sup.+) and highest in myeloid lineages
(Gr-1.sup.+ and Mac-1.sup.+), consistent with its ubiquitous
expression in bone, spleen and thymus (FIGS. 11 and 12).
[0135] For each of the miRNAs, specific expression was validated by
the reduction of correspondent miRNA expression in the reciprocal
lineage-depleted cell populations (FIG. 12, right panels). In
addition, expression of all four miRNAs was low in Lin cells
relative to their preferred Lin.sup.+ cell populations, suggesting
that these miRNAs were induced upon lineage commitment and
differentiation.
microRNAs are Capable of Modulating Hematopoietic Lineage
Commitment.
[0136] Hematopoietic progenitor cells from mouse bone marrow were
infected with the viral vectors expressing either miR-181, miR-223,
miR-132s, or a control miRNA, miR-30. Interestingly, ectopic
expression of each of these microRNAs in hematopoietic bone marrow
Lin.sup.- progenitor cells have differential effects on the
differentiation of bone marrow progenitor cells, particularly the
differentiation of T- or B-lymphoid lineages, which are indicated
by the expression of Thy-1.2 or CD-19 cell surface antigens (FIG.
13).
[0137] About 23.+-.2.5% (n=12) or 12.+-.2.3% (n=12) differentiated
cells expressed Thy-1.2 or CD-19 antigen, respectively, when
infected with control vector. Over-expression of miR-30 did not
significantly alter the ratio of T- and B-lymphoid lineage cells,
in that the marker expression was essentially unchanged compared to
that of cells infected with the empty vector. This indicated that
merely expressing an arbitrary microRNA did not markedly influence
lymphoid differentiation. In contrast, about 21.+-.4.2% (n=12) or
26.+-.1.7% (n=12) differentiated cells expressed Thy-1.2 or CD-19
antigen, respectively, when infected with miR-181 expression virus.
Thus, expression of miR-181 substantially affected the lineage
differentiation, resulting in more than a 2-fold increase in
B-lymphoid lineage with little change in T-lymphoid lineage. In
contrast, ectopic expression of miR-132s and miR-223 resulted in
opposite effects, with a 30.about.40% increase in the T-lymphoid
lineage and a slight reduction in the B-lymphoid lineage, and more
significant change in the ratio of T/B-lineage cells which
increased from .about.2-fold (vector) to 2.5 (miR-223) or 4.5-fold
(miR-132s), respectively.
[0138] Modest effects were seen when analyzing these cells for
myeloid lineage markers (FIG. 13c and d). Neutrophils are Mac-1 and
Gr-1 double-positive cells (Mac-1.sup.+ Gr-1.sup.+), whereas
monocytes are Mac-1 positive and Gr-1 negative-to-low (Mac-1.sup.+
Gr-1.sup.-/low). Non-myeloid cells were Mac-1 and Gr-1
double-negative (Mac-1.sup.- Gr-1.sup.-); they mostly expressed
Thy-1.2 or CD-19 lymphoid markers. Overexpression of the control
miR-30 had little if any effect on Mac-1 and Gr-1 expression.
Over-expression of either miR-132s or miR-181 led to a small
decrease in Mac-1.sup.+ Gr-1.sup.-/low cells but expression of
miR-181 led to a noticeable increase in Mac-1.sup.+ Gr-1.sup.+
cells.
[0139] The demonstration that certain miRNAs are differentially
expressed in hematopoietic lineages in vivo and are able to alter
lineage commitment in vitro provides solid evidence that microRNAs
represent a class of molecules that regulate mammalian development.
This supports the notion that the roles of translational regulation
in to hematopoiesis and, more broadly, vertebrate development might
have been under-appreciated. Furthermore, modulating the expression
of miRNAs can have therapeutic utility.
[0140] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. Various modifications of the invention in addition to
those shown and described herein will become apparent to those
skilled in the art from the foregoing description and fall within
the scope of the appended claims. The advantages and objects of the
invention are not necessarily encompassed by each embodiment of the
invention.
Sequence CWU 1
1
2816963DNAArtificial SequenceSynthetic oligonucleotide 1tgaaagaccc
cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 60ggaaaataca
taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
120gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc
agggccaaga 180acagatggtc cccagatgcg gtcccgccct cagcagtttc
tagagaacca tcagatgttt 240ccagggtgcc ccaaggacct gaaaatgacc
ctgtgcctta tttgaactaa ccaatcagtt 300cgcttctcgc ttctgttcgc
gcgcttctgc tccccgagct caataaaaga gcccacaacc 360cctcactcgg
cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat
420aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga
gggtctcctc 480agattgattg actgcccacc tcgggggtct ttcatttgga
ggttccaccg agatttggag 540acccctgcct agggaccacc gacccccccg
ccgggaggta agctggccag cggtcgtttc 600gtgtctgtct ctgtctttgt
gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg 660tactagttag
ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttctga
720acacccggcc gcaaccctgg gagacgtccc agggactttg ggggccgttt
ttgtggcccg 780acctgaggaa gggagtcgat gtggaatccg accccgtcag
gatatgtggt tctggtagga 840gacgagaacc taaaacagtt cccgcctccg
tctgaatttt tgctttcggt ttggaaccga 900agccgcgcgt cttgtctgct
gcagcgctgc agcatcgttc tgtgttgtct ctgtctgact 960gtgtttctgt
atttgtctga aaattagggc cagactgtta ccactccctt aagtttgacc
1020ttaggtcact ggaaagatgt cgagcggatc gctcacaacc agtcggtaga
tgtcaagaag 1080agacgttggg ttaccttctg ctctgcagaa tggccaacct
ttaacgtcgg atggccgcga 1140gacggcacct ttaaccgaga cctcatcacc
caggttaaga tcaaggtctt ttcacctggc 1200ccgcatggac acccagacca
ggtcccctac atcgtgacct gggaagcctt ggcttttgac 1260ccccctccct
gggtcaagcc ctttgtacac cctaagcctc cgcctcctct tcctccatcc
1320gccccgtctc tcccccttga acctcctcgt tcgaccccgc ctcgatcctc
cctttatcca 1380gccctcactc cttctctagg cgccggaatt agatcttata
tggggcaccc ccgccccttg 1440taaacttccc tgaccctgac atgacaagag
ttactaacag cccctctctc caagctcact 1500tacaggctct ctacttagtc
cagcacgaag tctggagacc tctggcggca gcctaccaag 1560aacaactgga
ccgaccggtg gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc
1620gccgacacca gactaagaac ctagaacctc gctggaaagg accttacaca
gtcctgctga 1680ccacccccac cgccctcaaa gtagacggca tcgcagcttg
gatacacgcc gcccacgtga 1740aggctgccga ccccgggggt ggaccatcct
ctagactgcc ggatcaattc ctaccgggta 1800ggggaggcgc ttttcccaag
gcagtctgga gcatgcgctt tagcggcccc gctgggcact 1860tggcgctaca
caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa
1920ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct
agtcaggaag 1980ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac
aaatggaagt agcacgtctc 2040actagtctcg tgcagatgga cagcaccgct
gagcaatgga agcgggtagg cctttggggc 2100agcggccaat agcagctttg
gctccttcgc tttctgggct cagaggctgg gaaggggtgg 2160gtccgggggc
gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg
2220aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct
cctcttcctc 2280atctccgggc ctttcgacct gcaccatggt gagcaagggc
gaggagctgt tcaccggggt 2340ggtgcccatc ctggtcgagc tggacggcga
cgtaaacggc cacaagttca gcgtgtccgg 2400cgagggcgag ggcgatgcca
cctacggcaa gctgaccctg aagttcatct gcaccaccgg 2460caagctgccc
gtgccctggc ccaccctcgt gaccaccttc acctacggcg tgcagtgctt
2520cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
tgcccgaagg 2580ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
aactacaaga cccgcgccga 2640ggtgaagttc gagggcgaca ccctggtgaa
ccgcatcgag ctgaagggca tcgacttcaa 2700ggaggacggc aacatcctgg
ggcacaagct ggagtacaac tacaacagcc acaacgtcta 2760tatcatggcc
gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
2820cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
tcggcgacgg 2880ccccgtgctg ctgcccgaca accactacct gagcacccag
tccgccctga gcaaagaccc 2940caacgagaag cgcgatcaca tggtcctgct
ggagttcgtg accgccgccg ggatcactct 3000cggcatggac gagctgtaca
agtaatgaat taattaagaa ttgcggccgc gtcgacctgc 3060agccaagctt
atcgataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga
3120aagaccccac ctgtaggttt ggcaagaatt cgtttaaacg ggctcgagtg
gtctcataca 3180gaacttataa gattcccaaa tccaaagaca tttcacgttt
atggtgattt cccagaacac 3240atagcgacat gcaaatattg ggatccgcta
gcttaagtaa cgccattttg caaggcatgg 3300aaaatacata actgagaata
gagaagttca gatcaaggtt aggaacagag agacagcaga 3360atatgggcca
aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac
3420agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc
agatgtttcc 3480agggtgcccc aaggacctga aaatgaccct gtgccttatt
tgaactaacc aatcagttcg 3540cttctcgctt ctgttcgcgc gcttctgctc
cccgagctca ataaaagagc ccacaacccc 3600tcactcggcg cgccagtcct
ccgatagact gcgtcgcccg ggtacccgtg tatccaataa 3660accctcttgc
agttgcatcc gacttgtggt ctcgctgttc cttgggaggg tctcctctga
3720gtgattgact acccgtcagc gggggtcttt catgggtaac agtttcttga
agttggagaa 3780caacattctg agggtaggag tcgaatatta agtaatcctg
actcaattag ccactgtttt 3840gaatccacat actccaatac tcctgaaata
gttcattatg gacagcgcag aagagctggg 3900gagaattaat tcgtaatcat
ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 3960aattccacac
aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
4020gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
gaaacctgtc 4080gtgccagctg cattaatgaa tcggccaacg cgcggggaga
ggcggtttgc gtattgggcg 4140ctcttccgct tcctcgctca ctgactcgct
gcgctcggtc gttcggctgc ggcgagcggt 4200atcagctcac tcaaaggcgg
taatacggtt atccacagaa tcaggggata acgcaggaaa 4260gaacatgtga
gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
4320gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
caagtcagag 4380gtggcgaaac ccgacaggac tataaagata ccaggcgttt
ccccctggaa gctccctcgt 4440gcgctctcct gttccgaccc tgccgcttac
cggatacctg tccgcctttc tcccttcggg 4500aagcgtggcg ctttctcata
gctcacgctg taggtatctc agttcggtgt aggtcgttcg 4560ctccaagctg
ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
4620taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
cagcagccac 4680tggtaacagg attagcagag cgaggtatgt aggcggtgct
acagagttct tgaagtggtg 4740gcctaactac ggctacacta gaaggacagt
atttggtatc tgcgctctgc tgaagccagt 4800taccttcgga aaaagagttg
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 4860tggttttttt
gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
4920tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
aagggatttt 4980ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
ttaaattaaa aatgaagttt 5040taaatcaatc taaagtatat atgagtaaac
ttggtctgac agttaccaat gcttaatcag 5100tgaggcacct atctcagcga
tctgtctatt tcgttcatcc atagttgcct gactccccgt 5160cgtgtagata
actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
5220gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
ccggaagggc 5280cgagcgcaga agtggtcctg caactttatc cgcctccatc
cagtctatta attgttgccg 5340ggaagctaga gtaagtagtt cgccagttaa
tagtttgcgc aacgttgttg ccattgctac 5400aggcatcgtg gtgtcacgct
cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 5460atcaaggcga
gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
5520tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
tggcagcact 5580gcataattct cttactgtca tgccatccgt aagatgcttt
tctgtgactg gtgagtactc 5640aaccaagtca ttctgagaat agtgtatgcg
gcgaccgagt tgctcttgcc cggcgtcaat 5700acgggataat accgcgccac
atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 5760ttcggggcga
aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
5820tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
ggtgagcaaa 5880aacaggaagg caaaatgccg caaaaaaggg aataagggcg
acacggaaat gttgaatact 5940catactcttc ctttttcaat attattgaag
catttatcag ggttattgtc tcatgagcgg 6000atacatattt gaatgtattt
agaaaaataa acaaataggg gttccgcgca catttccccg 6060aaaagtgcca
cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
6120gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
acctctgaca 6180catgcagctc ccggagacgg tcacagcttg tctgtaagcg
gatgccggga gcagacaagc 6240ccgtcagggc gcgtcagcgg gtgttggcgg
gtgtcggggc tggcttaact atgcggcatc 6300agagcagatt gtactgagag
tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag 6360gagaaaatac
cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg
6420atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
ctgcaaggcg 6480attaagttgg gtaacgccag ggttttccca gtcacgacgt
tgtaaaacga cggccagtgc 6540caagctcgct ctcccttatg cgactcctgc
attaggaagc agcccagtag taggttgagg 6600ccgttgagca ccgccgccgc
aaggaatggt gcatgcaagg agatggcgcc caacagtccc 6660ccggccacgg
ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
6720cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
cgcacctgtg 6780gcgccggtga tgccggccac gatgcgtccg gcgtagagga
tcgattagtc caatttgtta 6840aagacaggat atcagtggtc caggctctag
ttttgactca acaatatcac cagctgaagc 6900ctatagagta cgagccatag
ataaaataaa agattttatt tagtctccag aaaaaggggg 6960gaa
6963223DNAArtificial sequenceSynthetic microRNA 2caaagtgctg
ttcgtgcagg tag 23316DNAArtificial sequenceSynthetic microRNA
3gcaggcaaaa ccccaa 16421DNAArtificial sequenceSynthetic microRNA
4aacattcaac gctgtcggtg a 21519DNAArtificial sequenceSynthetic
microRNA 5cccaaaagag aaagcacac 19622DNAArtificial sequenceSynthetic
microRNA 6tgtaaacatc ctacactcag ct 22713DNAArtificial
sequenceSynthetic microRNA 7gaggagaggg agg 13813DNAArtificial
sequenceSynthetic microRNA 8gaggagagga cag 13915DNAArtificial
sequenceSynthetic microRNA 9agcagcacaa agggg 151017DNAArtificial
sequenceSynthetic microRNA 10agcagcacga aaaggcg 171118DNAArtificial
sequenceSynthetic microRNA 11ggcaaaccag caaaacga
181216DNAArtificial sequenceSynthetic microRNA 12aaaggcaagg caggag
161316DNAArtificial sequenceSynthetic microRNA 13acacagccag ggacca
161414DNAArtificial sequenceSynthetic microRNA 14cacagggcaa gcgc
141516DNAArtificial sequenceSynthetic microRNA 15agcaccacga aacgga
161617DNAArtificial sequenceSynthetic microRNA 16gaaacaccac acccagc
171715DNAArtificial sequenceSynthetic microRNA 17agcacagacg aggac
151820DNAArtificial sequenceSynthetic microRNA 18caacggaacc
caaaagcagc 201917DNAArtificial sequenceSynthetic microRNA
19ccgacgagcg ccccgag 172015DNAArtificial sequenceSynthetic microRNA
20aaaagcaaga gacga 152112DNAArtificial sequenceSynthetic microRNA
21gaggaagggc ga 122221DNAArtificial sequenceSynthetic microRNA
22accacaggga gaaccacgga c 2123163RNAArtificial sequenceSynthetic
precursor microRNA 23nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
gcuccannnn nnnnnnnnnn 60nnnnnnnnnn nnnnuguaau uaccugacnn nnnnnnnnnn
nnnnnnnnnn nnnnnncccg 120agcnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnn 1632464RNAArtificial sequenceSynthetic microRNA
24uccaaagugc uguucgugca gguaguguaa uuaccugacc uacugcugag cuagcacuuc
60ccga 642578RNAArtificial sequenceSynthetic microRNA 25ugggggcucc
aaagugcugu ucgugcaggu aguguaauua ccugaccuac ugcugagcua 60gcacuucccg
agccccca 782611DNAArtificial sequenceSynthetic DNA/microRNA
26ctcgagcucc a 112714DNAArtificial sequenceSynthetic DNA/microRNA
27uguaauuacc ugac 142812DNAArtificial sequenceSynthetic
DNA/microRNA 28cccgagctcg ag 12
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